Characterization of depth-related microbial communities in lake sediment by denaturing gradient gel electrophoresis of amplified 16S rRNA fragments

The characterization of microbial communities of different depth sediment samples was examined by a culture-independent method and compared with physicochemical parameters, those are organic matter （OM）, total nitrogen （TN）, total phosphorus （TP）, pH and redox potential （Eh）. Total genomic DNA was extracted from samples derived from different depths. After they were amplified with the GC-341 f/907r primer sets of partial bacterial 16S rRNA genes, the products were separated by denaturing gradient gel electrophoresis （DGGE）. The profile of DGGE fingerprints of different depth sediment samples revealed that the community structure remained relatively stable along the entire 45 cm sediment core, however, principal-component analysis of DGGE patterns revealed that at greater sediment depths, successional shifts in community structure were evident. The principle coordinates analysis suggested that the bacterial communities along the sediment core could be separated into two groups, which were located 0-20 cm and 21-45 cm, respectively. The sequencing dominant bands demonstrated that the major phylogenetic groups identified by DGGE belonged to Bacillus, Bacterium, Brevibacillus, Exiguobacterium, γ-Proteobacterium, Acinetobacter sp. and some uncultured or unidentified bacteria. The results indicated the existence of highly diverse bacterial community in the lake sediment core.

Analysis of interspecies adherence of oral bacteria using a membrane binding assay coupled with polymerase chain reaction-denaturing gradient gel electrophoresis profiling

Information on co-adherence of different oral bacterial species is important for understanding interspecies interactions within oral microbial community. Current knowledge on this topic is heavily based on pariwise coaggregation of known, cultivable species. In this study, we employed a membrane binding assay coupled with polymerase chain reaction-denaturing gradient gel electrophoresis （PCR-DGGE） to systematically analyze the co-adherence profiles of oral bacterial species, and achieved a more profound knowledge beyond pairwise coaggregation. Two oral bacterial species were selected to serve as ＂bait＂： Fusobacterium nucleatum （F. nucleatum） whose ability to adhere to a multitude of oral bacterial species has been extensively studied for pairwise interactions and Streptococcus mutans （S. mutans） whose interacting partners are largely unknown. To enable screening of interacting partner species within bacterial mixtures, cells of the ＂bait＂ oral bacterium were immobilized on nitrocellulose membranes which were washed and blocked to prevent unspecific binding. The ＂prey＂ bacterial mixtures （including known species or natural saliva samples） were added, unbound ceils were washed off after the incubation period and the remaining cells were eluted using 0.2 mol.L1 glycine. Genomic DNA was extraeted, subjeeted to 16S rRNA PCR amplification and separation of the resulting PCR produets by DGGE. Selected bands were recovered from the gel, sequenced and identified via Nucleotide BLAST searches against different databases. While few bacterial species bound to S. mutans, consistent with previous findings F.. nucleatum adhered to a variety of bacterial species including uncultivable and uneharacterized onesl This new approach can more effectively analyze the co-adherence profiles of oral bacteria, and could facilitate the systematic study of interbacterial binding of oral microbial species.

Variations in Laboratory-Scale Actinomycete Communities Exposed to Cadmium as Assessed by Denaturing Gradient Gel Electrophoresis Profiles

The actinomycete populations and functions in cadmium （Cd） contaminated soil were investigated by the cultivation- independent molecular methods. The genomic DNA was extracted and purified from soil adulterated with various con- centrations of Cd in the laboratory. The partial 16S rDNA genes were amplified by polymerase chain reaction （PCR） using specific primers bound to evolutionarily conserved regions within these actinomycete genes. The diversity in PCR- amplified products, as measured by denaturing gradient gel electrophoresis （EGGE）, was used as a genetic fingerprint of the population. Principle component analysis and Shannon-Weaver diversity index （H） analyses were used to analyze the DGGE results. Results showed that the two principal components accounted for only a low level of the total variance. The value H in contaminated soil was lower than that in the control at later stages of cultivation, whereas at earlier stages it was higher. Among the six sampling time points, the first, fifth and sixth weeks had the highest values of H. Significantly negative correlations between bioavallable Cd concentration and H values existed in the samples from weeks 2 （R = 0.929, P 〈 0.05） and 4 （R = 0.909, P 〈 0.05）. These results may shed light on the effect of Cd on the soil environment and the chemical behavior and toxicity of Cd to actinomycetes.

