DGGE和T-RFLP在堆肥微生物群落结构研究中的应用

对堆肥微生物种群分布及其动态变化的研究进行了分析,论述了分子生物技术中的变性梯度凝胶电泳和末端标记限制性片段长度多态性的原理和特点,以及用于研究堆肥微生物的群落结构演变规律,为分析和筛选堆肥中的微生物提供更加有效、快速的信息,促进堆肥技术的发展。     

变性梯度凝胶电泳在堆肥微生物研究中的应用*

对当前堆肥中微生物种群分布及其对有机物分解作用的研究进行分析 ,论述了分子生物技术中的变性梯度凝胶电泳 (DGGE)的特点。将DGGE与PCR扩增技术相结合 ,可用于研究自然菌种堆肥和人工培养驯化菌种堆肥过程中微生物的演替规律 ,为研究和筛选堆肥中的微生物提供更加有效、快速的信息 ,促进堆肥技术的发展。     

四种小鼠肠道微生物DNA提取方法比较

采用异硫氰酸胍(guandine thiocyanate,GITC)法、Tiangen DNA提取试剂盒、Omega DNA提取试剂盒和广泛应用的十六烷基三甲基溴化铵(hexadecyl trimethyl ammonium bromide,CTAB)法提取小鼠粪便微生物总DNA,通过比较所提取DNA的浓度和纯度,发现粪便DNA提取试剂盒提取的DNA纯度最高但浓度最低;CTAB法所得的DNA浓度最高但纯度最低;GITC法所得DNA的浓度高于粪便DNA提取试剂盒,纯度高于CTAB法。通过变性梯度凝胶电泳(16S r DNA-PCR-DGGE)指纹图谱分析技术进一步比较了各种提取方法所代表微生物群落的丰富度和多样性。结果表明,GITC法提取得到的DNA所代表细菌的丰富度和多样性显著高于其他3种方法。本实验所建立的GITC法可更全面地反映肠道微生物的多样性和群落结构,是一种较为理想的粪便微生物DNA提取方法。     

PCR-DGGE方法分析原油储层微生物群落结构及种群多样性

使用基于 16 S r DNA的 PCR- DGGE(变性梯度凝胶电泳 )图谱分析结合条带割胶回收 DNA进行序列分析 ,对新疆克拉玛依油田一中区注水井 (12 # 9- 11)和与该注水井相应的两个采油井 (12 # 9- 9S、13# 11- 8)井口样品微生物群落的多样性进行了比较并鉴定了部分群落成员。 DGGE图谱聚类分析表明注水井与两油井微生物群落的相似性分别为 30 %和 2 0 % ,而两油井间微生物群落结构的相似性为 5 4 %。DGGE图谱中优势条带序列分析表明注水井样品和油井样品中的优势菌群为未培养的环境微生物 ,它们与数据库中 α、γ、δ、ε变形杆菌 (Proteobacteria)和拟杆菌 (Bacteroidetes)有很近的亲缘关系。 DGGE与分子克隆相结合的分子生物学方法在研究微生物提高原油采收率 (MEOR)机理 ,以及指导 MEOR在油田生产中的应用有着重要的意义     

稀土尾矿土壤细菌群落结构对植被修复的响应

选用赣州-安远稀土弃废尾矿及其不同植被修复的堆浸田为研究对象,调查废弃尾矿及6种不同植被修复方案下土壤理化性质的变化,并利用变性凝胶梯度电泳(DGGE)技术,分析土壤微生物群落结构对植被修复的响应。结果表明:与未修复尾矿土壤相比,经不同植被修复后的土壤理化性质均得到明显改良,其中土壤含水量、有机质含量均比未修复尾矿土壤增加2—3倍。微生物群落结构分析表明,植被修复后土壤微生物群落与废弃尾矿土壤微生物群落亲缘度仅为0.21,表明植被修复后,土壤微生物群落结构发生了明显变化,且微生物多样性、均匀度、丰富度与未修复尾矿土壤相比均有了明显的提高。而在不同植被修复方案中,以湿地松和山胡椒为优势群落的两种植被修复方案对土壤改良效果最为明显,这两种修复方案不仅能显著改善土壤的固水性、有机质含量,并且对微生物群落的改善作用也最为显著。典范对应分析表明,废弃尾矿土壤微生物群落结构受土壤p H影响最为显著,而植被修复后土壤微生物群落的环境影响因子则转变为含水量、有机质、有机碳及总磷含量。进一步揭示了微生物在植被修复过程中所起到的重要作用,并为矿山生态重建过程中的土壤改良工作提供了丰富的理论依据。     

利用PCR-DGGE方法分析不同鸡群的盲肠微生物菌群结构变化

研究不同品种、饲养阶段的健康和不良鸡群对盲肠细菌种群结构和多样性的影响。使用基于16S rDNA的PCR-DGGE技术,结合割胶回收DNA进行克隆和测序,分别以4、6、10、16、20、40周龄蛋鸡及1、2、4、6、7、8周龄肉鸡健康、不良鸡群盲肠内容物为样本,研究其中特定细菌类群的16S rDNA序列片段指纹图谱,并进行聚类分析,鉴定特异性和共性种群。在两品种健康鸡群盲肠内容物的细菌群落中,Lactobacillus属菌株的相似性均高于不良鸡群,并且在不同饲养阶段的健康、不良鸡群间指纹图谱平均条带数差异显著(P<0.05);而Bacteroides属菌株在健康鸡群盲肠内容物细菌的相似性与不良鸡群较为相近,健康、不良鸡群间平均条带数差异显著(P<0.05);Clostridium属菌株在蛋鸡20、40周龄的平均条带数差异不显著(P>0.05),但肉鸡各周龄健康、不良鸡群间的平均条带数差异显著(P<0.05)。序列测序结果,在蛋鸡产蛋期健康、不良鸡群样本中均检测到能动乳杆菌(Lactobacillus agilis),而育雏和育成期中均检测到鸟乳杆菌(Lactobacillus aviaries)和不可培养细菌;两品种的健康、不良鸡群样本中均检测到Bacteroides属的生酸拟杆菌(Bacteroides acidifaciens)、不可培养物细菌(Uncultured bacterium);而健康、不良蛋鸡群样本中均检测到Clostridium属不可培养的变形菌(Uncultured proteobacterium),健康肉鸡群中检测到索氏志贺菌(Shigella sonnei),而两品种不良鸡群中均缺乏此类菌种。结果显示,不同品种、饲养阶段的鸡群,其盲肠细菌群落的组成差异显著,并且细菌种群结构对鸡群的生长发育影响较大。     

黄海北部不同站位海洋细菌群落分布特征

[目的]为揭示北黄海不同海域中真细菌群落分布的差异,[方法]采用16s rRNA基因文库和变性梯度凝胶电泳(DGGE)技术,对远海和近海两个站位的沉积物和水体中细菌群落特征进行了解析和评价.[结果]文库分析揭示海水及沉积物中微生物种类丰富,存在大量未被认知的类群.各站位中主要为变形菌门(Proteobacteria),沉积物中γ-Proteobacteria和δ-Proteobacteria亚门占优势,水中则以α-Proteobacteria亚门占优势,但各亚门微生物在两个站位中存在明显系统发育学分歧.DGGE图谱聚类分析显示,近海沉积物和海水中细菌群落优势类群相似性很高,而远海沉积物和海水中则相似性很低.[结论]研究结果表明,微生物种类在不同地理位置和生存介质中存在明显差异,环境因素对微生物的分布起主导作用.     

云南腾冲热海两热泉菌藻席细菌多样性的研究

应用显微形态观察和变性梯度凝胶电泳(DGGE)对云南腾冲热海两热泉菌藻席的细菌多样性进行了比较分析.直接从环境样品中提取总DNA,用两套细菌通用引物进行PCR扩增,得到包含V8和V9高变区的16S rDNA片段,进行DGGE分析,结合形态观察,结果显示,热泉菌藻席中存在丰富的细菌多样性,且不同温度范围的菌藻席细菌组成差异显著.     

