Effectiveness of oligonucleotide probes targeted against Thiothrix nivea and type 021n 16s rRNA for in situ identification and population monitoring in activated sludges

In situ hybridization (FISH) of two fluorescent oligonucleotide probes, TNI and 21N, directed against 16S rRNA fragments of Thiothrix nivea and type 021N filamentous bacteria, was used to study activated sludge samples of various origins. Comparison of the results obtained by this technique with morphological data enabled us to evaluate the sensitivity and specificity of the probes, as well as to propose practical classification criteria, in an effort to correlate the identification and ecology of these two microorganisms. We applied the two oligonucleotide probes to the study of Thiothrix sp. in pilot and laboratory batch reactors fed a substrate that was moderately deficient in rapidly available phosphorus. We monitored the growth dynamics of Thiothrix sp. in the pilot reactor, using various hydraulic configurations and after certain perturbations, such as transient substrate overloads. The results provided data which led us to question the importance of the length of time oxygen-deficient sludges spend in clarifier tanks and we discuss the effects of both moderate reductions in oxygen and of very intermittent feedings on the occurrence of Thiothrix sp. growth peaks. The identification and quantification of these filamentous bacteria by size class, made possible by in situ hybridization, are shown to be powerful tools for the early detection and evaluation of episodes of proliferation of filamentous bacteria, revealing them to be much more sensitive indicators than the sludge volume index (SVI).     

Variability of type 021n in activated sludge as determined by in situ substrate uptake pattern and in situ hybridization with fluorescent rRNA targeted probes

Bulking sludges were investigated in seven industrial or municipal activated sludge treatment plants from Denmark, Germany and Australia. The dominating filaments were all identified as type 021N according to the Eikelboom key. The extent of variability in the filament taxonomy was assessed using fluorescence in situ hybridization (FISH) with rRNA-targeted nucleic acid probes specific for type 021N, Thiothrix and Leucothrix. Not all of the filaments morphologically identified as type 021N hybridized with the 021N probe. In one treatment plant the predominant filament hybridized with the probe for Thiothrix and in one treatment plant the predominant filament did not hybridize with any of these probes. In none of the plants did filaments hybridize with the probe for Leucothrix. A study of the in situ uptake of different organic substrates by the various filaments was also conducted using microautogradiography. The uptake of 6 different organic substrates under aerobic conditions was studied by providing C-14 or H-3 labeled substrates (acetate, glucose, ethanol, glycine, leucme and oleic acid) in incubations of a period of 3 hours. No filaments took up all the tested substrates, and type 021N from the various treatment plants varied in their uptake abilities. The study demonstrated that strain differences with regard to substrate utilization are likely to occur among bacteria within the same genera and designated types which are indistinguishable on the basis of morphological observations alone and by the molecular probes used in this study for identification. Whether there is a clear correlation between type of wastewater and the capability of taking up the various organic substrates for the filaments remains to be elucidated.     

Virus removal in a membrane separation process

Recently, membrane technology has been considered an alternative to conventional water purification. To study the fate of viruses in membrane processes, indigenous coliphages in pilot scale membrane processes located in the eastern part of Tokyo Metropolitan area have been surveyed for 6 months. This plant used river water as its resource and had two microfiltration membrane processes which had different pore sizes (0.2 µm and 0.1 µm) and one ultrafiltration process which had 13,000 nominal molecular weight cut off. To detect indigenous coliphages, E. coli K12 F⁺(A/λ) and E. coli C were used as host bacteria. E. coli K12 F+(A/λ) can detect both DNA and RNA phages and E. coli C can only DNA phage. The resource water contained E. coli K12 phages at 200–1500 PFU/100 mL and the removal ratio of these DNA and RNA phages was lower than that of DNA phage by E. coli C in both MF membrane processes through 6 months. It is thought to be caused by difference of phage size, because DNA phage is bigger than RNA phage in general. The removal ratio of E. coli K12 and E. coli C phages reached 100% in the UF membrane process. According to the comparison of the concentration of phages in solution and eluted from suspended solid in resource and drain, it is thought that most phages concentrated in the drain were absorbed in suspended solids. To make certain of the removal ratio in UF and NF (nanofiltration) processes, high concentrations of coliphage Qβ and poliomyelitis virus vaccine were fed into these processes. The removal ratio of coliphage Qβ in UF and NF processes are 10⁻⁸³ and 10^−6.3 respectively, and the ratio of poliomyelitis virus vaccine in UF and NF are µ10^−6.7 and µ10^−7.3 respectively.     

