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ABSTRACT: A lab-scale expanded granular sludge bed (EGSB) reactor was seeded with granular sludge and operated to investigate the influence of temperature decrease on both process performance and the microbial community structure of the granular sludge. Synthetic wastewater containing sucrose and volatile fatty acids was used as feed. The EGSB reactor was brought online at a starting temperature of 15 degrees C and was reduced stepwise to a final temperature of 5 degrees C. The reactor exhibited sufficient COD removal efficiency between 10 degrees C and 15 degrees C. However at 5 degrees C serious deterioration of process performance was observed. The methane-producing activity of the retained sludge increased when it was compared to the activity of the seed sludge (day 0) during 10 degrees C to 15 degrees C operation. When hydrogen fed, sludge showed much higher methanogenic activity as compared with seed sludge activity at test temperatures of 15 degrees C and 20 degrees C on day 196 of reactor operation. At this time, proliferation of the genus Methanospirillum in the retained sludge was observed and a decrease in Methanobacterium species was also measured. Throughout the experiment, the genus Methanosaeta was detected in abundance and the community structure of the Domain Bacteria was stably maintained. The sugar-degrading acid-forming bacteria, Lactococcus and Anaerovibrio were detected in the retained sludge throughout the experiment as well and the propionate-degrading acetogen Syntrophobacter fumaroxidans was also detected, although its population size decreased at 5 degrees C.
Journal of Environmental Science and Health Part A 01/2009; 43(14):1650-6. · 1.19 Impact Factor
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ABSTRACT: Four obligately anaerobic, thermophilic, sulfate-reducing bacterial strains, designated TGE-P1(T), TDV(T), TGL-LS1 and TSL-P1, were isolated from thermophilic (operated at 55 degrees C) methanogenic sludges from waste and wastewater treatment. The optimum temperature for growth of all the strains was in the range 55-60 degrees C. The four strains grew by reduction of sulfate with a limited range of electron donors, such as hydrogen, formate, pyruvate and lactate. In co-culture with the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus DeltaH(T), strains TGE-P1(T), TGL-LS1 and TSL-P1 were able to utilize lactate syntrophically for growth. The DNA G+C contents of all the strains were in the range 34-35 mol%. The major cellular fatty acids of the strains were iso-C(17 : 0), iso-C(16 : 0), C(16 : 0) and anteiso-C(15 : 0). Phylogenetic analyses based on 16S rRNA gene sequences revealed that the strains belong to the Thermodesulfovibrio clade of the phylum 'Nitrospirae'. On the basis of their physiological, chemotaxonomic and genetic properties, strains TGL-LS1 (=JCM 13214) and TSL-P1 (=JCM 13215) were classified as strains of Thermodesulfovibrio islandicus. Two novel species of the genus Thermodesulfovibrio are proposed to accommodate the other two isolates: Thermodesulfovibrio aggregans sp. nov. (type strain TGE-P1(T) =JCM 13213(T) =DSM 17283(T)) and Thermodesulfovibrio thiophilus sp. nov. (type strain TDV(T) =JCM 13216(T) =DSM 17215(T)). To examine the ecological aspects of Thermodesulfovibrio-type cells in the sludge from which the strains were originally isolated, an oligonucleotide probe targeting 16S rRNA of all Thermodesulfovibrio species was designed and applied to thin sections of thermophilic sludge granules. Fluorescence in situ hybridization using the probe revealed rod- or vibrio-shaped cells as a significant population within the sludge, indicating their important role in the original ecosystem.
International journal of systematic and evolutionary microbiology 12/2008; 58(Pt 11):2541-8. · 2.27 Impact Factor
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ABSTRACT: Butyrate-degrading bacteria in four methanogenic sludges were studied by RNA-based stable isotope probing. Bacterial populations in the (13)C-labeled rRNA fractions were distinct from unlabeled fractions, and Syntrophaceae species, Tepidanaerobacter sp., and Clostridium spp. dominated. These results suggest that diverse microbes were active in butyrate degradation under methanogenic conditions.