2型糖尿病BKS-DB小鼠的小肠菌群结构差异性分析

研究肠道中的微生物群落与糖尿病之间的关系,对于预防治疗糖尿病具有十分重要的意义.以10只2型糖尿病小鼠和10只健康小鼠为对象,通过PCR-DGGE(polymerase chain reaction-denaturing gradient geleletrophoresis)分子技术分析小鼠小肠菌群结构.实验结果显示,实验组小鼠与对照组小鼠在多样性指数、丰富度指数及优势度指数上存在差异,对照组小鼠小肠内容物的菌群种类及数量明显高于实验组小鼠;而两组小鼠的小肠内容物的优势菌种类及相对含量却相似.特异性条带测序结果显示,正常小鼠小肠内含有乳杆菌,而实验组小鼠小肠内含量很低,甚至检测不到.该结果提示了乳杆菌和2型糖尿病之间的关系.     

综合养殖池塘中三角帆蚌和鱼类肠道细菌的组成

采用 PCR-DGGE (PCR-denaturing gradient gel electrophoresis)方法研究了综合养殖池塘中三角帆蚌(Hyriopsis cumingii)、草鱼(Ctenopharyngodon idella)、鳊(Parabramis pekinensis)、银鲫(Carassius auratus gibelio)、青鱼(Mylopharyngodon piceus)和鳙(Aristichthys nobilis)的肠道细菌组成,并分析了水体中的浮游细菌对鱼、蚌肠道细菌的影响。研究结果表明,三角帆蚌和混养鱼类肠道细菌属于厚壁菌门(Firmicutes)、变形菌门(Proteobacteria)、放线菌门(Actinobacteria)、疣微菌门(Verrucomicrobia)、蓝细菌(Cyanobacteria)、梭杆菌门(Fusobacteria)。其中,三角帆蚌肠道优势菌群为不动杆菌属(Acinetobacter)、志贺氏菌属(Shigella)和分枝杆菌属(Mycobacterium),草鱼肠道优势菌群为梭菌属(Clostridium),鳊肠道优势菌群为梭菌属和假单胞菌属(Pseudomonas),银鲫肠道优势菌群为不动杆菌属和志贺氏菌属,青鱼肠道优势菌群为梭菌属和不动杆菌属,鳙肠道优势菌群为不动杆菌属和弧菌属(Vibrio)。三角帆蚌与银鲫肠道细菌谱带相似性较高,草鱼与鳊肠道细菌谱带相似性较高,表明鱼类肠道细菌组成特点与食性存在一定的关系。水体浮游细菌优势菌群为类芽孢杆菌属(Paenibacillus)。三角帆蚌及鱼类肠道细菌群落与水体浮游细菌群落组成相似性较低,表明水体浮游细菌对三角帆蚌和鱼类肠道细菌优势菌群的影响有限。     

The fate of total petroleum hydrocarbons during oily sludge composting: a critical review

Reviews in Environmental Science and Bio/Technology - In this review, studies on petroleum sludge composting have been investigated in the aim to understand the sludge biotransformation and...     

Biodegradation of heavy oily sludge by a two-step inoculation composting process using synergistic effect of indigenous isolated bacteria

