Ester-linked polar lipid fatty acid pro®les of soil microbialcommunities: a comparison of extraction methods and evaluation

of interference from humic acids

Pernille Nielsen, Sùren O. Petersen*

Danish Institute of Agricultural Sciences, Department of Crop Physiology and Soil Science, P.O. Box 50, DK-8830 Tjele, Denmark

Received 7 July 1999; received in revised form 23 December 1999; accepted 21 February 2000

Abstract

Analyses of polar lipid fatty acids isolated from soil are frequently used for characterization of microbial communities, andany interference from fatty acids derived from dead organic material is assumed to be negligible. We studied the initialextraction of lipid material from eight di�erent soils and from puri®ed humic acids using four di�erent combinations of solvent

(chloroform or dichloromethane), methanol and bu�er (potassium phosphate, pH 7.4 or sodium citrate, pH 4). The quantitativeyields of polar lipid fatty acids (PLFA) and PLFA composition of soils and humic acids were compared with absorbance spectra(200±850 nm) of lipid extracts for evaluation of extraction e�ciency and potential interference. Chloroform + citrate bu�ergenerally gave the highest, and dichloromethane + phosphate bu�er the lowest PLFA yields, and it was estimated that <20%

of the yield di�erence between extraction methods could be explained by interference from humic acids. Principal componentanalyses of PLFA composition suggested an e�ect of extraction method for several soils, but when all soils were analyzedtogether the di�erences between soils were much more important than the choice of extraction method. Co-extraction of lipids

from living cells during preparation of humic acids was quanti®ed and, correcting for this, it was estimated that the interferencefrom non-microbial sources in PLFA analyses was probably not more than 5±10% with the extraction methodsemployed. 7 2000 Elsevier Science Ltd. All rights reserved.

Keywords: Phospholipid fatty acids; Extraction e�ciency; Phosphate; Citrate; Humic acid; PCA

1. Introduction

Fatty acid analyses of microorganisms are exten-sively used in studies of microbial ecology. The iso-lation of lipid material is typically achieved bymodi®cations of the single-phase extraction describedby Bligh and Dyer (1959) and introduced to environ-mental research by White and co-workers (King et al.,1977; White et al., 1979). The extraction mixture con-tains chloroform, methanol and an aqueous phase inproportions to give a single phase, which probably

improves the contact between extractant and cells incomplex matrices like soil or sediment compared toprocedures with separate aqueous and organic phases.Also, the inclusion of alcohol is crucial for the dissol-ution of the polar lipids in cell membranes (Kates,1986).

Brinch-Iversen and King (1990) proposed to usedichloromethane as an alternative to the more hazar-dous chloroform. This modi®cation was combinedwith the use of a strong sodium bromide solutionduring phase separation, which results in phase inver-sion with physical separation of the organic solventphase from sample residue following centrifugation.The composition of the aqueous phase was investi-gated by FrostegaÊ rd et al. (1991), who found a signi®-cant e�ect of bu�er type for an acid forest soil with a

Soil Biology & Biochemistry 32 (2000) 1241±1249

0038-0717/00/$ - see front matter 7 2000 Elsevier Science Ltd. All rights reserved.

PII: S0038-0717(00 )00041 -9

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* Corresponding author. Tel.: +45-8999-1723; fax: +45-8999-

1619.

E-mail address: soren.o.petersen@agrsci.dk (S.O. Petersen).

high organic matter content, but not for a neutral ara-ble soil.

Lipid extracts of environmental samples are typicallyyellow to brownish in color. The color intensity isapparently related to the amount of organic detritus inthe sample and presumably results from co-extractionof humic materials. Humic substances may containester-linked long-chain fatty acids (e.g., Schnitzer andNeyroud, 1975) and therefore represent a potentialsource of error in the characterization of living mi-crobial communities through fatty acid ®ngerprints.Higher yields of polar lipid fatty acids (PLFA) aretypically accompanied by higher intensity of color inthe crude lipid extract and, although microbial bio-mass has been shown to correlate with organic mattercontent (Anderson and Domsch, 1989), the yieldincrease could be partly derived from non-biomasssources.

Our study was designed to characterize and quantifythe interference from humic acids on ester-linkedPLFA pro®les of microbial communities when usingdi�erent lipid extraction methods. The study involvedeight cultivated or natural soils, humic acids (HA) iso-lated from selected soils as well as a HA reference de-rived from soil, and four di�erent versions of thesingle-phase extraction procedure.

