分子检测技术对活性污泥中氨氧化细菌的比较研究

采用PCR扩增、随机克隆测序等技术,分析处理含高浓度氨氮的废水处理系统不同驯化时期的4个活性污泥样品,对样品中氨氧化细菌（AOB）的种类和氨单加氧酶（AMO）的活性进行分析比较,并在国内首次采用PCR变性梯度凝胶电泳（DGGE）相结合的技术对样品中总的细菌类群的差异进行研究。结果表明所检测到的氨氧化细菌优势菌群均属于变形细菌的β亚类,与Nitrosomonas sp.具有较高的相似性。活性污泥驯化成熟后,废水处理系统中AMO的活性有明显提高,活性污泥中的细菌类群更加集中,优势菌群相对稳定,系统对废水的处理效率也相应提高。结果表明采用分子检测技术有利于更全面地了解AOB的类群和功能,进而改善废水处理系统的处理效果。     

污水处理活性污泥微生物群落多样性研究

为研究污水处理活性污泥微生物多样性,提取了活性污泥宏基因组DNA,并采用细菌通用引物27F和1492R扩增了上海污泥厂活性污泥细菌16S rDNA片段,构建了细菌16S rDNA克隆文库,并对该文库中的微生物群落进行了分析。共获得200条高质量序列并建立系统发育树,结果显示活性污泥主要的细菌类群为变形菌门(Proteobacteria)(91.9%)、厚壁菌门(Firmicures)(4.6%)、拟杆菌门(Bacteroidetes)(2%)、绿弯菌门(Chloroflexi)(0.5%)、硝化螺菌门(Nitrospirae)(1%)。其中,明显的优势菌群为Alcaligenes feacalis(55%)、Pseudomonas aeruginosa(12.8%)和Stenotrophomonas(12.8%),优势菌的产酶能力在活性污泥中显示生态修复功能菌的作用。     

ERIC-PCR-based strain-specific detection of phenol-degrading bacteria in activated sludge of wastewater treatment systems

Aims:  To evaluate the use of Enterobacterial Repetitive Intergenic Consensus PCR (ERIC-PCR)-derived probes and primers to specifically detect bacterial strains in an activated sludge microbial community.
Methods and Results:  ERIC-PCR was performed on two phenol-degrading bacterial strains, Arthrobacter nicotianae P1-7 and Klebsiella sp. P8-14. Their amplicons were DIG labelled for use as probes and then hybridized with ERIC-PCR fingerprints. The results showed the distinct band patterns for both bacterial strains. Strain-specific PCR primers were designed based on the sequences of ERIC-PCR bands. The DNA of each of these strains was successfully detected from its mixture with activated sludge DNA, either by using their respective ERIC-PCR-based probes for hybridization or by using species-specific primers for amplification, with higher sensitivity by latter method.
Conclusions:  Two phenol-degrading bacterial strains were identified from a mixture of activated sludge by using ERIC-PCR-based methods.
Significance and Impact of the Study:  The study demonstrated that the bacteria, which have important functions in complex wastewater treatment microbial communities, could be specifically detected by using ERIC-PCR fingerprint-based hybridization or amplification.     

Bacterial survival and association with sludge flocs during aerobic and anaerobic digestion of wastewater sludge under laboratory conditions.

The fate of indicator bacteria, a bacterial pathogen, and total aerobic bacteria during aerobic and anaerobic digestion of wastewater sludge under laboratory conditions was determined. Correlation coefficients were calculated between physical and chemical parameters (temperature, dissolved oxygen, pH, total solids, and volatile solids) and either the daily change in bacterial numbers or the percentage of bacteria in the supernatant. The major factor influencing survival of Salmonella typhimurium and indicator bacteria during aerobic digestion was the temperature of sludge digestion. At 28 degrees C with greater than 4 mg of dissolved oxygen per liter, the daily change in numbers of these bacteria was approximately -1.0 log10/ml. At 6 degrees C, the daily change was less than -0.3 log10/ml. Most of the bacteria were associated with the sludge flocs during aerobic digestion of sludge at 28 degrees C with greater than 2.4 mg of dissolved oxygen per liter. Lowering the temperature or the amount of dissolved oxygen decreased the fraction of bacteria associated with the flocs and increased the fraction found in the supernatant.     

