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Diversity evolution of functional bacteria and resistance genes (CzcA) in aerobic activated sludge under Cd(II) stress
Journal of Environmental Management 250 (2019) 109519
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Research article
Diversity evolution of functional bacteria and resistance genes (CzcA) in aerobic activated sludge under Cd(II) stress
T
Meixue Daib,1, Guangqing Zhoua,b,1, How Yong Ngc, Jinyong Zhangd, Yan Wange, Ning Lie, Xiaoyu Qia, Mingsheng Miaob, Qi Liub, Qiang Konga,c,∗ a College of Geography and Environment, Collaborative Innovation Center of Human-Nature and Green Development in Universities of Shandong, Shandong Normal University, Jinan, 250014, PR China b College of Life Science, Shandong Normal University, 88 Wenhua Donglu, Jinan, 250014, Shandong, PR China c Department of Civil and Environmental Engineering, National University of Singapore, Singapore, 117576, Singapore d Shandong Huankeyuan Environmental Engineering Co., Ltd, 50 Lishan Road, Jinan, 250013, Shandong, PR China e Dongying Customs, 101 Fuqian Dajie, Dongying, 257091, Shandong, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Activated sludge Cd(II) CzcA gene Bacterial community
An activated sludge sequencing batch reactor (SBR) was used to treat divalent cadmium (Cd(II)) wastewater for 60 d to investigate the overall treatment performance, evolution of the bacterial community, and abundance of the Cd(II) resistance gene CzcA and shifts in its potential host bacteria. During stable operation with a Cd(II) concentration of 20 mg/L, the average removal efficiencies of Cd(II) and chemical oxygen demand (COD) were more than 85% and that of total phosphorus was greater than 70%, while the total nitrogen (TN) was only about 45%. The protein (PN) content in the extracellular polymeric substances (EPS) increased significantly after Cd(II) addition, while polysaccharides displayed a decreasing trend (p < 0.05), indicating that EPS prefer to release PN to adsorb Cd(II) and protect bacteria from damage. Three-dimensional fluorescence spectral analysis showed that fulvic acid-like substances were the most abundant chemical components of EPS. The addition of Cd(II) adversely affected most denitrifying bacteria (p < 0.05), which is consistent with the low TN removal. In addition, quantitative polymerase chain reaction analysis revealed that CzcA gene abundance decreased as the Cd (II) concentration increased, possibly because expression of the CzcA gene was inhibited by Cd(II) stress. The majority of CzcA gene sequences were carried by Pseudomonas, making it the dominant genus among Cd(II)resistant bacteria.
1. Introduction
limit for Cd(II) emissions set by the World Health Organization is 0.01 mg/L; however, the average Cd(II) concentration in metal plating industry effluent ranges from 15 to 20 mg/L, while Cd(II) concentrations in acid mine drainage wastewater streams can be as high as 44 mg/L (Meng et al., 2019). Cd(II) possesses strong migration properties and causes various hazards when enriched in living organisms (Rahmanian et al., 2018). Cd(II)-containing wastewater flows into aquatic environments, where it is extremely toxic to aquatic organisms (Van et al., 2019). Moreover, Cd(II) also poses a serious risk to human health; chronic exposure to Cd(II) can result in hypertension, renal dysfunction, hepatic damage, bone fractions, and other diseases, and high levels of exposure are fatal (Ayangbenro and Babalola, 2017). Therefore, there is an urgent need to identify effective methods to remove this heavy metal from contaminated environments.
In recent years, heavy metals have increasingly entered the environment as a result of the continuous development of industry and agriculture (Shu et al., 2016). Heavy metals are not biodegradable and their toxicity, bioaccumulation, and environmental persistence pose a serious threat to the environment (Tang et al., 2019). As they accumulate in the food chain, heavy metals may be ingested by humans, and could lead to organ damage, neurological and respiratory diseases, and other adverse health conditions (Ebrahimi et al., 2018). Divalent cadmium (Cd(II)) is a heavy metal with high commercial value that has mainly been applied in industrial processes such as electroplating, metallurgy, Cd/Ni battery production, and plastics and fertilizer manufacturing, and is also found in mine drainage (Xu et al., 2019b). The ∗
Corresponding author. College of Geography and Environment, Collaborative Innovation Center of Human-Nature and Green Development in Universities of Shandong, Shandong Normal University, Jinan, 250014, PR China. E-mail address: [email protected] (Q. Kong). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.jenvman.2019.109519 Received 23 April 2019; Received in revised form 17 August 2019; Accepted 2 September 2019 0301-4797/ © 2019 Elsevier Ltd. All rights reserved.
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the aerobic aeration tank of a sewage treatment plant from Jinan Everbright Water (Shandong Province, China) was used as the inoculum. The mixed liquor suspended solids (MLSS) concentration was approximately 5000 mg/L. Synthetic wastewater with the following composition was added to the reactor (mg/L): bacto-peptone (188), sucrose (512), NH4Cl (100), KCl (15), KH2PO4 (25), and NaHCO3 (250). The pH was maintained at 6.8–7.0 with 1 M NaHCO3. The SBR was inoculated with activated sludge without added Cd(II) on day 0. In the acclimation period (five stages), Cd(NO3)2·4H2O was added to the influent wastewater at increasing concentrations of 2.8, 14, 28, 42, and 56 mg/L (corresponding to 1, 5, 10, 15, and 20 mg/L Cd (II), respectively). Concentrations were increased every 6 d, and the system thus ran continuously for 30 d. The system was then operated at the highest Cd(II) concentration of 20 mg/L for a further 30 d, which was referred to as the stable operation stage. The COD and TP and TN contents were analyzed every 3 d throughout the experimental period using the standard methods (Gilcreas, 1966). The gravimetric method was used to determine the MLSS content, while the influent and effluent Cd(II) concentrations were determined by flame atomic absorption spectrometer (Z-2000, Hitachi, Japan) (Ghorbani et al., 2018).
Various methods have been used to treat Cd(II)-containing heavy metal wastewater. The degradation of Cd(II) using physical methods such as sonication and biochar adsorption (Mohan et al., 2014a) and chemical methods such as ion exchange, chemical precipitation, and membrane filtration (Víctor-Ortega et al., 2016) have already been investigated. In addition, certain nanocomposite adsorbents such as hydrated manganese oxide nanoparticles impregnated into peanut shell-derived biochar have also been used to remove Cd(II) (Wan et al., 2018). Enhanced chitosan bead-supported zero-valent iron-nanoparticles have also been used in the removal of Cd(II) from electroplating wastewater in permeable reactive barriers (Liu et al., 2013). However, these technologies have many disadvantages such as high cost, high energy consumption, and partial exclusion and inefficiency at low heavy metal concentrations (Raj et al., 2018). Based on the principles of conservation and environmental protection, activated sludge processes have been widely used in the biological treatment of Cd(II)containing wastewater (Xu et al., 2017). Activated sludge contains a large amount of macromolecular organic substances (such as proteins (PN) and polysaccharides (PS)), and is therefore considered to be a good source of extracellular polymeric substances (EPS). EPS usually contain abundant charged functional groups (such as amino, carboxyl, phosphoric, and hydroxyl groups) and non-polar groups (such as hydrophobic regions in polysaccharides and aromatics, as well as aliphatic regions in PN) (More et al., 2014). EPS can adsorb heavy metal cations through hydrophobic interactions and can obstruct intracellular penetration of metals (Xu et al., 2017). Microorganisms can secrete EPS as biosorbents while also using mechanisms such as efflux pumps, metalbinding PN, metal ion chelation, and enzymatic detoxification to deal with Cd(II) present in the environment (Aktan et al., 2013). Most previous studies of Cd(II) wastewater treatment have focused on the effects of Cd(II) addition on pollutant removal and EPS adsorption (Bogusz et al., 2017; Xu et al., 2017, Xu et al., 2019b). However, few studies have investigated the diversity and population structure of the entire bacterial community, and no investigations of the expression of specific Cd(II) resistance genes or population analyses of these resistance genes host bacteria have been conducted to date. The CzcA gene is an efflux pump that acts as a reverse divalent cationproton transporter during chemical permeation and drives heavy metals (Co, Zn, and Cd) from the cytoplasm through the periplasmic space to the extracellular region (Nesler et al., 2017). The current study evaluated changes in the abundance of the typical Cd(II) resistance gene, CzcA, and further investigated the Cd(II)-tolerant bacterial community, thus making up for the deficiency of the effects of Cd(II) on the bacterial community at the genetic level. In the present study, activated sludge was used to treat Cd(II)-containing heavy metal wastewater. First, the effects of Cd(II) on the removal efficiencies of various pollutants (chemical oxygen demand (COD), total phosphorus (TP), total nitrogen (TN), and Cd(II)), and the content and chemical composition of EPS were studied to reflect the overall performance of an activated sludge sequencing batch reactor (SBR). The evolution of the bacterial community following the addition of Cd(II) was then studied. Next, evolution of the gene level was investigated with a focus on the abundance of the Cd(II) resistance gene CzcA in different reaction stages, while also exploring the population distribution of Cd(II)-resistant bacteria.
