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(2011)
Bioethanol production from lignocellulosics: an overview
Rishi Gupta, K. Sharma, R. Kuhad (2009)
Separate hydrolysis and fermentation (SHF) of Prosopis juliflora, a woody substrate, for the production of cellulosic ethanol by Saccharomyces cerevisiae and Pichia stipitis-NCIM 3498.Bioresource technology, 100 3
R. Kuhad, Rishi Gupta, Y. Khasa, A. Singh (2010)
Bioethanol production from Lantana camara (red sage): Pretreatment, saccharification and fermentation.Bioresource technology, 101 21
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Towards new enzymes for biofuels: lessons from chitinase research.Trends in biotechnology, 26 5
I. Ballesteros, M. Negro, J. Oliva, A. Cabañas, P. Manzanares, M. Ballesteros (2006)
Ethanol production from steam-explosion pretreated wheat strawApplied Biochemistry and Biotechnology, 130
R. Bollström, R. Koivunen, E. Jutila, P. Gane (2015)
TAPPI PaperCon 2015, Conference of Technical Association of the Pulp and Paper Industry, 19 - 22 April 2015 / U.S.A, Atlanta, GA
E. Tomás‐Pejó, J. Oliva, A. González, I. Ballesteros, M. Ballesteros (2009)
Bioethanol production from wheat straw by the thermotolerant yeast Kluyveromyces marxianus CECT 10875 in a simultaneous saccharification and fermentation fed-batch processFuel, 88
S. Khurana, M. Kapoor, S. Gupta, R. Kuhad (2007)
Statistical optimization of alkaline xylanase production from Streptomyces violaceoruber under submerged fermentation using response surface methodologyIndian Journal of Microbiology, 47
Rishi Gupta, Y. Khasa, R. Kuhad (2011)
Evaluation of pretreatment methods in improving the enzymatic saccharification of cellulosic materialsCarbohydrate Polymers, 84
David Hodge, M. Karim, Daniel Schell, James McMillan (2009)
Model-Based Fed-Batch for High-Solids Enzymatic Cellulose HydrolysisApplied Biochemistry and Biotechnology, 152
Zsófia Kádár, Z. Szengyel, K. Réczey (2004)
Simultaneous saccharification and fermentation (SSF) of industrial wastes for the production of ethanolIndustrial Crops and Products, 20
J. Weng, Xu Li, Nicholas Bonawitz, C. Chapple (2008)
Emerging strategies of lignin engineering and degradation for cellulosic biofuel production.Current opinion in biotechnology, 19 2
Johanna Söderström, M. Galbe, G. Zacchi (2005)
Separate versus Simultaneous Saccharification and Fermentation of Two‐Step Steam Pretreated Softwood for Ethanol ProductionJournal of Wood Chemistry and Technology, 25
T. Eriksson, J. Börjesson, F. Tjerneld (2002)
Mechanism of surfactant effect in enzymatic hydrolysis of lignocelluloseEnzyme and Microbial Technology, 31
T. Ghose (1987)
Measurement of cellulase activitiesPure and Applied Chemistry, 59
(2015)
Ann Microbiol
Rishi Gupta, Sanjay Kumar, J. Gomes, R. Kuhad (2012)
Kinetic study of batch and fed-batch enzymatic saccharification of pretreated substrate and subsequent fermentation to ethanolBiotechnology for Biofuels, 5
M. Chen, L. Xia, Pei-Jian Xue (2007)
Enzymatic hydrolysis of corncob and ethanol production from cellulosic hydrolysateInternational Biodeterioration & Biodegradation, 59
R. Kuhad, Ajay Singh (1993)
Lignocellulose Biotechnology: Current and Future ProspectsCritical Reviews in Biotechnology, 13
B. Lal, M. Reddy (2005)
Wealth from waste.
