3.2.2. TOC Values and C/N Ratio

An optimal C/N ratio in the feedstocks is important for the optimal growth of microorganisms [40], for the reduction of VFA accumulation [41], to prevent inhibition [39], to mitigate C and N emissions [42], to analyze organic matter in aquatic ecosystems [43], and for the production of lipids in yeasts [44], among other effects. For example, the optimal C/N ratio for anaerobic digestion [45] and composting [46] is between 20 and 30.

Sewage sludge is known for its lower C/N ratios due to high losses through ammonia emissions [47]. Similarly, low C/N ratios occur in slurries [41], manures [48] and slaughterhouse waste [49]. On the other hand, grass has a higher C/N ratio, between 10 and 25 [50]. For some biomass and waste sources, significantly higher values of C/N ratios have also been reported, up to more than 500 for wood shavings [50]. The C/N ratio is defined as the ratio between organic carbon (TOC) and total nitrogen (TN). Figure 3 shows the results for TOC values and the C/N ratio.

From Figure 3, it can be seen that the C/N ratios for untreated samples are within ranges as reported previously. As expected, the highest values were found in untreated grass samples (C/N ratio of 13.5). After thermal or biological pretreatment of the grass (G) samples, the C/N ratio decreased. There are various reasons for this decrease, one being that the C/N ratio decreased as a result of increased TN. Another reason for C/N decrease after biological pretreatment is that micro-organisms consume more carbon than nitrogen. The decrease in the C/N ratio could also be the result of a loss of carbon as CO2 by mineralization during the process [33].

The C/N ratio for all pretreated samples at both temperatures was between 4 and 7. For the mixtures with sludge (S + R, G + S, G + S + R) after thermal treatment, the C/N ratios were still higher compared to those from the sludge samples (S), indicating that the presence of grass and rumen fluid did positively affect the C/N ratio. However, C/N ratios in all samples after pretreatment were significantly lower than is suitable for anaerobic digestion and composting processes. Thus, the problem could be solved by mixing different organic substrates, such as grass and other organic waste.

TOC values in all samples containing sewage sludge (S) increased after thermal or biological pretreatment, and the highest value was detected in the sample thermally treated at 80 ◦C. These results confirmed that thermal pretreatment accelerates the biodegradation of materials such as sewage sludge and grass, and thus increases the TOC content in the liquid samples [39]. At a lower temperature (38.6 ◦C), the highest amount of organic carbon was released in the cases when rumen fluid was added to the reaction mixtures (S + R and G + S + R). This is in accordance with previous studies, where it has been reported that microbial cultures from rumen fluid have a great capacity to increase the hydrolysis of lignocellulosic substrates [51] such as grass [52]. Therefore, it can be concluded that rumen fluid significantly affects the biodegradation of organic materials. Thus, a combination of lower temperature thermal treatment and biological treatment could be interesting for such a purpose. Similar conclusions could be adopted for both the *t*-tests performed with the results of TOC measurements and those performed for TN concentration.

**Figure 3.** Total organic carbon (TOC) results (mg/L) and the carbon to nitrogen ratio (C/N).

## 3.2.3. Chemical Oxygen Demand (Soluble COD) Measurements

The COD represents the amount of oxygen required to oxidize organic material into water and CO2 and is therefore a measure of the quantity of organic material present in the material. Values of soluble COD measurements before and after pretreatment are shown in Figure 4. In general, as shown, the highest COD values were measured in all the samples exposed to thermal pretreatment at 38.6 ◦C, while at 80 ◦C the values were lower (except for sewage sludge).

The COD values for the grass samples significantly increased after thermal pretreatment at 38.6 ◦C as compared to the untreated sample and decreased after thermal pretreatment at 80 ◦C as compared to the value at 38.6 ◦C. However, both values were higher than in the untreated sample.

This increase is in agreement with the findings of previous studies, which state that thermal pretreatment breaks down the cell walls, which enables the transfer of organic material to the liquid phase and consequently increases the COD [39]. A study by Ariunbaatar et al. [53] also confirmed that thermal pretreatment increased solubilization of organic solids and/or increased hydrolysis, making the substrates more available for anaerobic microorganisms; thus, in the subsequent anaerobic digestion process, biomethane production was enhanced.

As in the grass samples, the sewage sludge samples also yielded increased COD values after thermal pretreatment. After pretreatment at 38.6 ◦C, the highest COD was detected in the mixture of grass and sewage sludge (G + S). On the other hand, the presence of rumen fluid in the samples with grass (biological pretreatment at 38.6 ◦C) caused a slight drop in COD values. This can be explained by the fact that the microorganisms from the rumen fluid hydrolyze the macromolecules (lignocellulose and proteins) in the grass, thus reducing the amount of organic material [54]. Again, statistical hypothesis testing showed similar conclusions for TN and TOC measurements.

**Figure 4.** The soluble chemical oxygen demand (COD) results (mg O2/L).
