Round 1

Reviewer 1 Report

This manuscript is entitled "Three-dimensional biofilm electrode reactors with polyure- 2

thane sponge carrier for highly-efficient treatment of pharma- 3 ceuticals wastewater containing tetrahydrofuran". These data are interesting, but some points still need to correct before publication.

 

1.     Please check spelling mistakes and the English language throughout the text.

2.     Abstract: please rewrite the main results and the purpose

3.     Introduction: The novelty and the advance added to the area must be clearly stated. Particularly Introduction could be enlarged. These things are missing.

4.     The figures' quality is very poor. Please provide clear images.

5.     Please check the whole manuscript to correct this type of mistake

6.     Please write details preparation method of all characterization techniques

7.     Please add an error bar in all figures

8.     Please check all units in order to be similar

9.     An environmental viability assessment should be added.

10.  References can be added from the host journal.

11.  Please check the format of the reference

 

12.  Conclusion: please add the key points with the further implication

Author Response

Response to Reviewer 1's Comments

This manuscript is entitled "Three-dimensional biofilm electrode reactors with polyurethane sponge carrier for highly-efficient treatment of pharmaceuticals wastewater containing tetrahydrofuran". These data are interesting, but some points still need to correct before publication.

Response: Thank you a lot for supporting our study. According to your valuable comments, we have revised this manuscript point by point, which is important in improving our knowledge and benefiting our next study. Thank you again.

(1) Please check spelling mistakes and the English language throughout the text.

Response: Thank you for your comments. We have checked and revised the language across the whole text.

The revisions were as follows:

Lines 26-30

whose degradation of THF intermediates was found by functional prediction, mainly through chemoheterotrophy, aerobic chemoheterotrophy, etc. Hopefully, this study will provide a solution to the practical treatment of pharmaceutical wastewater containing THF via this new 3D-BER system with polyurethane sponge carrier.

Lines 38-42

Among the pharmaceutical wastewater, THF is one of the most representative pollutants. However, the average treating ratio of pharmaceutical wastewater containing THF is less than 30%, and it has become a serious source of water pollution [2]. Pharmaceutical wastewater will cause serious detriments to both human and environment if it is discharged before proper treatment [3,4].

Lines 48-51

Electrochemical technology has obvious superiority in treating refractory wastewater due to its wide-adaptability in contaminant decomposition as well as simple equipment demand, small area occupation, and easy operation [8,9].

Lines 52-54

In recent years, electrochemical technology, especially 3D electrochemical tech-nology, has become the focus of study because of its easy operation, small footprint and short hydraulic retention time (HRT) [10]

Lines 81-86

3D-BERs were constructed to treat Rhodamine B (RhB) [14], and the results indicated that the application of voltage promoted the degradation of RhB. Three processes, including electro-adsorption, electro-chemical oxidation and elec-tro-biodegradation, were identified to contribute to RhB degradation. Biodegradation of ibuprofen has been recently demonstrated in a 3D-BER. The optimal conditions were identified at a current density of 12.73 A/m2 and an HRT of 3.5 h [11].

Lines 91-93

Electrodes (particle electrode, anode and cathode), which are important units of 3D-BERs, play critical roles in pollutant degradation, microbial attachment, electrons transfer, etc. [16].

Lines 139-141

The results can provide a solution to the practical treatment of pharmaceutical wastewater containing THF via this novel 3D-BERs filled by new particle electrodes with polyurethane sponge carrier.

Lines 148-154

A combined suspension ball polyurethane sponge carrier of Φ60 mm and Φ90 mm (filling rate of 50%) is filled between the electrode plates, and the filling rate of polyu-rethane sponge carrier in each individual suspension ball is 30-50% to form a 3D bio-film electrode by loading biofilm. Polyurethane sponge carrier is a medium-pore carri-er (20 mm). Suitable micropores can maintain not only an appropriate number of mi-croorganisms, but also a good biofilm structure and activity.

Lines 174-175

After mixing for electric Fenton pretreatment (electric Fenton operation parameters: HRT = 240 min, H₂O₂ = 50 mmol/L, Fe²⁺ injection amount determined as 10 mmol/L, initial pH = 3, an optimal current density of 15 mA/cm²), the effluent was then adjusted to pH between 7.5-9.0 to obtain 3D-BERs influent.

Lines 187-193

The COD_Cr of the electric Fenton effluent was diluted to about 1,000 mg/L, the dis-solved oxygen was controlled within 3-4.5 mg/L, and after continuous aeration, when the COD_Cr changed little and remained stable, water exchange was started when gradually increasing the influent concentration according to a gradient, and the efflu-ent COD_Cr removal rate was stabilized at 60±4% for one consecutive week after 21 d, marking the completion of the membrane hanging startup [11].

Lines 197-198

Under a specific voltage, COD_Cr changed slightly and remained basically stable (about 5 days), indicating that biological adaptation entered the next stage. Meanwhile, the control group was started in the same way as above, without powering up during the start-up and formal experiment phases.

Lines 201-204

During the sequencing batch experiment, the dissolved oxygen in the reactor was maintained within 5-8 mg/L, pole plate spacing was set to 150 mm, the voltage was maintained at 10 V for 39 days, and the HRT of reactor was operated for 24 h (includ-ing 0.2 h for inlet water, 23 h for aeration, 0.5 h for precipitation, and 0.3 h for dis-charge water).

Lines 223-227

During the biological stabilization, the measured biomass of polyurethane sponge carrier was 25-30 mg/cm³, at the same time, the packed biofilm near the anode (AB), the packed biofilm near the cathode (CB), and the control group activated sludge inoc-ulated in the reactor were taken (AS), and the microbial community structure was measured. All samples of COD_Cr, GC-MS and high-throughput sequencing were tested in triplicate.

Lines 294-296

After the operation of the 31st day, the effluent of 3D-BERs was detected by GC-MS. According to Fig. 3 (c) and (d), VOCs THF was detected in the effluent of 3D-BERs at the peak time of 2.71 min.

Lines 310-312

The THF concentrations in the influent and effluent water are 201847 µg/L and 4744 µg/L respectively. The removal rate of THF was as high as 97.65%, and the COD_Cr removal rate of the corresponding wastewater reached 98.14%.

Lines 323-324

which is finally further oxidized to succinicate, entering the tricarboxylic acid cycle and being thoroughly mineralized [29].

Lines 347-348

On the 39th day of stable operation, three groups of sludge samples from AB, CB and CG were taken, respectively. High-throughput sequencing was employed to analyze the abundance and the diversity of microbial communities, with the biofilm of anode (AB), cathode (CB), and the control group (CG).

Lines 388-391

In contrast, the relative abundance of Planctomycetota and Patescibacteria decreased in electric field, indicating that metabolism of those bacterial populations is inhibited by the high voltage. Myxococcota, Acidobacteriota, Gemmatimonadota almost disappeared under 10 V, indicating that the metabolism of those bacterial populations is significantly inhibited.

Lines 406-409

It is found that the genus Aureus is the dominant bacterial genus in the anode of microbial fuel cells, with exogenous electroactivity [30]. In addition, Chryseobacterium has excellent tolerance to toxic pollutants and can degrade various organic substances, and is rich in extracellular electron transfer genes.

Lines 424-425

Using microbial fuel cells to remove azide, Erysipelothrix was also found to be the dominant genus at the cathode [42].

Lines 428-430

The relative abundance of Pseudoxanthomonas reached 9.8%, which was 4.1% in anode samples and was rarely almost present in CG. Pseudoxanthomonas showed an important role in the process of degrading cyclic organic matter [48].

 

Lines 442-444

Paludibacter is a strict anaerobic bacterium belonging to the phylum Bacteroidetes, which can ferment a variety of monosaccharides and disaccharides to produce propionic acid, acetic acid, and a small amount of butyric acid [52].

Lines 454-456

At the same time, the COD_Cr range is 3438.30-4775.70 mg/L, and the threshold condition is greater than 0.9, we have also predicted the metabolic pathways.

