Yuichi Suwa

Diversity of ammonia monooxygenase operon in autotrophic ammonia-oxidizing bacteria

Abstract.Autotrophic ammonia-oxidizing bacteria use the essential enzyme ammonia monooxygenase (AMO) to transform ammonia to hydroxylamine. The amo operon consists of at least three genes, amoC, amoA, and amoB; amoA encodes the subunit containing the putative enzyme active site. The use of the amo genes as functional markers for ammonia-oxidizing bacteria in environmental applications requires knowledge of the diversity of the amo operon on several levels: (1) the copy number of the operon in the genome, (2) the arrangement of the three genes in an individual operon, and (3) the primary sequence of the individual genes. We present a database of amo gene sequences for pure cultures of ammonia-oxidizing bacteria representing both the β- and the γ-subdivision of Proteobacteria in the following genera: Nitrosospira (6 strains), Nitrosomonas (5 strains) and Nitrosococcus (2 strains). The amo operon was found in multiple (2–3) nearly identical copies in the β-subdivision representatives but in single copies in the γ-subdivision ammonia oxidizers. The analysis of the deduced amino acid sequence revealed strong conservation for all three Amo peptides in both primary and secondary structures. For the amoA gene within the β-subdivision, nucleotide identity values are approximately 85% within the Nitrosomonas or the Nitrosospira groups, but approximately 75% when comparing between these groups. Conserved regions in amoA and amoC were identified and used as primer sites for PCR amplification of amo genes from pure cultures, enrichments and the soil environment. The intergenic region between amoC and amoA is variable in length and may be used to profile the community of ammonia-oxidizing bacteria in environmental samples. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00203-001-0369-z.

Ammonia-oxidizing bacteria with different sensitivities to (NH4)2SO4 in activated sludges

Ammonia oxidizers were enumerated in 34 activated sludges, including sludges which were from sewage treatment plants (S-sludge), nightsoil treatment plants (N-sludge) and activated sludges cultivated with organic (O-sludge) or inorganic (I-sludge) artificial wastewaters in the laboratory. Two media were used for enumeration, one containing 0.76 mM (AL medium) and the other 37.9 mM (AH medium) of (NH4)2SO4. The MPN estimated with AL medium were higher than those with AH medium in S-sludges, N-sludges and O-sludges, while both media gave almost the same MPN for I-sludges. Ten ammonia oxidizers, all identified as Nitrosomonas spp, were isolated from sludge samples. Isolates obtained as predominants in S- and O-sludges were sensitive to (NH4)2SO4; they grew in medium containing 0.71 mM of (NH4)2SO4 but not in medium containing 35.7 mM of (NH4)2SO4. On the other hand, those obtained as predominants in an I-sludge, as well as ATCC strains grew in both media. The Monod equation described the relationship between (NH4)2SO4 concentration and nitrite production rates for (NH4)2SO4-insensitive strains but not for sensitive strains, which exhibited a relationship closer to the Haldane equation describing substrate inhibition kinetics. Among isolates obtained from S-sludge sample, Km and Vmax values of an (NH4)2SO4-sensitive strain were much lower than those of an insensitive strain. Similarly, among laboratory sludge isolates, Km and Km values of an (NH4)2SO4-sensitive strain from an O-sludge were lower than those of an insensitive strain from an I-sludge. These results suggest that (NH4)2SO4-sensitive strains had a growth advantage in lower (NH4)2SO4 concentrations, while insensitive strains had an advantage in higher (NH4)2SO4 concentrations.

Distribution of phospholipid ester-linked fatty acid biomarkers for bacteria in the sediment of Ise Bay, Japan

Abstract Surface sediments collected from 40 stations in Ise Bay, which is one of the most polluted bays in Japan, were subjected to phospholipid ester-linked fatty acid (PLFA) analysis. Thirty-five fatty acids were identified in the sediments; they included saturated, unsaturated and branched fatty acids. The major fatty acids included the even-numbered straight chain fatty acid 16:0 (20%), branched chain fatty acids i15:0 (8%) and a15:0 (11%),and monounsaturated fatty acids 16:1d9c (11%) and 18:1d11 (10%). Fatty acids which are common in bacterial membranes were found, and low amounts of longer chain fatty acids were detected in relatively constant amounts in the bay. One of the characteristic features of the PLFA analysis in Ise Bay is the virtual absence of polyunsaturated fatty acids in the sediments, except for 18:2. The PLFA profiles indicate that the microbial community structure is characterized by the absence of polyunsaturated fatty acids typical of microeukaryotes and high proportions of fatty acid biomarkers of prokaryotes in sediments. A wide distribution of aerobic bacteria. Gram-positive bacteria, anaerobic bacteria and sulfate-reducing bacteria in the microbial community structure was indicated by the presence of biomarker fatty acids. Similarity analysis of the PLFA profiles in sediments of all the stations showed that they were similar at the 90% level. The results of Tukeys test showed that a majority of the fatty acids in sediments were not significantly enriched in the bay. The absence of significant variation in the PLFA profiles in sediments revealed that the microbial community structure is similar throughout the bay, and this uniformity was attributed to the reported pollution and eutrophication in Ise Bay. Further, the significant PLFA patterns, with a high proportion of prokaryotic biomarker fatty acids and an absence of microeukaryotic biomarkers, indicate that PLFA analysis could be used as a measure of pollution in sediments.

