基于PLFA指纹图谱表征浓香型酒糟醅微生物群落结构

以9株白酒酿造过程中常见的菌株为对象,研究了不同种属菌株的细胞膜特征组分磷酸脂肪酸(PLFA)的特征,以及检出量与菌株生物量之间的关系.结果表明:供试细菌、放线菌、霉菌和酵母菌的PLFA指纹图谱存在显著差异,各菌株的PLFA指纹图谱可作为区别种属的依据.不同供试菌株生物量在一定范围内与检出的总PLFA量或16:0含量呈线性关系.将不同生物量的革兰氏阳性菌G+、革兰氏阴性菌G-和真菌分别加入糟醅后,检出的PLFA相对含量与对照差异显著.基于PLFA的指纹图谱能够定量或半定量地表征糟醅微生物群落结构特征及动态变化.经对多家酿酒企业糟醅PLFA组成的检测及微生物群落结构的剖析,该方法具有普适性.     

Microbial community structure in autotrophic nitrifying granules characterized by experimental and simulation analyses

This study evaluates the community structure in nitrifying granules (average diameter of 1600 μm) produced in an aerobic reactor fed with ammonia as the sole energy source by a multivalent approach combining molecular techniques, microelectrode measurements and mathematical modelling. Fluorescence in situ hybridization revealed that ammonia-oxidizing bacteria dominated within the first 200 μm below the granule surface, nitrite-oxidizing bacteria a deeper layer between 200 and 300 μm, while heterotrophic bacteria were present in the core of the nitrifying granule. Presence of these groups also became evident from a 16S rRNA clone library. Microprofiles of NH₄⁺, NO₂^–, NO₃^– and O₂ concentrations measured with microelectrodes showed good agreement with the spatial organization of nitrifying bacteria. One- and two-dimensional numerical biofilm models were constructed to explain the observed granule development as a result of the multiple bacteria–substrate interactions. The interaction between nitrifying and heterotrophic bacteria was evaluated by assuming three types of heterotrophic bacterial growth on soluble microbial products from nitrifying bacteria. The models described well the bacterial distribution obtained by fluorescence in situ hybridization analysis, as well as the measured oxygen, nitrite, nitrate and ammonium concentration profiles. Results of this study are important because they show that a combination of simulation and experimental techniques can better explain the interaction between nitrifying bacteria and heterotrophic bacteria in the granules than individual approaches alone.     

Microbial community structure in polluted Baltic Sea sediments

Nearly half the seabed of the Baltic Proper is incapable of supporting life of higher organisms as a consequence of oxygen depletion resulting from eutrophication. However, these areas are actually teeming with microbial life. Here we used terminal-restriction fragment length polymorphism (T-RFLP) to investigate the dominant archaeal and bacterial groups, with respect to community structure, in surface layers of bottom sediments of the Baltic Sea along a coastal pollution gradient. Both archaeal and bacterial communities formed distinct clusters along the pollution gradient and the community compositions were different at the polluted sites compared with the relatively clean reference sites. The structures of the bacterial communities were most strongly correlated to water depth, followed by organic carbon, oxygen, salinity and silicate levels. In contrast, the structures of the archaeal communities were most strongly correlated to oxygen, salinity, organic carbon, silicate and nitrate levels. Some members of the microbial communities were identified using a combination of traditional and molecular approaches. Isolates obtained on different culture media were identified by partial sequencing of their 16S rRNA genes and some novel species were found. In addition, we developed a computer program, aplaus, to elucidate the putative identities of the most dominant community members by T-RFLP.     

