17:15 〜 19:15
[MZZ42-P06] Do freshwater bacteria consume methylphosphonic acid?
キーワード：メチルホスホン酸、イオンクロマトグラフィー、淡水湖沼、リン循環、細菌による代謝、メタンパラドックス
1. Introduction
Methylphosphonic acid (MPn) is a phosphonic compound with a methyl group. Aquatic microorganisms primarily consume orthophosphate (Pi) but not MPn. However, in Pi depleted condition, MPn can be utilized to ingest Pi, and then methane is produced as the by-product of MPn decomposition. It might be one of the causes of methane paradox, namely the existence of high methane concentration layers in oxic environments (e.g. surface and subsurface layers of pelagic oceans and lakes). Methane is known as a potent greenhouse gas. Thus, MPn may have two important roles, such as “phosphorus supplement” and “methane production” on material cycling in aquatic environments. In this research, we tried to elucidate whether bacteria consume MPn in a freshwater lake by MPn quantification and bacterial isolation.
2. Material and Methods
Sampling and Quantification: Water and bacterial samples were collected in Lake Saiko basin and its inflow (Irisawa Stream) in summer of 2021, 2023 and 2024. Water temperature (℃) and DO (%) were measured by CTD in situ. The concentration of Pi (nmol/L) was determined by ion chromatography (IC) and CH4 (nmol/L) was measured by gas chromatography. Then, we concentrated MPn by Fe (OH)3 co-precipitation method and measured by IC (Yamamoto in 2021).
Isolation of bacteria with the ability of consuming MPn: We used four BG11 media with different condition for P (1: 1.8 µmol/L MPn, 2: 1.8 µmol/L MPn + Pi, 3: 1.8 µmol/L Pi, 4: No addition). We deal with bacteria in MPn medium as bacteria which has utilize MPn. Other media were “controlled”. The incubations were performed at 20 ℃ under 10 h light : 14 h dark cycle (light = 5-14 μmol-photon/m2・s). The incubation period was three months.
3. Results and Discussion
Quantification: MPn concentrations ranged from below the limit of detection (LOD: approx. 0.06 nmol/L) to 2.45 nmol/L in August and October during 2021 and 2023 (Fig.1). MPn concentrations tended to increase at the thermocline (10-13 m depth) in this term. The results may be attributed to three factors:
1. Sinking and decaying of High Molecular Weight Dissolved Organic Matter (HMWDOM) at the thermocline: HMWDOM with >1 kDa harvested through ultrafiltration (UF) can be easily degraded, resulting in the production of Low Molecular Weight Dissolved Organic Matter (LMWDOM) as has been observed in the Pacific Ocean. If HMWDOM sinks from upper layer to lower layers and is decayed by bacteria at the thermocline, it may produce LMWDOM such as MPn.
2. Input of MPn via inflow streams: Upstream rivers may supply MPn to the lake. In 2023, MPn concentration of Irisawa stream reached 1.2 nmol/L (October in 2023) and was higher than those of Lake Saiko. Inflow of MPn from streams thus might be an important source for MPn dynamics in lakes.
3. Formation of MPn by MPn-synthesizing bacteria: In an oligotrophic upper layer in the Pacific Ocean, Nitrosopumilus maritimus may produce MPn by MPn Synthase (MPnS). This species may utilize MPn instead of Pi. Similarly, when Pi is depleted in P-limited lakes, MPn may be produced by bacteria with the ability of MPn synthesis.
Contrasting to the observed pattern in 2021 and 2023, MPn was not detected at all depths in 2024. DO (%) was supersaturated exceeding 100 % at the depth of 9-13 m. Pi concentrations varied at the depth of 6-14 m, with the lowest concentration of 4.2 nmol/L. Thus, MPn and Pi concentrations were lower than those of 2023, suggesting that both P compounds have been seasonally consumed by bacteria and depleted in the lake waters.
Bacterial isolation: During the incubation of water of Lake Saiko, many bacterial colonies were observed in BG11 media with MPn as a sole P source. These colonies were formed not only on MPn media but also on control media. Although bacterial species are not yet clear, the result indicates that bacteria capable of consuming MPn exists in Lake Saiko. Such planktonic bacterial community may have an important role in phosphorus cycling in oligotrophic lake ecosystem.
