Bioresource Technology 101 (2010) 2623–2628

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Anaerobic digested dairy manure as a nutrient supplement for cultivation

of oil-rich green microalgae Chlorella sp.
Liang Wang, Yecong Li, Paul Chen, Min Min, Yifeng Chen, Jun Zhu, Roger R. Ruan *
Department of Bioproducts and Biosystems Engineering, University of Minnesota, 1390 Eckles Avenue, St. Paul, MN 55108, USA

a r t i c l e i n f o a b s t r a c t

Article history: The present study was to investigate the effectiveness of using digested dairy manure as a nutrient
Received 13 July 2009 supplement for cultivation of oil-rich green microalgae Chlorella sp. Different dilution multiples of
Received in revised form 19 October 2009 10, 15, 20, and 25 were applied to the digested manure and algal growth was compared in regard
Accepted 21 October 2009
to growth rate, nutrient removal efficiency, and final algal fatty acids content and composition. Slower
Available online 24 November 2009
growth rates were observed with less diluted manure samples with higher turbidities in the initial cul-
tivation days. A reverse linear relationship (R2 = 0.982) was found between the average specific growth
Keywords:
rate of the beginning 7 days and the initial turbidities. Algae removed ammonia, total nitrogen, total
Anaerobic digested dairy manure
Algae
phosphorus, and COD by 100%, 75.7–82.5%, 62.5–74.7%, and 27.4–38.4%, respectively, in differently
Nutrients removal diluted dairy manure. COD in digested dairy manure, beside CO2, proved to be another carbon source
Lipid content for mixotrophic Chlorella. Fatty acid profiles derived from triacylglyceride (TAG), phospholipid and free
Chlorella fatty acids showed that octadecadienoic acid (C18:2) and hexadecanoic acid (C16:0) were the two
most abundant fatty acids in the algae. The total fatty acid content of the dry weight increased from
9.00% to 13.7% along with the increasing dilution multiples. Based on the results from this study, a
process combining anaerobic digestion and algae cultivation can be proposed as an effective way to
convert high strength dairy manure into profitable byproducts as well as to reduce contaminations
to environment.
Published by Elsevier Ltd.

1. Introduction
Land disposal methods have traditionally been used to manage
animal manures (de Godos et al., 2009). However, the potential of
exporting tremendous nitrogen and phosphorus into water bodies
makes it not sustainable. Conventional biological treatments such
as activated sludge sequential-batch processes (Zhang et al.,
2006; Wang et al., 2009) provide potential approaches for a cor-
rect waste management, but the associated high energy inputs re-
tard their widespread implementation in rural areas (de Godos
et al., 2009). Algae based wastewater treatment processes have
been gaining tremendous attentions since 1960s (Golueke and
Oswald, 1962; Lau et al., 1995; Tam and Wong, 2000) because
they could potentially offer many advantages over the common
activated sludge process that requires high energy input for aer-
ation and high cost for subsequent sludge processing. The success
of an algae based system relies on the ability of the algal cells to
assimilate organic carbon (heterotrophic growth) (Burrell et al.,
1984) as well as inorganic nutrients such as nitrogen and phos-
* Corresponding author. Tel.: +1 612 625 1710; fax: +1 612 624 3005.
E-mail addresses: wangx739@umn.edu (L. Wang), ruanx001@umn.edu (R.R.
Ruan).
phorus (Lau et al., 1995) from the wastewater for their growth
without an aerobic environment being created and maintained.
The obtained algal biomass contain high ratios of hydrocarbons
in the form of extractable lipids such as free fatty acids, triacyl-
glyceride (TAG), phospholipid and glycolipid, and are ideal feed-
stocks for biodiesel production. Manure grown algae can also be
used as soil conditioner (Wilkie and Mulbry, 2002), animal feed
supplement (Barlow et al., 1975) and fermentation substrate (Ue-
noa et al., 1998). With all the above-mentioned merits, co-locat-
ing algae production with animal waste treatment could be
made environmentally effective as well as economically viable
in a not distant future.
Recovery of nutrients from swine manure wastewaters by mic-
roalgae has been extensively investigated by a number of research-
ers and a lot of experience has been gained in pilot-scale
operations (Barlow et al., 1975; Fallowfield and Garrett, 1985; de
Godos et al., 2009) with algal productivities varying from 7 to
33 g/m2/day and simultaneous reductions of COD, nitrogen and
phosphorus. The experience gained for dairy manure is not as
much as that for swine manure but still can be found in several lit-
eratures (Lincoln et al., 1996; Woertz, 2007; Mulbry et al., 2008).
From an outdoor algal turf scrubber (ATS) raceway system, Mulbry
et al. (2008) concluded that projected annual operation costs were

