The influence of soil bacteria on
aggregates and the possible effect of
tillage on bacterial communities
Laura Marteijn, University of Amsterdam, 5th July 2020 Supervisor: Elly Morriën, IBED
Abstract
This Bsc-thesis is part of a 4 year funded project on soil quality in the Netherlands. Research on soil quality is important to shift towards more sustainable agricultural practices. This literature review investigated the potential contribution of soil bacteria to soil structure and vice versa as well as the impact of different forms of tillage. Besides a literature review, a statistical analysis was performed on the relation between bulk density and bacterial biomass. This study found aggregates to have a significant effect on bacterial communities, through influencing their biomass, diversity, functioning and the F:B ratio. Bacteria were both abundant in micro- and macro-aggregates, but most studies found a dominance in micro-aggregates. The statistical analysis demonstrated that a lower bulk density yields a larger number of bacterial cells/mm 2
. Soil bacteria influence aggregates as well by influencing aggregation, aggregate turnover and stability. Some studies found extracellular polymeric substances (EPS) to have a significant impact on aggregates as binding agents. In the few studies conducted EPS was linked to improved water retention and aggregate stability. Tillage has negative effects on soil structure through mechanical breakdown of macro-aggregates. This is linked to a loss of bacterial biomass in the topsoil, in contrast with reduced or no-tillage where bacterial biomass increases in the topsoil. Tillage, aggregates and bacteria are connected through feedback loops and form a complex network of interactions. No tillage or reduced tillage could be a potential solution to shift towards more sustainable agriculture through decreased disruption of aggregates and associated bacterial communities. However, this research also demonstrates the lack of studies on the topic and the knowledge gaps in the field of research. Promising methods and more research are crucial for understanding soil processes better. This could pave the way towards more sustainable agricultural practices, where self-regulating processes, such as soil bacteria are the cornerstones of future agriculture.
Content
Introduction p. 3
Methods and data p. 5
Literature review p. 5
Experimental design p. 5
Statistical analysis p. 6
Chapter 1: Relation aggregates and bacteria p. 7
1.1 Soil structure: aggregates, bulk density and porosity p. 7
1.2 Micro-organisms p. 8
1.3 Relation aggregates and bacteria p. 9
1.4 Influence of tillage on aggregates and bacteria p. 11
Chapter 2: Effects of aggregates on bacteria p. 12
Chapter 3: Effects of bulk density on bacteria p. 15
Chapter 4: Effects of bacterial EPS on aggregates p. 17
Chapter 5: Effects of tillage on aggregates and bacteria p. 20
5.1 Effects of tillage on aggregates p. 20
5.2 Effects of tillage on bacteria p. 20
Chapter 6: Links between tillage, aggregates and micro-organisms p. 22
Chapter 7: Recommendations and further research p. 25
7.1 Discussion and knowledge gaps p. 25
7.2 Recommendations on further research p. 27
Conclusion p. 29
Acknowledgements p. 29
Introduction
Intensification of agriculture will cause major negative effects on diversity, composition and functioning of natural ecosystems and on their ability to provide essential ecosystem services (Tilman, 1987). In response, tendencies to reduce the impact of agriculture, such as decreasing the external inputs asks for a shift to a greater reliance on the self-regulating processes of the soil (D’hose et al., 2018). Soil biology is therefore becoming increasingly important, since it contributes to the quality of soils (D’hose et al., 2018). Microbial diversity is especifically vital and contributes by providing various ecosystem services, such as supply of nutrients to plants, maintenance of soil structure, water regulation, drought tolerance, fertility, disease suppressiveness and crop yield (Wilpiszeski al., 2019; D’hose et al., 2018; Upton et al., 2019).
Wim Reus, a farmer in Noord-Holland set the goal to produce his crops without damaging the environment. Together with other farmers he now collaborates with the University of Amsterdam by offering his cauliflower-farm as a living-lab. The University of Amsterdam and University of Applied Sciences Leeuwarden received funding to measure soil quality, biodiversity, water quality, emissions and to monitor meadow bird populations for four years. One part of this research aims to provide input for management guidelines to increase above- and belowground biodiversity, since this can potentially improve the quality and quantity of the harvest. These nature inclusive soil practices could potentially pave the way to more sustainable agriculture of the future with more reliance on self-regulating soil processes.
One part of this multi-year project is investigating the soil biology and its above- and belowground connections, processes and feedbacks. Despite the aforementioned services soil biota provide, indicators related to soil biodiversity, distribution and abundance are only measured rarely. This lack of knowledge can partly be attributed to the complexity of soil processes (Six et al., 2004). In spite of this complexity, previous studies demonstrate connections between soil biota and soil structure (aggregation) (Six et al., 2004; Lehmann et al., 2017; Upton et al., 2018). Lastly, the influence of agricultural practices should be considered since it is the most significant anthropogenic activity that alters soil characteristics (Jiang et al., 2011).
This research will focus on the role of soil bacteria in soil structural processes as well as the role of soil structure on bacterial communities. The functioning of a soil starts with its structure and influences a variety of soil processes (Wilpiszeski et al., 2019). Soil structure forms different habitats for soil biota, but soil biota also alter their own environment through decomposition of organic matter and production of binding agents for example. I specifically focus on soil bacteria, since these dominate in clay rich soils (Tang et al., 2011) which is the dominant soil type in Noord-Holland. Moreover, the influence of binding agents, known as extracellular polymeric substances (EPS) produced by bacteria are currently poorly investigated, making it an interesting topic to study. This will eventually be linked to different tillage practices, since these alter soil structure thus having an impact on the soil environment including its soil fauna.
Despite the complexity of soil processes, combining existing knowledge from previous studies can provide a general understanding of the relation between soil bacteria and their physical environment. This literature research will give an overview of the current knowledge on the connectivity between soil bacteria, structure and land management while it also indicates current knowledge gaps. The results from this research could potentially provide input for management guidelines which can improve the quality and quantity of the harvest as well as contribute to more sustainable agricultural practices to meet global goals to reduce damage caused by agricultural practices (D’hose et al., 2018).
This research will mainly focus on the role of soil bacteria, since this project is situated in Noord-Holland on clay-rich soils. Since bacteria dominate in clay-rich soils I expect a significant contribution of bacteria to soil processes. Six et al., (2004) concluded that despite the abundance of soil bacteria, the role of bacteria in many soil processes are still poorly understood. Therefore the following research question will be investigated: How are soil bacteria and aggregates linked and what are the responses of aggregates and associated bacterial communities to different tillage practices?
