ELSEVIER
FEMS Microbiology Ecology 2 1 (1996) 22 I-230
Effects on microbial activity by extraction of indigenous cells
from soil slurries
*, Kari Aa, Rolf A. Olsen
Viggo Lindahl
Department
of Biotechnological
Sciences, Agricultural
Utkersity
of Noma!,
P.O. Box 5040, N-1432 .&, Norway
Received 9 April 1996; revised 22 July 1996; accepted 14 August 1996
Abstract
Possible effects on the physiological
activity and cuhurability of soil microorganisms
by different soil dispersion
procedures, and effects on activity caused by extracting bacteria from soil, were investigated. There was no apparent
difference in cfu’s with dispersion of a silty loam soil and a loamy sand soil with pyrophosphate as compared to dispersion
in NaCI. Substrate-induced
respiration was reduced in the silty loam soil, and methanol oxidation was reduced in the loamy
sand soil with dispersion in pyrophosphate, and the soil pH was irreversibly increased by the treatment. Extracted bacterial
fractions had lower numbers of culturable cells as percentage of the total number of bacteria in each fraction, lower
respiration rates and no methanol oxidation activity as compared to the soil slurry both before and after extraction. The
physiological
activity was apparently not affected by the number of cells extracted. This indicates that the increased
extraction rate of indigenous soil bacteria obtained by effective disruption of aggregates and detachment of cells from
surfaces, only results in increased extraction of cells that have been physiologically changed as a result of the extraction
process.
Keywords;
Soil dispersion;
Bacterial
extraction;
Microbial
activity
1. Introduction
Extracting
indigenous
bacteria
from
soil is neces-
used in
the study of soil microbial ecology. Several problems
caused by the presence of soil particles are avoided
by extracting bacteria from soil. Humic material and
clay interfere with enzymatic reactions such as PCR
amplification [ 11, and nonspecific adsorption of antisary for the application
of many
techniques
* Corresponding
author. Tel: +47 64 94 77 44; Fax: +47
94 77 50: E-mail: [email protected]
0168-6496/96/$15.00
PII
Copyright
SO168-6496(96)00058-X
0 1996 Federation
64
of European
bodies to soil particles [2] makes both immunofluorescent microscopical
enumeration
and the use of
immunomagnetic
beads for extraction of species
populations from soil slurries difficult. Separation of
bacteria from soil has also made possible size fractionation 131 and flow cytometric investigation of soil
bacteria [4,5].
Physiological
studies of indigenous soil bacteria
have been possible using bacteria extracted from
soil. Fagri et al. [6] obtained constant respiratory
rates with bacteria extracted by the slow-speed centrifugation method. The bacterial suspension contained about 50-80% of the total bacteria in the soil,
Microbiological
Societies.
Published
by Elsevier Science B.V.
222
V. Lindahl et 01. / FEMS Microbiology
but constituted only 20-50% of the total respiration.
The same extraction procedure was also used to
estimate the growth rate of extracted bacteria by the
i3H]thymidine
incorporation
technique, and it was
found that the growth rate was little affected by the
perturbation induced by making a slurry [7].
A representative extraction of indigenous soil bacteria requires that cells are dislodged from particle
surfaces and released from within aggregates by an
efficient dispersion
method. Establishment
of the
strong binding between bacteria and soil particles
involves a variety of binding mechanisms [8]. The
initial attachment process is followed by strong binding within aggregates and between cells and surfaces, probably involving extracellular
polysaccharides [9]. These bonds must be overcome for an
effective separation of cells and soil particles; thus a
successful strategy for dispersion is a choice between
cell survival and dispersion efficiency. Lindahl and
Bakken [lo] demonstrated that physical dispersion in
the Waring blender was the most effective dispersion
method for extraction of bacteria, followed by separation of cells and soil particles with Nycodenz
density gradient centrifugation.
High extraction frequencies by repeated extractions increase the possibility of a more representative
sampling of indigenous bacteria for morphological and molecular biological studies, together with a more accurate enumeration of the total number of bacteria in the soil.
