Autotrophic ammonia-oxidizing bacteria contribute minimally to nitrification in a nitrogen-impacted forested ecosystem

Abstract

Deposition rates of atmospheric nitrogenous pollutants to forests in the San Bernardino Mountains range east of Los Angeles, California, are the highest reported in North America. Acidic soils from the west end of the range are N-saturated and have elevated rates of N-mineralization, nitrification, and nitrate leaching. We assessed the impact of this heavy nitrogen load on autotrophic ammonia-oxidizing communities by investigating their composition, abundance, and activity. Analysis of 177 cloned beta-Proteobacteria ammonia oxidizer 16S rRNA genes from highly to moderately N-impacted soils revealed similar levels of species composition; all of the soils supported the previously characterized Nitrosospira clusters 2, 3, and 4. Ammonia oxidizer abundance measured by quantitative PCR was also similar among the soils. However, rates of potential nitrification activity were greater for N-saturated soils than for soils collected from a less impacted site, but autotrophic (i.e., acetylene-sensitive) activity was low in all soils examined. N-saturated soils incubated for 30 days with ammonium accumulated additional soluble ammonium, whereas less-N-impacted soils had a net loss of ammonium. Lastly, nitrite production by cultivated Nitrosospira multiformis, an autotrophic ammonia-oxidizing bacterium adapted to relatively high ammonium concentrations, was significantly inhibited in pH-controlled slurries of sterilized soils amended with ammonium despite the maintenance of optimal ammonia-oxidizing conditions. Together, these results showed that factors other than autotrophic ammonia oxidizers contributed to high nitrification rates in these N-impacted forest soils and, unlike many other environments, differences in nitrogen content and soil pH did not favor particular autotrophic ammonia oxidizer groups.

FIG. 1.

Phylogenetic positions of cloned 1.1-kb 16S rRNA gene sequences. This phylogenetic tree was rooted with a 16S rRNA gene sequence for E. coli and was constructed using the neighbor-joining method and Kimura two-parameter model for nucleotide change. A mask of 733 nucleotides, including all nonambiguously aligned positions, was included. Bootstrap values over 75 (from 1,000 replications) generated using the maximum parsimony method (above) and neighbor-joining method (below) are indicated at the nodes (*). Italicized names are sequences from bacterial isolates, whereas nonitalicized names are cloned environmental gene sequences obtained from the GenBank database.

FIG. 2.

Daily accumulation of soluble NO_2⁻-N plus NO_3⁻-N in washed and buffered slurries of CP, SP, and DW soils collected in September 2002 and February 2003. Incubations without (black bars) and with (gray bars) acetylene are shown. Error bars represent 1 standard deviation from the mean (n = 3).

FIG. 3.

Daily accumulation of soluble NO₂⁻-N plus NO₃⁻-N in a slurry of fertilized, irrigated, sandy soil. Incubations without (black bars) and with (gray bars) acetylene are shown. Error bars represent 1 standard deviation from the mean (n = 3).

FIG. 4.

Daily accumulation of soluble NO₂⁻ by cultivated cells of N. multiformis inoculated onto sterilized, washed, and buffered soil slurries from CP (▴), DW (▪), and silica sand (⧫). Incubations containing acetylene (open symbols) and uninoculated slurries (open symbols with dashed lines) are shown. Error bars represent 1 standard deviation from the mean (n = 3).