To answer this, the following sub questions will be answered (1) how do aggregates influence soil bacteria? (2) what are the effects of bacterial produced EPS on soil aggregates? (3) What are the effects of reduced or no-tillage compared to conventional tillage on aggregates? (4) What are the effects of reduced or no-tillage compared to conventional tillage on bacterial communities?
Methods and data
This research contains an extensive literature research and a statistical analysis, which demand different methods and approaches.
Literature review
A total of 51 articles have been examined and compared. These articles are either easily accessible through Google Scholar or accessible through the online library of the University of Amsterdam (lib.uva.nl). To find suitable articles search terms such as soil bacteria, soil aggregates, binding agents, EPS, tillage, microorganisms, aggregate stability and agricultural practices were used in different combinations. The studies used, varied from old research to the newest studies on the topic from 2019/2020. The studies used different soil types, bacteria, aggregate size fractions, types of tillage and research methods,
Aggregate size, bacterial diversity and biomass were the response variables in most of the studies included in this review. A few recurring methods for measuring the distribution of aggregate fractions was dry-sieving and wet sieving as well as using X-rays to measure pore-space and aggregate sizes. Bacterial cells were mainly counted through PLFA analysis which measured phospholipids, in contrast to DNA phospholipids only occur in living cells thus indicating the amount of living microorganisms in the soil. (Briar et al., 2011).
Experimental design
The data used for analysis was published on figshare, a platform for sharing data and related research. The experiment was performed by Otten et al., (2020) at the University of Bremen. This dataset measured soil structural properties and bacterial cells/mm2 for two different bulk densities. Soil samples were collected
from a sandy-loam soil collected in Bullion field in Scotland. The soil was air-dried and sieved to aggregates of 1-2 mm, this aggregate size was chosen since it was in line with previous work. The same amount of Pseudomonas fluorescens bacterial cells were introduced to the samples. First two samples for different bulk densities were selected, with one having a bulk density of 1.3 g/cm³ and one of 1.5 g/cm³. These bulk densities resulted in different inter-aggregate pore-spaces and porosity in previous studies. The moisture content of the soil was adjusted to 60% filled pores for all samples. This was 0.224 cm3/g for a bulk density of 1.3 g/cm³ and 0.1569 cm³/g for a bulk density of 1.5 g/cm³. These samples were created in polyethene rings of size 3.4 cm³ and a total of 12 soil microcosms were used (4 samples for both bulk densities and 4 control samples with sterile inoculum pellet). The microcosms were kept at a temperature of 23 degrees for 14 days to allow the bacteria to grow and spread in the soil samples. To introduce the bacterial cells to the samples. A inoculum of washed cells (with 10ml sterile broth and 10 mL PBS solution) of 1000uL was mixed with 30 mL of 1.5% LMP agarose solution. To impregnate a mixture (2 L) was prepared with a combination of 1.4 L of polyester resin, with 2240 uL of Co-accelerator (1.6‰ (v/v) 1%-Cobalt Ocoate accelator) and 4480 uL of hardener (3.2‰ (v/v) cyclohexanone peroxide). 500 mL of the acetone mixture was added as a thinner, mixed well with the resin mixture and was kept under a vacuum (240 mbar) to remove gas bubbles before adding the mixture to the samples. The inoculum kept covered at room temperature for 9 days for the polymerization process. Finally, the samples were examined through X-ray CT-scans by a Metris X–Tek HMX CT scanner and the samples were scanned at 10.87 µm voxel resolution (200 keV,56 µA and 2000 angular projections). A volume processing software (VGStudio MAX V2.2) was used to reconstruct volumes and to export image stacks (*bmp format) for further analysis on soil structural properties. Bacterial cells were observed and counted using a Olympus BX61 fluorescence microscope. The cell counts were extrapolated to cell density i.e. cell counts per area of the counting spot.
Statistical analysis
For analysing the dataset a statistical analysis was performed using Matlab R2019. First, summary statistics were collected such as mean, median, minimum, maximum and IQR values. This was followed by a t-test for comparing two means for the results of the two bulk densities. To also compare the medians for the two bulk densities, a one-way ANOVA was also performed on the two bulk density samples and their related bacterial cell counts. This was followed by a Tukey HSD post-hoc test using the function multcompare. Results were visualized using Matlab 2019R as well.
Chapter 1: Relation aggregates and bacteria
1.1 Soil structure: aggregates, bulk density and porosity
Soil structure is an important soil property that plays a role in many soil processes and it can be measured in several ways, such as bulk density, porosity and aggregate arrangement. This thesis will mainly focus on the commonly used indicator: the size distribution of aggregates. However, bulk density and pore space will be evaluated as well.
Aggregates are sand, silt and clay particles bound together by inorganic and organic processes that are arranged in different size-fractions (Briar et al., 2011). The arrangement of aggregates creates unique physicochemical niches where solutes and substrates are dispersed over time and space (Fox et al., 2014). Aggregates are mainly separated into the spatial scales of microaggregates (<0.25 mm) and macroaggregates (0.25-2 mm) (Wilpizeski et al., 2019). Macro-aggregates are micro-aggregates bound together by organic compounds, nutrients and binding agents. Micro-aggregates bind soil organic carbon and protect soils from erosion while macro-aggregates limit oxygen diffusion and regulate water flow (Wilpizeski et al., 2019). Soil structure can change over time through aggregate turnover where macro-aggregates break down into micro-aggregates and aggregation where micro-aggregates are bound together to form one larger macro-aggregate (Six et al., 2004). Five major factors contributing to aggregation are (1) soil fauna, (2) microorganisms, (3) roots, (4) binding agents and (5) environmental variables (Six et al., 2004). Soil aggregation affects various soil processes, such as aeration, water infiltration and microbial activities (Six et al., 2004). Aggregate stability is often used to qualify soil quality as well, because stable aggregates will provide a positive influence on root growth, erodibility, water availability, aeration and enhances resistance to disruptive forces such as tillage (Arfarita et al., 2016). Furthermore, stable aggregates protect soil organic matter (SOM) as well (Six et al., 2004) which in turn has a positive effect on C sequestration among co-benefits such as improving soil quality and providing soil services (Cortrufo et al., 2019).
