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Unignorable toxicity of formaldehyde on electroactive bacteria in bioelectrochemical systems
Journal Pre-proof Unignorable toxicity of formaldehyde on electroactive bacteria in bioelectrochemical systems Tian Li, Fan Chen, Qixing Zhou, Xin Wang, Chengmei Liao, Lean Zhou, Lili Wan, Jingkun An, Yuxuan Wan, Nan Li PII:
S0013-9351(20)30035-9
DOI:
https://doi.org/10.1016/j.envres.2020.109143
Reference:
YENRS 109143
To appear in:
Environmental Research
Received Date: 8 November 2019 Revised Date:
31 December 2019
Accepted Date: 14 January 2020
Please cite this article as: Li, T., Chen, F., Zhou, Q., Wang, X., Liao, C., Zhou, L., Wan, L., An, J., Wan, Y., Li, N., Unignorable toxicity of formaldehyde on electroactive bacteria in bioelectrochemical systems, Environmental Research (2020), doi: https://doi.org/10.1016/j.envres.2020.109143. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.
Graphical abstract
Date:
Nov 8, 2019
Submitted to:
Environmental Research
Unignorable Toxicity of Formaldehyde on Electroactive Bacteria in Bioelectrochemical Systems Tian Li 1, Fan Chen 1, Qixing Zhou1*, Xin Wang 1, Chengmei Liao 1, Lean Zhou 1, Lili Wan 1, Jingkun An 2, Yuxuan Wan 1, and Nan Li 2 1
MOE Key Laboratory of Pollution Processes and Environmental Criteria / Tianjin Key Laboratory of Environmental Remediation and Pollution Control / College of Environmental Science and Engineering, Nankai University, No. 38 Tongyan Road, Jinnan District, Tianjin 300350, China
2
Tianjin Key Lab of Indoor Air Environmental Quality Control, School of Environmental Science and Engineering, Tianjin University, No. 92 Weijin Road, Nankai District, Tianjin 300072, China Corresponding Author: Phone: (86)22-58890402; fax: (86)22-23501117; E-mail:
[email protected]
1
Abstract Formaldehyde poses significant threats to the ecosystem and is widely used as a toxicity indicator to obtain electrical signal feedback in electroactive biofilm (EAB)-based sensors. Although many optimizations have been adopted to improve the performance of EAB to formaldehyde, nearly no studies have discussed the toxicity of formaldehyde to EAB. Here, EABs were acclimated with a stable current density (8.9 ± 0.2 A/m2) and then injected with formaldehyde. The current density decreased by 27% and 98% after the injection of 1 and 10 ppm formaldehyde, respectively, compared with that in the control. The ecotoxicity of formaldehyde caused the irreversible loss of current with 3% (1 ppm) and 81% (10 ppm). Confocal laser scanning microscopy and scanning electron microscopy results showed that the redox activity was inhibited by formaldehyde, and the number of dead/broken cells increased from 2% to 40% (1 ppm) and 91% (10 ppm). The contents of the total protein and extracellular polymer substances decreased by more than 28% (1 ppm) and 75% (10 ppm) because of the cleavage reaction caused by formaldehyde. Bacterial community analysis showed that the proportion of Geobacter decreased from 81% to 53% (1 ppm) and 24% (10 ppm). As a result, the current production was significantly impaired, and the irreversible loss increased. Toxicological analysis demonstrated that formaldehyde disturbed the physiological indices of cells, thereby inducing apoptosis. These findings fill the gap of ecotoxicology of toxicants to EAB in a bioelectrochemical system. Keywords: Bioelectrochemical systems; Electroactive biofilm; Formaldehyde; Ecotoxicity; Irreversible loss 2
1. Introduction Bioelectrochemical systems (BESs), as a green technology, have been widely studied in the fields of wastewater treatment and soil remediation (He et al., 2016; Li et al., 2016c). BES can directly convert waste or organic matter into electricity (Luo et al., 2018), hydrogen (Lu et al., 2015), or methane (Xiao et al., 2014) through electron transfer. Based on the direct feedback of bacterial activity with changes in current, BES has been demonstrated as a sensitive online monitoring biotoxicity sensor for early warning of water quality (Li et al., 2018). As the core part of BES, electroactive biofilms (EABs) are often regarded as the sensing element that responds to various toxicants, such as heavy metals, toxic volatile organic compounds, and pesticides (Prévoteau and Rabaey, 2017). Multiple optimized strategies are applied to promote the performance of EAB-based sensors to meet the requirement of water monitoring (Jiang et al., 2018). For example, Zhang et al. (Zhang et al., 2013) improved the stability of baseline in BES through the adjustment of the electrode material. Shen et al. (Shen et al., 2013) optimized the electrode structure and flow distribution to decrease the detection limit. Du et al. (Du et al., 2017) used polydopamine to act as the shell to protect EAB from extreme environment. As an online-monitoring sensor, many studies focus on how EAB-based sensors respond to toxicants and the aspects that can be improved. No studies have discussed the ecotoxicology of toxicants to EABs, which play an important role in degrading contaminants (Guo et al., 2017). Toxicants can cause the irreversible loss of EAB (Li et al., 2016a) and obstruct the performance of EAB-based sensor when applied in the field of water monitoring. 3
Different toxicants possess varied irreversible losses, and one toxicant also shows different irreversible losses in different EABs with different concentrations (Prévoteau and Rabaey, 2017) mainly because of the structure and composition of EABs, such as bacterial community and extracellular polymer substance (EPS). Formaldehyde has attracted great attention because of threats to ecological security when it enters the ecosystem. As such, monitoring formaldehyde in water is necessary. Formaldehyde, as a model toxicant, has been widely used to obtain electrical signal feedback in EAB-based sensors and lead to different irreversible losses because of the differentiation of EAB (Jiang et al., 2017; Wang et al., 2013). Previous studies reported that the irreversible loss increased with increasing formaldehyde concentration in the same sensor; moreover, irreversible loss caused by the same concentration of formaldehyde was different (Dávila et al., 2011; Wang et al., 2013; Yang et al., 2015). The sensor may be sufficient to obtain electrical signal feedback from formaldehyde. Considering the ecotoxicology of formaldehyde, the mechanism through which formaldehyde damages the EAB and the changes in EAB after exposure to formaldehyde remain unknown. In this study, formaldehyde was used as the indicator to fill the gap of ecotoxicology of toxicants to EAB. EABs were acclimated, and the electrochemical activities before and after formaldehyde injection were investigated through chronoamperometry and cyclic voltammetry (CV) analyses. The biofilm morphology, community characters, and EPS composition before and after formaldehyde injection were also evaluated by multiple biological methods. Furthermore, toxicological 4
