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The effect of continuous Zn (II) exposure on the organic degradation capability and soluble microbial products (SMP) of activated sludge
Journal of Hazardous Materials 244–245 (2013) 489–494
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Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
The effect of continuous Zn (II) exposure on the organic degradation capability and soluble microbial products (SMP) of activated sludge Jing-chao Han, Yan Liu ∗∗ , Xiang Liu ∗ , Yi Zhang, Yang-wei Yan, Rui-hua Dai, Xiao-song Zha, Cheng-shan Wang Department of Environmental Science and Engineering, Fudan University, 220 Handan Road, Shanghai 200433, China
h i g h l i g h t s After acclimation, the activated sludge could endure 400 mg/L Zn (II). A close correlation was found between SMP content and effluent COD. The change in DNA might reflect the Zn (II) toxicity to the biomass.
a r t i c l e
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Article history: Received 29 May 2012 Received in revised form 9 October 2012 Accepted 10 October 2012 Available online 3 November 2012 Keywords: Zinc (II) Activated sludge Toxicity Soluble microbial products (SMP) Organic degradation ability
a b s t r a c t This study describes the change of organic degradation capability and soluble microbial products (SMP) generated in activated sludge under continuous exposure to Zn (II) in a sequencing batch reactor (SBR). In 338 days of operation, the added Zn (II) concentrations were gradually increased from 50 to 100, 200, 400 to 600 and 800 mg/L. Results showed that after adaptation, the activated sludge could endure 400 mg/L Zn (II) without showing evident reduction in organic degradation ability (92 ± 1% of chemical oxygen demand (COD) removal in stable state). However, when 600 and 800 mg/L Zn (II) were applied, the effluent water quality significantly deteriorated. Meanwhile, under increasing Zn (II) concentrations, the SMP content in the activated sludge, together with its main biochemical constituents, first increased slightly below 400 mg/L of Zn (II), then rose sharply under 600 and 800 mg/L Zn (II). Furthermore, a close correlation was found between SMP content and effluent soluble COD in both the Experimental Reactor and Control Reactor. In addition, the Zn (II) concentrations in the effluent and SMP extraction liquid were further analyzed and discussed to reveal the role that SMP constituents played in defense and resistance to the toxicity of Zn (II). © 2012 Elsevier B.V. All rights reserved.
1. Introduction With the development of modern industries such as the electroplating, chemical manufacture, mining and mineral processing, a great amount of wastewater containing high concentrations of heavy metals is generated [1]. Due to the diversity in wastewater collection systems and policies, in many countries including China, this type of wastewater is partially transported into municipal wastewater treatment plant (WWTP) instead of specially designed industrial WWTP. As a result, many heavy metals such as lead and zinc are commonly found in wastewater and activated sludge. For example, the Zn (II) concentrations in actual influent of WWTP could reach 0.01–1.28 and 0.02–0.38 mg/L, respectively in the
∗ Corresponding author. Tel.: +86 21 6564 2018; fax: +86 21 6564 3597. ∗∗ Corresponding author. Tel.: +86 21 6564 3348; fax: +86 21 6564 3597. E-mail addresses: [email protected] (X. Liu),[email protected] (Y. Liu). 0304-3894/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jhazmat.2012.10.065
States and Japan [2,3]. In terms of China, according to the “Integrated Wastewater Discharge Standard” [4], the municipal and industrial wastewater must be pre-treated to a certain criterion (no higher than 5 mg/L for Zn ions) before flowing into sewer system. However, due to the bio-sorption and/or bioaccumulation effect as well as the circumfluence of sludge, the actual Zn (II) concentration which the activated sludge endured would be much higher than that in the influent of WWTP. It was reported that the quantity of zinc in excess sludge could reach as high as 2170.6 and 2841.2 mg/kg dry sludge in some municipal WWTPs of China [5,6]. Considering biological treatment is the most widely applied process in municipal WWTP, it is greatly needed to study the toxic effects of heavy metal ions on the properties of activated sludge. With respect to the effect of Zn (II), it is widely acknowledged that Zn (II) at low concentrations can perform as micronutrients and stimulate biological processes [7,8], while high concentrations of Zn (II) would pose toxic effect to microorganisms, reduce microbial diversity and inhibit biological treatment [8–10].
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Meanwhile, when exposed to metal toxicity, at least six mechanisms have been found to be utilized by microorganisms as instinctive self-protection [11]. For instance, when encountered with a single dosage of heavy metals, protein can sequestrate the metal within cells through binding and some enzymes can also mitigate the toxicity of the metal by catalyzing the detoxification process [11]. However, the continuous exposure of activated sludge to heavy metals may induce the defense and resistance mechanisms of biomass in a more complex way [12–14]. So far, most of the relevant researches have been focused on the response and performance of activated sludge subject to shock loading of zinc in a batch or a continuous-flow reactor [15–19], and the shock zinc concentrations range from 1 to 300 mg/L. Only a few researchers [10,20–22] have investigated the effect of continuous dosage of zinc on activated sludge in a continuous-flow reactor, in which the applied Zn (II) concentrations are generally between 1 and 50 mg/L [10,20–22]. Very limited literature has dealt with the toxic effect of continuous exposure of high concentrations (400 mg/L or above) zinc ions on activated sludge in a continuous-flow mode. It has been reported that the heavy metals in high concentrations can lead to production of unspecific complicated compounds within biomass cells and induce the cells to rupture [18]. Meanwhile, apart from the biomass growth, the biomass decay and cell lysis would also release substantial amount of soluble microbial products (SMP) [23]. Protein, polysaccharide, humic substances, and deoxyribonucleic acids (DNA) were the four main biochemical constituents of SMP [24]. To some extent, the changes in SMP quantity and components may reveal the response and resistance of activated sludge to the toxicity of zinc in high concentrations. However, most literature related to this topic only investigates the efficiency and mechanisms of biosorption of zinc by activated sludge or microorganisms [1,25–27], while the effects of Zn (II) toxicity on SMP production and its components change have rarely been examined. Moreover, a deeper knowledge of the role SMP plays in the resistance to Zn (II) toxicity is still needed. The aim of this study was to evaluate the effects of continuous high concentrations of zinc on the organic degradation ability (using chemical oxygen demand (COD) as indicator), SMP production and its components change in a sequencing batch reactor (SBR). The correlation between SMP and the effluent soluble COD, the change of SMP compositions were further examined to better understand the role which SMP played in Zn (II) toxicity resistance. In addition, the Zn (II) concentrations in the effluent and SMP extraction liquid were also analyzed to validate the function of SMP.
