Microbial Community Structure and Dynamics of Dark Fire-Cured Tobacco Fermentation

The Italian Toscano cigar production includes a fermentation step that starts when dark fire-cured tobacco leaves are moistened and mixed with ca. 20% prefermented tobacco to form a 500-kg bulk. The dynamics of the process, lasting ca. 18 days, has never been investigated in detail, and limited information is available on microbiota involved. Here we show that Toscano fermentation is invariably associated with the following: (i) an increase in temperature, pH, and total microbial population; (ii) a decrease in reducing sugars, citric and malic acids, and nitrate content; and (iii) an increase in oxalic acid, nitrite, and tobacco-specific nitrosamine content. The microbial community structure and dynamics were investigated by culture-based and culture-independent approaches, including denaturing gradient gel electrophoresis and single-strand conformational polymorphism. Results demonstrate that fermentation is assisted by a complex microbial community, changing in structure and composition during the process. During the early phase, the moderately acidic and mesophilic environment supports the rapid growth of a yeast population predominated by Debaryomyces hansenii. At this stage, Staphylococcaceae (Jeotgalicoccus and Staphylococcus) and Lactobacillales (Aerococcus, Lactobacillus, and Weissella) are the most commonly detected bacteria. When temperature and pH increase, endospore-forming low-G+C content gram-positive bacilli (Bacillus spp.) become evident. This leads to a further pH increase and promotes growth of moderately halotolerant and alkaliphilic Actinomycetales (Corynebacterium and Yania) during the late phase. To postulate a functional role for individual microbial species assisting the fermentation process, a preliminary physiological and biochemical characterization of representative isolates was performed.     

中等嗜热细菌浸出黄铜矿过程中pH对群落结构的影响

为了优化浸出工艺,研究了pH对浸矿过程主要微生物种群结构的影响。用中度嗜热混合菌槽浸黄铜矿精矿,在不控制pH,控制pH为2．5及控制pH为1．2时,应用PCR-RFLP（限制性酶切片段长度多态性）方法对上述浸出条件下的细菌群落动态变化进行研究。结果表明,浸出体系只有两种微生物,一种为Acidithiobacillus Caldus,一种为Leptospirillum ferriphilum。pH对群落结构有明显影响。不控制pH时,浸出开始阶段At．caldus是优势种群,占群落的96％,随着浸出的进行,L．ferriphilum增多,在浸出后期代替At．caldus成为优势菌种,占69％。控制pH时,L．ferriphilum始终占主导地位,同时发现pH为2．5时At．caldus在群落中的丰度比pH为1．2时高。     

Diversity of microbial communities in open mixed culture fermentations: impact of the pH and carbon source

Anaerobic fermentation by an open mixed culture was investigated at different pH values (4–8.5) and with three substrates (glucose, glycerol and xylose). The populations established in each condition were assessed by denaturing gradient gel electrophoresis analysis of the 16S ribosomal RNA gene fragments. The fermentation pattern and the composition of the microbial population were also evaluated when operational variations were imposed (increase of substrate concentration or introduction of a second substrate). The experimental results demonstrated that at low and high pH values, a clearly different fermentation pattern was associated with the dominance of a specialised group of clostridiae. At intermediate pH values, the product spectrum was rather variable and seemed to be sensitive to variations in the microbial community. Different substrates resulted in the establishment of different microbial communities. When fed with a mixture of two substrates, mixotrophic microorganisms (capable of degrading both substrates) were found to overgrow the originally dominant specialists. Overall, the experiments have shown that some operational variables have a clear impact on the fermentation pattern and on the population established. However, a uniform relationship between the process characteristics (associated to a metabolic response) and the microbial population present is not always possible. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

生物热对传统固态发酵菌群演替及其代谢影响的研究进展

解析传统固态发酵中产生的生物热对微生物菌群代谢的影响,是认识发酵机制、调控发酵过程、保证发酵效率的关键之一。固态发酵过程中,微生物菌群代谢活动所产生的生物热及传热效率低等问题引起微环境温度升高,进而影响微生物的生长与代谢。然而,关于传统固态发酵微生物受生物热的影响及其适应机制仍不明晰。因此,本文以传统固态发酵体系为研究对象,阐述持续生物热介导的高温对固态发酵过程中微生物群落演替和代谢功能的影响,并提出复杂群落中具有多层次调控微生物代谢以适应高温环境的方式,主要从微生物群体与个体层面介绍可能存在的耐热机制。了解生物热对传统固态发酵微生物的影响及潜在的耐热机制,有助于靶向调控发酵过程、强化高温发酵等,以满足未来的工业化需求。     

