*3.7. Assessment of the Evolution of the Anaerobic Process*

Figure 6 represents the biogas, methane and hydrogen generation curves, along with the pH evolution recorded during the CrM residue AD process, and the process as a whole, can be evaluated. It is observed that:

**Figure 6.** Evolution of the digestion process of residue CrM. Comparison of the generation of biogas, methane and hydrogen together with the evolution of pH.

During the first day (day 0–day 1), there is a delay in the generations without a trace of methane or hydrogen, so it is assumed that the phase of disintegration + hydrolysis occurs during the first day.

Between days 1 and 3 hydrogen generation begins until it reaches a peak on day 3. This means that during days 1 to 3 the phases of acidogenesis and acetogenesis occur. In fact, it is associated with a reduction in pH, indicating that acidic elements such as acetic acid and VFA have formed. At that point biogas and methane begin to appear. The growth of methane is slower (with a lower slope) as it is formed only by acetoclastic route.

During days 3–4 hydrogen disappears at a steady rate as methane and biogas continue to grow, indicating that methane is also generated by acetoclastic methanogenesis.

Between days 4 and 5 the reduction of hydrogen stops, and so do biogas and methane, so it is assumed that there is a slowdown of the process and not an inhibition, since it is subsequently resumed. In addition, being associated with a decrease in pH is assumed to be a slowdown by accumulation of acid elements, which is subsequently resumed when the pH is increased again during days 5 to 6. This is due to the small content in AN that is released and acts as a buffer, as expected as studied in Section 3.1.

From day 6 the second peak of generation and hydrogen appears, which is accompanied by a decrease in pH, so it is understood that the phases of acidogenesis and acetogenesis are resumed and the process of digestion resumes, In fact generations of biogas and methane are also resumed, which occurs mainly by acetoclastic route as the pH is increased by the transformation of the acid elements into methane.

From day 9 hydrogen reduction begins, so it is understood that hydrogenotrophic methanogenesis begins. This is confirmed by the generations of biogas and methane increasing its speed and slope, so methanogenesis is two-way, acetoclastic (pH increases) and hydrogenotrophic (hydrogen content is reduced).

On day 11 hydrogen is completely consumed, continuous methanogenesis by acetoclastic, pH is progressively increased to neutral values.

The process is stabilized on day 12–13 and ends without any inhibition, with correct pH parameters and with an evolution as expected and studied.

For all of the above, the process develops correctly, with a slight acidification by accumulation of acidic elements, but without impacts when it is dampened by the buffer effect of the AN released during the degradation of the small percentage of proteins. There are two phases of digestion, clearly identified by hydrogen peaks and the two pH changes. It is therefore assumed that during the first phase the organic matter is digested directly accessible, and during the second stage part of the non-soluble organic matter begins to hydrolyze.

## *3.8. Determinations and Mathematical Adjustment of The Anaerobic Process*

Depending on the COD values determined before the BMP test and once it has been completed, therefore the degraded COD values are known, along with the values obtained in terms of methane generation, the process can be mathematically adjusted (Table 4) as studied in Section 2.5, to extrapolate and compare it with other scenarios.


Based on the initial and final COD values in the reactor mixture, the theoretical production of methane is determined, that is, what is expected to be obtained if the process had been developed correctly and all that degraded COD would have been transformed into methane. This is estimated at 487.879 NmL of methane per 100 g of CrM residue introduced into the digester. The theoretical generation is only 6.396% higher than the average generation obtained, so it is understood that, within the degraded COD, the process has been complete since virtually all the COD consumed in methane has been transformed.

By adjusting the methane generation process to a kinetics of the first order, the maximum generation of methane obtained in all trials, quantified at 458.820 (±106.029%) is obtained. NmL of methane per 100 g of CrM residue, practically identical to the average generation of methane obtained. The disintegration constant, which provides information on the speed of the process and especially hydrolysis, can also be determined. In this case it is estimated at 0.164 days−<sup>1</sup> , a fairly fast constant compared to other constants of disintegration of other elements, and that allows to compare the speed of the process with that of other substrates. This disintegration constant is an indication of the speed of hydrolysis, but not the depth of the process, for this it is necessary to go to another parameter.

It has been seen that of all the COD digested, almost everything has been transformed into methane, so the process has been robust, and has also been rapid according to the hydrolysis constant, but now arises the question of whether the process has been deep and complete, that is, whether much of the organic matter available in the substrate has been degraded in the substrate, or only a small part has been degraded. To do this, the degradation coefficient of the substrate that provides information on how much COD of the substrate has been biodegraded in the anaerobic process is analyzed. In this case it has been determined at 15.282%, that is, although almost all the degraded COD has been transformed into methane, with respect to the total COD available on the CrM substrate only 15.282%. This indicates that the COD<sup>f</sup> which is directly accessible to microorganisms has probably been degraded, and subsequently, in the second stage of digestion, part of the COD that is encapsulated in the substrate is digested. It should be remembered that because of its high content of hemicellulose and lignin is a particulate substrate with strong outer membranes that makes it difficult to hydrolysate and release OM. In addition, it should be borne in mind that the nature of UASB sludge is granular, specially thought for wet digestion, combining two factors, that the substrate is strongly particulate by the high content of hemicellulose and lignin, and the granular nature of the inoculum, which complicates the adhesion of hydrolytic enzymes to the substrate. These low levels of degradation would also explain the methane content below 60%. These results open the door to process improvement through techniques such as pre-treated, which improve accessibility to the substrate, breaking the barriers created by lignocellulose and making the particulate and encapsulated COD solubilized and therefore directly accessible.

## *3.9. Comparison of Results with Previous Literature*

This section compares the results obtained with some of previous research. This is intended to check whether the test conditions have been adequate, whether the use of the new process indicators can be reliable, and whether the methodology applied is valid. They also serve to leave the main innovations developed in this manuscript marked. Publications with a considerable time lapse and similar test characteristics have been selected, however they are not the same, since precisely one of the novelties introduced is the use of UASB sludge as an inoculum. The approach of most research is microbiological, not process development, so it is also another point in favor of innovation and original input.

Table 5 shows the results of other research, and in the variables that can be checked, the results are quite similar. It is then confirmed that the new methodology used can be assumed as correct, that the changes introduced are valid and that new information is provided about:



