Microbiologic surveys for Baijiu fermentation are affected by experimental design.

The traditional Chinese alcoholic beverage Baijiu is produced by spontaneous fermentation of grains under anaerobic conditions. While numerous studies have used metagenomic technology to investigate the microbiome of Baijiu brewing, the microbial succession mechanism of Baijiu brewing has not been fully clarified, and metagenomic strategies for microecology surveys have not been comprehensively evaluated. Using the fermentation process of strong-flavor Baijiu as a model, we compared the data for bacterial communities based on short read 16S rRNA variable regions, V3-V4, and full-length 16S regions, V1-V9, to whole metagenomic shotgun sequencing (WMS) to measure the effect of technology selection on phylogenetic and functional profiles. The results showed differences in bacterial compositions and their relation to volatiles and physicochemical variables between sequencing methods. Furthermore, the percentage of V3-V4 sequences assigned to species level was higher than the percentage of V1-V9 sequences according to SILVA taxonomy, but lower according to NCBI taxonomy (P < 0.05). In both SILVA and NCBI taxonomies, the relative abundances of bacterial communities at both the genus and family levels were more positively correlated with WMS data in the V3-V4 dataset than in the V1-V9 dataset. The WMS identified changes in abundances of multiple metabolic pathways during fermentation (P < 0.05), including "starch and sucrose metabolism," "galactose metabolism," and "fatty acid biosynthesis." Although functional predictions derived from 16S data show similar patterns to WMS, most metabolic pathway changes are uncorrelated (P > 0.05). This study provided new technical and biological insights into Baijiu production that may assist in selection of methodologies for studies of fermentation systems.

Variation in microbiological heterogeneity in Chinese strong-flavor Baijiu fermentation for four representative varieties of sorghum.

The quality and composition of strong-flavor Baijiu (SFB), a type of Chinese liquor, depends on the variety of sorghum used in fermentation. However, comprehensive in situ studies measuring the effects sorghum varieties on the fermentation are lacking and the underlying microbial mechanisms remains poorly understood. We studied the in situ fermentation of SFB by using metagenomic, metaproteomic, and metabolomic techniques across four sorghum varieties. Sensory characteristics were best for SFB made from glutinous variety Luzhouhong, followed by glutinous hybrid Jinnuoliang and Jinuoliang, and those made with non-glutinous Dongzajiao. In agreement with sensory evaluations, the volatile composition of SFB samples differed between sorghum varieties (P < 0.05). Fermentation of different sorghum varieties varied in microbial diversity, structure, volatile compounds, and physicochemical properties (pH, temperature, starch, reducing sugar, and moisture) (P < 0.05), with most changes occurring within the first 21 days. Additionally, the microbial interactions and their relationship with volatiles, as well as the physicochemical factors that govern microbial succession, differed between varieties of sorghum. The number of physicochemical factors affecting bacterial communities outweighed those affecting fungal communities, suggesting that bacteria were less resilient to the brewing conditions. This correlates with the finding that bacteria play a major role in the differences in microbial communities and metabolic functions during fermentation with the different varieties of sorghum. Metagenomic function analysis revealed differences in amino acid and carbohydrate metabolism between sorghum varieties throughout most of the brewing process. Metaproteomics further indicated most differential proteins were found in these two pathways, related to differences in volatiles between sorghum varieties of Baijiu and originating from Lactobacillus. These results provide insight into the microbial principles underlying Baijiu production and can be used to improve the quality of Baijiu by selecting the appropriate raw materials and optimizing fermentation parameters.

Bioremediation of degraded pit mud by indigenous microbes for Baijiu production.

Microbes in pit mud play key roles in fermentation cellars for Chinese strong-flavor Baijiu (SFB) production. Pit mud, however, is frequently degraded during production, compromising the quality of the end product. In this study, a bioremediation method was used to restore degraded pit mud (DPM) using indigenous microbes derived from SFB production. Metabolomics and metagenomics were used to determine the dynamics of prokaryotes during DPM restoration and their link to SFB production. The composition of flavor compounds in SFB changed (P = 0.0001) before and after restoration of DPM. Consistent with the improved sensory quality, the ethyl caproate/ethyl lactate ratio, an SFB quality measure, increased after restoration (P < 0.001). The concentrations of humus, NH4+, available phosphorus, and available potassium in DPM increased during the restoration process (P < 0.05), which is consistent with high-quality pit mud. The relative abundance of microbes that are beneficial to SFB fermentation, such as Caproiciproducens, a bacterium that produces caproic acid, increased during the restoration process. Furthermore, a total of 18 metabolic pathways were enriched (P < 0.05) from DPM before and after restoration. This includes butanoate metabolism and pyruvate metabolism, which are related to the synthesis of key flavor esters in SFB.