The impact of two-step inoculation of indigenous strains and their synergistic effect in the scaling-up of petroleum hydrocarbons biodegradation from a mineral-based medium (MBM) to a two-phase composting process were investigated. After isolating the strains KA3 and KA4 from heavy oily sludge (HOS), their emulsification index (E₂₄), bacterial adhesion to hydrocarbon (BATH), and oil degradation efficiency were evaluated in the MBM. Then, they were inoculated twice into the composting bioreactors lasted for the primary 8 weeks as the first phase (FP) and subsequent 8 weeks as the second phase (SP). The results indicated that the consortium of the two strains degraded 16-61% of crude oil (1-5% concentration) in the MBM. In the composting reactors, removals of 20 g kg⁻¹ initial concentration of total petroleum hydrocarbons (TPH) were found to be 63.95, 61.00, and 89.35% for the strains KA3, KA4, and their consortium, respectively. The computed biodegradation constants indicated the synergistic effect of the two strains and the effectiveness of the second-step inoculation. The study demonstrated the successful scaling-up of HOS biodegradation from MBM to the two-phase composting process through two-step inoculation of the isolated strains.     

Comparison of bio-augmentation and composting for remediation of oily sludge: A field-scale study in China

Two bioremediation technologies were performed in order to explore a better treatment process for an oily sludge restoration in China during 2004. The bioremediation by augmentation of biopreparation was compared with a conventional composting. The oily sludge and oil-polluted soil were received from an oil production plant. The total hydrocarbon content (THC) varied from 327.7 to 371.2 g kg⁻¹ of dry sludge and the THC in contaminated soil was 151.0 g kg⁻¹. Before application of preparation, straw, sawdust, top sand and pure soil were added in different proportions to the sludge and soil and mixed thoroughly. Such sludge and soil composites were used for negative controls and for activation of indigenous oil degrading microorganisms with addition of fertilizer (positive controls). For composting, crude manure and straw were added to the oily sludge and the THC was 101.4 g kg⁻¹. The biopreparation was applied every 2 weeks and experiment lasted 56 days under the ambient temperature. The sludge was mixed and watered every 3 days. After three times of biopreparation application, the THC decreased by 46–53% in the oily sludge and soil, while in the positive controls (activation of indigenous microorganisms) the THC decreased by 13–23%, and there was no oil degradation in negative controls After composting, the THC decreased by 31% in the oily sludge. The planting of Tall Fescue (Festuca arundinace) revealed a decrease of sludge toxicity after application of both bioremediation technologies and additionally decreased the THC by 5–7%.     

Biotreatment of restaurant wastewater with an oily high concentration by newly isolated bacteria from oily sludge

DNA methylation has been introduced as a promising biomarker for different diseases. Alterations in macrophage DNA methylation status have been documented during Mycobacterium tuberculosis (Mtb) infection. We conducted this study using a human methylation PCR array kit, which comprised a panel of 22 genes in TLR2 signaling pathway, in order to gain insights into epigenetic interactions between drug-susceptible and -resistant Mtb strains and THP-1-derived macrophages (one of the main host immunity cells during TB infection). We also evaluated the expression of Rv1988 gene in the studied isolates. It was found that the methylation level of all of the studied inflammatory genes, except Irak-2 and Tbk-1, increased in THP-1 macrophages, which were infected by extensively drug-resistant (XDR) Mtb strains, compared with the mock cells (P?<?0.05). In susceptible strains, we only found hypomethylation in Irak-2 gene, in addition to a slight increase in the methylation levels of Ubev, Ube2n, and Traf6 genes. The present findings provide new insights into the potential role of resistant and susceptible Mtb strains in promoting aberrant epigenetic modifications in macrophages. Further investigations on the host epigenomes, infected with different Mtb isolates, are needed to elucidate their functions in immunological responses and to introduce new effective tools against Mtb infection.

     

Vermicomposting of tannery sludge mixed with cattle dung into valuable manure using earthworm Eisenia fetida (Savigny)

The present study revealed the role of earthworm in converting tannery sludge into a valuable product. Tannery sludge was toxic to earthworm, therefore it was mixed with cattle dung in different proportions viz. 0:100 (T₀), 10:90 (T₁₀), 25:75 (T₂₅), 50:50 (T₅₀) and 75:25 (T₇₅) on dry weight basis. The minimum mortality and highest population buildup of worms was in T₀ mixture. Nitrogen, sodium, phosphorus and pH increased from initial in the range of 7.3-66.6%, 16.90-70.58%, 8.57-44.8% and 2.8-13.65%, respectively. On the other hand potassium, organic carbon and electrical conductivity decreased in the range of 4.34-28.5%, 7.54-22.35% and 32.35-53.12%, respectively. C:N ratio decreased from 20.53% to 47.36% in the final products. Transition metals increased significantly from the initial value and within the permissible limit. The result indicated that vermicomposting with Eisenia fetida is better for changing this sludge into nutrient rich manure in a short period of time.     

