Screening and identification of microbial strains with lignocellulosic properties
Eleven bacteria and four fungi were isolated from other rice compost and were tested for their hydrolase ability. All bacteria and fungi showed xylanase, cellulase, and ligninase activity, except for the bacterial strains DSM141, IAM1221, and DSM1317 which lacked cellulase activity. Among the strains, 5 bacterial strains and 2 fungal strains showed amylase activity. The bacterial strains DSM13, DSM43377, and IAM12118, and also the fungal strain CBS298.64 showed the highest lignocellulosic activities (Table 1). The selected fungal and bacterial strains were identified using ITS and 16S rRNA gene amplification and sequencing, respectively. Phylogenetic analysis confirmed that bacteria DSM13, DSM43377, and IAM12118 were closely related to Bacillus licheniformis, Nocardiopsis alba, and Bacillus subtilis, respectively, and a fungal strain CBS398.64 was closely related to Thermoascus aurantiacus (Fig. 1-4) Finally, two groups of the selected microbial strains were used for the composting process; M1 (B. licheniformis, N. alba, B. subtilis, and T. aurantiacus) and M2 (B. licheniformis, N. alba, B. subtilis, and Trichoderma sp.). It should be noted that Trichoderma sp. was also obtained from the Agriculture Biotechnology Research Institute of Iran culture collection due to its suitable lignocellulosic activity.
Physicochemical analysis at the lab-scale
Temperature changes
The maximum temperature increase during the composting process was observed in treatments E and F (up to 59 oC) whereas the minimum increase was in treatments A and B. The treatments A and B failed to complete the known temperature phases of composting, while it was completed well for the treatments E and F from the third week of the experiment onwards (Fig. 5 and 6). Previous studies have shown that if the starting materials in the composting process are properly selected (based on the C/N ratio), consequently the composting process completely passes the mesophilic, thermophilic, and cooling phases [30-31]. So, the observed differences between the treatments in completing the composting phases in the present study should be due to the appropriate combination of treatments E and F compared to treatments A and B.
The results revealed that chicken manure had an important role in increasing the temperature of the composting process. The treatments E and F differ from treatments G and H in nitrogen source. Previous studies have shown that chicken manure helps composting process better than urea. This may be due to the biological activities, microbial flora, nitrogen, and other mineral elements contents present in the chicken manure compared to urea. Similar studies have shown that chicken manure and microorganisms could facilitate the composting process, increase the composting temperature during the thermophilic phase, accelerate the maturation of compost and finally, improve the quality of produced compost [9, 16, 32-33]. Our results showed that the presence of chicken manure alone is not enough to have qualified compost. Despite the similar contents of chicken manure in the control (A) and two other treatments (E and F), the temperature changes were significantly more in the E and F compared to that in the A. This could be related to the high hydrolase activity of the microbial additives (M1 and M2) used in the treatments E and F. It was also found that microorganisms (M1) used in E had higher lignocellulosic activities and resulted more temperature increase compares to that of F (M2). Similar results showed that the use of microorganisms such as Chytridiomycota, Mucoromycota, and Ascomycota led to an increase in temperature in the thermophilic phase of the composting process [8]. Comparison of treatments C and D with two treatments A and B showed the positive effect of olive pomace on temperature increase in temperature cycles in compost. The presence of beneficial microorganisms in olive pomace leads to an increase in temperature during composting [34].
EC changes
At the beginning of the composting process, the amount of salts and mineral phosphates is increased due to the decomposition of existing materials, which causes a significant increase in EC at the early stages [35]. Our results revealed that EC analysis was similar to that of temperature changes (Fig. 7). During the first week, EC increased in all treatments except for treatments A and B and continued until the fourth week due to the decomposition of materials in all treatments, the release of ions and salts, and the onset of activity of microorganisms [36]. The maximum EC was observed in treatment E (1623 ms/cm). From the fifth week, EC gradually decreased in the treatments. The volatilization of ammonia and the precipitation of mineral salts could be two key factors in reducing the amount of EC from the fifth week [35]. Our results showed that treatments E and G which contained microorganism group 1 (M1) had more EC compared to other treatments (F and H), which indicated higher activity of M1 compared to that of M2. Moreover, it was shown that treatments C and D had more EC due to the positive effect of olive pomace compared to treatments A and B. Comparison of EC in treatments A and B showed that the presence of chicken manure in treatment A could increase the amount of EC. Previous works have confirmed that the amount of EC during composting in treatments containing rice straw with chicken manure due to the increase of potassium and other ions and also release of mineral salts during decomposition, continuously is increased [37].