In aquatic systems bacteria attached to surfaces
make up a significant proportion of the total bacterial
numbers and account for most of the bacterial production and activity in areas where particulate material is common [ll]. Solid surfaces can apparently
modify the activity of attached bacteria, because of
the special physicochemical
conditions and forces at
the solid-liquid interface, and activity may be promoted through the adsorption (concentration)
of potential nutrients on surfaces, making them more accessible to attached bacteria [ 121. Soon after adsorption to a surface, many bacteria are seen to produce
large amounts of the extracellular material responsible for their strong adhesion [ 131. These extracellular
polysaccharides
can be expected to act in varying
degrees as diffusion barriers, molecular sieves and
adsorbents, and may have a protective role for excreted extracellular enzymes [ 141. Extracellular polymeric material may also affect survival through stor-
Ecology 21 11996) 221-230
age of water and buffering against water potential
fluctuations [ 151.
Extracting cells from soil alters the microenvironment surrounding the cells. By removing cells from
surfaces the advantages of surface adsorption will be
lost. Mechanical or chemical breakage of extracellular polymers will occur as a result of the soil dispersion procedures. Nutrients and extracellular enzymes
accumulated and entrapped within these extracellular
polymers will probably be lost, and many cells will
experience changes in the physicochemical
conditions. Unattached cells will probably not be subjected to such drastic changes. Thus, the physiological state of the extracted bacteria may not be
representative
of the physiological
state of indigenous bacteria in soil.
The aim of this work was to study the possible
effects on physiological
activity of indigenous microorganisms in soil by the use of different dispersion procedures, and effects on activity caused by
extracting bacteria from soil. The number of culturable bacteria and the respiration rate was measured
in the soil slurry, in the fraction of extracted bacteria
and in the sedimented
soil after extraction. The
activity of methylotrophic
bacteria was studied for
an evaluation of both extraction efficiency and possible effects on a specific physiological group by the
treatments.
2. Materials
and methods
2.1. Extraction
bacteria
and enumeration
of indigenous
soil
Initial experiments were carried out with an agricultural silty loam soil (Qsaker) with 7.6% organic
matter and an agricultural loamy sand soil (Larvik)
with 5.6% organic matter. The soils (20 g) were
dispersed (see below) by shaking or in the Waring
blender with 180 ml filter-sterilized 0.15% NaCl or
0.05 M pyrophosphate
(adjusted to pH 8.0 with
HCl), followed by centrifugation
in a GSA-rotor at
16000 X g for 30 min, before redispersion in 180 ml
0.15% NaCl by vortexing. Centrifugation
and redispersion were repeated 3 times, to minimize the pH
effect in soil of pyrophosphate
addition. The final
redispersion was done in 50 ml 0.15% NaCl.
V. Lindahl et al. / FEMS Microbiology
The silty loam soil (0saker) was chosen for further experiments due to the higher bacterial numbers
and apparent complexity with regard to microaggregates and clay content. These experiments were carried out by dispersion of 20 g agricultural silty loam
soil (0saker) in 180 ml 0.15% NaCl, by the following treatments:
Shaking of soil suspensions was done on a reciprocal shaker (125 cycles min- ’ ) for 2 h at 4”C, in
500-ml Pyrex flasks kept in a horizontal position
oriented along the movement axis of the shaker.
Waring blender treatments were done with the
80 12 S Waring commercial laboratory blender (Waring, New Hartford, CT), in the midi-container MCC3
(volume 50-250 ml) at maximum speed for 8 min
unless otherwise noted. The containers had cooling
jackets with constant circulation of ice-cold water,
securing low temperatures during the treatment.
Cells and soil particles from these treatments were
separated by high-speed centrifugation
with the density gradient medium Nycodenz (Nycomed Pharma
AS, Oslo, Norway), as described by Bakken and
Lindahl. Nycodenz is a water-soluble, nonionic and
nontoxic derivative of benzoic acid. A 20 ml Nycodenz cushion (1.3 g ml-i density) was placed below
a 200 ml soil suspension in 250-ml centrifugation
tubes, before centrifugation at 10 000 X g in a Sorval
HB-4 swing out rotor for 60 min at 4°C.
