Construction of a Synthetic Microbial Community for Enzymatic Pretreatment of Wheat Straw for Biogas Production via Anaerobic Digestion.

Biological pretreatment is a viable method for enhancing biogas production from straw crops, with the improvement in lignocellulose degradation efficiency being a crucial factor in this process. Herein, a metagenomic approach was used to screen core microorganisms (Bacillus subtilis, Acinetobacter johnsonii, Trichoderma viride, and Aspergillus niger) possessing lignocellulose-degrading abilities among samples from three environments: pile retting wheat straw (WS), WS returned to soil, and forest soil. Subsequently, synthetic microbial communities were constructed for fermentation-enzyme production. The crude enzyme solution obtained was used to pretreat WS and was compared with two commercial enzymes. The synthetic microbial community enzyme-producing pretreatment (SMCEP) yielded the highest enzymatic digestion efficacy for WS, yielding cellulose, hemicellulose, and lignin degradation rates of 39.85, 36.99, and 19.21%, respectively. Furthermore, pretreatment of WS with an enzyme solution, followed by anaerobic digestion achieved satisfactory results. SMCEP displayed the highest cumulative biogas production at 801.16 mL/g TS, which was 38.79% higher than that observed for WS, 22.15% higher than that of solid-state commercial enzyme pretreatment and 25.41% higher than that of liquid commercial enzyme pretreatment. These results indicate that enzyme-pretreated WS can significantly enhance biogas production. This study represents a solution to the environmental burden and energy use of crop residues.

Biocalorimetry-aided monitoring of fungal pretreatment of lignocellulosic agricultural residues.

The present study aimed to investigate whether and how non-invasive biocalorimetric measurements could serve for process monitoring of fungal pretreatment during solid-state fermentation (SSF) of lignocellulosic agricultural residues such as wheat straw. Seven filamentous fungi representing different lignocellulose decay types were employed. Water-soluble sugars being immediately available after fungal pretreatment and those becoming water-extractable after enzymatic digestion of pretreated wheat straw with hydrolysing (hemi)cellulases were considered to constitute the total bioaccessible sugar fraction. The latter was used to indicate the success of pretreatments and linked to corresponding species-specific metabolic heat yield coefficients (YQ/X) derived from metabolic heat flux measurements during fungal wheat straw colonisation. An YQ/X range of about 120 to 140 kJ/g was seemingly optimal for pretreatment upon consideration of all investigated fungi and application of a non-linear Gaussian fitting model. Upon exclusion from analysis of the brown-rot basidiomycete Gloeophyllum trabeum, which differs from all other here investigated fungi in employing extracellular Fenton chemistry for lignocellulose decomposition, a linear relationship where amounts of total bioaccessible sugars were suggested to increase with increasing YQ/X values was obtained. It remains to be elucidated whether an YQ/X range being optimal for fungal pretreatment could firmly be established, or if the sugar accessibility for post-treatment generally increases with increasing YQ/X values as long as "conventional" enzymatic, i.e. (hemi)cellulase-based, lignocellulose decomposition mechanisms are operative. In any case, metabolic heat measurement-derived parameters such as YQ/X values may become very valuable tools supporting the assessment of the suitability of different fungal species for pretreatment of lignocellulosic substrates. KEY POINTS: • Biocalorimetry was used to monitor wheat straw pretreatment with seven filamentous fungi. • Metabolic heat yield coefficients (YQ/X) seem to indicate pretreatment success. • YQ/X values may support the selection of suitable fungal strains for pretreatment.

Cellobionate production from sodium hydroxide pretreated wheat straw by engineered Neurospora crassa HL10.