2.2. Extraction and chemical analysis of EPS The EPS was collected from the 0-, 30-, and 60-d activated sludge samples (in triplicate) by the cation exchange resin method as described in Xu et al., 2019a and in the Supplementary Materials. The PN and PS contents in extracted EPS were analyzed using the Coomassie Brilliant Blue G-250 dye-binding method (based on bovine serum albumin) and the phenol-sulfuric acid method (based on glucose) (More et al., 2014), respectively. A fluorescence spectrometer (F-7000, Hitachi, Japan) was used to determine the three-dimensional excitation-emission matrix (3D-EEM) fluorescence spectrum of EPS. The specific parameters were as follows: 220- to 550-nm emission wavelengths (EM), 200- to 450-nm excitation wavelengths (EX), both with 10-nm increments; 5-nm emission and excitation slits; and 1200-nm/min scanning speed. The Origin 9.1 software (Origin Lab, USA) was used to conduct map analysis of the 3DEEM data. 2.3. Bacterial community analysis 2.3.1. High-throughput sequencing of 16S rRNA and CzcA gene The 0-, 30-, and 60-d activated sludge samples were taken from the SBR in sextuplicate, then analyzed in triplicate for microbial community analysis of the 16S rRNA and the Cd(II) resistance gene CzcA. A Soil DNA Kit (Omega D5625-01) was employed to extract the total genomic DNA, after which the DNA concentration was determined using an ultraviolet spectrophotometer (Nanodrop NC2000, USA). The 16S rRNA genes located in the hypervariable V3–V4 region were then amplified using universal primers (368F and 806R), as well as CzcA gene primers (CzcAF and CzcAR) designed based on gene sequences of the pattern bacteria Cupriavidus metallidurans CH34 (Janssen et al., 2010) (Table S1). A 25-μL reaction system was set up for each polymerase chain reaction (PCR) amplification, and the specific reaction systems and amplification conditions are given in the Supplementary Materials. The PCR amplification products were then electrophoresed on agarose gel (2% (w/v)) and recovered using a DNA gel extraction kit (AXYGEN AxyPrep). The amount of purified PCR product was measured with a FLx800 plate reader (BioTek, USA), after which the amplicons were normalized in equimolar amounts in the final mixture and pairedend sequencing was performed on the Illumina MiSeq platform (Illumina Inc., San Diego, CA, USA). Following removal of the chimeras, adapters, primers, and barcodes, the obtained sequences were further quality-filtered with the QIIME pipeline (v1.8.0, http://qiime.org/). The obtained raw data of the 16S rRNA and CzcA genes were uploaded to the National Center for Biotechnology Information (NCBI) Sequence
2. Materials and methods 2.1. Experimental setup and operation The SBR had a height of 500 mm, an inner diameter of 140 mm, and a total operating volume of 6 L. The filling, reacting, settling, drawing, and idle periods were 30, 210, 60, 45, and 15 min, respectively, with a total continuous cycle time of 6 h. The optimal operational parameters were set as an air intake of 0.3 ± 0.1 m3/h, temperature of 20 ± 3 °C, and hydraulic retention time of 12 h. Activated sludge gathered from 2
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Read Archive (SRA) database under accession numbers SRP8907301–SRP8907309 and SRP8919656–SRP8919664, respectively. 2.3.2. Data analysis The QIIME software was used to compare the most abundant 16S rRNA and CzcA gene sequences in the operational taxonomic unit (OTU) with sequences in the Greengenes and NCBI databases, respectively, and these sequences were assigned to the phylum, class, family, and genus levels. The Mothur program (http://www.mothur.org/) was used to calculate the alpha diversity indices, including the Simpson and Shannon indices for diversity, as well as the Chao1 estimator and abundance-based coverage estimator (ACE) for richness. Analysis of variance (ANOVA) was conducted using statistical software (SPSS 20.0, IBM, USA) to identify significant differences between microbes on days 0, 30, and 60 at the p < 0.05 level. 2.4. qPCR of Cd(II) resistance gene CzcA Triplicate analyses of the CzcA gene were conducted using quantitative polymerase chain reaction (qPCR). Briefly, the CzcA gene was cloned into a plasmid to acquire a calibration curve, after which its abundance was determined by the Cycle threshold (Ct) value. The specific qPCR reaction conditions are shown in the Supplementary Materials. The qPCR amplification specificity was further checked using melting curves. 3. Results and discussion 3.1. Performance of the SBR The performance of the SBR for Cd(II), COD, TP, and TN removal is shown in Fig. 1. During the stable operation stage, the average removal of Cd(II), COD, TP, and TN was 87.86% ± 0.18%, 85.96% ± 0.15%, 73.02% ± 0.28%, and 44.05% ± 0.41%, respectively. As shown in Fig. 1b, the COD removal decreased slightly as the Cd(II) concentration increased from 0 to 20 mg/L, but was never lower than 80%, indicating that Cd(II) stress had little effect on COD removal. The removal efficiencies of TP and TN decreased substantially during the acclimation stage, however, TP removal efficiency increased obviously and the TN removal remained at around 40% during the stable operation stage (Fig. 1c and d). An alternate aerobic–anaerobic environment provided from the SBR is beneficial to biological denitrification and biological dephosphorization (Zhou et al., 2019). The low TN removal was mainly because of the reduction of nitrite-oxidizing bacteria (NOB) and denitrifying bacteria (DNB) under the stress of Cd(II). Previous research has shown that the addition of Cd(II) can inhibit the proliferation of nitrifying bacteria and reduce the activity of nitrite oxidoreductase (Chen et al., 2014). Adding bio-accelerators (such as biotin, L-aspartic acid, and cytokinin) or carbon sources (such as glucose) could effectively restore the activity of nitrifying bacteria under Cd(II) stress, which could be used to improve the removal efficiency of TN (Wang et al., 2016b). The large accumulation of denitrifying polyphosphate-accumulating organisms (DNPAO) may explain the high TP removal. It has been reported that DNPAO achieve phosphorus removal through anoxic phosphorus uptake and anaerobic phosphorus release, during which nitrite or nitrate can be used as an electron acceptor instead of oxygen (Zhao et al., 2019). The MLSS increased gradually from 5000 to 7000 mg/L as the Cd(II) concentration increased from 0 to 20 mg/L. The increased MLSS provided superior conditions for the reproduction of DNPAO, which contributed to the accumulation and removal of TP (Wang et al., 2019). Cd(II) cannot be biodegraded, therefore the high Cd(II) removal in this study may be attributed to the biosorption of EPS and various removal mechanisms of microorganisms. It has been reported that the most prominent metal resistance system for Cd(II) is by
Fig. 1. Performance of SBR during 60 days of operation (stage I: acclimation, stage II: stable operation): (a) removal of Cd(II), (b) removal of COD, (c) removal of TP, and (d) removal of TN.
