[Show abstract][Hide abstract] ABSTRACT: In the methane production from waste activated sludge (WAS), complex bacterial interactions in WAS have been known as a major contribution to methane production. Therefore, the influence of bacterial community changes toward methane production from WAS was investigated by an application of antibiotics as a simple means for it. In this study, azithromycin (Azm) as an antibiotic was mainly used to observe the effect on microbial changes that influence methane production from WAS. The results showed that at the end of fermentation, Azm enhanced methane production about twofold compared to control. Azm fostered the growth of acid-producing bacterial communities, which synthesized more precursors for methane formation. DGGE result showed that the hydrolysis as well as acetogenesis stage was improved by the dominant of B1, B2 and B3 strains, which are Clostridium species. In the presence of Azm, the total population of archaeal group was increased, resulting in higher methane productivity achievement.
[Show abstract][Hide abstract] ABSTRACT: Since the actual role of Escherichia coli hydrogenases on fermentation from glycerol has not been clear, we evaluated the effect of inactivation of each E. coli hydrogenase on cell growth, hydrogen production, organic acids production, and ethanol production. Inactivation of hydrogenase 2 and hydrogenase 3 reduced cell growth, hydrogen and succinate production as well as glycerol utilization while acetate increased. Inactivation of hydrogenase 2 in minimal medium at pH 7.5 impaired hydrogen production, but no significant effect occurred at pH 6.5 or in complex medium. Inactivation of hydrogenase 3 impaired hydrogen production in minimal and rich medium, pH 6.5 and pH 7.5 accumulating formate in all conditions. Therefore during fermentation from glycerol, hydrogenase 3 is the main hydrogenase with hydrogen synthesis activity through the formate hydrogen lyase complex. Hydrogenase 2 seems mainly required for optimum glycerol metabolism rather than hydrogen synthesis. There were no significant impacts by inactivating hydrogenase 1 and hydrogenase 4.
International Journal of Hydrogen Energy 04/2013; 38(10):3905-3912. DOI:10.1016/j.ijhydene.2013.01.031 · 3.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We developed a detection method that uses quantitative real-time PCR (qPCR) and the TaqMan system to easily and rapidly assess the population of aniline-degrading bacteria in activated sludge prior to conducting a biodegradability test on a chemical compound. A primer and probe set for qPCR was designed by a multiple alignment of conserved amino acid sequences encoding the large (α) subunit of aniline dioxygenase. PCR amplification tests showed that the designed primer and probe set targeted aniline-degrading strains such as Acidovorax sp., Gordonia sp., Rhodococcus sp., and Pseudomonas putida, thereby suggesting that the developed method can detect a wide variety of aniline-degrading bacteria. There was a strong correlation between the relative copy number of the α-aniline dioxygenase gene in activated sludge obtained with the developed qPCR method and the number of aniline-degrading bacteria measured by the Most Probable Number method, which is the conventional method, and a good correlation with the lag time of the BOD curve for aniline degradation produced by the biodegradability test in activated sludge samples collected from eight different wastewater treatment plants in Japan. The developed method will be valuable for the rapid and accurate evaluation of the activity of inocula prior to conducting a ready biodegradability test.
[Show abstract][Hide abstract] ABSTRACT: Biohydrogen has gained importance as an alternative energy source, and advances in molecular biology and biotechnology have raised the quality and efficiency of biohydrogen production from various microorganisms and substrates. Here, Escherichia coli proteins YdjA and YhjY have been identified as essential in biohydrogen production from glucose. The mutations ydjA and yhjY reduced biohydrogen productivity compared to the parent strain from 40 to 4 and 29 mmol/mg protein, respectively. Through transcription analysis, it was determined that YdjA and YhjY are positive effectors of the FHL complex since their inactivation repressed fhlA. In addition, the FHL expression of the repressor gene, hycA, increased for the ydjA mutant, so YdjA reduces transcription of the HycA repressor. Hence, two new proteins have been identified that are important for biohydrogen production.
