This discrepancy could be due to misannotations in the genome or to the existence of alternative biosynthetic pathways. In any case, the addition of phenylalanine and histidine greatly improved the growth and phosphite oxidation rate of FiPS-3 cultures. The growth benefit conferred by these two amino acids was similar to that of yeast extract or casamino acids and was probably due to their ability to serve as organic carbon and nitrogen sources. FiPS-3 has previously been characterized as a strict anaerobe, and the presence of cytochrome c oxidase genes in its genome has been attributed to an uncharacterized mechanism of nitrite resistance, although this hypothesis was never tested . Here I show that FiPS-3 is not only capable of growing under aerobic conditions, but indeed grows better in the presence of oxygen than in controls containing no electron acceptor. This observation, coupled with the presence of cox genes in its genome, suggests that FiPS-3 is capable of oxygen respiration. This is an unexpected result given that oxygen tolerance is rare among sulfate reducing bacteria and that the capacity for aerobic respiration is rarer still, with only a handful of oxygen-respiring sulfate reducers having been documented so far . However,hydroponic indoor growing system the fact that FiPS-3 grew at a similar rate in the presence of oxygen as it did under sulfate-reducing conditions is puzzling, since oxygen is a substantially more favorable electron acceptor from a thermodynamic point of view and should, therefore, allow for faster growth. This may be indicative of a failure on the part of FiPS-3 to adequately cope with oxidative stress, which imposes a growth cost under aerobic conditions.
Its inability to grow in a well-aerated culture lends support to this hypothesis and highlights the fact that the actual dissolved oxygen concentrations it was exposed to in unmixed cultures were likely well below the concentrations present in the head space. As such, it may be more accurate to regard FiPS-3 as a microaerophile rather than a true facultative aerobe. Furthermore, it is also possible that FiPS-3 was not actually growing by aerobic respiration in oxygen-containing cultures but rather growing by sulfate reduction with the oxygen serving to re-oxidize any sulfide produced back to sulfate. Even though sulfate was not added to the aerobic cultures, even a small amount carried over from the inoculum could have supported the observed growth if the sulfide produced was continuously reacting with oxygen to replenish the sulfate pool available to the cells. Further experiments, such as quantification of cox gene expression levels during aerobic growth or assessment of aerobic growth in the presence of complex IV inhibitors, are needed in order to distinguish between these two scenarios. In my FiPS-3 cultures, growth by DPO led to the precipitation of struvite or hydroxyapatite crystals when magnesium or calcium were added to the media. Schink et al. have previously observed the formation of struvite crystals during phosphite oxidation by FiPS-3 cultures. They attributed this phenomenon to the accumulation of inorganic phosphate to high concentrations in the extracellular milieu and the subsequent reaction of this phosphate with magnesium ions present in the medium to form crystalline mineral precipitates. This process of DPO-dependent biomineralization exploits the large difference in solubility between phosphite and phosphate and could potentially be applied to the development of bio-concrete. Bio-concrete refers to ‘self-healing’ concrete that incorporates bio-mineralizing microorganisms in order to help seal cracks that develop over time and thus improve durability and strength .
Most of the research conducted in this area has so far focused on the precipitation of calcium carbonates by heterotrophic bacteria . However, the precipitation of calcium and magnesium phosphate minerals by means of a chemolithoautotrophic DPO-capable bacterium may present an alternate approach for the advancement of this technology. Although upregulation of the PtdF protein in the presence of phosphite has been previously reported , this is the first evidence of increased expression of the entire ptx-ptd gene cluster under phosphite-oxidizing conditions. This finding affirms the connection between the ptd genes and DPO and lends further support to the hypothesis that these genes are necessary for growth by means of phosphite oxidation. It is important to note that 1 mM phosphate was present in both growth conditions, so that the increased expression of these genes was not due to phosphate starvation, which is known to induce expression of ptx genes in APOcapable organisms . However, the substantial difference in expression levels seen between the ptx and ptd genes was unexpected and implies that ptxDE and ptdFCGHI may represent two distinct functional modules that are differentially regulated. The fact that ptxDE are also present in APO-capable organisms in combination with an alternate phosphite transporter also supports the notion that the ptx and ptd genes represent separate modules. As discussed in Chapter 1, PtxD is known to be the enzyme responsible for phosphite oxidation both in APO organisms and in FiPS-3, while the function of the predicted transcriptional regulator ptxE remains a mystery. It is possible that the role of ptxE is actually to promote transcription of ptdFCGHI in the presence of phosphite, which could explain the higher levels of expression seen for these genes. Given that PtxD is highly efficient at turning over phosphite , the transport and energy conservation steps may in fact represent the bottlenecks for growth by DPO. Increased expression of the ptd genes could, therefore, be a way to compensate for the relative inefficiency of these processes compared to the phosphite oxidation step itself. Furthermore, the exceptionally high level of ptdF expression compared to the other ptd genes suggests that this enzyme may catalyze the ratelimiting step in the pathway.
