ATETV takes viewers inside the classroom of a typical Biotechnology college course and finds that in addition to traditional math and science studies, hands-on lab training and business-focused projects are important components of today's Biotech curriculum.

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Biology: Biotechnology: Gene Cloning

Background:
We and other workers have shown that accessory enzymes, such as beta-glucosidase, xylanase and cellulase co-factors such as GH61 can considerably enhance the hydrolysis effectiveness of cellulase cocktails when added to pretreated lignocellulosic substrates. It is generally acknowledged that, among the several factors that hamper our current ability to attain efficient lignocellulosic biomass conversion yields at low enzyme loadings, a major problem lies in our incomplete understanding of the cooperative action of the different enzymes acting on pretreated lignocellulosic substrates.
Results:
The reported work assessed the interaction between cellulase and xylanase enzymes and their potential to improve the hydrolysis efficiency of various pretreated lignocellulosic substrates when added at low protein loadings. When xylanase were added to the minimum amount of cellulase enzymes required to achieve 70% cellulose hydrolysis of steam pretreated corn stover (SPCS) or used to partially replace the equivalent cellulase dose, both approaches resulted in enhanced enzymatic hydrolysis. However, the xylanase supplementation approach increased the total protein loading required to achieve significant improvements in hydrolysis (an additive effect), whereas the partial replacement of cellulases with xylanase resulted in similar improvements in hydrolysis without increasing enzyme loading (a synergistic effect). The enhancement resulting from xylanase-aided synergistic was higher when enzymes were added simultaneously at the beginning of hydrolysis. This co-hydrolysis of the xylan also influenced the gross fibre characteristics (e.g. fibre swelling) resulting in increased accessibility of the cellulose to the cellulase enzymes. These apparent increases in accessibility enhanced the SPCS digestibility resulting in three time's faster cellulose and xylan hydrolysis, a 7-fold decrease in cellulase loading and a significant increase in the hydrolysis performance of the optimized enzyme mixture. When a similar xylanase-aided enhancement strategy was assessed on other pretreated lignocellulosic substrates, equivalent increases in hydrolysis efficiency were also observed.
Conclusions:
It was apparent that the "blocking effect" of xylan was one of the major mechanisms that limited the accessibility of the cellulase enzymes to the cellulose. However, the synergistic interaction of the xylanase and cellulase enzymes was also shown to significantly improve cellulose accessibility through increasing fibre swelling and fibre porosity and also plays a major role in enhancing enzyme accessibility.Source:
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Background:
In order to generate biofuels, insoluble cellulosic substrates are pretreated and subsequently hydrolyzed with cellulases. One way to pretreat cellulose in a safe and environmentally friendly manner is to apply, under mild conditions, non-hydrolyzing proteins such as swollenin - naturally produced in low yields by the fungus Trichoderma reesei. To yield sufficient swollenin for industrial applications, the first aim of this study is to present a new way of producing recombinant swollenin. The main objective is to show how swollenin quantitatively affects relevant physical properties of cellulosic substrates and how it affects subsequent hydrolysis.
Results:
After expression in the yeast Kluyveromyces lactis, the resulting swollenin was purified. The adsorption parameters of the recombinant swollenin onto cellulose were quantified for the first time and were comparable to those of individual cellulases from T. reesei. Four different insoluble cellulosic substrates were then pretreated with swollenin. At first, it could be qualitatively shown by macroscopic evaluation and microscopy that swollenin caused deagglomeration of bigger cellulose agglomerates as well as dispersion of cellulose microfibrils (amorphogenesis). Afterwards, the effects of swollenin on cellulose particle size, maximum cellulase adsorption and cellulose crystallinity were quantified. The pretreatment with swollenin resulted in a significant decrease in particle size of the cellulosic substrates as well as in their crystallinity, thereby substantially increasing maximum cellulase adsorption onto these substrates. Subsequently, the pretreated cellulosic substrates were hydrolyzed with cellulases. Here, pretreatment of cellulosic substrates with swollenin, even in non-saturating concentrations, significantly accelerated the hydrolysis. By correlating particle size and crystallinity of the cellulosic substrates with initial hydrolysis rates, it could be shown that the swollenin-induced reduction in particle size and crystallinity resulted in high cellulose hydrolysis rates.
