Metagenomic analysis revealing the metabolic role of microbial communities in the free amino acid biosynthesis of Monascus rice vinegar during fermentation

Free amino acid(FAA)is the important component of vinegar that infl uences quality perception and consumer acceptance.FAA is one of the major metabolites produced by microorganisms;however,the microbial metabolic network on FAA biosynthesis remains unclear.Through metagenomic analysis,this work aimed to elucidate the roles of microbes in FAA biosynthesis during Monascus rice vinegar fermentation.Taxonomic profiles from functional analyses showed 14 dominant genera with high contributions to the metabolism pathways.The metabolic network for FAA biosynthesis was then constructed,and the microbial distribution in different metabolic pathways was illuminated.The results revealed that 5 functional genera were closely involved in FAA biosynthesis.This study illuminated the metabolic roles of microorganisms in FAA biosynthesis and provided crucial insights into the functional attributes of microbiota in vinegar fermentation.

CloudLCA: finding the lowest common ancestor in metagenome analysis using cloud computing

Estimating taxonomic content constitutes a key problem in metagenomic sequencing data analysis.However,extracting such content from high-throughput data of next-generation sequencing is very time-consuming with the currently available software.Here,we present CloudLCA,a parallel LCA algorithm that significantly improves the efficiency of determining taxonomic composition in metagenomic data analysis.Results show that CloudLCA(1)has a running time nearly linear with the increase of dataset magnitude,(2)displays linear speedup as the number of processors grows,especially for large datasets,and(3)reaches a speed of nearly 215 million reads each minute on a cluster with ten thin nodes.In comparison with MEGAN,a well-known metagenome analyzer,the speed of CloudLCA is up to 5 more times faster,and its peak memory usage is approximately 18.5%that of MEGAN,running on a fat node.CloudLCA can be run on one multiprocessor node or a cluster.It is expected to be part of MEGAN to accelerate analyzing reads,with the same output generated as MEGAN,which can be import into MEGAN in a direct way to finish the following analysis.Moreover,CloudLCA is a universal solution for finding the lowest common ancestor,and it can be applied in other fields requiring an LCA algorithm.

Lacticaseibacillus rhamnosus Probio-M9 may be vertically transmitted from mother to infant during lactation based on faeces metagenomics

Probiotics exert beneficial effects on the host.This study aimed to investigate whether maternally ingested Lacticaseibacillus rhamnosus Probio-M9 during pregnancy could access and colonize the infant gut.This study recruited one pregnant woman,who ingested Probio-M9 daily from 35 weeks of gestation to delivery.Feces of the mother-infant pair were regularly collected from one month before delivery to 6 months of infant's age for metagenomic sequencing.Probio-M9 genomes were mappable to all infant fecal samples,suggesting the ingested probiotics could be vertically transmitted from mother to infant.Infant-or mother-specific differential metabolic pathways were found between the maternal and infant's gut microbiome,implicating apparent differences in the intestinal metagenomic potential/function between the mother and the infant.In conclusion,maternal ingestion of Probio-M9 during the final weeks of gestation could deliver to the infant gut.The findings provided novel insights into shaping infant's gut microbiota.

Improvements on environmental DNA extraction and purification procedures for matagenomic analysis

Our previously described environmental DNA extraction method has been widely used in environmental microbial community analysis. However, residual humic substances may remain with obtained environmental DNA, which interferes downstream molecular analyses. To remedy this situation, two DNA extraction buffers （PIPES and Tris-HCl） and four purification strategies including our new modified low melting point gel purification method and three commercial kits from QIAEX, Omega and Promega were evaluated with diverse soil samples. The PIPES buffer （pH 6.5） is found to be more effective for removing the humic substances, but it leads to lower DNA yield and causes more severe DNA shearing than using the Tris-HC1 buffer （pH 8.0）. Gel purification and the Promega purification kit achieve much higher DNA recoveries than QIAEX or Omega kit, and higher purity of DNA is obtained by gel purification than by the Promega kit with both DNA extraction buffers mentioned above. Considering all results together, two alternative methods for DNA extraction and purification are proposed： one uses Tris-HCl buffer extraction and gel purification as the primary approach when the amount of soil or biomass is not a major concern, and the other uses PIPES buffer extraction and the Promega kit purification when severe DNA shearing and/or limited biomass occurs. Purified DNA samples by both methods are amenable for use as templates for whole community genome amplifications and PCR amplifications of bacterial 16S rRNA genes. It is demonstrated that these two alternative methods could be applied to a wide variety of environmental samples.

