APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 2005, p. 85738580 Vol. 71, No.

12
0099-2240/05/$08.000 doi:10.1128/AEM.71.12.85738580.2005
Copyright 2005, American Society for Microbiology. All Rights Reserved.

Effects of Elevated Atmospheric CO2 on Soil Microbial Biomass,

Activity, and Diversity in a Chaparral Ecosystem
David A. Lipson,* Richard F. Wilson, and Walter C. Oechel
Department of Biology, San Diego State University, San Diego, California 92182-4614
Received 28 May 2005/Accepted 13 September 2005

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-
sphere over the last century has led to an increased global
ecosystem C storage, at least temporarily, by stimulating pho-
tosynthesis (41). However, the fate of this C and its effects on
soil microbial communities are uncertain. Predicted changes in
atmospheric CO2 are small compared to the relatively high
CO2 concentrations in the pore space of active soils, so effects
of elevated atmospheric CO2 on soil microbes are generally
mediated by plant root production and exudation (22, 30, 50).
While plant responses to elevated atmospheric CO2 are fairly
well understood, the responses of soil microbial communities
are highly variable. In response to elevated CO2, microbial
biomass and activity have been observed to decrease (14, 34,
43), increase (15, 48), or remain unchanged (32, 35). No con-
sistent effects of increased CO2 on soil microbial community
composition have yet emerged. CO2-induced changes in mi-
crobial community composition have been detected in some
cases (15, 17, 28, 37), but none were found in others (16, 49).
Effects of global change on soil microbial communities are
potentially important in that microbes can control the re-
sponses of ecosystems through their effects on C and nutrient
cycling, yet little progress has been made in this area. Soil
microbial communities remain mysterious mainly because of
their extraordinary complexity (10, 13). However, this field of
study has recently benefited from the combination of molecu-
lar techniques to describe microbial communities (18, 33) with
more sophisticated phylogenetic techniques for analyzing and
* Corresponding author. Mailing address: Department of Biology,
San Diego State University, San Diego, CA 92182-4614. Phone: (619)
594-4460. Fax: (619) 594-5676. E-mail: dlipson@sciences.sdsu.edu.
comparing communities (6, 27). The present study investigated
the effects of 8 years of elevated CO2 treatment on root growth
and on microbial biomass, activity and community structure in
a chaparral ecosystem in southern California. The system af-
forded an opportunity to study how elevated CO2 interacts
with a complex, patchy landscape in a natural ecosystem with
strong water and nutrient limitations. We tested the hypothesis
that elevated CO2 increases root biomass, which in turn in-
creases microbial biomass and activity and alters microbial
community structure. This hypothesis was tested by comparing
how microbial parameters varied across the landscape inside a
free air CO2 enrichment (FACE) treatment ring relative to the
surrounding landscape.
MATERIALS AND METHODS
Site description and FACE treatment. The research was conducted at San
Diego State Universitys Sky Oaks Field Station in northeastern San Diego
County, California, (3323 N, 11637 W; 1,420 m above sea level). The chap-
arral at Sky Oaks Field Station is dominated by the shrubs Adenostoma fascicu-
latum H. & A., Adenostoma sparsifolium Torr., and Ceanothus greggii Gray. The
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)
(see Table 1 for other soil properties). The entire area used in this study was
burned in July 1992 prior to the establishment of the FACE treatment. The site
was dominated by A. fasciculatum, a species which quickly regenerates after fire
by resprouting from a lignotuber (9). The purpose of the burning treatment was
to minimize historical differences in vegetation and soil properties that might
have existed across the landscape. The FACE ring was 16 m in diameter,
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
so that gas was released only on the upwind side of the ring. The set point was
maintained within 10% for 87% of the time and within 20% for 96% of the time.
A detailed description of the construction and operation of the FACE facility is

8573
8574 LIPSON ET AL.
TABLE 1. Properties of soil from plant and interplant samples
of FACE and outside control plots
Mean value (SD)
Parameter Face Outside na
Plant Interplant Plant Interplant
% OM 4.2 (1.3) 2.8 (0.4) 5.2 (1.6) 4.2 (1.5) 103
% GWC 9.6 (7.0) 11.5 (2.4) 12.0 (7.3) 13.7 (5.6) 103
% Sand 73.32 (0.65) 74.00 (0.79) 77.44 (1.93) 72.03 (4.91) 17
% Silt 15.48 (1.54) 12.85 (0.39) 14.49 (1.43) 15.47 (1.60) 17
% Clay 11.20 (1.15) 13.15 (0.89) 8.07 (2.01) 12.50 (3.38) 17
a 16S rRNA clone libraries. Soil collected in February 2002 was used for the
n, sample size for analysis.
available elsewhere (38). The FACE treatment operated from January 1995 to
May 2003. The surrounding landscape (10 m beyond the ring) served as a
control with ambient levels of CO2 (360 ppm).
