Determination of microbial diversity in Daqu, a fermentation starter culture of Maotai liquor, using nested PCR-denaturing gradient gel electrophoresis

This study endeavored to investigate the diversity of microbes present during the shaping, ripening and drying of Daqu, a fermentation starter culture and substrata complex of Maotai alcoholic spirit. A nested PCR-denaturing gradient gel electrophoresis technique was utilized with different combinations of primers. The results showed the presence of bacteria, yeasts and molds. The microflora, which originate from wheat, were readily detectable during every stage of the fermentation process. However, the microbial structure had clear differences in the shaping, ripening and drying processes. In the shaping stage, there was a high level of diversity of the LAB (lactic acid bacteria) and fungi in the shaped samples. In the ripening stage, however, a reduction of diversity of fungi with a high level of diversity of the Bacilli was observed in the ripened samples. In the drying stage, the diversity of Bacilli and fungi, especially acid-producing bacteria, reduced dramatically. Interestingly, uncultured Lactococcus sp., Microbacterium testaceum, Cochliobolus sp., and Thermoascus crustaceus were the first to be detected in the fermentation starters used in liquor production. This study revealed the microbial diversity and distributions during the shaping, ripening and drying of Daqu-making, facilitating evaluation of the hygienic conditions and aiding in the design of specific starter and/or adjunct cultures.     

Characterization and comparison of microbial community of different typical Chinese liquor Daqus by PCR-DGGE

Aims: To identify and compare microbiota in Chinese liquor Daqu, which were produced in the different regions using different production process. Methods and Results: The DNA exacted from Daqu samples was used as a template for PCR with universal primers of 16S rRNA, 26S rRNA and 18S rRNA, respectively. The amplicons were analysed using denaturing gradient gel electrophoresis (DGGE). It was observed that the bacterial DGGE profile indicated high diversity and predominance of lactic acid bacteria. The results showed that Saccharomycopsis fibuligera and Pichia anomal were dominant yeast species and that several non‐Saccharomyces yeasts including Hanseniaspora guilliermondii, Debaryomyces hansenii, Issatchenkia orientalis and Trichosporon asahii were also detected. As for fungal DGGE, Aspergillus oryzae and Absidia blakesleeana were the most common species amongst different samples. Based on the DGGE analysis, a few differences in community structure were found between Daqu samples. Conclusions: A variety of bacteria, yeast and moulds were identified in Daqu samples, in addition to the present knowledge obtained mainly through the traditional culture‐dependent methods. Moreover, production temperature played a more decisive role on the formation of micro‐organism composition in Daqu than geographical region. Significance and Impact of the Study: PCR–DGGE technique was used in this study to fully observe and asses all microbial community (including bacteria, yeast and mould) in Chinese liquor Daqu for the first time and proved to be effective in profiling Daqu microbial diversity.     

Analysis of unculturable bacterial communities in tea orchard soils based on nested PCR-DGGE

The bacterial communities in the soils from tea orchards and their adjacent wasteland in Anhui Province, China were analysed by nested PCR-DGGE technique combined with sequencing. DGGE profiles revealed that the DGGE patterns of different soils were similar to each other and the most intensely bands appeared in all lanes. The bacterial genetic diversity index of tea orchard soils was lower than that of wasteland. For the tea orchard soils, Shannon’s diversity index decreased in the order: 45-year-old tea orchard >25-year-old tea orchard >7-year-old tea orchard >70-year-old tea orchard. The analysis of 16S rRNA gene sequences indicated that the fragments belong to Proteobacteria, Acidobacteria, TM7, Cyanobacteria and Firmicutes. A comprehensive analysis of the bacterial community structure in the tea orchard soils indicated the bacterial community was dominantly composed of Acidobacteria, followed by Proteobacteria (Gamma and Alpha), Firmicutes, Cyanobacteria and candidate division TM7. The RDA combined with UPGMA clustering analysis showed that the more similar the environmental variables were, the more similar the bacterial community structures in tea orchard soils were.     

