During anaerobic digestion, bacteria consume part of the organic matter to produce biogas and a residue known as digestate [1]. Biogas production from anaerobic digestion of organic matter has increased in recent years and thus, the application of digestates to soil as biofertilizers has become more common. Digestate is a very complex material therefore its application to soil has effect on the wide range of physical, chemical and biological properties of the soil, depending on the soil types. Generally, the application of digestate to soil have been shown to affect soil properties such as pH, macro and micro element contents, organic matter content, microbiological activities and the quality and yield of crops [2-9]. The application of digestate to agricultural soils may strongly influence various microbial groups in the soil. For example, Sapp et al. compared the effect of digestate and chemical fertilizer on soil bacterial community and plant yield, Garcia-Sanchez et al. studied the changes in soil microbial community functionality and structure and plant yield in a metal-polluted site as a result of digestate and fly ash application, while Caracciolo et al. studied the changes in microbial diversity and plant growth in a degraded agricultural soil after the addition of two composts obtained from cattle manure or pig waste slurry anaerobic digestate [4,7,9].

Microbial immobilization of labile nitrogen (N) after the application of organic matter to soil has been shown to reduce nitrification and thus can decrease nitrate leaching [10,11]. Alburquerque et al. reported that highly biodegradable digestates led to the immobilization of nitrogen (N) and retarded nitrification whereas less biodegradable digestates caused rapid nitrification [12]. Therefore, the biodegradability of digestates needs to be evaluated in order to more clearly understand nitrification processes in soils [13]. The oxidation of ammonium to nitrite is the rate limiting step in nitrification and is catalyzed by ammonia monoxygenase (AMO), which is encoded by the amoA genes harboured by both ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) [14]. However, the relative importance of AOA and AOB to the nitrification process is still under debate [15,16]. Using real-time PCR, it is showed that the application of a digestate from wet fermentation only increased AOB and not AOA amoA gene copies however, the relationship between amoA gene copies and nitrification rates currently remains unclear.

In one study, the effect of applying wet digestate (pH 8.2, C/N ratio 4.5), dry digestate (pH 8.8, C/N ratio 23.4) and a chemical fertilizer to Japanese paddy and upland soils on short-term nitrification under laboratory aerobic conditions were compared [17]. All applications led to rapid increases in nitrate nitrogen (NO3-N) contents in both soils and ammonia-oxidizing bacteria but not archaea may have played a critical role in nitrification in the amended soils. The net nitrification rates for both soils were the highest after the application of dry digestates, followed by wet digestates and then the chemical fertilizers in order of decreasing soil pH. Their result suggested that the immediate effects of applying digestates, especially the dry digestate with the highest pH, on nitrate leaching need to be considered when digestates are used as alternative fertilizers [13]. In this study, the effect of liquid digestate on the population dynamics of selected soil microbial groups during cultivation of maize (Zea mays) was investigated.

Collection and characterization of digestate and agricultural soil

The liquid digestate used to cultivate the test plant (Zea mays) was obtained from a pilot-scale anaerobic digester (ADH) treating organic fraction of municipal solid waste. The loamy soil used to cultivate the test plant (Zea mays) was obtained from an agricultural field at the Nigerian Institute for Oil Palm research (NIFOR) in Edo State, Nigeria. After collection, samples of the digestate and soil were taken to the laboratory to determine parameters such as moisture content (MC), pH, total organic carbon (TOC), biochemical oxygen demand (BOD), chemical oxygen demand (COD), ammonia nitrogen (NH4-N), total nitrogen (N), phosphorus (P), potassium (K), magnesium (Mg), calcium (Ca), micro elements (such as Mn, Zn, Fe, Cu, Co and Ni) and heavy metals (such as Pb, Cd, Cr and Hg) using the Standard Methods [18].

Experimental set-up

During cultivate of Zea mays (TZESR-W; early maturing streak resistant white grain variety), the plant was subjected to various concentrations (ranging from 0% to 72%) of the digestate using One-Factor Response Design (Design Expert version 9.0) with a total of ten (10) runs in 10L-capacity plastic pots (Table 1). After completely loading the pots with the loamy soil, two seeds of the Zea mays were sown into the soil in each of the pots at a depth of 5 cm per hole. After sowing, one set of experimental runs were treated with one-time application of the corresponding digestate concentration. This was tagged “One-time Digestate Application (OTDA)”. However, the other set of experimental runs were treated in such a way that the total amount of the liquid digestate required for each pot was divided into two equal halves and applied with respect to time instead of applying all of it at once. This was tagged “Two-time Digestate Application (TTDA)”. The first application of the liquid digestate (for OTDA and TTDA) was conducted two weeks after sowing while the second application (for TTDA alone) was conducted three weeks after the first application (Table 1). The liquid digestate (as bio-fertilizer) was applied by spraying it into the soil in the pots.

Table 1: One Factor Response Design for analysis of fertilizer potential of digestate.

Collection of soil samples

During the growth of maize plant in all set-ups, composite soil samples were collected with respect to time at depths between 0 cm and 20 cm from each of the set-ups into black polyethylene bags using sterilized cork-borer pushed vertically/horizontally into the soil in the pots. After collection, the polyethylene bags were sealed, labeled and immediately taken to the laboratory in order to estimate the population of soil microbes such as aerobic bacteria (AEB), strict anaerobic bacteria (SAB), ammonia oxidizing bacteria (AOB), nitrate reducing bacteria (NRB) and fungi (FUN) using the cultural techniques.

Determination of soil microbial populations

Aerobic Bacterial (AEB) populations were enumerated and isolated using the spread plate method as described [19]. Strict Anaerobic bacterial (SAB) populations were enumerated and isolated using the agar roll-tube technique described [20,21]. Ammonia Oxidizing Bacterial (AOB) populations were enumerated and isolated as described [22] Nitrate Reducing Bacteria (NRB) populations were enumerated and isolated as described [23]. Fungi (FUN) populations were enumerated and isolated using the soil dilution plate count and soil plate count methods as described [24]. Bacterial isolates were identified according to Bergey’s Manual of Determinative Bacteriology, using morphological and metabolic/biochemical tests [25,26]. Identification of the fungi that were isolated was based on the morphological characteristics of both colonial and microscopic examinations as described by [27-29], with help from additional literatures [24,30-33].

Properties of the digestate and the soil

The properties of the liquid digestate produced following anaerobic treatment of organic fraction of municipal solid waste (OFMSW) are presented in Table 2. The Table 2 also shows some properties of the loamy soil used to cultivate the test plant (Zea mays). It shows that elements (or compounds) such as carbon (as total organic carbon), total nitrogen, ammonium nitrogen, phosphorus, potassium, calcium, magnesium, manganese, zinc, copper, nickel, cobalt, iron, and molybdenum, which are known to be beneficial for plant growth and to soil microbes were higher in the digestate compared to the soil. Some heavy metals such as copper, zinc, lead, cadmium, mercury and chromium were also present in the digestate. However, their concentrations appear to be low. The Table 2 shows that the digestate contained both aerobic (most likely facultative anaerobic) bacteria and strict anaerobic bacteria [34]. The presence of macro and micro elements in most digestates make them excellent form of fertilizer when applied correctly [2-9,35].

Table 2: Characteristics of the liquid digestate and composite soil sample.

