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.

Diversity and abundance of ammonia-oxidizing bacteria and ammonia-oxidizing archaea during cattle manure composting.

Ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) play important roles in nitrification in various environments. They may also be key communities for ammonia oxidation in composting systems, although few studies have discussed their presence. We investigated the relative diversity and abundance of AOB and AOA using cloning procedures, denaturing gradient gel electrophoresis analysis, and real-time PCR during several stages in the process of cattle manure composting. Our results revealed that the AOB community structure changed during the process. At the high-temperature stage (>60°C), a member of the Nitrosomonas europaea/eutropha cluster dominated while the uncultured Nitrosomonas spp. cluster appeared after the temperature decreased. Additionally, our analysis indicated that AOA sequences, which were classified into a soil/sediment cluster, were present after the temperature decreased during the composting process. At these stages, the number of the archaeal amoA gene copies (3.2 or 3.9 × 10(7) copies per gram freeze-dried compost) was significantly higher than that of bacterial amoA gene copies (2.2-7.2 × 10(6) copies per gram freeze-dried compost). Our results suggest that both AOB and AOA are actively involved in nitrification of composting systems.

Isolation of bacteriocin-like substances producing bacteria from finished cattle-manure compost and activity evaluation against some food-borne pathogenic and spoilage bacteria.

A finished compost sample was examined for bacteriocin-like substance production against five pathogenic bacteria: Salmonella typhimurium EF 85-9, Escherichia coli O157:H7 ATCC 43888, Enterococcus faecalis JCM 8726, Staphylococcus aureus JCM 2151, and Yersinia enterocolitica JCM 7577. At the preliminary detection of bacterial strains exhibiting antimicrobial activity from the compost sample, thirteen strains could be isolated. Screening of the inhibitory activity was done using agar-well diffusion assay and Microtiter plate growth assay. Six bacterial strains from the compost showed an antimicrobial activity against one or more of the tested indicator strains. Four strains (M1-M4) belonged to Shigella species and the other two strains (M5 and M6) belonged to Salmonella species. The antimicrobial activity was sensitive for alpha-chymotrypsin and papain. The antimicrobial substances from M3, M4 and M6 were heat stable when heated for 15 min at 121 degrees C with 100% relative activity. The bacteriocin-like substance produced by strain M2 was partially characterized. It exhibited an inhibitory activity against the tested food-borne pathogenic and spoilage bacteria, except Enterobacter aerogenes JCM 1235 and Lactobacillus plantarum subsp. plantarum JCM 1149. It was stable at a wide range of pH (3-11). There was no loss of activity for up to 3 weeks when stored at 4 and -20 degrees C or for up to 2 weeks when stored at 28 and -80 degrees C. This is the first report indicating the presence of bacteriocin-like activity in animal manure compost.

Bacterial populations in epilithic biofilms along two oligotrophic rivers in the Tohoku region in Japan.

Bacterial populations in epilithic biofilms collected from two distinct oligotrophic rivers of Japan were studied using denaturing gradient gel electrophoresis (DGGE). PCR-DGGE of the 16S rRNA gene and subsequent sequencing analysis suggested that in freshwater biofilms, members of the Cytophaga-Flavobacterium-Bacteroides (CFB) group were the most dominant, followed by those of alpha, beta, gamma, and delta-Proteobacteria; Leptospiraceae; and unidentified bacteria. Members of the CFB group, alpha-Proteobacteria, and cyanobacteria/plastid DNA were also detected from the biofilms collected from the estuary site, but the species in these samples differed from those detected in biofilms in the freshwater areas of the rivers. A comparison between the determined sequences revealed that similar bacterial species existed in biofilms at different sites of a river, and identical species existed in biofilms of distinct rivers. The results suggested that bacterial species in biofilms found in the estuary were different from those found in the freshwater areas of the rivers; however, the common bacterial species were distributed in biofilms collected from not only different sites along the same river but also sites in distinct oligotrophic rivers.

Investigation of the microbial community in a microbiological additive used in a manure composting process.

