Investment in renewable energy sources is a reality nowadays (Atelge et al., 2020). Among the different renewable energy sources, the production of biogas from organic waste, generated in the complex microbial process of anaerobic digestion (AD), stands out due to its many advantages (Scarlat et al., 2018; Abanades et al., 2022; Wang et al., 2023). AD is an integrated biological process of the decomposition of organic matter in an oxygen-free environment that produces biogas and a digestate. Biogas is mainly composed of methane (CH₄), carbon dioxide (CO₂) and other minor molecules (H₂, H₂S, N₂, H₂O) (Madsen et al., 2011; Xu et al., 2018). Although it is a promising strategy, biogas production from AD still faces challenges in its process stability and efficiency (Appels et al., 2008; Xu et al., 2018). This process also deals with different groups of microorganisms that play crucial roles in AD (Deng et al., 2020) and the syntrophic relationship between these microorganisms, which have different growth requirements (Schnürer and Jarvis, 2018; Deng et al., 2020), is mandatory to achieve maximum biogas production (He et al., 2019). Thus, many strategies have been developed to improve microbial metabolic activities and biogas production in AD, such as co-digestion, pre-treatments and upgrades approaches (Patinvoh et al., 2017; Paritosh et al., 2018; Adnan et al., 2019; Abdelwahab et al., 2020; Anacleto et al., 2022).

The strategy of nanoparticles (NPs) addition to the AD process has shown results in improving the stability of the microbial community and the electric exchange between organic reductive bacteria and methanogenic archaea by enhancing direct interspecies electron transfer (DIET) efficiency (Rotaru et al., 2014; Park et al., 2018). Conductive NPs such as magnetite and nickel would be related to the increase in electron transfer efficiency between methane-producing microorganisms and other species that inhabit biogas reactors, a process also known as “electrical syntrophy” (Kato et al., 2012; Jin et al., 2019). It has also been suggested that the benefit of NPs addition may be related microbial growth requirements for trace elements, such as Fe, Ni, and Cu, for the enzymatic activity of the different microorganisms involved in AD.

NPs with different chemical compositions were studied for such purpose, like composed of silver (Grosser et al., 2021), zero valent iron (Abdelsalam et al., 2017b), cobalt (Abdelsalam et al., 2017a), nickel (Abdelsalam et al., 2017a; Hassanein et al., 2019), carbon (Baniamerian et al., 2019) and magnetite (Fe₃O₄) (Jin et al., 2019; Uysal and Mut, 2020; Amo-Duodu et al., 2021; Yu et al., 2022), among others (Abdelwahab et al., 2020). In 2019, Jin and others were successful in increasing 3–10 times the production of biogas by using chemically synthesized magnetite NPs. The increased yield was attributed to the conductive property of magnetite to increase the efficiency of DIET (Jin et al., 2019).

The benefit of the addition of NPs can also be a consequence of other NPs interactions such as the reduction of short-chain fatty acids accumulation, which in high amounts inhibits the AD process, organic matter overload, and the mitigation of H₂S and ammonia, which ends up directly increasing CH₄ production (Hernández et al., 2011; Abdelwahab et al., 2020; Farghali et al., 2020; Cui et al., 2022; Ziganshina et al., 2022). Thus, the NPs could be potentially used to accelerate microbial activities in AD and optimize CH₄ production. However, the outcome reported for NPs effect on biogas production is not consistent. Farghali et al. (2019) observed an increase in biogas and CH₄ production after adding iron oxide NPs that was lower than those previously observed by Abdelsalam et al. (2016). In their discussion, Farghali et al. (2019) suggested that this difference is due to different parameters in the methods between the studies, despite using NPs of similar composition. These differences include different specific composition and concentrations of NPs (Farghali used Fe₂O₃ at a concentration of 100 mg/L while Abdelsalam used Fe₃O₄ at 20 mg/L) and the substrates (Farghali tested cattle manure as substrate, while Abdelsalam used raw manure) (Abdelsalam et al., 2016; Farghali et al., 2019). This type of discrepancy in results emphasizes the importance of studies that allow comparison and systematizing the parameters and respective impacts of adding NPs to increase biogas production by AD.

Performing a systematic analysis is a suitable technique to integrate, in a reliable way, results from different studies to summarize its findings in more clear and robust conclusions (Van Houwelingen et al., 2002). In addition, conducting a meta-analysis contributes to resolving conflicting results from the literature and finding new patterns that bench experiments cannot achieve. Here, we performed a systematic review followed by a meta-analysis according to PRISMA 2020 statement (Page et al., 2021). Data collected was submitted to a series of statistical analysis to clarify NPs addition in the AD process and its implications for methane production improvement.

The main compositions of NPs with better average effects on CH₄ production were those of cobalt, zero valent iron and magnetite. It is worth noting that the number of experiments using magnetite NPs is much higher than for iron and cobalt. Magnetite NPs popularity and performance may be related to the diversity of effects reported for this composition, especially its driving role in DIET (Cruz Viggi et al., 2019; Jin et al., 2019). Magnetite is a metal oxide obtained relatively easily, most often by the process of co-precipitation or hydrothermal decomposition (Reddy et al., 2012). Despite being the most studied NPs, the majority of the studies that presented enough information about NPs did not get much more depth discussions about the microbiology perspective, being limited just to observations about chemical parameters (i.e., VFAs, pH, H₂S variations).

Analyzing both structural and functional changes in the community as well as the chemical characteristics is essential to a better understanding of magnetite effects in AD. This highlights the importance of interdisciplinary collaborations in this kind of studies. However, few studies did connect their NPs results with the microbiologic analysis. Huang et al. (2020) used 8 mM of magnetite in the AD of rice plantation residues and observed that there was a modulation of the microbial community with the enrichment of the Geobacter and Methanosaeta genera, which also perform DIET, and increase of substrate degradation in addition to increasing the methanogenic activity (Huang et al., 2020). Jin et al. (2019) applied 20 mM of magnetite into artificial wastewater with high sulfate concentration and increased CH₄ production and the number of bacteria genera related to sulfate reduction and methanogenic archaea.

These results indicate the stimulation of DIET among these microorganisms and therefore add a potential tool for “H₂S removal” to the possible advantages of using magnetite NPs in AD (Jin et al., 2019). In addition to the modulation of the microbial community by stimulating DIET, the release of Fe⁺² ions can favor the formation of iron sulfide salts, helping to mitigate the formation of H2S, which is toxic to a series of microorganisms. Increase in H₂S concentrations may result in pH decrease in the system and also in high corrosion of pipes and other facilities, in addition to presenting significant risks to human health when inhaled (Truong et al., 2006; Appels et al., 2008).

Even fewer studies explored both the relation between the NPs and the microbial community. Casals et al. (2014), reported changes in the magnetite structure throughout the municipal waste AD process. Authors observed that not only a NPs size decrease, but also a loss of Fe⁺² NPs content. The sustained release of iron from magnetite NPs stimulated the activity of methanogens without causing toxicity to the system, leading to an increase in the production of biogas by 180% and even the total CH₄ percentage (Casals et al., 2014). Fe⁺² ions are of great importance in methanogenic metabolism, but it is noteworthy that iron ions can also be released by iron oxides such as hematite, maghemite and magnetite, depending on their crystallographic characteristics, which are determinant in the dissolution of these minerals (Allen et al., 1988; Lagoeiro, 1998). In studies involving species of the genus Methanothermobacter, the importance of iron supply for the synthesis of a series of enzymes such as hydrogenases, dehydrogenases and reductases, in addition to ferredoxins, was observed (Kaster et al., 2011).

It is worth noting that merging different amounts of more than one composition of NPs might also display good improvements in CH₄ yields. Hassanein et al. (2019) tested different concentrations of Ni, Co, Fe and magnetite and then combinations between these compositions in the AD of poultry waste. These authors observed that the concentrations of 4.5 mg/L of Co, 100 mg/L of Fe and 15 mg/L of magnetite increased by circa 30% the CH₄ production, and significantly decreased H₂S production (Hassanein et al., 2019). Similar mixtures also promoted an increase of 23.7% in the production of biogas and a significant depletion in the production of H₂S in large scale digesters (30 L) (Hassanein et al., 2021). The benefits of NPs such as Co and Ni have been attributed to a possible supplementation of these elements as important components for some enzymatic processes for the microorganisms’ metabolism. For the Co NPs, for example, as it is necessary for synthesizing cobalamin and coenzyme B12, important parts of enzymatic complexes are involved in methanogenesis. Thus, supplementing this and other trace elements through NPs benefits metabolic improvements in AD (Zhang et al., 2009).

Depending on their concentration, smaller magnetite NPs seem to achieve a more effective result in the production of biogas (Figures 3A, B). The NPs’ surface area is inversely proportional to their average size, especially considering that most NPs usually have spherical shape (Juntupally et al., 2017; Liu et al., 2021; Zaidi et al., 2021; Yu et al., 2022). Thus, the addition of NPs with smaller dimensions would have comparably more advantages over larger ones, in addition to potentially presenting better dispersion with less material expense in the sludge present in the reactors.

However, it was observed that, even though <50 nm NPs have the highest individual effect on CH₄ production, NPs between 50 and 100 nm promote a mean larger increase in CH₄ production (Figure 3A), suggesting that intermediate NPs may be more predictable and less variable impacts on biogas production. The increase of CH₄ production of this size range does not have a significant correlation with its concentration, suggesting that more factors are involved in its effect on AD.

The findings that NPs concentrations below 100 mg/L with <50 nm sizes (Figure 3), suggest a possible “limit” in the concentration for the use of magnetite, that could be attributed to their probable dissolution over time, releasing Fe⁺² and Fe⁺³ ions. This could be potentially beneficial, especially considering that Fe is of great importance in the formation of enzymes important in the metabolism of most microorganisms involved in AD. However, it should always be remembered that iron becomes toxic to most microorganisms at higher concentrations (Goswami et al., 2016).

As NPs between 50 and 100 nm seem to be more resistant to dissolution and, therefore, less likely to cause overdose stress, they should be preferably used in AD. The lack of statistical correlation between concentration variables and the CH₄ production response for NPs between 50 and 100 nm not only suggests that there are more variables influencing this impact, but that their effect must go beyond the release of iron ions, since the high concentration could easily cause stress. Thus, it is likely that NPs in this size range have a driving influence as a stimulus to DIET rather than iron release. However, such deductions can only be confirmed with certainty from the use of analyzes of the microbial community linked to the conductivity profile of the digestate (Lovley, 2017; Fu et al., 2019).

An increase of studies in crystallographic characterization of the NPs applied to AD could be very enlightening to elucidate their effect, especially because of the existence of irregular faces in the crystals (e.g., the non-typical regular expected geometric morphologies like spherical, cuboid, etc.) that provide electronic instability in the molecular structure of the crystal. Apparently, it seems that NPs with shapes with inclusions that result in the largest contact surface would be of greater interest if the purpose of their addition were strictly the release of iron ions. However, stating that the impact of NPs on the system is solely caused by its iron release is only possible with refined characterization techniques of the nanomaterials added (i.e., analytical microscopy techniques). The crystallographic characteristics of the NPs materials used have an important hole in the resistance and hardness of these NPs. It means that depending on the crystallography of the NPs, their degradation inside the AD system may differ. It can be advantageous if the NPs benefits are related to the sustained release of some elements (i.e., Fe and Co), but it also means that some NPs compositions may persist for longer periods inside the AD than others. Magnetite, for example, has three crystallographic faces: [110], [111] and [100]. According to studies by Allen et al. (1988) and Yamazaki (2020), the [111] face is twice as prone to dissolution due to the compact molecular arrangement of oxygen atoms, which would be more easily protonated in acidified environments. This kind of characterization was rarely observed in the studies involving NPs addition in AD (Casals et al., 2014).

Hu et al. (2010) described that magnetite NPs with distinct surfaces, dissolution and aggregation potential presented different stability and aggregation/disaggregation behaviors according to organic matter concentrations and pH and that this also had a larger impact on particle surface charge status. This means that, depending on where and how NPs are released, their impact can vary from just being suspended particles, as an ions source, to potentially contaminants absorption. This result highlights the need for studies involving the nanotechnology application to AD. Even though a large amount of data based on the addition of magnetite NPs allowed the establishment of clear patterns, more experiments about their structure and other compositions could contribute to reach a deeper impact on all potential roles of NPs to AD.

The limited number of studies suggests a knowledge gap associated with the characterization of the changes promoted directly on the AD microbial community, but also the crystalline and chemical profiles of the NPs employed. Therefore, it is important to standardize the minimum information required in these types of research to allow the comparison, validation and reproducibility of the results described in the literature.

Here, we observed that some NPs have a direct and stimulatory effect on CH₄ production, especially magnetite NPs, while other chemical compositions had inhibitory and/or opposite effects depending on the substrate, chemical composition and concentration used in the AD process. Magnetite NPs showed greater benefits with <100 nm sizes and <100 mg/L concentrations; thus these are potentially the future of Nanotechnology in AD for enhancing biogas production. In addition, the fact that the increase in the concentration and size of the NPs used does not directly result in better results in the production of CH₄ has a significant impact on the cost of NPs addition in AD. The development of methods to increase CH₄ production in AD by the addition of NPs is a promising strategy. Future studies must include proper characterization of the NPs studied as well as microbial community analysis for better understanding of its effects. For a more in-depth discussion on the NPs crystallographic characteristics, it would be necessary to carry analytical electron microscopy techniques like high-resolution transmission electron microscopy (HRTEM), electron energy loss spectroscopy (EELS) analysis, X-ray diffraction (XRD), and X-ray absorption spectroscopy (XANES). These techniques would allow the understanding of the size, shape and structural behavior of the NPs, as well as its possible ion releases and chemical bondings during AD.