Effect of C/N ratio on biodegradation of ciprofloxacin and denitrification from Low C/N wastewater by a novel 3D-BER System

: Emerging pollutants as pharmaceuticals have been focusing international attention for few decades. Ciprofloxacin (CIP) is a common drug widely found in effluents from hospitals, industrial and different wastewater treatment plants, as well as rivers. In this work, the lab-scale 3D-BER system was established, and more than 90% of the antibiotic CIP removal from the Low C/N wastewater. Best results were obtained with current intensity, and different C/N ratio significantly improve the removal of CIP and nitrates, when the ideal conditions were; C/N = 1.5-3.5, pH =7.0-7.5, and I = 60 mA. The highest removal efficiency of CIP = 94.20 %, NO 3- -N= 95.53 % and total nitrogen (TN) = 84.27 %, respectively. In this novel system, the autotrophic-heterotrophic This system has the assortment and prosperous community revealed at the current intensity of 60 mA, and the analysis of bacterial community structure in effluent samples fluctuates under different condition of C/N ratios. According to the results of LC-MS/MS analysis, the intermediate products were proposed after efficient biodegradation of CIP. Microbial community on biodegrading was mostly found at phylum, and class level was dominantly responsible for the NO 3- -N and biodegradation of CIP. This work can provide some new insights towards the biodegradation of CIP the removal of nitrates from low C/N wastewater treatment by the novel 3D-BER system.


Introduction
Worldwide, the various kind of emerging pollutants such as pharmaceuticals compounds, antiinflammatory drugs, antibiotics, beta-blockers [1], and the massive amount of NO3-or NO2-have attracted international attention for the last few decades [2]. The antibiotic ciprofloxacin (CIP) is the 3rd-generation fluoroquinolone group usually used in humans and veterinarians [1,3,4]. The high-proportion of CIP is not fully metabolized in livestock and humans, but evacuated as a parent substance [5]. Hence, CIP can reach the environment through various pharmaceutical industries, sewage treatment plants, livestock activities, landfills, and application of sewage sludge [6], manure, or treated wastewater to agricultural land [3,7,8]. However, due to the potential development and dissemination of antibiotic resistance, this poses a potential threat to the ecosystems and human health [2,3]. Therefore, CIP removal must be considered before being released into the environment [9]. Antibiotic CIP is readily detectable in man aquatic-environment (usually found in surface water at ngL-1 to µg L-1 levels); [7], it occurs at high levels in the effluents of WWTPs (up to 6.55-31 mgL-1) receiving pharmaceutical wastewater and rivers polluted with industrial waste (up to 14 mgL-1) [6,10,11]. During wastewater treatment, 80-90 % of CIP was removed by adsorption to sludge, which stabilizes the substance [3]. Some scientific reports, data has been shown that biodegradation [7], and adsorption process [9] was the highest elimination methods for various kinds of antibiotic in WWTPs [4]. It has been shown in some studies around 50-100% adsorption is the main path of CIP removal by biodegradation in anaerobic sludge system [12,13]. This clearly shows the importance of sludge being released into the environment as a CIP reservoir and developing sludge management strategies [14]. In general, anaerobic digestion was a standard procedure for the stabilization of sludge; it was also meant to extract organic matter without particular regard for the removal of the antibiotics [8]. The biodegradation rate of absorbent CIP durability was 0-40% during sludge treatment [9,15]. A considerable amount of CIP persisted in the digested sludge contained in wastewater treatment plants [10]. Unfortunately, there were no reports on the microbial community structure of CIP degradation [16,17]. The traditional denitrification process depends on four basic denitrifying enzymes [18], including respiratory of nitrate reductase [19] has been shown: However, many processes reported in previous studies were CIP effects on the denitrifying enzymes and the activity of existing denitrifying bacteria [19][20][21]. In recent years, the biofilm electrode reactors (BERs), which combine biological and electrochemical techniques, can efficiently eliminate nitrogen, heavy metal, and antibiotics [10,22]. The autotrophic denitrify bacteria are immobilized on the cathode surface and the hydrogen produced from the water-electrolysis also used as an electron donor in the autotrophic denitrification process [21,22]. Therefore, the anode and cathode materials typically made of carbon and thus provide an inorganic-carbon source that can be used as a pH buffer solution, as shown in Eqs (1, 2 and 3): C+2H2O→ CO 2 + 4H + 4e − It was shown that, the process of denitrification uses hydrogen instead of an organic carbon source [23], to completely covert nitrate to N2 as per the following reaction [18,24], compared to the traditional BER consumes a large amount of energy and is relatively inefficient [28,29]. We recently introduced a 3D-BER system for the new advance technique in the potential benefits of low currents, and there is the best capability to treat toxic substances. Hence, an effective and economical methods for eliminating CIP antibiotics and nitrate or nitrite were developed. It is also imperious to improve the methodology to remove the residues of CIP antibiotics when high manipulative capacity of the anaerobic process occurred in 3D-BER system.
This study demonstrated that the removal efficiency of antibiotics CIP and nitrogen in the effluent of low C/N wastewater can be improved by a novel 3D-BER system. The major objective of this particular study were; (1) to examine the potential toxicity of ciprofloxacin and nitrogen removal; (2) Comprehensively examine the effect of pH on antibiotics-ciprofloxacin and denitrification; (3) to appraisal the degradation by microbial inhabitants and intermediate product of CIP were investigated. Therefore, this is the first study of the removal of CIP and enhanced the denitrification process for solving many problems in low C/N wastewater. As compared to traditional low C/N ratio in wastewater treatment systems, this technology is also designed to be a more efficient, useful complement or cost-effective alternative. The schematic view of the three-dimensional bioelectrochemical reactor system (3D-BERs) was used in this study to evaluate the biodegradation of ciprofloxacin and nitrates removal from Low C/N wastewater (Figure 1). The reactor was made of plexiglass cylindrical with a diameter of 10 cm and a height of 20 cm with a total volume of 1.2 L, and had a working volume is 0.785 L. The cathode consisting of eight graphite rods (height of 20 cm and diameter of 0.8 cm) were placed around the periphery of the reactor, while the one graphite rod (height of 20 cm and diameter 1.5 cm), as an anode was fixed in the centre of the reactor and were connected using insulated electrical copper wire by DC-power. A DC-controlled power supply: Germany) prior to testing. The pH of simulated influent wastewater is typically 7.5 ± 0.2, and no further adjustment is required.

