Deconstruction

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GLBRC's Deconstruction Research Area

Deconstruction

GLBRC's Deconstruction Lead takes top academic slot

Located at the intersection of the U.S.’s agricultural heartland and its northern forests, the GLBRC has access to a rich diversity of raw biomass for study. The Center's Deconstruction research focuses on identifying the best combinations of enzymes, chemicals, and physical processing methods for enhancing the digestibility of specific biomass sources.

Learn about the Center's research approach

Deconstruction Leadership

Deconstruction Lead

Dale is an expert on making ethanol from cellulose, plant stalks, grass, corn cobs and other woody plant parts and has developed a patented process called ammonia fiber expansion (AFEXTM), which makes the breakdown of cellulose more efficient, thus tackling...

Deconstruction Lead

Fox's research goals are to define the structure and the reactivity of the active site diiron center, to probe the catalytic contributions of the active site protein residues and to determine the consequences of protein-protein and protein-substrate interactions on the...

Project Overview

A biofuels reactor designed to produce ethanol at Michigan State University's Biomass Conversion Research Lab (BCRL)GLBRC Deconstruction research maintains a focus on the entire biofuels production pipeline: in addition to identifying and improving natural cellulose-degrading enzymes extracted from diverse environments, researchers apply unique biomass pretreatment technologies—such as ammonia fiber expansion (AFEX™), alkaline hydrogen peroxide (AHP), and extractive ammonia (EA)—that enable conversion technologies to maximize plant biomass utilization.. Researchers also explore strategies to add value to these processes by developing co-products from materials that would otherwise be treated as waste, such as lignin. Specific deconstruction projects include:

  • Pretreatment effects on biomass, alkaline peroxide pretreatment, fuel production from alkaline-pretreated biomass
  • Optimization of enzymes for biomass conversion, discovery of natural cellulolytic microbes, identification of novel microbial enzymes, and combinatorial discovery of enzymes and proteins

Deconstruction Publications

Mixture optimization of six core glycosyl hydrolases for maximizing saccharification of ammonia fiber expansion (AFEX) pretreated corn stover

Dahai Gao; Shishir P.S. Chundawat; Chandraraj Krishnan; Venkatesh Balan; Bruce E. Dale

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2010

In this work, six core glycosyl hydrolases (GH) were isolated and purified from various sources to help rationally optimize an enzyme cocktail to digest ammonia fiber expansion (AFEX) treated corn stover. The four core cellulases were endoglucanase I (EG I, GH family 7B), cellobiohydrolase I (CBH I, GH family 7A), cellobiohydrolase II (CBH II, GH family 6A) and β-glucosidase (βG, GH family 3). The two core hemicellulases were an endo-xylanase (EX, GH family 11) and a β-xylosidase (βX, GH family 3). Enzyme family and purity were confirmed by proteomics. Synergistic interactions among the six core enzymes for varying relative and total protein loading (8.25, 16.5 and 33 mg/g glucan) during hydrolysis of AFEX-treated corn stover was studied using a high-throughput microplate based protocol. The optimal composition (based on% protein mass loading) of the cocktail mixture was CBH I (28.4%): CBH II (18.0%): EG I (31.0%): EX (14.1%): βG (4.7%): βX (3.8%). These results demonstrate a rational strategy for the development of a minimal, synergistic enzymes cocktail that could reduce enzyme usage and maximize the fermentable sugar yields from pretreated lignocellulosics.

Multifaceted characterization of cell wall decomposition products formed during ammonia fiber expansion (AFEX) and dilute-acid based pretreatments

Shishir P.S. Chundawat; Ramin Vismeh; Lekh N. Sharma; James F. Humpula; Leonardo daCosta Sousa; C. K. Chambliss; A.D. Jones; Venkatesh Balan; Bruce E. Dale

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2010

Decomposition products formed/released during ammonia fiber expansion (AFEX) and dilute acid (DA) pretreatment of corn stover (CS) were quantified using robust mass spectrometry based analytical platforms. Ammonolytic cleavage of cell wall ester linkages during AFEX resulted in the formation of acetamide (25 mg/g AFEX CS) and various phenolic amides (15 mg/g AFEX CS) that are effective nutrients for downstream fermentation. After ammonolysis, Maillard reactions with carbonyl-containing intermediates represent the second largest sink for ammonia during AFEX. On the other hand, several carboxylic acids were formed (e.g. 35 mg acetic acid/g DA CS) during DA pretreatment. Formation of furans was 36-fold lower for AFEX compared to DA treatment; while carboxylic acids (e.g. lactic and succinic acids) yield was 100–1000-fold lower during AFEX compared to previous reports using sodium hydroxide as pretreatment reagent.

Process optimization to convert forage and sweet sorghum bagasse to ethanol based on ammonia fiber expansion (AFEX) pretreatment

Bing-Zhi Li; Venkatesh Balan; Ying-Jin Yuan; Bruce E. Dale

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2010

With growing demand for bio-based fuels and chemicals, there has been much attention given to the performance of different feedstocks. We have optimized the ammonia fiber expansion (AFEX) pretreatment and fermentation process to convert forage and sweet sorghum bagasse to ethanol. AFEX pretreatment was optimized for forage sorghum and sweet sorghum bagasse. Supplementing xylanase with cellulase during enzymatic hydrolysis increased both glucan and xylan conversion to 90% at 1% glucan loading. High solid loading hydrolyzates from the optimized AFEX conditions were fermented using Saccharomyces cerevisiae 424A (LNH-ST) without any external nutrient supplementation or detoxification. The strain was better able to utilize xylose at pH 6.0 than at pH 4.8, but glycerol production was higher for the former pH than the latter. The maximum final ethanol concentration in the fermentation broth was 30.9 g/L (forage sorghum) and 42.3 g/L (sweet sorghum bagasse). A complete mass balance for the process is given.

Rapid optimization of enzyme mixtures for deconstruction of diverse pretreatment/biomass feedstock combinations

Goutami Banerjee; Suzana Car; John Scott-Craig; Melissa Borrusch; Jonathan Walton

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2010

Enzymes for plant cell wall deconstruction are a major cost in the production of ethanol from lignocellulosic biomass. The goal of this research was to develop optimized synthetic mixtures of enzymes for multiple pretreatment/substrate combinations using our high-throughput biomass digestion platform, GENPLAT, which combines robotic liquid handling, statistical experimental design and automated Glc and Xyl assays. Proportions of six core fungal enzymes (CBH1, CBH2, EG1, beta-glucosidase, a GH10 endo-beta1,4-xylanase, and beta-xylosidase) were optimized at a fixed enzyme loading of 15 mg/g glucan for release of Glc and Xyl from all combinations of five biomass feedstocks (corn stover, switchgrass, Miscanthus, dried distillers' grains plus solubles [DDGS] and poplar) subjected to three alkaline pretreatments (AFEX, dilute base [0.25% NaOH] and alkaline peroxide [AP]). A 16-component mixture comprising the core set plus 10 accessory enzymes was optimized for three pretreatment/substrate combinations. Results were compared to the performance of two commercial enzymes (Accellerase 1000 and Spezyme CP) at the same protein loadings. RESULTS:When analyzed with GENPLAT, corn stover gave the highest yields of Glc with commercial enzymes and with the core set with all pretreatments, whereas corn stover, switchgrass and Miscanthus gave comparable Xyl yields. With commercial enzymes and with the core set, yields of Glc and Xyl were highest for grass stovers pretreated by AP compared to AFEX or dilute base. Corn stover, switchgrass and DDGS pretreated with AFEX and digested with the core set required a higher proportion of endo-beta1,4-xylanase (EX3) and a lower proportion of endo-beta1,4-glucanase (EG1) compared to the same materials pretreated with dilute base or AP. An optimized enzyme mixture containing 16 components (by addition of alpha-glucuronidase, a GH11 endoxylanase [EX2], Cel5A, Cel61A, Cip1, Cip2, beta-mannanase, amyloglucosidase, alpha-arabinosidase, and Cel12A to the core set) was determined for AFEX-pretreated corn stover, DDGS, and AP-pretreated corn stover. The optimized mixture for AP-corn stover contained more exo-beta1,4-glucanase (i.e., the sum of CBH1 + CBH2) and less endo-beta1,4-glucanase (EG1 + Cel5A) than the optimal mixture for AFEX-corn stover. Amyloglucosidase and beta-mannanase were the two most important enzymes for release of Glc from DDGS but were not required (i.e., 0% optimum) for corn stover subjected to AP or AFEX. As a function of enzyme loading over the range 0 to 30 mg/g glucan, Glc release from AP-corn stover reached a plateau of 60-70% Glc yield at a lower enzyme loading (5-10 mg/g glucan) than AFEX-corn stover. Accellerase 1000 was superior to Spezyme CP, the core set or the 16-component mixture for Glc yield at 12 h, but the 16-component set was as effective as the commercial enzyme mixtures at 48 h.CONCLUSION:The results in this paper demonstrate that GENPLAT can be used to rapidly produce enzyme cocktails for specific pretreatment/biomass combinations. Pretreatment conditions and feedstock source both influence the Glc and Xyl yields as well as optimal enzyme proportions. It is predicted that it will be possible to improve synthetic enzyme mixtures further by the addition of additional accessory enzymes.

Strategy for identification of novel fungal and bacterial glycosyl hydrolase hybrid mixtures that can efficiently saccharify pretreated lignocellulosic biomass

Dahai Gao; Shishir P.S. Chundawat; Tongjun Liu; Spencer Hermanson; Krishne Gowda; Phillip Brumm; Bruce E. Dale; Venkatesh Balan

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2010

A rational four-step strategy to identify novel bacterial glycosyl hydrolases (GH), in combination with various fungal enzymes, was applied in order to develop tailored enzyme cocktails to efficiently hydrolyze pretreated lignocellulosic biomass. The fungal cellulases include cellobiohydrolase I (CBH I; GH family 7A), cellobiohydrolase II (CBH II; GH family 6A), endoglucanase I (EG I; GH family 7B), and β-glucosidase (βG; GH family 3). Bacterial endocellulases (LC1 and LC2; GH family 5), β-glucosidase (LβG; GH family 1), endoxylanases (LX1 and LX2; GH family 10), and β-xylosidase (LβX; GH family 52) from multiple sources were cloned, expressed, and purified. Enzymatic hydrolysis for varying enzyme combinations was carried out on ammonia fiber expansion (AFEX)-treated corn stover at three total protein loadings (i.e., 33, 16.5, and 11 mg enzyme/g glucan). The optimal mass ratio of enzymes necessary to maximize both glucan and xylan yields was determined using a suitable design of experiments. The optimal hybrid enzyme mixtures contained fungal cellulases (78% of total protein loading), which included CBH I (loading ranging between 9-51% of total enzyme), CBH II (9-51%), EG I (10-50%), and bacterial hemicellulases (22% of total protein loading) comprising of LX1 (13%) and LβX (9%). The hybrid mixture was effective at 50°C, pH 4.5 to maximize saccharification of AFEX-treated corn stover resulting in 95% glucan and 65% xylan conversion. This strategy of screening novel enzyme mixtures on pretreated lignocellulose would ultimately lead to the development of tailored enzyme cocktails that can hydrolyze plant cell walls efficiently and economically to produce cellulosic ethanol.

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