Anshu Mathur

Enhanced cellulase production by Penicillium oxalicum for bio-ethanol application

Present study was focused on cellulase production from an indigenously isolated filamentous fungal strain, identified as Penicillium oxalicum. Initially, cellulase production under submerged fermentation in shake flasks resulted in cellulase activity of 0.7 FPU/mL. Optimization of process parameters enhanced cellulase production by 1.7-fold and resulted in maximum cellulase activity of 1.2 FPU/mL in 8 days. Cellulase production was successfully scaled-up to 7 L fermenter under controlled conditions and incubation time was reduced from 8 days to 4 days for achieving similar cellulase titer. Optimum pH and temperature for activity of the crude enzyme were pH 5 and 50 °C, respectively. At 50 °C the produced cellulase retained approximately 50% and 26% of its activity at 48 h and 72 h, respectively. Hydrolytic efficiency of P. oxalicum was comparable to commercial cellulase preparations which indicate its great potential for application in the lignocellulose hydrolysis.

Bioethanol production from wheat straw via enzymatic route employing Penicillium janthinellum cellulases.

This study concerns in-house development of cellulases from a mutant Penicillium janthinellum EMS-UV-8 and its application in separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes for bioethanol production from pre-treated wheat straw. In a 5L fermentor, the above strain could produce cellulases having activity of 3.1 FPU/mL and a specific activity of 0.83 FPU/mg of protein. In-house developed cellulase worked more efficiently in case of SSF as ethanol concentration of 21.6g/L and yield of 54.4% were obtained which were higher in comparison to SHF (ethanol concentration 12 g/L and 30.2% yield). This enzyme preparation when compared with commercial cellulase for hydrolysis of pre-treated wheat straw was found competitive. This study demonstrates that P. janthinellum EMS-UV-8 is a potential fungus for future large-scale production of cellulases.

Characterization of a new zeaxanthin producing strain of Chlorella saccharophila isolated from New Zealand marine waters

A fast growing strain of Chlorella saccharophila was isolated from the marine water of New Zealand and grown in heterotrophic conditions using glucose or glycerol as a carbon source. Biomass production was found to be higher in culture fed with glucose (2.14±0.08 g L(-1)) as compared to glycerol (0.378±0.04 g L(-1)). Lipid accumulation was similar for both carbon sources, at approximately 22% of dry cell weight. However, carotenoid yield was higher with glycerol (0.406±0.0125 mg g(-1)) than with glucose (0.21±0.034 mg g(-1)). Further optimization of the growth of the isolate gave maximal carotenoid production of 16.39±1.19 mg g(-1) total carotenoid, containing 11.32±0.64 mg g(-1) zeaxanthin and 5.07±0.55 mg g(-1) β-carotene. Comparison of various chemical and physical carotenoid extraction methods showed that ultrasonication was required for maximum extraction yields. The new strain has potential for biofuel, with carotenoid co-production.

Blending of cellulolytic enzyme preparations from different fungal sources for improved cellulose hydrolysis by increasing synergism

Cellulolytic enzymes were produced from the three fungal strains [P. janthinellum EMS-UV-8 (E), T. reesei Rut C-30 (R) and A. tubingenesis (A)] and used to prepare blends for the hydrolysis of avicel and acid treated wheat straw (A-WS). An enzyme blend prepared from three different crude preparations (E + R + A) on the basis of equivalent FPU or protein was found to be more synergistic and gave better hydrolysis of avicel or A-WS than the blend of two enzyme preparations (E + R, E + A and R + A) or individual enzyme preparations (E, R, and A). The triple blend gave two times higher hydrolysis of avicel or A-WS than the individual enzyme preparations at the same enzyme dosages. In all cases the individual or cumulative FPU or protein in blends was equal (10 FPU or 20 mg protein per g of substrate). Increased enzyme activities (CMCase and FPU) were found in the blends compared to the sum of individual enzyme activities added for the blend preparation. This reveals that the increased hydrolysis of cellulose by blends was a result of increased synergism between the same (endoglucanase) and/or different types of cellulases from different preparations. Enzyme blending is thus a facile, cost effective and sustainable approach for biomass saccharification for biofuels.

