Effect of bioaugmented inoculation on microbiota dynamics during solid-state fermentation of Daqu starter using autochthonous of Bacillus, Pediococcus, Wickerhamomyces and Saccharomycopsis.

Daqu, a traditional fermentation starter that is used for Chinese liquor and vinegar production, is still manufactured through a traditional spontaneous solid-state fermentation process with no selected microorganisms are intentionally inoculated. The aim of this work was to analyze the microbiota dynamics during the solid-state fermentation process of Daqu using a traditional and bioaugmented inoculation with autochthonous of Bacillus, Pediococcus, Saccharomycopsis and Wickerhamomyces at an industrial scale. Highly similar dynamics of physicochemical parameters, enzymatic activities and microbial communities were observed during the traditional and bioaugmented solid-state fermentation processes. Both in the two cases, groups of Streptophyta, Rickettsiales and Xanthomonadales only dominated the first two days, but Bacillales and Eurotiales became predominant members after 2 and 10 days fermentation, respectively. Phylotypes of Enterobacteriales, Lactobacillales, Saccharomycetales and Mucorales dominated the whole fermentation process. No significant difference (P > 0.05) in microbial structure was observed between the traditional and bioaugmented fermentation processes. However, slightly higher microbial richness was found during the bioaugmented fermentation process after 10 days fermentation. Our results reinforced the microbiota dynamic stability during the solid-state fermentation process of Daqu, and might aid in controlling the traditional Daqu manufacturing process.

Paenibacillus aceti sp. nov., isolated from the traditional solid-state acetic acid fermentation culture of Chinese cereal vinegar.

A Gram-stain-negative, rod-shaped, motile, endospore-forming, facultatively anaerobic bacterium, designated strain L14T, was isolated from the traditional acetic acid fermentation culture of Chinese cereal vinegars. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain L14T was affiliated to the genus Paenibacillus, most closely related to Paenibacillus motobuensis MC10T with 97.8 % similarity. Chemotaxonomic characterization supported the allocation of the strain to the genus Paenibacillus. The polar lipid profile of strain L14T contained the major compounds diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The predominant menaquinone was MK-7, and the major fatty acid components were anteiso-C15 : 0, iso-C15 : 0 and C16 : 0. The DNA G+C content of strain L14T was 49.9 mol%. The DNA-DNA relatedness value between strain L14T and P. motobuensis MC10T was 51.2 %. The results of physiological and biochemical tests allowed phenotypic differentiation of strain L14T from closely related species. On the basis of phenotypic and chemotaxonomic analyses, phylogenetic analysis and DNA-DNA relatedness values, strain L14T is considered to represent a novel species of the genus Paenibacillus, for which the name Paenibacillus aceti sp. nov. is proposed. The type strain is L14T (=CGMCC 1.15420T=JCM 31170T).

Microbiota Dynamics Associated with Environmental Conditions and Potential Roles of Cellulolytic Communities in Traditional Chinese Cereal Starter Solid-State Fermentation.

Traditional Chinese solid-state fermented cereal starters contain highly complex microbial communities and enzymes. Very little is known, however, about the microbial dynamics related to environmental conditions, and cellulolytic communities have never been proposed to exist during cereal starter fermentation. In this study, we performed Illumina MiSeq sequencing combined with PCR-denaturing gradient gel electrophoresis to investigate microbiota, coupled with clone library construction to trace cellulolytic communities in both fermentation stages. A succession of microbial assemblages was observed during the fermentation of starters. Lactobacillales and Saccharomycetales dominated the initial stages, with a continuous decline in relative abundance. However, thermotolerant and drought-resistant Bacillales, Eurotiales, and Mucorales were considerably accelerated during the heating stages, and these organisms dominated until the end of fermentation. Enterobacteriales were consistently ubiquitous throughout the process. For the cellulolytic communities, only the genera Sanguibacter, Beutenbergia, Agrobacterium, and Erwinia dominated the initial fermentation stages. In contrast, stages at high incubation temperature induced the appearance and dominance of Bacillus, Aspergillus, and Mucor. The enzymatic dynamics of amylase and glucoamylase also showed a similar trend, with the activities clearly increased in the first 7 days and subsequently decreased until the end of fermentation. Furthermore, ß-glucosidase activity continuously and significantly increased during the fermentation process. Evidently, cellulolytic potential can adapt to environmental conditions by changes in the community structure during the fermentation of starters.

Functional genes based analysis of sulfur-oxidizing bacteria community in sulfide removing bioreactor.

