Animal manures are microbe-rich environments in which bacteria constitute the largest fraction (around 70% of the total microbial biomass as assessed by PLFA analysis), with fungi mainly present as spores [24]. Thus, earthworm activity is expected to have a greater effect on bacteria than on fungi in these organic substrates in the short-term [79]. In line with this, a significant increase in the fungal biomass of pig manure, measured as ergosterol content, was detected in a short-term experiment (72 h) with the earthworm species E. fetida, and the effect depended on the density of earthworms [82]. A higher fungal biomass was found at intermediate and high densities of earthworms (50 and 100 earthworms per mesocosm, respectively), which suggests that there may be a threshold density of earthworms at which fungal growth is triggered. This priming effect on fungal populations was also observed in previous short-term experiments in the presence of the epigeic earthworms Eudrilus eugeniae and Lumbricus rubellus fed with pig and horse manure, respectively [16,86]. These contrasting short-term effects on bacterial and fungal populations are thus expected to have important implications on decomposition pathways during vermicomposting because important differences exist between both microbial decomposers related to resources requirements and exploitation [92]. This is based on the fact that fungi can immobilise great quantities of nutrients in their hyphal networks, whereas bacteria are more competitive in the use of readily decomposable compounds and have a more exploitative nutrient use strategy by rapidly using newly produced labile substrates [92].

The above-mentioned studies dealing with the effects of epigeic earthworms on microorganisms have focused on the changes before and after the active phase rather than those that occur throughout the whole vermicomposting process. Hence, in a current research study, and using a continuous-feeding vermicomposting system, we evaluated the different phases of interaction between earthworms and microorganisms and additionally, we monitored the stabilisation of the fresh manure during a period of 250 days. At the end of the experiment we obtained a profile of layers of increasing age, resembling a time profile, with a gradient of fresh-to-processed manure from the top to the bottom. This type of system allowed us to evaluate whether and when the samples reached an optimum value to be classified as vermicompost, as regards to the stabilisation of organic matter and the levels of microbial biomass and activity. Briefly, we used polyethylene reactors (n=5) with a volume of 1 m3, which were initially comprised of a 10 cm layer of mature vermicompost (a stabilised non- toxic substrate that serves as a bed for earthworms), on which earthworms (Eisenia fetida) were placed and a layer containing 5 kg of fresh rabbit manure, which was placed over a plastic mesh (5 mm pore size) to avoid sampling the earthworm bedding. New layers with the same amount of fresh manure were added to the vermireactor every fifty days according to the feeding activity of the earthworm population. This procedure allowed the addition of each layer to be dated within the reactors. The reactors were divided into 4 quadrants and two samples were taken at random from each quadrant with a cylindrical corer (8 cm diameter). Each corer sample was divided into five layers of increasing age and the samples from the same layer and each reactor were gently mixed to analyse the changes in microbial communities. The structure of the microbial communities was assessed by PLFA analysis; some specific PLFAs were used as biomarkers to determine the presence and abundance of specific microbial groups [93]. The sum of PLFAs characteristic of Gram-positive (iso/anteiso branched-chain PLFAs), and Gram-negative bacteria (monounsaturated and cyclopropyl PLFAs) were chosen to represent bacterial PLFAs, and the PLFA 18:2w6c was used as a fungal biomarker. Total microbial activity was also assessed by measuring the rate of evolution of CO2, as modified for [17] for samples with a high organic matter content. Dissolved organic carbon was determined colorimetrically in microplates after moist digestion (K2&2O7 and H2SO4) of aliquots of 0.5M K2SO4 extracts.

The earthworm species E. fetida had a strong effect in the decomposition of organic matter during vermicomposting, greatly modifying the structure of the microbial decomposer communities, as revealed by the phospholipid fatty acid analysis. The principal component analysis of the 27 identified PLFAs (10:0, 12:0, 13:0, 14:0, i14:0, 15:0, i15:0, a15:0, 16:0, i16:0, 17:0, a17:0, 18:0, 14:1w5c, 15:1w5c, 16:1w7c, 17:1w7c, 18:1w7c, 18:1w9c, 18:1w9t, 18:2w6c, 18:2w6t, 18:3w6c, 18:3w3c, cy17:0, cy19:0, 20:0) clearly differentiated between the samples in function of the age of layers, explaining 51% of the variance in the data (Figure 2). Thus, the upper layers (50 and 100 days old) along with the fresh manure were clearly distinguished from the intermediate (150 days old) and lower layers (200 and 250 days old) (Figure 2).

Such changes in the structure of microbial communities were accompanied by a decrease in the abundance of both Gram-positive and — negative bacterial populations with the depth of layers (Figure 3A, B), i. e. from upper to medium and lower layers; and the abundance of these groups were in the fresh rabbit manure 346 ± 49.0 and 336 ± 63 pg g-1 dw for Gram­positive and Gram-negative bacteria, respectively (Figure 3A, B). A similar trend was observed for fungal populations (Figure 3C), reaching an average value of 1.3 ± 0.1 at the end of the process (Figure 3C). These results are in accordance with previous studies based on PLFA profiles, with marked changes in the structure of microbial communities due to decreases in both bacterial and fungal populations throughout the process of vermicomposting [18, 89]. Recently, Fernandez-Gomez et al. [94] observed that the structure of fungal communities, assessed by DGGE profiles differed at the stage of maximum earthworm biomass the most, suggesting the existence of a strong gut passage effect on the microbial communities through a continuous-feeding vermicomposting system in the presence of E. fetida.

Decreases in microbial activity were also detected with depth of layer (Figure 4A) and, after a maturation period for 250 d, basal respiration values dropped below 100 mg CO2 kg-1 OM h-1 (Figure 4A), as previously shown by [18]. Accordingly, a reduction in the dissolved organic carbon content was detected from upper to lower layers (Figure 4B), reaching a value close to 7000 pg g-1 dw after 250 d of vermicomposting. In contrast, other authors [17] reported levels of DOC much more lower in a long-term experiment (252 days) with the epigeic earthworm E. fetida, with values below 1500 pg g-1 dw in the presence of earthworms. Such differences could be due to the composition of the parent material (pig slurry versus rabbit manure) and/or to the experimental setup conditions. Unlike compost — a limit value of 4000 mg kg-1 is suggested for a stable compost according to [34]- there is still no threshold level of DOC for which vermicompost is to be considered stable.

Overall, in the present study a higher degree of stabilisation was reached in the rabbit manure after a period of between 200 and 250 days, as indicated by the lower values of microbial biomass and activity that are indicative of stabilized materials. These results underscore the potential of epigeic earthworms in the stabilisation of this type of organic substrates, which is of great importance for the application of animal manures as organic amendments into agricultural soils because, as already mentioned, it is widely recognised that the overproduction of this type of substrate has led to inappropriate disposal practices, which may result in severe risks to the environment [6]. Furthermore, these findings constitute a powerful tool for the development of strategies leading to a more efficient process for the disposal and/or management of animal manures, thereby highlighting the continuous-feeding vermicomposting system as an environmentally sound management option for recycling such organic wastes, as previously reported by [94] for treating tomato — fruit waste from greenhouses. Ultimately, it should be borne in mind that the functioning of this type of reactors can lead to the gradual accumulation of layers and compaction of the substrate, thus minimizing earthworm — induced aeration, which can promote pathogen survival [89].