Polyphosphate (poly-P) is a chain of phosphate moieties linked through high-energy phosphoanhydride bond, and it plays an important role in regulatory functions in prokaryotic cells. Isotope effects of bacterial synthesis and degradation of poly-P may provide important insights into the roles of poly-P in environmental phosphorus (P) cycling. In this research, we investigated the enzymatic degradation of poly-P by cell-free enzyme solutions and bacterial cells (Escherichia coli JM103 and Pseudomonas putida KT2440) and analyzed phosphate oxygen isotope ratios (delta O-18(P)), enzyme activity, and dynamics of orthophosphate (P-i) and poly-P. Cell-free enzyme reaction results show that both acid and alkaline phosphatase enzymes are capable of catalyzing poly-P degradation, albeit with different efficiencies of >70 and <18%, respectively. Isotope fractionation factors during enzymatic degradation of poly-P were slightly positive [+0.59 (+/- 5.51) to +3.99 (+/- 15.86)%] for acid and alkaline phosphatase, respectively, which is rather uncommon in phosphatase enzyme-catalyzed degradation of many other organic P compounds. Poly-P is synthesized during the exponential growth phase of E. coli, causing an isotope fractionation of -5.65 (+/- 1.02)% leading to the enrichment of isotope in the residual P-i in the growth media. Degradation of poly-P occurred at the late stationary phase, with lighter isotope values of P-i in the growth media. These results imply that the specific isotopes, fractionation, and P dynamics during poly-P synthesis and degradation could serve as proxies to interpret poly-P dynamics in the environment.

Abiotic and biotic reactions operate side by side in the cycling of phosphorus (P) in the environment, but the relative roles of these two reactions vary both spatially and temporally. In biotic reactions, the uptake and release of P are catalyzed by enzymes and thus change phosphate oxygen isotope ratios, while in abiotic reactions, the absence of hydrolysis-condensation reactions results in no apparent changes in isotope composition, except short-term kinetic isotope effect due solely to preferential ion exchange. Therefore, isotope method could be a promising tool to differentiate relative roles of these two reactions in the environment but the relationship of the dynamic concentration and isotope exchange at the biota-water interface is largely unknown. In this study, we aimed to develop a process-based isotope model underpinning the competition of abiotic (sorption, desorption, and ion exchange) and biotic (uptake, metabolism, and release) reactions during uptake and recycling of ferrihydrite-bound P by E. coli. Our model comprises equations describing the partitioning relationship among different P pools and their corresponding oxygen isotope compositions and is based exclusively on oxygen isotope exchange at multiple sites including mineral surface, aqueous phase, and bacterial cells. The process-based model adequately reproduced the measured concentration and isotope compositions over time. Furthermore, parametric and sensitivity analyses using the model indicated that the rate of biological uptake of P was the major factor controlling the changes of phosphate isotope composition. In conclusion, our model provides new insights into a mechanistic aspect of isotope exchange and could be potentially useful for future efforts to understand the interplay of biotic and abiotic factors on phosphorus cycling in natural environments.

Oxygen isotope thermometry has been traditionally based on the ratio of ^18^O/^16^O in oxyanions of minerals such as carbonate (CaCO~3~) in shells/tests, and phosphate in bioapatites (Ca~5~(PO~4~)~3~OH) of marine invertebrates/vertebrates (for example, fish) and mammals. The requirement of mineral biomass, however, has restricted the application of oxygen isotope thermometry to only those organisms possessing biomineral hardparts. This has completely omitted from study, not only organisms lacking hard mineral tissues, but two entire Domains of life: Bacteria and Archaea. Prokaryotic organisms in the domains Bacteria and Archaea comprise the majority of earth9s biodiversity and also inhabit the most extreme environments on earth. This calls for a thermometry based on a more rudimentary component of biomass that is present in all organisms such as DNA. Our previous studies of the ubiquitous intracellular enzyme inorganic pyrophosphatase (PPase), which catalyzes oxygen isotope exchange between dissolved inorganic PO~4~ (P~i~) and water inside of cells, suggest that DNA may contain even more information than the blueprint for life. Here we show that PO~4~ moieties in DNA record the temperature at which life forms and evolves. Our results demonstrate that the ^18^O/^16^O ratio of PO~4~ (δ^18^O~P~) in DNA as well as in bulk biomass, reflects the temperature-dependent exchange of oxygen isotopes between PO~4~ and intracellular water. Thus, δ^18^O~P~ values of DNA may serve as both a new soft-tissue bio-thermometer and probe of intracellular PO~4~ or water during DNA synthesis and cellular growth. Results presented here of the first direct measurements of the oxygen isotopic composition of PO~4~ in microbial DNA, demonstrate systematic variation in δ^18^O~P~ of DNA from several different strains of microorganisms as a function of temperature, and also extend the PO~4~-water O-isotope thermometer to \>70 °C. A composite calibration curve based on several strains of bacteria is presented for DNA-PO~4~--water --temperature relations between 12 and 75 °C and suggests a "universal" DNA-based oxygen isotope thermometry for microorganisms that may further extend to all organisms. Our results open the possibility of connecting temperature with taxonomy and also expand the range of investigations of habitat temperatures and limits to life in extreme and diverse environments, from Earth9s subsurface deep biosphere to Antarctic ice sheets and extraterrestrial systems where life may have originated at extreme temperatures.