Metagenomic analysis to determine the characteristics of antibiotic resistance genes in typical antibiotic-contaminated sediments

Comprehensive studies of the effects of various physical and chemical variables(including heavymetals),antibiotics,and microorganisms in the environment on antibiotic resistance genes are rare.We collected sediment samples from the Shatian Lake aquaculture area and surrounding lakes and rivers located in Shanghai,China.The spatial distribution of sediment ARGs was assessed by metagenomic analysis that revealed 26 ARG types(510 subtypes),dominated by Multidrug,β-lactam,Aminoglycoside,Glycopeptides,Fluoroquinolone,and Tetracyline.Redundancy discriminant analysis indicated that antibiotics(SAs and MLs)in the aqueous environment and sediment along with water TN and TP were the key variables affecting the abundance distribution of total ARGs.However,the main environmental drivers and key influences differed among the different ARGs.For total ARGs,the environmental subtypes affecting their structural composition and distribution characteristics were mainly antibiotic residues.Procrustes analysis showed a significant correlation between ARGs and microbial communities in the sediment in the survey area.Network analysis revealed that most of the target ARGs were significantly and positively correlated with microorganisms,and a small number of ARGs(e.g.,rpoB,mdtC,and efpA)were highly significantly and positively correlated with microorganisms(e.g.,Knoellia,Tetrasphaera,and Gem-matirosa).Potential hosts for the major ARGs included Actinobacteria,Proteobacteria,and Gemmatimonadetes.Our study provides new insight and a comprehensive assessment of the distribution and abundance of ARGs and the drivers of ARG occurrence and transmission.

Metagenomic analysis on resistance genes in water and microplastics from a mariculture system

Microplastics existing widely in different matrices have been regarded as a reservoir for emerging contaminants.Mariculture systems have been observed to host microplastics and antibiotic resistance genes(ARGs).However,more information on proliferation of ARGs and metal resistance genes(MRGs)in mariculture system at the presence of microplastics is needed.This study used metagenomic analysis to investigate the distribution of ARGs and MRGs in water and microplastics of a typical mariculture pond.Total 18 types including 174 subtypes of ARGs were detected with the total relative abundances of 1.22/1.25 copies per 16S rRNA copy for microplastics/water.Chloramphenicol resistance genes were the dominant ARGs with the abundance of 0.35/0.42 copies per 16S rRNA copy for microplastics/water.Intergron intI1 was dominant gene among 6 detected mobile genetic elements(MGEs)with the abundance of 75.46/68.70 copies per 16S rRNA copy for water/microplastics.Total 9 types including 46 subtypes of MRGs were detected with total abundance of 5.02×10^(2)/6.39×10^(2)copies per 16S rRNA copy for water/microplastics while genes resistant to copper and iron served as the dominant MRGs.Proteobacteria,Bacteroidetes,and Actinobacteria accounted for 84.2%/89.5%of total microbial community in microplastics/water.ARGs with relatively high abundance were significantly positively related to major genera,MGEs,and MRGs.Microplastics in mariculture system could enrich most of MRGs and some ARGs to serve as potential reservoir for these pollutants.The findings of this study will provide important information on resistance gene pollution at presence of microplastics in the mariculture system for further proposing suitable strategy of environmental management.

Mechanisms of allicin exposure for the sludge fermentation enhancement:Focusing on the fermentation processes and microbial metabolic traits

As a frequently used product with antimicrobial activity,consumed allicin might be dis-charged and concentrated in waste-activated sludge(WAS).However,the influence of al-licin(as an exogenous pollutant)on WAS fermentation has not been clearly revealed.This study aimed to disclose the impacts of allicin on volatile fatty acid(VFA)generation dur-ing WAS fermentation.The results showed that the appropriate presence of allicin(10 mg/g TSS)significantly enhanced the VFA yield(1894 versus 575 mg COD/L in the control)with increased acetate proportion(24.3%).Further exploration found that allicin promoted WAS solubilization,hydrolysis and acidification simultaneously.Metagenomic analysis revealed that the key genes involved in extracellular hydrolysis metabolism(i.e.,CAZymes),mem-brane transport(i.e.,gtsA and ytfT),substrate metabolism(i.e.,yhdR and pfkC)and fatty acid synthesis(i.e.,accA and accD)were all highly expressed.Allicin also induced the bacteria to produce more signalling molecules and regulate cellular functions,thereby enhancing the microbial adaptive and regulatory capacity to the unfavourable environment.Moreover,the variations in fermentative microbes and their contributions to the upregulation of func-tional genes(i.e.,ytfR,gltL,INV,iolD and pflD)for VFA generation were disclosed.Overall,the simultaneous stimulation of functional microbial abundances and metabolic activities contributed to VFA production in allicin-conditioned reactors.