Microbial community dynamics in the forefield of glaciers

Retreating ice fronts (as a result of a warming climate) expose large expanses of deglaciated forefield, which become colonized by microbes and plants. There has been increasing interest in characterizing the biogeochemical development of these ecosystems using a chronosequence approach. Prior to the establishment of plants, microbes use autochthonously produced and allochthonously delivered nutrients for growth. The microbial community composition is largely made up of heterotrophic microbes (both bacteria and fungi), autotrophic microbes and nitrogen-fixing diazotrophs. Microbial activity is thought to be responsible for the initial build-up of labile nutrient pools, facilitating the growth of higher order plant life in developed soils. However, it is unclear to what extent these ecosystems rely on external sources of nutrients such as ancient carbon pools and periodic nitrogen deposition. Furthermore, the seasonal variation of chronosequence dynamics and the effect of winter are largely unexplored. Modelling this ecosystem will provide a quantitative evaluation of the key processes and could guide the focus of future research. Year-round datasets combined with novel metagenomic techniques will help answer some of the pressing questions in this relatively new but rapidly expanding field, which is of growing interest in the context of future large-scale ice retreat.     

An improved method for counting bacteria from sediments and turbid environments by epifluorescence microscopy

We present a new procedure for effectively detaching particle-associated bacteria by 10% (v/v) methanol and sonication which is particularly suitable for samples with a high particle load and sediments. We also optimized the sample preparation by applying the highly dsDNA-specific fluorescent stain SybrGreen I together with an optically brilliant mounting medium (polyvinylalcohol 4-88, 'moviol') in one step. The new protocol allows a much faster, easy and less toxic handling of samples as compared to other methods. Cells are stained directly on a black Nuclepore filter and show an intensive fluorescence signal with low background. The detachment procedure was optimized with respect to the temperature of the 10% methanol solution (35 degrees C), ultrasonication and centrifugation. The application of the new method in comparison with detachment procedures with pyrophosphate and Tween-80 with various types of marine samples including sediments always yielded higher numbers and/or higher fractions of particle-associated cells. Staining and mounting the samples with the moviol-SybrGreen I solution allowed an accurate and highly reproduceable enumeration of bacteria also in samples with high concentrations of SPM. Fixation of bacteria by glutardialdehyde resulted in a brighter fluorescence as compared to fixation by formalin. Because of the high specificity to dsDNA and bright fluorescence of SybrGreen I, the fast and easy handling and the possibility to store stained samples for at least several months at -20 degrees C without any loss in fluorescence intensity, the newly developed method is also an attractive alternative to DAPI staining of aquatic bacteria.     

Microbial community structure in methane hydrate-bearing sediments of freshwater Lake Baikal

Gas hydrates in marine sediments have been known for many years but recently hydrates were found in the sediments of Lake Baikal, the largest freshwater basin in the world. Marine gas hydrates are associated with complex microbial communities involved in methanogenesis, methane oxidation, sulfate reduction and other biotransformations. However, the contribution of microorganisms to the formation of gas hydrates remains poorly understood. We examined the microbial communities in the hydrate-bearing sediments and water column of Lake Baikal using pyrosequencing of 16S rRNA genes. Aerobic methanotrophic bacteria dominated the water sample collected at the lake floor in the hydrate-bearing site. The shallow sediments were dominated by Archaea. Methanogens of the orders Methanomicrobiales and Methanosarcinales were abundant, whereas representatives of archaeal lineages known to perform anaerobic oxidation of methane, as well as sulfate-reducing bacteria, were not found. Affiliation of archaea to methanogenic rather than methane-oxidizing lineages was supported by analysis of the sequences of the methyl coenzyme M reductase gene. The deeper sediments located at 85-90 cm depth close to the hydrate were dominated by Bacteria, mostly assigned to Chloroflexi, candidate division JS1 and Caldiserica. Overall, our results are consistent with the biological origin of methane hydrates in Lake Baikal.     

