污泥臭氧氧化处理过程中活菌抗药基因丰度的消减

臭氧氧化技术是一种广泛应用的污泥减量技术,然而臭氧处理能否对污泥中的抗生素抗药基因进行有效消减还不清楚。采用单叠氮溴化丙锭(propidium monoazide,PMA)预处理结合定量PCR(qPCR)方法对污泥臭氧减量过程中不同臭氧消耗量下活性污泥活菌中的四环素、氨基糖苷和大环内酯3大类共21种抗药基因的变化进行了研究。结果表明:臭氧氧化可以有效消减活性污泥活菌中21种抗药基因的绝对丰度(每毫升污泥的抗药基因拷贝数),在臭氧消耗量0.31 g·g~(-1)(TSS)情况下抗药基因总量降低了75.44%;尽管绝对丰度下降,污泥臭氧氧化处理过程中大量抗药基因的相对丰度(抗药基因拷贝数与细菌16S rRNA基因拷贝数的比例)逐渐增加,表明臭氧处理后污泥中耐药菌占总活菌比例可能增加,具有一定的抗性传播风险;Ⅰ型整合子是抗药基因水平转移的重要遗传元件,其绝对丰度同样随着臭氧消耗量增加而降低,而相对丰度逐渐上升。     

16S rDNA克隆文库解析AO-MBR系统中细菌种群多样性

采用分子生物学手段16S rDNA克隆文库方法对缺氧/好氧膜生物反应器(AO-MBR)的好氧池与缺氧池中细菌进行了多样性研究.实验结果表明,好氧池污泥样品的克隆文库包括9个类群,其中变形菌(Proteobacteria)和拟杆菌(Bacteroidetes)在文库中所占比例最大,分别为37.04％和14.81％;其次是酸杆菌(Acidobacteria)、未培养菌(uncultured bacterium)、绿菌(Chlorobi)和未培养的绿弯菌(uncultured Chloroflexi bacterium)、浮霉状菌(Planctomycetes),分别为11.11％、11.11％、7.41％、7.41％和5.56％;硝化螺旋菌(Nitrospirae)和裸藻门(Euglenozoa)所占比例相对较小,均为1.85％.缺氧池样品克隆文库包括10个类群,其中变形菌(Proteobacteria)、拟杆菌(Bacteroidetes)和未培养菌(uncultured bacterium)在文库中所占比例最大,分别为27.91％、13.95％和12.79％;其次是浮霉状菌(Planctomycetes)、酸杆菌(Acidobacteria)和绿弯菌(Chloroflexi),在文库中所占比例分别为9.3％、9.3％和9.3％;硝化螺旋菌(Nitrospirae)、裸藻门(Euglenozoa)、芽单胞菌(Gemmatimonadetes)和放线菌(Actinobacteria)所占比例相对较少,分别为6.98％、8.14％、1.16％和1.16％.两池细菌的主要类群相似,但菌属及比例有所差异,变形菌是系统中的主要脱氮菌属.     

活性污泥体系中聚糖菌的富集与鉴定

活性污泥体系中，聚糖菌(GAOs)在厌氧环境下与聚磷菌(PAOs)形成对底物的竞争关系，对聚糖菌的研究对于优化生物除磷工艺有重要意义。以葡萄糖为惟一碳源，在磷限制条件下，利用特殊运行方式对活性污泥进行驯化培养出了稳定的聚糖菌颗粒污泥，厌氧阶段磷释放量与有机物吸收量浓度(mg/L)比从7.4%下降为0.25%。从培养好的活性污泥反应器中分离获得2株聚糖菌，经菌落PCR和16S rRNA序列分析确定了所得聚糖菌菌株G1和菌株G2分别是枯草芽孢杆菌(Bacillus subtilis)和解鸟氨酸克雷伯氏菌(Klebsiella ornithinolytica)。     

全细胞脂肪酶(菌)对SBR系统中微生物群落演替的影响

将具有油脂污水处理功能的全细胞脂肪酶(菌)制剂,直接加入到SBR系统的活性污泥中,应用PCR-DGGE技术的应用,研究该制剂对活性污泥中微生物群落演替的影响。16S rDNA DGGE图谱表明投加全细胞脂肪酶(菌)制剂72h后,菌群丰富度Rs为57%、与空白组的48%相比变化不明显,不同样品相似性系数Cs在48.1%~74.4%之间,表明投加全细胞脂肪酶(菌)对活性污泥中原核微生物群落的演替没有明显影响。18S rDNA DGGE图谱表明,投加全细胞脂肪酶(菌)制剂72 h后,菌群丰富度Rs为72%、与空白组的22%相比存在显著性的差异,不同样品相似性系数Cs在14.5%~73.7%之间,而且优势菌体也发生动态变化,表明投加全细胞脂肪酶(菌)后,对活性污泥中的真核微生物群落演替影响明显。     

石河子市春季大气颗粒物TSP和PM_(10)中微生物群落特征

近来,有研究指出颗粒物中携带导致人类过敏、呼吸系统疾病或其他类疾病的微生物,其对公众健康的影响因为研究较少而一直被低估。采用16S rDNA、18S rDNA测序方法对石河子市2015年春季TSP和PM_(10)污染物中细菌、真菌的群落结构特征进行研究。结果表明:在门的水平上,变形菌门(Proteobacteria)和放线菌门(Actinobacteria)是石河子市春季TSP和PM_(10)中细菌的主要菌群,子囊菌门(Ascomycota)和担子菌门(Basidiomycota)是真菌的主要菌群。在属水平上,马赛菌属(Massilia)和不动杆菌属(Acinetobacter)在16S rDNA测序结果分析中相对含量较多,格孢腔菌属(Pleospora)和Penidiella在18S r DNA测序结果分析中相对丰度较高,仍然发现了一些可能导致人类及植物患病的菌属,期望此研究结果为人类生活提供参考。     

京杭运河杭州段水体污染和细菌群落结构特征

为了解人类活动对城市河道水体污染和细菌群落的影响,选取京杭运河杭州段进行了分季节采样研究。测定了水体的环境与水质参数,分析了细菌丰度、胞外酶活性和群落结构变化。结果显示,从上游到下游,水体中高锰酸盐指数、氨氮、总氮以及细菌丰度和β-葡萄糖苷酶活性大体呈递增趋势,冬季水体总体污染水平及细菌丰度均显著高于其他季节。水体中细菌的主要种类集中在变形菌门(Proteobacteria)、放线菌门(Actinobacteria)、拟杆菌门(Bacterioidetes)、厚壁菌门(Firmicutes)和蓝藻门(Cyanobacteria),所占比例分别为37.8%、24.3%、18.9%、10.8%和8.1%,属于黄杆菌(Flavobacterium)、厚壁菌(Firmicutes bacterium)和放线菌(Actinobacterium)的细菌是水体中大多数时间的优势细菌。限制性排序分析显示,水体细菌群落结构从上游到下游差异明显,高锰酸盐指数和总氮是关键影响因子。杭州市区由人类活动产生的污染物输入是京杭运河杭州段的主要污染源,水体细菌群落对水体污染物的变化有着明显的响应。     

生态修复工程对城市内河水体细菌多样性的影响

微生物是城市内河生态系统的重要组成部分,其多样性与水环境密切相关。以16S rRNA基因作为原核细菌分子标记,应用PCR-DGGE、定量PCR、克隆和测序方法,分析经生态修复工程处理后不同污染程度的城市内河水体细菌多样性特征。结果表明:生态修复工程明显提高了污染程度较低水体总氮(TN)、总磷(TP)和化学需氧量(CODMn)的消减能力,影响着城市内河水体中细菌群落的组成和含量,且随时间和空间不同而不同;生态修复工程显著地提高了重度污染水体细菌的含量;冗余分析(RDA)表明,水体中细菌群落组成与CODMn、TP和溶解氧(DO)等指标密切相关。     

人类特异性粪厌氧菌基因标记物实时荧光定量PCR检测方法的建立和应用

针对人类粪便污染,选择来源于拟杆菌和双歧杆菌16S rRNA的基因片段作为标记物,分别建立了相应的实时荧光定量PCR检测方法.选取不同来源的粪便样品进行检测,证实了引物的特异性.回收纯化从污水中扩增所得的目标PCR片段,经过连接转化,筛选阳性克隆提取其重组质粒作为实时荧光定量PCR的标准品.通过检测一系列梯度稀释的标准品,确定了拟杆菌基因标记物和双歧杆菌基因标记物定量PCR检测方法分别在1.98×102～1.98×107 copy/μL和2.12×101～2.12×107 copy/μL范围内具有良好的线性关系.     

有机负荷冲击对固定床厌氧反应器启动及古菌群落动态影响

为开发高效率、抗冲击能力强的有机废水沼气发酵技术,以人工配制的糖蜜废水为试材,在不同有机负荷(1.7~6.73 kg/(m3·d))的冲击方式下,对4个炭纤维膜为载体固定床厌氧反应器的启动进行了研究。通过COD去除率、p H、产气量、甲烷含量、变性梯度凝胶电泳(DGGE)、16S r DNA克隆文库技术、实时荧光定量PCR(Q-PCR)等指标和方法,分析炭纤维载体在抵御有机负荷冲击过程中的作用及对微生物定殖的贡献。结果表明,在不同有机负荷冲击下,4个炭纤维载体固定床厌氧反应器在第40天时均启动成功,其产气量稳定在21 L左右,出水p H在6.8~7.5之间,COD去除率在80%以上,甲烷含量在75%以上,其中冲击最大的R2反应器(COD每隔5 d增加5 000 mg/L)的有机负荷是固定床厌氧反应器的一个阈值。附着在炭纤维载体上的产甲烷古菌的多样性丰富,并且繁殖缓慢、容易附着的产甲烷微菌(MMB)的16S rRNA基因浓度最高,反应器中占优势的产甲烷菌是甲烷鬃毛菌(MST)和产甲烷微菌(MMB),说明炭纤维载体对微生物的定殖起到非常重要的作用,提高了污泥活性,从而提高了反应器的性能。     

Miseq测序分析活性污泥系统中细菌群落的动态变化

采用基于16S rRNA基因的Miseq测序方法,分析了某小试序批式反应器(SBR)中,细菌群落结构在1年中的动态变化。在研究中,系统运行稳定,COD出水浓度均在40 mg/L以下,总氮(TN)和总磷(TP)出水浓度分别在20 mg/L和0.3 mg/L之下。细菌的群落组成变化明显,在检测到的2 094个OTU中,有176个OTU只存在于3个以下的样品中。在385个属的细菌中,有54个属的微生物只存在于3个以下的样品中。移动窗口分析表明,系统中细菌群落的平均变化率Δt(30 d)为21.3%±6.2%,稳定的系统功能并未耦合稳定的细菌群落结构。Mantel test分析表明,溶解氧(DO),进水COD浓度和温度对细菌群落结构有显著影响。方差分解分析(VPA)结果表明,水质参数及运行参数可分别解释23.6%和21.5%的群落结构变化。     

有机碳源条件下厌氧氨氧化SBBR中的微生物多样性

研究有机碳源对SBBR厌氧氨氧化菌群等微生物的影响。采用16S rDNA序列与PCR-DGGE分析技术相结合的方法,对稳定运行的反应器内的活性污泥和生物膜样品,进行细菌多样性图谱分析,同时采用巢式PCR-DGGE技术对浮霉状菌属(Planctomycetes)细菌进行分析。结果表明,在有机碳源反应系统细菌条带数和多样性指数均高于无机系统,与活性污泥相比,生物膜表尤为明显。当进水不含有机碳源时,氨氧化细菌(ammonia oxidizing bacteria,AOB),厌氧氨氧化菌(anaerobic ammonia oxidizing bacteria,ANAMMOX)为优势功能菌;当进水含有机碳源时,系统中存在的AOB以亚硝化单胞菌(Nitrosomonas sp.)为优势菌群,同时存在反硝化菌,如索氏菌(Thauera sp.)以及厌氧氨氧化菌,它们共同作用完成N的去除。此外,与无机碳源系统相比,有机碳源的存在,有利于浮霉状菌的积累,但压缩了ANAMMOX的生存空间。本研究可为厌氧氨氧化工艺处理低C/N比有机废水提供了理论依据。     

Microbial transformation of synthetic estrogen 17α-ethinylestradiol

Natural estrogens such as estrone, 17β-estradiol, estriol, and the particularly recalcitrant synthetic estrogen 17α-ethinylestradiol used as oral contraceptive, accumulate in the environment and may give rise to health problems. The processes participating in their removal from soil, wastewater, water-sediments, groundwater-aquifer material, and wastewater or sewage treatment plant effluents may involve the action of bacterial and microbial consortia, and in some cases fungi and algae. This review discusses the different efficiencies of bacterial degradation of 17α-ethinylestradiol under aerobic and anaerobic conditions, the role of sulfate-, nitrate-, and iron-reducing conditions in anaerobic degradation, and the role of sorption. The participation of autotrophic ammonia oxidizing bacteria and heterotrophic bacteria in cometabolic degradation of estrogens, the estrogen-degrading action of ligninolytic fungi and their extracellular enzymes (lignin peroxidase, manganese-dependent peroxidase, versatile peroxidase, laccase), and of algae are discussed in detail.     

Bacterial cell monitoring in wastewater treatment plants by flow cytometry.

The activated sludge process is performed by a variable and mixed community of microorganisms in an aerobic aquatic environment, in which bacteria constitute the majority and represent the main microorganisms responsible for the degradation process in a plant. In this work, we monitored bacterial charge in different wastewater treatment plants by flow cytometry, also evaluating chlorination effects on bacterial viability, both by flow cytometry and traditional plate counts. Maximum values of bacterial charge were registered in the aeration tank of all plants monitored. Cell viability did not show significant differences (p > 0.05) in samples collected in "before chlorination" and "wastewater effluent" treatment steps; this suggests that the chlorination was not able to decrease total viable bacterial charge. In this work, we discuss the need to improve microbiological analyses, both in terms of measuring other potential pathogens and of using new methodological approaches in the traditional evaluation of the microbiological quality of effluents.     

Removal of petroleum pollutants and monitoring of bacterial community structure in a membrane bioreactor

The long-term operational stability (159 d) in removal of organics and ammonia from synthetic wastewater was investigated. The experiment was carried out in two identical plug flow membrane bioreactors (MBR) (each with a submerged A4 Kubota membrane) operated under aerobic conditions. The vacuum distillate of a crude oil fraction in the emulsified state, which was used to model the petroleum pollutants, was added into the feed medium. The performance of biological treatment was evaluated by physicochemical analyses such as nitrogen forms, COD, and BOD. Additionally, monitoring of PAHs in the wastewaters was performed using HPLC-diode array detector. Moreover, the community structure of bacteria was analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis. The MBR treatment was very effective with reduction by more than 90% of COD and Total Organic Carbon. Nearly complete removal of petroleum originated non-polar micropollutants was observed. The influence of the highest dosage of petroleum pollutants (1000 μLL(-1)) on the bacterial community was noted.     

Fungal bioremediation of the creosote-contaminated soil: influence of Pleurotus ostreatus and Irpex lacteus on polycyclic aromatic hydrocarbons removal and soil microbial community composition in the laboratory-scale study

The aim of this study was to determine the efficacy of selected basidiomycetes in the removing of polycyclic aromatic hydrocarbons (PAH) from the creosote-contaminated soil. Fungi Pleurotus ostreatus and Irpex lacteus were supplemented with creosote-contaminated (50-200 mg kg(-1) PAH) soil originating from a wood-preserving plant and incubated at 15 °C for 120 d. Either fungus degraded PAH with 4-6 aromatic rings more efficiently than the microbial community present initially in the soil. PAH removal was higher in P. ostreatus treatments (55-67%) than in I. lacteus treatments (27-36%) in general. P. ostreatus (respectively, I. lacteus) removed 86-96% (47-59%) of 2-rings PAH, 63-72% (33-45%) of 3-rings PAH, 32-49% (9-14%) of 4-rings PAH and 31-38% (11-13%) of 5-6-rings PAH. MIS (Microbial Identification System) Sherlock analysis of the bacterial community determined the presence of dominant Gram-negative bacteria (G-) Pseudomonas in the inoculated soil before the application of fungi. Complex soil microbial community was characterized by phospholipid fatty acids analysis followed by GC-MS/MS. Either fungus induced the decrease of bacterial biomass (G- bacteria in particular), but the soil microbial community was influenced by P. ostreatus in a different way than by I. lacteus. The bacterial community was stressed more by the presence of I. lacteus than P. ostreatus (as proved by the ratio of the fungal/bacterial markers and by the ratio of trans/cis mono-unsaturated fatty acids). Moreover, P. ostreatus stimulated the growth of Gram-positive bacteria (G+), especially actinobacteria and these results indicate the potential of the positive synergistic interaction of this fungus and actinobacteria in creosote biodegradation.     

Effects of wastewater disinfection on waterborne bacteria and viruses.

Wastewater disinfection is practiced with the goal of reducing risks of human exposure to pathogenic microorganisms. In most circumstances, the efficacy of a wastewater disinfection process is regulated and monitored based on measurements of the responses of indicator bacteria. However, inactivation of indicator bacteria does not guarantee an acceptable degree of inactivation among other waterborne microorganisms (e.g., microbial pathogens). Undisinfected effluent samples from several municipal wastewater treatment facilities were collected for analysis. Facilities were selected to provide a broad spectrum of effluent quality, particularly as related to nitrogenous compounds. Samples were subjected to bench-scale chlorination and dechlorination and UV irradiation under conditions that allowed compliance with relevant discharge regulations and such that disinfectant exposures could be accurately quantified. Disinfected samples were subjected to a battery of assays to assess the immediate and long-term effects of wastewater disinfection on waterborne bacteria and viruses. In general, (viable) bacterial populations showed an immediate decline as a result of disinfectant exposure; however, incubation of disinfected samples under conditions that were designed to mimic the conditions in a receiving stream resulted in substantial recovery of the total bacterial community. The bacterial groups that are commonly used as indicators do not provide an accurate representation of the response of the bacterial community to disinfectant exposure and subsequent recovery in the environment. UV irradiation and chlorination/dechlorination both accomplished measurable inactivation of indigenous phage; however, the extent of inactivation was fairly modest under the conditions of disinfection used in this study. UV irradiation was consistently more effective as a virucide than chlorination/dechlorination under the conditions of application, based on measurements of virus (phage) diversity and concentration. Taken together, and when considered in conjunction with previously published research, the results of these experiments illustrate several important limitations of common disinfection processes as applied in the treatment of municipal wastewaters. In general, it is not clear that conventional disinfection processes, as commonly implemented, are effective for control of the risks of disease transmission, particularly those associated with viral pathogens. Microbial quality in receiving streams may not be substantially improved by the application of these disinfection processes; under some circumstances, an argument can be made that disinfection may actually yield a decrease in effluent and receiving water quality. Decisions regarding the need for effluent disinfection must account for site-specific characteristics, but it is not clear that disinfection of municipal wastewater effluents is necessary or beneficial for all facilities. When direct human contact or ingestion of municipal wastewater effluents is likely, disinfection may be necessary. Under these circumstances, UV irradiation appears to be superior to chlorination in terms of microbial quality and chemistry and toxicology. This advantage is particularly evident in effluents that contain appreciable quantities of ammonia-nitrogen or organic nitrogen.     

Nitrifying community analysis in a single submerged attached-growth bioreactor for treatment of high-ammonia waste stream.