Applied and environmental microbiology 07/2008; 74(11):3610-4. · 3.69 Impact Factor
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ABSTRACT: Inactivation of indigenous indicator micro-organisms such as faecal coliforms, coliphages, and faecal streptococci was investigated in a full-scale biogas plant that mainly digested cow manure. The biogas plant consisted principally of a feed reservoir, fermentation tank (37 degrees C), heat-inactivation process (70 degrees C), and five reservoirs for the heat-inactivated, digested manure that was used by a local livestock farmer as liquid fertilizer. Although all the indicators tended to exhibit stepwise decreases with each stage of treatment, coliphages were found to be more capable of surviving than faecal coliforms and faecal streptococci under mesophilic anaerobic conditions as well as high temperature conditions (heat-inactivation at 70 degrees C). Liquid fertilizer produced at the biogas plant had faecal coliform densities less than the stipulations of the US EPA 40 CFR 503 Class A limits. Heat-inactivation tests indicated that although coliphages exhibited more tolerance than other bacterial indicators between 37 and 70 degrees C, they were more sensitive to continuous temperature increase than faecal coliforms and faecal streptococci.
Waste Management & Research 06/2008; 26(3):256-60. · 1.19 Impact Factor
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ABSTRACT: A novel mesophilic, hydrogenotrophic methanogen, strain SANAET, was isolated from an anaerobic, propionate-degrading enrichment culture, which was originally established from rice paddy soil. The cells were non-motile, Gram-negative and rod-shaped (1.8-2.4 microm long by 0.3-0.6 microm wide). Growth of strain SANAET was observed at 25-40 degrees C, with an optimum temperature range for growth of 35-37 degrees C. The pH range for growth was 6.5-7.8, with an optimum at pH 7.0. The salinity range for growth was 0-1 g NaCl l(-1) (0-17 mM). The isolate was able to utilize H2/CO2 and formate for growth and methane production. The G+C content of the genomic DNA was 56.6 mol%. Based on comparative 16S rRNA gene sequence analysis, strain SANAET was affiliated with a clone lineage of the Archaea, Rice Cluster I (RC-I), placing it between the orders Methanosarcinales and Methanomicrobiales within the class 'Methanomicrobia'. 16S rRNA gene sequence similarities between strain SANAET and members of Methanosarcinales were in the range 80.0-82.8 %, and those between the strain and members of Methanomicrobiales ranged from 77.5 to 82.4 %. In addition to 16S rRNA gene analysis, sequence analysis of the mcrA gene (encoding the alpha subunit of methyl-coenzyme M reductase, a key enzyme in the methane production pathway) also showed that strain SANAET was affiliated with the RC-I lineage. Here, we propose the name Methanocella paludicola gen. nov., sp. nov. for the isolate, the first of the RC-I lineage. The type strain is SANAET (=JCM 13418T=NBRC 101707T=DSM 17711T). In addition, we also propose the status of order for the RC-I lineage, for which we propose the name Methanocellales ord. nov.
International journal of systematic and evolutionary microbiology 05/2008; 58(Pt 4):929-36. · 2.27 Impact Factor
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ABSTRACT: Phenol degradation under methanogenic conditions has long been studied, but the anaerobes responsible for the degradation reaction are still largely unknown. An anaerobe, designated strain UI(T), was isolated in a pure syntrophic culture. This isolate is the first tangible, obligately anaerobic, syntrophic substrate-degrading organism capable of oxidizing phenol in association with an H(2)-scavenging methanogen partner. Besides phenol, it could metabolize p-cresol, 4-hydroxybenzoate, isophthalate, and benzoate. During the degradation of phenol, a small amount of 4-hydroxybenzoate (a maximum of 4 microM) and benzoate (a maximum of 11 microM) were formed as transient intermediates. When 4-hydroxybenzoate was used as the substrate, phenol (maximum, 20 microM) and benzoate (maximum, 92 microM) were detected as intermediates, which were then further degraded to acetate and methane by the coculture. No substrates were found to support the fermentative growth of strain UI(T) in pure culture, although 88 different substrates were tested for growth. 16S rRNA gene sequence analysis indicated that strain UI(T) belongs to an uncultured clone cluster (group TA) at the family (or order) level in the class Deltaproteobacteria. Syntrophorhabdus aromaticivorans gen. nov., sp. nov., is proposed for strain UI(T), and the novel family Syntrophorhabdaceae fam. nov. is described. Peripheral 16S rRNA gene sequences in the databases indicated that the proposed new family Syntrophorhabdaceae is largely represented by abundant bacteria within anaerobic ecosystems mainly decomposing aromatic compounds.
