Chinese Strong-flavor Liquor Research Articles

Yeast diversity in pit mud and related volatile compounds in fermented grains of chinese strong-flavour liquor

Chinese strong-flavour liquor is produced via a traditional solid-state fermentation strategy facilitated by live microorganisms in pit mud-based cellars. For the present analysis, pit mud samples from different spatial locations within fermentation cellars were collected, and the yeast communities therein were assessed via culture-based and denaturing gradient gel electrophoresis (DGGE) approaches. These analyses revealed significant differences in the composition of yeast communities present in different layers of pit mud. In total, 29 different yeast species were detected, and principal component analyses revealed clear differences in microbial diversity in pit mud samples taken from different cellar locations. Culture-dependent strategies similarly detected 20 different yeast species in these samples. However, while Geotrichum silvicola, Torulaspora delbrueckii, Hanseniaspora uvarum, Saturnispora silvae, Issatchenkia orientalis, Candida mucifera, Kazachstania barnettii, Cyberlindnera jadinii, Hanseniaspora spp., Alternaria tenuissima, Cryptococcus laurentii, Metschnikowia spp., and Rhodotorula dairenensis were detected via a PCR-DGGE approach, they were not detectable in culture-dependent analyses. In contrast, culture-based approaches led to the identification of Schizosaccharomyces pombe and Debaryomyces hansenii in these pit mud samples, whereas they were not detected using DGGE fingerprints profiles. An additional HS-SPME-GC-MS-based analysis of the volatile compounds present in fermented grains samples led to the identification of 66 such compounds, with the highest levels of volatile acids, esters, and alcohols being detected in fermented grains from lower layer samples. A canonical correspondence analysis (CCA) suggested they were significant correlations between pit mud yeast communities and associated volatile compounds in fermented grains.

Degradation of ethyl carbamate in strong-flavor Baijiu by the microbial combination culture

Ethyl carbamate (EC) is a potentially carcinogenic compound in Chinese Baijiu that can be reduced by adjusting the starter cultures. This work studied the effects of Bacillus amyloliquefaciens J10 and Wickerhamomyces anomalus M11 on EC and its precursors in Chinese Baijiu fermentation. Efficient strains were obtained, and the optimal mixed culture method was established for EC degradation via the comparative analysis of the enzyme activity and the EC concentrations in these strains. When Bacillus amyloliquefaciens J10 and Wickerhamomyces anomalus M11 were inoculated at a ratio of 1.5:1 into the Baijiu fermentation with additional EC, the EC concentration was reduced by up to 50.5%. During simulated fermentation, this method degraded 38.05% of the EC and improved the flavor of the liquor. In conclusion, this study found an effective method to reduce the EC content in Chinese strong-flavor liquor, providing a new reference for safely producing this product.

Comparison of the microbial communities in pits with different sealing methods for Chinese strong-flavor liquor production

Chinese liquor fermentation is a typical solid-state fermentation process driven by various natural microflora, which is susceptible to ambient influence. A new-type sealing method using stainless steel as the pit cover was adopted as an alternative solution to mitigate the influence. In this study, we investigated the microbial consortia and the flavor metabolites in both the new-type sealing pit (NSP) and the traditional sealing pit (TSP). The study found the concentrations of most flavor metabolites (39 out of 58) in NSP were higher than TSP, and most of them concentrated at the bottom of both pits. The comparison revealed the diversities of microbes in different layers of pits were different (p < 0.05). The compositions of microbiota in different pits were different, albeit Lactobacillus was the absolute dominant genus in both pits. There were 13 dominant genera in NSP and seven in TSP. Therefore, more correlations between microbes were found in NSP, in which the microbial community was more stable. The interplay between different microbes and metabolites consequently resulted in different association patterns in different pits. These findings elaborate on the reasons behind the preference of the new-type sealing method and are instrumental to the optimization of the fermentation process.

Fungal Diversity Profiles in Pit Mud Samples from Chinese Strong-Flavour Liquor Pit.

