Looking for syntrophic acetogen/methanogen interactions Caroline Van Steendam Final Report Course Directors: Dr. Jared Leadbetter and dr. Dianne Newman Microbial Diversity Course 2015 Caroline Van Steendam – Looking for syntrophic acetogen/methanogen interactions 1 Introduction Many natural environments (e.g., soil sediments) and engineered systems (e.g., rice paddy soils and wastewater treatment) are known to produce methane when anaerobically degrading organic matter. Uncontrolled release of methane in natural environments should be minimized as methane is 23 times more potent at retaining heat in the atmosphere than CO2. In contrast, methane production is often maximized in engineered systems because of its high calorific value and therefore its suitability as an (alternative) energy source. Even though the anaerobic degradation process of organic matter has been studied in detail for decades, new discoveries are still made. Fermenter acetogens and methanogens work together to convert intermediate degradation products, i.e., volatile fatty acids (VFAs), into methane. The fermentative conversion of VFAs is thermodynamically unfavorable under standard conditions (1 M concentration, or 105 Pa for gases) and requires a continuous removal of its reaction products by methanogens to overcome this energy barrier. On top of the well- known syntrophic interactions including interspecies H2, formate, and acetate transfer, it has recently been discovered that electrons can also be exchanged directly between the fermenter acetogen and methanogen (direct interspecies electron transfer, DIET) (Chen et al., 2014; Liu et al., 2012; Nagarajan et al., 2013; Shrestha et al., 2014; Shrestha et al., 2013; Summers et al., 2010; Zhao et al., 2015). The goal of this miniproject is to isolate different types of methanogens from Cedar Swamp and to identify what syntrophic interactions they are capable of. The first stage of the project consists of setting up different culture enrichments with inoculum from Cedar Swamp, to select for (i) hydrogenotrophic (utilizing H2 and CO2) and aceticlastic (utilizing acetate) methanogens, (ii) butyrate fermenting acetogens, and (iii) co-cultures of such acetogens and methanogens. Two bench-scale microbial fuel cells (MFCs), inoculated with a sample from Cedar Swamp, are set-up in parallel to select for acetogens and methanogens that are capable of directly exchanging electrons. A potentiostat is used in the final stage of the project to study the DIET capabilities of an enriched methanogenic culture. 2 Methods 2.1 Anaerobic Culturing A total of seven bottles were inoculated with Cedar Swamp samples, all of which containing the same basic liquid media (see Appendix A) but varying in gaseous headspace and acetate, butyrate, and bicarb concentrations (see Figure 1, liquid volume of 25 mL, gaseous volume of 132 mL). Table 1 shows the reactions related to the different types of metabolism targeted in these bottles (Dong et al., 1994). Table 1: Different reactions expected in cultures − − Aceticlastic 퐶퐻3퐶푂푂 + 퐻2푂 ↔ 퐻퐶푂3 + 퐶퐻4 Reaction 1 methanogenesis Hydrogenotrophic 퐶푂2 + 4퐻2 ↔ 퐶퐻4 + 2퐻2푂 Reaction 2 methanogenesis − − − + − Butyrate 퐶4퐻7푂2 + 2 퐻퐶푂3 ↔ 2퐶퐻3퐶푂푂 + 퐻 + 2 퐻퐶푂푂 Reaction 3 fermentation (1) − − + Butyrate 퐶4퐻7푂2 + 2퐻2푂 ↔ 2퐶퐻3퐶푂푂 + 퐻 + 2 퐻2 Reaction 4 fermentation (2) 2 Caroline Van Steendam – Looking for syntrophic acetogen/methanogen interactions The first bottle was given a 100% N2 headspace and was supplemented with 2 mM acetate, in order to enrich for Methanosaeta, an aceticlastic methanogen (Reaction 1). The second bottle also had 100% N2 as headspace but contained a higher acetate concentration for the more copiotrophic Methanosarcina, an aceticlastic methanogen often dominating in systems with an acetate concentration above 4 mM (Conklin et al., 2006). The third and fourth bottle had a headspace of 80% H2 and 20% CO2 to enrich for hydrogenotrophic methanogens (Reaction 2), while only the third contained 1 mM bicarbonate (in case some of the methanogens are heterotrophs and need an additional carbon source). A co-culture of a butyrate degrader and an aceticlastic methanogen was enriched for in the 5th bottle by adding 2 mM butyrate, 1 mM bicarb, and 0.01 mM acetate to the basic medium while supplying 100% N2 in the headspace (optimal thermodynamic concentration of acetate, as shown in Figure 2, and the relevant reactions are Reaction 1 and Reaction 3). The 6th bottle aimed at enriching a co-culture of a butyrate degrader and a hydrogenotrophic methanogen, by only supplying 2 mM butyrate (Reaction 2 and Reaction 4). Glass slides were also added to bottles #5 and 6 to promote biofilm growth and give the acetogens and methanogens the possibility to grow closer together. A butyrate fermenter was targeted in the last bottle, by supplying 2 mM butyrate and a 100% N2 headspace. Figure 1: Different cultures. 1,2: aceticlastic methanogens, 3, 4: hydrogenotrophic methanogens, 5: coculture of butyrate fermenter / aceticlastic methanogen, 6: coculture butyrate fermenter/hydrogenotrophic methanogen, 7: butyrate fermenter. Figure 2: Input (lefthand side) and output (righthand side) for thermodynamic excel sheet 2.2 Microbial fuel cells Two MFCs were built using available lab equipment, by connection two glass jars (referred to as chambers in the rest of the text) with a salt bridge for proton transfer and a copper wire for electron transfer (see Figure 3). All four chambers were inoculated with Cedar Swamp sample and had a headspace of 100% N2, however, different electron donors were supplied (total liquid volume of 50 mL). Both acetogenic chambers were supplemented with 2 mM butyrate, while one methanogenic chamber just consisted of 3 Caroline Van Steendam – Looking for syntrophic acetogen/methanogen interactions the basic methanogenic medium and the other methanogenic chamber had an additional 0.01 mM of acetate and 69 mM bicarb. Each chamber contained a submerged graphene rod attached to a wire with a resistor (470 kΩ) in the middle, allowing for electron transport. The connection of both chambers via a salt bridge, made by hardening a hot solution containing 2% agar and 1M KCl into a non-gas-permeable tube, allowed for proton transport between both chambers. Finally, some sampling ports were added and the chambers were connected to a N2-filled bottle to enhance the ability of the system to cope with pressure increase. Figure 3: Set-up of a microbial fuel cell 2.3 Potentiostat Another methanogenic chamber, containing the hyphomicrobium medium (see Appendix B) and a 100% N2 headspace, was inoculated with a culture enriched in methylotrophic methanosarcina (see Figure 4). Two graphene rods connected to the working and auxiliary electrode of a potentiostat were submerged in the chamber, as well as a reference electrode. The potentiostat was set-up in such a manner that the voltage through the working electrode equaled -0.5V vs standard hydrogen electrode. The chamber was, just as with the microbial fuel cells, connected to a N2-filled bottle to prevent gas leakage by providing more flexibility towards increased system pressure. Figure 4: Set-up of chamber connected to the potentiostat 4 Caroline Van Steendam – Looking for syntrophic acetogen/methanogen interactions 2.4 Chemical analyses The methane production in each bottle was quantified using a gas chromatograph (GC) connected to a flame ionization detector. A gas-tight syringe was flushed with N2 before it was used to inject 250 µL of sample into the GC. N2 was used as a carrier gas. A 12.7% methane standard was made by adding 20 ml of CH4 to a N2-flushed vial of 157 ml, and 17 ml was added to a 69.56 ml vial to obtain a 24.44% standard Both standards and a blank were run each time samples were analyzed, and all samples were run in triplicates. The acetate and butyrate concentrations were measured using a high pressure liquid chromatography (HPLC) connected to a UV detector. Butyrate standards, ranging from 20, 10, 5, 2.5, to 1.25 mM, were run once and used for the calibration of all samples. HPLC samples were prepared by centrifuging 900 µL of a sample together with 100 µL of a 5N H2SO4 solution, and adding 550 µL of the supernatant to a HPLC vial. 2.5 Molecular analyses Two attempts at creating a clone library were made. In the first try the genomic DNA was obtained by boiling the samples in ALP reagent (in order to take a sample from the liquid cultures, 750 µL of each sample was centrifuged and the pellet was picked with a toothpick) after which PCR amplification was performed with a mastermix solution (25 µL of GoTag green, 1 µL fwd primer, 1 µL rev primer 1341R, 20 µL NF H2O, 3 µL of the boiled template) and controlled with a gel extraction (1x TAE, 1% agarose LE, over 120 V). The PCR products were cloned into a plasmid vector (according to the pGEM T-easy cloning procedure) and then transformed into E. coli (utilizing the transformation procedure) and plated onto LB plates with ampicillin/IPTG/X-Gal. The second attempt included using a DNA extraction kit (‘Wizard Genomic DNA Purification Kit’) instead of performing boiling in an ALP solution. PCR amplification was performed with the same mastermix solution, after which the PCR products were extracted with a gel. 2.6 Microscopy The available Zeiss microscope was used to visualize liquid mounts with a 100X objective while applying phase contrast, while the measured excitation when transmitting Lucifer Yellow light was used to identify methanogens. The cultured samples were analyzed with CARD-FISH once, in order to identify and/or determine the relative abundance of the present microorganisms. After fixing these planktonic cells on a filter using a 2% PFA solution, the microorganisms were embedded with 0.1% agarose, and permeabilized using both a 1 mg/mL lysozyme solution and a fresh 15 µg/mL proteinase K solution. The endogenous peroxidases were inactivated using a 0.01 M HCl solution (3% H2O2) and the samples were hybridized using a hybridization buffer of the correct formamide concentration (e.g., probes ARCH 915 requires 35% formamide).