Research studies focused on the stabilization of animal manures through the aerobic processes of composting and vermicomposting.
Many environmental problems of current concern are due to the high production and local accumulations of organic wastes that are too great for the basic degradation processes inherent in nature. With adequate application rates, animal manure constitutes a valuable resource as a soil fertilizer, as it provides a high content of macro- and micronutrients for crop growth and represents a low-cost, environmentally- friendly alternative to mineral fertilizers . However, the intensification of animal husbandry has resulted in an increase in the production of manure - over 1500 million tonnes are produced yearly in the EU-27  as reported by Holm-Nielsen et al. - that need to be efficiently recycled due to the environmental problems associated with their indiscriminate and untimely application to agricultural fields. The potentially adverse effects of such indiscriminate applications include an excessive input of harmful trace metals, inorganic salts and pathogens; increased nutrient loss, mainly nitrogen and phosphorus, from soils through leaching, erosion and runoff-caused by a lack of consideration of the nutrient requirements of crops; and the gaseous emissions of odours, hydrogen sulphide, ammonia and other toxic gases . In fact, the agricultural contribution to total greenhouse gas emissions is around 10%, with livestock playing a key role through methane emission from enteric fermentation and through manure production. More specifically, around 65% of anthropogenic N2O and 64% of anthropogenic NH3 emissions come from the worldwide animal production sector .
The introduction of appropriate management technologies could thus mitigate the health and environmental risks associated with the overproduction of organic wastes derived from the livestock industry by stabilizing them before their use or disposal. Stabilisation involves the decomposition of an organic material to the extent of eliminating the hazards and is normally reflected by decreases in microbial biomass and its activity and in concentrations of labile compounds . Composting and vermicomposting have become two of the best-known environmentally appropriate technologies for the recycling of manures under aerobic conditions [6-7], by transforming them into safer and more stabilised products (compost and vermicompost) with benefits for both agriculture and the environment. Unlike composting, vermicomposting depends on the joint action between earthworms and microorganisms and does not involve a thermophilic phase . However, more than a century had to pass before vermicomposting was truly considered a field of scientific knowledge or even a real technology, despite Darwin  having already highlighted the important role of earthworms in the decomposition of dead plants and the release of nutrients from them.
Although microbial degradation under oxygen is usually faster and, as such aerobic processes are thermodynamically more favorable than anaerobic processes, in recent years, anaerobic digestion (AD) has become an upcoming technology for the treatment of animal manures [3, 10-13]. On the one hand, pretreatment of manure by anaerobic digestion can involve some advantages including malodor reduction, decreased biochemical oxygen demand, pathogen control, along with a reduction in the net global warming potential of the manure [4,14]. AD reduces the risk of water pollution associated with animal manure slurries (i.e., eutrophication) by removing 0.80–0.90 of soluble chemical oxygen demand and it improves human/farm cohabitation in rural regions by reducing odor emissions by 70–95% . This process has other direct advantages beyond these, which are related to biogas production for renewable energy and the enrichment of mineral fractions of N and P during digestion [4,10], resulting in a more balanced nutrient mix and increased nutrient bioavalability for plants compared with undigested manure .
Therefore, the purpose of this chapter is to give an overview of the three major management technologies of manure recycling, including the aerobic processes of composting and vermicomposting and the anaerobic digestion for biogas production. The main changes that occur in the substrate from a chemical and microbial viewpoint during the specific phases of each degradation process are addressed, as such changes determine the degree of stability of the end product and in turn its safe use as an organic amendment. Different methods that have been proposed to evaluate compost stability are summarised. Also, the influence of the end products derived from each process on the soil microbiota and disease suppressiveness are discussed.