2. Materials and methods

2.1. Reagents and glassware

Reagent bottles and 35 ml extraction tubes wereacid washed (10% HCl) and rinsed three times in deio-nized water. All other glassware was ignited at 5308Cfor 3 h. All solvents were LiCroSolv grade fromMerck (Darmstadt, Germany), chemicals were analyti-cal grade. Fatty acid methyl ester standards wereobtained from NuChekPrep (Elysian, MN).

2.2. Soils

The soils we used are characterized in Table 1. The

three arable soils (A1±A3), one heath soil (H) andfour forest soils (F1±F4) were sampled to 15 cmdepth, the forest soils after removal of the litter layer.Two arable soils (A2 and A3) were from di�erent®elds of the same location, and two of the forest soils(F1 and F2) were from deciduous forest areas, whileF3 and F4 were from coniferous forests. The organicmatter content ranged from 2.3 to 13.4% of the soildry wt. and the pHCaCl2

from 2.9 to 5.8. All soils weresieved (mesh size, 2 mm) and stored at 28C until used.Approximately 1 g (arable soils) or 0.5 g (natural soils)subsamples were used for lipid extractions.

2.3. Humic acids

Ha were prepared from two of the soils collected forthis study (A1 and F1) by a procedure modi®ed fromSwift (1996). Field moist soil was extracted in 0.1 MNaOH (NaOH:soil ratio 10:1, v/w) for 4 h under N2

and then left to settle overnight. After centrifugation(1890 � g for 10 min) the supernatant was decanted toa new centrifuge tube via a funnel with a plug of glass-wool. Following adjustment of the pH to 1.0 with 6 MHCl the extract was left for 12±16 h; precipitated HAwere concentrated by centrifugation. The HA was pur-i®ed twice by dissolution in NaOH and re-precipitationwith acid. This was followed by washing in deionizedwater and freeze-drying. The ®nal yield of HA corre-sponded to 30 and 20% of the soil organic matter forsoils A1 and F1, respectively.

A reference soil HA (Code 1S102H) was obtainedfrom the International Humic Substances Society(IHSS), which had been isolated by the proceduredescribed by Swift (1996). It was similar to the oneused in this study but with a more extensive puri®-cation of the precipitate to remove inorganic material.Lipid extractions were performed with 50±100 mg HA.

The ash contents of HA from the di�erent sourceswere as follows: Soil A1, 41%; Soil F1, 33%; IHSS,1%. In the following, yields of fatty acids in HA arecorrected for ash content.

Table 1

Characteristics of soils used in the present study

Key Soil type Clay (%) Organic matter (%) Total C (%) Total N (%) pHCaCl2Total CEC (meq 100 gÿ1)

A1 Arable 6.3 3.0 1.4 0.10 4.8 6.7

A2 Arable 8.7 4.1 1.9 0.17 5.8 9.8

A3 Arable 8.7 4.7 2.0 0.15 5.4 10.3

H1 Heath 4.1 2.3 0.8 0.04 3.5 4.9

F1 Forest, deciduous 5.2 7.1 3.4 0.20 4.7 11.8

F2 Forest, deciduous 5.3 9.0 3.9 0.18 3.3 9.9

F3 Forest, coniferous 9 7.1 3.3 0.19 3.6 14.8

F4 Forest, coniferous 3.7 13.4 6.7 0.17 2.9 20.4

P. Nielsen, S.O. Petersen / Soil Biology & Biochemistry 32 (2000) 1241±12491242

2.4. Lipid extraction

The extraction mixture recommended by White etal. (1979) for environmental samples consists ofchloroform, methanol and phosphate bu�er (50 mMK2HPO4, pH 7.4). FrostegaÊ rd et al. (1991) observedthat higher lipid yields were obtained from an acid soilwith a high organic matter content by replacing theneutral phosphate bu�er by an acid citrate bu�er (150mM Na3C6H5O7�2H2O, pH 4.0).

In our study we combined the use of chloroform (C)or dichloromethane (D) for lipid extraction with eitherphosphate or citrate bu�er; three of these four combi-nations have been used in published studies. Extrac-tions with dichloromethane employed sodium bromidefor the phase separation (see Petersen and Klug, 1994for details), while the separation of chloroform extractswas as described by FrostegaÊ rd et al. (1991). The fourextraction methods will be referred to as Dphos, Dcit,Cphos and Ccit:

Lipid extractions for PLFA analyses were performedon all soils and HA materials. The di�erent crude lipidextracts were dried under N2 and then exposed to thesame procedure, i.e., isolation of polar lipids (mainlyphospholipids) by solid phase extraction (100 mg silicicacid with [60 AÊ ] pore size; Varian, Harbor City, CA),mild alkaline transmethylation, and extraction ofmethyl esters with n-hexane (Dowling et al., 1986). Aninternal standard (IS), nonadecanoate methyl ester,was added during transmethylation. Fatty acid methylesters were analyzed by capillary gas chromatographywith settings as described by FrostegaÊ rd et al. (1993).A total of 38 fatty acids (see Table 4) were identi®edby a combination of retention times and parallel analy-sis of samples on a similar set-up with MS interface;unidenti®ed compounds were not used for the dataanalysis.

