Polyhydroxyalkanoate (PHA) accumulation potential and PHA-accumulating microbial communities in various activated sludge processes of municipal wastewater treatment plants.

AIMS: To clarify the polyhydroxyalkanoate (PHA) accumulation potential and the PHA-accumulating microbial community structure in activated sludge in municipal wastewater treatment plants (WWTPs) and to identify their influential factors. METHODS AND RESULTS: Nine activated sludge samples were collected from municipal WWTPs employing various biological treatment processes. In acetate-fed 24-h batch experiments under aerobic and nitrogen- and phosphorus-limited conditions, polyhydroxybutyrate (PHB) content of activated sludge increased from 0-1·3 wt% to 7·9-24 wt%, with PHB yields of 0·22-0·50 C-mol 3-hydroxybutyrate (C-mol acetate)(-1). Microbial community analyses found that activated sludge samples that accumulated >20 wt% of PHB after 24-h PHA accumulation experiments had >5·0 × 10(8) copies g(-1)-mixed liquor-suspended solid of phaC genes. CONCLUSIONS: Results indicated that (i) activated sludge in municipal WWTPs can accumulate up to approx. 20 wt% of PHA without enrichment processes, (ii) PHA accumulation potential of activated sludge varied depending on the operational conditions (treatment processes) of WWTPs, and (iii) phaC gene number can provide a simple indication of PHA accumulation potential. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to compare the PHA accumulation potential and PHA-accumulating microbial communities in activated sludge of various treatment processes. Our findings may be useful for enhancing the resource recovery potential of wastewater treatment systems.

Selenium recovery from kiln powder of cement manufacturing by chemical leaching and bioreduction.

A novel process by using chemical leaching followed by bacterial reductive precipitation was proposed for selenium recovery from kiln powder as a byproduct of cement manufacturing. The kiln powder at a slurry concentration of 10 w/v% with 0.25 M Na2CO3 at 28°C produced wastewater containing about 30 mg-Se/L selenium. The wastewater was diluted four-fold and adjusted to pH 8.0 as preconditioning for bioreduction. A bacterial strain Pseudomonas stutzeri NT-I, capable of reducing selenate and selenite into insoluble elemental selenium, could recover about 90% selenium from the preconditioned wastewater containing selenium of 5 mg-Se/L when supplemented with lactate or glycerol. The selenium concentrations in the treated wastewater were low around the regulated effluent concentration of 0.1 mg-Se/L in Japan.

A co-beneficial system using aquatic plants: bioethanol production from free-floating aquatic plants used for water purification.

A co-beneficial system using constructed wetlands (CWs) planted with aquatic plants is proposed for bioethanol production and nutrient removal from wastewater. The potential for bioethanol production from aquatic plant biomass was experimentally evaluated. Water hyacinth and water lettuce were selected because of their high growth rates and easy harvestability attributable to their free-floating vegetation form. The alkaline/oxidative pretreatment was selected for improving enzymatic hydrolysis of the aquatic plants. Ethanol was produced with yields of 0.14-0.17 g-ethanol/ g-biomass in a simultaneous saccharification and fermentation mode using a recombinant Escherichia coli strain or a typical yeast strain Saccharomyces cerevisiae. Subsequently, the combined benefits of the CWs planted with the aquatic plants for bioethanol production and nutrient removal were theoretically estimated. For treating domestic wastewater at 1,100 m(3)/d, it was inferred that the anoxic-oxic activated sludge process consumes energy at 3,200 MJ/d, whereas the conventional activated sludge process followed by the CW consumes only 1,800 MJ/d with ethanol production at 115 MJ/d.

Model analysis of energy consumption and greenhouse gas emissions of sewage sludge treatment systems with different processes and scales.

