Diversity Analysis Of Bacterial Community In Shrimp And Crab Shell-rich Soil And Preliminary Study On Core Functional Flora Degrading Shrimp And Crab Shell

With the production and consumption of crustacean aquatic products such as shrimp and crab,a large number of shrimp and crab shells(SCS)are piled up and discarded,which not only seriously affect the environment and human life,but also waste the available organic substances.However,the traditional chemical methods to treat SCS generate a large amount of acid wastewater,which is not conducive to environmental protection.The SCS treat by that microorganism is green and environment-friendly,the energy consumption of consumables is low.Therefore,it is necessary to tap the microbial resources that degrade SCS in the soil.In this study,the soil sample from the coastal mudflat silt of the North Sea coast and mangrove rhizosphere soil were mixed for SCS enrichment culture.The 16S rRNA Illumina Mi Seq Sequencing method was used to analyze the structure of soil bacterial community after enrichment of SCS.The functional flora in each sample was enriched by the separation of SCS using microbial culturable technology and its activity was measured.The chitinase gene and activity in the enriched samples were verified,and the fermentation conditions of one chitinase-producing strain were optimized.The validation study on the direct fermentation degradation of SCS by the core microflora constructed by the combination of the condition-optimized strain and other functional strains was conducted.This study provided a new idea for microbial degradation of SCS and microbial resources for the later improvement and construction of the core functional flora of degraded SCS.The main conclusions are as follows:(1)The enrichment of SCS changed that composition of soil bacterial community to a large extent.The characteristic OTUs enriched for days 0,15,45,75,105,and 135 d were 2024,7249,5724,4967,5003,5165, respectively.The results of?diversity significance analysis showed that?diversity was significant after enrichment for 45 d,and the enrichment process might lead to significant enrichment of dominant communities degrading SCS to some extent.(2)The enrichment of SCS changed that community structure and relative abundance of soil bacteria.At the phylum level,Proteobacteria,Firmicutes and Bacteroidetes were the dominant phylum.The relative abundance of bacterial community in different enriched substrate(shrimp shell and crab shell)was different,with Firmicutes and Bacteroidetes as the dominant phylum in the shrimp shell group and Actinobacteria as the dominant phylum in the crab shell group.At that genus level,the relative abundances of the genus Sulfurimonas,Desulfococcus,Fusibacter, Clostridium,Hydrogenophaga,Bacteroides,and Vibrio were significantly increased,Bacillus was the dominant genus in the shrimp shell group, Sulfurimonas was the dominant genus in the crab shell group,and Fusibacter in the control group was the dominant genus.The enrichment of SCS changed the living environment of bacteria in the soil,so that the dominant bacteria in the soil were dynamically changed.(3)The enrichment of SCS resulted in significant difference in that species composition and structure of bacteria in the soil.The changes of soil bacterial community structure were mainly affected by the nrichment substrate and enrichment time,which were the two main coordinate components.PCoA analysis showed that the cumulative ontribution rate of variance of the two principal coordinate components was 47.3%.LEf Se analysis(selective LDA>4 microorganisms) dentified 36,8,14,13,6,and 13 significantly different biomarkers for enrichment 0–135 d,respectively.(4)The SCS collected from 0,15,45,75,105 and 135 d soil samples were analyzed for culturable bacterial diversity.The culturable bacteria and functional strains were isolated and screened by four basic media(LB solid,R₂A,TSA,2216E)and four functional media(colloidal chitin edium,protease-producing medium,lipase-producing medium and cid-producing bacteria medium).A total of 272 culturable bacteria were obtained by co-isolation,purification and re-isolation of the four basic media,which were distributed in 95 Genera,45 Familiy,26 Orders,8 lasses,and 4 Phylum.There were 101 functional strains,including 38chitinase-producing strains,19 protease-producing strains,31 ipase-producing strains and 13 acid-producing strains,among which 19strains had more than two functions.(5)Unculturable combination with culturable was used to verify the chitinase gene and enzyme activity.First,the chitinase gene in SCS nriched soil was verified by RT-qPCR.The results showed that after 45d of enrichment,the chitinase gene in soil of the experimental group was significantly up-regulated,and the up-regulation in the shrimp shell group was more significant.The chitinase activity in enriched soil was detected by microplate fluorimetric assay.The results showed that after 45 d of nrichment,the activities of GlcNAc,(GlcNAc)₂and(GlcNAc)₃ lycosidases were significant,mainly the(GlcNAc)₃endonuclease,and the activity of(GlcNAc)₃endonuclease in the crab shell group was more significant than that in the shrimp shell group.After enrichment for 75 d,it is mainly GlcNAc exonuclease.(6)The fermentation conditions of chitinase-producing strain GXUN-20 were optimized in the culturable functional flora.After hysiological and biochemical identification and 16S rRNA sequence onstruction of phylogenetic tree,the strain was preliminarily determined to be Lysobacter firmicutimachus.Using chitinase activity as the index,the single factor condition optimization of fermentation enzyme roduction of GXUN-20 was conducted.The four-level orthogonal xperiment and orthogonal test analysis of variance were conducted on the basis of the four factors(nitrogen source concentration,initial pH of the fermentation broth,chitin powder concentration,and inoculation mount)that had significant results from the single factor optimization.The results showed that the concentration of nitrogen source had a ignificant effect on the enzyme production(P=0.013<0.05).Under the conditions of nitrogen source of 1 g/L soybean cake powder,chitin owder of 20 g/L,inoculation amount of 3%,initial pH of the ermentation broth of 8.4,30?,and fermentation at 200 r/min for 6 d,the chitinase activity in the fermentation broth was the largest,reaching0.965 U/mL,which was 6.89 times that before optimization.(7)The optimized strain is combined with the functional strain ombination obtained through culturable screening to directly ferment and degrade the SCS.The experimental results showed that the egradation of SCS by a single functional flora was not significant,but the degradation of SCS by mixed fermentation with multi-functional flora had the advantage.The chitinase activity in the fermentation broth ermented by mixing multiple functional flora for 14 d reached 0.6389U/mL,which was 6.48 times higher than that in the fermentation broth inoculated with only the chitinase-producing flora.The shrimp shells were almost completely degraded,and astaxanthin was dissolved in the fermentation broth,so the color of the fermentation broth was dark.The chitinase activity in the fermentation broth of mixed fermentation of crab shell with multifunctional flora for 14 d reached 0.3288 U/mL,which was2.84 times higher than that in the fermentation broth inoculated with only the chitinase-producing flora.Most of the crab shell was degraded,and the fermentation broth was dark in color.The comparison between the rimp shell group and the crab shell group revealed that the shrimp shell was more prone to degradation under the same fermentation conditions.