3.1 Results
3.1.1 The effect of mixing NH3-N and NO3-N under anoxic conditions
To establish reference points, we examined the concentration of pH, NH3-N, NO3-N, and TSS before the commencement of the experiment, and the results are shown in Fig. 2a. In the first batch (batch A), we measured the concentration of NH3-N and NO3-N in the anodic bulk solution daily, and the results showed a concurrent removal of the nutrients (Fig. 2b). Over the experimental period, we also calculated the average daily removal rate (ADRR) of NO3-N and NH3-N, and the results presented in Table 2 and Table 3 revealed the ADRR of 12. 9 ± 10.0 mg N L− 1 and 17. 1 ± 11.5 mg N L− 1 for NO3 and NH3, respectively. In this batch, we kept the ratio of NH3-N and NO3-N applied into the anolyte to 1.33 and achieved 96% and 95% removal efficiency, respectively.
Table 2
The average daily removal rate of nitrate-nitrogen in the anode chamber
| Anodic chamber | Count | Sum | Mean ± SD (mg N L− 1) | Variance | Mini d− 1 | Maxi d− 1 |
| A1 | 35 | 451.01 | 12.9 ± 10.0 | 100.4 | 0.0 | 50.64 |
| B1 | 35 | 429.47 | 12.3 ± 13.2 | 173.8 | 0.0 | 51.19 |
| C1 | 35 | 456.36 | 13 ± 15.9 | 251.9 | 0.0 | 54.00 |
| SD: standard deviation; Mini d− 1: minimum removal per day; Maxi d− 1: maximum removal per day |
Table 3
The average daily removal rate of ammonia-nitrogen in the anode chamber
| Anodic chamber | Count | Sum | Mean ± SD (mg N L− 1) | Variance | Mini d− 1 | Maxi d− 1 |
| A1 | 35 | 599.33 | 17.1 ± 11.5 | 132.4 | 0 | 62.13 |
| B1 | 35 | 505.23 | 14.4 ± 14.9 | 221.2 | 0 | 53.09 |
| C1 | 35 | 406 | 11.6 ± 14.6 | 212.4 | 0 | 59.00 |
| SD: standard deviation; Mini d− 1: minimum removal per day; Maxi d− 1: maximum removal per day |
In the second batch (batch B), we simultaneously removed NH3-N and NO3-N in the 35 d of operation (Fig. 2c). The ADRR of NO3-N and NH3-N was 12.3 ± 13.2 mg N L− 1 and 14.4 ± 14.9 mg N L− 1, respectively, as presented in Table 2 and Table 3. The mixing of NH3-N and NO3-N in the B1 showed a 97% and 94% removal efficiency, respectively (Fig. 2d). While in B2, the nitrate appeared in the bulk solution, and its concentration kept decreasing as the experiment continued.
In the third batch (batch C), the average daily removal rate of NO3-N and NH3-N were 13 ± 15.9 mg N L− 1 and 11.6 ± 14.6 mg N L− 1, respectively, as indicated in Table 2 and Table 3. In addition, the mixture of electron donors and acceptors present in the B1 had resulted in rapid NO3-N depletion with a removal efficiency of 99%. In contrast, a decrease to 67% in NH3-N removal efficiency was detected (Fig. 2d).
3.1.2 The effect of C. vulgaris and biochar mediation
In all three batches, the appearance of nitrate as a product of NH4+ conversion was monitored from the cathode chambers. The average daily production witnessed was 8.81 ± 6.53 mg N L− 1, 3.35 ± 1.85 mg N L− 1, and 3.73 ± 1.54 mg N L− 1 in batch A, B, and C, respectively (Table 4). The data from the analysis of variance indicated that C. vulgaris and biochar had a significant effect on the accumulation of NH4+ in cathodic chambers (P < 0.05), and then we further performed a variability analysis using Kruskal Wallis One-Way ANOVA (P < 0.001) to investigate the individual differences. The multiple pairwise comparisons (student-Newman-Keuls) results showed that the daily accumulation of NH4+-N in B2 was significantly lower compared to that of A2 and C2 (P < 0.001). This outcome confirmed the presence of C. vulgaris in the catholyte, as illustrated in Fig. 1a and b; nevertheless, the accumulation of NH4+-N in C2 did not significantly differ from A2 (control) at α = 0.1.
