Appl Microbiol Biotechnol (2006) 71: 875–880
DOI 10.1007/s00253-005-0245-x
APPLIED GENE TICS AN D MO LECULA R BIO TECH NOLOGY
H. Aoshima . A. Kimura . A. Shibutani .
C. Okada . Y. Matsumiya . M. Kubo
Evaluation of soil bacterial biomass using environmental DNA
extracted by slow-stirring method
Received: 5 September 2005 / Revised: 25 October 2005 / Accepted: 1 November 2005 / Published online: 4 March 2006
# Springer-Verlag 2006
Abstract A simple and rapid method (slow-stirring
method) for extracting environmental DNA (eDNA) from
soils was constructed by physical mild stirring with
chemical treatment. eDNA was extracted efficiently with
minimal damage from various kinds of soil. The amount of
eDNA and soil bacterial biomass showed a linear
proportional relation [Y=(1.70×108)X, r2=0.96], indicating
that bacterial biomass could be evaluated by quantifying
levels of eDNA. Consequently, the average bacterial
biomass in an agricultural field was calculated as
5.95×109 cells/g sample, approximately 10–100 times
higher than that in non- and oil-polluted fields.
Introduction
Chemical pollution of soil environments is a serious
problem worldwide, often occurring as a result of complex
contamination with multiple chemicals. Many analytical
methods for detecting chemicals have therefore been
developed.
Since environmental conditions affect soil microorgan-
isms, evaluation of soil bacterial biomass has been
attempted. A cultivation method using agar plates with
various growth media and cultural conditions was
previously suggested for counting numbers of microorgan-
isms (Barer and Harwood 1999; Barkay and Pritchard
1988; Olsen and Bakken 1987). However, 99.7–99.999%
of soil microorganisms do not grow on agar plates, but
rather they enter in a viable-but-non-culturable-state
(VBNC) (Barer and Harwood 1999; Oliver et al. 1991;
Rahman et al. 1994). Therefore, many studies have
H. Aoshima . A. Kimura . A. Shibutani .
C. Okada . Y. Matsumiya . M. Kubo (*)
Department of Bioscience and Technology,
Faculty of Science and Engineering,
Ritsumeikan University, Nojihigashi,
Kusatsu, Shiga 525-8577, Japan
e-mail: kubo@se.ritsumei.ac.jp
Tel.: +81-77-5613901
Fax: +81-77-5613901
investigated ways to evaluate soil microorganisms. For
example, substrate-induced respiration (SIR) and chloro-
form fumigation extraction (CFE) were developed based
on microbial respiration (Anderson and Domsch 1978;
Jenkinson and Powlson 1976), and recently, a 4′,6-
diamino-2-phenylindole (DAPI) staining method was de-
veloped for counting numbers of soil microorganisms by
vital staining (Otto and Tsou 1985; Poulin et al. 1994;
Schnedl et al. 1977; Taylor and Milthorpe 1980; Taylor et
al. 2002). However, many problems remain, such as
complications associated with operation, quantification,
reproducibility, and the high cost of many of these
techniques.
Analysis of environmental DNA (eDNA) has been
investigated as a new approach for evaluating numbers of
soil microorganisms (Leff et al. 1995; Miller et al. 1999;
Steffan et al. 1988; Tsai and Olson 1992a,b; Zhou et al.
1996). Since eDNA analysis does not require a cultivation
procedure, this technique is useful for detecting VBNC (Tsai
and Olson 1992a). Several methods for extraction of eDNA
from soil have been developed; however, the extraction
efficiency and level of DNA damage require improvements.
Our aim was to develop a technique whereby soil
environmental conditions could be evaluated based on
calculations of environmental microorganism biomass.
This paper describes the construction of an efficient
eDNA extraction method (slow-stirring method) and
presents estimations of soil bacterial biomass based on
this method.
Materials and methods
Bacterial strains and soil samples
Both gram-negative [Escherichia coli JM109 (Blattner et
al. 1997), Pseudomonas aeruginosa F201 (Oh et al. 2003),
Acinetobacter sp. ODDK71 (Koma et al. 2001)], and gram-
positive bacteria [Bacillus subtilis MT-2 (Imanaka et al.