Population dynamics of soil microbes

Aerobic bacteria (AEB): In the control set-up (treated with 0% digestate), the average population of soil aerobic bacteria (AEB) increased from 3.3 × 10⁶ CFU/g to 8.1 × 10⁶ CFU/g of soil after 70 days (Figure 1). In the set-ups treated with 8% digestate, the average population of soil AEB in OTDA and TTDA increased from 3.0 × 10⁶ CFU/g and 3.1× 10⁶ CFU/g to 9.8 × 10⁶ CFU/g and 9.5 × 10⁶ CFU/g respectively after 70 days (Figure 1). In the set-ups treated with 16% digestate, the average population of soil AEB in OTDA and TTDA increased from 3.1 × 10⁶ CFU/g and 3.0 × 10⁶ CFU/g to 14.5 × 10⁶ CFU/g and 13.7 × 10⁶ CFU/g respectively after 70 days (Figure 1). In the set-ups treated with 24% digestate, the average population of soil AEB in OTDA and TTDA increased from 3.2 × 10⁶ CFU/g and 3.1 × 10⁶ CFU/g to 17.6 × 10⁶ CFU/g and 15.9 × 10⁶ CFU/g respectively after 70 days (Figure 1). In the set-ups treated with 32% digestate, the average population of soil AEB in OTDA and TTDA increased from 3.3 × 10⁶ CFU/g and 3.2 × 10⁶ CFU/g to 30.5 × 10⁶ CFU/g and 29.5 × 10⁶ CFU/g respectively after 70 days (Figure 1). In the set-ups treated with 40% digestate, the average population of soil AEB in OTDA and TTDA increased from 3.2 × 10⁶ CFU/g and 3.0 × 10⁶ CFU/g to 36.1 × 10⁶ CFU/g and 38.4 × 10⁶ CFU/g respectively after 70 days (Figure 1). In the set-ups treated with 48% digestate, the average population of soil AEB in OTDA and TTDA increased from 3.4 × 10⁶ CFU/g and 3.1 × 10⁶ CFU/g to 50.2 × 10⁶ CFU/g and 58.5 × 10⁶ CFU/g respectively after 70 days (Figure 1). In the set-ups treated with 56% digestate, the average population of soil AEB in OTDA and TTDA increased from 3.3 × 10⁶ CFU/g and 3.2 × 10⁶ CFU/g to 40.3 × 10⁶ CFU/g and 69.1 × 10⁶ CFU/g respectively after 70 days (Figure 1). In the set-ups treated with 64% digestate, the average population of soil AEB in OTDA and TTDA increased from 3.4 × 10⁶ CFU/g and 3.1 × 10⁶ CFU/g to 32.8 × 10⁶ CFU/g and 73.4 × 10⁶ CFU/g respectively after 70 days (Figure 1). In the set-ups treated with 72% digestate, the average population of soil AEB in OTDA and TTDA increased from 3.1 × 10⁶ CFU/g and 3.3×10⁶ CFU/g to 27.9 × 10⁶ CFU/g and 78.6 × 10⁶ CFU/g respectively after 70 days (Figure 1).

Figure 1: Population dynamics of aerobic bacteria (AEB) with respect to digestate concentration in OTDA and TTDA set-ups before and after 70 days of applying the digestate.

