Qiaochong He

Erica A Dasi

Zhang Cheng

Emmanuel Talla

Kevan Main

Chuanping Feng

Sarina J Ergas
email: sergas@usf.edu

Wood and sulfur-based cyclic denitrification filters for treatment of saline wastewaters

Keywords: Saline wastewater, Nitrogen balance, Mixotrophic denitrification, Electron donor, Sulfate accumulation, Microbiome analysis

 should probably be removed and placed in supplementary material.

Highlights  High denitrification rates of saline wastewater achieved with wood-S 0 biofilters.

 Combined use of wood and S 0 resulted in lower SO4 2-accumulation than S 0 alone.

 Dynamic changes observed in autotrophic/heterotrophic denitrification mechanisms.

 WS-CDF microbiome contained a consortium of salt tolerant bacteria.

Detailed responses to reviewer's comments Manuscript Number: BITE-D-20-08810

Manuscript Title: Wood and sulfur based cyclic denitrification filters for treatment of saline wastewaters

We thank the reviewers for their careful review of our manuscript. The revisions have strengthened the paper. Our responses for each reviewer's comments are shown in italics and revisions are highlighted in red in the manuscript and below.

Reviewer #1:

The manuscript describes a system to promote N removal from the recirculation of a saline water aquaculture system. Wood chips and elemental sulfur are provided as electron donors. The manuscript evaluates the evolution of N and S species over time, as well as the capacity of the wood chips to provide sCOD for denitrification. The results are clearly explained, and the conclusions are realistic and consistent with the results.

Response:

We thank the reviewer for their review of the manuscript.

Specific comments:

1. Some of those conclusions (higher fish mortality than in their previous SAD system) should be included in the abstract. Even if the conclusion is somewhat negative, it is very relevant and abstract readers should be able to get a full picture when only reading the abstract.

Response:

We added the survival rate when using pine wood and sulfur for denitrification in the revised abstract.

Lines 24-27: Although fish tank water quality parameters, including ammonia, nitrite, nitrate and sulfide, were below the inhibitory levels for marine fish production, lower survival rates of Poecilia sphenops were observed compared with prior studies.

2. The accumulation of nitrite in these systems is indicative of incomplete denitrification (COD limitation), which leads to production of NO and N2O. Even though in this work nitrite depletes after a few weeks, the decrease in rbCOD in the woodchips overtime may lead to incomplete denitrification. I understand that the question of the GHGs may be out of the scope of this work but the authors should probably comment on it, especially when looking at how fast the soluble rbCOD depletes in the wood chips.

Response:

We added some discussion in the revised manuscript (Lines 294-297; 349-350). For example, before discussing the nitrite data observed in this study, we briefly mention the following as recommended by the reviewer: [START_REF] Apha | Standard Methods for the Examination of Water & Wastewater 23 rd Edition[END_REF] nitrite accumulation is an indication of incomplete denitrification; [START_REF] Bagarinao | The sulfide tolerance of milkfish and tilapia in relation to fish kills in farms and natural waters in the Philippines[END_REF] incomplete denitrification can lead to the formation of greenhouse gases, such as NO and N2O; and (3) measuring these GHGs was beyond the scope of this work. By doing Detailed Response to Reviewers so, the reader is informed about a potential concern of the WSHAD technology that future research can evaluate further. Please see the excerpt below for this example to receive further context.

Lines 294-297: NO2 -accumulation is an indicator of incomplete denitrification, which leads to production of NO and N2O, which are potent greenhouse gases [START_REF] Bertrand | Environmental Microbiology: Fundamentals and Applications[END_REF]. Although NO and N2O were not measured in this study, NO2 -was detected in the CDF effluent (Fig. 2b), especially on the first day (16.48 mg NO2 --N/L).

3. Lines 131-134: objective 1 includes N removal and the effect of WSHAD byproducts on fish growth. These seem to be two unrelated objectives unless the WSHAD byproducts they refer to are N compounds. Otherwise, please split.

Response: We separated objective 1 into two objectives (please see below):

The objectives of this study were to: 1) investigate nitrogen transformations (TAN, NO2 -, NO3 -) in the marine RASs; 2) investigate overall water quality and fish growth and survival in the systems; … (Lines 126-128)

4. 1 g/L equals 109 ppt. Therefore 15 g/L instant ocean means 15×109 ppt. Please correct.

Response:

The caption for Fig. 6 was revised. Fig. 5 was moved to the supplementary materials document. As a result, Fig. 6 in the original document that was submitted was renamed as Fig. [START_REF] Blandford | Epitheliocystis in fish: an emerging aquaculture disease with a global impact[END_REF]. In addition, the figure that quantifies the microbial community change on the genus level (originally Fig. S3), was moved from the supplementary materials to the revised manuscript and titled Fig. 6. This was done to provide the reader with additional context on notable genera that changed from the initial inoculum and WSHAD CDFs. Text describing the microbial community structure in the main manuscript was updated to reflect these changes.

A brief description of the phylum-level results is included to serve two purposes.

The first is to provide a general view of the microbial community structure and introduce a notable phylum identified in our analysis (i.e., Parcubateria). Parcubateria has not been classified beyond the phylum level to date and is discussed in detail in the genus-level results (Lines 408-410). The second purpose of the paragraph describing the phylum-level results is to transition the reader to the major themes that are discussed when the genus-level results are presented. Lines 395-399 feature the main conclusion of the phylum-level results, which states that "Variations in the abundance in the observed phyla between the inoculum, WS-CDF A, and WS-CDF B are likely to have occurred due to mixotrophic metabolism by the input of fish waste, woodchips, and/or S 0 changes in operation of the CDFs (lower HRT and more frequent backwashing), or the 16S amplicon sequencing process, which is elaborated further in the discussion of the genus-level results."

As noticed in the microbial community analysis (i.e., Section 3.3), we incorporate text to summarize the main take-home messages and/or provide a transition between paragraphs. These texts are present throughout Section 3.3, from the assessment of the microbial community analysis (Lines 380-483), phylum level analysis (Lines 395-398), and genus-level analysis (e.g., Lines 413-416; Lines 448-452). This approach is used to facilitate a discussion of the results in a way that clearly addresses the research gap presented in the introduction on how there are no previous reports on the microbial community structure of WSHAD biofilters operated under saline conditions (Lines 123-124).

Reviewer #2:

Dear Authors, the topic of the article is interesting, and the manuscript is well written. Please find attached some minor revisions I think are necessary to be performed before the acceptance on BITE journal.

Kind regards.

Response:

We thank the reviewer for their review of the manuscript.

