The Selenoproteome as a Dynamic Response Mechanism to Oxidative Stress in Hydrogenotrophic Methanogenic Communities.

Methanogenesis is a critical process in the carbon cycle that is applied industrially in anaerobic digestion and biogas production. While naturally occurring in diverse environments, methanogenesis requires anaerobic and reduced conditions, although varying degrees of oxygen tolerance have been described. Microaeration is suggested as the next step to increase methane production and improve hydrolysis in digestion processes; therefore, a deeper understanding of the methanogenic response to oxygen stress is needed. To explore the drivers of oxygen tolerance in methanogenesis, two parallel enrichments were performed under the addition of H2/CO2 in an environment without reducing agents and in a redox-buffered environment by adding redox mediator 9,10-anthraquinone-2,7-disulfonate disodium. The cellular response to oxidative conditions is mapped using proteomic analysis. The resulting community showed remarkable tolerance to high-redox environments and was unperturbed in its methane production. Next to the expression of pathways to mitigate reactive oxygen species, the higher redox potential environment showed an increased presence of selenocysteine and selenium-associated pathways. By including sulfur-to-selenium mass shifts in a proteomic database search, we provide the first evidence of the dynamic and large-scale incorporation of selenocysteine as a response to oxidative stress in hydrogenotrophic methanogenesis and the presence of a dynamic selenoproteome.

Assigning Function to Phylogeny: MAR-FISH.

Microautoradiography (MAR) is a technique by which assimilated radioactive tracers incorporated into the biomass can be detected by a film emulsion. This allows for the testing of cellular preferences in electron donors and acceptors of individual cells in complex microbial assemblages, as well as the ability to take up substrates under diverse environmental exposures.Combination with staining techniques such as fluorescence in situ hybridization (FISH) can be used to identify the involved cells. Here, the practical aspects of a combined microautoradiography and fluorescence in situ hybridization (MAR-FISH) approach are described.

Alternative strategies of nutrient acquisition and energy conservation map to the biogeography of marine ammonia-oxidizing archaea.

Ammonia-oxidizing archaea (AOA) are among the most abundant and ubiquitous microorganisms in the ocean, exerting primary control on nitrification and nitrogen oxides emission. Although united by a common physiology of chemoautotrophic growth on ammonia, a corresponding high genomic and habitat variability suggests tremendous adaptive capacity. Here, we compared 44 diverse AOA genomes, 37 from species cultivated from samples collected across diverse geographic locations and seven assembled from metagenomic sequences from the mesopelagic to hadopelagic zones of the deep ocean. Comparative analysis identified seven major marine AOA genotypic groups having gene content correlated with their distinctive biogeographies. Phosphorus and ammonia availabilities as well as hydrostatic pressure were identified as selective forces driving marine AOA genotypic and gene content variability in different oceanic regions. Notably, AOA methylphosphonate biosynthetic genes span diverse oceanic provinces, reinforcing their importance for methane production in the ocean. Together, our combined comparative physiological, genomic, and metagenomic analyses provide a comprehensive view of the biogeography of globally abundant AOA and their adaptive radiation into a vast range of marine and terrestrial habitats.

Microbial Production of the Off-Flavor Geosmin in Tilapia Production in Brazilian Water Reservoirs: Importance of Bacteria in the Intestine and Other Fish-Associated Environments.

