Literature DB >> 32428009

Pilot plant study on nitrogen and phosphorus removal in marine wastewater by marine sediment with sequencing batch reactor.

Jinsoo Kim1, Sangrim Kang2, Hyun-Sook Kim2, Sungchul Kim3, Sang-Seob Lee1,2.   

Abstract

Effective biological treatment of marine wastewater is not well-known. Accumulation of nitrogen and phosphorus from land-based effluent is a crucial cause of red-tide in marine systems. The purpose of the study is to reduce nitrogen and phosphorus in marine wastewater with a pilot plant-scale sequencing batch reactor (SBR) system by using marine sediment as eco-friendly and effective biological materials, and elucidate which bacterial strains in sludge from marine sediment influence the performance of SBR. By applying eco-friendly high efficiency marine sludge (eco-HEMS), the treatment performance was 15 m3 d-1 of treatment amount in 4.5 m3 of the reactor with the average removal efficiency of 89.3% for total nitrogen and 94.9% for total phosphorus at the optimal operation condition in summer. Moreover, the average removal efficiency was 84.0% for total nitrogen and 88.3% for total phosphorus in winter although biological treatment efficiency in winter is generally lower due to bacterial lower activity. These results were revealed by the DNA barcoding analysis of 16s rRNA amplicon sequencing of samples from the sludge in winter. The comparative analysis of the bacterial community composition in sludge at the high efficiency of the system showed the predominant genera Psychromonas (significantly increased to 45.6% relative abundance), Vibrio (13.3%), Gaetbulibacter (5.7%), and Psychroserpens (4.3%) in the 4 week adaptation after adding marine sediment, suggesting that those predominant bacteria influenced the treatment performance in winter.

Entities:  

Year:  2020        PMID： 32428009      PMCID： PMC7236998          DOI： 10.1371/journal.pone.0233042

Source DB:  PubMed          Journal:  PLoS One        ISSN： 1932-6203            Impact factor:   3.240


Introduction

Nitrogen (N) and phosphorus (P) released from land-based effluents is believed to be one reason which have increased N and P in the ocean [1]. Sequentially, accumulation of these nutrients could cause the harmful algal blooms (HABs) in marine coastal regions [2]. HABs as a pollution source bring out imbalanced marine ecosystems despite marine environmental precautions and protection efforts [2-5]. When HABs occur regularly in fishery areas, fishery production, marine ranching, and aquaculture in coastal or inland areas must be completely protected to prevent economic loss in the fishery and marine industries [6, 7]. In recent decades, scientific activities and government monitoring in South Korea have found that increasing nitrogen and phosphorus contents in seawater quality of coastal areas from the impact of inland pollution sources [8, 9]. Therefore, treatment of effluent from fish farms and coastal area is important in a long-term the prevention strategy. However, treatment of marine wastewater needs better understood because the high salinity of wastewater from land-based fish farms and fishery production facilities in the coastal area has hampered the formulation of an effective solution for biological treatment of wastewater [2, 10, 11]. For the reasons, physio-chemical methodologies such as ultraviolet irradiation/ozone, reverse osmosis, ion exchange, electro-dialysis, and photo catalysis have been applied to the marine wastewater, but they are very expensive and have caused secondary problems such occurrence of biofilm and derived chemicals [12, 13]. Thus, biological treatments have been proposed for more effective and safety methodology to remove nutrient salts in the high salinity of wastewater. The development of a sludge should have been preferred for effective application of biological method, because sludge should settle in highly saline water since marine wastewater can decrease settling of sludge that consists of various microorganisms by changing the bacterial community and lowering bacterial metabolism. Bacterial cell lysis occurs in unaccustomed bacteria in sludge to marine wastewater because of the osmotic difference in the treatment of marine wastewater [14-16]. Recent studies of the effective treatment of saline wastewater have utilized halo-tolerant marine bacteria, such as Halomonas sp., Aeromonas sp., Bacillus sp., Staphylococcus xylosus, and Vibrio diabolicus as the predominant microorganism. Our previous study found that Bacillus sp. KGN1 (KEMB 3401–006) efficiently removed nitrogen (N) with 86.0% removal efficiency (RE) for 10 h at initial 10 mg L-1 NH3-N and Vibrio sp. KGP1 (KEMB 3001–129) efficiently removed phosphorus (P) with 99.9% RE for 10 h at initial 10 mg/L PO43–P [17]. Moreover, aerobic granular sludge (AGS) instead of common activated sludge bacteria was used for improving settlement in the biological treatment of marine wastewater [10, 11, 17–19]. Thus, we also previously applied AGS to lab-scale SBR reactor, and found that AGS increased settlement of activated sludge for better efficiency in the biological treatment [18]. However, those studies showed the highly efficient performance in only the laboratory scale. The practitioners, managers and governors in marine industry always are longing to apply the economic solution to the field sites with more treatment amount. Meanwhile, biological materials also need more verification for well-adapted and economic value for the application to the field site. Thus, it is also required to study possible application of eco-friendly materials such marine sediment to the biological treatment. Moreover, understanding the predominant bacteria in the sludge is important to operate and manage for improving the performance of biological treatment of marine wastewater treatment. The nitrogen removal in wastewater treatment has well studied with nitrification bacteria such as ammonia-oxidizing bacteria (AOB), anaerobic ammonium oxidation (anammox) bacteria and denitrification bacteria [20-22]. Moreover, recent studies reported that phosphorus in wastewater treatment with high salinity was removed by denitrifying phosphorus removal (DPR) bacteria and phosphorus uptake metabolism [23, 24]. Thus, DNA barcoding has known as an useful metagenomics tool for how those effective bacteria in the bacterial community in sludge influenced and improved the treatment of marine wastewater [25]. In this study, we aim 1) to remove n class="Chemical">nitrogen and n class="Chemical">phosphorus in marine wastewater from a land based fish farm by marine sediment with a pilot plant-scale SBR; 2) to analyze the bacterial communities during adaptation of marine sediment to marine sludge (eco-HEMS). We hypothesize that the effective bacteria of the eco-HEMS improves the nitrogen and phosphorus removal and treatment performance of marine wastewater as bacteria adapted in the saline wastewater.

Materials and methods

Bacteria and eco-friendly high efficiency marine sludge (eco-HEMS)

Marine sediment in a volume of 0.5 m3 was collected from Jebudo Island, Hwaseong-si, South Korea (37°09’45.3” N, 126°37’19.5” E, S1(a) and S1(c) Fig) and sieved with 10 mesh (0.55 mm pore size) after removing tiny stones and debris. The sieved marine sediment was applied to a sequencing batch reactor (SBR) system in pilot plant-scale with SBR cycles (more description in the next section). Bacterial strains used in this study were described in our previous study [17], whereas Bacillus sp. KGN1 (KEMB 3401–006) and Vibrio sp. KGP1 (KEMB 3001–129) efficiently removes nitrogen and phosphorus. Those bacterial strains were routinely maintained and cultured in Difco 2216 marine broth (BD, Franklin Lakes, NJ, USA). Two bacterial strains were bulk-cultivated for 5 days in a 1.5 m3 bio-reactor (Nexus Co. Ltd., Seoul, South Korea) at a maintained temperature of 28°C and 1.5–2.0 mg L-1 of dissolved oxygen (DO), and then bacterial cells were collected with the high-speed tubular separator (Hanil SME Co. Ltd,, Seoul, South Korea) at 20,000 × g and a flow rate of 600 L h-1. In 2 week after adding marine sediment, the bacterial two strains pellets in 1 kg (wet weight) were added to the adapted marine sediment. During adaptation period for 2 weeks, marine sediment was maintained in an SBR cycle with a supplement of D-glucose, NH4Cl, and KH2PO4, and adjusted to a chemical oxygen demand of 100 mg L-1 by the dichromate method (CODCr) in the presence of 5 mg L-1 NH3-N and 1 mg L-1 PO4-3-P in the marine wastewater. The SBR cycling comprised four steps (influence/mix–aeration–settlement–idle/effluence). Meanwhile, aerobic granule sludge (AGS) in a volume of 1.0 m3 was employed in another setting as the control because AGS has high settlement with high nitrogen removal efficiency. The mixed liquor suspended solids (MLSS) concentration was adjusted to 1,500 mg L-1 for the setup since 1,500 mg L-1 of MLSS showed the highest efficiencies in the previous lab-scale reactors study.

Pilot plant-scale SBR system and operation conditions

The pilot plant-scale SBR biological system was designed as 10 m3d-1 of daily wastewater flow (Q) with 4.5 m3 of SBR reactor in a total volume (VT), 2.5 m3 of filling volume (VF), and 6 h cycle -1 of time per cycle (TC) from previously obtained parameters in lab-scale data [17, 18], and setup in Tongyeong-si, Gyeongsangnam-do, South Korea (34°49'31.82"N 128°20'10.17"E, S1(b) and S1(c) Fig). Tongyeong is located in the southern-coastal area of South Korea in which red tide frequently occurred according to the statistical data [26]. The optimal conditions obtained from the lab-scale reactor were applied to the pilot plant-scale SBR system at the setup. The pilot-sn class="Chemical">cale SBR biologin class="Chemical">cal system had been in operation for approximately 2 years (June 2015 to January 2017). Marine wastewater was used as the effluent from the land-based fish farms, Tongyeong. The system is depicted in Fig 1.
Fig 1

Schematic diagram of the SBR biological treatment system for effluent from the land-based fish farm.

