Kenichi Otawa

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 × 107 copies per gram freeze-dried compost) was significantly higher than that of bacterial amoA gene copies (2.2–7.2 × 106 copies per gram freeze-dried compost). Our results suggest that both AOB and AOA are actively involved in nitrification of composting systems.

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.

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.

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.

Bacteriophage control of vancomycin-resistant enterococci in cattle compost.

To isolate bacteriophage that infects vancomycin‐resistant enterococci (VRE) and to investigate the ability of this phage to diminish VRE number in vitro and in experimentally VRE‐inoculated compost.

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.

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.