Optimization of Anaerobic Digestion Processes of Organic Waste in Chiapas, Mexico, for Biogas Production

3.1. Characterization of Organic Waste

The organic waste used in this study was characterized through bromatological and physicochemical analysis, including the determination of Hydrogen Potential (pH), Chemical Oxygen Demand (COD), Total Solids (TS), Total Volatile Solids (TVS), Total Volatile Solids/Total Solids ratio (TVS/ TS), total carbon (C), total nitrogen (N), and the Carbon/ Nitrogen ratio (C/N) following the procedures established by the standards described in Table 1.

3.2. Experimental Design for Biogas Production

The experimental design strategy, as described in Table 2, consisted of a single-factor experimental design, performing the Analysis of Variance (ANOVA) using Fisher's, Tukey's, and Duncan's LSD tests, which corresponded to the methane production in mL during a digestion time of approximately 30 days, to determine significant differences between treatments. The experiment consisted of 10 treatments and 3 replicates for each treatment to obtain 30 experimental runs. In this study, a reliability analysis of the variance was conducted using RStudio software version 4.3.1 to get a single response variable.

The treatments M1, M2, and M3 consisted of 45 grams of fruit and vegetable waste—specifically pumpkin peel, tamarind peel, and corn cob, along with cow, sheep, and poultry manure in the same concentrations. For treatment M4, the three fruit and vegetable wastes were maintained at a concentration of 36 grams, including cow and sheep manure, at equal concentrations. Treatment M5 and M6 maintained the same conditions as treatment M4; however, for treatment M5, the fecal waste comprised cow and poultry manure, and in treatment M6, the fecal waste was made up of sheep and poultry manure. Treatment M7 incorporated the three types of fruit and vegetable waste and the three types of livestock fecal waste at a concentration of 30 grams per organic waste. Ultimately, treatments M8, M9, and M10 individually analyzed livestock fecal waste of cows, sheep, and poultry to evaluate the biogas production that each of these treatments could produce.

3.3. Establishment of Anaerobic Digestion

The anaerobic digestion process was carried out in 1.8 L tanks. For this purpose, a biodigester was designed based on a modified version of a discontinuous flow biodigester according to Cardenas C., 2022 [19], which consisted of a vacuum-sealed high-density polyethylene tank with a neoprene lid (to maintain anaerobic conditions). A working volume of 1.2 L was maintained, adding both the substrate and the an-

aerobic inoculum. In addition to using a fixed volume of 180 grams of organic matter from the mixture design described in Table 2 of the experimental design, the agitation applied to the biodigesters was performed manually at room temperature of 36 ± 2 °C, they were sealed with Teflon tape and heat-shrink seals for applying the thermofusion process, which consists of assembling the parts of the biodigesters with heat as mentioned by Camarena et al., 2024 [20], to avoid possible leaks, which can not only result in the loss of the biogas produced; but also in significant environmental and safety consequences.

3.4. Methane Quantification

The CH₄ gas quantification was monitored simultaneously throughout the entire biogas production kinetics, measuring each treatment daily during the 30 days of digestion retention. This process was carried out using the volumetric displacement method, which is based on the quantification of the volume of CH₄ produced during the anaerobic digestion retention process by using a 1N NaOH solution in a range of 15 to 20 g/L which allows the CO₂ present in the biogas to be converted into sodium carbonate (NA₂CO₃) [21].

3.5. Calculation of the Specific Methanogenic Activity of Anaerobic Inoculum

Specific Methanogenic Activity (SMA) corresponds to the maximum methane production and the rate of organic matter consumption expressed in g COD/g TVS*day [22]. SMA coefficients can be expressed in different ways, such as mL CH₄/g TVS*day or g COD/g TVS*day. The latter form will be used in this study, which is known as substrate organic loading based on COD, using Eq. (1).

(1)

Where:

SMA= Specific Methanogenic Activity in g COD CH₄/g TVS*day.

CMP= Cumulative Methane Production in mL CH₄/day.

TVS = Total Volatile Solids (TVS) concentration in the inoculum in g/L.

V = Volume of inoculum used in L.

SCFM = Stoichiometric Conversion Factor of Methane (350 mL CH₄/g COD).

4.1. Fromatological and Physicochemical Characterization of Organic Waste

The results of the carbon and nitrogen ratio of the tamarind husk and corn cob leaves waste are presented in Table 3, with a C/N ratio of 21.87 and 24.34, respectively. The study by Castro R. et al., 2021 [23] initially indicated that the anaerobic digestion process benefits from the adequate growth of the microorganisms in the anaerobic inoculum during the methanogenesis stage as the C/N ratio values fall within the optimal range established in the FAO Biogas Manual [6]. These findings positioned carbon as the indispensable source of energy for the microorganisms present in the anaerobic digestion process and nitrogen as the necessary element for their cellular duplication [24].

In contrast, the results of the bromatological and physicochemical characterization of the organic waste also included the TVS/TS ratio, which represents the percentages of biodegradable organic matter capable of being transformed into biogas. For this parameter, the values of 60, 92, and 95% were obtained for pumpkin peel, tamarind peel, and corn cob leaves, respectively. According to Ren et al. (2003) [25], the optimal value of the TVS/TS should be higher than 50%; therefore, the results indicate that the substrates contain a sufficient amount of assimilable organic matter to support anaerobic microbial activity and the production of biogas.

The assessment of the COD and the TVS/TS ratio for each substrate serves as an indicator of the biodegradability of the organic matter and its subsequent conversion into biogas. Table 3 shows that the corn cob leaf residue contained 1.13 grams of COD/L, a value higher than those obtained from the pumpkin peel and tamarind peel residues. The higher resulting value indicates that the corn cob leaf residue is the substrate that could present a better effect in terms of substrate/inoculum ratio for obtaining biogas [26]. However, the COD values of the initial characterization obtained in each of the residues do not necessarily indicate that all the organic matter contained is fully biodegradable. Therefore, the SMA of the anaerobic inoculum was calculated to determine the potential differences when each of the substrates was mixed with various residues used as anaerobic inocula.

4.2. Physicochemical Characterization of the Experimental Treatments

Table 4 presents the results of the physicochemical characterization for the parameters' including initial pH, TS, TVS, and the TVS/TS ratio for each of the formulated treatments at the onset of the anaerobic digestion process. Additionally, pH was monitored at the end of the retention time of the digestion to identify the decrease in this parameter and determine the potential changes that could modify the stages of the digestion process. Therefore, the pH was not regulated at the beginning of the anaerobic digestion. However, the pH of each material and the mixture was evaluated at the beginning of the digestion, as shown in Tables 3 and 4.

As mentioned above, pH adjustments are performed at the beginning of each anaerobic digestion process; however, for this experimental research, such adjustments were omitted because most of the treatments presented an initial pH between optimal ranges (6.8-7.4). When mixing substrate-anaerobic inoculum, the following presented optimal initial pH with values of M1: 6.98, M2: 7.11, M4: 7.07, M5: 7.12 and M6: 7.17, Treatment M3 presented a pH of 6.78 below the optimal range, and treatments M7, M8 and M9 with pH higher than the optimal range, with the following values of 7.73, 8.46 and 8.29, respectively. The pH value in biodigesters determines the production of biogas and its composition [6].

According to the study by Parra., 2014 [27], the pH determination in the anaerobic digestion process expresses the degree of acidity or alkalinity under which the organic matter is biodegradable. If the pH is not in the optimal range, adjustments are required to maintain the stability of the process. When the substrates present high concentrations of acidic organic matter, they are highly fermentable and tend to acidify the medium, which can inhibit the action of microorganisms and eventually cause the failure of the digestion process. These values also express the increase and production of Volatile Fatty Acids (VFAs) associated with the formation of long-chain or branched volatile fatty acids, which could prolong the adaptation times of microorganisms present in anaerobic inoculum during the hydrolysis stage [28]. According to Deubleien., 2008 [29], adjusting the pH between the optimal range of 6.8-7.4 is essential, not only for the hydrolysis stage but also for the methanogenesis stage. Generally, these pH values favor the adaptation conditions of bacteria such as Methanosarcina sp, capable of producing CH₄ even in pH conditions lower than 6.5.

The LSD, Tukey, and Duncan tests conducted at an alpha of 0.05 showed that the mixtures M2 and M5 do not present statistically significant differences in terms of optimal pH parameters. However, when relating these mixtures with the result of the rest of the treatments, there is a statistically significant difference resulting in a p-value < 0.05.

Table 4 shows a comparison between the initial and final pH parameters of the anaerobic digestion process for each of the experimental treatments analyzed, where a neutral pH was expected for all cases. The results indicate that the final pH of the treatments was not neutral and below the optimal value (pH: 6). According to the existing literature, a decrease in pH values below 6 may indicate that the biogas produced is low in methane content and, thereby, possessing energy characteristics that differ from the intended purpose of this research (generating biogas for injecting into the natural gas network) thus it is recommended to conduct a detailed analysis of the composition of the biogas generated in this experiment [30].