Monitoring of microbial community structure and succession in the biohydrogen production reactor by denaturing gradient gel electrophoresis(DGGE)

To study the structure of microbial communities in the biological hydrogen produc-tion reactor and determine the ecological function of hydrogen producing bacteria,anaerobic sludge was obtained from the continuous stirred tank reactor(CSTR)in different periods of time,and the diversity and dynamics of microbial communities were investigated by denaturing gra-dient gel electrophoresis(DGGE).The results of DGGE demonstrated that an obvious shift of microbial population happened from the beginning of star-up to the 28th day,and the ethanol type fermentation was established.After 28 days the structure of microbial community became stable,and the climax community was formed.Comparative analysis of 16S rDNA sequences from reamplifying and sequencing the prominent bands indicated that the dominant population belonged to low G+C Gram-positive bacteria(Clostridium sp.and Ethanologenbacterium sp.),β-proteobacteria(Acidovorax sp.),γ-proteobacteria(Kluyvera sp.),Bacteroides(uncultured bacte-rium SJA-168),and Spirochaetes(uncultured eubacterium E1-K13),respectively.The hydrogen production rate increased obviously with the increase of Ethanologenbacterium sp.,Clostridium sp.and uncultured Spirochaetes after 21 days,meanwhile the succession of ethanol type fer-mentation was formed.Throughout the succession the microbial diversity increased however it decreased after 21 days.Some types of Clostridium sp.Acidovorax sp.,Kluyvera sp.,and Bac-teroides were dominant populations during all periods of time.These special populations were essential for the construction of climax community.Hydrogen production efficiency was de-pendent on both hydrogen producing bacteria and other populations.It implied that the co-metabolism of microbial community played a great role of biohydrogen production in the reactors.

Denaturing gradient gel electrophoresis fingerprinting of soil bacteria in the vicinity of the Chinese Great Wall Station, King George Island, Antarctica

Bacterial diversity was investigated in soil samples collected from 13 sites around the Great Wall Station, Fildes Peninsula, King George Island, Antarctica, using denaturing gradient gel electrophoresis （DGGE） of 16S rRNA genes. The classes α-, β-, and γ-Proteobacteria, as well as the phylum Actinobacteria, were found to be the dominant bacteria in the soils around the Great Wall Station. Although the selected samples were not contaminated by oil, a relationship between soil parameters, microbial biodiversity, and human impact was still seen. Sample sites in human impacted areas showed lower bacterial biodiversity （average H′= 2.65） when compared to nonimpacted sites （average H′= 3.05）. There was no statistically significant correlation between soil bacterial diversity and total organic carbon （TOC）, total nitrogen, or total phosphorus contents of the soil. Canonical correlation analysis showed that TOC content was the most important factor determining bacterial community profiles among the measured soil parameters. In conclusion, microbial biodiversity and community characteristics within relatively small scales （1.5 km） were determined as a function of local environment parameters and anthropogenic impact.

3种植物精油对调理牛排优势腐败菌的抑菌性

为明确调理牛排的优势腐败菌并筛选出抑菌性、专一性强的植物精油,通过传统培养基法结合聚合酶链式反应-变性梯度凝胶电泳技术筛选出调理牛排的优势腐败菌,并通过抑菌圈、最小抑菌浓度、最小杀菌浓度及细菌生长曲线,从丁香精油、豆蔻精油、黑胡椒精油中选出抑菌性最强的植物精油。结果表明:优势腐败菌为深蓝紫色杆菌(Janthinobacterium lividum)、恶臭假单胞菌(Pseudomonas putida)和热杀索丝菌(Brochothrix thermosphacta);丁香精油的抑菌圈直径均大于10 mm,2~4μL/mL的精油添加量就能明显抑制优势腐败菌的生长繁殖;豆蔻精油抑菌性稍弱;黑胡椒精油对3株优势腐败菌均无明显抑菌效果。

GaAs太阳电池中减反射薄膜的梯度折射率调控

使用溶胶凝胶法制备了TiO_(2)和SiO_(2)两种材料,结合梯度折射率的结构设计将不同比例的TiO_(2)/SiO_(2)沉积在GaAs太阳能电池上实现宽带减反射效果,最终制备的6层减反射薄膜在200~1 800 nm的波长范围内平均反射率为8.7%,同时确定了300℃的退火温度和1 000μm/s的提拉速度为制备薄膜的最佳工艺条件.