Sulfur-oxidizing bacteria (SOB) are the main microorganisms that participate in the bioremediation of sulfide-rich wastewater. To reveal the SOB community structure and determine which members of SOB contribute to the sulfide oxidation in a sulfide-rich cloth printing and dyeing wastewater treatment plant, specific primer pairs dsrA 625F/877R, soxB 704F/1199R, and sqr 473F/982R based on the SOB functional genes encoding dissimilatory sulfite reductase, sulfate thioesterase/thiohydrolase, and sulfide: quinone oxidoreductase were designed. The restriction fragment length polymorphism analysis showed that the diversity indices and the abundance of each OTU have no significant changes after time, which suggested the SOB community in the sulfide removing bioreactor have high steady phylogenetic analysis of functional gene-based clone libraries detected the SOB from Chlorobia, α-proteobacteria, ß-proteobacteria, and γ-proteobacteria. The combined clone library showed the presence of dominant members of the SOB species closely related to families Halothiobacillaceae (17%), Hydrogenophilaceae (14%), and Rhodocyclaceae (13%), which may contribute to the sulfide oxidation in wastewater treatment process. This work provides a precise understanding of SOB microbial community within sulfide removing bioreactor, and the result gives assistance for the optimization of the treatment systems for sulfide biological degradation.

Comparison and characterization of microbial communities in sulfide-rich wastewater with and without propidium monoazide treatment.

A 16S rRNA gene-based culture-independent approach was used to study the bacterial and archaeal communities in a sulfide-rich wastewater. Propidium Monoazide (PMA) treatment was applied to limit the analysis to the fraction of viable cells in environment. A total of 104 and 68 clones respective from bacterial clone library and archaeal library were picked and analyzed by restriction fragment length polymorphism (RFLP). 35 RFLP patterns from bacterial clone library and 10 RFLP patterns from archaeal clone library were unique and the respective clones were selected for sequencing. BLAST analysis and RFLP analysis showed that the bacterial clone library mainly consisted of Gammaproteobacteria (73%), Anaerolineae (6%), Bacilli (5%), Deltaproteobacteria (7%), Clostridia (4%), Bacteroidetes (1%), and Chlorobia (1%); Methanomicrobia (99%) and Thermococci (1%) were the only two lineages of the archaeal domains. This study gave a first insight into the overall microbial structure in a cloth printing and dyeing wastewater treatment plant with high concentration of sulfide and increased knowledge on the applicability of the PMA treatment in combination with PCR-based molecular techniques to analyze only viable cells in microbial ecology.

Method to detect only viable cells in microbial ecology.

Propidium monoazide can limit the analysis of microbial communities derived from genetic fingerprints to viable cells with intact cell membranes. However, PMA treatment cannot completely suppress polymerase chain reaction (PCR) amplification when the targeted gene is too short. PMA treatment in combination with two-step nested PCR was designed to overcome this problem. Four experiments were performed to determine the limitation of PMA treatment and to evaluate the suitability of the method by applying the following samples: (1) pure cultures of Escherichia coli O157:H7, Enterobacter aerogenes, and Alcaligenes faecalis; (2) pond water samples spiked with heat-killed E. coli O157:H7 and E. aerogenes; (3) anaerobic sludge samples exposed to increasing heat stress; and (4) selected natural samples of estuarine sediment and lake mud. Results from the first two experiments show that PMA treatment cannot efficiently suppress dead cells from PCR amplification when the targeted gene is as short as 190 bp, however, the two-step nested PCR can overcome this problem. The last two experiments indicate the method that PMA treatment in combination with two-step nested PCR is useful for viable cells detection in microbial ecology.

Optimization of fermentation media for nitrite oxidizing bacteria using sequential statistical design.

The sequential statistical experimental design (Plackett-Burman, factorial, response surface and steepest ascent experiment) was applied to optimize the culture medium of nitrite oxidizing bacteria for improving the nitrite oxidizing rate. Estimated optimum medium composition of the nitrite oxidizing rate was as follows: NaHCO3, 1.86gl(-1); NaNO2, 2.04gl(-1); Na2CO3, 0.2gl(-1); NaCl, 0.2gl(-1); KH2PO4, 0.1gl(-1); MgSO4 x7H2O, 0.1gl(-1); and FeSO4 x 7H2O, 0.01gl(-1). The nitrite oxidizing rate was increased by 48.0% and reached a maximum at 859.5+/-8.4mgNO2-N/gMLSS.d as compared to 580.7+/-25.8mgNO2-N/gMLSS x d. In the field trial, 50L of nitrite oxidizing bacteria concentrate (1.99gVSS/L) with 850mgNO2-N/gMLSS x d were added to 0.6ha of the aquaculture water. Nitrite level in all treated ponds remained very low compared to the steady increase observed in all of the control ponds during 7 days.