Publications
Dr. Chundawat’s complete list of publications is available here and at . Current h-index is 39, i10-index is 72, and total citations are 8284 (updated 2024/01). Corresponding author/s highlighted by an asterisk (*). Peer-reviewed papers are available on the publisher’s website, RUcore, and some older papers are also posted on Dr. Chundawat’s personal ResearchGate account. Original preprints are available on the bioRxiv and chemRxiv websites. Patents are available on Google Patents website.
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Authors: Gao D, Haarmeyer C, Balan V, Whitehead TA, Dale BE, Chundawat SPS
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Abstract: 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 (binding to specific glycans) versus non-productive (binding to lignin) binding of cellulases to lignocellulose, there is currently a poor understanding of individual enzyme adsorption to lignin during the time course of pretreated biomass saccharification.
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Authors: Humpula J, Uppugundla N, Vismeh R, Sousa L, Chundawat SPS, Jones AD, Balan V, Dale BE, Cheh AM
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Abstract: 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 inhibition. Ultra-filtration of ultra-centrifuged AFEX-pretreated corn stover extractives using a 10kDa molecular weight cutoff (MWCO) membrane removed additional high molecular weight components that accounted for 24-28% of the total observed enzyme inhibition while a 3kDa MWCO membrane removed 60-65%, suggesting significant inhibition is caused by oligomeric materials. Solid phase extraction (SPE) of AFEX-pretreated corn stover extractives after ultra-centrifugation removed 34-43% of the inhibition; ultra-filtration with a 5kDa membrane removed 44-56% of the inhibition and when this ultra-filtrate was subjected to SPE a total of 69-70% of the inhibition were removed. Mass spectrometry found several phenolic compounds among the hydrophobic inhibition removed by SPE adsorption.
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Authors: Lim S, Chundawat SPS*, Fox BG
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Abstract: 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-E, was cloned into an Escherichia coli expression vector and expressed as a C-terminal fusion protein to GFP. The GFP-CBM2a fusion protein was purified from insoluble inclusion bodies and refolded. The solubilized protein was separated by size-exclusion chromatography into high molecular weight GFP-CBM2a multimers and monomeric GFP-CBM2a. Only the monomeric CBM2a protein was found to have high relative affinity (partition coefficient of 0.62ア0.04 liters/gram) to cellulose. Binding of monomeric CBM2a prepared in this manner exhibits fully reversible, high affinity binding to cellulose.
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Authors: Uppugundla N, Sousa L, Chundawat SPS, Xiurong Y, Simmons B, Singh S, Gao X, Kumar R, Wyman CE, Dale BE, Balan V
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Abstract: 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 comprehensive mass balances will help focus attention on necessary process improvements, and thereby help reduce the cost of biofuel production. Results An on-going collaboration between the three US Department of Energy (DOE) funded bioenergy research centers (Great Lakes Bioenergy Research Center (GLBRC), Joint BioEnergy Institute (JBEI) and BioEnergy Science Center (BESC)) has given us a unique opportunity to compare the performance of three pretreatment processes, notably dilute acid (DA), ionic liquid (IL) and ammonia fiber expansion (AFEXTM), using the same source of corn stover. Separate hydrolysis and fermentation (SHF) was carried out using various combinations of commercially available enzymes and engineered yeast (Saccharomyces cerevisiae 424A) strain. The optimal commercial enzyme combination (Ctec2: Htec2: Multifect Pectinase, percentage total protein loading basis) was evaluated for each pretreatment with a microplate-based assay using milled pretreated solids at 0.2% glucan loading and 15 mg total protein loading/g of glucan. The best enzyme combinations were 67:33:0 for DA, 39:33:28 for IL and 67:17:17 for AFEX. The amounts of sugar (kg) (glucose: xylose: total gluco- and xylo-oligomers) per 100 kg of untreated corn stover produced after 72 hours of 6% glucan loading enzymatic hydrolysis were: DA (25:2:2), IL (31:15:2) and AFEX (26:13:7). Additionally, the amounts of ethanol (kg) produced per 100 kg of untreated corn stover and the respective ethanol metabolic yield (%) achieved with exogenous nutrient supplemented fermentations were: DA (14.0, 92.0%), IL (21.2, 93.0%) and AFEX (20.5, 95.0%), respectively. The reason for lower ethanol yield for DA is because most of the xylose produced during the pretreatment was removed and not converted to ethanol during fermentation. Conclusions Compositional analysis of the pretreated biomass solids showed no significant change in composition for AFEX treated corn stover, while about 85% of hemicellulose was solubilized after DA pretreatment, and about 90% of lignin was removed after IL pretreatment. As expected, the optimal commercial enzyme combination was different for the solids prepared by different pretreatment technologies. Due to loss of nutrients during the pretreatment and washing steps, DA and IL pretreated hydrolysates required exogenous nutrient supplementation to ferment glucose and xylose efficiently, while AFEX pretreated hydrolysate did not require nutrient supplementation.
