Linking microbial community analysis and ecosystem studies: A rapid lipid analysis protocol for high throughput

Abstract

During the past two decades interest in linking soil microbial community composition and activity with ecosystem scale field studies of nutrient cycling or plant community response to disturbances has grown. Despite its importance there are challenges in making this linkage. Foremost is the question of analytical feasibility. In general, microbiological community-level methodologies have not been readily adaptable to the large sample sizes necessary for ecosystem-scale research. As a result, it has been difficult to generate compatible microbial and ecosystem data sets. Soil lipid analysis shows potential as a middle ground between simple biomass measures and molecular profiling. However, the two protocols that have most often been followed are either rapid but indiscriminate (total lipid analysis or fatty acid methyl ester analysis; FAME), or precise but time consuming (phospholipid fatty acid analysis; PLFA). In this paper we report results from a standardized soil used test a modified extraction method (the ‘hybrid’ method) developed to balance the speed of FAME and the precision of PLFA in order to increase sample throughput. In comparing the three methods, we find that FAME and PLFA are qualitatively and quantitatively distinct. The FAME method yielded the highest fatty acid abundance, but also had high variance resulting in low precision. The PLFA method had precision, but low yield. The ‘hybrid’ method fell midway between FAME and PLFA for quantitative fatty acid yield. In addition, the hybrid extraction can be completed in a fraction of the time it takes for PLFA. The hybrid protocol appears to provide an optimal balance between effort and accuracy and therefore is a good choice for large-scale ecosystem studies.

Keywords

Microbial community composition / Lipid analysis / PLFA / MIDI-FAME / Ecosystem studies

References

[1] Baas-Becking, L.G.M., 1934. Geobiologie of Inleiding tot de Milieukunde. Van Stockum & Zoon, The Hague.

[2] Bååth, E., 2003. The use of neutral lipid fatty acids to indicate the physiological conditions of soil fungi. Microbial Ecology 45, 373–383

[3] Bailey, V.L., Peacock, A.D., Smith, J.L., Bolton, H. Jr, 2002. Relationships between soil microbial biomass determined by chloroform fumigation-extraction, substrate-induced respiration, and phospholipid fatty acid analysis. Soil Biology & Biochemistry 34, 1385–1389

[4] Balser, T.C., Firestone, M.K., 2005. Linking microbial community composition and soil processes in a California annual grassland and mixed-conifer forest. Biogeochemistry 73, 395–415

[5] Balser, T.C., Gutknecht, J.L.M., Liang, C., 2010. How will climate change impact soil microbial communities? In: Dixon, G.R., Tilston, E. (Eds.), Soil Microbiology and Sustainable Crop Production. University of Reading Press, Reading, U.K.

[6] Balser, T.C., Kirchner, J.W., Firestone, M.K., 2002. Methodological variability in microbial community level physiological profiles. Soil Science Society of America Journal 66, 519–523

[7] Balser, T.C., Wixon, D.L., 2009. Investigating biological control over soil carbon temperature sensitivity. Global Change Biology 15, 2935–2949

[8] Bartelt-Ryser, J., Joshi, J., Schmid, B., Brandl, H., Balser, T., 2005. Soil feedbacks of plant diversity on soil microbial communities and subsequent plant growth. Perspectives in Plant Ecology, Evolution and Systematics 7, 27–49

[9] Ben-David, E.A., Zaady, E., Sher, Y., Nejidat, A., 2011. Assessment of the spatial distribution of soil microbial communities in patchy arid and semi-arid landscapes of the Negev Desert using combined PLFA and DGGE analyses. FEMS Microbiology Ecology 76, 492–503

[10] Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology 37, 911–917

[11] Buyer, J.S., Sasser, M., 2012. High throughput phospholipid fatty acid analysis of soils. Applied Soil Ecology 61, 127–130

[12] Cavigelli, M., Robertson, G., Klug, M., 1995. Fatty acid methyl ester (FAME) profiles as measures of soil microbial community structure. Plant and Soil 170, 99–113

[13] Cavigelli, M.A., Robertson, G.P., 2000. The functional significance of denitrifier community composition in a terrestrial ecosystem. Ecology 81, 1402–1414

[14] Cusack, D.F., Silver, W.L., Torn, M.S., Burton, S.D., Firestone, M.K., 2011. Changes in microbial community characteristics and soil organic matter with nitrogen additions in two tropical forests. Ecology 92, 621–632

[15] Jesus, E., Da, C., Liang, C., Quensen, J.F., Suliwati, E., Jackson, R., Balser, T., Tiedje, J., 2016. Influence of corn, switchgrass, and prairie cropping systems on soil microbial communities in the upper Midwest of the United States. Global Change Biology. Bioenergy 8, 481–494

