SOP for PLFA (Phospholipid Fatty Acid Analysis)

Microbial ID, Inc. Soil Microbial Biomass by High Throughput PLFA Analysis Extraction and Analysis Protocol

Approved by laboratory manager on 8-6-2021

Overview:

·  The following method describes sample storage, preparation of extraction solutions, extraction procedures, gas chromatograph analysis, and possible data analysis for soil microbial biomass through PLFA analysis.

·  The extraction process will take place in the Ag building Rm.1024, bottle top dispensers and glassware are located in the lab with appropriate labels.  Please keep the area clean, tidy and well stocked for the next researcher.

·  For scheduling of extraction procedure and GC analysis contact the laboratory manager at ecobgc@uwyo.edu or jessj@uwyo.edu.

Sample Prep

a.  Soil sample storage

1. Store 2mm sieved soil in collection vessels from the field at least overnight in -80°C freezer

2. Freeze-dry using Lyophilizer* for at least 24 hours.

3. Remove the cap from each vial after freeze-drying and record the dry weight of the soil and vial.

4. Use the Lyophilizer at least 24 hours prior to beginning the extraction procedure.

Extraction Solutions Protocol (for roughly 30 samples)

1. Phosphate buffer:

   1. PO4 buffer: 10.66 grams of Dipotassium Phosphate (K2HPO4) with 5.28 grams of Monopotassium Phosphate (KH2PO4) added to 2 liters of DI water (expiration date: 6 months from date made)

2. Internal Standard (19:0) 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine

   1. Avanti Polar Lipids Catalog # 850367P – white powder, stored in -20-degree C freezer.

   2. Dissolve 12.28 mg in 10 mL of chloroform (1.5 mM solution).

   3. Just before extracting, add to an appropriate volume of extractant at a rate of 0.5 μL internal standard per mL of extractant and mix, to Bligh-Dyer Extractant.

   4. This is equivalent to adding 6.1 nanomoles of 19:0.

3. Bligh-Dyer Extractant:

   1. 80 mL 50 mM PO4 buffer (step 1)

   2. 200 mL methanol

   3. 100 mL chloroform

   4. This reagent can be mixed in a ratio of 4:10:5 PO4 buffer: methanol: chloroform depending on how many samples are being extracted.

   5. Mix fresh daily and dispose of any extra.  Add internal standard from step 2.a-c just before extracting soil samples.

4. Transesterification Reagent

   1. 0.561 grams KOH

   2. 75 mL methanol

   3. 25 mL toluene

   4. Dissolve KOH in methanol and then add toluene.

   5. Expiration date: 6 months from date made.

5. Chromatography Wash Solution

   1. 50mL methanol

   2. 50mL chloroform

   3. 10mL DI H2O

   4. This reagent can be mixed in a ratio of 5:5:1 methanol: chloroform: DI H2O.

   5. Expiration date: 6 months from date made.

6. Calibration Solution

   1. Midi-Inc. part #1208 calibration mix, follow manufacturer’s instructions for working stock.

Part 1 Extraction of soil samples

1. Extraction (green laboratory tape)

a.     Use one additional test tube as a reagent blank per 20 samples.

b.    Remove between 0.50 – 1.02 grams of soil, record the weight, and place in a 23x85 mm test tube-short fat tubes (Fisher Part # 1495935AA).

c.     Add 4 mL of Bligh-Dyer extractant containing internal standard and cap tubes with 23x85 mm PTFE-lined screw caps.

d.    Vortex* for 5 seconds on high, then sonicate* for 10 minutes at room temperature, Place sonicator in the chemical fume hood for this step.

       i.  NOTE: Labels come off during sonicating, place labels on the caps, or write on the vials with a Sharpie.

e.     Repeat step c two more times, drying the outside of the tubes in between rounds of sonication, for a total of 30 minutes of sonication. 

f.      Centrifuge for 15minutes @ 3600 RPM ± 300.

g.     Transfer liquid phase to clean 23x85 mm (short fat tubes) test tubes using 5 or 9 inch Pasteur pipette. (label with orange laboratory tape)

Part 2 Separation

1. Separation (orange laboratory tape)

a.     Add 1mL each of chloroform and deionized water to each sample.

b.    Cap and vortex 5 seconds on high, then centrifuge 15minutes @ 3600 RPM ± 300.

c.     Transfer bottom phase to clean 13x55mm (8 mL narrow culture tubes with orange laboratory tape).

d.    For replicate samples: combine the bottom phases of both replicates into the same tube

e.     Use the nitrogen needle evaporator water bath at 40°C (for 30 min.~1hour).

f.      Stopping point:  samples may be stored, capped or covered, overnight in a -20°C freezer.