Bacterial adaptation phase
All experiments runs were inoculated with a mixed culture of acclimated autotrophicheterotrophic bacteria ( Table 2). These microbes were enriched from the anaerobic sludge (Mixed Liquor Suspended Sludge (MLSS.100g)) was taken as a source of inoculation from the Nanjing municipal wastewater treatment plant (WWTP). Before cultivation, 1.2 L of anaerobic sludge water was placed in a refrigerator with nutritious material at 4°C for seven-days. While the synthetic water, according to NaAc, Nitrates, and KH2PO4, were added into the anaerobic sludge with the ratio of C/N/P = 3:1:0.5. During the first three days, sludge water was circulated by a magnetic pump. The concentration of nitrate-nitrogen in the influent was maintained at approximately 30 mgL -1 . After 21 days, anaerobic sludge water was placed in the reactor, and 0.350 L of tap water was added to a total volume of 0.785 L. During the cultivation process, the electrical current was not supplied to the 3D-BER system after two weeks; the direct electrical current was gradually adjusted to 10 mA. introduced into cathode and anode layers of the 3D-BERs. However, the antibiotics CIP concentrations in the influent were 100 to 500µgL -1 , respectively. For the batch experiments, the biofilm in the anode/cathode surface, as well as the third electrode GAC, turned into dark grey at room temperature (27 ± 1.5 o C). The influent was renewed two times within 24 h to fast growth microorganism in the reactor. In the 3D-BER system, sustain an anaerobic condition to expand denitrifying bacteria. By observing the total nitrogen (TN) concentration in the reactor, when TN was exhausted, the residual substrate was removed, and fresh substrate was fed to the reactor to avoid endogenous metabolism of the microorganism.
Nitrite (N − NO 2 − ), was measured by N-(1-naphthyl) ethylenediamine dihydrochloride spectrophotometric method at λ = 540nm, and total nitrogen (TN performance of nitrate and CIP antibiotics was calculated based on the percentages of nitrate reduction and CIP as follows Eqs. (5) and (6): The removal efficiency of Nitrates reduction (%) = Nitrate in−Nitrate out Nitrate in × 100 (4) Where "in" is the initial concentration of nitrate, and the "out" is the final concentration of nitrate The removal efficiency of CIP reduction (%) = Where "in" and "out" are indications of CIP antibiotics concentration in the inlet and the outlet of the 3D-BERS, respectively. In phase A: water containing 0.1% formic acid and in the meantime in phase B-acetonitrile. To assess biodegradation products, thorough analysis of positive and negative ion modes was used.