An energy consumption model was developed for evaluating sewage sludge treatment plants (SSTPs) incorporating various treatment processes such as thickening, anaerobic digestion, dewatering, incineration, and melting. Based on data analyses from SSTPs in Osaka, Japan, electricity consumption intensities for thickening, anaerobic digestion, dewatering, incineration, and melting and heat consumption intensities for anaerobic digestion, incineration, and melting were expressed as functions of sludge-loading on each unit process. The model was applied for predicting the energy consumption and greenhouse gas (GHG) emissions of SSTPs using various treatment processes and power and heat generation processes using digestion gas. Results showed that SSTPs lacking incineration and melting processes but having power generation processes showed excess energy production at the high sludge-loading rate. Energy consumption of the SSTPs without incineration and melting processes were low, but their GHG emissions were high because of CH(4) and N(2)O emissions from sludge cake at the landfill site. Incineration and melting processes consume much energy, but have lower CH(4) and N(2)O emissions.

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.

White hard clam (Meretrix lyrata) shells media to improve phosphorus removal in lab-scale horizontal sub-surface flow constructed wetlands: Performance, removal pathways, and lifespan.

This work examined the phosphorus (P) removal from the synthetic pretreated swine wastewater using lab-scale horizontal sub-surface flow constructed wetlands (HSSF-CWs). White hard clam (Meretrix lyrata) shells (WHC) and Paspalum atratum were utilized as substrate and plant, respectively. The focus was placed on treatment performance, removal mechanisms and lifespan of the HSSF-CWs. Results indicated that WHC-based HSSF-CW with P. atratum exhibited a high P removal (89.9%). The mean P efluent concentration and P removal rate were 1.34 ± 0.95 mg/L and 0.32 ± 0.03 g/m2/d, respectively. The mass balance study showed that media sorption was the dominant P removal pathway (77.5%), followed by microbial assimilation (14.5%), plant uptake (5.4%), and other processes (2.6%). It was estimated the WHC-based bed could work effectively for approximately 2.84 years. This WHC-based HSSF-CWs technology will therefore pave the way for recycling Ca-rich waste materials as media in HSSF-CWs to enhance P-rich wastewater purification.

White hard clam (Meretrix lyrata) shells as novel filter media to augment the phosphorus removal from wastewater.

It is well recognized that filter media play a crucial role in constructed wetlands (CWs) for decontamination of phosphorus (P)-rich wastewater. This study investigates the suitability of raw white hard clam shells (WHC) and white hard clam shells thermally modified at 800 °C (WHC-M800) as potential media to enhance P treatment performance in CWs. The results indicated that both WHC and WHC-M800 displayed appropriate physicochemical properties, such as high porosity, excellent hydraulic conductivity, and rich Ca content. WHC-M800 exhibited a superior P adsorption capacity (38.7 mg/g) to WHC (12.8 mg/g). However, the practical utilization of WHC-M800 as filter media in CWs may be compromised, due to certain limitations, for example: extremely high pH values in the post-adsorption solutions; high weight losses during calcination and adsorption processes; low mechanical strength; and intensive energy consumption. In contrast, the WHC demonstrated significant advantages of reasonably high P adsorption capacity, locally abundant availability, low cost, and marginal side effects. The fractionation of inorganic P of WHC and WHC-M800 revealed that Ca-bounded P was the most dominant binding form, followed by loosely bound P, Fe-P, occluded P, and Al-P. The present study demonstrates that recycling of WHC shells as a potential substrate in CWs provides a feasible method for upgrading P removal in CWs. Additionally, it helps to reduce waste WHC shells in a simple, cheap, and eco-friendly way, thus can double environmental benefits.

Ethanol production from candidate energy crops: water hyacinth (Eichhornia crassipes) and water lettuce (Pistia stratiotes L.).