Applied and environmental microbiology 05/2008; 74(7):2051-8. · 3.69 Impact Factor
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ABSTRACT: A novel methane-producing archaeon, strain NOBI-1(T) was isolated from an anaerobic, propionate-degradation enrichment culture, which was originally obtained from a mesophilic methanogenic sludge digesting municipal sewage sludge. Cells were non-motile, rod-shaped, 0.7-1.0 microm by 2.0 microm, and formed multicellular filaments longer than 8 microm. Growth was observed between 35 and 55 degrees C (optimum 50 degrees C) and pH 6.7 and 8.0 (optimum pH 7.0). The G+C content of the genomic DNA was 56.3 mol%. The strain utilized H(2) and formate for growth and methane production. Based on comparative sequence analyses of the 16S rRNA gene and mcrA gene (encoding the alpha subunit of methyl-coenzyme M reductase, a key enzyme in the methane-production pathway), strain NOBI-1(T) was affiliated with the order Methanomicrobiales, but it was significantly distant from any other known species within the order. The most closely related species based on 16S rRNA and mcrA gene sequence similarity were respectively 'Candidatus Methanoregula boonei' (93.7% 16S rRNA gene sequence similarity) and Methanocorpusculum parvum (74.2% deduced McrA amino acid sequence similarity to the type strain). These phenotypic and genetic properties justified the creation of a novel species of a new genus for the strain, for which we propose the name Methanolinea tarda gen. nov., sp. nov. The type strain of Methanolinea tarda is strain NOBI-1(T) (=DSM 16494(T) =JCM 12467(T) =NBRC 102358(T)).
International journal of systematic and evolutionary microbiology 02/2008; 58(Pt 1):294-301. · 2.27 Impact Factor
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ABSTRACT: Several enzymatic permeabilization protocols (utilizing lysozyme, proteinase K, achromopeptidase, or recombinant pseudomurein endopeptidase [PeiW]) were evaluated for application of in situ hybridization with horseradish peroxidase-labeled oligonucleotide probes and catalyzed reporter deposition (CARD-FISH) to methanogens. In this study, twelve methanogens were selected that have typical cell surface structures: pseudomurein, surface layer, methanochondroitin and sheath. Among the treatments tested, PeiW treatment was observed to be the most effective one, although methanogens having a sheath were stained heterogeneously and methanogens having methanochondroitin were not permeabilized. On the other hand, lysozyme, proteinase K, and achromopeptidase treatments were ineffective or caused cell-lysis, resulting in weak or no signals. Applicability of PeiW treatment was further evaluated using an anaerobic granular sludge sample. The detection rate of Archaea by CARD-FISH increased remarkably after the treatment. Based on the results obtained in this study, we propose PeiW treatment as a novel permeabilization method for CARD-FISH application to methanogens.