Specific comments:

Response: Our responses to the comments that Reviewer #2 provided in the PDF file below are presented below.

1. Three highlights are the minimum required in BITE. Please add one/two more highlights.

Response: An additional highlight was provided:

 High denitrification rates of saline wastewater achieved with wood-S 0 biofilters.  Combined use of wood and S 0 resulted in lower SO4 2-accumulation than S 0 alone.  Dynamic changes observed in autotrophic/heterotrophic denitrification mechanisms.  WS-CDF microbiome contained a consortium of salt tolerant bacteria.  Sulfurimonas, Thioalbus, and Ornatilinea contributed to denitrification.

2. Lines 125-131, These lines should be moved to M&M section.

Response:

We have deleted these lines, as the WS-CDFs and pilot-scale marine RASs were described in detail in the Materials and Methods section.

3. Lines 235-245, The method description is too long. Please, be more synthetic.

You should cite one time the ref (APHA, 2012) for all the analytics.

Response:

The methods description section was carefully edited and shortened by citing the methods used in our previous study [START_REF] He | Biological denitrification in marine aquaculture systems: a multiple electron donor microcosm study: microcosm studies[END_REF], as follows:

Dissolved oxygen (DO), pH, and conductivity in the fish tanks were measured in situ daily using a portable DO (Mettler Toledo, USA), pH (Oakton™ Handheld Ion Meter), and conductivity (Oakton™ CON 6+ Portable Conductivity Meter) meters.

Water samples were collected from the fish tanks and WS-CDF effluent at least 3 times weekly for analysis of TAN, NO2 --N, NO3 --N, SO4 2-, S 2-, and COD, and weekly for alkalinity, total nitrogen (TN), total phosphorus (TP), TSS, and VSS according to Standard Methods (APHA, 2012). Samples were filtered through 0.45 µm membrane filters for TAN, NO2 --N, NO3 --N, TN, TP, sCOD, rbCOD and SO4 2- analysis, while samples were not filtered for S 2-and COD detection. Detailed analytical methods are described in [START_REF] He | Biological denitrification in marine aquaculture systems: a multiple electron donor microcosm study: microcosm studies[END_REF].

4. Lines 531-532, Specify the name of phylum or genus.

Response: Genera were added in the revised Conclusion.

The WS-CDF microbiome reflected a unique microbial consortium, including salt tolerant cellulose and sulfur utilizing denitrifiers, such as Sulfurimonas, Thioalbus, Ornatilinea, Mycobacterium and Parachlamydia. Results from this research can be applied to treatment of saline domestic, industrial and agricultural wastewaters.

Introduction

Saline wastewaters with high nitrate (NO3 -) concentrations are generated from a number of domestic and industrial processes. For example, seawater is used for toilet flushing in many arid countries, resulting in saline domestic wastewater [START_REF] Huang | Mechanism and performance of a self-flocculating marine bacterium in saline wastewater treatment[END_REF]. Ion-exchange resins used to treat NO3 -contaminated groundwater are regenerated using high NaCl concentration solutions, resulting in high-NO3 -brine wastes [START_REF] Trögl | Removal of nitrates from simulated ionexchange brines with Paracoccus denitrificans encapsulated in Lentikats Biocatalyst[END_REF]. Industrial and agricultural processes, such as aquaculture, seafood processing, tanneries, pulp and paper and textile dyeing, also generate saline wastewater containing high NO3 -levels [START_REF] Liang | Removal of nutrients in saline wastewater using constructed wetlands: Plant species, influent loads and salinity levels as influencing factors[END_REF].

In this project, we investigated the use of a novel cyclic denitrification filter (CDF), and applied this process for treatment of marine aquaculture wastewaters.

Aquaculture accounts for approximately half of the world's food fish production, and marine aquaculture contributes to 36% of global aquaculture production (FAO, 2018).

Recirculating aquaculture systems (RAS) have attracted considerable research and commercial interest due to their reduced water use and environmental impacts compared with conventional marine aquaculture using ponds, raceways or cages [START_REF] Van Rijn | Denitrification in recirculating systems: theory and applications[END_REF][START_REF] Hamlin | Comparing denitrification rates and carbon sources in commercial scale upflow denitrification biological filters in aquaculture[END_REF]. In RAS, water is circulated through filters and bioreactors, which remove solids, organic matter and total ammonia nitrogen (TAN), before being returned to the fish tanks. Aerobic biofilters are typically used for oxidation of organic carbon and nitrification, resulting in NO3 -accumulation in the RAS. Therefore, water exchanges are used to maintain NO3 -concentrations below levels that cause a chronic toxic effect on cultured fish [START_REF] Davidson | Effects of feeding a fishmeal-free versus a fishmeal-based diet on post-smolt Atlantic salmon Salmo salar performance, water quality, and waste production in recirculation aquaculture systems[END_REF]. Water exchanges consume large amounts of water and result in discharges of NO3 - containing wastewaters to the environment. Therefore, denitrification of saline wastewater is an inevitable problem to be solved in marine RAS practice.

Biological denitrification is an effective solution for NO3 -removal in marine RAS [START_REF] Van Rijn | Denitrification in recirculating systems: theory and applications[END_REF]. Although most RAS use organic substrates, such as methanol, to promote heterotrophic denitrification, autotrophic denitrification avoids carry-over of organic carbon (a source of oxygen demand) to the RAS and decreases the risk of NO2 -accumulation [START_REF] He | Biological denitrification in marine aquaculture systems: a multiple electron donor microcosm study: microcosm studies[END_REF]. Elemental sulfur (S 0 ) is a low-cost electron donor for S 0 autotrophic denitrification (SAD), which has been successfully used for NO3 -removal from freshwater (Sahinkaya and Dursun, 2012) and marine RAS [START_REF] He | A sulfur-based cyclic denitrification filter for marine recirculating aquaculture systems[END_REF]. One potential drawback of SAD is sulfate (SO4 2-) accumulation. In a prior study in our laboratory, NO3 -concentrations were maintained at a stable level in a pilot-scale marine RAS using a packed-bed denitrification bioreactor containing S 0 . However, SO4 2-concentrations increased from a background value of 1,400 to 2,322 mg SO4 2-/L after 146 days operation [START_REF] He | A sulfur-based cyclic denitrification filter for marine recirculating aquaculture systems[END_REF]. The effect of elevated SO4 2-on the health of marine aquacultured fish is not well understood.

