Stable Isotope Probing:Linking Functional Activity to Specific Members of Microbial Communities

Linking organisms or groups of organisms to specific functions within natural environments is a fundamental challenge in microbial ecology. Advances in technology for manipulating and analyzing nucleic acids have made it possible to characterize the members of microbial communities without the intervention of laboratory culturing. Results from such studies have shown that the vast majority of soil organisms have never been cultured, highlighting the risks of culture-based approaches in community analysis. The development of culture-independent techniques for following the flow of substrates through microbial communities therefore represents an important advance. These techniques, collectively known as stable isotope probing (SIP), involve introducing a stable isotope-labeled substrate into a microbial community and following the fate of the substrate by extracting diagnostic molecular species such as fatty acids and nucleic acids from the community and determining which specific molecules have incorporated the isotope. The molecules in which the isotope label appears provide identifying information about the organism that incorporated the substrate. Stable isotope probing allows direct observations of substrate assimilation in minimally disturbed communities, and thus represents an exciting new tool for linking microbial identity and function. The use of lipids or nucleic acids as the diagnostic molecule brings different strengths and weaknesses to the experimental approach, and necessitates the use of significantly different instrumentation and analytical techniques. This short review provides an overview of the lipid and nucleic acid approaches, discusses their strengths and weaknesses, gives examples of applications in various settings, and looks at prospects for the future of SIP technology.

Characterization of stable isotope fractionation during methane production in the sediment of a eutrophic lake,lake dagow,germany

We measured delta C-13 of CO2, CH4, and acetate-methyl in profundal sediment of eutrophic Lake Dagow by incubation experiments in the presence and absence of methanogenic inhibitors chloroform, bromoethane sulfonate (BES), and methyl fluoride, which have different specificities. Methyl fluoride predominantly inhibits acetoclastic methanogenesis and affects hydrogenotrophic methanogenesis relatively little. Optimization of methyl fluoride concentrations resulted in complete inhibition of acetoclastic methanogenesis. Methane was then exclusively produced by hydrogenotrophic methanogenesis and thus allowed determination of the fractionation factors specific for this methanogenic pathway. Acetate, which was then no longer consumed, accumulated and allowed determination of the isotopic signatures of the fermentatively produced acetate. BES and chloroform also inhibited CH4 production and resulted in accumulation of acetate. The fractionation factor for hydrogenotrophic methanogenesis exhibited variability, e. g., it changed with sediment depth. The delta C-13 of the methyl group of the accumulated acetate was similar to the delta C-13 of sedimentary organic carbon, while that of the carboxyl group was by about 12 parts per thousand higher. However, the delta C-13 of the acetate was by about 5 parts per thousand lower in samples with uninhibited compared with inhibited acetoclastic methanogenesis, indicating unusual isotopic fractionation. The isotope data were used for calculation of the relative contribution of hydrogenotrophic vs. acetoclastic methanogenesis to total CH4 production. Contribution of hydrogenotrophic methanogenesis increased with sediment depth from about 35% to 60%, indicating that organic matter was only partially oxidized in deeper sediment layers.

Food sources of three bivalves living in two habitats of Jiaozhou Bay (Qingdao, China): Indicated by lipid biomarkers and stable isotope analysis

Food Sources of three filter-feeding bivalves from two habitats (intertidal oyster Crassostrea gigas, mussel Mytilus galloprovincialis. and subtidal cultured scallop Chlamys farreri) of Jiaozhou Bay (Qingdao,China) were determined by fatty acid and stable isotope in analysis. Cultured scallop was characterized by significant diatom markets such as 16:1/16:0 close to 1 and high ratio of 20:5(n - 3)/22:6(n - 3), hence we assume that the scallop mainly feeds on diatoms. Fatty acid biomarkers specific to bacteria and terrestrial materials were also found in considerable amounts in scallop tissue, which suggested that there were Substantial bacterial and terrestrial input into the food of the species. Intertidal oyster and mussel, however, exhibited significant flagellate marker. 22:6(n - 3). and lower level of diatom markers. which indicated that flagellates are also part of intertidal bivalves' Planktonic food Sources: meanwhile, high level of Chlorophyta fatty acid marker, Sigma 18:2(n - 6) + 18:3(n - 3), suggested that Ulva pertusa (Chlorophyta) seaweed bed supplied important food sources to intertidal bivalves. Additionally, result of stable isotope analysis showed that phytoplankton contributed 86.2 to 89.0% to intertidal bivalves' carbon budget; macroalga U. pertusa origin source had a contribution of MIX, to 11.0%, which indicated its role Lis in important supplemental food source to intertidal bivalves. From this study. it is concluded that the dietary difference of three bivalves probably relates to the different potential food sources in the scallop farm and intertidal zone in Jiaozhou Bay.

