Process Biochemistry 40 (2005) 3025–3030

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Optimization of critical medium components using response

surface methodology for ethanol production from cellulosic
biomass by Clostridium thermocellum SS19
Ramesh Balusu, Ramamohan R. Paduru, S.K. Kuravi, G. Seenayya, G. Reddy *
Department of Microbiology, Osmania University, Hyderabad 500007, A.P., India
Received 21 September 2004; received in revised form 1 January 2005; accepted 14 February 2005

Abstract

The optimization of critical medium components for the production of ethanol from cellulose by Clostridium thermocellum SS19 in
anaerobic submerged fermentation was carried out using response surface methodology (RSM) based on central composite rotatable design
(CCRD). The design contains a total of 54 experimental trials with the first 32 organized in a fractional factorial design and from 33 to 40 and
51 to 54 involving the replications of the central points. The design was employed by selecting filter paper, corn steep liquor, cysteine
hydrochloride, magnesium chloride and ferrous sulphate as model factors. Among the five independent variables studied, all the nutrients
were found significant, except magnesium chloride. The concentrations of filter paper, corn steep liquor, cysteine hydrochloride and
ferrous sulphate in the medium, which have been found to be optimal for ethanol production, were 45, 8.0, 0.25, and 0.01 g/l, respectively.
The organism produced 0.41 g of ethanol/g of the substrate consumed (81% yield efficiency) in the nutritionally optimized medium.
The present study provides valuable information about the statistical optimization of media components for ethanol production from
cellulosic biomass.
# 2005 Elsevier Ltd. All rights reserved.

Keywords: Clostridium thermocellum; Cellulosic biomass; Response surface methodology; Ethanol; Anaerobic fermentation

1. Introduction the direct bioconversion of cellulose into ethanol by a

Over the last century, energy consumption has increased
progressively as the result of growing world population and
industrialization [1]. Ethanol is a renewable energy source
produced through fermentation of sugars unlike the fossil
fuels. Interest in the bioconversion of abundant and
renewable cellulosic biomass into fuel ethanol as an
alternative to petroleum is rising around the world owing to
the realization of diminishing natural oil and gas resources
[1–5]. Conventional techniques to achieve this bioconver-
sion include acid or enzyme hydrolysis of cellulose
followed by fermentation of the resulting soluble sugars
into ethanol [6]. A better alternative to this multiple step
process, from an economical and technical point of view, is
* Corresponding author. Tel.: +91 40 7682246; fax: +91 40 7098003.
E-mail address: gopalred@hotmail.com (G. Reddy).
bacterium such as Clostridium thermocellum [7,8]. C.
thermocellum is a cellulolytic, ethanologenic, thermophilic
and anaerobic bacterium that produces ethanol, acetic acid,
small amounts of lactic acid, CO2 and H2 as the
fermentation end products [8,9]. Wild strains of C.
thermocellum so far reported produced 0.08–0.37 g of
ethanol/g of glucose equivalents fermented [10–16]. With
this in mind, a wild strain of C. thermocellum SS19 [16] was
isolated and used in further studies for increased ethanol
production.
Selection of appropriate carbon, nitrogen and other
nutrients is one of the most critical stages in the
development of an efficient and economic bioprocess
[17,18]. Classical and statistical methodologies are
available for screening nutrients in bioprocess optimiza-
tion studies. Statistical methodologies involve use of
mathematical models for designing fermentation processes

1359-5113/$ – see front matter # 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2005.02.003
3026 R. Balusu et al. / Process Biochemistry 40 (2005) 3025–3030