On the other hand, the results shown in Table 4 concerning the TVS/TS ratio indicate that the content of available organic matter in most treatments is above 60% and there is a large amount of organic matter biodegradable for the consortium microorganisms present in the anaerobic inoculum [11]. Treatment with a value of 0.55 g/g equivalent of TVS/ TS at 55% is within the optimal range of 40-60% concerning TVS/TS. As mentioned above, the high content of organic matter in the substrates, when digested by microorganisms without prior dilution, would slow down the anaerobic digestion process; therefore, a working volume of 1.2 L was maintained so as not to affect the efficiency and production of biogas [31].

Finally, a detailed analysis of the results in Table 4 regarding the C/N ratio reveals that none of the treatments meets the initial requirements for this parameter. According to the FAO Biogas Manual, the optimal range to establish the anaerobic digestion process must be between 30:1 and 20:1. By having concentrations below the C/N ratio of 20:1, the process could be affected by the inhibition of the microbiological activity of the anaerobic inoculum and cause excessive production of ammonia content within the biodigester, which in large quantities is toxic and inhibits the digestion process [6]. In general, if the treatments do not meet this parameter, to achieve an optimal C/N ratio, it is necessary to mix them with other types of organic waste that contain the appropriate proportions to obtain the optimal C/N ratio.

4.3. Experimental Process of the Specific Methanogenic Activity of Anaerobic Inoculum

For this study, the results of the SMA were used to analyze and quantify the activity of the methanogenic population within the anaerobic inoculum in response to the biomass present in the different substrates. This evaluation aimed to assess microbial performance in the presence of potentially toxic or inhibitory compounds. On the other hand, De Lemos., 2020 [32] established that the calculation of the SMA can not only determine the degree of biodegradability of the substrates but also estimate possible pH changes in the medium, in addition to determining the maximum organic loading capacity applicable to any other type of anaerobic inoculum beyond those used in this research work.

To clarify differences in SMA between the different anaerobic inoculum, Table 5 compares the different inoculum that reached their exponential phase in CH₄ production from day 6 to day 11. However, for the calculation of SMA, data from days 8 and 9 were taken because they contained the highest values of CH₄ production in the exponential phase of anaerobic digestion of each treatment.

The maximum SMA of the anaerobic inoculum used in this study reached a conversion rate of 0.58 g COD CH₄/g TVS*day, as observed with the cattle manure substrate. According to Jiménez et al., 2015 [33] and Van L. et al., 1996 [34], the optimal values of the SMA range between 0.27 to 1.45 g COD CH₄/g TVS*day. Therefore, the three types of anaerobic inoculum used in this experiment are within the optimal range, as shown in the results in Table 5. In the study conducted by Arano et al., 2023 [35], a maximum SMA of 0.22 and 0.19 g COD CH₄/g TVS*day was presented for the cows and poultry manure inocula, respectively. When comparing these results, it is evident that the SMA values obtained in this work are higher than those reported by the referenced authors, indicating a high biodegradability capacity and potential to assimilate the greater organic load across various substrates. However, specific data on the SMA of sheep manure inoculum is limited in the literature. However, the 0.52 g COD CH₄ /g TVS*day of the SMA described in Table 4 for this type of inoculum is within the optimal range established for the anaerobic digestion process under optimal conditions.

Based on the statistical analysis performed using ANOVA with the LSD, Tukey, and Duncan tests, no statistically significant differences were found between treatments as the p-value is greater than 0.05. These results imply a high energy efficiency in the production of methane when mixing any of the three types of inoculum (cow, sheep, and poultry manure) with the three fruit and vegetable residues (pumpkin peel, tamarind peel, and corn cob leaves) to formulate the treatments of the proposed experimental design.

4.4. Methane Production Behavior During the Anaerobic Digestion Process

The kinetics of biogas production are closely related to the growth of microorganisms present in the anaerobic digestion process, which defines the rate of CH₄ production and the degradation of organic substrates. In addition, it is the basic tool to scale up laboratory biotechnological processes to an industrial scale, allowing to predict the yield and production of biogas [36].

Therefore, for each anaerobic digestion process, it is important to evaluate the bacterial growth inside the biodigesters from the start of the typical curve, graphed to have greater control over the biogas production process, where three stages are evaluated, the start-up, the stabilization, and the decline [6]. Fig. (1) presents the results of the methane production kinetics from the different treatments described in the experimental design of this study.

