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.     

Searching for links in the biotic characteristics and abiotic parameters of nine different biogas plants

To find links between the biotic characteristics and abiotic process parameters in anaerobic digestion systems, the microbial communities of nine full‐scale biogas plants in South Tyrol (Italy) and Vorarlberg (Austria) were investigated using molecular techniques and the physical and chemical properties were monitored. DNA from sludge samples was subjected to microarray hybridization with the ANAEROCHIP microarray and results indicated that sludge samples grouped into two main clusters, dominated either by Methanosarcina or by Methanosaeta, both aceticlastic methanogens. Hydrogenotrophic methanogens were hardly detected or if detected, gave low hybridization signals. Results obtained using denaturing gradient gel electrophoresis (DGGE) supported the findings of microarray hybridization. Real‐time PCR targeting Methanosarcina and Methanosaeta was conducted to provide quantitative data on the dominating methanogens. Correlation analysis to determine any links between the microbial communities found by microarray analysis, and the physicochemical parameters investigated was conducted. It was shown that the sludge samples dominated by the genus Methanosarcina were positively correlated with higher concentrations of acetate, whereas sludge samples dominated by representatives of the genus Methanosaeta had lower acetate concentrations. No other correlations between biotic characteristics and abiotic parameters were found. Methanogenic communities in each reactor were highly stable and resilient over the whole year.     

Quantification of Methanosaeta Species in Anaerobic Bioreactors Using Genus- and Species-Specific Hybridization Probes

A BSTRACTTo evaluate the role of Methanosaeta spp. in a variety of anaerobic environments, small-subunit rRNA targeted oligonucleotide hybridization probes were developed and experimentally characterized. The probes were designed to be genus specific for Methanosaeta and species specific for Methanosaeta concilii and Methanosaeta thermophila. The temperature of dissociation was determined for each probe. Probe specificities were determined using a diverse collection of Archaea and through an evaluation of probe nesting using samples from a variety of anaerobic bioreactors. Cell fixation and hybridization conditions for fluorescence in situ hybridizations were also evaluated. Although permeability of methanogens was variable, M. concilii cells could be permeabilized using a range of paraformaldehyde and ethanol based fixation conditions. Using the newly designed probes together with previously designed probes for methanogens, it was determined that Methanosaeta spp. were the dominant aceticlastic methanogens in a variety of anaerobic bioreactors when acetate concentrations were low. Their levels were higher in bioreactors with granular sludge than in those with flocculent sludge. In lab-scale upflow anaerobic sludge blanket reactors, the levels of M. concilii rRNA were as high as 30% of the total rRNA.     

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.     

Use of an alternative Archaea-specific probe for methanogen detection

An alternative 16S rRNA-targeted oligonucleotide probe specific for Archaea was developed and used for detection of methanogens in anaerobic reactors. The designed probe was checked for its specificity by computer-aided comparative sequence analysis. For in situ application, optimal stringency conditions were adjusted by performing whole cell hybridization using target and nontarget organisms. Anaerobic sludge samples were examined by in situ hybridization for methanogenic populations. The relative abundance of methanogens was monitored with epifluorescence microscopy. Individual cells could be visualized with strong fluorescence signals after hybridization with the newly developed probe.     

Acidophilic microbial communities catalyzing sludge bioleaching monitored by fluorescent in situ hybridization

Biological autotrophic sulfur oxidation processes have been proposed to remove heavy metals from wastewater treatment sludge by bioleaching. We made a characterization of the microbial population in batch and continuous sludge bioleaching reactors using fluorescent in situ hybridization of fluorescently-labeled oligonucleotidic probes targeting rRNA in a ‚top to bottom approach’. Batch incubations of sludge with 0.2% (w/v) elemental sulfur resulted in a pH value of 5. Alpha-Proteobacteria hybridizing with probe ALF1b were dominant in this incubation. Members of the Acidophilium-group (hybridizing with probe Acdp821) of Nitrospira/Leptospirillum phylum (Ntspa712 probe) and from the archaeal domain (ARCH915) were also detected. When sludge was incubated with 1% elemental sulfur in batch or continuous reactor experiments, final pH values were always below 2. Active microbial communities consisted almost exclusively of gamma-Proteobacteria (hybridizing with probe GAM42a). However, further hybridization experiments with probe Thio820 targeting Acidithiobacillus ferroxidans and Acidithiobacillus thioxidans gave negative results. A new probe, named THIO181, encompassing all known members of the genus was designed. Hybridization perfomed with THIO181 and GAM42a showed a perfect co-localization of the hybridization signals. Further hybridization experiments with probe THIO181 and THC642, specific for the species Acidithiobacillus caldus, confirmed that this bacteria was largely responsible for the sulfur oxidation reaction in our acidophilic sludge bioleaching reactors.     