The objectives of this study were to investigate the fate of microorganisms by using cultivation methods as well as DNA analyses in a commercial microbiological additive (MA) in the course of the composting. Almost all the predominant species in the microbial succession during composting process determined by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) were in disagreement with those determined by the clone library method. None of the microbial species in the composting stages corresponded to the microorganisms identified in the MA either by the cultivation method or DNA analysis. The results in regard to predominant microorganisms of the MA detected from the liquid medium by the PCR-DGGE did not correspond with those detected from the MA itself and composting processes. Although no evidence was found that predominant species in the MA itself dominate in the composting process, predominant species diversity in the MA itself was markedly changed after culturing at different thermophilic temperatures. These results suggested that cultivable microorganisms in the MA did not become predominant in the composting process: however, some microorganisms that are detected from the MA itself by the DNA analysis may act effectively in the composting process.

Population dynamics of phage-host system of Microlunatus phosphovorus indigenous in activated sludge.

Monitoring of the phage-host system of Microlunatus phosphovorus indigenous in activated sludge was attempted. A laboratory-scale activated sludge process was operated for 5 weeks with synthetic wastewater. The phage-host system population in the process was monitored by plaque assay and FISH methods at every 3 days. During the process operation, the phage-host system populations were more or less steady, except for 1 week in the middle of the operation. In that period, initially M. phosphovorus decreased significantly and its lytic bacteriophages increased, and then M. phosphovorus increased back to its original level while its lytic bacteriophages decreased. This observation suggests that lytic bacteriophages should be considered as one of the biological factors affecting the bacterial population dynamics in activated sludge processes.

Ammonia-oxidizing Archaea in laboratory-scale activated sludge systems for wastewater of low- or high-ammonium concentration.

Ammonia-oxidizing bacteria (AOB) is known as ammonia-oxidizer in wastewater treatment systems. However, ammonia-oxidizing Archaea (AOA) is found from various environments, including wastewater treatment systems. In this study, to investigate the relationships between AOA population and ammonia concentration, AOA was monitored in two laboratory-scale reactors treating artificial wastewater of different ammonium concentrations by denaturing gradient gel electrophoresis targeting ammonia monooxygenase genes. At day 60 of the operation, AOA populations dominant in each reactor differed, suggesting the importance of influent ammonia concentration in dominant AOA selection.

Establishment of evaluation method to determine effects of veterinary medicinal products on manure fermentation using small-scale composting apparatus.

To evaluate on a laboratory scale the influence of veterinary medicinal products (VMPs) excreted into feces on manure fermentation, we have developed an evaluation method that uses a small-scale composting apparatus. Each run is of approximately 3 kg scale and the operation can be conducted in an environmentally controlled laboratory. The main evaluation parameter is calorific value generated by aerobic fermentation. At the sulfadimethoxine (SDM) trial, the volume of CO(2) generated during fermentation and the disappearance of the inhibitory effect of immature manure on sprouting (using Komatsuna (Brassica rapa var. perviridis)) were measured. In addition, DNA of 16S rRNA was extracted from a manure sample and subjected to denaturing gradient gel electrophoresis (DGGE). The results suggest that the presence of such VMPs in feces affected the microbial community in manure fermentation, and indicate that the evaluation method may be used as a standard method to evaluate the effect of VMPs on the microbial community. Using the method, we obtained data of the influence of five VMPs approved for stockbreeding in Japan on swine manure fermentation. Erythromycin (EM) affected the calorific value even at a relatively low concentration (105 mg/3 kg manure). In contrast, oxytetracycline hydrochloride (OTC), norfloxacin (NFLX), and tylosin tartrate (TS) had no effect at that concentration. These VMPs also affected the increase of fermentation temperature when added at high concentrations.

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.

The first detection of Cryptosporidium deer-like genotype in cattle in Japan.