Improvements for comparative analysis of changes in diversity of microbial communities using internal standards in PCR-DGGE

The use of internal standards both during DNA extraction and PCR-DGGE procedure gives the opportunity to analyse the relative abundance of individual species back to the original sample, thereby facilitating relative comparative analysis of diversity. Internal standards were used throughout the DNA extraction and PCR-DGGE to compensate for experimental variability. Such variability causes decreased reproducibility among replicate samples as well as compromise comparisons between samples, since experimental errors cannot be differentiated from actual changes in the community abundance and structure. The use of internal standards during DNA extraction and PCR-DGGE is suitable for ecological and ecotoxicological experiments with microbial communities, where relative changes in the community abundance and structure are studied. We have developed a protocol Internal Standards in Molecular Analysis of Diversity (ISMAD) that is simple to use, inexpensive, rapid to perform and it does not require additional samples to be processed. The internal standard for DNA extraction (ExtrIS) is a fluorescent 510-basepair PCR product which is added to the samples prior to DNA extraction, recovered together with the extracted DNA from the samples and analysed with fluorescence spectrophotometry. The use of ExtrIS during isolation of sample DNA significantly reduced variation among replicate samples. The PCR internal standard (PCR(IS)) originates from the Drosophila melanogaster genome and is a 140-basepair long PCR product, which is amplified by non-competitive primers in the same PCR reaction tubes as the target DNA and analysed together with the target PCR product on the same DGGE gel. The use of PCR(IS) during PCR significantly reduced variation among replicate samples both when assessing total PCR product and when comparing bands representing species on a DGGE gel. The entire ISMAD protocol was shown to accurately describe changes in relative abundance in an environmental sample using PCR-DGGE. It should, however, be mentioned that despite the use of ISMAD some inherent biases still exist in DNA extraction and PCR-DGGE and these should be taken into consideration when interpreting the diversity in a sample based on a DGGE gel.     

Microorganisms in Daqu: a starter culture of Chinese Maotai-flavor liquor

Maotai-flavor liquor, a famous traditional Chinese drink, is distilled from fermented sorghum in the southern province of China. Moreover, it is of interest as one of the few examples of liquor distilled from the product of a fermentation using a wild microflora starter. Daqu is the starter of this fermentation process. Daqu is a mixture of two components: milled wheat and a complex microbial community. The composition and the effects of this microbial group are largely unknown. In this study, we have analysed the constituents of the microbial community and the development of microorganisms in the industrial Daqu preparation and ripening process. More than 200 colonies were isolated and characterized. The isolates were discriminated by phenotypic and conventional biochemical taxonomic methods. The results revealed the presence of bacteria, moulds and yeasts. Bacteria consist of Bacillus, Acetobacter, Lactobacillus, and Clostridium, among which Bacillus strains were found to be predominant. Moulds consisted of Aspergillus, Mucor, Rhizopus, Monascus and Trichoderma, and Aspergillus strains were found to be predominant in the six different biotypes. Yeasts comprised Saccharomyces, Hansenula, Candida, Pichia, and Torulaspora. The most frequently isolated yeasts belonged to the genus Saccharomyces. The microbial diversity shift showed that the microbial genera changed with increasing ripening time. Knowledge of the microbial diversity in Daqu provides a basis for microflora management and understanding of the role of microbes in the Daqu production process, and the contribution of Daqu performed as a starter culture to Maotai-flavor liquor.     