Biohydrogen production from a novel alkalophilic isolate Clostridium sp. IODB-O3

Hydrogen producing bacteria IODB-O3 was isolated from sludge and identified as Clostridium sp. by 16S rDNA gene analysis. In this study, biohydrogen production process was developed using low-cost agro-waste. Maximum H2 was produced at 37°C and pH 8.5. Maximum H2 yield was obtained 2.54±0.2mol-H2/mol-reducing sugar from wheat straw pre-hydrolysate (WSPH) and 2.61±0.1mol-H2/mol-reducing sugar from pre-treated wheat straw enzymatic-hydrolysate (WSEH). The cumulative H2 production (ml/L), 3680±105 and 3270±100, H2 production rate (ml/L/h), 153±5 and 136±5, and specific H2 production (ml/g/h), 511±5 and 681±10 with WSPH and WSEH were obtained, respectively. Biomass pre-treatment via steam-explosion generates ample amount of WSPH which remains unutilized for bioethanol production due to non-availability of efficient C5-fermenting microorganisms. This study shows that Clostridium sp. IODB-O3 is capable of utilizing WSPH efficiently for biohydrogen production. This would lead to reduced economic constrain on the overall cellulosic ethanol process and also establish a sustainable biohydrogen production process.

Untreated wheat straw: potential source for diverse cellulolytic enzyme secretion by Penicillium janthinellum EMS-UV-8 mutant.

Study describes the production of cellulases by Penicillium janthinellum EMS-UV-8 using untreated wheat straw (WS), treated WS (acid, alkali, steam exploded, organo-solv) and pure cellulosic substrates (avicel, cellulose-II and carboxymethyl cellulose). Severely pretreated WS and cellulose-II produced more cellulolytic enzymes than untreated samples. XRD and FTIR analysis revels that the increase in the amorphous structure of pretreated WS/cellulose increases enzyme production. Enzyme samples prepared using different substrates were used for the hydrolysis of dilute acid treated wheat straw (DATWS), steam exploded wheat straw (SEWS) and avicel. The enzyme prepared using untreated WS gave more hydrolysis of DATWS and SEWS than the enzyme prepared using pretreated WS or pure cellulosic substrates. This revels that more diverse/potential enzymes were secreted by P. janthinellum EMS-UV-8 mutant using untreated WS. This study may contribute in production of efficient enzyme mixture/cocktail by single fungal strain for economic conversion of biomass to sugars.

Improved saccharification of pilot-scale acid pretreated wheat straw by exploiting the synergistic behavior of lignocellulose degrading enzymes

Requirement of high enzyme dosage for lignocellulosic biomass hydrolysis is one of the challenges for the viability of the second generation bioethanol technology. Here, an optimal enzyme mixture was developed by partially replacing the cellulase proportion with accessory enzymes (β-glucosidase, xylanase, pectinase, laccase) and its hydrolytic performance was compared with different commercial counterparts for the saccharification of pretreated wheat straw (PWS) using a 250 kg per day continuous pilot plant. Maximum degree of synergism was observed with xylanase followed by pectinase, laccase, and β-glucosidase. The statistically optimized enzyme mixture enhanced hydrolysis by 51.23% and 40.66% in 6 h and 24 h, respectively. This study elucidates that presence of even small amount of oligomers and cellobiose pose a strong inhibition for the enzymes. Therefore, development of an optimal enzyme formulation is a sustainable approach to reduce overall enzyme loading for biomass saccharification.