analysis was utilized to assess the damage degree of EAB after injection with different concentrations of formaldehyde. 2. Experimental Section 2.1 Construction and operation of the BES Three electrode reactors were constructed to acclimate the EABs by using previously described methods (Zhou et al., 2017). The reactor consisted of a cylindrical glass container (diameter = 5 cm, height = 4 cm, and effective volume = 80 mL) and a polytetrafluoroethylene lid. The plain glassy carbon with an effective area of 0.19 cm2 was used as the working electrode. The counter electrode was made of platinum plate with an effective area of 1 cm2. Ag/AgCl with 4.0 M KCl was used as the reference electrode. All the electrodes were purchased from Aida Hengsheng Technology Co., Ltd., Tianjin, China. The positions of these electrodes are shown in supporting information. The reactors were connected to an eight-channel potentiostat (CHI 1000C, CH Instrument, Shanghai, China) at 0 V versus Ag/AgCl. The anode biofilms were inoculated from the medium with 25% (v/v) microbial fuel cells operated 3 years and 75% (v/v) 50 mM phosphate buffer solution (PBS: 4.576 g/L Na2HPO4, 2.132 g/L NaH2PO4, 0.31 g/L NH4Cl, and 0.13 g/L KCl), as described in previous studies (Du et al., 2018). The carbon source was acetate (1 g/L). The medium contained 5 mL/L vitamin solution and 12.5 mL/L trace mineral solution. Before inoculation, the medium was continuously bubbled with CO2/N2 (1/4, v/v) for 15 min to remove dissolved oxygen. The reactors were operated at 30 °C. 2.2 Electrochemical analyses 5
Currents were recorded every 100 s by using chronoamperometry, and a stable current was observed once the EAB was successfully accumulated on the anode. The CV was used to investigate the electrochemical activity of the EAB before and after formaldehyde injection. The potential window was −0.6 V to 0.2 V with a scan rate of 1 mV/s. Two cycles were measured each time, and the second cycle was used for CV plots. The first-order derivative of CVs were derived from CVs to analyze whether formaldehyde affects the redox species or the electron transfer pathways in EAB. 2.3 Biofilm topography and community analysis Scanning electron microscopy (SEM, Shimadzu SS-550, Japan) was employed to observe EAB before and after formaldehyde injection. The samples were pretreated by 2.5% glutaraldehyde solution for 4 h and washed by ethyl alcohol with different concentration gradients. The spatial live/dead topography of the biofilms before and after formaldehyde injection was obtained using a confocal laser scanning microscope (CLSM, Zeiss, LSM880, Germany). The samples were stained with a LIVE/DEAD BacLight Bacterial Viability Kit (L13152, ThermoFisher Scientific Inc., U.S.) according to standard protocols. Photo method was conducted as in a previous study (Li et al., 2016a). Autofluorescence of cytochrome C in the biofilm was also imaged by CLSM with excitation at 400 nm and emission at 435–485 nm (Esteve-Núñez et al., 2008). Biomass (protein) on the anode was measured by a BCA protein quantification assay kit (Solarbio, Beijing). The biofilms before and after formaldehyde injection were scraped from the electrode by using a sterile blade. NaOH solution was not 6
utilized to dissolve the membrane prior to investigating the effect of formaldehyde on the biofilm. EPS was extracted following the method of cation exchange resin (CER) (Bo et al., 1996). The biofilms were stirred at 2 000 g for 2 h at 4 °C with CER. The total protein concentration of EPS was measured by BCA protein quantification assay kit. Polysaccharide was evaluated by anthrone–sulfuric acid colorimetry. DNA was extracted from biofilms with or without formaldehyde treatment by using the Soil Genomic DNA Kit (CW2091S, 132 ComWin Biotech 133 Co., Ltd., China) according to standard protocols (Li et al., 2016b). Amplicons were quantified and sequenced on the MiSeq Illumina-sequencing platform by Novogene (Beijing, China). All sequence datasets are available at NCBI/Sequence Read Archive under the study with BioProject accession number PRJNA590323. 2.4 Formaldehyde toxicology test After approximately 3 days of acclimation in eight parallel reactors, a concentrated formaldehyde solution (40%) was injected into the reactors to achieve a final concentration of 10 ppm (0.0010%) and 1 ppm (0.0001%). Current was continuously recorded. The reactors were divided into control, 1 ppm, and 10 ppm groups. After the current decreased to the minimum value, the inhibition mechanism of formaldehyde on electroactive bacteria was thoroughly investigated by the following methods. Superoxide dismutase (SOD) activity was determined using a corresponding assay kit (A001-2, Nanjing Jiancheng Bioengineering Institute, China) to determine the relationship between formaldehyde and cell damage in EABs. The assay was 7
conducted
according
to
the
manufacturer’s
instructions,
and
a
UV–vis
spectrophotometer (TU-1900, Beijing Purkinje General Instrument, China) was used to read absorbance at 550 nm. Lactate dehydrogenase (LDH) assay kit (Wanleibio Co., Ltd. China) was used to evaluate the degree of cell damage in the biofilm after formaldehyde injection. The assay was conducted according to the manufacturer’s instructions. Absorbance was recorded at 450 nm by microplate reader (SPARK 10M, TECAN, Switzerland). Adenosine triphosphate (ATP) assay kit (Beijing Solarbio Science & Technology CO., Ltd) was used to measure the content of NADPH before and after formaldehyde injection to investigate the metabolic activity of EABs under the effect of formaldehyde. NADPH had a linear relationship to ATP. The manufacturer’s instructions of the assay kit were followed. Absorbance was recorded at 340 nm by using the UV–vis spectrophotometer. The inner active oxygen level of the biofilm was measured by reactive oxygen species (ROS) assay kit (Wanleibio Co., Ltd. China). 2ʹ,7ʹ-Dichlorodihydrofluorescein diacetate (DCFH-DA) was used as a fluorescence probe to measure intracellular ROS. The biofilm was scraped from the electrode and centrifuged three times. The biofilm was incubated with 10 µmol/L DCFH-DA in the dark at 25 °C for 30 min. Fluorescence intensity was measured using a microplate reader (SPARK 10M, TECAN, Switzerland) with an excitation wavelength of 502 nm and an emission wavelength of 530 nm. The relative ROS level was presented as the fluorescence intensity ratio of the formaldehyde groups to the control group. 2.5 Statistical analyses 8
Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference (HSD). The HSD results were reported only if the one-way ANOVA showed a significant effect (Srinivasan and Butler, 2017). 3. Results and discussion 3.1 Current decline after formaldehyde injection After 4.7 days of growth without substrate exchange, all reactors reached a stable current plateau (8.9 ± 0.2 A/m2; Fig. 1 and S2). These baselines remained stable for more than 5 h, and the current densities indicated that the EAB was well acclimated. Approximately no current drop was observed after the injection of PBS (Fig. S2). However, immediate current drop was observed in each reactor immediately after separate injection of the two concentrations of formaldehyde. The results showed a typical bacterial toxic response to 10 ppm (Fig. 1) and 1 ppm (Fig. S2) formaldehyde. Formaldehyde at 1 and 10 ppm induced current drops of 2.4 and 8.7 A/m2 after injection. This result indicated that the current density was inhibited completely, and EAB gave up resistance when 10 ppm formaldehyde was injected. The instantaneous current decline ratio (ICDR) was characterized by the slope of current density and time. The ICDR of 10 ppm formaldehyde was 16 mA/m2s, which was 7.2 times of 1 ppm formaldehyde (2.2 mA/m2s). Hence, the toxicity of formaldehyde on EAB increased with increasing exposure concentration to bacteria. The recovery current was produced after exchange solution. The unrecoverable current loss (Ucl) of 10 ppm was 7.2 A/m2, and another current plateau was obtained after 13 h. The recovery 9
