Priming effect was influenced by quantity and C/N ratio of added exudates
The resulting priming effect on peatland DOC decomposition was primarily affected by the exudate input: the low level of root exudatesto peatland water resulted in either a slightly negative (for C/N ratio 7) or no PE (for C/N ratio 25 and 50) on pre-existing peatland DOC (Fig 1c), whereas the higher level of root exudates a positive PEincreasing exudate C/N ratio from 7 to 50 (Fig 1d). Our results are in line with findings of [17] who found that low level of C addition lead to a negative or no PE during 7 week incubation, while the high level of C addition induced positive priming and of [40], who observed an increasing PE with an increasing C/N ratio of crop residues added to the soil.
Decomposition and microbial community dynamics after root exudates input
The priming effect was a dynamic process, closely related to the growth and succession of the present microbial community. The growth and the composition of the microbial communities in the samples were primarily controlled by the level of root exudates addition. While the low level of exudate addition enabled only limited microbial growth, the high exudate addition level resulted in a significant temporary increase in microbial, especially bacterial abundance. Therefore, the related growth nutrient demand and a capacity to produce extracellular enzymes and decompose the native DOC were larger after the high exudate input. The rapidly growing communities first took up the available nutrients, as shown by a pronounced depletion of ammonia and SRP from the incubated solutions. Later, the decomposition of native DOC increased in samples enriched by high exudate level as a result of co-metabolism and/or a targeted nutrient mining [21,23].
The two distinct phases of exudate and DOC utilization were connected with a significant successional development of bacterial community size (Fig. 4) and composition, similarly as observed by [18]. The highest shift in the bacterial community composition occurred between 4th and 11th day (Fig. 6). We compare the two stages (early stage and late stage) varying in exudate and DOC decomposition, microbial abundance and community composition in more detail below.
The early stage – a preferential use of exudates and negative PE
The early stage of incubation immediately following the exudate addition (till day 4) was characteristic by a preferential use of the added compounds by the microbial community, resulting in a strong negative PE (Fig. 1). The exudate addition induced transitional bacterial growth connected with a rapid uptake of SRP and inorganic N from the solution and subsequent depletion of simple exudates from the solution. The r-strategic bacteria, mainly the representatives of Gammaproteobacteria and Bacteriodetes, enhanced their relative abundance within the community. These are known as copiotrophic bacteria with a rapid turnover [41], which are often stimulated by the presence of labile substrates like root exudates [42–44]. The highest enrichment was observed for Burkholderia, Pseudomonas (Proteobacteria) and Mucilaginibacter (Bacteroidetes), which are typical by their large metabolic versatility [45,46]. Pseudomonas and Burkholderia are able to degrade complex aromatic compounds including lignin and other phenolics [47] and Pseudomonas also produce chitinolytic enzymes [48]. Pseudomonas belongs among the most efficient phosphate solubilizers due to production of organic acids [49] and can facilitate iron uptake of plants by producing pyoverdines [50]. Therefore, Pseudomonas could facilitate nutrients (mainly P) uptake by plants, especially in nutrient limited environment like peatlands. Mucilaginibacter is also able to degrade complex biopolymers like cellulose [51].
In accordance with the observed community enrichment by the efficient decomposers, our functional analysis showed a large potential of the present bacterial community to degrade complex organic compounds, including cellulose and aromatics. We therefore suggest that this community dominated by the r-strategic taxa with a large decomposition potential largely contributes to the positive PE, which was observed at 11th day (Fig. 2). This partly contradicts a classical understanding of the PE mechanism, which suggests that the main role in production of exoenzymes degrading complex SOC is played by K-strategists, which follow in the succession, when the r-strategic community dies after a depletion of simple substrates [27].
Because the largest PE occurred in the samples enriched by the high level of exudates (5% of pre-existing DOC) of the C/N ratio of 50, we focused on the specific bacterial taxa responding significantly under this treatment. We found that a facultative methanotroph Methylocella was enriched under these conditions (Table S3). Methylocella can grow on various simple C compounds including acetate, succinate, malate etc. [52]. It is able to fix atmospheric N2 [53,54], which may favour Methylocella species under N limiting conditions. Similarly, [55] found another methylotrophic and putative N2 fixing bacteria Methylobacterium enriched in treatments with high C/N ratio. Additionally, methanotrophs produce a unique enzyme methane monooxygenase (mmo). Monooxygenases produced by other bacteria are known to degrade aromatics. It was thus suggested that methanotrophs might also contribute to their degradation [56], although there has been no evidence that either Methylocella or Methylobacterium are capable of complex aromatic compounds degradation up to date [57].
