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Dep. of Crop and Soil Science, Oregon State Univ., Corvallis, OR 97331 USA
richard.dick{at}orst.edu
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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Abbreviations: EL, ester-linked • FAME, fatty acid methyl ester • GC, gas chromatography • MANOVA, multivariate analysis of variance • MIDI, Microbial ID, Inc. • NMS, non-metric multidimensional scaling • PCA, principal components analyses • PLFA, phospholipid fatty acid • TOC, total organic carbon
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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Although analysis of microbial PLFA profiles has proven extremely useful, the methods involved are time consuming. Microbial lipids are extracted from environmental samples in a phase-mixture of chloroform, methanol (MeOH), and water (Bligh and Dyer, 1959). Lipids associated with the organic phase are then fractionated into neutral, glyco-, and phospholipids on silicic acid columns, while the residue at the organic:aqueous interphase can be separated into lipopolysaccharides, teichoic acids, and muramic acid (Vestal and White, 1989). Finally, the phospholipids are subjected to alkaline methanolysis to produce fatty acid methyl esters (FAMEs) for analysis by gas chromatography (GC).
Recently, a simpler method has been developed to extract microbial fatty acids directly from soils. The MIDI protocol was designed to extract fatty acids from pure cultures of bacterial isolates for identification purposes, but it also has been applied to whole-soils. With this method, microbial cells in soil are saponified by heat and the addition of a strong base. Once fatty acids are cleaved from lipids, they are methylated to form FAMEs. The FAMEs are extracted in an organic solvent and analyzed by gas chromatography (Sasser, 1990). Since its introduction, the MIDI technique has been used to assess storage effects on bacterial isolates (Haldeman et al., 1994), to characterize root-associated microorganisms and arbuscular mycorrhizal fungi (Sicilaino and Germida, 1998; Graham et al., 1995), and to describe microbial communities of agricultural soils (Cavigelli et al., 1995; Buyer and Drinkwater, 1997; Ibekwe and Kennedy, 1998a) and soils amended with various organic and inorganic compounds (Fries et al., 1997; Macalady et al., 1998).
While the simplicity of the method is advantageous over that of the PLFA method, it is uncertain whether or not fatty acids extracted by the MIDI method originate only from living microorganisms. It is possible that this extraction procedure also may extract fatty acids associated with soil humic substances and plant roots. Because of this concern, efforts are being made to develop less harsh methods for the direct extraction of fatty acids from soil microorganisms. One potential method is the ester-linked (EL) procedure (Dr. Rhae Drijber, 1998, personal communication). This method uses a mild alkaline reagent to lyse cells and release fatty acids from lipids once the ester bonds are broken. In theory, only ester-linked and not free fatty acids are extracted with this method. Recently, the EL method successfully characterized microbial communities of several grass seed field soils and placed communities into groupings similar to those generated by a DNA-based method (Ritchie et al., 2000).
The effects of soil storage on FAME profiles also are of great concern to microbial community studies. For example, community structure may change in response to the temperature at which soils are stored (Petersen and Klug, 1994). To date, the effects of air drying or freezing on microbial community FAME structure have not been reported. Earlier studies have shown that microbial biomass and activities, including nitrogen mineralization, are less affected when stored at -20°C than at 2°C (Stenberg et al., 1998), and that storing air-dried soils at 21°C did not preserve biological activity compared with moist soils stored at cooler temperatures (Zelles et al., 1991). However, effects of storage are complicated by freeze-thaw processes, which can stimulate activity, and by the abilities of soil communities to withstand soil drying and wetting cycles (Stenberg et al., 1998).
The first objective of this study was to compare microbial community FAME profiles of different soils extracted with the MIDI and EL methods and to determine if both methods are able to discriminate between communities of different soil types. The second objective was to determine the effects of soil storage conditions on microbial community FAME structure. To satisfy the second objective, common storage protocols for soil research were chosen, including air-dried storage and cold storage at field capacity.
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Materials and methods |
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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Sample Preparation and Storage Procedures
After sampling, all soils were stored in coolers for transport to the laboratory, where they were stored at 4°C. Within five days of sampling, the agricultural and forest soils were passed through a 2- and 4.75-mm sieve, respectively. Subsamples of freshly sieved, moist soils were immediately analyzed for their FAME profiles according to the extraction methods described below. The remainder of each soil was then divided into four 200-g portions. Each portion was assigned to one of the following four storage treatments: field-capacity
at 4°C, field-capacity at -20°C, air-dried at 25°C, and partially-dried at 4°C. The air-dried soils were dried and stored at room temperature. For the fourth storage treatment, soils were slowly dried in a 4°C room until the gravimetric water content was in the range of 60 to 100 g kg-1 soil and thereafter stored at 4°C. This particular storage method was used in previous studies involving soil aggregates, where soil was required to be partially dry during the mechanical sieving/aggregate size separation procedure (Mendes and Bottomley, 1998).