A sandwich temperature control membrane inlet mass spectrometer for dissolved gases and volatile organic compounds in aqueous solution.

A sandwich temperature control membrane inlet system based on a miniature mass spectrometer is presented that demonstrates improved analytical performance for the measurement of dissolved gases and volatile organic compounds (VOCs) in aqueous solution. Aqueous solution is directly brought to the monolayer flat membrane interface at a constant flow rate. A heating resistor and a thermocouple are fixed on the side of the membrane and aqueous solution respectively. This new strategy allows for a temperature compensation method, affording an improvement of sensitivity and a reduction of response time compared with the conventional heating solution temperature control strategy. Furthermore, a static heating mode is applied to effectively remove the memory effect. Automatic sampling and measurement are achieved by using the membrane inlet system with silicone sheeting of 50 µm thickness. The vacuum is below 3 × 10-5 Torr, which can make the instrument work normally. A good linear response is observed for benzene in the range of 0.1 ppm-10 ppm and the detection limit is 50 ppb. The analytical capacity of this system is demonstrated by the on-line analysis of VOCs in aqueous solution, in which the dominant ions are detected rapidly. The results indicate that the sandwich temperature control membrane inlet mass spectrometer (STC-MIMS) has a potential application for on-line analyzing organic pollution in aquatic environments.

Structural and Functional Changes in Prokaryotic Communities in Artificial Pit Mud during Chinese Baijiu Production.

The Chinese alcoholic beverage strong-flavor baijiu (SFB) gets its characteristic flavor during fermentation in cellars lined with pit mud. Microbes in the pit mud produce key precursors of flavor esters. The maturation time of natural pit mud of over 20 years has promoted attempts to produce artificial pit mud (APM) with a shorter maturation time. However, knowledge about the molecular basis of APM microbial dynamics and associated functional variation during SFB brewing is limited, and the role of this variability in high-quality SFB production remains poorly understood. We studied APM maturation in new cellars until the fourth brewing batch using 16S rRNA gene amplicon sequencing, quantitative PCR, metaproteomics, and metabolomics techniques. A total of 36 prokaryotic classes and 195 genera were detected. Bacilli and Clostridia dominated consistently, and the relative abundance of Bacilli decreased along with the APM maturation. Even though both amplicon sequencing and quantitative PCR showed increased abundance of Clostridia, the levels of most of the Clostridium proteins were similar in both the first- and fourth-batch APM samples. Six genera correlated with eight or more major flavor compounds in SFB samples. Functional prediction suggested that the prokaryotic communities in the fourth-batch APM samples were actively engaged in organic acid metabolism, and the detected higher concentrations of proteins and metabolites in the corresponding metabolic pathways supported the prediction. This multi-omics approach captured changes in the abundances of specific microbial species, proteins, and metabolites during APM maturation, which are of great significance for the optimization of APM culture technique.IMPORTANCE Strong-flavor baijiu (SFB) accounts for more than 70% of all Chinese liquor production. In the Chinese baijiu brewing industry, artificial pit mud (APM) has been widely used since the 1960s to construct fermentation cellars for production of high-quality SFB. To gain insights at the systems level into the mechanisms driving APM prokaryotic taxonomic and functional dynamics and into how this variation is connected with high-quality SFB production, we performed the first combined metagenomic, metaproteomic, and metabolomic analyses of this brewing microecosystem. Together, the multi-omics approach enabled us to develop a more complete picture of the changing metabolic processes occurring in APM microbial communities during high-quality SFB production, which will be helpful for further optimization of APM culture technique and improvement of SFB quality.

Contrasting bacterial community structure in artificial pit mud-starter cultures of different qualities: a complex biological mixture for Chinese strong-flavor Baijiu production.

The complex starter culture for artificial pit mud (APMSC) hosts a wide variety of microbial communities that play a crucial role in Chinese strong-flavor Baijiu production. Based on its organoleptic properties, the quality of APMSC can be divided into normal and inferior quality grades. However, the relationship between the APMSC microbial community and APMSC quality is poorly understood. In this study, the bacterial community structure in normal and inferior APMSC derived from two different production batches was analyzed using denaturing gradient gel electrophoresis and Illumina MiSeq sequencing. Highly similar patterns of bacterial diversity and community structure were observed in the APMSC samples of the same quality, and a significant higher bacterial species diversity (Shannon index and Chao1) was detected in the normal compared to the inferior APMSC samples. Fifteen genera were detected in the APMSC samples, and seven (Caproiciproducens, Clostridium, Lactobacillus, Bacillus, Pediococcus, Rummeliibacillus, and Sporolactobacillus) were dominant, accounting for 92.12-99.89% of total abundance. Furthermore, the bacterial communities in the normal and inferior APMSC had significantly different structure and function. The normal APMSC was characterized by abundant Caproiciproducens and Clostridium and high caproic and butyric acid contents. In contrast, the inferior APMSC was overrepresented by Lactobacillus and Bacillus and lactic and acetic acids. This study may help clarify the key microbes sustaining APMSC ecosystem stability and functionality, and guide future improvements in APMSC production.

