Open Journal of Soil Science, 2012, 2, 64-69
http://dx.doi.org/10.4236/ojss.2012.21010 Published Online March 2012 (http://www.SciRP.org/journal/ojss)
Evaluation of Factors Influencing the Biomass of Soil
Microorganisms and DNA Content
Wolińska Agnieszka, Stępniewska Zofia, Bułaś Aleksandra, Banach Artur
Department of Biochemistry and Environmental Chemistry, Institute of Biotechnology, The John Paul II Catholic University, Lublin,
Poland.
Received November 10th, 2011; revised December 14th, 2011; accepted December 28th, 2011
ABSTRACT
The aim of the study was the statistical evaluation of the impact of water potential (pF), oxygen availability (ODR) and
the way of land use on microbial biomass (MB) and soil DNA content. Soil was extracted from the surface (0 - 20 cm)
and subsurface (20 - 40 cm) layers of Mollic Gleysol. Soil material was collected in July 2009 from the village
Kosiorów (SE part of Poland), from the two distinct neighbouring areas: agriculturally exploited (AE), and fallow land
(FL), which served as the control area. Moisture content was determined for a range of pF values (0, 1.0, 1.5, 2.0),
which corresponded to availability of water usable by microorganisms and plant roots. Finally, our results revealed sig-
nificant (p < 0.001) positive relationship between DNA and soil MB content, and negative correlations between soil MB
and both pF and ODR. Importantly, MB seemed significantly dependent on the different way of land use, and higher
MB content was noted in the soil agriculturally exploited (p < 0.05) in contrast to the control area.
Keywords: Microbial Biomass; Soil DNA; Water Potential; Agricultural Activity
1. Introduction
The term of microbial biomass is commonly used to de-
scribe the total mass of microorganisms present in soil
[1]. The importance of the MB in soil functioning is well
recognised [2]. Moreover, MB as an integrative measure
of the physiologically active part of the soil microflora is
recommended by 8 countries of European Union (e.g.,
Germany, United Kingdom, Austria, Switzerland), as im-
portant factor of soil quality, which is included in soil
microbial degradation monitoring program [3].
Soil MB is also considered to be a useful criterion for
an early indication of environmental stress [1,4] as vari-
ability in microbial communities can precede detectable
changes in soil properties. An example is the turnover
rate of the MB, which is much faster and takes, e.g., 1 - 5
years, than the turnover rate of total soil organic matter
[5]. It is partly due to the large pool of relatively inactive
and dormant microorganisms, having the potential to re-
flect the past [6]. Life in the soil environment is constantly
influenced by drying and rewetting cycles, as soils are
continually exposed to rainfall, wind and snow [7]. A part
of the microbial population dies during each drying-and-
wetting cycle resulting in the fluctuation of soil microbial
composition [8]. Soil water content as a function of the
soil water tension is described by pF curve, which pro-
vides information about the ability for water retaining by
the soil pores at any given water tension, or conversely,
how tightly a water is held between soil aggregates [9].
Therefore, also soil aeration status is strongly depended
from pF values. It has been also shown experimentally,
that ODR factor satisfactorily reflects the supply of oxy-
gen to the plant roots [10,11]. Oxygen availability is among
the most important factors affecting soil microbial active-
ties [12]. There is a specific gas demand for different soil
microbes including bacteria, fungi, and other microorga-
nisms [7]. ODR is affected by several factors. It increases
with reducing soil water content or increasing suction up
to a certain level and then declines with further depletion
of water [13]. Soil environment is the major reservoir of
microbial genetic diversity and thus should be particu-
larly protected. The breakthrough in soil biology was the
discovery of DNA, which is a carrier of biological infor-
mation and the best characteristics of every organism.
Thus, soil DNA analysis is considered to be important
and precise tool towards a better recognition of soil mi-
crobial functionality and interrelationships among them.
The size of MB was found to be strongly correlated
with content of base cations, base saturation, cation ex-
change capacity, and organic matter quality [14], as well
as with soil bulk density, nutrient contents and phospha-
tase and invertase activities [15]. However, there is a lack
of studies on relationships between MB and such impor-
tant soil factors like pF or ODR, what may be decisive

Evaluation of Factors Influencing the Biomass of Soil Microorganisms and DNA Content 65
for the course of processes responsible for plant devel-
opment, and soil fertility. Investigation of the effect of
human agricultural activity on MB content is also inter-
esting part of the current study. Thus, the aim of the work
was the statistical description of the impact of pF, ODR,
and human agricultural activities on MB and DNA con-
tent in soil.