Strict anaerobic bacteria (SAB): In the control set-up (treated with 0% digestate), the average population of soil SAB increased from 2.4 × 10⁴ CFU/g to 4.5 × 10⁴ CFU/g of soil after 70 days (Figure 2). In the set-ups treated with 8% digestate, the average population of soil SAB in OTDA and TTDA increased from 2.1 × 10⁴ CFU/g and 2.2 × 10⁴ CFU/g to 8.1 × 10⁴ CFU/g and 5.3 × 10⁴ CFU/g respectively after 70 days (Figure 2). In the set-ups treated with 16% digestate, the average population of soil SAB in OTDA and TTDA increased from 2.2 × 10⁴ CFU/g and 2.4 × 10⁴ CFU/g to 9.5 × 10⁴ CFU/g and 6.5 × 10⁴ CFU/g respectively after 70 days (Figure 2). In the set-ups treated with 24% digestate, the average population of soil SAB in OTDA and TTDA increased from 2.0 × 10⁴ CFU/g and 2.2 × 10⁴ CFU/g to 9.9 × 10⁴ CFU/g and 8.2 × 10⁴ CFU/g respectively after 70 days (Figure 2). In the set-ups treated with 32% digestate, the average population of soil SAB in OTDA and TTDA increased from 2.3 × 10⁴ CFU/g and 2.1 × 10⁴ CFU/g to 1.1 × 10⁵ CFU/g and 9.1 × 10⁴ CFU/g respectively after 70 days (Figure 2). In the set-ups treated with 40% digestate, the average population of soil SAB in OTDA and TTDA increased from 2.1 × 10⁴ CFU/g and 2.3 × 10⁴ CFU/g to 1.26 × 10⁵ CFU/g and 9.5 × 10⁴ CFU/g respectively after 70 days (Figure 2). In the set-ups treated with 48% digestate, the average population of soil SAB in OTDA and TTDA increased from 2.3 × 10⁴ CFU/g and 2.3 × 10⁴ CFU/g to 1.62 × 10⁵ CFU/g and 1.15 × 10⁵ CFU/g respectively after 70 days (Figure 2). In the set-ups treated with 56% digestate, the average population of soil SAB in OTDA and TTDA increased from 2.1 × 10⁴ CFU/g and 2.0 × 10⁴ CFU/g to 5.70 × 10⁵ CFU/g and 1.82 × 10⁵ CFU/g respectively after 70 days (Figure 2). In the set-ups treated with 64% digestate, the average population of soil SAB in OTDA and TTDA increased from 2.0 × 10⁴ CFU/g and 2.1 × 10⁴ CFU/g to 6.40 × 10⁵ CFU/g and 2.20 × 10⁵ CFU/g respectively after 70 days (Figure 2). In the set-ups treated with 72% digestate, the average population of soil SAB in OTDA and TTDA increased from 1.9 × 10⁴ CFU/g and 2.2 × 10⁴ CFU/g to 7.2 × 10⁵ CFU/g and 2.53 × 10⁵ CFU/g respectively after 70 days (Figure 2).

Figure 2: Population dynamics of strict anaerobic bacteria (SAB) with respect to digestate concentration in OTDA and TTDA set-ups before and after 70 days of applying the digestate.

Ammonia oxidizing bacteria (AOB): In the control set-up (treated with 0% digestate), the average population of soil AOB increased to 9.3 × 10³ MPN/g of soil after 70 days (Figure 3). In the set-ups treated with 8% digestate, the average population of soil AOB in OTDA and TTDA increased to 1.1 × 10⁴ MPN/g and 9.4 × 10³ MPN/g respectively after 70 days (Figure 3). In the set-ups treated with 16% digestate, the average population of soil AOB in OTDA and TTDA increased to 1.5 × 10⁴ MPN/g and 1.3 × 10⁴ MPN/g respectively after 70 days (Figure 3).  In the set-ups treated with 24% digestate, the average population of soil AOB in OTDA and TTDA increased to 1.6 × 10⁴ MPN/g and 1.4 × 10⁴ MPN/g respectively after 70 days (Figure 3).  In the set-ups treated with 32% digestate, the average population of soil AOB in OTDA and TTDA increased to 1.9 × 10⁴ MPN/g and 1.9 × 10⁴ MPN/g respectively after 70 days (Figure 3). In the set-ups treated with 40% digestate, the average population of soil AOB in OTDA and TTDA increased to 3.5 × 10⁴ MPN/g and 3.6 × 10⁴ MPN/g respectively after 70 days (Figure 3). In the set-ups treated with 48% digestate, the average population of soil AOB in OTDA and TTDA increased to 4.3 × 10⁴ MPN/g and 9.3 × 10⁴ MPN/g respectively after 70 days (Figure 3). In the set-ups treated with 56% digestate, the average population of soil AOB in OTDA and TTDA increased to 1.9 × 10⁴ MPN/g and 1.2 × 10⁵ MPN/g respectively after 70 days (Figure 3). In the set-ups treated with 64% digestate, the average population of soil AOB in OTDA and TTDA increased to 1.3 × 10⁴ MPN/g and 3.4 × 10⁵ MPN/g respectively after 70 days (Figure 3). In the set-ups treated with 72% digestate, the average population of soil AOB in OTDA and TTDA increased to 9.1 × 10³ MPN/g and 3.9 × 10⁵ MPN/g respectively after 70 days (Figure 3).

