Shishir Chundawat

Rutgers, The State University of New Jersey | Rutgers · Department of Chemical and Biochemical Engineering

With the transition toward continuous bioprocessing, process analytical technology (PAT) is becoming necessary for rapid and reliable in-process monitoring during biotherapeutics manufacturing. Bioprocess 4.0 is looking to build an end-to-end bioprocesses that includes PAT-enabled real-time process control. This is especially important for drug pro...

Protein adsorption to solid carbohydrate interfaces is critical to many biological processes, particularly in biomass deconstruction. To engineer more-efficient enzymes for biomass deconstruction into sugars, it is necessary to characterize the complex protein-carbohydrate interfacial interactions. A carbohydrate-binding module (CBM) is often assoc...

The analysis of particles bound to a surface by flexible tethers can facilitate understanding of various biophysical phenomena (e.g., molecular dynamics of DNA-protein or protein-ligand binding interactions, DNA extensibility and polymer biophysics). Being able to model such systems theoretically can aid in understanding experimentally observed mot...

Post-translational modification such as N-Glycosylation on biologics during the production of monoclonal antibody (mAb) based therapeutics is a critical quality attribute that dictates safety and efficacy. Variability is introduced in the cell culture process which influences, the glycosylation pattern which is known to be highly heterogenous and m...

The shifting of crude oil–based refinery to biomass-based biorefinery has enticed compelling scientific interest which focuses on the development of cellulosic ethanol as an alternative transportation fuel to fossil fuels. Therefore, conversion of plentiful lignocellulosic biomass to biofuel as transportation fuels will be a feasible alternative fo...

The biopharmaceutical industry is transitioning towards adoption of continuous biomanufacturing practices that are often more flexible and efficient than traditional batch processes. Regulatory agencies such as the Food and Drug Administration (FDA) are further urging use of advanced PAT to analyze the design space to increase process knowledge and...

Non-productive adsorption of cellulolytic enzymes to various plant cell wall components, such as lignin and cellulose, necessitates high enzyme loadings to achieve efficient conversion of pretreated lignocellulosic biomass to fermentable sugars. Carbohydrate-binding modules (CBMs), appended to various catalytic domains (CDs), promote lignocellulose...

To rationally engineer more efficient cellulolytic enzymes for cellulosic biomass deconstruction into sugars for biofuels production, it is necessary to better understand the complex enzyme-substrate interfacial interactions. Carbohydrate binding modules (CBM) are often associated with microbial surface-tethered cellulosomal or freely secreted cell...

Continuous bioprocessing is significantly changing the biological drugs (or biologics) manufacturing landscape by potentially improving product quality, process stability, and overall profitability, as was similarly seen during the adoption of advanced manufacturing processes for small molecule drugs in the past decade. However, the implementation...

Detection of azide-tagged biomolecules (e.g., azido sugars) inside living cells using "click" chemistry has been revolutionary to the field of chemical biology. However, we currently still lack suitable synthetic biology tools to autonomously and rapidly detect azide ions. Here, we have developed an engineered synthetic promoter system called cyn r...

Efficient enzymatic saccharification of cellulosic biomass into fermentable sugars can enable production of bioproducts like ethanol. Native crystalline cellulose, or cellulose I, is inefficiently processed via enzymatic hydrolysis, but can be converted into the structurally distinct cellulose III allomorph that is processed via cellulase cocktails...

Dissociation of nonproductively bound cellulolytic enzymes from cellulose is hypothesized to be a key rate‐limiting factor impeding cost‐effective biomass conversion to fermentable sugars. However, the role of carbohydrate‐binding modules (CBMs) in enabling nonproductive enzyme binding is not well understood. Here, we examine the subtle interplay o...

One of the stumbling blocks to advance the field of glycobiology has been the difficulty in synthesis of bespoke carbohydrate-based molecules like glycopolymers (e.g. human milk oligosaccharides) and glycoconjugates (e.g. glycosylated monoclonal antibodies). Recent strides towards using engineered Carbohydrate-Active enZymes (CAZymes) like glycosyl...

