See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/235643346

Growth rate of bacteria is affected by soil texture and extraction procedure

Article  in  Soil Biology and Biochemistry · February 2003

DOI: 10.1016/S0038-0717(02)00254-7

CITATIONS READS
27 347

2 authors:

Eva Kaštovská Hana Santruckova

University of South Bohemia in České Budějovice University of South Bohemia in České Budějovice
41 PUBLICATIONS   891 CITATIONS    142 PUBLICATIONS   5,133 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

The permafrost microbiome and its response to climate change View project

The role of nutrient availability in microbial soil organic matter formation and stabilization in agricultural soils of different C saturation status View project

All content following this page was uploaded by Eva Kaštovská on 07 January 2019.

The user has requested enhancement of the downloaded file.

 Soil Biology & Biochemistry 35 (2003) 217–224
www.elsevier.com/locate/soilbio

Growth rate of bacteria is affected by soil texture

and extraction procedure
E. Uhlı́řováa,b,*, H. Šantrůčkováa,b
a
Faculty of Biological Sciences, University of South Bohemia, Branišovská 31, 370 05 České Budějovice, Czech Republic
b
Institute of Soil Biology, Academy of Sciences of the Czech Republic, Na Sádkách 7, 370 05 České Budějovice, Czech Republic
Received 17 December 2001; received in revised form 28 October 2002; accepted 4 November 2002

Abstract
Effects of soil texture on the extraction efficiency of bacteria from soils and on biosynthetic activity of the extracted bacteria were studied.
Bacterial extracts were prepared from three soils of different texture by homogenization (ultrasonication and mixing) or by homogenization –
centrifugation at different speeds. Bacterial biosynthetic activity was estimated using thymidine and leucine incorporation techniques. In
each step of the extraction procedure, a higher extractability of bacteria was obtained in finer soils than in coarse soil. Also cell-specific
growth rates of bacteria were higher in the finer soils than in the coarse soil. However, in all soils, the extracted bacteria always had
significantly lower cell-specific thymidine and leucine incorporation rates than the bacteria in soil slurries and thus did not represent so well
the bacterial growth in the original soils. The total declines in cell-specific incorporation rates caused by the extraction were larger in fine soil
(96 – 98%) than in coarse soil (90%), but bacteria in the coarse soil were more responsive to only minor intervention. The homogenization –
centrifugation method eliminated the differences in bacterial biosynthesis found when working with soil slurries. Therefore, we recommend
using of soil slurries or, optionally, soil suspensions to compare bacterial biosynthetic activity among soils of different textures.
q 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Soil texture; Thymidine and leucine incorporation; Extraction of bacteria; Biosynthetic activity of bacteria; Turnover of bacteria

1. Introduction homogenization –centrifugation of a soil sample. When

Bacterial growth reflects biosynthetic activity of a cell. It
is the ability of the cell to create new biomass and to divide
(Pollard and Greenfield, 1997). The biosynthetic activity
characterizes an actual physiological state of the bacterial
population and its ability to survive and develop under the
given environmental conditions. Bacterial biosynthetic
activity can be expressed as growth rate, biomass pro-
duction g21 of soil, generation time, or turnover time.
Techniques of labeled thymidine (TdR) and leucine (Leu)
incorporation by bacteria offer a good possibility of
measuring the gross bacterial growth rate in soil, which is
not distorted by cell death and predation.
The TdR and Leu incorporation by soil bacteria can be
measured directly in a soil or in a soil extract obtained after
* Corresponding author. Present Address: Institute of Soil Biology,
Academy of Sciences of the Czech Republic, Na Sádkách 7, 370 05 České
Budějovice, Czech Republic. Tel.: þ 420-38-7775767; fax: þ 420-38-
5300133.
E-mail address: uhlirova@upb.cas.cz (E. Uhlı́řová).
0038-0717/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 3 8 - 0 7 1 7 ( 0 2 ) 0 0 2 5 4 - 7
working with soil, labeled TdR and Leu are added to a soil
slurry, therefore, the activity of the entire soil bacterial
community is taken into account (Christensen et al., 1989;
Bååth, 1990; Michel and Bloem, 1993). The use of the soil
extract, i.e. the homogenization – centrifugation method
(Bååth, 1992), deals only with an extracted portion of the
original soil bacterial community. Although it has been
demonstrated that the bacterial community obtained with
the homogenization –centrifugation method is not repre-
sentative of the total bacterial community of the soil (Bååth,
1996), the method has been routinely used in soil
microbiology because of its clear advantages. These include
the following: (i) the extract contains fewer soil particles
than the slurry, causing higher sample homogeneity and
lower sorption of TdR and Leu to soil particles, which
increases the accuracy of the measurement; (ii) the isotope
dilution is lower in the extract than in the soil slurry and,
therefore, the actual concentration of available labeled
substrate is higher and blank controls are very low; and (iii)
the method is markedly less time-consuming (Bååth, 1992).
218 E. Uhlı́řová, H. Šantrůčková / Soil Biology & Biochemistry 35 (2003) 217–224