and analyzing the process results [19–22]. There are Table 1

various advantages in using statistical methodologies in Central composite rotatable design consisting of 54 experiments for the
optimization of five nutrients (each at five levels) for production of ethanol
terms of rapid and reliable short listing of nutrients, from cellulose by C. thermocellum SS19 in anaerobic submerged fermenta-
understanding interactions among the nutrients at varying tion
concentrations and a tremendous reduction in total number C. no.a Coded values and real valuesb
of experiments, resulting in saving time, glassware,
Factor X1 Factor X2 Factor X3 Factor X4 Factor X5
chemicals and manpower [23–25]. Thus, the statistical
1 1 (20.0) 1 (4.0) 1 (0.2) 1 (0.02) 1 (1.0)
approach for optimization of media effectively tackles the
2 1 (40.0) 1 (4.0) 1 (0.2) 1 (0.02) 1 (1.0)
problem of cumbersomeness of classical designs. In spite 3 1 (20.0) 1 (8.0) 1 (0.2) 1 (0.02) 1 (1.0)
of various advantages, the statistical designs have been 4 1 (40.0) 1 (8.0) 1 (0.2) 1 (0.02) 1 (1.0)
applied to only a limited number of aerobic submerged and 5 1 (20.0) 1 (4.0) 1 (0.4) 1 (0.02) 1 (1.0)
solid-state fermentation [18,20–26] and anaerobic sub- 6 1 (40.0) 1 (4.0) 1 (0.4) 1 (0.02) 1 (1.0)
7 1 (20.0) 1 (8.0) 1 (0.4) 1 (0.02) 1 (1.0)
merged fermentation processes [17]. In the present study,
8 1 (40.0) 1 (8.0) 1 (0.4) 1 (0.02) 1 (1.0)
we report the optimization of concentrations of five 9 1 (20.0) 1 (4.0) 1 (0.2) 1 (0.04) 1 (1.0)
significant nutrients [17] using response surface metho- 10 1 (40.0) 1 (4.0) 1 (0.2) 1 (0.04) 1 (1.0)
dology (RSM), a central composite rotatable design 11 1 (20.0) 1 (8.0) 1 (0.2) 1 (0.04) 1 (1.0)
(CCRD), for the production of ethanol from cellulosic 12 1 (40.0) 1 (8.0) 1 (0.2) 1 (0.04) 1 (1.0)
13 1 (20.0) 1 (4.0) 1 (0.4) 1 (0.04) 1 (1.0)
biomass by C. thermocellum SS19 in anaerobic submerged
14 1 (40.0) 1 (4.0) 1 (0.4) 1 (0.04) 1 (1.0)
fermentation. 15 1 (20.0) 1 (8.0) 1 (0.4) 1 (0.04) 1 (1.0)
16 1 (40.0) 1 (8.0) 1 (0.4) 1 (0.04) 1 (1.0)
17 1 (20.0) 1 (4.0) 1 (0.2) 1 (0.02) 1 (2.0)
2. Materials and methods 18 1 (40.0) 1 (4.0) 1 (0.2) 1 (0.02) 1 (2.0)
19 1 (20.0) 1 (8.0) 1 (0.2) 1 (0.02) 1 (2.0)
20 1 (40.0) 1 (8.0) 1 (0.2) 1 (0.02) 1 (2.0)
2.1. Microorganism and culture conditions 21 1 (20.0) 1 (4.0) 1 (0.4) 1 (0.02) 1 (2.0)
22 1 (40.0) 1 (4.0) 1 (0.4) 1 (0.02) 1 (2.0)
The bacterial strain C. thermocellum SS19 was isolated 23 1 (20.0) 1 (8.0) 1 (0.4) 1 (0.02) 1 (2.0)
from goat fecal droppings using CMS medium [16]. 24 1 (40.0) 1 (8.0) 1 (0.4) 1 (0.02) 1 (2.0)
25 1 (20.0) 1 (4.0) 1 (0.2) 1 (0.04) 1 (2.0)
Fermentation studies were carried out in 120 ml serum
26 1 (40.0) 1 (4.0) 1 (0.2) 1 (0.04) 1 (2.0)
vials containing 20 ml of pre-reduced CMS medium with 27 1 (20.0) 1 (8.0) 1 (0.2) 1 (0.04) 1 (2.0)
