ABSTRACT
Hydrogen-entangled electron transfer has been verified as an important extracellular pathway of sharing reducing equivalents to regulate biofilm activities within a diversely anaerobic environment, especially in microbial electrosynthesis systems. However, with a lack of useful methods for in situ hydrogen detection in cathodic biofilms, the role of hydrogen involvement in electron transfer is still debatable. Here, a cathodic biofilm was constructed in CH4-produced microbial electrosynthesis reactors, in which the hydrogen evolution dynamic was analyzed to confirm the presence of hydrogen-associated electron transfer near the cathode within a micrometer scale. Fluorescent in situ hybridization images indicated that a colocalized community of archaea and bacteria developed within a 58.10-μm-thick biofilm at the cathode, suggesting that the hydrogen gradient detected by the microsensor was consumed by the collaboration of bacteria and archaea. Coupling of a microsensor and cyclic voltammetry test further provided semiquantitative results of the hydrogen-associated contribution to methane generation (around 21.20% ± 1.57% at a potential of −0.5 V to −0.69 V). This finding provides deep insight into the mechanism of electron transfer in biofilm on conductive materials.
IMPORTANCE Electron transfer from an electrode to biofilm is of great interest to the fields of microbial electrochemical technology, bioremediation, and methanogenesis. It has a promising potential application to boost more value-added products or pollutant degradation. Importantly, the ability of microbes to obtain electrons from electrodes and utilize them brings new insight into direct interspecies electron transfer during methanogenesis. Previous studies verified the direct pathway of electron transfer from the electrode to a pure-culture bacterium, but it was rarely reported how the methanogenic biofilm of mixed cultures shares electrons by a hydrogen-associated or hydrogen-free pathway. In the current study, a combination method of microsensor and cyclic voltammetry successfully semiquantified the role of hydrogen in electron transfer from an electrode to methanogenic biofilm.
INTRODUCTION
Conventionally, typical interspecies electron transfer was realized by hydrogen or formate acting as a carrier in an anaerobic digestion system, which is termed as interspecies hydrogen transfer or formate transfer in the methanogenic community (1). The hydrogen and formate were thermodynamically spontaneous (ΔG0′ = −1.3 kJ/mol) (2, 3), and the reactions were reversible under specific conditions. Commonly, the hydrogen is more important in suspended environments (4, 5) as interspecies hydrogen transfer is the basis of hydrogenotrophic methanogenesis in biogeochemical carbon recycling or artificial bioreactors and governs nearly ∼30% of methane emissions in these fields (6, 7).
In an electroactive bacterium-based microbial electrosynthesis system (MES) for methane production (8), a specific direct electron flow model has been found using physical contact with coculture of Geobacter and Methanosaeta or Methanosarcina (9, 10). Subsequently, a hydrogenase-independent mechanism was verified with a pure culture of Methanococcus maripaludis at the cathode (11). Moreover, the hydrogen molecule which is recovered on the cathode can also work as the electron transfer carrier for methanogens as well as other hydrogen scavengers (12–14). Presumably, hydrogen-mediated electron transfer could be essential to extend the biofilm growth of a mixed culture (15, 16). Overall, hydrogen could be a crucial mediator for electron transfer due to its being easily consumed by bacteria or methanogens with metabolic versatility (17). A clear distinctive classification for the electron transfer pathway could be constructed as either hydrogen associated or hydrogen free, which represents how hydrogen is deeply involved in electron transfer. The hydrogen-associated bacteria could be a representative of acetogens or methanogens, which can scavenge hydrogen as their electron donor (18–20). The hydrogen-free pathway was also shared by acetogens or methanogens, which has been verified by their ability to acquire electrons from the cathode. Electroactive bacteria, such as Geobacter or Shewanella, were capable of obtaining electrons from the cathode without hydrogen involvement (11, 21, 22).
