| Objective:Explore the diversity of fungi in turmeric residue and its stacking environment,obtain the fungi with high efficiency hydrolytic residue and establish synergistic fungal combinations to solve the environmental pollution of the stacking of turmeric residue and maintain ecological health;To improve the hydrolysis rate of pharmaceutical residue and lay a foundation for the reuse of biomass.Methods:1.To illustrate the decomposed dynamics during the stacking process,fungal diversity in anti-biodegradable herb residues stacked for 2 and 5 years was analyzed by using Pac Bio Sequel sequencing and using FUNGuild platform to predict fungal gene function and phenotype.2.Fungal strains were isolated from turmeric residue and its stacking environment by tissue separation and shaking table culture.Phylogenetic and morphological methods were used to identify and classify the isolated strains and evaluate their diversity.3.The isolated fungi were prescreened by growing on three media: carboxymethyl cellulose congo red agar,potato dextrose agar containing aniline blue,xylan congo red agar.The fungi showed potentially high enzyme activity from the agar plate screening were further cultured in 6% turmeric residues as the sole nutrient liquid medium and their lignocellulolytic activities from culture supernatants were evaluated.4.Gene annotation,GO and KEGG functional enrichment analysis and CAZymes gene expression prediction of Phaeophlebiopsis sp.(ZYJHYZ254)were performed using transcriptome.Results:1.A total of 43 753 effective sequences were detected from 6 samples of anti-biodegradable herb residues(3 for stacked 2 years and 3 for stacked 5 years).A total of 4 phyla,86 genera and 126 species in 4 phyla were detected from the residues stacked for 2 years with the dominant taxa of Arthrophylla,Conidia and Aspergillus.Three phyla,49 genera and 59 species were detected in the residues stacked for 5years and Chaetomium novozelandicum and Microcystiaceae were the dominant taxa.Functional composition analysis of antiphlogistic medicine residues showed that saprophytic fungi accounted for 46.97%,pathogenic fungi 32.26% and symbiotic fungi 20.77% in the residues stacked for 2 years.Saprophytic fungi accounted for96.60% and few symbiotic fungi(0.22%)of the residue stacked for 5 years.The pH of the residues stacked for 2 years was 4.9,and that of the residues stacked for 5 years was 3.6.The water content of residues stacked for 2(5)years was 49.15(20.82%).2.Isolated and identified 67 strains of fungi from turmeric and its stacking environment,belonging to 30 species belonging to 23 genera and 3 phyla.Bjerkandera Adusta was the dominant species with 11 strains,followed by Talaromyces Purpureogenus with 6strains.The dominant genus is Bjerkandera,followed by Aspergillus.3.From single polymer solid medium analysis,we were able to find that 35 strains produced transparent circles while 24 strains showed a ratio greater than or equal to two.All fungi produce different levels of enzymes with six different enzymes,viz.,β-glucosidase,cellulase,β-xylosidase,xylanase,laccase and peroxidase.Most fungi can produce higher β-glucosidase and cellulase activity on day 7-9,among which Aspergillus niger(ZYJHYZ242)and Fomitopsis palustris(ZYJHYZ247)produces the highest level.In the detection of enzymes related to lignin degradation,it was found that the peak of enzyme production of most strains occurred in 5-7 days,with Phaeophlebiopsis sp.(ZYJHYZ254)having the highest peroxidase enzyme activity,reaching 602.96 U/L on day 7,followed by Piloderma sp.(ZYJHYZ268)and Coniochaeta velutina(ZYJHYZ246).In the hemicellulase activity results,Phaeophlebiopsis sp.(ZYJHYZ254)had the highest xylanase production on the fifth day.Among all fungi,Coniochaeta Velutina(ZYJHYZ246)and Phaeophlebiopsis sp.(ZYJHYZ254)had the highest β-xylanase production.Nine strains were selected to construct a total of 12 synergistic fungal combinations,and the activities of five enzymes except lignin peroxidase were increased 2-3 times.The weight loss experiment found that No.10 had the most significant weight loss effect,with a weight loss rate of 60.34%.4.A total of 1 232 differentially expressed genes was detected from strain Phaeophlebiopsis sp.(ZYJHYZ254)in mycelia aged 3 and 7 day.Taking 3-day-old mycelia as the control,l 826 and 406 genes were significantly up-regulated and down-regulated,respectively.Gene annotation and GO and KEGG functional enrichment analysis showed that differentially expressed genes were mainly related to protein synthesis,metabolism and enzyme synthesis.In addition,a total of 387 CAZymes genes was expressed,and GH genes were the most abundant,accounting for 49.61%,and AA(97)and GT(62)ranked second and third,accounting for 25.06% and 16.02%,respectively.GH16(24),accounting for 12.50% of GH,is the most abundant in GH,mainly encoding glucosidase,xylanase,etc.AA3(37)is the most abundant in AA,accounting for 38.14%,encoding oxidase,dehydrogenase,etc.Further study found that GH16 and AA3 were most expressed in CAZymes in3-day-old and 7-day-old mycelia.Conclusions:Through high-throughput sequencing,it was found that there were many functional strains of lcellulose degradation in the turmeric residue,and a total of 302 strains of fungi were isolated.After initial screening and re-screening,four high-efficiency synergistic fungal combinations were constructed,and Phaeophlebiopsis sp.(ZYJHYZ254)was found CAZymes gene expression was abundant in CAZymes.This study has laid a foundation for solving the environmental pollution of pharmaceutical industry related to this kind of pharmaceutical residue and promoting ecological health.It is of great significance to degrade lignocellulose in special biomass,explore more resources for enzyme producing microorganisms,and lay a good foundation for biological enzyme industrial production. |
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