Table 4
An average daily accumulation of NH4+ in the cathode chamber
| Cathodic chamber | Count | Sum | Mean ± SD (mg N L− 1) | Variance | Mini d− 1 | Maxi d− 1 |
| A2 | 35 | 308.232 | 8.81 ± 6.53 | 42.6 | 1.976 | 30.40 |
| B2 | 35 | 117.3611 | 3.35 ± 1.85 | 3.43 | 0 | 6.135 |
| C2 | 35 | 130.466 | 3.73 ± 1.54 | 2.36 | 2.364 | 7.326 |
| SD: standard deviation; Mini d− 1: minimum accumulation per day; Maxi d− 1: maximum accumulation per day |
3.1.3 The community of microbial consortia
We analyzed the microbial diversity in the bulk sludge of each BRs at the end of the batch operations. The total sequencing reads based on the number of samples and OTUs detected by rarefaction measurement of samples A1, A2, B1, B2, C1, and C2, were 24,955, 7,097, 22,084, 15,132, 7,116, and 13,738, respectively. Furthermore, we plotted the rank-abundance distribution curve, and the number of OTUs detected in bulk sludge samples was 230, 219, 237, 227, 219, and 236. This study found that the 1st dominant phylum identified from the OTUs taxa in all samples (except sample B2, in which the proteobacteria was the first dominant phylum with an abundance of 49.98%) was Firmicutes with a diversity abundance of 56.64%, 57.09, 46.53%, 65.31%, and 68.58%, followed by Proteobacteria (16.65%, 20.54%, 25.6%, 21.01%, and 22.07%), and Planctomycetota (10.02%, 6.31%, 8.57%, 4.44%, 1.99%, and 1.78%) in sample A1, A2, B1, B2, C1, and C2, respectively.
3.1.4 The generation of biogas in the BRs
Here, we monitored biogas production in each chamber during the 35 d of the experiment, and the test of variation using Kruskal-Wallis one-way ANOVA demonstrated a statistically significant difference in biogas production in the BRs. Based on the unique nutrients’ reactions and microbial activities in anodic chambers, A1 showed the highest cumulative volume of biogas produced (3789.3 mL) with a linear, exponential increase starting from zero on day 1. At the same time, the C1 indicated a lower cumulative production (1791 mL), while the catholyte in B2, a special chamber where C. vulgaris was growing, recorded the highest volume of 6325 mL. Furthermore, the average volume of biogas produced in the BR A1, A2, B1, B2, C1, and C2 was 108.3 ± 88.1 mL, 111.8 ± 103.0 mL, 56.52 ± 62.1 mL, 180.7 ± 109. 9 mL, 51.2 ± 53.3 mL, and 110.0 ± 9.34 mL, respectively (Table 5). In contrast, the highest N2O production was detected in B1 (48%), followed by A1 (42%), while the lowest N2O generated was found in C2 (6%).