Bacterial community structure on two alpine debris-covered glaciers and biogeography of Polaromonas phylotypes

High-elevation cold environments are considered ideal places to test hypotheses about mechanisms of bacterial colonization and succession, and about bacterial biogeography. Debris-covered glaciers (glaciers whose ablation area is mainly covered by a continuous layer of rock debris fallen from the surrounding mountains) have never been investigated in this respect so far. We used the Illumina technology to analyse the V5 and V6 hypervariable regions of the bacterial 16S rRNA gene amplified from 38 samples collected in July and September 2009 at different distances from the terminus on two debris-covered glaciers (Miage and Belvedere—Italian Alps). Heterotrophic taxa-dominated communities and bacterial community structure changed according to ice ablation rate, organic carbon content of the debris and distance from the glacier terminus. Bacterial communities therefore change during downwards debris transport, and organic carbon of these recently exposed substrates is probably provided more by allochthonous deposition of organic matter than by primary production by autotrophic organisms. We also investigated whether phylotypes of the genus Polaromonas, which is ubiquitous in cold environments, do present a biogeographical distribution by analysing the sequences retrieved in this study together with others available in the literature. We found that the genetic distance among phylotypes increased with geographic distance; however, more focused analyses using discrete distance classes revealed that both sequences collected at sites <100 km and at sites 9400–13 500 km to each other were more similar than those collected at other distance classes. Evidences of biogeographic distribution of Polaromonas phylotypes were therefore contrasting.     

Scanning electron microscopy and epifluorescence investigation of bacterial colonization of marine sand sediments

Scanning electron microscopy (SEM) was employed for the investigation of microorganisms living in marine sand sediments. Epifluorescence, as well as sediment analyses, gave further data on the parameters of the sediment samples.SEM revealed a correlation between the site and density of bacterial colonization and the microtopography of the individual sand grains.Sand grains with a medium roundness showed the greatest density of bacterial colonization. Protected surface sites were favored in the colonization process. The mode of bacterial attachment varied; mostly the barren sand grain surface was colonized. However, bacteria were also observed close to or within detritus or attached to diatoms. Many of the attaching bacteria observed were found to produce polymer strands.In some cases special structures were discovered which could serve bacterial attachment. Entire colonies attached by means of polymer nets, and disc-shaped bacteria were observed.     

温州市境内城市河流底泥氨氧化菌富集培养物微生物群落结构分析

【背景】城市河流底泥含有丰富的微生物资源,底泥表面更是硝化作用的主要位点之一,其表面微生物在河流生态系统氮的转化过程中发挥着重要作用。【目的】以温州市境内的城市河流水系温瑞塘河茶山段舜岙河和横江河的4条河道作为采样点,比较分析4种不同环境下城市河流表层底泥氨氧化菌富集培养物的微生物群落结构。【方法】通过野外采样及室内培养对底泥中氨氧化功能菌进行富集培养,采用高通量测序技术分析微生物群落的组成、丰度和多样性。【结果】富集培养后主要优势类群为变形菌门(Proteobacteria)和拟杆菌门(Bacteroidetes)。4个样品共涉及氨氧化细菌3个属,分别为亚硝化单胞菌属(Nitrosomonas)、亚硝化螺菌属(Nitrosospira)、亚硝化球菌属(Nitrosococcus),涉及氨氧化古菌1个属为Nitrososphaera,其中所有样品均以Nitrosomonas为主。不同底泥富集样品氨氧化微生物可操作分类单元(Operational taxonomic unit,OTU)组成存在明显差异,栽种有水生植物的河道底泥样品DA2具有最高的氨氧化细菌OTU数量和相对丰度,而存在生活餐饮污染的河道底泥样品DA4具有最高的氨氧化古菌OTU数量和相对丰度;相较于滞留水体,采自相对流动水体的富集样品DA2、DA4具有更高的氨氧化微生物OTU数量和相对丰度。【结论】阐述了4种不同环境下城市河流底泥氨氧化菌富集培养物微生物群落结构的多样性,确定了富集培养之后的优势类群,为氨氧化微生物培养源的选择提供了参考,也为城市河流底泥中氨氧化菌进一步的筛选分离及其生理生态特征的研究提供了科学依据。     

