Documentos de Académico
Documentos de Profesional
Documentos de Cultura
Plenary Lectures:....................................................................................................................... 1
White Biotechnology
Session 1:
Session 2:
Session 3:
Bioeconomy................................................................................................................................ 22
Chair: Stanisaw Bielecki
Session 4:
Session 5:
Pharmaceutical biotechnology................................................................................................ 33
Chair: Alicja Jzkowicz, Katarzyna Kie-Kononowicz
Green Biotechnology
Session 6:
Session 7:
Environmental biotechnology................................................................................................. 75
Chair: Katarzyna Turnau
Session 8:
Session 9:
Session 10:
Session 11:
Satellite Panels
Session 1:
Session 2 :
Plenary lectures
PL1
PL2
Eurobiotech 2013
PL3
PL4
Stanisaw Karpiski
Warsaw University of Life Sciences,
Department of Genetics, Breeding and Plant Biotechnology,
Nowoursynowska 159, 02-776 Warsaw, Poland
Lectures
L1.2
L1.1
Eurobiotech 2013
Acknowledgements
This work was supported by National Centre for Science and Ministry
of Science and Higher Education of Poland under project No. MNiSW/
DPN/4878/TD/2010.
References
[1] Carp O., Huisman C.L., Reller A. (2004) Photoinduced reactivity of
titanium dioxide, Prog. Solid State Chem. 32: 33177.
[2] Nakata K., Fujishima A., (2012) TiO2 photocatalysis: design and
applications. J. Photochem. Photobiology C: Photochem. Rev. 13:
169189.
[3] Markowska-Szczupak A., Ulfig K., Morawski A.W. (2011) The
application of titanium dioxide for deactivation of bioparticulates: an
overview, Catal. Today, 169: 249257.
[4] Rozhkova E., Ulasov I., Lai B., Dimitrijevic N.M., Lesniak M.S., Rajh
T. (2009) Nanobio photocatalyst for targeted brain cancer therapy, Nano
Lett. 9: 33373342.
L1.3
Nanosilver a new product for disinfecting
treatment
Marcin Banach1, Jolanta Pulit1, Leszek Tymczyna2,
Anna Chmielowiec-Korzeniowska2
Institute of Inorganic Chemistry and Technology, Crakow
University of Technology; 2University of Life Sciences in Lublin
1
Eurobiotech 2013
References
[1] Burrell R. (1995) Ann. Agric. Environ. Med. 2, 11.
[2] Kowalski Z., Banach M., Powaka E. (2009) Przem. Chem. 88, 478.
[3] Konopka M., Kowalski Z., Wzorek Z. (2009) Arch. Environ. Prot.
35, 107.
[4] Banach M., Kowalski Z., Wzorek Z. (2007) Chemik 9, 435.
[5] Wright J.B., Lam K., Hansen D., Burrell R.E. (1999) Am. J. Infect.
Control 27, 344.
[6] Banach M., Pulit J. (2013) Przem. Chem. 92, 6, 1056.
L1.4
Pig manure treatment by filtration
Zygmunt Kowalski1, Agnieszka Makara1, Dalibor Matsek2,
Jzef Hoffmann3, Krystyna Hoffmann3
Institute of Chemistry and Inorganic Technology,
Crakow University of Technology, Warszawska 24,
31-155 Crakow, Poland; 2Institute of Geological Engineering,
VB-Technical University of Ostrava, 17. listopadu 15,
708 33 Ostrava-Poruba, Czech Republic; 3Institute of Inorganic
Technology and Mineral Fertilizers, Wroclaw University
of Technology, Wybrzeze Wyspianskiego 27, 50-370 Wroclaw,
Poland
1
Numerous livestock farms in Europe import fodders including the nitrogen (N) and phosphorus (P)-bearing
ingredients that these fodders contain. This results in
local nutrient surpluses because home-grown crops take
up less N and P than available in the manure. We have
developed in our study new pig manure treatment and
filtration process. The advantage of worked out technology is the method of incorporation in organic phase of the
manure of 4059% of crystalline phase. This resulted in
achievement of high filtration rate over 1000 L/m2/h with
the used pressure filters and good quality of filtrate. The
method is able to maintain an overall average pollutant
removal performance ~90% for the chemical oxide demand COD, > 99% for the suspended solids SS, to 47%
for the total nitrogen content determined with Kjeldahls
method TKN. Preliminary investigation indicated that
the mineralization process eliminates over 75% of the
odor intensity coming from the filtrate obtained in comparison to odor intensity coming from the pig manure.
Acknowledgements
This study has been carried out in the framework of the Development
Project No. 14-0003-10/2010 granted by the National Centre for
Research and Development.
Eurobiotech 2013
L1.5
L1.6
Eurobiotech 2013
Posters
P1.2
P1.1
Nanoemulsions offer several advantages for cosmeceutical use. They are easy to prepared, they characterized
by long term stability, high solubilization capacity for
hydrophilic and lipophilic actives and additionally improved transdermal active delivery [1, 2]. The formation
of O/W nano-emulsions suitable for cosmeceutical application was studied. Nano-emulsions were prepared by
using phase inversion composition (PIC) method, one of
low-energy emulsification methods. The process consist
of stepwise water addition to oil/surfactant mixture, at
25C. Caprylic/capric triglycerides, propylene glycol dicaprylate/dicaprate and oleic acid were applied as an oil
phase. Polisorbate 80 was used as the surfactant. Kinetic
stability of the nano-emulsions was analyzed by measuring droplet diameter as a function of time for different
oil/surfactant ratio. The particles size distribution was
analyzed by means DLS measurement technique (Dynamic Light Scattering), using Zetasizer Nano ZS (Malvern Instruments). One of triterpenoic acid, practically
non-water soluble substance was selected as an active and
incorporated into the stable formulation. The obtained
results proved that the nanoemulsion based on caprylic/
capric triglycerides was the most stable one and additionally showed the highest solubilisation for the triterpene.
The maximum concentration of the active achieved in
oil/surfactant mixture (20/80) was 2,5 mg/ml.
Acknowledgements
The research (work) was supported by the European Union through the
research scholarship Doctus Maopolski fundusz stypendialny dla
doktorantw.
References
[1] Maghraby G.M., Transdermal delivery of hydrocortisone from
eucalyptus oil microemulsion: effects of cosurfactants. International
Journal of Pharmaceutics, 2008, 355(12), 285292.
[2] Peltola S., Saarinen-Savolainen P., Kiesvaara J., Suhonen T.M., Urtii A.,
Microemulsions for topical delivery of estradiol, International Journal
of Pharmaceutics, 2003, 254, 99.
Eurobiotech 2013
P1.3
P1.4
Acknowledgements
The research (work) was supported by the European Union through
the European Social Fund within Cracow University of Technology
development program top quality teaching for the prospective
Polish engineers; University of the 21st century project (contract No.
UDA-POKL.04.01.01-00-029/10-00).
References
[1] Mller R.H., Petersen R.D., Hommoss A., Pardeike J. (2007)
Nanostructured lipid carriers (NLC) in cosmetic dermal products, Adv.
Drug Deliv. Rev. 59: 522530.
[2] Radtke M., Souto E., Mller R.H. (2005) Nanostructured lipid
carriers: a novel generation of solid lipid drug carriers, Pharm. Technol.
Eur. 17: 4550.
Eurobiotech 2013
P1.5
Carbon aerogels based on natural starches
synthesis and characterization
Monika Bakierska, Marcin Molenda, Magorzata Maria Zaitz,
Roman Dziembaj
Faculty of Chemistry, Jagiellonian University
Introduction. Carbon aerogels are a novel class of nanostructured and porous carbon materials which exhibit
unusual properties and can be used in a wide variety of
applications (eg. energy storage, catalysis, biomedicine
etc.) [13]. These materials can be obtained by the pyrolysis of organic aerogels at elevated temperatures under
inert atmosphere. The most widely used organics for the
fabrications of the aerogels via the sol-gel polycondensation are resorcinol and formaldehyde [4]. However, the
use of natural polysaccharides and their derivatives is
considered to be more attractive because of their stability,
availability, renewability, low cost and non toxicity [57].
The purpose of this study was to prepare and characterize carbon aerogels based on natural starches of different
botanical origin (potato, maize and rice). Moreover, to
decrease the process cost and shorten processing time, an
ambient pressure drying instead of supercritical drying
was applied.
Experimental. Carbon aerogels were prepared by the
carbonization of organic aerogels. Four main steps can
be distinguished in the preparation of these materials,
and the first was the gelation process in which different
types of starches (potato, maize and rice) were dissolved
in water and stirred while maintaining the appropriate
gelatinization temperature for each type of starch. The
second step was the solvent exchanging carried out either by soaking the gel directly in the new solvent ethanol. The third step was the wet gel drying performed at
50C for 1 day under ambient pressure and the fourth
was the carbonization of dried gel under constant flow
of argon at 600C and after that carbon aerogels were
obtained.
Optimal conditions for carbonization process of organic
aerogels were determined by thermal analysis methods
(TGA/DTG/SDTA/EGA-QMS). The structure and the
morphology of the prepared carbon aerogels were investigated using X-ray powder diffraction (XRD) and low
temperature N2 adsorption measurements (N2-BET).
Electrical properties of the obtained aerogels were examined by electrical conductivity studies (EC) using
4-probe method within temperature range of 20C to
+40C.
Conclusions. To conclude, starch-based carbon aerogels were synthesized by the pyrolysis of organic aerogels, which were prepared in gelatinization process of
natural starches (potato, maize and rice). This method
made it possible to obtain nanostructured materials
with well-developed porosity and high surface area. The
electrical conductivity measurements proved that pre-
10
Eurobiotech 2013
P1.6
P1.7
Acknowledgements
The research was funded from the budget for science for years
20132014 No. IP2012 062872.
References
[1] Weber Z., Kryczyski S. (red.) (2010) Fitopatologia, PWRiL, Pozna.
[2] Nair R., Varghese S.H., Nair B.G., Maekawa T., Yoshida Y.,
Kumar D.S. (2010) Plant Science, 179: 154163.
[3] Rai M., Yadav A., Gade A. (2009) Biotechnology Advances, 27:
7683.
[4] Zielinska A., Skwarek E., Zaleska A., Gazda M., Hupka J. (2009)
Procedia Chemistry 1: 15601566.
Eurobiotech 2013
Acknowledgements
The work is part of a project Synthesis and application of innovative
nanomaterials with antimicrobial properties supported by
National Centre for Research and Development under the contract
No. LIDER/03/146/L-3/11/NCBR/2012 for the period of 20122015.
References
[1] Vaidyanathan R., Kalishwaralal K., Gopalram S., Gurunathan
S., Nanosilver the burgeoning therapeutic molecule and its green
synthesis. Biotechnol. Adv. 2009, 27, 924.
[2] Sadeghi B., Jamali M., Kia S., Amininia A., Ghafari S., Synthesis and
characterization of silver nanoparticles for antibacterial activity. Int. J.
Nano. Dim. 2010, 1, 119.
[3] Pulit J., Banach M., Kowalski Z., Nanosilver making difficult
decisions. Ecol. Chem. Eng. S. 2011, 18, 185.
[4] Hakkinen S. H., Karenlampi S.O., Mykkanen H.M., Heinonen I.M.,
Torronen A.R., Ellagic acid content in berries: Influence of domestic
processing and storage. Eur. Food Res. Technol. 2000, 212, 75.
[5] Bellotti N., Salvatore L., Dey C., Del Panno M.T., del Amo B.,
Romagnoli R., The application of bioactive compounds from the
food industry to control mold growth in indoor waterborne coatings.
Colloids Surf. B, 2013, 104, 140.
11
P1.8
The impact of industrial nanoaerosols
on the pulmonary surfactant
Dorota Kondej1, Tomasz R. Sosnowski2
Central Institute for Labour Protection National Research
Institute/ Department of Chemical, Aerosol and Biological
Hazards/ Czerniakowska 16/ 00-701 Warsaw/ Poland;
2
Warsaw University of Technology/ Faculty of Chemical and
Process Engineering/ Warynskiego 1/ 00-645 Warsaw/ Poland
1
Acknowledgements
This paper has been prepared on the basis of the results of research
project No. I.B.10 carried out within the National Programme
Improvement of safety and working conditions partly supported
in 20112013 within the scope of research and development by the
Ministry of Science and Higher Education. CIOP-PIB has been the
Programme main coordinator.
12
Eurobiotech 2013
P1.9
The impact of nanosilver addition
on element ions release form light-cured
dental composite and compomer into
0,9% NaCl
Krzysztof Sokoowski1, Magorzata Iwona Szynkowska2,
Aleksandra Pawlaczyk2, Jerzy Sokoowski3
Department of Conservative Dentistry, Medical University
of Lodz, Pomorska 251, 92-213 Lodz, Poland; 2Institute
of General and Ecological Chemistry, Technical University
of Lodz, Zeromskiego 116, 90-924 Lodz, Poland; 3Department
of General Dentistry, Medical University of Lodz, Pomorska 251,
92-213 Lodz, Poland
1
Nowadays, a wide spectrum of dental materials is available, material properties depend on the basic material
type e.g. metals, ceramics or polymers. In fact, a detailed
understanding of the key concept for each of them gives
an insight into how each class of material can behave
as a restorative dental material, as well as an idea of the
potential of these materials if some of their limitations
can be overcome. There is no surprise that only certain
specific material properties determine their selection
for application in restorative dentistry. Basing on that
knowledge, it is possible to make a proper selection of
the material being the best compromise of desired properties versus inherent limitations. Due to the fact that
any class of basic type of presented materials possesses
all the desired properties, in many cases they are used in
combination limiting their usefulness [1]. The main objective of this study was to assess the release of metal ions
from dental fillings into 0.9% NaCl solution in different
time intervals. The investigated dental materials were
samples of flowable composite material with nanosilver
addition and flowable compomer material with nanosilver addition, both being a light-cured dental restorative
material with flow characteristics, which makes it ideal
material for filling small cavities in anterior and posterior teeth. Composite restorations seem to represent excellent aesthetics properties, but due to the fact that they
undergo polymerization shrinkage on setting, they are
associated with marginal leakage, which causes bacterial
penetration and, in consequence, the potential damage
to the tooth. Notwithstanding, fluoride releasing materials in combination with silver ions can influence the
surrounding micro environment, involving bacteria.
Each of the samples was first washed with ethanol and
then placed in a test-tube in 10 mL 0.9% NaCl solution.
A number of counts of metal ions released into NaCl
solution from the dental materials were determined using Optimass 8000 ICP-TOF MS spectrometer (GBC
Scientific Equipment, Australia) after a week, a month
and three months time of storage. The results confirmed
the significant increase in the number of counts of metal
ions originating form the main components of studied
dental material with the time of storage. The gathered
spectra were also compared with some previous out-
comes obtained for other restorative materials not containing silver in their composition.
Acknowledgements
The financial support of this work by the Polish Scientific Research
Council (grant N N209 343237 ) is gratefully acknowledged.
Lectures
L2.1
Metabolic Engineering of Non-Conventional
Yeasts for Construction of the Advanced
Producers of Lignocellulosic Ethanol
Andriy A. Sibirny1, 2
Department of Molecular Genetics and Biotechnology,
Institute of Cell Biology, NAS of Ukraine, Drahomanov Street,
14/16, Lviv 79005 Ukraine; 2Department of Biotechnology and
Microbiology, University of Rzeszow, Zelwerowicza 4, Rzeszow
35-601, Poland
1
Non-conventional yeasts are attractive ethanol producers from lignocellulose. One of the most promising
ethanol producer from lignocellulose is Hansenula polymorpha which belongs to the best studied non-conventional yeasts. Its genome has been sequenced and publicly available (http://genomeportal.jgipsf.org/Hanpo2/
Hanpo2.info.html) and methods of molecular genetics
and cell biology are well developed. It is a popular host
system for expression of heterologous proteins and some
metabolites as well as favorite model organism for studying peroxisome biogenesis and autophagic degradation, methanol catabolism and stress response. Besides,
H. polymorpha is apparently the most thermotolerant
yeast known with maximal growth temperature at 50C.
Several years ago, we have found that H. polymorpha is
capable of high-temperature xylose alcoholic fermentation though ethanol production is quite low. At the
same time is effectively ferments glucose and cellobiose
(Ryabova et al., 2003, Ishchuk et al., 2009). More recently, it was shown that H. polymorpha can effectively produce ethanol from glycerol, the byproduct of biodiesel
production (Suvannarangsee et al., 2010). To construct
effective ethanol producers from xylose, overexpression
of the modified gene XYL1m (encoding xylose reductase)
and the native genes XYL2 (xylitol dehydrogenase), XYL3
(xylulokinase) and PDC1 (pyruvate decarboxylase) and
additionally, the isolation of 3-bromopyruvate-resistant
mutants from metabolically engineered strains have been
conducted (Dmytruk et al., 2008; Ishchuk et al., 2008).
Using mentioned approaches, the strains of H. polymorpha have been constructed which accumulate elevated
amounts of ethanol from xylose, up to 1012 g/L or near
22 g/L after correction for ethanol evaporation. However,
to be economically feasible, available level of ethanol synthesis from xylose has to be increased for several times,
up to 3035 g/L. We have found that increase in ethanol
production from xylose can be achieved by either disrup-
14
Eurobiotech 2013
Posters
P2.1
The influence of phytase addition during
the enzymatic starch hydrolysis process
with the use of basic amylolytic enzymes
on yield and the course of alcoholic
fermentation process
Dawid Mikulski, Grzegorz Kosowski
Department of Biotechnology, Institute of Experimental Biology,
Kazimierz Wielki University, 85-667 Bydgoszcz,
Chodkiewicza 51 St., Poland
Acknowledgements
The study financed from the science resources in the years 20132014, as
an experimental study of the National Science Centre (No. 2012/05/N/
NZ9/02436).
References
[1] Garcia-Estepa R.M., Guerra-Hernndez E., Garcia-Villanova B.
(1999) Phytic acid content in milled cereal products and breads. Food
Research International 32, 217221.
[2] Lei X.G., Porres M. (2003) Phytase enzymology, applications, and
biotechnology. Biotechnology Letters, 25, 17871794.
Eurobiotech 2013
P2.2
Determination of the impact of lactose
suplementation of synthetic cellulolytic
media on the activity of cellulase complex
Dorota Macko, Beata Miklaszewska, Dawid Mikulski,
Grzegorz Kosowski
Department of Biotechnology, Institute of Experimental Biology,
Kazimierz Wielki University, 85-667 Bydgoszcz,
Chodkiewicza 51 St., Poland
15
[2] Mosier N., Wyman Ch., Dale B., Elander R., Lee Y.Y., Holtzapple M.,
Ladisch M. (2005) Features of promising technologies for pretreatment of
lignocellulosic biomass, Bioresource Technology 96: 673686.
[3] Ahmed S., Bashir A., Saleem H., Saadia M., Jamil A. (2009)
Production and purification of cellulose-degrading enzymes from
a filamentous fungus, Trichoderma harzianum Pakistan Journal of
Botany 41(3): 14111419.
16
Eurobiotech 2013
P2.3
P2.4
Anaerobic digestion is a process used in the most of sewage treatment plants. Volume reduction in relation to cost
is a big opportunity in the sludge management, both in
relation to less volume of digesters and less final product.
Volume reduction can be achieved by converting more of
the sludge to biogas and also by better mechanical dewatering of sludge.
Those aims are achieved with advanced digestion. Ultrasonic field as physical agent is very effective pretreatment
method to enhance the biodegradability of sludge in the
anaerobic digestion. Decomposition of organic (nutrient)
substances is limited mainly by the velocity of the first
step of methane fermentation process named as hydrolysis. During this hydrolysis the organic compounds can
be decomposed into liquid and finally becomes methane
and carbon dioxide. Intensification of the methane fermentation hydrolytic phase and the increase of biogas
generation depends on dispersion of sewage sludge solid
phase.
The aim of investigations was the preliminary treatment
process evaluation before the anaerobic digestion of excess sludge influencing on the amount of biogas generation and the energetic balance. The ratio of energy costs
during the sludge ultrasonication process to the amount
of biogas was evaluated. Excess sludge from the wastewater treatment plant of paper industry was the substrate
of investigations. For sewage sludge ultrasonication the
ultrasonic disintegrator VCX-1500 (Sonics Company)
of frequency 20 kHz was used. Ultrasonication of tested
sludge was made at four vibration amplitudes 16, 24, 32
and 39 m in the time of 300 seconds. The anaerobic digestion of preliminary modified sewage sludge with ultrasonic field was conducted in the digestion chamber at
temperature 37C during 25 days.
In the paper the effective preliminary method of sludge
pretreatment was presented, which improve degree of
sludge disintegration and biogas output. In the anaerobic digestion of tested excess sludge after this preliminary
treatment two time increase of biogas generation was
observed.
Keywords: anaerobic digestion, sewage sludge, ultrasonic field, biogas
Eurobiotech 2013
P2.5
P2.6
Tomasz Tokarz
Wydzia Inynierii I Technologii Chemicznej Politechniki
Krakowskiej, Instytut Chemii i Technologii Organicznej, Katedra
Technologii Organicznej i Procesw Rafineryjnych
Anhydrous ethanol from fermentation process of biomaterials is widely used in chemical, pharmaceutical and
cosmetic industries. This is also used as a fuel additive
in the internal combustion engines. Technologically ethanol production of a high concentration is a major problem, due to the presence in the system of ethanol/water
azeotropic point (p = 1,013 bar, T = 78,15C, 4,43% m/m
H2O). High energy consumption in the traditional azeotropic distillation is a main disadvantage of this process.
One of the most widely used alternative method is Pressure Swing Adsorption (PSA) in which zeolite nano-adsorbent is applied. The aim of this study is to find relations between surface area and pore structure for mass
production zeolite nano-adsorbents with their efficiency
in dehydration of raw ethanol.
Keywords: PSA, adsorption, separation, zeolite, ethanol
17
2,3-butanediol (2,3-BD) is a colorless and odorless polyhydroxyalcohol with a chemical formula C4H10O2. This
liquid is characterized by a high boiling temperature
and a low melting temperature. Because of attractive
physical properties and feasibility of diverse chemical
conversions this alcohol has found multiple applications
in cosmetic, pharmaceutical and fuel industries etc.
2,3-BD has been hitherto produced from crude oil by
chemical technologies, but due to the depletion of fossil
fuels and their growing prices its biosynthesis by microorganisms has recently become an interesting alternative. It is to note that the majority of microorganisms
synthesizing appreciable amounts of this compound are
pathogens, which cannot be used in industry. Therefore,
investigations on improvement of 2,3-BD biosynthesis
by non-pathogenic bacteria like Bacillus strains are so
important.
This study aimed at maximizing 2,3-BD biosynthesis by
a non-pathogenic Bacillus licheniformis strain through
fed-batch cultures and process up-scaling using bioreactors with volumes of 0.75 and 30 L.
Tested carbon sources used in 2,3-BD production
by this strain were by-products from food processing such as molasses and an enzymatic hydrolysate
of apple pomace. Fermentations conducted in 0.75 L
bioreactors enabled selection of the most appropriate
carbon source and optimum agitation intensity (tested rotation rates of a stirrer: 150, 250 or 450 rpm).
These results enabled efficient 2,3-BD production in
fed-batch cultures conducted in 30L bioreactors. The
most suitable carbon source both in batch and fedbatch cultures (irrespective of the scale) was found
to be molasses. Concentration of 2,3-BD synthesized
by the B. licheniformis strain in 30 L bioreactor (fedbatch cultures, fed 4-times with 50% glucose solution)
reached 80 g/L after 72 h. When carbon sources were
mixtures of the apple pomace hydrolysate and pure
glucose, the maximum 2,3-BD concentration was only
slightly lower (73 g/L) but it was achieved after 96 h of
fermentation.
These results are comparable to 2,3-BD yields presented
in literature for its most efficient pathogenic producer
Klebsiella pneumoniae strains. We found that among
tested by-products from food processing the most suitable for 2,3-BD production in industrial scale is molasses
from sugar beets. Advantages of this raw material are its
low price and good availability as well as, in contrast to
18
Eurobiotech 2013
the apple pomace, the lack of necessity of its earlier hydrolysis before fermentation.
Acknowledgements
Presented study was accomplished with the scope of the international
project Production and upgrading of 2,3-butanediol from biomass.
P2.7
Biogas from physical modified excess
sludge of pulp industry
Mariusz Baraski, Lidia Wolny, Iwona Zawieja
Institute of Environmental Engineering, Czestochowa University
of Technology
Eurobiotech 2013
19
P2.8
P2.9
Immobilization of enzymes used in the food, pharmaceutical and biofuels synthesis on polymeric supports significantly reduces the cost and improves the hydrolysis of
polysaccharides [1, 2].
The study involved polymer carriers based on crosslinked
N-vinyl formamide (NVF) synthesized by the reaction
of free radical suspension polymerization of N,N-methylenebisacrylamide (NMBA), optionally with ethylene
glycol dimethacrylate (EGDMA) [3].
Developed optimum parameters for copolymerization
NVF/NMBA and NVF/EGDMA in inverse suspension
giving spherical polymer grains having a low coefficient
of swelling in water. It has been shown that the most effective medium in this process is a silicone oil.
Selected six most preferred polymeric carriers characterized by physicochemical parameters and used it
to immobilize the enzyme mixture from the group of
carbohydrases under the trade name Viskozyme L
(Sigma-Aldrich). Received systems used in the test reactions of hydrolysis microcrystalline cellulose and hydrolysis of wood pulp as application process. Immobilization
of enzyme was carried out by means of glutaraldehyde,
as well as without its participation. The morphology of
carriers determined based on microscopic images. Physico-chemical methods for the characterization of obtained
catalytic polymer-carrier systems included CHN analysis,
FTIR spectroscopy and Raman spectroscopy.
It has been shown that reactive formamide groups do
not provide a permanent binding medium glutaraldehyde-enzyme, hence the catalytic tests showed less than
the immobilized enzyme activity of native enzyme.
Cellulases play an important role in specialized commercial applications such as fabric modifications, paper
and pulp industry, food industry [1] etc. Enzymatic hydrolysis of cellulose has been extensively studied in the
past decades since the utilization of cellulosic biomass
as a renewable resource has great potential for reducing
emissions of carbon dioxide and thereby prevents global
warming [2]. Cellulase can be used as the biocatalyst for
cellulose hydrolysis, but its activity is rather lower than
that of other hydrolases.
Cellulase was immobilized on N-vinylformamidecrosslinked with divinylbenzene as a result of copolymerization in reverse suspension [3]. The morphology of carriers determined based on microscopic images. Physico-chemical methods for the characterization of obtained
catalytic polymer-carrier systems included CHN analysis,
FTIR spectroscopy and Raman spectroscopy.
The enzyme Novozym 476 (Novozym) was covalently
bound directly to the supports or using glutaraldehyde
as a spacer [4]. The immobilization procedure was optimized involving such factors as temperature, pH, time,
sequence of reactions, and kind of carrier employed [5].
Received systems used in the test reactions of hydrolysis
microcrystalline cellulose and hydrolysis of paper as application process. The results of the immobilization were
evaluated in base on analyses of the enzyme activity and
stability prior and after immobilization, as well as on the
immobilization yield and stability.
Highly active biocomposites were designed that provided
their multiple application for cellulose hydrolysis.
Acknowledgements
The authors wish to thank the Ministry of Science and Higher Education
for the funding of the research. The grant N N204 354840.
References
[1] Konieczna-Molenda A., Kochanowski A., Walaszek A., Bortel E.,
Tomasik P. (2009) Chemical Engineering Journal, 146: 515519
[2] Farrell A.E., Plevin R.J., Turner B.T., Jones A.D., OHare M., Kammen
D.M. (2006) Science, 311 (5760), 506508
[3] Witek E. (2008) Polimery, 53: 477480.
Acknowledgements
The authors wish to thank the Ministry of Science and Higher Education
for the funding of the research. The grant N N204 354840.
References
[1] Li C., Yoshimoto M., Fukunaga K., Nakao K. (2007) Bioresearch
Technology, 98: 13661372.
[2] Li C., Yoshimoto M., Fukunaga K., Nakao K. (2004) J. Chem. Eng.
Jpn., 37: 680684.
[3] Witek E. (2008) Polimery, 53: 477480.
[4] Bryjak J., Bachmann K., Paww B., Maliszewska I., Trochimczuk A.,
Kolarz B.N. (1997) Chem. Eng. J.,65: 249.
[5] Konieczna-Molenda A., Kochanowski A., Grabiec, A., Bortel E.,
Tomasik P. (2008) PTTZ-Malopolska Branch, Krakow, Ch. 8: 103120.
20
Eurobiotech 2013
P2.10
P2.11
Roman Zagrodnik
Acknowledgements
The project was financed by the National Science Centre granted on the
basis of decision DEC-2012/05/N/NZ9/01577.
Acknowledgements
The research was supported by the the Polish Scientific Project
BS/ZBioch/Maria Curie-Skodowska University.
References
[1] Kunamneni A., Plou F.J., Ballesteros A., Alcalde M. (2008) Laccases:
a patent review. Recent Pat Biotechnol. 2: 1024.
[2] Kucharzyk K.H., Janusz G., Karczmarczyk I., Rogalski J. (2012)
Chemical modifications of laccase from white-rot basidiomycete
Cerrena unicolor. Appl Biochem Biotechnol. 168: 19892003.
Eurobiotech 2013
21
P2.12
P2.13
Classical methods for the extraction of active ingredients from the plant material are expensive, complicated
and often environmentally unfriendly. Popular methods
of extraction require the use of an expensive equipment,
a large quantity of electricity, heat, and large volumes of
flammable and toxic organic solvents such as methanol,
ethyl acetate or n-propanol [1, 2].
The micelle-mediated extraction method (MME) seems
to be a good alternative. The extraction temperatures are
relatively low and there is no need to apply organic solvents. In addition, this method enables to receive a higher
yield of polyphenols than the standard methods. The use
of surfactants that are suitable for human consumption
could also reduce costs of the process. The additional advantages of micelle-mediated extraction are no need for
separation of used surfactants from the polyphenols containing extract and protection of the polyphenols against
oxidation [2].
The aim of this work was the study of the possibility of
whey protein application as extraction agent. In this work,
extractions of elderberry blossoms (Flos Sambuci) were
performed. The micelle-mediated method using several
popular surfactants and whey protein was applied. The
results were compared with those obtained for extraction
with methanol. Antioxidative properties of the extract
and collected, after separation on the chromatographic
column, fractions were analyzed. Two different methods
were applied: reaction with DPPH reagent, Follins method. Futhermore, the flavonoid content was determined.