2. Materials and methods 2.1. Experimental methods Two SBRs with 4.0 L working volume each were operated for over 338 days (excluding the initial domestication period of 54 days) at 20 ± 5 ◦ C, one as Control Reactor (CR) and the other as Experimental Reactor (ER). The seed sludge was obtained from the returned sludge of a local WWTP in Shanghai city. The SBRs were operated with 12-h cycles, and each cycle comprised of five stages: fill (0.5 h), aeration (6.0 h), and settling (2.0 h), idle (3 h) and decant (0.5 h). In each cycle, at the decant stage, 2 L of the supernatant (effluent) was discharged from the reactor. Then 2.1 L of fresh synthetic wastewater (influent) was refilled in the fill stage. Afterwards, 100-mL mixed liquor was disposed at the beginning of aeration period. On each day, two cycles were operated and thereby aggregately 200-mL mixed liquor was eliminated to maintain the sludge retention time (SRT) at 20 days. The dissolved oxygen (DO)
in the aeration stage and pH were controlled at 2.0–2.5 mg/L and 6.5–8.0, respectively. For the nutrient supply, CH3 COONa was applied as the carbon source at a concentration of around 800 mg COD/L. The COD:N:P mass ratio of the synthetic wastewater was kept at 100:5:1, and mineral nutrients were added to each liter as follows: 15 mg CaCl2 , 15 mg MgSO4 , 37.5 mg NaHCO3 , 0.1118 mg FeCl3 ·6H2 O, 0.1118 mg H3 BO3 , 0.0224 mg CuSO4 ·5H2 O, 0.1342 mg KI, 0.0894 mg MnCl2 ·4H2 O, 0.0894 mg ZnSO4 ·7H2 O, and 0.1118 mg CoCl2 ·6H2 O. After 54 days of acclimation, the activated sludge in both reactors reached a steady state. The steady state in this study was identified by the situation that the standard deviation of COD removal efficiency was within 5% for at least 14 days. The mixed liquor suspended solids (MLSS) during that stage was maintained at 1700–2200 mg/L. The 55th day was designated as Day 1 for the toxicity study, after which the two reactors were further examined for another 11 days to re-confirm the steady state. Then ER was dosed with a ZnSO4 ·7H2 O solution of increasing concentrations (50, 100, 200, 400, 600 and 800 mg/L for Zinc ions). At first Zn (II) was added to the influent of ER on Day 13, resulting in a concentration of 50 mg/L in the synthetic wastewater. After a period of adaptation, the organic degradation ability in ER reached a steady state again. The stable state was maintained for at least 14 days before the concentration of Zn (II) was raised to 100 mg/L on Day 94. This process was repeated and the influent Zn (II) concentration was increased to 200 mg/L on Day 112, 400 mg/L on Day 168, 600 mg/L on Day 209 and 800 mg/L on Day 240, respectively. The COD, MLSS and SMP of the sludge were measured periodically to evaluate the toxic effect of Zn (II) and the corresponding responses of the activated sludge. Apart from the fact that no Zn (II) was added to CR, CR was operated in the same way as ER. 2.2. Analytical methods COD, Zn (II), MLSS and mixed liquor volatile suspended solids (MLVSS) were all analyzed according to the Standard Methods [28]. The effluent COD concentration in this study refers to the soluble COD, which was obtained by filtration of the sample through 0.45-m filter papers. Except that the Zn (II) sample in SMP extraction liquid had been filtered through 0.22-m filter papers in the extraction process of SMP, the rest of Zn (II) samples were all unfiltered. After all the Zn (II) samples were digested by HNO3 solution (50%, v/v), the Zn (II) concentration was determined with a P-4010 Inductively Coupled Plasma Emission Spectrometer (Hitachi Ltd., Japan). SMP was extracted from 50-mL mixed liquor after filtration through 0.22-m filter papers. The concentrations of the main components of SMP were analyzed as such: protein and humic acid by a modified Lowry method [29], polysaccharide by an anthrone colorimetric assay [30], and DNA through a diphenylamine method [31]. 3. Results and discussion 3.1. Organic degradation ability As seen in Fig. 1, the average influent COD concentration was 761 ± 34 mg/L for both CR and ER. From Day 1 to Day 338, the COD in the effluent of CR was comparatively steady, around a value of 34 ± 16 mg/L. However, for the ER, probably due to the continuous dosage of Zn (II), the effluent COD fluctuated in a large range, with a maximum and minimum values of 650 and 18 mg/L, respectively. At the beginning of the operation when 50 mg/L of Zn (II) was added, a sharp increase was observed in the effluent COD of ER. Then the effluent COD of ER gradually decreased. Around Days 49–56, another obvious temporary increase appeared in the
J.-c. Han et al. / Journal of Hazardous Materials 244–245 (2013) 489–494
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Zn (II) Concentration (mg/L) 0
50
100
900
400
200
800
600
800
COD(mg/L)
700 600 500 400 300
Influent Effluent of CR
200
Effluent of ER
100 0 0
30
60
90
120
150
180
210
240
270
300
330
360
Time (day) Fig. 1. Effect of Zn (II) on effluent COD under different Zn (II) concentrations: (♦) influent; () effluent of CR; () effluent of ER.