efflux pumping (Nesler et al., 2017). As the member of the resistance–nodulation–cell division PN family, the CzcA gene can perform its efflux pump function to discharge Cd(II) out of the cell (Wang et al., 2016c). Furthermore, the tertiary structure of metal-binding PN has a large amount of metal-coordinating amino acids, the residues of which could capture Cd ions (More et al., 2014). In addition, the increased
3
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MLSS concentrations enhanced the Cd(II) removal efficiency by increasing the surface area for Cd(II) adsorption (Mohan, 2014b). 3.2. PN and PS content and chemical composition of EPS under Cd(II) stress The EPS content increased significantly as the Cd(II) concentration increased during the acclimation stage. EPS have been reported to contribute to the removal of Cd(II) through bioadsorption, with the main components being PN and PS (Chen et al., 2015). After adding Cd (II), the PN content (in 1 g MLSS) increased significantly from 0.76 ± 0.10 mg on day 0 to 3.39 ± 0.75 mg on day 30 and 1.07 ± 0.64 mg on day 60, while the PS content decreased significantly from 20.91 ± 0.78 mg to 10.33 ± 0.45 mg (day 0 to day 60) (p < 0.05). Moreover, the PN/PS ratios increased from 0.04 on day 0 to 0.17 on day 30, indicating that bacteria prefer to secrete PN for protection. Higher PN content or PN/PS ratios provide higher EPS hydrophobicity because the amino acid compositions and secondary structures of the PN contribute substantially to hydrophobic interactions (Shi et al., 2017). It has also been shown that when adding extra EPS in rotating drum biofilm reactors, the PS fraction is consumed faster as a carbon source than the PN fraction in the supplemented EPS (Nouha et al., 2018). Cd(II) has a stronger binding affinity for the amino groups of PN, and an increasing amount of PN could protect bacteria from the toxic Cd(II) environment (He et al., 2017). The chemical compositions of EPS in sludge samples were analyzed by 3D-EEM fluorescence spectroscopy (Fig. 2). The results showed that the EPS on days 0, 30, and 60 did not display any difference in the functional groups and peak positions after the addition of Cd(II), whereas the fluorescence intensities increased significantly (p < 0.05). The EX and EM values on days 0, 30, and 60 were all 320–325 and 390–395 nm, respectively, which represent fulvic acid-like substances (Kong et al., 2017). However, the fluorescence intensity of the peaks showed an increasing trend, with values of 2602, 5267, and 6013 on days 0, 30, and 60, respectively, indicating that fulvic acid-like substances play an important role in resisting Cd(II) toxicity (Yue et al., 2015). The fulvic acid-like substances in EPS contained a large amount of hydroxyl and carbonyl groups that could interact with metal ions to complete the adsorption of Cd(II) (Wei et al., 2017). Therefore, the increased levels of fulvic acid-like substances may be related to the high Cd(II) removal efficiency. 3.3. Evolution of 16SrRNA gene bacterial community 16S rRNA high-throughput sequencing was used to study the evolution of the bacterial community. The results (Table S2) demonstrate that there was a marked reduction in bacterial diversity and richness as the Cd(II) concentration increased (p < 0.05). Based on the number of OTUs, the sludge sample from day 0 had the richest diversity, whereas samples from days 30 and 60 displayed decreasing diversity (P < 0.05). Moreover, in the presence of Cd(II), the diversity indices (Simpson and Shannon) and the richness indices (ACE and Chao1) were substantially lower than without Cd(II) addition (p < 0.05). These results indicate that the addition of Cd(II) had an adverse impact on the diversity of bacterial communities. Previous studies have shown that heavy metal pollution reduced microbial diversity because bacterial communities were sensitive to the heavy metals (Chen and Chang, 2017). It has also been reported that synthetic wastewater at a low Cd (II) concentration (10.5 mg/L) had toxic effects on the microbial community (Huang et al., 2017b), and that bacterial diversity decreased significantly as Cd(II) concentration increased to 20 mg/L (Zhang et al., 2018). The bacterial community composition of the sludge samples on days 0, 30, and 60 is shown in Fig. 3. Some taxa show a strong ability to adapt to high concentrations and long-term loading of Cd(II). At the phylum level, Proteobacteria showed the greatest abundance under Cd
Fig. 2. Excitation emission matrix (EEM) fluorescence spectra of extracellular polymeric substances (EPS) on days 0 (a), 30 (b) and 60 (c).
(II) stress, accounting for 53% and 59% of the total bacteria on days 30 and 60, respectively, in the SBR, which was consistent with the results of a previous study (Chen and Chang, 2017). This was followed by Bacteroidetes, which increased significantly in abundance from 13.22% ± 9.29% on day 0 to 46.58% ± 2.61% on day 30, then decreased slightly to 39.59% ± 2.10% on day 60 (p < 0.05). Both Proteobacteria and Bacteroidetes are Gram-negative bacteria and have outer membranes composed of lipopolysaccharide, which has the ability to resist Cd(II) toxicity (Huang et al., 2017b). Previous studies have shown that the abundance of Chloroflexi in metal-contaminated wastewater decreased (Chen et al., 2018), which was also observed in the present study. At the class level, both Bacteroidia and Gammaproteobacteria were dominant on days 30 and 60, indicating they can resist high concentrations of Cd(II). Gammaproteobacteria has been reported to play an 4
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Fig. 3. Relative abundance of 16S rRNA gene host bacteria at the phylum, class, family and genus levels.
(p < 0.05). This was an important reason for the inefficiency of TN removal. Nitrosomonas was reduced to a greater degree than Nitrospira as the Cd(II) concentration increased, indicating that AOB are more sensitive to high Cd(II) concentrations than NOB, which supports the results of a previous study (Wang et al., 2016c). However, for DNB, the OTU numbers of Flavobacterium, Acidovorax, Pseudoxanthomonas, and Zoogloea increased sharply after the addition of Cd(II) (p < 0.05), indicating these genera could withstand higher Cd(II) pressure to perform the functions of denitrification and nitrogen removal. Zoogloea has been reported to possess the property of EPS-production, which could promote microbial aggregation and denitrification (Kang et al., 2018). DNPAO had the ability to accumulate denitrifying phosphorus and PHA, which were the dominant functional bacteria for removal of phosphorus (Zhang et al., 2017). Evaluation of the DNPAO in the system revealed that the number of Dechloromonas and Candidatus_Accumulibacter OTUs under Cd(II) stress was significantly higher than that without Cd(II) addition (p < 0.05), which could explain the high TP removal efficiency in this study. Functional gene annotations were conducted using the KEGG pathway database (Fig. S1 and Fig. 4). There were six categories of functional bacteria observed, with those related to metabolism accounting for the greatest proportion (48.3%–51.7%; Fig. S1). These
important role in denitrification and phosphorus removal (He et al., 2016). There were three dominant families after Cd(II) was added, Burkholderiaceae, Flavobacteriaceae, and Rhodocyclaceae. Certain genera of Rhodocyclaceae, such as Dechloromonas, can use nitrite as an electron acceptor for phosphorus removal and simultaneous denitrification (Fu et al., 2019). At the genus level, Flavobacterium and Cloacibacterium were the dominant genera under Cd(II) stress, which accounted for 43.84% (on day 30) and 36.62% (on day 60) of all genera. A previous study showed that Flavobacterium could use nitrate for denitrification and anaerobic growth and have strong adaptability to Cd(II) stress (Li et al., 2019). 3.4. Functional analysis of the bacterial community Functional bacteria that have previously been reported were found in the SBR, including DNB, DNPAO, NOB, and ammonia-oxidizing bacteria (AOB) (Table 1), all of which play important roles in nitrogen and phosphorus removal from active sludge (Xu et al., 2018). Most genera associated with nitrogen removal, including Thauera, Paracoccus, Hyphomicrobium, Comamonas, Rhodobacter, Pseudomonas (DNB), Nitrosomonas (AOB), and Nitrospira (NOB), were inhibited by high Cd (II) stress, with OTU numbers all showing a sharply decreasing trend 5
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Table 1 Numbers of OTUs of functional bacteria in activated sludge on days 0, 30 and 60. Name of bacteria DNB
DNPAO
AOB NOB Total read
Level Thauera Paracoccus Hyphomicrobium Flavobacterium Comamonas Acidovorax Rhodobacter Pseudoxanthomonas zoogloea Pseudomonas Dechloromonas Candidatus_Accumulibacter Aeromonas Nitrosomonas Nitrospira
0d
30 d a
Genus Genus Genus Genus Genus Genus Genus Genus Genus Genus Genus Genus Genus Genus Genus
118 ± 72 77 ± 13a 224 ± 50a 1 ± 1a 93 ± 21a 28 ± 3a 156 ± 30a 2 ± 1a 5 ± 1a 12 ± 1a 4 ± 3a 2 ± 1a 7 ± 4a 35 ± 29a 443 ± 136a 31350
60 d b
16 ± 6 5 ± 3b 0 ± 0b 5384 ± 386b 87 ± 8a 492 ± 25b 13 ± 3b 1 ± 1a 240 ± 5b 5 ± 0b 261 ± 16b 10 ± 2b 1 ± 0b 0 ± 0b 12 ± 3b 37605
4 ± 1b 3 ± 1b 0 ± 0b 48 ± 1a 1 ± 1a 286 ± 36c 20 ± 7b 1217 ± 47b 39 ± 4c 0 ± 0c 36 ± 12c 4 ± 0a 0 ± 0c 0 ± 0b 3 ± 1b 37194
a, b, c: different letters at the same rows means significant difference at p = 0.05 level.
