バイオレメディエーションとバイオエネルギー生産のための細菌間相互作用を理解するための微生物群集動態解析

九州工業大学博士学位論文（要旨）学位記番号：生工博甲第275号 学位授与年月日：平成28年12月27

バイオレメディエーションとバイオエネルギー生産のための細菌間相互作用を理解するための微生物群集動態解析

Waste activated sludge (WAS) is a by-product that has been abundantly produced from wastewater treatment plant after secondary biological treatment. The disposal of WAS through landfill or incineration has created more vulnerable problems in term of cost and environmental issues. Many strategies have adapted bioremediation and bioenergy production in order to completely utilize this WAS for sustainable environment and biodiversity conservation. These processes are driven by a complex microorganisms in WAS. Therefore, the successful achievement of both strategies; bioremediation and bioenergy production can be clarified based on the microbial community profile through next-generation sequencing analysis, MiSeq. Bioremediation strategies utilize specific and powerful exogenous microorganisms to assist in the degradation of various harmful pollutants in WAS. However, this inoculant is unable to retain in WAS for a long period of time to carry out their specific functions in degradation. The interaction between this inoculant and indigenous microbes presence in WAS is suspected to be one of the main reasons. Therefore, through the microbial community analysis in this study, some indigenous bacteria have been identified as bacterial soldiers that are responsible in killing the inoculant bacteria. The enhanced green fluorescence protein (EGFP) expressing E. coli was used as the inoculant with the fluorescence marker and some antibiotic-resistant genes. The indigenous bacteria identified as bacterial soldiers were mainly from Comamonadaceae, Myxococcales and Sphingobacteriales communities as later proved by co-culture interaction with inoculant. Bioenergy production through methane fermentation is another approach that has been taken to utilize WAS. Many studies have been done in laboratory scale to find an efficient methane production approach including application of antibiotic, azithromycin. The effect of antibiotics on microbial diversity during anaerobic digestion stages is important to understand their mechanisms and functions in methane production. Therefore, in this study, different antibiotics that produced different methane profile were used to clarify the microbial interactions and regulatory systems in each stages of anaerobic digestion process. During anaerobic digestion, hydrolysis and fermentation stages were efficiently occurred due to the activation of hydrolytic and fermentative bacterial communities by all antibiotics used in this study. However, some antibiotics have shaped the unfavorable conditions for methanogens in methanogenesis stages. This unbalanced of microbial communities has affected on methane production. Thus, a balanced of microbial communities in all stages of anaerobic digestion is important for efficient methane production as shown by some antibiotics, including azithromycin. The importance of microbial communities was shown in bioremediation and methane fermentation processes. Another application using by-product taken from refining process of palm oil industry, phospholine gum was done for methane fermentation in WAS. It was found that phospholine gum has inhibited methane production but not affected other anaerobic digestion stages. Therefore, based on these phenomena, phospholine gum has potential to be applied as feed additive for ruminants in mitigating the enteric methane emission which contribute to greenhouse gases. In conclusion, various reflections and dynamics have been shown by microbial diversity due to the different effects and conditions in the biological processes. This has provided a better and clear understanding on their functions and mechanisms in ensuring a successful bioremediation process and bioenergy production as the excellent approaches for sustainable environment and biodiversity conservation.九州工業大学博士学位論文 学位記番号：生工博甲第275号 学位授与年月日：平成28年12月27日1 Introduction and literature review|2 General materials and methods|3 Bacterial soldiers in waste activated sludge – understanding of the bacterial natural selection for the future successful bioremediation –|4 Impact of different antibiotics on methane production using waste-activated sludge: mechanisms and microbial community dynamics|5 Effect of macrolides and lincosamides on methane production using waste activated sludge: mechanisms and microbial community dynamics|6 Inhibition of a mimic enteric methane fermentation by a palm oil industrial waste, phospholine gum – understanding of microbial community dynamics –|7 Concluding remarks and suggestions for further research九州工業大学平成28年

Effect of Aso limonite on anaerobic digestion of waste sewage sludge

The effect of Aso volcanic limonite was explored in anaerobic digestion using waste sewage sludge (WSS). In this study, methane and hydrogen sulfide were remarkably inhibited when Aso limonite was mixed with WSS as well as a significant reduction of ammonia. Although pH was lowered after adding Aso limonite, methane was still inhibited in neutralized pH condition at 7.0. Hydrolysis stage was not influenced by Aso limonite as supported by the result that a high protease activity was still detected in the presence of the material. However, acidogenesis stage was affected by Aso limonite as indicated by the different productions of organic acids. Acetic acid, was accumulated in the presence of Aso limonite due to the inhibition of methane production, except in the highest concentration of Aso limonite which the production of acetate may be inhibited. Besides, the production of propionate and butyrate reduced in accordance to the increased concentration of Aso limonite. In addition, Archaeal activity (methanogens) in WSS with Aso limonite was low in agreement with the low methane production. Thus, these results indicate that Aso limonite influences the acidogenesis and methanogenesis processes, by which the productions of methane and ammonia were inhibited. On the other hand, in the contactless of Aso limonite during the anaerobic digestion of WSS (Aso limonite was placed in the area of headspace in the vial), Aso limonite had the adsorptive ability for hydrogen sulfide from WSS, but not for methane. This contactless system of Aso limonite may be a practical means to remove hydrogen sulfide without inhibiting methane production as an important bioenergy source

