盐度对稀释平板法研究红树林区土壤微生物数量的影响

在使用稀释平板法分离潮间带红树林及其对照光滩土壤微生物以及计数时,多数情况下使用陈海水制作培养基和稀释水,很少考虑培养基和稀释水的盐度对最终计数结果的影响.使用稀释平板法研究了盐度对福建九龙江口红树林区与深圳福田红树林保护区土壤微生物平板计数的影响,结果表明培养基与稀释水盐度对微生物数量有明显的影响.统计分析显示细菌的海水稀释效果优于淡水,而放线菌与真菌则刚好相反(P<0.05,一个例外).海水不适合配制红树林区土壤微生物平板计数的培养基,从0～35,高盐度的平板培养基会降低微生物的数量,尤其是放线菌的数量,尽管培养基的盐度对真菌影响无规律,但细菌数量在低盐度时比在高盐度和不加氯化钠时要多.根据盐度效应,提出了稀释平板技术应用于潮间带的红树林及其相应光滩时的优化方法,认为细菌应该用海水作无菌稀释水,而放线菌和真菌则应用淡水作稀释水;包括光滩在内的红树林区土壤微生物分离与计数的培养基宜控制较低盐度范围.     

米酒曲细菌多样性研究

目的分析米酒曲中微生物群落组成。方法采集7个地区的米酒曲样品,通过PCR-DGGE与传统可培养方法对米酒曲中的细菌多样性进行解析。结果基于PCR-DGGE法,米酒曲中细菌由Enterococcus、Streptococcus、Lactobacillus、Pediococcus和Weissella等乳酸菌类群组成。基于传统纯培养方法,在厌氧条件下共分离到细菌24株,使用MRS培养基分离得到14株乳酸菌,其中Enterococcus类群乳酸菌最多,其次是Weissella,而Pediococcus最少;在厌氧条件下,通过LB培养基得到10株菌,经鉴定属于Cronobacter、Enterobacter、Klebsiella类群。结论米酒曲中存在着丰富的乳酸菌类群,同时也有有害微生物的存在。     

利用改良培养基探究西太平洋海水可培养细菌多样性

【目的】西太平洋复杂的海洋生态环境孕育了其独特的生物群落,蕴含着种类丰富的海洋微生物资源。本研究基于分离培养技术探究了西太平洋海域不同水深细菌的多样性,并尝试通过改良培养基提高海洋细菌可培养性。【方法】采用改良的2216E固体培养基(IMA)、R2A固体培养基(R2A)、MBM固体培养基(MBM)、TCBS固体培养基(TCBS)和改良的2216E液体富集培养基(IMB) 5种不同培养基进行微生物培养,通过菌株分离纯化、16SrRNA基因序列鉴定,分析西太平洋表层至6000m水深可培养细菌的多样性以及不同培养基在分离培养异养细菌方面的优势。【结果】本研究共获得1293株异养细菌,分属于4门7纲14目26科52属119种,其中变形菌门(Proteobacteria)为主导类群。纲水平上,γ-变形菌纲(Gammaproteobacteria)、α-变形菌纲(Alphaproteobacteria)和放线菌纲(Actinobacteria_c)为优势菌群。5种培养基所获得的最优势门都为变形菌门,最优势纲都为γ-变形菌纲,除TCBS培养基优势目是弧菌目(Vibrionales),最优势目都为交替单胞菌目(Alteromonadales)。此外,5种培养基在各分类水平上均体现出不同的选择性。5种培养基在种水平上可培养细菌多样性由高到低依次为R2A、IMA、MBM、TCBS以及IMB。分离自R2A的特有属数目最多,可达10个。随水深增加,可培养异养微生物属的数量呈减少趋势。分得菌株中共有68株为潜在新菌,新菌率在IMA、R2A和MBM中相对较高。【结论】本研究用5种不同培养基从西太平洋海水中获得大量可培养细菌,具有较高的多样性,同时揭示了不同培养基对可培养海洋细菌的选择性。本研究为进一步的生态学研究和分子生物学研究等提供了宝贵的种质资源,也为未来利用改良培养基分离难培养海洋微生物带来启发。     

中药渣堆肥中解磷细菌的筛选、鉴定及其拮抗作用

为开发新型、安全及高效的微生物菌肥,以堆肥2个月后的中药渣为试材,筛选解磷拮抗细菌.用有机磷细菌培养基初步筛选5株解磷细菌;采用溶磷圈法和磷钼蓝比色法测定初筛菌株的解磷作用,解磷作用最优的菌株是YP5;通过形态学特征观察、生理生化试验和16SrDNA序列分析鉴定,确定菌株YP5为铜绿假单胞菌(Pseudomonas a...     

