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Review Article
Volume 1 Issue 6 - 2017
Analysis of Soil Lipids for Studies of Microbial Communities
Alejandro E Ferrari* and Luis G Wall
Laboratorio de Bioquímica, Microbiología e Interacciones Biológicas en el Suelo, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, R. Sáenz Peña 352 B1876BXD Bernal, Buenos Aires, Argentina
*Corresponding Author: Alejandro E Ferrari, Laboratorio de Bioquímica, Microbiología e Interacciones Biológicas en el Suelo, Departamento de Ciencia y Tecnología, Universidad Nacional de Quilmes, R. Sáenz Peña 352 B1876BXD Bernal, Buenos Aires, Argentina.
Received: October 02, 2017; Published: October 16, 2017
Introduction
Lipids are a heterogenic group of natural chemical compounds, having in common low solubility in polar solvents and high in non-polar solvents. This definition includes steroids, carotenoids, terpenes and biliar acids. But also non-lipid substances like hydrocarbons. Chemical structure and biological functions of these compounds can be quite different. In a more restrictive definition, lipids are considered as fatty acids and their derivatives (amides and esters), together with compounds related through biosynthesis paths of biological function.
The most abundant lipid are the fatty acids linked by ester links to an alcohol (like glycerol), including acyl-glycerols (often called neutral lipids) and polar lipids, where one fatty acid is replaced by a sugar and a phosphate group (phospholipids).
Neutral lipids are important systems for storing energy in eukaryote organisms, while phospholipids are structural lipids in the cell membrane of every living being, constituting a double layer that allows polar molecules and ions passing through the cell.
Methodology
Lipids can be analyzed by gas or liquid chromatography.
Gas chromatography is fast, cheap and sensitive method for analysis lipids, but they must be derivatizated for increasing their volatility. It is done by alkaline saponification, where the fatty acids are converted in methyl esters fatty acids (FAMEs).
Two different approaches are used for lipid analysis:
The whole cell fatty acid (WCFA) method, sometimes called FAME or MIDI method (Sasser 1990, MIDI 2001) includes four steps: direct alkaline saponification, acid methylation, FAME extraction and extract washing.
The phospholipid fatty acid (PLFA) method (White., et al. 1979, Frostegård., et al. 1991) consists in a direct extraction from soil with a solvent mixture, followed by a fractionation step in a silica solid-phase extraction column (Zelles 1999). During the fractionation, three fractions are obtained: neutral lipid fatty acid (NLFA), glycolipid fatty acid (GLFA) and phospholipid fatty acid (PLFA) fractions. In general, only the first and the last fractions (NLFA and PLFA) are analyzed. A mild alkaline methanolysis step was applied to the fractions, where the ester-bond (but not the phosphate bond) is broken.
Though the WCFA lipids can be considered as the sum of the NLFA, GLFA and PLFA, both techniques are not comparable and must be considered different methods. However, both techniques are equally skillful for comparing soils under different agricultural management.
Applications
Identification of microorganisms in pure cultures
The WCFA or MIDI method has been developed for identification of pure cultures of unknown microorganisms on an artificial media by the Sherlock Microbial Identification System (MIS or MIDI) (MIDI Inc, Newark, DE, USA) (Sasser 1990). The method compares the lipid profile with a library and with reference strains of culturable microorganisms; especially aerobes and anaerobes bacteria and yeasts.
With the name WCFA, this method was later extended to analysis of soil samples, though some controversy about which fatty acids must be used as specific taxa markers still persists.
It is broadly accepted that the arbuscular mycorrhizal fungi have 16:1ω5c as a signature specific marker (Grigera., et al. 2007, Gryndler., et al. 2009).
The fatty acids 18:2ω6,9c, 18:1ω9c and 18:3ω6,9,12c were also used as markers of fungi (Montecchia., et al. 2011).
The branched fatty acids, especially 15:0iso, 15:0anteiso, 16:0iso, 17:0iso and 17:0anteiso are considered markers of Gram-positive bacteria (Hedrick., et al. 2010).