To investigate the methanogenic biofilm on the cathode surface, most studies were performed with pure cultures to show evidence for a direct electron transfer pathway. There was still a “black box” with respect to electron transfer in a mixed-culture cathodic biofilm, especially with regard to the role of hydrogen. Multisyntrophic levels in cathodic biofilm communities would be involved in the coexistence of hydrogen-mediated and hydrogen-free electron transfer. To elucidate the role of hydrogen in cathodic biofilms, we designed a combined method using a hydrogen microsensor system (MS) and cyclic voltammetry (CV) to achieve in situ hydrogen detection within the cathodic biofilm. The hydrogen dynamic change was analyzed near the cathode surface within a micrometer scale, which further illustrated the role of hydrogen in hydrogenotrophic methanogenesis. A comprehensive matrix, including a hydrogen gradient, current signal, and electrochemical potential, was evaluated within the cathodic methanogenic community.
RESULTS AND DISCUSSION
In situ hydrogen concentration within the cathodic biofilm.The microsensor provides details of the hydrogen concentration near the surface of the cathode. All data were collected under the working status of an external voltage of 0.80 V. The cathodic potential stayed around a value of −0.544 V (versus the reversible hydrogen electrode [RHE]) with minor variation, which favored hydrogen generation. The hydrogen concentration increased from ∼200 μmol/liter to ∼640 μmol/liter from a specific distance (∼1 mm) to the surface of the electrode (Fig. 1). In this study, 639.91 μmol/liter is the highest hydrogen concentration detected on the cathode surface, and it decreased to 514.13 μmol/liter at 100 μm. Subsequently, a smooth decline also occurred at the distance range of 100 to 400 μm. A further hydrogen concentration drop was observed until a distance of 700 μm from the electrode was reached. A slight increase was observed at 800 μm but the concentration fell again at 900 μm.
Hydrogen concentration around the cathode.
The diffusion simulation results are shown in Fig. S1 in the supplemental material. The detected hydrogen concentration at 100 μm and 31 s was lower than that of simulated results in water and biofilm, indicating that hydrogen diffusion could be affected by the microbial activity within 100 μm. Subsequently, the thickness of the biofilm was determined as 58.10 μm using the side fluorescence intensity (Fig. 2 and Fig. S2), which agrees with the result of biofilm thickness in a previous study (23). The biofilm appeared to be responsible for the sharp decrease of hydrogen that occurred within 100 μm. Within this range, the distribution of bacteria and archaea moderately coincided, which was revealed by the value of the confocal Pearson’s correlation coefficient (Pearson’s Rr) (Fig. 2) (24). The phenomenon potentially implied that the hydrogen influenced both bacteria and archaea, which were important to accelerate methane production (25). As shown in Fig. 2f and Table S1, 16S rRNA gene sequencing data provide the microbial composition (bacteria and archaea) of the biocathode. The methanogens accounted for nearly 87.12% of the total microbial community abundance. The second-largest functional microbial group was protein/amino acid degrader/fermenter organisms, showing the presence of multisyntrophic levels in cathodic biofilm. Therefore, there could be two pathways for bacteria to interact with methanogens to produce methane at the cathode, including a fermentation-methanogen and an acetogen-methanogen model. The former should be the result of fermentative bacteria interacting with methanogens, which could be suggested by the presence of fermentative bacteria (∼6.43%). A previous study also emphasized that this cooperation could be important for boosting methane production, indicating that a nearly 3.8-fold CH4 production rate was contributed by enriching with Proteobacteria, exoelectrogens, and putative producers (26). The acetogen-methanogen model has also been confirmed by our previous study (19); another study also showed that a coculture of acetogens and methanogens enhanced the methane production rate at the cathode (27). However, the acetogens detected in the current study were relatively low in abundance (∼0.55%), suggesting that the acetogen-methanogen model could be a minor pathway. According to the sequencing result, the multisyntrophic levels could lead to diverse electron transfer pathways, including hydrogen-associated and hydrogen-free pathways. Although the detection of a hydrogen gradient confirmed the involvement of hydrogen in cathodic electron transfer, the contribution of hydrogen to the whole process is uncertain with a single microsensor used for detection.