壳聚糖的提取与抗菌凝胶制备的实验教学设计

壳聚糖是甲壳素脱乙酰化后的产物,由于它具有良好的生物相容性和抗菌能力,被广泛用于抗菌消毒、药物递送等生物医用领域。本科普实验基于废物利用的可持续发展理念,选用生活中常见的厨余垃圾虾壳为原料,完成了对虾壳中甲壳素的提取与壳聚糖的制备。并通过一系列表征手段对文章中的观点加以佐证,同时根据本实验制备的壳聚糖衍生出一种壳聚糖抗菌消毒凝胶。我们采用梯度科普方案,让幼儿园小朋友及小学生、广大青少年、大学生与社会公众感受到化学的魅力所在,激发他们学习化学知识的兴趣,并在最后呼吁大家一起摒弃成见,重新审视化学,以更加深远的眼光领略化学之妙。

浓度梯度掺杂实现BiFeO_(3)薄膜自极化

BiFeO_(3)是一种非常有前途的无铅铁电材料,与大多数传统铁电材料相比,它具有更大的极化和更高的居里温度,为高温应用提供了可能。受到衬底强烈的夹持效应、较大的矫顽场和漏电流的影响,BiFeO_(3)薄膜难以被极化。自极化是解决这一问题的可行方法。本研究采用溶胶-凝胶法在Pt(111)/Ti/SiO_(2)/Si衬底上生长了BiFeO_(3)薄膜,向上梯度薄膜(从衬底BiFeO_(3)过渡到薄膜表面Bi_(0.80)Ca_(0.20)FeO_(2.90))以及向下梯度薄膜(从衬底Bi_(0.80)Ca_(0.20)FeO_(2.90)过渡到薄膜表面BiFeO_(3))。通过细致地调控薄膜内部缺陷的定向分布形成内置电场,从而导致薄膜具有自极化特性。压电力显微镜结果表明:在BiFeO_(3)薄膜中,Ca的梯度方向可以调控自极化的方向。此外,类似二极管的单向导通特性验证了薄膜的自极化是由Ca的浓度梯度掺杂导致。X射线光电子能谱结果表明,氧空位的梯度分布导致的内置电场可能是造成自极化现象的原因。本研究为实现铁电薄膜的自极化提供了一种新的策略,并在以自极化的内置电场为驱动,提高光伏或光敏器件性能方面具有潜在的应用前景。

Preparation and dielectric properties of compositionally graded (Ba,Sr)TiO_3 thin film by sol-gel technique

Compositional graded BaxSr1-xTiO3 (x=0.6, 0.7, 0.8, 0.9, 1.0) (BST) thin films (less than 400 nm) were fabricated on Si and Pt/Ti/SiO2/Si substrates by sol-gel technique. A special heating treatment was employed to form the uniform composition gradients at 700 ℃. The microstructures of the films were studied by means of X-ray diffraction, atomic force microscope and field emission scanning electron microscopy. The results show that the films have uniform and crack-free surface morphology with perovskite structure phase. The small signal dielectric constant (εr) and dielectric loss (tan δ) are found to be 335 and 0.045 at room temperature and 200 kHz. The dielectric properties change significantly with applied dc bias, and the graded thin film show high tunability of 42.3% at an applied field of 250 kV/cm. All the results indicate that the graded BST thin films prepared by sol-gel technique have a promising candidate for microelectronic device.