Real-time azide or azido-functionalized molecular detection inside living cells using bioorthogonal chemistry-based approaches has been revolutionary to advancing chemical-biology. These methods have enabled diverse applications ranging from understanding the role of cellular glycosylation pathways, identifying diseased cells, and targeting deliver...

Dissociation of non-productively bound cellulolytic enzymes from cellulose is hypothesized to be a key rate-limiting factor impeding cost-effective biomass conversion to fermentable sugars. However, the role of carbohydrate-binding modules (CBMs) in enabling non-productive enzyme binding is not well understood. Here, we examine the subtle interplay...

Chemoenzymatic approaches using carbohydrate‐active enzymes (CAZymes) offer a promising avenue for the synthesis of glycans like oligosaccharides. Here, we report a novel chemoenzymatic route for cellodextrins synthesis employed by chimeric CAZymes, akin to native glycosyltransferases, involving the unprecedented participation of a “non‐catalytic”...

Biochemical conversion of plant-based insoluble carbohydrate polymers, such as starch from corn grains or cellulose-hemicellulose from corn stover, into soluble fermentable sugars (e.g., glucose and xylose) for bioenergy production has seen tremendous research activity and commercial-scale biorefineries deployment over the last three decades, parti...

Cellulolytic microorganisms, like Trichoderma reesei or Clostridium thermocellum , frequently have non-catalytic carbohydrate-binding modules (CBMs) associated with secreted or cell surface bound multidomain carbohydrate-active enzymes (CAZymes) like cellulases. Mostly type-A family CBMs are known to promote cellulose deconstruction by increasing t...

Chemoenzymatic approaches using carbohydrate-active enzymes (CAZymes) offer a promising avenue for synthesis of glycans like oligosaccharides. Here, we report a novel chemoenzymatic route for cellodextrins synthesis employed by chimeric CAZymes, akin to native glycosyltransferases, involving the unprecedented participation of a ′non-catalytic′ lect...

Lignocellulosic materials are plant-derived feedstocks, such as crop residues (e.g., corn stover, rice straw, and sugar cane bagasse) and purpose-grown energy crops (e.g., miscanthus, and switchgrass) that are available in large quantities to produce biofuels, biochemicals, and animal feed. Plant polysaccharides (i.e., cellulose, hemicellulose, and...

Engineering of carbohydrate-active enzymes like glycosynthases for chemoenzymatic synthesis of bespoke oligosaccharides has been limited by the lack of suitable directed evolution based protein engineering methods. Currently there are no ultrahigh-throughput screening methods available for rapid and highly sensitive single cell-based screening of e...

Cellulose recalcitrance towards saccharification is a barrier for low-cost biofuels production. Ammonia-based pretreatments can alter the native cellulose-I allomorphic state to form an unnatural cellulose-III allomorph that is less recalcitrant towards enzymatic hydrolysis. Here, we characterize the hydrolytic activity of a thermophilic cellulolyt...

Here, we report a novel ammonia:ammonium salt solvent based pretreatment process that can rapidly dissolve crystalline cellulose into solution and eventually produce highly amorphous cellulose under near-ambient conditions. Pre-activating the cellulose I allomorph to its ammonia-cellulose swollen complex (or cellulose III allomorph) at ambient temp...

Ammonia-based pretreatments have been extensively studied in the last decade as one of the leading pretreatment technologies for lignocellulose biorefining. Here, we discuss the key features and compare performances of several leading ammonia-based pretreatments (e.g., soaking in aqueous ammonia or SAA, ammonia recycled percolation or ARP, ammonia...

Enzymatic conversion of cellulosic biomass into fermentable sugars such as glucose is a slow and catalytically inefficient process, largely due to limited accessibility of cellulose. Cellulose crystallinity can be reduced to increase substrate accessibility toward cellulolytic enzymes by either swelling or dissolving biomass in chemicals such as co...

Plant biomasses enriched in crystalline cellulose allomorphs, such as native cellulose I (CI), can be structurally altered using anhydrous liquid ammonia to form an enzymatically less recalcitrant cellulose III (CIII) allomorph. Here, we designed and implemented an advanced ammonia pretreatment reactor/sampler setup that allowed us to systematicall...

