Spatially-distributed microbial enzyme activities at intact, coated macropore surfaces in Luvisol Bt-horizons

Highlights

• Comparative analysis of enzyme activities at macropore surfaces.

• Xylanase and phenol oxidase assays using material from macropore walls and soil matrix.

• Zymography reveals xylanase but not phenol oxidase activities at macropore surfaces.

• Small-scale spatial distribution of enzyme activities in macroporous soils.

• Enzyme activities detected emphasise the role of macropores for C turnover processes.

Abstract

Soil macropores serve as preferential pathways for water and solute transport as well as for root growth. They are often coated with organic material and known as "hotspots" of nutrient and C turnover. Differences in the SOM composition between macropores and soil matrix as well as between macropore types (biopores, cracks, pinhole fillings) imply potential differences in the microbial community composition and enzymatic activities. The objective of this work was to detect and assess the spatial distribution of enzyme activities related to C turnover, xylanase (XYL) and phenol oxidase (POX), and the composition of microbial communities in structural components of Luvisol Bt-horizons, developed from loess and glacial till. We applied conventional enzyme assays and phospholipid fatty acids (PLFA) analysis to study materials separated from different types of macropores and soil components as well as bulk soil samples. The spatial distribution of enzyme activities on surfaces of large soil core slices (20 cm in diameter) was quantified by soil zymography. Higher XYL activities were detected in separated burrow wall materials, clay-organic coatings, and pinhole fillings from both sites, as compared to the respective soil matrix or bulk soil samples. The XYL activities correlated with bacteria-specific PLFAs. POX activities were solely found increased for earthworm burrow walls from the loess-derived Bt-horizon, but not for burrows from till samples. Zymograms revealed particularly increased XYL activities at rooted earthworm burrows, emphasising evidence for hotspots of enzyme activity and C turnover. The zymography of POX was hampered by methodological restrictions.

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.