The homogenization – centrifugation method selects only
a certain part of original bacterial community (Bååth, 1996).
If bacteria for subsequent TdR or Leu incorporation
measurement are extracted from differently textured soils,
one should also consider, whether soil texture can notably
affect the extraction efficiency of bacteria from soils and
especially their physiology. It is generally accepted that soil
texture affects bacterial movement and that bacteria can be
bound to soil particles in different ways and with different
strengths (Tan et al., 1991; Huysman and Verstraete, 1993;
Mayr et al., 1999). Bacteria that are more tightly bound to
soil particles have higher cell-specific growth rates than
bacteria readily released to an extractant by gentle shaking
or mixing a soil sample (Bååth, 1996). It suggests that soil
texture can affect not only the extraction efficiency of
bacteria from soil by homogenization –centrifugation but
also the resulting growth rates of the extracted bacteria. The
extraction of bacteria prior to an incorporation measurement
might not be appropriate, if it is necessary to compare
bacterial growth rates among soils of different texture.
Our goal was to test: (1) if soil texture affects the
extractability of bacteria and bacterial growth rates and (2)
whether homogenization –centrifugation is usable for soils
of different textures. We used the TdR and Leu incorpor-
ation techniques to measure total and cell-specific biosyn-
thetic activity of bacteria in soil slurries and soil extracts
prepared from three soils of different textures using different
treatments (homogenization or homogenization – centrifu-
gation at different speeds).
2. Materials and methods
2.1. Soils
Three soils of different texture were used in the
experiment (Table 1). Soils 1 and 2 were meadows, soil 3
was a forest soil. The soils were collected in different parts
of South Bohemia (Czech Republic), at depths of 5 –20 cm,
in October 2000. The samples did not contain litter material.
The fresh soils were sieved (2 mm) and stored in
polyethylene bags at 4 8C until used.
2.2. Sample treatment
Each soil was divided into six samples: a soil slurry, a
soil suspension and four soil extracts. Soil slurries were
prepared by vortexing 50 mg wet weight soil with 1 ml of
distilled water in plastic vials for 30 s. Soil suspensions and
soil extracts were prepared from 10 g wet weight of each
soil, which were homogenized with 200 ml distilled water
by ultrasonication for 3 min (Ramsay, 1984; Riis et al.,
1998) and mixing for 1 min with a hand mixer at the
maximum speed. The mixtures were left to sediment coarse
soil particles for 1 min. Soil suspensions without coarse
particles were divided into five parts. One part was used for
the incorporation measurement with no additional treatment
(‘soil suspension’). The other four parts (‘soil extracts’)
were centrifuged at 100, 250, 500 or 750g, respectively, for
10 min at 5 8C. One milliliter of the soil suspensions and the
soil extracts were placed into plastic vials, equilibrated for
10 min at 20 8C, and subsequently used for an incorporation
measurement.
2.3. TdR and Leu incorporation
Methyl-[3H]-thymidine (1.7 TBq mmol21, Lacomed,
CZ) and L-[U-14C]-leucine (10.3 GBq mmol21, Lacomed,
CZ) were added into plastic vials at 200 and 775 nM,
respectively, and the samples were mixed by vortexing. The
samples were incubated for 1 h at 20 8C, followed by
the addition of 100 ml of 36% formalin to stop the reaction.
The zero-time controls were always included where
formalin was added before the labeled substrate. Then
0.5 ml of trichloracetic acid (TCA, final concentration 5%)
was added to all samples and zero-controls, which were left
for 30 min at 0 8C. These were filtered through glass fiber
filters (Whatman GF/F, presoaked in 1% calgon), washed
three times with 5 ml ice-cold 80% (v/v) ethanol, and then
three times with 5 ml ice-cold 5% (w/v) TCA. The filters
were placed in scintillation vials. One ml of 0.1 M NaOH

