Yoshikuni Urushigawa

Parallel characterization of anaerobic toluene- and ethylbenzene-degrading microbial consortia by PCR-denaturing gradient gel electrophoresis, RNA-DNA membrane hybridization, and DNA microarray technology

ABSTRACT A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.

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.

Organochlorine Pesticides in Water, Sediment and Fish from the Nile River and Manzala Lake in Egypt

Abstract To assess the risks of organochlorine pesticides discharged into the hydrospheric environment of Egypt, river and lake water, drinking water, suspended solids, sediments and fish were collected during 1993—1994 from the Nile River and Manzala Lake in Egypt and were transported to Japan for chemical analysis. Among different organochlorine pesticides analyzed, p,p′-DDE was the most predominant in fish (7.6 to 67 ng/g wet wt.), sediments (3.2 to 432 ng/g dry wt.) and suspended solids (5.3 tc 138 pg/L). However, in the dissolved phase of water samples HCH compounds predominated (α-HCH, 71 to 2,815 pg/L). Concentrations of organochlorine pesticides, except chlordane, were higher in Manzala Lake than in the River Nile. Concentrations of organochlorine pesticides in fish corresponded with those in sediments from each location. Comparison of organochlorine concentrations in Nile River water with those reported in earlier studies suggested a decrease in concentrations during the last decade. However, concentrations of p,p′-DDE has increased in fish. It appears that the release of this metabolite from contaminated sediment is the major source of p,p′-DDE in fish during recent years.

Comparative description of microbial community structure in surface sediments of eutrophic bays

Comparisons of microbial community structure in sediments of eutrophic areas such as Hiroshima Bay, Etauchi Bay, Osaka Bay and Kojima Lake in Japan were undertaken by analysing the phospholipid fatty acid (PLFA) composition in sediments. In addition, the PLFA composition of aerobic bacteria, anaerobic bacteria and sulphate reducing bacteria (SRB) were analysed to assess their contribution to sedimentary lipid. The monounsaturated PLFA and branched PLFA were predominantly present in aerobic bacteria, and anaerobic bacteria and SRB, respectively. The PLFA composition of sediments showed significant regional differences among the four study areas, indicating the significant difference in microbial community structure. The aerobic prokaryotes and eukaryotes were predominantly present in all the study areas except in Osaka Bay where the gram-positive bacteria and anaerobic bacteria were predominant. The characteristic PLFA of aerobic prokaryotes and eukaryotes were significantly higher in Kojima Lake than other areas whereas the biomarker PLFA of gram-positive bacteria were predominantly present in Osaka Bay than that in other areas. These significant variations in microbial community structure can be attributed to the differences in environmental perturbation.

Major substrates for microbial sulfate reduction in the sediments of Ise Bay, Japan

To clarify the anaerobic microbial interactions in the process of carbon mineralization in marine eutrophic environments, the microbial sulfate reduction and methane production rates were examined in coastal marine sediments of Ise Bay, Japan, in autumn 1990. Sulfate reduction rates (51–210 nmol ml−1 day−1 at 24°C) were much higher than the methane production ones (<1.78 nmol ml−1 day−1) in the surface sediments (top 2 cm) at the six stations surveyed (water depth: 10.7–23.3 m). Substrates for sulfate-reducing bacteria (SRB) were estimated after the addition of a specific inhibitor for SRB (20 mmol l−1 molybdate) into the sediment slurry, from the substrate accumulation rates. In the presence of the inhibitor, sulfate reduction was completely stopped and volatile fatty acids (mainly acetate) were accumulated, although hydrogen was not. Methane production occurred markedly accompanied by consumption of the accumulated acetate from the third day after the addition of molybdate. The maximum rate of methane production was 1.2–1.9 μmol ml−1 day−1, which was similar to those in highly polluted freshwater sediments such as the Tama River, Tokyo, Japan. These results show that acetate is a common major substrate for sulfate reduction and methane production, and SRB competitively inhibit potential acetoclastic methanogenesis in coastal sediments. Methanogens may potentially inhabit the sediments at low levels of population density and activity.

Degradation of tri-n-buytltin in Ise Bay sediment

The vertical distribution patterns of the transformation products of tri-n-butyltin (TBT) in sediment cores collected from 6 sites in Ise Bay, Japan indicated that TBT was transformed by two pathways: methylation and debutylation. Anaerobic incubation of sediment spiked with TBT showed that the methylation was mainly supported by sulfate reducing activity and debutylation was mainly supported by nitrate reducing activity. These results showed that contribution of the two transformation reactions of TBT varied according to the dominant microbial activities in the sediment. The two types of vertical distribution of butyltins observed in the sediment cores appear to be the result of different dominant microbial activity in the sediment.

The distribution of chlorobenzenes in the bottom sediments of Ise bay

Abstract The concentration of dichloro- to hexachlorobenzene in the surficial bottom sediments in Ise Bay were determined as a preliminary study of the behavior of these compounds in this coastal environment. The sediment samples were collected from 10 stations on the west coast and in Yokkaichi Port in March 1987 and from 44 stations all over the bay in October 1987. 1, 2- and 1, 3-Dichlorobenzene and 1, 2, 3- and 1, 2, 4-trichlorobenzene levels were highest in the vicinity of Yokkaichi Port and Tsu-Matsuzaka Port. Higher levels of these compounds were also found in the middle of the bay. The concentrations of tetrachlorobenzene isomers were generally low but they were also higher in the vicinity of the two ports. 1, 3, 5-Trichlorobenzene was higher only in the vicinity of Yokkaichi. The levels of these compounds were not higher around Nagoya City, the largest city in the bay. The stations with higher dichloro- and trichlorobenzenes concentrations in the middle of the bay covered the area where the sediment particle size was small. This was also the area where downward water flow was estimated to exit. These findings indicated that these congeners were adsorbed by suspended particulates and transported by water flow. The distribution patterns, therefore, could be explained by the following effects: the distance from the source, the horizontal transport caused by the river inflows and the sedimentation caused by downward water flow. Pentachlorobenzene concentration was very low and its distribution was not clear. Hexachlorobenzene had quite a different distribution pattern from other congeners, indicating that its sources were different.

Biodegradation pathway of o-cresol by heterogeneous culture Phenol acclimated activated sludge

Abstract The biodegradation process of o -cresol by heterogeneous culture is presented in this paper. Metabolic intermediates of o -cresol by phenol acclimated activated sludge were analyzed by GC-MS. o -Cresol was first transformed to three dihydroxytoluenes; 3-methylcatechol, 4-methylresorcinol and methylhydroquinone. Three dihydroxytoluene biodegradation experiments with 3-methylcatechol, 4-methylresorcinol and methylhydroquinone revealed that 3-methylcatechol was the main route, and 3-methylcatechol was further degraded through at least two meta cleavage pathways. Various hydroxylated toluenes, namely trihydroxytoluenes and tetrahydroxytoluene were also found among the metabolites from 3-methylcatechol, 4-methylresorcinol and methylhydroquinone. These results indicated that various metabolism with main and side pathways coexisted in the biodegradation process by the heterogeneous culture.