Coexistance of acetogens, methanogens and sulfate reducing bacteria in enrichments from Trunk River María Consuelo Gazitúa Center for Genomics and Bioinformatics University Mayor - Chile [email protected] Introduction In the absence of oxygen, chemotrophic growth depends on alternative electron acceptors such as nitrate, iron, manganese, sulfate and CO2. In the presence of sulfate, such as in seawater, sulfate reduction is highly favorable, coupling the reduction of sulfate with the oxidation of H2 or simple organic compounds. In the absence of sulfate, methanogenic archaea are able to cleave acetate, producing CO2 and CH4, as well as reduce or disproportionate C1 compounds such as CO2, CO, formate and methanol, using H2 and formate as the main electron donor. Homoacetogenic bacteria use C-1 compounds as electron acceptors, catalyzing the reduction of two CO2 molecules to acetate. This is a highly variable group of strictly anaerobic bacteria, able to grow on a variety of substrates. In most anaerobic environments, hydrogen is present as an intermediate for which sulfate reducing bacteria (SRB), hydrogenotrophic methanogens and homoacetogens will compete. Under standard conditions, sulfate reduction and methanogenesis are thermodynamically more favorable than homoacetogenesis. However, Weijma et al. (2002) reported that when H2/CO2 is added as the sole substrate in sludge bioreactores, heterotrophic SRB and homoacetogens can coexist, suggesting the SRB growth depends on the growth kinetics of the homoacetogens. During the isolation of chemolithotrophic microorganisms during the course, specifically for SRB, homoacetogens and methanogens, the enrichment for SRB showed the coexistence of these three groups, based on measurements of methane, hydrogen sulfide and acetate. Some of the organisms growing in the enrichment formed aggregates, where the presence of methanogens could be inferred based on the blue autofluorescence, after excitation with AF430 (Doddema and Vogels 1978). Figure 1: aggregate formation in enrichments for sulfate reducing bacteria. Left: third transfer, right: second transfer. Scale bar 10 μm. The culture was transferred three times to fresh media, forming these kinds of aggregates every time. The main objective of this project is to follow up on the dynamics of this community during a 7 day time series, using the third transfer as an inoculum, determining the production of methane, acetate and hydrogen sulfide, as well as analyzing the community composition and distribution through fluorescent microscopy. Methods Enrichments: The initial inoculum for the enrichments used in this study comes from the sediments of Trunk River, in the first meters of the pond (after the river). Samples were stored in 50 mL falcon tubes, and used for inoculation during the same day. In the anaerobic chamber, a sample of approximately 0.5 cm3 was inoculated in 125 mL serum bottles, containing 25 ml of minimal media, specific for the enrichment of sulfate reducing bacteria. This media contained 342.2 mM NaCl, 14.8 mM MgCl2×6H2O, 1 mM CaCl2×2H2O, 6.71 mM KCl, 5mM NH4Cl, 1mM K phosphate, pH 7.2, 5mM MOPS buffer, pH 7.2, trace elements, 1% Resazurin, multivitamin solution, 28 mM NaHCO3 and 20 mM Na2SO4. The headspace gas is replaced with H2/CO2 (80%/20%) by five cycles of vacuum/pressurization. The bottle was incubated at 30ºC, and after 7 days, an inoculum of 1 mL was transferred to fresh media, using a sterile syringe. A total of four transfers were performed. For the last transfer, three conditions were evaluated: SRB media, incubated at 30ºC in the dark (4 bottles); SRB media at room temperature and 630 nm light (1 bottle) and methanogen enriching media (SRB media without sulfate) (1 bottle). The inoculum used for the fourth transfer was also used for the isolation of methanogens, acetogens and sulfate reducing bacteria in agar plates, using the same media described above. For the acetogens, the media was the same as for the methanogens, containing 0.01 M BES (inhibitor of methyl-coenzyme M reductase, a key enzyme in the methanogenic pathway). The plates were incubated at 30ºC in the Wolfe incubator, filled with N2/CO2/H2S gas. Methanogenesis, acetogenesis and sulfate reduction measurements The progress of the enrichments was monitored following the production of methane by gas chromatography, acetate by HPLC and sulfide by chemical assay. 