Influence of Molasses and Caesalpinia spinosa Meal Inoculums on Biogas Production from Cattle Manure

Abstract

The management of organic waste through anaerobic digestion is an alternative to energy recovery. This research focused on evaluating the production of biogas with different inoculums. For this purpose, two types of systems were implemented—one used a heating system controlled by an STC-1000 thermostat, while the other used a solar heating system under a polycarbonate parabolic trough. The experiment was carried out at laboratory level with 3 L PET bottle biodigesters and the biogas produced was collected with the water displacement technique in 3 L bottles, calibrated every 50 mL, over 43 days. Inoculums of the following manure concentrations were used: water (1:5, 1:2, 1:3) mixed with Caesalpinia spinosa meal and molasses. The results determined that the thermostat-controlled heating system generated 69.6 mL/day of biogas while the other system produced 610.9 mL/day. On the other hand, the T1 treatment with a manure:water ratio of 1:5 and molasses and Caesalpinia spinosa meal inoculums in both systems had a higher average biogas volume. In terms of methane (CH₄), the highest value of 76.9% was obtained through the T1 treatment under the controlled heating system. This allows the production of biogas with a high concentration of methane, which in future applications can be utilized for residential or industrial purposes, promoting economic, social and environmental development. Since the main challenge in the production of biogas is to reduce the digestion time, which is influenced by the temperature of the site, two types of inoculums with a low cost and easy access were used.

1. Introduction

Environmental concerns stemming from greenhouse gases generated by the decomposition of organic waste, together with the potential depletion of fossil fuels, have increased the demand for alternative energy sources [1]. Sustainable energy production is a global challenge driving research towards renewable and efficient technologies [2]. Among these technologies, biogas production through the anaerobic fermentation of organic wastes has emerged as a promising option due to its decentralized energy availability [3]; this allows its production according to the existence of organic sources, contributing to the mitigation of environmental pollution [4].

Biogas is considered a viable solution to reduce the use of fossil fuels and reduce greenhouse gas emissions, due to its short carbon cycle [5]. This fuel is mainly composed of methane (CH₄) and carbon dioxide (CO₂), with traces of hydrogen sulfide, water and carbon monoxide (CO) [6]. The efficiency of biogas production depends on several factors, including the characteristics of the substrates used and the presence of inoculums that promote microbial activity [7].

Bovine manure is a substrate used in the production of biogas, which is subjected to anaerobic conditions in a process known as biodigestion [8]. This process involves the biological transformation of the substrate through the actions of anaerobic microorganisms, creating biogas and organic fertilizer as the end products [9].

The use of specific inoculums has emerged as a strategy that can significantly improve biogas production [7]. Inoculums are materials that contain live microorganisms capable of accelerating the anaerobic digestion process by providing an active and adapted microbial population [10]. In this context, molasses, a by-product of the sugar industry that is rich in fermentable sugars, minerals and organic compounds, is an excellent supplement for anaerobic digestion [11]; its addition can increase the activity of methanogenic bacteria, improve process stability and increase biogas production.

Studies such as that of Romaniuk et al. [12] have shown that the addition of molasses can increase the methane production rate and the efficiency of substrate conversion; to improve biogas production, it is recommended to add molasses in a proportion of 30% of the volume of the substrate based on bovine manure. According to the author, this addition can reduce the economic recovery time of the biogas plant to 1.2 years. Since molasses can serve as an additional source of carbon and energy for microorganisms, favoring microbial growth and activity, it can be used as a source of energy for microorganisms [13]. Furthermore, another study [14] developed a two-stage anaerobic digestion system to produce methane and hydrogen from molasses wastewater; this system achieved a maximum production rate of 2.4 L of methane per liter of reactor per day, converting approximately 71.06% of the energy contained in the wastewater into methane and hydrogen.

On the other hand, Caesalpinia spinosa meal obtained from tara is a novel co-substrate for improving anaerobic digestion potential [15,16]. The combination of molasses and tara meal as inoculums for the anaerobic digestion of cattle manure is an innovative strategy that could maximize the efficiency of the process. Molasses provides a rapid source of energy, while Caesalpinia spinosa meal favorably modulates the anaerobic microbiota, resulting in increased biogas production and improved biogas quality [17].