Figure 3: Population dynamics of ammonia oxidizing bacteria (AOB) with respect to digestate concentration in OTDA and TTDA set-ups before and after 70 days of applying the digestate.

Nitrate reducing bacteria (NRB): In the control set-up (treated with 0% digestate), the average population of soil NRB increased to 4.3 × 10³ MPN/g of soil after 70 days (Figure 4). In the set-ups treated with 8% digestate, the average population of soil NRB in OTDA and TTDA increased to 6.1 × 10³ MPN/g and 4.6 × 10³ MPN/g respectively after 70 days (Figure 4). In the set-ups treated with 16% digestate, the average population of soil NRB in OTDA and TTDA increased to 6.1× 10⁴ MPN/g and 9.1 × 10³ MPN/g respectively after 70 days (Figure 4). In the set-ups treated with 24% digestate, the average population of soil NRB in OTDA and TTDA increased to 7.5 × 10⁴ MPN/g and 9.3 × 10³ MPN/g respectively after 70 days (Figure 4). In the set-ups treated with 32% digestate, the average population of soil NRB in OTDA and TTDA increased to 9.2 × 10⁴ MPN/g and 1.4 × 10⁴ MPN/g respectively after 70 days (Figure 4). In the set-ups treated with 40% digestate, the average population of soil NRB in OTDA and TTDA increased to 1.3 × 10⁵ MPN/g and 1.9 × 10⁴ MPN/g respectively after 70 days (Figure 4). In the set-ups treated with 48% digestate, the average population of soil NRB in OTDA and TTDA increased to 1.9 × 10⁵ MPN/g and 3.9 × 10⁴ MPN/g respectively after 70 days (Figure 4). In the set-ups treated with 56% digestate, the average population of soil NRB in OTDA and TTDA increased to 2.4 × 10⁵ MPN/g and 4.6 × 10⁴ MPN/g respectively after 70 days (Figure 4). In the set-ups treated with 64% digestate, the average population of soil NRB in OTDA and TTDA increased to 2.9 × 10⁵ MPN/g and 7.5 × 10⁴ MPN/g respectively after 70 days (Figure 4). In the set-ups treated with 72% digestate, the average population of soil NRB in OTDA and TTDA increased to 4.4 × 10⁵ MPN/g and 1.4 cc 10⁵ MPN/g respectively after 70 days (Figure 4).

Figure 4: Population dynamics of nitrate reducing bacteria (NRB) with respect to digestate concentration in OTDA and TTDA set-ups before and after 70 days of applying the digestate.

Fungi (FUN): In the control set-up (treated with 0% digestate), the average population of soil FUN increased from 1.6 × 10³ CFU/g to 5.9 × 10³ CFU/g of soil after 70 days (Figure 5). In the set-ups treated with 8% digestate, the average population of soil FUN in OTDA and TTDA increased from 1.6 × 10³ CFU/g and 1.4 × 10³ CFU/g to 6.1 × 10³ CFU/g and 6.4 × 10³ CFU/g respectively after 70 days (Figure 5). In the set-ups treated with 16% digestate, the average population of soil FUN in OTDA and TTDA increased from 1.4 × 10³ CFU/g and 1.5 × 10⁶ CFU/g to 7.0 × 10³ CFU/g and 7.3 × 10³ CFU/g respectively after 70 days (Figure 5). In the set-ups treated with 24% digestate, the average population of soil FUN in OTDA and TTDA increased from 1.5 × 10³ CFU/g and 1.3 × 10³ CFU/g to 7.2 × 10³ CFU/g and 7.8 × 10³ CFU/g respectively after 70 days (Figure 5). In the set-ups treated with 32% digestate, the average population of soil FUN in OTDA and TTDA increased from 1.3 × 10³ CFU/g and 1.4 × 10³ CFU/g to 8.7 × 10³ CFU/g and 8.3 × 10³ CFU/g respectively after 70 days (Figure 5). In the set-ups treated with 40% digestate, the average population of soil FUN in OTDA and TTDA increased from 1.4 × 10³ CFU/g and 1.3 × 10³ CFU/g to 9.3 × 10³ CFU/g and 8.8 × 10³ CFU/g respectively after 70 days (Figure 5). In the set-ups treated with 48% digestate, the average population of soil FUN in OTDA and TTDA increased from 1.5 × 10³ CFU/g and 1.3 × 10³ CFU/g to 1.1 × 10⁴ CFU/g and 9.4 × 10³ CFU/g respectively after 70 days (Figure 5). In the set-ups treated with 56% digestate, the average population of soil FUN in OTDA and TTDA increased from 1.3 × 10³ CFU/g and 1.5 × 10³ CFU/g to 1.8 × 10⁴ CFU/g and 9.8 × 10³ CFU/g respectively after 70 days (Figure 5). In the set-ups treated with 64% digestate, the average population of soil FUN in OTDA and TTDA increased from 1.5 × 10³ CFU/g and 1.6 × 10³ CFU/g to 2.2 × 10⁴ CFU/g and 7.1 × 10³ CFU/g respectively after 70 days (Figure 5). In the set-ups treated with 72% digestate, the average population of soil FUN in OTDA and TTDA increased from 1.3 × 10³ CFU/g and 1.2 × 10³ CFU/g to 2.6 × 10⁴ CFU/g and 6.2 × 10³ CFU/g respectively after 70 days (Figure 5).