江西部分地区微小扇头(牛)蜱中肠优势菌群分析

分析不同地区、不同宿主微小牛蜱中肠内容物优势菌群。利用变性梯度凝胶电泳(PCR-DGGE)技术对江西省宜春、萍乡、万载、新余四个地区的牛、羊体表微小牛蜱中肠细菌群落的结构进行分析。微小牛蜱体内细菌主要有8种,分别为立克次氏体、柯克斯氏体、犬埃立克体、克雷伯氏菌、肠杆菌、假单胞菌、孔雀立克次氏体、弯曲芽孢杆菌。牛体表微小牛蜱肠道优势菌主要为柯克斯氏体和立克次氏体。萍乡地区微小牛蜱除以上两种菌外,还含有犬埃立克体。克雷伯氏菌仅见于新余地区。山羊体表微小牛蜱肠道菌数量少于黄牛体表的微小牛蜱,其优势菌主要为假单孢菌。不同地区、不同宿主微小牛蜱中肠内容物优势菌群存在一定差异。

亚铁离子对罗非鱼片细菌生长的影响

采用2 g/100 mL的氯化亚铁溶液处理罗非鱼片,定期取样测定鱼片的感官评分、菌落总数、挥发性盐基氮(total volatile basic nitrogen,TVB-N)和三甲胺(trimethylamine,TMA)含量,结合聚合酶链式反应-变性梯度凝胶电泳(polymerase chain reaction-denaturing gradient gel electrophoresis,PCR-DGGE)分析亚铁离子对鱼片上细菌生长的影响。结果表明,氯化亚铁处理鱼片的感官较好,菌落总数、TVB-N和TMA含量较对照鱼片明显降低,对照鱼片和氯化钠处理鱼片在4℃冷藏9 d,菌落总数、TVB-N和TMA含量超过国家标准规定限量,而氯化亚铁处理鱼片的菌落总数、TVB-N和TMA含量未超标。通过PCR-DGGE分析可知,氯化亚铁处理鱼片在冷藏初期的菌相复杂,冷藏6 d后,腐败希瓦氏菌(Shewanella putrefaciens)、嗜冷杆菌(Psychrobacter)数量增多,假单胞菌(Pseudomonas)数量减少,但Pseudomonas在整个菌相中依旧数量最多,仍是优势菌。

红曲醋酿造过程中菌群动态分析

红曲醋是在多菌种共同作用下发酵酿造而成,其酿造周期与微生物有着重要关系。研究通过聚合酶链式反应-变性梯度凝胶电泳(polymerase chain reaction-denaturing gradient gel electrophoresis,PCR-DGGE)和高通量测序对红曲醋中的微生物菌群结构进行分析,并借助热图分析醋中的优势菌群,为揭示红曲醋中的微生物演替规律提供依据,并对缩短永春老醋酿造周期奠定基础。高通量测序研究结果发现,红曲醋中的优势菌群是乳酸菌(Lactobacillus)、醋酸菌(Acetobacter)、红球菌(Rhodococcus)、苍白杆菌(Ochrohactrum)、芽孢杆菌(Bacillus)、沉积物杆状菌(Sediminibacterium)、不动杆菌(Acinetobacter)、甲基杆菌(Methylobacterium)、酵母菌(Saccharomycetales sp.)、解脂耶氏酵母(Yarrowia lipolytica)、子囊菌(Ascomycota sp.)、假丝酵母菌(Candida xylopsoci)和毛孢壳属(Coniochaetaceae sp.)。芽孢杆菌(Bacillus)与酵母菌(Saccharomycetales sp.)有拮抗作用,红曲霉菌(Monascus purpureus)与克拉菌(Cryocola)、无氧芽孢菌(Anoxybacillus)、沙雷氏菌(Serratia)、微杆菌(Microbacterium)、甲基杆菌(Methylobacterium)有较强的促进作用。