G. Miller (1959)
Use of Dinitrosalicylic Acid Reagent for Determination of Reducing SugarAnalytical Chemistry, 31
M. Linde, E. Jakobsson, M. Galbe, G. Zacchi (2008)
Steam pretreatment of dilute H2SO4-impregnated wheat straw and SSF with low yeast and enzyme loadings for bioethanol production.Biomass & Bioenergy, 32
Ann Microbiol (2015) 65:423–429 DOI 10.1007/s13213-014-0875-2 ORIGINAL ARTICLE Simultaneous saccharification and fermentation of pretreated sugarcane bagasse to ethanol using a new thermotolerant yeast Parul Singh Antil & Rishi Gupta & Ramesh Chander Kuhad Received: 18 September 2013 /Accepted: 12 March 2014 /Published online: 7 May 2014 Springer-Verlag Berlin Heidelberg and the University of Milan 2014 Abstract Enzymatic hydrolysis of a cellulosic substrate is the structural recalcitrancy of lignocellulosics hinders its efficient most critical step for the production of bioethanol. In our enzymatic conversion to fermentable sugars (Eijsink et al. study, the hydrolysis of steam-exploded sugarcane bagasse 2008). Therefore, prior to enzymatic saccharification, it is (SESB) under optimized conditions (8 % substrate consisten- necessary to pretreat lignocellulosic materials to unlock the cy, 22.5 U filter paper cellulase, 0.55 % Tween 80) released a structure of lignocellulose and thereby facilitate enzymatic maximum of 461 mg per gram dry substrate sugars. We hydrolysis of the target polysaccharides. isolated a thermotolerant yeast strain, Blastobotrys Among the various pretreatments studied to date, steam adeninivorans RCKP 2012, from sugarcane bagasse collected explosion offers several advantages, such as a reduced need of from the Cooperative Sugar Mill, Sonepat, Haryana that was chemicals, or no chemicals at all , reduced generation of found to be capable of fermenting the enzymatic hydrolysate fermentation inhibitors and eco-friendliness (Kuhad et al. of SESB at 50 °C. When grown under simultaneous sacchar- 2011). During the steam explosion treatment, most of the ification and fermentation conditions, this yeast produced hemicellulosic fraction is extracted in the condensate, with −1 14.05 g L ethanol, which corresponds to a theoretical etha- the residual solid biomass containing mainly cellulose and nol yield of 46.87 %. lignin (Kuhad et al. 2011). The pretreated substrate is then enzymatically hydrolyzed using cellulases (endoglucanase, . . . Keywords Bioethanol Thermotolerant SSF cellobiohydrolase and β-glucosidase). The factors affecting Lignocellulose Steam explosion the enzymatic hydrolysis of cellulose include amount of sub- strate, cellulase activity, reaction conditions (temperature and pH) and additives (surfactants and metal ions), if any. Opti- Introduction mization of these factors is essential to improve the sugar yield and rate of enzymatic saccharification, which will eventually The rapid increase in industrialization and, consequently, in increase the bioethanol production. energy demand have been the driving forces behind the search Fermentation of the enzymatic hydrolysate can be carried for alternative energy sources. Among the various potential out using either of the two approaches, i.e. separate hydrolysis alternatives, bioethanol derived from lignocellulosics has been and fermentation (SHF) or simultaneous saccharification and considered a good choice due to its renewable nature and fermentation (SSF). SSF has been reported to have several carbon-balanced properties (Weng et al. 2008; Gupta et al. advantages over SHF (Soderstrom et al. 2005): (1) improved 2009). The major constituents of lignocellulosics are cellu- enzymatic hydrolysis rate and product concentration due to lose, hemicellulose and lignin (Kuhad and Singh 1993). The reduced end-product inhibition; (2) reduced production cost first step in the bioconversion of lignocellulosics to ethanol is due to both the hydrolysis and fermentation reactions being its conversion into the component sugars. However, the carried out in a single reactor (Chen et al. 2007). However, the difference in the temperature optima of the cellulases (45– : : P. S. Antil R. Gupta R. C. Kuhad (*) 50 °C) and that of the fermenting organism (28–35 °C) is a Lignocellulose Biotechnology Laboratory, Department critical factor in SSF (Kadar et al. 2004). Therefore there is an of Microbiology, University