Anaerobic Methane Oxidation: Occurrence and Ecology. ALEXANDER J. B. .... sealed with an aluminum seal (1), and made anaerobic ..... Bay Lagoon, Alaska.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 1980, p. 194-204 0099-2240/80/01-0194/11$02.00/0
Vol. 39, No. 1
Anaerobic Methane Oxidation: Occurrence and Ecology ALEXANDER J. B. ZEHNDER* AND THOMAS D. BROCK Department of Bacteriology, University of Wisconsin, Madison, Wisconsin 53706
Anoxic sediments and digested sewage sludge anaerobically oxidized methane to carbon dioxide while producing methane. This strictly anaerobic process showed a temperature optimum between 25 and 37°C, indicating an active
microbial participation in this reaction. Methane oxidation in these anaerobic habitats was inhibited by oxygen. The rate of the oxidation followed the rate of methane production. The observed anoxic methane oxidation in Lake Mendota and digested sewage sludge was more sensitive to 2-bromoethanesulfonic acid than the simultaneous methane formation. Sulfate diminished methane formation as well as methane oxidation. However, in the presence of iron and sulfate the ratio of methane oxidized to methane formed increased markedly. Manganese dioxide and higher partial pressures of methane also stimulated the oxidation. The rate of methane oxidation in untreated samples was approximately 2% of the CH4 production rate in Lake Mendota sediments and 8% of that in digested sludge. This percentage could be increased up to 90% in sludge in the presence of 10 mM ferrous sulfate and at a partial pressure of methane of 20 atm (2,027 kPa).
Measurements of methane in freshwater and marine environments made by several geochemists (2, 12, 19) have suggested that an anaerobic consumption of methane might occur. Although aerobic methane oxidation is a wellknown process (9, 18), the existence of any organisms which could oxidize methane anaerobically has been controversial (7, 13, 17, 24, 27). Recently, Zehnder and Brock (30) have shown that all methane-forming bacteria tested are also able to oxidize a small amount of methane anaerobically and that this oxidation occurs at the same time that methane is produced. With most of the methanogens, the only product formed from methane is carbon dioxide, but with Methanosarcina methanol and acetate are also formed and with the "acetate organism" (30) the major product of methane oxidation is acetate. Zehnder and Brock (30) presented evidence that methane oxidation by methanogens was not a simple back reaction, but involved intermediates different than those involved in methane production. In the present paper, we extend the studies on anaerobic methane oxidation to anaerobic habitats where methanogens are active. We show that anaerobic methane oxidation occurs routinely in such habitats and that the methane-producing bacteria might be actively involved in the process. However, a net consumption of methane, such as is required to explain the geochemical data (2, 12, 19), was not obtained, although in the presence of appropriate additions and under high pressures of methane, the oxidation rate was 0.9 of the production rate. In addition to our general studies describing
the nature of the anaerobic oxidation process, we have also carried out experiments in which various alternative electron acceptors [sulfate, iron (III), manganese (IV)] were added to determine whether they might be involved in the process. It can be seen from Table 1 that the free energy values for reaction of methane with some of these electron acceptors are almost as favorable as for reaction with 2. Although some of these additions markedly affect the process, we were unable to determine whether their influence was direct (on the methanogens) or indirect (on other organisms in the system). The present study should serve as a basis for further experiments on the nature and geochemical importance of the anaerobic methane oxidation process. MATERIALS AND METHODS Location and sampling. (i) Freshwater sediments. Freshwater sediments were obtained from Lake Mendota, a hard-water eutrophic dimictic lake with a mud bottom. From July until turnover in October, the water overlying the sediments is anoxic and contains hydrogen sulfide. These sediments actively produce methane during the entire year (32). Samples were taken in the deepest section of the lake with a water column depth of 23 m. Grab samples were taken with an Eckman dredge (Wildlife Supply Co., Saginaw, Mich.) and were immediately transferred into 200-ml Mason jars (ordinarily used for canning). The jars were filled up to the top with special attention to avoid entrapping air. The ferrous sulfide (5) in the sediments prevented harm to anaerobes by the brief exposure to oxygen while the sediments were dispensed (29). Sediment samples could be stored at 4°C for at least 2 weeks without any loss of methanogenic activity. 194
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TABLE 1. Gibbs free energy' for methane as electron donor and various inorganic electron acceptors at pH 7 and 25oCb AG Electron acceptor
kcal/e-
kJ/e-
CO2 SO42-c FeOOH(s)d
0 0 -2.8 -0.67 -22.6 -5.4 -81.48 -19.46 MnO2(s)' -22.77 -95.34 N03-f -24.6 -103.00 02 aCalculated from values in Wagman et al. (26) and Stumm and Morgan (25). b These equilibria have been calculated assuming the following concentrations: [H+] = 10-7 M; [HCO3-] = 10-3 M; [HS-] = 10-3 M; [SO42-] = 10-2 M; [NO3-] = 10-2 M; PCH, = 1 atm; Po2 = 0.2 atm. C Reduced form: H2S/HS- at pH 7. dAssuming the amorphous form in the oxidized state and FeCO3(s) (siderite) in the reduced state. In case of Fe(OH)2(s) or a-FeS(s) as the reduced state, the energy changes are 0.92 kcal/e- (3.9 kJ/e-) and -11.34 kcal/e- (47.5 kJ/e-), respectively. ' Manganate IV, "a-MnO2"(s), in the oxidized state and rhodochrosite MnCO3(s), in the reduced state. Between pe8 and 10.5 (470 and 620 mV) y-MnOOH(s) (manganite) and Mn3O4(s) (hausmannite) may be stable and act as possible electron acceptors. The Gibbs free energy for manganite reduction is -17.56 kcal/e(73.5 kJ/e-); the reduction of hausmannite yields -18.15 kcal/e- (78.0 kJ/e-). f Reduced form: N2. g Reduced form: H20.
