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Effect of aquaculture salinity on nitrification and microbial community in moving bed bioreactors with immobilized microbial granules
Journal Pre-proofs Effect of aquaculture salinity on nitrification and microbial community in moving bed bioreactors with immobilized microbial granules Yueshu Gao, Xupeng Wang, Jialun Li, Chew Tin Lee, Pei Ying Ong, Zhenjia Zhang, Chunjie Li PII: DOI: Reference:
S0960-8524(19)31657-8 https://doi.org/10.1016/j.biortech.2019.122427 BITE 122427
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Bioresource Technology
Received Date: Revised Date: Accepted Date:
18 September 2019 9 November 2019 12 November 2019
Please cite this article as: Gao, Y., Wang, X., Li, J., Tin Lee, C., Ying Ong, P., Zhang, Z., Li, C., Effect of aquaculture salinity on nitrification and microbial community in moving bed bioreactors with immobilized microbial granules, Bioresource Technology (2019), doi: https://doi.org/10.1016/j.biortech.2019.122427
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Effect of aquaculture salinity on nitrification and microbial community in moving bed bioreactors with immobilized microbial granules Yueshu Gaoa, Xupeng Wanga, Jialun Lia, Chew Tin Leeb, Pei Ying Ongc, Zhenjia Zhanga, Chunjie Li*a a
School of Environmental Science & Engineering, Shanghai Jiao Tong University,
Shanghai 200240, China bFaculty
of Chemical & Energy Engineering, Universiti Teknologi Malaysia, 81310
Johor Bahru, Johor, Malaysia
c
Innovation Center in Agritechnology For Advanced Bioprocessing (ICA), Universiti
Teknologi Malaysia, 84600 Pagoh, Johor, Malaysia
*Corresponding author, Chunjie Li, 800 Dong Chuan Road, School of Environmental Science and Engineering, Shanghai Jiao Tong University, Shanghai, P. R. China. E-mail: [email protected]. Tel.: +862113391048252.
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Abstract: The novel immobilized microbial granules (IMG) shows a significant effect of nitrification for freshwater aquaculture. However, there is lack of evaluation study on the performance of nitrification at high salinity due to the concentration of recycled water or seawater utilization. A laboratory scale moving bed bioreactor (MBBR) with IMG was tested on recycled synthetic aquaculture wastewater for the nitrification at 2.5 mg/L NH3-N daily. The results indicated that IMG showed a high salinity tolerance and effectively converted ammonia to nitrate up to 92% at high salinity of 35.0 g/L NaCl. As salinity increased from near zero to 35.0 g/L, the microbial activity of nitrite oxidation bacteria (NOB) in the IMG decreased by 86.32%. The microbial community analysis indicated that salinity significantly influenced the community structure. It was found that Nitrosomonas sp. and Nitrospira sp. were the dominant genera for ammonia oxidation bacteria (AOB) and NOB respectively at different salinity levels. Keywords: Aquaculture salinity, Nitrification, Immobilized microbes, Moving bed bioreactors, Microbial community
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1. Introduction Recirculating aquaculture systems (RAS) are viewed as a sustainable aquacultural practice to fulfill the rising global seafood demand (Sharada Navada et al., 2019). Compared to the flow-through production systems or the net-pens production systems, RAS use much less water, and the water can be recycled up to 90 - 99% by applying a series of water treatment methods (Badiola et al., 2012). In addition, RAS have a lower ecological impact than the marine fisheries by avoiding the direct discharge of nutrient and toxic waste into the sea (Zeller et al., 2018). In order to mass produce the high nutrient-dense aquaculture products, the optimum water quality control and wastewater management is highly needed for an effective operation of RAS (Goddek et al., 2018). The high level of ammonia and nitrite for instance poses a high risks to the life of the aquatic animals and decrease the productivity of aquaculture (Goddek et al., 2018). Kumar et al. (2010) reported that marine larva could not tolerate the concentration of the total ammonia nitrogen (TAN) and nitrite-N above 1.0 mg/L. Ebeling et al. (2006) also suggested that the NH3 concentration should be below 0.05 mg/L under long term exposure. On the other hand,
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the conversions of total ammonia and nitrate content are strongly related to the salinity level in wastewater. High salinity in wastewater treatment system can be considered as one of the common stress condition that might causes significantly negative impacts on the biological metabolic activity (Moussa et al., 2006), bacterial community structure (Kinyage et al., 2019), and settling properties of nitrifying bacteria, therefore causing the failure of nitrification processes (Yogalakshmi &Joseph, 2010). Extensive researches have been conducted and proved that microbial biomass and biodiversity of activated sludge may reduce significantly as increase of salinity level, while, the salt-tolerant microorganism which originally not the dominant species might increase gradually in the system (He et al., 2017). Hence, there is a need to maintain a low level of ammonia and nitrite content in the aquaculture water especially for the RAS (Jiang et al., 2019). Use of IMG for nitrification has been widely known as an effective biotechnology technique for wastewater treatment (Dong et al., 2017). The novel IMG was composed of gel-based polymer granules with immobilized nitrifying bacteria that was enriched from a municipal wastewater treatment plant. It has the advantages of high biological
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density, high volumetric metabolic activities, limited sludge production, and short hydraulic retention time (HRT) (Tabassum et al., 2015) due to facilitating cell separation and protection from extreme conditions (Isaka et al., 2007). In the previous report by
Tabassum et al. ( 2015), IMG could remove up to 95% of ammonia (3.5
mg/L) with a short HRT of 4 h. Li et al. (2009) also reported that IMG was able to maintain optimum fresh water quality by removing nitrogen pollutant at an efficiency of 71.8% for NH3-N and 51.5% for NO2-N in a tested laboratory-scale system (Li et al., 2019). However, the overall performance of IMG in the brackish effluent and other water sources containing seawater with high salinity has not been studied. Nitrifying bacteria from the brackish and marine water have been characterized using universal 16S rRNA gene and bacterial amoA gene sequencing (Kumar et al., 2010). They reported that the most dominant AOB included Nitrosomonas sp. and Nitrosospira sp and the most dominant NOB were Nitrobacter sp. and Nitrospira sp. Gonzalez-Silva et al. (2016) studied the microbial communities at different salinity using 454-pyrosequencing of 16S rRNA gene amplicons. The results indicated that Nitrosomonas sp was the sole AOB species in the three reactors. Nitrobacter sp. and
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Nitrospira sp. were the key NOB species in the freshwater and N. marine for the salt water reactors. However, a limited number of studies are available in the literature addressing the nitrification efficiency and microbial community of nitrifying bacteria using immobilized microbes under a range of salinity level. In this study, a laboratory-scale MBBR filled with IMG was developed to treat nitrogen pollutant in the simulated RAS with the ammonia concentration of 2.5 mg/L using brackish and seawater. The microbial activities and communities for AOB and NOB were monitored under different salinity levels in order to evaluate the nitrification process using IMG. The results from this study are expected to provide useful benchmarks for developing MBBR with IMG, advancing the knowledge of designing effective inoculum in IMG in the future work by knowing the dominant microbial community at different salinity levels and its corresponding performance.
2. Materials and methods 2.1 Bioreactor System
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The immobilized microbial granules (IMG) are the form of gel-polymer cubic granules that were immobilized with nitrifying bacteria (the side length is 2-5 mm, with a specific gravity of 1.02-1.08) (Fig. 1). The inoculated microbial cells were collected from a local wastewater treatment plant and enriched with ammonia-containing wastewater prior to the immobilization. The granules were comprised of enriched microbial culture, powdered activated carbon from bamboo pieces and waterborne polyurethane gel as described by Tabassum et al. (2015). 2.2 Setup of MBBR with IMG A lab-scale simulated RAS was setup using a MBBR with IMG and a recirculated holding tank (Fig.1). The bioreactor (volume of 13.3 L, 80 cm × 8 cm in diameter) was built by suspending the IMG in a 4-L working volume. The volume ratio of IMG over the recirculated effluent (100 L) was set at 1:25. The synthetic water (100 L) was prepared using tap water with added ammonia (NH4Cl, 2.5 mg/L NH4+-N). The synthetic water was filled in the recirculated holding tank (120 L). NH4+-N was supplemented to reach 2.5 mg/L daily to simulate the release of ammonia in the effluent of the RAS fish tank. The water was recirculated at a rate of 0.35 L/min between the
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holding tank and the MBBR. Aeration was supplied at a rate of 60 L/min using an air pump (ACO-003Sensen, China). Dissolved oxygen (DO) level was determined by the DO analyzer (HQ40d, HACH, USA) and maintained within the range of 6.0 - 8.0 mg/L. The pH value was maintained between 7.0 and 8.0 by adding 1 M NaHCO3 solution. The system was operated for 112 days at ambient temperature condition (24 ± 2℃). 2.3 Laboratory-scale MBBR tests IMG was initially cultured in the synthetic water without added salts (salinity near zero) as stated in Section 2.1. The salinity was gradually increased from near zero to 5.0, 15.0, 25.0 and 35.0 g/L NaCl by adding NaCl over a 112-day period. As the nitrification efficiency (i.e. the removal of NH3 -N and NO2--N) reached more than 90%, the salinity was then adjusted to a higher level. IMG samples were collected for different salinity levels at the end of each sampling period and named as S1, S2, S3, S4 and S5 for near zero, 5.0, 15.0, 25.0 and 35.0 g/L NaCl as indicated in Table 1. The samples were collected for the determination of microbial activities (section 2.4) and analysis of microbial communities (section 2.5).