Fluorescence images and sequencing result of the cathodic biofilm. (a to e) Fluorescence image (a) and fluorescence of Bacteria (b), Euryarchaea (c), and Methanobacteriaceae (d). (e) Pixel concentration image and quantitative coefficients calculated on the FISH images. (f) Functional classification of the microbial composition in a cathodic biofilm (others, sum of OTUs with abundance of ≤0.05%).
Hydrogen flow within the cathodic biofilm.The hydrogen variation and corresponding current were monitored under different cathodic potentials by an MS-CV test (Fig. 3). The abiotic cathode showed higher hydrogen concentration overall in the CV test than the biotic cathode, indicating that hydrogen was efficiently produced as electron acceptors in the absence of biofilm. The hydrogen concentration slowly increased when the cathode potential changed from −0.69 V to −0.16 V. The subsequent increase in potential led to a gradual decline in the detected hydrogen concentration. When the cathode potential exceeded +0.20 V, the hydrogen concentration fell sharply and approached the baseline level. The current of the abiotic cathode showed a significant change with the variation of potentials. Therefore, the main process here should be subject to Faraday’s law (28). A transient drop of current was visible at around +0.60 V before the potential became negative in abiotic biofilm, resulting in a low supply of hydrogen to the system. In contrast, the biotic cathode exhibited a stable hydrogen concentration around zero.
MS-CV results of the cathode without biofilm (abiotic condition) (a) and with the biofilm (biotic condition) (b). All data were detected at the zero position.
During the discharge process, the hydrogen concentration sharply increased from −0.15 V under the abiotic condition, representing the occurrence of hydrogen evolution. The absolute current value increased accordingly. In comparison, the biotic hydrogen concentration and the current value were unchanged from +0.69 V to −0.20 V. The phenomenon differed from results for the abiotic cathode. It was found that the hydrogen evolution current could stay at a lower value than that under the abiotic condition. The biotic hydrogen concentration initially increased at nearly −0.30 V of biocathode potential, which was lower than that under the abiotic condition. The onset hydrogen evolution potential was distinctly different, indicating a raised overpotential of hydrogen evolution for the biotic condition.
Cathodic electron transfer in cathodic biofilm remains controversial (29) even though direct electron transfer without hydrogenase has been proved with Methanococcus maripaludis (11). The typical electron transfer pathways via cathode that are analogous to the anodic process were proposed in a previous study, including cytochrome-mediated electron transfer and direct electron transfer by means of a cytochrome-hydrogenase partnership (21). Further research summarized the mechanisms of the direct and indirect pathways depending on the mediators (30). Direct electron transfer tended to rely on the presence of cytochrome; however, only the methanogens of the Methanosarcinales order contain cytochromes in almost all methanogens (31). Hydrogen could be crucial for indirect electron transfer, considering the role of various hydrogenases in methanogenesis (32, 33). However, different electron transfer pathways were allowed in mixed consortia due to the high diversity of cathodic biofilms. Therefore, how hydrogen is involved in the electron transfer of cathodic biofilms could be debatable. Here, hydrogen was proved to be the mediator for methane generation in cathodic methanogen-dominated biofilms according to the results from the microsensor. The MS-CV result also confirmed that hydrogen would contribute to methane generation and that it is not the unique electron shuttle for cathodic biofilms. A stoichiometric analysis could be beneficial to determine the underlying the role of hydrogen in cathodic electrosynthesis.