Monitoring the microbial community succession and diversity of Liangzhou fumigated vinegar during solid-state fermentation with next-generation sequencing

This study reveals the microbial community succession and diversity during the whole solid-fermentation processes of naturally fermented Liangzhou fumigated vinegar (LZFV). Dynamics and diversity of microbial community succession in “Daqu” starter and other fermentation stages (starch saccharification, alcoholic fermentation, and acetic acid fermentation) were monitored using a metagenomic approach involving high-throughput sequencing. Meanwhile, dynamic changes of characteristic flavor compounds of vinegar were determined by gas chromatograph (GC) analysis. The result showed that the microbiota composition exhibited rich diversity. Twenty-five bacterial and 18 fungal genera were found in the whole fermentation process where Lactobacillus, Acetobacter, Aspergillus, Saccharomyces, and Alternaria were the predominant microorganisms. Alpha diversity metrics showed that bacterial diversity in Daqu was greater than that in AF and AAF. By contrast, fungal diversity increased from Daqu to AF and decreased in the initial stage (5–8 days) of AAF then remained relatively steady. Hence, these results could help understand dynamics of microbial community succession in continuous fermentation of traditional Chinese vinegars. The LZFV fermentation is a continuous process with spontaneous growth that affects the dynamics of microbial communities. Continuous changes of micro-environment conditions in substrate affect the diversity and structure of microbiota. Microbial growth and metabolism were closely related to the changes in the physicochemical characteristics of the cultures. The microbial flora composition showed rich diversity, and with the increase in brewing time and the change in micro-ecological environmental conditions; the microbial community showed a complex dynamic changes.

     

Modeling growth and bacteriocin production by Lactobacillus amylovorus DCE 471 in response to temperature and pH values used for sourdough fermentations

The biokinetics of cell growth of Lactobacillus amylovorus DCE 471 and bacteriocin production by this strain were investigated as a function of the temperatures (28 to 44C) and pH values (pH 4.2 to 6.4) that are characteristic of a sourdough fermentation process. The influence of temperature and pH on microbial behavior is described by using a successfully validated predictive model.     

Bacterial community dynamics during the ensilage of wilted grass

Aims:  Grass silage is the product formed by a natural lactic acid bacterial fermentation when grass is stored under anaerobic conditions, and represents an important ruminant feedstuff on farms during winter. Of the two commonly employed methods of ensiling forage, baled silage composition frequently differs from that of comparable precision-chop silage reflecting a different ensiling environment. The aim of this study was to investigate the dynamics of the silage fermentation in wilted grass and between ensiling systems.
Methods and Results:  Fermentation dynamics were examined using traditional methods of silage analyses, including microbial enumeration and analysis of fermentation products, and culture-independent terminal restriction fragment length polymorphism (T-RFLP). A successful fermentation was achieved in both systems, with the fermentation (increase in lactic acid bacteria and lactic acid concentration, decrease in pH) proceeding rapidly once the herbage was ensiled.
Conclusions:  Under controlled conditions, little difference in silage quality and microbial composition were observed between ensiling systems and this was further reflected in the T-RFLP community analysis.
Significance and Impact of the Study:  T-RFLP proved a potentially useful tool to study the ensilage process and could provide valid support to traditional methods, or a viable alternative to these methods, for investigating the dynamics of the bacterial community over the course of the fermentation.     

施用韩国假单胞菌(Pseudomonas koreensis)CLP-7对连作烟田土壤质量及微生物群落功能多样性的影响

针对目前连作导至植烟土壤酸化和根茎病害发生重的问题,田间条件下,研究了嗜酸性韩国假单胞Pseudomonas koreensis CLP-7对酸化植烟土壤pH、养分、酶活性及微生物群落功能多样性的影响。结果表明:CLP-7可以提高酸化植烟土壤pH;同时,CLP-7显著提高根际土壤速效钾、有效磷、铵态氮、硝态氮和有机质含量,其中有机质含量增加最显著;根际土壤脲酶、蔗糖酶活性呈上升趋势且均高于未施菌土壤,过氧化氢酶活性变化不明显。CLP-7处理不同时间的土壤微生物代谢多样性指数表现出明显的差异,施菌30 d时Shannon指数、Simpson指数,Richness及McIntosh指数均达到最高;所有处理的Pielou指数变化较小。微生物碳代谢主成分分析结果显示,施菌30 d时,根际土壤微生物对碳源利用与其他处理差异显著,主要为羧酸类和糖类碳源;随着施菌时间增加,微生物对氨基酸类、羧酸类、双亲化合物类、聚合物类和糖类利用率明显提高,说明CLP-7有利于提高连作烟田根际土壤微生物对碳源的利用能力。综上所述,P.koreensis CLP-7能够明显提高土壤pH、土壤酶活性,增加土壤养分含量和土壤微生物群落功能多样性,进而改善酸化植烟土壤质量,在微生物修复酸化土壤和减轻根茎病害发生的烟草绿色防控中具有较大的应用潜力。     

