Phosphonates can also serve as a source of phosphorus or carbon for a variety of microorganisms and several pathways for phosphonate degradation have been characterized . For example, some bacteria can use methylphosphonate as a P source in a process that releases methane and inorganic phosphate . This process is catalyzed by the C-P lyase enzyme and involves a phosphate radical intermediate . Under mildly reducing conditions phosphate radicals can rearrange to form phosphite, making it a possible byproduct of methylphosphonate degradation in anaerobic environments . Moreover, phosphonates with carbonyl or hydroxyl groups at the α-carbon, such as phosphonoformic acid, tend to form phosphite rather than phosphate as the product of C-P cleavage even under oxidizing conditions . Given that C-P lyase enzymes are involved in the degradation of a variety of phosphonates, it is possible that these reactions are a significant source of environmental phosphite. Biological phosphate reduction has also been posited as a possible source of environmental phosphite. Devai and colleagues detected phosphine gas production in wastewater and marsh soils and showed that phosphine production was stimulated by the addition of inorganic phosphate and organic matter, leading them to conclude that phosphate was being reduced to phosphine by microorganisms present in their samples . Some of this phosphine could subsequently be oxidized to phosphite in the presence of O2or UV radiation . However,hydroponic grow kit the conclusion by Devai et al. that the phosphine they observed was derived from biological phosphate reduction has since been questioned by several researchers .

Roels and coworkers have noted that biological phosphate reduction is problematic from a thermodynamic standpoint, since there is no known biological electron donor with a low enough redox potential to make the reaction exergonic . Glindemann and coworkers have shown that phosphine can be produced during the corrosion of iron, even under sterile conditions . This is due to the fact that iron minerals often contain phosphorus impurities that can be abiotically reduced to iron phosphides during the industrial smelting process and these phosphides can then be released as phosphine gas during corrosion . Subsequent studies have likewise concluded that phosphine is released due to iron corrosion and that the higher rates of phosphine production observed in the presence of microorganisms is likely due to the microbial production of organic acids and hydrogen sulfide, which accelerate the corrosion process . Although evidence of biological phosphate reduction remains inconclusive, several theoretical mechanisms by which this process could occur have been proposed. Pasek and colleagues have suggested that in addition to being produced during phosphonate degradation, phosphite could also be formed as a byproduct of phosphonate biosynthesis in reducing environments . They determined that the reductive cleavage of phosphoenolpyruvate by H2 to form phosphite and pyruvate is thermodynamically feasible under standard cellular conditions . Given that phosphoenolpyruvate is a key intermediate in the production of phosphonates from inorganic phosphate, such a mechanism would be a way of indirectly converting phosphate to phosphite. A more direct mechanism of phosphate reduction has been proposed by Roels and colleagues, who note that the reduced molybdoferredoxin cofactor of the nitrogenase complex has a redox potential of -1.0 V, which is low enough to reduce phosphate to phosphite .

However, they question the usefulness of such a reaction since energy from ATP hydrolysis must be expended in order to achieve such a low reduction potential and the organism would gain nothing from the production of phosphite. Nevertheless, it is possible that phosphite may be formed as an unwanted product of nitrogenase function in the presence of phosphate. This sort of inadvertent phosphate reduction might also occur in photosynthetic organisms, since the redox potentials of excited reaction center chlorophyll molecules range from -0.8 V to -1.26 V . Environments dominated by anoxygenic phototrophs may therefore be potential hot spots of biological phosphite production since the absence of strong oxidants in these systems would favor the accumulation of reduced phosphorus species. produced would have to be diverted for use in anabolic reactions.FiPS-3, on the other hand, uses PtdC as its phosphite transporter instead of PtxABC. If PtdC does, in fact, function as a phosphite/phosphate antiporter as has been proposed, then there would be no energy cost associated with phosphite uptake in FiPS-3 . However, when both PtxD and PtdC were expressed in SaxT, it still did not gain the ability to grow by DPO, which indicates that an additional mechanism of energy conservation, possibly mediated by the ptdFGHI genes, is required in this organism. In contrast to P. stutzeri, FiPS-3 and SaxT growing by sulfate reduction would gain substantially less energy from NADH oxidation. During sulfate reduction 2 ATP must be initially expended in order to activate and reduce sulfate to sulfite, which can then be further reduced to sulfide in an exergonic reaction . Sulfate reducing bacteria growing on H2 typically generate 3 ATP from the sulfite reduction step for a net overall production of 1 mol ATP per mol sulfate reduced, which corresponds to the expected yield based on equation 4 .

However, if sulfite reduction were instead coupled to NADH oxidation according to equation 5 , the expected yield would only be 2 mol ATP per mol sulfite reduced, which would result in no net ATP production from the overall reduction of sulfate . In order to grow by DPO, therefore, FiPS-3 and SaxT would not only need to save energy on phosphite uptake, but also conserve more of the free energy available from the oxidation of phosphite. The NAD+ /NADH couple has a redox potential of -320 mV under standard physiological conditions , which means that the reduction of NAD+ coupled to phosphite oxidation releases 63.7 kJ.mol-1 phosphite. This additional energy is presumably lost in traditional APO-capable organisms, but there is evidence that it is conserved in FiPS-3. Schink and coworkers observed substantially higher cell yields when FiPS-3 was grown on phosphite and sulfate versus formate and sulfate . Since phosphite and formate both donate 2 electrons and the redox potential of the CO2/formate couple is actually higher than that of NAD+ /NADH , the higher yields seen on phosphite are not consistent with NADH oxidation being the sole means of ATP production during DPO. Furthermore,vertical farming racks the growth yield of FiPS-3 on phosphite and CO2 via the Wood-Ljungdahl pathway was about 10 times higher than the yields typically observed for other Wood-Ljungdahl acetogens growing on H2 and CO2, such as Acetobacterium woodiiand Acetogenium kivui . These results suggest that FiPS-3 can in fact take advantage of the extremely electronegative redox potential of phosphite, although it is unclear how this is accomplished since there are no known biological redox carriers that can accept electrons at such a low potential . Schink and coworkers have proposed that ATP is generated from phosphite oxidation by means of substrate level phosphorylation in addition to the reduction of NAD+ , thus yielding both energy and reducing equivalents for each molecule of substrate utilized . Such a reaction would be thermodynamically feasible according to equation 6 . Therefore, the function of the ptdFGHI genes may be to facilitate substrate level phosphorylation during phosphite oxidation . Relyea and van der Donk have suggested that one of the possible mechanisms of phosphite oxidation by PtxD may involve the creation of a phosphorylated enzyme intermediate that is subsequently hydrolyzed to release phosphate . PtdFGHI might interact with PtxD in order to facilitate the transfer of this phosphoryl group to ADP, either directly or by means of additional phosphorylated intermediates.

This is a promising avenue for future inquiry but more work is currently necessary in order to determine whether phosphite acts as a phosphoryl donor for ATP synthesis during DPO and what role, if any, the ptdFGHI genes play in this process. Over the last 20 years, the study of reduced phosphorus compounds and their role in nature has grown from a series of curious observations and intriguing theories into an exciting new frontier in biogeochemistry. In particular, recent discoveries regarding the geochemistry and biology of phosphite have highlighted the potential significance of this compound both as a facilitator for the emergence of life on ancient Earth and as a modern driver of microbial processes that continue to shape the global biosphere. Phosphite has been detected in several environments at concentrations that suggest the current existence of a phosphorus redox cycle occurring at short geological timescales. Several anthropogenic sources of phosphite have been identified, and there is evidence that phosphite may also be produced by natural processes such as biological phosphonate metabolism and geothermal phosphate reduction. The presence of the genes responsible for assimilatory phosphite oxidation in hundreds of microbial isolates from a variety of environments indicates that this process is widespread and may have a substantial impact on the global P cycle. Furthermore, the discovery of dissimilatory phosphite oxidation and its ability to sustain carbon fixation while providing an energetic benefit raises the possibility of phosphite as a key, though hitherto unrecognized, driver of primary productivity in the environment. Several milligrams of mineral precipitates were sampled from FiPS-3 cultures grown on phosphite with either calcium or magnesium in the media and mineral samples were ground into a powder. For SEM, a sample of the powdered mineral was suspended in several milliliters of distilled water to create a mineral slurry.A drop of the mineral slurry was fixed with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer, added to the silicon wafers, and allowed to settle for 1 h. Samples were then dehydrated for 10 min in 35%, 50%, 70%, 80%, 95%, and 100% ethanol, followed by critical point drying. Dehydrated samples were mounted onto stubs, sputter coated with palladium/gold, and visualized using a Hitachi S-5000 scanning electron microscope at 20 kV. For XRD, a sample of the powdered mineral was suspended in a few drops of amyl acetate to create a mineral slurry. The mineral slurry was then analyzed with a PANalytical X’Pert Pro diffractometer equipped with a Co x-ray tube and an X’Celerator detector. In accordance with previous observations, DPO-dependent growth of FiPS-3 was accompanied by the appearance of mineral precipitates in the medium several days after the onset of phosphite oxidation . The precipitates appeared to be crystalline and varied in size from several millimeters to several centimeters in length. Typically, some of the crystals would adhere to the bottom and sides of the glass culture tubes, although most would remain suspended in the medium. No precipitates were observed in cultures grown with fumarate as the electron donor instead of phosphite . Subsequent tests showed that DPO-dependent biomineralization could be used to consolidate a fine-grained calcium carbonate matrix at standard temperature and pressure and circumneutral pH. When FiPS-3 was grown in the presence of phosphite, all of the calcium carbonate present in the media was consolidated into a hardened mineral phase that adhered to the bottom of the glass culture bottles, whereas in FiPS-3 cultures grown on fumarate or in sterile phosphite-containing media the calcium carbonate particles remained suspended in the liquid phase . SEM imaging of precipitates from FiPS-3 cultures amended with either calcium or magnesium showed different mineral morphologies depending on which cation was present in the media . Analysis of the precipitates using XRD confirmed that they were crystalline phosphate minerals and that their chemical compositions varied based on the cation present. Hydroxyapatite 26) was produced in the presence of calcium, whereas struvite was produced in the presence of magnesium. The SEM images also appeared to show that some of the cells involved in the biomineralization process became embedded in the mineral phase . Genomic analysis suggested that FiPS-3 was incapable of synthesizing phenylalanine and histidine, and addition of these amino acids to the growth media did indeed result in a drastic reduction in cell doubling time and increase in maximum OD. However, FiPS-3 was still able to grow, albeit poorly, in the absence of phenylalanine and histidine, indicating that it is still able to make these amino acids even though it appears to be missing genes in both biosynthetic pathways.