**3. Results**

#### *3.1. Identification, and Salt-Tolerance in the Chitinolytic Bacterium*

Seven chitin degrading strains were newly isolated from the marine soil, where strain YHY1 showed maximum chitin hydrolysis activity with a zone diameter of 20 mm on chitin agar plates. The 16S rRNA sequencing data of strain YHY1 revealed 96% similarity with *Oceanisphaera arctica* strain V1-41; therefore, strain YHY1 was designated as *Oceanisphaera arctica* YHY1. The phylogenetic position of strain YHY1 and other allied strains is depicted in Figure 2a. The obtained gene sequence (989 bp) was submitted to Genbank under accession no. MH590704. Furthermore, the salt tolerance in strain YHY1 showed better growth on increasing the salt concentration from 5 g L−<sup>1</sup> to 40 g <sup>L</sup>−1, suggesting the necessity of salt for growth (Figure 2b). This strain was able to tolerate up to 80 g L−<sup>1</sup> of salt concentration; however, concentrations above 80 g L−<sup>1</sup> significantly inhibited the growth of the bacterium.

**Figure 2.** (**a**) Phylogenetic position of *O. arctica* YHY1; (**b**) Salt tolerance in strain YHY1.

#### *3.2. Electrochemical Assessment of MFCs Fueled with Chitin Waste*

Depleting non-renewable energy resources has enabled researchers to discover new sustainable energy assets. In MFCs, microbes act as the biocatalysts that transform the chemical energy of organic residues into electricity [10,15]. In this study, halotolerant *O. arctica* YHY1 was investigated for electricity production using seafood processing waste chitin as the carbon source. As shown in Figure 3a, the performance of MFCs fueled with 1% chitin biomass was studied, with a fixed external resistance of 1000 Ω and 40 g L−<sup>1</sup> NaCl. After inoculating the MFCs with *O. arctica* YHY1, a rapid increase in electricity generation was observed in the MFCs, with a maximum current output density reaching 0.302 mA/cm<sup>2</sup> at 12 h. Higher current production in the initial period could be related to the free GlcNAc (12.311 mg g<sup>−</sup><sup>1</sup> chitin) that was produced during the pre-treatment of the chitin. A similar mechanism has been reported earlier in *Aeromonas hydrophila*, *Bacillus circulans*, and *Shewanella oneidensis* MFCs using chitin as the carbon source [7,11,12]. However, the current output density slightly dropped to 0.210 mA/cm<sup>2</sup> after 20 h; this might be due to depletion in free GlcNAc. Thereafter, the electricity generation was enhanced and remained almost constant until 216 h (0.228 mA/cm2). Previously, chitin or pure GlcNAc has been reported as an electron donor in electricity production in MFCs using various microbial catalysts. For instance, *Aeromonas hydrophila* and *Shewanella oneidensis* fed with 0.2% chitin produced 8.77 μA/cm<sup>2</sup> and 4.24 μA/cm2, whereas using pure GlcNAc produced 6.65 μA/cm<sup>2</sup> and 6.17 μA/cm<sup>2</sup> of current output density, respectively [11,12]. Similarly, *Arenibacter palladensis* and *Bacillus circulans* produced 15.15 μA/cm<sup>2</sup> and 26.508 μA/cm<sup>2</sup> current output density, respectively, in MFCs fed with 1% chitin [7,10]. However, in the present study, a stable and higher current output density of 0.228 mA/cm<sup>2</sup> (216 h) was observed in *O. arctica* YHY1 using 1% chitin and 40 g L−<sup>1</sup> salt concentration. Salinity played an important role in the MFCs in the present study. The MFCs were supplied with 40 g L−<sup>1</sup> NaCl as the optimized salt concentration for *O. arctica* YHY1. The salinity of the media has a positive impact on electricity generation

as it decreases the internal resistance of the system and increases media conductivity. Furthermore, higher salinity can also prevent the acidification of the media due to the fast transfer of H+ ions from the anode to the cathode [9,16].