[16] Drenovsky, R.E., Elliott, G.N., Graham, K.J., Scow, K.M., 2004. Comparison of phospholipid fatty acid (PLFA) and total soil fatty acid methyl esters (TSFAME) for characterizing soil microbial communities. Soil Biology & Biochemistry 36, 1793–1800

[17] Drijber, R.A., Doran, J.W., Parkhurst, A.M., Lyon, D.J., 2000. Changes in soil microbial community structure with tillage under long-term wheat-fallow management. Soil Biology & Biochemistry 32, 1419–1430

[18] Fernandes, M.F., Saxena, J., Dick, R.P., 2013. Comparison of whole-cell fatty acid (MIDI) or phospholipid fatty acid (PLFA) extractants as biomarkers to profile soil microbial communities. Microbial Ecology 66, 145–157

[19] Fichtner, A., von Oheimb, G., Härdtle, W., Wilken, C., Gutknecht, J.L.M., 2014. Effects of anthropogenic disturbances on soil microbial communities in oak forests persist for more than 100 years. Soil Biology & Biochemistry 70, 79–87

[20] Fraterrigo, J.M., Balser, T.C., Turner, M.G., 2006. Microbial community variation and its relationship with nitrogen mineralization in historically altered forests. Ecology 87, 570–579

[21] Frostegård, A., Bååth, E., 1996. The use of phospholipid fatty acid analysis to estimate bacterial and fungal biomass in soil. Biology and Fertility of Soils 22, 59–65

[22] Frostegård, Å., Tunlid, A., Bååth, E., the Frostegård, 2011. Use and misuse of PLFA measurements in soils. Soil Biol Biochem 43, 1621–1625.

[23] Gutknecht, J.L.M., Field, C.B., Balser, T.C., 2012. Microbial communities and their responses to simulated global change fluctuate greatly over multiple years. Global Change Biology 18, 2256–2269

[24] Haack, S.K., Garchow, H., Odelson, D.A., Forney, L.J., Klug, M.J., 1994. Accuracy, reproducibility, and interpretation of Fatty Acid methyl ester profiles of model bacterial communities. Applied and Environmental Microbiology 60, 2483–2493

[25] Herman, D.J., Firestone, M.K., Nuccio, E., Hodge, A., 2012. Interactions between an arbuscular mycorrhizal fungus and a soil microbial community mediating litter decomposition. FEMS Microbiology Ecology 80, 236–247

[26] Jackson, R., Fierer, N., Schimel, J., 2007. New directions in microbial ecology. Ecology 88, 1343–1344

[27] Jandl, G., Leinweber, P., Schulten, H.R., Ekschmitt, K., 2005. Contribution of primary organic matter to the fatty acid pool in agricultural soils. Soil Biology & Biochemistry 37, 1033–1041

[28] Jin, V.L., Evans, R.D., 2010. Microbial 13C utilization patterns via stable isotope probing of phospholipid biomarkers in Mojave Desert soils exposed to ambient and elevated atmospheric CO₂. Global Change Biology 16, 2334–2344

[29] Jokela, W.E., Grabber, J.H., Karlen, D.L., Balser, T.C., Palmquist, D.E., 2009. Cover crop and liquid manure effects on soil quality indicators in a corn silage system. Agronomy Journal 101, 727–737

[30] Kao-Kniffin, J., Balser, T.C., 2007. Elevated CO₂ differentially alters belowground plant and soil microbial community structure in reed canary grass-invaded experimental wetlands. Soil Biology & Biochemistry 39, 517–525

[31] Kotas, P., Santruckova, H., Elster, J., Kastovska, E., 2018. Soil microbial biomass, acitivity and community composition along altitudinal gradients in the High Arctive (Billefjorden, Svalbard). Biogeosciences 15, 1879–1894

[32] Liang, C., Fujinuma, R., Balser, T.C., 2008. Comparing PLFA and amino sugars for microbial analysis in an Upper Michigan old growth forest. Soil Biology & Biochemistry 40, 2063–2065

[33] Liang, C., Gutknecht, J.L.M., Balser, T.C., 2015. Microbial lipid and amino sugar responses to long-term simulated global environmental changes in a California annual grassland. Frontiers in Microbiology 6, 385

[34] Mentzer, J., Goodman, R., Balser, T., 2006. Microbial response over time to hydrologic and fertilization treatments in a simulated wet prairie. Plant and Soil 284, 85–100

[35] Miura, T., Makoto, K., Niwa, S., Kaneko, N., Sakamoto, K., 2017. Compararision of atty acid methyl ester methods for characterization of microbial communities in forest and arable soil: Phospholipid fraction (PLFA) versus total ester linked fatty acids (EL-FAME). Pedobiologia 63, 14–18

[36] Ngosong, C., Gabriel, E., Ruess, L., 2012. Use of the signature Fatty Acid 16:1ω5 as a tool to determine the distribution of arbuscular mycorrhizal fungi in soil. J Lipids 2012, 236807.