Comparison of microbial communities in pilot-scale bioreactors treating Bayer liquor organic wastes

Western Australian bauxite deposits are naturally associated with high amounts of humic and fulvic materials that co-digest during Bayer processing. Sodium oxalate remains soluble and can co-precipitate with aluminium hydroxide unless it is removed. Removal of sodium oxalate requires a secondary crystallisation step followed by storage. Bioreactors treating oxalate wastes have been developed as economically and environmentally viable treatment alternatives but the microbial ecology and physiology of these treatment processes are poorly understood. Analysis of samples obtained from two pilot-scale moving bed biofilm reactors (MBBRs) and one aerobic suspended growth bioreactor (ASGB) using polymerase chain reaction- denaturing gradient gel electrophoresis of 16S rRNA genes showed that members of the α-, β- and γ-Proteobacteria subgroups were prominent in all three processes. Despite differing operating conditions, the composition of the microbial communities in the three reactors was conserved. MBBR2 was the only configuration that showed complete degradation of oxalate from the influent and the ASGB had the highest degradation rate of all three configurations. Several strains of the genus Halomonas were isolated from the bioreactors and their morphology and physiology was also determined.     

PCR-DGGE技术及其在微生物生态学中的应用

现代分子生物学技术PCR-DGGE是一种分析微生物群落的有效工具,可以用于研究生态系统中微生物多样性和群落动态性。本文简要介绍了PCR-DGGE技术原理及其在微生物生态学领域的应用,并对该技术的局限性进行了评价。     

Characterisation of culture-independent and -dependent microbial communities in a high-temperature offshore chalk petroleum reservoir

Recent studies have indicated that oil reservoirs harbour diverse microbial communities. Culture-dependent and culture-independent methods were used to evaluate the microbial diversity in produced water samples of the Ekofisk oil field, a high temperature, and fractured chalk reservoir in the North Sea. DGGE analyses of 16S rRNA gene fragments were used to assess the microbial diversity of both archaeal and bacterial communities in produced water samples and enrichment cultures from 4 different wells (B-08, X-08, X-18 and X-25). Low diversity communities were found when 16S rDNA libraries of bacterial and archaeal assemblages were generated from total community DNA obtained from produced water samples and enrichment cultures. Sequence analysis of the clones indicated close matches to microbes associated with high-temperature oil reservoirs or other similar environments. Sequences were found to be similar to members of the genera Thermotoga, Caminicella, Thermoanaerobacter, Archaeoglobus, Thermococcus, and Methanobulbus. Enrichment cultures obtained from the produced water samples were dominated by sheathed rods. Sequence analyses of the cultures indicated predominance of the genera Petrotoga, Arcobacter, Archaeoglobus and Thermococcus. The communities of both produced water and enrichment cultures appeared to be dominated by thermophilic fermenters capable of reducing sulphur compounds. These results suggest that the biochemical processes in the Ekofisk chalk reservoir are similar to those observed in high-temperature sandstone reservoirs.     

Microwave radiation and reactor design influence microbial communities during methane fermentation

The effect of reactor design and method of heating on the efficiency of methane fermentation and composition of microbial communities, especially methanogenic Archaea, were determined. The research was carried out using submerge- and trickling-bed reactors fed with wastewater and the heat supply into the reactors included a convection heating method and microwave radiation. The polymerase chain reaction-denaturing gradient gel electrophoresis and relative real-time PCR were used in order to assess the biofilm communities. The best fermentation results and the highest abundance of methanogenic Archaea in biomass were observed in microwave heated trickling-bed reactors. The research proved that in reactors of identical design, the application of microwaves enabled a higher fermentation efficiency to be obtained and simultaneously increased the diversity of methanogenic Archaea communities that favors process stability. All the identified sequences of Archaea belonged to Methanosarcina sp., suggesting that species from this genera are susceptible to non-thermal effects of microwaves. There were no effects from microwaves on the bacterial communities in both types of reactors, however, the bacterial species composition varied in the reactors of different design.     

