Tomonori Kindaichi

Ecophysiological interaction between nitrifying bacteria and heterotrophic bacteria in autotrophic nitrifying biofilms as determined by microautoradiography-fluorescence in situ hybridization.

ABSTRACT Ecophysiological interactions between the community members (i.e., nitrifiers and heterotrophic bacteria) in a carbon-limited autotrophic nitrifying biofilm fed only NH4+ 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 (α-Proteobacteria), 23%; γ-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. α- and γ-Proteobacteria dominated the utilization of [14C]acetic acid and 14C-amino acids in this biofilm. Despite their low abundance (ca. 2%) in the biofilm community, members of the CFB cluster accounted for the largest fraction (ca. 64%) of the bacterial community consuming N-acetyl-d-[1-14C]glucosamine (NAG). The GNSB accounted for 9% of the 14C-amino acid-consuming bacteria and 27% of the [14C]NAG-consuming bacteria but did not utilize [14C]acetic acid. 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.

Fate of 14C-Labeled Microbial Products Derived from Nitrifying Bacteria in Autotrophic Nitrifying Biofilms

ABSTRACT The cross-feeding of microbial products derived from 14C-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 [14C]bicarbonate, biofilm samples were incubated with and without NH4+ as a sole energy source for 10 days. The transfer of 14C 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 β-Proteobacteria showed significant uptake of 14C-labeled microbial products. In particular, the members of the Chloroflexi were strongly MAR positive in the culture without NH4+ 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 14C-labeled products in the culture with NH4+ 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 14C-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.

In situ activity and spatial organization of anaerobic ammonium-oxidizing (anammox) bacteria in biofilms.

ABSTRACT We investigated autotrophic anaerobic ammonium-oxidizing (anammox) biofilms for their spatial organization, community composition, and in situ activities by using molecular biological techniques combined with microelectrodes. Results of phylogenetic analysis and fluorescence in situ hybridization (FISH) revealed that “Brocadia”-like anammox bacteria that hybridized with the Amx820 probe dominated, with 60 to 92% of total bacteria in the upper part (<1,000 μm) of the biofilm, where high anammox activity was mainly detected with microelectrodes. The relative abundance of anammox bacteria decreased along the flow direction of the reactor. FISH results also indicated that Nitrosomonas-, Nitrosospira-, and Nitrosococcus-like aerobic ammonia-oxidizing bacteria (AOB) and Nitrospira-like nitrite-oxidizing bacteria (NOB) coexisted with anammox bacteria and accounted for 13 to 21% of total bacteria in the biofilms. Microelectrode measurements at three points along the anammox reactor revealed that the NH4+ and NO2− consumption rates decreased from 0.68 and 0.64 μmol cm−2 h−1 at P2 (the second port, 170 mm from the inlet port) to 0.30 and 0.35 μmol cm−2 h−1 at P3 (the third port, 205 mm from the inlet port), respectively. No anammox activity was detected at P4 (the fourth port, 240 mm from the inlet port), even though sufficient amounts of NH4+ and NO2− and a high abundance of anammox bacteria were still present. This result could be explained by the inhibitory effect of organic compounds derived from biomass decay and/or produced by anammox and coexisting bacteria in the upper parts of the biofilm and in the upstream part of the reactor. The anammox activities in the biofilm determined by microelectrodes reflected the overall reactor performance. The several groups of aerobic AOB lineages, Nitrospira-like NOB, and Betaproteobacteria coexisting in the anammox biofilm might consume a trace amount of O2 or organic compounds, which consequently established suitable microenvironments for anammox bacteria.

Biological oxidation of dissolved methane in effluents from anaerobic reactors using a down-flow hanging sponge reactor

Anaerobic wastewater treatment plants discharge dissolved methane, which is usually not recovered. To prevent emission of methane, which is a greenhouse gas, we utilized an encapsulated down-flow hanging sponge reactor as a post-treatment to biologically oxidize dissolved methane. Within 3 weeks after reactor start-up, methane removal efficiency of up to 95% was achieved with a methane removal rate of 0.8 kg COD m(-3) day(-1) at an HRT of 2 h. After increasing the methane-loading rate, the maximum methane removal rate reached 2.2 kg COD m(-3) day(-1) at an HRT of 0.5 h. On the other hand, only about 10% of influent ammonium was oxidized to nitrate during the first period, but as airflow was increased to 2.5 L day(-1), nitrification efficiency increased to approximately 70%. However, the ammonia oxidation rate then decreased with an increase in the methane-loading rate. These results indicate that methane oxidation occurred preferentially over ammonium oxidation in the reactor. Cloning of the 16S rRNA and pmoA genes as well as phylogenetic and T-RFLP analyses revealed that type I methanotrophs were the dominant methane oxidizers, whereas type II methanotrophs were detected only in minor portion of the reactor.

Dissolved methane oxidation and competition for oxygen in down-flow hanging sponge reactor for post-treatment of anaerobic wastewater treatment

Post-treatment of anaerobic wastewater was undertaken to biologically oxidize dissolved methane, with the aim of preventing methane emission. The performance of dissolved methane oxidation and competition for oxygen among methane, ammonium, organic matter, and sulfide oxidizing bacteria were investigated using a lab-scale closed-type down-flow hanging sponge (DHS) reactor. Under the oxygen abundant condition of a hydraulic retention time of 2h and volumetric air supply rate of 12.95m(3)-airm(-3)day(-1), greater than 90% oxidation of dissolved methane, ammonium, sulfide, and organic matter was achieved. With reduction in the air supply rate, ammonium oxidation first ceased, after which methane oxidation deteriorated. Sulfide oxidation was disrupted in the final step, indicating that COD and sulfide oxidation occurred prior to methane oxidation. A microbial community analysis revealed that peculiar methanotrophic communities dominating the Methylocaldum species were formed in the DHS reactor operation.