Table 5
The mean cumulative volume of biogas produced in all BRs
| BRs sample | Count | Sum | Mean ± SD (mL) | Variance | Mini | Maxi |
| A1 Biogas | 35 | 3789.3 | 108.3 ± 88.1 | 7764.0 | 0 | 283 |
| A2 Biogas | 35 | 3912.5 | 111.8 ± 103.0 | 10611.1 | 1 | 326 |
| B1 Biogas | 35 | 1978.2 | 56.52 ± 62.1 | 3861.9 | 0 | 184 |
| B2 Biogas | 35 | 6325 | 180.7 ± 109.9 | 12084.9 | 2 | 364 |
| C1 Biogas | 35 | 1791 | 51.2 ± 53.3 | 2844.9 | 0 | 165 |
| C2 Biogas | 35 | 3850.9 | 110 ± 92.4 | 8533.9 | 1.8 | 281 |
| SD: standard deviation; BRs: bioelectrochemical reactors; Mini: minimum production recorded; Maxi: maximum production recorded |
3.2 Discussion
3.2.1 Mechanisms and the viability of bioelectrochemical removal of nitrogen
The bioelectrochemical procedures for removing N in the BRs include nitrification, denitrification, anaerobic oxidation of ammonium, aerobic reduction reactions, hydrolysis, electron donor migration, and other biological and chemical reactions. In this study, we assessed the effects of NO3-N as an electron acceptor, microalga as a biocatalyst, and biochar as a carbon-based biocatalyst using three BRs with varied treatments under anaerobic and aerobic circumstances. The results from batch A showed that anaerobic bioelectrochemical removal of N was possible, indicating the use of electron acceptors in the specified ratio, operational conditions, inoculum/media proportions, and anolyte/catholyte volume ratio were all excellent determinants for N removal in wastewater treatment plants (WWTPs). Consequently, we compared the analyzed results of N removal in this batch to those obtained in batch B and C; hence this batch was designated as a control batch. Although the concentration of NO3-N sighted in the catholyte of this batch was high, it was evidence of NH4+ migration from the anode chamber. Zhang et al. [10] stated that depending on the baseline concentrations, they eliminated roughly 92% of NH3-N efficiently, compared to 57% when the lower baseline concentration was employed. Ordinarily, in WWTPs, autotrophic nitrification plays a pivotal role in N removal from wastewater. Thus, there is evidence that when electrons released by the organic matter are available, the activities and survival of DNB and NB in the presence of specific amounts of O2 increase the nitrification as well as denitrification process [5]. When comparing batch B to batch A, we found that using C. vulgaris considerably increased the removal of NH3-N in the anode chamber, and this phenomenon corroborated the increase in NH4+ displacement into the cathode via CEM. In this regard, Hu [18] affirmed that some microalgae have the assimilation potential and can convert inorganic N to its organic form. These findings exhibited the feasibility of using C. vulgaris to improve N removal bioelectrochemically.
Furthermore, we monitored the rate of N removal in batch C every day, noticing that the removal rate of NH3-N was relatively low in 35 consecutive operational days but high for NO3-N, as shown in Fig. 3a. The higher consumption of NO3-N could be attributed to the instability of the activities of microbial species [19], or it could mean that there was not enough NO3 available for the reaction. Although the NH3-N to NO3-N ratio remained unchanged as in previous batches, unfavorable conditions for AnB might be a significant cause. Consequently, the addition of biochar into C2 has indicated an effect on NH4+ transfer, lowering the bioelectrochemical efficiency of NH3 removal. To this effect, we witnessed a considerable reduction in the rate of NO3-N consumption in the cathode chamber compared to earlier batches; this has led to a decrease in the displacement of NH4+ through CEM into the C2. However, this trend could be attributable to the decline in NO3-N consumption and an increase in NO3-N accumulation in the anode and cathode, respectively.
3.2.2 The transfer and accumulation of NH4+-N into catholyte
The membrane utilized in this experiment allows NH4+ to flow from the anolyte to the catholyte. A2 samples showed a sharp variation in concentrations from the 5th to the 16th d, with concentrations ranging from 1.999 mg N L− 1 to 30 mg N L− 1, and then the values dropped from the 20th to the 35th d, ranging between 6.86 mg N L− 1 and 9.87 mg N L− 1. The appearance of NO3-N in B2, where C. vulgaris was growing, remained consistently low as the C. vulgaris continued to grow until roughly the 15th d after the start-up of operation. There was no nitrate in B2, but it suddenly spiked to around 3.5 mg N L− 1, fluctuating between 6 mg N L− 1 and 2 mg N L− 1 until the operation was completed.
