The stability of soil aggregates against shearing and compressive forces as
well as water-caused dispersion is an integral marker of soil quality. High
stability results in less compaction and erosion and has been linked to
enhanced water retention, dynamic water transport and aeration regimes,
increased rooting depth, and protection of soil organic matter (SOM) against
microbial degradation. In turn, particulate organic matter is supposed to
support soil aggregate stabilization. For decades the importance of biofilm
extracellular polymeric substances (EPSs) regarding particulate organic
matter (POM) occlusion and aggregate stability has been canonical because of
its distribution, geometric structure and ability to link primary particles.
However, experimental proof is still missing. This lack is mainly due to
methodological reasons. Thus, the objective of this work is to develop a
method of enzymatic biofilm detachment for studying the effects of EPSs on
POM occlusion. The method combines an enzymatic pre-treatment with different
activities of
Soil organic matter (SOM) comprises 50 % (
The stability of soil aggregates against shear and compression forces (Skidmore and Powers, 1982) as well as disaggregation caused by water (Tisdall and Oades, 1982) is an integral marker of soil quality (Bronick and Lal, 2005). Since aggregate stability implies pore stability, it results in less soil compactibility (Baumgartl and Horn, 1991; Alaoui et al., 2011) and a more dynamic water transport regime in the macropores that reduces erosion caused by surface runoff (Barthes and Roose, 2002). Other benefits in comparison to compacted soils are a higher aeration (Ball and Robertson, 1994) and lower penetration resistance (Bennie and Burger, 1988) causing increased rootability and rooting depth (Bengough and Mullins, 1990; Taylor and Brar, 1991). In addition, micropores within the aggregates enhance water retention.
The occlusion of POM within soil aggregates depends on the properties of the
aggregated components. The mineral part of the solid soil matrix is composed
of siliceous sand, silt and clay particles, oxides and hydroxides of Fe, Al
and Mn, and diverse minor minerals. Sticking together, pervaded and
coated with multivalent cations and organic constituents (like soluble
metabolic products, humic substances, black carbon and other POM)
macro-aggregates (
The structure-bearing primary particles, precipitates and adsorbed molecules
cohere by physico-chemical interactions between (i) permanent charge of
mainly the clay mineral fraction; (ii) multivalent cations with small hydrate
shells such as Ca
In addition, for a few decades biological structures like bacterial colonies, bacterial pseudomycelia, algae, fungal hyphae and their exudates (e.g., glomalin), roots, and soil fauna have been accepted as a major factor of soil aggregation (Tisdall, 1991; Oades, 1993; Wright and Upadhyaya, 1998; Brown et al., 2000; Chenu and Stotzky, 2002; Rillig, 2004; Bronick and Lal, 2005). Furthermore the role of extracellular polymeric substance (EPS) of bacterial biofilms as an adhesive between soil particles is seen to be of importance (Baldock, 2002; Ashman et al., 2009).
Proposed model of aggregate structure including biofilms in a soil aggregate: sand and silt (both grey) and organic particles (black) stick together by physico-chemical interactions and are bridged by EPS (striped), which additionally stabilizes the soil aggregate structure and the pore space (white).
Physical and chemical properties of soil mineral and organic matter allow one to
hypothesize a simple spacial model of the inner geometry of soil aggregates,
which includes biofilms as links between primary particles (Fig. 1). The
biofilm itself is a viscous microenvironment mainly comprised of 90–97 %
water (Zhang et al., 1998; Schmitt and Flemming, 1999; Pal and Paul, 2008).
The remaining dry mass contains differing ratios of polysaccharides,
extracellular DNA (eDNA), proteins and lipids in addition to 10–50 % cell biomass
(More et al., 2014). In contrast to “biofilm”, EPS terms the extracellular
polymeric matrix excluding cells. Extracellular polysaccharides cause the
EPS structural stability by means of entanglement and Ca
The composition of EPSs is highly variable depending on community composition
and environmental cues (Table 1): Redmile-Gordon et al. (2014) measured a
natural habitat extracellular polysaccharide concentration of 401
Concentrations and molar masses of biofilm stabilizing macromolecules (polysaccharides – PS, eDNA, lipids and proteins) in different environments.