On the other hand, temperature and pH are other critical factors that affect biogas production [18,19]. Anaerobic digestion of cattle manure is generally carried out under mesophilic conditions (25–35 °C), which are ideal for the activity of metallogenic bacteria [20]. The addition of inoculums such as molasses and Caesalpinia spinosa meal can influence the maintenance of these optimal conditions, improving the stability of the process. Furthermore, the integration of inoculums in biogas production has significant implications for the sustainability and economics of biodigestion [21]. The utilization of agricultural by-products and industrial wastes such as molasses and tara meal not only improves the efficiency of biogas production, but also offers a solution for the management of these waste products, thus promoting a circular economy [22,23]. This sustainable approach is crucial for the long-term viability of biogas plants and for the reduction of the carbon footprint associated with the production of energy [24,25].

The combination of resources such as molasses and Caesalpinia spinosa meal offers a promising approach for the production of biogas from livestock manure. Molasses, a by-product of the sugar industry, produces between 1 and 1.2 million tons of sugar per year [26] and Caesalpinia spinosa meal, grown on about 10,000 hectares, has an average yield of 10 to 12 tons of pods per hectare [27,28]. The addition of these inoculums in the process of anaerobic digestion of cattle manure can increase the efficiency of biogas generation, making maximum use of the available resources. Molasses and Caesalpinia spinosa meal not only optimize biogas production, but also represent a competitive advantage for Peru in the international renewable and sustainable energy market.

There are studies on biogas production at the laboratory level, where different biomass substrate ratios have been used, such as in the study by Barrena et al. [29]. They worked with a manure:water ratio of 1:5 associated with 4% (v/v) of whey and 3% (w/v) of cattail. In this case, by increasing the amounts of manure and cattail but decreasing the whey, a good biogas production rate was achieved. There have also been investigations using a water: manure ratio of 1:2; the drainage water at higher volumes than that established by the evaluated scale does not contribute to biogas production and, with higher quantities of potato peels, a higher production efficiency is obtained. These researchers also evaluated biogas production on a pilot scale, where they installed a 12 m³ PVC geomembrane tubular biodigester with a working volume of 9 m³. The biodigester was fed with manure:water mixtures at a ratio of 1:5 and the hydraulic retention time was 29 days at an average ambient temperature of 14. 4 °C, where the biogas produced met the demand as fuel for the daily cooking of food by a family.

This study focuses on evaluating the influence of molasses and Caesalpinia spinosa meal inoculums on the biogas produced from cattle manure at the laboratory level, in order to contribute to the field of bioenergy and sustainable agricultural waste management. By identifying effective strategies to improve biogas production using inoculums derived from natural resources, this work builds on the preliminary findings obtained in a previous study by Garcia Saldaña [30], where the influence of molasses and tara (Caesalpinia spinosa) meal inoculums on biogas production using cattle manure was explored. This is because, at present, the main challenge in biogas production is the reduction of digestion time, which is influenced by the temperature of the installation site of the anaerobic system used. For this reason, we sought to use two types of low-cost and easily accessible inoculums.

2. Materials and Methods

2.1. Anaerobic Digestion Substrates

2.1.1. Caesalpinia Spinosa Meal

To obtain Caesalpinia spinosa flour, three kilograms of Caesalpinia spinosa pods were collected in the outskirts of the city of Chachapoyas, Amazonas. The beans were then dried in a polycarbonate parabolic dryer until all moisture was removed. Next, a simple mechanical process of crushing the pods was carried out using a manual mill. The product was then sieved with a 1.7 mm sieve to obtain fine, light yellow particles.

2.1.2. Molasses

The molasses used was obtained through the physical process of grinding sugar cane at optimum maturity (six months) until the juice was extracted. Subsequently, the juice was subjected to a boiling process until it reached a high sugar concentration, resulting in molasses.

