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Environ. Eng. Res. 2021; 26(4): 200187 pISSN 1226-1025 https://doi.org/10.4491/eer.2020.187 eISSN 2005-968X Research Removal of biological organics in high-salinity wastewater produced from methylcellulose production and subsequent changes in the microbial community GueSoo Jo1, SeongWan Hong1,2, HyunGu Kim2, Zhuliping3, DaeHee Ahn1,2,† 1Department of Environmental Engineering and Energy, Myongji University, Yongin 17058, Republic of Korea 2BlueBank Co., Ltd., Business Incubator Center, Myongji University, Yongin 17058, Republic of Korea 3Wuxi YDS Environmental Protection & Energy Saving Co., Ltd., Environmental Protection Technology Building, Yixing 214200, P.R. China ABSTRACT The wastewater generated in methylcellulose (MC) production is characterized by high salinity and pH due to the residual sodium and chlorine separated from the methyl group. It is difficult to treat wastewater using the conventional activated sludge method because the high concentration of salt interferes with the microbial activity. This study confirms the biological removal of organic matter from MC wastewater using sludge dominated by Halomonas spp., a halophilic microorganism. The influent was mixed with MC wastewater and epichlorohydrin (ECH) wastewater in a 1:9 ratio and operated using a sequencing batch reactor with a hydraulic retention time of 27.8 d based on the MC wastewater. The removal efficiency of chemical oxygen demand (COD) increased from 80.4% to 93.5%, and removal efficiency had improved by adding nutrients such as nitrogen and phosphorus to the wastewater. In terms of microbial community change, Halomonas spp. decreased from 43.26% to 0.11%, whereas Marinobacter spp. and Methylophaga spp. increased from 0.50% to 15.12% and 7.51%, respectively. Keywords: Biological treatment, High salinity wastewater, Methylcellulose, Microbial community, Pyrosequencing 1. Introduction residual plasticizers and solvents. High concentrations of salt affect biological activity in the conven- tional activated sludge process [7], in which microorganisms have Although cellulose is a widely used natural polymer material, its a high osmotic pressure in their cells. High osmotic pressure can use is limited due to its low solubility. Cellulose derivatives have lead to the dehydration and degradation of cells and inhibit the been studied to address this issue and expand the scope of growth and activity of microorganisms [8]. However, halophiles applications. In particular, methylcellulose (MC) is a cellulose de- are known to dominate highly saline conditions and have the ca- rivative that resolves the insoluble properties of cellulose in organic pacity to remove organic compounds without inhibition [9–13]. and inorganic solvents, and has recently been used in diverse in- Although research on MC wastewater treatment has confirmed dustrial applications such as food, petrochemicals, and building the potential to substitute an external carbon source necessary materials [1-3]. for denitrification [14], there is insufficient research compared to MC wastewater is characterized by high levels of salinity and other cellulose-based wastewaters. A study on carboxymethyl cellu- organic matter. During MC production, chloride ions are separated lose (CMC) wastewater treatment, a cellulose ether (carboxy alkyl from chloromethane and sodium ions separated from sodium hy- series) similar to MC, was conducted using a physicochemical droxide are discharged as wastewater [4, 5]. In addition, MC pro- treatment method [15, 16] and a physicochemical-biological mixing duction involves the addition of a plasticizer (propylene glycol) method [17, 18]. Although these studies demonstrated effective and solvent (1-methoxy-2-propanol) [6], and the chemical oxygen organic matter removal, each of the physicochemical treatments demand (COD) concentration in the wastewater is increased by This is an Open Access article distributed under the terms Received April 20, 2020 Accepted September 09, 2020 of the Creative Commons Attribution Non-Commercial License † (http://creativecommons.org/licenses/by-nc/3.0/) which per- Corresponding author mits unrestricted non-commercial use, distribution, and reproduction in any Email: [email protected] medium, provided the original work is properly cited. Tel: +82-31-321-5901 Fax: +82-505-300-5901 Copyright © 2021 Korean Society of Environmental Engineers ORCID: 0000-0002-6031-3610 1 GueSoo Jo et al. used had the disadvantage of high energy consumption [16, 19]. 9, respectively. The SBR operation consisted of one cycle per Petrochemical and food processing wastewaters are similar to day under aerobic conditions, and air was introduced by an air MC wastewater with high salt and organic content [20, 21]. diffuser at the bottom of the reactor with a 2 L/min aeration High-salt wastewater is treated in parallel with physical and chem- flow rate. The SBR cycle times were controlled using a programable ical treatments [20, 22, 23], at great expense and energy ex- logic controller (PLC), and each cycle lasted for, with a feeding penditure [7]. Although studies using anaerobic sequencing batch of 10 min aeration of 23 h and 10 min, settling of 30 min, and biofilm reactors (AnSBBR) and membrane bioreactors (MBR) re- a decanting time of 10 min. The total operation period was 108 quire lower cost and energy consumption compared to phys- d, and the hydraulic retention time (HRT) was 2.78 d (exchange icochemical treatments [24, 21], extracellular polymeric sub- rate 36%). stances (EPS) released by microorganisms at high salt concen- In the initial operation, organic matter removal occurred without trations exhibit sub-optimal performance of the membrane the addition of phosphorus and nitrogen. The removal efficiency (biofilm and membrane) [25, 26]. in this study was less than 80% as the wastewater was nu- Other studies have applied several physicochemical methods trient-deficient and required for the biological reaction. To increase such as cohesion, distillation, photocatalysts, and membranes [15– removal efficiency, nutrients were administered as shown in Table 2 18, 20, 22-24]. However, there has been an absence of studies after 53rd day. The nutrients were used in the following reagents; demonstrating the removal of COD from MC wastewater using the nitrogen source being ammonium chloride (NH4Cl, SAMCHUN biological methods. The purpose of this study was to remove the Chemical, Korea), and the phosphorus source being potassium phos- COD of MC wastewater through a biological method under highly phate (KH2PO4, SAMCHUN Chemical, Korea). saline conditions using sludge dominated by halophiles. In this study, we aimed to identify the microbial community changes Table 2. The Change of Condition by Operating Period + -3 through pyrosequencing and provide useful insights into MC waste- Operation time NH4 -N PO4 -P water treatment. (d) (mg/L) (mg/L) Phase 1 0-53 - - 2. Material and Methods Phase 2 54-56 75 15 Phase 3 57-60 35.5 7.5 2.1. Properties and Characteristics of the Wastewater Phase 4 61-80 18.75 7.5 The wastewater used in the study was actual wastewater generated Phase 5 81-108 10 7.5 during MC and ECH manufacturing processes. MC wastewater has a high organic matter load of 40,000-60,000 mg/L COD; and its properties are shown in Table 1 [14]. In this study’s preliminary 2.3. Analysis of Microbial Community experiments, a foaming problem occurred when MC wastewater Changes in the microbial community were observed by py- was independently treated. The method of mixing ECH wastewater rosequencing analysis, carried out using the initial and end phases. was found to resolve this foaming problem. The ECH wastewater The supernatant was removed after centrifugation, and deoxy- is wastewater from other processes generated during MC manu- ribonucleic acid (DNA) was extracted using the FastDNA SPIN facturing and has lower salinity and COD concentrations than MC Kit for soil (MP Biomedicals). The polymerase chain reaction (PCR) wastewater. The ECH wastewater is characterized by a COD of (C1000 Touch thermal cycler, Bio-Rad) used 2 μL of extracted 150–1,500 mg/L, total nitrogen (TN) N.D., total phosphorus (TP) DNA and proceeded with the primary amplicon and secondary N.D., and an NaCl concentration of 2,000 mg/L. index. In the primary PCR amplicon, the initial denaturation was performed at 95°C for 3 min. After 25 cycles of denaturation (95°C, Table 1. The Compounds of MC Wastewater 30 s), the annealing (55°C, 30 s), extension (72°C, 30 s), and final Compound Composition(wt%) extension (72°C, 5 min) was conducted and finally fixed at 4°C. In the secondary index PCR, initial denaturation was performed Methanol 30 at 95°C for 3 min. After 8 cycles of denaturation (95°C, 30 sec), Propylene glycol 30 annealing (55°C, 30 s), extension (72°C, 30 s), final extension (72°C, 1-methyloxy-2-propanol 30 5 min) was performed and finally fixed at 4°C. Illumina MiSeq NaCl 12 was used for DNA sequencing, and the obtained nucleotide sequence was subsequently analyzed using CLcommunity™ (Chunlab Inc., Seoul, Korea). [27, 28]. 2.2. Reactor and Experimental Setup In this study, a laboratory-scale sequencing batch reactor (SBR) 2.4. Water Quality Analysis with a 5 L effective volume (length: 155 mm, width: 155 mm, Water quality analysis was performed with a filtrate using a glass + -3 height: 222 mm) was used (Fig. S1). The initial mixed liquor sus- fiber filter (GF/C). In addition, COD, NH4 -N, PO4 -P, and MLSS pended solids (MLSS) concentration was 6,000 mg/L and the seed were analyzed according to the standard method [29]. The shape sludge was dominated by Halomonas spp.. The influent used was of the sludge was observed at 40× magnification using an optical a mixture of MC and ECH wastewater, mixed at a ratio of 1 to microscope (OLYMPUS CX31). 2 Environmental Engineering Research 26(4) 200187 3. Results and Discussion a 3.1. Biological COD Removal Fig. 1 presents the concentration profiles and the COD removal efficiency in Phase 1. The average COD concentrations of the influent and effluent was 4,400 and 1,300 mg/L, respectively.

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