The extracellular matrix is exuded not only by soil bacteria and archaea but also by fungi and algae. It is engineered by grazing protozoa, small metazoa, and microbial extracellular enzymes (Battin et al., 2007; Flemming and Wingender, 2010).
The activity of EPS degrading enzymes in natural soils spans up to 2
orders of magnitude: the
Not much is known about the contribution of EPS to POM occlusion and aggregate stability in relation to other aggregate stabilizing factors. That is mainly due to methodological reasons: though, e.g., Tang et al. (2011) showed a significant contribution of bacterial growth on aggregate stability, the observations could not definitely be attributed to soil microbial exopolysaccharide production. Redmile-Gordon et al. (2014) subsequently found that the techniques previously used to measure extracellular polysaccharides in soil co-extracted large quantities of “random” soil organic matter which confounded estimates of EPS production. Owing to the widespread interest in the role of biofilms on soil fertility, the objectives of this work are (i) to design a selective method for enzymatic biofilm detachment with minor impact on other types of aggregate bonds and (ii) to apply the method to an agricultural soil to provide indications of the influence of biofilm cohesion on POM fixation, which is expected to contribute to aggregate stability (Six et al., 2004).
The method combines a modified enzymatic pre-treatment (Böckelmann et
al., 2003) with
We hypothesize that a destabilization of the EPS matrix occurs during enzymatic treatment. This should result in an increased cell detachment from aggregates. We also expect an increased fLF-SOC release from destabilized aggregates compared to the control and a shift of the oLF-SOC ratio from higher to lower binding strength (represented by ultrasonic energy levels) that is interpretable as alteration of soil aggregate stability.
Well-aggregated silty sand (Su3) of a plowed topsoil from a cropland near
Berge (Brandenburg, Germany) was air-dried and sieved to obtain a particle
size of 0.63 to 2.0 mm containing mainly macro-aggregates. The aggregates
have a pH
To estimate the soil microbial biomass, first
Four degradative enzymes were selected on the basis of soil pH and
temperature used for catalytic unit definition (
The literature shows a wide range of target concentrations related to these
enzymes in different soils. As we do not know target concentrations of our
soil (due to a lack of extraction methods), we considered the largest
published values (Table 2) of EPS content
Variables used for the calculation of enzyme units needed for biofilm target decomposition and scenario parameters shared by all variants.
Calculated by Eq. (1)
Specific scenario parameters of the variants E0, E1, E2, E3 and E4.
Fifteen grams of air-dried soil aggregates were incubated in five replicates per
scenario with 3.4 mL of highly concentrated artificial rainwater (ARW: 0.2 mM
NH
The release of bacterial cells into the solution was quantified using a FastDNA™ SPIN Kit for Soil and quantitative real-time PCR.
