processes

Article A Bioreactor Designed for Restricting Oversize of Aerobic Granular

Hongbo Feng 1 , Honggang Yang 2, Jianlong Sheng 1, Zengrui Pan 1 and Jun Li 1,*

1 Key Laboratory of Microbial Technology for Industrial Pollution Control of Zhejiang Province, College of Environment, Zhejiang University of Technology, Hangzhou 310014, China; [email protected] (H.F.); [email protected] (J.S.); [email protected] (Z.P.) 2 Jinhua Municipal Design Institute Company Limited, Jinhua 321017, China; [email protected] * Correspondence: [email protected]

Abstract: Aerobic granular sludge (AGS) with oversized diameter commonly affects its stability and pollutant removal. In order to effectively restrict the particle size of AGS, a (SBR) with a spiny aeration device was put forward. A conventional SBR (R1) and an SBR (R2) with the spiny aeration device treating tannery wastewater were compared in the laboratory. The result indicates that the size of the granular sludge from R2 was smaller than that from R1 with sludge granulation. The spines and air bubbles could effectively restrict the particle size of AGS by collision and abrasion. Nevertheless, there was no significant change in mixed liquor suspended solids (MLSS) and the sludge volume index (SVI) in either bioreactors. The removal (%) of chemical + oxygen demand (COD) and ammonia nitrogen (NH4 -N) in these two bioreactors did not differ from each other greatly. The analysis of biological composition displays that the proportion of Proteobacteria  decreased slightly in R2. The X-ray fluorescence (XRF) analysis revealed less accumulation of Fe and  Ca in smaller granules. Furthermore, a pilot-scale SBR with a spiny aeration device was successfully

Citation: Feng, H.; Yang, H.; Sheng, utilized to restrict the diameter of granules at about 300 µm. J.; Pan, Z.; Li, J. A Bioreactor Designed for Restricting Oversize of Aerobic Keywords: aerobic granular sludge; oversize; diameter; bioreactor; wastewater Granular Sludge. Processes 2021, 9, 374. https://doi.org/10.3390/ pr9020374 1. Introduction Academic Editors: Francesca Aerobic granular sludge (AGS) is a biological aggregate which is generally defined Raganati, Alessandra Procentese and as more than 90% of sludge with particle size greater than 200 µm [1,2]. It was first Bipro R. Dhar cultured by Mishima et al. [3] in an aerobic upflow sludge blanket (AUSB) with pure Received: 27 November 2020 oxygen aeration in 1911. AGS has excellent properties such as compact microbial structure, Accepted: 12 February 2021 Published: 18 February 2021 good settling performance, and tolerance to high organic loading [1,4]. Therefore, AGS has been evaluated for treatment of various industrial wastewaters such as textile [5],

Publisher’s Note: MDPI stays neutral rubber [6], brewery [7], and petroleum [8] wastewater. Moreover, AGS has been used with regard to jurisdictional claims in in municipal sewage, and it shows better performance than traditional . published maps and institutional affil- The AGS process is considered as a promising and alternative technology for wastewater iations. treatment. However, most of the reported AGS is still at the lab or pilot-scale [9,10], with only a few reported cases at full-scale [11,12]. The stability of AGS is one of the main reasons limiting its wide practical applica- tion [13]. Besides, the particle size of AGS is one of the major factors which exert influence on its stability. The selective pressure is necessary for the formation of AGS, generally in Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. the way of removing the flocculent sludge with poor settling performance and retaining This article is an open access article the sludge with good settling performance by regulating the settling time. The sludge with distributed under the terms and larger particle size shows better settling performance. The average particle size of AGS conditions of the Creative Commons in the reactor increases gradually under selective pressure. Granular sludge cultivated Attribution (CC BY) license (https:// by Jungles et al. [14] was about 3500 µm in diameter. Farooqi and Farrukh [15] cultivated creativecommons.org/licenses/by/ granular sludge of an average 2000–4000 µm in diameter in SBR with different selective 4.0/). pressures. The results showed that with the particle size increasing, the resistance of mass

Processes 2021, 9, 374. https://doi.org/10.3390/pr9020374 https://www.mdpi.com/journal/processes Processes 2021, 9, 374 2 of 12

which transfers from the surface to the interior would increase so that the anaerobic zone would expand, and the lack of nutrients in the interior would further lead to the disinte- gration of the sludge particles, and finally to the failure of operation [16]. Toh et al. [17] separated the AGS with different particle sizes in SBR to study its properties. They drew the conclusion that the most economical and effective particle size range is 1000–3000 µm. The effective restriction of the particle size of granular sludge is of great significance to the engineering application of it. A great number of studies have been conducted on the physicochemical properties and treatment effects of AGS with different particle sizes [18,19]. However, methods about restricting particle size have rarely been reported. A method for restricting the particle size of AGS is sieving. Long et al. [20] manually sieved to obtain the 2000–3000 µm granular sludge and returned it to the reactor. However, this is not easy to do in engineering applications. When the AGS was oversize, the efficiency of mass transfer would decrease and the stability would become worse. Therefore, there is a necessity to find an effective and convenient method to restrict particle size of AGS. A bioreactor which can restrict the particle size of AGS was proposed in this experiment. The spiny aeration device was adopted in this bioreactor to restrict the particle size of AGS to tackle the instability caused by its oversize diameter. The spiny aeration device could effectively restrict the particle size of AGS without additional equipment.

2. Materials and Methods 2.1. Inoculated Sludge and Wastewater The inoculated sludge was obtained from the aerobic tank of a tannery wastewater treatment plant in Haining, Zhejiang Province. The mixed liquor suspended solids (MLSS) of the inoculated sludge were about 8300 mg/L and the sludge volume index (SVI) with 30 min settling time (SVI30) was about 75 mL/g. The influent water was taken from pretreated tannery wastewater. The main parameters of the wastewater were as follows: + pH 6–8, (COD) 550–800 mg/L, ammonia nitrogen (NH4 -N) 105–160 mg/L, suspended solids (SS) 60–120 mg/L, and total phosphorous (TP) 1–4 mg/L.

2.2. Experimental Set-Up and Operation 2.2.1. Lab-Scale Sequencing Batch Reactors Aerobic granular sludge treating tannery wastewater was obtained in two lab-scale sequencing batch reactors (SBRs). The conventional aeration device was used in R1 and the spiny aeration device made of plastic material was used in R2 (Figure1). The SBRs each had a diameter of 20 cm and a working volume of 11 L with a volumetric exchange ratio of 7/11. One period was 12 h and including the following procedures, influent (5 min), aeration (11 h), settling (5 min), drainage (20 min), and standing (30 min). The lab-scale reactors were operated for a total period of 70 days. The aeration volume was controlled at about 0.7 m3/h. The laboratory temperature was 15~30 ◦C during the whole period. It is worth noting that bioreactors for wastewater treatment would usually be exposed to temperature differences in a real-life scenario (Table S1), so the temperature in the lab was not constant artificially. But R1 and R2 had the same temperature. The SBRs were cleaned weekly to prevent biofilm formation on the wall. Processes 2021, 9, 374 3 of 12 ProcessesProcesses 2021 2021, 9, ,x 9 FOR, x FOR PEER PEER REVIEW REVIEW 3 of3 12of 12

Figure 1. Schematic diagram of the sequencing batch reactors (SBRs). FigureFigure 1. Schematic1. Schematic diagram diagram of theof the sequencing sequencing batch batch reactors reactors (SBRs). (SBRs). 2.2.2. Pilot-Scale Sequencing Batch Reactors 2.2.2.2.2.2. Pilot Pilot‐Scale‐Scale Sequencing Sequencing Batch Batch Reactors Reactors A spiny aerationA spiny device aeration was installed device at was the installed bottom of at an the AGS bottom pilot of‐scale an AGS SBR system pilot-scale SBR sys- A spiny aeration device was installed at the bottom of an AGS pilot3 ‐scale SBR system tem [21] (Figure2). The system could3 dispose 120 m /d wastewater from a town. The [21][21] (Figure (Figure 2). 2). The The system system could could dispose dispose 120 120 m 3m/d /dwastewater wastewater from from a town.a town. The The pilot pilot‐ ‐ scale system pilot-scalewas composed system of two was parallel composed columns of two (diameter parallel columns of 2 m, (diameterheight of 6 of m, 2 m,and height of 6 m, scale system was composed of two parallel columns3 (diameter of 2 m, height of 6 m, and and H/D of3 2.5) with 31.4 m working volume and 50% volumetric exchange ratio. One H/DH/D of of2.5) 2.5) with with 31.4 31.4 m 3m working working volume volume and and 50% 50% volumetric volumetric exchange exchange ratio. ratio. One One period period period included fill (40 min), aeration (120 min), settling (60 min), and discharge (20 min). includedincluded fill fill (40 (40 min), min), aeration aeration (120 (120 min), min), settling settling (60 (60 min), min), and and discharge discharge (20 (20 min). min). The The The system was operated continuously for more than 400 days. The agnail aeration device systemsystem was was operated operated continuously continuously for for more more than than 400 400 days. days. The The agnail agnail aeration aeration device device and and and air pipe layout were shown in Figure2. airair pipe pipe layout layout were were shown shown in inFigure Figure 2. 2.

Figure 2. System of the pilot-scale reactor. FigureFigure 2. System2. System of theof the pilot pilot‐scale‐scale reactor. reactor. 2.3. Analytical Methods 2.3.2.3. Analytical Analytical Methods Methods The SVI30 andThe MLSS SVI were30 and measured MLSS were periodically. measured Liquid periodically. samples Liquid in the feed samples and inef‐ the feed and The SVI30 and MLSS were measured periodically. Liquid µsamples in the feed and ef‐ fluent were filteredeffluent periodically were filtered using periodically 0.45‐μm filters. using COD 0.45-Cr mwas filters. measured COD byCr fastwas diges measured‐ by fast fluent were filtered periodically using 0.45‐μm filters. CODCr was+ measured by fast diges‐ digestion-spectrophotometric+ method and NH -N was measured by Nesslerization. All tion‐spectrophotometric method and NH4 ‐N was measured4 by Nesslerization. All these tion‐spectrophotometric method and NH4+‐N was measured by Nesslerization. All these methods werethese in accordance methods were with in standard accordance methods with standard [22]. Samples methods were [22 taken]. Samples from werethe taken from methods were inthe accordance reactor at predetermined with standard time methods intervals [22]. for Samples analysis. were The taken pH was from monitored the using a pH reactorreactor at atpredetermined predetermined time time intervals intervals for for analysis. analysis. The The pH pH was was monitored monitored using using a pHa pH meter (PHS‐3D,meter Shanghai). (PHS-3D, The Shanghai). morphology The morphologyof sludge was of observed sludge was by an observed Olympus by CX31 an Olympus CX31 meter (PHS‐3D,microscope Shanghai). andThe morphology a digital camera of sludge (Canon was EOS observed 30D). The by an size Olympus of granules CX31 was analyzed by microscopemicroscope and and a digital a digital camera camera (Canon (Canon EOS EOS 30D). 30D). The The size size of ofgranules granules was was analyzed analyzed by by an image analysisan image system analysis (Image system‐Pro Plus (Image-Pro 6.0, Media Plus Cybernetics, 6.0, Media Cybernetics,Rockville, MD, Rockville, USA). MD, USA). an image analysisThe system system (Image reported‐Pro the Plus average 6.0, Media length Cybernetics, of diameters Rockville, measured MD, at 5◦ USA).intervals around the TheThe system system reported reported the the average average length length of ofdiameters diameters measured measured at at5° 5°intervals intervals around around the the

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centroid of each object and then output the statistics. The X-ray fluorescence (XRF) analysis was performed using ARL ADVANT’X IntelliPower TM 4200 (Thermo Fisher, Waltham, MA, USA). Three sludge samples acquired from the inoculation sludge and sludge from two bioreactors were freeze-dried for nucleotide extraction. Microbial DNA was extracted from AGS samples using the E.Z.N.A.® Mag-Bind Soil DNA Kit (Omega Bio-tek, Norcross, GA, USA) in accordance with the manufacturer’s protocols. The final DNA concentration and purification were determined by NanoDrop 2000 UV-vis spectrophotometer (Thermo Scientific, Wilmington, DE, USA), and DNA quality was checked by 1% agarose gel electrophoresis. The V3–V4 hypervariable regions of the bacteria 16S rRNA gene were amplified with primers 341F and 805R by thermocycle PCR system (GeneAmp 9700, ABI, Waltham, MA, USA). The PCR reactions were conducted using the following program: 3 min of denaturation at 94 ◦C; 20 cycles of 30 s at 94 ◦C, 20 s at 45 ◦C and 30 s at 65 ◦C; 20 cycles of 20 s at 94 ◦C, 20 s at 55 ◦C and 30 s at 72 ◦C; and a final extension at 72 ◦C for 5 min. The PCR product was recovered using an AxyPrep DNA Gel Extraction Kit (AXYGEN, Corning, NY, USA) and quantified with a QuantiFluor TM -ST system. The high- throughput sequencing of PCR products was performed using the Illumina MiSeq platform of Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). The operational taxonomic units (OTUs) were clustered with a distance limit of 0.03, using Usearch. Microbial alpha diversity indices such as Shannon, Simpson, Chao, and the abundance-based coverage estimator (ACE) were analyzed using the MOTHUR program. The RDP Classifier with a confidence threshold of 70% was used to classify effective sequences into different taxonomy units, while the community composition was analyzed at different taxonomy levels.

3. Results 3.1. Comparison of Sludge Characteristics 3.1.1. Sludge Particle Size The variation of sludge particle size is shown in Figure3. The same flocculent sludge was inoculated into the R1 and R2 bioreactors, respectively, and the MLSS in both reactors after inoculation was around 1750 mg/L. After 15 days’ operation, a few small particles began to appear in both reactors, but the sludge was still mainly flocs. The particle size and the number of granules in R1 were both slightly larger than those in R2. After 23 days of operation, aerobic sludge in R1 began to granulate, while AGS appeared in R2 six days later than in R1. On the thirty-fifth day, the granular sludge in both R1 and R2 had grown in size, but there was still plenty of flocculent sludge. After 62 days, the sludge in R1 was basically granular with a clear profile. The granular sludge in R2 was also clear but it still contained some flocculent sludge with a prominently smaller particle size than that in R1. The average particle size of the granular sludge was 285 µm in R1 and 190 µm in R2. The particle size distribution (PSD) was mainly within the range of 0–0.5 mm in SBR1 and SBR2. On day 62, and in SBR1, the PSD < 0.2 mm was 10.7%, while those at 0.2–0.5 mm reached 89.3%, respectively. In SBR2, the PSD < 0.2 mm was 36.2% and 0.2–0.4 mm was 63.8%. Processes 2021, 9, 374 5 of 12 Processes 2021, 9, x FOR PEER REVIEW 5 of 12

Figure 3. Variations in the morphology of sludge (scale bar: 200 μm). Figure 3. Variations in the morphology of sludge (scale bar: 200 µm).

3.1.2. Sludge3.1.2. Sedimentation Sludge Sedimentation The initialThe MLSS initial was MLSS 1750 was and SVI1750 was and 73 SVI mL/g was for73 mL/g both SBRfor both systems SBR in systems the first in the first phase. Onphase. the whole, On the the whole, MLSS the in bothMLSS reactors in both indicated reactors indicated an increasing an increasing trend while trend SVI while SVI indicatedindicated a decreasing a decreasing trend, with trend, both with the MLSS both the and MLSS SVI in and R1 beingSVI in lower R1 being than lower those than in those in R2 (FigureR24). (Figure In the first 4). In 4 days,the first due 4 days, to the due short to settling the short time settling of only time 5 min, of only sludge 5 min, with sludge with poor settlingpoor performance settling performance was discharged was discharged from the bioreactor. from the bioreactor. In consequence, In consequence, the MLSS the MLSS in R1 andin R2 R1 decreased and R2 decreased from 1750 from to 1733 1750 and to 1733 1730 and mg/L, 1730 respectively. mg/L, respectively. In the following In the following 4 4 days, MLSSdays, increased MLSS increased to 1819 and to 1819 1967 and mg/L 1967 in mg/L R1 and in R2, R1 respectively.and R2, respectively. On the eighth On the eighth day, the MLSSday, the decreased MLSS decreased both in R1 both and in R2,R1 and and R2, the and SVI the increased SVI increased on account on account of the of the de‐ deteriorationterioration of the sludge of the settlingsludge settling performance. performance. On the twenty-fifth On the twenty day,‐fifth the MLSSday, the in R1MLSS in R1 and R2 manifestedand R2 manifested an increasing an trendincreasing and augmented trend and to augmented 2003 and 2396 to 2003 mg/L, and respectively. 2396 mg/L, respec‐ After 25 days,tively. the After MLSS 25 in days, R1 sustained the MLSS growth in R1 in sustained a continuously growth stable in a statecontinuously at 2400 mg/L. stable state at The MLSS2400 in R2 mg/L. decreased The MLSS from in 2396 R2 decreased to 2105 mg/L from from 2396 day to 2105 25 to mg/L 45. After from 45 day days, 25 to the 45. After 45 MLSS in R2days, began the MLSS to increase, in R2 began and on to the increase, sixty-fifth and on day, the it sixty maintained‐fifth day, stable it maintained at about stable at 2450 mg/L.about The 2450 SVI in mg/L. R1 and The R2 SVI increased in R1 and to 100 R2 increased and 92 mL/g, to 100 respectively, and 92 mL/g, in the respectively, first 13 in the

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first 13 days of operation. After 13 days, the SVI in R1 continued to decrease and main‐ tained stable at about 45 mL/g after 65 days of operation, while the SVI in R2 increased to days of operation. After 13 days, the SVI in R1 continued to decrease and maintained stable 80 mL/g after 35 days of operation. After 35 days, the SVI in R2 began to decrease and was at about 45 mL/g after 65 days of operation, while the SVI in R2 increased to 80 mL/g basically stable at 50 mL/g on the sixty‐fifth day. The SVI of R2 decreased more slowly after 35 days of operation. After 35 days, the SVI in R2 began to decrease and was basically than that of R1, and eventually the SVI of the granular sludge in R2 was slightly higher stable at 50 mL/g on the sixty-fifth day. The SVI of R2 decreased more slowly than that of than that in R1. R1, and eventually the SVI of the granular sludge in R2 was slightly higher than that in R1.

FigureFigure 4. 4.Variations Variations in in the the mean mean particle particle size, size, mixed mixed liquor liquor suspended suspended solids solids (MLSS), (MLSS), and and sludge sludge volume volume index index (SVI) (SVI) of of sludge. ((a) mean particle size; (b) MLSS,sludge. (c )(( anda) mean SVI). particle size; (b) MLSS, (c) and SVI).

3.1.3.3.1.3. Microbial Microbial Community Community Analysis Analysis TheThe microbial microbial community community was was analyzed analyzed through through high-throughput high‐throughput sequencing. sequencing. As As shownshown in in Figure Figure5 ,5, the the results results indicated indicated that that two two bacterial bacterial phyla, phyla,Proteobacteria Proteobacteriaand andBac- Bac‐ teroidetesteroidetes,, were were the the most most abundant abundant in in the the inoculated inoculated sludge, sludge, accounting accounting for for 46.45% 46.45% and and 31.74%31.74% of of thethe total sequence sequence in in the the inoculated inoculated sludge sludge respectively, respectively, followed followed by Firmicutes by Firmi-, cutesDeinococcus–Thermus, Deinococcus–Thermus, Chloroflexi, Chloroflexi, Planctomycetes, Planctomycetes, Acidobacteria,, Acidobacteria, andand GemmatimonadetesGemmatimonadetes, ac,‐ accountingcounting for for 3.55%, 3.55%, 3.4%, 3.4%, 2.62%, 2.62%, 2.33%, 2.33%, 2.23%, 2.23%, and and 2.01% 2.01% in the in the inoculated inoculated sludge, sludge, respec re-‐ spectively.tively. The The microbial microbial community community of of R1 R1 sludge sludge was was mainly mainly constituted bybyProteobacteria Proteobacteria andandBacteroidetes Bacteroidetes,, accounting accounting for for 91.98% 91.98% and and 6.27% 6.27% of of the the total total sequence, sequence, respectively. respectively. R2 R2 waswas similar similar to to R1 R1 with withProteobacteria Proteobacteriaand andBacteroidetes, Bacteroidetes,accounting accounting for for 85.86% 85.86% and and 10.95% 10.95% ofof the the totaltotal sequences, respectively. respectively. Compared Compared with with inoculated inoculated sludge, sludge, the the amount amount of Pro of ‐ Proteobacteriateobacteria inin R1 R1 and and R2 R2 rolled rolled up, up, BacteroidetesBacteroidetes decreased, and otherother bacteriabacteria phyla phyla also also decreased.decreased.

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FigureFigure 5. 5.Distribution Distribution of of microorganisms microorganisms at at phyla phyla level level (( a(()a inoculated) inoculated sludge; sludge; (b ()b sludge) sludge of of R1; R1; (c )(c sludge) sludge of of R2). R2).

3.1.4.3.1.4. The The X-ray X‐Ray Fluorescence Fluorescence Analysis Analysis of of Sludge Sludge TheThe XRF XRF was was adopted adopted to to analyze analyze the the elementelement compositioncomposition of feed water water and and AGS. AGS. It Itshowed showed that that raw raw water water contains contains large large amounts amounts of ofNa, Na, Cl, Cl,Ca, Ca, Mg, Mg, and and P. The P. TheAGS AGS accu‐ accumulatesmulates elements elements such such as Fe, as Fe,Ca, Ca, Mg, Mg, and and P. As P. shown As shown in Figure in Figure 6a, 6influenta, influent samples samples were weremeasured measured for the for mass the mass fraction fraction of each of each element element in the in water the water and andthe mass the mass fraction fraction of the ofwater, the water, and andthe proportion the proportion of each of each element element in the in thetotal total solute solute mass mass was was obtained obtained by con by ‐ conversion.version. It can It can be beseen seen from from the the figure figure that that the the proportion proportion of the of the element element Na Nain the in thefeed feedwater water is 38%, is 38%, 27% 27% for Cl, for followed Cl, followed by 11%, by 11%, 11%, 11%,5%, 0.6%, 5%, 0.6%, and 0.3% and for 0.3% P, forMg, P, Ca, Mg, Si, Ca, and Si,Fe, and respectively, Fe, respectively, and 7.1% and for 7.1% the for other the otherelements. elements. The element The element distribution distribution of the ofAGS the in AGSR1 was in R1 shown was shown in Figure in Figure 6b. Ca6 b.and Ca Fe and account Fe account for a forlarger a larger proportion, proportion, 11.6% 11.6% and 10.4%, and 10.4%,respectively, respectively, followed followed by Na, by Cl, Na, Si, Cl, P, Si,Mg, P, Mg,Al, and Al, other and other elements elements as 5.6%, as 5.6%, 3.4%, 3.4%, 1.3%, 1.3%,1.2%, 1.2%, 0.9%, 0.9%, 0.4%, 0.4%, and 65.2%. and 65.2%. The element The element distribution distribution of AGS of AGSin R2 in was R2 shown was shown in Figure in Figure6c. The6c. elements The elements with with a larger a larger proportion proportion were were Fe, Fe,Na, Na, and and Ca Caat 8.4%, at 8.4%, 7%, 7%, and and 6%, 6%, fol‐ followedlowed by by Cl, Cl, Si, Si, P, P, Mg, Al, and otherother elementselements atat 4.4%, 4.4%, 1.2%, 1.2%, 1%, 1%, 0.8%, 0.8%, 0.3%, 0.3%, and and 70.9%, 70.9%, respectively.respectively. Comparing Comparing the the elements elements in in feed feed water water and and AGS, AGS, it it could could be be seen seen that that Fe Fe and and CaCa were were heavily heavily enriched enriched in in the the granular granular sludge. sludge. The The content content of of elements elements Fe Fe and and Ca Ca in in R1 R1 waswas greater greater than than that that in R2,in R2, but but the the content content of elements of elements Na andNa and Cl was Cl was less thanless than that inthat R2. in TheR2. proportions The proportions of other of other elements elements were similarwere similar in these in these two reactors. two reactors.

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Figure 6. Elemental composition ((a) influent; (b) sludge of R1; (c) sludge of R2).

3.2. Pollutant Removal Performance Figure 6. Elemental composition ((a) influent; (b) sludge of R1; (c) sludge of R2). + The removal effects of COD and NH4 -N were shown in Figure7a,b. The concen- 3.2.tration Pollutant of COD Removal in the Performance influent of R1 and R2 had maintained at 500–800 mg/L, while that in effluent had been kept below 300 mg/L since the outset of operation, and it stabilized The removal effects of COD and NH4+‐N were shown in Figure 7a,b. The concentra‐ at less than 200 mg/L and the removal (%) reached 79% after 50 days of operation. The tion of COD in the influent+ of R1 and R2 had maintained at 500–800 mg/L, while that in concentration of NH4 -N in the feed of R1 and R2 was 110–160 mg/L. After 49 days, this effluentfigure inhad the been effluent kept waterbelow was300 mg/L basically since less the than outset 70 mg/L.of operation, The concentration and it stabilized of COD at less than 200+ mg/L and the removal (%) reached 79% after 50 days of operation. The con‐ and NH4 -N in the effluent met the discharge standard [23] of water pollutants for the centration of NH4+‐N in the feed of R1 and R2 was 110–160 mg/L. After 49 days,+ this figure leather- and fur-making industry in China. In general, the COD and NH4 -N removal + ineffect the effluent of R1 and water R2 was did notbasically differ less from than each 70 other mg/L. significantly; The concentration namely, of spiny COD aerationand NH4 did‐ Nnot in the contribute effluent to met the the efficiency discharge of the standard pollutant [23] removal. of water pollutants for the leather‐ and + fur‐makingAfter industry aerobic sludge in China. granulation, In general, the the contamination COD and NH removal4 ‐N removal effect effect of R1 of and R1 and R2 in R2one did cycle not differ was analyzed, from each as other shown significantly; in Figure7 namely,c,d. The spiny situation aeration of R2 did was not similar contribute to R1 to the efficiency of the pollutant removal. + in that the concentration of COD and NH4 -N declined rapidly in both reactors within a shortAfter period aerobic of time, sludge with granulation, a slightly smaller the contamination decrease of COD removal and a effect slightly of largerR1 and decrease R2 in + onein NHcycle4 was-N at analyzed, the first 5 as h inshown R2 than in Figure in R1. The 7c,d. pH The of situation both R1 and of R2 R2 was was similar constantly to R1 rising, in thatwith the a concentration pH of 7.83 in theof COD influent and and NH4 8.63+‐N declined in the effluent. rapidly in both reactors within a short period of time, with a slightly smaller decrease of COD and a slightly larger decrease in NH4+‐N at the first 5 h in R2 than in R1. The pH of both R1 and R2 was constantly rising, with a pH of 7.83 in the influent and 8.63 in the effluent.

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800 a 160 b

700 140

600 120 500 Influent Influent

-N(mg/L) 100 Effluent(R ) Effluent(R ) + 4 1 400 1 Effluent(R ) COD(mg/L) COD(mg/L) Effluent(R ) 2 2 NH 80 300

200 60

100 40 0 10203040506070 0 10203040506070 Time(days) Time(days) 700 130 8.8 700 130 8.8 c d 120 600 120 8.6 600 8.6

110 110 500 8.4 500 8.4 100 100 COD COD

+ 400 + pH 8.2 400 8.2 -N(mg/L) pH

NH -N -N(mg/L) NH -N 4 4 + 4 90 + 4 pH 90 pH NH COD(mg/L) COD(mg/L) 300 NH 8.0 300 80 8.0 80 70 200 7.8 200 70 7.8 60 100 60 7.6 100 7.6 0246810 0246810 Time(hours) Time(hours) + + FigureFigure 7. Performance 7. Performance of theof the reactors reactors (( a(()a chemical) chemical oxygen oxygen demanddemand (COD); (COD); (b (b) )ammonia ammonia nitrogen nitrogen (NH (NH4 ‐N),4 -N),(c) COD, (c) COD, + NH4++‐N, and pH of R1; (d) COD, NH4+‐N, and pH of R2). NH4 -N, and pH of R1; (d) COD, NH4 -N, and pH of R2).

3.3.3.3. An An Attempt Attempt at at thethe Pilot-ScalePilot‐Scale System AfterAfter 7 7 days, days, small small aerobicaerobic granules could could be be observed. observed. On On the the twentieth twentieth day, day, loose loose sludgesludge began began to to conglomerate conglomerate into into lumps.lumps. On the fiftieth fiftieth day, day, there there was was mainly mainly AGS AGS with with a a compact structure and irregular outline in the reactor. As the reactor continued to oper‐ compact structure and irregular outline in the reactor. As the reactor continued to operate, ate, the average particle size of granular sludge remained at 300 μm and the SVI was 43 the average particle size of granular sludge remained at 300 µm and the SVI was 43 mL/g mL/g in the end. The pilot‐scale reaction was operated continuously for more than 400 in the end. The pilot-scale reaction was operated continuously for more than 400 days and days and maintained high removal efficiencies for pollutants. The removal of COD was maintained high removal efficiencies for pollutants. The removal of COD was maintained maintained at about 88% and almost all the NH4+‐N was removed. It could be seen that at about 88% and almost all the NH +-N was removed. It could be seen that the particle the particle size of AGS in SBR can 4be effectively restricted by using the spiny aeration size of AGS in SBR can be effectively restricted by using the spiny aeration tray, which tray, which indicated that the spiny aeration device plays an important role in restricting indicatedthe particle that size the in spiny the pilot aeration‐scale SBR. device plays an important role in restricting the particle size in the pilot-scale SBR. 4. Discussion 4. Discussion Through the analysis of sludge characteristics, the spiny aeration device effectively Through the analysis of sludge characteristics, the spiny aeration device effectively controlled the particle size of aerobic sludge by collision and abrasion. In the process of controlledaeration, the the granular particle sizesludge of aerobicwould collide sludge with by collision the agnail and on abrasion.the aeration In thedevice, process and of aeration,then the thesharp granular agnail would sludge abrade would large collide particles, with thecausing agnail them on to the be aeration broken (Figure device, 2). and thenThe the granular sharp sludge agnail with would a larger abrade particle large particles,size was more causing likely them to be to in be the broken lower (Figure half of 2). Thethe granular reactor so sludge that it with was a more larger likely particle to come size was into more contact likely with to the be inagnail. the lower At the half same of the reactortime, most so that of the it was small more particles likely could to come smoothly into contact pass through with the the agnail. space between At the same agnails time, mostwith of small the smallprobability particles of being could punctured. smoothly pass through the space between agnails with small probabilityDue to physical of being fragmentation, punctured. the sedimentation of the sludge showed no signifi‐ cantDue change to physical in both bioreactors. fragmentation, The theremoval sedimentation (%) of COD of and the sludgeNH4+‐N showed in these no two significant biore‐ + changeactors indid both not bioreactors.differ from each The other removal greatly. (%) of COD and NH4 -N in these two bioreactors did notIn differ this experiment, from each otherthe Proteobacteria greatly. was abundant and prior in the AGS of tannery wastewaterIn this experiment, treatment. This the resultProteobacteria was similarwas to abundant the research and of Zhao prior et in al. the [24] AGS and of Huang tannery wastewater treatment. This result was similar to the research of Zhao et al. [24] and Huang et al. [25]. It is reported that Proteobacteria is common in AGS and can produce excessive extracellular polymers to promote flocculation of flocculent sludge and formation of AGS.

Processes 2021, 9, 374 10 of 12

In addition, Proteobacteria have the ability to degrade COD and nitrogen. The number of Proteobacteria in AGS was much more than that in inoculated sludge, which indicated that Proteobacteria played an important role in the granulation of AGS. The analysis of biological composition displayed that the proportion of Proteobacteria decreased slightly in the R2. Most members of Proteobacteria are facultatively or obligately anaerobic. The larger anaerobic zone is formed in the bigger particle size of the sludge [26]. This indicated that the particle size of sludge in the R2 was effectively restricted. This experiment result of the X-ray fluorescence analysis indicates that the larger particle size of the granular sludge was enriched with more Fe and Ca, which was consistent with the study of Liu et al. [21], The ash in AGS contained more calcium salts (mainly calcium carbonate), and the content of calcium carbonate precipitate increased with the increase of the particle size of the granular sludge within a certain size range [27]. Other research and literature suggested that a large accumulation of calcium would undermine the biological activity [28]. Therefore, it could be seen that the particle size of AGS in SBR was effectively re- stricted by using the spiny aeration device, which indicated that the spiny aeration device played an important role in restricting the particle size in the SBR. Long et al. [20] en- hanced the stable operation of AGS in pilot-scale SBR by manually sieving to obtain the 2000–3000 µm granular sludge and returning it to the reactor to increase their proportion. Sieving manually can completely retain the structure of the AGS, but the high cost and low efficiency of this method make it unsuitable for engineering applications. Compared with the existing method of particle size control, using the spiny aeration device might damage the particle structure in some way, but its operability is suitable for engineering application. In addition, using the spiny aeration device did not exert influence on the removal (%) of + COD and NH4 -N in the system.

5. Conclusions A bioreactor with the spiny aeration device could effectively restrict the particle size of the AGS. The oversized AGS was more likely to collide and abrase with the spines and air bubbles. However, the spiny aeration device made no difference in the pollutant removal and sludge characteristics. By adding the spiny aeration device in the pilot-scale SBR system, the average particle size of AGS could be effectively restricted at about 300 µm.

Supplementary Materials: The following are available online at https://www.mdpi.com/2227-971 7/9/2/374/s1, Table S1: Basic climatic conditions of Hangzhou. Author Contributions: Conceptualization, H.F., H.Y., and J.L.; methodology, H.F. and H.Y.; software, H.F. and H.Y.; validation, H.F., Z.P., and J.S.; data curation, H.F. and H.Y.; writing—original draft preparation, H.F.; writing—review and editing, H.F., Z.P., and J.S.; funding acquisition, J.L. supervi- sion, J.L.; project administration, J.L. All authors have read and agreed to the published version of the manuscript. Funding: The research was funded by the Major Science and Technology Program for Water Pollution Control and Treatment (2017ZX07201003), the Zhejiang Key Research and Development Program (no. 2018C03006). Institutional Review Board Statement: “Not applicable” for studies not involving humans or animals. Informed Consent Statement: “Not applicable” for studies not involving humans. Data Availability Statement: Data sharing not applicable. Acknowledgments: This work was supported by the Major Science and Technology Program for Wa- ter Pollution Control and Treatment (2017ZX07201003), the Zhejiang Key Research and Development Program (no. 2018C03006). Conflicts of Interest: The authors declare no conflict of interest. Processes 2021, 9, 374 11 of 12

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