252 research outputs found
Profiling of Soluble Neutral Oligosaccharides from Treated Biomass using Solid Phase Extraction and Liquid Chromatography-Multiplexed Collision Induced Dissociation-Mass Spectrometry
No full textThermochemical pretreatment of cellulosic biomass improves cell wall enzymatic digestibility, while simultaneously releasing substantial amounts of soluble oligosaccharides. Profiling of oligosaccharides released during pretreatment yield 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 (AFEXTM)-pretreated corn stover. Our methodology employs solid phase extraction (SPE) enrichment of oligosaccharides based on porous graphitized carbon (PGC), followed by high performance liquid chromatography (HPLC) separation using a polymeric amine based column (Prevail Carbohydrate ES) and electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS) in both positive and negative modes. For structural elucidation on the chromatographic time scale, nonselective multiplexed collision-induced dissociation was performed for quasi-simultaneous acquisition of accurate molecular and fragment masses of neutral oligosaccharids in a single analysis. These analyses directly revealed presence of glucans up to degree of polymerization (DP) 22 without side-chain modifications. Additionally, arabinoxylans with DP up to 6 were detected in the pretreated biomass samples (post-enzymatic digestion). All linkages between sugar units in glucans and arabinoxylans were identified to be p-1-4 linkages based on cross-ring fragment masses. Comprehensive profiling of soluble oligosaccharides also demonstrated that arabinoxylan acetylation was reduced by greater than 85% post-AFEXTM treatment.Published version: Vismeh, Ramin, Humpula, James F., Chundawat, Shishir P. S., Balan, Venkatesh, Dale, Bruce E. & Jones, A. Daniel. (2013). Profiling of Soluble Neutral Oligosaccharides from Treated Biomass using Solid Phase Extraction and LC-TOF MS. Carbohydrate Polymers 94(2), 791-799. http://dx.doi.org/10.1016/j.carbpol.2013.02.00
Probing the nature of AFEX-pretreated corn stover derived decomposition products that inhibit cellulase activity
No full textSequential 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 10 kDa molecular weight cutoff (MWCO) membrane removed additional high molecular weight components that accounted for 24–28% of the total observed enzyme inhibition while a 3 kDa 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 5 kDa 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.Published version: Humpula, James F., Uppugundla, Nirmal, Vismeh, Ramin, Sousa, Leonardo, Chundawat, Shishir P. S., Jones, A. Daniel, Balan, Venkatesh, Dale, Bruce E. & Cheh, Albert M. (2014). Probing the nature of AFEX-retreated corn stover derived decomposition products that inhibit cellulase activity. Bioresource Technology 152, 38-45. http://dx.doi.org/10.1016/j.biortech.2013.10.08
Optimizing Harvest of Corn Stover Fractions Based on Overall Sugar Yields following AFEX Pretreatment and Enzymatic Hydrolysis
No full textBackground: Corn stover composition changes considerably throughout the growing season and also varies between the various fractions of the plant. These differences can impact optimal pretreatment conditions, enzymatic digestibility and maximum achievable sugar yields in the process of converting lignocellulosics to ethanol. The goal of this project was to determine which combination of corn stover fractions provides the most benefit to the biorefinery in terms of sugar yields and to determine the preferential order in which fractions should be harvested. Ammonia fiber expansion (AFEX) pretreatment, followed by enzymatic hydrolysis, was performed on early and late harvest corn stover fractions (stem, leaf, husk and cob). Sugar yields were used to optimize scenarios for the selective harvest of corn stover assuming 70% or 30% collection of the total available stover.
Results: The optimal AFEX conditions for all stover fractions, regardless of harvest period, were: 1.5 (g NH(3) g(-1) biomass); 60% moisture content (dry-weight basis; dwb), 90 degrees C and 5 min residence time. Enzymatic hydrolysis was conducted using cellulase, beta-glucosidase, and xylanase at 31.3, 41.3, and 3.1 mg g(-1) glucan, respectively. The optimal harvest order for selectively harvested corn stover (SHCS) was husk > leaf > stem > cob. This harvest scenario, combined with optimal AFEX pretreatment conditions, gave a theoretical ethanol yield of 2051 L ha(-1) and 912 L ha(-1) for 70% and 30% corn stover collection, respectively.