Identification and quantification of Gordona amarae strains in activated sludge systems using comparative rRNA sequence analysis and phylogenetic hybridization probes

Small subunit (SSU) ribosomal RNA (rRNA) genes of four Gordona (Nocardia) amarae strains were sequenced and compared to the sequence of the G. amarae type strain obtained from the Ribosomal Database Project (RDP). Comparative sequence analysis showed that the five strains represent two lines of evolutionary descent: Group I consists of strains NM23 and ASACI and Group 2 contains strains SE-6. SE 102, and ASF3. To determine the abundance of G. amarae in activated sludge systems, we designed three oligonucleotide probes: a species-specific probe for G. amarae, a probe specific for Group I and a probe targeting Group 2. The probes were characterized by performing dissociation temperature and specificity studies. Using these probes, two other strains, strains SE-149B and RBI, also were found to be part of Group I. We used these probes along with previously designed probes in membrane hybridizations to determine the abundance of G. amarae, Group I, Group 2, Bacteria, Mycobacterium complex, and Gordona in samples from foaming episodes. We demonstrated that the Mycobacterium complex, the genus Gordona, and G. amarae strains were present in significantly greater concentrations in activated sludge foam than in mixed liquor.     

In situ reciprocal transplants reveal species-specific growth pattern and geographic population differentiation among zebra and quagga mussels

For bivalves, somatic growth is often inferred from shell measurements alone. However, shell growth may not always reflect changes in soft tissue due to confounding factors such as seasonal or ontogenetic asynchrony between shell and tissue, flexible energy allocation, or population differences. This study compares the relationship between shell growth, changes in soft tissue mass, and RNA/DNA ratio in the zebra mussel (Dreissena polymorpha) and quagga mussel (Dreissena bugensis) from contrasting riverine and brackish estuarine environments. Reciprocal transplantation indicated that shell growth in late summer was consistently lower for the estuarine source zebra mussels while the RNA/DNA ratio was highest for zebra mussels independent of either geographic source or destination. Shell growth of the river source quagga mussels was almost two times greater than zebra mussels at the river site, but both shell growth and final tissue mass were lower in the estuarine environment. While there were no differences in final RNA/DNA ratios between zebra and quagga mussels from the same source, the RNA/DNA ratio of zebra mussels from the estuary and transplanted to the estuary was higher than that of all other zebra mussel treatments. This study suggests that shell growth does not always accurately reflect tissue growth and that the shell and tissue growth of quagga mussels is greater than that of zebra mussels in fresh but not brackish waters, and that physiological plasticity can have a fixed geographic component.     

Substrate utilization characteristics of the predominant filamentous and floc-forming bacteria isolated from a chemical fiber factory wastewater treatment plant

To supply theoretical verification of the function of a selector to control aerobic activated sludge bulking in the wastewater treatment plant for a chemical fiber factory in Taiwan, the filamentous and floc-forming bacteria in the aeration tank in the full-scale plant were examined microscopically and isolated. The kinetic characteristics of filamentous and floc-forming bacteria were also investigated.The predominant filamentous organism was Sphaerotilus natans. In addition to this organism, 21 strains of non-filamentous bacteria were isolated using the plate count method. These included Aeromonas jandaei DNA group 9, Acinetobacter johnsonii/genospecies 7, Bacillus pasteurii and Bacillus sp. (using the Biolog identification system). Nine strains showed the ability to form flocs when cultivated in glucose mineral salts medium.With glucose and acetate as sole substrates, the 4 floc-forming bacteria tested showed different substrate utilization characteristics. The 4 strains could be divided into 3 groups. The first group was the substrate degrading bacteria, the second group was the acid degrading bacteria and the other strains were those that had the highest substrate degradation rates at low substrate concentrations (below 800 mg/l). None of the floc-formers could utilize ethylene glycol, which is the major wastewater component. The kinetic characteristics of filamentous bacterium S. natans (Km = 4.0 mg glucose/l, Vm = 0.43 μl O₂/l) and the flocforming bacterium Aeromonas jandaei DNA group 9 (Km = 34.8 mg glucose/l, Vm = 0.59 μl O₂/l) provided information for selector design.     

