Molecular Identification of Microbial Community in Surface and Undersurface Douchi During Postfermentation

To find the reason for fermentation failure of surface Douchi during postfermentation, the microbial communities in undersurface and surface samples were investigated using cell counting method and denaturing gradient gel electrophoresis (DGGE). The results showed that the microbial biomass in surface Douchi was obviously different from that in undersurface Douchi even sampled from the same fermentation tanks, and a 10‐ to 100‐fold reduction of microbial cell counts in undersurface had been observed. The bacterial DGGE profile and principal component analysis (PCA) results indicated that only Lactococcus lacts subsp. lactis and Bacillus thermoamylovorans were detected from surface Douchi, while Lactococcus lacts subsp. lactis, Staphylococcus lentus and 2 uncultured strains occupied the dominant positions in undersurface Douchi; when amplified using Bacillus‐specific primers, Bacillus thermoamylovorans, Bacillus subtilis, and Enterobacter sp. were found in undersurface Douchi, while only Bacillus thermoamylovorans were detected from surface Douchi; compared to the bacteria and Bacillus, the DGGE profiles and PCA plot of fungi indicated that the fungal community between surface and undersurface Douchi was similar and mainly composed by yeasts. In this study, we detected the microbial biomass and species in postfermentation stage of Douchi, and the various microbial diversity in undersurface and surface samples might be the cause of the fermentation failure in surface fermentation tanks.     

Monitoring of population shifts in an enriched nitrifying system under gradually increased cadmium loading

The changes in nitrifying bacterial population under cadmium loading were monitored and evaluated in a laboratory scale continuous-flow enriched nitrification system. For this purpose, the following molecular microbiological methods were used: slot–blot hybridization, denaturing gradient gel electrophoresis (DGGE), real-time PCR followed by melting curve analysis, cloning and sequence analysis. The initial cadmium concentration was incrementally increased from 1 to 10 mg/l which led to a drop in ammonia removal efficiency from 99 to 10%. Inhibition was recovered when cadmium loading was stopped. During the second application of cadmium, nitrifying population became more tolerant. Even at 15 mg/l Cd, only a minor inhibition was observed. To investigate the variations in ammonia and nitrite oxidizing bacteria populations in a period of 483 days, ammonia monooxygenase (amoA) and 16S rRNA genes-based molecular techniques were used. An obvious shift was experienced in the diversity of ammonia oxidizers after the first application of 10 mg/l Cd. Metal-tolerant ammonia oxidizing species became dominant and the microbial diversity sharply shifted from Nitrosomonas and Nitrosococcus sp. to Nitrosospira sp. which were observed to tolerate higher cadmium loadings. This result indicated that the extent of nitrification inhibition was not only related to the metal concentration and quantity of microorganisms but also depended on the type of species.     

Identification of microorganisms involved in reductive dehalogenation of chlorinated ethenes in an anaerobic microbial community

In this study, we report on phylogenetic and physiological characterization of an anaerobic culture capable of reductive dehalogenation of tetrachloroethene (PCE) obtained from a PCE-contaminated site. The culture was enriched using different combinations of electron donors (hydrogen and acetate) and electron acceptors (PCE, cis-1,2-dichloroethene (cDCE) and controls without chlorinated ethenes). The resulting subcultures were analyzed using three different approaches: chemical analysis to document conversion of chlorinated ethenes; polymerase chain reaction (PCR) of 16S rRNA gene fragments and denaturing gradient gel electrophoresis (DGGE) to compare community compositions; fluorescence in situ hybridization (FISH) to quantify specific groups of microorganisms using oligonucleotide probes previously designed or newly designed based on the sequences retrieved from sequence analysis of specific DGGE bands. Members of two genera which contain bacteria capable of reductive dehalogenation were detected in the culture: Dehalococcoides and Desulfitobacterium. The combined analyses suggested that Dehalococcoides-like bacteria are associated with complete dehalogenation of chlorinated ethenes to ethene with hydrogen as electron donor; and Desulfitobacterium-like bacteria, in contrast, are associated with incomplete PCE dehalogenation to cDCE and appear to be able to use acetate as electron donor. In addition, Sporomusa-like bacteria were identified, which most likely act as homoacetogens. The results demonstrated that combination of culture enrichment with different substrates, DGGE, and FISH allowed a detailed qualitative and quantitative characterization of the dominant microorganisms associated with reductive dehalogenation.     