Table 1
Selected soil physico-chemical characteristics

Soil Soil texture (%)a Textural classa Cb (%) Nb (%) Cbactc (% C) pH (H2O)d

Sand .50 mm Silt 2–50 mm Clay ,2 mm

1 50 33 17 Loam 1.4 0.2 3.5 7.36

2 54 35 11 Sandy loam 6.8 0.5 1.5 4.15
3 80 14 6 Loamy sand 2.2 0.1 2.1 3.25
a
Particle-size distribution was analyzed with a hydrometer and soil fractions were classified according to the USDA.
b
C and N contents were measured using elemental NC 2100 Soil Analyzer (ThermoQuest, Italia).
c
Cbact was estimated from direct bacterial counts using the formula Cbact ¼ DAPI counts £ 218 £ V 0.86 (Loferer-Krößbacher et al., 1998), where V is an
average bacterial volume (0.7 mm3; Bloem et al., 1995).
d
Soil pH was measured in soil-distilled water paste of ratio 1:2.5 after short intensive mixing.
 E. Uhlı́řová, H. Šantrůčková / Soil Biology & Biochemistry 35 (2003) 217–224 219

was added to each vial, which were kept in a water-bath at
90 8C for 2 h to solubilize macromolecules (Bååth, 1992).
After cooling, 9 ml of scintillation cocktail (Rotiszint
EcoPlus, Roth) were added to the vials. Radioactivity was
counted in a liquid scintillation analyzer after 2 d. All
measurements were made in duplicate.
The values of Leu and TdR incorporation were corrected
to an isotopic dilution (Pollard and Moriarty, 1984). For
TdR, we measured the degree of partitioning of 0.70 in
loam, 0.47 in sandy loam, and 0.60 in loamy sand,
respectively, and for Leu we used the coefficients of 0.50
for the loam, 0.77 for sandy loam, and 0.87 for loamy sand,
respectively.
2.4. Incorporation into different macromolecules
The distribution of radioactivity among different macro-
molecules was determined in an additional experiment
using the method of Riemann and Sondergaard (1984) for
protein labeling and of Wicks and Robarts (1987) for
incorporation into DNA. The bacterial suspension was
divided into three parts after the incorporation step. To the
first part, ice-cold TCA (5% final concentration) was added
to precipitate DNA, RNA, and protein. The second part was
heated with TCA (20% final concentration) at 90 8C for
30 min to hydrolyze DNA and RNA. After cooling, the
protein fraction remained in the precipitate. All the samples
were then filtered through glass fiber filters presoaked in 1%
calgon as described earlier. To the third part (1 ml), 0.25 ml
5 M NaOH and 0.5 ml 50% ice-cold TCA were added and
the mixture was kept for 15 min at 0 8C to lyze bacterial
cells and precipitate macromolecules. The samples were
then filtered through glass fiber filters presoaked in 1%
calgon. These filters were washed three times with 5 ml of
5% ice-cold TCA, then with 50% phenol –chloroform
(5 ml) to remove labeled proteins. This was followed by
three washes with 5 ml of 80% ice-cold ethanol to remove
labeled lipids. All the filters were placed into scintillation
vials and the macromolecules were solubilized as described
earlier.
Regularly, about 50% of added TdR was found in the
DNA fraction and about 40% of Leu in the protein fraction.
The coefficient of variation was , 10%. These results of
specific labeling were used to estimate turnover of bacteria.
Bacteria were counted immediately with a fluorescent
microscope.
2.6. Statistical analysis
Statistical analysis was done using SigmaStat 2.0
(Illinois, USA). Effects of soil texture or the steps of
extraction procedure were tested using Kruskal – Wallis one
way ANOVA on Ranks. Student – Newman –Keuls (SNK)
test was used to compare the data within each group. Results
of statistical analysis are summarized in Table 2.
3. Results
3.1. Direct counts of bacteria
The highest direct counts of bacteria (6.3 £ 109 cells g21
dry soil) were found in the sandy loam, which coincided
with the highest C content. Loamy sand and loam contained
2.8 £ 109 and 3.0 £ 109 cells g21 dry soil, respectively.
Bacterial C (Cbact) made up 1.5 –3.5% of C content in the
studied soils (Table 1).
The soil-sample treatment considerably decreased bac-
terial counts in soil suspensions and extracts compared with
the bacterial abundance in soil slurries (Fig. 1). Extraction
efficiencies that were reached using individual steps of
sample treatment were dependent on soil texture (Table 2).
Bacterial counts declined significantly soon after homogen-
ization and sedimentation. The largest effect of this
treatment was found in loamy sand and the least in the
loam (Fig. 1). Centrifugation further decreased the bacterial
counts in all three soils, with a direct relationship between
centrifugation speed and the size of the decrease. The
extractability of bacteria by homogenization – centrifugation
at 750g was 20 –22% of total bacterial counts in sandy loam
and loamy sand, respectively, but 40% in loam.
Table 2
Summary of statistical analysis. Effects of soil texture and sample treatment
on selected microbial characteristics were tested using Kruskal– Wallis one
way ANOVA on ranks. Values of probability are given (n ¼ 4 for sandy
loam, n ¼ 2 for loam and loamy sand)
Probability, (P )

