Browsing by Author "Prabhu, Ashish A."
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Item Open Access Development of hypertolerant strain of Yarrowia lipolytica accumulating succinic acid using high levels of acetate(American Chemical Society, 2022-08-09) Narisetty, Vivek; Prabhu, Ashish A.; Bommareddy, Rajesh Reddy; Cox, Rylan; Agrawal, Deepti; Misra, Ashish; Ali Haider, M.; Bhatnagar, Amit; Pandey, Ashok; Kumar, VinodAcetate is emerging as a promising feedstock for biorefineries as it can serve as an alternate carbon source for microbial cell factories. In this study, we expressed acetyl-CoA synthase in Yarrowia lipolytica PSA02004PP, and the recombinant strain grew on acetate as the sole carbon source and accumulated succinic acid or succinate (SA). Unlike traditional feedstocks, acetate is a toxic substrate for microorganisms; therefore, the recombinant strain was further subjected to adaptive laboratory evolution to alleviate toxicity and improve tolerance against acetate. At high acetate concentrations, the adapted strain Y. lipolytica ACS 5.0 grew rapidly and accumulated lipids and SA. Bioreactor cultivation of ACS 5.0 with 22.5 g/L acetate in a batch mode resulted in a maximum cell OD600 of 9.2, with lipid and SA accumulation being 0.84 and 5.1 g/L, respectively. However, its fed-batch cultivation yielded a cell OD600 of 23.5, SA titer of 6.5 g/L, and lipid production of 1.5 g/L with an acetate uptake rate of 0.2 g/L h, about 2.86 times higher than the parent strain. Cofermentation of acetate and glucose significantly enhanced the SA titer and lipid accumulation to 12.2 and 1.8 g/L, respectively, with marginal increment in cell growth (OD600: 26.7). Furthermore, metabolic flux analysis has drawn insights into utilizing acetate for the production of metabolites that are downstream to acetyl-CoA. To the best of our knowledge, this is the first report on SA production from acetate by Y. lipolytica and demonstrates a path for direct valorization of sugar-rich biomass hydrolysates with elevated acetate levels to SA.Item Open Access Enhanced 2,3-Butanediol production by mutant Enterobacter ludwigii using Brewers’ spent grain hydrolysate: process optimization for a pragmatic biorefinery loom(Elsevier, 2021-06-18) Amraoui, Yassin; Prabhu, Ashish A.; Narisetty, Vivek; Coulon, Frederic; Chandel, Anuj Kumar; Willoughby, Nicholas; Jacob, Samuel; Koutinas, Apostolis; Kumar, Vinod2,3-Butanediol (BDO) is a fossil-based versatile bulk chemical with a multitude of applications. BDO can also be synthesized using microbial cell factories harnessing renewable feedstocks. However, the high cost of the substrate via microbial route impedes commercial manufacturing of BDO. Therefore, identification of cheaper substrates could make bio-based BDO production more cost-competitive. Brewers’ spent grain (BSG), a major by-product of breweries, is an inexpensive source of fermentable sugars and proteins. In the present study, we have attempted the bioproduction of BDO by Enterobacter ludwigii using BSG as feedstock. A random E. ludwigii mutant obtained after treatment with ethyl methane sulfonate (EMS) resulted in a BDO titer (9.5 g/L), ~30% higher in comparison to the wild type strain with a yield of 0.48 gBDO/gGlucose approaching the theoretical yield of 0.50 gBDO/gGlucose. The enzymatic hydrolysis of microwave-assisted alkali pretreated BSG was optimized using the statistical Taguchi design. The BSG hydrolysis under optimal conditions (pH: 6.0; temperature: 50 °C; BSG: 10% w/v; enzyme loading: 2% v/v) resulted in a glucose yield of 0.25 gGlucose/gBiomass. The uncontrolled pH was found to be more beneficial for BDO accumulation from BSG hydrolysate in batch bioreactor cultivation as compared with controlled one. The fed-batch cultivation with forced pH fluctuations at an aeration rate of 2.0 vvm resulted in BDO accumulation of 118.5 g/L from glucose-rich BSG hydrolysate with the yield and productivity of 0.43 g/g and 1.65 g/L.h, respectively. To the best of our knowledge, this is the first study on BDO