Effect of Magnet Powder (Fe3O4) on Aerobic Granular Sludge (AGS

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Effect of magnet powder (Fe3O4) on the aerobic granular sludge (AGS) formation and microbial community structure characteristics Xiaomin Ren, Liang Guo, Yue Chen, Zonglian She, Mengchun Gao, Yangguo Zhao, and Mengyu Shao ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b00670 • Publication Date (Web): 26 Jun 2018 Downloaded from http://pubs.acs.org on July 1, 2018

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ACS Sustainable Chemistry & Engineering

Effect of magnet powder (Fe3O4) on the aerobic granular sludge (AGS) formation and microbial community structure characteristics Xiaomin Ren1, Liang Guo1,2∗, Yue Chen1, Zonglian She1, Mengchun Gao1, Yangguo Zhao1, Mengyu Shao1 (1. College of Environmental Science and Engineering, Ocean University of China, Qingdao, 266100, China; 2. Key Laboratory of Marine Environmental and Ecology, Ministry of Educatin, Ocean University of China, Qingdao, 266100, China) Abstract Magnet powder (Fe3O4) could affect the growth and biodegradation ability of microbe by producing of magnetic field and iron ion. In this study, the enhancement of aerobic granulation by adding Fe3O4 was performed to evaluate the effects of different Fe3O4 concentrations (0, 0.4, 0.8, 1.2 and 1.6 g/L) on sludge granulation. Fe3O4 had a positive effect on the formation and growth of aerobic granular sludge (AGS) during the start-up period. In addition, the Fe3O4 concentration at 0.4-1.2 g/L promoted the COD removal compared to the SBR without Fe3O4. Three-dimensional-excitation emission matrix (3D-EEM) indicated that 0.8 g/L Fe3O4 addition could accelerate the granulation by stimulating the extracellular polymeric substances (EPSs) secretion which were advantage for enhancing granule size. Meanwhile, microbial richness and diversity of AGS was significantly affected with Fe3O4 addition by high throughput sequencing. Furthermore, dominant groups contributed to granules formation, COD removal, and nitrifying-denitrifying were identified under different Fe3O4 concentrations. Keywords: Fe3O4; Aerobic granular sludge (AGS); Aerobic granulation; Extracellular polymeric substances (EPSs); Microbial richness and diversity

∗

Corresponding author: Tel: +86 532-66781020 Fax: +86 532-66782810 E-mail address: [email protected] 1

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Introduction Aerobic granular sludge (AGS) has become a novel biotechnology in real wastewater treatment in recent decades 1, 2. It has been applied in wastewater widely due to good settling properties, dense and strong bacterial structure and high biomass retention 3, 4. But the mechanisms about aerobic granulation is lacking and this technology needs long start-up time 5. This is one of the shortcomings impeding the application of AGS. Granulation, as a complex process, is affected by some factors such as selection pressure, organic substrate loading rate, and hydraulic retention time5-8. Additionally, extracellular polymeric substances (EPSs) secreted by microbial contributed the formation of AGS 9. In addition, additives, dosing appropriate, might be contribute to the formation of AGS. It was reported that adding Ca2+ could decrease the time to cultivate AGS and affect the physical characteristics of AGS 10. The AGS adding Mg2+ was capable of the production of polysaccharides (PS), proteins (PN) and experienced a faster substrate biodegradation11. AGS formation time obviously shorted through Mg2+ and Al3+ addition, moreover, AGS possessed better simultaneously COD, NH4+-N and TP removal efficiencies12. It was reported that granular active carbon (GAC) which could provide nucleus for microbial accelerated the formation of AGS 13. Iron which is cheap and emerging effectively degrades contaminants helps in the synthesis of iron-containing proteins. Fe2+ was contributed to the microbial attachment of seed sludge 14 and Fe3+ was widely used in WWTPs as coagulating agents to promote activated sludge flocculation and precipitation15. It was reported that metal ion neutralized the overall negative charge present on the surface of microbial and EPSs, and thus helped to speed up the granulation process11, 16. As a magnetic material, magnet powder (Fe3O4) can produce magnetic field and be ionized into Fe(II) and Fe(III)17. 2


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Moreover, adding Fe3O4 into wastewater treatment could magnetize the iron compounds in activated sludge, and then the flock was enlarged and the sludge sedimentation efficiency was improved by combining their magnetic forces with each other 18, 19. However, it is lacking that the effects of Fe3O4 on the granulation of AGS. In this study, the effects of different Fe3O4 concentrations on the granulation of AGS in sequencing batch reactors (SBRs) were investigated. The start-up time, physicochemical characteristics of AGS were also analyzed. And EPSs compositions were investigated by the fluorescence spectra technology of three-dimensional excitation emission matrix (3D-EEM). Scanning electron microscope (SEM) was conducted to observe microbial of the AGS. The changes of microbial richness and diversity of the AGS with different Fe3O4 concentrations addition were evaluated using high-throughput sequencing. Thus, this study would be beneficial to AGS formation and understand the mechanism of Fe3O4 on AGS cultivation.

Material and Methods Set-up and operation of AGS reactors Five lab-scale SBRs (72 cm in height and 6 cm in diameter) with a working volume of 2 L were operated as R1, R2, R3, R4 and R5 which fed 0, 0.4, 0.8, 1.2 and 1.6 g/L Fe3O4, respectively, for aerobic granulation. Each SBR were performed for 18 days with a 2 h operating cycle which consisted of 7 min feeding, 100 min aeration, 2-10 min settling, and 1 min decanting. The settling time reduced from 10 min to 2 min gradually and the temperature of the reactors was maintained at room temperature. The exchange volume was 50% and an airflow rate of 3 L/min was applied.

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Inoculation and wastewater Activated sludge from Tuandao WWPT, Shandong, China, which had the initial mixed liquor suspended solids (MLSS) of 2580 mg/L and sludge volume index (SVI5) of 329 mL/g, respectively, was used as seed sludge for the five reactors. A synthetic wastewater used as feed to the five reactors with the following composition: COD (sodium acetate) 1500 mg/L, N (NH4Cl) 75 mg/L, P (KH2PO4) 15 mg/L, CaCl2.2H2O 15 mg/L, MgSO4.7H2O 12.5 mg/L, trace element solution 1.0 ml/L. The composition of the trace element solution was (mg/L): H3BO3 50, CuCl2 30, NiCl2 50, ZnCl2 50, MnSO4.H2O 50, (NH4)6Mo7O24.4H2O 50, AlCl3 50 and CoCl2.6H2O 50. The COD: NH4+-N: P ratio was always kept at 100: 5: 1. Analytical methods Measurement of COD, NH4+-N, NO2–-N, NO3–-N, MLSS and SVI5 were conducted using standard methods 20. Protein (PN) and polysaccharides (PS) content were analyzed through a modified Lowry and phenol-sulfuric acid method21, respectively. The concentration of Fe2+ and Fe3+ were determined by ortho phenanthroline spectrophotometry. The sludge samples taken from 15 cm above the reactor bottom were classified into four fractions by laboratory sieves with various openings (0.25, 0.5, 0.9, 2.0 mm) to estimate the size distribution 22. Granule surface structure was observed by the scanning electron microscope (SEM) (JSM-5600LV). The average of date that three samples were measured under identical condition could prevent any possible errors induced by sampling procedure.

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Table 1 Excitation and Emitting (Ex/Em) wavelengths of fluorescence region. Region

Substance

Ex/Em wavelengths (nm)

I

Tyrosine-like protein

200-250/200-330

II

Tryptophan-like protein

200-250/330-380

III

Fulvic acid-like organics

200-250/380-500

IV

Soluble microbial by-product

250-280/200-380

V

Humic acid-like organics

280-400/380-500

EPSs extraction and 3D-EEM fluorescence spectroscopy The thermal treatment was a method that used to extract the EPSs of sludge 23. The sludge was collected at 5000 r/min for 10 min and then removed the supernatant. And the left sludge was mixed with distilled water and then centrifuged which was conducted three times. The sludge that washed was heated at 80 °C for 10 min, then centrifuged at 8000 r/min for 10 min. The left supernatant were filtered using 0.45 µm cellulose acetate membrane for EPSs analysis. The extracted filtrate of EPSs was analyzed by EEM fluorescence spectroscopy by a Hitachi F-4500 spectrophotometer. The emission (Em) and excitation (Ex) wavelengths were set between 200-500 nm and 200-400 nm with 5 nm-step, respectively, and the scan speed was 1200 nm/min. EEM peaks were divided into five regions (Table 1). The fluorescence reginal integration (FRI) technique was adopted for EEM spectral data analysis and calculated percent fluorescence response (Pi, n,

%) according to the previous study 24.

High throughput sequencing Five AGS samples were collected at day 18 under the Fe3O4 concentrations of 0, 0.4, 0.8, 1.2 and 1.6 mg/L (R1, R2, R3, R4 and R5, denoted as G1, G2, G3, G4 and G5, respectively). DNA extraction, PCR amplification and high throughput sequencing of 5



the V2 -V3 region of bacterial16S rDNA were conducted sequentially using the bacterial universal primers BA101F (5’-TGGCGGACGGGTGAGTAA-3’) and BA534R (5’-ATTACCGCGG CTGCTGG-3’) as the procedures by 25. The microbial group of SBR was analyzed by the high-throughput sequencing on an Illumina HiSeq platform of Novogene (Beijing, China)26.

Results and Discussion Cultivation of AGS During the experiment, AGS was successfully achieved in the five reactors. Visible AGS first found in R2-R3 (0.4-0.8 g/L) on day 4 and became dominant after about 10 days, when the SVI5 was sharply decreased from about 278.5 to 74.5 mL/g (Fig. 1a). AGS in R4-R5 (1.2-1.6 g/L) first appeared on day 7 and the aerobic granulation was completed about 11days in view of the low SVI5. In contrast, the proportion of AGS in R1 did not increased significantly until day10 and reached full granulation on the 13th day. The result suggested that Fe3O4 which helped to reduce the start-up time had promoted AGS formation and growth. Compact granules with smooth surface dominated in the reactors on the 13th day, and SVI5 of the granules were stabilized at 35-44 mL/g in R2-R5 (0.4-1.6 g/L), and 56 mL/g in R1. It was concluded that mature AGS with Fe3O4 addition showed good settling ability. The changes in MLSS during granulation period are shown in Fig. 1b. Initially, the biomass concentration decreased significantly due to the activated sludge was washing out from the reactors. During the process of granulation, the MLSS concentrations in all reactors increased. The MLSS in R3-R5 (0.8-1.6 g/L) during steady-state were lower compared to 5.4 g/L in R1 (0 g/L) and reached to the value of 3.9 g/L, 3.7 g/L and 4.2 g/L, respectively, while it was increased to 5.6 g/L in R2 (0.4 g/L ). Moreover, Liu et al. 27 reported that the less MLSS resulted in 6


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the less frequent collision and attrition among particles. It was demonstrated that 0.8-1.6 g/L Fe3O4 had a negative effect on the increase of biomass, and low MLSS benefited the AGS formation and resulted in the bigger granule. 7

400

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1 0 -1 0

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Fig. 1. Variation in the process of aerobic granulation in R1 (0 g/L), R2 (0.4 g/L), R3 (0.8 g/L), R4 (1.2 g/L) and R5 (1.6 g/L) a.SVI5, b. MLSS

The COD and NH4+-N removals were evaluated at different Fe3O4 concentrations (Fig. 2). When AGS was achieved mature, the removals of COD and NH4+-N kept relatively stable in all reactors. The NH4+-N removals were found about 73.9%, 71.5%, 69.8%, 74.9% and 53.5% in R1 (0 g/L), R2 (0.4 g/L), R3 (0.8 g/L), R4 (1.2 g/L) and R5 (1.6 g/L), respectively (Fig. 2a), suggesting that high Fe3O4 addition could drastically deteriorated the removal of ammonia nitrogen. It was reported that with the Fe2+ and Fe3+ concentrations increased, NH4+-N removal was evidently deteriorated28. Compared to NH4+-N removal performance, COD removal rate showed noticeable increase with Fe3O4 addition, which were 85.5%, 94.4%, 94.4%, 96.7% and 91.9% in R1-R5 (0-1.6 g/L) (Fig. 2b), respectively. It indicated that nitrifiers had the higher sensitivity to iron ions compared to heterotrophs.

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Fig. 2. Effects of Fe3O4 on the COD and NH4+-N in R1 (0 g/L), R2 (0.4 g/L), R3 (0.8 g/L), R4 (1.2 g/L) and R5 (1.6 g/L) a. NH4+-N, b. COD

AGS morphology and size distributions Fig. 3 shows SEM observations of AGS from R1-R5 (0-1.6 g/L) collected at day 13. SEM images collected at day 13 showed that AGS in R2-R3 (0.4-0.8 g/L) had a relatively smooth and compact structure compared with that in R1 (0 g/L), R4 (1.2 g/L) and R5 (1.6 g/L) (Fig. 3A and Fig. 3C). The SEM observation showed the domination of rod-shaped bacteria on the surface and in the internal of the mature AGS, covered with EPSs (Fig. 3B and Fig. 3D). However, a large number of filamentous bacteria in R1 (0 g/L) were found near the AGS surfaces. This indicated that the addition of Fe3O4 could inhibit the growth of filamentous bacteria. With Fe3O4 concentration (1.2 and 1.6 g/L) increasing, SEM images of AGS revealed that damaged surfaces (green box) (Fig. 3C) appeared on the granular structure. The results possibly reflected that large AGS induced insufficient protection of the anaerobic microbial groups in the core, and thus AGS breakage. In addition, a similar phenomenon was also observed 29. Fig. 3D shows cavities presented (red circle) among the compacted bacteria, which were beneficial to allow mass transfer of substrates or metabolite products in and out of the AGS smoothly. The reason for forming cavities was that partial Fe2+ might be utilized to construct cells. It was reported that Ca precipitates was found to be mainly accumulated in the internal 8


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cross-section of the AGS 20. However, no iron precipitates were observed in the core of AGS at different Fe3O4 concentration. This finding was important in elucidating the mechanism of Fe3O4 on promoting AGS formation. A

B

C

9



D

Fig. 3. Morphology observation of aerobic granules at different Fe3O4 concentrations at low and high magnification by SEM A.

Surface view (magnification =5.0 k×50, bar = 1.0 mm);

B.

Surface view (magnification =5.0 k×10, bar = 5.0 µm);

C.

Section view (magnification =5.0 k×50, bar =1.0 mm);

D.

Section view (magnification =5.0 k×4.0, bar =10.0 µm)

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80 70

Size distribution (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


60 50 40 30 R1 R2 R3 R4 R5

20 10 0 0.125-0.25

0.25-0.5

0.5-0.9

0.9-2

Diameter (mm)

Fig. 4. Size distribution of AGS in R1 (0 g/L), R2 (0.4 g/L), R3 (0.8 g/L), R4 (1.2 g/L) and R5 (1.6 g/L) at day 13

The size distributions of AGS at day 13 in R1-R5 (0-1.6 g/L) are shown in Fig. 4. The proportion of AGS size larger than 0.9 mm in R1-R5 (0-1.6 g/L) were 7.2%, 18.9%, 42.3%, 70.2% and 70.4%, respectively. It could be inferred that Fe3O4 addition promoted a larger size of AGS than Fe2+ and Fe3+. Fe3O4 could be ionized into Fe (II) and Fe (III) in the reactor, and the effluent concentrations of Fe2+ and Fe3+ from reactors were lower than that in the feeds (Fig.S1). According to DLVO (Derjaguin-Landau-Verwey-Overbeek) theory, adding metal ions could neutralize and be bound to the negative charge present on the surface of microbial. Fe2+ and Fe3+ that ionized by Fe3O4 enhanced the aggregation of bacterial through charge neutralization and double-layer compression. It could be concluded that the Fe2+ and Fe3+ resulted in shorter start-up time. EPSs content and component of AGS EPSs, composed of PN and PS mainly, have a significant effect during the 11



bio-granulation process 30, 31. EPSs of AGS in five SBRs were measured after 13 days (Fig. 5). After complete full granulation, the production yield of PN and PS increased sharply with an increase in the concentration of Fe3O4 (especially the PN). At day 13, PS content was increased from 26.7 mg/g MLSS in R1 (0 g/L) to 50.6, 37.3, 46.7 and 62.7 mg/g MLSS in R2-R5 (0.4-1.6 g/L), respectively. In addition, PN content was increased from 92.8 mg/g MLSS in R1 (0 g/L) to 103.9, 161.8, 150.7 and 159.2 mg/g MLSS in R2-R5 (0.4-1.6 g/L), respectively. Compared with the case for 0-0.4 g/L Fe3O4, the content of PN was considerably greater in 0.8-1.6 g/L Fe3O4. In general, the presence of toxicity accelerated the production of PN over PS in AGS 32. Therefore, it natural to conclude that the increased PN could protect the cells from suffering the toxicity of Fe3O4.

200 PN PS

EPSs (mg/gMLSS)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

150

100

50

0 R1

R2

R3

R4

R5

Reactors

Fig. 5. Main EPSs production of the granular sludge cultivated in R1 (0 g/L), R2 (0.4 g/L), R3 (0.8 g/L), R4 (1.2 g/L) and R5 (1.6 g/L) at day 13

The results implied that AGS with Fe3O4 addition were able to promote the secretion of PN and PS. Fig. S2 summaries the 3D-EEM results of sludge EPSs at 1, 10 12


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and 13d during the granulation process in R1-R5 (0-1.6 g/L). The FRI technique which could better understand the EEM fluorescence characteristics of EPSs was used to quantitative assess the five Ex/Em regions change (Fig. 6). As shown in Fig. S2 and Fig. 6, Region II (tryptophan-like protein) and IV (soluble microbial-like material) in all reactors had the highest Pi, n with 30±5% and 30±5%, respectively. Region I (Tyrosine-like protein), III (fulvic acid-like substance) and V (humic-like organics) showed a slight variation in all granules. Humic-like organics were regarded as a dominant component in EPSs during the whole operation33. On day 4, the settling time was reduced to promote the formation of AGS, leading to the increase of EPSs. In addition, during the first few days, the biomass concentration decreased sharply due to the flocculated sludge. It was reported that lower MLSS could promote cell to produce more EPSs33. Therefore, the humic-like organics reached a peak content at day 4. When complete granulation of sludge occurred at day 13, the strength of Region I and II were 1464.6 and 974.6 au in R1 (0 g/L), while they obviously increased to 2082.6 and 1127.5 au in R3 (0.8 g/L), but had no obvious change in R2 (0.4 g/L), R4 (1.2 g/L) and R5 (1.6 g/L). The result revealed that protein-like substances were increased with 0.8 g/L Fe3O4 addition. Tyrosine-like and Tryptophan-like were one of amino acids 34, which was more hydrophobic and contributed to the stability of AGS. Meanwhile, the fluorescence intensity during the granulation process in R3 (0.8 g/L) increased from 5639 to 27177 au which was 1.5-fold, 1.2-fold, 1.6-fold and 2.3-fold of that in R1, R2 (0.4 g/L), R4 (1.2 g/L) and R5 (1.6 g/L), respectively. Thus, the results indicated that 0.8 g/L Fe3O4 resulted in the enhancement of hydrophobicity due to the increase of Tyrosine-like and Tryptophan-like in AGS, which were conducive to the stability of AGS.

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Fig. 6. Changes of FRI in EPSs during the granulation process in R1 (0 g/L), R2 (0.4 g/L), R3 (0.8 g/L), R4 (1.2 g/L) and R5 (1.6 g/L)

In this study, it was observed that AGS with Fe3O4 addition had a higher content of EPSs. Moreover, low concentration (0.4-0.8 g/L) of Fe3O4 were able to promote the higher secretion of PN and PS compared with high concentration (1.6 g/L Fe3O4). It might be that redundant magnetic Fe3O4 powder absorbed on the surface of microbial to induce the decline of mass transfer efficiency. Iron was involved in granulation by cell aggregation and interaction with EPSs 35. It was also reported that Fe3+ possessed a 14


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higher binding affinity and EPSs could bind tightly with Fe3+ 36. Thus, Fe2+ and Fe3+ enhance cell aggregation.

Microbial communities of AGS Bacterial community diversity The microbial diversity and community structure was assessed by High throughput sequencing. Table 2 summaries the richness and diversity of microbial group in the AGS at Fe3O4 concentration of 0 (G1), 0.4(G2), 0.8(G3), 1.2 (G4) and 1.6 g/L (G5), respectively. The effective sequences of AGS samples under steady states were 46,624, 57,560, 48,816, 49,586 and 51,999 in G1-G5, respectively. With a 3% nucleotide cutoff by the ribosomal database project, the effective sequence tags in G1-G5 were classified as 263, 163, 225, 238 and 232 operational taxonomic units (OTUs), respectively. High Good’s coverage varied between 0.997 and 0.999 in G1-G5, indicating the microbial diversity of the AGS samples could be well covered37. The higher richness and diversity in G1, G3, G4 and G5 than G2 indicated that low concentration of Fe3O4 (0.4 g/L) significantly reduced the richness and diversity, while high concentration of Fe3O4 (0.8-1.6 g/L) had minimal impact on AGS compared to G1. The results were also supported by rarefaction curves (Fig. 7a). The difference and similarity of microbial groups in G1-G5 were analyzed through Venn diagram (Fig. 7b). A total of 332 different OTUs in which 120 were shared among the five samples (36.1%), indicating that some microbes always existed with different Fe3O4 addition. Microbial population dynamics and key functional species At the phyla level (Fig. 8a), Proteobacteria and Bacteroidetes were the prominent phyla in the five samples, accounting for 96.5, 99.1, 97.3, 97.9 and 97.4% in G1-G5, respectively. The highly centralized microbial distributions in G2 led to the lower 15



richness and diversity than other samples. Firmicutes (0.7-3.2%) was the third dominant phyla in G1-G5. Other dominant phyla were Chloroflexi (0.01-0.04%) and

Actinobacteria (0.01-0.03%). At the class level (Fig. 8b), Betaproteobacteria increased from 59.9% in G1 to 83.8%, 84.8%, 78.6% and 79.3% in G2-G5, respectively. However,

Flavobacteriia, Gammaproteobacteria, and Alphaproteobacteria decreased in G2-G5 compared to G1. The relative abundance of Clostridia, Deltaproteobacteria,

Sphingobacteriia and Bacteroidia had no obvious variation in G1-G5. Thus, the changes of the relative abundance at phyla and class level suggested that Fe3O4 addition could affect the richness and diversity of microbial. At the genus level (Fig. 8c), 35 top abundant genus were analyzed to evaluate the function evolution of microbial groups with different Fe3O4 concentrations. Sharp fluctuations of microbes at genus level were observed. The relative abundance of the genus Zoogloea increased from 27.7% in G1 to 78.4, 48.8 and 29.1% in G2-G4, respectively, but decreased to 25.4% in G5. Compared to G1, the relative abundance of

Flavobacterium decreased with the increase of Fe3O4 concentration in G2-G5. In contrast, the relative abundance of Nitrosomonas showed stable in all samples. The relative abundance of Azonexus reduced from 0.8% in G1 to 0.4 and 0.7% in G2-G3, but increased to 31.7 and 9.1% in G4-G5. The relative abundance of Thauera increased from 21.2% in G1 to 25.5% in G3. The relative abundance of Dechloromonas in G2-G4 were very close to in G1, whereas it increased to 16.7% in G5. Therefore, not only the bacterial diversity and richness changed in G1-G5, the relative abundance of the most dominant microbial also varied obviously.

Proteobacteria, Bacteroidetes and Firmicutes were also the dominant groups at phyla level which were similar to some previous studies related with activated sludge. As Proteobacteria, Bacteroidetes and Firmicutes were universal in soil, it suggested that 16


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sludge characteristics was similar to soil 38. Proteobacteria was founded to be the most abundance in WWTPs 39. Betaproteobacteria, Gammaproteobacteria,

Alphaproteobacteria and Deltaproteobacteria, contributed to the reduction of nitrate and nitrite 40, among which Betaproteobacteria might resist shear force and be responsible for AGS formation 41. Enhanced granulation with 0.8 g/L Fe3O4 addition was supported by the greater abundance of Betaproteobacteria. The genera of Zoogloea, a floc forming bacteria, were contributed to secrete glue-like EPSs that could bind cells together42. The result was in consistent with the faster granulation with Fe3O4 addition, especially in G2 and G3. Besides, the group Azonexus, Thauera and Dechloromonas had the capable of denitrifying ability43-45. The genera Nitrosomonas could oxidize NH4+-N to NO2--N under autotrophic condition46.

Firmicutes was abundant in the research47, but the content varied dramatically in our study. It was reported that Firmicutes could be washed out from the reactor easily due to be vulnerable to hydraulic shear force 48. In our SBRs, strong shear force was an important factor for the formation of AGS. This could be the reason that low abundance of Firmicutes were observed in the present study. Besides, Bacteroidetes and Firmicutes were verified the denitrifying potential49, 50. Flavobacterium, belonging to the

Flavobacteriia subclass of the phylum of Bacteroidetes, was known for their ability to denitrify under anaerobic (or anoxic) environment51. Member of Chloroflexi played a key role in the biodegradation of carbohydrates and nitrifying-denitrifying process52. The phylum of Actinobacteria might contribute to COD removal53. The present results indicated that Fe3O4 addition could contribute to AGS formation and affect the microbial groups related to nitrifying-denitrifying process.

17


ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

Table 2 Richness and diversity of the microbial community in the SBR at different Fe3O4 concentrations. Samples

Raw

Raw

Effective

Reads

Sequences

Sequences

G1

50300

48680

46624

G2

61756

60162

G3

52640

G4 G5

OTUs

Chao1

ACE

Shannon

Simpson

Good’s

index

index

index

index

coverage

263

297.535

309.268

3.895

0.846

0.998

57560

163

177.091

182.507

1.651

0.378

0.999

51144

48816

225

299.286

292.85

2.875

0.695

0.997

56531

51922

49586

238

309.188

302.781

3.472

0.803

0.997

56531

54469

51999

232

266.459

274.378

3.887

0.869

0.998

18


Page 18 of 28

300

a

250 200 150 100

R1 R2 R3 R4 R5

50 0 0

5000

10000

15000

20000

25000

30000

Sequenes Number

Fig. 7. Rarefaction curves for OTUs (a) and Venn diagram (b) of granules at different Fe3O4 concentrations

Relative Abundance

1.0

a 0.8

0.6

Others Gracilibacteria Chlamydiae Actinobacteria Chloroflexi Chlorobi Spirochaetes Verrucomicrobia Firmicutes Bacteroidetes Proteobacteria

0.4

0.2

0.0 R1

R2

R3

R4

R5

Sample Name 1.0

Relative Abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Observed Species Number

Page 19 of 28

b

0.8

0.6

Others Erysipelotrichia WCHB1-32 Bacteroidia Sphingobacteriia Deltaproteobacteria Clostridia Alphaproteobacteria Gammaproteobacteria Flavobacteriia Betaproteobacteria

0.4

0.2

0.0 R1

R2

R3

R4

R5

Sample Name

19



Fig. 8. Microbial communities of granules at different Fe3O4 concentrations a. phylum level, b. Class level, and c. genus level

Acknowledgements The study was supported by the Natural Science Foundation of Shandong (Grant Number: ZR2017MEE067); Sciences and Technology Project of Qingdao (Grant Number:16-5-1-20-jch); Open Fund of Laboratory for Marine Ecology and Environmental Science, Qingdao National Laboratory for Marine Science and Technology(LMEES201805); Royal Society International Exchanges Scheme (IE140885); the authors also would like to thank the support by China Scholarship Council-International clean energy innovation talent (iCET) program and Ocean University of China-Auburn University (OUC-AU) grants program.

20


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Synopsis The iron metal is of low cost and emerging effectively degraded contaminants.


Page 28 of 28

Effect of Magnet Powder (Fe3O4) on Aerobic Granular Sludge (AGS

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