Estimated contribution of the hydrogen-associated pathway.The comparison between abiotic and biotic standardized hydrogen concentrations is shown in Fig. 4. The standardized hydrogen concentration increased along with the increase in potential from −0.2 to 0.69 V. The biotic value increased from ∼2.42 to ∼17.97 μmol/liter/mA. In contrast, the abiotic cathode increased from ∼9.83 to 71.61 μmol/liter/mA. The latter is significantly higher than the former value, suggesting that hydrogen production was been inhibited with the same current production. The inhibition could be due to the microbial activity of biofilm, which is absent under the abiotic condition. There was a clear difference in the increased rates of standardized hydrogen concentrations (Fig. 4). The abiotic value increased sharply with potentials from −0.2 V to −0.4 V, while the biotic value, in contrast, increase slowly and remained lower than the abiotic value at −0.2 V to −0.4 V; this was followed by a clear climb in the biotic value at potentials from −0.4 V to −0.5 V. The final stages of both biotic and abiotic standardized hydrogen production became stable at −0.5 V to −0.6 V. The increase in the biotic hydrogen concentration revealed that hydrogen production is sensitive at a potential lower than −0.4 V. The ratio of biotic to abiotic values, depicted in Fig. 4, first declined and then rapidly increased from ∼6.79% to ∼19.78%. Finally, the ratio was stabilized at around 22.35%. The calculated results of electron balance are shown in Fig. S3. The electrons provided from the current were proportionally increased as the potential declined. The hydrogen-associated electron transfer varied from ∼5% to ∼25% in the potential range of −0.20 V to −0.69 V, indicating that the major electron transfer was ascribed to the hydrogen-free process. The current increased at a potential of −0.40 V. The hydrogen-associated electron transfer also clearly increased from the −0.40 V point, and the average percentage was 21.20% ± 1.57%. Overall, the absolute value of the hydrogen contribution provided only a reference for evaluating the role of hydrogen in cathodic methanogenesis; i.e., the method is more useful for delivering a semiquantitative indicator for in situ detection of hydrogen.
The hydrogen concentration standardized by the current at a specific range of cathodic potentials.
Potential-dependent hydrogen production.The results of MS-CV showed the presence of hydrogen in cathodic methanogenesis, as well as implying the contribution of hydrogen to generate methane. Moreover, the hydrogen and methane yields were detected at different external voltages (1.00, 0.80, and 0.60 V); the cathodic potentials were accordingly −0.69, −0.54, and −0.39 V. The external voltages changed the cathodic potential, resulting in various methane and hydrogen yields. The average hydrogen yield was ∼63.60 ± 18.78 ml at 1.00 V. The values decreased to 4.78 ± 7.15 ml and 0.77 ± 0.96 ml for 0.80 V and 0.60 V, respectively. Little hydrogen was produced at a lower overpotential. The results were consistent with the linear regression of potentials and hydrogen production (Fig. S4). A greater negative potential intended to cause higher hydrogen yield with a higher standard deviation (SD) value. Moreover, the current results also showed a slight correlation with hydrogen yield (Fig. S5). It was noticeable that a higher negative potential (at −0.69 V) exhibited a stronger correlation than other values (R2 = 0.494), which could be due to a negative effect that could clearly cause hydrogen production (Fig. S4 and S6). Therefore, the current would convert to hydrogen at the higher potential. The electron ratio of molar methane to current exhibited an opposite trend to hydrogen yield (Fig. 5a). The average value was 0.29 ± 0.15 mol methane/mol electrons with an external voltage of 1.0 V (cathodic potential of −0.690 V), indicating that electron bifurcation to methane and hydrogen causes lower methane production. More electrons could flow into methane if the external voltage decreased to 0.8 V (cathodic potential of −0.544 V) and 0.6 V (cathodic potential of −0.391 V); the molar ratios were increased to 0.65 ± 0.21 and 0.69 ± 0.22 mol methane/mol electrons, respectively.
(a) Reactor performance in the molar ratio of methane to current. The boxes indicate 95% confidence intervals. (b) CV results of cathodic biofilm. (c) The dynamic of hydrogen concentration at the end of the CV scan along with cycle number. (d) The MS-CV result for cathodic biofilm.