effluent COD of ER. It was because on Day 48, the air pump used for aeration in ER stopped to work due to some accidental malfunction. The ceasing of aeration affected the normal metabolism of activated sludge and brought on the temporary rise in the effluent COD of ER. Ultimately it took about 2 days to buy and transport a new air pump to replace the broken one and another 5 days for the system to recover to the previously COD removal level (above 85% on Days 35–47). This was also why the addition period of 50-mg/L Zn (II) lasted longer than the others. As the operation progressed, the COD values gradually returned to a stable state (53 ± 16 mg/L in Days 63–91). Similarly, when the Zn (II) concentrations were increased to 100, 200 and 400 mg/L successively, the effluent COD all presented a quick increase first, and then recovered to a steady status, namely 58 ± 14, 62 ± 9 and 63 ± 9 mg/L during Days 99–112, Days 143–166 and Days 179–207, respectively. The results illustrated that even under as high as 400 mg/L Zn (II), after adaption and adjustment, the activated sludge in ER could still absorb and consume most of the organic matter and maintained the organic degradation ability at a high level (above 90% COD removal). Nevertheless, when the added Zn (II) concentration rose to 600 mg/L, the effluent COD values began to fluctuate significantly between 108 and 221 mg/L. After 16 days of acclimation the effluent COD stabilized, but the steady COD concentration in the effluent was 137 ± 27 mg/L, significantly higher than that achieved below 400 mg/L Zn (II). Moreover, under continuous dosage of 800 mg/L Zn (II), the effluent COD varied between 176 and 650 mg/L. The fluctuations in effluent COD were more pronounced than ever before. Although the ER was continuously operated for another 3 months, the organic degradation ability did not fully recover to the comparatively steady level under 400 mg/L Zn (II) or below. So far, many researches have been conducted on the toxic/inhibitory effect of Zn (II) on activated sludge. The Zn (II) concentrations documented which had brought on distinct inhibition (>10%) on organics removal and/or nitrification rates usually ranged from 0.08 to 100 mg/L [10,20,21,32–34]. However, to the best of our knowledge, after acclimation the highest Zn (II) concentration reported which the activated sludge could sustain was 100 mg/L [32]. There are scarcely any researchers who have investigated the organics removal capability of the activated sludge under 400 mg/L Zn (II) or above. This study indicated that through raising the Zn (II) concentrations gradually, the activated sludge could endure as high as 400 mg/L of Zn (II) without distinct reduction in organic degradation ability. The acclimated activated sludge would provide
important assistance to WWTP in treating wastewater containing high concentrations of Zn (II). 3.2. The total SMP production and its correlation with effluent COD As stated above, DNA, protein, humic substances and polysaccharide were the main biochemical components of SMP [24]. Therefore in the present study, the above four constituents were determined separately. The sum of these four substances was considered as the total SMP amount and referred to as the SMP content. Similar to that observed with effluent COD, each time when a new concentration of Zn (II) was introduced to ER, its SMP content exhibited a sharp increase first and then recovered to a relatively stable level. In the stable state under each Zn (II) concentration, the SMP content was determined for at least 3 times (once every 3 or 4 days). Then the SMP content and the corresponding effluent COD value in the stable state under each Zn (II) concentration were averaged and presented in Fig. 2, to reveal the correlation between SMP and effluent soluble COD. When no Zn (II) was added, as shown in Fig. 2, the SMP content was 49.2 ± 2.5 and 51.1 ± 3.5 mg/L for CR and ER, respectively. In the whole process, the SMP in CR did not change much. As for the ER, after the addition of 50 mg/L Zn (II), its SMP content was also comparatively stable (52.3 ± 3.5 mg/L). Even if further increasing the Zn (II) concentrations to 100, 200 and 400 mg/L, the SMP amount
Fig. 2. Effect of Zn (II) on the SMP and effluent COD under different Zn (II) con) SMP in CR; ( ) SMP in ER; () COD in CR; () COD in centrations: ( ER.
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Fig. 3. Effect of Zn (II) on (a) DNA, (b) protein, (c) humic substances and (d) polysaccharide production under different Zn (II) concentrations: (
in ER only increased in a very slight degree. However, when the synthetic wastewater with 600 mg/L of Zn (II) was added, the SMP content in ER began to rise rapidly and reached 90.3 ± 5.2 mg/L. When the additive Zn (II) concentration finally reached 800 mg/L, the SMP in ER had far exceeded that in CR. It was reported that heavy metals in high concentrations can disturb cellular functions, damage cell membranes and DNA structure [11], and even bring about significant lysis of biomass cells, which would inevitably induce the notable increase of SMP content [23]. Therefore, this sharp increase in SMP quantity under 600 and 800 mg/L Zn (II) was likely result of the mass death of activated sludge and rupture of cells. This result was consistent with the drastic rises in effluent COD under the same Zn (II) concentrations as shown in Fig. 1. It was widely acknowledged that SMP was an important part of soluble organic matter in effluent [35]. In this study, the effluent soluble COD in the stable state under each Zn (II) concentration was also presented in Fig. 2. Apparently, there was a close correlation between SMP and effluent COD, in both ER and CR. In addition, under the addition of 600 and 800 mg/L Zn (II), the effluent COD was found to rise more quickly than the SMP content in ER. This is probably because under these concentrations of Zn (II), the microorganisms began to die in large numbers and the substrate CH3 COONa was not adequately utilized. This part of unconsumed organics did not reflect in SMP content but substantially contributed to the sharp rise of the effluent COD. 3.3. Change of constituents in SMP It is commonly accepted that SMP is made up of two main parts: biomass-associated products (BAP) and utilizationassociated products (UAP) [23]. The BAP is generated due to biomass decay while UAP is produced from substrate metabolism and biomass growth [23,36]. In the present study, as CH3 COONa was utilized as the sole carbon source and it did not belong to any of the SMP categories, all the SMP detected was considered to be
) CR; (
) ER.