International Journal of Hydrogen Energy 12/2012; 37(23):17778-17787. DOI:10.1016/j.ijhydene.2012.08.115 · 3.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Synthesis-dependent strand-annealing (SDSA)-mediated homologous recombination replaces the sequence around a DNA double-strand break (DSB) with a copy of a homologous DNA template, while maintaining the original configuration of the flanking regions. In somatic cells at the 4n stage, Holliday-junction-mediated homologous recombination and nonhomologous end joining (NHEJ) cause crossovers (CO) between homologous chromosomes and deletions, respectively, resulting in loss of heterozygosity (LOH) upon cell division. However, the SDSA pathway prevents DSB-induced LOH. We developed a novel yeast DSB-repair assay with two discontinuous templates, set on different chromosomes, to determine the genetic requirements for somatic SDSA and precise end joining. At first we used our in vivo assay to verify that the Srs2 helicase promotes SDSA and prevents imprecise end joining. Genetic analyses indicated that a new DNA/RNA helicase gene, IRC20, is in the SDSA pathway involving SRS2. An irc20 knockout inhibited both SDSA and CO and suppressed the srs2 knockout-induced crossover enhancement, the mre11 knockout-induced inhibition of SDSA, CO, and NHEJ, and the mre11-induced hypersensitivities to DNA scissions. We propose that Irc20 and Mre11 functionally interact in the early steps of DSB repair and that Srs2 acts on the D-loops to lead to SDSA and to prevent crossoverv.
[Show abstract][Hide abstract] ABSTRACT: 2,4,6-Trinitrotoluene (TNT) is a highly energetic compound with the formula C6H2(NO2)3CH3 and best known as a useful explosive material with convenient handling properties which led to a relatively safe storage
due to the low sensitivity to impact shock and heat stimulation compared to other explosives and no metallic corrosion (Boileau
et al. 1987). Then, TNT has been used as an explosive for military and industrial purposes and the TNT production reached to its peak
during the two World Wars (Harter 1985). It is estimated that TNT is produced close to 1,000,000kg per year (Harter 1985). Therefore, a high concentration of TNT has been still found in soil and groundwater at former manufacturing sites (Fernando
et al. 1990; Hawari et al. 2000; Lewis et al. 2004; Maeda et al. 2006a). Presently soil and groundwater contamination by the explosive is a serious problem in the countries, mainly United States,
Germany and Canada (Pennington 1999; Fritsche et al. 2000). Sediments and soils beneath some industrial sites contain large amounts of nitro aromatics with up to 10g of TNT per kg
of soil being reported for some sites (Carpenter et al. 1978; Kaplan and Kaplan 1982; Fernando et al. 1990). The biodegradation studies have indicated that an explosive is highly recalcitrant for microbial biodegradation (Rieger
and Knackmuss 1995a, b). Among them, in particular, TNT is more recalcitrant than other nitroaromatic compounds (e.g. mono- and dinitrotoluenes),
because three nitro groups are located symmetrically on the aromatic ring which restrict the attack by classic dioxygenase
enzymes involved in the microbial metabolism of aromatic compounds. Hence, TNT has strong cytotoxicity and mutagenicity in
various living organisms (Won et al. 1976; Ahlborg et al. 1988; Tan et al. 1992; Berthe-Corti et al. 1998; Letzel et al. 2003; Padda et al. 2003; Saka 2004; Sun et al. 2005) and is listed as class C potential human carcinogen by the US Environmental Protection Agency. In addition, in TNT-exposed
humans, notable toxic manifestations have included aplastic anaemia, toxic hepatitis, cataracts, hepatomegaly, and liver cancer
(Sabbioni et al. 2007); therefore, it is significant to develop the bioremediation technology for TNT.