2.5. Absorption spectra

For both soils and HA, the absorption character-istics in the ultraviolet (200±400 nm) and visible range(400±800 nm) were determined for crude lipid extracts(one replicate only), as well as for the methanol frac-tion collected in the solid phase extraction of polarlipids. A Spectronic 2000 spectrophotometer (Bausch& Lomb, Rochester, NY) and a quartz cuvette(Hellma, MuÈ llheim, Germany) with a 1 cm light pathwas used. The absorbance at 400 nm was taken as aquantitative index of the concentration of humic sub-stances (Khan and Schnitzer, 1978; Sorouradin et al.,1993) after normalization with respect to the amountsof soil extracted and solvent used. For unknownreasons a few samples were slightly opaque, andabsorption spectra could not be obtained.

2.6. Co-extraction of microbial cells

Lipids originating from living organisms could bepartly included in the humic acid fraction isolated. Toinvestigate the potential for carry-over of cellular lipidmaterial during HA preparation, a control experimentwas carried out with a culture identi®ed as Rhodococ-cus erythropolis on the basis of morphology, mycolicacid composition, 16S rDNA analysis and fatty acidcomposition (Dr L. Elsgaard, pers. comm.); the fattyacids of Rhodococci are dominated by saturated andmonounsaturated fatty acids and, notably, tuberculos-tearic acid (10Me18:0) (Barton et al., 1989). Cells wereharvested by centrifugation (15,000 � g for 10 min)and washed once in autoclaved phosphate bu�er.

HA preparation + PLFA analysis was carried outfor (1) ca. 0.5 g dry wt. soil F1, and (2) ca. 0.5 g drywt. soil F1 + 100±200 mg fresh wt. cell material;exact weights were recorded. PLFA analysis was alsocarried out directly on 100±200 mg fresh wt. cell ma-terial and the seven dominant fatty acids of R. erythro-polis were used for calculation of recovery:

Recovery, FAj

���

HA-derived FAj

�soil � cells

ÿ �HA-derived FAj

�soil

��FAj

�cells

� 100%,

�1�where FAj is fatty acid j, The experiment was carriedout with Dphos and Ccit extraction procedures, respect-ively.

2.7. Statistics

All measurements were carried out in duplicate ortriplicate, except where indicated. The total amountsof PLFA extracted from soils or HA were calculatedfrom the IS. The yields with di�erent extractionmethods were compared by Tukey's multiple compari-sons test for each soil. The fatty acid compositionswere used for principal component analyses (PCA) fol-lowing log(n + 1) transformation of mole percentages;FrostegaÊ rd et al. (1997) found that the error involvedin neglecting the constraints associated with compo-sitional data (percentage distributions) were not im-portant with PLFA data due to the high number ofvariables. PCA analyses including all four extractionmethods were performed on each soil separately, aswell as on all eight soils combined. Statistical analyseswere carried out with SAS 6.11 for Windows (SASInst., 1998).

P. Nielsen, S.O. Petersen / Soil Biology & Biochemistry 32 (2000) 1241±1249 1243

3. Results

3.1. Quantitative yields

The four di�erent combinations of extraction sol-vent and bu�er Ð Dphos, Dcit, Cphos and Ccit Ð gavethe yields of PLFA listed in Table 2. Except for A2,i.e., the soil with the highest pH, yields with Ccit werealways signi®cantly higher than with Dphos, which con-stituted between 58 and 77% of the yield with Ccit.The other two combinations, Dcit and Cphos, gave in-termediate yields. Chloroform tended to give higheryields than dichloromethane, particularly with the for-est soils. When comparing extractions with dichloro-methane or chloroform separately, there were onlyminor e�ects of bu�er type.

Polar lipid ester-linked fatty acid yields of HA ma-terials with Dphos and Ccit extraction methods areshown in Table 3. The two HA materials produced inthis study gave much higher yields of PLFA than thereference HA. The di�erence between Dphos and Ccit

extraction methods was apparently similar to that

observed for the soils, although no replication wasgenerally included with these analyses.