Ciprofloxacin concentrations measurement and by-products identification
However, the molecular ions observed in positive and negative ions were subsequently applied in electrospray ionization techniques to provide a chromatographic shape of these putative biodegradation products. In order to extract LC-MS/MS spectra of the points determined in the final step, high collision energy was exclusively adjusted, and ion chromatographic response intensity was optimized in the range of 45 eV. From the literature, some researchers confirmed that the BES could improve the mechanism of antibiotic removal from the WWTPs due to adsorption techniques and which is a very economical system. The growth medium of the microbial community utilized the energy, C/N source [36,37]. However, the external calibration curve was used to determine the ciprofloxacin antibiotics concentration with a correlation coefficient (R 2 = 0.985).

Statistical data analysis
All sample data were analyzed using Microsoft Excel (2018) and also used with Origin pro 8.

DNA Sequencing and PCR amplification
The biofilm samples were collected from the 3D-BERs (anode /cathode) layers (1.0g) and effluent (100 mL) after long-term acclimation at different C/N ratios. The amplicons were isolated from 1.0% agarose gels and purified using DNA-extraction from the PowerSoil DNA ® Isolation Kit

Biofilms GAC samples analysis by SEM
The two biofilms samples of S1 and S2 particle electrodes surface morphology for bacteria in GACbiofilms were examined by scanning electron microscopy (SEM). Prior to SEM analysis, a series of batch experimental sample processing techniques (i.e., fixed, washed, dehydrated, dried, coating,  Figure. 2 the morphology of biofilm attached to granular activated carbon (GAC) particle electrodes in the 3D-BERs. The GAC had a high specific area about 450-900 m 2 g -1 and a porous structure to facilitate the growth of microbial attachment as reported [20,28,41]. The biofilm samples of the S2 were abundant compared to the S1, corresponding to the thicker bacterial community at 60 mA [42]. In the S1 zone, the microbes are less in quantity due to lacking electron donors. It was found that in the 3D-BERs with GAC, biofilm was imbedded in the abundance of microbial activity [41]. It was clearly observed in rod-shaped graphite anode and cathode, which was authentic common in the other denitrification system [20,43]. There also existed huge microbes, which may be hydrogenheterotrophic denitrifying bacteria.