**Figure 3.** (**a**) Performance of strain YHY1 in MFCs fed with 1% chitin, 40 g L−<sup>1</sup> NaCl, and external load of 1000 Ω; (**b**) CV of the MFCs fed with chitin. Plots represent the CV of media without inoculum (0 h), CV of media at 216 h of growth, CV of cell-free metabolites with a new anode.

CV studies were executed to reveal the electrochemical behavior of *O. arctica* YHY1 in MFCs fed with chitin. As depicted in Figure 3b, the initial CV profile of chitin media (0 h) without bacterial inoculation did not show any oxidation-reduction peaks, indicating a lack of redox mediators in the medium. However, the CV of MFCs at 216 h after inoculation showed distinct oxidation peaks at −0.15 V (vs. Ag/AgCl), and +0.20 V (vs. Ag/AgCl), reduction peaks at +0.10 V (vs. Ag/AgCl), and a broad peak at −0.40 V (vs. Ag/AgCl) (Figure 3b). During the forward scan, a higher current output density of 0.302 mA/cm<sup>2</sup> was observed, indicating higher oxidation reactions were recorded, as compared to the reductions reported earlier [7,17]. The CV of the cell-free degradation metabolites after the filtration and insertion of a new anode showed a broad oxidation-reduction peak at +,−0.40 V (vs. Ag/AgCl), suggesting production of a low quantity of soluble redox mediators in the chitin degraded medium. The observed formal potential of 0.200 V (vs. Ag/AgCl) can be related to the outer membrane-bound cytochrome, which can transfer the electrons directly to the electrode without any external redox mediators, as reported earlier in *Shewanella* [18]. Further, a formal potential of −0.15 V (vs. Ag/AgCl) was detected similar to *Geobacter sulfurreducens,* which generally use omcB to transfer electrons through the electrode/biofilm interface [19–22]. Similarly, the low amount of flavins might have been produced by *O. arctica* YHY1 with the formal potential of 0.40 V (vs. Ag/AgCl), as reported earlier [23]. Therefore, from the CV data, it could be predicted that *O. arctica* YHY1 mainly utilizes a direct electron transfer pathway to transfer electrons directly to the electrode surface using membrane-bound cytochromes. Furthermore, this bacterium also partly employs the indirect electron transfer pathway using extracellular redox mediators to shuttle electrons to the electrode. However, more detailed study at a molecular level is needed to find the exact mechanism used by *O. arctica* YHY1 to transfer electrons from bacteria to the electrode surface.

#### *3.3. Chitin Degradation By-Products and Other Metabolites*

Chitinolytic microbes can hydrolyze chitin biopolymer into monomeric or dimeric GlcNAc units and utilize them as the source of energy. In the present study, monomeric GlcNAc was the main by-product of chitin hydrolysis by *O. arctica* YHY1 in MFCs. The concentration of GlcNAc at 24 h was 58.21 mg g<sup>−</sup><sup>1</sup> chitin, which was significantly increased to 192.01 mg g<sup>−</sup><sup>1</sup> at 120 h. However, after 120 h, the concentration of GlcNAc was gradually decreased to 76.22 mg g<sup>−</sup><sup>1</sup> at 216 h. From these results, it could be concluded that until 120 h, chitin was efficiently degraded, with simultaneous utilization of GlcNAc. Thereafter, depletion in the chitin concentration in media might have terminated the chitinase-producing machinery or inhibited the enzyme activity due to the generation of toxic intermediate metabolites [7,24]. However, *O. arctica* YHY1 continued to utilize the produced GlcNAc with the concurrent electricity generation until 216 h. During hydrolysis of the chitin, several metabolites were detected in the MFCs, with lactate, acetate, propionate, and butyrate as the prominent metabolites. The highest concentration of acetate, 5.901 mM (144 h), was detected, followed by butyrate, 3.572 mM (96 h), lactate, 0.932 mM (96 h), and propionate, 0.157 (120 h) (Figure 4a). Acetate, lactate, and propionate were most preferred by *O. arctica* YHY1, with 1.593 mM, 0.110 mM, and 0.099 mM concentrations, respectively, remaining unutilized at 216 h. However, butyrate was strain YHY1's less favored electron donor (2.533 mM; 216 h). Although the GlcNAc content was depleted after 120 h of growth, the current output density remained stable as the bacterium also utilized produced metabolites like lactate and acetate for growth, which correlates with the previous report on chitin as the carbon source in MFCs [7]. In *Shewanella*, lactate is first produced, followed by pyruvate, and acetate, and thus the carbon source preference can be in the following order: lactate→pyruvat→acetate [25]. The lactate concentration was higher at 96 h whereas acetate was higher at 144 h, although both concentrations decreased after reaching the maximum concentration, suggesting lactate can be oxidized to acetate [26]. During chitin hydrolysis, the production of metabolites like lactate, acetate, butyrate, succinate, format, and propionate was reported earlier in *Shewanella oneidensis*, *Bacillus circulans*, *Arenibacter palladensis*, and *Aeromonas hydrophila* [7,10–12].

SEM investigation of the anodic biofilm of *O. arctica* YHY1 revealed a thick biofilm formation on the anode surface (Figure 4b). This result could also be correlated with the CV data, suggesting that membrane-bound cytochromes were principally involved in direct electron transfer to the anode.

#### *3.4. Investigating Structural Changes in Chitin Polymer in MFCs*

As shown in Figure 5a, chitin biopolymer has a typical band pattern for amide bonds at 1660 and 1630 cm<sup>−</sup><sup>1</sup> (amide I), 1558 cm<sup>−</sup><sup>1</sup> (amide II), 1318 cm<sup>−</sup><sup>1</sup> (amide III), and 693 cm<sup>−</sup><sup>1</sup> (amide V) [7,27]. Similarly, an IR peak at 1028 cm<sup>−</sup><sup>1</sup> assigned to stretching vibration for –C–O–C of the glucosamine ring, and a peak at 890 cm<sup>−</sup><sup>1</sup> related to ring stretching for

β-1,4 glycosidic bonds were detected in the chitin [28]. Nevertheless, on the degradation of chitin in MFCs, the band intensity at 1660 cm<sup>−</sup><sup>1</sup> (amide I), 1378 cm<sup>−</sup><sup>1</sup> (amide III), and 1080 cm<sup>−</sup><sup>1</sup> (C–O stretching) substantially decreased, suggesting breaking of the C–O and C–H bonds [7,10]. Likewise, the XRD analysis revealed alterations in the crystallinity of the chitin before and after degradation (Figure 5b). The diffraction pattern of chitin before degradation showed distinct peaks at lattice (020), (110), (120), (101), and (130) [7]. Strong reflections were detected at 2θ 9.45◦, 19.05◦, and 31.46◦, whereas other peaks were detected at 12.93◦, 20.09◦, 23.60◦, and 26.51◦. The crystalline index (CrI) of the chitin before and after degradation was calculated considering reflections at (020) and (110). Initial CrI(020) and CrI(110) of the chitin were 74.41% and 83.60%, respectively, which significantly decreased to 67.96% CrI(020) and 81.15%(CrI110), respectively, after the degradation of the chitin by *O. arctica* YHY1. Further, peaks at 2θ ∼= 23 related to the polysaccharide structure of chitin showed broad scattering due to the hydrolysis of the chitin, indicating a decrease in the crystallinity [7]. Likewise, a sharp peak detected at 31.46◦ was significantly reduced on the degradation of the chitin biomass.

**Figure 5.** Structural changes in the chitin before and after degradation. (**a**) FTIR; (**b**) XRD.