0960-8524/$ - see front matter Published by Elsevier Ltd.

doi:10.1016/j.biortech.2009.10.062
2624 L. Wang et al. / Bioresource Technology 101 (2010) 2623–2628
well below the costs cited for upgrading existing water treatment
plants in sensitive watersheds, indicating the algal technology for
dairy manure treatment very appealing from the environmental
standpoint.
The above studies mainly focused on the algae productivity and
nutrient recovery efficiency, and few have discussed the algal
growth type (heterotrophic or autotrophic) in a high organic
strength wastewater like diary manure, nor did concern about
the lipid content and fatty acid profile of the dairy manure grown
algae. The objective of this study was to test the effectiveness of
cultivating a wildly isolated green alga, Chlorella, sp. in diluted di-
gested diary manures sampled from a local dairy farm using batch-
wise experiments. Carbon usage related to the growth type, the
relationship between growth rates and turbidities, as well as the fi-
nal algal fatty acid content and composition will be discussed in
depth.
2. Methods
2.1. Algae strain and culture condition
Algae strain was a wild-type Chlorella sp. isolated from local
freshwater. It was preserved in Tris–Acetate–Phosphorus (TAP)
(Harris, 1989) media containing the following chemicals: NH4Cl
(400 mg/L), MgSO47H2O (100 mg/L), CaCl22H2O (50 mg/L),
K2HPO4 (108 mg/L), KH2PO4 (56 mg/L), Tris (hydroxymethyl) ami-
nomethane (2420 mg/L), glacial acetic acid (1 mL/L), and trace ele-
ments solution (1 mL/L), consisting of Na2EDTA 50 (g/L),
ZnSO47H2O (22 g/L), CaCl22H2O (0.05 g/L), H3BO3 (11.4 g/L),
MnCl24H2O (5.06 g/L), FeSO47H2O (4.99 g/L), CoCl26H2O (1.61 g/
L), CuSO45H2O (1.57 g/L), (NH4)6Mo7O244H2O (1.10 g/L), KOH
(16 g/L). Algae were inoculated at 10% (v/v) in 250 mL Erlenmeyer
flasks containing 100 mL liquid medium. The culture flasks were
incubated under stationary condition at 25 ± 2 °C and
200 lmolm2s1 continuous cool-white fluorescent light illumi-
nation on a shaker with 150 rpm rotation speed. All the experi-
ments were carried out in duplicate and average values were
reported.
2.2. Characteristics of dairy manure
Undigested and digested dairy manures were collected from
Haubenschild Farm, Princeton, Minnesota, where a plug-flow
anaerobic digester has been in operation since 1999. The charac-
teristics of the dairy manure are shown in Table 1. Ammonium
(NH4+–N), total nitrogen (TN), total phosphorus (TP) and chemical
oxygen demand (COD) were determined for both undigested and
digested dairy manures following the Hach DR 5000 Spectropho-
tometer Manual (Hach, 2008). Total solids (TS) and total volatile
solids (TVS) were performed following the standard methods
(APHA, 2005). The possible volatile fatty acids or organic sub-
stances in undigested and digested diary manures were deter-
mined using gas chromatography–mass spectrometry (GC–MS)
(Agilent 7890–5975C, USA). The GC was equipped with a flame
Table 1
Characteristics of the manure before and after digestion.
Parameters Before digestion
NH3–N (mg N/L) 1782
TKN (mg N/L) 3305
TP (mg PO4/L) 266
COD (mg/L) 38230
TS 9.70%
TVS 8.00%
ionization detector and a DB-5-MS capillary column. The oven
temperature was 80 °C, held for 5 min, raised to 290 °C at a rate
of 4 °C/min, and held at 290 °C for 5 min, while the injector and
detector temperature were set at 250 °C and 230 °C, respectively.
The carrier gas (helium) was controlled at 1.2 mL/min. Chromato-
graphic data were recorded and integrated using Agilent data anal-
ysis software. The compounds were identified in the NIST Mass
Spectral Database and quantified by comparing the peak area with
1000 mg/L acetic acid as an external standard (Sigma–Aldrich,
MO).
The digested dairy manure was used as the feedstock for grow-
ing algae. Four dilution multiples (10, 15, 20, 25) were applied to
the digested manure before inoculation. Diluted manure liquid
samples were filtered through glass microfiber filters (934-AH,
Whatman, USA) to remove large particles and indigenous bacteria.
Turbidities were determined before and after algal cultivation for
each sample using a nephelometer (Model 2100N Turbidimeter,
