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Chapter 3 : Phospholipid Fatty Acid Stable Isotope Probing Techniques in Microbial Ecology

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Abstract:

This chapter reviews the methodologies used to present stable isotopically labeled substrates to microbial communities present in a wide range of environmental materials, and also discusses the wet chemical and instrumental methods used to determine compound-specific δC values of individual phospholipid fatty acid (PLFA). The different ways in which the δ C values obtained can be used to assess a variety of properties and activities of microbial communities in the environment are discussed in this chapter. The most widely used labeling techniques for PLFA- stable isotope probing (SIP) utilize C-enriched gases as substrates. The PLFA-SIP approach significantly extends conventional PLFA profiling methods by identifying PLFAs diagnostic of specific functional groups through their enhanced C signatures derived from the assimilation of applied C-substrate(s). In this respect, the approach has key resonances with current trends in the way that environmental microbial communities are being considered in terms of the functioning and stability of ecosystems, especially in relation to the importance of ecosystem services in the context of sustainable environments. The relative ease of preparing PLFA fatty acid methyl esters (FAMEs), combined with the high sensitivity of the gas chromatography combustion-isotope ratio mass spectrometry (GC-C-IRMS) method, makes PLFA-SIP an extremely robust methodology, which allows very large numbers of environmental samples to be studied.

Citation: Maxfield P, Evershed R. 2011. Phospholipid Fatty Acid Stable Isotope Probing Techniques in Microbial Ecology, p 37-71. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch3

Key Concept Ranking

Microbial Ecology
0.7300024
Microbial Communities in Environment
0.51572007
Acetyl Coenzyme A
0.50581396
Cell Wall Components
0.42218503
Bacterial Cell Wall
0.40975738
0.7300024
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Figures

Image of FIGURE 1.
FIGURE 1.

General structure of a phospholipid. Sn denotes the C number position on the glycerol backbone. R and R represent long hydrocarbon chains. × represents one of the various substituents listed in Table 1 .

Citation: Maxfield P, Evershed R. 2011. Phospholipid Fatty Acid Stable Isotope Probing Techniques in Microbial Ecology, p 37-71. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch3
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Image of FIGURE 2.
FIGURE 2.

Pictorial representation of a cross section through a cell membrane displaying the phospholipid bilayer with integral (a) and peripheral (b) membrane proteins.

Citation: Maxfield P, Evershed R. 2011. Phospholipid Fatty Acid Stable Isotope Probing Techniques in Microbial Ecology, p 37-71. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch3
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Image of FIGURE 3.
FIGURE 3.

CO pulse labeling plant microcosm (adapted from ).

Citation: Maxfield P, Evershed R. 2011. Phospholipid Fatty Acid Stable Isotope Probing Techniques in Microbial Ecology, p 37-71. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch3
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Image of FIGURE 4.
FIGURE 4.

Static flux chamber (a) and continuous-flow flux chamber (b).

Citation: Maxfield P, Evershed R. 2011. Phospholipid Fatty Acid Stable Isotope Probing Techniques in Microbial Ecology, p 37-71. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch3
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Image of FIGURE 5.
FIGURE 5.

Freshwater sediment core incubation setup (adapted from ).

Citation: Maxfield P, Evershed R. 2011. Phospholipid Fatty Acid Stable Isotope Probing Techniques in Microbial Ecology, p 37-71. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch3
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Image of FIGURE 6.
FIGURE 6.

Continuous-flow gas diffusion core incubator schematic.

Citation: Maxfield P, Evershed R. 2011. Phospholipid Fatty Acid Stable Isotope Probing Techniques in Microbial Ecology, p 37-71. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch3
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Image of FIGURE 7.
FIGURE 7.

Partial gas chromatogram of the PLFA fraction of Bronydd Mawr upland grassland soil following derivatization with BF methanol. Separation was achieved using a Varian Factor Four VF23 ms (high cyanopropyl modified methyl polysiloxane) fused silica column (60 m by 0.32 mm internal diameter [ID]; 0.15-mm film thickness). The carrier gas was hydrogen, and the oven temperature was programmed from 50°C (held for 2 min) to 100°C at 15°C min, from 100 to 220°C at 4°C min, and from 220 to 240°C (held for 5 min) at 15°C min. PLFA assignments: 1 = C alkane, 2 = 14:0, 3 = 15:0, 4 = 15:0, 5 = 15:0, 6 = 16:0, 7 = 16:0, 8 = 16:111, 9 = 17:0, 10 = 16:17, 11 = 17:0 & 16:15, 12 = a17:0, 13 = 17:18, 14 = 17:0, 15 = 18:0, 16 = 19:0, 17 = 18:19c, 18 = 18:17c, 19 = 18:15, 20 = 18:23,6 ( ).

