Biogeosciences www.biogeosciences.net 1726-4170 1726-4189 4 5 2007 10.5194/bg-4-769-2007 http://www.biogeosciences.net/4/769/2007/ http://www.biogeosciences.net/4/769/2007/bg-4-769-2007.html http://www.biogeosciences.net/4/769/2007/bg-4-769-2007.pdf 769 779 2007-09-24 Community shifts and carbon translocation within metabolically-active rhizosphere microorganisms in grasslands under elevated CO₂ K. Denef karolien.denef@ugent.be H. Bubenheim K. Lenhart J. Vermeulen O. Van Cleemput P. Boeckx C. Müller Laboratory of Applied Physical Chemistry, Ghent University, Coupure Links 653, 9000 Gent, Belgium Justus-Liebig-University Gießen, Heinrich-Buff-Ring 26-32, 35392 Gießen, Germany School of Biology and Environmental Science, University College Dublin Belfield, Dublin 4, Ireland The aim of this study was to identify the microbial communities that are actively involved in the assimilation of rhizosphere-C and are most sensitive in their activity to elevated atmospheric CO₂ in a temperate semi-natural low-input grassland ecosystem. For this, we analyzed ¹³C signatures in microbial biomarker phospholipid fatty acids (PLFA) from an in-situ ¹³CO₂ pulse-labeling experiment in the Giessen Free Air Carbon dioxide Enrichment grasslands (GiFACE, Germany) exposed to ambient and elevated (i.e. 50% above ambient) CO₂ concentrations. Short-term ¹³C PLFA measurements at 3 h and 10 h after the pulse-labeling revealed very little to no ¹³C enrichment after 3 h in biomarker PLFAs and a much greater incorporation of new plant-C into fungal compared to bacterial PLFAs after 10 h. After a period of 11 months following the pulse-labeling experiment, the ¹³C enrichment of fungal PLFAs was still largely present but had decreased, while bacterial PLFAs were much more enriched in ¹³C compared to a few hours after the pulse-labeling. These results imply that new rhizodeposit-C is rapidly processed by fungal communities and only much later by the bacterial communities, which we attributed to either a fungal-mediated translocation of rhizosphere-C from the fungal to bacterial biomass or a preferential bacterial use of dead root or fungal necromass materials as C source over the direct utilization of fresh root-exudate C in these N-limited grassland ecosystems. Elevated CO₂ caused an increase in the proportional ¹³C enrichment (relative to the universal biomarker 16:0) of the arbuscular mycorrhizal fungal biomarker PLFA 16:1ω5 and one gram-positive bacterial biomarker PLFA i16:0, but a decrease in the proportional ¹³C enrichment of 18:1ω9c, a commonly used though questionable fungal biomarker PLFA. This suggests enhanced fungal rhizodeposit-C assimilation only by arbuscular mycorrhizal fungal species under elevated CO₂. Allen, A. S., Andrews, J. A., Finzi, A. C., Matamala, R., Richter, D. D., and Schlesinger, W. H.: Effects of free-air CO₂ enrichment (FACE) on belowground processes in a Pinus taeda forest, Ecol. Appl., 10, 437–448, 2000. Alley, R., Berntsen, T., Bindoff, N. L., Chen, Z., Chidthaisong, A., Friedlingstein, P., Gregory, J., Hegerl, G., Heimann, M., Hewitson, B., Hoskins, B., Joos, F., Jouzel, J., Kattsov, V., Lohmann, U., Manning, M., Matsuno, T., Molina, M., Nicholls, N., Overpeck, J., Qin, D., Raga, G., Ramaswamy, V., Ren, J., Rusticucci, M., Solomon, S., Somerville, R., Stocker, T. F., Stott, P., Stouffer, R. J., Whetton, P., Wood, R. A., and Wratt, D.: Climate change 2007: The Physical Science Basis, Contribution of working group I to the fourth assessment report of the Intergovernmental Panel on Climate Change, Paris, 2007. Arao, T.: In situ detection of changes in soil bacterial and fungal activities by measuring 13C incorporation into soil phospholipid