Primary production by anoxygenic bacteria

Primary production is defined as the synthesis of organic compounds from atmospheric (CO₂) or aquatic inorganic carbon (HCO₃^-). As described previously (see paragraphs 1.2.3), this process can occur through photosynthesis (using light as an energy source) or chemosynthesis by oxidation or reduction of various compounds. Organisms that contribute to primary production are known as autotrophs, and together they form the base of the food chain: photo- and chemo-trophic bacteria, phytoplankton, and plants. Unlike organisms that benefit from oxygenic photosynthesis, anoxygenic phototrophic bacteria may use instead of H2O as electron donor a number of organic substances as well as sulfide (H₂S), hydrogen (H₂), or ferrous iron (Fe⁺²).

Photoautotrophic anaerobic organisms are mainly found in redox transition zones of the freshwater and marine environments as well as in the part of sediments where light can penetrate. As the intensity of light in these environments is generally low, bacteria have evolved highly efficient photosynthetic pigments, which give cells a distinct color ranging from red-purple to green-brownish (see sections 1.2.2. and 1.2.3.1.).

5.1.1. Contribution of phototrophic sulfur bacteria to the primary production

Phototrophic sulfur bacteria represent one of the most important group of anoxygenic photosynthetic organisms and play a key role in the sulfur cycle (Imhoff 2004; Van Gemerden and Mas 2004). Several studies in different meromictic lakes have highlighted the importance of the dense populations of phototrophic sulfur bacteria in the CO2 economy of meromictic lakes, as well as their role as detoxifying agents when using toxic H2S as an electron donor during anoxygenic photosynthesis. Thus, in the presence of phototrophic sulfur bacteria, H2S concentrations in the upper layer of the chemocline tend to decrease during the day but increase again during the night (van Gemerden 1967; Takahashi and Ichimura 1968; Sorokin 1970;

Sorokin and Donato 1975; Parkin and Brock 1981; Guerrero et al. 1985; Overmann et al. 1991).

The overnight increase in H₂S was interpreted as respiration by phototrophic sulfur bacteria, and for this reason CO₂ assimilation was presumed to occur only in the presence of light. In fact, any CO₂ fixation that occurred in the dark was mostly ignored or subtracted from the carbon incorporation that occurred in the presence of light. However, measurable and, in some cases, high levels of CO2 fixation in the dark were reported in a number of meromictic lakes and marine environments (Jorgensen et al. 1979; Cloern et al. 1983; Pedros-Alio and Guerrero 1991;

Kuuppo-Leinikki and Salonen 1992; Sorokin et al. 1995), including: Lake Kinneret (Hadas et al.

2001), lakes from a karstic region in Spain (García-Cantizano et al. 2005; Casamayor et al.

2008), or in Arctic seawater (Alonso-Saez et al. 2010). Incidentally, these studies reported high rates of CO2 fixation in the dark at depths where dense populations of purple sulfur bacteria (PSB) occurred. Under laboratory conditions, various PSB strains showed a pronounced ability to incorporate CO2 in the dark (Van Gemerden and Mas 1995; Van Gemerden and Mas 2004).

However, to date, no significant correlation was made between CO2 fixation in absence of light, nutrient distribution and composition of microbial communities (Casamayor 2010). Yet, overall contribution of phototrophic sulfur bacteria to primary production in stratified lakes was estimated at ca. 28.7% (Overmann 2008).

5.1.2. CO₂ fixation in meromictic lakes in absence of light

Autotrophic processes other than photosynthesis were also described in various non-sulfurous freshwater environments (Gorlenko et al. 1983), it seems that alternative processes of CO₂ incorporation are more widespread than initially thought. Since dark CO2 fixation (herein referred to as “DCF”) was also reported to contribute significantly to overall inorganic carbon fixation in open marine waters (Prakash et al. 1991), DCF appears to be an important process in marine as well as in freshwater ecosystems. In meromictic lakes, DCF was mostly attributed to chemoautotrophs such as bacteria belonging to the genus Thiobacillus, which oxidize reduced inorganic compounds to obtain the energy to fix CO₂ in absence of photosynthesis. Although chemoautotrophic activities carried out by non-photosynthetic prokaryotes were reported for many habitats (Shively et al. 1998a), no potential chemo-autotrophe was detected in Lake Cadagno (Martinez et al. 1983; Tonolla et al. 2003; Gregersen et al. 2009). Yet, the ability of phototrophic sulfur bacteria to fix CO2 in the dark was initially shown using the Wood-Werkman reaction (Wood and Stjernholm 1962), and reported to occur at depths corresponding to dense populations of PSB (Cohen et al. 1977; Camacho et al. 2001; Casamayor et al. 2008). In this respect, our results confirmed that PSB, and in particular Candidatus “T. syntrophicum” strain Cad16^T and L. purpurea strain CadA31, exhibited important rates of DCF (Chapter 2).

5.1.3. Autotrophic inorganic carbon fixation pathways

Phototrophic sulfur bacteria are considered extremely versatile and capable of utilizing a multiplicity of metabolic pathways. In general, PSB are capable of both photo-autotrophy and

heterotrophy while green sulfur bacteria (GSB) are considered to be obligate photo-autotrophs (Parkin and Brock 1981; Van Gemerden and Mas 2004). In addition, some PSB strains are capable of chemotrophic growth in absence of light and under microaerophilic conditions (Kämpf and Pfennig 1986; de Wit and van Gemerden 1987). The mechanism of anoxygenic photosynthesis carried out by phototrophic sulfur bacteria has been studied extensively and is now well understood (see section 1.2.3.2.), with H2S being the most common electron donor for PSB and GSB in the process of CO2 assimilation process. In contrast, the chemo-autotrophic mechanisms by which these bacteria fix CO2 in the absence of light remain unclear. PSB are known to produce storage products in presence of light, including sulfur globules, polyhydroxyalkanoates (PHAs) granules, polyphosphate and glycogen (Van Gemerden and Mas 2004). These macromolecules may later serve as energy supplies for DCF processes, as suggested by the substantial levels of fixation retained by photosynthetic organisms once light disappears (García-Cantizano et al. 2005; Casamayor et al. 2008). To date, six distinct mechanisms of CO2 fixation were described: (i) photo- or chemo-autotrophy using the Calvin-Benson-Bassham cycle (CBB cycle), (ii) the reductive citric acid cycle (rTCA cycle), (iii) the reductive acetyl-CoA pathway (Wood-Ljungdahl pathway), (iv) the 3-hydroxypropionate cycle, (v) the 3-hydroxypropionate/4-hydroxybutyrate cycle (HP/HB cycle) and (vi) the dicarboxylate/4-hydroxybutyrate cycle (DC/HB cycle) mechanism (Rothschild 2008; Berg 2011;

Hanson et al. 2012). While increase in biomass remains the main purpose for these autotrophic pathways, an alternative function appears to be the disposal of excess reducing power as was observed in purple non-sulfur bacteria (Wang et al. 1993; Joshi et al. 2009). However, none of these six fixation mechanisms has been shown to be involved in DCF in phototrophic sulfur bacteria.