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Accepted Manuscript

Title: An integrative process for bio-ethanol productionemploying SSF produced cellulase without extraction

Author: Reeta Rani Singhania Reetu Saini Mukund Adsul

Jitendra Kumar Saini Anshu Mathur Deepak Tuli

PII: S1369-703X(15)00012-1

DOI: http://dx.doi.org/doi:10.1016/j.bej.2015.01.002

Reference: BEJ 6101

To appear in: Biochemical Engineering Journal

Received date: 10-11-2014

Revised date: 3-1-2015

Accepted date: 7-1-2015

Please cite this article as: Reeta Rani Singhania, Reetu Saini, Mukund Adsul,

Jitendra Kumar Saini, Anshu Mathur, Deepak Tuli, An integrative process for bio-

ethanol production employing SSF produced cellulase without extraction, Biochemical

Engineering Journal http://dx.doi.org/10.1016/j.bej.2015.01.002

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An integrative process for bio-ethanol production employing

SSF produced cellulase without extraction

Reeta Rani Singhania*, Reetu Saini, Mukund Adsul, Jitendra Kumar Saini, Anshu Mathur,

Deepak Tuli

DBT-IOC Advanced Bio-Energy Research Centre, Indian Oil Corporation; R&D Centre, Sector-13, Faridabad-121007, India

Abstract

Bio-ethanol production from pretreated biomass in a single vessel was investigated in which cellulase

production via SSF by Penicillium janthinellum EMS-UV-8, hydrolysis of biomass and ethanol

fermentation was carried out sequentially. In this study, feasibility of using whole fermented matter with

enzyme, fungal mycelia, spores and residual substrate, for the saccharification of pretreated wheat straw

or avicel and its further fermentation to ethanol was investigated. Whole fermented matter produced

during cellulase production via solid-state fermentation and extracted cellulase was compared for

hydrolysis efficiency towards cellulosic biomass. Ethanol fermentation by Kluveromyces marxianus

MTCC 4136 was investigated for both the above cases and no significant difference was observed. We

could obtain similar titers of ethanol by both methods. Employing the whole fermented matter offered

advantages over using extracted enzyme by reducing enzyme extraction step and thereby reducing

economic constraint.

Key words: Solid-State fermentation; Cellulase; Bioconversion; Biomass; Ethanol; Whole fermented

matter.

Introduction

Lignocellulosic (LC) biomass is the most abundant raw material available to mankind for value

addition. LC bio-ethanol is foreseen as one of the most feasible and eco-friendly renewable

alternatives of petroleum based fuels for transport sector. Availability of ethanol for blending

with gasoline, according to Indian biofuel policy [1] seems to be difficult unless LC bio-ethanol

is produced because molasses might not be enough to fulfill the ethanol need of whole

transportation sector for E85 [2].


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LC bio-ethanol production involves several steps such as pretreatment of biomass to increase

accessibility of enzyme, production of cellulase, enzymatic saccharification of biomass and then

ethanol fermentation of released sugars. Enzymatic hydrolysis of lignocellulosic biomass is

considered as the most efficient and least polluting method of generating glucose which is

constrained by the availability of cheaper cellulase [3]. Researchers all over the world have

adopted several strategies for decreasing the cost of cellulase production such as screening of

hyper cellulase producing strains, increasing cellulase titer and productivity by optimization of

fermentation process parameters, adopting cheaper bioprocess technology such as solid-state

fermentation (SSF), improving cellulase properties for efficient saccharification by protein

engineering or blending of different cellulase, etc. [4]. Cost effective bioprocess technology suchas SSF, which requires low cost substrates, has been a popular choice for cellulase production

with several benefits like high enzyme titer, low labor cost, lower capital input, etc. [5]. Onsite

cellulase production could further play an important role for decreasing the cost of overall bio-

ethanol production.