Cultivation and in situ detection of a thermophilic bacterium capable of oxidizing propionate in syntrophic association with hydrogenotrophic methanogens in a thermophilic methanogenic granular sludge

The thermophilic, anaerobic, propionate-oxidizing bacterial populations present in the methanogenic granular sludge in a thermophilic (55 degrees C) upflow anaerobic sludge blanket reactor were studied by cultivation and in situ hybridization analysis. For isolation of propionate-degrading microbes, primary enrichment was made with propionate as the sole energy source at 55 degrees C. After several attempts to purify the microbes, a thermophilic, syntrophic, propionate-oxidizing bacterium, designated strain SI, was isolated in both pure culture and coculture with Methanobacterium thermoautotrophicum. Under thermophilic (55 degrees C) conditions, strain SI oxidized propionate, ethanol, and lactate in coculture with M. thermoautotrophicum. In pure culture, the isolate was found to ferment pyruvate. 16S ribosomal DNA sequence analysis revealed that the strain was relatively close to members of the genus Desulfotomaculum, but it was only distantly related to any known species. To elucidate the abundance and spatial distribution of organisms of the strain SI type within the sludge granules, a 16S rRNA-targeted oligonucleotide probe specific for strain SI was developed and applied to thin sections of the granules. Fluorescence in situ hybridization combined with confocal laser scanning microscopy revealed that a number of rod-shaped cells were present in the middle and inner layers of the thermophilic granule sections and that they formed close associations with hydrogenotrophic methanogens. They accounted for approximately 1.1% of the total cells in the sludge. These results demonstrated that strain SI was one of the significant populations in the granular sludge and that it was responsible for propionate oxidation in the methanogenic granular sludge in the reactor.     

Detecting structural and functional differences in activated sludge bacterial communities originating from laboratory treatment of elementally and totally chlorine-free bleaching effluents

The ability to differentiate functional and structural diversity of bacterial communities present in activated sludges adapted to elementally (ECF) and totally (TCF) chlorine-free bleaching effluents was evaluated. Community function was evaluated through substrate utilization patterns in BiologGN microplates, and taxonomic structure was evaluated by fluorescent in situ hybridization using probes targeting the Eubacteria; the alpha, beta, and gamma subclasses of the Proteobacteria; and gram-positive bacteria with high GC content. Over 6-week sampling periods, ECF-and TCF-adapted sludge bacterial communities presented reproducible substrate utilization patterns that through principal components (PCs) analysis, separated the ECF samples from the TCF samples. Application of the fluorescent in situ hybridization technique was complicated by the intense autofluorescence of the bleaching effluent sludge samples that interfered with detection of specific hybridization signals. The most notable difference in community structure detected using the chosen set of probes was the relatively greater proportion of cells of the alpha subclass in TCF sludge (27%) than in ECF sludge (6%). Nonspecific hybridization with beta and gamma probes was relatively high, but both sludges appeared to have similar proportions of cells of the beta (20-22%) and gamma (11-12%) subclasses. The two sludges presented relatively few gram-positive cells with high GC content (<0.2% of eubacterial counts). Differences in both metabolic potential and taxonomic structure of the microbial communities in the ECF- and TCF-activated sludges were detected. The kinetics of the development of these differences in treatment plants and their relationships with treatment efficiency and production process conditions should now be evaluated.     

