Ecophysiological interactions between the community members (i.e., nitrifiers and heterotrophic bacteria) in a carbon-limited autotrophic nitrifying biofilm fed only NH 4 ؉ as an energy source were investigated by using a full-cycle 16S rRNA approach followed by microautoradiography (MAR)-fluorescence in situ hybridization (FISH). Phylogenetic differentiation (identification) of heterotrophic bacteria was performed by 16S rRNA gene sequence analysis, and FISH probes were designed to determine the community structure and the spatial organization (i.e., niche differentiation) in the biofilm. FISH analysis showed that this autotrophic nitrifying biofilm was composed of 50% nitrifying bacteria (ammonia-oxidizing bacteria [AOB] and nitrite-oxidizing bacteria [NOB]) and 50% heterotrophic bacteria, and the distribution was as follows: members of the alpha subclass of the class Proteobacteria (␣-Proteobacteria), 23%; ␥-Proteobacteria, 13%; green nonsulfur bacteria (GNSB), 9%; Cytophaga-Flavobacterium-Bacteroides (CFB) division, 2%; and unidentified (organisms that could not be hybridized with any probe except EUB338), 3%. These results indicated that a pair of nitrifiers (AOB and NOB) supported a heterotrophic bacterium via production of soluble microbial products (SMP). MAR-FISH revealed that the heterotrophic bacterial community was composed of bacteria that were phylogenetically and metabolically diverse and to some extent metabolically redundant, which ensured the stability of the ecosystem as a biofilm. ␣-and ␥-Proteobacteria dominated the utilization of Bacteria classified in the unidentified group accounted for 6% of the total heterotrophic bacteria and could utilize all organic substrates, including NAG. This showed that there was an efficient food web (carbon metabolism) in the autotrophic nitrifying biofilm community, which ensured maximum utilization of SMP produced by nitrifiers and prevented buildup of metabolites or waste materials of nitrifiers to significant levels.Microbial nitrification followed by denitrification for nitrogen removal is becoming more important due to strict nitrogen discharge regulations. Aerobic biofilm systems have been used for nitrogen removal because of a biomass retention time sufficiently long to achieve reliable nitrification. In such biofilm systems, the competitive interaction between heterotrophs and nitrifiers for dissolved oxygen and space is well known (31,32,33). In the presence of organic carbon, nitrifiers are usually outcompeted by heterotrophs due to the low growth rate and low growth yield of the former organisms. Autotrophic nitrifiers reduce inorganic carbon to form organic carbon in cell mass, produce and release soluble microbial products (SMP) into solution from substrate metabolism (usually with biomass growth), and decay biomass (41). Therefore, they also interact through the exchange of organic matter. Coexistence of a high level of heterotrophs with nitrifiers has been found often in autotrophic nitrifying biofilms cultured without an external organic carb...

To promptly establish anaerobic ammonium oxidation (anammox) reactors, appropriate seeding sludge with high abundance and activity of anammox bacteria was selected by quantifying 16S rRNA gene copy numbers of anammox bacteria by real-time quantitative PCR (RTQ-PCR) and batch culture experiments. The selected sludge was then inoculated into up-flow fixed-bed biofilm column reactors with nonwoven fabric sheets as biomass carrier and the reactor performances were monitored over 1 year. The anammox reaction was observed within 50 days and a total nitrogen removal rate of 26.0 kg-Nm(-3)day(-1) was obtained after 247 days. To our knowledge, such a high rate has never been reported before. Hydraulic retention time (HRT) and influent NH(4)(+) to NO(2)(-) molar ratio could be important determinant factors for efficient nitrogen removal in this study. The higher nitrogen removal rate was obtained at the shorter HRT and higher influent NH(4)(+)/NO(2)(-) molar ratio. After anammox reactors were fully developed, the community structure, spatial organization and in situ activity of the anammox biofilms were analyzed by the combined use of a full-cycle of 16S rRNA approach and microelectrodes. In situ hybridization results revealed that the probe Amx820-hybridized anaerobic anammox bacteria were distributed throughout the biofilm (accounting for more than 70% of total bacteria). They were associated with Nitrosomonas-like aerobic ammonia-oxidizing bacteria (AAOB) in the surface biofilm. The anammox bacteria present in this study were distantly related to the Candidatus Brocadia anammoxidans with the sequence similarity of 95%. Microelectrode measurements showed that a high in situ anammox activity (i.e., simultaneous consumption of NH(4)(+) and NO(2)(-)) of 4.45 g-N of (NH(4)(+)+NO(2)(-))m(-2)day(-1) was detected in the upper 800 microm of the biofilm, which was consistent with the spatial distribution of anammox bacteria.