production from BSG.Item Open Access Enhanced xylitol production using non-detoxified xylose rich pre-hydrolysate from sugarcane bagasse by newly isolated Pichia fermentans(BioMed Central / Springer Verlag, 2020-12-29) Prabhu, Ashish A.; Bosakornranut, Ekkarin; Amraoui, Yassin; Agrawal, Deepti; Coulon, Frederic; Vivekanand, Vivekanand; Thakur, Vijay Kumar; Kumar, VinodBackground Integrated management of hemicellulosic fraction and its economical transformation to value-added products is the key driver towards sustainable lignocellulosic biorefineries. In this aspect, microbial cell factories are harnessed for the sustainable production of commercially viable biochemicals by valorising C5 and C6 sugars generated from agro-industrial waste. However, in the terrestrial ecosystem, microbial systems can efficiently consume glucose. On the contrary, pentose sugars are less preferred carbon source as most of the microbes lack metabolic pathway for their utilization. The effective utilization of both pentose and hexose sugars is key for economical biorefinery. Results Bioprospecting the food waste and selective enrichment on xylose-rich medium led to screening and isolation of yeast which was phylogenetically identified as Pichia fermentans. The newly isolated xylose assimilating yeast was explored for xylitol production. The wild type strain robustly grew on xylose and produced xylitol with > 40% conversion yield. Chemical mutagenesis of isolated yeast with ethyl methanesulphonate (EMS) yielded seven mutants. The mutant obtained after 15 min EMS exposure, exhibited best xylose bioconversion efficiency. This mutant under shake flask conditions produced maximum xylitol titer and yield of 34.0 g/L and 0.68 g/g, respectively. However, under the same conditions, the control wild type strain accumulated 27.0 g/L xylitol with a conversion yield of 0.45 g/g. Improved performance of the mutant was attributed to 34.6% activity enhancement in xylose reductase with simultaneous reduction of xylitol dehydrogenase activity by 22.9%. Later, the culture medium was optimized using statistical design and validated at shake flask and bioreactor level. Bioreactor studies affirmed the competence of the mutant for xylitol accumulation. The xylitol titer and yield obtained with pure xylose were 98.9 g/L and 0.67 g/g, respectively. In comparison, xylitol produced using non-detoxified xylose rich pre-hydrolysate from sugarcane bagasse was 79.0 g/L with an overall yield of 0.54 g/g. Conclusion This study demonstrates the potential of newly isolated P. fermentans in successfully valorising the hemicellulosic fraction for the sustainable xylitol productionItem Open Access Integrated biorefineries for repurposing of food wastes into value-added products(Elsevier, 2022-09-01) Narisetty, Vivek; Adlakha, Nidhi; Singh, Navodit Kumar; Dalei, Sudipt Kumar; Prabhu, Ashish A.; Nagarajan, Sanjay; Kumar, A. Naresh; Nagoth, Joseph Amruthraj; Kumar, Gopalakrishnan; Singh, Vijai; Kumar, VinodFood waste (FW) generated through various scenarios from farm to fork causes serious environmental problems when either incinerated or disposed inappropriately. The presence of significant amounts of carbohydrates, proteins, and lipids enable FW to serve as sustainable and renewable feedstock for the biorefineries. Implementation of multiple substrates and product biorefinery as a platform could pursue an immense potential of reducing costs for bio-based process and improving its commercial viability. The review focuses on conversion of surplus FW into range of value-added products including biosurfactants, biopolymers, diols, and bioenergy. The review includes in-depth description of various types of FW, their chemical and nutrient compositions, current valorization techniques and regulations. Further, it describes limitations of FW as feedstock for biorefineries. In the end, review discuss future scope to provide a clear path for sustainable and net-zero carbon biorefineries.Item Open Access Microbial itaconic acid production from starchy food waste by newly