Brown rice as a potential feedstuff for poultry

Rice, especially brown rice, has the potential to replace corn as a feedstuff for poultry. It is an inexpensive local feed source with high availability and low production and processing costs. Two local varieties of brown rice, MR239 and MR257, were investigated for use as feedstuffs in the poultry industry, including their composition and TME values (using the force-feeding technique). The MR239 and MR257 varieties of brown rice contained nutrients such as CP, fat, ash, and carbohydrates. The energy content and amino acid profile of MR239 and MR257 are reported. The nonstarch polysaccharides in MR239 and MR257 consisted of CF, NDF, ADF, and acid detergent lignin. The β-glucan and arabinoxylan contents in MR239 and MR257 were determined. Both varieties of brown rice were found to be potential sources of feed for poultry

Seeking key microorganisms for enhancing methane production in anaerobic digestion of waste sewage sludge

Efficient approaches for the utilization of waste sewage sludge have been widely studied. One of them is to use it for the bioenergy production, specifically methane gas which is well-known to be driven by complex bacterial interactions during the anaerobic digestion process. Therefore, it is important to understand not only microorganisms for producing methane but also those for controlling or regulating the process. In this study, azithromycin analogs belonging to macrolide, ketolide, and lincosamide groups were applied to investigate the mechanisms and dynamics of bacterial community in waste sewage sludge for methane production. The stages of anaerobic digestion process were evaluated by measuring the production of intermediate substrates, such as protease activity, organic acids, the quantification of bacteria and archaea, and its community dynamics. All azithromycin analogs used in this study achieved a high methane production compared to the control sample without any antibiotic due to the efficient hydrolysis process and the presence of important fermentative bacteria and archaea responsible in the methanogenesis stage. The key microorganisms contributing to the methane production may be Clostridia, Cladilinea, Planctomycetes, and Alphaproteobacteria as an accelerator whereas Nitrosomonadaceae and Nitrospiraceae may be suppressors for methane production. In conclusion, the utilization of antibiotic analogs of macrolide, ketolide, and lincosamide groups has a promising ability in finding the essential microorganisms and improving the methane production using waste sewage sludge

Biochar enhanced the nitrifying and denitrifying bacterial communities during the composting of poultry manure and rice straw

Biochar has proven to be a feasible additive for mitigating nitrogen loss during the composting process. This study aims to evaluate the influence of biochar addition on bacterial community and physicochemical properties changes, including ammonium (NH4+), nitrite (NO2−) and nitrate (NO3−) contents during the composting of poultry manure. The composting was carried out by adding 20% (w/w) of biochar into the mixture of poultry manure and rice straw with a ratio of 2:1, and the same treatment without biochar was prepared as a control. The finished product of control compost recorded the high contents of NO2− and NO3− (366 mg/kg and 600 mg/kg) with reduced the total NH4+ content to 10 mg/kg. Meanwhile, biochar compost recorded a higher amount of total NH4+ content (110 mg/kg) with low NO2− and NO3− (161 mg/kg and 137 mg/kg) content in the final composting material. The principal component analysis showed that the dynamics of dominant genera related to Halomonas, Pusillimonas, and Pseudofulvimonas, all of which were known as nitrifying and denitrifying bacteria, was significantly correlated with the dynamic of NO2− and NO3− content throughout the composting process. The genera related to Pusillimonas, and Pseudofulvimonas appeared as the dominant communities as the NO2− and NO3− increased. In contrast, as the NO2− and NO3− concentration decreased, the Halomonas genus were notably enriched in biochar compost. This study revealed the bacterial community shifts corresponded with the change of physicochemical properties, which provides essential information for a better understanding of monitoring and improving the composting process

Inhibition of methane production by the palm oil industrial waste phospholine gum in a mimic enteric fermentation

The potential utilization of phospholine gum, a by-product of the palm oil industry was evaluated using waste sewage sludge (WSS) as a substrate as well as a microbial source to mimic methane production by enteric fermentation. Ruminant animals release enteric methane through their digestive process. The enteric methane is one of the greenhouse gasesthat can contribute to global warming and should be prevented. In this study, methane production was remarkably inhibited by adding phospholine gum to WSS, even at a low concentration. Phospholine gum reduced the activity of methanogens and Lactobacillus sp. and Megasphaera sp. which are known as important ruminal microorganisms were detected as bacterial species induced by the addition of phospholine gum to WSS. Also, the addition of phospholine gum triggered an increase in protein concentrations as well as proteaseactivities and stimulated to produce protease and cellulase by which phospholine gum may be degraded. Furthermore, a significant amount of propionate was produced in the presence of phospholine gum. Thus, phospholine gum inhibits methane production without inhibiting the stages of hydrolysis and acidogenesis/acetogenesis. Finally, methane fermentation using the rumen derived from a goat was also inhibited by phospholine gum. Therefore, these results indicate that the phospholine gum has great potential to inhibit methane production as a feed additive for ruminant animals

