Kazunari Sei

Occurrence of 4-tert-butylphenol (4-t-BP) biodegradation in an aquatic sample caused by the presence of Spirodela polyrrhiza and isolation of a 4-t-BP-utilizing bacterium

Although 4-tert-butylphenol (4-t-BP) is a serious aquatic pollutant, its biodegradation in aquatic environments has not been well documented. In this study, 4-t-BP was obviously and repeatedly removed from water from four different environments in the presence of Spirodela polyrrhiza, giant duckweed, but 4-t-BP persisted in the environmental waters in the absence of S. polyrrhiza. Also, 4-t-BP was not removed from autoclaved pond water with sterilized S. polyrrhiza. These results suggest that the 4-t-BP removal from the environmental waters was caused by biodegradation stimulated by the presence of S. polyrrhiza rather than by uptake by the plant. Moreover, Sphingobium fuliginis OMI capable of utilizing 4-t-BP as a sole carbon and energy source was isolated from the S. polyrrhiza rhizosphere. Strain OMI degraded 4-t-BP via a meta-cleavage pathway, and also degraded a broad range of alkylphenols with linear or branched alkyl side chains containing two to nine carbon atoms. Root exudates of S. polyrrhiza stimulated 4-t-BP degradation and cell growth of strain OMI. Thus, the stimulating effects of S. polyrrhiza root exudates on 4-t-BP-degrading bacteria might have contributed to 4-t-BP removal in the environmental waters with S. polyrrhiza. These results demonstrate that the S. polyrrhiza–bacteria association may be applicable to the removal of highly persistent 4-t-BP from wastewaters or polluted aquatic environments.

Comparative analysis of DNA-based microbial community composition and substrate utilisation patterns of activated sludge microorganisms from wastewater treatment plants operated under different conditions.

In this study, the microbial community structure and carbon source utilisation profile of activated sludge samples collected from full-scale municipal wastewater treatment plants (WWTPs) operated under different conditions were characterised and compared, respectively, using terminal-restriction fragment length polymorphism (T-RFLP) analysis and Biolog assay. Samples were collected from each biological treatment tank of six conventional activated sludge, two anaerobic-oxic, two anaerobic-anoxic-oxic, and one step-aeration processes in eight full-scale WWTPs in Osaka, Japan. Results of the T-RFLP analysis of eubacterial 16S rDNA showed that microbial communities of activated sludge differed greatly among samples, and that they were affected by process-based operational conditions. In contrast, the carbon source utilisation profiles of activated sludge samples were mutually similar, but appeared to be influenced slightly by aerated conditions at each reaction tank. Similar carbon source utilisation profiles among all samples suggest that the activated sludge community possesses functions that are necessary for wastewater treatment even if the phylogenetic composition is different. Different results from the T-RFLP analysis and Biolog assay suggest that the phylogenetic composition of microbial community might not necessarily reflect the wastewater treatment functions of the activated sludge.

Polyhydroxyalkanoate production potential of heterotrophic bacteria in activated sludge

This study was conducted to evaluate the polyhydroxyalkanoate (PHA) production potential of cultivable heterotrophic bacteria in activated sludge by genotypic and phenotypic characterizations. A total of 114 bacterial strains were isolated from four activated sludge samples taken from a lab-scale sequencing batch reactor and three wastewater treatment processes of two municipal wastewater treatment plants. PCR detection of the phaC genes encoding class I and II PHA synthase revealed that 15% of the total isolates possessed phaC genes, all of which had the closest similarities to known phaC genes of α- and β-Proteobacteria and Actinobacteria. PHA production experiments under aerobic and nitrogen-limited conditions showed that 68% of the total isolates were capable of producing PHA from at least one of the six substrates used (acetate, propionate, lactate, butyrate, glucose and glycerol). Genotypic and phenotypic characterizations revealed that 75% of the activated sludge bacteria had PHA production potential. Our results also indicated that short-chain fatty acids would be the preferable substrates for PHA production by activated sludge bacteria, and that there might be a variety of unidentified phaC genes in activated sludge.