Exploring omega-3 fatty acids, enzymes and biodiesel producing thraustochytrids from Australian and Indian marine biodiversity

The marine environment harbours a vast diversity of microorganisms, many of which are unique, and have potential to produce commercially useful materials. Therefore, marine biodiversity from Australian and Indian habitat has been explored to produce novel bioactives, and enzymes. Among these, thraustochytrids collected from Indian habitats were shown to be rich in saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs), together constituting 51–76 % of total fatty acids (TFA). Indian and Australian thraustochytrids occupy separate positions in the dendrogram, showing significant differences exist in the fatty acid profiles in these two sets of thraustochytrid strains. In general, Australian strains had a higher docosahexaenoic acid (DHA) content than Indian strains with DHA at 17–31 % of TFA. A range of enzyme activities were observed in the strains, with Australian strains showing overall higher levels of enzyme activity, with the exception of one Indian strain (DBTIOC‐1). Comparative analysis of the fatty acid profile of 34 strains revealed that Indian thraustochytrids are more suitable for biodiesel production since these strains have higher fatty acids content for biodiesel (FAB, 76 %) production than Australian thraustochytrids, while the Australian strains are more suitable for omega‐3 (40 %) production.

Second generation bioethanol production at high gravity of pilot-scale pretreated wheat straw employing newly isolated thermotolerant yeast Kluyveromyces marxianus DBTIOC-35

Second-generation bioethanol production by a newly isolated thermotolerant yeast strain was studied at 42 °C and above using pilot-scale dilute acid pretreated wheat straw (WS) as feedstock. This strain was identified as Kluyveromyces marxianus DBTIOC-35 by biochemical characterization as well as molecular phylogenetic analysis of the ITS-5.8S rRNA gene and D1/D2 domain of the 26S rRNA gene after PCR amplification and sequencing. Simultaneous saccharification and fermentation (SSF) at 42 °C and 45 °C using 10% biomass loading resulted in ethanol titers of 29.0 and 16.1 g L−1, respectively. At 42 °C ethanol productivity was higher during SSF (0.92 g L−1 h−1) than separate hydrolysis and fermentation (SHF) (0.49 g L−1 h−1) at 20% biomass loading. The results indicated that at 20% biomass loading, SSF without pre-saccharification led to more ethanol production (66.2 g L−1 with 83.3% yield) at a faster rate than SSF with pre-saccharification (PSSF) which produced an ethanol titer of 61.8 g L−1, 77.7% yield and productivity of 0.86 g L−1 h−1. Based on these findings, application of newly isolated yeast K. marxianus DBTIOC-35 in SSF of lignocellulosic biomass can eliminate the pre-saccharification step which is a novel advantage of thermotolerant yeasts in terms of cutting down the overall biomass to bioethanol process time and enhancing bioethanol titer, yields and productivities.

Lignocellulosic bioethanol production employing newly isolated inhibitor and thermotolerant Saccharomyces cerevisiae DBTIOC S24 strain in SSF and SHF

Bioethanol is a renewable alternative to fossil fuels which facilitate energy security and reduce greenhouse-gas emissions. High gravity fermentation employing a thermo and inhibitor tolerant strain is a promising technology to reduce fermentation time as well as cost. The present study investigates lignocellulosic ethanol production using inhibitor and thermotolerant S. cerevisiae DBTIOC S24 from non-detoxified and unsterilized rice straw hydrolysate. Efficient ethanol production was observed at a wide range of pH (3–7) and temperature (25–42 °C) using S. cerevisiae isolate. In the presence of lignocellulosic derived inhibitors, a maximum of 75.33 g L−1 (85.56%) and 73.30 g L−1 (79.93%) ethanol was produced at 30 °C and 42 °C, respectively. During fermentation, pH plays an important role in overcoming the synergistic effect of inhibitors. More than 80.65% and 73.5% ethanol yield was achieved employing this isolate with high solid loading (20%) and 20 FPU g−1 of solid loading via simultaneous saccharification and fermentation (SSF) and separate hydrolysis and fermentation (SHF), respectively. While, 91% ethanol yield was obtained during fermentation using rice enzymatic hydrolysate. These values are comparable to the best results reported. Therefore, this isolate has great potential due to its inhibitor and thermotolerant characteristics for lignocellulosic ethanol production at the industrial scale with a lower process time and cost.