After centrifugation,
the extracted cells were recovered in the supematant, including both the Nycodenz cushion and the supematant above the cushion.
Nycodenz could easily be removed by homogenizing
the supematant by shaking, and adding 50-100 ml
dispersion liquid for further dilution of the Nycodenz, before sedimentation of the cells by centrifugation in a Sorval GSA-rotor at 16000 X g for 60 min.
Repeated extractions were done by repeating the
procedure on the sedimented soil obtained after Nycodenz density gradient centrifugation.
Extractions by the slow-speed centrifugation
procedure were done according to Faegri et al. [6] with
minor modifications.
Soil slurries were treated for 3
nun in the Waring blender, before separating cells
and soil particles by centrifugation
in a Sorval
GSA-rotor at 1000 X g for 10 min. The procedure
was repeated 3 times before sedimentation
of the
cells in the supematants by centrifugation in a GSArotor at 10000 X g for 30 min.
Ecology 21 Cl 996) 221-230
223
Dispersion of soil together with the extraction
procedures resulted in three fractions (soil slurry,
extracted cells and the sedimented soil after extraction) which was compared by the physiological studies (see below). Extracted bacteria and the sedimented soil were redispersed in 50 ml dispersion
liquid. Extracted bacteria were first redispersed in a
small volume with a syringe and needle. The sedimented soil was redispersed by 1 min treatment in
the Waring blender.
Fluorescence
microscopical
enumeration
of indigenous cells was done by acridine orange direct
counting (AODC), as described by Hobbie et al.
[16], with counting of approximately
40 randomly
selected areas per filter.
Extraction efficiency was calculated as percentage
of cells extracted from the soil in relation to the total
number of cells in the soil slurry, as determined by
AODC.
2.2. Physiological
studies
The numbers of colony forming units (cfu’s) in
the different fractions were determined on water agar
( 15 g agar in 1000 ml distilled water), and GG agar
(IO g peptone, 10 g yeast extract, 5 g glucose, 0.2 g
MgSO,, 50 mg MnSO,, 1.5 g KI-12P0, and 15 g
agar in 1000 ml distilled water) diluted 1: 10 and
1: 100 (denoted l/ 10 GG and l/100 GG). Cycloheximide was added to a final concentration
of 50
pg ml-’ to inhibit fungal growth. Samples were
diluted in 0.15% NaCl, and five plates per dilution
were prepared. The plates were incubated at 20°C for
14 days.
The respiration rate was measured as CO, production from 50 ml samples in a 100 ml flask at
20°C with the Rosemount BINOS@lOO infrared gas
analyzer (Rosemount
& Co. GmbH, Hanau, Germany). Endogenous
respiration
was continuously
measured for 20-30 min before addition of 5 mg
glucose (final concentration 0.1 mg ml- ’ > for measurement of substrate-induced
respiration for 20-30
min. Measurements
were conducted with suspensions in NaCl. The samples were placed on ice and
measured both within 1 h and after storage overnight
at 2°C giving the same results. Respiration rates
were calculated from the mean values obtained.
Methanol oxidation was estimated by measuring
224
V, Lindahl et al. /FEMS
Microbiology
consumed methanol in 20-ml samples incubated in
120-ml serum flasks (sealed with butyl rubber stoppers) on a reciprocal shaker (125 cycles min-’ > at
2O”C, with the flasks kept in a horizontal position
oriented along the movement axis of the shaker. Ten
~1 methanol was added to each flask. Subsamples
(0.5 ml) were removed and filtered (through 0.2 pm
filters) during a ICday period (t = 0 was 15 min),
and the filtered subsamples were kept at -20°C
before measurements. The methanol concentration in
1 ~1 samples was measured with a FID detector
(13O”C, 0.9 min retention time) on a Shimadzu GC14A Gas Chromatograph
(Shimadzu
Corporation,
Kyoto, Japan) equipped with a Hayesep P column
MR 51375 (Supelco Inc., Bellefonte, PA) with N,
(3.0 kg cm3 _ I), HZ (0.6 kg cm3 ‘) and air (0.5 kg
was
cm 3 -‘I as carrier gasses. The chromatogram
recorded on a Shimadzu C-RSA integrator.