Microbial community composition in sediments resists perturbation by nutrient enrichment

Functional redundancy in bacterial communities is expected to allow microbial assemblages to survive perturbation by allowing continuity in function despite compositional changes in communities. Recent evidence suggests, however, that microbial communities change both composition and function as a result of disturbance. We present evidence for a third response: resistance. We examined microbial community response to perturbation caused by nutrient enrichment in salt marsh sediments using deep pyrosequencing of 16S rRNA and functional gene microarrays targeting the nirS gene. Composition of the microbial community, as demonstrated by both genes, was unaffected by significant variations in external nutrient supply in our sampling locations, despite demonstrable and diverse nutrient-induced changes in many aspects of marsh ecology. The lack of response to external forcing demonstrates a remarkable uncoupling between microbial composition and ecosystem-level biogeochemical processes and suggests that sediment microbial communities are able to resist some forms of perturbation.     

Microbial ecology of submerged marine caves and holes characterized by high levels of hydrogen sulphide

Submarine caves, cavities and niches characterised by H₂S elevated concentrations are particularly interesting for their inhabiting microflora as well as for the overall chemical, geological and biological parameters. These ecosystems are usually populated by well adapted living forms, physically distributed following the in situ concentration and gradient of micronutrients, O₂ and H₂S, and also according to the values of temperature and pH. The biota is primarily characterised by prokaryotes (both autotrophic and heterotrophic) adapted to anoxic and/or microaerophilic condition and capable to form extensive biofilms on the rocky surfaces and even on the bottom sediment. These habitats can be defined as extreme, because the scarcity or absence of solar irradiation, the chemo-physical traits and the fact that specialised prokaryotes are often the only inhabitants. This review is focused on the microbial ecology of marine caves and holes characterised by high levels of hydrogen sulphide. Ecological and geological data are already available but very few insights as far as regard microbiology were achieved in order to describe these fascinating habitats. The autochthonous mesophilic and thermotolerant microorganisms living in these caves may have interesting physiological traits and eventually may lead to potential application in biotechnological processes.     

Cholesterol in condensed and fluid phosphatidylcholine monolayers studied by epifluorescence microscopy

Epifluorescence microscopy was used to investigate the effect of cholesterol on monolayers of dipalmitoylphosphatidylcholine (DPPC) and 1 -palmitoyl-2-oleoyl phosphatidylcholine (POPC) at 21 +/- 2 degrees C using 1 mol% 1-palmitoyl-2-[12-[(7-nitro-2-1, 3-benzoxadizole-4-yl)amino]dodecanoyl]phosphatidylcholine (NBD-PC) as a fluorophore. Up to 30 mol% cholesterol in DPPC monolayers decreased the amounts of probe-excluded liquid-condensed (LC) phase at all surface pressures (pi), but did not effect the monolayers of POPC, which remained in the liquid-expanded (LE) phase at all pi. At low pi (2-5 mN/m), 10 mol% or more cholesterol in DPPC induced a lateral phase separation into dark probe-excluded and light probe-rich regions. In POPC monolayers, phase separation was observed at low pi when > or =40 mol% or more cholesterol was present. The lateral phase separation observed with increased cholesterol concentrations in these lipid monolayers may be a result of the segregation of cholesterol-rich domains in ordered fluid phases that preferentially exclude the fluorescent probe. With increasing pi, monolayers could be transformed from a heterogeneous dark and light appearance into a homogeneous fluorescent phase, in a manner that was dependent on pi and cholesterol content. The packing density of the acyl chains may be a determinant in the interaction of cholesterol with phosphatidylcholine (PC), because the transformations in monolayer surface texture were observed in phospholipid (PL)/sterol mixtures having similar molecular areas. At high pi (41 mN/m), elongated crystal-like structures were observed in monolayers containing 80-100 mol% cholesterol, and these structures grew in size when the monolayers were compressed after collapse. This observation could be associated with the segregation and crystallization of cholesterol after monolayer collapse.     

