https://orcid.org/0009-0000-8260-6476
Abstract
A two-chamber microbial fuel cell (MFC) with algal-film cathode was constructed. It showed good electric-generating performance with three electric-generating stages: start-up, development, and stable. An average output voltage reached ~0.412 V during the stable period. A maximum power density during continuous operation was 19.76 mW/m2. Bacterial samples were collected from the anode in the three stages (A1, A2, and A3), and their community structure and diversity were analyzed using Illumina MiSeq high-throughput sequencing technology. A total of 4238 operational taxonomic units were identified based on the number of taxa. At the phylum level, Proteobacteria and Bacteroidetes played a dominant role in the three stages and increased significantly during electricity generation. Compared with A1, the relative abundances of Proteobacteria in A2 and A3 increased by 23.30% and 32.06%, respectively, whereas those of Bacteroidetes in A2 and A3 increased by 5.56% and 14.50%, respectively. At the genus level, there were differences in the composition of bacterial communities among the three stages. Acinetobacter and Chlorobium became the dominant genera in A2, replacing Nitrospira and norank_f__Saprospiraceae in A1, and Sphingobacterium and Ochrobactrum became the dominant genera in A3. According to the sample cluster and principal component analyses, A1 was clustered into one class, and A2 and A3 were clustered into a second class. This work revealed bacterial community succession at the anode of an algal-film cathode MFC during the electricity generation process, which provides a theoretical basis for the subsequent promotion of electricity generation by algal-film cathode MFCs.
1. Introduction
Microbial fuel cells (MFCs) convert chemical energy into electrical energy by degrading organic matter and using microorganisms enriched in the anodes as catalysts [1]. MFCs have been widely studied in various countries as a new type of clean energy and owing to their advantages, such as high-energy conversion efficiency, synchronous wastewater treatment, and electric energy recovery [2]. A commonly used configuration is a two-chamber MFC reactor. Electrogenic bacteria oxidize and decompose organic matter in the anaerobic environment of the anode and produce electrons and protons, which are transferred to the cathode via an external circuit and a proton exchange membrane (PEM), respectively, and then interact with electron acceptors, such as O2, nitrate, permanganate, and other reduction reactions in the aerobic environment of the cathode [3]. MFC cathodes often use O2 as an electron acceptor, which requires continuous aeration during operation and incurs high operating costs [4].
Microalgae are rich in chlorophyl a, which can absorb nitrogen, phosphorus, and organic matter from wastewater and use CO2 released by the respiration of electrogenic bacteria for photosynthesis; nitrogen and phosphorus are converted into biomass, and O2 produced by microalgae can be used as an electron acceptor. This forms a self-sustainable system that reduces operating costs by using the oxygen produced by algae photosynthesis instead of aeration for the cathode [5]. An algae-cathode MFC with an appropriate hydraulic retention time can continuously generate electricity and simultaneously remove nutrients from real wastewater [6]. The application of microalgae in MFCs and the establishment of a bacterial-algal symbiotic system with electrogenic bacteria for material exchange can improve the tolerance of MFCs to the external environment, lower the cost of wastewater treatment, and have considerable future application prospects [7]. Anaerobic bacteria at the anode play an important role in the biodegradation and power generation of MFCs, and microbial activity can significantly affect the electricity generation performance of an MFC [8].
In our previous research, Oedogonium sp. exhibited high-performance nutrient removal efficiency from digested piggery wastewater compared to six other microalgae species [9]. Additionally, a method to create an algal film cathode using Oedogonium sp. was developed to enhance algae adhesion [10]. In this study, an MFC with the algal-film cathode (AC-MFC) was constructed. The electricity generation performance of the AC-MFC using synthetic biogas slurry as a substrate is discussed. The structure and diversity of the anode biota at different electric-generating stages were analyzed using high-throughput sequencing technology, and electricigen selection and biota characteristics of the AC-MFC were revealed.
2. Materials and methods
2.1 AC-MFC device
The AC-MFC device (Fig. 1) has a cylindrical two-chamber structure, and the cathode and anode chambers are made of plexiglass separated by a PEM. The volumes of the bottom anode and top cathode were 0.25 L. A graphite rod with a surface area of 24.3 cm2 was used as the anode to collect electrons. The algal-film cathode was prepared according to a patented method [10], and the inoculant algal seed was obtained from the Freshwater Microalgae Culture Collection of the Institute of Hydrobiology (FACHB Collection), Chinese Academy of Sciences, China. The cathode and anode electrodes were connected with titanium wire and an external 1 kΩ resistance forming a closed loop. The mixed bacteria for the MFC anode inoculation were obtained from the anaerobic tank of the Changsha Sewage Treatment Plant, and the recovered sludge was stored in an anaerobic sealed container. After precipitation, the supernatant was removed and the anaerobic sludge was inoculated onto the MFC anode at a volume ratio of 30%. The test temperature was maintained at 25°C with a 12-h photoperiod in the incubator. A biological light lamp was placed on the cathode side of the MFC, and the light intensity was controlled at 5500 lx.
