Isolation, characterization and Identification of nitrate reducing bacteria
The bacterial colonies grown under nitrate supplemented media were selected for purification, identification and characterization. The isolate was identified using MALDI-TOF-MS as Bacillus subtilis ssp. subtilis DSM 10 with 2.12 score which is interpreted as high confidence, NCBI identifier 135461. The Bacillus morphology identification, Mass Spectra and biochemical identification are reported in supplementary material (S1). This bacteria showed red and dry colonies on Congo red media which confirms the presence of fimbriae and indicates it has the ability to form biofilm. Structural characterization of Bacillus under study using X-ray diffraction showed peaks at 29 and 31 2ɵ which represents Fe-S and Mo (Fig. 1). Full Raman spectroscopy showed peaks at753, 1129, 1314 and 1583 cm-1 which correspond to cytochrome c subunit (Fig. 2), both suggest that this bacterium possess cofactors for NaR subunits.XRD and Raman spectroscopy can be used to characterize bacteria in single and biofilm forms, they provide a fast non-invasive technique for structural identification (Remoundak et al. 2007, Strola et al. 2014). The obtained results of XRD and raman spectroscopy confirm that a nitrate reductase enzyme complex is located in the outer membrane of Bacillus sp DN, this nitrate reductase consists of Mo cofactor as the alpha subunit, Fe-S cluster as the beta subunit, both are anchored to a heme containing cytochrome which represents the gamma subunit. The later receives electrons from a quinone pool and transfers them to the beta subunit and then to the electron acceptor (Coehlo et al. 2015). A previous study reported the structural shape of the membrane bound nitrate reductase heterotrimer as a “flower” (Balsco et al. 2001).
In order to confirm the electron transfer mechanism, cyclic voltammetry was used. The bacterium under study showed redox activities as indicated by the presence of anodic peaks at 0.18 mA and cathodic peak at -0.35 mA for the bacteria in PBS, while 2 anodic peaks were observed at 0.11 and 0.085 mA and cathodic peak of -00.114 mA when the bacterial culture filtrate was added to the bacteria (Fig. 3a). Electrochemical impedance spectroscopy (EIS) showed that the activation resistance (Rct) of the biofilm of the bacteria in the presence of the bacterial culture filtrate was less than that for the bacteria alone(Fig. 3b).This result confirms that Bacillus sp. DN possesses bacterial electron transfer mechanisms that depend on direct attachment to a substrate, this contact dependent mechanism is attributed to the multiheme cytochrome c proteins (MHCs) that shuttles electrons from the electron transport chain to the insoluble electron acceptor. This result is in agreement with Wringhton et al. (2011), Pankratova et al. (2018), Paquete (2020) who reported direct electron transfer in gram positive bacteria. In addition to the direct electron transfer, the presence of a soluble mediator in the media might have been the reason behind the second anodic peak and the decrease in activation resistance.
Biochemical and structural changes of Bacillus sp. in the presence of different nitrate concentrations
Fig. 4 represents the effect of nitrate on Bacillus sp. biofilm and three of its major components, EPS, proteins and eDNA. The biofilm components were stained using fluorescence dyes and were followed as fluorescence intensity. The use of concavalin A was used to identify carbohydrate moiety of the biofilm, this biofilm component showed an increase in the presence of nitrate that reached 2.16 fold when 12 mM nitrate were added to the media, however, the concavalin A decreased again at nitrate concentrations above that. Concavalin A is a fluorescent conjugate that binds with carbohydrate moiety, specifically glycoprotein (Drake et al 2006). This indicates that glycoprotein in the biofilms was affected by the nitrate concentration during biofilm formation. Methylene blue was used to follow the protein content (Liu et al 2010) as another biofilm component, the results showed an increase in protein that reached about 1.6 fold when the highest nitrate concentration was used. Ethidium bromide was used to detect the eDNA content in the biofilm in response to the nitrate concentrations used, the results showed almost no change in eDNA. (Iyer et al. 2012) described their work that eDNA and other components contribute to the formation of biofilm. On the other hand, when crystal violet was used to detect the overall biofilm, the crystal binding to biofilm increased and reached 2.5 fold when 50 mM nitrate was added. This result indicates that there is another component in the biofilm that was not detected using the abovementioned fluorescence dyes but is present and participates in the biofilm formation of the Bacillus sp. in the presence of nitrate. Shao et al. (2019) reported variation in polysaccharide and protein ratios of extracellular polymeric substances during a nitrification process. This confirms that nitrate presence modulates biofilm components.
SEM images shown in Fig. 5 represent all the samples at 2 magnifications, 750X and 5000X. The images reflect an increase in matrix binding the cells together, this matrix increases with the increase in nitrate concentration, consolidating the crystal violet result in Fig. 4.
The structural changes that took place when Bacillus sp. DN under study was incubated with different nitrate concentrations are shown in Fig. 6. FTIR spectra showed increased peaks as the concentration of nitrate increased, the evident peaks at 3247, 2972 cm-1 belong to CH2 and CH3 stretching, peak at 1735cm-1 belongs to ester carbonyl bond, while peaks located in the range from 1650 to 1000 cm-1belong to carbohydrate. A glucan or glycan, peak at 1542 cm-1 belong to protein in carbohydrate. Peaks in fingerprinting region below 1000 cm-1 belong to nucleic acids (Gieroba et al. 2020). The results indicate that a surface bound glycoprotein is present. The glycoproteins were reported as responsible for cell-cell recognition. The FTIR fingerprint for EPS highly resembles that obtained by Saravanan and Jayachandran (2008) except the peak at 1735 cm-1 that represents the ester carbonyl bond indicates the presence of para hydroxyl-butyrate compound (Tugarova et al. 2017).
