Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase (RubisCO) Is Essential for Growth of the Methanotroph Methylococcus capsulatus Strain Bath.

The ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) enzyme found in plants, algae, and an array of autotrophic bacteria is also encoded by a subset of methanotrophs, but its role in these microbes has largely remained elusive. In this study, we showed that CO2 was requisite for RubisCO-encoding Methylococcus capsulatus strain Bath growth in a bioreactor with continuous influent and effluent gas flow. RNA sequencing identified active transcription of several carboxylating enzymes, including key enzymes of the Calvin and serine cycles, that could mediate CO2 assimilation during cultivation with both CH4 and CO2 as carbon sources. Marker exchange mutagenesis of M. capsulatus Bath genes encoding key enzymes of potential CO2-assimilating metabolic pathways indicated that a complete serine cycle is not required, whereas RubisCO is essential for growth of this bacterium. 13CO2 tracer analysis showed that CH4 and CO2 enter overlapping anaplerotic pathways and implicated RubisCO as the primary enzyme mediating CO2 assimilation in M. capsulatus Bath. Notably, we quantified the relative abundance of 3-phosphoglycerate and ribulose-1,5-bisphosphate 13C isotopes, which supported that RubisCO-produced 3-phosphoglycerate is primarily converted to ribulose-1-5-bisphosphate via the oxidative pentose phosphate pathway in M. capsulatus Bath. Collectively, our data establish that RubisCO and CO2 play essential roles in M. capsulatus Bath metabolism. This study expands the known capacity of methanotrophs to fix CO2 via RubisCO, which may play a more pivotal role in the Earth's biogeochemical carbon cycling and greenhouse gas regulation than previously recognized. Further, M. capsulatus Bath and other CO2-assimilating methanotrophs represent excellent candidates for use in the bioconversion of biogas waste streams that consist of both CH4 and CO2. IMPORTANCE The importance of RubisCO and CO2 in M. capsulatus Bath metabolism is unclear. In this study, we demonstrated that both CO2 and RubisCO are essential for M. capsulatus Bath growth. 13CO2 tracing experiments supported that RubisCO mediates CO2 fixation and that a noncanonical Calvin cycle is active in this organism. Our study provides insights into the expanding knowledge of methanotroph metabolism and implicates dually CH4/CO2-utilizing bacteria as more important players in the biogeochemical carbon cycle than previously appreciated. In addition, M. capsulatus and other methanotrophs with CO2 assimilation capacity represent candidate organisms for the development of biotechnologies to mitigate the two most abundant greenhouse gases, CH4 and CO2.

The Soil Water Isotope Storage System (SWISS): An integrated soil water vapor sampling and multiport storage system for stable isotope geochemistry.

RATIONALE: Soil water stable isotopes are a powerful tool for tracking interactions among the hydrosphere, geosphere, atmosphere, and biosphere. The challenges associated with creating high-temporal-resolution soil water stable isotope datasets from a diversity of sites have limited the utility of stable isotope geochemistry in addressing a range of complex problems. A device that can enable further development of high-temporal-resolution soil water isotope datasets that are created with minimal soil profile disruption from remote sites would greatly expand the utility of soil water stable isotope analyses. METHODS: We designed a method for sampling and storing soil water vapor for stable isotope analysis that leverages recent advances in soil water sampling strategies. Here, we test the reliability of the storage system by introducing water vapor of known oxygen and hydrogen isotopic composition into the storage system, storing the water vapor for a predetermined amount of time, and then measuring the stable isotope composition of the vapor after the storage period. RESULTS: We demonstrate that water vapor stored in our flasks reliably maintains its isotope composition within overall system uncertainty (±0.5‰ for δ18 O values and ±2.4‰ for δ2 H values) for up to 30 days. CONCLUSIONS: This method has the potential to enable the collection of high-temporal-resolution soil water isotope datasets from remote sites that are not accessed daily in a time- and cost-effective manner. All the components used in the system can be easily controlled using open-source microcontrollers, which will be used in the future to automate sampling routines for remote field deployment. The system is designed to be an open-source tool for use by other researchers.

Long term geological record of a global deep subsurface microbial habitat in sand injection complexes.

There is extensive evidence from drilling into continental margins for microbial colonization of a deep biosphere. However it is difficult to prove deep biosphere activity in the geological record, where evidence for life is dominated by the remains of organic matter buried after deposition at the surface. Nevertheless we propose that natural injections of sand into muddy strata at continental margins represent an excellent habitat opportunity for deep microbial activity down to several kilometres' present day depth. Sulphur isotope data for iron sulphides precipitated soon after injection indicate consistent microbial sulphate reduction through the geological record. The complexes are favourable sites for colonization, because high permeability and extensive sand/mud interface allow ready availability of electron donors and nutrients. The measured examples of iron sulphide in injected sands extend back to the Proterozoic, and show that injected sand complexes have been a long-term environment for deep subsurface microbial colonization.