Conclusions:
Recombinant swollenin can be easily produced with the robust yeast K. lactis. Moreover, swollenin induces deagglomeration of cellulose agglomerates as well as amorphogenesis (decrystallization). For the first time, this study quantifies and elucidates in detail how swollenin affects different cellulosic substrates and their hydrolysis.Source:
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Background:
Ethanol production from paper sludge (PS) by simultaneous saccharification and fermentation (SSF) is considered to be the most appropriate way to process PS, as it contains negligible lignin. In this study, SSF was conducted using a cellulase produced from PS by the hyper cellulase producer, Acremonium cellulolyticus C-1 for PS saccharification, and a thermotolerant ethanol producer Saccharomyces cerevisiae TJ14 for ethanol production. Using cellulase of PS origin minimizes biofuel production costs, because the culture broth containing cellulase can be used directly.
Results:
When 50 g PS organic material (PSOM)/l was used in SSF, the ethanol yield based on PSOM was 23% (g ethanol/g PSOM), and was two times higher than that obtained by a separate hydrolysis and fermentation process. Cellulase activity throughout SSF remained at around 60% of the initial activity. When 50 to 150 g PSOM/l was used in SSF, the ethanol yield was 21-23% (g ethanol/g PSOM) in 500 ml Erlenmeyer flask scale. Ethanol production and theoretical ethanol yield based on initial hexose was 40 g/l and 66.3% (g ethanol/g hexose) at 80 h, respectively, when 161 g/l of PSOM, 15 FPU/g PSOM, and 20% inoculum were used for SSF, which was confirmed in the 2 liter scale experiment. This indicates that PS is a good raw material for bioethanol production.
Conclusion:
Ethanol concentration increased with increasing PSOM concentration. The ethanol yield was stable at PSOM concentrations of up to 150 g/l, but decreased at concentrations higher than 150 g/l because of mass transfer limitations. Based on 2-liter scale experiment, when 1000 kg PS was used, 3,182 kFPU cellulase was produced from 134.7 kg PS. Produced cellulase was used for SSF with 865.3 kg PS and ethanol production was estimated to be 51.1 kg. Increasing the yeast inoculum or cellulase concentration did not significantly improve the ethanol yield or concentration.Source:
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Background:
Microalgae are a promising feedstock for biofuel and bioenergy production due to their high photosynthetic efficiencies, high growth rates and no need for external organic carbon supply. In this study, utilization of Chlorella vulgaris (a fresh water microalga) and Dunaliella tertiolecta (a marine microalga) biomass was tested as a feedstock for anaerobic H2 and CH4 production.
Results:
Anaerobic serum bottle assays were conducted at 37 degrees C with enrichment cultures derived from municipal anaerobic digester sludge. Low levels of H2 were produced by anaerobic enrichment cultures, but H2 was subsequently consumed even in presence of 2-bromoethanesulfonic acid, an inhibitor of methanogens. Without inoculation, algal biomass still produced H2 due to the activities of satellite bacteria associated with algal cultures. CH4 was produced from both types of biomass with the anaerobic enrichments. Polymerase chain reaction-denaturing gradient gel electrophoresis profiling indicated the presence of H2-producing and H2-consuming bacteria in the anaerobic enrichment cultures and the presence of H2-producing bacteria among the satellite bacteria in both sources of algal biomass.
Conclusions:
H2 production by the satellite bacteria was comparable from D. tertiolecta (12.6 mL H2 g-volatile solids (VS)-1) and from C. vulgaris (10.8 mL H2 g-VS-1), whereas CH4 production was significantly higher from C. vulgaris (286 mL g-VS-1) than from D. tertiolecta (24 mL g-VS-1). High salinity of the D. tertiolecta slurry, prohibitive to methanogens, was the probable reason for lower CH4 production.Source:
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Background:
The main technological impediment to widespread utilization of lignocellulose for production of fuels and chemicals is the lack of low-cost technologies to overcome its recalcitrance. Organisms that hydrolyze lignocellulose and produce a valuable product such as ethanol at a high rate and titer could significantly reduce the costs of biomass conversion technologies, and will allow separate conversion steps to be combined in a consolidated bioprocess (CBP). Development of Saccharomyces cerevisiae for CBP requires the high level secretion of cellulases, particularly cellobiohydrolases.