AIM: To determine major sources of microbially produced geosmin in the commercially important aquaculture fish species tilapia. METHODS AND RESULTS: Abundance and composition of geosmin-producing bacteria in water and fish biosphere (intestine, digesta, and fins) of Nile tilapia (Oreachromis niloticus) raised in net cages in Brazilian freshwater farms were examined. By combining qPCR of the geosmin synthase geoA gene and 16S rRNA gene amplicon sequencing to identify potential geosmin-producing organisms, we observed that the proportion and composition of geosmin producers appeared to be rather similar in the water, digesta, intestinal mucous, and on skin, making up about 0.1-0.2% of the total bacterial densities. A high proportion of Cyanobacteria and other putative geosmin producers affiliated to the Actinomycetales were identified in the intestinal mucous layer. The main uptake site for geosmin in fish is traditionally assumed to be through the gill surface, but the present results suggest that uptake by the intestinal tract may represent a major source of geosmin uptake in fish. CONCLUSION: The high abundance of geosmin-producing bacteria in the intestinal mucous layer and digesta may indicate that the digestive system in fish is an important, but hitherto overlooked, source of geosmin and likely other off-flavors in fish. SIGNIFICANCE AND IMPACT OF STUDY: Tainting of fish by microbially produced off-flavors spoils fish quality and lowers consumer preferences for aquaculture-produced freshwater fish. Our results highlight the potential for the application of probiotic microorganisms for management of the intestinal microflora to improve the fish quality. HIGHLIGHTS: -Off-flavor producing bacteria are widely abundant in aquaculture.-Off-flavor producers found on skin surface of fish.-Off-flavor producing bacteria accumulate in the digestive system.-Off-flavor producers might release significant amounts of off-flavor during lysis in the gut.-Off-flavor uptake through the digestive system might be quantitatively significant.

Biogas upgrading with hydrogenotrophic methanogenic biofilms.

Hydrogen produced from periodic excess of electrical energy may be added to biogas reactors where it is converted to CH4 that can be utilized in the existing energy grid. The major challenge with this technology is gas-to-liquid mass transfer limitation. The microbial conversions in reactors designed for hydrogenotrophic methanogenesis were studied with microsensors for H2, pH, and CO2. The H2 consumption potential was dependent on the CO2 concentration, but could partially recover after CO2 depletion. Reactors with 3-dimensional biofilm carrier material and a large gas headspace allowed for a methanogenic biofilm in direct contact with the gas phase. A high density of Methanoculleus sp. in the biofilm mediated a high rate of CH4 production, and it was calculated that a reactor filled with 75% carrier material could mediate a biogas upgrading from 50 to 95% CH4 within 24 h when an equivalent amount of H2 was added.

The microbial community of the gut differs between piglets fed sow milk, milk replacer or bovine colostrum.

The aim of this study was to characterise the gut microbiota composition of piglets fed bovine colostrum (BC), milk replacer (MR) or sow milk (SM) in the post-weaning period. Piglets (n 36), 23-d old, were randomly allocated to the three diets. Faecal samples were collected at 23, 25, 27 and 30 d of age. Digesta from the stomach, ileum, caecum and mid-colon was collected at 30 d of age. Bacterial DNA from all samples was subjected to amplicon sequencing of the 16S rRNA gene. Bacterial enumerations by culture and SCFA analysis were conducted as well. BC-piglets had the highest abundance of Lactococcus in the stomach (P<0·0001) and ileal (P<0·0001) digesta, whereas SM-piglets had the highest abundance of Lactobacillus in the stomach digesta (P<0·0001). MR-piglets had a high abundance of Enterobacteriaceae in the ileal digesta (P<0·0001) and a higher number of haemolytic bacteria in ileal (P=0·0002) and mid-colon (P=0·001) digesta than SM-piglets. BC-piglets showed the highest colonic concentration of iso-butyric and iso-valeric acid (P=0·02). Sequencing and culture showed that MR-piglets were colonised by a higher number of Enterobacteriaceae, whereas the gut microbiota of BC-piglets was characterised by a change in lactic acid bacteria genera when compared with SM-piglets. We conclude that especially the ileal microbiota of BC-piglets had a closer resemblance to that of SM-piglets in regard to the abundance of potential enteric pathogens than did MR-piglets. The results indicate that BC may be a useful substitute for regular milk replacers, and as a feeding supplement in the immediate post-weaning period.

Identification of syntrophic acetate-oxidizing bacteria in anaerobic digesters by combined protein-based stable isotope probing and metagenomics.

Inhibition of anaerobic digestion through accumulation of volatile fatty acids occasionally occurs as the result of unbalanced growth between acidogenic bacteria and methanogens. A fast recovery is a prerequisite for establishing an economical production of biogas. However, very little is known about the microorganisms facilitating this recovery. In this study, we investigated the organisms involved by a novel approach of mapping protein-stable isotope probing (protein-SIP) onto a binned metagenome. Under simulation of acetate accumulation conditions, formations of (13)C-labeled CO2 and CH4 were detected immediately following incubation with [U-(13)C]acetate, indicating high turnover rate of acetate. The identified (13)C-labeled peptides were mapped onto a binned metagenome for improved identification of the organisms involved. The results revealed that Methanosarcina and Methanoculleus were actively involved in acetate turnover, as were five subspecies of Clostridia. The acetate-consuming organisms affiliating with Clostridia all contained the FTFHS gene for formyltetrahydrofolate synthetase, a key enzyme for reductive acetogenesis, indicating that these organisms are possible syntrophic acetate-oxidizing (SAO) bacteria that can facilitate acetate consumption via SAO, coupled with hydrogenotrophic methanogenesis (SAO-HM). This study represents the first study applying protein-SIP for analysis of complex biogas samples, a promising method for identifying key microorganisms utilizing specific pathways.

Genomic, Proteomic, and Metabolite Characterization of Gemfibrozil-Degrading Organism Bacillus sp. GeD10.

Gemfibrozil is a widely used hypolipidemic and triglyceride lowering drug. Excess of the drug is excreted and discharged into the environment primarily via wastewater treatment plant effluents. Bacillus sp. GeD10, a gemfibrozil-degrader, was previously isolated from activated sludge. It is the first identified bacterium capable of degrading gemfibrozil. Gemfibrozil degradation by Bacillus sp. GeD10 was here studied through genome sequencing, quantitative proteomics and metabolite analysis. From the bacterial proteome of Bacillus sp. GeD10 1974 proteins were quantified, of which 284 proteins were found to be overabundant by more than 2-fold (FDR corrected p-value ≤0.032, fold change (log2) ≥ 1) in response to gemfibrozil exposure. Metabolomic analysis identified two hydroxylated intermediates as well as a glucuronidated hydroxyl-metabolite of gemfibrozil. Overall, gemfibrozil exposure in Bacillus sp. GeD10 increased the abundance of several enzymes potentially involved in gemfibrozil degradation as well as resulted in the production of several gemfibrozil metabolites. The potential catabolic pathway/modification included ring-hydroxylation preparing the substrate for subsequent ring cleavage by a meta-cleaving enzyme. The identified genes may allow for monitoring of potential gemfibrozil-degrading organisms in situ and increase the understanding of microbial processing of trace level contaminants. This study represents the first omics study on a gemfibrozil-degrading bacterium.

Functional responses and adaptation of mesophilic microbial communities to psychrophilic anaerobic digestion.

Psychrophilic (<20°C) anaerobic digestion (AD) represents an attractive alternative to mesophilic wastewater treatment. In order to investigate the AD microbiome response to temperature change, with particular emphasis on methanogenic archaea, duplicate laboratory-scale AD bioreactors were operated at 37°C followed by a temperature drop to 15°C. A volatile fatty acid-based wastewater (composed of propionic acid, butyric acid, acetic acid and ethanol) was used to provide substrates representing the later stages of AD. Community structure was monitored using 16S rRNA gene clone libraries, as well as DNA and cDNA-based DGGE analysis, while the abundance of relevant methanogens was followed using qPCR. In addition, metaproteomics, microautoradiography-fluorescence in situ hybridization, and methanogenic activity measurements were employed to investigate microbial activities and functions. Methanomicrobiales abundance increased at low temperature, which correlated with an increased contribution of CH4 production from hydrogenotrophic methanogenesis at 15°C. Methanosarcinales utilized acetate and H2/CO2 as CH4 precursors at both temperatures and a partial shift from acetoclastic to hydrogenotrophic methanogenesis was observed for this archaeal population at 15°C. An upregulation of protein expression was reported at low temperature as well as the detection of chaperones indicating that mesophilic communities experienced stress during long-term exposure to 15°C. Overall, changes in microbial community structure and function were found to underpin the adaptation of mesophilic sludge to psychrophilic AD.

