Finzi and Schlesinger (2003) measured and analyzed pool sizes and fluxes of inorganic and organic nitrogen in the forest floor and top 30 cm of mineral soil during the first five years of differential atmospheric CO2 treatment of a stand of initially 13-year-old loblolly pine trees at the Duke Forest FACE facility in the Piedmont region of North Carolina (USA), where half of the experimental plots were maintained at a mean CO2 concentration that is 200 ppm above ambient. In doing so, they found that the extra CO2 significantly increased the input of carbon (C) and nitrogen (N) to the forest floor, as well as to the mineral soil in which the trees were growing. However, they report "there was no statistically significant change in the cycling rate of N derived from soil organic matter under elevated CO2" and that "neither the rate of net N mineralization nor gross ¹⁵NH4⁺ dynamics were significantly altered by elevated CO2." In fact, they could find "no statistically significant difference in the concentration or net flux of organic and inorganic N in the forest floor and top 30-cm of mineral soil after 5 years of CO2 fumigation," adding that "microbial biomass was not a larger sink for N." Based on these findings, they actually rejected their own original hypothesis, which was essentially the same as the PNL hypothesis, i.e., that the extra CO2 provided to the experimental plots would significantly increase the rate of N immobilization by the soil microbial communities found within the CO2-enriched FACE arrays and thereby lead to a reduction in the magnitude of the growth stimulation that was initially manifest in the CO2-enriched treatment.

Working at the same location, Schafer et al. (2003) measured net ecosystem exchange (NEE) and net ecosystem production (NEP) during the third and fourth years of the long-term CO2 enrichment study being conducted there. They found that the extra 200 ppm of CO2 supplied to the loblolly pine trees within the CO2-enriched FACE arrays increased the entire canopy's net uptake of CO2 (NEE) by fully 41%, and that canopy NEP was increased by 44%. In addition, they determined that 87% of the extra NEP "was sequestered in a moderately long-term C pool in wood." This large increase in solidly-sequestered carbon was truly amazing, especially in light of the declaration of Finzi and Schlesinger (2003) that the soil at the Duke Forest FACE site is in "a state of acute nutrient deficiency that can only be reversed with fertilization," which, of course, was not provided.

Three years later, Finzi et al. (2006) tested the PNL concept "using data on the pools and fluxes of C and N in tree biomass, microbes and soils," which were obtained from the first six years of the Duke Forest FACE study. As was the case three years earlier, it was once again found that "there was no reduction in the average stimulation of net primary production by elevated CO2," even though "significantly more N was immobilized in tree biomass and in the O [soil] horizon under elevated CO2." Also, and "in contrast to the PNL hypothesis," as they describe it, "microbial-N immobilization did not increase under elevated CO2, and although the rate of net N mineralization declined through time, the decline was not significantly more rapid under elevated CO2." In addition, the twelve researchers reported that "mass balance calculations demonstrated a large accrual of ecosystem N capital," and they said that the rate of extra N accrual was "much greater than the estimated rate of N input via atmospheric deposition or heterotrophic N fixation," noting further that "there are no plant species capable of symbiotic N fixation in this ecosystem." In other words, by some unknown means the loblolly pine trees obtained the extra N they needed to stave off the negative effects predicted by the PNL hypothesis, possibly, in the words of Finzi et al., by roots "actively taking up N and redistributing N from deeper in the soil profile."

After another two years had passed, Moore et al. (2006) reported finding "a sustained increase in basal area increment over the first 8 years of the experiment," which varied between 13 and 27% with variations in weather and the timing of growth. What is more, they said "there was no evidence of a decline in the relative enhancement of tree growth by elevated CO2 as might be expected if soil nutrients were becoming progressively more limiting," which would normally be expected, considering the unfertilized state of the soil in which the experiment was being conducted.

Two years later, Pritchard et al. (2008a) published the results they had obtained from minirhizotrons they employed to characterize the influence of the extra 200 ppm of CO2 on the fine roots of the Duke Forest loblolly pines over the six-year period 1998-2004. Averaged over all six years, they found that it had increased average fine-root standing crop by 23%, which compares well with the overall stimulation of tree net primary productivity of 18-24% observed over the period 1996-2002. Consequently, in light of their noting that "the positive effects of CO2 enrichment on fine root growth persisted 6 years following minirhizotron tube installation (8 years following initiation of the CO2 fumigation)," as they describe it, there was once again no hint of any progressive nitrogen limitation to the stimulatory effect of atmospheric CO2 enrichment in a situation where one would have expected to have encountered it. In partial explanation of this important positive finding, Pritchard et al. concluded their report by stating that the distal tips of fine roots are "the primary site for initiation of mycorrhizal partnerships which are critical for resource acquisition and could also influence whether or not forests can sustain higher productivity in a CO2-enriched world."

