Metagenomics and stable isotope probing reveal the complementary contribution of fungal and bacterial communities in the recycling of dead biomass in forest soil

Highlights

• Both fungi and bacteria actively degrade complex plant and microbial biomass.

• The pool of CAZymes was distinct in fungi and bacteria.

• Fungal communities encode more specific CAZymes that degrade plant biomass.

• Bacterial communities are richer in CAZymes that target microbial biomass.

• Bacteria use structurally variable but complementary enzymatic systems.

Abstract

Forest soils represent important terrestrial carbon (C) pools, where C is primarily fixed in plant biomass and then is incorporated in the biomass of fungi and bacteria. Although classical concepts assume that fungi are the main decomposers of the recalcitrant organic matter within plant and microbial biomass, whereas bacteria are considered to mostly utilize simpler compounds, recent studies have shown that fungi and bacteria overlap in substrate utilization. Here, we studied the microbial contribution to the recycling of dead biomass by analyzing the bacterial and fungal communities in soil microcosms supplemented with ¹³C-labeled biomass of plant, fungal, and bacterial origin using a combination of DNA-stable isotope probing and metagenomics. Both fungi and bacteria contributed actively to the degradation of complex components of plant and microbial biomass. Specific families of carbohydrate-active enzymes (CAZyme) were involved in the degradation of each biomass type. Moreover, the analysis of five bacterial metagenome-assembled genomes indicated the key role of some bacterial genera in the degradation of plant biomass (Cytophaga and Asticcacaulis) and microbial biomass (Herminiimonas). The enzymatic systems utilized by bacteria are highly complex and complementary but also highly diverse among taxa. The results confirm the importance of bacteria, in addition to fungi, as decomposers of complex organic matter in forest soils.

Introduction

Forests represent some of the most important carbon (C) pools and sinks on Earth. Since nearly half of the C stored in these ecosystems is contained in soils, understanding the processes involved in C cycling in forest soils is essential in the current context of global climate change (Pan et al., 2011). Microorganisms are the main players involved in the recycling and turnover of soil organic matter. As such, they contribute largely to the C flow in this habitat and have the potential to influence the feedback between climate and the global C cycle (Schimel and Schaeffer, 2012). Therefore, predicting how forests will respond to future environmental conditions is impossible without understanding the roles of soil microbes in C cycling (Graham et al., 2016; Trivedi et al., 2013).

The major sources of forest soil C are comprised of the C allocated by tree roots into soil and of the C contained in the dead plant biomass in the forms of litter and dead wood. This dead plant biomass is composed mostly of cellulose, hemicelluloses and lignin, forming a complex and recalcitrant matrix (Bomble et al., 2017). Microbial biomass represents another important pool of organic matter whose fate in the soil is far less understood. Forest soils are rich in ectomycorrhizal (ECM) and saprotrophic fungi and the decomposition of dead mycelia represents an important process for the cycling of C and other nutrients in these ecosystems (Ekblad et al., 2013; Fernandez and Koide, 2014). Dead fungal biomass is composed mainly of polysaccharides that can make up 80–90% of the total cell wall, but it also contains lipids and mannoproteins (Baldrian et al., 2013b; Fesel and Zuccaro, 2016; Free, 2013). The main components of the polysaccharide fraction include chitin, a polymer of N-acetylglucosamine units, different types of beta- and alpha-glucans, glucomannans and galactomannans. Dead bacterial biomass is considered to be equally abundant in forest soils, showing higher turnover rates than fungal biomass (Gunina et al., 2017). The composition of cell walls is highly diverse in bacteria (Silhavy et al., 2010). Peptidoglycan (PG), a polymer of N–acetylglucosamine and N-acetylmuramic acid units connected to chains of amino acids, is a major and universal component of bacterial cell walls (Egan et al., 2017; Scheffers and Pinho, 2005). In gram-positive bacteria, PG is densely functionalized with other polymers. Cell-wall glycopolymers such as teichoic, teichuronic and teichulosonic acids, which are attached either to the PG or to membrane lipids, are the most abundant (Brown et al., 2013; Schaffer and Messner, 2005; Weidenmaier and Peschel, 2008). Apart from PG and cell-wall glycopolymers, the bacterial cell wall includes proteins, glycosyl 1-phosphates and other sugar-containing polymers such as arabinogalactan, lipomannan and lipoarabinomannan (Hamedi and Poorinmohammad, 2017). Furthermore, gram-negative bacteria contain lipopolysaccharides and lipoproteins in their outer membranes (Silhavy et al., 2010). Many bacteria also produce a range of chemically different extracellular polysaccharides, which can be utilized as C sources by other microorganisms in soils (Bazaka et al., 2011; Mishra and Jha, 2013; Wang et al., 2015).