校园空气污染微生物的检测与评价

采用沉降平板法 ,选用牛肉膏培养基与察贝克培养基 (czpekmedium) ,分别在夏季与冬季检测了校园不同环境中空气污染细菌与霉菌的含量。结果表明 :校园不同环境的空气污染微生物类群与数量随季节的变化而变化。学生寝室、学生食堂、校区餐厅以及网吧的空气中污染微生物以细菌为主。其中 ,在冬季的细菌平均含菌量为 91 4 7.6 7个 /m3 空气 ,而以女生寝室的含菌量最高 ,达到 2 1 5 4 6个 /m3 空气 ,超出卫生标准的 3.1倍 ;在夏季时 ,细菌的平均含菌量为 1 0 6 6 1 .75个 /m3 空气 ,而以校园餐厅的含菌量最高 ,达到 4 35 1 1个 /m3 ,超出卫生标准的 6 .9倍。校图书馆、电影院、学生寝室及餐厅中的霉菌含量较多。其中 ,在冬季的霉菌平均含菌量为 5 6 8.4 4个 /m3 空气 ,以女生寝室的含菌量最高 ,84 6个 /m3 ;在夏季的霉菌含菌量为 85 5 .88个 /m3 ,最高时为 1 6 6 5个 /m3 ,这是由于卫生条件差 ,居室狭小 ,人口拥挤 ,空气潮湿 ,通风不良等因素造成的     

高灵敏假单胞菌铁载体的平板检测方法*

CAS蓝色检测平板是一种筛选、检测各类细菌铁载体的常用方法,而蔗糖-天冬酰氨培养基被用于假单胞菌产铁载体规律的研究。用天冬氨酸替代天冬酰氨,将CAS蓝色检测液与蔗糖-天冬氨酸培养基(MSA培养基)相结合,得到一种改进的MSA-CAS检测平板。通过对假单胞菌属7个种8个株进行荧光与非荧光铁载体检测方面的比较研究,结果表明MSA-CAS检测平板假单胞菌铁载体的检测灵敏度比通用CAS检测平板高,而且在检测荧光铁载体方面具有荧光背景低、荧光铁载体晕圈明显和晕圈与背景的对比度大的优点。     

东北旱田土壤中Anabaena伴生细菌的分离与鉴定

[背景]鱼腥藻(Anabaena)在农田土壤中广泛分布,具有固碳和固氮功能。明确伴生细菌与蓝细菌的关系,对提高农田土壤中Anabaena的功能具有重要意义。[目的]从东北不同旱田土壤中分离Anabaena sp.PCC7120的伴生细菌,初步鉴定伴生细菌的分类归属,推测伴生细菌的功能,为明确旱田土壤蓝细菌与伴生细菌的关系提供数据支撑。[方法]采用平板分离、PCR-DGGE、克隆测序技术测定并分析不同旱田土壤中伴生细菌的16S rRNA基因序列,确定伴生细菌的分类地位。[结果]PCR-DGGE图谱显示东北旱田14个土样中分离获得Anabaena sp.PCC7120伴生细菌数量和种类不同;PCR-克隆测序获得伴生细菌的16S rRNA基因序列37条,可鉴定到种水平的菌株36条,主要归为鞘氨醇盒菌属(Sphingopyxis)、贪噬菌属(Variovorax)、黄杆菌属(Flavobacterium)和红球菌属(Rhodococcus)等,推测这些伴生细菌具有适应寡营养、富集微量元素、清除毒素等功效。[结论]东北旱田不同土壤中Anabaena sp.PCC7120伴生细菌种类和数量各异,这些伴生细菌主要隶属于Sphingopyxis、Variovorax、Flavobacterium和Rhodococcus等属。     