Non-specific adsorption of cellulases to lignin hinders enzymatic biomass deconstruction. Here we tested the hypothesis that negatively supercharging cellulases could reduce lignin inhibition. Computational design was used to negatively supercharge the surfaces of Ruminoclostridium thermocellum family 5 CelE and a CelE-family 3a carbohydrate bindin...

Most cellulolytic enzyme blends, either procured from a commercial vendor or isolated from a single cellulolytic microbial secretome, do not efficiently hydrolyze ammonia-pretreated (e.g., ammonia fiber expansion, AFEX) lignocellulosic agricultural crop residues like corn stover to fermentable sugars. Typically reported commercial enzyme loading (3...

Consolidated bioprocessing (CBP) of pretreated lignocellulosic biomass using microbes like Clostridium thermocellum allows simultaneous polysaccharide saccharification and sugar fermentation to produce fuels or chemicals using a one-pot process. C. thermocellum is a thermophilic bacterium that deconstructs biomass using large multi-enzyme complexes...

Biological-mediated conversion of pretreated lignocellulosic biomass to biofuels and biochemicals is a promising avenue towards energy sustainability. However, a critical impediment to the commercialization of cellulosic biofuel production is the high cost of cellulase enzymes needed to deconstruct biomass into fermentable sugars. One major factor...

A method of alkaline pretreatment of biomass, in particular pretreating biomass with gaseous ammonia.

A new liquid ammonia pretreatment methodology called Extractive Ammonia (EA) was developed to simultaneously convert native crystalline cellulose Iβ (CI) to a highly digestible cellulose IIII (CIII) allomorph and selectively extract up to ∼45% of the lignin from lignocellulosic biomass with near-quantitative retention of all polysaccharides. EA pre...

Cellobiohydrolase 1 from Trichoderma reesei (TrCel7A) processively hydrolyses cellulose into cellobiose. Although enzymatic techniques have been established as promising tools in biofuel production, a clear understanding of the motor's mechanistic action has yet to be revealed. Here, we develop an optical tweezers-based single-molecule (SM) motilit...

Supplementary Figures 1-11, Supplementary Table 1, Supplementary Notes 1-2 and Supplementary Reference

A method for producing a microbial growth stimulant (MGS) from a plant biomass is described. In one embodiment, an ammonium hydroxide solution is used to extract a solution of proteins and ammonia from the biomass. Some of the proteins and ammonia are separated from the extracted solution to provide the MGS solution. The removed ammonia can be recy...

Isotopic enrichment of biomacromolecules is a widely used technique that enables the investigation of the structural and dynamic properties to provide information not accessible with natural abundance isotopic composition. This study reports an approach for deuterium incorporation into bacterial cellulose. A media formulation for growth of Acetobac...

During enzymatic hydrolysis of biomass, polysaccharides are cleaved by glycosyl hydrolases to soluble oligosaccharides and further hydrolyzed by β-glucosidase, β-xylosidase and other enzymes to monomeric sugars. However, not all oligosaccharides can be fully hydrolyzed and they may accumulate to 18-25% of the total soluble sugars at high solid load...

Cell walls, which constitute the bulk of plant biomass, vary considerably in their structure, composition, and architecture. Studies on plant cell walls can be conducted on both native and pre-treated plant biomass samples, allowing an enhanced understanding of these structural and compositional variations. Here glycome profiling was employed to de...

BACKGROUND: The fermentation inhibition of yeast or bacteria by lignocellulose-derived degradation products, during hexose/pentose co-fermentation, is a major bottleneck for cost-effective lignocellulosic biorefineries. To engineer microbial strains for improved performance, it is critical to understand the mechanisms of inhibition that affect fer...

Non-productive binding of enzymes to lignin is thought to impede the saccharification efficiency of pretreated lignocellulosic biomass to fermentable sugars. Due to a lack of suitable analytical techniques that track binding of individual enzymes within complex protein mixtures and the difficulty in distinguishing the contribution of productive (bi...

Commercial-scale biofuel production requires a deep understanding of the structure and dynamics of its principal target: cellulose. However, an accurate description and modeling of this carbohydrate structure at mesoscale remains elusive, particularly because of its overwhelming length scale and configurational complexity. We have derived a set of...