BAP. Meanwhile, polysaccharides, proteins and lipids are the dominating constituents of biomass cell walls [25] while DNA substance mainly distributes in cellular nucleus. Considering polysaccharide, protein and DNA are also the central components of SMP [24], large-scale biomass decay and cell lysis would inevitably induce a notable increase in the concentrations of the above three SMP components [23]. In order to uncover the change and role of the four main constituents in SMP, i.e. DNA, protein, humic substances and polysaccharide in resistance to high concentrations of Zn (II), their change pattern was further investigated and shown in Fig. 3. In terms of DNA content (Fig. 3a), with the increase of Zn (II) concentration from 0, 50, 100, 200 to 400 mg/L, a comparatively smooth trend was maintained. This phenomenon suggested that the toxic effect of Zn (II) under these concentration levels on the activated sludge was unapparent and did not cause significant cell rupture. However, when continuing to increase the Zn (II) concentrations to 600 and 800 mg/L, the DNA levels in ER significantly exceeded that in CR. Under 800 mg/L of Zn (II), the DNA content in ER and CR reached 16.56 ± 1.49 and 6.01 ± 0.45 mg/L, respectively. And the difference between the two values was 10.6 ± 0.4 mg/L, which was the highest DNA gap between ER and CR in the present study. As DNA substance is mainly located in cellular nucleus, it is probable that at this time the cell membrane as well as DNA structure were severely damaged due to the toxicity of Zn (II). As a result, many cells started to rupture and substantive DNA content was released into SMP. Combined with the change in effluent COD of ER, it was estimated that the change in DNA values would to a certain degree reflect the living state of biomass under the Zn (II) toxicity. Comparatively stable DNA value below 400 mg/L Zn (II) indicated the Zn (II) concentration was within the tolerance limit of activated sludge and the metabolism of microorganisms was maintained in a steady state. On the contrary, a dramatic increase in DNA under 600 and 800 mg/L Zn (II) implied that the Zn (II) concentration was beyond the tolerance of the biomass, which would lead to the mass death of
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Table 1 The Zn (II) concentrations in influent, effluent and SMP extraction liquid of ER in the stable state. Target (mg/L)
Influent (mg/L)
50 100 200 400 600 800
51.4 101 218 407 603 814
± ± ± ± ± ±
7.6 8 14 18 22 29
Effluent (mg/L) 4.07 4.12 3.40 3.93 148 314
± ± ± ± ± ±
0.30 0.68 0.54 0.53 7 13
microorganisms, sufficient release of DNA, and distinct decrease in the organic degradation capability of the system as shown in Fig. 1. Meanwhile, the protein, humic substances and polysaccharide content all varied in a trend similar to that of the DNA content, exhibiting a slowly increasing trend under 50–400 mg/L Zn (II) and then rose sharply under 600 and 800 mg/L. These data further confirmed that after long-term domestication, the activated sludge could endure 400 mg/L Zn (II) while 600 and 800 mg/L Zn (II) may cause large numbers of cells to die and break up. However, it should be noted that under 600–800 mg/L of Zn (II), the fluctuation in protein concentrations was more pronounced than that of humic substances, polysaccharide and DNA. For instance, the protein content under 800 mg/L Zn (II) was about 3.7 times as high as that under 400 mg/L Zn (II). The corresponding ratios for humic substances, polysaccharide and DNA were around 2.2, 2.6 and 2.7, respectively, all much lower than that of protein. Moreover, it has been well documented that protein plays a significant role in microorganisms’ adaptation to the presence of heavy metals, the mechanisms of which include sequestrating the metal through binding and mitigating the toxicity of metals by enzymatic detoxification [11]. This result suggested that the protein production in the activated sludge was likely more active under the excessive Zn (II) addition than the other three SMP constituents, which suggested the important role which protein played in resistance to Zn (II) toxicity. 3.4. The function of SMP compositions in resistance to Zn (II) To further uncover the function which the SMP played in resistance to the toxicity of Zn (II), the Zn (II) concentrations in the influent, effluent and SMP extraction liquid of ER in the steady state were determined and presented in Table 1. When the added Zn (II) concentration was below 400 mg/L, the Zn (II) concentration in the effluent of ER was comparatively low (around 4 mg/L) and did not changed significantly. Meanwhile, removing 200-mL mixed liquor from the 4-L reactor daily could just discharge a fairly small portion (about 5%) of Zn (II) out of ER. As a result, most of the added Zn (II) was retained in the system. However, the Zn (II) in SMP extraction liquid only increased from 0.72 ± 0.18 to 1.62 ± 0.29 mg/L when the added Zn (II) concentrations were raised from 50 to 400 mg/L. As mentioned above, the SMP extraction liquid was obtained by filtering the mixed liquor of ER through 0.22-m filter papers. Therefore, most of the retained Zn (II) in the ER was believed to be attached to the activated sludge through bio-sorption, bioaccumulation and/or precipitation. To support this estimation, the MLSS and MLVSS values were also measured and presented. Under 50, 100, 200 and 400 mg/L of Zn (II), the MLSS achieved 2.27 ± 0.81, 3.52 ± 0.78, 4.77 ± 0.23 and 11.5 ± 4.5 g/L, respectively, which rose rapidly and far exceeded the comparatively steady MLSS value (1.19 ± 0.74 g/L) at the same time (Day 13–Day 208) in CR. Furthermore, the ratios between MLVSS and MLSS under 50, 100, 200 and 400 mg/L of Zn (II) continued to decline from 0.65 ± 0.10, 0.56 ± 0.04 and 0.45 ± 0.08 to 0.35 ± 0.02. Differently, the corresponding ratio in CR was relatively stable, with an average value of 0.85 ± 0.10. The sharp increase in MLSS and
SMP extraction liquid (mg/L) 0.72 0.86 1.24 1.62 76.0 182
± ± ± ± ± ±
0.18 0.21 0.23 0.29 10.5 15
SMP extraction liquid/effluent (%) 17.7 20.9 36.5 41.2 51.3 58.0