生物有机肥对连作蕉园香蕉生产和土壤可培养微生物区系的影响

以连续种植香蕉12年的枯萎病高发病蕉园为试验点,通过平板计数和可培养微生物群落变性凝胶电泳(CD PCR-DGGE)等方法研究田间条件下连续两年施用化肥、牛粪、猪粪和生物有机肥对香蕉枯萎病的抑制作用,以及对香蕉产量、品质和土壤中可培养微生物区系的影响.结果表明:相比于其他处理,连续两年施用生物有机肥能够有效降低香蕉枯萎病发病率,显著提高大田香蕉单株质量、小区产量、果实可溶性糖含量及可溶性糖与可滴定酸的比值(糖酸比).可培养微生物区系分析结果表明,施用生物有机肥能够显著提高土壤微生物生物量,增加可培养细菌、芽孢杆菌和放线菌数量及细菌与真菌比值,降低尖孢镰刀菌数量.CD PCR-DGGE聚类分析表明,连续两年施用生物有机肥明显改变了土壤可培养细菌群落结构,增加了其丰度和多样性.切胶测序结果表明,连续两年施用生物有机肥的香蕉园土壤增加了类芽孢杆菌、伯克氏菌、未培养疣微菌及Bacillus aryabhattai的丰度,降低了青枯菌、粘金黄杆菌、Fluviicola taffensis、肠杆菌及巨大芽孢杆菌的丰度.表明连续施用生物有机肥能够优化连作蕉园土壤可培养微生物群落结构,防控香蕉枯萎病的发生,提高香蕉产量并改善果实品质.     

高灵敏假单胞菌铁载体的平板检测方法

CAS蓝色检测平板是一种筛选、检测各类细菌铁载体的常用方法,而蔗糖-天冬酰氨培养基被用于假单胞菌产铁载体规律的研究。用天冬氨酸替代天冬酰氨,将CAS蓝色检测液与蔗糖-天冬氨酸培养基（MSA培养基）相结合,得到一种改进的MSA-CAS检测平板。通过对假单胞菌属7个种8个株进行荧光与非荧光铁载体检测方面的比较研究,结果表明MSA-CAS检测平板假单胞菌铁载体的检测灵敏度比通用CAS检测平板高,而且在检测荧光铁载体方面具有荧光背景低、荧光铁载体晕圈明显和晕圈与背景的对比度大的优点。     

盐碱土壤与非盐碱土壤微生物的抗盐碱特性

为了比较分析盐碱土壤与非盐碱土壤微生物资源抗盐碱性差异,本研究利用含有不同浓度Na2CO3、NaHCO3和pH的培养基对盐碱土壤和非盐碱土壤细菌进行培养计数.结果显示:非盐碱土壤出菌数量随Na2CO3、pH和NaHCO3浓度升高而下降,盐碱土壤细菌出菌数量随着Na2CO3、pH和NaHCO3浓度升高先是升高然后下降,最高值分别出现在200 mmol/L NaHCO3、50 mmol/L Na2CO3和pH 9.0的分离平板上.此外,高Na2CO3、pH和NaHCO3浓度的平板中盐碱土壤出菌数量远高于非盐碱土壤;以上结果可见,耐盐碱细菌资源主要集中分布在盐碱土壤中,在非盐碱土壤中虽有分布,但是仅占有很少一部分.     

Direct profiling of the yeast dynamics in wine fermentations

We present a method to directly characterize the yeast diversity present in wine fermentations by employing denaturing gradient gel electrophoresis (DGGE) of polymerase chain reaction (PCR)-amplified 26S ribosomal RNA (rRNA) genes. PCR-DGGE of a portion of the 26S rRNA gene was shown to distinguish most yeast genera associated with the production of wine. With this method the microbial dynamics in several model wine fermentations were profiled. PCR-DGGE provided a qualitative assessment of the yeast diversity in these fermentations accurately identifying populations as low as 1000 cells ml(-1). PCR-DGGE represents an attractive alternative to traditional plating schemes for analysis of the microbial successions inherent in the fermentation of wine.     

