Bioresource Technology 100 (2009) 3613–3617

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

Optimization of culture conditions for production of yeast biomass

using bamboo wastewater by response surface methodology
Xin Li *, Jia Ouyang, Yong Xu, Mu Chen, Xiangyang Song, Qiang Yong, Shiyuan Yu
Key Laboratory of Forest Genetics and Biotechnology, Ministry of Education, Nanjing Forestry University, Nanjing, Jiangsu Province 210037, China

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

Article history: Response surface methodology (RSM) was applied to optimize culture conditions for the growth of Can-
Received 16 December 2008 dida utilis with bamboo wastewater. A significant influence of initial pH, fermentation time and yeast
Received in revised form 26 February 2009 extract on biomass of C. utilis was evaluated by Plackett–Burman design (PBD). These factors were further
Accepted 1 March 2009
optimized using a central composite design (CCD) and RSM. A combination of initial pH 6.1, fermentation
Available online 1 April 2009
time 69 h and yeast extract 1.17 g/L was optimum for maximum biomass of C. utilis. A 1.7-fold enhance-
ment of biomass of C. utilis was gained after optimization in shake-flask cultivation. The biomass of
Keywords:
C. utilis reached 19.17 g/L in 3 L fermentor.
Bamboo wastewater
Candida utilis
Ó 2009 Elsevier Ltd. All rights reserved.
Plackett–Burman design
Response surface methodology

1. Introduction
Bamboo biomass, defined as a renewable resource, was re-
corded 4.2 million ha of bamboo forests and plantations, and 3 mil-
lion ha of ‘‘mixed and mountain natural bamboo stands” (Mertens
et al., 2008), occupying 3% of total forest area in China (Scurlock
et al., 2000). Bamboo biomass has been used in the field of paper,
textile, food and reinforcing fibers due to its dual characteristics
of timber-oriented forests and edible-vegetable trees (Mertens
et al., 2008). In the field of paper, the bamboo pulping and paper-
making generate a considerable amount of wastewater character-
ized by biochemical oxygen demand and chemical oxygen
demand. Therefore, there is a need to develop a rapid, economical
and environmental method to treat with that wastewater. Contain-
ing oligosaccharide, monosaccharide, etc., the wastewater could be
applied for production of single cell protein.
C. utilis is mainly used in production of yeast biomass or single
cell protein because of its ability to utilize a variety of carbon
sources, such as rice polishings (Rajoka et al., 2006), potato starch
waste waters (Gélinas and Barrette, 2007), salad oil manufacturing
wastewater (Zheng et al., 2005) and molasses (Nigam and Vogel,
1991). And it has also been used as a host to produce several chem-
icals, such as glutathione (Liang et al., 2008), monellin (Kondo
et al., 1997) and ethyl acetate (Christen et al., 1999).
The variables for production of C. utilis biomass included
fermentation time, pH value, temperature, inoculum, potassium
dihydrogen phosphate, ammonium sulfate, calcium chloride, mag-
* Corresponding author. Fax: +86 25 85418873.
E-mail address: xin.lee@163.com (X. Li).
nesium sulfate and yeast extract (Rajoka et al., 2004; Tobajas and
Garcia-Calvo, 1999). Optimization of culture conditions has been
used in enhancing the biomass of many microorganisms. However,
there is no literature reported to optimize the culture conditions
for C. utilis in bamboo wastewater.
In the present work, a Plackett–Burman design (PBD) followed
by a central composite design (CCD) of response surface methodol-
ogy (RSM) has been used to optimize the culture conditions for C.
utilis in bamboo wastewater.
2. Methods
2.1. Strain and enzyme
C. utilis (CICC 1807) was purchased from China Center of Indus-
trial Culture Collection (CICC). It was maintained on slants of the
agar medium (w/v): glucose 2%, peptone 1%, yeast extract 0.5%
and agar 2% and kept at 4 °C.
Cellulase from Trichoderma reesei ATCC 26921 and Novozyme
188 were purchased from Sigma and kept at 4 °C. The cellulase
activity and b-glucosidase activity were measured according to
Ghose (1987).
2.2. Pretreatment of bamboo wastewater
Bamboo wastewater was a gift from Yaan Zhongzhu Paper Co.,
Ltd. (Sichuan Province, China). Enzymatic hydrolysis of bamboo
wastewater was performed with the commercial enzyme solutions
(Cellulase and Novozyme). The hydrolysis was performed in an

0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2009.03.001
3614 X. Li et al. / Bioresource Technology 100 (2009) 3613–3617