3.2. Absorbance spectra

The absorbance spectra of non-fractionated soillipid extracts are exempli®ed in Fig. 1A showingabsorbances with two extraction methods, Dphos andCcit, for soils A1 and F1. The absorbances increasedwith decreasing wave length, except that most curveshad shoulders at 400±450 nm and at 550±600 nm.Spectra of the polar lipid (methanol) fraction from

Table 2

Concentrations of PLFA in eight soils of di�erent origin, as deter-

mined using four di�erent extraction methods, Dphos (DCM + phos-

phate bu�er), Dcit (DCM + citrate bu�er), Cphos (chloroform +

phosphate bu�er) and Ccit (chloroform + citrate bu�er)a

Key Soil type Lipid extraction method

(nmol PLFA gÿ1 dry wt. soil)

Dphos Dcit Cphos Ccit

A1 Arable 27.6 c 33.5 bc 35.4 b 42.5 a

A2 Arable 33.9 a 42.9 a 43.7 a 48.1 a

A3 Arable 25.2 c 33.1 cb 42.0 ab 43.0 a

H1 Heath 16.7 b 22.0 ab 22.4 a 23.6 a

F1 Forest, deciduous 123.1 c 125.3 c 140.7 b 159.8 a

F2 Forest, deciduous 102.1 b 104.2 ab 152.5 ab 145.9 a

F3 Forest, coniferous 68.9 b 81.3 b 119.4 a 108.7 a

F4 Forest, coniferous 51.4 b 54.0 b 85.6 ab 74.6 a

a Di�erences between extraction methods are indicated by di�erent

letters behind the mean (n = 2).

Table 3

Concentrations of ester-linked fatty acids in humic acids (HA)

extracted from soils F1 and A1 (see Table 1) and from three com-

mercial HA materials, as determined with two di�erent extraction

methods. Dphos: extraction with dichloromethane and phosphate buf-

fer, Ccit: extraction with chloroform and citrate bu�er

HA source Dphos Ccit

(nmol gÿ1 ash-free dry wt)

Forest soil (F1) 292a 375

Arable soil (A1) 152 201

Standard soil (IHSS) 41 (3) 66 (23)

a No replication, except where indicated.

Fig. 1. Absorption spectra of lipid extracts based on soil (A) and

HA (B). The concentration of HA was quanti®ed on the basis of

abs400. Key to curves (soil types, see Table 1): 1. Soil A1, Ccit; 2. Soil

F1, Ccit; 3. Soil F1, Cphos; 4. Soil A1, Cphos; 5 and 6. Reagent blanks;

7. HA from soil F1, Ccit; 8. HA from soil A1, Ccit; 9. HA from

brown coal (should be disregarded), Ccit; 10 and 11. Reagent blanks.

P. Nielsen, S.O. Petersen / Soil Biology & Biochemistry 32 (2000) 1241±12491244

solid phase extraction were qualitatively similar,although weaker in absorbance (data not shown).

Absorbance spectra of lipid extracts of HA preparedfrom soils A1 and F1 are shown in Fig. 1B (thesmooth curve, which was obtained for a HA derivedfrom brown coal, should be disregarded). Lipidextracts of soil-derived HA had shoulders similar tothe whole-soil lipid extracts.

The absorbance at 400 nm was recorded as a quanti-tative index of humic material in the extracts. For allsoils there was consistently less absorbance in extrac-tions with phosphate than with citrate bu�er (data notshown). The abs400 readings in crude lipid extracts andin the polar lipid fraction were highly correlated (seeFig. 2). There was no correlation between soil PLFAconcentrations (Table 2) and absorbances in the crudelipid extracts (P> 0.10, n = 32), while the correlationbetween whole-soil PLFA and abs400 of the polar lipidfraction was signi®cant (P<0.025), although relativelyweak (r = 0.42). Hence, solid phase extractionremoved a pool of refractive material that was less clo-sely related to PLFA than the pool which was col-lected in the polar lipid fraction.

3.3. Fatty acid composition

The percentage distribution of fatty acids identi®edin PLFA analyses of soils A1 and F1 are shown inTable 4 for Dphos and Ccit, i.e., the two extractionmethods that were quantitatively most dissimilar. Thefatty acid composition was generally similar for the

two extraction methods, the most notable di�erencesbeing a relatively higher concentration of 16:0 andlower concentration of 18:1o7 when extracted withCcit, particularly in soil A1, and a relatively higherconcentration of cyclopropyl fatty acids with Dphos.

