Enrichment of Methylocystis dominant mixed culture from rice field for PHB production.

Presence of methanotrophs in diverse environmental habitats helps to reduce emissions of greenhouse gas like methane. Isolation and culture of undiscovered wealth of methanotrophic organisms can help in exploitation of these organisms in value added products. The present study focuses on the enrichment of methanotroph dominated mixed microbial community by use of three stage strategy of revival, proliferation, and segregation. During the enrichment process amplicon sequencing of 16 s rRNA V3-V4 region showed relative abundance of mixed culture comprising single methanotrophic species of Methylocystis genus (88.92%) along with only three other species. Methylocystis dominant mixed culture (MMI-11) was observed to produce polyhydroxyalkanoates (PHA). During studies to identify favourable culture conditions, nitrate was found to be preferred nitrogen source for growth and PHA production. Cell growth ability to produce PHA was also evaluated at 14 L fermentor by supplying gas using continuous bubbling and through pressurization in the headspace. The mixed methanotrophic culture was found to accumulate maximum of 22.20% polyhydroxybutyrate (PHB) under nitrate limited condition. The molecular weight of PHB was found to be 2.221 × 105 g mol-1 with polydispersity of 1.82.

Selection of methanotrophic platform for methanol production using methane and biogas.

To develop biotechnological process for methane to methanol conversion, selection of a suitable methanotrophic platform is an important aspect. Systematic approach based on literature and public databases was developed to select representative methanotrophs Methylotuvimicrobium alcaliphilum, Methylomonas methanica, Methylosinus trichosporium and Methylocella silvestris. Selected methanotrophs were further investigated for methanol tolerance and methanol production on pure methane as well as biogas along with key enzyme activities involved in methane utilization. Among selected methanotrophs M. alcaliphilum showed maximum methanol tolerance of 6% v/v along with maximum methanol production of 307.90 mg/L and 247.37 mg/L on pure methane and biogas respectively. Activity of methane monooxygenase and formate dehydrogenase enzymes in M.alcaliphilum was significantly higher up to 98.40 nmol/min/mg cells and 0.87 U/mg protein, respectively. Biotransformation trials in 14 L fermentor resulted in increased methanol production up to 418 and 331.20 mg/L, with yield coefficient 0.83 and 0.71 mg methanol/mg of pure methane and biogas respectively. The systematic selection resulted in haloalkaliphilic strain M. alcaliphilum as one of the potential methanotroph for bio-methanol production.

Critical factors affecting ethanol production by immobilized Pichia stipitis using corn cob hemicellulosic hydrolysate.

Fermentation of xylose from hydrolysate of acid-treated corn cob by Pichia stipitis is inhibited by acetic acid and lignin derivatives. In the present study, we have designed and implemented an immobilized cell culture for xylose to ethanol conversion from acid-treated corn cob hydrolysate without the removal of fermentation inhibitors. In this study, cultivations of suspended and immobilized Pichia were compared in terms of ethanol yield and productivity to investigate whether the cell immobilization could improve resistance to inhibitors. Cell immobilization clearly favored the fermentative metabolism in nondetoxified corn cob hydrolysate leading to an improvement of twofold ethanol productivity as compared to that achieved with suspension culture. Calcium alginate as an immobilization matrix was selected to immobilize Pichia cells. Concentrations of sodium alginate, calcium chloride, and fermentor agitation speed were optimized for ethanol production using statistical method. Statistical analysis showed that agitation speed had maximum influence on ethanol production by immobilized Pichia cells. In comparison to suspension culture, immobilization had a positive impact on the fermentative metabolism of Pichia, improving the ethanol yield from 0.40 to 0.43 g/g and productivity from 0.31 to 0.51 g/L/h for acid-treated corn cob hydrolysate.

Evaluation of fermentation kinetics of acid-treated corn cob hydrolysate for xylose fermentation in the presence of acetic acid by Pichia stipitis.

The efficient utilization of lignocellulosic biomass for ethanol production depends on the fermentability of the biomass hydrolysate obtained after pretreatment. In this work we evaluated the kinetics of ethanol production from xylose using Pichia stipitis in acid-treated corn cob hydrolysate. Acetic acid is one of the main inhibitors in corn cob hydrolysate that negatively impacts kinetics of xylose fermentation by P. stipitis. Unstructured kinetic model has been formulated that describes cell mass growth and ethanol production as a function of xylose, oxygen, ethanol, and acetic acid concentration. Kinetic parameters were estimated under different operating conditions affecting xylose fermentation. This is the first report on kinetics of xylose fermentation by P. stipitis which includes inhibition of acetic acid on growth and product formation. In the presence of acetic acid in the hydrolysate, the model accurately predicted reduction in maximum specific growth rate (from 0.23 to 0.15 h-1) and increase in ethanol yield per unit biomass (from 3 to 6.2 gg-1), which was also observed during experimental trials. Presence of acetic acid in the fermentation led to significant reduction in the cell growth rate, reduction in xylose consumption and ethanol production rate. The developed model accurately described physiological state of P. stipitis during corn cob hydrolysate fermentation. Proposed model can be used to predict the influence of xylose, ethanol, oxygen, and acetic acid concentration on cell growth and ethanol productivity in industrial fermentation.

Xylitol production by genetically modified industrial strain of Saccharomyces cerevisiae using glycerol as co-substrate.

