A pinboard by
Ninad Kothari

PhD Candidate, University of California - Riverside


Integration and optimization of thermochemical and biological processes for bioethanol production

The objective of my work is to design a cost effective benchmark process for the production of fuel ethanol from plant (lignocellulosic) biomass. Lignocellulosic biomass is the only sustainable option for powering transportation that dramatically reduces greenhouse gas emissions, lowers our dependence on imported petroleum, and creates domestic agricultural and manufacturing employment. However, plants have a complex architecture in their cell walls made up of cellulose, hemicellulose, and lignin for mechanical support and strength and to ward off attack by microbes. These self-preservation and structural attributes of lignocellulosic biomass make it extremely resistant to deconstruction into fermentable sugars. The major problem with converting plant sugars to ethanol is achieving high yields of product that are economically competitive. Lignocellulosic biomass must, therefore, be pretreated to overcome the resistance barrier for faster and more efficient degradation. The process of pretreatment opens up the complex cell wall structure of the plant biomass and makes cellulose sugar more accessible to further deconstruction. However, pretreatments introduce more variability in the biomass composition which affects further microbiological deconstruction. Therefore, the goal of my research is to optimize and integrate pretreatments with further microbial production of ethanol to achieve high yields. I apply a consolidated bioprocessing (CBP) microbe, Clostridium thermocellum, to directly convert switchgrass into ethanol after the pretreatment step. C. thermocellum produces its own enzymes to hydrolyze polysaccharides found in plant biomass into simple sugars and also ferments the sugars released to ethanol or other desired products. This process is economically attractive and inherently simple. With this work, I will help the biofuels community understand the mechanisms of thermo-chemical and biological deconstruction of lignocellulosic biomass. This research will also guide the understanding for rational design of processes for the production of ethanol at an industrial scale.


Impact of pretreated Switchgrass and biomass carbohydrates on Clostridium thermocellum ATCC 27405 cellulosome composition: a quantitative proteomic analysis.

Abstract: Economic feasibility and sustainability of lignocellulosic ethanol production requires the development of robust microorganisms that can efficiently degrade and convert plant biomass to ethanol. The anaerobic thermophilic bacterium Clostridium thermocellum is a candidate microorganism as it is capable of hydrolyzing cellulose and fermenting the hydrolysis products to ethanol and other metabolites. C. thermocellum achieves efficient cellulose hydrolysis using multiprotein extracellular enzymatic complexes, termed cellulosomes.In this study, we used quantitative proteomics (multidimensional LC-MS/MS and (15)N-metabolic labeling) to measure relative changes in levels of cellulosomal subunit proteins (per CipA scaffoldin basis) when C. thermocellum ATCC 27405 was grown on a variety of carbon sources [dilute-acid pretreated switchgrass, cellobiose, amorphous cellulose, crystalline cellulose (Avicel) and combinations of crystalline cellulose with pectin or xylan or both]. Cellulosome samples isolated from cultures grown on these carbon sources were compared to (15)N labeled cellulosome samples isolated from crystalline cellulose-grown cultures. In total from all samples, proteomic analysis identified 59 dockerin- and 8 cohesin-module containing components, including 16 previously undetected cellulosomal subunits. Many cellulosomal components showed differential protein abundance in the presence of non-cellulose substrates in the growth medium. Cellulosome samples from amorphous cellulose, cellobiose and pretreated switchgrass-grown cultures displayed the most distinct differences in composition as compared to cellulosome samples from crystalline cellulose-grown cultures. While Glycoside Hydrolase Family 9 enzymes showed increased levels in the presence of crystalline cellulose, and pretreated switchgrass, in particular, GH5 enzymes showed increased levels in response to the presence of cellulose in general, amorphous or crystalline.Overall, the quantitative results suggest a coordinated substrate-specific regulation of cellulosomal subunit composition in C. thermocellum to better suit the organism's needs for growth under different conditions. To date, this study provides the most comprehensive comparison of cellulosomal compositional changes in C. thermocellum in response to different carbon sources. Such studies are vital to engineering a strain that is best suited to grow on specific substrates of interest and provide the building blocks for constructing designer cellulosomes with tailored enzyme composition for industrial ethanol production.

Pub.: 23 Apr '09, Pinned: 30 Jun '17

Consolidated bioprocessing of transgenic switchgrass by an engineered and evolved Clostridium thermocellum strain.

Abstract: Switchgrass is an abundant and dedicated bioenergy feedstock, however its inherent recalcitrance is one of the economic hurdles for producing biofuels. The downregulation of the caffeic acid O-methyl transferase (COMT) gene in the lignin pathway of switchgrass reduced lignin content and S/G ratio, and the transgenic lines showed improved fermentation yield with Saccharomyces cerevisiae and wild-type Clostridium thermocellum (ATCC 27405) in comparison to the wild-type switchgrass.Here we examine the conversion and yield of the COMT transgenic and wild-type switchgrass lines with an engineered and evolved C. thermocellum (M1570) strain. The fermentation of the transgenic switchgrass by M1570 had superior conversion relative to the wild-type control switchgrass line with an increase in conversion of approximately 20% and ethanol being the primary product accounting for 90% of the total metabolites measured by HPLC analysis.The engineered and evolved C. thermocellum M1570 was found to respond to the apparent reduced recalcitrance of the COMT switchgrass with no substrate inhibition, producing more ethanol on the transgenic feedstock than the wild-type substrate. Since ethanol was the main fermentation metabolite produced by an engineered and evolved C. thermocellum strain, its ethanol yield on a transgenic switchgrass substrate (gram/gram (g/g) glucan liberated) is the highest produced thus far. This result indicates that the advantages of a modified feedstock can be combined with a modified consolidated bioprocessing microorganism as anticipated.

Pub.: 31 May '14, Pinned: 30 Jun '17

Characterization of Clostridium thermocellum (B8) secretome and purified cellulosomes for lignocellulosic biomass degradation

Abstract: The main goal of the present study was a complete proteomic characterization of total proteins eluted from residual substrate-bound proteins (RSBP), and cellulosomes secreted by Clostridium thermocellum B8 during growth in the presence of microcrystalline cellulose as a carbon source. The second goal was to evaluate their potential use as enzymatic blends for hydrolyzing agro-industrial residues to produce fermentable sugars. Protein identification through LC-MS/MS mass spectrometry showed that the RSBP sample, in addition to cellulosomal proteins, contains a wide variety of proteins, including those without a well-characterized role in plant cell wall degradation. The RSBP subsample defined as purified cellulosomes (PC) consists mainly of glycoside hydrolases grouped in families 5, 8, 9, 10 and 48. Dynamic light scattering, DLS, analysis of PC resulted in two protein peaks (pi1 and pi2) presenting molecular masses in agreement with those previously described for cellulosomes and polycellulosomes. These peaks weren’t detected after PC treatment with 1.0% Tween. PC and RSBP presented maximal activities at temperatures ranging from 60° to 70 °C and at pH 5.0. RSBP retained almost all of its activity after incubation at 50, 60 and 70 °C and PC showed remarkable thermostability at 50 and 60 °C. RSBP holocellullolytic activities were inhibited by phenolic compounds, while PC showed either increasing activity or a lesser degree of inhibition. RSBP and PC hydrolyze sugar cane straw, cotton waste and microcrystalline cellulose, liberating a diversity of saccharides; however, the highest concentration of released sugar was obtained for assays carried out using PC as an enzymatic blend and after ten days at 50 °C.

Pub.: 08 Nov '16, Pinned: 30 Jun '17

Biochemical characterization of microalgae collected from north east region of India advancing towards the algae-based commercial production

Abstract: Selection of suitable strain of microalgae is the crucial factor for large-scale production of algae-based products. Efforts have been made here for isolation, identification and biochemical characterization of five microalgae strains collected from Tripura (a small state in north-eastern region of India). Two Chlorococcum sp. (NITAAP008 and NITAAP019) demonstrate their high lipid (15–24%), equal amounts of carbohydrate and protein (35–40%), with specific growth rate of 0.13 day−1. These strains are potential resource for biofuel production. After lipid extraction, remaining biomass can be used as source of carbohydrate for the production of other biofuels. One isolated strain is identified as Chlorella sp. (NITAAP009) and shows 22–33% carbohydrate, 41–50% protein and 5% chlorophyll with specific growth rate of 0.125 day−1. Another Chlorella sp. (NITAAP011) isolated from lake area exhibits significant chlorophyll (5–6.4%), 30–50% carbohydrate, 48–60% protein and low lipid (1–10%) with lower specific growth rate (0.10 day−1). Both strains are having industrial competence for chlorophyll production due to their synthesizing ability of significant amount of chlorophyll (5–6.4%). The last one, Korshikoviella sp. (NITAAP017), has 15–18% lipid, 22–34% carbohydrate, 30–43% protein and 3–4% chlorophyll with specific growth rate of 0.12 day−1 and can be used for food supplement production or lipid synthesis. © 2017 Curtin University of Technology and John Wiley & Sons, Ltd.

Pub.: 29 Jun '17, Pinned: 30 Jun '17

Understanding Multiscale Structural Changes During Dilute Acid Pretreatment of Switchgrass and Poplar

Abstract: Structural and morphological changes over multiple scales in herbaceous and woody biomass during dilute sulfuric acid pretreatment were explored.Biofuels produced from lignocellulosic biomass hold great promise as a renewable alternative energy and fuel source. To realize a cost and energy efficient approach, a fundamental understanding of the deconstruction process is critically necessary to reduce biomass recalcitrance. Herein, the structural and morphological changes over multiple scales (5–6000 Å) in herbaceous (switchgrass) and woody (hybrid poplar) biomass during dilute sulfuric acid pretreatment were explored using neutron scattering and X-ray diffraction. Switchgrass undergoes a larger increase (20–84 Å) in the average diameter of the crystalline core of the elementary cellulose fibril than hybrid poplar (19–50 Å). Switchgrass initially forms lignin aggregates with an average size of 90 Å that coalesce to 200 Å, which is double that observed for hybrid poplar, 55–130 Å. Switchgrass shows a smooth-to-rough transition in the cell wall surface morphology unlike the diffuse-to-smooth transition of hybrid poplar. Yet, switchgrass and hybrid poplar pretreated under the same experimental conditions result in pretreated switchgrass producing higher glucose yields (∼76 wt %) than pretreated hybrid poplar (∼60 wt %). This observation shows that other aspects like cellulose allomorph transitions, cellulose accessibility, cellular biopolymer spatial distribution, and enzyme–substrate interactions may be more critical in governing the enzymatic hydrolysis efficiency.

Pub.: 05 Dec '16, Pinned: 29 Jun '17