fatty acids from $^13$C acetate, Soil Biol. Biochem., 31, 1015–1020, 1999. Beare, M. H.: Fungal and bacterial pathways of organic matter decomposition and nitrogen mineralization in arable soils, in: Soil Ecology in Sustainable Agricultural Systems, edited by: Brussaard, L. and Ferrera-Cerrato, R., Lewis Publishers, Boca Raton, FL, 37–70, 1997. Bailey, V. L., Smith, J. L., and Bolton, H.: Fungal-to-bacterial ratios in soils investigated for enhanced C sequestration, Soil Biol. Biochem., 34, 997–1007, 2002. Billings, S. A. and Ziegler, S. E.: Linking microbial activity and soil organic matter transformations in forest soils under elevated CO₂, Global Change Biol., 11, 203–212, 2005. Boschker, H. T. S., Nold, S. C., Wellsbury, P., Bos, D., de Graaf, W., Pel, R., Parkes, R. J., and Cappenberg, T. E.: Direct linking of microbial populations to specific biogeochemical processes by C-13-labelling of biomarkers, Nature, 392, 801–805, 1998. Bossio, D. A. and Scow, K. M.: Impact of Carbon and Flooding on the Metabolic Diversity of Microbial Communities in Soils, Appl. Environ. Microbiol., 61, 4043–4050, 1995. Bossuyt, H., Denef, K., Six, J., Frey, S. D., Merckx, R., and Paustian, K.: Influence of microbial populations and residue quality on aggregate stability, Appl. Soil Ecol., 16, 195–208, 2001. Bouillon, S., Moens, T., Koedam, N., Dahdouh-Guebas, F., Baeyens, W., and Dehairs, F.: Variability in the origin of carbon substrates for bacterial communities in mangrove sediments, FEMS Microbiol. Ecol., 49, 171–179, 2004. Brennan, P. J.: Mycobacterium and other actinomycetes, in: Microbial Lipids, edited by: Ratledge, C. and Wilkinson, S. G., London, Academic Press, 203–298, 1988. Butler, J. L., Williams, M. A., Bottomley P. J., and Myrold D. D.: Microbial community dynamics associated with rhizosphere carbon flow, Appl. Environ. Mircobiol., 69, 6793–6800, 2003. Carney, K. M., Hungate, B. A., Drake, B. G., and Megonigal, J. P.: Altered soil microbial community at elevated CO₂ leads to loss of soil carbon, Proc. Natl. Acad. Sci. USA, 104, 4990–4995, 2007. Chung, H., Zak, D. R., and Lilleskov, E. A.: Fungal community composition and metabolism under elevated CO₂ and O₃, Oecologia, 147, 143–154, 2006. Chung, H., Zak, D. R., Reich, P. B., and Ellsworth, D. S.: Plant species richness, elevated CO₂, and atmospheric nitrogen deposition alter soil microbial community composition and function, Global Change Biol., 13, 1–10, 2007. Cotrufo, M. F. and Gorissen, A.: Elevated CO₂ enhances below-ground C allocation in three perennial grass species at different levels of N availability, New Phytol., 137, 421–431, 1997. Cotrufo, M. F., Ineson, P., and Rowland, A. P.: Decomposition of tree leaf litters grown under elevated CO2: effect of litter quality, Plant Soil, 163, 121–130, 1994. Curtis, P. S. and Wang, X.: A meta-analysis of elevated CO₂ effects on woody plant mass, form and physiology, Oecologia, 113, 299–313, 1998. DeLucia, E. H., Hamilton, J. G., Naidu, S. L., Thomas, R. B., Andrews, J. A., Finzi, A., Lavine, M., Matamala, R., Mohan, J. E., Hendrey, G. R., and Schlesinger, W. H.: Net primary production of a forest ecosystem with experimental CO₂ enrichment, Science, 289, 1177–1179, 1999. Drijber, R. A., Doran, J. W., Parkhurst, A. M., and Lyon, D. J.: Changes in soil microbial community structure with tillage under long-term wheat-fallow management, Soil Biol. Biochem., 32, 1419–1430, 2000. Drissner, D., Blum, H., Tscherko, D., and Kandeler, E.: Nine years of enriched CO₂ changes the function and structural diversity of soil microorganisms in a grassland, Eur. J. Soil Sci., 58, 260–269, 2007. Ebersberger, D., Wermbter, N., Niklaus, P. A., and