As mentioned above, the biogas measurement was conducted using the volumetric displacement method to quantify the volume of methane produced during the anaerobic digestion process. This method involved the use of displacing solution–1N NaOH in a range of 15 to 20 g/L—due to its ability to react with the CO₂ present in the biogas and form sodium carbonate (Na₂CO₃) [21]. This reaction allowed an approximate measurement of the volume of methane produced in each of the treatments. The results in each of the graphs show that substrate–inoculum assimilation for biogas production from day 6 to 11. During this period, stability for the bacterial consortium is required to assimilate the load of organic matter present in the substrates. The results obtained by Briseño A. 2017 [21], in the same retention time of anaerobic fermentation (30 days), indicate that the assimilation of organic matter occurs between day 14 and day 25, similar to that reported by Quechulpa P. et al., 2021 [37], who report the assimilation of organic matter through the use of domestic biodigesters from day 13 to day 21, comparing the results, these would indicate accelerated assimilation for the methanogenic stage of the anaerobic digestion process.

To improve the results of the biogas production kinetics curves of this study presented in Fig. (1), a bacterial consortium of an active, stable biodigester rich in methanogenic bacteria should be added to shorten the start-up stage and improve the stabilization stage since the curves presented would be expressing low assimilation of the substrate with anaerobic inocula [6].

4.5. COD Removal Efficiency and Total Methane Production

Generally, COD indicates the amount of oxygen required to oxidize the organic and inorganic matter present in wastewater. In this study, it was important to evaluate the COD concentrations to identify the level and/or percentage of biodegradable organic matter in each designed treatment [38]. Therefore, the efficiencies of organic matter consumption during methane production in each treatment were evaluated using the initial and final COD of the anaerobic digestion process as a variable. The results described in Table 6 allowed for permanent control over each of the treatments, identification of the percentages of removal from the biodigesters, and evaluation of the efficiency and performance of biogas production during the digestion process.

The M3 and M10 treatments presented the highest efficiency in removing organic matter, with a value of 71% and 97%, respectively. This is attributed to the capacity of poultry manure to facilitate organic matter degradation, as demonstrated by the percentages of removal of this anaerobic inoculum.

Regarding methane production, the ANOVA used with the LSD, Tukey, and Duncan tests showed no statistically significant difference between the experimental treatments due to the normal distribution or randomness in the data extracted during the experiment. These results indicated that the 10 designed treatments could generate the same performance in terms of biogas production.

The M6 treatment obtained a 67% COD removal, exceeding the reported 64.9% COD removal in plant waste after 20 days of retention of the anaerobic digestion process [39]. In comparison, the rest of the treatments presented lower removal below 59% in COD degradation. Therefore, it is recommended to perform a detailed analysis regarding the chemical composition of these treatments (M1, M2, M4, M5, M7, M8, and M9), according to Crombet C. et al., 2016 [40], the variation in COD removal in the treatments attributed to the characteristics and composition of the anaerobic inoculum.

As observed in Table 6, the removal of COD in the treatments where only anaerobic inocula are evaluated presented a greater degradation of organic matter, which indicates that the treatments need to have a close substrate-inoculum relationship to be efficient and to optimize the degradation of organic matter to achieve high methane production [41].

Similarly, in the studies reported by Placencia R., 2014 [8], evaluations were conducted on methane production from cattle and pig manure digested with agroindustrial waste, which shows that cattle manure has a greater potential for methane production, presenting a biogas volume of 860 L of methane; on the other hand, a study carried out by Pimentel L., 2019 [42] suggested sheep manure to be the best substrate for biogas production with a yield of 50 L of methane. When comparing the results obtained in this research project, it is estimated that by generating mixtures between agroindustrial organic waste and any of the two types of manure already mentioned, high biogas production yields are produced with quantities of up to 850 L of CH₄ [8].

Although the percentage may vary by source, organic matter typically constitutes the majority component of Municipal Solid Waste (MSW). Chiapas' significant agricultural activity and the composition of its domestic waste represent both a challenge and an opportunity.

If not properly managed, this waste can contribute significantly to environmental problems such as soil and water pollution, greenhouse gas (methane) emissions, and public health issues.

However, given the large volume of organic waste generated, Chiapas has enormous potential to implement sustainable management strategies that can transform this “waste” into valuable resources. Anaerobic digestion, for instance, not only generates biogas—a renewable energy source—but also produces a digestate that can be used as fertilizer. Therefore, this study highlights the adequate management of agricultural waste through the control of variables that guarantee the appropriate production of biogas.

In addition, it promotes investment in research and development, technology transfer, financial support for municipalities, and the creation of a regulatory framework that incentivizes the management of organic waste, all of which are essential to strengthen the management of waste in Mexico and move toward a more circular and sustainable economy.