Part 3 Chromatography

1. Chromatography

a.     Dissolve samples in 1 mL chloroform and vortex for 5 seconds on low, leave on the bench for step 3d.

b.    100 mg/1mL silica gel SPE column tubes and place on vacuum apparatus collect waste into 10 mL culture tubes (Phenomenex Part # 8B-S012-EAK).

c.     Wash each well 3 times with 1 mL methanol then 3 times with 1 mL chloroform.

            i.  NOTE: Make sure the columns do not go completely dry. Make sure to go slow and steady, while draining.

d.    Add sample extract to wells. 

e.     Let sample drain into column.

f.      Repeat transfer from sample vial with another 1 mL chloroform add and drain into column.

g.     Wash with 1 mL of chloroform into column and discard waste vials.

h.    Place new 8 mL collection tube under column to collect the PLFA.

i.      Elute phospholipids with 0.5 mL of chromatography wash solution (5:5:1 methanol: chloroform: DI H₂O).

            i.  Extraction Solutions Protocol Part 5 a-e.

j.      Let sample drain out of the column into the collection tube with chromatography wash solution, until the column is completely dry.

k.    Use the nitrogen needle evaporator water bath at 40°C (for 30 min.~1hour).

l.      Stopping point:  samples may be stored, capped or covered, overnight in a -20°C freezer.

m.   Transesterification and Transfer to GC Vials

               i.  Add 0.2mL transesterification reagent (Extraction Solution Protocol step 4 a-e),cap with 13x100mm PTFE-lined caps, and vortex* 5 seconds on high.

               ii.  Incubate at 37°C for 15 minutes, with caps on and nitrogen turned off.

               iii.  Add 0.4 mL of 0.075 M acetic acid and 0.5 mL chloroform.  Vortex for 10 seconds on low and allow phases to separate.

1. Phase separation can take up to 15 minutes to separated.

               iv.  Transfer bottom phase to 13x100mm screw cap tubes.

               v.  Place the tubes into the nitrogen needle evaporator water bath at 40°C and dry for 15-30 minutes. If samples are not dry, continue additional 5-minute increments until dry. Do not over dry.

                vi.  Add 200 μL hexane to the tubes and cap.

1. For replicate samples: add 200 uL hexane.

                vii.  Vortex for 5 seconds on low.

                viii.  Transfer extract to a 200 uL limited volume glass vial insert (LabSphere Catalog # 05090129) and place the insert into the GC autosamplers vial.

                  ix.  Transfer GC vials to covered box, label and place in -20°C, fill out sample submission form at http://www.uwyo.edu/ecologybgc and email the form to ecobgc@uwyo.edu. 

Part 4 Cleaning and Notes

1. Glassware and Cap Cleaning

a.     All glassware is to be scrubbed carefully with Micro-9 laboratory detergent and thoroughly rinsed three times with DI water while wearing gloves (to avoid any lipids from forming a monolayer and spreading over the entire surface of the wet glass).

b.    Where necessary, bake glassware in a dedicated oven* at 220°C for at least 2 hours.

c.     DO NOT bake any volumetric glassware, as this would void certification

d.    Use an Ultrasonic Cleaning Bath with Micro-9 cleaner to clean the tube caps: Rinse and sonicate in DI water for 1 hour 3 additional times.

Notes

a.     All organic solvents should be HPLC grade or better.

b.     Contamination is a potential problem.

c.     Run at least 1 reagent blank per 20 samples.

d.     Wear nitrile gloves during the extraction process.

e.     GC caps must have PTFE/Silicone/PTFE Septa.

f.      Test tube caps must be PTFE-lined.

g.     Limited volume inserts must not have poly spring feet.

Part 4 Data Analysis Protocol

Overview

1)The MIDI Sherlock software is used to process the samples. It consists of three applications: Sample Processor used for analyzing samples on the Gas Chromatograph; CommandCenter used for inspecting processed samples and exporting data; and PLFA Tools used for transforming data into absolute moles based on an internal standard.