Treatment of plate count data and calculation of
confidence intervals were done as described by Baker
[17]. The experiments with dispersion methods and
activity measurements
(cfu, respiration
rates and
methanol oxidation) in the soil slurries from the two
different soils were done twice, and the data were
subjected to analysis of variance (ANOVA) followed
by Student’s t-test. The different fractions obtained
after extraction of cells from the Osaker soil by the
different dispersion treatments were treated as replicates when the data (cfu, respiration and methanol
oxidation) obtained after extraction were tested by
analysis of variance (ANOVA).
Table 1
Effect of dispersion
Eco1og.v 21 (19961 Z-230
3. Results
3.1. Bacterial
persion
actil:ity in the soil slurries after dis-
No significant difference (P < 0.05) in culturable
cells measured as cfu was observed in the two soils
after dispersion with 0.05 M pyrophosphate (pH 8.0)
and 0.15% NaCl (Table 1). The total number of
bacteria was approximately 50% higher in the Bsaker
soil than in the Larvik soil. Fluorescence microscopy
indicated that the Larvik soil contained fewer microaggregates, and relatively few bacteria were attached to the surfaces of the sand grains constituting
a considerable fraction of the soil (results not shown).
The number of cfu was higher with the diluted
media (l/100
GG and water agar) than with the
higher nutrient content medium (1 /lO GG). Colony
morphology was also different, with predominance
of small translucent
colonies on the low-nutrient
agars, as opposed to larger and more distinctive
colonies on the medium-nutrient
agar (I/ 10 GG).
The percentage culturable cells did not exceed 5%
under the conditions used.
The respiration rate measurements
for both soil
types showed no apparent difference with dispersion
in the Waring blender as compared to shaking, and
no difference in endogenous
respiration
was observed when dispersion was done in pyrophosphate
as compared to NaCl (Table 2). Increased substrateinduced respiration was observed in the silty loam
liquid on the number of cfu’s (per g dw soil) in soil slurries after dispersion
Agar
Soil type
in the Waring blender
Number of cfu’s X 10’
NaCl
Silty loam (0saker)
Loamy sand (Larvik)
’
b
l/IO GG
l/l00 GG
Water agar
l/lOGG
l/100 GG
Water agar
4.3
6. I
5.8
3.7
10.7
5.3
Pyrophosphate
(3.2-5.8)
(4.1-9. I)
(4.4-8.0)
(2.2-6.2)
(8.3-13.8)
(4.5-8.7)
4.9 (3.1%6.9)
Il.9 (5.3-26.7)
11.0(7.3-16.5)
4.0 (2.7-6.0)
8.1 (4.5-14.8)
4.4 (3.5-8.2)
' AODC = 4.2+ 0.5X 109/g dw soil.
h AODC = 2.8 f 0.3 X IO’/g dw soil.
All values are means with confidence intervals (95% confidence
level) in parenthesis.
AODC measured
after dispersion
in pyrophosphate.
225
V. Lindahl et al. / FEMS Microbiology Ecology 21 (1996) 221-230
soil (Osaker) with dispersion in NaCl as compared to
pyrophosphate (P < 0.05) while no significant difference was observed in the loamy sand soil (Larvik).
Dispersion in pyrophosphate
resulted in increased
soil pH, which was only slightly lowered by repeated
washings of the soils in NaCl. The increase in soil
pH was most pronounced
in the silty loam soil
((dsaker).
The rate of methanol oxidation was reduced in the
loamy sand soil (Larvik), dispersed with pyrophosphate as compared to NaCl (P < O.OS>, while no
such effect was observed in the silty loam soil
(Osaker) (Fig. 1).