Erlenmeyer flash (250 mL) containing 100 mL bamboo wastewater
at 50 °C for 48 h using a cellulase loading of 30 FPU/g reducing su-
gar and a Novozyme loading of 60 IU/g reducing sugar.
The hydrolyzed wastewater pH was adjusted to the designed
value with commercial calcium oxide in a 100 °C water bath and
keeping for 5 min. The treated wastewater was centrifuged at
4000 rpm for 30 min and the supernatant was used as carbon
source for culturing C. utilis.
2.3. Culture conditions
experimental design used for study is shown in Table 6. The bio-
mass of C. utilis was fitted using a second-order polynomial equa-
tion and a multiple regression of the data was carried out for
obtaining an empirical model related to the most significant fac-
tors. The general form of the second-order polynomial equation is
X X X
Y ¼ b0 þ bi xi þ bii x2i þ bij xi xj ð2Þ
where Y is the predicted response, xi and xj are independent factors,
b0 is the intercept, bi is the linear coefficient, bii is the quadratic
coefficient, and bij is the interaction coefficient.

The seed was prepared by transferring a loopful of cells to 2.5. Data analysis and software
50 mL seed culture medium containing (w/v): glucose 2%, peptone
1%, yeast extract 0.5% and grown at 30 °C on a rotary shaker incu- Design-Expert, Version 7.0 (STAT-EASE Inc., Minneapolis, USA)
bator at 150 rpm for 24 h. was used for the experimental designs and statistical analysis of
The seed was transferred into an Erlenmeyer flash (250 mL) the experimental data. The analysis of variance (ANOVA) was used
containing 50 mL of fermentation medium. The basal culture con- to estimate the statistical parameters.
ditions were initial pH, 7.0; fermentation time, 48 h; temperature,
30 °C; inoculum, 7.5%(v/v); KH2PO4, 5.0 g/L; (NH4)2SO4, 5.0 g/L; 2.6. Analytical methods
CaCl2, 0.13 g/L; MgSO4, 0.5 g/L; yeast extract, 0.5 g/L. The values
of culture conditions were varied based on the experimental de- According to laboratory analytical procedure (LAP) of National
sign. The rotary speed of shaker incubator was fixed to 150 rpm. Renewable Energy Laboratory (NREL), the concentrations of D-glu-
All experiments were performed in duplicate and results repre- cose, D-xylose, L-arabinose, acetic acid, formic acid and 5-hydroxy-
sented the mean values of two independent experiments. 2-furaldehyde (HMF) and furfural in bamboo wastewater were
determined using a Agilent high-performance liquid chromatogra-
2.4. Experimental designs phy (HPLC) with a refractive index (RI) detector and Bio-Rad Amin-
ex HPX-87H column (300  7.8 mm) at 55 °C (Sluiter et al., 2006).
2.4.1. Plackett–Burman design The eluent was 0.005 M H2SO4 at a flow rate of 0.6 mL/min. The in-
The independent variables of the culture conditions were initial jected volume of a sample was 10 lL. After diluted, the samples
pH, fermentation time, temperature, inoculum, potassium dihy- were filtered through a 0.22 lm membrane.
drogen phosphate, ammonium sulfate, calcium chloride, magne- The concentration of reducing sugar was determined by the
sium sulfate and yeast extract according to Rajoka et al. (2004) dinitrosalicylic acid (DNS) method with glucose as standard (Mill-
and Tobajas and Garcia-Calvo (1999). A Plackett–Burman design er, 1959).
is used for rapid screening multifactor to find the most significant Biomass of C. utilis was measured by dry cell weight of the har-
independent factors (Li et al., 2008a, b; Plackett and Burman, 1946; vested cells. The yeast biomass was collected following centrifuga-
Wang et al., 2007; Xiao et al., 2007). Then the nine factors were tion at 4000 rpm for 30 min. Dry yeast biomass was determined
investigated using the Plackett–Burman design with a first-order after the harvested cells were dried at 105 °C to a constant weight.
polynomial equation. Each factor was tested at low (1) and high
(+1) levels. Eleven variables (including two dummy variables) were 3. Results and discussions
screened in 12 experimental runs (Table 2). The fitted first-order
model is 3.1. Composition of bamboo wastewater
X
Y ¼ b0 þ b i xi ð1Þ
The main composition of the bamboo wastewater and hydroly-
Y is the predicted response, b0 and bi are constant coefficients, zate is shown in Table 1. Compared the composition between
and xi is the coded independent factors. wastewater and hydrolyzate, the hydrolyzate was rich in glucose
and xylose, and was reasonably used as a substrate for yeast cul-
2.4.2. Path of steepest ascent ture. However, the wastewater and the hydrolyzate both contained
The method of steepest ascent is a procedure for moving along higher concentrations of formic acid and acetic acid. And there
the direction of the maximum increase in the response (Chen et al., were fewer levulinic acid, HMF and furfural in the hydrolyzate.
2009; He and Tan, 2006). The direction of steepest ascent is the According to Larsson et al. (1999), the fermentation of Saccharomy-
direction in which biomass increased rapidly by increasing or ces cerevisiae was inhibited because of exiting acetic acid, formic
decreasing the condition of the significant factors. The factors acid, levulinic acid, furfural and HMF. Therefore, the hydrolyzate
screened by the Plackett–Burman design were further optimized was treated with commercial calcium oxide referred to Almeida
by this method. The path of steepest ascent was initiated from e Silva et al. (2003).
the center of the Plackett–Burman design. Experiments were per-
formed along the steepest ascent path until the response did not
increase any more. The experimental design and results of the Table 1
steepest ascent method are shown in Table 4. The main compositions of bamboo wastewater and hydrolyzate (g/L).
Item Cellobiose Glucose Xylose Arabinose Xylitol glycerol
2.4.3. Central composite designs and response surface analysis Wastewater 0.68 2.94 3.47 1.01 0.38 0.89
A CCD of RSM was employed to optimize the three most signif- Hydrolyzate 2.78 15.58 21.64 1.20 0.37 0.78
icant factors (initial pH, fermentation time, yeast extract) for
Item Formic acid Acetic acid Levulinic acid HMF Furfural
enhancing biomass of C. utilis, screened by Plackett–Burman de-
sign. The three independent factors were investigated at five differ- Wastewater 2.02 8.84 0.74 0.65 0.66
Hydrolyzate 2.17 9.86 0.23 0.65 0.56
ent levels (1.682, 1, 0, +1, +1.682) (Table 5) and the
 X. Li et al. / Bioresource Technology 100 (2009) 3613–3617 3615