Besides aggregates, pores are important for soil fauna as well. Pores are mainly separated into intergranular pores and inter-aggregate pores. Different aggregate sizes are connected to different pore spaces (1) macro-pores (2) pore-space between macro-aggregates (3) pore-space between micro-aggregates but within macro-aggregates and (4) pores within micro-aggregates (Six et al., 2004). These pores create different niches for organisms to occupy and are crucial for the aforementioned soil properties such as aeration and transport of nutrients and organic materials (Juyal et al., 2020). Bulk density has an influence on soil pores as well, mainly influencing larger invertebrates such as nematodes (Jonas & Thomasson, 1976). Lower bulk densities are associated with more pore space (Juyal et al., 2020).
A simplified visualization of micro-aggregates, macro-aggregates and pore space is demonstrated in Figure A.
Figure A: Simplified visualization of the soil environment on macro-and micro aggregate scale.
1.2 Microorganisms
Fungi and bacteria represent 98% of the soil microbial community with bacteria being the smallest and most numerous of the free-living microorganisms in the soil (Jiang et al., 2011; Clark, 1967). Bacteria dominate in the micro-aggregates and micro-pores while fungi dominate macro-aggregates and macro-pores (Wilpiszeski et al., 2019). In current studies the F:B (fungal-bacterial) ratio is often referred to when studying the dominance of fungi versus bacteria since bacteria and fungi are involved in different soil processes and live at different spatial scales (Wilpiszeski et al., 2019). Microorganisms are involved in many soil processes, including aggregate turnover and aggregation (Six et al., 2004). The relative importance of bacteria versus fungi for soil aggregation is site-specific and dependent on the soil texture and nutrient availability (Tang et al., 2011).
Understanding how bacteria are spatially structured is crucial for understanding soil microbiology (Upton et al., 2018). In general, bacteria contribute to the microbial diversity which is important for biodiversity across all trophic levels and can help create more resilience to stressors, improve soil quality, increase ecosystem services and promote sustainability (Upton et al., 2013; Zhang et al., 2013). Recent studies reveal that bacterial communities are related to several important soil processes, such as aggregation, decomposition of organic residues and carbon and nitrogen storage (Wilpiszeski et al., 2019; Zhang et al., 2013). Therefore, protecting and enhancing soil bacteria could potentially contribute to a more resilient self-regulating soil environment.
1.3 Relation aggregates and bacteria
Soil bacteria contribute to aggregation processes. The relation between soil bacteria and soil structure is complex with soil bacteria influencing aggregates through multiple feedbacks and vice-versa (Ceasar-TonThat et al., 2009). Soil bacteria for example contribute to aggregate turnover, stability and aggregation processes. Some essential traits of bacteria and their related processes that specifically influence soil aggregation processes are provided in Table 1 (Column 1).
Bacteria excrete extracellular polymeric substances (EPS) that serve as binding agents between soil particles. A fair amount of research has been conducted on aggregate binding agents, mainly on glomalin, a fungal equivalent of EPS (Fokom et al., 2012; Lehmann et al., 2017). These binding agents can enmesh particles together and increase C sequestration and aggregate stability (Bronick & Lal, 2005). EPS mainly consists of polysaccharides, but also proteins (Redmile-Gordon et al., 2020). Currently research on the role of EPS on soil aggregation is less accessible which might indicate a current knowledge gap in the relation between soil bacteria and aggregates. However, recently methodological challenges to quantify EPS have been overcome and therefore EPS is now receiving increased attention (Redmile-Gordon et al., 2020). Previous methods, such as 'hot sulphuric acid' failed to uncover any statistical significance due to not only extracting EPS but also non-target material (Redmile-Gordon et al., 2020). The only method found that does not extract non-target organic matter is the 'cation exchange resin' method, adapted and used by Redmile-Gordon et al., (2020).
Research suggests that EPS plays an important role in aggregation (Tang et al., 2011; Büks et al., 2016; Guo et al., 2018; Redmile-Gordon et al., 2020) since these biopolymers act as binding agents for aggregates for inter-particle cohesion on the micrometre scale (Lehmann et al., 2017; Tang et al., 2011). Bacterial EPS also improves aggregate stability (Tang et al., 2011). These studies suggest significant contribution of EPS to the formation and stability of aggregates. This BSc-thesis will look further into the potential role of EPS in soil aggregation.
The interaction between aggregates and soil bacteria is not a one-way connection, aggregates influence soil bacteria as well (Fox et al., 2014). In general soil biota depend on the distribution and size of aggregates, because they need adequate space for growth and activity (Briar et al, 2011; Wilpizeski et al., 2019). Soil biota can live attached to the aggregates and in the pores between the aggregates, and bacteria can even live in pores smaller than 3 µm (Briar et al., 2011). Bacteria often dominate over fungi in the micro-aggregates but can also be found in macro-aggregates (Briar et al., 2011). Spatial interactions between microaggregates, macroaggregates and pore-space are crucial for the function, structure, stability and evolution of microbial communities. Wilpizeski et al., (2019) even suggests that most complex soil interactions start at the formation of aggregates and continues to change as the soil matures. A list of essential traits of aggregates that influence bacterial soil communities is provided in Table 1 (Column 2). Some functions mentioned in Table 1 will be further reviewed and explained in the following chapters.
Table 1: Compilation of essential traits of bacteria positively influencing aggregates (Column 1) and a compilation of contributions of aggregates to soil bacteria (Column 2).
Contributions of soil bacteria to aggregates Contributions of aggregates to soil bacteria Particle adhering and orientation through
producing binding agents (Wilpizeski et al., 2019)
Providing space for transmission of viruses between microbial communities, activity and growth (Biar et al., 2017; Wilpizeski et al., 2019) Bacteria stimulate the turnover of organic matter
which influence micro-aggregate formation serving as building blocks for macro-aggregates (Vogel et al., 2014)
Micro-aggregates offer physical protection from pollutants (Six et al, 2004)
Bacteria produce hydrophobic EPS which leads to surface sealing of aggregates (Wilpizeski et al., 2019)
Aggregates offer a source of food through storing SOM and nutrients (Six et al., 2004). However, SOM is also protected by aggregates making it less accessible for bacteria that require fragmented and decomposed organic material (Six et al., 2004).