Results:
We expressed various cellobiohydrolases to identify enzymes that were efficiently secreted by S. cerevisiae. For enhanced cellulose hydrolysis, we engineered bi-modular derivatives of a well secreted enzyme that naturally lacks the carbohydrate-binding module, and constructed strains expressing combinations of cbh1 and cbh2 genes. Though there was significant variability in the enzyme levels produced, up to ~0.3 g/liter CBH1 and ~1 g/liter CBH2 could be produced in high cell density fermentations. Furthermore, we could show activation of the unfolded protein response as a result of cellobiohydrolase production. Finally, we report fermentation of microcrystalline Avicel cellulose to ethanol by CBH-producing S. cerevisiae strains with the addition of beta-glucosidase.
Conclusions:
Gene or protein specific features and compatibility with the host are important for efficient cellobiohydrolase secretion in yeast. The present work demonstrated that production of both CBH1 and CBH2 could be improved to levels where the barrier of CBH sufficiency in the hydrolysis of cellulose was overcome.Source:
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Background:
Caldicellulosiruptor saccharolyticus has attracted increased interest as an industrial hydrogen producer. The aim of the present study was to develop a kinetic growth model for this extreme thermophile. The model is based on Monod kinetics supplemented with the inhibitory effects of hydrogen and osmotic pressure, and the liquid-to-gas mass transfer of H2.
Results:
Mathematical expressions were developed to enable the simulation of microbial growth, substrate consumption and product formation. The model parameters were determined by fitting to experimental data. The derived model corresponded well with experimental data from batch fermentations in which the stripping rates and substrate concentrations were varied. The model was used to simulate the inhibition of growth by hydrogen and solute concentrations, giving a critical dissolved hydrogen concentration 2.2 mmol/L and an osmolarity of 0.27-0.29 osm/L of 2.2 mmol/L. The inhibition by hydrogen, being a function of the dissolved hydrogen concentration, was demonstrated to be mainly dependent on the hydrogen productivity and mass-transfer rate. The latter can be improved by increasing the stripping rate, thereby allowing higher hydrogen productivity. The experimentally determined degree of oversaturation of dissolved hydrogen was 12 to 34 times the equilibrium concentration, and was comparable to the values given by the model.
Conclusions:
The derived model is the first mechanistically based model for fermentative hydrogen production, and provides useful information to improve our understanding of the growth behavior of C. saccharolyticus. The model can be used to determine optimal operating conditions for hydrogen production regarding the substrate concentration and the stripping rate.Source:
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Background:
While advantages of biofuel have been widely reported, studies also highlight the challenges in large scale production of biofuel. Cost of ethanol and process energy use in cellulosic ethanol plants are dependent on technologies used for conversion of feedstock. Process modeling can aid in identifying techno-economic bottlenecks in a production process. A comprehensive techno-economic analysis was performed for conversion of cellulosic feedstock to ethanol using some of the common pretreatment technologies: dilute acid, dilute alkali, hot water and steam explosion. Detailed process models incorporating feedstock handling, pretreatment, simultaneous saccharification and co-fermentation, ethanol recovery and downstream processing were developed using SuperPro Designer. Tall Fescue (Festuca arundinacea Schreb) was used as a model feedstock.
Results:
Projected ethanol yields were 252.62, 255.80, 255.27 and 230.23 L/ dry metric ton biomass for conversion process using dilute acid, dilute alkali, hot water and steam explosion pretreatment technologies respectively. Price of feedstock and cellulose enzymes were assumed as $50/metric ton and 0.517/kg broth (10% protein in broth, 600 FPU/g protein) respectively. Capital cost of ethanol plants processing 250,000 metric tons of feedstock/year was $1.92, $1.73, $1.72 and $1.70/L ethanol for process using dilute acid, dilute alkali, hot water and steam explosion pretreatment respectively. Ethanol production cost of $0.83, $0.88, $0.81 and $0.85/L ethanol was estimated for production process using dilute acid, dilute alkali, hot water and steam explosion pretreatment respectively. Water use in the production process using dilute acid, dilute alkali, hot water and steam explosion pretreatment was estimated 5.96, 6.07, 5.84 and 4.36 kg/L ethanol respectively.