Single-stage, single-sludge nitrogen removal by an activated sludge process with cross-flow filtration

Nitrogen removal by a single-stage, single-sludge activated sludge process with cross-flow filtration was qualitatively studied. Artificial wastewater had a BOD/TKN ratio from 1.8 to 24.5. The BOD loading to the process ranged from 0.33 to 1.66 g l−1 d−1, the TKN loading from 0.032 to 0.268 g l−1 d−1 and the sludge retention time (SRT) was quite long (54–4200 d). In each experiment, more than 97% of organic carbon was removed. The sludge biomass concentration (MLVSS) was high at a high volumetric BOD loading. Total Kjeldahl nitrogen (TKN) was successfully nitrified and higher than 29% of the TKN which was loaded to the reactor was removed in many of the experimental runs. The nitrogen balance of each unit was calculated. Denitrification was estimated to be responsible for 39–86% of the removed nitrogen in an aeration vessel. Although denitrification was not observed up to 0.438 g l−1 d−1 of BOD loading, the denitrification rate increased as the BOD loading increased. The percentage of nitrogen removal was a function of the BOD/TKN ratio of the influent. Lower DO concentration did not increase the denitrification rate, but intermittent aeration-BOD loading did increase it; TKN was completely removed up to 0.21 TKN g l−1 d−1, and the denitrification rate reached 0.0074 gN VSS−1 d−1.

Anaerobic transformation of chlorophenols in methanogenic sludge unexposed to chlorophenols

Transformation of all 19 chlorophenol (CP) isomers was investigated in a laboratory anaerobic methanogenic sludge that had not been exposed to synthetic chemicals. Concentration of CP was analyzed over time to calculate disappearance rate constants using first-order reaction kinetics and all possible CP degradation pathways were estimated. The rate constants ranged between 0.46 x 10(-3) and 0.161 day(-1). CPs were transformed via dechlorination. The chlorine atom at the ortho-position was the most easily dechlorinated, whereas dechlorination rate at the para-position was lowest. The overall pathways of CP transformation were much less diverse than that we previously found for contaminated sediment. The Dolfing hypothesis of microbial selection of the most thermodynamically favorable pathways was not applicable for CP transformation in this study as well as previous study performed by our group.

Simultaneous removal of phenol and ammonia by an activated sludge process with cross-flow filtration

Attempts were made for removing ammonia from synthetic wastewater under the presence of phenol, which is inhibitory to nitrification, by using a single-stage activated sludge process with cross-flow filtration. Activated sludge biomass which had been acclimated with phenol for over 15 years was used for the inoculum, and synthetic wastewater was continuously supplied to the process retaining biomass at 8000 mg VSS l(-1). Phenol was completely removed, and ammonia was simultaneously nitrified to nitrate; nitrification rate reached 200 mg N l(-1) d(-1) when phenol was removed at a rate up to 300 mg l(-1) d(-1). It was observed that 0-13% of the ammonia was removed via denitrification. Intermittent aeration enhanced the denitrification rate to 160 mg N l(-1) d(-1) by utilizing phenol. and approximately 24% of the denitrified nitrogen was recovered as nitrous oxide. Methanol, which is the most commonly used electron donor in conventional nitrogen removal processes, did not enhance the denitrification rate of the phenol-acclimated activated sludge used in this study, however phenol did. The results suggest that this process potentially works as a space- and energy-saving nitrogen removal process by utilizing substances inhibitory to nitrifiers as electron donors for denitrification.

Effects of nutrient concentration on the growth of soil bacteria

Bacteria isolated from soil were cultivated on conventional nutrient broth at various dilutions (NB, NB/10, NB/100, NB/1,000, and NB/10,000). Based on the results obtained these organisms were divided into four groups by the range of dilution of the full strength nutrient broth (NB) on which growth was recognized; those which showed appreciable growth only on media within a range from NB to NB/1,000 were referred to as type I and those which grew well on media of a range involving further dilutions of NB, to type II. Among the isolates which did not grow well on NB, those which grew only on media within a range from NB/10 to NB/1,000 were assigned to type III, and those which grew well on media of a range involving further dilutions, to type IV. Some isolates of types II and IV showed appreciable growth even in distilled water. They were considered to have an ability of utilizing very small amounts of substances in liquid or air. In connection with these results we discussed the criteria for the identific...