Polycyclic aromatic hydrocarbons (PAHs) are principally derived from the incomplete combustion of fossil fuels. This study investigated the occurrence of PAHs in aquatic environments around the world, their effects on the environment and humans, and methods for their removal. Polycyclic aromatic hydrocarbons have a great negative impact on the environment and humans, and can even cause cancer in humans. Use of good methods and equipment are essential to monitoring PAHs, and GC/MS and HPLC are usually used for their analysis in aqueous solutions. In aquatic environments, the PAHs concentrations range widely from 0.03 ng/L (seawater; Southeastern Japan Sea, Japan) to 8,310,000 ng/L (Domestic Wastewater Treatment Plant, Siloam, South Africa). Moreover, bioaccumulation of ∑16PAHs in fish has been reported to range from 11.2 ng/L (Cynoscion guatucupa, South Africa) to 4207.5 ng/L (Saurida undosquamis, Egypt). Several physical/chemical and biological techniques have been reported to treat water contaminated by PAHs, but adsorption and combined treatment methods have shown better removal performance, with some methods removing up to 99.99% of PAHs.

The cross-feeding of microbial products derived from 14 C-labeled nitrifying bacteria to heterotrophic bacteria coexisting in an autotrophic nitrifying biofilm was quantitatively analyzed by using microautoradiography combined with fluorescence in situ hybridization (MAR-FISH). After only nitrifying bacteria were labeled with [ 14 C]bicarbonate, biofilm samples were incubated with and without NH 4 ؉ as a sole energy source for 10 days. The transfer of 14 C originally incorporated into nitrifying bacterial cells to heterotrophic bacteria was monitored with time by using MAR-FISH. The MAR-FISH analysis revealed that most phylogenetic groups of heterotrophic bacteria except the ␤-Proteobacteria showed significant uptake of 14 C-labeled microbial products. In particular, the members of the Chloroflexi were strongly MAR positive in the culture without NH 4 ؉ addition, in which nitrifying bacteria tended to decay. This indicated that the members of the Chloroflexi preferentially utilized microbial products derived from mainly biomass decay. On the other hand, the members of the Cytophaga-Flavobacterium cluster gradually utilized 14 C-labeled products in the culture with NH 4 ؉ addition in which nitrifying bacteria grew. This result suggested that these bacteria preferentially utilized substrate utilization-associated products of nitrifying bacteria and/or secondary metabolites of 14 C-labeled structural cell components. Our results clearly demonstrated that the coexisting heterotrophic bacteria efficiently degraded and utilized dead biomass and metabolites of nitrifying bacteria, which consequently prevented accumulation of organic waste products in the biofilm.Most bacteria in the natural environment and engineering systems are present in the form of complex multispecies biofilms attached to solid surfaces rather than as planktonic (freeswimming) isolated cells in the bulk water phase (10,27,37,38,41). Microbial life in such biofilms, which cannot be seen in a planktonic state, is mostly characterized by multiplicity (many species together), nutrient limitation, competition for oxygen, substrates, and/or space (beneficial or inhibitory interactions among microbial species), and a structured distribution of the microbial species. Therefore, several studies have been performed on wastewater treatment biofilms by combining fluorescence in situ hybridization (FISH) and microsensor technology to link the spatial organization of microbial communities and in situ functions at the community level (10,26,27,38). The coexistence of a high level of heterotrophic bacteria with nitrifying bacteria has often been found in autotrophic nitrifying biofilms cultured without an external organic carbon supply by applying a 16S rRNA approach (16,27,28). It has been hypothesized that heterotrophic bacteria scavenge organic matter derived from biomass decay and substrate metabolism of nitrifying bacteria. However, the in situ ecophysiology of heterotrophic bacteria in such autotrophic biofilms is still largely unknown because most of the h...

