Microbial biogeography of pit mud from an artificial brewing ecosystem on a large time scale: all roads lead to Rome.

IMPORTANCE: Baijiu is a typical example of how humans employ microorganisms to convert grains into new flavors. Mud cellars are used as the fermentation vessel for strong-flavor Baijiu (SFB) to complete the decomposition process of grains. The typical flavor of SFB is mainly attributed to the metabolites of the pit mud microbiome. China has a large number of SFB-producing regions. Previous research revealed the temporal profiles of the pit mud microbiome in different geographical regions. However, each single independent study rarely yields a thorough understanding of the pit mud ecosystem. Will the pit mud microbial communities in different production regions exhibit similar succession patterns and structures under the impact of the brewing environment? Hence, we conducted research in pit mud microbial biogeography to uncover the impact of specific environment on the microbial community over a long time scale.

Volatile Compound Abundance Correlations Provide a New Insight into Odor Balances in Sauce-Aroma Baijiu.

Sauce-aroma Baijiu (SAB) is one of the most famous Baijius in China; SAB has more than 500 aroma compounds in it. However, the key aroma compound in SAB flavor remains unclear. Volatiles play an important role in SAB aroma and are highly correlated to SAB quality. In the present study, 63 volatile compounds were quantified among 66 SAB samples using gas chromatography with flame ionization detector (GC-FID). The authors analyzed odor contributions and volatile compound correlations in two quality groups of SAB samples. Moreover, an odor activity value (OAV) ratio-based random forest classifier was used to explain the volatile compound relationship differentiations between the two quality groups. Our results proved higher quality SABs had richer aromas and indicated a set of fruity-like ethyl valerate, green- and malt-like isobutyraldehyde and malt-like 3-methylbutyraldehyde and sweet-like furfural, had closer co-abundance correlations in higher quality SABs. These results indicated that the aroma and contributions of volatile compounds in SABs should be analyzed not only with compound odor activity values, but also the correlations between different aroma compounds.

Comparative Genomics Unveils the Habitat Adaptation and Metabolic Profiles of Clostridium in an Artificial Ecosystem for Liquor Production.

Clostridium inhabiting pit mud (PM) is one of the important bacterial populations for synthesizing flavor compounds of Chinese strong-flavor baijiu. The long-term cereal fermentation with sorghum as the main raw material creates an environment rich in starch, ethanol, and organic acids (mainly lactic acid). However, the genetic factors underpinning Clostridium's adaptation to PM remain poorly understood. Here, we performed comparative genomic analysis between 30 pit mud-associated (PMA) and 100 non-pit mud-associated (NPMA) Clostridium strains. Comparison analysis of the enrichment of KEGG pathways between PMA and NPMA Clostridium strains showed two-component system, flagellar assembly, and bacterial chemotaxis pathways related to environmental adaptation were enriched in PMA strains. The number of genes encoding alcohol dehydrogenase and l-lactate dehydrogenase in PMA Clostridium strains was significantly higher than that in NPMA, which is helpful for them to adapt to the ethanol- and lactic acid-rich environment. The analysis of carbohydrate-active enzymes demonstrated that glycoside hydrolases (GHs) was the most abundant family in all Clostridium strains, and genes encoding GH4 and GH13, involved in starch and sucrose metabolism, were enriched in PMA Clostridium. Horizontal gene transfer analysis revealed that multiple genes encoding the enzymes involved in carbohydrate and amino acid metabolism were transferred from Bacillus to Clostridium in pit mud. Most of the PMA Clostridium strains had good potential for butyric acid synthesis from ethanol, lactic acid, and starch. Collectively, this study furthers our understanding of the habitat adaptation and metabolic potential of PMA Clostridium strains. IMPORTANCE Pit mud is a typical artificial ecosystem for Chinese liquor production. Clostridium inhabiting pit mud plays essential roles in the flavor formation of strong-flavor baijiu. The relative abundance of Clostridium increased with pit mud quality, further influencing the quality of baijiu. So far, the ecological adaptation of Clostridium to a pit mud-associated lifestyle is largely unknown. Here, comparative genomic analysis of pit mud-associated (PMA) and non-pit mud-associated (NPMA) Clostridium strains was performed. We found genes related to the metabolism of starch, ethanol, and lactic acid were enriched in PMA Clostridium strains, which facilitated their adaptation to the unique brewing environment. In addition, horizontal gene transfer contributed to the adaptation of Clostridium to pit mud. Our findings provide genetic insights on PMA Clostridium strains' ecological adaptation and metabolic characteristics.

Komagataeibacter europaeus improves community stability and function in solid-state cereal vinegar fermentation ecosystem: Non-abundant species plays important role.