In the present study, we have adopted solid-state fermentation (SSF) for cellulase

production and the whole fermented matter was employed as source of cellulase for hydrolyzing

biomass which was in turn fermented to ethanol. By utilizing whole fermented matter, the step of

enzyme extraction could be avoided which would further reduce the operating cost and avoid the

overall dilution of the enzymatic broth. Sequential saccharification and fermentation employing

fermented matter as well as extracted cellulase was compared for bio-ethanol production. In this

study, single vessel bio-ethanol production, including cellulase production, saccharification of

biomass and ethanol production was investigated. This could be a promising approach for cost

effective ethanol production in single vessel from biomass via enzymatic route.

1.

Materials and methods

2.1 Microorganisms and inoculum preparation

The fungal strain Penicillium janthinellum EMS-UV-8 was a kind gift from Dr D V Gokhale,

National Chemical Laboratory, Pune, and was maintained on potato dextrose agar slants. Spores

were used as inoculum and were obtained by dislodging them from the surface of a fully grown

PDA slants into sterile saline containing 0.05% Tween 80. Spores were counted on


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haemocytometer and 108 spores per 5 g substrate were inoculated into the shake flask for enzyme

production.

Thermotolerant yeast strain K. marxianus MTCC 4136 was procured from Microbial type culture

collection, IMTECH, Chandigarh, India and was maintained on malt-yeast extract agar (MYA)

medium containing (g/L): malt extract, 3; yeast extract, 3; peptone, 5; glucose, 10; agar, 20; pH

was adjusted to 7.0. For seed culture, cells were grown in 250 mL Erlenmeyer flask containing

50 mL yeast-extract peptone dextrose (YPD) medium and incubated in an orbital shaker

incubator at 30°C and 200 rpm. After 24 h incubation, broth was centrifuged; cells were washed

with sterile saline and were transferred in the enzymatic hydrolyzate/ethanol production media at

a concentration of 1.0 g/L cells on dry weight basis.

All the chemicals used in the medium were reagent grade from Himedia (India) and Sigma

(USA).

2.2 Medium for enzyme production, solid-state fermentation and extraction

Enzyme production was performed in 250ml Erlenmeyer flasks by SSF. Each flask contained 5 g

substrate (wheat bran + avicel in 4:1 ratio), moistened with 8 mL of nutrient solution with

following composition (g/L): KH2PO4-2.0, CaCl2.2H2O-0.3, urea-0.3, MgSO4.7H2O-0.3,

(NH4)2SO4-1.4, peptone-0.25, yeast extract-0.1, tween 80-0.1mL/L, FeSO4.7H2O-0.005,

MnSO4.H2O-0.0016, ZnSO4.7H2O- 0.0014, CoCl2.6H2O-0.002. Sterilization was done at 121⁰C

for 20 min. After cooling, SSF media was inoculated with 1 mL spore suspension containing 108

spores of P. janthinellum EMS-UV-8 and incubated at 30⁰C under static conditions. Experiments

were done in triplicates. After 6 d incubation, enzyme was either extracted with 50 mL of 0.05M

citrate buffer (pH 4.8) having 0.1% tween or the fermented matter was used as such without

extraction. For enzyme extraction, content of the shake flask was homogenized by proper mixing

with stirrer and then the shake flasks were kept at 180 rpm for 1 h in shaker so as to detach and

suspend the extracellular enzyme in the buffer. It was then centrifuged at 8000 rpm for 15 min to

separate the biomass and the supernatant was collected for analysis of the cellulase activity.

2.3 Enzyme assays


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Filter paper assay for total cellulase activity was done as per Ghosh [6] and β-glucosidase

activities were determined as reported earlier [7]. Beta-glucosidase activity was estimated using

PNPG (p-nitrophenyl β-D-glucopyranoside) as substrate. The assay mixture (1 mL) consisting of

0.9 mL of pNPG (1 mg/mL) and 0.1 mL of suitably diluted enzyme was incubated at 50⁰C for 30

min. The reaction was stopped with 2 mL of 2% sodium carbonate and the p-nitrophenol

liberated was measured at 410 nm. One unit (IU) of enzyme activity was defined as the amount

of enzyme required to liberate 1 µmol of glucose, xylose or p-nitrophenol from the appropriate

substrates per min under the standard assay conditions.

