Solid-state Fermentation Of Spectinomycin And Tylosin Mycelial Dregs And The Mechanisms Of Efficient Transformation Revealed Based On Integrated Meta-omics

Antibiotics are one of the greatest discoveries in the history of human medicine,which has improved our ability to fight bacterial infections.Antibiotics production has been surging for decades owing to their widespread use as growth promoters and therapeutic agents in humans and animals.The millions of tons of antibiotic mycelial dregs(AMDs)are produced annually during the enormous production of antibiotics.Residual antibiotics in the AMDs have led to the rapid emergence of antibiotic resistance genes(ARGs)and antibiotic resistant bacteria(ARB).After the untreated AMDs are discharged into the environment,the horizontal gene transfer mediated by mobile transfer elements(MGEs)will promote the widespread spread of ARGs and produce super-resistant bacteria.This will seriously threaten human health and disturb the dynamic balance of the ecological environment.So,antibiotic resistance,similar to climate warming,is a major global problem,which has attracted worldwide attention.The problem of antibiotic resistance can be achieved through a wide range of aspects,such as the production of antibiotics,utilization of antibiotics,and emission of AMDs.Among them,exploring green and efficient biological treatment technology is the key to solve this problem.AMDs has been listed as hazardous waste due to its large yield,high moisture content,residual antibiotics,and ARGs.It was forbidden to directly use in animal feed or field fertilizer.Therefore,AMDs has brought enormous production cost and environmental pressure to antibiotic manufacturers.Thus,a comprehensive understanding of the mechanism of antibiotic transformation,and further research on high-efficiency strategies for deactivating antibiotics and eliminating ARGs have become a pressing task.Solid-state fermentation technology is a reasonable way to resource utilization wastes.However,due to the complex raw materials,a wide variety of microorganisms,and extensive process control,an efficient and low-carbon production process has not been formed.In this study,integrated meta-omics technology was used to comprehensively understand the solid-state fermentation process of two AMDs and the removal mechanism of antibiotics and ARGs during the fermentation process,and provide a theoretical and technical basis for the harmlessness and greening of AMDs.The main achievements were listed as follows:1.Successfully realized the solid-state fermentation of spectinomycin mycelial dregs,and using integrated meta-omics to analyze the dynamic removal mechanism of antibiotic and antibiotic resistance genes.The solid-state fermentation of spectinomycin mycelial dregs with different moisture content under different storage time were carried out by using the methods of integrated meta-omics and qPCR to explore the dynamic removal mechanism of spectinomycin,ARGs,and functional microbial in the fermentation.The results showed that the removal efficiency of antibiotic in the fermentation of high moisture spectinomycin mycelial dregs reached up to 98%.The high abundance of aadA1(nucleotidyltransferase)gene encoded by Streptomyces,Lactobacillus,and Pseudomonas was associated with the efficient degradation of spectinomycin,and the inactivating enzymes secreted by degradative bacteria were identified.Furthermore,the dominant microbiota was impacted by moisture content significantly under high temperature environments.In the fermentation of low moisture spectinomycin mycelial dregs,Saccharopolyspora was the dominant microbiota which secreted S8 endopeptidase,M14,M15,S10,S13 carboxypeptidases,M1,M28,S15 aminopeptidases,and antioxidant enzymes,while in the fermentation of high moisture spectinomycin mycelial dregs,Bacillus and Cerasibacillus were dominant genera which mainly secreted S8 endopeptidase and antioxidant enzymes.The abundance of ARGs and MGEs decreased significantly at thermophilic phase,with maximum drops of 93.7%and 99.9%,respectively.Maintaining moisture content below 30%at the end phase could prevent the transmission of ARGs effectively.2.Effective compounding of raw materials and optimized process control could effectively remove the abundance of related antibiotic resistant bacteria and mobile transfer elements(Intl1)in the fermentation of tylosin mycelial dregs.The dynamics of tylosin residues,ARGs,microbial communities,and functions during the solid-state fermentation of tylosin mycelial dregs and three different bulking agents(corncob,wheat straw and rice husk)were tracked by integrated meta-omics,qPCR and HPLC methods.The results showed that the residual tylosin rapidly decreased,and the minimum detection limit was observed on day 14.The tylosin might have been rapidly removed through de-mycarose reaction of Saccharomonospora(GH3),esterase hydrolysis of Saccharomonospora(C7MYQ7),and catalase-peroxidase oxidation of Bacillus(A0A077JB13).The correlation between these bacteria and tylosin was further verified by network analysis.Correlation analysis showed that moisture content and MGEs were vital to control the rebound of ARGs.The removal efficiency of antibiotic resistant