Lines 459-461

Among them, Chryseobacterium and Desulfovibrio are very critical to the degradation of THF. In combination with electrode plate electro-oxidation, THF can be completely mineralized, which is a fundamental factor for 3D-BERs to improve the efficiency of microbial degradation of pharmaceutical wastewater.

 

• Abstract: please rewrite the main results and the purpose.

Response: Thank you for your comments. The detailed explanation have been added in Lines 14-30.

The revisions were as follows:

Line 14-30:

Abstract: Three-dimensional biofilm electrode reactors (3D-BERs) exhibit efficacy in the removal of refractory wastewater of pharmaceuticals due to the resistance of pharmaceutical wastewater to biodegradation. In this paper, a new 3D-BER with polyurethane sponge carrier is applied to the treatment of pharmaceutical wastewater containing tetrahydrofuran (THF) with an objective of exploring the removal efficiency, degradation pathway and main functions of microorganisms of 3D-BERs for wastewater containing THF. The results indicate that when the voltage is 10 V, the highest COD_Cr removal efficiency is (95.9±1.6)%. Compared to the control group, the removal rate has been increased by 21.97±4.69%. The main intermediates of THF, γ-butyrolactone and 4-hydroxybutyric acid, were detected respectively by Gas Chromatography–Mass Spectrometry (GC-MS), indicating that 3D-BERs contribute to the degradation of THF with electro-oxidation as well as microbial synergism. Microorganisms, such as Proteobacteria with extracellular electron transfer capacity, Bacteroidetes capable of degrading complex carbon sources and parthenogenic anaerobic bacteria Firmicutes, were found to be enriched by high-throughput sequencing analysis in 3D-BERs. which were conducive to the degradation of refractory pollutants. At the genus level, Chryseobacterium, Brevundimonas, Erysipelothrix, and Desulfovibrio were the main functional genera, whose degradation of THF intermediates was found by functional prediction, mainly through chemoheterotrophy, aerobic chemoheterotrophy, etc. Hopefully, this study will provide a solution to the practical treatment of pharmaceutical wastewater containing THF via this new 3D-BER system with polyurethane sponge carrier.

 

• Introduction: The novelty and the advance added to the area must be clearly stated. Particularly Introduction could be enlarged. These things are missing.

Response: Thank you for your comments. We have added a new overview of the development of particle electrodes, electroactive microorganisms, etc. The detailed extensions have been presented in Lines 52-141.(Lines 52-76: 3D electrochemical technology has been expanded, and added several mechanisms account for pollutants degradation in 3D-BERs, including adsorption, biodegradation, indicating its progressiveness; Lines 52-76: The reason why polyurethane sponge filler is used as particle electrode is proposed, but the conductive particle electrode such as GAC is not selected. In 3D-BERs, the electric field was conducted to improve EET capabilities and boosted biofilm formation, which produces a growing number of EAMs, and significantly improves con-ductivity for polyurethane sponge carrier. Therefore, it could be filled as particle electrodes in 3D-BERs because of the biofilm conductivity.)

The revisions were as follows:

Line 52-141:

In recent years, electrochemical technology, especially 3D electrochemical technology, has become the focus of study because of its easy operation, small footprint and short hydraulic retention time (HRT) [10]. However, the electrochemical process requires considerable energy input. Hence, it is a key to effectively reduce energy consumption for the future development of three-dimensional electrode reactors (3D-ERs). To address this problem, microorganisms are cultured on particle electrodes, and bipolar 3D-BERs have emerged. In addition, the response of different microorganisms to current intensity varies, therefore, functional microorganisms can be screened out by regulating current density. Under higher current density, the pollutants can be decomposed into easy components under the action of electricity, and further biodegraded by the biofilm attached on the particle electrode. Under lower current density, the activity of biofilm is improved by electrical stimulation, thereby enhancing the biodegradation of the pollutants. Bipolar 3D-BERs can also exert versatile targets by altering the operation conditions, such as electrolyzing water to produce hydrogen to reduce nitrate and sulfate [11].

Actually, 3D-BERs integrate biofilm technology with (3D-ERs), and take the advantage from both treatment processes. Several mechanisms account for pollutants degradation in 3D-BERs, including adsorption, biodegradation, electro-adsorption, electro-chemical oxidation and electro-biodegradation [12]. The accumulation of organisms and microorganisms induce biofouling on the electrode surfacedue to the presence of particle electrodes and the synergy of electricity and microorganisms. Among them, electro-biodegradation, i.e. the synergy of electricity and microorganisms, is critical for refractory pollutants removal. The mechanisms mainly include two aspects, one is the enhanced bio-degradation due to the stimulation of electric field on microbial metabolism and acclimation of microbial community [13], the other is the positive contribution of the intermediates generated from electrochemical process to biodegradation .

With the synergy of electricity and microorganisms, 3D-BERs also exhibit efficacy in the removal of refractory wastewater [14]. Recently, 3D-BERs have been developed for refractory wastewater treatment by taking the advantage of both electrochemical and bio-logical technologies [12]. Because of their lower operation costs than that of 3D-ERs, 3D-BERs is considered mainly for the pretreatment of pharmaceutical wastewater [15]. 3D-BERs were constructed to treat Rhodamine B (RhB) [14], and the results indicated that the application of voltage promoted the degradation of RhB. Three processes, including electro-adsorption, electro-chemical oxidation and electro-biodegradation, were identified to contribute to RhB degradation. Biodegradation of ibuprofen has been recently demonstrated in a 3D-BER. The optimal conditions were identified at a current density of 12.73 A/m² and an HRT of 3.5 h [11]. Recently, 3D-BERs have been developed for the treatment of wastewater containing antibiotics, and its potential to the removal of sulfamethoxazole (SMX) and tetracycline (TC) was evaluated, and 88.9-93.5% of SMX removal rate and 89.3-95.6% of TC removal rate were obtained, respectively [9].

Electrodes (particle electrode, anode and cathode), which are important units of 3D-BERs, play critical roles in pollutant degradation, microbial attachment, electrons transfer, etc. [16]. Filled particles are the important units of 3D-BERs. When external potential is applied, they are polarized to form a large amount- of micro-electrodes, and the area of the main electrode is enlarged, namely, particle electrode in 3D-BERs. Particle electrode can improve the performance of 3D-BERs, not only because of its participation of electrochemical reaction, but also due to its function of microbial carriers for colonizing organisms to promote associated microbial growth and reproduction. Currently, granular activated carbon (GAC) has a porous structure and a large specific surface area, which is beneficial to mass transfer and microbial attachment [17,18]. A 3D-BER packed with GAC particle electrodes was developed to treat reactive brilliant red X-3B (RBRX-3B) dying wastewater [19]. Under the condition of HRT 24 h, 90% of average decolorization efficiency and 80% of COD_Cr removal efficiency were obtained when the initial concentration of RBRX-3B was 1000 mg/L. In recent years, some other particle electrodes such as zeolite, lithium slag, steel slag, ceramist and sulfonated cation-exchange resin have been applied in 3D-BERs to treat wastewater [11]. Different particle electrodes resulted in distinct interaction with microorganisms in terms of biocompatibility, electrical conductivity, microbial activity and specific metabolic functions.

The particle electrode in 3D-BERs is an important place for pollutants degradation. Therefore,  its materials need to have advantages such as good conductivity, high corrosion resistance, nontoxicity and good biocompatibility. GAC is most widely used due to good conductivity. However, bioreactors filled with GAC can be easily blocked with the increased biomass. The biofilm on particle electrode is usually considered as weak conductivity, but a growing number of microorganisms have been shown to have electroactivity, which can be attached to biofilm formation [20]. The microorganisms with electroactivity are called electroactive microorganisms (EAMs), which have the ability to carry out the flow and exchange of electrons between intracellular and extracellular redox-active electron donors and acceptors. In add-ition, EAMs have efficient extracellular electron transfer (EET) capabilities [21]. EAMs can be used for pollutant degradation and environmental remediation via their EET capabilities, which have been enhanced by modifying EAMs using synthetic biology and material engineering strategies. For example, the synthesis of electron shuttles can be enhanced through synthetic biology to improve the EET, and boosting biofilm formation can be improved by material engineering to increase conductivity of EAMs [22]. 

