Ester-linked polar lipid fatty acid pro®les of soil microbial
communities: 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, and any interference from fatty acids derived from dead organic material is assumed to be negligible. We studied the initial extraction of lipid material from eight dierent soils and from puri®ed humic acids using four dierent combinations of solvent (chloroform or dichloromethane), methanol and buer (potassium phosphate, pH 7.4 or sodium citrate, pH 4). The quantitative yields 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 eciency and potential interference. Chloroform + citrate buer generally gave the highest, and dichloromethane + phosphate buer the lowest PLFA yields, and it was estimated that <20% of the yield dierence between extraction methods could be explained by interference from humic acids. Principal component analyses of PLFA composition suggested an eect of extraction method for several soils, but when all soils were analyzed together the dierences 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 interference from non-microbial sources in PLFA analyses was probably not more than 5±10% with the extraction methods employed.72000 Elsevier Science Ltd. All rights reserved.
Keywords:Phospholipid fatty acids; Extraction eciency; 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 by modi®cations of the single-phase extraction described by 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 in proportions to give a single phase, which probably
improves the contact between extractant and cells in complex matrices like soil or sediment compared to procedures 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 use dichloromethane as an alternative to the more hazar-dous chloroform. This modi®cation was combined with the use of a strong sodium bromide solution during phase separation, which results in phase inver-sion with physical separation of the organic solvent phase from sample residue following centrifugation. The composition of the aqueous phase was investi-gated by FrostegaÊrd et al. (1991), who found a signi®-cant eect of buer type for an acid forest soil with a
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* Corresponding author. Tel.: 1723; fax: +45-8999-1619.
high organic matter content, but not for a neutral ara-ble soil.
Lipid extracts of environmental samples are typically yellow to brownish in color. The color intensity is apparently related to the amount of organic detritus in the sample and presumably results from co-extraction of humic materials. Humic substances may contain ester-linked long-chain fatty acids (e.g., Schnitzer and Neyroud, 1975) and therefore represent a potential source of error in the characterization of living mi-crobial communities through fatty acid ®ngerprints. Higher yields of polar lipid fatty acids (PLFA) are typically accompanied by higher intensity of color in the crude lipid extract and, although microbial bio-mass has been shown to correlate with organic matter content (Anderson and Domsch, 1989), the yield increase could be partly derived from non-biomass sources.
Our study was designed to characterize and quantify the interference from humic acids on ester-linked PLFA pro®les of microbial communities when using dierent lipid extraction methods. The study involved eight cultivated or natural soils, humic acids (HA) iso-lated from selected soils as well as a HA reference de-rived from soil, and four dierent versions of the single-phase extraction procedure.
2. Materials and methods
2.1. Reagents and glassware
Reagent bottles and 35 ml extraction tubes were acid washed (10% HCl) and rinsed three times in deio-nized water. All other glassware was ignited at 5308C
for 3 h. All solvents were LiCroSolv grade from Merck (Darmstadt, Germany), chemicals were analyti-cal grade. Fatty acid methyl ester standards were obtained 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) and four forest soils (F1±F4) were sampled to 15 cm depth, the forest soils after removal of the litter layer. Two arable soils (A2 and A3) were from dierent ®elds of the same location, and two of the forest soils (F1 and F2) were from deciduous forest areas, while F3 and F4 were from coniferous forests. The organic matter content ranged from 2.3 to 13.4% of the soil dry wt. and the pHCaCl2from 2.9 to 5.8. All soils were
sieved (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 for this study (A1 and F1) by a procedure modi®ed from Swift (1996). Field moist soil was extracted in 0.1 M NaOH (NaOH:soil ratio 10:1, v/w) for 4 h under N2
and then left to settle overnight. After centrifugation (1890g for 10 min) the supernatant was decanted to a new centrifuge tube via a funnel with a plug of glass-wool. Following adjustment of the pH to 1.0 with 6 M HCl the extract was left for 12±16 h; precipitated HA were concentrated by centrifugation. The HA was pur-i®ed twice by dissolution in NaOH and re-precipitation with acid. This was followed by washing in deionized water and freeze-drying. The ®nal yield of HA corre-sponded to 30 and 20% of the soil organic matter for soils A1 and F1, respectively.