Marine wastewater inpan> the wastewater storage tank flowed into the influence storage tank [1.2 m (W) × 1.7 m (L) × 1.8 m (H)] and then was pumped to the SBR reactor [2.0 m (W) × 1.7 m (L) × 1.8 m (H)], which was where the eco-HEMS sludge biologically reacted to remove nutrient salts in the marine wastewater. The treated water then was decanted to the effluent storage tank [1.2 m (W) × 1.7 m (L) × 1.8 m (H)] for external discharge. The pilot-scale SBR system was regulated with an auto-control system comprising an electronic touch panel placed in the container (3.0 m × 9.0 m). The SBR reactor is the single reactor with 4.5 m3 of VT housing nine aerators and a decant system. Unlike expectation with 6 h cycle-1 of TC, eco-HEMS showed higher treatment performance, so the pilot plant-scale SBR operated 4 h cycle-1 of TC with following four stages: the influence stage (0.5 h) within the aeration/ mixing stage (3.0 h), settlement stage (0.5 h), and idle/effluence stage (0.5 h). The system operated with 7.2 h hydraulic retention time (HRT), 24.5 d solids retention time (SRT) and exchange ratio (VF/VT) of 0.56, since the filling volume (VF) was 2.5 m3 cycle-1. As reducing reaction time, Q was 15 m3 d-1. Based on previous studies, MLSS was adjusted and routinely maintained at 1,500 mg L-1, and the mixed liquor volatile suspended solids (MLVSS) were adjusted and routinely maintained at approximately 1,200 mg L-1 (Table 1).
Table 1

Optimal operation conditions of pilot plant-scale SBR biological treatment for marine wastewater with eco-HEMS during operation period (2 months, n = 360 cycles).

Operation conditionseco-HEMSAGS
Q (m3 d-1)15.010.0
VT (m3)4.54.5
VF (m3)2.52.5
TC (h • cycle-1)4.06.0
Cycles • d-16.04.0
HRT(h)7.210.8
SRT (d)a24.525.0
F/M (g COD • g MLSS-1 d-1)0.2780.185
MLSS (mg L-1)1,5001,500
MLVSS (mg L-1)1,2001,200

* a, SRT maintained 24.5 d in the winter, but 20.0 d in the summer (AGS also maintained 20.0 d in the summer).

* a, SRT maintained 24.5 d in the winter, but 20.0 d in the summer (n class="Chemical">AGS also mainpan>tainpan>ed 20.0 d inpan> the summer). Although the nutrient quality in the marine wastewater varied, the inpan>fluence was adjusted with the daily supplementation of D-glucose (acetate for AGS), NH4Cl, and KH2PO4 depending on the purpose of the study. During the operating period, profiling of eco-HEMS and AGS was done in the pilot-scale SBR system at various CODCr: N: P (below C: N: P) ratios. Data were collected in all seasons.

Analysis of environmental factors, nutrients, and kinetic parameters

During the operation periods, analysis of chemicals including the MLSS was routinely performed. The environmental factors of marine wastewater such as temperature, salinity, DO, and pH were also routinely determined by each portable equipment. CODCr, MLSS, MLVSS, N, and P were analyzed as following the standard methods [27]. CODCr analysis was carried out by soluble CODCr after filtration. CODCr, ammonia N (NH3-N), nitrate N (NO3-N), total N (T-N), phosphate P (PO43-P), and total P (T-P) were determined using a model DR4000 spectrophotometer (HACH Co., Frederick, MD, USA) followed as the HACH manual. The REs of CODCr, NH3-N, and PO43-P were quantified using the initial and final concentrations. For evaluation of sludge settlement, sludge volume in settlement for 30 min (SV30) was determined as following the standard methods [27]. For the operational evaluation of the SBR system in the pilot plant-scale, analytical data were used to calculate the kinetic parameters of HRT, SRT, bacterial growth yield (Y), specific growth rate (μ), and solid volume index (SVI) using the appropriate formula. For comparative analysis of the efficiency between eco-HEMS and AGS, specific efficiency component per unit sludge (MLSS) was determined for NH3-N, NO3-N, and P removal according to reaction time in response to bacterial growth. Specific substrate utilization rate (SSR, Eq 1), specific nitrification rate (SNR, Eq 2), specific denitrification rate (SdNR, Eq 3), and specific phosphate uptake rate (SPR, Eq 4) were calculated using the measured concentrations as follows: Where S0, is the initial CODCr concentration; S is the final CODCr concentration; N0 is the initial NH3-N concentration; N is the final NH3-N concentration; dN0 is the initial NO3--N concentration; dN is the final NO3--N concentration; P0 is the initial PO43--P concentration; P is the final PO43--P concentration; X0 is the initial MLSS; and X is the final MLSS.

Analysis of bacterial community

Each 100 mL of sludge sampled from the 10 sites of the SBR reactor was collected and mixed, and then 50 mL of them was used for every bacterial community analysis. Sampling was performed every week after the marine sediment had adapted to the eco-HEMS. The bacterial community obtained in the summer and winter using the optimal operating conditions were compared. Comparative analysis of the bacterial community of AGS in the summer was performed. All bacterial communities were analyzed by DNA barcoding sequencing outsourced to Macrogen Inc. (Seoul, South Korea). Sequencing was performed on the Miseq 15027617 system with V4 region in 16S ribosomal RNA (Illumina Inc., San Diego, CA, USA) and the generated raw images were analyzed MiSeq Control Software v2.2 for system control and base calling through Real Time Analysis. v1.18 integrated primary analysis software. The base calls binary was converted into FASTQ utilizing the bcl2fastq (v1.8.4) package (Illumina). Adapters were trimmed away from the reads [28]. Taxonomic assignment of the sequenced reads was carried out using previously described methodology [18]. From the obtained data, a statistical analysis of the bacterial community composition was weekly performed during marine sludge adapted Eco-HEMS sludge from the beginning to fifth week. The relative difference in bacterial composition during the activation of the marine sediment was analyzed by principle component analysis (PCoA) to evaluate the bacterial community shift.

Results and discussion

1. Performance of the SBR biological treatment system with eco-HEMS

The pilot plant-scale study was performed to scale up the treatment volume with high efficiency and to provide a system that would be useful for fish aquaculture managers and practitioners using effluent from the land-based fish farms. Daily wastewater treatment amount (Q) is important. The SBR biological treatment system was designed and setup with a Q value of 10.0 m3 d-1 based on the data of the lab-scale study. Unexpectedly, the Q of the pilot plant-scale SBR biological treatment with eco-HEMS increased from 10.0 m3 d-1 to 15.0 m3 d-1, since the reaction time in the SBR cycle shorten from 6 h cycle-1 to 4 h cycle-1 (Table 1). As a result, HRT could decrease to 7.2 h. SRT was maintained and operated for 20 d, except in winter when it increased to 24.5 d.

Environmental factors and seasonal performance

During the operation period, the environmental factors of marinpan>e wastewater from land-based fish farms were investigated. These included temperature, pH, and salinity, which were important parameters to be considered when operating and maintaining the activated sludge (both AGS and eco-HEMS) in the pilot plant-scale SBR system. Trends of temperature and salinity showed seasonal fluctuation, but there was no significant difference in seasonal pH trends on average (Table 2 and S2 Fig).
Table 2

Environmental factors of marine wastewater from land-based farming on each season during operation periods.

Spring, from March to May; Summer, June to 21 September; Autumn, from 22 September to November; Winter, from December to February.

Season Environmental factorSpringSummerAutumnWinter
pHAVE7.87.78.07.7
min-MAX7.3–8.36.5–8.27.5–8.77.4–8.3
Temperature (°C)AVE14.222.714.59.02
min-MAX8.0–21.019.7–27.010.0–22.32.0–13.0
Salinity (PSU)AVE33.631.431.233.9
min-MAX32.4–34.530.2–32.630.3–32.632.6–34.9

Environmental factors of marine wastewater from land-based farming on each season during operation periods.

Spring, from Marn class="Chemical">ch to May; Summer, Junpan>e to 21 September; Autumn, from 22 September to November; Winter, from December to February. From monitoring results of environmental factors, the sprinpan>g and autumn temperatures were similar. Interestinpan>gly, seasonal salinpan>ity decreased from spring to summer and autumn, then increased again from winter to spring. Salinity in summer was similar to that in autumn, and salinity in winter was similar to that in spring. The average salinity in spring and winter was lower (average 2.2–2.7 PSU) than that in winter and autumn. The pH was highest in autumn and the pH difference was greatest in summer. The pH and salinity values were due to the typhoon and rainy season that occurs in the summer and early autumn on the south coastal area of South Korea. The concentration of DO, which is another influential environmental factor, was not considered because the equalization tank of the SBR system takes role of storage and equalization of marine wastewater and air was supplied to the SBR tank of the system (Fig 1). From all data, the seasonal performance results of the pilot plant-scale SBR biological treatment indicated that eco-HEMS showed similar REs in both spring and autumn with on the average, 75.8% of CODCr (initial: 149.0 mg L-1), 54.9% of total nitrogen (T-N, initial: 9.3 mg L-1), 65.5% of total phosphorus (T-P, initial: 1.8 mg L-1). In summer, eco-HEMS showed, on the average REs, 51.2% of CODCr (initial: 100.8 mg L-1), 70.9% of T-N (initial: 5.9 mg L-1), 47.9% of T-P (initial: 1.8 mg L-1). Surprisingly, in winter, eco-HEMS showed higher average REs with 65.5% of CODCr (initial: 158.0 mg L-1), 82.0% of T-N (initial: 9.7 mg L-1), 79.7% of T-P (initial: 2.0 mg L-1). Meanwhile, AGS showed lower average REs in winter with 67.4% of CODCr (initial: 164.8 mg L-1), 53.3% of T-N (initial: 6.7 mg L-1), 52.5% of T-P (initial: 2.0 mg L-1) (S1 Table and S3 Fig). CODCr concentration in effluent was below 40.0 mg L-1, T-N was below 5.0 mg L-1, and T-P was below 0.6 mg L-1. Common sea water was analyzed as averagely 42.0–43.0 mg L-1 of CODCr, 5.0 mg of T-N, and 0.5 mg L-1 of T-P.