2. Aerobic degradation: Composting and vermicomposting processes
Under aerobic conditions, the degradation of organic matter is an exothermic process during which oxygen acts as a terminal electron acceptor and the organic materials are transformed into more stable products, carbon dioxide and water are released, and heat is evolved. Under field conditions, aerobic degradation takes place slowly at the soil surface, without reaching high temperatures; but this natural breakdown process can be accelerated by heaping the material into windrows to avoid heat losses and thus allowing for temperature increases (composting) or by using specific species of earthworms as agents for turning, fragmentation, and aeration (vermicomposting). Although both aerobic processes, composting and vermicomposting, have been widely used for processing different types of animal manure either separately or in combination with each other (see Table 1), most of the studies are not comparable mainly due to differences in the applied experimental designs, parent material, earthworm species, as well as the length of the experiments and the parameters used for analysis, among others. Despite these limitations, all these findings have largely contributed to better understand the changes that the material undergoes during these biological stabilisation processes, which is of great importance for their optimisation, and ultimately to obtain a high quality final product. In line with this, certain chemical characteristics of the animal manures can limit the efficiency of these processes, such as an excess of moisture, low porosity, a high N concentration in relation to the organic C content or high pH values . Therefore, different aeration strategies, substrate conditioning-feedstock formulation, bulking agents and process control options have been considered in manure composting and vermicomposting so as to reduce the time and costs of both processes and enhance the quality of the end-products [6-7].
2.1. The composting process
Composting is defined as a bio-oxidative process involving the mineralization and partial humification of the organic matter, leading to a stabilised final product, free of phytotoxicity and pathogens and with certain humic properties, which can be used to improve and maintain soil quality and fertility . Composting of animal manures has been traditionally carried out by the farmers after manure collection for better handling, transport and management . Frequently, the wastes were heaped up and very little attention was paid to the process conditions (aeration, temperature, ammonia loss, etc.) and using rudimentary methodology.
From a microbial viewpoint continuous composting processes may be described as a sequence of continuous cultures, each of them with their own physical (temperature), chemical (the available substrate), and biological (i.e., the microbial community composition) properties and feedback effects. These changes make it difficult to study the process, which is virtually impossible to simulate in the laboratory since temperature, moisture, aeration, etc., are directly related to the surface/volume ratio. However, in general, composting may be described as a four-phase process in which the energy-rich, abundant and easily degradable compounds like sugars and proteins are degraded by fungi and bacteria (referred to as primary decomposers) during an initial phase called the
When a temperature exceeding 55 ºC is reached in a compost pile, fungal growth is usually inhibited and the thermophilic bacteria and Actinobacteria are the main degraders during this peak-heating phase. Moreover, oxygen supply affects fungi to a greater extent than bacteria, and even in force-aerated systems, temporary anoxic conditions may occur. Hence, fungi play a negligible role during this phase, except for the composting of lignocellulosic residues. Bacteria of the genus
The final temperature increase may exceed 80 ºC and it is mainly due to the effect of abiotic exothermic reactions in which temperature-stable enzymes of Actinobacteria might be involved. Such high temperatures are crucial for compost hygienisation in order to destroy human and plant pathogens, and kill weed seeds and insect larvae . The disadvantage of temperatures exceeding 70 ºC is that most mesophiles are killed, and therefore the recovery of the decomposer community is retarded after the temperature peak. The inoculation with matter from the first mesophilic stage might, however, solve this problem.
When the activity of thermophilic organisms ceases due to the exhaustion of substrates, the temperature starts to decrease. This constitutes the beginning of the third stage of composting, called the
2.1.1. Evaluation of compost stability and maturity
The stability and maturity of compost is essential for its successful application, particularly for composts used in high value horticultural crops . Both terms are usually used interchangeably to describe the degree of decomposition and transformation of the organic matter in compost , despite the fact that they describe different properties of the composting substrate. Stability is strongly related to the degree to which composts have been decomposed to more stable organic materials . Unstable compost, in contrast, contains a high proportion of biodegradable matter that may sustain a high microbial activity . Typically, compost stability is evaluated by different respirometric measurements and/or by studying the transformations in the chemical characteristics of compost organic matter . On the other hand, compost maturity generally refers to the degree of decomposition of phytotoxic organic substances produced during the active composting stage and to the absence of pathogens and viable weed seeds . This property is often characterised by germination indexes  and/or by nitrification  and has been related to the degree of compost humification. Wu et al.  reported that the low CO2 evolution is not always an indicator of a non-phytotoxic compost, which suggests that a stable compost may not always be at a level of maturity suitable for its use as a growing medium for certain species of plant.