Core Ideas Positive correlation between phytate P, inorganic P, and PMB along the creek. Bacteria with BPP genes about four orders of magnitude higher in water than sediment. High inorganic P in the creek most likely derived partly from phytate degradation. Phytate is often the most common and recalcitrant form of organic P in soils. Given that extracellular phytase activities are attributed to soil microorganisms, the distribution and characterization of phytate‐mineralizing bacteria (PMB) and quantification of phytate genes across an environmental gradient can address questions related to the degree of relative recalcitrancy of phytate P and microbial response to phytate loading. In this study, we analyzed paired sediment and water samples from different sites along a stretch of East Creek, a coastal tributary to the Chesapeake Bay, for the abundance of PMB as well as β‐propeller phytase (BPP) genes, a gene for the most common of the phytase enzymes, using culture‐dependent and ‐independent methods. We found that a larger proportion of isolates from sites near the headwaters were PMB than at the mouth of the creek. Our quantitative polymerase chain reaction (qPCR) results show that the total number of bacteria (both with and without BPP genes) in water and sediment samples was highest at the agricultural headwaters but decreased significantly at wetland sites. Similarly, 16S rRNA‐normalized BPP copies show that the abundance of bacteria capable of phytate degradation was about four orders of magnitude higher in water than in sediment. These results probably indicate that the presence of phytate promotes the proliferation of phytate‐degrading microorganisms with a potentially higher rate of phytate degradation in water than in sediments. Overall, these results provide important insights into phytate loading and PMB abundance in a coastal tributary and P export to the Chesapeake Bay.

Phytate, the salt of myo-inositol hexakisphosphate, and its partially dephosphorylated products are commonly present in the environment, but their origins and bioavailability are not well understood. This research applied phosphate O isotope ratios (delta(OP)-O-18) in combination with nuclear magnetic resonance (NMR) and high-performance liquid chromatography (HPLC) methods to characterize the kinetics and pathways of phytate degradation by wheat phytase and the O isotopic composition during progressive degradation of phytate. Our results show that phytate degradation undertakes two pathways: D-inositol-1,2,3,5,6-pentakisphosphate, D-inositol-1,2,5,6-tetrakisphosphate, D-inositol-1,2,6-trisphosphate, d-inositol-1,2-bisphosphate, D-inositol-1-phosphate; and d-inositol-1,2,4,5,6-pentakisphosphate, D-inositol-1,2,5,6-tetrakisphosphate, D-inositol-1,5,6-trisphosphate. The first pathway is similar to that previously known, while the other one is a new pathway. Isotope results show that the cleavage of the P-O bond during phytate degradation is accompanied by the introduction of one O atom solely from water to the released inorganic orthophosphate. Interestingly, isotopic compositions of all phosphate moieties in K phytate used in this study were found to be the same. This means that the original source of phytate can be tracked from its partially dephosphorylated products. Overall, these results provide improved insights into the mechanisms and pathways of phytate degradation and highlight the importance of an isotopic tool that can potentially be used for tracking phytate source and its degradation products in the environment.