The polyurethane sponge carrier with medium-pore can not only increase the biodiversity of microorganisms in 3D-BERs, but also ensure an excellent biofilm structure and, provide excellent treatment performance. However, the weak electrical conductivity was the shortcoming for polyurethane sponge as particle electrodes. In 3D-BERs, the electric field was conducted to improve EET capabilities and boosting biofilm formation, which produces a growing number of EAMs, and significantly improves conductivity for polyurethane sponge carrier, which, therefore, could be filled as particle electrodes in 3D-BERs because of biofilm conductivity .

In this study, polyurethane sponge carrier is adopted to build a new particle electrode in 3D-BERs, which aims to develop the biomass and activity for the particle electrode with boosting EAMs, and which is used in the advanced treatment of pharmaceutical wastewater containing THF. The treatment efficiency for pharmaceutical wastewater in the 3D-BER system was explored, and the degradation process of THF was identified. Moreover, the function and microbial community of the new 3D-BERs was investigated. The results can provide a solution to the practical treatment of pharmaceutical wastewater containing THF via this novel 3D-BERs filled by new particle electrodes with polyurethane sponge carrier.

References:

[11] Wu, Z.Y.; Xu, J.; Wu, L.; Ni, B.J. Three-dimensional biofilm electrode reactors (3D-BERs) for wastewater treatment. Bioresour Technol. 2021, 344, 126274. https://doi.org/10.1016/j.biortech.2021.126274.

[12] Feng, L.; Li, X.; Gan, L.; Xu, J. Synergistic effects of electricity and biofilm on Rhodamine B (RhB) degradation in three-dimensional biofilm electrode reactors (3D-BERs). Electrochimica Acta. 2018, 290, 165–175. https://doi.org/10.1016/j.electacta.2018.09.068.

[13] Zeyoudi, M.; Altenaiji, E.; Ozer, L.Y.; Ahmed, I.; Yousef, A.F.; Hasan, S.W. Impact of continuous and intermittent supply of electric field on the function and microbial community of wastewater treatment electro-bioreactors. Electrochimica Acta. 2015, 181, 271–279. https://doi.org/10.1016/j.electacta.2015.04.095.

[14] Liu, F.; Luo, S.; Wang, H.; Zuo, K.; Wang, L.; Zhang, X.; Liang, P.; Huang, X. Improving wastewater treatment capacity by optimizing hydraulic retention time of dual-anode assembled microbial desalination cell system. Sep Purif Technol. 2019, 226 39–47. https://doi.org/10.1016/j.seppur.2019.05.071.

[15] Feng, Y.; Long, Y.; Wang, Z.; Wang, X.; Shi, N.; Suo, N.; Shi, Y.; Yu, Y. Performance and microbial community of an electric biological integration reactor (EBIR) for treatment of wastewater containing ibuprofen. Bioresour Technol. 2018, 274, 447–458. https://doi.org/10.1016/j.biortech.2018.12.015.

[16] Chen, M.; Xu, J.; Dai, R.; Wu, Z.; Liu, M.; Wang, Z. Development of a moving-bed electrochemical membrane bioreactor to enhance removal of low-concentration antibiotic from wastewater. Bioresour Technol. 2019, 293, 122022. https://doi.org/10.1016/j.biortech.2019.122022.

[17] Tang, Q.; Sheng, Y.; Li, C.; Wang, W.; Liu, X. Simultaneous removal of nitrate and sulfate using an up-flow three-dimensional biofilm electrode reactor: Performance and microbial response. Bioresour Technol. 2020, 318, 124096. https://doi.org/10.1016/j.biortech.2020.124096.

[18] Wei, V.; Oleszkiewicz, J.A.; Elektorowicz, M. Nutrient removal in an electrically enhanced membrane bioreactor. Water Sci Technol. 2009, 60, 3159–3163. https://doi.org/10.2166/wst.2009.625.

[19] Liu, S.; Feng, X.; Gu, F.; Li, X.; Wang, Y. Sequential reduction/oxidation of azo dyes in a three-dimensional biofilm electrode reactor. Chemosphere. 2017, 186, 287–294. https://doi.org/10.1016/j.chemosphere.2017.08.001.

[20] Zhao, J.; Li, F.; Cao, Y.; Zhang, X.; Chen, T.; Song, H.; Wang, Z. Microbial extracellular electron transfer and strategies for engineering electroactive microorganisms. Biotechnology Advances. 2021, 53, 107682. https://doi.org/10.1016/j.biotechadv.2020.107682.

[21] Koch, C.; Harnisch, F. What Is the Essence of Microbial Electroactivity?. Front Microbiol. 2016, 7. https://doi.org/10.3389/fmicb.2016.01890.

[22] Glaven, S.M. Bioelectrochemical systems and synthetic biology: more power, more products. Microb Biotechnol. 2019, 12 819–823. https://doi.org/10.1111/1751-7915.13456.

 

• The figures' quality is very poor. Please provide clear images.

Response: Thank you for your comments. We have modified all the figures and improved the resolution.

 

Figure 1. Schematic diagram of 3D-BERs.

 

 

 

 

 

• Please check the whole manuscript to correct this type of mistake.

Response: Thank you for your comments. We have modified all the figures.

Figure 3. GC-MS spectra of pharmaceutical wastewater degradation process. (a) Electric Fenton effluent of VOCs; and (b) SVOCs; (c) 3D-BERs effluent of VOCs; and (d) SVOCs.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Figure 4. Degradation process of tetrahydrofuran in 3D-BERs.

 

 

Figure 5. Microbial communities at phylum level (relative abundance over 1%).

 

 

 

Figure 6. Microbial communities heat map analysis of 3D-BERs at genus level.

 

 

 

 

 

 

 

 

 

 

 

Figure 7. Network analysis diagram of microorganisms and metabolic pathways.

 

• Please write details preparation method of all characterization techniques.

Response: Thank you for your comments. We have made supplementary instructions on the testing methods and instruments. The most important GC-MS detection step was added. The detailed explanation have been added in Lines 209-222.

The revisions were as follows:

Line 209-222:

Measuring instrument: pH meter (PHS-3C, Leici, Shanghai); Glass rod thermometer (Chenhua, Shanghai); DO instrument (HQ30d, HACH, USA). CODCr determination (6B-12 digestion instrument /6B-201 rapid analyzer, Shengaohua, Jiangsu); GC-MS (7890A-5977A, Agilent, USA).

The intermediate compound analysis of pharmaceutical wastewater sample was carried out using GC-MS (7890A-5977A, Agilent, USA) by extracting wastewater samples (100 mL) with dichloromethane. The GC was equipped with DB-WAX ms capillary column (L × D × T = 30 m × 0.25 mm × 0.25 μm). The chromatographic conditions included the following temperature programs: the column temperature was initially held at 30°C for 6 min, this was subsequently increased to 60°C at 4°C/min, then increased to 110°C at 10°C/min kept constant for 5 min, and then increased to 250°C at 20°C/min, the final temperature was held for 2 min. A voltage of 1.2 kV was used, with electron impact ionization at 70 eV for molecular fragmentation. The mass spectra of the peaks were compared with the GC–MS database with a standard library for compound identification.

During the biological stabilization, the measured biomass of polyurethane sponge carrier was 25-30 mg/cm3, at the same time, the packed biofilm near the anode (AB), the packed biofilm near the cathode (CB), and the control group activated sludge inoculated in the reactor were taken (AS), and the microbial community structure was measured. All samples of COD_Cr, GC-MS and high-throughput sequencing were tested in triplicate.

 

• Please add an error bar in all figures.

Response: Thank you for your comments. For the first time, we plotted according to the average value. This time, we added error bars and redraw and adjusted all figures.

 

 

 

 

Figure 2. COD_Cr removal rate of pharmaceutical wastewater in 3D-BERs of (a) under different voltages; (b) a voltage of 10 V; and (c) control group (no power).

 

• Please check all units in order to be similar.

Response: Thank you for your comments. We have unified all unit forms, such as mg/L, kWh/m³, A/m², etc.

The revisions were as follows:

Line 86-87:

The optimal conditions were identified at a current density of 12.73 A/m² and an HRT of 3.5 h [11].