Mixotrophic denitrification, a combination of heterotrophic and autotrophic denitrification, is a potential strategy for marine RAS applications. Under mixotrophic conditions, a portion of NO3 -is removed heterotrophically and the remainder is denitrified by SAD, resulting in increased NO3 -removal rates and decreased SO4 2- production and alkalinity consumption [START_REF] Oh | Effect of organics on sulfur-utilizing autotrophic denitrification under mixotrophic conditions[END_REF]. Liquid organic carbon sources have been used previously for mixotrophic denitrification, including methanol [START_REF] Oh | Effect of organics on sulfur-utilizing autotrophic denitrification under mixotrophic conditions[END_REF][START_REF] Sahinkaya | Simultaneous heterotrophic and sulfur-oxidizing autotrophic denitrification process for drinking water treatment: control of sulfate production[END_REF] and ethanol [START_REF] Zhang | Sulfur-based mixotrophic denitrification corresponding to different electron donors and microbial profiling in anoxic fluidized-bed membrane bioreactors[END_REF]). An alternative is the use of a slow release solid organic carbon substrate, such as wood chips, which avoids the need for liquid chemical feed systems. Krayzelova [START_REF] Robertson | Nitrate removal rates in woodchip media of varying age[END_REF]. However, wood chips can cause an initial spike in readily biodegradable chemical oxygen demand (rbCOD) concentrations, which decreases over time. High rbCOD concentrations can increase RAS oxygen demands and result in sulfide (S 2-) production, posing a toxicological threat to the cultured product [START_REF] Spotte | Seawater aquariums: the captive environment[END_REF]. At low rbCOD concentrations; however, nitrite (NO2 -) accumulation can occur resulting in brown blood disease, which reduces the oxygen carrying capacity of the fish's blood [START_REF] Hamlin | Comparing denitrification rates and carbon sources in commercial scale upflow denitrification biological filters in aquaculture[END_REF]. However, there are no reports about the adverse effects of substrates released from wood on marine aquacultured fish production.

The microbial communities of WSHAD bioreactors are still poorly understood.

Campylobacteriales was observed in the saline bioreactors, which contains

Arcobacter -a genus that has been shown to oxidize S 2-to S 0 (De Gusseme et al., 2009). To the best of our knowledge, there are no studies of the microbiome in WSHAD bioreactors used to treat saline wastewaters.

In this study, wood-sulfur cyclic denitrification filters (WS-CDFs) were investigated in pilot-scale marine RASs. The objectives of this study were to: 1) investigate nitrogen transformations (TAN, NO2 -, NO3 -) in the marine RASs; 2) investigate overall water quality and fish growth and survival in the systems; 3) evaluate the changes in dominant electron donors in the WS-CDF; 4) reveal the microbial community structure in a WS-CDF treating marine RAS water.

Materials and Methods

Materials

Water and fish

The study was carried out in a pilot-scale RAS, which was modified from a previous study [START_REF] He | A sulfur-based cyclic denitrification filter for marine recirculating aquaculture systems[END_REF]. Saline wastewater was prepared by adding 15 g/L Instant Ocean Sea Salt (Instant Ocean®) to tap water. According to the manufacturer, Instant Ocean Sea Salt contains major, minor, and trace elements necessary for marine fish health and is free of NO3 -and phosphate. Poecilia sphenops (mollies) were initially stocked in the fish tanks at a density of 1.97 kg/m 3 and an initial weight of 6.70 ± 0.97 g. Mollies were fed fish food pellets (0.6 mm Purina AquaMax Fingerling 300) twice daily at a rate of 2.5 grams/(tank-day) using an automatic feeder (Fish Mate F14, with 14 individual meals). The pellets contain a minimum of 50% crude protein.

Media materials

Elemental sulfur pellets (4.0-6.0 mm) were obtained from Southern Aggregates in Palmetto, Florida. Crushed oyster shells (2.0-4.0 mm) were added as an alkalinity source and were obtained from Myco Supply (Pittsburgh, Pennsylvania). Lightweight expanded clay aggregate in the size range of 3-5 mm was used as a biofilm carrier and was obtained from Trinity Lightweight (Livingston, AL). Expanded clay was washed with tap water and dried in the air at room temperature (~23 °C) prior to use.

White pine (Pinus strobus) lumber was obtained from a supplier in Tampa, Florida. The wood was cut into cubes with side length of 7.5 ± 1.4 mm. Pretreatment of the wood cubes was carried out to remove initial high levels of rbCOD using the following method: 1) 642 g of wood cubes were soaked in 3 L of tap water with added Instant Ocean (15 g/L) for 4 days; 2) the soaking solution was poured off and the wood cubes were rinsed 3 times in tap water; 3) wood cubes were soaked in 3 L of fresh saline water of for another 4 days; 4) the cubes were rinsed again before addition to the WS-CDF. After soaking, the side length of the wood cubes was 8.0 ± 1.6 mm.

Inoculum

Inoculum was obtained from a S 0 -based CDF, which had been operated in a pilot-scale marine RAS for 250 days [START_REF] He | A sulfur-based cyclic denitrification filter for marine recirculating aquaculture systems[END_REF]. This biomass was selected as the inoculum because the microbes were adapted to saline conditions and had received a mix of S 0 and fish waste, which promoted both SAD and heterotrophic denitrification [START_REF] He | A sulfur-based cyclic denitrification filter for marine recirculating aquaculture systems[END_REF]. Media from the S 0 -based CDF were washed with saline water to obtain 2 L of saline sludge with a total suspended solids (TSS) concentration of 8,178 mg/L and a volatile suspended solids (VSS) concentration of 2,714 mg/L. This sludge was inoculated into the CDF units in the two parallel pilot-RASs as described below.

Pilot-scale marine RAS

Two pilot-scale marine RAS (Fig. 1) were set up in parallel at the University of South Florida (USF), Tampa (WS-CDF A and WS-CDF B). The design of the pilotscale marine RAS is fully described in [START_REF] He | A sulfur-based cyclic denitrification filter for marine recirculating aquaculture systems[END_REF]. Briefly, each RAS was comprised of a fish tank (62 cm H × 30 cm W × 76 cm L) with two water-treatment loops. The first treatment loop included an upflow solids filter (I) (5.2 cm D, 35 cm H), a downflow solids filter (II) (5.0 cm D, 20 cm H), and an aerated Moving Bed Biofilm Reactor (MBBR) (8.5 cm D, 30 cm H). The second treatment loop was a WS-CDF (12.7 cm D, 60 cm H), with a working volume of 2.5 L, which was maintained using a 20 cm height standpipe (Fig. 1). Each WS-CDF was packed with a mixture of 312 g elemental sulfur pellets, 156 g crushed oyster shells, 312 g soaked wood cubes (dry weight), and expanded clay. The filling ratio of sulfur, oyster shells, and wood cubes was based on our previous microcosm study [START_REF] He | Biological denitrification in marine aquaculture systems: a multiple electron donor microcosm study: microcosm studies[END_REF], while the expanded clay was used to make up the remaining working volume. The pore volume was 1.1 L. Each CDF was seeded with 1 L saline sludge after all the media materials were packed.