2.1.3. Cattle Manure

Fresh cattle manure was obtained from the stable at the university campus of the Universidad Nacional Toribio Rodríguez de Mendoza and was collected the same day that the biodigesters were loaded. Rainwater was used to mix the manure with the other substrates, because this water does not contain chlorine and also for the purpose of promoting the harvesting of rainwater, since in the study area there is a dry season and a rainy season.

2.2. Experimental System

The experimental research arrangement consisted of the implementation of biodigesters at laboratory level, a process which was adapted from Barrena et al. A total of 36 biodigesters, 36 gasometers and 36 calibrated 10 mL containers were used to measure biogas. These containers were 3 L polyethylene terephthalate (PET) bottles. A connection was made between the biodigester, the gasometer and the biogas meter by using a 1/4-inch hose to connect the edge of the biodigester lid to the gasometer, thereby allowing the biogas to pass through. Two 6 mm holes were drilled in the gasometer: one for the biogas inlet and the other to allow the water to move to a third container.

2.3. Implementation of the Experimental System

The biodigesters were filled to 75% of their total capacity. The lids were hermetically sealed with silicone and secured with a clamp to prevent gas leakage. Six treatments were carried out in every sistem, using manure:water proportion in ratios of 1:5, 1:3 and 1:2, as specified in

Table 1. Proportions of substrate for each treatment in systems A and B.

Systems	Treatments
A	T1	T2	T3	T4	T5	T6
	(1:5)	(1:3)	(1:2)	(1:5)	(1:3)	(1:2)
	M + HT	M + HT	M + HT
B	(1:5)	(1:3)	(1:2)	(1:5)	(1:3)	(1:2)
	M + HT	M + HT	M + HT

Note: ratio 1:5, 1:3 and 1:2, ratio manure:water; M: cane molasses; HT: Caesalpinia spinosa meal.

and

Table 2. Proportions of substrate for each treatment.

Treatments	Variables				Useful Capacity of the Biodigester mL
	Manure	Molasses	Caesalpinia spinosa Meal	Water
	g	mL	g	mL
T1	400	300	30	2000	2800
T2	400	120	18	1200	2800
T3	400	40	12	800	2800
T4	400	-	-	2000	2800
T5	400	-	-	1200	2800
T6	400	-	-	800	2800

. Treatments 1, 2 and 3 included molasses in a v/v ratio and Caesalpinia spinosa meal in a w/w ratio, while treatments 4, 5 and 6 contained only the manure ratio. Manure and Caesalpinia spinosa meal were weighed on an OHAUS balance, while molasses and water were measured with a graduated cylinder, considering that 1 L of water equals 1 kg (Figure 1).

2.4. Biogas Production Operating Systems

The study was carried out using two systems with 6 treatments and 3 replicates each, under different temperature conditions. One system operated with temperature control by means of an STC-1000 thermostat (Elitech Technology, Inc., San Jose, CA, USA), maintaining temperatures between 25 °C and 35 °C; for this system, a rectangular plywood box was constructed that was 3 m long, 40 cm wide and 60 cm high, insulated with Technopor and equipped with three spotlights to provide continuous heat. The second system used a polycarbonate parabolic cylindrical tubular design, constructed with a concrete base, an iron frame and a polycarbonate roof, optimizing the concentration of ambient heat. Both systems were subjected to the same treatments and repetitions.

2.5. Measurement of Base Parameters

2.5.1. pH

A Multi 3620 IDS pH meter (Xylem Analytics, Kawasaki, Japan) was used to measure the pH of each substrate before loading the biodigesters and after mixing the treatments (

Table 3. Initial pH values.

Samples and Treatments	Initial pH
Water	7.27
Manure	7.60
Molasses	4.98
Caesalpinia spinosa meal	5.90
T1	6.5
T2	7.00
T3	6.8
T4	6.6
T5	6.5
T6	6.6

). To ensure the correct functioning of the anaerobic digestion system, the pH should be maintained at an optimal range of 6.8 to 7.5.