Microbial Degradation of Xenobiotics, 10/2011: pages 213-233;
[Show abstract][Hide abstract] ABSTRACT: A novel protease secreted by Brevibacillus sp. KH3 isolated from excess sludge at 50 °C and used as a sludge-lysing strain was investigated in this study. Sludge reduction was minimized by protease inhibitors and a 40-kDa protease, which significantly contributed to this sludge-reducing activity, was purified as the target protein. The final purified protease demonstrated 92-fold higher specific activity than the initial crude extracts. The sludge-reducing efficiency deteriorated relative to decreased protease activity triggered by EDTA; thus, the purified protease was a causative agent in reducing excess sludge. The 40-kDa protease was a serine metalloprotease and showed the highest activity at 50 °C and pH 8.0, and the activity was enhanced in the presence of calcium ions, indicating that the purified protease contained calcium ion. Furthermore, this 40-kDa protease inhibited biofilm formation in excess sludge. These results imply that sludge reduction is because of reduction of biofilm formation in excess sludge.
[Show abstract][Hide abstract] ABSTRACT: Escherichia coli DNA-unwinding protein RecQ has roles in the regulation of general recombination and the processing of stalled replication forks. In this study, we found that knockout of the recQ gene in combination with xonA xseA recJ mutations, which inhibit methyl-directed mismatch repair (MMR), caused about 100-fold increase in sensitivity to a purine analog 2-aminopurine (2AP). Intriguingly, inactivation of a MMR initiator due to the either mutation mutS or uvrD completely suppressed the 2AP sensitivity caused by recQ xonA xseA recJ mutations, suggesting that RecQ helicase might act on the DNA structures that are generated by the processing of DNA by the MutSLH complex and UvrD helicase. Moreover, the recQ gene knockout in combination with xonA xseA recJ mutations enhanced 2AP-induced filament formation, and increased by twofold the rate of spontaneous forward mutations in the thyA locus but did not increase the rate of rifampicin-resistant mutations. We discuss about the possible interplay between E. coli RecQ helicase and mismatch recognition factors.
[Show abstract][Hide abstract] ABSTRACT: The development of a facile technology for utilizing effectively and/or reducing excess sludge is one of the urgent problems since a large quantity of sewage sludge is formed by activated sludge processes. Excess sludge containing 50 mM sucrose was fermented at 50 °C using endogenous bacteria in excess sludge, resulting in a high lactic acid production (8.45 g/L) and in an increased sludge reduction (38.2%). Conversion rate to lactic acid was up to 106.0% by standard fermentation at 50 °C compared to 43.8% at 30 °C and this phenomenon that conversion rate was higher was observed only at 50 °C as the fermentation at less or more than 50 °C had lower conversion rate than that at 50 °C. Lactic acid bacteria increased at 50 °C during 1-d fermentation whereas the number of total viable bacteria only increased slightly, indicating that lactic acid bacteria in sludge at 50 °C were preferentially able to utilize the sucrose for producing lactic acid. Finally, pH-vibration fermentation at 50 °C enabled to completely consume residual sucrose in the normal fermentation, resulting in the maximum production of lactic acid. Lactate fermentation by a purely cultured lactic acid bacterium TS1 with autoclaved excess sludge containing 50 mM sucrose had more than 100% of conversion rate to lactic acid, indicating that a part of sludge was converted into lactic acid during the fermentation. Our technique is useful as a facile engineering for reducing excess sludge concomitantly with producing lactic acid by lactate fermentation.
[Show abstract][Hide abstract] ABSTRACT: A strain of sludge-lysing bacteria was isolated from waste activated sludge (WAS) in this study. The result of 16S rRNA gene analysis demonstrated that it was a species of new genus Brevibacillus (named Brevibacillus sp. KH3). The strain could release the protease with molecule weight of about 40 kDa which could enhance the efficiency of sludge thermophilic aerobic digestion. During the sterilized sludge digestion experiment inoculated with Brevibacillus sp. KH3, the maximum protease activity was 0.41 U/ml at pH 8 and 50 degrees C, and maximum TSS removal ratio achieved 32.8% after 120 h digestion at pH 8 and 50 degrees C. In the case of un-sterilized sludge digestion inoculated with Brevibacillus sp. KH3, TSS removal ratio in inoculated-group was 54.8%, increasing at 11.86% compared with un-inoculation (46.2%). The result demonstrated that inoculation of Brevibacillus sp. KH3 could help to degrade the EPS and promote the collapse of cells and inhibit the growth of certain kinds of microorganisms. It indicated that Brevibacillus sp. KH3 strain had a high potential to enhance WAS-degradation efficiency in thermophilic aerobic digestion.