bacteria(Streptomyces,Pseudomonas,norank_f_Sphingobacteriaceae,and Paenalcaligenes)and Intl1(98.8%)in the fermentation treatment TC21 with corncob as the bulking agent was significantly higher than that in the other three fermentation treatments(88.3%).3.The mixed fermentation of spectinomycin and tylosin mycelial dregs was tracked by integrated meta-omics technology,which indicated that the mixed fermentation of these two kinds of different properties was feasible.Spectinomycin mycelial dregs and tylosin mycelial dregs were two kinds of different properties of mycelial dregs.The results showed that the two kinds of antibiotics did not affect the fermentation process,and the two kinds of antibiotics can be effectively removed.The analysis of microbial community diversity showed that there was no significant difference in the diversity of microbial communities after the mixed fermentation and separate fermentation of AMDs.The analysis at the microbial phylum level found that the succession of the microbial communities in the first 5 days of mixed fermentation was similar to that of the tylosin mycelial dregs fermentation,which may be related to the composition of the raw materials.After the 14th day of fermentation,the microbial communities of the three treatments tended to be consistent.Bacillus was identified as the dominant genus in the middle and late fermentation of the three groups at the genus level.Meanwhile,there was no positive correlation between Bacillus,ARGs,and MGEs,suggesting that Bacillus would not cause the increase of resistance during fermentation.4.Successfully realized the industrial application of hundreds tons of antibiotic mycelial dregs for large-scale production fermentation by optimizing the process and using the integrated meta-omics study.The pilot fermentation and large-scale fermentation were carried out using integrated meta-omics technology to compare the removal of antibiotics,the dynamics of ARGs and bacterial community composition under the same raw material ratio and different scale fermentations.The results showed that the tylosin and spectinomycin residues in both fermentation scales could be effectively reduced over time.However,there were differences in the remove of ARGs between the two groups under different fermentation scales.Compared with the pilot fermentation,the varieties and abundance of ARGs detected in large-scale production fermentation were lower.Through the analysis of bacterial community diversity,it is found that there were significant differences between the pilot and large-scale production fermentation processes,and the microbial communities in large-scale production fermentation could reach stability in a shorter time.On the 10th day of large-scale production fermentation,95%of Firmicutes became the dominant bacteria.The co-occurrence network of ARGs and bacterial communities showed that the dominant Bacillaceae in the late stages of large-scale production,did not increase the abundance of ARGs.And,it might play an important role in inhibiting the horizontal transfer of ARGs.5.The genomes of two dominant microbial strains in antibiotic mycelial dregs fermentation were analyzed,the mode of cooperation between flora in the efficient degradation of protein was preliminarily determined.Two dominant strains,Bacillus thermoamylovorans and Saccharopolyspora rectivirgula,which were representative and produce proteases in two dominant genera of microbial communities during AMDs were selected.The types and quantities of proteases and lignocellulose-degrading enzymes of the two strains were analyzed by using the genomic data in the database,and the specific recognition patterns of key proteases on the proteome were also analyzed.The results showed that the analysis of genomic data was consistent with the proteome data,and the proteases of Bacillus thermoamylovorans and Saccharopolyspora rectivirgula were complementary.Bacillus thermoamylovorans mainly secreted S8 family endopeptidases initially hydrolyze complex substrate proteins into peptides;Saccharopolyspora rectivirgula mainly secreted a large amount of M1,M28,S15 family aminopeptidases and M14,M15,S10 and S13 family carboxypeptidases,which can further degrade the peptide into amino acids under the action of these peptidases.6.Through solid-state fermentation technology to realize the green and efficient transformation of antibiotic mycelial dregs,and the fermentation products met the relevant national standards of organic fertilizer.In this study,by adding bulking agents,the separate fermentation and mixed fermentation of spectinomycin and tylosin mycelial dregs were successfully fermented in solid state.On this basis,the mixed fermentations were fermented on a pilot scale of 6 tons and a large scale of 100 tons.In the end,the residues of antibiotics and ARGs were eliminated effectively from the fermentation products of 100 tons of industrial production.Tylosin was not detected in large-scale fermentation products,and spectinomycin was trace,which passed the residues determination.Other physical and chemical indexes are in line with the relevant national standards of organic fertilizer(NY525-2021),realizing the green and efficient transformation of AMDs.