Conclusions:
Ethanol price and energy use were highly dependent on process conditions used in the ethanol production plant. Potential for significant ethanol cost reductions exist in increasing pentose fermentation efficiency and reducing biomass and enzyme costs. The results demonstrated the importance of addressing the tradeoffs in capital costs, pretreatment and downstream processing technologies.KeywordsGrass straw, cellulosic ethanol, pretreatment, process model, process economics.Source:
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Background:
The trichothecene mycotoxin deoxynivalenol (DON) may be concentrated in dried distillers grains with solubles (DDGS), a co-product of fuel ethanol fermentation, when grain containing DON is used to produce fuel ethanol. Even low levels of DON ([less than or equal to] 5ppm) in DDGS sold as feed pose a significant threat to the health of monogastric animals. New and improved strategies to reduce DON in DDGS need to be developed and implemented to address this problem. Enzymes known as trichothecene 3-O-acetyltransferases convert DON to 3-acetyldeoxynivalenol (3ADON) and reduce its toxicity in plants and animals.
Results:
Two Fusarium trichothecene 3-O-acetyltransferases (FgTRI101 and FfTRI201) were cloned and expressed in yeast (Saccharomyces cerevisiae) during a series of small-scale barley (Hordeum vulgare) ethanol fermentations. DON was concentrated 1.6 to 8.2 times in DDGS compared to the starting ground grain. During the fermentation process, FgTRI101 converted 9.2% to 55.3% of DON to 3ADON, resulting in DDGS with reductions in DON and increases in 3ADON when Virginia winter barley cultivars Eve, Thoroughbred, and Price and experimental line VA06H-25 were used. Barley mashes from the barley line VA04B-125 showed that yeast expressing FfTRI201 were more effective at acetylating DON than FgTRI101; DON conversion for FfTRI201 ranged from 26.1% to 28.3%, while FgTRI101 ranged from 18.3% to 21.8% in VA04B-125 mashes. Ethanol yields were highest with the industrial yeast strain Ethanol Red (R), which also consumed galactose when present in the mash.
Conclusions:
This study demonstrates the potential of using yeast expressing a trichothecene 3-O-acetyltransferase to modify DON during commercial fuel ethanol fermentation.Source:
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Background:
Cellulases and related hydrolytic enzymes represent a key cost factor for biochemical conversion of cellulosic biomass feedstocks to sugars for biofuels and chemicals production. The United States Department of Energy (DOE) is cost-sharing projects to decrease the cost of enzymes for biomass saccharification. The performance of benchmark cellulase preparations produced by Danisco, DSM, Novozymes and Verenium to convert pretreated corn stover (PCS) cellulose to glucose was evaluated under common experimental conditions and is reported here in a non-attributed manner.
Results:
Two hydrolysis modes were examined, enzymatic hydrolysis (EH) of PCS whole slurry or washed PCS solids at pH 5 and 50degreesC, and simultaneous saccharification and fermentation (SSF) of washed PCS solids at pH 5 and 38degreesC. Enzymes were dosed on a total protein mass basis, with protein quantified using both the bicinchoninic acid (BCA) assay and the Bradford assay. Substantial differences were observed in absolute cellulose to glucose conversion performance levels under the conditions tested. Higher cellulose conversion yields were obtained using washed solids compared to whole slurry, and estimated enzyme protein dosages required to achieve a particular cellulose conversion to glucose yield were extremely dependent on the protein assay used. All four enzyme systems achieved glucose yields of 90% of theoretical or higher in SSF mode. Glucose yields were reduced in EH mode, with all enzymes achieving glucose yields of at least 85% of theoretical on washed PCS solids and 75% in PCS whole slurry. One of the enzyme systems ("Enzyme B") exhibited the best overall performance. However in attaining high conversion yields at lower total enzyme protein loadings, the relative and rank ordered performance of the enzyme systems varied significantly depending upon which hydrolysis mode and protein assay were used as bases for comparison.
Conclusions:
This study provides extensive information about the performance of four pre-commercial cellulase preparations. Though test conditions were not necessarily optimal for some of the enzymes, all were able to effectively saccharify PCS cellulose. Large differences in estimated enzyme dosage requirements depending on the assay used to measure protein concentration highlight the need for better consensus methods to quantify enzyme protein.Source:
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Background:
Biochemical conversion of lignocellulose hydrolysates remains challenging largely because most microbial processes have significantly reduced efficiency in the presence of both hexoses and pentoses. Thus, identification of microorganisms capable of efficient and simultaneous utilization of both glucose and xylose is pivotal.