1.2. Comparative analytical profile between eco-HEMS and AGS in the optimal conditions

In winter, n class="Chemical">eco-HEMS showed higher performann class="Chemical">ce in the pilot plant-scale SBR treatment system. Thus, the profiling of eco-HEMS was performed in winter with 100: 5: 1, 200: 5: 1 and 300: 5: 1 of C: N: P ratios, respectively. The eco-HEMS profile in the SBR system displayed optimal treatment efficiency in the winter when the C: N: P was 100: 5: 1 (Fig 2).
Fig 2

Analytical profiles of eco-HEMS in the winter season, with COD:N:P of 100:5:1 as the optimal condition.

Environmental factors (a), CODCr and MLSS (b), T-N, NH3-N, and NO3-N(c), and T-P and PO4 3--P(d).

Analytical profiles of eco-HEMS in the winter season, with COD:N:P of 100:5:1 as the optimal condition.

Environmental fan class="Chemical">ctors (a), n class="Chemical">CODCr and MLSS (b), T-N, NH3-N, and NO3-N(c), and T-P and PO4 3--P(d). During the profiling of eco-HEMS with nutrient removal, the temperature was not significantly changed, but DO and pH sharply decreased from 0 min to 75 min and increased again from 75 min to 180 min in SBR stage II (aeration and mixing). At the start of SBR stage III (settlement in anaerobic condition), pH was not change and DO spontaneously decreased. CODCr (initial: 103.5 ± 2.0 mg L-1) decreased in the first 60 min, with no change thereafter (final: 51.2 ± 5.9 mg L-1). The concentration of MLSS increased, with 1,460 ± 40 mg L-1 to 1,620 ± 20 mg L-1 during the total profile time of 240 min. MLSS displayed a logarithmic increase from 0 min to 60 min, and slightly increased from 60 min to 240 min. T-N (initial: 7.1 ± 0.5 mg L-1) constantly decreased from the initial time to 180 min in SBS stage II, and slightly decreased in the SBR stage III (final: 1.1 ± 0.5 mg L-1). NH3-N sharply decreased from the initial time to 180 min. NH3-N decreased and NO3--N simultaneously increased by nitrification from the initial time to 60 min, and NO3--N decreased from 60 min to 240 min by denitrification (initial: 5.2 ± 0.3 mg L-1, NO3- -N, 1.9 ± 0.4 mg L-1; final: NH3-N, 0.2 ± 0.3 mg L-1; NO3--N, 0.9 ± 0.6 mg L-1). P readily decreased from the initial time to 30 min, steadily decreased at the end of SBR stage II, and slightly increased in SBR stage III (initial: T-P 1.1 ± 0.1 mg L-1, PO43--P 0.8 ± 0.1 mg L-1; final: T-P 0.1 ± 0.1 mg L-1; PO43--P, 0.1 ± 0.1 mg L-1). The profiles of eco-HEMS when the C: N: P ratio was 200: 5: 1 and 300: 5: 1 are shown in the S4 and S5 Figs. The initial CODCr was higher and at the time when DO was zero became longer, and pH was also lower. The CODCr was reduced from the initial time to the end of SBR stage II, and the MLSS increased from 0 min to 60 min, and slightly increased from 60 min to 240 min. As the initial CODCr concentration was higher, the RE of T-N decreased and that of P slightly decreased. As the ratio of CODCr in the C: N: P was higher, removal of NH3-N was greater, but the removal of NO3--N was increased more by the nitrification of ammonia. P steadily decreased from the initial time to the end. Overall, the condition when C: N: P was 100:5:1 was optimal because REs of CODCr, N, and P were higher with consumption of DO by microbial growth, metabolism, and activity. Moreover, the N treatment efficiency by eco-HEMS was highest because the efficiency of nitrification and denitrification was the highest with lower biomass production. The P treatment efficiency by eco-HEMS was also highest. Comparatively, at the same condition (C: N: P is 100: 5: 1) in winter, the profile of AGS revealed lower microbial activity and treatment efficiency of organic C, N, and P with lower DO consumption (Table 3).
Table 3

Comparative results of CODCr, T-N, NH3-N, NO3- -N, T-P, PO4 3- -P Removal Efficiency (RE) between AGS and eco-HEMS in summer and winter season, when COD:N:P was 100:5:1 in the optimal condition.

SeasonSummerWinter
Applying Sludgeeco-HEMSAGSeco-HEMSAGS
Reaction Time (h) in a cycle04060406
CODCr (mg L-1)AVE110.940.1122.546.0103.551.2111.876.3
STD10.41.79.19.62.05.99.510.2
RE (%)63.962.550.631.8
T-N (mg L-1)AVE7.50.85.71.47.11.16.43.2
STD0.80.20.50.80.50.50.80.8
RE (%)89.375.584.049.6
NH3-N (mg L-1)AVE.6.00.15.20.05.20.25.01.2
STD0.90.10.60.00.30.30.30.9
RE (%)99.0100.095.876.8
NO3- -N (mg L-1)AVE.1.50.70.01.41.90.91.42.1
STD0.50.20.00.90.40.60.60.1
RE (%)50.6-51.5-46.8
T-P (mg L-1)AVE.1.70.11.71.11.10.11.40.7
STD0.30.10.20.10.10.10.20.2
RE (%)94.935.588.349.0
PO4 3- -P (mg L-1)AVE.1.20.11.30.90.80.11.00.6
STD0.20.10.20.10.10.10.10.2
RE (%)94.431.686.845.2
MLSS (mg L-1)AVE1535.01800.01520.01710.01460.01620.01543.31631.7
STD115.050.049.729.440.020.033.035.7
dX*265.0190.0160.088.3

(Summer: approximately 4 months data (n = 120), winter: approximately 3 months data; n = 90). AVE stands for average, STD stands for standard deviation, and dX* indicates the change of MLSS from the initial to the final reaction time.

(Summer: approximately 4 months data (n = 120), winter: approximately 3 months data; n = 90). AVE stands for average, STD stands for standard deviation, and dX* indin class="Chemical">cates the n class="Chemical">change of MLSS from the initial to the final reaction time. AGS showed, on the average, REs of 31.8% for CODCr, 49.6% for T-N, 76.8% for NH3-N, -46.8% for NO3--N, 49.0% for T-P, and 45.2% for PO43--P. In the same condition, the profiles of eco-HEMS and AGS in the summer showed different trends in the S6 and S7 Figs and Table 3. The temperature was slightly changed (23 ± 2°C) in both conditions. DO steadily decreased from the initial time to the end of an SBR cycle (eco-HEMS: 4 h cycle-1, AGS: 6 h cycle-1), and pH trends were similar to that in winter, in which pH decreased from the initial time to 120 min in the profile of eco-HEMS (180 min in the profile of AGS during the time when CODCr sharply decreased, pH increased again from 120 min to the end. The profile of eco-HEMS in the summer showed an averagely increase of 260 mg L-1 for MLSS (increase of MLSS in the profile of AGS: 190 mg L-1). Microbial growth that was evident as an increase of MLSS was much higher in summer than in winter, with the removal of CODCr (eco-HEMS: 110.9 ± 10.4 mg L-1 to 40.1 ± 1.7 mg L-1, 63.9% RE; AGS: 122.5 ± 9.1 mg L-1 to 46.0 ± 9.6 mg L-1, 62.5% RE). Interestingly, the trend of N removal in the eco-HEMS profile indicated effective nitrification and denitrification, with a higher RE of T-N compared to that of AGS (eco-HEMS: 7.5 ± 0.8 mg L-1 to 0.8 ± 0.2 mg L-1, 89.3% RE; AGS: 5.7 ± 0.5 mg L-1 to 1.4 ± 0.8 mg T-N L-1, 75.5% RE). Although the AGS showed a 100% RE of NH3-N, the RE of T-N was lower because concentration of NO3--N was high in the effluence (a slight decrease of NO3--N was evident from 120 min to 240 min). However, both AGS and eco-HEMS showed effective nitrogen activity in the summer using the same optimal condition. Regarding the removal of P, eco-HEMS displayed a markedly higher RE of P than AGS. The use of eco-HEMS resulted in the prompt removal of T-P and PO43--P within the first 30 min, while the use of AGS produced a slight decrease of both T-P and PO43--P during SBR cycles.

1.3. Kinetic parameters in the optimal conditions

The kinetin class="Chemical">c parameters were obtainpan>ed from data n class="Chemical">collected during the run of the SBR biological treatment system using optimal conditions (Table 4).
Table 4

Kinetic parameters’ average values obtained from the profile data between AGS and eco-HEMS application at each condition.