This study investigated cellobionate production from a lignocellulosic substrate using Neurospora crassa HL10. Utilizing NaOH-pretreated wheat straw as the substrate obviated the need for an exogenous redox mediator addition, as lignin contained in the pretreated wheat served as a natural mediator. The low laccase production by N. crassa HL10 on pretreated wheat straw caused slow cellobionate production, and exogenous laccase addition accelerated the process. Cycloheximide induced substantial laccase production in N. crassa HL10, enabling the strain to yield approximately 57 mM cellobionate from pretreated wheat straw (equivalent to 20 g/L cellulose), shortening the conversion time from 8 to 6 days. About 92% of the cellulose contained in the pretreated wheat straw is converted to cellobionate. In contrast to existing methods requiring pure cellobiose or cellulase enzymes, this process efficiently converts a low-cost feedstock into cellobionate at a high yield without enzyme or redox mediator supplementation.

Valorization of wheat straw into paper by ultrafiltered enzymatic bleaching approach.

In this study, the potential of ultrafiltered xylano-pectinolytic enzymatic bleaching approach was investigated, for manufacturing wheat straw-based paper. The enzymatic step was found to be most effective, with xylanase-pectinase dose of 4-1.7 IU/g pulp and time period of 180 min. The absorption spectra of the pulp free filtrate samples obtained after treatment of the pulp with ultrafiltered enzymes showed the removal of more impurities, in comparison to the treatment with crude enzymes. Microscopic analysis also showed the removal of lignin impurities in enzymatically bleached pulp samples. This bleaching approach using enzymes resulted in 27% reduction in ClO2 dose. Ultrafiltered enzymes treated pulp samples also showed improved quality-related parameters, and Gurley porosity, burst index, breaking length, double fold, tear index, and viscosity increased by 19.05, 13.70, 8.18, 29.27, 4.41, and 13.27%, respectively. The lignin content, TDS, TSS, BOD and COD values also decreased in the effluent samples obtained after enzymatic bleaching plus 73% chemical bleaching dose. The BOD and COD values of the effluent samples improved by 23.01 and 23.66%, respectively. Thus, indicating the potential of ultrafiltered xylano-pectinolytic enzymes in reducing pollution during bleaching of wheat straw. This is the first study, mentioning the efficacy of ultrafiltered enzymes in the bleaching of wheat straw-based paper with better optical-strength-related properties and effluent characteristics.

The Synergetic Reduction of the Condensation Degree of Dissolved Lignin (DL) during the Refining Process of Wheat Straw Biomass Based on the MA/O3 System.

Lignin, a natural pol2ymer with a complex structure that is difficult to separate, is prone to C-C bond condensation during the separation process. To reduce the condensation of lignin, here, a novel method is proposed for separating the components by using a combination of maleic acid (MA)/ozone (O3) to co-treat wheat straw. The removal of lignin, glucan, and xylan was 38.07 ± 0.2%, 31.44 ± 0.1%, and 71.98 ± 0.1%, respectively, under the conditions of ball-milling of wheat straw for 6 h, reaction temperature of 60 °C, and O3 holding time of 9 min. Lignin-rich solutions were collected to extract the dissolved lignin (DL) after washing the treated samples. The DL obtained under MA/O3 conditions had a carboxyl group (-COOH) content of 2.96 mmol/g. The carboxyl group of MA underwent esterification with the hydroxyl group (-OH) at the γ position of lignin and O3 reacted on the positions of the lignin side chain or the phenolic ring, resulting in a break in the side chain and the opening of the phenolic ring to introduce the carboxyl group. The 2D-HSQC-NMR results revealed that the phenolic ring-opening reaction of lignin in the presence of O3 was essentially free of ß-ß and ß-5 condensation bonds.

Exploration of the Linkages between Lignin and Carbohydrates in Kraft Pulp from Wheat Straw Using a 13C/2H Isotopic Tracer.