A PCA analysis was carried out for each soil separ-ately, which included the transformed mole percentagedistributions from all extraction methods, and furtheran analysis was done with all soils and extractionmethods combined. When soils were analyzed indivi-dually, there were indications of qualitative di�erencesbetween extraction methods for six of the eight soils,

Table 4

Mole percentage distribution of phospholipid fatty acids from two

soils, as determined using two di�erent extraction methods, Dphos

(dichloromethane and phosphate bu�er) or Ccit (chloroform and

citrate bu�er)a

Fatty acid Soil A1 (arable) Soil F1 (forest, deciduous)

Dphos Ccit Dphos Ccit

i14:0 0.65 (0.06) 0.60 (0.05) 0.66 (0.00) 0.65 (0.03)

14:0 2.07 (0.12) 1.91 (0.43) 1.18 (0.11) 1.31 (0.03)

i15:0 7.78 (0.42) 7.77 (0.12) 7.61 (0.19) 7.93 (0.27)

a15:0 4.77 (0.24) 4.31 (0.06) 4.72 (0.11) 4.48 (0.13)

15:0 0.89 (0.02) 0.99 (0.05) 0.59 (0.02) 0.74 (0.03)

br16:0 0.60 (0.03) 0.50 (0.01) 0.16 (0.00) 0.14 (0.00)

14:0 2OH 1.29 (0.25) 0.73 (0.19) 0.36 (0.02) 0.29 (0.02)

i16:0 2.10 (0.08) 2.69 (0.08) 1.98 (0.04) 3.31 (0.11)

16:1 0.67 (0.12) 0.57 (0.06) 0.60 (0.03) 0.59 (0.01)

16:1o7c 5.91 (0.64) 5.24 (0.42) 5.71 (0.18) 5.94 (0.07)

16:1o7t 0.36 (0.04) 0.33 (0.02) 0.64 (0.06) 0.85 (0.02)

16:1o5c 2.40 (0.20) 2.31 (0.23) 3.70 (0.11) 3.72 (0.10)

16:0 16.71 (0.43) 22.36 (0.98) 12.11 (0.32) 12.89 (0.05)

br17:0 0.28 (0.01) 0.30 (0.01) 0.16 (0.01) 0.21 (0.01)

10Me16:0 2.72 (0.27) 2.37 (0.17) 1.93 (0.09) 2.01 (0.05)

i17:1 4.49 (0.05) 3.75 (0.61) 4.08 (0.12) 3.85 (0.35)

i17:0 1.64 (0.07) 1.71 (0.06) 1.83 (0.07) 1.90 (0.09)

a17:0 2.18 (0.09) 2.11 (0.44) 1.31 (0.01) 1.38 (0.02)

17:1o8 0.66 (0.09) 0.68 (0.01) 0.39 (0.02) 0.40 (0.01)

cy17:0 3.86 (0.15) 3.19 (0.14) 2.96 (0.13) 2.60 (0.06)

17:0 0.52 (0.02) 0.62 (0.05) 0.46 (0.07) 0.51 (0.03)

br18:0 0.85 (0.02) 0.92 (0.05) 0.69 (0.00) 0.92 (0.02)

10Me17:0 0.90 (0.14) 0.97 (0.05) 0.69 (0.01) 0.85 (0.02)

18:3 0.95 (0.08) 1.05 (0.47) 3.19 (0.03) 2.69 (0.01)

18:2o6c 2.02 (0.48) 2.00 (0.28) 2.47 (0.70) 2.89 (0.46)

18:1o9c 6.27 (0.52) 5.69 (0.38) 6.06 (0.34) 5.91 (0.23)

18:1o7 8.24 (0.58) 6.75 (0.28) 13.60 (0.31) 12.99 (0.06)

18:1o5c 0.96 (0.16) 0.95 (0.05) 0.59 (0.03) 1.11 (0.03)

18:0 3.22 (0.26) 3.59 (0.10) 2.76 (0.45) 2.63 (0.14)

19:1 0.82 (0.06) 0.76 (0.08) 0.96 (0.06) 0.84 (0.04)

10Me18:0 2.15 (0.22) 2.44 (0.16) 1.86 (0.07) 2.18 (0.07)

cy19:0 5.43 (0.17) 4.17 (0.27) 8.42 (0.29) 6.76 (0.11)

20:4 0.22 (0.09) 0.25 (0.05) 0.31 (0.02) 0.48 (0.05)

20:3 0.08 (0.14) 0.29 (0.01) NDb 0.17 (0.04)

20:1 2.36 (0.55) 1.50 (0.50) 2.82 (0.39) 1.31 (0.23)

20:0 1.36 (0.09) 1.41 (0.09) 0.88 (0.02) 0.92 (0.11)

22:6 0.07 (0.12) 0.14 (0.24) NDb NDb

22:0 1.59 (0.16) 2.07 (0.08) 1.51 (0.03) 1.63 (0.05)

a Numbers indicate mean2S.D. (n = 3).b ND: not detected.

