Soil Biology & Biochemistry 57 (2013) 644e653

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Soil Biology & Biochemistry

journal homepage: www.elsevier.com/locate/soilbio

Microbial community response to varying magnitudes of desiccation in soil:

A test of the osmolyte accumulation hypothesis
Madhavi L. Kakumanu a, Charles L. Cantrell b, Mark A. Williams a, *
a
b

Rhizosphere and Soil Microbial Ecology Laboratory, Virginia Polytechnic and State University, 301 Latham Hall, Blacksburg, VA 24060, USA
Natural Products Utilization Research, University of Mississippi, 38677, USA

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 19 March 2012
Received in revised form
9 August 2012
Accepted 13 August 2012
Available online 10 September 2012

Numerous studies have observed the physiological responses of soil microorganisms to water stress
caused by soil drying, however, only a few have attempted to assess the microbial response in soil in situ.
An experiment was conducted to analyze the change in extractable metabolites, particularly sugars and
amino acids, in soil and the associated microbial community at various intensities of soil desiccation.
Water potential was manipulated in two soils, Marietta and Sumter, representing relatively moist and
drought-prone water regimes, respectively. The matric potential of the soils was maintained relatively
moist at
0.03 MPa or lowered to
1.5,
4.5,
10,
20 and
40 MPa by air drying over w3 days. We
hypothesized that microbial communities inhabiting the drought-prone Sumter would accumulate more
osmolytes, and that the soil with a relatively moist water regime, the Marietta, may have communities
less adaptable to water stress, have fewer osmolytes, and show evidence for greater microbial turnover
and death. However, there was no evidence that the soils responded to drying by accumulating osmolytes or that there was greater microbial turnover and death related to soil type. Microbial community
structure did change with drying, however, with greater fungal-to-bacterial biomass in the Sumter but
not in Marietta soil. A signicant increase of w10e25% in phenol sulfuric acid analyzable sugars (PSAsugars) at intermediate levels (
4.5 MPa) of drying was observed compared to dryer and more moist
conditions. However, the GCeMS derived quantities of polyols (glucitol, inositol and xylitol), sugars, and
amino acids showed few strong and consistent patterns with level of desiccation. These results provide
some of the rst evidence that microbial communities in soil in situ do not strongly rely on these basic
osmolytes to cope with typical soil water decits. In natural soils, we propose that microbial communities respond differently to water decits perhaps through re-allocation of C to cell wall mucilage,
exopolysaccharides (EPS), and phospholipids, than organisms in culture, perhaps a consequence of low
energy and limiting supplies of N.
2012 Elsevier Ltd. All rights reserved.

Keywords:
Osmolytes
Compatible solutes
Matric water potential
Soil
EPS
GCeMS

1. Introduction
The uctuations in soil water potential caused by episodic
dryerewet events in terrestrial ecosystems exert physiological
and energetic challenges to microbial communities. Extremely
large uxes of C, important to the global C cycle, have also been
linked to soil drying and re-wetting in ecosystems (Birch, 1958;
Fierer and Schimel, 2002). Maintenance of cell turgor, which is
vital to microbial cell growth and survival, is strongly regulated by
extracellular water dynamics (Bremer and Krmer, 2000; Schimel
et al., 2007). Numerous hypotheses have emerged on the adaptation strategies that soil microorganisms utilize to cope with

* Corresponding author. Tel.: 1 540 231 2547; fax: 1 540 231 3083.
E-mail address: markwill@vt.edu (M.A. Williams).
0038-0717/$ e see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.soilbio.2012.08.014

declining and low water potentials. Perhaps the most common

hypothesis, supported by observations of soil C ux and microbial
biomass dynamics, is that microbial cells accumulate and release
intracellular osmolytes to rapidly respond to water dynamics
(Brown, 1976; Harris, 1981; Miller and Wood, 1996; Mikha et al.,
2005).
Microorganisms exposed to low osmotic potentials in laboratory
culture have been shown to accumulate inorganic and/or organic
osmolytes in their cytoplasm to maintain cell turgor. The osmolytes
include K ions and a group of organic solutes like glutamate,
proline, peptides, N-acetylated amino acids (amino acids and their
derivatives), sucrose, trehalose (carbohydrates), polyols, glycine
betaine, carnitine (quaternary amines) and tetrahydropyramidines
like ectoines (Killham and Firestone, 1984; Csonka, 1989; Blomberg
and Adler, 1992; Galinski and Truper, 1994; Kempf and Bremer,
1998; Poolman and Glaasker, 1998) rich in C and N. Among these