Identification of Putative Genes Involved in Bisphenol A Degradation Using Differential Protein Abundance Analysis of Sphingobium sp. BiD32.

Discharge of the endocrine disrupting compound bisphenol A (BPA) with wastewater treatment plant (WWTP) effluents into surface waters results in deleterious effects on aquatic life. Sphingobium sp. BiD32 was previously isolated from activated sludge based on its ability to degrade BPA. This study investigated BPA metabolism by Sphingobium sp. BiD32 using label-free quantitative proteomics. The genome of Sphingobium sp. BiD32 was sequenced to provide a species-specific platform for optimal protein identification. The bacterial proteomes of Sphingobium sp. BiD32 in the presence and absence of BPA were identified and quantified. A total of 2155 proteins were identified; 1174 of these proteins were quantified, and 184 of these proteins had a statistically significant change in abundance in response to the presence/absence of BPA (p ≤ 0.05). Proteins encoded by genes previously identified to be responsible for protocatechuate degradation were upregulated in the presence of BPA. The analysis of the metabolites from BPA degradation by Sphingobium sp. BiD32 detected a hydroxylated metabolite. A novel p-hydroxybenzoate hydroxylase enzyme detected by proteomics was implicated in the metabolic pathway associated with the detected metabolite. This enzyme is hypothesized to be involved in BPA degradation by Sphingobium sp. BiD32, and may serve as a future genetic marker for BPA degradation.

Nitrosospira lacus sp. nov., a psychrotolerant, ammonia-oxidizing bacterium from sandy lake sediment.

A Gram-negative, spiral-shaped, chemolithotrophic, ammonia-oxidizing bacterium, designated APG3(T), was isolated into pure culture from sandy lake sediment collected from Green Lake, Seattle, WA, USA. Phylogenetic analyses based on the 16S rRNA gene sequence showed that strain APG3(T) belongs to cluster 0 of the genus Nitrosospira, which is presently not represented by described species, with Nitrosospira multiformis (cluster 3) as the closest species with a validly published name (identity of 98.6 % to the type strain). Strain APG3(T) grew at 4 °C but could not grow at 35 °C, indicating that this bacterium is psychrotolerant. Remarkably, the strain was able to grow over a wide range of pH (pH 5-9), which was greater than the pH range of any studied ammonia-oxidizing bacteria in pure culture. The DNA G+C content of the APG3(T) genome is 53.5 %, which is similar to that of Nitrosospira multiformis ATCC 25196(T) (53.9 %) but higher than that of Nitrosomonas europaea ATCC 19718 (50.7 %) and Nitrosomonas eutropha C71 (48.5 %). The average nucleotide identity (ANI) calculated for the genomes of strain APG3(T) and Nitrosospira multiformis ATCC 25196(T) was 75.45 %, significantly lower than the value of 95 % ANI that corresponds to the 70 % species-level cut-off based on DNA-DNA hybridization. Overall polyphasic taxonomy study indicated that strain APG3(T) represents a novel species in the genus Nitrosospira, for which the name Nitrosospira lacus sp. nov. is proposed (type strain APG3(T) = NCIMB 14869(T) = LMG 27536(T) = ATCC BAA-2542(T)).

Rapid TaqMan-based quantification of chlorophyll d-containing cyanobacteria in the genus Acaryochloris.