3.2. Plackett–Burman design Table 5

Levels of the factors tested in the central composite design.

The Plackett–Burman design is a powerful method for screening Factor Levels of factors
significant factors. Twelve runs were carried out to analyze the ef- 1.682 1 0 +1 +1.682
fect of 11 variables (including two dummy variables) on yeast bio-
Initial pH (A) 5.49 5.7 6.0 6.3 6.50
mass production and the results are demonstrated in Table 2. Fermentation time (B, h) 45.82 54 66 78 86.18
Analyzed by Design-Expert, a first-order model was fitted to the re- Yeast extract (C, g/L) 0.76 0.9 1.1 1.3 1.44
sults obtained from the twelve experiments:
Yðg=LÞ ¼ 6:11 þ 1:40X 1 þ 1:27X 2  0:60X 3 þ 0:21X 4
3.3. Path of steepest ascent
 0:10X 5 þ 0:33X 6  0:42X 7  0:082X 8 þ 0:70X 9 ð3Þ
The coefficient R2 of the first-order model was 0.9896, indicat- Based on the results obtained from the Plackett–Burman design,
ing that nearly 99% of the variability in the response could be ex- initial pH, fermentation time and yeast extract were the major fac-
plained by the model. The t-test was used to identify the effect tors influenced the biomass of C. utilis. The path of steepest ascent
of every factor on yeast biomass. Table 3 shows that initial pH, fer- was determined to find the proper direction of changing variables
mentation time and yeast extract are the most significant factors by increasing or decreasing the value of the main factors. The path
(P < 0.05). And then, initial pH, fermentation time and yeast extract of steepest ascent was based on the center of the Plackett–Burman
were selected for further optimization to obtain a maximum design and moved along the path in which initial pH, fermentation
response. time and yeast extract increased. The experimental design and re-

Table 2
Plackett–Burman design for screening of significant factors affecting biomass of Candida utilis*.