SeasonSludgeCOD:N:PF/M (g CODCr • g MLSS-1•d-1)Y (g MLVSS • g CODCr-1 •d-1)μ (g• m-3d-1)SV30 (mL•L-1)SVI (mL•g1)SSR (g CODCr removal • g MLVSS-1• d-1)SNR (g NH3-N removal • g MLVSS-1• d-1)SdNR (g NO3–-N removal • g MLVSS-1• d-1)SPR (g PO43–-P removal • g MLVSS-1• d-1)
Wintereco-HEMS100:5:10.2360.9170.6248054.81.0910.10440.02030.0136
200:5:10.4060.5340.8029057.01.8740.0662-0.01090.0234
300:5:10.6970.3951.0809058.42.5310.0831-0.00330.0172
AGS100:5:10.2410.7450.223200129.60.8940.0968-0.01670.0117
Summereco-HEMS100:5:10.2411.1230.9568052.10.8900.07420.00950.0143
AGS100:5:10.2690.7450.47115098.70.8950.0608-0.01640.0047

(n = 4 cycles)

(n = 4 n class="Chemical">cyn class="Chemical">cles) The optimal operation conditions (C: N: P = 100: 5: 1; F/M, 0.236–0.241 g CODCr • g MLSS-1 • d-1) were as follows: Q, 15.0 m-1d-1; TC, 4 h; numbers of cycles per day (NC), 6; HRT, 7.2 h; and SRT, 20.0 d (winter 24.5 d). As the COD ratio increased, the specific growth rate (μ) and SSR increased, but bacterial growth yield (Y) and SNR decreased. The SdNR and SPR were not significantly related. In the N and P removals, the use of eco-HEMS in winter produced higher SNR and SdNR at the C: N: P ratio of 100: 5: 1 than at the other ratios, but SPR was slightly lower at the 100:5:1 ratio than at the other ratios. Thus, a C: N ratio of 20: 1 was the optimal condition for the eco-HEMS-mediated removal of N. The kinetic parameters of eco-HEMS were much higher than those of AGS. At the optimal 100:5:1 ratio, the obtained kinetic parameters of eco-HEMS in winter were: Y, 0.917 g MLVSS • g CODCr-1 •d-1; μ, 0.624 g m-3 d-1; SV30, 80 mL L-1; SVI, 54.8 mL g-1; SSR, 1.091 g CODCr removal • g cell-1 • d-1; SNR, 0.1044 g NH3-N removal • g cell-1 • d-1; SdNR, 0.0203 g NO3- -N removal • g cell-1 • d-1; and SPR, 0.0136 g PO43--P removal • g cell-1 • d-1. The kinetic parameters of eco-HEMS in the summer were higher than those in the winter: Y, 1.123 g MLVSS • g CODCr-1 • d-1; μ, 0.956 g • m-3 d-1; SV30, 80 mL L-1, SVI, 52.1 mL g-1; SSR, 0.890 g CODCr removal • g MLVSS-1 • d-1; SNR, 0.0742 g NH3-N removal • g MLVSS-1 • d-1; SdNR, 0.0095 g NO3- -N removal • g MLVSS-1 • d-1; and SPR, 0.0143 g PO43--P removal • g MLVSS-1 • d-1. Interestingly, marine wastewaters generally decrease the settling of sludge during wastewater treatment due to high salinity [14]. Presently, the eco-HEMS marine sediment sludge displayed pronounced higher settling with low SVI values, even in the winter season. Subsequent sections in this paper provide further details of why the bacterial community in the eco-HEMS condition resulted in effective N and P removal with higher settling in winter.

2. Bacterial community diversity and richness

2.1. Adaptation and stabilization of marine sludge to eco-HEMS

As the marine sediment adapted and changed to eco-HEMS in winter, the bacterial community changed and stabilized with stable diversity and richness. Firstly, an analysis of the operational taxonomic units (OTUs) revealed the most diverse with 286 OTUs, 286 Chao1 and 7.57 Shannon’s index in the initial marine sediment, with a decrease to 126 OTUs by week 2 when the nitrogen and phosphorus removal bacterial strains applied (Table 5).
Table 5

Comparative results of diversity and abundance values (OTUs, Chao1, Shannon, Simpson) from bacteria community analysis.

SludgeSeasonWeekOTUsChao1ShannonSimpsonGoods Coverage
Eco-HEMSWinter02862867.570.991.00
11331415.070.921.00
21261264.330.891.00
31681744.650.921.00
41831873.690.771.00
52112113.040.631.00
Summer1902115.420.941.00
The values of OTUs and Chao1 inpan>creased again, but Shannon’s index decreased from week 3 as the SBR reactor system began operating. As the marine sediment adapted and changed to eco-HEMS, the Shannon index for the abundance and diversity of the bacterial community decreased from 7.57 to 3.04, and the Simpson index decreased from 0.99 to 0.63. These findings indicated the stabilization of the bacterial composition. PC plots revealed relative differences of the bacterial communities during the period of adaptation of the marine sediment to eco-HEMS, with the bacterial communities shifting relatively closer in the S8 Fig

2. 2. Relative abundance and taxonomic assignment of bacterial community

In the adaptation period of the marine sediment, the marine sediment changed to effective marine sludge with high performance including nitrogen and phosphorus removal. Bacterial community analysis revealed how effective bacteria (Bacillus sp. and Vibrio sp.) had dominated in the eco-HEMS during operation. In the phylum level analysis of the bacterial community, phylum Proteobacteria was the most abundant, with the relative abundance increasing to 76.8% in week 4, followed by Bacteroidetes, Planctomycetes, and Firmicutes (Fig 3 and S2 Table).
Fig 3

Analysis of bacterial community at the phylum level during the adaptation of marine sediment to eco-HEMS sludge.

Phylum Ban class="Chemical">cteroidetes n class="Chemical">consisted comprised approximately 20% of the bacterial community of eco-HEMS. In week 1, the relative abundance of phylum Cyanobacteria was 24.4% and phylum Firmicutes was 25.4%. In more detail, the most abundant 20 genera indicated a change of the eco-HEMS bacterial community, with the most pronounced change being the treatment of effluent from land-based fish farms (Fig 4 and S2 Table).
Fig 4

Top 20 genera concerning abundance rate during the adaptation of marine sediment to marine sludge.

(a) week 0, (b) week 1, (c) week 2, (d) week 3, (e) week 4, and (f) week 5.

Top 20 genera concerning abundance rate during the adaptation of marine sediment to marine sludge.

(a) week 0, (b) week 1, (n class="Chemical">c) week 2, (d) week 3, (e) week 4, and (f) week 5. There was no overwhelmingly predominpan>ant genus above 10% of relative abundance in the marine sediment in the beginning (week 0). Thirteen genera displayed relative abundance ranging from 1.1% to 5.6%. After week 1, Genus Bacillariophyta was predominant (24.4%), followed by Thioprofundum (7.8%), Robiginitalea (5.5%), and Sulfurovum (4.7%). In week 2, following the application of the high efficiency bacteria to eco-HEMS, the genera of Vibrio and Bacillus were predominant (relative abundance of 35.3% and 20.1%, respectively). Other dominant genera in terms of relative abundance were Thalassomonas (7.8%), Marinomonas (5.8%), and Pseudoalteromonas (5.0%). In week 3, nitrogen removal bacteria Vibrio was still predominant, but the relative abundance had decreased to 22.7%. Another predominant genus was Psychromonas (13.9%), followed by Gaetbulibacter (9.1%), Psychroserpens (5.4%), Cobetia (5.0%), and Bacillus (5.0%). In week 4, genus Psychromonas was predominant with a relative abundance of 45.6%. The genus Vibrio was also predominant with a relative abundance of 13.3%, which was a decrease from the week before. Other dominant genera in terms of relative abundance were Gaetbulibacter (5.7%) and Psychroserpens (4.3%). In week 5, the predominant genus was again Psychromonas (60.0%), followed by Gaetbulibacter (6.0%), Glaciecola (4.7%), and Psychroserpens (4.3%). The relative abundance of Vibrio sharply decreased to 1.0%. The data indicate that the predominant genera Psychromonas, Vibrio, Gaetbulibacter, and Psychroserpens could influence the treatment efficiency of eco-HEMS in winter. Especially, genus Psychromonas includes halophilic (high-salt adapted) and psychrophilic (low temperature adapted) species, which display various chemotrophic metabolic activities including nitrification and denitrification, and the synthesis of polyunsaturated fatty acids, such as eicosapentaenoic acid and docosahexaenoic acid [29-31]. In comparison to the bacterial community that developed in summer (S3 Table), phylum Proteobacteria was the most abundant in the eco-HEMS bacterial communities, with a relative abundance of 75.9%. Analytical results of the eco-HEMS summer bacterial community revealed the predominant phyla in terms of relative abundance were Planctomycetes (9.9%), Bacteroidetes (6.3%), Parcubacteria (1.3%), and Chloroflexi (1.1%). Unlike the bacterial community in winter, 11 of the top 20 genera displayed a relative abundance of more than 1% in the eco-HEMS summer bacterial community. The predominant genera with a relative abundance of more than 10% were Denitromonas (17.5%) and Vibrio (14.7%). Other genera of family Rhodobacteraceae had a relative abundance rate of 10.4%. Dominant genera were family Phycisphaeracea (9.7%), genus Arcobacter (8.9%), Roseovarius (4.5%), Leucothrix mucor (2.6%), and Bacillus (2.1%). Among them, genus Denitromonas is important in the settling of sludge in marine wastewater with pronounced reduction of nitrate [32, 33]. Finally, the bacterial community analyses revealed that the predominant genera in eco-HEMS were different between winter and summer, but the some genera in the marine sediment had mainly adapted to the marine wastewater for efficient removal of N and P. Thus, each predominant genus in summer or winter could be an important influence on the treatment efficiency and settlement. Moreover, the predominant phylum was Proteobacteria. The approximately 70% relative abundance rate of Proteobacteria revealed the presence of stable sludge with high RE in the SBR biological treatment for effluent from the land-based fish farms. Interestingly, the high efficiency bacteria (Vibrio and Bacillus) did adapt, but did not remain predominant for long-term. The high efficiency bacteria could not be determined how influential to the bacterial community, but bacterial community in eco-HEMS has been quickly stabilized after added those bacterial strains. Otherwise, anammox bacteria did not show in the bacterial community data. Lin et al. (2018) described that low temperature inhibited anammox process to treat wastewater [34]. However, denitrification and phosphorus uptake rate were high from the treatment data in optimal condition with above predominant genera, that is, those predominant genera Psychromonas, Vibrio, Gaetbulibacter, and Psychroserpens (in winter), Denitromonas, Vibrio, other genera of family Rhodobacteraceae, Phycisphaeracea, genus Arcobacter, Roseovarius (in summer) in eco-HEMS could make role for denitrifying phosphorus removal (DPR). Mandel et al. (2018) reported that denitrifying phosphorus removal bacteria adapted and increased in the sludge during biomass adaption [22, 24].