gradual decrease in [MLVSS]/[MLSS] ratio indicated that a large number of enthetic substances had entered into the MLSS. This result further confirmed that below 400 mg/L of Zn (II), most of the added Zn (II) had transferred into the activated sludge and this part of Zn (II) should be responsible for the significant increase in MLSS of ER. Nevertheless, when the Zn (II) concentrations in synthetic wastewater were raised to 600 and 800 mg/L, its concentrations in the effluent exhibited a large increase. The effluent Zn (II) concentrations accounted for approximately 25% and 39% of added Zn (II), respectively. It suggested that the current Zn (II) concentrations might have exceeded the adsorption and accumulation capabilities of the activated sludge. Meanwhile, the MLSS in ER under 600 and 800 mg/L Zn (II) were 13.7 ± 2.4 and 16.3 ± 2.1 g/L, respectively. Although the MLSS value still kept an upward trend, its growth rate had clearly slowed down. The corresponding [MLVSS]/[MLSS] ratios under 600 and 800 mg/L Zn (II) were 0.34 ± 0.02 and 0.33 ± 0.01, respectively, which also did not changed much compared to that under 400 mg/L. From these data it could be inferred that the growth–adsorption–growth equilibrium state in the activated sludge had been destroyed under 600 and 800 mg/L Zn (II), and the toxicity of Zn (II) became evident. This result was consistent with the conclusion that the activated sludge could stand 400 mg/L Zn (II) while 600 and 800 mg/L of Zn (II) exceeded the tolerance limit of the activated sludge in ER. Meanwhile, it should be noted that the ratio in Zn (II) concentrations between SMP extraction liquid and effluent gradually increased (Table 1), which demonstrated that more zinc ions were released into SMP extraction liquid. Meanwhile, a sharp rise in Zn (II) concentration of SMP extraction liquid was concomitant with the addition of 600 and 800 mg/L Zn (II). There were three possible sources for these emerging zinc ions: (1) after biomass decay and cell lysis, a portion of Zn (II) may still be attached to the SMP components such as polysaccharide, protein and humic substances through adsorption, ion exchange, and/or complexation [25,27,37]; (2) with the increase in SMP amount, some newly-released zinc ions by cell rupture and some newly-added zinc ions may be freshly bound and/or complexed by the chelating functional groups, such as carboxylates, hydroxyls and sulfydryls ( SH) in SMP constituents [23,38]; (3) some newly-added Zn (II), which had exceeded the adsorption and complexation ability of activated sludge and its SMP components, may still keep a unbound dissolved state and contribute to the sharp rise in Zn (II) concentration of SMP extraction liquid. It had been proven that the protein could bind heavy metals such as Zinc with different metal binding sites [11]. Moreover, the humic substances and polysaccharide were also reported to be able to bind Zn (II) through complexation and/or adsorption [39–42]. Therefore, the rise of Zn (II) concentrations in SMP extraction liquid may at least partly be attributed to the above three components of SMP. The preliminary results further indicated that protein, humic substances and polysaccharide might play an important role in the resistance to Zn (II) toxicity. Meanwhile, as the protein component increased in a larger degree than the humic substances and polysaccharide when the Zn (II) concentrations were raised from 400 to
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600 and 800 mg/L (Fig. 3), protein was postulated to play a more important role in resistance to heavy metal toxicity. More in-depth research and analysis are still needed to confirm the speculation. 4. Conclusion (1) After long-term acclimatization, the activated sludge in SBR could tolerate 400 mg/L Zn (II) while 600 and 800 mg/L of Zn (II) would led to the mass death of activated sludge and cell rupture. (2) A close correlation was observed between SMP and effluent soluble COD, in both ER and CR. (3) Under increasing Zn (II) concentrations, the SMP content, together with its four components, first maintained a slight increasing trend under 50–400 mg/L and rose rapidly between 600 and 800 mg/L. The quick increase was probably due to the mass death of activated sludge and cell lysis. (4) The Zn (II) concentrations in the effluent and SMP extraction liquid varied in a similar way as SMP, further confirming that the domesticated activated sludge could stand as high as 400 mg/L Zn (II). Acknowledgments The authors wish to thank National Key Technologies for Water Pollution Control (no. 2009ZX07318-007-01), State Key Laboratory of Pollution Control and Resources Reuse Foundation (no. PCRRF10003) and National Natural Science Foundation of China (no. 51078091) for the financial support of this study. References ˜ [1] A. Hammaini, F. González, A. Ballester, M.L. Blázquez, J.A. Munoz, Biosorption of heavy metals by activated sludge and their desorption characteristics, J. Environ. Manage. 84 (2007) 419–426. [2] EPA, Local Limits Development Guidance, EPA/833/R-04/002A, U.S. Environmental Protection Agency, Washington, DC, 2004. [3] H. Yamagata, M. Yoshizawa, M. Minamiyama, Assessment of current status of zinc in wastewater treatment plants to set effluent standards for protecting aquatic organisms in Japan, Environ. Monit. Assess. 169 (2010) 67–73. [4] General Administration of Quality Supervision, Inspection and Quarantine of PR China, Integrated Wastewater Discharge Standard, GB 8978 1996, China Environmental Science Press, Beijing, 1996. [5] X.F. Zeng, X.M. Yu, Z.W. Wang, D.B. Wei, Research on removal and recovery of heavy metals from municipal sludge, China Water Wastewater 25 (2009) 81–84. [6] F.M. Ren, Y.S. Zhou, M.C. Niu, Z.Y. Xu, Characteristics analysis and environmental assessment on heavy metals in the sludge of sewage, J. Beijing Jiaotong Univ. 31 (2007) 102–105. [7] W. Liang, H.Y. Hu, H. Wang, Y.F. Gu, Y.D. Song, Y.L. Che, Effects of micronutrients on biological treatment efficiency of textile wastewater, Fresenius Environ. Bull. 16 (2007) 1578–1582. [8] T.A. Özbelge, H. Önder Özbelge, P. Altınten, Effect of acclimatization of microorganisms to heavy metals on the performance of activated sludge process, J. Hazard. Mater. 142 (2007) 332–339. [9] X. Bing, Analysis of the effect of zinc ions on activated sludge microbes by random amplified polymorphism DNA (RAPD), Int. J. Environ. Pollut. 21 (2004) 533–546. [10] X.H. Zhou, T. Yu, H.C. Shi, H.M. Shi, Temporal and spatial inhibitory effects of zinc and copper on wastewater biofilms from oxygen concentration profiles determined by microelectrodes, Water Res. 45 (2011) 953–959. [11] M.R. Bruins, S. Kapil, F.W. Oehme, Microbial resistance to metals in the environment, Ecotoxicol. Environ. Saf. 45 (2000) 198–207. [12] E. Peltier, J. Vincent, C. Finn, D.W. Graham, Zinc-induced antibiotic resistance in activated sludge bioreactors, Water Res. 44 (2010) 3829–3836. [13] J. Li, T. Zhang, L. Wang, Y. Liu, R.H. Dai, X. Liu, Characterization and quantification of the nickel resistant microbial community in activated sludge using 16S rDNA and nickel resistance genes, Environ. Technol. 32 (2011) 533–542.