appropriate concentrations of nutrients under study. In all 28 1(40.0) 1 (8.0) 1 (0.2) 1 (0.04) 1 (2.0)
experiments, a 5% (v/v) inoculum, grown on 0.4% cellulose 29 1 (20.0) 1 (4.0) 1 (0.4) 1 (0.04) 1 (2.0)
for 72 h in CMS medium was added and incubated 30 1 (40.0) 1 (4.0) 1 (0.4) 1 (0.04) 1 (2.0)
31 1 (20.0) 1 (8.0) 1 (0.4) 1 (0.04) 1 (2.0)
anaerobically at 60 8C for 5 days.
32 1 (40.0) 1 (8.0) 1 (0.4) 1 (0.04) 1 (2.0)
Five critical nutrients (g/l) filter paper 10–50, corn steep 33–40 0 (30.0) 0 (6.0) 0 (0.3) 0 (0.03) 0 (1.5)
liquor 2–10, cysteine HCl 0.1–0.5, FeSO4
7H2O 0.01–0.05, 41 2 (10.0) 0 (6.0) 0 (0.3) 0 (0.03) 0 (1.5)
and MgCl2
6H2O 0.5–2.5 with 30, 6.0, 0.3, 0.03 and 1.5, 42 2 (50.0) 0 (6.0) 0 (0.3) 0 (0.03) 0 (1.5)
respectively, as their central points were studied. The 43 0 (30.0) 2 (2.0) 0 (0.3) 0 (0.03) 0 (1.5)
44 0 (30.0) 2 (10.0) 0 (0.3) 0 (0.03) 0 (1.5)
concentration range for each nutrient was fixed based on the
45 0 (30.0) 0 (6.0) 2 (0.1) 0 (0.03) 0 (1.5)
literature and experience gained. The medium prepared and 46 0 (30.0) 0 (6.0) 2 (0.5) 0 (0.03) 0 (1.5)
used was essentially the same as described earlier [17], 47 0 (30.0) 0 (6.0) 0 (0.3) 2 (0.01) 0 (1.5)
except that the nutrients under study were added at varying 48 0 (30.0) 0 (6.0) 0 (0.3) 2 (0.05) 0 (1.5)
concentrations as given in Table 1. 49 0 (30.0) 0 (6.0) 0 (0.3) 0 (0.03) 2 (0.5)
50 0 (30.0) 0 (6.0) 0 (0.3) 0 (0.03) 2 (2.5)
51–54 0 (30.0) 0 (6.0) 0 (0.3) 0 (0.03) 0 (1.5)
2.2. Estimation of growth and fermentation products a
Combination number.
b
Real values (given in parentheses) are in g/l; Factors X1, X2, X3, X4 and
Cell growth was determined by measuring the absor- X5 are filter paper, corn steep liquor, cysteine HCl, ferrous sulphate and
bance of culture broth at 660 nm [26]. For the estimation of magnesium chloride, respectively.
ethanol and acetic acid, 10 ml of fermented broth was
centrifuged at 10,000  g for 30 min at 4 8C. The 2.3. Experimental design and statistical analysis
supernatant solution was acidified with 1 ml of 2N
phosphoric acid and 2 ml was injected into a Chromosorb The RSM used in the present study is a central composite
101 column in a CIC gas chromatograph equipped with rotatable design (CCRD) involving five different factors.
flame ionization detector. The following parameters were Experiments were conducted in a randomized fashion. The
chosen for analysis: oven temperature, 160 8C; injector CCRD contains a total of 54 experiments with the first 32
temperature, 170 8C; carrier gas, N2; and flow rate, 20 ml experiments organized in a fractional factorial design, with
per min [17]. the experimental trails from 33 to 40 and 51 to 54 involving
 R. Balusu et al. / Process Biochemistry 40 (2005) 3025–3030 3027