Anaerobic digestion of sludge by different pretreatments:Changes of amino acids and microbial community

Many studies have investigated the effects of different pretreatments on the performance of anaerobic digestion of sludge.However,the detailed changes of dissolved organic nitrogen,particularly the release behavior of proteins and the byproducts of protein hydrolysis-amino acids,are rarely known during anaerobic digestion of sludge by different pretreatments.Here we quantified the changes of three types of proteins and 17 types of amino acids in sludge samples solubilized by ultrasonic,thermal,and acid/alkaline pretreatments and their transformation during anaerobic digestion of sludge.Tryptophan protein,aromatic protein I,aromatic protein II,and cysteine were identified as the key dissolved organic nitrogen responsible for methane production during anaerobic digestion of sludge,regardless of the different pretreatment methods.Different from the depletion of other amino acids,cysteine was resistant to degradation after an incubation period of 30 days in all sludge samples.Meanwhile,the“cysteine and methionine metabolism(K00270)”was absent in all sludge samples by identifying 6755 Kyoto Encyclopedia of Genes and Genomes assignments of genes hits.Cysteine contributed to the generation of methane and the degradation of acetic,propionic,and n-butyric acids through decreasing oxidation-reduction potential and enhancing biomass activity.This study provided an alternative strategy to enhance anaerobic digestion of sludge through in situ production of cysteine.

Insights from metagenomic,metatranscriptomic,and molecular ecological network analyses into the effects of chromium nanoparticles on activated sludge system

The objectives of this study were to investigate the influence of chromium nanoparticles(Cr NPs)on the nitrogen and phosphorus removal performance and the bacterial structures of an activated sludge(AS)system.Also,we through molecular ecological networks(MENs)discussed the bacterial interactions.At last we researched the change of the functional genes and their expression patterns related to nitrogen and phosphorus removaT in an AS system.The results showed that long-term exposure to 1 mg/L Cr NPs significantly promoted the denitrifying process and phosphorus removal in the AS system.The relative abundance of denitrifying and phosphorus removal microorganisms,such as Denitratisoma,Thauera,Dechloromonas,and Defluviicoccus,increased significantly.Candidatus Accumulibacter,well-known as polyphosphate-accumulating organisms(PAOs),increased significantly;the relative abundance of Candidatus Competibacter,known as glycogen-accumulating organisms(GAOs),decreased significantly.Furthermore,metagenomic and metatranscriptomic analysis revealed that most of the genera related to denitrifying and phosphorus removal had greatly increased,according to the quantities of denitrifying and phosphorus genes,and the corresponding transcription likewise greatly increased.Lastly,MENs analysis showed that although the overall network became smaller and looser in the presence of Cr NPs,the microbial connections among members related to nitrogen and phosphorus removal were enhanced.The abundance increases of denitrifiers and PAOs,and their increased transcription of functional genes,together with the enhanced interactions may be associated with the promotion of the denitrifying process and phosphorus removal.

Unveiling degradation mechanism of PAHs by a Sphingobium strain from a microbial consortium

Polycyclic aromatic hydrocarbons(PAHs)are a class of persistent pollutants with adverse biological effects and pose a serious threat to ecological environments and human health.The previously isolated phenanthrene‐degrading bacterial consortium(PDMC)consists of the genera Sphingobium and Pseudomonas and can degrade a wide range of PAHs.To identify the degradation mechanism of PAHs in the consortium PDMC,metagenomic binning was conducted and a Sphingomonadales assembly genome with 100%completeness was obtained.Additionally,Sphingobium sp.SHPJ‐2,an efficient degrader of PAHs,was successfully isolated from the consortium PDMC.Strain SHPJ‐2 has powerful degrading abilities and various degradation pathways of high‐molecular‐weight PAHs,including fluoranthene,pyrene,benzo[a]anthracene,and chrysene.Two ring‐hydroxylating dioxygenases,five cytochrome P450s,and a pair of electron transfer chains associated with PAH degradation in strain SHPJ‐2,which share 83.0%–99.0%similarity with their corresponding homologous proteins,were identified by a combination of Sphingomonadales assembly genome annotation,reverse‐transcription quantitative polymerase chain reaction and heterologous expression.Furthermore,when coexpressed in Escherichia coli BL21(DE3)with the appropriate electron transfer chain,PhnA1B1 could effectively degrade chrysene and benzo[a]anthracene,while PhnA2B2 degrade fluoranthene.Altogether,these results provide a comprehensive assessment of strain SHPJ‐2 and contribute to a better understanding of the molecular mechanism responsible for the PAH degradation.