Spatially-distributed microbial enzyme activities at intact, coated macropore surfaces in Luvisol Bt-horizons
Introduction
In structured soils, macropores such as biopores, root channels, shrinkage cracks, and inter-aggregate spaces form complex networks. Beside their role as preferential flow paths for water and solutes (Beven and Germann, 1982; Jarvis, 2007) as well as root growth (Angers and Caron, 1998; Kautz, 2014), they are denoted as "hotspots" of nutrient and C turnover (e.g., Bundt et al., 2001; Kuzyakov and Blagodatskaya, 2015).
Biopores, in particular earthworm burrow walls were found to be enriched in soil organic matter (SOM) (Don et al., 2008; Pagenkemper et al., 2015; Leue et al., 2018). In addition, the SOM composition of the drilosphere (i.e., the soil influenced by earthworms) differed to that of the surrounding soil matrix due to increased contents of aliphatic-C (Leue et al., 2016) derived from incorporated litter, mucilage, and extracellular polymeric substances (EPS). Recent research revealed strongly increased enzymatic activities along earthworm burrows (Hoang et al., 2016a; Lipiec et al., 2016) as well as along root channels (Razavi et al., 2016; Ma et al., 2018). Correspondingly, the content of phospholipid fatty acids (PLFAs) as a chemotaxonomic marker for living biomass was found to be strongly increased in the drilosphere as compared to the bulk soil (Stromberger et al., 2012; Giacometti et al., 2013).
Clay-illuvial horizons of Luvisols (Bt-horizons) feature cracks characteristically coated by clay-organic material (IUSS, 2006) as a result of clay migration and SOM adsorption to the accumulated clay and other soil minerals (e.g., Schulten and Leinweber, 2000). Crack coatings and clay-organic fillings of single, disconnected pores (‘pinholes’) revealed similarly high SOC levels as found for biopores (Leue et al., 2018). Furthermore, the composition of SOM in cracks and pinholes was significantly different from that of biopores (Leue et al., 2016, 2017). In Luvisol Bt-horizons, crack coatings and pinhole fillings comprised increased contents of relatively stable, high-molecular compounds. In part, these fillings consisted of combustion residues such as benzonitrile and naphthalene. In contrast, biopore wall material was found to be enriched in aliphatic-C compounds (Leue et al., 2016, 2017). These local differences in the SOM composition may imply also small-scale differences in microbial community composition and enzymatic activities between different macropore types of Bt-horizons, such as earthworm burrows and cracks. This implies differences in intensities of decomposition processes and C turnover. However, information on biogeochemical functions of macropore-associated microbial communities and associated microbially-driven processes remains sparse. To a certain extent, this lack in information is probably due to the fact that most soil samples are taken as bulk soil samples. Any measurement of mixed or bulk samples only yields insufficient signals from thin macropore surfaces (‘interfaces’). Consequently, the quantitative role of macropores and small but important structures and components on transport and turnover processes in soils is frequently underestimated. Analyses of microbial hotspots could generally improve our understanding of C turnover in structured soils.
Microbially produced extracellular enzymes catalyse C-turnover in soils and can be grouped into hydrolytic and oxidative enzymes. Hydrolytic enzymes such as cellobiohydrolase, ß-glucosidase or xylanase (XYL) are associated with the turnover of readily decomposable C-compounds such as (hemi-) cellulose, mucilage, and EPS particularly in the drilosphere and rhizosphere (Hoang et al., 2016a; Ma et al., 2018). These enzymes can therefore be expected to be predominant around biopores. In contrast, oxidative enzymes such as phenol oxidase (POX) are associated with more recalcitrant, aromatic C-compounds such as lignin and secondary compounds (Veum et al., 2014). Increased POX activities can be expected around crack coatings and pinhole fillings. However, POX is considered to be less stable in soil as compared to extracellular hydrolases. Furthermore, high spatiotemporal variation often obscures their relationships with environmental variables and ecological processes (Sinsabaugh, 2010). SOM decomposition is mainly driven by microorganisms. Thus, differences in SOM composition between biopores and crack coatings or pinhole fillings are likely to be reflected in different microbial community composition and enzyme patterns. Kravchenko et al. (2020) reported that microorganisms localised in large pores respond to new C inputs with faster turnover, increased growth, and more intensive enzyme production compared to those inhabiting the small pores.