秸秆还田与施肥对稻田土壤微生物生物量及固氮菌群落结构的影响

利用氯仿熏蒸法和变性梯度凝胶电泳法(PCR-DGGE)研究了秸秆覆盖还田与施肥对灰棕冲积水稻土0—10cm和10—20cm土层土壤微生物生物量碳、氮和固氮菌群落结构的影响。结果表明:土壤微生物量碳、氮和固氮菌多样性从0—10cm土层到10—20cm土层均呈现降低趋势。无秸秆覆盖处理(对照组)的土壤微生物生物量碳(SMB-C)和微生物生物量氮(SMB-N)量最小。在秸秆覆盖还田处理中,低氮和无钾处理的SMB-C和SMB-N都显著低于全量氮磷钾肥处理。虽然无磷处理的SMB-N低于全量氮磷钾处理,但差异不显著。说明秸秆覆盖还田配施充足氮磷钾肥能显著提高土壤微生物生物量碳、氮。由DGGE图谱多样性指数分析得知,配施充足氮磷钾肥的处理土壤的固氮菌多样性最丰富。UPGMA聚类分析显示,10种不同处理的聚类图也不同,对照(无秸秆)处理0—10cm和10—20cm的微生物不同于其它处理单独聚在了一个群里。DGGE条带测序得知,14个条带的近缘种大部分为非培养细菌nifH基因片段,主要优势菌群其归属于变形菌门(Proteobacteria)的β-变形菌纲(Betaproteobacteria)。应用PCR-DGGE技术可以解释灰棕冲积水稻土秸秆覆盖不同肥料用量固氮菌分子群落结构特点。     

Estimation of dominant microbial population sizes in the anaerobic granular sludge of a full-scale UASB treating streptomycin wastewater by PCR-DGGE

Although the dominant members of microbial communities in wastewater bio-treatment systems were often paid attention due to their possible important roles in treatment performance, their population sizes, especially the unculturable species, were still little known. Then PCR-DGGE was used in an attempt to estimate the dominant microbial population sizes in the anaerobic granular sludge treating streptomycin wastewater, coupled with an inoculated strain (Esherichia coli) with known population sizes as an internal standard. The results indicated that the band intensities of the inoculated strain in DGGE profiles showed good correlation with population sizes. Then it was possible to estimate the dominant microbial population sizes by means of comparing their DGGE band intensities with the inoculated strain. The estimated results demonstrated that the sizes of major dominant microbial populations in the sludge sample were at the level of 10⁷–10⁸ CFU/g. The sizes of secondary dominant microbial populations were at the level of 10⁵–10⁶ CFU/g. The microbial populations with the size level lower than 10³ CFU/g were undetectable by PCR-DGGE. These results provided a potential approach to evaluate dominant microbial population sizes in complex microbial communities.     

Seasonal and Diel Variability in Dissolved DNA and in Microbial Biomass and Activity in a Subtropical Estuary

Dissolved DNA and microbial biomass and activity parameters were measured over a 15-month period at three stations along a salinity gradient in Tampa Bay, Fla. Dissolved DNA showed seasonal variation, with minimal values in December and January and maximal values in summer months (July and August). This pattern of seasonal variation followed that of particulate DNA and water temperature and did not correlate with bacterioplankton (direct counts and [³H]thymidine incorporation) or phytoplankton (chlorophyll a and ¹⁴CO₂ fixation) biomass and activity. Microautotrophic populations showed maxima in the spring and fall, whereas microheterotrophic activity was greatest in late summer (September). Both autotrophic and heterotrophic microbial activity was greatest at the high estuarine (low salinity) station and lowest at the mouth of the bay (high salinity station), irrespective of season. Dissolved DNA carbon and phosphorus constituted 0.11 ± 0.05% of the dissolved organic carbon and 6.6 ± 6.5% of the dissolved organic phosphorus, respectively. Strong diel periodicity was noted in dissolved DNA and in microbial activity in Bayboro Harbor during the dry season. A noon maximum in primary productivity was followed by an 8 p.m. maximum in heterotrophic activity and a midnight maximum in dissolved DNA. This diel periodicity was less pronounced in the wet season, when microbial parameters were strongly influenced by episodic inputs of freshwater. These results suggest that seasonal and diel production of dissolved DNA is driven by primary production, either through direct DNA release by phytoplankton, or more likely, through growth of bacterioplankton on phytoplankton exudates, followed by excretion and lysis.     

Polymerase chain reaction-denaturing gradient gel electrophoresis analysis of bacterial community structure in the food,intestines, and feces of earthworms

The bacterial communities in the food, intestines, and feces of earthworms were investigated by PCR-denaturing Gradient gel electrophoresis (DGGE). In this study, PCR-DGGE was optimized by testing 6 universal primer sets for microbial 16S rRNA in 6 pure culture strains of intestinal microbes in earthworms. One primer set effectively amplified 16S rRNA from bacterial populations that were found in the food, intestines, and feces of earthworms. Compared with the reference markers from the pure culture strains, the resulting DGGE profiles contained 28 unique DNA fragments. The dominant microorganisms in the food, intestines, and feces of earthworms included Rhodobacterales bacterium, Fusobacteria, Ferrimonas marina, Aeromonas popoffii, and soil bacteria. Other straisn, such as Acinetobacter, Clostridium, and Veillonella, as well as rumen bacteria and uncultured bacteria also were present. These results demonstrated that PCR-DGGE analysis can be used to elucidate bacterial diversity and identify unculturable microorganisms.     

Microbial diversity in lake sediments detected by PCR-DGGE

In this study,PCR-denaturing gradient gel electrophoresis (DGGE) was applied to analyze the microbial communities in lake sediments from Lake Xuanwu,Lake Mochou in Nanjing and Lake Taihu in Wuxi.Sediment samples from seven locations in three lakes were collected and their genomic DNAs were extracted.The DNA yields of the sediments of Lake Xuanwu and Lake Mochou were high (10 μg/g),while that of sediments in Lake Taihu was relatively low.After DNA purification,the 16S rDNA genes (V3 to V5 region) were amplified and the amplified DNA fragments were separated by parallel DGGE.The DGGE profiles showed that there were five common bands in all the lake sediment samples indicating that there were similarities among the populations of microorganisms in all the lake sediments.The DGGE profiles of Lake Xuanwu and Lake Mochou were similar and about 20 types of micro-organisms were identified in the sediment samples of both lakes.These results suggest that the sediment samples of these two city lakes (Xuanwu,Mochou) have similar microbial communities.However,the DGGE profiles of sediment samples in Lake Taihu were significantly differ-ent from these two lakes.Furthermore,the DGGE pro-files of sediment samples in different locations in Lake Taihu were also different,suggesting that the microbial communities in Lake Taihu are more diversified than those in Lake Xuanwu and Lake Mochou.The differences in microbial diversity may be caused by the different environmental conditions,such as redox potential,pH,and the concentrations of organic matters.Seven major bands of 16S rDNA genes fragments from the DGGE profiles of sediment samples were further re-amplified and sequenced.The results of sequencing analysis indicate that five sequences shared 99%-100% homology with known sequences (Bacillus and Brevibacillus,uncultured bacteria),while the other two sequences shared 93%-96% homology with known sequences (Acinetobacter,and Bacillus).The study shows that the PCR-DGGE tech-nique combined with sequence analysis is a feasible and efficient method for the determination of microbial com-munities in sediment samples.     

Microbial community in anaerobic hydrogen-producing microflora enriched from sludge compost

Hydrogen production by thermophilic anaerobic microflora enriched from sludge compost was studied by using an artificial medium containing cellulose powder. Hydrogen gas was evolved with the formation of acetate, ethanol, and butyrate by decomposition of the cellulose powder. The hydrogen production yield was 2.0 mol/mol-hexose by either batch or chemostat cultivation. A medium that did not contain peptone demonstrated a lower hydrogen production yield of 1.0 mol/mol-hexose with less formation of butyrate. The microbial community in the microflora was investigated through isolation of the microorganisms by both plating and denaturing gradient gel electrophoresis (DGGE) of the' PCR-amplified V3 region of 16S rDNA. Sixty-eight microorganisms were isolated from the microflora and classified into nine distinct groups by genetic fingerprinting of the PCR-DGGE or by a random amplified polymorphic DNA analysis and determination of the partial sequence of 16S rDNA. Most of the isolates belonged to the cluster of the thermophilic Clostridium/Bacillus subphylum of low G+C gram-positive bacteria. Product formation by most of the isolated strains corresponded to that produced by the microflora. Thermoanaerobacterium thermosaccharolyticium was isolated in the enrichment culture with or without added peptone. and was detected with strong intensity by PCR-DGGE. Two other thermophilic cellulolytic microorganisms, Clostridium thermocellum and Clostridium cellulosi, were also detected by PCR-DGGE, although they could not be isolated. These findings imply that hydrogen production from cellulose by microflora is performed by a consortium of several species of microorganisms.     

Variability of the microbial community in the western Antarctic Peninsula from late fall to spring during a low ice cover year

Although winter conditions play a major role in determining the productivity of the western Antarctic Peninsula (WAP) waters for the following spring and summer, a few studies have dealt with the seasonal variability of microorganisms in the WAP in winter. Moreover, because of regional warming, sea-ice retreat is happening earlier in spring, at the onset of the production season. In this context, this study describes the dynamics of the marine microbial community in the Melchior Archipelago (WAP) from fall to spring 2006. Samples were collected monthly to biweekly at four depths from the surface to the aphotic layer. The abundance and carbon content of bacteria, phytoplankton and microzooplankton were analyzed using flow cytometry and inverted microscopy, and bacterial richness was examined by PCR–DGGE. As expected, due to the extreme environmental conditions, the microbial community abundance and biomass were low in fall and winter. Bacterial abundance ranged from 1.2 to 2.8 × 10⁵ cells ml^?1 showing a slight increase in spring. Phytoplankton biomass was low and dominated by small cells (<2 μm) in fall and winter (average chlorophyll a concentration, Chl-a, of, respectively, 0.3 and 0.13 μg l^?1). Phytoplankton biomass increased in spring (Chl-a up to 1.13 μg l^?1), and, despite potentially adequate growth conditions, this rise was small and phytoplankton was still dominated by small cells (2–20 μm). In addition, the early disappearing of sea-ice in spring 2006 let the surface water exposed to ultraviolet B radiations (UVBR, 280–320 nm), which seemed to have a negative impact on the microbial community in surface waters.     

草原土壤微生物受放牧的影响及其季节变化

以内蒙古克什克腾旗西部的典型草原为对象,研究轻度放牧区(LG)、中度放牧区(MG)、重度放牧区(HG)土壤中的微生物数量、微生物生物量和土壤呼吸强度的季节变化以及放牧强度对它们的影响。结果表明,微生物数量、微生物生物量以及土壤的呼吸作用强度均有较明显的季节性变化,峰值均出现在8月份,而且三者之间具有极显著的正相关关系;轻度和中度放牧有利于土壤中的微生物数量、生物量的增加,而重度放牧则导致土壤中微生物数量和生物量的减少。     

Influence of Season and Plant Species on the Abundance and Diversity of Sulfate Reducing Bacteria and Ammonia Oxidizing Bacteria in Constructed Wetland Microcosms

Constructed wetlands offer an effective means for treatment of wastewater from a variety of sources. An understanding of the microbial ecology controlling nitrogen, carbon and sulfur cycles in constructed wetlands has been identified as the greatest gap for optimizing performance of these promising treatment systems. It is suspected that operational factors such as plant types and hydraulic operation influence the subsurface wetland environment, especially redox, and that the observed variation in effluent quality is due to shifts in the microbial populations and/or their activity. This study investigated the biofilm associated sulfate reducing bacteria and ammonia oxidizing bacteria (using the dsrB and amoA genes, respectively) by examining a variety of surfaces within a model wetland (gravel, thick roots, fine roots, effluent), and the changes in activity (gene abundance) of these functional groups as influenced by plant species and season. Molecular techniques were used including quantitative PCR and denaturing gradient gel electrophoresis (DGGE), both with and without propidium monoazide (PMA) treatment. PMA treatment is a method for excluding from further analysis those cells with compromised membranes. Rigorous statistical analysis showed an interaction between the abundance of these two functional groups with the type of plant and season (p?<?0.05). The richness of the sulfate reducing bacterial community, as indicated by DGGE profiles, increased in planted vs. unplanted microcosms. For ammonia oxidizing bacteria, season had the greatest impact on gene abundance and diversity (higher in summer than in winter). Overall, the primary influence of plant presence is believed to be related to root oxygen loss and its effect on rhizosphere redox.