Temporal microbial diversity changes in solvent-degrading anaerobic granular sludge from low-temperature (15 degrees C) wastewater treatment bioreactors

Anaerobic sludge granules were obtained from laboratory-scale anaerobic bioreactors used to treat pharmaceutical-like (methanol-, acetone- and propanol-contaminated) wastewater under low-temperature conditions (15 degrees C). The microbial diversity and diversity changes of the sludge samples were ascertained by applying 16S rRNA gene cloning and terminal restriction fragment length polymorphism (TRFLP) analyses, respectively, and using sludge samples from the inoculum, throughout and at the conclusion of the bioreactor trial. Data from genetic fingerprinting correlated well with those from physiological activity assays of the reactor biomass. Specifically, for example, TRFLP profiles indicated the dominance of hydrogenotrophic methanogens within the archaeal community, thus supporting the findings of specific methanogenic activity measurements. TRFLP data supported the hypothesis that the deviation between the replicated reactors, in terms of treatment efficiency, was associated with succession within the microbial communities present, and indicated that community development was linked to both operating temperature and wastewater composition. Fluorescence in situ hybridization (FISH) was also applied, to quantitatively assess the abundance of selected microbial groups, and revealed the underestimation of the abundance Methanosarcina by gene cloning analysis and demonstrated the spatial arrangement of these organisms within the architecture of the low-temperature solvent-degrading anaerobic biofilms.     

Carbon source — A strong determinant of microbial community structure and performance of an anaerobic reactor

Industrial effluents differ in their organic composition thereby providing different carbon sources to the microbial communities involved in its treatment. This study aimed to investigate the correlation of microbial community structure with wastewater composition and reactor's performance. Self-immobilized granules were developed in simulated wastewater based on different carbon sources (glucose, sugarcane molasses, and milk) in three hybrid anaerobic reactors operated at 37 °C. To study archaeal community structure, a polyphasic approach was used with both qualitative and quantitative analysis. While PCR-denaturing gradient gel electrophoresis of 16S rRNA gene did not reveal major shifts in diversity of archaea with change in substrate, quantification of different groups of methanogens and total bacteria by real-time PCR showed variations in relative abundances with the dominance of Methanosaetaceae and Methanobacteriales. These data were supported by differences in the ratio of total counts of archaea and bacteria analyzed by catalyzed reporter deposition – fluorescence in situ hybridization. During hydraulic and organic shocks, the molasses-based reactor showed the best performance followed by the milk- and the glucose-based reactor. The study indicates that carbon source shapes the microbial community structure more in terms of relative abundance with distinct metabolic capacities rather than its diversity itself.     

The microbial community composition of a nitrifying-denitrifying activated sludge from an industrial sewage treatment plant analyzed by the full-cycle rRNA approach

The composition of the microbial community present in the nitrifying-denitrifying activated sludge of an industrial wastewater treatment plant connected to a rendering facility was investigated by the full-cycle rRNA approach. After DNA extraction using three different methods, 94 almost full-length 16S rRNA gene clones were retrieved and analyzed phylogenetically. 59% of the clones were affiliated with the Proteobacteria and clustered with the beta- (29 clones), alpha- (24), and delta-class (2 clones), respectively. 15 clones grouped within the green nonsulfur (GNS) bacteria and 11 clones belonged to the Planctomycetes. The Verrucomicrobia, Acidobacteria, Nitrospira, Bacteroidetes, Firmicutes and Actinobacteria were each represented by one to five clones. Interestingly, the highest 'species richness' [measured as number of operational taxonomic units (OTUs)] was found within the alpha-class of Proteobacteria, followed by the Planctomycetes, the beta-class of Proteobacteria, and the GNS-bacteria. The microbial community composition of the activated sludge was determined quantitatively by using 36 group-, subgroup-, and OTU-specific rRNA-targeted oligonucleotide probes for fluorescence in situ hybridization (FISH), confocal laser scanning microscopy and digital image analysis. 89% of all bacteria detectable by FISH with a bacterial probe set could be assigned to specific divisions. Consistent with the 16S rRNA gene library data, members of the beta-class of Proteobacteria dominated the microbial community and represented almost half of the biovolume of all bacteria detectable by FISH. Within the beta-class, 98% of the cells could be identified by the application of genus- or OTU-specific probes demonstrating a high in situ abundance of bacteria related to Zoogloea and Azoarcus sensu lato. Taken together, this study provides the first encompassing, high-resolution insight into the in situ composition of the microbial community present in a full-scale, industrial wastewater treatment plant.     

Changes in the structure and function of microbial communities in drinking water treatment bioreactors upon addition of phosphorus

Phosphorus was added as a nutrient to bench-scale and pilot-scale biologically active carbon (BAC) reactors operated for perchlorate and nitrate removal from contaminated groundwater. The two bioreactors responded similarly to phosphorus addition in terms of microbial community function (i.e., reactor performance), while drastically different responses in microbial community structure were detected. Improvement in reactor performance with respect to perchlorate and nitrate removal started within a few days after phosphorus addition for both reactors. Microbial community structures were evaluated using molecular techniques targeting 16S rRNA genes. Clone library results showed that the relative abundance of perchlorate-reducing bacteria (PRB) Dechloromonas and Azospira in the bench-scale reactor increased from 15.2% and 0.6% to 54.2% and 11.7% after phosphorus addition, respectively. Real-time quantitative PCR (qPCR) experiments revealed that these increases started within a few days after phosphorus addition. In contrast, after phosphorus addition, the relative abundance of Dechloromonas in the pilot-scale reactor decreased from 7.1 to 0.6%, while Zoogloea increased from 17.9 to 52.0%. The results of this study demonstrated that similar operating conditions for bench-scale and pilot-scale reactors resulted in similar contaminant removal performances, despite dramatically different responses from microbial communities. These findings suggest that it is important to evaluate the microbial community compositions inside bioreactors used for drinking water treatment, as they determine the microbial composition in the effluent and impact downstream treatment requirements for drinking water production. This information could be particularly relevant to drinking water safety, if pathogens or disinfectant-resistant bacteria are detected in the bioreactors.     

Site-specific variation in Antarctic marine biofilms established on artificial surfaces

The community structure and composition of marine microbial biofilms established on glass surfaces was investigated across three differentially contaminated Antarctic sites within McMurdo Sound. Diverse microbial communities were revealed at all sites using fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) techniques. Sequencing of excised DGGE bands demonstrated close affiliation with known psychrophiles or undescribed bacteria also recovered from the Antarctic environment. The majority of bacterial sequences were affiliated to the Gammaproteobacteria, Cytophaga/Flavobacteria of Bacteroidetes (CFB), Verrucomicrobia and Planctomycetales. Principal components analysis of quantitative FISH data revealed distinct differences in community composition between sites. Each of the sites were dominated by different bacterial groups: Alphaproteobacteria, Gammaproteobacteria and CFB at the least impacted site, Cape Armitage; green sulfur and sulfate reducing bacteria near the semi-impacted Scott Base and Planctomycetales and sulfate reducing bacteria near the highly impacted McMurdo Station. The highest abundance of archaea was detected near Scott Base (2.5% of total bacteria). Multivariate analyses (non-metric multidimensional scaling and analysis of similarities) of DGGE patterns revealed greater variability in community composition between sites than within sites. This is the first investigation of Antarctic biofilm structure and FISH results suggest that anthropogenic impacts may influence the complex composition of microbial communities.     

Microbial composition of the activated sludge of Moscow wastewater treatment plants

The contribution of the major technologically important microbial groups (ammonium- and nitrite-oxidizing, phosphate-accumulating, foam-inducing, and anammox bacteria, as well as planctomycetes and methanogenic archaea) was characterized for the aeration tanks of the Moscow wastewater treatment facilities. FISH investigation revealed that aerobic sludge were eubacterial communities; the metabolically active archaea contributed insignificantly. Stage II nitrifying microorganisms and planctomycetes were significant constituents of the bacterial component of activated sludges, with Nitrobacter spp. being the dominant nitrifiers. No metabolically active anammox bacteria were revealed in the sludge from aeration tanks. The sludge from the aeration tanks using different wastewater treatment technologies were found to have differing characteristics. Abundance of the nitrifying and phosphate-accumulating bacteria in the sludge generally correlated with microbial activity in microcosms and with efficiency of nitrogen and phosphorus removal from wastewater. The highest microbial numbers and activity were found in the sludge of the tanks operating according to the technologies developed in the universities of Hannover and Cape Town. The activated sludge from the Novokur’yanovo facilities, where abundant growth of filamentous bacteria resulted in foam formation, exhibited the lowest activity. The group of foaming bacteria included Gordonia spp. and Acinetobacter spp utilizing petroleum and motor oils, Sphaerotilus spp. utilizing unsaturated fatty acids, and Candidatus ‘Microthrix parvicella’. Thus, the data on abundance and composition of metabolically active microorganisms obtained by FISH may be used for the technological control of wastewater treatment.     

FISH荧光原位杂交技术在污水生物脱氮研究中的应用

简要介绍了荧光原位杂交（FISH）的基本原理,着重讨论近年来该技术在污水生物脱氮研究中的应用现状和特点。研究表明：FISH技术能够准确地表现污水处理反应器中脱氮菌群落的类型和结构形态。但在关于SRT、DO、C／N比等工艺参数的变化对脱氮反应器中微生物类型、数量和结构的影响等方面的研究还有待深入。FISH技术与PCR—DGGE和16SrRNA／rDNA序列分析等技术相结合是对污水处理构筑物中生物脱氮群落深入研究的发展方向。     

Ecophysiology of abundant denitrifying bacteria in activated sludge

The abundance of potential denitrifiers in full-scale wastewater treatment plants with biological nitrogen and phosphorus removal was investigated by FISH and various oligonucleotide probes. The potential denitrifiers were characterized as probe-defined populations that were able to consume radiolabelled substrate with oxygen, nitrate and nitrite as electron acceptor as determined by microautoradiography. The most abundant potential denitrifiers were related to the genera Aquaspirillum, Azoarcus, Thauera and Rhodocyclus, all within the Betaproteobacteria. They made up 20-49% of all bacteria in most of the 17 nitrogen removal plants investigated and were hardly present in four plants without denitrification. The ecophysiology of Aquaspirillum, Azoarcus and Thauera-related bacteria was consistent within each probe-defined group in the plants investigated. These three groups showed distinct physiological differences, with the Aquaspirillum-related bacteria appearing as the most specialized one, consuming only amino acids among the substrates tested, and Thauera as the most versatile consuming some volatile fatty acids, ethanol and amino acids. The coexistence of Aquaspirillum, Azoarcus and Thauera-related bacteria in a range of treatment plants with differences in wastewater, design and operation suggest that the populations ensure a functional stability of the plants by occupying different ecological niches related to the carbon transformation.     

毛乌素沙地固碳功能菌群落随生物结皮发育阶段的演变特征

生物结皮是干旱区生态系统光合固碳的重要贡献者,固碳功能菌是生物结皮碳固定过程中的关键功能群,而毛乌素沙地生物结皮有关固碳功能菌群落多样性的研究未见详细报道。通过qPCR和高通量测序,研究了毛乌素沙地固碳功能菌基因丰度、群落多样性在生物结皮发育过程中的演变规律及其关键环境因子。结果表明,form IAB、IC和ID的基因丰度均随生物结皮发育呈升高趋势,在地衣结皮最高,在藓结皮又明显降低;form IAB的Chao1和form IC的Shannon指数随生物结皮演替呈显著上升趋势,而 form IC和ID的Chao1则在藻结皮达到峰值。物种组成上,IAB型固碳功能菌以蓝藻门的颤藻目占优势,其相对丰度在藻结皮最高,随生物结皮演替逐渐降低;IC型固碳功能菌在裸沙中以土壤红杆菌目为主, 而在三种生物结皮中以生丝微菌目、亚硝化单胞菌目和红螺菌目占优势; ID型固碳菌则以硅藻门的舟形藻目占优势,其丰度在裸沙中最高。IAB和IC型固碳功能菌群落结构在藻结皮和地衣结皮类似,而与裸沙和藓结皮差异显著,裸沙与藓结皮亦具显著差异。生物结皮演替过程中生物类群和土壤理化特征的改变为固碳功能菌提供了不同的生态位,土壤总有机碳、全氮、铵态氮、全磷、速效磷和pH对固碳功能菌群落进行综合调控,通过对固碳功能菌的筛选作用,最终改变固碳功能菌群落组成和结构。为深入理解荒漠生态系统生物结皮的光合固碳功能的微生物学机制提供了理论支撑。     

Link between microbial composition and carbon substrate-uptake preferences in a PHA-storing community

The microbial community of a fermented molasses-fed sequencing batch reactor (SBR) operated under feast and famine conditions for production of polyhydroxyalkanoates (PHAs) was identified and quantified through a 16 S rRNA gene clone library and fluorescence in situ hybridization (FISH). The microbial enrichment was found to be composed of PHA-storing populations (84% of the microbial community), comprising members of the genera Azoarcus, Thauera and Paracoccus. The dominant PHA-storing populations ensured the high functional stability of the system (characterized by high PHA-storage efficiency, up to 60% PHA content). The fermented molasses contained primarily acetate, propionate, butyrate and valerate. The substrate preferences were determined by microautoradiography-FISH and differences in the substrate-uptake capabilities for the various probe-defined populations were found. The results showed that in the presence of multiple substrates, microbial populations specialized in different substrates were selected, thereby co-existing in the SBR by adapting to different niches. Azoarcus and Thauera, primarily consumed acetate and butyrate, respectively. Paracoccus consumed a broader range of substrates and had a higher cell-specific substrate uptake. The relative species composition and their substrate specialization were reflected in the substrate removal rates of different volatile fatty acids in the SBR reactor.     

Microbial community composition and dynamics of moving bed biofilm reactor systems treating municipal sewage

Moving bed biofilm reactor (MBBR) systems are increasingly used for municipal and industrial wastewater treatment, yet in contrast to activated sludge (AS) systems, little is known about their constituent microbial communities. This study investigated the community composition of two municipal MBBR wastewater treatment plants (WWTPs) in Wellington, New Zealand. Monthly samples comprising biofilm and suspended biomass were collected over a 12-month period. Bacterial and archaeal community composition was determined using a full-cycle community approach, including analysis of 16S rRNA gene libraries, fluorescence in situ hybridization (FISH) and automated ribosomal intergenic spacer analysis (ARISA). Differences in microbial community structure and abundance were observed between the two WWTPs and between biofilm and suspended biomass. Biofilms from both plants were dominated by Clostridia and sulfate-reducing members of the Deltaproteobacteria (SRBs). FISH analyses indicated morphological differences in the Deltaproteobacteria detected at the two plants and also revealed distinctive clustering between SRBs and members of the Methanosarcinales, which were the only Archaea detected and were present in low abundance (<5%). Biovolume estimates of the SRBs were higher in biofilm samples from one of the WWTPs which receives both domestic and industrial waste and is influenced by seawater infiltration. The suspended communities from both plants were diverse and dominated by aerobic members of the Gammaproteobacteria and Betaproteobacteria. This study represents the first detailed analysis of microbial communities in full-scale MBBR systems and indicates that this process selects for distinctive biofilm and planktonic communities, both of which differ from those found in conventional AS systems.     

Spatial distribution of ammonia-oxidizing bacteria and archaea across a 44-hectare farm related to ecosystem functioning

Characterization of spatial patterns of functional microbial communities could facilitate the understanding of the relationships between the ecology of microbial communities, the biogeochemical processes they perform and the corresponding ecosystem functions. Because of the important role the ammonia-oxidizing bacteria (AOB) and archaea (AOA) have in nitrogen cycling and nitrate leaching, we explored the spatial distribution of their activity, abundance and community composition across a 44-ha large farm divided into an organic and an integrated farming system. The spatial patterns were mapped by geostatistical modeling and correlations to soil properties and ecosystem functioning in terms of nitrate leaching were determined. All measured community components for both AOB and AOA exhibited spatial patterns at the hectare scale. The patchy patterns of community structures did not reflect the farming systems, but the AOB community was weakly related to differences in soil pH and moisture, whereas the AOA community to differences in soil pH and clay content. Soil properties related differently to the size of the communities, with soil organic carbon and total nitrogen correlating positively to AOB abundance, while clay content and pH showed a negative correlation to AOA abundance. Contrasting spatial patterns were observed for the abundance distributions of the two groups indicating that the AOB and AOA may occupy different niches in agro-ecosystems. In addition, the two communities correlated differently to community and ecosystem functions. Our results suggest that the AOA, not the AOB, were contributing to nitrate leaching at the site by providing substrate for the nitrite oxidizers.     

Plant Communities, Soil Microorganisms, and Soil Carbon Cycling: Does Altering the World Belowground Matter to Ecosystem Functioning?

Soil microorganisms mediate many critical ecosystem processes. Little is known, however, about the factors that determine soil microbial community composition, and whether microbial community composition influences process rates. Here, we investigated whether aboveground plant diversity affects soil microbial community composition, and whether differences in microbial communities in turn affect ecosystem process rates. Using an experimental system at La Selva Biological Station, Costa Rica, we found that plant diversity (plots contained 1, 3, 5, or > 25 plant species) had a significant effect on microbial community composition (as determined by phospholipid fatty acid analysis). The different microbial communities had significantly different respiration responses to 24 labile carbon compounds. We then tested whether these differences in microbial composition and catabolic capabilities were indicative of the ability of distinct microbial communities to decompose different types of litter in a fully factorial laboratory litter transplant experiment. Both microbial biomass and microbial community composition appeared to play a role in litter decomposition rates. Our work suggests, however, that the more important mechanism through which changes in plant diversity affect soil microbial communities and their carbon cycling activities may be through alterations in their abundance rather than their community composition.     

Enumeration and identification of dominant types of sulfate-reducing bacteria in pulp from a paper-recycling plant: a multiphasic approach

Multiple independent approaches were applied for monitoring the abundance and identity of sulfate-reducing bacteria (SRB) in pulp of a paper-recycling plant suffering from excessive sulfide emission. The methods applied included most-probable-number (MPN) enumeration of cultivable SRB, rate measurements, FISH and PCR-based retrieval of the functional marker genes dsrA and B (encoding the two major subunits of dissimilatory bisulfite reductase) and 16S rRNA genes. The SRB community was composed of phylogenetically highly different lineages all of low abundance relative to the total microbial community in the pulp, which hampered the applicability of FISH. It was also demonstrated that dsrA- or B -targeted PCR primers commonly used for denaturing gradient gel electrophoresis and real-time PCR analyses were biased. However, using a novel approach combining MPN-PCR and terminal restriction fragment length polymorphism analysis of dsrAB amplicons generated from serially diluted DNA extracts allowed the enumeration and identification of the quantitatively most important members of the SRB community. For fast quantification of SRB in the pulp, the dsrAB -MPN-PCR assay and sulfate reduction rate measurements were found to be most suitable.     

Effects of plant and influent C:N:P ratio on microbial diversity in pilot-scale constructed wetlands

Microbial processes within the rhizosphere of constructed wetlands are crucial to wastewater treatment, but the relation between microbial community diversity in rhizosphere, plant growth and water quality are unclear at present. The effects of plant growth, water C:N:P ratio and their interaction on microbial diversity in the rhizosphere were studied in synthetic wastewater in planted and unplanted wetlands during three different seasons. The physiological profile of microbial community-level in each wetland was assessed using substrate utilization patterns gathered via BIOLOG? ECO plates. Plant had a significant effect on AWCD parameter, since the planted wetlands usually had a higher the total microbial activity than the unplanted over the study period. The Shannon, Simpson and McIntosh indices in the planted wetlands were apparently higher than those in the unplanted wetlands under any C:N:P ratio influent condition especially in summer. It was also shown that the unplanted wetlands have a greater shift of the interstitial microbial community than the planted at different seasons, since plant rhizospheres produce a more ecologically stable system in order to resist against shifts in microbial community composition in response to C:N:P ratio change in wastewater. Principal component analysis and clustering analysis indicated that influent C:N:P ratio would induce similar microbial species in the planted wetlands and detach them from the unplanted wetlands.     

Diversity and population structure of sewage‐derived microorganisms in wastewater treatment plant influent

The release of untreated sewage introduces non‐indigenous microbial populations of uncertain composition into surface waters. We used massively parallel 454 pyrosequencing of hypervariable regions in rRNA genes to profile microbial communities from eight untreated sewage influent samples of two wastewater treatment plants (WWTPs) in metropolitan Milwaukee. The sewage profiles included a discernible human faecal signature made up of several taxonomic groups including multiple Bifidobacteriaceae, Coriobacteriaceae, Bacteroidaceae, Lachnospiraceae and Ruminococcaceae genera. The faecal signature made up a small fraction of the taxa present in sewage but the relative abundance of these sequence tags mirrored the population structures of human faecal samples. These genera were much more prevalent in the sewage influent than standard indicators species. High‐abundance sequences from taxonomic groups within the Beta‐ and Gammaproteobacteria dominated the sewage samples but occurred at very low levels in faecal and surface water samples, suggesting that these organisms proliferate within the sewer system. Samples from Jones Island (JI – servicing residential plus a combined sewer system) and South Shore (SS – servicing a residential area) WWTPs had very consistent community profiles, with greater similarity between WWTPs on a given collection day than the same plant collected on different days. Rainfall increased influent flows at SS and JI WWTPs, and this corresponded to greater diversity in the community at both plants. Overall, the sewer system appears to be a defined environment with both infiltration of rainwater and stormwater inputs modulating community composition. Microbial sewage communities represent a combination of inputs from human faecal microbes and enrichment of specific microbes from the environment to form a unique population structure.     