• An environmental viability assessment should be added.

Response: Thank you for your valuable comments. As for environmental viability assessment, we determined the unit power consumption of 3D-BERs under 10V voltage, which is acceptable in terms of refractory wastewater. The detailed explanation have been added in Lines 268-273.

The revisions were as follows:

Line 268-273:

Based on the above effects, in terms of environmental feasibility assessment, it is proved that 3D-BERs can effectively degrade the pharmaceutical wastewater containing tetrahydrofuran, which is 21.97 ± 4.69% higher than that of the CG. Although the power consumption is increased, the power consumption under 10 V voltage is 10 kWh/m³, which is acceptable in terms of energy consumption for refractory wastewater treatment.

 

• References can be added from the host journal.

Response: Thank you for your comments. We referred to the water journal and added relevant reference.

Line 171-172:

The raw wastewater had a poor biodegradability as a consequence of its complexity and toxicity[23]

[23]Lee, K.H.; Wie, Y.M.; Lee, Y.-S. Characterization of 1,4-Dioxane Biodegradation by a Microbial Community. Water. 2020, 12, 3372. https://doi.org/ 10.3390/w12123372.

 

• Please check the format of the reference.

Response: Thank you for your comments. According to the format of water journal, we have unified the format of references.

 

• Conclusion: please add the key points with the further implication.

Response: Thank you for your comments. We revised the conclusion and emphasized the bacteria that are conducive to THF degradation. The detailed explanation have been added in Lines 472-481.

The revisions were as follows:

Line 472-481:

The detection of intermediate products γ-butyrolactone and γ-hydroxybutyrate inferred the degradation pathway of tetrahydrofuran in 3D-BERs. Electrochemical oxidation can break the structure of THF. Electrical acclimation enriches EAMs with EET on the surface of PU. Typical EAMs includes Chryseobacterium and Desulfovibrio. Electrochemical oxidation and electro-biodegradation work together to achieve effective degradation of THF, which is an important factor for 3D-BERs to improve the efficiency of microbial degradation of pharmaceutical wastewater. This study provides a reasonable analysis and a decision-making mechanism to guide and standardize the practice of comprehensive treatment of pharmaceutical wastewater in the future.

 

Author Response File: Author Response.docx

Reviewer 2 Report

In the manuscript “Three-dimensional biofilm electrode reactors with polyurethane sponge carrier for highly-efficient treatment of pharmaceuticals wastewater containing tetrahydrofuran”, the authors employed the 3D-BERs with polyurethane carriers to treat THF in the pharmaceutical wastewater. The system performance was clearly presented. The degradation products and the microbial composition were analyzed. The topic is interesting and relevant to the scope of the journal. I would suggest a minor revision before the manuscript is accepted.

 

The detailed comments are as follows:

1.     Extensive editing of the English language and style is required, especially the Abstract. e.g., L15~18 should be more clear and concise.

2.     L26, “their degradation”? Why were the microbes degraded?

3.     The resolution of all the figures should be improved.

4.     Did the authors repeat the experiments or run the reactors in triplicate? Please include the information.

5.     L361, please avoid using absolute words.

6.     L390, please clarify the “shock load resistance” and offer the threshold conditions.

 

7.     L394, please clarify which genera are considered responsible for the degradation of THF.

Author Response

Response to Reviewer 2's Comments

In the manuscript “Three-dimensional biofilm electrode reactors with polyurethane sponge carrier for highly-efficient treatment of pharmaceuticals wastewater containing tetrahydrofuran”, the authors employed the 3D-BERs with polyurethane carriers to treat THF in the pharmaceutical wastewater. The system performance was clearly presented. The degradation products and the microbial composition were analyzed. The topic is interesting and relevant to the scope of the journal. I would suggest a minor revision before the manuscript is accepted.

Response: Thank you a lot for supporting our study. According to your valuable comments, we have revised this manuscript point by point, which is important in improving our knowledge and benefiting our next study. Thank you again.

• Extensive editing of the English language and style is required, especially the Abstract. e.g., L15~18 should be more clear and concise.

Response: Thank you for your comments. We have checked and revised the language across the whole text. 

The revisions were as follows:

Lines 26-30

whose degradation of THF intermediates was found by functional prediction, mainly through chemoheterotrophy, aerobic chemoheterotrophy, etc. Hopefully, this study will provide a solution to the practical treatment of pharmaceutical wastewater containing THF via this new 3D-BER system with polyurethane sponge carrier.

Lines 38-42

Among the pharmaceutical wastewater, THF is one of the most representative pollutants. However, the average treating ratio of pharmaceutical wastewater containing THF is less than 30%, and it has become a serious source of water pollution [2]. Pharmaceutical wastewater will cause serious detriments to both human and environment if it is discharged before proper treatment [3,4].

Lines 48-51

Electrochemical technology has obvious superiority in treating refractory wastewater due to its wide-adaptability in contaminant decomposition as well as simple equipment demand, small area occupation, and easy operation [8,9].

Lines 52-54

In recent years, electrochemical technology, especially 3D electrochemical tech-nology, has become the focus of study because of its easy operation, small footprint and short hydraulic retention time (HRT) [10]

Lines 81-86

3D-BERs were constructed to treat Rhodamine B (RhB) [14], and the results indicated that the application of voltage promoted the degradation of RhB. Three processes, including electro-adsorption, electro-chemical oxidation and elec-tro-biodegradation, were identified to contribute to RhB degradation. Biodegradation of ibuprofen has been recently demonstrated in a 3D-BER. The optimal conditions were identified at a current density of 12.73 A/m2 and an HRT of 3.5 h [11].

Lines 91-93

Electrodes (particle electrode, anode and cathode), which are important units of 3D-BERs, play critical roles in pollutant degradation, microbial attachment, electrons transfer, etc. [16].

Lines 139-141

The results can provide a solution to the practical treatment of pharmaceutical wastewater containing THF via this novel 3D-BERs filled by new particle electrodes with polyurethane sponge carrier.

Lines 148-154

A combined suspension ball polyurethane sponge carrier of Φ60 mm and Φ90 mm (filling rate of 50%) is filled between the electrode plates, and the filling rate of polyu-rethane sponge carrier in each individual suspension ball is 30-50% to form a 3D bio-film electrode by loading biofilm. Polyurethane sponge carrier is a medium-pore carri-er (20 mm). Suitable micropores can maintain not only an appropriate number of mi-croorganisms, but also a good biofilm structure and activity.

Lines 174-175

After mixing for electric Fenton pretreatment (electric Fenton operation parameters: HRT = 240 min, H₂O₂ = 50 mmol/L, Fe²⁺ injection amount determined as 10 mmol/L, initial pH = 3, an optimal current density of 15 mA/cm²), the effluent was then adjusted to pH between 7.5-9.0 to obtain 3D-BERs influent.

Lines 187-193

The COD_Cr of the electric Fenton effluent was diluted to about 1,000 mg/L, the dis-solved oxygen was controlled within 3-4.5 mg/L, and after continuous aeration, when the COD_Cr changed little and remained stable, water exchange was started when gradually increasing the influent concentration according to a gradient, and the efflu-ent COD_Cr removal rate was stabilized at 60±4% for one consecutive week after 21 d, marking the completion of the membrane hanging startup [11].

Lines 197-198

Under a specific voltage, COD_Cr changed slightly and remained basically stable (about 5 days), indicating that biological adaptation entered the next stage. Meanwhile, the control group was started in the same way as above, without powering up during the start-up and formal experiment phases.

Lines 201-204

During the sequencing batch experiment, the dissolved oxygen in the reactor was maintained within 5-8 mg/L, pole plate spacing was set to 150 mm, the voltage was maintained at 10 V for 39 days, and the HRT of reactor was operated for 24 h (includ-ing 0.2 h for inlet water, 23 h for aeration, 0.5 h for precipitation, and 0.3 h for dis-charge water).

Lines 223-227

During the biological stabilization, the measured biomass of polyurethane sponge carrier was 25-30 mg/cm³, at the same time, the packed biofilm near the anode (AB), the packed biofilm near the cathode (CB), and the control group activated sludge inoc-ulated in the reactor were taken (AS), and the microbial community structure was measured. All samples of COD_Cr, GC-MS and high-throughput sequencing were tested in triplicate.

Lines 294-296

After the operation of the 31st day, the effluent of 3D-BERs was detected by GC-MS. According to Fig. 3 (c) and (d), VOCs THF was detected in the effluent of 3D-BERs at the peak time of 2.71 min.

Lines 310-312

The THF concentrations in the influent and effluent water are 201847 µg/L and 4744 µg/L respectively. The removal rate of THF was as high as 97.65%, and the COD_Cr removal rate of the corresponding wastewater reached 98.14%.

Lines 323-324

which is finally further oxidized to succinicate, entering the tricarboxylic acid cycle and being thoroughly mineralized [29].

Lines 347-348

On the 39th day of stable operation, three groups of sludge samples from AB, CB and CG were taken, respectively. High-throughput sequencing was employed to analyze the abundance and the diversity of microbial communities, with the biofilm of anode (AB), cathode (CB), and the control group (CG).

Lines 388-391

In contrast, the relative abundance of Planctomycetota and Patescibacteria decreased in electric field, indicating that metabolism of those bacterial populations is inhibited by the high voltage. Myxococcota, Acidobacteriota, Gemmatimonadota almost disappeared under 10 V, indicating that the metabolism of those bacterial populations is significantly inhibited.

Lines 406-409

It is found that the genus Aureus is the dominant bacterial genus in the anode of microbial fuel cells, with exogenous electroactivity [30]. In addition, Chryseobacterium has excellent tolerance to toxic pollutants and can degrade various organic substances, and is rich in extracellular electron transfer genes.

Lines 424-425

Using microbial fuel cells to remove azide, Erysipelothrix was also found to be the dominant genus at the cathode [42].

Lines 428-430

The relative abundance of Pseudoxanthomonas reached 9.8%, which was 4.1% in anode samples and was rarely almost present in CG. Pseudoxanthomonas showed an important role in the process of degrading cyclic organic matter [48].

 

Lines 442-444

Paludibacter is a strict anaerobic bacterium belonging to the phylum Bacteroidetes, which can ferment a variety of monosaccharides and disaccharides to produce propionic acid, acetic acid, and a small amount of butyric acid [52].

Lines 454-456

At the same time, the COD_Cr range is 3438.30-4775.70 mg/L, and the threshold condition is greater than 0.9, we have also predicted the metabolic pathways.

Lines 459-461

Among them, Chryseobacterium and Desulfovibrio are very critical to the degradation of THF. In combination with electrode plate electro-oxidation, THF can be completely mineralized, which is a fundamental factor for 3D-BERs to improve the efficiency of microbial degradation of pharmaceutical wastewater.

 

• L26, “their degradation”? Why were the microbes degraded?

Response: Thank you for your comments. The detailed explanation have been added in Lines 125-141 and Lines 154-157.

The revisions were as follows: 

The polyurethane sponge carrier with medium-pore can not only increase the biodiversity of microorganisms in 3D-BERs, but also ensure an excellent biofilm structure and, provide excellent treatment performance. However, the weak electrical conductivity was the shortcoming for polyurethane sponge as particle electrodes. In 3D-BERs, the electric field was conducted to improve EET capabilities and boosting biofilm formation, which produces a growing number of EAMs, and significantly improves conductivity for polyurethane sponge carrier, which, therefore, could be filled as particle electrodes in 3D-BERs because of biofilm conductivity.

In this study, polyurethane sponge carrier is adopted to build a new particle electrode in 3D-BERs, which aims to develop the biomass and activity for the particle electrode with boosting EAMs, and which is used in the advanced treatment of pharmaceutical wastewater containing THF. The treatment efficiency for pharmaceutical wastewater in the 3D-BER system was explored, and the degradation process of THF was identified. Moreover, the function and microbial community of the new 3D-BERs was investigated. The results can provide a solution to the practical treatment of pharmaceutical wastewater containing THF via this novel 3D-BERs filled by new particle electrodes with polyurethane sponge carrier.

Line 154-157:

2.1. Experimental setup

…… 

The high surface area provides a huge breeding ground for EAMs. During enrichment, electrical acclimation helps to form EAMs with EET, which plays the same role as conductive particles, thus increasing conductivity and forming a new type of 3D-BERs, which is beneficial to the degradation of refractory pollutants

• The resolution of all the figures should be improved.

Response: Thank you for your comments. We have modified all figures to improve the resolution.

 

Figure 1. Schematic diagram of 3D-BERs.

 

 

 

Figure 2. COD_Cr removal rate of pharmaceutical wastewater in 3D-BERs of (a) under different voltages; (b) a voltage of 10 V; and (c) control group (no power).

 

Figure 3. GC-MS spectra of pharmaceutical wastewater degradation process. (a) Electric Fenton effluent of VOCs; and (b) SVOCs; (c) 3D-BERs effluent of VOCs; and (d) SVOCs.

 

 

 

 

 

 

 

 

 

 

 

Figure 4. Degradation process of tetrahydrofuran in 3D-BERs.

 

 

 

Figure 5. Microbial communities at phylum level (relative abundance over 1%).

 

 

Figure 6. Microbial communities heat map analysis of 3D-BERs at genus level.

 

 

 

 

 

 

 

 

 

 

 

Figure 7. Network analysis diagram of microorganisms and metabolic pathways.

 

• Did the authors repeat the experiments or run the reactors in triplicate? Please include the information.

Response: Thank you for your comments. We belong to the repeated experiment, in duplicate, with the control group without power.

The revisions were as follows:

Line 201-204:

During the sequencing batch experiment, the dissolved oxygen in the reactor was maintained within 5-8 mg/L, pole plate spacing was set to 150 mm, the voltage was maintained at 10 V for 39 days, and the HRT of reactor was operated for 24 h (including 0.2 h for inlet water, 23 h for aeration, 0.5 h for precipitation, and 0.3 h for discharge water) The inlet and discharge water samples were taken after daily monitoring of water pH, temperature and dissolved oxygen.

 

• L361, please avoid using absolute words.

Response: Thank you for your comments. We have added almost before present and replace “essential” with “important”.

The revisions were as follows:

Line 428-430:

The relative abundance of Pseudoxanthomonas reached 9.8%, which was 4.1% in anode samples and was rarely almost present in CG. Pseudoxanthomonas showed an important role in the process of degrading cyclic organic matter [48].

• L390, please clarify the “shock load resistance” and offer the threshold conditions.

Response: Thank you for your comments. We define the threshold conditions according to the experimental results.

The revisions were as follows:

Line 454-459:

At the same time, the COD_Cr range is 3438.30-4775.70 mg/L, and the threshold condition is greater than 0.9, we have also predicted the metabolic pathways. As shown in Fig. 7, Chryseobacterium, Brevundimonas in the anode and Erysipelothrix, Pseudoxanthomonas, Desulfovibrio, and other dominant bacteria genera in the anode utilize chemoheterotrophy, aerobic chemoheterotrophy, fermentation, and aromatic compound degradation.

• L394, please clarify which genera are considered responsible for the degradation of THF.

Response: Thank you for your comments. We checked the data related to refractory wastewater, and combined with our research, we determined that Chryseobacterium and Desulfovibrio belong to EAMs, and they are beneficial to THF degradation.

The revisions were as follows:

Line 459-463:

Among them, Chryseobacterium and Desulfovibrio are very critical to the degradation of THF. In combination with electrode plate electro-oxidation, THF can be completely mineralized, which is a fundamental factor for 3D-BERs to improve the efficiency of microbial degradation of pharmaceutical wastewater.

References:

Zhao, J.; Li, F.; Cao, Y.; Zhang, X.; Chen, T.; Song, H.; Wang, Z. Microbial extracellular electron transfer and strategies for engineering electroactive microorganisms. Biotechnology Advances. 2021, 53, 107682. https://doi.org/10.1016/j.biotechadv.2020.107682. 

Koch, C.; Harnisch, F. What Is the Essence of Microbial Electroactivity?. Front Microbiol. 2016, 7. https://doi.org/10.3389/fmicb.2016.01890.