In a study designed to further explore this aspect of the ongoing long-term Duke Forest FACE experiment, Pritchard et al. (2008b) focused their attention on the role played by ectomycorrhizal (ECM) fungi over a period of five years, based on minirhizotron observations of fungal dynamics. Summed across all years of the study, the five researchers found that the extra 200 ppm of CO2 enjoyed by the trees in the high-CO2 treatment did not influence mycorrhizal production in the top 15 cm of the forest soil, but that it increased mycorrhizal root-tip production by 194% throughout the 15-30 cm depth interval. In addition, they report that production of soil rhizomorph length was 27% greater in the CO2-enriched plots than it was in the ambient-air plots.

In discussing their findings, Pritchard et al. stated that the CO2-induced "stimulation of carbon flow into soil has increased the intensity of root and fungal foraging for nutrients," and that "the shift in distribution of mycorrhizal fungi to deeper soils may enable perennial plant systems to acquire additional soil nitrogen to balance the increased availability of ecosystem carbohydrates in CO2-enriched atmospheres," which additional acquisition of nitrogen in the CO2-enriched plots of the Duke Forest FACE study has been determined to be approximately 12 g N per m² per year.

In further commenting on the results of their work, Pritchard et al. write that "the notion that CO2 enrichment expands the volume of soil effectively explored by roots and fungi, and that foraging in a given volume of soil also seems to intensify, provides compelling evidence to indicate that CO2 enrichment has the potential to stimulate productivity (and carbon sequestration) in N-limited ecosystems more than previously expected." On the other hand, they also say "it is unlikely that ecosystem productivity will be stimulated by CO2 enrichment indefinitely." Be that as it may, nature has so far proved such negative hunches wrong, nearly every step of the way, as scientists have probed ever deeper into this particular subject.

Most recently, in the introduction to their paper summarizing nine years of work at the Duke Forest FACE facility, Lichter et al. (2008) again warned that progressive nitrogen limitation may "accompany carbon sequestration in plants and soils stimulated by CO2 fertilization, gradually attenuating the CO2 response," after which they went on to describe what they had learned about the PNL hypothesis over the prior nine years. They noted, first of all, that their data pertaining to forest-floor carbon pools indicate the existence of "a long-term steady-state sink" of about 30 g C per m² per year, which represents, in their words, "a substantial increase in forest-floor C storage under elevated CO2 (i.e. 29%)," and which they attribute to "increased litterfall and root turnover during the first 9 years of the study." Secondly, down below the forest floor, they say that of the mineral soil carbon formed during the past 9 years, "approximately 20% has been allocated to stable pools that will likely remain protected from microbial activity and associated release as CO2."

A third important finding of the research team was "a significant widening of the C:N ratio of soil organic matter in the upper mineral soil under both elevated and ambient CO2," which suggests, as they describe it, that "enhanced rates of soil organic matter decomposition are increasing mineralization and uptake to provide the extra N required to support the observed increase in primary productivity under elevated CO2." At the Duke Forest FACE site, Pritchard et al. (2008) say this CO2-induced increase in productivity amounts to approximately 30% annually; and they add that there is "little evidence to indicate a diminished response through time," citing the analysis of Finzi et al. (2007), who found the same to be true at the long-term forest FACE studies being conducted at Rhinelander, Wisconsin (USA), Oak Ridge National Laboratory (USA), and Tuscania (Italy).

Contrary to the early expectations of many scientists, it would thus appear that many of earth's forests that are thought to have access to less-than-adequate soil nitrogen supplies may indeed be able to acquire the extra nitrogen they need to maintain the sizable increases in their growth rates that are driven by elevated concentrations of atmospheric CO2. In the case of North Carolina's Duke Forest, for example, "even after nine years of experimental CO2 fertilization," as Lichter et al. describe it, "attenuation of the CO2-induced productivity enhancement has not been observed," as was also found to be the case by Finzi et al. (2006). And this finding at this location is extremely significant, because the growth of pine-hardwood forests in the southeastern United States often removes so much nitrogen from the soils in which they grow that they induce what Finzi and Schlesinger (2003) have described as "a state of acute nutrient deficiency that can only be reversed with fertilization," which operation, as noted earlier in this section, was not employed at the Duke Forest FACE study.

References
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