Reports of the chlorophyll (Chl) d-containing cyanobacterium Acaryochloris have accumulated since its initial discovery in 1996. The majority of this evidence is based on amplification of the gene coding for the 16S rRNA, and due to the wide geographical distribution of these sequences, a global distribution of Acaryochloris species was suggested. Here, we present a rapid, reliable, and cost-effective TaqMan-based quantitative PCR (qPCR) assay that was developed for the specific detection of Acaryochloris species in complex environmental samples. The TaqMan probe showed detection limits of ~10 16S rRNA gene copy numbers based on standard curves consisting of plasmid inserts. DNA from five Acaryochloris strains, i.e., MBIC11017, CCMEE5410, HICR111A, CRS, and Awaji-1, exhibited amplification efficiencies of >94% when tested in the TaqMan assay. When used on complex natural communities, the TaqMan assay detected the presence of Acaryochloris species in four out of eight samples of crustose coralline algae (CCA), collected from temperate and tropical regions. In three out of these TaqMan-positive samples, the presence of Chl d was confirmed via high-performance liquid chromatography (HPLC), and corresponding cell estimates of Acaryochloris species amounted to 7.6 × 10(1) to 3.0 × 10(3) per mg of CCA. These numbers indicate a substantial contribution of Chl d-containing cyanobacteria to primary productivity in endolithic niches. The new TaqMan assay allows quick and easy screening of environmental samples for the presence of Acaryochloris species and is an important tool to further resolve the global distribution and significance of this unique oxyphototroph.

Draft Genome Sequence of Nitrosospira sp. Strain APG3, a Psychrotolerant Ammonia-Oxidizing Bacterium Isolated from Sandy Lake Sediment.

Bacteria in the genus Nitrosospira play vital roles in the nitrogen cycle. Nitrosospira sp. strain APG3 is a psychrotolerant betaproteobacterial ammonia-oxidizing bacterium isolated from freshwater lake sediment. The draft genome revealed that it represents a new species of cluster 0 Nitrosospira, which is presently not represented by described species.

Community structure of bacteria and fungi in aerosols of a pig confinement building.

Modern intensive husbandry practices can create poor indoor air quality, with high levels of airborne dust, endotoxins, ammonia, and microorganisms. Air in a sow breeding barn was investigated to determine the biomass composition of bioaerosols using molecular methods supplemented with microscopic and cultivation-dependent approaches. A total of 2.7 ± 0.7 × 10(7)  bacterial cells m(-3) air and 1.2 ± 0.3 × 10(6)  fungi spores m(-3) were detected, corresponding to the fungal biovolume constituted 98% of the total microbial biovolume (fungal and bacterial). Fifty-two percent of all 4',6-diamidino-2-phenyl indole-stained cells were detectable with fluorescence in situ hybridization (FISH) with a general bacterial probe mixture. Quantitative FISH of the bacterial consortium revealed Firmicutes as the dominant group with Streptococcus as the major genus, while Actinobacteria constituted 10% of the detectable bacteria. Additionally, the study revealed an abundant and diverse fungal community including species not previously found in similar environments. The most abundant fungal 18S rRNA gene clone sequences identified affiliated with the Aspergillus-Eurotium cluster, but among others, species of Wallemia, Mucorales, and Russulales were detected. For both fungi and anaerobic bacteria, a hitherto undescribed diversity was found in bioaerosols from a modern sow breeding barn, which potentially could create poor indoor air quality, although their effect on the health of farmworkers and stock still is not resolved.

Butyric acid- and dimethyl disulfide-assimilating microorganisms in a biofilter treating air emissions from a livestock facility.