[Show abstract][Hide abstract] ABSTRACT: Anaerobic bacteria have been used to produce 2,4-dihydroxylamino-nitrotoluene (2,4DHANT), a reductive metabolite of 2,4,6-trinitrotoluene (TNT). Here, an aerobic TNT biodegrader Pseudomonas sp. strain TM15 produced 2,4DHANT as evidenced by the molecular ion with m/z of 199 identified from LC-TOFMS analyses. TNT biodegradation with a high cell concentration (10(9) cells/ml) led to a significant accumulation of 2,4DHANT in the culture medium, as well as hydroxylamino-dinitrotoluenes (HADNTs), although these products were not accumulated when a low cell concentration was used; also, the accumulation of diamino-nitrotoluene and of an unidentified metabolite were observed in the culture medium with the high cell concentration (10(10) cells/ml). 2,4DHANT overproduction was a function of the aeration speed since cultures with low aeration speeds (30 rpm) had a 19-fold higher DHANT productivity than those aerated with high speeds (180 rpm); this indicates that molecular oxygen was related to the formation of 2,4DHANT. The quantification of dissolved oxygen (DO) in the media demonstrated that the productivity of 2,4DHANT was increased at low DO values. Moreover, supplying oxygen to the culture media produced a remarkable decrease of 2,4DHANT accumulation; these results clearly indicate that high 2,4DHANT production was a consequence of the oxygen deficit in the culture medium. This finding is useful for understanding the TNT biodegradation (bioremediation technology) in an anoxic environment.
[Show abstract][Hide abstract] ABSTRACT: Two kinds of hydroxylamino-dinitrotoluenes, 2-hydroxylamino-4, 6-dinitrotoluene (2HADNT) and 4-hydroxylamino-2, 6-dinitrotoluene (4HADNT), are known to be major metabolites produced from 2,4,6-trinitrotoluene (TNT) by bacteria. The amounts of 2HADNT and 4HADNT in distilled water are found to spontaneously decrease with time, albeit abiotic conditions, and many white precipitates are educed in the processes of this phenomenon; however, how these compounds are converted is unclear. We evinced the mystery of this phenomenon by using thin-layer chromatography (TLC) and laser time-of-flight mass spectrometry (TOFMS). TLC analyses in the spontaneous conversion processes of 2HADNT, 4HADNT, and 2HADNT plus 4HADNT demonstrats that three novel spots emerge on the TLC plate, respectively. These products are individually extracted into acetonitrile by collecting each spot. The purity of these extracts, which have retention times of 14.0, 17.7, and 15.4 min, is approximately 98%, judging from the results of high-performance liquid chromatographic analyses. The spontaneous conversion products of 2HADNT, 4HADNT, and 2HADNT plus 4HADNT are identified as 4,4',6,6'-tetranitro-2,2'-azoxytoluene (2,2'AZT), 2,2',6,6'-tetranitro-4,4'-azoxytoluene (4,4'AZT), and 4,2',6, 6'-tetranitro-2,4'-azoxytoluene (2,4'AZT) by obtaining their mass spectra with laser TOFMS. It is confirmed that most of the spontaneous conversion products are 2,2'AZT, 4,4'AZT, or 2,4'AZT, judging from the results of mass balance in the spontaneous conversion processes of 2HADNT, 4HADNT, and 2HADNT plus 4HADNT.