Hach, USA). Results are reported in Nephelometric Ratio Turbidity
Units (NTRU’s).
2.3. Determination of algal growth
Optical density (OD) of the inoculated manure at 680 nm was
measured daily as the algal density indicator using a spectropho-
tometer (Genesys 5, Spectronic Instruments, UK). A linear relation-
ship between OD and dry weight (DW, g/L) was determined for this
strain:
Dry weightðg=LÞ ¼ 0:3477OD680  0:0068; R2 ¼ 0:997
The growth rate (GR, d1) was calculated by fitting the OD for
the first 5 days of culture to an exponential function:
GR ¼ ðln ODt  ln OD0 Þ=t
where OD0 is the optical density at initial day, ODt is the optical
density for day t and t is the time between the two measurements.
2.4. Fatty acid analysis
Algae cells were harvested by centrifugation and then dried by a
freeze dryer (Savant Instruments Inc., USA) before being analyzed
for lipid content. Fatty acid content and composition analysis
was performed by following two consecutive steps including prep-
aration of fatty acid methyl ester (FAME) and GC–MS analysis.
FAME was prepared by a one-step extraction–transesterification
method as described by (Indarti et al., 2005), after suitable modifi-
cation. Dried algae samples (about 100 mg) were weighed into
clean, 25 mL screw-top glass bottles, to which 10 mL mixture of
methanol, concentrated sulfuric acid, and chloroform
(4.25:0.75:5) was added. Transesterification was carried out in
90 °C water bath (Cole-Parmer, USA) for 90 min. Upon completion
of the reaction, the chloroform layer containing FAME was care-
fully collected and subject to GC–MS analysis. The GC was
equipped with a flame ionization detector and a DB-5-MS capillary
column. The oven temperature was 80 °C, held for 5 min, raised to
290 °C at a rate of 4 °C/min, and held at 290 °C for 5 min, while the
After digestion injector and detector temperature were set at 250 °C and 230 °C,
2232
respectively. The carrier gas (helium) was controlled at 1.2 mL/
3456 min. Chromatographic data were recorded and integrated using
249.7 Agilent data analysis software. The compounds were identified in
23760 the NIST Mass Spectral Database and quantified by comparing
6.60%
the peak area with that of the external standard (C18:2) (Sigma–
5.10%
Aldrich, MO).
 L. Wang et al. / Bioresource Technology 101 (2010) 2623–2628
2.5. Physico-chemical analysis
Liquid samples for nutrient consumption analysis were col-
lected on the first day and the last day of the experiment. The
whole experiment ended at the 21st day when all the 4 growth
curves started to level off. Samples were centrifuged at 5000 rpm
for 15 min and supernatants were collected for analyses of ammo-
nium (NH4+–N), total nitrogen (TN), total phosphorus (TP) and
chemical oxygen demand (COD). Measurements of NH4+, TN, TP
and COD were performed following the Hach DR 5000 Spectropho-
tometer Manual (Hach, 2008). Removal rates for those parameters
were calculated by dividing the difference between the first day
and final day concentrations by the first day concentration, and
then multiplied by 100. The carbon, hydrogen and nitrogen content
of dried algae samples were measured using an elementary ana-
lyzer (CE-440, Exeter Analytical Inc., USA).
3. Results and discussion
3.1. Dairy manure characteristics before and after digestion
The Haubenschild Farms digester is a covered 350,000-gallon
concrete tank installed in the ground, the temperature of which
was controlled 95–105 °F by suspended heating pipes. The digester
was designed with a maximum treating capacity of 1000 cows and
a retention time of 20 days. Table 1 shows the characteristics of the
manure before and after digestion. The total nitrogen and total
phosphorus did not change much since anaerobic digesters are
known to reduce negligible amounts of nutrients (Lusk, 1998),
while ammonium increased obviously, due to the anaerobic bio-
conversion of proteins contained in manure into amino acids and
then to ammonia (Cheng and Liu, 2002). The organic substances
in the undigested diary manure detected by GC–MS were acetic
acid and propionic acid, accounting for 31.3% and 27.5% of the total
undigested COD, respectively. Isopropoxycarbamic acid ethyl ester
was the only organic substance detected in the digested sample,