Microorganisms carry out biochemical transformations of nutrients that make up their cells. Therefore, understanding how these nutrients are transformed or cycled in natural environments requires knowledge of microbial activity. Commonly used indicators for microbial activity typically include determining microbial respiration by O2/CO2 measurements, cell counts, and measurement of enzyme activities. However, coupled studies on nutrient cycling and microbial activity are not given enough emphasis. Here we apply phosphate oxygen isotope ratios (δ18OP) as a tool for measurement of microbial activity and compare the rate of isotope exchange with methods of measuring microbial activities that are more commonly applied in environmental studies including respiration, dehydrogenase activity, alkaline phosphatase activity, and cell counts. Our results show that different bacteria may have different strategies for P uptake, storage and release, their respiration and consequently expression of DHA and APase activities, but in general the trend of their enzyme activities are comparable. Phosphate δ18OP values correlated well with these other parameters used to measure microbial activity with the strongest linear relationships between δ18OP and CO2 evolution (r = −0.99). Even though the rate of isotope exchange for each microorganism used in this study is different, the rate per unit CO2 respiration showed one general trend, where δ18OP values move towards equilibrium while CO2 is generated. While this suggests that P cycling among microorganisms used in this study can be generalized, further research is needed to determine whether the microorganism-specific isotope exchange trend may occur in natural environments. In summary, phosphate oxygen isotope measurements may offer an alternative for use as a tracer to measure microbial activity in soils, sediments, and many other natural environments.

A key question to address in the development of oxygen isotope ratios in phosphate (δ(18)O(p)) as a tracer of biogeochemical cycling of phosphorus in ancient and modern environments is the nature of isotopic signatures associated with uptake and cycling of mineral-bound phosphate by microorganisms. Here, we present experimental results aimed at understanding the biotic and abiotic pathways of P cycling during biological uptake of phosphate sorbed to ferrihydrite and the selective uptake of sedimentary phosphate phases by Escherichia coli and Marinobacter aquaeolei. Results indicate that a significant fraction of ferrihydrite-bound phosphate is biologically available. The fraction of phosphate taken up by E. coli attained an equilibrium isotopic composition in a short time (<50 h) due to efficient O-isotope exchange (between O in PO(4) and O in water; that is, actual breaking and reforming of P-O bonds) (biotic pathway). The difference in isotopic composition between newly equilibrated aqueous and residual sorbed phosphate groups promoted the ion exchange (analogous to isotopic mixing) of intact phosphate ions (abiotic pathway) so that this difference gradually became negligible. In sediment containing different P phases, E. coli extracted loosely sorbed phosphate first, whereas M. aquaeolei preferred Fe-oxide-bound phosphate. The presence of bacteria always imprinted a biotic isotopic signature on the P phase that was taken up and cycled. For example, the δ(18)O(p) value of loosely sorbed phosphate shifted gradually toward equilibrium isotopic composition. The δ(18)O(p) value of Fe-oxide-bound phosphate, however, showed only slight changes initially but, when new Fe-oxides were formed, coprecipitated/occluded phosphate retained δ(18)O values of the aqueous phosphate at the time of formation of new Fe oxides. Concentrations and isotopic compositions of authigenic and detrital phosphates did not change, suggesting that these phosphate phases were not utilized by bacteria. These findings support burgeoning applications of δ(18)O(p) as a tracer of phosphorus cycling in sediments, soils, and aquatic environments and as an indicator of paleo- environmental conditions.

We present oxygen isotope, micro-textural, and molecular evidence of microbial activity in the formation of hydroxylapatite (HAP) in three limestone caves (Gosu, Sungryu, and Ssang caves) in South Korea. HAP typically forms as crusts (0.1 to 0.5mm thick) coating carbonates of speleothems and host rock surfaces, on and near bat habitats. Micro-textures within HAP crusts indicate that a metastable apatite precursor (AP) is initially precipitated on and near the surfaces of sulfur-bearing microbial filaments and then transforms to HAP. Analysis of DNA extracted from the HAP crusts confirms that sulfur-oxidizing bacteria are present in some of the HAP samples. The δ18O values of phosphate (δ18OP) in HAP precipitated in the caves range from 14.6 to 15.6‰ and are close to isotopic equilibrium with the weighted mean annual δ18O value of rain water (= cave water) at the mean annual air temperature (= measured cave temperature). The difference in oxygen isotopic composition between speleothem carbonate (δ18OC) and phosphate (δ18OP) in adjacent apatitic crusts is similar to that of co-existing carbonate and phosphate in modern biogenic apatite. These results suggest that phosphate, likely derived from bat excretions, was metabolized by microorganisms and has undergone extensive oxygen isotope exchange with cave drip water by intense biological turnover of phosphate, and then precipitated as HAP in near-equilibrium with water and carbonate in the cave ecosystem. Results from these studies of δ18OP values of HAP crusts in limestone caves demonstrate the utility of δ18OP as an environmental temperature proxy and signature of microbiological processes.