Journal of Microbiological Methods 02/2008; 72(1):54-9. · 2.09 Impact Factor
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ABSTRACT: We previously reported that partial nitrification in the down-flow hanging sponge (DHS) system was satisfactorily accomplished under oxygen-limited conditions [Chuang et al., Water Res., 41, 295-302 (2007)]. In this study, we investigated the microbes that are responsible for the partial nitrification in this unique system by 16S rRNA- and amoA-based cloning analyses and fluorescence in situ hybridization. Microbes related to Nitrosomonas species were found to be chiefly responsible for catalyzing the partial nitrification. Microbes affiliated with the uncultivated phyla OP10 and Bacteroidetes were also numerous in the DHS, but their ecological niches are still unknown.
Journal of Bioscience and Bioengineering 01/2008; 104(6):525-8. · 1.79 Impact Factor
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ABSTRACT: Thermophilic (strain GOMI-1(T)) and mesophilic (strain KOME-1(T)) strains were isolated from two different cultures of propionate-degrading consortia obtained from thermophilic digester sludge and rice paddy soil, respectively. The two strains were non-spore-forming, non-motile and Gram-negative. Both strains were obligately anaerobic micro-organisms, showing multicellular filamentous morphotypes more than 100 mum in length. The cell width for strain GOMI-1(T) was 0.2-0.4 mum and that of strain KOME-1(T) was 0.4-0.6 mum. Strain GOMI-1(T) could grow at 45-65 degrees C with a pH range of 6.0-7.5 (optimum growth at 55 degrees C, pH 7.0). The temperature range for growth of strain KOME-1(T) was 30-40 degrees C and the pH range was pH 5.0-8.5 (optimum growth around 37 degrees C, pH 7.0). Yeast extract was required for growth of both strains. Strain GOMI-1(T) was able to grow with a number of carbohydrates in the presence of yeast extract. In yeast extract-containing medium, strain KOME-1(T) could utilize proteins and a limited range of sugars for growth. The G+C contents of the DNA of strains GOMI-1(T) and KOME-1(T) were respectively 54.7 and 57.6 mol%. Major fatty acids of strain GOMI-1(T) were C(16 : 0), C(14 : 0) and iso-C(15 : 0), whereas those of strain KOME-1(T) were iso-C(15 : 0), anteiso-C(15 : 0) and C(14 : 0). Based on comparative analysis of 16S rRNA gene sequences of strains GOMI-1(T) and KOME-1(T), the strains were placed in different phylogenetic positions in the class Anaerolineae of the bacterial phylum Chloroflexi. Their phenotypic and genetic traits strongly supported the conclusion that the strains should be described as two independent taxa in the class Anaerolineae. Hence, we propose the names Bellilinea caldifistulae gen. nov., sp. nov., and Longilinea arvoryzae gen. nov., sp. nov., for strains GOMI-1(T) and KOME-1(T). The type strains of Bellilinea caldifistulae and Longilinea arvoryzae are respectively GOMI-1(T) (=JCM 13669(T) =DSM 17877(T)) and KOME-1(T) (=JCM 13670(T) =KTCC 5380(T)).
International journal of systematic and evolutionary microbiology 11/2007; 57(Pt 10):2299-306. · 2.27 Impact Factor
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ABSTRACT: A mesophilic, syntrophic, fatty-acid-oxidizing anaerobic strain, designated MPA(T), was isolated from granular sludge in a mesophilic upflow anaerobic sludge blanket reactor used to treat palm oil mill effluent. Cells were slightly curved, non-motile rods. Spore formation was not observed. The optimal temperature for growth was around 37 degrees C and optimal pH for growth was 7.0. Strain MPA(T) was able to grow on crotonate or pentenoate plus butyrate in pure culture. In co-culture with the hydrogenotrophic methanogen Methanospirillum hungatei, strain MPA(T) was able to oxidize straight-chain saturated fatty acids with carbon chain lengths of C4-C18. The strain was unable to utilize sulfate, sulfite, thiosulfate, nitrate, fumarate, iron(III) or DMSO as an electron acceptor. The G+C content of the DNA was 45.0 mol%. Based on comparative 16S rRNA gene sequence analysis, strain MPA(T) was found to be a member of the genus Syntrophomonas and was most closely related to the type strains of Syntrophomonas curvata and Syntrophomonas sapovorans (sequence similarities of 94 %). Genetic and phenotypic characteristics demonstrated that strain MPA(T) represents a novel species, for which the name Syntrophomonas palmitatica sp. nov. is proposed. The type strain is MPA(T) (=JCM 14374(T)=NBRC 102128(T)=DSM 18709(T)).