Several authors highlight that there is no one single method that can be applied successfully for determining compost stability mainly due to the wide range of raw materials used to produce compost, as reviewed by . Therefore, the integration of different parameters seems to be the most reliable option for evaluating the stability/maturity stage of composted materials. For instance, physical parameters including temperature, odor and color constitute a very simple and rapid method for stability evaluation, giving a general idea of the decomposition stage reached; however, little information is achieved as regards to the degree of maturation. In addition, chemical parameters including pH, electrical conductivity, cation exchange capacity (CEC), the ratios of C to N and NH4+ to NO3- and humification parameters have also been widely used as indicators of stability . Nevertheless, several drawbacks have been found regarding these parameters, thereby preventing their use as accurate indicators. According to Wu et al. , pH and electrical conductivity may be used to monitor compost stabilisation, as long as the source waste composition is relatively consistent and other stability tests are conducted. Moreover, Namkoong et al.  established that the C to N ratio could not be considered as a reliable index of compost stability, as it changed irregularly with time. In fact, when wastes rich in nitrogen are used as the source material for composting, like sewage sludges or manures, the C to N ratio can be within the values of a stable compost even though it may still be unstable. Zmora-Nahum et al.  reported a C to N ratio lower than the cutoff value of 15 very early during the composting of cattle manure, while important stabilisation processes were still taking place. The increase in CEC with composting time is related to the formation of carboxyl and phenolic functional groups during the humification processes; however, the wide variation in CEC values among the initial substrates prevent to establish a threshold level and to use it as a stability indicator .
Biological parameters such as respiration rates (CO2 evolution rate and/or O2 uptake rate) and enzyme activities have been proposed to measure compost stability [6-39]. The principle of the respirometric tests is that unstable compost has a strong demand for O2 and high CO2 production rates as a consequence of the intensive microbial development due to the presence of easily biodegradable compounds in the raw material. Then, as composting proceeds, the decrease in the amount of degradable organic matter is accompanied by a decline in both O2 and CO2 respirometry. The Solvita test, which measures CO2 evolution and ammonia emissions simultaneously have been found to be a simple and easily used procedure for quantifying soil microbial activity in comparison with both titration and infrared gas analysis ; this test has also been used for determining the stability degree in diverse composts . Enzymatic activities have also been found suitable as indicators of the state and evolution of the organic matter during composting, as they are implicated in the biological and biochemical processes through which the initial organic substrates are transformed (Tiquia, 2005). Important enzymes during composting are related to the C-cycle (cellulases, β-glucosidase, β-galactosidase), the N-cycle (protease, urease, amidase) and/or the P-cycle (phosphatase) . These latter authors established that the formation of a stable enzymatic complex, either in moist or air-dried compost samples, could represent a useful index of stabilisation. Additionally, enzymatic activities, especially dehydrogenases, are considered easy, quick and cheap stability measurements ; however, the wide range of organic substrates involved in the composting process makes it difficult to establish general threshold values for these parameters. The hydrolysis of fluorescein diacetate (FDA), which is a colourless fluorescein conjugated that is hydrolyzed by both free (exoenzymes) and membrane bound enzymes , has been suggested as a valid parameter for measuring the degree of biological stability of the composting material, as it showed a good correlation with other important stability indexes . The analysis of phospholipid fatty acid (PLFA) composition has also been proposed for determining compost stability [47-48]. These authors found a positive correlation between the proportion of PLFA biomarkers for Gram-positive bacteria and the germination index during the maturation of composting of poultry manure and cattle manure, respectively. The strength of this lipid-based approach, as compared to other microbial community assays, is that PLFAs are rapidly synthesized during microbial growth and quickly degraded upon microbial death and they are not found in storage molecules, thereby providing an accurate ‘fingerprint’ of the current living community . The potential ability of the microbial community to utilise select carbon sources by determining the community-level physiological profiles (CLPPs) with the Biolog® Ecoplate has also been considered for compost stability testing . The principle is that compost extracts are inoculated onto microtiter plates that contain 31 different C substrates [51-52]. Ultimately, molecular techniques are becoming increasingly useful in composting research. For example, as in reference  the authors used three different cultivation-independent techniques based on 16S rRNA gene sequences, i.e. PCR-denaturing gradient gel electrophoresis (DGGE), clone libraries, and an oligonucleotide microarray (COMPOCHIP), in order to evaluate the dynamics of microbial communities during the compost-curing phase. Specific compost-targeted microarrays are suitable to investigate bacterial [53-54] and fungal community patterns , including plant growth promoting organisms and plant and human pathogens.
2.1.2. Influence of compost amendments on the soil microbiota
Composted materials have gained a wide acceptance as organic amendments in sustainable agriculture, as they have been shown to provide numerous benefits whereby they increase soil organic matter levels, improve soil physical properties (increased porosity and aggregate stability and reduced bulk density) and modify soil microbial communities . Substantial evidence indicates that the use of compost amendments typically promotes an increase in soil microbial biomass and activity, as reviewed in [56-57]. This enhancing effect may be attributed to the input of microbial biomass as part of the amendments ; however, the quantity of organic matter applied with the compost is very small in comparison with the total organic matter present in the soil and, in turn it is believed that the major cause is the activation of the indigenous soil microbiota by the supply of C-rich organic compounds contained in the composting materials . Such effects on microbial communities were reported to be dependent on the feedstocks used in the process . However, other authors did not find significant differences between soil plots that had been amended with four different compost types (green manure compost, organic waste compost, manure compost and sewage sludge compost) over 15 years . Similarly, Ros et al.  observed that different types of composts had a similar effect on the fungal community and microbial biomass in soil in a long-term field experiment. This fact suggests that the soil itself influences the community diversity more strongly than the compost treatments. Such discrepancies between the previous findings may be due to differences in soil properties, land-use and compost type (i.e., different starting material and process parameters), frequency and dose of application, length of the experiment and parameters chosen for analysis, among others.
Furthermore, C addition to soil seems to select for specific microbial groups that feed primarily on organic compounds. Therefore, it can be expected that the addition of organic amendments not only increases the size of the microbial community but also changes its composition, as has already been observed in previous experiments [61-63]. As shown by  higher amounts of composts resulted in a more pronounced and faster effect in the structure of microbial communities, as revealed by PLFA analysis, indicating that the compost application rate is a major factor regarding the impact of compost amendments on soil microbiota. Carrera et al.  found that soil PLFA profiles were influenced by both the treatment with poultry manure compost and the sampling date. Ros et al.  observed that the date of sampling contributed more to modifications in fungal community structure, assessed by PCR-DGGE analysis, than treatment effects. However, in contrast to fungi, the bacterial community structure, both on the universal and the Streptomycetes group-specific level, were influenced by compost amendments, especially the combined compost and mineral fertilisers treatments. This seems reasonable, as bacteria have a much shorter turnover time than fungi and can react faster to the environmental changes in soil. Bacterial growth is often limited by the lack of readily available C substrates, even in soils with a high C/N ratio, and are the first group of microorganisms to assimilate most of the readily available organic substrates after they are added to the soil . CLPP profiles have also been used to evaluate the impact of compost amendments on the potential functional diversity of soil microbiota, as they are considered suitable indicators for detecting soil management changes . As shown by  different types of compost (household solid waste compost and manure compost) affected differently the substrate utilization patterns of the soil microbial community relative to unamended control soils. Contrarily, no significant changes in CLPP profiles were found by . Other authors also reported that the sampling date had more weight on CLPP results than compost treatments [63,67]. All these studies together highlight the importance of a multi-parameter approach for determining the influence of compost amendments on the soil microbiota, which is of utmost importance to understand the disease suppressive activity of compost and the mechanisms involved in such suppression .