Endolithic microbial habitats hosted in carbonate nodules currently forming within sediment at a high methane flux site in the sea of Japan

Concretionary carbonates in deep-sea methane seep fields are formed as a result of microbial methane degradation, called anaerobic oxidation of methane (AOM). Recently, active microorganisms, including anaerobic methanotrophic archaea, were discovered from methane seep-associated carbonate outcroppings on the seafloor. However sedimentary buried carbonate nodules are a hitherto unknown microbial habitat. In this study, we investigated the microbial community structures in two carbonate nodules collected from a high methane flux site in a gas hydrate field off the Oki islands in the Sea of Japan. The nodules were formed around sulfate-methane interfaces (SMI) corresponding to 0.7 and 2.2 m below the seafloor. Based on a geochemical analysis, light carbon isotopic values ranging from −54.91‰ to −37.32‰ were found from the nodules collected at the shallow SMI depth, which were attributed to the high contributions of AOM-induced carbonate precipitation. Signatures of methanotrophic archaeal populations within the sedimentary buried nodule were detected based on microbial community composition analyses and quantitative real-time PCR targeted 16S rRNA, and functional genes for AOM. These results suggest that the buried carbonate nodule currently develops AOM-related microbial communities, and grows depending on the continued AOM under high methane flux conditions

Complete genome sequence of fowl adenovirus-8b UPM04217 isolate associated with the inclusion body hepatitis disease in commercial broiler chickens in Malaysia reveals intermediate evolution

The main aim of our study was to explore the genome sequence of the inclusion body hepatitis associated Fowl adenovirus serotype 8b (FAdV-8b) UPM04217 and to study its genomic organisation. The nucleotide sequence of the whole genome of FAdV-8b UPM04217 was determined by using the 454 Pyrosequencing platform and the Sanger sequencing method. The complete genome was found to be 44,059 bp long with 57.9% G + C content and shared 97.5% genome identity with the reference FAdV-E genome (HG isolate). Interestingly, the genome analysis using ORF Finder, Glimmer3 and FGENESV predicted a total of 39 open reading frames (ORFs) compared to the FAdV-E HG that possessed 46 ORFs. Fourteen ORFs located within the central genomic region and 16 ORFs located within the left and right ends of the genome were assigned as being the high protein-coding regions. The fusion of the small ORFs at the right end terminal specifically in ORF22 and ORF33 could be the result of gene truncation in the FAdV-E HG. The frame shift mutation in ORF25 and other mutations in ORF13 and ORF17 might have lead to the emergence of genes that could have different functions. Besides, one of the minor capsid components, pVI, in FAdV-8b UPM04217 shared the highest similarity of 93% with that of FAdV-D, while only 47% similarity was found with FAdV-E. From the gene arrangement layout of the FAdV genome, FAdV-8b UPM04217 showed intermediate evolution between the FAdV-E HG and the FAdV-D although it was apparently more similar to the FAdV-E HG

Dynamics of microbial populations responsible for biodegradation during the full-scale treatment of palm oil mill effluent

Despite efforts to address the composition of the microbial community during the anaerobic treatment of palm oil mill effluent (POME), its composition in relation to biodegradation in the full-scale treatment system has not yet been extensively examined. Therefore, a thorough analysis of bacterial and archaeal communities was performed in the present study using MiSeq sequencing at the different stages of the POME treatment, which comprised anaerobic as well as facultative anaerobic and aerobic processes, including the mixed raw effluent (MRE), mixing pond, holding tank, and final discharge phases. Based on the results obtained, the following biodegradation processes were suggested to occur at the different treatment stages: (1) Lactobacillaceae (35.9%) dominated the first stage, which contributed to high lactic acid production; (2) the higher population of Clostridiaceae in the mixing pond (47.7%) and Prevotellaceae in the holding tank (49.7%) promoted acetic acid production; (3) the aceticlastic methanogen Methanosaetaceae (0.6-0.8%) played a role in acetic acid degradation in the open digester and closed reactor for methane generation; (4) Syntrophomonas (21.5-29.2%) appeared to be involved in the degradation of fatty acids and acetic acid by syntrophic cooperation with the hydrogenotrophic methanogen, Methanobacteriaceae (0.6-1.3%); and (5) the phenols and alcohols detected in the early phases, but not in the final discharge phase, indicated the successful degradation of lignocellulosic materials. The present results contribute to a better understanding of the biodegradation mechanisms involved in the different stages of the full-scale treatment of POME