International journal of systematic and evolutionary microbiology 10/2007; 57(Pt 9):2137-42. · 2.27 Impact Factor
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ABSTRACT: This paper evaluates the total annual cost including capital and operation and maintenance (O&M) costs for the up-flow anaerobic sludge blanket (UASB) and waste stabilization pond (WSP) systems operated in India. It also compares UASB and WSP systems with the activated sludge process (ASP) and biological aerated filter (BAF) systems in terms of total annual cost and chemical oxygen demand (COD) removal cost by assuming various annual interest rates and land prices. It was found that the relationship between capital and O&M costs per unit size of a UASB or WSP system and its treatment capacity can be established by a first-order equation. The relation between the cost of organic removal and capital or O&M cost for various sewage treatment systems at various annual interest rates revealed that, for the Indian context, UASB could be the most suitable option in terms of expenses and treatment efficiency.
Journal of Environmental Management 10/2007; 84(4):447-60. · 3.24 Impact Factor
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ABSTRACT: Long-chain fatty acid (LCFA) degradation is a key step in methanogenic treatment of wastes/wastewaters containing high concentrations of lipids. However, despite the importance of LCFA-degrading bacteria, their natural diversity is little explored due to the limited availability of isolate information and the lack of appropriate molecular markers. We therefore investigated these microbes by using RNA-based stable isotope probing. We incubated four methanogenic sludges (mesophilic sludges MP and MBF and thermophilic sludges TP and JET) with (13)C-labeled palmitate (1 mM) as a substrate. After 8 to 19 days of incubation, we could detect (13)C-labeled bacterial rRNA. A density-resolved terminal restriction fragment length polymorphism fingerprinting analysis showed distinct bacterial populations in (13)C-labeled and unlabeled rRNA fractions. The bacterial populations in the (13)C-labeled rRNA fractions were identified by cloning and sequencing of reverse-transcribed 16S rRNA. Diverse phylogenetic bacterial sequences were retrieved, including those of members of the family Syntrophaceae, clone cluster MST belonging to the class Deltaproteobacteria, Clostridium clusters III and IV, phylum Bacteroidetes, phylum Spirochaetes, and family Syntrophomonadaceae. Although Syntrophomonadaceae species are considered to be the major fatty acid-degrading syntrophic microorganisms under methanogenic conditions, they were detected in only two of the clone libraries. These results suggest that phylogenetically diverse bacterial groups were active in situ in the degradation of LCFA under methanogenic conditions.
Applied and Environmental Microbiology 08/2007; 73(13):4119-27. · 3.83 Impact Factor
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ABSTRACT: Despite the fact that rice paddy fields (RPFs) are contributing 10 to 25% of global methane emissions, the organisms responsible for methane production in RPFs have remained uncultivated and thus uncharacterized. Here we report the isolation of a methanogen (strain SANAE) belonging to an abundant and ubiquitous group of methanogens called rice cluster I (RC-I) previously identified as an ecologically important microbial component via culture-independent analyses. To enrich the RC-I methanogens from rice paddy samples, we attempted to mimic the in situ conditions of RC-I on the basis of the idea that methanogens in such ecosystems should thrive by receiving low concentrations of substrate (H(2)) continuously provided by heterotrophic H(2)-producing bacteria. For this purpose, we developed a coculture method using an indirect substrate (propionate) in defined medium and a propionate-oxidizing, H(2)-producing syntroph, Syntrophobacter fumaroxidans, as the H(2) supplier. By doing so, we significantly enriched the RC-I methanogens and eventually obtained a methanogen within the RC-I group in pure culture. This is the first report on the isolation of a methanogen within RC-I.