Fermentation modes and microorganisms related to two typical free-floating aquatic plants, water hyacinth and water lettuce, were investigated for their use in ethanol production. Except for arabinose, sugar contents in water lettuce resembled those in water hyacinth leaves. Water lettuce had slightly higher starch contents and lower contents of cellulose and hemicellulose. A traditional strain, Saccharomyces cerevisiae NBRC 2346, produced 14.4 and 14.9 g l(-1) ethanol, respectively, from water hyacinth and water lettuce. Moreover, a recombinant strain, Escherichia coli KO11, produced 16.9 and 16.2 g l(-1) ethanol in the simultaneous saccharification and fermentation mode (SSF), which was more effective than the separated hydrolysis and fermentation mode (SHF). The ethanol yield per unit biomass was comparable to those reported for other agricultural biomasses: 0.14-0.17 g g-dry(-1) for water hyacinth and 0.15-0.16 g g-dry(-1) for water lettuce.

Removal of arsenic from groundwater by arsenite-oxidizing bacteria.

The presence of arsenic in groundwater has been of great public concern because of its high toxicity. For purification of arsenic-contaminated groundwater, bacterial oxidation of arsenite, As(III), with a chemical adsorption process was examined in this study. After As(III) oxidation to arsenate, As(V), arsenic is easily removable from contaminated groundwater because As(V) is more adsorptive to absorbents than As(III). By acclimation to As(III) of high concentrations, a mixed culture of heterotrophic bacteria with high As(III)-oxidizing activity was obtained from a soil sample that was free from contamination. With initial concentration up to 1,500 mg l(-1) As(III), the mixed culture showed high As(III)-oxidizing activity at pH values of 7-10 and at temperatures of 25-35 degrees C. The mixed culture contained several genera of heterotrophic As(III)-oxidizing and arsenic-tolerant bacteria: Haemophilus, Micrococcus, and Bacillus. Activated alumina was added to the basal salt medium containing 75 mg l(-1) As(III) before and after bacterial oxidation. Arsenic removal by activated alumina was greatly enhanced by bacterial oxidation of As(III) to As(V). The isotherms of As(III) and As(V) onto activated alumina verified that bacterial As(III) oxidation is a helpful pretreatment process for the conventional adsorption process for arsenic removal.

Application of real-time polymerase chain reaction (PCR) coupled with ethidium monoazide treatment for selective quantification of viable bacteria in aquatic environment.

Ethidium monoazide (EMA) was used to quantify DNA selectively from viable cells with healthy membrane/cell wall system, but not from dead cells, of a target bacterium in the aquatic environment using real-time PCR. Spiking experiments to determine the EMA treatment conditions showed that EMA treatment with EMA at 10-25 microg/ml and subsequent halogen light exposure for 2 min was suitable for selective quantification of DNA from viable cells in an aquatic sample using real-time PCR coupled with EMA treatment (real-time EMA-PCR). Optimized real-time EMA-PCR was applied in combination with culture-based method and conventional real-time PCR without EMA treatment to elucidate the behavior of an Escherichia coli strain inoculated into a pond water microcosm. Quantification results obtained using real-time EMA-PCR were lower than those by conventional real-time PCR without EMA treatment and higher than those by culture-based method. The results suggest that quantification by real-time EMA-PCR seemed to represent the viable population, which would partly include viable but non-culturable state bacteria. Real-time EMA-PCR optimized here can be a useful tool for selective monitoring of the viable population of a target bacterium in the aquatic environment, and thereby contribute to assessment of potential microbial risks generated from waterborne pathogenic bacteria.

Effects of light intensity and water temperature on oxygen release from roots into water lettuce rhizosphere.

The oxygen release rate into the rhizosphere by a floating aquatic plant-water lettuce-was determined under various light intensities (0.0-1.2x10(5)lx) and water temperatures (10-35 degrees C). The net specific oxygen release rate was expressed by a model equation comprising the gross oxygen release rate and the rhizosphere respiration terms. Experimental and simulated results show that the net specific oxygen release rate increased with light intensity up to the optimal value, but slight inhibition by higher light intensities was observed at 10-20 degrees C. With increased water temperature, the respiration rate became larger than the gross oxygen release rate. The maximum net specific oxygen release rate of 11.0-12.5mg-O(2)kg-wet(-1)h(-1) was obtained at the optimal condition of about 25 degrees C and 9.0x10(4)-1.1x10(5)lx. The net oxygen release rate was negligible at 35 degrees C at any light intensity because the respiration rate was much greater than the gross oxygen release rate into the rhizosphere.

Specific interaction of arabinose residue in ginsenoside with egg phosphatidylcholine vesicles.

The interaction of the specific sugar residue in ginsenosides with egg phosphatidylcholine vesicles was investigated by ESR spectrometry using phosphatidic acid spin-labeled at the polar head groups. Ginsenoside-Rc, which has an alpha-L-arabinofuranose residue and agglutinability toward egg yolk phosphatidylcholine vesicles (Fukuda, K. et al. (1985) Biochim. Biophys. Acta 820, 199-206), caused the restriction of the segmental motion of spin-labeled phosphatidic acid in egg phosphatidylcholine vesicles, indicating that the saponin interacted with the polar head groups of vesicles. Other ginsenosides-Rb2, Rb1, Rd and p-nitrophenyl glycoside derivatives which have less or no agglutinability were also investigated in the same manner. Only ginsenoside-Rb2 and p-nitrophenyl alpha-L-arabinofuranoside which have the specific sugar residue (arabinose) showed a strong interaction with the polar head groups of vesicles. To gain an insight into the mechanism of agglutination by ginsenoside-Rc, the interaction with the fatty acyl groups was also studied by using phosphatidylcholine spin-labeled at the fatty acyl groups. Ginsenoside-Rc increased the order parameter of the spin-labeled phosphatidylcholine, indicating that the saponin was inserted into lipid bilayers. In other saponins investigated, only ginsenoside-Rb2 interacted with the fatty acyl part of vesicles. The process of expression of agglutination by ginsenoside-Rc was discussed on the basis of the ESR studies.

Characterizing kinetics of transport and transformation of selenium in water-sediment microcosm free from selenium contamination using a simple mathematical model.

This study developed a seven-compartment model for predicting the fate of selenium (Se) in an aquatic environment containing a water-sediment boundary. Speciation of Se in water-sediment microcosms under microaerobic conditions was measured to evaluate first-order kinetics of Se transportation and transformation. The microcosm consisted of a 10-ml solution containing 1mM soluble Se as selenate (Se6+) or selenite (Se4+) and 8 g wet sediment that was free from Se contamination, sampled from the Senri, Yamato, or Yodo Rivers in Osaka, Japan. Stepwise reaction coefficients describing transportation and transformation were determined using an inverse method on this model which includes: selenate (Se(W)6+) and selenite (Se(W)4+) in ponded water; selenate (Se(S)6+) and selenite (Se(S)4+), elemental Se (Se0), organic Se (Se2-) in sediment; and gaseous Se (DMSe). During this 1-month experiment, soluble Se was transported from ponded water to the sediment and Se was transformed sequentially to other Se species through biochemical reactions. Experimental and kinetic analyses indicated quantitatively that the Yamato River microcosm, with its high organic matter content, had a high adsorption rate of soluble Se. The Yodo River microcosm had a low adsorption rate for Se6+ and a low Se reduction rate. The Senri River microcosm had an apparent high volatilization rate of DMSe. The model developed in this study is extremely useful for predicting fate of Se in aquatic environment in the field.

Adsorption of bacterial cells onto activated sludge flocs.

The adsorption of 9 species of bacteria onto laboratory-activated sludge flocs were investigated and a kinetic model describing the adsorption process was proposed in order to design an effective bioaugmentation strategy. The typical time course of bacterial adsorption, which is a triphasic process, consisted of lag, rapid adsorption, and stationary phases. The equilibrium of the cells in the stationary phase obeyed the Freundlich isotherm. The reversible and nonlinear model could describe the process to a certain degree and the Freundlich parameters and specific sorption rates were estimated for each bacterial strain. There was no apparent relationship between the estimated parameters and characteristics of the bacterial strains, such as specific growth rate, hydrophobicity of the cells, and flocculation activity against kaolin clays. However, the high floc formation ability of the bacterial strains was observed to be related to high cell concentrations although a longer lag time was required.

Application of a floc-forming genetically engineered microorganism to a sequencing batch reactor for phenolic wastewater treatment.

For enhancing the survival of genetically engineered microorganisms (GEMs) in activated sludge processes, the use of a floc-forming bacterium as the host for a recombinant plasmid was proposed. The floc-forming and phenol-degrading GEM Sphingomonas paucimobilis 551 (pS10-45) was cultured to demonstrate this proposal. Although the maximum growth rate of the host strain S. paucimobilis 551 was low and the recombinant plasmid pS10-45 was unstable in the host, the resultant GEM S. paucimobilis 551 (pS10-45) was difficult to wash out together with the effluent, and it maintained population 3-4 times higher than the non-floc-forming GEM Escherichia coli HB101 (pS10-45) in a model activated sludge process operated in a sequencing batch mode. In the long run, the GEM-inoculated activated sludge process showed better phenol removal ability by the recombinant plasmid and better sludge settlement by the host strain.

Decolorization of heat-treatment liquor of waste sludge by a bioreactor using polyurethane foam-immobilized white rot fungus equipped with an ultramembrane filtration unit.

A bench-scale bioreactor using immobilized fungal cells equipped with an ultramembrane filtration unit was developed as a means of decolorizing brown color components (melanoidins) arising from the heat-treatment liquor (HTL) of waste sludge. Artificial HTL containing 4200 color units of synthetic melanoidin supplemented with 1000 mg/l ethanol was first subjected to decolorization by the fungus Coriolus hirsutus IFO4917 immobilized onto polyurethane foam cubes. Then, the resultant biologically treated HTL was subjected to ultrafiltration to obtain the permeate (filtrate) as the effluent. The retentate (concentrate) of the filtration unit, containing the remaining melanoidin of high molecular weight and extracellular decolorizing enzymes, was returned to the fungal bioreactor to allow further decolorization. This system was operated in a sequencing batch mode under nonsterile conditions. Contamination of the bioreactor with air/water-born microbes markedly lowered the decolorization efficiency. However, this problem was solved by heating the returned concentrate at 50 degrees C for 10 min. Under the almost stable condition of a hydraulic retention time of 2 d in a 1-d cycle sequencing batch mode, about 70% decolorization was routinely achieved using the entire system (bioreactor + ultrafiltration), while the contribution of the fungal bioreactor alone to the decolorization was about 45%.

Gitelman's syndrome first diagnosed as Bartter's syndrome.

A 29-year-old man, who had been treated with potassium, spironolactone and indomethacin for over 9 years, was admitted because of nausea, vomiting, diarrhea and tetany manifestation. At the age of 20, he had been diagnosed as having Bartter's syndrome according to the criteria of the Japanese Ministry of Health and Welfare. Findings on admission were hypokalemia, hypomagnesemia and hypocalciuria. Renal distal fractional reabsorption rates of sodium, chloride and calcium were markedly decreased by administration of furosemide but there was no obvious change with administration of thiazide. These findings indicate that the patient had Gitelman's syndrome rather than Bartter's syndrome.

[A case of gas gangrene following operation of cervical esophageal carcinoma].

A 74-year-old woman underwent surgery for cervical esophageal carcinoma. After operation, she had abdominal distension, high fever, and suddenly fell into shock and died. Autopsy revealed necrosis and multiple gas blebs in the entire digestive tract and liver. Direct smear specimens Gram-stained disclosed clostridium septicum and the cause of death was found to be gas gangrene. This is the first of clostridium infection localized in the digestive tract and liver after operation. Abdominal radiogram showed massive gas in the intestine, intramural bowel gas and gas in the portal vein, and these are very suggestive of gas gangrene of the intestinal tract.