Long-term cathode performance and the microbial communities that develop in microbial fuel cells fed different fermentation endproducts

To better understand how cathode performance and substrates affected communities that evolved in these reactors over long periods of time, microbial fuel cells were operated for more than 1 year with individual endproducts of lignocellulose fermentation (acetic acid, formic acid, lactic acid, succinic acid, or ethanol). Large variations in reactor performance were primarily due to the specific substrates, with power densities ranging from 835 ± 21 to 62 ± 1 mW/m³. Cathodes performance degraded over time, as shown by an increase in power of up to 26% when the cathode biofilm was removed, and 118% using new cathodes. Communities that developed on the anodes included exoelectrogenic families, such as Rhodobacteraceae, Geobacteraceae, and Peptococcaceae, with the Deltaproteobacteria dominating most reactors. Pelobacter propionicus was the predominant member in reactors fed acetic acid, and it was abundant in several other MFCs. These results provide valuable insights into the effects of long-term MFC operation on reactor performance.     

Continuous acetone-butanol-ethanol fermentation using SO2-ethanol-water spent liquor from spruce

SO₂–ethanol–water (SEW) spent liquor from spruce chips was successfully used for batch and continuous production of acetone, butanol and ethanol (ABE). Initially, batch experiments were performed using spent liquor to check the suitability for production of ABE. Maximum concentration of total ABE was found to be 8.79 g/l using 4-fold diluted SEW liquor supplemented with 35 g/l of glucose. The effect of dilution rate on solvent production, productivity and yield was studied in column reactor consisting of immobilized Clostridium acetobutylicum DSM 792 on wood pulp. Total solvent concentration of 12 g/l was obtained at a dilution rate of 0.21 h⁻¹. The maximum solvent productivity (4.86 g/l h) with yield of 0.27 g/g was obtained at dilution rate of 0.64 h⁻¹. Further, to increase the solvent yield, the unutilized sugars were subjected to batch fermentation.     

Characterization of microbial communities in anaerobic bioreactors using molecular probes

The microbial community structure of twenty-one single-phase and one two-phase full-scale anaerobic sewage sludge digesters was evaluated using oligonucleotide probes complementary to conserved tracts of the 16S rRNAs of phylogenetically defined groups of methanogens and sulfate-reducing bacteria. These probe results were interpreted in combination with results from traditional chemical analyses and metabolic activity assays. It was determined that methanogens in healthy mesophilic, single-phase sewage sludge digesters accounted for approximately 8–12% of the total community and thatMethanosarcinales andMethanomicrobiales constituted the majority of the total methanogen population.Methanobacteriales andMethanococcales played a relatively minor role in the digesters. Phylogenetic groups of mesophilic, Gram-negative sulfate-reducing bacteria were consistently present at significant levels:Desulfovibrio andDesulfobulbus spp. were the dominant sulfate-reducing populations,Desulfobacter andDesulfobacterium spp. were present at lower levels, andDesulfosarcina, Desulfococcus, andDesulfobotulus spp. were absent. Sulfate reduction by one or more of these populations played a significant role in all digesters evaluated in this study. In addition, sulfate-reducing bacteria played a role in favoring methanogenesis by providing their substrates. The analysis of the two-phase digester indicated that true phase separation was not accomplished: significant levels of active methanogens were present in the first phase. It was determined that the dominant populations in the second phase were different from those in the single-phase digesters.     

Effects of acid pre-treatment on bio-hydrogen production and microbial communities during dark fermentation

Optimal conditions for acid pre-treatment were investigated for the enrichment of hydrogen-producing bacteria (HPB) in a mixed culture using three strong acids: HCl, HNO(3), and H2SO4 x HCl was selected as a suitable acid for the enrichment of HPB in the fermentation process. The volume of bio-hydrogen produced when the mixed culture was pre-treated using HCl at pH 2 was 3.2 times higher than that obtained without acid pre-treatment. Changes in the microbial community during acid pre-treatment were monitored using images obtained by the fluorescent in situ hybridization (FISH) method and the Live/Dead cell viability test. The tests clearly indicated that the Clostridium species of cluster I were the predominant strains involved in bio-H(2) fermentation, and could be selectively enriched by HCl pre-treatment.     