Actinobacteria associated with Glycyrrhiza inflata Bat. are diverse and have plant growth promoting and antimicrobial activity.

Many of the plant associated microbes may directly and indirectly contribute to plant growth and stress resistance. Our aim was to assess the plant growth-promoting and antimicrobial activities of actinobacteria isolated from Glycyrrhiza inflata Bat. plants to find strains that could be applied in agricultural industry, for example in reclaiming saline soils. We isolated 36 and 52 strains that showed morphological characteristics of actinobacteria from one year old and three year old G. inflata plants, respectively. Based on 16S rRNA gene sequence analysis, the strains represented ten actinobacterial genera. Most of the strains had plant growth promoting characteristics in vitro, tolerated 200 mM NaCl and inhibited the growth of at least one indicator organism. The eight selected Streptomyces strains increased the germination rate of G. inflata seeds under salt stress. In addition, the four best seed germination promoters promoted the growth of G. inflata in vivo. The best promoters of G. inflata growth, strains SCAU5283 and SCAU5215, inhibited a wide range of indicator organisms, and may thus be considered as promising candidates to be applied in inoculating G. inflata.

Actinobacteria associated with Chinaberry tree are diverse and show antimicrobial activity.

Many actinobacteria produce secondary metabolites that include antimicrobial compounds. Since most of the actinobacteria cannot be cultivated, their antimicrobial potential awaits to be revealed. We hypothesized that the actinobacterial endophyte communities inside Melia toosendan (Chinaberry) tree are diverse, include strains with antimicrobial activity, and that antimicrobial activity can be detected using a cultivation independent approach and co-occurrence analysis. We isolated and identified actinobacteria from Chinaberry, tested their antimicrobial activities, and characterized the communities using amplicon sequencing and denaturing gradient gel electrophoresis as cultivation independent methods. Most of the isolates were identified as Streptomyces spp., whereas based on amplicon sequencing the most abundant OTU was assigned to Rhodococcus, and Tomitella was the most diverse genus. Out of the 135 isolates, 113 inhibited the growth of at least one indicator organism. Six out of the 7577 operational taxonomic units (OTUs) matched 46 cultivated isolates. Only three OTUs, Streptomyces OTU4, OTU11, and OTU26, and their corresponding isolate groups were available for comparing co-occurrences and antimicrobial activity. Streptomyces OTU4 correlated negatively with a high number of OTUs, and the isolates corresponding to Streptomyces OTU4 had high antimicrobial activity. However, for the other two OTUs and their corresponding isolate groups there was no clear relation between the numbers of negative correlations and antimicrobial activity. Thus, the applicability of co-occurrence analysis in detecting antimicrobially active actinobacteria could not be proven.

Deep sequencing reveals high bacterial diversity and phylogenetic novelty in pit mud from Luzhou Laojiao cellars for Chinese strong-flavor Baijiu.

The pit mud (PM) in fermentation cellar is a complex ecosystem that hosts diverse microbial communities that contribute to the production of Chinese strong-flavor Baijiu (CSFB). However, the microbial ecology of PM, particularly the extent of their phylogenetic novelty remains poorly understood. Here we conducted Illumina MiSeq sequencing to explore the diversity and novelty patterns of PM bacterial communities from Luzhou Laojiao cellars in use for 40 and 400years. High diversity indices were found in the PM with 16 phyla and 105 genera. Interestingly, the compositions of dominant genera of the PM were significantly different than that reported previously for PM sampled from other geographic sites, suggesting greater microbial diversity of PM. The dominant genus of Caproiciproducens, a caproic acid-producing bacterium, is the first reported for Chinese Baijiu production. Our results demonstrate that the PM hosts a large number of novel taxa, with 26% of the total OTUs (operational taxonomic units) distant to cultured counterparts. The class Clostridia within Firmicutes presented the highest proportion of novel OTUs. Most novel OTUs were initially isolated from diverse environments, the most abundant of which came from Chinese Baijiu brewing ecosystems, highlighting the huge culturing gap within the PM, but at the same time suggesting the importance of these OTUs in CSFB production. The data presented in this study significantly increases the number of bacteria known to be associated with CSFB production and should help guide the future exploration of microbial resources for biotechnological applications.