Stable isotope ratios indicate diet and habitat use in New World monkeys.

This paper demonstrates the use of stable isotope ratios of carbon and nitrogen in animal tissue for indicating aspects of species behavioral strategy. We analyzed hair from individuals representing four species of New World monkeys (Alouatta palliata, the mantled howler; Ateles geoffroyi, the spider monkey; Cebus capucinus, the capuchin; and Brachyteles arachnoides, the woolly-spider monkey or muriqui) for delta 13C and delta 15N using previously developed methods. There are no significant differences in either carbon or nitrogen ratios between sexes, sampling year, or year of analysis. Seasonal differences in delta 13C reached a low level of significance but do not affect general patterns. Variation within species was similar to that recorded previously within single individuals. The omega 13C data show a bimodal distribution with significant difference between the means. The two monkey populations living in an evergreen forest were similar to each other and different from the other two monkey populations that inhabited dry, deciduous forests. This bimodal distribution is independent of any particular species' diet and reflects the level of leaf cover in the two types of forest. The delta 15N data display three significantly different modes. The omnivorous capuchins were most positive reflecting a trophic level offset. The spider monkeys and the muriquis were similar to one another and significantly more positive than the howlers. This distribution among totally herbivorous species correlates with the ingestion of legumes by the howler monkey population. In combination, these data indicate that museum-curated primate material can be analyzed to yield information on forest cover and diet in populations and species lacking behavioral data.

Effects of formalin preservation on stable carbon and nitrogen isotope signatures in Calanoid copepods: implications for the use of Continuous Plankton Recorder Survey samples in stable isotope analyses

Preserved and archived organic material offers huge potential for the conduct of retrospective and long-term historical ecosystem reconstructions using stable isotope analyses, but because of isotopic exchange with preservatives the obtained values require validation. The Continuous Plankton Recorder (CPR) Survey is the most extensive long-term monitoring program for plankton communities worldwide and has utilised ships of opportunity to collect samples since 1931. To keep the samples intact for subsequent analysis, they are collected and preserved in formalin; however, previous studies have found that this may alter stable carbon and nitrogen isotope ratios in zooplankton. A maximum ~0.9‰ increase of δ15N and a time dependent maximum ~1.0‰ decrease of δ13C were observed when the copepod, Calanus helgolandicus, was experimentally exposed to two formalin preservatives for 12 months. Applying specific correction factors to δ15N and δ13C values for similarly preserved Calanoid species collected by the CPR Survey within 12 months of analysis may be appropriate to enable their use in stable isotope studies. The isotope values of samples stored frozen did not differ significantly from those of controls. Although the impact of formalin preservation was relatively small in this and other studies of marine zooplankton, changes in isotope signatures are not consistent across taxa, especially for δ15N, indicating that species-specific studies may be required. Copyright © 2011 John Wiley & Sons, Ltd.

Combining metagenomics with metaproteomics and stable isotope probing reveals metabolic pathways used by a naturally occurring marine methylotroph

A variety of culture-independent techniques have been developed that can be used in conjunction with culture-dependent physiological and metabolic studies of key microbial organisms, in order to better understand how the activity of natural populations influences and regulates all major biogeochemical cycles. In this study, we combined DNA-stable isotope probing with metagenomics and metaproteomics to characterize an as yet uncultivated marine methylotroph that actively incorporated carbon from 13C-labeled methanol into biomass. By metagenomic sequencing of the heavy DNA, we retrieved virtually the whole genome of this bacterium and determined its metabolic potential. Through protein-stable isotope probing, the RuMP cycle was established as the main carbon assimilation pathway, and the classical methanol dehydrogenase-encoding gene mxaF, as well as three out of four identified xoxF homologues were found to be expressed. This proof-of-concept study is the first in which theculture-independent techniques of DNA- and protein-stable isotope probing have been used to characterize the metabolism of a naturally-ocurring Methylophaga-like bacterium in the marine environment (i.e. M. thiooxydans L4) and thus provides a powerful approach to access the genome and proteome of uncultivated microbes involved in key processes in the environment