Solid-state fermentation of Chinese traditional cereal vinegar is a complex and retractable ecosystem with multi-species involved, including few abundant and many non-abundant species. However, the roles of non-abundant species in vinegar fermentation remain unknown. Here, we studied the assembly and co-occurrence patterns for abundant and non-abundant bacterial sub-communities using Zhenjiang aromatic vinegar fermentation as a model system. Our results showed that the change of reducing sugar and total titratable acid were the main driving forces for the assembly of abundant and non-abundant sub-communities, respectively. The non-abundant sub-community was more sensitive to the environmental variation of acetic acid fermentation (AAF) process. Integrated co-occurrence network revealed that non-abundant sub-communities occupied most of the nodes in the network, which play fundamental roles in network stability. Importantly, non-abundant species-Komagataeibacter europaeus, showed the highest value of degree in the co-occurrence network, implying its importance for the metabolic function and resilience of the microbial community. Bioaugmentation of K. europaeus JNP1 verified that it can effectively modulate bacterial composition and improve the robustness of co-occurrence network in situ, accompanied by (i) increased acetic acid content (14.78%) and decreased reducing sugar content (40.38%); and (ii) increased the gene numbers of phosphogluconate dehydratase (212.24%) and aldehyde dehydrogenase (192.31%). Overall, the results showed that non-abundant bacteria could be used to regulate the desired metabolic function of the community, and might play an important ecological significance in traditional fermented foods.

Mining the Factors Driving the Evolution of the Pit Mud Microbiome under the Impact of Long-Term Production of Strong-Flavor Baijiu.

The mud cellar creates a unique microenvironment for the fermentation of strong-flavor baijiu (SFB). Recent research and long-term practice have highlighted the key roles of microbes inhabiting pit mud in the formation of SFB's characteristic flavor. A positive correlation between the quality of SFB and cellar age was extracted from practice; however, the evolutionary patterns of pit mud microbiome and driving factors remain unclear. Here, based on the variation regularity analysis of microbial community structure and metabolites of samples from cellars of different ages (∼30/100/300 years), we further investigated the effects of lactate and acetate (main microbial metabolites in fermented grains) on modulating the pit mud microbiome. Esters (50.3% to 64.5%) dominated the volatile compounds identified in pit mud, and contents of the four typical acids (lactate, hexanoate, acetate, and butyrate) increased with cellar age. Bacteria (9.5 to 10.4 log10 [lg] copies/g) and archaea (8.3 to 9.1 lg copies/g) mainly constituted pit mud microbiota, respectively dominated by Clostridia (39.7% to 81.2%) and Methanomicrobia (32.8% to 92.9%). An upward trend with cellar age characterized the relative and absolute abundance of the most predominant bacterial and archaeal genera, Caproiciproducens and Methanosarcina. Correlation analysis revealed significantly (P < 0.05) positive relationships between the two genera and major metabolites. Anaerobic fermentation with acetate and lactate as carbon sources enhanced the enrichment of Clostridia, and furthermore, the relative abundance of Caproiciproducens (40.9%) significantly increased after 15-day fed-batch fermentation with lactate compared with the initial pit mud (0.22%). This work presents a directional evolutionary pattern of pit mud microbial consortia and provides an alternative way to accelerate the enrichment of functional microbes. IMPORTANCE The solid-state anaerobic fermentation in a mud cellar is the most typical feature of strong-flavor baijiu (SFB). Metabolites produced by microbes inhabiting pit mud are crucial to create the unique flavor of SFB. Accordingly, craftspeople have always highlighted the importance of the pit mud microbiome and concluded by centuries of practice that the production rate of high-quality baijiu increases with cellar age. To deepen the understanding of the pit mud microbiome, we determined the microbial community and metabolites of different-aged pit mud, inferred the main functional groups, and explored the forces driving the microbial community evolution through metagenomic, metabolomic, and multivariate statistical analyses. The results showed that the microbial consortia of pit mud presented a regular and directional evolutionary pattern under the impact of continuous batch-to-batch brewing activities. This work provides insight into the key roles of the pit mud microbiome in SFB production and supports the production optimization of high-quality pit mud.

Deciphering the succession patterns of bacterial community and their correlations with environmental factors and flavor compounds during the fermentation of Zhejiang rosy vinegar.

