Saprativ P. Das

Enhanced Cellulase Production from Bacillus subtilis by Optimizing Physical Parameters for Bioethanol Production

Effect of physical parameters such as initial pH, agitation (rpm), and temperature (°C) for cellulase production from Bacillus subtilis AS3 was investigated. Central composite design of experiments followed by multiple desirability function was applied for the optimization of cellulase activity and cell growth. The effect of the temperature and agitation was found to be significant among the three independent variables. The optimum levels of initial pH, temperature, and agitation for alkaline carboxymethylcellulase (CMCase) production predicted by the model were 7.2, 39°C, and 121 rpm, respectively. The CMCase activity with unoptimized physical parameters and previously optimized medium composition was 0.43 U/mL. The maximum activity (0.56 U/mL) and cell growth (2.01 mg/mL) predicted by the model were in consensus with values (0.57 U/mL, 2.1 mg/mL) obtained using optimized medium and optimal values of physical parameters. After optimization, 33% enhancement in CMCase activity (0.57 U/mL) was recorded. On scale-up of cellulase production process in bioreactor with all the optimized conditions, an activity of 0.75 U/mL was achieved. Consequently, the bacterial cellulase employed for bioethanol production expending (5%, w/v) NaOH-pretreated wild grass with Zymomonas mobilis yielded an utmost ethanol titre of 7.56 g/L and 11.65 g/L at shake flask and bioreactor level, respectively.

Lignocellulosic fermentation of Wild grass employing recombinant hydrolytic enzymes and fermentative microbes with effective bioethanol recovery.

Simultaneous saccharification and fermentation (SSF) studies of steam exploded and alkali pretreated different leafy biomass were accomplished by recombinant Clostridium thermocellum hydrolytic enzymes and fermentative microbes for bioethanol production. The recombinant C. thermocellum GH5 cellulase and GH43 hemicellulase genes expressed in Escherichia coli cells were grown in repetitive batch mode, with the aim of enhancing the cell biomass production and enzyme activity. In batch mode, the cell biomass (A 600 nm) of E. coli cells and enzyme activities of GH5 cellulase and GH43 hemicellulase were 1.4 and 1.6 with 2.8 and 2.2 U·mg−1, which were augmented to 2.8 and 2.9 with 5.6 and 3.8 U·mg−1 in repetitive batch mode, respectively. Steam exploded wild grass (Achnatherum hymenoides) provided the best ethanol titres as compared to other biomasses. Mixed enzyme (GH5 cellulase, GH43 hemicellulase) mixed culture (Saccharomyces cerevisiae, Candida shehatae) system gave 2-fold higher ethanol titre than single enzyme (GH5 cellulase) single culture (Saccharomyces cerevisiae) system employing 1% (w/v) pretreated substrate. 5% (w/v) substrate gave 11.2 g·L−1 of ethanol at shake flask level which on scaling up to 2 L bioreactor resulted in 23 g·L−1 ethanol. 91.6% (v/v) ethanol was recovered by rotary evaporator with 21.2% purification efficiency.

Statistical Optimization of Fermentation Process Parameters by Taguchi Orthogonal Array Design for Improved Bioethanol Production

The statistical optimization of different fermentation process parameters in SSF of mixed MAA and organosolv pretreated 1% (w v−1) wild grass, namely, recombinant Clostridium thermocellum hydrolytic enzymes’ volume (GH5 cellulase, GH43 hemicellulase), fermentative microbes’ inoculum volume (Saccharomyces cerevisiae, Candida shehatae), pH, and temperature, was accomplished by Taguchi orthogonal array design. The optimized parameters in 100 mL of fermentation medium were (%, v v−1) as follows: 1.0, recombinant GH5 cellulase (5.7 mg−1, 0.45 mg mL−1); 2.0, recombinant GH43 hemicellulase (3.7 U mg−1, 0.32 mg mL−1); 1.5, S. cerevisiae (3.9 × 108 cells mL−1); 0.25, C. shehatae (2.7 × 107 cells mL−1); pH, 4.3; and temperature, 35∘C. pH with p-value 0.001 was found to be the most significant factor affecting SSF. The ethanol titre obtained in Taguchi optimized shake flask SSF was 2.0 g L−1 implying a 1.3-fold increase as compared to ethanol titre of 1.5 g L−1 in unoptimized shake flask SSF. A 1.5-fold gain in ethanol titre (3.1 g L−1) was obtained with the same substrate concentration in lab scale bioreactor on scaling up the shake flask SSF with Taguchi optimized process parameters.