Soils were stored at their respective moisture contents and temperatures for 90 d. After 30 and 90 d, four subsamples were removed from each treatment and analyzed for their FAME profiles by both extraction methods.
MIDI Extraction Method
Four analytical replications of each fresh and stored soil were extracted by the MIDI procedure (Sasser, 1990; Ibekwe and Kennedy, 1998a). This method uses four reagents and consists of four steps: saponification, methylation, extraction, and a wash. Three grams of soil contained in a 20- by 125-mm teflon-lined, screw-cap test tube were mixed with 3 mL of 3.75 M NaOH in MeOH:H2O (1:1). Test tubes were vortexed and placed in a 100°C water bath for 30 min, during which cells were lysed and saponified (fatty acids cleaved from the cell lipids and converted to sodium salts). To convert fatty acids to FAMEs for increased volatility, 6 mL of 6.00 M HCl:MeOH (1:0.85) were added to the tubes. The tubes were then incubated in a water bath for 10 min at 85°C. Next, 2 to 3 mL of hexane:methyl-tert butyl ether (1:1) were added to extract the FAMEs from the acidic, aqueous phase into the organic phase. Soil samples were centrifuged at this point to separate soil organic matter from the organic phase. The contents of each tube were transferred to a teflon-lined, screw-cap, 35-mL glass centrifuge tube and centrifuged at 480 x g for 10 min. Afterwards, the clear organic phase was transferred to a 13- by 100-mm teflon-lined, screw-cap glass tube. The organic phase was washed of residual acidic reagents by adding 3 mL of mild base (0.3 M NaOH), followed by gentle mixing for 5 min. Finally, the organic phase was transferred from the glass tube to a vial for gas chromatography (GC) analysis with a Hewlett-Packard 5890 Series II (Palo Alto, CA) equipped with an HP Ultra 2 capillary column (5% diphenyl-95% dimethylpolysiloxane, 25 m by 0.2) and a flame ionization detector. The temperature program ramped from 170 to 270°C at 5°C per min, with 2 min at 270°C between samples to clean the column. Fatty acids were identified and their relative peak areas were determined using the MIS Aerobe chromatographic program and peak naming table as supplied by MIDI.
In cases where samples were concentrated to improve detection of FAMEs, a portion of the organic solvent was evaporated under a stream of N2. The remaining organic phase was transferred into a 250-µL glass insert and placed in a GC vial. Because low amounts of total FAME were recovered from the Walla Walla silt loam soils by the MIDI method, Walla Walla FAME samples from fresh and stored soils were concentrated prior to GC analysis. Chehalis soil FAME samples were concentrated after the third month of soil storage, when FAME recovery was low.
A blank (reagents only) and a positive control were included in each set of MIDI extractions. The positive control was Stenotrophomonas maltophilia (American Type Culture Collection, #13637).
Ester-Linked (EL) Extraction Method
The second FAME extraction procedure employs a mild alkaline methanolysis method, which is thought to extract ester-linked fatty acids but not free fatty acids. This particular method was developed by Dr. Rhae Drijber, University of Nebraska, Lincoln, NE (Drijber, 1998, personal communication) and consists of four reagents and four steps. Four analytical replications were extracted per fresh and stored soil. In the first step, 15 mL of 0.2 M KOH in methanol were added to a 35-mL teflon-lined, screw-cap glass centrifuge tube containing 3 g of soil. The contents of the tubes were mixed and incubated at 37°C for 1 h, during which ester-linked fatty acids were released and methylated. Samples were vortexed every 10 min during the incubation period. In the second step, 3 mL of 1.0 M acetic acid were added to neutralize the pH of the tube contents. FAMEs were partitioned into an organic phase by adding 10 mL of hexane followed by centrifugation at 480 x g for 10 min. No washing step was needed at this point in contrast to the MIDI method, which requires that all acidic residues be removed from the organic phase to prevent damage to the GC column. After the hexane layer was transferred to a clean glass test tube, the hexane was evaporated under a stream of N2. In the final step, FAMEs were dissolved in 0.5 mL of 1:1 hexane:methyl-tert butyl ether and transferred to a GC vial for analysis; results were analyzed by the MIDI system as described above. It was not necessary to concentrate any of the FAME samples from fresh or stored soils with this method.