Schematic diagram of AC-MFC reaction device.
2.2 Test scheme
Synthetic biogas slurry was pumped into the cathode as fuel, the composition of which was the same as that described in a previous work [11]. The fuel hydraulic retention time was 24 h, the anode effluent was reused at the cathode, and the cathode effluent was discharged directly from the top outlet of the AC-MFC. The output voltage data of the AC-MFC were collected in real time using an online monitoring system. The anode biota was collected at day 0 and every 2 days while the cell was in operation, and each sample included three parallel samples. After centrifugation, the supernatant was discarded, and the pellet collected and precipitated in 2 ml centrifuge tubes and stored in a freezer at −80°C.
2.3 Data collection and index measurement
2.3.1 Data acquisition system
The voltage data were collected under the external 1 kΩ resistance in real time, and the current density (C, mA m−2) calculated as follows:
where R is the external resistance, Ω; Ac is the anode surface area, m2, and I is the current, mA.
2.3.2 Illumina MiSeq sequencing
High-throughput sequencing of the 16S rRNA gene of the microbial community was performed by Shanghai Meiji Biomedical Technology Co., Ltd. Total DNA from the microbial communities was extracted by 1% agarose gel electrophoresis. The concentration and purity of the DNA were determined using a NanoDrop2000. The primers 27F (5ʹ-AGAGTTTGATCCTGGCTCAG-3ʹ) and 533R (5ʹ-TTACCGCGGCTGCTGGCAC-3ʹ) were used to amplify the variable region of bacterial 16S rRNA gene V1–V3. The PCR instrument used was an ABI GeneAmp® 9700. Sequencing was performed using Illumina’s MiSeq PE300 platform once the target band size was correct and the concentration appropriate.
3. Results and analysis
3.1 AC-MFC electricity generation performance
The output voltage characteristics of the MFC under continuous operation are shown in Fig. 2. During the first 10 h, the maximum output voltage of the battery was only 0.01 V. In continuous-operation experiments, microorganisms can continuously use substrates for electricity generation. The output voltage increased to 0.178 V on the third day and stayed in the range of 0.165–0.223 V for the next 3 days. The output voltage increased rapidly on the seventh day and the average output voltage was ~0.412 V during the stable period. The maximum power density during continuous operation was 19.76 mW/m2. The reported two-chamber MFC with an algal cathode uses domestic sewage, aniline wastewater, pharmaceutical wastewater, landfill leachate, and aquaculture wastewater as substrates, with a maximum output voltage of 0.254–0.756 V and a maximum power density of 3.07–838.68 mW/m2 [12–15]. More bioelectricity was obtained from constructed wetlands-microbial fuel cells (CW-MFC) with anodic biofilm using Juncus effusus root exudates as endogenous substrates compared to using Philodendron cordatum macrophytes [16]. Further, MFCs with an inoculum of anaerobic granular sludge showed a 10% greater coulombic efficiency compared to those with an inoculum of stream sediments [17]. This suggests that substrates and inoculum might be important factors affecting the electricity generation performance of MFCs. Additionally, improved electrochemical performance was obtained in a CW-MFC by increasing the oxygen reduction reaction of the cathodes through surface modification [18, 19]. An increase in oxygenation by algae in this study could improve the speed of oxygen reduction and the cathodic potential. Therefore, under the experimental conditions of this study, the AC-MFC had good electricity generation performance with three clear stages: start-up (first 10 h), development (2–6 days), and stable (day 7 onward) with increasing electricity generation. Environmental factors affecting the electricity generation performance, such as temperature, pH, dissolved oxygen concentration, and hydraulic retention time [20], will be researched in further AC-MFC studies.
Voltage (A) and power generation performance (B) of AC-MFC.
