Process Biochemistry 40 (2005) 857863

High-carbohydrate wastewater treatment by IAL-CHS

with immobilized Candida tropicalis
Yongming Zhang
a
, Bruce E. Rittmann
b
, Jianlong Wang
c,
, Yuhong Sheng
d
,
Juntang Yu
e
, Hanchang Shi
d
, Yi Qian
d
a
Department of Environmental Engineering, School of Urban Science, Shanghai Normal University, Shanghai 200234, PR China
b
Department of Civil and Environmental Engineering, Northwestern University, Evanston, IL 60208-3109, USA
c
Laboratory of Environmental Technology, INET, Tsinghua University, Beijing 100084, PR China
d
State Key Joint Laboratory of Environmental Simulation and Pollution Control, Tsinghua University, Beijing 100084, PR China
e
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China
Received 8 February 2004; accepted 19 February 2004
Abstract
Anovel immobilized-cell bioreactor was developed and utilized for treatment of high-carbohydrate wastewater and production of single-cell
protein (SCP) simultaneously. The system is the internal airlift loop reactor with ceramic honeycomb support for biolm (IAL-CHS), and
the microorganisms tested was Candida tropicalis, which has a high protein yield. The optimum temperature for C. tropicalis was 3540

C,
and bioreactor experiments were performed at 35

C. The internal airlift loop reactor with ceramic honeycomb supports allowed a large
and dense accumulation of biomass, but also generated signicant SCP. Mass-transport resistance did not limit the overall COD-removal
kinetics and oxygen mass transfer may have been accelerated to the biolm. Fermentation of carbohydrates to organic acids lowered the pH
below the optimum range of 4.56.5, and pH below 4.5 retarded or stopped the oxidation reactions for the organic acids. Since organic-acid
oxidation is necessary for attaining COD removal from the wastewater, pH control may be essential for effective treatment in some cases with
high-carbohydrate wastewaters. The addition of 0.35 g/L urea could increase the COD removal rate. Continuous wastewater was carried out
and the organic loading reached 15 kg COD/(m
3
day).
2004 Elsevier Ltd. All rights reserved.
Keywords: Airlift biolm bioreactor; Ceramic honeycomb support; High-carbohydrate wastewater; Immobilized cells; Single-cell protein
1. Introduction
Wastewaters containing high-carbohydrate are produced
widely in industries such as food processing and fermenta-
tion [1]. This kind of wastewater can cause serious pollution
if released into the natural environment without proper
treatment or disposal. Many reports document the treat-
ment of high-carbohydrate wastewater [2,3]. Conventional
biological treatment, such as the activated sludge process,
often occurs some operational problems, including sludge
bulking [4]. Furthermore, discharge strictly as a treated
waste eliminates the resource value of the carbohydrate,
such as for producing single-cell protein (SCP).

Corresponding author. Tel.: +86-10-62784843;

