The raw materials used for the preparation of digestate were catering waste and crop residues maize crop residues. The catering waste samples were taken from local food restaurants and banquet halls of Rawalpindi city, while the maize residues were collected from PMAS Arid Agriculture University Research Farm located at Koont, Pakistan. The initial physico-chemical characterization of catering waste and maize crop residues are described in Table 1.

Prior to anaerobic digestion maize crop residues were supplied with the pretreatment steps, which were selected based on a partial level of treatment with minimal energy prerequisite, and that avoids the complete degradation (Taherzadeh and Karimi, 2008). Sequential physical pretreatments, microwave irradiation and steam water were applied to the maize crop residues in order to enhance the accessibility of lignocellulose of maize crop residues for biological degradation. For microwave irradiation pretreatment a household-type DW-105-G microwave oven (Dawlance, Pakistan). Maize crop residues (50 g of each) were placed in the microwave oven for 3 min supplying energy of 800 J/g/min. Subsequently, irradiated substrate was placed in a high-pressure steam unit (Nanolytik Nanoclave 1, Germany) for 40 min at 121°C for steam treatment (Anjum et al., 2016). After pretreatments, maize crop residues were dried in air for 6 h, and later in a digital oven for 12 h prior to use in co-digestion.

Anaerobic digestion experiments were performed in two steps. First, the optimization of feeding composition was performed and secondly the analysis of optimized co-digestion treatment was conducted during biogas production. At first set of experiment anaerobic co-digestion experiments were performed using catering waste as a main substrate mixed with partially pre-treated maize crop residues at different compositions. Maize crop residues (M) were used in 20, 30, 40, and 50% of total fraction with catering waste, while similar experiments were repeated with untreated maize crop residues. Anaerobic glass batch bottles of 500 ml capacity were used with total substrate volume of 350 and 150 ml empty head space. All mixtures of substrates were prepared on the basis of total solids. The seed anaerobic microorganisms were applied using previously digested organic waste added at a rate of 100 g/L (Anjum et al., 2012). A suitable amount of water was added keeping the moisture content of reactors fixed between ranges of the 70 and 75%. Co-digestion experiment took place for 6 weeks 42 at mesophilic temperature 30 ± 1°C. To examine the degradation efficiency of co-digestion, samples were analyze at different interval changes in COD and VS.

In the second set of experiment, selected treatments (40 % maize crop residues and 60% catering waste) (TEM3) was subjected to biogas production in a lab scale, anaerobic static batch reactor and compared with control. Two similar reactor of dimensions 13 × 30 cm (diameter × width) each, having capacity of 2,500 ml were designed. One reactor was used for co-digestion (TEM3), while the second was used as control reactor having catering waste only. About 2,000 ml of the reactor volume was filled with the substrates, while 500 ml head space was left empty for biogas accumulation. Biogas was collected in biogas collection bag connected at the top biogas valve, through the flow tube built in with an internal valve to control the one-way flow of biogas. The digestate samples were taken from the lower part of the reactor where a sampling valve was set. Both reactors were placed in an incubator at fixed temperature 30 ± 1°C. The biogas was analyzed using liquid displacement apparatus.

Two digestate samples were collected from anaerobic co-digestion of catering waste and maize crop residues. One digestate sample is supplied with an aerobic post treatment in order to reduce toxic substances exist in digestate, while second digestate sample did not supply with any post treatment. Aerobic pretreatment was performed using continuous stirred aerobic reactor supplied by continuous air diffusion from the bottom and string of 150 rpm. The temperature was maintained in range of 32–35°C and reaction was continued for 20 days. The digestate quality was analyzed by measuring carbon content, volatile solids, pH and EC using standard methods. The digestate was dried in a digital oven for 24 h at 70°C. The final obtained digestate materials were preserved in the cold storing unit at temperature >50°C for further utilization in experiments.

The phytotoxicity test of both digestate was conducted by using 72 h seed germination assay. Germination assay is a rapid and widely applied method for evaluating phytotoxicity of organic product (Da Ros et al., 2018). Three dilution of each digestate material (post treated and untreated digestate) were prepared using 5, 10, and 20 g of dried digestate in 100 ml of distilled water. Wheat seed were used to testing seed germination assay. For the germination assay, Petri dishes of 10 cm diameter were lined with watt man filter paper. Each dish received 5 ml of the digestate solution using a micropipet. Ten wheat seeds (sterilized) were placed in each petri dish and sealed with the parafilm. The petri dishes were incubated at 25°C for 72 h in the dark conditions. The germinated seeds were counted and, root length and shoot length were measured were at regular interval during 72 h. The seed germination percentage (G%), germination index (GI) and vigor index (VI) were analyzed to estimate the phytotoxicity of digestate.