2.5.2. Temperature

The temperatures of the controlled heating system and the solar heating system under a parabolic trough were measured three times a day for 43 days using a BOECO Germany thermometer, which measures temperatures from −50 to +70 °C. Temperatures of 35.0 °C (maximum) and 25.0 °C (minimum) were recorded for the controlled heating system. For the solar heating system, the temperatures were 51.3 °C (maximum at 13:00) and 17.4 °C (minimum at 7:00).

2.5.3. Total Solids

For biodigesters operating with cattle manure, a total solids (TS) content between 10% and 12% is recommended for optimal contact between methanogenic bacteria and the substrate (Moncayo, 2013). In our study, total solids were determined following the methodology of Varnero (2015). A representative sample of the mixture used in the biodigesters was taken and analyzed at the soil and water laboratory of the Universidad Nacional Toribio Rodríguez de Mendoza. The results obtained are presented in

Table 4. Total solids values (%).

	Treatments
Total solids (%)	T1	T2	T3	T4	T5	T6
	13.29	22.5	39.2	11.37	20.3	36.3

.

The TS concentration was calculated using the following formula:

$$\mathrm{\%}\mathrm{S}\mathrm{T}={\displaystyle \frac{{\mathrm{M}}_2}{{\mathrm{M}}_1}} \ast 100$$

where

• ST: total solids concentration (%),
• M₁: fresh weight of the sample (g),
• M₂: dry weight of the sample at 65 °C (g).

2.5.4. Measurement of Biogas Volume and Methane Content

Biogas volume measurements were performed daily for 43 days at 07:00, 13:00 and 18:00 h, at which points the temperatures of both systems were also recorded. The volume of biogas produced was measured using the liquid displacement method, as used by Barrena et al. [29]. This method consists of conducting biogas through a hose into a vessel with liquid; the biogas displaces the liquid and the volume of liquid displaced is equivalent to the volume of biogas produced. The methane content of the biogas in the biodigesters was measured after 30 days of fermentation using a Sewerin Multitec 545 portable biogas analyzer (Sewerin, Gütersloh, Germany).

2.6. Statistical Analysis

In this study, an experimental design featuring completely randomized blocks (DBCA) was used, with a 2 × 6 factorial order (2 blocks by 6 treatments) and 3 replications, evaluated 3 times a day for 43 days. The statistical analysis of the volume of biogas produced was carried out with “R” software version 4.4.1. To determine whether there were differences in biogas production between treatments, the Kruskal–Wallis test was carried out. Differences between treatments were analyzed with Conover’s test, which compares multiple means or medians with a probability of p < 0.05. To evaluate the differences in biogas production between the two blocks (controlled heating system and solar heating system), Student’s t-test or the Mann–Whitney test were used.

3. Results

3.1. Evaluation of the Biogas Production of the Systems

Table 5. Differences in biogas production between systems.

Systems	Minimum	Maximum	Median	Media	Standard Deviation
Controlled heating	0.0	2135.0	595.0	619.6	419.1
Solar heating	0.0	2350.0	560.0	610.9	426.8

shows that the system with solar heating under a polycarbonate parabolic trough yielded the highest biogas production, with a value equivalent to 2350 mL/day, while the system with controlled heating by a STC-1000 thermostat yielded the equivalent of 2135 mL/day of biogas during the 43-day digestion period.

Figure 2 shows that the thermostatically controlled heating system generated the highest average volume of biogas, reaching 619.6 mL/day in 43 days; in contrast, the solar heating system (under the polycarbonate parabolic dryer) produced 610.9 mL/day, showing that there is no significant difference between the two systems.

Figure 3 shows the average biogas production according to the time of evaluation. The system with solar heating under the polycarbonate parabolic dryer achieved a higher average biogas production rate, with 648.5 mL/day recorded at 13:00 h, compared to the controlled heating system, which obtained a maximum production of 625.9 mL/day at the same hour.

3.2. Evaluation of the Influence of Inoculums on Biogas Production by System and Treatment

3.2.1. Biogas Production with a Controlled Heating System

The treatment that presented the highest average biogas production rate was T1, composed of a 1:5 manure:water ratio, which reached an average production rate of 731.9 mL/day when associated with inoculums; on the other hand, T4, composed of the same manure:water ratio but without inoculums, obtained an average production rate of 632.9 mL/day during the 43 days of anaerobic digestion (Figure 4). Likewise, Figure 5 shows that T1 achieved an average biogas production rate of 739 mL/day.