Characterization of microbial communities distributed in the groundwater pumped from deep tube wells in the Kathmandu Valley of Nepal

Although groundwater is a major water supply source in the Kathmandu Valley of Nepal, it is known that the groundwater has significant microbial contamination exceeding the drinking water quality standard recommended by the World Health Organization (WHO), and that this has been implicated in causing a variety of diseases among people living in the valley. However, little is known about the distribution of pathogenic microbes in the groundwater. Here, we analysed the microbial communities of the six water samples from deep tube wells by using the 16S rRNA gene sequences based culture-independent method. The analysis showed that the groundwater has been contaminated with various types of opportunistic microbes in addition to fecal microbes. Particularly, the clonal sequences related to the opportunistic microbes within the genus Acinetobacter were detected in all samples. As many strains of Acinetobacter are known as multi-drug resistant microbes that are currently spreading in the world, we conducted a molecular-based survey for detection of the gene encoding carbapenem-hydrolysing β-lactamase (bla(oxa-23-like) gene), which is a key enzyme responsible for multi-drug resistance, in the groundwater samples. Nested polymerase chain reaction (PCR) using two specific primer sets for amplifying bla(oxa-23-like) gene indicated that two of six groundwater samples contain multi-drug resistant Acinetobacter.

Effects of planting Phragmites australis on nitrogen removal, microbial nitrogen cycling, and abundance of ammonia-oxidizing and denitrifying microorganisms in sediments

We examined the effect of planting an emergent aquatic plant (Phragmites australis) on nitrogen removal from sediments using a 42-d pot experiment. The experimental pot systems comprised two types of sediments planted with and without young P. australis. Total nitrogen (total N), total dissolved N, and NH4–N in the sediments decreased markedly after planting. In contrast, those levels decreased only slightly in the unplanted sediments. The decrease in total N in the P. australis-planted sediments was 7–20 times those in the unplanted sediments. Abundances of bacterial 16S rRNA, archaeal 16S rRNA, ammonia-oxidizing bacterial ammonia monooxygenase (amoA), ammonia-oxidizing archaeal amoA, and denitrifying bacterial nitrite reductase (nirK) genes increased significantly in sediments after planting. Phragmites australis appears to have released oxygen and created a repeating cycle of oxidizing and reducing conditions in the sediments. These conditions should promote mineralization of organic N, nitrification, and denitrification in the sediments. Phragmites australis absorbed bioavailable nitrogen generated by microbial nitrogen metabolism. During the 42-d period after planting, 31–44% of total N was removed by microbial nitrogen cycling, and 56–69% was removed via absorption by P. australis. These results suggest that planting P. australis can increase microbial populations and their activities, and that nitrogen removal can be accelerated by the combined functions of P. australis and microorganisms in the sediment. Thus, planting P. australis has considerable potential as an effective remediation technology for eutrophic sediments.

1,4-Dioxane degradation potential of members of the genera Pseudonocardia and Rhodococcus

In recent years, several strains capable of degrading 1,4-dioxane have been isolated from the genera Pseudonocardia and Rhodococcus. This study was conducted to evaluate the 1,4-dioxane degradation potential of phylogenetically diverse strains in these genera. The abilities to degrade 1,4-dioxane as a sole carbon and energy source and co-metabolically with tetrahydrofuran (THF) were evaluated for 13 Pseudonocardia and 12 Rhodococcus species. Pseudonocardia dioxanivorans JCM 13855T, which is a 1,4-dioxane degrading bacterium also known as P. dioxanivorans CB1190, and Rhodococcus aetherivorans JCM 14343T could degrade 1,4-dioxane as the sole carbon and energy source. In addition to these two strains, ten Pseudonocardia strains could degrade THF, but no Rhodococcus strains could degrade THF. Of the ten Pseudonocardia strains, Pseudonocardia acacia JCM 16707T and Pseudonocardia asaccharolytica JCM 10410T degraded 1,4-dioxane co-metabolically with THF. These results indicated that 1,4-dioxane degradation potential, including degradation for growth and by co-metabolism with THF, is possessed by selected strains of Pseudonocardia and Rhodococcus, although THF degradation potential appeared to be widely distributed in Pseudonocardia. Analysis of soluble di-iron monooxygenase (SDIMO) α-subunit genes in THF and/or 1,4-dioxane degrading strains revealed that not only THF and 1,4-dioxane monooxygenases but also propane monooxygenase-like SDIMOs can be involved in 1,4-dioxane degradation.

Next-generation sequencing identification of pathogenic bacterial genes and their relationship with fecal indicator bacteria in different water sources in the Kathmandu Valley, Nepal