Biofiltration has proven an efficient tool for the elimination of volatile organic compounds (VOCs) and ammonia from livestock facilities, thereby reducing nuisance odors and ammonia emissions to the local environment. The active microbial communities comprising these filter biofilms have not been well characterized. In this study, a trickle biofilter treating air from a pig facility was investigated and proved efficient in removing carboxylic acids (>70% reduction), mainly attributed to the primary filter section within which reduced organic sulfur compounds were also depleted (up to 50%). The secondary filter eliminated several aromatic compounds: phenol (81%), p-cresol (89%), 4-ethylphenol (68%), indole (48%), and skatole (69%). The active butyric acid degrading bacterial community of an air filter sample was identified by DNA stable-isotope probing (DNA-SIP) and microautoradiography, combined with fluorescence in situ hybridization (MAR-FISH). The predominant 16S rRNA gene sequences from a clone library derived from "heavy" DNA from [(13)C(4)]butyric acid incubations were Microbacterium, Gordonia, Dietzia, Rhodococcus, Propionibacterium, and Janibacter, all from the Actinobacteria. Actinobacteria were confirmed and quantified by MAR-FISH as being the major bacterial phylum assimilating butyric acid along with several Burkholderiales-related Betaproteobacteria. The active bacterial community assimilating dimethyl disulfide (DMDS) was characterized by DNA-SIP and MAR-FISH and found to be associated with the Actinobacteria, along with a few representatives of Flavobacteria and Sphingobacteria. Interestingly, ammonia-oxidizing Betaproteobacteria were also implicated in DMDS degradation, as were fungi. Thus, multiple isotope-based methods provided complementary data, enabling high-resolution identification and quantitative assessments of odor-eliminating Actinobacteria-dominated populations of these biofilter environments.

Thaumarchaeotes abundant in refinery nitrifying sludges express amoA but are not obligate autotrophic ammonia oxidizers.

Nitrification is a core process in the global nitrogen cycle that is essential for the functioning of many ecosystems. The discovery of autotrophic ammonia-oxidizing archaea (AOA) within the phylum Thaumarchaeota has changed our perception of the microbiology of nitrification, in particular since their numerical dominance over ammonia-oxidizing bacteria (AOB) in many environments has been revealed. These and other data have led to a widely held assumption that all amoA-encoding members of the Thaumarchaeota (AEA) are autotrophic nitrifiers. In this study, 52 municipal and industrial wastewater treatment plants were screened for the presence of AEA and AOB. Thaumarchaeota carrying amoA were detected in high abundance only in four industrial plants. In one plant, thaumarchaeotes closely related to soil group I.1b outnumbered AOB up to 10,000-fold, and their numbers, which can only be explained by active growth in this continuous culture system, were two to three orders of magnitude higher than could be sustained by autotrophic ammonia oxidation. Consistently, (14)CO(2) fixation could only be detected in AOB but not in AEA in actively nitrifying sludge from this plant via FISH combined with microautoradiography. Furthermore, in situ transcription of archaeal amoA, and very weak in situ labeling of crenarchaeol after addition of (13)CO(2), was independent of the addition of ammonium. These data demonstrate that some amoA-carrying group I.1b Thaumarchaeota are not obligate chemolithoautotrophs.

Growth kinetics of hydrogen sulfide oxidizing bacteria in corroded concrete from sewers.

Hydrogen sulfide oxidation by microbes present on concrete surfaces of sewer pipes is a key process in sewer corrosion. The growth of aerobic sulfur oxidizing bacteria from corroded concrete surfaces was studied in a batch reactor. Samples of corrosion products, containing sulfur oxidizing bacteria, were suspended in aqueous solution at pH similar to that of corroded concrete. Hydrogen sulfide was supplied to the reactor to provide the source of reduced sulfur. The removal of hydrogen sulfide and oxygen was monitored. The utilization rates of both hydrogen sulfide and oxygen suggested exponential bacterial growth with median growth rates of 1.25 d(-1) and 1.33 d(-1) as determined from the utilization rates of hydrogen sulfide and oxygen, respectively. Elemental sulfur was found to be the immediate product of the hydrogen sulfide oxidation. When exponential growth had been achieved, the addition of hydrogen sulfide was terminated leading to elemental sulfur oxidation. The ratio of consumed sulfur to consumed oxygen suggested that sulfuric acid was the ultimate oxidation product. To the knowledge of the authors, this is the first study to determine the growth rate of bacteria involved in concrete corrosion with hydrogen sulfide as source of reduced sulfur.

