ANAEROBIC METHANE OXIDATION - RATE DEPTH DISTRIBUTIONS IN SKAN BAY SEDIMENTS

Abstract A radiotracer method that measures rates of oxidation of methane to carbon dioxide has been applied to anoxic marine sediments. The results confirm the occurrence of anaerobic methane oxidation and agree with model predictions of a zone of intense anaerobic methane oxidation at the base of the sulfate-reducing zone.


Introduction
Anaerobic methane oxidation has been suggested as a process occurring in marine sediments [1][2][3]. The process appears to be a general feature of anoxic marine sediments and has been identified as a major sink for methane [4]. It also accounts for a substantial fraction of sulfate reduction in marine sediments [5]. Fig. 1 [4] schematically summarizes depth distributions of methane (CH4), total carbon dioxide (X;CO2), sulfate (SO~-) and the stable carbon isotope ratio of carbon dioxide (613CO2) from a number of anoxic marine sediments. The distributions of all species show slope changes or minima at the same depth, suggesting that most of the methane consumption takes place in a thin subsurface depth interval.
The 61aCO2 minimum appears to be the result of local injection of methane-derived carbon dioxide. Diagenetic models indicate the process occurs at rates in the 1-10 mM/yr range [1,2].
The apparent absence of anaerobic methane oxidation in freshwater (low sulfate) sediments suggests the involvement of sulfate reducers [6].
Anaerobic methane oxidation has been described geochemically by the following net reaction: CH4 + SO42-+ 2H ÷ ~ H2S + C02 + 2H20 (1) This reaction has not been observed directly and organisms capable of mediating it have not been isolated. Laboratory evidence favoring [7] and disputing [8] anaerobic methane oxidation has been presented. Zehnder and Brock [9] reported simultaneous methane production and anaerobic consumption by nine methanogen strains. Since the amounts oxidized were less than 1% of the CH4 formed, this process is an unlikely explanation for the large CH4 losses observed in geochemical studies of marine sediments. Panganiban et al. [10] showed that enrichment cultures of organisms from Lake Mendota surface sediments were capable of oxidizing CH4 anaerobically. These organisms required sulfate, acetate and methane for growth, assimilating acetate and oxidizing methane. Kosiur and Warford [ 11 ] added labelled acetate and lactate to Santa Barbara Basin sediments in in-vitro experiments. Changes with time in the specific activity of the 14CH4 and 14C02 produced indicated that anaerobic methane oxidation occurred in the 100/IM/yr range. This report describes "quasi-in-situ" measurements of anaerobic methane oxidation and sulfate reduction in Skan Bay sediments. These measurements provide more direct evidence that anaerobic methane oxidation does occur and give rate depth distributions and rates that agree with diagenetic models [ 1,2].