1983), Streptmyces sp. MF20 (Hasegawa et al. 2001)] were
used in this study. All bacteria were cultured in Luria–
876
Bertani medium (LB medium; 1% peptone, 0.5% yeast
extract, 0.5% NaCl). Soil samples were collected from
different environments, namely, an agricultural field, sandy
beach, forest and oil-polluted field, based on the Food and
Agriculture Organization (FAO) of the United Nations
classification (Table 1; FAO 1988). They were stored at
4°C until analysis in the laboratory within 1 week.
Extraction of eDNA from soil using the
slow-stirring method
Soil samples (1.0 g) were mixed with 8.0 ml of DNA
extraction buffer [100 mmol/l Tris–HCl (pH 8.0),
100 mmol/l sodium EDTA 100 mmol/l sodium phosphate
1.5 mol/l NaCl, and 1% hexadecylmethylammonium
bromide (CTAB)] and 1.0 ml of autoclaved 20% (w/v)
sodium dodecyl sulfate (SDS) solution. The sample
solution was mixed in a 50-ml tube with propeller agitation
at 1,500 rpm for 20 min at room temperature. Next, 1.5 ml
of sample solution was transferred to a 1.5-ml microtube.
After centrifugation at 6,000×g for 10 min at 20°C, 700 μl
of supernatant was transferred into a 1.5-ml microtube into
which an equal volume of chloroform–isoamylalcohol
[24:1(v/v)] was then added before centrifugation at
18,000×g for 10 min at 20°C. The aqueous phase
(500 μl) was recovered into a 1.5-ml microtube by
centrifugation at 18,000×g for 20 min at 20°C and mixed
with 300 μl of isopropanol. The pellet was then washed
with 1.0 ml of cold 70% ethanol and centrifuged at
18,000×g for 5 min at 20°C. The pellet of crude nucleic
acid was dissolved in 50 μl of TE buffer (Tris/EDTA =
10:1 mmol/l) after drying under reduced pressure (eDNA
solution).
Analysis of eDNA
Spectrophotometric characterization of the extracted
eDNA solution was conducted using a spectrophotometer
(UV-T160A, Shimazu, Kyoto, Japan). The amount of
eDNA was quantified using KODAK 1D 3.5 imaging
software after electrophoresis on 1.0% agarose gel.
Table 1 Sampling locations Experimenta
and properties of the soils used
in this study
(1) eDNA extraction efficiency
(2) Relationship between the bacterial number obtained using
DAPI staining and the amount of eDNA
FAO Food and Agriculture Or-
ganization, eDNA environmental (3) Distribution of bacterial biomass in an agricultural,
DNA, DAPI 4′,6-diamino-2- nonagricultural, and oil-polluted field
phenylindole
a
See text for details
Analysis of eDNA extraction from alive
and dead bacteria
Acinetobacter sp. ODDK71 (1.6×109 cells/g sample) was
inoculated into an autoclaved soil. The soil was kept at
room temperature for 3 months, and eDNA was monitored.
DAPI staining
Soil samples (1.0 g) were mixed and diluted with sterilized
water then added to sterilized 25% glutaraldehyde (Wako
Pure Chemical Industries, Ltd., Osaka, Japan) solution
(final 1%) by a disposable syringe filter unit (diameter
0.22 μm; ADVANTEC TOYO, Tokyo, Japan). Fixed soil
microorganisms were mixed well with 5.0 μg/ml DAPI
staining solution (final volume 1 μg/ml; Nacalai Tesque
Co., Kyoto, Japan), sterilized as mentioned above and
stored in the dark at 4°C for 24 h before microscopic
analysis. The stained sample solution (1.0 ml) was
absorbed into and stacked on Nuclepore filters (Filter
Type GTBP, Millipore Co. Ltd., USA). Filters containing
stained sample solutions were then placed on a slide, which
was consequently covered after being rinsed with emulsion
oil. Slides were examined with a 1,000× objective on an
epifluorescence microscope (BX50 BX-FLA, OLYMPUS,
Tokyo, Japan; Schnedl et al. 1977; Taylor and Milthorpe
1980).