Bacteria dominate the micro-aggregate environment and contribute to stable micro-aggregate formation (Fox et al., 2014)
Aggregates physically protect bacteria against predation (Six et al., 2004)
Bacteria interact with roots and fungal hyphae increasing the aggregate stability (Wilpizeski et al., 2019)
Soil aggregate size determines organic matter, carbon and nitrogen availability for
microorganisms (Upton et al., 2018) Bacteria increase aggregate stability through EPS
(Tang et al., 2011; Büks et al., 2016) Stable aggregates retain more water which decreases the risk of drought, which can be a stressor for microorganisms (Six et al., 2004) Bacteria decompose soil organic matter (SOM)
which changes the soil structure (Upton et al., 2018)
Aggregates adsorb and desorb nutrients which offers a source of nutrients for bacteria living around and in the pore-spaces of aggregates(Six et al., 2004; Zhang et al., 2013)
Micro-aggregates are more stable because of the presence of bacterial cells and EPS surrounded by clay particles (Ceasar-TonThat et al., 2009)
Turnover of aggregates, from macro- to micro-aggregates influence microbial communities (Upton et al., 2018)
1.4 Influence of tillage on aggregates and bacteria
Agricultural management strongly influences both aggregate size and distribution (Trivedi et al., 2015; Zhang et al., 2012) as well as the diversity and abundance of soil fauna (Briar et al., 2011; D’hose et al., 2018). Changes in management regulate the soil physical and chemical properties and consequently influences the distribution of microbial communities (Trivedi et al., 2015). Tillage is one agricultural practice that has a significant impact on soil structure. Some advantages of tillage are aerating and loosening the soil as well as distributing the organic residues evenly throughout the soil column. However, tillage has mostly negative effects on soil quality. Therefore converting from conventional agriculture including regular tillage practices and mono-culture to a more diversified cropping system with reduced tillage almost always benefits soil biota, with only few studies contradicting this (Upton et al., 2018). Sustainable agricultural practices that enhance soil biology, such as reducing tillage, are the cornerstones of sustainable agriculture (D’hose et al., 2018).
This research specifically focuses on tillage, since this directly disrupts soil aggregates by altering their distribution and size (Jiang et al., 2011). Thereby, bacterial communities are disturbed as well, since aggregates and bacterial communities are connected (Table 1). Tillage mainly disrupts soil structure by breaking down macro-aggregates, resulting in reduced bacterial biomass due to decreased protection by these larger aggregates (Helgason et al., 2010; Lupwayi et al., 2019). Macro- and micro-aggregates are related to different soil processes and have their own benefits and limitations (Helgason et al., 2010). Therefore you expect the quality of the soil to improve when macro- and micro-aggregates are both present in the soil.
The soil disturbance caused by tillage can affect bacteria through desiccation, mechanical destruction, soil compaction, reduced pore volume, limited access to food and reduced soil moisture (Wilpiszeski et al., 2019). Throughout this research several tillage practices are compared and Table 2 clarifies the terminology used in the reviewed literature.
Table 2: Terms used for tillage and their definitions
Terms Definition
Conventional tillage Incorporates most or all of organic residues into the soil leaving the surface mostly bare. This is often conducted with a moldboard plough and is often repeated at least every year and sometimes multiple times a year
Reduced tillage (referred to as conservation tillage in some studies)
Minimizing the frequency and intensity of tillage practices
Zero tillage All plant residues are left on top of the soil without any ploughing
Chapter 2: Effects of aggregates on bacteria
Multiple studies, going back to the 1950s have already linked aggregates to microorganisms (Six et al., 2004). This chapter will review and explain where current knowledge in this study-field is at now and what needs to be investigated more thoroughly.
Most studies indicate that aggregate size has significant effects on the bacterial community structure (Fox et al., 2014; Wei et al., 2014, Heijnen & Van Veen, 1991; Briar et al., 2011; Ranjard et Richaume., 2001). Fox et al., (2014) found that the bacterial communities in the smallest fraction (silt and clay) differ significantly from the larger aggregate fractions. Moreover, Fox et al., (2014) also found that the bacterial richness was highest in the silt-clay fraction as well compared to the larger fractions (Fox et al., 2014). These results are in line with the understanding that bacteria are most abundant in the micro pores, since these provide a safe environment to avoid predation (Heijnen & Van Veen, 1991). However, not all studies found the same results. Briar et al., (2011) also found significant differences of micro-organisms for different aggregate fractions, but these results showed a greater abundance of microorganisms in the larger macro-aggregates than in the micro-aggregates and pore space as this study measured the overall abundance of microorganisms and did not solely investigate the abundance of bacteria. Therefore, the abundance of bacteria in different aggregate sizes could differ from the overall results.
Besides the macro- and micro-aggregates, the pore space influences micro-organisms as well. The size of pore space for example plays an important role in the transport and availability of water and oxygen (Wilpiszeski et al., 2019). The smaller the pore-space the more water and oxygen will be restricted. This limited diffusive transport can also limit the signaling molecules bacteria produce to interact (Wilpiszeski et al., 2019). Especially in dry conditions, bacteria have limited access to nutrients due to decreased solute diffusion. This shortage of nutrients can affect the functioning of bacteria through changing microbial pathogenesis, biofilm capabilities and motility (Wilpiszeski et al., 2019). Studies do not mention the impact nutrient deficiencies have on EPS production, indicating another knowledge gap.
Hence, pore space can impact the functioning of bacteria. Another example of this is demonstrated by Wilpiszeski et al (2019). Water is important for the exchange of nutrients, genetic material and metabolites between bacterial communities. During wet periods, communities living in the pore space between aggregates can interact through gene transfer and nutrient exchange. In contrast to dry periods, when communities cannot interact and have to function by themselves (Wilpiszeski et al., 2019). Therefore you expect the size distribution of aggregates to have a significant effect on the water availability through the arrangement of aggregates and pores where diffusion can take place. Briar et al., (2011) found a larger abundance of microorganisms attached to macro-aggregates than in the pore space. This might be related to the higher C content in macro-aggregates, since these are bound together by C-rich organic matter which is an important source of food for microorganisms (Briar et al., 2011). Bacteria occupying the pore space rely more on water which transports nutrients and enables exchange with other communities. The reliable source of energy in aggregates explains why greater abundances of microorganisms live attached to aggregates, rather than in the pore space between aggregates.
