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Robustness of an aerobic granular sludge sequencing batch reactor for low strength and salinity wastewater treatment at ambient to winter temperatures
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Robustness of an aerobic granular sludge sequencing batch reactor for low strength and salinity wastewater treatment at ambient to winter temperatures Qiulai Hea, , Hongyu Wangb, Li Chenc, Shuxian Gaod, Wei Zhangb, Jianyang Songb, Jian Yua ⁎
a
Key Laboratory of Building Safety and Energy Efficiency, Ministry of Education, Department of Water Engineering and Science, College of Civil Engineering, Hunan University, Changsha, 410082, China b School of Civil Engineering, Wuhan University, Wuhan, 430072, China c State Key Laboratory of Pollution Control and Resources Reuse, College of Environmental Science and Engineering, Tongji University, Shanghai, 200092, China d CAS Key Laboratory of Urban Pollutant Conversion, Department of Applied Chemistry, University of Science and Technology of China, Hefei 230026, China
GRAPHICAL ABSTRACT
ARTICLE INFO
ABSTRACT
Editor: R Teresa
Acclimation performances and characteristics of aerobic granular sludge to salt and temperature were investigated in a sequencing batch reactor (SBR) performing simultaneous nitrification, denitrification and phosphorus removal (SNDPR). The aerobic granular SNDPR system was firstly subjected to low salinity (0.5%, w/v) at moderate temperature (> 15 ℃) and subsequent low temperature (< 15 ℃). The shock loading of salinity temporarily deteriorated biological phosphorus removal, while dual stresses of salinity and low temperature induced temporary inhibition on both nitrogen and phosphorus removal. Both salinity and low temperature stimulated the settleability of aerobic granules, accompanied with decreased ratios of protein to polysaccharide (PN/PS). Illumina MiSeq sequencing revealed that salinity rarely affected bacterial richness, but significantly decreased the diversity. Whereas low temperature strengthened both bacterial richness and diversity. Phyla Proteobacteria, Chloroflexi and their sub-groups acted as the main halophilic bacteria while Proteobacteria was also psychrophilic. The functional bacteria such as nitrifiers, denitrifiers, and phosphorus removal bacteria exhibited greater tolerance to salt and low temperature than glycogen accumulating organisms (GAOs). Overall, the present study demonstrated the resilience and robustness of aerobic granular sludge toward salinity and low temperature, which could aid the knowledge of saline wastewater treatment by aerobic granular sludge.
Keywords: Aerobic granular sludge Nitrogen and phosphorus removal Low strength and salinity Low temperature Illumina MiSeq sequencing
⁎
Corresponding author. E-mail address: [email protected] (Q. He).
https://doi.org/10.1016/j.jhazmat.2019.121454 Received 22 August 2019; Received in revised form 9 October 2019; Accepted 9 October 2019 0304-3894/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Qiulai He, et al., Journal of Hazardous Materials, https://doi.org/10.1016/j.jhazmat.2019.121454
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1. Introduction
simultaneous nitrification, denitrification and phosphorus removal (SNDPR) was configured to treat saline wastewater (0.5%, w/v) from ambient to low temperatures. Comprehensive comparisons were explored in terms of characteristics of granules, process performance as well as the microbial population dynamics.
The superior advantages of aerobic granular sludge such as much better settleability, smaller footprint, lower energy consumption and higher biomass retention over conventional activated sludge make it an attractive alternative for future wastewater treatment (Zhang et al., 2016; Sarma et al., 2017; Sarma and Tay, 2018). However, the longterm stability of aerobic granules is of primary concern since instable episodes directly hinder the application of this technology (Sarma and Tay, 2018). Multiple factors like overloading, unbalanced substrates, inappropriate operational configurations, intrusion of stressing compounds are responsible for granular instability (Zheng et al., 2006; Sguanci et al., 2019; Yuan et al., 2017). It is of vital importance to maintain the aerobic granular integrity in order to keep the superiorities of this promising technology (Franca et al., 2018). Two important parameters with negative effects on aerobic granular sludge performance and stability are salt addition and low wastewater temperature (Remy et al., 2011; Ramos et al., 2015; Bao et al., 2009). Salinity stress has been reported to be one of the main causative parameters for failure in biological nutrients removal since it may pose detrimental effects on sludge aggregation and bioactivity (Pronk et al., 2014; Zhang and Huang, 2011; Lujan-Facundo et al., 2018). Aerobic granules could tolerate and acclimate to saline conditions at certain concentrations (He et al., 2017a; Hou et al., 2019; Sudmalis et al., 2018; Wang et al., 2017a). Multiple works have been conducted to evaluate the main and side effects of salinity on aerobic granules (Pronk et al., 2014; He et al., 2017a; Chen et al., 2018), which were mainly focused on the biological nutrients removal and physiochemical characteristics dynamics. Whereas, it is interestingly reported that certain concentrations of salt could eventually enhance the granular performance such as nutrients removal and settleability (Ou et al., 2018). However, comprehensive and systematic understanding on the acclimation of mature aerobic granular sludge to saline wastewater was still necessary in terms of other aspects such as the structural integrity and the biological aspects, since it might specify the direction of applying aerobic granular sludge for municipal wastewater intruding by industrial wastewater or totally industrial wastewater treatment (Wang et al., 2017b; van den Akker et al., 2015). Therefore, inner understanding on the responses of structural and functional stability of aerobic granular sludge to salinity stress have to be improved. Temperature as one of the main operational parameters largely affects the long-term stability of aerobic granules especially on the microorganisms (Fontenot et al., 2007; Rebolledo et al., 2018). Improper temperature such as winter temperature was always accompanied with poor process performance and granular integrity (Munoz-Palazon et al., 2018; Rodriguez-Sanchez et al., 2019; de Kreuk et al., 2005). Despite numerous studies have revealed the effects of temperature especially moderate temperature, the long-term effects of winter temperatures still lack enough information (Winkler et al., 2012; Bassin et al., 2012). Previous works have summarized that gradual acclimation to low wastewater temperatures could gain better performance than shock or direct stress (Munoz-Palazon et al., 2018). Besides, cold-adapted sludge could tolerate low working temperatures (Gonzalez-Martinez et al., 2017). It is of common situations for the aerobic granules to be suffered from both saline and low operational temperatures especially during the winter conditions. For the wastewater treatment plants, operational durations from the moderate temperatures to winter temperatures occurred yearly. Therefore, the responses of aerobic granules treating saline wastewater should also be considered at low temperatures, when both salinity and low temperature posed stress. However, to the best of our knowledge, limited studies have focused on the acclimation of aerobic granules to simultaneous saline and low temperature conditions. Therefore, the main objective of the present work was to evaluate the effects of salt addition and temperature on aerobic granules. An aerobic granular sequencing batch reactor (SBR) performing
2. Materials and methods 2.1. Experimental setup and operation The plexiglass reactor was with an inner diameter of 100 mm and a total height of 500 mm, giving a working volume of 3.6 L. Air was introduced into the reactor from the right bottom of the reactor at a constant aeration rate of 200 mL/min, corresponding to the superficial gas velocity (SGV) of 0.11 cm/s (He et al., 2017b). Influent was pumped into the reactor from top of the reactor at the beginning of each cycle after equal amount of effluent was discharged from the middle height of the effective height, making an exchange ratio of 50%. A mechanical stirrer was fixed with a constant speed of 150 rpm. The reactor was run in a 6-h cycle including feeding (2 min), anaerobic reaction (120 min), oxic reaction (90 min), anoxic reaction (144 min), settling (2 min) and discharge (2 min), respectively. The reactor was run continuously and automatically by a programable logic controller (PLC) targeting influent and effluent pumps, aeration pump and stirrers, respectively. The hydraulic retention time was about 12 h ignoring the time for feeding, settling and discharge. No manual sludge disposal was conducted during the whole operation of the reactor with the observed sludge retention time (SRT) of about 25days (He et al., 2020). The reactor was placed near the window in the indoor location, where was vulnerable to dynamics of seasonal temperatures. Based on the water temperature, the whole operation could be divided into two separate phases, namely phase Ⅰ from day 1 to 49, and phase Ⅱ from day 50 to 75. The water temperature of 15 ℃ was utilized as the watershed for separating ambient and low temperatures (de Kreuk et al., 2005; Chu et al., 2005). 2.2. Synthetic wastewater The SBR was initially seeded with mature aerobic granules from the previous work by He et al. (2017b). Synthetic wastewater was used as the influent in present study, which was pumped into the reactor at the beginning of each cycle. The compositions of the synthetic wastewater were listed in Table 1. Detailed components of the trace solution could be referenced to He et al. (2018a). The low concentrations of COD and nutrients were used to simulate the low-strength domestic wastewater in China especially south China (Ni et al., 2009). The salinity level of 0.5% (w/v) was applied through adding 50 g of commercially purchased NaCl into 1 L synthetic wastewater as described above. The pH of the wastewater was adjusted to around 7.5 using HCl or NaOH solutions. Table 1 Compositions of synthetic wastewater used in the present work.
