Linking autotrophic activity in environmental samples with specific bacterial taxa by detection of ¹³C‐labelled fatty acids

Abstract

A method for the detection of physiologically active autotrophic bacteria in complex microbial communities was developed based on labelling with the stable isotope 13C. Labelling of autotrophic nitrifying, sulphur-oxidizing and iron-oxidizing populations was performed in situ by incubation with NaH[13C]O3. Incorporated label into fatty acid methyl esters (FAMEs) was detected and quantified using gas chromatography-mass spectrometry in single ion monitoring mode. Before the analyses of different environmental samples, the protocol was evaluated in pure culture experiments. In different environmental samples a selective labelling of fatty acids demonstrated which microbial taxa were responsible for the respective chemolithoautotrophic activity. The most strongly labelled fatty acids of a sample from a sulphide treating biofilter from an animal rendering plant were cis-7-hexadecenoic acid (16:1 cis7) and 11-methyl hexadecanoic acid (16:0 11methyl), which are as-yet not known for any sulphide-oxidizing autotroph. The fatty acid labelling pattern of an experimental biotrickling filter sample supplied with dimethyl disulphide clearly indicated the presence and activity of sulphide-oxidizing bacteria of the genus Thiobacillus. For a third environmental sample from an acid mining lake sediment, the assignment of autotrophic activity to bacteria of the genus Leptospirillum but not to Acidithiobacillus could be made by this method, as the fatty acid patterns of these bacteria show clear differences.

Dynamics of a Pasture Soil Microbial Community after Deposition of Cattle Urine Amended with [ 13 C]Urea

“…The greatest amounts of 13 C were found in the common fatty acids 16:0, 16:17c, and 18:17c, which predominate in Nitrosomonas and Nitrobacter cell membranes (4,27,28) but are also present in many other organisms. Fixation of CO 2 by heterotrophic bacteria may have accounted for some incorporation of 13 C into PLFAs, although a more uniform distribution of label within a range of microbial PLFAs would have been expected if this had been the main mechanism for urea C incorporation (44).…”

“…Fixation of CO 2 by heterotrophic bacteria may have accounted for some incorporation of 13 C into PLFAs, although a more uniform distribution of label within a range of microbial PLFAs would have been expected if this had been the main mechanism for urea C incorporation (44). Both cy17:0 and cy19:0 can be synthesized by autotrophic NO 2 Ϫ oxidizers, including Nitrobacter winogradski (27,28). If Nitrobacter was the main source of de novo CFA synthesis, then this might explain why 13 C depletion was so extreme in these compounds.…”

“…Even though isotope fractionation during lipid biosynthesis is a matter of concern (5), this approach can potentially link specific substrates with individual populations in situ (6,16,22). Substrate-derived 13 C has been traced in membrane lipids of heterotrophic (1,7) and autotrophic (27) organisms. Further, 13 C enrichment of phospholipid fatty acid (PLFA) profiles has been used to investigate microbial community dynamics (9).…”

Influence of CH<sub>4</sub> and H<sub>2</sub>S availability on symbiont distribution, carbon assimilation and transfer in the dual symbiotic vent mussel <i>Bathymodiolus azoricus</i>

“…Incubations with 13 C-enriched substrates are commonly used to study the activity of pure bacterial cultures as well as the activity of different complex bacterial communities in natural and artificial ecosystems and soils. Autotrophic SOB were already described performing such tracer experiments with 13 C-labelled bicarbonate (Knief et al, 2003a), as were communities of physiologically active MOB using 13 CH 4 (Knief et al, 2003b). In the present study, we investigated symbiont populations and mussel nutrition in specimens of B. azoricus collected from the MAR and kept under various conditions in a controlled laboratory environment (LabHorta facility in the Azores, Colaço and Santos, 2002b).…”

Butyric Acid- and Dimethyl Disulfide-Assimilating Microorganisms in a Biofilter Treating Air Emissions from a Livestock Facility

“…Compre-hensive characterization and understanding of air biofilter metabolism requires knowledge about in situ metabolic function and activity. The use of stable-isotope probing (SIP) in combination with phospholipid fatty acid analysis on lab-scale biofilter samples identified Pseudomonas as a part of the styrenedegrading community (1) and Thiobacillus as an active member of the autotrophic community in a dimethyl disulfidetreating biofilter (25). Findings by Friedrich and Lipski (13), who also combined isotope labeling and PLFA to a full-scale oil mill biofilter, revealed a central role of Gordonia in hexane transformations.…”