The results confirmed that the MME method with using
whey protein might be an alternative and a convenient
method for the preparation of rich in natural antioxidants
plant extracts.
Reference
[1] Katsoyannos E., Chatzilazarou A. (2006) Fresenius Environmental
Bulletin, 15: 1122.
[2] Chatzilazarou A., Katsoyannos E. (2010) Journal of The Air & Waste
Management Association, 60: 454.
Introduction. 2,3-Butanediol (2,3-BD) is a chiral bivalent alcohol which exhibits a wide range of potential utilizations as a solvent, liquid fuel or precursor for 1,3-butadiene an intermediate for synthetic rubber production.
Several microorganisms are able to synthesis 2,3-BD.
So far the highest yields were reached using risk class 2
strains e.g. Klebsiella species, however an industrial process with risk class 1 microorganisms are cheaper and less
recalcitrant. Based on this fact, the present studies deal
with the production of 2,3-BD from sugar beet pulp hydrolysates by risk class 1 bacteria, namely Raoultella planticola CECT 843 strain.
AIM. The main study objective was to statistically optimise the biosynthesis process of 2,3-BD from sugar beet
pulp hydrolysates by Raoultella planticola CECT 843
strain.
Materials and methods. Response surface methodology
(RSM) was used in this work to optimise the biosynthesis of 2,3-Butanediol by Raoultella planticola CECT 843
strain. Sugar beet pulp hydrolysates were used as a medium culture. The optimisation experiment was planned
according to D-optimal design and consisted of 25 runs.
The effect of yeast extract (YE) and concentration of
CH3COO, Fe2+ and Mg2+ ions was evaluated on the final level of 2,3-butanodiol. Sugars and other carbon compounds
concentration were determined preferably by HPLC
or GC.
Results. Obtained results showed statistically significant
influence (p < 0,05) of the yeast extract, CH3COO and
Mg2+ ions concentration on the studied process. On the
basis of statistic analysis, optimal values of analysed variables were determined as follows (gdm3): yeast extract
4; CH3COONH4 4; FeSO47H2O 0,1; MgSO4H2O
0,3; that corresponded to 19,7 gdm3 final 2,3-Butanediol
concentration.
Conclusions. Under optimised conditions Raoultella
planticola CECT 843 can effectively convert fermentable
sugars from sugar beet pulp hydrolysates to 2,3-BD. Production of diol achieved nearly 20 gdm3 and efficiency
of production reached 0,42 gg1glucose in 24h of cultivation.
It shows that risk class 1 bacteria strains are promising
direction in research of microbial production of 2,3-Butanediol.
Acknowledgements
This work was supported by ERA-IB EU-project called Production and
Upgrading of 2,3-Butanediol from Biomass (ERA-NET-IB/03/2009).
Bioeconomy
Lectures
L3.2
L3.1
Andrzej Okruszek
Lodz University of Technology, Institute of Technical
Biochemistry
The aim of the project (acronymBIOMASA) is utilization of various kinds of plant biomass and textile waste
materials by their transformation with biotechnological
methods, involving either enzymatic or microbial processes, into fibrous polymer materials. The intermediate
products in those transformations are: cellulose nanofibres, tactic polylactide and aliphatic-aromatic co-polyesters, which all are known to be important raw-materials for the production of biodegradable fibrous materials
as well as other kinds of biodegradable polymer composites.
For the preparation of cellulose nanofibres, a cellulose-rich plant biomass is being utilized, including grass
and straw of various cereals as well as waste fibres from
textile industry (cotton, linen). The biomass is first pretreated with physical and/or chemical methods including
boiling, steam-explosion or treatment with certain chemicals. Multienzyme complex obtained from Aspergillus
niger mould is utilized as the main enzymatic tool. The
fibrous materials and composites prepared within this
project on the basis of abovementioned intermediates will
be further utilized for obtaining new functional textiles
and nonwovens with potential sanitary or technical applications, such as sweat-absorbing textile inserts, sanitary
textiles, filtration materials, geotextiles and agrotextiles.
Within this project, the processes of ageing and controlled
biodegradation of prepared materials will be studied, as
well as the conditions of their recycling and possible use
of degradation products in agriculture.
The synthesis of tactic polylactide is being performed by
chemical polymerization of L,L-lactide, prepared from
L-lactic acid. The latter is obtained by stereoselective fermentation of plant biomass, after its saccharization by
ppropriate enzymes (Aspergillus niger preparations). The
microorganisms (bacteria), used for the fermentation,
were selected by classical microbiology methods from the
environment. In this case patatoes, cereal grains or beet
pulp are employed as starting biomass. The tactic polylactide will be further utilized for fiber formation and hermoforming.
The third path involves utilization of various oil-plant biomass, which on sequential treatment with lipase prepara-
Eurobiotech 2013
tions obtained from Mucor circinelloides and Mucor racemosus moulds (transesterification with 2-methylbutanol)
and dimerization of obtained esters (cycloaddition) are
transformed into dimeric esters containing fatty acid
residues. These will be co-polymerized with appropriate
reagents in order to produce new biodegradable aliphatic-aromatic co-polyesters. The polyesters will be utilized
as components in the preparation of various fibrous polymers and composites.
The project is being realized by nine research groups
from Poland belonging to four different research establishments, with the Lodz University of Technology being the leader. The elaboration within this project new
methods of preparation of polymer fibrous materials and
composites will positively influence development of science-based economy and will increase the innovativeness
of connected areas of research and production. The main
recipients of elaborated methods will be producers of fibers and nonwovens from thermoplastic materials, sanitary textiles, filtration materials, geotextiles, agrotextiles
and packing materials.
Acknowledgements
This research is realized within the project BIOMASA (POIG 01.01.0210-123/09) and partially financed by the European Union from the
European Regional Development.
23
L3.3
Biotransfromations as a tool
for Biorafineries
Pawel Kafarski
Department of Bioorganic Chemistry, Faculty of Chemistry,
Wroclaw University of Technology, Wybrzeze Wyspianskiego 27,
50-370 Wroclaw, Poland
The biorefinery concept is analogous to petrolium refinery, which produce multiple fuels and products from petroleum. At the beginning this term was used to describe
a facility that integrates biomass conversion processes and
equipment to produce fuels, power, heat, and value-added chemicals. Athough still being related to biofuel production in a broader sense biorefining is the sustainable
processing of biomass into a spectrum of marketable
products, especially high-value fine chemicals. Ideally it
is focused on integrated production for food, feed and
non-food applications, while aiming for zero waste and
getting maximum added value from co-products. It is believed that in 2030 many key chemicals using biological
and/or chemical processes.
Biotransformations are chemical reactions that are catalyzed by microorganisms in terms of growing or resting
cells or that are catalyzed by isolated enzymes. Because
of the high stereo- or regioselectivity combined with
high product purity and high enantiomeric excesses, biotransformations are considered as technically superior to
traditional chemical synthesis. They appeare to be a superiour tool in production of high-value chemicals upon
biorefining processes. For example, the Department of
Energy, USA has identified 30 platform chemicals composed of one to six carbon atoms as potential candidates
for biobased production with biotransformations being
leading processes in this respect.
Acknowledgements
This work was supportedby the project Biotransformations for
pharmaceutical and cosmetics industry No. POIG.01.03.01-00-158/09,
which is partly-financed by the European Union within the European
Regional Development Fund for the Innovative Economy.
24
Eurobiotech 2013
L3.4
L3.5
Tomasz Kapela
Patricia Osseweijer
Biotechnika
The main goal of the presentation is to outline in general the current shape of bioindustries in Poland, briefly define what are the foundations of this new industrial branch and try to outline what pushes it forward and
what holds it back. An author will try, basing on his own
experience, to draw a picture of current biobusinesses in
Poland and characterise major fields of activities. On one
hand Poland is a country of great opportunities and expectations in terms of its bioresources. But on the other
it is still struggling numerous problems of various nature
(legal, financial, public awareness related, etc.). Hopefully the presentation will flash some light on positives as
well as those issues that still need to be tackled in order to
ensure, that Poland takes its chance to built strong, biobased economy. Several case studies will help the guests
to have a complete picture of discussed ideas
There are many reasons why we should replace our present fossil-based products such as plastics, chemicals and
carpets to products made from biorenewable sources.
One of them is that we have to be much more sustainable
if we wish to mitigate climate change and feed a growing world population. In that sense biobased production
does contribute to societal challenges. However, the way
we do this will matter, so far integrated approaches for
production of materials, chemicals and energy do seem
to provide the best economical, environmentally sustainable and social benefits.
However to introduce such practices successfully, we
not only require innovation in industrial biotechnology
to deliver the best routes to develop new materials from
waste products but also a better management of our agricultural production and novel collaboration between
agricultural, chemical and energy sectors.
In order to achieve this and the adequate policy measures
to support this we need to have the support of citizens
and consumers as well. That is not straightforward as for
example the debate on food versus (bio)fuel shows. The
presentation will address the different views on sustainability and the importance of (scientists to be involved
in) communication to achieve a supported and well governed transition to a biobased society.
Eurobiotech 2013
Posters
P3.1
Isolation of bacterial strains capable
of converting glycerol into succinic acid
Marcin Podleny, Piotr Jarocki, Jakub Wyrostek,
Tomasz Czernecki, Zdzisaw Targoski
Department of Biotechnology, Human Nutrition and Food
Commodities, University of Life Sciences in Lublin
Succinic acid is currently used mainly in industries producing food, pharmaceutical products, detergents, surfactants and ingredients stimulating plant and animal
growth [1]. Enormous versatility of succinic acid and
its derivatives applications leads to treatment of this dicarboxylic acid as one of the top twelve building block
chemicals of great interest according to the United States
Department of Energy [2]. Development of economically efficient biotechnology process for succinic acid
production could extend application market for this
chemical to biodegradable plastics and green solvents.
Present production scheme of succinic acid is based on
petroleum as the starting source. Attempts for petroleum substitution by renewable low-cost raw material
are intensively made [3, 4]. These promising efforts are
inseparable connected with the usage of high-yield producing microorganisms. Therefore searching for microorganisms capable of efficient utilization of renewable
low-cost substrates during succinic acid production is
of the great interest [5]. Among considered inexpensive
compounds for succinic acid production glycerol gained
significant attention. This byproduct generated during
biodiesel production process is desirable substrate because having the same level of reduction as succinic
acid. Bioconversion of glycerol to succinic acid seems
therefore a redox balanced pathway [6].
Present study describes isolation and screening of microorganisms for succinic acid production on glycerol. Isolation of bacteria from different eight eco-niches
was carried out. A total number of 187 bacterial isolates
were obtained using microbiological techniques. After
96 hours of anaerobic cultivation on glycerol containing
medium isolates were screened for succinic acid production by HPLC. Succinic acid was not detected in nearly
half of the examined bacterial strains. About eight percent of bacterial isolates screened showed succinic acid
production rates exceeding 2 g/l. Succinic acid was not
the only product of glycerol utilization in anaerobic condition. Ethanol and acetic acid were the most common
byproducts followed by formic acid. Isolate KRBGAL,
afacultative anaerobe isolated from Holstein Freisian cow
rumen fluid, showed maximum yield of 4,6 g/l of succinic
acid from 20 g/l of glycerol. The glycerol utilization rate
during growth of KRBGAL isolate was nearly 68%. This
isolate was identified as Escherichia coli by 16S rDNA sequence analysis followed by biochemical identification
using API 20E. This strain may become a candidate for
25
Acknowledgements
This study was co-funded by The European Union project No. PO
IG 01.01.02-00-074/09 from The European Regional Development
Fund within the framework of the Innovative Economy Operational
Programme 20072013.
References
[1] McKinlay J.B., Vieille C., Zeikus J.G. (2007) Prospects for a biobased succinate industry. Appl Microbiol Biotechnol 76: 72740.
[2] Werpy T., Petersen G. Eds. (2004) Top Value Added Chemicals from
Biomass. US DoE.
[3] Du C., Lin S.K.C., Koutinas A., Wang R., Webb C. (2007) Succinic
acid production from wheat using biorefining strategy. Appl Microbiol
Biotechnol 76: 126370.
[4] Liu Y.P., Zheng P., Sun Z.H., Ni Y., Dong J.J., Zhu L.L. (2008)
Economical succinic acid production from cane molasses by
Actinobacillus succinogenes. Biores Technol 99: 173642.
[5] Scholten D., Daegele D. (2008) Succinic acid production by a newly
isolated bacterium. Biotechnol Lett 30: 214346.
[6] Yazdani S.S., Gonzalez R. (2007) Anaerobic fermentation of glicerol:
a path to economic viability for the biofuels industry. Current Opinion
in Biotechnology 18: 213-19.
Lectures
L4.1
Novel bioplastic fibres for tissue
engineering and biodegradable packaging
materials
Magdalena Wrbel-Kwiatkowska1, Jan Szopa1, 2, 3
Linum Foundation, Wroclaw, Poland; 2Faculty of Biotechnology,
University of Wroclaw, Wroclaw, Poland; 3WCB EIT+, Wroclaw,
Poland
1
Flax (Linum usitatissimum L.) is best known as a plant cultivated for industrial purposes. Flax seeds are the source
of oil used as the basal component or an additive for various paints or polymers, flax stems are the source of bres
that can be used in the textile and paper industries. A new
generation of novel bioplastic bres contain polyhydroxybutyrate (PHB) were obtained from transgenic ax plants.
Transgenic plants were generated by introduction of three
bacterial genes necessary for PHB synthesis [1]. Biochemical analysis of bioplastic bres revealed presence of several antioxidative compounds of hydrophilic (phenolics)
and hydrophobic (cannabidiol CBD, lutein) nature, indicating their high antioxidant potential.
IR spectra of transgenic ax bres revealed structural
changes in comparison to unmodified fibres, i.e. observed
higher ordering of cellulose in transgenic bres resulted
from the intra- and/or intermolecular hydrogen bonds of
glucopyranosyl residues and from the formation of the
PHB-cellulose composite [2]. The presence of PHB naturally incorporated into the bres increases their compatibility with other polymers of biodegradable (polyhydroxyalconate) and non-biodegradable (polypropylene,
polystyrene) nature.
Natural bioplastic ax fibres with stronger physical and
higher bioactive properties may serve as the source of
material for industry (automotive industry, packaging
materials) or for biomedical application (tissues scaffold,
implant).
Thus biodegradable and bioactive composites with polylactic acid (PLA) or poly-e-caprolactone (PCL) as the
matrix and bioplastic ax bres as reinforcement were
prepared and analyzed [3]. FTIR analysis of composites
showed intermolecular hydrogen bonds between the
constituents in composite PLA-ax bers which were
not detected in PCL-based composite. Mechanical analysis of prepared composites revealed improved stiffness
and a decrease in tensile strength. The viability of human
dermal broblasts on the surface of composites made of
PLA and transgenic ax bres was the same as for cells
References
[1] Wrbel M., ebrowski J., Szopa J. (2004) Polyhydroxybutyrate
synthesis in transgenic ax. J. Biotech. 107: 4154.
[2] Wrbel-Kwiatkowska M., Zuk M., Szopa J. et al. (2009) Poly3-hydroxy butyric acid interaction with the transgenic flax fibers:
FT-IR and Raman spectra of the composite extracted from a GM flax.
Spectrochim Acta A Mol Biomol Spectrosc 73: 286294.
[3] Wrbel-Kwiatkowska M., Czemplik M., Kulma A., uk M., Kaczmar
J., Dymiska L., Hanuza J., Ptak M., Szopa J. (2012) New biocomposites
based on bioplastic flax fibres and biodegradable polymers. Biotech.
Prog. 13361316.
[4] Gredes T., Kunert-Keil C., Dominiak M., Gedrange T., Wrbel-Kwiatkowska M., Szopa J. (2010) The influence of biocomposites
containing genetically modified flax fibers on gene expression in rat
skeletal muscle. Biomedizinische Technik. Biomedical engineering
55(6): 3239.
Eurobiotech 2013
27
L4.2
L4.3
Bacterial nanocellulose (BNC), synthesized by Gluconacetobacter strain, is the natural biopolymer of exceptional
purity and outstanding biocompatibility. Bionanocellulose is the material of known properties being already investigated in a wide range of industrial and medical applications, including clinical trials for wet wound dressing
and as a potential implant e.g. in vascular surgery (small
vascular grafts), bone, dental fillings, peripheral nerves
regeneration and many others. Many publications confirm that as a nondegradable biomaterial BNC implants
fulfil their function by appropriate high hydrophilicity,
inertness, lack of cytotoxicity, outstanding biocompatibility and stability in a wide range of temperatures and
pH. It was recently investigated that the neurotubes made
of BNC are of very good biocompatibility and allow the
accumulation of neurotrophic factors inside, thus, facilitating the process of peripheral nerves regeneration. The
successful application of BNC fragments in trachea reconstruction was also described. Following the obtained
results the direction of the current research is set to the
improvement of BNC scaffolds in vivo functionality.
A new method of production of novel composites made
of BNC and polymeric or titanium meshes has been developed and investigated. The potential application of
such constructs include cranioplasty and herniorraphy.
Also a cartilage-like BNC scaffolds mechanical properties
were evaluated together with in vitro biocompatibility
confirmation. The results suggest the possible application
of such a material in cartilage reconstruction. Although
still, the stable connection with the growing cells is crucial for the effective tissue regeneration. Since the properly adjusted structure of BNC membrane determines the
success of such constructs adaptation, it is necessary to
improve the manufacturing process to produce a number
of both suitably dense and porous BNC scaffolds, in order
to create the efficient drug reservoir system, as well as the
proper conditions for the proliferation of host and in vitro
introduced tissue-engineered cells.
References
Kowalska-Ludwicka K. et al. (2013) Arch Med Sci 9(3), 527534.
Bielecki S. et al. (2012) In Bacterial Nanocellulose: A Sophisticated
Multifunctional Material; Eds. Boca Raton, Florida: CRC Press,
157174.
Kolodziejczyk M. et al. (2010) Military Pharmacy and Medicine 3(1),
6265.
Barbara Klajnert-Maculewicz
28
Eurobiotech 2013
L4.4
L4.5
Crystalline cellulose nanoparticles, such as microfibrillated cellulose (MFC), have been recently considered for
manufacturing of short fiber reinforced polymeric composite materials as an alternative for artificial fibers, especially glass fibers. Due to submicronic dimensions and
crystalline structure cellulose nanofibers present higher
mechanical strength and better thermal stability as compared to natural fibers. However, initial degradation temperature of MFC is still lower than processing temperatures of numerous engineering polymers and restrain
wide implementation of green composites in structural
applications. With respect to that problem a heterogeneous surface modification of never dried MFC was
carried out by acetylation with acetic acid as well as esterification with hexanoyl and decanoyl chlorides. The
effect of molar ratio of total cellulose hydroxyl groups
to modifying agent (OH:MA) and time of reaction on
the thermal stability, structure, morphology and surface
properties of cellulose fibrils were discussed. As a result of
modification an increase in thermal stability of MFC was
achieved while crystalline structure and fibrous morphology of the cellulose filler was preserved. Moreover, modified MFC displayed significantly improved compatibility
with a new bio-based engineering polymer polyamide
4,10. Thus blending of modified MFC with polymer melt
was possible at temperatures as high as 285C. PA 4,10/
acetylated MFC composites showed significantly better
structural and morphological properties than polyamide
4,10/neat MFC system. The composite structure clearly
depended on the processing conditions.
Acknowledgements
Authors are grateful to the PolishNational Science Center for financial
support under contract No. DEC-2011/01/M/ST8/06834. J. Rettenmeier
& Shne GmbH +CO KG is kindly acknowledged for sending free
samples of microfibrillated cellulose.
Eurobiotech 2013
Posters
P4.2
P4.1
The enzymatic oxidative polymerization of five technical lignins with different molecular properties, i.e. Soda
Grass/Wheat straw Lignin, Organosolv Hardwood Lignin, Soda Wheat straw Lignin, Alkali pretreated Wheat
straw Lignin, and Kraft Softwood was studied. All lignins were previously fractionated by acetone/water 50:50
(v/v) and the polymerisation of the low molecular weight
fractions (Mw < 4000 g/mol) was carried out in the same
solvent system. Reactivity of lignin substrates in laccase-catalysed reactions was determined by monitoring
the oxygen consumption. The oxidation reactions in 50%
acetone in water mixture proceed with high rate for all
tested lignins but the enzyme lost its catalytic activity at
acetone concentration above 55%. The syringyl type Organosolv Hardwood Lignin had the highest reactivity and
the reaction proceeded with 19,66 (M/min) initial rate.
Polymerisation products were analysed by Size Exclusion
Chromatography, Fourier Transform Infrared Spectroscopy, 31P-NMR. The results provide evidence of important
lignin modifications after incubation with laccase. Structural oxidation and a notably molecular weight increase
(Mw up to 17500 g/mol) were attained. 31P-NMR spectral
analysis revealed a decrease of the total phenolic hydroxyl
groups and a substantial increase of the condensed phenolic OH content for all the polymerisation products. The
obtained polymers have potential for applications in bioplastics, adhesives and polymeric dispersants production.
Acknowledgements
The work of I.F. Fiigu was partially supported by the strategic grant
POSDRU 107/1.5/S/77265, inside POSDRU Romania 20072013
co-financed by the European Social Fund Investing in People.
The work at WUR FBR was carried out in the frame of the ERA-IB
project Products from Lignocellulose (EIB 10.013).
29
30
Eurobiotech 2013
P4.3
P4.4
Itaconic acid (IA) is a one of twelve most important platfrom chemicals that can be produced from sugars via
biological conversions. Large scale fermentation processes using molasses as carbohydrate source and fungus Aspergillus Terreus have been developed. The acid
and its derivatives are frequently used in production of
various polymers, which are viable alternatives to acrylic and methacrylic acid based polymers. Recently, due
to non-toxic nature of the acid and biocompatibility,
IA-based copolymers have been synthesized in form of
hydrogels for biomedical applications. Itaconic acid is
a well-know, unsaturated monomer which polymerizes
via radical mechanism with difficulties. Persulfates and
water are used as a standard initiators and the most common solvent, respectively. However, the process takes
quiet a long time and for that reason, persulfate activators
such as amino alcohols (N,N-dimethylenethanoloamine) or sodium hypophosphite have been developed to
increase the polymerization rate or decrease the process
temperature. As stated above, polymerization IA is usually carried out in aqueous phase and to the best of our
knowledge application of ionic liquids as the polymerization medium or the polymerization catalysts has not
been previously reported. In this study, a preliminary results of application choline salts or choline-based ionic
liquids as polymerization catalyst or solvents, respectively, were shown. The catalytic effect of choline cation on
decomposition rate of the polymerization initiator was
demonstrated. Additionally, possibility of polymerization
of ionic liquid choline hydrogen-itaconate to obtain
poly(itaconic acid) was presented.
Acknowledgements
This work was supported by the European Union Funds under Human
Capital Programme within Bioinynier chemiczny (BINC) project
(contract number POKL.04.01.02-00-217/11-00).
Acknowledgements
The study was realized within the scope of the project POIG 01.01.0210-123/09 Application of biomass in production of environmentally
friendly polymer materials tasks 3.2., co-financed from the funds
of European Fund of Regional Development within the frames of
Operation Program Innovative Economy 20072013.
References
[1] Szczesna-Antczak M. et al. (2009) Renew. Energy 34, 11851194.
[2] Szczesna-Antczak M. et al. (2006) Enzyme Microb. Technol. 39,
12141222.
[3] Szczesna-Antczak M. et al. (2004) J. Mol. Cat. B, Enzymatic, 29, 163171.
[4] Antczak T. et al. (2002) J. Mol. Cat. B Enzymatic, 19-20, 287294.
[5] Lowry R.R., Tinsley I.J. (1976) J Am. Oil Chem. Soc, 53, 470472.
Eurobiotech 2013
P4.5
P4.6
Acknowledgements
The study was realized within the scope of the project POIG 01.01.0210-123/09 Application of biomass in production of environmentally
friendly polymer materials task PZ 3.2., co-financed from the funds
of European Fund of Regional Development within the frames of
Operation Program Innovative Economy 20072013.
References
[1] Lowry R.R., Tinsley I.J. (1976) J Am. Oil Chem. Soc, 53, 470472.
31
32
Eurobiotech 2013
P4.7
P4.8
Application of selectively-hydrogenated
rapeseed oil for modification of flexible
foams
Microwave-enhanced chemical
modification of chitosan
Pharmaceutical biotechnology
Lectures
L5.2
L5.1
Biosynthesis of delta-9-tetrahydrocannabinolic
acid from biotransformation studies
in Cannabis sativa L. to biotechnological
prodution in S. cerevisiae
Oliver Kayser
Technical University Dortmund
delta-9-Tetrahydrocannabinolic acid (THCA) is an important drug from Cannabis sativa L. with psychomimetic activities. THCA and related herbal products are
mostly misued and illegal use is dominating despite the
fact that THCA has a significant medicinal value (e.g. chemotherapy, multiple sclerosis, anorexia). Today, breeding
and cultivation of high content plants is not legal for extraction, but also organic synthesis is too expensive and
not efficient to obtain high amount of THCA and it corresponding decarboxylated THC derivative for industrial
production. Biotechnology may provide new avenues for
biosynthesis and production strategies, but the biosynthesis in planta is not fully eleucidated, why heterologous expression of biosynthetic genes is complicated. Herein we
provide an overview of recent developments on the elucidation of the pathway towards cannabinoids and updated
attempts for construction of a recombinant host for the
production of THCA and its main precursors CBGA and
GPP. In short plants genetics, transciptomics and metabolimcs of trichomes where biosynthesis is localised will be
discussed [1], principles of gene expression of the main
important genes (CBGA-Synthase, THCA-Synthase) will
be explained, and into basic concepts of bioengineering of
yeasts as potential hosts will be introduced [2].
Acknowledgements
This research was funded by Deutsche Bundesstiftung Umwelt (DBU,
Frderkennzeichen 13252) and Arbeitsgemeinschaft Industrielle Forschung (AIF). Further information on heterologous THC production
on www.pharma-biotechnologie.de.
References
[1] Happyana N. et al. (2013) Analysis of cannabinoids in laser
microdissected trichomes of medicinal Cannabis sativa using LCMS and
cryogenic NMR. Phytochermistry 87: 5159.
[2] Muntendam R et al. (2009) Perspectives and limits of engineering
the isoprenoid metabolism in heterologous hosts Appl. Microbiol
Biotechnol. 84: 10031019.
34
Eurobiotech 2013
were able to show that in the A2BAR only the highly conserved C3.25-C45.50 disulfide bond is essential for ligand
binding and receptor activation [4]. We additionally exchanged the whole ECL2 of the A2BAR for the ECL2 of
the A2AAR and demonstrated that the ECL2 is involved in
ligand binding and subtype selectivity and modulates agonist-bound receptor conformations, thereby controlling
signaling efficacy [3]. The interdisciplinary approach presented in this study using both, experimental data and
computational predictions, provides valuable information for the rational design of desired highly potent and
selective ligands, which are required to validate and exploit their therapeutic potential, and to further elucidate
the (patho)physiological roles of GPCRs.
References
[1] Thimm et al., Biochemistry, 2013, 52, 726740.
[2] Sherbiny et al., J. Comput. Aided Mol. Des., 2009, 23, 80728.
[3] Seibt et al., Biochem. Pharmacol., 2013, 85, 13171329.
[4] Schiedel et al., Biochem. Pharmacol., 2011, 82, 389399.
[5] Borrmann et al., J Med Chem, 2009, 52, 39944006.
L5.3
Monoclonal antibodies targeting
angiogenesis and lymphangiogenesis
in cancer therapies
Monika Bzowska, Magorzata Kulesza, Tomasz Klaus,
Karolina Ossysek, Renata Myk-Kope, Joanna Bereta
Faculty of Biochemistry, Biophysics and Biotechnology,
Jagiellonian University
Monoclonal antibodies (mAbs) are one of the fastest developing branches of the pharmaceutical industry. Due to
a very high affinity and selectivity of binding to antigens,
mAbs are used for specific targeting of certain molecules,
e.g. soluble cytokines or tumor-associated antigens expressed by cancer cells. Presently, therapeutic mAbs are
applied: (i) to prevent transplant rejection, (ii) to inhibit the progression of chronic inflammatory diseases and
(iii) to fight cancer (usually in combination with other
therapies). Apart from mAbs that directly target cancer
cells, another approach which aims at angiogenesis has
also been introduced to the clinic to inhibit tumor growth
and spreading (metastasis). However, long-term studies
indicate limited beneficial effects of this anti-angiogenic
approach. Since tumor cells can escape from the primary
site by entering not only blood- but also lymphatic vessels, the attempts to inhibit lymphangiogenesis in order
to hamper tumor metastasis seem reasonable [1].
The aim of our project is to generate mAbs able to bind
and inhibit VEGF-C, the major growth factor for lymphatic endothelium. We use a phagemid library Tomlinson I+J and a phage display method to select the set of
phages expressing anti-VEGF-C antibodies in a form of
scFv (single chain variable fragment). The procedure of
phage selection and obtaining of specific mAbs as well as
the evaluation of their inhibitory activity will be presented. The potential of anti-VEGF-C antibodies to limit tumor development and spreading will be discussed.
Acknowledgements
This work was supported by a grant from the Polish-Swiss Research
Programme (PSPB-057/2010 to JB).
References
[1] Antibody-based antiangiogenic and antilymphangiogenic therapies
to prevent tumor growth and progression. Bzowska M., Myk-Kope
R., Prchnicki T., Kulesza M., Klaus T., Bereta J., Acta Biochimica
Polonica, Paper in Press, No. 2013_481.
Eurobiotech 2013
L5.4
Bacterial cell wall assembly:
new antibiotics and industrial
biopolymers
Douglas Weibel2, 3, 4, George Auer3, Hannah Tuson4, Nate Cira4,
KC Huang5
Department of Chemistry, University of Wisconsin-Madison;
Department of Biomedical Engineering,
University of Wisconsin-Madison; 3Department of Biochemistry,
University of Wisconsin-Madison; 4Department of Bioengineering,
Stanford University
1
2
This presentation describes how we are using our discovery of bacterial cell wall assembly and maintanence to
develop new chemotherapeutics and produce new classes
of industrial biopolymers. This presentation builds a connection between two connected areas of research in our
lab that center on the bacterial cell wall: 1) identifying,
studying, and exploiting the machinery that bacteria use
to synthesize their polymer exoskeleton (the peptidoglycan, PG) as a new feedstock for industrial polymers; and
2) and developing new clinical antibiotics and that target
the bacterial cell wall and protecting these valuable biomedical resources.