Under ultraviolet irradiation, pale fluorescent substances
were counted as bacteria, but radiated yellow substances,
which represent minerals in the soil, were excluded. A
10×10-squares (50×50 μm) eye micrometer was defined as
the field of vision. Bacterial numbers were counted in 20
fields of vision, twice per square, i.e., in a total of 40 fields
of vision, and the total number of soil bacteria per 1.0 g of
soil was calculated using the following equation:


Total number of soil bacteria cells g sample
¼ Average number of bacteria in the field of vision

Valid filtration area
Area of the eye micrometer

Filtration volume
Dilution ratio:
Soil texture (FAO Number of
classification) or type samples
Clay and humus 3
(xanthic ferralsol)
Sandy loam (orthic 2
luvisol)
Loam (albic luvisol) 2
Agricultural field 16
Nonagricultural field 31
Oil-polluted field 10
Agricultural field 86
Nonagricultural field 60
Oil-polluted field 63

The valid filtration area represents the filter area of the
filtrated pass.
Statistical analysis of the relationship between
the amount of eDNA, soil bacteria biomass,
and soil characteristics
The amount of eDNA was calculated using the amount of
eDNA recovery and efficiency of eDNA extraction from
each soil sample using the following equation:
Amount of eDNA 

¼ Amount of eDNA recovery cells g sample

100 Efficiency of eDNA extractionð%Þ:
Results
Extraction of nucleic acid from soils
and quantification of eDNA
To evaluate the bacterial biomass in soil using eDNA, a
procedure of eDNA extraction from soils was investigated.
Zhou et al. (1996) developed an efficient soil eDNA
extraction method using heat treatment at 65°C (the heat
treatment method); however, this method requires an
expensive enzyme and takes a long time to extract
eDNA. Physical extraction using beads (the bead method)
is a simple and rapid method for extracting eDNA from
soils (Burgmann et al. 2001; Cornejo et al. 1998), and an
efficient eDNA extraction system based on this method is
used widely for investigating soil environments (FastDNA
Spin Kit for Soil; MP Biomedicals, Irvine, CA, USA).
However, to quantify eDNA accurately using the band
intensity of agarose gel electrophoresis, physical damage of
eDNA should be suppressed. We therefore attempted to
construct a rapid soil eDNA extraction method (slow-
stirring method) that does not induce physical damage.
The efficiency of the bead method, heat treatment
method, and slow-stirring method were compared using
agarose gel electrophoresis (Fig. 1). The amount of eDNA
extracted with the bead method could not be determined
because the resulting damaged eDNA appeared as a
smeared band on the agarose gel (Fig. 1). The amounts
of eDNA extracted with the heat treatment and slow-
stirring methods were 9.53±1.55 and 13.09±0.64 μg/g
sample, respectively. Absorbances at 465 nm, which
represents the specific absorbance of humic substances,
with the slow-stirring and heat treatment methods were
0.09±0.01 and 1.01±0.01, respectively (Matsuda and
Schnitzer 1971; Tsai and Olson 1992b). The eDNA
extracted from soil with the slow-stirring method was
available to use for polymerase chain reaction (PCR)
amplification and restriction enzyme treatment without
further purification procedures (data not shown). These
results indicate that the slow-stirring method was able to
877
Fig. 1 Agarose gel electrophoresis of environmental DNA (eDNA)
extracted from soil in an agricultural field using various eDNA
extraction methods. Lane 1 0.2- to 10-kbp smart ladder (mass
marker), Lane 2 slow-stirring method, Lane 3 heat treatment
method, Lane 4 the bead method
efficiently extract high-purity eDNA without physical
digestion.
