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The diseases related to the cardiovascular system significantly contribute towards global mortality. Dysregulation/malfunctioning of coagulation and fibrinolytic pathways especially under pathophysiological conditions disturbs the homeostasis and causes cardiovascular complications. Thrombosis is one such disorder wherein normal blood flow is obstructed due to an abnormal fibrin clot. Serious issues with the available therapeutics (thrombolytics) viz. fatal side effects, low specificity and efficacy and high cost, motivate the research for discovery of potentially novel, safe, efficacious and cost-effective thrombolytics. In the present study, an effectual fibrin specific fibrinolytic protease (thrombolytic) was produced from a newly isolated fungus Aspergillus flavus SH71 by using low-cost agricultural residues as substrates. Molasses as a sole carbon source and linseed cake as an exclusive nitrogen source supported the protease production of 188.99 U/mL and 215.67 U/mL respectively which were higher than that obtained in control (133.91 U/mL). Also, some other agro-based substrates (orange peel, rice bran, malt, mixed fruit brunch and soymeal) supported higher enzyme titre than control. Furthermore, a bioprocess was developed for fibrinolytic protease production by optimization of process variables i.e. concentration of molasses and linseed cake, medium pH and incubation time based on design of experiments and a substantial yield enhancement was achieved (1.97 folds).

Quest for renewable/eco-friendly energy sources has received immense focus in recent years. Current study involved consolidated bioprocessing of Saccharum spontaneum biomass (SSB) for biofuel-ethanol generation in a 'one pot consolidated bioprocess' (OPCB). SSB was pretreated with protic ionic liquid, triethylamine-bisulfate ([TEA][HSO

The efficacy of ionic liquid (IL)-based pretreatment of lignocellulosic biomass (LB) can be enhanced by simultaneous application of surfactants/salts/deep eutectic solvent (DES) systems which may realize more effectual biorefining of LB to biofuels or other commodities. However, due to inhibitory nature of IL, IL-stable saccharification enzymes (cellulase/xylanase) are desired for enzymatic hydrolysis of IL-pretreated biomass. Bacillus spp. are considered as the super microbial factories for production of commercially important robust enzymes. The current study presents the enhanced production (1.438-fold) of an IL-stable cellulase from a newly isolated IL-tolerant Bacillus amyloliquefaciens SV29 by statistical optimization using agroindustrial residues as carbon (groundnut shell) and nitrogen source (mustard cake). The process variables such as groundnut shell and mustard cake, incubation time, and inoculum size were optimized. The enzyme preparation (cellulase/xylanase) was evaluated for its saccharification potential of Prosopis sp. (twigs/pods) biomass that was pretreated either with IL (1-ethyl-3-methylimidazolium methanesulfonate, EMIMS) standalone or IL along with surfactants/salts/DES systems in a consolidated bioprocess (CBP), i.e., one pot consolidated bioprocess, due to several technoeconomic advantages of the latter. No reported studies are available on bioconversion of Prosopis sp. biomass using IL or CBP. Sugar yield was enhanced when IL was used along with either DES choline chloride glycerol (54.4%) or with FeSO4 (51%). Thus, the pretreatment efficacy of EMIMS is substantially enhanced when used in combination with choline chloride glycerol or FeSO4 for getting increased sugar yield upon enzymatic hydrolysis of Prosopis sp. biomass with IL-stable enzymes.