Modeling Growth and Bacteriocin Production by Lactobacillus amylovorus DCE 471 in Response to Temperature and pH Values Used for Sourdough Fermentations

The biokinetics of cell growth of Lactobacillus amylovorus DCE 471 and bacteriocin production by this strain were investigated as a function of the temperatures (28 to 44°C) and pH values (pH 4.2 to 6.4) that are characteristic of a sourdough fermentation process. The influence of temperature and pH on microbial behavior is described by using a successfully validated predictive model.     

Metabolomic profiling elucidates community dynamics of the Ketogulonicigenium vulgare–Bacillus megaterium consortium

Many microbial consortia are established upon metabolic interactions. Elucidating such interactions is a priority in understanding the population dynamics of these microbial consortia. In this study, we investigated the interaction dynamics of the vitamin C biosynthesis consortium comprising of Ketogulonicigenium vulgare and Bacillus megaterium. We systematically quantified the dynamic evolution of the ecosystem??s population and metabolism in response to a wide range of seeding concentrations and compositions of the two microorganisms. The consortium population dynamics was determined by quantitative PCR. The metabolomic profile of the community was systematically investigated by gas chromatography coupled with time-of-flight mass spectrometry. Our results showed that B. megaterium was responsible for initiating the reproduction of K. vulgare, meanwhile, K. vulgare could promote the growth of B. megaterium. Principal component analysis of the metabolomic profiling elucidated variations of intermediates in central carbon metabolism, nucleotide and amino acids metabolism in this microbial consortium. These findings provided new insights into the characterization of the community dynamics and the optimization of co-culture fermentation for vitamin C biosynthesis.     

Regime shift and microbial dynamics in a sequencing batch reactor for nitrification and anammox treatment of urine

The microbial population and physicochemical process parameters of a sequencing batch reactor for nitrogen removal from urine were monitored over a 1.5-year period. Microbial community fingerprinting (automated ribosomal intergenic spacer analysis), 16S rRNA gene sequencing, and quantitative PCR on nitrogen cycle functional groups were used to characterize the microbial population. The reactor combined nitrification (ammonium oxidation)/anammox with organoheterotrophic denitrification. The nitrogen elimination rate initially increased by 400%, followed by an extended period of performance degradation. This phase was characterized by accumulation of nitrite and nitrous oxide, reduced anammox activity, and a different but stable microbial community. Outwashing of anammox bacteria or their inhibition by oxygen or nitrite was insufficient to explain reactor behavior. Multiple lines of evidence, e.g., regime-shift analysis of chemical and physical parameters and cluster and ordination analysis of the microbial community, indicated that the system had experienced a rapid transition to a new stable state that led to the observed inferior process rates. The events in the reactor can thus be interpreted to be an ecological regime shift. Constrained ordination indicated that the pH set point controlling cycle duration, temperature, airflow rate, and the release of nitric and nitrous oxides controlled the primarily heterotrophic microbial community. We show that by combining chemical and physical measurements, microbial community analysis and ecological theory allowed extraction of useful information about the causes and dynamics of the observed process instability.     

传统豆酱发酵过程中细菌多样性动态

细菌在豆酱发酵过程中起到非常重要的作用,并与豆酱的风味和质量密切相关,因此研究豆酱中细菌的多样性具有重要意义。以自然发酵的豆酱样品为研究对象,采用细菌16S rDNA的部分可变区的PCR-DGGE技术对自然发酵豆酱样品的细菌群落组成和优势菌群进行研究。结果表明,传统豆酱发酵过程细菌群体中既有原始种群的减少和增长,也有次级种群的增多和演变。在整个发酵过程中,初期和末期以不可培养细菌为主,初期细菌群体快速演替,细菌种群多样性指数在发酵42 d和56 d达到两次高峰。     

氨基酸对青稞酒酿造微生物群落演替及风味代谢的驱动

[背景]环境因素是微生物生长代谢的重要驱动因素,因此,解析其对白酒酿造过程中微生物群落演替的影响对青稞酒的可控化生产具有重要作用.氨基酸作为微生物生长代谢的重要营养底物以及环境因素,其对微生物群落演替的作用尚不明确.[目的]揭示环境因素对青稞酒发酵过程中微生物群落演替及风味代谢的驱动作用.[方法]通过气相色谱-质谱联用...     