Quantification of Hyphomicrobium populations in activated sludge from an industrial wastewater treatment system as determined by 16S rRNA analysis

The bacterial community structure of the activated sludge from a 25 million-gal-per-day industrial wastewater treatment plant was investigated using rRNA analysis. 16S ribosomal DNA (rDNA) libraries were created from three sludge samples taken on different dates. Partial rRNA gene sequences were obtained for 46 rDNA clones, and nearly complete 16S rRNA sequences were obtained for 18 clones. Seventeen of these clones were members of the beta subdivision, and their sequences showed high homology to sequences of known bacterial species as well as published 16S rDNA sequences from other activated sludge sources. Sixteen clones belonged to the alpha subdivision, 7 of which showed similarity to Hyphomicrobium species. This cluster was chosen for further studies due to earlier work on Hyphomicrobium sp. strain M3 isolated from this treatment plant. A nearly full-length 16S rDNA sequence was obtained from Hyphomicrobium sp. strain M3. Phylogenetic analysis revealed that Hyphomicrobium sp. strain M3 was 99% similar to Hyphomicrobium denitrificans DSM 1869(T) in Hyphomicrobium cluster II. Three of the cloned sequences from the activated sludge samples also grouped with those of Hyphomicrobium cluster II, with a 96% sequence similarity to that of Hyphomicrobium sp. strain M3. The other four cloned sequences from the activated sludge sample were more closely related to those of the Hyphomicrobium cluster I organisms (95 to 97% similarity). Whole-cell fluorescence hybridization of microorganisms in the activated sludge with genus-specific Hyphomicrobium probe S-G-Hypho-1241-a-A-19 enhanced the visualization of Hyphomicrobium and revealed that Hyphomicrobium appears to be abundant both on the outside of flocs and within the floc structure. Dot blot hybridization of activated sludge samples from 1995 with probes designed for Hyphomicrobium cluster I and Hyphomicrobium cluster II indicated that Hyphomicrobium cluster II-positive 16S rRNA dominated over Hyphomicrobium cluster I-positive 16S rRNA by 3- to 12-fold. Hyphomicrobium 16S rRNA comprised approximately 5% of the 16S rRNA in the activated sludge.     

16S rRNA gene-based oligonucleotide microarray for environmental monitoring of the betaproteobacterial order "Rhodocyclales"

For simultaneous identification of members of the betaproteobacterial order "Rhodocyclales" in environmental samples, a 16S rRNA gene-targeted oligonucleotide microarray (RHC-PhyloChip) consisting of 79 probes was developed. Probe design was based on phylogenetic analysis of available 16S rRNA sequences from all cultured and as yet uncultured members of the "Rhodocyclales." The multiple nested probe set was evaluated for microarray hybridization with 16S rRNA gene PCR amplicons from 29 reference organisms. Subsequently, the RHC-PhyloChip was successfully used for cultivation-independent "Rhodocyclales" diversity analysis in activated sludge from an industrial wastewater treatment plant. The implementation of a newly designed "Rhodocyclales"-selective PCR amplification system prior to microarray hybridization greatly enhanced the sensitivity of the RHC-PhyloChip and thus enabled the detection of "Rhodocyclales" populations with relative abundances of less than 1% of all bacteria (as determined by fluorescence in situ hybridization) in the activated sludge. The presence of as yet uncultured Zoogloea-, Ferribacterium/Dechloromonas-, and Sterolibacterium-related bacteria in the industrial activated sludge, as indicated by the RHC-PhyloChip analysis, was confirmed by retrieval of their 16S rRNA gene sequences and subsequent phylogenetic analysis, demonstrating the suitability of the RHC-PhyloChip as a novel monitoring tool for environmental microbiology.     

Bioaugmentation as a tool for improving the start-up and stability of a pilot-scale partial nitrification biofilm airlift reactor

The effectiveness of bioaugmentation in the improvement of the start-up of a biofilm airlift reactor to perform partial nitrification was investigated. Two identical biofilm airlift reactors were inoculated. The non-bioaugmented reactor (NB-reactor) was inoculated with conventional activated sludge, whereas the bioaugmented reactor (B-reactor) was seeded with the same conventional activated sludge but bioaugmented with nitrifying activated sludge from a pilot plant performing full nitritation under stable conditions (100% oxidation of influent ammonium to nitrite). The fraction of specialized nitrifying activated sludge in the inoculum of the B-reactor was only 6% (measured as dry matter). To simplify comparison of the results, operational parameters were equivalent for both reactors. Partial nitrification was achieved significantly faster in the B-reactor, showing a very stable operation. The results obtained by fluorescence in situ hybridization assays showed that the specialized nitrifying biomass added to the B-reactor remained in the biofilm throughout the start-up period.     

