We have carefully considered Dr. Dolfing's comments on the issue of free-energy calculations. We agree that we made an error in the calculations of the ΔG° by using 1 atm rather than molar concentrations and that the ΔG° should have been corrected to pH 8. We thank him for his corrections. However, the consequences of these corrections, with much lower values of [NO2−]/[NO3−]0.5, are to support even more strongly our conclusions on the relative energy yields of nitrite and nitrate as substrates for conversion to N2O.
We attempted to use a thermodynamic argument to indicate whether, under certain conditions, nitrite conversion to N2
O might be energetically advantageous to nitrate conversion to N2
O and, hence, in an energy-limited environment to favor those organisms using this pathway. However, although thermodynamics can indicate whether a particular reaction should proceed under given conditions of temperature, pressure, concentration, etc., it tells us nothing about the reaction mechanisms or the kinetics. The overall reactions from nitrite or nitrate to N2
O or to N2
are catalyzed by different enzymes, such as nitrate reductase, nitrite reductase, nitrous oxide reductase, etc., which may vary phylogenetically. It is true to say that, as Dolfing puts it, “it is, from a thermodynamic point of view, not logical that N2
O was emitted from the Colne estuary” because of higher free-energy production from conversion of N2
O to N2
than its formation from nitrate or nitrite. However, whether or not N2
O is emitted largely depends on the balance of the rates of its formation and removal. If formation is faster than removal, N2
O may accumulate. Conversion of N2
O to N2
by nitrous oxide reductase can be inhibited by a number of compounds (3
). For instance, sulfide, commonly present in high-sulfate marine and estuarine sediments, inhibits nitrous oxide reductase, so that N2
O accumulates. This can be a major factor in the production of N2
O in some estuarine sediments (7
) and operates over and above any thermodynamic considerations. Also, some denitrifying bacteria may be genetically able only to use nitrite (not nitrate) as the electron acceptor and produce N2
O (not N2
) as the sole end product (1
). Thus, factors other than thermodynamics may also influence whether N2
O is formed in estuaries.
Dr. Dolfing argues for a more direct thermodynamic model for nitrate/nitrite reduction that explicitly considers concentrations of electron donors and acceptors. However, nitrate reducers can use a wide array of different electron donors, whose in situ concentrations are unknown. An explicit model would have to make assumptions about these unknown electron donor concentrations. The advantage of our approach is that, by using nitrite/nitrate ratios ([NO2−]/[NO3−]0.5), we can make conclusions about the relative free energy without having to make any assumptions about the unknown electron donor concentrations.
Dr. Dolfing (paragraph 4, sentence 4) makes the comment that if the concentrations of all reactants, including electron donors, is such that the reaction is endergonic, this might be missed by our approach. Considering the high standard free-energy release from nitrite or nitrate reduction to N2O and the actual concentrations of the reactants and products in the sediment, the reduction of nitrate or nitrite to N2O is extremely unlikely to be endergonic. For a given sediment, the concentrations of reducing substrates present in a sediment to drive nitrate/nitrite reduction is unknown but may be assumed to be relatively constant, at least compared to electron acceptor concentrations, which may vary tidally. So, evaluating energy yields by using nitrite or nitrate as an electron acceptor becomes a matter of evaluating the relative concentrations of nitrite and nitrate, i.e., the ratio [NO2−]/[NO3−]0.5. At pH 8 and 25°C, if the ratio [NO2−]/[NO3−]0.5 is >0.00000012 at [N2O] = 1 nM or >0.00000070 at [N2O] = 1,000 nM, the reaction using nitrite as the electron acceptor should be energetically more favorable. This approach has a wider justification and can be easily used for other sediments.
Dr. Dolfing comments in his last paragraph that, as addition of nitrate/nitrite stimulated N2
O formation, N2
O formation was not limited by the availability of reducing equivalents (i.e., the electron donor concentration) and, hence, that there is no merit in looking at the energetics of the reactions involved in its formation from nitrate/nitrite. This assumes that there is a single limiting factor (e.g., the nitrate or nitrite concentration) controlling such reactions in sediments, whereas in many sediments there is substantial evidence of simultaneous regulation of microbial activities by a number of different environmental variables, including both electron donor and electron acceptor concentrations, and physical factors such as temperature also (5
). Egli et al. (2
) have discussed models for simultaneous regulation of growth by a variety of organic substrates under oligotrophic conditions (and see reference 4
also), and the same models can be applied to simultaneous regulation by electron acceptors also. Addition of nitrate may stimulate denitrification in sediment, but addition of nitrate plus acetate or glucose usually stimulates rates even more. Thus, in many sediments, both electron donors and acceptors are simultaneously at limiting concentrations and there will be interaction between multiple factors that limit rates of denitrification and nitrous oxide production. Measurements of potentials for denitrification, when both electron donors and acceptors are provided in excess, gives rates of denitrification much greater than measured in situ rates. Thus, that addition of nitrate and nitrite stimulated N2
O formation may not necessarily mean that N2
O formation was not also limited by the availability of organic substrates.
The further assumption by Dr. Dolfing in his last paragraph that “whether nitrate or nitrite was the energetically more favorable electron acceptor for N2O production is not an issue” because all electron acceptors possible will be used is not correct. In a closed system, it is true to say “under conditions of electron acceptor limitation, the organisms will tend to use every electron acceptor available, with the qualification that the energetically most favorable electron acceptor will be used first.” A sediment is an open system; nitrate and nitrite can be used by denitrifying bacteria but be continuously resupplied by inputs of nitrate from overlying water or by internal nitrification and would never be completely exhausted by the microorganisms. Therefore, the question of which electron acceptor, nitrite or nitrate, would be energetically more favorable in the process of nitrous oxide formation remains important.