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-
tion measurements were taken (EGM-4 gas analyzer with SRC-1 chamber; PP
Systems, Amesbury MA) before samples were collected from the same area. Soil
samples were generally collected with a 5-cm-diameter polyvinylchloride pipe to
a depth of 12 to 15 cm, except in March 2003, when samples were collected for
root biomass measurements by using a 10-cm-diameter metal soil coring device
to a depth of 30 cm. Holes were back-filled to minimize disturbance in the plots.
Samples were collected directly under plant canopies and in gaps between plants,
from the FACE ring and from the control area. For root biomass measurements,
the samples were taken at 10 cm and 30 cm from the bases of plants located
within or outside the FACE ring. Soil samples were sieved (2 mm), and roots
were separated from soil and sorted into size classes. Roots were rinsed in
deionized water and dried at 60C to constant weight. Soil organic matter (OM)
(weight loss on combustion at 500C for 24 h) and gravimetric water content
(GWC) (100C until constant weight) were determined for all soil samples. Soil
texture was measured for a subset of the samples by using sieving and sedimen-
tation. The samples used for molecular analysis of bacterial diversity were kept
frozen (80C) until analysis. The samples used for measurements of microbial
activity and biomass were kept cool (0 to 4C) until analysis (up to 1 week).
Microbial biomass and activity. Substrate-induced respiration (SIR) measure-
ments using glucose were done as described earlier (24). Briefly, using sidearm
flasks (Bellco Glass, Vineland, NJ), enough glucose was added to soils to max-
imize respiration (2 mg C g1 soil), along with [14C]glucose (0.1 Ci g1).
Evolved CO2 was trapped in 1 ml NaOH (1 M) in the sidearm portion of each
flask, and radioactivity was measured by liquid scintillation counting. Glucose
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.
APPL. ENVIRON. MICROBIOL.
SIR is commonly used to represent general heterotrophic microbial activity and
biomass (2, 24, 42). SIR measurement was performed with soils near optimal
water content (60% of field capacity). Microbial biomass C was measured by
the chloroform fumigation-extraction method (21) with modifications (23). The
activities of extracellular enzymes in breaking down carboxymethylcellulose (a
soluble cellulose analog) and starch (amylase activity) were measured as de-
scribed earlier (23). Fungal biomass was measured by direct microscopic obser-
vation, using the grid-intersection method to estimate fungal length (7). Hyphal
length was converted to biomass by using an estimated average hyphal diameter
of 5 m and a factor of 0.26 g biomass cm3 cell volume (7). Bacteria stained
with DAPI (4,6-diamidino-2-phenylindole) (Molecular Probes, Eugene, OR)
were counted by fluorescence microscopy and converted to biomass by assuming
0.27 pg/cell.
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
type were pooled, producing four clone libraries (under plants or in gaps from
FACE and control plots). Soil was extracted using a modified bead beating
protocol (29). Tubes containing approximately 5 g soil samples, 2.0 g zirconia/
silica beads (0.1 mm; BioSpec Products, Bartlesville, OK), and 10 ml lysis buffer
(Tris-EDTA with 0.2% sodium dodecyl sulfate) were vortexed (Vortex Genie II;
Fisher Scientific) at maximum speed for 5 min. To the resultant mixtures, 30
units of proteinase K and 10 units lysozyme (Fisher Bioreagents) were added,
and the samples were incubated in a shaking incubator (37C, 100 rpm) for 1 h.
Standard protocols were used to purify DNA by cetyltrimethylammonium bro-
mide extraction (5). DNA was further purified by agarose gel extraction (Qiaex
II; QIAGEN). Bacterial 16S rRNA genes were amplified using universal bacte-
rial primers f8-27 (5-AGAGTTTGATCCTGGCTCAG-3) and r1510 (5-GGT
TACCTTGTTACGACTT-3). The PCR mixture consisted of 3.0 mM MgCl2,
0.2 mM of each deoxynucleoside triphosphate, 1 M of each primer, 1 g/liter
bovine serum albumin, 50 mM betaine, 1 unit Fisher Taq polymerase, and buffer
A supplied with the enzyme (Fisher Biosciences). After an initial denaturation
step of 4 min at 94C, reactions were run for 32 cycles (1 min at 94C, 45 s at
56C, and 1 min at 72C), followed by a final 10-min extension step at 72C. The
PCR product was gel purified (Quiex II; QIAGEN). The purified product was
cloned using the TOPO TA cloning kit (Invitrogen). Clones from the four
libraries were partially sequenced on a Prism 3100 capillary electrophoresis DNA
sequencer (ABI) at the San Diego State University Microchemical Core Facility,
using universal bacterial primer r1111 (5-TTGCGCTCGTTGCGGGACT-3).
Statistical and phylogenetic analyses. Regression analysis showed that most
measured variables were significantly related to OM. To control for variation in
OM across the landscape and between treatments, the effect of CO2 on most
variables was tested by analysis of covariance (ANCOVA). These analyses in-
cluded data from several dates, so a full general linear model was first used to test
VOL. 71, 2005
FIG. 2. Variation in soil respiration with (A) soil organic matter
(grams OM gram1 soil) and (B) soil water content (grams H2O
gram1 soil) in FACE and outside control plots. The P values are for
the interaction terms in the ANCOVA.
effects of date, CO2 treatment, and OM on microbial activity and biomass. As
can be seen by the reasonably good fits of the regressions in the figures discussed
below, OM generally accounted for the majority of the variance. The CO2-date
interaction was not significant for any of the variables tested. For these reasons
and because the primary purpose of this study was not to address seasonal
changes, date was not factored into the analysis. When the effect of OM was not
significant, a simple analysis of variance (ANOVA) was used to compare FACE
and control samples. ANCOVA analysis was used to detect different responses
of soil GWC in the FACE and control treatments and to detect differences in
fungal biomass per unit microbial biomass. In some cases, data were log trans-
formed to fit the assumptions of the analysis. Statview software (SAS Institute)
was used for statistical analyses.
Sequences from the clone libraries were screened for chimeras by using
Chimera_Check (http://rdp.cme.msu.edu). A total of 156 sequences (69 from
inside the FACE ring and 87 from outside) were produced and identified using
BLAST searches. Sequences were aligned using ARB software and placed into
a neighbor-joining phylogenetic tree using Phylip software. Sequences with
greater than 98% similarity were considered duplicates and were removed from
the analysis. This resulted in 129 unique sequences (60 from inside the FACE
ring and 69 from outside). The number of sequences that appeared only once
versus the number that appeared twice was used to calculate the Chao1 estimate
of diversity by using EstimateS software (12). Neighbor-joining analysis used 100
bootstrap replicates and the Jin-Nei method with a gamma factor of 0.1 to allow
for different substitution rates between sites. The bacterial communities from the
four clone libraries were compared by using permutation tail probability (PTP)
and the Fst statistic (27), using PAUP and Arlequin software, respectively. In the
PTP test, the hypothesis that phylogeny covaries with community type was tested
by generating 10,000 randomly permuted trees and calculating the tree length
needed to evolve the different communities. The tree length of the original data
set was compared to this frequency distribution to produce a P value. The Fst
ELEVATED CO2 AND CHAPARRAL SOIL MICROBES 8575
FIG. 3. Variation in (A) microbial biomass C and (B) glucose SIR
with soil OM (grams OM gram1 soil) in FACE and control plots. The
P values are for the CO2-OM interaction term in the ANCOVA.
statistic compares genetic diversity between samples to overall diversity, using
the formula Fst (T W)/T, where T is total genetic diversity and W is
average within-sample diversity for all samples This produces values that vary
from 0 to 1 and which can be considered pairwise distances. The statistical
significance of Fst was determined by comparing the actual Fst value with a
distribution created by randomly permuting the communities 1,000 times.
Nucleotide sequence accession numbers. The sequences used for phylogenetic
analysis have been submitted to GenBank (www.ncbi.nlm.nih.gov) under acces-
sion numbers DQ201647 through DQ201772.
RESULTS
Plant root biomass and soil respiration. The concentration
of root biomass in soil taken from inside the FACE ring was
greater than that in soil from outside the ring (Fig. 1). The
higher root biomass in FACE soils could be attributed to
increased roots close to the bases of plants, rather than to
roots found in canopy gaps, where root biomass was gener-
ally lower. This root distribution is characteristic of the
patchy nature of the chaparral. While soil texture does not
vary widely over the landscape, soil OM is highly variable
and is consistently lower in canopy gaps than under plant
canopies (Table 1). Most of the microbial biomass and ac-
tivity parameters were strongly correlated with soil OM, as
is commonly observed (4, 19, 46). To control for variability
in OM across the landscape and to correct for any system-
atic biases between the FACE plot and the rest of the
8576 LIPSON ET AL. APPL. ENVIRON. MICROBIOL.

FIG. 5. Variation of fungal biomass with microbial biomass C

(chloroform fumigation-extraction) in FACE and control plots. The P
value is for the interaction term in the ANCOVA.

