Establishment of direct interspecies electron transfer through ethanol supply during azo dye Reactive Red 2 anaerobic degradation

Establishment of direct interspecies electron transfer through ethanol supply during azo dye Reactive Red 2 anaerobic degradation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Establishment of direct interspecies electron transfer through ethanol supply during azo dye Reactive Red 2 anaerobic degradation Zisheng Zhao, Yixin Li, Kang Wang, Yu An, guangyi Zhang, Long Huang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3989947/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Azo dye, for example reactive red 2, threatened the environment and human health when directly discharging into waters, and appropriate treatment methods are urgently required for such contaminants. In this study, ethanol was added to the digesters to promote azo dye Reactive Red 2 (RR2) anaerobic digestion efficiency. Results showed that the COD removal and RR2 removal efficiency were 37.0% and 63.2% in cycle 6 (only RR2 used as the substrates) in ethanol co-digested reactor, which was 16.0% and 54.5% higher than that in control reactor, and 14.5% and 52.0% higher than that in acetate co-digested reactor, respectively. Mechanisms exploration found that the electron transfer system (ETS) activity, specific methanogenic activity (SMA) and Coenzyme F420 of the sludge were effectively improved in ethanol co-digested reactor, which indicated that the addition of ethanol to anaerobic digester could enhance the activity of the microbial. Microbial community analysis showed that the electroactive microbial ( Geobacter and Methanothrix ) were more enriched in ethanol co-digested reactor. It was speculated that the direct interspecies electron transfer (DIET) process was possible established between Geobacter and Methanothrix , which played an important role for the improvement of RR2 removal efficiency in ethanol co-digested reactor. Anaerobic digestion RR2 removal direct interspecies electron transfer Ethanol stimulation Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Azo dyes, as the persistent organics containing azo and aromatic groups, are extensively used in industry. However, during textile production process, a certain amount of the azo dyes can’t be consumed and discharged together with the textile wastewater eventually(Cai et al. 2017 ). Previous study found that many synthetic azo dyes were proved to be toxic and carcinogenic(de Aragão Umbuzeiro et al. 2005 ), which will induce potential environmental pollution and threaten human health when directly discharging into natural waters(Dai et al. 2016 , Tang et al. 2016 ). Many physiochemical methods are used to treating dyestuff wastewater and received efficient performance, but the high energy consumption and equipment investment were unacceptable(Chhabra, Mishra and Sreekrishnan 2015 , Saratale et al. 2011 , Nouren and Bhatti 2015 ). Anaerobic digestion, as a low costs and energy recovery methods, exhibit potential advantage for treating dye wastewater(Meng et al. 2014 ). During textile wastewater anaerobic digestion process, the azo dyes could be degraded biologically with the participation of azo-reductases enzymes(Wang et al. 2018 ). However, the property of azo dye (persistent and high toxicity) was bacteriostatic, which would limit the efficiency of electron transfer between microbials and deteriorate anaerobic performance(van der Zee and Villaverde 2005 ). It was reported that electron transfer efficiency closely related to the organics anaerobic digestion performance(Kouzuma, Kato and Watanabe 2015 , Schink 1997 ). Previous study indicated that strengthening electron transfer efficiency could enhance microbial metabolism, which would promote organics anaerobic degradation(Zhao et al. 2021 ). During anaerobic methanogenesis, the electron transfer mode was widely considered as interspecies H 2 /formate transfer (IHT/IFT) with H 2 /formate serving as the electron carrier in recent decades(Stams and Plugge 2009 , Sieber, McInerney and Gunsalus 2012 ). In order to keep growth of the syntrophic partners, the electron carriers should be consumed efficiently with the participation of methanogens. It was reported that only when the concentration of H 2 /formate is kept very low, the substrates syntrophic oxidation was thermodynamically feasible, since this process is endergonic under standard conditions(Stams and Plugge 2009 ). Direct Interspecies Electron Transfer (DIET) has recently been considered as the new interspecies electron transfer mode between bacterial and methanogens(Rotaru et al. 2014b , Rotaru et al. 2014a ). It was reported during DIET process microbial use the c-type cytochromes(Lovley et al. 2011 ) and/or conductive pili(Lovley et al. 2011 ) as the mediator for extracellular electron transfer, which not rely on H2/formate as the electron carriers, thus making it potentially more efficient and energy-conserving mode for methane production(Viggi et al. 2014 , Lovley 2017 ). Originally, DIET was only reported in few defined co-cultures, such as co-cultures of two Geobacter species(Summers et al. 2010 ) and co-cultures of Geobacter with methanogens(Rotaru et al. 2014a , Lei et al. 2016 ) with ethanol as electron donor. Subsequently, DIET was found in mixed culture with propionate(Viggi et al. 2014 ), leachate(Lei et al. 2016 ) and phenols(Li et al. 2021b ) as the substrates. However, in the absence of any amendments, DIET was only existed as the predominant electron transfer mode in treating brewery wastes. Thereafter, researchers found that varieties conductive materials could improve anaerobic digestion at the certain extent, and they ascribed the enhancement to accelerating of DIET(Zhao et al. 2017 ). However, the enriched bacteria were Synaatrophomonas , Proteiniclasticum , Syntrophomonadaceae and Clostridium , which were not assertive evidence for DIET(Zhao et al. 2017 ). For example, in Li’s study(Li et al. 2015a , Li et al. 2015b ), Syntrophomonadaceae was reported as the bacteria to metabolize butyrate to acetate via IHT rather than DIET. It was reported that ethanol could effectively improve the abundance of Geobacter and Methanothrix or Methanosarcina species, and form the aggregates with high conductivity(Zhao et al. 2016 ). Ethanol was used as the trigger for DIET to dispose varieties organics (such as propionate, waste activated sludge), and received satisfactory performance(Zhao et al. 2016 , Li et al. 2020 ). The potential mechanism was that during anaerobic digestion with the presence of ethanol, Geobacter could be enriched, which as the most important exoelectrogens during DIET process was reported that can utilize a broad of organics, such as alcohols, volatile fatty acids, phenols and benzene as substrates(Lovley et al. 2011 ). However, researches about ethanol as the trigger for DIET to promote persistent organics degradation had been few reported. Based on these considerations, research about ethanol using as the specific substrate to perform DIET was conducted to investigated if RR2 degradation could be enhanced through stimulating the microbial communities. 2. Material and methods 2.1 Chemicals and inoculum Ethanol, sodium acetate, RR2 and other experimental chemicals were purchased from Aladdin reagent CO. Ltd. (Shanghai, China). The sludge collected from Wulongkou waste sludge anaerobic treatment plant of Zhengzhou (China) was used as the inoculant sludge in this experiment, and cultured in an anaerobic digester before the experiment. The substrates and operating conditions for the culturing were according to the reference(Li et al. 2021a ). 2.2 Batch experiment operation In this study, batch experiments were conducted to investigate the effects of ethanol on RR2 anaerobic removal. To exclude the co-substrates effects of ethanol, a unique control reactor was set up with equal amount COD of sodium acetate as the co-substrates. Therefore, in this experiments, three group reactors were operated, i.e. control reactor with RR2 as the sole substrates during the entire experiment process, hereafter referred as R1; co-digestion of RR2 with ethanol at the initial stage of the experiment, hereafter referred as R2; co-digestion of RR2 with acetate at the initial stage of the experiment, hereafter referred as R3. All groups were conducted in 250 ml serum bottle for six cycles, and each cycle lasted for six days. During experiment, 100 mL mixture (containing 15 mL inoculum and 85 mL substrates) were added into each bottle. In phase I (the first two cycles) 100 mg/L RR2 was used as the substrates in R1, the substrates in R2 were 100 mg/L RR2 and 1000 mg COD/L ethanol, the substrates in R3 were 100 mg/L RR2 and 1000 mg COD/L acetate. In phase II (the third and fourth cycles) the concentration of RR2 in all reactors were increased to 200 mg/L, and the concentration of acetate and ethanol in R2 and R3 were unaltered. In phase Ⅲ (the last two cycles), only 200 mg/L RR2 were used as the substrates in all reactors with the removal of acetate and ethanol from R2 and R3. In addition, the COD: N: P ratio is kept at 200:5:1 with the addition of NH4Cl and KH2PO4. 