the replications of the central points (Table 1). Once the Table 2
experiments are performed, the coefficient of polynomial Central composite rotatable design for the production of ethanol from
cellulose by C. thermocellum SS19 in anaerobic submerged fermentation;
model is calculated using the equation [27]: mathematically predicted yields and experimental yields
X
k X
k k X
X k C. no.a Ethanol yield (g/l)
Y ¼ b0 þ b i Xi þ bi j Xi2 þ b i j Xi X j þ e Predicted yield Experimental yield Residual value
i¼1 i¼1 ii < j j
1 9.137 9.248 0.111
2 11.996 11.757 0.239
where, i, j are linear, quadratic coefficients, respectively,
3 8.887 10.108 1.221
while ‘b’ is regression coefficient, k the number of factors 4 12.405 12.044 0.361
studied and optimized in the experiment and ‘e’ is random 5 11.274 9.165 2.109
error. The significance of each coefficient was determined 6 10.511 11.342 0.831
using Student’s t-test [28]. Model terms were selected or 7 11.113 10.616 0.497
8 11.764 12.219 0.455
rejected based on the student t-value or significance. The
9 9.482 8.493 0.989
results were analyzed by using ‘Indostat’ statistical software 10 10.683 10.679 0.004
developed by Indostat Services, Hyderabad, India. Three- 11 8.277 8.585 0.308
dimensional plots and their respective contour plots were 12 11.243 10.198 1.045
obtained based on the effect of the levels of two parameters 13 8.188 8.068 0.120
14 10.219 9.921 0.298
(at five different levels each) and their interactions on the
15 8.880 8.750 0.130
yield of ethanol by keeping the other three parameters at 16 9.309 10.030 0.721
their optimal concentrations (as obtained through ANOVA). 17 10.428 9.501 0.927
From these three-dimensional plots, the interaction of one 18 11.118 11.132 0.014
parameter with another parameter was studied. The opti- 19 11.242 11.075 0.167
20 11.636 12.133 0.497
mum concentration of each parameter was identified based
21 8.448 9.439 0.991
on the hump in the three-dimensional plots. 22 11.078 10.736 0.342
23 11.285 11.604 0.319
24 11.976 12.328 0.352
3. Results and discussion 25 8.233 7.708 0.525
26 8.537 9.015 0.478
27 9.014 8.513 0.501
In earlier studies, various nutrients were screened using a 28 7.791 9.247 1.456
statistical methodology, Plackett–Burman design [17]. Based 29 7.022 7.302 0.280
on the product promoting ability, availability, cost and the 30 9.740 8.277 1.463
need to keep the number of factors as low as possible for 31 9.051 8.699 0.352
32 8.709 9.100 0.391
optimization studies using response surface methodology,
33 11.249 10.970 0.279
five key nutrients, viz. filter paper, corn steep liquor, cysteine 34 9.827 10.970 1.143
hydrochloride, magnesium chloride and ferrous sulphate have 35 10.430 10.970 0.540
been identified as most effective. When medium was 36 11.726 10.970 0.756
optimized with the concentrations of these selected key 37 10.689 10.970 0.281
38 11.033 10.970 0.063
nutrients using response surface methodology, C. thermo-
39 9.570 10.970 1.400
cellum SS19 produced 13.66 g/l ethanol with 33.66 g/l 40 11.596 10.970 0.626
degraded substrate in anaerobic submerged fermentation. 41 6.288 7.626 1.338
42 11.463 10.536 0.927
3.1. Response surface analysis for the optimization of 43 6.809 8.759 1.950
44 11.982 10.443 1.539
nutrient levels
45 8.485 8.615 0.130
46 8.105 8.385 0.280
The actual yields of ethanol obtained in the experiments 47 11.250 10.969 0.281
and the yields predicted by the model equation are given in 48 6.294 6.986 0.692
Table 2. The regression coefficients and significance levels of 49 9.742 10.609 0.867
50 10.389 9.933 0.456
the terms are given in Table 3. It is evident from Table 3 that
51 11.115 10.137 0.978
the model used in the present study gave a satisfactory fit 52 9.132 10.137 1.005
( p < 0.00035). The significant factors and their interactions 53 11.492 10.137 1.355
were identified and considered for selecting the best fits. It can 54 10.861 10.137 0.724
a
be seen from the degree of significance (Table 3) that the linear Combination number.
terms of concentrations of filter paper and ferrous sulphate
have greatest effect, followed by the concentrations of corn significant effect (Table 3). From these observations, the
steep liquor and square terms of the concentrations of cysteine intercept regression coefficient equation for the ethanol was
HCl. By contrast magnesium chloride did not have any calculated as: 11.8042 + 0.7276A + 0.4208B  0.9958D
3028 R. Balusu et al. / Process Biochemistry 40 (2005) 3025–3030