Population dynamics and metabolite analysis of yeasts involved in a Chinese miscellaneous-flavor liquor fermentation

Baiyunbian liquor, the most popular miscellaneous-flavor liquor in China, is mainly produced in four successive fermentation batches every year. Sensory analysis based on the trained expert panel test revealed that the raw Baiyunbian liquor distilled from the last two fermentation batches always has higher quality than that distilled from the first two batches. In this study, the yeast composition, volatile compounds, and fermentation conditions associated with each fermentation batch were monitored. Significant differences were observed in the yeast community structure and fermentation parameters among different batches, which influenced the volatile profiles of the resulting liquors. Redundancy discriminate analysis and single-strain fermentation revealed that Pichia kudriavzevii and Candida humilis play important roles in enriching volatile compounds. Saccharomyces cerevisiae and Zygosaccharomyces bailii were the dominant yeasts in the last two fermentation batches. The sensorial characteristics of the liquors produced by single-strain fermentation were also analyzed. This study provides an in-depth understanding of the factors that influence liquor production and is beneficial in the development of new fermentation techniques with stable liquor quality.     

纳豆激酶发酵液的脱色工艺研究

从11种待选脱色介质中筛选出330(OH)型树脂对纳豆激酶发酵液进行脱色研究.结果表明:(1)330(OH)型树脂对纳豆激酶发酵液中的色素为优吸型吸附,其动力学模型符合扩散方程,其动力学拟和方程为-ln(1-F)=0.0223t+0.0511,R2=0.9978,其中F=Qt/Qe,Qt为脱色时间为t时330(OH)型...     

Monitoring gene expression in mixed microbial communities by using DNA microarrays

A DNA microarray to monitor the expression of bacterial metabolic genes within mixed microbial communities was designed and tested. Total RNA was extracted from pure and mixed cultures containing the 2,4-dichlorophenoxyacetic acid (2,4-D)-degrading bacterium Ralstonia eutropha JMP134, and the inducing agent 2,4-D. Induction of the 2,4-D catabolic genes present in this organism was readily detected 4, 7, and 24 h after the addition of 2,4-D. This strain was diluted into a constructed mixed microbial community derived from a laboratory scale sequencing batch reactor. Induction of two of five 2,4-D catabolic genes (tfdA and tfdC) from populations of JMP134 as low as 10(5) cells/ml was clearly detected against a background of 10(8) cells/ml. Induction of two others (tfdB and tfdE) was detected from populations of 10(6) cells/ml in the same background; however, the last gene, tfdF, showed no significant induction due to high variability. In another experiment, the induction of resin acid degradative genes was statistically detectable in sludge-fed pulp mill effluent exposed to dehydroabietic acid in batch experiments. We conclude that microarrays will be useful tools for the detection of bacterial gene expression in wastewaters and other complex systems.     

Isolation of deoxynivalenol-transforming bacteria from the chicken intestines using the approach of PCR-DGGE guided microbial selection

Background  

Contamination of grains with trichothecene mycotoxins, especially deoxynivalenol (DON), has been an ongoing problem for Canada and many other countries. Mycotoxin contamination creates food safety risks, reduces grain market values, threatens livestock industries, and limits agricultural produce exports. DON is a secondary metabolite produced by some Fusarium species of fungi. To date, there is a lack of effective and economical methods to significantly reduce the levels of trichothecene mycotoxins in food and feed, including the efforts to breed Fusarium pathogen-resistant crops and chemical/physical treatments to remove the mycotoxins. Biological approaches, such as the use of microorganisms to convert the toxins to non- or less toxic compounds, have become a preferred choice recently due to their high specificity, efficacy, and environmental soundness. However, such approaches are often limited by the availability of microbial agents with the ability to detoxify the mycotoxins. In the present study, an approach with PCR-DGGE guided microbial selection was developed and used to isolate DON -transforming bacteria from chicken intestines, which resulted in the successful isolation of several bacterial isolates that demonstrated the function to transform DON to its de-epoxy form, deepoxy-4-deoxynivalenol (DOM-1), a product much less toxic than DON.