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In addition, the liquid samples were collected daily for the analyses of NH4+-N, NO2--N and NO3--N concentrations prior to the supplementation of NH4Cl solution. Ammonia and nitrate was measured using the method of Nessler Reagent Spectrophotometry and UV Spectrophotometry respectively and while nitrite was determined by N-(1-naphthyl)-ethylenediamine Spectrophotometry (NEPA, 2002). Duplicated samples were generated, analyzed and the average values were recorded. 2.4 Metabolic activity analysis Metabolic activities of IMG were analyzed based on the specific oxygen uptake rates (SOUR) of the nitrifying bacteria at different salinity levels. For the analysis, IMG samples (40 mL) were rinsed with deionized water. The SOUR of the nitrifying bacteria was measured using a DO probe (HQ40d, Hach, USA) according to the method by Ni et al. (2018). Meanwhile, the solutions for the SOUR were supplemented with added salts to match the respective salinities. 2.5 Microbial community analysis
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In order to characterize the microbial communities and to established the profile of major groups related to nitrification, high-throughput sequencing technology including Illumina sequencing was used. The IMG samples were collected from the reactor at different salinity levels (near zero, 5.0, 15.0, 25.0, and 35.0 g/L NaCl) and were immediately stored in the sterilized centrifuge tubes at -20 ℃. The total genomic DNA was extracted from each sample using the E.Z.N.ATM Mag-Bind Soil DNA Kit (Norcross, Georgia, USA). Meanwhile, agarose gel (1.5% w/v) electrophoresis was used to test the quality of the extracted DNA. By using the universal bacterial primers 341F and 805R, the V3-V4 region of the 16S rRNA gene was amplified in the Miseq sequencing platform (Illumina Inc., California, USA) (Lu et al., 2015). Representative operational taxonomy units (OTUs) were bolted using the default of Uparse for further analysis (Edgar, 2013). Uchime was used to detect the reference-based chimera (Edgar et al., 2011) and to compare against the RDP database (Cole et al., 2014). The OTU table was made up using the Usearch global alignment algorithm at 3% dissimilarity (Edgar, 2010). The samples and the OTU information
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were listed in the columns and rows in the OTU Table as indicated in Table 2. The data analysis included the indexes of Shannon, Simpson, Chao1, observed OTUs, and the representing Alpha diversity for each sample. Representative sequences for each OTU were analyze against training classifier RDP database version 2.10 to determine the phylogeny (RDP database version 2.10, Michigan State University, USA) with bootstrap values at 80%.
3 Results and discussions 3.1 The effect of salinity on nitrification The changes in the concentrations of ammonia, nitrite and nitrate in the MBBR at different salinities are shown in Fig.2. Over 14 d of operation at a salinity of near zero, the NH3-N was converted to NO3--N completely with the final concentration of 0.2 mg /L. The removal efficiency increased gradually and then stabilized at 84.9-92.0% (Fig.2a) without nitrite accumulation (Fig. 2b). The average rate of nitrate accumulation was found at 2.2 g/(L·d) (Fig. 2c). The salinity increased to 5.0 mg/L NaCl (d 15-28) and further increase to 15.0 mg/L NaCl (d 29-49); the results shows that the removal
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efficiency of NH4+-N was maintained above 94.0% and subsequently disappeared at low levels of nitrite which peaked at 0.8 and 1.3 mg/L. In both phases, it took about 9 to 11 d for the accumulated nitrite converted to nitrate completely. This showed that NOB grew slower to reach complete activity for the conversion of nitrite to nitrate. The results indicated that complete oxidation of ammonia to nitrate can be achieved at salinity of 15.0 g/L NaCl or lower. As the salinity increased to 25.0 g/L NaCl (d 50-77), the removal efficiency of NH4+-N still reached about 91 % over a 14 d period (Fig. 2a) and nitrite concentration increased continuously to 21.1 mg/L on day 63. Nitrite accumulated at a rate of 1.4g/(L·d). Subsequently, the nitrite-containing water was replaced with fresh water at salinity of 25.0 g/L NaCl. The nitrite initially accumulated and then declined to near zero (0.1 mg/L) on d 77 while nitrate concentration reached 31.4 mg/L with a 89.7 % conversion efficiency of ammonia to nitrate (Fig. 2c). The results indicated that AOB was not inhibited, nevertheless opposite results were observed for NOB by increasing the salinity to 25.0 g/L NaCl. The improvement of NOB performance by replacing the water suggested the inhibition may cause by both the salinity and the nitrite produced.
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At this salinity level, controlling the level of nitrite becomes critical to maintain the NOB activity and performance. As the salinity further increased to 35.0 g/L NaCl (d 78-112), the average NH4+-N removal efficiency was achieved 99.0% within 14 d while the nitrite concentration increased continuously to 25.0 mg/L (Fig. 1a and 1b). The rate of nitrate accumulation was 0.53 g/(L·d). Nitrate concentration increased to only 7.1 mg/L. On d 92, the nitrite-containing water in the system was replaced with fresh water. However, the accumulation of nitrite remained at a similar rate of 0.47 g/(L·d) and the nitrate concentration increased slowly as shown in Fig. 2b and 2c. The results indicated that AOB activity was not impacted by the increased salinity while NOB activity and growth were inhibited severely at salinity of 35.0 g/L NaCl. The results demonstrated that a salinity up to 35.0 g/L NaCl, which is the average level of seawater, has no negative impact on the ammonia oxidization and AOB activity of IMG in the MBBR. The NOB activity was impacted as the salinity increased above 15.0 g/L NaCl, the activity could be recovered at 25.0 g/L NaCl, and severely inhibited at 35.0 g/L NaCl. Therefore, it is safe to use the MBBR with IMG at salinity below the
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range of 15.0 to 25.0 g/L NaCl. The impact of salinity can be dependent on the source of microbial culture. In general, high salinity has a negative impact on nitrification. Cortes-Lorenzo et al. (2015) reported that a high salt concentration (⩾ 24.1 g/L NaCl) in the influent water resulted in a remarkable decrease of the ammonia oxidation capacity compared to that of zero NaCl in a submerged fixed bed bioreactor. Zhao et al. (2016) reported that at the salinity of 2.5% (25.64 g/L NaCl) and 3.0% (30.93 g/L NaCl), the removal rate of ammonia decreased to 65% and 56% in a sequencing batch reactor, respectively. Compared with these previous results, the IMG from this study exhibits a satisfying nitrification efficiency, where high ammonia oxidization ability was maintained even at the salinity of 35.0 g/L NaCl. It may be due to the immobilization that protected the cell from extreme conditions. The IMG used in this study were inoculated with microbial culture from municipal wastewater which contains freshwater and soil microorganisms. The microbiome in the IMG might be lacking of marine microorganisms which has a higher tolerance to high salinity. Further research should be conducted to investigate the effect of different microbial inocula in response to the high level of salinity equivalent to the
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seawater level or even higher. 3.2 Microbial activity of IMG The microbial activities of AOB and NOB of IMG were determined at different operational levels of salinity. The specific activities of AOB and NOB are presented based on SOUR as shown in Fig. 3. The metabolic activity of AOB was higher than that of NOB at all salinity levels as expected and both were negatively impacted as salinity increased (Fig.3). The SOUR of AOB decreased from 16.88 to 3.60 mg O2/(L·h) as the salinity increased from near zero to 5.0 g/L NaCl. Subsequently, the AOB activity increased dramatically with the further increase in salinity. At a salinity of 35.0 g/L NaCl, the SOUR of AOB reached the highest level of 17.71 mg O2/(L·h). The results suggested that the ammonia removal efficiency could be achieved at a high salinity which equaled to that of the sea water. The SOUR of NOB was different from AOB where it continuously decreased with the increase in salinity. At a salinity of 35.0 g/L NaCl, the NOB activity decreased by 86.42 % compared to that at salinity near zero (Fig. 3). This verified that salinity had less impact on AOB activities but exerted a negative effect on the metabolic activity of
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NOB as reported by other researchers (Ge et al., 2019). They reported that SOUR of AOB remained high as salinity reached 3.0% (30.93 g/L NaCl) while lower SOUR of NOB was observed at salinity of 3.0% (30.93 g/L NaCl) and 5.0% (52.63 g/L NaCl) compared with salinity near zero. NOB was more sensitive than AOB to high salinity. 3.3 Microbial community of IMG 3.3.1 Alpha diversity After data processing, about 173,194 raw sequences were produced by the 16S rRNA gene V3-V4 region sequencing, and 165,511 high-quality sequences were obtained for all five samples, with an average of 33,102 per sample. Alpha diversity analysis of five IMG samples collected at different salinity were characterized (Table 2). The operational taxonomic units (OTUs) were examined for each sample at distance levels of 0.03 to compare the bacterial species richness among the five samples. The observed OTUs number declined as the salinity increased. Shannon index, ACE index and other diversity indicators corresponded well with the result of OTUs number. These results indicated that microbial diversity in the immobilized microbial granules declined as salinity increased, which showed a similar trend in the activated sludge (Zhao et al.