fax: +86-10-62771150.
E-mail address: wangjl@tsinghua.edu.cn (J. Wang).
An economical and efcient way of handling high-
carbohydrate wastewater is to treat it with immobilized
cells with conversion into SCP at the same time. Wastewa-
ter treatment by immobilized cells can eliminate operating
problems encountered with the activated sludge process
[59]. Production of SCP captures the resource value of the
carbohydrate.
The overall objective of this work was to investigate the
treatment of high-carbohydrate wastewater and production
of SCP simultaneously using Candida tropicalis in a novel
immobilized-cell bioreactor, the internal airlift loop-ceramic
honeycomb support (IAL-CHS) biolmreactor. C. tropicalis
is an excellent choice for SCP production, because it has a
high protein content and biomass yield [10]. Successfully us-
ing C. tropicalis in an immobilized-cell bioreactor demands
a bioreactor that has excellent biomass retention, but does
not incur too much mass-transport resistance. The IAL-CHS
is promising in this regard, because the ceramic honeycomb
0032-9592/$ see front matter 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2004.02.010
858 Y. Zhang et al. / Process Biochemistry 40 (2005) 857863
support offers little resistance to liquid and gas transfer
through it. The specic objectives of this work were to doc-
ument that the IAL-CHS can be used with C. tropicalis; to
evaluate mass-transport advantages or disadvantages when
treating high-carbohydrate wastewater; to determine the op-
timal conditions for temperature and pH control when us-
ing immobilized C. tropicalis, because pH and temperature
of the medium were usually critical factors for cells growth
and COD removal [11]; and to evaluate the balance between
biomass retention and SCP production in the IAL-CHS.
2. Materials and methods
2.1. Biolm reactor
A novel internal airlift-loop biolm reactor in which cells
are immobilized onto a ceramic honeycomb support was
used in this study. This type of reactor has been used to
treat quinoline-containing wastewater [5]. The bioreactor
was made of plexiglass with an internal diameter of 100 mm,
a height of 250 mm, a draft-tube diameter of 60 mm, a
draft-tube height of 200 mm, and total working volume of
1500 mL. The ceramic honeycomb support, which t inside
the draft tube, had a diameter of 58 mm, a height of 150 mm
and pore holes with 2 mm 2 mm cross-section.
The airlift biolm reactor was aerated by compressed air
so that water circulated through the draft tube from bottom
to top. Based on tracer studies [12,13], the circulation rate
was approximately 4 L/min through the draft tube.
2.2. Microorganism
C. tropicalis was kindly provided by Professor YU Jun-
tang, at East China University of Science and Technology. It
was grown to about 10
7
cells/mL in batch culture in a shake
ask before being used for immobilization in the ceramic
honeycomb support. In order to immobilize the cells in the
ceramic honeycomb support for batch experiments, 1800 mL
of suspension including the C. tropicalis, which contained
about 3800 mg/L (dry weight), was added to the bioreactor
and the cells immobilized onto the ceramic honeycomb sup-
port for 10 min. The suspension was then ushed out leaving
approximately 60% of the inoculated biomass retained in
the ceramic honeycomb support. Microorganisms were in-
oculated once, and then the different experiments were run
one after the other.
2.3. Wastewaters
Two kinds of wastewaters were used. Wastewater 1 (W1)
was grains-washing wastewater froma brewery and included
mainly wort, which is made up mainly of maltose together
with small amounts of glucose and fructose [14]. Wastewater
2 (W2) was a synthetic carbohydrate wastewater composed
of glucose, ammonium chloride and potassium dihydrogen
phosphate dissolved in tap water and made sterilised by
boiling.
2.4. Batch experiments with suspended cells
Grains-washing wastewater (W1) was treated with sus-
pended C. tropicalis in the same bioreactor, but without
ceramic honeycomb support at 30, 35, or 40

C to investi-
gate the effect of temperature on cell growth and chemical
oxygen demand (COD) removal. Samples of efuent were
taken and used for analyzing COD, saccharides, organic
carboxylic acids and cell density at various time intervals.
In some experiments, the pH was not controlled, in other
experiments, the pH was controlled by addition of 0.05 M
sodium hydroxide by an auto-titrator (Model: Bellopon
GX14-P5TEX128SP, NIKISSO EIKO Co. Ltd.).
2.5. Batch experiments with immobilized cells
C. tropicalis immobilized onto the ceramic honeycomb
support were used to treat both wastewaters in batch exper-
iments conducted at 35

C. Samples of treated wastewater

were taken for the analysis of COD, saccharides, and organic
carboxylic acids at various time intervals. The bioreactor
was washed with sterilised tap water before each experiment
in order to remove suspended cells. In some experiments
with W1, urea at 0.35 g/L was added into the wastewater to
investigate the effect of nitrogen source on COD removal
rate during the experiment.
2.6. Continuous-ow experiment with immobilized cells
Grains-washing wastewater (W1) was treated by the
IAL-CHS in continuous-ow at 35