2.4 Saccharification of biomass by extracted cellulase and whole fermented matter

After 6 d SSF for cellulase production, extraction was done in one set of flask to analyze the

amount of cellulase produced, so as to calculate the enzyme dosage (FPU/g biomass) and the

biomass loading for the hydrolysis of pretreated wheat straw. In the initial experiment 10% solid

biomass loading and enzyme dosage of 10 FPU/g was employed. Further, two different solid

loadings (5% and 10%) with two different cellulase dosages (10 FPU/g and 20 FPU/g) were

studied (Table 1) for comparing saccharification efficiency of both form of cellulase (extracted

and fermented matter) as well as maximal sugar liberation.

Both fermented matter (FM) and extracted enzyme (EE) were evaluated for hydrolysis using

avicel (AV) and pre-treated wheat straw (PWS) as substrates. Experimental set 1 and 2 employed

extracted cellulose, whereas in set 3 and 4 whole fermented matter was used as enzyme source

(Table 1). Saccharification was carried out at 50 ⁰C, 180 rpm for 72 h. Samples of the enzyme

hydrolysate were withdrawn at 24, 48 and 72 h and reducing sugar was analyzed by DNS

method [8].

2.5 Ethanol fermentation of hydrolyzed sugars

Experimental set 1c, set 2c, set 3c and set 4c were further examined for ethanol fermentation.

After 72 h, temperature of the hydrolysate was brought down to 42⁰C and inoculated with K.

marxianus MTCC 4136 (details are given in section 2.1) for ethanol fermentation which was

continued till 72h. Fermented broth was analyzed by YSI model 2950D Analyzer (YSI,USA) for

ethanol and glucose, and total reducing sugars were analyzed by DNS method. % Ethanol yield

and biomass to sugar conversion efficiency was derived as per Dowe and McMillan, [9].


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3. Results and discussion

3.1 Enzyme production via SSF

After 6 d incubation, enzyme was extracted from one set of SSF experiment and analyzed for

FPU as well as BGL which were 20±0.1 U/gds and 25±0.5 U/gds respectively. Thus, single flask

containing 5 g substrate had total 100 FPUs. Optimum temperature and pH for this particular

cellulase was 50 ⁰C and 4.8 respectively (result not shown). SSF has been preferred over

submerged fermentation for cellulase production for bioethanol applications for higher titers,

concentrated production and cost effective process [4, 5]. There are many reports on cellulase

production via SSF by filamentous fungi which vary significantly from each other based onsubstrate, microorganisms, culture conditions, nutritional parameters employed etc. Pirota et al,

[10] reported 1.0 FPU/gds via SSF by Trichoderma reesei RUT C 30. T reesei RUT C 30 was

employed for cellulase production via SSF using wheat bran and 3.68 IU/gds was reported [11]

where as on dried kinnow pulp supplemented with wheat bran (4:1), 13.4 IU/gds was reported

[12]. Mixed culture of filamentous fungi, T. reesei and A. niger when employed for cellulase

production via SSF with rice chaff could produce maximum of 5.64 IU/gds [13]. Trichoderma

koningii produced maximum of 6.9 IU/gds cellulase using waste from vinegar industry via SSF

[14]. P janthinellum EMS-UV-8 was reported for cellulase production via SmF in bioreactor

exhibiting 3.2 IU/ml which is 91IU/g carbon source [15]. Also one study by Adsul et al, [16]

reported 61IU/gds by same organism but with longer incubation time. Main aim of this study was

to establish utilization of whole fermented matter for hydrolysis of biomass rather than extracted

cellulase.

3.2 Saccharification of biomass (AV and PWS) by extracted cellulase and whole

fermented matter

In each flask of all sets containing 5 g fermented matter (FM) 10 g of either steam pretreated

wheat straw (PWS) or avicel (AV) was added with 100 mL of 0.05M citrate buffer (pH 4.8) to

have 10% solid loading and 10 FPU/g biomass. As a control the same experiment was done

simultaneously with extracted enzyme instead of whole fermented matter. Figure 1 shows that the

hydrolysis efficiency increased for AV as well as PWS with time, maximum being at 72 h and

was 28.1% for AV and 22.1% for PWS using fermented matter. Percentage hydrolysis with


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extracted cellulase was 27.9% for AV and 20.7% for PWS. This shows that overall hydrolysis

efficiency in both the cases was similar. This proves the efficacy of the idea of utilizing whole

fermented matter rather than extracting enzyme which will reduce a step of enzyme extraction.