硅藻变温发酵生产二十碳五烯酸的研究

本文考察不了同温度（10℃～30℃）对硅藻(Nitzschia laevis)合成二十碳五烯酸(EPA)的影响,对不同温度下的发酵过程进行动力学特性分析。在此基础上,提出了EPA合成的变温培养方法：延滞期及对数期初期温度控制在25℃,从对数期中期开始在20℃条件下进行培养。采用此变温培养进行发酵,EPA的含量和产量分别达到了6.00%和291.60 mg·L-1,较采用单一温度(25℃)发酵的最大值分别提高了24.07%和18.81%。     

水稻秸秆低温复合菌系多样性及发酵动态

【目的】为解决中国寒冷地区水稻秸秆大面积废弃问题,加快低温地区水稻秸秆饲料转化,本文筛选了可以低温下加速秸秆发酵过程的微生物复合菌系,研究其微生物组成并跟踪其发酵动态。【方法】通过5℃下连续定向富集筛选,获得低温复合菌系。采用克隆文库方法分析复合菌系的组成。将复合菌系和商业接种剂(由Lactobacillus plantarum,Enterococcus faecium,L.salivarilus,Pediococcus acidilactici组成)分别接入稻秸进行10℃发酵。气质联机(GC-MS)测定发酵产物的同时,通过变性梯度凝胶电泳检测微生物在发酵体系的定殖情况。采用定量PCR方法追踪复合菌系组成菌在发酵过程中的动态。【结果】16S rDNA克隆文库分析结果表明复合系主要由两种微生物组成,一种属乳酸杆菌(Lactobacillus),一种属乳酸球菌(Leuconostoc)。10℃稻秸发酵结果表明,在发酵第6天接种复合菌系处理的pH已经下降到4.3,乳酸菌菌落形成单位为2.9×109CFU/g鲜样,而接种商业接种剂的处理pH为5.3,乳酸菌菌落形成单位为3.6×108 CFU/g鲜样;在发酵30 d时,接种复合菌系处理的乳酸含量为8.1 g/kg鲜样,接种商业接种剂处理的乳酸含量为2.0 g/kg鲜样。变性梯度凝胶电泳结果表明,在接种复合菌系的稻秸中,从发酵的第6天开始,检测到的微生物主要为L.sakei和Leuconostoc inhae,在整个发酵过程中,两菌一直存在;在商业接种剂处理中,发酵第6天检测到的微生物除其四种组成菌外,还包括Uncultured bacterium;而在发酵第16天和第30天,只检测到组成菌中的L.plantarum和E.faecium。定量PCR结果显示,接种复合菌系处理中,L.sakeiDNA在发酵第6天达到41.0%,在发酵第16天已达到65%,Le inhae在发酵的第6天达到整个发酵过程中的最大值(5.5%)。【结论】接种复合菌系,可以有效促进水稻秸秆的低温发酵进程。复合菌系组成菌可以定殖在发酵体系中,并占据优势。复合菌系的关键菌为L.sakei。     

Effects of Elevated Tetracycline Concentrations on Aerobic Composting of Human Feces: Composting Behavior and Microbial Community Succession

The effects of antibiotics on aerobic composting are investigated by dosing of tetracycline (TC) in fresh human feces with sawdust as biomass carrier. Variability in process parameters such as temperature, pH, water-soluble carbon, germination index (GI) and dehydrogenase activity (DHA) are evaluated at TC dosages of 0, 100, 250 and 500 mg/kg in a 21-day composting. Moreover, microbial community succession is examined by high-throughput 16S rRNA gene sequencing. Findings indicate significant impacts to the process parameters with the increase of TC concentration such as inhibition of temperature increases during aerobic composting, lowering of pH, increasing of water-soluble carbon residue, a decrease of GI, and hindering of DHA. Furthermore, elevated TC concentrations significantly alter the microbial community succession and reduce the community diversity and abundance. Therefore, interference in microbial community structures and a hindrance to biological activity are believed to be the main adverse effects of TC on the composting process and maturity of the composting products.     