Fig. 2. The absorbance at 400 nm of crude lipid extracts and the

polar lipid fraction (isolated by solid phase extraction) was highly

correlated.

P. Nielsen, S.O. Petersen / Soil Biology & Biochemistry 32 (2000) 1241±1249 1245

i.e., distances between replicates in the score plots ofPC1 and PC2 were smaller than distances betweenextraction methods (data not shown). The di�erenceswere more pronounced with forest soils and particu-larly depended on the choice of extraction solvent(dichloromethane or chloroform). Hence, these obser-vations corroborate the trends observed with respectto quantitative yields (Table 2).

When all soils were included in one PCA (see Fig. 3),almost identical patterns in the distribution of soilswere obtained with the four extraction methods, show-ing that di�erences between soils were much more im-portant than any di�erences between extractionmethods. In Fig. 3, scores along PC1 and PC2 of eachextraction method are presented in separate plots forclari®cation, but all data were derived from the samePCA.

The composition of ester-linked fatty acids in thepolar lipid fraction of HA from soils A1 and F1 werecompared to that of parent soils in a separate PCAanalysis, and both Dphos and Ccit extraction data wereincluded. The results are presented in Fig. 4 and,although the HA data were generally not replicated,some interesting trends were suggested; the plot isscaled in proportion to the fractions of the total varia-bility explained by PC 1 (48.5%) and PC 2 (23.0%).Firstly, the di�erences in PLFA patterns between iso-lated HA and the corresponding soil explained moreof the variation than did di�erences between soils. Sec-

ondly, the results suggest that the choice of extractionmethod (in Fig. 4 indicated by di�erent ®ll colorwithin a group) was more critical for the HA fractionthan for the whole-soil samples, i.e., that choice ofextraction method was relatively more important forthe yield of fatty acids from the HA fraction.

3.4. Interference from cellular lipids

A bacterial culture was added to soil and the mix-ture carried through HA extraction followed by PLFApreparation, along with unamended soil samples. Forseven selected fatty acids, the enrichment due to thecell material was determined and compared to the con-centrations of these fatty acids in PLFA analyses ofthe cell material (Table 5). The experiment was carriedout with both Dphos and Ccit extraction methods. Theco-extraction of cellular PLFA averaged ca. 2% withboth extraction methods, with some variation for indi-vidual fatty acids.

In this control experiment HA was extracted from

Fig. 4. Score plot of a PCA analysis including soils A1 and F1 and

the corresponding HA materials. The lighter colour within a group

indicates that Dphos was used, the darker color that Ccit was used for

the extraction of lipids.

Table 5

Recovery of PLFA from a bacterial pure culture after HA extrac-

tion. The cell material was added to soil F1 and carried through HA

extraction and PLFA analysis; the enrichment relative to soil F1

alone was then calculated and expressed as percentages of PLFA

concentrations in the cell material when analyzed directly

Fatty acid Recovery of cellular lipids (%)

Dphos Ccit

14:0 1.0 (0.2) 0.9 (0.0)

16:1o7c 3.2 (0.8) 1.5 (0.2)

16:0 2.4 (0.0) 2.8 (0.7)

18:1o9c 5.0 (1.1) 1.8 (0.3)

18:1o7 2.6 (0.0) 4.3 (0.1)

18:0 0.4 (0.0) 3.4 (2.3)

10Me18:0 1.0 (0.1) 1.2 (0.3)

All seven fatty acids 2.1 (0.2) 1.8 (0.4)

Fig. 3. A principal component analysis was carried out based on the

mole percentage distribution of polar lipid fatty acids in all eight

soils with all four extraction methods. The scores along the ®rst two

principal components, explaining 51% of the total variability, are

presented; results for each extraction method are shown separately

for clari®cation.