In order to more economically process cellulosic feedstocks using a biochemical pathway for fuel production, it is necessary to develop a detailed understanding of plant cell wall characteristics, pretreatment reaction chemistry, and their complex interactions. However given the large number of thermochemical pretreatment methods that are currently...

Background In a biorefinery producing cellulosic biofuels, biomass pretreatment will significantly influence the efficacy of enzymatic hydrolysis and microbial fermentation. Comparison of different biomass pretreatment techniques by studying the impact of pretreatment on downstream operations at industrially relevant conditions and performing compr...

Isotopic enrichment has been widely used for investigating the structural and dynamic properties of biomacromolecules to provide information that cannot be carried out with molecules composed of natural abundance isotopes. A media formulation for controlled incorporation of deuterium in bacterial cellulose synthesized by Gluconacetobacter xylinus s...

Streptomyces sp. SirexAA-E (ActE) has been identified as a highly cellulolytic actinobacterium capable of deconstructing lignocellulosic biomass. SirexAA-E CAZymes most frequently contain a carbohydrate-binding module from family 2a (CBM2a). The DNA encoding the CBM2a from gene locus SACTE_0237, the most abundantly expressed cellulase from SirexAA-...

Sequential fractionation of AFEX-pretreated corn stover extracts was carried out using ultra-centrifugation, ultra-filtration, and solid phase extraction to isolate various classes of pretreatment products to evaluate their inhibitory effect on cellulases. Ultra-centrifugation removed dark brown precipitates that caused no appreciable enzyme inhibi...

A method is provided comprising converting at least a portion of native cellulose I to cellulose III by treating plant biomass with liquid ammonia and/or one or more organic solvents to produce a treated plant biomass containing lignin and a lignin fraction; and extracting lignin and/or other plant cell wall components from the lignin fraction to p...

Recalcitrance of grasses to enzymatic digestion arises to a significant degree from a complex array of phenolic crosslinks between cell wall polysaccharide chains that inhibit their conversion to biofuels and lower their nutritive value for animal feed applications. Polysaccharide esters of ferulic acid are abundant in plant cell walls. Crosslinks...

The invention related to methods for treating lignocellulosic biomass to obtain useful products there from.

Substrate binding is typically one of the rate-limiting steps preceding enzyme catalytic action during homogeneous reactions. However, interfacial-based enzyme catalysis on insoluble crystalline substrates, like cellulose, has additional bottlenecks of individual biopolymer chain decrystallization from the substrate interface followed by its proces...

Note that article is freely available online through the PNAS open access option (http://www.pnas.org/content/110/27/10922.long). Substrate binding is typically one of the rate-limiting steps preceding enzyme catalytic action during homogeneous reactions. However, interfacial-based enzyme catalysis on insoluble crystalline substrates, like cellulo...

We provide an extensive review of ammonia fiber expansion (AFEX) pretreatment from a techno-economical and environmental sustainability perspective. A brief historical perspective on concentrated ammonia-based pretreatments is provided followed by a detailed overview of the AFEX process and its physicochemical impact on lignocellulosic plant cell w...

A process for producing sugars from lignocellulosic materials such as corn stover by the addition of corn stillage as a carbon source is disclosed. The sugars are formed by treating the combination of the corn stillage and the lignocellulosic materials with hydrolytic enzymes. The sugars can be fermented to ethanol, and the process improves ethanol...

The ammonia fiber expansion or AFEX™ process is a leading thermochemical pretreatment process that facilitates the rapid deconstruction of lignocellulosic biomass to fermentable sugars in an economical fashion. Two major limitations of the conventional AFEX™ process are; (i) it is unable to reduce the inherent recalcitrance of crystalline cellulose...

Extractive AFEXTM (E-AFEXTM) is a novel pretreatment technology that is able to simultaneously convert cellulose I to cellulose III and partially extract lignin from lignocellulosic biomass. As a result, pretreated feedstocks contain highly digestible carbohydrate content and significantly reduced lignin content. Depending on the pretreatment condi...

Substrate binding affinity is typically thought to be one of the primary rate-limiting steps preceding enzyme catalytic action during most homogeneous reactions. However, interfacial-based enzyme catalysis on insoluble crystalline substrates, like cellulose, has an additional step comprising of individual biopolymer chain decrystallization from the...

We present the results of Langevin dynamics simulations on a coarse-grained model for a structural transition in crystalline cellulose pertinent to the cellulose degradation problem. We analyze two different cellulose crystalline forms: cellulose I(β) (the natural form of cellulose) and cellulose III(I) (obtained after cellulose I(β) is treated wit...

AFEX pretreatment was found to substantially improve overall enzymatic digestibility by 4–20 fold for both untreated guayule shrub and latex-extracted bagasse. Maximum glucan and xylan conversion achieved for the latex-extracted bagasse was 40% and 50%, respectively. The yeast was readily able to ferment both glucose and xylose to ethanol from the...

With dwindling oil reserves and growing environmental concerns, researchers are looking at producing sustainable biofuels and chemicals from renewable resources like switchgrass. Biofuels and biochemicals will be produced in the near future from switchgrass in biorefineries using both biochemical and thermochemical platforms. We have summarized rec...

Simultaneously achieving economic, environmental and social sustainability is a major challenge for the emerging renewable fuel industry. We approach this problem by demonstrating a cellulosic biorefinery paradigm which produces ethanol and food precursors using lignocellulosic biomass as the exclusive source for carbohydrates and minerals. Enzymat...

In this quantum chemical study, we explore hydrogen bonding (H-bonding) and stacking interactions in different crystalline cellulose allomorphs; namely, cellulose I(β) and cellulose III(I). We consider a model system representing a cellulose crystalline core made from six cellobiose units arranged in three layers with two chains per layer. We calcu...

The depolymerization of cellulose to monomeric glucose is the key step for cellulosic ethanol biorefinery process. Cellulases binding on biomass is the essential first step for effective breakdown of biomass to sugars. A new fast flow liquid chromatography (FPLC) based method has been developed to quantify endoglucanase I (EG I, GH family 7B), cell...

There are two major problems in current cellulosic ethanol production process: low ethanol productivity and high enzyme loading. Based on the current Separate Hydrolysis and Fermentation (SHF) process, it takes at least 96h to perform enzymatic hydrolysis to convert AFEX-CS to fermentable sugars at 6% glucan loading. It will then take another 96-16...

One of the limitations of screening enzyme combinations using microplate based assays (Gao et al. 2010, Bioresour Technol 101, 8:2770) is that these are typically carried out using milled biomass (<500 mms) at low solids loading (<1%, w/v). Biomass particle size and solids loading can both influence the enzyme set necessary for efficient hydrolysis...

The benefits of delignification on plant cell wall digestibility by hydrolytic enzymes are well described in the literature. The presence of lignin increases non-productive binding of cellulases, resulting in enzyme inhibition and decrease in the sugar yields. Moreover, lignin acts as an anti-microbial agent, reducing cell viability and ethanol yie...

Several degradation products are formed or released during AFEX pretreatment process, which can be divided into five groups: 1) carbohydrates, 2) aliphatic acids, 3) nitrogenous compounds, 4) furans, 5) aromatic compounds. Some of these degradation products are expected to be inhibitory to Saccharomyces cerevisiae 424A(LNH-ST), thus affecting the x...

Enzymatic conversion of cellulose can be improved via either reducing cellulose crystallinity by thermochemical treatments or enhancing cellulase activity by protein engineering. A third approach is via reorganizing the hydrogen bonding network within crystalline cellulose to produce a less recalcitrant allomorph. Cellulose III is one such allomorp...

Various chemicals are being explored for catalyzing efficient lignocellulose deconstruction. In particular, when liquid ammonia is used to convert the naturally occurring cellulose crystalline phase I(β), to cellulose III(I), the rearrangement of the hydrogen bond network in cellulose III(I) results in enhanced hydrolysis yields. We use molecular d...

Studying the binding properties of cellulases to lignocellulosic substrates is critical to achieving a fundamental understanding of plant cell wall saccharification. Lignin auto-fluorescence and degradation products formed during pretreatment impede accurate quantification of individual glycosyl hydrolases (GH) binding to pretreated cell walls. A h...