Since the 1980s a large number of experiments have been addressed describing a wide array of pathosystems and composts from a broad variety of raw materials. Interestingly, Noble and Coventry  evaluated the suppression of soilborne plant pathogens by compost in both laboratory and field scale experiments. In general, they found that the effects in the field were smaller and more variable than those observed at lab-scale. Termorshuizen et al.  compared the effectiveness of 18 different composts on seven pathosystems and interestingly they found significant disease suppression in 54% of the cases, whereas only 3% of the cases showed significant disease enhancement. They highlighted that the different composts did not affect the pathogens in the same way and that no single compost was found to be effective against all the pathogens. Furthermore, in a study carried out with 100 composts produced from various substrates under various process conditions, it was found that those composts that had undergone some anaerobic phase showed the best results in terms of suppressing plant disease .
However, up to now, there is still a general lack of understanding concerning the suppressivity of compost , as it depends on a complex range of abiotic and biotic factors. Such factors are reviewed by . Briefly, the main mechanisms by which compost amendments exert their suppressivity effect against soil-borne plant pathogens include hyperparasitism; antibiosis; competition for nutrients (carbon and/or iron); and induced systemic resistance in the host plant . The first three affect the pathogen directly and reduce its survival, whilst the latter one acts indirectly via the plant and affect the disease cycle.
Two mechanisms of biological control, based on antibiosis, hyperparasitism, competition and induced protection, have been reported for compost amendments. On the one hand, diseases caused by plant pathogens such as
The addition of antagonistic microorganisms to compost is also a promising technique to improve its suppressivity. Already in 1983, Nelson et al.  increased the suppressive potential of compost by adding selected
2.2. The vermicomposting process
Vermicomposting is defined as a bio-oxidative process in which detritivore earthworms interact intensively with microorganisms and other fauna within the decomposer community, accelerating the stabilization of organic matter and greatly modifying its physical and biochemical properties . Epigeic earthworms with their natural ability to colonize organic wastes; high rates of consumption, digestion and assimilation of organic matter; tolerance to a wide range of environmental factors; short life cycles, high reproductive rates, and endurance and resistance to handling show good potential for vermicomposting . The earthworm species
Vermicomposting systems sustain a complex food web that results in the recycling of organic matter and release of nutrients . Biotic interactions between decomposers (i.e., bacteria and fungi) and earthworms include competition, mutualism, predation and facilitation, and the rapid changes that occur in both functional diversity and in substrate quality are the main properties of these systems . The biochemical decomposition of organic matter is primarily accomplished by the microbes, but earthworms are crucial drivers of the process as they may affect microbial decomposer activity by grazing directly on microorganisms [78-79], and by increasing the surface area available for microbial attack after the comminution of organic matter . Furthermore, earthworms are known to excrete large amounts of casts, which are difficult to separate from the ingested substrate . The contact between worm-worked and unworked material may thus affect the decomposition rates , due to the presence of microbial communities in earthworm casts different from those contained in the material prior to ingestion . In addition, the nutrient content of the egested materials differ from that in the ingested material , which may enable better exploitation of resources, because of the presence of a pool of readily assimilable compounds in the earthworm casts. Therefore, the decaying organic matter in vermicomposting systems is a spatially and temporally heterogeneous matrix of organic resources with contrasting qualities that result from the different rates of degradation that occur during decomposition .
The impact of earthworms on the decomposition of organic waste during the vermicomposting process is initially due to
Overall, vermicomposting includes two different phases regarding earthworm activity: (i) an
Collectively, the aforementioned studies highlight the importance of monitoring the changes in microbial communities during vermicomposting, because if the earthworms were to stimulate or depress microbiota or modify the structure and activity of microbial communities, they would have different effects on the decomposition rate of organic matter, thereby influencing the vermicompost properties, which is critical to guarantee a safe use of this end-product as an organic amendment and thus benefit both agriculture and the environment.
2.2.1. Effects of earthworms on microbial communities during vermicomposting: a case study.
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 . Thus, earthworm activity is expected to have a greater effect on bacteria than on fungi in these organic substrates in the short-term . 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
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 (
The earthworm species
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 µg g-1 dw for Gram-positive 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, Fernández-Gómez et al.  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
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 . Accordingly, a reduction in the dissolved organic carbon content was detected from upper to lower layers (Figure 4B), reaching a value close to 7000 µg g-1 dw after 250 d of vermicomposting. In contrast, other authors  reported levels of DOC much more lower in a long-term experiment (252 days) with the epigeic earthworm
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 . 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  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 .