M.L. Kakumanu et al. / Soil Biology & Biochemistry 57 (2013) 644e653

organic solutes, fungi tend to accumulate polyols whereas bacteria

utilize amino acids and sugars to cope with water decit (Brown,
1976). When exposed to hypo-osmotic conditions, microorganisms expel the accumulated solutes extracellularly to maintain
equilibrium (Tschichholz and Trper, 1990; Halverson et al., 2000).
The sudden ush of C and N mineralization following the
rewetting of dry soil has been reported by many researchers and
more recently was hypothesized as representing microbial release
of intracellular osmolytes (Birch, 1958; Sorensen, 1974; Schimel
et al., 1999; Franzluebbers et al., 2000; Chowdhury et al., 2011).
Although isotopic studies have revealed that at least part of the C
released during the short-term pulse following the rewetting of dry
soil is microbial in origin (Kieft et al., 1987; Van Gestel et al., 1992;
Magid et al., 1999), it is unclear whether microbial cell lysis, the
regulated expulsion of intracellular microbial osmolytes, or other
mechanisms are responsible for the pulse.
The response of soil microorganisms to low water potential has
been studied by exposing soil isolates to salt induced hyperosmotic
stress and through desiccation on a simulated soil matrix (Killham
and Firestone, 1984, 1984b; Schimel et al., 1989; Roberson and
Firestone, 1992; Halverson et al., 2000). Several of these studies
observed increased levels of amino acids, sugars and the accumulation of extracellular polysaccharides in response to declining
water potential. Enhanced pools of cytoplasmic C and N content
with water potential decit were also reported. However, to our
knowledge no study has attempted to directly measure the pool of
soil microbial compatible solutes in response to drying under in situ
soil conditions. Laboratory cultures are so vastly different from soil
conditions that studies under these latter scenarios are greatly
needed to understand microbial adaptations to water stress and the
controls of soil C and nutrient dynamics in ecosystems.
An experiment was designed to test the physiological and
structural response of soil microbial communities to drying across
a water decit gradient using two soils that developed within
contrasting water regimes. The rst and primary objective was to
determine if microorganisms adapt to soil drying by accumulating
compatible solutes. It was hypothesized that as the soils were dried,
greater amounts of organic osmolytes would be detected. We expected an increase in fungal polyols, and bacterial derived sugars
and amino acids in response to water decit. The second objective
was to assess whether microbial communities in two different
soil types with contrasting water regime histories would respond
differently to drying and whether changes in fungal to bacterial
ratios would coincide with the composition of fungal and bacterial
osmolyte pools. It was hypothesized that microbial communities
inhabiting the drought-prone soil would accumulate more osmolytes, and that the soil with a relatively moist water regime may
have communities less adaptable to water stress, have fewer
osmolytes, and show evidence for greater microbial turnover and
death.
2. Materials and methods
2.1. Site description
The experiment was conducted on two soils, the wetter lowland
Marietta and dryer upland Sumter series located near Mississippi
State University, Mississippi, USA (33 280 N and 088 470 W) in Fall
2009. These soils have been previously sampled across a 5 county
area in northern Mississippi and were shown to have similar pH,
texture, color, and hue as predicted by the soil type. For the purposes
of the work in this paper, a 100-m transect across a 2e5 Ha forest
was used within each of the respective soil types to collect soil to
a 10-cm depth. Five-kg of soil was collected every 20-m from within
a central location of each forest. At sampling, the soils were

645

relatively moist with water content of 34e36% (w
150 kPa). The

soils were sieved to 4 mm and thoroughly cleaned of obvious plant
litter and rocks, and refrigerated at 4  C until use.
Rainfall across the area, for both soil types, averages 51 inches
and the mean annual temperature is 17.7  C. The Marietta soils are
(ne-loamy, siliceous, active, thermic Fluvaquentic Eutrudepts)
derived from deep alluvial deposits near streams in the blackland
prairie region of Mississippi. These soils drain areas of the mixed
uplands of the Southern Coastal Plain. The soils are subject to
frequent ooding. Mottles and stains starting at the depth of 10-cm
and shallow water table are indicators of the generally moist water
status of these soils. The site was forested with >50-y old deciduous vegetation dominated by Carya illinoinensis. The C:N content
of the Marietta soils was 2.35% and 0.17% respectively with pH of
6.2. The Sumter soil is silty clay, with medium granular structure,
moderately deep, well drained, with rapid runoff. These upland
soils were formed in marly clays and chalk of the blackland prairies
(ne-silty, carbonatic, thermic Rendollic Eutrudepts) and drain to
lowlands (e.g. Marietta). The water table is deep and the permeability of the soil is slow. The soil has a pH of 6.3 with C and N
content of 2.56 and 0.15% respectively. Total soil organic C and N
contents were measured on a Vario MAX CNS macro elemental
analyzer (Elementar Americas, Inc., Mt. Laurel, NJ). Soil pH was
measured after shaking the soil with 0.01 M CaCl2 (1:1, mass:
volume) suspension for 30 min.
2.2. Experimental setup
A laboratory experiment was conducted to study the physiological response of soil microbial communities to water potential
decits (matric) caused by air drying. Keeping in view of the
textural differences in the soil types and their water holding
capacities, we used water potential to measure the drying effect
on the soil microbial communities. Each treatment (soil, n 2;
water potential, n 6) was replicated three times, thus resulting
in a total of 36 separate samples. Each sample consisted of 10 g
(dry weight) of well homogenized soil weighed into 150 ml
volume specimen cups. This experimental setup was repeated 5
to have enough material for all the analysis (e.g. Phospholipid
fatty acids (PLFA), osmolytes, respiration, microbial C, N and
soluble C). Keeping the soil mass low within each specimen cup
also allowed for control and homogeneity of the soil drying
process. The water content of all the samples was adjusted to their
respective eld capacities (
0.03 MPa) by adding sterile distilled
water. Soils were pre-incubated at room temperature (22  C)
for ve days to reduce disturbance effects related to sampling,
sieving, and storage.
The pre-incubated soils were either maintained moist
(
0.03 MPa) or slowly air dried to ve different water potentials
of
1.5,
4.5,
10,
20, and
40 MPa at 22  C. For three consecutive days, soils were dried for 6e12 h each day until reaching
target water potential. The soils took approximately 16, 22, 29, 33
and 36 drying hours to reach the water potentials of
1.5 MPa,

4.5 MPa,
10 MPa,
20 MPa,
40 MPa, respectively. The relation
between the soil water content and water potential were analyzed
prior to the experiment by lter paper method (McInnes et al., 1994).
As well, the water potentials of the soils were cross-validated and
monitored using a WP4 dewpoint potentiameter by (Decagon, Inc.,
Pullman, WA).
The soils remained at respective water potentials for 24 h before
any further analysis. PLFA analysis was done to understand the
structural and physiological changes in the community with
drying. The respective soil samples were stored at
80  C until
analysis. All the other extractions except for the PLFAs were done
the following day. Microbial osmolytes are known to comprise