Functional amyloid in Pseudomonas.

Amyloids are highly abundant in many microbial biofilms and may play an important role in their architecture. Nevertheless, little is known of the amyloid proteins. We report the discovery of a novel functional amyloid expressed by a Pseudomonas strain of the P. fluorescens group. The amyloid protein was purified and the amyloid-like structure verified. Partial sequencing by MS/MS combined with full genomic sequencing of the Pseudomonas strain identified the gene coding for the major subunit of the amyloid fibril, termed fapC. FapC contains a thrice repeated motif that differs from those previously found in curli fimbrins and prion proteins. The lack of aromatic residues in the repeat shows that aromatic side chains are not needed for efficient amyloid formation. In contrast, glutamine and asparagine residues seem to play a major role in amyloid formation as these are highly conserved in curli, prion proteins and FapC. fapC is conserved in many Pseudomonas strains including the opportunistic pathogen P. aeruginosa and is situated in a conserved operon containing six genes, of which one encodes a fapC homologue. Heterologous expression of the fapA-F operon in Escherichia coli BL21(DE3) resulted in a highly aggregative phenotype, showing that the operon is involved in biofilm formation.

Adhesion characteristics of nitrifying bacteria in activated sludge.

Nitrifiers are known to form relatively dense and strong microcolonies in activated sludge flocs, but little is known about their adhesion characteristics and how these are relative to other floc components. The size distribution of ammonia and nitrite-oxidizing bacteria (Nitrosomonas oligotropha and Nitrospira spp.) in activated sludge from a nutrient removal plant showed that the majority of N. oligotropha cells formed microcolonies with a diameter of 13-22.5 microm, and most Nitrospira spp. cells formed microcolonies with a diameter of 9-22.5 microm. By applying high shear forces (2200 s(-1)), the largest microcolonies of N. oligotropha fragmented to a level well below the Kolmogorov microscale (approx. 15-25 microm). Only very little erosion of single cells took place. Nitrospira spp. microcolonies were generally slightly stronger than N. oligotropha. In order to characterize the adhesion/binding mechanisms for the individual microcolonies, a number of different physico-chemical treatments were combined with shear, and even though this did not lead to any explicit characterization of the species-specific adhesion mechanisms, entanglement of extracellular polymers was proposed as a plausible important adhesion mechanism. When compared to other floc components, the deflocculated fractions of N. oligotropha and Nitrospira spp. were much lower than those of cells in general (total cell count, DAPI) or the organic matter. Deflocculation of N. oligotropha ranged from 3% to 11% of the total N. oligotropha biovolume, Nitrospira spp. from 1% to 4% of the total Nitrospira spp. biovolume, whereas the number of deflocculated cells was 9-54% of the total cell count, and the deflocculated organic matter constituted 8-43% of the total amount of organic matter. These results clearly showed that activated sludge contained a large pool of loosely attached cells and extracellular polymeric substances, and that the nitrifiers and some other microcolony formers were very strong and remained almost intact even under extreme physical and chemical conditions.

Ecophysiology of the Actinobacteria in activated sludge systems.

This review considers what is known about the Actinobacteria in activated sludge systems, their abundance and their functional roles there. Participation in processes leading to the microbiological removal of phosphate and in the operational problems of bulking and foaming are discussed in terms of their ecophysiological traits. We consider critically whether elucidation of their nutritional requirements and other physiological properties allow us to understand better what might affect their survival capabilities in these highly competitive systems. Furthermore, how this information might allow us to improve how these processes work is discussed.