MIDISherlock.  Software system for analyzing GC samples.

a.     Includes:Sherlock Method PLFAD2 containing the instrument method as well as the peak naming table for PLFA compounds.

b.     Sample Processor for automatically processing samples through the Gas Chromatograph using method PLFAD2.

c.     CommandCenter for manually inspecting sample results and generating Access databases containing necessary data and.csv files.

d.     PLFA Tools TransformSamps for automatically converting raw data into absolute moles based on an internal standard. The D2_MOLE file isprovided with the tools.

e.     MIDI, Inc. 1208 Calibration mixture, used both to calibrate the Gas Chromatograph and used as the positive control for each run.

CSV Export and RTF file will be generated and sent to each researcher.

Soil Microbial Biomass by High Throughput PLFA Analysis Calculations

a.     Introduction

                                               i.     The MIDI, Inc. Sherlock PLFA Analysis System is designed to automate the process of analyzing soil sample extracted through the PLFA procedure.  Samples for the project are analyzed using a combination of the standard Sherlock PLFA analysis.  As an aid to quantitation, an internal standard (19:0 PLFA) is added at a specific concentration in the first step of the extraction procedure; this standard both determines the scaling for the data and is used to evaluate the extraction efficiency.  Once the extract is placed on the Gas Chromatograph (GC), the main Sherlock software manages the characterization of the compounds and their relative amounts.  After Sherlock creates sample information in relative amounts, the PLFA Tools add-on uses the internal standard to scale to absolute amounts, and adjusts for the amount of soil actually used, giving values in picomoles/gram.

b.     Sherlock GC Analysis 

                                               i.     The Sherlock PLFA Analysis System automatically names fatty acids by calculating an Equivalent Carbon Length (ECL) for each peak using a calibration mix processed with the batch, and comparing peaks to the peak naming table. (Sasser, 1990; Ford, 2019). The ECL method begins by assigning integral ECLs to the straight-chain fatty acids with C10 being 10.000, C11 being 11.000 and so on. A peak’s ECL is determined by a simple linear interpolation, comparing the peak’s Retention Time (RT) with that of the previous and following calibration compound. The formula is a standard interpolation: 𝐸𝐶𝐿𝑠 = 𝐸𝐶𝐿𝑝 + (𝐸𝐶𝐿𝑓 − 𝐸𝐶𝐿𝑝) × (𝑅𝑇𝑠 − 𝑅𝑇𝑝) ÷ (𝑅𝑇𝑓 − 𝑅𝑇𝑝) where once the ECL is calculated for a peak, the compound name is determined by a lookup in the peak naming table. For example, a compound such as 15:0 iso is described in the peak naming table as ECL 14.6170 ± 0.0200, so any peak whose ECL is calculated between 14.5970 and 14.6370 is assigned the name “15:0 iso”.  ECLs is the calculated ECL for the sample peak ECLp is the ECL for the previous calibration compound ECLf is the ECL for the following calibration compound RTs is the RT for the sample peakRTp is the RT for the previous calibration compound RTf is the RT for the following calibration compound

                                             ii.     While the Flame Ion Detector (FID) for a GC is a nearly universal detector, it operates by burning carbon and as such is selective: responding higher to compounds with more carbons. The Sherlock software automatically adjusts for this effect by calculating a response factor based on a quantitative calibration mix run with every batch.  For each quantitative compound in the calibration mix, a response factor is determined using the following formula:𝑅𝐹𝑖 = (𝐴𝑀𝑇𝑖 ÷ 𝑅𝑆𝑃𝑖) × ( 𝑆𝑢𝑚𝑅𝑆𝑃 ÷ 𝑆𝑢𝑚𝐴𝑀𝑇)

                                           iii.     Where the response factor for each compound is listed on the calibration report.  When a sample is processed, the responses for all compounds, those in the calibration mix and those not in the calibration mix, are multiplied by the response factor to yield an “amount” for that compound. Note that this is a relative weight amount, that is, it gives an amount for that compound which corresponds to its relative weight compared to the other compounds in the sample.