To analyze the eDNA extraction from alive and dead
bacteria by the slow-stirring method, Acinetobacter sp. was
inoculated into the autoclaved soil, and eDNA from the soil
was monitored for 3 months. The colony forming unit
(CFU) was decreased from 1.6×109 CFU/g sample to
7.2×108 CFU/g sample soil for 3 months, and the smear
band correspondingly increased. Analysis of eDNAs
(initial and 3 months incubated soil) is shown in Fig. 2.
The bacterial biomass evaluated from the nondegraded
eDNA and CFU was almost the same. The result indicates
that the smear band was from dead bacteria, and the slow-
stirring method could evaluate alive bacterial biomass.
Efficiency of eDNA extraction from various soil
samples
To analyze the efficiency of eDNA extraction from various
soil samples using the slow-stirring method, three types of
soil were used. Soil samples were classified as clay and
humus (Xanthic ferralsol), sandy loam (Orthic luvisol), and
loam (Albic luvisol), according to the FAO classification
(Table 1; FAO 1988). No eDNA was detected in any soil
sample after sterilization at 121°C for 15 min. E. coli
JM109 (1×1010 CFU/ml) was added to each sterilized soil
sample and mixed well, and then eDNA was extracted
using the slow-stirring method and quantified. As a result,
878
Fig. 2 Analysis of the eDNA extracted from the soil inoculated
with Acinetobacter sp. Lane 1 0.2- to 10-kbp smart ladder (mass
marker), Lane 2 initial soil, Lane 3 3-month incubated soil
almost the same amount of eDNA was extracted from
various types of soil. In addition, the amount of eDNA
from soil and water containing the same amount of strain
JM109 was also identical. According to calculations of the
whole genome size based on the E. coli K12 genome,
approximately 65% of the DNA was recovered from the
various types of soil using the slow-stirring method
(Blattner et al. 1997). Identical results were obtained
using P. aeruginosa F201, B. subtilis MT2, and Strepto-
myces sp. MF20. However, eDNA from fungi and yeasts
was not extracted (data not shown). These results indicate
that extraction of eDNA using the slow-stirring method can
be applied with various types of soil and various kinds of
bacteria. Furthermore, eDNA was efficiently recovered
even from oil-polluted soil (data not shown), suggesting
that this method is applicable in monitoring the bacterial
biomass during bioremediation.
The relationship between bacterial numbers
and the amount of eDNA
Fifty-seven soil samples were used to evaluate the
bacterial biomass in soils using eDNA (Table 1). Viable
bacterial numbers were counted based on bacterial shape
using a fluorescence microscope after DAPI staining. The
relationship between bacterial numbers and the amount of
eDNA is shown in Fig. 3. The amounts of bacteria and
eDNA in the soil samples were 5.19×107–8.96×109 cells/g
sample and 0.30–52.55 μg/g sample, respectively. The
amount of eDNA was highly correlated with the bacterial
number obtained using the DAPI staining method; the
equation of the calibration curve was Y=(1.70×108)X, and
the correlation coefficient (r2) was 0.96. Using this
calibration curve, bacterial biomass can be calculated
according to the amount of eDNA. More than 42 ng/g
sample (7.3×106 cells/g sample) of eDNA could be
detected using KODAK 1D 3.5. The calibration curve is
able to use as a universal equation to evaluate the bacterial
biomass when eDNA was extracted by the slow-stirring
method.
Evaluation of the bacterial biomass in various soils
Application of the proposed eDNA extraction method was
further evaluated by analyzing the bacterial biomass of
various soil samples. A total of 209 soil samples from
agricultural, nonagricultural, and oil-polluted fields were
analyzed using the slow-stirring method (Fig. 4). In the
nonagricultural field, the range was 3.25×108–9.63×109 cells/
g sample, and the average was 1.88×109 cells/g sample;
however, in the oil-polluted field, the range was less than
1×109 cells/g sample. The bacterial biomass in the
agricultural field was therefore higher than that in the oil-
polluted field.