Results:
In this work, we showed that the oleaginous yeast Trichosporon cutaneum AS 2.571 assimilated glucose and xylose simultaneously, and accumulated intracellular lipid up to 59 wt% with a lipid coefficient up to 0.17 g/g sugar, upon cultivation on a 2:1 glucose/xylose mixture in a 3-liter stirred tank bioreactor. In addition, no classical diauxic growth behavior was observed as microbial cell mass was increasing during the whole culture process without any lag periods. During shake flask cultures with different initial glucose/xylose ratios, glucose and xylose were consumed simultaneously at rates roughly proportional to their individual concentrations in the medium, leading to complete utilization of both sugars at the same time. Simultaneous utilization of glucose and xylose was also observed during corn stover hydrolysate fermentation with lipid content and coefficient of 39.2% and 0.15 g/g sugar, respectively. Lipid produced herein had fatty acid compositional profile similar to those of conventional vegetable oil, indicating that it could be explored as raw material for biodiesel production.
Conclusion:
Efficient lipid production with simultaneous consumption of glucose and xylose was achieved in this study. It provides an exciting opportunity to transform lignocellulosic materials into biofuel molecules and should also provoke further study to elucidate this unique sugar assimilation mechanism.Source:
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Background:
Neocallimastix patriciarum is one of the common anaerobic fungi in the digestive tract of ruminants that can actively digest cellulosic materials and its cellulases are of great potential for hydrolyzing cellulosic feedstocks. Due to the difficulty in culture and lack of a genome database, it is not easy to gain a global understanding of the glycosyl hydrolases (GHs) produced by this anaerobic fungus.
Results:
We have developed an efficient platform that uses a combination of transcriptomic and proteomic approaches on N. patriciarum to accelerate gene identification, enzyme classification, and application in rice straw degradation. By complementary studies of transcriptome (Roche 454 GS and Illumina GA IIx) and secretome (ESI-Trap LC-MS/MS), we identified 219 putative glycosyl hydrolase (GH) contigs and classified them into 25 GH families. The secretome analysis identified four major enzymes involved in rice straw degradation: beta-glucosidase, endo-1,4-beta-xylanase, xylanase B and Cel48A exo-glucanase. From the sequences of assembled contigs we cloned 19 putative cellulase genes, including GH1, GH3, GH5, GH6, GH9, GH18, GH43 and GH48 gene families, which were highly expressed in N. patriciarum cultures grown on different feedstocks.
Conclusions:
These GH genes were expressed in Pichia pastoris and/or Saccharomyces cerevisiae for functional characterization. At least five novel cellulases displayed cellulytic activity for glucose production. One beta-glucosidases (W5-16143) and one exo-cellulase (W5-CAT26) showed strong activities and could potentially be developed into commercial enzymes.Keywordsanaerobic fungi, biomass, rice straw, sugarcane, napiergrass, GH, next-generation sequencingSource:
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Background:
To efficiently deconstruct recalcitrant plant biomass to fermentable sugars in industrial processes, biocatalysts of higher performance and lower cost are required. The genetic diversity found in the metagenomes of natural microbial biomass decay communities may harbor such enzymes. Our goal was to discover and characterize new glycoside hydrolases (GHases) from microbial biomass decay communities, especially those from unknown or never-been-cultivated microorganisms.
Results:
From the metagenome sequences of an anaerobic microbial community actively decaying poplar biomass, we identified approximately 4,000 GHase homologues. Based on homology to GHase families/activities of interest and the quality of the sequences, candidates were selected for full length cloning and subsequent expression. As an alternative strategy, a metagenome expression library was constructed and screened for GHase activities. These combined efforts resulted in the cloning of four novel GHases that could be successfully expressed in E. coli. Further characterization showed that two enzymes showed significant activity on p-nitrophenyl-alpha-L-arabinofuranoside, one enzyme had significant activity against p-nitrophenyl-beta-D-glucopyranoside, and one enzyme showed significant activity against p-nitrophenyl-beta-D-xylopyranoside. Enzymes were also tested in the presence of ionic liquids.
Conclusions:
Metagenomics provides a good resource for mining novel biomass degrading enzymes and for screening of cellulolytic enzyme activities. The four GHases that were cloned may have potential application for deconstruction of biomass pretreated with ionic liquids, as they remain active in the presence of up to 20% ionic liquid (except for 1-ethyl-3-methylimidazolium diethyl phosphate). Alternatively, ionic liquids might be used to immobilize or stabilize these enzymes for minimal solvent processing of biomass.

