3.2.1. Results of N, P and K Analysis

Concentrations of nitrogen, potassium and phosphorus (NPK) in the samples are expressed in the form of N, P2O5 and K2O, since NPK values in the organic substrates are often expressed in that way [28]. Table 3 shows the results for P2O5 and K2O content, and Figure 2 shows the content of TN.


**Table 2.** Basic characteristics of the materials in use.

Results in Table 3 show that the lowest concentrations of K2O and P2O5 values were generally obtained in untreated samples (samples G or S). After lower temperature thermal pretreatment at 38.6 ◦C, concentrations of K2O and P2O5 in all samples increased as compared to untreated samples (Table 3). The highest values of K2O were obtained in the mixture of grass and sludge (sample G + S), while the highest P2O5 value occurred in the sludge sample (S). It is also interesting to note that in the case of biologically-pretreated samples with the addition of rumen fluid (samples G + R and G + S + R), the K2O and P2O5 content decreased, compared to the same samples without rumen fluid. Based on these results, it can be concluded that enzymes and bacteria in rumen fluid break down the cell membranes and degrade these nutrients [29]. In addition, if anaerobic fermentation occurs because of the presence of enzymes in the sewage sludge, any released polyphosphate can be completely degraded to PO4 <sup>3</sup>−−P [30].

In the case of higher temperature thermal pretreatment (80 ◦C), concentrations of K2O decreased, while the concentration of P2O5 increased slightly in comparison with the lower temperature pretreatment. Among all samples, the concentration of P2O5 was highest in the samples treated at 80 ◦C, especially for the S and G + S samples; this shows that thermal pretreatment deformed the chemical bonds in the sludge and grass, and thus P is released from the raw substrates. According to Zou and Li [30], the cell membranes of sludge could be disrupted via thermal pretreatment, so that P (mainly in the form of polyphosphate) could easily diffuse out of the cytoplasm. Kuroda et al. [31], on the other hand, discovered that nearly all the polyphosphate could be released from activated sludge simply by heating it at 70 ◦C for only a few hours.

**Table 3.** Concentration of potassium in the form of K2O and phosphorus expressed as P2O5 in the samples.


For the purpose of the further use of these substrates for biofuels and biochemicals (e.g., for production of biogas and biofertilizer), a combination of grass and sludge is suggested rather than using mono substrates, owing to the high NPK content, which is efficient for biofertilizer production. However, it should be noted that such pretreated materials that contain sewage sludge have restricted further applications [32]. Figure 2 shows the results for total nitrogen concentrations (TN). The highest amount of TN was observed in the sewage sludge sample and its mixtures, which is in accordance with the fact that the raw sewage sludge contained more TN than grass or rumen fluid (as shown in Table 2).

**Figure 2.** Concentrations of total nitrogen (mg/L).

With thermal pretreatment of samples at 38.6 ◦C, the amount of TN increased. The increase can be explained by the decomposition of proteins under the effect of heat and the action of microorganisms [33]. In the case of sewage sludge, the higher the temperature, the higher the TN concentration. The presence of anammox and denitrifying bacteria in the sludge (and also rumen fluid [34]) contribute importantly to the conversion of ammonium and nitrite into N2 [35].

For mixtures of sludge and rumen fluid (S + R), concentration of TN decreased, compared to the sample containing only sludge (S) under the same conditions (38.6 ◦C). On the other hand, in the case of mixtures of grass, sludge and rumen fluid (G + S + R), the concentration of TN after pretreatment was higher than in the same mixture without rumen fluid (G + S). This indicates that rumen fluid actively participated in the degradation of grass, although the degradation mechanisms are still quite unclear, since the composition of rumen fluid is complex [36]. The main microbial population of rumen fluid includes bacteria, fungi, archaea, and protozoa, of which bacteria and fungi are mainly involved in lignocellulose degradation, while archaea are related to CH4 formation [37]. The degradation process occurs via lignocellulolytic enzymes that are capable of digesting lignocellulosic materials (mainly consisting of cellulose, hemicellulose and lignin) into proteins, volatile fatty acids (VFAs) and gases [38].

At the highest pretreatment temperature (80 ◦C), TN concentrations were similar to those at the lower temperature (38.6 ◦C), while in the sample of sewage sludge (S) the concentrations were even higher. Similar results regarding TN release during thermal pretreatment have been reported previously [39].

Statistical *t*-tests performed for TN concentration show significant differences in the values between treated and untreated samples (at 90% confidence interval). The tests also showed significant differences among the different feedstock materials (sludge, grass, and rumen fluid). On the other hand, less significant differences were found in the case of biologically treated samples when compared to the same biologically untreated samples. The t*-*tests comparing results of the higher and lower temperature thermal pretreatment provided similar results, since in many cases the differences were insignificant.