2
Main compositions
Concentration (mg/L)
Trace compositions
Concentration (mg/L)
CH3COONa NH4Cl KH2PO4 CaCl2 MgSO4·7H2O
256 76.5 14.6 10 10
FeCl3 H3BO3 KI CuSO4∙5H2O MnCl2∙4H2O ZnSO4∙7H2O CoCl2∙6H2O Na2MoO4∙2H2O EDTA
0.9 0.15 0.18 0.03 0.06 0.12 0.15 0.06 10
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2.3. Analytical methods In this experiment, the concentrations of chemical oxygen demand (COD), ammonia nitrogen (NH4+-N), nitrite (NO2−-N) and nitrate (NO3−-N), total phosphorus (TP) of the influents and effluents, mixed liquor suspended solids (MLSS) and mixed liquor volatile suspended solids (MLVSS), sludge volume index at 2 min (SVI2) were measured according to the standard method (APHA, 2005). The pH and dissolved oxygen (DO) in the bulk solution were measured using a pHS-25 m (pHS-25, Shanghai Leici Instrument Factory, China) and YSI5000 m (YSI, Yellow Springs, Ohio, USA), respectively. Aerobic granules were collected at the end of each phase and pretreated with 0.9% NaCl solution and subsequent centrifugation at 4000 rpm for 5 min to separate the solids and supernatant for three times. The residual pellet was suspended with 0.9% NaCl solution again and then heated under 80 ℃ for 30 min. The supernatant after centrifugation at 10,000 rpm for 20 min was filtered through 0.45 μm water filter membrane for further analysis. The Bradford method and sulfuric acid-anthrone colorimetric method were utilized for PN and PS quantifications, respectively. EPS were referred to the sum to PN and PS (He et al., 2018b). Three-dimensional excitation and emission matrix (3D-EEM) analysis of EPS extracts was conducted according to previous procedures by He et al. (2018b) using a luminescence spectrometry FS5 Spectrophotometer (Edinburgh, UK). The corresponding scanning emission spectra from 200 to 500 nm and excitation wavelengths from 200 to 400 nm at 5 nm intervals were used. The excitation and emission slits were kept at 10 nm with a constant scanning speed of 1200 nm/min. Deionized Milli-Q water was excluded from the spectrum of EPS samples. The elliptical shape of contours was present as the final results of 3D-EEM spectra.
Fig. 1. Biomass concentrations and settleability of aerobic granules in different phases: the data in day 1 are of granules without salt addition at ambient temperature; data in day 18 and 49 are of granules with 0.5% salinity at ambient temperature; data in day 55 and 66 are of granules with 0.5% salinity at winter temperature.
Though under the salinity stress, there witnessed a significant enrichment of biomass (MLSS and MLVSS) (p < 0.05) with similar MLVSS/ MLSS (∼0.9) (p > 0.05) in contrast to the initially seeding sludge. However, both MLSS and MLVSS decreased slightly under low temperature (with 0.5%, w/v) (p > 0.05). Therefore, the combination of salinity and low temperature posed an adverse effect on biomass accumulation, while individual salinity had the positive influence. Similar effect was also seen toward the settleability. Since SVI2 decreased with 0.5% salinity, but slightly increased under synergistic effects of salinity and low temperature. Salinity, however, improved the settleability of activated sludge (Chen et al., 2018) and granular sludge (Pronk et al., 2014) at much wider ranges up to 20 g/L. Therefore, the aerobic granular sludge showed its robustness toward salinity and low temperature in terms of biomass concentrations and the settleability.
2.4. Biological analysis Aerobic granular samples were taken from the reactor at the end of each phase, namely the seeded granular samples S0, samples at the end of phases Ⅰ and Ⅱ (denoted as S1-2). The biomass samples were pretreated and stored for further DNA extraction. Biological experiments including DNA extraction, PCR amplification and subsequent sequencing were conducted according to the manufactures’ protocols. Hypervariable V3-4 regions of the 16S rRNA gene of the bacteria were amplified with the forward primer 338 F (5’-ACTCCTACGGGAGGCA GCA-3’) and reverse primer 806R (5’-GGACTACHVGGGTWTCTAAT-3’). The Illumina MiSeq apparatus (Illumina, Hayward, CA, USA) were applied for the bacterial community analysis by Shanghai MajorBio BioPharm Technology Co., Ltd, China. Data collection and statistical analysis were carried out according to previous researches (He et al., 2020, 2019) using the online platform of MajorBio Cloud Platform. The original gene sequences are available at NCBI Sequence Read Archive (SRA, http://www.ncbi.nlm.nih.gov/sra/) with a newly established BioProject ID PRJNA574902.
3.2. Content of EPS Profiles of EPS production over operation were shown in Fig. 2. It was elucidated that the introduction of salinity resulted in significant decrease in both PN and PS, as well as constant decrease of PN/PS (p < 0.05). However, with the decrease of temperature, contents of PN and PS raised sharply and even exceeded the initial levels, though still with low PN/PS values. It was noted that PS rather than PN varied significantly during the whole operation periods, implying the adjusting roles of PS in response to salinity and low temperature stresses. Chen et al. (Chen and Murata, 2002) also reported that the accumulation of compatible solutes including PS was a widespread response against environmental stress. Besides, the constant decrease of PN/PS indicated increase of cell hydrophobicity, protecting the aerobic granules from the strongly stressing conditions (He et al., 2017a).