of Delhi South Campus, urge to use for thermotolerant yeast for improved performance Benito Juarez Road, New Delhi 110021, India of SSF system. e-mail: kuhad85@gmail.com 424 Ann Microbiol (2015) 65:423–429 The aim of our study was to optimize conditions for the To identify the potent thermotolerant fermenting yeast enzymatic saccharification of steam-exploded sugarcane ba- isolate (LBLY 2), we amplified a 500-bp region of the 18S gasse (SESB) and to enhance its saccharification yield. We rRNA gene in a thermocycler (G-Storm; BMG Labtech, also attempted to simultaneouslysaccharify andferment Aylesbury, UK) using the universal primers ITS1 (TCCGTA SESB under optimized saccharification conditions using GGTGAACCTGCGG) and ITS4 (TCCTCCGCTTATTGAT thermotolerant yeast. ATGC). The PCR products were purified and sequenced as described earlier (Khurana et al. 2007), and the nucleotide sequence has been deposited in the GenBank database. The Materials and methods sequence data were analyzed for homology with the similar existing sequences available in the database of the National Raw materials and chemicals Center for Biotechnology Information (NCBI) using BLAST. Steam-exploded sugarcane bagasse was a kind gift from Dr. Cellulase production using Trichoderma citrinoviridae A.J. Varma, Polymer Chemistry Division, National Chemical RCK2012 under SSF Laboratory, Pune, India; the wheat bran was procured locally. 3,5-Dinitrosalicylic acid (DNS) was purchased from Sigma (St. Trichoderma citrinoviridae RCK2012 was used for cellulase Louis, MO). Ethanol for standard preparation was obtained production under solid-state cultivation conditions. The inoc- from Merck India Pvt. Ltd (Mumbai, India). Other chemicals ulum for enzyme production was prepared by inoculating four and media components used were purchased locally. fungal discs (diameter 8 mm) removed from the periphery of a 8-day-old potato dextrose agar plate into a 250-mL Erlenmey- Chemical composition analysis of SESB er flask containing 100 mL of potato dextrose broth and incubating the flask at 30°C under static culture conditions The chemical composition of SESB was analyzed for for 4 days. holocellulose, Klason lignin, pentosans, ash and moisture The enzyme production process was carried out in 250-mL content. The plant material was extracted with alcohol–ben- Erlenmeyer flasks, with each containing 5.0 g of dry wheat zene (1:2, v:v) to remove wax and resin. The extractive-free −1 bran moistened with a mineral salt solution [(g L ): soybean wood dust was processed for chemical analysis following meal, 24; (NH ) SO ,0.3; KH PO , 0.6; yeast extract, 5.0; pH 4 2 4 2 4 TAPPI (1992) protocols. 5.5] to attain a final substrate-to-moisture ratio of 1:3. The flasks were sterilized by autoclaving at 121°C (15 psi); fol- Isolation, screening and identification of the thermotolerant lowing cooling to room temperature, they were inoculated yeast strain −1 with crushed fungal mass (20 % w v , on dry weight basis) obtained from the fungal mat in the inoculum flask. The The environmental samples consisted of sugarcane bagasse contents of the flasks were mixed well with a sterilized glass was collected from the Cooperative Sugar Mill, Sonepat, rod to distribute the inoculum evenly throughout the substrate Haryana, Balma, the starter culture of the traditional beverage and incubated at 30 °C. After the wheat bran had been of the Bhootiya tribe was procured from Uttrakhand and fermented by the fungus for an appropriate interval, it was starter culture of alcoholic beverages was obtained from aseptically removed from the flasks, suspended in 25 ml of Kangra Herb Pvt. Ltd, Kangra, Himachal Pradesh. All sam- 50 mM citrate phosphate buffer (pH 5.0) and stirred for ples were serially diluted and spread on Sabouraud’s agar 10 min. The extrudates were squeezed through muslin cloth −1 medium [(g L ): peptone, 10.0; glucose, 40.0; agar, 15.0; for maximizing the enzyme extraction and centrifuged at sodium propionate, 3.5; pH 6.0]. The plates were incubated at 10,000 g at 4 °C for 10 min. The enzyme solution thus 30 °C for 3 days. Pure cultures were obtained from the obtained was assayed for cellulase activities. colonies which developed through repeated transfer of the −1 cultures on MGYP agar plates [ (g L ): malt extract, 3; glucose, 20; yeast extract, 