The microbial community structure of different permeable sandy sediments characterized by the investigation of bacterial fatty acids and fluorescence in situ hybridization

This study describes the microbial community structure of three sandy sediment stations that differed with respect to median grain size and permeability in the German Bight of the Southern North Sea. The microbial community was investigated using lipid biomarker analyses and fluorescence in situ hybridization. For further characterization we determined the stable carbon isotope composition of the biomarkers. Biomarkers identified belong to different bacterial groups such as members of the Cytophaga-Flavobacterium cluster and sulfate-reducing bacteria (SRB). To support these findings, investigations using different fluorescent in situ hybridization probes were performed, specifically targeting Cytophaga-Flavobacterium, gamma-Proteobacteria and different members of the SRB. Depth profiles of bacterial fatty acid relative abundances revealed elevated subsurface peaks for the fine sediment, whereas at the other sandy sediment stations the concentrations were less variable with depth. Although oxygen penetrates deeper into the coarser and more permeable sediments, the SRB biomarkers are similarly abundant, indicating suboxic to anoxic niches in these environments. We detected SRB in all sediment types as well as in the surface and at greater depth, which suggests that SRB play a more important role in oxygenated marine sediments than previously thought.     

Microbial community structure and biogeochemistry of Miocene subsurface sediments: implications for long-term microbial survival

Thirty closely spaced cores were obtained from Miocene-aged fluvial, lacustrine and palaeosol subsurface sediments ranging in depth from 173 to 197 m at a site in south-central Washington to investigate the size and composition of the microbial community in relation to sediment geochemical and geophysical properties. Total phospholipid fatty acid (PLFA) analysis indicated that the greatest concentrations of microbial bio-mass were in low-permeability lacustrine sediments that also contained high concentrations of organic carbon. Community structure, based on lipid analyses and on in situ hybridization of bacterial cells with 16S RNA-directed DNA probes, also revealed the presence of metabolically active bacteria that respire sulphate and/or Fe(III) in the lacustrine sediments. Concentrations of pore water sulphate were low (4–8 mg/L) and HCI-extractable Fe was predominantly Fe(II) in the same samples where total biomass and organic carbon were highest. The low hydraulic conductivity (10^-6 to < 10^-9 cm/s) of these sediments has likely contributed to the long term maintenance of both bacteria and organic carbon by limiting the supply of soluble electron acceptors for microbial respiration. These results suggest that the current subsurface microbial population was derived from organisms that were present during lake sedimentation = 6–8 million years ago.     

Microenvironments and microbial community structure in sediments

The aim of this study was to explore the potential of a combined chemical and microbiological approach as part of a study of organic carbon oxidation processes in sediments. An assessment of microbiological diversity using molecular techniques was carried out in combination with high resolution chemical measurements at the sediment-water interface of a coastal lagoon affected by eutrophication in autumn 2000. There was a 0.2 mm overlap between the O2 and H2S profiles. pH showed a maximum just above the sediment-water interface coinciding with an oxygen maximum, suggesting photosynthetic activity, and a minimum coinciding with the O2-H2S interface. The redox potential was high in bottom water and surface sediment, reflecting the presence of oxygen and oxides, and reached low values after a step-wise decrease at -18 mm. Reduction of Fe occurred within the biofilm at the O2-H2S interface and was mostly due to reduction by H2S. The elevated concentrations of dissolved Mn in the oxic water may have been caused either by in situ production within organic aggregates or lateral water flow from sites nearby at which Mn2+ diffuses out of the sediment. Sequences related to sulphur chemolitotrophs were retrieved from the biofilm samples, which is consistent with the small overlap between O2 and H2S observed in this biofilm. Although the resolution of techniques used was different, sequencing results were consistent with chemical data in delineating the same horizons according to redox, pH or ecological properties.     