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2016). They reported that approximately 50% of OTU numbers decreased when salinity level increased from 0 to 1.5 % (15.23 g/L NaCl) while only 23.67 % decreased in this study. IMG performed more tolerances to salinity changes, which might be ascribed to the immobilization of the microbial bacteria, facilitating the protection from high salt pressure (Tabassum et al., 2018). The diversity and richness of IMG at near zero salinity were much higher (OTU:1445 ) compared to those previously reported for the industry wastewater treatment plant (OTU: 451) (Ma et al., 2015) and municipal wastewater treatment systems with activated sludge (OTU: 352) (Zhao et al., 2016). It may be due to the high-density of microbes lived in the IMG. Venn diagram is a useful tool to visualize and demonstrate the distribution of the shared and unique OTUs under different salinity levels (Fig 4a). A total of 2,531 representative OTUs were acquired by the Miseq sequencing platform. There were significant differences of unique OTUs between near zero and other salinity levels. Only 13.43 % of the OTUs were found to be shared OTUs. It meant that a large number of OTU disappeared due to the salinity added. The microbial community structure that shift trajectory at different salinity levels has been traced using a tridimensional PCA
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plot based on the OTUs. It is found that an apparent shift was observed in the overall microbial community structure (Fig. 4b). The microbial community structure deviated greatly in the PCA1 direction after salt added. In addition, it was confirmed that the microbial community structure of lower salinity (5.0 g/L NaCl and 15.0 g/L NaCl) were more similar and the groups from higher salinity (25.0 g/L NaCl and 35.0 g/L NaCl) were more similar.
3.3.2 Dynamics of microbial community structures The taxonomic analysis results at levels of phylum, class and species of microbial community from five samples of different salinity levels in the IMG are presented in Fig. 5. At the phylum level, more than 15 known bacterial phyla were detected from the initial IMG which belong to Proteobacteria, Bacteroidetes, Planctomycetes, Acidobacteria, Actinobacteria, Firmicutes, Nitrospirae, Chloroflexi and Euryarchaeota (Fig.5a). As salinity level gradually increased from near zero to 35.0 g/L NaCl, some bacterial phyla were declined including Proteobacteria (reduced from 64.07% to 47.84 %), Actinobacteria (reduced from 4.33% to 1.05%) and Euryarchaeota (reduced
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from 1.13% to 0.03%) and some bacterial phyla Crenarchaeota and Thaumarchaeota was disappeared. However, over all salinity levels Proteobacteria was determined to be the dominant phylum. The finding is generally consistent with the fact that Proteobacteria was detected as the most prominent phylum in wastewater treatment, regardless under saline or unsalted condition (He et al., 2015; Qiu & Ting, 2013; Jeong et al., 2018). Many nitrogen removal related microbes belong to this phylum. Recently, phylum Nitrospira was detected as the dominant and specialized NOB found in some wastewater treatment plants (Harms et al., 2003). In this study, Nitrospirae was the second most abundant phylum at low salinity levels (5.0 g/L NaCl and 15.0 g/L NaCl). As salinity increased, the Bacteroidetes became more abundant. The phylum Bacteroidetes was considered as one of the most abundant heterotrophic bacterial groups in both the fresh and marine environments (Zhang et al., 2013). They play a key role in the degradation of particulate organic matter (Díez-Vives et al., 2012). Differences in community structure in the IMG could also be detected at the class level. More than 25 bacterial classes were identified and Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Nitrospira and Cytophagia were found to
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be the dominant groups (Fig.5b). As the salinity level increased from near zero to 35.0 g/L NaCl, Alphaproteobacteria and Gammaproteobacteria gradually reduced from 33.86% to 16.54% and from 13.94% to 3.25%, respectively. Betaproteobacteria increased from 11.40% to 24.25% when salinity increased from 0 to 35 g/L NaCl. At the high salinity (35 g/L NaCl), Betaproteobacteria was an abundant class within the Proteobacteria phylum, which conforms with the results obtained by Kwon et al. (2010). Betaproteobacteria had been observed a sharp decrease and then an obvious enrichment from 0 to 24.1 g/L NaCl in a biofilm reactor (Cortés-Lorenzo et al., 2014), showing that although salinity has a strong inhibition effect on betaproteobacteria, this class can adapt the salty environment gradually. A sharp increase from 1.01% to 25.81% for the class of Cytophagia were displayed. This is the first time, to our knowledge, the class Cytophagia has been identified as a major phylotype in a wastewater treatment system at high salinity (over 30 g/L). Microbial analysis at the genus level provided more comprehensive information on the shift of microbial functional structure (Fig.5c). Nitrosomonas, which belongs to AOB, and Nitrospira, a typical NOB, became the dominant genus as the salinity
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increased. Some genus including Aridibacter, Phycisphaera, Gp3, Hoeflea and Ohtaekwangia became more abundant in the IMG with increased salinity. A large number of bacteria genus decreased including the Pseudomonas, Pseudoxanthomonas, Kofleria, Sphingomonas when the salinity increased to 35.0 g/L NaCl. Some genus even vanished at a low level of salinity (5.0 g/L NaCl), including Sphingorhabdus, Blastomonas and Arthrobacter. More unclassified genera (nearly 40% relative abundance) were found at high salinity levels (25.0 and 35.0 g/L NaCl) compared with less genus (only 20% relative abundance) at low salinity levels (0, 5.0 and 15.0 g/L NaCl). Some genus including Aridibacter, Phycisphaera, Gp3, Hoeflea and Ohtaekwangia became more abundant in the IMG with increased salinity. These genera were widely detected in the active sludge and biofilm system under fresh or saline/marine conditions (Babatsouli et al., 2015; Wang et al., 2016). Proliferation of halotolerant/halophilic microbes in IMG samples stabilized biological performance of MBBR in response to the rising salinity. 3.3.3 Response of nitrifying bacteria on salinity changes
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Based on the taxonomic information, the OTUs belonging to nitrifying bacteria and the relative abundances were listed in the Table 3. Of the total 2531 OTUs, 38 OTUs of AOB which were classified as Nitrosomonas (32 OTUs) and Nitrososphaera (6 OTUs); and 27 OTUs of NOB were classified as Nitrospira (20 OTUs) and Nitrolancea (5 OTUs). In addition, 2 OTUs were classified as genus Nitrobacter. The abundance of AOB and NOB responses differently to the salinity increase. When the salinity level increased to 5.0 g/L NaCl, the abundance of AOB decreased from 3.93% (near 0 g/L) to 1.46%. Similar trend was reported by other researchers (Wang et al., 2017), i.e., 5.0 g/L NaCl salinity resulting in the decrease of relative abundance of AOB from 10.50% (salinity of zero) to 3.40%. It is likely that the energy obtained from substrate degradation is required to resist the threats from the salt-stressed environment and maintain normal cell activities (Moussa et al., 2005). Through a period of acclimation, AOB in the system gradually adapted to the salty environment. After the salinity increased to 25.0 g/L NaCl, the relative abundance of AOB increased to 11.23%, nearly twice of that at zero salinity. At salinity of 35.0 g/L NaCl, the relative abundance of AOB continued to increase to 20.93%. These result was consistent with the report of
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Ge et al. (2019), that the abundance of AOB in sludge sample under 3.0% salinity was 9.42%, nearly two times of these under salinity of zero. The inhibition effect of salt for AOB vary in the literatures. Mosquera-Corral et al. (2005) reported that ammonia oxidizing bacteria had a stable activity at a concentration up to 25 g/L NaCl. Corsino et al. (2016) found that the acclimated aerobic granules could adapt to hypersaline wastewater (50 g/L NaCl). In this study, the most abundant genus of AOB in all samples under different salinity levels was Nitrosomonas, which was found in fresh water, soil, and marine environments (Brown et al., 2013; Ramirez-Vargas et al., 2015). Many species such as “N. europaea” in this genus are reported to possess higher salt endurance compared with other AOB species (Wang, et al., 2017). The differences between the researchers is due to different reactor configurations, operational parameters, the way of introducing the salinity to the reactors and the bacteria existing in the system (Bassin et al., 2011; Gonzalez-Silva et al., 2016). The NOB sequences contributed only 2.13% at zero salinity (S1 or without NaCl addition). When the salinity increased to 5.0 g/L NaCl, the abundance of NOB increased rapidly to 28.33%., but when the salinity increased to 35.0 g/L NaCl, it
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decreased to 8.28 %. Similar results were also reported by Ye et al. (2009), i.e. NOB could survive in the salty environment with decreased activity. Salinity had a significant negative effect on the abundance of NOB bacteria. Nitrospira was the dominant NOB genus in all samples under different salinity levels, which was reported as the major nitrite oxidizer in salt reactors for marine RAS (Keuter et al., 2011). Discrepancies in the microbial species involved may be the main reason for the different trends in activities and relative abundance (Wang et al., 2017). Nevertheless, these studies all proved that the presence of salt can change the NOB population.