A solenoid valve controlled by a timer was used to change the flow direction between the Loop 1 and Loop 2 (Fig. 1). Normally, water from the fish tank was circulated in Loop 1 at a rate of 220 ± 20 mL/min. The flow direction was switched to Loop 2 every 12 hr, resulting in water being pumped from the outlet of Solids Filter I to the WS-CDF. After 10 min, the flow was switched back to Loop 1, and the WS-CDF was allowed to react undisturbed until the next cycle. Treated effluent from the WS-CDF overflowed to the MBBR to oxidize any residual NO2 -, rbCOD, or reduced sulfur compounds before recirculating the flow back to the fish tank. The time interval between cycles was 12 hr, which was defined as the hydraulic retention time (HRT) of the WS-CDF.

Fresh tap water was added to the fish tanks to make up for the water losses due to solids wasting, sampling, and evaporation. Instant Ocean was added as needed to maintain a salinity of approximately 1.5%. NaHCO3 was periodically added into fish tanks to maintain the alkalinity > 100 mg CaCO3/L, which is considered in the healthy range for mollies. The MBBR, tubing, and solids filters I and II were cleaned periodically as described previously [START_REF] He | A sulfur-based cyclic denitrification filter for marine recirculating aquaculture systems[END_REF]. Backwashing of the CDF was carried out monthly as described previously to keep the systems from clogging (He et al., 2020).

Wood chip COD release

Wood chip soluble COD (sCOD) and rbCOD release tests were performed on fresh wood cubes, wood cubes that had been soaked in saline water for 6 days, and periodically using cubes removed from the WS-CDF. Each test was performed in duplicate; 20 wood cubes were submerged in 100 mL of saline water for 2 days. The supernatant was drawn off and the sCOD and rbCOD concentrations were measured as described below. After each test, the wood cubes that had been removed were returned to the CDF.

Analytical methods

Dissolved oxygen (DO), pH, and conductivity in the fish tanks were measured in situ daily using a portable DO (Mettler Toledo, USA), pH (Oakton™ Handheld Ion Meter), and conductivity (Oakton™ CON 6+ Portable Conductivity Meter) meters.

Water samples were collected from the fish tanks and WS-CDF effluent at least 3 times weekly for analysis of TAN, NO2 --N, NO3 --N, SO4 2-, S 2-, and COD, and weekly for alkalinity, total nitrogen (TN), total phosphorus (TP), TSS, and VSS according to Raw sequencing data processing and analysis were performed using software available on the Galaxy server. In-house perl scripts were used to compare the duplicates of the inoculum, WS-CDF A, and WS-CDF B. Operational taxomonic units (OTUs) of duplicates were organized into separate files representing each taxonomic rank from phylum to genus (files can be accessed after the conclusion section).

Relative microbial community structure for the phylum and genus ranks are shown as stacked bar charts. Mean abundances of genera not characterized to the genus level were combined by taxonomic rank to represent phylum_unclassified, class_unclassified, order_unclassified, and family_unclassified. Changes in microbial community structure between the inoculum and WS-CDFs were shown in bar charts expressing the fold changes of selected phyla and genera. A brown color was observed in the RAS water, most likely due to the combined effect of the fish food [START_REF] Schuster | The effect of fish meal content in trout food on water colour in a closed recirculating aquaculture system[END_REF] and substances released from wood. The color was not quantified; however, the water was visibly darker than in our previous study with a S 0 -based CDF (He et al., 2020). [START_REF] Hartz | Wood chip denitrification bioreactors can reduce nitrate in tile drainage[END_REF] observed that effluent from a wood chip-denitrification reactor was dark colored for the first several weeks of operation due to tannins that leached from the woodchips. Clear water could enhance the ability of fish to capture feed and allow the farmer to observe fish health, behavior, and feeding activity [START_REF] Davidson | Effects of feeding a fishmeal-free versus a fishmeal-based diet on post-smolt Atlantic salmon Salmo salar performance, water quality, and waste production in recirculation aquaculture systems[END_REF]. Water exchanges during the initial operation, activated carbon filtration, or advanced oxidation processes (e.g., ozone) could be used to remove water color [START_REF] Schuster | The effect of fish meal content in trout food on water colour in a closed recirculating aquaculture system[END_REF].

Results and Discussion

Nitrogen removal

Nitrogen entered the system with the fish feed, which contained 50% protein.

Dissolved nitrogen compounds, including TAN, NO2 -, and NO3 -, were generated in the RAS by fish excretion, decomposition of uneaten feed, and nitrification. Fig. 2a shows changes in TAN, NO2 -, and NO3 -concentrations in fish tanks during RAS operation. Average TAN, NO2 -, and NO3 -concentrations were maintained at 0.17 ± 0.05 mg N/L, 0.41 ± 0.17 mg NO2 --N/L, and 56.77 ± 2.72 mg NO3 --N/L, respectively.

Average TN and total organic nitrogen (TON) concentrations in the fish tanks were ~81 mg N/L and ~25 mg N/L, respectively. The steady performance over the entire operational period indicates that nitrogen added on a daily basis was removed by assimilation into fish and microbial biomass, solids wasting, nitrification, and denitrification. A nitrogen mass balance [START_REF] Cheng | Model of a Sulfur-based Cyclic Denitrification Filter for Marine Recirculating Aquaculture Systems[END_REF] showed that passive denitrification in the first loop contributed to 5% of the nitrogen removal in the RAS.

Passive denitrification occurs within anoxic environments in solids filters, as well as biofilms in MBBRs and fish tanks where water contains organic carbon and NO3 -

(Van Rijn et al., 2006).

NO3 -, NO2 -, and TAN concentrations in WS-CDF effluent over time are shown in Fig. 2b. The short acclimation period (1 day) shows that the inoculum from the S 0based CDF quickly adapted to using a combination of wood and S 0 as electron donors. After acclimation, the average NO3 -removal rate was 102.6 ± 8.5 g N/(m 3 •d) in the WS-CDF (removal efficiency of 90.3 ± 4.3%). The mass balance showed that nitrogen removal in the WS-CDF accounted for 57% of the total nitrogen removed.