Evidence for degradation of 2-chlorophenol by enrichment cultures under denitrifying conditions.

Although chlorophenol (CP) degradation has been studied, no bacterium responsible for degradation of CP under denitrifying conditions has been isolated. Moreover, little substantial evidence for anaerobic degradation of CPs coupled with denitrification is available even for mixed cultures. Degradation of CP [2-CP, 3-CP, 4-CP, 2,4-dichlorophenol (DCP) or 2,6-DCP] under denitrifying conditions was examined in anaerobic batch culture inoculated with activated sludge. Although 3-CP, 4-CP, 2,4-DCP and 2,6-DCP were not stably degraded, 2-CP was degraded and its degradation capability was sustained in a subculture. However, the rate of 2-CP degradation was not significantly enhanced by subculturing. In 2-CP-degrading cultures, nitrate was consumed stoichiometrically and concomitantly during 2-CP degradation, and a dechlorination intermediate was not detected, suggesting that 2-CP degradation was coupled with nitrate reduction. A 2-CP-degrading enrichment culture degraded 2-CP in the presence of nitrate, but did not in the absence of nitrate or the presence of sulfate. This suggests that the enrichment culture strictly requires nitrate for degradation of 2-CP. The apparent specific growth rate of the 2-CP degrading species was 0.0139 d(-1). Thus the apparent doubling time of the 2-CP-degrading population in the enrichment culture was greater than 50 d, which may explain difficulty in enrichment and isolation of micro-organisms responsible for CP degradation under denitrifying conditions.

Rate determination and distribution of anammox activity in activated sludge treating swine wastewater.

This paper presents a quantitative investigation and analysis of anammox activity in sludge taken from biological swine wastewater treatment plants. An incubation experiment using a (15)N tracer technique showed anammox activity in sludge taken from 6 out of 13 plants with the rate ranging from 0.0036 micromol-N(2)/g-VSS/h to 3.1 micromol-N(2)/g-VSS/h, and in a biofilm with the highest activity at 25.8 micromol-N(2)/g-VSS/h. It is notable that 9 out of 11 sludges in which the pH was maintained between 6.6 and 8.1 retained anammox activity, while those with either a lower or higher pH did not. Moreover, anammox-positive sludge had a significantly higher concentration of NO(2)(-)-N plus NO(3)(-)-N than did anammox-negative sludge. A significant difference was not observed between anammox-positive and -negative sludge regarding BOD/NH(4)(+)-N in the influent, DO concentration in aeration tanks, and the concentrations of NH(4)(+)-N, free nitric acid, and free ammonia.

N2O Emission from Degraded Soybean Nodules Depends on Denitrification by Bradyrhizobium japonicum and Other Microbes in the Rhizosphere

A model system developed to produce N2O emissions from degrading soybean nodules in the laboratory was used to clarify the mechanism of N2O emission from soybean fields. Soybean plants inoculated with nosZ-defective strains of Bradyrhizobium japonicum USDA110 (ΔnosZ, lacking N2O reductase) were grown in aseptic jars. After 30 days, shoot decapitation (D, to promote nodule degradation), soil addition (S, to supply soil microbes), or both (DS) were applied. N2O was emitted only with DS treatment. Thus, both soil microbes and nodule degradation are required for the emission of N2O from the soybean rhizosphere. The N2O flux peaked 15 days after DS treatment. Nitrate addition markedly enhanced N2O emission. A 15N tracer experiment indicated that N2O was derived from N fixed in the nodules. To evaluate the contribution of bradyrhizobia, N2O emission was compared between a nirK mutant (ΔnirKΔnosZ, lacking nitrite reductase) and ΔnosZ. The N2O flux from the ΔnirKΔnosZ rhizosphere was significantly lower than that from ΔnosZ, but was still 40% to 60% of that of ΔnosZ, suggesting that N2O emission is due to both B. japonicum and other soil microorganisms. Only nosZ-competent B. japonicum (nosZ+ strain) could take up N2O. Therefore, during nodule degradation, both B. japonicum and other soil microorganisms release N2O from nodule N via their denitrification processes (N2O source), whereas nosZ-competent B. japonicum exclusively takes up N2O (N2O sink). Net N2O flux from soybean rhizosphere is likely determined by the balance of N2O source and sink.