Analysis of very-high surface area 3D-printed media in a moving bed biofilm reactor for wastewater treatment

Gabriel Proano-Pena; Andres L Carrano; David M Blersch

doi:10.1371/journal.pone.0238386

. 2020 Aug 27;15(8):e0238386. doi: 10.1371/journal.pone.0238386

Analysis of very-high surface area 3D-printed media in a moving bed biofilm reactor for wastewater treatment

Gabriel Proano-Pena ^1,^#, Andres L Carrano ^2,^#, David M Blersch ^3,^*,^#

Editor: Leonidas Matsakas⁴

PMCID: PMC7451639 PMID: 32853235

Abstract

Moving Bed Biofilm Reactors (MBBRs) can efficiently treat wastewater by incorporating suspended biocarriers that provide attachment surfaces for active microorganisms. The performance of MBBRs for wastewater treatment is, among other factors, contingent upon the characteristics of the surface area of the biocarriers. Thus, novel biocarrier topology designs can potentially increase MBBR performance in a significant manner. The goal of this work is to assess the performance of 3-D-printed biofilter media biocarriers with varying surface area designs for use in nitrifying MBBRs for wastewater treatment. Mathematical models, rendering, and 3D printing were used to design and fabricate gyroid-shaped biocarriers with a high degree of complexity at three different levels of specific surface area (SSA), generally providing greater specific surface areas than currently available commercial designs. The biocarriers were inoculated with a nitrifying bacteria community, and tested in a series of batch reactors for ammonia conversion to nitrate, in three different experimental configurations: constant fill ratio, constant total surface area, and constant biocarrier media count. Results showed that large and medium SSA gyroid biocarriers delivered the best ammonia conversion performance of all designs, and significantly better than that of a standard commercial design. The percentage of ammonia nitrogen conversion at 8 hours for the best performing biocarrier design was: 99.33% (large SSA gyroid, constant fill ratio), 94.74% (medium SSA gyroid, constant total surface area), and 92.73% (large SSA gyroid, constant biocarrier media count). Additionally, it is shown that the ammonia conversion performance was correlated to the specific surface area of the biocarrier, with the greatest rates of ammonia conversion (99.33%) and nitrate production (2.7 mg/L) for manufactured gyroid biocarriers with a specific surface area greater than 1980.5 m²/m³. The results suggest that the performance of commercial MBBRs for wastewater treatment can be greatly improved by manipulation of media design through topology optimization.

Introduction

Moving Bed Biofilm Reactors (MBBRs) are used for nutrient removal and recovery applications in secondary and tertiary levels of wastewater treatment. These applications include pharmaceutical wastewater [1, 2], petroleum contaminated waters [3, 4], pulp and paper industry waste streams [5, 6], piggery wastewater treatment [7], and sustainable nutrient remediation in municipal wastewater treatment [8]. For applications with high nitrogen concentrations, MBBRs are commonly used for nitrification, the aerobic microbial bioconversion of ammonia nitrogen to nitrate nitrogen. Air supply injects oxygen to enhance nitrogen oxidation, while at the same time achieving mixing of the treated water and biocarrier media that carry the nitrifying microbial community [9, 10]. The biocarrier media in MBBRs provide a surface on which a functional microbial biofilm attaches, and mixing in the reactor replenishes nutrient-rich wastewater to the active biofilm surface on each biocarrier. The rate of reaction, determining the overall performance of the reactor, is determined in part by the surface area characteristics of the biocarrier media, including the media size, shape, surface area per unit volume (the specific surface area, or SSA), and the fill ratio in the reactor vessel [9, 11].

An approach to improve the treatment process efficiency of a MBBR may consider the role of the biocarrier media. For example, by increasing the media fill ratio (the volume of biocarriers relative to the total volume in the container vessel), the total surface area available in the reactor is increased, which should improve the reaction yields. Another alternative is to increase the SSA of the biocarrier (the ratio of the total surface area of the biocarrier to its own volume), usually attained by increasing the geometric complexity and features of the surface. However, the optimization of the biocarrier SSA is critical to the performance of MBBR technology, as it determines the functional characteristics of the biofilm through defining mass transfer characteristics, biofilm thickness, and biofilm attachment strength. For example, in nitrification MBBRs, where the microbial biofilm on the carriers performs the ammonia oxidation, excess biofilm growth limits oxygen mass transfer to deeper biofilm layers, leading to biofilm detachment through sloughing from the surface of the biocarrier [12] and consequently reducing nutrient removal performance. In this regard, previous research efforts [13] attempted to develop biocarrier geometries that control the biofilm thickness and thus the ammonia oxidation process. Otherwise, the lack of biofilm thickness control may transform the MBBR design into a hybrid unit (MBBR and suspended sludge) [14], which in turn affects the overall performance and operation. Improving on the design of biocarrier geometry is therefore seen as critical for maximizing the performance of the MBBRs and, consequentially, its economic viability.

Several research efforts have been directed at exploring variations on the design of the biocarrier, with the goal of improving the performance in MBBRs through the amount of bacterial biofilm retained [15]. These variations include shapes, sizes, and materials that can be varied to affect biocarrier performance [16–19]. Shape and size have been shown to control the oxygen mass transfer into the attached biofilm, demonstrating its key influence to shorten the start-up period on MBBRs [19]. Avoiding media clogging through excess biofilm is also an important consideration for the geometry of the biocarrier, and the effect of geometry on biofilm thickness and water flow regime has been shown to be a major factor [20]. Typically, any improvement in reactor performance is limited by the balance between available surface area per unit volume and topographical complexity, itself limited by the manufacturability of more complex designs.

Among the potential fabrication technologies for manufacturing artificial biocarriers, additive manufacturing (AM) is an emerging technology that provides the capability to fabricate biocarriers with high levels of geometric complexity. The feasibility of achieving microbial colonization on 3D printed substrata has already been previously determined [21, 22], where AM was successfully used to produce both cubic gyroids and replicates of commercial carriers [22] that showed biofilm development in phototrophic biofilm reactors. Additional research demonstrated the effect of specialized topographic surfaces fabricated using AM on the species composition [23], colonization patterns [24], and functional growth and productivity [25] of phototrophic microbial biofilms. Other studies reported the fabrication of three fullerene-type microbial biocarriers using Laser Selective Sintering (SLS) technology and evaluated biofilm growth performance [26]. Under simulated sewage wastewater conditions in a sequencing batch reactor, the 3D-printed biocarriers developed a thicker biofilm than that found on the outer regions of the conventional Kaldnes K3 media, but developed thinner biofilms compared to those found in the inner walls. Tang et al. [2] used stereolithography technology (SLA) to fabricate a semi-suspended spindle-shaped biocarrier with synthetic material (isocyanate and polyhydric alcohols), which demonstrated the effects of shaped microhabitats on biofilm growth and microbial community diversity [2]. In preliminary work on the topic, Elliott et al. [27] demonstrated the manufacture of 3D-printed gyroid-based biocarriers using material jetting technologies and established the feasibility of implementing such technology for nitrification MBBRs [27].

To date, very little research has focused on the evaluation of novel 3D printed carriers in nitrifying MBBRs to reduce ammonia from wastewater and evaluate conversion rates as a function of the SSA or the fill ratio of biocarrier media. The 3D printed carriers reported in previous research [27] have the potential to accomplish faster nutrient conversion rates due to the large, mathematically modulated SSA. Additionally, gyroid-type carriers have geometric complexity that can define local hydrodynamics that are beneficial to biofilm formation rates and thus improve start-up times for MBBRs [28]. Given the fidelity of material jetting technology, and the easy scaling of gyroid geometry through parameter manipulation, different levels of void space can be designed in such a way that an optimal value of SSA can be determined that maximizes ammonia removal rates. Thus, the main objective of this research is to evaluate the performance of 3D printed gyroid biocarriers at different scaling parameters, SSA, and fill ratio in nitrification of wastewater in a MBBR.

Materials and methods

Biocarrier design and fabrication

Carriers were fabricated with additive manufacturing (i.e., 3D printing) technologies to produce complex geometries that span the limits on requirements for bacterial development in moving beds. Such requirements include a high SSA, an optimal void size (resulting in minimum clogging) and sufficient topographical sheltering (to protect bacterial biofilm from premature sloughing).