No distinct effect of centrifugation
on bacterial
activity was found (results not shown). Almost equal
numbers of culturable cells were found in the soil
slurries both before and after centrifugation. No clear
difference was found in either respiration rate or
methanol oxidation rate, when measurements
were
done after one washing of the soils in NaCl as
compared to several consecutive washings.
3.2. Bacterial
activity in the difSerent soil fractions
The number of culturable cells was significantly
lower in the extracted bacterial fractions than in the
sedimented soil after extraction (P < 0.05) and never
exceeded 1% of the total number of bacteria in the
fraction (Table 3). The highest number of cfu’s in all
fractions was obtained with 1/lOO GG and water
agar, but the number never exceeded 2-4% even in
the sedimented
soil after extraction. The mildest
Table 2
Respiration
Soil
0saker
measured
as pg CO> min-’
Dispersion
treatment
Shaking
Waring blender
Larvik
Shaking
Waring blender
produced
Dispersion in
NaCI:
-oSilty loam (0saker)
---o--. Loamy sand (Larvik)
Pyrophosphate
----+-
0
2
4
:
Silty loam (Osaker)
Loamy sand (Larvik)
6
Days
Fig. 1. Methanol oxidation
of soil slurries (pmol
oxidized
methanol ml - ’ ) after dispersion with 0.15% NaCl and 0.05 M
pyrophosphate
(pH 8.0) in the Waring blender, and addition of
12.5 pmol methanol ml-‘. All samples were washed and redispersed in 0.15% NaCl. Standard error bars smaller than symbols
(n = 2).
dispersion treatment (shaking) resulted in the lowest
number of cfu’s (9 X 105) but the percentage cfu’s
of the number of cells extracted after one extraction
was significantly higher on l/ 100 GG by this treatment than with the other treatments (P < 0.05) while
there was no difference on 1/ 10 GG and water agar.
Repeated extractions with Nycodenz density gradient
centrifugation apparently resulted in the highest total
number of cfu’s from the extracted bacteria (1.4 X
lo’), together with the highest percentage cfu’s of
the number of cells extracted. No significant difference was observed between the soil slurry and the
sedimented soil after extraction.
Both endogenous
and substrate-induced
respiration was significantly
higher in the soil slurry and
sedimented soil after extraction than in the bacterial
in 50.ml soil slurries (20 g soil) after dispersion
Dispersion
liquid
NaCl
Pyrophosphate
NaCl
Pyrophosphate
NaCl
Pyrophosphate
NaCl
Pyrophosphate
Endogenous
1.4
0.7
1.2
1.7
0.7
0.5
1.2
1.0
*
+
+
+
+
+
i
f
0.1
0.4
0.1
0.5
0.1
0.2
0.4
0.1
* Total respiration after addition of glucose to final concentration 0.1 mg ml-’
All samples were washed and redispersed in 0.15% NaCl. All values are means + S.E.
Substrate-induced
2.4
0.5
2.5
2.0
1.0
0.6
0.8
1.6
+
*
+
f
+
+
*
f
0.3
0.2
0.4
0.3
0.1
0.3
0.3
0.2
a
PH
6.2
7.2
5.8
7.5
6.9
7.1
6.7
7.3
226
V. Lindahl et al. / FEMS Microbiology
Ecology 21 (1996) 221-230
Table 3
Number of viable and culturable cells (cfu) in different fractions of a silty loam soil (@s&r) as percentage of the number of bacteria (as
AODC per g dw soil) in each fraction. Soil dispersion was done in NaCl by shaking or by Waring blender treatments
Treatment
Centrifugation
Fraction
AODC X lo9
l/lOGG
l/100
Waring blender
Shaking
No
Nycodenz
Waring blender
Nycodenz
Repeated Waring blender
Nycodenz
Waring blender
Slow speed
Soil slurry
Extract. bact.
Pellet
Extract. bact.
Pellet
Extract 1
Extract 2
Extract 3
Pellet
Extract. bact.