Effect of Effect of sample

2.5. Bacterial enumeration soil texture treatment

Five milliliters of each sample were stored in 3% Direct counts of bacteria (DAPI) 0.003 ,0.001
formalin at 4 8C until counted. Samples of 102 – 104 dilution TdR incorporation 0.013 ,0.001
Leu incorporation ,0.001 0.007
(5 – 500 mg soil ml21) were used for direct bacterial Cell-specific TdR incorporation, 0.015 0.001
counting using the DAPI staining method (Bloem et al., Turnover time calculated from
1992). Briefly, 1 ml of the diluted sample was put on a cell-specific TdR incorporation rate
0.2 mm black Nucleopore filter (Poretics Products, Osmo- Cell-specific Leu incorporation, ,0.001 0.014
Turnover time calculated from
nics) and stained with DAPI (50 ml) for 2 min. Then 1 ml of
cell-specific Leu incorporation rate
sterile filtered water was added and the sample filtered.
220 E. Uhlı́řová, H. Šantrůčková / Soil Biology & Biochemistry 35 (2003) 217–224
Fig. 1. Bacterial counts in soil slurries, suspensions, and extracts subjected to different centrifugation speeds, prepared from soils of different texture. Data are
expressed in percentages of total bacterial numbers of the soil slurries, which were 1.26 £ 108 cells ml21 in loam, 2.05 £ 108 cells ml21 in sandy loam, and
1.16 £ 108 cells ml21 in loamy sand. Mean values (n ¼ 2) and standard deviations are shown ( loam,
3.2. TdR and Leu incorporation
Soil texture and the sample treatment affected the rates of
TdR and Leu incorporation into total bacterial macromol-
ecules (Table 2). The highest TdR and Leu incorporation
rates were found in the soil slurries of all three soil types,
being highest in the loam and lowest in the loamy sand
(Fig. 2).
Homogenization and subsequent centrifugation
decreased the TdR and Leu incorporation rates in a
similar way as bacterial counts. In the loam, the most
considerable decrease of TdR and Leu incorporation in
comparison with the slurry (by 92– 93%) was found in
the soil extract centrifuged at 100g. In loamy sand, there
was a large decrease in incorporation after homogeniz-
ation (by 95%); the effect of further centrifugation was
negligible. The effect in the sandy loam was intermediate
of the other two soils. A large decline in incorporation
rates occurred after homogenization (by 60%) and a
second one after centrifugation at 100g (an additional
32%) (Fig. 2). Bacteria in the soil extracts incorporated
only 1 – 7% of TdR and 2 – 6% of Leu, in comparison
with total bacteria in the soil slurries.
3.3. Cell-specific TdR and Leu incorporation
Cell-specific TdR and Leu incorporation rates were
affected by the sample treatment as well as by soil texture
(Table 2). The cell-specific TdR and Leu incorporation rates
in soil slurries decreased in order: loam . sandy loam .
loamy sand (Fig. 3). Generally, the bacteria in soil slurries
had the highest cell-specific TdR and Leu incorporation
rates, being one or two orders of magnitude higher than the
bacteria in the soil extracts. The effect of sample treatment
was most pronounced in the loam and lowest in the loamy
sand (Fig. 3). In the loam and sandy loam, each step of
sandy loam, A loamy sand).
the sample treatment caused a significant decrease in the
cell-specific Leu incorporation (SNK test, P , 0.05). The
cell-specific TdR incorporation rates declined considerably
after homogenization and after centrifugation at 100g. In the
coarse-textured loamy sand, the cell-specific incorporation
of TdR and Leu decreased significantly only after
homogenization.
These effects of soil treatment and soil texture on
bacterial biosynthesis were reflected in an estimation of
bacterial turnover (Table 3). Turnover times calculated from
the cell-specific Leu incorporation were always shorter than
those calculated from TdR. Bacterial turnover times were
shortest in the loam and longest in the loamy sand, as
counted from the cell-specific TdR and Leu incorporations
in soil slurries. Bacteria in soil suspensions and soil extracts
always had longer turnover times than bacteria from the soil
slurries (Table 3).
4. Discussion
4.1. Effect of soil texture
Soil texture significantly affected extraction efficien-
cies of bacteria by the homogenization – centrifugation
method as well as bacterial biosynthetic activity,
measured by TdR and Leu incorporation. In each
extraction step, higher extraction efficiencies were
obtained in finer soils, i.e. in the loam and the sandy
loam, than in the coarse-textured loamy sand, as found
by Ramsay (1984). She showed that ultrasonication
released bacteria not only from coarse particles but also
from finer soil particles due to effective disruption of soil
aggregates. Sonication also considerably increased the
amount of clay particles floating in homogenized
suspensions of fine soils, which could contain additional
 E. Uhlı́řová, H. Šantrůčková / Soil Biology & Biochemistry 35 (2003) 217–224 221