Therefore 45
Amplification of 10-fold diluted DNA samples was performed using a C1000
Touch Thermal Cycler (BioRad). According to the reference for SG qPCR Master
Mix (Roboklon) thermocycling comprised an initial denaturation at
95
For evaluation of the light fraction SOC (LF-SOC) release, mean values, and standard deviations were calculated. Parallels of each variant were
positively tested to provide normal distribution and evidence of variance
homogeneity (Shapiro–Wilk test, Levene test, both
The relative LF carbon release from soil aggregate samples after different
enzymatic treatments is shown in Fig. 2. The proportionate C of each
captured fraction is defined as C
Relative POC release of treatments (E0, E1, E2, E3, E4) at
different energy levels (0, 50, 100, 150 J mL
None of the enzymatic treatments altered the quantity of fLF-SOC released in
the absence of sonication (0 J mL
The sediment represents the SOC remaining unextractable at
Cumulating LF-SOC releases of all energy levels, E1 shows a reduction by
16 % compared to the control (3.3 % of C
The relative DNA release after enzymatic treatment, as pictured with the
treatments E0, E1 and E4 in Fig. 3, is defined as the ratio of extracted DNA
from suspended bacterial cells (DNA
Relative bacterial DNA release from soil aggregates after treatments
E0, E1 and E4 defined as 100
We found that increasing the quantity of enzymes applied to aggregates led
to increased release of LF-SOC when aggregates were sonicated. This
detachment is explained by the following mechanism: the enzyme mix flows
into the unsaturated pore space. From there
In the following, LF-SOC is interpreted as SOC from released POM, since the share of both adsorbed DOM and colloids on captured dry mass is considered to be negligible after SPT treatment. Furthermore, LF-SOC transferred from the sediment fraction to light fractions due to enzymatic treatment is also interpreted as POM; in contrast mineral-associated organic matter of the HF is not assumed to be extractable at the applied energies (Cerli et al., 2012).
In accordance with the model, measured oLF-SOC releases indicate a trend for
increased POM release with increasing enzyme addition (Fig. 2). The E4
scenario shows that relative oLF-SOC release increased by 63 % (5 % of
C
The relation of LF-SOC release with enzymatic biofilm digestion is supported by the comparison of bacterial DNA releases between the treatments (Fig. 3). This indicates that applied enzymes are targeting biofilm components and release bacterial cells: the E4 scenario shows EPS digestion and additional cell release leading to a doubled relative DNA release compared with the control and E1. However, considering that most of the soil bacteria are expected to live in biofilms (Davey and O'toole, 2000), the total DNA release of only 5.6 % in the E4 scenario is too low for total biofilm digestion. Hence, biofilm detachment caused by E4 is still likely to be incomplete and the increased oLF-SOC release of E4 only results from a partial soil biofilm detachment. We conclude that there is a slight influence of enzymatic treatment on the occlusion of POM at enzyme concentrations exceeding natural concentrations. This conforms to results of Böckelmann et al. (2003), which indicate that a treatment with enzyme concentrations of near that of E4 is sufficient to destabilize biofilms within 1 h.
The incomplete biofilm detachment can be explained by the reduction of enzyme activity due to interaction with the soil matrix. Based on our calculations enzyme concentrations of mix E1 should have been sufficient for total biofilm digestion within time of application (1 h) – as long as there are no other factors reducing enzyme efficiency. As surveys of natural soils show enzyme concentrations up to mix E3 (Cooper and Morgan, 1981; Eivazi and Tabatabai, 1988; Margesin et al., 1999; Acosta-Martinez and Tabatabai, 2000; Margesin et al., 2000), such factors might be reasonably assumed. After addition to the soil sample, enzymes must enter the EPS matrix by diffusion. Therefore parts of the enzymes probably do not reach the biofilm due to inhibited diffusion. Beside diffusion, sorption and decomposition could play a major role in reducing enzyme efficiency. Whereas turn-over rates of soil enzymes have not yet been assessed, extended stabilization of active enzymes over time on soil mineral and organic surfaces is reported (Burns et al., 2013). This mechanism could explain immobilization of enzymes off the biofilm and high measured soil enzyme concentrations from the literature in face of still existing biofilms. After penetration of biofilms, (macro)molecules interfere with EPS components depending on molecular size, charge and biofilm structure (Stewart, 1998; Lieleg and Ribbeck, 2011), which strongly influences decay rates of enzymes. Due to these boundary conditions, quantification of the relation of enzyme concentration and POC release was not possible in this work.
