高通量测序技术解析扳倒井浓香型白酒窖泥原核微生物群落结构

通过Illumina Miseq 2×300bp高通量测序技术对山东扳倒井股份有限公司浓香型白酒窖泥原核微生物群落结构进行了研究。发现:窖泥中细菌主要为梭菌纲(Clostridia)、拟杆菌纲(Bacteroidia)、甲烷杆菌纲(Methanobacteria)和芽孢杆菌纲(Bacilli);古菌主要为甲烷微菌纲(Methanomicrobia)、甲烷杆菌纲(Methanobacteria)和热原体纲(Thermoplasmata)。同时得出窖池位置对窖泥原核微生物群落结构的丰富度和多样性影响最大,压池时间越长窖泥菌落结构的丰富度和多样性越高,多粮发酵窖泥菌落结构与单粮发酵窖泥菌落结构既有特异性也保持相关性。     

酱香型窖泥古菌群落结构的研究

采用ARDRA免培养手段研究酱香型窖泥中的古菌群落结构,通过对窖泥总DNA的提取,扩增古菌16SrDNA序列,构建古菌16S rDNA文库。随机挑选39个阳性克隆子,通过HhaI限制性内切酶酶切,选择酶切图谱不同的25个克隆子用于后续测序,测序结果与NCBI数据库比对分析,获得其分类信息。克隆文库分析结果表明,酱香型窖泥中古菌主要分布于广古菌门中的甲烷袋状菌属（Methanoculleus）、甲烷八叠球菌属（Methanosarcina）、甲烷鬃毛菌（Methanosaeta）和甲烷杆菌属（Methanobacterium）,分别占44%、41%、3%、9%。     

浓香型白酒窖泥古菌群落结构及其多样性

以泸州老窖1、50、100和400年窖泥为研究对象,采用变性梯度凝胶电泳(DGGE)研究浓香型白酒窖泥古菌的群落结构及其多样性。DGGE图谱显示,同窖池窖泥多样性指数(H)和均匀度指数(EH)均以窖底泥较高。不同窖池同部位窖泥的H值差异较大,窖壁泥和窖底泥H值分别为2.54².86和2.762.86和2.76³.23;EH值差异较小,且随窖龄延长而上升,分别为0.9853.23;EH值差异较小,且随窖龄延长而上升,分别为0.985⁰.991和0.9910.991和0.991⁰.995。此外,同窖池窖泥的群落结构相似性系数(S C)水平较高,为0.360.995。此外,同窖池窖泥的群落结构相似性系数(S C)水平较高,为0.36⁰.74;其次是窖底泥,为0.420.74;其次是窖底泥,为0.42⁰.63;窖壁泥为0.360.63;窖壁泥为0.36⁰.52。测序结果显示,与DGGE条带序列亲缘最近的种属均为产甲烷菌,归于甲烷短杆菌属(Methanobrevibacter)、甲烷杆菌属(Methanobacterium)、甲烷鬃菌属(Methanosaeta)和甲烷囊菌属(Methanoculleus)。其中,甲烷短杆菌属(Methanobrevibacter)在不同窖泥中占优势,优势度为7.23%0.52。测序结果显示,与DGGE条带序列亲缘最近的种属均为产甲烷菌,归于甲烷短杆菌属(Methanobrevibacter)、甲烷杆菌属(Methanobacterium)、甲烷鬃菌属(Methanosaeta)和甲烷囊菌属(Methanoculleus)。其中,甲烷短杆菌属(Methanobrevibacter)在不同窖泥中占优势,优势度为7.23%³9.64%。实验结果表明,浓香型白酒窖泥古菌群落结构及其多样性因窖龄和空间分布的不同而存在差异,其中以同窖池古菌种类较相似,窖底泥群落结构较稳定;窖泥中蕴藏着产甲烷菌资源,具有进一步开发研究的价值。     