The coexistence of uncultured heterotrophic bacteria belonging to the phylum Chloroflexi has often been observed in anaerobic ammonium oxidation (anammox) reactors fed with synthetic nutrient medium without organic carbon compounds. To determine if coexisting Chloroflexi in anammox reactors scavenge organic matter derived from anammox bacterial cells, the present study was conducted to investigate the substrate uptake pattern of the uncultured Chloroflexi present in an anammox reactor and to clarify if they take up microbial products derived from anammox bacterial cells. To accomplish this, combined microautoradiography and fluorescence in situ hybridization (MAR-FISH) was conducted. Phylogenetic analysis revealed that 36% of the clones analyzed in this study were affiliated with Chloroflexi. The sequence similarities to Anaerolinea thermophila and Caldilinea aerophila within the phylum Chloroflexi were only 81.0-88.7% and 80.3-83.8%, respectively. The uncultured Chloroflexi were found to incorporate sucrose, glucose, and N-acetyl-glucosamine. The (14)C-tracing experiment revealed that the uncultured Chloroflexi were clearly MAR-positive, indicating the utilization of decaying anammox bacterial cell materials. Taken together, these results indicate that coexisting uncultured Chloroflexi in anammox reactors scavenge organic compounds derived from anammox bacterial cells.

To date, six candidate genera of anaerobic ammonium-oxidizing (anammox) bacteria have been identified, and numerous studies have been conducted to understand their ecophysiology. In this study, we examined the physiological characteristics of an anammox bacterium in the genus 'Candidatus Jettenia'. Planctomycete KSU-1 was found to be a mesophilic (20-42.5°C) and neutrophilic (pH 6.5-8.5) bacterium with a maximum growth rate of 0.0020 h(-1) . Planctomycete KSU-1 cells showed typical physiological and structural features of anammox bacteria; i.e. (29) N2 gas production by coupling of (15) NH4 (+) and (14) NO2 (-) , accumulation of hydrazine with the consumption of hydroxylamine and the presence of anammoxosome. In addition, the cells were capable of respiratory ammonification with oxidation of acetate. Notably, the cells contained menaquinone-7 as a dominant respiratory quinone. Proteomic analysis was performed to examine underlying core metabolisms, and high expressions of hydrazine synthase, hydrazine dehydrogenase, hydroxylamine dehydrogenase, nitrite/nitrate oxidoreductase and carbon monoxide dehydrogenase/acetyl-CoA synthase were detected. These proteins require iron or copper as a metal cofactor, and both were dominant in planctomycete KSU-1 cells. On the basis of these experimental results, we proposed the name 'Ca. Jettenia caeni' sp. nov. for the bacterial clade of the planctomycete KSU-1.

The anaerobic ammonium-oxidizing (ANAMMOX) bacteria were enriched from a rotating disk reactor (RDR) biofilm in semi-batch cultures. Based on fluorescence in situ hybridization (FISH) analysis, this enrichment led to a relative population size of 36% ANAMMOX bacteria. Phylogenetic analysis revealed that all the detected clones were related to the previously reported ANAMMOX bacteria, Candidatus Brocadia anammoxidans (AF375994), with 92% sequence similarity. Furthermore, we successfully developed a real-time polymerase chain reaction (PCR) assay to quantify populations of ANAMMOX bacteria in the enrichment cultures. For this real-time PCR assay, PCR primer sets targeting 16S ribosomal RNA genes of ANAMMOX bacteria were designed and used. The quantification range of this assay was 6 orders of magnitude, from 8.9x10(1) to 8.9x10(6) copies per PCR, corresponding to the detection limit of 3.6x10(3) target copies mL(-1). A significant correlation was found between the increase in copy numbers of 16S rRNA gene of ANAMMOX bacteria and the increase in nitrogen removal rates in the enrichment cultures. Quantifying ANAMMOX bacterial populations in the enrichment culture made it possible to estimate the doubling time of the enriched ANAMMOX bacteria to be 3.6 to 5.4 days. The real-time PCR assay gave comparable population sizes in the enrichment cultures with the FISH results. These results suggest that the real-time PCR assay developed in this study is useful and reliable for quantifying the populations of ANAMMOX bacteria in environmental and engineering samples.

bThe phylogenetic affiliation and physiological characteristics (e.g., K s and maximum specific growth rate [ max ]) of an anaerobic ammonium oxidation (anammox) bacterium, "Candidatus Scalindua sp.," enriched from the marine sediment of Hiroshima Bay, Japan, were investigated. "Candidatus Scalindua sp." exhibits higher affinity for nitrite and a lower growth rate and yield than the known anammox species.