Cultivation and In Situ Detection of a Thermophilic Bacterium Capable of Oxidizing Propionate in Syntrophic Association with Hydrogenotrophic Methanogens in a Thermophilic Methanogenic Granular Sludge

The thermophilic, anaerobic, propionate-oxidizing bacterial populations present in the methanogenic granular sludge in a thermophilic (55°C) upflow anaerobic sludge blanket reactor were studied by cultivation and in situ hybridization analysis. For isolation of propionate-degrading microbes, primary enrichment was made with propionate as the sole energy source at 55°C. After several attempts to purify the microbes, a thermophilic, syntrophic, propionate-oxidizing bacterium, designated strain SI, was isolated in both pure culture and coculture with Methanobacterium thermoautotrophicum. Under thermophilic (55°C) conditions, strain SI oxidized propionate, ethanol, and lactate in coculture with M. thermoautotrophicum. In pure culture, the isolate was found to ferment pyruvate. 16S ribosomal DNA sequence analysis revealed that the strain was relatively close to members of the genus Desulfotomaculum, but it was only distantly related to any known species. To elucidate the abundance and spatial distribution of organisms of the strain SI type within the sludge granules, a 16S rRNA-targeted oligonucleotide probe specific for strain SI was developed and applied to thin sections of the granules. Fluorescence in situ hybridization combined with confocal laser scanning microscopy revealed that a number of rod-shaped cells were present in the middle and inner layers of the thermophilic granule sections and that they formed close associations with hydrogenotrophic methanogens. They accounted for approximately 1.1% of the total cells in the sludge. These results demonstrated that strain SI was one of the significant populations in the granular sludge and that it was responsible for propionate oxidation in the methanogenic granular sludge in the reactor.     

Detection and quantification of Desulforhabdus amnigenus in anaerobic granular sludge by dot blot hybridization and PCR amplification

A species-specific 16S rRNA oligonucleotide probe (ASRB1) was developed for the detection of Desulforhabdus amnigenus in anaerobic granular sludge. The presence of nucleic acids from cells of D. amnigenus in granular sludge was determined using ASRB1 as a specific primer for polymerase chain reaction (PCR) amplification or as a probe for dot blot hybridizations. The detection threshold and the reproducibility of these two methods were determined with sludge amended with 10⁴–10¹⁰ D. amnigenus cells per gram of volatile suspended solids (VSS). For D. amnigenus cells with a ribosomal RNA content of 15 fg cell⁻¹, the lowest number of target cells detected by hybridization was 1 × 10⁸ cells g⁻¹ VSS. With the PCR amplification method the lowest number of target cells which could be detected was 1 × 10⁷ g⁻¹ VSS. This corresponds to a threshold level for hybridization of 0·1–0·001‰ of the total bacterial sludge population, while the threshold level obtained with the PCR approach amounted to 0·01–0·0001‰. The rRNA content of D. amnigenus was found to be affected by the growth rate and the growth phase, and it ranged from 19 fg cell⁻¹ in slow-growing cultures to 90 fg cell⁻¹ in fast-growing cultures. Therefore, the detection threshold of the dot blot hybridization method for fast-growing cells is lower than for slow-growing cells.     

Combination of fluorescent in situ hybridization and microautoradiography-a new tool for structure-function analyses in microbial ecology

A new microscopic method for simultaneously determining in situ the identities, activities, and specific substrate uptake profiles of individual bacterial cells within complex microbial communities was developed by combining fluorescent in situ hybridization (FISH) performed with rRNA-targeted oligonucleotide probes and microautoradiography. This method was evaluated by using defined artificial mixtures of Escherichia coli and Herpetosiphon aurantiacus under aerobic incubation conditions with added [3H]glucose. Subsequently, we were able to demonstrate the potential of this method by visualizing the uptake of organic and inorganic radiolabeled substrates ([14C]acetate, [14C]butyrate, [14C]bicarbonate, and 33Pi) in probe-defined populations from complex activated sludge microbial communities by using aerobic incubation conditions and anaerobic incubation conditions (with and without nitrate). For both defined cell mixtures and activated sludge, the method proved to be useful for simultaneous identification and analysis of the uptake of labeled substrates under the different experimental conditions used. Optimal results were obtained when fluorescently labeled oligonucleotides were applied prior to the microautoradiographic developing procedure. For single-cell resolution of FISH and microautoradiographic signals within activated sludge flocs, cryosectioned sample material was examined with a confocal laser scanning microscope. The combination of in situ rRNA hybridization techniques, cryosectioning, microautoradiography, and confocal laser scanning microscopy provides a unique opportunity for obtaining cultivation-independent insights into the structure and function of bacterial communities.     