3.2 Alpha diversity of bacterial community
To clarify the characteristics of the changes in the anode bacterial community in the three stages, microbial samples collected on days 0, 4, and 10, designated A1, A2, and A3, respectively, were analyzed by Illumina MiSeq sequencing. The alpha diversity of the bacterial communities is shown in Table 1. Relevant indices can be used to estimate the species abundance and diversity of the environmental community. The coverage indices of the three samples were 99.95%, 99.88%, and 99.84%. Therefore, the test results effectively reflected the genuine information regarding the microbial species in the samples. The ACE and Chao1 indices represent the abundance of microbial communities; the higher the ACE and Chao1 values, the higher the abundance [21]. Compared with A1, the ACE and Chao1 indices of A2 decreased slightly, whereas the ACE and Chao1 indices of A3 increased by 10.01% and 7.93%, respectively, indicating that the abundance of the anode microbial community increased during the stable period. The Shannon and Simpson indices indicate microbial community diversity. The greater the Shannon index and the lower the Simpson index, the higher the microbial community diversity [14]. Throughout the electricity generation stage, the Shannon index showed an evident decrease initially and then a slight increase, whereas the Simpson index showed the opposite trend, indicating that the diversity of the anode microbial community in the middle and later stages of electricity generation was significantly lower than that of the initial sludge.
Analysis of alpha diversity index of microbial samples.
| Sample | Shannon | Simpson | Ace | Chao | Coverage |
|---|---|---|---|---|---|
| A1 | 4.5224 | 0.0220 | 430.5397 | 426.9767 | 0.9995 |
| A2 | 3.6584 | 0.0737 | 427.8137 | 415.4800 | 0.9988 |
| A3 | 3.7478 | 0.0629 | 473.6642 | 460.8630 | 0.9984 |
| Sample | Shannon | Simpson | Ace | Chao | Coverage |
|---|---|---|---|---|---|
| A1 | 4.5224 | 0.0220 | 430.5397 | 426.9767 | 0.9995 |
| A2 | 3.6584 | 0.0737 | 427.8137 | 415.4800 | 0.9988 |
| A3 | 3.7478 | 0.0629 | 473.6642 | 460.8630 | 0.9984 |
Analysis of alpha diversity index of microbial samples.
| Sample | Shannon | Simpson | Ace | Chao | Coverage |
|---|---|---|---|---|---|
| A1 | 4.5224 | 0.0220 | 430.5397 | 426.9767 | 0.9995 |
| A2 | 3.6584 | 0.0737 | 427.8137 | 415.4800 | 0.9988 |
| A3 | 3.7478 | 0.0629 | 473.6642 | 460.8630 | 0.9984 |
| Sample | Shannon | Simpson | Ace | Chao | Coverage |
|---|---|---|---|---|---|
| A1 | 4.5224 | 0.0220 | 430.5397 | 426.9767 | 0.9995 |
| A2 | 3.6584 | 0.0737 | 427.8137 | 415.4800 | 0.9988 |
| A3 | 3.7478 | 0.0629 | 473.6642 | 460.8630 | 0.9984 |
A rarefaction curve was constructed using the microbial alpha diversity index of each sample at different sequencing depths to reflect the microbial diversity of each sample at different sequencing quantities. The rarefaction curves of the bacteria at different stages of electricity generation in the AC-MFC are shown in Fig. 3A. According to the analysis of the observed species index, when the sequencing depth was less than 20 000, the diversity index increased significantly with the increasing number of sequences; however, when the number of sequences was between 20 000 and 40 000, the diversity index increased slowly. The curve was flat, indicating that the amount of sequencing data was reasonable.
Rarefaction curve of sobs (A) and Venn diagram of bacterial distribution (B) in the anode of AC-MFC in three stages.
3.3 Analysis of bacterial community distribution
The effective sequence numbers (reads) obtained from the three samples were clustered at 97.0% similarity to obtain operational taxonomic units (OTUs). A total of 4238 OTUs were obtained from samples A1–A3, which assigned to 1266 genera. As shown in Fig. 3B, samples A1, A2, and A3 contained 423, 404, and 439 genera, respectively, among which 263 genera overlapped, accounting for 43.76% of the bacteria on genus level, indicating high similarity of bacteria at different stages of electricity generation. In addition, samples A2 and A3 had 64 genera that overlapped, which was higher than the overlap of A1 and A2 and that of A1 and A3. The unique genus numbers for A1, A2, and A3 were 85, 38, and 76, respectively. The maximum proportion of unique genus was 14.14% (A1), and the lowest proportion was 6.32% after 4 days of operation of the AC-MFC (A2). The proportion then increased to 12.65% (A3), indicating that the anodic bacterial community structure and species underwent substantial changes under the experimental conditions. The abundance of the bacterial community first decreased and then increased, which was consistent with the results of the alpha diversity analysis.