P. Nielsen, S.O. Petersen / Soil Biology & Biochemistry 32 (2000) 1241±12491246

known quantities of soil F1, and therefore the concen-trations of PLFA in HA (without cell material) couldbe compared with the PLFA concentrations of the soilwhen analyzed directly. These results are shown inTable 6 for seven selected fatty acids; when all 38 fattyacids were considered, the HA-derived fatty acids con-stituted 4.0 2 0.7% and 2.9 2 0.3% of whole-soilPLFA with Dphos and Ccit, respectively.

4. Discussion

The main objective of our experiments was to esti-mate the interference from HA in pro®les of ester-linked polar lipid fatty acids, which are assumed tocome primarily from the membranes of living organ-isms (Tunlid and White, 1992). The material isolatedin the HA fraction does not represent soil organic mat-ter in general, but it does represent an empiricallywell-de®ned mixture of organic compounds of rela-tively high complexity (Piccolo et al., 1990).

Schnitzer and Neyroud (1975) found that up to 10%of humic materials could be accounted for by fattyacids, albeit only after exhaustive extraction steps.They proposed that both physical adsorption andchemical binding via ester-linkages was involved in thestabilization. In general the degradation of lipids isretarded with increasing acidity (Dinel et al., 1990),and this increase is mainly accounted for by polymer-ized polar lipids of high complexity (Moucawi et al.,1981). Together these observations suggest that there isa potential for interference from soil organic matter inlipid analyses of microbial communities, and that thisinterference could be accentuated with decreasing pH.

The group of lipids considered in this study, i.e.,14C±20C fatty acids, is generally not persistent in thesoil environment (Dinel et al., 1990) and would prob-ably be rapidly oxidized if not protected by interactionwith other soil constituents. The di�erences in quanti-

tative yields between the four fairly mild extractionmethods used in our study (Table 2) indicate thatmicroorganisms and, conceivably, their decompositionproducts can indeed be physically protected in the soilenvironment. The PLFA composition of soil and HAmaterial (Fig. 4) was more dissimilar with the Ccit

extraction method than with Dphos, which suggeststhat Ccit extracted a pool of material that was notaccessible with Dphos.

The di�erences in PLFA yield observed betweenextraction methods support the observations made byFrostegaÊ rd et al. (1991) who compared an organic soil(79% organic matter, pH 4.1) with an arable soil(4.7% organic matter, pH 7.8). They found that thephosphate bu�er gave 30% lower yields of PLFA thanthe citrate bu�er with the organic soil, but not withthe arable soil. In our study the only soil where no sig-ni®cant di�erence was observed was the one with thehighest pH. However, FrostegaÊ rd et al. (1991) alsonoted that yields were not exclusively a re¯ection ofpH, since an acetate bu�er at pH 4 was not e�ective,and this was attributed to the higher concentration ofsodium ions introduced with the trivalent citrate ion.In our study, where all four combinations of solventand bu�er were compared, the organic solvent,dichloromethane or chloroform, seemed to be moreimportant than bu�er type for the di�erence in yields.

With respect to PLFA composition, FrostegaÊ rd etal. (1991) did not ®nd any discrimination as a functionof bu�er choice. But fatty acids in humic and fulvicacids are dominated by 14C±34C fatty acids (Schnitzerand Schulten, 1989) and partly of microbial origin,and any interference from soil organic matter maytherefore be di�cult to detect on the basis of fattyacid composition. In this study we also found onlyminor qualitative di�erences between the four extrac-tion methods used, as revealed by PCA analyses(Fig. 3). The higher proportion of cyclopropyl fattyacids with Dphos (Table 4) is in accordance with theobservation by Khan and Schnitzer (1972) thatbranched-cyclic fatty acid were more easily removedfrom HA than straight-chain fatty acids, since the lesse�cient extractant would then be enriched with thesefatty acids. The absorbance spectra (Fig. 1) had dis-tinct shoulders that may have been derived from so-called P type humic acids (Kumada, 1987). The quali-tative similarity between absorption spectra of intactsoils (Fig. 1A) and soil HA (Fig. 1B) suggests that thecolour of the whole-soil lipid extracts was in fact dueto co-extraction of HA-like material, although theharsh conditions of HA extraction may lead to chemi-cal changes (Stevenson, 1994; Christie, 1982) that com-plicate the use of HA characteristics for extrapolationto organic material in the undisturbed soil.

