Evaluation of Factors Influencing the Biomass of Soil Microorganisms and DNA Content

doi:10.4236/ojss.2012.21010

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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.

Email: [email protected]

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

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

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

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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