Standard nomenclature is used to describe FAMEs detected by both extraction methods. Numbering of carbons begins at the aliphatic (
) end of the fatty acid molecule. The number of double bonds within the molecule is given after the colon. Cis and trans conformations are designated with the suffixes "c" and "t", respectively. Other notations are "Me" for a methyl group, "OH" for hydroxy, "cy" for cyclopropane groups, and the prefixes "i" and "a" for iso- and anteiso-branched FAMEs.
Statistical Analysis
Overall effects of extraction method and storage protocol on relative amounts of FAMEs detected in each soil type were determined by multivariate analysis of variance (MANOVA) and ANOVA procedures (SAS Institute, 1996). All data transformations and multivariate analyses were performed using the PC-ORD program (McCune and Mefford, 1997).
To determine the overall effects of soil type, extraction method, and storage on community structure, non-metric multidimensional scaling (NMS) was performed on a data set containing relative FAME amounts for all four soil types, fresh and stored, extracted with the MIDI and EL methods. FAMEs present in only one replicate of one soil sample within the entire data set were deleted prior to NMS; the deleted FAMEs were i10:0, 11:0 2OH, i15:1/ 13:0 3OH, 16:17c alcohol, i19:0, and i19:1. FAME data also were transformed by the arcsine squareroot transformation option of PC-ORD to reduce the non-normality of the data set.
For the above analysis, NMS was performed rather than principal components analysis because of the non-normality of the FAME data set that occurred when EL and MIDI-extracted soils were analyzed together. NMS is an iterative technique that ordinates samples in a lower-dimensional space so that the distances between objects in this space match as closely as possible the distances between objects in the original p-dimensional space, where p equals the number of variables (i.e., the number of FAMEs in the data set) (McCune and Mefford, 1997). Examples of NMS applied to ecological studies include Prentice (1977), Field et al. (1982), Oksanen (1983), Tuomisto et al. (1995), and Neitlich and McCune (1997). Distance was measured as Sørensen distance, and 150 iterations were performed to ensure stress was minimized (stress is a measure of the dissimilarity between ordinations in the original p-dimensional space and in the reduced dimensional space). Stability of the reduced-dimensional ordination pattern was assessed by plotting values of stress vs. iteration number (not shown), and two dimensions were selected for final analysis.
For each extraction method, fresh and stored soils from the agricultural systems were analyzed by principal components analysis (PCA) to determine the overall effects of management, soil type, and storage on microbial communities. To assess specific effects of storage on community profiles, PCA was performed on FAME data from fresh and stored soils of each site extracted with the MIDI or EL method.
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Results |
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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. Separation of communities by soil type also occurred; communities from the forested McKenzie River soil were unique from communities of the agricultural soils, regardless of extraction method.
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8c, 16:15c, and 17:17c. The FAME 17:17c was present in especially high amounts in the MIDI extracts of the McKenzie River soil (Table 2). For polyunsaturated FAMEs, 20:46,9,12,15c was detected at significantly greater amounts when soils were extracted by the EL method rather than the MIDI method (Table 3), but an opposite trend occurred for 18:36,9,12c (Table 2). Detection of branched FAMEs of different C-chain lengths also was dependent on the method employed. The MIDI procedure resulted in equal or greater abundances of shorter-chain, iso- and anteiso-branched FAMEs, including i11:0, a12:0, i13:0, a13:0, and i14:0 (Table 2 and 4). Of these, i11:0, a12:0, and a13:0 were found exclusively in MIDI-extracted soils. All other longer-chain branched FAMEs were more abundant in the EL soil extracts (Table 3).
Hydroxylated FAMEs generally were more abundant when soils were extracted with the MIDI method rather than the EL method. Three hydroxylated FAMEs (10:0 3OH, 12:0 2OH, and 12:0 3OH) were detected only in MIDI soil extracts, and 16:0 2OH and 16:0 3OH were present in equal or significantly greater amounts in MIDI versus EL soil extracts (Table 2). In addition, 16:0 N alcohol was detected at significantly greater amounts in MIDI extracts (Table 2). In contrast, soils extracted with the EL method were more abundant in FAMEs with cyclopropane or methyl groups (Table 3), with the exception of 10Me19:0.
There were a total of 11 FAMEs unique to the EL method and 17 unique to the MIDI method. Three of the 11 FAMEs unique to the EL method were present in only one replicate of one soil sample (11:0 2OH, i15:1 H/I/13:0 3OH, and i19:1 I) and were deleted from the data sets prior to NMS or PCA analysis. For FAME i15:1 H/I/13:0 3OH, MIDI cannot differentiate between the compounds because of identical retention times (such FAMEs are identified as summed features by the MIDI program). The other EL-unique FAMEs were i15:1 at 5, a15:1 A, 17:0 3OH, i18:0, i18:1H, 19:111c/unnamed, 20:19t, and one unnamed FAME. Most of these FAMEs were present in two of the four soil types at concentrations less than 0.1% (Tables 3 and 4). Of the 17 FAMEs unique to MIDI, three were present in only one soil sample (i10:0, 16:17c alcohol, and i19:0) and were subsequently deleted from the data sets. Of the remaining 14 FAMEs, two were present in all four soil types (9:0 and i17:1 I/a17:1 B), and most others were present in multiple replicates of one to three soil types at relative concentrations greater than 0.1% (10:0 3OH, i11:0, a12:0, a13:0, 12:0 2OH, 12:0 3OH, 15:1 8c, i15:0 3OH, i16:0 3OH, 17:16c, i17:1 10c, and 10Me19:0) (Tables 2 and 4).
Effect of Soil Properties and Storage
It was clear that both the EL and MIDI method could differentiate between forest and agriculture soil communities (Fig. 1). Therefore, only the agricultural soils were analyzed further to determine if both extraction methods could separate communities of soils differing in properties and management. Results are shown in Fig. 2
. Overall, soil type and management had greater influences on soil community profiles compared with storage effects. Regardless if soils were fresh or stored, the EL and MIDI methods were able to distinguish between the communities from the Chehalis and two Walla Walla soils (Fig. 2).
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7c/9t/12t and 18:19c, which decreased after storage, and i11:0 3OH, which increased upon storage (data not shown). For some soils, FAME profiles continued to change during the 90-d storage period (McKenzie River, Fig. 3C and 3D; manure-amended Walla Walla soils, data not shown). For other soils, there were relatively little changes in FAME profiles after 30 d of storage (EL-extracted Chehalis soil, Fig. 3A, MIDI-extracted N-amended Walla Walla soils, data not shown). For Chehalis soil extracted by MIDI (Fig. 3B), the similarity between profiles of fresh soil and soils stored for 90 d is an artifact; after 90 d of storage, MIDI-extracted FAME levels were below detection limits so samples were concentrated prior to GC analysis.
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9c, and a17:19c. Shifts in FAME structure for the MIDI-extracted Chehalis soils also can be explained by overall changes in extractable-FAME concentration. After 30 d of storage, the relative amounts of several FAMEs, including 10:0, 15:0, 18:0, a13:0, 10:0 3OH, 16:0 N alcohol, and 10Me16:0, declined (not shown), corresponding to the rightward shift observed in Fig. 3B. After 90 d of storage, it was necessary to concentrate the MIDI extracts, thereby increasing FAME diversity and resulting in a shift in FAME profiles towards that of the fresh soils. |
Discussion |
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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5c may be a marker for arbuscular mycorrhizal fungi and specific bacteria such as Cytophaga (Olsson et al., 1998; Frosteg6,9,12,15c (Vestal and White, 1989; Ringelberg et al., 1997) were more abundant when soils were extracted with the EL method. Also, abundances of several markers for Gram-negative bacteria differed between the two extraction methods. Relative amounts of hydroxylated FAMEs were greater in MIDI extracts, whereas EL extracts contained relatively greater amounts of cyclopropane FAMEs. Although both methods were able to differentiate among the four soil types, inferences regarding community structure clearly may vary according to the method employed. Alternatively, an extraction method may be chosen based on a FAME marker of interest if a particular group of microorganisms is being studied. There are practical considerations which suggest that the EL-FAME method has advantages compared with the MIDI method. Although approximately equal numbers of samples can be extracted in a given period of time, the EL method lacks the 100°C saponification step and the washing step. The saponification step employed by MIDI can be troublesome as tube contents may boil up and leak out of the test tube. This is of concern, especially if fatty acids are volatilized and escape. Heating a strong alkaline solution also can be hazardous to laboratory personnel. Although the concentration of extracted FAMEs was not quantified, MIDI-extracted samples often had to be concentrated to reach minimal threshold detection limits.
Differences in FAME composition based on extraction method may be due to differences in extraction efficiencies or differences in FAME sources. With the strong saponification and methylation steps, it is conceivable that the MIDI procedure may yield some FAMEs derived from humic substances. We did find that the NMS distance between MIDI- and EL-extracted soils of a given site increased as the TOC content of the soil increased (Fig. 1), suggesting that the effect of extraction method on soil FAME profiles may be related to soil TOC content. However, without further studies we cannot conclude whether or not FAMEs unique to MIDI extracts originated from living microorganisms or soil organic matter. Unfortunately, distinguishing between humic and bacterial fatty acids has proven difficult so far. For example, ß-hydroxy and iso- and anteiso-branched fatty acids have been extracted from purified humin fractions, but because they are also found in bacterial membranes, these fatty acids were considered to originate from bacteria rather than the actual humin (Almendros and Sanz, 1991).
In our study, storing soils for even 30 d resulted in changes in extractable FAME profiles. It is not clear whether or not these changes were due to changes in microbial populations over time or to autooxidation of microbial lipids. Autooxidation is the process by which unsaturated fatty acids react with O2, forming labile hydroperoxides that are readily converted to several secondary oxidation products (Gunstone, 1986). In all cases of this study, soils were stored aerobically, and the effects of autooxidation were unknown. Future studies may consider storing soils anaerobically or comparing FAME profiles from stored soils (sterile and unsterile) to distinguish between biological and autooxidation effects on FAME profiles.
There does not appear to be one particular storage method that is best for consistently producing FAME profiles that are highly similar to fresh FAME profiles. For 30-d stored soils, the percentage of FAMEs significantly affected by storage protocol ranged from 7 to 26% for EL-extracted soils and 6 to 45% for MIDI-extracted soils. Corresponding values for soils stored for 90 d ranged from 16 to 44% and 6 to 43%. However, there was no one storage protocol that resulted in the fewest changes in FAME numbers and relative amounts among the four soils. For example, after 30 d of storage, relative amounts of 19:0 cy 8c was lowest in Chehalis soil stored moist at -20°C compared with the other storage protocols, but in McKenzie River soil, it was lowest in the partially dry soil stored at 4°C. Because of these inconsistencies, it is recommended that community-level analyses be conducted on fresh rather than stored soils.
Another study has examined specifically the influence of storage on microbial community structure, although storage treatments differed from those of this study (Petersen and Klug, 1994). Moist soils were stored at 4.5, 10, or 25°C for 3 wk, during which community PLFAs were measured. At the two lower temperatures, changes in PLFA profiles over a 3-wk period were inconsistent, with no clear effect of time. However, there was a rapid change in profiles for soils stored at 25°C: PLFAs i15:0, i17:0, and 18:0 increased but 16:17c, 18:17c, and 18:26,9c decreased over time. In our study, the method of storage was not as critical as the length of time a soil was stored, and changes in response to storage were due mainly to changes in the number of FAMEs extracted. In contrast to our study, an increase in PLFA numbers was not observed by Petersen and Klug (1994).
In conclusion, both the EL and MIDI methods were able to separate fresh soils of differing texture and TOC content. Certain FAMEs were unique to each method, but the relative amounts of unique FAMEs generally were minute. Interpretations of community structure may differ depending on the extraction method employed because of differences in the types and relative amounts of fatty acids extracted. Overall, effects of storage method on soil FAME profiles were small compared with effects of extraction method and soil type.Neitlich McCune 1992; Siciliano Germida 1998
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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Received for publication November 8, 1999.
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TOPNOTESABSTRACTINTRODUCTIONMaterials and methodsResultsDiscussionREFERENCES |
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., Fritze H. Soil bacterial biomass, activity, phospholipid fatty acid pattern, and pH tolerance in an area polluted with alkaline dust deposition. Appl. Environ. Microbiol. 1992;58:4026-4031.rd ., Bth E., Tunlid A. Shifts in the structure of soil microbial communities in limed forests as revealed by phospholipid fatty acid analysis. Soil Biol. Biochem. 1993;25:723-730.th E. Microbial community dynamics during composting of straw material studied using phospholipid fatty acid analysis. FEMS Microbiol. Ecol. 1998;27:9-20.th E., Jakobsen I. Phosphorus effects on the mycelium and storage structures of an arbuscular mycorrhizal fungus as studied in the soil and roots by analysis of fatty acid signatures. Appl. Environ. Microbiol. 1997;63:3531-3538.[Abstract]th E. Structure of a microbial community in soil after prolonged addition of low levels of simulated acid rain. Appl. Environ. Microbiol. 1998;64:2173-2180.rd ., Fritze H., Bth E. Phospholipid fatty acid composition and heavy metal tolerance of soil microbial communities along two heavy metal-polluted gradients in coniferous forests. Appl. Environ. Microbiol. 1996;62:420-428.[Abstract]
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. The variance explained by the first principal component axis (PC) is shown in parentheses