In this effort, a gyroid model was used to design the surface. This surface is generated from a mathematical equation with terms of sine and cosine functions (Eq 1) of an infinitely connected periodic minimal surface [29]. The equation model allows for manipulation of void size and overall surface area per unit volume by scaling the periodicity of the variables in the function.

sin x * cos y + sin y * cos z + sin z * cos x = 0

(1)

Out of the infinite possible parameter combinations, three gyroid biocarrier geometries were designed based on previous work [27] as nominal spheres with 20 mm diameter, representing different configurations of SSA (large, medium, and small). The performance of these in nitrification MBBR was to be experimentally compared against a control group of Kaldnes K1 media, a commonly-used commercial biocarrier design. The three gyroid designs were tessellated, converted into STL file format, and fabricated using an Objet-30 machine (Stratasys^® Ltd., Eden Prairie, Minnesota), which uses a layer thickness of 28 μm of UV-light cured acrylic polymer to build the objects.

The SSA of the carriers was obtained with the aid of Netfabb^®, an additive manufacturing software that calculates spatial information on the 3D models, including the total surface area, the true volume, and the volume of the work envelope of the carrier. True volume is defined as the consumed material (expressed as volume units) required to fabricate a single biocarrier object. The work envelope is the minimum closed surface that encompasses the entire geometry of the biocarrier. For the gyroid-based topologies used in this study, it consists of the minimum sphere that would theoretically wrap or enclose the entire biocarrier. For the Kaldnes K1 topology, it consists of the minimum cylinder that would encompass the entire biocarrier. The determination of the unit SSA for the gyroid biocarrier is the ratio of the surface area to the work envelope volume.

In a similar way, the unit SSA for the Kaldnes K1 commercial carriers was estimated, shown to be greater than the nominal value of 500 m²/m³ reported both in commercial and scientific literature [10]. To estimate the entire surface area of the commercial biocarrier, a reverse engineering approach was used, where the dimensions of the Kaldnes K1 biocarrier features were measured and the part was rebuilt via computer model using Solidworks^® (Dassault Systèmes SolidWorks Corp., Waltham, Massachusetts). The file was then imported into Netfabb^® and the information regarding its surface area retrieved. Images of example specimens of all biocarrier designs examined in this research are shown in Fig 1. Table 1 depicts the geometric parameters and material properties for all biocarrier designs that were used in this study.

Fig 1 — (a) Kaldnes K1; (b) small specific surface area gyroid; (c) medium specific surface area gyroid; (d) large specific surface area gyroid.

Table 1. Biocarrier designs and their geometric and material characteristics.

Name	Diameter (mm)	Surface area (m²)	Volume of Work envelope (m³)	Specific Surface Area (SSA)^* (m²/m³)	Material density (kg/m³)
Small SSA gyroid	20.00	21.94 x10^-04	4.189 x 10⁻⁶	523.8	1,033
Medium SSA gyroid	20.00	42.42 x10^-04	4.189 x 10⁻⁶	1013	1,033
Large SSA gyroid	20.00	82.96 x10^-04	4.189 x 10⁻⁶	1981	1,033
Kaldnes K1	10.39	9.44 x10^-04	6.215 x 10⁻⁷	1519	950^**

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*Calculated specific surface area from design rendering models.

**Referenced material density from [30].

Biofilter reactor design and setup

The experiments were conducted in a bench-scale Moving Bed Biofilm Sequencing Batch Reactor (MBBSBR) system, a sub-category of the broader category of MBBR. The experimental apparatus comprised two identical systems, each containing 6 reactors (Fig 2). Each reactor had a volume capacity for treating 1.3 liters of synthetic wastewater, and these were placed in a water bath to maintain constant temperature of 30°C [30].

Water and environmental conditions were controlled so that the nitrification processes were the main biological mechanisms to take place. The reactors were built by using commercial 2L jars (height: 15 cm) to which an 11-cm aeration ring and sampling ports were incorporated. Aeration was necessary to provide aerobic conditions suitable for the nitrifying bacteria, and to provide motion and agitation to the carriers. To maintain a concentration of dissolved oxygen adequate for aerobic conditions to support nitrification (5 ppm, as in [10]), the air supply pressure for the bioreactors was kept constantly at an operational level of 1 psi, monitored with an analog pressure gage. An air relief and check valve was designed into the lid of each reactor jar to maintain constant pressure. Samples of water were obtained through a sampling port in each lid. When sampling was required, the air supply was stopped and the contents of the reactor were allowed to settle for 30 minutes; then the port plug was removed, and the sample was extracted with a 100 mL syringe.

The performance of the biocarriers was assessed by monitoring the concentrations of total ammonia and nitrate in a prepared solution of synthetic wastewater. The recipe and procedure for the preparation of the synthetic wastewater is based on the presence of dissolved ammonia supplied by ammonium chloride at a concentration of 10 mg NH₄Cl L^-1. The theoretical minimum volume required in the commercial (Kaldnes K1) carriers for complete removal of ammonia within an 8-hour treatment process was estimated at 1.86 mL. This was supported by design calculations [31–33] for dissolved oxygen conditions within the range of 5–6 mg/L and an average temperature of 30°C [10].

Wastewater media preparation and analysis

The preparation of the synthetic wastewater media was based on procedures for assessing aquaculture systems found in literature [34]. The media recipe involved 17L of dechlorinated water, into which was dissolved ammonium chloride (170 mg), calcium carbonate (340 mg) and sodium bicarbonate (595 mg), plus trace elements necessary for nitrification provided by a low concentration (0.2% solution, 34 ml) marine salt solution (Seachem, Inc., Madison, Ga) [35]. Excess alkalinity of 27 mg/L (as CaCO₃) was added by calcium carbonate (15 mg/L) and sodium bicarbonate (35 mg/L), providing a buffering capacity to maintain pH in the range of 7.0–7.5 throughout an 8-hour long nitrification process. In addition, stock water was prepared to replenish evaporative losses. The stock water was dechlorinated tap water conditioned with off-the-shelf tap water conditioner (API, MARS Fishcare North America, Chalfont, PA) commonly used to dechlorinate aquarium waters. During the acclimatization period of multiple days, reactors ran continuously and evaporative losses were replenished with stock water every morning. Following preliminary operation and trials, synthetic wastewater was supplied using a high ammonia dilution of this media, resulting in an initial total ammonia nitrogen (TAN) concentration of 5 mg/L, typical for the lower range of concentrations for wastewaters.

Prior to introducing the carriers into the reactors for experimentation, they were exposed to bacterial inoculation and, hence, biofilm formation for approximately two months. Four buckets (each one per biocarrier type), each with 6 liters of synthetic wastewater and an air stone diffuser and a recirculation pump, were built for the purpose of inoculation.

A volume of 1.5 mL of Nitromax (Tropical Science Biolabs, Inc), an-off-the-shelf mixture of live Nitrosomonas and Nitrobacter, was added to each of the four buckets the first day followed by 750 μL every other day. Daily evaporation losses were replenished with dechlorinated water.

Ammonia, ammonium, nitrite, nitrate, pH, chlorine, hardness and alkalinity were inspected daily with test strips (Tetra Test Strips, Spectrum Brands Pet, Blacksburg, Virginia) to monitor water quality status during the inoculation period. All parameters remained consistent within broad ranges indicated by test strips throughout the inoculation period. The pH remained within a constrained range (7.48–7.75) because of carbonate buffering; as such, ammonia and nitrite were below detection throughout inoculation and experiment periods.

After two months and once biofilm formation was achieved, the gyroid and Kaldnes K1 carriers were placed into their respective reactors with synthetic wastewater for acclimatization. Reactors were placed in the water baths at 30°C nominal (measured at a 28.25±0.99°C, n = 12), air diffusion was kept at 1 PSI on the pressure gauge, hence maintaining approximately 5–6 mg O₂/L in each reactor. A volume of 163 μL of Nitromax was added to each reactor every other day. This acclimatization process required daily inspection that was performed with test strips, digital thermometer, pH and DO probes to make sure that the conditions for nitrification were optimal. Two weeks after the start of the acclimatization process, the system started to cycle and to produce nitrates, as determined through daily inspection with test strips.

Concentrations of ammonia and nitrate were determined by colorimetry using a photometer (YSI 9500, Yellow Springs, Ohio) that measures the color intensity of the sample after the addition of extra reagents. The water quality was monitored using standard colorimetric methods. Ammonia nitrogen determination was based on an indophenol method using manufacturer reagents (Ammonia Reagent, VWR Catalog No. 55407–204). Nitrate nitrogen determination was performed with zinc-based methods (Nitratest Tablets, VWR Catalog No. 55407–152). Determinations were done on triplicate samples from each one of the six reactors and three times during each experimental trial: at the beginning, at the middle and at the end of an 8-hour run. Prior to each sampling process, the reactors were set to idle for 30 minutes to let the water stand, while the sampling process itself took 15 minutes. Because of this, by the time the samples were fully processed and stored for later analysis, they corresponded to the following time stamps: 00:45, 04:45, and 08:45 (hr:min). However, for practical purposes, results were reported to nominal values of 0 hours, 4 hours and 8 hours of treatment. The dissolved oxygen concentration, pH, and temperature measurements were obtained both at the beginning and at the end of every experimental trial, with pH remaining within the prescribed range (7.48–7.75), dissolved oxygen controlled (4.9–5.6 ppm), and temperature constrained (30°C). The water dechlorination process to prepare the synthetic wastewater was done 24 hours prior to use. Most of the water analyses were performed immediately after sample collection.

Experimental design and analysis of results

The overall guiding hypothesis for each experiment is that that a larger total surface area, whether provided on a media unit basis or spread across the bulk of the media, promotes faster rate of conversion of total ammonia nitrogen to nitrate nitrogen through the nitrification microbial process. In the research, three separate experiments were performed to measure the effect of biocarrier design on the performance of ammonia conversion to nitrate while maintaining constant across all reactors: (1) the biocarrier fill ratio and packing volume; (2) the total biocarrier surface area; and (3) the number (count) of biocarriers units (Fig 3). The packing volume, which is used to estimate the fill ratio, refers to the volume occupied by a predetermined count of biocarrier elements and is dependent on the overall geometry and surface topology nesting characteristics, which would allow for a denser packing arrangement.

Fig 3 — (a) Experiment 1, testing constant biocarrier fill ratio; (b) Experiment 2, testing constant total biocarrier surface area; (c) Experiment 3, testing constant number of biocarriers. Diagram not to scale.

In every experiment, three trials were conducted. In every trial, each of the four different biocarrier designs were replicated three times thus employing twelve reactors, each treating synthetic ammonia-laden wastewater for 8-hour trials. In all experiments, sizing was determined based on calculations of minimum required biocarrier surface area for complete nitrification, as reported in [36, p.95] and validated through trial and error in preliminary trials (data not reported here). From preliminary trials it was identified that 2.00x10^-04 m³ was a workable volume of packed biocarriers (for all types) that provided an observable mobility inside the reactor. Such volume selection consequently derived into the selection of the number of each carrier of each type for each of the experimental treatments described below.

For Experiment 1, the fill ratios were kept constant across trials (Table 2). Again, the fill ratio is the percentage of the reactor volume occupied by the bulk media when initially dry-loaded and packed tightly in the reactor. This required different quantities (count) of biocarrier elements depending upon the biocarrier design. The chosen fill ratio of 15% was consistent with values found in literature [12, 37, 38].

Table 2. Parameters for Experiment 1 with constant fill ratio (15%).

Treatment/Biocarrier type	Surface area per biocarrier unit (m²)	Count per reactor	Total surface area per reactor (m²)
Kaldnes K1	9.44 x10^-04	148	0.140
Small SSA gyroid	21.94 x10^-04	25	0.055
Medium SSA gyroid	42.42 x10^-04	25	0.106
Large SSA gyroid	82.96 x10^-04	25	0.207

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For Experiment 2, the total biocarrier surface area was kept constant across all trials (Table 3). The total biocarrier surface area is the summation of the available surface area of all biocarriers. Because of the different specific surface area for each type of biocarrier, a different number (count) of biocarriers was required for each trial unit. The total surface area per reactor of 0.055 m² was chosen based on the minimum amount of surface area for gyroids needed for a measurable rate of nitrification throughout the time of the trial, as determined through trial and error preliminary investigations and confirmed through repetition in other experiments (for example, Experiment 1, Table 2).

Table 3. Parameters for Experiment 2 with constant total biocarrier surface area (0.055 m²).

Treatment/Biocarrier Type	Packing Volume (m³)	Surface Area per Biocarrier Unit (m²)	Count per reactor	Fill Ratio per Reactor (%)
Kaldnes K1	6.91x 10⁻⁰⁵	9.44 x10^-04	58	5
Small SSA gyroid	2.00x10^-04	21.94 x10^-04	25	15
Medium SSA gyroid	1.04x10^-04	42.42 x10^-04	13	8
Large SSA gyroid	5.30x10^-05	82.96 x10^-04	7	4

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Finally, for Experiment 3, the total number (count) of carriers was kept constant across all trials (Table 4). Because of the different specific surface area for each type of biocarrier, the same count resulted in different total surface area and fill ratio per reactor for different biocarriers. The constant count value of 7 units of biocarriers in each reactor was chosen based on the minimum number of Kaldnes K1 biocarriers needed for a detectable amount of total ammonia conversion during the 8-hour trials, as determined through observations in trial and error preliminary investigations (data not included here).

Table 4. Parameters for Experiment 3 with constant total number (count) of biocarriers (7 biocarriers each).

Treatment/Biocarrier Type	Packing Volume (m³)	Surface Area per Biocarrier Unit (m²)	Total surface area per reactor (m²)	Fill ratio per reactor (%)
Kaldnes K1	8.33x10^-06	9.44 x10^-04	0.007	<1
Small SSA gyroid	5.30x10^-05	21.94 x10^-04	0.015	4
Medium SSA gyroid	5.30x10^-05	42.42 x10^-04	0.030	4
Large SSA gyroid	5.30x10^-05	82.96 x10^-04	0.055	4

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For each experiment and trial, the experimental design was a full factorial with three replicate reactors (4 factors x 3 reps = 12 reactors/trial) and over three experimental trials for a total of 36 observations (9 per biocarrier treatment). The experimental design was conducted by blocking the reactors into two separate baths with six reactors each. The locations of the reactors within and between baths were randomly assigned in each trial and throughout the entire experiment to guard against uncontrolled factors. An analysis of homoscedasticity and a Kolmogorov-Smirnov normality test showed that the data do not follow the underlying assumptions for an Analysis of Variance. Consequently, the statistical analysis was conducted with a non-parametric Kruskal-Wallis statistical test on the medians. Additional analysis was performed with a Mood’s Median test to compare the medians of pairwise samples to determine significance. Additionally, before each experimental trial, all carriers of the same type were mixed together in a bag and randomly selected and replaced into the reactors. All conditions were reset after each trial and new assignments of reactor locations and biocarrier sets were randomly conducted. For all experiments, water samples were taken regularly and analyzed for ammonia and nitrate nitrogen concentrations, for comparison across all treatments of nitrification performance. Ammonia concentration conversion percentage was calculated for each replicate, where the pooled ammonia nitrogen concentration at the 8^th hour was deducted from the concentration at the start of the water treatment, and the difference divided by the starting concentration of ammonia nitrogen. These are reported and compared for all trials and all experiments.

Results and discussion

Experiment 1: Constant fill ratio of biocarriers

The results from Experiment 1, constant Fill Ratio of biocarriers, are presented in Fig 4a, with parameters expressed as pooled Total Ammonia Nitrogen (TAN) and Nitrate Nitrogen concentrations over time. Results show that gyroids with medium and large SSA convert significantly more ammonia than the commercial Kaldnes K1 and the gyroid with small SSA (p<0.001). Two separate Mood’s median tests (5% significance level) were performed on the total ammonia nitrogen and nitrate nitrogen. These tests could not differentiate between the performances of large SSA and medium SSA gyroids at 4 and 8 hours, so these were statistically found to provide the same performance. Similarly, from the same statistical test, the performances of the small SSA gyroid and the Kaldnes K1 were found to be statistically the same at 4 and 8 hours. The observed variation in the ammonia conversion performance when manipulating the SSA parameter reinforces previously reported findings [39] that SSA is an important parameter for MBBR design. Furthermore, these findings strongly suggest that SSA can be used as a proxy parameter when seeking to optimize MBBR performance. Also, the amounts of ammonia conversion by the medium and large SSA gyroid types were similar (close to 100% of ammonia removed from solution) with the same biocarrier volume (as expressed by fill ratio) in the reactors. This suggests that the subsidy of increased surface area is limited, preventing a continued increase in ammonia conversion rate despite the increased SSA. Previous studies suggest that a smaller biocarrier pore size results in slower rates of substrate and oxygen transport to inner regions inside the voids [28]. This might apply to the large SSA gyroid, which has the smallest pore sizes of the gyroid biocarriers, thus preventing an increase in nitrification performance proportional to the increase in SSA.

The observed trends in nitrate concentration were similar, where the gyroids with medium and large SSA increased nitrate concentrations at a greater rate than the small SSA gyroid and Kaldnes K1. By comparing the changes in concentration of ammonia nitrogen and nitrate concentration over time, a conservation of nitrogen was observed within the process. In all trials, the amount of ammonia nitrogen consumed matched the amount of nitrate nitrogen produced, confirming the nitrification performance of the biofilters.

Percentage change for total ammonia nitrogen for all trials was calculated and is shown in Fig 5. In Experiment 1, the 8-hour percentage change of total ammonia nitrogen was nearly 100% conversion for the highest-performing treatments (Gyroids with Large and Medium SSA), whereas the small SSA gyroid and Kaldnes K1 media had significantly lower TAN change percentage (42% and 30%, respectively). The significantly higher TAN conversion percentage correlates with the higher specific surface area, and thus total surface area, in the reactors with the medium and large SSA gyroid.

Results on the performance of the different media in Experiment 1 also help to understand the role of surface area conformation in establishing performance. From results of Experiment 1, it is readily observed that the percentage conversion of ammonia nitrogen correlates with the SSA per biocarrier type. Such relationship was identified between the treatments with small (low) and medium SSA gyroids. In such treatments, the amount of ammonia nitrogen converted by the medium SSA gyroids was approximately twice that converted by the small SSA gyroids, corresponding to the doubling of the SSA for medium gyroids (Fig 6). The performance in TAN conversion did not necessarily correlate with total surface area in the reactor, where the Kaldnes K1 media had the second largest total surface area but the lowest TAN percent change (Fig 6). The amount of ammonia nitrogen converted by Kaldnes K1 was not greater than the amount of ammonia nitrogen converted by the medium or small SSA gyroids, even though the SSA of the Kaldnes K1 (1519 m²/m³) is nominally larger than the other two (1013 and 524 m²/m³ respectively). However, the amount of ammonia nitrogen converted by the Kaldnes K1 biocarrier was consistent with its reported surface area of 500 m²/m³ as communicated by the manufacturer [40, 41]. This suggests that the performance of the gyroid media is enhanced beyond the subsidy of greater SSA, with this enhancement coming from materials considerations or particular characteristics of the gyroid shape.

Experiment 2: Constant biocarrier total surface area

The results from Experiment 2, constant total surface area, are presented in Fig 4b, with parameters expressed as pooled total ammonia nitrogen (TAN) and nitrate nitrogen concentrations over time. Results showed a decrease in ammonia nitrogen concentrations over time for all trials (Fig 4b). The greatest rate of conversion of ammonia was seen for the medium SSA gyroids; the next greatest rates of conversion seen for large and small SSA gyroids; and the smallest rate of conversion observed for the Kaldnes K1 media. Results were significant (p<0.001, Kruskal-Wallis test) for the differences in 8-hour ammonia nitrogen conversion between all gyroids and the Kaldnes K1 media. Similarly, nitrate nitrogen concentration increases followed the same pattern (Fig 4b), with the greatest change observed for the medium SSA gyroid and the least for the Kaldnes K1, with significance (p<0.001). Two separate Mood’s median tests (5% significance level) were performed on the total ammonia nitrogen and nitrate nitrogen. These tests could not differentiate between the performances of large SSA, medium SSA, and small SSA gyroids at 4 and 8 hours, so these were statistically found to provide the same performance.

Percentage change of ammonia nitrogen concentration after 8 hours for Experiment 2 (Fig 5) shows a greater performance for all gyroids than Kaldnes K1 (p<0.001). It is important to highlight the performance of medium SSA gyroids, where almost 100% of ammonia nitrogen was converted within 8 hours, approaching the same conversion rate as the high SSA gyroids in Experiment 1 (0.40 mg/L/hour), even though the number of biocarriers was decreased (25 to 13) in these trials. A potential reason for this continued high performance is that, under the conditions in Experiment 2, the decrease in the number of biocarriers in the reactor allowed for increased agitation and increased mixing intensities, enhancing mass transfer of oxygen or substrate [40, 41]. Additionally, the high performance of the medium SSA gyroid, compared to others, suggests that an optimum configuration of surface area exists, given that the total surface area in all trials is the same. The lower performance the large SSA gyroid suggest the limitation of SSA on performance, likely as a result of mass transfer limitations on smaller features and pore sizes of the large SSA gyroid. This result suggests the perspective that SSA and total surface area of media in a reactor could be optimized for best performance through a combination of media SSA complexity and total count (fill ratio) of media in the reactor.

Experiment 3: Constant biocarrier count

The results from Experiment 3, constant biocarrier count, are presented in Fig 4c, with parameters expressed as pooled total ammonia nitrogen (TAN) and nitrate nitrogen concentrations over time. As before, results showed a decrease in ammonia nitrogen concentration over time for all trials. The greatest rate of conversion of ammonia was seen for the large SSA gyroids; a moderate rate of conversion seen for medium SSA gyroid; and the smallest rate of conversion observed for both the small SSA gyroid and Kaldnes K1 biocarriers. As before, results were significant (p<0.001, Kruskal-Wallis test) for differences in 8-hour ammonia concentration decrease between all gyroids and the Kaldnes K1 media. Similarly, nitrate nitrogen concentration increases followed the same pattern (Fig 4c), with the greatest change observed for the large SSA gyroid and the least for the Kaldnes K1, with significance (p<0.001). Two separate Mood’s median tests (5% significance level) were performed on the total ammonia nitrogen and nitrate nitrogen. These tests could not differentiate between the performances of large SSA, medium SSA, and small SSA gyroids at 4 and 8 hours so these were statistically found to provide the same performance.

Percentage change of ammonia nitrogen concentration over 8 hours for Experiment 3 (Fig 5) shows a greater rate of total TAN concentration decrease for all gyroids than for Kaldnes K1 media (p<0.001). TAN percentage change was 71% for the large SSA gyroid, 45% for medium SSA gyroid, 30% for small SSA gyroid, and 22% for Kaldnes K1 biocarrier. The results show the subsidy of greater specific surface area of the gyroid media, where TAN percentage conversion performance increases with increasing specific surface area of the biocarrier media. All gyroid trials had a greater TAN conversion performance than the Kaldnes K1 media at the equivalent number. The TAN percentage change from gyroids was measurably greater than conventional biocarriers even when the biocarrier count was notably less than that compared to other experiments. This suggests that the performance of the gyroid biocarriers is enhanced by the high specific surface area. In addition, the amount of increases in performance for the medium and large SSA gyroids, despite the relatively small number of biocarriers in this experiment, suggests a subsidy in performance resulting from the high amount of internal surface area, a result directly related to the conformation of the topographical design of the biocarriers.

Summary of all results

Overall, the manufactured gyroids of all SSA showed high performance in nitrification MBBR applications. In all experiments, the nitrification rate was directly related to the specific surface area of the biocarrier, and the cumulative nitrification was influenced by the total surface area in the reactor, accumulated by the combination of the specific surface area of the media and the total count of the individual media carriers. In all cases, even when reactors were normalized for total surface area available, total fill ratio, and total count, manufactured gyroid biocarriers had a greater nitrification performance than commercial Kaldnes K1 media. It is expected that the generally greater SSA with more shape conformation, providing a large amount of surface area that could protect an established active biofilm, contributes to the overall subsidy of performance. Optimization of the surface area complexity for the gyroids is necessary to understand the effects of long-term operation on biofilm accumulation, mass transport dynamics, and ultimately reactor-scale performance in operations. Additionally, performance of the biocarriers should be investigated for applications to wastewater streams, including municipal or aquaculture sources, where higher concentrations in organic carbon and nutrient concentrations differentially impact the accumulation of biofilm on the carriers themselves, and ultimately the performance of the bioreactor in any given application. Life span considerations are important, too, as the material and shape integrity of the biocarriers over long-term repeated use is unknown. The approach for manufacture and application of gyroid geometry as a biocarrier, however, is demonstrated through the significant performance increases observed in these experiments.

Conclusions

The levels of geometrical complexity of the design and fabrication of biocarriers for water treatment processes were manipulated with the capabilities of additive manufacturing. Three mathematical designs with increasing specific surface area (SSA) were used to fabricate biocarriers and test their performance in three different scenarios: constant fill ratio, constant total surface area, and constant biocarrier media count. For all the scenarios, the gyroid media designs presenting large and medium SSA provide the best nitrification performances: that is, the lowest final concentration of pooled total ammonia nitrogen as well the highest concentration of pooled nitrate nitrogen. The best performing design for the scenario of constant fill ratio is the large SSA gyroid with 99.33% of ammonia nitrogen conversion at 8 hours. The best performing design for the scenario of constant total surface area is the medium SSA gyroid with 94.74% of ammonia nitrogen conversion at 8 hours. The best performing design for the scenario of constant biocarrier media count is the large SSA gyroid with 92.73% of ammonia nitrogen conversion at 8 hours. From a practitioner’s standpoint, Experiment 1 perhaps provides the most relevant information for selection of biofilter media, as one of the most practical considerations in bioreactors loading conditions is the fill ratio in the tanks. In this regard, and with manufacturing considerations aside, the results indicate that either large or medium gyroid SSA would be the best choice. However, this study was focused on the early stages of nitrification and thus limited to an 8-hour period. Therefore, it ignores some of the steady-state operational issues (e.g. clogging, biofilm build-up, sloughing, etc.) of bioreactors that would likely give a functional advantage to a design with larger pore openings while maintaining optimal performance (i.e. medium SSA gyroid). Additionally, results from Experiments 2 and 3 indicate that further optimization on the geometry and its proxy SSA can be pursued. Future work will aim at this through the use of statistical response surface methodologies over a longer period of bioreactor performance.

Data Availability

All data files are publicly available from the Digital Commons at Georgia Southern University (DOI 10.20429/data.2020.1).

Funding Statement

This work was partially supported by Grant Number SU836122, from the U.S. Environmental Protection Agency (www.epa.gov) awarded to DMB and ALC. Material in-kind support provided to DMB by the Alabama Agricultural Experiment Station of Auburn University (aaes.auburn.edu). Partial support was from the Ecuadorian scholarship funded by the Secretaria de Educación Superior, Ciencia y Tecnología e Innovación-Senescyt (www.educacionsuperior.gob.ec), awarded to GPP. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. doi: 10.1371/journal.pone.0238386.r001

Decision Letter 0

Leonidas Matsakas

4 Jun 2020

PONE-D-20-13545

Analysis of Very-High Surface Area 3D-printed Media in a Moving Bed Biofilm Reactor for Wastewater Treatment

PLOS ONE

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Reviewer #1: PONE-D-20-13545

Analysis of Very-High Surface Area 3D-printed Media in a Moving Bed Biofilm Reactor for Wastewater Treatment

The study is about the wastewater treatment specific to ammonia/TAN using a high surface area 3D fabricate gyroid-shaped bio-carrier. Treatment performance was compared with the market available commercial bio-carrier. Wastewater treatment results in terms of ammonia removal are interesting with 3D fabricated gyroid-shaped bio-carriers compared to conventional bio-carrier. However, the experiments were carried out using synthetic wastewater. The actual performance/potential of bio carriers can be examined with real field wastewater even at the lab scale. The discussion on removal is weak, not focused the treatment efficiency.

Comments

• The photograph showed for commercial bio-carrier is actually not such bad as shown here, it can be accepted that 3D-Printed bio carrier is better but portraying such images for commercial bio-carrier is not acceptable.

• Real field wastewater treatment is always advisable to evaluate the potential of designed bio carriers. The process may be examined with real field wastewater if possible.

• What is the life of designed bio carriers and their interaction with microbes?

• Quality of graphs are very poor to read the contents.

• Why the initial concentration of nitrates and TAN was chosen below 5 mg/L as wastewater typically contains more than 20 mg/L.

• Any figure can be produced with biofilm formation on designed and conventional bio carrier.

• Ammonia, ammonium, nitrite, nitrate, pH, chlorine, hardness, and alkalinity were also inspected but values were not mentioned in the study.

• Line 303: it cannot be claimed that SSA is the main parameter for maximizing the performance of MBBRs as many other factors influence the whole treatment process.

• Ammonia removal close to 100% might be due to the adaption of microbes at low concentration of ammonia being fed in the bioreactor- the case might be opposite with real field wastewater or when the concentration of ammonia is more than 5 mg/L.

• Any relation found for nitrate production and TAN removal, as nitrate production is almost the same whereas trend for TAN removal is quite opposite.

Reviewer #2: Abstract: Some data must be provided to support your statements.

Page 3, lines 41-44: Introduce the following current reference regarding the applications of MBBR in order to complement the literature cited: “Moving bed biofilm reactor as an alternative wastewater treatment process for nutrient removal and recovery in the circular economy model”.(https://doi.org/10.1016/j.biortech.2019.122631).

Page 3, line 53: Include the following reference in relation to the filling ratio: “Effect of the filling ratio, MLSS, hydraulic retention time, and temperature on the behavior of the hybrid biomass in a hybrid moving bed membrane bioreactor plant to treat urban wastewater”. (10.1061/(ASCE)EE.1943-7870.0000939)”.

Page 3, line 60: Correct “MMBR”.

Page 6, line 128: you mention reference 23, but in introduction you say that this kind of design had not been carried out previously for MBBR. Could you justify this contradictory information?

Figure 1: Explain why you introduce this figure in the paper.

Page 7, line 154: Why do you indicate “Table 1” in bold?

Table 1: some lines of the table are missing. Specify the meaning of SSA and include the abbreviation in brackets. Indicate the reference where you have obtained the value of SSA and density for K1 carrier from. The meaning of “work envelope” must be explained.

Figures 2 - 6: They have not enough quality. Please improve them.

Page 8, line 174: 5 ppm for dissolved oxygen is excessive.

Section “Wastewater media preparation” is too long. You must divide it into more sections. Furthermore, some descriptions of analytical methods can be summarized or included in Supplementary Information.

Table 2: remove it and indicate the volume in the text (since the concentrations are given).

Page 10, line 221: What does “uL” mean?

Page 12, line 267: Justify the choice of 0.055 m2. It is not clear in the text.

Table 4: Explain the meaning of “packing volume” in the text in order to understand the information supplied in this table.

Page 12, line 276: justify with some reference the number of 7 carriers.

Section “Results and Discussion” must be divided in several sub-sections according to the three experiments carried out.

Page 13, line 300: do not use capital letters and use “total ammonia nitrogen” without “-N”.

Page 13, line 302: it is not logical to give a p-value with three significant numbers. Revise p-values in manuscript and introduce the statistical method in “Materials and Methods”. I can not revise the results from Figure 4 as I can not see them (poor quality of the figure). Please, do not use abbreviations in X or Y axis.

When you say “ammonia removed”, what do you refer to? In your systems, there is no an anoxic zone or anoxic time. Explain it better in order to get a better comprehension for potential readers. I suppose that you consider the transformation of ammonium nitrogen into nitrate nitrogen (nitrification only). Specify clearly it (“remove” is confusing). Additionally, explain why you analyze both parameters TAN and nitrate concentration.

Page 15, lines 345-349: compare with the values of protected surface area for biofilm growth corresponding to your biocarriers.

Figure 6: it is not easily interpretable. Moreover, use superscripts.

Page 16, lines 394-395: Justify what you say.

Pages 16-17, lines 396-400: Explain it with more detail.

Conclusions: this section must be summarized, you must give conclusions without repeating information from Materials and Methods for example. You can number the main conclusions for each experiment carried out. Include some data to support your conclusions. Explain the last paragraph: you state 1013 m2/m3 as the most suitable carrier, which carrier does this value correspond to? Justify this last paragraph.

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Reviewer #2: No

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PLoS One. 2020 Aug 27;15(8):e0238386. doi: 10.1371/journal.pone.0238386.r002

Author response to Decision Letter 0

13 Jul 2020

Response to Reviewers

We present the response to reviewers in detail in this communication. We thank the reviewers for their helpful and constructive comments that will ultimately improve this manuscript. We believe we have addressed all comments in this letter and/or in the manuscript.

In this response letter, reviewer comments are shown in italicized font, followed by our response in standard text. The additions or edits of significance to the manuscript are in shown in bold text as part of the response. The location for the changes made to the document is also indicated within the response.

Reviewer #1:

Specific Comments

Thank you for your comment and observation on the figure. Indeed, the figure is of poor quality, and our random selection does not adequately reflect all the biocarrier designs examined in this research. We have produced a new image of the commercial bio-carrier, and added images of each of the fabricated biocarriers for an improved overview presentation of our research biocarriers. Along with this, we have updated the caption for Figure 1 (line 166-168, Manuscript Markup) to read as follows:

Figure 1: Images of commercial (K1) and fabricated (gyroid) biocarrier designs examined in this research. (a) Kaldnes K1; (b) small specific surface area gyroid; (c) medium specific surface area gyroid; (d) large specific surface area gyroid.

• Real field wastewater treatment is always advisable to evaluate the potential of designed bio carriers. The process may be examined with real field wastewater if possible.

We thank the reviewer for this comment. Indeed, additional value into understanding the performance of the novel biocarriers would be generated from investigating with real wastewater. We chose not to investigate this for this initial investigation, however, because of the desire to precisely control the chemistry of the input water to reduce experimental noise in a constrained experimental design. The potential variability in water chemistries, including variability in both nutrient and organic concentrations, of most wastewaters was undesirable for planning our initial investigations here. As such, our media was not intended to fully emulate municipal sourced wastewater, but more so a high-nutrient low organic carbon wastewater that might be found in aquaculture applications, where mixed bed bioreactor technologies are often used for nitrification. We strongly feel that future work on these biocarriers should include investigations with real wastewater sourced from multiple sources and of varying water quality and nutrient and organics concentration. We have added the following sentence (lines 474-478, Manuscript Markup) to indicate this:

Additionally, performance of the biocarriers should be investigated for applications to wastewater streams, including municipal or aquaculture sources, where variations in organic carbon and nutrient concentrations differentially impact the accumulation of biofilm on the carriers themselves, and ultimately the performance of the bioreactor in any given application.

• What is the life of designed bio carriers and their interaction with microbes?

We appreciate this comment as it relates to one of our main concerns and interests. We agree that life span of any biocarrier is an important operational consideration that would impact use, utility and potential commercial success of the biocarrier in any application. In this research, however, we are focused on the fundamental aspects of design of the shape and the performance aspects in the early initiation stages of biofilm development and application (i.e. the first 8 hours of operation). Further research is indeed required to determine the durability and lifespan aspects of these biocarriers.. To address this concern, we have added the following sentence (lines 478-479, Manuscript Markup) to our summary paragraph:

Life span considerations are important, too, as the material and shape integrity of the biocarriers over long-term repeated use is unknown.

• Quality of graphs are very poor to read the contents.

Thank you for pointing this out to us.. We apologize for the poor quality of the graphs, which is suspected to have occurred during file conversion upon upload of images. We have re-formatted all figure images with high resolution files, which is expected to improve the quality of all figures.

• Why the initial concentration of nitrates and TAN was chosen below 5 mg/L as wastewater typically contains more than 20 mg/L.

Thank you for your comment. The reviewer is correct that wastewater often has TAN concentrations higher than 5 mg/L. We chose the concentration of TAN to reflect a low-end concentration, that is often typical of wastewaters from biofloc aquaculture production. The reviewer comment is important and formative, however, for future work regarding the optimization of performance for our biofilter media. We have added clarification of this in the Methods section (lines 214-217, Manuscript Markup):

Following preliminary operation and trials, synthetic wastewater was supplied using a high ammonia recipe (Table 2), resulting in an initial total ammonia nitrogen (TAN) concentration of 5 mg/L, typical for the lower range of concentrations for wastewaters.

Also, we have added this consideration to recommendations for future research (lines 474-478, Manuscript Markup):

Additionally, performance of the biocarriers should be investigated for applications to wastewater streams, including municipal or aquaculture sources, where higher concentrations in organic carbon and nutrient concentrations differentially impact the accumulation of biofilm on the carriers themselves, and ultimately the performance of the bioreactor in any given application.

• Any figure can be produced with biofilm formation on designed and conventional bio carrier.

Thank you for your comment. Although we confirmed the presence of biofilm on all biocarriers with microscopy, we do not have any images showing this, and chose to not report that investigation in this manuscript.

• Ammonia, ammonium, nitrite, nitrate, pH, chlorine, hardness, and alkalinity were also inspected but values were not mentioned in the study.

Thank you for your comment. We will attempt to clarify. Most parameters mentioned here were monitored to ensure that they remained within prescribed ranges known to be favorable for nitrification. Many parameters (chlorine, hardness, alkalinity) were measured via colorimetric test strips, which provide a broad range for precision, and thus numerical reporting for these would be rather imprecise. pH was controlled during all experiments through carbonate buffering, and remained within a narrow range (7.48-7.75). At this pH range, ammonia was measured below detection; also, nitrite was measured below detection, suggesting complete nitrification processes. As then reported in our manuscript, ammonium and nitrate concentrations were measured via photometer as the dependent variables reported in our results. We have added clarification in two places in the Methods, including the statement (lines 228-231, Manuscript Markup):

All parameters remained consistent within broad ranges indicated by test strips throughout the inoculation period. The pH remained within a constrained range (7.48-7.75) because of carbonate buffering; as such, ammonia and nitrite were below detection throughout inoculation and experiment periods.

And the later statement amended as such (lines 253-256, Manuscript Markup):

The dissolved oxygen concentration, pH, and temperature measurements were obtained both at the beginning and at the end of every experimental trial, with pH remaining within the prescribed range (7.48-7.75), dissolved oxygen controlled (4.9-5.6 ppm), and temperature constrained (30°C).

• Line 303: it cannot be claimed that SSA is the main parameter for maximizing the performance of MBBRs as many other factors influence the whole treatment process.

Indeed, the reviewer is correct in that the text can be misconstrued as currently expressed. We thank the reviewer for pointing this out. We have replaced this statement and sharpened the language for clarification. The revised statements now suggest a more nuanced meaning of our interpretation (lines 347-350, Manuscript Markup):

The observed variation in the ammonia removal performance when manipulating the SSA parameter reinforces previously reported findings (36) that SSA is an important parameter for MBBR design. Furthermore, these findings strongly suggest that SSA can be used as a proxy parameter when seeking to optimize MBBR performance.

Thank you for your statement. We agree with your interpretation of the processes occurring. Indeed, it is possible that the microbes are well-adapted to the comparatively lower ammonia concentrations in the wastewater simulant. However, it should be noted that the ammonia concentrations are scaled for a particular wastewater from an aquaculture system, where ammonia concentrations are typically lower than that found in municipal wastewater because of the particular dilutions. Results on performance of the biofilter system and media may indeed be different for higher concentrations of ammonia in the input wastewater. We support the idea that these sorts of studies should be done in the future to expand the knowledge of performance for these particular biofilter media in other applications. As noted previously, an indication of the need for future research using higher wastewater concentrations is inserted in the Discussion (lines 474-478, Manuscript Markup):

• Any relation found for nitrate production and TAN removal, as nitrate production is almost the same whereas trend for TAN removal is quite opposite.

Thank you for your comment. Indeed, removal of TAN and production of nitrate is expected to be nearly equivalent amounts, according to common understanding of the nitrification process. As such, we believe that is reflected in our data as presented, where, in all cases and treatments, nitrate concentration increases proportionally to the decrease in TAN.

Reviewer #2:

Abstract: Some data must be provided to support your statements.

Thank you for your comment. We agree that more precise language and presentation of data in the Abstract is best practice for support of our statements. We have rewritten portions of the Abstract to have more detail with support from data, found in the associated lines (lines 31-40 Manuscript Markup):

Results showed that large and medium SSA gyroid biocarriers delivered the best ammonia removal performance of all designs, and significantly better than that of a standard commercial design. The percentage of ammonia nitrogen conversion at 8 hours for the best performing biocarrier design was: 99.33% (large SSA gyroid, constant fill ratio), 94.74% (medium SSA gyroid, constant total surface area), and 92.73% (large SSA gyroid, constant biocarrier media count). Additionally, it is shown that the ammonia conversion performance was correlated to the specific surface area of the biocarrier, with the greatest rates of ammonia conversion (99.33%) and nitrate production (2.7 mg/L) for manufactured gyroid biocarriers with a specific surface area greater than 1980.5 m2/m3.

Thank you for pointing us to this article. We have added a reference to the paper (line 49-50, Manuscript Markup).

Thank you for your comment. We have added a reference to the paper (line 59, Manuscript Markup).

Page 3, line 60: Correct “MMBR”.

We are grateful for letting us know. We have corrected the text (line 66, Manuscript Markup).

Page 6, line 128: you mention reference 23, but in introduction you say that this kind of design had not been carried out previously for MBBR. Could you justify this contradictory information?

Thank you for your comment. That particular reference demonstrated preliminary work on the production of the gyroid production, but did not explore the performance aspects in a replicated experimental design. We have clarified the language in various places to reflect the preliminary nature of that work, and the follow-on nature of this manuscript, such as in the introductory text (lines 105-108, Manuscript Markup):

In preliminary work on the topic, Elliott et al. (23) demonstrated the manufacture of 3D-printed gyroid-based biocarriers using material jetting technologies and established the feasibility of implementing such technology for nitrification MBBRs.

Likewise, in the line following (line 109-111, Manuscript Markup):

To date, very little research has focused on the evaluation of novel 3D printed carriers in MBBRs to remove ammonia from wastewater and evaluate removal rates as a function of the SSA or the fill ratio of biocarrier media.

Figure 1: Explain why you introduce this figure in the paper.

Thank you for your comment. Based upon your comment, we have reconsidered the information best presented in Figure 1. We have now presented images of each of the four types of biocarrier designs tested in this experiment. The cross-reference for Figure 1 is now rewritten as follows (lines 158-159, Manuscript Markup):

Images of example specimens of all biocarrier designs examined in this research are shown in Figure 1.

Page 7, line 154: Why do you indicate “Table 1” in bold?

Thank you for your comment. Bold-face labels of this type were a legacy of a previous formatting style. We have removed the bold type from all cross-reference labels of this type globally.

Thank you for your comments. We have reformatted the table to address the problems with lines. We have spelled out “Specific Surface Area (SSA)” in the table headings. We have added notes on the table referencing the source for values for SSA (calculated using design rendering and mesh models). Also, we have added an explanation of the work envelope in the text prior to the table (lines 146-149, Manuscript Markup):

The work envelope is the minimum closed surface that encompasses the entire geometry of the biocarrier. For the gyroid-based topologies used in this study, it consists of the minimum sphere that would theoretically wrap or enclose the entire biocarrier. For the Kaldnes K1 topology, it consists of the minimum cylinder that would encompass the entire biocarrier.

Figures 2 - 6: They have not enough quality. Please improve them.

Thank you for your comment. We have included high resolution files of all figures now for improving.

Page 8, line 174: 5 ppm for dissolved oxygen is excessive.

Thank you for your comment. We chose a concentration of 5 mg/l of dissolved oxygen was due to the intention to test TAN removal rates at their maximum performance level, and the way to gain control on this parameter was to avoid potential scenarios of dissolved oxygen limitation, as referred to in literature. We have added an explanation of this with a citation as follows (lines 186-189, Manuscript Markup):

To maintain a concentration of dissolved oxygen adequate for aerobic conditions to support nitrification (5 ppm, as in (9)), the air supply pressure for the bioreactors was kept constantly at an operational level of 1 psi, monitored with an analog pressure gage.

Thank you for your comments. We have divided the entire “Methods’ section into more subsections, to include the following: Biocarrier Design and Fabrication; Biofilter Reactor Design and Setup; Wastewater Media Preparation and Analysis; Experimental Design and Analysis of Results. We believe this organizes better the entire section for clarity of presentation.

Table 2: remove it and indicate the volume in the text (since the concentrations are given).

Thank you for your comment. We have removed Table 2, and indicated the media recipe in detail in the text, as follows (lines 204-207, Manuscript Markup):

The media recipe involved 17L of dechlorinated water, into which was dissolved ammonium chloride (170 mg), calcium carbonate (340 mg) and sodium bicarbonate (595 mg), plus trace elements necessary for nitrification provided by a low concentration (0.2% solution, 34 ml) marine salt solution (Seachem, Inc., Madison, Ga).

Page 10, line 221: What does “uL” mean?

Thank you for your comment. That abbreviation is supposed to represent ‘micro-liters’. We have edited it to reflect the proper symbol (line 236, Manuscript Markup).

Page 12, line 267: Justify the choice of 0.055 m2. It is not clear in the text.

Thank you for your comment. We determined this surface area through preliminary investigations not reported here. To that, we have attempted to clarify the writing in this section, which now reads as follows (lines 296-299, Manuscript Markup):

The total surface area per reactor of 0.055 m2 was chosen based on the minimum amount of surface area for gyroids needed for a measurable rate of nitrification through the trial, as determined through preliminary investigation and confirmed repeated in other experiments (for example, Experiment 1, Table 2).

We have also added clarification of the information determined from preliminary investigation earlier in the Methods section, with the following statements (lines 278-283, Manuscript Markup):

In all experiments, sizing was determined based on results from preliminary trials (not reported here). From preliminary trials it was identified that 2.00x10-04 m3 was a workable volume of packed biocarriers (for all types) that provided an observable mobility inside the reactor. Such volume selection consequently derived into the selection of the number of each carrier of each type for each of the experimental treatments described below.

Table 4: Explain the meaning of “packing volume” in the text in order to understand the information supplied in this table.

Thank you for your comment. We have made changes to the text in an attempt to clarify, including the following (lines 266-269, Manuscript Markup):

The packing volume, which is used to estimate the fill ratio, refers to the volume occupied by a predetermined count of biocarrier elements and is dependent on the overall geometry and surface topology nesting characteristics, which would allow for a denser packing arrangement.

Page 12, line 276: justify with some reference the number of 7 carriers.

Thank you for your comment. We determined the sufficiency of 7 carriers through preliminary investigations that are not reported here. We have added this clarification in the text as follows (lines 307-310, Manuscript Markup):

The constant count value of 7 units of media available in each reactor was chosen based on the minimum number of Kaldnes K1 biocarriers needed for a detectable amount of total ammonia conversion during the 8-hour trials, as determined through observations in preliminary investigations (data not shown).

Section “Results and Discussion” must be divided in several sub-sections according to the three experiments carried out.

Thank you for your comment and suggestion. We have now added the following subsection headings: “Experiment 1: Constant Fill Ratio of Biocarriers” (line 337, Manuscript Markup); “Experiment 2: Constant Biocarrier Total Surface Area” (line 404, Manuscript Markup); “Experiment 3: Constant Biocarrier Count” (line 433, Manuscript Markup); “Summary of All Results” (line 462, Manuscript Markup).

Page 13, line 300: do not use capital letters and use “total ammonia nitrogen” without “-N”.

Thank you for your comment. We have corrected the terminology in that location and elsewhere when applicable.

Page 13, line 302: it is not logical to give a p-value with three significant numbers.

Thank you very much for pointing this out. We revised the accepted format for the levels of p-values and have changed it in multiple places to the journal accepted notation of p<0.001 (instead of p=0.000).

Revise p-values in manuscript and introduce the statistical method in “Materials and Methods”.

Thank you for your comment. We have revised p-values globally through the document. Additionally, we have added a subsection heading in the Methods section entitled “Experimental Design and Analysis of Results” (line 259, Manuscript Markup). We have added the following explanation of statistical analyses in this section (lines 320-325, Manuscript Markup):

An analysis of homoscedasticity and a Kolmogorov-Smirnov normality test showed that the data do not follow the underlying assumptions an Analysis of Variance. Consequently, the statistical analysis was conducted with a non-parametric Kruskal-Wallis statistical test on the medians. Additional analysis was performed with a Mood’s Median test to compare the medians of pairwise samples to determine significance.

I can not revise the results from Figure 4 as I can not see them (poor quality of the figure).

Thank you for your comment. We have revised Figure 4 and reproduced it at a high resolution for quality.

Please, do not use abbreviations in X or Y axis.

Thank you for your comment. We have revised all figures to remove abbreviations for clarity.

Thank you for your comment. We agree that the language can be confusing, as indeed there is no removal of total nitrogen, as might occur if anoxic zones are present to allow for possible denitrification. Rather, we are discussing conversion of ammonia nitrogen to nitrate nitrogen through the well-known process of nitrification, which we are measuring indirectly through the expected decrease in ammonia concentrations and proportionate increase in nitrate concentrations. To that, we have amended the language in various locations throughout, especially accentuating that the process being described is nitrification as represented by the conversion of ammonia nitrogen to nitrate nitrogen (for example, line 51, Manuscript Markup; also, line 264, Manuscript Markup), and by the replacement of the word “removal” with the words “ ammonia nitrogen conversion”, globally in multiple places distributed throughout the text.

Page 15, lines 345-349: compare with the values of protected surface area for biofilm growth corresponding to your biocarriers.

Thank you for this comment. The protected area of a biocarrier is not a design parameter but rather a limitation of the design as expressed by the Kaldnes K1 manufacturer. There is no unique definition of protected area but it is loosely used in commercial terminology to explain why biofilm develops only on selected areas. Because this term is not well defined, and also because it is not critical to any discussion in the manuscript, we have removed all verbiage regarding protected surface areas from the manuscript.

Figure 6: it is not easily interpretable. Moreover, use superscripts.

Thank you for your comments. We have recrafted Figure 6 into two separate panels to better demonstrate the relationships. Also, we have corrected the superscripts in the figure.

Page 16, lines 394-395: Justify what you say.

Thank you for your comment. We believe that this statement is redundant with the meaning of the previous sentence, where the performance results of each of the biocarrier types is discussed in detail. Because of this, we have chosen to remove this sentence.

Pages 16-17, lines 396-400: Explain it with more detail.

Thank you for your comments. We have attempted to clarify our writing here with more detail. It now reads as follows (lines 455-461, Manuscript Markup):

The TAN percentage change from gyroids was measurably greater than conventional biocarriers even when the biocarrier count was notably less than that compared to other experiments. This suggests that the performance of the gyroid biocarriers is enhanced by the high specific surface area. In addition, the amount of increases in performance for the medium and large SSA gyroids, despite the relatively number of biocarriers in this experiment, suggests a subsidy in performance resulting from the high proportion of protected surface area, a result directly related to the conformation of the topographical design of the biocarriers.

Thank you for your comment. We have rewritten the Conclusions based upon your recommendations. The Conclusions section now reads as follows (lines 483-505, Manuscript Markup):

The levels of geometrical complexity of the design and fabrication of biocarriers for water treatment processes were manipulated with the capabilities of additive manufacturing. Three mathematical designs with increasing specific surface area (SSA) were used to fabricate biocarriers and test their performance three different scenarios: constant fill ratio, constant total surface area, and constant biocarrier media count. For all the scenarios, the gyroid media designs presenting large and medium SSA provide the best nitrification performances: that is, the lowest final concentration of pooled total ammonia nitrogen as well the highest concentration of pooled nitrate nitrogen. The best performing design for the scenario of constant fill ratio is the large SSA gyroid with 99.33% of ammonia nitrogen conversion at 8 hours. The best performing design for the scenario of constant total surface area is the medium SSA gyroid with 94.74% of ammonia nitrogen conversion at 8 hours. The best performing design for the scenario of constant biocarrier media count is the large SSA gyroid with 92.73% of ammonia nitrogen conversion at 8 hours. From a practitioner’s standpoint, experiment 1 perhaps provides the most relevant information for selection of biofilter media, as one of the most practical considerations in bioreactors loading conditions is the fill ratio in the tanks. In this regard, and with manufacturing considerations aside, the results indicate that either large or medium gyroid SSA would be the best choice. However, this study was focused on the early stages of nitrification and thus limited to an 8-hour period. Therefore, it ignores some of the steady-state operational issues (e.g. clogging, biofilm build-up, sloughing, etc.) of bioreactors that would likely give a functional advantage to a design with a larger pore openings while maintaining optimal performance (i.e. medium SSA gyroid). Additionally, results from experiment 2 and 3 indicate that further optimization on the geometry and its proxy SSA can be pursued. Future work will aim at this through the use of a statistical response surface methodologies over a longer period of bioreactor performance.

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PLoS One. doi: 10.1371/journal.pone.0238386.r003

Decision Letter 1

Leonidas Matsakas

27 Jul 2020

PONE-D-20-13545R1

Analysis of very-high surface area 3D-printed media in a moving bed biofilm reactor for wastewater treatment

PLOS ONE

Dear Dr. Blersch,

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Reviewers' comments:

Reviewer's Responses to Questions

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

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Reviewer #2: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Reviewer #1: Authors have responded to all the comments and Manuscript has been significantly revised with scientific contents.

Reviewer #2: Authors have addressed most of comments. However, figures are presented in different sub-figures (in several pages); it would be more suitable if each figure (with all sub-figures) is included in one page. Table 1 contains several symbols "*" and "**" but the meaning of them is missing. Finally, when you speak about the surface area (0.055 m2) and the numbers of carriers (7) it is necessary to specify the references used (it is not sufficient to say "preliminary investigation").

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PLoS One. 2020 Aug 27;15(8):e0238386. doi: 10.1371/journal.pone.0238386.r004

Author response to Decision Letter 1

12 Aug 2020

We present the response to reviewers in detail in this communication. We thank the reviewers again for their helpful and constructive comments that continue to improve this manuscript. We have addressed all comments in this letter and/or in the manuscript.

Reviewer #2:

Authors have addressed most of comments. However, figures are presented in different sub-figures (in several pages); it would be more suitable if each figure (with all sub-figures) is included in one page.

Thank you for your comments and observations on the figures. We have re-combined all the sub-figures as panels in each overall figure, formatted to fit on one page.

• Table 1 contains several symbols "*" and "**" but the meaning of them is missing.

Thank you for catching that error resulting from formatting the table for submission. We have now included the table footnotes that are indicated by those symbols (Table 1, p. 7, markup manuscript).

Finally, when you speak about the surface area (0.055 m2) and the numbers of carriers (7) it is necessary to specify the references used (it is not sufficient to say "preliminary investigation").

Thank you for your comment. The theoretically required surface area was initially calculated using performance metrics estimated from literature, and then validated through trial-and-error experimentation in preliminary work in the laboratory, the data for which are not reportable. However, the calculations that support the initial planning are reported in a published document. We have included this reference in the text, and indicated more clearly the preliminary aspects of the trials, in the following location (lines 278-280, Manuscript Markup):

"In all experiments, sizing was determined based on calculations of minimum required biocarrier surface area for complete nitrification, as reported in [36, p.95] and validated through trial and error in preliminary trials (data not reported here)."

Where the reference that is newly included as a citation (line 629-630, Manuscript Markup):

Proaño Peña GF. 3D-printed Custom Substratum for Fast Functional Responses from Microbial Colonization. Auburn, Alabama, USA: Auburn University; 2018.

Also, in the following location (lines 297-300, Manuscript Markup):

"The total surface area per reactor of 0.055 m2 was chosen based on the minimum amount of surface area for gyroids needed for a measurable rate of nitrification throughout the time of the trial, as determined through trial and error preliminary investigations and confirmed through repetition in other experiments (for example, Experiment 1, Table 2)."

Also, in the following location (line 310-313, Manuscript Markup):

"The constant count value of 7 units of biocarriers in each reactor was chosen based on the minimum number of Kaldnes K1 biocarriers needed for a detectable amount of total ammonia conversion during the 8-hour trials, as determined through observations in trial and error preliminary investigations (data not included here)."

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PLoS One. doi: 10.1371/journal.pone.0238386.r005

Decision Letter 2

Leonidas Matsakas

17 Aug 2020

Analysis of very-high surface area 3D-printed media in a moving bed biofilm reactor for wastewater treatment

PONE-D-20-13545R2

Dear Dr. Blersch,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Leonidas Matsakas

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

PLoS One. doi: 10.1371/journal.pone.0238386.r006

Acceptance letter

Leonidas Matsakas

19 Aug 2020

PONE-D-20-13545R2

Analysis of very-high surface area 3D-printed media in a moving bed biofilm reactor for wastewater treatment

Dear Dr. Blersch:

I'm 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.

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on behalf of

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

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Data Availability Statement

All data files are publicly available from the Digital Commons at Georgia Southern University (DOI 10.20429/data.2020.1).

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PERMALINK

Analysis of very-high surface area 3D-printed media in a moving bed biofilm reactor for wastewater treatment

Gabriel Proano-Pena

Andres L Carrano

David M Blersch

Roles

Abstract

Introduction

Materials and methods

Biocarrier design and fabrication

Fig 1. Images of commercial (K1) and fabricated (gyroid) biocarrier designs examined in this research.

Table 1. Biocarrier designs and their geometric and material characteristics.

Biofilter reactor design and setup

Fig 2. Bench-scale MBBSBRs used for experimentation; (a) photograph of one system containing 6 reactors; (b) side-view schematic; (c) top-view schematic.

Wastewater media preparation and analysis

Experimental design and analysis of results

Fig 3. Schematic of the experimental design for the three experiments.

Table 2. Parameters for Experiment 1 with constant fill ratio (15%).

Table 3. Parameters for Experiment 2 with constant total biocarrier surface area (0.055 m2).

Table 4. Parameters for Experiment 3 with constant total number (count) of biocarriers (7 biocarriers each).

Results and discussion

Experiment 1: Constant fill ratio of biocarriers

Fig 4. Pooled results for total ammonia nitrogen and nitrate nitrogen concentrations over time for (a) Experiment 1, constant biocarrier fill ratio; (b) Experiment 2, constant biocarrier total surface area; (c) Experiment 3, constant biocarrier count.

Fig 5. 8-hour total ammonia nitrogen (TAN) percentage change for each biocarrier type in each experiment (error bars represent standard deviation).

Fig 6. Total ammonia conversion for each biocarrier as a function of (a) total surface area per treatment, and (b) specific surface area of the biocarrier, for Experiment 1, constant fill ratio.

Experiment 2: Constant biocarrier total surface area

Experiment 3: Constant biocarrier count

Summary of all results

Conclusions

Data Availability

Funding Statement

References

Decision Letter 0

Leonidas Matsakas

Roles

Author response to Decision Letter 0

Decision Letter 1

Leonidas Matsakas

Roles

Author response to Decision Letter 1

Decision Letter 2

Leonidas Matsakas

Roles

Acceptance letter

Leonidas Matsakas

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Table 3. Parameters for Experiment 2 with constant total biocarrier surface area (0.055 m²).