Pellet
4.0
0.1
2.1
0.9
3.3
1.1
1.0
0.8
3.1
3.4
3.8
1.0
0.2
0.6
0.1
0.9
0.1
0.3
0.3
1.2
0.1
0.7
2.7
0.9
1.6
0.3
3.7
0.3
0.5
0.6
2.1
0.3
0.9
All values are means with confidence
intervals (95% confidence
(0.8-1.2)
(0. I-0.3)
(0.3-1.4)
(0.1-0.1)
(0.6-1.3)
(0.1-0.1)
(0.2-0.4)
(0.1-0.7)
(0.9-l .6)
(0.1-0.1)
(0.6-0.7)
GG
(2.1-3.5)
(0.4- 1.9)
(0.6-4.7)
(0.2-0.5)
(2.5-5.5)
(0.2-0.3)
(0.4-0.5)
(0.6-0.7)
(1.6-2.7)
(0.2-0.4)
(0.9-l .O)
Water agar
1.0
0.4
1.1
0.1
1.3
0.3
0.2
0.3
1.3
0.1
0.5
(0.5-1.8)
(0.2-0.8)
(0.8-1.4)
(0. I-0.2)
(1.0-1.6)
(0.2-0.7)
(0.1-0.5)
(0.3-0.3)
(0.5-3.2)
(0.1-0.2)
(0.4-0.7)
level) in parenthesis
fractions (P < 0.05). The respiration rates in the
extracted bacterial fractions were low and apparently
equal (Table 4). There was no statistically significant
difference in both endogenous and substrate-induced
respiration between the soil slurries and the sedimented soil after extraction. There was no increase
in respiration rate when glucose was added to extracted bacteria. The respiration per bacterium was
probably highest in extracted bacteria obtained after
dispersion by shaking (with the lowest number of
extracted bacteria), and lowest after slow-speed cen-
trifugation extraction (with the highest number of
extracted bacteria). No difference in activity was
observed by storage of samples at 2°C for 20 h
before measurements
compared to immediate measurements after dispersion and extraction (results not
shown).
No methanol oxidation was observed in any of the
fractions of extracted bacteria (Fig. 2). The oxidation
rate in the sedimented
soils after extraction was
significantly (P < 0.05)lower than the oxidation in
the soil slurry before extraction (Fig. 2). The oxida-
Table 4
Total respiration (Fg CO, min-’ ) and respiration per cell (g CO, X lo- ” min- ’ ) after dispersion and extraction of bacteria
silty loam soil (asaker). Dispersions of soil with 0.15% NaCl were done by shaking or by Waring blender treatments
Treatment
Sample
Shaking + Nycodenz
Waring blender + Nycodenz
Repeated Waring blender + Nycodenz
Waring blender + Slow speed centrifugation
a Total respiration
Bacteria
Pellet
Soil slurry
Bacteria
Pellet
Soil slurry
Bacteria
Pellet
Soil slurry
Bacteria
Pellet
Soil slurrv
AODC X 10’”
0.17
3.9
4.0
1.2
4.8
6.0
4.2
4.5
8.5
4.9
5.5
10.5
after addition of glucose to final concentration
0.1 mg ml-‘.
Total respiration
Respiration
from 20 g
per cell
Endogenous
Substrateinduced ’
Endogenous
Substrateinduced ’
0.4
1.4
1.5
0.5
1.1
0.6
0.2
1.o
0.9
0.3
0.9
0.6
0.4
2.8
3.3
0.4
2.0
2.1
< 0.1
2.4
2.0
0.1
2.0
3.3
23.5
3.6
3.8
4.2
2.3
1.0
0.5
2.2
1.1
0.6
1.6
0.6
27.8
7.2
8.3
3.3
4.2
3.5
< 0.2
5.3
2.4
0.2
3.7
3.1
V. Lindahl et al./ FEMS Microbiology Ecology 21 (1996) 221-230
Resultant soil
Extracted bacteria
0
0
2
4
6
8
10 12 14 16
Days
Fig. 2. Methanol oxidation in the extracted bacterial fractions and
the sedimented soil after bacterial extraction, after addition of 12.5
pmol methanol ml-‘. Bacteria extracted by Nycodenz gradient
centrifugation
after dispersion by shaking (+), Waring blender
(A ) and repeated extractions (0). and extracted by slow speed
centrifugation
(0). Soil slurry without treatment (0) as control.