Background:
As the supply of starch grain and sugar cane, currently the main feedstocks for bioethanol production, become limited, lignocelluloses will be sought as alternative materials for bioethanol production. Production of cellulosic ethanol is still cost-inefficient due to the low final ethanol concentration and the addition of nutrients. Here, simultaneous saccharification and co-fermentation (SSCF) of lignocellulosic residues from commercial furfural production (furfural residue, FR) and corn kernels were carried out to compare different nutritional media. The final ethanol concentration, the yield, the amount of live yeast cells and yeast cell death ratio were investigated to evaluate the effectiveness of integrating cellulosic and starch ethanol.
Results:
Both the ethanol yield and amount of live yeast cells increased with increasing corn kernels concentration, while the yeast cell death ratio decreased in the SSCF of FR and corn kernels. An ethanol concentration of 73.1 g/L at 120 h, which corresponded to a 101.1% ethanol yield based on FR cellulose and corn starch, was obtained in the SSCF of 7.5% FR and 14.5% corn kernels with mineral salt medium. SSCF could simultaneously convert cellulose into ethanol from both corn kernels and FR, and the SSCF ethanol yield was similar between the organic and mineral salt media.
Conclusions:
Starch ethanol promotes cellulosic ethanol by providing important nutrients for fermentative organisms whereby cellulosic ethanol promotes starch ethanol by providing cellulosic enzymes that convert the cellulosic polysaccharides in starch materials into additional ethanol. It is feasible to produce ethanol in SSCF of FR and corn kernels with mineral salt medium. It would be cost-efficient to produce ethanol in SSCF of high water insoluble solid (WIS) of lignocellulosic materials and corn kernels. Compared with prehydrolysis and fed-batch strategy using lignocellulosic materials, addition of starch hydrolysates to cellulosic ethanol production is a more suitable method to improve the final ethanol concentration.

Background:
Isobutanol can be a better biofuel than ethanol due to its higher energy density and lower hygroscopicity. Furthermore, the branched-chain structure of isobutanol gives a higher octane number than the isomeric n-butanol. Saccharomyces cerevisiae was chosen as the production host because of its relative tolerance to alcohols, robustness in industrial fermentations, and the possibility for future combination of isobutanol production with fermentation of lignocellulosic materials.
Results:
The yield of isobutanol was improved from 0.16 to 0.97 mg per g glucose by simultaneous overexpression of biosynthetic genes ILV2, ILV3, and ILV5 in valine metabolism in anaerobic fermentation of glucose in mineral medium in S. cerevisiae. Isobutanol yield was further improved by two times by the additional overexpression of BAT2, encoding the cytoplasmic branched-chain amino acid aminotransferase. Overexpression of ILV6, encoding the regulatory subunit of Ilv2, in the ILV2 ILV3 ILV5 overexpression strain decreased isobutanol production yield by three times. In aerobic cultivations in shake flasks in mineral medium the isobutanol yield of the ILV2 ILV3 ILV5 overexpression strain and the reference strain were 3.86 and 0.28 mg per g glucose, respectively. They were increased to 4.12 and 2.4 mg per g glucose in YPD complex medium under aerobic conditions, respectively.
Conclusions:
Overexpression of genes ILV2, ILV3, ILV5, and BAT2 in valine metabolism led to an increase in isobutanol production in S. cerevisiae. Additional overexpression of ILV6 in the ILV2 ILV3 ILV5 overexpression strain had a negative effect, presumably by increasing the sensitivity of Ilv2 to valine inhibition, thus weakening the positive impact of overexpression of ILV2, ILV3, and ILV5 on isobutanol production.Aerobic cultivations of the ILV2 ILV3 ILV5 overexpression strain and the reference strain showed that supplying amino acids in cultivation media gave a substantial improvement in isobutanol production for the reference strain, but not for the ILV2 ILV3 ILV5 overexpression strain. This result implies that other constraints besides the enzyme activities for the supply of 2-ketoisovalerate may become bottlenecks for isobutanol production after ILV2 ILV3 and ILV5 have been overexpressed, and it most probably includes the valine inhibition to Ilv2.

Background:
The use of energy crops and agricultural residues is expected to increase to fulfill the legislative demands of bio-based components in transport fuels. Ensiling methods, adapted from the feed sector, are suitable storage methods to preserve fresh crops throughout the year for e.g. biogas production. Various preservation methods, namely ensiling with and without acid addition for whole crop maize, fiber hemp and faba bean were investigated. For the drier fiber hemp, alkaline urea treatment was studied as well. These treatments were also explored as mild pre-treatment methods to improve the disassembly and hydrolysis of these lignocellulosic substrates.