[14] J. Li, Y. Liu, T. Zhang, L. Wang, X. Liu, R.H. Dai, The effect of Ni (II) on properties of bulking activated sludge and microbial analysis of sludge using 16S rDNA gene, Bioresour. Technol. 102 (2011) 3783–3789. [15] I.R. Leighton, C.F. Forster, The adsorption of heavy metals in an acidogenic thermophilic anaerobic reactor, Water Res. 31 (1997) 2969–2972. [16] A. Cabrero, S. Fernandez, F. Mirada, J. Garcia, Effects of copper and zinc on the activated sludge bacteria growth kinetics, Water Res. 32 (1998) 1355–1362. [17] C.A. Lajoie, S.C. Lin, C.J. Kelly, Comparison of bacterial bioluminescence with activated sludge oxygen uptake rates during zinc toxic shock loads in a wastewater treatment system, J. Environ. Eng. ASCE 129 (2003) 879–883. [18] P. Principi, F. Villa, M. Bernasconi, E. Zanardini, Metal toxicity in municipal wastewater activated sludge investigated by multivariate analysis and in situ hybridization, Water Res. 40 (2006) 99–106. [19] J. Scullion, M. Winson, R. Matthews, Inhibition and recovery in a fixed microbial film leachate treatment system subject to shock loading of copper and zinc, Water Res. 41 (2007) 4129–4138. [20] S.S. S¸engör, P. Gikas, J.G. Moberly, B.M. Peyton, T.R. Ginn, Comparison of single and joint effects of Zn and Cu in continuous flow and batch reactors, J. Chem. Technol. Biotechnol. 87 (2012) 374–380. [21] H. Chua, P.H.F. Yu, S.N. Sin, M.W.L. Cheung, Sub-lethal effects of heavy metals on activated sludge microorganisms, Chemosphere 39 (1999) 2681–2692. [22] S. Sirianuntapiboon, T. Hongsrisuwan, Removal of Zn2+ and Cu2+ by a sequencing batch reactor (SBR) system, Bioresour. Technol. 98 (2007) 808–818. [23] D.J. Barker, D.C. Stuckey, A review of soluble microbial products (SMP) in wastewater treatment systems, Water Res. 33 (1999) 3063–3082. [24] Y.Y. Wei, Y. Liu, Y. Zhang, R.H. Dai, X. Liu, J.J. Wu, Q. Zhang, Influence of soluble microbial products (SMP) on wastewater disinfection byproducts: trihalomethanes and haloacetic acid species from the chlorination of SMP, Environ. Sci. Pollut. Res. 18 (2011) 46–50. [25] R.M. Pérez Silva, A.Á. Rodríguez, J.M. Gómez Montes De Oca, D.C. Moreno, Biosorption of chromium, copper, manganese and zinc by Pseudomonas aeruginosa AT18 isolated from a site contaminated with petroleum, Bioresour. Technol. 100 (2009) 1533–1538. [26] A. Ahmad, R. Ghufran, W.M. Faizal, Cd (II), Pb (II) and Zn (II) removal from contaminated water by biosorption using activated sludge biomass, Clean-Soil Air Water 38 (2010) 153–158. [27] E. Katsou, S. Malamis, M. Loizidou, Performance of a membrane bioreactor used for the treatment of wastewater contaminated with heavy metals, Bioresour. Technol. 102 (2011) 4325–4332. [28] APHA (American Public Health Association), Standard Methods for the Examination of Water and Wastewater, 19th ed., APHA, Washington, DC, 1995. [29] M. Ras, E. Girbal-Neuhauser, E. Paul, M. Spérandio, D. Lefebvre, Protein extraction from activated sludge: an analytical approach, Water Res. 42 (2008) 1867–1878. [30] A.F. Gaudy, Colorimetric determination of protein and carbohydrate, Ind. Water Wastes 7 (1962) 7–22. [31] B. Frlund, R. Palmgren, K. Keiding, P.H. Nielsen, Extraction of extracellular polymers from activated sludge using a cation exchange resin, Water Res. 30 (1996) 1749–1758. [32] C.P.C. Poon, K.H. Bhayani, Metal toxicity to sewage organisms, J. Sanit. Eng. Div. ASCE Proc. 97 (1981) 161–169. [33] P. Madoni, D. Davoli, L. Guglielmi, Response of SOUR and AUR to heavy metal contamination in activated sludge, Water Res. 33 (1999) 2459–2464. [34] S.R. Juliastuti, J. Baeyens, C. Creemers, D. Bixio, E. Lodewyckx, The inhibitory effects of heavy metals and organic compounds on the net maximum specific growth rate of the autotrophic biomass in activated sludge, J. Hazard. Mater. B100 (2003) 271–283. [35] C. Jarusutthirak, G. Amy, Understanding soluble microbial products (SMP) as a component of effluent organic matter (EfOM), Water Res. 41 (2007) 2787–2793. [36] E. Namkung, B.E. Rittmann, Soluble microbial products (SMP) formation kinetics by bioflms, Water Res. 20 (1986) 795–806. [37] B. Volesky, Biosorbent materials, Biotechnol. Bioeng. Symp. 16 (1986) 121–126. [38] W.C. Kuo, G.F. Parkin, Characterization of soluble microbial products from anaerobic treatment by molecular weight distribution and nickel-chelating properties, Water Res. 30 (1996) 915–922. [39] A. Terbouche, S. Djebbar, O. Benali-Baitich, D. Hauchard, Complexation study of humic acids extracted from forest and Sahara soils with zinc (II) and cadmium (II) by differential pulse anodic stripping voltammetry (DPASV) and conductimetric methods, Water Air Soil Pollut. 216 (2011) 679–691. [40] E. Lipczynska-Kochany, J. Kochany, Effect of humate on biological treatment of wastewater containing heavy metals, Chemosphere 77 (2009) 279–284. [41] G. 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Journal of Hazardous Materials journal homepage: www.elsevier.com/locate/jhazmat
The effect of continuous Zn (II) exposure on the organic degradation capability and soluble microbial products (SMP) of activated sludge Jing-chao Han, Yan Liu ∗∗ , Xiang Liu ∗ , Yi Zhang, Yang-wei Yan, Rui-hua Dai, Xiao-song Zha, Cheng-shan Wang Department of Environmental Science and Engineering, Fudan University, 220 Handan Road, Shanghai 200433, China
h i g h l i g h t s After acclimation, the activated sludge could endure 400 mg/L Zn (II). A close correlation was found between SMP content and effluent COD. The change in DNA might reflect the Zn (II) toxicity to the biomass.
a r t i c l e
i n f o
Article history: Received 29 May 2012 Received in revised form 9 October 2012 Accepted 10 October 2012 Available online 3 November 2012 Keywords: Zinc (II) Activated sludge Toxicity Soluble microbial products (SMP) Organic degradation ability
a b s t r a c t This study describes the change of organic degradation capability and soluble microbial products (SMP) generated in activated sludge under continuous exposure to Zn (II) in a sequencing batch reactor (SBR). In 338 days of operation, the added Zn (II) concentrations were gradually increased from 50 to 100, 200, 400 to 600 and 800 mg/L. Results showed that after adaptation, the activated sludge could endure 400 mg/L Zn (II) without showing evident reduction in organic degradation ability (92 ± 1% of chemical oxygen demand (COD) removal in stable state). However, when 600 and 800 mg/L Zn (II) were applied, the effluent water quality significantly deteriorated. Meanwhile, under increasing Zn (II) concentrations, the SMP content in the activated sludge, together with its main biochemical constituents, first increased slightly below 400 mg/L of Zn (II), then rose sharply under 600 and 800 mg/L Zn (II). Furthermore, a close correlation was found between SMP content and effluent soluble COD in both the Experimental Reactor and Control Reactor. In addition, the Zn (II) concentrations in the effluent and SMP extraction liquid were further analyzed and discussed to reveal the role that SMP constituents played in defense and resistance to the toxicity of Zn (II). © 2012 Elsevier B.V. All rights reserved.