4. Conclusions The novel Immobilized microbial granules (IMG) were characterized in response to the increased salinity up to 35.0 g/L NaCl in the MBBR in terms of nitrifying metabolic activities and microbial communities. It was found that ammonia was effectively removed and converted to nitrate and nitrite with conversion efficiency up to 92% with salinity of 35.0 g/L NaCl. Inhibition occurred for the conversion of nitrite to nitrate at the salinity above 15.0 to 25.0 g/L NaCl, resulting in the accumulation of
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nitrite. The results also indicated that the microbial community structure was significantly impacted by the salinity change where AOB Nitrosomonas.sp and NOB Nitrospira sp were the dominant nitrifying genus at different salinity levels.
Acknowledgments This research is financially supported by Natural Science Foundation Council of China (No. 21808141), National Science and Technology Commission of Shanghai Municipality (No. 18230743000) and Startup Fund for Youngman Research at SJTU (17X100040069). We appreciate help and suggestions from Dr. Weimin Wu, Department of Civil & Environmental Engineering, Stanford University, USA and Dr. Haslenda Hashim, Faculty of Chemical & Energy Engineering, Universiti Teknologi Malaysia, Malaysia.
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effect of influent salinity and inhibition time on partial nitrification in a sequencing batch reactor treating saline sewage. Desalination, 246(1-3), 556-566. Zeller, D., Cashion, T., Palomares, M., & Pauly, D. 2018. Global marine fisheries discards: A synthesis of reconstructed data. Fish Fish., 19(1), 30-39. Zhang, L., Gao, G., Tang, X., Shao, K., Bayartu, S., Dai, J., 2013. Bacterial community changes along a salinity gradient in a Chinese wetland. Can. J. Microbiol. 59 (9), 611–619. Zhang, T., Shao, M.F., Ye, L., 2012. 454 pyrosequencing reveals bacterial diversity ofactivated sludge from 14 sewage treatment plants. ISME J. 6, 1137–1147. Zhao, Y., Park, H.-D., Park, J.-H., Zhang, F., Chen, C., Li, X., Zhao, D., Zhao, F. 2016. Effect of different salinity adaptation on the performance and microbial community in a sequencing batch reactor. Bioresour. Technol., 216, 808-816.
Figure captions Fig.1 (a) Schematic diagram of MBBR with IMG; (b) Photo of IMG used in the MBBR.
35
Fig.2 Operational results of bioreactor tests at different salinities. (a) ammonia concentrations, (b) nitrite and (c) nitrate. Fig.3 The metabolic activities of AOB and NOB of IMG. Fig.4 Venn diagram showing a total of 2531 OUT (a) and PCA plot (b) under different salinity levels (S1 to S5). Fig.5 Microbial communities of IMG varied as salinity increased. Taxonomic identification at (a) phylum; (b) class; and (c) genus at salinity levels of near zero (S1), 5.0 (S2), 15.0 (S3), 25.0 (S4) and 35.0 g/L (S5).
36
Effluent
a
b IMG
Pump
Holding tank
Influent
Aerator
Fig.1 (a) Schematic diagram of MBBR with IMG; (b) Photo of IMG used in the MBBR.
37
a
2.5
Ⅰ
NH4+ (mg/L)
2.0
Ⅲ
Ⅱ
Ⅴ
Ⅳ
30
NaCl (g/L)
1.5 20 1.0
Ammonia Nacl 10
0.5 0.0
0 0
14
28
42
56
70
84
98
112
Time (d)
Ⅰ
Nitrite (mg/L)
30
Ⅱ
Ⅳ
Ⅲ
Ⅴ
30
20
20 Nitrite NaCl 10
0
NaCl (g/L)
b
10
0 0
14
28
42
56
70
84
98
112
Time (d)
c 50
Ⅱ
Ⅰ
Ⅲ
Ⅴ
Ⅳ
30
20
30 Nitrate
20
NaCl (g/L)
Nitrate (mg/L)
40
10
NaCl 10 0
0 0
14
28
42
56
70
84
98
112
Time (d)
Fig. 2 Operational results of bioreactor tests at different salinities. (a) ammonia concentrations, (b) nitrite and (c) nitrate.
38
AOB NOB
SOUR (mg O2/(L·h))
20
15
10
5
0
0
5.0
15.0
25.0
35.0
NaCl (g/L)
Fig. 3 The metabolic activities of AOB and NOB of IMG.
39
a
b
A
B
Fig 4 Venn diagram showing a total of 2531 OUT (a) and PCA plot (b) under different salinity levels (S1 to S5).
40
a Abundance of phylum(%)
A
100 90 80 70 60 50 40 30 20 10 0 S1
S2
S3
S4
S5
Sample ID
b Abundance of class(%)
B
100 90 80 70 60 50 40 30 20 10 0 S1
S2
S3
S4
S5
Sample ID
100
C
80
Abundance of genus (%)
c
Others Thaumarchaeota Deinococcus-Thermus Spirochaetes Crenarchaeota candidate division WPS-1 Candidatus Saccharibacteria Chlamydiae Euryarchaeota Armatimonadetes Verrucomicrobia Gemmatimonadetes Chloroflexi Firmicutes Actinobacteria unclassified Planctomycetes Acidobacteria Bacteroidetes Nitrospirae Proteobacteria
Others Thermoprotei Epsilonproteobacteria Bacteroidia Subdivision3 Clostridia Gemmatimonadetes Bacilli Anaerolineae Acidobacteria_Gp3 Actinobacteria Sphingobacteriia Deltaproteobacteria Phycisphaerae Planctomycetia Acidobacteria_Gp4 unclassified Gammaproteobacteria Cytophagia Betaproteobacteria Nitrospira Alphaproteobacteria
Others Noviherbaspirillum Blastomonas Labilithrix Sphingorhabdus Sphingomonas Ohtaekwangia Lacibacterium Hydrogenophaga Chryseolinea Gp3 Hyphomicrobium Aridibacter Pseudomonas Nitrospira
90
70 60 50 40 30 20 10
Arthrobacter Arcobacter Pedomicrobium Blastocatella Novosphingobium Gemmatimonas Kofleria Hoeflea Bradyrhizobium Pseudoxanthomonas Phycisphaera Piscinibacter Sphingopyxis Nitrosomonas unclassified
0 S1
S2
S3
S4
S5
Sample ID
Fig.5 Microbial communities of IMG varied as salinity increased. Taxonomic identification at (a) phylum; (b) class; and (c) genus at salinity levels of near zero (S1), 5.0 (S2), 15.0 (S3), 25.0 (S4) and 35.0 g/L (S5).
41
42
Table 1 Operational phases of different salinity levels
Phase
Salinity /(g/L)
Sample ID
Period (d)
Ⅰ
Near zero
S1
0-14
Ⅱ
5.0
S2
15-28
Ⅲ
15.0
S3
29-49
Ⅳ
25.0
S4
50-77
Ⅴ
35.0
S5
78-112
43
Table 2 Sequences of the five IMG samples based on 0.03 distances ID (g/L of
Sequence
OTU
Shannon
ACE
Chao1
Coverage
Simpson
salinity)
number
number
index
index
Index
(%)
number
S1 (0)
20,098
1445
5.36
1637.35 1532.31
98.59
0.017
S2 (5)
38,868
1013
4.04
1291.82 1230.88
99.26
0.088
S3(15)
38,324
1103
4.54
1327.28 1278.05
99.29
0.050
S4(25)
29,741
916
4.02
1275.52 1219.34
98.94
0.071
S5(35)
24,496
873
3.71
1232.28 1167.14
98.72
0.099
44
Table 3 The relative abundances of AOB and NOB at salinity levels of zero (S1), 5.0 (S2), 15.0 (S3), 25.0 (S4), and 35.0 g/L (S5)
AOB
NOB
Genus
S1
S2
S3
S4
S5
Nitrosomonas
3.75
1.43
1.94
11.23
20.93
Nitrosopumilus
0.06
0.03
0.01
-
-
Nitrososphaera
0.12
-
-
-
-
Nitrospira
2.05
28.26
20.86
9.40
8.24
Nitrolancea
0.07
0.07
0.08
0.08
0.04
Nitrobacter
0.01
-
-
-
-
45
Highlights
Immobilized microbial granules improved salinity tolerance.