3; peptone, 5 agar, 20; pH 6.0]. Optimization of enzymatic saccharification of SESB All yeast isolates were screened for their thermotolerance and fermenting capability by culture in 50-mL culture tubes, Enzymatic saccharification of SESB was carried in a 50 mM each containing 10 mL MGYP broth. The tubes were inocu- citrate phosphate buffer (pH 5.0). Prior to enzyme loading, the lated with a loop-full of the appropriate yeast isolate and slurry was preincubated at 50 °C on a rotatory shaker (Innova incubated for 72 h at 50 °C and 200 rpm in a rotatory incubator 4400; New Brunswick Scientific) at 200 rpm for 2 h. Enzyme shaker (Innova 4400; New Brunswick Scientific, Nürtingen, and the non-ionic surfactant (Tween 80) were then added to Germany). Samples were taken out at 6-h intervals an analyzed the preincubated slurry and the reaction was allowed to con- for residual sugars, ethanol content and biomass produced. tinue at 50°C and 200 rpm. Ann Microbiol (2015) 65:423–429 425 In order to optimize the enzymatic saccharification of commercial grade ethanol (Merck, Darmstadt, Germany). Ni- −1 SESB, we performed 17 runs of response surface methodolo- trogen at a flow rate of 0.5 mL min was used as the carrier gy (RSM)-based Box–Behnkhen design (BBD) experiments gas. The saccharification yield was calculated as: (Design Expert, ver. 6.1; Stat-Ease, Minneapolis, MN), with ðÞ Amount of reducing sugars released 100 HydrolysisðÞ % ¼ ; substrate consistency, enzyme dosage and Tween 80 dosage as Holocellulose content of pretreated substrate factors and saccharification yield as response. The range of each variable is shown in Table 1. Samples were withdrawn at While the ethanol yield was calculated as: 4-h intervals and centrifuged at 10,000 g for 10 min in a refrigerated centrifuge (Sigma) at 4°C; the supernatant was Ethanol concentration Ethanol yieldðÞ % ¼ 100 used for further analysis. Holocellulose content SSF of SESB All of the experiments were done in triplicate, and the results are presented as the mean ± standard deviation. The primary inoculum was prepared by inoculating a loop full of yeast culture from 24-h-old MGYP agar plates into the −1 inoculation medium [(g L ): glucose, 30.0; yeast extract, 3.0; Results and discussion peptone, 5.0; (NH ) HPO , 0.25; pH 6.0±0.2] (Chen et al. 4 2 4 2007; Kuhad et al. 2010). The secondary inoculum was de- Proximate chemical composition of the SESB veloped by inoculating 2 % of primary inoculum into the inoculation medium and culturing the yeast cells until an The SESB contained 52.97 % α-cellulose, 7.51 % pentosans, optical density (OD )of 0.6. 32.83 % Klason lignin, 4.66 % moisture and 2.02 % ash The SSF experiments were performed in 250-mL capped content (Fig. 1). The carbohydrate content in the SESB is conical flasks (SCHOTT DURAN, Mainz, Germany) contain- comparable to the holocellulose content of other cellulosic ing 50 mL reaction volume under the optimum enzymatic feedstocks used for bioethanol production, such as Prosopis saccharification conditions. Nutrients (malt extract, yeast ex- juliflora (67 %), Lantana camara (61 %) and corn cob tract and peptone) were added to the medium to a final −1 (71.6 %) (Gupta et al. 2009, 2011; Kuhad et al. 2010). The concentration of 3.0, 3.0 and 5.0 g L , respectively. Prior to considerably high carbohydrate content (holocellulose SSF, a prefermentation saccharification step was carried out 60.48 %) of the SESB qualified it as potential feedstock for for 2 h at 50 °C and 150 rpm, following which 6 % inoculum bioethanol production. of the thermotolerant yeast was added. Samples were with- drawn at 6-h intervals, centrifuged at 10,000 g for 10 min at 4 ºC and the supernatant was assayed for Isolation, screening and identification of the potent sugars and ethanol. hexose-fermenting thermotolerant yeast isolate Analytical methods Out of total 41 yeast isolates grown from three types of samples, 17 showed the ability to ferment glucose; of these The enzyme assays were carried out using standard Interna- 17 isolates, isolate LBLY 2 exhibited the ability to ferment tional Union of Pure and Applied Chemistry methods (Ghose glucose at a higher temperature (50 °C). Yeast isolate LBLY 2 1987). Total reducing sugars were estimated by the DNS was identified based on the results of nucleotide BLAST