Author Response File: Author Response.docx

Reviewer 3 Report

The reviewed article concerns the application of three-dimensional biofilm electrode reactors with a polyurethane sponge carrier for the highly efficient treatment of pharmaceutical wastewater containing tetrahydrofuran. The research and results presented are interesting and relevant to the field of water protection and wastewater treatment. The large scope of the research, and the state-of-the-art research methods used in the paper are to be admired. The cited references are mostly recent publications (within the last 5 years) and relevant.
However, the research presented is not clear, methods section is missing. Also, the excessive number of variables in the research conducted makes it difficult to draw final conclusions and justify them.

1. The first problem is the experimental design. Authors decided to use polyurethane sponge carrier as an particle electrode in 3B-BER system. In classic 3D-BER systems the carrier should also be a particle electrode with high electric conductivity to improve the efficiency of the system. While polyurethane sponge is a very good carrier materials for microorganisms growth, it is also considered to be natural insulator. Therefore, I am not certain the reactor applied by the Authors should be called 3D-BER (maybe „modified”?), unless the Authors modified the polyurethane to change its properties (but there is no information about it).
2. Abstract. In lines 15-17 the sentence is misleading, indicating that the distance between electrode plates affected the outcome. However, the distance between the electrode plates was constant during whole research, so it was not affecting the final efficiency of the reactor. The sentence should be rewritten.
3. Line 67: The word „biodegradation” is repeated twice at the end of the sentence.
4. Line 71-72: The sencence should be: „Biodegradation of ibuprofen has been recently demonstrated in a 3D-BER”
5. 2.2. Wastewater source: The data presented in table 1 (COD 4190±159 mg/L) do not match the data described in results (lines 182-183): „the influent CODCr fluctuated within 3438.30-4775.70 mg/L”.
6. Experimental procedure is unclear. How many days was BER operated at a specific voltage? How was adaptation period for microorganisms measured? How was the control reactor prepared?
7. Materials and methods. There are no information concerning the physical, chemical and microbiological analyses: pH, temperature, COD measurement, GC-MS analysis, molecular analysis. How they were conducted? What methods and equipment was used?
8. Results: 3.1. COD removal. It is unclear if the data described in the text and shown in Figure 2 are the average data (if yes, from which period?) or the maximum efficiency or after stabilisation of the system?
9. Figure 2 requires improvement. The numbers are almost invisible – higher resolution is required.
10. Lines 182-183 indicated that there were at least three different variables during research: water temperature fluctuated within 15.1-25.0℃, the influent CODCr fluctuated within 3438.30- 4775.70 mg/L and the composition of effluent discharges from pharmaceutical plants also differed. While this is the situation typical for the technical scale operation, it is vary hard to draw conclusions concerning the impact of each variable, especially temperature changes (not shown anywhere).
11. 3.2.1. Identification of intermediates using GC-MS. How many samples were taken for GC analysis? When were they taken?
12. Figure 3 requires improvement. The numbers are almost invisible – higher resolution is required.
13. 3.2.2. Analysis of degradation process of THF in 3D-BERs – there are no research data shown to prove the analysis presented in this subchapter. Did Authors perform additional research?
14. 3.3.1. Microbial community analysis at the phylum level. How many samples were taken and when for molecular analysis?
15. Figure 4 requires improvement. The names are almost invisible – higher resolution is required.
16. Lines 276-278. Authors discuss Shannon and Chao1 values, but they are not presented anywhere in the text.
17. Lines 343-344: „Using microbial fuel cells to remove azide, Erysipelas was also found to be the dominant genus at the cathode [38].” Erysipelas is the name of illness, not the name of bacteria.
18. Figures 5 and 6 requires improvement. The names are almost invisible – higher resolution is required.

Author Response

Response to Reviewer 3's Comments

The reviewed article concerns the application of three-dimensional biofilm electrode reactors with a polyurethane sponge carrier for the highly efficient treatment of pharmaceutical wastewater containing tetrahydrofuran. The research and results presented are interesting and relevant to the field of water protection and wastewater treatment. The large scope of the research, and the state-of-the-art research methods used in the paper are to be admired. The cited references are mostly recent publications (within the last 5 years) and relevant.

However, the research presented is not clear, methods section is missing. Also, the excessive number of variables in the research conducted makes it difficult to draw final conclusions and justify them.

Response: Thank you a lot for supporting our study. According to your valuable comments, we have revised this manuscript point by point, which is important in improving our knowledge and benefiting our next study. Thank you again.

• The first problem is the experimental design. Authors decided to use polyurethane sponge carrier as an particle electrode in 3B-BER system. In classic 3D-BER systems the carrier should also be a particle electrode with high electric conductivity to improve the efficiency of the system. While polyurethane sponge is a very good carrier materials for microorganisms growth, it is also considered to be natural insulator. Therefore, I am not certain the reactor applied by the Authors should be called 3D-BER (maybe “modified”?), unless the Authors modified the polyurethane to change its properties (but there is no information about it).

Response: Thank you for your valuable comments. The question you raised are very critical, and our team attached great importance to it. The particle electrode in 3D-BERs have an important role in pollutants degradation. Therefore, its materials need to have advantages such as good conductivity, high corrosion resistance, nontoxicity and good biocompatibility. GAC is most widely used due to good conductivity. However, bioreactors filled with GAC can be easily blocked with the increased biomass. The biofilm on particle electrode is usually considered to be weak conductivity, but a growing number of microorganisms have been shown to have electroactivity, which can be attached to biofilm formation (Zhao et al., 2021). The microorganisms with electroactivity are called electroactive microorganisms (EAMs), which have the ability to carry out the flow and exchange of electrons between intracellular and extracellular redoxactive electron donors and acceptors. In addition, EAMs have efficient extracellular electron transfer (EET) capabilities (Koch et al., 2016). EAMs can be used for pollutant degradation and environmental remediation via their EET capabilities, which have been enhanced by modifying EAMs using synthetic biology and material engineering strategies. Thank you again. The revisions above have been presented in Lines 109-141.

The revisions were as follows:

Line 109-141:

The particle electrode in 3D-BERs is an important place for pollutants degradation. Therefore, its materials need to have advantages such as good conductivity, high corrosion resistance, nontoxicity and good biocompatibility. GAC is most widely used due to good conductivity. However, bioreactors filled with GAC can be easily blocked with the increased biomass. The biofilm on particle electrode is usually considered as weak conductivity, but a growing number of microorganisms have been shown to have electroactivity, which can be attached to biofilm formation [20]. The microorganisms with electroactivity are called electroactive microorganisms (EAMs), which have the ability to carry out the flow and exchange of electrons between intracellular and extracellular redox-active electron donors and acceptors. In add-ition, EAMs have efficient extracellular electron transfer (EET) capabilities [21]. EAMs can be used for pollutant degradation and environmental remediation via their EET capabilities, which have been enhanced by modifying EAMs using synthetic biology and material engineering strategies. For example, the synthesis of electron shuttles can be enhanced through synthetic biology to improve the EET, and boosting biofilm formation can be improved by material engineering to increase conductivity of EAMs [22].

The polyurethane sponge carrier with medium-pore can not only increase the biodiversity of microorganisms in 3D-BERs, but also ensure an excellent biofilm structure and, provide excellent treatment performance. However, the weak electrical conductivity was the shortcoming for polyurethane sponge as particle electrodes. In 3D-BERs, the electric field was conducted to improve EET capabilities and boosting biofilm formation, which produces a growing number of EAMs, and significantly improves conductivity for polyurethane sponge carrier, which, therefore, could be filled as particle electrodes in 3D-BERs because of biofilm conductivity .

In this study, polyurethane sponge carrier is adopted to build a new particle electrode in 3D-BERs, which aims to develop the biomass and activity for the particle electrode with boosting EAMs, and which is used in the advanced treatment of pharmaceutical wastewater containing THF. The treatment efficiency for pharmaceutical wastewater in the 3D-BER system was explored, and the degradation process of THF was identified. Moreover, the function and microbial community of the new 3D-BERs was investigated. The results can provide a solution to the practical treatment of pharmaceutical wastewater containing THF via this novel 3D-BERs filled by new particle electrodes with polyurethane sponge carrier.

References:

[11] Wu, Z.Y.; Xu, J.; Wu, L.; Ni, B.J. Three-dimensional biofilm electrode reactors (3D-BERs) for wastewater treatment. Bioresour Technol. 2021, 344, 126274. https://doi.org/10.1016/j.biortech.2021.126274.

[12] Feng, L.; Li, X.; Gan, L.; Xu, J. Synergistic effects of electricity and biofilm on Rhodamine B (RhB) degradation in three-dimensional biofilm electrode reactors (3D-BERs). Electrochimica Acta. 2018, 290, 165–175. https://doi.org/10.1016/j.electacta.2018.09.068.

[13] Zeyoudi, M.; Altenaiji, E.; Ozer, L.Y.; Ahmed, I.; Yousef, A.F.; Hasan, S.W. Impact of continuous and intermittent supply of electric field on the function and microbial community of wastewater treatment electro-bioreactors. Electrochimica Acta. 2015, 181, 271–279. https://doi.org/10.1016/j.electacta.2015.04.095.

[14] Liu, F.; Luo, S.; Wang, H.; Zuo, K.; Wang, L.; Zhang, X.; Liang, P.; Huang, X. Improving wastewater treatment capacity by optimizing hydraulic retention time of dual-anode assembled microbial desalination cell system. Sep Purif Technol. 2019, 226 39–47. https://doi.org/10.1016/j.seppur.2019.05.071.

[15] Feng, Y.; Long, Y.; Wang, Z.; Wang, X.; Shi, N.; Suo, N.; Shi, Y.; Yu, Y. Performance and microbial community of an electric biological integration reactor (EBIR) for treatment of wastewater containing ibuprofen. Bioresour Technol. 2018, 274, 447–458. https://doi.org/10.1016/j.biortech.2018.12.015.

[16] Chen, M.; Xu, J.; Dai, R.; Wu, Z.; Liu, M.; Wang, Z. Development of a moving-bed electrochemical membrane bioreactor to enhance removal of low-concentration antibiotic from wastewater. Bioresour Technol. 2019, 293, 122022. https://doi.org/10.1016/j.biortech.2019.122022.

[17] Tang, Q.; Sheng, Y.; Li, C.; Wang, W.; Liu, X. Simultaneous removal of nitrate and sulfate using an up-flow three-dimensional biofilm electrode reactor: Performance and microbial response. Bioresour Technol. 2020, 318, 124096. https://doi.org/10.1016/j.biortech.2020.124096.

[18] Wei, V.; Oleszkiewicz, J.A.; Elektorowicz, M. Nutrient removal in an electrically enhanced membrane bioreactor. Water Sci Technol. 2009, 60, 3159–3163. https://doi.org/10.2166/wst.2009.625.

[19] Liu, S.; Feng, X.; Gu, F.; Li, X.; Wang, Y. Sequential reduction/oxidation of azo dyes in a three-dimensional biofilm electrode reactor. Chemosphere. 2017, 186, 287–294. https://doi.org/10.1016/j.chemosphere.2017.08.001.

[20] Zhao, J.; Li, F.; Cao, Y.; Zhang, X.; Chen, T.; Song, H.; Wang, Z. Microbial extracellular electron transfer and strategies for engineering electroactive microorganisms. Biotechnology Advances. 2021, 53, 107682. https://doi.org/10.1016/j.biotechadv.2020.107682.

[21] Koch, C.; Harnisch, F. What Is the Essence of Microbial Electroactivity?. Front Microbiol. 2016, 7. https://doi.org/10.3389/fmicb.2016.01890.

[22] Glaven, S.M. Bioelectrochemical systems and synthetic biology: more power, more products. Microb Biotechnol. 2019, 12 819–823. https://doi.org/10.1111/1751-7915.13456.

 

• In lines 15-17 the sentence is misleading, indicating that the distance between electrode plates affected the outcome. However, the distance between the electrode plates was constant during whole research, so it was not affecting the final efficiency of the reactor. The sentence should be rewritten.

Response: Thank you for your comments. we have revised this sentence.

The revisions were as follows:

Line 14-18:

 In this paper, a new 3D-BER with polyurethane sponge carrier is applied to the treatment of pharmaceutical wastewater containing tetrahydrofuran (THF) with an objective of exploring the removal efficiency, degradation pathway and main functions of microorganisms of 3D-BERs for wastewater containing THF. The results indicate that when the voltage is 10 V, the highest COD_Cr removal efficiency is (95.9±1.6)%. Compared to the control group, the removal rate has been increased by 21.97±4.69%.

 

• Line 67: The word “biodegradation” is repeated twice at the end of the sentence.

Response: Thank you for your comments. We have revised and deleted the duplicate “biodegradation”.

The revisions were as follows:

Line 73-76:

The mechanisms mainly include two aspects, one is the enhanced bio-degradation due to the stimulation of electric field on microbial metabolism and acclimation of microbial community [13], the other is the positive contribution of the intermediates generated from electrochemical process to biodegradation.

 

• Line 71-72: The sencence should be: “Biodegradation of ibuprofen has been recently demonstrated in a 3D-BER”.

Response: Thank you for your comments. We have made changes according to your opinions, thank you again.

The revisions were as follows:

Line 83-84:

Biodegradation of ibuprofen has been recently demonstrated in a 3D-BER.

 

• 2. Wastewater source: The data presented in table 1 (COD 4190±159 mg/L) do not match the data described in results (lines 182-183): “the influent COD_Crfluctuated within 3438.30-4775.70 mg/L”.

Response: Thank you for your comments. The average value is shown in the table, and we also realized that it was wrong. So we revised the COD_Cr value to keep the data consistently.

The revisions were as follows:

Line 180-181:

Table 1. Source characteristics of pharmaceutical wastewater.

Parameter	pH	Temperature (℃)	NH4+-N(mg/L)	CODCr (mg/L)	THF (mg/L)
Raw wastewater	5.5~7.0	25±5	9±1	5750±750	304±39
Electric Fenton effluent	3.0~4.0	20±5	8±1	4190±585	195±7

 

• Experimental procedure is unclear. How many days was BER operated at a specific voltage? How was adaptation period for microorganisms measured? How was the control reactor prepared?

Response: Thank you for your comments. 3D-BERs startup and power on domestication are very critical. We usually adjust the time for each stage to about 5 days, depending on the change of COD_Cr in effluent.

The revisions were as follows:

Line 193-200:

2.3. Experimental procedure

The reactor voltage was increased from 0 V to 14 V, and a charge of 2 V was started on the 21st d. After holding for 5 days, it was increased to 4 V. This operation was continued, with each voltage increase of 2 V, until the voltage was increased to 14 V. After one week of continuous 14 V operation, we entered the experimental phase. Under a specific voltage, COD_Cr changed slightly and remained basically stable (about 5 days), indicating that biological adaptation entered the next stage.

 

• Materials and methods. There are no information concerning the physical, chemical and microbiological analyses: pH, temperature, COD measurement, GC-MS analysis, molecular analysis. How they were conducted? What methods and equipment was used?

Response: Thank you for your comments. We have added pH, temperature, COD_Cr, GC-MS measurement methods. we listed the measurement instruments and specific steps of GC-MS.

The revisions were as follows:

Line 209-222:

Measuring instrument: pH meter (PHS-3C, Leici, Shanghai); Glass rod thermometer (Chenhua, Shanghai); DO instrument (HQ30d, HACH, USA). CODCr determination (6B-12 digestion instrument /6B-201 rapid analyzer, Shengaohua, Jiangsu); GC-MS (7890A-5977A, Agilent, USA).

The intermediate compound analysis of pharmaceutical wastewater sample was carried out using GC-MS (7890A-5977A, Agilent, USA) by extracting wastewater samples (100 mL) with dichloromethane. The GC was equipped with DB-WAX ms capillary column (L × D × T = 30 m × 0.25 mm × 0.25 μm). The chromatographic conditions included the following temperature programs: the column temperature was initially held at 30°C for 6 min, this was subsequently increased to 60°C at 4°C/min, then increased to 110°C at 10°C/min kept constant for 5 min, and then increased to 250°C at 20°C/min, the final temperature was held for 2 min. A voltage of 1.2 kV was used, with electron impact ionization at 70 eV for molecular fragmentation. The mass spectra of the peaks were compared with the GC–MS database with a standard library for compound identification.

 

• Results: 3.1. COD removal. It is unclear if the data described in the text and shown in Figure 2 are the average data (if yes, from which period?) or the maximum efficiency or after stabilisation of the system?

Response: Thank you for your comments. We started by plotting on average data. This time we adjusted the figure 2 and added error bars. The numerical measurement starts when the system is started, the acclimation is completed and the effluent is stable.

The revisions were as follows:

Line 187-193:

The COD_Cr of the electric Fenton effluent was diluted to about 1,000 mg/L, the dissolved oxygen was controlled within 3-4.5 mg/L, and after continuous aeration, when the COD_Cr changed little and remained stable, water exchange was started when gradually increasing the influent concentration according to a gradient, and the effluent COD_Cr removal rate was stabilized at 60±4% for one consecutive week after 21 d, marking the completion of the membrane hanging startup [11].

• Figure 2 requires improvement. The numbers are almost invisible – higher resolution is required.

Response: Thank you for your comments. We redrew and adjusted the resolution of the figures.

 

Figure 2. COD_Cr removal rate of pharmaceutical wastewater in 3D-BERs of (a) under different voltages; (b) a voltage of 10 V; and (c) control group (no power).

 

• Lines 182-183 indicated that there were at least three different variables during research: water temperature fluctuated within 15.1-25.0℃, the influent COD_Crfluctuated within 3438.30- 4775.70 mg/L and the composition of effluent discharges from pharmaceutical plants also differed. While this is the situation typical for the technical scale operation, it is vary hard to draw conclusions concerning the impact of each variable, especially temperature changes (not shown anywhere).

Response: Thank you for your comments. At the experiment stage in the laboratory. In the all experiment stage, we insisted on measuring the water temperature of influent and effluent. The temperature maintained was 15.1-25℃, and there was no extreme condition, so the overall temperature had no obvious impact on the experiment.

Line 246-249:

Dissolved oxygen was maintained within 5.07-7.92 mg/L during the whole phase of 3D-BERs operation; water temperature fluctuated maintained 15.1-25.0℃, and the influent COD_Cr fluctuated within 3438.30-4775.70 mg/L due to different effluent discharges from pharmaceutical plants.

• 2.1. Identification of intermediates using GC-MS. How many samples were taken for GC analysis? When were they taken?

Response: Thank you for your comments. GC-MS is one of the important detection methods in our experiment. We added detection methods of GC-MS and delivery time in the materials and methods part.

The revisions were as follows:

Line 223-227:

During the biological stabilization, the measured biomass of polyurethane sponge carrier was 25-30 mg/cm³, at the same time, the packed biofilm near the anode (AB), the packed biofilm near the cathode (CB), and the control group activated sludge inoculated in the reactor were taken (AS), and the microbial community structure was measured. All samples of COD_Cr, GC-MS and high-throughput sequencing were tested in triplicate.

Line 294:

3.2.1. Identification of intermediates using GC-MS

After the operation of the 31st day, the effluent of 3D-BERs was detected by GC-MS.

• Figure 3 requires improvement. The numbers are almost invisible – higher resolution is required.

Response: Thank you for your comments. We have readjusted resolution improvement for all figures and make the names of organic compounds clearly visible.

Figure 3. GC-MS spectra of pharmaceutical wastewater degradation process. (a) Electric Fenton effluent of VOCs; and (b) SVOCs; (c) 3D-BERs effluent of VOCs; and (d) SVOCs.

 

• 2.2. Analysis of degradation process of THF in 3D-BERs – there are no research data shown to prove the analysis presented in this subchapter. Did Authors perform additional research?

Response: Thank you a lot for supporting our study and providing such valuable comments. To be honest, we must admit the imbalance of data (covering “γ-butyrolactone” , “γ-hydroxybutyrate” and “4-hydroxybutyric acid”) and some shortcomings of the paper design and expression after reading your comments carefully. For this, we have increased the concentration change of THF in the influent and effluent water of 3D-BERs. We will continue to perfect the determination of THF intermediates in the follow-up work.

The imbalance of data led us to influence our speculations of THF degradation pathway, but in previous studies, we found that these speculations were verified (Wang et al., 2021).

Our research team deeply regrets the imbalance in the before and after THF intermediates data and sincerely hopes to gain your understanding. Your advice is of great importance in improving our following study.

The revisions were as follows:

Line 308-312:

In order to confirm the removal effect of 3D-BERs on THF in pharmaceutical wastewater, the influent and the effluent were further subjected to GC-MS quantitative analysis. The THF concentrations in the influent and effluent water are 201847 µg/L and 4744 µg/L respectively. The removal rate of THF was as high as 97.65%, and the COD_Cr removal rate of the corresponding wastewater reached 98.14%.

References:

Wang, P. Cometabolic degradation of 1,4-dioxane by a tetrahydrofuran-growing Arthrobacter sp. WN18. Ecotoxicol Environ Saf. 2021, 217, 112206. https://doi.org/10.1016/j.ecoenv.2021.112206. 

 

• 3.1. Microbial community analysis at the phylum level. How many samples were taken and when for molecular analysis?

Response: Thank you for your comments. The determination of sludge samples is very critical, including biological monitoring of sludge samples and delivery time. We have added of the materials and methods as well as the results and discussions. Thank you again for your comments

The revisions were as follows:

Line 226-227:

All samples of COD_Cr, GC-MS and high-throughput sequencing were tested in triplicate.

Line 347-348:

3.3.1. Microbial community analysis at the phylum level

On the 39th day of stable operation, three groups of sludge samples from AB, CB and CG were taken, respectively.

 

 

 

 

 

 

 

 

 

 

 

 

 

• Figure 4 requires improvement. The names are almost invisible – higher resolution is required.

Response: Thank you for your comments. We readjusted the figure to ensure that all organic compounds are clearly visible.

 

Figure 4. Degradation process of tetrahydrofuran in 3D-BERs.

 

• Lines 276-278. Authors discuss Shannon and Chao1 values, but they are not presented anywhere in the text.

Response: Thank you for your comments. We extracted Shannon values and Chao1 values from the biological monitoring of sludge report and added them to the article.

The revisions were as follows:

Line 350-355:

Based on the threshold of operational taxonomic unit (OTUs) > 0.97, the abundance and species diversity of the aerobic bioreactor was relatively high, suggesting that the microbes in 3D-BERs grew well using pharmaceutical wastewater containing THF as substrates. The Shannon values of sludge samples AB, CB and CG obtained from the high-throughput sequencing report were 3.9009, 4.7747 and 4.6567 respectively. Chao1 values were 463.01, 674.98 and 887.86, respectively.

 

• Lines 343-344: „Using microbial fuel cells to remove azide, Erysipelas was also found to be the dominant genus at the cathode [38].” Erysipelas is the name of illness, not the name of bacteria.

Response: We are sorry for this mistake. "Erysipelas" is a spelling error. We have rechecked and changed it to "Erysipelothrix". Thank you for your comments.

The revisions were as follows:

Line 424-425:

Using microbial fuel cells to remove azide, Erysipelothrix was also found to be the dominant genus at the cathode.

 

• Figures 5 and 6 requires improvement. The names are almost invisible – higher resolution is required.

Response: Thank you for your comments. We have readjusted quality of the figures to make all bacteria clearly visible. 

 

 

 

Figure 5. Microbial communities at phylum level (relative abundance over 1%).

 

 

 

 

 

 

 

 

 

 

 

 

Figure 6. Microbial communities heat map analysis of 3D-BERs at genus level.

 

 

 

 

 

 

 

 

Figure 7. Network analysis diagram of microorganisms and metabolic pathways.

 

Author Response File: Author Response.docx

Round 2

Reviewer 3 Report

The authors have answered all my previous comments and concerns in a complete and satisfactory manner. I have no further comments.