Gram-negative bacteria are characterized by hydroxylated, cyclic and some MUFAs, but there exist strong differences in the selection of them. Most authors choose 17:0cy y 19:0cy as markers of Gram-negative (Pankhurst., et al. 2001).
For actinomycetes, the fatty acids methylated in carbon 10 are considered signature biomarkers, especially 16:010Me, 17:010Me and 18:010Me(Zelles, 1999).
The fatty acid 20:4 is a general marker of eukaryotes, especially protozoa (Ravnskov., et al. 2006).
Assessment of microbial biomass
Because of their high turnover as biomolecules, phospholipid fatty acids are thought to provide information about the active soil microbial community in soil samples (Tunlid & White, 1992). The PLFAs showed good correlation with total viable biomass, as these fatty acids do not have energy storage functions and they quickly degrade after cell death. Neutral lipid fatty acids, which mainly consist in energy reserve substances in living organisms, may provide information about the physiological status of soil biota.
It has been demonstrated that biomass measured as total PLFA show good correlations with other method of assessment of microbial biomass, like fumigation-extraction (Bailey., et al. 2002), Fumigation-incubation (Feng., et al. 2003), substrate induced respiration (Rinklebey Langer 2010).
There is only one limitation in this topic, when the soil is contaminated. So, toxic substances in the soil can kill the microbes and inhibit the enzymes that degrade PLFAs, delaying the degradation process (Frostegård., et al. 2011).
PLFAs as markers of cell stress
Bacterial cells have defined strategies for adapting to environmental stress, including the modification of their membrane fatty acids (Hedrick., et al. 2010, Frostegård., et al. 2011) in order to decrease the permeability.
In this way, many stress markers based on lipids have been proposed, like the precursor/substrate ratios; applied to different stress conditions like acidity, toxicity, anaerobiosis, high temperatures, starvation, osmotic stress y low nutrient availability (Meriles., et al. 2009, Romaniuk., et al. 2011).
According to known metabolic paths of lipid biosynthesis, the formation of trans and cyclo fatty acids from their precursors cis and MUFA, respectively, indicate environmental stress (Borjesson., et al. 2012), being the conversions cisMUFA → transMUFA y cisMUFA→ ciclo the most used (Hedrick., et al. 2010).
A long exposition to conditions that induce the stationary face growth has produced higher levels of cyclic PLFAs (Bossio y Scow 1998). For example, with protobacteria, the 17:0cy/16:1ω7c y 19:0cy/18:1ω7c ratios increased from about 0.05 in the log face to about 2.5 in the stationary face (Hedrick., et al. 2010).
Lipid as markers of agricultural management
Analysis of lipid in soil samples is a key tool for studies of microbial communities because they do not depend of microbial culture. It is supposed that only 1% of the microbial population in soil can be cultivated in the lab (Kirk., et al. 2004).
Either the WCFA or the PLFA methods have been used in studies of agricultural soils all over the world. The different soil agricultural managements, as levels of crop rotation, fertilization and pest control, should produce shifts in the microbial community structure and these shifts affect the lipid profiles. In this way, lipid profiles would allow the detection of specific fatty acids that can show high sensitivity to changes in agricultural management, in fields with different use history (Ferrari., et al. 2014).
Lipid analysis by both WCFA and PLFA methods have been use to study the effects of fertilization (Zhao., et al. 2015, Börjesson., et al. 2012), crop rotation (Meriles., et al. 2009, Montecchia., et al. 2011, Ferrari., et al. 2014), tillage (Ritchie., et al. 2000, Meriles., et al. 2009, Ibekwe., et al. 2002, van Groenigen., et al. 2010, Feng., et al. 2003), fumigants (Ibekwe., et al. 2001), pollution  and bioremediation (Macnaughton., et al. 1999) and benefic-pathogen microbe interactions (Ravnskov., et al. 2006, Chen., et al. 2001, Ruess., et al. 2007).
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Citation: Alejandro E Ferrari and Luis G Wall. “Analysis of Soil Lipids for Studies of Microbial Communities”. Innovative Techniques in Agriculture 1.6 (2017): 262-266.
Copyright: © 2017 Alejandro E Ferrari and Luis G Wall. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.