Table 3
Significance of regression coefficients of ethanol production model
Variable Regression t-value Significance
coefficient level
Intercept 11.8042 19.9444
A (filter paper) 0.7276 4.2586 ***
B (corn steep liquor) 0.4208 2.4632 **
C (cysteine HCl) 0.0576 0.3368 I
D (FeSO4
7H2O) 0.9958 5.8284 ***
E (MgCl2
6H2O) 0.1692 0.9900 I
A2 0.2639 1.4134 I
B2 0.1339 0.7172 I
C2 0.4090 2.1906 *
D2 0.2898 1.5520 I
E2 0.0336 0.1798 I
AB 0.1433 0.7502 I Fig. 2. Response surface plot of ethanol production by C. thermocellum
AC 0.0832 0.4358 I SS19: filter paper vs. ferrous sulphate with constant levels of (g/l): corn
AD 0.0808 0.4231 I steep liquor (6.0), cysteine HCl (0.3) and magnesium chloride (1.5).
AE 0.2197 1.1501 I
BC 0.1477 0.7731 I
BD 0.1921 1.0058 I
BE 0.1785 0.9345 I From the response surface plots, it is easy and convenient
CD 0.0857 0.4486 I to understand the interactions between two nutrients and
CE 0.0051 0.0265 I
also to locate their optimum levels. It can be seen from the
DE 0.2596 1.3591 I
Block 0.8338 2.4070 * response surface plots (Figs. 1–3) that the yield of ethanol
Significant levels of regression coefficients are given as ***99.9%, **99.0%
increased upon increasing the concentration of filter paper
and *95% by t-test; ‘F-ratio’ for the model was 13.94 (degrees of freedom from 10 to 50 g/l. The yield of ethanol was significantly
were 21, 32) (F Prob. 0.00035), R2 adj. 0.83684. I: insignificant. affected by the concentration of ferrous sulphate (Figs. 1 and
5) when the concentrations of filter paper are more and
 0.4090C2  0.8338Block, where (g/l) A, filter paper (10– cysteine HCl concentrations in the range of 0.2–0.3 g/l
50 with 30 as central value); B, corn steep liquor (2–10 with 6 (Figs. 1 and 5). In these cases, lower concentrations of
as central value); C, cysteine HCl (0.1–0.5 with 0.3 as central ferrous sulphate enhanced the yield of ethanol. Thus, it was
value); D, FeSO4
7H2O (0.01–0.05 with 0.03 as central implied that a low concentration (0.01 g/l) of ferrous
value); E, MgCl2
6H2O (0.5–2.5 with 1.5 as central value) sulphate was favourable for the production of ethanol. It is
with a multiple correlation of 0.71490. clear from Figs. 3 and 4 that there is a gradual increase in the
ethanol yields upon increasing the concentrations of corn
3.2. Interaction among the nutrients steep liquor upto 8.0 g/l. Therefore, lower levels of ferrous
sulphate and higher levels of filter paper and corn steep
Figs. 1–5 are the response surface curves for variation in liquor with 0.2–0.3 g/l cysteine HCl are considered to be
the yields of ethanol, as a function of concentrations of two optimum for the production of ethanol by C. thermocellum
nutrients with the other three nutrients being at their constant SS19 in anaerobic submerged fermentation
levels (obtained through analysis of variance).

