S-ZVI@biochar constructs a directed electron transfer channel between dechlorinating bacteria, *Shewanella oneidensis* MR-1 and trichloroethylene

Trichloroethylene (TCE) is a widespread industrial pollutant requiring effective remediation strategies. Current methods, such as using zero-valent iron (ZVI) or dechlorinating bacteria (DB), suffer limitations. ZVI, while highly reactive, readily passivates and agglomerates, reducing its efficiency. DB-based bioremediation is often slow and requires additional electron donors. This research investigates a synergistic approach combining the chemical reactivity of modified ZVI with the biological activity of DB, aiming to overcome the individual limitations of each method. Specifically, the study explores the use of sulfurized zero-valent iron (S-ZVI) incorporated into a biochar matrix (S-ZVI@biochar) to enhance electron transfer between the chemical reductant and the microbial community, including the iron-reducing bacteria *Shewanella oneidensis* MR-1, for more efficient TCE degradation. The successful development of such a system could offer a sustainable and effective solution for long-term TCE remediation in anaerobic environments, addressing the current challenges in both chemical and biological dechlorination methods.

Numerous studies have explored the use of ZVI for TCE remediation. However, ZVI's susceptibility to passivation and agglomeration limits its long-term effectiveness. Modifications to ZVI, such as sulfurization, have been shown to enhance reactivity and electron transfer. The incorporation of biochar, a porous carbonaceous material, further improves dispersion and biocompatibility. Research on the synergistic combination of ZVI and DB has demonstrated enhanced TCE removal. The use of *Shewanella* species, known for their iron-reducing capabilities, has shown promise in reactivating passivated ZVI and promoting DB growth. The literature, however, lacks a comprehensive investigation into the precise mechanisms and extent of enhanced electron transfer within such integrated chem-bio systems, particularly with the inclusion of biochar to bridge the chemical and biological components. This study aims to fill this gap by thoroughly investigating the synergistic effects of the S-ZVI@biochar composite, DB, and *S. oneidensis* MR-1 on TCE degradation and elucidating the underlying mechanisms involved.

The study involved several key steps: **1. Materials Preparation:** Biochar was produced by pyrolysis of pine wood chips under nitrogen atmosphere. mZVI (400 mesh) was sulfurized using sodium dithionite (Na2S2O4) to produce S-ZVI. S-ZVI@biochar was then synthesized by ball milling S-ZVI with biochar. **2. Microorganism Cultivation:** Dechlorinating bacteria (DB) were isolated from soil samples, and *Shewanella oneidensis* MR-1 was obtained commercially. Both were cultured under anaerobic conditions using specific media. **3. Batch Experiments:** Batch experiments were conducted in sealed vials to assess TCE removal under various conditions: DB alone, DB + MR-1, ZVI, S-ZVI, S-ZVI@biochar, and combinations of DB/MR-1 with ZVI or S-ZVI@biochar. Sodium acetate was used as an electron donor. The effects of S-ZVI@biochar dosage and sodium acetate concentration were optimized. **4. Analytical Methods:** TCE and its dechlorination byproducts were analyzed using gas chromatography (GC-FID). Total and dissolved iron concentrations were measured using ICP-AES and the phenanthroline method, respectively. Hydrogen concentration was determined by GC-TCD. pH and oxidation-reduction potential (ORP) were monitored. SEM, XRD, and XPS were used to characterize the materials before and after reactions. **5. Metagenomic Sequencing:** Metagenomic sequencing was performed on samples from different reaction systems to analyze changes in microbial community composition and gene expression.

The study revealed several significant findings: **1. Enhanced TCE Removal:** The DB + MR-1 + S-ZVI@biochar system exhibited the highest TCE removal efficiency (96.5% after 30 days), substantially surpassing other systems. The S-ZVI@biochar composite demonstrated superior performance compared to ZVI and S-ZVI alone. **2. Improved Electron Transfer:** Electrochemical analyses (EAC, EDC, CV, GCD) demonstrated the superior electron transfer capacity of S-ZVI@biochar compared to ZVI and S-ZVI. Biochar acted as an effective electron shuttle, facilitating electron transfer between S-ZVI and microorganisms. **3. Microbial Community Shifts:** Metagenomic sequencing revealed shifts in microbial community composition and diversity across the different experimental systems. The addition of S-ZVI@biochar and MR-1 significantly influenced the abundance of different bacterial genera, notably enhancing the proportion of *Pseudomonas*, a known TCE degrader. The presence of MR-1 was associated with an increase in the Fe(II)/Fe(III) ratio, consistent with its role in iron reduction and recycling. **4. Dechlorination Pathways:** The study identified both hydrogenolysis and β-elimination pathways for TCE dechlorination. The S-ZVI@biochar system primarily exhibited β-elimination, while the DB systems favored hydrogenolysis. The combination of the system resulted in a combined effect of both pathways. MR-1 enhanced the hydrogenolysis pathway by indirectly supporting the activity of dechlorinating bacteria. **5. Iron Recycling:** The presence of MR-1 facilitated iron recycling by converting Fe(III) (corrosion product) back to Fe(II), enhancing the overall reductive capacity of the system. XPS analysis showed the relative amounts of Fe(II) and Fe(III) on the surface of S-ZVI@biochar during the process. This cycle was evidenced by changes in the Fe(II)/Fe(III) ratio observed through XPS analysis. **6. Synergistic Effects:** The study conclusively demonstrated the synergistic effects of combining S-ZVI@biochar, DB, and MR-1, significantly enhancing TCE removal efficiency compared to the individual components acting alone.

The results demonstrate the successful development of a highly effective chem-bio hybrid system for TCE degradation. The key to its success lies in the improved electron transfer facilitated by the S-ZVI@biochar composite. Biochar acts as a conductive bridge, enhancing the interaction between the chemical and biological components. The increase in the proportion of *Pseudomonas* in the microbial community, a known dechlorinating bacteria, further supports this synergy. The iron recycling process, driven by MR-1, contributes to the sustainability and long-term performance of the system. The findings highlight the importance of considering integrated chem-bio approaches for environmental remediation, moving beyond solely chemical or biological strategies. This system offers advantages over traditional methods, promising more efficient, sustainable, and cost-effective TCE remediation in groundwater.

This study successfully demonstrated a highly effective and sustainable chem-bio hybrid system for TCE degradation utilizing S-ZVI@biochar, DB, and MR-1. The S-ZVI@biochar composite enhanced electron transfer efficiency, improved microbial community structure, and facilitated iron recycling. This innovative system offers a promising approach for long-term and cost-effective TCE remediation in anaerobic environments. Future research should explore the scalability and applicability of this approach in real-world settings, investigating factors like varying contaminant concentrations and groundwater conditions.

While the study demonstrates significant promise, several limitations should be noted. The experiments were conducted under controlled laboratory conditions, and the results may not fully reflect the complexities of real-world groundwater environments. Further research is needed to evaluate the long-term stability and effectiveness of the system under diverse environmental conditions, including the effects of other co-contaminants. The specific mechanisms of electron transfer between S-ZVI@biochar and microorganisms require further investigation. Scaling up the system for practical applications also needs further study.