Chemolithoautotrophic carbon dioxide fixation in lakes

Chemolithoautotrophic prokaryotes fix carbon dioxide (CO2 ) through energy not acquired from light but from inorganic substrates. To date, six different mechanisms are known by which chemolithoautotrophic bacteria and archaea assimilate CO 2 . While the biochemistry and molecular biology of CO 2 fixation pathways are well understood for many isolates under laboratory conditions, the driving forces and biogeochemical processes determining the occurrence of diverse chemolithoautotophic CO 2 fixation strategies in the environment are mostly unexplored. In this project, we will assess which CO 2 fixation pathways are used by chemolithoautotrophic prokaryotic communities, studying three lakes with distinct stratification patterns. These lakes, characterized by different concentration gradients of oxygen and redox states of elements such as carbon, nitrogen, sulfur and iron, are particularly suitable for the investigation of chemoautotrophic microbial communities in a well-defined framework of habitat heterogeneity. Our research will focus on the analysis of genes coding for selected key enzymes for the assimilation of CO 2 in the Calvin-Benson-Bassham cycle, the reductive tricarboxylic acid cycle, and the recently discovered 3-hydroxypropionate/4-hydroxybutyrate pathway. From preliminary investigations, we anticipate that these three CO 2 fixation strategies are of significance in different zones of the lakes. We are especially interested to determine which CO 2 assimilation pathways are used under in situ conditions and plan to ascertain which prokaryotes are responsible for specific chemoautotrophic activities. Methods based on mRNA sequence analysis of functional genes, quantitative reverse transcription-polymerase chain reaction (RT-qPCR) and DNA stable-isotope probing (DNA-SIP) after 13C-bicarbonate incorporation are powerful tools by which we plan to identify active CO 2 fixation pathways in association with the phylogenetic profile of the respective organisms. Our proposal is unique because it will link the genetic potential and in situ activities of chemolithoautotrophic microbes with their distribution in lakes. Moreover, the comprehensive analysis of genes coding for key enzymes of a variety of CO 2 fixation pathways will provide fundamental data for the development of molecular assays for the study of chemolithoautotrophic microorganisms in lakes and other environments. Ultimately, we expect to gain insight on whether the diversity of CO 2 fixation pathways correspond to ecologically distinct groups and to identify which drivers and processes are most influential for the dynamic of chemolithoautotrophic prokaryotic community structures and carbon fixation strategies in aquatic environments.

Primary production based on photosynthesis is a major biological process on earth and of fundamental importance in the global carbon cycling. The presence of light, however, is not a prerequisite for the assimilation of inorganic carbon, as also chemosynthesis can provide energy for this process. A variety of reduced compounds is used for the generation of energy in order to enable a chemolithoautotrophic lifestyle of microorganisms, which are also significantly involved in the biogeochemical cycling of nitrogen, sulphur and other elements. The ecological significance for the distribution of diverse CO2 fixation strategies used by chemoautotrophs in the environment is, however, mostly unexplored. The overall objective of this project was to gain insight whether the diversity of CO2 assimilation pathways corresponds to ecologically distinct groups and to identify which drivers and processes are most influential for the distribution and diversity of CO2 fixation strategies in stratified lakes. Investigations based on molecular analysis in combination with stable isotope probing (SIP) after 13C-bicarbonate incorporation and fluorescent in situ hybridization provided powerful tools by which an active CO2 fixation metabolism was identified in association with the phylogenetic profile of the respective organism. Different forms of RubisCO, the key enzyme in the Calvin-Benson-Bassham (CBB) cycle, were widely distributed at the study sites and encompassed a wide range of potential physiologies in taxonomic diverse chemolithoautotrophs. These results suggest that CO2 fixation via the CBB pathway is a major pathway in lakes and is operational under different environmental conditions. Ammonia oxidizing Thaumarchaea using an energy efficient variant of the 3-hydroxypropionate/4- hydroxybutyrate pathway dominated the aphotic zone of deep and oligotrophic lakes. The reductive tricarboxylic acid (rTCA) cycle was often detected in anoxic or microoxic water layers and used by chemoautotrophs mostly involved in the oxidation of reduced sulfur compounds in the sulfur cycle. However, nitrifying Nitrospira were the most abundant group of chemoautotrophs harboring the rTCA cyle. In these bacteria, enzymatic adaptations strengthen the O2 robustness of the cycle that allows the pathway to function also in aerobic habitats. The distribution pattern of Nitrospira closely followed the presence of Thaumarchaea in large lakes. Overall, this study deciphered the niche differentiation of different guilds of chemoautotrophs and therefore provided a fundamental insight on how physico-chemical characteristics in lakes are associated to the distribution of chemoautotrophic bacteria and archaea based on an autotrophic perspective.