造纸废水活性污泥的微生物群落结构及多样性分析

为探究造纸废水活性污泥中微生物群落结构多样性以及对造纸废水处理效果的影响，利用Illumina MiSeq 高通量测序方法，分析在处理造纸废水过程中，同一运行阶段两个并联氧化沟内活性污泥的微生物群落与多样性组成。结果表明，系统中处理造纸废水的活性污泥在同一废水条件下微生物群落结构总体稳定，优势细菌为绿弯菌门(Chloroflexi)、拟杆菌门（Bacteroidota）、变形菌门（Proteobacteria）、Myxococcota、放线菌门(Actinobacteria）、厚壁菌门(Firmicutes）等。最重要的优势细菌类群为Chloroflexi，相对丰度占比为47.67%~48.22%，远远高于其他废水中Chloroflexi的占比，其中厌氧绳菌纲（Anaerolineae）是其主要成员，占比84.39%~88.34%，可针对性地去除造纸废水中的污染物。造纸废水活性污泥样品中存在大量特殊功能菌群，其在废水中污染物尤其是木质素的去除中发挥着重要作用。     

Suitability of Sludge Biotic Index (SBI), Sludge Index (SI) and filamentous bacteria analysis for assessing activated sludge process performance: the case of piggery slaughterhouse wastewater

Piggery slaughterhouse wastewater poses serious issues in terms of disposal feasibility and environmental impact, due to its huge organic load and variability. It is commonly treated by means of activated sludge processes, whose performance, in case of municipal wastewater, can be monitored by means of specific analyses, such as Sludge Biotic Index (SBI), Sludge Index (SI) and floc and filamentous bacteria observation. Therefore, this paper was aimed at assessing the applicability of these techniques to piggery slaughterhouse sewage. A plant located in Northern Italy was monitored for 1 year. Physical, chemical and operation parameters were measured; the activated sludge community (ciliates, flagellates, amoebae and small metazoa) was analysed for calculating SBI and SI. Floc and filamentous bacteria were examined and described accordingly with internationally adopted criteria. The results showed the full applicability of the studied techniques for optimizing the operation of a piggery slaughterhouse wastewater treatment plant.     

Isolation of an aboriginal bacterial community capable of utilizing cyanide, thiocyanate, and ammonia from metallurgical plant wastewater

An aboriginal bacterial community capable of degrading cyanide (10 mg/l) and thiocyanate (2 g/l) and eliminating ammonia (120 mg/l) had been isolated from recycled water samples after blast-furnace gas purification of a metallurgical plant wastewater. It was shown that the optimal conditions for this bacterial community were as follows: temperature, 34 degrees C; pH, 8.8-9.0; available organic matter concentration (glucose equivalent), 5 g/l; and dissolved O2 concentration, 8-10 mg/l. This aboriginal community was formed by the bacteria belonging to the genus Pseudomonas.     

Bacterial community dynamics during the winter-spring transition in the North Sea

Bacterioplankton dynamics at Helgoland Roads (54 degrees 11.3'N, 7 degrees 54.0'E) in the North Sea over the winter-spring transition were investigated. The bacterial community was analyzed and correlated with phytoplankton community data and abiotic parameters. The community structure was analyzed by ribosomal intergenic spacer analysis (RISA) and by denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes followed by DNA sequence analysis. The linkage of abiotic and biotic environmental factors and bacterial community as well as phylotypes (sequenced DGGE bands) was analyzed by the ordination technique of canonical correspondence analysis (CCA). Generally, an influence of temperature and phytoplankton on the bacterial community during the sampling period was observed. Additionally, multivariate analysis by factors revealed an influence on specific bacterial phylotypes of these factors. Overall, results indicate that changes in the bacterial community were caused not only by abiotic factors but also by the phytoplankton community.     

Microbial community analysis and performance of a phosphate-removing activated sludge

The microbial community of a phosphate-removing activated sludge was analyzed according to the extracted 16S rDNA sequences. The sludge, which accumulated 5.6% P by weight, was obtained from a sequencing batch reactor treating a fatty-acid rich wastewater containing 108 mg l(-1) total organic carbon (TOC), 14.0 mg l(-1) N and 16.2 mg l(-1) P. The reactor at 25 degrees C and pH 7.6 removed over 96% TOC and 99.9% P from the wastewater. According to the 16S rDNA analysis of the 114 clones developed, the sludge had a diverse population, mainly comprising Proteobacteria (71.0%) and the Cytophaga Flavobacterium Bacteroides group (23.7%), plus a few species of Planctomycetales (2.6%), Verrucomicrobiales (1.8%) and Firmicutes (0.9%). Of the 114 clones, 36 (31.6%) were closely affiliated with Acinetobacter. However, Acinetobacter did not accumulate phosphate judging from the images of sludge samples hybridized with an Acinetobacter-specific probe and stained with a phosphate-specific dye. The identities of the phosphate-removing bacteria remain unclear.     

Impacts of Multiwalled Carbon Nanotubes on Nutrient Removal from Wastewater and Bacterial Community Structure in Activated Sludge

Background

The increasing use of multiwalled carbon nanotubes (MWCNTs) will inevitably lead to the exposure of wastewater treatment facilities. However, knowledge of the impacts of MWCNTs on wastewater nutrient removal and bacterial community structure in the activated sludge process is sparse.

Aims

To investigate the effects of MWCNTs on wastewater nutrient removal, and bacterial community structure in activated sludge.

Methods

Three triplicate sequencing batch reactors (SBR) were exposed to wastewater which contained 0, 1, and 20 mg/L MWCNTs. MiSeq sequencing was used to investigate the bacterial community structures in activated sludge samples which were exposed to different concentrations of MWCNTs.

Results

Exposure to 1 and 20 mg/L MWCNTs had no acute (1 day) impact on nutrient removal from wastewater. After long-term (180 days) exposure to 1 mg/L MWCNTs, the average total nitrogen (TN) removal efficiency was not significantly affected. TN removal efficiency decreased from 84.0% to 71.9% after long-term effects of 20 mg/L MWCNTs. After long-term exposure to 1 and 20 mg/L MWCNTs, the total phosphorus removal efficiencies decreased from 96.8% to 52.3% and from 98.2% to 34.0% respectively. Further study revealed that long-term exposure to 20 mg/L MWCNTs inhibited activities of ammonia monooxygenase and nitrite oxidoreductase. Long-term exposure to 1 and 20 mg/L MWCNTs both inhibited activities of exopolyphosphatase and polyphosphate kinase. MiSeq sequencing data indicated that 20 mg/L MWCNTs significantly decreased the diversity of bacterial community in activated sludge. Long-term exposure to 1 and 20 mg/L MWCNTs differentially decreased the abundance of nitrifying bacteria, especially ammonia-oxidizing bacteria. The abundance of PAOs was decreased after long-term exposure to 20 mg/L MWCNTs. The abundance of glycogen accumulating organisms (GAOs) was increased after long-term exposure to 1 mg/L MWCNTs.

Conclusion

MWCNTs have adverse effects on biological wastewater nutrient removal, and altered the diversity and structure of bacterial community in activated sludge.     

Sequencing bands of ribosomal intergenic spacer analysis fingerprints for characterization and microscale distribution of soil bacterium populations responding to mercury spiking

Two major emerging bands (a 350-bp band and a 650-bp band) within the RISA (ribosomal intergenic spacer analysis) profile of a soil bacterial community spiked with Hg(II) were selected for further identification of the populations involved in the response of the community to the added metal. The bands were cut out from polyacrylamide gels, cloned, characterized by restriction analysis, and sequenced for phylogenetic affiliation of dominant clones. The sequences were the intergenic spacer between the rrs and rrl genes and the first 130 nucleotides of the rrl gene. Comparison of sequences derived from the 350-bp band to The GenBank database permitted us to identify the bacteria as being mostly close relatives to low G+C firmicutes (Clostridium-like genera), while the 650-bp band permitted us to identify the bacteria as being mostly close relatives to beta-proteobacteria (Ralstonia-like genera). Oligonucleotide probes specific for the identified dominant bacteria were designed and hybridized with the RISA profiles derived from the control and spiked communities. These studies confirmed the contribution of these populations to the community response to the metal. Hybridization of the RISA profiles from subcommunities (bacterial pools associated with different soil microenvironments) also permitted to characterize the distribution and the dynamics of these populations at a microscale level following mercury spiking.     

Pyrosequencing analysis of bacterial diversity in 14 wastewater treatment systems in china

To determine if there is a core microbial community in the microbial populations of different wastewater treatment plants (WWTPs) and to investigate the effects of wastewater characteristics, operational parameters, and geographic locations on microbial communities, activated sludge samples were collected from 14 wastewater treatment systems located in 4 cities in China. High-throughput pyrosequencing was used to examine the 16S rRNA genes of bacteria in the wastewater treatment systems. Our results showed that there were 60 genera of bacterial populations commonly shared by all 14 samples, including Ferruginibacter, Prosthecobacter, Zoogloea, Subdivision 3 genera incertae sedis, Gp4, Gp6, etc., indicating that there is a core microbial community in the microbial populations of WWTPs at different geographic locations. The canonical correspondence analysis (CCA) results showed that the bacterial community variance correlated most strongly with water temperature, conductivity, pH, and dissolved oxygen (DO) content. Variance partitioning analyses suggested that wastewater characteristics had the greatest contribution to the bacterial community variance, explaining 25.7% of the variance of bacterial communities independently, followed by operational parameters (23.9%) and geographic location (14.7%). Results of this study provided insights into the bacterial community structure and diversity in geographically distributed WWTPs and discerned the relationships between bacterial community and environmental variables in WWTPs.     

Detection of anaerobic processes and microorganisms in immobilized activated sludge of a wastewater treatment plant with intense aeration

Attached activated sludge from the Krasnaya Polyana (Sochi) wastewater treatment plant was studied after the reconstruction by increased aeration and water recycle, as well as by the installation of a bristle carrier for activated sludge immobilization. The activated sludge biofilms developing under conditions of intense aeration were shown to contain both aerobic and anaerobic microorganisms. Activity of a strictly anaerobic methanogenic community was revealed, which degraded organic compounds to methane, further oxidized by aerobic methanotrophs. Volatile fatty acids, the intermediates of anaerobic degradation of complex organic compounds, were used by both aerobic and anaerobic microorganisms. Anaerobic oxidation of ammonium with nitrite (anammox) and the presence of obligate anammox bacteria were revealed in attached activated sludge biofilms. Simultaneous aerobic and anaerobic degradation of organic contaminants by attached activated sludge provides for high rates of water treatment, stability of the activated sludge under variable environmental conditions, and decreased excess sludge formation.     

Biodegradability evaluation of dairy effluents originated in selected sections of dairy production

Main goal of the study was present the results of some respirometric measurements of activated sludge biodegrading the substrate in the wastewater originated in selected sections of the dairy processing line. The following dairy production effluents were analyzed in the research: the pumping station wastewater (combined wastewater from all the sections of the dairy factory), the apparatus room wastewater, the butter section wastewater, the milk reception point wastewater, the cheese section wastewater and the cottage cheese section wastewater. Apart from that, sweet and sour whey, which are secondary products of hard cheese and cottage cheese production, respectively, was the subject of the research. The amount of organic matter being oxidized during a 5-day measurement session was calculated on 1g of the activated sludge biomass. The research was conducted at the temperature of 20 degrees C and 35 degrees C at the applied sludge loading rate of A'=0.2 g BOD g(-1) dry mass d(-1), which ensured complete biodegradation. The results indicated a correlation between a technological process of dairy processing, an ultimate outcome of which was the wastewater analyzed, and dairy wastewater biodegradability. The results confirmed that all dairy processing effluents can be treated together, with the exception of whey, whose complex biodegradation demands may cause too much burden to any wastewater treatment technological system and thus should be managed within a separate installation.     

In situ detection of protein-hydrolysing microorganisms in activated sludge

Protein hydrolysis plays an important role in the transformation of organic matter in activated sludge wastewater treatment plants, but no information is currently available regarding the identity and ecophysiology of protein-hydrolysing organisms (PHOs). In this study, fluorescence in situ enzyme staining with casein and bovine serum albumin conjugated with BODIPY dye was applied and optimized to label PHOs in activated sludge plants. A strong fluorescent labeling of the surface of microorganisms expressing protease activity was achieved. Metabolic inhibitors were applied to inhibit the metabolic activity to prevent uptake of the fluorescent hydrolysates by oligopeptide-consuming bacteria. In five full-scale, nutrient-removing activated sludge plants examined, the dominant PHOs were always different morphotypes of filamentous bacteria and the epiflora attached to many of these. The PHOs were identified by FISH using a range of available oligonucleotide probes. The filamentous PHOs belonged to the candidate phylum TM7, the phylum Chloroflexi and the class Betaproteobacteria. In total they comprised 1-5% of the bacterial biovolume. Most of the epiflora-PHOs hybridized with probe SAP-309 targeting Saprospiraceae in the phylum Bacteroidetes and accounted for 8-12% of the total bacterial biovolume in most plants and were thus an important and dominant part of the microbial communities.     

Microbial Diversity and Activity along the Forefields of Two Receding Glaciers

Forefields of two receding glaciers were sampled along either a 150 or 200 m long transect at identical spatial intervals for assessment of soil microbial activity and community diversity trends. The forefields belonged to the Dammaglacier (forefield area is 157 ha, 2000 m above sea level) and Rotfirnglacier (100 ha, 2200 m) and at the time of sampling were receding at an estimated rate of 8 and 10 m yr(-1) over the past 5 years, respectively. Direct counting of bacteria (DAPI staining), assessment of dehydrogenase activity (DH), and fluorescein diacetate hydrolysis activity (FDA) were performed to estimate bacteria number and soil microbial activity. Along the Dammaglacier forefield (from youngest to oldest soil), bacteria number (8.21 x 10(7) to 1.49 x 10(9) cells g(-1) soil), DH activity (0 to 61 mg TTC reduced g(-1) soil h(-1)), and FDA activity (0 to 100 mg fluorescein produced g-1 soil h-1) increased, suggesting the development of microbial populations increasing in number and activity. The Rotfirn forefield exhibited similar trends per gram of soil in bacteria number (1.13 x 10(8) to 5.93 x 10(9) cells), DH activity (0 to 36 mg TTC reduced), and FDA activity (2 to 70 mg fluorescein produced), but with more variability among samples than the Damma forefield samples. Molecular assessment of bacterial diversity included denaturing gradient gel electrophoresis (DGGE) and ribosomal intergenic spacer analysis (RISA) of soil DNA. DGGE and RISA revealed that the composition and succession of bacterial populations were different in both forefields. Comparison of Shannon diversity index values indicated that all populations sampled from the Damma forefield were significantly different (p < 0.05). Conversely, similar populations existed in the Rotfirn forefield succession. Overall, the results indicate that diverse bacterial assemblages increasing in number and activity characterize these glacier forefield soils with both forefield successions exhibiting differing modes of bacterial community establishment.     

Pyrosequencing analysis of bacterial community and assembly in activated sludge samples from different geographic regions in China

In order to investigate if there are geographic differences of bacterial community in the activated sludge collected from different geographic regions (eastern, northwestern, and northern parts) of China and to determine the co-occurrence patterns of bacterial community, activated sludge samples were collected from 10 municipal wastewater treatment plants located in 8 cities in China. High-throughput pyrosequencing combined with the bioinformatics analysis were used to examine the bacterial community compositions in the activated sludge samples. The result of taxonomy classifier indicated that a total of 76 genera were commonly shared by more than 7 samples, which accounted for 62 to 96 % of the classified sequences in each sample. Even though some core genera existed in all examined activated sludge samples regardless of the sampling geographic location and treatment process, significant geographic differences of bacterial community compositions among the activated sludge samples were revealed by the nonmetric multidimensional scaling (NMDS) and analyses of similarity (ANOSIM) analysis. A total of 165 pairs of significant and robust correlations (positive and negative) were identified from 61 bacterial genera based on the network analysis. The data obtained in this study could provide useful information to understand the bacterial community composition in geographically distributed wastewater treatment plants and discern the co-occurrence patterns of bacterial community.     