Run X1 X2 (h) X3 (°C) X4 (%(v/v)) X5 (g/L) X6 (g/L) X7 (g/L) X8 (g/L) X9 (g/L) X10 X11 Biomass (g/L)
1 4 24 25 10 3 7.5 0.16 0.25 0.7 1 1 5.27
2 4 72 35 10 3 2.5 0.1 0.75 0.3 1 1 5.21
3 4 72 25 10 7 2.5 0.16 0.75 0.7 1 1 6.34
4 6 72 25 10 7 7.5 0.1 0.25 0.3 1 1 9.85
5 4 24 35 5 7 7.5 0.1 0.75 0.7 1 1 4.11
6 6 24 35 10 3 7.5 0.16 0.75 0.3 1 1 4.85
7 6 72 35 5 3 2.5 0.16 0.25 0.7 1 1 8.33
8 6 72 25 5 3 7.5 0.1 0.75 0.7 1 1 10.42
9 6 24 35 10 7 2.5 0.1 0.25 0.7 1 1 6.39
10 6 24 25 5 7 2.5 0.16 0.75 0.3 1 1 5.22
11 4 24 25 5 3 2.5 0.1 0.25 0.3 1 1 3.16
12 4 72 35 5 7 7.5 0.16 0.25 0.3 1 1 4.13
*
X1-initial pH; X2-fermentation time (h); X3-temperature (°C); X4-inoculum (%(v/v)); X5-KH2PO4 (g/L); X6-(NH4)2SO4 (g/L); X7-CaCl2 (g/L); X8-MgSO4 (g/L); X9-yeast extract (g/
L); X10, X11-dummy variables.

Table 3
Statistical analysis of the model.

Source Sum of squares Degrees of freedom Mean square F-value Prob > F
Model 57.53 9 6.39 21.05 0.0462*
Initial pH 23.63 1 23.63 77.80 0.0126*
Fermentation time 19.46 1 19.46 64.05 0.0153*
Temperature 4.37 1 4.37 14.38 0.0630
Inoculum 0.54 1 0.54 1.77 0.3148
KH2PO4 0.12 1 0.12 0.40 0.5939
(NH4)2SO4 1.32 1 1.32 4.35 0.1725
CaCl2 2.08 1 2.08 6.86 0.1201
MgSO4 0.08 1 0.08 0.26 0.6588
Yeast extract 5.94 1 5.94 19.54 0.0475*
*
Statistically significant at 95% of confidence level.

Table 4
Experiment design and results of the steepest ascent path.

Run Initial pH Fermentation time (h) Yeast extract (g/L) Biomass (g/L)
Origin 5 48 0.5 8.48
1 5.3 54 0.7 10.23
2 5.6 60 0.9 11.31
3 5.9 66 1.1 14.73
4 6.2 72 1.3 12.07
5 6.5 78 1.5 10.84
6 6.8 84 1.7 9.54
7 7.1 90 1.9 7.56
3616 X. Li et al. / Bioresource Technology 100 (2009) 3613–3617

Table 6
The central composite design of RSM for optimization of biomass production of Candida utilis.

Run A (pH) B (Fermentation time) C (Yeast extract) Biomass (g/L)

Observed Predicted
1 1 1 1 9.18 9.30
2 1 1 1 10.68 11.08
3 1 1 1 9.46 9.87
4 1 1 1 10.73 11.13
5 1 1 1 10.36 10.62
6 1 1 1 11.89 12.19
7 1 1 1 12.02 12.30
8 1 1 1 12.75 13.26
9 1.682 0 0 10.96 10.61
10 1.682 0 0 13.62 12.95
11 0 1.682 0 11.43 11.09
12 0 1.682 0 13.21 12.53
13 0 0 1.682 9.63 9.18
14 0 0 1.682 12.75 12.19
15 0 0 0 14.83 14.49
16 0 0 0 14.64 14.49
17 0 0 0 14.44 14.49
18 0 0 0 14.48 14.49
19 0 0 0 13.84 14.49
20 0 0 0 14.56 14.49