Conversely, the accumulation of NH4+-N in the catholyte of batch C was a different scenario since there was little or no NH4+-N over time. We reasoned that the presence of biochar in the cathode chamber inhibited the migration of NH4+ into the catholyte, and Zhang et al. [10] found that when the cathode chamber did not contain a carbon-based biocatalyst, about 97% of the NH4+-N in the intermediate chamber migrated. Besides, recent research has revealed that biochar might alter microbial activity by stressing both microorganisms and enzymes, resulting in poor organic matter decomposition as well as a decrease in ammonification and nitrification processes due to low nitrate concentrations [20–22]. High pH biochar leads to ammonia volatility, while biochar with higher anion exchange capacity and lower pH can increase the amount of NO3 [20, 23, 24]. In addition, biochar produced from lower pyrolysis temperatures tends to promote denitrification but owing to more carbon availability since it has the capacity to capture and absorb NO3; and a lower C: N ratio promotes rapid mineralization, which can lead to ammonia loss, and as a result, a lower amount of NO3 can be generated [20, 21, 24].
In the current study, the lowest production of NH4+ per d in the A2 was 1.98 mg N L− 1 d− 1, reaching a maximum of 30.4 mg N L− 1 d− 1 when the migration and accumulation of NH4+ (microbially transformed to NO2-N and NO3-N) was not affected. However, in B2 and C2, having the catholyte supplemented with microalgae and biochar, a minimum daily accumulation ranged from 0 to 6.13 mg N L− 1 d− 1 and 2.36 to 7.33 mg N L− 1 d− 1, respectively (Table 4).
3.2.3 Anammox-like reaction, nitrification, and denitrification in the BRs
Initially (within the first 5 d), we did not notice the presence of NH4+-N in the cathode chambers of all batches, as shown in Fig. 3b. A related study mentioned that because of their affinity for the substrate present in the anodic chamber, “AnB enriched in the BRs fed with organic compounds” like acetate (C2H3O2) and propionate (C3H5O2) “are capable of competing with denitrifying heterotrophic bacteria for electron acceptor (NO3)” [25, 19]. Strous et al. [26] discovered that AnB had a low growth rate and cellular yield compared to denitrifying heterotrophic bacteria. Generally, denitrifiers are heterotrophic bacteria that recover energy from organic matter and use NO2− or NO3− as an electron acceptor in cellular respiration [12]. As a result, organic compounds must provide adequate electron donors before NO3 removal in WWTPs may be considered practical. In the first week of operation, the amount of NO3-N in all anodes increased dramatically, and removal was exceedingly slow (Fig. 3c); this behavior might be due to the continuous input of NO3-N or bacteria stabilization/growth rate in the BRs.
3.2.4 The effect of physicochemical parameters on the efficiency of bioelectrochemical removal of nitrogen
In the first, second, and third successive batches, we started the bioelectrochemical operation with TSS concentrations of 6100 mg L− 1, 5900 mg L− 1, and 5800 mg L− 1, respectively. The concentrations fell dramatically until stabilizing at 700 mg L− 1, 1650 mg L− 1, and 1500 mg L− 1, respectively (Fig. 3d). The BR had an average pH of 7.5 0.6 and a DO of 0 to 0.9 mg L− 1 in the anolyte of the first batch. The pH of the medium solution was slightly alkaline (8.11–9.31) in the first 4 d of the experiment and remained constant at a neutral level until the 24th d (Fig. 4a), whereas we observed a slightly acidic (6.57–6.98) from the 25th to 28th d. On the other hand, the average pH value in the second batch was 7.4 ± 0.33. We also controlled the DO between 0 and 0.8 mg L− 1 to maintain the anaerobic microbial respiration in the anolyte, except for the 18th − 23rd d, when we detected minor acidic pH values (6.54–6.79) as shown in Fig. 4a, the pH was consistently neutral throughout the analysis. However, in the third batch, all anodic activities of microorganisms in the sludge and electrochemical reactions of physicochemical parameters and nutrients were carried out under anaerobic settings with a neutral pH value (7.3 ± 0.4) depicted in Fig. 4a.
Electrode spacing and surface area, COD and DO levels, and NH3-N concentration all have a massive effect on the removal efficacy of N [10]. It is worth noting that DO levels play a pivotal role in the bioelectrochemical removal of N, as witnessed in the study of Jiang and Co. [27] when they discovered that the removal efficiency of N decreased from 87.42–75.39% in reactor 1 and 92.38–84.23% in reactor 2 with the DO level increased from 4.0 ± 0.5 to 7.0 ± 0.5 mg L− 1. In the present study, the pH dropped further as the operation progressed and eventually stabilized (Fig. 4a). Still, the pH continued to fall in lockstep with the removal of NH3-N and NO3-N, except when additional variables were present. We have also seen that the ammonia-nitrogen concentration in anode chambers exhibits a gradual reduction (Fig. 4b) and, according to Joicy et al. [5] “Bicarbonate, a major component of alkalinity at neutral pH, provides a carbon source for autotrophs.” This indication suggested that the AOB and NRB present in the BRs might be autotrophs.
3.2.5 Algal growth and nitrogen removal
Immediately after pre-culture, the experimental alga was inoculated and grown in the B2 under aerobic conditions with sufficient illumination and nutrients for optimal growth (BG11 medium). In Fig. 4c, the pattern of C. vulgaris’ growth profile was estimated as cell biomass in gram per liter and related to optical density from UV-Vis Spectrometer (OD680). We carefully monitored the growth of C. vulgaris daily, and the results revealed three distinct phases: in the first 3 d, there was a lag in production ranging from 0.24 g L− 1 d− 1 and 0.47 g L− 1 d− 1 in the BR; following that, the algal biomass increased exponentially from 0.47 g L− 1 d− 1 to 0.97 g L− 1 d− 1 until the 10th d when it reached a stationary phase for nearly 4 d; and subsequently from the 20th d onwards, there was a steady decline (Fig. 4c).
The N removal rate in batch B had been high and strikingly consistent in the first 14 d, which coincided with the viable time for C. vulgaris growth, as previously described in subsection 3.1. However, when the microalgae growth slowed, a high amount of NO3-N appeared immediately in the BR, and this situation is considered evidence of N assimilation in the B2. Another reason for the formation of NO3-N could be due to the activities of ANB and DNB, which converted the migrating NH4+ into NO2, NO3, and then N2. Perhaps the microalgae could not directly assimilate all of the migrating NH4+ that was deposited in the anode chamber; thus, the function of ANB and DNB is enormous at this stage because they made it readily available in the form of NO3 and ultimately assimilable; in addition, the working of electrodes in the catholyte may increase the diversity of bacteria responsible for that function. Similarly, Jia and Yuan [12] stated that certain microbial strains such as Pseudomonas vesicularis (P. vesicularis) and Pseudomonas diminuta (P. diminuta) promoted the growth of Chlorella sp. and demonstrated N removal when compared to a previous study that used heterotrophic bacterial nitrification and denitrification.
3.2.6 Composition and volume of the generated biogas
The daily biogas generated in the BRs of all batches was shown in Fig. 4d, revealing that biogas output was low initially but rose and stabilized as the experiment progressed. Nonetheless, Fernández-Rodrguez et al. [28] used a co-digestion mixture of 25% solid waste from an Olive mill and 25% microalgae sp. as a co-substrate for digestion and obtained a faster and higher production of biomethane (48.1 mL CH4/(g VS.d)). In C2, where the biochar was applied, this research found a relatively low gas volume (3850.9 mL), as shown in Table 5. These findings indicate that the C. vulgaris might have improved the biogas production or augmented bacterial communities to generate more biogas and vice visa in the case of the carbon-based biocatalyst.