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.     

Diversity, localization, and physiological properties of filamentous microbes belonging to Chloroflexi subphylum I in mesophilic and thermophilic methanogenic sludge granules

We previously reported that the thermophilic filamentous anaerobe Anaerolinea thermophila, which is the first cultured representative of subphylum I of the bacterial phylum Chloroflexi, not only was one of the predominant constituents of thermophilic sludge granules but also was a causative agent of filamentous sludge bulking in a thermophilic (55 degrees C) upflow anaerobic sludge blanket (UASB) reactor in which high-strength organic wastewater was treated (Y. Sekiguchi, H. Takahashi, Y. Kamagata, A. Ohashi, and H. Harada, Appl. Environ. Microbiol. 67:5740-5749, 2001). To further elucidate the ecology and function of Anaerolinea-type filamentous microbes in UASB sludge granules, we surveyed the diversity, distribution, and physiological properties of Chloroflexi subphylum I microbes residing in UASB granules. Five different types of mesophilic and thermophilic UASB sludge were used to analyze the Chloroflexi subphylum I populations. 16S rRNA gene cloning-based analyses using a 16S rRNA gene-targeted Chloroflexi-specific PCR primer set revealed that all clonal sequences were affiliated with the Chloroflexi subphylum I group and that a number of different phylotypes were present in each clone library, suggesting the ubiquity and vast genetic diversity of these populations in UASB sludge granules. Subsequent fluorescence in situ hybridization (FISH) of the three different types of mesophilic sludge granules using a Chloroflexi-specific probe suggested that all probe-reactive cells had a filamentous morphology and were widely distributed within the sludge granules. The FISH observations also indicated that the Chloroflexi subphylum I bacteria were not always the predominant populations within mesophilic sludge granules, in contrast to thermophilic sludge granules. We isolated two mesophilic strains and one thermophilic strain belonging to the Chloroflexi subphylum I group. The physiological properties of these isolates suggested that these populations may contribute to the degradation of carbohydrates and other cellular components, such as amino acids, in the bioreactors.     

Polyphosphate kinase from activated sludge performing enhanced biological phosphorus removal

A novel polyphosphate kinase (PPK) was retrieved from an uncultivated organism in activated sludge carrying out enhanced biological phosphorus removal (EBPR). Acetate-fed laboratory-scale sequencing batch reactors were used to maintain sludge with a high phosphorus content (approximately 11% of the biomass). PCR-based clone libraries of small subunit rRNA genes and fluorescent in situ hybridization (FISH) were used to verify that the sludge was enriched in Rhodocyclus-like beta-Proteobacteria known to be associated with sludges carrying out EBPR. These organisms comprised approximately 80% of total bacteria in the sludge, as assessed by FISH. Degenerate PCR primers were designed to retrieve fragments of putative ppk genes from a pure culture of Rhodocyclus tenuis and from organisms in the sludge. Four novel ppk homologs were found in the sludge, and two of these (types I and II) shared a high degree of amino acid similarity with R. tenuis PPK (86 and 87% similarity, respectively). Dot blot analysis of total RNA extracted from sludge demonstrated that the Type I ppk mRNA was present, indicating that this gene is expressed during EBPR. Inverse PCR was used to obtain the full Type I sequence from sludge DNA, and a full-length PPK was cloned, overexpressed, and purified to near homogeneity. The purified PPK has a specific activity comparable to that of other PPKs, has a requirement for Mg(2+), and does not appear to operate in reverse. PPK activity was found mainly in the particulate fraction of lysed sludge microorganisms.     