The prokaryotic community of subglacial bottom sediments of Antarctic Lake Untersee: Detection by cultural and direct microscopic techniques

The heterotrophic mesophilic microbial component was studied in microbial communities of the samples of frozen regolith collected from the glacier near Lake Untersee collected in 2011 during the joint Russian-American expedition to central Dronning Maud Land (Eastern Antarctica). Cultural techniques revealed high bacterial numbers in the samples. For enumeration of viable cells, the most probable numbers (MPN) method proved more efficient than plating on agar media. Fluorescent in situ hybridization with the relevant oligonucleotide probes revealed members of the groups Eubacteria (Actinobacteria, Firmicutes) and Archaea. The application of the methods of cell resuscitation, such as the use of diluted media and prevention of oxidative stress, did not result in a significant increase in the numbers of viable cells retrieved from subglacial sediment samples. Our previous investigations demonstrated the necessity for special procedures for efficient reactivation of the cells from microbial communities of replace with buried soil and permafrost samples collected in the Arctic zone. The differing responses to the special resuscitation procedures may reflect the differences in the physiological and morphological state of bacterial cells in microbial communities subject to continuous or periodic low temperatures and dehydration.     

Microbial diversity and community respiration in freshwater sediments influenced by artificial light at night

An increasing proportion of the Earth''s surface is illuminated at night. In aquatic ecosystems, artificial light at night (ALAN) may influence microbial communities living in the sediments. These communities are highly diverse and play an important role in the global carbon cycle. We combined field and laboratory experiments using sediments from an agricultural drainage system to examine how ALAN affects communities and alters carbon mineralization. Two identical light infrastructures were installed parallel to a drainage ditch before the start of the experiment. DNA metabarcoding indicated that both sediment communities were similar. After one was lit for five months (July–December 2012) we observed an increase in photoautotroph abundance (diatoms, Cyanobacteria) in ALAN-exposed sediments. In laboratory incubations mimicking summer and winter (six weeks each), communities in sediments that were exposed to ALAN for 1 year (July 2012–June 2013) showed less overall seasonal change compared with ALAN-naive sediments. Nocturnal community respiration was reduced in ALAN-exposed sediments. In long-term exposed summer-sediments, we observed a shift from negative to positive net ecosystem production. Our results indicate ALAN may alter sediment microbial communities over time, with implications for ecosystem-level functions. It may thus have the potential to transform inland waters to nocturnal carbon sinks.     

Microbial community structure of surface sediments from a tropical estuarine environment using next generation sequencing

Microbial community structure was analyzed from tropical monsoon influenced Mandovi-Zuari (Ma-Zu) estuarine sediment by means of Next Gen Sequencing (NGS) approach using Ion Torrent PGM™. The sequencing generated 80,282 raw sequence reads. Barcoding with Ion Tags allowed multiplex analysis of microbial community and helped in identifying shifts in microbial community structure. Analysis of sequence data revealed that sediment at both the stations in the Mandovi estuary was dominated by Archaeal group, Euryarchaeota (53.1% and 64.01%). Among Euryarchaeota, Methanomicrobia was dominant. Methanococci was present only at the mouth and Methanopyri was detected at the mid-estuarine station. Whereas, both the stations of Zuari estuary were dominated by Bacteria, Proteobacteria, mainly Gammaproteobacteria (97.67% and 54.41%). A clear influence of mangrove ecosystem on the bacterial diversity was evident in the Zuari estuary. These results suggest that the two estuaries have a very distinct microbial community structure. Characterization of microbial communities in this study area using NGS for the first time points out that even within geographically close habitats, the microbial population structure is significantly influenced by localized interactions. The signatures obtained from sediments can thus be used to reconstruct habitat characteristics and serve as biomarkers. Future studies should focus on the functional gene profiling of different microbial communities and the influence of seasons and tide in such monsoon influenced estuaries.