宮永 一彦

ABSTRACT Escherichia coli O157:H7 is a globally important foodborne pathogen with implications for food safety. Antibiotic treatment for O157 may potentially contribute to the exacerbation of hemolytic uremic syndrome, and the increasing prevalence of antibiotic-resistant strains necessitates the development of new treatment strategies. In this study, the bactericidal effects and resistance development of antibiotic and bacteriophage monotherapy were compared with those of combination therapy against O157. Experiments involving continuous exposure of O157 to phages and antibiotics, along with genetic deletion studies, revealed that the deletion of glpT and uhpT significantly increased resistance to fosfomycin. Furthermore, we found that OmpC functions as a receptor for the PP01 phage, which infects O157, and FhuA functions as a receptor for the newly isolated SP15 phage, targeting O157. In the glpT and uhpT deletion mutants, additional deletion in ompC , the receptor for the PP01 phage, increased resistance to fosfomycin. These findings suggest that specific phages may contribute to antibiotic resistance by selecting the emergence of gene mutations responsible for both phage and antibiotic resistance. While combination therapy with phages and antibiotics holds promise for the treatment of bacterial infections, careful consideration of phage selection is necessary. IMPORTANCE The combination treatment of fosfomycin and bacteriophages against Escherichia coli O157 demonstrated superior bactericidal efficacy compared to monotherapy, effectively suppressing the emergence of resistance. However, mutations selected by phage PP01 led to enhanced resistance not only to the phage but also to fosfomycin. These findings underscore the importance of exercising caution in selecting phages for combination therapy, as resistance selected by specific phages may increase the risk of developing antibiotic resistance.

There is an urgent need to develop phage therapies for multidrug-resistant bacterial infections. However, although bacteria have been shown to be susceptible to phage therapy, phage therapy is not sufficient in some cases. PhiMR003 is a methicillin-resistant Staphylococcus aureus phage previously isolated from sewage influent, and it has demonstrated high lytic activity and a broad host range to MRSA clinical isolates in vitro. To investigate the potential of phiMR003 for the treatment of MRSA infection, the effects of phiMR003 on immune responses in vivo were analysed using phiMR003-susceptible MRSA strains in a mouse wound infection model. Additionally, we assessed whether phiMR003 could affect the immune response to infection with a nonsusceptible MRSA strain. Interestingly, wounds infected with both susceptible and nonsusceptible MRSA strains treated with phiMR003 demonstrated decreased bacterial load, reduced inflammation and accelerated wound closure. Moreover, the infiltration of inflammatory cells in infected tissue was altered by phiMR003. While the effects of phiMR003 on inflammation and bacterial load disappeared with heat inactivation of phiMR003. Transcripts of proinflammatory cytokines induced by lipopolysaccharide were reduced in mouse peritoneal macrophages. These results show that the immune modulation occurring as a response to the phage itself improves the clinical outcomes of phage therapy.

Tonle Sap Lake, the largest freshwater body in Southeast Asia, plays an important role in lives and environment. The lake is reportedly under anthropogenic pressure and suffers from eutrophication. The floating villagers suffer from waterborne diseases. However, the shift in bacterial community due to human activities in this great lake has not yet been reported. We aimed to determine the dynamics of the bacterial community and their concentration in the lake using 67 surface waters, 53 sub-layer waters and 59 sediment samples by Next Generation Sequencing (NGS). The bacterial communities in the surface water and sub-layer water were similar but they differed from the sediment however, their abundance showed spatiotemporal variations. The bacterial diversity reached the highest value in the dry season but lowest value in the rainy season in the surface water and sediment. Their diversity in the sub-layer water was highest in the transition from dry to rainy season. The total 16S rRNA gene copy number in the sediment were &gt 100 times higher than that measured in the water. The Cyanobacteria, Actinobacteria, and Proteobacteria concentrations in the lake water increased in the dry season and reached a peak in the transition from dry to rainy season. The concentrations of Proteobacteria and Firmicutes elevated in the lake water and sediment, respectively, in the floating villages which were &gt 10 times higher than the places with non-point sources. The bacterial concentration and its diversity in the Tonle Sap Lake changed based on the lake water volume between rainy and dry season. The bacterial concentration in the Tonle Sap Lake diluted with the water inflow from Mekong River and its tributaries in the rainy season. As influenced by the fecal waste, the bacterial community in the floating villages differed from the places with non-point source.

The application of nitrate-mediated souring control has been proposed as a promising tool. However, the efficiency of this method remains controversial due to several unresolved issues, such as the determination of effective nitrate concentration, the optimal injection period, etc. In this study, we investigated the depletion of specific hydrocarbons as the electron donor for sulfate-reducing bacteria (SRB) and nitrate-reducing bacteria (NRB) by varying the nitrate concentration (1.5 mM and 4.5 mM) and injection timing (single, Ns and multiple Nm) to control souring. Based on analysis of bacterial communities, genus Arcobacter was predominant, followed by NRB of family Rhodospirillaceae, which includes Thalassospira sp. By contrast, in the condition without nitrate addition (Nw/o), genus Desulfotignum was dominant. Both SRB and NRB share similar hydrocarbon preferences: toluene, ethylbenzene, and xylene. At the limiting nitrate concentration to suppress SRB activity, 1 mM, SRB could co-exist with NRB and promote a more diverse bacterial community.

Phage therapy is getting considerable attention as a method for prophylaxis of food poisoning caused by Escherichia coli O157:H7, an important pathogen causing life-threatening bloody diarrhea. Despite previous studies have shown the feasibility of phage therapy to E. coli O157:H7, promising results have not been obtained in vivo yet. A major drawback of phage therapy is that bacteriophages have high specificity and cannot infect all the sub-strains of a particular pathogenic strain. To overcome this hurdle, we thought to establish a way to artificially expand the host-range of E. coli O157:H7-specific phages. To develop a proof-of-concept for this method, we focused on T2 phage, which cannot infect E. coli O157:H7 strains, and PP01 phage, which displays broad infectivity to them, and attempted to make T2 phage able to infect E. coli O157:H7 as efficiently as PP01. We report the trials of T2 genome editing using the CRISPR/Cas9 system and the modification of both long and short tail fibers of this phage based on comparison with PP01. The resultant recombinant showed the adsorption rate comparable to PP01. Thus, we provided the evidence that the short tail fiber of PP01 plays an important role in adsorption to E. coli O157:H7.

Biochemical reaction kinetics for enzyme and cell (including microbe, plant, and animal cells) are reviewed. Fundamental concept and/or background for the kinetics model construction and the parameter determination of model using experimental data are assessed. Factors affecting the kinetics such as inhibitor, temperature, pH, product, and substrate are also discussed. Stoichiometric consideration of cell reaction is made in order to clarify the elemental balance of the process. Yields of biochemical reaction process occurring in cells are defined in terms of various variables such as biomass, product, heat of reaction as a function of substrate, oxygen, adenosine triphosphate (ATP), etc. Empirical, but universal relationships are suggested in that the elemental composition of microbial cell is similar among various microbial species, and that the cell mass yield referred to ATP is quite similar independently of microbial species and of utilized substrates.

Seawater injection into oil reservoirs for secondary oil recovery is frequently accompanied by souring. Souring causes various problems, such as microbiologically influenced corrosion (MIC) and deterioration of crude oil. Sulfate-reducing bacteria (SRB) are considered to be major players in souring. Nitrate injection has been widely used to control the growth of SRB. The aim of this study was to investigate the changes in the bacterial community in response to nitrate addition to control biological souring. We investigated the effect of nitrate addition in an artificial souring experiment, using diluted crude oil as substrate and electron donor. Desulfotignum sp. was the predominant SRB under all conditions tested. Addition of nitrate at the beginning (N0) repressed the growth of SRB, concomitant with significant growth of the nitrate-reducing bacteria (NRB) Thalassospira sp. Nitrate addition after SRB growth (at day 28, N28) successfully remediated the sulfide produced by SRB, but no significant reduction in sulfate was observed subsequently. In the N28 experiment might be the result of the role of Arcobacter sp., which are nitrate-reducing sulfide-oxidizing bacteria, and/or the ability of Desulfotignum sp. to reduce nitrate and/or nitrite as a stress response. Thus, SRB might persist after nitrate addition, potentially causing subsequent SRB outbreaks.

Tracing the fate of pathogens in environmental water, particularly in wastewater, with a suitable methodology is a demanding task. We investigated the fate of Escherichia coli K12 in sewage influent and activated sludge using a novel approach that involves the application of a biologically stable dialysis device. The ion concentrations inside the device could reach that of surrounding solution when it was incubated in phosphate buffered saline for 2 h. E. coli K12 above 107 CFU mL1 (inoculated in distilled water, influent, activated sludge) were introduced into the device and incubated in influent and activated sludge for 10 days. Without indigenous microorganisms, E. coli K12 could survive even with the limited ions and nutrients concentrations in influent and activated sludge. E. coli K12 abundance in influent and activated sludge were reduced by 60 and 85%, respectively, after just 1 day. The establishment of microbial community in wastewater played an important role in reducing E. coli K12. Bacteriophage propagated in filtered influent or activated sludge when E. coli K12 was introduced, but not in raw influent or activated sludge. The methodology developed in this study can be applied in the actual environmental water to trace the fate of pathogens.

Nitrate injection has been widely used to minimize the production of biological hydrogen sulfide in oil and gas field industry, by controlling the growth of sulfate-reducing bacteria (SRB) chemically and biologically. This study aimed to investigate the changes in the bacterial community in response to nitrate addition used to control biological souring. Specifically, we examined the effect of nitrate addition in an artificial souring experiment, using diluted crude oil as substrate and electron donor. Desulfotignum sp. was the predominant SRB under all conditions tested. Addition of nitrate at the beginning (N0) repressed the growth of SRB, as revealed by chemical and bacterial community analysis, concomitant with significant growth of the nitrate-reducing bacteria (NRB) Thalassospira sp. Nitrate addition after SRB growth (at day 28, N28) successfully remediated the sulfide produced by SRB, but no significant reduction in sulfate was observed subsequently moreover, the bacterial communities before and after nitrate addition remained identical. Isolation of Desulfotignum YB01 (D. YB01) proved the resistance of this predominant SRB in high nitrate environment. Simultaneous reduction of sulfate and nitrate by D. YB01 was also observed in this study. Therefore, the phenomenon in the N28 experiment might be the result of the role of Arcobacter sp. which are nitrate-reducing sulfide-oxidizing bacteria, and/or the ability of Desulfotignum sp. to reduce nitrate and/or nitrite as a stress response. Thus, SRB might persist after nitrate addition, potentially causing subsequent SRB outbreaks.

Seawater injection into oil reservoirs for secondary oil recovery is frequently accompanied by souring (increased sulfide concentrations) in crude oil. The hydrogen sulfide produced by microbiological sulfate reduction in the seawater causes various problems, including corrosion of tubing materials and deterioration of crude oil. Sulfate-reducing bacteria (SRBs) play major roles in souring. However, under high pH (>9), most microbes (including SRBs) cannot grow. Moreover, it is known that iron corrosion is theoretically negligible under the alkaline condition. To investigate new approaches to simultaneously control souring and metal corrosion, we analyzed souring under high-pH conditions. NaOH was added to adjust the pH clean seawater (ca. pH 8) to 11, or 13. Then, a carbon steel test coupon was incubated for 123 d and supplemented with microbes separated from oil field water (OFW) and crude oil. At pH 11 and pH 13, the corrosion rate of the test coupon was decreased. Additionally, souring did not occur at pH 11 and 13, although it took place at pH 8 with microbes. Next-generation sequencing analysis of the 16S rRNA gene revealed drastic changes in the microbial consortia for pH 8 after incubating for 111 d. Desulfotignum, which shows a high identity compared to that of toluene-utilizing SRB, became dominant. It is thought to contribute a biological souring by utilizing toluene in the crude oil at pH 8. On the other hand, at pH 11, the microbial consortia did not change significantly after 111 d of incubation. At pH 13, the microbial consortia drastically changed compared with that of initial condition (OFW) due to cell lysis. That is, even under strict conditions (e.g., pH 13), some bacteria are not lysed, increasing their relative ratio without growth. Alkaline addition could inhibit not only metal corrosion but also biological souring.

In this study, we analyzed coevolution between Staphylococcus aureus strain SA003 and its lytic bacteriophage Phi SA012 to obtain novel insights into S. aureus-its phage interaction. Coevolution was induced in batch co-cultures, repeated 46 times with serial transfer. Although five replicate co-cultures ultimately resulted in bacterial survival and phage extinction, SA003 and Phi SA012 stably coexisted for 260 days (until the end of 45th batch). Strains and phages were periodically isolated from each co-culture replicate until the phage extinction. In the spot testing, isolates showed an antagonistic coevolution with cumulative development of phage resistance and infectivity against the host strain. The center of infection (COI) assays quantitatively revealed that coevolution entailed a gradual reduction of interaction between hosts and phages. The COI value of the isolates from 38th batch was less than 1% of that of the wild-type pair, and this value was still sufficient for their sustained coexistence. Adsorption assays and single-step growth experiments revealed that coevolution involved the changes in both adsorption rate and phage productivity. A fitness cost of phage resistance was observed as a reduction of the specific growth rate. However, it tended to recover during the long-term coevolution. (C) 2017 Elsevier B.V. All rights reserved.

In this study, Desulfotignum species (Desulfotignum YB01) was isolated with roll-tube method. Desulfotignum YB01 can grow on toluene, lacatate, acetate and formate as carbon source with sulfate reduction to sulfide. Genus Desulfotignum, that was dominant in this study, was suggested to be toluene degrading SRB and also fatty acid sweeper. Those roles are significant in souring. Furthermore, Whole genome draft sequence revealed that Desulfotignum YB01 possess upper pathway of toluene degradation, which is toluene to benzoyl-CoA pathway of bssABCDEF (ben-zylsuccinate synthase gene cluster) and bbsABCDEFGH (beta oxidation of benzyl succinate gene cluster). It is similar to gene cluster of Thauera aromatica Azoarcus EbN1, well-known facultative anaerobic toluene degrading nitrate reducing bacteria.

Thanks to their wide host range and virulence, staphylococcal bacteriophages (phages) belonging to the genus Twortlikevirus (staphylococcal Twort-like phages) are regarded as ideal candidates for clinical application for Staphylococcus aureus infections due to the emergence of antibiotic-resistant bacteria of this species. To increase the usability of these phages, it is necessary to understand the mechanism underlying host recognition, especially the receptor-binding proteins (RBPs) that determine host range. In this study, we found that the staphylococcal Twort-like phage Phi SA012 possesses at least two RBPs. Genomic analysis of five mutant phages of Phi SA012 revealed point mutations in orf103, in a region unique to staphylococcal Twort-like phages. Phages harboring mutated ORF103 could not infect S. aureus strains in which wall teichoic acids (WTAs) are glycosylated with alpha-N-acetylglucosamine (alpha-GlcNAc). A polyclonal antibody against ORF103 also inhibited infection by Phi SA012 in the presence of alpha-GlcNAc, suggesting that ORF103 binds to alpha-GlcNAc. In contrast, a polyclonal antibody against ORF105, a short tail fiber component previously shown to be an RBP, inhibited phage infection irrespective of the presence of alpha-GlcNAc. Immunoelectron microscopy indicated that ORF103 is a tail fiber component localized at the bottom of the baseplate. From these results, we conclude that ORF103 binds alpha-GlcNAc in WTAs, whereas ORF105, the primary RBP, is likely to bind the WTA backbone. These findings provide insight into the infection mechanism of staphylococcal Twort-like phages.

The objective of this research is to investigate mechanisms of souring from the view of Volatile fatty acids (VFA) and sulfide production by the crude oil biodegradation. A microbial consortium corrected from oil-water separator was suspended to seawater. Toluene, n-hexadecane, and mixture of them was added as carbon source to the culture medium, independently. After anaerobic incubation for a month, acetate was produced from all the samples. However, according to the production of sulfide, produced acetate was disappeared. High productivity of acetate and sulfide was observed when toluene was used as a carbon source. Co addition of n-hexadecane did not promote souring. Detection of acetate indicated that the oil-field microorganisms excreted acetate as a by-product and the subsequent decreases indicated that other microorganisms such as SRB consume acetate and produce sulfide. To investigate the biological pathway of fatty-acid production under anaerobic conditions, PCR to detect the BssA gene (bssA) encoding benzylsuccinate synthase was conducted. Sequencing revealed that clones were most similar to the bssA of Desulfobacula tol-uolica, a toluene-degrading SRB. However, similarities were low (85%). Relative abundance of bssA to 16SrDNA was slightly increased according to the procession of souring. Community analysis by pyrosequencing revealed that abundant Genus in day 56 was Desulfotignum toluenicum.

Staphylococcus aureus causes a variety of diseases, including bovine mastitis. The standard treatment regimen is treating it with antibiotics. However this approach has drawbacks, including high cost and the incidence of antibiotic-resistant bacteria. As a result, there has been renewed interest in the use of bacteriophages (phages) to control S. aureus. However, aggregation facilitates this bacterium's persistence in the host, and the ability to aggregate has been increasingly recognized as an important Staphylococcus virulence factor. This study revealed that polyclonal bovine IgG enhanced aggregation of S. aureus (SA003), isolated from raw-milk samples from cows with mastitis. IgG-dependent aggregation delayed lysis of SA003 by the specific bacteriophage (Phi SA012), originally isolated from sewage influent. Addition of IgG also lowered the phage host adsorption rate constant (k(a)). The k(a) value of the IgG-containing condition was approximately 1/8 of that of the IgG-free condition. In addition to the decrease of ka value, increase of the minimum inhibitory concentration (MIC) of Phi SA012 toward SA003 was observed. Fluorescence microscopical and confocal laser microscopical observations revealed that phages were mainly localized on the surfaces of cell aggregates. IgG-dependent S. aureus aggregation in crude milk is a problem that must be solved before phage therapy can be successfully used to treat bovine mastitis caused by this bacterium. (C) 2015 Elsevier B.V. All rights reserved.

Seawater injection into oil reservoirs for secondary oil recovery is frequently accompanied by souring (increased sulfide concentrations). Production of hydrogen sulfide causes various problems, such as microbiologically influenced corrosion, deterioration of crude oil. Sulfate-reducing bacteria (SRB) are considered to be major players in souring. The most common method to control souring is injection of biocide and/or nitrate into reservoir to inhibit SRB. However, biocides are expensive and require repeated or continuous injection to be effective. Nitrate injection exacerbates corrosion due to its oxidizing potential. In order to investigate new approaches to controlling souring we analyzed souring under the high pH condition. Corrosion of iron under the high pH (&gt 9) is theoretically negligible and most of the microbes includes SRB cannot grow. The pH of clean sea water was adjusted to 8, 11, and 13 by NaOH addition. Carbon steel test coupon was incubated in the sea water with different pH condition supplemented with microbes separated from oil field water and crude oil for 120 days. Corrosion rate of the test coupon under pH=13 was almost negligible and no souring was observed under the pH 11 and 13 conditions. The 16S rRNA gene was amplified for pyrosequencing. The microbial communities were clearly different under the different pH condition. The most important problem of the idea to controlling souring under high pH condition was the production of fine particle. The analysis of fine particle and method to remove it also investigated in this study.

Synanthropic flies have been implicated in the rapid dissemination of antibiotic-resistant bacteria and resistance determinants in the biosphere. These flies stably harbor a considerable number of bacteria that exhibit resistance to various antibiotics, but the mechanisms underlying this phenomenon remain unclear. In this study, we investigated the persistence of antibiotic-resistant bacteria in the digestive tract of houseflies and green bottle flies, using Proteus mirabilis as a model microorganism. One resistant strain carried the blaTEM and aphA1 genes, and another carried a plasmid containing qnrD gene. Quantitative PCR and 454 pyrosequencing were used to monitor the relative abundance of the Proteus strains, as well as potential changes in the overall structure of the whole bacterial community incurred by the artificial induction of Proteus cultures. Both antibiotic-resistant and -sensitive P. mirabilis strains persisted in the fly digestive tract for at least 3 days, and there was no significant difference in the relative abundance of resistant and sensitive strains despite the lower growth rate of resistant strains when cultured in vitro. Therefore, conditions in the fly digestive tract may allow resistant strains to survive the competition with sensitive strains in the absence of antibiotic selective pressure. The composition of the fly-associated bacterial community changed over time, but the contribution of the artificially introduced P. mirabilis strains to these changes was not clear. In order to explain these changes, it will be necessary to obtain more information about bacterial interspecies antagonism in the fly digestive tract.

Seawater injection into oil reservoirs for purposes of secondary oil recovery is frequently accompanied by souring (increased sulfide concentrations). Production of hydrogen sulfide causes various problems, such as microbiologically influenced corrosion (MIC) and deterioration of crude oil. Sulfate-reducing bacteria (SRB) are considered to be major players in souring. Volatile fatty acids (VFAs) in oil-field water are believed to be produced by microbial degradation of crude oil. The objective of this research was to investigate mechanisms of souring, focusing specifically on VFA production via crude oil biodegradation. To this end, a microbial consortium collected from an oil-water separator was suspended in seawater; crude oil or liquid n-alkane mixture was added to the culture medium as the sole carbon source, and the culture was incubated under anaerobic conditions for 190 days. Physicochemical analysis showed that preferential toluene degradation and sulfate reduction occurred concomitantly in the culture containing crude oil. Sulfide concentrations were much lower in the alkane-supplemented culture than in the crude oil-supplemented culture. These observations suggest that SRB are related to the toluene activation and VFA consumption steps of crude oil degradation. Therefore, the electron donors for SRB are not only VFA, but many components of crude oil, especially toluene. Alkanes were also degraded by microorganisms, but did not contribute to reservoir souring. (C) 2014 Elsevier B.V. All rights reserved.

Green bottle flies occur frequently around human environments in Japan. Many species of green bottle flies have been studied with regard to their importance in forensic examinations or clinical therapies, but the bacterial communities associated with this group of flies have not been comprehensively investigated. In this research, 454 pyrosequencing was used to reveal the bacterial communities in green bottle flies collected in different seasons. Meanwhile, the bacteria were screened with selective media and tested for antibiotic susceptibility. Samples collected in three different seasons harbored distinctive bacterial communities. The predominant genera associated with green bottles flies were Staphylococcus in spring, Ignatzschineria in summer, and Vagococcus, Dysgonomonas, and an unclassified Acetobacteraceae in autumn. An upward trend in bacterial community diversity was observed from spring to autumn. Changes in climatic conditions could be the cause of these seasonal variations in fly-associated bacterial communities. The species of isolated antibiotic-resistant bacteria also differed across seasons, but it was difficult to correlate seasonal changes in antibiotic-resistant bacteria with changes in whole communities. A number of multiple-antibiotic-resistant bacteria were isolated, and some of these strains were closely affiliated with pathogens such as Enterococcus faecalis and Enterococcus faecium, which could cause serious threats to public health. Overall, this research provided us with information about the composition and seasonality of bacterial communities in green bottle flies, and highlighted the risks of fly-mediated dissemination of antibiotic-resistant pathogens.

Oil souring has important implications with respect to energy resources. Understanding the physiology of the microorganisms that play a role and the biological mechanisms are both important for the maintenance of infrastructure and mitigation of corrosion processes. The objective of this study was to identify crude-oil components and microorganisms in oil-field water that contribute to crude-oil souring. To identify the crude-oil components and microorganisms that are responsible for anaerobic souring in oil reservoirs, biological conversion of crude-oil components under anaerobic conditions was investigated. Microorganisms in oil field water in Akita, Japan degraded alkanes and aromatics to volatile fatty acids (VFAs) under anaerobic conditions, and fermenting bacteria such as Fusibacter sp. were involved in VFA production. Aromatics such as toluene and ethylbenzene were degraded by sulfate-reducing bacteria (Desulfotignum sp.) via the fumarate-addition pathway and not only degradation of VFA but also degradation of aromatics by sulfate-reducing bacteria was the cause of souring. Naphthenic acid and 2,4-xylenol were not converted.

Seawater injection into oil reservoirs for secondary oil recovery is frequently accompanied by souring (increased sulfide concentrations). Production of hydrogen sulfide causes various problems, such as microbiologically influenced corrosion (MIC), deterioration of crude oil. Sulfate-reducing bacteria (SRB) are considered to be major players in souring. Volatile fatty acids (VFAs) in oil field water are assumed to be produced by microbial degradation of crude oil. The objective of this research is to investigate mechanisms of souring from the view of VFA production by the crude oil biodegradation. A microbial consortium collected from oil-water separator was suspended to seawater. Crude oil or liquid n-alkane mixture was added to the culture medium as sole carbon source. Anaerobic incubation was conducted for 190 days. Physicochemical analysis showed that preferable toluene degradation and sulfate reduction occurred concomitantly in crude oil amended condition. Sulfide concentration was much lower in alkane mixture amended condition than that of crude oil amended condition. These observations suggest that SRB are related to toluene activation and VFA consumption steps in crude oil degradation. Therefore, the electron donors for SRB were not only VFA, but a lot of crude oil components, especially toluene. Alkanes were also degraded by microorganisms, but did not so contribute to reservoir souring.

Microbial activities in brine, seawater, or estuarine mud are involved in iodine cycle. To investigate the effects of the microbiologically induced iodine on other bacteria in the environment, a total of 13 bacteria that potentially participated in the iodide-oxidizing process were isolated from water or biofilm at a location containing 131 mu g ml(-1) iodide. Three distinct strains were further identified as Roseovarius spp. based on 16 S rRNA gene sequences after being distinguished by restriction fragment length polymorphism analysis. Morphological characteristics of these three Roseovarius spp. varied considerably across and within strains. Iodine production increased with Roseovarius spp. growth when cultured in Marine Broth with 200 mu g ml(-1) iodide (I-). When 10(6) CFU/ml Escherichia coli, Pseudomonas aeruginosa, and Bacillus pumilus were exposed to various concentrations of molecular iodine (I-2), the minimum inhibitory concentrations (MICs) were 0.5, 1.0, and 1.0 mu g ml(-1), respectively. However, fivefold increases in the MICs for Roseovarius spp. were obtained. In co-cultured Roseovarius sp. IOB-7 and E. coli in Marine Broth containing iodide (I-), the molecular iodine concentration was estimated to be 0.76 mu g ml(-1) after 24 h and less than 50 % of E. coli was viable compared to that co-cultured without iodide. The growth inhibition of E. coli was also observed in co-cultures with the two other Roseovarius spp. strains when the molecular iodine concentration was assumed to be 0.52 mu g ml(-1).

The housefly (Musca domestica) is an important host for a variety of bacteria, including some pathogenic and antibiotic-resistant strains. To further investigate the relationship between the housefly and the bacteria it harbors, it is necessary to understand the fate of microorganisms during the larval metamorphosis. The major bacterial communities in three developmental stages of the housefly (maggot, pupa, and adult fly) were investigated by a culture-independent method, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of 16S rRNA genes. The bacteria that were identified using DGGE analysis spanned phyla Proteobacteria, Firmicutes, and Bacteroidetes. Changes in the predominant genera were observed during the housefly development. Bacteroides, Koukoulia, and Schineria were detected in maggots, Neisseria in pupae, and Macrococcus, Lactococcus, and Kurthia in adult flies. Antibiotic-resistant bacteria were screened using a selective medium and tested for antibiotic susceptibility. Most resistant isolates from maggots and pupae were classified as Proteus spp., while those from adult flies were much more diverse and spanned 12 genera. Among 20 tested strains across the three stages, 18 were resistant to at least two antibiotics. Overall, we demonstrated that there are changes in the major bacterial communities and antibiotic-resistant strains as the housefly develops.

Bioclogging is a phenomenon that causes reduced pore space due to the accumulation of microbial biomass in a porous medium, which consequently leads to a decrease in the hydraulic conductivity of the medium. In geotechnical engineering, bioclogging can be used to reduce the erosion of drain channels, reduce the migration of heavy metals and organic pollutants, and prevent piping of earth dams and dikes. However, one negative aspect of bioclogging is that it can cause plugging at produced water injection well, resulting in the subsequent loss of well injectivity. Iodine recovery at a natural gas production plant in Japan involved the addition of sulfuric acid for pH adjustment, resulting in an additional ca. 200 mg/L of sulfate in the waste brine after iodine recovery. Bioclogging occurred at the waste brine injection well, causing a decrease in well injectivity. To examine the factors that contribute to bioclogging, an on-site experiment was conducted by amending 10 liters of brine with different conditions and then incubating the brine for 5 months under open air. The control case was exposed to open air but did not receive additional chemicals. When sulfate addition was coupled with low iodine, there was a drastic increase in the total amount of accumulated biomass (and subsequently the risk of bioclogging) that was nearly six times higher than the control. The bioclogging-associated corrosion rate of carbon steel was 84.5 μm per year, which is four times higher than that observed under other conditions. Analysis of the microbial communities by denaturing gradient gel electrophoresis revealed that the additional sulfate established a sulfur cycle and induced the growth of phototrophic bacteria, including cyanobacteria and purple bacteria. In the presence of sulfate and low iodine levels, cyanobacteria and purple bacteria bloomed and the accumulation of abundant biomass may have created a more conducive environment for anaerobic sulfatereducing bacteria. It is believed that the higher corrosion rate was caused by a differential aeration cell that was established by the heterogeneous distribution of the biomass that covered the surface of the test coupons.

The emission of hydrogen sulfide (H2S) near the estuary of a regional river (Tokyo, Japan), which is thought to be caused by biological reduction of sulfate in seawater mixing with overflow wastewater, poses a severe environmental problem. In order to investigate vertically the biochemical alteration of the river under the long-time treatment for the sulfide problem, we set up a vertical column simulator composed of artificial sewage-filled columns and packed-bed columns. During operation, we carried out chemical and biological analyses to elucidate vertical distributions of sulfide concentration, dissolved oxygen concentration, oxidation/reduction potential and microbial consortia. H2S was not detected in the top section, which was supplied with continuous aerated artificial wastewater, whereas H2S was formed at high concentrations under anaerobic conditions. After 1 year, we changed the supply position for aerated sewage from the top to the middle of the simulator; subsequently, the sulfide concentrations in all sections, especially in the sediment region, dropped to negligible levels. Furthermore, under each of these 2 conditions, pyrosequencing revealed that the microbial consortia differed significantly following the change of the aerated sewage supply position. On the basic of these results, purging oxygen at the border of the water column and the sediment could help to solve the sulfide problem more effectively and might enhance the growth of bacteria involved in the sulfide oxidation process.

Phylogenetic diversity of [FeFe]-hydrogenase (HydA) in termite guts was assessed by pyrosequencing PCR amplicons obtained using newly designed primers. Of 8,066 reads, 776 hydA phylotypes, defined with 97% nucleotide sequence identity, were recovered from the gut homogenates of three termite species, Hodotermopsis sjoestedti, Reticulitermes speratus, and Nasutitermes takasagoensis. The phylotype coverage was 92–98%, and the majority shared only low identity with database sequences. It was estimated that 194–745 hydA phylotypes existed in the gut of each termite species. Our results demonstrate that hydA gene diversity in the termite gut microbiota is much higher than previously estimated.<br>

This study investigated the effect of heat-alkaline treatment (HAT) at pH 11 and 60 degrees C on volatile fatty acid (VFA) production and protein degradation in excess sludge, soluble and insoluble proteins, and pure cultures. In addition, quantification of bacteria present in the sludge was also examined. Experimental results showed that following acid fermentation under pH 7 and 37 degrees C, HAT enhanced VFA production in excess sludge, albumin, and Gram-negative bacteria, but not in casein or Gram-positive bacteria. Protein solubility was therefore found not to be the main criteria for VFA production. In the protein analysis, it was shown that the outer membrane protein (OmpC) of Escherichia coli K12 was resistant to chemical and enzymatic hydrolysis. Gram staining revealed that Gram-negative bacteria were predominant in the activated sludge used in this study. In addition, the bacteria present in the activated sludge comprised only 10% of mixed liquor suspended solids (MLSS) by quantitative PCR. (c) 2012 Elsevier Ltd. All rights reserved.

The simple two-chamber diffusion method was improved to study the diffusion properties of bacteriophage (phage) T4 through a model biofilm agarose gel membrane (AGM) embedded with dead host Escherichia coli K12 cells. The apparent diffusion coefficient (Dapp) of phage T4 was calculated to be 2.4 X 10-12 m2/s in 0.5% AGM, which was lower than the coefficient of 4.2 X 10-12 m2/s in 0.5% AGM without host cells. The phage adsorption process by dead host cells slowed the apparent phage diffusion. The Langmuir adsorption equation was used to simulate phage adsorption under different multiplicity of infections (MOIs); the maximum adsorbed phage MOI was calculated to be 417 PFU/CFU, and the Langmuir adsorption constant KL was 6.9 X 10-4 CFU/PFU. To evaluate the effects of phage proliferation on diffusion, a simple syringe-based biofilm model was developed. The phage was added into this homogenous biofilm model when the host cells were in an exponential growth phase, and the apparent diffusion coefficient was greatly enhanced. We concluded that Dapp of phages through biofilms could be distinctly affected by phage adsorption and proliferation, and that the idea of Dapp and these methods can be used to study diffusion properties through real biofilms. (c) 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2012

Biosurfactants are produced by fermenting pure strains but not treating excess sludge. For reducing the excess sludge, heat and alkaline treatment was reported as a feasible process. To develop a biosurfactant from treated sludge, the surface activity of heat-alkaline treated sludge was investigated in this study. Sludge, Escherichia coli, and extracted E. coli lipids were treated under pH 7, pH 11 and pH 13 conditions that were then combined with or without heat treatment. When the sludge was treated at pH 13 and then heat-treated for 18 h, the surface tension of the supernatant of the treated sludge was reduced by 26.6% from 72.5 mN/m to 53.0 mN/m. As a whole, the alkaline treatment more effectively reduced surface tension than heat treatment because the surface tension of the supernatant was lower than 55.4 mN/rn after the sludge was treated at pH 13. N-hexadecane droplets were emulsified by the supernatant of treated sludge, and then collected using a designed sampling device. The droplet size distribution and cumulative frequency curve of n-hexadecane droplets showed that over half of the droplets were smaller than 80 mu m after they were emulsified by supernatant from alkaline-treated sludge at pH 13 treatment condition no matter with or without heat treatment. These findings indicate that heat-alkaline treated sludge has both surface activity and emulsifying capability. (C) 2011 Elsevier B.V. All rights reserved.

Biochemical reaction kinetics for enzyme and cell (including microbe, plant, and animal cells) are reviewed. Fundamental concept and/or background for the kinetics model construction and the parameter determination of model using experimental data are assessed. Factors affecting the kinetics such as inhibitor, temperature, pH, product, and substrate are also discussed. Stoichiometric consideration of cell reaction is made in order to clarify the elemental balance of the process. Yields of biochemical reaction process occurring in cells are defined in terms of various variables such as biomass, product, heat of reaction as a function of substrate, oxygen, adenosine triphosphate (ATP), etc. Empirical, but universal relationships are suggested in that the elemental composition of microbial cell is similar among various microbial species, and that the cell mass yield referred to ATP is quite similar independently of microbial species and of utilized substrates.

Iodine recovery at a natural gas production plant in Japan involved the addition of sulfuric acid for pH adjustment, resulting in an additional about 200 mg/L of sulfate in the waste brine after iodine recovery. Bioclogging occurred at the waste brine injection well, causing a decrease in well injectivity. To examine the factors that contribute to bioclogging, an on-site experiment was conducted by amending 10 L of brine with different conditions and then incubating the brine for 5 months under open air. The control case was exposed to open air but did not receive additional chemicals. When sulfate addition was coupled with low iodine, there was a drastic increase in the total amount of accumulated biomass (and subsequently the risk of bioclogging) that was nearly six times higher than the control. The bioclogging-associated corrosion rate of carbon steel was 84.5 mu m/year, which is four times higher than that observed under other conditions. Analysis of the microbial communities by denaturing gradient gel electrophoresis revealed that the additional sulfate established a sulfur cycle and induced the growth of phototrophic bacteria, including cyanobacteria and purple bacteria. In the presence of sulfate and low iodine levels, cyanobacteria and purple bacteria bloomed, and the accumulation of abundant biomass may have created a more conducive environment for anaerobic sulfate-reducing bacteria. It is believed that the higher corrosion rate was caused by a differential aeration cell that was established by the heterogeneous distribution of the biomass that covered the surface of the test coupons.

This study examined the effects of combining heat-alkaline treatment (HAT) with an acclimation process on sludge reduction Changes in sludge components and microbial communities in both the mixed liquor suspended solids (MLSS) and supernatant fractions were monitored throughout the process HAT was performed under different pH conditions (pH 7 pH 11 and pH 13) at 60 C Approximately 42-62% of the released materials were proteins After an 8-day acclimation of sludge the protein concentration in the supernatant had significantly decreased under all conditions Treatment conditions at pH 11 were optimal for sludge reduction due to the increased efficiency and reduced consumption of chemicals to adjust the pH A molecular analysis showed that the microbial consortia in both fractions after the cell lysis differed depending on the pH and temperature and only a few types of bacteria were resistant under extreme conditions The microbial communities in the MISS under different conditions were similar after the 8-day acclimation (C) 2010 Elsevier B V All rights reserved

A simple two-chamber diffusion method was developed to study the diffusion properties of bacteriophages (phages). The apparent diffusion coefficients (D(app)) of Myoviridae phage T4 and filamentous phage fNEL were investigated, and the diffusion of the phages was found to he much slower than the diffusion of three antibiotics, ciprofloxacin, penicillin G. and tetracycline. D(app) of T4 and fNEL in water through filter paper were calculated to be 2.8 x 10(-11) m(2)/s and 6.8 x 10(-12) m(2)/s, respectively, and D(app) of fNEL through agarose gel membrane, an artificial biofilm, was also calculated to be smaller than that of T4. In addition, D(app) of phages through agarose gel was dependent on agarose concentration due to the similar size of phage and agarose gel mesh. We concluded that D(app) of phages through an artificial biofilm is dependent on both phage morphology and biofilm density, and suggest the use of this method to study diffusion properties through real biofilms. (C) 2010 American Institute of Chemical Engineers Biotechnol. Prog., 26: 1213-1221, 2010

The water samples collected from two non water-flooded oil fields contained a variety of organic acids, sulfate-reducing bacteria (SRB) and little sulfate. Acetate and propionate were the major components of organic acids. Over 6 weeks of artificial souring experiment, a maximum of 3 mM of sulfide was produced when oil field water was mixed with seawater at 25 degrees C. Propionate was completely consumed under soured conditions. This indicated that the propionate-consuming SRB underwent souring in this experiment. Significant cell growth was confirmed at 25 degrees C with no relation to souring. The dominant SRB species were shifted from Desulfomicrobium thermophilum to Desulfobacter vibrioformis and uncultured Desulfobacter.

Cathodic protection (CP) is known as a reliable method to protect steel from corrosion. However, the influence of CP on biofilm formation and maturation is not well known. There are three possible effects of CP on biofilm formation and maturation. Those are: 1) deprivation of anchorage sites for bacterial adhesion, 2) increase of electrostatic repulsion between the steel surface and negatively charged bacteria, 3) elimination of bacteria in the biofilm by increasing pH. To investigate these possible effects of CP on biofilm, carbon steel coupons were immersed in artificial seawater with and without CP. CP retarded steel corrosion, as a consequence of inhibition of biofilm formation. CP also inhibited initial attachment of Pseudomonas aeruginosa PAO1, a strain well known for its negative charge and pioneering adhesive characteristics. Since the inhibitive effect of cell adhesion was greater in low ionic conditions than in high ionic conditions, CP increased electrical repulsion between the steel surface and bacteria. The increase of pH in an artificial biofilm composed of 0.5% agar and 0.9% NaCl was also investigated theoretically and experimentally. The pH profile in the artificial biofilm was analyzed using a capillary pH electrode. After three hours of CP, the biofilm pH increased and as a consequence sterilized P. aeruginosa PAO1 imbedded in the film. However, pH at the edge of the biofilm decreased sharply and reached the same value as that in the bulk-water. Therefore, bacteria located at the edge of the biofilm survived during the application of CP. © 2007 by NACE International.

Recently, excess sludge from wastewater treatment plant has been one of the serious problems. They are disposed by landfill, incineration, bio-gas production and so on. However, these conventional methods contain drawbacks such as short of landfill, dioxin emissions or inefficient production. A countermeasure against the problems is to minimize the sludge production in the wastewater treatment process. In this study, a biochemical sludge-reduction system was proposed. The microbes were experienced in the aerobic and anaerobic conditions alternately. By the change of microbial growth condition, it is thought that obligate aerobic and anaerobic microbes are to be dead, resulting in their solubilization under alternated anaerobic and aerobic conditions, respectively. The concentrations of organic carbonaceous and nitrogenous substrates and mixed liquor suspended solid (MLSS) were more greatly reduced under the alternate aerobic-anaerobic conditions than under the continuous aerobic or anaerobic conditions. The MLSS mainly consisted of protein (more than 50%w/w) and the amount of protein in the suspension fluctuated during the cultivation without any additions of substrates. These results indicate that the microbial solubilization and utilization of protein lysate seem to take place in this system. So far, however, the effects of growth condition on the microbial viabilities in the solubilization have not been investigated. To clarify them, carboxyfluorescein diacetate (CFDA) that was substrate of intracellular esterase was used as an indicator of microbial viability. As a further experiment, the relationships between sludge solubilization, bacterial viabilities and substrate removal are to be investigated in the continuous wastewater treatment process.