Detection of filamentous and nitrifying bacteria in activated sludge with 16S rRNA probes

Activated sludge samples from wastewater treatment plants from potato starch and starch derivatives factory and from a municipal sewage treatment plant were analyzed with DNA probes specific for several filamentous bacteria. It was found that Haliscomenobacter hydrossis, Sphaerotilus natans, Thiothrix sp. and Eikelboom Type 021N were common in the activated sludges. Fluorescent in situ Hybridization (FISH) analysis could detect more types of sheathed bacteria and yielded a more accurate quantification of bacteria than conventional microscopy.In a pilot and a full scale wastewater treatment plant (WWTP) clear correlations were found between the SVI and growth of a Sphaerotilus natans and a Thiothrix sp. Addition of chlorine to the bulking sludge resulted in an improved SVI of the sludge but only damaged filamentous cells outside the floc. Nitrification was measured with substrate depletion and FISH analysis. Signal interpretation of FISH analysis was demonstrated both manually and with automated image analysis.     

Genetic diversity and population dynamics of Hyphomicrobium spp. in a sewage treatment plant and its receiving lake

Hyphomicrobium spp. were counted and isolated for 12 months in a sewage treatment plant with a combination of simultaneous and intermittent nitrification and denitriftcation using Most-Probable-Number methods. Genomic DNA of these hyphomicrobia was investigated by Southern or dot blot hybridizations with gene probes specific for genes of dissimilatory nitrate reduction (nitrate reductase, narG; cytochrome c,d-containing nitrite reductase, nirS; Cu-containing nitrite reductase, nirK; nitrous oxide reductase, nosZ), nitrification (ammonia monooxygenase, antoA), and N₂-fixation (nitrogenase, nifH). In particular, the Hyphomicrobium DNA/DNA-hybridization group HG 27 constituted one of the dominant denitrifying Hyphomicrobium populations in the activated sludge of this sewage treatment plant. A species-specific gene probe (Hvu-1) for HG 27 was generated from a transposon Tn5-132 insertion mutant defective in methanol oxidation using the inverse polymerase chain reaction. With this probe the abundance of this group in activated sludge of the sewage treatment plant and its receiving lake was determined as a subfraction of the total cultivable hyphomicrobia. Fragments of the mxaF gene encoding for the α-subunit of the methanol dehydrogenase of Hyphomicrobium spp, were amplified by PCR and analysed by denaturing gradient gel electrophoresis (DGGE). The DGGE analysis pattern showed a substantial separation of these fragments according to their nucleic acid sequences.     

Molecular biological methods to detect “Microthrix parvicella” and to determine its abundance in activated sludge