Fig. 1. Response surface plot of ethanol production by C. thermocellum Fig. 3. Response surface plot of ethanol production by C. thermocellum
SS19: filter paper vs. cysteine HCl with constant levels of (g/l): corn steep SS19: filter paper vs. corn steep liquor with constant levels of (g/l): cysteine
liquor (6.0), ferrous sulphate (0.03) and magnesium chloride (1.5). HCl (0.3), ferrous sulphate (0.03) and magnesium chloride (1.5).
 R. Balusu et al. / Process Biochemistry 40 (2005) 3025–3030
Fig. 4. Response surface plot of ethanol production by C. thermocellum
SS19: corn steep liquor vs. cysteine HCl with constant levels of (g/l): filter
paper (30.0), ferrous sulphate (0.03) and magnesium chloride (1.5).
3.3. Selection of optimum concentrations of nutrients
and their verification
As per the main observations made from the interactions,
it is evident that maximum ethanol production is obtained
when concentrations of filter paper, CSL, cysteine HCl,
FeSO4
7H2O, and MgCl2
6H2O are 45, 8.0, 0.25, 0.01 and
2.0 g/l, respectively, in the medium. With these levels the
model predicted 13.06, 5.19 and 35.69 g of ethanol, acetic
acid, and substrate degradation, respectively, per liter. These
values were experimentally verified by taking the above
media components in their respective concentrations. The
strain C. thermocellum SS19 produced 13.66 g ethanol,
5.15 g acetic acid with substrate degradation of 33.69 g
per liter. To validate and confirm these predictions, an
experiment was designed with random but moderate levels
of the nutrients (g/l) filter paper, 40; CSL, 7.0; cysteine HCl,
0.3; FeSO4
7H2O, 0.02 and MgCl2
6H2O, 1.0. The strain
SS19 degraded 35 g of substrate and produced 12.2 g of
ethanol and 5.32 g of acetic acid per lit culture broth, which
are almost similar to system predicted values (12.5 and
5.24 g of ethanol and acetic acid with 35.6 g of degraded
substrate per liter).
Although there are studies on the nutritional requirements
of C. thermocellum for the production of ethanol in
anaerobic submerged fermentation [11,12,26,29–32], there
are no reports available for the systematic approach to screen
and optimize the nutritional requirements of the micro-
organisms for ethanol production from biomass. Conven-
tional medium formulation studies are time consuming and
expensive [21]. To overcome these problems, a Plackett–
Burman design was used earlier to shortlist a few effective
nutrients for ethanol production by C. thermocellum SS19 in
anaerobic submerged fermentation and five nutrients had
been identified as the most significant for promoting ethanol
yields [17].
From the present study, it is evident that the use of
statistical media optimization approach, response surface
methodology has helped to locate the most significant
3029
Fig. 5. Response surface plot of ethanol production by C. thermocellum
SS19: cysteine HCl vs. ferrous sulphate with constant levels of (g/l): filter
paper (30.0), corn steep liquor (6.0) and magnesium chloride (1.5).
nutrients optimum levels with minimum effort and time. In
addition, it has also proved to be useful in increasing ethanol
yields from 0.32 to 0.41 g per g of substrate consumed which
is about 18% more using a very limited number of nutrients
and experiments.
Acknowledgement
We gratefully acknowledge the Council of Scientific and
Industrial Research (CSIR), New Delhi, India for financial
support.
References
[1] Sun Y, Cheng J. Hydrolysis of lignocellulosic materials for ethanol
production: a review. Bioresour Technol 2002;83:1–11.
[2] Lowe SE, Jain MK, Zeikus JG. Biology, ecology and biotechnological
applications of anaerobic bacteria adapted to environmental stresses in
temperature, pH, salinity or substrates. Microbiol Rev 1993;57:
451–509.
[3] Beguin P, Aubert JP. The biological degradation of cellulose. FEMS
Microbiol Rev 1994;13:25–58.
[4] Lin WR, Peng Y, Lew S, Lee CC, Hsu JJ, Hamel F, Demain AL.
Purification and characterization of acetate kinase from Clostridium
thermocellum. Tetrahedron Lett 1998;54:15915–25.
[5] Stevenson DM, Weimer PJ. Isolation and characterization of a Tri-
choderma strain capable of fermenting cellulose to ethanol. Appl
Microbiol Biotechnol 2002;59:721–6.
[6] Zertuche L, Zall RR. A study of producing ethanol from cellulose
using Clostridium thermocellum. Biotechnol Bioeng 1982;24:57–68.
[7] Slapack GE, Russell I, Stewart GG. Project Report submitted to
division of Energy, NRCC No. 2441, Ottava, ON 1985, p. 1–404.
[8] Lovitt RW, Kim BH, Shen GJ, Zeikus JG. Solvent production by
microorganisms. CRC Crit Rev Biotechnol 1988;7:107–86.