Figure 5: Population dynamics of fungi (FUN) with respect to digestate concentration in OTDA and TTDA set-ups before and after 70 days of applying the digestate.

Figures 1-5 show that the application of liquid digestate (as bio-fertilizer) to the soil in the different set-ups during cultivation of maize plant actually affected the population of soil microbes [6-9]. The population of all the microbial groups considered in this study increased with time after the application of the liquid digestate. This could partly be attributed to the nutrient content of the liquid digestate shown in Table 1. Most of the nutrients are just as beneficial to soil microbes as they are to plants [2-4,8,35]. The result also shows that the one-time digestate application (OTDA) treatment seemed to be less favourable to the growth of aerobic bacteria (AEB) and ammonia oxidizing bacteria (AOB) populations compared to the two-time digestate application (TTDA) treatment which appears to have favoured the populations of AEB and AOB respectively with time. Because these groups of bacteria (especially AOB) are the drivers of nitrification in the soil, it therefore indicates that applying the liquid digestate periodically at smaller quantities (instead of applying it all at once) may have favoured nitrification which is actually beneficial to the plants to some degree [10-14]. The one-time mode of digestate application (OTDA) seemed to have favoured the populations of strict anaerobic bacteria (SAB) and nitrate reducing bacteria (NRB) with time especially at higher concentrations when compared to the two-time mode of digestate application (TTDA).

This is because, application of higher concentrations of the liquid digestate to the soil inside the pots at once (instead of periodically) may have displaced some or most of the oxygen dissolved in the soil solution and this could have promoted some levels of oxygen-limiting condition (or anaerobiosm) which usually favours the populations of SAB and NRB [36,37]. An increase in the population of SAB and especially NRB may indicate an increase in the formation of organic acids, methane (if methanogens are present) and oxides of nitrogen as a result of denitrification which usually leads to the loss of nitrogen from the soil [5,6,12,36-38]. The loss of nitrogen from the soil in the form of oxides (such as NO2, NO or N2O) contributes to global warming because these oxides of nitrogen are known to be potent greenhouse gases [36,37]. This condition may be quite unfavourable to plants, soil microbes and the environment at large [36,37]. Therefore, it will be necessary to consider the concentration and mode of application of liquid digestate to agricultural fields in order to prevent the emission of more potent greenhouse gases such as oxides of nitrogen and methane [13].

The result further shows that both (OTDA and TTDA) methods of digestate application increased the growth of fungi (FUN) population to some degree with time. However, the one-time digestate application (OTDA) treatment appeared to have favoured the population of fungi (FUN) more than the two-time digestate application (TTDA) treatment with time especially at higher concentrations (%). This may be possible because the OTDA treatment method appeared to have slightly acidified the soil at higher concentrations of the digestate than the TTDA treatment method. Because fungi appear to thrive better under acidic (or lower) pH, it may be reasonable to assume that this would have been a possible reason why their population appeared to have been slightly higher in the OTDA treatment than in the TTDA treatment with time [17].

Distribution and characteristics of soil microbes isolated

Generally, a total of 21 bacteria species and 13 fungi species were isolated. The bacteria include species which belong to genera such as Arthrobacter, Azotobacter, Flavobacterium, Nocardia, Bacillus, Clostridium, Cellulomonas, Micrococcus and Pseudomonas respectively. In the control set-up, a total of 2 Arthrobacter sp., 1 Azotobacter sp., 1 Flavobacterium sp., 2 Bacillus sp., 1 Cellulomonas sp., and 1 Pseudomonas sp. were isolated (Figure 6). In the OTDA set-up, a total of 1 Arthrobacter sp., 1 Azotobacter sp., 2 Flavobacterium sp., 2 Bacillus sp., 3 Clostridium sp., 2 Cellulomonas sp., 2 Micrococcus sp. and 1 Pseudomonas sp. were isolated (Figure 6). In the TTDA set-up, a total of 2 Arthrobacter sp., 1 Azotobacter sp., 2 Flavobacterium sp., 2 Nocardia sp., 4 Bacillus sp., 1 Clostridium sp., 1 Cellulomonas sp., 2 Micrococcus sp. and 2 Pseudomonas sp. were isolated (Figure 6).

Figure 6: Bacteria species isolated from digestate-treated and untreated soil samples.

The fungi include species which belong to genera such as Aspergillus, Cladosporium, Fusarium, Geotrichum, Penicillium, Rhizopus and Trichoderma respectively (Figure 7). In the control set-up, a total of 2 Aspergillus sp., 1 Fusarium sp., 1 Geotrichum sp., 1 Penicillium sp., 1 Rhizopus sp. and 1 Trichoderma sp. were isolated (Figure 7). In the OTDA set-up, a total of 3 Aspergillus sp., 1 Cladosporium sp., 2 Fusarium sp., 1 Geotrichum sp., 2 Penicillium sp., 1 Rhizopus sp. and 1 Trichoderma sp. were isolated (Figure 7). In the TTDA set-up, a total of 2 Aspergillus sp., 2 Fusarium sp., 1 Geotrichum sp., 2 Penicillium sp., 1 Rhizopus sp. and 1 Trichoderma sp. were isolated (Figure 7).

Figure 7: Fungi species isolated from digestate-treated and untreated soils samples.

The characteristics of bacteria and fungi isolated from composite soil sample in the control set-up (with 0% digestate application), OTDA set-up and TTDA set-up are presented in Table 3-6 respectively.

Table 3: Characteristics of bacteria isolated from digestate-treated soil and the control.

Table 4: Characteristics of bacteria isolated soil from digestate-treated and the control.

Table 5: Characteristics of bacteria isolated from digestate-treated soil and the control.

Table 6: Characteristics of fungi isolated from digestate-treated set-up and the control.

In conclusion, the two-time mode of digestate application (TTDA) appeared to have favoured the population of aerobic bacteria (AEB) and ammonia-oxidizing bacteria (AOB) better than the one-time mode of digestate application (OTDA) with time. This suggests that the TTDA treatment method may have favoured nitrification better than the OTDA treatment method. In addition, the TTDA treatment method appeared to have been less favourable to the population of strict anaerobic bacteria (SAB) and nitrate reducing bacteria (NRB) compared the OTDA treatment method with time. This suggests that the OTDA method may have promoted soil denitrification more than the TTDA treatment method with time. From the current study, it appears that the two-time mode of digestate application (TTDA) to agricultural soils may have been more beneficial to the growth of the test plants (Zea mays) as well as minimizing greenhouse gas (GHG) emission compared to one-time digestate application (OTDA) treatment which appeared to have promoted conditions which may have favoured the emission of greenhouse gases such as oxides of nitrogen (and may be methane as well).