sults are shown in Table 4. It is shown that the highest response reliable. The lack of fit F-value of 4.96 implied that there was a
was 14.73 g/L when the significant factors were: initial pH 5.9, fer- 5.17% chance that the lack of fit F-value could occur due to noise.
mentation time 66 h and yeast extract 1.1 g/L. It suggested that The response surface curves are plotted to explain the interac-
this point was near the optimal point and was chosen for further tion of the variables and to determine the optimum level of each
optimization. variable for maximum response. The response surface curves are
shown in Figs. 1–3. Each figure demonstrates the effect of two fac-
tors while the other factors were fixed at zero level. The model pre-
3.4. Central composite designs and response surface analysis
dicted the optimal values (coded) of the three most significant
variables were X1 = 0.33, X2 = 0.25 and X9 = 0.35. Correspondingly,
A response surface design is further applied when the optimal
the values of initial pH, fermentation time and yeast extract were
region for running the process has been identified (Li et al., 2007;
6.1, 69 h and 1.17 g/L, respectively. The maximum predicted bio-
Wang et al., 2007; Xiao et al., 2007). Based on the Plackett–Burman
mass of C. utilis was 14.82 g/L. By optimization of culture condi-
design and the path of steepest ascent, RSM using CCD was applied
tions, biomass of C. utilis was enhanced from 8.48 to 14.82 g/L.
to determine the optimal levels of the three selected variables (ini-
tial pH, fermentation time and yeast extract) which significantly
3.5. Validation of the optimized condition
influenced the growth of C. utilis. The respective low and high levels
with the coded levels for the three variables are defined in Table 5.
In order to confirm the optimized culture conditions, three
A total of 20 runs with different combination of initial pH (A), fer-
additional experiments in shake flasks were performed using the
mentation time (B) and yeast extract (C) was designed in Table 6.
predicted culture conditions. The mean value of yeast biomass
The observed and predicted responses of the twenty experiments
was 14.67 g/L, which agreed well with the predicted value. This re-
are also presented in Table 6. The experimental results were ana-
sult demonstrates the validity of the response model. An additional
lyzed by standard ANOVA and the CCD was fitted with the sec-
batch experiment was carried to obtain 17.19 g/L C. utilis biomass
ond-order polynomial equation:

Yðg=LÞ ¼ 14:49 þ 0:70A þ 0:43B þ 0:89C  0:13AB

 0:064AC þ 0:27BC  0:96A2  0:95B2  1:35C 2 ð4Þ
where A, B and C are the coded factors of initial pH, fermentation
time and yeast extract, respectively.
The fit of the model was checked by the coefficient of determi-
nation R2, which was calculated to be 0.9507, indicating that
95.07% of the variability in the response could be explained by
the model. The statistical significance of the model equation was
evaluated by the F-test for ANOVA. The model F-value of 21.41 im-
plied the model was significant. There was only a 0.01% chance
that the model F-value could occur due to noise. The P-value was
also very low (P < 0.0001) indicating the significance of the model.
Values of ‘‘Prob > F” less than 0.05 indicated that model terms were
significant. In this case, six model terms (A, B, C, A2, B2, C2) were sig-
nificant. The coefficient of variation (CV) indicates the degree of
precision with which the treatments are compared. A lower CV
means a higher reliability of the experiment. The lower value of Fig. 1. Response surface curve for biomass of Candida utilis showing the interaction
CV (4.73%) demonstrated the performed experiments were highly between initial pH and fermentation time at C = 0.
 X. Li et al. / Bioresource Technology 100 (2009) 3613–3617
Fig. 2. Response surface curve for biomass of Candida utilis showing the interaction
between initial pH and yeast extract concentration at B = 0.
Fig. 3. Response surface curve for biomass of Candida utilis showing the interaction
between fermentation time and yeast extract concentration at A = 0.
in 3 L fermentor under the optimized conditions. The residual com-
position of the hydrolyzed bamboo wastewater was (g/L): cellobi-
ose, 0.51; glucose, 0.74; xylose, 1.25; arabinose, 0.79; xylitol, not
detected; glycerol, 0.73; formic acid, 1.75; acetic acid, 0.39; levu-
linic acid, 0.21; HMF, 0.11; furfural, not detected. The optimal cul-
ture conditions by RSM were: initial pH 6.1, fermentation time
69 h, temperature 30 °C, inoculum 7.5%(v/v), potassium dihydro-
gen phosphate 5 g/L, ammonium sulfate 5 g/L, calcium chloride
0.13 g/L, magnesium sulfate 0.5 g/L and yeast extract 1.17 g/L.
4. Conclusion
Plackett–Burman and RSM designs have been proved to be
effective in optimizing biomass of C. utilis using bamboo wastewa-
ter. The final composition of the optimized culture conditions was
as follows: initial pH 6.1, fermentation time 69 h, temperature
30 °C, inoculum 7.5%(v/v), potassium dihydrogen phosphate 5 g/
L, ammonium sulfate 5 g/L, calcium chloride 0.13 g/L, magnesium
sulfate 0.5 g/L and yeast extract 1.17 g/L, which resulted in an over-
all 1.7-fold increase compared with that using the original condi-
tions in shake-flask cultivation. Thus, such a C. utilis strain may
have highly potential application for industrial production of yeast
biomass using bamboo wastewater.
3617
Acknowledgements
This work was supported by the Natural Science Foundation of
the Jiangsu Higher Education Institutions of China (08KJB220002),
the Major Basic R&D Program of China (2007CB707801) and Hi-
Tech R&D Program of China (2007AA02Z213 and 2006AA020204).
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