pH changes
It has been confirmed that by the beginning of the microorganisms activities at the primary steps of the composting process, the pH value is gradually decreasing [38]. The presence of chicken manure reduces the pH at the beginning of the composting process, while during the process, by converting mineral nitrogen to ammonia; the pH value consequently is decreasing and makes the environment alkaline. Elevation of pH due to ammonia formation is associated with protein degradation and decomposition of organic acids, which coincides with the maximum activity of microorganisms, which is especially observed in the thermophilic phase [39]. During the first week, the treatments containing chicken manure were associated with a decrease in pH, and treatment H showed a minimum pH value (5.8). From the second week, the pH value was gradually increased, and in some treatments reached up to 8 (Fig. 8). The pH value of treatments E and F were reduced after the thermophilic stage. Other treatments had fluctuations during the composting period. However, the treatments with M1 microorganisms (E and G) had higher pH compared to those treatments containing M2 microorganisms (F and H) which showed the effect of M1 microorganisms on increasing the pH in these treatments. Research conducted by Duan et al. [8] also showed that microorganisms such as Chytridiomycota, Mucoromycota, Ascomycota, Basidiomycota, and Neocallimastigomycota were forced to increase the pH of the compost [8].
C/N ratio changes
C/N ratio is one of the most important factors in evaluating the quality of compost. The C/N ratio reduction during the composting process is known as one of the most important factors affecting the maturity of compost. C/N ratio reduction of more than 40% indicates the maturation of the compost [40]. The higher the C/N ratio in the biomass at starting scale, shows the higher carbon contents and less nitrogen content, which causes a longer process time, immaturity and low quality of the final produced compost due to lower microbial activity [41]. The C/N analysis revealed that the treatments containing chicken manure (A, C, E, and F) instead of urea (B, D, G, and H) as a source of nitrogen showed a greater reduction in the C/N ratio. The treatments E and F recorded the highest C/N ratio reduction and the treatments A and B had the minimum reduction. Moreover, the treatments with M1 microorganisms showed a significantly more C/N ratio decrease compared to those contained M2 microorganisms, which indicates their higher lignocellulosic activity. Ttreatment E with the maximum C/N ratio reduction (76.66%) is evidence that the combination of chicken manure and M1 microorganisms had the greatest effect on the decomposition of rice straw and accelerating the compost maturation time (Table 2 and Fig. 9). The presence of olive pomace in treatments C and D resulted in a more significant C/N ration reduction compared to treatments A and B., followed by increasing the quality and maturity of compost. The results of a similar study showed that co-application of chicken manure and olive pomace caused a greater C/N ratio reduction compared to other treatments containing only chicken manure[42].
Ash and volatile solid changes
The volatile solids (VS) represent the organic content [43]. During composting, the amount of volatile solids decreases from the beginning to maturity. The rate of reduction of volatile solids indicates the rate of decomposition of organic matter during composting [28]. Microorganisms play an important role in the decomposition of organic matter during composting, which is considered a key factor in reducing the amount of volatile solids during composting [44]. The highest reduction of VS (%) was observed in treatment E (32.26%) and the lowest reduction of VS (%) was observed in treatment A (11.44%) (Table 3). The VS analysis revealed that the treatments with microorganisms M1 and M2 (E, F, G, and H) showed significantly more reduction of VS (%) compared to other treatments (A, B, C, and D), which indicates microorganisms have played an effective role in the decomposition of organic matter. Comparison of treatment E with F and also a comparison of treatment G with H showed that M1 microorganisms have the greatest effect on the decomposition of organic matter compared to M2 microorganisms. The treatments containing chicken manure (A, C, E, and F) instead of urea (B, D, G, and H) as a source of nitrogen showed a greater reduction in the C/N ratio. Comparison of the treatments E and F with G and H as well as comparison of the A with B revealed that the treatments containing chicken manure instead of urea as a source of nitrogen showed a greater reduction in VS (%) that the presence of microorganisms in chicken manure could cause of this fact. A comparison of treatment C with A as well as B with D revealed that the presence of microorganisms in olive-mill waste could enhance the reduction in VS (%).