Applied and Environmental Microbiology 08/2007; 73(13):4326-31. · 3.83 Impact Factor
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ABSTRACT: An anaerobic, mesophilic, syntrophic, propionate-oxidizing bacterium, strain MGP(T), was isolated as a defined co-culture with Methanospirillum hungatei from the methanogenic sludge of a mesophilic upflow anaerobic sludge blanket (UASB) reactor. The strain grew in the presence of propionate, but only in co-culture with methanogens, suggesting that it is an obligately syntrophic bacterium. The optimum temperature for growth was 37 degrees C, and the optimum pH was between 6.5 and 7.2. Based on comparative 16S rRNA gene sequence analysis, strain MGP(T) was affiliated with subcluster Ih of 'Desulfotomaculum cluster I', in which it was found to be moderately related to known species of the genera Pelotomaculum and Cryptanaerobacter. Similar to known species of the genus Pelotomaculum, strain MGP(T) could degrade propionate in syntrophy, but had no ability to reduce sulfate, sulfite and thiosulfate. Further phenotypic and genetic studies supported the affiliation of the strain as a novel species in this genus, for which the name Pelotomaculum propionicicum sp. nov. is proposed. The type strain is MGP(T) (=DSM 15578(T)=JCM 11929(T)). The strain has been deposited in the DSM and JCM culture collections as a defined co-culture with Methanospirillum hungatei.
International journal of systematic and evolutionary microbiology 08/2007; 57(Pt 7):1487-92. · 2.27 Impact Factor
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ABSTRACT: A fatal bulking phenomenon was found to occur occasionally in the methanogenic granular sludge of a mesophilic (35-40 degrees C), full-scale upflow anaerobic sludge blanket (UASB) reactor treating organic wastewater discharged from a sugar manufacturing factory. A vast number of filamentous cells were observed in the bulking sludge that were morphologically distinct from the previously recognized anaerobic bulking agent Anaerolinea thermophila. 16S rRNA gene-based analyses of the microbial populations in the bulking sludge revealed that the dominant filamentous organisms were members of proposed candidate bacterial phylum, KSB3. Fluorescence in situ hybridization (FISH) analysis of the healthy sludge granules showed that the KSB3 filaments were the dominant granule surface population suggesting that they are fundamental constituents of the sludge granules and that they occasionally overgrow in the reactor, possibly triggering the filamentous bulking. We surveyed 10 additional mesophilic and thermophilic anaerobic sludges for the presence and diversity of KSB3 populations. Bacteria closely related to the characterized KSB3 filaments were present in two types of mesophilically grown UASB sludge granules treating actual wastewater discharged from sugar-processing industries.
The ISME Journal 08/2007; 1(3):246-55. · 7.38 Impact Factor
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ABSTRACT: We investigated long-chain fatty acid (LCFA)-degrading anaerobic microbes by enrichment, isolation, and RNA-based stable isotope probing (SIP). Primary enrichment cultures were made with each of four LCFA substrates (palmitate, stearate, oleate, or linoleate, as the sole energy source) at 55 degrees C or 37 degrees C with two sources of anaerobic granular sludge as the inoculum. After several transfers, we obtained seven stable enrichment cultures in which LCFAs were converted to methane. The bacterial populations in these cultures were then subjected to 16S rRNA gene-based cloning, in situ hybridization, and RNA-SIP. In five of seven enrichment cultures, the predominant bacteria were affiliated with the family Syntrophomonadaceae. The other two enrichment cultures contained different bacterial populations in which the majority of members belonged to the phylum Firmicutes and the class Deltaproteobacteria. After several attempts to isolate these dominant bacteria, strain MPA, belonging to the family Syntrophomonadaceae, and strain TOL, affiliated with the phylum Firmicutes, were successfully isolated. Strain MPA converts palmitate to acetate and methane in syntrophic association with Methanospirillum hungatei. Even though strain TOL assimilated [(13)C]palmitate in the original enrichment culture, strain TOL has not shown the ability to degrade LCFAs after isolation. These results suggest that microbes involved in the degradation of LCFAs under methanogenic conditions might not belong only to the family Syntrophomonadaceae, as most anaerobic LCFA-degrading microbes do, but may also be found in phylogenetically diverse bacterial groups.