2. Material and Method
2.1. Soil and Investigated Area Description
The soil used in the experiment was Mollic Gleysol (Ta-
ble 1). Soil was sampled in July 2009 from the village
Kosiorów, situated in the Wilków community (SE part of
Poland) from depths of 0 - 20 and 20 - 40 cm. To make
possible an estimation of the effect of different way of
land use on MB, soil samples were collected from two
neighbouring plots: one of them was agriculturally exploit-
ed with systematic fertilization and pasturage (under hu-
man activity), whilst the other one was classified as fal-
low land and used as a control area (without any human
impact).
2.2. Assaying of Soil Retention Curves
The instrument used for determining water retention cur-
ves was a steel pressure chamber, inside of which a po-
rous plate saturated with water was located. At the bot-
tom, soil samples, continuously exposed to atmospheric
pressure, make the hydraulic contact with the porous plate
[16]. The chamber was closed and the desired air pres-
sure P was applied, driving away the soil water retained
at pressures below P, until equilibrium was reached [16].
Soil samples were collected using plastic containers and
placed in an airtight chamber (for 10 days), part of a la-
boratory set LAB o12 (Soil Moisture Equipment Compa-
ny, USA), before a pressure was applied. The moisture
content was determined via the drying process, for the
range of water potentials (0, 1.0, 1.5 and 2.0 pF), corre-
sponding to availability of water usable by microorgan-
isms and plant roots.
2.3. ODR Measurement
After determination of proper pF values, ODR was mea-
sured by ODR-meter manufactured by the Institute of
Agrophysics, Polish Academy of Soil Sciences (Lublin),
using Malicki and Bieganowski [17] method. The ODR
technique consists of the measurement of the electric cur-
rent intensity corresponding to the reduction of oxygen
on a platinum cathode placed in the soil and negatively
polarized with respect to the reference electrode (calomel).
As oxygen is consumed at the microelectrode, more oxy-
gen needs to diffuse radially to the electrode in response
to the accumulated gradient. This is analogous to oxygen
consumption by respiration of root surface or by micro-
bial respiration. Four platinum wire electrodes (0.5 mm ×
4 mm) were placed at the depth of 2 cm and polarized to
–0.65 V versus saturated calomel electrode for 4 min. The
data were recorded in three replicates, for each sample.
2.4. Microbial Biomass
Soil MB was determined by a fumigation-extraction me-
thod using CHCl3 as an agent responsible for the cellular
death of microorganisms, according to Joregensen [18]
procedure. Concentration of head-space CO2 released by
microorganisms which survived incubation with CHCl3
was measured by a gas chromatograph (Varian CP-3800,
equipped with a TCD detector).
2.5. DNA Extraction
Soil DNA was extracted from samples at pF 0 (full water
capacity conditions) and pF 2.0 (field capacity conditions),
using the GeneMatrix soil DNA isolation kit (EURx 1.4,
Poland), according to the manufacturer’s instructions.
This kit was designed specifically for the rapid isolation
of pure, humic-free microbial DNA from soil samples,
and guaranteed the proper DNA isolation procedure. Con-
centration of DNA was determined spectrophotometri-
cally at 260 nm (Shimadzu, UV-1800, Japan).
2.6. Data Analyses
Statistical analyses were performed by means of Statis-
tica 8.0 software (STATSOFT, USA). One-way ANOVA
test was used to investigate significant (p < 0.05) effect
of pF, ODR, DNA content on soil MB quantity. Results
of the significance differences analyses are presented in
Table 2 only. In Figures 1-4 the average values with stan-
dard deviations are demonstrated.
Table 1. Basic characteristics of the soil.
Granulometric composition (%, dia in mm)
Place Depth (cm)
1 - 0.1 0.1 - 0.02 0.02 - 0.002 <0.002
pH (H2O) TOC (%)
0 - 20 87 8 3 2 6.7 22.5
Agriculturally exploited
(AE) 20 - 40 90 7 2 1 6.4 1.4
0 - 20 91 6 3 0 6.2 20.1
Fallow land (FL) 20 - 40 95 3 2 0 6.5 1.3

Evaluation of Factors Influencing the Biomass of Soil Microorganisms and DNA Content
66
Table 2. Statistically significant relationships between MB
and analyzed parameters, N = 12.
Object
investigated Depth (cm) pF ODR c DNA
0 - 20 –0.68* –0.20 n.s. 0.97**
AE 20 - 40 –0.53* –0.52 n.s. 0.99***
0 - 20 –0.94*** –0.83*** 0.86*
FL 20 - 40 –0.74** –0.59* 0.89*
*, **, ***—indicate significance at the p < 0.05, p < 0.01, and p < 0.001,
respectively; n.s.—not significant difference.