[Show abstract][Hide abstract] ABSTRACT: Although many studies have reported that nitroaromatics such as 2,4,6-trinitrotoluene (TNT) have strong mutagenicity, the mechanism of mutagenic activity in these compounds has not yet been reported. We examined the mutagenicity versus reactivity and biodegradation by bacteria using TNT and its analogs (1,3,5-trinitrobenzene [TNB], 2,4,6-trinitroaniline [TNA], 2,4,6-trinitro-phenol [TNP], N,2,4,6-tetranitro-N-methyl-aniline [tetryl], 2,4-dinitrotoluene [24DNT], and 2,6-dinitrotoluene [26DNT]). Aromatic compounds harboring three nitro groups (except TNP) have high mutagenicity, judging from the results of the umu test using luminescent bacteria (Salmonella typhimurium strain TA1535/pTL210). Single-electron reduction potentials for these chemicals were -530, -555, -565, -575, -640, -674, and -764 mV for TNA, tetyl, TNT, TNB, 24DNT, 26DNT, and TNP, respectively, indicating that trinitro-aromatics (except TNP) were more reducible than other compounds. Pseudomonas sp. strain TM15, which was isolated from TNT-contaminated soils in the Yamada Green Zone, Kitakyushu City, Japan, could efficiently biotransform TNT, TNB, TNA, and tetryl; 24DNT, 26DNT, and TNP were less biodegradable. This strain converted all TNT analogs into reduction products; nitro groups were reduced to amino groups. We revealed that the mutagenicity of nitroaromatics correlate with reactivity and biodegradability. This finding may contribute to the elucidation of mutagenic expression of nitroaromatic compounds in organisms.
[Show abstract][Hide abstract] ABSTRACT: A rapid spectrophotometric determination for 2,4,6-trinitrotoluene (TNT) is significant because this method is suitable for simultaneous analyses of the numerous samples. We found one problem that TNT reduction products interfere with the TNT detection in this assay, and we overcame this problem by heating the samples at 95 degrees C, resulting in the production of compounds that did not interfere.
[Show abstract][Hide abstract] ABSTRACT: Two kinds of hydroxylamino-dinitrotoluenes (HADNTs), 2-hydroxylamino-4,6-dinitrotoluene (2HADNT) and 4-hydroxylamino-2,6-dinitrotoluene (4HADNT), are known to be major metabolites produced from 2,4,6-trinitrotoluene (TNT) by bacteria. These chemicals could not be identified as TNT metabolites produced by Pseudomonas sp. strain TM15 because the mass spectra of these chemicals could not be obtained by liquid chromatography-mass spectrometry (MS) or gas chromatography-MS, which are the classic methods for identifying the metabolites of xenobiotics. However, these problems are overcome by isolating 2HADNT and 4HADNT from TNT metabolites with one-step thin-layer chromatography using dichloromethane as the developing solvent, and individually extracting them into acetonitrile by collecting spots of 2HADNT and 4HADNT. The purity of each HADNT was approximately 98%, based on the results of high-performance liquid chromatographic analyses. 2HADNT and 4HADNT are identified by obtaining their mass spectra with laser time-of-flight MS. 2HADNT and 4HADNT dissolve in distilled water and are spontaneously broken down with time. Also, heat treatment (increasing temperatures) and dissolved oxygen accelerate the destruction of HADNTs. This technique may be applicable for the identification and exact quantitative analysis of unstable and fragile compounds such as HADNTs.
[Show abstract][Hide abstract] ABSTRACT: This study has determined the conditions for operation of a phosphorus-removal system using coagulation–filtration to gain high efficiency and has confirmed its practicability. Sand of 0.6 mm in diameter, anthracite of 1.2 mm in diameter and a mixture of both were tested as filter media. The dual filter bed was proved to be superior in terms of pressure drop and breakthrough. This system was operated continuously for over 20 h. A blocked filter bed can be recovered by backwashing. Over 80% phosphorus removal efficiency is achieved at an LV of under 5.0 m h−l, when the PAC dose is controlled so that the Al/P mole ratio would be 3.0 for the first period, and then subsequently around 2.0. Flocs caught in the filter further adsorb the soluble phosphorus in the wastewater. Thus, the chemical requirement can be reduced compared to the chemical precipitation method.