which was about 18.8% of the total digested COD. Anaerobic diges-
tion reduced COD, TS and TVS by 30–40%, which was comparable
to the typical performance level of anaerobic digesters treating
dairy manure (Martin, 2003; Demirer and Chen, 2004). Therefore,
critical nutrients were still retained in the dairy manure after
anaerobic digestion, making it a good supplement for growing
algae.
3.2. Algal growth and nutrient removal efficiencies
Algal growth as indicated by optical density measured at
680 nm in 10, 15, 20 and 25 diluted digested dairy manure
6
Optical Density at 680 nm
5 180
4 140
3 100
2 R2=0.982
1
0 0
0 2 4 6 8 10 12 14 16
Days of Cultivation
Fig. 1. Growth curves of Chlorella sp. in digested dairy manure with different
dilution multiples.
2625
were shown in Fig. 1. This wild-isolated Chlorella sp. could survive
in all of the diluted dairy manure samples and no obvious lag
phases were observed. Comparing between the serial dilutions, it
is noticed that algae grew faster in 20 and 25 diluted samples
in the first 7 days. After the 7th day, the growth curve of algae in
25 diluted sample started to level off due to earlier exhaustion
of less amount of nutrients. The algae in 10 diluted samples
picked up their growth rate in the latter part of the cultivation per-
iod. The average specific growth rates under the four conditions in
the first 7 days were 0.282, 0.350, 0.407 and 0.409 d1, respec-
tively. These growth rates were comparable to that grown in the
commercial Bristol medium (0.3644 d1) under axenic conditions
(Lau et al., 1994).
Algal growth is directly affected by the availability of nutrients
(El-Nabarawy and Welter, 1984), light (Sorokin and Krauss, 1958),
the stability of pH (Azov and Shelef, 1987) and temperature (Talbot
and de la Noiie, 1993) and the initial inoculation density (Lau et al.,
1995). It is expected that the higher the initial inoculum density,
the better the growth and the higher the nutrient removal effi-
ciency (Lau et al., 1995) while problems such as self-shading, accu-
mulation of auto-inhibitors (Darley, 1982) are not present. Lau
et al. (1995) studied the effect of initial algal inoculum sizes on
the nutrient removal efficiency for the primary settled sewage
and found that the superconcentrated culture of Chlorella vulgaris
(with an initial inoculum of 1  107 cells m11) did not exhibit
any self-shading limitation of growth and nutrient removal. The
inoculation size of present study was about 1.2  107 cells m11,
which was considered to be an optimal level for most efficient
nutrient reduction (Lau et al., 1995) compared with inoculation
sizes of 106 (medium) and 105 (low) cells m11.
The relationship between initial 7-day average specific growth
rates and original sample turbidities was plotted in Fig. 2. The ini-
tial slower growth rate in less diluted manure sample correlated
well with their higher turbidities (R2 = 0.982), indicating that tur-
bidity is an important parameter determining the suitability of a
wastewater for algae growth. Turbidity, caused by suspended mat-
ter or impurities that interfere with the clarity of the water, ex-
presses the extent of the light being scattered and absorbed
rather than transmitted in straight lines through a water sample.
The impurities responsible for turbidity may include clay, silt, fi-
nely divided inorganic and organic matter, soluble colored organic
compounds, and plankton and other microscopic organisms (USEP-
A, 2009). Since the manure was diluted and filtered before testing,
the high turbidities of 10 and 15 diluted samples should be lar-
gely attributed to some finely divided inorganic and organic matter
instead of particles and microorganisms. The 20 and 25 diluted
manures with turbidities of 95 and 75 NTRU, respectively, did not
show much difference in the growth rate, suggesting that the light
200
160
Turbidity (NTRU)
120
y = -789.32x + 393.83
10*dilution 80
15*dilution 60
20*dilution 40
25*dilution 20
18 20 22 0.25 0.3 0.35 0.4
Specific growth rate (1/d)
Fig. 2. Correlation between sample turbidities and average algal specific growth
rates in the first 7 cultivation days.
2626 L. Wang et al. / Bioresource Technology 101 (2010) 2623–2628