646

M.L. Kakumanu et al. / Soil Biology & Biochemistry 57 (2013) 644e653

amino acids (e.g. proline, glutamic acid), sugars (e.g. sucrose,

trehalose) and polyols (e.g. sorbitol, mannitol). The physiological
and biomass responses of microbial communities were assessed by
ninhydrin analysis (amino acids) and phenol sulfuric acid (PSA)
analysis for sugars. Metabolite composition was analyzed using
GCeMS.
2.3. Extraction of metabolites from soil and associated
microorganisms
Microbial metabolites from soil were extracted using a mixture
of chloroform and 0.01 M K2SO4 (1:4; v/v). Soluble organics were
extracted using 0.01 M K2SO4 only. The principle behind the
extraction of microbial metabolites was based on chloroform lyses
of microbial cells and the consequent release and dissolution of
intracellular cytosol into the 0.01 M K2SO4. We utilized the chloroform slurry method for extraction of microbial metabolites
instead of a traditional fumigationeextraction assay for two
reasons. First, fumigation of dried soils has been shown to give
erratic results (Sparling and West, 1989) that would then make it
difcult to compare between drying treatments. Second, the
activity of hydrolytic enzymes on proteins and polysaccharides in
soils during fumigation (Renella et al., 2002) would result in the
contamination of the cytosolic pool of organics and exaggerate
concentrations of sugars and amino acids.
For microbial metabolite extraction, 10 ml chloroform was
added to 10 g (dry weight) of soil that was transferred to separate
160 ml serum bottle. After one minute, the K2SO4 solution was
added to bring each bottle up to 40 ml of 0.01 M K2SO4 and
vigorously agitated for 2 h (250 rpm) on an orbital shaker. Soluble
organics were extracted identically, but without chloroform. To
separate the chloroform and aqueous phase, the samples were
allowed to settle for 15 min and then centrifuged at 1500 rpm for
10 min on IEC bucket centrifuge. The aqueous supernatant was
taken and ltered through whatman #1 lter paper and the
solution was lyophilized and stored at
80  C until further
analysis.
For measuring microbial and soluble C and N concentrations,
soil samples were extracted, ltered, and analyzed on a Shimadzu
TOC analyzer. Microbial C and N were determined by subtracting
soluble C and N values from chloroform-K2SO4 extracts. Unlike the
fumigation method (Vance et al., 1987) no correction factor was
applied to the values.
2.4. Analysis of soil extracts by colorimetric methods
Reducing sugars in the soil extracts were analyzed by the PSA
method (Dubois et al., 1956). Briey, 50 ml of 80% phenol solution
followed by addition 5 ml of concentrated H2SO4 (w18 M) solution
was added to a small volume of soil extract. The mixture was
allowed to stand at room temperature for 45 min. The absorbance
was measured at 480 nm on UV spectrometer. Absorbance values
were used to calculate the concentration of reducing sugars based
on an 8-point standard curve of glucose.
Amino acids were determined using the ninhydrin method
(Stevenson, 1982). Though ninhydrin also reacts with peptides,
proteins, ammonium and other compounds with free a-amino
groups it is generally considered sensitive and useful for quantication of amino acids (Jorgenson and Brooks, 1990). The soil
extracts along with 0.5 ml of citric acid and 2 ml of ninhydrin
reagent were incubated at 100  C for 25 min. The solution was
cooled, diluted with 5 ml of 50% ethanol, and the absorbance was
measured at 570 nm on UV spectrometer. A standard curve was
made by measuring the absorbance at different concentrations of
L-Leucine-N.

2.5. Analysis of extractable metabolites by Gas Chromatographye

Mass Spectroscopy
The sugars and amino acids in the extracts were characterized
using GCeMS. Prior to analysis the samples were derivatized.
Derivatization was carried out in 1 ml reaction vials treated with
5% dimethyldichlorosilane in toluene. For analysis of sugars, the
soil extracts were derivatized by N, O-bis-(trimethylsilyl) trifuroacetamide (BSTFA) solution. Approximately 250 ml of the aliquots
of soil extract were taken in silylated reaction vials and dried
completely under nitrogen. The dried residues were converted to
trimethylsilyl derivatives by adding BSTFA containing 1% trimethylchlorosilane (TMCS) and pyridine in 2:1 ratio and incubating
them for 3 h at 70  C (Medeiros et al., 2006). The samples were
allowed to stay overnight at room temperature and completely
dried under pure nitrogen. The dried residues were redissolved in
110 ml of hexane and sent for GC analysis.
Derivatization of amino acids was done as per the method given
by Fan (1996). Approximately 500 ml of soil extracts were taken and
the pH was lowered to 2 by adding equal volumes 1 M HCl in
reaction vials. The solution was dried completely under pure
nitrogen. Dried extracts were sonicated with 1:1 mixture of NMethyl-N-(Tert-Butyldimethylsilyl) triuoroacetamide (MTBSTFA)
and acetonitrile for 2 h at 60  C. The solution was left at room
temperature overnight and dried under nitrogen. The derivatized
extracts were redissolved in 110 ml of hexane before GC analysis.
The samples were analyzed on Varian CP-3800 Gas Chromatograph coupled to a Varian Saturn 2000 MS/MS. The GC was equipped with a DB-5 fused silica capillary column (30 m  0.25 mm, with
lm thickness of 0.25 mm) operated using the following conditions:
injector temperature, 240  C, column temperature, 60e280  C at
8  C/min then held at 280  C for 5 min; carrier gas, He; injection
volume, 1 mL (splitless). The MS mass ranged from 40 to 650 m/z,
lament delay of 3 min, target TIC of 20,000, a prescan ionization
time of 100 ms, an ion trap temperature of 150  C, manifold
temperature of 60  C, and a transfer line temperature of 170  C.
Individual sugars were identied by comparison of mass spectra
with literature and library data, comparison of mass spectra and GC
retention times with those authentic standards and/or interpretation of mass spectrometric fragmentation patterns. Standard solutions of glucose and other compounds like trehalose, sorbitol,
sucrose, proline, glutamine which are commonly expected as
microbial osmolytes, were analyzed (Williams and Xia, 2009).
Compounds were quantied using total ion current (TIC) peak area
and converted to compound mass using calibration curves of the
external standards (glucose for monosaccharides, sorbitol for sugar
alcohols and sucrose for disaccharides).
2.6. PLFA analysis
Total lipids were extracted according to the procedure of White
and Ringelberg (1998) as modied by Butler et al. (2003). Briey,
w10 g (dry weight) of soil samples from 3 replications of all the
treatments were transferred to sterilized 160 ml serum bottles. The
soils were extracted overnight using a mixture of 50 mM phosphate
buffer (pH 7.1), chloroform and methanol (0.8:1:2). The samples
were centrifuged the following day at 1000 rpm for 5 min and
ltered using Whatman # 1 lter paper. The ltrate was added with
3 M NaCl solution and a pinch of Na2SO4 salt and allowed to stand
for w8 h for phase separation. The chloroform phase was collected
into separate glass tubes and dried completely under stream of
nitrogen. Dried lipids were redissolved and fractionated into
neutral, glyco- and phospholipids using silicic acid bonded phase
extraction columns (Supelco, cat. No. 505048). The neutral and
glyco lipids were eluted using chloroform and acetone respectively.

