We believe that biogeochemical cycles on Earth can only be understood if the complex networks of microbial activities are unraveled and linked to the organisms involved. Our research on the physiology of marine subsurface microbes is a combination of descriptive studies on environmental samples, isotopic tracer experiments, microbial cultivation, and modeling.

Descriptive studies

To examine in situ activity of microbes, we measure concentrations and isotopic compositions of key metabolites in environmental samples. Metabolites analyzed include O₂, nitrate, Fe(II), sulfate, methane, dissolved inorganic carbon, in addition to volatile fatty acids, H₂, carbon monoxide and methanol. To complement this geochemical data, we study the phylogenetic composition and concentrations of functional genes (DNA) and functional gene transcripts (mRNA) that allow us to link microbes to microbially-driven processes.  Currently, we are focusing on genes diagnostic of sulfate reduction (dsrAB), methanogenesis and anaerobic methane oxidation (mcrA), reductive dehalogenation (rdh), and CO₂ fixation via the reductive acetyl CoA pathway (acsB).

Isotopic tracer experiments

By adding radioactive tracers (e.g. ³⁵S) to sediments and measuring their conversion to metabolic end products, we obtain estimates of net metabolic rates of microbial processes, such as sulfate reduction. In addition, we study microbial substrate use via stable isotope probing (SIP). In this method, isotopically labeled microbial substrates are added to samples at environmentally relevant concentrations. Energy production pathways and substrate assimilation are then examined by monitoring the incorporation of isotopic label into metabolic end products, biomass of single cells and bulk nucleic acids (DNA or RNA). Isotopic compositions of metabolic end products and single cells are measured by isotope ratio mass spectroscopy and NANO-SIMS. Isotopically labeled nucleic acids are extracted and microbes that have assimilated added substrates identified via PCR assays or pyrosequencing followed by phylogenetic analyses.

Microbial cultivation

Cultivation and isolation remain the only means by which the physiology of any given microbial strain can be thoroughly characterized. In cultivating subseafloor microbes, we are particularly interested in organisms adapted to life conditions in deeply buried sediments. This implies slow growth, low maintenance energy and adaptation to low substrate concentrations. A key question is whether or not unique guilds of subseafloor microbes exists that are physiologically adapted to the low-energy conditions that prevail in the subsurface.

Modeling of activity

We model net reaction rates of microbial metabolic pathways, e.g. oxic respiration, sulfate reduction or methane oxidation, based on porewater concentration profiles of electron acceptors and electron donors.  In addition, using thermodynamic calculations, we determine the in situ energy yields of metabolic reactions in the subsurface. The combination of modeling with quantitative data on cell abundances is then used to investigate in situ energy availability to microbes in the subsurface.

• Lever MA, Heuer VB, Morono Y, Masui N, Schmidt F, Alperin MJ, Inagaki F, Hinrichs K-U, Teske A (2010) "Acetogenesis in deep subseafloor sediments of the Juan de Fuca Ridge Flank: a synthesis of geochemical, thermodynamic, and gene-based evidence" Geomicrobiology Journal 27:183-211.
• Høgslund S, Revsbech NP, Kuenen JG, Jørgensen BB, Gallardo VA, van de Vossenberg J, Nielsen JL, Holmkvist L, Arning ET, Nielsen LP (2009) "Physiology and behaviour of marine Thioploca" The ISME Journal 3: 647-657.
• Leloup J, Fossing H, Kohls K, Holmkvist L, Borowski C, Jørgensen BB (2009) "Sulfate-reducing bacteria in marine sediment (Aarhus Bay, Denmark): abundance and diversity related to geochemical zonation" Environmental Microbiology 11: 1278-1291.
• Knab NJ, Cragg BA, Holmkvist L, Borowski C, Parkes RJ, Jørgensen BB (2009) "Regulation of anaerobic methane oxidation in sediments of the Black Sea" Biogeosciences 5: 2305-2341.