Effect of Combined Particle Size Reduction and Fe₃O₄ Additives on Biogas and Methane Yields of Arachis hypogea Shells at Mesophilic Temperature

Abstract

Enzymatic hydrolysis of lignocellulose materials has been identified as the rate-limiting step during anaerobic digestion. The application of pretreatment techniques can influence the biodegradability of lignocellulose substrate. This study combined Fe₃O₄ nanoparticles, which serve as a heterogeneous catalyst during anaerobic digestion, with different particle sizes of Arachis hypogea shells. Batch anaerobic digestion was set up at mesophilic temperature for 35 days. The results showed that 20 mg/L Fe₃O₄ additives, as a single pretreatment, significantly influence biogas and methane yields with an 80.59 and 106.66% increase, respectively. The combination of 20 mg/L Fe₃O₄ with a 6 mm particle size of Arachis hypogea shells produced the highest cumulative biogas yield of 130.85 mL/gVS_added and a cumulative methane yield of 100.86 mL/gVS_added. This study shows that 20 mg/L of Fe₃O₄ additive, combined with the particle size pretreatment, improved the biogas and methane yields of Arachis hypogea shells. This result can be replicated on the industrial scale to improve the energy recovery from Arachis hypogea shells.

1. Introduction

Effective environmental management and proper use of the available resources have caught the attention of global research and developmental policies in the last decades [1]. In the same way, the production and application of renewable energy, free from impurities, from readily available resources is of paramount interest to the stakeholders in the energy industry [2]. This is more prominent, as there is a need to search for alternatives to fossil-based fuels, which are non-renewable and have resultant challenges of environmental degradation, global warming, and climate change, among others [3]. These ecological challenges of fossil fuel combustion have spurred researchers in energy and sustainable growth to identify new, novel, and sustainable energy sources that can positively impact the development of society and provide economic stability [4]. As part of the global energy transition from fossil fuels to green energy, different energy sources other than fossil fuels are being considered [5]. To attain this, the utilization of lignocellulose materials from agricultural residues, for the production of versatile and clean energy, has been reported in many research works in the last decades [6]. Biogas, which is a product of the anaerobic digestion of various biomasses, is one of the energy sources that has been proposed [7,8]. Biogas can be upgraded to biomethane when siloxanes and other organic and inorganic materials are removed [9].

Biogas production from various agricultural residues, via anaerobic digestion, has increased globally because it is environmentally friendly and does not contend with the food supply. In addition, this process offers other benefits such as reducing pollution caused by organic waste, waste ceasing to be garbage, and suitable products converted into clean energy [10]. The non-edible parts of agricultural residues that can be used as feedstock for biogas production include leaves, corn stover, groundnut shell, cocoa pod, cassava peel, straw, etc. They are available in abundance globally and are cost-effective, with a vast capacity for biotransformation into biofuels and other ancillary products [11,12]. Hydrolysis, acidogenesis, acetogenesis, and methanogenesis are the four vital biological and chemical stages of anaerobic digestion. The first stage of anaerobic digestion is hydrolysis, and it occurs outside the microbial cells. At this stage, extracellular enzymes, which are primary fermenting bacteria, are either attached to the surface of the feedstock or hydrolyze the feedstock. Hydrolysis rate is determined by the structure and composition of the feedstock. Complex carbohydrates such as celluloses, hemicelluloses, and lignin degrade more slowly when compared with the simple ones—for instance, proteins [13]. The general low digestion efficiency of the lignocellulose feedstock, along with the longer retention time (30–40 days), results in limited efficiency [14]. The efficiency and proportion of the hydrolysis and morphological characteristics of lignocellulosic feedstocks can be adapted by biological, chemical, thermal, and mechanical pretreatment methods [15,16,17]. Combining two or more pretreatment techniques has been investigated, and different researchers have recommended its application [18,19,20]. Nevertheless, the most effective and economical pretreatment methods among these techniques have not been established for some lignocellulose materials [21].

The use of nanoparticles in environmental protection has recently gained special attention. The application of nanoparticles was investigated in the flexible and sensitive energy harvester, and it was recorded to be a novel means of energy harvesting [22]. It is being used in biofuel production, waste management, water treatment, and soil remediation [23]. The efficiency of organic biodigestion microorganisms is enhanced when fed with nanoparticles, and it improves biogas yields. Due to nanoparticles’ fluid nature, the result can be assumed as regulated, leading to optimum biogas generation [24]. The addition of trace metals into anaerobic digestion provides essential cofactors and enzymes that have been shown to stimulate and stabilize anaerobic digestion process performance [25,26]. It was reported that the application of Fe nanoparticles significantly reduced the hydrogen sulphide yield in biogas and increased the methane yield in most cases [27]. The addition of Fe²⁺/Fe³⁺ ions, in the form of Fe₃O₄ nanoparticles in the digester, could be taken as growth supplements for the anaerobic microorganisms and influence their performance [28]. The physicochemical characteristics of Fe₃O₄ have been reported to consist of magnetite and a small quantity of goethite, where magnetite produces bioavailable ions (Fe²⁺ and Fe³⁺) that have been recorded as an essential nutrient for microbial power production [29]. Fe₃O₄ enhances the hydrogenotrophic methanogenesis by supplying electrons or hydrogen evolution from the iron corrosion, which increases methane yield from carbon dioxide consumption, as shown in Equations (1)–(3) [26,30].