Pit mud, a specific fermented soil, is an essential material for the fermentation of Chinese strong-flavour liquor. However, few studies to date have sought to characterize the spatial profiles of pit mud fungal communities in fermentation cellars from Chinese strong-flavour liquor distilleries. In this analysis, differences in fungal community structures and physicochemical properties in pit mud samples from different spatial positions within fermentation cellars were analyzed, revealing unique characteristic multidimensional pit mud fungal community profiles. Penicillium roqueforti, Pichia kudriavzevii, Aotearoamyces nothofagi, Penicillium robsamsonii, Alternaria arborescens, Trichosporon insectorum, Seltsamia ulmi, Trichosporon coremiiforme, Malassezia restricta were dominant in the pit mud samples form the upper cellar wall, whereas Metarhizium frigidum, Calonectria pseudoreteaudii, Penicillium clavigerum, Fusarium equiseti, Simplicillium chinense, Aspergillus intermedius, Trichosporon coremiiforme, Fusarium circinatum, Alternaria radicina, Aspergillus heterocaryoticus were predominant in the middle cellar wall. Alternaria radicina, Cladosporium chasmanthicola, Alternaria helianthiinficiens, Penicillium argentinense, Antarctomyces psychrotrophicus, and Trichosporon inkin are majorly present in the down cellar wall layer. Bipolaris axonopicola, Ramgea ozimecii, Penicillium argentinense, Calonectria queenslandica, Metarhizium robertsii, and Penicillium roqueforti were identified as the dominant fungi in pit mud samples from the cellar bottom. Additionally, Alternaria destruens and Alternaria doliconidium are present at notably high levels in all layers of pit mud samples. Moisture, pH, PO43-, acetic acid, humus, K+, Mg2+, Ca2+, butyric acid, and caproic acid levels in these different pit mud positions exhibited a rising incremental pattern from the upper wall layer to the bottom layer, whereas lactic acid levels were significantly lower in the bottom pit mud layer relative to these other layers. Moisture, pH, and NH4+-N were identified as the three most significant factors associated with fungal community composition through a redundancy analysis. Overall, these findings may offer a theoretical foundation for future efforts to improve or standardize artificial pit mud.

Description of a moderately acidotolerant and aerotolerant anaerobic bacterium Acidilutibacter cellobiosedens gen. nov., sp. nov. within the family Acidilutibacteraceae fam. nov., and proposal of Sporanaerobacteraceae fam. nov. and Tepidimicrobiaceae fam. nov.

A Gram-stain positive, moderately thermophilic, acidotolerant and aerotolerant anaerobic bacterium, designated JN-28 T, was isolated from the pit mud of Chinese strong-flavor liquor. Growth was observed at 25–50 °C and pH 5.5–8.0 in the presence of 0–25 g l−1 NaCl (optimally at 45 °C, pH 6.0, without NaCl). Strain JN-28 T was heterotrophic, requiring yeast extract for growth. The major cellular fatty acids were iso-C15:0 and C14:0. The DNA G + C content of genomic DNA was 33.54 mol%. The strain was resistant to vancomycin (10 mg l−1). Genome analysis revealed the presence of genes involved in the response to mild acid stress and oxidative stress, and resistance to vancomycin. 16S rRNA gene-based phylogenetic analysis showed that strain JN-28 T shares ≤ 89.3 % sequence similarity with its closest relatives Sporanaerobacter acetigenes DSM 13106 T and other members in the order Tissierellales. Based on phenotypic and phylogenetic characteristics, Acidilutibacter cellobiosedens gen. nov., sp. nov. is proposed for the new genus and novel species with the type strain JN-28 T (=CCAM 418 T = JCM 39087 T). Further phylogenetic and phylogenomic analyses suggested strain JN-28 T represents a novel family within the order Tissierellales, for which Acidilutibacteraceae fam. nov. is proposed. In addition, the family Tissierellaceae was reclassified, Sporanaerobacteraceae fam. nov. and Tepidimicrobiaceae fam. nov. were formally proposed. Emended description of the family Tissierellaceae is also provided.