C and pH 6 for 88 h.
The ow rate was 3.1 mL/min.
2.7. Analytical methods
Samples assayed for COD, glucose, and organic car-
boxylic acids were centrifuged at 10,000 rpm to remove
suspended matters. COD was determined by oxidation with
potassium dichromate under strongly acidic conditions and
at an elevated temperature for 2 h [15]. Glucose was assayed
with an automatic biochemical analyzer (model: VITALAB
Selectra 2, The Netherlands). Organic carboxylic acids
were measured by titration with sodium hydroxide, because
carboxylic acids would form while maltose or glucose was
degraded. The quantity of sodium hydroxide consumed for
neutralization of carboxylic acids is regarded as the equiv-
alent carboxylic acids [16]. Cell density was measured by
spectrophotometer as absorbance at 600 nm and compared
with a standard curve to obtain dry weight.
2.8. Scanning electron micros
Scanning electron micros (SEM) was used to observe the
biomass in the ceramic support (Hitachi (Japan) S-570). The
Y. Zhang et al. / Process Biochemistry 40 (2005) 857863 859
sample of ceramic support with cells was rst removed by
cutting it out with a saw. The sample was then sequentially
dehydrated with 30, 50, 70, 85, and 95% ethanol one time
each for 20 min and then 100%ethanol twice for 20 min. The
dehydrated sample was mounted on a sample plate with an
electrically conducting paster and sputter-coated with gold
[17]. The samples were viewed with a scanning electron
microscope.
3. Results and discussion
3.1. Effect of temperature on cell growth and
COD removal
The grains-washing wastewater (W1) was treated batch-
wise with suspended cells at 30, 35, or 40

C. The pH was
controlled in the range 5.56.5. The changes of cells and
COD concentrations are shown in Figs. 1 and 2, respectively.
After 5 ml of cells were inoculated into 1500 ml of W1, it
took about 16 h for the cells to enter a logarithmic growth
phase at 30

C, but only 6 h to enter the logarithmic growth

phase at 35 and 40

C (Fig. 1). The COD was not removed

signicantly until the cells entered the logarithmic growth,
as revealed by comparing Figs. 1 and 2. Since these experi-
ments showed that the optimum temperature was 3540

C,
later experiments were all performed at 35

C.
10
100
1000
10000
0 12 18 24 30 36
T / h
C
e
l
l
/
m
g

L
-
1
Cell (30 C)
Cell (35 C)
Cell (40 C)
6
Fig. 1. Effect of temperature on suspended-cell growth during batch
incubation with W1.
0
2000
4000
6000
8000
0 6 12 18 24 30 36
T / h
C
O
D
/
m
g

L
-
1
COD (30 C)
COD (35 C)
COD (40 C)
Fig. 2. Effect of temperature on COD removal by suspended cells during
batch incubation with W1.
Ci = 7296.3e
-0.0659t
Cs = 5859.2e
-0.0134t
0
2000
4000
6000
8000
0 4 8 12 16 20 24 28
T / h
C
O
D
/
m
g

h
-
1
COD removal by immobilized cells
COD removal by suspended cells
Fig. 3. COD removal by suspended and immobilized cells in batch
experiments with W1 at 35

C, pH was controlled at approximately 6.0,

and the biomass concentration was about 2600 mg/L.
3.2. Suspended cells versus immobilized cells
In order to compare immobilized and suspended cells for
COD removal, the same biomass concentration was neces-
sary in both systems. Fig. 3 shows the COD removal results
for both systems when the biomass concentration was ap-
proximately 2600 mg/L. For the suspended cells, this corre-
sponds to the time from 12 to 36 h in Figs. 1 and 2. The
initial biomass in the immobilized cell system was about
2600 mg/L and remained roughly constant, since the cells
escaped from the ceramic support constantly to approach a
steady state. In both cases, the pH was controlled to approx-
imately 6.0 and the temperature was 35

C.
The comparison of W1 treated with suspended cells and
immobilized cells (Fig. 3) shows that the immobilized cells
reduced the COD more rapidly. The immobilized cells re-
moved COD approximately 4.9 times faster than the sus-
pended cells over the course of the experiments.
Dense biolm packing could induce strong mass-transfer
limitation and concentration gradients for the organic sub-
strate, oxygen, or both. Strong mass-transfer limitation
inside the support could slow COD-removal kinetics. The
fact that the COD-removal rate was higher for the immobi-
lized cells (Fig. 3) indicated that mass-transport resistance
was not a signicant limitation in these experiments. In
contrast, the faster rate suggests that the mass transfer rate
to the immobilized cells may have been faster, perhaps
due to direct transfer of gaseous O
2
to the biolm, a phe-
nomenon observed earlier for the biological aerated lter
[18].
3.3. The pH change during the treatment of wastewaters
without pH control
W1 and W2 were treated with immobilized cells with-
out pH control and the changes of organic acids and pH
are shown in Figs. 4 and 5. The corresponding COD val-
ues are shown in Fig. 6. Fig. 4 showed that the pH fell
steadily to below 4 over the rst 6 h as organic acids were
formed by fermentation of the carbohydrates in W1. Fig. 6
860 Y. Zhang et al. / Process Biochemistry 40 (2005) 857863
0
2
4
6
0 2 4 6 8
T / h
p
H
0
3
6
9
12
15
18

