Ryoki Asano

Archaeal community dynamics and detection of ammonia-oxidizing archaea during composting of cattle manure using culture-independent DNA analysis

The composting process is carried out under aerobic conditions involving bacteria, archaea, and fungi. Little is known about the diversity of archaeal community in compost, although they may play an important role in methane production and ammonia oxidation. In the present study, archaeal community dynamics during cattle manure composting were analyzed using a clone library of the archaeal 16S rRNA gene. The results indicated that methane-producing archaea (methanogen) and ammonia-oxidizing archaea (AOA) may be the dominant microbes throughout the composting. The community consisted primarily of Methanocorpusculum-like and Methanosarcina-like sequences until day 2, while the number of Candidatus Nitrososphaera-like sequences increased from day 6 to day 30. Methanosarcina thermophila-like sequences were dominant from day 2, suggesting that M. thermophila-like species can adapt to increasing temperature or nutrient loss. A denaturant gradient gel electrophoresis analysis of the archaeal amoA genes revealed that the dominant amoA gene sequence with 99% homology to that of Candidatus Nitrososphaera gargensis was identical to those obtained from a different composting facility. These data suggested that AOA may play a role in ammonia oxidation in several composting practices. Our results provide fundamental information regarding archaeal community dynamics that will help in understanding the collective microbial community in compost.

Reduction in excess sludge production in a dairy wastewater treatment plant via nozzle-cavitation treatment : Case study of an on-farm wastewater treatment plant

Nozzle-cavitation treatment was used to reduce excess sludge production in a dairy wastewater treatment plant. During the 450-d pilot-scale membrane bioreactor (MBR) operation, when 300 l of the sludge mixed liquor (1/10 of the MBR volume) was disintegrated per day by the nozzle-cavitation treatment with the addition of sodium hydrate (final concentration: 0.01% W/W) and returned to the MBR, the amount of excess sludge produced was reduced by 80% compared with that when sludge was not disintegrated. On the basis of the efficiency of CODCr removal and the ammonia oxidation reaction, it was concluded that the nozzle-cavitation treatment did not have a negative impact on the performance of the MBR. The estimation of the inorganic material balance showed that when the mass of the excess sludge was decreased, the inorganic content of the activated sludge increased and some part of the inorganic material was simultaneously solubilized in the effluent.

Development and analysis of microbial characteristics of an acidulocomposting system for the treatment of garbage and cattle manure

An acidulocomposting system for the treatment of cattle manure with little emission of ammonia gas was developed, and the structure of its microbial community was investigated by denaturing gradient gel electrophoresis (DGGE) and clone library construction. An acidulocomposting apparatus (BC20, 20 L) was operated for 79 days to treat 2 kg (wet wt) of garbage per 1 or 2 days. On day 80 of operation, the substrate was changed from garbage to cattle manure (1 kg of beef cattle manure was added to the apparatus every 2 or 3 days), and the system continued operating from days 80 to 158. The compost in the vessel was under acidic conditions at pH 5.2-5.8, and ammonia emission was below the detectable level (<5 ppm) throughout the period of cattle manure feeding. Total nitrogen and total carbon in the compost were 26-29 and 439-466 mg/g of dry weight, respectively, which are higher than those in general cattle manure compost. The main acids accumulated during operation were lactic and acetic. Sequencing analysis targeting the 16S rRNA gene revealed the stable dominance of the bacterial phylum Firmicutes, with a high proportion of the isolates belonging to the genus Bacillus. Using a culturing method with MRS agar, we isolated lactic acid bacteria (LAB) related to Pediococcus acidilactici, Weissella paramesenteroides, and Lactobacillus salivarius, indicating the existence of LAB in the system. These results indicate that acidulocomposting treatment of cattle manure is not accompanied by ammonia emission and that Bacillus and LAB may be the key components in the system.

Change in the community structure of ammonia-oxidizing bacteria in activated sludge during selective incubation for MPN determination.

We investigated the changes in the community structure of ammonia-oxidizing bacteria (AOB) in activated sludge during incubation of the sludge in a medium selective for AOB. The number of AOB present in the activated sludge sample was enumerated by the most-probable-number (MPN) method. Both the activated sludge sample and the incubated samples for MPN determination were analyzed by polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE). Universal PCR-DGGE indicated that even after 40-d incubation in a medium selected for AOB, the MPN samples were predominantly composed of heterotrophic bacteria and not AOB. Denitrification by heterotrophic bacteria might lead to the underestimation of the MPN count of AOB. Not dominated in whole bacteria, one species of AOB was detected in both original activated sludge and samples after MPN incubation by PCR-DGGE targeting AOB. Furthermore, two new species of AOB were detected only after incubation. Therefore, the community structure of AOB in the MPN samples partially resembled that in the original activated sludge.

Middle-thermophilic sulfur-oxidizing bacteria Thiomonas sp. RAN5 strain for hydrogen sulfide removal

Hydrogen sulfide (H2S) is one of the most toxic and offensively odorous gases and is generated in anaerobic bioreactors. A middle-thermophilic sulfur-oxidizing bacterium (SOB), Thiomonas sp. strain RAN5, was isolated and applied for H2S removal from both artificial and anaerobically digested gas. When a bioreactor containing medium inoculated with RAN5 was aerated continuously with artificial gas (containing 100 ppm H2S) at 45 ° C for 156 hr, the H2S concentration in the vented gas was reduced by 99%. This was not affected by the presence of other microbes in the bioreactor. The H2S removal efficiency of the RAN5 bioreactor for anaerobically digested gas was greater than 99% at influent H2S concentrations ranging from 2 to 1800 ppm; the efficiency decreased to 90% at influent H2S concentrations greater than 2000 ppm. Thiomonas sp. strain RAN5 cannot survive at room temperature, and thus its leakage from a wastewater treatment plant would not damage sewage systems. These data suggest that Thiomonas sp. strain RAN5 may be a useful microorganism for H2S removal. Implications: The hydrogen sulfide removal efficiency of the RAN5 bioreactor was greater than 99% over the long term, for influent H2S concentrations of up to 2000 ppm. Removal was not affected by the presence of other microbes. Leakage of strain RAN5 would not damage sewage systems.

Detection of Escherichia coli in a cattle manure composting process by selective cultivation and colony polymerase chain reaction

Livestock manure is suitable for use as a composting material. However, various intestinal microbes, such as Escherichia coli, are significant components of such manures. Thus, it is desirable that the level of intestinal microbes, and particularly opportunistic pathogens, in compost is inspected and counted regularly. The sensitivity and specificity of detection of E. coli in compost have been improved by selective cultivation followed by colony polymerase chain reaction (PCR) using the ECO primer. Indeed, the sensitivity of this method is higher than that of DNA extraction from compost and PCR. In this study, changes in numbers of E. coli present in a field-scale composting process over time was assessed using selective cultivation and colony PCR. Numbers of ECO-positive colonies after 24 h decreased, with a concomitant rise in compost temperature. ECO-positive colonies were not detected from 33 to 48 h. However, ECO-positive colony numbers increased beginning on day 4 and continuing until day 42. Thus, it seems likely that the high temperatures reached during the composting process did not affect E. coli numbers in the final compost. Additionally, selective cultivation followed by colony PCR using specific primers is an appropriate method of determining levels of cultivable pathogens in composted materials.