High ammonia removal (92 %) was achieved at salinity up to 35.0 g/L.
Nitrite oxidation bacteria was depressed at salinity above 15.0 g/L.
Microbial community structure was shifted as salinity increased.
Nitrosomonas sp. and Nitrospira sp. were the dominant genera.
46
Author Contributions Section Gao Yueshu: Conceptualization, Methodolog, Writing- Original draft preparation Wang Xupeng: Investigation, Visualization Li Jialun: Investigation, Software Lee Chew Tin: Visualization, Reviewing and Editing Ong Pei Ying: Software, Validation Zhang Zhenjia: Supervision, Resource Li Chunjie: Writing- Reviewing and Editing, Project administration
47
48
S0960-8524(19)31657-8 https://doi.org/10.1016/j.biortech.2019.122427 BITE 122427
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
18 September 2019 9 November 2019 12 November 2019
Please cite this article as: Gao, Y., Wang, X., Li, J., Tin Lee, C., Ying Ong, P., Zhang, Z., Li, C., Effect of aquaculture salinity on nitrification and microbial community in moving bed bioreactors with immobilized microbial granules, Bioresource Technology (2019), doi: https://doi.org/10.1016/j.biortech.2019.122427
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© 2019 Elsevier Ltd. All rights reserved.
Effect of aquaculture salinity on nitrification and microbial community in moving bed bioreactors with immobilized microbial granules Yueshu Gaoa, Xupeng Wanga, Jialun Lia, Chew Tin Leeb, Pei Ying Ongc, Zhenjia Zhanga, Chunjie Li*a a
School of Environmental Science & Engineering, Shanghai Jiao Tong University,
Shanghai 200240, China bFaculty
of Chemical & Energy Engineering, Universiti Teknologi Malaysia, 81310
Johor Bahru, Johor, Malaysia
c
Innovation Center in Agritechnology For Advanced Bioprocessing (ICA), Universiti
Teknologi Malaysia, 84600 Pagoh, Johor, Malaysia
*Corresponding author, Chunjie Li, 800 Dong Chuan Road, School of Environmental Science and Engineering, Shanghai Jiao Tong University, Shanghai, P. R. China. E-mail: [email protected]. Tel.: +862113391048252.
1
Abstract: The novel immobilized microbial granules (IMG) shows a significant effect of nitrification for freshwater aquaculture. However, there is lack of evaluation study on the performance of nitrification at high salinity due to the concentration of recycled water or seawater utilization. A laboratory scale moving bed bioreactor (MBBR) with IMG was tested on recycled synthetic aquaculture wastewater for the nitrification at 2.5 mg/L NH3-N daily. The results indicated that IMG showed a high salinity tolerance and effectively converted ammonia to nitrate up to 92% at high salinity of 35.0 g/L NaCl. As salinity increased from near zero to 35.0 g/L, the microbial activity of nitrite oxidation bacteria (NOB) in the IMG decreased by 86.32%. The microbial community analysis indicated that salinity significantly influenced the community structure. It was found that Nitrosomonas sp. and Nitrospira sp. were the dominant genera for ammonia oxidation bacteria (AOB) and NOB respectively at different salinity levels. Keywords: Aquaculture salinity, Nitrification, Immobilized microbes, Moving bed bioreactors, Microbial community
2
1. Introduction Recirculating aquaculture systems (RAS) are viewed as a sustainable aquacultural practice to fulfill the rising global seafood demand (Sharada Navada et al., 2019). Compared to the flow-through production systems or the net-pens production systems, RAS use much less water, and the water can be recycled up to 90 - 99% by applying a series of water treatment methods (Badiola et al., 2012). In addition, RAS have a lower ecological impact than the marine fisheries by avoiding the direct discharge of nutrient and toxic waste into the sea (Zeller et al., 2018). In order to mass produce the high nutrient-dense aquaculture products, the optimum water quality control and wastewater management is highly needed for an effective operation of RAS (Goddek et al., 2018). The high level of ammonia and nitrite for instance poses a high risks to the life of the aquatic animals and decrease the productivity of aquaculture (Goddek et al., 2018). Kumar et al. (2010) reported that marine larva could not tolerate the concentration of the total ammonia nitrogen (TAN) and nitrite-N above 1.0 mg/L. Ebeling et al. (2006) also suggested that the NH3 concentration should be below 0.05 mg/L under long term exposure. On the other hand,
3
the conversions of total ammonia and nitrate content are strongly related to the salinity level in wastewater. High salinity in wastewater treatment system can be considered as one of the common stress condition that might causes significantly negative impacts on the biological metabolic activity (Moussa et al., 2006), bacterial community structure (Kinyage et al., 2019), and settling properties of nitrifying bacteria, therefore causing the failure of nitrification processes (Yogalakshmi &Joseph, 2010). Extensive researches have been conducted and proved that microbial biomass and biodiversity of activated sludge may reduce significantly as increase of salinity level, while, the salt-tolerant microorganism which originally not the dominant species might increase gradually in the system (He et al., 2017). Hence, there is a need to maintain a low level of ammonia and nitrite content in the aquaculture water especially for the RAS (Jiang et al., 2019). Use of IMG for nitrification has been widely known as an effective biotechnology technique for wastewater treatment (Dong et al., 2017). The novel IMG was composed of gel-based polymer granules with immobilized nitrifying bacteria that was enriched from a municipal wastewater treatment plant. It has the advantages of high biological
4
density, high volumetric metabolic activities, limited sludge production, and short hydraulic retention time (HRT) (Tabassum et al., 2015) due to facilitating cell separation and protection from extreme conditions (Isaka et al., 2007). In the previous report by
Tabassum et al. ( 2015), IMG could remove up to 95% of ammonia (3.5
mg/L) with a short HRT of 4 h. Li et al. (2009) also reported that IMG was able to maintain optimum fresh water quality by removing nitrogen pollutant at an efficiency of 71.8% for NH3-N and 51.5% for NO2-N in a tested laboratory-scale system (Li et al., 2019). However, the overall performance of IMG in the brackish effluent and other water sources containing seawater with high salinity has not been studied. Nitrifying bacteria from the brackish and marine water have been characterized using universal 16S rRNA gene and bacterial amoA gene sequencing (Kumar et al., 2010). They reported that the most dominant AOB included Nitrosomonas sp. and Nitrosospira sp and the most dominant NOB were Nitrobacter sp. and Nitrospira sp. Gonzalez-Silva et al. (2016) studied the microbial communities at different salinity using 454-pyrosequencing of 16S rRNA gene amplicons. The results indicated that Nitrosomonas sp was the sole AOB species in the three reactors. Nitrobacter sp. and
5
Nitrospira sp. were the key NOB species in the freshwater and N. marine for the salt water reactors. However, a limited number of studies are available in the literature addressing the nitrification efficiency and microbial community of nitrifying bacteria using immobilized microbes under a range of salinity level. In this study, a laboratory-scale MBBR filled with IMG was developed to treat nitrogen pollutant in the simulated RAS with the ammonia concentration of 2.5 mg/L using brackish and seawater. The microbial activities and communities for AOB and NOB were monitored under different salinity levels in order to evaluate the nitrification process using IMG. The results from this study are expected to provide useful benchmarks for developing MBBR with IMG, advancing the knowledge of designing effective inoculum in IMG in the future work by knowing the dominant microbial community at different salinity levels and its corresponding performance.
2. Materials and methods 2.1 Bioreactor System
6
The immobilized microbial granules (IMG) are the form of gel-polymer cubic granules that were immobilized with nitrifying bacteria (the side length is 2-5 mm, with a specific gravity of 1.02-1.08) (Fig. 1). The inoculated microbial cells were collected from a local wastewater treatment plant and enriched with ammonia-containing wastewater prior to the immobilization. The granules were comprised of enriched microbial culture, powdered activated carbon from bamboo pieces and waterborne polyurethane gel as described by Tabassum et al. (2015). 2.2 Setup of MBBR with IMG A lab-scale simulated RAS was setup using a MBBR with IMG and a recirculated holding tank (Fig.1). The bioreactor (volume of 13.3 L, 80 cm × 8 cm in diameter) was built by suspending the IMG in a 4-L working volume. The volume ratio of IMG over the recirculated effluent (100 L) was set at 1:25. The synthetic water (100 L) was prepared using tap water with added ammonia (NH4Cl, 2.5 mg/L NH4+-N). The synthetic water was filled in the recirculated holding tank (120 L). NH4+-N was supplemented to reach 2.5 mg/L daily to simulate the release of ammonia in the effluent of the RAS fish tank. The water was recirculated at a rate of 0.35 L/min between the
7
holding tank and the MBBR. Aeration was supplied at a rate of 60 L/min using an air pump (ACO-003Sensen, China). Dissolved oxygen (DO) level was determined by the DO analyzer (HQ40d, HACH, USA) and maintained within the range of 6.0 - 8.0 mg/L. The pH value was maintained between 7.0 and 8.0 by adding 1 M NaHCO3 solution. The system was operated for 112 days at ambient temperature condition (24 ± 2℃). 2.3 Laboratory-scale MBBR tests IMG was initially cultured in the synthetic water without added salts (salinity near zero) as stated in Section 2.1. The salinity was gradually increased from near zero to 5.0, 15.0, 25.0 and 35.0 g/L NaCl by adding NaCl over a 112-day period. As the nitrification efficiency (i.e. the removal of NH3 -N and NO2--N) reached more than 90%, the salinity was then adjusted to a higher level. IMG samples were collected for different salinity levels at the end of each sampling period and named as S1, S2, S3, S4 and S5 for near zero, 5.0, 15.0, 25.0 and 35.0 g/L NaCl as indicated in Table 1. The samples were collected for the determination of microbial activities (section 2.4) and analysis of microbial communities (section 2.5).
8
In addition, the liquid samples were collected daily for the analyses of NH4+-N, NO2--N and NO3--N concentrations prior to the supplementation of NH4Cl solution. Ammonia and nitrate was measured using the method of Nessler Reagent Spectrophotometry and UV Spectrophotometry respectively and while nitrite was determined by N-(1-naphthyl)-ethylenediamine Spectrophotometry (NEPA, 2002). Duplicated samples were generated, analyzed and the average values were recorded. 2.4 Metabolic activity analysis Metabolic activities of IMG were analyzed based on the specific oxygen uptake rates (SOUR) of the nitrifying bacteria at different salinity levels. For the analysis, IMG samples (40 mL) were rinsed with deionized water. The SOUR of the nitrifying bacteria was measured using a DO probe (HQ40d, Hach, USA) according to the method by Ni et al. (2018). Meanwhile, the solutions for the SOUR were supplemented with added salts to match the respective salinities. 2.5 Microbial community analysis
9
In order to characterize the microbial communities and to established the profile of major groups related to nitrification, high-throughput sequencing technology including Illumina sequencing was used. The IMG samples were collected from the reactor at different salinity levels (near zero, 5.0, 15.0, 25.0, and 35.0 g/L NaCl) and were immediately stored in the sterilized centrifuge tubes at -20 ℃. The total genomic DNA was extracted from each sample using the E.Z.N.ATM Mag-Bind Soil DNA Kit (Norcross, Georgia, USA). Meanwhile, agarose gel (1.5% w/v) electrophoresis was used to test the quality of the extracted DNA. By using the universal bacterial primers 341F and 805R, the V3-V4 region of the 16S rRNA gene was amplified in the Miseq sequencing platform (Illumina Inc., California, USA) (Lu et al., 2015). Representative operational taxonomy units (OTUs) were bolted using the default of Uparse for further analysis (Edgar, 2013). Uchime was used to detect the reference-based chimera (Edgar et al., 2011) and to compare against the RDP database (Cole et al., 2014). The OTU table was made up using the Usearch global alignment algorithm at 3% dissimilarity (Edgar, 2010). The samples and the OTU information
10
were listed in the columns and rows in the OTU Table as indicated in Table 2. The data analysis included the indexes of Shannon, Simpson, Chao1, observed OTUs, and the representing Alpha diversity for each sample. Representative sequences for each OTU were analyze against training classifier RDP database version 2.10 to determine the phylogeny (RDP database version 2.10, Michigan State University, USA) with bootstrap values at 80%.
3 Results and discussions 3.1 The effect of salinity on nitrification The changes in the concentrations of ammonia, nitrite and nitrate in the MBBR at different salinities are shown in Fig.2. Over 14 d of operation at a salinity of near zero, the NH3-N was converted to NO3--N completely with the final concentration of 0.2 mg /L. The removal efficiency increased gradually and then stabilized at 84.9-92.0% (Fig.2a) without nitrite accumulation (Fig. 2b). The average rate of nitrate accumulation was found at 2.2 g/(L·d) (Fig. 2c). The salinity increased to 5.0 mg/L NaCl (d 15-28) and further increase to 15.0 mg/L NaCl (d 29-49); the results shows that the removal
11
efficiency of NH4+-N was maintained above 94.0% and subsequently disappeared at low levels of nitrite which peaked at 0.8 and 1.3 mg/L. In both phases, it took about 9 to 11 d for the accumulated nitrite converted to nitrate completely. This showed that NOB grew slower to reach complete activity for the conversion of nitrite to nitrate. The results indicated that complete oxidation of ammonia to nitrate can be achieved at salinity of 15.0 g/L NaCl or lower. As the salinity increased to 25.0 g/L NaCl (d 50-77), the removal efficiency of NH4+-N still reached about 91 % over a 14 d period (Fig. 2a) and nitrite concentration increased continuously to 21.1 mg/L on day 63. Nitrite accumulated at a rate of 1.4g/(L·d). Subsequently, the nitrite-containing water was replaced with fresh water at salinity of 25.0 g/L NaCl. The nitrite initially accumulated and then declined to near zero (0.1 mg/L) on d 77 while nitrate concentration reached 31.4 mg/L with a 89.7 % conversion efficiency of ammonia to nitrate (Fig. 2c). The results indicated that AOB was not inhibited, nevertheless opposite results were observed for NOB by increasing the salinity to 25.0 g/L NaCl. The improvement of NOB performance by replacing the water suggested the inhibition may cause by both the salinity and the nitrite produced.
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At this salinity level, controlling the level of nitrite becomes critical to maintain the NOB activity and performance. As the salinity further increased to 35.0 g/L NaCl (d 78-112), the average NH4+-N removal efficiency was achieved 99.0% within 14 d while the nitrite concentration increased continuously to 25.0 mg/L (Fig. 1a and 1b). The rate of nitrate accumulation was 0.53 g/(L·d). Nitrate concentration increased to only 7.1 mg/L. On d 92, the nitrite-containing water in the system was replaced with fresh water. However, the accumulation of nitrite remained at a similar rate of 0.47 g/(L·d) and the nitrate concentration increased slowly as shown in Fig. 2b and 2c. The results indicated that AOB activity was not impacted by the increased salinity while NOB activity and growth were inhibited severely at salinity of 35.0 g/L NaCl. The results demonstrated that a salinity up to 35.0 g/L NaCl, which is the average level of seawater, has no negative impact on the ammonia oxidization and AOB activity of IMG in the MBBR. The NOB activity was impacted as the salinity increased above 15.0 g/L NaCl, the activity could be recovered at 25.0 g/L NaCl, and severely inhibited at 35.0 g/L NaCl. Therefore, it is safe to use the MBBR with IMG at salinity below the
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range of 15.0 to 25.0 g/L NaCl. The impact of salinity can be dependent on the source of microbial culture. In general, high salinity has a negative impact on nitrification. Cortes-Lorenzo et al. (2015) reported that a high salt concentration (⩾ 24.1 g/L NaCl) in the influent water resulted in a remarkable decrease of the ammonia oxidation capacity compared to that of zero NaCl in a submerged fixed bed bioreactor. Zhao et al. (2016) reported that at the salinity of 2.5% (25.64 g/L NaCl) and 3.0% (30.93 g/L NaCl), the removal rate of ammonia decreased to 65% and 56% in a sequencing batch reactor, respectively. Compared with these previous results, the IMG from this study exhibits a satisfying nitrification efficiency, where high ammonia oxidization ability was maintained even at the salinity of 35.0 g/L NaCl. It may be due to the immobilization that protected the cell from extreme conditions. The IMG used in this study were inoculated with microbial culture from municipal wastewater which contains freshwater and soil microorganisms. The microbiome in the IMG might be lacking of marine microorganisms which has a higher tolerance to high salinity. Further research should be conducted to investigate the effect of different microbial inocula in response to the high level of salinity equivalent to the
14
seawater level or even higher. 3.2 Microbial activity of IMG The microbial activities of AOB and NOB of IMG were determined at different operational levels of salinity. The specific activities of AOB and NOB are presented based on SOUR as shown in Fig. 3. The metabolic activity of AOB was higher than that of NOB at all salinity levels as expected and both were negatively impacted as salinity increased (Fig.3). The SOUR of AOB decreased from 16.88 to 3.60 mg O2/(L·h) as the salinity increased from near zero to 5.0 g/L NaCl. Subsequently, the AOB activity increased dramatically with the further increase in salinity. At a salinity of 35.0 g/L NaCl, the SOUR of AOB reached the highest level of 17.71 mg O2/(L·h). The results suggested that the ammonia removal efficiency could be achieved at a high salinity which equaled to that of the sea water. The SOUR of NOB was different from AOB where it continuously decreased with the increase in salinity. At a salinity of 35.0 g/L NaCl, the NOB activity decreased by 86.42 % compared to that at salinity near zero (Fig. 3). This verified that salinity had less impact on AOB activities but exerted a negative effect on the metabolic activity of
15
NOB as reported by other researchers (Ge et al., 2019). They reported that SOUR of AOB remained high as salinity reached 3.0% (30.93 g/L NaCl) while lower SOUR of NOB was observed at salinity of 3.0% (30.93 g/L NaCl) and 5.0% (52.63 g/L NaCl) compared with salinity near zero. NOB was more sensitive than AOB to high salinity. 3.3 Microbial community of IMG 3.3.1 Alpha diversity After data processing, about 173,194 raw sequences were produced by the 16S rRNA gene V3-V4 region sequencing, and 165,511 high-quality sequences were obtained for all five samples, with an average of 33,102 per sample. Alpha diversity analysis of five IMG samples collected at different salinity were characterized (Table 2). The operational taxonomic units (OTUs) were examined for each sample at distance levels of 0.03 to compare the bacterial species richness among the five samples. The observed OTUs number declined as the salinity increased. Shannon index, ACE index and other diversity indicators corresponded well with the result of OTUs number. These results indicated that microbial diversity in the immobilized microbial granules declined as salinity increased, which showed a similar trend in the activated sludge (Zhao et al.
16
2016). They reported that approximately 50% of OTU numbers decreased when salinity level increased from 0 to 1.5 % (15.23 g/L NaCl) while only 23.67 % decreased in this study. IMG performed more tolerances to salinity changes, which might be ascribed to the immobilization of the microbial bacteria, facilitating the protection from high salt pressure (Tabassum et al., 2018). The diversity and richness of IMG at near zero salinity were much higher (OTU:1445 ) compared to those previously reported for the industry wastewater treatment plant (OTU: 451) (Ma et al., 2015) and municipal wastewater treatment systems with activated sludge (OTU: 352) (Zhao et al., 2016). It may be due to the high-density of microbes lived in the IMG. Venn diagram is a useful tool to visualize and demonstrate the distribution of the shared and unique OTUs under different salinity levels (Fig 4a). A total of 2,531 representative OTUs were acquired by the Miseq sequencing platform. There were significant differences of unique OTUs between near zero and other salinity levels. Only 13.43 % of the OTUs were found to be shared OTUs. It meant that a large number of OTU disappeared due to the salinity added. The microbial community structure that shift trajectory at different salinity levels has been traced using a tridimensional PCA
17
plot based on the OTUs. It is found that an apparent shift was observed in the overall microbial community structure (Fig. 4b). The microbial community structure deviated greatly in the PCA1 direction after salt added. In addition, it was confirmed that the microbial community structure of lower salinity (5.0 g/L NaCl and 15.0 g/L NaCl) were more similar and the groups from higher salinity (25.0 g/L NaCl and 35.0 g/L NaCl) were more similar.
3.3.2 Dynamics of microbial community structures The taxonomic analysis results at levels of phylum, class and species of microbial community from five samples of different salinity levels in the IMG are presented in Fig. 5. At the phylum level, more than 15 known bacterial phyla were detected from the initial IMG which belong to Proteobacteria, Bacteroidetes, Planctomycetes, Acidobacteria, Actinobacteria, Firmicutes, Nitrospirae, Chloroflexi and Euryarchaeota (Fig.5a). As salinity level gradually increased from near zero to 35.0 g/L NaCl, some bacterial phyla were declined including Proteobacteria (reduced from 64.07% to 47.84 %), Actinobacteria (reduced from 4.33% to 1.05%) and Euryarchaeota (reduced
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from 1.13% to 0.03%) and some bacterial phyla Crenarchaeota and Thaumarchaeota was disappeared. However, over all salinity levels Proteobacteria was determined to be the dominant phylum. The finding is generally consistent with the fact that Proteobacteria was detected as the most prominent phylum in wastewater treatment, regardless under saline or unsalted condition (He et al., 2015; Qiu & Ting, 2013; Jeong et al., 2018). Many nitrogen removal related microbes belong to this phylum. Recently, phylum Nitrospira was detected as the dominant and specialized NOB found in some wastewater treatment plants (Harms et al., 2003). In this study, Nitrospirae was the second most abundant phylum at low salinity levels (5.0 g/L NaCl and 15.0 g/L NaCl). As salinity increased, the Bacteroidetes became more abundant. The phylum Bacteroidetes was considered as one of the most abundant heterotrophic bacterial groups in both the fresh and marine environments (Zhang et al., 2013). They play a key role in the degradation of particulate organic matter (Díez-Vives et al., 2012). Differences in community structure in the IMG could also be detected at the class level. More than 25 bacterial classes were identified and Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Nitrospira and Cytophagia were found to
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be the dominant groups (Fig.5b). As the salinity level increased from near zero to 35.0 g/L NaCl, Alphaproteobacteria and Gammaproteobacteria gradually reduced from 33.86% to 16.54% and from 13.94% to 3.25%, respectively. Betaproteobacteria increased from 11.40% to 24.25% when salinity increased from 0 to 35 g/L NaCl. At the high salinity (35 g/L NaCl), Betaproteobacteria was an abundant class within the Proteobacteria phylum, which conforms with the results obtained by Kwon et al. (2010). Betaproteobacteria had been observed a sharp decrease and then an obvious enrichment from 0 to 24.1 g/L NaCl in a biofilm reactor (Cortés-Lorenzo et al., 2014), showing that although salinity has a strong inhibition effect on betaproteobacteria, this class can adapt the salty environment gradually. A sharp increase from 1.01% to 25.81% for the class of Cytophagia were displayed. This is the first time, to our knowledge, the class Cytophagia has been identified as a major phylotype in a wastewater treatment system at high salinity (over 30 g/L). Microbial analysis at the genus level provided more comprehensive information on the shift of microbial functional structure (Fig.5c). Nitrosomonas, which belongs to AOB, and Nitrospira, a typical NOB, became the dominant genus as the salinity
20
increased. Some genus including Aridibacter, Phycisphaera, Gp3, Hoeflea and Ohtaekwangia became more abundant in the IMG with increased salinity. A large number of bacteria genus decreased including the Pseudomonas, Pseudoxanthomonas, Kofleria, Sphingomonas when the salinity increased to 35.0 g/L NaCl. Some genus even vanished at a low level of salinity (5.0 g/L NaCl), including Sphingorhabdus, Blastomonas and Arthrobacter. More unclassified genera (nearly 40% relative abundance) were found at high salinity levels (25.0 and 35.0 g/L NaCl) compared with less genus (only 20% relative abundance) at low salinity levels (0, 5.0 and 15.0 g/L NaCl). Some genus including Aridibacter, Phycisphaera, Gp3, Hoeflea and Ohtaekwangia became more abundant in the IMG with increased salinity. These genera were widely detected in the active sludge and biofilm system under fresh or saline/marine conditions (Babatsouli et al., 2015; Wang et al., 2016). Proliferation of halotolerant/halophilic microbes in IMG samples stabilized biological performance of MBBR in response to the rising salinity. 3.3.3 Response of nitrifying bacteria on salinity changes
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Based on the taxonomic information, the OTUs belonging to nitrifying bacteria and the relative abundances were listed in the Table 3. Of the total 2531 OTUs, 38 OTUs of AOB which were classified as Nitrosomonas (32 OTUs) and Nitrososphaera (6 OTUs); and 27 OTUs of NOB were classified as Nitrospira (20 OTUs) and Nitrolancea (5 OTUs). In addition, 2 OTUs were classified as genus Nitrobacter. The abundance of AOB and NOB responses differently to the salinity increase. When the salinity level increased to 5.0 g/L NaCl, the abundance of AOB decreased from 3.93% (near 0 g/L) to 1.46%. Similar trend was reported by other researchers (Wang et al., 2017), i.e., 5.0 g/L NaCl salinity resulting in the decrease of relative abundance of AOB from 10.50% (salinity of zero) to 3.40%. It is likely that the energy obtained from substrate degradation is required to resist the threats from the salt-stressed environment and maintain normal cell activities (Moussa et al., 2005). Through a period of acclimation, AOB in the system gradually adapted to the salty environment. After the salinity increased to 25.0 g/L NaCl, the relative abundance of AOB increased to 11.23%, nearly twice of that at zero salinity. At salinity of 35.0 g/L NaCl, the relative abundance of AOB continued to increase to 20.93%. These result was consistent with the report of
22
Ge et al. (2019), that the abundance of AOB in sludge sample under 3.0% salinity was 9.42%, nearly two times of these under salinity of zero. The inhibition effect of salt for AOB vary in the literatures. Mosquera-Corral et al. (2005) reported that ammonia oxidizing bacteria had a stable activity at a concentration up to 25 g/L NaCl. Corsino et al. (2016) found that the acclimated aerobic granules could adapt to hypersaline wastewater (50 g/L NaCl). In this study, the most abundant genus of AOB in all samples under different salinity levels was Nitrosomonas, which was found in fresh water, soil, and marine environments (Brown et al., 2013; Ramirez-Vargas et al., 2015). Many species such as “N. europaea” in this genus are reported to possess higher salt endurance compared with other AOB species (Wang, et al., 2017). The differences between the researchers is due to different reactor configurations, operational parameters, the way of introducing the salinity to the reactors and the bacteria existing in the system (Bassin et al., 2011; Gonzalez-Silva et al., 2016). The NOB sequences contributed only 2.13% at zero salinity (S1 or without NaCl addition). When the salinity increased to 5.0 g/L NaCl, the abundance of NOB increased rapidly to 28.33%., but when the salinity increased to 35.0 g/L NaCl, it
23
decreased to 8.28 %. Similar results were also reported by Ye et al. (2009), i.e. NOB could survive in the salty environment with decreased activity. Salinity had a significant negative effect on the abundance of NOB bacteria. Nitrospira was the dominant NOB genus in all samples under different salinity levels, which was reported as the major nitrite oxidizer in salt reactors for marine RAS (Keuter et al., 2011). Discrepancies in the microbial species involved may be the main reason for the different trends in activities and relative abundance (Wang et al., 2017). Nevertheless, these studies all proved that the presence of salt can change the NOB population.