                                            iv.     For compounds that were part of the calibration mix, the multiplying response factor is obvious. For a sample compound that is not part of the calibration mix, the response factor for the compound is calculated by interpolating the immediately previous and the immediately following calibration compounds’ response factors, using the relative distance of the sample compound from the two calibration compounds.

                                             v.     As described above, the Sherlock software converts Retention Times (RT) to Equivalent Carbon Lengths (ECL) for peak identification; it also uses those units for interpolation calculation of response factor (though RT could be used with insignificant differences):RFi is the calculated response factor for compound i, AMTi is the actual amount of compound i in the calibration mix RSPi is the GCFID response (area) for the peak associated with compound i.  SumRSP is the sum of the responses of all calibration compounds in the calibration mix SumAMT is the sum of the actual amounts of all of the compounds in the calibration mix𝑅𝐹𝑠 = 𝑅𝐹𝑝 + (𝑅𝐹𝑓 − 𝑅𝐹𝑝) × (𝐸𝐶𝐿𝑠 − 𝐸𝐶𝐿𝑝) ÷ (𝐸𝐶𝐿𝑓 − 𝐸𝐶𝐿𝑝) where to discern that this formula is correct, note that if ECLs = ECLp then RFs = RFp; and if ECLs = ECLf then RFs = RFp + (RFf – RFp) thus RFp.

                                            vi.     The relative amount of each compound is then computed by simply multiplying its response by its response factor.RFs is the calculated response factor for the sample compound RFp is the response factor for the previous calibration compound RFf is the response factor for the following calibration compound ECLs is the ECL for the sample compound.  ECLp is the ECL for the previous compound.  ECLf is the ECL for the following compound

c.     Sherlock PLFA Tools Scaling

                                               i.     For each sample a list of named compounds with relative weight amounts. The PLFA Tools application converts those into absolute moles using the information available from the internal standard.  While the Buyer/Sasser PLFA method (Buyer, 2012) suggests adding 10,000 picomoles to each sample, for historical reasons the Microbial ID method adds 6,100 picomoles. The PLFA Tools software has a parameter that is set appropriately for this use.  There are two aspects to scaling a sample to absolute moles; first, the relative weights for each compound needs to be converted to relative moles; then the mole values need to be scaled to take into account the known amount of the internal standard and the weight of soil used in the original sample.  Because the internal standard used is 19:0, relative scaling is achieved using 1.000 as the scale for 19:0. Other compounds are scaled based on the ratio of the molecular weight of 19:0 to their molecular weight. For example, the 15:0 iso (FAME) has molecular weight 256.4 while 19:0 (FAME) has weight 312.5; thus the 15:0 iso is scaled by a factor of 312.5/256.4 or 1.219.  As stated above 6,100 picomoles of internal standard is added to each sample. Having scaled to relative moles, the sample is scaled so that the value for 19:0 will be 6,100. Now all values are in absolute picomoles. However, if an amount of soil other than 1.00 grams is used, that amount is recorded with the sample and induces a final scaling by 1/weight. In this manner the final result is in absolute picomoles per gram.  All of the above calculations are handled automatically by the PLFA Tools application, yielding a new data file with values in absolute picomoles per gram.

d.     Summary

                                               i.     Using the MIDI, Inc. Sherlock Chemical Analysis Software with PLFA Tools add-on allows the bulk of the analysis of data to be interpreted without human manipulation in the software.  An .RTF file and .CSV will be sent to each researcher after analysis.

References

1. Buyer, J., Sasser, M (2012). High throughput phospholipid fatty acid analysis of soils.

Applied Soil Ecology, 61, 127-130. https://doi.org/10.1016/j.apsoil.2012.06.005

1. Ford, W. F., Harmon, A. D., Tucker, A. O., Sasser, M., Jackoway, G., Albornoz, G., Grypa, R. D., Pratt, J. L., & Cardellina, J. H. (2019). Cinnamon – differentiation of four species by linking classical botany to an automated chromatographic authentication system.

Journal of AOAC International, 102 (2), 363-368. https://doi.org/10.5740/jaoacint.18-0343

1. Sasser, M. (1990)

Identification of bacteria by gas chromatography of cellular fatty acids; Technical Note # 101 for Microbial ID, Inc.: Newark, DE USA.

1. Soil PLFA and NLFA Testing. http://SoilPLFA.com