Experimental
These experiments were conducted on sediment cores from Skan Bay (57°37'N, 167°03'W, 65 m), a  0  4  8  12  CH4(mM)  '1  I  I  I  -20  -15  -I0  -5  0  5 813 C02 (%*)  [  i  I  I  I  i  0  I0  20  50  SO4(mM)   I  I  I  I   0  I0  20  30  40  '"  basin with an intermittently anoxic water column on the northwest side of Unalaska Island [ 12]. Skan Bay has a sill depth of 10 m and a maximum depth of 65 m. The sediments are rich in organic material (kelp fragments) and are anoxic. Sediments were sampled with a Benthos gravity corer used without a core catcher. Sediment samples were extruded from the cores into squeezers and the interstitial waters were analyzed for CH4 and ~CO2 by gas chromatography [ 14]. Sulfate samples were collected in pre-weighed bottles containing HC1. Sulfate was separated using ion exchange and evaporation [15] and was analyzed by conductometric titration [16]. The CH4, ~;CO2 and SO~-depth distributions in Skan Bay sediments are shown in Fig. 2.
Anaerobic methane oxidation rates and sulfate reduction rates were measuredusing modifications of the technique described by J¢rgensen and Tenchel [17]. Solution of 14CH4 and NazaSSO4 were injected into selected depth intervals of minimally disturbed sediment cores. Following incubation, the products COz and H2S were stripped from solution and assayed for radioactivity. Three separate cores were used in the experiments reported here, one for the determination of CH4, ECOz and SO~-and one each for the methane oxidation and sulfate reduction tracer experiments. Reeburgh [14] and Mattisofet al. [18] have established that interstitial water distributions in parallel cores from a single station are subject to small lateral variations and much larger vertical variations. Errors introduced by this compromise are expected to be small.
Freshly collected gravity cores were transferred to an experimental core tube for these experiments. The experimental core tube was joined with plastic electrical tape to the top of a sediment core and the sediment was extruded upward into the experimental core tube, overflowing the overlying water and avoiding contact with air. The experimental core tube consisted of segments (6.5 cm I.D. X 2.5 cm) of plastic core liner joined together with plastic electrical tape. Alternate segments of the experimental core tube were equipped with septa of RTV silicone rubber for syringe injection of the tracer solutions. Depth intervals of interest in the sediment core were positioned within experimental core tube segments for the tracer experiments.
Both tracers were dissolved in a sterile, oxygenfree NaC1 solution with a density close to that of the interstitial water to avoid adding electron acceptors and to minimize migration of the added tracer during incubation. 14CO2 impurities were removed from the 14CH4 used in the methane oxidation rate measurements 6y admitting the 14CH4 to an evacuated manifold and toepler pumping through a trap containing Mallcosorb (20-50 mesh). A portion of 14CH4 was transferred to an evacuated reservoir bulb and the tracer solution was formed by admitting the heliumstripped NaC1 solution. Aliquots of the 14CH4 solution were displaced from the reservoir bulb into Chaney adapter syringes by syringe addition of pure mercury. Mercury added to sediments with the 14CH4 tracer solution should be rapidly immobilized by excess sulfide in the sediments [ 19]. Portions of the 14CH4 solution were injected into a carbon dioxide absorbing scintillation cocktail [20] and stripped with helium. Lack of activity in the stripped solution demonstrated that the 14CH4 solution was 14COzfree. Fifty microliters of the 14CH4 tracer solution (0.15/~Ci, 4.25 mCi/mmole, 0.7 raM) were injected into the center of each experimental core segment.
The Na235SO4 used in the sulfate reduction rate measurements was obtained carrier-free and was dissolved in a similar NaC1 solution. Twenty microliters of the 3SSO~-tracer solution (0.1 ~Ci, 55.5 mCi/ mmole, 0.09 mM) were injected into the center of each experimental core segment.
Following incubation in a water bath near in-situ temperatures for 24-36 hours, the experimental core tubes were clamped upright and dismantled by removing the plastic tape and isolating each segment with sheets of stainless steel shim stock. Each labelled core segment was transferred to a helium-flushed Mason jar containing a magnetic stir bar. The jars were capped with rubber gasketed lucite lids fitted with a gas inlet, a gas outlet and a septum modified from Swagelok fittings. The lids were sealed with electrical tape and held in place with threaded metal binding rings. Five milliliters of 2N KOH were added to kill the samples and to retain the CO2 and H2 S formed [21]. The samples were frozen until analysis (4-6 weeks) in the laboratory. The frozen samples were attached to the absorption line, thawed, and a 347 stirrable sediment slurry was formed by adding distilled water through the septum with a syringe. Methane was removed by stripping the basic sediment slurry with helium (100 ml/min) and was trapped as CO2 after oxidation on a CuO column at 600°C in a series of three LSC vial strippers containing a carbon dioxide-absorbing cocktail [20]. Following removal of the CH4, the CO2 was collected by acidifying the slurry and stripping with helium. The CO2 was trapped in another series of LSC vial strippers. The large quantities of H2S produced on acidification cause severe quenching; this HaS was removed in a gas phase trap consisting of CuSO4 dried on Celite. The trapped 14CO2 was counted to 1% precision in the LSC vials used for stripping. Quench corrections were determined with external standard ratios and the counts from three stripper vials were summed. The initial activity of the added 14 CH4 was determined in the laboratory by oxidizing 50/11 aliquots of the 14CH4 tracer solution in a circulating combustion and trapping system [21 ]. The carbon dioxide formed was trapped in phenethylamine and counted in a toluene cocktail. Hydrogen sulfide from the sulfate reduction rate measurements was stripped from an acidified sediment slurry and was trapped as CdS in a series of three LSC vial strippers containing CdCl2 solution. Weighed portions of this CdS was counted in a gas flow proportional counter; a self-absorption curve was prepared using constant specific activity Cd3SS obtained by chemical reduction [22] of 3SSO24-and carrier sulfate. The stock 3SSO~-solution activity was measured when the recovered activity was counted, avoiding decay corrections [23].