Our prior study with a S 0 -based CDF incorporated into a similar RAS achieved an average denitrification rate of 86.7 ± 5.1 g N/(m 3 •d) [START_REF] He | A sulfur-based cyclic denitrification filter for marine recirculating aquaculture systems[END_REF]. The higher denitrification rate obtained in this study was likely due to higher influent NO3 - concentration and the combined use of S 0 and wood chips. NO2 -accumulation is an indicator of incomplete denitrification, which leads to production of NO and N2O, which are potent greenhouse gases [START_REF] Bertrand | Environmental Microbiology: Fundamentals and Applications[END_REF].

Although NO and N2O were not measured in this study, NO2 -was detected in the CDF effluent (Fig. 2b), especially on the first day (16.48 mg NO2 --N/L). As mentioned previously, bacteria needed to adapt to the new environment at the beginning of RAS operation, resulting in incomplete denitrification. However, the effluent NO2 -concentration quickly stabilized at 0.47 ± 0.22 mg NO2 --N/L. Pine wood contains complex organic molecules, which can sometimes be utilized by fermentative bacteria capable of reducing NO3 -to NO2 - [START_REF] Beauchamp | Carbon sources for bacterial denitrification[END_REF]. In a prior microcosm study with a variety of electron donors [START_REF] He | Biological denitrification in marine aquaculture systems: a multiple electron donor microcosm study: microcosm studies[END_REF]), NO2 - accumulation was higher in wood-containing bioreactors than in SAD bioreactors.

As shown in Fig. 2b, TN concentrations in the WS-CDF effluent were 14.57 ± 7.24 mg N/L; effluent TAN concentrations ranged from 0.5 to 1.6 mg N/L. TAN can be generated in the WS-CDF through ammonification of nitrogen containing organic matter (wood or fish waste) or dissimilatory nitrate reduction to ammonium (DNRA).

Influent TON concentrations decreased from 24.59 ± 4.70 to 7.48 ± 3.83 mg N/L in the effluent (Figs. 2a and2b), indicating degradation of organic nitrogen. DNRA was not quantified; however, it has been previously observed in wood chip denitrification reactors [START_REF] Greenan | Comparing carbon substrates for denitrification of subsurface drainage water[END_REF].

Table 1 summarizes studies that combined S 0 and organic carbon sources as electron donors for denitrification. Differences in denitrification rates in different studies are likely due to differences in labilities of organic matter for heterotrophic denitrifying bacteria. Higher mixotrophic denitrification rates were obtained when methanol was used compared with solid carbon sources [START_REF] Oh | Effect of organics on sulfur-utilizing autotrophic denitrification under mixotrophic conditions[END_REF][START_REF] Sahinkaya | Simultaneous heterotrophic and sulfur-oxidizing autotrophic denitrification process for drinking water treatment: control of sulfate production[END_REF]. However, as discussed previously, the methanol dosage needs to be strictly controlled to avoid carry-over of rbCOD into the RAS.

In terms of solid carbon sources, a relatively higher denitrification rate was observed with tire chips than wood chips. Tire chips not only provided a carbon source for heterotrophic denitrification, but was also shown to adsorb NO3 -when the influent NO3 -loading exceeded the denitrification capacity of the biofilm [START_REF] Krayzelova | A tire-sulfur hybrid adsorption denitrification (T-SHAD) process for decentralized wastewater treatment[END_REF]. The current study achieved the highest denitrification rate of the studies carried out with wood-based media. Von Ahnen et al. (2018) used Salix viminalis (basket willow), which is a hardwood. The pine used in this study is a softwood, which has been shown to have a higher permeability than hardwoods, making it prone to water capture and enhanced contact between microorganisms and the substrate [START_REF] Van Driel | Denitrification of agricultural drainage using wood-based reactors[END_REF]. In addition, the wood used by Von Ahnen et al. (2018) had been in reactors treating aquaculture effluent for approximately one year. The bioavailability of organic carbon released from wood was shown to decrease over time with denitrifying bioreactor operation [START_REF] He | Biological denitrification in marine aquaculture systems: a multiple electron donor microcosm study: microcosm studies[END_REF]. [START_REF] Robertson | Nitrate removal rates in woodchip media of varying age[END_REF] investigated woodchip denitrification performance in reactors of varying ages and found that woodchips lost about 50% of their reactivity after one year of operation due to leaching of soluble organic compounds.

Dominant electron donor variations in the CDFs

As shown in Fig. 3a, COD concentrations in the WS-CDF effluent were higher than those in fish tanks during the first 60 days, indicating that wood chips initially released excess organic carbon not used for heterotrophic denitrification. The decrease in effluent COD concentrations on Day 61 may have been due to the second backwashing on Day 60, which floated some wood cubes to the top of the WS-CDFs.

Tests were carried out to investigate the release of sCOD and rbCOD over time from the wood cubes. rbCOD consists of simple organic molecules, which can pass through cell membrane of denitrifying bacteria and be metabolized within minutes [START_REF] Mamais | A rapid physical-chemical method for the determination of readily biodegradable soluble cod in municipal wastewater[END_REF]. As shown in Fig. 4, fresh pine wood had a relatively high sCOD and rbCOD release (116.7 ± 1.7 mg sCOD/(g wood) and 94.7 ± 13.5 mg rbCOD/(g wood)), which decreased after being submerged in saline water for 8 days (19.2 ± 0.5 mg sCOD/(g wood) and 15.7 ± 0.7 mg rbCOD/(g wood) when they were packed in the WS-CDF. The decrease in rbCOD in the woodchips over time may lead to NO and N2O production due to incomplete denitrification [START_REF] Bertrand | Environmental Microbiology: Fundamentals and Applications[END_REF]. sCOD and rbCOD release decreased exponentially according to the following: sCOD (mg sCOD/(g wood))=117×e -0.267×t (R 2 =0.9932)

rbCOD (mg COD/(g wood))=95×e -0.404×t (R 2 =0.9768)

In our previous study, significant SO4 2-accumulation was observed in the fish tanks when only S 0 was used as the electron donor for denitrification [START_REF] He | A sulfur-based cyclic denitrification filter for marine recirculating aquaculture systems[END_REF].

In this study, fish tank SO4 2-concentrations stabilized at around 2,057 ± 122 mg SO4 2- /L (Fig. 3b). The use of WSHAD successfully avoided high SO4 2-accumulation in the marine RAS (i.e., SO4 2-production due to SAD was balanced by SO4 2-removal due to water losses during sampling and solids wasting). To understand the dominant electron donor variations in the WS-CDFs over time, the data were divided into four stages: 1) days 0-30; 2) days 31-60; 3) days 61-90; and 4) days 91-125. SO4 2- generation during Stages 1 through 4 was 3.73 mg SO4 2-/mg NO3 --N, 3.07 mg SO4 2- /mg NO3 --N, 5.76 mg SO4 2-/mg NO3 --N, and 6.65 mg SO4 2-/mg NO3 --N, respectively.

