Nitrogen Mass Balance in Caves Ecosystems

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2 NO3 + H2O → 2 HNO3 + 0.5 O2

2 CaCO3 + H2CO3 + 2 HNO3 → 2 Ca2+ + 3 HCO3- + N2 + H2O + 2.5 O2 (Eq.1)

- Heterotrophic metabolism (respiratory NO3 reduction): Microbes can use dissolved nitrogen to oxidize natural organic matter in anaerobic conditions under the water table. Contrarily to the autotrophic scenario that has been previously described (microbes feeding on the inorganic substrate through carbonate dissolution), heterotrophic denitrifiers will feed on allochthonous organic carbon loads (animal and human wastes). Richards, 1965 suggested the following reaction to explain denitrification in anoxic basins.

(CH2O)106(NH3)16(H3PO4)x+ 84.8 HNO3 → 106 CO2 + 42.4 N2 + 148.4 H2O + 16 NH3 + x H3PO4 (Eq.2)

Engel et al., 2001 measured chemoautotrophic fixation that occurs on microbial mats in Cesspool Cave in Virginia (30.4 ± 12.0 ng C/mg dry wt/hr). They estimated that heterotrophic denitrifiers consumed only 0.17 ± 0.02 ng C/ mg dry wt/hr (1.7% of autotrophic uptake). Considering that 2 mg of microbial mat aliquot was used to estimate autotrophic productivity, we can express chemoautotrophic fixation in kgC/yr (60.8 ng C/hr = 60.8 10-12 kg C/hr = 5.2 10-7 kgC/yr) and heterotrophic uptake (0.34 ngC/hr = 0.34 10-12 kgC/hr = 2.9 10-9 kgC/ yr). Using the carbon to nitrogen ratio of the microbial mats average of 13.5 (Engel et al., 2001), we can translate chemoautotrophic and heterotrophic fixations in terms of nitrogen fluxes: chemoautotrophic fixation=0.39 10-7 kgN/yr/unit surface of microbial mats (order of m2) =0.039 kgN/yr/km2 =3.9 10-4 kgN/ha/yr for autotrophic fixation; heterotrophic uptake ~ 1.7 % autotrophic fixation = 6.6 10-6 kgN/ha/yr

- Macroinvertebrates assimilation

Lee and Childress (1994) estimated assimilation of nitrogen by marine invertebrates. With the assumption that invertebrates in caves show similar metabolism with the marine ones, the N assimilation flux by Macroinvertebrates can be estimated at 236 mgN/m2/day (0.85 kgN/ha/yr) and the N excretion = 136 mgN/m2/day (0.49 kgN/ha/yr), which yields a total of 0.85-0.49 = 0.36 kg N/ha/yr cycled by invertebrates (~ 2.8 % of total N inputs; Fig.1).

2.3.2. TIN outputs:

Based on microbial and macroinvertebrates’ uptake, we expect a yearly output of 13.8 kg N/ha/yr (Fig.1).

- Description of perturbation: interannual crop changes

We define perturbation as any external process that can change allochthonous N input and therefore influence N uptake by cave organisms and the amount of N released to the atmosphere through autotrophic uptake (Eq.1) and heterotrophic metabolism (Eq.2). As terrestrial N inputs are largely dominated by fertilizers (>70%), changes in crops and resulting changes in fertilizers application rates will constitute a major process affecting N flux and N cycling within the cave. Crops show a variability in nutrient use efficiency, therefore, in watersheds dominated with crops coverage (agriculture), seasonal changes (corn to soybeans rotation) and interannual changes (transition from corn to cereals/wheat) can affect the N input released from fertilizers. Panno and Kelly (2003) estimated annual rate of fertilizers application for corn (135–168 kg NO3-N/ha/yr) and for wheat and other small grains cereals (90–112 kg NO3-N/ha/yr). Integrating to a total cropland area of 1000 ha, they calculated fertilizer inputs of 135,000-168,000 kg NO3-N/yr in the case of dominant corn production. Similarly, we can estimate fertilizers inputs range of 90,000 to 112,000 kg NO3-N/yr for a dominant cereal production. Using the later value, and the NO3/N ratio (0.225), we can estimate fertilizer flux of 6.1 kgN/ha/yr and total terrestrial inputs of 9.76 kgN/ha/yr under cereal production (Fig.2), which yields outputs of 11.2 kgN/ha/yr if we consider a metastable system.

- Box-model mass balance

Pre-perturbation mass balance model:

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Fig.1. Dissolved Inorganic Nitrogen-NO3 mass balance in caves aquifers in agricultural and farming areas (Pre-perturbation). *Inputs from atmospheric depositions (particulate N) and atmospheric N2 fixation and outputs to the atmosphere through volatilization are not considered in the present model.

Post perturbation mass-balance model:

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Fig.2. Dissolved Inorganic Nitrogen-NO3 mass balance in caves aquifers in agricultural and farming areas with perturbation (change corn to cereals).

With a 20% decrease in terrestrial inputs from crop changes (Fig. 2), we expect:

- Microbial uptake (arrows 3 and 4; Fig. 2) to remain consistently low (10-4 and 10-6 ranges) because even with the decrease in N inputs, microbial fluxes are significantly lower than terrestrial inputs.

- Macroinvertebrates assimilation and excretion fluxes