Effect of dung burial by the dung beetle Bubas bison on numbers and viability of Cryptosporidium oocysts in cattle dung

Cryptosporidium oocysts were inoculated into fresh dung (∼1.2 × 10⁴ oocysts per gram wet weight) and fed to dung beetles to assess the effect of dung burial by the dung beetle Bubas bison on the distribution of the oocysts in small cores of soil in the laboratory. The experiment consisted of five replicates of each of two treatments; controls (dung but no dung beetles) and the experimental treatment (inoculated dung and seven pairs of dung beetles). After 5 days, when approximately 90% of the dung was buried, the surface and buried dung was recovered and subsampled. The oocysts in the subsamples were recovered and enumerated using qPCR. Oocyst viability was evaluated using an assay based on the exclusion or inclusion of two fluorogenic vital dyes, 4′,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI). Results revealed that overall 13.7% of oocysts remained on the surface and 86.3% of oocysts were buried. The viability of oocysts in buried dung was only 10% compared to oocysts the surface dung (58%). Therefore, widespread dung burial by B. bison during the winter months could substantially reduce the numbers of Cryptosporidium oocysts available to be washed into waterways following winter rains.     

Molecular analyses of methanogen diversity associated with cattle dung

Methanogen communities were characterized in cattle dung of different ages by using a culture-independent approach. Community structures were determined by the phylogenetic analyses of methyl-coenzyme M reductase A (mcrA) clones of fresh, 8-month-old, and 24-month-old-dry dung samples. The clones in the mcrA libraries of fresh and 8-month old dung samples were identified as belonging to Methanomicrobiales, Methanobacteriales, and Methanosarcinales. However, clones in the library of 24-month-old dung were not affiliated to Methanomicrobiales. Anaerobic digestion of 2-month-old dung produced only 15% less methane compared to fresh dung which indicated the possibility of using dry dung to fuel the biogas plants in areas where unavailability of fresh dung hinders their continuous functioning. Our results first time showed the presence of viable methanogens in dry cattle dung stored for prolonged periods of time.     

Biodegradation of oily sludge in Norwegian soils

Summary The degradation rate of oily sludge in soil under Norwegian conditions has been studied in field experiments during a 32 months' period. The experimental plots were added 0, 200, 400, and 600 kg N per ha. In uncultivated soil the oil content was reduced by respectively 4, 9, 22, and 26% during the first 9 months. In the same period the corresponding biodegradation in cultivated soil were 10, 15, 39, and 45%. At the end of the experiment only minor differences between the two different soil types were found. The mean degradation in percent was now 35, 50, 74, and 83, respectively. The optimum temperature for oil degradation in soil was found to be about 18°C. About 2/3 of the optimum activity was retained at 12°C. No leaching of oil or lead through soil columns infiltrated with oily sludge could be observed. A close relationship between oil content of the soil and the rate of water infiltration was found. Due to the extremely high content of lead in this oily sludge, a second application of sludge could not be recommended.     

Biodegradation of oily sludge in Kuwait soil

Soil microorganisms were not inhibited by mixing oily sludge in soil up to 8.7% (w/w) oil (15% sludge). Adding NH ₄ ⁺ and phosphate increased microbial activity. Microbial activity was also affected by seasonal variation. Thermotolerant microorganisms were more predominant during the summer. After 29 months, 72%, 84%, and 83% of the soil was degraded in fertilized soils dosed with 2.9, 5.8 and 8.7% oil, respectively.