3.4 Analysis of bacterial community composition
3.4.1 Analysis of bacterial community composition at the phylum level
Changes in the bacterial community structure at the phylum level during electricity generation are shown in Fig. 4. Five bacterial phyla had relative abundances >5%: Proteobacteria, Bacteroidetes, Chloroflexi, Firmicutes, and Patescibacteria. Proteobacteria and Bacteroidota were the dominant bacteria in the electricity generation process, and their relative abundance increased considerably during the process, accounting for 16.18%–48.24% and 18.54%–33.04%, respectively. Proteobacteria and Bacteroidota have been reported as the dominant phyla in many MFC studies and are some of the most common denitrifying phyla; many denitrifying and methyl-oxidizing bacteria belong to these two phyla [22, 23]. Proteobacteria are electroactive bacteria involved in electron transfer to electrodes. Most use flagella to attach to the electrodes, and the generated electrons are transferred to the electrode via the flagella [24]. The enrichment of Proteobacteria plays a decisive role in improving the electricity generation capacity of AC-MFCs. Bacteroidota are anaerobic and can be coupled with methane-oxidizing bacteria to convert methane into short-chain fatty acids [25]. The abundance of Bacteroidota increases considerably, which can promote the removal of nitrate and sulfate and the dissociation of methane.
Bacterial community composition at the phylum level in the anode of AC-MFC in three stages.
Chloroflexi widely exist in anaerobic environments, such as the seabed, lakes, and soil, and their main role is to degrade aromatic compounds in the environment through their own metabolism [26]. In addition, Chloroflexi and Actinobacteria are involved in the degradation of complex organic matter in MFCs supplied with sewage sludge [27]. Firmicutes are Gram-positive bacteria with good electrochemical activity through direct electron transfer [28].
Firmicutes are highly efficient denitrifying bacteria that catalyze the denitrification process under anaerobic conditions, degrade complex organic matter into simple organic matter, and perform sulfate reduction under the action of sulfate-reducing bacteria [29]. Patescibacteria is a supergroup proposed in 2018 [30]. Few studies have been conducted on Patescibacteria, which often coexist with denitrifying bacteria in groundwater and play an important role in nitrogen removal [31].
3.4.2 Analysis of bacterial community composition at the genus level
As shown in Fig. 5, the bacterial communities of compositions A1, A2, and A3 differed significantly at the genus level. Compared with A1, the relative abundances of Chlorobium and Ochrobactrum increased in A2 and A3, accounting for 13.55% and 1.29% in A2 and 8.04% and 12.4% in A3, respectively. Chlorobium are photoenergetic, inorganic, and autotrophic bacteria that often grow in environments with light and no oxygen. Chlorobium can use H2S to perform photosynthesis without releasing oxygen to fix nitrogen and sulfur, to remove nitrogen without destroying the anoxic environment of the water body [32]. Chlorobium can also utilize reduced sulfur compounds as electron donors to a photosynthetic electron transport chain that provides energy and reduced ferredoxin to drive carbon fixation, biosynthesis, and cell growth [33]. Ochrobactrum was recently isolated from an MFC and have independent electron transport capabilities [34]. The relative abundance of Sphingobacterium increased to 0.06% and 16.6% in A2 and A3, respectively. Sphingobacterium has a special metabolic regulatory mechanism to adapt to nutrient-poor environments and resists many adverse environmental changes by adjusting their growth [35]. Sphingobacterium can utilize a wide range of substrates, from polymers, such as petroleum and chloroethane, to simple inorganic substances, allowing them to grow and reproduce in an autotrophic environment [36].
Bacterial community composition at the genus level in the anode of AC-MFC in three stages.
3.4.3 Heatmap analysis of different generation stages based on genus level
A heatmap uses color gradients to represent the data size in two-dimensional matrices or tables and presents information about community species composition and species abundance. The variation in the abundance of different species in samples is shown through color block gradients. A heatmap of the AC-MFC at the three different stages is shown in Fig. 6. At the genus level, the top 5 bacteria in total abundance were Chlorobium, Acinetobacter, Sphingobacterium, Ochrobactrum, and norank _f__norank_o__Candidatus_Moran. The dominant bacterial genera in the three stages were Nitrospira, Acinetobacter, and Sphingobacterium, which accounted for 7.17%, 20.80%, and 16.58% in the three stages of electricity generation, respectively. The second most dominant bacterial genera in the three stages were also different, namely norank_f__Saprospiraceae, Chlorobium, and Ochrobactrum, accounting for 4.96%, 13.05%, and 12.45%, respectively. Differences are evident in the composition of bacterial communities at the different stages. Although some bacteria are similar, their contents differ. This result was similar to previous studies. The bacterial community in the anode changed substantially during MFC operation using synthetic or real wastewater as fuel [37, 38]. Microbial adaptation to the substrate had a notable effect on microbial community diversity and species richness in CW-MFC [39]. This may explain the composition changes of bacterial communities at different stages of the AC-MFC.
