Enhancing biogas yield of Xyris capensis grass using different nanoparticles additive

To enhance the efficiency of the anaerobic digestion process of lignocellulose feedstocks, there is a need for appropriate pretreatment methods. The influence of nanoparticles additive on biogas yield of new novel Xyris capensis grass as biogas feedstock was investigated. A laboratory-batch experiment was set up at mesophilic conditions (37 ± 2° C) to study the biogas production potential of Xyris capensis, and different nanoparticles were added as a means of pretreatment. 20 mg/L of Fe3O4, 1.4 mg/L of CuO, 10 mg/L of ZnO, and 10 mg/L of MgO were added to the anaerobic digestion process, and their influence on biogas and methane yield was compared with the untreated substrate. Biogas yield of 376.67, 156.86, 175.34, 190.00, and 290.00 mL/g VSadded was recorded for Fe3O4, CuO, ZnO, MgO additives, and untreated substrate, respectively, while methane yield of 282.50, 156.86, 97.66, 116.33, and 198.51 mLCH4/g VSadded were observed, respectively. It can be observed that only 20 mg/L of Fe3O4 increased biogas and methane yields by 29.89 and 42.31%, respectively.


Introduction
The rapid increase in industrialization during the fourth industrial revolution has increased the energy demand globally.Most of the energy used during this period is from the combustion of fossil fuels [1].Reliance on fossil fuels has led to the monopoly of some countries that manipulate petroleum prices and create artificial scarcity.Global warming and other environmental challenges can also be traced to the carbon dioxide and other greenhouse gases released during fossil fuel combustion [2].The worsening ecological challenges due to fossil fuel combustion have necessitated the search for renewable, clean, and sustainable energies with minimal antecedent environmental effects.Biomass has been identified as a sustainable source of feedstock that can be used to produce renewable energies that can serve as an alternative source to fossil fuels [3].Lignocellulose materials are readily available and can be converted into bioenergy products such as biodiesel, bioethanol, biogas, biohydrogen, etc [4].Biogas, renewable energy produced from lignocellulose biomass, has been observed to be sustainable and environmentally friendly [3].Biogas can be generated through the anaerobic digestion of organic materials and can serve as a versatile energy source for lighting, electricity, heat generation, and transport fuel.It can also be purified and injected into the grid [5].Biogas production provides other merits like organic waste control, lowering greenhouse gas release, and generating another economically viable fertilizer.
Lignocellulose materials are the most abundant renewable bioresource feedstock available on earth.It mainly consists of hemicellulose, cellulose, and lignin, which are strongly intertwined.This structural arrangement of lignocellulose materials made them recalcitrant and unavailable for enzymatic hydrolysis during anaerobic digestion, making the process uneconomical [6].Therefore, removing the sturdy and rugged lignin portion that hinders solubilization is necessary to make it to bacteria.Lignocellulose feedstock pretreatment before anaerobic digestion has been identified as a means to separate the complexly interlinked fractions and improve feedstock availability The challenges associated with the application of some of these pretreatment techniques, such as the production of inhibitory compounds, elimination of unreacted solvents, neutralization, etc., have shown the need to identify another novel, sustainable, and green means of breaking down the cell wall of lignocellulose feedstock [7].Nanoparticle additives have been observed as the foremost choice, being the brightest biocatalysts for the environmentally benign and effective debasement of lignocellulose feedstock into simple sugar, considering their selectivity, high specificity, and their catalytic strength for enzyme activities.This process encourages a green approach because no chemical is required, and toxic inhibitors are produced minimally.The influence of Fe3O4 nanoparticle additives was experimented with during the biogas production from Arachis hypogea shells, and biogas and methane released increased by 80.59 and 106.66%, respectively [9].Adding 20 mg/L of Fe3O4 to cow dung during the anaerobic digestion enhances biogas yield by 73% [10].Adding 10 mg/g TSS of ZnO on waste-activated sludge does not significantly influence the biogas released [11].But when 100 mg/g TSS of Fe2O3 was investigated on waste-activated sludge, the methane yield was improved by 117% [12].
A perennial grass with a rush-like appearance called Xyris capensis grows from a rhizome that creeps.This grass is a common species that poses no hazard and is considered to be of the least concern (LC); thus, there is no immediate threat of extinction.It can be found in riverbanks, marshes, and grassy seepage areas up to 2400 m high.There is still little literature on using this affordable grass that is readily available and does not compete with food [13].Due to its microstructural arrangement illustrated in Figure 1, Xyris capensis can be regarded as a lignocellulose material.This substrate can be a feedstock for biogas production and an energy source.As previously reported, lignocellulose materials are complex and unavailable to microorganisms during biogas production.Pretreatment before anaerobic digestion is required to alter the cell walls and make it accessible to bacteria.Therefore, this study investigated the potential of Xyris capensis as biogas feedstock and the influence of different nanoparticle additives on the biogas and methane released.

Materials and method 2.1 Substrate and inoculum collection
Locally grown Xyris capensis from South Africa's Limpopo Province was used for this experiment.The stabled inoculum was obtained in the digester, where cow manure and kitchen waste were co-digested.Before physicochemical property analysis and anaerobic digestion, the feedstock and inoculum were kept in a well-ventilated laboratory at 4 °C.According to the Association of Official Analytical Chemists (AOAC) [14]standard protocol, the volatile solids (VS), total solids (TS), hemicellulose, lignin, cellulose, carbon, ash content, nitrogen, and Sulphur concentration of the Xyris capensis and inoculum were examined.Nanoparticles used were procured locally from Sigma-Aldrich (pty), Limited, South Africa.These nanoparticles was selected based on the previous studies on the application of nanoparticles pretreatment [15-17].

Ultimate methane yield calculation
Methane stoichiometry and the empirical formula of the organic matter contents of the substrate were used to determine the ultimate methane yield (UMY) of the Xyris capensis, as shown in equations 1 and 2. Equation 1 is mainly referred to as Buswell equation [18].This equation does not consider the energy demand of the microorganism's community and non-digestible organics; therefore, the equation is optimistic in some cases.
Where a, x, y, and z, are the stoichiometry ratios of carbon, hydrogen, oxygen, and nitrogen, respectively.

Anaerobic digestion
The anaerobic digestion was carried out in a laboratory batch as prescribed by European standards [16], as shown in Figure 2. 1000 ml round bottom narrow neck flack bottles served as the digester.The gas bottle used to collect the gas released was made from a 500 ml ultra-clear calibrated polypropylene cylinder.The gas was measured through the water displacement method.A silicon pipe connected the gas bottles to 500 ml laboratory bottles containing distilled water.Eight digesters were fed with the calculated amount of stable inoculum and substrate in a ratio 1: 2 (I: S) using volatile solid content using equation 3.
As recommended in previous research, the experiment was repeated twice, and the participants were tagged, as shown in Table 1.The experiment was duplicated twice, as recommended in the earlier studies, and was labeled as presented in Table 1.The experiment was aimed at investigating the influence of different nanoparticles on biogas yield; therefore, nanoparticles were added to the digesters already loaded with inoculum and substrate, as recommended in previous studies.The concentration of 20 mg/L of Fe3O4 (<50 nm) was added to two digesters [9], 1.4 mg/L of CuO (40 nm) was added to another two digesters [19], for ZnO, 10 mg/L ( 30 nm,) was introduced to another two digesters [11], and 10 mg/L of MgO (50 nm) was added to another set of two digesters [20].Two sets of digesters without nanoparticle additives were also run as control experiments.Two parallel experiments with only inoculum were run.
The gas released was recorded as the inoculum yield and deducted from other digesters to ascertain the exact volume of gas generated.The digesters' headspace was purged with helium gas to set anaerobic conditions for the process.The digesters were carefully arranged in a 40-L thermostatic water batch preset at mesophilic temperature (37 ± 2 °C), which remained constant throughout.To break sediment, scum, and lumps, the digesters were shaken manually daily, and the digestion process was terminated on day 25 when it was observed that the daily gas released was less than 1% of the total yield.Daily readings of the amount of water displaced on the calibrated gas bottles were used to calculate the gas released.The constituent of the gas released was determined at intervals using Biogas 5000 gas analyzer (Geotech, GA5000, Warwichshire, UK) depending on the volume of gas liberated.Date, time, atmospheric pressure, and temperature were also recorded daily.The volume of biogas released was standardized and ascertained using equations 4 -9 [21].

Physicochemical characteristics of Xyris capensis
Table 2 illustrates the physicochemical properties of Xyris capensis.The total solids (TS) and volatile solids (VS) of the substrate are 84.62 and 95.00%, respectively, according to the Table .The VS result shows that more organic matter is available for producing biogas, a positive indication of prospective feedstock.It can be seen that the substrate has more considerable potential for producing biogas when compared to other lignocellulose feedstocks, such as peanut shells 91.27% [9], corn straw (75.05 0.3%), rice straw (74.9 0.2%), and dairy manure (84.7 2.4%) [22].It shows that microorganisms will have an enormous capacity for buffering during the anaerobic digestion of Xyris capensis.It has been noted that the volatile solid content of the feedstock affects the biogas and methane yield of the anaerobic digestion process [23].On the other hand, higher TS may prevent the substrate from being compacted enough inside the digester and may encourage anaerobic decomposition at the digester exit [24].The ideal TS for the best anaerobic digestion is frequently thought to be between 28 and 40%.Water was added to the substrate and inoculum to lower the TS to the acceptable limit because the feedstock and the inoculum's TS were higher than the necessary standards of 30 and 98% [21].

Ultimate Methane Yield
The elemental composition was utilized to identify the organic content as C7.52H11.70O6.26N,and this was used to calculate the UMY using equation 2.
The organic content was determined to be C7.52H11.70O6.26Nusing the elemental composition, and this was used to calculate the UMY using equation 2.Where x = 11.70, a = 7.52, y = 6.26 and z = 1 The theoretical methane yield of Xyris capensis was calculated to be 338.95mL CH4/gVSadded which is higher when compared with some other lignocellulose feedstocks.TMY of 286.08 mLCH4/gVSadded was reported for Arachis hypogea shells [8] and 212.30mLCH4/gVSadded for organic fraction of municipal solid waste and biological sludge [25].But the yield is lower than what was reported (415 mLCH4/gVSadded) in a similar study [26].

Effects of nanoparticles additive on cumulative biogas yield of Xyris capensis
The cumulative biogas yield of Xyris capensis after 24 days is presented in Figure 3.It can be observed that cumulative biogas yields 376.67, 156.86, 190.00, 173.34, and 290.00 ml/g VSadded, for Fe3O4, CuO, MgO, ZnO, and control, respectively.It can be noticed that all the nanoparticle additives have varying influences on the biogas yield of Xyris capensis.This agreed with what was previously observed that nanoparticle additives could influence the catalytic activities of microorganisms during the anaerobic digestion process [27].Compared with the control experiment, it can be observed that Fe3O4 additives improve the biogas yield by 29.89%.In contrast, CuO, MgO, and ZnO nano additives reduced the cumulative biogas yield by 45.91, 34.48, and 40.23% respectively.The increase in biogas yield from the Fe3O4 nanoparticle additive supported the earlier observation that Fe nanoparticles can improve biogas yield [9].The addition of Fe3O4 was noticed to enhance the performance of microorganisms and the release of biogas.Due to its fluid nature, this can be traced to its ability to regulate the process [28].Fe 2+ /Fe 3+ nanoparticle additives in the form of Fe3O4 during anaerobic digestion could serve as growth supplements for the microbes and enhance their activities.The physicochemical properties of Fe3O4 have been noticed to have magnetite and some percentage of goethite, where magnetite releases bioavailable ions (Fe 3+ and Fe 2+ ) that have been identified as a vital nutrient for microbe's power generation [29].When Fe 2+ was added to the anaerobic co-digestion of Phragmites, and manure, an 18.10% increase in biogas was observed.This increase in biogas yield is lower compared to what was observed (29.89%) in this study.Fe3O4 nanoparticles were added during the anaerobic digestion of Arachis hypogea shells as a single pretreatment and combined with particle size reduction.It was noticed that single pretreatment with Fe3O4 increased the biogas yield by 80.59%, and when combined with a 6 mm particle size, it increased the biogas yield by 273.96% [9].The results reported in that study are higher than what was observed in this experiment.This can be linked to their variation in lignin percentage, and combining it with other pretreatment boosts the effectiveness further as this increases the surface area for the nanoparticle and bacteria attachment.Iron oxide additive during the anaerobic digestion of sorghum stover and winery solid was also observed to enhance biogas yield [30]. Biogas yield of fresh raw manure was improved by 73% when 20 mg/L of Fe3O4 was applied as nanoparticle additives [12].

Figure 3: Influence of different nanoparticle additives on cumulative biogas yield.
On the contrary, using CuO reduced the cumulative biogas yield by 45.91% compared to the control experiment.Contrary to what was noticed in this study, copper has been reported as an essential trace element that enhances metabolism [27].This result agreed with what was observed when 1.4 mg/L of 37 nm CuO was used, and the biogas yield was reduced by 25% [19].Two different sizes of CuO, micro and nano-sized (5 nm -30 mm), were experimented with on cattle manure, and it was observed that nano-sized produced a negative result, but micro-sized had no effect [31].This can be linked to the toxic behavior of CuO on methanogenic bacteria.It has been noticed to produce inhibitory compounds related to membrane breakdown and oxidative stress responses [32].The 5.8 to 84% inhibitory effect was observed when the CuO concentration varied from 5 mg/g to 1000 mg/g TS.This indicates that the concentration of CuO determines the level of the inhibitory compound released [33].Magnesium oxide additive was observed to reduce the cumulative biogas yield of Xyris capensis by 34.48%.This can be traced to the characteristics of the MgO that destroy the cell membrane, especially when high concentration is applied [34].Different concentrations of MgO were experimented with on wasteactivated sludge, and it was noticed that 1, 10, and 100 mg/g TSS did not significantly improve biogas yield.Biogas yield was reduced by 108% when 500 mg/g TSS was experimented with [34].It can be inferred from this study that the application of MgO as an additive to enhance biogas yield is harmful to both sludge and lignocellulose materials.As presented in Figure 2, it can be observed that cumulative biogas yield was reduced by 40.23% when ZnO was considered.This agreed with what was previously noticed when 850 nm ZnO was used as an additive on sludge from USAB; 10 mg/L reduced the biogas yield by 8%.When 1000 mg/L of 850 nm was added to the sludge from USAB, the biogas released decreased by 65% [35].On the contrary, there was no effect when 10 mg/g TSS of ZnO was applied to waste-activated sludge [11].It can be inferred that the effect of ZnO nanoparticle additives on biogas generation relies on the particle size, concentration, and substrate type.However, when comparing earlier results with this study, it seems that ZnO does not favor the biogas yield of both sludge and lignocellulose materials.The variation in the yield from this study compared to the previous literature can be linked to the difference in the microstructural composition of the feedstock used.This study has established that it is only Fe3O4 that can enhance the biogas yield of Xyris capensis.

Effects of nanoparticles additive on cumulative methane yield of Xyris capensis
The total methane yield liberated during the anaerobic digestion of Xyris capensis after 24 days of retention time is illustrated in Figure 4. Cumulative methane yield of 282.50, 156.86, 116.33, 97.66, and 198.51 mL CH4/g VSadded was recorded for Fe3O4, CuO, MgO, ZnO nanoparticle additives, and control, respectively.The average daily methane yield for Fe3O4, CuO, MgO, ZnO nanoparticle additives, and control after 24 days is 11.77, 6.54, 4.85, 4.07, and 8.27 mL CH4/g VSadded daily.It can be noticed from Figure 3 that Fe3O4 and CuO improve the startup of methane yield at day 2. The results from this study indicate that all the nanoparticles considered in this experiment have a varying degree of influence on methane yield, which also agrees with the previous research [7].This result shows that Fe3O4 improves the methane yield by 42.31% while CuO, MgO, and ZnO reduce methane yield by 20.98, 41.40, and 50.80%, respectively, compared to the control.Although the effects were adverse, it can be observed that CuO negative effect is higher in biogas yield than methane.
Still, methane's negative influence is more heightened than in biogas yield for MgO and ZnO.Fe3O4 increased cumulative methane result supported the previous study that reported that iron nanoparticles could improve methane yield.The methane yield of organic wastes was enhanced by 234% when 100 mg/L of Fe3O4 (7 nm) was used as a nano-additive during anaerobic digestion [36].A methane yield of 49.66 mL/g VSadded was observed when 20 mg/l Fe3O4 was experimented on Arachis hypogea shells.This yield represents a 106.66% increase, and the improvement increased to 247.44% when combined with 4 mm particle size [9].The methane yield of waste-activated sludge was enhanced by 117% when it was pretreated with 100 mg/g TSS (>30 nm) of Fe2O3 [34].When 20 g/L was experimented with on organic waste, methane yield was improved by 43.5% [35].When comparing this study with previous works, it can be inferred that the influence of iron nanoparticles differs, which can be linked to the difference in concentration, particle sizes, and the level of recalcitrant properties of the feedstock [37,38].CuO, MgO, and ZnO nanoparticles were noticed to reduce cumulative methane yields with varying degrees, as presented in Figure 3.This supports previously reported that methane yield decreased by 8 and 15% when ZnO and CuO were experimented with [19,35].Adding heavy metal ions, such as Cu, Zn, Mg, Ni, Co, etc., to the anaerobic digestion process of biodegradable materials is fundamental for several Nonetheless, when the concentration of these elements is high, the tendency to generate inhibitory compounds is high [31].This could be the reason for the harmful effect of these elements on microorganisms and the subsequent decrease in cumulative methane yield.Cumulative methane yield decreased by 65% when 1000 mg/l of ZnO was experimented on organic feedstock [39].A similar study observed that CuO nanoparticles were more toxic than Ag and CeO2 nanoparticles during a more extended retention period.This led to Archaea inhibition and methane reduction of 84% at 1000 mg per g TS [33].The influence of MgO was harmful to the process because of its toxic properties on microorganisms.This result contradicts what was observed when MgO nanoparticle was combined with microwave pretreatment during the anaerobic digestion of Enteromorpha.It was noticed that MgO nanoparticles combined with microwave pretreatment improved the cumulative yield by 1.38 folds [40].This indicates that combining MgO nanoparticles with other pretreatment methods can have a positive effect.When the inhibitory influence of some nanoparticles was investigated on methanogenic bacteria activities of anaerobic granular sludge, it was discovered that the likes of CuO, MgO, and ZnO produced severe methanogenic inhibition.The Cu 2+ , Mg 2+ , and Zn 2+ produced similar inhibition considering the equilibrium soluble metal concentrations recorded during the process.This indicates that the toxicity was because of metal ions released from the nanoparticles during corrosion [32].The addition of nanoparticles of iron origin during anaerobic digestion is a vital cofactor and enzyme that has been identified to stabilize and stimulate the performance of the anaerobic digestion process [41].Studies have noticed that using Fe nanoparticles lowers the hydrogen sulphide in the biogas mixture and improves the methane released in most processes [9].Fe3O4 improves hydrogenotrophic methanogenesis by producing hydrogen or electron evolution through iron corrosion, which enhances the methane released from carbon dioxide consumption, as presented in equations 8 -10 [41].
effectively.Different 1322 (2024) 012001 IOP Publishing doi:10.1088/1755-1315/1322/1/012001 2 pretreatment methods have been experimented with in biogas production and are classified as biological, chemical, nanoparticle, additives, thermal, mechanical, and a combination of two or more methods [7].The study has shown that pretreatment methods have varying influence on different feedstocks depending on the microstructural arrangement and pretreatment conditions [8].This necessitates extensive research in developing different pretreatment techniques that are easy, available, and cost-efficient for different feedstock.

Figure 1 :
Figure 1: SEM image showing the microstructural arrangement of Xyris capensis.

Figure 4 :
Figure 4: Influence of different nanoparticles additive on cumulative methane yield.

13) 4. Conclusion This
study investigated the influence of different nanoparticle additives on the biogas and methane yield of Xyris capensis.Fe3O4 nano-additives improved biogas and methane yield by 29.89 and 42.31%, whereas CuO, MgO, and ZnO additives were observed to reduce the biogas and methane yield.Therefore, appropriate pretreatment of Xyris capensis can assist in the conversion of its enormous potential into biogas and methane that can substitute for fossil fuels.Hafiz Dzarfan Othman M, Hashim H, Matsuura T, Ismail A F, Rezaei-DashtArzhandi M and Wan Azelee I 2017 Biogas as a renewable energy fuel -A review of biogas upgrading, utilisation and storage Energy Convers Manag 150 277-94 [6] Olatunji K O, Madyira D M, Ahmed N A and Ogunkunle O 2022 Biomethane production from Arachis hypogea shells: effect of thermal pretreatment on substrate structure and yield Biomass Conversion and Biorefinery 1-14 [7] Olatunji K O, Ahmed N A and Ogunkunle O 2021 Optimization of biogas yield from lignocellulosic materials with different pretreatment methods: a review Biotechnology for Biofuels 14 1-34 [8] Olatunji K O, Madyira D M, Ahmed N A and Ogunkunle O 2022 Influence of alkali pretreatment on morphological structure and methane yield of Arachis hypogea shells Biomass Conversion and Biorefinery, 1-12 [9] Olatunji K O, Madyira D M, Ahmed N A and Ogunkunle O 2022 Effect of Combined Particle Size Reduction and Fe3O4 Additives on Biogas and Methane Yields of Arachis hypogea Shells at Mesophilic Temperature Energies, 15 3983 [10] Abdelsalam E, Samer M, Attia Y A, Abdel-Hadi M A, Hassan H E and Badr Y 2016 Comparison of nanoparticles effects on biogas and methane production from anaerobic digestion of cattle dung slurry Renew Energy 87 592-8 [11] Mu H, Zheng X, Chen Y, Chen H and Liu K 2012 Response of anaerobic granular sludge to a shock load of zinc oxide nanoparticles during biological wastewater treatment Environ Sci Technol 46 5997-6003 [12] Abdelsalam E, Samer M, Attia Y A, Abdel-Hadi M A, Hassan H E and Badr Y 2016 Comparison of nanoparticles effects on biogas and methane production from anaerobic digestion of cattle dung slurry Renew Energy 87 592-8 [13] De Wet H, Struwig M and Van Wyk B E 2014 Taxonomic notes on the genus Stephania (Menispermaceae) in southern Africa South African Journal of Botany 95 146-51 [14] Anon Official Methods of Analysis, 21st Edition (2019) -AOAC INTERNATIONAL [15] Hassaan M A, Pantaleo A, Tedone L, Elkatory M R, Ali R M, Nemr A El and Mastro G De 2021 Enhancement of biogas production via green ZnO nanoparticles: experimental results of selected herbaceous crops Chem Eng Commun 208 242-55