Table 2
Turbidities and removal rates for different samples.

Parameters 10*dilution 15*dilution 20*dilution 25*dilution

Turbidity before treatment (NRTU) 180 120 95 75
Turbidity after treatment (NRTU) 57.5 45 31 24.5
Removal rate 68.0% 62.5% 67.4% 67.3%

penetration would be affected only when the turbidity went be-
yond a certain level. As time moved on, algae removed those par-
ticles accounted for turbidity (Table 2), which was another
reason, why the growth in less diluted samples caught up in the
latter part of the cultivation, beside the aspect that continued algal
growth could also be supported by the higher nutrient concentra-
tions in less diluted samples.
3.3. Nutrient removal efficiencies
For all eukaryotic algae, the only forms of inorganic nitrogen
that are directly assimilable are nitrate (NO 
3 ), nitrite (NO2 ), and
þ
ammonium (NH4 ) (Barsanti and Gualtieri, 2006).
Most of the nitrogen in the digested dairy manure was in the
form of ammonium nitrogen, and therefore was readily available
to algae. Results from this experiment showed that ammonium
was completely removed within the 21-day growth period for all
of the four diluted manure samples (Fig. 3a) regardless how high
the initial concentration (81–178 mg/L) was, which is in agreement
with results reported by De la Noüe and Bassères (1989). Therefore
the possible toxicity or inhibition of high levels of NH4 to the mic-
roalgae (Abeliovich and Azov, 1976) was not present for this strain
in tested samples, proving the strain to be ammoniacal-N tolerant.
Total nitrogen was greatly reduced by 75.7–82.5% but not com-
pletely removed (Fig. 3b), indicating there were still some organic
compounds that could not be converted to ammonium nitrogen
and assimilated by algae. A significant reduction (62.5–74.7%) of
total phosphorus was also found for all of the four samples
(Fig. 3c), which was also observed by Woertz (2007) who achieved
around 76% phosphate reduction when treating digested dairy
manure with algae but starting at a lower concentration of
7.7 mg PO4/L. CODs in the manure were utilized by algae as one
of carbon sources to some extent (27.4–38.4%) but not as efficient
as nitrogen and phosphorus (Fig. 3d). The experiment was per-
formed in axenic condition and therefore the reduction of COD
could only be attributed to consumption by algae. In photoautotro-
phic culture of microalgae, CO2 is the sole carbon source and can be
fixed into organic compounds such as protein, sugars and lipids
(Hirataa and Hayashitania, 1998). However, it is not the case for
some species of Chlorella, facultative algae, whose metabolic path-
way can alter with supply of organic substrates such as organic
acids or glucose (Eny, 1951). Endo et al. (1977) reported that the

220
35
200 100% (a) NH3-N_initial
70.1%
removal (b) TP_initial
removal
Total phosphorus concentrations
NH3-N concentrations (mgN/L)

180 NH3-N_final 30
TP_final
62.5%
160 100%
25 removal
removal
140 71.6%
(mg PO4/L)

100%
removal 20 removal 74.7%
120
100% removal
100 removal
15
80
60 10

40
5
20
0 0
10*dilution 15*dilution 20*dilution 25*dilution 10*dilution 15*dilution 20*dilution 25*dilution

300 1600
82.5%
removal (c) TKN_initial 1400
38.4% (d) COD_initial
TKN concentrations (mg N/L)

removal
COD concentrations (mg/L)

250 27.4% COD_final

TKN_final
1200 removal
75.7%
200 removal 34.3%
1000 removal
78.3% 77.6% 27.9%
150 removal removal 800 removal