Illumina-MiSeq sequencing
The 16R r DNA gene sequence obtained from Illumina-MiSeq at Novogene Co., Ltd., Beijing, China. Using this program, truncate the adapter with low-quality sequences <Q20 and short <300-bp were trimmed. Disposing of potential chimeric sequences using the Mothur chimeric UCHIME algorithm. The taxonomic classification of the sequences was conducted using the cutting indicating a significant increase in denitrifying bacteria [19], and the anoxic condition had achieved [43]. It was observed that the removal efficiency of nitrogen increases with the amplified C/N ratios.
In particular, the denitrification performance of 3D-BERs with a current intensity of 60 mA was slightly higher in the system was compared with published previous work, as shown in Table   3. When the ratio of C/N increased from 0 to 1.5 and 2.5 to 3.5, the average concentration of total  [45]. Similar results were also reported [18,28,46]. In 3D-BERS, the average removal efficiency was respectively 96.81 % and 97.82 % when C/N ratio was 1. In addition, the C/N ratio was 1.5 and 3.5; the final effluent concentrations respectively were 0.44 mgL -1 and 0.39 mgL -1 . However, under these conditions, the initial NO3 --N loading of denitrification was high, so NO2 --N in the wastewater remains almost zero and constant [19,45]. In addition, the final effluent of NH3-N in the 3D-BER system decreased from 4.12 mgL -1 to 2.29 mgL -1 , 4.06 mgL -1 to 1.49 mgL -1 , and the different C/N ratios from 0 to 1.5 and 2.5 to 3.5, and the average NH3-N concentration in the final effluent was 4.06 mgL -1 to 1.49 mgL -1 , and the C/N ratio was 1.5 to 3.5, compared to other techniques previously studied [27,43,45]. . As the C/N ratio increased between 0.5 and 1.5, the effluent NO2 --N reduced rapidly 2.52 mgL -1 to 0.242 mgL -1 . At ratio C/N=1.5, then NO2 --N continues to decline at lower levels.
Heterotrophic denitrification may play a pivotal role at an advanced level of the C/N ratio, and the rate of denitrification was high. When C/N ratio was 1.5 and 3.5, the removal efficiency increased by 95.53% ̶ 85.73% and 80.27% ̶ 73.85% for NO3 --N and TN. However, NH3-N tends to be opposite to NO3 --N and TN. These results are in good compared with previous findings [19,21,22]. The NO3 -was commonly used as an acceptor of electrons and could be reduced to NH4 + under anoxic and electron-acceptor conditions [20,43].
The 3D-BER system was an anoxic condition, and the force of the electric field prevents NO3 -from moving to the surface of the anode, so there may be no electron acceptors near the surface of the cathode. In this study, ratio C/N is very close to the complete denitrification in the range of 1.5 to 3.5 and is established by single heterotrophic denitrification [27,29,48].
Several scientific studies reported on the elimination of nitrogen were conducted by autotrophic denitrification [47,49]. This method, elimination of NO3 --N, NH3-N and total nitrogen increased while accumulation of NO2 --N decreased gradually. In this study, the complete denitrification was obtained in C/N= 1.5 and may be due to system temperature and denitrification removal rate. However, a similar observation was reported by other researchers shown in

Effects of pH on antibiotics CIP, nitrate and total nitrogen removal
As shown in Figure.  respectively as shown in Figure. 5 [48,54,55]. It was described that denitrification inhibited with CIP and development more obvious during the prolonged microbial cultivation period was reported [6,56,57].

Effect of biodegradation mechanism of antibiotic ciprofloxacin
Generally, the ideal pH for Autotrophic-Heterotrophic denitrification systems was chosen to be 7.5, because recent reports have shown that increasing antibiotics CIP levels can significantly reduce the performance of autotrophic denitrification systems [6,57]. For another factor that was selected as the ultimate circumstance, the final result under optimal conditions was pH = 7.5. Therefore, the latest novel 3D-BER system was introduced to enhance the denitrification process and achieves the highest removal efficiency of nitrogen. However, Eq.1 and Eq.3 were initiated to indicate that the pH illustrates by the OHproduced during the denitrification process and water electrolysis. In our research work, we found that the pH level of the final effluent maintained at 7.5 ± 0.2 [46,49]. Moreover, it indicates that CO2 produced by the graphite anode is not only dissolved in the carbonate but also dissolved in the hydrogen carbonate, as shown in Eq. 3, and reacts with OHso that the pH in following equations shows: The pH of the system was always in a favourable condition for the biological activity of the denitrifying bacteria. Some scientific research suggests that CIP was predominantly eliminated by adsorption during biological wastewater treatment [7,13]. Previous publications demonstrated that According to Pearson's bivariate correlation analysis, CIP biodegradation was significantly correlated (p < 0.01, R 2 = 0.996). To better describe the CIP metabolic degradation pathways [4,23] at the gene and predominant enzyme levels, superior isolates of ciprofloxacin in anaerobic sludge can be used by transcriptomics and metagenomics studies [31,59]. The information obtained from LC-MS/MS was insufficient for the fully characterize and the chemical structure of these metabolites.
Whereas, the realistic standards will be used in future work to confirm the formation of Ciprofloxacin biotransformation pathways.

