*3.1. Characterization of Samples, Substrates, Inoculum and Mixtures in the Digester*

Studying the characterization of substrates, of the inoculum and from the mixture that exists in the digester, before and at the end of the BMP test, is essential to understand the development of the process and to be able to make decisions about the results obtained during the AD process. All tests have been conducted as described in Section 2.2, and the results are shown below.

Table 2 shows characterization results of the substrate (CrM), of the inoculum (S) and the mixture in the digester (CrM + S) before the BMP test, and after completion of the latter at 20 days. All results are indicated in the respective units, per gram of substrate, or per milliliter of sludge or reactor mix.


**Table 2.** Characterization results for BMP tests of residue CrM, at the start and after completion of the test.

CrM substrate is presented as a substrate with a high humidity level (62.60%) which makes its solubilization, in principle, easy and the process fast and profound. If the LPCH content is compared, it is observed that it is a carbohydrate-type substrate, especially rich in simple cellulose-type carbohydrates. The digestion of this type of substrate is stable and fast, but with the likelihood of releasing VFA during its digestion from the acid digestion of monosaccharides and other simple carbohydrates. However, because of the presence of a certain protein content, a small amount of ammoniacal nitrogen is likely to be released that can compensate by acting as a buffer for the slight acidification caused by VFA. In terms of COD it is a relatively rich substrate in carbonous matter and organic matter, with a particularly high solubility (of 58.81%), indicating that, of the entire COD, almost 60% is directly accessible to microorganisms without the need to hydrolyze or release from encapsulation due to being a particulate substrate. For this easy accessibility, a rapid degradation is expected, even though the inoculum is a granular UASB sludge, difficult to solubilize. In terms of nitrogen content, it is not very high. It is distributed in ON that comes from the light protein content, and in AN, whose content is not excessive, in fact, is below the limit of accumulation studied (2 g/L), so it is expected that the AN will be released as a buffer for pH control and compensate for possible acidifications, without accumulating and ending up inhibiting the process by excess ammonia in the reactor. The CrM material has a C/N ratio of 12.33, which is close to the optimal C/N ratio (approx. 20) [33,35,36] to ensure stable digestion so it is expected that the process will have alterations, but without impact, such as a release of VFA due to the carbohydrate content, compensated with a slight release of ammoniacal nitrogen that will act as buffer dampening its effect.

Analyzing changes in the composition of the mixture in the reactor, before and after the BMP test, it can be observed that the humidity has been slightly reduced, by −8.50%, which is logical when it comes to anaerobic, closed, and wet digestion digesters, as is the case of a UASB digester specially designed for liquid substrates. The reduction of vs. and TS is very remarkable, of −51.82% and −55.11% respectively, which gives an idea that the process has developed correctly because the organic matter present in solids form has been digested. The reduction of COD has been very low, of −10.31%, indicating that, although the digestion process has been correct, the methanization has not been completely profound (later it will be analyzed with the content in methane, since the carbon contained in the COD is the one that is transformed into methane). However, the reduction in COD<sup>f</sup> has been very noticeable, of −55.61%, indicating that, although only 10% of the COD has been reduced to be converted into methane, this has been practically a reduction in COD that is not encapsulated, and is therefore directly accessible to micro-strategies. This means that the methanization process has not been complete, by a failure of the disintegration + hydrolysis stage, which will provide a lower-than-expected methane content, below 60% which is considered the stable development limit. According to the nitrogen content, TKN is slightly increased by 3.89% by the release of some of the nitrogen encapsulated in the proteins, the ON. In fact, the ON is reduced by −9.75% precisely by the release of this nitrogen, by degrading the proteins. For its part, the AN is increased by 28.29% when the ON is released, until it reaches a value of 1.24 mg/mL, falling below the accumulation limit (2 g/L) in this way it is expected that the released ammoniacal nitrogen will act as a buffer, dampening any acidification, for example, that coming from the release of VFA when digesting carbohydrates. The initial and final pH values are very similar; however, they are expected to have varied during the 20 days of process. As for alkalinity, TA is increased by 43.92%, giving an idea of the stability of the process. Intermediate alkalinity is also increased by 64.94%, indicating that either no VFA has been released, or on the contrary these have been neutralized by the buffer effect of the released AN.

All these assumptions should be checked later with the analysis of the process that develops.