C
O
O
H
/
m
g

L
-
1
pH
COOH
Fig. 4. Changes of organic acid and pH during batch treatment of W1 at
35

C without pH control.
0
2
4
6
8
0 2 4 6
T / h
p
H
0
3
6
9
12
15

C
O
O
H
/
m
g

L
-
1
pH
COOH
Fig. 5. Changes of organic acid and pH during batch treatment of W2 at
35

C without pH control.
showed that the COD was reduced by only a small fraction
during this initial fermentation period. For W2, the pH de-
creased to approximately 5 over 6 h, and COD removal was
negligible.
The lack of COD removal was probably caused by
suppression of oxidation reactions associated with the
tricarboxylic-acid cycle. This suppression could have been
due to dissolved-oxygen limitation, low-pH inhibition, or
a combination of both. Comparison of the results in Fig. 6
with those in Fig. 3 suggested that inhibition by low pH
was the dominant cause for W1, because COD removal oc-
0
1000
2000
3000
4000
5000
0 2 4 6 8
T / h
C
O
D
/
m
g

L
-
1
COD(pH4.0) COD(pH4.5) COD(pH5.0)
COD(pH5.5) COD(pH6.0) COD(pH6.5)
COD(pH7.0)
Fig. 7. COD removal under different pH values for W1.
0
1000
2000
3000
4000
0 2 4 6 8
T / h
C
O
D
/
m
g

L
-
1
COD (W1)
COD (W2)
Fig. 6. Changes of COD during batch treatment of W1 and W2 when
pH was not regulated.
curred immediately when the pH was controlled at 5.56.5.
No similar comparison can be made for W2.
3.4. Optimal pH
The pH was controlled at different values from 4 to 7
in a series of experiments with immobilized cells treat-
ing W1. The changes of COD concentration over 8 h for
the different pH values are shown in Fig. 7, while Fig. 8
summarized the percentage removals of COD at the end
of each experiment. The percentage COD removal in the
pH range 4.56.5 was higher than at pH 4 and 7. Thus,
the optimal pH range was from 4.5 to 6.5 for immobilized
C. tropicalis.
3.5. Relationships among glucose and COD
removals and pH change
W2 was treated with immobilized cells without pH con-
trol. Fig. 9 showed the changes of COD, glucose, and pH for
the 6 h experiments. The COD was reduced in parallel to the
removal of glucose, but not as much as for glucose. Glucose
was continuously degraded completely within 4 h, while the
CODdeclined from3500 to about 1500 mg/L within 6 h. The
pH declined steadily over the rst 2 h and then stabilized at
Y. Zhang et al. / Process Biochemistry 40 (2005) 857863 861
14.8
50.5
52.4
51.2
55
50.6
38
0
20
40
60
4 4.5 5 5.5 6 6.5 7
pH
C
O
D

r
e
m
o
v
a
l
r
a
t
i
o

/

%
COD removal ratio
Fig. 8. COD removal percentages at the end of each experiment shown
in Fig. 7.
5.2 to 5.4 once the glucose was depleted. The results shown
in Fig. 9 conrmed that good COD removal was achieved
as long as the pH remained within the optimal pH range,
where the oxidation reactions of the tricarboxylic-acid cycle
functioned well.
3.6. Effect of added nitrogen source
One of the characteristics of grains-washing wastewater is
the lack of a nitrogen source [1]. Therefore, urea was added
to the wastewater as a supplementary nitrogen source during
some batch experiments with W1. The effect of urea addition
is shown in Fig. 10. The rst-order removal rate was more
than 4 times faster with added urea, which indicated that
treating high-carbohydrate waste benets from an added N
source.
The most likely explanation for how urea increased the
COD removal is that it accelerated biomass growth by
supplying the growth-limiting N source. Fig. 11 shows the
efuent biomass concentration from the IAL-CHS corre-
sponding to the COD results in Fig. 10. The efuent biomass
is hardly affected. However, this lack of difference does
not mean that urea did not stimulate more biomass growth,
since most of the biomass accumulated in the honeycomb
medium.
0
1000
2000
3000
4000
0 2 4 6
T / h
C
O
D

a
n
d
G
l
u
c
o
s
e

/
m
g

L
-
1
0
2
4
6
8
p
H
COD
Glucose
pH
Fig. 9. COD and glucose removals, along with the pH, during the batch
treatment of W2 by immobilized cells without pH control.
C =6 791.3e
-0.057t
C =6 605.1e
-0.014t
0
2000
4000
6000
8000
0 6 12 18 24 30 36
T / h
C
O
D
/
m
g