2.2.2. Influence of vermicompost amendments on the soil microbiota
As occurred with compost amendments, vermicompost has also been found to provide manifold benefits when used as a total or partial substitute for mineral fertiliser in peat-based artificial greenhouse potting media and as a soil amendment in field studies . Among the advantages of vermicompost as a soil amendment is its potential to maintain soil organic matter, foster nutrient availability, suppress plant diseases and increase soil microbial abundance and activity. However, although several studies have tried to disentangle the complex interactions between vermicompost application and soil microbial properties, most of them are frequently not comparable to each other due to differences in the experimental design, the land-use and vermicompost type (i.e., different starting material and earthworm species), the dose of application as well as the duration of experiments, among others. Despite these limitations, some recent findings have been made, thereby contributing to better understand whether and to what extent vermicompost amendments affect soil microbial biomass, activity and community structure. For instance, Arancon et al.  observed that a single application of vermicompost to a strawberry crop resulted in a significantly higher increase in soil microbial biomass than the application of an inorganic fertiliser, regardless of the dose used. Increases in the microbial activity and in the activity of the soil enzymes involved in the release of the main plant macronutrients with vermicompost amendments, have also been signalled in several studies [96-98]. Such increase could be due to the fact that soil microorganisms degrade organic matter through the production of a variety of extracellular enzymes and, in turn an input of organic matter is expected to be accompanied by a higher enzymatic activity. Moreover, the added material may contain intra- and extracellular enzymes and may also stimulate microbial activity in the soil . Additionally, vermicompost has been found to promote the establishment of a specific microbial community in the rhizosphere different from that of plants supplemented with mineral fertilisers or other types of organic fertilisers such as manure . Inorganic fertilisation only supplies N, P and K, whereas organic fertilisers also supply different amounts of C and macro- and micronutrients, which can select for microbial communities with different nutritional requirements . Moreover, microbial communities in vermicompost are metabolically more diverse than those in manure , and may be incorporated, at least in the short-term, to soils . Interestingly, Aira et al.  observed that the effect of the addition of vermicompost occurred despite the low dose used (25% of total fertilisation), and despite the short duration of the experiment (four months). Jack et al.  also examined how different organic transplant media amendments, including vermicompost, thermogenic compost and industry standard amendments affected the rhizosphere bacterial communities of organically produced tomato plants. They found differences in the bacterial community structure between the different amendments and these differences persisted for at least one month after seedlings were transplanted to the field. Since both compost and vermicompost were made from the same parent material, such differences could be due to the way in which the organic matter was processed prior to the amendment . Previous comparisons between vermicompost and compost with respect to microbial communities [103-105] are difficult to interpret because different feedstocks were used for each process. Compost feedstocks are known to alter the material’s effects on the structure of the microbial communities , so it is essential to use composts made from the same feedstock in order to draw valid comparisons between the two biological processes. Furthermore, it may be expected that different hybrids or plant genotypes will respond differently to vermicompost, considering that plant genotype determines important differences in nutrient uptake capacity, nutrient use efficiency and resource allocation within the plant. Different genotypes may therefore enhance root growth or modify root exudation patterns in order to increase nutrient uptake , and all of these strategies will determine the establishment of different interactions with the microbial communities at the rhizosphere level. Indeed, after the application of vermicompost to sweet corn crops, these authors found important differences in the rhizosphere microbial community of two genotypes from cultivars of maize, with the sugary endosperm mutation (
Furthermore, recent studies have demonstrated the presence of various bacteria, which are useful for different biotechnological purposes, in diverse vermicomposts [106-107], reinforcing that the biological component (i.e., the microbial community composition) of a vermicompost largely determines its usefulness in agriculture and other applications, such as soil restoration and bioremediation. For instance, Fernández-Gómez et al.  detected the presence of
3. Anaerobic digestion
The process of anaerobic digestion (AD) has been extensively studied in natural and engineered ecosystems for more than a century. In natural habits, the anaerobic degradation of organic matter takes place in sediments, waterlogged soils and animal intestinal tracts, in which the oxygen access is restricted; whilst in engineered environments it refers to the biotechnological process by which organic matter (i.e., organic waste, wastewater and/or a renewable resource) is degraded in the absence of oxygen for the commercial production of biogas that can be used as an eco-friendly energy source , thereby representing an important asset in times of decreasing fossil fuel supplies. According to , the anaerobic digestion from swine, bovine and poultry slurries resulted in the production of biogas at average rates of 0.30, 0.25 and 0.48 L/g volatile solids, respectively. Another valuable co-product derived from this process is the anaerobic digested slurry, which can be applied as an organic amendment into soil either in agricultural and non-agricultural lands (section 3.1). It is for this reason along with the production of biogas and the reduction in greenhouse gas emissions  that anaerobic digestion is becoming increasingly popular as a methodological alternative for manure recycling, which in turn has increased the number of farm-scale anaerobic bioreactors up to 4200 in central and northern Europe .
Bacteria represent over 80% of the total diversity in anaerobic digesters , and they are mainly composed by the phyla Firmicutes, Proteobacteria and Bacteroidetes; whereas most of the archaeal representatives belong to the phylum Euryarchaeota, which includes all the known methanogens. Anaerobic eukaryotes - particularly, fungi and protozoa- have received less attention probably because they are slower growers than bacteria and, as such, their abundance is lower in anaerobic reactors. As occurs with composting, anaerobic digestion may be described as a four-phase microbiologically driven process. Briefly, the first and rate limiting step of the anaerobic food chain is the
The monomeric compounds released after the hydrolysis of biopolymers can be taken up by microbial cells, in which they are either fermented or anaerobically oxidised into alcohols, short-chain fatty acids, CO2 and molecular hydrogen (H2). This step is known as
3.1. Influence of anaerobic digested slurry on the soil microbiota
The changes that occur in the parent waste during the process of anaerobic digestion largely depend on the dynamics of the abovementioned microbial groups, ultimately influencing the quality of the final products, i.e. biogas and anaerobic digested slurry. As mentioned before, anaerobic slurries are rich in partially stable organic carbon and can be used as organic amendments for crop production. However so far, many environmental issues relevant when these co-products are applied to agricultural land still have to be studied, especially those related to the impact of the anaerobic digested slurry on the soil microbiota. In a recent work, Walsh et al.  observed that its application affected the fungal and bacterial growth in a very similar way to the application of mineral fertilizers in a 16- week greenhouse experiment. They found a pronounced shift towards a bacterial dominated microbial decomposer community, and such effects were consistent in different soils and different crop types. They conclude that mineral fertiliser could be thus exchanged for anaerobic digested slurry with limited impact on the actively growing soil microbial community, which is of great importance in the regulation of soil processes and consequently in soil fertility and crop yield. Recently, Massé et al.  gave an overview of the agronomic value of anaerobic digestion treated manures. In line with this, previous findings showed that the AD of animal slurries improved their fertilizer value , thereby leading to an increased forage yield and N uptake relative to raw liquid swine manure and mineral fertilizers . Bougnom et al.  found a 20% increase in grass yield compared to conventional manure. Liedl et al.  also found that digested poultry litter resulted in similar or superior grass and vegetable yields versus N fertilizers. Therefore, all of the above provides evidence that the anaerobic digested slurry acts similarly to mineral fertiliser and should be considered as such in its application to land. Additionally, the enrichment of mineral fractions of N and P during digestion ultimately results in a higher concentration of plant-available nutrients compared with undigested manure and a subsequently elevated plant growth promotion ability, suggested to be similar to mineral fertilisers [4,123]. These latter authors also found that AD reduced swine manure total and volatile solid concentrations by up to 80% resulting in improved manure homogeneity and lowered viscosity allowing more uniform land application . Nevertheless, the higher levels of mineral N found in the slurry, mainly ammonia, may also lead to an increase in the level of phytotoxicity of the slurry, thereby affecting seed germination and plant growth after land- spreading of this co-product into soils . The presence of other phytotoxic substances, such as volatile fatty acids (i.e., acetic, propionic and butyric acids), as well as the high content of soluble salts may contribute to the slurry phytotoxicity . Furthermore, Goberna et al.  found that amending soils with slurry resulted in greater nitrate losses during the first 30 d of a 100 d incubation period in 20 cm-depth lysimeters. In fact, around 23 and 45% of the total N contained in the soil (natural + added) was lost from soils amended with cattle manure and anaerobic slurry, respectively. Other authors also observed that N leaching was, along with NH3 volatilisation, one of the most important sources of N leakage to the environment in a field-scale experiment, after having quantified the amount of mineral N at 1.7 m depth from grass cultivated plots amended with anaerobic digested slurry and mineral fertiliser . Thus, the use of this co-product as an organic amendment should accurately match crop N demand because if not taken by the plants, nitrates could be drained to surface waters, leached to ground waters or denitrified into gaseous forms and emitted to the atmosphere.
The presence of pathogenic bacteria in agricultural amendments also represents a potential threat and their screening is thus of great importance mainly in those produced from animal manures, as it has been shown that such pathogenic organisms constitute a common fraction of the microbial community in manure . In fact, it has been shown that some pathogenic bacteria can survive the process of anaerobic digestion and persist in the slurry, as previously reported by . In line with this, those microorganisms with a spore-forming capacity such as
In a current study, we evaluated, at a microcosm level, the short and long-term effects of the anaerobic digested slurry on soil chemical and microbiological properties compared to its ingestate (i.e., raw manure) and the two widely-recognized products, compost and vermicompost. All of the organic substrates were mixed with soil by turning at a rate of 40 mg N kg-1 soil (dry weight). A control treatment that consisted of soil without the addition of any organic amendment was also included. A total of 45 experimental units (5 amendment levels x 3 incubation times x 3 replicates) were set-up in the present study. After an equilibration period of 4 days at 4 °C, 15 columns were dismantled and the sample was collected to analyze (incubation time 0 days). The remaining thirty columns were then maintained in a room at 22 °C, which is the average temperature of the hottest and wettest month in this area and the most suitable for the survival of pathogens. These columns were destructively sampled after 15 and 60 d incubation corresponding to short and medium-term effects. The survival of selected pathogens was then determined according to standard protocols [138-140] (ISO 16649-2, 2001 for
Briefly, culturable forms of both faecal coliforms and
However, the spore-forming
The intensity and concentrated activity of the livestock industry generate huge amounts of biodegradable wastes, which must be managed with appropriate disposal practices to avoid a negative impact on the environment. Composting is one of the best-known processes for the biological stabilization of solid organic wastes under aerobic conditions. Vermicomposting, i.e. the processing of organic wastes by earthworms under aerobic and mesophilic conditions, has also proven to be a low-cost and rapid technique. Although aerobic processes are thermodynamically more favorable, the manure treatment by anaerobic digestion has become increasingly important due to its energetic potential. A stabilised end product that can be used as an organic amendment is obtained under both aerobic and anaerobic conditions. A multi-parameter approach applying diverse methods constitutes the best option for evaluating the stability/maturity degree of the organic matter, which is of utmost importance for its safe use for the agriculture and the environment. During biodegradation all organic matter goes through the microbial decomposer pool and thus, further knowledge about the changes occurring during the process from a microbial viewpoint will contribute to further develop efficient strategies for the management of animal manures.
Waste management continues to be a topic of increasing importance. Deeper knowledge of the different biological processes involved in the recycling and recovery of waste components is thus of utmost importance in order to contribute towards more sustainable production and consumption systems. For example, biowaste may be used as a resource to produce high quality lactic acid and protein, as well as biogas in a cascade procedure. Briefly, biowaste is separated into two phases, i.e. a solid phase that is used to feed
Marina Fernández- Delgado Juárez is in receipt of a PhD fellowship from Doktoratsstipendium aus der Nachwuchsförderung der Universität Innsbruck. María Gómez Brandón is financially supported by a postdoctoral research grant from Fundación Alfonso Martín Escudero. The authors acknowledge Paul Fraiz for his highly valuable help in language editing.