Robin Sutka

Distinguishing Nitrous Oxide Production from Nitrification and Denitrification on the Basis of Isotopomer Abundances

ABSTRACT The intramolecular distribution of nitrogen isotopes in N2O is an emerging tool for defining the relative importance of microbial sources of this greenhouse gas. The application of intramolecular isotopic distributions to evaluate the origins of N2O, however, requires a foundation in laboratory experiments in which individual production pathways can be isolated. Here we evaluate the site preferences of N2O produced during hydroxylamine oxidation by ammonia oxidizers and by a methanotroph, ammonia oxidation by a nitrifier, nitrite reduction during nitrifier denitrification, and nitrate and nitrite reduction by denitrifiers. The site preferences produced during hydroxylamine oxidation were 33.5 ± 1.2‰, 32.5 ± 0.6‰, and 35.6 ± 1.4‰ for Nitrosomonas europaea, Nitrosospira multiformis, and Methylosinus trichosporium, respectively, indicating similar site preferences for methane and ammonia oxidizers. The site preference of N2O from ammonia oxidation by N. europaea (31.4 ± 4.2‰) was similar to that produced during hydroxylamine oxidation (33.5 ± 1.2‰) and distinct from that produced during nitrifier denitrification by N. multiformis (0.1 ± 1.7‰), indicating that isotopomers differentiate between nitrification and nitrifier denitrification. The site preferences of N2O produced during nitrite reduction by the denitrifiers Pseudomonas chlororaphis and Pseudomonas aureofaciens (−0.6 ± 1.9‰ and −0.5 ± 1.9‰, respectively) were similar to those during nitrate reduction (−0.5 ± 1.9‰ and −0.5 ± 0.6‰, respectively), indicating no influence of either substrate on site preference. Site preferences of ∼33‰ and ∼0‰ are characteristic of nitrification and denitrification, respectively, and provide a basis to quantitatively apportion N2O.

Biphenyl-utilizing bacteria and their functional genes in a pine root zone contaminated with polychlorinated biphenyls (PCBs)

Bacteria and functional genes associated with biphenyl (BP) degradation in the root zone of an Austrian pine (Pinus nigra L.) growing naturally in polychlorinated-BP (PCB)-contaminated soil were identified using stable isotope probing (SIP) integrated with comprehensive functional gene analyses. SIP revealed 75 different genera that derived carbon from 13C-BP, with Pseudonocardia, Kribella, Nocardiodes and Sphingomonas predominating carbon acquisition. Rhodococcus spp. were not detected with SIP, despite being the most abundant BP utilizers isolated from agar plates. Only one organism, an Arthrobacter spp., was detected as a BP utilizer by both cultivation and SIP methods. Time-course SIP analyses indicated that secondary carbon flow from BP-utilizing bacteria into other soil organisms may have occurred largely between 4 and 14 days incubation. Functional gene contents of the BP-utilizing metagenome (13C-DNA) were explored using the GeoChip, a functional gene array containing 6465 probes targeting aromatic degradative genes. The GeoChip detected 27 genes, including several associated with catabolism of BP, benzoate and a variety of aromatic ring hydroxylating dioygenase (ARHD) subunits. Genes associated with the β-ketoadipate pathway were also detected, suggesting a potential role for this plant aromatic catabolic pathway in PCB degradation. Further ARHD analyses using targeted polymerase chain reaction primers and sequence analyses revealed novel dioxygenase sequences in 13C-DNA, including several sequences that clustered distantly from all known ARHDs and others that resembled known Rhodococcus ARHDs. The findings improve our understanding of BP degradation and carbon flow in soil, reveal the extent of culture bias, and may benefit bioremediation research by facilitating the development of molecular tools to detect, quantify and monitor populations involved in degradative processes.

Isotopologue fractionation during N2O production by fungal denitrification

Identifying the importance of fungi to nitrous oxide (N2O) production requires a non-intrusive method for differentiating between fungal and bacterial N2O production such as natural abundance stable isotopes. We compare the isotopologue composition of N2O produced during nitrite reduction by the fungal denitrifiers Fusarium oxysporum and Cylindrocarpon tonkinense with published data for N2O production during bacterial nitrification and denitrification. The fractionation factors for bulk nitrogen isotope values for fungal denitrification were in the range -74.7 to -6.6 per thousand. There was an inverse relationship between the absolute value of the fractionation factors and the reaction rate constant. We interpret this in terms of variation in the relative importance of the rate constants for diffusion and enzymatic reduction in controlling the net isotope effect for N2O production during fungal denitrification. Over the course of nitrite reduction, the delta(18)O values for N2O remained constant and did not exhibit a relationship with the concentration characteristic of an isotope effect. This probably reflects isotopic exchange with water. Similar to the delta(18)O data, the site preference (SP; the difference in delta(15)N between the central and outer N atoms in N2O) was unrelated to concentration during nitrite reduction and, therefore, has the potential to act as a conservative tracer of production from fungal denitrification. The SP values of N2O produced by F. oxysporum and C. tonkinense were 37.1 +/- 2.5 per thousand and 36.9 +/- 2.8 per thousand, respectively. These SP values are similar to those obtained in pure culture studies of bacterial nitrification but quite distinct from SP values for bacterial denitrification. The large magnitude of the bulk nitrogen isotope fractionation and the delta(18)O values associated with fungal denitrification are distinct from bacterial production pathways; thus multiple isotopologue data holds much promise for resolving bacterial and fungal production. Our work further provides insight into the role that fungal and bacterial nitric oxide reductases have in determining site preference during N2O production.

Stable nitrogen isotope dynamics of dissolved nitrate in a transect from the North Pacific Subtropical Gyre to the Eastern Tropical North Pacific

Abstract The stable nitrogen isotopic composition of nitrate, concentrations of inorganic nitrogen and phosphorus, dissolved oxygen and nitrification rates were determined at six stations ranging from the oligotrophic North Pacific Subtropical Gyre (NPSG) to the more productive Eastern Tropical North Pacific (ETNP). Nitrification rates increased along the transect from a maximum rate of 1 nmol L −1 d −1 at station ALOHA to 23.7 nmol L −1 d −1 at station 6. In oxic surface waters, nitrate isotopically enriched in 15 N (maximum δ 15 N-NO 3 − value of 12.5‰) was most likely the result of assimilatory nitrate reduction. In contrast, high δ 15 N-NO 3 − values (maximum of 12.3‰) in association with high nitrate deficits and anoxic conditions supported the interpretation of isotopic fractionation due to denitrification. A one-dimensional vertical advection and diffusion model was used to estimate the fractionation factor for denitrification at two stations in the ETNP. A comparison of modeled to observed δ 15 N-NO 3 − data indicated an isotopic enrichment factor (e) of 30‰ at station 4 and 30 to 35‰ at station 5. Isotopically light nitrate (1.1 and 3.2‰) was observed in the upper 200 m of the water column at stations in the ETNP. Tracer studies of 15 NH 4 and biogeochemical indicators of nitrogen fixation supported the interpretation of nitrification as the most plausible explanation for low δ 15 N-NO 3 − values observed in water column samples. Our results are consistent with the occurrence of nitrification within the euphotic zone and for the first time provide corroborating stable nitrogen isotopic evidence for this process.