To further our understanding of the change in association between lignin and carbohydrates after kraft pulping, isotope-labeled kraft pulp (KP) was prepared using 13C and D double-isotope-labeled wheat straw, and it was subjected to enzymatic hydrolysis and ionic liquid treatment to explore the linkages between lignin and carbohydrate complexes in wheat straw. Isotope abundance determination showed that 13C and D abundances in the experimental groups were substantially higher than those in the control group, indicating that the injected exogenous coniferin-[α-13C], coniferin-[γ-13C], and d-glucose-[6-D2] were effectively absorbed and metabolized during wheat internode growth. Solid-state CP/MAS 13C-NMR spectroscopy showed that lignin was mainly linked to polysaccharides via acetal, benzyl ether, and benzyl ester bonds. Kraft pulp (KP) from the labeled wheat straw was degraded by cellulase. The obtained residue was fractionated using the ionic liquid DMSO/TBAH to separate the cellulose-lignin complex (KP-CLC) and xylan-lignin complex (KP-XLC). X-ray diffractometer determination showed that the KP-CLC regenerated cellulose type II from type I after the ionic liquid conversion. The 13C-NMR spectrum of Ac-En-KP-CLC showed that the cellulose-lignin complex structure was chemically bonded between the lignin and cellulose through acetal and benzyl ether bonds. The 13C-NMR spectrum of En-KP-XLC showed a lignin-hemicellulose complex structure, wherein lignin and xylan were chemically bonded by benzyl ether and acetal bonds. These results indicate that the cross-linking between lignin and carbohydrates exists in lignocellulosic fibers even after kraft pulping.

Manganese promoted wheat straw decomposition by regulating microbial communities and enzyme activities.

AIMS: This study investigated the dose-effect of manganese (Mn) addition on wheat straw (WS) decomposition, and explored the potential mechanisms of Mn involved in the acceleration of WS decomposition in regards to the soil microbial communities and enzyme activities. METHODS AND RESULTS: A 180-day incubation experiment was performed to examine the decomposition of WS under four Mn levels, that is, 0, 0.25, 1 and 2 mg g-1 . The effects of microbial communities and enzyme activities were evaluated using control (0 mg g-1 ) and Mn (0.25 mg g-1 ) treatments. Our results revealed that Mn (0.25 mg g-1 ) addition significantly increased WS decomposition, and enhanced the release of carbon and nitrogen. Optimal Mn addition (0.25 mg g-1 ) also caused significant increases in the activity of neutral xylanase (NEX), laccase (Lac), manganese peroxidase (MnP) and lignin peroxidase (LiP) within the incubation period. Mn (0.25 mg g-1 ) addition also enriched some operational taxonomic units (OTUs) that, in turn, had the potential ability to decompose crop straw, such as secreting lignocellulolytic enzymes. CONCLUSIONS: Mn (0.25 mg g-1 ) could promote WS decomposition through enrichment of the microbial species involved in biomass decomposition, which enhanced the lignocellulose-degrading enzyme activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides evidence for Mn to promote WS biodegradation after Mn application, opening new windows to improve the utilization efficiency of crop residues.

Full components conversion of lignocellulose via a closed-circuit biorefinery process on a pilot scale.

This study developed a closed-circuit biorefinery process for full conversion of lignocellulose into ethanol, biogas and organic fertilizer with zero waste on a pilot scale. In the process, subcritical water pretreatment could effectively break the structure of wheat straw (WS), and ethanol was obtained from pretreated wheat straw (PWS) using two batches of simultaneous saccharification and fermentation (SSF). The pretreatment and ethanol fermentation wastes were reused for biogas and organic fertilizer production by anaerobic digestion (AD), whereas the pretreatment and ethanol conversion efficiency were reduced when supernatant after AD was recovered for next batch pretreatment. The yields of ethanol (0.08-0.09 g/g), biogas (0.05-0.10 L/g) and organic fertilizer (0.55-0.79 g/g) were demonstrated through mass balance. Furthermore, the hidden problems were exposed on pilot-scale conversion process, and several strategies were provided for optimizing the biorefinery process in the future.

Enzymatic hydrolysis of lignocellulosic biomass using native cellulase produced by Aspergillus niger ITV02 under liquid state fermentation.