M.L. Kakumanu et al. / Soil Biology & Biochemistry 57 (2013) 644e653

-1

PSA analyzable C (g g soil)

160

Marietta
Sumter

140

120

*
100

b
-1

2.7. Statistical analysis

Two-way ANOVA was done on all the data to test for effects of
soil type, water potential and their interaction. However, irrespective of the interaction effects we looked at each factor separately using one-way ANOVA (Randomized complete block design
with factors: soil type, water potential and 3 replications) and the
treatments were considered signicant at p < 0.05 (Microsoft Excel,
2007; SAS 9.2, Sysstat software, 2008). Data were transformed
when necessary to meet the assumptions of normality. We used
PC-ORD version 4 software (MJM Software, Gleneden Beach, OR)
for ordination and multivariate analysis of the data as per McCune
and Grace (2002).

80

Total PLFA- C (g g soil)

Phospholipids were nally eluted with methanol into separate

tubes and completely dried under nitrogen. The dried phospholipids fraction was transformed into fatty acids methyl esters under
alkaline conditions and extracted twice in 1:4 chloroformehexane
solution. The chloroformehexane mixture was completely evaporated under stream of ultra high purity 99.999% N2 gas and the
residue was resuspended in 500 ml of hexane for GC analysis.
The fatty acids were quantied and detected on Agilent 6890
Series gas chromatograph (Santa Clara, CA) equipped with a ame
ionization detector, an Ultra-2 column (19091B-102; 0.2 mm by
25 m), and controlled by a computer loaded with ChemStation and
Sherlock software. Ultra high purity H2 was the carrier gas at
a column head pressure of 20 kPa, septum purge of 5 ml min
1,
a split ratio of 40:1, injection temperature of 300  C, injection
volume of 2 ml. The oven temperature ramps from 170  C to 288  C
at 28  C min-1 and the analysis time of each sample was 6 min.
Peak identication was carried out by the Microbial Identication
System (MIDI, Inc.) following calibration with a standard mixture of
17 fatty acid methyl esters (1300A calibration mix).
The PLFAs i15:0, a15:0, i16:0, a16:0, i17:0, a17:0 (gram-positive),
16:1u9, 16:1u7, 18:1u7 and cy19:0 (gram-negative) were considered as bacterial biomarkers, 10:Me 16:0 and 10:Me 18:0 for actinomycetes and 18:1u9 and 18:2u6 as fungal biomarkers (Frostegrd
and Bth, 1996; Zhang et al., 2005; Liang et al., 2008). The ratio of
fungal to bacteria biomarker fatty acids were used to indicate
change in the fungal to bacterial biomass ratio (Bossio et al., 1998).

647

36
32
28
24

-20

-40

20
16
-0.03

-1.5

-4.5

-10

Water Potential (MPa)

Fig. 1. Concentrations of (a) reducing sugar carbon (mg g
1soil) in chloroformeK2SO4
extracts as determined by the phenol sulfuric acid (PSA) method and (b) total PLFA
carbon (nmol g
1soil) at different levels of water potentials in two soils (Marietta and
Sumter). Total PLFA-C was quantied using the detector response to 16:0 methyl ester
to calculate the concentrations of all fatty acids. Error bars represent the mean standard error between the replicates. Asterisks (*) denote signicant difference between
the corresponding drying treatment and the control (
0.03 MPa) within each soil
(n 3; P < 0.05). Differences between soils were signicantly different (n 3;
P < 0.05).

3. Results

0.30

3.1. Quantication of sugars in microbial extracts

3.2. PLFAs and fungal to bacterial ratio

Fungal/Bacterial Ratio

Phenol sulfuric acid analyzable sugars (PSA-sugars) in the

(chloroform labile) soil extracts indicated that Marietta had significantly greater (w30%) amounts of PSA-sugars than Sumter soil
(Fig. 1a). Moreover, PSA concentrations in the two soils responded
similarly to water potential decit. For instance, in Marietta soil PSAsugars increased signicantly (w20e25%) up to
10 MPa, while
Sumter had w15% greater PSA-sugar in moderately (
4.5 MPa)
dry soil compared with the amounts in moist and extremely dry soil.

Marietta
Sumter

0.28

-4.5

-10

0.26

0.24
0.22
0.20
0.18
0.16

The overall abundance of PLFA in Marietta compared to Sumter

(Fig. 1b) were in agreement with differences in the size of the PSAsugar pool, however, the general response to drying tended to
contrast with the results of PSA-sugar. Indeed, Marietta tended to
show declines and Sumter showed signicant increases in PLFA due
to drying.
There was no signicant effect of water potential decit on the
fungal to bacterial ratio in Marietta; however, there was a signicant increase with drying in the Sumter soil (Fig. 2). Sumter had

0.14
-0.03

-1.5

-20

-40

Water Potential (MPa)

Fig. 2. The changes in fungal/bacterial ratio across the water potential gradient in two
soils (Marietta and Sumter). Error bars represent the standard error between the
replicates. Asterisks (*) denote signicant difference between the drying treatments
and the control (
0.03 MPa) within each soil (n 3; P < 0.05). Differences between
soils were signicantly different (n 3; P < 0.05).

M.L. Kakumanu et al. / Soil Biology & Biochemistry 57 (2013) 644e653

3.3. Microbial C, N, and soluble C

Microbial C and N were both signicantly greater in the Marietta
than Sumter. In general, microbial biomass C was not affected
by drying (Fig. 4), however, there was an increase at
20 MPa
compared to
0.03 MPa in Sumter. Microbial N in Sumter soil
decreased when dried to
40 MPa but otherwise was unaffected by
drying. In the Marietta soil, in contrast, microbial N tended to
decrease during moderate drying compared to moist and extremely
dry soil. Sumter had greater levels of soluble C compared to Marietta soil. Soluble C concentrations across the drying gradient
changed similarly for both soil types (Fig. 5) increasing signicantly
at the dryer end of the gradient. Microbial C:N ratios tended to
maintain values of w12, however, when soils dried to the two driest
states, ratios increased considerably in the drought-prone Sumter
but not the mesic Marietta soil (Fig. 6). Our soil respiration data (not
shown) indicated a very typical response to drying and rewetting
whereby the respiration declined several fold between moist and

Carbon

Marietta
Sumter

140
120

-1

signicantly greater fungal to bacterial ratios than Marietta soil. The

increase in fungal to bacterial ratio with soil drying is consistent
with higher microbial C:N ratio at dry treatments in Sumter. The
relative abundance of PLFA biomarkers indicative of gram-positive
bacteria and actinomycetes showed decreases with water decit
whereas fungal biomarkers increased across the water potential
gradient of the two soils (Fig. 3).

Microbial biomass carbon and nitrogen (g g soil)

648

100

*
80
60
0

Nitrogen

20

15

10

*
*
*

20

Marietta

-1.5
-4.5
-10
-20
-40

15

-1.5

-4.5

-10

-20

-40

Water Potential (MPa)

Fig. 4. The amount of microbial biomass (a) carbon (b) nitrogen extracted from two
soils (Marietta and Sumter), at different levels of water potential. Microbial C and N
was calculated as (C and N in choloroformeK2SO4 extracts) minus (C and N in soluble
extracts). No conversion factor was applied. Error bars represent the standard error
between the replicates. Asterisks (*) denote signicant difference between the drying
treatments and the control (
0.03 MPa) treatment within each soil (n 3; P < 0.05).
Differences between soils were signicantly different (n 3; P < 0.05).

0
-5

25
-10

Soluble Carbon (g g soil)

Sumter

15

-1

%Variation in microbial composition

10

-0.03

10
5
0

Marietta
Sumter

20

*
*

15

10

-5
-10

GramPositive

GramNegative

Actinomycetes

Fungi

Fig. 3. Percent variation in the relative abundance of PLFA biomarkers (indicative of

bacteria and fungi) across the water potential gradient of
1.5,
4.5,
10,
20
and
40 MPa in a) Marietta b) Sumter soils. The bars below and above the zero
indicate the percent decrease and increase in the biomarkers at respective water
potentials relative to moist control (
0.03 MPa).

-0.03

-1.5

-4.5

-10

-20

-40

Water Potential (MPa)

Fig. 5. The amount of soluble carbon extracted from two soils (Marietta and Sumter),
at different levels of water potentials. Error bars represent the standard error between
the replicates. Asterisks (*) denote signicant difference between the drying treatments and the control (
0.03 MPa) treatment within each soil (n 3; P < 0.05).
Differences between soils were signicantly different (n 3; P < 0.05).

M.L. Kakumanu et al. / Soil Biology & Biochemistry 57 (2013) 644e653

40

C/N ratio of microbial

biomass

Marietta
Sumter

30

20

10

0
-0.03

-1.5

-4.5

-10

-20

-40

Water Potential (MPa)

Fig. 6. The C:N ratio of microbial extracts across the matric water potential gradient in
two soils (Marietta and Sumter). Error bars represent the standard error between the
replicates. Asterisks (*) denote signicant difference between the drying treatments
and the control (
0.03 MPa) treatment within each soil (n 3; P < 0.05). Differences
between soils were signicantly different except at
4.5 MPa (n 3; P < 0.05).

very dry soil and a 20e50% increase in CO2 upon rewetting

compared to continuously moist soil.
3.4. Ninhydrin reactive nitrogen (NRN)
Ninhydrin reactive-N (NRN) in chloroformeK2SO4 extracts at
various intensities of matric stress showed different dynamics in
Marietta and Sumter soil (Fig. 7). The initial concentration of NRN in
Marietta (7.46 mg g
1 soil) was considerably greater than in Sumter
soil (4.62 mg g
1), resembling the relative differences observed for
the PSA-sugars. Moreover, the amounts of NRN declined signicantly in Marietta but not in the Sumter soil as a result of soil
drying.
3.5. GCeMS characterization of soil microbial extracts
The GCeMS analysis of trimethylsilyl (TMS) derivatives of
chloroform labile soil extracts showed the presence of several

-1

Ninhydrin Reactive N (g g soil)