Kandeler, E.: Effects of long term CO₂ enrichment on microbial community structure in calcareous grassland, Plant Soil, 264, 313–323, 2004. Federle, T. W.: Microbial distribution in soil – new techniques, in: Perspectives in Microbial Ecology, edited by: Megusar, F. and Ganthar, M., Slovene Society for Microbiology, Ljubljana, 493–498, 1986. Fierer, N., Schimel, J. P., and Holden, P. A.: Variations in microbial community composition through two soil depth profiles, Soil Biol. Biochem., 35, 167–176, 2003. Frankland, J. C., Dighton, J., and Boddy, L.: Methods for studying fungi in soil and forest litter, Meth. Microbiol., 22, 343–404, 1990. Frey, S. D., Elliott, E. T., Paustian, K., and Peterson, G. A.: Fungal translocation as a mechanism for soil nitrogen inputs to surface residue decomposition in a no-tillage agroecosystem, Soil Biol. Biochem., 32, 689–698, 2000. Frostegård, Å. and Bååth, E.: The use of phospholipid fatty acid analysis to estimate bacterial and fungal biomass in soil, Biol. Fertil. Soils, 22, 59–65, 1996. Ghannoum, O., Von Caemmerer, S., Ziska, L. H., and Conroy, J. P.: The growth response of C4 plants to rising atmospheric CO₂ partial pressure: a reassessment, Plant Cell Environ., 23, 931–942, 2000. Gill, R. A., Polley, H. W., Johnson, H. B., Anderson, L. J., Maherali, H., and Jackson, R. B.: Nonlinear grassland responses to past and future atmospheric CO₂, Nature, 417, 279–282, 2002. Griffiths, B. S., Ritz, K., Ebblewhite, N., and Dobson, G.: Soil microbial community structure: effects of substrate loading rates, Soil Biol. Biochem., 30, 369–378, 1999. Griffiths, R. I., Manefield, M., Bailey, M. J., Whitely, A. S., Ostle, N., McNamara, N., and O'Donnell, A. G.: $^13$CO₂ pulse labeling of plants in tandem with stable isotope probing: methodological considerations for examining microbial function in the rhizosphere, J. Microbiol. Meth., 58, 119–129, 2004. Harwood, D. L. and Russell, N. J.: Lipids in Plants and Microbes, George Allen and Unwin Ltd., London, 1984. Hu, S., Chapin, F. S., Firestone, M. K., Field, C. B., and Chiariello, N. R.: Nitrogen limitation of microbial decomposition in a grassland under elevated CO₂, Nature, 409, 188–191, 2001. Jäger, H. -J., Schmidt, S. W., Kammann, C., Grünhage, L., Müller, C., and Hanewald, K.: The University of Giessen Free-Air Carbon Dioxide Enrichment Study: Description of the Experimental Site and of a New Enrichment System, J. Appl. Bot., 77, 117–127, 2003. Jastrow, J. D., Miller, R. M., Matamala, R., Norby, R. J., Boutton, T. W., Rice, C. W., and Owensby, C. E.: Elevated atmospheric carbon dioxide increases soil carbon, Global Change Biol., 11, 2057–2064, 2005. Johnson, D., Leake, J. R., and Read, D. J.: Transfer to recent photosynthate into mycorrhizal mycelium of an upland grassland: short-term respiratory losses and accumulation of $^14$C, Soil Biol. Biochem., 34, 1521–1524, 2002. Jongen, M., Jones, M. B., Hebeisen, T., Blum, H., and Hendrey, G.: The effects of elevated CO₂ concentrations on the root growth of Lolium perenne and Trifolium repens grown in a FACE system, Global Change Biol., 1, 361–371, 1995. Kammann, C., Grünhage, L., Grüters, S., Janze, S., and Jäger, H.