Besides standardised ex situ assays for the quantification of potential soil enzyme activities (German et al., 2011), the zymography technique was developed for in situ detection and mapping of the two-dimensional distribution of enzyme activities in the rhizosphere or drilosphere (Spohn et al., 2013; Spohn and Kuzyakov, 2014; Heitkötter and Marschner, 2018). This technique is based on enzyme-specific substrates, from which fluorescent products are released in case of enzyme activities. In order to determine the mm-scale distributions of these activities, thin gels or membranes have been equipped with substrates and incubated on the surfaces of exposed rhizoboxes (e.g., Spohn et al., 2013; Razavi et al., 2016; Ma et al., 2018), or flow cells (Heitkötter and Marschner, 2018). Applications on intact surfaces of earthworm burrows (Hoang et al., 2016a) are particularly challenged by the actual surface roughness or micro-topography of natural macropores. Such roughness is known from non-destructive spectroscopic approaches using intact structural surfaces (Leue et al., 2011; Leue and Gerke, 2016). Considering the microscale local distribution of organic matter at intact soil structural surfaces, we assume that the enzymatic activity will be related to the distribution of macropores.
The objective of this work was to detect and compare the spatial distributions of potential enzymatic activities, and microbial community compositions of different types of macropore surfaces or soil components (earthworm burrows, coated cracks, pinhole fillings, fine-porous soil matrix). For this purpose, Luvisol Bt-horizons developed from different parent materials, i.e. loess and glacial till, were examined. Soil material was separated from intact macropore surfaces and used to determine potential activities of XYL and POX ex situ. In parallel, soil microbial biomass and community composition were assessed by PLFA signatures. The spatial distributions of enzyme activities on macropore walls and in the matrix of the structured soils were observed in situ by zymography at soil slices from intact large-scale soil cores.
Section snippets
Soils and sampling
Soil samples were collected from two Haplic Luvisols (IUSS, 2006) which had a similar genesis and were under arable land use but have been developed from different parental materials. The soil developed from loess (L) was located in Northern Bohemia (Hnevceves, near Hradec Kralove, Czech Republic; 15°43′03″E, 50°18′47″N; mean annual precipitation 618 mm; mean annual temperature 8.5 °C). The soil developed from glacial till (T) was located in North-Eastern Germany (Holzendorf, near Prenzlau,
Xylanase and phenol oxidase activities in separated macropore surfaces
Enzyme assays revealed highest XYL activity in earthworm burrow walls (EB) at both study sites (Fig. 1a). For the loess-Bt samples, the XYL activities along the burrows as well as in clay-organic material of crack coatings (CS + C) and pinhole fillings (PIN) were three times higher (p ≤ 0.05) compared to those of uncoated cracks (CS). The EB values were 2.5-times higher compared to the bulk soil (with a non-significant trend considering PIN and the bulk soil). For the till-Bt samples, the XYL
Xylanase
The clear difference in XYL activities between earthworm burrow walls and bulk soil or the soil matrix of the till-derived Bt-horizon indicates the relevance of biopores as microbiological hotspots for this site. The results basically confirm findings of Athmann et al. (2017), reasoning that XYL is mainly bound to particulate organic matter which is likely to be enriched in the drilosphere. The XYL activity levels of the separated earthworm burrows, crack coatings, and pinholes were at a
Conclusions
Our results suggest that macropores show type-specific and site-specific differences in XYL and POX activities as well as in PLFA-related microbial communities. Soil areas or structures with increased XYL activities were not necessarily related to the walls of earthworm burrows but rather to the presence of roots and were dominated by bacterial communities. The spatial distribution of XYL activity detected by zymography on soil core slices was strongly related to rooting intensity which can be
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
We thank Petra Lange and Ulrike Klauß for laboratory support, and Ingrid Onasch, Lidia Völker, Jennika Hammar, Norbert Wypler (ZALF Müncheberg) and Radka Kodešová and Miroslav Fer (Czech Agricultural University, Prague) for kind support during the soil sampling. Special thanks to Bahar Razavi, Stefanie Heinze, and Felix Heitkötter for help and advice with the zymography. We thank three anonymous reviewers for improving the manuscript. The study was financially supported by the Deutsche
References (54)
- et al.
Six months of L. terrestris L. activity in root-formed biopores increases nutrient availability, microbial biomass and enzyme activity
Applied Soil Ecology
(2017) - et al.
Measuring phenol oxidase and peroxidase activities with pyrogallol, L-DOPA, and ABTS: effect of assay conditions and soil type
Soil Biology and Biochemistry
(2013) - et al.
Preferential flow paths: biological ‘hot spots’ in soils
Soil Biology and Biochemistry
(2001) - et al.