CH4, ECO2 and S024 -
The distributions of CH4, ~CO2 and SO42-shown in Fig. 2 are similar to those shown schematically in Fig. 1, indicating that Skan Bay is similar to other marine environments. Methane bubbles formed at depths below 17 cm in the cores; decreases in methane concentration at depth and the lack of a distinct low-CH4 surface zone as shown in Fig. 1 probably reflect the effects of CH4 ebullition and dissolution after core collection.

Rate measurements
These tracer experiments are intended to show depth distributions of the rates of anaerobic methane oxidation and sulfate reduction in minimally disturbed sediment cores. Two main points must be addressed in experiments of this type: (1) the tracer added and product formed must be contained in the experimental core segment and recovered completely, and (2) the form and fate of the tracer and products must be known. Equal amounts of tracer were injected into the experimental core segments and identical procedures were used in the addition of the tracer and extraction of the products. Care was taken to insure tracer purity and both were added in small quantities in a biologically passive supporting solution. The tracer solutions in both rate measurements were added to a small volume in the center of a core segment in concentrations that did not exceed ambient concentrations [24]. No attempt was made to homogenize the tracer solution and sediment. The experimental volumes of each core segment were well removed from the effects of sediment smearing along the walls during coring and core transfer. Tracer diffusion distances of less than a centimeter are indicated by characteristic diffusion coefficients and incubation times, so diffusive losses of tracer during incubation and transfer to the Mason jars are not expected to be a problem [25]. By the same reasoning, diffusive inputs of oxygen will be too small to allow aerobic methane oxidation [21] to bias the results. J~rgensen [26] has experimentally confirmed this analysis with 3Sso~-. The stripping process in the methane oxidation experiments was conducted in two stages, one with the sediment slurry at high pH to allow removal of the unreacted 14CH4 and the other at low pH to recover the product 14CO2. The CuSO4 trap removes inorganic and organic sulfides [27], so that the gas collected in an acidic, carboncontaining gas, and must be COs. The sediment showed no activity after the low-pH stripping, indicating that the added 14CH4 was not converted to a non-volatile form [10]. The sulfate reduction rate measurements can be biased by rapid pyrite formation [28], which would produce lower rates. Skan Bay sediments contain large quantities of acid labile iron sulfides, indicating that conditions are unfavorable for rapid pyrite formation.
It is difficult to perform meaningful poisoned control experiments in studies involving intact undisturbed cores like these. Previous work on sulfate reduction rates has apparently involved no control experiments [17,26]. The main question is how to poison the experimental volume without physical or chemical disruption. One approach to this problem is to immediately dismantle and preserve the experimental core segment, but this precludes incubation and defeats one of the main points of control experiments. J~brgensen [26] has considered tracer measurements of sulfate reduction rates in undisturbed sediments and indicates that tracer methods give absolute rates when points 1 and 2 raised earlier are carefully considered.
A troublesome point is the low recoveries of 14CH4, which were about 1% of the amount added. Methane is the only member of this tracer-product system that is not either chemically trapped or nonvolatile. Conditions in the stored samples are too basic to permit aerobic oxidation of the 14CH4 during storage to bias the results. Several of the lucite Mason jar lids leaked during stripping, so it appears that the unreacted ~4CH4 was lost during storage or the initial stages of stripping. Subsequent work has involved use of regular metal Mason jar lids and O-seal Swaglock fittings for the gas inlet, outlet and septum. Fig. 3 shows measurable anaerobic methane oxidation at all depths in Skan Bay cores with a maximum rate of 3.4 mlgl/yr in the 24.8-to 27.2-cm depth interval. Both maxima are bracketed by depth intervals over which CH4, ECO2 and SO24 - (Fig. 2) show slope changes. The distribution of the anaerobic methane oxidation rate is a mirror image of the 613CO~ distribution shown in Fig. 1 and is consistent with the suggested injection of methane-derived CO2 in a subsurface zone near the bottom of the sulfatereducing zone. The maximum in the methane oxidation rate and its location are consistent with model predictions and the rate lies within the range predicted [ 1,2]. Anaerobic methane oxidation rates in sediments are about the same magnitude as aerobic methane oxidation rates reported in water columns of lakes [21]. Sulfate reduction rates are shown in Fig. 4. Although there is more scatter in the upper portions of these distributions, the general distributions and rates are similar to other reported results [17,26]. Sulfate must be the principal oxidant in this system [5,29]; sulfate reduction rates are up to a factor of 4 higher than anaerobic methane oxidation rates at the depth of the methane oxidation maximum.