PROCESS BIOCHEMISTRY 07/2005; 40(8-40):2623-2627. DOI:10.1016/j.procbio.2004.06.056 · 2.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The product of the BLM gene, which is mutated in Bloom syndrome in humans, and the Saccharomyces cerevisiae protein Sgs1 are both homologous to the Escherichia coli DNA helicase RecQ, and have been shown to be involved in the regulation of homologous recombination. Mutations in these genes result in genome instability because they increase the incidence of deletions and translocations. We present evidence for a genetic interaction between SGS1 and YKU70, which encodes the S. cerevisiae homologue of the human DNA helicase Ku70. In a yku70 mutant background, sgs1 mutations increased sensitivity to DNA breakage induced either by treatment with camptothecin or by the expression of the restriction enzyme EcoRI. The yku70 mutation caused a fourfold increase in the rate of double-strand break (DSB)-induced target integration as that seen in the sgs1 mutant. The combination of yku70 and sgs1 mutations additively increased the rate of the targeted integration, and this effect was completely suppressed by deletion of RAD51. Interestingly, an extra copy of YKU70 partially suppressed the increase in targeted integration seen in the sgs1 single mutant. These results suggest that Yku70 modulates the repair of DSBs associated with homologous recombination in a different way from Sgs1, and that the inactivation of RecQ and Ku70 homologues may enhance the frequency of gene targeting in higher eukaryotes.
[Show abstract][Hide abstract] ABSTRACT: The adsorption of a fixed bed filled with bone char was investigated in terms of its efficiency and capacity by determining operational conditions for the purpose of further reduction of organic matter and removal of phosphorus using a continuous flow of real secondary effluent. Simultaneous removals of phosphorus and chemical oxygen demand (COD) were sufficiently achieved by this fixed-bed method. Stable performance was maintained even at a linear flow velocity (abbreviated as LV) of 1.5 m h–1. Appropriate backwashing and regeneration were required to operate the system continuously for a long period of time. During the regeneration, the use of treated water including Ca2+ ion was so effective that phosphorus removal efficiency increased from about 50 to 80%, and afterwards maintained over 65%, until inflow water of the volume up to 150 times as large as the volume of bone char had passed through. Even when the inflow water of the volume rose up to 200 times, the phosphorus removal efficiency could be maintained over 50%. During this operation, the adsorptions of phosphorus and COD onto the bone char surface were observed to be over 6.7 and 35 gL–1, respectively.
Water Air and Soil Pollution 10/2004; 159(1):313-324. DOI:10.1023/B:WATE.0000049181.63852.98 · 1.55 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An experiment of anaerobic filtration using a floating media was carried out in this study. In the present system, a bench-scale column of 50 mm in diameter and of 1500 mm in height and a floating media consisting of S-shaped polystyrene pieces were employed. The purpose of this study was to collect the basic data of anaerobic biological filtration using a floating media. Under the laboratory conditions, it was found that the start-up of an anaerobic biological filter took about half month at 20 degrees C and a lower BOD loading was favorable for this start-up. The BOD removal efficiency over 60% could be achieved at a BOD volumetric loading of the filter bed under 6 kg/m3/d. An effluent BOD concentration became high when the flow rate was high, especially with circulation of treated water, which afforded a large effect on an effluent BOD concentration. As for the mechanism of BOD removal by anaerobic filtration, it was evident that long retention time worked in favor for organic acid generation, and the circulation of treated water promoted decomposition of organic acids.
Journal of Environmental Science and Health Part A 02/2004; 39(1):77-87. DOI:10.1081/ESE-120027369 · 1.16 Impact Factor