Based on the theoretical SO4 2-productivity of SAD of 7.54 mg SO4 2-/mg NO3 --N, The phylum level microbial community composition of the inoculum and WS-CDFs were similar, with slight differences in the abundance of specific phyla. Figures that depict the relative abundance of bacteria within these samples and quantify the changes that occurred can be accessed after the conclusion section. Proteobacteria, Actinobacteria, and Bacteriodetes were the three largest phyla observed in the samples and accounted for between 21-33%, 23-30%, and 8.3-32%, respectively.

Notable changes between the inoculum and WS-CDF systems include those occurring in Chlorobi and Parcubacteria, which represented 7.9% and 2.4%, respectively, in the

Link between CDF media, operation, and microbiome

The relative abundance of bacteria on the genus level in the inoculum, WS-CDF A, and WS-CDF B is shown in Fig. 5. Notable genera in the inoculum include Prosthecochloris, Sulfurovum, and Chlorobium, which represented 5.3%, 4.4%, and 1.0% of the total population, respectively. Bacteria belonging to Sulfurovum are obligate autotrophs and might have contributed to nitrogen removal through SAD (Inagaki et al., 2004). Furthermore, Prosthecochloris and Chlorobium include species capable of autotrophic growth using sulfide as an electron donor [START_REF] Kumar | Prosthecochloris indica sp. nov., a novel green sulfur bacterium from a marine aquaculture pond, Kakinada, India[END_REF][START_REF] Vogel | Chlorobium chlorochromatii sp. nov., a symbiotic green sulfur bacterium isolated from the phototrophic consortium "Chlorochromatium aggregatum[END_REF]. SO4 2-produced during SAD may have migrated to lower redox potential zones in the CDF and been used to form sulfide by SO4 2--reducing bacteria belonging to the phylum Parcubacteria, which today remains unclassified.

Desulfocapsa, a sulfur-disproportionating bacterium whose abundance was 0.15%, may have also provided S 2-for the cultivation of Prosthecochloris and Chlorobium [START_REF] Finster | Elemental sulfur and thiosulfate disproportionation by Desulfocapsa sulfoexigens sp. nov., a new anaerobic bacterium isolated from marine surface sediment[END_REF]. Microbial community analysis of the initial inoculum indicates that the S 0 -based CDF was enriched with a diverse consortium of salt-tolerant organisms including S 0 -oxidizing denitrifiers, SO4 2--reducing bacteria, and sulfurdisproportionating bacteria.

Differences in media composition, HRT and backwashing frequency likely contributed to the changes in the microbial community structure between the initial inoculum from the S 0 -based CDF and WS-CDF microbiome. Less S 0 was provided in the WS-CDFs compared to the S 0 -based CDF (312 vs. 1200 g, respectively) to compensate for the addition of woodchips. The reactor volume for the WS-CDFs was smaller than that applied in the S 0 -based CDF. This was done to decrease the HRT and minimize spatial and/or temporal redox reactions that generate S 2-. In addition, the CDFs were backwashed monthly to avoid clogging due to biofilm accumulation. Fig. 6 quantifies the microbial community change that occurred in the WS-CDFs, compared to the inoculum. Analysis of the microbial community change at the genus level indicates that a lower quantity of S 0 in the WS-CDFs appears to have been insufficient to maintain the growth of Sulfurovum, causing this genera to decrease by more than 16 times (Log2 WS-CDF:Inoculum = -4.4 ± 1.3; Fig. 6). The microbial community change also suggests that reducing the HRT and increasing the backwashing frequency resulted in less favorable conditions for SO4 2--reducing bacteria. As a result, SO4 2--reducing bacteria belonging to Parcubacteria decreased by more than four times (Log2 WS-CDF:Inoculum = -2.5 ± 1.8; Fig. 6) and subsequently Chlorobium and Prosthecochloris were undetected in one of the WS-CDFs (Fig. 5).

Chlorobium and Prosthecochloris are members of the phylum Chlorobi; thus, the decrease of these populations to a point below detection is in agreement with the phylum level observations mentioned previously.

Changes made to the environmental conditions within the CDFs by augmenting the media and operating conditions, seems to have allowed for the cultivation of other bacteria that initially represented much lower percentages of the inoculum.

Noteworthy genera that increased by more than two times include Sulfurimonas, Thioalbus, and Ornatilinea (Log2 WS-CDF:Inoculum = 1.2 ± 0.86, 2.2 ± 0.52, and 2.9 ± 0.79, respectively; Fig. 6). Sulfurimonas and Thioalbus include species of sulfur oxidizing bacterium that are capable of denitrification, while Ornatilinea includes Mycobacteriaceae; thus, it is probable that this genus also represents one of the largest genera in WS-CDF A. Variations between the populations in WS-CDF A and B may be attributed to differences in TSS and VSS. The inverse simpson index, or invsimpson, is a diversity metric that accounts for both the total number of OTUs and abundance.WS-CDF A had a higher invsimpson value, suggesting that its microbial community structure was more diverse that WS-CDF B.

Another source of variation between the WS-CDFs may have resulted from the 16S amplicon sequencing process. Sequencing of samples collected from WS-CDF A and B occurred at different time periods. Therefore, technical bias may have been introduced due to sample preparation and/or instrumentation.

Fish growth and performance

The initial fish weight was 6.70 ± 0.97 g and reached 9.38 ± 5.78 g at the end of the study, with a survival rate of 62 ± 4%. The survival rate was significantly lower than that obtained in our previous study (88%), where S 0 was the only added electron donor for denitrification [START_REF] He | A sulfur-based cyclic denitrification filter for marine recirculating aquaculture systems[END_REF]. In addition, side swimming, rapid swimming, and body bending were observed during the operation, especially during the initial period. Fish that showed these behaviors were euthanized according to USF's Institutional Animal Care and Use Committee (IACUC) protocols. It was noticed that these symptoms were relieved during the last month of operation.

As summarized in Table 2, nitrogen concentrations, including NO3 -, NO2 -and TAN, were in the healthy range and were similar to those achieved in our prior study [START_REF] He | A sulfur-based cyclic denitrification filter for marine recirculating aquaculture systems[END_REF]. Therefore, RAS nitrogen species concentrations were not responsible for the high fish mortality. Other water quality parameters were also in the healthy range, including TP, S 2-, TSS, VSS, alkalinity, pH, salinity, DO and temperature.

