Response to Comment on “Abiotic Controls on H2 ... - ACS Publications

James P. McKinley. Battelle, Pacific Northwest Division P.O. Box 999 Richland, Washington 99352. Environ. Sci. Technol. , 2001, 35 (7), pp 1558–1559...
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Environ. Sci. Technol. 2001, 35, 1558-1559

Response to Comment on “Abiotic Controls on H2 Production from Basalt-Water Reactions and Implications for Aquifer Biogeochemistry” SIR: We have proposed that microorganisms in Columbia River Basalt (CRB) aquifers carry out primary production using H2 produced during anaerobic weathering of basalt (1, 2). This hypothesis needs to be tested in a variety of ways. In our recent paper (2), we described environmental controls on the H2-producing reactions and identified the reactive components in basalt. Our intent was to demonstrate the feasibility of this mechanism as a source of H2 for microbial metabolism. Anderson et al. previously claimed to have disproved our hypothesis by conducting a similar solidwater interaction experiment with basalt and groundwater from the Snake River Plain (3). They reported no observations of the CRB system nor experiments with CRB materials; we briefly noted (2) that they provided no evidence for their claim. Their paper also contained a number of arguments that were not based on their study and which we feel are all essentially incorrect. Anderson et al. repeat some of those arguments here (11). We strongly disagree with all of those arguments. Although they are outside of the scope of our Environ. Sci. Technol. paper (2), or are addressed in our previous publications, we discuss them here briefly in order of presentation. (i) Anderson et al. (11) claim that the community structure of CRB microorganisms is inconsistent with our hypothesis of in situ primary production. First, it should be noted that the community structure of microorganisms in CRB aquifers has never been observed directly, so any argument on that basis is rather weak. All observations have been of organisms in well water, which are likely a subset of CRB organisms detached from surfaces in situ, mixed with an unknown proportion of contaminating organisms growing in the well structures (1, 4). [However, the biogeochemical signatures of microbial metabolism in the groundwaters (1) and putative microfossils in fracture-fill material (5) indicate that anaerobic microorganisms are native to the CRB aquifers.] Using dilution culture techniques, we observed that groundwater emitted from wells in high-methane aquifers tended to be dominated by methanogens, while those from wells in highsulfate aquifers tended to be dominated by sulfate-reducing microorganisms (1, 4). Homoacetogenic bacteria were abundant in both types of groundwater, and smaller numbers of heterotrophic microorganisms were also present. Fry et al. (6) extracted nucleic acids from two wells tapping CRB aquifers and obtained somewhat different results. Anderson et al. (11) consider that the results of Fry et al. (6) are without bias and use them as a basis to refute our results. However, extraction of nucleic acids from environmental samples is not without bias (e.g., ref 7 and references therein). For instance, extraction efficiencies and rRNA gene copy numbers vary widely and could easily introduce order of magnitude errors into attempted quantitation. In addition, Fry et al. (6) did not sample well waters directly but used a concentrated colloidal paste obtained by circulating large volumes of water through a circulating hollow-fiber filter unit. This unit was “sterilized” by storing it in 100 ppm bleach. However, biofilms growing within the apparatus were not removed by this treatment. When we previously tested the exact same apparatus, we found that while few cells were eluted from the filter after the bleach treatment, abundant bacterial cells and protozoa could subsequently be dislodged with a 1558

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pyrophosphate solution. It is clear that the samples of Fry et al. were grossly contaminated with such extraneous organisms. They reported that more than 14% of the DNA they recovered was eukaryotic. Such a large proportion of the population should certainly be visible by microscopic observation, yet no eukaryotic organism has ever been observed in direct examination of numerous CRB well water samples. (ii) Anderson et al. (11) state that organic matter is “a more likely energy source for microorganisms” in the CRB, stating that the observed DOC is sufficient to support the observed biomass. However, the DOC concentrations of 1-5 ppm are of the same order as the observed entrained biomass (ca. 0.25-1 ppm organic carbon, assuming 3 × 10 -13 g dry wt cell-1), and attached biomass in situ should be at least an order of magnitude larger, so the DOC is at least as likely to be a product of in situ primary production. Furthermore, there is no trend in DOC concentration with depth, as would be expected if DOC from outside the system were being consumed to produce methane. In any case, this amount of DOM is orders of magnitude too low to support the observed methane in the system. Mere observation of this low concentration of DOM provides no information for or against our hypothesis. As we previously reported (1), the trends in concentration and stable isotopes of dissolved inorganic carbon in CRB aquifers indicate that methanogenic CO2 reduction (using H2 as an electron donor) is the main process affecting the DIC pool. As groundwaters age, DIC declines and becomes isotopically heavier, while methane increases. For the available dataset, the ∂13CDIC can be predicted quite well by assuming that the only process affecting the pool is removal of CO2 by methanogens. If degradation of DOM were the source of methane, then DIC would increase with time, not decrease, and the resulting ∂13CDIC would not conform to the CO2reduction model. (iii) Anderson et al. (11) make a claim about the availability of reducing power from iron in basalt. It should be obvious that rates measured in laboratory flasks cannot be extrapolated to derive reaction rates in natural environments in simplistic ways such as their calculation. Using this sort of reasoning, one could easily “prove” for instance that Escherichia coli does not oxidize glucose since, at the rate used in vitro, it would rapidly consume all carbon on earth. A trivial disproof of the claim can be found by observing that H2 production from serpentinization proceeds for many millions of years, in seafloor ultramafic rocks and continental ophiolites, without depleting Fe in the rocks (see refs 1 and 2). These rocks contain about 1 wt % more FeO than CRB basalt but not orders of magnitude more. In making their calculation, Anderson et al. (11) appear to assume that the entire mass of the CRB (>130 000 km3) is composed of finely ground material that is all reacting with water at once. In reality, the CRB are composed of massive bodies of crystalline rock with relatively very little wetted surface area. A detailed evaluation of reaction rates in the CRB system might prove a good test of our hypothesis, but most of the necessary data do not yet exist. To make such calculations one would need data such as: in situ reaction rates, the wetted surface area and fluid volume in the CRB, the stoichiometry of the H2 production reaction(s), the Fe2+ content of the rocks, the kinetics of fracture propagation and healing, in situ pH and temperature, and information about how all of these parameters change through space and time. This is a tractable but large problem. Some crude approximations can be made with available data, although we can have little confidence in the resulting values. The rates 10.1021/es001926+ CCC: $20.00

 2001 American Chemical Society Published on Web 02/23/2001

of H2 production in our laboratory experiments [ca. 1000 m2 of basalt (L of fluid)-1, yielding 1-5 nmol of H2 (day)-1 (m2 of basalt)-1] correspond well to a first approximation with the amount required to produce the highest methane concentrations observed in CRB groundwaters [ca. 7 m2 of basalt (L of fluid)-1, yielding ca. 5 nmol of H2 (day)-1 (m2 of basalt)-1] (2). Using the effective porosity data from a single field site [ca. 0.25 L (m3 of basalt)-1] (8) and rough values for iron content [ca. 5000 mol of Fe (m3 of basalt)-1] (9) and stoichiometry [ca. 2-10 Fe (H2)-1] (10) and assuming that all rates are constant throughout the formation (unlikely) and over geological time (very unlikely), we can estimate that the current rate of H2 production might proceed between 1.5 × 108 and 3.8 × 109 yr before depleting the available Fe in a given volume of basalt. This is in rough agreement with observations that CRB basalt, ca. 107 yr old, contains ca. 1-5% alteration products (9). While these crude estimates are unlikely to be correct, they suggest that the CRB subsurface ecosystem, as we hypothesize it, could feasibly persist over geological time. (iv) The statements in the fourth argument used by Anderson et al. (11) are simply false. They claim that our data (2) show no H2 production from basalt at pH values found in situ in the CRB. In fact, while H2 production rates were lower at alkaline pH than at pH ca. 6, they always attained concentrations (>100 nM) well within the range useful to microorganisms (>5-10 nM). They merely appear low in comparison to the low pH rates plotted in the same figures. (As we noted, the data from nonbasalt or nonwetted control experiments were left out of these figures because their values as well as the analytical detection limit would be indistiguishable from the baseline of the graph, at the scale plotted.) In addition, our Figure 3 (2) showed that when the ferrous iron concentration was adjusted to near in situ values, the rates of H2 production were similar at pH 8 and pH 6, although we did not determine whether it was because soluble Fe(II) was a reactant or played some other role such as scrubbing traces of O2 from the experiment. Anderson et al. claim that it was “misleading” for us to conduct experiments at pH 6. In Figure 1 (2), we compared H2 production from different mineral samples to determine which components of basalt were reactants. We used pH 6 for reasons of experimental tractablility because it was previously determined to yield the fastest results. The only danger in this procedure is that a completely different mechanism might be active at high pH than at low pH. Figures 2 and 3 (2) compare the effect of pH on H2 generation rates and compare H2 production at high and low pH. It is hardly misleading to include low pH samples in experiments that investigate the effect of pH. Furthermore, the range of rates used in our simple extrapolating calculation encompassed rates from alkaline experiments. In any case, as we noted, the uncertainties in such calculations easily span orders of magnitude, so that the rate differences between alkaline and acidic samples become relatively small. That such a crude estimate comes close to our observed values seems remarkable; that is why we alerted the readers to the uncertainty with multiple qualifiers. (v) The final argument by Anderson et al. (11) is selfcontradictory. They state that “studies with actual aquifer material and groundwater, incubated under conditions that approximate those in situ, will provide the best insights into the potential for abiotic hydrogen production in basalt aquifers”. However, they use this argument to justify selecting uncharacterized materials collected hundreds of kilometers away from the aquifers in question. They also imply that the Snake River Plain basalts have a uniform composition, such that a sample collected at random by them and uncharacterized would have a composition identical to the mineralogically characterized sample that we used; they then imply

that CRB basalts are so heterogeneous that our samples, from a well-characterized outcrop of the same flow present at depth in methanogenic aquifers, are irrelevant to those methanogenic aquifers. Anderson et al. (11) criticize us for implying that the phenomena we hypothesize for the CRB occur in all basaltic aquifers; what we said was that they are probably not unique to the CRB (2). In fact, there are important differences between CRB and SRP aquifers that could make such a system less likely in the SRP. The CRB is a series of massive nearly monolithic horizontal flows that cover hundreds of square kilometers. Groundwater is confined between these flows for long periods and reacts with rock over tens of thousands of years. The groundwaters are generally anoxic. The H2-producing reactions that we observed occur only in the absence of O2. The SRP is a series of overlapping flows characteristic of shield volcanoes, with rubble zones that are highly porous and permeable to water. For example, two rivers entering the SRP (The Big and Little Lost Rivers) subside completely into the subsurface. Groundwater flow times are relatively short, and groundwaters are generally aerobic. The summation presented by Anderson et al. (11) is incorrect. We feel that the data presented in our publications (1, 2) are most consistent with a community based on primary production supported by H2 from water-rock interactions. In particular, the stable isotope and DIC trends are not “consistent with a microbial community that is supported from the degradation of organic matter”, and a source of sufficient organic matter to support the observed biomass and methane in the CRB has not been identified. Anderson et al. (11) have not tested our hypothesis, so cannot have disproved it.

Literature Cited (1) Stevens, T. O.; McKinley, J. P. Science 1995, 270, 450. (2) Stevens, T. O.; McKinley, J. P. Environ. Sci. Technol. 2000, 34, 826. (3) Anderson, R. T.; Chapelle, F. H.; Lovley, D. R. Science 1998, 281, 976. (4) Stevens, T. O.; McKinley, J. P.; Fredrickson, J. K. Microb. Ecol. 1993, 25, 35. (5) McKinley, J. P.; Stevens, T.O.; Westall, F. W. Geomicrobiol. J. 2000, 17, 43. (6) Fry, N. K.; Fredrickson, J. K.; Fishbain, S.; Wagner, M.; Stahl, D. A. Appl. Environ. Microbiol. 1997, 63, 1498. (7) Frostegard, A.; Courtois, S.; Ramisse, V.; Clerc, S.; Bernillon, D.; Le Gall, F.; Jeannin, P.; Nesme, X.; Simonet, P. Appl. Environ. Microbiol. 1999, 65, 5409-5420. (8) Nuclear Waste Policy Act, Section 113. Consultation Draft, Site Characterization Plan. Reference Repository Location, Hanford Site, Washington. 1988; U.S. Department of Energy: Washington, DC, 1988; DOE/RW-0164. (9) Allen, C. C.; Johnston, R. G.; Strope, M. B. Characterization of Reference Umtanum and Cohassett Basalt; SD-BWI-DP-05; U.S. Department of Energy: Richland, WA, 1985. (10) O’Hanley, D. S. Serpentinites; Records of Tectonic and Petrological History; Oxford Monographs on Geology and Geophysics 34; Oxford: New York, 1996. (11) Anderson, R. T.; Chapelle, F. H.; Lovley, D. R. Environ. Sci. Technol. 2001, 35, 1556-1557.

Todd O. Stevens* 1710 State Road Mosier, Oregon 97040

James P. McKinley Battelle, Pacific Northwest Division P.O. Box 999 Richland, Washington 99352 ES001926+ VOL. 35, NO. 7, 2001 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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