1 mL stock solution of trace element was added to 1 L substrates, which was prepared according to previous study(Zhao et al. 2020 ). At the end of each cycle, another 85 ml fresh substrate as described above was added to each reactors to replace the residual liquid in the reactors. Before starting of each cycle, oxygen in the bottles was removed with 99.9% nitrogen to maintain anaerobic condition. Subsequently, these sealed reactors were placed in an homothermal incubator (37 ◦C). During experiment, COD and RR2 concentration were measured every day, and at the end of each cycle the methane production was analyzed. All groups were performed in triplicate. 2.3 Analytical methods The COD and biogas components (CH 4 and CO 2 ) were measured as the references(Zhang et al. 2020 ). A visible spectrophotometer (DR3900, HACH, USA) were used to measure the RR2 concentration by analyzing the absorbance at the 512 nm. Before the measurement of COD and RR2 concentration, liquid collected from the reactors were filtered by the membrane filters with 0.45 µm aperture. The COD value of unit mass substrates were as follows: 0.61 g-COD/g RR2, 2.05 g-COD/g ethanol, 0.49 g-COD/g sodium acetate. The intermediate products of RR2 during anaerobic digestion process were analyzed by a liquid chromatography-mass spectrometer (LC- MS, Agilent 6410B, Palo Alto, USA). The electron transport system (ETS) activity was measured by the methods of 2-(piodophenyl) − 3-(p-nitrophenyl) − 5-phenyl tetrazolium chloride (INT) reduction, and the detailed methods was according to the reference(Zhang et al. 2020 ). The specific methanogenic activity (SMA) of the sludge in the reactors after anaerobic digestion was measured according to the methods described by Hu et al.(Hu et al. 2020 ). Coenzyme F420 was measured by a fluorescence spectrophotometer (Hitachi, F-4500, Japan) according to Tian et al. (Tian et al. 2017 ). At the end of experiment, microbial community was analyzed by High-throughput gene pyrosequencing. Genomic DNA in the suspended sludge was extracted with a DNA extraction kit (BioTeke Corporation, Beijing, China) as the manufacturer’s instructions described. Detailed methods were same as previous study(Zhu et al. 2021 ). 3. Results and discussion 3.1 Organics removal and methane production This experiment included three phases (phase I: co-digestion of acetate/ethanol and 100 mg/L RR2, phase II: co-digestion of acetate/ethanol and 200 mg/L RR2, phase Ⅲ: mono-digestion of 200 mg/L RR2), and each phase containing two cycles, and each cycle proceeded for 6 days. During this experiment, the COD in each cycle was measured and the removal efficiency was showed in Fig. 1 a. From the figure, it can be seen that in phase I and phase II both co-digesting with ethanol and acetate could promote the COD removal. Exactly, in cycle 2 (phase Ⅰ), the COD removal efficiency in R1 (control reactor), R2 (ethanol-co-digested reactor) and R3 (acetate-co-digested reactor) were 41.0%, 80.3% and 69.0%, respectively. The reason of higher removal efficiency in R2 and R3 was the removal of ethanol and acetate. And in cycle 4 (phase II), the COD removal efficiency in R1, R2 and R3 were 34.9%, 78.5% and 69.2%, respectively. Compared with control reactor, in cycle 4 the COD removal efficiency in ethanol-co-digested reactor and acetate-co-digested reactor were increased about 43.6% and 34.3%, respectively. However, in phase Ⅲ, with the removal of ethanol/acetate from the inflow, the COD removal rate in R2 and R3 were all deteriorated. Exactly, in cycle 6, the COD in R3 was 22.5% after six days digestion, which was almost same with the control reactor. However, in R2 the COD removal could also reach 37.0%. It could be seen that the COD removal efficiency in R2 was higher than that in R3, especially in phase Ⅲ. The potential mechanism of relatively better performance in R2 may be that supplying ethanol would change the structure of microbial community, which was more stable during organic variation process(Zhao et al. 2016 ). The CH 4 production accumulated in each reactor is shown in Fig. 1 b, which was in agreement with the COD removal. Results showed that when co-digesting with ethanol/acetate the methane production was increased in R2 and R3. It was worth to mention that in phase Ⅲ, with the removal of ethanol/acetate from the inflow, the methane production in R2 and R3 reactors was decreased obviously, which implied that the methane production in R2 and R3 was closely related to the participation of ethanol/acetate. The RR2 removal efficiency was shown in Fig. 1 c, which performed similar trend with the COD removal. Generally, in phase Ⅰ and phase Ⅱ the RR2 removal efficiency was increased in co-digested reactors (i.e. R2 and R3). Exactly, in cycle 2 (phase Ⅰ), the RR2 removal rate was 74.0%, 94.3% and 83.1% in R1, R2 and R3, respectively, which was all exceed 70%. In phase Ⅱ, with the increment of RR2 concentration from 100 mg/L to 200 mg/L, the RR2 removal efficiency in R1, R2 and R3 was 21.3%, 90.8% and 75.6%, respectively. It can be seen that the RR2 removal efficiency in co-digested reactors had only slight decrement compared with phase Ⅰ. Nevertheless, the RR2 removal efficiency in R1 was declined sharply, which dropped from 74.0–21.3%. The feasible reason might be that in phase Ⅱ the RR2 load was exceed the degradation capacity of the reactors, which was disadvantageous to microbial metabolis(Li et al. 2017 ). In co-digested reactors, compared with RR2, the easier degraded organics (ethanol or acetate) was more available for microbial growth and provide energy for microbial to degrade RR2(Donoso-Bravo et al. 2009 ). In phase Ⅲ, ethanol/acetate were removed from the inflow, and then RR2 was used as the only carbon source in all reactors. Results show that the RR2 removal efficiency in R3 (acetate co-digested reactor) was declined drastically (from 75.6–11.0%). It might because that in R3 when the microbial preferred carbon source (i.e., acetate) was removed from the inflow, microbial could not metabolize RR2 efficiently, which resulted in the sharp reduction of RR2 removal efficiency. Nevertheless, the RR2 removal efficiency in R2 (ethanol co-digested reactor) could still keep over 60% in cycle 6, which was 54.5% higher than R1 and 52.1% than R3, respectively. The potential mechanisms for the best performance in R2 would be ascribed to the change of microbial community with the supplying of ethanol. 3.2 Microbial activity and LC-MS analysis The ETS activity can be used to embody the bioactivity of sludge and the respiratory activity of microorganisms, which could be quantified by monitoring the microbial respiration chain electron transfer rate. In this experiment, the ETS activity was measured at the end of phase Ⅲ, which was shown in Fig. 2 a. Results showed that ETS activity closely related to the RR2 degradation efficiency, which was in agreement with the reference(Zhao et al. 2020 ). The relative ETS activity in R3 (ethanol co-digested reactor) was about 66.9% and 29.5% higher than R1(control reactor) and R3 (acetate co-digested reactor), respectively. It can be seen that the co-digestion of ethanol and RR2 could tremendously increase the ability of the electron transfer of anaerobic systems.The SMA of the system could reflect the methanogenic ability of sludge, which was considered as an important physiological indicator of methanogenic bacteria activity. In this study, the SMA of R1, R2 and R3 were measured at the end of experiment and shown in Fig. 2 b, which was 86.3 mgCOD-CH 4 /gVSS·d, 213.8 mgCOD-CH 4 /gVSS·d and 149.9 mgCOD-CH 4 /gVSS·d, respectively. Results indicated that SMA in R2 (ethanol co-digested reactor) was the highest, which was about 147.7% and 42.6% higher than that in R1(control reactor) and R3(acetate co-digested reactor), respectively. Coenzyme F420, as another indicator could quantitatively and feasibly reflect the activity of methanogens, was also monitored at the end of the experiment (Fig. 5b). It can be seen that the coenzyme F420 in R2 was about 33.2% higher than R1 and 13.7% higher than R3. In general, during RR2 anaerobic digestion process, adding ethanol as the co-substrates could effectively improve the ETS, Coenzyme F420 and SMA, which played an important role for the enhancement of the organics degradation and methane production. At the cycle 6 of phase Ⅲ, the samples were collected from each reactor and analyzed by LC-MS. According to the LC-MS results, the potential pathway of RR2 degradation was shown in Fig. 3 . Generally, during the degradation process, chromophoric group was destructed with the interruption of the conjugated double-bond. Subsequently, RR2 was degraded into benzodiazepine, triazine, aniline, and naphthalene rings with the participation of anaerobic bacteria. And then the produced triazine and naphthalene were further degraded into esters, hydrocarbons and aldehydes, whereas some of the produced aniline was degraded into phenol. Finally, through the reduction and oxidation the aromatic amine compounds were transferred into naphthalene rings, benzene and other intermediates, and then under the effects of microbial theses intermediates were further degrades into smaller alcohols, phenols and lipid compounds. 3.3 Microbial community analysis After operation of six cycles, the samples in the three reactors were collected and identified for the microbial community at genus level. The results were shown in Fig. 4 . From Fig. 4 a, it can be seen that the most prominent archaea genus in the three reactors were Methanothrix and Methanomassiliicoccus , which totally account for over 80% of the archaea 16S rRNA gene sequences in all reactors. Exactly, the abundance of Methanothrix in R2 and R3 were 74.2% and 67.1%, which was 77.1% and 60.1% higher than the abundance in the R1, respectively. Previous study found that Methanothrix species as the acetate-utilizing methanogens can reduce carbon dioxide into methane through direct interspecies electron transfer (DIET) process(Jin, Zhao and Zhang 2019 ). It was reported that digesters fed with ethanol could effectively maintains a stable syntrophic metabolism through improving the abundance of DIET-based syntrophic partners(Zhao et al. 2016 ), which was the main reason for the improvement of the abundance of Methanothrix in R2 (ethanol co-digested reactor). In R3, the increment of the Methanothrix abundance would be ascribed to the supplying acetate to substrates. Methanothrix as the acetate-utilizing methanogens would be enriched when acetate concentration increased in the substrates. Methanomassiliicoccus were categorized as H 2 -utilizing methanogens, which could use H 2 as the electron donor to reduce CO 2 into methane(Zhang, Loh and Zhang 2019 ). From Fig. 4 a, it can be seen that the abundance of Methanomassiliicoccus in R1 (control reactor) and R3 (acetate co-digested reactor) were 40.6% and 28.1%, which was only 10.0% in R2 (ethanol co-digested reactor). This result means that the reduction of CO 2 into methane through H 2 served as the electron donor with the participation of Methanomassiliicoccus played more important role in R1 and R3 reactors compared with R2. The genus-level community structure of bacteria was identified and showed in Fig. 4 b. Results showed that the most predominant genus were Bellilinea , Aminicenant es and Smithella , which total account for about 25%-31% in the three reactors. These three genera were common bacteria during anaerobic digestion process and all related to volatile fatty acids oxidizing(Saha et al. 2019 , Luis et al. 2019 , Lam et al. 2020 ). These genera in the three reactors had no obvious difference. It was necessary to mention that the abundance of Desulfomicrobium and Geobacter in R2 (ethanol co-digested reactor) were 3.38% and 1.77%, which were 3.1 and 7.4 times higher than that in R1, and 5.8 and 16.7 times higher than that in R3, respectively. It was reported that all the members of the Desulfomicrobium genus are able to oxidize ethanol to acetate with sulfate as the electron acceptor(Zeng et al. 2019 ). And the enrichment of Desulfomicrobium in R2 reactor would be ascribed to the supplying ethanol to the substrates. And the enrichment of Desulfomicrobium would be beneficial to the removal of sulfur-containing organics which was produced during RR2 degradation process. Geobacter as the electroactive bacteria was extensively reported owing to the ability of direct accepting electron from conductive materials or syntrophic partners through conductive pili. Previous study found that Geobacter could oxide the organics and transfer electron to syntrophic archaea (i.e. Methanothrix or Methanosarcina) through direct interspecies electron transfer (DIET) process to reduce CO2 to methane(Rotaru et al. 2014b ). In this study, considering the enrichment of Methanothrix in R2, it would be reasonably speculated that the DIET process was established between Methanothrix and Geobacter in this reactor. The results presented in this study indicated that the addition of ethanol to the anaerobic digester could stimulate microbial community and then enhance the syntrophic metabolism. Especially, in phase Ⅲ when RR2 was the only substrates, the organics removal efficiency in R2 (ethanol co-digested reactor) was significantly higher than that in R1 and R3. In this study, evidences indicated that DIET was potentially promoted in R2, which would be the main reason for the improvement of RR2 degradation. Firstly, the ETS activity in R3 significantly increased, which could embody the bioactivity of sludge and reflect the electron transfer efficiency of the anaerobic system. The medium of electron transferring via DIET process was the electrically conductive pili of the Geobacter , which was more efficiency than traditional IHT. The higher ETS activity in R3 might be ascribed to the enrichment of Geobacter species which could proceed interspecies electron transfer via conductive pili. The second potential evidence for the establishment of DIET was the microbial community structure variation. In R1(control reactor) and R3(acetate co-digested reactor), the abundance of Geobacter (as the confirmed electrobacteria which could proceed DIET) was only 0.2% (or even much less), which meant the effects of DIET process could be ignored. Archaea analysis indicated that the abundance of Methanomassiliicoccus in R1 (control reactor) and R3 (acetate co-digested reactor) were over 28%, which meant that methane production through CO 2 reduction by Methanomassiliicoccus with H 2 serving as the electron donor played an important role in methane production in these two reactors. In R2, the abundance of Methanothrix (74.23%) and Geobacter (1.77%) were obviously enriched, combined with organics removal efficiency and methane production improvement, which can be reasonably speculated that DIET was established in R2. It can be concluded that adding ethanol to anaerobic digester could stimulate the enrichment of electroactive bacteria, which were more efficient during electron transfer through DIET process and played predominant role for RR2 removal. 4. Conclusion The results of this study indicate that the addition of ethanol into RR2 anaerobic digester could improve the organics removal and methane production. Mechanisms analysis indicated that electron transfer system (ETS) activity, specific methanogenic activity (SMA) and Coenzyme F420 of the sludge were effectively improved in ethanol co-digested reactor, which indicated that the addition of ethanol to anaerobic digester could enhance the activity of the microbial. Microbial community analysis indicated that the DIET process was potentially established, which was an alternative to IHT and could keep high efficiency during RR2 concentration variation. Declarations Author Contribution Z.Z. : Conceptualization, Methodology, Visualization, Writing - review & editing; Y.L. : Data curation, Figure edited; K.W. : Data curation, Figure edited; Y.A. : Data curation; G.Z. : Resources, Funding acquisition; L.H. : Resources, Methodology, Funding acquisition Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 52200109), National Natural Science Foundation of China (No. 52000162), Open Foundation of Key Laboratory of Industrial Ecology and Environmental Engineering MOE (KLIEE-21-10). References Cai, Z., Y. Sun, W. Liu, F. Pan, P. Sun & J. Fu (2017) An overview of nanomaterials applied for removing dyes from wastewater. Environmental Science and Pollution Research, 24 , 15882-15904. https://doi.org/10.1007/s11356-017-9003-8. Chhabra, M., S. Mishra & T. R. 