A reference soil HA (Code 1S102H) was obtained from the International Humic Substances Society (IHSS), which had been isolated by the procedure described by Swift (1996). It was similar to the one used 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 dierent sources were as follows: Soil A1, 41%; Soil F1, 33%; IHSS, 1%. In the following, yields of fatty acids in HA are corrected for ash content.
Table 1
Characteristics of soils used in the present study
Key Soil type Clay (%) Organic matter (%) Total C (%) Total N (%) pHCaCl2 Total CEC (meq 100 g ÿ1)
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
2.4. Lipid extraction
The extraction mixture recommended by White et al. (1979) for environmental samples consists of chloroform, methanol and phosphate buer (50 mM K2HPO4, pH 7.4). FrostegaÊrd et al. (1991) observed
that higher lipid yields were obtained from an acid soil with a high organic matter content by replacing the neutral phosphate buer by an acid citrate buer (150 mM Na3C6H5O72H2O, pH 4.0).
In our study we combined the use of chloroform (C) or dichloromethane (D) for lipid extraction with either phosphate or citrate buer; three of these four combi-nations have been used in published studies. Extrac-tions with dichloromethane employed sodium bromide for the phase separation (see Petersen and Klug, 1994 for details), while the separation of chloroform extracts was as described by FrostegaÊrd et al. (1991). The four extraction methods will be referred to as Dphos, Dcit,
CphosandCcit:
Lipid extractions for PLFA analyses were performed on all soils and HA materials. The dierent crude lipid extracts were dried under N2 and then exposed to the
same procedure, i.e., isolation of polar lipids (mainly phospholipids) by solid phase extraction (100 mg silicic acid with [60 AÊ] pore size; Varian, Harbor City, CA), mild alkaline transmethylation, and extraction of methyl esters withn-hexane (Dowling et al., 1986). An internal standard (IS), nonadecanoate methyl ester, was added during transmethylation. Fatty acid methyl esters were analyzed by capillary gas chromatography with settings as described by FrostegaÊrd et al. (1993). A total of 38 fatty acids (see Table 4) were identi®ed by 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 data analysis.
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 extracfrac-tion of polar lipids. A Spectronic 2000 spectrophotometer (Bausch & Lomb, Rochester, NY) and a quartz cuvette (Hellma, MuÈllheim, Germany) with a 1 cm light path was used. The absorbance at 400 nm was taken as a quantitative index of the concentration of humic sub-stances (Khan and Schnitzer, 1978; Sorouradin et al., 1993) after normalization with respect to the amounts of soil extracted and solvent used. For unknown reasons a few samples were slightly opaque, and absorption spectra could not be obtained.
2.6. Co-extraction of microbial cells
Lipids originating from living organisms could be partly included in the humic acid fraction isolated. To investigate the potential for carry-over of cellular lipid material during HA preparation, a control experiment was carried out with a culture identi®ed as Rhodococ-cus erythropolis on the basis of morphology, mycolic acid composition, 16S rDNA analysis and fatty acid composition (Dr L. Elsgaard, pers. comm.); the fatty acids of Rhodococci are dominated by saturated and monounsaturated fatty acids and, notably, tuberculos-tearic acid (10Me18:0) (Barton et al., 1989). Cells were harvested by centrifugation (15,000 g for 10 min) and washed once in autoclaved phosphate buer.
HA preparation + PLFA analysis was carried out for (1) ca. 0.5 g dry wt. soil F1, and (2) ca. 0.5 g dry wt. soil F1 + 100±200 mg fresh wt. cell material; exact weights were recorded. PLFA analysis was also carried out directly on 100±200 mg fresh wt. cell ma-terial and the seven dominant fatty acids of R. erythro-poliswere used for calculation of recovery:
Recovery,FAj
where FAj is fatty acid j, The experiment was carried out with Dphos and Ccit extraction procedures,
respect-ively.