[37] Nielsen, P., Petersen, S.O., 2000. Ester-linked polar lipid fatty acid profiles of soil microbial communities: a comparison of extraction methods and evaluation of interference from humic acids. Soil Biology & Biochemistry 32, 1241–1249

[38] Pankhurst, C.E., Yu, S., Hawke, B.G., Harch, B.D., 2001. Capacity of fatty acid profiles and substrate utilization patterns to describe differences in soil microbial communities associated with increased salinity or alkalinity at three locations in South Australia. Biology and Fertility of Soils 33, 204–217

[39] Pei, Z., Eichenberg, D., Bruelheide, H., Kröber, W., Kühn, P., Li, Y., von Oheimb, G., Purschke, O., Scholten, T., Buscot, F., Gutknecht, J.L.M., 2016. Soil and tree species traits both shape soil microbial communities during early growth of Chinese subtropical forests. Soil Biology & Biochemistry 96, 180–190

[40] Schimel, J., 2004. Playing scales in the methane cycle: from microbial ecology to the globe. Proceedings of the National Academy of Sciences of the United States of America 101, 12400–12401

[41] Schmidt, J., Schulz, E., Michalzik, B., Buscot, F., Gutknecht, J.L.M., 2015. Carbon input and crop-related changes in microbial biomarker levels strongly affect the turnover and composition of soil organic carbon. Soil Biology & Biochemistry 85, 39–50

[42] Schutter, M.E., Dick, R.P., 2000. Comparison of Fatty Acid Methyl Ester (FAME) methods for characterizing microbial communities. Soil Science Society of America Journal 64, 1659–1668

[43] Smithwick, E.A.H., Turner, M.G., Metzger, K.L., Balser, T.C., 2005. Variation in NH₄⁺ mineralization and microbial communities with stand age in lodgepole pine (Pinus contorta) forests, Yellowstone National Park (USA). Soil Biology & Biochemistry 37, 1546–1559

[44] Tiedje, J.M., Asuming-Brempong, S., Nuslein, K., Marsh, T.L., Flynn, S.J., 1999. Opening the black box of soil microbial diversity. Applied Soil Ecology 13, 109–122

[45] Ushio, M., Wagai, R., Balser, T.C., Kitayama, K., 2008. Variations in the soil microbial community composition of a tropical montane forest ecosystem: Does tree species matter? Soil Biology & Biochemistry 40, 2699–2702

[46] Vestal, J.R., White, D.C., 1989. Lipid analysis in microbial ecology: quantitative approaches to the study of microbial communities. Bioscience 39, 535–541

[47] Wallenstein, M., Hall, E., 2012. A trait-based framework for predicting when and where microbial adaptation to climate change will affect ecosystem functioning. Biogeochemistry 109, 35–47

[48] Willers, C., Jansen van Rensburg, P.J., Claassens, S., 2015a. Phospholipid fatty acid profiling of microbial communities--a review of interpretations and recent applications. Journal of Applied Microbiology 119, 1207–1218

[49] Willers, C., Jansen van Rensburg, P.J., Claassens, S., 2015b. Microbial signature lipid biomarker analysis- an approach that is still preferred, even amid various method modifications. Journal of Applied Microbiology 118, 1251–1263

[50] Zak, D.R., Holmes, W.E., White, D.C., Peacock, A.D., Tilman, D., 2003. Plant diversity, soil microbial communities, and ecosystem function: are there any links? Ecology 84, 2042–2050

[51] Zelles, L., 1999. Faty acid patterns of phospholipids and lipopolysaccharides in the characterization of microbial communities in soil: a review. Biology and Fertility of Soils 29, 111–129

Acknowledgments

This study was supported by grants from the Kearney Foundation, the Andrew W. Mellon Foundation and the Department of Energy-Great Lakes Bioenergy Research Center DOE-GLBRC) funding. We would also like to thank the Matson laboratory (Stanford University) and Balser laboratory (University of Wisconsin-Madison) where method testing was performed. We are also grateful for the assistance of Carrie Nielsen and Phil Barak.

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