Fig. 2. TdR and Leu incorporation rates by bacteria in soil slurries, suspensions and extracts subjected to different centrifugation speeds from three soils of
different texture. Average values (n ¼ 2–4) and standard deviations are given for TdR incorporation and A Leu incorporation rates. (Note the different
scale of y-axis for TdR incorporation in loamy sand and for Leu incorporation in all three soil).

bacteria. In both these finer soils, we also found larger
total as well as cell-specific TdR and Leu incorporation
rates. An explanation could be that loamy soils, in
general, offer their microflora more habitable pore-space
due to a higher surface area of clay and organic particles
than sandy soils (Rutherford and Juma, 1992; Hassink
et al., 1993; Huysman and Verstraete, 1993) and higher
nutrient availability. In such soils, often more than 80%
of the microbes, including bacteria, are associated with
soil aggregates consisting of clay and silt particles mixed
with organic material (Van Es et al., 1984). There are
indications from observations in aquatic and soil
environments that it is the adhesion of bacteria to
surfaces, which positively affects their activity (Kirchman
and Mitchell, 1982; Bååth, 1996). We suggest that the
higher cell-specific bacterial biosynthesis in fine soils as
well as some other results from our study support this
statement (as we will discuss later).
222 E. Uhlı́řová, H. Šantrůčková / Soil Biology & Biochemistry 35 (2003) 217–224

Fig. 3. Cell-specific TdR and Leu incorporation rates by bacteria in soil slurries, suspensions, and extracts subjected to different centrifugation speeds. The
samples were prepared from three soils of different texture. Average values (n ¼ 2–4) and standard deviations are given for loam, sandy loam, A
loamy sand.