Trichococcus was found to be relatively abundant in the genera explored in this study. We also discovered that they are tolerant fermenters capable of producing acetate and lactate from glucose under anoxic conditions; and are known to produce formate, ethanol, and methane in the absence of oxygen. The result obtained in this study indicates that these genera contributed to methane generation, although other factors hindered more generation of the gas. Figure 5a represents the volume (%) of the specific biogas (N2O, CH4, and CO2) produced during the study, showing that the anaerobic state was maintained, and DNB was detected even though the number was negligible in B1. Interestingly, the high N2O observed in B2 could be due to the availability of DO, the presence of NO3, and the formation of NO2 driven by microalgae, and this phenomenon has demonstrated the penetration of NH4+ and subsequent conversion to dinitrogen in the presence of oxygen; whereas, the lowest N2O detected in C2 could imply that biochar has an inhibitory effect on NH4+ migration.
The sample of methane gas taken from B1 (24%) was also the highest in this group, followed by 22% collected from C1 and 0.20 and 0.18% from B2 and C2, respectively. Here, the growth of microalgae in the cathode chamber might be the purpose for low CH4, as N2O was already a dominant gas in the anode. Furthermore, carbon dioxide was highest in B2 (49%) and lowest in B1 (1.82%), and microalgae required more CO2 during photosynthesis, which could be reduced in the cathode and converted to glucose by microalgae. This process may increase the abundance of CO2 on the B2 side, and generally, the biogas obtained in this study, especially methane content, was relatively low, indicating a conversion of most of the depleted NH3-N and the NO3-N to N2 or nitrous oxide [5].
3.2.7 The relationship between changes in algal cell biomass, nutrients, physicochemical parameters, and generated biogas
The relationship between nutrients, algal cell biomass, physicochemical characteristics, and produced biogas was determined using Pearson correlation analysis (a 2-tailed test of significance). The correlation is considered significant at the 0.05 level in this study. In batch A, the pH values in the anode chamber revealed a meaningful association (0.69) with NH3-N, even though the pH was a little high at the start of the experiment and NH3 removal was slow, but as the values declined, so did the concentration of NH3-N. These variations could indicate that the NH3-N reduction in the anode chamber was pH-dependent. We also observed that the decrease in TSS level went along with the removal of NH3-N, signifying a strong correlation (0.96). Similarly, a reduction in NO3-N concentration considerably affects the NH3-N removal rate in the anode chambers.
In batch B, the removal rate was not significantly affected by the ratio of two nitrogen forms during the operation (Fig. 5b). Still, the C. vulgaris grown in the cathode chamber had a negative correlation (-0.7) with the nitrate concentration. This phenomenon is a shred of evidence that the C. vulgaris assimilated nitrate and supported the increase in algal cell biomass; yet, nitrate in the cathode showed a substantial positive relationship (0.7) with biogas production. The high amount of CO2 detected in this chamber could be the reason for this correlation because the C. vulgaris quickly formed the assimilated nitrate. More so, we previously discovered that C. vulgaris impacted NH4+ migration from the anode chamber and has improved the pollutant removal capability; thus, this is one of the bioelectrochemical characteristics of an anode chamber where organic matter is oxidized. Despite the fact that algal growth was exponential while DO fluctuated, the growth of microalgae was interestingly shown to be highly correlated (0.8) with DO concentration (Fig. 5d). According to a study carried out by Mouget et al. as cited in Jia and Yuan [12], some microbial strains enhance the growth of green algae without releasing any growth-promoting substance. At the same time, the degree of dependency between nitrate-ammonia and ammonia-nitrogen was depicted in Fig. 5c, while the statistical test revealed a significant correlation between nitrate-ammonia and ammonia-nitrogen removal rates.
In the analysis of batch C, we saw that whenever the amount of produced biogas increased, the DO decreased and thus showing a negative (-0.6) relationship. We also observed that during the simultaneous removal of ammonia and nitrate in the anode chambers, the nutrients did not rely only on the 1.33 ratio at a starting point (Fig. 6a); the subsequent ratio may not be a concern, but still further study is needed to investigate this scenario. Traditionally, ammonia and nitrate are nutrients that can be present in water, and excessive nutrient levels in water can cause severe problems to human health.