Community heatmap analysis on genus level.
3.5 Beta diversity of bacterial communities
To study the similarities or differences in the community structure of the MFCs at different stages, a sample-level cluster analysis was carried out on the community distance matrix at the three stages of electricity generation. As shown in Fig. 7A, compared with A1, the bacterial community structures of A2 and A3 are more similar, and the three stages of electricity generation can be clustered into two types: A2 and A3 are clustered into one type, and A1 is clustered into another type. The growth and metabolism of anode microorganisms exhibit significant changes during the process of electricity generation, resulting in differences in bacterial abundance.
Hierarchical clustering tree (A) and PCA (B) on genus level.
At the genus level, principal component analysis (PCA) was performed on the anode bacteria of the AC-MFC. The results of this analysis are shown in Fig. 7B. In PCA, the greater the distance between each point, the greater the difference between them. In contrast, the closer the sample points, the more similar the species compositions of the two samples. The distance between each point in the PCA analysis was large, indicating that the microbial community structure of each sample was different. Additionally, PC1 (66.47%) was much higher than PC2 (33.53%). On the PC1 axis, the distance between the A2 and A3 sample groups is smaller, and both have a larger distance from A1, indicating that there is a large difference in the bacterial community structure between the start-up stage and the electricity generation stage of the AC-MFC, which further supports the results of the sample-level cluster analysis.
4. Conclusion
In this study, an AC-MFC was constructed to process a biogas slurry and recover electrical energy. In the continuous-operation experiment, the AC-MFC exhibited good electric production performance, and its electric production cycle showed three typical stages: start-up, development, and a stable period. The average output voltage increased to ~0.412 V during the stable period, up from a maximum of 0.223 V during the development period. The maximum power density during continuous operation was 19.76 mW/m2.
The anodes in the three stages of MFC generation were analyzed using Illumina MiSeq sequencing, and 4238 OTUs were identified according to the number of taxa. At the phylum level, the relative abundance of Proteobacteria and Bacteroidetes increased with electricity generation and played a leading role. Genera-level analysis showed that there were some differences in the composition of the bacterial community at different stages of electricity generation. Although some bacteria had similar structures, their composition differed. Ochrobactrum and Sphingobacterium were dominant during the stable generation period. Compared to A1, the bacterial community structures of A2 and A3 were more similar. According to the sample-level cluster analysis, A2 and A3 were clustered into one type and A1 was clustered into another type.
The operation of the AC-MFC changed the structure of the sludge microbial community, increased the diversity of the microbial community, increased the abundance of dominant bacteria, changed the dominant bacterial genus, increased the number of specific functional bacteria, and improved electric production performance and electron transfer ability. This study reveals the dynamic succession of the anode bacterial community during electricity generation in an AC-MFC, providing a theoretical basis for the subsequent promotion of MFC electricity generation.
Acknowledgements
We gratefully acknowledge anonymous reviewers for their constructive comments and suggestions.
Author contributions
Haiping Wang (Data curation [lead], Formal analysis [lead], Funding acquisition [lead], Methodology [lead], Project administration [lead], Writing—original draft [lead], Writing—review & editing [lead]), Liguo Zheng (Data curation [equal], Methodology [equal], Writing—original draft [equal]), Changyin Tan (Data curation [equal], Methodology [equal], Writing—review & editing [equal]), Ling Li (Software [equal], Writing—review & editing [equal]), Feng Liu (Methodology [equal], Writing—review & editing [equal]) and Hui Feng (Funding acquisition [equal], Writing—review & editing [equal]).
Conflict of interest statement
None declared.
Funding
This study was supported by the Natural Science Foundation of Hunan Province (2022JJ60097 and 2022JJ60094), the Research Project of Education Department of Hunan Province (22C1420).
Data availability
The data underlying this article will be shared on reasonable request to the corresponding author.