Increased mortalities observed in this study may have occurred due to high SO4 2- concentrations, pathogenic bacteria, or the release of toxic compounds from wood.

High SO4 2-concentrations (> 2000 mg SO4 2-/L) may have chronic toxic effect on freshwater fish health [START_REF] Elphick | An aquatic toxicological evaluation of sulfate: the case for considering hardness as a modifying factor in setting water quality guidelines[END_REF]. [START_REF] Davidson | Comparing the effects of high vs. low nitrate on the health, performance, and welfare of juvenile rainbow trout Oncorhynchus mykiss within water recirculating aquaculture systems[END_REF] discussed potential toxicity of water parameters and mentioned that SO4 2-may cause a mild chronic effect to rainbow trout. Mycobacterium and Parachlamydia, which were identified during the microbial community analysis (Fig. 6), include suspected agents of epitheliocystis and mycobacteriosis -two infectious diseases common in freshwater and marine species, especially those raised in intensive aquaculture systems [START_REF] Blandford | Epitheliocystis in fish: an emerging aquaculture disease with a global impact[END_REF]. Higher abundances of these bacteria in the WS-CDFs than the S 0 -based CDFs may have contributed to the higher mortalities observed in this study (Pawlikowska-Warych and Deptula, 2016). Wood releases compounds, such as phenols, resin acids, tannins, lignin and volatile fatty acids, that can be toxic to animals [START_REF] Hedmark | Review of environmental effects and treatment of runoff from storage and handling of wood[END_REF]. It was reported that water leachate from woodchips was toxic to trout [START_REF] Taylor | Toxicity of aspen wood leachate to aquatic life: laboratory studies[END_REF]. Additional research is needed to understand the effect of high SO4 2-and chemicals released from wood on marine RAS species and whether these conditions trigger stress that promotes a high susceptibility to epitheliocystis and mycobacteriosis.

Conclusions

This study is the first to apply the WSHAD process for saline wastewater treatment and elucidate the microbiomes of these mixotrophic biofilters. WS-CDFs achieved high denitrification rates, with lower SO4 2-accumulation than SAD alone.

Changes in rbCOD release from wood chips over time resulted a shift in the dominant denitrification mechanism from heterotrophic to SAD. The WS-CDF microbiome reflected a unique microbial consortium, including salt tolerant cellulose and sulfur utilizing denitrifiers, such as Sulfurimonas, Thioalbus, Ornatilinea, Mycobacterium

  et al. (2014) developed a tire-sulfur hybrid adsorption denitrification (T-SHAD) process where tire chips were used as a slow release source of organic substrate to enhance NO3 -removal in decentralized wastewater treatment applications. Li et al. (2016) investigated a wood-sulfur heterotrophic-autotrophic denitrification (WSHAD) process for water treatment, and observed decreased SO4 2-accumulation compared with SAD. In a microcosm study carried out in our laboratory with saline wastewater, lower SO4 2-production and higher NO3 -removal rates were observed in a WSHAD system compared with SAD alone (He et al., 2018). Wood is mainly composed of cellulose (45-50%), hemicellulose (25-35%), and lignin (25-35%) (Malherbe and Cloete, 2002). It has been used as a carbon source for denitrification of wastewater, stormwater and agricultural drainage (see Lopez-Ponnada et al., 2017 for review), aquaculture effluents (Von Ahnen et al., 2018) and RAS wastewater (Von Ahnen et al., 2019). Wood has been shown to deliver long term (5-15 years) denitrification rates (1-20 g N/(m 3 •d)) in onsite wastewater applications with minimum maintenance

  Li et al. (2016) performed WSHAD batch reactor experiments with NO3 -contaminated water. The authors identified the genera Thiobacillus and Hydrogenophaga, which include obligate and facultative autotrophs that can use reduced sulfur compounds or hydrogen as electron donors and NO3 -as an electron acceptor (Bertrand et al., 2011; Lin et al. 2017). However, previous research suggests that salinity affects the microbial community composition of denitrification systems (Von Ahnen et al., 2019). Von Ahnen et al. (2019) investigated the effect of salinity on microbial community structure and denitrification in horizontal-flow woodchip bioreactors. The presence of heterotrophic denitrifying bacteria, such as those belonging to the orders Betaproteobacteriales, Rhizobiales, and Xanthomonadales, decreased with increasing salinity, suggesting that decreased NO3 -removal rate was linked to the abundance of these organisms (Von Ahnen et al., 2019).

  Standard Methods[START_REF] Apha | Standard Methods for the Examination of Water & Wastewater 23 rd Edition[END_REF]. Samples were filtered through 0.45 µm membrane filters for TAN, NO2 --N, NO3 --N, TN, TP, sCOD, rbCOD and SO4 2- analysis, while samples were not filtered for S 2-and COD measurements. Detailed analytical methods are described in[START_REF] He | Biological denitrification in marine aquaculture systems: a multiple electron donor microcosm study: microcosm studies[END_REF].

2. 5

 5 Microbial community analysis and visualizationSamples were collected in duplicate from the initial inoculum and the center of the CDFs at the end of the 125-day operational period. Genomic DNA was extracted from each sample using the AllPrep PowerViral DNA/RNA Kit (QIAGEN, INC.), according to the manufacturer's protocol and stored at -80 C until PCR amplification. PCR amplification, library preparation, and sequencing were performed by Applied Biological Materials, Inc. (Vancouver, Canada). The 337F (5'-GAC TCC TAC GGG AGG CWG CAG-3') and 805R (5'-GAC TAC CAG GGT ATC TAA TC-3') universal bacterial primers were used to target the V3-V4 hypervariable region of the 16S rRNA gene. Library preparation proceeded by two-step PCR amplification and all amplicons were pooled together and sequenced on an Illumina MiSeq platform using a 2x300 bp paired end sequencing run. The raw reads were deposited into the NCBI Sequence Read Archive database under the following accession number: PRJNA695006.

3. 1 .

 1 Marine RAS performance 3.1.1. Solids and color TSS and VSS concentrations in the fish tanks ranged from 20 to 35 mg/L and from 12 to 21 mg/L, respectively, showing that solids were effectively removed in the solids filters. Accumulation of solids within aquaculture tanks can promote an environment that harbors fish pathogens and leaches nutrients that adversely affect the health of fish (Davidson and Summerfelt, 2005).