method (Miller 1959). Ethanol was estimated by gas chroma- similarity search against existing 18S rRNA gene sequences tography Clarus 500; PerkinElmer, Waltham, MA) with in the NCBI database, revealing that the isolate was closely an elite-wax (cross bond-polyethylene glycol) column related to the genus Blastobotrys and species adeninivorans (30.0 m×0.25 mm) at an oven temperature of 90 °C, an (Fig. 2). The organism was therefore termed Blastobotrys injector temperature of 150 °C and flame ionization detection adeninivorans RCKP 2012, and the sequence was submitted at 200 °C. The ethanol standards were prepared using to the NCBI GenBank as accession no. HE657273. Table 1 Range of each variable Factor Name Units −1 Level 0 Level +1 Level used for response surface meth- odology-based Box–Behnkhen −1 A Enzyme dose (FPU g ds ) 17.5 20.0 22.5 design experiments −1 B Substrate consistency (% w v ) 7.5 10.0 12.5 −1 FPU filter paper unit; g ds gram C Surfactant dose (% v v ) 0.4 0.5 0.6 dry substrate 426 Ann Microbiol (2015) 65:423–429 -Cellulose error and indicates that the influence (significant or not) of Pentosans each controlled factor on the tested model was significant at a Klason lignin high confidence level. The model was highly significant, as Ash manifested by an F value and a probability value (P > F)of total Moisture <0.0001 (Table 2). The adequate precision of 48.49 signifies that the model has desirable values. The goodness of fit was 2 2 manifested by the determination coefficient (R ), and the R value of 0.9971 indicated that the response model could explain 99.71 % of the total variation; in addition, the value of the adjusted R was also sufficiently high (0.9934) to indicate the significance of the model (Table 2). The R values Fig. 1 Composition of steam exploded sugarcane bagasse (SESB) provide a measure of variability in the observed response values that can be explained by the experimental factors and 2 2 Cellulase production using Trichoderma citrinoviridae their interactions. The adjusted R corrects the R value for RCK2012 during SSF sample size and number of terms in the model. If there is a large number of values in the model and sample size is small, The soft rot fungus T. citrinoviridae RCK2012 when grown the adjusted R may be significantly smaller than the predicted under solid state fermentation cultivation condition produced R . The purpose of statistical analysis is to determine the a maximum of 50 U filter paper cellulase (FPase), 75 U experimental factors which generate signals that are large in carboxymethyl cellulase (CMCase), and 150 U comparison to noise. The adequate precision, a measure of −1 β-glucosidase g ds after 6 days of incubation. signal-to-noise ratio was 48.49 (Table 2). A signal-to-noise ratio greater than 4 is desirable. Based on these results, we concluded that the model was fit and that it could be used to Statistical optimization for enzymatic saccharification navigate the design space. of SESB using RSM The result of the RSM experiment on the effect of the three independent variables [enzyme dose (A), substrate consisten- Response surface methodology was used to study the effect of cy (B) and surfactant dose (C)] together with the mean pre- three independent variables: enzyme dose (A), substrate con- dicted series of experiments that were designed and conducted sistency (B) and surfactant dose (C). From multiple regression are shown in Table 3. The three-dimensional response surface analysis, we obtained the quadratic equation that plots were employed to determine the interaction of the pa- explained the saccharification yield irregardless of the rameters and their effect on saccharification yield. The plots significance of the coefficients: were generated by plotting the response using the z-axis against two independent variables while keeping the other 2 2 Y ¼ 427:59 þ 18:72*A − 18:63*B − 4:39*C − 15:50* A − 17:30* B independent variables at their 0 level. The coordinates of the − 5:45*C − 6:55*A* B þ 17:43*A*C − 3:25*B*C central point within the highest contour levels in each of the figures correspond to the optimum concentrations of the re- −1 where Y is the saccharification yield (mg g ), and A, B spective components. and C represent the coded levels of enzyme dose Figure 3 describes the effects of substrate consistency