1. Introduction With the development of modern industries such as the electroplating, chemical manufacture, mining and mineral processing, a great amount of wastewater containing high concentrations of heavy metals is generated [1]. Due to the diversity in wastewater collection systems and policies, in many countries including China, this type of wastewater is partially transported into municipal wastewater treatment plant (WWTP) instead of specially designed industrial WWTP. As a result, many heavy metals such as lead and zinc are commonly found in wastewater and activated sludge. For example, the Zn (II) concentrations in actual influent of WWTP could reach 0.01–1.28 and 0.02–0.38 mg/L, respectively in the
∗ Corresponding author. Tel.: +86 21 6564 2018; fax: +86 21 6564 3597. ∗∗ Corresponding author. Tel.: +86 21 6564 3348; fax: +86 21 6564 3597. E-mail addresses: [email protected] (X. Liu),[email protected] (Y. Liu). 0304-3894/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jhazmat.2012.10.065
States and Japan [2,3]. In terms of China, according to the “Integrated Wastewater Discharge Standard” [4], the municipal and industrial wastewater must be pre-treated to a certain criterion (no higher than 5 mg/L for Zn ions) before flowing into sewer system. However, due to the bio-sorption and/or bioaccumulation effect as well as the circumfluence of sludge, the actual Zn (II) concentration which the activated sludge endured would be much higher than that in the influent of WWTP. It was reported that the quantity of zinc in excess sludge could reach as high as 2170.6 and 2841.2 mg/kg dry sludge in some municipal WWTPs of China [5,6]. Considering biological treatment is the most widely applied process in municipal WWTP, it is greatly needed to study the toxic effects of heavy metal ions on the properties of activated sludge. With respect to the effect of Zn (II), it is widely acknowledged that Zn (II) at low concentrations can perform as micronutrients and stimulate biological processes [7,8], while high concentrations of Zn (II) would pose toxic effect to microorganisms, reduce microbial diversity and inhibit biological treatment [8–10].
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Meanwhile, when exposed to metal toxicity, at least six mechanisms have been found to be utilized by microorganisms as instinctive self-protection [11]. For instance, when encountered with a single dosage of heavy metals, protein can sequestrate the metal within cells through binding and some enzymes can also mitigate the toxicity of the metal by catalyzing the detoxification process [11]. However, the continuous exposure of activated sludge to heavy metals may induce the defense and resistance mechanisms of biomass in a more complex way [12–14]. So far, most of the relevant researches have been focused on the response and performance of activated sludge subject to shock loading of zinc in a batch or a continuous-flow reactor [15–19], and the shock zinc concentrations range from 1 to 300 mg/L. Only a few researchers [10,20–22] have investigated the effect of continuous dosage of zinc on activated sludge in a continuous-flow reactor, in which the applied Zn (II) concentrations are generally between 1 and 50 mg/L [10,20–22]. Very limited literature has dealt with the toxic effect of continuous exposure of high concentrations (400 mg/L or above) zinc ions on activated sludge in a continuous-flow mode. It has been reported that the heavy metals in high concentrations can lead to production of unspecific complicated compounds within biomass cells and induce the cells to rupture [18]. Meanwhile, apart from the biomass growth, the biomass decay and cell lysis would also release substantial amount of soluble microbial products (SMP) [23]. Protein, polysaccharide, humic substances, and deoxyribonucleic acids (DNA) were the four main biochemical constituents of SMP [24]. To some extent, the changes in SMP quantity and components may reveal the response and resistance of activated sludge to the toxicity of zinc in high concentrations. However, most literature related to this topic only investigates the efficiency and mechanisms of biosorption of zinc by activated sludge or microorganisms [1,25–27], while the effects of Zn (II) toxicity on SMP production and its components change have rarely been examined. Moreover, a deeper knowledge of the role SMP plays in the resistance to Zn (II) toxicity is still needed. The aim of this study was to evaluate the effects of continuous high concentrations of zinc on the organic degradation ability (using chemical oxygen demand (COD) as indicator), SMP production and its components change in a sequencing batch reactor (SBR). The correlation between SMP and the effluent soluble COD, the change of SMP compositions were further examined to better understand the role which SMP played in Zn (II) toxicity resistance. In addition, the Zn (II) concentrations in the effluent and SMP extraction liquid were also analyzed to validate the function of SMP.