Conclusion: Changing the proportion of stover fractions collected had a smaller impact on theoretical ethanol yields (29 - 141 L ha(-1)) compared to the effect of altering pretreatment and enzymatic hydrolysis conditions (150 - 462 L ha(-1)) or harvesting less stover (852 - 1139 L ha(-1)). Resources may be more effectively spent on improving sustainable harvesting, thereby increasing potential ethanol yields per hectare harvested, and optimizing biomass processing rather than focusing on the selective harvest of specific corn stover fractions.Peer reviewed
'Cradle-to-grave' assessment of existing lignocellulose pretreatment technologies
No full textPretreatment is considered to be a central unit process in a biorefinery to convert lignocellulosic biomass into fuels and chemicals, affecting all other operations in the process. A variety of technologies to pretreat lignocellulosic biomass are available today, which encompass a wide range of physical, chemical, and biological based processes. Among these, chemical based pretreatments are considered to be the most promising for future biorefineries. However, several key criteria regarding technical, economical, and environmental considerations should be critically analyzed when adapting these technologies for the nascent biorefinery industry. This review will discuss the most important pretreatment methods available today and will highlight key criteria for the development of a future ideal pretreatment.Peer reviewed
Strategy for identification of novel fungal and bacterial glycosyl hydrolase hybrid mixtures that can efficiently saccharify pretreated lignocellulosic biomass
No full textWe have applied a rational four-step strategy to identify novel bacterial glycosyl hydrolases (GH), in combination with various fungal enzymes, in order to define an efficient enzyme cocktail to 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 ß-glucosidase (ßG; GH family 3). Bacterial endocellulases (LC1 and LC2; GH family 5), ß-glucosidase (LßG; GH family 1), endoxylanases (LX1 and LX2; GH family 10) and ß-xylosidase (LßX; GH family 52) from multiple sources were cloned, expressed and purified as well. Enzymatic hydrolysis for various enzyme combinations was performed on Ammonia Fiber Expansion (AFEX) treated corn stover at various total protein loadings (30, 15 and 10 mg enzyme/g glucan). The optimal mass ratio of enzymes necessary for obtaining high glucan and xylan yields was determined using a suitable mixture design of experiments. The optimal hybrid enzyme mixtures contain fungal cellulases (78% of total protein loading), which include CBH I (loading ranging between 9-51% of total enzyme load), CBH II (9-51%), EG I (10-50%), and bacterial hemicellulases (22% of total protein loading) comprising of LXl (13%) and LßX (9%). The hybrid mixture works effectively at 50 °C and pH 4.5 to efficiently saccharify AFEX treated corn stover giving as high as 95% glucan and 65% xylan conversion, respectively. This strategy of screening enzyme mixtures on pretreated lignocellulose will ultimately help develop enzyme cocktails that can hydrolyze plant cell walls efficiently and economically to produce cellulosic ethanol.The final publication (Version of Record) is available at Springer via http://dx.doi.org/10.1007/s12155-009-9066-
Overview to Ammonia Pretreatments for Lignocellulosic Biorefineries
No full textDevelopment of environmentally sustainable and economically viable technologies for plant cell wall deconstruction to fermentable sugars has been impeded due to native plant cell wall recalcitrance to thermochemical and biological based processing. Lower severity alkaline-based pretreatment processes like Ammonia Fiber Expansion (AFEX™) can overcome several limitations of traditional pretreatment approaches (e.g., acidic pretreatments) to producing cellulosic biofuels and biochemicals. Here, we give an overview of chemical reactions taking place during alkaline pretreatments including reactions between ammonia and polysaccharides/lignin (e.g., ammonolysis, hydrolysis and Maillard-type reactions). AFEX™ based pretreatments enhance enzymatic digestibility and fermentability of lignocellulosic biomass through various chemical and ultra-structural modifications within the cell wall. An improved mechanistic understanding of the AFEX™ process has led to the development of novel alkaline pretreatments that are briefly discussed in this review.Peer reviewed
Biochemical and Thermochemical Conversion of Switchgrass to Biofuels
No full textWith 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 recent literature pertaining to different processing steps within the biochemical platform (pretreatment, enzyme hydrolysis, microbial fermentation, protein extraction) and thermochemical platform (pyrolysis, bio-oil, gasification, combustion, hydrothermal process) in this chapter. Though we have improved our fundamental understanding on the different processing steps to produce biofuels, several challenges still have to be overcome to create a bioeconomy and produce fuels and chemicals from biomass in an economic and sustainable manner.Part of the Green Energy and Technology book series
The quest for alternatives to microbial cellulase mix production: corn stover-produced heterologous multi-cellulases readily deconstruct lignocellulosic biomass into fermentable sugars
No full textBACKGROUND: Production of cellulosic ethanol is still expensive compared with corn (maize) grain ethanol due to the high costs of bulk production of microbial cellulases. At least three cellulases including endo-cellulase, exo-cellulase and cellobiase are needed to convert cellulosic biomass into fermentable sugars. All these cellulases could be self-produced within cells of transgenic bio-energy crops. The production of heterologous Acidothermus cellulolyticus (E1) endo-cellulase in endoplasmic reticulum and mitochondria of green tissues of transgenic corn plants was recently reported, and it was confirmed that the heterologous E1 converts cellulose into fermentable sugars.