Organic carbon sequestration in earthworm burrows
Soil Biology and Biochemistry
(2008) - et al.
ABTS assay of phenol oxidase activity in soil
Journal of Microbiological Methods
(2007) - et al.
Microbial biomass measured as total lipid phosphate in soil of different organic content
Journal of Microbiological Methods
(1991) - et al.
Optimization of hydrolytic and oxidative enzyme methods for ecosystem studies
Soil Biology and Biochemistry
(2011) - et al.
Chemical and microbiological soil quality indicators and their potential to differentiate fertilization regimes in temperate agroecosystems
Applied Soil Ecology
(2013) - et al.
Quantitative soil zymography: mechanisms, processes of substrate and enzyme diffusion in porous media
Soil Biology and Biochemistry
(2018) - et al.
Soil zymography as a powerful tool for exploring hotspots and substrate limitation in undisturbed subsoil
Soil Biology and Biochemistry
(2018)
Earthworm burrows: kinetics and spatial distribution of enzymes of C-, N- and P- cycles
Soil Biology and Biochemistry
Co-occurring increased phosphatase activity and labile P depletion in the rhizosphere of Lupinus angustifolius assessed with a novel, combined 2D-imaging approach
Soil Biology and Biochemistry
Xylanase, invertase and protease at the soil±litter interface of a loamy sand
Soil Biology and Biochemistry
Microbial hotspots and hot moments in soil: concept & review
Soil Biology and Biochemistry
Two-dimensional distribution of soil organic carbon at intact macropore surfaces in Bt-horizons
Soil and Tillage Research
Spatial patterns of enzyme activities in the rhizosphere: effects of root hairs and root radius
Soil Biology and Biochemistry
The effect of earthworm activity on soil bioporosity – investigated with X-ray computed tomography and endoscopy
Soil and Tillage Research
Rhizosphere shape of lentil and maize: spatial distribution of enzyme activities
Soil Biology and Biochemistry
Soil zymography: simple and reliable? Review of current knowledge and optimization of the method
Rhizosphere
Phenol oxidase, peroxidase and organic matter dynamics of soil
Soil Biology and Biochemistry
Soil zymography: a novel in situ method for mapping distribution of enzyme activity in soil
Soil Biology and Biochemistry
Distinct microbial and faunal communities and translocated carbon in Lumbricus terrestris drilospheres
Soil Biology and Biochemistry
Potentials and limitations of quantification of fungi in freshwaterenvironments based on PLFA profiles
Fungal Ecology
Phospholipid fatty acid profiles in selected members of soil microbial communities
Chemosphere
Plant-induced changes in soil structure: processes and feedbacks
Biogeochemistry
High-throughput fluorometric measurement of potential soil extracelluar enzyme activities
Journal of Visualized Experiments
Macropores and water-flow in soils
Water Resources Research
Cited by (9)
Soil zymography: A decade of rapid development in microbial hotspot imaging
2024, Soil Biology and BiochemistryStructural heterogeneity of soil clods: Correlating Weibull parameters to fracture surface topography
2022, GeodermaCitation Excerpt :Samples were collected from distinct soil horizons of two arable soils, one is a Haplic Luvisols (haLV) and the other a Haplic Regosol (calcaric) (haRG) (IUSS Working Group WRB, 2006). The haLV soil profile (Leue et al., 2020) was located in northern Bohemia in the village of Hněvčeves, near Hradec Králové, Czech Republic (50°18′47″ N, 15°43′03″ E; mean annual precipitation: 618 mm; annual mean temperature: 8.5 °C); here the samples were from the Bt horizon (Bt-haLV) with sand, silt, and clay contents of 9, 62, 29 %, respectively, and a soil organic carbon content (SOC) of 0.48 % (Leue et al., 2021). The haRG soil profile (Rieckh et al., 2012) was located in the hummocky arable soil landscape of the Uckermark region in northeastern Germany (53°23′ N, 13°47′ E); here the samples were from the C horizon (C-haRG) with sand, silt, and clay contents of 61, 27 and 12 %, respectively, and SOC 0.10 % (Rieckh et al., 2012).
Soil oxidoreductase zymography: Visualizing spatial distributions of peroxidase and phenol oxidase activities at the root-soil interface
2022, Soil Biology and BiochemistryCitation Excerpt :It can be simply calibrated using standard solutions of commercially available resorufin. It has clear advantage over ABTS-based light adsorption zymography, which lacks appropriate calibration standards (Leue et al., 2021). In the Amplex Red-based assay, however, special requirements for the reagent's preparation and analysis in a dark place need to be considered.