2.7. Statistics
3. Results
3.1. Quantitative yields
The four dierent combinations of extraction sol-vent and buer Ð Dphos, Dcit, Cphos and Ccit Ð gave
the yields of PLFA listed in Table 2. Except for A2, i.e., the soil with the highest pH, yields with Ccit were
always 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 higher yields than dichloromethane, particularly with the for-est soils. When comparing extractions with dichloro-methane or chloroform separately, there were only minor eects of buer type.
Polar lipid ester-linked fatty acid yields of HA ma-terials with Dphos and Ccit extraction methods are
shown in Table 3. The two HA materials produced in this study gave much higher yields of PLFA than the reference HA. The dierence between Dphos and Ccit
extraction methods was apparently similar to that
observed for the soils, although no replication was generally included with these analyses.
3.2. Absorbance spectra
The absorbance spectra of non-fractionated soil lipid extracts are exempli®ed in Fig. 1A showing absorbances with two extraction methods, Dphos and
Ccit, for soils A1 and F1. The absorbances increased
with decreasing wave length, except that most curves had 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 dierent origin, as deter-mined using four dierent extraction methods, Dphos(DCM +
phos-phate buer), Dcit (DCM + citrate buer), Cphos (chloroform +
phosphate buer) and Ccit(chloroform + citrate buer) a
Key Soil type Lipid extraction method (nmol PLFA gÿ1dry 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
Dierences between extraction methods are indicated by dierent 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 dierent extraction methods. Dphos: extraction with dichloromethane and phosphate
buf-fer, Ccit: extraction with chloroform and citrate buer
HA source Dphos Ccit
(nmol gÿ1ash-free dry wt)
Forest soil (F1) 292a 375
Arable soil (A1) 152 201
Standard soil (IHSS) 41 (3) 66 (23)
aNo 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
solid phase extraction were qualitatively similar, although weaker in absorbance (data not shown).
Absorbance spectra of lipid extracts of HA prepared from soils A1 and F1 are shown in Fig. 1B (the smooth curve, which was obtained for a HA derived from brown coal, should be disregarded). Lipid extracts of soil-derived HA had shoulders similar to the whole-soil lipid extracts.
The absorbance at 400 nm was recorded as a quanti-tative index of humic material in the extracts. For all soils there was consistently less absorbance in extrac-tions with phosphate than with citrate buer (data not shown). The abs400readings in crude lipid extracts and
in the polar lipid fraction were highly correlated (see Fig. 2). There was no correlation between soil PLFA concentrations (Table 2) and absorbances in the crude lipid extracts (P> 0.10, n= 32), while the correlation between whole-soil PLFA and abs400 of the polar lipid
fraction was signi®cant (P< 0.025), although relatively weak (r = 0.42). Hence, solid phase extraction removed 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®ed in PLFA analyses of soils A1 and F1 are shown in Table 4 for Dphos and Ccit, i.e., the two extraction
methods that were quantitatively most dissimilar. The fatty acid composition was generally similar for the
two extraction methods, the most notable dierences being a relatively higher concentration of 16:0 and lower concentration of 18:1o7 when extracted with
Ccit, particularly in soil A1, and a relatively higher
concentration of cyclopropyl fatty acids with Dphos.
A PCA analysis was carried out for each soil separ-ately, which included the transformed mole percentage distributions from all extraction methods, and further an analysis was done with all soils and extraction methods combined. When soils were analyzed indivi-dually, there were indications of qualitative dierences between extraction methods for six of the eight soils,
Table 4
Mole percentage distribution of phospholipid fatty acids from two soils, as determined using two dierent extraction methods, Dphos
(dichloromethane and phosphate buer) or Ccit (chloroform and
citrate buer)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).
bND: not detected.
i.e., distances between replicates in the score plots of PC1 and PC2 were smaller than distances between extraction methods (data not shown). The dierences were 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 respect to quantitative yields (Table 2).