SAD accounted for 49 . 3 . 3

 4933 4% of NO3 -removal during Stage 1, decreased to 40.7% in Stage 2, and then increased to 76.4% in Stage 3 and 88.2% in Stage 4. As mentioned previously, the seed sludge was from a S 0 -based CDF, which has been operated for > 6 months. This may have resulted in the high initial SO4 2-generation observed in Stage 1. As the bacteria adapted to the WSHAD environment, heterotrophic denitrification became more dominant during Stage 2. However, with the decrease in rbCOD released from pine wood chips, the contributions of SAD to NO3 -removal increased in Stages 3 and 4. Microbial community structure in the CDFs 3.3.1 CDF phylum-level microbial community structure Information from the 16S rRNA gene libraries for the initial inoculum, WS-CDF A, and WS-CDF B samples can be accessed after the conclusion section. As stated previously, the initial inoculum was sludge collected from a S 0 -based CDF integrated into a pilot-scale marine RAS (He et al., 2020). Good's coverage suggested that the microbial composition for each sample was well represented by the constructed sequence libraries and thus reflect the true bacterial profile. The observed OTUs representing specific phyla or genera for each sample were used to examine the microbial community structure and infer the main taxonomic groups responsible for CDF performance at the phylum and genus levels, respectively.

inoculum.

  Parcubacteria decreased by more than four times (Log2 WS-CDF:Inoculum = -2.5 ± 0.91) and Chlorobi was undetected in WS-CDF A and decreased by more than four times in WS-CDF B (Log2 WS-CDF B:Inoculum = -4.6). Variations in the abundance in the observed phyla between the inoculum, WS-CDF A, and WS-CDF B are likely to have occurred due to mixotrophic metabolism by the input of fish waste, woodchips, and/or S 0 , changes in operation of the CDFs (lower HRT and more frequent backwashing), or the 16S amplicon sequencing process, which is elaborated further in the discussion of the genus level results.

5 .

 5 heterotrophic bacteria capable of utilizing cellulose as a carbon source[START_REF] Bertrand | Environmental Microbiology: Fundamentals and Applications[END_REF][START_REF] Podosokorskaya | Ornatilinea apprima gen. nov., sp. nov., a novel cellulolytic representative of class Anaerolineae[END_REF]. The increase in abundance of Ornatilinea within the WS-CDFs indicates the presence of specialized bacteria that utilize organic carbon from the pine wood chips supplied. Genus level analysis of the WS-CDF shows that the microbial community included multiple autotrophic and heterotrophic denitrifiers including Defluviimonas[START_REF] Liu | Defluviimonas nitratireducens sp. nov., isolated from surface seawater[END_REF], which contributed to a higher NO3 -removal rate than that observed in our prior S 0 -based CDF study[START_REF] He | A sulfur-based cyclic denitrification filter for marine recirculating aquaculture systems[END_REF].The operation of the WS-CDFs may have also played a role in their individual microbial community composition. As shown in Fig.5, Mycobacterium and Parachlamydia were the largest genera observed in both the inoculum and WS-CDF B, representing between 18-22% and 1.1-4.0% of the population, respectively. Members belonging to the Mycobacterium and Parachlamydia genera are heterotrophic and the former includes salt-tolerant species capable of reducing NO3 -[START_REF] Bertrand | Environmental Microbiology: Fundamentals and Applications[END_REF]. Therefore, the presence of these bacteria indicates their involvement in COD removal and/or heterotrophic denitrification within the CDFs.While Parachlamydia represented one of the largest genera in WS-CDF A (2.0%), Mycobacterium accounted for only 1.6%. However, members belonging to the family Mycobacteriaceae accounted for the majority (22%) of the total bacteria in WS-CDF A that were classified to only the family rank (30%), or the family_unclassified group shown in Fig.Mycobacteriumis the only known genus belonging to

Fig. 1 .

 1 Fig. 1. Schematic of the pilot-scale marine RAS (WS-CDF: wood-sulfur cyclic

Fig. 2 .

 2 Fig. 2. Profiles of dissolved nitrogen species concentrations in: a) fish tanks and b)

Fig. 3 .

 3 Fig. 3. Changes in a) sCOD and b) SO4 2-concentrations in the fish tanks and WS-

Fig. 4 .

 4 Fig. 4. sCOD and rbCOD released from wood in WS-CDFs over time.

Fig. 5 .

 5 Fig. 5. Relative abundance of bacteria present in the inoculum and WS-CDF systems

Fig. 6 .

 6 Fig. 6. Relative change in the microbial community composition on genus level

Figure

  Figure Click here to access/download;Figure;Figures-Revised-01-25-2021.docx

Fig. 1

 1 Fig.1

Fig. 2

 2 Fig.2

Fig. 3

 3 Fig.3

Fig. 4

 4 Fig.4

Fig. 5

 5 Fig.5

Fig. 6

 6 Fig.6

Table 1 .

 1 Sulfur-based mixotrophic denitrification performance in other studies and this study.

	NO3 --contaminated water	Carbon source	Alkalinity source	Influent NO3 -concentration (mg NO3 --N/L)	HRT (h)	Removal efficiency (%)	Denitrification N/(m 3 •d)) rate (g	Reference
	Marine RAS	Pine blocks	Oyster shells	56.8 ± 2.7	12	90.3 ± 4.3	102.6 ± 8.5	This study
	Freshwater	Wood chips	Seashells	30	10	-	8.12 ± 0.49	Von Ahnen et
	aquaculture							al. (2018)
	Synthetic	Tire chips	Oyster shells	50	6	89.5	368.8	Krayzelova et
	nitrified							al. (2014)
	wastewater
	Synthetic NO3 --	Methanol/	NaHCO3	100	7.5	>97	1,920~2,700	Oh et al. (2001)
	contaminated	landfill
	water	leachate
	Groundwater	Methanol	Limestone	75	11	75	164	Sahinkaya et al.
								(2011)
	Groundwater	Methanol	NaHCO3	75	4	100	450	Sahinkaya and
								Dursun (2012)

Table

  Click here to access/download;Table;Tables revised 1-30-2021.docx

and Parachlamydia. Results from this research can be applied to treatment of saline domestic, industrial and agricultural wastewaters.

Acknowledgements

This research work was supported by China Scholarship Council (CSC, NO. 201606400027) and the Chateaubriand Fellowship Program. This material is based upon work supported by the National Science Foundation under Grant No. 1243510 and Florida Sea Grant. Any opinions, findings, conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation or Florida Sea Grant. The authors would like to thank Allan Feldman for help with wood preparation, Lance Wood and Daniel Delgado for help with DNA extraction and Ileana Bermudez for help with RAS operation and wood COD release studies.

Appendix A: Supplementary data E-supplementary data of this work can be found in the online version of the paper.

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: