Identification of polyphosphate-accumulating organisms and design of 16S rRNA-directed probes for their detection and quantitation

Laboratory-scale sequencing batch reactors (SBRs) as models for activated sludge processes were used to study enhanced biological phosphorus removal (EBPR) from wastewater. Enrichment for polyphosphate-accumulating organisms (PAOs) was achieved essentially by increasing the phosphorus concentration in the influent to the SBRs. Fluorescence in situ hybridization (FISH) using domain-, division-, and subdivision-level probes was used to assess the proportions of microorganisms in the sludges. The A sludge, a high-performance P-removing sludge containing 15.1% P in the biomass, was comprised of large clusters of polyphosphate-containing coccobacilli. By FISH, >80% of the A sludge bacteria were beta-2 Proteobacteria arranged in clusters of coccobacilli, strongly suggesting that this group contains a PAO responsible for EBPR. The second dominant group in the A sludge was the Actinobacteria. Clone libraries of PCR-amplified bacterial 16S rRNA genes from three high-performance P-removing sludges were prepared, and clones belonging to the beta-2 Proteobacteria were fully sequenced. A distinctive group of clones (sharing >/=98% sequence identity) related to Rhodocyclus spp. (94 to 97% identity) and Propionibacter pelophilus (95 to 96% identity) was identified as the most likely candidate PAOs. Three probes specific for the highly related candidate PAO group were designed from the sequence data. All three probes specifically bound to the morphologically distinctive clusters of PAOs in the A sludge, exactly coinciding with the beta-2 Proteobacteria probe. Sequential FISH and polyphosphate staining of EBPR sludges clearly demonstrated that PAO probe-binding cells contained polyphosphate. Subsequent PAO probe analyses of a number of sludges with various P removal capacities indicated a strong positive correlation between P removal from the wastewater as determined by sludge P content and number of PAO probe-binding cells. We conclude therefore that an important group of PAOs in EBPR sludges are bacteria closely related to Rhodocyclus and Propionibacter.     

Discrepancies in the widely applied GAM42a fluorescence in situ hybridisation probe for Gammaproteobacteria

A bacterial culture collection of 104 strains was obtained from an activated sludge wastewater treatment plant to pursue studies into microbial flocculation. Characterisation of the culture collection using a polyphasic approach indicated seven isolates, phylogenetically affiliated with the deep-branching Xanthomonas group of the class Gammaproteobacteria, were unable to hybridise the GAM42a fluorescence in situ hybridisation (FISH) probe for Gammaproteobacteria. The sequence of the GAM42a probe target region in the 23S rRNA gene of these isolates was determined to have mismatches to GAM42a. Probes perfectly targeting the mismatches (GAM42a_T1038_G1031, and GAM42a_T1038 and GAM42a_A1041_A1040) were synthesised, and used in conjunction with GAM42a in FISH to study the Gammaproteobacteria community structure in one full-scale activated sludge plant. Several bacteria in the activated sludge biomass bound the modified probes demonstrating their presence and the fact that these Gammaproteobacteria have been overlooked in community structure analyses of activated sludge.     

rRNA-targeted寡核苷酸探针识别活性污泥微生物群落结构与功能研究进展

活性污泥中微生物群落内部关系非常复杂 ,及时对活性污泥中优势菌群和群落内部关系进行监测是污水处理中采取正确措施的关键。历史研究表明传统培养方法经常导致活性污泥优势菌群检测的失败 ,而r RNA- targeted寡核苷酸探针作为一种快速原位监测活性污泥微生物群落结构和功能的新工具被引入 ,使我们对参与污水净化的微生物群落结构和优势菌群能有较全面的了解。就该方法在识别除磷污泥、脱氮污泥、污泥泡沫和膨胀污泥中微生物群落结构和功能的典型应用进行综述 ,分析了该方法存在的优点和缺点 ,并对目前已建立且应用于活性污泥微生物检测的 r RNA- targeted寡核苷酸探针进行了详细总结     

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 diversity of bacteria associated with the marine sponge Rhopaloeides odorabile

Molecular techniques were employed to document the microbial diversity associated with the marine sponge Rhopaloeides odorabile. The phylogenetic affiliation of sponge-associated bacteria was assessed by 16S rRNA sequencing of cloned DNA fragments. Fluorescence in situ hybridization (FISH) was used to confirm the presence of the predominant groups indicated by 16S rDNA analysis. The community structure was extremely diverse with representatives of the Actinobacteria, low-G+C gram-positive bacteria, the beta- and gamma-subdivisions of the Proteobacteria, Cytophaga/Flavobacterium, green sulfur bacteria, green nonsulfur bacteria, planctomycetes, and other sequence types with no known close relatives. FISH probes revealed the spatial location of these bacteria within the sponge tissue, in some cases suggesting possible symbiotic functions. The high proportion of 16S rRNA sequences derived from novel actinomycetes is good evidence for the presence of an indigenous marine actinomycete assemblage in R. odorabile. High microbial diversity was inferred from low duplication of clones in a library with 70 representatives. Determining the phylogenetic affiliation of sponge-associated microorganisms by 16S rRNA analysis facilitated the rational selection of culture media and isolation conditions to target specific groups of well-represented bacteria for laboratory culture. Novel media incorporating sponge extracts were used to isolate bacteria not previously recovered from this sponge.     

Phylogenetic analysis and in situ identification of bacteria community composition in an acidic Sphagnum peat bog

The Bacteria community composition in an acidic Sphagnum peat bog (pH 3.9 to 4.5) was characterized by a combination of 16S rRNA gene clone library analysis, rRNA-targeted fluorescence in situ hybridization (FISH), and cultivation. Among 84 environmental 16S rRNA gene clones, a set of only 16 cloned sequences was closely related (>or=95% similarity) to taxonomically described organisms. Main groups of clones were affiliated with the Acidobacteria (24 clones), Alphaproteobacteria (20), Verrucomicrobia (13), Actinobacteria (8), Deltaproteobacteria (4), Chloroflexi (3), and Planctomycetes (3). The proportion of cells that hybridized with oligonucleotide probes specific for members of the domains Bacteria (EUB338-mix) and Archaea (ARCH915 and ARC344) accounted for only 12 to 22% of the total cell counts. Up to 24% of the EUB338-positive cells could be assigned by FISH to specific bacterial phyla. Alphaproteobacteria and Planctomycetes were the most numerous bacterial groups (up to 1.3x10(7) and 1.1x10(7) cells g-1 peat, respectively). In contrast to conventional plating techniques, a novel biofilm-mediated enrichment approach allowed us to isolate some representatives of predominant Bacteria groups, such as Acidobacteria and Planctomycetes. This novel strategy has great potential to enable the isolation of a significant proportion of the peat bog bacterial diversity.     

Combined Molecular and Conventional Analyses of Nitrifying Bacterium Diversity in Activated Sludge: Nitrosococcus mobilis and Nitrospira-Like Bacteria as Dominant Populations

The ammonia-oxidizing and nitrite-oxidizing bacterial populations occurring in the nitrifying activated sludge of an industrial wastewater treatment plant receiving sewage with high ammonia concentrations were studied by use of a polyphasic approach. In situ hybridization with a set of hierarchical 16S rRNA-targeted probes for ammonia-oxidizing bacteria revealed the dominance of Nitrosococcus mobilis-like bacteria. The phylogenetic affiliation suggested by fluorescent in situ hybridization (FISH) was confirmed by isolation of N. mobilis as the numerically dominant ammonia oxidizer and subsequent comparative 16S rRNA gene (rDNA) sequence and DNA-DNA hybridization analyses. For molecular fine-scale analysis of the ammonia-oxidizing population, a partial stretch of the gene encoding the active-site polypeptide of ammonia monooxygenase (amoA) was amplified from total DNA extracted from ammonia oxidizer isolates and from activated sludge. However, comparative sequence analysis of 13 amoA clone sequences from activated sludge demonstrated that these sequences were highly similar to each other and to the corresponding amoA gene fragments of Nitrosomonas europaea Nm50 and the N. mobilis isolate. The unexpected high sequence similarity between the amoA gene fragments of the N. mobilis isolate and N. europaea indicates a possible lateral gene transfer event. Although a Nitrobacter strain was isolated, members of the nitrite-oxidizing genus Nitrobacter were not detectable in the activated sludge by in situ hybridization. Therefore, we used the rRNA approach to investigate the abundance of other well-known nitrite-oxidizing bacterial genera. Three different methods were used for DNA extraction from the activated sludge. For each DNA preparation, almost full-length genes encoding small-subunit rRNA were separately amplified and used to generate three 16S rDNA libraries. By comparative sequence analysis, 2 of 60 randomly selected clones could be assigned to the nitrite-oxidizing bacteria of the genus Nitrospira. Based on these clone sequences, a specific 16S rRNA-targeted probe was developed. FISH of the activated sludge with this probe demonstrated that Nitrospira-like bacteria were present in significant numbers (9% of the total bacterial counts) and frequently occurred in coaggregated microcolonies with N. mobilis.     

Identification of some of the major groups of bacteria in efficient and nonefficient biological phosphorus removal activated sludge systems.

To investigate the bacteria that are important to phosphorus (P) removal in activated sludge, microbial populations were analyzed during the operation of a laboratory-scale reactor with various P removal performances. The bacterial population structure, analyzed by fluorescence in situ hybridization (FISH) with oligonucleotides probes complementary to regions of the 16S and 23S rRNAs, was associated with the P removal performance of the reactor. At one stage of the reactor operation, chemical characterization revealed that extremely poor P removal was occurring. However, like in typical P-removing sludges, complete anaerobic uptake of the carbon substrate occurred. Bacteria inhibiting P removal overwhelmed the reactor, and according to FISH, bacteria of the beta subclass of the class Proteobacteria other than beta-1 or beta-2 were dominant in the sludge (58% of the population). Changes made to the operation of the reactor led to the development of a biomass population with an extremely good P removal capacity. The biochemical transformations observed in this sludge were characteristic of typical P-removing activated sludge. The microbial population analysis of the P-removing sludge indicated that bacteria of the beta-2 subclass of the class Proteobacteria and actinobacteria were dominant (55 and 35%, respectively), therefore implicating bacteria from these groups in high-performance P removal. The changes in operation that led to the improved performance of the reactor included allowing the pH to rise during the anaerobic period, which promoted anaerobic phosphate release and possibly caused selection against non-phosphate-removing bacteria.     

Microbial community structures in conventional activated sludge system and membrane bioreactor (MBR)

The microbial community structures of a conventional activated sludge and MBR systems treating the municipal wastewater were studied using Fluorescent in-situ Hybridization (FISH) analysis to identify differences in both systems. The oligonucleotide probes specific for overall bacteria, including α-, β-, and γ-subclasses of Proteobacteria, ammonia-oxidizing bacteria (Nitrosomonas), and nitrite-oxidizing bacteria (Nitrobacter) were used to compare the microbial community structure of both systems. A trend of less hybridization with bacteria-specific probe EUB338 was observed in MBR systems operated under aerobic condition, compared to conventional activated sludge system. The less hybridization trend with the probes could be associated with low ribosomal RNA (rRNA) content in the biomass, which suggests that the biomass in the MBR system was not in a physiological state characteristic for growth due to low substrate per unit biomass     

Quantification of Chloroflexi Eikelboom morphotype 1851 for prediction and control of bulking events in municipal activated sludge plants in Japan

The dominant filamentous bacteria associated with bulking incidents in Japanese activated sludge plants with nutrient removal were identified and their quantitative correlations with sludge settleability were assessed, with the aim of controlling bulking incidents by specifically suppressing bacterial growth. Fluorescence in situ hybridization (FISH) analyses using existing oligonucleotide FISH probes indicated that the presence of Eikelboom type 1851 filamentous bacteria belonging to the phylum Chloroflexi is correlated with biomass settleability in the municipal wastewater treatment plants examined. Real-time quantitative PCR (qPCR) assays developed in this study also showed a linear correlation between type 1851 filament members and sludge settleability, with the exception of some winter samples. The real-time qPCR assays and 16S ribosomal RNA gene amplicon sequencing to reveal the microbial community of activated sludge showed that the abundance of type 1851 at 200 mL g⁻¹ of sludge volume index was estimated to be about 1.9% of the total microbial cells. The abundance of type 1851 served as a bulking indicator in plants where type 1851 was dominant.

     

Bacterial diversity in an industrial wastewater bioreactor

Industrial wastewater bioreactors are potentially important sources of novel biocatalysts. However, the microbial populations in these bioreactors are not well characterized. The microbial community in an industrial wastewater bioreactor was surveyed by extracting DNA from a sample of activated sludge, followed by PCR amplification and sequencing of cloned 16S rRNA genes. A total of 407 cloned 16S rRNA gene sequences were compared with 88 bacterial isolates cultured from the same sample of sludge using a variety of standard media. Most of the bacteria detected by the PCR-based approach were -subdivision Proteobacteria, whereas most of the cultured bacteria were -subdivision Proteobacteria. Only a few types of bacteria were detected by both approaches. These observations indicate that multiple techniques are necessary to characterize the microbial diversity in any complex ecosystem.     

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.     

海水养殖尾水处理系统中微生物群落对水处理阶段的响应

为了阐明南美白对虾高位池养殖尾水处理系统中不同水处理阶段微生物群落演替机制, 利用16S rRNA基因高通量测序技术分析了水体和生物膜的微生物群落结构。结果显示, 在水处理系统中主要是变形菌门(Proteobacteria)、浮霉菌门(Planctomycetes)、拟杆菌门(Bacteroidetes)、蓝细菌门(Cyanobacteria)、放线菌门(Actinobacteria)及酸杆菌门(Acidobacteria), 平均占细菌总OTU的88.61%。生物膜中生物多样性指数普遍高于水样, 与水体的共有菌为320种, 载体不同是造成群落结构差异的主要原因, 黏土陶粒和北美海蓬子(Salicornia bigelovii)根系是硝化作用的主要反应场所。在属水平上筛选出160种微生物, 主要属于变形菌门、拟杆菌门、浮霉菌门、蓝细菌门、厚壁菌门(Firmicutes)及放线菌门, 它们能够较好地区分菌群的来源及水处理的反应阶段。研究揭示了不同水处理阶段以及不同生物填料中微生物动态变化情况, 为今后的海水养殖尾水处理提供理论依据和技术参考。     

Phylogenetic diversity of bacteria in an earth-cave in Guizhou province, southwest of China

The objective of this study was to analyze the phylogenetic composition of bacterial community in the soil of an earth-cave (Niu Cave) using a culture-independent molecular approach. 16S rRNA genes were amplified directly from soil DNA with universally conserved and Bacteria-specific rRNA gene primers and cloned. The clone library was screened by restriction fragment length polymorphism (RFLP), and representative rRNA gene sequences were determined. A total of 115 bacterial sequence types were found in 190 analyzed clones. Phylogenetic sequence analyses revealed novel 16S rRNA gene sequence types and a high diversity of putative bacterial community. Members of these bacteria included Proteobacteria (42.6%), Acidobacteria (18.6%), Planctomycetes (9.0%), Chloroflexi (Green nonsulfur bacteria, 7.5%), Bacteroidetes (2.1%), Gemmatimonadetes (2.7%), Nitrospirae (8.0%), Actinobacteria (High G+C Gram-positive bacteria, 6.4%) and candidate divisions (including the OP3, GN08, and SBR1093, 3.2%). Thirty-five clones were affiliated with bacteria that were related to nitrogen, sulfur, iron or manganese cycles. The comparison of the present data with the data obtained previously from caves based on 16S rRNA gene analysis revealed similarities in the bacterial community components, especially in the high abundance of Proteobacteria and Acidobacteria. Furthermore, this study provided the novel evidence for presence of Gemmatimonadetes, Nitrosomonadales, Oceanospirillales, and Rubrobacterales in a karstic hypogean environment.     

Dominance of endospore-forming bacteria on a Rotating Activated Bacillus Contactor biofilm for advanced wastewater treatment

The bacterial diversity inherent to the biofilm community structure of a modified rotating biological contactor wastewater treatment process, referred to as the Rotating Activated Bacillus Contactor (RABC) process, was characterized in this study, via both culture-dependent and culture-independent methods. On the basis of culture-dependent methods, Bacillus sp. were found to exist in large numbers on the biofilm (6.5% of the heterotrophic bacteria) and the microbial composition of the biofilms was quite simple. Only three phyla were identified-namely, the Proteobacteria, the Actinobacteria (High G+C Gram-positive bacteria), and the Firmicutes (Low G+C Gram-positive bacteria). The culture-independent partial 16S rDNA sequence analysis revealed a considerably more diverse microbial composition within the biofilms. A total of eight phyla were recovered in this case, three of which were major groups: the Firmicutes (43.9%), the Proteobacteria (28.6%), and the Bacteroidetes (17.6%). The remaining five phyla were minor groups: the Planctomycetes (4.4%), the Chlorobi (2.2%), the Actinobacteria (1.1%), the Nitrospirae (1.1%), and the Verrucomicrobia (1.1%). The two most abundant genera detected were the endospore-forming bacteria (31.8%), Clostridium and Bacillus, both of which are members of the Firmicutes phylum. This finding indicates that these endospore-forming bacteria successfully colonized and dominated the RABC process biofilms. Many of the colonies or clones recovered from the biofilms evidenced significantly high homology in the 16S rDNA sequences of bacteria stored in databases associated with advanced wastewater treatment capabilities, including nitrification and denitrification, phosphorus accumulation, the removal of volatile odors, and the removal of chlorohydrocarbons or heavy metals. The microbial community structures observed in the biofilms were found to correlate nicely with the enhanced performance of advanced wastewater treatment protocols.     

Use of stable-isotope probing, full-cycle rRNA analysis, and fluorescence in situ hybridization-microautoradiography to study a methanol-fed denitrifying microbial community

A denitrifying microbial consortium was enriched in an anoxically operated, methanol-fed sequencing batch reactor (SBR) fed with a mineral salts medium containing methanol as the sole carbon source and nitrate as the electron acceptor. The SBR was inoculated with sludge from a biological nutrient removal activated sludge plant exhibiting good denitrification. The SBR denitrification rate improved from less than 0.02 mg of NO(3)(-)-N mg of mixed-liquor volatile suspended solids (MLVSS)(-1) h(-1) to a steady-state value of 0.06 mg of NO(3)(-)-N mg of MLVSS(-1) h(-1) over a 7-month operational period. At this time, the enriched microbial community was subjected to stable-isotope probing (SIP) with [(13)C]methanol to biomark the DNA of the denitrifiers. The extracted [(13)C]DNA and [(12)C]DNA from the SIP experiment were separately subjected to full-cycle rRNA analysis. The dominant 16S rRNA gene phylotype (group A clones) in the [(13)C]DNA clone library was closely related to those of the obligate methylotrophs Methylobacillus and Methylophilus in the order Methylophilales of the Betaproteobacteria (96 to 97% sequence identities), while the most abundant clone groups in the [(12)C]DNA clone library mostly belonged to the family Saprospiraceae in the Bacteroidetes phylum. Oligonucleotide probes for use in fluorescence in situ hybridization (FISH) were designed to specifically target the group A clones and Methylophilales (probes DEN67 and MET1216, respectively) and the Saprospiraceae clones (probe SAP553). Application of these probes to the SBR biomass over the enrichment period demonstrated a strong correlation between the level of SBR denitrification and relative abundance of DEN67-targeted bacteria in the SBR community. By contrast, there was no correlation between the denitrification rate and the relative abundances of the well-known denitrifying genera Hyphomicrobium and Paracoccus or the Saprospiraceae clones visualized by FISH in the SBR biomass. FISH combined with microautoradiography independently confirmed that the DEN67-targeted cells were the dominant bacterial group capable of anoxic [(14)C]methanol uptake in the enriched biomass. The well-known denitrification lag period in the methanol-fed SBR was shown to coincide with a lag phase in growth of the DEN67-targeted denitrifying population. We conclude that Methylophilales bacteria are the dominant denitrifiers in our SBR system and likely are important denitrifiers in full-scale methanol-fed denitrifying sludges.     

Studies on the in situ physiology of Thiothrix spp. present in activated sludge

The in situ physiology of the filamentous sulphur bacterium Thiothrix spp. was investigated in an industrial wastewater treatment plant with severe bulking problems as a result of overgrowth of Thiothrix. Identification and enumeration using fluorescence in situ hybridization (FISH) with species-specific 16S and 23S rRNA probes revealed that 5–10% of the bacteria in the activated sludge were Thiothrix spp. By using a combination of FISH and microautoradiography it was possible to study the in situ physiology of probe-defined Thiothrix filaments under different environmental conditions. The Thiothrix filaments were very versatile and showed incorporation of radiolabelled acetate and/or bicarbonate under heterotrophic, mixotrophic and chemolithoautotrophic conditions. The Thiothrix filaments were active under anaerobic conditions (with or without nitrate) in which intracellular sulphur globules were formed from thiosulphate and acetate was taken up. Thiothrix -specific substrate uptake rates and growth rates in activated sludge samples were determined under different conditions. Doubling times of 6–9 h under mixotrophic conditions and 15–30 h under autotrophic conditions were estimated. The key properties that Thiothrix might be employing to outcompete other microorganisms in activated sludge were probably related to the mixotrophic growth potential with strong stimulation of acetate uptake by thiosulphate, as well as stimulation of bicarbonate incorporation by acetate in the presence of thiosulphate.     

Diversity and dynamics of Archaea in an activated sludge wastewater treatment plant

ABSTRACT: BACKGROUND: The activated sludge process is one of the most widely used methods for treatment of wastewater and the microbial community composition in the sludge is important for the process operation. While the bacterial communities have been characterized in various activated sludge systems little is known about archaeal communities in activated sludge. The diversity and dynamics of the Archaea community in a full-scale activated sludge wastewater treatment plant were investigated by fluorescence in situ hybridization, terminal restriction fragment length polymorphism analysis and cloning and sequencing of 16S rRNA genes. RESULTS: The Archaea community was specialized and dominated by Methanosaeta-like species. During a 15 month period major changes in the community composition were only observed twice despite seasonal variations in environmental and operating conditions. Water temperature appeared to be the process parameter that affected the community composition the most. Several terminal restriction fragments also showed strong correlations with sludge properties and effluent water properties. The Archaea were estimated to make up 1.6-% of total cell numbers in the activated sludge and were present both as single cells and colonies of varying sizes. CONCLUSIONS: The results presented here show that Archaea can constitute a constant and integral part of the activated sludge and that it can therefore be useful to include Archaea in future studies of microbial communities in activated sludge.     

Investigation of an acetate-fed denitrifying microbial community by stable isotope probing, full-cycle rRNA analysis, and fluorescent in situ hybridization-microautoradiography

The acetate-utilizing microbial consortium in a full-scale activated sludge process was investigated without prior enrichment using stable isotope probing (SIP). [13C]acetate was used in SIP to label the DNA of the denitrifiers. The [13C]DNA fraction that was extracted was subjected to a full-cycle rRNA analysis. The dominant 16S rRNA gene phylotypes in the 13C library were closely related to the bacterial families Comamonadaceae and Rhodocyclaceae in the class Betaproteobacteria. Seven oligonucleotide probes for use in fluorescent in situ hybridization (FISH) were designed to specifically target these clones. Application of these probes to the sludge of a continuously fed denitrifying sequencing batch reactor (CFDSBR) operated for 16 days revealed that there was a significant positive correlation between the CFDSBR denitrification rate and the relative abundance of all probe-targeted bacteria in the CFDSBR community. FISH-microautoradiography demonstrated that the DEN581 and DEN124 probe-targeted cells that dominated the CFDSBR were capable of taking up [14C]acetate under anoxic conditions. Initially, DEN444 and DEN1454 probe-targeted bacteria also dominated the CFDSBR biomass, but eventually DEN581 and DEN124 probe-targeted bacteria were the dominant bacterial groups. All probe-targeted bacteria assessed in this study were denitrifiers capable of utilizing acetate as a source of carbon. The rapid increase in the number of organisms positively correlated with the immediate increase in denitrification rates observed by plant operators when acetate is used as an external source of carbon to enhance denitrification. We suggest that the impact of bacteria on activated sludge subjected to intermittent acetate supplementation should be assessed prior to the widespread use of acetate in the wastewater industry to enhance denitrification.     

Quantification of Target Molecules Needed To Detect Microorganisms by Fluorescence In Situ Hybridization (FISH) and Catalyzed Reporter Deposition-FISH

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes is a method that is widely used to detect and quantify microorganisms in environmental samples and medical specimens by fluorescence microscopy. Difficulties with FISH arise if the rRNA content of the probe target organisms is low, causing dim fluorescence signals that are not detectable against the background fluorescence. This limitation is ameliorated by technical modifications such as catalyzed reporter deposition (CARD)-FISH, but the minimal numbers of rRNA copies needed to obtain a visible signal of a microbial cell after FISH or CARD-FISH have not been determined previously. In this study, a novel competitive FISH approach was developed and used to determine, based on a thermodynamic model of probe competition, the numbers of 16S rRNA copies per cell required to detect bacteria by FISH and CARD-FISH with oligonucleotide probes in mixed pure cultures and in activated sludge. The detection limits of conventional FISH with Cy3-labeled probe EUB338-I were found to be 370 ± 45 16S rRNA molecules per cell for Escherichia coli hybridized on glass microscope slides and 1,400 ± 170 16S rRNA copies per E. coli cell in activated sludge. For CARD-FISH the values ranged from 8.9 ± 1.5 to 14 ± 2 and from 36 ± 6 to 54 ± 7 16S rRNA molecules per cell, respectively, indicating that the sensitivity of CARD-FISH was 26- to 41-fold higher than that of conventional FISH. These results suggest that optimized FISH protocols using oligonucleotide probes could be suitable for more recent applications of FISH (for example, to detect mRNA in situ in microbial cells).