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Authors: He J, Pingali SV, Chundawat SPS, Pack A, Jones AD, Langan P, Davison BH, Urban V, Evans B, O’Neill H
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Abstract: 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 subsp. sucrofermentans is reported. The purified cellulose was characterized using Fourier Transform Infra-Red spectrophotometry and mass spectrometry which revealed that the level of deuterium incorporation in the perdeuterated cellulose was greater than 90 %. Small-angle neutron scattering analysis demonstrated that the overall structure of the cellulose was unaffected by the substitution of deuterium for hydrogen. In addition, by varying the amount of D-glycerol in the media it was possible to vary the scattering length density of the deuterated cellulose. A large disk model was used to fit the curves of bacterial cellulose grown using 0 and 100 % D-Glycerol yielding a lower bound to the disk radii, R min = 1,132 ± 6 and 1,154 ± 3 Å and disk thickness, T = 128 ± 1 and 83 ± 1 Å for the protiated and deuterated forms of the bacterial cellulose, respectively. This agrees well with the scanning electron microscopy analysis which revealed stacked sheets in the cellulose pellicles. Controlled incorporation of deuterium into cellulose will enable new types of experiments using techniques such as neutron scattering to reveal information about the structure and dynamics of cellulose and its interactions with proteins and other (bio) polymers.
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Authors: Gao D, Chundawat SPS*, Sethi A, Gnanakaran S, Balan V, Dale BE.
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Abstract: 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 processive depolymerization to soluble sugars. This additional decrystallization step has ramifications on the role of enzyme–substrate binding and its relationship to overall catalytic efficiency. We found that altering the crystalline structure of cellulose from its native allomorph Iβ to IIII results in 40–50% lower binding partition coefficient for fungal cellulases, but surprisingly, it enhanced hydrolytic activity on the latter allomorph. We developed a comprehensive kinetic model for processive cellulases acting on insoluble substrates to explain this anomalous finding. Our model predicts that a reduction in the effective binding affinity to the substrate coupled with an increase in the decrystallization procession rate of individual cellulose chains from the substrate surface into the enzyme active site can reproduce our anomalous experimental findings.
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Authors: Vismeh R, Lu F, Chundawat SPS, Humpula J, Azarpira A, Ralph J, Balan V, Dale BE, Jones AD
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Abstract: 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 between polysaccharides are formed through oxidative dehydrodimerization of ferulates, producing dehydrodiferulates (henceforth termed diferulates). Such ferulates and diferulates further crosslink plant cell walls by radical coupling cross-reactions during lignification. Although cell wall digestibility can be improved by cell wall metabolic engineering, or post-harvest by various pretreatment processes, a more comprehensive understanding of the role and impact of ferulate crosslinking on polysaccharide hydrolysis would be accelerated by availability of analytical methods that can distinguish the various diferulates released during biomass pretreatments, many of which are isomers. In this report, we present an ultrahigh-performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) strategy for comprehensive separation and identification of diferulate isomers. Collision-induced dissociation (CID) mass spectra of [M + H]+ ions distinguished various isomers without requiring derivatization. Characteristic product ions for 8-O-4-, 8-8-non-cyclic, 8-8-cyclic, 8-5-cyclic, 8-5-non-cyclic, and 5-5-linked isomers were identified. All diferulates were identified either as di-acids in extracts of NaOH-hydrolyzed corn stover, or as a diverse group of diferulate mono- and di-amides in extracts of Ammonia Fiber Expansion (AFEX™)-treated corn stover. This approach allows for direct analysis of released diferulates with minimal sample preparation, and can serve as the foundation for high-throughput profiling and correlating pretreatment conditions with biomass digestibility in biorefineries producing biofuels and biochemicals.