10
Marietta
Sumter

9
8

649

sugars (monosaccharides and disaccharides) and polyols. The

compounds that were conrmed include glucose, galactose, trihydroxy butyric acid, arabinose, glycerol, glucitol, xylitol, inositol,
myo-inositol, turanose and sucrose. The total sugars detected by
GCeMS varied from approximately 20 mg g
1soil to 120 mg g
1soil,
an amount w10e50% the size of the total pool of sugars (Table 1).
The changes in the concentration of glucose, polyols and other
saccharides at different intensities of matric stress in Marietta
and Sumter soil are shown in Fig. 8. Glucose was the most
abundant monosaccharide found in all treatments and accounted
for 45e60% of total GCeMS metabolites. Compounds like glycerol, galactose, glucose, glucitol, myo-inositol and turanose were
also found in all treatments. However, certain compounds were
not consistently present along the moisture regime. For example,
inositol was found only in Marietta soil and not Sumter. Similarly,
sugars like arabinose, fructose and polyols like xylitol were
found in detectable amounts only in some replications of dried
treatments.
The composition and the relative abundance of the saccharides
and polyols vary with the soil type (Fig. 8). In this regard, Sumter
had signicantly greater concentrations of cellular metabolites.
In general, drying tended to reduce the metabolite content in
Sumter soil and tended to change little in the Marietta soil except
for an increase at intermediate levels of drying (
4.5 MPa).
Metabolites were thus a greater proportion of total biomass in
Sumter compared to Marietta soil.
Amino acids, the other important group of osmolytes were
found only in low concentrations in continuously moist treatments
but were not detected in dry soils. We identied the amino acids
valine, proline, leucine, isoleucine, glutamine, glutamic acids, and
some fatty acids in continuously moist treatments. Both NRN and
spectroscopic NMR analysis supported the relatively low abundance of amino acids in the extracts (data not shown).
4. Discussion
The primary aim of the research was to test the microbial
osmolyte accumulation hypothesis (OAH) in soil. We hypothesized
that microbial communities inhabiting the drought-prone soil
would accumulate more osmolytes, and that the soil with a relatively moist water regime may have communities less adaptable to
water stress, have fewer osmolytes, and show evidence for greater
microbial turnover and death. Sugars, polyols and amino acids have
been reported as common osmolytes that accumulate within
microorganisms; however, these reports are overwhelmingly based
on microbial responses to salt stress when grown in cultures
(Galinski and Truper, 1994; Kempf and Bremer, 1998; Poolman and
Glaasker, 1998). Generally speaking, the results from this study did

*
*

Table 1
Total amount of sugars (mg g
1 soil) identied in GCeMS analysis in Marietta and
Sumter soils at different water potentials.
Treatment

4
3
-0.03

-1.5

-4.5

-10

-20

-40

Water Potential (MPa)

Fig. 7. Concentrations (mg g
1 soil) of Ninhydrin Reactive Nitrogen (NRN) by chloroformeK2SO4 extracts at different water potentials in (Marietta and Sumter) soil. Error
bar represents the mean standard error between the replicates and Asterisks (*) denote
signicant difference between the corresponding drying treatment and the control
(
0.03 MPa) within each soil (n 3; P < 0.05).

0.03

1.5

4.5

10

20

40

Marietta

Sumter

Sugars
(mg g
1 soil)

% Detecteda

Sugars
(mg g
1 soil)

% Detecteda

34.3
37.8
69.4
37.7
21.4
21.2

14.3
15.2
15.0
12.2
7.7
7.7

93.5
46.2
46.1
58.0
39.5
27.0

50.5
26.6
21.1
30.4
23.2
16.4

(15.9)
(23.1)
(16.3)
(4.1)
(1.1)
(7.5)

(6.8)
(9.6)
(5.1)
(1.2)
(2.7)
(2.7)

(13.8)
(10.6)
(6.9)
(18.5)
(14.8)
(3.4)

(9.6)
(5.4)
(2.61)
(10.2)
(8.8)
(2.0)

Values are means and the gures in the parenthesis indicate the standard error with
in the treatments.
Amount of sugars detected in GC
MS
a
 100
% detected
Sugars detected in PSA analysis

650

M.L. Kakumanu et al. / Soil Biology & Biochemistry 57 (2013) 644e653

Glucose
Polyols
Other sugars

60

Marietta

50
40

20

-1

g metabolites g soil

30

10
0

b Sumter

60
50
40
30

*
20

10
0
-0.03

-1.5

-4.5

-10

-20

-40

Water potential (MPa)

Fig. 8. The amount of metabolite (mg g
1 soil) pools detected by GCeMS at different of
water potentials in (a) Marietta and (b) Sumter soils. The metabolites include glucose,
polyols (sum of glycerol, xylitol, glucitol, inositol, myo-inositol) and others (sum of
trihydroxy butyric acid, galactose, turanose). Error bar represents the mean standard
errors (n 3).

not support the OAH as a primary mechanism for microbial adaptation to soil drying. Specic pools of metabolites such as sugars
and alcohols did not increase consistently in soil microorganisms
due to drying. However, two other insights are important to note.
First, the pool of microbial metabolites was relatively large,
accounting for a large proportion (20e50%) of the total microbial
biomass C (not including the use of k-factors to estimate
microbial biomass) and suggesting that these molecules are
a physiologically important pool of high energy compounds.
Second, there was considerable change in the pools of microbial
derived PLFA, microbial C, and PSA-sugars that may be important
indications of community structure and intracellular re-allocations
or C resulting from microbial adaptation to soil drying.

4.1. Metabolite composition

The composition of the metabolite pool was consistent with the
types of compounds expected to allow for microbial cytoplasmic
adjustment to water potential decits (Brown and Simpson, 1972;
Hocking, 1986; Al-Hamdani and Cooke, 1987; Kelly and Budd, 1991;
Witteveen and Visser, 1995; Shen et al., 1999). Yet, it is important to
also note that these compounds are very common components of
cellular metabolism (Fig. 8) and energy cycling. The amounts of
metabolites, if utilized for physiological adaptation to water decit,
would have increased and have occurred in near molar concentrations within microbial cells due to desiccation, thus making their

detection straightforward (Yancey et al., 1982; Schimel et al., 1989).

Overall, the results suggest that microorganisms are not accumulating large concentrations of typical osmolytes during soil drying
(Fig. 8), and thus do not support OAH as a primary mechanism of
microbial adaptation to soil drying.
It was expected that drought-prone Sumter soils would
respond differently to drying than the more mesic Marietta soil. It
was therefore interesting that the Sumter soil which contained
half the microbial biomass had 2 the concentration of metabolites relative to the Marietta even when soils were moist (Fig. 8).
The microbial metabolite response to drying was also different
between the two soils but was inconsistent with the main idea that
the drought-prone Sumter would accumulate more osmolytes
with drying and that the Marietta soil would show more evidence
of microbial death and lysis (Fig. 1a,b; Fig. 4). Indeed, related to the
issue of microbial turnover, bulk pools of PLFA and PSA did not
change in ways consistent with microbial lysis, and it is thus
concluded that microbial lysis in soils did not result during soil
drying.
If soil microbial communities are not primarily reliant upon
osmolyte accumulation, but are nevertheless resistant and adaptable to drying, then other mechanisms are needed to explain soil
microbial responses to desiccation. Rather than having unlimited
resources like that typically found during early microbial growth in
culture, soil microorganisms under nutrient limited and oligotrophic energy conditions may not be able to physiologically afford the
costs of intracellular accumulation of osmolytes. In contrast, more
efcient utilization (Tiemann and Billings, 2011) and re-allocation
of resources from within microbial cells may be a potentially
important mechanism for efciently coping with water decit in
soils. For example, the production of exopolysaccharides (EPS) has
been shown to be an important mechanism for bacterial adaptation
to desiccation and other environmental stresses (Roberson and
Firestone, 1992).
4.2. Microbial pools of PLFA, sugars & NRN
The PSA-sugar concentrations along the drying gradient tended
to increase in both soil types during moderate levels of drying
(Fig. 1a). The PSA methodology measures both monomeric and
oligosaccharide sugars and thus is not a specic determinate of
monomer-type sugars that have so often been shown to play a role
in osmolyte accumulation (Safarik and Santruckova, 1992; Gallardo
and Schlesinger, 1995). It is clear, however, that microbial cells are
adapting to soil drying either by increasing microbial biomass or
specic pools of cell biomass, a result that is often reported to occur
during desiccation in soil (e.g. Lundquist et al., 1999; Williams and
Xia, 2009; Schimel et al., 2010). Similarly, it is notable that the
droughty Sumter soil had increased levels of NRN at moderate
levels of drying and that the Marietta soil showed signicant
declines in NRN as result of drying. The ninhydrin method,
however, much like the PSA method must be interpreted cautiously
because it is not specic to known osmloytes, and is also sensitive
to peptides, proteins, and ammonium, for example. The concentrations of NRN are also rather low and the changes due to drying
were relatively small compared to the size of the water potential
decit (Fig. 7). While the greater PSA and NRN concentrations at
intermediate levels of drying could be construed as consistent with
a microbial response to desiccation, the small changes observed
would explain only a small part of the response needed to conrm
a general OAH hypothesis (Killham and Firestone, 1984; Schimel
et al., 2007).
The results presented here contrast with the 11e60% increase in
N and amino acids by cultivated soil microorganisms (Killham and
Firestone, 1984b; Schimel et al., 1989) in response to matric decit.

M.L. Kakumanu et al. / Soil Biology & Biochemistry 57 (2013) 644e653

However, unlike that of most culture conditions, C and N are

limiting nutrients for microbial biomass production in most nonagricultural soils and may help to explain the small pool of
amino-type N found in our study. Moreover, under dry conditions
the thinning of water lms and the discontinuity between lms can
result in substantial reductions in substrate diffusion (Schimel
et al., 1989) and nutrient availability to microorganisms. Under
such conditions the limitations in nutrient (e.g. nitrogen) and
energy availability could lower the capacity for an organism to
produce/accumulate appropriate concentrations of osmolytes to
counterbalance cellular water loss (Stark and Firestone, 1995).
Could microorganisms in C and nutrient limited soil environments
have evolved different mechanisms to cope with soil drying (and
re-wetting) than mechanisms observed to occur by microbes in
nutrient-rich laboratory culture (Tschichholz and Trper, 1990;
Schleyer et al., 1993)?
Microbial respiration rates in this study (data not shown)
declined in a typical manner with degree of drying and following
the re-wetting of soil (e.g. Birch, 1958; Williams and Xia, 2009). If
other microbial responses are similarly tied to desiccation level,
then it might be expected that intracellular osmolyte molecule
concentrations might show similar patterns of change to desiccation intensity. Indeed, the ush of soluble C (Fig. 5) released
following re-wetting of soil has been widely hypothesized to
represent the intracellular release of microbial osmolytes and also
tends to correlate well with the degree of drying (Schimel et al.,
1999; Williams and Xia, 2009; Chowdhury et al., 2011), however,
the chemical composition of this pool has not been observed to
contain high concentrations of putative osmolytes (Williams and
Xia, 2009).
The increase in PSA-sugars and NRN in the moderately dry
compared to both dry and moist soils may be interpreted to arise
from completely different microbial adaptations. Under extremely
dry conditions (
20 and
40 MPa) the costs of cell maintenance
rise and the availability of substrate to sustain microbial acclimation would tend to decline, perhaps favoring lower cost solutions
such as re-allocation of resources or transition into inactive and
dormant states. Cell maintenance activities under low water
potential, for example, are used to maintain cell wall uidity,
metabolite regulation, and reallocation of resources (Potts et al.,
2005). In this regard, the dynamics of sugar and nitrogen provide
clues to understanding how soil communities respond to desiccation and that may not be necessarily tied to osmolyte accumulation. The shifting of resources, such as PLFA, microbial C, and N
within a desiccation resistant microbial community suggests that
microbes could be re-allocating resources to cope with soil drying
(Singh et al., 2002).
It has been hypothesized that the increases in microbial C that
often occur during soil drying may be the result of re-distribution of
cellular C from structural to cytoplasmic cellular material (Schimel
et al., 1989; Williams and Xia, 2009). However, the re-allocation in
the opposite direction, from cytoplasmic to structural cell wall
material may also occur (Kieft et al., 1994; Singh et al., 2002). In this
regard, it is interesting that the abundance of PLFA showed
opposing trends to PSA-sugar abundance, for example, increasing
the PLFA concentration in the drought-prone Sumter and declining
in the more mesic Marietta soil in response to drought (Fig. 1b). The
different response of the cellular sugar (carbohydrate) and PLFA
pool sizes further conrm the different community responses, but
are also consistent with a cellular re-allocation hypothesis in
response to water decit. The reallocation of resources between
high energy carbohydrates and wall membranes may be an
important means of cellular adaptation to desiccation for microbes
in soils (Roberson and Firestone, 1992; Potts, 2001; Gustavs et al.,
2009; Hocking et al., 2012).