and −1 −1 (U g ds ), substrate consistency (% w v ) and surfac- surfactant dose on lignocellulose saccharification when the −1 −1 tant dose (% v v ), respectively. enzyme dose is fixed at its ‘0’ level (20 U FPase g ds ). The The statistical significance of the regression model was figure reveals that the saccharification yield increased with checked by the F test. The model F (269.471) value is a ratio Table 2 Analysis of of the mean square due to regression to the mean square due to Factors Value variance of the response surface methodology- R 0.997122 based model for sacchar- ification yield Adjusted R 0.993422 Predicted R 0.953952 Adequate precision 48.4926 PRESS 451.781 Coefficient of variance. 0.490345 Prob > F <0.0001 PRESS predicted residu- Fig. 2 Dendrogram of Blastobotrys adeninivorans RCKP 2012 showing F value 269.471 al sums of squares the similarity with other Blastobotrys cultures Ann Microbiol (2015) 65:423–429 427 Table 3 Experimental design Std Run Independent variables affecting enzymatic saccharification of Y: Saccharification yield and results of the response surface −1 SESB (mg g ) methodology-based model for optimization of saccharification A: Enzyme dosage B: Substrate C: Surfactant Actual Predicted of steam-exploded sugarcane −1 −1 (U g ds ) consistency (%) dosage (% v v ) bagasse 1 2 17.5 7.5 0.5 389.33 388.16 2 6 22.5 7.5 0.5 436.60 438.69 3 10 17.5 12.5 0.5 366.08 363.99 4 14 22.5 12.5 0.5 387.17 388.34 5 17 17.5 10 0.4 407.30 409.74 6 11 22.5 10 0.4 413.14 412.32 7 13 17.5 10 0.6 365.29 366.11 8 4 22.5 10 0.6 440.85 438.41 9 9 20 7.5 0.4 425.89 424.62 10 15 20 12.5 0.4 394.21 393.86 11 12 20 7.5 0.6 421.98 422.34 12 1 20 12.5 0.6 377.31 378.58 13 16 20 10 0.5 427.59 427.59 14 5 20 10 0.5 427.59 427.59 15 7 20 10 0.5 427.59 427.59 16 8 20 10 0.5 427.59 427.59 SESB, Steam-exploded sugarcane 17 3 20 10 0.5 427.59 427.59 bagasse increased substrate consistency up to 8 % and decreased saccharification yield and the amount of enzyme dosage, with thereafter. However, the enhancement of surfactant concen- saccharification yield increasing regularly with increases in tration did not significantly increase the saccharification yield the enzyme dose. Interestingly, surfactant concentration had a (Fig. 3). The interaction between surfactant and enzyme dos- significant effect on saccharification yield at higher enzyme age on the saccharification of SESB is shown in Fig. 4.This levels but not at lower enzyme doses (Fig. 4). The increase in graph shows a direct proportional relationship between the saccharification yield with the addition of surfactant might be due to the reduced surface tension or reduced thermal deactivation of the enzyme. Similar observations of Fig. 3 Response curve of the response surface methodology (RSM)- −1 based experiment showing the effect of substrate consistency (% w v ) Fig. 4 Response curve of the RSM-based experiment showing the effect −1 −1 −1 −1 and surfactant dose (% v v ) on saccharification yield (mg g ds )of of enzyme dose (FPU g ds ) and surfactant dose (% v v ) on sacchar- −1 SESB ification yield (mg g ds )ofSESB. FPU Filter paper dose 428 Ann Microbiol (2015) 65:423–429 The optimal conditions (enzyme dose of 22.5 FPU −1 gds , 8 % substrate consistency, surfactant dose of 0.55 %) for the maximum predicted saccharification −1 yield(448mggds ) were validated experimentally. The validation results showed that the maximum sac- −1 charification of 461 mg g ds was in close agreement with the predicted values. SSF of SESB During the time course of SSF of SESB, we noted a regular increase in ethanol production up to 90 h, which remained almost constant thereafter (Fig. 6). However, a residual sugar −1 content of approximately 17 g L was also observed after 90 h of incubation. The high amount of glucose available during the early stage of fermentation was due to the pre- saccharification of SESB (Fig. 6). The maximum ethanol −1 production (14.05 g L ) that was obtained after 96 h of Fig. 5 Response curve of the RSM-based experiment showing the effect −1 −1 fermentation corresponds to a theoretical yield of 46.87 % of substrate consistency (% w v ) and enzyme dose (FPU g ds )on −1 saccharification yield (mg g ds )of SESB based on total carbohydrates present in the SESB, which is an ethanol conversion of 14 g ethanol/100 