
Full text loading...
Category: Environmental Microbiology; Applied and Industrial Microbiology
Phospholipid Fatty Acid Stable Isotope Probing Techniques in Microbial Ecology, Page 1 of 2
< Previous page | Next page > /docserver/preview/fulltext/10.1128/9781555816896/9781555815370_Chap03-1.gif /docserver/preview/fulltext/10.1128/9781555816896/9781555815370_Chap03-2.gifAbstract:
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 δ13C values of individual phospholipid fatty acid (PLFA). The different ways in which the δ 13C 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 13C-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 13C signatures derived from the assimilation of applied 13C-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.
Full text loading...
General structure of a phospholipid. Sn denotes the C number position on the glycerol backbone. R1 and R2 represent long hydrocarbon chains. × represents one of the various substituents listed in Table 1 .
Pictorial representation of a cross section through a cell membrane displaying the phospholipid bilayer with integral (a) and peripheral (b) membrane proteins.
13CO2 pulse labeling plant microcosm (adapted from Nakano-Hylander and Olsson, 2007 ).
Static flux chamber (a) and continuous-flow flux chamber (b).
Freshwater sediment core incubation setup (adapted from Deines et al., 2007 ).
Continuous-flow gas diffusion core incubator schematic.
Partial gas chromatogram of the PLFA fraction of Bronydd Mawr upland grassland soil following derivatization with BF3 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-1, from 100 to 220°C at 4°C min-1, and from 220 to 240°C (held for 5 min) at 15°C min-1. PLFA assignments: 1 = C19 alkane, 2 = 14:0, 3 = i15:0, 4 = a15:0, 5 = 15:0, 6 = i16:0, 7 = 16:0, 8 = 16:1ω11, 9 = br17:0, 10 = 16:1ω7, 11 = i17:0 & 16:1ω5, 12 = a17:0, 13 = 17:1ω8, 14 = 17:0, 15 = 18:0, 16 = cyc19:0, 17 = 18:1ω9c, 18 = 18:1ω7c, 19 = 18:1ω5, 20 = 18:2ω3,6 ( Maxfield et al., 2006 ).
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-1, then 150 to 260°C (held for 5 min) at 4°C min-1. PLFA assignments: 1 = i15:0, 2 = a15:0, 3 = i16:0, 4 = 16:0, 5 = br17:0, 6 = i17:0, 7 = a17:0, 8 = 17:0, 9 = 18:0, 10 = 19:0, 11 = cyc19:0, 12 = 18:1ω13, 13 = 15:1ω11, 14 = 16:1ω12, 15 = 18:1ω7, 16 = 16:1ω11 & 16:1ω9, 17 = 16:1ω7, 18 = 16:1ω5, 19 = 17:1ω8, 20 = 18:1ω9c, 21 = 18:1ω7c, 22 = 18:1ω5, 23 = 19:1ω6, 24 = 19:1ω7. All unsaturated compounds were analyzed as DMDS derivatives.
Mass spectra of monounsaturated C18 fatty acid DMDS derivatives (18:1ω7 and 18:1ω9) 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.
Mass spectra of picolinyl esters of (a) straight-chain 17:0 fatty acid and (b) 10Me16:0 fatty acid. Note loss of ion at m/z 262 in panel b, associated with branching at position 7 on the aliphatic C chain.
Origin of major fragment ions in the EI mass spectra of picolinyl esters of PLFAs methyl esters.
Generalized schematic of a GC-C-IRMS configured for determining δ13C 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 CO2 and H2O; H2O is removed; and the CO2 is introduced into an MS equipped with a triple collector comprising three Faraday cups monitoring simultaneously m/z 44, 45, and 46, corresponding to 12C16O2, 13C16O2, and 12C18O16O, respectively. The output currents are amplified and integrated to allow calculation of δ13C values. Reference CO2 and FAs of known δ13C values are utilized to monitor instrument performance and standardize determinations.
The m/z 44 ion current (below) and instantaneous ratio of m/z 45/44 ions (above) recorded for the PLFAs extracted from a soil following incubation with 13CH4.PLFA assignments: 1 = C19 alkane, 2 = 14:0, 3 = i15:0, 4 = a15:0, 5 = 15:0, 6 = i16:0, 7 = 16:0, 8 = 16:1ω11, 9 = br17:0, 10 = 16:1ω7, 11 = i17:0 and 16:1ω5, 12 = a17:0, 13 = 17:1ω8, 14 = 17:0, 15 = i18:0, 16 = 18:0, 17 = cyc19:0, 18 = 18:1ω9c, 19 = 18:1ω7c, 20 = 18:1ω5, 21 = 18:2ω3,6.
Concentrations of the total extracted PLFAs and comparison of the 13C-label concentration incorporated into each PLFA as nanograms of 13C per gram of soil dry weight following 17 to 18 weeks incubation under 2 ppmv 13CH4 for Bronydd Mawr NCaPK, CaPK, and Nil-graze soils. Error bars represent ±1 standard deviation (Maxfield et al., 2008a).
Comparison of Tenerife Andisol 13C-labeled PLFA distributions with published PLFA compositions of pure cultures of methanotrophic bacteria ( Bowman et al., 1993 ; Dedysh, 2002 ; Bodelier et al., 2009 ). 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 ( Maxfield et al., 2009 ).
Landfill soil PLFA δ13C values following up to 27 days of incubation under 5,000 ppmv 13CH4 (1% enriched 13C) indicating primary (a), secondary (b), and tertiary (c) C assimilation ( Dildar, 2010 ).
The structure and distribution of major phospholipids a
Nomenclature of some naturally occurring fatty acids
Classes of PLFAs associated with particular taxonomic or functional groups of microorganisms a
Summary of PLFA 13C-labeling studies of environmental microbial communities