current reached the initial current, indicating that EAB did not sustain irreparable damage when exposed to 1 ppm formaldehyde. On the contrary, a higher concentration (10 ppm) induced irreversible influence on EAB, which may damage EAB structure and cause microbial community change. Hence, the response mechanism of EAB to formaldehyde and some suggestions for improvement on EAB-based sensor were mentioned in this paper. Redox property changes of EAB influenced by formaldehyde were revealed by turnover CVs (Fig. 2). Before formaldehyde addition, CVs with the same sigmoidal waveform reached the same limiting current density of 8.9 A/m2 (Fig. 2 and S3). The limiting current density decreased by 95% for formaldehyde concentration of 10 ppm, and the curve switched to nonturnover CV (Fig. 2 insert figure). Nonturnover CV in EAB can be explained by the substrate depletion or the existence of respiratory inhibitors, which caused the redox enzyme to be in a transient state corresponding with the potential positions (Marsili et al., 2008). In this study, the substrate was sufficient to maintain EAB growth, and formaldehyde could act as the respiratory inhibitor when influenced on the animal as previous study mentioned (Bansal et al., 2011). Thus, the results of nonturnover CV may be due to inhibition of respiration by formaldehyde. However, the recovery current in CV did not reach the former current when the formaldehyde was removed, which exhibited the same trend shown in Fig. 1. Furthermore, the redox window decreased 50% in peak height and 44% in peak width after formaldehyde worked on the biofilm according to the derivative of current 10
density (Fig. S3). Hence, formaldehyde was a hypertoxic factor, which led to protein inactivation in the biofilm and bacteria structure destruction or even death. 3.2 Morphological assessments corresponding with current profiles After accumulating bacteria on the anode, SEM images were prepared before and after 1 ppm formaldehyde injection. The EAB developed well on the anode surrounded with EPS (Fig. 3A). The bacteria in EAB maintained a complete and healthy state with cell division process (Fig. 3B). However, the EAB structure was destroyed by 1 ppm formaldehyde. The EPS decreased under the influence of formaldehyde, and bacteria on the anode broke down (Fig. 3C and 3D). EPS was considered as the protective umbrella of bacteria (Sheng et al., 2010); as such, the decrease in EPS indicates that EAB is attacked by formaldehyde and that the bacteria exposed to formaldehyde then cleave. The changes in EAB finally influenced the output of current density (Fig. 1). Spatial images in Fig. 4A showed that EAB was colonized on the anode surface after 5 days of acclimation, where green and red were considered as living and dead bacteria, respectively, based on the difference in their cell membrane permeability (Sun et al., 2015). The surface of the control group was dominated by green (Fig. 4A), showing that nearly all bacteria acclimated in the biofilm were active before formaldehyde addition. The number of dead cells increased with increasing formaldehyde concentration (Fig. 4B and 4C), which gradually invaded the biofilm 11
toward the bacteria–electrode interface. For example, dead cells (40%) mixed with live cells (60%) (color turned to yellow, data shown in Fig. S4) appeared on the anode for 1 ppm formaldehyde treatment as a partial inactivation; thus, the current recovered immediately (Fig. S2). However, dead cells nearly occupied the whole anode (91%) for 10 ppm formaldehyde injection, resulting in an unrecoverable damage, corresponding to an unrecoverable current loss (Ucl) >70% (Fig. 1). Formaldehyde at 10 ppm clearly decreased the biomass of the anode (Fig. 4C), which could be explained by the decrease in EPS and detached cells from EAB. The convalescent EAB was changed from live/dead state (Fig. 4B) to live state with a loss of biomass (Fig. 4D) after removing the 1 ppm formaldehyde. These results exhibited similar trend to that of current (Fig. 1 and S2). The shape of EAB in control was a whole membrane when it was scraped with a sterile knife (Fig. S5), but the shape turned into tiny fragments after 1 ppm formaldehyde injection, thereby indicating that formaldehyde cleaved the EAB structure. The convalescent EAB turned to a whole membrane again when formaldehyde was removed. This result confirmed the ability of self-healing in EAB along with current recovery. Previous studies reported the importance of biomass in supporting the current output, and EPS acted as an agglomerant during EAB formation (Li et al., 2017; Sheng et al., 2010). In the current study, formaldehyde inhibited current because of the decreased EPS and the destruction of bacteria. 3.3 Biological changes after formaldehyde injection 12
Given that the EAB on the anode was confirmed to be exposed to formaldehyde, the biological changes were dominant reasons for the response. The EAB without formaldehyde contained 225 ± 15 µg/mL protein, which is 55% higher than that (145 ± 14 µg/mL) of EAB influenced by 1 ppm formaldehyde and 436% higher than that (42 ± 7 µg/mL) of EAB influenced by 10 ppm formaldehyde (Table. S1). The convalescent EAB from 1 ppm formaldehyde reached 199 ± 15 µg/mL of protein with current recovery, but 10 ppm formaldehyde inhibited protein recovery from the low level. Compared with 10 ppm irreversible damage to EAB, 1 ppm only caused a small amount of protein loss. When low concentration (1 ppm) formaldehyde was removed from EAB, the biomass and activity of protein were restored, which was in keeping with current changes. Previous study reported that EPS was involved in extracellular electron transfer by C-type cytochrome and electron medium (Sheng et al., 2010). Therefore, the formaldehyde may change EPS result in a pool long-distance electron transfer in EAB. Total EPS was 177 ± 13 µg/mL in control EAB, which was 1.4 times of EAB reacted by 1 ppm (128 ± 17 µg/mL) and 4.0 times of EAB reacted by 10 ppm (44 ± 8 µg/mL) (Table. S2). The extracellular protein decreased from 57 ± 12 µg/mL in control to 32 ± 10 and 10 ± 3 µg/mL handled by 1 and 10 ppm, respectively. These changes were related to the concentration of formaldehyde and corresponded to the current changes. However, the recovery extracellular polysaccharide influenced by 1 ppm (110 ± 17 µg/mL) was similar to that (120 ± 15 µg/mL) in the control, which was higher than that (34 ± 12 µg/mL) influenced by 10 ppm. This result may be due to the damage of the EAB structure under 10 ppm (Fig. 4C). Low concentration 13