The objective of this work was to evaluate the biochemical characteristics of an enzymatic extract obtained from autochthonous fungus Aspergillus niger ITV02 and its application in the enzymatic hydrolysis of wheat straw and corn stubble pretreated by steam explosion. The enzymatic extract was obtained by submerged fermentation using delignified sweet sorghum bagasse as a carbon source. The results obtained showed that the enzymatic extract had ß-glucosidase and endoglucanase activities. The effects of pH and temperature on cellulase activity were evaluated and its thermostability was determined. The optimal parameters of the ß-glucosidase and endoglucanase activities obtained were pH 5 and 70 °C. The enzymatic extract of A. niger ITV02 was used to hydrolyze wheat straw and corn stubble, and the hydrolysis yields were compared with those obtained by a commercial cellulase (Celluclast 1.5L NS 50013) and CellicCTec3. The results showed that with the use the mixture of Celluclast 1.5L-A. niger ITV02 and CellicCTec3-A. niger ITV02 in the hydrolysis, conversions of 86.36% and 67.8% were obtained, respectively. Glucose production for the mixture extract increased 2.15 times more than when the enzyme was used independently alone. The present work shows that A. niger ITV02 has a potential as an enzyme producer for lignocellulosic hydrolysis.

Pretreatment of Wheat Straw Lignocelluloses by Deep Eutectic Solvent for Lignin Extraction.

In order to increase the fractionation efficiency of the wheat straw, a deep eutectic solvent (DES) system consisting of chlorine/lactic acid was used in this study for wheat straw pretreatment. The outcomes exhibited that DES pretreatment significantly enhanced the capability to extract lignin, retain cellulose, and remove hemicellulose. The best condition for the pretreatment of wheat straw was 150 °C for 6 h. The process retained most cellulose in the pretreated biomass (49.94-73.60%), and the enzymatic digestibility of the pretreatment residue reached 89.98%. Further characterization of lignin showed that the high yield (81.54%) and the high purity (91.33%) resulted from the ether bond cleavage in lignin and the connection between hemicellulose and lignin. As for application, the enzymatic hydrolysis of the best condition reached 89.98%, and the lignin also had suitable stability. The investigation exhibited that DES pretreatment has the potential to realize an efficient fractionation of lignocellulosic biomass into high-applicability cellulose and lignin of high-quality.

Lytic Polysaccharide Monooxygenase from Talaromyces amestolkiae with an Enigmatic Linker-like Region: The Role of This Enzyme on Cellulose Saccharification.

The first lytic polysaccharide monooxygenase (LPMO) detected in the genome of the widespread ascomycete Talaromyces amestolkiae (TamAA9A) has been successfully expressed in Pichia pastoris and characterized. Molecular modeling of TamAA9A showed a structure similar to those from other AA9 LPMOs. Although fungal LPMOs belonging to the genera Penicillium or Talaromyces have not been analyzed in terms of regioselectivity, phylogenetic analyses suggested C1/C4 oxidation which was confirmed by HPAEC. To ascertain the function of a C-terminal linker-like region present in the wild-type sequence of the LPMO, two variants of the wild-type enzyme, one without this sequence and one with an additional C-terminal carbohydrate binding domain (CBM), were designed. The three enzymes (native, without linker and chimeric variant with a CBM) were purified in two chromatographic steps and were thermostable and active in the presence of H2O2. The transition midpoint temperature of the wild-type LPMO (Tm = 67.7 °C) and its variant with only the catalytic domain (Tm = 67.6 °C) showed the highest thermostability, whereas the presence of a CBM reduced it (Tm = 57.8 °C) and indicates an adverse effect on the enzyme structure. Besides, the potential of the different T. amestolkiae LPMO variants for their application in the saccharification of cellulosic and lignocellulosic materials was corroborated.

Polysaccharides Release in a Laboratory-Scale Batch Hydrothermal Pretreatment of Wheat Straw under Rigorous Isothermal Operation.