g ds. This yield agrees enhancement in saccharification efficiency with the addition with those reported earlier by Ballesteros et al. (2006)and of a non-ionic surfactant has also been observed by other Linde et al. (2008). Linde et al. (2008) observed an ethanol researchers (Eriksson et al. 2002; Gupta et al. 2009). conversion of 18 g/100 g steam exploded wheat straw, while A similar linear increase in saccharification yield was ob- Ballesteros et al. (2006) achieved an ethanol conversion of served with increased enzyme dose (Fig. 5). Moreover, opti- 10 g/100 g acid-pretreated wheat straw. Interestingly, during mum saccharification was observed at 8 % substrate consis- the late phase of fermentation, a continuous decline in the rate tency, and deviations from this level resulted in decreased of fermentation was also observed, which subsequently result- saccharification yield (Fig. 5). The decrease in saccharifica- ed in the accumulation of glucose in the fermentation broth tion yield with increased substrate consistency might be due to (Fig. 6). This trend was also reported by Tomás-Pejó an increase in viscosity or rheological problems, such as et al.( 2009), whoobserveda declineinfermentation improper mixing of substrate or improper temperature control rate after 48 h and subsequent enhancement in the (Hodge et al. 2009; Gupta et al. 2012). accumulated residual sugars. Fig. 6 Time courses of ethanol concentration, glucose concentration and ethanol yield during the simultaneous saccharification and fermentation process Ann Microbiol (2015) 65:423–429 429 Gupta R, Khasa YP, Kuhad RC (2011) Evaluation of pretreatment Conclusion methods in improving the enzymatic saccharification of cellulosic materials. Carbohydr Polym 84:1103–1109 Optimization of enzymatic saccharification using a statistical Gupta R, Kumar S, Gomes J, Kuhad RC (2012) Kinetic study of approach allows maximum utilization of the substrate, which batch and fed-batch enzymatic saccharification of pretreated substrate and subsequent fermentation to ethanol. Biotechnol will ultimately improve the economics of the process. We Biofuel 5:16 have shown that the thermotolerant yeast Blastobotrys Hodge DB, Karim MN, Schell DJ, McMillan JD (2009) Model-based adeninivorans RCKP2012 has a good potential for fermenting fed-batch for high-solids enzymatic cellulose hydrolysis. Appl sugars in the SSF process in a single reactor. This may be a Biochem Biotechnol 152:88–107 Kadar Z, Szengyel Z, Reczey K (2004) Simultaneous saccharification better approach to enhance the process efficiency. However, and fermentation (SSF) of industrial wastes for the production of further detailed studies on the bioprocessing of ethanol fer- ethanol. Ind Crop Prod 20:103–110 mentation are needed. Khurana S, Kapoor M, Gupta S, Kuhad RC (2007) Statistical optimiza- tion of alkaline xylanase production from Streptomyces violaceoruber under submerged fermentation using response sur- Acknowledgments The authors acknowledge the support from the face methodology. Indian J Microbiol 47:144–152 University of Delhi for providing the facilities and finances to carry out Kuhad RC, Singh A (1993) Lignocellulose biotechnology: current and the work. The authors also acknowledge the technical support of Mr. future prospects. Crit Rev Biotechnol 13:151–172 Subhojit Chakraborty. Rishi Gupta also acknowledges the Council of Kuhad RC, Gupta R, Khasa YP, Singh A (2010) Bioethanol production Scientific and Industrial Research for providing a Senior Research from Lantana camara (red sage): pretreatment, saccharification and Fellowship. fermentation. Bioresour Technol 101:8348–8354 Kuhad RC, Gupta R, Khasa YP (2011) Bioethanol production from lignocellulosics: an overview. In: Lal B, Sarma PM (eds) Wealth from waste. 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Fuel 88:2142–2147 mentation (SHF) of Prosopis juliflora, a woody substrate, for the Weng JK, Li X, Bonawitz ND, Chapple C (2008) Emerging strategies of production of cellulosic ethanol by Saccharomyces cerevisiae and lignin engineering and degradation for cellulosics biofuel produc- Pichia stipitis-NCIM 3498. Bioresour Technol 100:1214–1220 tion. Curr Opin Biotechnol 19:166–172
Annals of Microbiology – Springer Journals
Published: May 7, 2014
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