Bacterial diversity and community composition
Evolution of ciprofloxacin degradation and denitrifying bacteria under long-term acclimation from low C/N wastewater. Summarizes in Table 4 the microbial species richness and diversity indices for these two samples. The high-throughput sequence analysis of 63,376 classifiable sequences obtained from S2 samples belonged to 35 class, 21 phyla, and 205 genera. The 73,382 classification sequences in S1 samples were 39 class, 23 phyla, and it belonged to 195 genera. S2 and S1, but their relative abundance was different in each sample (Figure 7a). Although in the samples S2 and S1, bacteria dominated, the bacteria in the sample S2 were more abundant than the S1 samples. According to the contract, the number of S1samples of Firmicutes (1.8%) was obtained in less abundant [15], than in sample S2 (5.6%). In the taxonomic classification, the relative abundance of class levels was shown in Figure 7b. Table 4. Diversity indices of bacterial communities in S1 and S2 sludge samples of 3D-BERS at different C/N ratio and applied electric current 60 mA However, the average richness of sample S1 is less than 2.0% for the classes of Gammaproteobacteria Clostridia (11.47%) [2,59]. It was evident that the distribution of the main classes in these two samples was significantly different. Nevertheless, the amount of Gammaproteobacteria present in sample S2 was more significant than that of sample S1 compared to the distribution of Alphaproteobacteria, Bacteroidia, Deltaproteobacteria, Bacillus, Actinobacteria and Clostridia [1,3], in the S1 sample was relatively low compared to the sample S2 anaerobic sludge from the 3D-BER system. For the understanding of the similarities and some variations between sample S1 and S2, shown in Figure 7c that the genus levels of more than 65 abundant genera. In the abundant genus with a relative richness of more than 2.5%, the abundance of Pseudomonas was significantly higher in the sample S1 (50.93%) [9], while the relative abundance of the sample S2 was only 35.44% [4,57]. In general, it has been widely found in previous studies (such as Pseudomonas aeruginosa), especially in terms of the possibility of denitrification [19,60]. Also, Pseudomonas stutzeri [41,61], and the certain type of Pseudomonas bacteria belonging to the genus Pseudomonas sp. C27 have autotrophic and heterotrophic denitrifiers that utilize an assortment of electron donors [40]. As the C/N ratio increases, the impact on bacteria output involved in autotrophic denitrification, the microbial growth yield of denitrifying autotrophic microbes is smaller than that of heterotrophic bacteria. Despite a gradual increase in the C/N ratio, the denitrifying autohydrogenotrophic bacteria may have been gradually domesticated to heterotrophic denitrifying bacteria for effective nitrates and antibiotic CIP C S1 S2 Genus Level a b S1 S1 S2 S2 Phylum Level Class Level 8 elimination. Therefore, Pseudomonas abundance in sample S1 indicates that overall similarity to autotrophic and heterotrophic denitrifying species is related to Pseudomonas in the occurrence of organic substance and applied electrical currents. Moreover, significantly higher amounts of Thauera were detected in sample S2 (64.66%) compared to sample S1 (0.80%). In contrast to S2 samples, Bacillus (11%), Thiobacillus (28.8%), Flavobacterium (3.81%) [4] and Acinetobacter (9.59%) are the most important genus of S2 samples, but the abundance of S1 example was Thiobacillus (23.34%), Bacillus (5.6%), Acinetobacter (4.53%) and Flavobacterium (0.99%) [2,52]. Thiobacillus has been identified as a widespread autotrophic bacterium in recent years, closely related to the oxidation of nitrates to nitrogen. [40,49]. In recent years, several spices belonging to the genus Thiobacillus, Bacillus, and Thauera have been suggested to play an important role in antibiotic CIP and removal of nitrates [57].
Whereas, Thiobacillus and Thauera have the capability to transfer electron directly from the carbon electrode and promote nitrate reduction [16,59]. The richness and abundance of Pseudomonas (35.4%), Thiobacillus (28.88%), and Thauera (64.66%) [24,62] were enriched in the 3D-BER system. The removal of CIP and nitrates was due to the distinction between the dominant Thauera, Pseudomonas, and Thiobacillus, which helped to merge heterotrophic denitrification and autotrophic denitrification processes. In conclusion, the main bacteria enhanced in the third electrode (GAC) will continue in both heterotrophic-autotrophic denitrifications, whereas enriched bacteria in S1 are primarily involved in autotrophic denitrification. Therefore, further research is needed to characterize the denitrifying bacteria in the 3D-BER system.

Conclusion
In this study, the simultaneous removal of ciprofloxacin and nitrates from Low C/N wastewater by a novel 3D-BER System. It has proved to be more economically and technically attractive than traditional denitrification process due to the more than 90% reduction in antibiotics, 55% sludge production. However, the removal performance increased significantly with increasing