600
100
400
50
200

0 0
10*dilution 15*dilution 20*dilution 25*dilution 10*dilution 15*dilution 20*dilution 25*dilution

Fig. 3. (a) Initial and final ammonium concentrations and removal rates; (b) initial and final TKN concentrations and removal rates; (c) initial and final TP concentrations and
removal rates and (d) initial and final COD concentrations and removal rates.
 L. Wang et al. / Bioresource Technology 101 (2010) 2623–2628 2627

Table 3 ergy for cell division and proliferation (Adams and Early, 2004).
Results from elementary analysis of dried algae samples grown under the four It is no doubt that the growth rate can be accelerated significantly
dilution multiples.
if some organic carbon sources can be used directly by microalgae
10*dilution 15*dilution 20*dilution 25*dilution in the oxidative assimilation process for storage material produc-
C/N 2.64 3.16 3.3 3.81 tion. As the result from elemental analysis suggested that the algae
C% 41.6 42.8 44.5 45.6 cells had C/N ratios varying between 2.64 and 3.81 (Table 3), com-
H% 6.42 6.63 6.78 6.92 pared with the C/N ratios (0.83–1.12) consumed in the dairy man-
(C + H)% 48.02 49.43 51.28 52.52
ure supplemented growth media by algae in Table 4, it is
concluded that algae used the organic carbon in the digested dairy
manure only as part of their carbon source while CO2 was also
Table 4
assimilated through the process of autotrophic photosynthesis.
Calculated ratios of carbon, nitrogen and phosphorus removed by algae (the
difference of initial and final concentration in the sample was the amount removed).
3.4. Algal fatty acids content and composition
Quotient Values
COD/TKN 2.48 2.24 2.99 2.21 Based on Murakami et al. (1997), lipids in C. vulgaris grown on
a
C/N 0.93 0.84 1.12 0.83 both organic and inorganic media included neutral lipid, phospho-
COD/TP 23.6 20.0 22.5 17.0 lipid, glycolipid, and Trans-hexadecanoic acid. The percentages of
TKN/TP 9.54 8.95 7.51 7.68
these lipids in their algae samples (dry weight base) are shown
a
C in C/N is the carbon equivalence of COD (O2) consumed. in Table 5. The percentage of TAG of the total lipid generally varies
with species and physiological state of the culture, ranging be-
tween 38% and 65% (Tambiev et al., 1989), while lipid of some spe-
cies consists mainly of esters of sterols and hydrocarbons (to 60%)
Table 5
Lipids in Chlorella vulgaris grown on both organic and inorganic medium (MURAKAMI
(Tambiev et al., 1989). Biodiesel, by definition, is the methyl ester
et al., 1997). produced from transesterification of TAG in plant oils or animal
fats. When taking a close look at the lipid composition in Chlorella
Lipid Percentage (%) of dry weight
cells, it is noticed that methyl esters derived from free fatty acids
Organic Inorganic and phospholipids also fall into the biodiesel category. Thus, the
media media
one-step extraction–transesterification process used in this study
Neutral lipid (waxester 80% and TAG 20%) 2.8 1.88 may provide a direct measurement of an algal species/strain’s po-
Phospholipid 8.6 6.2
tential as a biodiesel feedstock. The FAMEs obtained in this study
Glycolipid 5.7 5.7
Trans-hexadecanoic acid 1.6 2.3 should be derived from several lipid species including free fatty
acids, TAGs, and phospholipid in the algal bodies.
Fatty acids are primary metabolites of acetyl CoA pathway,
growth rate was approximately the same as the sum of growth which is genetically determined, evolutionarily very old, and
rates in the autotrophic and heterotrophic cultures when the cells therefore conservative (Petkov and Garcia, 2007). For green alga
were cultured under mixotrophic condition (in light with acetate). Chlorella, the fatty acid composition of 14:0, 16:0, 16:1, 16:2,
Definite growth due to the presence of the organic acids was also 16:3, 18:0, 18:1, 18:2, a-18:3 is confirmed under all kinds of con-
observed by Eny (1949), indicating that they are used by Chlorella ditions such as photoautotrophic and heterotrophic cultivation,
as a source of carbon. Organic acids are known to exist as interme- nitrogen starvation, and outdoor in a photobioreactor (Petkov
diates in the pathway of carbohydrate metabolism of most animals and Garcia, 2007). Table 6 shows the fatty acid profiles derived
and bacteria (Eny, 1951). The synthesis of storage materials such as from TAG, phospholipid and free fatty acids in algae cultivated
carbohydrate and protein may result either from photosynthesis or on diluted digested dairy manure samples in our study. Except
from oxidative assimilation of an organic substrate (Myers and for those mentioned by Petkov and Garcia (2007), 15:0, 17:0 and
Cramer, 1947). Respiration of the storage materials provides en- 20:0 fatty acids were also observed in small quantities in our algae