Hydrothermal pretreatment (HP) is an eco-friendly process for deconstructing lignocellulosic biomass (LCB) that plays a key role in ensuring the profitability of producing biofuels or bioproducts in a biorefinery. At the laboratory scale, HP is usually carried out under non-isothermal regimes with poor temperature control. In contrast, HP is usually carried out under isothermal conditions at the commercial scale. Consequently, significant discrepancies in the values of polysaccharide releases are found in the literature. Therefore, laboratory-scale HP data are not trustworthy if scale-up or retrofitting of HP at larger scales is required. This contribution presents the results of laboratory-scale batch HP for wheat straw in terms of xylan and glucan release that were obtained with rigorous temperature control under isothermal conditions during the reaction stage. The heating and cooling stages were carried out with fast rates (43 and -40 °C/min, respectively), minimizing non-isothermal reaction periods. Therefore, the polysaccharide release results can be associated exclusively with the isothermic reaction stage and can be considered as a reliable source of information for HP at commercial scales. The highest amount of xylan release was 4.8 g/L or 43% obtained at 180 °C and 20 min, while the glucan release exhibited a maximum of 1.2 g/L or 5.5%. at 160 °C/180 °C and 30 min.

Durability of Flame-Retarded, Co-Extruded Profiles Based on High-Density Polyethylene and Wheat Straw Residues.

At present, little information is available in the scientific literature related to the durability (weathering resistance) of fire-retarded wood and natural fiber-reinforced thermoplastics. In this work, thermoplastic profiles for façade applications based on high-density polyethylene, wheat straw particles, and fire-retardants were extruded and their reaction-to-fire performance before and after artificial weathering evaluated. Profile geometries were either solid or hollow-core profiles, and fire-retardants (FR) were added either in the co-extruded layer or in the bulk. Various FR for inclusion in the co-extruded layer were screened based on UL-94 tests. For profile extrusion, two types of FR were chosen: a coated intumescent combination based on ammonium polyphosphate (APP) and an APP coated with melamine and without formaldehyde. Before weathering, the peak heat release rate (pHRR) and the total heat release (THR), which were determined using cone calorimeter measurements, were reduced by up to 64% and 67% due to the FR. However, even before weathering, pHRR of the profiles was relatively high, with best (lowest) values between 230 and 250 kW/m2 under the test conditions. After 28 days of artificial weathering, changes in reaction-to-fire performance and color were evaluated. Use of the APP in the co-extruded layer worsened color change compared to the formulation without APP but the pHRR was not significantly changed. The influence of weathering on the fire behavior was small compared to the difference between fire-retarded and non-fire-retarded materials. Results from the cone calorimeter were analyzed with regard to ETAG 028, which provides requirements related to the durability of fire performance of building products. In many formulations, increase in THR was less than 20% compared to before weathering, which would place some of the profiles in class C or better (EN 13501-1). However, due to the high pHRR, at best, class D was obtained under the conditions of this study. In addition to cone calorimeter measurements, results from the single flame source test, limiting oxygen index determination and thermogravimetric analysis, are shown and discussed. Strength properties, water uptake and swelling of the profiles, thermal conductivity, and energy dispersive X-ray data are also presented.

Comparative Analysis of Herbaceous and Woody Cell Wall Digestibility by Pathogenic Fungi.

Fungal pathogens have evolved combinations of plant cell-wall-degrading enzymes (PCWDEs) to deconstruct host plant cell walls (PCWs). An understanding of this process is hoped to create a basis for improving plant biomass conversion efficiency into sustainable biofuels and bioproducts. Here, an approach integrating enzyme activity assay, biomass pretreatment, field emission scanning electron microscopy (FESEM), and genomic analysis of PCWDEs were applied to examine digestibility or degradability of selected woody and herbaceous biomass by pathogenic fungi. Preferred hydrolysis of apple tree branch, rapeseed straw, or wheat straw were observed by the apple-tree-specific pathogen Valsa mali, the rapeseed pathogen Sclerotinia sclerotiorum, and the wheat pathogen Rhizoctonia cerealis, respectively. Delignification by peracetic acid (PAA) pretreatment increased PCW digestibility, and the increase was generally more profound with non-host than host PCW substrates. Hemicellulase pretreatment slightly reduced or had no effect on hemicellulose content in the PCW substrates tested; however, the pretreatment significantly changed hydrolytic preferences of the selected pathogens, indicating a role of hemicellulose branching in PCW digestibility. Cellulose organization appears to also impact digestibility of host PCWs, as reflected by differences in cellulose microfibril organization in woody and herbaceous PCWs and variation in cellulose-binding domain organization in cellulases of pathogenic fungi, which is known to influence enzyme access to cellulose. Taken together, this study highlighted the importance of chemical structure of both hemicelluloses and cellulose in host PCW digestibility by fungal pathogens.

Novel strategy to improve the colonizing ability of Irpex lacteus in non-sterile wheat straw for enhanced rumen and enzymatic digestibility.

Pretreatment with white rot fungi is a promising method to enhance the digestibility of lignocelluloses; however, sterilization of feedstocks prior to inoculation is one of the costliest steps. To improve the colonizing ability of white rot fungi under non-sterile condition, Irpex lacteus, Pleurotus ostreatus, and Phanerochaete chrysosporium were inoculated in the wheat straw ensiled for 28 days and incubated for 56 days to determine the changes in microbe counts, organic acid content, chemical composition, and rumen and enzymatic digestibility. Results showed that ensiling produced abundant organic acids and suppressed most microbes in wheat straw. Significant growth of I. lacteus was observed after 3 days of incubation, and molds were only detectable at day 7 in the group. At the end of incubation, aerobic bacteria and lactic acid bacteria decreased by 18% and 38% in the wheat straw treated with I. lacteus, but molds, aerobic bacteria, and lactic acid bacteria thrived in those treated with P. ostreatus and P. chrysosporium. Even more, P. ostreatus and P. chrysosporium increased the lignin content of the ensiled wheat straw by 34% and 65%. However, I. lacteus selectively degraded lignin by 28% and improved the rumen and enzymatic digestibility by 18% and 34%. The finding indicates that ensiling prior to fermentation with I. lacteus is an effective method to control spoilage microbes and to enhance the rumen and enzymatic digestibility of wheat straw.

Substitution of wheat straw with sugarcane bagasse in low-forage diets fed to mid-lactation dairy cows: Milk production, digestibility, and chewing behavior.

Sugarcane bagasse (SB) is a low-quality roughage source that is often plentiful during times of forage shortage. It is generally less costly compared with other conventional sources of forage. We hypothesized that SB could be used as a source of roughage for dairy cattle by replacing wheat straw (WS), another low-quality forage. This study evaluated the effects of replacing WS with SB in diets offered to mid-lactation dairy cows on milk production and fatty acid profile, intake, digestibility, chewing activity, and ruminal fermentation. Nine multiparous Holstein cows averaging (mean ± standard deviation) 105 ± 12 d in milk, 42.1 ± 2.9 kg of milk/d, and 617 ± 59 kg of body weight were used in a replicated 3 × 3 Latin square with 21-d periods. Treatments were (% of dietary dry matter, DM): (1) 0SB, diet containing 0% SB and 27% WS, (2) 9SB, diet containing 9% SB and 18% WS, and (3) 18SB, diet containing 18% SB and 9% WS. Sugarcane bagasse had greater organic matter (OM; 94.1 vs. 85.1% of DM), neutral detergent fiber (NDF; 86.2 vs. 76.4% of DM), acid detergent fiber (ADF; 62.9 vs. 45.2% of DM), and lignin (19.9 vs. 10.3% of DM) concentration, but less crude protein (CP; 2.63 vs. 3.72% of DM) concentration than WS. Sugarcane bagasse also had greater physically effective NDF (total dietary NDF multiplied by % of TMR on the 8-mm + 19-mm sieves, peNDF8; 63.2 vs. 40.6% of DM) and undegraded NDF after 288 h of incubation (uNDF288; 35.5 vs. 21.2% of DM) contents than WS. The undegraded NDF after 30 h of incubation (uNDF30) content was similar for all diets; however, peNDF8 concentration and proportion of long particles (retained on a 19-mm sieve) increased linearly as SB inclusion in the diets increased. Cows increasingly sorted against long particles as SB replaced WS. Intakes of DM (26.53 kg/d) and NDF (8.58 kg/d) did not differ among the treatments, but intakes of OM and CP decreased, whereas ADF and uNDF288 intakes increased with SB inclusion level. Total-tract digestibilities of OM, CP, and NDF decreased linearly as SB replaced WS. Milk yield (37.0 kg/d), energy-corrected milk yield (ECM; 38.2 kg/d), feed efficiency (1.44 kg ECM yield/kg DM intake), and milk composition (fat, 3.89%; true protein, 2.90%) did not differ among diets. Increasing SB concentration of the diet linearly increased rumination time, but ruminal pH (ruminocentesis, 4 h after feeding) decreased. Total volatile fatty acid concentration increased linearly, whereas acetate:propionate decreased linearly, as SB replaced WS. The results indicate that replacement of WS with increasing levels of SB in low-forage diets with similar uNDF30 concentrations did not affect performance of mid-lactation dairy cows. We conclude that SB can be used as a fiber source in diets fed to dairy cows in mid-lactation; however, the decrease in total-tract digestibility of diets may decrease lactational performance when fed to high-producing dairy cows.

Direct Precipitation of Lignin Nanoparticles from Wheat Straw Organosolv Liquors Using a Static Mixer.

Micro- and nanosize lignin shows improved properties compared to standard lignin available today and has been gaining interest in recent years. Lignin is the largest renewable resource with an aromatic skeleton on earth but it is used for relatively low-value applications. Lignin in micro- to nanoscale; however, could facilitate rather valuable applications. Current production methods consume high amounts of solvents for purification and precipitation. The process investigated in this work uses the direct precipitation of lignin nanoparticles from organosolv pretreatment extract in a static mixer and can reduce solvent consumption drastically. The pH value, ratio of antisolvent to organosolv extract and flowrate in the mixer were investigated as precipitation parameters in terms of the resulting particle properties. Particles with dimensions ranging from 97.3 to 219.3 nm could be produced, and at certain precipitation parameters, carbohydrate impurities reach values as low as in purified lignin particles. Yields were found independent of the precipitation parameters with 48.2 ± 4.99%. Results presented in this work can be used to optimize precipitation parameters with emphasis on particle size, carbohydrate impurities or the solvent consumption.

The enrichment of anaerobic fungi and methanogens showed higher lignocellulose degrading and methane producing ability than that of bacteria and methanogens.

In this study, rumen content was used to obtain three enrichments of anaerobic fungi and methanogens (F + M enrichment), bacteria and methanogens (B + M enrichment), and whole rumen content (WRC enrichment), to evaluate their respective ability to degrade lignocellulose and produce methane. Among the treatments, F + M enrichment elicited the strongest lignocellulose degradation and methane production ability with both rice straw and wheat straw as substrates. Quantitative real-time PCR analysis and diversity analyses of methanogens in the three enrichment treatments demonstrated that F + M had larger number of 16S rRNA gene copies of methanogens and higher relative abundance of Methanobrevibacter, the predominant methanogen found in all enrichments. Caecomyces was the main anaerobic fungal genus for co-culturing to provide substrates for methanogens in this enrichment. Importantly, the F + M enrichment was stable and could be maintained with transfers supplied every 3 days, confirming its potential utility in anaerobic digestion for lignocellulose degradation and methane production.

Metabolically engineered Lactobacillus gasseri JCM 1131 as a novel producer of optically pure L- and D-lactate.

High-quality environmentally-friendly bioplastics can be produced by mixing poly-L-lactate with poly-D-lactate. On an industrial scale, this process simultaneously consumes large amounts of both optically pure lactate stereoisomers. However, because optimal growth conditions of L-lactate producers often differ from those of D-lactate producers, each stereoisomer is produced in a specialised facility, which raises cost and lowers sustainability. To address this challenge, we metabolically engineered Lactobacillus gasseri JCM 1131T, a bioprocess-friendly and genetically malleable strain of homofermentative lactic acid bacterium, to efficiently produce either pure L- or pure D-lactate under the same bioprocess conditions. Transformation of L. gasseri with plasmids carrying additional genes for L- or D-lactate dehydrogenases failed to affect the ratio of produced stereoisomers, but inactivation of the endogenous genes created strains which yielded 0.96 g of either L- or D-lactate per gram of glucose. In this study, the plasmid pHBintE, routinely used for gene disruption in Bacillus megaterium, was used for the first time to inactivate genes in lactobacilli. Strains with inactivated genes for endogenous lactate dehydrogenases efficiently fermented sugars released by enzymatic hydrolysis of alkali pre-treated wheat straw, an abundant lignocellulose-containing raw material, producing 0.37-0.42 g of lactate per gram of solid part of alkali-treated wheat straw. Thus, the constructed strains are primed to serve as producers of both optically pure L-lactate and D-lactate in the next-generation biorefineries.

Isolation and identification of cellulolytic bacteria from gastrointestinal tract of Arabian horse and investigation of their effect on the nutritional value of wheat straw.

AIMS: This study was conducted to isolate and identify the cellulolytic bacteria from gastrointestinal tract of Arabian horse and investigate their effect on the nutritional value of wheat straw (WS). METHODS AND RESULTS: Fresh faeces were collected from four Arabian horses. The cellulose-hydrolytic bacteria were isolated by using a medium amended with carboxymethyl cellulose (CMC). The activity of CMC was determined by measuring the release of reducing sugars from CMC. Among the isolates, four isolates (L11, L12, L2 and Z2) showed maximum hydrolysis capacity. 16S rRNA sequence analysis showed that these isolates possessed 99, 99, 99 and 98% similarity with Paenibacillus polymyxa, Paenibacillus polymyxa, Enterobacter cloacae and Escherichia coli respectively. Wheat straw was incubated with the isolated bacteria in liquid medium. Disappeared dry matter (DM) and crude protein (CP), neutral detergent fibre (NDF), indigestible NDF (iNDF) and acid detergent fibre (ADF) contents of WS were affected by microbial treatments (P < 0·05). The highest disappearance of DM and CP contents and the lowest NDF, iNDF and ADF contents of WS were observed by treating with P. polymyxa L11. The values of NDF, iNDF and ADF were the highest and CP was the lowest in control. Gas production parameters and digestibility of processed samples were determined. Bacterial treatments enhanced (P < 0·05) DM, OM, CP, NDF and ADF digestibility of WS. The highest and lowest (P < 0·05) DM, OM, CP, NDF and ADF digestibilities were observed for WS treated with isolates L12 and control respectively. Potential of gas production (b), truly degraded organic matter (TDOM) and microbial biomass of bacteria treatments were significantly higher (P < 0·05) compared to control. CONCLUSIONS: Generally, the results of this study showed that the isolated bacteria from horse faeces are capable of changing the chemical composition, increasing digestibility as well as enhancing nutrition value of WS. SIGNIFICANCE AND IMPACT OF THE STUDY: Wheat straw is a major agro-residue fed to ruminants. However, high fibre contents reduce digestibility and limit animal productivity. It seems that enhancement of ruminal degradation of these by-products is necessary. The results of this study revealed that cellulolytic bacteria isolated from gastrointestinal tract of horse can be used for biological treatment of WS.