Hydrogen redox peak and decay with biofilm.Further evidence was reflected by the CV test. In the first cycle, a clear peak was shown at the range of 0 V to +0.15 V. It was assumed to be the hydrogen oxidation reaction. The corresponding reaction could be described by the following equation (equation 1):
As shown in Fig. 5b to d, the abiotic hydrogen concentration was around 80 μmol/liter/mA at the end of the CV scan; a slight increase was found at several of the initial cycles. The biotic value was only half that of the abiotic state in the first and second cycles, and then the value declined to 20 μmol/liter/mA. The decay of hydrogen could be ascribed to the delay in activating the microbes that attached on the surface of the electrode. The electrochemical response should be more sensitive than the biofilm. Therefore, the hydrogen could be accumulated at initial cycles while being available to methanogens. Importantly, the hydrogen concentration was stable in the last three CV cycles, indicating that the capacity of methanogens to consume all of the hydrogen was limited.
The hydrogen decay proved the role of microbes in consuming hydrogen as well as revealing the potential activation time for microbes. Additionally, the ability of the microbes to utilize hydrogen as an electron donor was also influenced by the electrical stimulation, which can potentially affect the metabolic (34) and catabolic (35) activities. Therefore, the microbes consumed hydrogen only under negative potential rather than under the positive potential, which would be due to the thermodynamic barrier formed for hydrogen oxidizers in the latter case. Since hydrogen was present on the cathode, another limitation could also be related to the hydrogen partial pressure, which had been involved in microbial thermodynamic and kinetic principles. The hydrogen accumulation at the cathode would improve the hydrogen utilization threshold for methanogens, which would be selective for specific methanogens (33). In a previous study, Methanobacterium was found to be dominant in cathodic biofilm (36). However, few methanogens that were capable of directly exchanging electrons with the electrode were detected. The hydrogen could be the key to benefit methanogens with lower hydrogen thresholds (37). This could be proved further.
In this study, semiquantitative detection of hydrogen transfer within a cathodic biofilm was investigated. The results implied the significant role of hydrogen in electron transfer within a methanogenic biofilm in an MES. Hydrogen transfer is nonsensitive to physical distance, unlike a direct electron transfer pathway, which means that the enhanced hydrogen-mediated route can expand the ability of the electrode to shift the microbial community involved in anaerobic digestion. As the biomass retention on the electrode is important for methane generation, the hydrogen produced would change the microenvironment around the cathode, further contributing to the growth of microorganisms to improve the biomass near the cathode (38). The ratio of electrode surface area to the effective volume where biomass has been enhanced would lead to different conclusions regarding the importance of biofilm and bulk sludge (39). By understanding the role of hydrogen for electron transfer, more effective regulation strategies may be developed to enhance methane production for use in MES.
MATERIALS AND METHODS
Reactor construction for methane production.Six single-chamber microbial electrosynthesis reactors (microbial electrolysis cells) were built to achieve stable methane generation with a mature cathodic methanogenic community. All reactors were made of glass cylinders and had a total volume of 150 ml. A gas bag (200 ml; Delin, China) was connected on the top of each reactor to collect the produced gas. A carbon brush was employed as the anode to enrich exoelectrogens. A pretreatment method for the brush anode was applied to improve anode performance according to a previous report (40) in which the brush anode was immersed in acetone for 24 h and heated at 450°C for 30 min. A carbon cloth cathode was fabricated with a Pt/C catalyst for the formation of the methanogenic community, as described previously (19).