Zhejiang Rosy Vinegar (ZRV) is a traditional condiment in Southeast China, produced using semi-solid-state fermentation techniques under an open environment, yet little is known about the functional microbiota involved in the flavor formation of ZRV. In this study, 43 kinds of volatile flavor substances were identified by HS-SPME/GC-MS, mainly including ethyl acetate (relative content at the end of fermentation: 1104.1 mg/L), phenylethyl alcohol (417.6 mg/L) and acetoin (605.2 mg/L). The most abundant organic acid was acetic acid (59.6 g/L), which kept rising during the fermentation, followed by lactic acid (7.0 g/L), which showed a continuously downward trend. Amplicon sequencing analysis revealed that the richness and diversity of bacterial community were the highest at the beginning and then maintained decreasing during the fermentation. The predominant bacteria were scattered in Acetobacter (average relative abundance: 63.7%) and Lactobacillus (19.8%). Both sequencing and culture-dependent analysis showed Lactobacillus dominated the early stage (day 10 to 30), and Acetobacter kept highly abundant from day 40 to the end. Spearman correlation analysis displayed that the potential major groups involved in the formation of flavor compounds were Acetobacter and Lactobacillus, which were also showed strong relationships with other bacteria through co-occurrence network analysis (edges attached to Acetobacter: 61.7%; Lactobacillus: 14.0%). Moreover, structural equation model showed that the contents of ethanol, titratable acid and reducing sugar were the major environmental factors playing essential roles in influencing the succession of bacterial community and their metabolism during the fermentation. Overall, these findings illuminated the dynamic profiles of bacterial community and flavor compounds and the potential functional microbes, which were expected to help us understand the formation of flavor substances in ZRV.

Dynamic change of bacterial community during dairy manure composting process revealed by high-throughput sequencing and advanced bioinformatics tools.

The aim of this work was to study the dynamic change in structure and potential function of bacterial community during dairy manure composting process using high-throughput sequencing and advanced bioinformatics tools. Alpha diversity of microbial community significantly decreased during the thermophilic phase and then recovered gradually. Beta diversity analysis showed unique community structures in different composting phases. Keystone microbes such as genus Corynebacterium, Bacillus, Luteimonas and Nonomuraea were identified for different composting phases. Six functional modules were identified for bacterial community during the composting process using co-occurrence analysis. These modules were significantly associated with temperature, pH, degradation of organic matter and maturation of compost. Predicted metagenomics analysis showed that the relative abundance of amino acid, lipid, energy and xenobiotics metabolism increased during the composting process. These results provide valuable insights into the microbiota during dairy manure composting and how the structures and metabolic functions changed in response to composting phases.

Insight into the microbiology of nitrogen cycle in the dairy manure composting process revealed by combining high-throughput sequencing and quantitative PCR.

Nitrogen cycling during composting process is not yet fully understood. This study explored the key genes involved in nitrogen cycling during dairy manure composting process using high-throughput sequencing and quantitative PCR technologies. Results showed that nitrogen fixation occurred mainly during the thermophilic and cooling phases, and significantly enhanced the nitrogen content of compost. Thermoclostridium stercorarium was the main diazotroph. Ammonia oxidation occurred during the maturation phase and Nitrosomonas sp. was the most abundant ammonia oxidizing bacteria. Denitrification contributed to the greatest nitrogen loss during the composting process. The nirK community was dominated by Luteimonas sp. and Achromobacter sp., while the nirS community was dominated by Alcaligenes faecalis and Pseudomonas stutzeri. The nosZ community varied in a succession of Halomonas ilicicola, Pseudomonas flexibili and Labrenzia alba dominated communities according to different composting phases. Based on these results, nitrogen cycling models for different phases of the dairy manure composting process were established.

Minimizing ammonia emissions from dairy manure composting by biofiltration using a pre-composted material as the packing media.

Compost-based biofiltration is a method widely used to mitigate ammonia emissions during composting. To improve the efficiency of a composting-biofiltration system, it is necessary to determine the most effective degree of composting at which to process the packing media used in the biofiltration system. In this study, materials pre-composted for 20 and 30 d (C20 and C30, respectively), and mature compost (CM) that had been treated for 60 d, were applied as biofilter media to remove ammonia from dairy manure composting exhaust gases. A comparison of the results revealed that the C30 biofilter not only completely removed ammonia, but also produced the least nitrogen loss (1.84%). The C20 biofilter exhibited an inferior performance, indicating that enough pre-composted time is necessary for material used as the packing media. Though the CM biofilter displayed good performance with regard to ammonia removal (97.8%), it had a high nitrogen loss (6.46%). A spearman rank correlation matrix revealed that the abundance of nitrogen cycle genes including amoA, nosZ, nirK, and nirS, had a strong correlation with the physicochemical properties such as nitrate content, carbon source, moisture content, and pH of the biofilter media. C30 provided advantageous conditions and contained a relatively high abundance of nitrifiers and the lowest abundance of denitrifiers. As a result, C30 rather than CM was a more appropriate biofilter media for ammonia removal. Moreover, the occurrence of biological nitrification during the dairy manure composting process indicates the effectiveness of a material for use as biofilter media.

A comparative study of composting the solid fraction of dairy manure with or without bulking material: Performance and microbial community dynamics.

