Transformation mechanism of methylphosphonate to methane by Burkholderia sp: Insight from multi-labeled water isotope probing and transcriptomic.

Methylphosphonate (MPn), has been identified as a likely source of methane in aerobic ocean and may be responsible for the "ocean methane paradox", that is oversaturation of dissolved methane in oxic sea waters. However, the mechanism underlying the cleavage of C-P bonds during microbial degradation is not well understood. Using multi-labeled water isotope probing (MLWIP) and transcriptome analysis, we investigated the phosphate oxygen isotope systematics and mechanisms of microbial-mediated degradation of MPn in this study. In the aerobic culture containing MPn as the only phosphorus source, there was a significant release of inorganic phosphate (149.4 µmol/L) and free methane (268.3 mg/L). The oxygen isotopic composition of inorganic phosphorus (δ18OP) of accumulated released phosphate was 4.50‰, 23.96‰, and 40.88‰, respectively, in the corresponding 18O-labeled waters of -10.3‰, 9.9‰, and 30.6‰, and the slope obtained in plots of δ18OP versus the oxygen isotopic composition of water (δ18OW) was 0.89. Consequently, 89% of the oxygen atoms (Os) in phosphate (PO4) were exchanged with 18O-labeled waters in the medium, while the rest were exchanged with intracellular metabolic water. It has been confirmed that the C-P bond cleavage of MPn occurs in the cell with both ambient and metabolic water participation. Moreover, phn gene clusters play significant roles to cleave the C-P bond of MPn for Burkholderia sp. HQL1813, in which phnJ, phnM and phnI genes are significantly up-regulated during MPn decomposition to methane. In conclusion, the aerobic biotransformation of MPn to free methane by Burkholderia sp. HQL1813 has been elucidated, providing new insights into the mechanism that bio-cleaves C-P bonds to produce methane aerobically in aqueous environments for representative phosphonates.

An optimized method for extraction and purification of inorganic phosphate from plant material for oxygen isotope ratio analysis.

Compound-specific stable isotope ratio analysis of oxygen isotopes in inorganic phosphate can be used to study biological phosphorus cycling and the transformation processes controlling the fate of phosphorus. However, methods for extraction of inorganic phosphate from plant tissue for oxygen isotope ratio analysis are not consistent. Further, the purification into solid silver phosphate can be challenging and laborious. In this work, a detailed and optimized method to provide a more consistent, easily implementable and reproducible extraction using trichloroacetic acid and subsequent purification of inorganic phosphate from plant material for oxygen isotope ratio analysis is presented. Key focus points were: uniform extraction of inorganic phosphate from barley leaves, removal of dissolved organic material, flexibility in regards to the amount of inorganic phosphate extracted for the purification into silver phosphate, reduced use of chemicals and, removal of co-precipitated oxygen-bearing compounds before analysis. Most notable optimizations to the method and associated effects were:•Drying of plant material before inorganic phosphate extraction increases the method applicability to a broader range of plant sample types.•Removal of dissolved organic matter improves inorganic phosphate purification.•Sample volume adjustment according to inorganic phosphate content is vital for effective and quantitative precipitations.

Phosphate oxygen isotope evidence for methylphosphonate sources of methane and dissolved inorganic phosphate.

The ocean is an important source of methane, however, the sources of oceanic methane and mechanisms of its release to the atmosphere have only recently begun to be understood. Recent studies have identified methylphosphonate (MPn) as a previously unknown and likely source of methane in the aerobic ocean (Karl et al., 2008), as well as shown the biosynthesis of methylphosphonic acid to be a widespread trait in marine microbes (Metcalf et al., 2012). The mechanisms and reaction pathways from MPn to free methane, however, have not been well studied. Here we present results of laboratory studies on the photo-degradation of MPn, a likely mechanism of methane release to the atmosphere and phosphate release to the surface oceans. Phosphonoacetic acid was also studied as an additional model compound for comparison. We used the multi-labeled water isotope probing (MLWIP) approach, involving 18O-labeled waters to probe the photolytic mechanism of CP bond cleavage in phosphates through analysis of P released from MPn as PO4. These studies identified distinct reaction pathways involving phosphates compared with other common organophosphorus compounds (e.g., phosphoesters), as well as suggest the involvement of both ambient water and atmospheric oxygen in CP bond cleavage. There is only a small amount of water oxygen incorporated into product PO4 after cleavage of the CP bond in MPn, suggesting atmospheric O2 or radicals formed from O2 under Ultra Violet Radiation (UVR), as the primary source of O that replaces C in the CP bond of MPn. Model calculations suggest that the δ18OP signature of phosphate released via UV-degradation of phosphates is largely (75%) inherited from the original phosphate substrate. This opens up the possibility of tracing and differentiating specific phosphate sources of dissolved phosphate from other organophosphorus (Porg) sources (e.g., phosphoesters) used in primary production, as well as for tracing specific MPn sources of atmospheric methane.