4:30 PM - 4:45 PM
[MIS14-11] Exploring regulators for anammox abundance in the relationship between anammox and coexisiting-bacteria
Keywords:anammox, denitrification, metabolic potential analysis
[Background]
Anaerobic ammonia-oxidizing (anammox) bacteria can couple the anaerobic NH4+ oxidation to NO2- reduction to produce N2, which are significantly involved in the global nitrogen cycle. The anammox bacteria have not yet been successfully isolated despite many years of attempts, and therefore, the influence of the coexisting bacterial community on the growth of anammox bacteria is attracting attention.
In recent years, some studies have been conducted on anammox-enriched systems using culture-independent metagenomic and metatranscriptomic analyses to estimate the interactions between anammox and coexisting bacteria. However, few studies have examined the stable interactions between anammox and coexisting bacteria based on variations in the composition and metabolic potential of anammox consortia over time. In most cases, competitive and cooperative relationships have been inferred by reconstructing the individual genomes of dominant species from metagenomic sequences and intercomparing the functional potentials at the bacterial species level. However, the presence rate and functional importance are not always proportional, as in the case of a bacterial group with a small abundance determining the heavy oil decomposition performance of the entire reactor (Sato et al., 2019).
[Objective]
In this study, we used MAPLE (Takami et al., 2016), a metabolic potential evaluation software based on the KEGG functional module, to evaluate the presence and abundance of metabolic capacity of bacterial communities and the microbial composition responsible for each function. We conducted metagenomic analyses using two anammox reactors supplied with inorganic medium to explore the metabolic potential of coexisting bacteria that showed similar variation to the variation in the abundance of anammox bacteria. Based on the results, we aimed to estimate the functions of the coexisting bacteria that are important for the growth of anammox bacteria.
[Methods]
Biomass collected from an up-flow column reactor using non-woven fabric as a carrier. Collected biomass was homogenized by passing through a sieve (53 µm pore diameter). DNA was extracted by a method based on the protocol provided by ISOIL for beads beating (Nippon Gene), with the addition or modification of enzymatic treatment and bead beating procedure. 250 bp paired end sequencing was performed on NovaSeq, and approximately 3 million reads of amino acid sequence per sample were available for analysis in MAPLE. Bacterial community composition was evaluated based on the composition of sequences annotated on bacterial ribosomal proteins. We also performed BLASTp homology searches on the nr database for sequences classified in the phylum Planctomycetes to evaluate the composition of the anammox community. The presence or absence of each metabolic potentials were defined as the number of reaction steps (%) in which the functional gene (group) responsible for each reaction was detected in relation to all reaction steps in each metabolic module. In addition to this, the process with the lowest number of reads per sequence length of the functional genes responsible for each reaction step was considered as the rate-limiting step, and the abundance of this functional potentials (abundance) was compared.
[Results and Discussion]
Anammox and denitrification are in competition for NO2- as a substrate, while several studies have suggested a cooperative relationship via NO as an intermediate product. In both reactors, the abundance of the enzyme gene responsible for NO reduction was the lowest in each reaction step of denitrification, remaining below 20% of the NO production potential. It suggested that the relationship in which a portion of the NO produced by the denitrifying bacteria is supplied to the anammox bacteria may function. In addition, several pathways for the synthesis of amino acids, cofactors, and vitamins were found that were not satisfied only by the anammox bacteria, but had synthetic potential in the entire bacterial community. Among them, the metabolic abundance of the menaquinone synthesis (futalosine pathway) and the tetrahydrofolate synthesis pathways showed a significant positive correlation (p < 0.05) with the relative abundance of anammox bacteria. Both menaquinone and tetrahydrofolate are essential for the carbon fixation capacity of anammox bacteria, suggesting that anammox bacteria grow in a dependent or constrained manner on the production and release of these substances by coexisting bacteria.
Anaerobic ammonia-oxidizing (anammox) bacteria can couple the anaerobic NH4+ oxidation to NO2- reduction to produce N2, which are significantly involved in the global nitrogen cycle. The anammox bacteria have not yet been successfully isolated despite many years of attempts, and therefore, the influence of the coexisting bacterial community on the growth of anammox bacteria is attracting attention.
In recent years, some studies have been conducted on anammox-enriched systems using culture-independent metagenomic and metatranscriptomic analyses to estimate the interactions between anammox and coexisting bacteria. However, few studies have examined the stable interactions between anammox and coexisting bacteria based on variations in the composition and metabolic potential of anammox consortia over time. In most cases, competitive and cooperative relationships have been inferred by reconstructing the individual genomes of dominant species from metagenomic sequences and intercomparing the functional potentials at the bacterial species level. However, the presence rate and functional importance are not always proportional, as in the case of a bacterial group with a small abundance determining the heavy oil decomposition performance of the entire reactor (Sato et al., 2019).
[Objective]
In this study, we used MAPLE (Takami et al., 2016), a metabolic potential evaluation software based on the KEGG functional module, to evaluate the presence and abundance of metabolic capacity of bacterial communities and the microbial composition responsible for each function. We conducted metagenomic analyses using two anammox reactors supplied with inorganic medium to explore the metabolic potential of coexisting bacteria that showed similar variation to the variation in the abundance of anammox bacteria. Based on the results, we aimed to estimate the functions of the coexisting bacteria that are important for the growth of anammox bacteria.
[Methods]
Biomass collected from an up-flow column reactor using non-woven fabric as a carrier. Collected biomass was homogenized by passing through a sieve (53 µm pore diameter). DNA was extracted by a method based on the protocol provided by ISOIL for beads beating (Nippon Gene), with the addition or modification of enzymatic treatment and bead beating procedure. 250 bp paired end sequencing was performed on NovaSeq, and approximately 3 million reads of amino acid sequence per sample were available for analysis in MAPLE. Bacterial community composition was evaluated based on the composition of sequences annotated on bacterial ribosomal proteins. We also performed BLASTp homology searches on the nr database for sequences classified in the phylum Planctomycetes to evaluate the composition of the anammox community. The presence or absence of each metabolic potentials were defined as the number of reaction steps (%) in which the functional gene (group) responsible for each reaction was detected in relation to all reaction steps in each metabolic module. In addition to this, the process with the lowest number of reads per sequence length of the functional genes responsible for each reaction step was considered as the rate-limiting step, and the abundance of this functional potentials (abundance) was compared.
[Results and Discussion]
Anammox and denitrification are in competition for NO2- as a substrate, while several studies have suggested a cooperative relationship via NO as an intermediate product. In both reactors, the abundance of the enzyme gene responsible for NO reduction was the lowest in each reaction step of denitrification, remaining below 20% of the NO production potential. It suggested that the relationship in which a portion of the NO produced by the denitrifying bacteria is supplied to the anammox bacteria may function. In addition, several pathways for the synthesis of amino acids, cofactors, and vitamins were found that were not satisfied only by the anammox bacteria, but had synthetic potential in the entire bacterial community. Among them, the metabolic abundance of the menaquinone synthesis (futalosine pathway) and the tetrahydrofolate synthesis pathways showed a significant positive correlation (p < 0.05) with the relative abundance of anammox bacteria. Both menaquinone and tetrahydrofolate are essential for the carbon fixation capacity of anammox bacteria, suggesting that anammox bacteria grow in a dependent or constrained manner on the production and release of these substances by coexisting bacteria.