formaldehyde (1 ppm) did not cause irreversible structural damage, which resulted in biomass and EPS reconstruction with negligible loss. By contrast, the high concentration formaldehyde (10 ppm) destroyed EAB. The contents of protein and EPS provided solid evidence that the current decline caused by different concentrations formaldehyde was attributed to the damage of EAB. Microbial community was further analyzed (Fig. 5) to assess whether formaldehyde influences bacteria. Illumina sequencing revealed that the formaldehyde influenced the EAB microbial community. The phylogenetic analysis showed a high bacterial diversity in different biofilm samples, with Geobacter accounting for the highest population (Fig. 5). The proportion of Geobacter decreased from 81% in the control to 53% and 24% after formaldehyde treatment, showing that formaldehyde inhibited the growth of exoelectrogens, such as Geobacter, in a mixed EAB. The decrease in the number of exoelectrogens resulted in current decline. The injection of formaldehyde at 1 and 10 ppm increased the Stenotrophomonas abundance from 5% (control) to 20% and 36%, respectively.
Previous studies reported that
Stenotrophomonas had great adaptability and showed strong vitality after resistance selection (Oviasogie and Igbinosa, 2014). The increase in the number of Stenotrophomonas indicated that EAB was retained in a cruel environment because of the toxicity of formaldehyde. Formaldehyde inhibited the electroactivity of Geobacter and reconstructed EAB with a higher proportion of other bacteria from 19% (control) to 47% (1 ppm) and 76% (10 ppm), resulting in different unrecoverable current losses. For bioelectrochemical sensors, tolerance of EAB was a key indicator to evaluate the 14
performance. When the high concentration toxicant broke the tolerance, unrecoverable influence would against the application of bioelectrochemical sensors. Therefore, the toxicity of formaldehyde to EAB should be further analyzed in terms of intracellular changes. 3.4 Physiological indices changes after formaldehyde injection Intracellular changes reflect the damage status of EAB under different concentrations of formaldehyde. The evaluation of oxidative stress was conducive to optimize the bioelectrochemical sensor and provide data support on the damage of EAB. As shown in Fig. 6, the generation of ROS level was increased by 26% and 125% after the injection of formaldehyde with 1 ppm and 10 ppm, respectively, compared with the control. The increase in the ROS level removed the balance between ROS and antioxidant, resulting in stress response (Brinkman et al., 2016). This result proved the destruction of the structure of EAB suffering formaldehyde. SOD is a critical antioxidant enzyme that maintains redox balance (Hu et al., 2014). No significant differences were observed in SOD activity between control and 1 ppm, but 10 ppm was 1.6 times of the control. The upregulation of SOD levels was consistent with the increase in ROS from exposure to formaldehyde. A high level of oxidative stress is suggested to influence the redox reactions (Hu et al., 2015), thereby resulting in the decline of current density. Formaldehyde can be used to fix cells with other reagents because of its ability to dissolve lipid substances in the membrane (Takatori et al., 2014). As presented in Fig. 6, the LDH influenced by 1 and 10 ppm formaldehyde 15
with were 1.8- and 6.0 times of control. This result indicated that EAB was destroyed by formaldehyde, and the extent of the damage was increased with increasing formaldehyde concentration (Fig. 3). The devastated EAB needed longer time to complete self-repair when damaged by 10 ppm than 1 ppm, which could be explained by the process of current recovery. Moreover, the large area destruction by 10 ppm formaldehyde might induce the reconstruction of microbial community, leading to the low level of recovery current. The change in the ATP level further showed the influence of formaldehyde on organelle after breaking through cell membrane. As displayed in data, ATP level was increased by 34% after 1 ppm formaldehyde and 92% after 10 ppm formaldehyde than the control, thereby indicating that formaldehyde destroyed the balance between ATP and ADP. The toxicity of formaldehyde to EAB increased with concentration, and finally EAB suffered tremendous damage from the organelles to the membrane with the formaldehyde ranging from 1 ppm to 10 ppm. Hence, concentration control of formaldehyde is crucial to the performance protection of bioelectrochemical sensor. Furthermore, this work provides a foundation to promote the tolerance of EAB to extreme environment under the notion that the performance is unaffected. 4. Conclusions Formaldehyde with different concentrations brought significant influence on EAB, including the inhibition of current, destruction of morphology, change in microbial community, and upregulation of physiological indices. As a model toxicant, 16
formaldehyde with 1 ppm and 10 ppm decreased by 27% and 98% of current, respectively. The various current losses under different concentrations of formaldehyde reflected the damage degree of the EAB, which was confirmed by the results of CV and biofilm viability, including protein content. The content of dominant phylum Proteobacteria was found to be closely related to the EAB performance. The number of Geobacter family decreased from 81% to 53% and 24% after the influence of 1 ppm and 10 ppm formaldehyde, respectively. The toxicity of formaldehyde deeply disturbed the physiological index of EAB with the increase of ROS, SOD, LDH, and ATP. In terms of EAB-based sensors, our findings showed the inhibition mechanism of formaldehyde on EAB and contributed by establishing the relationship between BES and ecotoxicology of other contaminants. Acknowledgments This research work was financially supported by National Natural Science Foundation of China (No. 21577068, 21677080 and 21876090), the Tianjin Research Program of Application Foundation and Advanced Technology (18JCZDJC39400 and S19ZC60133), the Postdoctoral Science Foundation of China (2019M660985), the Fundamental Research Funds for the Central Universities, and the 111 Program of the Ministry of Education of China (T2017002). Appendix A. Supplementary data Supplementary data to this article can be found online at References Bansal, N., et al., 2011. Toxic effect of formaldehyde on the respiratory organs of rabbits: A light and electron microscopic study. Toxicol Ind Health. 27, 563-569. 17
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Figure Captions Fig. 1. Current density before and after 10 ppm formaldehyde addition. Fig. 2. CVs before and after 10 ppm formaldehyde addition. Fig. 3. SEM images of EAB in control (A and B) and under 1 ppm formaldehyde (C and D). No EAB was formed under 10 ppm formaldehyde. Fig. 4. CLSM images of EABs in control (A), under 1 ppm formaldehyde (B), under 10 ppm formaldehyde (C) and recovery from 1ppm formaldehyde (D). Fig. 5. Taxonomic classification of bacterial DNA sequences from communities of EAB at the genus level. Fig. 6. Oxidative effects in EABs exposed to different concentration formaldehyde.* Significant difference (p < 0.05) compared to the control group.