$${\mathrm{CO}}_2+4{\mathrm{Fe}}^0+8{\mathrm{H}}^{+}\to {\mathrm{CH}}_4+4{\mathrm{Fe}}^{2+}+2{\mathrm{H}}_2\mathrm{O}$$

(1)

$${\mathrm{Fe}}^0+2{\mathrm{H}}_2\mathrm{O}\to {\mathrm{Fe}}^{2+}+{\mathrm{H}}_2+2{\mathrm{OH}}^{-}$$

(2)

$${\mathrm{CO}}_2+4{\mathrm{H}}_2\to {\mathrm{CH}}_4+2{\mathrm{H}}_2\mathrm{O}$$

(3)

Cost-effective renewable energy generation, for domestic and industrial consumption, can be improved with the application of nanotechnologies. They can enhance the conversion of feedstock to chemical intermediates, particular chemicals, fuels, and they can be used as an essential instrument for improving the efficiency of bioenergy production. The methane and biogas yield concentration is enhanced by directing modified nano-particles to optimize the anaerobic digestion process [31].

Groundnut (Arachis hypogea L.) is a leguminous crop known for its oil production and as a significant subsistence and food crop cultivated in nearly all world countries [32]. It is mostly referred to as the “king of oilseed” and grown in over 100 countries globally [33], and about one-quarter of the global production is produced in sub-Saharan Africa [32]. Between 2010 and 2019, groundnut was cultivated on an average of 27,489,741 hectares, with an average cumulative yield of 45,755,248 tons [34]. The principal producers of Arachis hypogea globally are China, India, Nigeria, USA, and Myanmar. It has been reported that Arachis hypogea can produce modest yields even under severe conditions where other crops cannot survive [35]. Almost all the parts of Arachis hypogea are useful: the seeds, stems, leaves, and shells. Arachis hypogea shells form between 25–35%, by mass, of the pod [36], they belong to lignocellulose materials because of their high carbohydrate contents, and they have been utilized for different purposes. It is an organic material that can be used to produce various bio-products like biofuels and nano-sheets. It can also be employed in enzyme production, dye, and heavy metal degradation. The engineering use of the Arachis hypogea shells is limited due to their high lignocellulosic content [37]. The shells have been reported to consist of 37% cellulose, 18% hemicellulose, and 27% lignin [17]. Arachis hypogea shells’ surface morphological examination is shown in Figure 1. The morphological structure image showed that Arachis hypogea shells had a fine, intact arrangement, with a firm and fibrillary morphology unaltered by particle size reduction. It has a regular complex and rigid structure that does make it easily accessible to the microorganisms during the anaerobic digestion process unless other pretreatment methods are added. Therefore, Arachis hypogea shells require other pretreatment methods, after particle size reduction, for easy accessibility of the microorganisms. If adequately harnessed, the higher percentage of the sugar content in the shell could function as a suitable substrate for the hydrolysis stage of anaerobic digestion, thereby releasing the optimum yield of biogas. Jekayinfa et al. [38] earlier reported that particle size reduction improves the biogas and methane yield of Arachis hypogea shells. Still, the SEM image of the feedstock shows that particle size reduction alone is not sufficient as a pretreatment technique to enhance the biogas and methane yields of Arachis hypogea shells. However, suitable pretreatment methods must be applied to enhance their biodegradability by anaerobic microbes. Therefore, there is a need for further research on the potential of Arachis hypogea shells for biogas and methane production.

This work is thereby aimed at the production of biogas and methane, from the anaerobic digestion of Arachis hypogea shells, through the application of combined pretreatment methods, i.e., mechanical and nanoparticle pretreatment, as well as the establishment of their responses in terms of biogas and methane yields that are missing in the literature. The result from this study is expected to serve as a baseline for subsequent research on the anaerobic digestion of the lignocellulose biomass.

2. Materials and Methods

2.1. Materials

Arachis hypogea shells were procured locally in the Johannesburg market, and they were stored in a ventilated area before pretreatment. The inoculum used in this research was produced from co-digestion of cow dung and kitchen wastes in a batch digester for 60 days at ambient temperature. The Arachis hypogea shells were milled with a hammer mill (SSY 4000902, South Africa) using screen sizes of 2, 4, 6, and 8 mm. The particle sizes were selected based on the recommended particle size by earlier researchers [38,39]. These particle sizes were chosen with some modification to the earlier reports in particle size selection of lignocellulose feedstock that were reported to have similar structural arrangements with Arachis hypogea shells. Fe₃O₄ (<50 NM, 544884—Merck (pty) Limited, Darmstadt, Germany) was procured for the experiment was procured from Sigma-Aldrich (pty) Limited, Johannesburg, South Africa.

2.2. Sample Analysis

The cellulose, hemicellulose, lignin, total solids, volatile solids, ash content, and other elemental compositions of the substrate and inoculum were analyzed in accordance with the Association of Official Analytical Chemist methods [40], and the result is as shown in

Table 1. Physicochemical composition of the substrate and inoculum.

Parameters	Substrate	Inoculum
Cellulose (%)	38.21	NA
Hemicellulose (%)	18.22	NA
Lignin (%)	27.68	NA
Total Solid (TS) (%)	95.51	4.01
Volatile Solid (VS) (%)	91.27	84.83
Ash content (%)	5.96	13.07
Moisture content (%)	4.49	95.99
Nitrogen (%)	1.50	1.48
Carbon (%)	36.39	42.57
Hydrogen (%)	4.79	5.50
Sulphur (%)	0.53	0.60

.

2.3. Experimental Set Up

A laboratory batch anaerobic digestion process was set up to investigate the effect of pretreatment techniques on biogas and the methane yield of Arachis hypogea shells, according to Standard Methods VDI 4630 [41]. Flasks with a round bottom narrow neck bottles of a 1-L inner volume (Schott Duran 10091871, Germany) were used as the digester. They were connected to calibrated gas bottles, with an internal volume of 0.5-L, made from an ultra-clear polypropylene graduated cylinder (LMS Boro 3.3, Germany). The gas produced was collected using water displacement methods, and the gas bottles were connected to laboratory bottles with the inner volume of 0.5-L (Schott Duran 10093435, Germany) using a silicon pipe. EcoBath thermostatic water batch (30 L) (ECOBATH LABOTECH Model 132 A, South Africa), with the control unit software, maintained the temperature at mesophilic conditions (37 ± 0.02 °C) to control the digestion temperature. The substrate and inoculum measured into the digester were calculated using Equation 4, based on volatile solids content. A control sample with inoculum alone was also digested, and the volume of the gas produced was subtracted, afterward, from the substrate sample yield. The digesters were loaded and labeled, as shown in

Table 2. Mechanical and nanoparticle pretreatment of the substrate.

Digester	Treatment
A	2 mm particle size
B	4 mm particle size
C	6 mm particle size
D	8 mm particle size
E	Non-mechanical
F	Untreated substrate

. Next, 20 mg/L of Fe₃O₄ (<50 nm) was added separately to each digester, as recommended by Abdelsala et al. [28], except for a set that served as control. Additionally, 20 mg/L of Fe3O4 nanoparticles were used because of their better ability to improve the biogas and methane yields when compared with other dosages and nanoparticles, as earlier reported. The digester performance was measured for the total volume of biogas yield, and it was corrected to the standard temperature (273 K) and pressure (760 mmHg). The experiments were replicated twice, as recommended by Linke and Schelle [42]. The volume of biogas produced was recorded daily, through the ultra-clear graduated cylinder, by considering the volume of water displaced by the gas yield. Likewise, the date, time, temperature, and atmospheric pressure were also recorded daily when taking the gas reading. The composition of the gas yield (CH₄, CO₂, H₂S, and O₂) was measured at intervals using the BioGas 5000 gas analyzer (Geotech, GA5000, Warwichshire, UK). The experiment was terminated at 35 days when it was discovered that the volume of biogas released daily was below 1% of the total gas yield.

$${\mathrm{M}}_{\mathrm{s}}=\frac{{\mathrm{M}}_{\mathrm{i}}{\mathrm{C}}_{\mathrm{i}}}{2{\mathrm{C}}_{\mathrm{s}}}$$

(4)

where: M_s = Mass of substrate (g), M_i = Mass of inoculums (g), C_s = Concentration of substrate (%), C_i = Concentration of inoculum (%). Inoculum required is 80% of the reactor volume [41].

Gas factor (F), biogas, and methane yields were calculated, as prescribed by VDI 4630 [41]. The quantity of biogas yield was converted to standard conditions (273.15 °C and 1013.25 mbar), and Equations (5) and (6) were used to determine the gas factor, while Equations (7)–(13) were used to determine the biogas and methane yield in organic dry matter and fresh form [41].

$$\mathrm{F}=\frac{\left(\mathrm{P}-{\mathrm{P}}_{{\mathrm{H}}_2\mathrm{O}}\right){\times \mathrm{T}}_{\mathrm{O}}}{\left(\mathrm{t}+273.15\right){\times \mathrm{P}}_{\mathrm{O}}}$$

(5)

where: T_O = 273.15 °C (Normal Temperature), t = Gas Temperature in °C, P_O = 1013.25 mbar (standard pressure), P = Air pressure [41].

The vapor pressure of water P_H2O is dependent on the gas temperature and amounts to 23.4 mbar at 20 °C. The respective vapor pressure of water, as a function of temperature for describing the range between 15 and 30 °C, is given in Equation (6).

$${\mathrm{P}}_{{\mathrm{H}}_2\mathrm{O}}={\mathrm{y}}_{\mathrm{o}}+\mathrm{a}·{\mathrm{e}}^{\mathrm{bt}}$$

(6)

where: y_o = −4.3905; a = 9.762 and b = 0.0521.