浓香型白酒窖泥古菌群落结构研究及其系统发育学分析

本试验分别提取30、100和200年窖龄窖泥样品总DNA,采用PCR-DGGE和16S rDNA测序技术探索浓香型白酒不同窖龄窖泥古菌群落演替规律,结果表明:不同窖龄窖泥的古菌DGGE图谱均出现14~17条较清晰的条带,其中第4、7、11号条带在所有样品中均有检出,且优势度较高,均在4%以上;不同窖龄窖泥的古菌群落多样性指数都在1.91~2.29之间,且随窖龄的增加而呈现上升趋势;不同窖龄窖泥的古菌相似性指数在0.43~0.61之间,30年与100年窖泥古菌群落相似性指数达到了0.61且聚为一类,与200年古菌群落相似性指数相比较却分为两类。PCR-DGGE图谱优势条带割胶测序结果显示:他们分属于产甲烷古菌(Methanogenic archaeon)、甲烷袋状菌属(Methanoculleus sp)、甲烷八叠球菌属(Methanosarcinales archaeon)、瘤胃古菌属(Rumen archaeon)四个类群。本研究结果对浓香型白酒窖泥古菌群落有了一定的认识。     

基于高通量测序的浓香型窖泥原核微生物群落的窖池空间分布

利用Illumina Miseq高通量测序技术解析河南某酒企6年和12年窖泥微生物群落多样性和组成。结果表明,两类窖龄窖池中窖泥原核微生物群落组成具有相似的空间分布特征,且物种丰度（Chao1指数）和多样性（Shannon指数）均分别在窖壁中层和下层窖泥最高;乳杆菌属（Lactobacillus）、产己酸菌属（Caproiciproducens）、嗜蛋白菌属（Proteiniphilum）及甲烷囊菌属（Methanoculleus）等14个属为窖泥优势属,其中78.6%优势属在两类窖龄窖池下部（下层和窖底）窖泥中含量均高于窖池上部（上层和中层）窖泥,且主要隶属于梭状芽胞杆菌纲（Clostridia）和拟杆菌纲（Bacteroidia）;57.6%（19/33）Top10属的空间分布特征与窖龄无关;随窖龄增加,Top10属产生了132个变化,其中75.6%的变化均较小（＜1%）;冗余分析（RDA）结果推测,铵态氮和有效磷可能是影响供试窖泥原核微生物菌群空间分布的主要环境因子。     

高通量测序技术解析五粮液窖泥原核微生物群落结构

为清晰认识五粮液特殊窖泥微生态群落结构及功能,利用Illumina NextSeq 500高通量深度测序平台首次分析了五粮液不同窖泥原核微生物群落16S rDNA V4高变区序列;优化了QIIME程序并整理和统计样品序列数目和操作分类单元(OTUs)的数量,分析了不同窖泥中原核微生物群落的丰度、多样性;利用系统发育分析确定了优势菌群的系统发育地位。该研究结果表明,从五粮液窖泥中共获得7 555 164条高质量目标片段。在相同的测序深度条件下(300 000条),五粮液窖泥原核微生物主要由细菌和古菌构成,共检出22个门,52个纲,89个目,168个科,330个属原核微生物,细菌占92.3%,古菌占7.6%。厚壁菌门(Fimicutes)是所有样品的绝对优势菌群(85.6%),共检出74个属,Clostridium、Lactobacillus,Coprococcus,Caldicoprobacter,Soehngenia是丰度最高的细菌属。结合系统发育分析确定窖泥中优势菌群为Lactobacillus,Caldicoprobacter,Caloramater,Clostridium,Caloribacterium,Garciella,Eubacterium,Syntrophomonas,Sedimentbacter,Sporanaerobacter,Tissierella,Methanosarcina,Methanobacterium,Methanobrevibacter,Methanoculleus。五粮液窖泥中蕴藏了显著区别于其他浓香型白酒窖泥的原核微生物群落,尤其是群系复杂的厚壁菌门细菌,较高丰度的产己酸菌群和促进己酸生成的甲烷菌群。     

浓香型白酒窖泥变质前后古菌群落差异分析

窖泥是浓香型白酒的生命线,窖泥变质则为酒厂带来巨大损失。采用Illumina Miseq高通量测序DNA技术对窖泥变质前后古菌群落结构进行分析。通过构建古菌16S r DNA基因文库,结果表明,变质后窖泥共扩增出52876条序列,变质前窖泥扩增出11703条序列,古菌丰度有明显提高。对序列进行聚类、测序、分析,窖泥变质前优势古菌为:甲烷短杆菌属(35.66%)、甲烷杆菌属(10.25%)、类芽孢杆菌属(4.27%)、芽孢杆菌属(3.39%)、甲烷八叠球菌属(3.27%)等;窖泥变质后优势菌为:产甲烷袋菌属(38.18%)、甲烷杆菌属(34.21%)、甲烷袋状菌属(6.90%)等。结果揭示了甲烷菌是窖泥变质前后古菌的优势菌,为进一步研究窖泥微生物全貌及窖泥培养、养护提供依据。     

浓香型白酒窖池糟醅原核微生物区系的分类研究

为了探讨中国传统白酒窖池糟醅中的原核微生物区系的构成及变化,运用PCR扩增技术和16SrDNA序列同源性分析等方法测定糟醅中原核微生物的16SrDNA基因全序列,根据与基因数据库中相似菌群16SrDNA序列的同源性,分析窖池糟醅中原核微生物的生理特性和区系分布。结果发现,发酵60d的浓香型白酒糟醅中,真细菌主要分为6个菌群：低GKmol％革兰氏阳性菌、高G Cmol％革兰氏阳性放线菌群、革兰氏阴性拟杆菌群、革兰氏阴性变形杆菌群、革兰氏阳性纤毛菌群和耵Ⅵ7门,其中β-变形杆菌群约占窖池糟醅中原核微生物的80％;古菌主要为产甲烷古细菌,包括Methanoculleus群和Methanospirillum群。通过不同窖池的比较分析得出,微生物的多样性与窖池微生态密切相关。     

剑南春天益老号窖泥特殊功能菌的选育及应用

研究了剑南春不同窖龄窖泥的微生物群落结构,解析其主导微生物菌群,并在天益老号老窖泥中分离选育窖泥特殊功能微生物。结果表明,窖泥中优势种群均分布在厚壁菌门、广古菌门、拟杆菌门。梭菌属、甲烷八叠球菌属在老窖池窖泥中含量远远高于新窖池窖泥,这两种微生物是窖泥老熟度的标志性微生物。采用微生物分离技术选育到1株产己酸能力强的菌株,产己酸能力可达到18000 mg/L以上,经16S rDNA序列分析和BIOLOG全自动微生物鉴定仪鉴定,定性为生孢梭菌(Clostridium sporogense),并将该功能菌发酵液用于人工窖泥的培养及窖池的维护保养,均取得了显著的效果。     

浓香型白酒窖池窖泥中原核微生物菌群的研究

提取浓香型窖池窖壁泥和窖底泥样品总DNA,构建窖泥细菌16S rRNA文库,经过16S r RNA扩增片段的限制性酶切分析(ARDRA)、DNA序列测定和系统发育关系分析,对比研究了窖壁泥和窖底泥细菌群落组成。数据分析结果表明,窖池窖壁泥和窖底泥中细菌种类及数量的差异性较大。窖壁泥中优势菌属为Clostridiumsp、Caloramatorsp和Desulfotoma-culum carboxydivorans,所占比例分别为17%、8%和6%。窖底泥优势菌属为Clostridium acidurici、Ruminococcus bromii、Proteiniphilumsp和Parabacteroides merdae,所占比例分别为18%、8%、7%和6%。初步揭示了窖池窖壁泥和窖底泥中细菌系统发育多样性及相互之间存在的差异。     

Analyses of archaeal communities in Doenjang and Ganjang using a culture-independent manner based on 16S rRNA sequences

The archaeal diversity in the soybean fermented food was analysed by culture-independent methods based on the 16S rRNA sequences. The 21 doenjang archaea — clones from the doenjang library were grouped into 5 groups: uncultured archaeon clone AE34 (42.8%), uncultured archaeon clone AS17 (28.6%), uncultured compost archaeon clone 4A29 (4.8%), uncultured compost archaeon clone 0A10 (19.0%), and uncultured compost archaeon clone 5A16 (4.8%). The 21 ganjang archaea — clones from the ganjang library were grouped into 5 groups: Halophilic archaeon MH1-34-1 (19.0%), H. archaeon MH1-16-3 (61.9%), Halococcus thailandensis (4.8%), Haloplanus sp. RO5-8 (9.5%), and H. archaeon MH1-136-2 (4.8%).     

甲烷囊菌属特异性引物的设计及其在古井贡酒窖泥质量评价中的初步应用

甲烷囊菌（Methanoculleus）作为优势甲烷菌存在于浓香型白酒的窖泥中,该研究对古井贡酒窖泥中含量最多的30个属的 16S rDNA全长代表序列进行傅里叶转换多序列比对（MAFFT）,得到114个可能的甲烷囊菌特异性单核苷酸多态性（SNP）位点。 对 SNP位点在甲烷囊菌属内和属外5 000多条序列进一步甄别,找出了3个效果最好的SNP位点,利用其中1个位点和半随机引物-聚合 酶链反应（SAP-PCR）技术原理设计了两对特异性引物。 以窖泥宏基因组为模板,成功扩增出了甲烷囊菌的相应序列。 将引物应用于18个 古井窖泥样本的实时定量聚合酶链反应（RT-QPCR）分析,结果表明,老厂优质窖泥中甲烷囊菌的相对含量远远高于新厂普通窖泥（最高达4 000多倍）。 利用本研究的方法原则上可以设计任意其他菌属在复杂菌群背景下的特异性引物。     

基于高通量测序解析赊店老酒浓香型白酒大曲古菌群落多样性

该研究采用高通量测序技术对赊店老酒制作的浓香型白酒大曲的古菌群落多样性进行分析,并进行蛋白直系同源簇（COG）功能预测。结果表明,大曲的优势古菌门（相对丰度＞1%）为广古菌门（Euryarchaeota）,相对丰度为98.51%;优势古菌属（相对丰度＞1%）为甲烷短杆菌属（Methanobrevibacter）、甲烷囊菌属（Methanoculleus）、甲烷发菌属（Methanothrix）、盐碱球菌属（Halalkalicoccus）、Methanomassiliicoccus、甲烷球形菌属（Methanosphaera）、甲烷杆菌属（Methanobacterium）、甲烷八叠球菌属（Methanosarcina）、Methanospirillum、甲烷嗜热杆菌属（Methanothermobacter）,相对丰度分别为61.92%、10.51%、7.17%、6.30%、3.84%、1.72%、1.61%、1.07%、1.04%、1.00%。此外,首次在大曲中发现盐碱球菌属（Halalkalicoccus）、热球菌属（Thermococcus）、甲烷绳菌属（Methanolinea）、亚硝化侏儒菌属（Nitrosopumilus）。COG功能主要富集在翻译、核糖体结构和生物发生,能源生产与转化,氨基酸转运和代谢,辅酶运输和代谢,复制、重组和修复,转录,反映了大曲中各种酶系的合成活跃。     

Characterization of archaeal community in Luzhou‐flavour pit mud

The aim of this study was to investigate the archaeal community in different ages of pit mud by a combined fluorescence in situ hybridization (FISH) and polymerase chain reaction–denaturing gradient gel electrophoresis (PCR‐DGGE) method. Four probes were used to detect the major methanogenic archaea by FISH experiments, and the results showed that orders Methanosarcinales, Methanobacteriales, Methanomicrobiales and Methanococcales were detected in the various ages (50, 100 and 300 year) of pit mud, except for the 1 year‐old pit and the 100 year‐old sample, which exhibited the highest numbers of archaea. The amounts of the four methanogenic archaea significantly increased from the 50 to 100 year‐old pit mud and slightly decreased in the 300 year‐old pit mud. Results of PCR‐DGGE analysis suggest that all archaeal 16S rRNA gene sequences fall into the phylum Euryarchaeota, and Methanobacteriales and Methanomicrobiales dominated in low‐age (1 and 50 year) and old age (100 and 300 year) of pit mud, respectively. Analysis of the community diversity based on the DGGE profiles showed that the 100 and 300 year samples exhibited similar diversity indices compared with the 1 and 50 year samples. This is the first report about the archaeal community structure in different ages of pit mud determined by both FISH and PCR‐DGGE analysis. Copyright © 2015 The Institute of Brewing & Distilling     

Microbial community analysis of mesophilic anaerobic protein degradation process using bovine serum albumin (BSA)-fed continuous cultivation

Two mesophilic anaerobic chemostats, one without added Ni2+ and Co2+ (chemostat 1) and the other with added Ni2+ and Co2+ (chemostat 2), were supplied with synthetic wastewater containing bovine serum albumin (BSA) as the sole carbon and energy source in order to study the capacity of protein degradation, microbial community structure and the effects of the addition of trace metals. Volatile fatty acids and ammonia were the main products of chemostat 1, while methane, CO2 and ammonia were the main products of chemostat 2, and critical dilution rates of 0.15 d-1 and 0.08 d-1 were obtained, respectively. Fluorescence in situ hybridization (FISH) with archaeal and bacterial domain-specific probes showed that archaeal cells were very limited in chemostat 1 while large populations of several types of archaeal cells were present in chemostat 2. Phylogenetic analyses based on 16S rRNA gene clonal sequences, DGGE, and quantitative real-time polymerase chain reaction (PCR) showed that, within the domain Archaea, methanogens affiliated with the genera Methanosaeta and Methanoculleus were predominant in chemostat 2. Within the domain Bacteria, rRNA genes obtained from chemostat 1 were affiliated with the three phyla; Firmicutes (43%), Bacteroidetes (50%) and Proteobacteria (7%). A total of 56% of rRNA genes obtained from chemostat 2 was affiliated with the three phyla, Firmicutes (32%), Bacteroidetes (11%) and Proteobacteria (13%) while 44% of rRNA genes remained unclassified. Phylogenetically distinct clones were obtained in these two chemostats, suggesting that different protein degradation pathways were dominant in the two chemostats: coupled degradation of amino acids via the Stickland reaction in chemostat 1 and uncoupled degradation of amino acids via syntrophic association of amino acid degraders and hydrogenotrophic methanogens in chemostat 2.     

Effect of dilution rate on structure of a mesophilic acetate-degrading methanogenic community during continuous cultivation

The community structures of two mesophilic acetate-degrading methanogenic consortia enriched at dilution rates of 0.025 and 0.6 d(-1) were analyzed by fluorescence in situ hybridization (FISH) and phylogenetic analyses based on 16S rDNA clonal sequences and quantitative real-time polymerase chain reaction (PCR). FISH experiments with archaeal and bacterial domain-specific probes showed that archaeal cells were predominant and only a small number of bacterial cells were detected at both dilution rates. In the domain Archaea, the number of cells closely related to Methanosarcina barkeri was shown to be greater at the high dilution rate using FISH with species-specific probes. Taxonomic analyses based on rDNA clonal sequences obtained at the low and high dilution rates showed that 43% of 100 clones and 72% of 92 clones, respectively, were affiliated with the domain Archaea and the remainders at each dilution rate were affiliated with the domain Bacteria. Within the domain Archaea, all rDNA clones at both dilution rates were affiliated with the genera Methanosaeta or Methanosarcina of the aceticlastic methanogens. Within the domain Bacteria, the rDNA clones obtained at the low dilution rate were affiliated with four phyla, Firmicutes (36%), Bacteroidetes (9%), Chloroflexi (6%) and candidate division OP12 (5%). The rDNA clones obtained at the high dilution rate were affiliated with four phyla, Firmicutes (16%), Bacteroidetes (8%), Proteobacteria (1%) and candidate division OP12 (3%). Real-time quantitative PCR experiments showed that the number of rDNA sequences affiliated with the genus Methanosarcina was greater at the high dilution rate. In addition, a significant number of rDNA sequences affiliated with the genus Methanoculleus were detected only at the low dilution rate. Detection of a hydrogenotrophic methanogen at the low dilution rate suggests that the syntrophic acetate oxidation by hydrogenotrophic methanogens and acetate-oxidizing bacteria could occur at the low dilution rate.     

浓香习酒窖泥微生物菌群多样性及系统发育分析

窖池窖泥中微生物的分类鉴定对于分析窖泥的时间长短及其对酒的香味和风味影响发挥着非常重要的作用。运用多种微生物分子生态学技术手段对习酒酿酒窖泥细菌进行研究分析,结果表明,夏季老窖多粮窖壁泥中的细菌主要分为4个菌群:Petrimonas sulfuriphila,Thermacetogenium phaeum,Caloramator及不可培养的杆菌属;古菌主要分为2个簇:Methanoculleus和Methanoculleus palmolei。所有细菌都是厌氧的且大多数是嗜热或嗜温的,窖泥中细菌和古菌共同作用生成多种活性物质降解复杂的有机物,其代谢产物在窖池特定的环境中通过复杂的生理生化反应能形成浓香型白酒的特征风味因子。