The trend for increased POM release with increasing enzyme addition was only
broken by the control treatment. This could probably be explained by
pre-incubation of soil aggregates given 0.2 mM NH
Enzyme C in E1 to E4 could be used as microbial C source. The addition of C
increases the C
Under certain conditions POC release is indicative of soil aggregate
stability. Generally, aggregate stability is characterized by determining
the reduction in aggregate size after application of mechanical force. The
commonly used methods are dry and wet sieving. However, the destruction of
soil aggregates by ultrasonication has an advantage over these methods,
which is the quantification of the applied energy (North, 1976). It is used
for studying reduction of aggregate size (Imeson and Vis, 1984) as well as
detachment of occluded POC (Golchin et al., 1994). Kaiser and Berhe (2014) reviewed 15 studies using ultrasonication of soil aggregates in
consideration of its destructiveness to the soil mineral matrix and occluded
POM. They found destruction of POM at applied energy levels
Although our results give slight evidence for the influence of biofilms on
aggregate stability, they have to be recognized with restrictions to full
quantifiability:
The enzyme concentration hypothetically needed to
disperse the whole soil sample EPS matrix depends on diverse boundary
conditions like the concentration of enzyme targets, environmental conditions
such as pH, temperature as well as ion activity and delay factors such as low
diffusion, kinetic influence or metabolization of enzymes by soil organisms. Underlying enzyme kinetics were measured by the producer using pure
targets for unit definition, while biofilm targets are much more diverse and
soil matrix could interfere. Alternative enzyme targets might be
reasonably assumed within the complex chemism of the soil matrix. Released
organic cytoplasm molecules of lysed cells can be excluded to be an
additional enzyme target due to their low concentration. On the other hand,
enzyme specificity to EPS targets in face of the organic soil matrix is
unbeknown. The decrease of extracted POM mass due to biofilm erasement
from surfaces is suggested to be low but could cause underestimation of POM
release especially in scenario E4. In contrast, a direct contribution of
enzyme C to the POC release can be refuted. Even in the case of complete
adsorption to the POM of only one fraction, the highest enzyme concentration
(E4) would result in additional 13.5 Regarding DNA release
measurement as well, data are semi-quantitative, since quantification of the
detachment effect is limited by a potential adherence of detached cells to
soil particles after washing (Absolom et al., 1983; Li and Logan, 2004).
Thus, cell release could be underestimated as biofilm detachment increases.
Many of these uncertainties are owed to the high complexity of the soil
system. Enzymes were applied in concentrations 4 orders of magnitude
higher than calculated from actual C
However, these results give a first though still vague insight into the fundamental processes underlying POM occlusion. A slight release of occluded POM coupled with increased bacterial DNA release after treatment with high enzyme concentrations underpins the assumption that biofilm is involved in POM occlusion being a stabilizing agent of soil aggregates as proposed in a review by Or et al. (2007). The apparent increase of POC release caused by the digestion of EPS components suggests biofilm relevance in soil ecosystems, e.g., in terms of soil-aggregate related functions like soil water and C dynamics, mechanical stability and rootability. However, the statistical power of this introductory work is low and a more quantitative analysis of the relation of enzymatic EPS detachment and POM release would require deeper knowledge of enzyme dynamics in soil, more replicate samples, additional enzyme concentrations and probably inclusion of soils from different land use. However, this was beyond the scope of the present study.
Extracellular polymeric substance (EPS) was shown to be a promising
candidate factor of aggregate stability. Our experimental results suggest
that EPS contributes to occlusion and attachment of particulate organic
matter (POM) in sandy soil aggregates. The application of a highly
concentrated mix of
This project was financially supported by the Leibniz Association (SAW Pact for Research, SAW-2012-ATB-3). We also are grateful to Ulrich Szewzyk and the helpful staff of the Chair of Environmental Microbiology (Department of Environmental Technology, TU Berlin) for the unbureaucratic possibility to use their laboratories. In addition our thanks go out to Lara Schneider. Her bachelor thesis helped us to get an overview in the early phase of this experiment. And thanks both to Dennis Prieß for initiating a good idea during a very good Taiji training and to Tom Grassmann for his help in handling the R coding.Edited by: S. Doerr Reviewed by: M. Redmile-Gordon and one anonymous referee