Though mycelia and spores remained in the medium but at 50 ⁰C these did not germinate or grow

and hence, the sugars generated during hydrolysis were not likely to be utilized by the fungus for

its growth. This experiment proves the feasibility of utilizing fermented matter for hydrolysis of

biomass, however, ethanol production from hydrolyzed sugars would validate it completely. In

case of avicel hydrolysed by enzyme or fermented matter as such, liberated sugars were 28 g/L,

which would be quite less for further fermentation.

3.3 Sequential saccharification and fermentation

For further increasing the sugar yield experiments were done by varying solid biomass loading as

well as enzyme dosage. Table 1 shows the scheme of experiments and the shaded sets were

carried over for sequential ethanol fermentation. It also shows the release of reducing sugar which

increased with time and increase in enzyme dosage. Figure 2 depicts amount of total reducing

sugar and as well as glucose yield after 72 h hydrolysis. There was difference in total reducing

sugar released and glucose available. Amount of glucose released was lower than total sugars as

cellobiose, xylose, arabinose or oligosaccharides may also be present. Maximum sugars were

obtained at 10% solid loading and 20 FPU/g biomass in each set, irrespective of the biomass (AV

or PWS) employed for hydrolysis. So, the sets having maximum released sugars (Table 1, set1c,

set2c, set3c and set4c) were carried over for ethanol fermentation. Due to higher solid loadings

(10%) higher sugar concentration was present in the medium compared to sets having 5% solid

loading. In above experiments, glucose was estimated before fermentation using YSI analyzer in

addition to reducing sugar by DNS so as to estimate ethanol conversion efficiency as per glucose

available rather than overall reducing sugar (Figure 2). Reducing sugar estimation by DNS

accounts overall sugars like glucose, xylose, cellobiose, arabinose or other oligomers which are

not fermented by K. marxianus. However, in total reducing sugar, amount of glucose was more

than 92% which is a positive indicator of having an efficient enzyme cocktail with sufficient β-

glucosidase for hydrolysis. Table 2 shows ethanol production at 72 h and its % yield on the basis

of sugar released after hydrolysis. Set1c and 3c produced maximum of 11.0 and 11.2 g/L ethanol

from 100 g/L cellulose using extracted cellulase and fermented matter, respectively, which proves


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the efficacy of the bio-ethanol process from biomass by utilizing fermented matter. Park et al,

[17] achieved similar ethanol yield of 0.12-0.19 g/g cellulose in their study by inoculating

Acremonium cellulolyticus and S. cerevisae together. In their case, cellulase was produced by

SmF and also was a simultaneous saccharification study. In our study, cellulase was produced via

SSF, which is a cheaper technology as compared to SmF [5]. We could achieve maximum ethanol

yield of 0.11 g/g cellulose and 0.265 g/g glucose (set3c) in this study using whole fermented

matter which was very similar to set1a when extracted cellulase was employed (Table 1). Set 2c

and 4c also resulted in quite similar amounts of the released sugars as well as ethanol yield. Table

3 depicts % ethanol yield in all sets based on cellulose content as well as glucose released, which

shows space for further improvement in hydrolysis part. It could be improved by blending ofheterologous cellulase as reported by Adsul et al, [7] who achieved upto 80% hydrolysis of

biomass.

We have adopted sequential process for bio-ethanol fermentation because P. janthinellum

is highly aerobic and K. marxianus requires anaerobic condition for ethanol fermentation unlike

Park et al, [10] who inoculated A. cellulolyticus and S. cerevisae together in SmF. Ours is a rare

report for cellulase production (via SSF), hydrolysis and ethanol fermentation done in single

vessel in a sequential manner. The study by Khokhar et al [18], though seems to have similar

interest but there were many lacunae as the details of cellulase production related to amount of

FPU produced or utilized for saccharification were not reported. In our study we have tried to

address these issues showing a complete process and analyzing its proper feasibility. Though our

study scheme shows similarity to Pirota et al, [10] also at first sight, but the cellulase production

via SSF (1FPU/gds) is quite poor comparative to our study, also they have added 100g/L of

glucose during fermentation, which according to us makes the whole study insignificant. Glucose

can be easily fermented into ethanol and robust yeast strains are available to do the job, however,

we need a sustainable technology for bio-ethanol from LC biomass.

Conclusion:

Whole fermented matter containing cellulase, when directly utilized for biomass hydrolysis shows

similar hydrolysis potential as that of extracted cellulase. The sequential approach of cellulase

production till ethanol fermentation leads to an economically viable technology reducing the step


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of enzyme extraction which is energy intensive. The fungal biomass present in the fermented

matter does not have adverse effect on hydrolysis and ethanol fermentation due to higher

temperature and anaerobic condition which is not favorable for its germination. This study opens

an avenue for cost effective bio-ethanol production in a single vessel by onsite cellulase

production via SSF.

Acknowledgement

All authors acknowledge Department of Biotechnology, Govt. of India and Indian Oil

Corporation R & D Centre for providing financial support. Authors RRS and MA thank DBT for

DBT-Energy Bioscience Overseas Fellowship. All authors are very thankful to Dr. D.V. Gokhale

for providing P janthinellum EMS-UV-8 strain for our study.

References

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21/10/2014.2. M. Sengupta, A. Poddar, National policy on biofuels under the scanner. Int. J. Emerg.

Technol. Adv. Eng. 3 (2013), 521-526.

3. R.K. Sukumaran, R.R. Singhania, A. Pandey, Microbial cellulases-production,

applications and challenges. J. Sci. Ind. Res. 64 (2005), 832–844.

4. R.R. Singhania, R.K. Sukumaran, A.K. Patel, C. Larroche, A., Pandey, Advancement and

comparative profiles in the production technologies using solid-state and submerged

fermentation for microbial cellulases. Enzyme Microb. Tech. 46 (2010) 541-549.

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R.R. Singhania, A.K. Patel, C.R. Soccol, A, Pandey, Recent advances in solid-state

fermentation, Biochem. Eng. J. 44 (2009), 13-18.

6. Ghose, T.K., 1987. Measurement of cellulase activities. Pure Appl. Chem. 59: 257–68.

7. M. Adsul, B. Sharma, R.R. Singhania, J.K. Saini, A. Sharma, A. Mathur, R. Gupta, and

D.K. Tuli, Blending of cellulolytic enzyme preparations from different fungal sources for

improved cellulose hydrolysis by increasing synergism. RSC Advances 4 (2014), 44726-

44732.


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8. G.L. Miller, Use of Dinitrosalicylic acid reagent for determination of reducing sugar.

Anal. Chem. 31 (1959) 426–428.

9.

N. Dowe, J. McMillan, SSF experimental protocols – lignocellulosic biomass hydrolysis

and fermentation-laboratory analytical procedure (LAP), National Renewable Energy

Laboratory Technical Report (2008) NREL/TP-510-42630.

10. R.D.P.B. Pirota, P.S. Delabona, C.S. Farinas, Simplification of the biomass to ethanol

conversion process by using the whole medium of filamentous fungi cultivated under

solid-state fermentation. Bioenerg. Res. 7(2014), 744-752.

11. R.R. Singhania, R.K. Sukumaran, A. Pandey,. Improved cellulase production by

Trichoderma reesei RUT C30 under SSF through process optimization. Appl. Biochem.Biotechnol. 142 (2007), 60-70.

12. H.S. Oberoi, Y. Chavan, S. Bansal, G.S. Dhillon, Production of cellulases through solid

state fermentation using kinnow pulp as a major substrate. Food Bioprocess Technol. 3

(2008) 528-36.

13. Y.H. Yang, B.C. Wang, Q.H. Wang, L.J. Xiang, C.R. Duan, Research on solid-state

fermentation on rice chaff with a microbial consortium, colloids and surfaces B;

Biointerphases 34, (2004) 1-6.

14. J. Liu, J. Yang, Cellulase production by T. koningii, Food Technol. Biotechnol. 45,

(2007) 420-425.

15. R.R. Singhania, J.K. Saini, R. Saini, M. Adsul, A. Mathur, R. Gupta, D.K. Tuli,

Bioethanol production from wheat straw via enzymatic route employing Penicillium

janthinellum cellulases, Bioresource Technol. 168 (2014) 490-945.

16. M. Adsul, A.P. Terwadkar, A.J. Verma, D.V. Gokhale, Cellulases and ionic liquids.

Bioresources 4 (2009) 1670-1681.

17.

E.Y. Park, K. Naruse, T. Kato, One-pot bioethanol production from cellulose by co-

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Figure captions:

Figure 1: Comparison of hydrolysis efficiencies of extracted cellulase as well as fermented

matter.

FM=Fermented matterPWS=Pretreated wheat strawEE=extracted enzyme

AV=Avicel

Figure 2: Sugar content in the hydrolysate after enzymatic saccharification (72h)

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

FM+ PWS FM+AV EE+PWS EE+AV

H y d r o l y s i s e f f i c i e n c y ( % )

24h

48h

72h

0

10

20

30

40

50

a b c d a b c d a b c d a b c d

R e d u c i n g s u g a r s o r

g l u c o s e i n g / L

Reducing…Glucose in g/L


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Table 1: Reducing sugars profile with time during saccharification with different combinations ofsolid loading, cellulase loading and biomass (AV or PWS)

Form ofcellulase

employed

Experimentcode

Biomassadded for

hydrolysis

Solidloading

FPUsloading

24 hSugars

g/ L

48 hSugars

g/ L

72 hSugars

g/L%

Hydrolysis

E x t r a c t e d c e l l u l a s e

Set 1 a AV 5 10 13.6 16.3 18.54 33.70

b AV 5 20 15.29 19.61 22.21 40.39

c AV 10 20 33 40.7 43.00 39.20

d AV 10 10 25 31.9 36.20 32.90

Set 2 a PWS 5 10 8.4 13.7 11.40 29.70

b PWS 5 20 6.0 7.3 8.70 22.60

c PWS 10 20 19.2 22.5 31.20 40.20

d PWS 10 10 18.5 19.5 19.60 25.50

W h o l e f e r m e n t e d m a t t e r Set 3 a AV 5 10 12.8 14.14 17.70 32.16

b AV 5 20 15.05 19.9 23.50 42.78

c AV 10 20 39.3 43.7 46.20 42.05

d AV 10 10 27 31.7 37.74 34.31

Set 4 a PWS 5 10 9.2 11.5 11.50 29.60

b PWS 5 20 15.1 14.8 13.40 34.90

c PWS 10 20 27.9 30.9 34.80 45.20

d PWS 10 10 18.5 19.5 21.40 27.80 Note: Cellulose content of PWS is 70%

*% Hydolysis was calculated after 72 h for avicel with formula

Total sugar released x 100 (considering 110 mg glucose/g avicel)

110

Total sugar released x 100 (considering 770 mg glucose/g PWS)770

Table 2: Ethanol fermentation of selected sets of experiment with maximum reducing sugars

Biomassadded for

hydrolysis

Solidloading

FPUsloading

72 hSugars

g/L

Glucose in g/Lbefore

fermentation

Ethanolyield

g/L

%Ethanol

yield

Set 1c AV 10 20 43.00 41.00 11.00±0.5 52.5

Set 2c PWS 10 20 31.20 27.00 6.16±0.2 44.6

Set 3c AV 10 20 46.20 42.20 11.20±0.6 51.9

set 4c PWS 10 20 34.80 29.10 6.45±0.3 43.4

Note: PWS contains 70% cellulose, estimated as per NREL protocol

% ethanol yield on basis of glucose available

an integrative process for bio-ethanol production employing ssf produced cellulase without...

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