Cellulosic biomass is an abundant and underused substrate for biofuel production. The inability of many microbes to metabolize the pentose sugars abundant within hemicellulose creates specific ...challenges for microbial biofuel production from cellulosic material. Although engineered strains of Saccharomyces cerevisiae can use the pentose xylose, the fermentative capacity pales in comparison with glucose, limiting the economic feasibility of industrial fermentations. To better understand xylose utilization for subsequent microbial engineering, we sequenced the genomes of two xylose-fermenting, beetle-associated fungi, Spathaspora passalidarum and Candida tenuis. To identify genes involved in xylose metabolism, we applied a comparative genomic approach across 14 Ascomycete genomes, mapping phenotypes and genotypes onto the fungal phylogeny, and measured genomic expression across five Hemiascomycete species with different xylose-consumption phenotypes. This approach implicated many genes and processes involved in xylose assimilation. Several of these genes significantly improved xylose utilization when engineered into S. cerevisiae, demonstrating the power of comparative methods in rapidly identifying genes for biomass conversion while reflecting on fungal ecology.
Empty palm fruit bunch fiber (EPFBF), a readily available cellulosic biomass from palm processing facilities, is investigated as a potential carbohydrate source for cellulosic ethanol production. ...This feedstock was pretreated using ammonia fiber expansion (AFEX) and enzymatically hydrolyzed. The best tested AFEX conditions were at 135 °C, 45 min retention time, water to dry biomass loading of 1:1 (weight ratio), and ammonia to dry biomass loading of 1:1 (weight ratio). The particle size of the pretreated biomass was reduced post-AFEX. The optimized enzyme formulation consists of Accellerase (84 μL/g biomass), Multifect Xylanase (31 μL/g biomass), and Multifect Pectinase (24 μL/g biomass). This mixture achieved close to 90% of the total maximum yield within 72 h of enzymatic hydrolysis. Fermentation on the water extract of this biomass affirms that nutrients solely from the pretreated EPFBF can support yeast growth for complete glucose fermentation. These results suggest that AFEX-treated EPFBF can be used for cellulosic biofuels production because biomass recalcitrance has been overcome without reducing the fermentability of the pretreated materials.
•AFEX hydrolysate storage temperature, pH and time were investigated.•Hydrolysate composition and fermentability before and after storage were examined.•Precipitates formed during storage increased ...with increasing pH and time.•Hydrolysate stored at 4°C and pH 4.8 formed little precipitate.•Little change of composition and fermentability was found after storage.
To minimize the change of lignocellulosic hydrolysate composition during storage, the effects of storage conditions (temperature, pH and time) on the composition and fermentability of hydrolysate prepared from AFEX™ (Ammonia Fiber Expansion – a trademark of MBI, Lansing, MI) pretreated corn stover were investigated. Precipitates formed during hydrolysate storage increased with increasing storage pH and time. The precipitate amount was the least when hydrolysate was stored at 4°C and pH 4.8, accounting for only 0.02% of the total hydrolysate weight after 3-month storage. No significant changes of NMR (Nuclear Magnetic Resonance) spectra and concentrations of sugars, minerals and heavy metals were observed after storage under this condition. When pH was adjusted higher before fermentation, precipitates also formed, consisting of mostly struvite (MgNH4PO4·6H2O) and brushite (CaHPO4·2H2O). Escherichia coli and Saccharomyces cerevisiae fermentation studies and yeast cell growth assays showed no significant difference in fermentability between fresh hydrolysate and stored hydrolysate.
► Guayule shrub and bagasse (after latex extraction) are potent cellulosic feedstock for biofuel production. ► Ammonia fiber expansion (AFEX) pretreatment improved carbohydrate enzymatic ...digestibility by up to 20 fold. ► Fermentation of hydrolyzed sugars (glucose, xylose) to ethanol was achieved without external nutrient supplementation.
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 (AFEX11AFEX: ammonia fiber expansion.) 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/ARS22USDA/ARS: United States Department of Agriculture/Agricultural Research Service. 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 NREL33NREL: National Renewable Energy Laboratory. 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.
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 guayule bagasse hydrolyzate with or without external nutrient supplementation (i.e., yeast extract and tryptone). Our results highlight the possible utilization of guayule as a feedstock for lignocellulosic refineries co-producing natural rubber latex and biofuels. However, further process improvements (e.g., lignin/resin extraction and cellulose decrystallization using a modified AFEX process) are necessary to increase the effectiveness of ammonia-based pretreatments for further enhancing enzymatic digestibility of guayule-derived hardwood biomass.
► Xylose fermentation was poor in 6h pre-hydrolysis SSCF on AFEXTM corn stover. ► Xylose fermentation was improved by extending the pre-hydrolysis time. ► Glucose concentration after pre-hydrolysis ...was the critical factor affecting xylose fermentation. ► Low temperature and ethanol inhibition limited the hydrolysis rates during SSCF.
Xylose consumption by Saccharomyces cerevisiae 424A(LNH-ST) during simultaneous saccharification and co-fermentation (SSCF) of AFEXTM pretreated switchgrass was inhibited by unhydrolyzed solids. Such inhibitory effects were not found in unhydrolyzed solids from AFEXTM pretreated corn stover (AFEXTM-CS). However, the xylose consumption was still unsatisfactory during 6h pre-hydrolysis SSCF. By extending the pre-hydrolysis time to 24h or longer, the xylose consumption was improved significantly. In order to better understand the reasons for such improvement, the hydrolysate slurries after 6h pre-hydrolysis and 24h pre-hydrolysis were studied and compared. We found that the glucose concentration after pre-hydrolysis was the critical factor that determined cell viability and hence xylose consumption during SSCF. Low temperature (30°C) and ethanol inhibition were shown to be the factors limiting hydrolysis rate and hence productivity during SSCF.
Reduced xylose fermentation performance has been an issue during fermentation of AFEXTM hydrolysate using Saccharomyces cerevisiae 424A (LNH-ST) or Escherichia coli KO11. To better understand why ...fermentation performance is reduced, we quantitatively studied the effects of compounds present in the fermentation broth on xylose consumption. The compounds include biomass degradation products, ethanol and fermentation metabolites. The xylose consumption capability of E. coli KO11 was almost totally inhibited by the presence of both degradation products and ethanol. On the other hand, for S. cerevisiae 424A, 89% reduction of xylose consumption rate was found during hydrolysate fermentation. Degradation products, ethanol and fermentation metabolites were responsible for 32%, 24% and 33% of such reduction, respectively. Those results suggest that to further improve the xylose fermentation in hydrolysate, strains should be selected not only for degradation products tolerance but also for ethanol and fermentation metabolites tolerance.
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