Molecular biological methods were evaluated in attempts to detect and quantitate levels of “Microthrix parvicella” in activated sludges. Approximately 66% of the 23S rRNA gene sequence of a strain (Ben43) of the Gram positive bulking and foaming organism “Microthrix parvicella” was determined, while a lesser amount was determined for “M. parvicella” strain RNI. The high mo1%G+C Gram positive bacteria (HGCGPBs) possess two powerfully diagnostic regions in the 23S rDNA and these were investigated in both strains. Firstly, the 18 nucleotide HGCGPB probe sequence (HGC69a) varied in at least two nucleotides with the sequence from both strains of “M. parvicella”. Secondly, an approximately 100 nucleotide stable insert between helices 54 and 55 in the 23S rRNA of HGCGPBs was discovered to be present in “M. parvicella”, but in both strains it was unique in length (79 nucleotides) and sequence. The region of the 23S rDNA with the stable insert was exploited to develop a polymerase chain reaction assay in which amplicons from “M. parvicella” were larger than those from nonHGCGPBs (i.e. all Bacteria except the HGCGPBs), and smaller than those from HGCGPBs. This assay was evaluated with DNAs extracted from activated sludges but although “M. parvicella” was morphologically identified, and was a dominant filament in at least one of the samples, no “M. parvicella” specific sized amplicons could be recovered from it. Amplicons of sizes generated by nonHGCGPBs and HGCGPBs were routinely produced in the stable insert PCR with DNAs from activated sludges where the highest yield was of amplicons from nonHGCGPBS.A second series of experiments were undertaken with the objective of evaluating the use of a non-radioactive hybridization method, based on extraction of bacterial RNA, for quantifying “M. parvicella” in activated sludge samples. Total nucleic acids were extracted from activated sludge samples and immobilized on nylon membranes. Probing with 16S rRNA-directed DIG-labelled oligonucleotide probes, detection of chemiluminescent signals on membranes and densitometry allowed hybridization signals to be quantified. The relationship between the amount of nucleic acid hybridized and the hybridization signal intensity observed was found to be linear over a specified range of signal intensities. A range of activated sludge samples were analysed for “M. parvicella” and variations in levels could be distinguished.     

Effect of Fasting under Different Temperature Conditions on Nucleic Acid Ratios in the Opossum Shrimp Mysis relicta: a Calibration Approach

The opossum shrimp Mysis relicta is an important component of the diet of benthivorous and planktivorous fish in the Great Lakes. The invasion of the Great Lakes by exotic invertebrates (Bythotrephes longimanus, Cercopagis pengoi, Dreissena polymorpha, and D. bugensis) has altered the base and intermediate levels of the foodweb. Thus, information about the condition of M. relicta may reveal the extent of indirect effects of these changes on this trophically-important invertebrate. Biochemical indices based on nucleic acid ratios have been shown to be suitable proxies for the growth and condition of aquatic organisms. These indices are affected by multiple factors, such as; food level, temperature, body size, sex/life stage, maturation, and moult stage and need to be calibrated before field data can be interpreted on a quantitative basis. In this study, we investigated the effect of fasting under different temperature conditions on the nucleic acid ratios RNA/DNA, RNA/protein and protein/DNA in M. relicta. Juvenile M. relicta were exposed to fasting conditions for 11 and 21 d in two controlled laboratory experiments at 3°C and 8°C. Several effects of time and temperature on the condition indices of fasting M. relicta were observed; however, we concluded that, of the various metrics tested, only RNA/DNA ratios provide a suitable index of metabolism and condition in fasting animals. RNA concentrations declined in response to fasting on the order of 3–4 d at 8°C and between 4 and 11 d at 3°C. Juvenile M. relicta with RNA/DNA ratios < 1.5–1.8 were clearly identified as fasting animals. Field-caught animals having RNA/DNA ratios near these levels are demonstrating clear signs of metabolic stress.     

Combining fluorescent in situ hybridization (fish) with cultivation and mathematical modeling to study population structure and function of ammonia-oxidizing bacteria in activated sludge

16S rRNA-targeted oligonucleotide probes for phylogenetically defined groups of autotrophic ammonia-oxidizing bacteria were used for analyzing the natural diversity of nitrifiers in an industrial sewage treatment plant receiving sewage with high ammonia concentrations. In this facility discontinuous aeration is used to allow for complete nitrification and denitrification. In situ hybridization revealed a yet undescribed diversity of ammonia oxidizers occurring in the plant. Surprisingly, the majority of the ammonia oxidizers were detected with probe combinations which indicate a close affiliation of these cells with Nitrosococcus mobilis. In addition, low numbers of ammonia-oxidizers related to the Nitrosomonas europaea - Nitrosomonas eutropha cluster were present. Interestingly, we also observed hybridization patterns which suggested the occurrence of a novel population of ammonia oxidizers. Confocal laser scanning microscopy revealed that all specifically stained ammonia oxidizers were clustered in microcolonies formed by rod-shaped bacteria. Combination of FISH and mathematical modeling was used to investigate diffusion limitation of ammonia and O₂ within these aggregates. Model simulations suggest that mass transfer limitations inside the clusters arc not as significant as the substrate limitations due to the activity of surrounding heterotrophic bacteria. To learn more about the ammonia-oxidizers of the industrial plant, we enriched and isolated ammonia-oxidizing bacteria from the activated sludge by combining classical cultivation techniques and FISH. Monitoring the isolates with the nested probe set allowed us to specifically identify those ammonia oxidizers which were found in situ to be numerically dominant. The phylogenetic relationship of these isolates determined by comparative 165 rDNA sequence analysts confirmed the affiliation suggested by FISH.     