2.5. Statistical analysis The results of this study were shown in the form of mean value ± standard error. To statistically compare the data of the experiment, an analysis of variance (ANOVA) at the significance level of p < 0.05 was adopted using the GraphPad Prism software (Version 5.0.3, Graphpad Software Inc., San Diego, CA, USA) (He et al., 2019). 3. Results and discussion 3.1. Biomass retentions and settleability The biomass accumulation of aerobic granular SNDPR system and the changes of settleability in terms of SVI2 were shown in Fig. 1.
Fig. 2. Profiles of EPS within aerobic granules at different phases. 3
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3.3. 3D-EEM spectra
3.5. Microbial diversity
3D-EEM spectra was utilized to provide information on the compositional changes in EPS subject to salinity and temperature variations. As shown in Fig. 3, it could be obviously told that three separate peaks A, B and C could be identified in three aerobic granular samples. According to He et al. (2018b), Peak A was assigned to tryptophan & protein-like substances, while Peaks B and C were identified as humic acid-like substances. Therefore, it could be concluded that apart from the protective role of PS as discussed above, other components within EPS including tryptophan & protein-like and humic acid-like substances also responded and contributed to aerobic granular stability and performance. Further parameters of peak from the 3D-EEM were given in Table 2. Individual salinity stress and synergistic effects of salinity and low temperature witnessed variations of peak locations and specific intensities, suggesting the responsive actions of EPS toward stress conditions (Su et al., 2019). It should be noticed that the peak intensity ratios of EPS sample from the individual salinity stress was much different with others, implying that salinity might be the primary factor shaping the fluorescence components of EPS (He et al., 2018b). It could be concluded that both contents and compositions of EPS changed in response to stresses induced by salinity and low temperature, which might be the main contributor for structural stability of aerobic granules.
Three aerobic granular samples collected within the reactor with salinity-free, 0.5% salinity at moderate temperature, and 0.5% at low temperature (denoted S0-2, respectively) were analyzed using Illumina MiSeq sequencing. The alpha-diversity results were shown in Fig. 5. The dosage of salinity rarely changed the bacterial richness in terms of similar Ace and Chao estimators (p > 0.05). However, decrease in temperature along with salinity of 0.5% significantly enhanced the bacterial richness (p < 0.05). The consistent orders of diversity indices Shannon (S2 > S0 > S1) and Simpson (S2 < S0 < S1) implied that salinity only decreased the bacterial diversity, while combined stress of salinity and low temperature led to highest diversity. The rarefaction curves indicated that three samples held similar number of sequences (∼30,000) and the curves all tended to plateau, suggesting that enough data were collected for sequencing. Similar evenness of three samples were observed from the rank-abundance curves and the serval core microbes possessed much proportions (He et al., 2017b). Venn diagram revealed that the three samples shared a high percent of OTUs. Besides, less unique OTUs were held with the gradual stresses of salinity and low temperature. 3.6. Microbial compositions Major phyla and classes with an abundance over 1% were shown in Fig. 6A–B, while the top 50 genera were collected and mapped in heatmap in Fig. 6C. Proteobacteria were the most abundant phylum under salinity-free condition, accounting for 35.76%. 0.5% salinity stress shifted the predominant phylum to Chloroflexi (41.72%). Interestingly, Proteobacteria recovered to the highest relative abundance of 54.22% under dual stresses of salinity and low temperature. It could be indicated that Proteobacteria and Chloroflexi were halophilic bacteria, which the former was psychrophilic (Ou et al., 2018). Other predominant phyla with dramatically varying relative abundances including Bacteroidetes (8.08, 7.18, and 19.32%), Parcubacteria (20.70, 0.19, and 0.02%), Gracilibacteria (11.14, 0.08, and 0.01%), Chlorobi (0.20, 3.84, and 1.26), Planctomycetes (2.91, 1.35, and 0.87), Nitrospirae (0.73, 1.33, and 2.03), Acidobacteria (0.51, 1.77, and 0.91), and Verrucomicrobia (0.28, 1.02, and 0.91) within reactor under the salinityfree at moderate temperature, 0.5% salinity at moderate temperature, and 0.5% salinity at low temperature conditions, respectively. Concludingly, only the halophilic and psychrophilic bacteria could survive the hypersaline and low temperature conditions. The sub-groups of Proteobacteria including Gamma-, Beta-, Alpha- and Delta-proteobacteria classes occupied large proportions and were the dominant microorganisms. Other abundant classes within the above predominant phyla distributed diversely within the reactor. Gamma-, Beta- and Alphaproteobacteria and Anaerolineae were the typical halophilic classes, while Alphaproteobacteria and Anaerolineae were susceptible to low temperature and decayed under low temperature. Candidatus_Moranbacteria from the phylum Parcubacteria were sensitive to salinity, which dropped from 20.48% to nearly undetectable levels under saline
3.4. Process performance Variations of nutrients removal performance along operation were elucidated in Fig. 4, as well as the dynamics of water temperatures. Reliable COD removal rates over 90% were obtained under salinity at ambient temperature, and low temperature even stimulated COD removal efficiencies. Superb removal performance toward NH4+-N and TIN could be achieved over operation, though low temperature shock led to temporary decrease in NH4+-N removal. This could be indicated that nitrification process was more vulnerable to stresses by salinity and low temperature, while denitrification remained unaffected. Sudden dosage of 0.5% salinity at ambient temperature deteriorated TP removal during the first several days, which could be soon recovered afterward. After the acclimation to salinity stress, TP removal rates only decreased slightly when the temperature dropped from ambient to low levels. Although both nitrogen and phosphorus removal could be influenced by salinity and low temperature, reliable performances could be eventually obtained after different durations of acclimation. In addition, the acclimation process under salinity stress even strengthened the robustness of the aerobic granular SNDPR toward the combination of salinity and low temperature, especially for the enhanced biological phosphorus removal (EBPR). Therefore, much shorter durations (5 days vs 15 days for TP removal as shown in Fig. 4D) for lower removal rates could be observed in response to salinity at low temperature than at moderate temperature.