Temperature, regulated by an STC-1000 thermostat in a mesophilic range of 25 to 35 °C, showed variations during evaluations at 7:00 a.m., 1:00 p.m. and 6:00 p.m. The highest temperature at 7:00 a.m. was 28 °C on day 13, coinciding with a peak in biogas production. At 1:00 p.m., the temperature remained constant, favoring biogas production, especially in treatment T1, which had its highest yield between days 11 and 19. From day 25, biogas production decreased, until the evaluation ended when one replicate reached zero production. At 6:00 p.m., the temperature remained constant, reaching a maximum of 33 °C on day 28.

3.2.2. Biogas Production with Solar Heating System (Under Polycarbonate Parabolic Trough)

Treatment T1, with a (1:5) manure:water ratio and associated with inoculums, obtained the highest average biogas production rate, with an equivalent of 736.3 mL/day. On the other hand, T4, constituted by the same ratio (1:5) of manure:water but with an absence of inoculums, obtained an average production rate of 646.7 mL/day (Figure 6), indicating that in T1 the inoculums exerted an influence on the average biogas production rate. On the other hand, Figure 7 shows an average production rate of 739.0 mL/day over the 43 days evaluated.

Figure 8 shows the biogas production rate for each treatment and system applied. It can be observed that treatments T1 and T4 present a higher percentage of methane volume, evidencing that the controlled system has a better production rate than the solar system.

3.3. Production Start Time and Total Biogas Quantification

• Start of biogas production: Start-up occurred 14 h after starting up the biodigesters, which were loaded at 17:00 h and measured the next day at 07:00 h. This process was applied for both systems.
• Biogas production: During the 43-day evaluation, the total volume of biogas produced by the controlled heating system (T1) was 31,736 L, measured at 07:00 h (Figure 9). In contrast, the system with solar heating under a parabolic trough produced 32,933 L of biogas at T1, measured at 13:00 h (Figure 10), resulting in a difference of 1197 L in favor of the solar system. Small-scale biogas technology can replace fossil fuels in homes, allowing the use of environmentally friendly gas.

4. Discussion

The study of biogas production at the laboratory level is important because it elucidates the mixing concentrations of the substrates, as stated by Kabaivanova et al. [31]. In this study, two low-cost systems were constructed using 3-L PET bottles for biodigesters and gasometers, connected in series. Controlled heating was implemented in one system, operating within the mesophilic range of 25 °C to 35 °C—maintaining this stable temperature avoids drastic variations, which allows methanogenic bacteria to work more efficiently during the anaerobic digestion process [32].

Biogas production under specific substrate and temperature conditions is crucial for optimizing the efficiency of the anaerobic digestion process [33]. In this study, the results show biogas production levels of 1080.17 mL from cattle manure, 836.8 mL from vegetable waste and 714.7 mL from the mixture of manure with vegetable waste. It was concluded that the best substrate for use in the production of biogas is manure and vegetable waste; this is due to the fact that the main substrate for biogas production is cattle manure, as stated by Barrena et al. [29]. The Caesalpinia spinosa meal inoculum is a metagenomic battery selector, while molasses helps to accelerate anaerobic digestion, as mentioned by Helguero et al. [33]. This indicates that both Caesalpinia spinosa meal inoculum and molasses are accelerators in biogas production from bovine manure.

Likewise, in a study by Postigo et al. [34] it was noted that, as in beer production, where the selection of different non-Saccharomyces yeast strains can significantly influence the fermentation yield and sensory profile of the final product, in biogas production, the choice of substrates and inoculums, such as Caesalpinia spinosa meal and molasses, can optimize the efficiency and quality of the biogas produced. Different strains of non-Saccharomyces yeasts show how variations in biological components can lead to significant inclusion of specific inoculums in the anaerobic digestion process, which can improve the efficiency of the process and the quality of the biogas generated. Controlled environmental and physicochemical parameters are important for adequate biogas production because they have a direct relationship with the biodigester temperature, as stated by Mamani et al. [35] and Onen et al. [36]. Another important factor in anaerobic digestion is the pH, the working range of which during our research was 6.5 to 7.0 [37,38], with the cattle manure having pH values of 6.5 to 7.5, which is considered optimal for biogas production since this range allows the growth of methanogenic bacteria [18].

