Qun Wang

Evaluation of bacterial expansin EXLX1 as a cellulase synergist for the saccharification of lignocellulosic agro-industrial wastes.

Various types of lignocellulosic wastes extensively used in biofuel production were provided to assess the potential of EXLX1 as a cellulase synergist. Enzymatic hydrolysis of natural wheat straw showed that all the treatments using mixtures of cellulase and an optimized amount of EXLX1, released greater quantities of sugars than those using cellulase alone, regardless of cellulase dosage and incubation time. EXLX1 exhibited different synergism and binding characteristics for different wastes, but this can be related to their lignocellulosic components. The cellulose proportion could be one of the important factors. However, when the cellulose proportion of different biomass samples exhibited no remarkable differences, a higher synergism of EXLX1 is prone to occur on these materials, with a high proportion of hemicellulose and a low proportion of lignin. The information could be favorable to assess whether EXLX1 is effective as a cellulase synergist for the hydrolysis of the used materials. Binding assay experiments further suggested that EXLX1 bound preferentially to alkali pretreated materials, as opposed to acid pretreated materials under the assay condition and the binding preference would be affected by incubation temperature.

Genetic engineering of microorganisms for biodiesel production

Biodiesel, as one type of renewable energy, is an ideal substitute for petroleum-based diesel fuel and is usually made from triacylglycerides by transesterification with alcohols. Biodiesel production based on microbial fermentation aiming to establish more efficient, less-cost and sustainable biodiesel production strategies is under current investigation by various start-up biotechnology companies and research centers. Genetic engineering plays a key role in the transformation of microbes into the desired cell factories with high efficiency of biodiesel production. Here, we present an overview of principal microorganisms used in the microbial biodiesel production and recent advances in metabolic engineering for the modification required. Overexpression or deletion of the related enzymes for de novo synthesis of biodiesel is highlighted with relevant examples.

Sweetpotato vines hydrolysate induces glycerol to be an effective substrate for lipid production of Trichosporon fermentans.

By co-fermented with sweetpotato vines hydrolysate (SVH), glycerol can be used as an effective substrate for Trichosporon fermentans to produce lipids. Submerged fermentation results showed that T. fermentans exhibited a maximum lipid yield of 7.45 g l(-1) with a biomass of 17.95 g l(-1) on 10% SVH added glycerol mineral medium (Glycerol MM-10% SVH), which was 4.34-fold higher than that on glycerol mineral medium. Lipids produced on glycerol based media exhibited a significantly different fatty acid composition profile from that produced on sugar based media accompanying by a sharp increase in polyunsaturated fatty acids content. Biochemical behaviors characterization further demonstrated that SVH has a remarkable promoting effect on the biomass formation and glycerol uptake. The extremely high lipid yield on Glycerol MM-10% SVH was mainly attributed to the enhancement of SVH on biomass, although SVH is an excellent nutrient supplier for lipid accumulation due to its low nitrogen availability.

Sweetpotato vines hydrolysate promotes single cell oils production of Trichosporon fermentans in high-density molasses fermentation.

This study investigated the co-fermentation of molasses and sweetpotato vine hydrolysate (SVH) by Trichosporon fermentans. T. fermentans showed low lipid accumulation on pure molasses; however, its lipid content increased by 35% when 10% SVH was added. The strong influence of SVH on lipid production was further demonstrated by the result of sensitivity analysis on effects of factors based on an artificial neural network model because the relative importance value of SVH dosage for lipid production was only lower than that of fermentation time. Scanning electron microscope observation and flow cytometry of yeast cells grown in culture with and without SVH showed that less deformation cells were involved in the culture with SVH. The activity of malic enzyme, which plays a key role in fatty acid synthesis, increased from 2.4U/mg to 3.7U/mg after SVH added. All results indicated SVH is a good supplement for lipid fermentation on molasses.