651

4.3. Effect of water decit on microbial communities and fungal/

bacterial (F/B) ratio
The response of the microbial community to desiccation may
simultaneously result in physiological re-allocation between
cellular pools and compositional community change. Shifts in
community composition were much larger for the Sumter than the
Marietta (Fig. 3) soil, indicating that the soils contain communities
with different desiccation coping mechanisms. In particular, the
greater community dynamics (greater F/B ratio) in the droughtprone Sumter may suggest the presence of adaptations that allow
greater levels of activity and functioning during desiccation, an
observation consistent with the idea that fungi are more likely to
remain active at very low water potentials than bacteria (Harris,
1981; Shipton and Burggraaf, 1982). The more mesic and less
drought-prone Marietta soil communities may adapt to drying, in
contrast, by reducing activity and overall community change. This
interpretation is consistent with the 50e75% reduction in microbial
respiration in Marietta relative to the Sumter soil (data not shown).
Related to the EPS hypothesis, it was observed that desiccation
increased the F/B ratio of Sumter but not the Marietta soil. Bacteria
are well known for EPS production and fungi are likely to modify
cell wall composition during stress periods (Csonka and Hanson,

1991; Blomberg and Adler, 1992; Kieft et al., 1994; Sajbidor,
1997;
Vargas, 2005). In this regard, the greater F/B ratio and increasing
pool size of membrane PLFA is consistent with fungal cell
membrane based responses to water potential decit in the
drought-prone Sumter. Though the relatively high activity of fungi
may increase cellular turnover and perhaps result in the loss of
some modular biomass and hyphae when dry soil is re-wetted
(Williams, 2007), it also provides the opportunity for foraging by
hyphae and access to resources with less competition from relatively localized and sessile taxa, such as bacteria.
Change in microbial C and N pool sizes to desiccation can also be
explained by community changes. Because fungi generally require
less N than bacteria to build cellular biomass, the increasing PLFAbased fungal to bacterial biomass ratio (Fig. 2) is consistent with the
greater microbial C:N ratio with drying in the Sumter soil (Williams
and Xia, 2009). Decreasing the water potential, furthermore, may
also increase cell wall growth relative to cytoplasmic growth, thus
increasing the C/N ratio of fungal organisms (Paustian and
Schnrer, 1987) and provide an explanation for the increasing
microbial C:N ratios with drying. This mechanism assumes limiting
resources are re-directed to hyphal growth to enhance resource
exploitation. The difference in microbial N demand in fungi
compared to bacteria could thus effect microbial physiological
responses and explain some of the different community responses
to drying between soils.
Among the bacterial groups, gram-positive bacteria, and to
some extent actinomycetes showed declines in their relative
abundance, an observation that goes against the idea that these
bacteria, because of their enhanced peptidoglycan layer should be
more resistant to drying (Potts, 1994). Indeed, recent studies have
shown opposite trends, that gram-negative are more well adapted
than gram-positive bacteria to water stress (Williams, 2007;
Williams and Rice, 2007; Aanderud and Lennon, 2011, Fig. 3.). It is
similarly possible that changes in methyl-branched PLFA, for
example, are indicative of physiological change in cell membranes
used to cope with drying (Nordstrm, 1993).
Though fungi beneted relative to bacterial biomass as a result
of desiccation in the drought-prone Sumter soil, there was again no
indication that specic fungal osmolytes, such as alcohols, were
produced to adapt to drying soil. In contrast, fungi may alter the
allocation of energy and C to cell wall production, and especially
change fatty acid synthesis during desiccation (Fig. 2; Blomberg

652

M.L. Kakumanu et al. / Soil Biology & Biochemistry 57 (2013) 644e653

and Adler, 1992). Bacteria, in contrast, may respond to desiccation

by increasing the allocation of C to EPS on the outer surface of the
cell wall (Roberson and Firestone, 1992), an interpretation consistent with increases in PSA-sugars in the Marietta soil (Fig. 1a).
5. Conclusion
The study was focused on testing the osmolyte accumulation
hypothesis (OAH) in soil by characterizing the concentrations of
common microbial osmolytes across a gradient of desiccation stress
under in situ soil conditions. Eleven putative osmolytes (sugars and
alcohols) were characterized but showed no consistent increases
with water potential decit that would link their production to
microbial adaptation to water decit. In contrast, the dynamics in
microbial PLFA, microbial C, and PSA-sugars indicate that microorganisms are responding to water decit. It is hypothesized that
microbes are adapting to desiccation, perhaps through the reallocation of molecular resources to the cell wall (fungi) and
extracellular cell wall EPS (bacteria). Some of the variability in
microbial responses to soil drying may also be explained by
differences in microbial community composition, structure, and
activity. The utilization of EPS production by bacteria to adapt to
desiccation has been described previously, however, in a native soil
context it has not been explicitly tested as a means of microbial
adaptation. Compared to OAH, these hypothesized mechanisms of
microbial response may be more consistent with the oligotrophic
energy and nutrient conditions typically found in soils. The
research suggests that current conceptual models of microbial
adaptation to water decit need re-evaluation.
Acknowledgments
Research was supported in part by NSFePCE and DOE awards to
M.A. Williams
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.soilbio.2012.08.
014.
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