The inoculum was prepared at a ratio of 1:1 (vol/vol) by mixing waste activated sludge from a secondary settlement (Gaobeidian wastewater treatment plant in Beijing) and effluent from bioelectrochemistry reactors operated over a long time. The artificial influent was 50 mM phosphate-buffered saline (PBS) with 1.50 g/liter acetate, as well as minerals and a vitamin solution, as previously described (40). The reactors were operated in batch mode at room temperature (20 ± 5°C). All reactors were started up and operated for nearly 1 month with 0.80 V external voltage until they achieved stable performance of methane production (41). The current variation of the reactors, as provided in Fig. S8 in the supplemental material, showed that the cathodic biofilm had matured to generate methane in the replicates. For further detection and microbial sampling, three reactors were used to test CV, MS-CV, and the fluorescent in situ hybridization (FISH). The other three reactors were kept running over four batch cycles, and then the external voltage was successively changed to 1.00 V and 0.60 V. The values of 1.00 V and 0.60 V were selected to ensure that the cathodic potential was beyond or under the standard threshold of hydrogen evolution to evaluate whether hydrogen production was affected by the change in potential of the cathode.
Hydrogen detection and measurement in a cathodic biofilm.The gas collected from each reactor was analyzed by gas chromatography (A4000; East and West Analytical Instruments, Inc., China) with a thermal conductivity detector (TCD) to analyze the methane and hydrogen contents. The hydrogen concentration was detected by the microsensor system (Manual MicroProfiling System; Unisense A/S, Denmark). The molar ratios were calculated according to equations 2 and 3:
A hydrogen microelectrode (H2-10-709465; Unisense A/S, Denmark) was employed to detect the hydrogen in the interfacial solution (42). To obtain the transient hydrogen change in the biofilm, the microelectrode was pushed into the solution to approach the surface of biocathode until the electrode moved slightly , which ensured the physical contact between microprobe and electrode (Fig. S9). The contacted site was used to confirm the zero position (details are shown in the Results section). The microelectrode was manually operated to gradually change the position following the scale of the manual micromanipulator (MM33; PreSens). The whole process was protected by the N2 flow to exclude oxygen disturbance.
We used the one-dimensional diffusion equation of Fick’s law to calculate the hydrogen concentration at a specific time and position (43). The diffusion simulation was completed by Matlab software. The one-dimensional equation was as follows (equation 4):
The initial and boundary values are according to equations 5 and 6:
Electrochemical tests.The CV test was completed from the reactor which operated with an external voltage of 0.80 V. All electrochemical tests were completed with an electrochemical workstation (CHI660; CH Instruments, Inc., Austin, TX). The Ag/AgCl electrode was employed as a reference. All potentials in the context have been converted versus the potential of the RHE according to the following equation: E versus RHE = 0.059 × (pH + E versus Ag/AgCl) + 0.197 V (47). The scan range was set from −1.30 V to 0 V versus that of Ag/AgCl, which was equal to −0.69 V to 0.61 V of RHE. The scan rate was 2 mV/s (48).
Abiotic/biotic MS-CV detection.To reveal how the hydrogen dynamic was entangled with the potential change, the microsensor was used alongside the CV test (at zero position) to record the hydrogen on the surface of the cathode. The original reactor was biotic; for the abiotic situation, a new cathode without any biofilm was used to replace the original cathode with biofilm, ensuring an effective comparison between the absence and presence of biofilm. During the test, the microsensor was set to automatically record the data every 2 s, while the CV test was processed with a scan rate of 2 mV/s.
