Feasibility analyses of $\textit{Methylosinus trichosporium}$ OB3b bioaugmentation in environmental applications

Abstract

Methanotrophs are widely distributed in the various environments generating methane in very reduced condition. There are many places from which methane gas is being emitted such as rice paddy, landfill and wastewater treatment plant. From researches of these places, this microbial group has received attention in that the microbe mitigates the amounts of methane emitted to the atmosphere. Besides the methane consumption, they have various metabolic activities. They could induce accumulation of nitrous oxide with their copper chelator, called methanobactin, in co-culture with denitrifying bacteria and bioremediation removing ethylene chloride and could produce PHA which is a sort of bio-degradable plastics. To analyze whether their activities would be sustained under natural conditions, this study researched the feasibility of applying methane-oxidizing bacteria to environmental processes. Through applying two aerobic methanotrophs, $\textit{Methylosinus trichosporium}$ OB3b and $\textit{Methylomicrobium album}$ BG8, into aeration tank sample from wastewater treatment plant with bioaugmentation method, the effects of bioaugmentation on microbial communities and their ecological niches were identified. Under 5% of $CH_4$ and $O_2$ , OB3b-amended culture showed the fastest gas consumption rate among the all three microcosms. The OB3b-amended culture also accumulated nitrous oxide significantly while the other two cultures did not. The nitrous oxide production could be a double-edged sword in that the gas should be one of the greenhouse gases and the nitrous oxide could be used as industrial materials such as medial anesthetic. To identify whether the rapid gas consumption was derivated from increasing ratio of the strain OB3b to total bacteria, using quantitative polymerase chain reaction, it was observed that the ratio of the strain OB3b to total bacterial group had been increased within the strain OB3b-amended cultures. Th ratio of the strain BG8 to total bacterial group within the strain BG8-amended cultures had been constant. The absence of the strain OB3b in the strain BG8-amended cultures was assured with primer sets (1055yF/1392R and recAF/recAR for each strain). According to the microbial community analyses, family $\textit{Beijerinkiaceae}$ occupied significant relative abundance within the strain OB3b-amended microcosm meaning the bioaugmentation of the strain OB3b to environmental processes has potential to be realistic.

Another interesting observation is that when wastewater microcosms were incubated with only $O_2$ and $N_2$ without $CH_4$, $O_2$ consumption and $OD_{600nm}$ by indigenous microbes was constant without any increment. However, according to the analyses of microbial communities, ASVs affiliated to non-methanotrophic organisms also enriched. From these results, it can be said that methane-oxidizing bacterial group affected the other non-methanotrophs during consuming methane while supplying various nutrients to their surrounding environments. To get to know which principle exists for the growth of specific microbial taxa, fresh cultures were incubated under 5% of $O_2$. After identifying constant $O_2$ concentration, the pre-incubated control, the strain BG8, OB3b-aemnded microcosms with 5% of $CH_4$ and $O_2$ were centrifugated (10min, 4,000 × $\textit{g}$) and separated into supernatant and cell pellet. The cellular exudates of each pre-incubated cultures were injected into the no-$CH_4$ controls. Without any microbial growth and $O_2$ consumption, the no-CH4 controls also were treated with cell lysates from each pre-incubated cultures. Only one no-$CH_4$ control culture which received cellular metabolites from lysates of pre-incubated the strain OB3b-amended culture represent $O_2$ consumption and microbial growth. Analyses of inorganic nitrogens showed that concentrations of nitrate, nitrite was constant and concentration of ammonium was increased and decreased when microbial growth occurred in the no-$CH_4$ control treated with the strain OB3b-amended culture lysates. With the results and fact that concentration of ammonium was increased at the next day of lysates addition not right after, candidates of cellular metabolites which induced microbial growth include amine which has potential to be used carbon and nitrogen sources such as amino acids.