Determination of the fungal community of pit mud in fermentation cellars for Chinese strong-flavor liquor, using DGGE and Illumina MiSeq sequencing.

Chinese strong-flavor liquor (CSFL) is fermented in cellars lined with pit mud (PM). This PM, specific fermented clay, contains microbes that play important roles in CSFL production. However, there is limited information about fungal community structure and cellar-age-related changes in PM. In this study, PM samples were removed from the cellars used for 5 and 100years and characterized using denaturing gradient gel electrophoresis (DGGE) and Illumina MiSeq sequencing. Both methods revealed there were no significant differences in fungal species diversity (Shannon index, Chao1, and observed species) between the 5- and 100-year PM samples (p>0.05), but the communities were more stable in the 100-year PM samples (UPGMA). Illumina MiSeq sequencing allowed identification of 111 fungal genera belonging to 4 phyla (Ascomycota, Zygomycota, Basidiomycota, and Chytridiomycota) in the PM samples, with the predominant phylum being Ascomycota. The results also indicated that the compositions of dominant genera in the PM samples were significantly changed during long-term CSFL fermentation. There were relatively more Rhizopus, Phoma, and Trichosporon in the 5-year PM samples, and Aspergillus and Candida were most highly represented in the 100-year PM samples (p<0.05). Of these, Candida increased its relative abundance significantly in the 100-year samples (p<0.05). Overall, the results provide novel insights into the fungal community associated with CSFL production, and may suggest why fermentation in a cellar with PM that has been in usage for a longer time allows better quality CSFL production.

Isolation and antimicrobial activities of actinobacteria closely associated with liquorice plants Glycyrrhiza glabra L. and Glycyrrhiza inflate BAT. in Xinjiang, China.

A total of 218 actinobacteria strains were isolated from wild perennial liquorice plants Glycyrrhiza glabra L. and Glycyrrhiza. inflate BAT. Based on morphological characteristics, 45 and 32 strains from G. inflate and G. glabra, respectively, were selected for further analyses. According to 16S rRNA sequence analysis, most of the strains belonged to genus Streptomyces and a few strains represented the rare actinobacteria Micromonospora, Rhodococcus and Tsukamurella. A total of 39 strains from G. inflate and 27 strains from G. glabra showed antimicrobial activity against at least one indicator organism. The range of the antimicrobial activity of the strains isolated from G. glabra and G. inflate was similar. A total of 34 strains from G. inflate and 29 strains from G. glabra carried at least one of the genes encoding polyketide synthases, non-ribosomal peptide synthetase and FADH2-dependent halogenase. In the type II polyketide synthase KSα gene phylogenetic analysis, the strains were divided into two major clades: one included known spore pigment production-linked KSα sequences and other sequences were linked to the production of different types of aromatic polyketide antibiotics. Based on the antimicrobial range, the isolates that carried different KSα types were not separated from each other or from the isolates that did not carry KSα. The incongruent phylogenies of 16S rRNA and KSα genes indicated that the KSα genes were possibly horizontally transferred. In all, the liquorice plants were a rich source of biocontrol agents that may produce novel bioactive compounds.

Maize rhizosphere in Sichuan, China, hosts plant growth promoting Burkholderia cepacia with phosphate solubilizing and antifungal abilities.

Plant growth-promoting rhizobacteria promote plant growth by direct and indirect mechanisms. We isolated twelve bacterial strains showing different degrees of phosphate solubilizing activity from maize rhizosphere. Four isolates solubilized over 300 µg mL⁻¹ phosphate from insoluble Ca3(PO4)2, with isolate SCAUK0330 solubilizing over 450 µg mL⁻¹. Based on the 16S rRNA gene sequence analysis SCAUK0330 was identified as Burkholderia cepacia. SCAUK0330 grew at 10-40 °C and pH 4.0-10.0, tolerated up to 5% NaCl, and showed antagonism against nine pathogenic fungi. SCAUK0330 promoted the growth of both healthy and Helminthosporium maydis infected maize plants, indicating that the isolate was a good candidate to be applied as a biofertilizer and a biocontrol agent under a wide range of environmental conditions.The expression of a single SCAUK0330 gene gave E. coli a pH decrease linked ability to solubilize phosphate. The nucleotide and the deduced amino acid sequences of this phosphate solubilization linked gene showed high degree of sequence identity with B. cepacia E37gabY. The production of gluconic acid is considered as the principle mechanism for phosphate solubilization. In agreement with the proposed periplasmic location of the gluconic acid production, the predicted signal peptide and transmembrane regions implied that GabY is membrane bound.