The normalized amount of biogas volume is given as:

Biogas [NmL] = Biogas [mL] × F

(7)

Normalized by the amount of biogas, the amount of gas taken off the control batch is given as:

Biogas [NmL] = (Biogas [NmL] − Control [NmL])

(8)

The mass of biogas yield, in standard liters/kg fresh mass (FM), is based on the weight.

The following apply:

1 standard mL/g FM = 1 standard liters/kg FM = 1m³/t FM

$$\mathrm{Mass} \mathrm{of} \mathrm{Biogas} \mathrm{Yield}={\displaystyle \sum }\frac{\mathrm{Biogas}\left[\mathrm{NmL}\right]}{\mathrm{Mass}\left[\mathrm{g}\right]}$$

(9)

The oDM biogas yield, based on the percentage of volatile solids (VS) in the substrate, is:

$$\mathrm{oDM} \mathrm{biogas} \mathrm{yield}={\displaystyle \sum }\frac{\mathrm{Biogas}\left[\mathrm{NmL}\right]}{\mathrm{Mass}\left[\mathrm{g}\right]}$$

(10)

The corrected CH₄ depends on the composition of CH₄, CO₂, and the mass of the substrate, which determines the methane yield in both fresh and organic form.

$${\mathrm{CH}}_{4 \mathrm{corr}.}=\frac{{\mathrm{CH}}_4\left[\mathrm{vol}\right]\times 100}{\left(\mathrm{Mass}\left[\mathrm{g}\right]+{\mathrm{CO}}_2\left[\mathrm{vol} \%\right]\right)}$$

(11)

$$\mathrm{Fresh} \mathrm{mass} \mathrm{Methane} \mathrm{yield}=\frac{\mathrm{Fresh} \mathrm{mass} \mathrm{biogas} \mathrm{yield}\times {\mathrm{CH}}_{4 \mathrm{corr}.}}{100}$$

(12)

$$\mathrm{oDM} \mathrm{Methane} \mathrm{Yield}=\frac{\mathrm{oDM} \mathrm{biogas} \mathrm{yield}\times {\mathrm{CH}}_{4 \mathrm{corr}.}}{100}$$

(13)

The anaerobic process’s digestion rate can be determined using the reaction kinetics model of chemical reaction control reported by Luo and Wu [43]. Assuming the biodigestion process takes place on the surface of the substrate evenly, and at an equal rate, the substrate decomposition rate can be expressed in Equation (14). The substrate’s surface area, determined by the particle size, plays a significant role in digestion.

$$\mathrm{V}=-\frac{\mathrm{dm}}{\mathrm{dt}}={\mathrm{kAC}}^{\mathrm{n}}$$

(14)

where: V = liquid-solid reaction rate (g s⁻¹), m = mass of the substrate (g), t = retention time of the digestion (s), A = the surface area of the substrate where digestion take place (m²), k = digestion rate constant of the substrate biodigestion (g¹⁻ⁿ s⁻¹ m³ⁿ⁻²), n = rate constant (dimensionless).

3. Results and Discussions

3.1. Effects of Pretreatment on Cumulative Biogas Yield

Biogas yield of Arachis hypogea shells was enhanced by Fe₃O₄ and its combination with different particle sizes, as shown in

Table 3. Cumulative biogas yield for pretreated and untreated substrate (mL/g VS_added).

Days	2 mm	4 mm	6 mm	8 mm	Single Treatment	Control
1	0.00	0.00	0.00	0.00	0.00	0.00
2	4.07	3.37	5.80	4.46	3.50	1.47
3	8.51	7.94	10.75	9.59	7.05	3.29
4	13.67	13.06	17.75	16.12	10.08	5.16
5	20.13	19.68	25.11	19.52	15.87	5.65
6	22.76	22.19	29.41	23.37	18.13	6.88
7	26.59	26.28	34.81	28.75	21.11	8.97
8	32.30	33.78	42.84	35.22	25.55	11.31
9	38.42	39.59	50.04	38.48	29.92	11.85
10	40.91	41.69	52.04	40.62	31.18	11.90
11	42.94	43.03	54.65	43.06	33.05	12.53
12	45.77	48.05	60.04	46.88	34.12	13.89
13	49.77	54.62	65.55	49.14	35.98	17.45
14	52.34	62.00	72.56	55.43	37.39	18.90
15	58.24	66.60	76.80	62.57	39.42	20.88
16	65.36	70.72	83.20	68.67	40.06	21.78
17	71.26	75.88	88.36	72.26	41.13	23.26
18	75.01	82.04	92.84	77.71	42.49	25.25
19	80.48	86.63	95.14	79.73	44.17	27.11
20	82.54	89.16	97.87	82.33	46.28	27.63
21	82.17	92.03	101.47	85.13	47.51	28.03
22	87.81	94.71	105.10	87.66	49.54	28.59
23	90.34	98.32	107.16	90.53	50.86	29.65
24	93.40	101.55	109.55	93.45	52.40	30.31
25	96.35	104.12	112.21	96.11	53.72	31.35
26	99.19	107.89	114.82	98.81	55.95	32.22
27	101.78	109.87	117.42	101.14	57.51	33.01
28	104.21	112.96	121.07	103.91	59.25	33.67
29	106.78	115.43	123.88	106.19	60.64	34.02
30	108.93	117.32	126.88	107.84	61.53	34.49
31	110.37	118.86	128.72	109.35	62.17	34.82
32	111.44	120.02	130.25	110.17	62.66	34.99
33	112.16	120.92	130.85	110.25	63.08	34.99
34	112.16	120.92	130.85	110.25	63.17	34.99
35	112.16	120.92	130.85	110.25	63.19	34.99

and Figure 2. It can be noticed that both single treatment (treatment E) and combined treatment affected cumulative biogas yield compared with the control experiment (treatment F). The cumulative biogas yield of 112.16, 120.92, 130.85, 110.25, 63.19, and 34.99 mL/gVS_added for treatments A, B, C, D, E, and F, respectively. This represents a 220.55, 245.58, 273.96, 215.09, and 80.59% increase compared with the control experiment (treatment F). At the end of the 35 day retention period, the average daily biogas yield recorded was 3.20, 3.45, 3.74, 3.15, 1.81, and 1.00 mL/gVS_added for treatments A, B, C, D, E, and F, respectively. When the single treatment of Fe₃O₄ is compared with the control, it can be deduced that the Fe₃O₄ additive improves the biogas yield of Arachis hypogea shells by 80.59%. This result corroborates what was earlier reported—that trace metals are important in anaerobic digestion because they stimulate the activities of the methanogenic bacteria. Metals like nickel, iron, cobalt, zinc, etc., serve as nutrients for methanogenic bacteria [44]. Fe₃O₄ can influence the activities of immobilized enzymes since they provide sufficient surface area for the enzyme attachments, which improves enzyme loading per unit mass of substrate particles [45,46]. This supports what was earlier reported by Abdelsalam et al. [28]—that the addition of 20 mg/L of Fe₃O₄ improves the biogas yield of cow slurry by 73%. In a similar experiment, where iron oxide nanoparticles were used to pretreat winery solid and sorghum stover, it was reported that the addition of iron oxide improved the biogas yield [47]. Biogas and hydrogen yield of green algae was improved when iron oxide was experimented with as an additive for biogas optimization [48]. This result agrees with what Suanon et al. reported when the iron powder was experimented with during the anaerobic digestion of sludge [49]. Fe₃O₄ additive was reported to improve the biogas yield during the anaerobic digestion of Ulva intestinalis Linnaeus [29]. During the anaerobic co-digestion of Phragmites straw and cow manure, it was reported that Fe²⁺ increased the cumulative biogas yield by 18.1% and extended the gas production peak stage by influencing the cellulase activities [50]. Results from most of this literature are lesser compared with what is recorded in this experiment. However, when cow slurry was pretreated with waste iron powder, there was no effect on the biogas yield of the process [51]. The impact of zero-valent iron nanoparticles on the biogas potential of blackwater, during anaerobic digestion, was also negligible [26]. There was no effect on biogas yield when 10 mg/g TSS of ZnO was added to waste-activated sludge [52], but when iron oxide was added to the same waste-activated sludge, an increase in biogas yield was recorded [53]. The effect of nanoparticles on biogas yield depends on the particle size and concentration of the nanoparticles, as well as the structure of the feedstock [21]. However, Arachis hypogea shells were not the feedstock used by earlier researchers, which may be the reason for the differences in the result. Iron oxide nanoparticles have been reported to influence hydrogenotrophic and syntrophic methanogenesis, improving the methanogenesis rate [50]. Fe²⁺ served as a special source that disintegrates the volatile solid and improves the biogas yield during anaerobic digestion [31]. Therefore, it can be deduced that Fe₃O₄ has a positive influence on the anaerobic digestion of some feedstock, especially Arachis hypogea shells, and has diverse influences on the anaerobic digestion of different feedstock/systems.

The combination of Fe₃O₄ with particle size reduction shows a better biogas yield of Arachis hypogea shells compared to the single treatment of Fe₃O₄. Particle size reduction improves the lysis rate, which leads to improvements in biogas yield [38]. Particle size reduction improves the surface area of the feedstock that hydrolyzes the glycosidic bond in the carbohydrates and polysaccharides, which produces simple sugars. The dissolution of the Arachis hypogea shell’s cell wall by mechanical pretreatment was noticed, as elucidated by the results in Figure 2. The optimum yield ( 130.85 mL/gVS_added) of cumulative biogas yield was recorded when 6 mm particle size was combined with Fe₃O₄ (Treatment C). This was followed by treatments B (120.92 mL/gVS_added), A (112.16 mL/gVS_added), and D (110.25 mL/gVS_added), respectively. Mechanical pretreatment, in this case, was noticed to enhance the biogas yield of the Arachis hypogea shells. The size reduction process, duration, and substrate structure will enhance the specific surface area, total polymerization level, and the final cellulose crystallinity reduction [54]. In this research, particle size reduction increased the surface area of the substrate, thereby creating sufficient space for the Fe₃O₄ attachment to the substrate and improving the hydrolysis and methanogenesis stage. The results show that particle size reduction, from whole pods of Arachis hypogea shells, started to improve the biogas yield until 6 mm particle size is reached, where the optimum yield is recorded; below this size, the yield started declining. The results here support what was reported earlier—that combined pretreatment improves the biogas yields and reduces the retention period [55]—which is a major benefit of the process. An earlier report of the single pretreatment of Arachis hypogea shells showed that a 6 mm particle size released the highest fresh mass of biogas yield [56]. Menardo et al. [57] previously reported that particle size reduction improves lignocellulose materials’ biogas yield. Likewise, it was reported that combining nanoparticle pretreatment with other pretreatment methods improves the biogas yield [29,48]. Treatment C with a 6 mm particle size produced the optimum biogas yield during combined pretreatment. This supports what was reported earlier by Herrmann et al.—that 6 mm particle sizes of some crop residues produce the highest biogas yield. Below 6 mm, the biogas yield started to decline [58]. Motta et al. reported that a bigger particle size produced the best biogas yield [59], but in this case, the best yield is recorded in between the particle sizes. The result recorded is higher than what was reported (51.5% increase) when microwave pretreatment was combined with Fe₃O₄ during anaerobic digestion of Green Algae [48]. This result supports reports that combining other pretreatment methods with Fe₃O₄ increases the biogas yield [48]. Microwave pretreatment was combined with Fe₃O₄ nanoparticles during the anaerobic digestion of green Algae, and it was reported that combined pretreatment produced a better biogas yield than individual single pretreatment. Compared to individual treatments, the combined pretreatment of thermal alkali and a steam explosion of lignocellulose waste improved biogas yield [60]. The results here are contrary to what was previously reported by some literature—that smaller particle sizes released the optimum biogas yield [61,62]. The lower biogas yield from 2 mm particle size may be due to the substrate’s loss of some carbon content and the production of an inhibitory substance that hinders the biogas production. Smaller particles improve the surface area of the substrate and enhance the attachment of the nanoparticles. This improves the hydrolysis rate, resulting in over-accumulation of volatile fatty acids (VFAs). Over accumulation of VFAs will affect the pH of the process, which will, in turn, have a negative influence on the methanogens that release biogas. The anaerobic digestion process is most efficient when the pH of the process is closer to neutral points (6–8) [63]. Lower or higher pH of the process, due to VFA accumulation, will affect the methanogenic bacteria development, which will eventually lower gas yield. It is mainly a result of overloading due to fast hydrolysis of smaller particle sizes [64]. This result agrees with what was recorded by earlier researchers when compared with other, larger particle sizes [65,66,67,68]. The substrate considered in the earlier literature is not Arachis hypogea shells, which may be the reason for the difference in the yield. However, when Arachis hypogea shells were pretreated with only particle size, it was reported that a 4 mm particle size produced the highest biogas yield [17]. It can be assumed, from this result, that a certain percentage of carbon held by the 6 mm particle size, compared to the smaller sizes, was accessible with the addition of Fe₃O₄ to the process. This result has shown that adding Fe₃O₄ to the particle size of Arachis hypogea shells during anaerobic digestion enhances biogas yield. When considering combined pretreatment for Arachis hypogea shells and other lignocellulose materials, this must be considered. This result also indicates a particular particle size, whereby further reduction will produce inhibitory materials that will not benefit the biogas yield, and the addition of Fe₃O₄ cannot cushion the said effect.

3.2. Effects of Pretreatment on Cumulative Methane Yield

The cumulative methane yield produced from the combined pretreatment, single pretreatment, and control is presented in

Table 4. Cumulative methane yield for treated and untreated substrate (CH₄ mL/g VS_added).

Days	2 mm	4 mm	6 mm	8 mm	Single Treatment	Control
1	0.00	0.00	0.00	0.00	0.00	0.00
2	3.21	2.41	4.47	2.63	2.71	1.05
3	7.04	5.50	8.27	5.42	5.49	2.33
4	11.79	9.10	13.64	8.74	7.81	3.64
5	14.30	13.75	19.34	12.93	12.35	3.96
6	17.02	15.57	22.65	14.52	14.15	4.81
7	20.91	18.46	26.80	15.10	16.54	6.24
8	25.54	23.86	32.98	18.87	20.06	7.90
9	27.96	27.92	38.56	22.94	23.51	8.27
10	29.62	29.25	40.11	24.36	24.50	8.29
11	31.40	30.15	42.11	25.48	26.01	8.65
12	33.93	33.95	46.29	27.17	26.87	9.54
13	35.41	38.74	50.59	29.70	28.34	12.06
14	39.48	44.45	55.44	31.19	29.46	13.08
15	45.19	47.16	59.23	35.11	31.07	14.52
16	50.08	49.63	64.15	39.91	31.53	15.11
17	52.97	52.98	68.12	43.83	32.30	16.09
18	56.95	57.30	71.54	46.19	33.36	17.51
19	58.94	59.99	73.32	49.79	34.63	18.86
20	60.79	61.78	75.81	50.93	36.29	19.20
21	62.76	63.82	78.53	52.48	37.27	19.27
22	64.54	65.74	81.87	54.05	38.93	19.62
23	66.72	68.15	83.01	55.52	40.00	20.37
24	69.00	70.22	84.87	57.38	41.23	20.81
25	71.06	71.90	86.90	59.19	42.27	21.52
26	73.10	74.45	88.92	62.21	44.08	22.15
27	74.87	75.72	90.90	63.74	45.31	22.68
28	76.78	77.98	93.71	65.13	46.60	23.13
29	78.48	79.64	95.82	66.65	47.69	23.37
30	79.61	80.98	98.01	67.83	48.34	23.69
31	80.41	82.06	99.38	68.50	48.82	23.93
32	80.92	82.90	100.50	68.90	49.22	24.03
33	80.93	83.49	100.86	69.04	49.56	24.03
34	80.93	83.49	100.86	69.04	49.66	24.03
35	80.93	83.49	100.86	69.04	49.66	24.03

and Figure 3. Cumulative methane yield recorded was 80.93, 83.49, 100.86, 69.04, 49.66, and 24.03 mL/gVS_added for treatments A, B, C, D, E, and F, respectively. This result represents a 236.78, 247.44, 319.73, 187.31 and 106.66% increase for treatments A, B, C, D, and E, respectively, compared with the control (treatment F). The average daily methane yield for the period of 35 days was 2.31, 2.39, 2.88, 1.97, 1.42, and 0.69 mL/gVS_added daily. The pretreatment was noticed to enhance the startup of methane yield and lower the lag phase. This result has shown that the Fe₃O₄ additive influences the methane yield of Arachis hypogea shells. This agreed with what was reported—that inhibition of dechlorination by iron nanoparticles influences methane yield [69]. The result agreed with that of Casals et al., showing that adding Fe₃O₄ during anaerobic digestion improves the methane yield [31]. Organic waste was pretreated with 100 mg/L of Fe₃O₄ (7 nm) during anaerobic digestion, and a 234% methane increase was recorded, which is within the range of the percentage increase recorded in this work. Methane yield was increased by 1.25 and 0.9 times, when 0.75 and 1.5 g per 0.5 L of Fe₃O₄ were added to wastewater sludge during the anaerobic digestion in the batch digester at a mesophilic temperature [49]. The addition of Fe₃O₄, at the rate of 20 mg/L, was reported to increase the methane of fresh raw manure by 115.6% [28], which is within the range of values recorded in this work. Waste activated sludge, pretreated with 100 mg/g-TSS (>30 nm) of Fe₂O₃ at a mesophilic temperature, in a batch digester for 40 days, was reported to improve the methane yield by 117% [53]. A methane increase of about 234% was recorded when iron oxide was used as an additive during anaerobic digestion [31]. It was reported that the addition of 20 mg/L of Fe₃O₄ into the cow slurry improved the methane yield [28]. This result buttressed what was reported: nanoparticles influence the anaerobic digestion process and encourage slurry digestion, leading to increased methane yield, but only at a specific dosage [70]. The result also agreed with Ni et al., who reported that the negative effect recorded when 50 mg/L magnetic nanoparticles’ performance were not significant and concluded that magnetic nanoparticles could be said to be non-toxic to bacteria during long term process showed mild toxicity at the beginning [71]. The result here showed that 20 mg/L Fe₃O₄ additives improve the microbes’ performance from the beginning to the end of the process. The range of percentage increase (106.66–319.73%) in methane, produced when 20 mg/L of Fe₃O₄ was added, was higher than what was reported (100.16%) when the same quantity of nanoparticles was investigated [28]. When 20 g/L was investigated, a 43.5% increase in methane yield was recorded, which was lower than the ranges recorded here [72]. Men et al. [73] experimented with using 10 g/L of zero-valent iron on the anaerobic digestion of lignocellulose materials, and there was a 10.3% decrease in cumulative methane yield. This result is contrary to what was reported when a lower concentration, of 5 and 10 g/mL, was applied during the anaerobic digestion of durum wheat [70].

When compared with other metal oxides like CeO₂, ZnO, and CuO, our result agreed with [74], who reported an 11% increase in yield but disagreed with Duc [74] and Otero-González et al. [75] who reported an 8 and 15% decrease in methane yield for ZnO and CuO. Our results confirmed that 20 mg/L Fe₂O₃ additive was effective for methane production, which agrees with Abdelsalam et al. [28].

Methane production was influenced by the combination of Fe₃O₄ with different particle sizes of Arachis hypogea shells, as shown in Figure 3. It can be deduced that all particle sizes increase the methane production compared to single pretreatment. The total methane recorded was 80.93, 83.49, 100.86, and 69.04 ml/gVS_added, representing 62.97, 68.12, 103.10, and 39.03% increases for treatment A, B, C, and D, respectively, compared with single treatment (E). This work further established what Zaidi et al. [48] have reported—that the combination of nanoparticle pretreatment with other pretreatment methods increases nanoparticle pretreatment effectiveness. This result agreed with what was reported by Menardo et al. [57]—that mechanical pretreatment improves the methane yield of lignocellulose materials. Mechanical pretreatment was reported to improve the methane yield of agricultural by-products by more than 80%, which is higher than what is reported here, when compared with single pretreatment. Olatunji et al. [56] reported that the particle size of Arachis hypogea shells significantly influences methane yield, and the expected yield will determine the choice of particle size. When Arachis hypogea shells were pretreated with a single pretreatment of particle size reduction, it was reported that a 6 mm particle size produced the optimum fresh mass methane yield [17]. Improvement in methane yield was reported when microwave pretreatment was combined with Fe₃O₄ during the anaerobic digestion of macroalgae [29]. Fe₃O₄ nanoparticles were combined with ultrasonic and ozone pretreatments during the anaerobic digestion of Ulva intestinalis Linnaeus. It was reported that their combined effects produced better results than individual pretreatments [29]. Multi-pretreatment techniques have been investigated and confirmed to improve enzymatic hydrolysis and the corresponding methane yield [21]. They cannot be referred to as biological, chemical, mechanical, or thermal because they combine methods. Although it is more complicated than a typical single pretreatment, they are the more successful methods. The results show that the optimum yield was recorded when the particle size was 6 mm (treatment C). It can be inferred, from the result, that the methane production increases with a reduction in particle size until the 6 mm size is reached, but below that size, the methane yield starts to decline with further particle size reduction. This can be traced to the loss of some carbon content of the substrate with further size reduction. It could also be a result of the release of inhibitory compounds that can hinder the production of methane. The result agreed with what was earlier reported—that particle size below 6 mm resulted in the methane yield declining [58]. Another related study reported that bigger substrate particle sizes produce the optimum methane yields [59], which aligned with our result on methane yield.

On the contrary, it was reported, in some earlier literature, that the highest methane yield was produced from 1–2 mm particle sizes [62]. In similar research, it was reported that 2 mm particle size released the highest methane content [65,66,67,76]. The substrates considered in those results were not Arachis hypogea shells, and they were evaluated in a single pretreatment, which may be the reason for the difference in results recorded. This difference in results from the same lignocellulose feedstocks can also be traced to the addition of Fe₃O₄ nanoparticles. This further confirmed the ability of iron oxide to lose or gain electrons, making it an efficient additive that can be used to enhance the anaerobic digestion process, if not applied excessively. The highest methane yield recorded from 6 mm particle size shows that Arachis hypogea shells belong to the category of the lignocellulose materials and that size reduction to smaller particle sizes does not improve their methane yield [57,58].

4. Conclusions

The single and combined effects of Fe₃O₄ nanoparticle additives, with different particle sizes of Arachis hypogea shells, were investigated, and it showed a significant improvement in biodegradability. The single effect of 20 mg/L Fe₃O₄ nanoparticle additives improved biogas yield by 80.5% and methane yield by 106.66%. The optimum cumulative biogas (130.85 mL/gVS_added) and cumulative methane (100.86 mL/gVS_added) yields were recorded from the combination of 20 mg/L Fe₃O₄ nanoparticles with the 6 mm particle size of Arachis hypogea shells.

In contrast, the 2 mm particle size of Arachis hypogea shells, with 20 mg/L of Fe₃O₄ nanoparticle additives, produced the low biogas and methane yield in a combined pretreatment as opposed to some reports that smaller particle sizes produce the optimum biogas and methane yield. This can result from fast hydrolysis of the process, leading to over-accumulation of VFAs, and adversely affecting the pH and methanogenic bacteria. Experimental results indicated that the 20 mg/L Fe₃O₄ nanoparticle additive enhances the solubility of Arachis hypogea shells to improve the biogas and methane yields, during anaerobic digestion processes, or accelerate the digestion with a reduction in process period. Further investigation showed that particle size reduction in lignocellulose materials is required before the Fe₃O₄ nanoparticle additive to improve the hydrolysis process and enhance biogas and methane yields. The choice of particle sizes of Arachis hypogea shells will be determined by the structural arrangement of the lignocellulose feedstock. Therefore, using these combined pretreatments poses a novel biotechnological way of biogas production. This study can be replicated with other lignocellulose materials, as well as feedstocks with resistant cell walls or cellulose arrangement, to enhance the hydrolysis and methanogenesis stages to optimize energy recovery. This efficient optimizing energy recovery from lignocellulose materials can be easily scaled-up for industrial-scale biogas production.