Xylitol is commercially used in chewing gum and dental care products as a low calorie sweetener having medicinal properties. Industrial yeast strain of S. cerevisiae was genetically modified to overexpress an endogenous aldose reductase gene GRE3 and a xylose transporter gene SUT1 for the production of xylitol. The recombinant strain (XP-RTK) carried the expression cassettes of both the genes and the G418 resistance marker cassette KanMX integrated into the genome of S. cerevisiae. Short segments from the 5' and 3' delta regions of the Ty1 retrotransposons were used as homology regions for integration of the cassettes. Xylitol production by the industrial recombinant strain was evaluated using hemicellulosic hydrolysate of the corn cob with glucose as the cosubstrate. The recombinant strain XP-RTK showed significantly higher xylitol productivity (212 mg L-1 h-1) over the control strain XP (81 mg L-1 h-1). Glucose was successfully replaced by glycerol as a co-substrate for xylitol production by S. cerevisiae. Strain XP-RTK showed the highest xylitol productivity of 318.6 mg L-1 h-1 and titre of 47 g L-1 of xylitol at 12 g L-1 initial DCW using glycerol as cosubstrate. The amount of glycerol consumed per amount of xylitol produced (0.47 mol mol-1) was significantly lower than glucose (23.7 mol mol-1). Fermentation strategies such as cell recycle and use of the industrial nitrogen sources were demonstrated using hemicellulosic hydrolysate for xylitol production.

Xylitol production by Saccharomyces cerevisiae overexpressing different xylose reductases using non-detoxified hemicellulosic hydrolysate of corncob.

Xylitol production was compared in fed batch fermentation by Saccharomyces cerevisiae strains overexpressing xylose reductase (XR) genes from Candida tropicalis, Pichia stipitis, Neurospora crassa, and an endogenous gene GRE3. The gene encoding a xylose specific transporter (SUT1) from P. stipitis was cloned to improve xylose transport and fed batch fermentation was used with glucose as a cosubstrate to regenerate NADPH. Xylitol yield was near theoretical for all the strains in fed batch fermentation. The highest volumetric (0.28 gL-1 h-1) and specific (34 mgg-1 h-1) xylitol productivities were obtained by the strain overexpressing GRE3 gene, while the control strain showed 7.2 mgg-1 h-1 specific productivity. The recombinant strains carrying XR from C. tropicalis, P. stipitis, and N. crassa produced xylitol with lower specific productivity of 14.3, 6.8, and 6.3 mgg-1 h-1, respectively, than GRE3 overexpressing strain. The glucose fed as cosubstrate was converted to biomass and ethanol, while xylose was only converted to xylitol. The efficiency of ethanol production was in the range of 38-45 % of the theoretical maximum for all the strains. Xylitol production from the non-detoxified corncob hemicellulosic hydrolysate by recombinant S. cerevisiae was reported for the first time. Xylitol productivity was found to be equivalent in the synthetic xylose as well as hemicellulosic hydrolysate-based media showing no inhibition on the S. cerevisiae due to the inhibitors present in the hydrolysate. A systematic evaluation of heterologous XRs and endogenous GRE3 genes was performed, and the strain overexpressing the endogenous GRE3 gene showed the best xylitol productivity.

Enhancement of over expression and chaperone assisted yield of folded recombinant aconitase in Escherichia coli in bioreactor cultures.

A major portion of the over expressed yeast mitochondrial aconitase, a large 82 kDa monomeric TCA cycle enzyme, in Escherichia coli led to the formation of inclusion bodies. Bacterial chaperonin GroEL mediated the correct folding of aconitase with the assistance of its co-chaperonin GroES in an ATP dependent manner. Till date the chaperonin assisted folding of aconitase was limited to the shake flask studies with relatively low yields of folded aconitase. No attempt had yet been made to enhance the yield of chaperone mediated folding of aconitase using a bioreactor. The current report deals with the effect of co-expression of GroEL/GroES in the production of soluble, biologically active recombinant aconitase in E. coli by cultivation in a bioreactor at different temperatures under optimized conditions. It revealed that the yield of functional aconitase was enhanced, either in presence of co-expressed GroEL/ES or at low temperature cultivation. However, the outcome from the chaperone assisted folding of aconitase was more pronounced at lower temperature. A 3-fold enhancement in the yield of functional aconitase from the bioreactor based chaperone assisted folding was obtained as compared to the shake flask study. Hence, the present study provides optimized conditions for increasing the yield of functional aconitase by batch cultivation in a bioreactor.

Secretory expression of interferon-alpha 2b in recombinant Pichia pastoris using three different secretion signals.

Human interferon-alpha 2b (IFN-alpha2b) was cloned and expressed in Pichia pastoris under the control of alcohol oxidase promoter (AOX1) using three different secretion signals. Native secretion signal of IFN-alpha2b, Saccharomycescerevisiae MF-alpha factor prepro sequence and a mutated alpha prepro sequence without the Glu-Ala (EAEA) repeats were used separately for directing the secretion of IFN-alpha2b into the culture medium of P. pastoris. The native secretion signal of IFN-alpha2b did not secrete protein into the culture medium of P. pastoris. The alpha prepro sequence without the EAEA repeats directed the secretion of maximum amount of IFN-alpha2b (200 mg/l) into the culture medium, with the same amino acid sequence as that of the native IFN-alpha2b secreted by human lymphocytes. The full alpha prepro sequence, having both the protease cleavage sites for KEX2 and STE13 gene products, also secreted an equivalent amount of IFN-alpha2b into the culture medium. However, two interferon bands with similar molecular masses were observed, when full alpha prepro sequence was used for the secretion of IFN-alpha2b. The difference in the molecular masses of the two bands was found to arise due to the difference in the molecular masses of the N-terminal fragment, and the inefficient processing of secretion signal.