The diversity of bacterial communities is influenced by aggregate size as well. Micro-aggregates show the greatest microbial diversity (Upton et al., 2018; Bach et al., 2018). This can be attributed to the fact that micro-aggregates host a variety of different habitat types supporting a range of microbial communities compared to one larger macro-aggregate (Upton et al., 2018). Another explanation is that micro-aggregates are thought to be stable for decades or centuries (Upton et al., 2018), while macro-aggregates naturally turnover into smaller aggregates (Six et al., 2004). This stability could benefit species since it offers a stable habitat where microorganisms are less disturbed. However, bacteria are abundant in all aggregate fractions (Fox et al., 2014). Fox et al., (2014) found that the most abundant bacterial species are not predated by nematodes explaining why bacteria are abundant in all horizons and aggregate fractions. However, less abundant bacteria that are predated escape the macro-aggregate environment and occupy the micro-aggregates which explains the significant difference in bacterial community structure between these environments (Fox et al., 2014). The distinct species found in the silt- and clay fraction have in common
that they occupy anaerobic environments (Fox et al., 2014). The micro-pore environment restricts oxygen permeability in contrast to macro-pores (Fox et al., 2014). Nevertheless, the micro-aggregates offer other benefits besides the protection against predators such as stable water retention and restricted access of pollutants with toxic elements (Fox et al., 2014). It should be emphasized that the majority of bacterial species are found in all aggregate size fractions. Fox et al., (2014) therefore concluded that aggregate size had a greater influence on the bacterial biomass than the bacterial diversity.
Aggregates also have a significant impact on the functional diversity of microorganisms. An example to amplify this is nutrient cycling. Nitrogen is shown to be related to distinct portions of soil structure (Wilpiszeski et al., 2019; Ranjard & Richaume, 2001). Nitrifiërs have been found most productive in the micro-aggregates (2-20um) while nitrogen-fixing bacteria are most abundant in the smallest micro-aggregates (<2 um) (Wilpiszeski et al., 2019). This indicates that specific processes related to nutrient cycling are taking place in different aggregate fractions. Taking aggregate-size and arrangement in the soil into account for soil management would therefore most likely have a positive effect on the nutrient cycling. It remains to be tested whether other biogeochemical processes are influenced by bacterial functioning across different aggregate structures, indicating a knowledge gap.
Similar to the nitrogen cycle, carbon turnover can also be impacted by aggregate size. Trivedi et al., (2015) found that different aggregate size fractions influence the microbial community structure as well as the carbon turnover (Trivedi et al., 2015). The highest amounts of nutrients (C and N) were found in micro-aggregates (Trivedi et al., 2015). This study also found that all enzymatic activities of microorganisms increase with decreasing aggregate-size. Micro-aggregates are thus important since they enable increased enzymatic activity as well as containing the highest amounts of nutrients. One explanation for this can be the abundance of bacteria and the higher C-content in micro-aggregates (Trivedi et al., 2015). Bacteria are most abundant in micro-aggregates since the relation between bacteria, clay particles and organic matter is crucial for their existence (Trivedi et al., 2015). Nevertheless, microbial communities in macro-aggregates show strongest response to organic matter input, indicating the positive effects adding organic matter can have on microbial activity. Thus, the microbial response to changes in organic matter input decline as aggregate size declines as well. Therefore changes in management are expected to mainly influence bacteria living in the macro-aggregates.
The aforementioned observations of higher amounts of nutrients in micro-aggregates is not supported by all studies. Wei et al., (2014) found macro-aggregates to contain higher concentrations of nutrients than micro-aggregates (Wei et al., 2014). However, Wei et al., (2014) also acknowledges the fact that, other studies claim the opposite, having observed a higher nutrient content in micro-aggregates. Trivedi et al., (2017) also found macro-aggregates and their associated microbial communities to contribute most to C sequestration. Compared to micro-aggregates, macro-aggregates contain a higher amount of fresh organic matter, leading to a higher amount of SOM which is an important energy and carbon source for microorganisms (Wei et al., 2014). This source of organic matter for micro-organisms stimulates the production of binding agents (Wei et al., 2014). The relationship between aggregate-size and nutrient availability is thus not straightforward and studies reveal contradicting results. This contradiction can be attributed to different soils or research methods yielding different results. Further research is needed to gain a better understanding of the linkage between microorganisms, nutrient cycling and aggregate size. Yet again, it is important to emphasize that both macro- and micro-aggregates enable different benefits and limitations. However, the benefits, limitations and specific processes related to micro-and macro-aggregates are poorly understood and contradicting, indicating a potential knowledge gap.
Multiple studies found that aggregate size influences the F:B ratio in soils with fungi dominating at the larger particle size classes and bacteria in the finer fractions (Sessitsch et al., 2001; Jiang et al., 2011, Six et al., 2004). This is in line with the aggregation theory that states that fungi are involved with forming macro-aggregates and therefore macro-aggregates show the highest F:B ratios (Six et al., 2004). Sessitsch et al., (2001) showed that aggregate size had a greater influence on the community structure of bacteria and fungi than adding organic matter had. This indicates the importance of aggregate size on the ratio of microorganisms. Liao et al., (2020) also found that the fungal-bacterial network was more complicated in
macro-aggregates compared to micro-aggregates and the silt/clay fraction, because of more cooperation and competition.
In conclusion, multiple studies show the influence of aggregate size on microbial communities. Most studies are in line with the idea that different aggregate sizes are associated with different benefits and limitations (Six et al., 2004, Trivedi et al., 2015). It should be emphasized that some of the studies mentioned in this chapter investigated microorganisms as a whole, rather than separating bacteria and fungi. Therefore, some results might be different when investigating bacteria and fungi separately. For further research separating these groups would be a recommendation, since fungi and bacteria are involved with different soil processes and their abundance, biomass and functionality is dependent on different aggregate-fractions among other environmental conditions. Nevertheless, studies such as Liao et al., (2020) investigating the connectivity between fungi and bacteria are important as well, to understand their cooperation and competition better.
The direct and indirect effects of aggregates on soil bacteria are demonstrated in Figure B. Direct connections are effects where aggregate size, distribution or stability influences soil bacteria directly while indirect connections are effects caused by aggregates that influence other soil properties that might have an effect on soil bacteria.
Figure B: a framework for the direct and indirect effects of aggregates on soil bacteria
Chapter 3: Effects of bulk density on bacteria
This chapter reviews collected data on bacterial cell density for two different bulk densities by using a shared dataset from Otten et al., (2020). Results show an increase of number of bacterial cells/mm 2 for a
bulk density of 1.3 g/cm³ compared to a higher bulk density of 1.5 g/cm³. The bulk density of 1.3 showed a mean cell-count of 174.13 while the bulk density of 1.5 showed a mean cell-count of 99.29. A statistical t-test for comparing two means was applied and showed a significant difference in bacterial cell counts between the two bulk densities (P<0.01). The average cell density of Pseudomonas bacteria was 42% higher for the lower bulk-density.
However, using the means for bacterial cell counts is not necessarily the best method for evaluating the data due to a high standard deviation (103.21 and 69.85 respectively) and outliers. Using the median as a measure for spread is more reliable when dealing with large spread of data and outliers. Figure C demonstrates the median and IQR for this dataset. Bulk density of 1.3 g/cm³ has a median value of 158.73 which is lower than the mean cell-count. The median for bulk density of 1.5 is lower as well with a value of 95.23. The IQR of bulk density 1.3 is a lot bigger compared to a bulk density of 1.5. An one-way ANOVA for the bacterial cell counts for the two bulk densities returns a p-value <0.01 which means we can reject the zero hypothesis that there is no difference in bacterial cell density for the two bulk densities. This is thus still in accordance with the results of the t-test for comparing means, since both comparing the mean and median result in a significant difference (P<0.01) in bacterial cell counts for the two different bulk densities. At last, a Tukey HSD post-hoc test was performed which demonstrated the significant difference in means, which is visualized in Figure D.
Figure C: Box plots displaying the abundance of bacteria for two different two different bulk densities . Red line indicating the median values, blue lines indicating the 25% and 75% quartiles, black lines indicating the range and red crosses indicating outliers.
Figure D: comparison for significance of the ANOVA-test for mean bacterial cell density using the function multcompare.
In this experiment bulk density was used as a measure for soil structure instead of the arrangement of aggregates (Yuval et al., 2020). Since bulk density is related to the amount of pore space in the soil, with lower bulk densities showing more porosity, with 48% porosity for a bulk density of 1.3 g/cm³ and a 40% porosity for a bulk density of 1.5 g/cm³ (Yuval et al., 2020). The results from this analysis shows that bacteria grow and spread faster in soils with lower bulk density (associated with higher porosity) compared to a higher bulk density. The difference in cell density could be explained by the limited access to nutrients in soils with a higher bulk density. Another explanation could be attributed to the fact that pore-space is important for the transport of water, air and other substrates, which are important for bacterial growth and and abundance (Six et al., 2004). Whether bulk density is connected to aggregation is poorly understood.
García-Orenes et al., (2005) linked an increase in aggregate stability to a decrease in bulk density. Increasing the aggregate stability could therefore result in higher lower bulk densities with higher numbers of bacterial cells/mm2 . One way to increase aggregate stability is by adding organic matter to the soil
(García-Orenes et al., 2005).
This dataset thus demonstrates the significant effect of bulk density on bacterial density. Therefore bulk density can be used as a measure for soil structure as well that can be linked to the functioning of microorganisms. However, currently studies on the relation between bulk density and soil bacteria as well as the relation between bulk density and aggregates is lacking. The dynamics of bulk density, aggregates and soil bacteria are lacking in most research, illustrating a knowledge gap.
Chapter 4: Effects of bacterial EPS on aggregates
Bacteria influence aggregates in multiple ways. The influence of micro-organisms on aggregates has been extensively reviewed before (Six et al., 2004; Oades, 1993; Wilpizeski et al., 2019). For example, research demonstrated the influence of bacteria on decomposition of organic materials by attaching themselves to soil particles (Six et al., 2004). Since these processes are already investigated, this chapter mainly reviews current knowledge on the influence of extracellular polymeric substances (EPS) on aggregates, which remains poorly understood thus far.
The composition of EPS varies among different bacterial species (Guo et al., 2018) and the environmental conditions under which it was formed (Zhang et al., 2014; Guo et al., 2018). EPS can consist of proteins, polysaccharides, lipids, extracellular DNA and surfactants (Guo et al., 2018). There are three main mechanisms how EPS can influence the soil moisture, through (1) holding water directly within its polymeric matrix, (2) promoting the formation of soil aggregates creating smaller pores which reduces water drainage and (3) creating more hydrophobic micropores that inhibit evaporation (Guo et al., 2018). These substances and their structure enable connections to soil surfaces, immobilization, cell-to-cell communication, water retention and degradation of larger molecules for use by cells (Guo et al., 2018). Some of these processes are further discussed in this chapter. Since this thesis focuses on the connection between bacteria to soil surfaces, we focus mainly on the role of EPS on soil structure in this chapter.
Guo et al. (2018) investigated the influence of EPS on water content variability on different pore scales. Results show that even small amounts of EPS from Sinorhizobium meliloti, a commonly found soil bacteria in the rhizosphere, significantly increases water content and water variability at the micropore scale. Small amounts of EPS limit water evaporation at narrow pore throats, located close to the surface of aggregates. However, on the macropore scale EPS had no effect on the water retention. Furthermore, EPS influenced the variability of moisture in local micro-environments as well by creating small disconnected water films in unsaturated soils. This local variability enables small microhabitats for organisms, allowing them to coexist in the soil, while not competing. This moisture variability enhanced by EPS may therefore have positive effects on microbial diversity through reduction of competition and creating favorable niches. Enhancing soil microbial diversity can eventually contribute to better resilience of terrestrial ecosystems (Guo et al., 2018; Upton et al., 2018). However, the effects of EPS on microbial communities through water films should be studied more extensively, to prove the greater effects of EPS on ecosystem resilience and biodiversity (Guo et al., 2018). Nevertheless, this study shows the potential of EPS, since only small amounts significantly amplified the water content in the micro-pores. It should be emphasized that this study only used EPS, without introducing the Sinorhizobium meliloti to the soil, to solely measure the effects of EPS (Guo et al., 2018). Therefore, biological growth and redistribution of bacteria did not influence the results of this study, contributing to more reliable knowledge on the effects of EPS. More extensive research on the role of EPS in the macropore and macro-aggregate environment is required, since this research did not find significant effects on moisture retention. It should also be emphasized that this research was conducted on a sandy-loamy soil type, so depending on the soil types EPS might show different effects on soil moisture and other soil properties and therefore effects of EPS on other soil types would be crucial as well.
Amellal et al., (1999) used a different approach by inoculating EPS-producing Pantoea agglomerans bacteria on the aggregation in the rhizosphere of wheat fields. Instead of solely using EPS, this study looked at the effects by introducing bacteria to the soils. The samples that were enhanced with the EPS-producing bacteria showed striking results as they had a significant positive effect on the mean aggregate diameter, macro-porosity and mechanical stability of the soil (Amellal et al., 1999). This can be explained by the adhesive effect of EPS mainly found in micro-aggregate pores. Secondly, the hydrophobic character of EPS can improve the water retention of the soil (Amellal et al., 1999). One point of discussion would be that inoculating bacteria to measure the effect of EPS can provide mixed results, since the
bacteria themselves can affect the outcomings of the research by biological growth and redistribution, since bacteria influence soil processes multiple ways, not solely by producing EPS.
Multiple studies researched the effects of EPS on aggregate stability. These studies show varying results on the effects of EPS on aggregate stability. Degens et al., (1997) found that organic binding agents, such as EPS, increase the aggregate stability especially in sandy soils where aggregates are less stable than more clay-rich soils. Molope & Page (1986) also found that adhesive substances produced by fungi and bacteria can enhance the aggregate stability. More recent studies, such as Harahap et al. (2018) also show a significant effect of different bacteria producing EPS on the aggregate stability especially in combination with addition of organic matter. This research also found that adding sucrose to the soil samples increased the production of EPS, which resulted in more stable aggregates (Harahap et al., 2018). A recent study from Redmile-Gordon et al., (2020) revealed that there is a differentiation between EPS-protein and EPS-polysaccharides. The protein is closer related to aggregate stability than the polysaccharides, but both have a positive effect on the aggregate stability (Redmile-Gordon et al., 202). EPS is calculated by the sum of protein and polysaccharides. There are however several studies that fail to prove the positive effects of EPS on aggregate stability. Tang et al., (2011) for example found lower soil aggregate stability in the samples containing most EPS. These contradicting results are possibly caused by the different methods used to investigate the effect of EPS on aggregate stability. Tang et al., (2011) used a dilute acid-extractable carbohydrate to measure EPS, but this might not stand the stress of the fast wet-sieving procedure. Redmile-Gordon succeeded by using the 'cation exchange resin' method to find significant effects of EPS. This is however only recently discovered and therefore this method still needs to be further developed and tested. Therefore, finding a suitable method for measuring the contribution of EPS in soils remains a knowledge gap.
Few studies have investigated the differences in EPS structure among different microbial communities. Despite the fact that all biofilms consist of the aforementioned components such as extracellular polysaccharides, the composition can vary among different species (Büks et al., 2016). The same organism can even produce different forms of EPS under varying environmental conditions in the soil (Büks et al., 2016). Researchers therefore expect to see differences in EPS productions among different microbial community structures which could potentially result in different aggregate stabilities (Büks et al., 2016). However, proof for this hypothesis is still missing and Büks et al., (2016) found no changes in aggregate stability among different microbial community structures. This research concludes that aggregate stability does not seem to alter when comparing multiple microbial communities that produce different forms and quantities of EPS.
Tang et al., (2011) and Büks et al., (2016) stated that more accurate methods for measuring aggregate stability are still missing. Abiven et al., (2007) for example found that the influence of binding agents such as EPS on the aggregate stability depends on the organic matter available or added. This study also found that different methods, such as fast wetting, slow wetting and mechanical breakdown, generated different results of aggregate stability and therefore selecting the appropriate method for measuring aggregate stability is rather complicated (Abiven et al., 2007). Finding more precise methods is thus important for further research, since even small changes in aggregate stability can improve multiple soil properties (Büks et al., 2016). However, it should also be considered that EPS might not be an effective binding agent for stabilization of aggregates in some soil environments. Nevertheless, EPS can still play an important role in a variety of soils and related soil processes and I recommend to investigate EPS across different soil environments in the future.
Figure E demonstrates a the direct and indirect effects soil bacteria have on aggregates. The indirect effects alter the environment which effects aggregates and aggregation processes and conditions. Direct effects broadcast the influence bacteria directly have on aggregates and associated processes such as turnover and aggregation.
Figure E: a framework for the direct and indirect effects of soil bacteria on aggregates. The green arrow demonstrates the positive feedback loop between soil bacteria and aggregates.
EPS is potentially not solely important as a binding agent, but has a positive influence on the bacteria themselves as well. Microbes are dramatically affected by soil structure and chemical soil properties, but through producing EPS they can also change their microenvironment (Guo et al., 2018). Therefore, this can be considered a positive feedback loop, since EPS has a positive influence on aggregates, which in return has a positive effect on soil bacteria. This is demonstrated by the green arrow in Figure E.
Chapter 5: Effects of tillage on aggregates and
bacteria
Land use change is known to strongly impact soil aggregates (Spohn & Giani, 2011) and this chapter discusses the influence of tillage practices on soil structure and bacteria.
5.1 Effects of tillage on aggregates
The majority of studies found that conventional tillage caused a strong reduction in macro-aggregates compared to reduced tillage, and no-tillage (Wei et al., 2014; Helgason et al., 2010; Six et al., 2004; Lupwayi et al, 2001; Jiang et al., 2011; Zhang et al., 2014; Jiang et al., Briar et al., 2011; Ceasar-TonThat). Wei et al., (2014) for example found a strong reduction in macro-aggregates when applying conventional tillage, with a reduction of 68.9 and 44.7% for the >2 mm and 1-2 mm aggregates respectively in the soil surface layer. The subsurface soil (20-40 cm) showed a similar decrease of 64.3 and 37.2% respectively for the two aggregate fractions (Wei et al., 2014). Jiang et al., (2011) also found a reduction of macro-aggregates of 67% under conventional tillage practices. Helgason et al., (2010) found a similar reduction in macro-aggregates when applying conventional tillage compared to no-tillage. Wei et al., (2014) and Helgason et al., (2010) both explained this phenomena with the theory that macro-aggregates are less stable than micro-aggregates since they are not bounded as strongly which makes them more vulnerable for land-use changes, in particular agricultural changes. Besides the reduction in macro-aggregates, the amount of micro-aggregates increased significantly (Wei et al., 2014).
Moreover, Wei et al., (2014) also described a rapid decrease in associated nutrients with decreasing aggregation under conventional tillage. C and N content decreased due to accelerated decomposition of SOM under conventional tillage practices. This is in line with earlier research that found a 51% decrease of total particulate organic matter (POM) under conventional tillage compared to no-tillage (Six et al., 1999). POM is organic matter is coarse organic matter between 0.053 and 2 mm and thus often associated with macro-aggregates. Lupwayi et al., (2001) also found increased mineralizable C in macro-aggregates compared to smaller aggregates in no-tillage practices. This can be explained by the physical protection of organic matter by macro-aggregates (Lupwayi et al., 2001). Tillage therefore not only reduced the amount of macro-aggregates, but can also affect the organic matter associated with these aggregates (Six et al., 1999; Lupwayi et al., 2001). Caesar-TonThat et al., (2010) not only found an increase in the 4.75- 2mm (macroaggregate) fraction under no-tillage practices, but also noticed that in the third year the fraction of macro-aggregates was significantly larger than in the first and second year. This multi-year study would suggest that long-term no-tillage practices have even greater effects on the abundance of macro-aggregates.