Micro-macro elements and heavy metals analysis
The amounts of heavy metals were acceptable in all treatments. Treatments containing chicken manure including E, F, and C showed the maximum scales of Mn, Mg, Fe, K, P, and Ca compared to other treatments. Treatment D also had higher scales of elements these elements compared to the treatments G and H, which all contained urea as a source of nitrogen. Olive-mill waste had a direct positive effect on the amount of these micro and macro elements (Table 4). Previously, it has been confirmed that the presence of chicken manure and olive pomace have many micro-macro elements which play a positive role in the quality of the final compost [42].
Physicochemical analysis at the pilot-scale
Temperature changes
The temperature changes started after one week in treatments E and F, and all three phases of compost production were completed during the process, while treatment A (control) did not show any significant temperature changes. The mesophilic phase was performed for two weeks, and the process was entered into the thermophilic phase at the end of the second week. The maximum temperature of the thermophilic phase was in treatments E (68.9 oC) and F (66.2 oC), respectively (Fig. 10). The temperature accumulative analysis of the treatments also showed that the maximum and minimum accumulated temperatures were for treatments E and A, respectively (Fig. 11).
EC changes
The results of the pilot experiments on EC of the selected treatments showed similar results to those of the lab-scale experiments. During the process, from the first to fourth weeks, the EC content was increased, while from the fifth to eighth weeks it was gradually decreasing. The highest amount of EC was observed in treatment E (1756 ms/cm) (Fig. 12). Given that the only difference between the two treatments E and F was the type of microorganisms used in them, so, our results showed that treatment E which contained microorganisms group 1 (M1) had more EC compared to treatment F.
pH changes
The results obtained at the pilot-scale confirmed the lab results. The highest pH was observed for treatment E (8.4). In the last two weeks, the pH scale in both E and F reached 7 (Fig. 13). Treatment E had higher pH compared to treatment F which showed more efficiency of the M1 microorganisms compared to M2 in increasing the pH.
C/N ratio changes
The maximum (73.48 %) and minimum (13.03%) C/N ratio reduction were observed for the treatments E and t A, respectively (Table 5 and Fig. 14). These results coordinated with the results of the lab-scale experiments, in which the presence of chicken manure and M1 microorganisms had significant effects on composting process and C/N ratio reduction.
Ash, volatile solid changes
At the pilot scale, the results were similar to lab samples (Table 6). The maximum (32.67 %) and minimum (12.52%) VS reduction were observed in the treatments E and A, respectively. The difference in the reduction of VS (%) between the treatments E and F compared to A showed that microorganisms were a key factor in the decomposition of organic matter during composting, and M1 microorganisms have a more significant effect on the decomposition of organic matter compared to the M2 microorganisms.
Micro-macro elements and heavy metals analysis
Examination of micro and macro elements at the pilot scale showed that the amount of these components was the same as the results at the lab scale, with the difference that among the three treatments evaluated, the nutritional value of treatment E was higher than that of other two treatments (Table 7). Treatment E had the maximum scales of Mn, Mg, Fe, K, P, and Ca compared to other treatments.
Phytotoxicity tests
Phytotoxicity is an important factor in determining whether the fertilizer produced is a barrier to plant growth or not. Ammonium and organic acids are important inhibitors in organic fertilizers [33, 45]. The maximum germination rate was observed in two treatments E and F (96% and 88%, respectively), while the control showed minimum germination (53%). The use of poultry manure improves the germination rate in the final compost [46]. Similar studies showed that a reduction in the C/N ratio led to a reduction in toxicity of rice straw compost [37], in this study also showed that treatment E recorded the highest C/N ratio reduction.
Effect of compost on wheat growth
Both treatments E and F significantly showed higher fertilizing properties on wheat growth indices compared to the control (A). However, treatment E showed the maximum positive effect on wheat growth indices. There were no significant differences between the treatments of 10 and 20% of compost for both E and F treatments (Table 8). Determination of the effects of final composts on plant growth is known as one of the most informative tests for compost quality evaluation. Humic substances present in compost improve shoot biomass through hormonal effects on root elongation and plant development. A very important point in this field is the quality and maturity of the produced compost, which has a great impact on plant growth. Microorganisms with enzymatic activity have a great contribution to the decomposition of cellulosic materials and the improvement of the composting process [47].