Sasidhar Varanasi

Mitigation of Cellulose Recalcitrance to Enzymatic Hydrolysis by Ionic Liquid Pretreatment

Efficient hydrolysis of cellulose-to-glucose is critically important in producing fuels and chemicals from renewable feedstocks. Cellulose hydrolysis in aqueous media suffers from slow reaction rates because cellulose is a water-insoluble crystalline biopolymer. The high-crystallinity of cellulose fibrils renders the internal surface of cellulose inaccessible to the hydrolyzing enzymes (cellulases) as well as water. Pretreatment methods, which increase the surface area accessible to water and cellulases are vital to improving the hydrolysis kinetics and conversion of cellulose to glucose. In a novel technique, the microcrystalline cellulose was first subjected to an ionic liquid (IL) treatment and then recovered as essentially amorphous or as a mixture of amorphous and partially crystalline cellulose by rapidly quenching the solution with an antisolvent. Because of their extremely low-volatility, ILs are expected to have minimal environmental impact. Two different ILs, 1-n-butyl-3-methylimidazolium chloride (BMIMC1) and 1-allyl-3-methylimidazolium chloride (AMIMC1) were investigated. Hydrolysis kinetics of the IL-treated cellulose is significantly enhanced. With appropriate selection of IL treatment conditions and enzymes, the initial hydrolysis rates for IL-treated cellulose were up to 90 times greater than those of untreated cellulose. We infer that this drastic improvement in the “overall hydrolysis rates” with IL-treated cellulose is mainly because of a significant enhancement in the kinetics of the “primary hydrolysis step” (conversion of solid cellulose to soluble oligomers), which is the rate-limiting step for untreated cellulose. Thus, with IL-treated cellulose, primary hydrolysis rates increase and become comparable with the rates of inherently faster “secondary hydrolysis” (conversion of soluble oligomers to glucose).

Comparative study of pyrolysis of algal biomass from natural lake blooms with lignocellulosic biomass

Pyrolysis experiments were performed with algal and lignocellulosic feedstocks under similar reactor conditions for comparison of product (bio-oil, gas and bio-char) yields and composition. In spite of major differences in component bio-polymers, feedstock properties relevant to thermo-chemical conversions, such as overall C, H and O-content, C/O and H/C molar ratio as well as calorific values, were found to be similar for algae and lignocellulosic material. Bio-oil yields from algae and some lignocellulosic materials were similar; however, algal bio-oils were compositionally different and contained several N-compounds (most likely from protein degradation). Algal bio-char also had a significantly higher N-content. Overall, our results suggest that it is feasible to convert algal cultures deficient in lipids, such as nuisance algae obtained from natural blooms, into liquid fuels by thermochemical methods. As such, pyrolysis technologies being developed for lignocellulosic biomass may be directly applicable to algal feedstocks as well.

Enhanced ethanol fermentation of brewery wastewater using the genetically modified strain E. coli KO11

We have used liquid waste obtained from a beer brewery process to produce ethanol. To increase the productivity, genetically modified organism, Escherichiacoli KO11, was used for ethanol fermentation. Yeast was also used to produce ethanol from the same feed stock, and the ethanol production rates and resulting concentrations of sugars and ethanol were compared with those of KO11. In the experiments, first the raw wastewater was directly fermented using two strains with no saccharification enzymes added. Then, commercial enzymes, α-amylase, pectinase, or a combination of both, were used for simultaneous saccharification and fermentation, and the results were compared with those of the no-enzyme experiments for KO11 and yeast. Under the given conditions with or without the enzymes, yeast produced ethanol more rapidly than E. coli KO11, but the final ethanol concentrations were almost the same. For both yeast and KO11, the enzymes were observed to enhance the ethanol yields by 61–84% as compared to the fermentation without enzymes. The combination of the two enzymes increased ethanol production the most for the both strains. The advantages of using KO11 were not demonstrated clearly as compared to the yeast fermentation results.

Fermentation of biomass sugars to ethanol using native industrial yeast strains

In this paper, the feasibility of a technology for fermenting sugar mixtures representative of cellulosic biomass hydrolyzates with native industrial yeast strains is demonstrated. This paper explores the isomerization of xylose to xylulose using a bi-layered enzyme pellet system capable of sustaining a micro-environmental pH gradient. This ability allows for considerable flexibility in conducting the isomerization and fermentation steps. With this method, the isomerization and fermentation could be conducted sequentially, in fed-batch, or simultaneously to maximize utilization of both C5 and C6 sugars and ethanol yield. This system takes advantage of a pH-dependent complexation of xylulose with a supplemented additive to achieve up to 86% isomerization of xylose at fermentation conditions. Commercially-proven Saccharomyces cerevisiae strains from the corn-ethanol industry were used and shown to be very effective in implementation of the technology for ethanol production.

Helicobacter pylori survival in gastric mucosa by generation of a pH gradient.

Helicobacter pylori has been established as the major causative agent of human active gastritis and is an essential factor in peptic ulcer disease and gastric cancer. The mechanism that has been proposed for H. pylori to control its inhospitable microenvironment happens to coincide with the pH control technique developed by us. This technique was developed to separate an acidic environment from a basic environment for a sequential enzymatic reaction by the hydrolysis of urea within a thin layer of immobilized urease. In this paper, a mathematical model is presented to consider how H. pylori survives the gastric acidity. The computed results explain well the experimental data available involving H. pylori.

High yield aldose–ketose transformation for isolation and facile conversion of biomass sugar to furan

Traditional approaches to producing furfural from C5 biomass sugars have several limitations which include high reaction temperatures/pressures, significant sugar loss to side-reactions, modest furfural yields, and high purification costs. We present a novel method for converting the C5 sugar xylose to furfural at facile conditions in very high yield. In this approach, we isomerize xylose to its ketose isomer in high yield via a simultaneous-isomerization-and-reactive-extraction (SIRE) scheme, concentrate and purify xylulose by back-extraction (BE) into an acid medium, and then rapidly dehydrate the xylulose sugar to furfural at relatively low temperature (∼110 °C) with no additional catalyst. To our knowledge, production of furfural from concentrated xylulose (30 g l−1) has not been reported previously; this is likely due to the difficulty of producing relatively large quantities of high-purity xylulose in a cost-effective manner. Through simple strategies, such as addition of an aprotic solvent to the aqueous medium or in situ extraction of furfural during the dehydration, furfural yields of up to 90% were achieved. The mild process conditions associated with each of the steps in the process (SIRE, BE and dehydration), along with the ability to concentrate the incoming sugar stream and recycle process streams and catalysts, results in minimal chemical and energy inputs and have a significant favorable impact on the overall process economics.

An algebraic equation for the steady-state response of enzyme-pH electrodes and field effect transistors

Abstract The response of enzyme-pH electrodes and field effect transistors (pH-ENFETs) is strongly affected by the pH-buffers present in the test solution, the test solutions pH, and the degree of dissociation of the acidic and/or basic products formed in the enzymic reaction. It is shown that a theoretical model for the sensors steady-state response, which incorporates alt the above factors, leads to a single transcendental equation which provides the sensors response. However, when the concentration of the analyte at the face of the enzymic film in contact with the pH sensor (Cos) is negligible compared to its concentration at the face in contact with the test solution (Cbs), the transcendental equation reduces to an algebraic equation. This simple equation is independent of the actual kinetics of the enzymic reaction and the diffusion coefficients of the various species. By controlling (i) the Thiele modulus of the enzymic film and (ii) the concentration of the pH-buffer in the test solution, one can design a sensor so as to have Cos ⪡ Cbs. The aforementioned transcendental equation allows one to compute the required values for these two design variables in order to satisfy the above strong inequality. The response behavior predicted by the algebraic equation is in excellent agreement with the experimental data available on penicillinase—pH and urease—pH sensors

Cross-metathesis approach to produce precursors of nylon 12 and nylon 13 from microalgae

A two-step synthesis for producing methyl 12-aminododecanoate and 13-aminotridecanoate, the precursors of nylon 12 and nylon 13, from methyl oleate is described. First, methyl 11-cyano-9-undecenoate or 12-cyano-9-dodecenoate were prepared by cross metathesis of methyl oleate with either allyl cyanide or homoallyl cyanide, respectively. Subsequently, all the unsaturation of the two intermediates was hydrogenated to deliver the final products. This method represents the first synthesis of nylon 12 and 13 precursors from methyl oleate, an ester of an abundant and renewable natural fatty acid present in vegetable oil or microalgae. It also represents the shortest synthesis of nylon precursors from fatty acids, and as demonstrated in this study, can be directly applied to crude fatty acid methyl ester extracts from microalgae.

Demonstration of pH control in a commercial immobilized glucose isomerase

The synthesis of a variety of important biochemicals involves multistep enzyme-catalyzed reactions. In many cases, the optimal operating pH is much different for the individual enzymatic steps of such synthesis reactions. Yet, it may be beneficial if such reaction steps are combined or paired, allowing them to occur simultaneously, in proximity to one another, and at their respective optimal pH. This can be achieved by separating the micro-environments of the two steps of a reaction pathway using a thin urease layer that catalyzes an ammonia-forming reaction. In this article, the pH control system in a commercial immobilized glucose (xylose) isomerase pellet, which has an optimal pH of 7.5, is demonstrated. This system allows the glucose isomerase to have near its optimal pH activity when immersed in a bulk solution of pH 4.6. A theoretical analysis is also given for the effective fraction of the immobilized glucose isomerase, which remains active when the bulk pH is at 4.6 in the presence of 20 mM urea versus when the bulk pH is at its optimal pH of 7.5. Both theoretical and experimental results show that this pH control system works well in this case. (c) 1996 John Wiley & Sons, Inc.

A FEASIBILITY ANALYSIS OF A NOVEL APPROACH FOR THE CONVERSION OF XYLOSE TO ETHANOL

Economic production of ethanol from plant biomass could be significantly increased if the feedstock for the fermentation is more completely utilized. Currently, simple sugars (mostly D-glucose and D-xylose) can be recovered from lignocellulose by enzymatic or acid hydrolysis. However, while glucose can be readily converted to ethanol by yeasts, the xylose is not fermentable by many of the same species of yeasts that are able to convert glucose into ethanol. Nevertheless, xylose can be converted to its ketose isomer, xylulose, by the enzyme xylose isomerase and this isomer can be converted to ethanol. A major obstacle, however, in converting the xylose to xylulose and then simultaneously converting the xylulose to ethanol is that the pH at which xylose isomerase displays its optimal activity (pH of 7.0-8.0) is much different from the pH at which the fermentation of the xylulose and glucose is best carried out (pH of 4.0-5.0). Herein we propose a novel scheme to provide a means by which the isomerization an...