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. [120] 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, Masse et al. [4] 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 [3], thereby leading to an increased forage yield and N uptake relative to raw liquid swine manure and mineral fertilizers [121]. Bougnom et al. [122] found a 20% increase in grass yield compared to conventional manure. Liedl et al. [123] 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 [4]. 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 [124]. 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 [125]. Furthermore, Goberna et al. [126] 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 [127]. Thus, the use of this co­product 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 [128]. 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 [129]. In line with this, those microorganisms with a spore­forming capacity such as Clostridium and Bacillus species, which are commonly found in the intestinal flora of most warm-blooded animals and can harbor some highly pathogenic members for animals and humans [12,129], cannot be reduced during the process [130]. Accordingly, Olsen and Larsen [131] observed that the spores of Clostridium perfringens were not inactivated in either mesophilic or thermophilic biogas digesters. Similar results were observed by other authors [132-133] in a reactor operating under mesophilic and thermophilic conditions, respectively. It is acknowledged that bacterial spores can survive in extreme conditions and germinate after long periods, when the conditions become more favourable [131]. The non-hygienic conditions of the storage/transporting tanks can also favour pathogen regrowth [134]. The composition of the substrate fed into the reactor, as well as the reactor conditions such as pH, digestion temperature, slurry hydraulic retention time, ammonium concentration, volatile fatty acids content and nutrient supply are expected to have a significant influence on the sanitation of the end-product [130]. This indicates that there exists a potential risk of spreading potentially pathogenic microbes after the application of anaerobic slurries into soil. Indeed, Crane and Moore [135] stated that amending soils with raw and treated manures, even with a low pathogenic load, still posed a threat for the environment because a period of regrowth of some pathogens including Escherichia coli, enterococci, faecal streptococci and Salmonella enterica have been shown after manure deposition to soil [136]. Goberna et al. [126] also found that the levels of Listeria in soils amended with either cattle manure or anaerobic slurry were significantly higher than those in the control treatment after having been incubated for a month. They observed, however, that the cultivable forms of Listeria in the studied soils could correspond to L. innocua instead of L. monocytogenes, as shown by the polymerase chain reaction assays. However, as recently summarised by [137], anaerobic digestion generally reduces the pathogen risk when compared to untreated substrates.

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 Escherichia coli; ISO 4832, 1991 for faecal coliforms; and ISO 7937, 2004 for Clostridium perfringens) in all the organic materials and amended soils.

Briefly, culturable forms of both faecal coliforms and E. coli were isolated from all the initial materials, although their levels were greatly lower in compost relative to the other substrates (Figure 5). This is not surprising taking into account that the composting process, unlike vermicomposting, involved a four-day thermophilic phase, during which the process reached a temperature of 70 °C. Those pathogens were also detected in the anaerobic digested slurry after 40 days of anaerobic digestion (Figure 5). This fact suggests that feeding the reactor with four to five m3 cattle manure d-1 could have provided enough nutrients to maintain a large population of the studied pathogens. Indeed, nutrient availability is one of the major factors influencing pathogen survival in biogas digesters, as previously reported by [130]. Once applied to soils, E. coli CFUs were detected in manure — amended soils at the start of the experiment and after incubation for 15 d (Figure 5A); whilst faecal coliforms CFUs were recorded in both manure and slurry-amended soils in the short­term, even though at lower values in comparison with the start of incubation (Figure 5B).

However, the spore-forming C. perfringens persisted in all the amended soils (Figure 6), which supports the fact that this bacterium has more resistance to environmental stresses and the capacity to outcompete the native soil microbiota. After 60 d CFU of C. perfringens were much closer to those in the control in the slurry-amended soils (Figure 6), which suggests that this time period could be considered as a safe delay between land-spreading the product into soil and crop harvesting with respect to its pathogenic load.