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Bruto, C. Marchand, E. Record & M. Hugoni (2019) Contrasted ecological niches shape fungal and prokaryotic community structure in mangroves sediments. Environmental Microbiology, 21 , 1407-1424. https://doi.org/10.1111/1462-2920.14571. Meng, X. M., G. F. Liu, J. T. Zhou & Q. S. Fu (2014) Effects of redox mediators on azo dye decolorization by Shewanella algae under saline conditions. Bioresource Technology, 151 , 63-68. https://doi.org/10.1016/j.biortech.2013.09.131. Nouren, S. & H. N. Bhatti (2015) Mechanistic study of degradation of basic violet 3 by Citrus limon peroxidase and phytotoxicity assessment of its degradation products. Biochemical Engineering Journal, 95 , 9-19. https://doi.org/10.1016/j.bej.2014.11.021. Rotaru, A. E., P. M. Shrestha, F. Liu, B. Markovaite, S. Chen, K. P. Nevin & D. R. Lovley (2014a) Direct Interspecies Electron Transfer between Geobacter metallireducens and Methanosarcina barkeri. Applied and Environmental Microbiology, 80 , 4599-4605. https://doi.org/10.1128/AEM.00895-14. Rotaru, A. E., P. M. Shrestha, F. H. Liu, M. Shrestha, D. Shrestha, M. Embree, K. Zengler, C. Wardman, K. P. Nevin & D. R. Lovley (2014b) A new model for electron flow during anaerobic digestion: direct interspecies electron transfer to Methanosaeta for the reduction of carbon dioxide to methane. Energy & Environmental Science, 7 , 408-415. https://doi.org/10.1039/c3ee42189a. Saha, S., B. H. Jeon, M. B. Kurade, S. P. Govindwar, P. K. Chatterjee, S. E. Oh, H. S. Roh & S. S. Lee (2019) Interspecies microbial nexus facilitated methanation of polysaccharidic wastes. Bioresource Technology, 289. https://doi.org/10.1016/j.biortech.2019.121638. Saratale, R. G., G. D. Saratale, J. S. Chang & S. P. Govindwar (2011) Bacterial decolorization and degradation of azo dyes: A review. Journal of the Taiwan Institute of Chemical Engineers, 42 , 138-157. https://doi.org/10.1016/j.jtice.2010.06.006. Schink, B. (1997) Energetics of syntrophic cooperation in methanogenic degradation %J Microbiology and Molecular Biology Reviews Vol.61 , 262-280. https://doi.org/10.1128/.61.2.262-280.1997. Sieber, J. R., M. J. McInerney & R. P. Gunsalus (2012) Genomic Insights into Syntrophy: The Paradigm for Anaerobic Metabolic Cooperation. Annual Review of Microbiology, Vol 66, 66 , 429-452. https://doi.org/10.1146/annurev-micro-090110-102844 Stams, A. J. M. & C. M. Plugge (2009) Electron transfer in syntrophic communities of anaerobic bacteria and archaea. Nature Reviews Microbiology, 7 , 568-577. https://doi.org/10.1038/nrmicro2166. Summers, Z. M., H. E. Fogarty, C. Leang, A. E. Franks, N. S. Malvankar & D. R. Lovley (2010) Direct Exchange of Electrons Within Aggregates of an Evolved Syntrophic Coculture of Anaerobic Bacteria. Science, 330 , 1413-1415. https://doi.org/10.1126/science.1196526. Tang, J. H., M. K. Wu, J. G. Huang, J. J. Chen, W. Han & Z. M. Xie (2016) Effects of different henna plant parts on enhanced removal of an azo dye Orange II: Biotic and abiotic contributions. Environmental Progress & Sustainable Energy, 35 , 404-410. https://doi.org/10.1002/ep.12248. Tian, T., S. Qiao, C. Yu, Y. H. Tian, Y. Yang & J. T. Zhou (2017) Distinct and diverse anaerobic respiration of methanogenic community in response to MnO nanoparticles in anaerobic digester sludge. Water Research, 123 , 206-215. https://doi.org/10.1016/j.watres.2017.06.066. van der Zee, F. P. & S. Villaverde (2005) Combined anaerobic-aerobic treatment of azo dyes - A short review of bioreactor studies. Water Research, 39 , 1425-1440. https://doi.org/10.1016/j.watres.2005.03.007. Viggi, C. C., S. Rossetti, S. Fazi, P. Paiano, M. Majone & F. Aulenta (2014) Magnetite Particles Triggering a Faster and More Robust Syntrophic Pathway of Methanogenic Propionate Degradation. Environmental Science & Technology, 48 , 7536-7543. https://doi.org/10.1021/es5016789. Wang, Z. Z., Q. D. Yin, M. Q. Gu, K. He & G. X. Wu (2018) Enhanced azo dye Reactive Red 2 degradation in anaerobic reactors by dosing conductive material of ferroferric oxide. Journal of Hazardous Materials, 357 , 226-234. https://doi.org/10.1016/j.jhazmat.2018.06.005. Zeng, D. F., Q. D. Yin, Q. Du & G. X. Wu (2019) System performance and microbial community in ethanol-fed anaerobic reactors acclimated with different organic carbon to sulfate ratios. Bioresource Technology, 278 , 34-42. https://doi.org/10.1016/j.biortech.2019.01.047. Zhang, G. Y., Y. H. Shi, Z. S. Zhao, X. W. Wang & M. Dou (2020) Enhanced two-phase anaerobic digestion of waste-activated sludge by combining magnetite and zero-valent iron. Bioresource Technology, 306. https://doi.org/10.1016/j.biortech.2020.123122. Zhang, L., K. C. Loh & J. X. Zhang (2019) Jointly reducing antibiotic resistance genes and improving methane yield in anaerobic digestion of chicken manure by feedstock microwave pretreatment and activated carbon supplementation. Chemical Engineering Journal, 372 , 815-824. https://doi.org/10.1016/j.cej.2019.04.207. Zhao, Z. Q., Y. B. Zhang, Y. Li, Y. Dang, T. T. Zhu & X. Quan (2017) Potentially shifting from interspecies hydrogen transfer to direct interspecies electron transfer for syntrophic metabolism to resist acidic impact with conductive carbon cloth. Chemical Engineering Journal, 313 , 10-18. https://doi.org/10.1016/j.cej.2016.11.149. Zhao, Z. Q., Y. B. Zhang, Q. L. Yu, Y. Dang, Y. Li & X. Quan (2016) Communities stimulated with ethanol to perform direct interspecies electron transfer for syntrophic metabolism of propionate and butyrate. Water Research, 102 , 475-484. https://doi.org/10.1016/j.watres.2016.07.005. Zhao, Z. S., Y. Cao, S. Y. Li & Y. B. Zhang (2021) Effects of biowaste-derived biochar on the electron transport efficiency during anaerobic acid orange 7 removal. Bioresource Technology, 320. https://doi.org/10.1016/j.biortech.2020.124295. Zhao, Z. S., G. Y. Zhang, Y. B. Zhang, M. Dou & Y. Li (2020) Fe3O4 accelerates tetracycline degradation during anaerobic digestion: Synergistic role of adsorption and microbial metabolism. Water Research, 185. https://doi.org/10.1016/j.watres.2020.116225. Zhu, T. T., Y. B. Zhang, Y. W. Liu & Z. S. Zhao (2021) Electrostimulation enhanced ammonium removal during Fe(III) reduction coupled with anaerobic ammonium oxidation (Feammox) process. Science of the Total Environment, 751. https://doi.org/10.1016/j.scitotenv.2020.141703. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3989947","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":275864873,"identity":"9db6b808-2cdc-4da5-9467-3ddcb32e40a5","order_by":0,"name":"Zisheng Zhao","email":"","orcid":"","institution":"Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Zisheng","middleName":"","lastName":"Zhao","suffix":""},{"id":275864874,"identity":"bb0c45fa-48e0-47aa-9b24-560c9a3616ce","order_by":1,"name":"Yixin Li","email":"","orcid":"","institution":"Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Yixin","middleName":"","lastName":"Li","suffix":""},{"id":275864875,"identity":"4a4ee6b5-9eab-4d3e-b06b-f4974d867c43","order_by":2,"name":"Kang Wang","email":"","orcid":"","institution":"Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Kang","middleName":"","lastName":"Wang","suffix":""},{"id":275864876,"identity":"b99741a6-f2f9-4481-8c2e-ea7e8cd565f3","order_by":3,"name":"Yu An","email":"","orcid":"","institution":"Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"An","suffix":""},{"id":275864877,"identity":"2171ac82-85f6-4ec6-9cfd-f0cfd96101bb","order_by":4,"name":"guangyi Zhang","email":"","orcid":"","institution":"Zhengzhou University","correspondingAuthor":false,"prefix":"","firstName":"guangyi","middleName":"","lastName":"Zhang","suffix":""},{"id":275864878,"identity":"5d9399ff-a778-42c7-bef8-ffc353974fc6","order_by":5,"name":"Long Huang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAs0lEQVRIiWNgGAWjYBACA3bmAwc/GNjw8LM3EKuFmS3xsERFmoxkzwGitfAYH+A5c9jG4IYDkVrMmRkMDki2nedhuMHA+OFjDhFaLJsZEg4Utt3mYZzdwCw5cxsxDjvMcABoy20eZpkDbMy8xGlhbDjA23aOh00igWgtzAxA7x/g4SFBCxsDMJCTeSR4DjYT6Zfj/Z8/fjCws7c/3nzww0ditCABxgbS1I+CUTAKRsEowA0AnOI3F+Is1YYAAAAASUVORK5CYII=","orcid":"","institution":"Zhengzhou University","correspondingAuthor":true,"prefix":"","firstName":"Long","middleName":"","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2024-02-26 05:03:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3989947/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3989947/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":52022040,"identity":"0b384826-13d8-4531-a64a-b722e5201f3e","added_by":"auto","created_at":"2024-03-05 15:00:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":117759,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Fe\u003csub\u003e3\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003e on anaerobic digestion performance. (a) COD concentration; (b) Accumulated CH\u003csub\u003e4\u003c/sub\u003e production; (c) RR2 removal efficiency.