Category Archives: Biotechnology

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.

Background:
Termites are highly effective in lignocelluloses degradation thus can be used as model for studying plant cell wall degradation in biological systems. However, the process of lignin deconstruction and/or degradation in termite is still not well understood.
Results:
We have investigated the associated structural modification by termite in the lignin biomolecular assembly in softwood tissues critical for cell wall degradation. Comparative studies on the termite digested (termite feces) and native (control) softwood tissues with the aid of advanced analytical techniques; such as, 13C cross polarization magic angle spinning (CP/MAS) nuclear magnetic resonance (NMR) spectroscopy, pyrolysis gas chromatography mass spectrometry (Py-GC/MS), and flash pyrolysis in presence of tetramethylammonium hydroxide (TMAH) were conducted. The 13C CP/MAS NMR spectroscopic analysis revealed elevated level of guaiacyl derived (G unit) polymeric frame work in the termite digested softwood (feces), while providing specific evidence of cellulose degradation. The Py-GC/MS data were in agreement with the 13C CP/MAS NMR spectroscopic studies, thus indicated dehydroxylation and modification of selective intermonomer side-chain linkages in the lignin proper in the termite feces. Moreover, Py (TMAH)-GC/MS analysis revealed significant differences in the product distribution between control and termite feces. This strongly suggests that the structural modification in lignin proper could be associated with the formation of additional condensed inter-unit linkages.
Conclusion:
Collectively, these data further establish: (1) the conservation of the major beta-O-4' (beta-aryl ether), albeit with sub-structure degeneracy, and (2) the nature of resulting polymer in termite feces retained most of its original aromatic moieties (G unit derived). Overall, these results provide insight into lignin unlocking mechanisms for understanding plant cell wall deconstruction towards development of new enzymatic pretreatment processes mimicking termite system for biochemical conversion of lignocellulosic biomass to fuels and chemicals.

Background:
Pretreatment is a critical step in the conversion of lignocellulose to fermentable sugars. Although many pretreatment processes are currently under investigation, none of them are entirely satisfactory in regard to effectiveness, cost, or environmental impact. The use of hydrogen peroxide at pH 11.5 (alkaline hydrogen peroxide or AHP) was shown by Gould and co-workers to be an effective pretreatment of grass stovers and other plant materials in the context of animal nutrition and ethanol production. Our earlier experiments indicated that AHP performed well when compared against two other alkaline pretreatments. Here we explored several key parameters to test the potential of AHP for further improvement relevant to lignocellulosic ethanol production.
Results:
The effects of biomass loading, hydrogen peroxide loading, residence time, and pH control were tested in combination with subsequent digestion with a commercial enzyme preparation, optimized mixtures of four commercial enzymes, or optimized synthetic mixtures of pure enzymes. AHP pretreatment was performed at room temperature (23C ) and atmospheric pressure, and after AHP pretreatment the biomass was neutralized with HCl but not washed before enzyme digestion. Standard enzyme digestion conditions were 0.2% glucan loading, 15 mg protein/g glucan, and 48 h digestion at 50degreesC. Higher pretreatment biomass loadings (10% - 20%) gave higher monomeric glucose (Glc) and xylose (Xyl) yields than the 2% loading used in earlier studies. An H2O2 loading of 0.25 g/g biomass was almost as effective as 0.5 g/g, but 0.125 g/g was significantly less effective. Optimized mixtures of four commercial enzymes substantially increased post-AHP-pretreatment enzymatic hydrolysis yields at all H2O2 concentrations compared to any single commercial enzyme. At a pretreatment biomass loading of 10% and an H2O2 loading of 0.5 g/g biomass, an optimized commercial mixture at total protein loadings of 8 or 15 mg/g glucan gave monomeric Glc yields of 83% or 95%, respectively. Yields of Glc and Xyl after pretreatment at a low hydrogen peroxide loading (0.125 g H2O2/g biomass) could be improved by extending the pretreatment residence time to 48 h and readjusting the pH to 11.5 every 6 h during the pretreatment. A Glc yield of 77% was obtained using a pretreatment of 15% biomass loading, 0.125 g H2O2/g biomass, and 48 h with pH adjustment, followed by digestion with an optimized commercial enzyme mixture at an enzyme loading of 15 mg protein/g glucan.
Conclusion:
Alkaline peroxide is an effective pretreatment for corn stover. Particular advantages are the use of reagents with low environmental impact and avoidance of special reaction chambers. Reasonable yields of monomeric Glc can be obtained at an H2O2 concentration one-fourth of that used in previous AHP research. Additional improvements in the AHP process, such as peroxide stabilization, peroxide recycling, and improved pH control, could lead to further improvements in AHP pretreatment.

Background:
Biomass use for the production of bioethanol or for the production of platform chemicals requires the efficient biomass breakdown to fermentable monosaccharides. Lignocellulosic feedstocks often require a physicochemical pretreatment prior to enzymatic hydrolysis. Optimal pretreatments can be different for different feedstocks and should not lead to biomass destruction and the formation of toxic products. The influence of six mild sulfuric acid or water pretreatments at different temperatures on the enzymatic degradability of sugar beet pulp was examined.
Results:
An optimal pretreatment of 15 min at 140 degreesC in water can solubilize 60 w/w% of the total carbohydrates present, mainly pectins. Higher severities lead to the destruction of solubilized sugars and to the subsequent production of the sugar degradation products furfural, hydroxy methyl furfural, acetic acid and formic acid. The pretreated samples were enzymatically degraded successfully with an experimental cellulase preparation.
Conclusions:
Pretreatment of sugar beet pulp greatly facilitates the subsequent enzymatic degradation within economically feasible times ranges and enzyme dosages. In addition, pretreatment of sugar beet pulp can be useful to fractionate functional ingredients like arabinans and pectins from cellulose. The optimal combined severity factor to enhance the enzymatic degradation of sugar beet pulp is between Log R'(0) = -2.0 and Log R' (0) = -1.5. Optimal pretreatment and enzyme treatment solubilized up to 80% of all sugars present in optimally pretreated sugar beet pulp, including > 90% of the cellulose.