Cores (6.6-cm diameter) were ordinarily taken by a scuba diver. After the cores were removed, they were sealed with black rubber stoppers at the lake bottom and immediately taken to the laboratory in their liners. Sediment samples from the different depths were taken with a syringe through holes which were drilled at 1-cm intervals into the liners before core sampling in the lake (the holes were sealed with adhesive tape to avoid loss of liquid during transport). In some cases a Benthos core sampler was used, taking into account that the uppermost 20 cm of the sediment was considerably compressed by this method. Subsamples from these cores were taken as described above. (ii) Marine sediments. A 20-cm core of sediment, black and smelling strongly of hydrogen sulfide, was used in the marine sediment study. The core was collected by Michael Klug in the Izembek Bay which is adjacent to Cold Bay, Alaska. The bay supports one of the most productive sea grass beds (Zostera marina) in the world. The core originated from an area devoid of grasses, but having a heavy detrital mat over the sediments. The area is about 2 m deep at high tide and never completely exposed at the lowest tides. Syringes could not be used to take subsamples, because broken shells and coarse sand caused clogging even when large-bore needles were used. Therefore, we applied the following method: the lower part of the core liner was sealed with a one-hole rubber stopper. Oxygen- and sulfate-free ocean water (see below for
195
composition) was added slowly through the hole with a slight overpressure to force the sediment core to the top of the liner. Subsamples were taken by carefully pushing a glass tube (1-cm inner diameter) 1 or 2 cm into the sediment. To avoid compression, a slight vacuum was applied to the tube. The subsamples were transferred to serum vials which were immediately sealed and made anaerobic (see below). This procedure allowed only minimal exposure of a minor part of the sediment to the air. Traces of oxygen which nevertheless difused into the sediment were immediately removed in the serum vials (see below). (iii) Digested sewage sludge. Digested sewage sludge was obtained from the digestor of the Madison metropolitan sewage treatment plant. This slightly overloaded digestor runs on an average loading of 3 kg/m3 (as volatile solids) with a mean retention time of 8 to 10 days. Incubation procedure. All incubations were made in the dark in 35-ml serum vials with 20 ml of liquid. The vials were closed with black-lip rubber stoppers, sealed with an aluminum seal (1), and made anaerobic by evacuating and flushing alternately several times with the gas mixture desired. Additions of nonvolatile substances were made before the flushing procedure. To dilute samples, we used either a mineral salt medium or synthetic ocean water. Mineral salt medium consisted of the following (in grams per liter of distilled water): KH2PO4, 0.41; Na2HPO4, 0.43; NH2Cl, 0.48; NaCl, 0.48; CaCl2-2H20, 0.18; MgCl2.6H20, 0.16. The free bicarbonate concentrations in lake sediments and digested sludge differ greatly. Therefore, sodium bicarbonate was added in such amounts that the final concentration in the incubation vials was the same as in the natural sample. The pH was kept at neutrality by means of carbon dioxide in the headspace. The anaerobic metabolism of different substrates supplied often increased the pH, which was detected by measuring the carbon dioxide concentration in the headspace. When necessary, the pH was readjusted by injecting carbon dioxide into the headspace. Synthetic ocean water. The amounts of the chemicals in grams per liter of distilled water were calculated from the total ion composition of seawater given by Riley and Chester (23): NaCl, 23.8; MgCl2. 6H20, 11; Na2SO4, 4; CaCl2.2H20, 1.5:, KCI, 0.76; NaHCO3, 0.2; NaBr, 0.082; SrCl2.6H20, 0.024; KF. 2H20, 0.0066; H3BO3, 0.0037. For most of our studies, we omitted the sodium sulfate. To keep the ionic strength constant, 3 g/liter of NaCl were added instead. The pH was kept at neutrality as described above. Colloidal MnO2. To precipitate colloidal manganese dioxide, the procedure described by Morgan and Stumm (15) was used. The "B-MnO2" (manganate IV) (25) formed was washed five times with and subsequently stored in distilled water. Determination of methane oxidation. Radioactive 14CH4 was injected into the headspace with unlabeled methane (2 ml). Radioactive methane was made from ['4C]bicarbonate or [2-'4C]acetate by means of methanogenic organisms (30). This methane was rigorously tested for radiochemical purity, and no contaminations have been detected (30). To analyze for
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APPL. ENVIRON. MICROBIOL.