Effects of selected pharmaceutically active compounds on treatment performance in sequencing batch reactors mimicking wastewater treatment plants operations

The impact of four pharmaceutically active compounds (PhACs) introduced both individually and in mixtures was ascertained on the performance of laboratory-scale wastewater treatment sequencing batch reactors (SBRs). When introduced individually at concentrations of 0.1, 1 and 10 μM, no significant differences were observed with respect to chemical oxygen demand (COD) and ammonia removal. Microbial community analyses reveal that although similarity index values generally decreased over time with an increase in PhAC concentrations as compared to the controls, no major microbial community shifts were observed for total bacteria and ammonia-oxidizing bacteria (AOB) communities. However, when some PhACs were introduced in mixtures, they were found to both inhibit nitrification and alter AOB community structure. Ammonia removal decreased by up to 45% in the presence of 0.25 μM gemfibrozil and 0.75 μM naproxen. PhAC mixtures did not however affect COD removal performance suggesting that heterotrophic bacteria are more robust to PhACs than AOB. These results highlight that the joint action of PhACs in mixtures may have significantly different effects on nitrification than the individual PhACs. This phenomenon should be further investigated with a wider range of PhACs so that toxicity effects can more accurately be predicted.     

Quantitative and qualitative analyses of methanogenic community development in high-rate anaerobic bioreactors

Methanogenic community structure and population dynamics were investigated in two anaerobic reactors treating a dairy wastewater, an Inverted Fluidized Bed (IFB) and Expanded Granular Sludge Bed (EGSB). A combination of real-time PCR, denaturing gradient gel electrophoresis and statistical techniques was employed. Distinct methanogenic communities developed in the IFB and EGSB reactors reflecting step-wise reductions in the applied hydraulic retention time from 72 to 12 h during the 200-day trial. The aceticlastic family Methanosarcinaceae was only detected in the IFB and the order Methanomicrobiales was also much more abundant in this reactor, while the aceticlastic family Methanosaetaceae was more abundant in the EGSB. The hydrogenotrophic order, Methanobacteriales, predominated in both reactors under all applied operational conditions. Non-metric multidimensional scaling (NMS) and moving-window analyses, based on absolute and relative abundance quantification data, demonstrated that the methanogenic communities developed in a different manner in the IFB, compared to the EGSB reactor. In our study, relative abundance-based quantification by NMS and moving-window analysis appeared to be a valuable molecular approach that was more applicable to reflect the changes in the anaerobic digestion process than approaches based either on qualitative analysis, or solely on absolute quantification of the various methanogenic groups. The overall results and findings provided a comparative, quantitative and qualitative insight into anaerobic digestion processes, which could be helpful for better future reactor design and process control.     

耐除草剂转基因大豆对根际土壤固氮微生物nifH基因多样性的影响

通过大田试验,以两种耐除草剂转基因大豆（PAT、ALS）及相应非转基因亲本大豆（PAT1、ALS1）和当地主栽大豆中黄13为材料,采用聚合酶链式反应-变性梯度凝胶电泳（PCR-DGGE）技术及扩增产物序列分析方法,探讨耐除草剂转基因大豆种植对根际土壤固氮微生物nifH基因多样性的影响。结果表明, PAT、ALS与相应亲本大豆PAT1、ALS1相比,根际土壤固氮微生物群落组成相似度均在60%左右。PAT、ALS根际土壤固氮微生物nifH基因多样性指数（H）和均匀度指数（EH）与相应亲本大豆相比差异均不显著（p〉0.05）。测序结果表明,PAT、ALS与相应亲本大豆根际土壤中固氮微生物主要隶属于蓝藻门（Cyanobacteria）、变形菌门（Proteobacteria）和厚壁菌门（Firmicutes）。不同处理下nifH基因与理化因子的典范对应分析结果表明,速效磷（AP）、硝态氮（NO3--N）对固氮微生物区系的影响达到显著水平（p〈0.05）。以上结论表明,与PAT1、ALS1相比,两种耐除草剂转基因大豆对根际土壤固氮微生物nifH基因多样性的影响不显著。     