Error bars not visible when smaller than symbols.
tion rate in the sedimented soil after shaking was not
significantly
higher than the soils obtained by the
other treatments.
4. Discussion
Recovery of l-5%
of culturable cells by the
spread-plate method is within the expected range of
l-10% of the number of cells obtained by direct
microscopic counts [ 181 (Table 1). The highest number of cfu’s were observed on the low-nutrient media, indicating an oligotrophic nature of the majority
of culturable soil bacteria. Growth on the water-agar
is probably supported by organic material contaminating the agar [ 191, as well as volatile carbon material from the air [20].
No apparent difference in respiration rate with
dispersion of soil in the Waring blender compared to
shaking (Table 2) indicates that Waring blender
treatment does not affect the metabolic status or
integrity of the cells. The reduced substrate-induced
respiration (Table 2) in the silty loam soil (0saker)
after dispersion with pyrophosphate as compared to
NaCl, together with an apparent reduction in the rate
of methanol oxidation in the loamy sand soil (Larvik)
(Fig. l), might m
. d’rca te negative effects of pyrophosphate on metabolic integrity. This could be a result
of the increase in soil pH by the treatment. Higher
221
both endogenous and substrate-induced
respiration in
the silty loam soil (Osaker) than in the loamy sand
soil (Larvik) after dispersion with NaCl is probably a
result of the higher bacterial numbers in this soil.
No observable effect on bacterial activity of centrifugation suggests that procedures including several
centrifugation steps may be applied to soil slurries.
We found that microscopic counts were enhanced
by extraction of bacteria, because the more cells
extracted the fewer cells remained undetected in the
soil during microscopical
examination
as observed
by Lindahl [21]. Repeated extractions therefore ensured a relatively correct enumeration
of the total
number of bacteria in the soil. This is probably a
result of the disruptive effects on aggregates and
increased detachment of cells from surfaces by this
treatment. Dispersion of the soil by shaking resulted
in low microscopic counts of bacteria in the soil
slurry, and a low extraction efficiency.
The higher extraction
efficiency
obtained by
slow-speed centrifugation
extraction as compared to
Nycodenz density gradient centrifugation
(Table 3)
may be explained by the findings of Hopkins et al.
[22] that the majority of cells extracted by slow-speed
centrifugation extraction were not completely dissociated from soil particles. The extracts we obtained
were associated with relatively dense soil material,
even obscuring microscopical detection of cells. Extracts obtained by Nycodenz density gradient centrifugation were almost devoid of soil material by
comparison.
Extracted bacterial fractions had a much lower
proportion of cfu’s to the total number of cells than
both the soil slurry and the sedimented soil after
extraction (Table 3). Repeated extractions result in
the highest number of extracted cells [21], and also
the apparent highest number of cfu’s. The low percentage of cfu’s in the extracted bacterial fractions
may partly be a result of altering the microenvironment surrounding the cells in soil by the extraction
procedures, but breaking up of aggregates and detaching cells from surfaces probably also affects the
culturability
of the extracted cells. Both the autochthonous and the zymogenous flora were affected
by extraction, since the low percentage of cfu’s in
the extracted bacterial fractions was observed with
ail the media used. BIHth [23] obtained a higher
percentage of cfu’s after extraction of bacteria by
228
V. Lindnhl
et nl. / FEMS Micmhiokqq
slow-speed centrifugation,
and this might be due to
the different soils used.
Respiration per cell was much higher in extracted
bacterial fractions obtained by shaking than by the
other treatments, as opposed to the findings of B%ath
[23] that the [ 3H]thymidine
incorporation
rate per
cell in extracted bacteria was lower after shaking
than after the use of a blender. This may indicate that
low cellular maintenance
activity (of both the nongrowing and the growing population)
contributes
more to the thymidine incorporation
measurements
than the respiration activity measured in this work.
