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0099-2240/05/$08.000 doi:10.1128/AEM.71.12.85738580.2005
Copyright 2005, American Society for Microbiology. All Rights Reserved.
This study reports the effects of long-term elevated atmospheric CO2 on root production and microbial
activity, biomass, and diversity in a chaparral ecosystem in southern California. The free air CO2 enrichment
(FACE) ring was located in a stand dominated by the woody shrub Adenostoma fasciculatum. Between 1995 and
2003, the FACE ring maintained an average daytime atmospheric CO2 concentration of 550 ppm. During the
last two years of operation, observations were made on soil cores collected from the FACE ring and adjacent
areas of chaparral with ambient CO2 levels. Root biomass roughly doubled in the FACE plot. Microbial
biomass and activity were related to soil organic matter (OM) content, and so analysis of covariance was used
to detect CO2 effects while controlling for variation across the landscape. Extracellular enzymatic activity
(cellulase and amylase) and microbial biomass C (chloroform fumigation-extraction) increased more rapidly
with OM in the FACE plot than in controls, but glucose substrate-induced respiration (SIR) rates did not. The
metabolic quotient (field respiration over potential respiration) was significantly higher in FACE samples,
possibly indicating that microbial respiration was less C limited under high CO2. The treatments also differed
in the ratio of SIR to microbial biomass C, indicating a metabolic difference between the microbial commu-
nities. Bacterial diversity, described by 16S rRNA clone libraries, was unaffected by the CO2 treatment, but
fungal biomass was stimulated. Furthermore, fungal biomass was correlated with cellulase and amylase
activities, indicating that fungi were responsible for the stimulation of enzymatic activity in the FACE
treatment.
The rapid increase of carbon dioxide (CO2) in the atmo- comparing communities (6, 27). The present study investigated
sphere over the last century has led to an increased global the effects of 8 years of elevated CO2 treatment on root growth
ecosystem C storage, at least temporarily, by stimulating pho- and on microbial biomass, activity and community structure in
tosynthesis (41). However, the fate of this C and its effects on a chaparral ecosystem in southern California. The system af-
soil microbial communities are uncertain. Predicted changes in forded an opportunity to study how elevated CO2 interacts
atmospheric CO2 are small compared to the relatively high with a complex, patchy landscape in a natural ecosystem with
CO2 concentrations in the pore space of active soils, so effects strong water and nutrient limitations. We tested the hypothesis
of elevated atmospheric CO2 on soil microbes are generally that elevated CO2 increases root biomass, which in turn in-
mediated by plant root production and exudation (22, 30, 50). creases microbial biomass and activity and alters microbial
While plant responses to elevated atmospheric CO2 are fairly community structure. This hypothesis was tested by comparing
well understood, the responses of soil microbial communities how microbial parameters varied across the landscape inside a
are highly variable. In response to elevated CO2, microbial free air CO2 enrichment (FACE) treatment ring relative to the
biomass and activity have been observed to decrease (14, 34, surrounding landscape.
43), increase (15, 48), or remain unchanged (32, 35). No con-
sistent effects of increased CO2 on soil microbial community
MATERIALS AND METHODS
composition have yet emerged. CO2-induced changes in mi-
crobial community composition have been detected in some Site description and FACE treatment. The research was conducted at San
Diego State Universitys Sky Oaks Field Station in northeastern San Diego
cases (15, 17, 28, 37), but none were found in others (16, 49). County, California, (3323 N, 11637 W; 1,420 m above sea level). The chap-
Effects of global change on soil microbial communities are arral at Sky Oaks Field Station is dominated by the shrubs Adenostoma fascicu-
potentially important in that microbes can control the re- latum H. & A., Adenostoma sparsifolium Torr., and Ceanothus greggii Gray. The
sponses of ecosystems through their effects on C and nutrient soil in the study area is a loamy sand, Ultic Haploxeroll, with a bulk density of
1.04g cm3, containing 32% rocks, and belonging to the Sheephead series (8)
cycling, yet little progress has been made in this area. Soil
(see Table 1 for other soil properties). The entire area used in this study was
microbial communities remain mysterious mainly because of burned in July 1992 prior to the establishment of the FACE treatment. The site
their extraordinary complexity (10, 13). However, this field of was dominated by A. fasciculatum, a species which quickly regenerates after fire
study has recently benefited from the combination of molecu- by resprouting from a lignotuber (9). The purpose of the burning treatment was
lar techniques to describe microbial communities (18, 33) with to minimize historical differences in vegetation and soil properties that might
have existed across the landscape. The FACE ring was 16 m in diameter,
more sophisticated phylogenetic techniques for analyzing and occupying an area of 178 m2 of chaparral. The CO2 concentration was main-
tained near 550 ppm during the daylight hours by releasing compressed CO2 gas
from pipes at the perimeter of the ring. Wind direction was continuously sensed
* Corresponding author. Mailing address: Department of Biology, so that gas was released only on the upwind side of the ring. The set point was
San Diego State University, San Diego, CA 92182-4614. Phone: (619) maintained within 10% for 87% of the time and within 20% for 96% of the time.
594-4460. Fax: (619) 594-5676. E-mail: dlipson@sciences.sdsu.edu. A detailed description of the construction and operation of the FACE facility is
8573
8574 LIPSON ET AL. APPL. ENVIRON. MICROBIOL.
TABLE 1. Properties of soil from plant and interplant samples SIR is commonly used to represent general heterotrophic microbial activity and
of FACE and outside control plots biomass (2, 24, 42). SIR measurement was performed with soils near optimal
water content (60% of field capacity). Microbial biomass C was measured by
Mean value (SD) the chloroform fumigation-extraction method (21) with modifications (23). The
Parameter Face Outside na activities of extracellular enzymes in breaking down carboxymethylcellulose (a
soluble cellulose analog) and starch (amylase activity) were measured as de-
Plant Interplant Plant Interplant scribed earlier (23). Fungal biomass was measured by direct microscopic obser-
vation, using the grid-intersection method to estimate fungal length (7). Hyphal
% OM 4.2 (1.3) 2.8 (0.4) 5.2 (1.6) 4.2 (1.5) 103
length was converted to biomass by using an estimated average hyphal diameter
% GWC 9.6 (7.0) 11.5 (2.4) 12.0 (7.3) 13.7 (5.6) 103
of 5 m and a factor of 0.26 g biomass cm3 cell volume (7). Bacteria stained
% Sand 73.32 (0.65) 74.00 (0.79) 77.44 (1.93) 72.03 (4.91) 17
with DAPI (4,6-diamidino-2-phenylindole) (Molecular Probes, Eugene, OR)
% Silt 15.48 (1.54) 12.85 (0.39) 14.49 (1.43) 15.47 (1.60) 17
were counted by fluorescence microscopy and converted to biomass by assuming
% Clay 11.20 (1.15) 13.15 (0.89) 8.07 (2.01) 12.50 (3.38) 17
0.27 pg/cell.
a 16S rRNA clone libraries. Soil collected in February 2002 was used for the
n, sample size for analysis.
construction of clone libraries. To obtain a spatially averaged measure of bac-
terial diversity for each treatment (and because of the high cost of producing and
sequencing multiple clone libraries), four spatial replicates from each sample
available elsewhere (38). The FACE treatment operated from January 1995 to
type were pooled, producing four clone libraries (under plants or in gaps from
May 2003. The surrounding landscape (10 m beyond the ring) served as a
FACE and control plots). Soil was extracted using a modified bead beating
control with ambient levels of CO2 (360 ppm).
protocol (29). Tubes containing approximately 5 g soil samples, 2.0 g zirconia/
Soil collection and analysis. Soil samples were collected on various dates from
2001 to 2003, mainly during spring and summer. On dates in 2003, soil respira- silica beads (0.1 mm; BioSpec Products, Bartlesville, OK), and 10 ml lysis buffer
tion measurements were taken (EGM-4 gas analyzer with SRC-1 chamber; PP (Tris-EDTA with 0.2% sodium dodecyl sulfate) were vortexed (Vortex Genie II;
Systems, Amesbury MA) before samples were collected from the same area. Soil Fisher Scientific) at maximum speed for 5 min. To the resultant mixtures, 30
samples were generally collected with a 5-cm-diameter polyvinylchloride pipe to units of proteinase K and 10 units lysozyme (Fisher Bioreagents) were added,
a depth of 12 to 15 cm, except in March 2003, when samples were collected for and the samples were incubated in a shaking incubator (37C, 100 rpm) for 1 h.
root biomass measurements by using a 10-cm-diameter metal soil coring device Standard protocols were used to purify DNA by cetyltrimethylammonium bro-
to a depth of 30 cm. Holes were back-filled to minimize disturbance in the plots. mide extraction (5). DNA was further purified by agarose gel extraction (Qiaex
Samples were collected directly under plant canopies and in gaps between plants, II; QIAGEN). Bacterial 16S rRNA genes were amplified using universal bacte-
from the FACE ring and from the control area. For root biomass measurements, rial primers f8-27 (5-AGAGTTTGATCCTGGCTCAG-3) and r1510 (5-GGT
the samples were taken at 10 cm and 30 cm from the bases of plants located TACCTTGTTACGACTT-3). The PCR mixture consisted of 3.0 mM MgCl2,
within or outside the FACE ring. Soil samples were sieved (2 mm), and roots 0.2 mM of each deoxynucleoside triphosphate, 1 M of each primer, 1 g/liter
were separated from soil and sorted into size classes. Roots were rinsed in bovine serum albumin, 50 mM betaine, 1 unit Fisher Taq polymerase, and buffer
deionized water and dried at 60C to constant weight. Soil organic matter (OM) A supplied with the enzyme (Fisher Biosciences). After an initial denaturation
(weight loss on combustion at 500C for 24 h) and gravimetric water content step of 4 min at 94C, reactions were run for 32 cycles (1 min at 94C, 45 s at
(GWC) (100C until constant weight) were determined for all soil samples. Soil 56C, and 1 min at 72C), followed by a final 10-min extension step at 72C. The
texture was measured for a subset of the samples by using sieving and sedimen- PCR product was gel purified (Quiex II; QIAGEN). The purified product was
tation. The samples used for molecular analysis of bacterial diversity were kept cloned using the TOPO TA cloning kit (Invitrogen). Clones from the four
frozen (80C) until analysis. The samples used for measurements of microbial libraries were partially sequenced on a Prism 3100 capillary electrophoresis DNA
activity and biomass were kept cool (0 to 4C) until analysis (up to 1 week). sequencer (ABI) at the San Diego State University Microchemical Core Facility,
Microbial biomass and activity. Substrate-induced respiration (SIR) measure- using universal bacterial primer r1111 (5-TTGCGCTCGTTGCGGGACT-3).
ments using glucose were done as described earlier (24). Briefly, using sidearm Statistical and phylogenetic analyses. Regression analysis showed that most
flasks (Bellco Glass, Vineland, NJ), enough glucose was added to soils to max- measured variables were significantly related to OM. To control for variation in
imize respiration (2 mg C g1 soil), along with [14C]glucose (0.1 Ci g1). OM across the landscape and between treatments, the effect of CO2 on most
Evolved CO2 was trapped in 1 ml NaOH (1 M) in the sidearm portion of each variables was tested by analysis of covariance (ANCOVA). These analyses in-
flask, and radioactivity was measured by liquid scintillation counting. Glucose cluded data from several dates, so a full general linear model was first used to test
FIG. 1. Fine and coarse plant root dry masses in cores from FACE and control soils collected at two distances from bases of plants. Each bar
represents the mean and standard error of 9 or 10 measurements.
VOL. 71, 2005 ELEVATED CO2 AND CHAPARRAL SOIL MICROBES 8575
FIG. 2. Variation in soil respiration with (A) soil organic matter FIG. 3. Variation in (A) microbial biomass C and (B) glucose SIR
(grams OM gram1 soil) and (B) soil water content (grams H2O with soil OM (grams OM gram1 soil) in FACE and control plots. The
gram1 soil) in FACE and outside control plots. The P values are for P values are for the CO2-OM interaction term in the ANCOVA.
the interaction terms in the ANCOVA.
FIG. 7. Neighbor-joining phylogenetic tree of 16S rRNA sequences in clone libraries from plant and interplant samples of FACE and outside
control plots. Firmi, Firmicutes; CFB, Cytophaga-Bacteroides-Flexibacter; Actino, Actinobacteria; BD, Gemmatimonadetes/BD; Verruco, Verrucomi-
crobia; NS, Nitrospira; GNS, green nonsulfur bacteria.
the metabolic state of microbial biomass. These variables were lated significantly with the activities of amylase and cellulase
both log transformed before the analysis in Table 2, which enzymes in the soil (Fig. 6), whereas bacterial biomass did not
shows that this ratio is lower in FACE soils. These respiratory (data not shown).
parameters were not correlated with soil OM, and so both were Bacterial diversity. The soil bacterial community was dom-
analyzed by one-way ANOVA. inated by the Acidobacteria and Proteobacteria phyla (Fig. 7 and
The ratio of fungal biomass to total microbial biomass C was 8), as is typical for soils (18). Based on the Chao1 parameter,
higher in FACE soils than in control soils (Table 2), and this there were 445 225 distinct bacterial ribotypes in this com-
differential between FACE and control plots increased with munity. There was no obvious clustering of sequences from the
increasing biomass (Fig. 5), indicating that the stimulatory same sample type in Fig. 7, and PTP analysis confirms that the
effect of elevated CO2 on microbial biomass disproportionately bacterial community is not different between the FACE and
affected fungi. Bacterial biomass was not significantly different control plots (P 0.310) or between plant and gap samples
in FACE and control plots (Table 2). Fungal biomass corre- (P 0.561). Similarly, the Fst statistic showed that the FACE
8578 LIPSON ET AL. APPL. ENVIRON. MICROBIOL.
responses of mycorrhizal and saprotrophic fungi are beyond less sensitive to drought than control soil respiration, possibly
the scope of this study. Stimulation of arbuscular mycorrhizae extending ecosystem C loss during dry periods. On the other
(AM) and changes in AM species composition by elevated hand, a continued shift toward a fungus-dominated microbial
CO2 have been reported for A. fasciculatum and other chap- community with lower potential respiration rates per unit bio-
arral plants (36, 44), and higher plant allocation to mycorrhizae is mass could lead to a decreased ability of the microbial com-
consistent with the observed increase in root growth. The data munity to respond to C inputs and could change the relation-
strongly suggest that saprotrophic fungi are stimulated as well. ship between soil respiration and microbial biomass in this
The increased extracellular enzymatic activities under elevated ecosystem. This result emphasizes the need to understand the
CO2 correlated with fungal biomass, indicating that saprotro- relationship between microbial community structure and soil
phic fungi responded to increased root biomass with growth respiration under current and future conditions.
and exoenzyme production. Other researchers have found that
ACKNOWLEDGMENTS
fungi increase in response to inputs of live and dead plant roots
(51). Dark septate fungal hyphae were commonly observed Thanks go to Michelle Blair, Yufu Cheng, Steve Hastings, Pablo
in soil samples (unpublished observation). Based on mor- Bryant, and Joe Verfaillie for field, laboratory, and logistical assistance
and to Scott Kelley for assistance with the phylogenetic analyses.
phology, this type is clearly not an AM fungus, although
Thanks also go to the anonymous reviewers, who provided many de-
such morphotypes have also been observed in possible as- tailed and helpful comments.
sociations with A. fasciculatum roots (1). Increased root
REFERENCES
biomass probably stimulated fungi that thrive on both dead
1. Allen, M. F., L. M. Egerton-Warburton, E. B. Allen, and O. Karen. 1999.
and live roots. Mycorrhizae in Adenostoma fasciculatum Hook. & Arn.: a combination of
The stimulation of root growth by elevated CO2 is consistent unusual ecto- and endo-forms. Mycorrhiza 8:225228.
with the increased photosynthesis and leaf and stem area index 2. Anderson, J. P. E., and K. H. Domsch. 1978. A physiological method for the
quantitative measurement of microbial biomass in soils. Soil Biol. Biochem.
observed in the FACE treatment by others (11) and has been 10:215221.
widely reported for many ecosystems (22, 31). Additionally, 3. Anderson, T.-H., and K. H. Domsch. 1985. Determination of eco-physiolog-
ical maintenance requirements of soil microorganisms in a dormant state.
leaf tissue chemistry was altered in the FACE plots (26). It Biol. Fert. Soils. 1:8189.
follows that microbial biomass and extracellular enzymatic ac- 4. Anderson, T-H., and K. H. Domsch. 1989. Ratios of microbial biomass
tivity would respond to increased root growth and altered leaf carbon to total organic carbon in arable soils. Soil Biol. Biochem. 21:471
479.
chemistry, as observed in this study, although this is not always 5. Ausubel, F. M. (ed.) 1994. Current protocols in molecular biology, John
the case. Occasionally a smaller, more active pool of microbial Wiley and Sons, New York, N.Y.
biomass has been reported to result from elevated CO2 (15, 40, 6. Bohannan, B. J. M., and J. Hughes. 2003. New approaches to analyzing
microbial biodiversity data. Curr. Opin. Microbiol. 6:282287.
45, 50). In the present study, the respiratory physiology of the 7. Bottomley, P. J. 1994. Light microscopic methods for studying soil mi-
microbial community shifted in response to elevated CO2. The croorganisms, p. 81104. In S. H. Mickelson (ed.), Methods of soil anal-
ysis, part 2. Microbiological and biochemical properties. Soil Science
increased ratio of soil respiration to glucose SIR could have Society of America, Madison, Wis.
been caused by a better-fed microbial community functioning 8. Bowman, R. H. 1973. Soil survey of the San Diego area, California, part I.
closer to its respiratory potential (3, 20, 47). This effect could USDA Soil Conservation Service and Forest Service, Washington, D.C.
9. Canadell, J., and P. H. Zedler. 1995. Underground structures of woody
also be caused by stimulated root respiration: although soil plants in Mediterranean regions of California, Chile, and Australia, p. 177
respiration in the FACE treatment was not significantly in- 210. In M. T. Kalin-Arroyo, P. H. Zedler, and M. D. Fox (ed.), Ecology
creased in the present study, higher respiration rates were and biogeography of Mediterranean ecosystems in Chile, California, and
Australia. Springer-Verlag, New York, N.Y.
observed in a more temporally intensive study (11). The mi- 10. Chatzinotas, A., R. A. Sandaa, W. Schoenhuber, R. Amann, F. L. Daae, V.
crobial community in the FACE samples had a markedly lower Torsvik, J. Zeyer, and D. Hahn. 1998. Analysis of broad-scale differences in
microbial community composition of two pristine forest soils. Syst. Appl.
ratio of glucose SIR to microbial biomass C. This could be Microbiol. 21:579587.
attributed to the higher proportion of fungi in FACE samples. 11. Cheng, Y., W. C. Oechel, P. J. Bryant, S. J. Hastings. The impacts of elevated
Filamentous fungi have the ability to shift resources through- CO2 on carbon flux of southern California chaparral using free-air CO2
enrichment. Submitted for publication.
out their mycelial network to exploit areas of high resources, 12. Colwell, R. K. 1997. EstimateS: statistical estimation of species richness
while maintaining viable but inactive hyphae elsewhere in the and shared species from samples, version 5. Users guide and application.
soil. Higher levels of viable, inactive biomass in fungus- [Online.] http://viceroy.eeb.uconn.edu/estimates.
13. Curtis, T. P., W. T. Sloan, and J. W. Scannall. 2002. Estimating prokaryotic
dominated soils would result in lower SIR activity per unit diversity and its limits. Proc. Natl. Acad. Sci. USA 99:1049410499.
microbial biomass C as measured by fumigation-extraction. 14. Diaz, S., J. P. Grime, J. Harris, and E. McPherson. 1993. Evidence of a
feedback mechanism limiting plant response to elevated carbon dioxide.
Bacteria and fungi generally have different growth kinetics Nature 364:616617.
(25), and bacterium/fungus ratios have been linked to vari- 15. Grayston, S. J., C. D. Campbell, J. L Lutze, and R. M. Gifford. 1998. Impact
ations in specific respiration (respiration per unit biomass) of elevated CO2 on the metabolic diversity of microbial communities in N
limited grass swards. Plant Soil 203:289300.
across soil types (39). 16. Griffiths, B. S., K. Ritz, N. Ebblewhite, E. Paterson, and K. Killham. 1998.
This study strongly suggests that fungi, and not bacteria, Ryegrass rhizosphere microbial community structure under elevated carbon
respond to increased root growth under elevated CO2 and that dioxide concentrations, with observations on wheat rhizosphere. Soil Biol.
Biochem. 30:315321.
fungi and bacteria differ significantly in their respiratory prop- 17. Horz, H. P., A. Barbrook, C. B. Field, and B. J. M. Bohannan. 2004. Am-
erties. These results may also have implications for the C monia-oxidizing bacteria respond to multifactorial global change. Proc. Natl.
Acad. Sci. USA 101:1513615141.
balance of the chaparral ecosystem under elevated CO2. While 18. Hugenholtz, P., B. M. Goebel, and N. R. Pace. 1998. Impact of culture-
root biomass was stimulated, this potential sink is likely to be independent studies on the emerging phylogenetic view of bacterial diversity.
offset by increased decomposition activity, as demonstrated by J. Bacteriol. 180:47654774.
19. Insam, H., and K. H. Domsch. 1988. Relationship between soil organic C and
higher cellulase and amylase activities in the FACE plot. Fur- microbial biomass on chronosequences of reclamation sites. Microb. Ecol.
thermore, soil respiration under elevated CO2 appeared to be 15:177188.
8580 LIPSON ET AL. APPL. ENVIRON. MICROBIOL.
20. Insam, H., and K. Haselwandter. 1989. Metabolic coefficient of the soil 37. Rillig, M. C., K. M. Scow, J. N. Klironomos, and M. F. Allen. 1997. Microbial
microflora in relation to plant succession. Oecologia 79:174178. carbon substrate utilization in the rhizosphere of Gutierrezia sarothrae grown
21. Jensen, L. S., and J. Sorensen. 1994. Microscale fumigation-extraction and in elevated atmospheric carbon dioxide. Soil Biol. Biochem. 29:13871394.
substrate-induced respiration methods for measuring microbial biomass in 38. Roberts, S. W., W. C. Oechel, P. J. Bryant, S. J. Hastings, J. Major, and V.
barley rhizosphere. Plant Soil 162:151161. Nosov. 1998. A field fumigation system for elevated carbon dioxide exposure
22. Korner, C., M. Diemer, B. Schappi, P. Niklaus, and J. Arnone, III. 1997. The in chaparral shrubs. Func. Ecol. 12:708719.
responses of alpine grassland to four seasons of CO2 enrichment: a synthesis. 39. Sakamoto, K., and Y. Oba. 1994. Effect of fungal to bacterial biomass ratio
Acta Oecologica 18:165175. on the relationship between CO2 evolution and total soil microbial biomass.
23. Lipson, D. A., C. W. Schadt, and S. K. Schmidt. 2002. Changes in microbial Biol. Fertil. Soils 17:3944.
community structure and function following snow melt in an alpine soil. 40. Santruckova, H., and M. Simek. 1994. Soil microorganisms at different CO2
Microb. Ecol. 43:307314.
and O2 tensions. Folia Microbiol. 39:225230.
24. Lipson, D. A., S. K. Schmidt, and R. K. Monson. 1999. Links between
41. Schimel, D. S., J. Melillo, H. Tian, A. D. McGuire, D. Kicklighter, T. Kittel,
microbial population dynamics and N availability in an alpine ecosystem.
N. Rosenbloom, S. Running, P. Thornton, D. Ojima, W. Parton, R. Kelly, M.
Ecology 80:16231631.
25. Lipson, D. A., and S. K. Schmidt. 2002. Kinetics of microbial processes and Sykes, R. Neilson, and B. Rizzo. 2000. Contribution of increasing CO2 and
population growth in soil, p. 17481757. In G. Bitton (ed.) The encyclopedia climate to carbon storage by ecosystems in the United States. Science 287:
of environmental microbiology. Wiley and Sons, New York, N.Y. 20042006.
26. Marriott, A. M. 2003. Effects of elevated carbon dioxide on Adenostoma 42. Schmidt, S. K. 1992. A substrate-induced growth-response (SIGR) method
fasciculatum leaf nutrients. M.S. thesis. San Diego State University, San for estimating the biomass of microbial functional groups in soil and aquatic
Diego, Calif. systems. FEMS Microbiol. Ecol. 101:197206.
27. Martin, A. P. 2002. Phylogenetic approaches for describing and comparing 43. Schortemeyer, M., U. A. Hartwig, G. R. Hendrey, and M. J. Sadowsky. 1996.
the diversity of microbial communities. Appl. Environ. Microbiol. 68:3673 Microbial community changes in the rhizospheres of white clover and pe-
3682. rennial ryegrass exposed to free air carbon dioxide enrichment (FACE). Soil
28. Mayr, C., M. Miller, and H. Insam. 1999. Elevated CO2 alters community Biol. Biochem. 28:17171724.
level physiological profiles and enzyme activities in alpine grassland. J. Mi- 44. Treseder, K. K., L. M. Egerton-Warburton, M. F. Allen, Y. F. Cheng, and
crobiol. Methods 36:3543. W. C. Oechel. 2003. Alteration of soil carbon pools and communities of
29. Miller, D. N., J. E. Bryant, E. L. Madsen, and W. C. Ghiorse. 1999. Evalu- mycorrhizal fungi in chaparral exposed to elevated carbon dioxide. Ecosys-
ation and optimization of DNA extraction and purification procedures for tems 6:786796.
soil and sediment samples. Appl. Environ. Microbiol. 65:47154724. 45. Van Ginkel, J. H., A. Gorissen, and J. A. van Veen. 1996. Long term decom-
30. Montealegre, C. M., C. van Kessel, M. P. Russele, and M. J. Sadowsky. 2002. position of grass roots as affected by elevated atmospheric carbon dioxide. J.
Changes in microbial activity and composition in a pasture ecosystem ex- Environ. Qual. 25:11221128.
posed to elevated atmospheric carbon dioxide. Plant Soil 243:197207. 46. Wardle, D. A. 1992. A comparative assessment of factors which influence
31. Norby, R. J., and R. B. Jackson. 2000. Root dynamics and global change: microbial biomass carbon and nitrogen levels in soils. Biol. Rev. 67:321358.
seeking an ecosystem perspective. New Phytol. 147:312. 47. Wardle, D. A., and A. Ghani. 1995. A critique of the microbial metabolic
32. ONeill, E. G. 1994. Response of soil biota to elevated atmospheric carbon quotient (qC02) as a bioindicator of disturbance and ecosystem develop-
dioxide. Plant Soil 165:5565.
ment. Soil Biol. Biochem. 27:16011610.
33. Pace, N. R. 1997. A molecular view of microbial diversity and the biosphere.
48. Williams, M. A., C. W. Rice, and C. E. Owensby. 2000. Carbon dynamics and
Science 276:734740.
34. Prior, S. A., H. A. Torbert, G. B. Runion, H. H. Rogers, C. W. Wood, B. A. microbial activity in tallgrass prairie exposed to elevated CO2 for 8 years.
Kimball, R. L. Lamorte, P. J. Winter, and G. W. Wall. 1997. Free air carbon Plant Soil 227:127137.
dioxide enrichment of wheat: soil carbon and nitrogen dynamics. J. Environ. 49. Zak, D. R., K. S. Pregitzer, P. S. Curtis, W. E. Holmes. 2000. Atmospheric
Qual. 26:11611166. CO2 and the composition and function of soil microbial communities. Ecol.
35. Randlett, D. L., D. R. Zak, K. S. Pregitzer, and P. S. Curtis. 1996. Elevated Appl. 10:4759.
atmospheric carbon dioxide and leaf litter chemistry: influences on microbial 50. Zak, D. R., K. S. Pregitzer, J. S. King, and W. E. Holmes. 2000. Elevated
respiration and net nitrogen mineralization. Soil Sci. Soc. Am. J. 60:1571 atmospheric CO2, fine roots and the response of soil microorganisms: a
1577. review and hypothesis. New Phytol. 147:201222.
36. Rillig, M. C., and M. F. Allen. 1998. Arbuscular mycorrhizae of Gutierrezia 51. Zhu, W., J. G. Ehrenfeld, R. W. Parmelee, W. F. J. Parsons, X. Han. 1996.
sarothrae and elevated carbon dioxide: evidence for shifts in C allocation to The effects of live and dead roots on soil fungi in spodosolic soils of the New
and within the mycobiont. Soil Biol. Biochem. 30:20012008. Jersey Pinelands. Biol. Fertil. Soils 21:215226.