Lignocellulosic biomass (LB) despite its huge potential as a renewable bioenergy resource faces bottlenecks due to its recalcitrance and lack of appropriate pretreatment approaches. The current study evaluates the combinatorial application of alkali and acid pretreatment of pine needle biomass (PNB), for achieving high sugar release upon enzymatic saccharification. Pine needle accumulation poses a big threat to the forest soil fertility and overall ecosystem and environment. However, pine needle waste can be valorized after appropriate pretreatment and enzymatic saccharification for production of renewable energy, i.e. biofuel–ethanol. In combinatorial pretreatment strategy, first PNB was subjected to ammonium carbonate pretreatment, and parameters like ammonium carbonate concentration, incubation time and pretreatment temperature were optimized using design of experiment (DoE) approach. The relative influence of parameters on efficacy of pretreatment was established individually and in interactive terms. Based on DoE, sugar yield of 7.56 mg/g of PNB was obtained. Furthermore, DoE-based pretreated PNB was subjected to sulphuric acid pretreatment, followed by enzymatic saccharification. The sugar released during various steps was pooled (8.19 g/100 g), concentrated and subjected to ethanol fermentation with dual yeast cultures using Saccharomyces cerevisiae and Pichia stipitis. An ethanol yield of 8.8%, v/v (6.94% w/v), was obtained. This represents the process efficiency of 19.34% for bioethanol production from PNB.

Lignocellulosic biomass (LB) based ethanol-biofuel represents a promising, sustainable, clean and renewable energy resource to meet the ever-growing global energy demand, and to mitigate the environmental issues related to excessive fuel usage. However, the technology faces multifaceted challenges like high cost/energy intensive and environmentally-unsafe LB pretreatments, lack of appropriate saccharification enzymes, poor process efficacy, and others. Deep-eutectic-solvent (DES) based LB pretreatment has recently emerged as an eco-benign approach which offers multitude of advantages over conventional methods of biomass conversion. Consolidated bioprocessing, CBP provides discreet benefits over traditional multi-reactor biomass processing. Scanty reports are available on application of Saccharum spontaneum biomass (SSB) as a promising biorefinery feedstock despite its huge potential. No studies have yet been undertaken on CBP of SSB by using DESs, and the present study is first such report. In the current study, SSB after pretreatment with DES i.e. choline chloride-glycerol (ChCl-G) and Ca(OH)2 was subjected to in-situ enzymatic hydrolysis with DES-compatible cellulase-xylanase enzymes (Bacillus subtilis G2) followed by ethanol fermentation of sugars, in one pot consolidated bioprocess. Optimization of CBP for pretreatment and enzymatic saccharification via design of experiment resulted in substantially increased sugar yield (4.94-fold) i.e. from 75.25 mg/g biomass (unoptimized) to 372.3 mg/g biomass (after optimization). Fermentation of sugars under CBP yielded an ethanol content of 173.61 mg/g of SSB (60.28% of the theoretical yield). Extensive characterization of pretreated SSB based on SEM, FT-IR, XRD, NMR, surface area measurements, cellulase adsorption isotherms, and water retention capacity further provided molecular insights into the pretreatment mechanisms.

A novel strategy involving sodium dodecylsulfate (SDS) (SDS assisted tris (2-hydroxyethyl) methyl- ammonium methyl sulphate ([TMA][MeSO4], ionic liquid) pretreatment of Saccharum spontaneum biomass (SSB) following its enzymatic saccharification, and conversion into ethanol-biofuel in a consolidated bioprocess (CBP) was developed. Ionic liquid stable enzyme preparation developed from Bacillus subtilis G2 was used for saccharification. Optimized pretreatment and saccharification variables enhanced the sugar yield (2.35-fold), which was fermented to ethanol content of 104.42 mg/g biomass with an efficiency of 35.73%. The pretreated biomass was examined for textural/ultrastructural alterations by scanning electron microscopy (SEM), 1H/13C nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), surface area measurements, water retention value, and cellulase adsorption isotherms. The combined [TMA][MeSO4] and SDS pretreatment disrupted the lignocellulosic microfibrils, and increased the porosity and surface area. The study provides new mechanistic insights on combined IL and surfactant pretreatment of biomass for its efficient conversion to biofuel.