Results:
The investigated storage treatments increased the availability of the substrates for biogas production from hemp and in most cases from whole maize but not from faba bean. Ensiling of hemp, without or with addition of formic acid, increased methane production by more than 50% compared to fresh hemp. Ensiling resulted in substantially increased methane yields also from maize, and the use of formic acid in ensiling of maize further enhanced methane yields by 16%, as compared with fresh maize. Ensiled faba bean, in contrast, yielded somewhat less methane than the fresh material.Acidic additives preserved and even increased the amount of the valuable water-soluble carbohydrates during storage, which affected most significantly the enzymatic hydrolysis yield of maize. On the other hand, preservation without additives decreased the enzymatic hydrolysis yield especially in maize, due to its high content of soluble sugars that were already converted to acids during storage.Urea-based preservation significantly increased the enzymatic hydrolysability of hemp. Hemp, preserved with urea, produced the highest carbohydrate increase of 46% in enzymatic hydrolysis as compared to the fresh material. Alkaline pretreatment conditions of hemp improved also the methane yields.
Conclusions:
The results showed that ensiling and alkaline preservation of fresh crop materials are useful pre-treatment methods for methane production. Improvements in enzymatic hydrolysis were also promising. While all three crops still require a more powerful pre-treatment to release the maximum amount of carbohydrates, anaerobic preservation is clearly a suitable storage and pre-treatment method prior to production of platform sugars from fresh crops.

Background:
The BioEnergy Science Center (BESC) developed a high-throughput screening method to rapidly identify low-recalcitrance biomass variants. Because the customary separation and analysis of liquid and solids between pretreatment and enzymatic hydrolysis used in conventional analyses is slow, labor-intensive and very difficult to automate, a streamlined approach we term 'co-hydrolysis' was developed. In this method, the solids and liquid in the pretreated biomass slurry are not separated, but instead hydrolysis is performed by adding enzymes to the whole pretreated slurry. The effects of pretreatment method, severity and solids loading on co-hydrolysis performance were investigated.
Results:
For hydrothermal pretreatment at solids concentrations of 0.5 to 2%, high enzyme protein loadings of about 100 mg/g of substrate (glucan plus xylan) in the original poplar wood achieved glucose and xylose yields for co-hydrolysis that were comparable with those for washed solids. In addition, although poplar wood sugar yields from co-hydrolysis at 2% solids concentrations fell short of those from hydrolysis of washed solids after dilute sulfuric acid pretreatment even at high enzyme loadings, pretreatment at 0.5% solids concentrations resulted in similar yields for all but the lowest enzyme loading.
Conclusions:
Overall, the influence of severity on susceptibility of pretreated substrates to enzymatic hydrolysis was clearly discernable, showing co-hydrolysis to be a viable approach for identifying plant-pretreatment-enzyme combinations with substantial advantages for sugar production.

Background:
Hemicellulose is often credited with being one of the important physical barriers to enzymatic hydrolysis of cellulose by blocking enzyme access to the cellulose surface. In addition to that, our recent research suggested that hemicelluloses, particularly in the form of xylan and its oligomers, can more strongly inhibit cellulase activity than glucose and cellobiose. Removal of hemicelluloses or elimination of their negative impacts can, therefore, become especially pivotal to achieving higher cellulose conversion with lower enzyme doses.
Results:
In this study, cellulase was supplemented with xylanase and beta-xylosidase to boost conversion of both cellulose and hemicellulose in pretreated biomass through conversion of xylan and xylooligomers to less inhibitory xylose. Although addition of xylanase and beta-xylosidase did not necessarily enhance Avicel hydrolysis, glucan conversions increased by 27% and 8% for AFEX and dilute acid pretreated corn stover, respectively. In addition, adding hemicellulase several hours prior to adding cellulase was more beneficial than later addition, possibly as a result of a higher adsorption affinity of cellulase and xylanase to xylan than glucan.
Conclusions:
This key finding elucidates a possible mechanism for cellulase inhibition by xylan and xylooligomers and advances the need to optimize the enzyme formulation for each pretreated substrate. More research is needed to identify advanced enzyme systems designed to hydrolyze different substrates with the maximum overall enzyme efficacy.