Soil Signature Lipid Biomarker Analysis

Introduction

Signature lipid biomarker (SLB) analysis can be employed to give detailed insights into soil microbial community structure. All living cells are surrounded by a plasma membrane, which is a barrier against the environment. The plasma membrane is constituted by a lipid bilayer, containing a diverse array of lipids, which are essential structural components of the cell membranes, comprising, among others, glycerolipids, sphingolipids, and sterols.

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Lipids can also serve as storage compounds (triacylglycerols) and participate in many biochemical processes such as signaling (jasmonic acid, lyso-phosphatidylcholine), protein modification (dolichol) and plant-microbe interactions (Siebers et al., 2016). They are defined according to their solubility properties, being insoluble in water, but soluble in non-aqueous solvents such as chloroform or alcohols. Lipids are characterized by a high structural diversity and therefore, suitable biomarkers to monitor adaptations to altered environmental conditions. Phospholipids are composed of two fatty acids esterified at sn-1 and sn-2 position to a glycerol backbone and a polar headgroup, which is attached at sn-3 position. After cell death, phospholipids are characterized by a rapid degradation into neutral lipids such as diacylglycerol (DAG) or into phosphatidic acid (PA) due to enzymatic hydrolysis. Hence, the amount of intact phospholipids in soil is highly correlated with the living microbial biomass in soil and can be employed as a quantitative measure of the viable biomass (White et al., 1993). The diacylglycerol moiety of PA is incorporated into different phospholipids by phosphate ester condensation. Distinct amino-alcohols [e.g. phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and phosphatidylinositol (PI)] can be attached to the phosphate moiety of the headgroups. The fact that the different headgroups can be combined with a large number of fatty acids, varying in chain length and degree of desaturation leads to a vast number of different phospholipid molecular species. In addition there are also bacteria that ubiquitously occur in soil having ether-linked glycerophospholipids as membrane lipids which are characterized by an ether-linked acyl chain at the sn-1 and sn-2 position of the glycerol backbone. These ether bond phospholipids are most prominent in Archaea (Albers et al., 2000) and are more resistant to oxidation and high temperatures than ester bonds. Since the fatty acid composition in phospholipids varies widely among different microorganisms, their distribution profiles can be employed reflecting the soil microbial community structure (White et al., 1993). Phospholipid-derived fatty acid (PLFA) analysis is a widely used method to measure the microbial biomass and the community structure composition (Buyer and Sasser, 2012). Advantages and disadvantages of PLFA analysis to elucidate soil microbial community structures were subject of several excellent publications (Frostegard et al., 2010; Zelles, 1999; Buyer and Sasser, 2012).

A tremendous increase in our knowledge of the role of lipids in plant-microbe-soil interactions has been achieved since the availability of highly sensitive and quantitative analytical technologies utilizing mass-spectrometry (MS), gas chromatography (GC), and high-pressure liquid chromatography (HPLC) (Wewer et al., 2011; Welti et al., 2004). To give a reliable community fingerprint of soil microorganisms, several analytical methods has to be assessed.

Lipid Extraction from Soil Samples

The described procedure for phospholipid extraction from soil is based on Bligh and Dyer (1959) with slight modification. To minimize contamination only glass- and teflon-ware is used. All solvents are HPLC or GC grade and contain 0.01% (w/v) butylated hydroxytoluene as antioxidant (Welti et al., 2002). Glassware is washed with chloroform prior to usage. For lipid extraction from soil, samples (fine earth <2 mm, ~5 g wet weight) are placed into a 50 mL glass vial with a teflon-lined screw cap. Total lipids are extracted in two steps. Lipid-extract A: 10 mL of chloroform/methanol/formic acid (1:1:0.1) is added to the sample and incubated under continuous shaking at 150 rpm for 1 h at room temperature. The presence of formic acid in the solvent prevents lipid degradation by lipases during lipid isolation (Browse et al., 1986). After centrifugation at 1500 x g for 10 min, the supernatant is transferred to another 50 mL glass vial with teflon-lined screw cap and evaporated under N2. Lipid-extract B: the residue of extraction A is re-extracted with 8 mL of chloroform/methanol (2:1). After incubation for 30 min at RT and subsequent centrifugation at 1500 x g for 10 min, the supernatant are combined with lipid extract A. 2 mL of 1 M KCl/0.2 M H3PO4 is added to the lipid extract and the mixture is shaken vigorously. Phase separation is achieved by centrifugation at 1500 x g for 10 min. The lower organic phase containing the total lipids is harvested completely evaporated and eluted in chloroform. Samples can be stored at -20 °C for later preparation or directly used. Finally, samples are dried for 48 h at 105 °C to determine the dry weight.

Lipid Fractionation by Normal-Phase SPE and FAME Analysis

A commonly used method to separate phospholipids from other lipids is solid phase extraction (SPE). The normal phase SPE typically involves a polar analyte, a mid- to nonpolar matrix (e.g. acetone, chloroform, or hexane), and a polar stationary phase. Silica is the sorbent of choice for lipid fractionation, since lipids are first recovered in nonpolar solvents such as chloroform. The retention of an analyte under normal phase conditions is primarily due to the interactions between the polar functional groups of the analyte (e.g. lipids) and the polar groups on the sorbent surface (e.g. silica), for example due to hydrogen bonding. A compound which is adsorped by these mechanisms is eluted by passing a solvent that disrupts the binding – usually a solvent that is more polar than the sample`s original matrix. Hence, the polar silanol groups at the surface of the silica sorbent will more strongly adsorb polar compounds such as phospholipids, rather than neutral lipids such as triacylglycerol or sterols. Several procedures to isolate distinct lipid classed by this method have been described (Hamilton and Comai, 1988; Kim and Salem, 1990).