Rates and rate depth distributions
These results show that methane distributions in marine sediments are controlled by intense local anaerobic methane oxidation combined with anaerobic methane oxidation at lower rates throughout the sulfate-reducing zone.
First-order reaction kinetics have been used to describe concentration changes in both methane [3] and sulfate [30] over a range of concentrations in marine sediments. This approach predicts methane oxidation rate depth distributions that parallel the depth distributions o f meth ane concentration. Unless 349 a lower boundary is assigned, this approach predicts high rates of methane consumption at depths where the principal oxidant, sulfate, is depleted. A zone of maximum methane oxidation has been predicted from depth distributions ofCH4, Y~COz, SO~-and 513CO2, and second-order reaction kinetics, i.e. the product of methane and sulfate concentrations, have been suggested as controlling the location of the maximum rate [4]. If equation (1) can be used as a guide, it seems reasonable that the reaction responsible for anaerobic methane oxidation is second-or higher-order overall, and that the reaction kinetics for methane and sulfate are actually pseudo-first order. The methane oxidation rate depth distributions from this study show a maximum bracketed by slope changes in CH4, ~CO 2 and SO42-, but apparent redistribution of methane leads to no [CH4] [SO24 -] maximum. We have considered the location of the anaerobic methane oxidation maximum rate in a mathematical model [3 1], but more information on the organisms responsible for anaerobic methane oxidation and their substrate requirements will be required to fully resolve this point.

Conclusions
Techniques have been developed that permit the use of 14CH4 as a tracer in studies of anaerobic methane oxidation in minimally disturbed sediment cores. Preliminary application of these techniques shows that anaerobic methane oxidation occurs throughout the sulfate-reducing zone in anoxic marine sediments, reaching a maximum at the base of the sulfate-reducing zone. The low-methane concentration zone observed at the surface of marine sediments results from the distribution of anaerobic methane oxidation rates in the sulfate-reducing zone. Oxidation at high rates at the base of the zone [ 1,2,6] consumes the majority of the upward methane flux, while oxidation at lower rates [3,11 ] removes the small quantities of methane formed in the remainder of the sulfate reducing zone. "Qausi-in-situ" studies using 14CH4 solutions reveal structure not visible in in-vitro studies employing headspace gases. These studies suggest that the search for the organism responsible for anaerobic methane oxidation should be directed toward depth intervals of marine cores chosen on the basis of CH4, ZCO2 and SO~-depth distributions.