4.2. Effect of sample treatment
Homogenization– centrifugation is a method selective
for the original bacterial community (Bååth, 1996). As
expected, each step of the sample treatment used led to a
decrease in bacterial numbers in soil extracts to 20 –40% of
total cell counts in the slurries, depending on soil texture.
This decrease was followed by a decline of total bacterial
biosynthetic activity to 1 – 2% of the values in the slurries.
The greater the centrifugation speed, the lower the bacterial
numbers and the total TdR and Leu incorporation rates by
the extracted bacterial community (Figs. 1 and 2).
However, the bacteria in treated samples always had
significantly lower cell-specific TdR and Leu incorporation
rates in comparison with the cell-specific biosynthesis of
bacteria in soil slurries in each of the three soils (Fig. 3).
This was a result of an obvious disproportion between the
slower decreases of bacterial numbers and the larger falls of
total bacterial biosynthetic activity, caused by the extraction
procedure. It showed that the extraction of bacteria was
 E. Uhlı́řová, H. Šantrůčková / Soil Biology & Biochemistry 35 (2003) 217–224
Table 3
Turnover times of bacteria in soil slurries, suspensions, and extracts prepared from three soils of different soil texture. Mean values (n ¼ 2–4) and standard
deviations are shown
Turnover (days)a
Loam
Calculated from TdR Calculated from Leu
Slurry 2.8 ^ 0.1 1.0 ^ 0.1
Suspension 3.5 ^ 0.6 1.1 ^ 0.1
100g 39.1 ^ 1.2 10.5 ^ 0.1
250g 31.3 ^ 2.8 8.0 ^ 0.1
500g 133.6 ^ 15.9 17.1 ^ 0.1
750g 242.0 ^ 50.6 28.4 ^ 0.3
a
Turnover times were calculated from the cell-specific TdR and Leu incorporation rates assuming that 50% of TdR was incorporated into DNA and 40% of
Leu into protein. Conversion factors 2 £ 1018 cells mol21 TdR (Christensen, 1993) and 7.1 £ 1016 cells mol21 Leu (Bååth, 1994) were used.
a selective step. The extracted bacteria inadequately
represented the biosynthetic activity of the total soil
bacterial community. This finding is in agreement with
Bååth (1992, 1996), except that the difference between soil
slurries and soil extracts centrifuged at 750g as observed by
Bååth (1992) was less than that observed in our study. He
found only a 30 – 40% decrease in cell-specific incorpor-
ation rates while we found it decreased by 80 –99%. We
propose the following explanation. First, we found the
largest effect of homogenization – centrifugation on bac-
terial growth rates was in the fine-textured loam. Such fine
soils were not tested by Bååth, who worked with soils of a
coarser texture. Secondly, we used a more effective
technique to homogenize the soil samples, sonication and
mixing, in comparison to only mixing (Ramsay, 1984; Fry,
1990). It produced higher extraction efficiencies of bacteria
in soil extracts (20 –40% depending on soil texture) than
those usually published (10 – 30%) (Bååth, 1992), but
probably could have a stronger negative effect on bacterial
biosynthesis (see later).
The cell-specific biosynthetic activity of bacteria
declines significantly in soil suspensions indicating that
homogenizing a soil slurry has an indispensable effect on
the cell-specific biosynthetic activity of bacteria in treated
soils. A homogenization step is unavoidable, because it
brings a significant portion of the bacterial community into
solution before it is centrifuged. This is obtained by
disrupting soil aggregates and bacterial binding to soil
particles and releasing bacteria to the solution. In this case,
the effect of sonication or blending would be greater than
the effect of mixing (Ramsay, 1984; Riis et al., 1998). The
considerable changes in cell-specific bacterial physiology
found after the homogenization indicate that the adhesion of
bacteria to soil particles, and the strength (durability) of
these bonds, are very important factors affecting bacterial
activity. Attached bacteria are known to have a considerably
higher cell-specific activity than free-living ones (Kjelle-
berg et al., 1983; Kjelleberg and Hermansson, 1984;
Vanloosdrecht et al., 1990; Fletcher, 1991). Bååth (1996)
223
Sandy loam Loamy sand
Calculated from TdR Calculated from Leu Calculated from TdR Calculated from Leu
4.8 ^ 0.1 4.6 ^ 0.1 8.3 ^ 0.8 7.0 ^ 0.1
6.6 ^ 0.6 3.7 ^ 0.1 56.2 ^ 2.2 53.7 ^ 1.7
24.9 ^ 0.8 24.2 ^ 0.2 76.5 ^ 2.9 72.8 ^ 0.7
39.9 ^ 4.1 40.4 ^ 0.1 103.0 ^ 19.2 97.1 ^ 1.0
