Bacterial strain for bast fiber crops degumming and its bio-degumming technique.

The research and development of bio-degumming technology is under a slow progress due to the shortage of proper efficient bacterial strains and processes. A degumming bacterial strain-Pectobacterium wasabiae (PW)-with broad-spectrum degumming abilities was screened out in this study. After the fermentation for 12 h, the residual gum contents of kenaf bast, ramie bast, hemp bast, flax bast, and Apocynum venetum bast were all lower than 15%. This bacterial strain could realize the simultaneous extracellular secretion of pectinase, mannase, and xylanase with the maximum enzyme activity levels of 130.25, 157.58, and 115.24 U/mL, respectively. The optimal degumming conditions of this bacterial strain were as follows: degumming time of 12 h, bath ratio of 1:10, temperature of 33 °C, and inoculum size of 2%. After the bio-degumming through this bacterial strain, the COD in wastewater was below 4000 mg/L, which was over 60% lower than that in boiling-off wastewater generated by chemical degumming. This technology achieves higher efficiency, higher quality, and lower pollution.

Comparative transcriptomics of Pleurotus eryngii reveals blue-light regulation of carbohydrate-active enzymes (CAZymes) expression at primordium differentiated into fruiting body stage.

Blue light is an important environmental factor which could induce mushroom primordium differentiation and fruiting body development. However, the mechanisms of Pleurotus eryngii primordium differentiation and development induced by blue light are still unclear. The CAZymes (carbohydrate-active enzymes) play important roles in degradation of renewable lignocelluloses to provide carbohydrates for fungal growth, development and reproduction. In the present research, the expression profiles of genes were measured by comparison between the Pleurotus eryngii at primordium differentiated into fruiting body stage after blue light stimulation and dark using high-throughput sequencing approach. After assembly and compared to the Pleurotus eryngii reference genome, 11,343 unigenes were identified. 539 differentially expressed genes including white collar 2 type of transcription factor gene, A mating type protein gene, MAP kinase gene, oxidative phosphorylation associated genes, CAZymes genes and other metabolism related genes were identified during primordium differentiated into fruiting body stage after blue light stimulation. KEGG results showed that carbon metabolism, glycolysis/gluconeogenesis and biosynthesis of amino acids pathways were affected during blue light inducing primordia formation. Most importantly, 319 differentially expressed CAZymes participated in carbon metabolism were identified. The expression patterns of six representative CAZymes and laccase genes were further confirmed by qRT-PCR. Enzyme activity results indicated that the activities of CAZymes and laccase were affected in primordium differentiated into fruiting body under blue light stimulation. In conclusion, the comprehensive transcriptome and CAZymes of Pleurotus eryngii at primordium differentiated into fruiting body stage after blue light stimulation were obtained. The biological insights gained from this integrative system represent a valuable resource for future genomic studies on this commercially important mushroom.

Effects of Different Substrates on Lignocellulosic Enzyme Expression, Enzyme Activity, Substrate Utilization and Biological Efficiency of Pleurotus Eryngii.

BACKGROUND/AIMS: Pleurotus eryngii is one of the most valued and delicious mushrooms which are commercially cultivated on various agro-wastes. How different substrates affect lignocellulosic biomass degradation, lignocellulosic enzyme production and biological efficiency in Pleurotus eryngii was unclear. METHODS AND RESULTS: In this report, Pleurotus eryngii was cultivated in substrates including ramie stalks, kenaf stalks, cottonseed hulls and bulrush stalks. The results showed that ramie stalks and kenaf stalks were found to best suitable to cultivate Pleurotus eryngii with the biological efficiency achieved at 55% and 57%, respectively. In order to establish correlations between different substrates and lignocellulosic enzymes expression, the extracellular proteins from four substrates were profiled with high throughput TMT-based quantitative proteomic approach. 241 non-redundant proteins were identified and 74 high confidence lignocellulosic enzymes were quantified. Most of the cellulases, hemicellulases and lignin depolymerization enzymes were highly up-regulated when ramie stalks and kenaf stalks were used as carbon sources. The enzyme activities results suggested cellulases, hemicellulases and lignin depolymerization enzymes were significantly induced by ramie stalks and kenaf stalks. CONCLUSION: The lignocelluloses degradation, most of the lignocellulosic enzymes expressions and activities of Pleurotus eryngii had positive correlation with the biological efficiency, which depend on the nature of lignocellulosic substrates. In addition, the lignocellulosic enzymes expression profiles during Pleurotus eryngii growth in different substrates were obtained. The present study suggested that most of the lignocellulosic enzymes expressions and activities can be used as tools for selecting better performing substrates for commercial mushroom cultivation.

Secretome analysis of Pleurotus eryngii reveals enzymatic composition for ramie stalk degradation.

Pleurotus eryngii (P. eryngii) can secrete large amount of hydrolytic and oxidative enzymes to degrade lignocellulosic biomass. In spite of several researches on the individual lignolytic enzymes, a direct deconstruction of lignocellulose by enzyme mixture is not yet possible. Identifying more high-performance enzymes or enzyme complexes will lead to efficient in vitro lignocelluloses degradation. In this report, secretomic analysis was used to search for the new or interesting enzymes for lignocellulose degradation. Besides, the utilization ability of P. eryngii to ramie stalk substrate was evaluated from the degradation of cellulose, hemicellulose, and lignin in medium and six extracellular enzymes activities during different growth stages were discussed. The results showed that a high biological efficiency of 71% was obtained; cellulose, hemicelluloses, and lignin decomposition rates of P. eryngii were 29.2, 26.0, and 51.2%, respectively. Enzyme activity showed that carboxymethyl cellulase, xylanase, laccase, and peroxidase activity peaks appeared at the primordial initiation stage. In addition, we profiled a global view of the secretome of P. eryngii cultivated in ramie stalk media to understand the mechanism behind lignocellulosic biomass hydrolysis. Eighty-seven nonredundant proteins were identified and a diverse group of enzymes, including cellulases, hemicellulases, pectinase, ligninase, protease, peptidases, and phosphatase implicated in lignocellulose degradation were found. In conclusion, the information in this report will be helpful to better understand the lignocelluloses degradation mechanisms of P. eryngii.