The later stage of the incubation – shift from r- to K-strategy after exudate depletion
Exudate depletion from the solution occurred before day 4 as indicated by a sharp decrease in microbial respiration rates (Fig. 1) and was followed by a decline of bacterial abundance between days 4 and 11 mainly. Consequently, the large original community of r-strategists was replaced by a new, smaller community composed of different groups of bacteria mostly belonging to Alphaproteobacteria and Acidobacteria. Acidobacteria in general are reported as the dominant microbial group in peatlands because they prefer low pH and oligotrophic conditions [58,59]. Members of Acidobacteria are also efficient cellulose decomposers [45,51,60,61] and their high abundance in microbial community may drive the litter degradation in acidic Sphagnum peat [62]. From Acidobacteria, the Bryocella species, enriched specifically at day 11, was shown to have high enzymatic activity [45] and thus may contribute to the significant positive PE observed at day 11 (Fig. 2). Another Acidobacteria, Candidatus Solibacter, was one of a few genera enriched under high C/N ratio at the end of the incubation (day 25). This genus is capable of cellulose, hemicellulose and chitin degradation and may contribute to the DOC decomposition in the later stage of incubation. We thus suggest that the enhanced DOC decomposition and the observed positive PE at days 11 and 25 can be attributed to a mixture of enzymes produced by both the r-strategists activated after exudate addition and by later incoming K-strategists. In the samples amended by the high level of exudates with C/N ratio of 50, where the microbial growth was the most dynamic, the community was further enriched in anaerobes in the late stage of the incubation (25th day). This may relate to the largest die back of the community grown on exudates and a consequent release of simple compounds during its necromass decomposition [63].
Except of the above mentioned heterotrophic bacteria, the community characteristic for the later stage of incubation, specifically for the 11th day, was further enriched in chemolithotrophic ammonia oxidizers (Fig. 9). Their larger presence was likely enabled by a depletion of simple organic substrates and a die-back of fast-growing bacteria, which could otherwise over compete these slow growing microbes. Moreover, the biomass die-back and the DOC decomposition enhanced the availability of NH4+, which is used in their energetic metabolism.
Upscale to ecosystem level (a potential role of plant exudates in stimulation of peatland DOC decomposition in situ)
In the study [26] was estimated that root exudates of peatland vascular plants, which were easily degradable, could contribute from 1% up to 5% to the pre-existing peatland DOC in situ. Our results evoke that a lower input of root exudates, achieving around 2% of DOC has a significant effect on composition of rhizosphere microbial community, but is insufficient to induce a significant positive PE on recalcitrant peatland DOC. However, when the exudate input increases to the level of 5% of the present DOC, it may induce a transient positive priming effect lasting several days. With exudates poor for N (C/N ratio of 50), the induced positive priming may persist for more than two weeks and result in an enhanced decomposition of the pre-existing DOC. According to [26], the situation, when the exudation input is high enough, occurs at the top of the season especially in the presence of graminoid species such as Eriophorum vaginatum. However, the exudates at that time were relatively rich in N, therefore likely not causing significant peatland DOC losses. We suggest that the plants may rather benefit from the changes in the composition of microbial community, which the exudates induce in their rhizosphere. According to our results, root exudates input supports a growth of r-strategic species, which are able to immobilize high amounts of nutrients in their biomass, keep them in the vicinity of plant roots, protect them from losses with the leaching DOC and potentially release them for the plant uptake during their fast turnover. The rhizosphere community is enriched in species like Pseudomonas, which can mobilize P and others (e.g. Burkholderia and Mucilaginibacter) with high metabolic potential. The presence of microbial communities able to keep nutrients in the rhizosphere of peatland vascular plants is further supported by our previous results from the field. In [64] we showed that the soil microbial biomass associated with Eriophorum vaginatum and Vaccinium myrtillus immobilizes large amounts of N and P present in the system. Additionally, the association of ericaceous shrubs with mycorrhizal fungi increases P mobilization and increases its availability to the plants (Read et al. 2004).
Differently from root exudates of vascular plants, Sphagnum “exudates” is not expected to cause a significant positive priming effect on DOC decomposition. Although the Sphagnum-released compounds can contribute up to 20% to the peatland DOC, they are of low degradability being only around 15% [26]. We expect that these would not stimulate a growth of specific bacterial communities and their enzymatic production. Additionally, the compounds leached from Sphagnum were shown to immobilize phosphorus by its incorporation to the high molecular weight complexes and by co-precipitation with metals [65] and they are known by their antimicrobial effects [66].
In accordance with the study of [8], we suggest that an input of root exudates from vascular plants may induce a positive PE on organic matter decomposition in the peatlands, but its importance for C transformation and ecosystem C balance is likely minor under current conditions. Current level of root exudation rather helps vascular plants to keep nutrients immobilized in the microbial biomass with fast turnover in the vicinity of the roots and thus facilitates their survival in the nutrient limited environment. However, if ongoing climate changes will result in a significant spread of vascular plants over peatlands, a priming effect caused by the enhanced root exudation could lead to higher dynamics of C cycle in peatlands and larger C mineralization.