Microbial community composition controls carbon flux across litter types in early phase of litter decomposition

Leaf litter decomposition is a major carbon input to soil, making it a target for increasing soil carbon storage through microbiome engineering. We expand upon previous findings to show with multiple leaf litter types that microbial composition can drive variation in carbon flow from litter decomposition and specific microbial community features are associated with synonymous patterns of carbon flow among litter types. Although plant litter type selects for different decomposer communities, within a litter type, microbial composition drives variation in the quantity of dissolved organic carbon (DOC) measured at the end of the decomposition period. Bacterial richness was negatively correlated with DOC quantity, supporting our hypothesis that across multiple litter types there are common microbial traits linked to carbon flow patterns. Variation in DOC abundance (i.e. high versus low DOC) driven by microbial composition is tentatively due to differences in bacterial metabolism of labile compounds, rather than catabolism of non-labile substrates such as lignin. The temporal asynchrony of metabolic processes across litter types may be a substantial impediment to discovering more microbial features common to synonymous patterns of carbon flow among litters. Overall, our findings support the concept that carbon flow may be programmed by manipulating microbial community composition.     

Soil microbial and chemical responses to foliar Epichloë fungal infection in Lolium perenne,Hordeum brevisubulatum and Achnatherum inebrians

We tested whether the host species identity in grass-Epichloë symbioses affected soil chemical and microbial properties. We grew endophyte infected (E+) and endophyte free (E−) Lolium perenne, Hordeum brevisubulatum and Achnatherum inebrians for 18 months in field plots. In E+ soil of all three grasses, available phosphorus was lower whereas total soil nitrogen was higher. Endophyte effects on soil pH, microbial biomass nitrogen, total carbon and organic carbon as well as bacteria and fungi abundance were host species dependent. Ammonia oxidizing bacteria abundance was higher in E+ soils for all species. Bacterial community composition of E+ and E− soils were different only for Lolium perenne with soil pH being the key factor. Fungal community composition of E+ and E− soils was not different for the three grasses. This study confirmed that the effects of foliar Epichloë infection on belowground properties depended on host species identity.     

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。     

Phylogenetic Composition, Spatial Structure, and Dynamics of Lotic Bacterial Biofilms Investigated by Fluorescent in Situ Hybridization and Confocal Laser Scanning Microscopy

Abstract The phylogenetic composition, three-dimensional structure and dynamics of bacterial communities in river biofilms generated in a rotating annular reactor system were studied by fluorescent in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Biofilms grew on independently removable polycarbonate slides exposed in the reactor system with natural river water as inoculum and sole nutrient and carbon source. The microbial biofilm community developed from attached single cells and distinct microcolonies via a more confluent structure characterized by various filamentous bacteria to a mature biofilm rich in polymeric material with fewer cells on a per-area basis after 56 days. During the different stages of biofilm development, characteristic microcolonies and cell morphotypes could be identified as typical features of the investigated lotic biofilms. In situ analysis using a comprehensive suite of rRNA-targeted probes visualized individual cells within the alpha-, beta-, and gamma-Proteobacteria as well as the Cytophaga–Flavobacterium group as major parts of the attached community. The relative abundance of these major groups was determined by using digital image analysis to measure specific cell numbers as well as specific cell area after in situ probing. Within the lotic biofilm community, 87% of the whole bacterial cell area and 79% of the total cell counts hybridized with a Bacteria specific probe. During initial biofilm development, beta-Proteobacteria dominated the bacterial population. This was followed by a rapid increase of alpha-Proteobacteria and bacteria affiliated to the Cytophaga–Flavobacterium group. In mature biofilms, alpha-Proteobacteria and Cytophaga–Flavobacteria continued to be the prevalent bacterial groups. Beta-Proteobacteria constituted the morphologically most diverse group within the biofilm communities, and more narrow phylogenetic staining revealed the importance of distinct phylotypes within the beta1-Proteobacteria for the composition of the microbial community. The presence of sulfate-reducing bacteria affiliated to the Desulfovibrionaceae and Desulfobacteriaceae confirmed the range of metabolic potential within the lotic biofilms. Received: 24 September 1998; Accepted: 17 February 1999