Also, León et al. [39] worked with total solids contents of 11% and 13% during a pilot study, using a hydraulic retention time of 21 days. In a similar approach to our study, a ratio of manure and water was used, together with molasses inoculums and Caesalpinia spinosa meal, with a total solids content of 13.3%. In this case, the anaerobic digestion process lasted for 43 days and biogas production started the day after the biodigesters were loaded. These data suggest that to optimize the operation of biodigesters, it is advisable to dilute the mixture, which allows better contact between the methanogenic bacteria and the substrate.

Finally, the quality of the biogas is reflected in the concentrations of its components. Methane (50% to 70%), carbon dioxide (30% and 45%) and traces of other gases such as H₂ S (0.005–2%), NH₃ < 1%), N₂ (0–2%), H₂ (0–4%) and H₂O (5–10%) were found in a study by Atelge et al. [40], where the methane concentration obtained from the mixture ratio (1:5) manure:water, associated with the inoculums (400 g (w/w) manure, 2000 mL (v/v) rainwater, 30 g (w/w) tara flour (Caesalpinia spinosa) and 300 mL (v/v) molasses), was 76.9%. Those results show that the biogas quality was superior to that obtained by Murillo et al. [41] from manure–water mixtures which were subjected to a mesophilic temperature of 35 °C during a hydraulic retention time of 20 days, where an average methane value of 31% was obtained for the product. Likewise, Sharma et al. [42] evaluated manure–water substrates associated with inoculums at two temperature conditions, mesophilic and psychrophilic, where they obtained methane concentrations of 62.33% to 69.16% and 65.21% to 69.15%, respectively.

The optimization of biological processes is crucial for the production of energy by combining different microorganisms and regulating the culture conditions [43]. The interaction between these microorganisms and the control of environmental factors can significantly improve the efficiency and sustainability of bioenergy production [44]. In this study, the necessary microorganisms were already present in the bovine manure, so it was not necessary to add them. The results of this research allow the valorization of organic waste, such as manure, in the production of biogas, demonstrating that the anaerobic process is a viable alternative for organic waste management [45]. The concentrations of the inoculums can be used in other research at pilot or industrial levels, contributing to the production of renewable energy from biomass, which favors the energy transition and promotes social, economic and environmental development [46,47].

5. Conclusions

Two thermal systems were evaluated: one with controlled heating and another with a parabolic solar system. The controlled system achieved a maximum average biogas production value of 625.9 mL/day and the solar system reached 648.5 mL/day, measured daily at 13:00 h for 43 days. These values, which represent the average of all treatments in each system, indicate that solar heating is more economically most promising in the production of biogas. Treatment t1, with a manure:water ratio of 1:5 and the presence of molasses and Caesal-pinia spinosa meal inoculums, produced an average of 731.6 mL/day in the controlled system and 736.3 mL/day in the solar system; both systems were evaluated for 43 days and proved to be suitable for pilot or industrial scale applications. The physicochemical and environmental parameters are also crucial in biogas production, and the temperatures used, in the range of 25 °C to 35 °C in the controlled system and 41.6 °C in the solar system, together with the pH levels of 6.5 to 7 and total solids content of 13%, optimized production. Likewise, the biogas quality was high, reaching 76.9% CH4 in the T1 treatment, making it suitable for residential or industrial applications, promoting economic and environmental development. This study demonstrates that solar and controlled heating systems are viable and efficient for biogas production; however, long-term studies are suggested to evaluate the sustainability and consistency of the results, to explore seasonal variability and applications in different climatic conditions, to optimize production with different inoculums and mixtures and to investigate the integration of these systems into larger energy grids.