The present study compared the development of various physicochemical properties and the composition of microbial communities involved in the composting process in the solid fraction of dairy manure (SFDM) with a sawdust-regulated SFDM (RDM). The changes in several primary physicochemical properties were similar in the two composting processes, and both resulted in mature end-products within 48days. The bacterial communities in both composting processes primarily comprised Proteobacteria and Bacteroidetes. Firmicutes were predominant in the thermophilic phase, whereas Chloroflexi, Planctomycetes, and Nitrospirae were more abundant in the final mature phase. Furthermore, the succession of bacteria in both groups proceeded in a similar pattern, suggesting that the effects of the bulking material on bacterial dynamics were minor. These results demonstrate the feasibility of composting using only the SFDM, reflected by the evolution of physicochemical properties and the microbial communities involved in the composting process.

Aerobic composting of distilled grain waste eluted from a Chinese spirit-making process: The effects of initial pH adjustment.

Aerobic composting of distilled grain waste (DGW) at different initial pH values adjusted by CaO addition was investigated. Three pH-adjusted treatments with initial pH values of 4 (R1), 5 (R2) and 6 (R3) and a control treatment (R0) with a pH value of 3.5 were conducted simultaneously. The results showed that R0 had an unsuccessful start-up of composting. However, the pH-adjusted treatments produced remarkable results, with a relatively high initial pH being beneficial for the start-up. Within 65days of composting, the degradation of volatile solids (VS) and the physicochemical properties of R2 and R3 displayed similar tendencies, and both produced a mature end-product, while R1 exhibited a lower VS degradation rate, and some of its physicochemical properties indicated the end-product was immature. Quantitative PCR analysis of ammonia oxidizers indicated that the occurrence of nitrification during the composting of DGW could be attributed to the activity of ammonia-oxidizing bacteria.

Aerobic composting of digested residue eluted from dry methane fermentation to develop a zero-emission process.

Digested residue remained at the end of a process for the production of fuel ethanol and methane from kitchen garbage. To develop a zero-emission process, the compostability of the digested residue was assessed to obtain an added-value fertilizer. Composting of the digested residue by adding matured compost and a bulking agent was performed using a lab-scale composting reactor. The composting process showed that volatile total solid (VTS) degradation mainly occurred during the first 13days, and the highest VTS degradation efficiency was about 27% at the end. The raw material was not suitable as a fertilizer due to its high NH4+ and volatile fatty acids (VFAs) concentration. However, the composting process produced remarkable results; the physicochemical properties indicated that highly matured compost was obtained within 62days of the composting process, and the final N concentration, NO3- concentration, and the germination index (GI) at the end of the composting process was 16.4gkg-1-TS, 9.7gkg-1-TS, and 151%, respectively. Real-time quantitative PCR (qPCR) analysis of ammonia oxidizers indicated that the occurrence of nitrification during the composting of digested residue was attributed to the activity of ammonia-oxidizing bacteria (AOB).

Production of nitrate-rich compost from the solid fraction of dairy manure by a lab-scale composting system.

In the present study, we developed an efficient composting process for the solid fraction of dairy manure (SFDM) using lab-scale systems. We first evaluated the factors affecting the SFDM composting process using different thermophilic phase durations (TPD, 6 or 3days) and aeration rates (AR, 0.4 or 0.2 lmin(-1)kg(-1)-total solid (TS)). Results indicated that a similar volatile total solid (VTS) degradation efficiency (approximately 60%) was achieved with a TPD of 6 or 3days and an AR of 0.4 l min(-1) kg(-1)-TS (hereafter called higher AR), and a TPD of 3days resulted in less N loss caused by ammonia stripping. N loss was least when AR was decreased to 0.2 l min(-1) kg(-1)-TS (hereafter called lower AR) during the SFDM composting process. However, moisture content (MC) in the composting pile increased at the lower AR because of water production by VTS degradation and less water volatilization. Reduced oxygen availability caused by excess water led to lower VTS degradation efficiency and inhibition of nitrification. Adding sawdust to adjust the C/N ratio and decrease the MC improved nitrification during the composing processes; however, the addition of increasing amounts of sawdust decreased NO3(-) concentration in matured compost. When an improved composting reactor with a condensate removal and collection system was used for the SFDM composting process, the MC of the composting pile was significantly reduced, and nitrification was detected 10-14days earlier. This was attributed to the activity of ammonia-oxidizing bacteria (AOB). Highly matured compost could be generated within 40-50days. The VTS degradation efficiency reached 62.0% and the final N content, NO3(-) concentration, and germination index (GI) at the end of the composting process were 3.3%, 15.5×10(3)mg kg(-1)-TS, and 112.1%, respectively.