Fig. 3. 3D-EEM spectra of EPS extracted from aerobic granules: A, aerobic granular sludge at ambient temperature without salt addition (day 1); B, aerobic granules at ambient temperature with 0.5% salinity (day 49); C, aerobic granules at winter temperature with 0.5% salinity (day 66). 4
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Table 2 Fluorescence spectral parameters of peaks identified from three aerobic granular samples S0-2 by 3D-EEM spectra. Samples
S0 S1 S2
Peak A
Peak B
Peak C
Peak intensity ratios
Ex/Em
Intensity
Ex/Em
Intensity
Ex/Em
Intensity
A/B
A/C
B/C
270/350 280/355 280/355
7424.38 7424.38 12068.79
270/440 270/435 270/440
4093.43 9515.64 6921.92
345/445 350/445 350/440
1821.74 4177.07 3152.36
1.81 0.78 1.74
4.08 1.78 3.83
2.25 2.28 2.20
conditions at moderate and low temperatures. The most dominant genera shifted with the operational conditions. Candidatus_Competibacter (24.18%), norank_f__Anaerolineaceae (37.61%) and Acinetobacter (20.78%) held the highest relative abundances under salinity-free, 0.5% salinity, and 0.5% salinity at low temperature conditions, suggesting their vital roles under different stressing circumstances. norank_c__Candidatus_Moranbacteria dropped from 20.48% to 0.01% or even lower under hypersaline conditions, implying it was sensitive and vulnerable to salt. Conversely, Thauera increased from 0.10% to 11.69%, which could be both halophilic and psychrophilic. Other predominant genera included Azospira, Nitrospira, Dechloromonas, and Hydrogenophaga, which varied frequently in relative abundances over operation. The individual effect of salinity or dual effects of
salinity and low temperature largely shaped the microbial compositions at various levels. 3.7. Susceptibility of functional groups The mainly functional groups within the aerobic granular SNDPR system included those involved in nitrogen and phosphorus removal, such as nitrifiers (including ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB)), dentrifiers (DNB), phosphorus accumulating organisms (PAOs), and denitrifying PAOs (DNPAOs). Glycogen accumulating organisms (GAOs) were always taken into consideration since they were the primary competitors for carbon source with PAOs/DNPAOs and DNB, which largely affected the
Fig. 4. Process performance during different phases for the aerobic granular SNDPR system: A, COD removal; B, NH4+-N removal; C, TIN removal; D, TP removal and E, profile of temperature. 5
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Fig. 5. Microbial diversity based on the OTU classifications on the three separate aerobic granular samples S0-2: A, diversity estimators; B, rarefaction curves; C, rank-abundance curves; and D, Venn diagram.
nitrogen and phosphorus removal efficiencies. The list of the above functional bacteria was shown in Fig. 7. Generally, salinity and temperature severely changed the functional bacteria in terms of the compositions and relative abundances. Two major AOB including Nitrosomonas (0.02-0.04%) and
norank_f_Nitrosomonadaceae (0.01-0.07%) were detected within different conditions, leading to a slight increase in the sum of abundances. However, NOB Nitrospira grew constantly from 0.73 to 2.03% with salinity and subsequent low temperature, suggesting that NOB might be more tolerant to salinity and low temperature. The cooperation of AOB
Fig. 6. Microbial structure of aerobic granular samples S0-2 at A, phylum level; B, class level; and C, genus level. The heatmap was drawn with relative abundances (%). 6
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Fig. 7. Relative abundances of functional groups: A, AOB, NOB; B, GAOs, PAOs and DNPAOs; C, DNB in three separate samples S0-2. The heatmap was drawn with the logarithmic values of relative abundances (%) and the excluded values from the color bar were in white color.
the major halophilic bacteria, while Proteobacteria was also psychrophilic. Functional groups involved in nitrogen and phosphorus removal demonstrated various tolerance capacities toward salinity and temperature and the superiority of such as NOB, DNB, PAOs, DNPAOs, and AOB over GAOs under the hard environment was the biological basis for robust reactor performance.
and NOB ensured excellent nitrifying efficiency. Multiple bacteria capable of denitrifying pathway were found with Acinetobacter (0.31–20.78%), Thauera (0.10–11.69%) being the predominant ones (He et al., 2019), which got significantly accumulated with salinity and low temperature. The various DNB laid the biological basis for reliable denitrification rate. Two kinds of GAOs Candidatus_Competibacter (24.18-1.92%) and Defluviicoccus (0.58-0.18%) were detected and found decreased sharply with individual or dual stressing conditions. In contrast, PAOs (1.00–3.71%) and DNPAOs (0.40–2.48%) enriched by times simultaneously. This implied that PAOs/DNPAOs offered stronger competitiveness and capacities for adaption than GAOs. Wang et al. (2017a) reported contrary conclusion in much higher salinity concentrations. Therefore, the tolerance toward salinity might also be dosage-dependent. In addition, the decay of GAOs and increase of PAOs/ DNPAOs and DNB was favorable for nitrogen and phosphorus removal (He et al., 2017b).
Declaration of Competing Interest The authors declare that there are no conflicts of interest. Acknowledgement This work was finally supported by the Fundamental Research Funds for the Central Universities (No. 531118010401), the Key Research and Development Program of Hunan Province (No. 2019SK2111), and the National Natural Science Foundation of China (NSFC, China) (No. 51878517).
4. Conclusions
References
Individual stress by 0.5% salinity and dual effects of 0.5% salinity and low temperature below 15 ℃ were posed to an aerobic granular SNDPR system. Aerobic granular sludge offered capacity for withstanding stressing conditions in terms of granular stability and nutrients removal performance. Salinity temporarily deteriorated EBPR while strengthened the adaptability toward subsequent hypersaline and low temperature conditions. Nitrification was slightly affected by dual stresses and could recover soon to reliable levels. Both contents and compositions of EPS altered in response to salinity and temperature, which was the main mechanism for the structural stability of aerobic granules. Proteobacteria and Chloroflexi phyla and their sub-groups were
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removal performance and activated sludge characteristics in sequencing batch reactors. Bioresour. Technol. 249, 890–899. Chu, L.B., Yang, F.L., Zhang, X.W., 2005. Anaerobic treatment of domestic wastewater in a membrane-coupled expended granular sludge bed (EGSB) reactor under moderate to low temperature. Process Biochem. 40, 1063–1070. de Kreuk, M.K., Pronk, M., van Loosdrecht, M.C., 2005. Formation of aerobic granules and conversion processes in an aerobic granular sludge reactor at moderate and low temperatures. Water Res. 39, 4476–4484. Fontenot, Q., Bonvillain, C., Kilgen, M., Boopathy, R., 2007. Effects of temperature, salinity, and carbon: nitrogen ratio on sequencing batch reactor treating shrimp aquaculture wastewater. Bioresour. Technol. 98, 1700–1703. Franca, R.D.G., Pinheiro, H.M., van Loosdrecht, M.C.M., Lourenco, N.D., 2018. Stability of aerobic granules during long-term bioreactor operation. Biotechnol. Adv. 36, 228–246. Gonzalez-Martinez, A., Munoz-Palazon, B., Rodriguez-Sanchez, A., Maza-Marquez, P., Mikola, A., Gonzalez-Lopez, J., Vahala, R., 2017. Start-up and operation of an aerobic granular sludge system under low working temperature inoculated with cold-adapted activated sludge from Finland. Bioresour. Technol. 239, 180–189. He, H., Chen, Y., Li, X., Cheng, Y., Yang, C., Zeng, G., 2017a. Influence of salinity on microorganisms in activated sludge processes: a review. Int. Biodeter. Biodegrad. 119, 520–527. He, Q., Zhang, W., Zhang, S., Wang, H., 2017b. Enhanced nitrogen removal in an aerobic granular sequencing batch reactor performing simultaneous nitrification, endogenous denitrification and phosphorus removal with low superficial gas velocity. Chem. Eng. J. 326, 1223–1231. He, Q., Chen, L., Zhang, S., Chen, R., Wang, H., Zhang, W., Song, J., 2018a. Natural sunlight induced rapid formation of water-born algal-bacterial granules in an aerobic bacterial granular photo-sequencing batch reactor. J. Hazard. Mater. 359, 222–230. He, Q., Song, Q., Zhang, S., Zhang, W., Wang, H., 2018b. Simultaneous nitrification, denitrification and phosphorus removal in an aerobic granular sequencing batch reactor with mixed carbon sources: reactor performance, extracellular polymeric substances and microbial successions. Chem. Eng. J. 331, 841–849. He, Q., Zhang, J., Gao, S., Chen, L., Lyu, W., Zhang, W., Song, J., Hu, X., Chen, R., Wang, H., Yu, J., 2019. A comprehensive comparison between non-bulking and bulking aerobic granular sludge in microbial communities. Bioresour. Technol. 294, 122151. He, Q., Song, J., Zhang, W., Gao, S., Wang, H., Yu, J., 2020. Enhanced simultaneous nitrification, denitrification and phosphorus removal through mixed carbon source by aerobic granular sludge. J. Hazard. Mater. 382, 121043. Hou, M., Li, W., Li, H., Li, C., Wu, X., Liu, Y., 2019. Performance and bacterial characteristics of aerobic granular sludge in response to alternating salinity. Int. Biodeter. Biodegrad. 142, 211–217. Lujan-Facundo, M.J., Fernandez-Navarro, J., Alonso-Molina, J.L., Amoros-Munoz, I., Moreno, Y., Mendoza-Roca, J.A., Pastor-Alcaniz, L., 2018. The role of salinity on the changes of the biomass characteristics and on the performance of an OMBR treating tannery wastewater. Water Res. 142, 129–137. Munoz-Palazon, B., Pesciaroli, C., Rodriguez-Sanchez, A., Gonzalez-Lopez, J., GonzalezMartinez, A., 2018. Pollutants degradation performance and microbial community structure of aerobic granular sludge systems using inoculums adapted at mild and low temperature. Chemosphere 204, 431–441. Ni, B.J., Xie, W.M., Liu, S.G., Yu, H.Q., Wang, Y.Z., Wang, G., Dai, X.L., 2009. Granulation of activated sludge in a pilot-scale sequencing batch reactor for the treatment of lowstrength municipal wastewater. Water Res. 43, 751–761. Ou, D., Li, W., Li, H., Wu, X., Li, C., Zhuge, Y., Liu, Y.D., 2018. Enhancement of the
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Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
Robustness of an aerobic granular sludge sequencing batch reactor for low strength and salinity wastewater treatment at ambient to winter temperatures Qiulai Hea, , Hongyu Wangb, Li Chenc, Shuxian Gaod, Wei Zhangb, Jianyang Songb, Jian Yua ⁎
a
Key Laboratory of Building Safety and Energy Efficiency, Ministry of Education, Department of Water Engineering and Science, College of Civil Engineering, Hunan University, Changsha, 410082, China b School of Civil Engineering, Wuhan University, Wuhan, 430072, China c State Key Laboratory of Pollution Control and Resources Reuse, College of Environmental Science and Engineering, Tongji University, Shanghai, 200092, China d CAS Key Laboratory of Urban Pollutant Conversion, Department of Applied Chemistry, University of Science and Technology of China, Hefei 230026, China
GRAPHICAL ABSTRACT
ARTICLE INFO
ABSTRACT
Editor: R Teresa
Acclimation performances and characteristics of aerobic granular sludge to salt and temperature were investigated in a sequencing batch reactor (SBR) performing simultaneous nitrification, denitrification and phosphorus removal (SNDPR). The aerobic granular SNDPR system was firstly subjected to low salinity (0.5%, w/v) at moderate temperature (> 15 ℃) and subsequent low temperature (< 15 ℃). The shock loading of salinity temporarily deteriorated biological phosphorus removal, while dual stresses of salinity and low temperature induced temporary inhibition on both nitrogen and phosphorus removal. Both salinity and low temperature stimulated the settleability of aerobic granules, accompanied with decreased ratios of protein to polysaccharide (PN/PS). Illumina MiSeq sequencing revealed that salinity rarely affected bacterial richness, but significantly decreased the diversity. Whereas low temperature strengthened both bacterial richness and diversity. Phyla Proteobacteria, Chloroflexi and their sub-groups acted as the main halophilic bacteria while Proteobacteria was also psychrophilic. The functional bacteria such as nitrifiers, denitrifiers, and phosphorus removal bacteria exhibited greater tolerance to salt and low temperature than glycogen accumulating organisms (GAOs). Overall, the present study demonstrated the resilience and robustness of aerobic granular sludge toward salinity and low temperature, which could aid the knowledge of saline wastewater treatment by aerobic granular sludge.
Keywords: Aerobic granular sludge Nitrogen and phosphorus removal Low strength and salinity Low temperature Illumina MiSeq sequencing
⁎
Corresponding author. E-mail address: [email protected] (Q. He).
https://doi.org/10.1016/j.jhazmat.2019.121454 Received 22 August 2019; Received in revised form 9 October 2019; Accepted 9 October 2019 0304-3894/ © 2019 Elsevier B.V. All rights reserved.
Please cite this article as: Qiulai He, et al., Journal of Hazardous Materials, https://doi.org/10.1016/j.jhazmat.2019.121454
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1. Introduction
simultaneous nitrification, denitrification and phosphorus removal (SNDPR) was configured to treat saline wastewater (0.5%, w/v) from ambient to low temperatures. Comprehensive comparisons were explored in terms of characteristics of granules, process performance as well as the microbial population dynamics.
The superior advantages of aerobic granular sludge such as much better settleability, smaller footprint, lower energy consumption and higher biomass retention over conventional activated sludge make it an attractive alternative for future wastewater treatment (Zhang et al., 2016; Sarma et al., 2017; Sarma and Tay, 2018). However, the longterm stability of aerobic granules is of primary concern since instable episodes directly hinder the application of this technology (Sarma and Tay, 2018). Multiple factors like overloading, unbalanced substrates, inappropriate operational configurations, intrusion of stressing compounds are responsible for granular instability (Zheng et al., 2006; Sguanci et al., 2019; Yuan et al., 2017). It is of vital importance to maintain the aerobic granular integrity in order to keep the superiorities of this promising technology (Franca et al., 2018). Two important parameters with negative effects on aerobic granular sludge performance and stability are salt addition and low wastewater temperature (Remy et al., 2011; Ramos et al., 2015; Bao et al., 2009). Salinity stress has been reported to be one of the main causative parameters for failure in biological nutrients removal since it may pose detrimental effects on sludge aggregation and bioactivity (Pronk et al., 2014; Zhang and Huang, 2011; Lujan-Facundo et al., 2018). Aerobic granules could tolerate and acclimate to saline conditions at certain concentrations (He et al., 2017a; Hou et al., 2019; Sudmalis et al., 2018; Wang et al., 2017a). Multiple works have been conducted to evaluate the main and side effects of salinity on aerobic granules (Pronk et al., 2014; He et al., 2017a; Chen et al., 2018), which were mainly focused on the biological nutrients removal and physiochemical characteristics dynamics. Whereas, it is interestingly reported that certain concentrations of salt could eventually enhance the granular performance such as nutrients removal and settleability (Ou et al., 2018). However, comprehensive and systematic understanding on the acclimation of mature aerobic granular sludge to saline wastewater was still necessary in terms of other aspects such as the structural integrity and the biological aspects, since it might specify the direction of applying aerobic granular sludge for municipal wastewater intruding by industrial wastewater or totally industrial wastewater treatment (Wang et al., 2017b; van den Akker et al., 2015). Therefore, inner understanding on the responses of structural and functional stability of aerobic granular sludge to salinity stress have to be improved. Temperature as one of the main operational parameters largely affects the long-term stability of aerobic granules especially on the microorganisms (Fontenot et al., 2007; Rebolledo et al., 2018). Improper temperature such as winter temperature was always accompanied with poor process performance and granular integrity (Munoz-Palazon et al., 2018; Rodriguez-Sanchez et al., 2019; de Kreuk et al., 2005). Despite numerous studies have revealed the effects of temperature especially moderate temperature, the long-term effects of winter temperatures still lack enough information (Winkler et al., 2012; Bassin et al., 2012). Previous works have summarized that gradual acclimation to low wastewater temperatures could gain better performance than shock or direct stress (Munoz-Palazon et al., 2018). Besides, cold-adapted sludge could tolerate low working temperatures (Gonzalez-Martinez et al., 2017). It is of common situations for the aerobic granules to be suffered from both saline and low operational temperatures especially during the winter conditions. For the wastewater treatment plants, operational durations from the moderate temperatures to winter temperatures occurred yearly. Therefore, the responses of aerobic granules treating saline wastewater should also be considered at low temperatures, when both salinity and low temperature posed stress. However, to the best of our knowledge, limited studies have focused on the acclimation of aerobic granules to simultaneous saline and low temperature conditions. Therefore, the main objective of the present work was to evaluate the effects of salt addition and temperature on aerobic granules. An aerobic granular sequencing batch reactor (SBR) performing
2. Materials and methods 2.1. Experimental setup and operation The plexiglass reactor was with an inner diameter of 100 mm and a total height of 500 mm, giving a working volume of 3.6 L. Air was introduced into the reactor from the right bottom of the reactor at a constant aeration rate of 200 mL/min, corresponding to the superficial gas velocity (SGV) of 0.11 cm/s (He et al., 2017b). Influent was pumped into the reactor from top of the reactor at the beginning of each cycle after equal amount of effluent was discharged from the middle height of the effective height, making an exchange ratio of 50%. A mechanical stirrer was fixed with a constant speed of 150 rpm. The reactor was run in a 6-h cycle including feeding (2 min), anaerobic reaction (120 min), oxic reaction (90 min), anoxic reaction (144 min), settling (2 min) and discharge (2 min), respectively. The reactor was run continuously and automatically by a programable logic controller (PLC) targeting influent and effluent pumps, aeration pump and stirrers, respectively. The hydraulic retention time was about 12 h ignoring the time for feeding, settling and discharge. No manual sludge disposal was conducted during the whole operation of the reactor with the observed sludge retention time (SRT) of about 25days (He et al., 2020). The reactor was placed near the window in the indoor location, where was vulnerable to dynamics of seasonal temperatures. Based on the water temperature, the whole operation could be divided into two separate phases, namely phase Ⅰ from day 1 to 49, and phase Ⅱ from day 50 to 75. The water temperature of 15 ℃ was utilized as the watershed for separating ambient and low temperatures (de Kreuk et al., 2005; Chu et al., 2005). 2.2. Synthetic wastewater The SBR was initially seeded with mature aerobic granules from the previous work by He et al. (2017b). Synthetic wastewater was used as the influent in present study, which was pumped into the reactor at the beginning of each cycle. The compositions of the synthetic wastewater were listed in Table 1. Detailed components of the trace solution could be referenced to He et al. (2018a). The low concentrations of COD and nutrients were used to simulate the low-strength domestic wastewater in China especially south China (Ni et al., 2009). The salinity level of 0.5% (w/v) was applied through adding 50 g of commercially purchased NaCl into 1 L synthetic wastewater as described above. The pH of the wastewater was adjusted to around 7.5 using HCl or NaOH solutions. Table 1 Compositions of synthetic wastewater used in the present work.
2
Main compositions
Concentration (mg/L)
Trace compositions
Concentration (mg/L)
CH3COONa NH4Cl KH2PO4 CaCl2 MgSO4·7H2O
256 76.5 14.6 10 10
FeCl3 H3BO3 KI CuSO4∙5H2O MnCl2∙4H2O ZnSO4∙7H2O CoCl2∙6H2O Na2MoO4∙2H2O EDTA
0.9 0.15 0.18 0.03 0.06 0.12 0.15 0.06 10
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2.3. Analytical methods In this experiment, the concentrations of chemical oxygen demand (COD), ammonia nitrogen (NH4+-N), nitrite (NO2−-N) and nitrate (NO3−-N), total phosphorus (TP) of the influents and effluents, mixed liquor suspended solids (MLSS) and mixed liquor volatile suspended solids (MLVSS), sludge volume index at 2 min (SVI2) were measured according to the standard method (APHA, 2005). The pH and dissolved oxygen (DO) in the bulk solution were measured using a pHS-25 m (pHS-25, Shanghai Leici Instrument Factory, China) and YSI5000 m (YSI, Yellow Springs, Ohio, USA), respectively. Aerobic granules were collected at the end of each phase and pretreated with 0.9% NaCl solution and subsequent centrifugation at 4000 rpm for 5 min to separate the solids and supernatant for three times. The residual pellet was suspended with 0.9% NaCl solution again and then heated under 80 ℃ for 30 min. The supernatant after centrifugation at 10,000 rpm for 20 min was filtered through 0.45 μm water filter membrane for further analysis. The Bradford method and sulfuric acid-anthrone colorimetric method were utilized for PN and PS quantifications, respectively. EPS were referred to the sum to PN and PS (He et al., 2018b). Three-dimensional excitation and emission matrix (3D-EEM) analysis of EPS extracts was conducted according to previous procedures by He et al. (2018b) using a luminescence spectrometry FS5 Spectrophotometer (Edinburgh, UK). The corresponding scanning emission spectra from 200 to 500 nm and excitation wavelengths from 200 to 400 nm at 5 nm intervals were used. The excitation and emission slits were kept at 10 nm with a constant scanning speed of 1200 nm/min. Deionized Milli-Q water was excluded from the spectrum of EPS samples. The elliptical shape of contours was present as the final results of 3D-EEM spectra.
Fig. 1. Biomass concentrations and settleability of aerobic granules in different phases: the data in day 1 are of granules without salt addition at ambient temperature; data in day 18 and 49 are of granules with 0.5% salinity at ambient temperature; data in day 55 and 66 are of granules with 0.5% salinity at winter temperature.
Though under the salinity stress, there witnessed a significant enrichment of biomass (MLSS and MLVSS) (p < 0.05) with similar MLVSS/ MLSS (∼0.9) (p > 0.05) in contrast to the initially seeding sludge. However, both MLSS and MLVSS decreased slightly under low temperature (with 0.5%, w/v) (p > 0.05). Therefore, the combination of salinity and low temperature posed an adverse effect on biomass accumulation, while individual salinity had the positive influence. Similar effect was also seen toward the settleability. Since SVI2 decreased with 0.5% salinity, but slightly increased under synergistic effects of salinity and low temperature. Salinity, however, improved the settleability of activated sludge (Chen et al., 2018) and granular sludge (Pronk et al., 2014) at much wider ranges up to 20 g/L. Therefore, the aerobic granular sludge showed its robustness toward salinity and low temperature in terms of biomass concentrations and the settleability.
2.4. Biological analysis Aerobic granular samples were taken from the reactor at the end of each phase, namely the seeded granular samples S0, samples at the end of phases Ⅰ and Ⅱ (denoted as S1-2). The biomass samples were pretreated and stored for further DNA extraction. Biological experiments including DNA extraction, PCR amplification and subsequent sequencing were conducted according to the manufactures’ protocols. Hypervariable V3-4 regions of the 16S rRNA gene of the bacteria were amplified with the forward primer 338 F (5’-ACTCCTACGGGAGGCA GCA-3’) and reverse primer 806R (5’-GGACTACHVGGGTWTCTAAT-3’). The Illumina MiSeq apparatus (Illumina, Hayward, CA, USA) were applied for the bacterial community analysis by Shanghai MajorBio BioPharm Technology Co., Ltd, China. Data collection and statistical analysis were carried out according to previous researches (He et al., 2020, 2019) using the online platform of MajorBio Cloud Platform. The original gene sequences are available at NCBI Sequence Read Archive (SRA, http://www.ncbi.nlm.nih.gov/sra/) with a newly established BioProject ID PRJNA574902.
3.2. Content of EPS Profiles of EPS production over operation were shown in Fig. 2. It was elucidated that the introduction of salinity resulted in significant decrease in both PN and PS, as well as constant decrease of PN/PS (p < 0.05). However, with the decrease of temperature, contents of PN and PS raised sharply and even exceeded the initial levels, though still with low PN/PS values. It was noted that PS rather than PN varied significantly during the whole operation periods, implying the adjusting roles of PS in response to salinity and low temperature stresses. Chen et al. (Chen and Murata, 2002) also reported that the accumulation of compatible solutes including PS was a widespread response against environmental stress. Besides, the constant decrease of PN/PS indicated increase of cell hydrophobicity, protecting the aerobic granules from the strongly stressing conditions (He et al., 2017a).
2.5. Statistical analysis The results of this study were shown in the form of mean value ± standard error. To statistically compare the data of the experiment, an analysis of variance (ANOVA) at the significance level of p < 0.05 was adopted using the GraphPad Prism software (Version 5.0.3, Graphpad Software Inc., San Diego, CA, USA) (He et al., 2019). 3. Results and discussion 3.1. Biomass retentions and settleability The biomass accumulation of aerobic granular SNDPR system and the changes of settleability in terms of SVI2 were shown in Fig. 1.
Fig. 2. Profiles of EPS within aerobic granules at different phases. 3
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3.3. 3D-EEM spectra
3.5. Microbial diversity
3D-EEM spectra was utilized to provide information on the compositional changes in EPS subject to salinity and temperature variations. As shown in Fig. 3, it could be obviously told that three separate peaks A, B and C could be identified in three aerobic granular samples. According to He et al. (2018b), Peak A was assigned to tryptophan & protein-like substances, while Peaks B and C were identified as humic acid-like substances. Therefore, it could be concluded that apart from the protective role of PS as discussed above, other components within EPS including tryptophan & protein-like and humic acid-like substances also responded and contributed to aerobic granular stability and performance. Further parameters of peak from the 3D-EEM were given in Table 2. Individual salinity stress and synergistic effects of salinity and low temperature witnessed variations of peak locations and specific intensities, suggesting the responsive actions of EPS toward stress conditions (Su et al., 2019). It should be noticed that the peak intensity ratios of EPS sample from the individual salinity stress was much different with others, implying that salinity might be the primary factor shaping the fluorescence components of EPS (He et al., 2018b). It could be concluded that both contents and compositions of EPS changed in response to stresses induced by salinity and low temperature, which might be the main contributor for structural stability of aerobic granules.
Three aerobic granular samples collected within the reactor with salinity-free, 0.5% salinity at moderate temperature, and 0.5% at low temperature (denoted S0-2, respectively) were analyzed using Illumina MiSeq sequencing. The alpha-diversity results were shown in Fig. 5. The dosage of salinity rarely changed the bacterial richness in terms of similar Ace and Chao estimators (p > 0.05). However, decrease in temperature along with salinity of 0.5% significantly enhanced the bacterial richness (p < 0.05). The consistent orders of diversity indices Shannon (S2 > S0 > S1) and Simpson (S2 < S0 < S1) implied that salinity only decreased the bacterial diversity, while combined stress of salinity and low temperature led to highest diversity. The rarefaction curves indicated that three samples held similar number of sequences (∼30,000) and the curves all tended to plateau, suggesting that enough data were collected for sequencing. Similar evenness of three samples were observed from the rank-abundance curves and the serval core microbes possessed much proportions (He et al., 2017b). Venn diagram revealed that the three samples shared a high percent of OTUs. Besides, less unique OTUs were held with the gradual stresses of salinity and low temperature. 3.6. Microbial compositions Major phyla and classes with an abundance over 1% were shown in Fig. 6A–B, while the top 50 genera were collected and mapped in heatmap in Fig. 6C. Proteobacteria were the most abundant phylum under salinity-free condition, accounting for 35.76%. 0.5% salinity stress shifted the predominant phylum to Chloroflexi (41.72%). Interestingly, Proteobacteria recovered to the highest relative abundance of 54.22% under dual stresses of salinity and low temperature. It could be indicated that Proteobacteria and Chloroflexi were halophilic bacteria, which the former was psychrophilic (Ou et al., 2018). Other predominant phyla with dramatically varying relative abundances including Bacteroidetes (8.08, 7.18, and 19.32%), Parcubacteria (20.70, 0.19, and 0.02%), Gracilibacteria (11.14, 0.08, and 0.01%), Chlorobi (0.20, 3.84, and 1.26), Planctomycetes (2.91, 1.35, and 0.87), Nitrospirae (0.73, 1.33, and 2.03), Acidobacteria (0.51, 1.77, and 0.91), and Verrucomicrobia (0.28, 1.02, and 0.91) within reactor under the salinityfree at moderate temperature, 0.5% salinity at moderate temperature, and 0.5% salinity at low temperature conditions, respectively. Concludingly, only the halophilic and psychrophilic bacteria could survive the hypersaline and low temperature conditions. The sub-groups of Proteobacteria including Gamma-, Beta-, Alpha- and Delta-proteobacteria classes occupied large proportions and were the dominant microorganisms. Other abundant classes within the above predominant phyla distributed diversely within the reactor. Gamma-, Beta- and Alphaproteobacteria and Anaerolineae were the typical halophilic classes, while Alphaproteobacteria and Anaerolineae were susceptible to low temperature and decayed under low temperature. Candidatus_Moranbacteria from the phylum Parcubacteria were sensitive to salinity, which dropped from 20.48% to nearly undetectable levels under saline
3.4. Process performance Variations of nutrients removal performance along operation were elucidated in Fig. 4, as well as the dynamics of water temperatures. Reliable COD removal rates over 90% were obtained under salinity at ambient temperature, and low temperature even stimulated COD removal efficiencies. Superb removal performance toward NH4+-N and TIN could be achieved over operation, though low temperature shock led to temporary decrease in NH4+-N removal. This could be indicated that nitrification process was more vulnerable to stresses by salinity and low temperature, while denitrification remained unaffected. Sudden dosage of 0.5% salinity at ambient temperature deteriorated TP removal during the first several days, which could be soon recovered afterward. After the acclimation to salinity stress, TP removal rates only decreased slightly when the temperature dropped from ambient to low levels. Although both nitrogen and phosphorus removal could be influenced by salinity and low temperature, reliable performances could be eventually obtained after different durations of acclimation. In addition, the acclimation process under salinity stress even strengthened the robustness of the aerobic granular SNDPR toward the combination of salinity and low temperature, especially for the enhanced biological phosphorus removal (EBPR). Therefore, much shorter durations (5 days vs 15 days for TP removal as shown in Fig. 4D) for lower removal rates could be observed in response to salinity at low temperature than at moderate temperature.
Fig. 3. 3D-EEM spectra of EPS extracted from aerobic granules: A, aerobic granular sludge at ambient temperature without salt addition (day 1); B, aerobic granules at ambient temperature with 0.5% salinity (day 49); C, aerobic granules at winter temperature with 0.5% salinity (day 66). 4
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Table 2 Fluorescence spectral parameters of peaks identified from three aerobic granular samples S0-2 by 3D-EEM spectra. Samples
S0 S1 S2
Peak A
Peak B
Peak C
Peak intensity ratios
Ex/Em
Intensity
Ex/Em
Intensity
Ex/Em
Intensity
A/B
A/C
B/C
270/350 280/355 280/355
7424.38 7424.38 12068.79
270/440 270/435 270/440
4093.43 9515.64 6921.92
345/445 350/445 350/440
1821.74 4177.07 3152.36
1.81 0.78 1.74
4.08 1.78 3.83
2.25 2.28 2.20
conditions at moderate and low temperatures. The most dominant genera shifted with the operational conditions. Candidatus_Competibacter (24.18%), norank_f__Anaerolineaceae (37.61%) and Acinetobacter (20.78%) held the highest relative abundances under salinity-free, 0.5% salinity, and 0.5% salinity at low temperature conditions, suggesting their vital roles under different stressing circumstances. norank_c__Candidatus_Moranbacteria dropped from 20.48% to 0.01% or even lower under hypersaline conditions, implying it was sensitive and vulnerable to salt. Conversely, Thauera increased from 0.10% to 11.69%, which could be both halophilic and psychrophilic. Other predominant genera included Azospira, Nitrospira, Dechloromonas, and Hydrogenophaga, which varied frequently in relative abundances over operation. The individual effect of salinity or dual effects of
salinity and low temperature largely shaped the microbial compositions at various levels. 3.7. Susceptibility of functional groups The mainly functional groups within the aerobic granular SNDPR system included those involved in nitrogen and phosphorus removal, such as nitrifiers (including ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB)), dentrifiers (DNB), phosphorus accumulating organisms (PAOs), and denitrifying PAOs (DNPAOs). Glycogen accumulating organisms (GAOs) were always taken into consideration since they were the primary competitors for carbon source with PAOs/DNPAOs and DNB, which largely affected the
Fig. 4. Process performance during different phases for the aerobic granular SNDPR system: A, COD removal; B, NH4+-N removal; C, TIN removal; D, TP removal and E, profile of temperature. 5
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Fig. 5. Microbial diversity based on the OTU classifications on the three separate aerobic granular samples S0-2: A, diversity estimators; B, rarefaction curves; C, rank-abundance curves; and D, Venn diagram.
nitrogen and phosphorus removal efficiencies. The list of the above functional bacteria was shown in Fig. 7. Generally, salinity and temperature severely changed the functional bacteria in terms of the compositions and relative abundances. Two major AOB including Nitrosomonas (0.02-0.04%) and
norank_f_Nitrosomonadaceae (0.01-0.07%) were detected within different conditions, leading to a slight increase in the sum of abundances. However, NOB Nitrospira grew constantly from 0.73 to 2.03% with salinity and subsequent low temperature, suggesting that NOB might be more tolerant to salinity and low temperature. The cooperation of AOB
Fig. 6. Microbial structure of aerobic granular samples S0-2 at A, phylum level; B, class level; and C, genus level. The heatmap was drawn with relative abundances (%). 6
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Fig. 7. Relative abundances of functional groups: A, AOB, NOB; B, GAOs, PAOs and DNPAOs; C, DNB in three separate samples S0-2. The heatmap was drawn with the logarithmic values of relative abundances (%) and the excluded values from the color bar were in white color.
the major halophilic bacteria, while Proteobacteria was also psychrophilic. Functional groups involved in nitrogen and phosphorus removal demonstrated various tolerance capacities toward salinity and temperature and the superiority of such as NOB, DNB, PAOs, DNPAOs, and AOB over GAOs under the hard environment was the biological basis for robust reactor performance.
and NOB ensured excellent nitrifying efficiency. Multiple bacteria capable of denitrifying pathway were found with Acinetobacter (0.31–20.78%), Thauera (0.10–11.69%) being the predominant ones (He et al., 2019), which got significantly accumulated with salinity and low temperature. The various DNB laid the biological basis for reliable denitrification rate. Two kinds of GAOs Candidatus_Competibacter (24.18-1.92%) and Defluviicoccus (0.58-0.18%) were detected and found decreased sharply with individual or dual stressing conditions. In contrast, PAOs (1.00–3.71%) and DNPAOs (0.40–2.48%) enriched by times simultaneously. This implied that PAOs/DNPAOs offered stronger competitiveness and capacities for adaption than GAOs. Wang et al. (2017a) reported contrary conclusion in much higher salinity concentrations. Therefore, the tolerance toward salinity might also be dosage-dependent. In addition, the decay of GAOs and increase of PAOs/ DNPAOs and DNB was favorable for nitrogen and phosphorus removal (He et al., 2017b).
Declaration of Competing Interest The authors declare that there are no conflicts of interest. Acknowledgement This work was finally supported by the Fundamental Research Funds for the Central Universities (No. 531118010401), the Key Research and Development Program of Hunan Province (No. 2019SK2111), and the National Natural Science Foundation of China (NSFC, China) (No. 51878517).
4. Conclusions
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