5.2 Effects of tillage on bacteria
In general, most studies demonstrate a greater microbial biomass under no-tillage versus conventional tillage systems (Helgason et al., 2010; Lupwayi et al., 2001; Jiang et al., 2011; Muruganandam et al., 2010; Ceasar-TonThat et al., 2010). Jiang et al., (2011) for example found an increase in bacterial biomass of 52% under no-tillage compared to conventional tillage practices. The same study also found that the distribution of microorganisms throughout the soil was similar for the two tillage practices (Jiang et al., 2011). These results therefore mainly showed an effect of tillage on the microbial biomass.
Besides the effects of tillage on microbial biomass, tillage practices can also influence the bacterial diversity. Lupwayi et al., (2001) found an increased bacterial diversity under no tilled soils compared to conventional tilled soils. However, research on the influence of tillage on bacterial diversity is currently lacking. Ceasar-TonThat et al., (2010) found that the number of soil aggregating bacterial species in
micro-aggregates was 2.2 times higher under no-tillage compared to conventional tillage in the topsoil (0-5 cm).
Research on the effect of tillage on the production of EPS is currently lacking as well. However, research on the effects on fungal binding agents showed a positive effect when shifting from conventional tillage to reduced tillage (Zhang et al., 2014). Whether, reduced or no-tillage have the same effect positive on bacterial EPS is unknown. This is therefore another knowledge gap. However, since greater numbers of bacterial biomass are observed under reduced tillage, EPS production is expected to increase as well. Ceasar-TonThat et al., (2010) confirms this and observed an increase in soil aggregating bacteria in the topsoil (0-5 cm). Since the amount of soil aggregating bacteria increases, soil aggregation and aggregate stability should improve as well (Ceasar-TonThat et al., 2010). Another recent study investigated the effects of tillage on EPS-production as well, finding increased EPS-production under reduced tillage practices (Cania et al., 2020).
Despite the fact that most studies show a positive effect on total microbial biomass as well as on solely bacterial biomass, tillage effects on microorganisms remain complicated. This can be attributed to the indirect links between tillage and soil biota (Jiang et al., 2011). The next chapter will shine light upon this connectivity and complexity.
Chapter 6: Links between tillage, aggregates and
micro-organisms
As mentioned before conventional tillage causes a reduction in macro-aggregates and no-tillage practices increased the bacterial biomass and total amount of microorganisms (Helgason et al., 2010; Jiang et al., 2011). Rather than demonstrating two individual findings, these findings are connected to one another. This chapter will review the connectivity between tillage, aggregates and micro-organisms.
Many studies found a relation between aggregate size, microbial biomass and tillage practices (Helgason et al., 2010; Lupwayi et al., 2001; Jiang et al., 2011; Murunganandam et al., 2010; Briar et al., 2011; Ceasar-TonThat et al., 2010). Helgason et al., (2010) for example found that microbial biomass varied for different aggregate fractions under different tillage practices which demonstrates the role of aggregate arrangement on soil biota once again. Lupwayi et al. (2001) even found the observations on bacterial biomass was only significant when taking aggregate arrangement in account, since bacterial biomass was not significantly affected by different tillage practices when looking at the soil as a whole. Jiang et al., (2011) also investigated the effects of different tillage practices on bacteria associated with aggregate size and found similar results. These studies therefore emphasize the importance of taking aggregates in account when investigating the effects of tillage on soil biota.
Not all studies found similar relations. Lupwayi et al., (2001) found a higher microbial biomass in larger aggregates which is explained by the the physical protection of organic matter in macro-aggregates which serves as a source of food for microorganisms. By deteriorating soil structure, mainly affecting macro-aggregates, tillage practices have negative effects on soil microbial dynamics. In contrast, Jiang et al., (2011) found that no-tillage had greatest effects on the aggregates <1 mm (micro-aggregates) where the bacterial biomass was 54% higher under no-tillage compared to conventional tillage. This is contradictory, as you expect no-tillage practices to mainly impact the macro-aggregates since these break down faster than micro-aggregates (Six et al., 2004). Nevertheless, results from Jiang et al., (2011) show that no-tillage practices can even positively affect smaller aggregates. Jiang et al., (2011) concludes that since soil total microbial biomass was mainly located in macro-aggregates which decreased by 67% under conventional tillage practices, it is assumed that the deterioration of macro-aggregates is the main driver of reduced microbial biomass under conventional tillage.
Some studies found a difference in microbial biomass between the topsoil and soil layers below under different tillage practices. Microbial biomass often increased in the topsoil (0-10 cm) under no tillage while some studies demonstrate a decrease in microbial biomass in the layer below (10-20 cm) (Jiang et al.,, 2011). This is likely due to the increased plant residues left on top of the soil, increasing the organic matter which is a food source for microorganisms. Normally, residues would be ploughed into the soil, which would supply plant residues into the deeper soil layers as well. However, when no-tillage is applied, these layers do not get plant residues mixed in, which could potentially be the reason for the observed decrease in microbial biomass in the 10-20 cm soil layer.
Besides the change in microbial biomass Helgason et al., (2010) found a difference between the inhibition of bacteria and fungi in the soil under different tillage practices. Macro-aggregates had a higher F:B ratio and higher relative abundance of fungi, which is in agreement with Six et al., (2004) where fungi are associated with the macro-aggregation processes (Helgason et al., 2010). Thus the F:B ratio decreases with decreasing aggregate size (Helgason et al., 2004). Since macro-aggregates are broken down by conventional tillage practices, fungi are expected to thrive in reduced or no-tillage soils (Six et al., 2004). Fungi therefore seem to have a greater effect on the aggregate stability in no tillage soils compared to conventional tilled soils, since they enable macro-aggregate formation (Helgason et al., 2010). This increase in F:B ratio can be at the expense of bacteria (Helgason et al., 2010). However, total bacterial biomass was still higher under no-tillage practices (Helgason et al., 2010).