Figure 1. The relationship between soil water content (%
v/v) and pF values. The curves are related to two depths (0 -
20; 20 - 40 cm) of the investigated soil areas.
Figure 2. Variability of ODR values as an effect of water
potential at two depths of the investigated soil areas. Aver-
age values with standard deviations are presented.
microorganisms
microorganisms
Figure 3. Variability of MB levels as an effect of water po-
tential at two depths of the investigated soil areas. Average
values with standard deviations are presented.
Figure 4. DNA content at two depths of the investigated soil
areas as an effect of water potential. Average values with
standard deviations are presented.
3. Results and Discuss
3.1. Soil Retention Abilities
An incubation of the soil samples under different control-
led moisture conditions altered significantly ODR, MB
and DNA concentration. The relationships between soil
water content (%, v/v) and pF for the two layers of Mol-
lic Gleysol, for both tested areas (AE and FL) are pre-
sented in Figure 1.

Evaluation of Factors Influencing the Biomass of Soil Microorganisms and DNA Content 67
Generally, the investigated soils demonstrated similar
abilities to retain water, even though soil from the control
area (FL) displayed slightly higher (c.a. 3% more than
soil agriculturally exploited) capacity for water keeping.
This might be connected with beginning of decay process
on AE object, what resulted in looseness of soil structure
and higher intensity of mineralization and humification
of the soil organic compounds. Moreover, higher ability
of holding water in the subsurface layers were noted, and
equalled 34% - 47% v/v and 41% - 50% v/v in AE and
FL objects, respectively. Similar capability of Mollic Gley-
sol for water maintaining was also indicated [19]. The
soil-water interactions are greatly or extremely important
to soil fertility and therefore are the subject of interest to
agricultural engineers and farmers. Furthermore, infor-
mation about water holding capacity is important for
agronomic and hydrologic characteristic of soils. It ex-
presses, how much water can be stored in the soil for
plant use during periods without rain or irrigation [20].
This provides an indication of soil sensitivity to drou-
ght and could be used to calculate the probability of oc-
currence of deep drainage or groundwater recharge proc-
ess. It was also reported that macropore continuity is
very important to the aeration status of the soil, thus the
effect of soil compaction on other aeration properties de-
pends on soil hydro-physical status [13,21].
3.2. Oxygen Availability in AE and FL Objects
Based on the performed measurements it was found, that
pF constitutes a significant factor (p < 0.001) determin-
ing ODR levels in the soil environment. Soil desiccation,
occurring in the direction from pF 0 to pF 2.0, was the
reason of stimulation of ODR (Figure 2).
The oxygen availability in relation to the soil water
potential indicates, that ODR values at surface layer of
AL object fluctuated from 50 to 233 μg O2/(m2·s) at pF 0
and pF 2.0, respectively. At deeper layer (20 - 40 cm) of
agriculturally exploited Mollic Gleysol ODR ranged be-
tween 26 till to 240 μg O2/(m2·s), for as follows pF 0 and
pF 2.0. Stronger variety of ODR values in the FL object
was observed, we noted values between 60 and 266 μg
O2/(m2·s) as in the surface layer, whilst in the subsurface
80% decrease of ODR was found and registered values
oscillated from 23 to 81 μg O2/(m2·s), for pF 0 and pF
2.0, respectively. We assume that way of land use and
sys- tematically applied ploughing at AE object may be
the reason of higher ODR level as well in surface as in
sub- surface layer. FL on the other hand, despite the fact
of comparable level of ODR in surface layer, in the sub-
sur- face was characterized by rapid reduction of oxygen
availability, below 35 μg O2/(m2·s), which is the minimum
level of ODR necessary for proper root growth [11,22].
This might be caused by lack of human agricultural ac-
tivities, as e.g. ploughing may contribute in soil ventila-
tion improvement. On the contrary, [19] Walczak et al.
(2001) noted tendency for higher oxygen availability in
the deeper layers, rather than in surface of the Mollic
Gleysol, whereas [23] Stępniewska et al. (2003) observed
analogous trend in the Eutric Cambisol.
It may be caused either by methodical limitations, as
the water barriers or water films present on the surface of
electrode could be broken off [9], or by the differences in
granulometric composition of analyzed soil samples (Ta-
ble 1), as the fact, that large granulation favourable for
forming of aeration pores was noted in the subsurface
layers.
3.3. Soil Microbial Biomass
Soil MB content was also strongly influenced by pF con-
ditions and the way of land use (Figure 3). The signifi-
cantly higher values (0.0077 and 0.0058 g/g of soil) were
stated in the surface layers at full water capacity condi-
tions (pF 0) for AL and FL objects, respectively, as com-
pared to the deeper layers of soil. In subsurface layers the
reduction of MB content, c.a. 5 times, was observed.