isolated thermotolerant Aspergillus terreus strain(Elsevier, 2021-06-17) Narisetty, Vivek; Prabhu, Ashish A.; Al-Jaradah, Khalid; Gopaliya, Deeksha; Hossain, Abeer H.; Khare, Sunil Kumar; Punt, Peter J.; Kumar, VinodIn the present study, we have explored the potential of newly isolated Aspergillus terreus BD strain, which can accumulate itaconic acid (IA) at higher temperature. The shake flask cultivation of thermotolerant strain with medium optimized using Box-Behnken Design at 45 °C resulted in IA accumulation of 28.9 g/L with yield of 0.27 g/g. The enzymatic saccharification of the synthetic food waste (SFW) consisting of potatoes, rice & noodles were optimized using Taguchi method of orthogonal array to maximize the release of fermentable sugar. The maximum glucose release of 0.60 g/g was achieved with 10% biomass loading, 5% enzyme concentration, pH 5.5 and temperature 60 0C. The sugars obtained from SFW was integrated with IA production and maximum IA titer achieved with SFW hydrolysate during bioreactor cultivation was 41.1 g/L with conversion yield of 0.27 g/g while with pure glucose IA titer and yield were 44.7 g/L and 0.30 g/g, respectively.Item Open Access Prospects on bio-based 2,3-butanediol and acetoin production: recent progress and advances(Elsevier, 2021-06-29) Maina, Sofia; Prabhu, Ashish A.; Vivek, Narisetty; Vlysidis, Anestis; Koutinas, Apostolis; Kumar, VinodThe bio-based platform chemicals 2,3-butanediol (BDO) and acetoin have various applications in chemical, cosmetics, food, agriculture, and pharmaceutical industries, whereas the derivatives of BDO could be used as fuel additives, polymer and synthetic rubber production. This review summarizes the novel technological developments in adapting genetic and metabolic engineering strategies for selection and construction of chassis strains for BDO and acetoin production. The valorization of renewable feedstocks and bioprocess development for the upstream and downstream stages of bio-based BDO and acetoin production are discussed. The techno-economic aspects evaluating the viability and industrial potential of bio-based BDO production are presented. The commercialization of bio-based BDO and acetoin production requires the utilization of crude renewable resources, the chassis strains with high fermentation production efficiencies and development of sustainable purification or conversion technologies.Item Open Access Recent advances in bioethanol production from lignocelluloses: a comprehensive review with a focus on enzyme engineering and designer biocatalysts(Green Wave Publishing of Canada, 2020-12-01) Lugani, Yogita; Rai, Rohit; Prabhu, Ashish A.; Maan, Poonam; Hans, Meenu; Kumar, Vinod; Kumar, Sachin; Chandel, Anuj K.; Sengar, R. S.Many countries have their biofuel policy programs in place as part of their overall strategy to achieve sustainable development. Among biofuels, bioethanol as a promising alternative to gasoline is of substantial interest. However, there is limited availability of a sufficient quantity of bioethanol to meet demands due to bottlenecks in the present technologies to convert non-edible feedstocks, including lignocelluloses. This review article presents and critically discusses the recent advances in the pretreatment of lignocellulosic biomass, with a focus on the use of green solvents, including ionic liquids and deep eutectic solvents, followed by enzymatic saccharification using auxiliary proteins for the efficient saccharification of pretreated biomass. Different techniques used in strain improvement strategies to develop hyper-producing deregulated lignocellulolytic strains are also compared and discussed. The advanced techniques employed for fermentation of mixed sugars contained in lignocellulosic hydrolysates for maximizing bioethanol production are summarized with an emphasis on pathway and transporters engineering for xylose assimilation. Further, the integration of different steps is suggested and discussed for efficient biomass utilization and improved ethanol yields and productivity.