Applied and Environmental Microbiology 03/2007; 73(4):1332-40. · 3.83 Impact Factor
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ABSTRACT: Combining the processes of partial nitrification and anaerobic ammonium oxidation (ANAMMOX) is an attractive wastewater-treatment technology for nitrogen removal. In this study we investigated partial nitrification by implementing a closed down-flow hanging sponge (DHS) reactor operated at controlled oxygen concentrations. Basic concept of DHS process is similar to that of trickling filter, in which oxygen concentration can be easily manipulated by controlling airflow to the reactor. The closed reactor was fed with artificial wastewater containing NH(4)Cl and operated with an HRT of 1.5h at 30 degrees C. Oxygen inside the reactor was maintained below 3% (1.2mgDO x L(-1)) (DO-dissolved oxygen) except during the startup periods. Five months of continuous operation showed that there was a strong relationship between oxygen concentration and nitrite production. The ratio of nitrite produced relative to ammonium oxidized increased by decreasing oxygen concentration. Partial nitrification was satisfactorily accomplished under oxygen limitation at around 0.5% in the gas phase (0.2mgDOL(-1)). The system showed a high ammonium-removal rate, at a maximum of 1.46kg NH(4)(+)-Nm(-3)day(-1), even at limited oxygen concentration. We also found that oxygen concentration played an important role in the production of nitrous oxide, which increased with decreasing oxygen concentration.
Water Research 02/2007; 41(2):295-302. · 4.86 Impact Factor
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ABSTRACT: Two-pass tyramide signal amplification-fluorescence in situ hybridization (two-pass TSA-FISH) with a horseradish peroxidase (HRP)-labeled oligonucleotide probe was applied to detect prokaryotic mRNA. In this study, mRNA of a key enzyme for methanogenesis, methyl coenzyme M reductase (mcr), in Methanococcus vannielii was targeted. Applicability of mRNA-targeted probes to in situ hybridization was verified by Clone-FISH. It was observed that sensitivity of two-pass TSA-FISH was significantly higher than that of TSA-FISH, which was further increased by the addition of dextran sulphate in TSA working solution. Signals from two-pass TSA-FISH were more reliable compared to the weak, spotty signals yielded by TSA-FISH.
Journal of Microbiological Methods 10/2006; 66(3):521-8. · 2.09 Impact Factor
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ABSTRACT: Low signal intensity due to poor probe hybridization efficiency is one of the major drawbacks of rRNA-targeted in situ hybridization. There are two major factors affecting the hybridization efficiency: probe accessibility and affinity to the targeted rRNA molecules. In this study, we demonstrate remarkable improvement in in situ hybridization efficiency by applying locked-nucleic-acid (LNA)-incorporated oligodeoxynucleotide probes (LNA/DNA probes) without compromising specificity. Fluorescently labeled LNA/DNA probes with two to four LNA substitutions exhibited strong fluorescence intensities equal to or greater than that of probe Eub338, although these probes did not show bright signals when they were synthesized as DNA probes; for example, the fluorescence intensity of probe Eco468 increased by 22-fold after three LNA bases were substituted for DNA bases. Dissociation profiles of the probes revealed that the dissociation temperature was directly related to the number of LNA substitutions and the fluorescence intensity. These results suggest that the introduction of LNA residues in DNA probes will be a useful approach for effectively enhancing probe hybridization efficiency.
Applied and Environmental Microbiology 09/2006; 72(8):5311-7. · 3.83 Impact Factor