The rhizosphere in terrestrial systems is the region of soil surrounding plant roots where there is increased microbial activity; in aquatic plants, this definition may be less clear because of diffusion of nutrients in water, but there is still a zone of influence by plant roots in this environment [1]. Within that zone chemical conditions differ from those of the surrounding environment as a consequence of a range of processes that were induced either directly by the activity of plant roots or by the activity of rhizosphere microflora. Recently, there are a number of new studies related to rhizospheres of aquatic plants and specifically their increased potential for remediation of contaminants, especially remediation of metals through aquatic plant-microbial interaction.

Certain plants are known to accumulate heavy metals, and can be used in remediation of polluted soil or water. Plant-associated bacteria, especially those that are metal tolerant, may enhance the total amount of metal accumulated by the plant, but this process is still unclear. In this study, we investigated metal enhancement vs. exclusion by plants, and the phytoprotective role plant-associated bacteria might provide to plants exposed to heavy metal. We isolated cadmium-tolerant bacteria from the roots of the aquatic plant Lemna minor grown in heavy metal-polluted waters, and tested these isolates for tolerance to cadmium. The efficiency of plants to accumulate heavy metal from their surrounding environment was then tested by comparing L. minor plants grown with added metal tolerant bacteria to plants grown axenically to determine, whether bacteria associated with these plants increase metal accumulation in the plant. Unexpectedly, cadmium tolerance was not seen in all bacterial isolates that had been exposed to cadmium. Axenic plants accumulated slightly more cadmium than plants inoculated with bacterial isolates. Certain isolates promoted root growth, but overall, addition of bacterial strains did not enhance plant cadmium uptake, and in some cases, inhibited cadmium accumulation by plants. This suggests that bacteria serve a phytoprotective role in their relationship with Lemna minor, preventing toxic cadmium from entering plants.

The volcanic Sulphur Springs, St. Lucia, present an extreme environment due to high temperatures, low pH values, and high concentrations of sulfate and boron. St. Lucia offers some unique geochemical characteristics that may shape the microbial communities within the Sulphur Springs area. We chose six pools representing a range of geochemical characteristics for detailed microbial community analyses. Chemical concentrations varied greatly between sites. Microbial diversity was analyzed using 16S rRNA gene clone library analyses. With the exception of one pool with relatively low concentrations of dissolved ions, microbial diversity was very low, with Aquificales sequences dominating bacterial communities at most pools. The archaeal component of all pools was almost exclusively Acidianus spp. and did not vary between sites with different chemical characteristics. In the pool with the highest boron and sulfate concentrations, only archaeal sequences were detected. Compared with other sulfur springs such as those at Yellowstone, the microbial diversity at St. Lucia is very different, but it is similar to that at the nearby Lesser Antilles island of Montserrat. While high elemental concentrations seem to be related to differences in bacterial diversity here, similarities with other Lesser Antilles sites suggest that there may be a biogeographical component as well.

ABSTRACT In this study we present the comparative molecular analysis of bacterial communities of the aquatic plant Lemna minor from a contaminated site (RCP) and from a laboratory culture (EPA), as well as each of these with the addition of cadmium. Plants were identified as L. minor by analysis of the rpl 16 chloroplast region. Comparative bacterial community studies were based on the analyses of 16S rRNA clone libraries, each containing about 100 clones from the root surfaces of plants. Bacterial communities were compared at three phylogenetic levels of resolution. At the level of bacterial divisions, differences in diversity index scores between treatments, with and without cadmium within the same plant type (EPA or RCP), were small, indicating that cadmium had little effect. When we compared genera within the most dominant group, the β-proteobacteria, differences between unamended and cadmium-amended libraries were much larger. Bacterial diversity increased upon cadmium addition for both EPA and RCP libraries. Analyses of diversity at the phylotype level showed parallel shifts to more even communities upon cadmium addition; that is, percentage changes in diversity indices due to cadmium addition were the same for either plant type, indicating that contamination history might be independent of disturbance-induced diversity shifts. At finer phylogenetic levels of resolution, the effects of cadmium addition on bacterial communities were very noticeable. This study is a first step in understanding the role of aquatic plant-associated microbial communities in phytoremediation of heavy metals.