37.5 ^ 2.3 32.4 ^ 0.1 105.3 ^ 9.3 91.2 ^ 1.2
35.0 ^ 2.0 23.3 ^ 0.1 107.0 ^ 1.6 86.4 ^ 2.2
also measured 40– 60% higher cell-specific TdR incorpor-
ation rates by bacteria more tightly bound to soil particles
than by bacteria easily released to soil solution. The
described negative effect of homogenization (either by
ultrasonication or mixing), together with the additional loss
of attached bacteria due to sedimentation and centrifu-
gation, should result in a decrease of cell-specific incorpor-
ation rates in soil extracts as found by Bååth (1992, 1996)
and in our study.
4.3. Combined effects of soil texture and sample treatment
In general, bacterial biosynthesis was reduced after
homogenization –centrifugation in all three soils. How-
ever, the different soil textures of the three soils clearly
affected (i) bacterial extractability, and (ii) the overall
biosynthetic activity of bacteria, as discussed earlier, but
also (iii) the degree of changes in bacterial activities after
individual steps of sample treatment. The gross effect of
sample treatment was larger in finer soils because it
resulted in much greater fall of cell-specific TdR and Leu
incorporation rates than in coarse-textured loamy sand
(Fig. 3). On the other hand, it appeared that the bacterial
communities of coarser soils could be more responsive to
minor intervention. For example, homogenization of the
loamy sand led to decreases in bacterial counts and cell-
specific TdR and Leu incorporation rates of 60 and 95%,
respectively, but in the loam, it caused ‘only’ a 20% loss
of bacterial counts and a 30– 40% decrease in TdR and
Leu incorporation rates. The most critical step in the loam
was centrifugation at 100g (Fig. 3). Another important
thing was that homogenization – centrifugation prior to
incorporation of TdR and Leu almost eliminated the
differences among bacterial activities, which we found in
non-treated soil slurries (Fig. 3). To compare soils of
different texture, we recommend either working with soil
slurries or with soil suspensions. It must be considered
that bacterial biosynthetic activities measured in soil
suspensions will be significantly lower than real values
224 E. Uhlı́řová, H. Šantrůčková / Soil Biology & Biochemistry 35 (2003) 217–224
but the differences among differently textured soils will
still be apparent. The preparation of soil suspensions is
less time-consuming, which could be advantageous for big
sets of samples.
5. Conclusions
Soil texture affects the extraction efficiency of bacteria
from soils and also their biosynthetic activity. The finer the
soil the higher the extraction efficiency of bacteria and the
higher the biosynthetic activity and turnover of bacteria.
Soil texture in combination with the homogenization –
centrifugation cause a distortion of cell-specific TdR and
Leu incorporation rates of bacteria. The finer the soil
texture, the higher the underestimation of biosynthetic
activity of the extracted bacteria. Homogenization –cen-
trifugation diminishes the differences among bacterial
biosynthetic activities in soils of different textures. Thus,
working with soil slurries or, optionally, with soil suspen-
sions, which is less time-consuming, is more appropriate for
differently textured soils.
Acknowledgements
We thank T. Picek for valuable suggestions and
K. Edwards for correcting the English language. The
study was supported by the grants structure and functioning
of the ecosystem water – soil – plant from MŠMT ČR and
MIDAIR from EU.
References
Bååth, E., 1990. Thymidine incorporation into soil bacteria. Soil Biology &
Biochemistry 22, 803–810.
Bååth, E., 1992. Thymidine incorporation into macromolecules of bacteria
extracted from soil by homogenization–centrifugation. Soil Biology &
Biochemistry 24, 1157–1165.
Bååth, E., 1994. Measurement of protein synthesis by soil bacterail
assemblages with the leucine incorporation technique. Biology and
Fertility of Soils 17, 147 –153.
Bååth, E., 1996. Thymidine incorporation of bacteria sequentially extracted
from soil using repeated homogenization–centrifugation. Microbial
Ecology 31, 153 –166.
Bloem, J., Deruiter, P.C., Koopman, G.J., Lebbink, G., Brussaard, L., 1992.
Microbial numbers and activity in dried and rewetted arable soil under
integrated and conventional management. Soil Biology & Biochemistry
24, 655 –665.