PCR-DGGE技术分析韩式大酱与中式大酱中微生物多样性

微生物在大酱发酵过程中起到至关重要的作用,并与大酱的风味与质量密切相关,因此研究大酱中微生物的多样性有重要意义。该研究选择加工工艺不同的韩式大酱与中式大酱为对象,采用聚合酶链式反应-变性梯度凝胶电泳(polymerase chain reaction-denaturing gradient gel electrophoresis,PCR-DGGE)技术,通过PCR扩增、切胶回收、PCR测序等分析不同大酱中的微生物多样性。结果表明,大酱中的微生物由于制作工艺的不同存在明显差异。在韩式大酱中,细菌如芽孢杆菌属(Bacillus)、不动杆菌属(Acinetobacter)、四联球菌属(Tetragenococcus)、嗜盐单胞菌属(Halomonas),真菌如根毛霉属(Rhizomucor)、青霉菌属(Penicillium)、毛霉属(Mucor)、外瓶霉属(Exophiala)、曲霉属(Aspergillus)等分布广泛。在中式大酱中,乳酸菌如片球菌属(Pediococcus)、乳杆菌属(Lactobacillus)、明串珠菌属(Leucanostoc)、肠杆菌属(Enterobacter),酵母如鲁氏接合酵母(Zygosaccharomyces rouxii)分布较多。与中式大酱相比,韩式大酱中的真菌种类更丰富。研究结果为进一步探讨传统发酵大酱品质提供了理论依据。

PCR-DGGE Analysis of Bacterial Communities Structure in Babylonia areolata Culture Systems of The Subtidal Zone and The Pond Mulched Plastic Film and Sand in Bottom

To know the bacterial communities structure in Babylonia areolata culture systems and to research and optimize the management pattem of Babylonia areola-ta culture systems of the pond mulched plastic film and sand in bottom, the bacte- rial communities in Babylonia areolata culture systems of the sub-tidal zone and the pond mulched plastic film and sand in bottom were analyzed at molecular level by adopting the denaturing gradient gel electrophoresis （DGGE）. The results indicated that the dominant bacterial communities in Babylonia areolata culture systems of the sub-tidal zone and the pond mulched plastic film and sand in bottom, which were built on the basis of the seawater in East-island of Zhanjiang, included Proteobac- teda Chloroflexi, Cyanobacteria and Actinobacteria. The dominant bacterial groups in the above pond culture system were Garnmaproteobacteria, Alphaproteobacteria, Deltaprotecbacteda, Epsilonproteobacteda, Anaerolineae, Cyanobacteria and Acti- nobacteda. The dominant bacterial communities in the subtidal zone culture system were Gammaprotecbacteda, Alphaproteobacteria, Deltaproteobacteria, Anaerolineae and Cyanobacteda, and there were less Epsilonproteobacteria and Actinobacteria in the culture system. The higher diversity was detected in the above two culture sys- tems. The results of unweighted pair group method with arithmetic average （UPG- MA） showed that the bacterial communities of the sediment samples S1 and S2 in the above two culture systems were a cluster, the similarity of bacterial communities was 54.5%. The bacterial communities of seawater samples S3 and S4 in the above culture systems were in clusters, and the similarity of the bacterial communi- ties was 84.0%. The results showed that the microorganism ecological level in the Babylonia areolata culture systems of the pond mulched plastic film and sand in bottom could be similar to the sub-tidal zone culture systems through changing the pond seawater and monitoring the microbial population.

PCR-DGGE技术用于湖泊沉积物中微生物群落结构多样性研究

采用PCR-DGGE分子指纹图谱技术比较南京市玄武湖、奠愁湖和太湖不同位置的表层沉积物微生物群落结构，研究结果表明，三湖泊沉积物微生物的16SrDNA的PCR扩增结果约为626bp，为16S rDNA V3～V5区特异性片段。玄武湖和莫愁湖表层沉积物中大约有20种优势菌群，且同一湖泊不同采样点DGGE图谱的差异性不大，细菌群落结构具有较高的相似性，而太湖样品DGGE条带的数目和位置表现出明显差异，且不同采样点图谱的差异性较大。三湖泊除具有特征性的微生物种属外，还分布约5个相同的细菌种群，可能与沉积物的理化性质和水生植被的影响相关。对DGGE图谱中7条主带进行回收、扩增和测序，结果显示其优势菌群具有不同的序列组成，其中5个序列与Genebank中已登录的细菌种群的同源性≥99％，2个序列的同源性为96％和93％，其中2个相似的细菌类群目前尚未获得纯培养。

PCR-DGGE技术在农田土壤微生物多样性研究中的应用

变性梯度凝胶电泳技术 ( DGGE)在微生物生态学领域有着广泛的应用。研究采用化学裂解法直接提取出不同农田土壤微生物基因组 DNA,并以此基因组 DNA为模板 ,选择特异性引物 F357GC和 R518对 1 6Sr RNA基因的 V3区进行扩增 ,长约 2 30 bp的 PCR产物经变性梯度凝胶电泳 ( DGGE)进行分离后 ,得到不同数目且分离效果较好的电泳条带。结果说明 ,DGGE能够对土壤样品中的不同微生物的 1 6Sr RNA基因的 V3区的 DNA扩增片断进行分离 ,为这些 DNA片断的定性和鉴定提供了条件。与传统的平板培养方法相比 ,变性梯度凝胶电泳 ( DGGE)技术能够更精确的反映出土壤微生物多样性 ,它是一种有效的微生物多样性研究技术。

秸秆还田后土壤微生物群落结构变化的初步研究

通过实验室条件下的小麦秸秆粉和油菜秸秆粉的还土试验,结合常规的分析手段和基于DNA的分子技术(变性梯度凝胶电泳(DGGE)及测序技术),初步研究了秸秆粉还田后土壤物理化学特性及生物学特性的变化。结果表明,培养60d后,秸秆还田土壤的肥力明显提高,其纤维素酶活性明显增强。DGGE图谱表明,对照土壤(S)以及处理土壤(SW和SR),在培养过程中,β-Proteobacteria类细菌组成都在发生变化。对其中一个样品的DGGE条带进行了割胶测序,测序结果表明,大部分条带代表的微生物是未培养的或不可培养的。采用传统分离培养技术,从秸秆还田土壤中分离了2株纤维素降解菌。以上结果说明,秸秆还田具有良好的土壤综合效应。

大港孔店油田水驱油藏微生物群落的分子分析

通过多聚酶链式反应 温度梯度凝胶电泳(PCR TGGE)和构建16SrRNA基因克隆文库两种方法对比研究了大港油田孔二北断块注水井和采油井的微生物群落结构。16SrDNAV3区PCR扩增产物的TGGE图谱分析表明,这两个油井的微生物群落结构差异很大。注水井样品的TGGE图谱中有6条主要条带,而采油井样品中只有一个条带占绝对优势。同时,建立了两个样品的16SrRNA基因克隆文库,从中分别挑选了10 8和5 0个克隆进行限制性酶切片段长度多样性分析(ARDRA)。注水井样品有33个操作分类单元(OTU) ,其中6个OTU是优势类型;而采油井样品只有8个OTU ,有1个OTU在文库中占绝对优势。克隆文库和TGGE的研究结果一致,均表明注水井样品的微生物多样性比采油井丰富很多。每个OTU的代表克隆序列分析结果表明,注水井样品中的细菌主要属于α、β、γ变形菌纲和放线菌纲,尤其是红细菌亚纲( 4 7% )。采油井样品的细菌主要属于α、β、γ变形菌纲,尤其是假单胞菌属( 6 2 % )。

堆肥化过程中微生物群落的动态

通过变性梯度凝胶电泳（DGGE）和平板计数法对堆肥化过程中微生物的区系动态变化进行了研究.结果表明,在堆肥化过程中,微生物数量总的趋势是细菌的数量最多,放线菌次之,真菌的数量最少,中温微生物的数量始终高于高温微生物.当发酵结束后,中温微生物的数量低于发酵初始水平,高温放线菌和高温真菌的数量试验结束后高于初始水平,高温细菌的数量在整个堆肥化过程中变化不大.通过DGGE分析表明,发酵过程中细菌的种类发生了明显的更迭现象.发酵初期Bdellovibrio、Clostridiabacterium、Bacillus、Clostridium等占优势,中期Beta proteobacterium、Petrobacter succinimandens、Nitrospiraebacterium、Clostridium等占优势,后期Clostridium、Beta proteobacterium、Paenibacillus等占优势,而在整个堆肥化过程中Clostridium都是优势种.