Table 6
Fatty acid profiles derived from triacylglycerol, phospholipid and free fatty acids in algae.
a
Fatty acid 10*dilution (%) 15*dilution (%) 20*dilution (%) 25*dilution (%)
Total fatty acid/dry weight 9.00 11.0 13.6 13.7
Saturated fatty acids (% of total fatty acids) 27.1 28.3 30.7 33.0
14:0 0.10 0.30 0.30 0.40
15:0 0.60 0.40 0.30 0.40
16:0 20.6 22.2 24.3 26.0
17:0 3.40 3.00 2.60 2.20
18:0 2.40 2.10 3.00 3.80
20:0 0.30 0.20 0.20
Monounsaturated fatty acids (% of total fatty acids) 19.1 22.2 29.9 27.7
16:1 6.60 10.80 9.10 7.50
18:1 12.50 11.40 20.80 20.20
Polyunsaturated fatty acids (% of total fatty acids) 53.8 49.4 39.2 39.1
16:2 10.4 8.60 6.80 5.70
16:3 6.00
18:2 27.2 30.6 32.4 33.4
18:3 10.2 10.2
a
Fatty acids are defined by the number of carbon atoms followed by the number of double bonds after the colon, classified to (1) SFA, saturated fatty acids
(C14:0 + C15:0 + C16:0 + C17:0 + C18:0 + C20:0); (2) MUFA, monounsaturated fatty acids (C16:1 + C18:1); (3) PUFA, polyunsaturated fatty acids (C16:2 + C16:3 + C18:2 +
C18:3).
2628 L. Wang et al. / Bioresource Technology 101 (2010) 2623–2628
samples. Octadecadienoic acid (C18:2) was the most abundant
fatty acid in algae bodies under all conditions, ranging from
27.2% to 33.4% of the total fatty acids. Hexadecanoic acid (C16:0)
was the second abundant fatty acid, ranging from 20.6% to 26.0%.
Both acids increased in their contents when cultivated under more
diluted samples, indicating their accumulation as storage materials
could be enhanced when subject to low/scare nutrient environ-
ment. The total fatty acid contents increased from 9.00% to 13.7%
of dry weight with increasing dilution multiples. Taking the cell
dry weight density (1.57, 1.47, 1.71, 1.48 g/L, respectively) into ac-
count, the final total fatty acids concentrations were 0.141, 0.162,
0.233, and 0.203 g/L, respectively, for 10, 15, 20, 25 diluted
samples. The saturated fatty acids increased from 27.1% to 33.0%
of total fatty acids while the polyunsaturated fatty acids decreased
from 53.8% to 39.1% of total fatty acids with dilution rates from 10
to 25. The monounsaturated fatty acids did not show a similar
trend but generally were accumulated more in more diluted
samples.
4. Conclusions
Vast amounts of animal manures produced from concentrated
animal feeding operations present opportunities for economic
gains if proper utilization strategies are employed (Demirer and
Chen, 2004). Anaerobic digestion has been widely employed to
partially treat dairy manure and generate on-site energy. A process
of combining anaerobic digestion further with algae cultivation can
offer many environmental benefits with the production of abun-
dant algal biomass to serve as a biofuel feedstock, a fertilizer or
an animal feed and thus provide a valuable solution to the refrac-
tory dairy wastes.
Acknowledgements
The authors are grateful to Richard Huelskamp for supplying
manure samples and Blanca C. Martinez for providing help in the
labs. The study was supported in part by grants from the University
of Minnesota Initiative for Renewable Energy and the Environment
(IREE) and the Center for Biorefining.
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