Citation: Maxfield P, Evershed R. 2011. Phospholipid Fatty Acid Stable Isotope Probing Techniques in Microbial Ecology, p 37-71. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch3
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Image of FIGURE 8.
FIGURE 8.

Partial gas chromatogram of the PLFA fraction of Bronydd Mawr upland grassland soil following derivatization with DMDS to separate and identify the unsaturated PLFAs. Separation was achieved with a Chrompack CPSIL5-CB (100% dimethyl polysiloxane) fused silica column (50 m by 0.32 mm ID; 0.12 mm film thickness). The carrier gas was hydrogen and the oven was programmed from 40°C (held for 2 min) to 150°C at 12°C min, then 150 to 260°C (held for 5 min) at 4°C min. PLFA assignments: 1 = 15:0, 2 = 15:0, 3 = 16:0, 4 = 16:0, 5 = 17:0, 6 = 17:0, 7 = 17:0, 8 = 17:0, 9 = 18:0, 10 = 19:0, 11 = 19:0, 12 = 18:113, 13 = 15:111, 14 = 16:112, 15 = 18:17, 16 = 16:111 & 16:19, 17 = 16:17, 18 = 16:15, 19 = 17:18, 20 = 18:19c, 21 = 18:17c, 22 = 18:15, 23 = 19:16, 24 = 19:17. All unsaturated compounds were analyzed as DMDS derivatives.

Citation: Maxfield P, Evershed R. 2011. Phospholipid Fatty Acid Stable Isotope Probing Techniques in Microbial Ecology, p 37-71. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch3
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Image of FIGURE 9.
FIGURE 9.

Mass spectra of monounsaturated C fatty acid DMDS derivatives (18:17 and 18:19) extracted from Bronydd Mawr upland grassland soil. The fragment ions denoted D, E, and F are used to determine the position of the double bonds in the original PLFA.

Citation: Maxfield P, Evershed R. 2011. Phospholipid Fatty Acid Stable Isotope Probing Techniques in Microbial Ecology, p 37-71. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch3
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Image of FIGURE 10.
FIGURE 10.

Mass spectra of picolinyl esters of (a) straight-chain 17:0 fatty acid and (b) 10Me16:0 fatty acid. Note loss of ion at 262 in panel b, associated with branching at position 7 on the aliphatic C chain.

Citation: Maxfield P, Evershed R. 2011. Phospholipid Fatty Acid Stable Isotope Probing Techniques in Microbial Ecology, p 37-71. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch3
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Image of FIGURE 11.
FIGURE 11.

Origin of major fragment ions in the EI mass spectra of picolinyl esters of PLFAs methyl esters.

Citation: Maxfield P, Evershed R. 2011. Phospholipid Fatty Acid Stable Isotope Probing Techniques in Microbial Ecology, p 37-71. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch3
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Image of FIGURE 12.
FIGURE 12.

Generalized schematic of a GC-C-IRMS configured for determining δC values of individual compounds. Inset (a) details the optimized connection of the fused silica capillary to the combustion reactor. Mixtures of compounds are separated by GC; combusted online, generating CO and HO; HO is removed; and the CO is introduced into an MS equipped with a triple collector comprising three Faraday cups monitoring simultaneously 44, 45, and 46, corresponding to CO, CO, and COO, respectively. The output currents are amplified and integrated to allow calculation of δC values. Reference CO and FAs of known δC values are utilized to monitor instrument performance and standardize determinations.

Citation: Maxfield P, Evershed R. 2011. Phospholipid Fatty Acid Stable Isotope Probing Techniques in Microbial Ecology, p 37-71. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch3
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Image of FIGURE 13.
FIGURE 13.

The 44 ion current (below) and instantaneous ratio of 45/44 ions (above) recorded for the PLFAs extracted from a soil following incubation with CH.PLFA assignments: 1 = C19 alkane, 2 = 14:0, 3 = 15:0, 4 = 15:0, 5 = 15:0, 6 = 16:0, 7 = 16:0, 8 = 16:111, 9 = 17:0, 10 = 16:17, 11 = 17:0 and 16:15, 12 = 17:0, 13 = 17:18, 14 = 17:0, 15 = 18:0, 16 = 18:0, 17 = 19:0, 18 = 18:19c, 19 = 18:17c, 20 = 18:15, 21 = 18:23,6.