Deciphering the intra- and inter-kingdom networks of microbiota in the pit mud of Chinese strong-flavor liquor

Microbes have central roles in the pit mud (PM) ecosystems and flavor formation, yet specific ecological interactions and keystone taxa among these taxa are largely unknown. Here, we used network analyses to explore the PM microbial intra- and inter-kingdom networks based on Illumina MiSeq sequencing data. The results showed that Firmicutes (mainly Caproiciproducens, Lactobacillus and Bacillus), Ascomycota (mainly Thermoascus, Eurotium and Aspergillus) and Euryarchaeota (mainly Methanobacterium, Methanosarcina, Methanobrevibacter and Methanocorpusculum) were the most dominant taxa detected in PM microbiota. The complexity of PM bacterial networks was much higher than that of fungal and archaeal networks, the hubs were mainly derived from bacteria rather than fungi and archaea in each inter-kingdom network, and the positive correlations dominated in bacterial (>75%), fungal (>90%) and inter-kingdom (>80%) networks. In addition, Mantel test showed that PM properties, especially pH, humic acid, available P and organic matter, were closely correlated with compositional structure of microbial communities and their main ecological clusters. The findings will contribute to a better understanding of PM microbiome structure and the ecological rules guiding community assembly, and have important implications for manipulating microbial resources and controlling PM environmental factors for pit management and sustainable brewing.

Distinct succession of abundant and rare fungi in fermented grains during Chinese strong-flavor liquor fermentation

The traditional spontaneous solid-state fermentation of Chinese strong-flavor liquor is a complex and unique ecosystem with few abundant and many rare taxa. However, the rare and keystone taxa of fermented grains (FGs) during liquor fermentation are still not completely clear. In this study, we used Illumina MiSeq sequencing to investigate the succession of abundant and rare fungi during Chinese strong-flavor liquor fermentation. The results showed that the changes in alpha and beta diversity of abundant and rare taxa were significantly different during liquor fermentation. There were 9 abundant fungal genera and 176 rare fungal genera during liquor fermentation, and the dominant fungal genera changed from Thermoascus within 0 day’ FG to Torulaspora and Saccharomyces within others. Co-occurrence network analysis identified 14 keystone taxa during liquor fermentation, and displayed that positive correlations (95.82%) was dominate in fungal network. Moreover, environmental fungi (Daqu, pit mud and air fungi) and FG characteristics (mainly reducing sugar) together drove the succession of the abundant and rare fungi during Chinese strong-flavor liquor fermentation. Our study provides new insights for understanding the dynamic succession of fungal communities during liquor fermentation and improving the quality and controllability in liquor production.

Identification of core microbiota in the fermented grains of a Chinese strong-flavor liquor from Sichuan

In traditional fermentation process of Chinese strong-flavor liquor, composition, dynamic succession, and interaction of microbial community in the fermented grains (FG) are fluctuant, resulting in instability and inconsistency of liquor flavor and quality. It is therefore important to reveal the core microbiota of FG from different pit ages and layers of fermentation cellars in this study. Firstly, Venn analysis showed that 14 and 13 ubiquitous bacterial and fungal genera were respectively observed. Meanwhile, Lactobacillus, Aquabacterium, Thermoactinomyces, Aspergillus, Thermoascus, Kazachstania, Pichia, and Rhizomucor with a relative abundance over 1% in all samples were identified as dominant microbiota. Additionally, 8 bacterial and 3 fungal genera were significantly correlated with flavor compounds including esters, alcohols, acids, and aldehydes by RDA, indicating these genera were flavor-associated microbiota. Furthermore, co-occurrence network analysis demonstrated that 7 bacterial and 5 fungal genera were highly connected with other genera, which were defined as co-occurring microbiota. Taken together, Lactobacillus, Thermoactinomyces, Aquabacterium, Aspergillus, and Kazachstania were identified as the core microbiota in Chinese strong-flavor liquor brewing process. Core microbiota of liquor brewing microecosystem were defined in this work, it is of paramount important to construct synthetic microbiota for stable and homogenous production of high-quality liquor.