The media components and addition of some compounds can affect the structural biofilm development (Pamp and Neilsen 2007). Nitrate in specific is very ubiquitous, it has been known to inhibit biofilm formation in Burkholderia pseudomallei, nitrate inhibited the secondary signaling compound c-diGMP which affected the biofilm formation through a signaling process through a nitrate transporter, Nar-K1 (Mangalea et al. 2017). This indicates that nitrate acts as a signaling molecule. An association between nitrate reduction and biofilm extracellular matrix was established in bacteria colonizing urinary tract highlighting the role of NarL, a response regulator in modulating the biofilm regulator CsgD in Escherichia coli (Martín-Rodríguez et al. 2020). Despite that the abovementioned information is for bacteria different than the one under study, yet it proves that nitrate can act as a signal transducer in Bacillus as well since Bacillus possesses a two-component system (Fabret et al. 1999) and this is what they all have in common.
Biophysical changes of Bacillus sp. in the presence of different nitrate concentrations
The cells are surrounded by cell wall with different layers each with different compositions and porous structure to allow exchange small molecules with their surroundings. The cell wall contains large amounts of polysaccharides and other natural polymers, which are charged groups that give the wall their electrical properties (Markx & Davey1999). The dielectric properties of cells' suspension represent a tool to investigate the behaviour of the membrane as a whole under different treatment conditions. They can provide a description through membrane conductivity and membrane permittivity about the dynamic and steric properties of membrane structure. These two parameters (membrane conductivity and permittivity) are important in investigating any occurring modifications in the overall membrane structure as a results of external treatment. The relative permittivity decreases as a function of frequency giving the well-known dispersion curve. The dielectric loss factor expresses the rate of conversion of electric energy to heat in the sample and appears as guassian curve all over the frequency range considered in this study. The area under the loss curve is proportional to the total concentration of dipoles in the material (Pethig, 1979). The relative permittivity, dielectric loss and conductivity at 1 kHz decreased from the control sample for 6, 12 and 25 mM nitrate concentration, then increase as the nitrate concentration increased to 50 mM (Fig. 7 a, b and c). The capacitance determines the amount of charge that can be stored across the membrane when the cell is exposed to an electric field, and depends strongly on the structure of cell wall and membrane. Each subunit of these structures acts as capacitor, their effective capacitance (Ceff) is determined by their relative positions. The response of the cell membrane to different concentration of nitrate treatment varies according to the nitrate concentration. In this study the effective capacitance (Ceff) showed decrease as the nitrate concentration increase up to 12 mM, and then increase to reach 3.8 fold the control value for the 50 mM nitrate concentration as shown in Fig. 7d.
The effect of nitrate on the decolorization of congo red
The choice of this strain was based on its ability to form biofilms, decolorize dyes in the presence of nitrate. The presence of nitrates affected the decolorization of congo red by Bacillus under study. Results in Fig. 8 shows that the ratio of biodegradation to adsorption was 80:20 in the absence of nitrates and 75:25 in the presence of 6 mM nitrates but shifted to 50:50 at 12, 25 and 50 mM nitrate concentration. The results show that azo reduction is at its best at low nitrate concentrations, whereas high nitrate concentrations affected the biodegradation, this result is in agreement with Cirik et al. (2013) who reported an adverse effect on azo dye reduction at high nitrate concentrations. Competition between nitrate and another electron acceptor compound such as azo dyes have been reported (Gomaa et al. 2017). The same competition was reported for a co-polluted media containing nitrate and perchlorate suggesting that nitrate has a lower energy barrier for proton and electron transfer (Lv et al. 2020). This electron competition affects the biodegradation performance of the bacteria. In the present study, this competition was compensated with an increase in biofilm formation that varied in thickness and components, providing functional groups that act as an adsorptive matrix, making, at the end, the treated wastewater clear.
Proposed model for sustainable bioremediation
Fig. 9 represents the SEM images of Bacillus sp DN growing on carbon granules and glass beads in the presence and absence of nitrates. The images show that the cells can form granular biofilm using 12 mM nitrate which was optimal for production of EPS (Fig. 4). EPS has been reported to be responsible for facilitating cell adhesion and cell-cell recognition for biofilm formation.
Biofilms are known to form with a thickness, the structure of the biofilm usually consist of 1) distal region which is the outer layer and its exposed to oxygen, and is suitable for nitrate reduction 2) medial bulk region which has less oxygen and can be termed anoxic, it is very suitable for microaerophilic dye decolorization and 3) proximal region which is located closest to the substrate on which the biofilm grows, the latter is anaerobic and is very suitable for butyrate or any other SCFA production or biopolymer (such as PHB), and would provide energy source for bacterial cells. This configuration of bacterial growth in the 3 layers would make the same bacteria act as nitrate reducing, azo dye reducing and butyrate producing, rendering the biofilm granules as a complete system suitable for textile wastewater bioremediation and ensure longevity of performance. Butyrate was produced by Bacillus subtilis under anaerobic fermentation (Rahimi et al. 2020). The spherical architecture proposed has been previously stated as an acceptable conceptual model for aggregates used in bioremediation under different oxygen levels (Aqeel et al. 2019). Fig. 10 represents the proposed model. This proposed model is currently the focus of undergoing investigation and work will be published soon.