Discussion
The attention paid to environmental microorganisms has
increased; however, information about flora of micro-
organisms, and VBNC in nature remains limited. Physical
extraction method using beads or heating with a combina-
tion of physical, chemical, and biochemical treatments was
developed to extract eDNA directly from soils. Even
though eDNA is extracted efficiently in a short time using
Fig. 3 Relationship between the bacterial number obtained using
4′,6-diamino-2-phenylindole (DAPI) staining and the amount of
eDNA in 57 soils. The line shows the correlation between bacterial
number, squares the amount of eDNA in an agricultural field,
rhombuses oil-polluted field, triangles nonagricultural field. The
lower detection limit of this calibration curve represents the amount
of eDNA; 42 ng/g sample bacteria (7.8×106 cells/g sample)

Fig. 4 Evaluation of the bacte-
rial biomass in various kinds of
soils. a Agricultural field.
b Nonagricultural field. c Oil-
polluted field (total: 209 soil
samples)
the bead method, physical damage of eDNA often occurs.
The heat treatment method, on the other hand, needs a long
time to extract undamaged eDNA and requires an
expensive enzyme. Moreover, the extracted eDNA con-
tains impurities such as humic substances (Tsai and Olson
1992b). With the method proposed here, contact between
the soil and chemicals is increased by slow-stirring at a low
temperature, resulting in extraction of undamaged high-
purity eDNA in a short time. The extracted eDNA is useful
for denaturing gradient gel electrophoresis (DGGE) anal-
ysis of flora of bacteria in soils.
eDNA from both dead and living bacteria was extracted
with this method. eDNA from dead cells is degraded via
autolysis; therefore, it appears as a low molecular weight
band with agarose gel electrophoresis; high molecular
weight eDNA corresponds to eDNA of living bacteria. A
close correlation between the bacterial number counted
using DAPI staining and the amount of eDNA from living
bacteria was found in this study. eDNA extraction from
fungi and yeasts was extremely poor using the slow-stirring
method because of the different cytoarchitecture of fungi
and yeast compared to bacteria. In the future, extraction of
eDNA from fungi and yeasts in soils using the slow-stirring
method might be possible using lytic enzymes.
879
The bacterial biomass in agricultural fields by the
cultivation method was around 1×107–8 cells/g sample of
bacteria (Hattori et al. 1996; Janssen et al. 2002), while
above 1×109 cells/g sample of bacteria were counted by the
vital staining direct counting methods (DAPI or acridine
orange staining methods; Markus et al. 1998; Miller et al.,
1999). The difference of the bacterial biomass by the
cultivation method and the vital staining direct counting
method may be the existence of VBNC. The bacterial
biomass by the slow-stirring method was almost the same
level as the vital staining direct counting method. There-
fore, the slow-stirring method could evaluate the bacterial
biomass including VBNC.
The bacterial biomass in almost all agricultural field soil
samples was above 1 billion cells/g sample, and several
contained above 10 billion cells/g sample. In nonagricul-
tural fields, some samples from areas of rich vegetation
contained high levels of bacterial biomass; however, the
bacterial biomass in oil-polluted soils was relatively low
compared with agricultural fields (Luo et al. 2004; Mintie
et al. 2003; Mowat and Bundy 2001; Sikkema et al. 1995).
The soil bacterial biomass and flora are affected by soil
condition (Hattori et al. 1996; Luo et al. 2004; Mintie et al.
2003; Mowat and Bundy 2001; Sikkema et al. 1995).
880
Several attempts have been carried out to understand and
evaluate soil condition by the soil bacterial biomass based
on the bacterial respiration and/or mineralization activity
(Anderson and Domsch 1978; Jenkinson and Powlson
1976). However, these methods require a long time and
have several problems for accurate evaluation of bacterial
biomass. On the other hand, the bacterial biomass was
correctly evaluated by the eDNA extracted with the slow-
stirring method in a short time. If databases about eDNA vs
pollutions, eDNA vs mineralization activity, and eDNA vs
agricultural harvest, etc. are constructed, the diagnosis of
soil condition would be possible by eDNA. A similar
system might also be useful for analyzing the condition of
the hydrosphere and wastewater treatment.
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