When comparing Dphos and Ccit extraction methods,Ccit had higher PLFA yields but, as indicated by the

Table 6

Yields of selected PLFA in the HA fraction isolated from soil F1,

expressed as percentages of the yields obtained with PLFA analysis

of soil F1 directly

Fatty acid Whole-soil fatty acids recovered in HA

fraction (%)

Dphos Ccit

14:0 6.6 (3.1) 5.0 (1.7)

16:1o7c 3.7 (0.6) 2.3 (0.2)

16:0 4.7 (0.6) 3.2 (0.3)

18:1o9c 2.4 (0.2) 1.6 (0.2)

18:1o7 2.3 (0.3) 1.9 (0.1)

18:0 8.3 (0.5) 6.0 (0.6)

10Me18:0 2.4 (0.3) 2.2 (0.4)

All seven fatty acids 3.7 (0.4) 2.6 (0.3)

P. Nielsen, S.O. Petersen / Soil Biology & Biochemistry 32 (2000) 1241±1249 1247

absorbance readings, also more HA. And as shownabove (Table 3) HA contains ester-linked fatty acids.Accordingly, HA can account for some of the di�er-ence in whole-soil PLFA yield Ð but how much? Thecontribution from HA to the di�erence was ®rst esti-mated for soils A1 and F1 by comparing the ratiobetween DPLFA and Dabs400, i.e.:

DPLFA

Dabs400���

PLFACcit

�ÿ �PLFADphos

��ÿabs400, Ccit

ÿabs400, Dphos

�,

�2�

for HA and soil, respectively. The ratio for HA consti-tuted between 8 and 17% of the ratio for soil, bothwhen crude lipid extracts and when methanol fractionswere considered (data not shown). Assuming theabsorbance of the soil extracts was due to HA alone,only 8±17% of the di�erence in PLFA yield betweenCcit and Dphos could therefore be accounted for byfatty acids derived from HA.

The interference from non-microbial sources wasalso estimated for soils A1 and F1 assuming all soil or-ganic matter (Table 1) had an extractable fatty acidcontent similar to that determined for the isolated HAmaterials (Table 3). This may be a crude approxi-mation, although humic materials do constitute 70±80% of soil organic matter (Schnitzer, 1978). For thetwo extraction methods and soils employed, fatty acidsderived from soil organic matter would then corre-spond to 14±17% of the whole-soil PLFA. Hence, thetwo alternative calculation approaches gave similarestimates of the contribution from HA to the di�er-ence between the two extraction methods.

We wanted to quantify the co-extraction of cellularlipids during the procedure for preparation of HAemployed. It is known that strong alkali may dissolveprotoplastic and structural cell components, althoughsome hydrolysis of lipids may also occur (Ratledgeand Wilkinson, 1988; Stevenson, 1994). The controlexperiment with HA preparation followed by PLFAanalysis of soil F1 alone or soil F1 + a bacterial cul-ture showed a carry-over of cellular PLFA of ca. 2%(Table 5). Comparing this value to the calculated totalinterference from the HA extracted of 3±4% (Table 6),the data suggest that cellular lipids may account for 1/2±2/3 of the interference, while the `real' interferencefrom soil organic matter accounts for the rest. Thiscould partly explain the di�erence in PLFA concen-trations between HA isolated from soils in this studyand the IHSS reference soil with its more extensivepuri®cation (Table 3). If it is assumed that the carry-over of PLFA from soil microbial biomass corre-sponds to that determined above for added cells, andthat the interference of all soil organic matter is similarto that derived from the 20% consituted by HA, then

the total interference from non-biomass sources wouldbe in the order of 5±10%.

5. Conclusion

We compared four di�erent modi®cations of aBligh±Dyer single-phase extraction for PLFA analysis.Signi®cant quantitative di�erences were observed thatmay have been related to the degree of soil particledispersion, and the combination of chloroform andcitrate bu�er gave the highest yields. The measure-ments have indicated that there was a contribution ofHA-derived fatty acids in PLFA analyses, even withthe mild extraction methods we employed, and thatthis contribution was slightly higher with the combi-nation of chloroform and citrate bu�er. It is not sur-prising that the most e�ective extractant also has thehighest potential for interference, but it was estimatedthat this interference amounted to, at most, 5±10% ofthe total PLFA yield. Qualitative di�erences betweenthe four extraction methods were small compared todi�erences between soils, and with respect to PLFAcomposition the choice of extraction method thereforedid not seem to be critical. In contrast, quantitativedi�erences should be considered when results based ondi�erent extraction methods are compared.

Acknowledgements

The GC analyses were carried out at Aalborg Uni-versity. We thank L. Elsgaard for providing the bac-terial culture, and C. Lohse for helpful suggestions inplanning this study. Also, J. Larsen and B.T. Christen-sen are acknowledged for valuable comments to themanuscript.

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