Phosphate recovery as concentrated solution from treated wastewater by a PAO-enriched biofilm reactor

Phosphorus recovery from wastewaters and its recycling are of importance for sustaining agricultural production. During the conventional enhanced biological phosphorus removal process, phosphorus is removed by withdrawing excess sludge from wastewater. However, excess sludge disposal is costly and energy intensive. A proposed novel process for phosphorus recovery from sewage treatment will result in no excess sludge if a polyphosphate accumulating organisms (PAOs) enrichment biofilm can be applied to effluents containing phosphate. This process allows the recovery of phosphate as phosphate-concentrated solutions by controlling PAOs to absorb and release phosphate. A reactor consisting of a modified trickling filter with a synthetic substrate (5 mg P L⁻¹) was operated to form a PAO-enriched biofilm. As a result of the enrichment, the concentration of phosphate of >100 mg P L⁻¹ was successfully achieved. During this experiment, no sludge withdrawal was carried out over the duration of the operation of 255 days. To highlight the new process, the principle of enriching PAOs on biofilm and concentrating phosphate from treated sewage is explained, and a discussion on phosphate recovery performance is given.

Loading and removal of PAHs in a wastewater treatment plant in a separated sewer system.

The loading and removal of polycyclic aromatic hydrocarbons (PAHs) were measured and estimated in a wastewater treatment plant in a separated sewer system in a suburban area of Japan. The influent 16 PAHs concentration was 219 ± 210 ng L(-1), whereas the effluent concentration was 43.5 ± 42.5 ng L(-1) (mean ± sd). No clear diurnal or weekly fluctuation was observed. However, evaluation of long-term changes revealed PAH fluctuations continuing for more than 1 week. Half of the PAHs (63%) were biologically or chemically transformed, or vaporized in the treatment plant, while the remainder were discharged with effluent (28%) and excess sludge (9%). Measurement of the per capita loading of the treatment plant revealed values of 142 ± 53 and 28 ± 11 μg person(-1)day(-1) (mean ± 95% confidence interval) for influent and effluent, respectively. Isomer ratio analysis revealed that the PAHs originated from a mixture of petroleum, petroleum combustion, and burning of biomass residues.

PAHs concentration and toxicity in organic solvent extracts of atmospheric particulate matter and sea sediments

The concentration of polycyclic aromatic hydrocarbons (PAHs) and the toxicity to marine bacteria (Vibrio fischeri) were measured for the organic solvent extracts of sea sediments collected from an urban watershed area (Hiroshima Bay) of Japan and compared with the concentrations and toxicity of atmospheric particulate matter (PM). In atmospheric PM, the PAHs concentration was highest in fine particulate matter (FPM) collected during cold seasons. The concentrations of sea sediments were 0.01-0.001 times those of atmospheric PM. 1/EC50 was 1-10 L g(-1) PM for atmospheric PM and 0.1-1 L g(-1) dry solids for sea sediments. These results imply that toxic substances from atmospheric PM are diluted several tens or hundreds of times in sea sediments. The ratio of the 1/EC50 to PAHs concentration ((1/EC50)/16PAHs) was stable for all sea sediments (0.1-1 L μg(-1) 16PAHs) and was the same order of magnitude as that of FPM and coarse particulate matter (CPM). The ratio of sediments collected from the west was more similar to that of CPM while that from the east was more similar to FPM, possibly because of hydraulic differences among water bodies. The PAHs concentration pattern analyses (principal component analysis and isomer ratio analysis) were conducted and the results showed that the PAHs pattern in sea sediments was quite different to that of FPM and CPM. Comparison with previously conducted PAHs analyses suggested that biomass burning residues comprised a major portion of these other sources.

Nitrogen removal using an anammox membrane bioreactor at low temperature

Membrane bioreactors (MBRs) have the ability to completely retain biomass and are thus suitable for slowly growing anammox bacteria. In the present study, an anammox MBR was operated to investigate whether the anammox activity would remain stable at low temperature, without anammox biomass washout. The maximum nitrogen removal rates were 6.7 and 1.1 g-N L⁻¹ day⁻¹ at 35 °C and 15 °C, respectively. Fluorescence in situ hybridization and 16S rRNA-based phylogenetic analysis revealed no change in the predominant anammox species with temperature because of the complete retention of anammox biomass in the MBR. These results indicate that the predominant anammox bacteria in the MBR cannot adapt to a low temperature during short-term operation. Conversely, anammox activity recovered rapidly after restoring the temperature from the lower value to the optimal temperature (35 °C). The rapid recovery of anammox activity is a distinct advantage of using an MBR anammox reactor.

PAH contents in road dust on principal roads collected nationwide in Japan and their influential factors

Fifty-four road dust samples were collected from principal roads (n = 37) and residential roads (n = 17) nationwide in Japan from March 2010 to November 2012. Sixteen polycyclic aromatic hydrocarbons (PAHs) and ignition loss (IL) were determined. The total PAH contents ranged from 62 to 6,325 ng g(-1) with a geometric mean of 484 ng g(-1). The IL ranged from 0.8 to 17% with a mean of 6%. The PAH contents were correlated with the IL contents, and the IL contents were dependent on the population density. From the PAH pattern analysis, the PAHs from road dust are considered to be mainly from diesel emissions.