Characterization of Cellulase Secretion and Cre1-Mediated Carbon Source Repression in the Potential Lignocellulose-Degrading Strain Trichoderma asperellum T-1

Trichoderma asperellum, a traditional bio-control species, was demonstrated to be an excellent candidate for lignocellulose degradation in this work. Comparing to the representatively industrial strain of Trichoderma reeseiQM6a, T. asperellum T-1 showed more robust growth, stronger spore production, faster secretion of lignocellulose-decomposing enzymes and better pH tolerance. The reducing sugar released by strain T-1 on the second day of fermentation was 87% higher than that of strain QM6a, although the maximum reducing sugar yield and the cellulase production persistence of the strain T-1 were lower. Our experiment found that the cellulase secretion was strongly inhibited by glucose, suggesting the existence of carbon source repression pathway in T. asperellum T-1. The inhibiting effect was enhanced with an increase in glucose concentration and was closely related to mycelium growth. SDS-PAGE and secondary mass-spectrum identification confirmed that the expression of endo-1,4-β-xylanase I in T. asperellum T-1 was down-regulated when glucose was added. The factor Cre1, which plays an important role in the down-regulation of the endo-1,4-β-xylanase I gene, was investigated by bioinformatics methods. The protein structure of Cre1, analyzed using multiple protein sequence alignment, indicates the existence of the Zn-fingers domain. Then, the binding sites of Cre1 on the endo-1,4-β-xylanase I gene promoter were further elucidated. This study is the first report about Cre1-mediated carbon repression in the bio-control strain T. asperellum T-1. All of the above results provided good references for better understanding T. asperellum T-1 and improving its application for lignocellulose degradation.

Application of methanol and sweet potato vine hydrolysate as enhancers of citric acid production by Aspergillus niger

BackgroundAgricultural waste is as an alternative low-cost carbon source or beneficial additives which catch most people’s eyes. In addition, methanol and sweet potato vine hydrolysate (SVH) have been reported as the efficient enhancers of fermentation according to some reports. The objective of the present study was to confirm SVH as an efficient additive in CA production and explore the synergistic effects of methanol and SVH in fermentation reactions.ResultsThe optimal fermentation conditions resulted in a maximum citric acid concentration of 3.729 g/L. The final citric acid concentration under the optimized conditions was increased by 3.6-fold over the original conditions, 0.49-fold over the optimized conditions without methanol, and 1.8-fold over the optimized conditions in the absence of SVH. Kinetic analysis showed that Qp, Yp/s, and Yx/s in the optimized systems were significantly improved compared with those obtained in the absence of methanol or SVH. Further, scanning electron microscopy (SEM) revealed that methanol stress promoted the formation of conidiophores, while SVH could neutralize the effect and prolong Aspergillus niger vegetative growth. Cell viability analysis also showed that SVH might eliminate the harmful effects of methanol and enhance cell membrane integrity.ConclusionsSVH was a superior additive for organic acid fermentation, and the combination of methanol and SVH displayed a significant synergistic effect. The research provides a preliminary theoretical basis for SVH practical application in the fermentation industry.

Engineering Aspergillus oryzae A-4 through the Chromosomal Insertion of Foreign Cellulase Expression Cassette to Improve Conversion of Cellulosic Biomass into Lipids

A genetic modification scheme was designed for Aspergillus oryzae A-4, a natural cellulosic lipids producer, to enhance its lipid production from biomass by putting the spotlight on improving cellulase secretion. Four cellulase genes were separately expressed in A-4 under the control of hlyA promoter, with the help of the successful development of a chromosomal genetic manipulation system. Comparison of cellulase activities of PCR-positive transformants showed that these transformants integrated with celA gene and with celC gene had significantly (p<0.05) higher average FPAase activities than those strains integrated with celB gene and with celD gene. Through the assessment of cellulosic lipids accumulating abilities, celA transformant A2-2 and celC transformant D1-B1 were isolated as promising candidates, which could yield 101%–133% and 35.22%–59.57% higher amount of lipids than the reference strain A-4 (WT) under submerged (SmF) conditions and solid-state (SSF) conditions, respectively. Variability in metabolism associated to the introduction of cellulase gene in A2-2 and D1-B1 was subsequently investigated. It was noted that cellulase expression repressed biomass formation but enhanced lipid accumulation; whereas the inhibitory effect on cell growth would be shielded during cellulosic lipids production owing to the essential role of cellulase in substrate utilization. Different metabolic profiles also existed between A2-2 and D1-B1, which could be attributed to not only different transgene but also biological impacts of different integration. Overall, both simultaneous saccharification and lipid accumulation were enhanced in A2-2 and D1-B1, resulting in efficient conversion of cellulose into lipids. A regulation of cellulase secretion in natural cellulosic lipids producers could be a possible strategy to enhance its lipid production from lignocellulosic biomass.

Excellent waste biomass-degrading performance of Trichoderma asperellum T-1 during submerged fermentation

The random disposal and incineration of agricultural residues cause resources waste and environmental pollution. The potential of waste biomass for the production of alternative liquid fuels is increasing and the bioconversion of lignocellulose to fermentable monomeric sugars is essential for second-generation biofuel production. Here, natural and pretreated switch grass or rice straw were fermented by both Trichoderma asperellum T-1 and Trichoderma reesei QM6a, with the fermentation results highlighted the potential of T. asperellum T-1 in the degradation of natural waste lignocellulosic materials. In fermenting different substrates, the filter paper activity, β-glucosidase activity, xylanase activity and carboxymethyl cellulase activity of T-1 can respectively reach 1.88, 8.00, 7.15 and 20.52 times that of QM6a. Although acid pretreatment could improve the enzyme activities of both T-1 and QM6a, its effect on T-1 was much smaller than that on QM6a. Moreover, strain T-1 fermented the natural rice straw better than the pretreated rice straw. Therefore, T-1 is considered to be more suitable for the degradation of natural biomass, especially for the degradation of rice straw. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and scanning electron microscopy (SEM) showed that the cellulase series secreted by T. asperellum T-1 was more abundant, and its substrate deconstruction ability was stronger than T. reesei QM6a. All these results suggest the potential of T. asperellum T-1 in the degradation of natural waste lignocellulosic material, with practical benefits in terms of cost and pollution reduction.

Comparative genome analysis of the oleaginous yeast Trichosporon fermentans reveals its potential applications in lipid accumulation

In this work, Trichosporon fermentans CICC 1368, which has been shown to accumulate cellular lipids efficiently using industry-agricultural wastes, was subjected to preliminary genome analysis, yielding a genome size of 31.3 million bases and 12,702 predicted protein-coding genes. Our analysis also showed a high degree of gene duplications and unique genes compared with those observed in other oleaginous yeasts, with 3-4-fold more genes related to fatty acid elongation and degradation compared with those in Rhodosporidium toruloides NP11 and Yarrowia lipolytica CLIB122. Phylogenetic analysis with other oleaginous microbes suggested that the lipogenic capacity of T. fermentans was obtained during evolution after the divergence of genera. Thus, our study provided the first draft genome and comparative analysis of T. fermentans, laying the foundation for its genetic improvement to facilitate cost-effective lipid production.

The contrasting effects of N -( n -butyl) thiophosphoric triamide (NBPT) on N 2 O emissions in arable soils differing in pH are underlain by complex microbial mechanisms

The urease inhibitor, N-(n-butyl) thiophosphoric triamide (NBPT), has been proposed to reduce synthetic fertilizer-N losses, including nitrous oxide (N2O) emissions from agricultural soils. However, the response of N2O emission to NBPT amendment is inconsistent across soils and associated microbial mechanisms remain largely unknown. Here we performed a meta-analysis of the effects of NBPT on N2O emissions and found NBPT significantly reduced N2O emissions in alkaline soils whereas no obvious effects exhibited in acid soils. Based on the finding of meta-analysis that pH was a key modifier in regulating the effect of NBPT on N2O emissions, we selected two arable soils differing in pH and conducted a microcosm study. In conjunction with measurement of N2O emission, community structure and abundance of functional guilds were assessed using T-RFLP and qPCR. Our results showed NBPT retarded urea hydrolysis and inhibited nitrification, but stimulated N2O emission in alkaline soil, whereas it exhibited no remarkable effects in acid soil, thereby only partly confirming the results of meta-analysis. Abundances of AOB and ureC-containing bacteria decreased, while abundance of AOA increased in both soils with NBPT addition. For acid soil, N2O emissions were significantly correlated with both abundances and community structures of AOA and ureC-containing bacteria, as well as abundance of AOB; for alkaline soil, abundances and community structures of AOB were correlated with N2O emission, as well as community structures of ureC-containing bacteria and archaea, indicating an inconsistent response pattern of community traits of N2O emissions-related functional guilds to NBPT between alkaline soil and acid soil. Our findings suggest that (i) efficacy of NBPT in N2O emission was mainly influenced by soil pH and (ii) variable effects of NBPT on N2O emission might originate not only from the direct effect of NBPT on community traits of urease-positive microbes, but from the indirect effect on ammonia oxidizers.