[9] Lynd LR. Production of ethanol from lignocellulosic materials using
thermophilic bacteria: critical evaluation of potential and review. Adv
Biochem Eng Biotechnol 1989;38:1–52.
[10] Bender J, Vatcharapijarn Y, Jeffries TW. Characteristics and adapt-
ability of some new isolates of Clostridium thermocellum. Appl
Environ Microbiol 1985;49:475–7.
3030 R. Balusu et al. / Process Biochemistry 40 (2005) 3025–3030
[11] Freier D, Mothershed CP, Wiegel J. Characterization of Clostridium
thermocellum JW 20. Appl Environ Microbiol 1988;54:204–11.
[12] Mori Y. Characterization of a symbiotic coculture of Clostridium
thermohydrosulfuriculm YM3 and Clostridium thermocellum YM4.
Appl Environ Microbiol 1990;56:37–42.
[13] Sai Ram M, Rao CV, Seenayya G. Characteristics of Clostridium
thermocellum strain SS8: a broad saccharolytic thermophile. World J
Microbiol Biotechnol 1991;7:272–5.
[14] Sai Ram M, Seenayya G. Production of ethanol from straw and
bamboo pulp by primary isolates of Clostridium thermocellum. World
J Microbiol Biotechnol 1991;7:372–8.
[15] Sato K, Goto S, Yonemura K, Sekine E, Okuma T, Takagi KH, Saiki T.
Effect of yeast extract and vitamin B12 on ethanol production from
cellulose by Clostridium thermocellum I-1-B. Appl Environ Microbiol
1992;58:734–6.
[16] Sudha Rani K, Swamy MV, Seenayya G. High ethanol production by
new isolates of Clostridium thermocellum. Biotechnol Lett 1996;18:
957–62.
[17] Ramesh B, Reddy PRM, Seenayya G, Reddy G. Production of ethanol
from cellulosic biomass by Clostridium thermocellum SS19 in sub-
merged fermentation: screening of nutrients using Plackett–Burman
design. J Biochem Biotechnol 2004;113:133–41.
[18] Reddy PRM, Reddy G, Seenayya G. Production of thermostable b-
amylase and pullulanase by Clostridium thermosulfurogenes SV2 in
solid-state fermentation: screening of nutrients using Plackett–Bur-
man design. Bioprocess Eng 1999;21:175–9.
[19] Akhnazarova S, Kafarov V. Experimental optimization in chemistry
and chemical engineering. Moscow: MIR Publishers, 1982.
[20] Strobel RJ, Nakatsukasa WM. Response surface methods for optimiz-
ing Saccharopolyspora spinosa, a macrolide producer. J Ind Microbiol
1993;11:121–7.
[21] Srinivas MRS, Nagin C, Lonsane BK. Use of Plackett–Burman design
for rapid screening of several nitrogen sources, growth/product pro-
moters, minerals and enzyme inducers for the production of alpha-
galactosidase by Aspergillus niger MRSS 234 in solid-state fermenta-
tion system. Bioprocess Eng 1994;10:139–44.
[22] Carvalho CML, Serralheiro MLM, Cabral JMS, Aires-Barros MR.
Application of factorial design to the study of transesterification
reactions using cutinase in AOT-reversed micelles. Enzyme Microbial
Technol 1997;21:117–23.
[23] Reddy PRM, Mrudula S, Ramesh B, Reddy G, Seenayya G. Produc-
tion of thermostable pullulanase by Clostridium thermosulfurogenes
SV2 in solid-state fermentation: optimization of enzyme leaching
conditions using response surface methodology. Bioprocess Eng
2000;23:107–12.
[24] Poorna V, Neelesh RS. Statistical designs for optimisation of process
parameters for alpha amylase production by A. flavus under solid state
fermentation of Amaranthus paniculatas grains as a new source of
starch. J Basic Microbiol 2001;41:57–64.
[25] Qasim Khalil B, Vikram S, Rani G. Statistical media optimization and
alkaline protease production from Bacillus mojavensis in a bioreactor.
Process Biochem 2003;39:203–9.
[26] Weimer PJ, Zeikus JG. Fermentation of cellulose and cellobiose by
Clostridium thermocellum in the absence and presence of Methano-
bacterium thermoautotrophicum. Appl Environ Microbiol 1977;33:
289–97.
[27] Maddaox IS, Richert SH. Use of response surface methodology for the
rapid optimization of microbiological media. J Appl Bacteriol 1977;
43:197–204.
[28] Needler JA, Mead R. Factorial design. Comput J 1965;7:308–10.
[29] Ng TK, Weimer PJ, Zeikus JG. Cellulolytic and physiological proper-
ties of Clostridium thermocellum. Arch Microbiol 1977;114:1–7.
[30] Ng TK, Ben-Bassat A, Zeikus JG. Appl Environ Microbiol 1981;41:
1337–43.
[31] Lamed R, Bayer EA. The cellulosome concept: exocellular
extracellular enzyme reactor centers for efficient binding and
cellulolysis. In: Aubert JP, Beguin P, Millet J, editors. Biochemistry
and genetics of cellulose degradation. London: Academic Press; 1988.
p. 101–16.
[32] Tailliez P, Girard H, Millet J, Beguin P. Enhanced cellulose fermenta-
tion by an asporogenous and ethanol tolerant mutant of Clostridium
thermocellum. Appl Environ Microbiol 1989;55:207–11.