cChinese strong-flavored liquor (CSFL) accounts for more than 70% of all Chinese liquor production. Microbes in pit mud play key roles in the fermentation cellar for the CSFL production. However, microbial diversity, community structure, and cellar-agerelated changes in pit mud are poorly understood. Here, we investigated the prokaryotic community structure and diversity in pit-mud samples with different cellar ages (1, 10, 25, and 50 years) using the pyrosequencing technique. Results indicated that prokaryotic diversity increased with cellar age until the age reached 25 years and that prokaryotic community structure changed significantly between three cellar ages (1, 10, and 25 years). Significant correlations between prokaryotic communities and environmental variables (pH, NH 4 ؉ , lactic acid, butyric acid, and caproic acid) were observed. Overall, our study results suggested that the long-term brewing operation shapes unique prokaryotic community structure and diversity as well as pit-mud chemistry. We have proposed a three-phase model to characterize the changes of pit-mud prokaryotic communities. C hinese strong-flavored liquor (CSFL), also called "Luzhou flavor liquor," accounts for more than 70% of Chinese liquor production (1). It is produced by the unique and traditional Chinese solid-state fermentation technique, which has a history of several thousand years. In brief, a cellar is constructed by digging a rectangular soil pit in which the entire inner wall is covered with precultured pit mud. The precultured pit mud is usually prepared by mixing aged pit mud (as an inoculum), fresh common soil, and water and incubating the mixture for about a year in an anaerobic cellar before use. The raw materials for the fermentation, including wheat, sorghum, and corn, are mixed, crushed, and distilled by steaming. The steamed raw material is supplied with 2% to 3% (wt/wt) Daqu-starter, which mainly includes mold and yeast, and placed into the cellar. The cellar is sealed with common mud, and fermentation is allowed to proceed for 60 days. Fermented material is then taken out of the cellar and distilled to make Chinese liquor. The process described above is periodically repeated after new fermentation materials are supplied.Microbes in the pit mud produce various flavor components such as butyric acid, caproic acid, and ethyl caproate. In particular, ethyl caproate is recognized as a key component affecting the CSFL flavor and quality. In general, CSFL quality improves with increasing cellar age. High-quality liquor is produced only in old cellars, which are maintained at least for 20 years by continuous use (2, 3). In particular, some long-aged cellars have been used for several hundred years without interruption, and well-known CSFLs such as Wuliangye, Jiannanchun, and Luzhoulaojiao are brewed in such long-aged cellars (1, 4). High CSFL quality is attributed to the maturing process of pit mud, which results in a well-balanced microbial community structure and diversity in the pit mud to produce distinctive flavo...

A unique microbiome that metabolizes lactate rather than ethanol for n-caproate production was obtained from a fermentation pit used for the production of Chinese strong-flavour liquor (CSFL). The microbiome was able to produce n-caproate at concentrations as high as 23.41 g/L at a maximum rate of 2.97 g/L/d in batch trials without in-line extraction. Compared with previous work using ethanol as the electron donor, the n-caproate concentration increased by 82.89%. High-throughput sequencing analysis showed that the microbiome was dominated by a Clostridium cluster IV, which accounted for 79.07% of total reads. A new process for n-caproate production was proposed, lactate oxidation coupled to chain elongation, which revealed new insight into the well-studied lactate conversion and carbon chain elongation. In addition, these findings indicated a new synthesis mechanism of n-caproate in CSFL. We believe that this efficient process will provide a promising opportunity for the innovation of waste recovery as well as for n-caproate biosynthesis.

Background n-Caproic acid (CA), as a medium-chain carboxylic acid, is a valuable chemical feedstock for various industrial applications. The fermentative production of CA from renewable carbon sources has attracted a lot of attentions. Lactate is a significant intermediate waste in the anaerobic breakdown of carbohydrates that comprise 18–70% of the chemical oxygen demand (COD) in municipal and some industrial wastewaters. Recently, researchers (including our own group) reported the CA production using lactate as electron donor with newly identified microbiome systems. However, within such processes, it was hard to determine whether the CA production was completed by a single strain or by the co-metabolism of different microorganisms.ResultsHere, we report the CA production using lactate as electron donor using the strain CPB6, which we isolated from a microbiome for CA production as described previously. Strain CPB6 is affiliated with Clostridium cluster IV of the family of Ruminococcaceae based on 16S rRNA gene sequence analysis. The strain prefers acidic initial pH condition (pH 5.0–6.5), and the temperature ranging from 30 to 40 °C for CA production. In a fed-batch fermentation with non-sterilized lactate-containing organic wastewater as feedstock, strain CPB6 produced 16.6 g/L CA (from 45.1 g/L lactate) with a maximum productivity of 5.29 g/L/day. Enzyme assays with crude cell extract showed that CPB6 can metabolize acetate and butyryl-CoA to produce n-butyric acid, and acetate/n-butyrate and caproyl-CoA to produce CA, respectively.ConclusionThis study demonstrated that high concentration of CA production can be obtained by a newly isolated pure culture CPB6. This strain can be employed as a powerful workhorse for high-efficient CA recovery from lactate-containing waste streams. Our preliminary investigation suggested that the CA production from lactate in strain CPB6 might be via the chain elongation pathway of the reverse β-oxidation; the detailed mechanism, however, warrants further investigation using various molecular microbiology techniques.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0788-y) contains supplementary material, which is available to authorized users.