3.2.8 Microbial Diversity
The richness and evenness estimation showed that the diversities in the samples were slightly differed from each other and were found to be lower than in earlier studies conducted using activated sludge [29]. Anammox, nitrification, and denitrification mechanisms and the activities of many functional microorganisms have played an essential part in all of the phases analyzed. The phylum group of Firmicutes makes an outstanding contribution to the conversion and release of N in anaerobic conditions, and other dominating phyla displayed synergistic interactions and could potentially improve the removal of NH3-N and NO3-N in the BRs. The phylum, Planctomycetota, which was detected in abundance in all batches of this study, is well known for its function in the anammox process [19]. As a result, to successfully remove N from wastewater, anaerobic ammonium oxidation in the BRs’ anodic chamber requires abundant AnB such as Planctomycetota, and as well the nitrogen high removal efficiency seen in this research confirmed the activities of these phyla. In addition, Actinobacteriota was also abundant in all of the tested samples, and this phylum represents a critical community of microorganisms that aid in the remediation of polluted environments [29]; decomposing many types of organic molecules is one of the Actinobacteriota’s numerous valuable functions. Moreover, the phylum Proteobacteria was also found in every sample (Fig. 6b), and these facultative anaerobic microbes can be employed to reduce NO3 and ferment glucose to produce propionic acid.
The specific information from Fig. 6c revealed that sporosarcina and Tissierella are the two significant genera that constituted the abundance of Firmicutes and Proteobacteria based on the genus taxonomic categorization. In contrast, Massilia, a member of the Proteobacteria phylum, was found in abundance in B2 and was the reason for Proteobacteria’s dominance over Firmicutes in that sample. On the other hand, Pseudomonas, a Proteobacteria family, is an aerobic bacterium that actively participates in denitrification, and its presence in C2 and A2 suggests that nitrate was reduced to dinitrogen. Ultimately, the existence of the studied taxa and phyla revealed a redox reaction and DNB activity [30].
3.2.8.1 Alpha diversity indices
In a nutshell, alpha diversity refers to the microbiological variety present in a specific population [31, 32]. In this study, we looked at the diversity of species within one particular community sample, and statistical analysis (Two-way ANOVA) revealed a significant difference in columns while insignificant in rows among the samples of observed species; Chao, Shannon, and Simpson (= 0.05). The study identified phylum Nitrospirota in most instances with relatively high abundance in batch B; its presence signals nitrite oxidation because it is a chemolithoautotrophic nitrite-oxidizing bacterium commonly found in freshwater and plays a critical biogeochemical function in marine habitats. The activities of Nitrospira had indicated a significant difference between the operating condition in batch B and other batches, despite the fact that it was deficient in the current study. The anaerobic chambers had the most commonalities within each group, indicating that the anoxic condition altered the structure of the microbial community. Hence, the samples of the same BRs had shown a remarkable similarity, which could be due to the inclusion of microalgae and biochar in the cathode chambers of batch B and C.
3.2.9 The effect of biofilm on graphite electrode
To monitor the microstructure of the electrodes, we use SEM to microscopically scan the graphite used as an electrode in the A1, B1, and C1. The formation of biofilm and the sticking of microbes such as fungi, bacteria, or actinomycetes-based communities on the surface of graphite electrodes can potentially affect the long-term functioning of the electrodes [33]. Previous studies reported that the embedded attachment on the anode electrode could adversely affect the function of electrodes in the microbial electrolytic system and often influences metal corrosion as opposed to in the microbial fuel cells [33–35]. Conversely, when inoculating an anode chamber with activated sludge, the inactive, damaged, or dead cells could form a biofilm membrane and accumulate on the surface of the anode electrode over time, which as a result, degrade the quality of the electrodes and decrease their electrochemical performance [33–35]. Although the electrodes’ type, purity, size, and installation orientations all have a part in defining the life-quality of electrodes in terms of biofilm-embedment effect, there is still a need to avoid embedment as much as possible precautionarily. To prevent a poor performance influence on the graphite in this research, we purposely chose pure-grade graphite, pre-treated and appropriately installed it; ultimately, the SEM results revealed a few or no adhering bacteria on the surface of the graphite electrodes, as shown in Fig. 6d.