significant (P 0.055), as was the difference in intercept

FIG. 4. Variation in the activities of the soil extracellular enzymes
(A) cellulase and (B) amylase with soil OM (grams OM gram1 soil)
in FACE and control plots. The P values are for the CO2-OM inter-
action term in the ANCOVA.
landscape, ANCOVA was used to detect differences be-
tween FACE and control soils in the relationships between
microbes and soil OM. Soil respiration was significantly
correlated with soil OM (P 0.02), but the slightly higher
slope in FACE soils was not significant (Fig. 2A). However,
respiration in FACE and control soils differed in the re-
sponse to soil water content (Fig. 2B). Respiration from
control soils was significantly related to water content (P
0.005), while there was no such relationship in FACE soils
(P 0.817). In the ANCOVA of respiration versus GWC
and CO2 treatment, the difference in slope was marginally
(P 0.052).
Microbial biomass and activity. Microbial biomass C (mea-
sured by chloroform fumigation-extraction) increased more
rapidly with soil OM in FACE soils than in controls (Fig. 3A).
However, the corresponding trend for glucose SIR was smaller
and nonsignificant (Fig. 3B). Soil cellulase and amylase activ-
ities both increased more steeply with OM in FACE soils than
in controls (Fig. 4). These results show that elevated CO2
stimulated microbial biomass production and activity, particu-
larly in high-OM areas (i.e., under plants).
The ratio of soil respiration to glucose SIR was significantly
higher in FACE soils than in controls (Table 2). This param-
eter is similar to a metabolic quotient and to specific res-
piration used to indicate the metabolic state of microbial
biomass (3, 20, 47). However, it should be noted that soil
respiration was measured in the field and included root respi-
ration, while SIR was measured in the laboratory without
roots. The glucose SIR/microbial biomass ratio also describes

TABLE 2. Means (and standard errors) of selected respiratory,

bacterial and fungal biomass parameters in soils from
FACE and control plots
Mean value (SE)
Parameter (units) Pa nb
Control FACE

Respiration/SIR (unitless) 0.291 (0.026) 0.419 (0.052) 0.032 68

Log SIR/log MBC c (h1) 0.363 (0.022) 0.297 (0.013) 0.0095 59
Bacterial biomass (g g1) 607 (114) 546 (67) 0.640 30
Bacteria/fungi (unitless) 3.57 (1.09) 2.46 (0.44) 0.329 30
Fungi/MBC (unitless) 3.84 (0.62) 6.73 (1.37) 0.0024 55
a
The P value listed is for the one-way ANOVA, performed on log- or square-
root-transformed data in some cases. FIG. 6. Correlations of extracellular enzymatic activities with fun-
b
n, total sample size for analysis. gal biomass measurements in soils. The P values are given for both
c
MBC, microbial biomass C determined by chloroform fumigation-extraction. regressions.
VOL. 71, 2005 ELEVATED CO2 AND CHAPARRAL SOIL MICROBES 8577

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
both log transformed before the analysis in Table 2, which
shows that this ratio is lower in FACE soils. These respiratory
parameters were not correlated with soil OM, and so both were
analyzed by one-way ANOVA.
The ratio of fungal biomass to total microbial biomass C was
higher in FACE soils than in control soils (Table 2), and this
differential between FACE and control plots increased with
increasing biomass (Fig. 5), indicating that the stimulatory
effect of elevated CO2 on microbial biomass disproportionately
affected fungi. Bacterial biomass was not significantly different
in FACE and control plots (Table 2). Fungal biomass corre-
lated significantly with the activities of amylase and cellulase
enzymes in the soil (Fig. 6), whereas bacterial biomass did not
(data not shown).
Bacterial diversity. The soil bacterial community was dom-
inated by the Acidobacteria and Proteobacteria phyla (Fig. 7 and
8), as is typical for soils (18). Based on the Chao1 parameter,
there were 445 225 distinct bacterial ribotypes in this com-
munity. There was no obvious clustering of sequences from the
same sample type in Fig. 7, and PTP analysis confirms that the
bacterial community is not different between the FACE and
control plots (P 0.310) or between plant and gap samples
(P 0.561). Similarly, the Fst statistic showed that the FACE
8578 LIPSON ET AL.
FIG. 8. Frequencies of major bacterial taxa in (A) all four clone
libraries and (B) weighted means and standard errors of the plant and
interplant (gap) libraries from FACE and control plots. Acido, Acido-
bacteria; Alpha, Beta, Gamma, and Delta, classes of Proteobacteria;
CFB, Cytophaga-Bacteroides-Flexibacter; Firmi, Firmicutes; Verruco,
Verrucomicrobia.
and control communities were very similar in composition (dis-
tance of 0.0027 on a scale of 0 to 1) and not significantly
different from each other (P 0.223). The PTP and Fst anal-
yses address whether two communities are phylogenetically
distinct and are not sensitive to the abundance of species.
However, there was also remarkable similarity in the frequency
of major bacterial taxa in the four clone libraries (Fig. 8). The
only group that showed consistent differences between the two
FACE and two control libraries was the Gammaproteobacteria,
and this change, if real, was relatively minor in terms of the
whole community.
DISCUSSION
The patchy plant distribution in the study area gave rise to a
similar pattern in soil OM, with highest levels occurring di-
rectly under plants. While there was high variability in micro-
bial biomass and activity across the landscape, soil OM ex-
plained most of the variation in these data. This allowed us to
overcome the lack of replicate FACE rings (the cost in com-
pressed CO2 gas alone was about $40,000 per year for a single
ring). Because the variation across the landscape was well
understood and controlled for by using ANCOVA with soil
OM as a covariate, differences between FACE and control
samples could be attributed to CO2 effects rather than random
landscape effects. From a microbial perspective, the 178-m2
APPL. ENVIRON. MICROBIOL.
region within the FACE ring represents a vast landscape that
includes similar extremes in soil OM found elsewhere across
the landscape. The FACE ring soil also did not differ in texture
or pH from other areas in the landscape where control samples
were collected. Hence, the elevated CO2 treatment is the most
likely explanation for the different relationships between soil
OM and microbial activity in the FACE and control plots.
Furthermore, an increase in root biomass and microbial activ-
ity in response to elevated CO2 is consistent with the literature
discussed below.
Whereas soil OM significantly affected microbial biomass
and activity, its effect on bacterial diversity was minimal. The
frequency of the bacterial taxa in clone libraries varied some-
what between higher-OM samples under plants and lower-OM
samples in gaps (Fig. 8A), but the PTP analysis showed that
these communities were not phylogenetically distinct. A more
spatially intensive study of bacterial diversity would be re-
quired to address whether the abundance of these groups truly
varies with OM, but we can conclude from the PTP analysis
that similar species exist at both extremes.
The compositions of the clone libraries of FACE and control
plots were strikingly similar, both in the abundance of major
taxa (Fig. 8B), and in the phylogenetic placement of species
(Fig. 7). The only group that possibly showed a consistent
response to elevated CO2 was the Gammaproteobacteria, a
relatively minor component of the community. A spatially in-
tensive approach would be required to see if this increase from
5% to 9% was a consistent effect of elevated CO2 on the
Gammaproteobacteria of this ecosystem. The PTP and Fst anal-
yses verified that the bacterial communities in the FACE and
control plots were not phylogenetically distinct. The insignifi-
cant PTP and Fst results can best be understood by noting that
most major branches on the phylogenetic tree (Fig. 7) have
representatives from all four libraries.
The 16S rRNA clone libraries were designed to represent
the spatially averaged bacterial communities within each treat-
ment block, and therefore spatial replicates were combined.
This design does not provide enough replicates to compare the
abundances of each bacterial taxon with statistical rigor, but it
does provide well-mixed samples of diversity from each treat-
ment type that can be compared with phylogenetic tools, such
as the PTP and Fst tests. It is generally not practical to exhaus-
tively sequence all 16S rRNA ribotypes in a soil community.
However, the 129 unique sequences generated in this study
represent a significant subsample of the total diversity: about
29%, based on the Chao1 estimate of 445 225 distinct ri-
botypes for this community. In order for further sequencing to
alter the results of the PTP or Fst analyses, entirely new clades
would have to emerge, consisting solely of FACE or outside
sequences. The phylogenetic tree (Fig. 7) represents the most
common phyla found in soils (18) and has sequences from both
FACE and outside plots in most clades. Therefore, it is un-
likely that the remaining unsequenced ribotypes would drasti-
cally change the results.
The lack of response of the bacterial community to elevated
CO2 was consistent with the finding that bacterial biomass was
not significantly different between FACE and control plots,
despite the significant effects on total microbial biomass. In
contrast, fungal biomass responded markedly. Whether the
fungal community also changed in composition and the relative
VOL. 71, 2005
responses of mycorrhizal and saprotrophic fungi are beyond
the scope of this study. Stimulation of arbuscular mycorrhizae
(AM) and changes in AM species composition by elevated
CO2 have been reported for A. fasciculatum and other chap-
arral plants (36, 44), and higher plant allocation to mycorrhizae is
consistent with the observed increase in root growth. The data
strongly suggest that saprotrophic fungi are stimulated as well.
The increased extracellular enzymatic activities under elevated
CO2 correlated with fungal biomass, indicating that saprotro-
phic fungi responded to increased root biomass with growth
and exoenzyme production. Other researchers have found that
fungi increase in response to inputs of live and dead plant roots
(51). Dark septate fungal hyphae were commonly observed
in soil samples (unpublished observation). Based on mor-
phology, this type is clearly not an AM fungus, although
such morphotypes have also been observed in possible as-
sociations with A. fasciculatum roots (1). Increased root
biomass probably stimulated fungi that thrive on both dead
and live roots.
The stimulation of root growth by elevated CO2 is consistent
with the increased photosynthesis and leaf and stem area index
observed in the FACE treatment by others (11) and has been
widely reported for many ecosystems (22, 31). Additionally,
leaf tissue chemistry was altered in the FACE plots (26). It
follows that microbial biomass and extracellular enzymatic ac-
tivity would respond to increased root growth and altered leaf
chemistry, as observed in this study, although this is not always
the case. Occasionally a smaller, more active pool of microbial
biomass has been reported to result from elevated CO2 (15, 40,
45, 50). In the present study, the respiratory physiology of the
microbial community shifted in response to elevated CO2. The
increased ratio of soil respiration to glucose SIR could have
been caused by a better-fed microbial community functioning
closer to its respiratory potential (3, 20, 47). This effect could
also be caused by stimulated root respiration: although soil
respiration in the FACE treatment was not significantly in-
creased in the present study, higher respiration rates were
observed in a more temporally intensive study (11). The mi-
crobial community in the FACE samples had a markedly lower
ratio of glucose SIR to microbial biomass C. This could be
attributed to the higher proportion of fungi in FACE samples.
Filamentous fungi have the ability to shift resources through-
out their mycelial network to exploit areas of high resources,
while maintaining viable but inactive hyphae elsewhere in the
soil. Higher levels of viable, inactive biomass in fungus-
dominated soils would result in lower SIR activity per unit
microbial biomass C as measured by fumigation-extraction.
Bacteria and fungi generally have different growth kinetics
(25), and bacterium/fungus ratios have been linked to vari-
ations in specific respiration (respiration per unit biomass)
across soil types (39).
This study strongly suggests that fungi, and not bacteria,
respond to increased root growth under elevated CO2 and that
fungi and bacteria differ significantly in their respiratory prop-
erties. These results may also have implications for the C
balance of the chaparral ecosystem under elevated CO2. While
root biomass was stimulated, this potential sink is likely to be
offset by increased decomposition activity, as demonstrated by
higher cellulase and amylase activities in the FACE plot. Fur-
thermore, soil respiration under elevated CO2 appeared to be
ELEVATED CO2 AND CHAPARRAL SOIL MICROBES 8579
less sensitive to drought than control soil respiration, possibly
extending ecosystem C loss during dry periods. On the other
hand, a continued shift toward a fungus-dominated microbial
community with lower potential respiration rates per unit bio-
mass could lead to a decreased ability of the microbial com-
munity to respond to C inputs and could change the relation-
ship between soil respiration and microbial biomass in this
ecosystem. This result emphasizes the need to understand the
relationship between microbial community structure and soil
respiration under current and future conditions.
ACKNOWLEDGMENTS
Thanks go to Michelle Blair, Yufu Cheng, Steve Hastings, Pablo
Bryant, and Joe Verfaillie for field, laboratory, and logistical assistance
and to Scott Kelley for assistance with the phylogenetic analyses.
Thanks also go to the anonymous reviewers, who provided many de-
tailed and helpful comments.
REFERENCES
1. Allen, M. F., L. M. Egerton-Warburton, E. B. Allen, and O. Karen. 1999.
Mycorrhizae in Adenostoma fasciculatum Hook. & Arn.: a combination of
unusual ecto- and endo-forms. Mycorrhiza 8:225228.
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.
10:215221.
3. Anderson, T.-H., and K. H. Domsch. 1985. Determination of eco-physiolog-
ical maintenance requirements of soil microorganisms in a dormant state.
Biol. Fert. Soils. 1:8189.
4. Anderson, T-H., and K. H. Domsch. 1989. Ratios of microbial biomass
carbon to total organic carbon in arable soils. Soil Biol. Biochem. 21:471
479.
5. Ausubel, F. M. (ed.) 1994. Current protocols in molecular biology, John
Wiley and Sons, New York, N.Y.
6. Bohannan, B. J. M., and J. Hughes. 2003. New approaches to analyzing
microbial biodiversity data. Curr. Opin. Microbiol. 6:282287.
7. Bottomley, P. J. 1994. Light microscopic methods for studying soil mi-
croorganisms, p. 81104. In S. H. Mickelson (ed.), Methods of soil anal-
ysis, part 2. Microbiological and biochemical properties. Soil Science
Society of America, Madison, Wis.
8. Bowman, R. H. 1973. Soil survey of the San Diego area, California, part I.
USDA Soil Conservation Service and Forest Service, Washington, D.C.
9. Canadell, J., and P. H. Zedler. 1995. Underground structures of woody
plants in Mediterranean regions of California, Chile, and Australia, p. 177
210. In M. T. Kalin-Arroyo, P. H. Zedler, and M. D. Fox (ed.), Ecology
and biogeography of Mediterranean ecosystems in Chile, California, and
Australia. Springer-Verlag, New York, N.Y.
10. Chatzinotas, A., R. A. Sandaa, W. Schoenhuber, R. Amann, F. L. Daae, V.
Torsvik, J. Zeyer, and D. Hahn. 1998. Analysis of broad-scale differences in
microbial community composition of two pristine forest soils. Syst. Appl.
Microbiol. 21:579587.
11. Cheng, Y., W. C. Oechel, P. J. Bryant, S. J. Hastings. The impacts of elevated
CO2 on carbon flux of southern California chaparral using free-air CO2
enrichment. Submitted for publication.
12. Colwell, R. K. 1997. EstimateS: statistical estimation of species richness
and shared species from samples, version 5. Users guide and application.
[Online.] http://viceroy.eeb.uconn.edu/estimates.
13. Curtis, T. P., W. T. Sloan, and J. W. Scannall. 2002. Estimating prokaryotic
diversity and its limits. Proc. Natl. Acad. Sci. USA 99:1049410499.
14. Diaz, S., J. P. Grime, J. Harris, and E. McPherson. 1993. Evidence of a
feedback mechanism limiting plant response to elevated carbon dioxide.
Nature 364:616617.
15. Grayston, S. J., C. D. Campbell, J. L Lutze, and R. M. Gifford. 1998. Impact
of elevated CO2 on the metabolic diversity of microbial communities in N
limited grass swards. Plant Soil 203:289300.
16. Griffiths, B. S., K. Ritz, N. Ebblewhite, E. Paterson, and K. Killham. 1998.
Ryegrass rhizosphere microbial community structure under elevated carbon
dioxide concentrations, with observations on wheat rhizosphere. Soil Biol.
Biochem. 30:315321.
17. Horz, H. P., A. Barbrook, C. B. Field, and B. J. M. Bohannan. 2004. Am-
monia-oxidizing bacteria respond to multifactorial global change. Proc. Natl.
Acad. Sci. USA 101:1513615141.
18. Hugenholtz, P., B. M. Goebel, and N. R. Pace. 1998. Impact of culture-
independent studies on the emerging phylogenetic view of bacterial diversity.
J. Bacteriol. 180:47654774.
19. Insam, H., and K. H. Domsch. 1988. Relationship between soil organic C and
microbial biomass on chronosequences of reclamation sites. Microb. Ecol.
15:177188.
8580 LIPSON ET AL.
20. Insam, H., and K. Haselwandter. 1989. Metabolic coefficient of the soil
microflora in relation to plant succession. Oecologia 79:174178.
21. Jensen, L. S., and J. Sorensen. 1994. Microscale fumigation-extraction and
substrate-induced respiration methods for measuring microbial biomass in
barley rhizosphere. Plant Soil 162:151161.
22. Korner, C., M. Diemer, B. Schappi, P. Niklaus, and J. Arnone, III. 1997. The
responses of alpine grassland to four seasons of CO2 enrichment: a synthesis.
Acta Oecologica 18:165175.
23. Lipson, D. A., C. W. Schadt, and S. K. Schmidt. 2002. Changes in microbial
community structure and function following snow melt in an alpine soil.
Microb. Ecol. 43:307314.
24. Lipson, D. A., S. K. Schmidt, and R. K. Monson. 1999. Links between
microbial population dynamics and N availability in an alpine ecosystem.
Ecology 80:16231631.
25. Lipson, D. A., and S. K. Schmidt. 2002. Kinetics of microbial processes and
population growth in soil, p. 17481757. In G. Bitton (ed.) The encyclopedia
of environmental microbiology. Wiley and Sons, New York, N.Y.
26. Marriott, A. M. 2003. Effects of elevated carbon dioxide on Adenostoma
fasciculatum leaf nutrients. M.S. thesis. San Diego State University, San
Diego, Calif.
27. Martin, A. P. 2002. Phylogenetic approaches for describing and comparing
the diversity of microbial communities. Appl. Environ. Microbiol. 68:3673
3682.
28. Mayr, C., M. Miller, and H. Insam. 1999. Elevated CO2 alters community
level physiological profiles and enzyme activities in alpine grassland. J. Mi-
crobiol. Methods 36:3543.
29. Miller, D. N., J. E. Bryant, E. L. Madsen, and W. C. Ghiorse. 1999. Evalu-
ation and optimization of DNA extraction and purification procedures for
soil and sediment samples. Appl. Environ. Microbiol. 65:47154724.
30. Montealegre, C. M., C. van Kessel, M. P. Russele, and M. J. Sadowsky. 2002.
Changes in microbial activity and composition in a pasture ecosystem ex-
posed to elevated atmospheric carbon dioxide. Plant Soil 243:197207.
31. Norby, R. J., and R. B. Jackson. 2000. Root dynamics and global change:
seeking an ecosystem perspective. New Phytol. 147:312.
32. ONeill, E. G. 1994. Response of soil biota to elevated atmospheric carbon
dioxide. Plant Soil 165:5565.
33. Pace, N. R. 1997. A molecular view of microbial diversity and the biosphere.
Science 276:734740.
34. Prior, S. A., H. A. Torbert, G. B. Runion, H. H. Rogers, C. W. Wood, B. A.
Kimball, R. L. Lamorte, P. J. Winter, and G. W. Wall. 1997. Free air carbon
dioxide enrichment of wheat: soil carbon and nitrogen dynamics. J. Environ.
Qual. 26:11611166.
35. Randlett, D. L., D. R. Zak, K. S. Pregitzer, and P. S. Curtis. 1996. Elevated
atmospheric carbon dioxide and leaf litter chemistry: influences on microbial
respiration and net nitrogen mineralization. Soil Sci. Soc. Am. J. 60:1571
1577.
36. Rillig, M. C., and M. F. Allen. 1998. Arbuscular mycorrhizae of Gutierrezia
sarothrae and elevated carbon dioxide: evidence for shifts in C allocation to
and within the mycobiont. Soil Biol. Biochem. 30:20012008.
APPL. ENVIRON. MICROBIOL.
37. Rillig, M. C., K. M. Scow, J. N. Klironomos, and M. F. Allen. 1997. Microbial
carbon substrate utilization in the rhizosphere of Gutierrezia sarothrae grown
in elevated atmospheric carbon dioxide. Soil Biol. Biochem. 29:13871394.
38. Roberts, S. W., W. C. Oechel, P. J. Bryant, S. J. Hastings, J. Major, and V.
Nosov. 1998. A field fumigation system for elevated carbon dioxide exposure
in chaparral shrubs. Func. Ecol. 12:708719.
39. Sakamoto, K., and Y. Oba. 1994. Effect of fungal to bacterial biomass ratio
on the relationship between CO2 evolution and total soil microbial biomass.
Biol. Fertil. Soils 17:3944.
40. Santruckova, H., and M. Simek. 1994. Soil microorganisms at different CO2
and O2 tensions. Folia Microbiol. 39:225230.
41. Schimel, D. S., J. Melillo, H. Tian, A. D. McGuire, D. Kicklighter, T. Kittel,
N. Rosenbloom, S. Running, P. Thornton, D. Ojima, W. Parton, R. Kelly, M.
Sykes, R. Neilson, and B. Rizzo. 2000. Contribution of increasing CO2 and
climate to carbon storage by ecosystems in the United States. Science 287:
20042006.
42. Schmidt, S. K. 1992. A substrate-induced growth-response (SIGR) method
for estimating the biomass of microbial functional groups in soil and aquatic
systems. FEMS Microbiol. Ecol. 101:197206.
43. Schortemeyer, M., U. A. Hartwig, G. R. Hendrey, and M. J. Sadowsky. 1996.
Microbial community changes in the rhizospheres of white clover and pe-
rennial ryegrass exposed to free air carbon dioxide enrichment (FACE). Soil
Biol. Biochem. 28:17171724.
44. Treseder, K. K., L. M. Egerton-Warburton, M. F. Allen, Y. F. Cheng, and
W. C. Oechel. 2003. Alteration of soil carbon pools and communities of
mycorrhizal fungi in chaparral exposed to elevated carbon dioxide. Ecosys-
tems 6:786796.
45. Van Ginkel, J. H., A. Gorissen, and J. A. van Veen. 1996. Long term decom-
position of grass roots as affected by elevated atmospheric carbon dioxide. J.
Environ. Qual. 25:11221128.
46. Wardle, D. A. 1992. A comparative assessment of factors which influence
microbial biomass carbon and nitrogen levels in soils. Biol. Rev. 67:321358.
47. Wardle, D. A., and A. Ghani. 1995. A critique of the microbial metabolic
quotient (qC02) as a bioindicator of disturbance and ecosystem develop-
ment. Soil Biol. Biochem. 27:16011610.
48. Williams, M. A., C. W. Rice, and C. E. Owensby. 2000. Carbon dynamics and
microbial activity in tallgrass prairie exposed to elevated CO2 for 8 years.
Plant Soil 227:127137.
49. Zak, D. R., K. S. Pregitzer, P. S. Curtis, W. E. Holmes. 2000. Atmospheric
CO2 and the composition and function of soil microbial communities. Ecol.
Appl. 10:4759.
50. Zak, D. R., K. S. Pregitzer, J. S. King, and W. E. Holmes. 2000. Elevated
atmospheric CO2, fine roots and the response of soil microorganisms: a
review and hypothesis. New Phytol. 147:201222.
51. Zhu, W., J. G. Ehrenfeld, R. W. Parmelee, W. F. J. Parsons, X. Han. 1996.
The effects of live and dead roots on soil fungi in spodosolic soils of the New
Jersey Pinelands. Biol. Fertil. Soils 21:215226.