Magnetite-assisted in situ microbial oxidation of H₂S to S⁰ during anaerobic digestion: A new potential for sulfide control

Highlights

• The effect of magnetite on anaerobic digestion of sulfur-rich biomass was studied.

• Magnetite addition did not induce an apparent enhancement of CH₄ production.

• The production of H₂S in biogas was significantly reduced by magnetite addition.

• Magnetite promoted the anaerobic microbial oxidation of sulfide to S⁰ through DIET.

• A novel electric syntrophy between unknown ASOBs and methanogens is proposed.

Abstract

Direct interspecies electron transfer (DIET) between exoelectrogenic fatty acid-oxidizing bacteria and electrotrophic methanogens has recently been discovered, and studies have suggested that promoting DIET by adding electrically conductive material can effectively enhance the methanogenic performance and stability of anaerobic digestion (AD). This study investigated the effect of conductive magnetite (Fe₃O₄) addition on the AD of a sulfur-rich organic waste mixture, with an emphasis on the fate of sulfur and on H₂S production. In contrast to previous findings, methanogenic performance under magnetite-added conditions was not significantly enhanced within the tested dose range of up to 20 mM Fe. When magnetite was added, H₂S production decreased remarkably along with the extracellular accumulation of S⁰. Moreover, the H₂S content in biogas was below 100 ppmv at magnetite doses of 8 mM Fe or higher (>6000 ppmv under control conditions, i.e., without magnetite). The reduced H₂S production appears to be due to the anaerobic oxidation of sulfide to S⁰ because sulfate reduction remained active, whereas FeS was not produced under magnetite-added conditions. Based on microbial community analysis results and thermodynamic calculations, the electric syntrophy via DIET between exoelectrogenic anaerobic sulfide-oxidizing bacteria and electrotrophic methanogens is suggested to have been promoted by magnetite. To the best of our knowledge, this study is the first to propose an electro-syntrophic association that couples the oxidation of sulfide to S⁰ with the reduction of CO₂ to CH₄ in methanogenic environments. The present findings open a new possibility for in situ H₂S control and sulfur recovery in AD processes for sulfur-rich waste treatment.

Introduction

Anaerobic digestion (AD) is a well-established technology that has been widely used for the treatment and valorization of different organic wastes. Recently, biogas production by AD has received increasing attention as an alternative energy source for sustainable development. AD involves a series of biological reactions whereby complex organic compounds are degraded and converted into methane and carbon dioxide by the concerted activity of different microbial groups. Due to the mixed-culture nature of AD microbial communities, AD processes typically involve diverse microorganisms with different metabolic functions particularly in waste treatment environments. Some of the community members likely have functions that are not beneficial or even detrimental to effective AD and biogas production. A representative example is the dissimilatory sulfate reduction by sulfate-reducing bacteria (SRBs), a process that generates hydrogen sulfide (H₂S) as end product. H₂S is toxic at low concentrations (>50 mg/L) to microorganisms involved in biogas production [1]; furthermore, the SRBs compete with methanogens for common substrates (i.e., H₂ and acetate). Therefore, dissimilatory sulfate reduction directly affects methanogenic activity in AD processes, and the adverse effect is multiplied when treating sulfur-rich waste streams, such as those coming from pharmaceutical, petrochemical, and food processing industries [1]. Additionally, the malodorous and highly corrosive properties of H₂S can cause serious health and hygiene issues and mechanical damages. The H₂S content in biogas typically ranges from several hundred to several thousand parts per million volume (ppmv), and costly biogas cleaning is required to prevent corrosion problems according to the use of biogas, for example, <1000 ppmv for heaters and <500 ppmv for engines [2]. As described above, H₂S generation is inevitable in anaerobic decomposition of sulfur-containing compounds and is a major concern for the stable and economical operation of an AD process.

Many efforts have been made to control the generation and emission of H₂S in anaerobic digesters in order to create a favorable environment for methanogenesis and to reduce the load in the biogas desulfurization step. Several in situ sulfide control measures based on chemical, physical, and biological approaches have been developed and applied at full scale, and among the most commonly employed approaches today are the precipitation of sulfide using iron salts and the oxidation of sulfide by microaeration [2]. Both methods effectively remove sulfide in situ; however, they still have several drawbacks related to the continuous consumption of chemicals, increase in sludge production, loss of digester capacity, possibility of methane oxidation, or need for energy-intensive aeration. SRBs are metabolically versatile and highly tolerant to environmental stresses, making them difficult to selectively suppress by controlling the operating conditions (e.g., temperature and pH) of a digester [3], [4]. Inhibitors, such as biocides and molybdate, have also been used to suppress SRBs; however, continuous feeding of inhibitors above a critical concentration is expensive and may affect other microorganisms, including those involved in methanogenesis [5]. Moreover, studies have reported that SRBs can recover from and adapt to the effects of different biocides [6], [7]. Because of the versatile and resilient characteristics of SRBs, it becomes difficult to apply preventive approaches (i.e., inhibition of sulfate reduction) for in situ sulfide control.

Recent studies have revealed the widespread existence of electric syntrophy mediated by direct interspecies electron transfer (DIET) in diverse anoxic environments, and the exchange of electrons directly between syntrophic partners is believed to be a significant advantage under certain conditions [8]. DIET between exoelectrogenic fatty acid-oxidizing bacteria and electrotrophic methanogens has been found to be an alternative for indirect interspecies electron transfer (IIET) that involves reduced electron carrier molecules, such as hydrogen and formate, in methanogenic environments [9], [10]. DIET is energetically and kinetically advantageous over IIET in methanogenesis as it does not require complex steps for the synthesis of hydrogen or formate [11]. Increasing interest is being given to the potential of promoting DIET as a new strategy to enhance methanogenic activity in AD processes [12]. Particularly, adding a conductive material, such as magnetite, biochar, activated carbon, or carbon cloth, which can serve as electrical conduit, has been applied to conveniently promote DIET and has been proven to effectively enhance AD performance and stability in recent studies [13], [14], [15], [16]. Given that it is not unlikely that mixed-culture AD microbial communities include diverse electroactive microorganisms, such approaches could affect other microbial redox processes, including those related to sulfur metabolism, either directly (i.e., stimulation of electric syntrophy) or indirectly (i.e., alteration of electron flow). H₂S is a major electron sink in anaerobic environments, and the effect of conductive material addition on the flow of electrons is supposed to be complex in AD processes employed in sulfur-rich waste treatment. However, little is known about such possibilities and their implications.

To address this knowledge gap, this study investigated the effects of promoting DIET by adding magnetite, which has been most used in previous studies on the promotion of DIET in methanogenic environments due to its widely available, non-toxic, and highly conductive (ca. 2.0 × 10⁵ μS/cm) nature [12], on the methanogenic and sulfidogenic activities in the AD of sulfur-rich waste, with a focus on the fate of sulfide. The microbial communities were characterized by high-throughput sequencing (HTS) of the 16S rRNA and 16S rRNA genes to gain a deeper insight into the process behavior under different operating conditions. The findings of this study offer a new approach for in situ sulfide control and for possible recovery of elemental sulfur.