Conclusions

Eco-HEMS from marinpan>e sediment as a biological resource was applied to the SBR biological treatment system in a 2-year pilot plant-scale study for marine wastewater from land-based fish farms. The results demonstrate that eco-HEMS can improve the treatment efficiency of N and P in the high salinity marine wastewater with a daily treatment volume of 15 m3. The treatment efficiency of eco-HEMS is maintained in the winter as well as other seasons. The RE of N is markedly enhanced, as is settlement. The bacterial community appears capable of adapting and optimizing to eco-HEMS during the operation period of the SBR biological treatment. This eco-friendly and economic biological resource could contribute to an effective solution to reduce the cause of HABs by reducing the nutrients presence in the effluent from land-based fish farms or wastewater treatment in coastal areas. The lon class="Chemical">cations of marinpan>e-sedimenpan>t samplinpan>g site (a) and pilot plant setup (b) inpan> the South Korea (n class="Chemical">c). (n class="Chemical">DOn class="Chemical">CX) n class="Chemical">Clin class="Chemical">ck here for additional data file.

Analytical data of environment factors such as temperature, pH, and salinity in marine wastewater for the pilot plant-scale SBR treatment system during all operation period.

(n class="Chemical">DOn class="Chemical">CX) n class="Chemical">Clin class="Chemical">ck here for additional data file. Treatment of organin class="Chemical">c n class="Chemical">carbon (CODCr, (a)), total nitrogen (b), total phosphorus (c) by the eco-friendly high efficiency marine sludge (eco-HEMS) and the aerobic granule sludge (AGS) in the pilot plant-scale SBR biological treatment system during operation period. (n class="Chemical">DOn class="Chemical">CX) n class="Chemical">Clin class="Chemical">ck here for additional data file.

The analytical profile of eco-HEMS applying SBR treatment system in winter when COD: N: P ratio was 200: 5: 1.

(a), environment factors; (b), CODCr and MLSS, (c), NH3-N, NO3- -N, and T-N; (d) PO43- -P and T-P. SBR stages consisted of follow 4 stages: I, influence; II, aeration and mixing reaction; III, anaerobic and settlement, IV, decant-effluence and idle. (n class="Chemical">DOn class="Chemical">CX) n class="Chemical">Clin class="Chemical">ck here for additional data file.

The analytical profile of eco-HEMS applying SBR treatment system in winter when COD: N: P ratio was 300:5:1.

(a), environment factors; (b), CODCr and MLSS, (c), NH3-N, NO3- -N, and T-N; (d) PO43- -P and T-P. SBR stages consisted of follow 4 stages: I, influence; II, aeration and mixing reaction; III, anaerobic and settlement, IV, decant-effluence and idle. (n class="Chemical">DOn class="Chemical">CX) n class="Chemical">Clin class="Chemical">ck here for additional data file.

The analytical profile of eco-HEMS applying SBR treatment system in summer when COD: N: P ratio was 100: 5: 1.

(a), environment factors; (b), CODCr and MLSS, (c), NH3-N, NO3- -N, and T-N; (d) PO43- -P and T-P. SBR stages consisted of follow 4 stages: I, influence; II, aeration and mixing reaction; III, anaerobic and settlement, IV, decant-effluence and idle. (n class="Chemical">DOn class="Chemical">CX) n class="Chemical">Clin class="Chemical">ck here for additional data file.

The analytical profile of AGS applying SBR treatment system in winter when COD: N: P ratio was 100: 5: 1.

(a), environment factors; (b), CODCr and MLSS, (c), NH3-N, NO3- -N, and T-N; (d) PO43- -P and T-P. SBR stages consisted of follow 4 stages: I, influence; II, aeration and mixing reaction; III, anaerobic and settlement, IV, decant-effluence and idle. (n class="Chemical">DOn class="Chemical">CX) n class="Chemical">Clin class="Chemical">ck here for additional data file.

The Principle Component Analysis (PCA) with PC plots for bacterial communities during adaptation period from the marine sludge to eco-HEMS.

(a) PCA analysis based on PC1 vs PC2, (b) PCA analysis based on PC1 vs PC3, (c) PCA analysis based on PC3 vs PC2; 0 week (●), 1 week (▪), 2 week(▶), 3week (▲),4week (◀), 5 week (▼). (n class="Chemical">DOn class="Chemical">CX) n class="Chemical">Clin class="Chemical">ck here for additional data file.

Comparative analytical data of CODCr, T-N, T-P between eco-friendly high efficiency marine sludge (eco-HEMS) and aerobic granule sludge (AGS) application in pilot plant-scale SBR treatment system during all operation period.

(n class="Chemical">DOn class="Chemical">CX) n class="Chemical">Clin class="Chemical">ck here for additional data file.

Comparative results of diversity and abundance values (OTUs, Chao1, Shannon, Simpson) from bacteria community analysis.

(XLSX) n class="Chemical">Clin class="Chemical">ck here for additional data file.

Comparative analysis of bacterial community between eco-HEMS and AGS in summer.

(XLSX) n class="Chemical">Clin class="Chemical">ck here for additional data file. 19 Nov 2019 PONE-D-19-18032 Pilot Plant Study on n class="Chemical">Nitrogen and n class="Chemical">Phosphorus Removal in Marine Wastewater by Marine Sediment with Sequencing Batch Reactor PLOS ONE Dear Professor Lee, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. We would appreciate receiving your revised manuscript by Jan 03 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ Additional Editor n class="Chemical">Comments (if provided): Dear Sang-Seob Lee, Thank you for sending us your article and givinpan>g us the chance to consider your work. Your article was carefully reviewed by external reviewers for the consideration of publication in PLOS ONE. Unfortunately, the manuscript was not able to accept in the present form. However, we consider your article after complete revision of your manuscript. You are advised to address the comments raised by the reviewers and revise your manuscript. I strongly encourage you to seek the support of a native English speaker language expert and advised to make a new version of your manuscript. [n class="Chemical">Note: HTML markup is below. 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Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: No Reviewer #2: Yes Reviewer #3: No ********** 5. Review n class="Chemical">Comments to the Author Please use the space provided to explainpan> your answers to the questions above. You may also inpan>clude additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This article reports on the results of a pilot plant study that treats agricultural marine wastewater via a sequencing batch reactor. This seems to be the follow up to a previous laboratory scale SBR study. Overall, I find the study to be of high interest and overall it was well done. The manuscript is organized well. The main problem I see is that the authors are not specific with regards to what was done in replicate analysis. I strongly advise that the authors be clear in the methods with what kind of replicates was done. The authors should be more n class="Chemical">clear inpan> the inpan>trodun class="Chemical">ction or abstract that this builds on their previous study. I am not a fan of the ‘en class="Chemical">co’ part of the ‘n class="Chemical">eco-HEMS’ name – it seems a little bit like salesmanship and all wastewater treatment can be called ‘eco-friendly’. However, since the term was used in prior studies, the authors could also consider keeping it for consistency purposes. Though I had no problems understanding the authors, there are many areas in which grammar could be improved (I point to some cases in details below), especially in the abstract. The authors and should carefully review the final version for grammar, especially since PLOSONE won’t help. I hope the authors find my comments on some of these grammar errors are helpful. I think the authors n class="Chemical">could be more spen class="Chemical">cific with how they determined “optimal operational conditions”. What was the basis of ‘optimal’, and what variables were tested? Spen class="Chemical">cifin class="Chemical">c comments: Line 2. Is it more difficult because of the salinity or do we just know less about it. I think it is a reach to say it is difficult, since whole oceans are out there with all sorts of P and N cycling processes being done on a massive scale. I recommend the authors stating something more like ”Effective biological treatment of marine wastewater is not well-known.” Line 2-3. The grammar on this sentenn class="Chemical">ce is odd. “An class="Chemical">ccumulation of nitrogen and phosphorus from land-based effluent is a crucial cause of red-tide in marine systems” makes more sense. Line 4: Add “The” before purpose Line 5. “a” instead of “the” before “pilot plant-sn class="Chemical">cale sequenpan>n class="Chemical">cing…” Line 6. After the n class="Chemical">common, edit to “ elun class="Chemical">cidate which bacterial strains in sludge from marine sediment influence the performance of the SBR”. Line 9: “Improved by” n class="Chemical">doesnpan>’t really make senpan>se, maybe delete it? Line 11. After summer, use a period and start a new sentenn class="Chemical">ce with “moreover”. Line 12: “Unlike the n class="Chemical">common treatmenpan>t” would be better grammar, and what is the n class="Chemical">common treatment? Please specify here so that the reader knows the main point of reference Line 16: This isn’t specific enough, please state the method. (i.e. state something like “From 16S rRNA amplicon sequencing, the predominant genera was found to be ….” ). Too often, scientists are becoming careless with regards to what high throughput sequencing data gives us. If there is a PCR amplification, you don’t have a ‘true’ relative abundance, what you have a messy estimate. Thus, it needs to be clearer when we scientists are getting composition estimates based on sequencing of amplicons, vs when we are using a method that gives us a much better estimate of relative abundance (like from total rRNA direct sequencing, or true metagenomics sequencing, etc). Line 28. Inland aquan class="Chemical">culture and fish produn class="Chemical">ction may have cause HAB in some marine coastal regions, but the way this is stated grammatically implied is the main cause, which I don’t think is true. Line 33: This needs revised, it reads that government and scientists are beinpan>g monitored! A suggested edit would be “In recent decades, scientific and government monitoring in South Korea have found that increasing…” Line 36: A simplifin class="Chemical">cation would be “important inpan> a long-term prevenpan>tion strategy.” Line 37. This sentenn class="Chemical">ce should be edited as well. “Treatmenpan>t of marinpan>e wasten class="Chemical">water needs better understood because the …..” Line 60: Suggested edit “…to study possible applin class="Chemical">cation of …” Line 61. I suggest deleting “more” Line 71: use “a” instead of “the” to refer to the land based fish farm. Line 72: Replan class="Chemical">ce “with a” inpan>stead of “and the” before pilot plant SBR. I thinpan>k everythinpan>g inpan> that senpan>tenpan>n class="Chemical">ce after SBR can be deleted. Line 74. Put a period after (n class="Chemical">eco-HEMS), and start “we” as a new senpan>tenpan>n class="Chemical">ce. Line 75. “in ban class="Chemical">cterial n class="Chemical">community” is redundant information and can be deleted. Line 76. Delete ”and pren class="Chemical">dominpan>ated” Line 86 and 88. Instead of “is effin class="Chemical">cienpan>t remove”, just write “effin class="Chemical">ciently removes” Lind 107. “Was” instead of “ware” and the rest of this sentenn class="Chemical">ce needs some help (the n class="Chemical">MLSS was chosen based on the lab scale reactors?) Line 180. “as the n class="Chemical">control” n class="Chemical">doesn’t make sense to me, I would just delete these words. Line 326. “stable” n class="Chemical">can be deleted Line 340. This sentenn class="Chemical">ce is n class="Chemical">confusing. Line 343. Instead of “effen class="Chemical">ctive ban class="Chemical">cteria”, can you use a more specific term (i.e. genera names? Are these the added bacteria? Line 349. Is this also week 1? Line 350. The plural of genus is genera. How many reads were gathered in these samples? The read depth should be stated somewhere. Line 389-390. This sentenn class="Chemical">ce is n class="Chemical">confusing and needs edited. Line 398. Not sure how anammox connects to the information preceeding this sentence. The Anammox bacteria are Planctomycetes, which was observed (as reported in lines 379 – 9.9% Planctomycetes is actually relatively high for a non anammox reactor). Thus, I am not sure why they also say that they did not shown in the bacterial community data, unless these Planctomycetes are all from classes not known to have anammox, in which case, this should be better stated. Reviewer #2: The baits in mariculture inpan>dustry brinpan>g a lot of nutrients to the offshore area and destroy the balance of the marine ecosystem, causing red tide and other phenomena. An efficient device has been developed in this study to deal with nutrients such as N and P. This study has a high application value. The research methods and results of this paper are very detailed and logical. I think it is suitable for publication in this journal. There is only one suggestion, i.e. almost all the text in “results and discussion” is only “results”, there is almost no “discussion”. For example, what causes seasonal changes in microbial diversity? How tolerant are these strains to the different environment? I think the readers are also very interested in these mechanisms. However, mechanisms may be difficult to explain because it is not the focus of this research. Anyway, it is worthy to make more discussion. Reviewer #3: Summary of the manusn class="Chemical">cript In this paper the overall idea was really good, but the methods were doesn’t have clarity and not well organized. Manuscript has lots of short comings and more related discussions should be included. It can’t be considered for the publication in the same format. Spen class="Chemical">cifin class="Chemical">c corrections The manusn class="Chemical">cript needs an extenpan>sive review of the English language, manusn class="Chemical">cript format and also spelling mistakes. Authors have mentioned that 89.3% & 94.9% removal during summer and 84% & 88% during winter, but stated that higher removal observed during winter. Avoid n class="Chemical">controversy statemenpan>ts. Avoid earlier study’s results disn class="Chemical">cussions inpan> the methon class="Chemical">dology part (line no. 89-93); it can be included in the introduction or discussion part. The authors stated that they used the ban class="Chemical">cterial strainpan>s of n class="Species">Bacillus sp. KGN1and Vibrio sp. KGP1 for the treatment of TN and TP. Since, these strains are very potential why the authors are not taken any effort to confirm the species? Supporting evidence for the seasonal fluctuation of environmental parameters is missing. Parameters such as Temperature, Salinity and pH are closely related parameter but in the present study, authors have not observed any correlation between the parameters. The study n class="Chemical">can be improved by usinpan>g statistin class="Chemical">cal tools The salinity high during winter and was den class="Chemical">creased durinpan>g sprinpan>g and summer, how it possible? Disn class="Chemical">cussionpan> part to be improved The references are not inpan> the n class="Chemical">correct format; lots of punctuations errors are noticed. ********** 6. PLOS authors have the option to publish the peer review history of their article (what n class="Chemical">does this mean?). If published, this will include your full peer review and any attached files. If you n class="Chemical">choose “no”, your idenpan>tity will remainpan> anonymous but your review may still be made publin class="Chemical">c. n class="Chemical">Do you want your idenpan>tity to be publin class="Chemical">c for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: No Reviewer #3: No [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Enginpan>e (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org. Please note that Supporting Information files do not need this step. 21 Mar 2020 Dear Dr. n class="Chemical">Arumugam Sunpan>daramanin class="Chemical">ckam, Thank you for giving me the opportunity to submit a revised draft of my manuscript titled “ PONE-D-19-18032: Pilot plant study on nitrogen and phosphorus removal in marine wastewater by marine sediment with sequencing batch reactor” to PLOS ONE. We appreciate the time and effort that you and the reviewers have dedicated to providing your valuable feedback on our manuscript. We are grateful to the reviewers for their insightful comments on our paper. We have been able to incorporate changes to reflect most of the suggestions provided by the reviewers. We have attached followings: your letter and a point-by point response to the reviewers’ comments and concerns. Sinn class="Chemical">cerely, Sang-Seob Lee Professor, Department of Life Sn class="Chemical">cienpan>n class="Chemical">ces, n class="Chemical">College of n class="Chemical">Convergence and Integrated Science Kyonggi University PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. Please ensure that your manusn class="Chemical">cript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at http://www.journals.plos.org/n class="Chemical">plosone/s/file?id=wjVg/n class="Chemical">PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf Response: Thank you for your n class="Chemical">commenpan>ts inpan>formation. Therefore, we revised the manusn class="Chemical">cript as PLOS ONE’s style. 1. Thank you for includinpan>g your n class="Chemical">competing interests statement; "The authors have declared that no competing interests exist." We note that one or more of the authors are employed by a n class="Chemical">commern class="Chemical">cial company: "Researn class="Chemical">ch & Developmenpan>t Institute of Invenpan>tory n class="Chemical">Co. Ltd" Response: Thank you for your comments. ‘R&D inpan>stitute of Inventory Co. Ltd’ did not play a role with any funding. Also, The authors, Jinsoo Kim and Sungchul Kim, are not employed by Inventory Co. Ltd any more. Thus, we decided to erase the affiliation ‘R&D institute of Inventory Co. Ltd’ but remained the affiliation ‘Kyonggi university’. We are not related to the following comments “1, 2”. 1. Please provide an amended Funding Statement declarinpan>g this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form. Please also inn class="Chemical">clude the followinpan>g statemenpan>t withinpan> your amenpan>ded Funpan>dinpan>g Statemenpan>t. “The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.” If your n class="Chemical">commern class="Chemical">cial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement. 2. Please also provide an updated Competinpan>g Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc. Within your Competinpan>g Interests Statement, please confirm that this commercial affiliation does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If this adherence statement is not accurate and there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. Please include both an updated Fundinpan>g Statement and Competing Interests Statement in your cover letter. We will change the online submission form on your behalf. Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests 2. Please include captions for your Supporting Information files at the end of your manuscript, and update any in-text citations to match accordingly. Please see our Supporting Information guidelines for more information: http://journals.plos.org/plosone/s/supporting-information. Response: Thank you for your n class="Chemical">commenpan>ts and we inpan>cluded captions for supporting information files at the manusn class="Chemical">cript and update inpan>-text n class="Chemical">citations to match accordingly as you commented and gave information. 3. PLOS requires an ORCID iD for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager. To do this, go to ‘Update my Information’ (in the upper left-hand corner of the main menu), and click on the Fetch/Validate link next to the ORCID field. This will take you to the ORCID site and allow you to create a new iD or authenticate a pre-existing iD in Editorial Manager. Please see the following video for instructions on linking an ORCID iD to your Editorial Manager account: https://www.youtube.com/watch?v=_xcclfuvtxQ Response: Thank you for your n class="Chemical">commenpan>ts and the n class="Chemical">corresponding author ‘Sang-Seob Lee’ has updated information with ORCID ID. Additional Editor n class="Chemical">Comments (if provided): Dear Sang-Seob Lee, Thank you for sending us your article and givinpan>g us the chance to consider your work. Your article was carefully reviewed by external reviewers for the consideration of publication in PLOS ONE. Unfortunately, the manuscript was not able to accept in the present form. However, we consider your article after complete revision of your manuscript. You are advised to address the comments raised by the reviewers and revise your manuscript. I strongly encourage you to seek the support of a native English speaker language expert and advised to make a new version of your manuscript. Response: Thank you for your review. We revised the manuscript as reviewer’s comments, and we have completed the revision of the manuscript with correction and improving English. We also have taken an English editing service from Editage company, which is a recommended English editing company by PLOSONE. [n class="Chemical">Note: HTML markup is below. Please n class="Chemical">do not edit.] Reviewers' n class="Chemical">comments: Reviewer's Responses to Questions n class="Chemical">Comments to the Author 1. Is the manusn class="Chemical">cript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: No Response: Thank you for your review. We revised the n class="Chemical">conclusion from obtained data in the study. Additionally, we repeatedly performed and analyzed in every day and every cycle with more than 2 weeks at the same operational condition of the pilot plant SBR system. ________________________________________ 2. Has the statistin class="Chemical">cal analysis been performed appropriately and rigorously? Reviewer #1: I n class="Chemical">Don't Know Reviewer #2: Yes Reviewer #3: No Response: Thank you for your responses. We re-n class="Chemical">chen class="Chemical">cked the statistical analysis in the study and revised the manuscript. ________________________________________ 3. Have the authors made all data underlying the findings in their manusn class="Chemical">cript fully available? The PLOS Data policy requires authors to make all data underlyinpan>g the finpan>dinpan>gs described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes Response: Thank you for your review. ________________________________________ 4. Is the manusn class="Chemical">cript presenpan>ted inpan> an inpan>telligible fashion and writtenpan> inpan> standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: No Reviewer #2: Yes Reviewer #3: No Response: Thank you for your review. We revised the manuscript as reviewer’s comments, and we have completed the revision of the manuscript with correction and improving English. We also have taken an English editing service from Editage company, which is a recommended English editing company by PLOSONE. ________________________________________ 5. Review n class="Chemical">Comments to the Author Please use the space provided to explainpan> your answers to the questions above. You may also inpan>clude additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This article reports on the results of a pilot plant study that treats agricultural marine wastewater via a sequencing batch reactor. This seems to be the follow up to a previous laboratory scale SBR study. Overall, I find the study to be of high interest and overall it was well done. The manuscript is organized well. The main problem I see is that the authors are not specific with regards to what was done in replicate analysis. I strongly advise that the authors be clear in the methods with what kind of replicates was done. Response: Thank you for your review. We repeatedly performed and analyzed in every day and every cycle with more than 2 weeks at the same operational condition of the pilot plant SBR system. Moreover, we commented the replicates for the experiment data as well as methods in detail. The authors should be more n class="Chemical">clear inpan> the inpan>trodun class="Chemical">ction or abstract that this builds on their previous study. - Thank you for your n class="Chemical">commenpan>ts, we make clear in the introduction or abstract about the previous study. I am not a fan of the ‘en class="Chemical">co’ part of the ‘n class="Chemical">eco-HEMS’ name – it seems a little bit like salesmanship and all wastewater treatment can be called ‘eco-friendly’. However, since the term was used in prior studies, the authors could also consider keeping it for consistency purposes. - Thank you for your comments, inpan>deed, ‘eco-HEMS’ does not imply as commercial purpose, but it emphasizes the meaning of ‘eco-friendly’ and ‘high efficiency’. We used marine sediment and high efficiency bacteria in the previous study (lab-scale study), so we wanted to use both meaning and abbreviate the words, ‘eco-friendly high efficiency marine sludge (eco-HEMS). Though I had no problems understanding the authors, there are many areas in which grammar could be improved (I point to some cases in details below), especially in the abstract. The authors and should carefully review the final version for grammar, especially since PLOSONE won’t help. I hope the authors find my comments on some of these grammar errors are helpful. - Thank you for your kind comments, we revised the manuscript as your specific comments. Indeed, we had confirmed English by Editage company, which is a recommended English editing company by PLOSONE. I think the authors n class="Chemical">could be more spen class="Chemical">cific with how they determined “optimal operational conditions”. What was the basis of ‘optimal’, and what variables were tested? - Thank you for your kind comments, we searched the operational condition of SBR system for high removal efficiency of N, P. Indeed, we already obtained the optimal conditions about variables (Aerobic/anaerobic time, Q, Tc, HRT, SRT, COD:N:P ratio, F/M, MLSS/MLVSS concentration, and so on) in the previous study (lab-scale reactor), and the data were not shown in this paper. The variables of optimal operational conditions were showed in the Table 1. We repeatedly performed in every operational conditions. Spen class="Chemical">cifin class="Chemical">c comments: Line 2. Is it more difficult because of the salinity or do we just know less about it. I think it is a reach to say it is difficult, since whole oceans are out there with all sorts of P and N cycling processes being done on a massive scale. I recommend the authors stating something more like ”Effective biological treatment of marine wastewater is not well-known.” -> I agree with you and thank you for your n class="Chemical">commenpan>ts. We revised the sentenn class="Chemical">ce as you ren class="Chemical">commended. Line 2-3. The grammar on this sentenn class="Chemical">ce is odd. “An class="Chemical">ccumulation of nitrogen and phosphorus from land-based effluent is a crucial cause of red-tide in marine systems” makes more sense. -> I learned from you so mun class="Chemical">ch. Thank you for your n class="Chemical">commenpan>ts and we revised the senpan>tenpan>n class="Chemical">ce as you recommended. Line 4: Add “The” before purpose -> Thank you for your n class="Chemical">commenpan>ts and we revised the senpan>tenpan>n class="Chemical">ce as you recommended. Line 5. “a” instead of “the” before “pilot plant-sn class="Chemical">cale sequenpan>n class="Chemical">cing…” -> Thank you for your n class="Chemical">commenpan>ts and we revised the senpan>tenpan>n class="Chemical">ce as you recommended. Line 6. After the n class="Chemical">common, edit to “ elun class="Chemical">cidate which bacterial strains in sludge from marine sediment influence the performance of the SBR”. -> Thank you for your n class="Chemical">commenpan>ts and we revised the senpan>tenpan>n class="Chemical">ce as you recommended. Line 9: “Improved by” n class="Chemical">doesnpan>’t really make senpan>se, maybe delete it? -> Thank you for your n class="Chemical">commenpan>ts and we delete “improved by” as you ren class="Chemical">commended. Line 11. After summer, use a period and start a new sentenn class="Chemical">ce with “moreover”. -> Thank you for your n class="Chemical">commenpan>ts and we revised it as you ren class="Chemical">commended. Line 12: “Unlike the n class="Chemical">common treatmenpan>t” would be better grammar, and what is the n class="Chemical">common treatment? Please specify here so that the reader knows the main point of reference. -> Thank you for your n class="Chemical">commenpan>ts and we revised the senpan>tenpan>n class="Chemical">ce as you mentioned. Biological treatment efficiency is lower in winter because general bacteria activity is lower at the lower temperature. Line 16: This isn’t specific enough, please state the method. (i.e. state something like “From 16S rRNA amplicon sequencing, the predominant genera was found to be ….” ). Too often, scientists are becoming careless with regards to what high throughput sequencing data gives us. If there is a PCR amplification, you don’t have a ‘true’ relative abundance, what you have a messy estimate. Thus, it needs to be clearer when we scientists are getting composition estimates based on sequencing of amplicons, vs when we are using a method that gives us a much better estimate of relative abundance (like from total rRNA direct sequencing, or true metagenomics sequencing, etc). -> I agree with you, thank you for your n class="Chemical">commenpan>ts and we revised the senpan>tenpan>n class="Chemical">ce as you mentioned. Line 28. Inland aquan class="Chemical">culture and fish produn class="Chemical">ction may have cause HAB in some marine coastal regions, but the way this is stated grammatically implied is the main cause, which I don’t think is true. -> Thank you for your n class="Chemical">commenpan>ts and we revised the senpan>tenpan>n class="Chemical">ce. Line 33: This needs revised, it reads that government and scientists are beinpan>g monitored! A suggested edit would be “In recent decades, scientific and government monitoring in South Korea have found that increasing…” -> Thank you for your n class="Chemical">commenpan>ts and we revised the senpan>tenpan>n class="Chemical">ce as you suggested. Line 36: A simplifin class="Chemical">cation would be “important inpan> a long-term prevenpan>tion strategy.” -> Thank you for your n class="Chemical">commenpan>ts and we revised the senpan>tenpan>n class="Chemical">ce as you mentioned. Line 37. This sentenn class="Chemical">ce should be edited as well. “Treatmenpan>t of marinpan>e wasten class="Chemical">water needs better understood because the …..” -> Thank you for your n class="Chemical">commenpan>ts and we revised the senpan>tenpan>n class="Chemical">ce as you mentioned. Line 60: Suggested edit “…to study possible applin class="Chemical">cation of …” -> Thank you for your n class="Chemical">commenpan>ts and we revised the senpan>tenpan>n class="Chemical">ce as you mentioned. Line 61. I suggest deleting “more” -> Thank you for your n class="Chemical">commenpan>ts and we deleted it as you menpan>tioned. Line 71: use “a” instead of “the” to refer to the land based fish farm. -> Thank you for your n class="Chemical">commenpan>ts and we revised it as you menpan>tioned. Line 72: Replan class="Chemical">ce “with a” inpan>stead of “and the” before pilot plant SBR. I thinpan>k everythinpan>g inpan> that senpan>tenpan>n class="Chemical">ce after SBR can be deleted. -> Thank you for your n class="Chemical">commenpan>ts and we revised it as you menpan>tioned. Line 74. Put a period after (n class="Chemical">eco-HEMS), and start “we” as a new senpan>tenpan>n class="Chemical">ce. -> Thank you for your n class="Chemical">commenpan>ts and we revised it as you menpan>tioned. Line 75. “in ban class="Chemical">cterial n class="Chemical">community” is redundant information and can be deleted. -> Thank you for your n class="Chemical">commenpan>ts and we deleted it as you menpan>tioned. Line 76. Delete ”and pren class="Chemical">dominpan>ated” -> Thank you for your n class="Chemical">commenpan>ts and we deleted the senpan>tenpan>n class="Chemical">ce as you mentioned. Line 86 and 88. Instead of “is effin class="Chemical">cienpan>t remove”, just write “effin class="Chemical">ciently removes” -> Thank you for your n class="Chemical">commenpan>ts and we revised it as you menpan>tioned. Lind 107. “Was” instead of “ware” and the rest of this sentenn class="Chemical">ce needs some help (the n class="Chemical">MLSS was chosen based on the lab scale reactors?) -> Thank you for your n class="Chemical">commenpan>ts and we revised it as you menpan>tioned. 1,500 mg L-1 of n class="Chemical">MLSS showed the highest efficiencies in the previous lab-scale reactors study. Line 180. “as the n class="Chemical">control” n class="Chemical">doesn’t make sense to me, I would just delete these words. -> Thank you for your n class="Chemical">commenpan>ts and we deleted it as you menpan>tioned. Line 326. “stable” n class="Chemical">can be deleted -> Thank you for your n class="Chemical">commenpan>ts and we deleted it as you menpan>tioned. Line 340. This sentenn class="Chemical">ce is n class="Chemical">confusing. -> Thank you for your n class="Chemical">commenpan>ts and we revised the senpan>tenpan>n class="Chemical">ce as you mentioned. Line 343. Instead of “effen class="Chemical">ctive ban class="Chemical">cteria”, can you use a more specific term (i.e. genera names? Are these the added bacteria? -> Thank you for your n class="Chemical">commenpan>ts and we added ban class="Chemical">cteria names (Bacillus sp. and Vibrio sp.). Yes, we added these ban class="Chemical">cteria for improvinpan>g effin class="Chemical">ciency. Line 349. Is this also week 1? -> Yes, the result was also week 1. Line 350. The plural of genus is genera. -> Thank you for your n class="Chemical">commenpan>ts and we revised it as you menpan>tioned. How many reads were gathered in these samples? The read depth should be stated somewhere. Line 389-390. This sentenn class="Chemical">ce is n class="Chemical">confusing and needs edited. -> Thank you for your n class="Chemical">commenpan>ts and we revised it. Line 398. Not sure how anammox connects to the information preceeding this sentence. The Anammox bacteria are Planctomycetes, which was observed (as reported in lines 379 – 9.9% Planctomycetes is actually relatively high for a non anammox reactor). Thus, I am not sure why they also say that they did not shown in the bacterial community data, unless these Planctomycetes are all from classes not known to have anammox, in which case, this should be better stated. -> Thank you for your n class="Chemical">commenpan>ts and we re-stated the results and disn class="Chemical">cussion. Reviewer #2: The baits in mariculture inpan>dustry brinpan>g a lot of nutrients to the offshore area and destroy the balance of the marine ecosystem, causing red tide and other phenomena. An efficient device has been developed in this study to deal with nutrients such as N and P. This study has a high application value. The research methods and results of this paper are very detailed and logical. I think it is suitable for publication in this journal. There is only one suggestion, i.e. almost all the text in “results and discussion” is only “results”, there is almost no “discussion”. For example, what causes seasonal changes in microbial diversity? How tolerant are these strains to the different environment? I think the readers are also very interested in these mechanisms. However, mechanisms may be difficult to explain because it is not the focus of this research. Anyway, it is worthy to make more discussion. -> Thank you so mun class="Chemical">ch for your n class="Chemical">comments. We added more discussion including your comments as your brilliant comment. Reviewer #3: Summary of the manusn class="Chemical">cript In this paper the overall idea was really good, but the methods were doesn’t have clarity and not well organized. Manuscript has lots of short comings and more related discussions should be included. It can’t be considered for the publication in the same format. -> Thank you so mun class="Chemical">ch for your n class="Chemical">comments. We revised the methods and discussion as your brilliant comment. Spen class="Chemical">cifin class="Chemical">c corrections The manusn class="Chemical">cript needs an extenpan>sive review of the English language, manusn class="Chemical">cript format and also spelling mistakes. -> Thank you for your n class="Chemical">commenpan>ts and we revised and checked many mistakes and English problems. Authors have mentioned that 89.3% & 94.9% removal during summer and 84% & 88% during winter, but stated that higher removal observed during winter. Avoid n class="Chemical">controversy statemenpan>ts. -> Thank you for your n class="Chemical">commenpan>ts and we revised it. Avoid earlier study’s results disn class="Chemical">cussions inpan> the methon class="Chemical">dology part (line no. 89-93); it can be included in the introduction or discussion part. -> Thank you for your n class="Chemical">commenpan>ts and we inpan>cluded the previous methodology and results into the introduction as you commented. The authors stated that they used the ban class="Chemical">cterial strainpan>s of n class="Species">Bacillus sp. KGN1and Vibrio sp. KGP1 for the treatment of TN and TP. Since, these strains are very potential why the authors are not taken any effort to confirm the species? -> Thank you for your n class="Chemical">commenpan>ts. Indeed, we already idenpan>tified the strainpan>s inpan> the earlier previous study. Supporting evidence for the seasonal fluctuation of environmental parameters is missing Parameters such as Temperature, Salinity and pH are closely related parameter but in the present study, authors have not observed any correlation between the parameters. The study can be improved by using statistical tools The salinity high during winter and was den class="Chemical">creased durinpan>g sprinpan>g and summer, how it possible? Disn class="Chemical">cussionpan> part to be improved -> Thank you for your comments. Salinpan>ity values were decreased due to the typhoon and rainy season occurs in the summer and early autumn on the south coastal area of South Korea. We revised and improved the environmental parameters’ part as you commented. The references are not inpan> the n class="Chemical">correct format; lots of punctuations errors are noticed. -> Thank you for your n class="Chemical">commenpan>ts. We n class="Chemical">corrected the references as you commented. Submitted filename: View Letter.n class="Chemical">don class="Chemical">cx n class="Chemical">Clin class="Chemical">ck here for additional data file. 28 Apr 2020 Pilot plant study on n class="Chemical">nitrogen and n class="Chemical">phosphorus removal in marine wastewater by marine sediment with sequencing batch reactor PONE-D-19-18032R1 Dear Dr. Lee, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. With kind regards, n class="Chemical">Arumugam Sunpan>daramanin class="Chemical">ckam, PhD An class="Chemical">cademin class="Chemical">c Editor PLOS ONE Additional Editor n class="Chemical">Commenpan>ts (optional): Dear Dr Sang-Seob Lee, I am pleased to inform you that the reviewers now ren class="Chemical">commenpan>d  your manusn class="Chemical">cript for publication in PLOS ONE. Thank you for submitting your work to this journal. With regards A. Sundaramanin class="Chemical">ckam An class="Chemical">cademin class="Chemical">c Editor PLOS ONE Reviewers' n class="Chemical">comments: Reviewer's Responses to Questions n class="Chemical">Comments to the Author 1. If the authors have adequately addressed your comments raised inpan> a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All n class="Chemical">comments have been addressed Reviewer #3: All n class="Chemical">comments have been addressed Reviewer #4: All n class="Chemical">comments have been addressed ********** 2. Is the manusn class="Chemical">cript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #3: Yes Reviewer #4: (n class="Chemical">No Responpan>se) ********** 3. Has the statistin class="Chemical">cal analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #3: Yes Reviewer #4: (n class="Chemical">No Responpan>se) ********** 4. Have the authors made all data underlying the findings in their manusn class="Chemical">cript fully available? The PLOS Data policy requires authors to make all data underlyinpan>g the finpan>dinpan>gs described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #3: Yes Reviewer #4: (n class="Chemical">No Responpan>se) ********** 5. Is the manusn class="Chemical">cript presenpan>ted inpan> an inpan>telligible fashion and writtenpan> inpan> standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #3: Yes Reviewer #4: (n class="Chemical">No Responpan>se) ********** 6. Review n class="Chemical">Comments to the Author Please use the space provided to explainpan> your answers to the questions above. You may also inpan>clude additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The authors improved upon their previous submission and appears to have addressed previous reviewer comments. I am satisfied with the revisions and believe it is suitable for publication. It is an interesting area of study and appreciate the authors work studying and developing wastewater treatment processes in this niche area. Reviewer #3: n class="Chemical">Comments Overall the manusn class="Chemical">cript has revised well, all the n class="Chemical">comments were addressed in the nice manner. So that I would recommend this manuscript for the acceptance. Spen class="Chemical">cifin class="Chemical">cally, author has corrected grammatical and spelling errors. Introdun class="Chemical">ctionpan> had improved with previous study results. Author n class="Chemical">clarified all the questions and n class="Chemical">doubts which were raised by me. Referenn class="Chemical">ces also revised inpan> the same format as I menpan>tioned. Reviewer #4: (n class="Chemical">No Responpan>se) ********** 7. PLOS authors have the option to publish the peer review history of their article (what n class="Chemical">does this mean?). If published, this will include your full peer review and any attached files. If you n class="Chemical">choose “no”, your idenpan>tity will remainpan> anonymous but your review may still be made publin class="Chemical">c. n class="Chemical">Do you want your idenpan>tity to be publin class="Chemical">c for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #3: No Reviewer #4: No 4 May 2020 PONE-D-19-18032R1 Pilot plant study on n class="Chemical">nitrogen and n class="Chemical">phosphorus removal in marine wastewater by marine sediment with sequencing batch reactor Dear Dr. Lee: I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. For any other questions or n class="Chemical">conn class="Chemical">cerns, please email plosone@plos.org. Thank you for submitting your work to PLOS On class="Chemical">NE. With kind regards, PLOS On class="Chemical">NE Editorial Offin class="Chemical">ce Staff on behalf of Professor n class="Chemical">Arumugam Sunpan>daramanin class="Chemical">ckam An class="Chemical">cademin class="Chemical">c Editor PLOS ONE
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1.  In-situ remediation of nitrogen and phosphorus of beverage industry by potential strains Bacillus sp. (BK1) and Aspergillus sp. (BK2).

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