Bioethanol production from hemicellulose rich Populus nigra involving recombinant hemicellulases from Clostridium thermocellum.

Bioethanol was produced from poplar leafy biomass rich in hemicelluloses content involving recombinant Clostridium thermocellum hemicellulases and pentose sugar utilizing Candida shehatae. FT-IR analysis revealed effective AFEX pretreatment of poplar leaves. Repetitive batch strategy yielded ∼1.5-fold rise in cell biomass and specific activity of both, acetylxylanesterase (Axe) and GH43 hemicellulase. TLC and HPAEC exhibited xylose and arabinose release from hydrolyzed biomass. SSF trial with 1% (wv(-1)) pretreated poplar and mixed enzymes showed ∼1.5-fold higher ethanol titre as compared with SHF. The shake flask SSF with 5% (wv(-1)) pretreated poplar furnished 4.56 and 5.43gL(-1) ethanol with Axe and mixed enzymes, respectively. Whereas, bioreactor scale-up exhibited ∼1.25-fold increase in ethanol titres (5.68, 6.75gL(-1)) as compared with shake flask with an yield of 0.295 (gg(-1)) and 0.351 (gg(-1)), respectively with Axe and mixed enzymes.

BIOETHANOL PRODUCTION FROM LEAFY BIOMASS OF MANGO (Mangifera indica) INVOLVING NATURALLY ISOLATED AND RECOMBINANT ENZYMES

The present study describes the usage of dried leafy biomass of mango (Mangifera indica) containing 26.3% (w/w) cellulose, 54.4% (w/w) hemicellulose, and 16.9% (w/w) lignin, as a substrate for bioethanol production from Zymomonas mobilis and Candida shehatae. The substrate was subjected to two different pretreatment strategies, namely, wet oxidation and an organosolv process. An ethanol concentration (1.21 g/L) was obtained with Z. mobilis in a shake-flask simultaneous saccharification and fermentation (SSF) trial using 1% (w/v) wet oxidation pretreated mango leaves along with mixed enzymatic consortium of Bacillus subtilis cellulase and recombinant hemicellulase (GH43), whereas C. shehatae gave a slightly higher (8%) ethanol titer of 1.31 g/L. Employing 1% (w/v) organosolv pretreated mango leaves and using Z. mobilis and C. shehatae separately in the SSF, the ethanol titers of 1.33 g/L and 1.52 g/L, respectively, were obtained. The SSF experiments performed with 5% (w/v) organosolv-pretreated substrate along with C. shehatae as fermentative organism gave a significantly enhanced ethanol titer value of 8.11 g/L using the shake flask and 12.33 g/L at the bioreactor level. From the bioreactor, 94.4% (v/v) ethanol was recovered by rotary evaporator with 21% purification efficiency.

Scale up and efficient bioethanol production involving recombinant cellulase (Glycoside hydrolase family 5) from Clostridium thermocellum

BackgroundLignocellulose degrading fungal enzymes have been in use at industrial level for more than three decades. However, the main drawback is the high cost of the commercially available Trichoderma reesei cellulolytic enzymes.ResultsThe hydrolytic performance of a novel Clostridium thermocellum cellulolytic recombinant cellulase expressed in Escherichia coli cells was compared with the naturally isolated cellulases in different modes of fermentation trials using steam explosion pretreated thatch grass and Zymomonas mobilis. Fourier transform infrared (FT-IR) spectroscopic analysis confirmed the efficiency of steam explosion pretreatment in significant release of free glucose moiety from complex lignocellulosic thatch grass. The recombinant GH5 cellulase with 1% (w v-1) substrate and Z. mobilis in shake flask separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) trials demonstrated highest ethanol titre (0.99 g L-1, 1.2 g L-1) as compared to Bacillus subtilis (0.51 g L-1, 0.72 g L-1) and Trichoderma reesei (0.67 g L-1, 0.94 g L-1). A 5% (w v-1) substrate with recombinant enzyme in shake flask SSF resulted in a 7 fold increment of ethanol titre (8.8 g L-1). The subsequent scale up in a 2 L bioreactor with 1 L working volume yielded 16.13 g L-1 ethanol titre implying a 2 fold upturn. The rotary evaporator based product recovery from bioreactor contributed 94.4 (%, v v-1) pure ethanol with purification process efficiency of 22.2%.ConclusionsThe saccharification of steam exploded thatch grass (Hyparrhenia rufa) by recombinant cellulase (GH5) along with Z. mobilis in bioethanol production was studied for the first time. The effective pretreatment released substantial hexose sugars from cellulose as confirmed by FT-IR studies. In contrast to two modes of fermentation, SSF processes utilizing recombinant C. thermocellum enzymes have the capability of yielding a value-added product, bioethanol with the curtailment of the production costs in industry.

Water Hyacinth as a Potential Source of Biofuel for Sustainable Development

Water hyacinth (Eichhornia crassipes), a noxious weed and fast growing perennial aquatic plant found in lakes and ponds all over Guwahati is affecting the ecosystem in a deleterious manner. It tampers with aquatic life by deoxygenating the water and depleting nutrients for young fish in sheltered bays. It also blocks supply intakes for the hydroelectric plant, interrupting electrical power. Contrastingly, owing to high cellulose and hemicellulose content in its biomass and having a wide distribution in Assam, it was selected for our study. Therefore, using water hyacinth for bioethanol production can tackle the pollution problem owing to fossil fuel emissions and control its growing extent in water bodies. In the present research, water hyacinth was used for bioethanol production involving simultaneous hydrolysis and fermentation at shake flask and reactor level using different saccharifying enzymes and potential fermentative microbes. Candida shehatae, utilizing pentoses was used along with Saccharomyces cerevisiae in simultaneous saccharification and fermentation (SSF) experiments. The substrate was subjected to three pretreatments viz. wet oxidation, phosphoric acid–acetone treatment and ammonia fibre expansion (AFEX). Recombinant E. coli BL21 (DE3) and BL21 (plysS) cells harbouring expressing Glycoside hydrolase family 5 (GH5) and family 43 (GH43) genes from Clostridium thermocellum were employed for cellulase and hemicellulase production respectively. Trichoderma reesei cellulase and Bacillus subtilis AS3 producing thermostable cellulases were also engaged in saccharification process. The wet oxidation-treated water hyacinth conferred an ethanol titre of 0.90 g/L with B. subtilis cellulase and 1.26 g/L with T. reesei cellulase amid a fermentative microbial combination of S. cerevisiae and C. shehatae. A higher ethanol titre (1.69 g/L) was achieved with an enzymatic consortium of GH5 cellulase and GH43 hemicellulase along with S. cerevisiae and C. shehatae. Contrastingly, AFEX pretreatment yielded a maximum ethanol titre of 1.98 g/L with same enzymatic consortium and bioethanol producers. Consequently, on scaling up the SSF experiments with 5% (w/v) AFEX-pretreated substrate in shake flask and bioreactor along with S. cerevisiae and C. shehatae, an ethanol titre of 9.78 and 17.97 g/L were obtained respectively.