Methylphosphonic acid (MPn) is a phosphonic compound with a methyl group. Aquatic microorganisms primarily consume orthophosphate (Pi) but not MPn. However, in Pi depleted condition, MPn can be utilized to ingest Pi, and then methane is produced as the by-product of MPn decomposition. It might be one of the causes of methane paradox, namely the existence of high methane concentration layers in oxic environments (e.g. surface and subsurface layers of pelagic oceans and lakes). Methane is known as a potent greenhouse gas. Thus, MPn may have two important roles, such as “phosphorus supplement” and “methane production” on material cycling in aquatic environments. In this research, we tried to elucidate whether bacteria consume MPn in a freshwater lake by MPn quantification and bacterial isolation.
2. Material and Methods
Sampling and Quantification: Water and bacterial samples were collected in Lake Saiko basin and its inflow (Irisawa Stream) in summer of 2021, 2023 and 2024. Water temperature (℃) and DO (%) were measured by CTD in situ. The concentration of Pi (nmol/L) was determined by ion chromatography (IC) and CH4 (nmol/L) was measured by gas chromatography. Then, we concentrated MPn by Fe (OH)3 co-precipitation method and measured by IC (Yamamoto in 2021).
Isolation of bacteria with the ability of consuming MPn: We used four BG11 media with different condition for P (1: 1.8 µmol/L MPn, 2: 1.8 µmol/L MPn + Pi, 3: 1.8 µmol/L Pi, 4: No addition). We deal with bacteria in MPn medium as bacteria which has utilize MPn. Other media were “controlled”. The incubations were performed at 20 ℃ under 10 h light : 14 h dark cycle (light = 5-14 μmol-photon/m2・s). The incubation period was three months.
3. Results and Discussion
Quantification: MPn concentrations ranged from below the limit of detection (LOD: approx. 0.06 nmol/L) to 2.45 nmol/L in August and October during 2021 and 2023 (Fig.1). MPn concentrations tended to increase at the thermocline (10-13 m depth) in this term. The results may be attributed to three factors:
1. Sinking and decaying of High Molecular Weight Dissolved Organic Matter (HMWDOM) at the thermocline: HMWDOM with >1 kDa harvested through ultrafiltration (UF) can be easily degraded, resulting in the production of Low Molecular Weight Dissolved Organic Matter (LMWDOM) as has been observed in the Pacific Ocean. If HMWDOM sinks from upper layer to lower layers and is decayed by bacteria at the thermocline, it may produce LMWDOM such as MPn.
2. Input of MPn via inflow streams: Upstream rivers may supply MPn to the lake. In 2023, MPn concentration of Irisawa stream reached 1.2 nmol/L (October in 2023) and was higher than those of Lake Saiko. Inflow of MPn from streams thus might be an important source for MPn dynamics in lakes.
3. Formation of MPn by MPn-synthesizing bacteria: In an oligotrophic upper layer in the Pacific Ocean, Nitrosopumilus maritimus may produce MPn by MPn Synthase (MPnS). This species may utilize MPn instead of Pi. Similarly, when Pi is depleted in P-limited lakes, MPn may be produced by bacteria with the ability of MPn synthesis.
Contrasting to the observed pattern in 2021 and 2023, MPn was not detected at all depths in 2024. DO (%) was supersaturated exceeding 100 % at the depth of 9-13 m. Pi concentrations varied at the depth of 6-14 m, with the lowest concentration of 4.2 nmol/L. Thus, MPn and Pi concentrations were lower than those of 2023, suggesting that both P compounds have been seasonally consumed by bacteria and depleted in the lake waters.
Bacterial isolation: During the incubation of water of Lake Saiko, many bacterial colonies were observed in BG11 media with MPn as a sole P source. These colonies were formed not only on MPn media but also on control media. Although bacterial species are not yet clear, the result indicates that bacteria capable of consuming MPn exists in Lake Saiko. Such planktonic bacterial community may have an important role in phosphorus cycling in oligotrophic lake ecosystem.