Near-Bottom Pelagic Bacteria at a Deep-Water Sewage Sludge Disposal Site

The epibenthic bacterial community at deep-ocean sewage sludge disposal site DWD-106, located approximately 106 miles (ca. 196 km) off the coast of New Jersey, was assessed for changes associated with the introduction of large amounts of sewage sludge. Mixed cultures and bacterial isolates obtained from water overlying sediment core samples collected at the deep-water (2,500 m) municipal sewage disposal site were tested for the ability to grow under in situ conditions of temperature and pressure. The responses of cultures collected at a DWD-106 station heavily impacted by sewage sludge were compared with those of samples collected from a station at the same depth which was not contaminated by sewage sludge. Significant differences were observed in the ability of mixed bacterial cultures and isolates from the two sites to grow under deep-sea pressure and temperature conditions. The levels of sludge contamination were established by enumerating Clostridium perfringens, a sewage indicator bacterium, in sediment samples from the two sites. The results of hybridization experiments in which DNAs extracted directly from the water overlying sediment core samples were used indicate that the reference site epibenthic community, the disposal site epibenthic community, and the community in a surface sludge plume share many members. Decreased culturability of reference site mixed cultures in the presence of sewage sludge was observed. Thus, the culturable portions of both the autochthonous and allochthonous bacterial communities at the disposal site may be inhibited in situ, the former by sewage sludge and the latter by high pressure and low temperature.     

Detection of nitrifying bacteria in activated sludge by fluorescent in situ hybridization and fluorescence spectrometry

16S rRNA-targeted oligonucleotide probes for eubacteria (EUB338), ammonium-oxidizing bacteria (Nsm156) and nitrite-oxidizing bacteria (Nb1000) were used for the rapid detection of nitrifying bacteria in the activated sludge of a pilot nitrifying reactor by whole-cell, fluorescent in situ hybridization (FISH). Emission scanning and synchronous scanning fluorescence spectrometry were used to measure the hybridization. The binding of the probes at a temperature significantly lower than the melting temperature of the hybrids was conventionally considered as non-specific. Total binding of the probes at a temperature significantly higher than the melting temperature of the hybrids was conventionally considered as the sum of non-specific and specific binding (hybridization). Non-specific binding of the oligonucleotide probes with a biomass of activated sludge was 37% of the total binding of the EUB338 probe, 54% of the total binding of the Nsm156 probe, and 69% of the total binding of the Nb1000 probe. The ratio of the specific binding of the Nsm156 and Nb1000 probes was 2.3:1. The ratio of the numbers of ammonium-oxidizing bacteria to nitrite-oxidizing bacteria, determined by microbiological methods, was 2.4:1. Measuring fluorescent in situ hybridization by fluorescence spectrometry appears to be a practical tool for monitoring the microbial communities that contain nitrifying bacteria. However, a method that accounts for the non-specific binding of the probes more easily and reliably should be developed for practical application.     

Round robin investigation of methods for recovering human enteric viruses from sludge

To select a tentative standard method for detection of viruses in sludge the American Society for Testing and Materials D19:24:04:04 Subcommittee Task Group initiated round robin comparative testing of two procedures that, after initial screening of several methodologies, were found to meet the basic criteria considered essential by the task group. Eight task group member laboratories agreed to perform round robin testing of the two candidate methods, namely, The Environmental Protection Agency or low pH-AlCl3 method and the Glass or sonication-extraction method. Five different types of sludge were tested. For each particular type of sludge, a single laboratory was designated to collect the sludge in a single sampling, make samples, and ship it to the participating laboratories. In most cases, participating laboratories completed all the tests within 48 h of sample arrival. To establish the reproducibility of the methods, each laboratory tested each sludge sample in triplicate for the two candidate virus methods. Each processed sludge sample was quantitatively assayed for viruses by the procedures of each individual round robin laboratory. To attain a more uniform standard of comparison, a sample of each processed sample from all laboratories was reassayed with one cell line and passage number by a single laboratory (Environmental Protection Agency Environmental Monitoring and Support Laboratory, Cincinnati, Ohio). When the data were statistically analyzed, the Environmental Protection Agency method was found to yield slightly higher virus recoveries for all sludge types, except the dewatered sludge. The precisions of both methods were not significantly different.(ABSTRACT TRUNCATED AT 250 WORDS)