Bioremediation of the tobacco waste-contaminated soil by Pseudomonas sp. HF-1: nicotine degradation and microbial community analysis

The highly effective nicotine-degrading bacterium Pseudomonas sp. HF-1 was augmented into the tobacco waste-contaminated soil to degrade nicotine and evaluate the effect of the bioremediation. Comparing with non-adding (NA) systems, the treatments with addition of strain HF-1 (TA) exhibited considerably stronger pollution disposal abilities and higher stability of pH value and moisture content, especially in groups containing a large quantity of tobacco waste. The denaturing gradient gel electrophoresis (DGGE) profiles showed that the Shannon–Wiener index decreased with increasing wastes in the NA treatments, while a gradual increase was found in the TA groups. A comparison of sequences from DGGE bands demonstrated that there were differences in the dominant microbial species between the two treatments, suggesting that strain HF-1 could persist in the soil and enhance the efficiency of tobacco waste disposal. The results of real-time fluorescence quantitative PCR (RT-qPCR) also indicated that strain HF-1 existed in the TA systems and grew with relative high quantities. In conclusion, the nicotine-degrading strain HF-1 played a leading role in the bioremediation of the tobacco waste-contaminated soil and influenced the dynamics and structure of the microbial community.     

Enhanced isomer purity of lactic acid from the non-sterile fermentation of kitchen wastes

In order to improve the purity of lactic acid isomers, the effects of pH, temperature, fermentation time and their interactions on l(+) or d(-)-lactic acid production were evaluated during lactic acid fermentation of the non-sterile kitchen wastes. The results showed that l(+)-lactic acid was the main isomeric form. The isomer purity was much higher at acidic or alkalic pH (non-controlled pH, pH 5 and pH 8) than neutral pH (pH 6 and pH 7). Increasing the fermentation temperature from 35 degrees C to 45 degrees C at pH 7 enhanced the isomer purity from 60:40 to 83:17. The optimal fermentation time for the purity of lactic acid isomers was found to depend on the corresponding pH and temperature. From the response surface analysis, the optimized combination of pH and temperature could obviously increase the l(+)-isomer concentration. It is confirmed that the variation of the isomer purity with pH, temperature and fermentation time change resulted from the substitution of microbial community composition. The lactic acid bacteria and Clostridium sp. dominated the fermentation of non-sterile kitchen wastes, and the emergence and disappearance of lactic acid bacteria which produced l(+)-isomer and Clostridium sp. resulted in the variations of the isomer purity.     

柱式反应器处理黄铜矿过程中等嗜热微生物群落变化

从中国的多个铜矿取样,在45°C条件下富集获得了一种高效的中等嗜热浸矿富集物,探讨了该富集物在柱式反应器中浸出低品位黄铜矿的pH变化以及与Cu2+浸出的关系,并采用限制性片段长度多态性(RFLP)技术分析了微生物的群落结构和种群动态变化规律。结果表明在整个浸出过程中pH变化较为明显,且一直在1.8以上,60 d内回收了13.6%的铜。RFLP结果表明:在初期,嗜铁钩端螺旋菌(Leptospirillum ferriphilum)在浸出前期占有很高比例(81%),随后逐渐降低,至后期只有13%,而耐温氧化硫化杆菌(Sulfobacillus thermotolerans)和喜温硫杆菌(Acidithiobacillus caldus)的比例逐渐升高,在中期分别达到32%和23%;至末期,耐温氧化硫化杆菌达到了79%,成为优势种群。研究加深了对中等嗜热微生物浸矿特性的了解,也为中等嗜热菌处理低品位黄铜矿的工业应用提供了可供借鉴的数据。     

High predictability of the seasonal dynamics of a species-like Polynucleobacter population in a freshwater lake

One of the key questions in microbial ecology is if seasonal patterns of bacterial community composition (BCC) observed in one year repeat in the following years. We have investigated if the recorded annual dynamics of a species-like Polynucleobacter (subcluster PnecB) population allowed the prediction of the population dynamics in another year. The abundance of PnecB bacteria in the pelagic of temperate Lake Mondsee was investigated by fluorescence in situ hybridization (FISH) over three consecutive years. The PnecB bacteria formed a persistent population, and were present in the entire water body of the lake. Two of the three investigated years differed strongly in summer temperatures and precipitation, which resulted in markedly different growth conditions. But despite of these different environmental conditions, the PnecB population demonstrated remarkably similar seasonal dynamics in the three investigated years. Water temperature was the best predictor of the population dynamics during the first half of the annual cycles. Statistical analysis also indicated influences of phytoplankton and metazooplankton successions on the PnecB population dynamics. Furthermore, 65 lakes and ponds were investigated for the presence of PnecB bacteria. They were detected in the majority (78%) of circum-neutral and alkaline freshwater habitats, but not in any investigated acidic or saline habitat.