1.15 × 108 ± 0.62 × 108 on day 0, then decreased significantly to 6.00 × 106 ± 0.32 × 106 as the Cd(II) loadings increased on day 30 (p < 0.05), after which they increased slightly to 6.30 × 106 ± 0.42 × 106 on day 60, which agreed with the results of the predicted function. The abundance of CzcA decreased sharply in the acclimation stage, indicating the expression of the CzcA gene was inhibited by Cd(II) stress, which may have been a result of selection of CzcA-containing resistant bacteria that could withstand high concentrations of Cd(II). Previous studies have also shown that the abundance of the CzcA gene decreased as the Cd(II) concentration increased (Miao et al., 2018). However, the quantitative increase in the CzcA gene during stable operation may indicate that more Cd(II)-resistant bacteria adapted to the high Cd(II) stress environment and propagated, causing expression of the CzcA gene to increase. It has been reported that quantitative increases in gene diversity may occur because some species could tolerate Cd(II) and subsequently thrive in bioreactor of wastewater treatment plants that receive Cd(II) (Chen et al., 2018). 3.6. Diversity of CzcA potential bacterial hosts under Cd(II) stress Cd(II)-resistant bacteria were acclimated under high-concentration and long-term Cd(II) stress. The population distribution of these acclimated bacteria related to the CzcA gene is shown in Fig. 5. At the phylum level (Fig. 5a), there were four organisms containing CzcA genes: Proteobacteria, Nitrospirae, Verrucomicrobia, and Cyanobacteria. Among these, the CzcA gene had the highest expression in Proteobacteria, with a relative abundance up to 96.87% ± 0.69% on day 30, indicating Proteobacteria was the dominant Cd(II)-resistant phyla. A previous study also showed that Proteobacteria (89.82%) became the dominant phylum under Cd(II) stress (Huang et al., 2017b). At the class level (Fig. 5b), Betaproteobacteria and Gammaproteobacteria were found to have the highest abundance with Cd(II) loading. It has also been reported that Betaproteobacteria and Gammaproteobacteria have the ability to reduce nitrate and nitrite, and their abundances are positively correlated with Cd(II) concentration (Li et al., 2017). At the family level (Fig. 5c), the relative abundances of Pseudomonadaceae and Comamonadaceae on days 30 and 60 were significantly higher than those on day 0 without Cd(II) addition (p < 0.05), which indicates that they can withstand high concentrations of Cd(II). Comamonadaceae has been reported to play an important role in nitrogen removal via denitrification (Fu et al., 2019). At the genus level (Fig. 5d), Pseudomonas had the highest abundance of CzcA genes in all samples and significantly higher relative abundance on days 30 and 60 than on day 0, suggesting that the efflux pump function of CzcA gene in Pseudomonas plays an important role in resisting Cd(II) stress. Furthermore, the cell wall of Pseudomonas is negatively charged and can thus bind a large
Fig. 4. Predicting the relative abundance of functional gene content.
bacteria played an important role in resisting Cd(II) stress (Zeng et al., 2019). The specific functions are shown in Fig. 4. When compared with samples collected on day 0, the samples collected on days 30 or 60 contained more functional bacteria related to membrane transport, signal transduction, transport and catabolism, and xenobiotic biodegradation and metabolism, which may have been related to Cd(II) resistance. For example, the CzcA gene is an efflux pump that conducts membrane transport that drives Cd(II) out of the cell (Wang et al., 2016a). The results of the current study indicated that, with long-term acclimation and stable operation under Cd(II) stress, the bacterial community in active sludge has evolved an adaption strategy imparting it with resistance to Cd(II) (Chen et al., 2018). 3.5. Cd(II) effects on resistance gene CzcA The abundance of the Cd(II) efflux gene CzcA decreased as the Cd (II) concentration increased. Functional prediction included Co/Zn/Cd cation transporters, for which the number of OTUs decreased significantly from day 0 to day 30, then increased slightly on day 60 (p < 0.05). The CzcA gene is a member of the Co/Zn/Cd cation transporters (Nesler et al., 2017), which abundance was quantified by qPCR. The results showed that its abundance (copies per g sludge) was 6
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from long-term heavy metal-contaminated areas have the ability to resist and reduce heavy metals (Huang et al., 2017a). Finally, the relative abundance of Variovorax increased significantly from 0.24% ± 0.02% on day 0–3.67% ± 0.34% on day 30 and 28.37% ± 0.81% on day 60 (p < 0.05), suggesting that it is also an important genus that is resistant to high concentrations of Cd(II). 4. Applications and prospects This experiment systematically investigated the removal efficiency of various pollutants, the evolution of EPS and bacterial communities under Cd(II) stress, and the evolution of host bacteria of the Cd(II) resistance gene CzcA. The results presented herein provide theoretical and technical support for the treatment of Cd(II)-contaminated wastewater, further promoting the improvement of water quality and the restoration of heavy metal-polluted wastewater. The findings also highlight the potential development of applying the dominant host bacteria of resistance genes to the bioaugmentation of wastewater treatment. However, additional studies to improve the removal efficiency of Cd(II) and recovery of Cd(II) from activated sludge (such as adding new biosorbent) are warranted. 5. Conclusion In this study, the average removal efficiencies of Cd(II), COD, and TP were all more than 70%, while the TN levels were relatively lower (about 50%). The abundance of most functional bacteria, including DNB, AOB, and NOB, decreased under Cd(II) stress, which may be an important reason for the inefficiency of TN removal. EPS preferred to release PN and fulvic acid to adsorb Cd(II) and protect bacteria from Cd (II) stress. Investigation of the resistance gene showed that Pseudomonas was the main host of the CzcA gene and was able to tolerate long-term and high-concentration Cd(II) stress. Compliance with ethical standards Conflict of interest The authors declare no conflicts of interest. Ethical approval This article does not contain any studies with human participants or animals performed by the authors. Acknowledgements This work was supported by the Key Research and Development Program of Shandong Province, P.R. China (No. 2019GSF109103), National Natural Science Foundation of China (No. 51708340), International Postdoctoral Exchange Fellowship Program (No. 20180063), Special Financial Grant from the China Postdoctoral Science Foundation (No. 2015T80738), and National Major Science and Technology Program for Water Pollution Control and Treatment (No. 2017ZX07101001). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jenvman.2019.109519.
Fig. 5. Relative abundance of CzcA gene host bacteria at the phylum (a), class (b), family (c), and genus (d) levels.
References amount of positively charged Cd ions to immobilize Cd(II) and stop the Cd(II) influx by associating with the extracellular pump system (Deb et al., 2013). It has also been reported that Pseudomonas strains isolated
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Research article
Diversity evolution of functional bacteria and resistance genes (CzcA) in aerobic activated sludge under Cd(II) stress
T
Meixue Daib,1, Guangqing Zhoua,b,1, How Yong Ngc, Jinyong Zhangd, Yan Wange, Ning Lie, Xiaoyu Qia, Mingsheng Miaob, Qi Liub, Qiang Konga,c,∗ a College of Geography and Environment, Collaborative Innovation Center of Human-Nature and Green Development in Universities of Shandong, Shandong Normal University, Jinan, 250014, PR China b College of Life Science, Shandong Normal University, 88 Wenhua Donglu, Jinan, 250014, Shandong, PR China c Department of Civil and Environmental Engineering, National University of Singapore, Singapore, 117576, Singapore d Shandong Huankeyuan Environmental Engineering Co., Ltd, 50 Lishan Road, Jinan, 250013, Shandong, PR China e Dongying Customs, 101 Fuqian Dajie, Dongying, 257091, Shandong, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Activated sludge Cd(II) CzcA gene Bacterial community
An activated sludge sequencing batch reactor (SBR) was used to treat divalent cadmium (Cd(II)) wastewater for 60 d to investigate the overall treatment performance, evolution of the bacterial community, and abundance of the Cd(II) resistance gene CzcA and shifts in its potential host bacteria. During stable operation with a Cd(II) concentration of 20 mg/L, the average removal efficiencies of Cd(II) and chemical oxygen demand (COD) were more than 85% and that of total phosphorus was greater than 70%, while the total nitrogen (TN) was only about 45%. The protein (PN) content in the extracellular polymeric substances (EPS) increased significantly after Cd(II) addition, while polysaccharides displayed a decreasing trend (p < 0.05), indicating that EPS prefer to release PN to adsorb Cd(II) and protect bacteria from damage. Three-dimensional fluorescence spectral analysis showed that fulvic acid-like substances were the most abundant chemical components of EPS. The addition of Cd(II) adversely affected most denitrifying bacteria (p < 0.05), which is consistent with the low TN removal. In addition, quantitative polymerase chain reaction analysis revealed that CzcA gene abundance decreased as the Cd (II) concentration increased, possibly because expression of the CzcA gene was inhibited by Cd(II) stress. The majority of CzcA gene sequences were carried by Pseudomonas, making it the dominant genus among Cd(II)resistant bacteria.
1. Introduction
limit for Cd(II) emissions set by the World Health Organization is 0.01 mg/L; however, the average Cd(II) concentration in metal plating industry effluent ranges from 15 to 20 mg/L, while Cd(II) concentrations in acid mine drainage wastewater streams can be as high as 44 mg/L (Meng et al., 2019). Cd(II) possesses strong migration properties and causes various hazards when enriched in living organisms (Rahmanian et al., 2018). Cd(II)-containing wastewater flows into aquatic environments, where it is extremely toxic to aquatic organisms (Van et al., 2019). Moreover, Cd(II) also poses a serious risk to human health; chronic exposure to Cd(II) can result in hypertension, renal dysfunction, hepatic damage, bone fractions, and other diseases, and high levels of exposure are fatal (Ayangbenro and Babalola, 2017). Therefore, there is an urgent need to identify effective methods to remove this heavy metal from contaminated environments.
In recent years, heavy metals have increasingly entered the environment as a result of the continuous development of industry and agriculture (Shu et al., 2016). Heavy metals are not biodegradable and their toxicity, bioaccumulation, and environmental persistence pose a serious threat to the environment (Tang et al., 2019). As they accumulate in the food chain, heavy metals may be ingested by humans, and could lead to organ damage, neurological and respiratory diseases, and other adverse health conditions (Ebrahimi et al., 2018). Divalent cadmium (Cd(II)) is a heavy metal with high commercial value that has mainly been applied in industrial processes such as electroplating, metallurgy, Cd/Ni battery production, and plastics and fertilizer manufacturing, and is also found in mine drainage (Xu et al., 2019b). The ∗
Corresponding author. College of Geography and Environment, Collaborative Innovation Center of Human-Nature and Green Development in Universities of Shandong, Shandong Normal University, Jinan, 250014, PR China. E-mail address: [email protected] (Q. Kong). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.jenvman.2019.109519 Received 23 April 2019; Received in revised form 17 August 2019; Accepted 2 September 2019 0301-4797/ © 2019 Elsevier Ltd. All rights reserved.
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the aerobic aeration tank of a sewage treatment plant from Jinan Everbright Water (Shandong Province, China) was used as the inoculum. The mixed liquor suspended solids (MLSS) concentration was approximately 5000 mg/L. Synthetic wastewater with the following composition was added to the reactor (mg/L): bacto-peptone (188), sucrose (512), NH4Cl (100), KCl (15), KH2PO4 (25), and NaHCO3 (250). The pH was maintained at 6.8–7.0 with 1 M NaHCO3. The SBR was inoculated with activated sludge without added Cd(II) on day 0. In the acclimation period (five stages), Cd(NO3)2·4H2O was added to the influent wastewater at increasing concentrations of 2.8, 14, 28, 42, and 56 mg/L (corresponding to 1, 5, 10, 15, and 20 mg/L Cd (II), respectively). Concentrations were increased every 6 d, and the system thus ran continuously for 30 d. The system was then operated at the highest Cd(II) concentration of 20 mg/L for a further 30 d, which was referred to as the stable operation stage. The COD and TP and TN contents were analyzed every 3 d throughout the experimental period using the standard methods (Gilcreas, 1966). The gravimetric method was used to determine the MLSS content, while the influent and effluent Cd(II) concentrations were determined by flame atomic absorption spectrometer (Z-2000, Hitachi, Japan) (Ghorbani et al., 2018).
Various methods have been used to treat Cd(II)-containing heavy metal wastewater. The degradation of Cd(II) using physical methods such as sonication and biochar adsorption (Mohan et al., 2014a) and chemical methods such as ion exchange, chemical precipitation, and membrane filtration (Víctor-Ortega et al., 2016) have already been investigated. In addition, certain nanocomposite adsorbents such as hydrated manganese oxide nanoparticles impregnated into peanut shell-derived biochar have also been used to remove Cd(II) (Wan et al., 2018). Enhanced chitosan bead-supported zero-valent iron-nanoparticles have also been used in the removal of Cd(II) from electroplating wastewater in permeable reactive barriers (Liu et al., 2013). However, these technologies have many disadvantages such as high cost, high energy consumption, and partial exclusion and inefficiency at low heavy metal concentrations (Raj et al., 2018). Based on the principles of conservation and environmental protection, activated sludge processes have been widely used in the biological treatment of Cd(II)containing wastewater (Xu et al., 2017). Activated sludge contains a large amount of macromolecular organic substances (such as proteins (PN) and polysaccharides (PS)), and is therefore considered to be a good source of extracellular polymeric substances (EPS). EPS usually contain abundant charged functional groups (such as amino, carboxyl, phosphoric, and hydroxyl groups) and non-polar groups (such as hydrophobic regions in polysaccharides and aromatics, as well as aliphatic regions in PN) (More et al., 2014). EPS can adsorb heavy metal cations through hydrophobic interactions and can obstruct intracellular penetration of metals (Xu et al., 2017). Microorganisms can secrete EPS as biosorbents while also using mechanisms such as efflux pumps, metalbinding PN, metal ion chelation, and enzymatic detoxification to deal with Cd(II) present in the environment (Aktan et al., 2013). Most previous studies of Cd(II) wastewater treatment have focused on the effects of Cd(II) addition on pollutant removal and EPS adsorption (Bogusz et al., 2017; Xu et al., 2017, Xu et al., 2019b). However, few studies have investigated the diversity and population structure of the entire bacterial community, and no investigations of the expression of specific Cd(II) resistance genes or population analyses of these resistance genes host bacteria have been conducted to date. The CzcA gene is an efflux pump that acts as a reverse divalent cationproton transporter during chemical permeation and drives heavy metals (Co, Zn, and Cd) from the cytoplasm through the periplasmic space to the extracellular region (Nesler et al., 2017). The current study evaluated changes in the abundance of the typical Cd(II) resistance gene, CzcA, and further investigated the Cd(II)-tolerant bacterial community, thus making up for the deficiency of the effects of Cd(II) on the bacterial community at the genetic level. In the present study, activated sludge was used to treat Cd(II)-containing heavy metal wastewater. First, the effects of Cd(II) on the removal efficiencies of various pollutants (chemical oxygen demand (COD), total phosphorus (TP), total nitrogen (TN), and Cd(II)), and the content and chemical composition of EPS were studied to reflect the overall performance of an activated sludge sequencing batch reactor (SBR). The evolution of the bacterial community following the addition of Cd(II) was then studied. Next, evolution of the gene level was investigated with a focus on the abundance of the Cd(II) resistance gene CzcA in different reaction stages, while also exploring the population distribution of Cd(II)-resistant bacteria.
2.2. Extraction and chemical analysis of EPS The EPS was collected from the 0-, 30-, and 60-d activated sludge samples (in triplicate) by the cation exchange resin method as described in Xu et al., 2019a and in the Supplementary Materials. The PN and PS contents in extracted EPS were analyzed using the Coomassie Brilliant Blue G-250 dye-binding method (based on bovine serum albumin) and the phenol-sulfuric acid method (based on glucose) (More et al., 2014), respectively. A fluorescence spectrometer (F-7000, Hitachi, Japan) was used to determine the three-dimensional excitation-emission matrix (3D-EEM) fluorescence spectrum of EPS. The specific parameters were as follows: 220- to 550-nm emission wavelengths (EM), 200- to 450-nm excitation wavelengths (EX), both with 10-nm increments; 5-nm emission and excitation slits; and 1200-nm/min scanning speed. The Origin 9.1 software (Origin Lab, USA) was used to conduct map analysis of the 3DEEM data. 2.3. Bacterial community analysis 2.3.1. High-throughput sequencing of 16S rRNA and CzcA gene The 0-, 30-, and 60-d activated sludge samples were taken from the SBR in sextuplicate, then analyzed in triplicate for microbial community analysis of the 16S rRNA and the Cd(II) resistance gene CzcA. A Soil DNA Kit (Omega D5625-01) was employed to extract the total genomic DNA, after which the DNA concentration was determined using an ultraviolet spectrophotometer (Nanodrop NC2000, USA). The 16S rRNA genes located in the hypervariable V3–V4 region were then amplified using universal primers (368F and 806R), as well as CzcA gene primers (CzcAF and CzcAR) designed based on gene sequences of the pattern bacteria Cupriavidus metallidurans CH34 (Janssen et al., 2010) (Table S1). A 25-μL reaction system was set up for each polymerase chain reaction (PCR) amplification, and the specific reaction systems and amplification conditions are given in the Supplementary Materials. The PCR amplification products were then electrophoresed on agarose gel (2% (w/v)) and recovered using a DNA gel extraction kit (AXYGEN AxyPrep). The amount of purified PCR product was measured with a FLx800 plate reader (BioTek, USA), after which the amplicons were normalized in equimolar amounts in the final mixture and pairedend sequencing was performed on the Illumina MiSeq platform (Illumina Inc., San Diego, CA, USA). Following removal of the chimeras, adapters, primers, and barcodes, the obtained sequences were further quality-filtered with the QIIME pipeline (v1.8.0, http://qiime.org/). The obtained raw data of the 16S rRNA and CzcA genes were uploaded to the National Center for Biotechnology Information (NCBI) Sequence
2. Materials and methods 2.1. Experimental setup and operation The SBR had a height of 500 mm, an inner diameter of 140 mm, and a total operating volume of 6 L. The filling, reacting, settling, drawing, and idle periods were 30, 210, 60, 45, and 15 min, respectively, with a total continuous cycle time of 6 h. The optimal operational parameters were set as an air intake of 0.3 ± 0.1 m3/h, temperature of 20 ± 3 °C, and hydraulic retention time of 12 h. Activated sludge gathered from 2
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Read Archive (SRA) database under accession numbers SRP8907301–SRP8907309 and SRP8919656–SRP8919664, respectively. 2.3.2. Data analysis The QIIME software was used to compare the most abundant 16S rRNA and CzcA gene sequences in the operational taxonomic unit (OTU) with sequences in the Greengenes and NCBI databases, respectively, and these sequences were assigned to the phylum, class, family, and genus levels. The Mothur program (http://www.mothur.org/) was used to calculate the alpha diversity indices, including the Simpson and Shannon indices for diversity, as well as the Chao1 estimator and abundance-based coverage estimator (ACE) for richness. Analysis of variance (ANOVA) was conducted using statistical software (SPSS 20.0, IBM, USA) to identify significant differences between microbes on days 0, 30, and 60 at the p < 0.05 level. 2.4. qPCR of Cd(II) resistance gene CzcA Triplicate analyses of the CzcA gene were conducted using quantitative polymerase chain reaction (qPCR). Briefly, the CzcA gene was cloned into a plasmid to acquire a calibration curve, after which its abundance was determined by the Cycle threshold (Ct) value. The specific qPCR reaction conditions are shown in the Supplementary Materials. The qPCR amplification specificity was further checked using melting curves. 3. Results and discussion 3.1. Performance of the SBR The performance of the SBR for Cd(II), COD, TP, and TN removal is shown in Fig. 1. During the stable operation stage, the average removal of Cd(II), COD, TP, and TN was 87.86% ± 0.18%, 85.96% ± 0.15%, 73.02% ± 0.28%, and 44.05% ± 0.41%, respectively. As shown in Fig. 1b, the COD removal decreased slightly as the Cd(II) concentration increased from 0 to 20 mg/L, but was never lower than 80%, indicating that Cd(II) stress had little effect on COD removal. The removal efficiencies of TP and TN decreased substantially during the acclimation stage, however, TP removal efficiency increased obviously and the TN removal remained at around 40% during the stable operation stage (Fig. 1c and d). An alternate aerobic–anaerobic environment provided from the SBR is beneficial to biological denitrification and biological dephosphorization (Zhou et al., 2019). The low TN removal was mainly because of the reduction of nitrite-oxidizing bacteria (NOB) and denitrifying bacteria (DNB) under the stress of Cd(II). Previous research has shown that the addition of Cd(II) can inhibit the proliferation of nitrifying bacteria and reduce the activity of nitrite oxidoreductase (Chen et al., 2014). Adding bio-accelerators (such as biotin, L-aspartic acid, and cytokinin) or carbon sources (such as glucose) could effectively restore the activity of nitrifying bacteria under Cd(II) stress, which could be used to improve the removal efficiency of TN (Wang et al., 2016b). The large accumulation of denitrifying polyphosphate-accumulating organisms (DNPAO) may explain the high TP removal. It has been reported that DNPAO achieve phosphorus removal through anoxic phosphorus uptake and anaerobic phosphorus release, during which nitrite or nitrate can be used as an electron acceptor instead of oxygen (Zhao et al., 2019). The MLSS increased gradually from 5000 to 7000 mg/L as the Cd(II) concentration increased from 0 to 20 mg/L. The increased MLSS provided superior conditions for the reproduction of DNPAO, which contributed to the accumulation and removal of TP (Wang et al., 2019). Cd(II) cannot be biodegraded, therefore the high Cd(II) removal in this study may be attributed to the biosorption of EPS and various removal mechanisms of microorganisms. It has been reported that the most prominent metal resistance system for Cd(II) is by
Fig. 1. Performance of SBR during 60 days of operation (stage I: acclimation, stage II: stable operation): (a) removal of Cd(II), (b) removal of COD, (c) removal of TP, and (d) removal of TN.
efflux pumping (Nesler et al., 2017). As the member of the resistance–nodulation–cell division PN family, the CzcA gene can perform its efflux pump function to discharge Cd(II) out of the cell (Wang et al., 2016c). Furthermore, the tertiary structure of metal-binding PN has a large amount of metal-coordinating amino acids, the residues of which could capture Cd ions (More et al., 2014). In addition, the increased
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MLSS concentrations enhanced the Cd(II) removal efficiency by increasing the surface area for Cd(II) adsorption (Mohan, 2014b). 3.2. PN and PS content and chemical composition of EPS under Cd(II) stress The EPS content increased significantly as the Cd(II) concentration increased during the acclimation stage. EPS have been reported to contribute to the removal of Cd(II) through bioadsorption, with the main components being PN and PS (Chen et al., 2015). After adding Cd (II), the PN content (in 1 g MLSS) increased significantly from 0.76 ± 0.10 mg on day 0 to 3.39 ± 0.75 mg on day 30 and 1.07 ± 0.64 mg on day 60, while the PS content decreased significantly from 20.91 ± 0.78 mg to 10.33 ± 0.45 mg (day 0 to day 60) (p < 0.05). Moreover, the PN/PS ratios increased from 0.04 on day 0 to 0.17 on day 30, indicating that bacteria prefer to secrete PN for protection. Higher PN content or PN/PS ratios provide higher EPS hydrophobicity because the amino acid compositions and secondary structures of the PN contribute substantially to hydrophobic interactions (Shi et al., 2017). It has also been shown that when adding extra EPS in rotating drum biofilm reactors, the PS fraction is consumed faster as a carbon source than the PN fraction in the supplemented EPS (Nouha et al., 2018). Cd(II) has a stronger binding affinity for the amino groups of PN, and an increasing amount of PN could protect bacteria from the toxic Cd(II) environment (He et al., 2017). The chemical compositions of EPS in sludge samples were analyzed by 3D-EEM fluorescence spectroscopy (Fig. 2). The results showed that the EPS on days 0, 30, and 60 did not display any difference in the functional groups and peak positions after the addition of Cd(II), whereas the fluorescence intensities increased significantly (p < 0.05). The EX and EM values on days 0, 30, and 60 were all 320–325 and 390–395 nm, respectively, which represent fulvic acid-like substances (Kong et al., 2017). However, the fluorescence intensity of the peaks showed an increasing trend, with values of 2602, 5267, and 6013 on days 0, 30, and 60, respectively, indicating that fulvic acid-like substances play an important role in resisting Cd(II) toxicity (Yue et al., 2015). The fulvic acid-like substances in EPS contained a large amount of hydroxyl and carbonyl groups that could interact with metal ions to complete the adsorption of Cd(II) (Wei et al., 2017). Therefore, the increased levels of fulvic acid-like substances may be related to the high Cd(II) removal efficiency. 3.3. Evolution of 16SrRNA gene bacterial community 16S rRNA high-throughput sequencing was used to study the evolution of the bacterial community. The results (Table S2) demonstrate that there was a marked reduction in bacterial diversity and richness as the Cd(II) concentration increased (p < 0.05). Based on the number of OTUs, the sludge sample from day 0 had the richest diversity, whereas samples from days 30 and 60 displayed decreasing diversity (P < 0.05). Moreover, in the presence of Cd(II), the diversity indices (Simpson and Shannon) and the richness indices (ACE and Chao1) were substantially lower than without Cd(II) addition (p < 0.05). These results indicate that the addition of Cd(II) had an adverse impact on the diversity of bacterial communities. Previous studies have shown that heavy metal pollution reduced microbial diversity because bacterial communities were sensitive to the heavy metals (Chen and Chang, 2017). It has also been reported that synthetic wastewater at a low Cd (II) concentration (10.5 mg/L) had toxic effects on the microbial community (Huang et al., 2017b), and that bacterial diversity decreased significantly as Cd(II) concentration increased to 20 mg/L (Zhang et al., 2018). The bacterial community composition of the sludge samples on days 0, 30, and 60 is shown in Fig. 3. Some taxa show a strong ability to adapt to high concentrations and long-term loading of Cd(II). At the phylum level, Proteobacteria showed the greatest abundance under Cd
Fig. 2. Excitation emission matrix (EEM) fluorescence spectra of extracellular polymeric substances (EPS) on days 0 (a), 30 (b) and 60 (c).
(II) stress, accounting for 53% and 59% of the total bacteria on days 30 and 60, respectively, in the SBR, which was consistent with the results of a previous study (Chen and Chang, 2017). This was followed by Bacteroidetes, which increased significantly in abundance from 13.22% ± 9.29% on day 0 to 46.58% ± 2.61% on day 30, then decreased slightly to 39.59% ± 2.10% on day 60 (p < 0.05). Both Proteobacteria and Bacteroidetes are Gram-negative bacteria and have outer membranes composed of lipopolysaccharide, which has the ability to resist Cd(II) toxicity (Huang et al., 2017b). Previous studies have shown that the abundance of Chloroflexi in metal-contaminated wastewater decreased (Chen et al., 2018), which was also observed in the present study. At the class level, both Bacteroidia and Gammaproteobacteria were dominant on days 30 and 60, indicating they can resist high concentrations of Cd(II). Gammaproteobacteria has been reported to play an 4
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Fig. 3. Relative abundance of 16S rRNA gene host bacteria at the phylum, class, family and genus levels.
(p < 0.05). This was an important reason for the inefficiency of TN removal. Nitrosomonas was reduced to a greater degree than Nitrospira as the Cd(II) concentration increased, indicating that AOB are more sensitive to high Cd(II) concentrations than NOB, which supports the results of a previous study (Wang et al., 2016c). However, for DNB, the OTU numbers of Flavobacterium, Acidovorax, Pseudoxanthomonas, and Zoogloea increased sharply after the addition of Cd(II) (p < 0.05), indicating these genera could withstand higher Cd(II) pressure to perform the functions of denitrification and nitrogen removal. Zoogloea has been reported to possess the property of EPS-production, which could promote microbial aggregation and denitrification (Kang et al., 2018). DNPAO had the ability to accumulate denitrifying phosphorus and PHA, which were the dominant functional bacteria for removal of phosphorus (Zhang et al., 2017). Evaluation of the DNPAO in the system revealed that the number of Dechloromonas and Candidatus_Accumulibacter OTUs under Cd(II) stress was significantly higher than that without Cd(II) addition (p < 0.05), which could explain the high TP removal efficiency in this study. Functional gene annotations were conducted using the KEGG pathway database (Fig. S1 and Fig. 4). There were six categories of functional bacteria observed, with those related to metabolism accounting for the greatest proportion (48.3%–51.7%; Fig. S1). These
important role in denitrification and phosphorus removal (He et al., 2016). There were three dominant families after Cd(II) was added, Burkholderiaceae, Flavobacteriaceae, and Rhodocyclaceae. Certain genera of Rhodocyclaceae, such as Dechloromonas, can use nitrite as an electron acceptor for phosphorus removal and simultaneous denitrification (Fu et al., 2019). At the genus level, Flavobacterium and Cloacibacterium were the dominant genera under Cd(II) stress, which accounted for 43.84% (on day 30) and 36.62% (on day 60) of all genera. A previous study showed that Flavobacterium could use nitrate for denitrification and anaerobic growth and have strong adaptability to Cd(II) stress (Li et al., 2019). 3.4. Functional analysis of the bacterial community Functional bacteria that have previously been reported were found in the SBR, including DNB, DNPAO, NOB, and ammonia-oxidizing bacteria (AOB) (Table 1), all of which play important roles in nitrogen and phosphorus removal from active sludge (Xu et al., 2018). Most genera associated with nitrogen removal, including Thauera, Paracoccus, Hyphomicrobium, Comamonas, Rhodobacter, Pseudomonas (DNB), Nitrosomonas (AOB), and Nitrospira (NOB), were inhibited by high Cd (II) stress, with OTU numbers all showing a sharply decreasing trend 5
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Table 1 Numbers of OTUs of functional bacteria in activated sludge on days 0, 30 and 60. Name of bacteria DNB
DNPAO
AOB NOB Total read
Level Thauera Paracoccus Hyphomicrobium Flavobacterium Comamonas Acidovorax Rhodobacter Pseudoxanthomonas zoogloea Pseudomonas Dechloromonas Candidatus_Accumulibacter Aeromonas Nitrosomonas Nitrospira
0d
30 d a
Genus Genus Genus Genus Genus Genus Genus Genus Genus Genus Genus Genus Genus Genus Genus
118 ± 72 77 ± 13a 224 ± 50a 1 ± 1a 93 ± 21a 28 ± 3a 156 ± 30a 2 ± 1a 5 ± 1a 12 ± 1a 4 ± 3a 2 ± 1a 7 ± 4a 35 ± 29a 443 ± 136a 31350
60 d b
16 ± 6 5 ± 3b 0 ± 0b 5384 ± 386b 87 ± 8a 492 ± 25b 13 ± 3b 1 ± 1a 240 ± 5b 5 ± 0b 261 ± 16b 10 ± 2b 1 ± 0b 0 ± 0b 12 ± 3b 37605
4 ± 1b 3 ± 1b 0 ± 0b 48 ± 1a 1 ± 1a 286 ± 36c 20 ± 7b 1217 ± 47b 39 ± 4c 0 ± 0c 36 ± 12c 4 ± 0a 0 ± 0c 0 ± 0b 3 ± 1b 37194
a, b, c: different letters at the same rows means significant difference at p = 0.05 level.
1.15 × 108 ± 0.62 × 108 on day 0, then decreased significantly to 6.00 × 106 ± 0.32 × 106 as the Cd(II) loadings increased on day 30 (p < 0.05), after which they increased slightly to 6.30 × 106 ± 0.42 × 106 on day 60, which agreed with the results of the predicted function. The abundance of CzcA decreased sharply in the acclimation stage, indicating the expression of the CzcA gene was inhibited by Cd(II) stress, which may have been a result of selection of CzcA-containing resistant bacteria that could withstand high concentrations of Cd(II). Previous studies have also shown that the abundance of the CzcA gene decreased as the Cd(II) concentration increased (Miao et al., 2018). However, the quantitative increase in the CzcA gene during stable operation may indicate that more Cd(II)-resistant bacteria adapted to the high Cd(II) stress environment and propagated, causing expression of the CzcA gene to increase. It has been reported that quantitative increases in gene diversity may occur because some species could tolerate Cd(II) and subsequently thrive in bioreactor of wastewater treatment plants that receive Cd(II) (Chen et al., 2018). 3.6. Diversity of CzcA potential bacterial hosts under Cd(II) stress Cd(II)-resistant bacteria were acclimated under high-concentration and long-term Cd(II) stress. The population distribution of these acclimated bacteria related to the CzcA gene is shown in Fig. 5. At the phylum level (Fig. 5a), there were four organisms containing CzcA genes: Proteobacteria, Nitrospirae, Verrucomicrobia, and Cyanobacteria. Among these, the CzcA gene had the highest expression in Proteobacteria, with a relative abundance up to 96.87% ± 0.69% on day 30, indicating Proteobacteria was the dominant Cd(II)-resistant phyla. A previous study also showed that Proteobacteria (89.82%) became the dominant phylum under Cd(II) stress (Huang et al., 2017b). At the class level (Fig. 5b), Betaproteobacteria and Gammaproteobacteria were found to have the highest abundance with Cd(II) loading. It has also been reported that Betaproteobacteria and Gammaproteobacteria have the ability to reduce nitrate and nitrite, and their abundances are positively correlated with Cd(II) concentration (Li et al., 2017). At the family level (Fig. 5c), the relative abundances of Pseudomonadaceae and Comamonadaceae on days 30 and 60 were significantly higher than those on day 0 without Cd(II) addition (p < 0.05), which indicates that they can withstand high concentrations of Cd(II). Comamonadaceae has been reported to play an important role in nitrogen removal via denitrification (Fu et al., 2019). At the genus level (Fig. 5d), Pseudomonas had the highest abundance of CzcA genes in all samples and significantly higher relative abundance on days 30 and 60 than on day 0, suggesting that the efflux pump function of CzcA gene in Pseudomonas plays an important role in resisting Cd(II) stress. Furthermore, the cell wall of Pseudomonas is negatively charged and can thus bind a large
Fig. 4. Predicting the relative abundance of functional gene content.
bacteria played an important role in resisting Cd(II) stress (Zeng et al., 2019). The specific functions are shown in Fig. 4. When compared with samples collected on day 0, the samples collected on days 30 or 60 contained more functional bacteria related to membrane transport, signal transduction, transport and catabolism, and xenobiotic biodegradation and metabolism, which may have been related to Cd(II) resistance. For example, the CzcA gene is an efflux pump that conducts membrane transport that drives Cd(II) out of the cell (Wang et al., 2016a). The results of the current study indicated that, with long-term acclimation and stable operation under Cd(II) stress, the bacterial community in active sludge has evolved an adaption strategy imparting it with resistance to Cd(II) (Chen et al., 2018). 3.5. Cd(II) effects on resistance gene CzcA The abundance of the Cd(II) efflux gene CzcA decreased as the Cd (II) concentration increased. Functional prediction included Co/Zn/Cd cation transporters, for which the number of OTUs decreased significantly from day 0 to day 30, then increased slightly on day 60 (p < 0.05). The CzcA gene is a member of the Co/Zn/Cd cation transporters (Nesler et al., 2017), which abundance was quantified by qPCR. The results showed that its abundance (copies per g sludge) was 6
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from long-term heavy metal-contaminated areas have the ability to resist and reduce heavy metals (Huang et al., 2017a). Finally, the relative abundance of Variovorax increased significantly from 0.24% ± 0.02% on day 0–3.67% ± 0.34% on day 30 and 28.37% ± 0.81% on day 60 (p < 0.05), suggesting that it is also an important genus that is resistant to high concentrations of Cd(II). 4. Applications and prospects This experiment systematically investigated the removal efficiency of various pollutants, the evolution of EPS and bacterial communities under Cd(II) stress, and the evolution of host bacteria of the Cd(II) resistance gene CzcA. The results presented herein provide theoretical and technical support for the treatment of Cd(II)-contaminated wastewater, further promoting the improvement of water quality and the restoration of heavy metal-polluted wastewater. The findings also highlight the potential development of applying the dominant host bacteria of resistance genes to the bioaugmentation of wastewater treatment. However, additional studies to improve the removal efficiency of Cd(II) and recovery of Cd(II) from activated sludge (such as adding new biosorbent) are warranted. 5. Conclusion In this study, the average removal efficiencies of Cd(II), COD, and TP were all more than 70%, while the TN levels were relatively lower (about 50%). The abundance of most functional bacteria, including DNB, AOB, and NOB, decreased under Cd(II) stress, which may be an important reason for the inefficiency of TN removal. EPS preferred to release PN and fulvic acid to adsorb Cd(II) and protect bacteria from Cd (II) stress. Investigation of the resistance gene showed that Pseudomonas was the main host of the CzcA gene and was able to tolerate long-term and high-concentration Cd(II) stress. Compliance with ethical standards Conflict of interest The authors declare no conflicts of interest. Ethical approval This article does not contain any studies with human participants or animals performed by the authors. Acknowledgements This work was supported by the Key Research and Development Program of Shandong Province, P.R. China (No. 2019GSF109103), National Natural Science Foundation of China (No. 51708340), International Postdoctoral Exchange Fellowship Program (No. 20180063), Special Financial Grant from the China Postdoctoral Science Foundation (No. 2015T80738), and National Major Science and Technology Program for Water Pollution Control and Treatment (No. 2017ZX07101001). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jenvman.2019.109519.
Fig. 5. Relative abundance of CzcA gene host bacteria at the phylum (a), class (b), family (c), and genus (d) levels.
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