Coupled arsenotrophy in a hot spring photosynthetic biofilm at Mono Lake, California

Abstract

Red-pigmented biofilms grow on rock and cobble surfaces present in anoxic hot springs located on Paoha Island in Mono Lake. The bacterial community was dominated (∼ 85% of 16S rRNA gene clones) by sequences from the photosynthetic Ectothiorhodospira genus. Scraped biofilm materials incubated under anoxic conditions rapidly oxidized As(III) to As(V) in the light via anoxygenic photosynthesis but could also readily reduce As(V) to As(III) in the dark at comparable rates. Back-labeling experiments with 73 As(V) demonstrated that reduction to 73 As(III) also occurred in the light, thereby illustrating the cooccurrence of these two anaerobic processes as an example of closely coupled arsenotrophy. Oxic biofilms also oxidized As(III) to As(V). Biofilms incubated with [ 14 C]acetate oxidized the radiolabel to 14 CO 2 in the light but not the dark, indicating a capacity for photoheterotrophy but not chemoheterotrophy. Anoxic, dark-incubated samples demonstrated As(V) reduction linked to additions of hydrogen or sulfide but not acetate. Chemoautotrophy linked to As(V) as measured by dark fixation of [ 14 C]bicarbonate into cell material was stimulated by either H 2 or HS − . Functional genes for the arsenate respiratory reductase ( arrA ) and arsenic resistance ( arsB ) were detected in sequenced amplicons of extracted DNA, with about half of the arrA sequences closely related (∼98% translated amino acid identity) to those from the family Ectothiorhodospiraceae . Surprisingly, no authentic PCR products for arsenite oxidase ( aoxB ) were obtained, despite observing aerobic arsenite oxidation activity. Collectively, these results demonstrate close linkages of these arsenic redox processes occurring within these biofilms. Oxyanions of the group 15 element arsenic, arsenate [As(V)] and arsenite [As(III)], have been known for millennia to be potent poisons. Despite its well-established toxicity to life, the phenomenon of arsenic resistance was discovered whereby some microorganisms maintain an otherwise “normal” existence in the presence of high concentrations of As(V) or As(III) (17, 29, 31). More recently it has become recognized that certain representatives from the bacterial and archaeal domains can actually exploit the electrochemical potential of the As(V)/As(III) redox couple (+130 mV) to gain energy for growth. This can be achieved either by employing As(III) as an autotrophic electron donor or by using As(V) as a respiratory electron acceptor (18, 21, 34). The latter phenomenon, although most commonly associated with chemoheterotrophy, can also employ inorganic substances like sulfide or H 2 . Indeed, As(V)-respiring anaerobes displaying a capacity for chemoautotrophy with these electron donors have been isolated and described (5, 7, 16). We recently reported that photoautotrophy is supported by As(III) in anoxic biofilms located in hot springs on Paoha Island in Mono Lake, CA (15). This process represented a novel means of As(III) oxidation achieved via anoxygenic photosynthesis occurring in certain photosynthetic bacteria (i.e., Ectothiorhodospira ) and possibly within some cyanobacteria as well (e.g., “ Oscillatoria ”). Whether or not a microbial habitat is overtly oxic or anoxic, or temporally shifts between these two states over a diel cycle, critical energy linkages between aerobes and anaerobes have long been known for the biogeochemical cycles of key elements, such as sulfur, iron, and nitrogen. Most prominently studied is the case of nitrogen, whereby an ecological coupling exists between the processes of nitrification and denitrification (9, 10, 28). The former process provides energy to aerobic nitrifiers, while the latter process consumes the nitrate produced by this reaction, thereby meeting the energy needs of the denitrifiers. For arsenic, the detection of both As(III) oxidation and As(V) reduction in oxic and anoxic incubations of freshly collected periphyton suggested that an analogous coupled process may also occur for this element (12). Similarly, several uncontaminated soils in Japan displayed a capacity for either As(V) reduction or As(III) oxidation upon arsenic oxyanion amendment and whether they were incubated under oxic or anoxic conditions (39). A defined coculture consisting of an aerobic As(III) oxidizer (strain OL1) and an anaerobic As(V) respirer (strain Y5) was shown to function in this fashion under manipulated laboratory conditions of oxygen tension (26). We pursued the phenomenon of coupled arsenic metabolism further by using materials collected from the hot spring biofilms in Mono Lake, but we focused on examination of the cycling of arsenic under anoxic conditions. In this paper we report results obtained by manipulated incubations of red-pigmented biofilms found in the hot springs of Paoha Island. Preliminary community characterizations of these biofilms show that they are dominated by B acteria from the genus Ectothiorhodospira but also harbor an assemblage of Archaea related to the Halobacteriacaea . Incubation results have demonstrated the presence of the following arsenic metabolic activities: respiratory As(V) reduction, photosynthetic anaerobic As(III) oxidation, and aerobic As(III) oxidation, along with the ecophysiological conditions under which they occur. Surprisingly, we were unable to obtain authentic PCR products for arsenite oxidase genes ( aoxB ), despite observing aerobic As(III) oxidation activity. These biofilms serve as a model system for how anaerobic cycling of arsenic can be sustained with oxidation of As(III) by anoxygenic photosynthesis coupled to regeneration of this electron donor via dissimilatory As(V) reduction. The significance that such a light-driven anaerobic ecosystem may have played in the Archean Earth is discussed.