The extracted bacterial fractions obtained after
Waring blender treatment also had reduced endogenous respiration rates and showed no substrate-induced respiration, as opposed to the soil slurries and
the sedimented soil after extraction (Table 4). The
similar respiration rates in the soil slurries and the
sedimented soil after extraction indicates little influence on respiration in soil by extraction. The reduced
respiration rates in extracted bacterial fractions may
partly be a result of alteration of the physicochemical
conditions, together with the effects caused by detachment of cells from surfaces and disruption of
extracellular
polymeric materials. This may partly
explain the findings of B%%th[7] that the [3H]thymidine incorporation per bacterium was approximately
30% lower in the extracted bacterial fraction than in
the whole soil. Both reduced respiration rates and
low proportion of cfu’s in the extracted bacterial
fractions may indicate that only bacteria with low
activity are extracted, but this is unlikely since 3045% of bacteria were extracted from the soil with the
repeated extraction treatments.
Nycodenz density gradient centrifugation
did not
seem to affect bacterial activity, since the respiration
rates were almost equal in the soil slurries and in the
sedimented soil after extraction with Nycodenz.
Increased substrate uptake has been found by cells
after biofilm formation compared to their free-living
counterparts [ 121, indicating increased substrate uptake after attachment to surfaces, but this may only
partly explain the observed respiration rates after
extraction. The need for resuscitation
is probably
more important. A proportion of the metabolically
active cells in the environment
cannot be cultured
[24], but some of these viable but nonculturable cells
may be recoverable after storage due to resuscitation
Ecology
21 (lYY6,61221-230
of the cells [25]. The storage process needed for
resuscitation of extracted soil bacteria will probably
result in alteration of the community structure in the
extracted bacterial fractions [26], but physiological
studies of certain microbial species may still be
possible.
The lack of methanol oxidation in all the extracted bacterial fractions (Fig. 2) indicates either
extraction of very few methylotrophic
bacteria, or
physiological
changes in the cells as a result of
extraction. Methanol oxidation should have been detected even after the reduction in bacterial activity
observed after extraction, if extraction of these cells
had been successful. These cells either remain firmly
attached to surfaces after dispersion, or they lose
methylotrophic activity because of extraction. Lower
methylotrophic
activity in the sedimented soil after
extraction than in the soil slurries before extraction
(Fig. 21 may indicate that some of the methylotrophic bacteria had been extracted. This reduced
activity was not a result of dispersion. since the soil
slurry was also dispersed.
There are indications that the highest percentage
of cfu’s in the extracted bacterial fraction was obtained after dispersion by shaking. The respiration
rate in this fraction was equal to the respiration rates
in the other extracted bacterial fractions that contained more cells. Only the most loosely bound cells
are obtained after dispersion by shaking. and these
cells might be responsible for most of the cfu’s and
the measured respiration in all the fractions of extracted bacteria. The cells extracted after dispersion
by shaking are probably not representative
of the
cells found in soil, because most of the indigenous
bacteria in soil are firmly attached to surfaces or
found in aggregates. Thus, indigenous soil bacteria
obtained after detachment from surfaces and disruption of aggregates
have possibly
been physiologically changed as a result of the extraction procedures used.
Increased number of cfu’s and total bacterial
counts by dispersion of soil in pyrophosphate indicates that dispersion is more efficient with the use of
pyrophosphate instead of NaCl. We have not found
evidence for reduced bacterial activity after disper-
V. Lindahl et al. /FEMS
Microbiology
sion of soil in pyrophosphate. Bacterial activity measured as cfu’s, respiration and methanol oxidation, is
lower in extracted bacterial fractions than in soil
slurries and the sedimented
soil after extraction.
Measurement
of bacterial activity after extraction
should probably only be done after an inefficient
dispersion procedure like shaking. Measurements will
then be restricted to the loosely attached bacteria in
soil, which are the cells that appears least affected
physiologically
by the treatments. The value of such
measurements for studies of the soil community will
unfortunately
be limited, even though certain other
bacterial groups may be studied after a resuscitation
period.
Acknowledgements
This work was financially
search Council of Norway.
supported
by the Re-
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