The turnover of carbohydrates in plant and microbial biomass can be tracked by analyzing the microbial enzymes that take part in the C turnover—the carbohydrate-active enzymes (CAZymes) (Žifčáková et al., 2017). CAZymes, classified into a hierarchy of families based on their structure and function, act on oligosaccharides, polysaccharides and glycoconjugates (Lombard et al., 2014). Among the CAZYmes, glycoside hydrolases (GHs), which hydrolytically cleave the glycosidic bonds within carbohydrates or between a carbohydrate and a noncarbohydrate moiety, are the most important in decomposition. In this sense, cellulases, β-glucosidases and hemicellulases such as endoxylanases, β-xylosidases, xyloglucanases, endomannanases, mannosidases, fucosidases, and arabinosidases from several GH families are the main enzymes that degrade plant biomass (Bomble et al., 2017). Beside them, lytic polysaccharide monooxygenases (LPMOs), classified as enzymes with auxiliary activities (AA) in the CAZy database, have also been found to play an important role in the degradation of cellulose (Vaaje-Kolstad et al., 2017). In addition, several AA families including peroxidase, oxidoreductase and laccase activities participate either directly or indirectly in the degradation of lignin (Levasseur et al., 2013). Finally, carbohydrate esterases (CEs) from several families participate in the decomposition of hemicelluloses. In the case of fungal biomass, chitinases and N-acetylglucosaminidases from three GH families are involved in the degradation of chitin, and glucanases from several GH families, which degrade glucans, are highlighted as main players involved in its degradation. The lysozymes and PG lytic transglycosylases are important enzymes involved in the degradation of PG in bacterial biomass. Catalytically active CAZYmes may contain carbohydrate-binding modules (CBMs), which are essential for effective hydrolysis because they mediate binding to cellulose, xylan, chitin or other carbohydrates (Donohoe and Resch, 2015). Many GH families include enzymes that are structurally similar but have wider substrate specificity, and associating one family to the degradation of one type of compound is not always easy (Nguyen et al., 2018). Moreover, the complex, diverse and not fully characterized composition of dead biomass in forest soils, especially in the case of fungal and bacterial biomass, may entail the implication of more CAZyme families than those currently proposed.

For a long time, fungi were assumed to be the major decomposers of complex organic matter in forest soils due to their filamentous nature, which allows them to colonize substrates efficiently, their ability to produce a rich battery of extracellular enzymes and their limited requirements of N, which is rather rare in cell wall biopolymers. This assumption led to underestimation of the role of bacteria in decomposition, and bacteria were typically expected to target simple substrates (de Boer et al., 2005; Rousk and Frey, 2015). Different studies have indicated that bacteria play a more important role in the transformation and mineralization of organic matter and contribute significantly to decomposition in forest soils (Eichorst and Kuske, 2012; Štursová et al., 2012; Verastegui et al., 2014). The high percentage of bacteria that potentially decompose cellulose found in forest soil and the high frequency of genes involved in the degradation of structural plant polysaccharides found in bacterial genomes support that the involvement of bacteria in plant biomass decomposition is relatively common (Berlemont and Martiny, 2015; López-Mondéjar et al., 2016a; Wilhelm et al., 2019). In addition, analyses of forest soil metatranscriptomes show significant contribution of bacteria to CAZyme production (Hesse et al., 2015; Lladó et al., 2019; Žifčáková et al., 2017). Moreover, Brabcova et al. (2016) showed that decomposing mycelium in forest soil presents hotspots of bacterial abundance, maintaining bacterial over fungal decomposers. In our previous work, we demonstrated that both fungi and bacteria are involved in the assimilation and mineralization of C from complex sources existing in soil. In addition, we showed that fungi may be better suited for the utilization of plant biomass, whereas most bacteria prefer microbial biomass (López-Mondéjar et al., 2018).

The aim of this study was to describe the enzymatic toolbox used for the decomposition of various biomass types by forest soil bacteria and fungi. To accomplish this, we prepared soil microcosms with the addition of ¹³C-labeled biomass of plant, fungal, and bacterial origin. We used DNA-SIP and metagenomics to analyze the enzymatic tools of microbial decomposers. We hypothesized that although the CAZyme pool will be different for each type of biomass, fungi and bacteria will encode similar CAZyme families involved in the degradation of biomass of the same origin. Additionally, in line with the preference of fungi for plant biomass, we hypothesized that the number of fungal CAZymes involved in the degradation of plant biomass will be higher than the number of those targeting microbial biomass. Importantly, this study also provides a comprehensive answer about the CAZyme families involved in the degradation of various biomass types, including the involvement of minor CAZy families that might have been overlooked so far.