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Authors: Vismeh R, Humpula J, Chundawat SPS, Balan V, Dale BE, Jones AD
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Abstract: Thermochemical pretreatments of cellulosic biomass are known to improve cell wall enzymatic digestibility, while simultaneously releasing substantial amounts of soluble oligosaccharides. Profiling of oligosaccharides released during pretreatment yields information essential for choosing glycosyl hydrolases necessary for cost-effective conversion of cellulosic biomass to desired biofuel/biochemical end-products. In this report we present a methodology for profiling of soluble neutral oligosaccharides released from ammonia fiber expansion (AFEX™)-pretreated corn stover. Our methodology employs solid phase extraction (SPE) enrichment of oligosaccharides using porous graphitized carbon (PGC), followed by high performance liquid chromatography (HPLC) separation using a polymeric amine based column and electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS). For structural elucidation on the chromatographic time scale, nonselective multiplexed collision-induced dissociation was performed for quasi-simultaneous acquisition of oligosaccharide molecular and fragment masses in a single analysis. These analyses revealed glucans up to degree of polymerization (DP) 22 without modifications. Additionally, arabinoxylans up to DP=6 were detected in pretreated biomass extracts (post-enzymatic digestion). Cross-ring fragment ion abundances were consistent with assignment of linkages between sugar units in glucans and also xylose backbone in arabinoxylans as 1-4 linkages. Comprehensive profiling of soluble oligosaccharides also demonstrated decreases in levels of acetate esters of arabinoxylan oligosaccharides with concomitant increases in nonacetylated oligosaccharides that were consistent with earlier observations of 85% release of acetate esters by AFEX™ pretreatment.
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Authors: Bellesia G, Chundawat SPS, Langan P, Redondo A, Dale BE, Gnanakaran S
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Abstract: 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 Iβ (the natural form of cellulose) and cellulose IIII (obtained after cellulose Iβ is treated with anhydrous liquid ammonia). Cellulose IIII has been the focus of wide interest in the field of cellulosic biofuels, as it can be efficiently hydrolyzed to readily fermentable glucose (its enzymatic degradation rates are up to 5-fold higher than those of cellulose Iβ). The coarse-grained model presented in this study is based on a simplified geometry and on an effective potential mimicking the changes in both intracrystalline hydrogen bonds and stacking interactions during the transition from cellulose Iβ to cellulose IIII. The model reproduces both structural and thermomechanical properties of cellulose Iβ and IIII. The work presented herein describes the structural transition from cellulose Iβ to cellulose IIII as driven by the change in the equilibrium state of two degrees of freedom in the cellulose chains. The structural transition from cellulose Iβ to cellulose IIII is essentially reduced to a search for optimal spatial arrangement of the cellulose chains.
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Authors: Lau M*, Bals B, Chundawat SPS, Jin M, Gunawan C, Jones AD, Balan V, Dale BE
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Abstract: 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. Enzymatic hydrolysate from Ammonia Fiber Expansion (AFEX)-pretreated corn stover at 18% w/w solids loading was found to be nutrient-rich. This hydrolysate was fermented completely within 48 h in two stages to produce ethanol and native yeast cells. An in-house saccharolytic enzyme production using AFEX-pretreated corn stover as carbohydrate source greatly reduces the dependence on commercial enzymes. The inducer mixture is 2.5–7 times more potent than lactose, a common enzyme inducer. Economic analysis indicates that the proposed paradigm is substantially more cost-effective relative to the 2005 NREL model. This improvement is largely attributed to the native yeast cells co-production and the reduction of enzyme cost through the in-house production.
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Authors: Chundawat SPS*, Chang L, McMahan C, Gunawan C, Balan V, Dale BE
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Abstract: Natural rubber latex extraction from guayule leaves behind greater than 90% (by weight) of agricultural residue as a feedstock suitable for conversion to biofuels via a thermochemical or biochemical route. Untreated guayule shrub and bagasse (after latex extraction) has shown to be very recalcitrant to enzymatic hydrolysis, necessitating application of a chemical pretreatment to enhance cellulase accessibility. The objective of this work was to carry out detailed compositional analysis, ammonia fiber expansion (AFEX1) pretreatment, enzymatic hydrolysis and ethanol fermentation for various guayule-derived biomass fractions. Plant feedstocks tested were derived from two sources; (a) a mature 2007 AZ-2 whole guayule shrub plant obtained from USDA/ARS2 research fields, and (b) the guayule latex-extracted commercial grade bagasse (62505) from Yulex Corporation. Compositional analysis and enzymatic hydrolysis were carried out using standard NREL3 protocols (www.nrel.gov/biomass/analytical_procedures.html). AFEX pretreatment was carried out using concentrated ammonium hydroxide at elevated temperatures for desired residence times in a pressurized reactor. Yeast fermentations on biomass hydrolyzates were carried out micro-aerobically using Saccharomyces cerevisiae (424A strain) in shake flasks.