For lipid fractionation by normal phase SPE, silica columns (e.g. Strata Si, 100 mg), are equilibrated with 2 x column volume of chloroform. The lipid-extracts are subsequently loaded onto the column and the non-polar lipids are eluted with 4 mL of chloroform. In a second step the more polar glycolipids are eluted with 4 mL of acetone/isopropanol [1:1]. Finally, the polar lipid fraction, including the phospholipids, is eluted by applying 4 mL of methanol. The methanol phase, which is highly enriched in phospholipids is then evaporated under N2 and dissolved in the respective solvent (e.g. chloroform) prior to further analysis. Direct analysis of phospholipids and free fatty acids via gas chromatography (GC) is impaired due to their high polarity. Therefore, derivatization to the more volatile fatty acids methyl esters (FAMEs) of lipids and fatty acids by acid-catalyzed methylation is necessary for the analysis by GC. FAME synthesis can be done according to Browse et al. (1986). On this account ~150 μL lipid samples is incubated at 80 °C for 1 h in 1 mL 1 N HCl in methanol. After cooling down to RT, 1 mL hexane and 1 mL 0.9 % NaCl is added and vortexed thoroughly. To achieve phase separation samples are centrifuged for 3 min at 1000 x g. The upper hexane phase, containing the FAMEs, is completely evaporated under N2. Subsequently, FAMEs are dissolved in a defined volume of hexane. The quantification of fatty acids by GC depends on the availability of suitable internal standards, which must be absent in the sample. Therefore, a defined amount of an internal standard (e.g. pentadecanoid acid, 15:0) is added to the sample.

TLC of Phospholipids

For the PLFA analysis by GC the extracted and separated phospholipids are methanolyzed and the acyl groups are converted into their methyl esters after cleavage. In this approach only the acyl groups but not the headgroups can be analyzed. The GC-based methods are unable to provide information about the different molecular species of phospholipids. To this end the separation by thin layer chromatography (TLC) prior to FAME synthesis is required (Wu et al., 1994). On this account silica gel is most commonly used combined with different solvent systems (e.g. acetone/toluene/water [91:30:8]) (Kahn and Williams, 1977). Once separated, the acyl groups can be quantified within the different phospholipid classes by isolating individual lipids from the silica material (Benning and Somerville, 1992).

For TLC, silica gel plates and a suitable mobile phase can be used as a simple method for the separation of the different phospholipid classes. The one-dimensional TLC system described here is a commonly used method to separate the different phospholipid classes extracted from soil. First, TLC plates are submerged in 0.15 M ammonium sulfate and dried at RT for several days before activation at 120 °C for 2.5 h prior to use. Lipids, eluted in chloroform/methanol (2:1) are loaded onto the plate as 1 or 2 cm parallel streaks in the concentration zone. Plates are short air-dried in a fume hood and subsequently placed in the chromatography tank containing acetone/toluene/water [91:30:8]. After ~45 to 60 min migration time the plates are dried in a fume hood and bands are visualized using different staining methods. The lipids are identified with the help of co-migrating standards.

Two-dimensional (2D) TLC using two different solvent systems is performed to further separate lipid extracts with complex lipid compositions. On this account, the lipid extract is applied to the lower left corner of the silica plate, without concentration zone. After developing the first dimension the plate is air dried in a fume hood and placed in a second chromatography chamber containing a different solvent for the development of the second direction. The most efficient solvent system is to use a neutral or basic solvent in the first direction followed by an acidic solvent in the second direction, e.g. chloroform/methanol/conc ammonia 65:35:4 for the first dimension and butanol/acetic acid/water (60:20:20) for the second dimension. The main disadvantages of this method are the time-consuming double development and that no standard sample can be used to appreciate the variations, which may occur from one run to another. Lipids are visualized by spraying with p-anisaldehyde (ANS, 0.2 % (w/v) 8-anilino-1-naphtalene sulfonic acid in Methanol), which is a general-purpose stain, particularly good with groups having nucleophilic properties. ANS is also particularly good for preparative TLC, because it does not interfere with subsequent analyses by GC or mass spectrometry (MS).

Lipid Analysis by Direct Infusion Nanospray Q-TOF MS

Quadrupole Time-of-Flight Mass Spectrometry (Q-TOF MS) represents a method for the non-destructive identification and quantification of intact lipids such as phospholipids. The analysis by mass spectrometry requires the ionization of the molecules by an ion source. On this account the lipids are directly infused into the Q-TOF through an infusion chip (HPLC-chip), which is supplied with a nano-capillary. The molecules are ionized by nanospray ionization. Subsequently, the ions are transferred into the mass analyzer and separated according to their m/z in the quadrupole. The ions of a specific m/z value are selected and characteristic fragment ions of the molecular ions are generated by collision induces dissociation in the MS/MS mode. In the time-of-flight analyzer ions are accelerated, separated, and finally detected at the photomultiplier plate. The quantification of the different lipids is based on precursor ion or neutral loss scanning in relation to internal standards of known concentrations (Gasulla et al., 2013). For each lipid class at least two different internal standards should be selected, which are absent from the sample, enabling a very precise and reliable lipid quantification. The Q-TOF MS/MS based analysis of lipids extracted from soil samples offers several advantages over the GC-based analysis as reviewed by Kruse et al. (2015).

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