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

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

3. 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)

4. What is a Phospholipid?

5. Structure of the lipid bi-layer

6. 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.

7. 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).

8. 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

9. 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:
Using a database of pure cultures and known metabolic pathways

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

11. 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

12. 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

13. 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)

14. Sample chromatograph

15. Sample Chromatograph

16. 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 ).
Griffiths et al (1999) SBB 31: suggest possibility that cy19:0 may represent Gram + bacterial groups more then cy17:0

17. 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

18. 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

19. 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

21. 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)

22. 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

23. 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

24. 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

25. 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

26. 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 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

27. PLFAs in the literature
Waldrop et al., (2000. SBB 32: ) 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.

28. 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, AEM 60: ).