The general perception is that cattle are major reservoirs for Cryptosporidium parvum infections in humans and that C. parvum is a major cause of diarrhea and production loss in cattle. Adult cattle may play an important role as cryptic carrier of the infection. Cryptosporidium spp. in asymptomatic adult dairy cattle from some farms around Osaki area, Miyagi prefecture, Japan, was examined on a field visit during August, 2007, by polymerase chain reaction techniques for detection, genotyping, and subtyping. Cryptosporidium oocysts were detected in the feces of five out of 50 animals. Of the five Cryptosporidium-positive specimens available for molecular analysis, C. parvum was identified in three specimens, Cryptosporidium deer-like genotype in one, and Cryptosporidium andersoni in one specimen. Amplification of Cpgp60 from C. andersoni and Cryptosporidium deer-like genotype samples revealed that these samples have light concurrent C. parvum infection. Sequence analysis of the 60-kDa glycoprotein gene indicated that all C. parvum samples are IIa subtype. Detection of Cryptosporidium deer-like genotype is geographically unique in Japan. The genetic diversity of Cryptosporidium in dairy cattle in Japan may be much greater than that reported before.

Abundance, diversity, and dynamics of viruses on microorganisms in activated sludge processes.

We examined the abundance of viruses on microorganisms in activated sludge and the dynamics of their community structure. Direct counting with epifluorescence microscopy and pulsed-field gel electrophoresis (PFGE) were applied to 20 samples from 14 full-scale wastewater treatment plants (wwtps) treating municipal, industrial, or animal wastewater. Furthermore, to observe the dynamics of viral community structure over time, a laboratory-scale sequencing batch reactor was operated for 58 days. The concentrations of virus particles in the wwtps, as quantified by epifluorescence microscopy, ranged from 4.2 x 10(7) to 3.0 x 10(9) mL-1. PFGE, improved by the introduction of a higher concentration of Tris-EDTA buffer in the DNA extraction step, was successfully used to profile DNA viruses in the activated sludge. Most of the samples from different wwtps commonly had bands in the 40-70 kb range. In the monitoring of viral DNA size distribution in the laboratory-scale reactor, some bands were observed stably throughout the experimental period, some emerged during the operation, and others disappeared. Rapid emergence and disappearance of two intense bands within 6 days was observed. Our data suggest that viruses--especially those associated with microorganisms--are abundant and show dynamic behavior in activated sludge.

Molecular analysis of ammonia-oxidizing bacteria community in intermittent aeration sequencing batch reactors used for animal wastewater treatment.

Bacterial communities and betaproteobacterial ammonia-oxidizing bacteria (AOB) communities were evaluated seasonally in an intermittent-aeration sequencing batch process (SBR, plant A) and in 12 other livestock wastewater treatment plants (WWTP): eight SBRs and four conventional activated-sludge systems. Microbial communities were analysed by reverse transcription polymerase chain reaction followed by denaturing-gradient gel electrophoresis (DGGE) and the construction of clone libraries for 16S rRNA and ammonia monooxygenase (amoA) genes. In plant A, the dominant bacteria were as-yet-uncultured bacteria of Bacteroidetes and Proteobacteria, and the DGGE profiles showed that the bacterial communities were stable during a given treatment cycle, but changed seasonally. In betaproteobacterial AOB communities, two AOB phylotypes (members of the Nitrosomonas ureae-oligotropha-marina cluster) were dominant during the seasons in plant A. Although the dominant AOB phylotypes differed among the 13 WWTPs, dominance by one or two AOB phylotypes was commonly observed in all plants. Sequencing of the DGGE bands indicated that amoA sequences belonging to the Nitrosomonas europaea-eutropha cluster were dominant in 11 plants, where the ammonia-nitrogen concentration was high in the raw wastewater, whereas those belonging to the Nitrosomonas ureae-oligotropha-marina cluster were dominant in two plants where the concentration was relatively low. Even though we detected many minor amoA sequences by means of five clone libraries for the A to D plants, no libraries comprised both amoA sequences belonging to the two clusters, indicating that the dominant AOBs were defined by cluster level in each plant.