Bloem, J., Veniga, M., Shepherd, J., 1995. Fully-automatic determination
of soil bacterium numbers, cell volumes, and frequencies of dividing
cells by confocal laser-scanning microscopy and image-analysis.
Applied and Environmental Microbiology 61, 926–936.
Christensen, H., 1993. Conversion factors for the thymidine incorporation
technique estimated with bacteria in pure culture and on seedling roots.
Soil Biology & Biochemistry 25, 1085–1096.
Christensen, H., Funk-Jensen, D., Kjoller, A., 1989. Growth rate of
rhizosphere bacteria measured directly by tritiated thymidine incorpor-
ation technique. Soil Biology & Biochemistry 21, 113 –117.
View publication stats
Fletcher, M., 1991. The physiological-activity of bacteria attached to solid
surfaces. Advances in Microbial Physiology 32, 53 –85.
Fry, J.C., 1990. Direct methods and biomass estimation. In: Grigorova, R.,
Norris, J.R. (Eds.), Methods in Microbiology: Techniques in Microbial
Ecology, Academic Press, London, pp. 41–85.
Hassink, J., Bouwman, L.A., Zwart, K.B., Bloem, J., Brussaard, L., 1993.
Relationships between soil texture, physical protection of organic-
matter, soil biota, and C-mineralization and N-mineralization in
grassland soils. Geoderma 57, 105–128.
Huysman, F., Verstraete, W., 1993. Effect of cell-surface characteristics on
the adhesion of bacteria to soil particles. Biology and Fertility of Soils
16, 21–26.
Kirchman, D., Mitchell, R., 1982. Contribution of particle-bound bacteria
to total microheterotrophic activity in five ponds and two marshes.
Applied and Environmental Microbiology 42, 802 –810.
Kjelleberg, S., Hermansson, M., 1984. Starvation-induced effects on
bacterial surface characteristics. Applied and Environmental Micro-
biology 48, 497 –503.
Kjelleberg, S., Humphrey, B.A., Marshall, K.C., 1983. Initial phases of
starvation and activity of bacteria at surfaces. Applied and Environ-
mental Microbiology 46, 978–984.
Loferer-Krößbacher, M., Klima, J., Psenner, R., 1998. Determination of
bacterial cell dry mass by transmission electron microscopy and
densitometric image analysis. Applied and Environmental Micro-
biology 64, 688 –694.
Mayr, C., Winding, A., Hendriksen, N.B., 1999. Community level
physiological profile of soil bacteria unaffected by extraction method.
Journal of Microbiological Methods 36, 29–33.
Michel, P.H., Bloem, J., 1993. Conversion factors for estimation of
cell production-rates of soil bacteria from [3H] thymidine and
[3H] leucine incorporation. Soil Biology & Biochemistry 25,
943 –950.
Pollard, P.C., Greenfield, P.F., 1997. Measuring in situ bacterial specific
growth rates and population dynamics in wastewater. Water Research
31, 1074–1082.
Pollard, P.C., Moriarty, D.J.V., 1984. Validity of the tritiated thymidine
method for estimating bacterial growth rates: measurement of isotope
dilution during DNA synthesis. Applied and Environmental Micro-
biology 48, 1076–1083.
Ramsay, A.J., 1984. Extraction of bacteria from soil: efficiency of shaking
or ultrasonication as indicated by direct counts and autoradiography.
Soil Biology & Biochemistry 16, 475–481.
Riemann, B., Sondergaard, M., 1984. Measurements of diel rates of
bacterial secondary production in aquatic environments. Applied and
Environmental Microbiology 47, 632 –638.
Riis, V., Lorbeer, H., Babel, W., 1998. Extraction of microorganisms
from soil: evaluation of the efficiency by counting methods and
activity measurements. Soil Biology & Biochemistry 30,
1573–1581.
Rutherford, P.M., Juma, N.G., 1992. Influence of texture on habitable pore-
space and bacterial–protozoan populations in soil. Biology and Fertility
of Soils 12, 221 –227.
Tan, Y., Bond, W.J., Rovira, A.D., Brisbane, P.G., Griffin, D.M., 1991.
Movement through soil of a biological-control agent, Pseudomonas-
fluorescens. Soil Biology & Biochemistry 23, 821–825.
Van Es, F.B., Laanbroek, H.J., Veldkamp, H., 1984. Microbial ecology: an
overview. In: Codd, G.A., (Ed.), Aspects of Microbial Metabolism and
Ecology. Society for General Microbiology, Academic Press, London,
pp. 1–33.
Vanloosdrecht, M.C.M., Lyklema, J., Norde, W., Zehnder, A.J.B., 1990.
Influence of interfaces on microbial activity. Microbiological Reviews
54, 75–87.
Wicks, R.J., Robarts, R.D., 1987. The extraction and purification of DNA
labeled with [methyl-3H]thymidine in aquatic bacterial production
studies. Journal of Plankton Research 9, 1159–1166.