芝麻香型白酒高温大曲微生物总DNA提取的改良方法

从常温研磨、蛋白酶K和R nase的加入对传统CTAB法进行改良,并结合试剂盒法,提出了一种高效提取高温大曲微生物总DNA的方法。该方法获得的总基因片段大小约21Kb;A260/A280=1.878;A260/A230=1.706;PCR反应抑制物少,可直接用于16S rDNA的扩增;并通过构建克隆文库技术和变性梯度凝胶电泳(DGGE)分析后发现高温大曲中细菌多样性丰富,能一定程度上反映微生物群落结构和组成。研究结果表明该方法提取的DNA适用于芝麻香高温大曲中微生物的分子生态学研究。     

Effect of electrokinetic remediation on indigenous microbial activity and community within diesel contaminated soil

Electrokinetic remediation has been successfully used to remove organic contaminants and heavy metals within soil. The electrokinetic process changes basic soil properties, but little is known about the impact of this remediation technology on indigenous soil microbial activities. This study reports on the effects of electrokinetic remediation on indigenous microbial activity and community within diesel contaminated soil. The main removal mechanism of diesel was electroosmosis and most of the bacteria were transported by electroosmosis. After 25 days of electrokinetic remediation (0.63 mA cm^− 2), soil pH developed from pH 3.5 near the anode to pH 10.8 near the cathode. The soil pH change by electrokinetics reduced microbial cell number and microbial diversity. Especially the number of culturable bacteria decreased significantly and only Bacillus and strains in Bacillales were found as culturable bacteria. The use of EDTA as an electrolyte seemed to have detrimental effects on the soil microbial activity, particularly in the soil near the cathode. On the other hand, the soil dehydrogenase activity was enhanced close to the anode and the analysis of microbial community structure showed the increase of several microbial populations after electrokinetics. It is thought that the main causes of changes in microbial activities were soil pH and direct electric current. The results described here suggest that the application of electrokinetics can be a promising soil remediation technology if soil parameters, electric current, and electrolyte are suitably controlled based on the understanding of interaction between electrokinetics, contaminants, and indigenous microbial community.     

阿特拉津微污染原水生物活性炭处理系统种群动态演替分析

采用降解菌柱、土壤悬液柱和对照柱3种活性炭柱处理阿特拉津微污染原水,考察了阿特拉津去除效率,并利用PCR-DGGE技术分析炭柱运行期间微生物菌群动态变化。结果表明140 d后对照柱去除率下降至30％～40％,而降解菌柱的去除效率保持在65%～75％。DGGE分析表明3个炭柱中都有自来水中微生物生长,土壤悬液柱中微生物在贫营养状态时种群多样性降低,实验室分离的阿特拉津降解菌接入降解菌柱后在运行期间可以保持相对优势,延长了炭的使用时间。     

石油降解混合菌群的降解特性研究及群落结构分析

从新疆克拉玛依油田石油污染土壤中分离获得石油降解菌群KO5-2,该菌群在30°C条件下培养7 d对10 g/L总石油烃（TPH）的去除率为56.9%,并且培养3 d ,7 d和9 d后能分别去除100%的芴、98.93%的菲和65.73%的芘。从KO5-2分离得到的来自6个不同菌属的12株可培养单菌,其中只有8株具有降解原油的能力。运用变性梯度凝胶电泳（DGGE）分析不同碳源条件（包括TPH、饱和烃、芴、菲和芘）下KO5-2的群落结构,结果表明,在分别以原油和饱和烃为碳源的培养条件下菌群组成大致相同,在三种不同多环芳烃（PAHs）为碳源的培养条件下,混合菌群的群落结构存在差异。红球菌属（Rhodococcus sp.）和假单胞菌属（Pseudomonas sp.）在五种不同的碳源中均能生存,芽孢杆菌（Bacillus sp.）、鞘氨醇单胞菌属（Sphingomonas sp.）和苍白杆菌（Ochrobactrum sp.）分别是降解饱和烃、PAHs和菲的关键菌。该研究表明,生物修复过程中,混合菌群的群落结构与不同的污染物和单菌株之间的相互作用有关。