When all soils were included in one PCA (see Fig. 3), almost identical patterns in the distribution of soils were obtained with the four extraction methods, show-ing that dierences between soils were much more im-portant than any dierences between extraction methods. In Fig. 3, scores along PC1 and PC2 of each extraction method are presented in separate plots for clari®cation, but all data were derived from the same PCA.
The composition of ester-linked fatty acids in the polar lipid fraction of HA from soils A1 and F1 were compared to that of parent soils in a separate PCA analysis, and both Dphosand Ccit extraction data were
included. The results are presented in Fig. 4 and, although the HA data were generally not replicated, some interesting trends were suggested; the plot is scaled in proportion to the fractions of the total varia-bility explained by PC 1 (48.5%) and PC 2 (23.0%). Firstly, the dierences in PLFA patterns between iso-lated HA and the corresponding soil explained more of the variation than did dierences between soils.
Sec-ondly, the results suggest that the choice of extraction method (in Fig. 4 indicated by dierent ®ll color within a group) was more critical for the HA fraction than for the whole-soil samples, i.e., that choice of extraction method was relatively more important for the 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 PLFA preparation, along with unamended soil samples. For seven selected fatty acids, the enrichment due to the cell material was determined and compared to the con-centrations of these fatty acids in PLFA analyses of the cell material (Table 5). The experiment was carried out with both Dphos and Ccit extraction methods. The
co-extraction of cellular PLFA averaged ca. 2% with both 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 Dphoswas used, the darker color that Ccitwas 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
known quantities of soil F1, and therefore the concen-trations of PLFA in HA (without cell material) could be compared with the PLFA concentrations of the soil when analyzed directly. These results are shown in Table 6 for seven selected fatty acids; when all 38 fatty acids were considered, the HA-derived fatty acids con-stituted 4.02 0.7% and 2.9 20.3% of whole-soil PLFA with Dphosand 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 to come primarily from the membranes of living organ-isms (Tunlid and White, 1992). The material isolated in the HA fraction does not represent soil organic mat-ter in general, but it does represent an empirically well-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 fatty acids, albeit only after exhaustive extraction steps. They proposed that both physical adsorption and chemical binding via ester-linkages was involved in the stabilization. In general the degradation of lipids is retarded 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 is a potential for interference from soil organic matter in lipid analyses of microbial communities, and that this interference 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 the soil environment (Dinel et al., 1990) and would prob-ably be rapidly oxidized if not protected by interaction with other soil constituents. The dierences in
quanti-tative yields between the four fairly mild extraction methods used in our study (Table 2) indicate that microorganisms and, conceivably, their decomposition products can indeed be physically protected in the soil environment. The PLFA composition of soil and HA material (Fig. 4) was more dissimilar with the Ccit
extraction method than with Dphos, which suggests
that Ccit extracted a pool of material that was not
accessible with Dphos.
The dierences in PLFA yield observed between extraction methods support the observations made by FrostegaÊrd 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 the phosphate buer gave 30% lower yields of PLFA than the citrate buer with the organic soil, but not with the arable soil. In our study the only soil where no sig-ni®cant dierence was observed was the one with the highest pH. However, FrostegaÊrd et al. (1991) also noted that yields were not exclusively a re¯ection of pH, since an acetate buer at pH 4 was not eective, and this was attributed to the higher concentration of sodium ions introduced with the trivalent citrate ion. In our study, where all four combinations of solvent and buer were compared, the organic solvent, dichloromethane or chloroform, seemed to be more important than buer type for the dierence in yields.
With respect to PLFA composition, FrostegaÊrd et al. (1991) did not ®nd any discrimination as a function of buer choice. But fatty acids in humic and fulvic acids are dominated by 14C±34C fatty acids (Schnitzer and Schulten, 1989) and partly of microbial origin, and any interference from soil organic matter may therefore be dicult to detect on the basis of fatty acid composition. In this study we also found only minor qualitative dierences between the four extrac-tion methods used, as revealed by PCA analyses (Fig. 3). The higher proportion of cyclopropyl fatty acids with Dphos (Table 4) is in accordance with the
observation by Khan and Schnitzer (1972) that branched-cyclic fatty acid were more easily removed from HA than straight-chain fatty acids, since the less ecient extractant would then be enriched with these fatty 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 intact soils (Fig. 1A) and soil HA (Fig. 1B) suggests that the colour of the whole-soil lipid extracts was in fact due to co-extraction of HA-like material, although the harsh conditions of HA extraction may lead to chemi-cal changes (Stevenson, 1994; Christie, 1982) that com-plicate the use of HA characteristics for extrapolation to organic material in the undisturbed soil.
When comparing Dphosand 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 (%)
10Me18:0 2.4 (0.3) 2.2 (0.4)
absorbance readings, also more HA. And as shown above (Table 3) HA contains ester-linked fatty acids. Accordingly, HA can account for some of the dier-ence in whole-soil PLFA yield Ð but how much? The contribution from HA to the dierence was ®rst esti-mated for soils A1 and F1 by comparing the ratio betweenDPLFA andDabs400, i.e.:
for HA and soil, respectively. The ratio for HA consti-tuted between 8 and 17% of the ratio for soil, both when crude lipid extracts and when methanol fractions were considered (data not shown). Assuming the absorbance of the soil extracts was due to HA alone, only 8±17% of the dierence in PLFA yield between Ccit and Dphos could therefore be accounted for by
fatty acids derived from HA.
The interference from non-microbial sources was also estimated for soils A1 and F1 assuming all soil or-ganic matter (Table 1) had an extractable fatty acid content similar to that determined for the isolated HA materials (Table 3). This may be a crude approxi-mation, although humic materials do constitute 70± 80% of soil organic matter (Schnitzer, 1978). For the two extraction methods and soils employed, fatty acids derived from soil organic matter would then corre-spond to 14±17% of the whole-soil PLFA. Hence, the two alternative calculation approaches gave similar estimates of the contribution from HA to the dier-ence between the two extraction methods.
We wanted to quantify the co-extraction of cellular lipids during the procedure for preparation of HA employed. It is known that strong alkali may dissolve protoplastic and structural cell components, although some hydrolysis of lipids may also occur (Ratledge and Wilkinson, 1988; Stevenson, 1994). The control experiment with HA preparation followed by PLFA analysis 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 total interference 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' interference from soil organic matter accounts for the rest. This could partly explain the dierence in PLFA concen-trations between HA isolated from soils in this study and the IHSS reference soil with its more extensive puri®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, and that the interference of all soil organic matter is similar to that derived from the 20% consituted by HA, then
the total interference from non-biomass sources would be in the order of 5±10%.
5. Conclusion
We compared four dierent modi®cations of a Bligh±Dyer single-phase extraction for PLFA analysis. Signi®cant quantitative dierences were observed that may have been related to the degree of soil particle dispersion, and the combination of chloroform and citrate buer gave the highest yields. The measure-ments have indicated that there was a contribution of HA-derived fatty acids in PLFA analyses, even with the mild extraction methods we employed, and that this contribution was slightly higher with the combi-nation of chloroform and citrate buer. It is not sur-prising that the most eective extractant also has the highest potential for interference, but it was estimated that this interference amounted to, at most, 5±10% of the total PLFA yield. Qualitative dierences between the four extraction methods were small compared to dierences between soils, and with respect to PLFA composition the choice of extraction method therefore did not seem to be critical. In contrast, quantitative dierences should be considered when results based on dierent 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 in planning this study. Also, J. Larsen and B.T. Christen-sen are acknowledged for valuable comments to the manuscript.
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