High-throughput method for simultaneous quantification of N, C and S stable isotopes and contents in organics and soils.

RATIONALE: Information about the sulfur stable isotope composition (δ(34) S value) of organic materials and sediments, in addition to their nitrogen (δ(15) N value) and carbon (δ(13) C value) stable isotope compositions, can provide insights into mechanisms and processes in different areas of biological and geological research. The quantification of δ(34) S values has traditionally required an additional and often more difficult analytical procedure than NC dual analysis. Here, we report on the development of a high-throughput method that simultaneously measures the elemental and isotopic compositions of N, C and S in a single sample, and over a wide range of sample sizes and C/N and C/S ratios. METHODS: We tested a commercially available CHNOS elemental analyzer in line with an isotope ratio mass spectrometer for the simultaneous quantification of N, C and S stable isotope ratios and contents, and modified the elemental analyzer in order to overcome the interference of (18) O in δ(34) S values, to minimize any water condensation that could also influence S memory, and to achieve the complete reduction of nitrogen oxides to N2 gas for accurate measurement of δ(15) N values. A selection of organic materials and soils was analyzed with a ratio of 1:1.4 standards to unknowns per run. RESULTS: The modifications allowed high quality measurements for N, C and S isotope ratios simultaneously (1 SD of ±0.13‰ for δ(15) N value, ±0.12‰ for δ(13) C value, and ±0.4‰ for δ(34) S value), with high throughput (>75 unknowns per run) and over a wide range of element amount per capsule (25 to 500 µg N, 200-4000 µg C, and 8-120 µg S). CONCLUSIONS: This method is suitable for widespread use and can significantly enhance the application of δ(34) S measurements in a broad range of soils and organic samples in ecological and biogeochemical research. Copyright © 2016 John Wiley & Sons, Ltd.

Isotopologue fractionation during N(2)O 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.

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 beta-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.