In the first part of this presentation, I describe a gel-based
force clamp technique that we recently developed to measure the mechanical properties of live bacterial cells [1].
We recently used this technique to measure how single
gene deletions in bacteria alter cell stiffness [Auer et al.
in preparation]. Using this approach we have identified
machinery that is essential for regulation of the mechanical properties of the PG and are now engineering the
biochemistry of these proteins to tailor PG assembly as
asource of biopolymers.
In the second part of my talk, I introduce the cell wall machinery as the most important targets of clinical antibiotics developed to date. Protecting these valuable resources
requires providing health care workers with informed
data on how to best treat pathogens at the point-of-need.
To facilitate this process, we have developed a very simple, inexpensive, and small plastic cartridge, which we refer to as QuickChip that uses an isothermal, DNA-based
amplification technique to identify antibiotic resistance
elements in bacteria in clinical samples and determines
antibiotic susceptibility [24]. Integrating QuickChip
with a smart phone or tablet-powered manifold enables
its hands-free operation: the manifold heats the cartridge
and measures the fluorescence output from the isothermal assay, the app sends the data to the cloud for analysis and diagnosis, and the cloud returns information on
the bacterial strain and optimal antibiotic dosing to guide
the effective treatment.
We envision that the integration of these technologies
will have an important impact on biomaterials science
and clinical medicine by drawing new connections between the structure, assembly, and mechanical properties
of the bacterial cell wall.
35
Acknowledgements
This research was supported by the Aspen Center for Physics (NSF
grant 1066293), the NIH (grants T32 GM07215, DP2OD006466, and
DP2OD008735), DuPont, and the Alfred P. Sloan Foundation.
References
[1] Tuson H.H., Auer G.K., Renner L.D., Hasebe M., Salick M., Crone
W.C., Gopinathan A., Huang K.C., Weibel D.B. (2012) Measuring the
stiffness of bacterial cells from growth rates in hydrogels of tunable
elasticity. Mol. Microbiol. 84, 874891.
[2] Cira N.J., Dueck M.E., Weibel D.B. (2012) A self-loading microfluidic
device for determining antibiotic toxicity. Lab Chip 12, 10521059.
[3] Cira N.J., Weibel D.B., Self-loading microfluidic device and methods
of use. 13/303,982. Filed: November 23.2011
[4] Ho J., Cira N.J., Crooks J., Weibel D.B. (2012) Rapid identification of
bacterial pathogens in an autonomous microfluidic device. PLoS One.
7, e41245.
36
Eurobiotech 2013
L5.5
L5.6
Hepatitis B is still one of the most common human infectious diseases, despite the fact that effective anti-HBV
subunit vaccines have been available for 30 years. Orally
administered plant-based vaccines have been considered
as alternatives or supplements for standard injection vaccines, due to assumed low-cost production and simplified
vaccination. First trials showed prospects of mucoso-intestinal immunisation. Unfortunately, despite the unquestionable milestone, edible vaccines were impractical or insufficiently effective. Professional anti-HBV plant-derived
vaccine would require an appropriate plant producer as
well as vaccine composition and administration protocol.
Herbicide-resistant lettuce lines expressing native HBV
antigens, HBsAg subunits (S, M, L) and HBcAg, at levels
5200 g/g FW were engineered. Vegetative propagation
of efficient plant lines enabled production scaling-up of an
antigen-bearing material for an oral vaccine preparation.
The prototype manufacture technology of the vaccine has
been developed, based on freeze-drying of plant material.
Powdered lyophilised tissue facilitated controlled antigen
delivery and enabled conversion into oral formulas, e.g.
tablets.
Exclusively oral delivery of lyophilised tissue containing
S-HBsAg induced systemic humoral response in mice at
the minimal protection level ( 10 mIU/ml of anti-HBs
antibodies) with higher S-IgA response in intestinal mucosa and immediate stimulation of Treg lymphocytes.
Combination of injection priming and low-dose oral
boosting triggered significant immune response in mice,
as high as classical three-dose intramuscular vaccination.
Titre of anti-HBs antibodies reaction reached hundreds
mIU/ml, as well as stimulation of specific lymphocyte
subpopulations and production of cytokines were clearly visible. In turn, tablets administered to humans previously immunised with the standard pattern, increased
anti-HBs level from 0 up to 100 mIU/ml or 2 3-fold
when pre-immune anti-HBs were detectable.
The study provides some basics regarding parenteral-oral
immunisation strategy using an efficacious and convenient
plant-derived formula which would serve as a booster vaccine for possible human vaccination against hepatitis B.
Acknowledgements
This study was supported by grants No. 2 P04B 001 27 and No. N N302
157837 from the Polish Ministry of Science and Higher Education and
under the auspices of Medana Pharma Inc., Sieradz, Poland.
Eurobiotech 2013
L5.7
Posters
P5.1
Jerzy Dobrucki
Jagiellonian University, Faculty of Biochemistry, Biophysics
and Biotechnology, Laboratory of Cell Biophysics
Only a few microscopy methods of visualizing extracellular matrix (ECM) in live tissues in situ and in freshly
excised tissues are available. Collagen fibers polymerized
under laboratory conditions can be imaged in an optical microscope, using dark field, differential interference
(Nomarski), or polarization contrast, however these
methods are not ECM specific. Two sophisticated imaging techniques can image collagen detecting second
harmonic generation (SHG), and a coherent anti-Stokes
Raman scattering (CARS). There are no simple, inexpensive, or widely available techniques for 3-dimensional
imaging of collagen and elastin fibers in live animals, or
excised metabolically active tissues.
A new low molecular weight fluorescent probe, Col-F,
that exhibits affinity to collagen and elastin, was used
successfully in confocal imaging of extracellular matrix
in freshly excised animal tissues. The dye readily penetrates between live cells into tissues and binds to fibers of
collagen and elastin by a noncovalent mechanism. Col-F
provides a simple and convenient tool for fluorescence
three-dimensional imaging of intricate collagenous and
elastic structures in live and fixed animal tissues, as well
as in collagen-containing biomaterials.
Acknowledgements
This research was supported by a grant 2067/P01/2007/32 from Ministry
of Science and Higher Education in Warsaw.
37
Eurobiotech 2013
38
P5.2
P5.3
Medicinal plants are used in folk medicine for centuries due to biological activities of bioactive metabolites.
Physalis peruviana L. (Solanaceae) is one of the common
medicinal herb used for its properties as anticancer, antimicrobial, antipyretic, diuretic, and anti-inammatory
immunomodulator. For this reason we aimed to evaluate
in vitro antibacterial and antioxidant activites and total
phenolic content of P. peruviana ethanol extracts.
Disk diffusion assay was used to determine antibacterial
effect. In antibacterial activity maximum inhibition zone
was determined in Lactococcus lactis. The lowest MIC
value was 100 microgram/disc for Staphylococcus aureus
A950277, the highest MIC value was 700 microgram/disc
for Escherichia coli DH5-alpha. The antioxidant activity
was evaluated by Cuprac method using trolox as standard and extracts exhibit total antioxidant capacity. Total
phenolic content was measured as gallic acid equivalents.
The highest TEAC and total phenolic content values of
the leaf and the shoot extracts are 0.291 0.04 and 0.192
0.015 and 40.69 0.21 and 30.21 0.71, respectively.
These results indicate that P. peruviana ethanol extracts
may include effective compounds to be used as therapeutic agents and give a great interest for future reseach.
Eurobiotech 2013
P5.4
Arbutin production via biotransformation
of hydroquinone in in vitro cultures
of Aronia melanocarpa (Michx.) Elliott
Inga Kwiecie, Agnieszka Szopa, Kornelia Madej, Halina Ekiert
Chair and Department of Pharmaceutical Botany,
Jagiellonian University, Collegium Medicum, 9 Medyczna Street,
30-688 Crakow, Poland
39
40
Eurobiotech 2013
P5.5
The accumulation of phenolic acids
in agitating cultures of Ruta graveolens L
Agnieszka Szewczyk, Marzena Surzyn, Halina Ekiert
Department of Pharmaceutical Botany,
Jagiellonian University, Collegium Medicum, 9 Medyczna Street,
30-688 Crakow, Poland
References
[1] Ekiert H., Czygan F.Ch. (2007) In: Biotechnology Secondary
metabolites. Plants and Microbes (Ramawat K.G. and Merillon J.M.
ed.), Science Publishers, Enfield, New Hampshire, USA, 445482.
[2] Ekiert H., Szewczyk A., Ku A. (2009) Pharmazie 64: 694696.
[3] Linsmaier E.M., Skoog F. (1965) Physiol. Plant. 18: 100127.
[4] Ellnain-Wojtaszek M., Zgrka G. (1999) J. Liq. Chrom. Tech. 22:
14571471.
Eurobiotech 2013
P5.6
The influence of L-phenylalanine, methyl
jasmonate and sucrose concentration
on the accumulation of phenolic acids
in Exacum affine Balf. f. shoot culture
Ewa Skrzypczak-Pietraszek, Joanna Sota
Chair and Department of Pharmaceutical Botany,
Jagiellonian University, Collegium Medicum, 9 Medyczna Street,
30-688 Crakow, Poland
Phenolic acids are an important group of plant secondary metabolites with different, valuable therapeutic
properties (e.g. antioxidant, antiphlogistic, immunostimulating, antiseptic) [1]. Besides plants growing in
the open air, tissue cultures can be an alternative source
of secondary metabolites. Yield of their accumulation in in vitro cultures can be increased by different
methods, including culture medium supplementation
with precursors, elicitors and changing the standard
amounts of the medium components. The purpose of
this study was to investigate the influence of precursor (L-phenylalanine), elicitor (methyl jasmonate) and
different sucrose concentration on phenolic acids accumulation in agitated shoot cultures of Exacum affine
Balf. f. (Gentianaceae).
Cultures were maintained in conical flasks with Murashige and Skoog medium [2] supplemented with plant
growth regulators: BAP (6-benzylaminopurine), 1 mg/l,
NAA (-naphthaleneacetic acid), 0,5 mg/l and GA3 (gibberelic acid), 0,25 mg/l. Variant A contained 3% of sucrose (standard amount) and the other six variants (A-F)
6% of sucrose. After two weeks L-phenylalanine (1,6g/l
of medium) and/or methyl jasmonate (two concentrations: 100 M or 800 M) were added to B-F variants.
Variants A and A were treated as references. Plant materials were collected after 1, 3 and 7 days after the addition
of the precursor and/or the elicitor. Control samples were
collected too. Phenolic acids were assayed in the collected
biomass before and after acid hydrolysis (2 M HCl, 2 h).
Qualitative and quantitative analysis of phenolic acids in
methanolic extracts from biomass were conducted by an
HPLC method [3].
Fourteen phenolic acids (protocatechuic, gallic, gentisic,
chlorogenic, p-hydroxybenzoic, vanillic, caffeic, syringic,
p-coumaric, ferulic, sinapic, salicylic, o-coumaric, rosmarinic) and cinnamic acid were found in all samples.
The total content of free phenolic acids increased from
approximately 0,368% to 0,838% (2,3-times) and the total
content of the whole phenolic acids (free and bound)
from 0,866% to 1,390% (1,6times) depending on the
MS medium variants. The studies show that the best variant contained 6% of sucrose (double amount of the standard), L-phenylalanine 1,6 g/l of medium and methyl
jasmonate 100 M.
Analysis of the results in the described experiment
showed that it is possible to increase the accumulation of
41
42
Eurobiotech 2013
P5.7
P5.8
Biotechnology in cosmetology:
Introduction of Argireline an anti-aging
peptide. Study of cellular cytotoxicity
of Argireline solution
Argireline is an anti-aging peptide synthesized by company: Lipotec S.A. In cosmetology it is used as synthetic
cosmetic ingredient in form of a powder or more commonly 0,05% argireline solution. This acetyl hexapeptide-3 is mimicking the N-terminal end of SNAP-25 protein, that plays important role in formation of neuronal
SNARE complex. Argireline prevents formation of skin
lines and wrinkles in a very similar way as botulinum
toxin (Botox). It competes with SNAP-25 for a position
in the SNARE complex, destabilizing it. As a result, the
neuronal vesicles cannot release neurotransmitters from
the axon endings. In the end that causes attenuation of
skin muscle contraction, especially in the forehead and
around the eyes. It also inhibits overproduction of catecholamines, which can induce formation of wrinkles.
Argireline can be incorporated in cosmetic formulations
such as emulsions, gels or sera. Its effectiveness has been
confirmed in several anti-wrinkle tests performed in vitro
using chromaffin cells and in vivo on healthy volunteers
[1]. The aim of presented study is to elaborate the proper methodology of cytotoxity study of cosmetics active
ingredients. Tests were performed on argireline solution
in its declared by producer concentration. In order to estimate cytotoxity of the argireline solution MTS test was
used. This test allows to measure the efficiency of mitochondrial oxidative activity in living cells [2]. In the experiments both HEK293 cells and human fibroblasts were
treated with argireline solution. Examined were: short
time cytotoxic effect of argireline as well as its influence
on cellular proliferation features. Considered methods
result in dose dependent argireline cytotoxicity effect.
Argireline solution, dissolved more than six times from
original concentration decreases cellular proliferation
rate and metabolic activity. In conclusion it was shown
that the MTS test can be used in order to determine cytotoxic properties of cosmetics active ingredients.
Acknowledgements
We would like to thank: Przedsibiorstwo Produkcyjno-Handlowe
Ryszard Kaczmarek i Synowie Sp. z o.o. Spka Komandytowa company,
that is the only official distributor of Lipotec S.A. products in Poland,
for providing argireline solution sample, that was used in the presented
experiments.
This work was supported by K/ZDS/001915.
References
[1] Lipotec S.A. (2002) Neuronal exocytosis inhibiting peptides and
cosmetic and pharmaceutical compositions containing said peptides,
WO 00/64932.
[2] Cory J.G., Owen A.H., Barltrop T.C. (1991) Use of an aqueous
soluble tetrazolium/formazan assay for cell growth assays in culture.
Cancer Communications, 3(7), 207212.
Eurobiotech 2013
43
P5.9
Cell wall proteins of Candida albicans
and non-albicans Candida species as the
binders for human proteins and potential
therapeutic targets
Justyna Karkowska-Kuleta1, Sylwia Kedracka-Krok2,
Karolina Seweryn1, Maria Rapala-Kozik1, Andrzej Kozik1
Jagiellonian University, Faculty of Biochemistry, Biophysics
and Biotechnology, Department of Analytical Biochemistry;
2
Jagiellonian University, Faculty of Biochemistry, Biophysics
and Biotechnology, Department of Physical Biochemistry
1
The development of infections caused by pathogenic fungi is inseparably connected with the specific properties
of pathogen cell surface. In the yeasts of genus Candida,
comprising a number of species pathogenic for humans,
the cell wall is composed of -1,3- and -1,6-glucans, chitin, mannoproteins anchored to the polysaccharide scaffold through various types of linkages, loosely attached
moonlighting proteins and small amount of lipids.
As is well known, the surface-connected proteins of
C. albicans, the most extensively characterized fungal
pathogen, are often involved in the adhesion to host proteins and cells, contributing to yeast virulence, whereas so
far nothing is known about the adhesins of non-albicans
Candida species, particularly two emerging pathogens
C. tropicalis and C. parapsilosis.
In this work, with the use of affinity chromatography
and tandem mass spectrometry coupled with high-performance liquid chromatography (LC-MS/MS), we were
able to isolate and identify fungal proteins responsible for
tight binding of a human serum protein, high molecular
mass kininogen (HK), a component of kinin-generating
system involved in the regulation of many physiological
and pathological processes such the coagulation and fibrinolysis, inflammation, blood pressure control, neovascularization and apoptosis.
In the case of unicellular, yeast-like forms of C. albicans,
the interaction with HK occurred mainly via the moonlighting proteins such as glycoamidase (Png2p), enolase
(Eno1p), phosphoglycerate mutase (Gpm1p) and triosephosphate isomerase (Tpi1p). In C. tropicalis and C. parapsilosis, in their most adhesive hyphal forms, the typical
adhesins, agglutinin-like sequence proteins Als3p and
Als7p, respectively, were identified as the key elements
that strongly bound HK. Because of multifunctionality of
HK, HK-binding proteins which occur on the pathogen
surface can be considered as potentially useful targets for
new therapeutic approaches.
Acknowledgements
This work was supported in part by the National Science Centre, Poland
(the grant No. 2012/07/B/NZ1/02867 to A.K.)
44
Eurobiotech 2013
P5.10
P5.11
Application of immobilized
ethylbenzene dehydrogenase and
whole-cell recombinant phenylethanol
dehydrogenase system for synthesis
of chiral alcohols
Monika A. Papie
Department of Cytobiology, Faculty of Pharmacy,
Jagiellonian University Medical College, Crakow, Poland
Eurobiotech 2013
Acknowledgements
Authors acknowledge financial support of the project
Biotransformations for pharmaceutical and cosmetics industry No.
POIG.01.03.01-00-158/09-04.
References
[1] Knack D., Hagel C., Szaleniec M., Dudzik A., Salwinski A., Heider J.,
Appl. Environ. Microb., 78 (2012) 64756482.
[2] Hffken H.W., Duong M., Friedrich T., Breuer M., Hauer B.,
Reinhardt R., Rabus R., Heider J., Biochemistry, 45 (2006) 82.
45
P5.12
Studies on sintering process
of bone-derived hydroxyapatite
Dagmara Malina, Kamila Biernat, Agnieszka Sobczak-Kupiec
Institute of Inorganic Chemistry and Technology,
Crakow University of Technology, 24 Warszawska St.,
31-155 Crakow, Poland
46
Eurobiotech 2013
P5.13
P5.14
Eurobiotech 2013
References
[1] Martins A., Amaral L. (2012) Screening for Efflux Pump Systems
of Bacteria by the New Acridine Orange Agar Method, In vivo 26:
203206.
[2] Martins M., Viveiros M., Couto I., Costa S.S., Pacheco T., Fanning S.,
Pags J.-M., Amaral L. (2011) Identification of Efflux Pump-mediated
Multidrug-resistant Bacteria by the Ethidium Bromide-agar Cartwheel
Method, In vivo 25: 171178.
47
P5.15
Production of triterpenoids with cell
and tissue cultures
Magdalena Malinowska, Elbieta Sikora, Jan Ogonowski
Crakow Univeristy of Technology
48
Eurobiotech 2013
References
[1] Dzubak P., Hajduch M., Vydra D., Hustova A., Kvasnica M.,
Biedermann D., Nat Prod Rep, 2006, 23, 394.
[2] Mufflera K., Leipolda D., Schellera M.C., Haasb C., Steingroewerb
J., Bleyb T., Neuhausc H.E., Miratad M.A., Schraderd J., Ulbera R.,
Process Biochem., 2011, 46, 11.
[3] Cheng Z.-H., Yu B.-Y., Cordell G.A., Qiu S.-X., Org Lett, 2004, 6,
3163.
[4] Chen Q.-H., Liu J., Zhang H.-F., He G.-Q., Fu M.-L., Enzyme
Microb. Tech., 2009, 45, 175.
[5] Qian L.-W., Zhang J., Liu J.-H., Yu B.-Y., Tetrahedron Lett, 2009,
50, 2193.
[6] Parra A., Rivas F., Garcia-Granados A., Martinez A., Mini-Rev. Org.
Chem., 2009, 6, 307.
[7] Chatterjee P., Kouzi S.A., Pezzuto J.M., Hamann M.T., Appl Environ
Microb, 2000, 66, 3850.
[8] Vanisree M., Lee C.Y., Lo S.F., Nalawade S.M., Lin C.Y., Tsay H.S.,
Bot Bull Acad Sin, 2004, 42, 1.
[9] Ramachandra Rao S., Ravishankar G.A., Biotechnol Adv., 2002, 20,
101.
[10] Wink M., Charlwood B.V., Rhodes M.J.C. (Eds.), Clarendon Press,
Oxford, 1990.
[11] Pavlov A., Georgiev M., Bley T., Z Naturforsch C, 2007, 62, 439.
[12] Drnenburg H., Process Biochem, 2004, 39, 1369.
[13] Lee M., Jeong J., Seo J., Shin C., Kim Y., J. In, Plant Cell Physiol,
2004, 45, 976.
P5.16
Determination of antioxidant activity of
black tea extract
Magdalena Malinowska, Kamil Kurleto, Grzegorz Kurowski,
Barbara Laskowska, Elbieta Sikora, Otmar Vogt
Crakow Univeristy of Technology
Tea has been consumed all over the World for over
two thousand years and now it is the most popular caffeine-containing beverage. The first written records of the
tea can be found in VIII century BC, where a brews of the
tea leaves were treated as a medicine. Over the centuries,
tea plantations have been established and the methods of
tea collection and processing have been improved [14].
The tea is not only important because of its popularity
but also due to its beneficial influence on human health.
Therapeutic effects of tea have been extensively examined
in many in vitro and in vivo studies. It was confirmed
that tea leaves ingredients has antibacterial, antifungial,
antiviral properties, they also prevent cell mutations and
they inhibit progress of heart diseases. Moreover, tea can
stimulate neural system and regulate its functions. Positive effect of the tea drinking is associated mainly with
high content of polyphenols, mainly catechins that act as
an antioxidants. Their amount in tea infusion depends
not only on the type of tea, but also on brewing process
[519].
The subject of this work was to investigate the influence
of the brewing conditions: temperature, time and degree
of leaf fragmentation, on antioxidant activity of black tea.
The total antioxidant activity (TAA) of the tea infusions
was studied using three different test methods: DPPH
free radical reduction, Folin-Ciocalteu and thiocyanate.
UV-VIS spectrophotometer was applied to measure the
absorbance of the samples.
The obtained results showed that brewing conditions
have a significant effect on the TAA of the black tea infusions. The brewing temperature is the most important parameter influencing on polyphenols content in black tea
infusions.. Results of all three applied methods showed
that the highest antioxidant activity was characterized
the infusions obtained at the 90 and 95C. Brewing time
also determines TAA of the tea infusions and the optimal
brewing time was 5 to 10 minutes. Recommended 23
minutes of brewing time is not sufficient to obtain the
maximum antioxidant activity of the tea extracts. Also
the leaf fragmentation has a positive impact on TAA of
the tea infusions.
References
[1] uczaj W., Skrzydlewska E., Prev. Med., 2005, 40, 910.
[2] Bykbalci A., Sedef Nehir E., Plant Foods Hum Nutr., 2008, 63, 27.
[3] Caprari M., Herbata, Warszawa 2009.
[4] Sharangi A.B., Food Res. Intern., 2009, 42, 529.
[5] Gramza A., Korczak J., Amarowicz R., Pol. J. Food Nutr. Sci., 2005,
3, 219.
[6] Wei K., Wang L., Zhou J., He W., Zeng J., Jiang Y., Cheng H., Food
Chem., 2011, 125, 44.
[7] Leung L.K., Su Y., Chen R., Zhang Z., Huang Y., Chen Z.Y., J. Nutr.,
2001, 131, 2248.
Eurobiotech 2013
[8] Wright L.P., Biochemical analysis for identification of quality in
black tea, University of Pretoria, Pretoria, 2002.
[9] Friedman M., Mol. Nutr. Food Res., 2007, 51, 116.
[10] Davies M.J., Judd J.T., Baer D.J., Clevidence B.A., Paul D.R.,
Edwards A.J., Wiseman S.A., Muesing R.A., Chen S.C., J. Nutr., 2003,
133, 3298.
[11] Ferrazzano G.F., Amato I., Ingenito A., de Natale A., Pollio A.,
Fitoterapia, 2009, 80, 255.
[12] Robak J., Gryglewski R.J., Pol. J. Pharmacol., 1996, 48, 555.
[13] Saha P., Das S., Asian Pac. J. Cancer Prev., 2002, 3, 225.
[14] Cicho Z., Miniakiewicz M., Zesz. Nauk. AE Krak., 2005, 678,
103.
[15] Yang D.J., Hwang L.S., Lin J.T., J. Chromatogr. A, 2007, 1156, 312.
[16] Gramaza A., Korczak J., Trends Food Sci. Tech., 2005, 16, 351.
[17] Thanaraj S.N., Seshardi R., J. Sci. Food Agric., 1990, 51, 57.
[18] Wei K., Wang L., Zhou J., He W., Zeng J., Jiang Y., Cheng H., Food
Chem., 2011, 125, 44.
[19] Obanda M., Owuor P.O., Mangoka R., Food Chem., 2004, 85, 163.
49
P5.17
Homology Modeling of Steroid C25
Dehydrogenase
Agnieszka Rugor1, Stefan Mordarski2, Jakub Staro2,
Andrzej Bojarski2, Maciej Szaleniec1
Jerzy Haber Institute of Catalysis and Surface Chemistry, PAS,
Niezapominajek 8, 30-239 Crakow, Poland;
2
Instytute of Pharmacology, PAS, Smetna 12, 31-343 Crakow,
Poland
1
50
Eurobiotech 2013
P5.18
P5.19
Steroid C25 dehydrogenase (S25DH) from Sterolibacterium denitrificans is a member of the DMSO reductase
family of molybdenum enzymes with a bis-MGD cofactor in its active site. It catalyzes the oxygen independent
hydroxylation of the tertiary C25 atom of the side chain
of cholesterol and other steroid compounds to the respective tertiary alcohols [1].The ability of S25DH to introduce hydroxyl groups into the C25 atom of cholesterol
and its derivatives has the potential for application in the
production of pharmaceuticals such as activated vitamin
D3 or 25-hydroxycholesterol [2].
Up to date, S25DH was purified by a multi-step anaerobic
protocol developed by Dermer and Fuchs. However, it has
been shown that EBDH-like enzymes can be stabilized in
aerobic conditions by addition of a suitable electron acceptor (e.g. ferrocenium (III) ions) [3]. In the poster we
present the shortened anaerobic and aerobic enzyme purification procedures that delivers an enzymatic formulation with defined biological activity.
To confirm the application potential of S25DH, it was
used as a catalyst in the synthesis of 25-hydroxy-cholest4-en-3-one. The synthesis was carried out in a batch reactor system with homogenous and immobilized enzyme.
Admittedly, the S25DH purification from S. denitrificans
is still not efficient enough for industrial purposes. Therefore, the experiments aiming at the development of a heterologous expression system were undertaken. An artificial operon containing all the three S25DH subunits and
the chaperone gene was generated and the genes overexpressed in E.coli. The poster will discuss the method used
for construction of the artificial operon and its efficiency
in production of the enzyme.
Acknowledgements
The authors acknowledge the Polish National Center of Research and
Development under grant LIDER/33/147/L-3/11/NCBR/2012.
References
[1] Dermer J., Fuchs G. (2012) J. Biol. Chem. 287: 3690536916.
[2] Chiang Y.R., Ismail W., Mller M., Fuchs G. (2007) J. Biol. Chem.
282: 1324013249.
[3] Szaleniec M. et al. (2007) Biochemistry, 46 7637-764653: 10851091.
Eurobiotech 2013
51
P5.20
P5.21
Acknowledgements
This research was supported by Ministry of Science and Higher
Education project C-4/257/2013/DS-M.
References
[1] Bajpai A.K., Shukla S.K., Bhanu S., Kankane S., Prog Polym Sci 33
(2008) 10881118.
[2] Liu Z., Jiao Y., Wang Y., Zhou C., Zhang Z., Adv Drug Deliv Rev 60
(2008) 165062.
[3] Agnihotri S.A., Mallikarjuna N.N., Aminabhavi T.M., J Control
Release 100 (2004) 528.
[4] Nafea E.H., Marson A., Poole-Warren L.A., Martens P.J., J Control
Release 154 (2011) 110122.
[5] Lofmark S., Edlund Ch., Nord C. E., Clin Infect Dis 50 (2010) 1623.
[6] Herculano R.D., Alencar de Queiroz A. A., Kinoshita A., Oliveira
O.N. Jr., Graeff C.F.O., Mat Sci Eng C 31 (2011) 272275.
52
Eurobiotech 2013
P5.22
P5.23
Monika Papie
PriB is a primosomal protein that catalyzes DNA replication in Procaryota. The replication pathway starts with
PriA protein the initiator protein that binds to a DNA
replication fork, unwinds double-stranded DNA and role
of PriB is to stabilize PriA on the DNA. However there are
many biochemical differences in replication mechanism
in bacteria and only some of them use PriB proteins.
A few of PriB proteins were published and only three
structures of them were resolved (Escherichia coli, Klebsiella pneumoniae and Neisseria gonorrhoeae). All upto-date known PriB proteins have one OB domain per
monomer and they are homodimers in solution.
Recently, we have published the crystal structure of PriB
protein from Thermoanaerobacter tengcongensis that
represents new class of PriB with two oligonucleotide/
oligosaccharide-binding domain (OB) per monomer that
means it exists as monomer in solution.
The aim of this study is identification and characterization
of the primosomal protein B (PriB) from an anaerobic,
thermophilic bacterium Clostridium thermocellum (CthPriB). This PriB protein consisting 238 amino acid residues and a calculated molecular mass is 27,5 kDa. What is
more CthPriB protein contains two single-stranded DNA
binding domain (OB-fold) and functions as monomer
like PriB protein from bacterium Thermoanaerobacter
tengcongensis.Therefore, our studies suggest that we discovered new classes of PriB which werent published recently.
Eurobiotech 2013
P5.24
Salvia lavandulifolia from spain: aromatic
profile by enantioselective gas
chromatography-mass spectrometry
Ana Belen Cutillas5, Alejandro Carrasco1, Vanessa Ortiz1,
Ramiro Martinez-Gutierrez3, Francisco Javier Martinez4,
Mariano Sanchez5, Virginia Tomas2, Jose Tudela1
GENZ-Grupo de Investigacion Enzimologia (www.um.es/genz),
Departamento de Bioquimica y Biologia Molecular-A, Campus
de Excelencia Internacional Regional Campus Mare Nostrum,
Universidad de Murcia, Murcia, Spain; 2Departamento
de Quimica Analitica, Universidad de Murcia; 3NOVOZYMES
SPAIN S.A. (www.novozymes.com); 4Esencias Martinez-Lozano
S.A. (www.esenciaslozano.com); 5Europermanent S.L.
1
Objectives. The identification and determination of biochemicals of the essential oil of Salvia lavandulifolia,
grown from organic farming in Murcia (Spain). The oil
has been eco-extracted, by steam distillation with a portable still, next to cultivation, and with a boiler feed with
biomass, from plants previously distilled.
Methodology (www.um.es/genz/e00605/serv03.htm).
Fast Gas Chromatography on a non-polar fast column
(SLB-5ms) 15 m 0.1 mm 0.1 m, and Enantioselective
Gas Chromatography on a chiral column Chiraldex
B-DM 30m x 0.25 mm 0.12 m, were carried out on
a GC-chromatograph (Agilent 7890), with hydrogen as
gas carrier (PDH). The Mass Spectrometry Detector used
an electronic impact ionizer (70 eV), and a single quadrupole analyzer (Agilent 5975 MSD). Sandwich split/
splitless injections, with hexadecane as inner standard,
and calibration straigths with standards (Gerstel MPS2XT autosampler).
Results. The chromatographic results showed that the
S. lavandulifolia oil from Murcia is especially rich in
some biomolecules like (mM concentration, % enantiomers): Camphor (4068, +76, 24), Eucalyptol (1299),
Camphene (811, +26, 74), -Pinene (483, +52, 48),
-Pinene (365, +42, 58), Limonene (333, +81, 19),
Borneol (319, +34, 66), Myrcene (233), Linalool
(142, +4, 96), Linalyl acetate (138), -Terpineol
(96, +8, 92), -Terpinyl acetate (66, +6, 94), Bornyl acetate (61, +4, 96), and other minor components.
Conclusions. Salvia lavandulifolia oil has a very high
concentration of Camphor, Eucalyptol and Camphene,
about 1020 times higher than that of many other
biomolecules. Near pure ()-enantiomers are present for Linalool, -Terpineol, -Terpinyl acetate and
Bornyl acetate. There are mainly (+)-Camphor and
(+)-Limonene, whereas there are mainly ()-Camphene
and ()-Borneol. Furthermore, -Pinene and -Pinene
have similar proportions of both enantiomers. This biochemotype is markedly different than that of Salvia lavandulifolia oil, with similar cultivation and extraction
procedures in Castilla-La Mancha (Spain). The essential
oil from Murcia has higher concentrations than that
stated in the corresponding ISO standard for Camphor,
Eucalyptol and Limonene. This oil is a good source of
53
Acknowledgements
This work has been partially supported by grants from several Spanish
organizations. Projects BIO2009-12956 (MINECO, Madrid) and
08856/PI/08 (Fundacin Seneca, CARM, Murcia). AC has a fellowship
from Esencias Martinez Lozano S.A. (Murcia). VO has a FPU fellowship
(AP2010-4300).
54
Eurobiotech 2013
P5.25
P5.26
Acknowledgements
Original article with the same title will be submitted to Acta Biochimica
Polonica. Submitted abstract and article are a part of Prof. Florian
Ryszkas contribution.
PriB is a primosomal protein that catalyzes DNA replication in Procaryota. The replication pathway starts with
PriA protein the initiator protein that binds to a DNA
replication fork, unwinds double-stranded DNA and role
of PriB is to stabilize PriA on the DNA. However there are
many biochemical differences in replication mechanism
in bacteria and only some of them use PriB proteins.
A few of PriB proteins were published and only three
structures of them were resolved (Escherichia coli, Klebsiella pneumoniae and Neisseria gonorrhoeae). All upto-date known PriB proteins have one OB domain per
monomer and they are homodimers in solution.
Recently, we have published the crystal structure of PriB
protein from Thermoanaerobacter tengcongensis that
represents new class of PriB with two oligonucleotide/
oligosaccharide-binding domain (OB) per monomer that
means it exists as monomer in solution.
The aim of this study is identification and characterization of the primosomal protein B (PriB) from an anaerobic, thermophilic bacterium Clostridium thermocellum
(CthPriB). This PriB protein consisting 238 amino acid
residues and a calculated molecular mass is 27,5 kDa.
What is more CthPriB protein contains two single-stranded DNA binding domain (OB-fold) and functions as
monomer like PriB protein from bacterium Thermoanaerobacter tengcongensis.Therefore, our studies suggest
that we discovered new classes of PriB which werent
published recently.
Eurobiotech 2013
55
P5.27
P5.28
56
Eurobiotech 2013
P5.29
The effect of new hydantoin derivatives
on the increase of ciprofloxacin efficacy
in drug-resistant E. coli
Anna Matys, Ewa Otrbska, Jakub Mazurkiewicz,
Daria Studnicka, Beata Mastek, Jadwiga Handzlik,
Katarzyna Kie-Kononowicz
Department of Technology and Biotechnology of Drugs,
Jagiellonian University, Collegium Medicum, Faculty
of Pharmacy, Crakow, Poland
Acknowledgements
The authors wish to thank Professor Leonard Amaral and Professor
Isabel Couto (Grupo de Micobacterias, Unidade de Microbiologia,
Instituto de Higiene e Medicina Tropical, Universidade Nova de
Lisboa, Lisbon, Portugal) and dr hab. Anna Biaecka (Centrum
Bada Mikrobiologicznych i Autoszczepionek, Cracow, Poland) for
providing the strains used in this study. The study was financed from
the programme K/ZDS/001915.
References
[1] Handzlik J., Szymaska E., Alibert S., Chevalier J., Otrbska E.,
Pkala E., Pags J.-M., Kie-Kononowicz K., Search for new tools to
combat Gram-negative resistant bacteria among amine derivatives of
5-arylidenehydantoin. Bioorg. Med. Chem. 21 (2013) 135145.
Eurobiotech 2013
P5.30
Laccase activity and stability in the
presence of menthol-based ionic liquids
Joanna Feder-Kubis1, Jolanta Bryjak2
Wroclaw University of Technology, Faculty of Chemistry,
Department of Chemical Engineering; 2Wroclaw University
of Technology, Faculty of Chemistry, Department of Bioorganic
Chemistry
1
57
58
Eurobiotech 2013
P5.31
P5.32
Eurobiotech 2013
References
[1] Siehler S., Cell-based assays in GPCR drug discovery. J Biotech, 2008,
3: 471483.
[2] Hermans E., Generation of model cell lines expressing recombinant
G-protein-coupled receptors. Methods Mol Biol, 2004, 259: 137153.
[3] Oda T. et al., Molecular cloning and characterization of a novel type
of histamine receptor preferentially expressed in leukocytes. J Biol Chem,
2000, 275: 3678136786.
[4] Nguyen T. et al., Discovery of a novel member of the histamine
receptor family. Mol Pharmacol, 2001, 59(3): 42733.
[5] Jablonowski J., Carruthers N., Thurmond R., The histamine H4
receptor and potential therapeutic uses for H4 ligands. Mini Rev Med
Chem, 2004, 4: 9931000.
59
P5.33
The effect of egg yolk lipids enriched with
CLA on cytotoxicity of human melanoma
cell line WM793
Dominik Domagaa1, Aneta Koronowicz1, Jarosaw Oczkowicz1,
Elbieta Sikora1, Piotr Laidler2, Teresa Leszczyska1
University of Agriculture, Department of Human Nutrition,
Crakow, Poland; 2Jagiellonian University Medical College,
Crakow, Poland
1
Lectures
L6.2
L6.1
Engineering of a retrotransposon
for insertion mutagenesis in plants
Nikola Winter, Eneda Xhikaj, Lilian Nehlin, Andrea Tramontano,
Andreas Bachmair
Dept. of Biochemistry and Cell Biology, Center for Molecular
Biology, Max F. Perutz Laboratories, Univ. of Vienna,
Dr. Bohr Gasse 9, A-1030 Vienna, Austria
Petr Smkal
Department of Botany, Slechtitelu 11, Palacky University
in Olomouc, 783 71 Oolomouc, Czech Republic
Eurobiotech 2013
L6.3
High through output phenotyping and
genotyping stepsfor drought tolerance
improvement in barley
Marcin Rapacz1, Magdalena Wjcik-Jaga1, Anna Fiust1,
Bartomiej Kozera1, Mirosaw Tyrka2
University of Agriculture in Crakow; 2Rzeszow University
of Technology
1
61
Acknowledgements
The research were supported by Polish National Research and
Development Center (PBZ-2/3/2006/17 and GENMARK PBS1/
A8/1/2012).
62
Eurobiotech 2013
L6.4
L6.5
Eurobiotech 2013
Posters
P6.1
New sources of phenolic compounds
and anthocyanins for biotechnolgy
Nijol Anisimovien1, Jurga Jankauskien1, Milda Jodinskien1,
Vidmantas Bendokas2, Vidmantas Stanys2,
Tadeuas iknianas2
Institute of Botany of Nature Research Centre;
Institute of Horticulture, Lithuanian Research Centre for
Agriculture and Forestry
1
2
Introduction. One of the current problems in biotechnology is a search for plant original bioactive compounds
important due to their benefits to human health (He,
Guisti, 2010; Oancea, Oprean, 2011). Anthocyanins from
berries have received considerable interest as dietary
antioxidants among the different bioactive substances
in human health (Tsuda, 2012). These compounds are
being able to capture reactive oxygen species, delay the
initiation or propagation of oxidative chain reactions.
The prevention role of anthocyanins in cardiovascular
disease, cancer, diabetes and other chronic diseases was
indicated (Pascual-Teresa, Sanches-Ballesta, 2008; Shipp,
Abdel-Aal, 2010; Tsuda, 2012).
The growing interest in impact of antioxidants on human
health has triggered our study of berries from different
Ribes and Prunus species. Also, complex interspecific hybrids were created in order to obtain plants with higher
phenolic compound and anthocyanin amount, as well
as clarification of anthocyanin composition in order to
identify cultivars or hybrids with highest antioxidant activity.
Material and Methods. Four different Ribes species:
R. nigrum L. Ben Tirran (black berries), R. aureum
Au Gs5 (yellow berries) and Corona (black berries),
R. petreum Jonkher van Tets (red berries), R. uva-cripsa
Liai (red berries) and iornyj negus (black berries)
and four interspecific Ribes hybrids were studied. Anthocyanin and phenol compound content, and antioxidative
activity were evaluated in P. avium, P. cerasus cultivars,
P. machii wild form and 4 their interspecific hybrids, too.
All berries were collected at technical maturity phase
and immediately frozen at 70C. Anthocyanins and
other phenolics from frozen berries were extracted by
90% aqueous methanol, acidified by HCl to 0.1 N, at ratio 1:20 (g/ml). Total phenolics content in extracts was
determined by Folin-Ciocelteu method, using galic acid
as standard. Antioxidant activity (capacity) was evaluated spectrophoretically, by radical scavenging assay,
according DPPH test (Anisimovien et al., 2009). The
anthocyanin content was determined using a spectrophotometric differential pH method (Wrolstad et al., 2005)
and calculated using a molar extinction coefficient of cyanidin-3-glucoside 29600 (Cy-3-Glu). The composition
of anthocyanins was determined by HPLC procedure
(Durst, Wrolstad, 2001; Liobikas et al., 2009).
63
Acknowledgements
Research was funded by a grant No. SVE-01/2011 from the Research
Council of Lithuania.
References
[1] Anisimovien, N., Rubinskien M., Vikelis P., Stackeviien E., Stanys
V., iknianas T., Jankovska, E., Sasnauskas A. (2009) Anthocyanins
in currants, cherries, blueberries, and antioxidative activity of berry
extracts. Zemdirbyste=Agriculture, 96(3): 158167.
64
Eurobiotech 2013
P6.2
Selection efficiency of converted DArT
markers in spring barley breeding for
drought tolerance
Magdalena Wjcik-Jaga1, Anna Fiust1, Marcin Rapacz1,
Mirosaw Tyrka2
Department of Plant Physiology, University of Agriculture
in Crakow; 2Department of Biochemistry and Biotechnology,
Rzeszow University of Technology
1
Eurobiotech 2013
65
P6.3
P6.4
Alicja Chuda
University of Agriculture in Crakow, Faculty of Horticulture,
Institute of Plant Biology and Biotechnology, Department
of Genetics, Plant Breeding and Seed Science,
al. 29 Listopada 54, 31-425 Crakow, Poland
66
Eurobiotech 2013
P6.5
P6.6
Eurobiotech 2013
67
P6.7
Intergenic Spacer length variability
in cultivated, weedy and wild rye species
Lidia Skuza, Ewa Filip, Izabela Szuko
Chair of Cell Biology, University of Szczecin, Waska 13,
PL-70415 Szczecin, Poland
68
Eurobiotech 2013
The variation in the total size of the IGS among the species detected in this work could be due to dissimilarity in
the sequence of the repetitive elements or in their tandem
repeat number (Saghai-Maroof et al. 1984; Barker et al.
1988; May and Zhiyong 1996). Highly inter specific polymorphisms for rDNA IGS region suggesting the IGS will
be a useful molecular marker for studies of Secale species.
Acknowledgements
This work is supported by the State Committee for Scientific Research
grant No. N N310 435498 Degree of relatedness within the genus
Secale using non-coding chloroplast and mitochondrial sequences, and
nuclear rDNA IGS regions.
P6.8
Intra-population genetic diversity
of cultivated carrot (Daucus carota L.)
assessed by analysis of microsatellite
markers
Anna Maksylewicz, Rafal Baranski
University of Agriculture in Crakow, Institute of Plant Biology
and Biotechnology, Unit of Genetics, Plant Breeding and Seed
Science, al. 29 Listopada 54, 31-425 Crakow, Poland
Eurobiotech 2013
69
P6.9
Detection of Plasmodiophora brassicae
in soil by PCR method
Anna Czubatka, Jozef Robak, Agnieszka Czajka,
Wojciech Szczechura, Miroslawa Staniaszek
Research Institute of Horticulture, Konstytucji 3 Maja 1/3 St.,
96-100 Skierniewice, Poland
70
Eurobiotech 2013
P6.10
P6.11
Eurobiotech 2013
71
P6.12
DNA markers as a tool for selection
of tomato plants with resistance to
Fusarium oxysporum f.sp. radicis-lycopersici
Mirosawa Staniaszek, Wojciech Szczechura, Elbieta Kozik,
Marzena Nowakowska, Hanna Habdas
Research Institute of Horticulture, Department of Genetic
Breeding and Biotechnology Vegetable Plants,
96-100 Skierniewice, Poland
Tomato is one of the most important and popular vegetable grown in many world regions. In modern breeding of
this species more frequently are using molecular markers,
especially based on the polymerase chain reaction (PCR).
These methods allowing to selection of plants with specific trait such as resistance to fungal pathogens. Fusarium
oxysporum f.sp. radicis-lycopersici (Forl) is an important
tomato pathogen. This pathogen causes fusarium crown
and root rot, one of the most destructive disease of tomato in crops under cover. Resistance to Forl is determined
by single dominant gen, named Frl, located on the long
arm of chromosome 9 tomato genome. In studies undertaken in Research Institute of Horticulture, Skierniewice,
CAPS C2-251100 marker was identified which is linked to
Frl gene. The amplification products was digested by XapI
restriction enzyme to obtain a DNA polymorphism. This
marker can be useful in marker assisted selection in tomato breeding programs.
This work was performed in the frame of Multi-annual
Programme Development of sustainable methods of
horticultural production to ensure high biological and
nutritional quality of horticultural products and to preserve the biodiversity of the environment and to protect
its resources, finance by Polish Ministry of Agriculture
and Rural Development; Task 6.6.
72
Eurobiotech 2013
P6.13
Effect of different conditions for cell
immobilization and phytosulfokine
supplementation on somatic
embryogenesis in protoplast cultures
of Daucus species
Katarzyna Makowska, Ewa Grzebelus
University of Agriculture in Crakow, Dept. of Genetics,
Plant Breeding and Seed Science
Eurobiotech 2013
P6.14
P6.15
73
74
Eurobiotech 2013
References
[1] Anamthawat-Jnsson (2004) Preparation of chromosomes from
plant leaf meristems for karyotype analysis and in situ hybridization.
Methods in Cell Science 25(3-4): 9195.
Environmental biotechnology
Lectures
L7.2
L7.1
Metallophytes: a biodiversity
and phytotechnological resource for
soil clean-up, phytomining and mine site
restoration
Alan J M Baker
School of Botany, The University of Melbourne, Australia;
Centre for Mined Land Rehabilitation, The University
of Queensland, Australia; Department of Animal and Plant
Sciences, The University of Sheffield, UK
Metallophytes plants that have evolved on metal-enriched soils have key values that must drive research
on their unique properties, and ultimately their conservation. The ability of metallophytes to tolerate extreme metal
concentrations commends them as the optimal choice for
phytostabilization and ecological restoration of mineral
wastes and metal-contaminated soils. Metallophytes, and
in particular metal-hyperaccumulating plants, have also
spawned several novel phytotechnologies, including phytoremediation, phytoextraction and phytomining. Other
new potentials for exploiting their unique properties are
emerging. The last decade has seen an ever-increasing
interest in metal-tolerant and metal-accumulating plants
both from an academic standpoint and in developing their
potentials in phytotechnologies. Few studies have highlighted the need to conserve these species. This presentation identifies future research needs for the conservation
and utilization of the global metallophyte biodiversity.
76
Eurobiotech 2013
L7.3
L7.4
Establishment of a vegetation cover on mine waste substrate is one of the crucial but also one of the most difficult parts of the remediation process. This study tests
two different approaches to facilitate plant growth on
toxic and oligotrophic substrates, i.e., the addition of
coarse mineral particles and the inoculation with putative
growth supporting bacteria.
Extremely fine grained mine waste from Ingortosu/Italy
containing high amounts of Zn, Cu, Cd and other toxic elements was supplemented with a mixture of sand
and volcanic clay, with a bacterial consortium (Bacillus
cereus, Curtobacterium flaccumfaciens and eight other
strands) or with a combination of both, a control was left
untreated. On these substrates, Euphorbia pithyusa, Helianthus annuus, Agrostis capillaris, Deschampsia flexuosa
and Festuca rubra were grown in pots under greenhouse
conditions.
The bacteria established well and reduced the extractability of most metals. Their effect on plant growth, however, was ambiguous and usually negative. The addition of
sand and volcanic clay, on the other hand, had a positive
effect on all plant species except E. pithyusa. The effects
of a double treatment with both bacteria and sand and
volcanic clay were insignificant or negative.
It is concluded that manipulations of the soil texture of
mine substrates may play a key role in the process of
bioremediation.
Acknowledgement
This study was supported by funding of the the EU project Umbrella
(EU 226870), of the Austrian Ministery for Science and Research, and
the Appear project Biorem. We acknowledge the use of the green
house facility of the University of Vienna and we are thankful for the
help of the gardeners, Thomas Joch and Andreas Schrfl. The soil from
Ingurtosu/Sardinia was kindly provided by Giovanni de Giudici from
the University of Cagliari/I.
Eurobiotech 2013
L7.5
L7.6
Mosses are frequently regarded as tolerant to toxic metals and as accumulators of such metals regardless of their
original habitats. The reasons for this tolerance are poorly
understood. Unlike vascular plants, mosses absorb nutrients via the entire surface, and do not possess exclusion mechanisms like an exodermis or an endodermis. In
preliminary experiments, we confirmed an exceptionally
high zinc tolerance of moss gametophytes from heavy
metal contaminated mining sites, and also for Physcomitrella patens Hedw. (Funariaceae) which avoids metal
enriched habitats in nature.
Gametophytes of P. patens were cultivated on sterile
agar plates. We offered zinc (as Na2ZnEDTA, ZnCl2 and
ZnSO4) and copper (as Na2CuEDTA, CuCl2 and CuSO4).
After five weeks of cultivation the moss samples were
washed, air-dried, mounted and carbon-coated. During
sample preparation, great attention was paid to avoid
contamination with the metal rich growth medium. Copper and zinc uptake of P. patens leaves were semi-quantitatively analysed by X-raymicroanalysis in a scanning
electron microscope.
We observed differences in tolerance and uptake of copper and zinc. A gradient elevation of the heavy metal content of leaf tissues corresponding to the added amount of
zinc and copper in the growth media could be observed.
Furthermore, our results indicate strong differences in
uptake and resistance depending on the anion. E.g., metals coordinated with EDTA proved to be significantly less
toxic for P. patens. The relevance of coordinating agents
for metal toxicity is discussed.
Acknowledgements
This research was supported by the Gesellschaft zur Frderung der
Pflanzenwissenschaften, the Hochschuljubilumsstiftung der Stadt Wien
(grant H-1939/2008), the University of Vienna (Forschungsstipendium
2010 to S.S.) and Appear (Biorem)
77
Mining and ore processing frequently results in the formation of metal rich drainage water, representing a serious threat for people and environment. The remediation
of such waters is difficult and expensive, and alternatives
to conventional water treatment are intensively investigated.
At a historic mining site in the Austrian Alps, a Cu contaminated creek is remediating via constant binding of
metals to microbial mats. This study deals with the localisation of the absorbed copper, as well as the isotopic
signature and species composition of the microbial community.
The biofilm is almost exclusively dominated by the Cyanobacterium Phormidum sp. and contains 3.9 1.8%
Cu. The constant absorption from the water reduces
the Cu content of the creek from 0.6 0.1 mg l1 to
0.2 0.2 mg kg1 after only 300 m, thereby exceeding
abiotic Cu precipitation by far. Ancient dead layers of biofilm covered by humus but still rich in Cu indicate that the
immobilisation is permanent. In spite of the variable Cu
content of the biofilm, it exhibits a remarkably constant
65Cu of 0.50.1 which is significantly higher than in
the contaminated water (0.90.2) making uptake of
the Cu into the living cell improbable. This is confirmed
by the ubiquitous occurrence of electron dense mineral
particles in the gelatinous sheath of Phormidium, partly
identified as the secondary Cu mineral sampleite.
Fixed mats of Phormidium have been frequently used
for metal absorption under laboratory conditions. The
spontaneous self-remediation of a creek by Phormidium,
however, is a novel feature and suggests new strategies for
the application of Cyanobacteria in the field of bioremediation.
Acknowledgements
We are grateful to Prof. Dr. A. Beran (University of Vienna) for the
identification of sampleite. Thanks are due to Forstmeister R. Schilcher,
who made scientific research in the Schwarzwand possible. This study
was supported by the EU project Umbrella (EU226870), the Appear
project Biorem and the OEAD project Promote.
78
Eurobiotech 2013
L7.7
FISH detection of autochthonous bacteria
of ceramic clayey raw materials
Paulina Supel1, Pawe Kaszycki1, Joanna Brzeszcz2,
Piotr Kapusta2, Piotr Wyszomirski3
Departament of Biochemistry, Institute of Plant Biology and
Biotechnology, Faculty of Horticulture, University of Agriculture
in Crakow; 2Department of Microbiology, Oil and Gas Institute,
Crakow, 3AGH University of Science and Technology, Faculty
of Materials Science and Ceramics, Crakow
1
Contamination of clay raw materials with organic compounds causes severe problems in ceramic industry. Occurrence of organic fraction leads to lowered aesthetic
value, profoundly reduced mechanical strength, and
thus decreases the overall industrial suitability. Therefore, quality requirements along with increasingly strict
environmental standards give strong reason to elaborate
innovative methods of beneficiation of ceramic clays.
Among these, biotechnological methods based on microbiological enzymatic activities seem to be the most challenging and efficient. The main principle of these methods is to degrade undesirable organic admixtures by heterotrophic microorganisms capable of bioremediation of
an organic fraction as well as to remove iron compounds
by bioleaching.
Autochthonous microorganisms appear as the best natural candidates for biological improvement of ceramic
industry clay materials. If present, they are best fitted
and adapted to the clayey microenvironment and can
possibly utilize material organic impurities as a source
of carbon.
The aim of the study was to prove the occurrence of
autochthonous bacteria in three selected ceramic clay
materials, obtained from different areas and differing in
the content and form of an organic fraction: (1) Lower
Jurassic (Liassic) clay from arnw, with a low organic substance content, (2) Tertiary material form Turw,
characterized by a moderate level of carbon compounds, especially the so called fine-dispersed brown
coal substances, obtained from a site adjacent to brown
coal deposits, and (3) a clay from the Radzymin deposit,
belonging to the relatively young, Quaternary materials,
used mainly in building ceramics due to the high content of organic compounds.
Standard microbiological plating techniques showed the
occurrence of autochthons in materials (2) and (3). Identification of several colony morphotypes indicated high
biological diversity of the strains. The FISH (fluorescence
in situ hybridization) technique enabled to reveal even
greater biodiversity, including clay (1). The visualisation
method was based on several specific molecular probes
directed at conservative genes 16S rRNA (EUB338I, II,
III-Cy3, ALF968-Cy3) and 23S rRNA (BET42a-Cy3,
GAM42a-Cy3, HGC69a-Fluos). The above sets of oligonucleotide probes were constructed to identify bacteria
belonging to the following systematic groups: -Prote-
obacteria (ALF 968), -Proteobacteria (BET42a), -Proteobacteria (GAM42a), Actinobacteria (HGC69a), and
Eubacteria (a mix of three probes EUB 338I, II, III.)
The above data bring for the first time the information
of bacterial consortia inhabiting unique environments of
clayey minerals. We claim the research that we have just
launched will bring interesting and valuable applications
in terms of treatment of ceramic industry materials prior
to their processing.
Acknowledgements
The work was financially supported by the grant for scientific research
No. 3500, approved by the Polish Ministry of Science and Higher
Education.
Eurobiotech 2013
L7.8
Posters
P7.1
Prasad M.N.V.
Department of Plant Sciences, University of Hyderabad,
Hyderabad 500 046, India
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Eurobiotech 2013
Acknowledgements
The study financed from the science resources in the years 20132014, as
an experimental study of the National Science Centre (No. 2012/05/N/
NZ9/02436).
References
[1] Garcia-Estepa R.M., Guerra-Hernndez E., Garcia-Villanova B.
(1999) Phytic acid content in milled cereal products and breads. Food
Research International 32, 217221.
[2] Lei X.G., Porres M. (2003) Phytase enzymology, applications, and
biotechnology. Biotechnology Letters, 25, 17871794.
P7.2
The Kinetic reduction of Cr(VI) by yeast
cells of Saccharomyces cerevisiae,
Phaffia rhodozyma and their protoplasts
Jarosaw Chwastowski, Henryk Kooczek
Crakow University of Technology, Department of Inorganic
Technology and Environmental Biotechnology
Contamination with heavy metals is a serious environmental problem. Pollution by chromium originating
from several industrial processes such as leather tanning, electroplating, textile industries, nuclear power
plant, water cooling, pulp production, ore and petroleum refining processes is a reason for deterioration of
midland water quality. In trace amounts, chromium is
beneficial to living organisms (M. Pas et al., 2004). However, at higher concentrations it is extremely toxic, causing allergies, egzema, and respiratory track disorders. It
is also a strongly mutagenic and cancerogenic agent, especially in its oxidized form, Cr(VI) (Barceloux, 1999).
Once inside the cell Cr(VI) is being reduced to the
Cr(V) and can react with H2O2 what leads to the production of hydroxyl radicals (OH) via the Fenton-like
reaction. Cr(V) causes DNA breaks and various mutations in chromosomes.
Chromium on the sixth oxidation state can enter easily
into the living cells because Cr(VI) ion has tetrahedral
symmetry (same as SO42 and HPO42, unlike Cr(III)
which has octahedral symmetry) and can enter the cell
membrane through non-specific sulfate transporters by
facilitated diffusion.
The adverse health effects and diverse cellular and molecular reactions make the studies on chromium toxicology and metabolism very crucial in terms of environmental protection and clinical medicine. Such
studies are performed using eukaryotic organisms,
mainly yeast, plants, mammalian cells and transgenic
mice (Cervantes et al., 2000). Among these, yeast has
proved to be a very suitable model for the research of
eukaryotic cell response to chromium stress and bioremediation pathways (Ksheminska, Koloczek et al., 2001,
2005). Microbiological bioremediation of chromium
contamination is performed by means of the reduction
of chromate to the less toxic Cr(III) form which can be
easily removed by precipitation. The mechanisms of
chromate reduction and its transport to the yeast cells
are the subject of the studies.
The kinetic reduction of Cr(VI) presented in this study
is determined in intact yeast cells and their protoplasts.
The study evaluates the behavior of yeasts: Saccharomyces
cerevisiae and Phaffia rhodozyma cells and their isolated
protoplasts to reduce Cr(VI) to lower oxidation states.
The viability of yeast cells was carried out using the methylene blue staining method and the plate method (after
the incubation with different concentration of Cr(VI),
which varied from 0,5 mM to 10 mM).
Eurobiotech 2013
81
P7.3
The citricacid-modified enzyme-resistant
dextrin from potato starch
as potentialprebiotic
Katarzyna liewska1, Renata Barczyska2, Janusz Kapuniak2
Institute of Fermentation Technology and Microbiology,
Faculty of Biotechnology and Food Sciences, Technical
University of Lodz, 171173 Wolczanska Street, 90-924 Lodz,
Poland; 2Institute of Chemistry, Environmental Protection and
Biotechnology, Jan Dlugosz University, 13/15 Armii Krajowej
Avenue, 42-200 Czestochowa, Poland
1
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Eurobiotech 2013
P7.4
Comparison of ARDRA and ARISA
fingerprinting patterns analysis
as a methods in early bulking activated
sludge symptoms detection
Dagna Sotysik, Daria Matczyska, Ilona Bednarek
Department of Biotechnology and Genetic Engineering, Medical
University of Silesia in Katowice, Narcyzow 1,
41-200 Sosnowiec, Poland
Eurobiotech 2013
P7.5
P7.6
83
This study presents the results of a laboratory experiments, including a phytofiltration prototype system, to
reduce the uranium (U) concentration in contaminated
waters. The species Callitriche stagnalis Scop., Potamogeton natans L. and Potamogeton pectinatus L. of the native plant community of the uraniferous region of Beiras
(Central Portugal) were selected because they are autochthonous and they show high accumulation levels and/or
high biomass production. Fluorometry was adopted for
the determination of the U content in the water and plant
samples using a Fluorat-022M analyzer (Lumex, Russia). The installed prototype consists of a closed circuit
of channels. The system was initially contaminated with
500 g/L of U as uranyl. The performance of this system was very effective. The U concentration in the water
dropped to 220 g/L in 24 hours and after two weeks it
had decreased to 72.3 g/L, thus representing an efficiency of 85.5%. The U concentration in C. stagnalis increased
from 0.98 to 1567 mg/kg, in P. natans increased from 3.46
to 271 mg/kg and in P. pectinatus increased from 2.63 to
1588 mg/kg. The results show the effectiveness of these
plants to remove U from the water.
Lipopolysaccharide (LPS, endotoxin) is an integral component of the outer membrane of the cell envelope of
Gram-negative bacteria as well as some cyanobacteria.
LPS is the major virulence factor of bacteria and a strong
stimulator of innate immunity in diverse of eukaryotic
species. LPS massively released by pathogenic bacteria infecting the organism, induces overproduction of inflammatory mediators, leading to the systemic inflammatory
response (sepsis) and potentially death. Cyanobacterial
lipopolysaccharides, compared with pathogenic bacterial LPS such as Vibrio cholerae, Neisseria meningitidis,
Yersinia pestis, Plesiomonas shigelloides, have been less
studied [1, 2].
The purpose of this study was to investigate biological activities of lipopolysaccharide isolated from cyanobacterium Synechococcus PCC 7002. LPS was tested by the chromogenic kinetic Limulus amebocyte lysate assay (LAL
test) and by cell culture assay the induction of proinflammatory cytokines, tumor necrosis factor a (TNF-a)
and interleukin 6 (IL-6) by mouse macrophage cell line
J774A.1. Lipopolysaccharide Synechococcus PCC 7002,
activated of the clotting factors released by amebocytes
Limulus and has the ability to induction of the inflammatory cytokines in immunocompetent cells. However LPS
Synechococcus PCC 7002 was less active than this from
the pathogenic Escherichia coli O55.
References
[1] ukasiewicz J., ugowski C. (2003) Post. Hig. Med. Dosw. 57: 3353.
[2] Bernardova K. et al. (2008) J. Appl. Toxicol. 28: 7277.
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P7.7
Influence of imidazolium ionic liquids
on activated sludge process
Dorota Gendaszewska1, Ewa Liwarska-Bizukoj1,
Cedric Maton2, Chris V. Stevens2
Technical University of Lodz, Institute of Fermentation
Technology and Microbiology, Lodz, Poland; 2Ghent University,
Department of Sustainable Chemistry and Technology, Ghent,
Belgium
1
Ionic liquids (ILs) have attracted great interest in scientists and industrial centers during the last decade. This
compounds due to their specific features could be a green
alternative for organic solvents. Unfortunately, their
properties like high chemical and thermal stability and
solubility in water can affect on the quality and quantitative composition of the population of activated sludge.
As a consequence, biological wastewater treatment can
be difficult, not fully effective and cause a threat for
aquatic environment. Therefore, it is important to carry
out research on the impact of these compounds on biological wastewater treatment. The aim of the study is to
investigate the effect of the ILs on activated sludge process. To achieve it, the selected imidazolium liquids were
tested with respect to their influence on morphology of
activated sludge flocs, biomass concentration, degrees
of ILs and COD removal. Four different ionic liquids
were tested:1-ethyl-2H-3-methyl-4,5-dimethylimidaziolium iodide (IL 1), 1-ethyl-2-methyl-3-methyl-4,5-dimethylimidazolium iodide (IL 2), 1-ethyl-2-isopropyl-3-methyl-4,5-dimethylimidazolium iodide (IL 4),
l-hexyl-2H-3-methyl-4,5-dimethylimidazolium iodide
(IL 6). The activated sludge was taken from the aeration
chamber at the Combined Wastewater Treatment in Lodz
(Poland). All experiments were conducted in the shake
flasks (300 ml) under aerobic conditions. The following
concentrations of ILs were applied: 1, 5, 25 and 50 mg/l.
The flasks were incubated at 200,5C in a thermostated rotary shaker at 130 min1 for 24 hours. For each ILs
test was performed in triplicate. In addition, control tests
without ionic liquid were conducted. The concentration
of ILs, Chemical Oxygen Demand (COD) and volatile
suspended solids (VSS) were determined at the beginning
and at the end of the tests. Moreover, three independent
slides from each sample were prepared and approximately 40 images were taken. They were processed and analysed using the automated image analysis procedure. As
a result the basic morphological parameters of the flocs
were measured. Ionic liquids influenced on activated
sludge flocs morphology, particularly with regard to flocs
size. The mean projected area of flocs for each sample was
lower than the control, especially at the high initial concentrations of ILs in wastewater (25 and 50 mg/l). The
smallest flocs were found in the tests with IL 6 (50 mg/l).
Also, the presence of ILs in wastewater at higher concentrations caused to the decrease of the concentrations of
activated sludge biomass. The above mentioned observa-
Eurobiotech 2013
P7.8
Treatment of fibreboard wastewater
by combined ozone, membrane bioreactor
and activated carbon process
Dorota Krzemiska, Magdalena Madea, Ewa Neczaj
Czestochowa University of Technology, Institute
of Environmental Engineering, Brzeznicka 60a,
42-200 Czestochowa, Poland
The aim of this work was to estimate the influence of ozonation pretreatment of fibreaboard wastewater (FBM) on
anaerobic treatment efficiency in membrane bioreactor
(MBR). Moreover, granular activated carbons (GAC)
were used as a tertiary treatment step.
The influent fed into the anaerobic bioreactor was
collected from the raw wastewater generated in fibreboard company (Silesian Region, Poland). Ozone
gas was generated from air using an ozone generator
(CH-KTB-3G with a power on 0,075 KW). The gas
mixture of air and ozone was fed into the glass tank
containing wastewater via a porous diffuser. Batch experiments were conducted with the use of 1 L wastewater with natural pH and temperature room (2023C).
A 23,5 mg/L of ozone dose in different time was applied for ozonation experiments with an air ow rate of
0,18 m3/h. Anaerobic treatment experiment was conducted by means of 12 L capacity laboratory scale submerged membrane reactor with the capillary ultrafiltration module. The reactor was filled up with granular
sludge from industrial anaerobic wastewater treatment
plant (UASB) from brewing industry. Experiments
were performed under temperature of 37oC with granular sludge concentration of 10 g/l. After acclimatization period reactor was fed with mixture of synthetic
wastewater (80%, v/v) and FBM wastewater (20%, v/v).
Membrane bioreactor was operated at organic loading
rates (OLR) of 1.51 and 1.24 kg COD/m3d and hydraulic retention time (HRT) of 2,4d. In the next step
of this investigations a two types of activated carbons:
WG-12 and Picabiol with different characteristic were
used as a post-treatment step.
The results of our investigations indicated that ozonation irradiation improves the treatability of fibreaboard
wastewater resulting in a higher percent COD removals
and biogas yield. Under the optimal condition, the investigated dose of 19,5 mgO3/L/h and reaction time of
10min, anaerobic biodegradability of FBM wastewater in
MBR reactor was increased by 19%. Moreover the higher
biogas production (0.750.78 m3/kgCODrem.d) was also
observed under ozonation pretreatment. Therefore ozone
pretreatment of fibreboard wastewater is a promising
method to enhance fermentation efficiency. Moreover,
the results obtained showed that GAC can be used in the
removal of COD from fibreboard wastewater. The higher
reduction of COD (67%) was obtained for carbon WG-12
with adsorbent dose of 6 g/l.
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Eurobiotech 2013
P7.9
Treatment of fibreboard wastewater by
combined fentons process, membrane
bioreactor and activated carbon process
Dorota Krzemiska, Magdalena Madea, Ewa Neczaj
Czestochowa University of Technology, Institute
of Environmental Engineering, Brzeznicka 60a,
42-200 Czestochowa, Poland
Eurobiotech 2013
P7.10
P7.11
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Eurobiotech 2013
P7.12
P7.13
The conducted study regarded the assessment of sorption properties of moss Pleurozium shreberi and lichen
Hypogymnia physodes in reference to heavy metal cations: Mn2+, Ni2+, Cu2+, Zn2+, Cd2+ and Pb2+. The aim of the
study was to confirm the thesis that moss reveals better
sorption properties regardless of the type of sorbed cations. The sorption process of the studied analytes was
conducted under static conditions (at changing analytes
concentration in solution) and under dynamic conditions (at constant analytes concentration in solution). To
describe the equilibrium, the Langmuir isotherm model
was applied.
On the basis of the conducted study, it was found out that
the equilibrium is attained after approximately 30min. In
the experiment conditions, 9095% of metals are sorbed
within the first 10 min. It was also stated that moss
Pleurozium shreberi is characterised by better sorption
properties as compared to lichen Hypogymnia physodes,
and that they sorb heavy metals proportionally to their
amount in the solution, in which they were immersed.
On the basis of the conducted study, the affinity of the
studied cations with moss and lichen sorption structures
was determined.
Keywords: lichen Hypogymnia physodes, moss Pleurozium shreberi,
heavy metals, sorption kinetics and equilibrium, Langmuir isotherm
Eurobiotech 2013
Acknowledgements
Project Nr POIG.01.03.01-00-158/09 Biotransformations useful in
pharmaceutical and cosmetic industries.
Project partially financed by the European Regional Funds within the
scope of Operation Program Innovative Economy.
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P7.14
Changes in dynamics of ascorbateglutathione metabolism in response
to trace metal stress in Pisum sativum
Aneta Piechalak1, Arleta Malecka1, Agnieszka Kutrowska1,
Anetta Hanc2, Danuta Baralkiewicz2, Barbara Tomaszewska1
Department of Biochemistry, Faculty of Biology,
Adam Mickiewicz University, Umultowska 89, 61-614 Poznan,
Poland; 2Department of Trace Element Analysis by
Spectroscopy Method, Faculty of Chemistry, Adam Mickiewicz
University, Umultowska 90, 61-614 Poznan, Poland
1
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Eurobiotech 2013
P7.15
P7.16
The wastewater originating from explosives manufacturing plants are characterized by a high concentration of
nitrates, sulphates and low pH. The determined composition of treated wastes was: 3200 mg N L1, 48 mg L1
chlorides, 1470 mg L1 sulfates. The DNC6 consortium
bacterial cell samples collected during denitrification of
industrial wastewater in the bioreactor were subjected
to separation with the use of the FACS Aria III flow cytometer (Becton Dickinson, USA). Cytometric analysis
of the metabolic activity, expressed as the red-ox potential, allowed for the evaluation of specific single microbial
cells. Separation of cells allowed for the isolation of pure
monocultures, which were used for sequencing and subsequent genetic identification. The microorganisms present in the bacterial population used for the denitrification
were identified at the first, second, fourth and sixth day
of the process. The combination of flow cytometry and
genetic identification allowed for determination of bacterial species present in both areas of metabolic activity.
The low metabolic activity area accounted for 32% of the
total cell count, while the high metabolic activity area
constituted for 19% of the total cell count. In the area of
highest activity P. stutzeri accounted for 43% of total cell
count, while P. putida accounted for 39%. The remaining 18% included freundi, P. alcaligenes and A. xylosoxidans. The composition of cells present low activity area
was more diverse, with 79% accounting for four main
species O. intermedium, R. rubber, S. multivorum and
S. maltophila). The highest difference in metabolic activity between these two areas was observed during the
second day of the denitrification process. The microorganisms in the high activity area exhibited a fluorescence
intensity at 12500, while the value for microbes in the low
activity area was at 960.
The industrial development and expansion of urban agglomerations, which have been constantly progressing
since the 18th century, contribute to the accumulation
of compounds from the group of polycyclic aromatic
hydrocarbons (PAH) in different areas of environment
and to their subsequent deposition into surface waters.
The scale of the phenomenon is evidenced by numerous
exceeding of standards recorded during the diagnostic
monitoring of priority substances conducted under the
State Environmental Monitoring. Since it was found that
the prolonged effects of PAH on living organisms are
highly unfavorable, there has been an increasing need to
develop technologies of their effective degradation. This
study aimed to determine the effect of ozonation-induced
radical reactions on the biodegradation kinetics of selected PAH compounds by a microbial consortium isolated
from soils contaminated with organic substances. The
degradation was carried out under aerobic conditions at
25C for 240 h in aqueous mineral solutions containing
naphthalene, phenanthrene or pyrene at concentrations
of 50 mg/dm3. The ozonation was performed before microbial inoculation and lasted 60 minutes (at an efficiency of 25 g ozone/h). Measurement of PAH removal was
performed using the photoluminescence method with
prior hexane extraction. It was found that ozonation had
no lethal effects on the microbial consortium, reflecting
the lack of toxicity of radical reactions products. In addition, the biodegradability of particular PAH in ozonated
systems showed similarity to the reference tests. The increase in the number of aromatic rings was related with
a decrease in the degradation rate, which was associated
with a decrease in water solubility and lower bioavailability of the studied compounds. Assessment of biodegradation kinetics (based on the comparison of PAH degradation half-times) showed that ozonation increased the
biodegradation efficiency by approx. 25%. These results
demonstrate the possible application of combined techniques: ozonation and biodegradation in environment
protection.
Acknowledgements
This work was financially supported by the Ministry of Science and
Higher Education, Poland (Grant No. N N523 419 837).
Eurobiotech 2013
P7.17
Different patterns of SOD generation
and scavenging in barley SSD lines
during drought
Renata Bczek-Kwinta1, Magorzata Borek2,
Janusz Kocielniak3, Katarzyna muda4
University of Agriculture in Crakow, Faculty of Agriculture and
Economics, Plant Physiology Department; 2University
of Agriculture in Crakow, Faculty of Agriculture and Economics,
Plant Physiology Department; 3University of Agriculture
in Crakow, Faculty of Agriculture and Economics, Plant
Physiology Department; 4University of Agriculture in Crakow,
Faculty of Agriculture and Economics, Plant Physiology
Department
1
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Eurobiotech 2013
P7.18
P7.19
The study focused on assessing the influence of bioaugmentation and addition of rhamnolipids on diesel oil biodegradation efficiency under environmental conditions.
Initial laboratory studies (measurement of emitted CO2
and dehydrogenase activity) were carried out in order
to select the consortium for biougmentation as well as
to evaluate the most appropriate concentration of rhamnolipids. The selected consortium consisted of following
bacterial taxa: Aeromonas sp., Alcaligenes sp., Gordonia sp., Pseudomonas fluorescens, Pseudomonas putida,
Rhodococcus sp., Stenotrophomonas maltophilia, Xanthomonas sp. It was established that the application of the
rhamnolipids at 150 mg/kg of soil was most appropriate
in terms of dehydrogenase activity. Based on the obtained
results, four treatment methods were designed and tested
during long-term field studies: I) natural attenuation; II)
addition of biosurfactants III) bioaugmentation; IV) bioaugmentation and addition of biosurfactants. It was observed that bioaugmentation contributed to the highest
diesel oil biodegradation efficiency, whereas the addition
of rhamnolipids did not notably influence the treatment
process.
Acknowledgements
This work was supported by the grant number 32-400/2013 DS-MK.
Eurobiotech 2013
P7.20
P7.21
Surface-active compounds of biological origin have attracted much attention and their popularity seems to
steadily increase during recent years. Like every other
natural substance, biosurfatants have numerous advantages compared to their synthetic counterparts. They
are equally effective as synthethic surfactants in terms of
solubilization and emulsification, however they are readily biodegradable, less toxic and more environmentally
friendly. Moreover, biosurfactants can be obtained from
waste materials, which renders their production favorable on account of economics. All these relevant traits
contribute to a high applicability of biosurfactants, which
currently stems to several branches of industry as well
as remediation. This review summarizes the recent revelations in the field of biosurfactant amended bioremediation, focusing mainly on a critical approach towards
potential limitations and causes of failure, while studying
the effects of biosurfactants on the efficiency of biodegradation and phytoextraction processes.
Acknowledgements
This work was supported by the grant number 32-400/2013 DS-MK.
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Eurobiotech 2013
P7.22
P7.23
Jakub Idkowiak2, Bogdan Wyrwas2, Agnieszka Zgoa-Grzekowiak2, Anna Syguda1, Joanna Wojtera-Kwiczor3,
Anna Gielnik 3, ukasz awniczak 1, ukasz Chrzanowski 1
Institute of Chemical Technology and Engineering, Poznan
University of Technology, Poznan, Poland; 2Institute
of Chemistry, Poznan University of Technology, Poznan,
Poland; 3Institute of Molecular Biology and Biotechnology,
Adam Mickiewicz University in Poznan, Poznan, Poland
1
Eurobiotech 2013
P7.24
P7.25
Biodiesel is considered as an attractive alternative to petroleum derived diesel fuel. However its environmental impact has not been fully investigated yet. Many researches concerning the emission of green-house gases
and exhaust emission have been conducted, but little attention has been paid towards the fate of biodiesel fuels
in case of an environmental spill. As the production and
consumption of biodiesel grows, the threat of its spills
into the environment increases as well. In fact, biodiesel
is mainly used in the form of blends with petro-diesel.
Therefore simultaneous presence of fatty acids alkyl esters
and petroleum hydrocarbons in the fuel contaminated
site is highly probable. Although it has been confirmed
by several authors that biodiesel is more readily biodegraded than petroleum diesel in general, the impact of
biodiesel on the biodegradation of hydrocarbons remains
unclear. Some authors suggest that the presence of a readily available carbon source may stimulate the growth of
microorganisms and hence increase the dissipation of
hydrocarbons. On the other hand, other researchers do
not confirm this hypothesis. The aim of this work was
to investigate the influence of biodiesel on the biodegradation of hydrocarbons in the water environment. For
this purpose, a set of aqueous microcosms was prepared.
Mixtures of model hydrocarbons paraffins, aromatics
and naphthenes served as a sole carbon source in one
half of the microcosms. The other half was supplemented with 20% addition of biodiesel. During the 3 weeks
lasting experiment, sets of samples were sacrificed periodically for chromatographic determination of carbon
source residues. The results of the research show that
the applied bacterial consortium isolated from crude oil
polluted site exhibits great abilities to decompose various
chemical groups of hydrocarbons and biodiesel, although
branched alkanes and naphthenes were degraded in lesser extent. On the other hand, the most readily degraded
compounds were fatty acids methyl esters and straight
chain alkanes, which were dissipated in a similar rate.
There was no significant change of hydrocarbons biodegradation rate in the presence of biodiesel. This suggests
that biodiesel addition to diesel fuel will not affect the
biodegradation of the petroleum fraction of such fuels.
Acknowledgements
This work was supported by the grant number 32-400/2013 DS-MK.
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Eurobiotech 2013
P7.26
P 7.27
The aim of our study was to investigate the effect of Triton X-100 on the biodegradation efficiency of hexadecane and phenanthrene carried out by two bacterial consortia isolated from soil. It was established that the tested
consortia were not able to directly uptake compounds
entrapped in micelles, since biodegradation extents of
both hydrocarbons were limited by the presence of Triton
X-100. In micellar systems, the nonionic synthetic surfactant was preferentially degraded (the degradation efficiency of Triton X-100 after 21 days was 70% of the initial
concentration 500 mg/l), followed by a lesser decomposition of hydrocarbon released from the micelles (30% for
hexadecane and 20% for phenanthrene). In comparison,
when hydrocarbons were used as the sole carbon source,
70% of hexadecane and 30% of phenanthrene were degraded. Those results confirmed limited bioavailability of
hydrocarbons entrapped in micelles. In conclusion, one
can observe that Triton X-100 was the preferred carbon
source compared to hydrocarbons. The degradation of
the surfactant did not contribute to notable shifts in bacterial community dynamics, as determined by Real-Time
PCR. The obtained results suggest that if surfactant-supplementation is to be used as an integral part of a bioremediation process, then possible bioavailability decrease
due to entrapment of the contaminant into surfactant
micelles should also be taken into consideration, as this
phenomenon may have a negative impact on the biodegradation efficiency. Surfactant-induced mobilization of
otherwise recalcitrant hydrocarbons may contribute to
the spreading of contaminants in the environment and
prevent their biodegradation.
Acknowledgements
This work was supported by the grant number 32-400/2013 DS-MK.
Acknowledgements
This work was supported by the grant number 32-400/2013 DS-MK.
Eurobiotech 2013
P7.28
P7.29
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P7.31
Eurobiotech 2013
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P7.32
P7.33
Biodegradation of rhamnolipids
and its effect on bacterial community
composition during diesel oil degradation
Ionic liquids (IL) are considered as a novel and promising group of chemical compounds which exhibits
vast structural diversity as well as high applicability in
several branches of the industry (i.e. organic synthesis,
phase transfer catalysis, electrochemistry, formation of
functional polymers, textile softening and production
of hygiene-related products). These peculiar chemicals
are composed of an ionic pair, in which both the cation
and the anion may exhibit certain properties, enabling
the synthesis of highly specific agents. Ionic liquids have
gained immense popularity over the past decades, mostly due to their abundant applications and high efficiency, hence their high-scale production and introduction
into commonly used products is becoming a fact. While
there are certainly many advantages of employing ionic liquids for every-day use, there are also numerous
environmental concerns regarding these compounds.
Certain IL are characterized by a high biological activity, which is why the presence of such xenobiotics may
contribute to negative interactions with micro and macroorganisms in case of environmental contaminations.
Furthermore, several types of IL are excellent pesticides
and herbicides such applications correspond to a direct introduction of these compounds into the environment. Considering the fact, that IL exhibit surface active
properties and may therefore easily penetrate the soil
matrix and transport via groundwater, it should be clear
that assessment of their influence of the environment is
a priority.
The aim of this study was to investigate the biodegradation
rate of selected ionic liquids (ammonium and phosphonium-based IL) by autochthonous soil microorganisms and
determine the qualitative changes in bacterial soil populations as a response to the presence of such compounds
during long-term (300 days) soil microcosm studies. Biodegradability of ionic liquids was assessed by employing
HPLC-MS and the Disulphine Blue Active Substances
(DBAS) method as well as respirometric measurements
(determination of emitted CO2). Total bacterial DNA
was extracted from the soil prior to the experiments and
upon finishing the experiments. Subsequent molecular
analyzes (16s rRNA) allowed for identification of residual
dominant autochthonic species, which were most likely
involved in the biodegradation of the selected ionic liquids. Interestingly, the obtained results suggest that the
100
Eurobiotech 2013
Acknowledgements
This work was supported by the grant number 2011/03/B/NZ9/00731
from The National Science Centre.
P7.34
Phytotoxic effect of rhamnolipids
supplementation during biodegradation
of diesel oil in soil
Anna Parus1, Marta Woniak1, Mateusz Sydow1, Alicja Szulc1,
Katarzyna Mucha2, Joanna Wojtera-Kwiczor3, Anna Gielnik3,
ukasz Chrzanowski1
Institute of Chemical Technology and Engineering,
Poznan University of Technology, Poznan, Poland;
2
Faculty of Materials Science, Technology and Design,
Kazimierz Pulaski University of Technology and Humanities
in Radom, Radom, Poland; 3Institute of Molecular Biology and
Biotechnology, Adam Mickiewicz University in Poznan, Poznan,
Poland
1
This study aimed at assessing the influence of rhamnolipids on the phytotoxicity of diesel oil-contaminated soil
samples. Standard tests evaluating the seed germination
and growth inhibition of alfalfa, sorghum, mustard and
cuckooflower were carried out at rhamnolipids concentrations ranging from 0 to 1.200 mg/kg (wet soil). Diesel
oil content ranged from 0 to 25 ml/kg of wet soil. Obtained results confirmed that the sole presence of rhamnolipids may be phytotoxic at various levels, which is especially notable for sorghum (the germination index decreased to 41%). The supplementation of diesel oil-contaminated soil samples with rhamnolipids contributed
to a significant increase of their phytotoxicity. The most
notable toxic effect was observed after a rhamnolipids-supplemented diesel oil biodegradation, carried out
with the use of a hydrocarbon degrading bacteria consortium. The supplemention of rhamnolipids (600 mg/kg
of wet soil) resulted in a decrease of seed germination of
all studied plant species and an inhibition of microbial
activity, which was measured by the 2,3,5-triphenyltetrazolium chloride (TTC) tests. These findings indicate that
the presence of rhamnolipids may considerably increase
the phytotoxicity of diesel oil. Therefore, their use at high
concentrations during in situ bioremediation processes
should be carefully planned in a terrestrial environment.
Acknowledgements
This work was supported by the grant number 32-400/2013 DS-MK.
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The microbial community and physiochemical processes were monitored in two SBR systems differ in operational parameters. PCR-DGGE of total bacterial community and Anammox bacteria together with sequencing of the dominant Anammox genotypes were used as
a tool for bacterial biocenoses analysis. Both SBRs were
treating reject water with high ammonia concentration
(> 600 mg/l) for over 370 days. After this period of time
the ammonia concentration decreased to 250300 mg/l.
PCR-DGGE monitoring enabled to present the biodiversity shifts linked with community rearrangement and
bacterial biocenoses qualitative change. The analysis revealed that the drastic decrease of ammonia concentration in the influent to adapted biocenoses caused slight
qualitative change in community composition, mainly
in low GC part of the bacteria genotypes but biodiversity index slightly responded to such a change. It was
also stated that bacteria which DNA was amplified with
Anammox-bacteria targeted primers and underwent sequencing were not identified as Planctomycetes probably
because this group of bacteria are still not well known and
Genbank still does not possess enough 16S rRNA gene
sequences belonging to these microorganisms. Difference
in the SBRs working scheme can be the reason that the
community structure differs but with no drastic influence
on its performance.
Acknowledgements
This work was supported by Ministry of Science and Higher Education.
Grant number N523751740.
Karina Krzciuk
The interest in the study of hyperaccumulation increases from year to year. Natural plant species that are able
to take up and accumulate high concentrations of trace
elements are called hyperaccumulators [1]. Hyperaccumulators have found their application in environmental
biotechnology (phytoremediation, phytomining) [2], as
well as in nanotechnology [3]. According to the recently published data, about 577 plant species show hyperaccumulative properties [4]. Most of them, about 78%,
are hyperaccumulators of Ni. Other known species hyperaccumulate Cu, Co, Se, Pb, Zn, Mn, As and Cd [4].
However, there is a need to widen the scope of data for
plants accumulating other elements, including anthropogenic pollutants (Cr, Pb, B) or elements of commercial
value (Cu, Au, rare earth elements). Semiquantitative
mode in inductively coupled plasma mass spectrometry
(ICP-MS) is a good tool for the search of new hyperaccumulators. Semiquantitative analysis is usually used for
arapid screening before quantitative multi-element analysis of unknown samples and it can be used for preselection of the samples with high-concentrations of elements.
This approach may help both for identification of new hyperaccumulators and for pinpointing a larger number of
elements accumulated by plants. The reliability of semiquantitative analysis by ICP-MS is high due to its good
accuracy (typically 3050%) and reproductibility.
Acknowledgements
This work was supported by project from the program Knowledge and
Economics a development of scientific and business competence for
the growth of regional economy competitiveness.
References
[1] Baker A.J.M., Brooks R.R., Terrestrial higher plants which
hyperaccumulate metallic elements A review of their distribution,
ecology and phytochemistry. Biorecovery 1987; 1: 81126.
[2] Brooks R.R., Plants that hyperaccumulate heavy metals. Their role in
phytoremediation, microbiology, archaeology, mineral exploration and
phytomining. CAB International: Wallingford, 1998.
[3] Qu J., Luo C., Cong Q., Yuan X., Carbon nanotubes and CuZn
nanoparticles synthesis using hyperaccumulator plants. Environmental
Chemistry Letters 2012; 10: 153158.
[4] Van der Ent A., Baker A.J.M., Reeves R.D., Pollard A.J., Schat H.,
Hyperaccumulators of metal and metalloid trace elements: Facts and
fiction. Plant Soil 2013; 362(1-2): 319334.
[5] Chen H., Dabek-Zlotorzynska E., Rasumssen P.E., Hassan N.,
Lanouette M., Evaluation of semiquantitative analysis mode in ICP-MS.
Talanta 2008; 74: 15471555.
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Acknowledgements
The work is realized with financial support from Polish National Science
Center, grant No. NN523614839, using equipment purchased in the
project: BIO-FARMA Silesia. Centre for Biotechnology, Bioengineering
and Bioinformatics, co-financed by ERDF OP IG, 20072013.
W. Dec is a scholar in the Project SWIFT (Supporting Innovative Grants
Technology Forum).
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Immobilization of cellobiose
dehydrogenase from Cerrena unicolor
to silica beads supports
Laccase, (EC 1.10.3.2) is multi-copper-containing enzyme which catalyses oxidation of a wide range of substrates including phenolic and aromatic compounds
which may be transformed to potentially textile dyes.
First of all, laccase is widespread in ligninolytic fungi,
especially in white -rot strains of Basidiomycota which
produce extracellular laccase during the growth on media about low pH value, optimal for the activity of ligninolytic enzymes. Nevertheless there is an exception like
asoil-dwelling and plant-pathogenic fungus Rhizoctonia
praticola. This species is well known because of producing a large amount of extracellular laccase which is active
in alkaline conditions.
The aim of presented work was the screening of different organic precursors to obtain the best substrates for
transformation by alkalic laccase to coloured products.
The process was conducted on solid growing medium
with addition one or mixture of two different phenolic
compounds as dyes precursors. During the transformation the dyes appearance and the growth of the mycelium
were measured. As it came out not every phenolic precursor may be an suitable substrate for laccase from R. praticola. Some of the precursors could not be transformed to
dyes probably because of their structure. Additionally, the
efficiency of the transformation and mycelium growth
were different for each precursor or their mixture. What
is more interesting the process conducted only in places
where the ground was covered by the mycelium.
This study allowed qualify which precursors may be used
in further studies of the transformation by laccase from
R. praticola and define potentially what kind of structure
is decisive in this process.
Acknowledgements
This work was partially supported by the National Science Centre
(NN 302 633040).
References
[1] Zamocky M., Ludwig R., Peterbauer C.K., Hallberg B.M.,
Divne C., Nicholls D., Haltrich D. (2006) Cellobiose dehydrogenase
A flavocytochrome from wood-degrading, phytopathogenic
and saprotropic fungi, Current Protein and Peptide Science, 7 (3):
255280.
[2] Stoica L., Dimcheva N., Haltrich D., Ruzgas T., Gorton L. (2005)
Electrochemical investigation of cellobiose dehydrogenase from new
fungal sources on Au electrodes, Biosensors and Bioelectronics, 20 (10):
20102018.
[3] Desriani, Ferri S., Sode K. (2010) Functional expression of
Phanerochaete chrysosporium cellobiose dehydrogenase flavin domain
in Escherichia coli, Biotechnology letters, 32: 8559.
Eurobiotech 2013
[4] Harreither W., Sygmund C., Augustin M., Narciso M., Rabinovich
M.L., Gorton L., Haltrich D., Ludwig R. (2011) Catalytic properties and
classification of cellobiose dehydrogenases from Ascomycetes. Appl
Environ Microbiol 77: 18041815
Acknowledgements
This research was supported by the National Science Centre in
Poland (Grant No. 2011/01/N/NZ1/03458) and the research program
BS/UMCS.
105
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The use of Miscanthus gigantues
and Phalaris arundinacea in the process
of phytoremediation of soils
Karolina Rosiko, Magorzata Kacprzak
Czestochowa University of Technology, Institute
of Environmental Engineering, Brzeznicka 60a,
42-200 Czestochowa, Poland
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Conventional methods for removing metals from aqueous solutions include chemical precipitation, chemical
oxidation or reduction, ion exchange, filtration, electrochemical treatment, reverse osmosis, membrane technologies, and evaporation recovery. These processes may
be ineffective or extremely expensive;especially when
the metals in solution are in the range of 1100 mg/l,
the production of toxic chemical sludge and its disposal/
treatment becomes a costly affair and is not ecofriendly.
Therefore, removal of toxic heavy metals to an environmentally safe level in a costeffective and environment
friendly manner was of great importance. The use of biosorbents with microbial origin especially bacteria, algae,
fungi and yeasts is a considerable alternative to the existing methods because of their good performance, low cost
and large availability. This work presents some results
on the use of Saccharomyces cerevisiae cells biomass for
removal of lead, zinc, nickel, cadmium and copper ions
from contaminated industrial wastewater. Yeast biomass
was immobilized in the process of flocculation by using
calcium chloride and hydrogen peroxide. Process conditions: pH 5.0, temperature 20C, the concentration of
biomass, 1.0 g/l, contact time 30 minutes of flocculation,
aeration and continuous operation. The initial content of
metal ions ranged from 2.2 mg/l (cadmium) to 83.1 mg/l
(lead). Analysis of microbiological and physicochemical
industrial wastewaters showed high efficiency the methods immobilization of insoluble aggregates of cells with
calcium chloride and hydrogen peroxide in removing
heavy metals. Reduction of concentration of Pb(II) was
98.0%, Cd (II) 96.0%, Cu (II) 95.9%, Zn (II) 95.2%
and Ni (II) 92.7%. The results indicate the possibility of
using the aggregation of cells in flocs during flocculation
in wastewater treatment from heavy metal ions.
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The microbial populations inhabiting soil or aquatic environments are composed of diverse, synergistic or antagonistic communities. The presence of multiple bacterial
species in an environmental community allows for an
efficient removal of a wide range of compounds. Largescale pollution due to man-made chemical substances is
the current global concern. From the viewpoint of man
and his impact on the environment, the most important form of pollution are oil spills. Diesel oil is still the
worlds first volume-produced fuel. The composition of
this mixture comes close to 3,000 different hydrocarbons.
Among them are aliphatic, cycloaliphatic and aromatic
compounds. The straight-chain alkanes fraction is the
easiest to decompose by bacteria, whereas cyclic and aromatic compounds are degraded over a relatively long period of time. Moreover, if the pollution consists of many
chemical compounds, as in the case of diesel oil, gradual
changes in mixture composition are observed.
Relatively little is known regarding the determinants of
microbial population dynamics in soil contaminated
with complex hydrocarbon mixtures such as diesel oil
or petroleum. The ability to metabolize oil is displayed
by many different microorganisms and some of them are
more versatile than others. Many bacterial species prefer
specific carbon sources over complex mixtures of compounds. Other bacteria are capable of using many carbon
sources.
The aim of this study was to evaluate quantitative and
qualitative changes within the composition of a microbial consortium resulting from selective cultivation on
the chosen diesel oil fractions and to assess the impact
of such changes on the biodegradation efficiency of commercial diesel oil. During the research, specific substrate
preferences as well as quantitative and qualitative changes within the microbial consortium were determined by
using relative quantitative real-time PCR method. Moreover, all mineralization efficiencies of diversified consortia were compared to utilization capacity of the initial
bacterial community with the use of mathematical modeling methods. The obtained results demonstrate that cultivation on specific carbon sources did not significantly
affect diesel oil biodegradation efficiency in soil.
Acknowledgements
This work was supported by the grant number 32-400/2013 DS-MK.
Acknowledgements
This work was supported by the grant number 32-400/2013 DS-MK.
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Acknowledgements
This study was prepared within the framework of the project PO IG
01.01.02-00-074/09, co-funded by The European Union from The
European Regional Development Fund within the framework of the
Innovative Economy Operational Programme 20072013.
P7.52
Metal cross-talk in Indian mustard results
in induced uptake of Cd and Pb when
supplemented with Zn
Agnieszka Kutrowska1, Aneta Piechalak1, Arleta Maecka1,
Anetta Ha2, Danuta Barakiewicz2, Barbara Tomaszewska1
Department of Biochemistry, Faculty of Biology,
Adam Mickiewicz University, Umultowska 89,
61-614 Poznan, Poland; 2Department of Trace Element Analysis
by Spectroscopy Method, Faculty of Chemistry,
Adam Mickiewicz University, Umultowska 90, 61-614 Poznan,
Poland
1
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References
[1] Jadia C.D., Fulekar M.H., Phytoremediation of heavy metals: Recent
techniques. African Journal of Biotechnology, 2009, 8(6): 921928.
[2] Rascio N., Navari-Izzo F., Heavy metal hyperaccumulating plants:
How and why do they do it? And what makes them so interesting? Plant
Science, 2011, 180: 169181.
[3] Singh V.P., Metal Toxicity and Tolerance in Plants and Animals.
Sarup & Son, 2008.
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High density polyethylene as a carbon
source for Achromobacter xylosoxidans
Anna Kowalczyk1, Marek Chyc2, Przemysaw Ryszka3,
Kazimierz Strzaka1, Dariusz Latowski1
Department of Plant Physiology and Biochemistry,
Faculty of Biochemistry, Biophysics and Biotechnology,
Jagiellonian University, Gronostajowa 7, 30-387 Crakow,
Poland; 2Silesian Environmental Doctoral Study, Plac Gwarkow 1,
40-166 Katowice, Poland; 3Institute of Environmental Sciences,
Faculty of Biology and Earth Sciences, Gronostajowa 7,
30-387 Crakow, Poland
1
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The influence of vermicomposting
on heavy metals contained in sewage
sludge
Hanine Suleiman1, Agnieszka Rorat2, Barbara Pytycz3,
Magorzata Kacprzak2, Franck Vandenbulcke1
Univ Lille Nord de France, LGCgE, EA 4515, Univ Lille 1,
EcoNum-Ecotox, F-59655 Villeneuve dAscq, France;
2
Institute of Environmental Engineering, Czestochowa
University of Technology, Czestochowa, Poland;
3
Institute of Zoology, Jagiellonian University, Crakow, Poland
1
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The effect of phosphate starvation
and mycorrhizae on the gene expression
of phosphate transporters in greenhouse
tomato
Iwona Kowalska1, Marzena Wiska-Krysiak2, Anna Konieczny1
Department of Soil Cultivation and Fertilization of Horticultural
Plants, Faculty of Horticulture, University of Agriculture
in Crakow, al. 29 Listopada 54, 31-425 Crakow, Poland;
2
Laboratory of Basic Research in Horticulture, Warsaw
University of Life Sciences SGGW, Nowoursynowska 159,
02-776 Warsaw, Poland
1
113
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Sewage sludge is a product of wastewater treatment processes; nowadays, about 8 million tones are produced
each year in the EU member states. A preferred method
of sewage sludges utilization is its application in soil fertilization and reclamation. The above has an unfavorable
impact on the environment, it opens the gates to environment pollution by organic and inorganic compounds and
pathogens. Sewage sludge pollution with polycyclic aromatic hydrocarbons is common. Their content in sewage
sludge can range across very wide borders. The standards
valid in the European Union forbid the usage of sewage
sludge with the sum content of 11 PAHs exceeding 6 mg
kg1. This article presents results of many authors, who
composted organic wastes such as sewage ludge, green
wastes, organic fraction of municipal solid wastes. Studies
have shown (Semple et al., 2001; Antizar-Ladislao et al.,
2004) that composting of soils polluted with PAH results
in a significant reduction of PAH content. Some recent
studies (Lazzari et al., 2000; Amir et al., 2005; Oleszczuk,
2006) showed also that sewage sludge composting can
significantly reduce PAH content in this material. The
above issue should be considered not only in the case of
sewage sludges in which valid norms are exceeded but
also in the case of sludges and organic fraction of municipal solid waste in which the content level of these compounds is increased. Such an approach will allow for the
limiting of PAH accumulation in fertilised soil and, at the
same time, influence the improvement of sewage sludge
fertilising properties. Microbial degradation represents
the major route suitable for the ecological recovery of
PAH-contaminated sites, with better results obtained
for the degradation of low molecular weight PAHs (24
rings). The refractory nature of some PAHs is related to
their limited water solubility, to their diverse and complex structure and to the requirement of providing oxygen to start degradation. Environmental conditions
which can affect the biodegradation rate are: high temperature which increases the rate of enzymatic reactions
and water solubility as well, good availability of nutrients,
high moisture level, good oxygenation, presence of alternative substrates required for fulfilling co-oxidation reactions, increased microbial variety and pH near neutrality.
It seems that composting process is very good to remove
some organic compounds from wastes or change them to
less toxic form.
Acknowledgements
This article was financed by the projects own Minister of Science and
Higher Education BS/MN-401-313/11.
Plant growth-promoting rhizobacteria (PGPR) are naturally occurring soil bacteria and benefit plants by providing growth promotion. This study was designed to
isolate and characterize PGPR bacteria from Arabidopsis
thaliana (L.) Heynh. and Morus alba L. (white mulberry). Plants were grown under extreme soil contamination with heavy metals (Zn 980 mg/kg, Pb 1400 mg/kg,
Cd 16 mg/kg) near zinc smelter area in Silesia region.
A large number of bacteria were isolated from roots with
three time levels of surface sterilization (0, 2, 10 min.)
using ethanol. For enrichment and isolation of root-associated bacteria the following media were used: Congo
Red agar and nitrogen-free base (NFb) media to isolate
afree-living diazotrophic bacteria, Luria agar (LA) to isolate nutritionally demanding bacteria, and yeast extract
mannitol agar (YEMA) to isolate Rhizobiaceae bacteria.
Isolates were grown until exponential growth phase to
evaluate the atmospheric nitrogen fixation, phosphate
solubilization, siderophores, indole-3-acetic acid production, as well as antifungal, protease, and 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. A total
number of 26 for A. thaliana and 25 for mulberry isolates
were obtained from the enrichment cultures and tested.
All bacteria were able to grow and to produce IAA, and
the highest concentrations were found for bacteria isolated from mulberry plants. Only few of them were able to
produce siderophores, and solubilize phosphate. ACC deaminase activity was positive solely for three strains. The
growth of Alternaria alternta, Fusarium oxysosporum
and F. culmorum was inhibited over 50% for eight bacteria strains. To determine and confirm the plant growth
promoting potential, some bacteria strains were chosen
for plant inoculation in the growth chamber experiment.
Acknowledgements
The project was supported by National Science Centre grant
UMO-2011/ 03/N/ NZ9/ 02034.
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From a great group of environmental processes participating on the natural material circle is possible to use
mainly the processes of bioleaching and biogenesis in
raw materials processing. The bio-oxidation reactions are
the main basis for bioleaching procedures often parallel
participating in the leaching processes. During leaching
processes of the polycomponent sulphide substrates also
the factor of processes selection plays an important role
being in a direct relation to the electric properties and
galvanic effect occurring between the individual components of the leaching substrate.
This work gives summary of results from research focused on possibilities of using biotechnological procedures for Slovak sulphides ores treatment. The object of
the research is an extraction of valuable metals, undesirable admixtures and degradation of crystal lattice of
sulphides for subsequent chemical leaching processing of
precious metals.
Further, the results of experiments on existence of biogenic processes in situ on waste dumps from exploitation
containing residual sulphides are presented. Outcome of
this processes is acid mine drainage waters generation.
These waters are strongly mineralized and of low pH
thats why they are very aggressive. The heavy and toxic
metals contents as well as Cu, Zn, Fe, As, Cd etc. in out
flowed waters from old mines loadings are high over the
lawful limits.
Acknowledgements
This work was supported by the Operational Programme Research and
Development through the project: Centre of Excellence for Integrated
Research of the Earths Geosphere (ITMS: 26220120064), which is
co-financed through the European Regional Development Fund,
SGA project No. VEGA-2/0086/10 and within the SRDA project No.
APVV-0252-10.
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Crude glycerol is the waste byproduct of biodiesel production process and its conversion mainly to organic
acids brings significant economic benefits. Fungus of the
genus Rhizopus are well known producers of key metabolites such as lactic acid, fumaric acid and ethanol as
co-products. Most of researches was focused on simple
carbon sources like glucose or fructose to study metabolic profiles of this fungus. Previous cultures that were
conducted with supplementation of pure glycerol showed
minimal or no consumption of this carbon source by investigated fungi, which affected on low fumaric acid production. Crude glycerol contains lots of inorganic salts
and MONG (matter organic non glycerol) which can be
used for enhancing biomass growth and modify metabolic profiles.
In this study ability to utilization of crude glycerol by six
strains of Rhizopus oryzae was studied. Preselected cultures showed that optimal amount of crude glycerol in
fermentation medium is 30 g/L. Each of selected fungus
were grown in shaking flask culture in triplicate for 168h.
To avoid excessive acidification of cultures sodium carbonate as a neutralization factor was added to reach 2%
as the final concentration. After HPLC analysis of broth
samples the glycerol consumption rate was observed to
reach 0,16 g/L/h. Best result of final amount of fumaric
acid was observed in R oryzae ATCC 20344 culture, that
concentration reached 15 g/L. Some of studied fungus
showed weak biomass growth in crude glycerol supplemented medium. HLPC analysis of those samples proved
that neither significant utilization of propanetriol nor fumaric acid production was occurred.
Acknowledgements
This study was prepared within the framework of the project PO IG
01.01.02-00-074/09, co-funded by The European Union from The
European Regional Development Fund within the framework of the
Innovative Economy Operational Programme 20072013.
Eurobiotech 2013
P7.62
Effectiveness of eco-friendly,
natural products in in vitro rooting
of Prunus domestica L. microshoots
Alina Wiszniewska, Barbara Nowak, Anna Koton
Department of Botany and Plant Physiology,
Faculty of Horticulture, University of Agriculture in Crakow
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Acknowledgements
This study was prepared within the framework of the project PO IG
01.01.02-00-074/09, cofunded by The European Union from The
European Regional Development Fund within the framework of the
Innovative Economy Operational Programme 20072013.
Eurobiotech 2013
P7.65
Biostimulation of xenobiotic-degrading
bacterial consortia with oxygen-releasing
compo
Pawe Kaszycki, Paula Bana, Przemysaw Petryszak
Departament of Biochemistry, Institute of Plant Biology
and Biotechnology, Faculty of Horticulture, University
of Agriculture in Crakw
Bioremediation is a powerful method to deal with environmental pollution. Particularly toxic and recalcitrant
xenobiotics can be biodegraded employing microbial
communities that reveal specialized metabolic pathways. Biochemical processes leading to the reduction of
total load of chemical pollutants such as oily products
are very complex. In general, the maximum contaminant removal efficiency is obtained at aerobic conditions where oxygen serves as the final electron acceptor
in biooxidation reactions. Since water and soil are main
targets for hydrocarbon discharge by industrial activities, oxygen level often becomes a limiting factor hampering biodegradation activity of autochthonous and/or
allochthonous microorganisms. To overcome the bottle-neck effect of oxygen consumption some methods of
soil bioremediation suggest microflora biostimulation
with oxygen-releasing compounds. Wastewaters can be
treated using consortia aerated with air-compressors,
however this approach may not applicable for in-situ
cleanup projects.
In this study a microbial consortium ZB-01 was tested
for its susceptibility for aerobic metabolism stimulation with two variant oxygen-supplementing commercial products, differing in the mechanism and intensity of oxygen release. The ZB-01 is a highly specialized
biocenosis constructed at Biochemistry Department
of University of Agriculture in Crakow. It consists of
anumber of strains able to biodegrade aliphatic and aromatic hydrocarbons, both in water and soil environments. The two sources of oxygen were: an EHC-OTM
preparation (Adventus Americas Inc.), applied predominantly for reclamation of soil environment, and oxygen
tablets, OxyTABS (JBL GmbH & Co. KG), used mainly
to maintain proper aeration for live in small ponds and
fish tanks. For both substances, optimized concentrations were established as determined by bacterial survivability experiments. The preparations showed a stimulating influence on microorganisms metabolism measured with triphenyltetrazolium chloride (TTC) used to
reveal dehydrogenase activity. Also, bacterial frequency
determinations proved proliferation of microbial cells
under oxygen supplementation conditions. Oxygen level was monitored as a DO (dissolved oxygen) parameter.
Based on the comparative tests the best effect was found
for EHC-OTM applied at 0.1%.
It is concluded that the use of oxygen-releasing compounds to provide aerobic conditions in aqueous media
appear favorable in terms of both, short-time bacterial
119
metabolism stimulation prior to application at contaminated sites, and optimization of long-term consortia
storage.
Acknowledgements
The work was financially supported by the grant for scientific research
No. 3500, approved by the Polish Ministry of Science and Higher
Education.
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Introduction. Using crude glycerol from a biodiesel production process for producing 1,3-PD is a good solution
from the economical as well as ecological point of view.
Biotechnological production of 1,3-PD from waste biomass is a promising and attractive alternative to the traditional chemical synthesis. The productivity of 1,3-PD
can be improved through the application of fermentation
with cell immobilization on porous carriers in packed
bed reactor (PBR).
AIM. The aim of this study was to investigate the influence of physico-chemical properties of porous carriers
used in PBR to immobilize Clostridium butyricum cells.
Materials and methods. An analysis of the surface properties was made using scanning electron microscopy
(SEM) and energy-dispersive X-ray spectroscopy (EDS).
SEM is a type of electron microscope that produces images of a sample by scanning it with a focused beam of
electrons used for analysis of samples surface topography
and composition. EDS is an analytical technique used for
the elemental analysis or chemical characterization of
a sample. All samples were cleaned of any organic residues (300oC for 3h), cut to appropriate size, and mounted
on a specimen holder for viewing in the SEM.
Results. Using the signal of secondary electrons (accelerating voltages of 515 kV), image of: pumice, expanded
clay (LCA), Sera Siporax Mini, Eheim Substrat Pro and
coral gravel were made. Images allowed for assessment
of topography and porosity of each carrier, which are related to the adsorption of bacterial cells to the surface.
EDS analysis have shown that most common element was
silicon (Si).
Conclusions. The area of porous carriers available for the
adhesion of microorganisms, affect the amount of immobilized biomass, which in turn affects the efficiency of the
bioconversion process. Impact on the bacterial adhesion
efficiency has a content of such elements as magnesium
and calcium, and any scratches and surface irregularities.
Acknowledgements
This work was funded within the framework of project No. 01.01.0200-074/09 co-funded by The EU from the European Regional
Development Fund within the framework of the Innovative Economy
Operational Programme 20072013.
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The physiological response of perennial
grasses grown on heavy-metal polluted
soilSesja:
Krystyna Rybka1, Grzegorz urek1, Marta Pogrzeba2,
Jacek Krzyak2, Kamil Prokopiuk1
Plant Breeding and Acclimatization Institute IHAR-PIB,
Radzikow, 05-870 Blonie, Poland; 2Phytoremediation Team,
Institute for Ecology of Industrial Areas, 40-844 Katowice,
Poland
1
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Utilisation of betaine in vinasse stillage by
a mixed culture of aerobic bacteria under
non-controlled pH
Agnieszka Ryznar-Luty, Edmund Cibis, Magorzata Krzywonos
Wroclaw University of Economics, Department of Bioprocess
Engineering
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Presence of beneficial organisms in degraded soil is usually limited, especially in soil polluted with heavy metals
or in soil previously used for agricultural purposes. The
main obstacle is usually the lack of well adapted mycorrhizal fungi. Bioremediation and reforestration can be
supported by inoculum production of mycorrhizal fungi.
The introduction of the particular strain should be preceded, however, by proper knowledge of fungal species in
a given habitat, known receptivity of the soil understood
as the ability of the fungus to survive under given condition and to establish long lasting existence. Further, the
succession of fungal taxa should be estimated and controlled. In such studies it is not possible to rely only on
conventional studies but the use of molecular tools is necessary. The research carried out on Abies alba seedlings
regenerating in forest sites established on agricultural
land are presented. We aimed to determine whether the
fungal communities of seedlings from degraded land differ from fungal communities of seedlings growing in natural forest and whether there was the need for some treatments like inoculation. Mean mycorrhizal colonization
of seedlings was over 90%. A total of 49 ectomycorrhizal
taxa were identified, 36 of which were identified in natural forest and 23 in forest on degraded area. According to
the analysis of similarity, the ECM fungal communities
were different between these forests. Only 20% symbionts
common to both sites suggests that succession of ECM
fungus specific to fir regenerating on post-arable soil is
still in progress. The use of mycorrhizal inocula offers the
possibility to improve the reforestation process.
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Lectures
L8.2
L8.1
Biotechnology of Flax
The development of molecular biology, especially the sequencing of nucleic acid, resulted in the stream of data
on gene structure. Study of gene structure forced the development of research on identification of their function.
For evaluation of gene function, the most effective and
actually the only method is the generation of an organism
with overexpression or/and repression or/and complementation of repression/overexpression of the gene with
the subsequent analysis of modified organism. GM plants,
generated for over three decades, provided the valuable
information of gene function, and simultaneously indicated the advantages of transgenic plants.
According to the European Commission, the challenge
for biotechnology, as a scientific discipline, is searching
for tools for agriculture and industry development and
providing such a plant productivity, that will satisfy growing food demand.
To rise this challenge, the generation and application of
GM plants is justified. At the turn of 2010/2011 the European Commission released a study summing up the tenyear (20012010) research on GM plants (A decade of
EU-funded GMO research). The aim of the study was to
estimate the influence of transgenic plants on the broadly- taken environment, study of the risks connected with
their release into the environment, determination of the
putative horizontal gene transfer between GMO and nonGMO, study of the safety of food produced from transgenic plants, etc. 50 projects realised for over 200 million
of Euro by over 400 research groups were summarised.
The main conclusion summarizing these research is, that
the cultivation, processing and using of GM plants is not
more risky than those of conventional cultivation. Even
though, another study released by the European Commission (Europeans and Biotechnology in 2010) says,
that over 60% (6190% depending of the country) of the
society does not accept GMO.
From two decades we (Linum Foundation, www.leczenielnem.pl) have been generating GM potatoes and nowadays
GM flax. At first, the aim was to identify the genes function
and their significance for plant productivity and metabolism. The most attention was given to those genes, that
are putatively key genes for plant infection resistance, are
crucial for regulation of the synthesis of the compounds
of biomedical functions (phenolic acids, flavonoids, terpenes) or are applicable in industry (fatty acids) and in the
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L8.3
RNAi in cereal functional genomics
Waclaw Orczyk1, Anna Nadolska-Orczyk2, Sebastian Gasparis2,
Wojciech Zalewski2, Yuliya Yanushevska1
Dept. of Genetic Engineering, Acclimatization
and Plant Breeding Institute Natl Research Institute,
Radzikow, 05-870 Blonie, Poland; 2Dept. of Functional
Genomics, Acclimatization and Plant Breeding Institute Natl
Research Institute, Radzikow, 05-870 Blonie, Poland
1
Eurobiotech 2013
known. We found that HvCKX1 and HvCKX2 were specifically induced in developing barley spikes. Silencing
of the genes in barley strongly correlated with increased
productivity shown as higher number and higher mass of
kernels. The trait was inherited in the next generations.
The results confirm the hypothesis that organ specific
expression of the CKX genes influences traits related to
plant productivity [3].
Comparison of two different transformation methods
(Agrobacterium-based and particle bombardment) revealed that the experimental design might have a big
impact in final results. Introduction of the same silencing cassette via Agrobacterium and by particle bombardment gave clearly different results. In our case depression
of the CKX transcript level was observed only in Agro
transformed plants. Particle delivery of the silencing
construct led to inconsistent and self-contradictory results [5].
Current project, and unpublished results, is focused on
identification and functional analysis of putative barley
ortholog of brassinosteroid regulators OsGSK1 in rice
and BIN2 / AtSK2 in Arabidopsis. Depressing transcript
level of the tested gene to 0.09 0.22 of the transcript in
control plants revealed the clear correlation with elevated
salt tolerance of the seedlings. The structure of the gene
and biological function of the gene will presented and
discussed.
Acknowledgements
The research was financed by grants: 620/N-COST/09/2010/0
(AN-O), UMO-2011/03/B/NZ9/01383 (AN-O), N302 013 31/1517
(AN-O), 718/N-COST/2010/0 (WO), UMO-2011/01/B/NZ9/02387
(WO).
References
[1] Gasparis S., Bregier C., Orczyk W., Nadolska-Orczyk A. (2008)
Agrobacterium-mediated transformation of oat (Avena sativa L.)
cultivars via immature embryo and leaf explants. Plant Cell Rep, 27:
17211729.
[2] Binka A., Orczyk W., Nadolska-Orczyk A. (2012) The Agrobacteriummediated transformation of common wheat (Triticum aestivum L.) and
triticale (x Triticosecale Wittmack): role of the binary vector systems and
selection cassettes. J Appl Genet, 53: 18.
[3] Zalewski W., Galuszka P., Gasparis S., Orczyk W., Nadolska-Orczyk
A. (2010) Silencing of the HvCKX1 gene decreases the cytokinin oxidase/
dehydrogenase level in barley and leads to higher plant productivity.
J Exp Botany, 61: 18391851.
[4] Gasparis S., Orczyk W., Zalewski W., Nadolska-Orczyk A. (2011)
The RNA-mediated silencing of one of the Pin genes in allohexaploid
wheat simultaneously decreases the expression of the other, and
increases grain hardness. J Exp Botany, 62: 40254036.
[5] Zalewski W., Orczyk W., Gasparis S., Nadolska-Orczyk A. (2012)
HvCKX2 gene silencing by biolistic or Agrobacterium-mediated
transformation in barley leads to different phenotypes. BMC Plant
Biology, 12: 206.
127
L8.4
Expression of three diadinoxanthin
de-epoxidase genes of Pheodacylum
tricornutum in Escherichia coli Origami
b strain
Monika Olchawa-Pajor1, Monika Bojko1, Wojciech Strzaka2,
Paulina Kuczyska1, Dariusz Latowski1, Kazimierz Strzaka1
Department of Plant Physiology and Biochemistry,
Faculty of Biochemistry, Biophysics and Biotechnology,
Jagiellonian University, Gronostajowa 7, 30-387 Crakow,
Poland; 2Department of Plant Biotechnology, Faculty
of Biochemistry, Biophysics and Biotechnology,
Jagiellonian University, Gronostajowa 7, 30-387 Crakow, Poland
1
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L8.5
The role of ubiquitins in cucumber
(C. sativus L.) flower morphogenesis
Magdalena Pawekowicz, Cezary Kowalczuk, Pawe Osipowski,
Micha Wojcieszek, Grzegorz Kojder, Katarzyna Kosik,
Zbigniew Przybecki
Department of Plant Genetics, Breeding & Biotechnology,
Faculty of Horticulture and Landscape Architecture,
Warsaw University of Life Sciences- SGGW,
Nowoursynowska 166 St., 02-776, Warsaw, Poland
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129
Posters
P8.2
P8.1
Hairy root cultures obtained after Agrobacterium rhizogenes-mediated genetic transformation can serve as model
system for studying plant metabolism and physiology or
can be utilized for the production of secondary metabolites. So far sugar beet hairy roots were used mainly for
studying resistance to BNYVV, Heterodera schachtii or
Tetanops myopaeformis, but no efficient protocol of hairy
root production has been publically released. Usually explants of diploid or tetraploid donor sugar beet plants were
used for transformation however, the use of haploids could
be more desirable. Diploidization of genetically modified
haploid hairy roots could result in fast development of tissue that is homozygous at the transgene locus.
Here we report an efficient system for sugar beet genetic
transformation leading to the development of hairy roots.
The study included two A. rhizogenes strains (A4T and
LBA1334) carrying a binary vector pBIN-m-gfp5-ER or
pCAMBIA1301 possessing gfp and uidA reporter genes, respectively. Petiole and midrib explants of two haploid and
two diploid sugar beet genotypes were inoculated in five
treatment combinations including time of explants exposure
to ultrasound, application of bacteria suspension before or
after sonication and time of bacteria-explant co-culture.
The formation of hairy roots was observed as early as two
weeks after inoculation at the site of tissue wounding.
Hairy roots appeared on 0% to 36% explants depending on the treatment combination. The highest frequency was achieved when explants of a haploid genotype
No. 169 were sonicated for 15 s in the A4T pCAMBIA1301
inoculum of OD600 = 0.5 followed by a 3-day co-culture.
Using the same treatment combination the explants of
the remaining genotypes developed hairy roots with the
frequency ranging from 10% to 30%.
The T-DNA presence in hairy roots was confirmed by PCR
and the copy number of transgenes was assessed by Southern hybridization and Real-Time PCR and determined
as one to several copies depending on the transformation
event. Expression of the transgenes in hairy roots was confirmed visually i.e., beta-glucuronidase activity was verified by histochemical staining and the synthesis of green
fluorescent protein by its fluorescence in UV light. 54% of
the used hairy root cultures showed transgene expression.
The results indicate that the developed protocol allows production of 42 hairy roots per one hundred explants.
Acknowledgements
This work was supported by the Polish Ministry of Agriculture and
Rural Development (Decision No. HOR hn-801-10/13).
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P8.3
DesC desaturase of cyanobacterium
Synechococcus vulcanus expression
in canola plants does not improve the low
positive temperature growth
Mariia Slyvets1, 2, Liudmyla Sakhno1, Yuri Sheludko1
Institute of Cell Biology and Genetic Engineering NAS
of Ukraine; 2National Technical University of Ukraine
Kyiv Polytechnic Institute
1
One of the plant adaptation mechanisms to cold is the increase in the unsaturation of fatty acid residues in cellular
membranes, sustaining the required membrane fluidity.
The important role in this process is attributed to fatty
acid desaturases, catalyzing the transformation of a single
bond between carbon atoms in acyl chains (CC) into the
double bond (C=C).
Earlier we have obtained spring canola (Brassica napus L.,
cv Obreey) lines bearing hybrid gene of acyl-lipid 9-desaturase (des) from cyanobacterium Synechococcus vulcanus translationally fused with termostable lichenase
from Clostridium thermocellum (licBM3) reporter gene
in their nuclear genome using Agrobacterium tumefaciens-mediated leaf disk transformation. Desaturase gene
expression was estimate qualitatively (lichenase plate
test) and quantitatively (photometry) on the basis of activity of licBM3 gene product as a part of hybrid protein.
Gas-chromatographic data revealed that cyanobacterium
DesC expression did not lead to qualitative changes in
canola leaf fatty acid composition but resulted in increase
in the lipid content (up 48%) and in the quantitative alteration in the fatty acid composition. Trienoic (16:3 and
18:3) fatty acid content was increased up 33% and saturated fatty acid content (16:0) was decreased up 16%.
These alterations allows suppose that desC canola lines
might be more resistant to chilling damage in comparison with control ones.
The present work was aimed at investigation of transgenic line growth under low positive temperature (+4C).
During experiments control and des expressing canola
lines (primary transformants 18, 18 and plants of first
generation Bn18/6, Bn18/25) were analysed after propagation by cutting under aseptic conditions. Plants were
grown during four weeks in termal room (16/8 photoperiod, +23) in Sigma 25150 (Sigmawave) tubes
containing 15 ml agar solidified MS medium without
hormones. Then they were transferred to chamber (16/8
photoperiod, +4) for 5 days. After growth under low
temperature plants were returned under initial conditions for four weeks. Stress effect on plant growth was
evaluated using measurement of fresh weight, total soluble protein content and superoxide dismutase activity.
We documented that desC canola plants did not differ
significantly from control under all tested temperature
regimes. It seems trienoic fatty acid content increase in
our canola due to DesC expression was not sufficient for
Acknowledgements
The work was supported with the grant of National Academy of Science
of Ukraine 0110U006062.
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P8.4
P8.5
132
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codons bias of Escherichia coli gene. During the optimization process the following cis-acting sequence motifs
were omitted: internal TATA-boxes, chi-sites and ribosomal entry sites; AT-rich or GC-rich sequence stretches; repeat sequences and RNA secondary structures. The
optimized gene was inserted to pet-15b vector using ligation kit. Purified plasmid containing AtVDE named
pet-15b/AtVDE was received.
Both plasmids were used to transform E. coli Origami b
strain cells. PtVDE and AtVDE effective expression after
IPTG induction were evidenced by SDS-Page electrophoresis and Western-Blot methods. VDE proteins were purified using TALON super flow Metal Affinity resin. The
stable expression in E. coli (Origami b strain) provided
enzymes with molecular weight about 42 kDa for VDE
from A. thaliana and 49 kDa for VDE from Ph. tricornutum. Both obtained enzymes were active relative to Vx
although AtVDE exhibited higher activity than PtVDE.
P8.6
Identification and analysys of WUSCHEL
(WUS/WOX) and CLAVATA genes
in cucumber (Cucumis sativus L.)
Micha Wojcieszek1, Magdalena Pawekowicz1, Marcin Olszak1,
Agata Jdrzejuk2, Zbigniew Przybecki1, Wojciech Burza1
Department of Plant Genetics, Breeding & Biotechnology,
Faculty of Horticulture and Landscape Architecture,
Warsaw University of Life Sciences- SGGW,
Nowoursynowska 166 St., 02-776, Warsaw, Poland;
2
Department of Ornamental Plants, Faculty of Horticulture
and Landscape Architecture, Warsaw University of Life
Sciences- SGGW, Nowoursynowska 166 St., 02-776, Warsaw,
Poland
1
Eurobiotech 2013
P8.7
P8.8
Interspecific hybridization between various species of genus Lathyrus is difficult. Conventional methods of crossing have been successful only in several instances. This
obstructs obtaining of hybrids and introduction of new
desirable features to crops. Application of biotechnological methods seems to be the only solution of this obstacle.
Currently, among the available biotechnology techniques,
protoplast fusion is often used for obtaining somatic hybrids. Besides, protoplasts are excellent explants for genetic transformation due to the absence of barrier that
is cell wall. Prerequisite to use these methods is development of efficient procedures of protoplasts isolation,
their cultivation and plant regeneration. Lathyrus species
belong to large-seeded Fabaceae plants that are very sensitive to in vitro manipulations and very recalcitrant to
regeneration.
In the study, protoplasts were isolated from mesophyll of
two Lathyrus species: L. tingitanus and L. cicera. Leaves,
originating from either 3 week-old ex vitro seedlings or
2 month-old plants growing in the field conditions, were
used as explants. Different enzymatic mixtures (composed of Cellulase, Pectinase, Macerozyme and osmotic
agent (sorbitol) in different concentration) as well as various time of tissue incubation were investigated. Effectiveness of applied isolation conditions was evaluated on the
basis of the number of protoplasts per 1 g plant tissue,
protoplasts viability and the rate of cell wall digestion.
These studies will determine the optimal conditions for
efficient isolation of viable protoplasts that will be a contributive research to perform the fusion between protoplasts of Lathyrus species in the future.
Acknowledgements
This work was supported by the Ministry of Science and Higher
Education of Republic of Poland (BM 4541).
133
is concomitant with the altered expression of growth arrest-, oxidative stress-, cell pluripotency-, apoptosis- and
lipid metabolism-related genes (Bock et al. 2010, Siqueira Filho et al. 2011, Bogliolo et al. 2011). Huang et al.
2009a showed HP related changes in the proteome of
treated porcine spermatozoa that was detected after the
HP treatment, but remained throughout the cryopreservation procedure and after thawing as well, and might be
associated with improved survival and fertilizing capacity
resulting in higher pregnancy rate and litter size upon insemination. Studies indicate that HP treatment results in
significant changes of gene expression and highlight the
need for a comprehensive transcriptome analysis in mammalian cells. Recently, global gene expression profiling of
HP-treated porcine oocytes, and the parthenogenetically
activated or cloned embryos developed from these oocytes, identified several HP-responsive genes.
Preconditioning gametes and embryos with a predefined,
controlled sublethal stress (hydrostatic pressure) seems to
enhance significantly cell survival and function after various in vitro procedures such as cryopreservation, in vitro
culture or cloning.
References
Bogliolo L., Ariu F., Leoni G., Uccheddu S. & Bebbere D. (2011) High
hydrostatic pressure treatment improves the quality of in vitro-produced ovine blastocysts. Reproduction, Fertility, and Development 23,
809817.
Callesen H. (2010) Challenge testing of gametes to enhance their viability. Reproduction, Fertility, and Development 22, 4046.
Du Y., Pribenszky C., Molnar M., Zhang X., Yang H., Kuwayama M.,
Pedersen A.M., Villemoes K., Bolund L. & Vajta G. (2008) High hydrostatic pressure: a new way to improve in vitro developmental competence of porcine matured oocytes after vitrification. Reproduction 135,
1317.
Huang S.Y., Pribenszky C., Kuo Y.H., Teng S.H., Chen Y.H., Chung M.T.
& Chiu Y.F. (2009a) Hydrostatic pressure pre-treatment affects the protein profile of boar sperm before and after freezing-thawing. Animal
Reproduction Science 112, 136149.
Lin L., Luo Y., Srensen P., Prtorius H., Vajta G., Callesen H., Pribenszky C., Bolund L. & Kristensen T.N. (2013) Effects of high hydrostatic
pressure on genomic expression profiling of porcine parthenogenetic activated and cloned embryos. Reproduction, Fertility, and Development
[in press] (http://dx.doi.org/10.1071/RD13037).
Lin L., Pribenszky C., Molnar M., Kragh P.M., Du Y., Zhang X., Yang H.,
Bolund L., Callesen H., Machaty Z. et al. (2010) High hydrostatic pressure treatment of porcine oocytes induces parthenogenetic activation. Cellular Reprogramming 12, 475480.
Matyas S., Pribenszky C., Kovacs P., Rajczy K., Molnar K., Losonczi E.,
Molnar M., Kaali S.G. & Vajta G. (2010) Preconditioning oocytes by
sublethal hydrostatic pressure stress in order to improve cryosurvival
animal models and human application. Proceedings of The 1st International Congress on Controversies in Cryopreservation of Stem Cells,
Reproductive Cells, Tissue and Organs Valencia, Spain.
Pribenszky C. & Vajta G. (2010) Cells under pressure: how sublethal
hydrostatic pressure stress treatment increases gametes and embryos
performance? Reproduction, Fertility, and Development 23, 4855.
Pribenszky C., Du Y., Molnar M. & Vajta G. (2008b) Sublethal stress
on porcine oocytes enhances the efficacy of ART procedures. Human
Reproduction 23, 161.
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Pribenszky C., Du Y., Molnar M., Harnos A. & Vajta G. (2008a) Increased stress tolerance of matured pig oocytes after high hydrostatic
pressure treatment. Animal Reproduction Science 106, 200207.
Pribenszky C., Lin L., Du Y., Losonczi E., Dinnyes A. & Vajta G. (2012)
Controlled stress improves oocyte performance-cell preconditioning in
assisted reproduction. Reproduction in Domestic Animals 47, 197206.
Pribenszky C., Matyas S., Losonczi E., Stanca C., Bock I. & Vajta G.
(2010b) Stress for stress tolerance: improving cell survival by sublethal
stress treatment of eggs before vitrification pilot study. Fertility and
Sterility 9, S32.
Pribenszky C., Molnar M., Cseh S. & Solti L. (2005) Improving postthaw survival of cryopreserved mouse blastocysts by hydrostatic pressure challenge. Animal Reproduction Science 87, 143150.
Pribenszky C., Vajta G., Molnar M., Du Y., Lin L., Bolund L. & Yovich
J. (2010a) Stress for stress tolerance? A fundamentally new approach in
mammalian embryology. Biology of Reproduction 83, 690697.
Siqueira Filho E., Caixeta E.S., Pribenszky C., Molnar M., Horvath A.,
Harnos A., Franco M.M. & Rumpf R. (2011) Vitrification of bovine blastocysts pretreated with sublethal hydrostatic pressure stress: evaluation
of post-thaw in vitro development and gene expression. Reproduction,
Fertility, and Development 23, 585590.
135
L9.2
Large animal models in biomedical
research
Marlena Szalata1, 2, Daniel Lipiski1, 2, Joanna Zeyland1,
Magdalena Boksa1, Hanna Przystaowska1,
Bartosz Grzekowiak1, 3, Karol Tunio1, 3,
Marzena Skrzypczak-Zieliska2, Ryszard Somski1, 2, 3
Department of Biochemistry and Biotechnology,
Poznan University of Life Science, Poland; 2Institute
of Human Genetics, Polish Academy of Sciences, Poznan,
Poland; 3NanoBioMedical Centre (NBMC) at Adam Mickiewicz
University in Poznan, Poland
1
136
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L9.3
L9.4
Eurobiotech 2013
137
L9.5
Retroviral restriction factor gene
expression in normal human dermal
fibroblasts after porcine endogenous
retrovirus infection
Magorzata Kimsa, Magdalena Kimsa, Barbara Strzaka-Mrozik,
Joanna Gola, Jolanta Adamska, Urszula Mazurek
Department of Molecular Biology, Medical University of Silesia,
Narcyzow 1, 41-200 Sosnowiec, Poland
138
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Posters
P9.2
P9.1
The therapeutic usage of porcine material in xenotransplantation is not confirmed to be completely safe
because of the presence of various pathogens such as
porcine endogenous retroviruses (PERVs). Numerous
studies have demonstrated that PERVs are transmitted
to different human cell lines but it is still necessary to
conduct research to explain the interaction between
PERVs and human cells and to search for diagnostic
markers of infection.
Toll like receptors (TLRs) are specific immunologic
markers. The understanding of the molecular details
of effector immune responses and signal transduction
activated by TLRs may provide guidelines for the development of novel diagnostic and therapeutic strategies in
the aspect of PERV infection during xenotransplantation.
The present study focuses on the identification the
effect of porcine endogenous retroviruses on human
dermal fibroblasts by investigating the changes of the
aspect of the expression of TLR and TLR-dependent
genes.
The PERV infectivity was analyzed in the co-culture system. Normal human dermal fibroblasts (NHDF cell line)
were co-cultured for 5 days with normal porcine kidney
epithelial cells (PK15 cell line). Genomic DNA was isolated from harvested cells using the salting out extraction
method. Total RNA was extracted from cells using TRIzol
reagent. The infectivity of PERVs was determined using
real-time Q-PCR and QRT-PCR assay. The analysis of
the expression profile of TLR and TLR-dependent genes
was performed using HG-U133A 2.0 oligonucleotide microarrays.
The copy number of PERV A and PERV B RNA in NHDF
cells (637.30363.0; 77.0058.0, respectively) were revealed after co-culture. However, only the copy number
of PERV A DNA was observed (10.147.6). The study
indicated that after PERV infection, NHDF cells showed
a statistically significant increase of expression of TLR3
(p = 0.008, FC = 1.46). Among TLR3-dependent genes,
45 genes were differentially regulated with a fold change
above 1.1 and pOf these 45 genes, 22 were up-regulated,
23 were down-regulated and 6 were found to be regulated
by more than 1.5-fold.
Eurobiotech 2013
Acknowledgements
This study was supported by the project No. KNW-2-032/D/3/N from
Medical University of Silesia, Katowice, Poland.
139
P9.3
Cyclosporine A and expression of genes
associated with cell cycle in normal human
dermal fibroblasts cultured in vitro
Grzegorz Hibner1, Adam Wilczok1, Tomasz Janikowski2,
Urszula Mazurek2
Department of Biopharmacy, Medical University of Silesia,
Narcyzow 1, 41-200 Sosnowiec, Poland; 2Department
of Molecular Biology, Medical University of Silesia, Narcyzow 1,
41-200 Sosnowiec, Poland
1
140
Eurobiotech 2013
P9.4
P9.5
Background. The interaction between PERVs and human cells in inflammatory conditions is still unclear. The
key factors of the immune response in the presence of infectious agents are many cytokines, including tumour necrosis factor (TNF). TNF acts through receptors TNFR1
and TNFR2, inducing MAPK, NFkB and caspase signalling cascades.
Aim. The aim of this work was to assess the changes in
the transcriptome of genes related to TNF signal transduction pathways infected with PERVs with and without
lipopolysaccharide stimulation.
Methods. Human fibroblasts (NHDF) were co-cultured
with normal epithelial porcine kidney cells (PK15 cell
line) in the presence of lipopolysaccharide (LPS). Total
RNA was extracted with the use of phenol-chlorophorm
method. The expression profile of genes related to the
TNF signal transduction pathways was appointed with
the use of oligonucleotide microarrays HG-U133A 2.0
(Affymetrix). Data analysis was performed with the use
of GeneSpring 12.0 platform (Agilent Technologies).
Genes were considered differentiating when p0,001 and
FC3,0 (fold change).
Results. In PERV infected fibroblasts in the presence of lipopolysaccharide six genes were differentiating: MAP3K7, MAP3K8, MAP4K3, BIRC5 (survivin),
MEF2C and TANK, comparing to control. The only
downregulated gene was BIRC5 survivin (FC = 4.09).
Conclusion. PERV infection and the presence of inflammatory factor lead to significant changes in the mRNA
level of survivin and MAPK genes.
Eurobiotech 2013
P9.6
Abundance of blastocysts developed
in vitro from porcine cloned embryos
reconstructed with cell nuclei of adult bone
marrow-descended mesenchymal stem
cells
Marcin Samiec, Jolanta Opiela, Jurij Koseniuk
National Research Institute of Animal Production,
Department of Biotechnology of Animal Reproduction,
Balice near Crakow, Poland
141
Acknowledgements
The research project was funded by the Polish National Science Centre
resources allocated on the basis of decision number DEC-2011/03/D/
NZ9/05537.
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P9.7
Scriptaid-induced epigenetic modulation
of adult dermal fibroblast cells before their
use for somatic cell nuclear transfer
in pigs
Marcin Samiec, Maria Skrzyszowska
National Research Institute of Animal Production, Department
of Biotechnology of Animal Reproduction, Balice near Crakow,
Poland
Eurobiotech 2013
P9.8
P9.9
143
Alloferon for the first time was isolated from the blowfly
Calliphora vicina larvae. This compound was characterized by a strong antibacterial activity which protected
blow fly larvae against adverse environmental conditions.
In the last few years, increased an interest in using alloferon in biomedicine. This is caused by discovery of antitumor activities and inhibition of Human herpesvirus,
Papillomaviruses and Coxsackieviruses replication by
alloferon. Furthermore, this peptide stimulated cytotoxic
activity of natural killer cells, haemocytes apoptosis and
interferon synthesis. For many biological activities, the
most important is discovering the active structures which
will affect the immune and circulatory system.
In presented research were used alloferon and its 10
structural analogues, which were characterized by the
highest proapoptotic activities. The significant differences
in the actions of alloferon analogues on cellular and humoral responses were observed, but only two analogues
decreased both immune responses. These compounds
had structural modification in position 1 where native
amino acid histidine was changed on synthetic aromatic amino acid Phe(p-Cl) or Phe(p-OMe). Both structural
modifications in position 1 probably increase proteolytic
stability of alloferon analogues. In addition, long-term
immunomodulation effect may suggest a long half-life of
these peptides in the haemolymph of T. molitor. Alloferon and its analogues didnt affect the heart beat frequency. This result indicates a high specify activity of these
compounds in T. molitor. Presented results enhance the
knowledge about relationship between bioactivity and
structure of alloferon. The discovery of alloferon active
core is necessary for the design and synthesis of peptidomimetics, which can be used for pest control and new
antitumor therapy.
Over the past several years insects have gained interest as model organisms used in studies of fundamental
physiological processes. Some beneficial features, such as
short life cycle, large number of offspring and low costs
of rearing, but also genetic simplicity and less complex
metabolism make insects promising alternative to mammalian models in studying mechanisms of many human
diseases.
The aim of this study was to analyze the relationships in
the heart performance during ageing between beetle Tenebrio molitor L. and humans.
In vitro bioassays of a semi-isolated myocardium and
video microscopic techniques combined with dynamic
image analysis were used to trace mechanic and hemodynamic parameters of ageing beetle heart. We detected
that parameters, such as ejection fraction, stroke volume
and cardiac output of myocardium of ageing beetles undergo changes similar to the decline of cardiac function
in elderly people with heart failure.
Our results suggest that beetle myocardium seems to be
an appropriate candidate as a model to study cardiac performance decline with age and disease and can be used
e.g. in pharmacological or sport medicine researches.
144
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P.9.10
Identification of 77 novel SNPs within
entire intron A of the pregnancy-associated
glycoprotein 2-like gene subfamily
(PAG2-L) in cross-breed pigs*
Martyna Bieniek, Grzegorz Panasiewicz, Aleksandra Zamojska,
Bozena Szafranska
Department of Animal Physiology, Faculty of Biology
and Biotechnology, University of Warmia and Mazury in Olsztyn,
10-719 Olsztyn-Kortowo, Poland
Eurobiotech 2013
P9.11
Identification of 5 novel SNPs within intron
E & F of porcine Pregnancy-Associated
Glycoprotein 2-like subfamily (pPAG2-L)
in two hybrid lines*
Grzegorz Panasiewicz1, Aleksandra Zamojska1,
Martyna Bieniek1, Roman Jdryczko2, Bozena Szafranska1
Department of Animal Physiology, Faculty of Biology
and Biotechnology, University of Warmia and Mazury in Olsztyn,
10-719 Olsztyn-Kortowo; 2Veterinary Diagnostic Laboratory,
11-036 Gietrzwald
1
145
146
Eurobiotech 2013
P9.12
Novel glycoprotein family within human
placenta detected with polyclonals
against recombinant or native porcine
Pregnancy-Associated Glycoproteins*
Marta Majewska1, aleksandra Zamojska2,
Grzegorz Panasiewicz2, Martyna Bieniek2,
Zbigniew aganowski3, Bozena Szafranska2
Department of Human Physiology, Faculty of Medical
Sciences, University of Warmia and Mazury in Olsztyn,
10-082 Olsztyn; 2Department of Animal Physiology,
Faculty of Biology and Biotechnology, University of Warmia and
Mazury in Olsztyn, 10-719 Olsztyn-Kortowo; 3The Womans
Health Clinic, 10-684 Olsztyn, Poland
1
Multiple PAG family (PAGs) has been previously identified in various domestic and wild species (Szafranska
et al. 2006). So far, we detected the PAGs in the pig (Majewska et al. 2005, 2006), the European bison (Majewska
et al. 2008), and three Camelidae species: the alpaca, C.
bactrianus and C. dromedarius (Majewska et al. 2009,
2011, 2013). The PAGs belong to aspartic proteinase
superfamily (AP), which includes different proteolytic and lysosomal enzymes (i.e. pepsins or cathepsins).
All AP members possess a two-bilobe structure with a
cleft capable for short peptide binding. As a role of the
PAGs is still unclear, it is however involved in proper
embryo-maternal interaction and placenta development
(Szafranska et al. 2007; Panasiewicz et al. 2007). Thus
abnormal PAG expression reflects placental disorders.
Therefore, the PAGs are routinely used as prenatal diagnostic markers for various pregnancy tests (similarly to
hCG-tests), based on the PAG concentration in peripheral maternal blood or milk of ruminant species (US
Patents; USA).
The aim of this study was to identify PAG-like expression
within hemochorial human placenta by heterologous detection with the use of two different rabbit polyclonals
raised against recombinant porcine PAG2 antigen (anti-RpPAG2) or polyvalent polyclonals raised against native pPAG-like (pPAG-L). Late placental tissues of three
healthy women were collected after caesarean section and
immediately stored at 70oC. The PAG-like immunoreactivity was examined by immunohistochemistry (IHC)
and Western blotting. Total proteins were extracted from
homogenized placental explants and concentrated by
ultrafiltration (MWCO >10 kDa). Extracted placental
proteins were directly used for Western dot-blotting or
separated by SDS-PAGE. Proteins were semi-dry transferred onto nitrocellulose membranes and used for
Western. Primary rabbit polyvalent anti-pPAG-L (1:300)
or anti-RpPAG2 (1:50) polyclonals, characterised previously (Szafranska et al. 2002), were used to identify
molecular mass of human PAG-like proteins. The immuno-complexes were detected with mouse anti-rabbit IgG
monoclonals (1:100 000)conjugated with alkaline phosphatase and then visualized with BCIP/NBT substrates.
Cryosectioned human placental tissues were fixed, dehydrated and used for heterologous dual fluorescent IHC
(htdF-IHC) with the same polyclonals as for Western
blotting. The PAG immuno-complexes within human
placental sections were visualized with secondary goat
anti-rabbit immunoglobulins (1:1000)conjugated with
Alexa 488 dye (495ext/519em nm), as the visualizing
fluorochrome (green). Finally placental sections were
counterstained with propidium iodide (red) to visualize
nuclei.
This is the first study identifying cellular localization of
the PAGs in human placental proteome. The human PAG
family (hPAGs) expression was found to be restricted to
various trophectoderm (chorionic epithelium) cells only.
The separated human placental proteins weakly reacted
with polyvalent anti-pPAG-L polyclonals, but strongly
with anti-recombinant pPAG2 polyclonals. Western blotting revealed dominant approx. 60 kDa hPAG isoform.
The cross-reactivity of the anti-RpPAG2 polyclonals with
the hPAGs revealed structural epitope similarities of the
PAG isoforms in human placenta and other eutherian
species.
Acknowledgements
Supported by UWM (WBiB#528-0206-806 and WNM#1501.801).
Eurobiotech 2013
P9.13
Antibacterial peptides from WPC
hydrolisate
Paulina Worsztynowicz, Dagmara Leniak, Wodzimierz Grajek
Poznan University of Life Sciences
147
used for the antibacterial assay. The growth medium consisted of (w/v) 1% peptone, 0.5% yeas extract and 0.5%
sodium chloride. Each tube was filled with 4 ml growth
medium, 0.5 ml bacterial inoculum and 0.5 ml WPC hydrolysate (10 mg/ml). WPC hydrolysate was dissolved
in physiological saline which was sterilised by filtration
through a0.22 mm membrane. Control assays contained
all components except WPC hydrolysate. The mixtures
were incubated at for 37C 12 h. Viable bacterial counts
of E. coli were obtained using the plate count method
after being cultured on nutrient agar. Plates were incubated at 37C for 24 h. The antibacterial effect of WPC
hydrolysate and its fraction was defined as antibacterial
rate again E. coli, L. monocytogenes and S. enteritidis was
calculated by the following equation: Antibacterial rated
(%) = (Nc Ns)/Nc x 100 where Nc and Ns are the bacteria numbers of the control group and the sample group,
respectively.
SEM analysis. Scanning electron microscopy (SEM) was
used to examine the ultrastructural changes in bacteria
induced by antimicrobial peptides.
Results. It has been shown that the studied strain
L. lactis produced three kinds of proteases: cell wall-associated proteinase, intracellular and extracellular proteinase. The antimicrobial potential of WPC hydrolyzed on
E. coli, L. monocytogenes and S. enteritidis were determined. While hydrolysates for 8 h, 16 h, 22 h and
28h did not show antibacterial activity, whey proteins
hydrolyzed for 40 h by cell wall-associated proteinase,
intracellular and extracellular proteinase exhibited antibacterial activity.
Eurobiotech 2013
L10.2
Development of non-antibiotic treatment
to prevent animal digestive tract from
bacterial infections
Stefan Kwiatkowski1, Karl Dawson1, Maciej Gryszel2
1
2
References
[1] Wellens A. et al., PLOS one 3(4):e2040 (2008).
[2] Neeser J-R. et al., Infection and Immunity 52(2): 433.
[3] Parks C.W. et al., Poultry Science 84(12):196773.
149
150
Eurobiotech 2013
L10.3
L10.4
Zofia Madej
University of Life Sciences Poznan, Department of Genetics
and Animal Breeding
Animal models represent important tools for investigating the physiological and pathological mechanisms of
metabolic processes. The main reason of using animal
models for regulation of metabolism is that the results
can be extrapolated of the humans. Traditionally, animal models can be grouped into the following categories: spontaneous models; genetically modified models;
induced or experimental models and negative models
[3]. Continuous development of genomics, proteomics,
biotechnology and bioinformatics changed trends in applying of animal models. For instance, transgenic technique allows to study the role of specific gene products
involved in the physiological regulation, development or
pathogenesis [5].
Most of the available models are based on rodents because
of their small size, short generation interval, easy availability and economic considerations [1]. Unfortunately,
rodents models differ from human metabolic processes
in many aspects. For example, normal mouse lipoprotein
profiles have essentially atheroprotective HDL, whereas
human lipoprotein profiles contain originally atherogenic low-density lipoproteins (LDL) [7]. For modelling the
metabolic disorder, obese mice combined with dyslipidemia, hypertension and/or elevated glucose levels has
been determined. In recent years, large number of new
genetically modified animal models including transgenic,
generalized knock-out and tissue-specific knockout mice
or rat have been engineered for the study of metabolic
processes and disorders (e.g. mice IRS2-KO, AG4KO,
MG4KO, AMG4KO, IR/IRS-1) [2]. Unfortunately, none
rodents model can exactly mimic all aspects of human
metabolism [7].
The selection of an animal model is difficult and might
lead to misinterpretation of data or even to the wrong
conclusions. Therefore, non-rodent models of metabolic
diseases are urgently needed as a valuable supplement to
rodents for improving an extrapolation of study results.
The pig has many similarities in structure and function to
humans, including size, feeding patterns, digestive physiology, dietary habits, propensity to obesity and social
behaviours [6]. Additionally, pig cardiovascular anatomy and physiology, in combination with the porcine response to atherogenic diets, have made them a universally standard model for the study of atherosclerosis. Their
gastrointestinal anatomy has some significant differences from that of humans; however, the physiology of the
digestive processes has made them a valuable model for
studying disorders. Therefore, the Gottingen, Yucatan,
Eurobiotech 2013
and Ossabaw pigs breeds have been used widely for investigations of adipose tissue activities, glucose and lipids
metabolism, obesity or cardiovascular diseases [4].
Undoubtedly, there are some limitations like expensiveness, practical difficulties and ethical considerations associated with the use of large animal species (pigs, dogs,
monkey). However, in order to better understanding the
regulation of metabolism mechanisms; discover new targets for the treatment of metabolic diseases in humans,
these models are required [5].
Acknowledgements
Supported by DS 3243/KFiEZ/13.
References
[1] Artinamo A., Castro M. (2009) Experimental rat models to study the
metabolic syndrome. Br. J. Nutr. 102(9): 124653.
[2] Gilliam L.A., Neufer P.D. (2012) Transgenic mouse models resistant
to diet-induced metabolic disease: is energy balance the key? Pharmacol.
Exp. Ther. 342(3): 6316.
[3] McMurray F., Moir L., Cox R.D. (2012) From mice to humans. Curr.
Diab. Rep. 12(6): 6518.
[4] Neeb Z.P., Edwards J.M., Allosh M., Long X., Mokelke E.A., Sturek
M. (2010) Metabolic syndrome and coronary artery disease in Ossabaw
compared with Yucatan swine. Comp. Med. 60(4): 30015.
[5] Speakman J., Hambly C., Mitchell S., Krol E. (2008) The contribution
of animal models to the study of obesity. Lab. Anim. 42(4): 41332.
[6] Spurlock M.E., Gabler N.K. (2008) The development of porcine
models of obesity and metabolic syndrome. J. Nutr. 138(2): 397402.
[7] Tschop M., Heiman M.L. (2001) Rodent obesity models: an
overview. Exp. Clin. Endocrinol. Diab. 109(6): 30719.
151
Posters
P10.1
Cytogenetic mapping of the HSPB genes
in the domestic Bovids
Barbara Danielak-Czech, Katarzyna Kruczek,
Anna Kozubska-Sobocika, Barbara Rejduch, Agnieszka Bk
Department of Animal Cytogenetics and Molecular Genetics,
National Research Institute of Animal Production, Krakowska 1,
32-083 Balice near Crakow, Poland
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P10.3
The small heat shock proteins (sHSPs) exert housekeeping role in normal cell metabolism as well as protective
functions under stress conditions such as exposure to
elevated temperature or toxic environmental and metabolic products. Developmentally regulated expression
of sHSPs is required for differentiation of nervous cells
whereas the mutations of genes encoding these proteins
are responsible for desmin-related myopathies or neurodegenerative and autoimmune diseases. The aim of this
study was the chromosomal localization of five sHSPs
genes involved in neurodegenerative processes in the
pig genome. Clones, containing sequences of the HspB1,
HspB2, HspB5, HspB6 and HspB8 genes, were obtained
from the CHORI-242 Porcine BAC library (http://bacpac.
chori.org/librariesphp). The clones, selected based on information about BAC-end sequences (BES) (http://www.
sanger.ac.uk/Projects/S_scrofa/BES.shtml), were used
as probes in the FISH experiments. It was not possible
to select separate BAC clones for the HspB2 and HspB5
genes due to their close proximity in the pig genome, thus
the same clone containing sequences of both these genes
was used. The genes were assigned to the following pig
chromosome (SSC) regions: HspB1 (SSC3p15), HspB2
(SSC9p21), HspB5 (SSC9p21), HspB6 (SSC6q12) and
HspB8 (SSC14q21). The HspB2 and HspB5 loci, clustered
at the distance of above 0.6 kb in the pig genome (http://
www.ncbi.nlm.nih.gov/gene/), were identified in the same
SSC9p21 chromosome band. Similarly in humans, these
two related genes are located in HSA11q22-q23 genome
region and arranged in a head-to-head manner with an
intergenic sequence of less than 1 kb, raising a possibility
of shared regulatory elements for their expression. The
human HspB2 and HspB5 genes (composed of two and
three exons, respectively) are transcribed in the opposite
direction and expressed in most cells under physiological
conditions. However, recent studies point out that they
both are dispensable for cardiac function and maintenance of myocardial integrity as well as suggest their important neuronal function under stress conditions. The
experiments involving genomic context and structure of
porcine sHSPs genes provide a compelling rationale of future biomedical studies to define the complete interaction
of small heat shock proteins in cardiac and neuronal cells
under both basal and stress conditions.
Acknowledgements
Research work financed from National Science Centre funds, authors
project No. N N311 082540.
Acknowledgements
This study was conducted as part of NRIAP statutory activity, projects
No. 04-2.01.1, 04-6.03.1.
Eurobiotech 2013
P10.4
P10.5
Visfatin is a protein with several suggested functions: cytokine, enzyme as well as adipokine. This adipokine, also
known as extracellular pre-B-cell colony-enhancing factor (PBEF) and nicotinamide phosphoribosyltransferase
(Nampt), is peptide whose circulating levels are enhanced
during metabolic disorders [1]. The study has demonstrated that visfatin may exert direct destructive actions
on the cardiovascular system, including cell proliferation,
monocyte/macrophage activation, vascular inflammation and remodeling, all of them leading to the development of atherosclerosis [2].
Thus, the aim of the study was to determine the synthesis and secretion of visfatin from endothelial cells in response to hyperglycemia. Additionally, the adipokine was
estimated as an indicator of endothelium dysfunction.
Human aortic endothelial cells (HAEC) were cultured in
a standard medium (M 199) supplemented with LSGS
kit (Invitrogen). The experiment was performed two
times on fourth passage of HAEC culture. The cells were
incubated for 24 hours with glucose in concentrations
22.2mmol/l. After completing the experiment, total RNA
was isolated from cells using Ambion Cells-to-CT Kits
(Life Technologies). Reverse transcription was undertaken using High Capacity Reverse Transcription Kit (Life
Technologies). Quantitative PCR analysis was performed
using StepOnePlus Real-Time PCR System (Life Technologies) with the Universal Master Mix and TaqMan
chemistry (Life Technologies). Visfatin concentration
was measured using a commercial ELISA kit (BioVendor
R&D), according to the manufacturers protocol.
The activity of aortic endothelial cell negatively changed
in response to hyperglycemia. The cells proliferation was
decreased by 36,9% compare to control group (p
Acknowledgements
Supported by DS 3243/KFiEZ/13.
References
[1] Fukuhara A., Matsuda M., Nishizawa M. (2005) Visfatin: a protein
secreted by visceral fat that mimics the effects of insulin. Science.
307(5708): 426430.
[2] Stam F., van Guldener C., Schalkwijk C.G. (2003) Impaired renal
function is associated with markers of endothelial dysfunction and
increased inflammatory activity. Nephrology Dialysis Transplantation
18(5): 892898.
153
5-Vic ATTCCGCAATAAAAC
The primers and the probe that detect horse components
were taken from TANABE et al. (2007), Biosci. Biotechnol. Biochem., 71; 3131-3135. A 75-bp cytB fragment of
horse mtDNA was amplified. The limit of quantitation
(LOQ) for the beef and horse component in pork meat
is 0.2%.
The small size of the amplicon used in the above analyses
is useful for detecting species composition of processed
food products with degraded DNA. For this reason, the
method is applied to analyse adulterations in both raw
and processed meat.
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P10.6
P10.7
In order to determine species composition of food products for humans as well as pet food, a method for rapid and sensitive identification of their components was
developed. Species specific primers and TaqMan probes
were designed on mitochondrial DNA: ATPase6 for cattle, cox1 for sheep and pigs, and 16SrRNA for chickens.
In the case of chicken and sheep identification, the proposed primers amplify the DNA of the species for which
they were designed. When identifying cattle and pigs,
across-reaction was found between these species during
the 35th reaction cycle. However, this did not hamper the
analysis of the results because during this cycle a reaction
takes place for the identified product at a level of 0.05%
and at 100% for the second species.
The limit of quantitation for chickens and sheep has not
been determined, but it is lower than 0.1%. The limit of
quantitation for cattle and pigs, due to the cross-reaction,
is 0.1%.
The developed method is suitable for species identification of raw meat, processed meat and components of
meat-and-bone meals subjected to thermobaric treatment at a temperature of 133C and pressure of 3 bar for
20 minutes.
The calpains are the enzymes belong to calcium-dependent, non-lysosomal cysteine proteases expressed in
mammals and many other organisms. The CAPN3 gene
encodes a major intracellular protease, which is muscle
specific. In chickens it is localized on chromosome 5. The
calpains have a high capacity degradation of cytoskeletal
and muscle fibers proteins. Therefore they play an important role in fusion of mioblasts, proliferation, growing
and migration of cells. The CAPN3 has been chosen as
candidate gene associated with meat quality in chickens.
Consequently the aim of our study was to identified new
polymorphisms in regulatory region of CAPN3 gene and
examine their impact on CAPN3 transcript abundance in
breast muscles. In experiment used broilers of two genetic lines: fast and slow-growing. As a screening method
the High Resolution Melting (HRM) was used. The polymorphisms were identified with on Beckman Coulter sequencer based on the Sanger method. The CAPN3 gene
expression was estimated on Real Time 7500 Applied Biosystems using succinate dehydrogenase complex; subunit
A (SDHA) and 60S ribosomal protein L4 (RPL4) genes
as endogenous control. Statistical and linkage analyses
were performed using SAS Enterprise. Four new polymorphisms were found. One in promoter region c. 450
G > A and three in 3`UTR (c. 176* C>T, c. 137*_147* del.,
c. 144* G > C) region. One of them in 3`UTR region was
deletion of 11 nucleotides (CAGCCCTGCTT). New polymorphisms were identified by used restriction enzymes
ScrFI, BslI, AcuI, HpyHV, respectively. The frequency of
polymorphisms found in 3`UTR region was similar in
both lines. Nevertheless, in fast growing line appeared
more genotypes than in slow growing according to c.
C > T 176* and c. G > C 144* polymorphisms. Moreover,
both broiler lines were in Hardy-Weinberg disequilibrium of c. 450 G > A polymorphism, although this polymorphism does not change any transcription binding
site. Analysis of effect of new polymorphisms on CAPN3
gene expression showed, that in fast growing lines the
chickens with GG genotype according to c. 450 G > A
polymorphism characterized with the highest CAPN3
gene expression. Other polymorphisms in 3`UTR region
seem not effect on CAPN3 gene expression.
Acknowledgements
This work was supported from National Research Institute of Animal
Production statutory activity, research project No. 01-4.02.1
Eurobiotech 2013
P10.8
Different culture media affect growth rate
and morphology of porcine bone
marrow-derived mesenchymal stromal
cells- the preliminary results of media
screening suitable for both porcine MSCs
and bovine embryo in vitro culture
Jolanta Opiela, Micha Bochenek, Joanna Romanek,
Zdzisaw Smorg
Department of Biotechnology of Animal Reproduction, National
Research Institute of Animal Production, 32-083 Balice near
Crakow, Poland
155
B2 and SOF, which are the media routinely used for bovine embryo culture.
The choice of MSCs expansion medium can have a significant influence on both growth and cell morphology
which is of fundamental importance for their implementation in other biotechnological procedures like e.g.
embryo in vitro culture. The in vitro co-culture system of
embryos with MSCs requires further research.
Acknowledgements
This research was supported by the Statutory Activity of National
Research Institute of Animal Production 02-4.03.1 and by the Polish
National Science Centre resources allocated on the basis of decision
number DEC-2011/03/D/NZ9/05537.
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P10.9
P10.10
Next Generation Sequencing RNA-seq technology is a powerful tool which creates new possibilities in
whole-transcriptome analysis. The RNA analysis using
high-throughput sequencing method allows to measure
gene expression on a genome-wide scale. In our study,
RNA-seq method was applied to analyze global changes
in transcriptome form the muscle tissue (m. semimembranosus) in two pig breeds (Pietrain 5 pigs, Puawska
7pigs). The total RNA wasisolated using TRI-Reagent.
The quantity and quality of RNA were evaluated with the
NanoDrop 2000 and by 2% agarose gel electrophoresis.
cDNA libraries were prepared by using the TruSeq RNA
Sample Preparation Kit v2 (Illumina) according to standard protocol. Sequencing by synthesis of the libraries
was performed on a HiScanSQ System with 50 single-end
cycles using TruSeq SBS Kit v 3 HS chemistry (Illumina).
Using two different approaches by using DESeq, egdeR
freewares and considered the most restrictive criteria
we identified 184 differentially expressed genes between
Pietrain and Puawska pigs. In both breeds, the most
abundant were: transcripts encoding ribosomal proteins,
genes associated with metabolic processes, which play
akey role in respiratory electron transport chain in mitochondrion, intercellular or cation transport (NDUFS6,
SYPL2, CHCHD3, ATP5A1, DLD).
In Puawska pigs we indicated up-regulation of several
genes that play a variety of roles crucial for metabolic
processes metabolism of polysaccharides an carbohydrates (CHID1, HS3ST5, MFSD10), amino acid and protein metabolic processes (NUBP2, ALDH16A1, OTUB1).
Furthermore, when compare to Pietrain pigs, the significantly higher expression was obtained for genes encoding proteins associated with cell adhesion and cell cycle
(EMILIN1, EMILIN3, GADD45G, H1FX). In Pietrain
breed, only 37 genes were over-expressed and majority
of them play an important role in cell developmental processes i.a. regulation of apoptosis, mitosis, cell-cell signaling and adhesion (SGPP1, HTRA2,SLITRK2, MAFB,
ACTL6B), as well as in the muscle contraction and cation/anion transport (GLRB, SLC9A7). The different expression profile of selected genes obtained for two pig
breeds may be the result of long-term selection to improve the meat content in carcasses conducted especially
in Pietrain breed.
Acknowledgements
This study was supported by the National Research Institute of Animal
Production statutory activity, Research Project No. 01-5.03.1.
Eurobiotech 2013
P10.11
P10.12
157
158
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P10.13
P10.14
High Hydrostatic Pressure (HHP) by stimulating the defensive reaction to stress increases cells resistance to another adverse factor. Cells after HHP treatment overexpress resistance proteins, therefore they can quicker adapt
to next adverse factor like cryopreservation. As a result,
a higher survival rates after cryopreservation can be obtained. As a beneficial effect of HHP on sperm, oocytes
and embryos cryotolerance is already documented, we
assume that the HHP treatment may also have a positive
effect on the Mesenchymal Stem Cells (MSCs) quality after cryopreservation.
The aim of this study was to examine the influence of
varied HHP values on porcine MSCs to increase survival
rate and quality after cryopreservation. The survival rate
and phosphatidylserine (PS) exposure in external surface
of the cells were analyzed to indicate the most optimal
HHP value for further experiments. In normal cells PS
is located on the cytosolic side of cell membranes but
during the early stages of apoptosis PS becomes exposed
on the surface of the cell. Annexin V has the highest affinity for PS, therefore it is used as a probe to detect cells
that have expressed PS.
The MSCs were isolated from bone morrow. Aspirate was
diluted with PBS and subjected to centrifugation with
Ficoll. The mononuclear cells were isolated from the interface, washed twice in PBS and resuspended in DMEM
supplemented with 10% FCS. The MSCs were cultured for
about 2 weeks until 80% confluence was reached. Then,
MSCs were detached with 0.25% trypsin/EDTA, centrifuged at 300 g and aspirated into 0.5 ml straws with M199
Hepes modification supplemented with 2% FCS. Such
prepared straws were subjected to the five different HHP
treatments (20 MPa, 30 MPa, 40 MPa, 50 MPa, 60MPa)
for 1 h in 24C. After HHP treatment, MSCs were suspended in 10% DMSO and stored in liquid nitrogen for
at least 24 h. Thawed MSCs were used for survival rate
and early apoptosis detection by trypan blue staining and
Annexin V-Biotin Apoptosis Detection Kit, respectively.
Regarding MSCs viability, the high significant difference
(P < 0.001) was noted between MSCs subjected to HHP
ranging for 30 MPa to 60 MPa and control MSCs. The
significant difference (P < 0.05) was noted between MSC
subjected to 20 MPa HHP and control. No significant difference was observed in PS exposure in any of analyzed
experimental groups.
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159
P10.15
Analysis of the changes in the expression
profile of liver transcriptome by RNA-seq
in pigs fed with various diet supplements
Maria Oczkowicz1, Katarzyna Ropka-Molik1,
Magorzata witkiewicz2, Kacper ukowski3, Artur Gurgul1
National Research Institute of Animal Production, Laboratory
of Genomics; 2National Research Institute of Animal Production,
Department of Animal Nutrition And Feed Science;
3
National Research Institute of Animal Production, Department
of Animal Breeding and Genetics
1
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P10.16
Regulation of Tenebrio molitor heart
beating by monoamines and tropane
alkaloids
Szymon Chowaski, Jan Lubawy, Arkadiusz Urbaski,
Marta Spochacz, Grzegorz Smykalla, Grzegorz Rosiski
Department of Animal Physiology and Development,
Faculty of Biology, Adam Mickiewicz University in Poznan
Eurobiotech 2013
161
P10.17
P10.18
Leptin and leptin receptor genes are considered as markers of production traits in dairy or beef cattle. The aim
of this study was to verify the associations of polymorphisms in bovine LEP and LEPR genes with production
and reproduction traits in Slovak Spotted and Pinzgau
cows. Evaluated were long life production: milk, protein, and fat yield and reproduction traits: age at first
calving, calving interval, days open, and insemination interval. In total 296 blood samples of Slovak Spotted and
85 hair roots samples of Pinzgau cows were analyzed. In
order to detect LEP/Sau3AI (BTA 4, inron 2) and LEPR/
T945M (BTA 3, exon 20) genotypes PCR-RFLP method
was used. In Slovak Spotted and Pinzgau cows were the
allele frequencies 0.838/0.162 and 0.694/0.306 for A and
B LEP variants, and 0.954/0.046 and 0.912/0.088 for C
and T LEPR variants, respectively. For testing the associations between SNPs LEP/Sau3AI and LEPR/T945M
and evaluated traits the General Linear Model (GLM)
procedure in SAS Software was used. Statistical analysis
showed that the SNP LEP/Sau3AI significant affected
milk, protein and fat yield (P
Acknowledgements
This work was supported by the Slovak Research and Development
Agency under the contract No. APVV-0636-11.
Acknowledgements
This work was supported by the Slovak Research and Development
Agency under the Contract No. APVV-0636-11.
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P10.19
P10.20
Acknowledgements
This work was supported by the Slovak Research and Development
Agency under the contract No. APVV-0636-11.
Acknowledgements
This work has been supported by the grants: The Slovak Research
and Development Agency under the contract No. LPP-0220-09 and
No. APVV-0636-11.
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P10.21
P10. 22
The aim of this work was to optimize fast, reliable and efficient modification of the PCR methods for identification
of polymorphism for candidate genes calpain 1 (CAPN1)
and calpastatin (CAST) and to analyze the genetic structure in population of 145 animals of Slovak Spotted
breed and 137 animals of Slovak Pinzgau breed. Genetic
structure for the selected breeds was evaluaeted by using
three markers of CAPN1 gene (CAPN316, CAPN4685,
CAPN4751) and three markers of CAST gene (CAST-T1,
CAST-UoG, CAST-WSU). Genomic DNA was isolated
from whole blood, sperm and hairs by using commercial
column kit. The study of CAPN1 and CAST gene polymorphisms was done by using molecular-genetics methods such as PCR-RFLP, ARM-PCR and HRMA. Genetic
analysis confirmed a higher frequency of preferred alleles
(C; C; A; C) and genotypes (CC; CC; AA; CC) of markers CAPN316, CAPN4751, CAST-T1 and CAST-UoG associated with meat tenderness, in Slovak Pinzgau breed
unlike Slovak Spotted breed, which was characterized by
the lowest frequence of preferred alleles and genotypes
of these markers. For marker CAPN4685 associated with
ahigher lean share in valuable cuts was detected the prevalence of favourable allele T and genotype TT in Slovak
Pinzgau breed too. Our primary results suggest, that the
Slovak Pinzgau breed has favourable genetic base for improvement of quality meat such as tenderness in compare
with the Slovak Spotted breed, which has markedly prevalency of favourable allele C for marker CAST-WSU which
is associated with longevity and reproduction traits.
Acknowledgements
This work has been supported by the projects APVV No. LPP-0220-09
and APVV-0636-11.
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Eurobiotech 2013
P10.23
Relationships between type traits
and functional productive life in Slovak
Holstein Cows
Eva Strapkov, Peter Strapk, Juraj Candrk
Slovak University of Agriculture in Nitra
The relationships between type traits and functional productive life were analyzed in 60208 Slovak Holstein cows
first calved from 1996 to 2010. Functional productive
life was defined as the number of days from first calving
to death, culling or censoring. All cows were scored for
conformation during the first lactation. Weibull models
were fitted to analyze the data. The strongest relationship
between estimated breeding values for survival and type
traits were found for rear legs side view, rump width, angularity, fore udder attachment, dairy strenght, body condition and udder depth. Analyses were done one at atime
for each of those 7 type traits. Based on analyses was
found that cows with shallower udder, moderate fore udder attachment, marked angularity, correct rear legs side
wiev or narrower rump achieved longer productive life.
Evaluation of body condition confirmed that very thin or
very fat cows reached higher involuntary risk of culling.
Acknowledgements
Financial support was provided by the project APVV-0636-11 and
KEGA 027SPU-4/2012.
L11.2
L11.1
Andrew Tommey
DuPont Pioneer, Avenue des Arts 44, 1040 Brussels, Belgium
166
Eurobiotech 2013
L11.3
L11.4
Tomasz Twardowski
Green biotechnology plays an important role in bioeconomy strategies in both developed and developing
countries. According to the Strategy Europe 2020 bioeconomy is a key element for smart and green growth
in Europe. Now it is obvious that Europe needs new
approaches to production and consumption in order to
meet global environmental and economical challenges.
In 2012 genetically modified plants were cultivated in 28
countries worldwide and its role in food/feed production
is increasing steadily in the last years. On the other hand
Europe is very reluctant in adoption of GM plants but
remains big GM feed importer. Poland is one of the few
EU countries where GM plants have been cultivated in
the past years on commercial scale. In South of Poland
where infestation with the European corn borer (Ostrinia nubilalis) pose severe damages to maize production
Polish farmers used to grow authorized for cultivation in
EU genetically modified maize varieties. As from 28th of
January 2013 regulation of Polish Council of Ministers
prohibited the use of MON810 maize seeds and Amflora
potato the only GM events approved for cultivation in
EU. Poland along with Austria, France, Hungary become
one of the European countries where GM plants are not
being cultivated. Wide adoption of genetically modified
plants in European and Polish agriculture could be important not only to ensure food security, reduce dependence on non-renewable resources but also to create jobs
and maintain competitiveness on the world market. Currently GM plant varieties are not available to Polish farmers but recent advancements in innovative plant breeding
techniques brings new insight to application of genetic
engineering in Polish and European agriculture.
It is too late to close the door on GM products: biomaterials, bioenergy, biopharmaceuticals as well as food and
feed. The political economy of biotechnology policies
depends on consumers. In the United Europe the consumers (= the voters) said no to the GM farming giants in
several states, however that didnt stop millions of tones
of GM soya and corn entering the European food chain.
Legislation including IPR, innovative technology, public
acceptance and many more factors are critical for the further development of biotechnology.
Eurobiotech 2013
L11.4
Posters
P11.1
Ewa Waszkowska
Sebastian Kwiatkowski1, 2, 3
167
Human embryonic stem cells hold great promise for advancing human health. However, patents for the use of
human embryonic stem cells have been the subjects of
extensive public discussion and are regarded as extremely
problematical. The ethical and legal problems are associated.
In October 2011, The Court of Justice of the European Union issued its decision in Brstle v Greenpeace
(C-34/10), which relates to the patentability of technology based on the use of human embryonic stem cells.
The European court ruled that German scientists could
not patent a technique based on human embryonic stem
cells because it involved the destruction of something
capable of commencing the process of development of
ahuman being in other words a human embryo.
The definition of a human embryo used by the court included types of artificially created embryos covering
these that are not capable of developing into a fetus.
Is this definition too broad?
The initial aim of patents is to promote innovation and
reward scientific efforts.
The ban on patenting human stem cell research could
harm its future development in Europe?
Lectures
L1.2
L1.1
Human induced pluripotent stem cells (hiPS) are generated by epigenetic reprogramming of somatic cells through
the exogenous expression of four transcription factors
(Oct4, Klf4, Sox2, c-Myc). These cells has the all characteristic features of human embryonic stem cells (hES)
as self-renewal and ability to differentiate to cells of all
three germ layers. Growing body of published data clearly
shows that that epigenetic mechanisms are involved in the
process of reprogramming of somatic cells into pluripotent state. TRIM28/KAP1 protein is one of the epigenetic regulators that regulate heterochromatin formation
through histone modifications and DNA methylation.
Over 500 unique zinc finger proteins containing KRAB
domain recruit TRIM28/KAP1 protein to genomic DNA
in sequence-specific fashion thus leading to transcriptional repression of neighboring genes. Here, we showed
that TRIM28/KAP1 protein controls self-renewal of hES
and hiPS cells. Stable knockdown of TRIM28/KAP1 gene
expression in both hES and hiPS cells achieved using lentiviral vectors carrying specific shRNAs resulted in progressive loss of pluripotent phenotype as confirmed by
analysis of surface embryonic markers expression using
immunofluorescence and FACS analyses. Moreover, we
have documented progressive loss of expression of selected embryonic transcription factors in hES and hiPS cells
depleted of TRIM28/KAP1 exkpression. Our ongoing experiments are focused on detailed analysis of the molecular mechanisms that are involved in the observed phenotype and may shed new light on epigenetic mechanisms
that preserve self renewal of hES and hiPS cells.
Embryonic stem cells (ESCs), derived from prelimplantation embryos at the blastocyst stage, are unique in unlimited self-renew ability and pluripotency allowing their
differentiation into any cell type. Until the beginning of
XXI century ESCs were considered as the only cell lines
which pluripotency was proved both in vitro and in vivo.
In 2006 first paper describing reprogramming of fibroblasts into sa called induced pluripotent cells (iPSCs) was
published. The basis of this spectacular achievement done
by Yamanaka and his collaborators was establised in 50s
and 60s of the XX century. The short history of the cellular
reprogramming and pluripotent stem cells research that
led to the Nobel Prize for Yamanaka and Gurdon will be
a topic of this talk.
Eurobiotech 2013
169
L1.3
L1.4
Maciej Wiznerowicz1, 2
Gene Therapy Laboratory, Department of Cancer Immunology,
Poznan University of Medical Sciences, Chair of Medical
Biotechnology, Poland; 2Gene Therapy Laboratory,
Department of Cancer Immunology, Greater Poland Cancer
Centre, Poland
1
Induced pluripotent stem cells (iPS) cells are generated by dedifferentiation of adult cells through the forced
expression of few embryonic transcription factors. The
reprogramming process involves ordered and global
epigenetic alterations including histone modifications
and DNA methylation that progressively silence expression of lineage-specific genes whilst enabling transcription of embryonic factors genes. We explored the role
of KRAB-containing zinc finger proteins (KRAB-ZFPs)
and their cofactor TRIM28/KAP1 during the reprogramming process. The results of our work strongly suggest
that knockdown of TRIM28/KAP1 expression facilitate
de-differentiation of human primary fibroblasts towards
the iPS cells. In the other hand the inhibition of TRIM28/
KAP1 function in established human pluripotent cells results in progressive loss of their self-renewal potential in
contrast the the wild-type human iPS cells. Additionally,
our parallel lines of research clearly demonstrated that
binding of TRIM28/KAP1 through KRAB-containing
transcriptional repressors results in specific methylation
of the cellular promoters. Finally, we have revealed that
epigenetic factors recruited by TRIM28/KAP1 controls
retrotransposition events during the reprogramming of
human somatic cells to pluripotency. Taken together, our
results demonstrated novel role for TRIM28/KAP1 protein in the reprogramming process and self-renewal of
human iPS cell as well as their involvement in modeling
landscape of genomic DNA methylation during that process. In-depth understanding of epigenetic mechanisms
involved in the cellular reprogramming and self-renewal of human iPS cells will have implications for basic research and may pave ways to novel therapies.
170
Eurobiotech 2013
L1.5
L1.6
Leonora Buzanska
Eurobiotech 2013
Posters
P1.2
P1.1
171
172
Eurobiotech 2013
P1.3
P1.4
Human induced pluripotent stem cells (IPS) are generated by reprogramming of somatic cells through enforced
expression of embryonic transcription factors. However,
clinical applications require that expression of introduced
transgenes must be permanently switched off in the IPS
cells and obtained differentiated progenies. Here, we took
advantage of epigenetic switch that relies on reversible
binding of tTRKRAB transrepressor to tetO element,
which results in tight transcriptional repression of proximal promoter through heterochromatin formation. In
order to apply this system for reprogramming, the tetO
element was inserted into pSTEMCCA lentiviral vector
carrying OCT4, SOX2, KLF4 and cMYC under control
of EF-1alpha promoter. Transduction of human skin fibroblasts with obtained pSTEMCCA-tetO allowed for
expression of reprogramming factors and thus efficient
generation of human iPS clones. Obtained clones were
picked and further cultured until establishing stable IPS
cell lines. Then cells were then transduced with lentiviral
vector pLV-HK carrying tTRKRAB, in order to switch off
reprogramming transgene expression.
Tight repression of introduced transgenes in all human
IPS clones was analyzed by RT-PCR and confirmed
full functionality of our system. Obtained IPS cell lines
showed no abnormalities in karyotypes. Pluripotent phenotype of IPS cells was revealed by analysis of endogenous embryonic genes expression using RT-PCR and
immunofluorescence staining. Analyzed cells were also
able to form embryonic bodies in vitro and teratomas in
immunocompromised mice, which proved their ability
to differentiate into cells derived from three germ layers. tTRKRAB-mediated epigenetic repression persisted
through prolonged culture of obtained IPS cell lines. Importantly, expression of introduced transgenes remained
undetectable after differentiation into embryonic bodies.
In order to confirm molecular homogeneity of obtained
IPS cell lines, high throughput molecular profiling including RNA-Seq and global DNA methylation analysis
are currently being performed.
Our results confirm that our epigenetic switch effectively
prohibits re-expression of embryonic transgenes in human IPS cells and their differentiated progenies paving
the way for their applications in various fields of regenerative medicine, disease modeling and drug discovery.
Eurobiotech 2013
P1.5
P1.6
173
174
Eurobiotech 2013
P1.7
P1.8
Eurobiotech 2013
P1.9
TRIM28/KAP1 protein controls self-renewal
of human induced pluripotent stem cells
Wojciech Barczak1, Katarzyna Kulcenty1, Maciej Wiznerowicz1, 2
Gene Therapy Laboratory, Department of Cancer Immunology,
Poznan University of Medical Sciences, Chair of Medical
Biotechnology, Poland; 2Gene Therapy Laboratory, Department
of Cancer Immunology, Greater Poland Cancer Centre, Poland
1
Human induced pluripotent stem cells (hiPS) are generated by epigenetic reprogramming of somatic cells through
the exogenous expression of four transcription factors
(Oct4, Klf4, Sox2, c-Myc). These cells has the all characteristic features of human embryonic stem cells (hES)
as self-renewal and ability to differentiate to cells of all
three germ layers. Growing body of published data clearly
shows that that epigenetic mechanisms are involved in the
process of reprogramming of somatic cells into pluripotent state. TRIM28/KAP1 protein is one of the epigenetic regulators that regulate heterochromatin formation
through histone modifications and DNA methylation.
Over 500 unique zinc finger proteins containing KRAB
domain recruit TRIM28/KAP1 protein to genomic DNA
in sequence-specific fashion thus leading to transcriptional repression of neighboring genes. Here, we showed
that TRIM28/KAP1 protein controls self-renewal of hES
and hiPS cells. Stable knockdown of TRIM28/KAP1 gene
expression in both hES and hiPS cells achieved using lentiviral vectors carrying specific shRNAs resulted in progressive loss of pluripotent phenotype as confirmed by
analysis of surface embryonic markers expression using
immunofluorescence and FACS analyses. Moreover, we
have documented progressive loss of expression of selected embryonic transcription factors in hES and hiPS
cells depleted of TRIM28/KAP1 exkpression. Our ongoing experiments are focused on detailed analysis of the
molecular mechanisms that are involved in the observed
phenotype and may shed new light on epigenetic mechanisms that preserve self renewal of hES and hiPS cells.
175