This study investigated the nitrifying community structure in a single-stage submerged attached-growth bioreactor (SAGB) that successfully achieved stable nitrogen removal over nitrite of a high-strength ammonia wastewater. The reactor was operated with intermittent aeration and external carbon addition (methanol). With influent ammonia and total Kjeldahl nitrogen ranging from 537 to 968 mg/L and 643 to 1510 mg/L, respectively, 85% nitrogen removal was obtained, and effluent was dominated by nitrite (NO2-/NOx > 0.95). Nitrifying community analysis using fluorescence in situ hybridization (FISH), with a hierarchical set of probes targeting known ammonia-oxidizing bacteria (AOB) within beta-proteobacteria, showed that the AOB community of the biofilter consists almost entirely of members of the Nitrosomonas europaea/eutropha and the Nitrosococcus mobilis lineages. Image analysis of FISH pictures was used to quantify the identified AOB, and it was estimated that Nitrosomonas europaea/eutropha-like AOB accounted for 4.3% of the total volume of the biofilm, while Nitrosococcus mobilis-like AOB made up 1.2%; these numbers summed up to a total AOB fraction of 5.5% of the total volume on the biofilm. Nitrite-oxidizing bacteria (NOB) were not detectable in the biofilm samples with probes for either Nitrospira sp. or Nitrobacter sp., which indicated that NOB were either absent from the biofilters or present in numbers below the detection limit of FISH (< 0.1% of the total biofilm). Nitrite oxidizers were likely outcompeted from the system because of the free ammonia inhibition and the possibility that the aeration period (from intermittent aeration) was not sufficiently long for the NOB to be released from the competition for oxygen with heterotrophs and AOB. The nitrogen removal via nitrite in a SAGB reactor described in this study is applicable for high-ammonia-strength wastewater treatment, such as centrate or industrial wastes.     

Structural changes in soil communities after triclopyr application in soils invaded by Acacia dealbata Link

Triclopyr is a commonly used herbicide in the control of woody plants and can exhibit toxic effects to soil microorganisms. However, the impact on soils invaded by plant exotics has not yet been addressed. Here, we present the results of an 18-month field study conducted to evaluate the impact of triclopyr on the structure of fungal and bacterial communities in soils invaded by Acacia dealbata Link, through the use of denature gradient gel electrophoresis. After triclopyr application, analyses of bacterial fingerprints suggested a change in the structure of the soil bacterial community, whereas the structure of the soil fungal community remained unaltered. Bacterial density and F:B ratio values changed across the year but were not altered due to herbicide spraying. On the contrary, fungal diversity was increased in plots sprayed with triclopyr 5 months after the first application. Richness and diversity (H´) of both bacteria and fungi were not modified after triclopyr application.     

菌源对多环芳烃降解菌的筛选及降解性能的影响

高效降解菌的筛选对利用生物修复技术有效去除环境中的多环芳烃具有重要意义。分别以石油污染土壤和焦化废水活性污泥为菌源,分离出芘降解菌和混合PAHs(菲、荧蒽和芘)降解菌共14株并对其降解性能进行对比研究。结果表明,筛选得到的菌株分别属于9个菌属,其中2种菌源共有的菌属为Mycobacterium sp.、Ralstonia sp.和Shinella sp.。芘和PAHs的高效降解菌(CP16和CM32)均属于分支杆菌属(Mycobacterium),来源于焦化废水活性污泥;菌株CP16对芘(50mg/L)的7 d降解率为74.99%,CM32对PAHs(菲50 mg/L、荧蒽和芘各10 mg/L)的7 d降解率为100%。因此,以焦化废水活性污泥为菌源更有利于获得高效的多环芳烃降解菌。     

PCR-DGGE技术解析A2/O工艺好氧单元中微生物群落结构

应用PCR-DGGE方法,追踪了汉沽工业废水处理中好氧工艺的活性污泥系统中微生物群落结构动态变化过程及其微生物群落结构组成。研究结果表明:系统中的微生物群落结构随水质变化而变化,随着培养时间的延长,微生物群落结构趋于稳定,分别属于5大类群,与γ、δ、α、ε变形杆菌(Proteobacterias)、芽孢杆菌(Bacilli)的亲缘关系较近。其中γ变形杆菌是该废水处理过程中的主要菌群,包括Pseudomonas sp.、Rheinheimera sp.、Citrobacter sp.、Klebsiella sp.、Enterbacte-riaceae、Stenotrophomonas maltophilia、Acinetobacter。在整个系统中uncultured Pseudomonas sp.、Halobacillus sp.、Pseudomonassp.、Pseudomonas stutzeri、Acinetobacter sp.可稳定存在于系统中,为该污水处理系统中的优势微生物。因此,提高Halobacillussp.、Pseudomonas sp.、Pseudomonas stutzeri、Acinetobacter sp.菌属在系统中的数量和质量,有利于提高废水生化处理的效果。