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Figures
Fig. 1
Fig. 2 21
Fig. 3
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Fig. 4
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Fig. 5
Fig. 6
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Highlights 1. The ecotoxicology of formaldehyde caused irreversible loss on EAB. 2. Morphology and structure of EAB were damaged by formaldehyde. 3. Formaldehyde decreased the proportion of Geobactor. 4. The physiological indexes of cell on EAB was distrubed by formaldehyde.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
S0013-9351(20)30035-9
DOI:
https://doi.org/10.1016/j.envres.2020.109143
Reference:
YENRS 109143
To appear in:
Environmental Research
Received Date: 8 November 2019 Revised Date:
31 December 2019
Accepted Date: 14 January 2020
Please cite this article as: Li, T., Chen, F., Zhou, Q., Wang, X., Liao, C., Zhou, L., Wan, L., An, J., Wan, Y., Li, N., Unignorable toxicity of formaldehyde on electroactive bacteria in bioelectrochemical systems, Environmental Research (2020), doi: https://doi.org/10.1016/j.envres.2020.109143. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Inc.
Graphical abstract
Date:
Nov 8, 2019
Submitted to:
Environmental Research
Unignorable Toxicity of Formaldehyde on Electroactive Bacteria in Bioelectrochemical Systems Tian Li 1, Fan Chen 1, Qixing Zhou1*, Xin Wang 1, Chengmei Liao 1, Lean Zhou 1, Lili Wan 1, Jingkun An 2, Yuxuan Wan 1, and Nan Li 2 1
MOE Key Laboratory of Pollution Processes and Environmental Criteria / Tianjin Key Laboratory of Environmental Remediation and Pollution Control / College of Environmental Science and Engineering, Nankai University, No. 38 Tongyan Road, Jinnan District, Tianjin 300350, China
2
Tianjin Key Lab of Indoor Air Environmental Quality Control, School of Environmental Science and Engineering, Tianjin University, No. 92 Weijin Road, Nankai District, Tianjin 300072, China Corresponding Author: Phone: (86)22-58890402; fax: (86)22-23501117; E-mail:
[email protected]
1
Abstract Formaldehyde poses significant threats to the ecosystem and is widely used as a toxicity indicator to obtain electrical signal feedback in electroactive biofilm (EAB)-based sensors. Although many optimizations have been adopted to improve the performance of EAB to formaldehyde, nearly no studies have discussed the toxicity of formaldehyde to EAB. Here, EABs were acclimated with a stable current density (8.9 ± 0.2 A/m2) and then injected with formaldehyde. The current density decreased by 27% and 98% after the injection of 1 and 10 ppm formaldehyde, respectively, compared with that in the control. The ecotoxicity of formaldehyde caused the irreversible loss of current with 3% (1 ppm) and 81% (10 ppm). Confocal laser scanning microscopy and scanning electron microscopy results showed that the redox activity was inhibited by formaldehyde, and the number of dead/broken cells increased from 2% to 40% (1 ppm) and 91% (10 ppm). The contents of the total protein and extracellular polymer substances decreased by more than 28% (1 ppm) and 75% (10 ppm) because of the cleavage reaction caused by formaldehyde. Bacterial community analysis showed that the proportion of Geobacter decreased from 81% to 53% (1 ppm) and 24% (10 ppm). As a result, the current production was significantly impaired, and the irreversible loss increased. Toxicological analysis demonstrated that formaldehyde disturbed the physiological indices of cells, thereby inducing apoptosis. These findings fill the gap of ecotoxicology of toxicants to EAB in a bioelectrochemical system. Keywords: Bioelectrochemical systems; Electroactive biofilm; Formaldehyde; Ecotoxicity; Irreversible loss 2
1. Introduction Bioelectrochemical systems (BESs), as a green technology, have been widely studied in the fields of wastewater treatment and soil remediation (He et al., 2016; Li et al., 2016c). BES can directly convert waste or organic matter into electricity (Luo et al., 2018), hydrogen (Lu et al., 2015), or methane (Xiao et al., 2014) through electron transfer. Based on the direct feedback of bacterial activity with changes in current, BES has been demonstrated as a sensitive online monitoring biotoxicity sensor for early warning of water quality (Li et al., 2018). As the core part of BES, electroactive biofilms (EABs) are often regarded as the sensing element that responds to various toxicants, such as heavy metals, toxic volatile organic compounds, and pesticides (Prévoteau and Rabaey, 2017). Multiple optimized strategies are applied to promote the performance of EAB-based sensors to meet the requirement of water monitoring (Jiang et al., 2018). For example, Zhang et al. (Zhang et al., 2013) improved the stability of baseline in BES through the adjustment of the electrode material. Shen et al. (Shen et al., 2013) optimized the electrode structure and flow distribution to decrease the detection limit. Du et al. (Du et al., 2017) used polydopamine to act as the shell to protect EAB from extreme environment. As an online-monitoring sensor, many studies focus on how EAB-based sensors respond to toxicants and the aspects that can be improved. No studies have discussed the ecotoxicology of toxicants to EABs, which play an important role in degrading contaminants (Guo et al., 2017). Toxicants can cause the irreversible loss of EAB (Li et al., 2016a) and obstruct the performance of EAB-based sensor when applied in the field of water monitoring. 3
Different toxicants possess varied irreversible losses, and one toxicant also shows different irreversible losses in different EABs with different concentrations (Prévoteau and Rabaey, 2017) mainly because of the structure and composition of EABs, such as bacterial community and extracellular polymer substance (EPS). Formaldehyde has attracted great attention because of threats to ecological security when it enters the ecosystem. As such, monitoring formaldehyde in water is necessary. Formaldehyde, as a model toxicant, has been widely used to obtain electrical signal feedback in EAB-based sensors and lead to different irreversible losses because of the differentiation of EAB (Jiang et al., 2017; Wang et al., 2013). Previous studies reported that the irreversible loss increased with increasing formaldehyde concentration in the same sensor; moreover, irreversible loss caused by the same concentration of formaldehyde was different (Dávila et al., 2011; Wang et al., 2013; Yang et al., 2015). The sensor may be sufficient to obtain electrical signal feedback from formaldehyde. Considering the ecotoxicology of formaldehyde, the mechanism through which formaldehyde damages the EAB and the changes in EAB after exposure to formaldehyde remain unknown. In this study, formaldehyde was used as the indicator to fill the gap of ecotoxicology of toxicants to EAB. EABs were acclimated, and the electrochemical activities before and after formaldehyde injection were investigated through chronoamperometry and cyclic voltammetry (CV) analyses. The biofilm morphology, community characters, and EPS composition before and after formaldehyde injection were also evaluated by multiple biological methods. Furthermore, toxicological 4
analysis was utilized to assess the damage degree of EAB after injection with different concentrations of formaldehyde. 2. Experimental Section 2.1 Construction and operation of the BES Three electrode reactors were constructed to acclimate the EABs by using previously described methods (Zhou et al., 2017). The reactor consisted of a cylindrical glass container (diameter = 5 cm, height = 4 cm, and effective volume = 80 mL) and a polytetrafluoroethylene lid. The plain glassy carbon with an effective area of 0.19 cm2 was used as the working electrode. The counter electrode was made of platinum plate with an effective area of 1 cm2. Ag/AgCl with 4.0 M KCl was used as the reference electrode. All the electrodes were purchased from Aida Hengsheng Technology Co., Ltd., Tianjin, China. The positions of these electrodes are shown in supporting information. The reactors were connected to an eight-channel potentiostat (CHI 1000C, CH Instrument, Shanghai, China) at 0 V versus Ag/AgCl. The anode biofilms were inoculated from the medium with 25% (v/v) microbial fuel cells operated 3 years and 75% (v/v) 50 mM phosphate buffer solution (PBS: 4.576 g/L Na2HPO4, 2.132 g/L NaH2PO4, 0.31 g/L NH4Cl, and 0.13 g/L KCl), as described in previous studies (Du et al., 2018). The carbon source was acetate (1 g/L). The medium contained 5 mL/L vitamin solution and 12.5 mL/L trace mineral solution. Before inoculation, the medium was continuously bubbled with CO2/N2 (1/4, v/v) for 15 min to remove dissolved oxygen. The reactors were operated at 30 °C. 2.2 Electrochemical analyses 5
Currents were recorded every 100 s by using chronoamperometry, and a stable current was observed once the EAB was successfully accumulated on the anode. The CV was used to investigate the electrochemical activity of the EAB before and after formaldehyde injection. The potential window was −0.6 V to 0.2 V with a scan rate of 1 mV/s. Two cycles were measured each time, and the second cycle was used for CV plots. The first-order derivative of CVs were derived from CVs to analyze whether formaldehyde affects the redox species or the electron transfer pathways in EAB. 2.3 Biofilm topography and community analysis Scanning electron microscopy (SEM, Shimadzu SS-550, Japan) was employed to observe EAB before and after formaldehyde injection. The samples were pretreated by 2.5% glutaraldehyde solution for 4 h and washed by ethyl alcohol with different concentration gradients. The spatial live/dead topography of the biofilms before and after formaldehyde injection was obtained using a confocal laser scanning microscope (CLSM, Zeiss, LSM880, Germany). The samples were stained with a LIVE/DEAD BacLight Bacterial Viability Kit (L13152, ThermoFisher Scientific Inc., U.S.) according to standard protocols. Photo method was conducted as in a previous study (Li et al., 2016a). Autofluorescence of cytochrome C in the biofilm was also imaged by CLSM with excitation at 400 nm and emission at 435–485 nm (Esteve-Núñez et al., 2008). Biomass (protein) on the anode was measured by a BCA protein quantification assay kit (Solarbio, Beijing). The biofilms before and after formaldehyde injection were scraped from the electrode by using a sterile blade. NaOH solution was not 6
utilized to dissolve the membrane prior to investigating the effect of formaldehyde on the biofilm. EPS was extracted following the method of cation exchange resin (CER) (Bo et al., 1996). The biofilms were stirred at 2 000 g for 2 h at 4 °C with CER. The total protein concentration of EPS was measured by BCA protein quantification assay kit. Polysaccharide was evaluated by anthrone–sulfuric acid colorimetry. DNA was extracted from biofilms with or without formaldehyde treatment by using the Soil Genomic DNA Kit (CW2091S, 132 ComWin Biotech 133 Co., Ltd., China) according to standard protocols (Li et al., 2016b). Amplicons were quantified and sequenced on the MiSeq Illumina-sequencing platform by Novogene (Beijing, China). All sequence datasets are available at NCBI/Sequence Read Archive under the study with BioProject accession number PRJNA590323. 2.4 Formaldehyde toxicology test After approximately 3 days of acclimation in eight parallel reactors, a concentrated formaldehyde solution (40%) was injected into the reactors to achieve a final concentration of 10 ppm (0.0010%) and 1 ppm (0.0001%). Current was continuously recorded. The reactors were divided into control, 1 ppm, and 10 ppm groups. After the current decreased to the minimum value, the inhibition mechanism of formaldehyde on electroactive bacteria was thoroughly investigated by the following methods. Superoxide dismutase (SOD) activity was determined using a corresponding assay kit (A001-2, Nanjing Jiancheng Bioengineering Institute, China) to determine the relationship between formaldehyde and cell damage in EABs. The assay was 7
conducted
according
to
the
manufacturer’s
instructions,
and
a
UV–vis
spectrophotometer (TU-1900, Beijing Purkinje General Instrument, China) was used to read absorbance at 550 nm. Lactate dehydrogenase (LDH) assay kit (Wanleibio Co., Ltd. China) was used to evaluate the degree of cell damage in the biofilm after formaldehyde injection. The assay was conducted according to the manufacturer’s instructions. Absorbance was recorded at 450 nm by microplate reader (SPARK 10M, TECAN, Switzerland). Adenosine triphosphate (ATP) assay kit (Beijing Solarbio Science & Technology CO., Ltd) was used to measure the content of NADPH before and after formaldehyde injection to investigate the metabolic activity of EABs under the effect of formaldehyde. NADPH had a linear relationship to ATP. The manufacturer’s instructions of the assay kit were followed. Absorbance was recorded at 340 nm by using the UV–vis spectrophotometer. The inner active oxygen level of the biofilm was measured by reactive oxygen species (ROS) assay kit (Wanleibio Co., Ltd. China). 2ʹ,7ʹ-Dichlorodihydrofluorescein diacetate (DCFH-DA) was used as a fluorescence probe to measure intracellular ROS. The biofilm was scraped from the electrode and centrifuged three times. The biofilm was incubated with 10 µmol/L DCFH-DA in the dark at 25 °C for 30 min. Fluorescence intensity was measured using a microplate reader (SPARK 10M, TECAN, Switzerland) with an excitation wavelength of 502 nm and an emission wavelength of 530 nm. The relative ROS level was presented as the fluorescence intensity ratio of the formaldehyde groups to the control group. 2.5 Statistical analyses 8
Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference (HSD). The HSD results were reported only if the one-way ANOVA showed a significant effect (Srinivasan and Butler, 2017). 3. Results and discussion 3.1 Current decline after formaldehyde injection After 4.7 days of growth without substrate exchange, all reactors reached a stable current plateau (8.9 ± 0.2 A/m2; Fig. 1 and S2). These baselines remained stable for more than 5 h, and the current densities indicated that the EAB was well acclimated. Approximately no current drop was observed after the injection of PBS (Fig. S2). However, immediate current drop was observed in each reactor immediately after separate injection of the two concentrations of formaldehyde. The results showed a typical bacterial toxic response to 10 ppm (Fig. 1) and 1 ppm (Fig. S2) formaldehyde. Formaldehyde at 1 and 10 ppm induced current drops of 2.4 and 8.7 A/m2 after injection. This result indicated that the current density was inhibited completely, and EAB gave up resistance when 10 ppm formaldehyde was injected. The instantaneous current decline ratio (ICDR) was characterized by the slope of current density and time. The ICDR of 10 ppm formaldehyde was 16 mA/m2s, which was 7.2 times of 1 ppm formaldehyde (2.2 mA/m2s). Hence, the toxicity of formaldehyde on EAB increased with increasing exposure concentration to bacteria. The recovery current was produced after exchange solution. The unrecoverable current loss (Ucl) of 10 ppm was 7.2 A/m2, and another current plateau was obtained after 13 h. The recovery 9
current reached the initial current, indicating that EAB did not sustain irreparable damage when exposed to 1 ppm formaldehyde. On the contrary, a higher concentration (10 ppm) induced irreversible influence on EAB, which may damage EAB structure and cause microbial community change. Hence, the response mechanism of EAB to formaldehyde and some suggestions for improvement on EAB-based sensor were mentioned in this paper.
density (Fig. S3). Hence, formaldehyde was a hypertoxic factor, which led to protein inactivation in the biofilm and bacteria structure destruction or even death.
toward the bacteria–electrode interface. For example, dead cells (40%) mixed with live cells (60%) (color turned to yellow, data shown in Fig. S4) appeared on the anode for 1 ppm formaldehyde treatment as a partial inactivation; thus, the current recovered immediately (Fig. S2). However, dead cells nearly occupied the whole anode (91%) for 10 ppm formaldehyde injection, resulting in an unrecoverable damage, corresponding to an unrecoverable current loss (Ucl) >70% (Fig. 1). Formaldehyde at 10 ppm clearly decreased the biomass of the anode (Fig. 4C), which could be explained by the decrease in EPS and detached cells from EAB. The convalescent EAB was changed from live/dead state (Fig. 4B) to live state with a loss of biomass (Fig. 4D) after removing the 1 ppm formaldehyde. These results exhibited similar trend to that of current (Fig. 1 and S2). The shape of EAB in control was a whole membrane when it was scraped with a sterile knife (Fig. S5), but the shape turned into tiny fragments after 1 ppm formaldehyde injection, thereby indicating that formaldehyde cleaved the EAB structure. The convalescent EAB turned to a whole membrane again when formaldehyde was removed. This result confirmed the ability of self-healing in EAB along with current recovery. Previous studies reported the importance of biomass in supporting the current output, and EPS acted as an agglomerant during EAB formation (Li et al., 2017; Sheng et al., 2010). In the current study, formaldehyde inhibited current because of the decreased EPS and the destruction of bacteria.
Given that the EAB on the anode was confirmed to be exposed to formaldehyde, the biological changes were dominant reasons for the response. The EAB without formaldehyde contained 225 ± 15 µg/mL protein, which is 55% higher than that (145 ± 14 µg/mL) of EAB influenced by 1 ppm formaldehyde and 436% higher than that (42 ± 7 µg/mL) of EAB influenced by 10 ppm formaldehyde (Table. S1). The convalescent EAB from 1 ppm formaldehyde reached 199 ± 15 µg/mL of protein with current recovery, but 10 ppm formaldehyde inhibited protein recovery from the low level. Compared with 10 ppm irreversible damage to EAB, 1 ppm only caused a small amount of protein loss. When low concentration (1 ppm) formaldehyde was removed from EAB, the biomass and activity of protein were restored, which was in keeping with current changes. Previous study reported that EPS was involved in extracellular electron transfer by C-type cytochrome and electron medium (Sheng et al., 2010). Therefore, the formaldehyde may change EPS result in a pool long-distance electron transfer in EAB. Total EPS was 177 ± 13 µg/mL in control EAB, which was 1.4 times of EAB reacted by 1 ppm (128 ± 17 µg/mL) and 4.0 times of EAB reacted by 10 ppm (44 ± 8 µg/mL) (Table. S2). The extracellular protein decreased from 57 ± 12 µg/mL in control to 32 ± 10 and 10 ± 3 µg/mL handled by 1 and 10 ppm, respectively. These changes were related to the concentration of formaldehyde and corresponded to the current changes. However, the recovery extracellular polysaccharide influenced by 1 ppm (110 ± 17 µg/mL) was similar to that (120 ± 15 µg/mL) in the control, which was higher than that (34 ± 12 µg/mL) influenced by 10 ppm. This result may be due to the damage of the EAB structure under 10 ppm (Fig. 4C). Low concentration 13
formaldehyde (1 ppm) did not cause irreversible structural damage, which resulted in biomass and EPS reconstruction with negligible loss. By contrast, the high concentration formaldehyde (10 ppm) destroyed EAB. The contents of protein and EPS provided solid evidence that the current decline caused by different concentrations formaldehyde was attributed to the damage of EAB. Microbial community was further analyzed (Fig. 5) to assess whether formaldehyde influences bacteria. Illumina sequencing revealed that the formaldehyde influenced the EAB microbial community. The phylogenetic analysis showed a high bacterial diversity in different biofilm samples, with Geobacter accounting for the highest population (Fig. 5). The proportion of Geobacter decreased from 81% in the control to 53% and 24% after formaldehyde treatment, showing that formaldehyde inhibited the growth of exoelectrogens, such as Geobacter, in a mixed EAB. The decrease in the number of exoelectrogens resulted in current decline. The injection of formaldehyde at 1 and 10 ppm increased the Stenotrophomonas abundance from 5% (control) to 20% and 36%, respectively.
Previous studies reported that
Stenotrophomonas had great adaptability and showed strong vitality after resistance selection (Oviasogie and Igbinosa, 2014). The increase in the number of Stenotrophomonas indicated that EAB was retained in a cruel environment because of the toxicity of formaldehyde. Formaldehyde inhibited the electroactivity of Geobacter and reconstructed EAB with a higher proportion of other bacteria from 19% (control) to 47% (1 ppm) and 76% (10 ppm), resulting in different unrecoverable current losses. For bioelectrochemical sensors, tolerance of EAB was a key indicator to evaluate the 14
performance. When the high concentration toxicant broke the tolerance, unrecoverable influence would against the application of bioelectrochemical sensors. Therefore, the toxicity of formaldehyde to EAB should be further analyzed in terms of intracellular changes.
with were 1.8- and 6.0 times of control. This result indicated that EAB was destroyed by formaldehyde, and the extent of the damage was increased with increasing formaldehyde concentration (Fig. 3). The devastated EAB needed longer time to complete self-repair when damaged by 10 ppm than 1 ppm, which could be explained by the process of current recovery. Moreover, the large area destruction by 10 ppm formaldehyde might induce the reconstruction of microbial community, leading to the low level of recovery current. The change in the ATP level further showed the influence of formaldehyde on organelle after breaking through cell membrane. As displayed in data, ATP level was increased by 34% after 1 ppm formaldehyde and 92% after 10 ppm formaldehyde than the control, thereby indicating that formaldehyde destroyed the balance between ATP and ADP. The toxicity of formaldehyde to EAB increased with concentration, and finally EAB suffered tremendous damage from the organelles to the membrane with the formaldehyde ranging from 1 ppm to 10 ppm. Hence, concentration control of formaldehyde is crucial to the performance protection of bioelectrochemical sensor. Furthermore, this work provides a foundation to promote the tolerance of EAB to extreme environment under the notion that the performance is unaffected.
formaldehyde with 1 ppm and 10 ppm decreased by 27% and 98% of current, respectively. The various current losses under different concentrations of formaldehyde reflected the damage degree of the EAB, which was confirmed by the results of CV and biofilm viability, including protein content. The content of dominant phylum Proteobacteria was found to be closely related to the EAB performance. The number of Geobacter family decreased from 81% to 53% and 24% after the influence of 1 ppm and 10 ppm formaldehyde, respectively. The toxicity of formaldehyde deeply disturbed the physiological index of EAB with the increase of ROS, SOD, LDH, and ATP. In terms of EAB-based sensors, our findings showed the inhibition mechanism of formaldehyde on EAB and contributed by establishing the relationship between BES and ecotoxicology of other contaminants. Acknowledgments This research work was financially supported by National Natural Science Foundation of China (No. 21577068, 21677080 and 21876090), the Tianjin Research Program of Application Foundation and Advanced Technology (18JCZDJC39400 and S19ZC60133), the Postdoctoral Science Foundation of China (2019M660985), the Fundamental Research Funds for the Central Universities, and the 111 Program of the Ministry of Education of China (T2017002). Appendix A. Supplementary data Supplementary data to this article can be found online at References Bansal, N., et al., 2011. Toxic effect of formaldehyde on the respiratory organs of rabbits: A light and electron microscopic study. Toxicol Ind Health. 27, 563-569. 17
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Figure Captions Fig. 1. Current density before and after 10 ppm formaldehyde addition. Fig. 2. CVs before and after 10 ppm formaldehyde addition. Fig. 3. SEM images of EAB in control (A and B) and under 1 ppm formaldehyde (C and D). No EAB was formed under 10 ppm formaldehyde. Fig. 4. CLSM images of EABs in control (A), under 1 ppm formaldehyde (B), under 10 ppm formaldehyde (C) and recovery from 1ppm formaldehyde (D). Fig. 5. Taxonomic classification of bacterial DNA sequences from communities of EAB at the genus level. Fig. 6. Oxidative effects in EABs exposed to different concentration formaldehyde.* Significant difference (p < 0.05) compared to the control group.
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Figures
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Fig. 3
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Fig. 5
Fig. 6
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Highlights 1. The ecotoxicology of formaldehyde caused irreversible loss on EAB. 2. Morphology and structure of EAB were damaged by formaldehyde. 3. Formaldehyde decreased the proportion of Geobactor. 4. The physiological indexes of cell on EAB was distrubed by formaldehyde.
Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: