**1. Introduction**

The seafood processing industry produces a substantial amount of wastewater, mainly containing soluble, colloidal, and particulate matter. Among crustaceans, the total production of shrimp reached 5.03 million tons in 2020 and is estimated to increase to 7.28 million tons by 2025, with a compound annual growth rate of 6.1% from 2020 to 2025 and 67.6 billion USD turnover [1]. Asia alone contributes more than 80% of the global shrimp production, where Thailand is the major exporter of cultivated shrimp to the USA, Canada, Europe, South Korea, and Japan [2]. Depending on the market requirements, shrimp is exported or stored in frozen conditions with or without an outer shell. For shrimp processing, a huge amount of water is required, generating around 1000 L of highly polluted wastewater per ton of shrimp [1,3]. Shrimp processing produces 50–60% of solid waste comprising the head, viscera, and shell, which are discarded as the by-products generated in processing. The biochemical composition of shrimp waste mainly contains 15–46% chitin, 30–60% minerals, 10–40% protein, and 10–40% lipids [1,4]. Similarly, the salinity of the seafood processing wastewater is another important factor that mainly depends on the products or species being processed. The precooking or

**Citation:** Gurav, R.; Bhatia, S.K.; Choi, T.-R.; Kim, H.-J.; Lee, H.-J.; Cho, J.-Y.; Ham, S.; Suh, M.-J.; Kim, S.-H.; Kim, S.-K.; et al. Seafood Processing Chitin Waste for Electricity Generation in a Microbial Fuel Cell Using Halotolerant Catalyst *Oceanisphaera arctica* YHY1. *Sustainability* **2021**, *13*, 8508. https://doi.org/10.3390/su13158508

Academic Editor: Dino Musmarra

Received: 15 June 2021 Accepted: 26 July 2021 Published: 29 July 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

brine treatment for the canning of shrimp generates wastewater, with the NaCl concentration ranging between 20–30 g L−<sup>1</sup> [5].

Chitin is the second most abundant natural biopolymer, consisting of monomeric units of GlcNAc linked with (1,4)-β-linkages [6]. The microbial chitinase (EC 3.2.2.14) is a glycoside hydrolytic enzyme capable of hydrolyzing chitin into GlcNAc or oligomers and utilizing it as a source of carbon for growth and development [7]. Chitin waste has been partly used as animal feed, as a component of aquaculture feed formulation, and for the recovery of bioactive molecules. However, a large amount of chitin biomass is being wasted, which increases environmental pollution [1]. However, particulate substrates like chitin can be inexpensive, easily available, and renewable feedstock in microbial fuel cells (MFCs) for electricity production [8]. The MFC is a bio-electrochemical system that can break and utilize chitin more effectively compared to normal fermentation conditions, owing to the anode working as an electron acceptor [7,8]. Several natural and synthetic substrates, including acetate, glucose, lactate, amino acids, butyrate, formate, fumarate, alcohols, or complex carbohydrates like cellulose, sucrose, molasses, starch, or industrial and domestic wastewater, were widely researched as fuels for MFCs [8,9]. However, high production costs and easy depletion have constrained the use of synthetic substrates in MFCs. Nevertheless, chitin waste can be an alternative synthetic substrate source for sustainable energy production by using bacteria as a catalyst to oxidize organic substrates directly into electrical energy. Previously, chitin has been utilized as a substrate by *Bacillus circulans*, *Arenibacter palladensis*, *Shewanella oneidensis*, *Aeromonas hydrophila*, or as sediment wastewater-based systems for electricity production [7,10].

Therefore, in the present study, a newly isolated halotolerant marine bacterium *O. arctica* YHY1 capable of hydrolyzing chitin waste was studied as a biocatalyst for electricity generation in MFCs. Furthermore, the electrochemical parameters, the structural changes in chitin before and after degradation, and the by-products or metabolites of degradation were analyzed.

## **2. Materials and Methods**

#### *2.1. Chitin Preparation, Isolation, and Identification of Chitin Degrading Microbes*

The shrimp shell chitin was obtained from the local seafood processing unit near Seoul, South Korea. The obtained chitin biomass was washed, dried, and pre-treated according to the method reported earlier by Gurav et al. [10]. The resulting colloidal chitin was dried and stored in a refrigerator for further applications. Isolation of chitinolytic microbes was performed using marine soil collected from a beach near Geoje (34◦51-16.5-- N 128◦43-43.9-- E), Eastern Sea of South Korea. In brief, one gram of soil was serially diluted and smeared on the chitin agar plates containing (g <sup>L</sup>−1) 0.7-KH2PO4, 0.3-K2HPO4, 5.0-NaCl, 0.5-MgSO4, 0.0001-ZnSO4, 0.0001-MnSO4, 10-chitin, and 25-agar [6,10]. Seven morphologically different strains showing clear zones were isolated and cultured as a monoculture on chitin-agar plates. These seven strains were initially tested for electric current production in MFCs supplemented with 1% chitin. Based on its higher chitin degradation and electricity generation ability, strain YHY1 was selected for identification by using 16S rRNA gene sequencing.

#### *2.2. MFC Setup and Electrochemical Analysis*

As shown in Figure 1, a dual-chamber MFC consisting of cathodic and anodic chambers separated by a proton exchange membrane (Nafion 212, Omniscience, Yongin, Korea) was assembled. The anodic chamber was equipped with 2.25 cm<sup>2</sup> carbon felt and a silver reference electrode (Ag/AgCl), whereas the cathodic chamber contained 4 cm<sup>2</sup> platinumcoated carbon felt. Degassed growth media containing (g <sup>L</sup>−1) 0.7-KH2PO4, 0.3-K2HPO4, 40-NaCl, 0.5-MgSO4, 0.0001-ZnSO4, 0.0001-MnSO4, and 10-chitin was inoculated with 1% *v/v* inoculum with an optical density of 0.9 ± 0.05 at the anodic chamber, whereas 50 mL phosphate buffer (pH 7.0; 50 mM) containing 50 mM ferricyanide was filled at the cathode chamber. The anode and cathode were connected through a potentiostat (WizECM-8100

premium, Wizmac, Daejeon, Korea) and operated as a closed-circuit using an external resistance of 1000 Ω [7,8,11,12]. The current output density (mA/cm2) of the system was recorded and plotted versus time (h). To investigate the electrocatalytic behavior and interaction between the redox mediator, electrode, and anodic biofilm, the CV was performed using a three-electrode system including working, counter, and reference electrodes with a 10 mV/S scan rate and a +1 V to −1 V potential range.

**Figure 1.** Setup of the dual-chamber MFC.

#### *2.3. Analysis of Degradation Products*

HPLC (Young Lin, YL-9100, Seoul, Korea) investigation was performed to quantify free GlcNAc in the degradation media using a C18 column (ZORBAX, SBC18) and acetonitrile: water (20:80) as the mobile phase [7,10]. Further, the metabolic profiling of the chitin degradation media was performed using HPLC (Bio-Rad, Hercules, CA, USA) equipped with Bio-Rad Aminex HPX-87H column and 5 mM H2SO4 as the solvent phase [13].

#### *2.4. SEM, XRD and FTIR Analysis*

SEM (Hitachi TM4000Plus, Tokyo, Japan) was performed to investigate the anodic biofilm [9]. The FTIR (Nicolet 6700, Thermo Fisher Scientific, Waltham, MA, USA) was executed to verify the structural changes in the chitin before and after degradation in MFCs [7,10], whereas XRD (D8 ADVANCE-DAVINCI, Bruker, Bremen, Germany) was examined to determine changes in the crystallinity of chitin. The crystalline index (CrI, %) was calculated from the XRD data using the following equations [10,14].

$$\text{CrI}\_{020} = \left[ (\text{I}\_{020} - \text{Iam}) / \text{I}\_{020} \times 100 \right] \tag{1}$$

where I020 and Iam is the maximum intensity at 2θ ∼= 9◦ and the intensity of amorphous diffraction at 2θ ∼= 16◦, respectively.

$$\text{CrI}\_{110} = \left[ (\text{I}\_{110} - \text{I}\_{\text{am}}) / \text{I}\_{110} \times 100 \right] \tag{2}$$

where I110 is the maximum intensity at 2θ ∼= 20◦.