Yeasts from Chinese strong flavour Daqu samples: isolation and evaluation of their potential for fortified Daqu production

In total, 16 yeast were isolated from Chinese strong flavour Daqu samples and underwent RAPD analysis and identification. Totally, 11 different species were identified among these isolates including Saccharomyces cerevisiae, Hanseniaspora vineae, Pichia kluyveri, Trichosporon asahii, Wickerhamomyces anomalus, Kluyveromyces lactis, Yarrowia lipolytica, Wickerhamomyces mori, Galactomyces geotrichum, Dabaryomyces hansenii, and Saccharomyces kudriavzevii. To understand the impact of these yeast strains on the quality and flavour of Daqu, we then assessed volatile compounds associated with Daqu samples fermented with corresponding strains. These analyses revealed strain YE006 exhibited the most robust ability to produce ethanol via fermentation but yielded relatively low quantities of volatile compounds, whereas strain YE010 exhibited relatively poor fermentation efficiency but produced the greatest quantity of volatile compounds. These two yeast strains were then utilized in a mixed culture to produce fortified Daqu, with the optimal inoculum size being assessed experimentally. These analyses revealed that maximal fermentation, saccharifying, liquefying, and esterifying power as well as high levels of volatile compounds were achieved when using a 2% inoculum composed of YE006/YE010 at a 1:2 (v/v) ratio. When the liquor prepared using this optimized fortified Daqu was compared to unfortified control Daqu, the former was found to exhibit significantly higher levels of flavour compounds and better sensory scores. Overall, our findings may provide a reliable approach to ensuring Daqu quality and improving the consistency and flavour of Chinese strong-flavour liquor through bioaugmentation.

Blautia liquoris sp. nov., isolated from the mud in a fermentation cellar used for the production of Chinese strong-flavour liquor.

A novel Gram-positive, non-motile, non-flagellated, strictly anaerobic, non-spore-forming and dumbbell-shaped, coccoid- or chain-shaped bacterium, designated strain LZLJ-3T, was isolated from a mud fermentation cellar which has been used for the production of Chinese strong-flavour liquor for over 100 years. Strain LZLJ-3T grew at 20-40 °C (optimum, 37 °C), at pH 6.0-8.0 (optimum, pH 8.0) and with NaCl concentrations up to 1 % (w/v; optimum, 0 %). Phylogenetic trees established based on 16S rRNA gene sequences showed that strain LZLJ-3T belonged to the genus Blautia of the family Lachnospiraceae, with the highest sequence similarity to Blautia stercoris GAM6-1T (91.7 %) and Blautia faecicola KGMB01111T (91.7 %). Comparative genome analysis showed that the orthologous average nucleotide identity (OrthoANI) and genome-to-genome distance (GGD) values between strain LZLJ-3T and B. stercoris GAM6-1T were respectively 69.1 and 22.9 %; the OrthoANI and GGD values between strain LZLJ-3T and B. faecicola KGMB01111T were respectively 70.86 and 36 % . The DNA G+C content of strain LZLJ-3T genome was 42.1 mol%. The predominant celluar fatty acids (>10 %) of strain LZLJ-3T were C16 : 0 FAME (27.9 %), C14 : 0 FAME (17.6 %) and C16 : 0 DMA (13.0 %). Arabinose, glucose and maltose could be utilized by strain LZLJ-3T as sole carbon sources for growth, with weak utilization of raffinose and l-fucose. API ZYM analysis gave positive reactions with α-galactosidase, β-galactosidase, α-glucosidase and β-glucosidase. The major end product of glucose fermentation was acetic acid. Based on the results of phenotypic, genotypic and phylogenetic analyses, strain LZLJ-3T is considered to represent a novel species of Blautia, for which the name Blautia liquoris sp. nov. is proposed. The type strain is LZLJ-3T (=KCTC 25163T=CGMCC 1.5299T=JCM 34225T).

Metagenome and analysis of metabolic potential of the microbial community in pit mud used for Chinese strong-flavor liquor production

Complex microbiomes of pit mud (PM) play significant roles in imbuing flavors and qualities of Chinese strong-flavor liquor (CSFL) during fermentation. However, understanding both of the taxonomic and functional diversity of the whole microorganisms in PM still remain a major challenge. Here, PM microbiomes were investigated based on metagenomic sequencing, assembly and binning. Metagenomic data revealed that Euryarchaeota was the predominant phylum, followed by Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria. For further functional exploration, 703 metagenome-assembled genomes (MAGs), including 304 novel strains, 197 novel species, and 94 novel genera were reconstructed. Three primary groups of Firmicutes (n = 406), Euryarchaeota (n = 130) and Bacteroidetes (n = 74), particularly genus of them Syntrophomonas, Thermacetogenium and Clostridium, methanogens (Methanobacterium, Methanoculleus, and Methanosarcina), Proteiniphilum and Prevotella, contained most of metabolic potential genes. Additionally, Chloroflexi was firstly reported to have potential to be involved in the caproic acid (CA) production. Bacteroidetes could be the key phylum to synthesize terpenes, and Armatimonadetes, Firmicutes, Ignavibacteriae and Verrucomicrobia may possess the same metabolic potential as well. Overall, this study will significantly improve our understanding of the diverse PM microbiome and help guide the future exploration of microbial resources for modifying PM fermentation processes.

Detection of Viable and Total Bacterial Community in the Pit Mud of Chinese Strong-Flavor Liquor Using Propidium Monoazide Combined With Quantitative PCR and 16S rRNA Gene Sequencing.

Microbiota in the pit mud (PM) plays a crucial role in the production of Chinese strong-flavor liquor (CSFL), the most popular distilled liquor in China. However, previous studies used total microbes, instead of viable ones, for the characterization of the microbial community in this environment. In this study, we used propidium monoazide (PMA) combined with quantitative polymerase chain reaction (qPCR) and 16S rRNA gene sequencing to verify the effect of non-viablee bacteria on the characterization of PM bacteria. After PMA concentration optimization, 50 μM PMA was chosen to pretreat 5 and 20 years PMs. The qPCR results showed that there were 50.78 and 71.84% of non-viable bacteria in the 5-year PM and 20-year PM, respectively. Both copy numbers of total bacteria and viable bacteria were significantly higher in 20-year PM than those in 5-year PM. Nevertheless, in terms of bacterial diversity and composition analyses at the operational taxonomic unit (OTU), phylum, class, and genus levels, 16S rRNA gene sequencing results displayed no significant differences between total bacteria and viable bacteria in both PM types. In conclusion, it is necessary for non-viable bacteria to be considered in determining absolute biomass of bacteria in PM, but not necessary in the analysis of diversity and composition of PM bacteria. To the best of our knowledge, our study is the first attempt to analyze viable bacteria in the PM of CSFL and provides useful information on how to accurately characterize a microbial community in a PM environment.

Co-occurrence patterns among prokaryotes across an age gradient in pit mud of Chinese strong-flavor liquor.

It is widely believed that the quality and characteristics of Chinese strong-flavor liquor (CSFL) are closely related to the age of the pit mud; CSFL produced from older pit mud tastes better. This study aimed to investigate the alteration and interaction of prokaryotic communities across an age gradient in pit mud. Prokaryotic microbes in different-aged pit mud (1, 6, and 10 years old) were analyzed by Illumina MiSeq sequencing of the 16S rRNA gene. Analysis of the 16S rRNA gene indicated that the prokaryotic community was significantly altered with pit mud age. There was a significant increase in the genera Methanosarcina, Methanobacterium, and Aminobacterium with increased age of pit mud, while the genus Lactobacillus showed a significant decreasing trend. Network analysis demonstrated that both synergetic co-occurrence and niche competition were dominated by 68 prokaryotic genera. These genera formed 10 hubs of co-occurrence patterns, mainly under the phyla Firmicutes, Euryarchaeota, and Bacteroidetes, playing important roles on ecosystem stability of the pit mud. Environmental variables (pH, NH4+, available P, available K, and Ca2+) correlated significantly with prokaryotic community assembly. The interaction of prokaryotic communities in the pit mud ecosystem and the relationship among prokaryotic communities and environmental factors contribute to the higher quality of the pit mud in older fermentation pits.

Effect of Pit Mud on Bacterial Community and Aroma Components in Yellow Water and Their Changes during the Fermentation of Chinese Strong-Flavor Liquor.

As the main by-product of Chinese strong-flavor liquor, yellow water plays an important role in the formation of flavor components. Yellow water from different fermentation periods (30th day, 45th day, 60th day) was selected to analyze the aroma components by Headspace solid phase micro-extraction Gas Chromatography–Mass Spectrometry, and the microorganism community was evaluated by high-throughput sequencing technology and bioinformatics analysis of DNA. As the fermentation time was prolonged, the main flavor components significantly increased, and the amount of the common microbial population between yellow water and pit mud increased gradually. Among the common microorganisms, Lactobacillus accounted for the largest proportion, at about 56.96%. The microbes in the yellow water mainly belonged to Firmicutes. The abundance of Bacilli (the main bacteria) gradually decreased with time, at 87.60% at the 30th day down to 68.87% at the 60th day, but Clostridia gradually increased from 10.29% to 27.48%. At the genus level, some microbes increased significantly from the 30th day to 60th day, such as Caproiciproducens, which increased from 2.65% to 6.30%, and Sedimentibacter, increasing from 0.47% to 2.49%. RDA analysis indicated that the main aroma components were positively correlated with Clostridia and negatively correlated with Bacilli.

Bioturbation effect of fortified Daqu on microbial community and flavor metabolite in Chinese strong-flavor liquor brewing microecosystem.

Traditional spontaneous fermentation in microecosystem with microbial successions and environmental variables results in inconsistent quality of fermented foods. We therefore propose the directional bioturbation for microbiota regulation and metabolites production in food fermentation. Here, we revealed the bioturbation effect of fortified Daqu on microbial community based on taxonomic composition, co-occurrence network, and metabolic potential, using Chinese strong-flavor liquor fermentation as a microecosystem. According to principal coordinate analysis, microbial communities were obviously influenced by the bioturbation of fortified Daqu. More specifically, bioturbation increased the abundances of Caproiciproducens, Clostridium, Aspergillus, Candida, Methanobacterium, and Methanosarcina, while decreased that of Lactobacillus. Meanwhile, higher abundances of most genes that encoding enzymes involved in interspecies hydrogen transfer between hexanoic acid bacteria and methanogens were observed in the bioturbated ecosystem by PICRUSt approach. Additionally, co-occurrence analysis showed that bioturbation increased the diversity and complexity of interspecies interactions in microecosystem, which contributed to higher production of flavor metabolites such as hexanoic acid, ethyl hexanoate, and hexyl hexanoate. These results indicated that the bioturbation of fortified Daqu is feasible for flavor metabolism by interspecies interactions of functional microbiota in liquor fermentation. Taken together, it is of great importance for regulating Chinese liquor and even other foods fermentation by bioturbation.

Effect of Fortified Daqu on the Microbial Community and Flavor in Chinese Strong-Flavor Liquor Brewing Process.

Daqu, an important fermentation starter for the production of Chinese liquor, as used in the current study included traditional Daqu and fortified Daqu inoculated with Bacillus velezensis and Bacillus subtilis. To evaluate the effect of fortified Daqu on strong-flavor liquor production, the differences of microbial communities among three inoculation patterns of fermented grains (FG) were analyzed by the Illumina MiSeq platform. A higher relative abundance of dominant genera including Bacillus, Lactococcus, Aspergillus, and Candida, and lower relative abundance of Lactobacillus, were observed in FG50, in which mixed Daqu (traditional: fortified Daqu = 1: 1, w/w, 50% fortified Daqu) was used as the starter. Then, volatile compounds of their distillations were also examined by HS-SPME-GC-MS. The results showed that the contents of skeleton flavor components, mainly including important esters and aromatic compounds, were higher in the corresponding liquor L50, which distillated from FG50. Moreover, most esters mainly positively correlated with Lactobacillus and Candida in the bottom layer of FG fermented with 50% fortified Daqu (FG50-B). Aromatic compounds were strongly positively correlated with Bacillus and Aspergillus in the middle layer of FG with 50% fortified Daqu used (FG50-M). In particular, hexyl hexanoate showed a positive correlation with higher abundances of Ruminococcus in the FG with addition of 100% fortified Daqu (FG100). This study observed microbial compositions in the FG with fortified Daqu addition, and it further revealed the correlations between pivotal microbes and main flavor compounds. These results may help to develop effective strategies to regulate microbes for the brewing process and further improve the flavors of Chinese liquor.

Bacterial and fungal diversity in the traditional Chinese strong flavour liquor Daqu

Bacterial and fungal diversity in the traditional Chinese strong flavour liquor <i>Daqu</i>

Improvement of caproic acid production in a Clostridium kluyveri H068 and Methanogen 166 co-culture fermentation system

The aim of this study was to develop a bioprocess capable of producing caproic acid using a binary fermentation system consisting of Clostridium kluyveri H068 and Methanogen 166 which could then be applied to the brewing of Chinese strong flavor liquor. We initially explored the mechanism by which the Methanogen 166 strain facilitates caproic acid accumulation, revealing its ability to convert accumulated H2 that is produced by C. kluyveri H068 into methane, thereby eliminating the hydrogen-mediated feedback inhibition that normally constrains C. kluyveri H068 and thus enhancing caproic acid production. In addition, laboratory experiments were conducted to optimize this binary fermentation system, allowing us to determine that the optimum conditions for caproic acid production are a mixed inoculum size of 10% with a C. kluyveri H588/Methanogen 166 inoculation ratio of 2:1 (v/v), a sodium acetate concentration of 20 g/L, a 4% ethanol content (v/v), and a yeast extract concentration of 10 g/L. We further scaled this optimized condition up to use in a 1000 L fermenter and the obtained caproic acid broth was subjected to pit-entry fermentation. Our results demonstrated that this pit-entry fermentation approach was an efficient means of improving the quality of Chinese strong flavor liquor.

Complete Genome Sequence of Clostridium kluyveri JZZ Applied in Chinese Strong-Flavor Liquor Production.

Chinese strong-flavor liquor (CSFL), accounting for more than 70% of both Chinese liquor production and sales, was produced by complex fermentation with pit mud. Clostridium kluyveri, an important species coexisted with other microorganisms in fermentation pit mud (FPM), could produce caproic acid, which was subsequently converted to the key CSFL flavor substance ethyl caproate. In this study, we present the first complete genome sequence of C. kluyveri isolated from FPM. Clostridium kluyveri JZZ contains one circular chromosome and one circular plasmid with length of 4,454,353 and 58,581bp, respectively. 4158 protein-coding genes were predicted and 2792 genes could be assigned with COG categories. It possesses the pathway predicted for biosynthesis of caproic acid with ethanol. Compared to other two C. kluyveri genomes, JZZ consists of longer chromosome with multiple gene rearrangements, and contains more genes involved in defense mechanisms, as well as DNA replication, recombination, and repair. Meanwhile, JZZ contains fewer genes involved in secondary metabolites biosynthesis, transport, and catabolism, including genes encoding Polyketide Synthases/Non-ribosomal Peptide Synthetases. Additionally, JZZ possesses 960 unique genes with relatively aggregating in defense mechanisms and transcription. Our study will be available for further research about C. kluyveri isolated from FPM, and will also facilitate the genetic engineering to increase biofuel production and improve fragrance flavor of CSFL.

Prokaryotic diversity and biochemical properties in aging artificial pit mud used for the production of Chinese strong flavor liquor

At present, artificial pit mud (APM) is widely used in Chinese liquor-making industry and plays a particular role in the production of Chinese strong flavor liquor (CSFL). However, APM frequently ages during fermentation, thus becoming unsuitable for sustainable use due to its low-quality. The reasons behind, and results of, APM aging during the production of CSFL are not yet understood. Sequencing the V4 region of the 16S rRNA gene shows that prokaryotic diversity is significantly decreased (Shannon's diversity index, P<0.01) and community composition is distinctly changed (from 1197 to 865 OTUs) in aging APM. On the phylum level, the increase of Firmicutes and decrease of Proteobacteria are the main consequences of APM aging during the production of CSFL. The counting of cultivatable bacteria confirmed that there was a large increase in Lactobacilli and aerobic spore-forming bacteria in aging low-quality APM (more than twofold). Unexpectedly, the total number of caproic acid-producing bacteria, mainly Clostridia, did not change significantly between the two kinds of APM. Furthermore, biochemical analysis indicates that the pH and the levels of NH4+ and K+ are decreased in aging low-quality APM (P<0.01). The results obtained in this study support the possibility that environmental factors (pH, nutrients) induce the decrease of prokaryotic diversity, and the changed community composition influences the environmental properties. Therefore, through interfering with the cycle, APM aging can be controlled potentially by adjustment of environmental factors and/or supplementation of diminished or missed microorganisms.