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3989947/v1/c27a1a594395c74152966098.png"},{"id":52021804,"identity":"c3218534-a908-404d-bb99-4025b158b52a","added_by":"auto","created_at":"2024-03-05 14:52:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":108143,"visible":true,"origin":"","legend":"\u003cp\u003eMicrobial activity of the system. (a) the ETS activity in different reactors; (b) SMA in different reactors; (c) relative activity of F420 in different reactors.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3989947/v1/1d569934e56428cb3479256a.png"},{"id":52021803,"identity":"03ea2213-0122-4de2-8077-14fa3860ec27","added_by":"auto","created_at":"2024-03-05 14:52:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":98202,"visible":true,"origin":"","legend":"\u003cp\u003eProposed degradation pathway of RR2 and the chemical structure of intermediates\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3989947/v1/e3fc5c1bb9176845c956a1eb.png"},{"id":52021801,"identity":"75c98d83-2e17-4bdd-a0ab-d6922091c443","added_by":"auto","created_at":"2024-03-05 14:52:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":24736,"visible":true,"origin":"","legend":"\u003cp\u003eHigh-throughput 16S rRNA gene pyrosequencing analyses of the microbial community. (a) Archaea ommunity structure at genus level; (b) Bacterial community structure at genus level.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3989947/v1/ee800631cefa653314eb7ecb.png"},{"id":52042326,"identity":"da428cd1-3c63-473d-98d1-d26884e6169c","added_by":"auto","created_at":"2024-03-05 18:33:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":556688,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3989947/v1/23b76a24-84ec-4b0d-885d-5d6bf496870b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Establishment of direct interspecies electron transfer through ethanol supply during azo dye Reactive Red 2 anaerobic degradation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eAzo dyes, as the persistent organics containing azo and aromatic groups, are extensively used in industry. However, during textile production process, a certain amount of the azo dyes can\u0026rsquo;t be consumed and discharged together with the textile wastewater eventually(Cai et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Previous study found that many synthetic azo dyes were proved to be toxic and carcinogenic(de Arag\u0026atilde;o Umbuzeiro et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), which will induce potential environmental pollution and threaten human health when directly discharging into natural waters(Dai et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Tang et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Many physiochemical methods are used to treating dyestuff wastewater and received efficient performance, but the high energy consumption and equipment investment were unacceptable(Chhabra, Mishra and Sreekrishnan \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Saratale et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, Nouren and Bhatti \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAnaerobic digestion, as a low costs and energy recovery methods, exhibit potential advantage for treating dye wastewater(Meng et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). During textile wastewater anaerobic digestion process, the azo dyes could be degraded biologically with the participation of azo-reductases enzymes(Wang et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). However, the property of azo dye (persistent and high toxicity) was bacteriostatic, which would limit the efficiency of electron transfer between microbials and deteriorate anaerobic performance(van der Zee and Villaverde \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). It was reported that electron transfer efficiency closely related to the organics anaerobic digestion performance(Kouzuma, Kato and Watanabe \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e, Schink \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Previous study indicated that strengthening electron transfer efficiency could enhance microbial metabolism, which would promote organics anaerobic degradation(Zhao et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). During anaerobic methanogenesis, the electron transfer mode was widely considered as interspecies H\u003csub\u003e2\u003c/sub\u003e/formate transfer (IHT/IFT) with H\u003csub\u003e2\u003c/sub\u003e/formate serving as the electron carrier in recent decades(Stams and Plugge \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2009\u003c/span\u003e, Sieber, McInerney and Gunsalus \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). In order to keep growth of the syntrophic partners, the electron carriers should be consumed efficiently with the participation of methanogens. It was reported that only when the concentration of H\u003csub\u003e2\u003c/sub\u003e/formate is kept very low, the substrates syntrophic oxidation was thermodynamically feasible, since this process is endergonic under standard conditions(Stams and Plugge \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDirect Interspecies Electron Transfer (DIET) has recently been considered as the new interspecies electron transfer mode between bacterial and methanogens(Rotaru et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2014b\u003c/span\u003e, Rotaru et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014a\u003c/span\u003e). It was reported during DIET process microbial use the c-type cytochromes(Lovley et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) and/or conductive pili(Lovley et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2011\u003c/span\u003e) as the mediator for extracellular electron transfer, which not rely on H2/formate as the electron carriers, thus making it potentially more efficient and energy-conserving mode for methane production(Viggi et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e, Lovley \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Originally, DIET was only reported in few defined co-cultures, such as co-cultures of two \u003cem\u003eGeobacter\u003c/em\u003e species(Summers et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and co-cultures of \u003cem\u003eGeobacter\u003c/em\u003e with methanogens(Rotaru et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2014a\u003c/span\u003e, Lei et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) with ethanol as electron donor. Subsequently, DIET was found in mixed culture with propionate(Viggi et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), leachate(Lei et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) and phenols(Li et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e) as the substrates. However, in the absence of any amendments, DIET was only existed as the predominant electron transfer mode in treating brewery wastes.\u003c/p\u003e \u003cp\u003eThereafter, researchers found that varieties conductive materials could improve anaerobic digestion at the certain extent, and they ascribed the enhancement to accelerating of DIET(Zhao et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, the enriched bacteria were \u003cem\u003eSynaatrophomonas\u003c/em\u003e, \u003cem\u003eProteiniclasticum\u003c/em\u003e, \u003cem\u003eSyntrophomonadaceae\u003c/em\u003e and \u003cem\u003eClostridium\u003c/em\u003e, which were not assertive evidence for DIET(Zhao et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). For example, in Li\u0026rsquo;s study(Li et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015a\u003c/span\u003e, Li et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2015b\u003c/span\u003e), \u003cem\u003eSyntrophomonadaceae\u003c/em\u003e was reported as the bacteria to metabolize butyrate to acetate via IHT rather than DIET. It was reported that ethanol could effectively improve the abundance of \u003cem\u003eGeobacter\u003c/em\u003e and \u003cem\u003eMethanothrix\u003c/em\u003e or \u003cem\u003eMethanosarcina\u003c/em\u003e species, and form the aggregates with high conductivity(Zhao et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Ethanol was used as the trigger for DIET to dispose varieties organics (such as propionate, waste activated sludge), and received satisfactory performance(Zhao et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e, Li et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The potential mechanism was that during anaerobic digestion with the presence of ethanol, \u003cem\u003eGeobacter\u003c/em\u003e could be enriched, which as the most important exoelectrogens during DIET process was reported that can utilize a broad of organics, such as alcohols, volatile fatty acids, phenols and benzene as substrates(Lovley et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). However, researches about ethanol as the trigger for DIET to promote persistent organics degradation had been few reported. Based on these considerations, research about ethanol using as the specific substrate to perform DIET was conducted to investigated if RR2 degradation could be enhanced through stimulating the microbial communities.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Chemicals and inoculum\u003c/h2\u003e \u003cp\u003eEthanol, sodium acetate, RR2 and other experimental chemicals were purchased from Aladdin reagent CO. Ltd. (Shanghai, China). The sludge collected from Wulongkou waste sludge anaerobic treatment plant of Zhengzhou (China) was used as the inoculant sludge in this experiment, and cultured in an anaerobic digester before the experiment. The substrates and operating conditions for the culturing were according to the reference(Li et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Batch experiment operation\u003c/h2\u003e \u003cp\u003eIn this study, batch experiments were conducted to investigate the effects of ethanol on RR2 anaerobic removal. To exclude the co-substrates effects of ethanol, a unique control reactor was set up with equal amount COD of sodium acetate as the co-substrates. Therefore, in this experiments, three group reactors were operated, i.e. control reactor with RR2 as the sole substrates during the entire experiment process, hereafter referred as R1; co-digestion of RR2 with ethanol at the initial stage of the experiment, hereafter referred as R2; co-digestion of RR2 with acetate at the initial stage of the experiment, hereafter referred as R3. All groups were conducted in 250 ml serum bottle for six cycles, and each cycle lasted for six days. During experiment, 100 mL mixture (containing 15 mL inoculum and 85 mL substrates) were added into each bottle. In phase I (the first two cycles) 100 mg/L RR2 was used as the substrates in R1, the substrates in R2 were 100 mg/L RR2 and 1000 mg COD/L ethanol, the substrates in R3 were 100 mg/L RR2 and 1000 mg COD/L acetate. In phase II (the third and fourth cycles) the concentration of RR2 in all reactors were increased to 200 mg/L, and the concentration of acetate and ethanol in R2 and R3 were unaltered. In phase Ⅲ (the last two cycles), only 200 mg/L RR2 were used as the substrates in all reactors with the removal of acetate and ethanol from R2 and R3. In addition, the COD: N: P ratio is kept at 200:5:1 with the addition of NH4Cl and KH2PO4. 1 mL stock solution of trace element was added to 1 L substrates, which was prepared according to previous study(Zhao et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). At the end of each cycle, another 85 ml fresh substrate as described above was added to each reactors to replace the residual liquid in the reactors. Before starting of each cycle, oxygen in the bottles was removed with 99.9% nitrogen to maintain anaerobic condition. Subsequently, these sealed reactors were placed in an homothermal incubator (37 ◦C). During experiment, COD and RR2 concentration were measured every day, and at the end of each cycle the methane production was analyzed. All groups were performed in triplicate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Analytical methods\u003c/h2\u003e \u003cp\u003eThe COD and biogas components (CH\u003csub\u003e4\u003c/sub\u003e and CO\u003csub\u003e2\u003c/sub\u003e) were measured as the references(Zhang et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). A visible spectrophotometer (DR3900, HACH, USA) were used to measure the RR2 concentration by analyzing the absorbance at the 512 nm. Before the measurement of COD and RR2 concentration, liquid collected from the reactors were filtered by the membrane filters with 0.45 \u0026micro;m aperture. The COD value of unit mass substrates were as follows: 0.61 g-COD/g RR2, 2.05 g-COD/g ethanol, 0.49 g-COD/g sodium acetate. The intermediate products of RR2 during anaerobic digestion process were analyzed by a liquid chromatography-mass spectrometer (LC- MS, Agilent 6410B, Palo Alto, USA). The electron transport system (ETS) activity was measured by the methods of 2-(piodophenyl)\u0026thinsp;\u0026minus;\u0026thinsp;3-(p-nitrophenyl)\u0026thinsp;\u0026minus;\u0026thinsp;5-phenyl tetrazolium chloride (INT) reduction, and the detailed methods was according to the reference(Zhang et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The specific methanogenic activity (SMA) of the sludge in the reactors after anaerobic digestion was measured according to the methods described by Hu et al.(Hu et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Coenzyme F420 was measured by a fluorescence spectrophotometer (Hitachi, F-4500, Japan) according to Tian et al. (Tian et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). At the end of experiment, microbial community was analyzed by High-throughput gene pyrosequencing. Genomic DNA in the suspended sludge was extracted with a DNA extraction kit (BioTeke Corporation, Beijing, China) as the manufacturer\u0026rsquo;s instructions described. Detailed methods were same as previous study(Zhu et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results and discussion","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Organics removal and methane production\u003c/h2\u003e \u003cp\u003eThis experiment included three phases (phase I: co-digestion of acetate/ethanol and 100 mg/L RR2, phase II: co-digestion of acetate/ethanol and 200 mg/L RR2, phase Ⅲ: mono-digestion of 200 mg/L RR2), and each phase containing two cycles, and each cycle proceeded for 6 days. During this experiment, the COD in each cycle was measured and the removal efficiency was showed in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea. From the figure, it can be seen that in phase I and phase II both co-digesting with ethanol and acetate could promote the COD removal. Exactly, in cycle 2 (phase Ⅰ), the COD removal efficiency in R1 (control reactor), R2 (ethanol-co-digested reactor) and R3 (acetate-co-digested reactor) were 41.0%, 80.3% and 69.0%, respectively. The reason of higher removal efficiency in R2 and R3 was the removal of ethanol and acetate. And in cycle 4 (phase II), the COD removal efficiency in R1, R2 and R3 were 34.9%, 78.5% and 69.2%, respectively. Compared with control reactor, in cycle 4 the COD removal efficiency in ethanol-co-digested reactor and acetate-co-digested reactor were increased about 43.6% and 34.3%, respectively. However, in phase Ⅲ, with the removal of ethanol/acetate from the inflow, the COD removal rate in R2 and R3 were all deteriorated. Exactly, in cycle 6, the COD in R3 was 22.5% after six days digestion, which was almost same with the control reactor. However, in R2 the COD removal could also reach 37.0%. It could be seen that the COD removal efficiency in R2 was higher than that in R3, especially in phase Ⅲ. The potential mechanism of relatively better performance in R2 may be that supplying ethanol would change the structure of microbial community, which was more stable during organic variation process(Zhao et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The CH\u003csub\u003e4\u003c/sub\u003e production accumulated in each reactor is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb, which was in agreement with the COD removal. Results showed that when co-digesting with ethanol/acetate the methane production was increased in R2 and R3. It was worth to mention that in phase Ⅲ, with the removal of ethanol/acetate from the inflow, the methane production in R2 and R3 reactors was decreased obviously, which implied that the methane production in R2 and R3 was closely related to the participation of ethanol/acetate.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe RR2 removal efficiency was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec, which performed similar trend with the COD removal. Generally, in phase Ⅰ and phase Ⅱ the RR2 removal efficiency was increased in co-digested reactors (i.e. R2 and R3). Exactly, in cycle 2 (phase Ⅰ), the RR2 removal rate was 74.0%, 94.3% and 83.1% in R1, R2 and R3, respectively, which was all exceed 70%. In phase Ⅱ, with the increment of RR2 concentration from 100 mg/L to 200 mg/L, the RR2 removal efficiency in R1, R2 and R3 was 21.3%, 90.8% and 75.6%, respectively. It can be seen that the RR2 removal efficiency in co-digested reactors had only slight decrement compared with phase Ⅰ. Nevertheless, the RR2 removal efficiency in R1 was declined sharply, which dropped from 74.0\u0026ndash;21.3%. The feasible reason might be that in phase Ⅱ the RR2 load was exceed the degradation capacity of the reactors, which was disadvantageous to microbial metabolis(Li et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In co-digested reactors, compared with RR2, the easier degraded organics (ethanol or acetate) was more available for microbial growth and provide energy for microbial to degrade RR2(Donoso-Bravo et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). In phase Ⅲ, ethanol/acetate were removed from the inflow, and then RR2 was used as the only carbon source in all reactors. Results show that the RR2 removal efficiency in R3 (acetate co-digested reactor) was declined drastically (from 75.6\u0026ndash;11.0%). It might because that in R3 when the microbial preferred carbon source (i.e., acetate) was removed from the inflow, microbial could not metabolize RR2 efficiently, which resulted in the sharp reduction of RR2 removal efficiency. Nevertheless, the RR2 removal efficiency in R2 (ethanol co-digested reactor) could still keep over 60% in cycle 6, which was 54.5% higher than R1 and 52.1% than R3, respectively. The potential mechanisms for the best performance in R2 would be ascribed to the change of microbial community with the supplying of ethanol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Microbial activity and LC-MS analysis\u003c/h2\u003e \u003cp\u003eThe ETS activity can be used to embody the bioactivity of sludge and the respiratory activity of microorganisms, which could be quantified by monitoring the microbial respiration chain electron transfer rate. In this experiment, the ETS activity was measured at the end of phase Ⅲ, which was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea. Results showed that ETS activity closely related to the RR2 degradation efficiency, which was in agreement with the reference(Zhao et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The relative ETS activity in R3 (ethanol co-digested reactor) was about 66.9% and 29.5% higher than R1(control reactor) and R3 (acetate co-digested reactor), respectively. It can be seen that the co-digestion of ethanol and RR2 could tremendously increase the ability of the electron transfer of anaerobic systems.The SMA of the system could reflect the methanogenic ability of sludge, which was considered as an important physiological indicator of methanogenic bacteria activity. In this study, the SMA of R1, R2 and R3 were measured at the end of experiment and shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, which was 86.3 mgCOD-CH\u003csub\u003e4\u003c/sub\u003e/gVSS\u0026middot;d, 213.8 mgCOD-CH\u003csub\u003e4\u003c/sub\u003e/gVSS\u0026middot;d and 149.9 mgCOD-CH\u003csub\u003e4\u003c/sub\u003e/gVSS\u0026middot;d, respectively. Results indicated that SMA in R2 (ethanol co-digested reactor) was the highest, which was about 147.7% and 42.6% higher than that in R1(control reactor) and R3(acetate co-digested reactor), respectively. Coenzyme F420, as another indicator could quantitatively and feasibly reflect the activity of methanogens, was also monitored at the end of the experiment (Fig.\u0026nbsp;5b). It can be seen that the coenzyme F420 in R2 was about 33.2% higher than R1 and 13.7% higher than R3. In general, during RR2 anaerobic digestion process, adding ethanol as the co-substrates could effectively improve the ETS, Coenzyme F420 and SMA, which played an important role for the enhancement of the organics degradation and methane production.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt the cycle 6 of phase Ⅲ, the samples were collected from each reactor and analyzed by LC-MS. According to the LC-MS results, the potential pathway of RR2 degradation was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Generally, during the degradation process, chromophoric group was destructed with the interruption of the conjugated double-bond. Subsequently, RR2 was degraded into benzodiazepine, triazine, aniline, and naphthalene rings with the participation of anaerobic bacteria. And then the produced triazine and naphthalene were further degraded into esters, hydrocarbons and aldehydes, whereas some of the produced aniline was degraded into phenol. Finally, through the reduction and oxidation the aromatic amine compounds were transferred into naphthalene rings, benzene and other intermediates, and then under the effects of microbial theses intermediates were further degrades into smaller alcohols, phenols and lipid compounds.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Microbial community analysis\u003c/h2\u003e \u003cp\u003eAfter operation of six cycles, the samples in the three reactors were collected and identified for the microbial community at genus level. The results were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. From Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, it can be seen that the most prominent archaea genus in the three reactors were \u003cem\u003eMethanothrix\u003c/em\u003e and \u003cem\u003eMethanomassiliicoccus\u003c/em\u003e, which totally account for over 80% of the archaea 16S rRNA gene sequences in all reactors. Exactly, the abundance of \u003cem\u003eMethanothrix\u003c/em\u003e in R2 and R3 were 74.2% and 67.1%, which was 77.1% and 60.1% higher than the abundance in the R1, respectively. Previous study found that \u003cem\u003eMethanothrix\u003c/em\u003e species as the acetate-utilizing methanogens can reduce carbon dioxide into methane through direct interspecies electron transfer (DIET) process(Jin, Zhao and Zhang \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It was reported that digesters fed with ethanol could effectively maintains a stable syntrophic metabolism through improving the abundance of DIET-based syntrophic partners(Zhao et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), which was the main reason for the improvement of the abundance of \u003cem\u003eMethanothrix\u003c/em\u003e in R2 (ethanol co-digested reactor). In R3, the increment of the \u003cem\u003eMethanothrix\u003c/em\u003e abundance would be ascribed to the supplying acetate to substrates. \u003cem\u003eMethanothrix\u003c/em\u003e as the acetate-utilizing methanogens would be enriched when acetate concentration increased in the substrates. \u003cem\u003eMethanomassiliicoccus\u003c/em\u003e were categorized as H\u003csub\u003e2\u003c/sub\u003e-utilizing methanogens, which could use H\u003csub\u003e2\u003c/sub\u003e as the electron donor to reduce CO\u003csub\u003e2\u003c/sub\u003e into methane(Zhang, Loh and Zhang \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). From Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, it can be seen that the abundance of \u003cem\u003eMethanomassiliicoccus\u003c/em\u003e in R1 (control reactor) and R3 (acetate co-digested reactor) were 40.6% and 28.1%, which was only 10.0% in R2 (ethanol co-digested reactor). This result means that the reduction of CO\u003csub\u003e2\u003c/sub\u003e into methane through H\u003csub\u003e2\u003c/sub\u003e served as the electron donor with the participation of \u003cem\u003eMethanomassiliicoccus\u003c/em\u003e played more important role in R1 and R3 reactors compared with R2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe genus-level community structure of bacteria was identified and showed in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb. Results showed that the most predominant genus were \u003cem\u003eBellilinea\u003c/em\u003e, \u003cem\u003eAminicenant\u003c/em\u003ees and \u003cem\u003eSmithella\u003c/em\u003e, which total account for about 25%-31% in the three reactors. These three genera were common bacteria during anaerobic digestion process and all related to volatile fatty acids oxidizing(Saha et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Luis et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e, Lam et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These genera in the three reactors had no obvious difference. It was necessary to mention that the abundance of \u003cem\u003eDesulfomicrobium\u003c/em\u003e and \u003cem\u003eGeobacter\u003c/em\u003e in R2 (ethanol co-digested reactor) were 3.38% and 1.77%, which were 3.1 and 7.4 times higher than that in R1, and 5.8 and 16.7 times higher than that in R3, respectively. It was reported that all the members of the \u003cem\u003eDesulfomicrobium\u003c/em\u003e genus are able to oxidize ethanol to acetate with sulfate as the electron acceptor(Zeng et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). And the enrichment of \u003cem\u003eDesulfomicrobium\u003c/em\u003e in R2 reactor would be ascribed to the supplying ethanol to the substrates. And the enrichment of \u003cem\u003eDesulfomicrobium\u003c/em\u003e would be beneficial to the removal of sulfur-containing organics which was produced during RR2 degradation process. \u003cem\u003eGeobacter\u003c/em\u003e as the electroactive bacteria was extensively reported owing to the ability of direct accepting electron from conductive materials or syntrophic partners through conductive pili. Previous study found that \u003cem\u003eGeobacter\u003c/em\u003e could oxide the organics and transfer electron to syntrophic archaea (i.e. Methanothrix or Methanosarcina) through direct interspecies electron transfer (DIET) process to reduce CO2 to methane(Rotaru et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2014b\u003c/span\u003e). In this study, considering the enrichment of \u003cem\u003eMethanothrix\u003c/em\u003e in R2, it would be reasonably speculated that the DIET process was established between \u003cem\u003eMethanothrix\u003c/em\u003e and \u003cem\u003eGeobacter\u003c/em\u003e in this reactor.\u003c/p\u003e \u003cp\u003eThe results presented in this study indicated that the addition of ethanol to the anaerobic digester could stimulate microbial community and then enhance the syntrophic metabolism. Especially, in phase Ⅲ when RR2 was the only substrates, the organics removal efficiency in R2 (ethanol co-digested reactor) was significantly higher than that in R1 and R3. In this study, evidences indicated that DIET was potentially promoted in R2, which would be the main reason for the improvement of RR2 degradation. Firstly, the ETS activity in R3 significantly increased, which could embody the bioactivity of sludge and reflect the electron transfer efficiency of the anaerobic system. The medium of electron transferring via DIET process was the electrically conductive pili of the \u003cem\u003eGeobacter\u003c/em\u003e, which was more efficiency than traditional IHT. The higher ETS activity in R3 might be ascribed to the enrichment of \u003cem\u003eGeobacter\u003c/em\u003e species which could proceed interspecies electron transfer via conductive pili. The second potential evidence for the establishment of DIET was the microbial community structure variation. In R1(control reactor) and R3(acetate co-digested reactor), the abundance of \u003cem\u003eGeobacter\u003c/em\u003e (as the confirmed electrobacteria which could proceed DIET) was only 0.2% (or even much less), which meant the effects of DIET process could be ignored. Archaea analysis indicated that the abundance of \u003cem\u003eMethanomassiliicoccus\u003c/em\u003e in R1 (control reactor) and R3 (acetate co-digested reactor) were over 28%, which meant that methane production through CO\u003csub\u003e2\u003c/sub\u003e reduction by \u003cem\u003eMethanomassiliicoccus\u003c/em\u003e with H\u003csub\u003e2\u003c/sub\u003e serving as the electron donor played an important role in methane production in these two reactors. In R2, the abundance of \u003cem\u003eMethanothrix\u003c/em\u003e (74.23%) and \u003cem\u003eGeobacter\u003c/em\u003e (1.77%) were obviously enriched, combined with organics removal efficiency and methane production improvement, which can be reasonably speculated that DIET was established in R2. It can be concluded that adding ethanol to anaerobic digester could stimulate the enrichment of electroactive bacteria, which were more efficient during electron transfer through DIET process and played predominant role for RR2 removal.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eThe results of this study indicate that the addition of ethanol into RR2 anaerobic digester could improve the organics removal and methane production. Mechanisms analysis indicated that electron transfer system (ETS) activity, specific methanogenic activity (SMA) and Coenzyme F420 of the sludge were effectively improved in ethanol co-digested reactor, which indicated that the addition of ethanol to anaerobic digester could enhance the activity of the microbial. Microbial community analysis indicated that the DIET process was potentially established, which was an alternative to IHT and could keep high efficiency during RR2 concentration variation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eZ.Z. : Conceptualization, Methodology, Visualization, Writing - review \u0026amp; editing; Y.L. : Data curation, Figure edited; K.W. : Data curation, Figure edited; Y.A. : Data curation; G.Z. : Resources, Funding acquisition; L.H. : Resources, Methodology, Funding acquisition\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Natural Science Foundation of China (No. 52200109), National Natural Science Foundation of China (No. 52000162), Open Foundation of Key Laboratory of Industrial Ecology and Environmental Engineering MOE (KLIEE-21-10).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eCai, Z., Y. 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In this study, ethanol was added to the digesters to promote azo dye Reactive Red 2 (RR2) anaerobic digestion efficiency. Results showed that the COD removal and RR2 removal efficiency were 37.0% and 63.2% in cycle 6 (only RR2 used as the substrates) in ethanol co-digested reactor, which was 16.0% and 54.5% higher than that in control reactor, and 14.5% and 52.0% higher than that in acetate co-digested reactor, respectively. Mechanisms exploration found that the electron transfer system (ETS) activity, specific methanogenic activity (SMA) and Coenzyme F420 of the sludge were effectively improved in ethanol co-digested reactor, which indicated that the addition of ethanol to anaerobic digester could enhance the activity of the microbial. Microbial community analysis showed that the electroactive microbial (\u003cem\u003eGeobacter\u003c/em\u003e and \u003cem\u003eMethanothrix\u003c/em\u003e) were more enriched in ethanol co-digested reactor. It was speculated that the direct interspecies electron transfer (DIET) process was possible established between \u003cem\u003eGeobacter\u003c/em\u003e and \u003cem\u003eMethanothrix\u003c/em\u003e, which played an important role for the improvement of RR2 removal efficiency in ethanol co-digested reactor.\u003c/p\u003e","manuscriptTitle":"Establishment of direct interspecies electron transfer through ethanol supply during azo dye Reactive Red 2 anaerobic degradation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-05 14:52:43","doi":"10.21203/rs.3.rs-3989947/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a6609403-69c1-4682-8f16-774481553390","owner":[],"postedDate":"March 5th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-03-05T18:25:50+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-05 14:52:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3989947","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3989947","identity":"rs-3989947","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}