Probing biofilm.To probe biofilm composition in situ, three typical primers (5′-GCTGCCTCCCGTAGGAGT-3′, 5′-CACAGCGTTTACACCTAG-3′, and 5′-TACCGTCGTCCACTCCTTCCTC-3′) were employed to mark the Bacteria, Euryarchaeota, and Methanobacteriaceae. The samples of biofilm were taken from the reactors when they were performing under steady-state conditions. The samples were pretreated to fix the microbes and hybridized with specific fluorescence probes in a dark room as previously described (49). Subsequently, the prepared sample was observed by confocal microscopy (SP8; Leica, Germany). The images were processed with ImageJ software (version 1.52a [https://imagej.nih.gov/ij/]). Colonization was analyzed by the plug-in Colocalization Finder (https://imagej.nih.gov/ij/plugins/colocalization-finder.html) (24). Three biofilm samples were collected from three replicate reactors; a 515F-806R primer was used to amplify the original DNA, which was extracted from samples using a soil DNA kit (MP Biomedicals) as previously described (50). The sequencing was completed using an Illumina MiSeq platform, and the fastq files were subsequently submitted to the online Galaxy system (http://mem.rcees.ac.cn:8080/) (51). The Uparse program was used to generate operational taxonomic units (OTUs), and the Ribosomal Database Project (RDP; release 11.5) classifier was used to assign all OTUs to microbial taxa. To confirm the function of each taxon at the genus level, the Microbial Database for Activated Sludge (MiDAS) browser and previous studies were employed as references to identify the potential function of each genus (52). The comparative results are shown in Table S1. All visualizations were completed by R software.
Semiquantitative hydrogen contribution.All electrons that originated from the current are supposed to be converted into hydrogen under abiotic conditions. The hydrogen was detected once hydrogen was formed on the surface of the cathode because the microsensor directly contacted the electrode. According to the following equation (equation 7), the electron balance could be constructed:
As the reaction is reversible, the vforward (rate from proton and electron to hydrogen) and vbackward (rate from hydrogen to proton and electron) values could be related to the cathodic potential. The direction of the equation depends on the negative or positive state of the electrode, which determines the energy of the electrons. To eliminate the effect of vbackward on the electron balance, we chose the range from −0.20 V to −0.69 V to calculate the electron balance as the vforward value greatly exceeds that of vbackward. The hydrogen production will be predominant in this range.
The electron balance equation (equation 8) is as follows:
The overall balance in the whole CV test was also calculated by equation 6 (within the range of −0.69 V to +0.69 V and from +0.69 V to −0.69 V). The analysis of the electron balance can be equilibrated under the assumption that all electrons were transferred to hydrogen. Thereby, the only unknown factor, hequivalent, can be calculated from the abiotic balance.
There was a little difference in the electron balance equation for the biotic cathode. The presence of biofilm expanded the potential electron fluxes; the equation can be expressed as follows (equation 9):
Although the microbes were capable of consuming hydrogen to influence the amount of hydrogen detected, the physical contact can capture hydrogen as soon as possible to avoid the consumption of hydrogen by the microbes, which would decrease the amount detected; i.e., the hydrogen can be detected once it is produced from the surface of the cathode. Another negative effect is that the electrode potential was consecutive while the hydrogen detection was discrete; i.e., the detection of the microsensor was achieved over an interval of time while the CV test made the potential change in a fairly short time interval. The time lag could make an impact on the hydrogen concentration as the microbes would instantly consume the hydrogen. Hence, the time interval for each detection point was set to 2 s to eliminate this disadvantage. The unknown factor could be the hequivalent, which could be predicted by the abiotic equation (equation 8). Therefore, the portion of hydrogen that contributed to the total electron transfer could be obtained through analyzing the balance. To achieve the goal of a semiquantitative result for the hydrogen contribution in the current study, we therefore used a comparison between the biotic and abiotic methods to show the difference. The hydrogen concentration was standardized by the current value at specific potentials. Due to the constant interval of potentials in a fixed time interval, there is no need to standardize time to evaluate the comparison.
Data availability.All raw sequencing data have been deposited in the National Center for Biotechnology Information (NCBI) under BioProject accession number PRJNA629292.
ACKNOWLEDGMENTS
This research was supported by National Natural Science Foundation of China (grants 51908030 and 51778607) and Beijing Outstanding Young Scientist Program (BJJWZYJH01201910004016).
We declare that we have no competing financial interests.
FOOTNOTES
- Received 6 May 2020.
- Accepted 13 June 2020.
- Accepted manuscript posted online 19 June 2020.
Supplemental material is available online only.
- Copyright © 2020 American Society for Microbiology.