Bacteriological analysis of drinking water leads to detection of only conventional fecal indicator bacteria. This study aimed to explore and characterize bacterial diversity, to understand the extent of pathogenic bacterial contamination, and to examine the relationship between pathogenic bacteria and fecal indicator bacteria in different water sources in the Kathmandu Valley, Nepal. Sixteen water samples were collected from shallow dug wells (n=12), a deep tube well (n=1), a spring (n=1), and rivers (n=2) in September 2014 for 16S rRNA gene next-generation sequencing. A total of 525 genera were identified, of which 81 genera were classified as possible pathogenic bacteria. Acinetobacter, Arcobacter, and Clostridium were detected with a relatively higher abundance (>0.1% of total bacterial genes) in 16, 13, and 5 of the 16 samples, respectively, and the highest abundance ratio of Acinetobacter (85.14%) was obtained in the deep tube well sample. Furthermore, the blaOXA23-like genes of Acinetobacter were detected using SYBR Green-based quantitative PCR in 13 (35%) of 37 water samples, including the 16 samples that were analyzed for next-generation sequencing, with concentrations ranging 5.3-7.5logcopies/100mL. There was no sufficient correlation found between fecal indicator bacteria, such as Escherichia coli and total coliforms, and potential pathogenic bacteria, as well as the blaOXA23-like gene of Acinetobacter. These results suggest the limitation of using conventional fecal indicator bacteria in evaluating the pathogenic bacteria contamination of different water sources in the Kathmandu Valley.

High-throughput DNA microarray detection of pathogenic bacteria in shallow well groundwater in the Kathmandu Valley, Nepal.

Because of heavy dependence on groundwater for drinking water and other domestic use, microbial contamination of groundwater is a serious problem in the Kathmandu Valley, Nepal. This study investigated comprehensively the occurrence of pathogenic bacteria in shallow well groundwater in the Kathmandu Valley by applying DNA microarray analysis targeting 941 pathogenic bacterial species/groups. Water quality measurements found significant coliform (fecal) contamination in 10 of the 11 investigated groundwater samples and significant nitrogen contamination in some samples. The results of DNA microarray analysis revealed the presence of 1–37 pathogen species/groups, including 1–27 biosafety level 2 ones, in 9 of the 11 groundwater samples. While the detected pathogens included several feces- and animal-related ones, those belonging to Legionella and Arthrobacter, which were considered not to be directly associated with feces, were detected prevalently. This study could provide a rough picture of overall pathogenic bacterial contamination in the Kathmandu Valley, and demonstrated the usefulness of DNA microarray analysis as a comprehensive screening tool of a wide variety of pathogenic bacteria.

Removal characteristics of retinoic acids and 4-oxo-retinoic acids in wastewater by activated sludge treatment

Retinoic acid (RA) receptor (RAR) agonists are potential teratogens to various vertebrates. Their contamination has been detected in municipal wastewater in different countries. This study involved field investigations and laboratory batch treatment experiments to elucidate the removal characteristics by activated sludge treatment of RAs (all-trans RA and 13-cis RA) and 4-oxo-RAs (4-oxo-all-trans RA and 4-oxo-13-cis RA), which were identified as major RAR agonists in municipal wastewater. Results obtained in this study show that currently employed activated sludge treatments can remove RAs, 4-oxo-RAs and overall RAR agonist contamination effectively from municipal wastewater in general, although high RAR agonistic activity might sometimes remain in the effluent. Laboratory experiments revealed that RAs were removed rapidly from the aqueous phase by adsorption to the sludge, after which they were removed further by biological and/or chemical degradation. Aside from adsorption to the sludge, 4-oxo-RAs were also apparently removed by biological and chemical degradation. Biodegradation contributed greatly to the removal. Results of additional experiments indicated that novel non-identifiable RAR agonists can occur through the biodegradation of 4-oxo-RAs by activated sludge and that they can persist for a long period.

Polyhydroxyalkanoate accumulation ability and associated microbial community in activated sludge-derived acetate-fed microbial cultures enriched under different temperature and pH conditions

The influence of temperature and pH during enrichment on the polyhydroxyalkanoate (PHA) accumulation ability and composition of PHA-accumulating microorganisms (PHAAMOs) in enrichment cultures was investigated. Enrichment of PHAAMOs from activated sludge was conducted in acetate-fed sequencing batch reactors using a feast-famine regime under different temperature (20°C, 28°C, and 36°C) and pH (controlled at 7.2 or not) conditions. PHA accumulation ability, which was evaluated in nitrogen- and phosphorus-deficient 24-h single-batch cultures, was greatly enhanced by enrichment, irrespective of the temperature and pH. Enrichment at 20°C or 28°C and without pH control seemed most appropriate for strong PHA accumulation. Analyses of the PHAAMO composition by the clone library method targeting phaC genes, which encode the class I and II PHA synthases, revealed that Burkholderiales were the dominant PHAAMOs in the seed sludge, while Rhodocyclales, specifically Azoarcus spp. and Thauera spp., were dominant after enrichment without pH control, showing a strong ability to accumulate PHA. The results indicated that Azoarcus spp. and Thauera spp. are key PHAAMOs in an enrichment culture based on the feast-famine method, with high PHA accumulation ability.