Analysis of rDNA Regions of Five Freshwater Unionid Mussel Species in Presque Isle Bay,Southeastern Lake Erie

Population structure and fecundity of freshwater mussels can be difficult to assess due to the benthic habitat and complex life cycles of these organisms. However, rapid and reliable classification of unionids can be accomplished with polymerase chain reaction if species-specific DNA primers are established. In this report we describe the sequence analysis of the ITS1 and ITS2 regions from five mussel species (Amblema plicata, Fusconia flava, Lampsilis siliquoidea, Ligumia nasuta, and Pyganodon grandis) isolated from a refuge in Lake Erie. Sequence comparison revealed strong similarities between A. plicata and F. flava and between L. siliquoidea and L.nasuta. P. grandis contained regions of additional DNA not present within the other species. Our study confirmed the placement of Ligumia nasuta within the Lampsilinae. The analysis also revealed DNA sequences within the ITS1 and ITS2 regions that are species-specific. Thus, the development of species-specific PCR primers can be utilized for analysis of adult populations and for glochidia on host fish.     

Population dynamics and biofilm composition in a new three-phase circulating bed reactor

The biofilm characteristics of a novel three-phase reactor, the circulating bed reactor (CBR), were studied using industrial prototype fed with primary and secondary settled effluent in conditions of tertiary N and secondary C+N nitrification. The results showed a high nitrification rate close to the intrinsic values for N and C+N conditions: up to 2 and 0.6 kgN-NH₄ m^-3 d^-1, or 1.88±0.26 and 0.22±0.07 gN g^-1 PR d^-1, respectively. The application of an integrated approach for biofilm analysis enabled the better understanding of biofihn dynamics. The biofilm remained relatively thin, below 100 μm, indicating an effective control of the biofilm development. Protein, measured by the conventional colometric method and pyrolysis-GCMS, was the major fraction accounting for up to 35% of the biomass dry weight and 58% of the biopolymer content. The polysaccharide's fraction remained very low (<3%). The ribosomal RNA probes analysis confirmed the predominance of bacterial cells in the CBR biofilm (80–86% of bacteria versus the universal probe) showing a high proportion of nitrifying bacteria accounting for up to 50% and 27% in the N and C+N removal respectively. Nitrosomonas predominated in tertiary nitrification whereas carbon input led to the appearance of other ammonia oxidizers. This particular composition was characterized by a high state of oxidation of the biomass, expressed by the low COD/DW ratio of about 0.85. In conclusion, it can be stated that this new three-phase bioreactor ensures a high nitrification rate through an effective biofilm control promoting the development of bacterial cells, especially nitrifying bacteria, and minimizing exopolysaccharides production.     

Formation and characteristics of nitritation granules cultivated in sequencing batch reactor by stepwise increase of N/C ratio

The cultivation of nitritation granules in sequencing batch reactor (SBR) by seeding conventional floccular activated sludge was investigated using ethanol-based synthetic wastewater. Reducing settling time offers selection pressure for aerobic granulation, and stepwise increase of influent N/C ratio can help to selectively enrich ammonia oxidizing bacteria (AOB) in aerobic granules. The spherical shaped granules were observed with the mean diameter of 1.25 mm, average settling velocity of 1.9 cm s(-1) and the sludge volume index (SVI) of 18.5-31.4 ml g(-1). After 25 days of operation, the nitrogen loading rate reached 0.0455 kg NH(4)(+)-N (kg MLSS·d)(-1), which was 4.55 times higher than that of the start-up period. The mature granules showed high nitrification ability. Ammonia removal efficiency was above 95% and nitrite accumulation ratio was in the range of 80-95%. The nitrifying bacteria were quantified by fluorescence in situ hybridization analysis, which indicated that AOB was 14.9 ± 0.5% of the total bacteria and nitrite oxidizing bacteria (NOB) was 0.89 ± 0.1% of the total bacteria. Therefore, AOB was the dominant nitrifying bacteria. It was concluded that the associated inhibition of free ammonia at the start of each cycle and free nitrous acid during the later phase of aeration may be the key factors to start up and maintain the stable nitritation.     

Isolation and characterization of Lake Erie bacteria that degrade the cyanobacterial microcystin toxin MC-LR

Microcystin-LR (MC-LR) is a cyclic hepatotoxin produced by cyanobacteria, including Microcystis sp. and Planktothrix sp. Harmful algal blooms (HABs) in Lake Erie have become a major human health concern in recent years, highlighted by the August 2014 City of Toledo, Ohio, municipal water “do not drink” order that affected nearly 500,000 residents for 3?days. Given that microcystin degrading bacteria have been reported from HAB-affected waters around the world, we hypothesized that MC-LR degrading bacteria could be isolated from Lake Erie. To test this hypothesis, 13 water samples were collected from various Lake Erie locations during the summers of 2014 and 2015, MC-LR was continuously added to each water sample for 3 to 5?weeks to enrich for MC-LR-degrading bacteria, and MC-LR was quantitated over time. Whereas MC-LR was relatively stable in sterile-filtered lake water, robust MC-LR degradation (up to 19?ppb/day) was observed in some water samples. Following the MC-LR selection process, 67 individual bacterial isolates were isolated from MC-LR degrading water samples and genotyped to exclude potential human pathogens, and MC-LR degradation by smaller groups of bacterial isolates (e.g., groups of 22 isolates, groups of 11 isolates, etc.) was examined. Of those smaller groups, selected groups of four to five bacterial isolates were found to degrade MC-LR into non-toxic forms and form robust biofilms on siliconized glass tubes. Taken together, these studies support the potential use of isolated bacterial isolates to remove MC-LR from drinking water.     

Diet effects on lipid composition,somatic growth potential,and survival of the benthic amphipod Diporeia spp.

The benthic amphipod Diporeia represents a crucial trophic link that conveys vital nutrients and energy to predators at higher trophic levels. The current decline of Diporeia populations, mostly in the North American Great Lakes, may, in part, be related to concurrent declines in food quantity and/or quality. We hypothesized that somatic growth and survival of Diporeia would be positively related to dietary supply and subsequent retention of polyunsaturated fatty acids (PUFA); a class of chemicals known to affect diet quality. We examined how different algal PUFA concentrations in; a) Ankistrodesmus falcatus (Chlorophyta), b) a naturally occurring diatom assemblage from Lake Ontario, c) a non-toxic strain of Microcystis aeruginosa (Cyanophyta), and, d) fasting for 30 d, affected PUFA concentrations, somatic growth, and survival of Diporeia. Total PUFA concentrations were significantly higher in A. falcatus than in diatoms and Microcystis, but only diatoms contained considerable amounts of eicosapentaenoic (EPA) and docosahexaenoic acid (DHA). EPA, DHA, and arachidonic acid (ARA) were highly retained in Diporeia even in the absence of dietary supply with ARA being the most efficiently bioaccumulated PUFA. Survivorship of Diporeia ranged from 60% (diatom-fed), 68% (A. falcatus-fed), to 70% (fasting treatment), but was 0% in the M. aeruginosa diet treatment. Nucleic acid ratios (RNA:DNA), commonly used as proxies for somatic growth potential, were highest in Diporeia feeding on diatoms and lowest in fasting animals. We conclude that overall condition of Diporeia improved with dietary access to EPA and DHA, but survival was not related to this food quantity and/or quality.     