Lignocellulosic-bioethanol-fuel (LBF) technology faces challenges due to cost/energy intensive nature of lignocellulosic biomass (LB) pretreatment and saccharification. Ionic liquid (IL) based pretreatment of LB has recently emerged as an environmentally friendly approach. However, for saccharification of IL pretreated LB, IL stable enzymes must be used else the latter may be inhibited. Furthermore, availability of IL stable enzymes may help designing novel consolidated process by in situ coupling of IL mediated pretreatment of LB and enzymatic saccharification processes in a single vessel, that is, one pot consolidated bioprocess (OPCB). OPCB may potentially offer new avenues for technoeconomic feasibility of LBF. In this study, an OPCB was developed and optimized for the first time for bioethanol production from pine needle biomass (PNB) using in situ IL pretreatment and enzymatic saccharification in a single pot. Various process parameters, namely, biomass loading, reaction time, and cellulase/xylanase enzyme dose were optimized, and sugar yield of 1.88 g per five gram of PNB was obtained. The sugar hydrolysate obtained was subjected to ethanol fermentation using dual yeast culture, that is, Saccharomyces cerevisiae and Pichia stipitis. The maximum ethanol yield of 0.148 g/g PNB was obtained after 72 h of fermentation representing an efficiency of 41.39%. The study shows that consolidated processing of PNB may be an efficient, sustainable, and feasible approach for valorization of PNB for industrial production of second generation ethanol-biofuel. © 2017 American Institute of Chemical Engineers Environ Prog, 2017

Enzyme-based processes promise a sustainable and environmentally-friendly solution to the pollution that results from industrial technologies. Thermozymes are extremely temperature-stable biocatalysts that broaden the application horizon of enzymes. Thermophiles and hyperthermophiles belonging to several genera viz. Bacillus, Clostridium, Pyrococcus, Thermus, Thermotoga, and Aquifex have been explored for thermozymes. Thermozymes, in addition to offering thermostability, are also equipped with properties such as solvent tolerance, resistance to denaturants, substrate-selectivity, and higher reaction rates; thus, making them potentially more suitable for applications as compared with mesozymes. Adaptive mechanisms of thermozymes include modifications in amino acid sequence/composition, hydrogen bonding patterns, electrostatic interactions, disulfide bonds, hydrophobic interactions, and metal binding ability, resulting in superior conformational structure. Investigation of thermostable biocatalysts could help simplify the molecular basis of stability, and may help in crafting designer enzymes via bioengineering and computational approaches. Such tailored biocatalysts may be candidates for potentially novel industrial process applications.

Ionic liquid (IL) based pretreatment of lignocellulosic biomass for facilitating efficient enzymatic saccharification has emerged as an environmentally benign approach that offers several advantages over conventional strategies. However, residues of ionic liquid left in the pretreated biomass may cause inactivation of saccharifying enzymes thus, necessitating the requirement of ionic liquid-stable enzymes. Cost-effective production of industrial enzymes is always desired to enhance the overall process economy. Current study reports IL-stable cellulase production from a newly isolated bacterium Bacillus subtilis G2. Design of experiment (DoE) based on response surface methodology was used in sequential manner for optimizing cultural and environmental variables to enhance cellulase production by 2.66-fold. IL-stable cellulase was used for saccharification of IL-pretreated pine needle biomass (PNB) with 1-ethyl-3-methylimidazolium methanesulfonate in a consolidated single pot process i.e. one pot consolidated bioprocess (OPCB). The saccharification efficiency of 23.57 % was observed under OPCB. The hydrolsate obtained was fermented by dual culture of yeast i.e. Saccharomyces cereviasie NCIM 3078 and Pichia stipitis NCIM 3497, and a yield of 0.092 g ethanol/g of PNB was obtained with fermentation efficiency of 25.62 %. This study is first ever where-in IL-stable cellulase production is accomplished using agroindustrial residues by employing DoE, and assessed for its application potential under OPCB for saccharification of IL-pretreated PNB. IL-stable cellulases would not only preclude expensive washing step following IL-pretreatment of biomass, but their application in a consolidated single pot process (OPCB) offers numerous technoeconomic advantages over conventional multi pot processes. Production of ionic liquid (IL) tolerant cellulase from Bacillus subtilis G2 was enhanced by 2.66-fold using response surface methodology. Cellulase was assessed for its saccharification potential on IL-pretreated pine needle biomass under one pot consolidated bioprocess, and the hydrolysate was fermented to ethanol using Saccharomyces cereviasie and Pichia stipitis.