Chemical oxygen demand (COD) was analyzed using the closed, reflux titrimetric method applying the standard method of American Public Health Association (APHA (American Public Health Association), 2005). COD was calculated using the following equation (Equation 1):

The factor X-B is ml of FAS used for blank subtracted from ml of FAS used for sample. M denotes the molarity of FAS, whereas the value 8,000 is the weight of oxygen mill equivalent 1,000 ml/l. Solid fractions i.e., TS and VS were analyzed using the standard method # 1684 (US EPA, 2001). TS was determined by measuring the difference in mass of the sample before and after drying in oven at temperature 105°C, whereas VS was measured as an ignition loss at high temperature (550°C) in the muffle furnace. The C:N ratio was calculated by taking the total mass of carbon divided by mass of nitrogen in grams (Smith and Holtzapple, 2011; Anjum et al., 2012). pH, EC, and TDS values were recorded on Crison Multimeter (Model number: “CRISON MM-40þ”). In anaerobic digesters, the biogas production was measured as methane production through liquid a displacement methods for duration of 80 days using the method described by Elaiyaraju and Partha (2012). During phytotoxicity experiment, seed germination assay was conducted where seed germination index, germination percentage, seedling length, and vigor index were measured. The germination index (GI) was calculated using the following formula (Equation 2) (Mitelut and Popa, 2011):

where L_o and G_o are root growth and germination percentage. Data interpretation, calculation, averages and standard deviations were calculated using MS Office Excel 2010.

In order to prepare anaerobic digestate bio-product as low phytotoxic organic fertilizer, aerobic post treatment was applied to the anaerobic digestate of co-digested catering waste and wheat crop residues (TCM3). Figure 4A expressed the results regarding effect on VS and carbon content due aerobic post treatment. VS in the digestate was decreased from 209 g/L to 113 g/L after 20 days where as carbon content was lowered to 17% from 24% in fresh digestate. The lowering in VS and carbon content is attributed degradation of toxic organic compound especially organic acids produced during anaerobic digestion process. The lowering in VS content in post treatment make digestate more stable (Wojnowska-Baryła et al., 2018). These means that aerobic post treatment for 20 days could lower the phytotoxic compounds in digestate and make more feasible bio-product for soil application.

In case of pH, aerobic post treatment increases an overall pH of the digestate up 8.32 which is very favorable for soil application (Figure 4B). The basic reason of low pH in anaerobic digestion production of organic acid in high quantity which inhibit the digestion process (Yang et al., 2015; Zhang et al., 2018), but in the present study the pH of 7.29 showed that there were limit accumulation of organic acids and aerobic post treatment has further improved the pH of the digestate. Electrical conductivity is another important indicator of quality of digestate which mainly represents the concentration of salts and ions. EC values of 3.62 dS/m was found in anaerobic fresh digestate which is further lowered to 1.81 dS/m after aerobic digestion process (Figure 4B). EC can influence seed germination in contact with digestate, where lower EC is more favorable for seed germination (Coelho et al., 2018). This showed that EC of the digestate could be improved by post treatment of digestate in order to cause least negative effect in soil due to higher EC.

Phytotoxicity is widely applied and promising parameter to evaluate digestate application on plants which describes the index its overall eco-toxicological impact (Da Ros et al., 2018). The phytotoxicity test of both untreated and post treated digestate at varying digestate solution (5, 10, and 20 g per 100 ml water) was performed using seed germination assay on wheat seeds. The results regarding seed germination percentage are described in Figure 5. Seed germination was continuously increased in case of control (Water only) where after 72 h 91% of seed were observed fully germinated. When digestate was applied G% was lowered where only 17 % of germination was found even after 72 h in D20 (20 g untreated digestate/100 ml water) (Figure 5A). However, a slightly higher G% (38 and24%) was found in D5 and D10, respectively after 72 h. This shows that the digested without post treatment cause swear toxicity to wheat seed which in increased as the concentration of digestate increase.

In case of post treated digestate an improvement in G% was observed with up to 85% seed germination was attained after 72 h in PD5 (5 g/100 ml) (Figure 5B). These finding also confirmed by the Germination Index (GI) values with a maximum GI of 46 was shown by PD5 and followed by PD10 (GI = 39) and PD20 (GI = 37) (Figure 6). However, the lower germination index values in un-treated digestate was observed which is probably due to low high EC values (Tang et al., 2018).

The seedling length was measured after 72 h of incubation and illustrated in Figure 7. Both shoot length and rood length were significantly increased with post treated digestate solution. A maximum root length of 6.01 cm and 5.19 cm were achieved by PD5 and PD10 which were 4.9 and 4.8 times higher than respective treatments with untreated digestate. Similarly, shoot length of 7.34 cm was the highest in PD5 with the slight lowing in PD10 (6.06 cm) and PD20 (5.60 cm). The improvement in seedling length is attributed to the post treatment of digestate which supply more plants essential nutrients [N, P, and K (Coelho et al., 2018)] to seedling with least effect of toxic materials.

Vigor index is one the known indicator used in analyses toxicity in plants (Shaikh et al., 2013). The vigor of seeds was described as a vigor index (VI) as illustrated in Figure 8. Seeds treated with post treated digestate showed the higher VI i.e., up to 609 in PD5, where with the increase in concentration of digestate from 5 g/100 ml to 20 g/100 ml, VI was decreased to 377. On the other hand the seed grown with untreated digestate shows significantly lower vigor i.e., even with a value of zero with D20 (20 g/100 ml).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.