Liquid hot water pretreatment combined with high-solids enzymatic hydrolysis and fed-batch fermentation for succinic acid sustainable processed from sugarcane bagasse.

In order to sustainable process of bio-succinic acid (SA), response surface methodology (RSM) was applied to optimize liquid hot water pretreatment pretreatment of sugarcane bagasse (SCB), followed by high-solids enzymatic hydrolysis of pretreated residual that without washing, then the hydrolysates and partial pretreatment liquid were used as carbon sources for SA fermentation. Results showed that the highest sugars yield could be achieved at pretreatment conditions of temperature 186 °C, time 25 min and solid-to-liquid ratio 0.08; enzymatic digestion the pretreated residuals at 20 % (w/v) solid content via enzymes reconstruction and fed-batch strategy, the obtained sugars reached to 121 g/L; by controlling the nutrition and conditions of the fermentation process, most of the C5 and C6 sugars in the hydrolysate and pretreatment liquid were converted into SA with a conversion rate high to 280 mg/g SCB. This study can provide a novel clue for clean and efficient biorefining of chemicals.

Comparative secretome of white-rot fungi reveals co-regulated carbohydrate-active enzymes associated with selective ligninolysis of ramie stalks.

In the present research, Phanerochaete chrysosporium and Irpex Lacteus simultaneously degraded lignin and cellulose in ramie stalks, whereas Pleurotus ostreatus and Pleurotus eryngii could depolymerize lignin but little cellulose. Comparative proteomic analysis of these four white-rot fungi was used to investigate the molecular mechanism of this selective ligninolysis. 292 proteins, including CAZymes, sugar transporters, cytochrome P450, proteases, phosphatases and proteins with other function, were successfully identified. A total of 58 CAZyme proteins were differentially expressed, and at the same time, oxidoreductases participated in lignin degradation were expressed at higher levels in P. eryngii and P. ostreatus. Enzyme activity results indicated that cellulase activities were higher in P. chrysosporium and I. lacteus, while the activities of lignin-degrading enzymes were higher in P. eryngii and P. ostreatus. In addition to the lignocellulosic degrading enzymes, several proteins including sugar transporters, cytochrome P450 monooxygenases, peptidases, proteinases, phosphatases and kinases were also found to be differentially expressed among these four species of white-rot fungi. In summary, the protein expression patterns of P. eryngii and P. ostreatus exhibit co-upregulated oxidoreductase potential and co-downregulated cellulolytic capability relative to those of P. chrysosporium and I. lacteus, providing a mechanism consistent with selective ligninolysis by P. eryngii and P. ostreatus.

Co-fermentation of succinic acid and ethanol from sugarcane bagasse based on full hexose and pentose utilization and carbon dioxide reduction.

The full utilization of carbohydrates in lignocellulosic biomass is essential for an efficient biorefining process. In this study, co-fermentation was performed for processing ethanol and succinic from sugarcane bagasse. By optimizing the co-fermentation conditions, nutrition and feeding strategies, a novel process was developed to make full utilization of the glucose and xylose in the hydrolysate of sugarcane bagasse. The achieved concentrations of succinic acid and ethanol reached to 22.1 and 22.0 g/L, respectively, and could realize the conversion of 100 g SCB raw material into 8.6 g ethanol and 8.7 g succinic acid. It is worth mentioning that the CO2 released from S. cerevisiae in co-fermentation system was recycled by A. succinogenes to synthesize succinic acid, realized CO2 emission reduction in the process of lignocellulosic biomass biorefinery. This study provided a clue for efficient biorefinery of lignocellulosic biomass and reduction greenhouse gas emissions.

Mapping the Secretome and Its N-Linked Glycosylation of Pleurotus eryngii and Pleurotus ostreatus Grown on Hemp Stalks.

Our previous research showed that Pleurotus eryngii and Pleurotus ostreatus were effective fungi for pretreatment of industrial hemp stalks to improve enzymatic saccharification. The secretomes of these two fungi were analyzed to search for the effective enzyme cocktails degrading hemp lignin during the pretreatment process. In total, 169 and 155 proteins were identified in Pleurotus eryngii and Pleurotus ostreatus, respectively, and 50% of the proteins involved in lignocellulose degradation were CAZymes. Because most of the extracellular proteins secreted by fungi are glycosylated proteins, the N-linked glycosylation of enzymes could be mapped. In total, 27 and 24 N-glycosylated peptides were detected in Pleurotus eryngii and Pleurotus ostreatus secretomes, respectively. N-Glycosylated peptides of laccase, GH92, exoglucanase, phenol oxidase, α-galactosidase, carboxylic ester hydrolase, and pectin lyase were identified. Deglycosylation could decrease enzymatic saccharification of hemp stalks. The activities of laccase, α-galactosidase, and phenol oxidase and the thermal stability of laccase were reduced after deglycosylation.