4. Conclusions The novel Immobilized microbial granules (IMG) were characterized in response to the increased salinity up to 35.0 g/L NaCl in the MBBR in terms of nitrifying metabolic activities and microbial communities. It was found that ammonia was effectively removed and converted to nitrate and nitrite with conversion efficiency up to 92% with salinity of 35.0 g/L NaCl. Inhibition occurred for the conversion of nitrite to nitrate at the salinity above 15.0 to 25.0 g/L NaCl, resulting in the accumulation of
24
nitrite. The results also indicated that the microbial community structure was significantly impacted by the salinity change where AOB Nitrosomonas.sp and NOB Nitrospira sp were the dominant nitrifying genus at different salinity levels.
Acknowledgments This research is financially supported by Natural Science Foundation Council of China (No. 21808141), National Science and Technology Commission of Shanghai Municipality (No. 18230743000) and Startup Fund for Youngman Research at SJTU (17X100040069). We appreciate help and suggestions from Dr. Weimin Wu, Department of Civil & Environmental Engineering, Stanford University, USA and Dr. Haslenda Hashim, Faculty of Chemical & Energy Engineering, Universiti Teknologi Malaysia, Malaysia.
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Figure captions Fig.1 (a) Schematic diagram of MBBR with IMG; (b) Photo of IMG used in the MBBR.
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Fig.2 Operational results of bioreactor tests at different salinities. (a) ammonia concentrations, (b) nitrite and (c) nitrate. Fig.3 The metabolic activities of AOB and NOB of IMG. Fig.4 Venn diagram showing a total of 2531 OUT (a) and PCA plot (b) under different salinity levels (S1 to S5). Fig.5 Microbial communities of IMG varied as salinity increased. Taxonomic identification at (a) phylum; (b) class; and (c) genus at salinity levels of near zero (S1), 5.0 (S2), 15.0 (S3), 25.0 (S4) and 35.0 g/L (S5).
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Effluent
a
b IMG
Pump
Holding tank
Influent
Aerator
Fig.1 (a) Schematic diagram of MBBR with IMG; (b) Photo of IMG used in the MBBR.
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a
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Ⅰ
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NaCl (g/L)
1.5 20 1.0
Ammonia Nacl 10
0.5 0.0
0 0
14
28
42
56
70
84
98
112
Time (d)
Ⅰ
Nitrite (mg/L)
30
Ⅱ
Ⅳ
Ⅲ
Ⅴ
30
20
20 Nitrite NaCl 10
0
NaCl (g/L)
b
10
0 0
14
28
42
56
70
84
98
112
Time (d)
c 50
Ⅱ
Ⅰ
Ⅲ
Ⅴ
Ⅳ
30
20
30 Nitrate
20
NaCl (g/L)
Nitrate (mg/L)
40
10
NaCl 10 0
0 0
14
28
42
56
70
84
98
112
Time (d)
Fig. 2 Operational results of bioreactor tests at different salinities. (a) ammonia concentrations, (b) nitrite and (c) nitrate.
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AOB NOB
SOUR (mg O2/(L·h))
20
15
10
5
0
0
5.0
15.0
25.0
35.0
NaCl (g/L)
Fig. 3 The metabolic activities of AOB and NOB of IMG.
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a
b
A
B
Fig 4 Venn diagram showing a total of 2531 OUT (a) and PCA plot (b) under different salinity levels (S1 to S5).
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a Abundance of phylum(%)
A
100 90 80 70 60 50 40 30 20 10 0 S1
S2
S3
S4
S5
Sample ID
b Abundance of class(%)
B
100 90 80 70 60 50 40 30 20 10 0 S1
S2
S3
S4
S5
Sample ID
100
C
80
Abundance of genus (%)
c
Others Thaumarchaeota Deinococcus-Thermus Spirochaetes Crenarchaeota candidate division WPS-1 Candidatus Saccharibacteria Chlamydiae Euryarchaeota Armatimonadetes Verrucomicrobia Gemmatimonadetes Chloroflexi Firmicutes Actinobacteria unclassified Planctomycetes Acidobacteria Bacteroidetes Nitrospirae Proteobacteria
Others Thermoprotei Epsilonproteobacteria Bacteroidia Subdivision3 Clostridia Gemmatimonadetes Bacilli Anaerolineae Acidobacteria_Gp3 Actinobacteria Sphingobacteriia Deltaproteobacteria Phycisphaerae Planctomycetia Acidobacteria_Gp4 unclassified Gammaproteobacteria Cytophagia Betaproteobacteria Nitrospira Alphaproteobacteria
Others Noviherbaspirillum Blastomonas Labilithrix Sphingorhabdus Sphingomonas Ohtaekwangia Lacibacterium Hydrogenophaga Chryseolinea Gp3 Hyphomicrobium Aridibacter Pseudomonas Nitrospira
90
70 60 50 40 30 20 10
Arthrobacter Arcobacter Pedomicrobium Blastocatella Novosphingobium Gemmatimonas Kofleria Hoeflea Bradyrhizobium Pseudoxanthomonas Phycisphaera Piscinibacter Sphingopyxis Nitrosomonas unclassified
0 S1
S2
S3
S4
S5
Sample ID
Fig.5 Microbial communities of IMG varied as salinity increased. Taxonomic identification at (a) phylum; (b) class; and (c) genus at salinity levels of near zero (S1), 5.0 (S2), 15.0 (S3), 25.0 (S4) and 35.0 g/L (S5).
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42
Table 1 Operational phases of different salinity levels
Phase
Salinity /(g/L)
Sample ID
Period (d)
Ⅰ
Near zero
S1
0-14
Ⅱ
5.0
S2
15-28
Ⅲ
15.0
S3
29-49
Ⅳ
25.0
S4
50-77
Ⅴ
35.0
S5
78-112
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Table 2 Sequences of the five IMG samples based on 0.03 distances ID (g/L of
Sequence
OTU
Shannon
ACE
Chao1
Coverage
Simpson
salinity)
number
number
index
index
Index
(%)
number
S1 (0)
20,098
1445
5.36
1637.35 1532.31
98.59
0.017
S2 (5)
38,868
1013
4.04
1291.82 1230.88
99.26
0.088
S3(15)
38,324
1103
4.54
1327.28 1278.05
99.29
0.050
S4(25)
29,741
916
4.02
1275.52 1219.34
98.94
0.071
S5(35)
24,496
873
3.71
1232.28 1167.14
98.72
0.099
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Table 3 The relative abundances of AOB and NOB at salinity levels of zero (S1), 5.0 (S2), 15.0 (S3), 25.0 (S4), and 35.0 g/L (S5)
AOB
NOB
Genus
S1
S2
S3
S4
S5
Nitrosomonas
3.75
1.43
1.94
11.23
20.93
Nitrosopumilus
0.06
0.03
0.01
-
-
Nitrososphaera
0.12
-
-
-
-
Nitrospira
2.05
28.26
20.86
9.40
8.24
Nitrolancea
0.07
0.07
0.08
0.08
0.04
Nitrobacter
0.01
-
-
-
-
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Highlights
Immobilized microbial granules improved salinity tolerance.
High ammonia removal (92 %) was achieved at salinity up to 35.0 g/L.
Nitrite oxidation bacteria was depressed at salinity above 15.0 g/L.
Microbial community structure was shifted as salinity increased.
Nitrosomonas sp. and Nitrospira sp. were the dominant genera.
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Author Contributions Section Gao Yueshu: Conceptualization, Methodolog, Writing- Original draft preparation Wang Xupeng: Investigation, Visualization Li Jialun: Investigation, Software Lee Chew Tin: Visualization, Reviewing and Editing Ong Pei Ying: Software, Validation Zhang Zhenjia: Supervision, Resource Li Chunjie: Writing- Reviewing and Editing, Project administration
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