methane oxidation products, 2 ml of liquid was re- not exceed 5% under the condition used (e.g., very moved with a syringe and injected through a rubber carefully bubbling and scintillation counting at least septum into a 10-ml serum vial containing 1 ml of 1.5 up to 2,000 cpm). Tubes for higher pressures. For pressures higher N NaOH. Subsequently the vial was vigorously shaken to absorb all C02 or volatile thiols. To fractionate the than 3 atm, the methods of Balch and Wolfe (1) were sample, first a short needle was inserted into the gas modified by using autoclavable stainless steel tubes space of the serum vial. To obtain volatile amines, air and a special device for pressurizing them (Fig. 1). The was bubbled through the vial and sequentially bubbled tubes were shaken during pressurization to allow gases through two scintillation vials, in series, which con- to dissolve. Gas analysis. Oxygen and carbon dioxide were tained 0.1 N H2SO4 (23). Scintillation vials were subsequently replaced by three others in series. The first measured with a Packard model 419 gas chromatovial contained 4 ml of an acid (pH < 2) 3% (wt/vol) graph equipped with a thermal conductivity detector. HgCl2 solution to absorb volatile thiols such as meth- Oxygen was quantified with a column (70 cm long, 2.5 ane thiol and hydrogen sulfide. The next two vials mm inside diameter) held at room temperature and each contained 2 ml of phenethylamine (scintillation packed with molecular sieve (100/120 mesh). Helium grade) and 2 ml of methanol to trap the carbon dioxide. as carrier gas had a flow rate of 30 ml/min. For the To liberate these acid-volatile compounds, 2 ml of a 6 quantitation of carbon dioxide, the same settings were N H2SO4 solution was carefully injected into the serum used, except that Poropack QS (80/100 mesh) was vial containing the alkaline sample. After the C02 was used as stationary phase. The detection linits for bubbled off, the acidified sample was filtered through a glass fiber filter (Whatman glass microfiber paper, grade GF/C, Scientific Products), and the filtrate was collected. Then, 4 ml of the filtrate, the 0.1 N H2S04 trapping solution, and the HgCl2 solution were each mixed with Aquasol (New England Nuclear Corp., Boston, Mass.). The scintillation vials with phenethylamine-methanol solution received 10 ml of a toluene fluor mixture with 0.375 g of PPO (2,5-diphenyloxazole, Beckman Instruments, Inc., Fullerton, Calif.) and 0.1 g of dimethyl-POPOP (1,4-bis[2(4 methyl-5-phenyloxazolyl)]-benzene, Packard Instrument Co., Inc.) per 1,000 ml of toluene. All 14C radioactivity was counted with a Tri-Carb 3375 scintillation spectrome5 ter (Packard) with the window set at 40 to 1,000 and the gain set at 12%. Quench corrections were made by the channels ratio method. 3. In the course of incubation some samples produced methane, and the ['4C]methane was therefore continuously diluted. The specific activity of methane was determined for each time point by the method described by Zehnder et al. (31). The total amount of CH4 oxidized was calculated by the method of Zehnder and Brock (30). In a time course experiment it was necessary to determine the distribution of the carbonate species to calculate the total amount of "'4C02" [CO2 refers to the total of C02(gas), C02(dissolved), HC03-, and CO32-] formed from methane. For this purpose, control samples were prepared exactly as those in which the methane oxidation had to be followed, but instead of radioactive methane 0.5 uCi of NaH'4C03 (50 mCi/mmol) was added. These controls were sampled simultaneously with the other vials to FIG. 1. High pressure tube and device for pressurdetermine the distribution of the carbonate species. The radioactive bicarbonate controls were not neces- ization. All parts in stainless steel if not otherwise sary if an entire incubation mixture was made alkaline stated. Part 1, Cap 5/8 inch (ca. 15.9 mm) with hole to trap all of the CO2 in the liquid phase. In this case in the center (Swagelok). Part 2, Black rubber stopper samples for the amine analysis were taken directly no. 0 with the top cut off. Part 3, Tube 18 cm long, from the headspace and injected into vials containing outer diameter 5/8 inch, with a 0.65-inch (ca. 16.5 0.1 N H2SO4. In all cases, the dissolved radioactive mm) wall. Part 4, Same cap as under I but without methane was bubbled off with air. Freshly prepared hole. This tube should hold up to 250 atm (25,331 ['4C]bicarbonate controls were taken to check the kPa). Part 5, Tube with 1/4-inch (ca. 6.4-mm) outer reproducibility and efficiency of the radioactive CO2 diameter filled with cotton to use as sterile filter. Part recovery. Before the analysis the vials were shaken for 6, Female connector (Swagelok). Part 7, Connector to 30 min to allow the carbonate species to equilibrate. Luer Lock made out of Kel-F (Hamilton, Co., part no. The standard deviation of the C02 measurements did 86536). Part 8, Disposable needle.
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oxygen were 0.1 nmol and those for carbon dioxide were 2.1 umol. Methane was assayed with a gas chromatograph with a flame ionization detector (31). At high methane partial pressures it was necessary to
dilute samples before injection. Control experiments. Two different "killed" controls were made for each experiment and addition. In the one, high temperature (900C) was used to prevent biological activity. The other control received 10 mg of mercury bichloride per ml of solution. This fairly high concentration was needed because mercury forms an insoluble salt with sulfide which is present in considerable amounts in sediments and digested sludge. These two control experiments were chosen based on the consideration that a system should undergo the least changes while all biologically mediated reactions were inactivated (4, 30). Chemicals, radiochemicals, and gases used. 2Bromoethanesulfonic acid sodium salt was obtained from Eastman Kodak Co., Rochester, N.Y. All chemicals were of reagent grade. ['4C]Sodium bicarbonate (50 mCi/mmol), [2-'4C]sodium acetate (2 mCi/mmol), ['4C]sodium formate (3.9 mCi/mmol), ['4C]methanol (0.85 mCi/mmol), [14C]methanethiol (14.5 mCi/ mmol), and [methyl-'4C]methionine (10 mCi/mmol) were purchased from New England Nuclear, Boston, Mass. Gases and gas mixtures were purchased from Matheson Gas Products, Joliet, Ill. in anaerobic purity. We never detected in these gases oxygen contamination.
RESULTS
Methane formation and methane oxidation in anaerobic habitats. When anaerobic sediments or digested sludge are incubated anaerobically under a headspace of nitrogen and carbon dioxide which contains ['4C]methane, a production of 14CO2 can be observed. The amount of 14C02 formed increases with time in parallel with the build-up of "endogenous" methane (Fig. 2). A typical time course of such an experiment is shown in Fig. 2. The oxidation product from methane was >99% carbon dioxide. The rest of the radioactivity was acid soluble, and no labeled amines or thiols were found. To ensure that the observed formation of carbon dioxide from methane was not due to oxygen leaking into the vials through the black rubber stoppers, serum vials with anaerobic black mud from Lake Mendota were either incubated just as they were or immersed upside down in a sealed anaerobe jar (BBL Microbiology Systems, Cockeysville, Md.) filled to the top with 10 mM neutralized titanium (III) citrate solution (33) (water containing sulfide as an anaerobic barrier is not suitable because the aluminum seals of the serum vials react with sulfide and are subsequently dissolved). This setup made it unlikely that oxygen might penetrate into the vials containing the sediments. The results of this test are summarized in Table 2 and clearly
0
4
12
8
16
20
0
2
DAY
6
10
DAY6
FIG. 2. Typical time course of methane formation and simultaneous anaerobic methane oxidation by anoxic Lake Mendota surface sediment collected at the end of the summer stagnation period and by digested sewage sludge. With both materials, 10 ml was diluted with an additional 10 ml of mineral salts medium. Initial specific activity of methane, 39 ,iCi/ mmol. A heat inactivated and HgCl2 killed sample served as controls. TABLE 2. Methane formation and oxidation by lake sedimentsa Sample Immersedd 37°C Not immersed
370C 370C + HgCl2
CH4
CH4 oxidized (dpm)b
formed (ml)
14C02
7.31 ± 0.52
62,400 + 4,800
180 ± 48
7.25 ± 0.61
63,500 ± 5,300 450 ± 63
210 ± 41 120 ± 50