L
-
1
no added urea
added urea
Fig. 10. Effect of urea addition on the COD-removal rate in batch exper-
iments with W1. Exponential regression gives the rst-order constants in
1/h: 0.014 (r
2
= 0.97) for no added urea and 0.057 (r
2
= 0.99) for
the added urea test.
10
1000
0 4 8 12 16 20 24 28
T/ h
C
e
l
l
/
m
g

L
-
1
no added urea
added urea
Fig. 11. Efuent biomass concentrations with and without urea addition
show little difference.
3.7. Continuous treatment of wastewater
The grain-washing wastewater (W1) added with 0.33 g/L
of urea was continuously treated in IAL-CHS reactor for 8 h
hydraulic residence time. Fig. 12 showed that the COD re-
moval was 33% initially, but increased to more than 60% at
steady level after 64 h. The organic loading rate tracked the
COD removal rate and reached 15 kg COD/(m
3
day). This
is a high volumetric loading rate compared to typical values
for aerobic biolm treatment of less than 1 kg COD/(m
3
day)
for conventional loading and up to 6 kg COD/(m
3
day) for
0
3000
6000
9000
0 8 16 24 32 40 48 56 64 72 80 88 96
T / h
C
O
D

/
m
g

L
-
1
influent COD
effluent COD
Fig. 12. Cell growth and COD removal during the continuous treatment
of grain-washing wastewater with 0.33 g/L of added urea.
862 Y. Zhang et al. / Process Biochemistry 40 (2005) 857863
Fig. 13. Cells immobilized onto ceramic support as shown by SEM with
a magnication of 3000.
roughing treatment [19]. Excellent oxygen transfer, per-
haps directly from gas bubbles to the biolm, may account
for the high loading possible.
3.8. Immobilized and released C. tropicalis
Immobilization of cells prevented washout of biomass,
but also generated SCP. C. tropicalis cells immobilized
densely in the macropores of the ceramic support, as shown
in Fig. 13. Clearly, the ceramic macropores provided an
excellent microenvironment for the cells to proliferate.
Fig. 14 shows efuent biomass data from two experiments
in the sequencing-batch mode. During the sequencing batch
experiments, the IAL-CHS reactor was ushed with ster-
ile water between cycles and then run in the batch mode.
The cells proliferating in the macropores escaped into the
efuent, yielding SCP. The yield of biomass for the two
experiments averaged about 0.26 g cells/g COD by the end
of both experiments in Fig. 14. Although biomass retention
0
2000
4000
6000
8000
0 4 8 12 16 20 24 28
T / h
C
O
D

/
m
g

L
-
1
0
400
800
1200
1600
C
O
D

/
m
g

L
-
1
COD-1 COD-2
Cells-1 Cells-2
Fig. 14. Relationship between cell yield and COD removal during two
sequencing-batch experiments.
was dense in the macropores, the IAL-CHS produced a
signicant yield of SCP.
4. Conclusions
The airlift-loop reactor containing porous ceramic sup-
ports can be used for the treatment of high-carbohydrate
wastewater by C. tropicalis with the concurrent production
of SCP. The optimum temperature for C. tropicalis was
3540

C. The airlift reactor with ceramic honeycomb sup-

ports allowed a large and dense accumulation of biomass and
relatively high reaction rates. Mass-transport resistance and
concentration gradients apparently did not limit the overall
COD-removal kinetics, and the oxygen mass transfer may be
accelerated through direct transfer to the biolm. The volu-
metric loading rate was as high as 15 kg COD/(m
3
day), sup-
porting that the IAL-CHS has outstanding oxygen-transfer
kinetics. Supplying urea as a nitrogen source signi-
cantly increased the COD removal from grain-washing
wastewater.
The pH of the medium was a critical environmental
parameter governing the activities of cells biodegrading
glucose or complex carbohydrates during the treatment of
high-carbohydrate wastewater. Fermentation of carbohy-
drates to organic acids lowered the pH below the optimum
range of 4.56.5, and pH below 4.55 retarded or stopped
the oxidation reactions for the organic acids. Organic-acid
oxidation is necessary for COD removal, therefore, pH
control may be essential for good treatment in some cases
with high-carbohydrate wastewaters.
In summary, the IAL-CHS provided good biolm reten-
tion, oxygen mass transfer, and volumetric loading, while
also producing SCP from high-carbohydrate wastewater.
Acknowledgements
The one of authors, Dr. Yongming Zhang acknowledges
the nancial support of Visiting Scholar Foundation of Key
Laboratory at Tsinghua University and Open Project Pro-
gram of State Key Laboratory of Bioreactor Engineering
at East China University of Science and Technology. This
project was also nanced by the National Natural Science
Foundation of China to Professor Jianlong Wang (Grant No.
29637010).
References
[1] Zhang Z. Handbook of environmental engineeringvolume of water
pollution prevention. Beijing: Chinese Higher Education Press; 1996.
p. 91363.
[2] Kumar V, Wati L, Nigam P, Banat IM, Yadav BS, Singh D, et al. De-
colorization and biodegradatioin of anaerobically digested sugarcane
molasses spent ash efuent from biomethanation plants by white rot
fungi. Process Bio Cycle 1998;39:6472.
Y. Zhang et al. / Process Biochemistry 40 (2005) 857863 863
[3] Pawar NJ, Pondhe GM, Patil SF. Groundwater pollution due to
sugar-mill efuent, at Sonai, Maharashtra. India Environ Geol
1998;34:15160.
[4] Forster CF. Biotechnology and wastewater treatment. Cambridge:
Cambridge University Press; 1985. p. 22031.
[5] Zhang Y, Han L, Wang J, Shi H, Qian Y. An internal airlift
loop bioreactor with Burkholderia pickttii immobilized onto ceramic
honeycomb support for degradation of quinoline. Biochem Eng J
2002;11:14957.
[6] Wang J, Quan X, Han L, Qian Y, Werner H. Microbial degradation
of quinoline by immobilized cells of Burkholderia pickettii. Water
Res 2002;36:28896.
[7] Wang J, Qian Y. Wastewater treatment by a hybrid biological reactor
(HBR): effect of loading rates. Process Biochem 2000;36:297303.
[8] Wang J, Qian Y. Microbial degradation of 4-chlorophenol by mi-
croorganisms entrapped in carrageenan-chitosan gels. Chemosphere
1999;38:310917.
[9] Wang J, Hou W, Qian Y. Immobilization of microbial cells us-
ing polyvinyl alcohol (PVA)-polyacrylamide gels. Biotechnol Tech
1995;10:2038.
[10] Chen J. Environmental biological technology. Beijing: Chinese Light
Industry Press; 1999. p. 22432.
[11] Aloice WM. Effects of temperature and pH on the kinetic growth
of unialga Chlorella vulgaris cultures containing bacteria. Water
Environ Res 1997;69:6472.
[12] Levenspiel O. Chemical reaction engineering. 2nd ed. New York:
Wiley; 1972.
[13] Zhang Y, Yu J, Wang J, Shi H, Qian Y. Hydraulic property of
internal airlift loop bioreactor with cells immobilized onto ceramic
honeycomb support. Chin J Environ Sci 2001;22:536 [in Chinese].
[14] Atkinson B, Mavituna F. Biochemical engineering and biotechnology
handbook. 2nd ed. Macmillan Publishers Ltd.; 1999.
[15] Xi D, Sun Y, Liu X. Environmental monitoring. 2nd ed. Beijing:
High Education Press; 1995. p. 7881.
[16] Jin S. Course of Organic Chemistry Analysis. Beijing: Higher Edu-
cation Press; 1992. p. 2578.
[17] Yu Y, Wu G, Meng X. Inspection handbook of environmental engi-
neering microbiology. Beijing: Chinese Environmental Science Press;
1990. p. 518.
[18] Lee KM, Stensel HD. Aeration and substrate utilization in a
sparged packed-bed biolm reactor. J Water Pollut Control Fedn
1986;58:106672.
[19] Rittmann BE, McCarty PL. Environmental biotechnology: principles
and application. New York: McGraw-Hill Book Co.; 2001.