Characterization of filamentous foaming in activated sludge systems using oligonucleotide hybridization probes and antibody probes

A quantitative method was developed for estimating Gordona mass in activated sludge foam and mixed liquor samples. The technique involves in situ hybridization with a genus-specific fluorescently labeled oligonucleotide probe calibrated on pure cultures of Gordona. The immunofluorescent technique of Hernandez et al. was modified to allow staining with fluorescently labeled antibody and hybridization probes. The results of this technique were compared to those from membrane hybridization studies using radioactively-labeled oligonuelcotide probes. Quantitative membrane hybridizations, in situ hybridizations, and antibody staining resulted in significantly different levels of Gordona in activated sludge foam, activated sludge mixed liquor, return activated sludge, and anaerobic digester sludge. Simultaneous staining with labeled antibodies and oligonucleolide probes provide a definitive identification for Gordona, and represents a new approach for in situ studies of this organism's role to foaming.     

Evaluation of filamentous microorganism growth factors in an industrial wastewater activated sludge system

An investigation comprising four studies was undertaken to determine possible factors affecting the growth of several different types of filamentous microorganisms present in a bulking industrial wastewater activated sludge. Results from laboratory-scale continuous-flow and full-scale studies suggested that DO concentration and F:M ratio were the likely key factors affecting filamentous growth in the activated sludge. From the results of two laboratory studies isolating the effects of DO concentration and F:M ratio on filamentous growth, favorable growth ranges of DO concentration or F:M ratio were estimated for the following filaments: Microthrix parvicella, Nocardia spp., Nostocoida limicola II, and Types 0041, 1851, and 1863. Most of the bacteria causing filamentous bulking of the activated sludge were found to be filaments typically associated with low F:M, and increasing the F:M ratio appeared to cause N. limicola II to lose its competitive advantage in the activated sludge system. Type 1863, on the other hand, was found to be a low DO filament, as DO concentrations of 0.1 mg O₂/l or less appeared to be a necessary condition for its filamentous growth. Though Nocardia was found to be a low F:M filament, its growth also seemed to be affected by DO concentration, as its growth was stimulated by concentrations of 1.0 mg O₂/l or greater, with a near linear relationship up to at least 5 mg O₂/l.     

Effect of co-substrate, biomass and sulfate concentration on the performance of a control strategy used to determine the anaerobic stage length of an anaerobic/aerobic SBR degrading p-nitrophenol.

The effect of the p-nitrophenol to propionic acid ratio, the biomass concentration, and the presence of sulfates on the performance of a control strategy of an anaerobic/aerobic SBR degrading p-nitrophenol was studied. The duration of the anaerobic stage was controlled by an algorithm based on the on-line measurement of the oxidation-reduction potential, which indicates the end of the reduction of p-nitrophenol to p-aminophenol. It was observed that no significant influence on the performance of the algorithm was found when the co-substrate, the biomass concentration and the presence of sulfates were varied, indicating the robustness of the control strategy. Only for the case when a low concentration of cosubstrate was used, was there no transformation of p-nitrophenol.     

Gradients in the taxonomic composition of different microbial systems: Comparison between biofilms for advanced waste treatment and lake sediments

The application of in situ hybridization with group specific oligonucleotide probes detected by epifluorescence microscopy and confocal laser scanning microscopy was tested to identify spatial gradients in the distribution of bacteria in biofilms of plug flow reactors and in the bottom sediment layer of a drinking water reservoir. The two tubular biofihn reactors were fed with the effluent from a full scale biological wastewater treatment plant to which were added the chlorophenols whole degradation was being investigated. One was operated as a continuous-flow reactor and the other as a sequencing batch reactor.The vertical gradients in the microbial colonization of the sediment were analyzed by means of glass slides exposed to the sediment.In the biofilms of both reactors the beta-Proteobacteria dominated. The Cytophaga-Flavobacterium group and the Gram-positive bacteria were also abundant. Only small amounts of gamma-bacteria could be detected. This is contrary to findings using traditional cultivation methods. Unlike the biofilms in the reactor, the bacterial Aufwuchs on the glass slides in the sediment presented a surprising diversity of morphological types and size classes of bacteria.