Bacterial community dynamics in long‐term operation of a pilot plant using aerobic granular sludge to treat pig slurry

Aerobic granular sludge represents an interesting approach for simultaneous organic matter and nitrogen removal in wastewater treatment plants. However, the information about microbial communities in aerobic granular systems dealing with industrial wastewater like pig slurry is limited. Herein, bacterial diversity and dynamics were assessed in a pilot scale plant using aerobic granular sludge for organic matter and nitrogen elimination from swine slurry during more than 300 days. Results indicated that bacterial composition evolved throughout the operational period from flocculent activated sludge, used as inoculum, to mature aerobic granules. Bacterial diversity increased at the beginning of the granulation process and then declined due to the application of transient organic matter and nitrogen loads. The operational conditions of the pilot plant and the degree of granulation determined the microbial community of the aerobic granules. Brachymonas, Zoogloea and Thauera were attributed with structural function as they are able to produce extracellular polymeric substances to maintain the granular structure. Nitrogen removal was justified by partial nitrification (Nitrosomonas) and denitrification (Thauera and Zoogloea), while Comamonas was identified as the main organic matter oxidizing bacteria. Overall, clear links between bacterial dynamics and composition with process performance were found and will help to predict their biological functions in wastewater ecosystems improving the future control of the process. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1212–1221, 2016     

Microbial community analysis of field-grown soybeans with different nodulation phenotypes

Microorganisms associated with the stems and roots of nonnodulated (Nod(-)), wild-type nodulated (Nod(+)), and hypernodulated (Nod(++)) soybeans [Glycine max (L.) Merril] were analyzed by ribosomal intergenic transcribed spacer analysis (RISA) and automated RISA (ARISA). RISA of stem samples detected no bands specific to the nodulation phenotype, whereas RISA of root samples revealed differential bands for the nodulation phenotypes. Pseudomonas fluorescens was exclusively associated with Nod(+) soybean roots. Fusarium solani was stably associated with nodulated (Nod(+) and Nod(++)) roots and less abundant in Nod(-) soybeans, whereas the abundance of basidiomycetes was just the opposite. The phylogenetic analyses suggested that these basidiomycetous fungi might represent a root-associated group in the Auriculariales. Principal-component analysis of the ARISA results showed that there was no clear relationship between nodulation phenotype and bacterial community structure in the stem. In contrast, both the bacterial and fungal community structures in the roots were related to nodulation phenotype. The principal-component analysis further suggested that bacterial community structure in roots could be classified into three groups according to the nodulation phenotype (Nod(-), Nod(+), or Nod(++)). The analysis of root samples indicated that the microbial community in Nod(-) soybeans was more similar to that in Nod(++) soybeans than to that in Nod(+) soybeans.     

A soil microscale study to reveal the heterogeneity of Hg(II) impact on indigenous bacteria by quantification of adapted phenotypes and analysis of community DNA fingerprints

The short term impact of 50 μM Hg(II) on soil bacterial community structure was evaluated in different microenvironments of a silt loam soil in order to determine the contribution of bacteria located in these microenvironments to the overall bacterial response to mercury spiking. Microenvironments and associated bacteria, designated as bacterial pools, were obtained by successive soil washes to separate the outer fraction, containing loosely associated bacteria, and the inner fraction, containing bacteria retained into aggregates, followed by a physical fractionation of the inner fraction to separate aggregates according to their size (size fractions). Indirect enumerations of viable heterotrophic (VH) and resistant (Hg(R)) bacteria were performed before and 30 days after mercury spiking. A ribosomal intergenic spacer analysis (RISA), combined with multivariate analysis, was used to compare modifications at the community level in the unfractionated soil and in the microenvironments. The spatial heterogeneity of the mercury impact was revealed by a higher increase of Hg(R) numbers in the outer fraction and in the coarse size fractions. Furthermore, shifts in RISA patterns of total community DNA indicated changes in the composition of the dominant bacterial populations in response to Hg(II) stress in the outer and in the clay size fractions. The heterogeneity of metal impact on indigenous bacteria, observed at a microscale level, is related to both the physical and chemical characteristics of the soil microenvironments governing mercury bioavailability and to the bacterial composition present before spiking.