The highest values of MB estimations in AE object were
undoubtedly connected with total organic carbon (TOC)
content, what favoured the microorganisms’ abundance
by supplying sources of energy necessary for activity of
soil biota and growing crops. This observation is consis-
tent with other studies [24-26]. Registered almost 85%
reduction of MB in the subsurface layers come out of
distribution of microorganisms in the soil profile, since
microorganisms are mostly confined to the surface soil
layer owing to better aeration and greater nutrient avail-
ability. Anthropogenic activities and soil management in
particular, are mostly responsible for disturbing the che-
mico-physical and biological equilibrium of soil [27]. A
particularly serious problem is the decrease in the orga-
nic matter content of agricultural soils, which may endan-
ger soil fertility and enhance erosion. The MB, as a small
fraction of soil organic matter, is a source and sink of
nutrients and controls soil organic matter mineralization
[27,28] Fisk and Fahey (2001), analogically at current
study noted higher content of MB (by 20% - 30%) at ag-
ricultural areas in response to fertilization, than at fallow
ones. Also, other findings pointed out a distinct relation-
ship between soil fertility and soil MB, suggesting that
MB measurements provide a valid estimate of soil qual-
ity [14,29].
3.4. DNA Content
Similarly to MB distribution of DNA concentrations in
surface layers were 2 times higher in AE object (Figure
4), than in control soil (not under cultivation) at pF 0.
Even more differentiation in subsurface layers were stated,
where DNA level reached 12 fold higher values in AE, in

Evaluation of Factors Influencing the Biomass of Soil Microorganisms and DNA Content
68
relation to FL area. The usage of GeneMatrix soil DNA
isolation kit let us to receive 0.4 - 1.8 µg/g and 0.5 - 1.2
µg/g of soil DNA concentrations in AE and FL objects,
respectively and revealed that the quality (fragment size
and purity) of the extracted DNA was generally very
good. However, one should always realize that extraction
of DNA from soil samples is never 100% efficient and
can vary from a few µg to almost 200 µg DNA per g dry
weight of soil. Most of authors, however, reported that
the obtained values ranged from 1 to about 50 µg of total
DNA per g dry weight of soil depending on the method
applied and soil sample studied [30,31].
3.5. Statistical Relationships between
Parameters Analyzed
Statistical relationships between MB and investigated pa-
rameters (pF, ODR, DNA content, way of land use) de-
scribed by correlations coefficient (r) are presented in
Table 2. Significant influence (p < 0.05) of tested para-
meters on MB was found. The positive relationships be-
tween MB and DNA content, and negative correlations
between MB and soil physical factors like pF and ODR
were revealed.
Both our and other studies demonstrated that MB can
be highly sensitive to environmental factors. Although,
study by [32] Singh and Yadava (2006) confirmed nega-
tive correlation between MB and soil moisture, even so
prior to our study, rather little attention has been paid to
the influence of pF, ODR, DNA content and way of land
use on soil MB. Therefore, our work was focused on the-
se relationships and determination of statistical correla-
tions. The positive relationships between MB and DNA
content suggest the domination of intercellular DNA form
at both AE and FL objects, and MB is considered to be a
good factor relating to the total mass of microorganisms
present in soil. Our results are supported by the findings
of [33] Blagodatskaya et al. (2003), who noted that DNA
content correlated strongly with the total MB in Paleosol
soils from Southern Urals (R2 = 0.97), as well as by work
of [34] Hartmann et al. (2005), who described analogical
correlation (r = 0.91***). Nevertheless, the interpretation
of our results has been challenging because of the lack of
enough publications in available literature concerning the
determination of r coefficients as a goodness of fit be-
tween investigated soil factors and MB. However, deter-
mined relationships demand further investigations and
selection of the other soil types, for better explanation and
confirmation of the obtained correlations.
4. Conclusion
Importantly, the way of land use significantly (p < 0.05)
influenced on MB and DNA content, and higher content
of MB and DNA concentration were noted in the AE (p <
0.05), in contrast to the FL area. Significant (p < 0.05)
positive relationships between soil MB and DNA con-
centration were revealed, whereas pF and ODR were ne-
gatively correlated to MB. Oxygen availability values of
30 - 255 μg O2/(m2·s) and 20 - 80 μg O2/(m2·s) in the AE
and FL objects, respectively, suggested higher oxygen
availability in the subsurface layer of AE area (p < 0.05),
what might be connected with human agricultural prac-
tices, e.g. traditional regular ploughing, which signifi-
cantly improves soil aeration status.
5. Acknowledgements
This work was supported by the IUVENTUS PLUS grant
(No IP 2010 001070).
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