World has witnessed most unprecedented economic/industrial growth during past few decades. But this resulted in massive depletion in the fossil fuel reserves, and grave environmental concerns like green house gas emissions, climate change etc. Keeping in view the serious consideration there is a paradigm shift towards the exploration of renewable energy resources, and development of processes/products that are green, clean and ecobenign. Lignocellulosic biomass, being an inexpensive and abundant energy source could be exploited for the production of bioenergy and other oleochemicals. But due to recalcitrant nature of lignocellulosic biomass, which is attributed to presence of lignin and hemicelluloses making the substrate inaccessible to hydrolytic enzymes. Therefore, the major challenge in biomass to biofuel/bio-actives is conversion delignification of lignocellulosic biomass. With the application of appropriate pretreatment technique, the complex biomass can be partially loosened and made accessible for hydrolysis. Environment friendly and cost effective biological pretreatment method using microorganisms offers advantages in getting the desired results in energy efficient manner. Appropriate combination of hydrolytic enzymes is required for complete degradation of cellulose and hemicelluloses into simpler sugars which served as raw material for further transformation. Successful saccharification of lignocellulosic biomass results in release of fermentable sugars which could act as starting material for production of bioenergy (Bioethanol, biobutanol, biohydrogen, biogas etc.) and other value-added products (Bioplastic, animal feed, composites, enzymes, xylooligosaccharides etc.). With the advancement in technology (green biotechnology), the conversion costs of lignocellulosic biomass could be lowered and product yields could be enhanced making the production processes more economical and alleviating the deleterious effects of harsh chemicals and fossil fuels on environment.

The cost-effective bulk production of cellulases with desirable characteristics e.g., ionic liquid (IL)-stability, thermal, and pH stability is highly desirable. This study reports the optimization of cultural and environmental variables for enhanced production of an IL-stable, broad pH range, and thermo-stable cellulase from Bacillus subtilis I-2 employing low-cost agro-industrial wastes. Furthermore, combined interactive effects of different variables on enzyme yield were investigated using response surface methodology. The optimal levels of carbon and nitrogen sources were determined (% w/v, wheat bran 2.0, potato peel 1.5, cotton seed cake 0.8, and soybean meal 0.8) for enhanced cellulase yield. Further, optimization of environmental variables (temperature 48.41 °C, pH 7.0, and agitation rate 180 rpm) lead to overall 4.1-fold (76– 315.90 U/ml) enhancement of cellulase yield.

Pretreatment is the requisite step for the bioconversion of lignocellulosics. Since most of the pretreatment strategies are cost/energy intensive and environmentally hazardous, there is a need for the development of an environment-friendly pretreatment process. An ionic liquid (IL) based pretreatment approach has recently emerged as the most appropriate one as it can be accomplished under ambient process conditions. However, IL-pretreated biomass needs extensive washing prior to enzymatic saccharification as the enzymes may be inhibited by the residual IL. This necessitated the exploration of IL-stable saccharification enzymes (cellulases). Current study aims at optimizing the bioprocess variables viz. carbon/nitrogen sources, medium pH and fermentation time, by using a Design of Experiments approach for achieving enhanced production of ionic liquid tolerant cellulase from a bacterial isolate Bacillus subtilis SV1. The cellulase production was increased by 1.41-fold as compared to that under unoptimized conditions. IL-stable cellulase was employed for saccharification of IL (1-ethyl-3-methylimidazolium methanesulfonate) pretreated pine needle biomass in a newly designed bioprocess named as “one pot consolidated bioprocess” (OPCB), and a saccharification efficiency of 65.9% was obtained. Consolidated bioprocesses, i.e., OPCB, offer numerous techno-economic advantages over conventional multistep processes, and may potentially pave the way for successful biorefining of biomass to biofuel, and other commercial products.