Citation: Maxfield P, Evershed R. 2011. Phospholipid Fatty Acid Stable Isotope Probing Techniques in Microbial Ecology, p 37-71. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch3
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Citation: Maxfield P, Evershed R. 2011. Phospholipid Fatty Acid Stable Isotope Probing Techniques in Microbial Ecology, p 37-71. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch3
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Citation: Maxfield P, Evershed R. 2011. Phospholipid Fatty Acid Stable Isotope Probing Techniques in Microbial Ecology, p 37-71. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch3
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Citation: Maxfield P, Evershed R. 2011. Phospholipid Fatty Acid Stable Isotope Probing Techniques in Microbial Ecology, p 37-71. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch3
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Citation: Maxfield P, Evershed R. 2011. Phospholipid Fatty Acid Stable Isotope Probing Techniques in Microbial Ecology, p 37-71. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch3
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Citation: Maxfield P, Evershed R. 2011. Phospholipid Fatty Acid Stable Isotope Probing Techniques in Microbial Ecology, p 37-71. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch3
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Citation: Maxfield P, Evershed R. 2011. Phospholipid Fatty Acid Stable Isotope Probing Techniques in Microbial Ecology, p 37-71. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch3
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Image of FIGURE 14.
FIGURE 14.

Concentrations of the total extracted PLFAs and comparison of the C-label concentration incorporated into each PLFA as nanograms of C per gram of soil dry weight following 17 to 18 weeks incubation under 2 ppmv CH for Bronydd Mawr NCaPK, CaPK, and Nil-graze soils. Error bars represent ±1 standard deviation (Maxfield et al., 2008a).

Citation: Maxfield P, Evershed R. 2011. Phospholipid Fatty Acid Stable Isotope Probing Techniques in Microbial Ecology, p 37-71. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch3
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Image of FIGURE 15.
FIGURE 15.

Comparison of Tenerife Andisol C-labeled PLFA distributions with published PLFA compositions of pure cultures of methanotrophic bacteria ( ). The PLFA compositions employed were the mole percentages of the PLFAs of pure cultures and the labeled PLFAs extracted from the soils. A hierarchical tree was produced by cluster analysis performed with the R v2.8.1 statistical package. Bootstrap probabilities were based on 1,000 repetitions ( ).

Citation: Maxfield P, Evershed R. 2011. Phospholipid Fatty Acid Stable Isotope Probing Techniques in Microbial Ecology, p 37-71. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch3
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Image of FIGURE 16.
FIGURE 16.

Landfill soil PLFA δC values following up to 27 days of incubation under 5,000 ppmv CH (1% enriched C) indicating primary (a), secondary (b), and tertiary (c) C assimilation ( ).

Citation: Maxfield P, Evershed R. 2011. Phospholipid Fatty Acid Stable Isotope Probing Techniques in Microbial Ecology, p 37-71. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch3
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Tables

Generic image for table
TABLE 1

The structure and distribution of major phospholipids

Citation: Maxfield P, Evershed R. 2011. Phospholipid Fatty Acid Stable Isotope Probing Techniques in Microbial Ecology, p 37-71. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch3
Generic image for table
TABLE 2

Nomenclature of some naturally occurring fatty acids

Citation: Maxfield P, Evershed R. 2011. Phospholipid Fatty Acid Stable Isotope Probing Techniques in Microbial Ecology, p 37-71. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch3
Generic image for table
TABLE 3

Classes of PLFAs associated with particular taxonomic or functional groups of microorganisms

Citation: Maxfield P, Evershed R. 2011. Phospholipid Fatty Acid Stable Isotope Probing Techniques in Microbial Ecology, p 37-71. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch3
Generic image for table
TABLE 4

Summary of PLFA C-labeling studies of environmental microbial communities

Citation: Maxfield P, Evershed R. 2011. Phospholipid Fatty Acid Stable Isotope Probing Techniques in Microbial Ecology, p 37-71. In Murrell J, Whiteley A (ed), Stable Isotope Probing and Related Technologies. ASM Press, Washington, DC. doi: 10.1128/9781555816896.ch3

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