Overall, my RNAseq results highlight the importance of the ptd genes during DPO-dependent growth of FiPS-3, but exactly what their functional roles are and how they are regulated remains to be elucidated. It is essential, going forward, to determine the minimal set of genes that are necessary and sufficient for growth by DPO,vertical rack system either through targeted knockouts in FiPS-3 or through expression of candidate genes in heterologous hosts. DPO enzymes could then be purified and characterized in vitro in order to determine their mechanisms of action. Unfortunately, my attempts to carry out targeted gene deletions in FiPS-3 as well as to heterologously express the full ptx-ptd gene cluster in D. balticum SaxT and in E. coli have sofar proven unsuccessful. Nonetheless, I hope that my efforts to improve the growth of FiPS-3 under both anaerobic and aerobic conditions will aid in the development of genetic tools in this organism that will allow future researchers to address some of these unanswered questions. Furthermore, the ability of FiPS-3 to precipitate phosphate minerals as a byproduct of DPO holds great promise as a potential mechanism for bioconcrete production. However, before this process can be applied on an industrial scale, further research must be undertaken in order to establish the optimal conditions for biomineralization by FiPS-3 as well as to assess the material properties of the different mineral products.Killed controls showed no phosphite oxidation even after 29 weeks and the rate of phosphite oxidation increased over time. The persistence of phosphite under abiotic conditions is congruent with its kinetic stability resulting from the high activation energy needed to break the P-H bond in the phosphite molecule . Moreover, the extent of phosphite oxidation observed in our enrichments is evidence of a dissimilatory process involved in energy metabolism as opposed to a purely assimilatory process. Phosphate accumulated in the media over time at a rate that corresponded to that of phosphite depletion and reached a final concentration that matched the amount of phosphite consumed . Assuming a phosphorus content of ~30 fg/cell , a bacterial culture would have to assimilate only about 300 µM phosphate in order to reach a density of ~1×109 cells/mL. This means that the phosphite consumed in our enrichments far exceeded what would have been required for use as a phosphorus source. In fact, there was more than enough phosphate initially present in the cultures to fulfill the phosphorus needs of growing cells in the absence of phosphite supplementation. The fact that microbial growth rates were significantly higher in phosphite-oxidizing cultures compared to no-phosphite controls provides further evidence that phosphite was acting as an electron donor and energy source for cellular metabolism. Phosphite oxidation and growth only occurred in media amended with CO2 and/or HCO3 – indicating that DPO was coupled to CO2 reduction, as has been shown for FiPS-3 . The rates of phosphite oxidation seen in our rumen fluid amended enrichments are comparable to those seen in cultures of FiPS-3 . However, our enrichments were incubated at 37o C and FiPS-3 can only grow at temperatures between 15 and 30o C . Indeed, our microbial community analysis confirmed the absence of any 16S rDNA reads belonging to the Desulfotignum genus in our enrichments.
There was, however, a clear shift in community composition in the presence of phosphite, with OTU 33 , OTU 21 , and OTU 28 being the most enriched taxa. Addition of rumen fluid to the enrichments greatly enhanced the rate of phosphite oxidation and also substantially altered the microbial community composition. Interestingly, OTUs 33 and 28 were no longer observed in the community. Instead, OTU 21 was further enriched to about 40% of the total community. The tight correlation seen between phosphite oxidation and Phox-21 abundance strongly supports its functional role in DPO. It remains unclear why OTUs 33 and 28 were enriched in our original cultures but not in the presence of rumen fluid. Given that rumen fluid was added to the cultures in an attempt to enhance the growth of OTU 33, its decline was particularly surprising. It is possible that these organisms benefited from the presence of phosphite under low-nutrient conditions, but were then outcompeted when nutrient-rich rumen fluid was added. Also, the fact that these taxa had very low initial abundances means that even moderate amounts of growth would have led to large increases in fold change, which would have magnified the effect of any ancillary growth benefits that these organisms may have received. In any case, the addition of rumen fluid did enhance the growth of Phox-21, which was an unexpected outcome, but perhaps not too surprising given the rich variety of nutrients and cofactors present in rumen fluid .The strong inhibitory effect of molybdate on phosphite oxidation and Phox-21 abundance in a culture not dependent on sulfate reduction was likewise unexpected, since molybdate is known to be a potent and selective inhibitor of sulfate reduction. It acts as a futile substrate for the enzyme ATP sulfurylase, resulting in depletion of the cell’s ATP pool . However, the addition of sulfate did not enhance phosphite oxidation, stimulate sulfidogenesis, or enhance the growth of Phox-21. It is possible that molybdate is targeting the phosphite oxidation pathway directly or affecting some other unrelated pathway, but further work is needed in order to confirm the mechanism of inhibition. Sulfite also appears to inhibit phosphite oxidation, which is further evidence of a potential incompatibility of the sulfate reduction pathway and phosphite oxidation in our enrichment. It is not clear whether the inhibition by sulfite was due to a direct interaction with the phosphite oxidation pathway or to a broader toxicity. PtxD, the phosphite oxidase found in FIPS-3 as well as in most APO-capable bacteria, is known to be inhibited by sulfite in vitro . This enzyme, if present in Phox-21, would be a likely target of sulfite inhibition. However, sulfite is also known to have a broad antimicrobial activity at concentrations as low as 600 µM , which may have also played a role in its inhibition. Phox-21 belongs to the GW-28 candidate order, a poorly studied clade within the Deltaproteobacteria with no known cultured representatives. Its closest relatives are all uncultured clones from different anaerobic waste treatment sites. Clone GW-28 was found in a household biogas digester in China , clone POMEbac42 was found in a food waste digester in Singapore , clone De3155 was found in alkaline landfill leachate sediment in China , and clone QEEB2BG06 was found in a mesophilic wastewater digester in Germany .