PLFA/FAME Analyses for Microbial Community Assessment CSS 645 Jennifer Moore 16 Oct 2003

Outline Lipid biochemistry 101 Justification for use Procedure for ester-linked phospholipid fatty acids Interpretation Advantages/Disadvantages Future outlook

What is a Phospholipid? Lipids are organic molecules are nondissolvable in H2O They are comprised of many C-H bonds which store energy Phospholipids: Glycerol, 2 fatty acids, & 1 phosphate group (PO4)

What is a Phospholipid?

Structure of the lipid bi-layer

Fatty Acid Nomenclature Fatty acids are designated by the total number of carbon atoms:number of double bonds, followed by the position of the dbl bond from the methyl end of the molecule, indicated by w and a number, or from the carboxyl end, indicated by D. Example18:2w6 is an 18-C FA with 2 double bonds beginning at the 6th C from the methyl end (same as 18:2D12). The prefixes i and a indicate iso and anteiso branching, respectively. Example: i16:0 has a methyl branch on the first C from the w end.

Fatty Acid Nomenclature -cy indicates a cyclopropane fatty acid -Me indicates methyl branching from the carboxyl end of the chain Example: 10Me16:0 has a methyl branch on the 10th C from the carboxylic end. The abbreviations t and c indicate trans and cis configuration of the double bond (cis is more common).

Why use fatty acids (in particular Phospholipid FA)? They are essential components of every living cell and are useful biomarkers because: they are and have great structural diversity and are coupled with high biological specificity. Used as a proxy for the ‘living’ and possibly the ‘active’ microbial biomass The phosphate group is quickly consumed when an organism dies They are not found in storage products Make up a relatively constant proportion of the biomass

PLFA resolution Compare total PLFA pattern by multivariate statistics => changes in pattern = changes in microbial community Changes in specific PLFAs can be used as indicators of changes in specific organism groups. Baath, E. (2003) Microbial Ecology 45:373-383 Using a database of pure cultures and known metabolic pathways

Procedure Initial Extraction Lipid Separation Saponification/Methylation GC analysis Data Analysis and Interpretation

Procedure Initial extraction 1-25g soil (dw) is typically used Chloroform:Methanol:Phosphate buffer (2.0:1.0:0.8) Centrifuge, filter, and remove chl phase Dry under N2

Procedure Lipid Separation Use solid-phase extraction silicic acid columns Lipids separate by eluting with the following chemicals: Neutral lipids => with chloroform Glycolipids => with acetone Phospholipids => with methanol

Procedure Saponification/Methylation Use 2M KOH dissolved in MeOH Separate FAMES on GC-FID (redissolve FAME with hexane) Use standards to determine quantitative amounts and 37-FAME std to id peaks Use multivariate statistics to evaluate data (namely PCA or NMS)

Sample chromatograph

Sample Chromatograph

Interpretation Fatty Acid Microbial Group 15:0i, 17:0i, 15:0a, etc.. Gram positive bacteria cy17:0, cy19:0, 18:1D11c Gram negative bacteria (also cy19:0 gm+) 10 Me18:0, 10 Me17:0, 10 Me16:0 Actinomycetes 18:2w6,9, 18:1w9c Fungi 20:4 w6 Protozoan 16:1 w5 Arbuscular mycorrhizal fungi 18:1w8c Methanotrophs the fungal fatty acids have been positively correlated with ergosterol content and have been found to comprise up to 95% of total fatty acid content in fungi (Stahl and Klug (1996) AEM 4136-4146). Griffiths et al (1999) SBB 31:145-153 suggest possibility that cy19:0 may represent Gram + bacterial groups more then cy17:0

Interpretation Caution A particular fatty acid might be misinterpreted because its occurrence might be representative of other organisms previously unknown Therefore, it is important to know the fatty acid composition of the individual species which make up the community. Can also evaluate using full “groups” of fatty acids instead of a signal signature

Interpretation Sum the following PLFAs for bacterial biomass: i15:0, a15:0, i16:0, 16:1w9, 16:1w7t, i17:0, a17:0, 17:0, cy17:0, 18:1w7 and cy19:0 Use 18:2w6,9 and 18:1w9c for fungal biomass Can use fungi:bacteria ratios

Interpretation Cyclopropyl fatty acids are formed by a modification of existing membrane lipids, often as the organisms enter the stationary phase.....it’s suggested that this class of PLFAs is formed under stressful conditions 16:1w7c and 18:1w7c are the precursors to cy17:0 and cy19:0

New Help for Fungal Marker? Use NLFA/PLFA ratio as indicator of the nutrient status or physiological state of fungi (neutral lipids are used for storage for eukaryotes but not by bacteria) but must be cautious (E. Baath, 2003)

PLFA Advantages Detects microbial community in an environmental sample (i.e., avoids problems associated with cultures and direct counting methods) Can be used to detect (rapid) changes in a wide range of soil type, sediments, water, and humus Relatively easy and ‘quick’ so large number of samples can be processed

PLFA Advantages Provides more accurate and precise estimates of the viable microbial biomass compared to CFM. PLFA profiles contain detailed info on lipid structure that can be used to investigate microbial community structure and metabolic condition. Provides a fingerprint of microbial diversity at time of sampling Relatively inexpensive if you already own a GC

PLFA Disadvantages PLFA profiles do not reveal species-level information Archae bacteria can not be determined with this method Can’t detect unusual FA or those found in low concentrations => but these may represent a functionally, very important group Interpretation needs further work as fatty acids in community structure can apply to more then one group of organisms

PLFA Disadvantages Linking PLFA profiles and function of ecosystems has not been well established. Spatial and temporal variability can be high, thus, making interpretation difficult Database is centered around fatty acids from microorganisms from pure cultures

Optimistic Future? Database is constantly growing which should enhance interpretation of biomarkers Correlation with novel methods focusing on function will enhance interpretation Perhaps full fractionation of lipids would shed the most light??? Estimated that 50 FA represent about 10000 species...more accurate determination of fatty acids may lead to the detection of unique FA of single strains of bacteria or biomarker FA also present in low amounts of organisms

PLFAs in the literature Waldrop et al., (2000. SBB 32:1837-1846) found a positive correlation with enzyme activity and community composition using PLFA in tropical forest soils of differing ages and management (BIOLOG was not correlated). Zelles et al (1995) found differences in the monounsaturated FA fraction when comparing soils from grassland and cultivated systems. Wander et al (1995) showed highest total PLFA in organic cover cropped soil when compared to an organic-manure amended soil.

Interpretation Changes in lipid composition, particularly decreases in unsaturation, typically occur only as a result of biosynthesis of new lipids; since biosynthesis is energetically expensive it is probable that portions of the microbial community will experience metabolic stress and death as temperature increases (Peterson and Klug, 1994. AEM 60:2421-2430).