1 mL samples were taken daily, and H2/CO2 was added every second day. Methane was measured taking 250 μl of the headspace and injecting it to the gas chromatograph (Shimadzu). The moles of methane present in the headspace were subsequently transformed to moles of methane per mL of culture, considering the variable volume of headspace/culture, due to daily sampling. These values were finally corrected to reflect accumulated methane, taking into account that after every H2/CO2 vacuum/pressurization event, the total methane was eliminated. The acetate was measured in the HPLC (Shimadzu), using 450 μl of culture fixed with 50 μl of 5N H2SO4. - 2- The concentration of dissolved hydrogen sulfide (H2S, HS , S ) was measured colorimetrically using the Cline assay (REF). Briefly, 300 μl of culture were fixed with 600 μl of Zinc Acetate 1M and stored at -20ºC. The Zinc Sulfide produced was diluted in MilliQ water (10 to 50 times) dissolved with 10 μl of the first reagent of the Hach kit, followed by the addition of 10 μl of the second reagent (N,N-dimethyl-p- phenylenediamine sulfate and ferric chloride). After 5 minutes the absorbance was determined spectrophotometrically at 670 nm. The concentration of H2S was corrected, considering a) the Cline assay measures only the aqueous forms of the dissolved hydrogen sulfide, excluding the gaseous H2S b) the pK value of the reaction H2S(aq) ! H2S(g) at 30ºC is -1.05, giving a ratio of H2S(g): H2S(aq) of 11.22 - + c) the pKa for the reaction H2S "! HS + H at 30ºC is 7.01 and the pH of the - media is 7.2, deriving ratio coefficients for H2S and HS of 0.39 and 0.61, respectively. d) After each H2/CO2 (80%/20%) vacuum/pressurization event, the H2S(g) is evacuated from the sample. The mmoles lost were added to the total dissolved hydrogen sulfide of the following two days. CARD-FISH Finally, 250 μl of culture was fixed with 250 μl of PFA 4%, and incubated at 4ºC overnight. The cells were washed with 10 mL of 1x PBS, filtered through a membrane filter (0.2 μm pore size, 47 mm diameter, polycarbonate (GTTP), Milipore). The cells were washed one more time, passing 15 mL of PBS through the filter. The filters from day 7 of the 6 bottles, and those from the first, second and third transfers, were used for CARD-FISH analysis. The filters were embedded with warm low gelling point agarose (0.2%) using a spray dispenser. The cells were permeabilized with lysozyme (100,000 U ml-1 in 0.05 M EDTA, pH 8.0; 0.1 M Tris-HCl, pH 8.0) for 30-60 minutes at 37ºC. After washing in excess MilliQ water, the cells were treated with proteinase K (15 μg ml-1 in 0.05 M EDTA, pH 8.0; 0.1 M Tris-HCl, pH 8.0, 0.5 M NaCl) for 2-5 minutes at room temperature. Next, endogenous peroxidases were inactivated with 0.01 M HCl for 10-20 minutes at room temperature, followed by a wash in excess MilliQ water and an additional incubation in 3% H2O2 in water for 10 minutes at room temperature. The filters were washed in excess MilliQ water and 96% ethanol, and dried at room temperature. Pieces of filters were incubated with the selected probes in hybridization solution at 46ºC for 3 hours, followed by a 10 minute incubation in washing buffer at 48º and a 15 minute incubation in 1x PBS at room temperature. The filters were subsequently incubated in amplification buffer with 0.15% H2O2 solution in a ratio 100:1 and fluorescently labeled tyramide in a ratio 1000:1, at 46ºC for 30 minutes in the dark. Finally, filters were incubated in 1x PBS at room temperature in the dark, and washed in excess MilliQ water and in 96% ethanol, letting them dry in the dark. Those filters that were subjected to only one CARD-FISH probe were counterstained with DAPI. Those who were subjected to a second hybridization followed the same protocol described above, beginning from the inactivation of peroxidases. The filters were analyzed through fluorescent microscopy (AXIO Imager A2, Zeiss), exciting the cells at 365 nm, 470 ± 20 nm and 550 ± 12,5 nm, for DAPI, Alexa Fluor 488 tyramide and Alexa Flour 594 tyramide signals, respectively. SEQUENCING Two clone libraries, one for bacteria and one for archaea, were prepared with DNA extracted from the culture used as an inoculum for the fourth transfer. A total of 8 clones were sequenced and aligned to the NCBI database. RESULTS The cultures from the six bottles showed consistent growth during the week of incubation. Cell density was qualitatively measured during day 2, 5 and 7 by microscopy, revealing cell growth and the formation of small aggregates. In this case, the size of the aggregates was between 3 and 5 μm (figure 2). Figure 2: DAPI staining of cells from day 5, present in the enrichment from a bottle in SRB media (left) and Metgen media (right), both incubated at 30º in the dark. Scale bar: 10 μm. The production of methane, acetate and hydrogen sulfide was measured daily. Figure 3 shows the moles of each compound, per ml of culture. Hydrogen sulfide increased consistently in those bottles that contained sulfate. The bottle that was incubated at room temperature and 630 nm produced approximately 1 mmole l-1 less than those grown at 30ºC in the dark.