RESULTS: Biologically active A. cellulolyticus E1, Trichoderma reesei 1,4-β-cellobiohydrolases I (CBH I) exo-cellulase and bovine rumen Butyrivibrio fibrisolvens cellobiase were expressed in corn plant endoplasmic reticulum (ER), apoplast (cell wall areas) and vacuole respectively. Results show that the ratio 1 : 4 : 1 (E1 : CBH I : cellobiase) of crude heterologous cellulases is ideal for converting ammonia fiber explosion (AFEX) pretreated corn stover into fermentable sugars.
CONCLUSIONS: Corn plants that express all three biologically active heterologous cellulases within their cellulosic biomass to facilitate conversion of pretreated corn stover into fermentable sugars is a step forward in the quest for alternatives to the present microbial cellulase mix production for cellulosic biofuels.Peer reviewed
Enzymatic digestibility and ethanol fermentability of AFEX-treated starch-rich lignocellulosics such as corn silage and whole corn plant
No full textBackground: Corn grain is an important renewable source for bioethanol production in the USA. Corn ethanol is currently produced by steam liquefaction of starch-rich grains followed by enzymatic saccharification and fermentation. Corn stover (the non-grain parts of the plant) is a potential feedstock to produce cellulosic ethanol in second-generation biorefineries. At present, corn grain is harvested by removing the grain from the living plant while leaving the stover behind on the field. Alternatively, whole corn plants can be harvested to cohydrolyze both starch and cellulose after a suitable thermochemical pretreatment to produce fermentable monomeric sugars. In this study, we used physiologically immature corn silage (CS) and matured whole corn plants (WCP) as feedstocks to produce ethanol using ammonia fiber expansion (AFEX) pretreatment followed by enzymatic hydrolysis (at low enzyme loadings) and cofermentation (for both glucose and xylose) using a cellulase-amylase-based cocktail and a recombinant Saccharomyces cerevisiae 424A (LNH-ST) strain, respectively. The effect on hydrolysis yields of AFEX pretreatment conditions and a starch/cellulose-degrading enzyme addition sequence for both substrates was also studied.
Results: AFEX-pretreated starch-rich substrates (for example, corn grain, soluble starch) had a 1.5-3-fold higher enzymatic hydrolysis yield compared with the untreated substrates. Sequential addition of cellulases after hydrolysis of starch within WCP resulted in 15-20% higher hydrolysis yield compared with imultaneous addition of hydrolytic enzymes. AFEX-pretreated CS gave 70% glucan conversion after 72 h of hydrolysis for 6% glucan loading (at 8 mg total enzyme loading per gram glucan). Microbial inoculation of CS before ensilation yielded a 10-15% lower glucose hydrolysis yield for the pretreated substrate, due to loss in starch content. Ethanol fermentation of AFEX-treated (at 6% w/w glucan loading) CS hydrolyzate (resulting in 28 g/L ethanol at 93% metabolic yield) and WCP (resulting in 30 g/L ethanol at 89% metabolic yield) is reported in this work.
Conclusions: The current results indicate the feasibility of co-utilization of whole plants (that is, starchy grains plus cellulosic residues) using an ammonia-based (AFEX) pretreatment to increase bioethanol yield and reduce overall production cost.Peer reviewed
Enzymatic digestibility and pretreatment degradation products of AFEX-treated hardwoods (Populus nigra)
No full textThere is a growing need to find alternatives to crude oil as the primary feed stock for the chemicals and fuel industry and ethanol has been demonstrated to be a viable alternative. Among the various feed stocks for producing ethanol, poplar (Populus nigra × Populus maximowiczii) is considered to have great potential as a biorefinery feedstock in the United States, due to their widespread availability and good productivity in several parts of the country. We have optimized AFEX pretreatment conditions (180°C, 2:1 ammonia to biomass loading, 233% moisture, 30 minutes residence time) and by using various combinations of enzymes (commercical celluloses and xylanases) to achieve high glucan and xylan conversion (93 and 65%, respectively). We have also identified and quantified several important degradation products formed during AFEX using liquid chromatography followed by mass spectrometry (LC-MS/MS). As a part of degradation product analysis, we have also quantified oligosaccharides in the AFEX water wash extracts by acid hydrolysis. It is interesting to note that corn stover (C4 grass) can be pretreated effectively using mild AFEX pretreatment conditions, while on the other hand hardwood poplar requires much harsher AFEX conditions to obtain equivalent sugar yields upon enzymatic hydrolysis. Comparing corn stover and poplar, we conclude that pretreatment severity and enzymatic hydrolysis efficiency are dictated to a large extent by lignin carbohydrate complexes and arabinoxylan cross-linkages for AFEX.Peer reviewed
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