-J.: Response of aboveground grassland biomass and soil moisture to moderate long-term CO2 enrichment, Basic Appl. Ecol., 6, 351–365, 2005. Kandeler, E., Mosier, A. R., Morgan, J. A., Milchunas, D. G., King, J. Y., Rudolph, S., and Tscherko, D.: Response of soil microbial biomass and enzyme activities to the transient elevation of carbon dioxide in a semi-arid-grassland, Soil Biol. Biochem., 38, 2448–2460, 2006. Klironomos, J. N., Rillig, M. C., and Allen, M. F.: Below-ground microbial and microfaunal responses to Artemisia tridentata grown under elevated atmospheric CO₂, Funct. Ecol., 10, 527–534, 1996. Körner, C.: Biosphere responses to CO₂ enrichment, Ecol. Appl., 10, 1590–1619, 2000. Kroppenstedt, R. M.: Fatty acid and Menaquinone analysis of actinomycetes and related organisms, in: Chemical Methods in Bacterial Systematics, edited by: Goodfellow, M. and Minnikin, D. E., Academic Press, London, 173–199, 1985. Lipson, D. A., Wilson, R. F., and Oechel, W. C.: Effects of elevated atmospheric CO₂ on soil microbial biomass, activity and diversity in a chaparral ecosystem, Appl. Environ. Mircobiol., 71, 8573–8580, 2005. Lu, Y., Rosencrantz, D., Liesack, W., and Conrad, R.: Structure and activity of bacterial community inhabiting rice roots and the rhizosphere, Environ. Microbiol., 8, 1351–1360, 2006. Lu, Y., Abraham, W-R., and Conrad, R.: Spatial variation of active microbiota in the rice rhizosphere revealed by in situ stable isotope probing of phospholipid fatty acids, Environ. Microbiol., 9, 474–481, 2007. McMahon, S. K., Williams, M. A. Bottomley, P. J., and Myrold, D. D.: Dynamics of microbial communities during decomposition of carbon-13 labeled ryegrass fractions in soil, Soil Sci. Soc. Am. J., 69, 1238–1247, 2005. Montealegre, C. M., van Kessel, C., Russelle, M. P., and Sadowsky, M. J.: Changes in microbial activity and composition in a pasture ecosystem exposed to elevated atmospheric carbon dioxide, Plant Soil, 243, 197–207, 2002. Moscatelli, M. C., Lagomarsino, A., de Angelis, P., and Grego, S.: Seasonality of soil biological properties in a polar plantation growing under elevated atmospheric CO₂, Appl. Soil Ecol., 30, 162–173, 2005. Niklaus, P. A., Alphei, J., Ebersberger, D., Kampichler, C., Kandeler, E., and Tscherko, D.: Six years of in situ CO₂ enrichment evoke changes in soil structure and soil biota of nutrient-poor grassland, Global Change Biol., 9, 585–600, 2003. Olsson, P. A. and Johnson, N. C.: Tracking carbon from the atmosphere to the rhizosphere, Ecol. Let., 8, 1264–1270, 2005. Olsson, P. A., Bååth, E., Jakobsen, I., and Söderström, B.: The use of phospholipid and neutral lipid fatty acids to estimate biomass of arbuscular mycorrhizal fungi in soil, Mycol. Res., 99, 623–629, 1995. Phillips, R. L., Zak, D. R., Holmes, W. E., and White, D. C.: Microbial community composition and function beneath temperate trees exposed to elevated atmospheric carbon dioxide and ozone, Oecologia, 131, 236–244, 2002. Prosser, J. I., Rangel-Castro, J. I., and Killham, K.: Studying plant-microbe interactions using stable isotope technologies, Curr. Opin. Biotechnol., 17, 98–102, 2006. Rangel-Castro, J. I., Killham, K., Ostle, N., Nicol, G. W., Anderson, I. C., Scrimgeour, C. M., Ineson, P., Meharg, A., and Prosser, J. I.: Stable isotope probing analysis of the influence of liming on root exudates utilization by soil microorganisms, Environ. Microbiol., 7, 828–838, 2005. Rillig, M. C., Field, C. B., and Allen, F. A.: Soil biota responses to long-term atmospheric CO₂ enrichment in two California annual grasslands, Oecologia, 119, 572–577, 1999. Rillig, M. C., Wright, S. F., and Eviner, V.T.: The role of arbuscular mycorrhizal fungi and glomalin in soil aggregation: comparing effects of five plant species, Plant Soil, 238, 325–333, 2002. Rogers, H. H., Runion, G. B., and Krupa, S. V.: Plant responses to atmospheric CO$_2 $enrichment with emphasis on roots and the rhizosphere, Environ. Pollut., 83, 155–189, 1994. Rønn, R., Gavito, M., Larsen, J., Jakobsen, I., Frederiksen, H., and Christensen, S.: Response of free-living soil protozoa and microorganisms to elevated atmospheric CO₂ and presence of mycorrhiza, Soil Biol. Biochem., 34, 923–932, 2002. Sadowsky, M. J. and Schortemeyer, M.: Soil microbial response to increased concentrations of atmospheric CO₂, Global Change Biol., 3, 217–224, 1997. Schäppi, B. and Körner, C.: Growth responses of an alpine grassland to elevated CO₂, Oecologia, 1, 43–52, 1996. Schortemeyer, M., Hartwig, U. A., Hendrey, G. R., and Sadowsky, M. J.: Microbial community changes in the rhizosphere of white clover and perennial ryegrass exposed to free air carbon dioxide enrichment (FACE), Soil Biol. Biochem., 28, 1717–1724, 1996. Sonnemann, I. and Wolters, V.: The microfood web of grassland soils responds to a moderate increase in atmospheric CO₂, Global Change Biol., 11, 1148–1155, 2005. Stahl, P. D. and Klug, M. J.: Characterization and differentiation of filamentous fungi based on fatty acid composition, Appl. Environ. Microb., 62, 4136–4146, 1996. Stöcklin, J., Schweizer, K., and Körner, C.: Effects of elevated CO₂ and phosphorus addition on productivity and community composition of intact monoliths from calcareous grassland, Oecologia, 116, 50–56, 1998. Treonis, A. M., Ostle, N. J., Stott, A. W., Primrose, R., Graystone, S. J., and Ineson, P.: Identification of groups of metabolically-active rhizosphere microorganisms by stable isotope probing of PLFAs, Soil Biol. Biochem., 36, 533–537, 2004. Treseder, K. K.: A meta-analysis of mycorrhizal responses to nitrogen, phosphorus, and atmospheric CO₂ in field studies, New Phytol., 164, 347–355, 2004. Treseder, K. K. and Allen, M. F.: Mycorrhizal fungi have a potential role in soil carbon storage under elevated CO₂ and nitrogen deposition. New Phytol., 147, 189–200, 2000. van Groenigen, K. J., Six, J., Hungate, B. A., de Graaff, M. A., van Breemen, N., and van Kessel, C.: Element interactions limit soil carbon storage, Proc. Natl. Acad. Sci. USA, 103, 6571–6574, 2006. Vestal, J. R. and White, D. C.: Lipid analysis in microbial ecology, BioScience, 39, 535–541, 1989. Waldrop, M. P. and Firestone, M. K.: Microbial community utilization of recalcitrant and simple carbon compounds: impact of oak-woodland plant communities, Oecologia, 138, 275–284, 2004. Wardle, D. A., Brown, V. K, Behan-Pelletier, V., St. John, M., Wojtowicz, T., Bardgett, R. D., Brown, G. G., Ineson, P., Lavelle, P., van der Putten, W. H., Anderson, J. M., Brussaard, L., Hunt, W. H., Paul, E. A., and Wall, D. H.: Vulnerability to global change of ecosystem goods and services driven by soil biota, in: Sustaining Biodiversity and Ecosystem Services in Soils and Sediments, edited by: Wall, D. H., Island Press, Washington DC, 101–136, 2004. White, D. C., Davis, W. M., Nickels, J. S., King, J. D., and Bobbie, R. J.: Determination of the sedimentary microbial biomass by extractable lipid phosphate, Oecologia, 40, 51–62, 1979. Williams, M. A., Myrold, D. D., and Bottomley, P. J.: Carbon flow from $^13$C-labeled straw and root residues into the phospholipid fatty acid of a soil microbial community under field conditions, Soil Biol. Biochem., 38, 759–768, 2006. Zak, D. R., Pregitzer, K. S., Curtis, P. S., and Holmes, W. E.: Elevated atmospheric CO₂ and feedback between carbon and nitrogen cycles, Plant Soil, 151, 105–117, 1993. Zak, D. R., Ringelberg, D. B., Pregitzer, K. S., Randlett, D. L., White, D. C., and Curtis, P. S.: Soil microbial communities beneath Populus grandidentata grown under elevated atmospheric CO₂, Ecol. Appl., 6, 257–262, 1996. Zak, D. R., Pregitzer, K. S., Curtis, P. S., and Holmes, W. E.: Atmospheric CO₂ and the composition and function of soil microbial communities, Ecol. Appl., 10, 47–59, 2000. Zelles, L.: Phospholipid fatty acid profiles in selected members of soil microbial communities, Chemosphere, 35, 275–294, 1997.