2. Materials and methods 2.1. Experimental methods Two SBRs with 4.0 L working volume each were operated for over 338 days (excluding the initial domestication period of 54 days) at 20 ± 5 ◦ C, one as Control Reactor (CR) and the other as Experimental Reactor (ER). The seed sludge was obtained from the returned sludge of a local WWTP in Shanghai city. The SBRs were operated with 12-h cycles, and each cycle comprised of five stages: fill (0.5 h), aeration (6.0 h), and settling (2.0 h), idle (3 h) and decant (0.5 h). In each cycle, at the decant stage, 2 L of the supernatant (effluent) was discharged from the reactor. Then 2.1 L of fresh synthetic wastewater (influent) was refilled in the fill stage. Afterwards, 100-mL mixed liquor was disposed at the beginning of aeration period. On each day, two cycles were operated and thereby aggregately 200-mL mixed liquor was eliminated to maintain the sludge retention time (SRT) at 20 days. The dissolved oxygen (DO)
in the aeration stage and pH were controlled at 2.0–2.5 mg/L and 6.5–8.0, respectively. For the nutrient supply, CH3 COONa was applied as the carbon source at a concentration of around 800 mg COD/L. The COD:N:P mass ratio of the synthetic wastewater was kept at 100:5:1, and mineral nutrients were added to each liter as follows: 15 mg CaCl2 , 15 mg MgSO4 , 37.5 mg NaHCO3 , 0.1118 mg FeCl3 ·6H2 O, 0.1118 mg H3 BO3 , 0.0224 mg CuSO4 ·5H2 O, 0.1342 mg KI, 0.0894 mg MnCl2 ·4H2 O, 0.0894 mg ZnSO4 ·7H2 O, and 0.1118 mg CoCl2 ·6H2 O. After 54 days of acclimation, the activated sludge in both reactors reached a steady state. The steady state in this study was identified by the situation that the standard deviation of COD removal efficiency was within 5% for at least 14 days. The mixed liquor suspended solids (MLSS) during that stage was maintained at 1700–2200 mg/L. The 55th day was designated as Day 1 for the toxicity study, after which the two reactors were further examined for another 11 days to re-confirm the steady state. Then ER was dosed with a ZnSO4 ·7H2 O solution of increasing concentrations (50, 100, 200, 400, 600 and 800 mg/L for Zinc ions). At first Zn (II) was added to the influent of ER on Day 13, resulting in a concentration of 50 mg/L in the synthetic wastewater. After a period of adaptation, the organic degradation ability in ER reached a steady state again. The stable state was maintained for at least 14 days before the concentration of Zn (II) was raised to 100 mg/L on Day 94. This process was repeated and the influent Zn (II) concentration was increased to 200 mg/L on Day 112, 400 mg/L on Day 168, 600 mg/L on Day 209 and 800 mg/L on Day 240, respectively. The COD, MLSS and SMP of the sludge were measured periodically to evaluate the toxic effect of Zn (II) and the corresponding responses of the activated sludge. Apart from the fact that no Zn (II) was added to CR, CR was operated in the same way as ER. 2.2. Analytical methods COD, Zn (II), MLSS and mixed liquor volatile suspended solids (MLVSS) were all analyzed according to the Standard Methods [28]. The effluent COD concentration in this study refers to the soluble COD, which was obtained by filtration of the sample through 0.45-m filter papers. Except that the Zn (II) sample in SMP extraction liquid had been filtered through 0.22-m filter papers in the extraction process of SMP, the rest of Zn (II) samples were all unfiltered. After all the Zn (II) samples were digested by HNO3 solution (50%, v/v), the Zn (II) concentration was determined with a P-4010 Inductively Coupled Plasma Emission Spectrometer (Hitachi Ltd., Japan). SMP was extracted from 50-mL mixed liquor after filtration through 0.22-m filter papers. The concentrations of the main components of SMP were analyzed as such: protein and humic acid by a modified Lowry method [29], polysaccharide by an anthrone colorimetric assay [30], and DNA through a diphenylamine method [31]. 3. Results and discussion 3.1. Organic degradation ability As seen in Fig. 1, the average influent COD concentration was 761 ± 34 mg/L for both CR and ER. From Day 1 to Day 338, the COD in the effluent of CR was comparatively steady, around a value of 34 ± 16 mg/L. However, for the ER, probably due to the continuous dosage of Zn (II), the effluent COD fluctuated in a large range, with a maximum and minimum values of 650 and 18 mg/L, respectively. At the beginning of the operation when 50 mg/L of Zn (II) was added, a sharp increase was observed in the effluent COD of ER. Then the effluent COD of ER gradually decreased. Around Days 49–56, another obvious temporary increase appeared in the
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Zn (II) Concentration (mg/L) 0
50
100
900
400
200
800
600
800
COD(mg/L)
700 600 500 400 300
Influent Effluent of CR
200
Effluent of ER
100 0 0
30
60
90
120
150
180
210
240
270
300
330
360
Time (day) Fig. 1. Effect of Zn (II) on effluent COD under different Zn (II) concentrations: (♦) influent; () effluent of CR; () effluent of ER.
effluent COD of ER. It was because on Day 48, the air pump used for aeration in ER stopped to work due to some accidental malfunction. The ceasing of aeration affected the normal metabolism of activated sludge and brought on the temporary rise in the effluent COD of ER. Ultimately it took about 2 days to buy and transport a new air pump to replace the broken one and another 5 days for the system to recover to the previously COD removal level (above 85% on Days 35–47). This was also why the addition period of 50-mg/L Zn (II) lasted longer than the others. As the operation progressed, the COD values gradually returned to a stable state (53 ± 16 mg/L in Days 63–91). Similarly, when the Zn (II) concentrations were increased to 100, 200 and 400 mg/L successively, the effluent COD all presented a quick increase first, and then recovered to a steady status, namely 58 ± 14, 62 ± 9 and 63 ± 9 mg/L during Days 99–112, Days 143–166 and Days 179–207, respectively. The results illustrated that even under as high as 400 mg/L Zn (II), after adaption and adjustment, the activated sludge in ER could still absorb and consume most of the organic matter and maintained the organic degradation ability at a high level (above 90% COD removal). Nevertheless, when the added Zn (II) concentration rose to 600 mg/L, the effluent COD values began to fluctuate significantly between 108 and 221 mg/L. After 16 days of acclimation the effluent COD stabilized, but the steady COD concentration in the effluent was 137 ± 27 mg/L, significantly higher than that achieved below 400 mg/L Zn (II). Moreover, under continuous dosage of 800 mg/L Zn (II), the effluent COD varied between 176 and 650 mg/L. The fluctuations in effluent COD were more pronounced than ever before. Although the ER was continuously operated for another 3 months, the organic degradation ability did not fully recover to the comparatively steady level under 400 mg/L Zn (II) or below. So far, many researches have been conducted on the toxic/inhibitory effect of Zn (II) on activated sludge. The Zn (II) concentrations documented which had brought on distinct inhibition (>10%) on organics removal and/or nitrification rates usually ranged from 0.08 to 100 mg/L [10,20,21,32–34]. However, to the best of our knowledge, after acclimation the highest Zn (II) concentration reported which the activated sludge could sustain was 100 mg/L [32]. There are scarcely any researchers who have investigated the organics removal capability of the activated sludge under 400 mg/L Zn (II) or above. This study indicated that through raising the Zn (II) concentrations gradually, the activated sludge could endure as high as 400 mg/L of Zn (II) without distinct reduction in organic degradation ability. The acclimated activated sludge would provide
important assistance to WWTP in treating wastewater containing high concentrations of Zn (II). 3.2. The total SMP production and its correlation with effluent COD As stated above, DNA, protein, humic substances and polysaccharide were the main biochemical components of SMP [24]. Therefore in the present study, the above four constituents were determined separately. The sum of these four substances was considered as the total SMP amount and referred to as the SMP content. Similar to that observed with effluent COD, each time when a new concentration of Zn (II) was introduced to ER, its SMP content exhibited a sharp increase first and then recovered to a relatively stable level. In the stable state under each Zn (II) concentration, the SMP content was determined for at least 3 times (once every 3 or 4 days). Then the SMP content and the corresponding effluent COD value in the stable state under each Zn (II) concentration were averaged and presented in Fig. 2, to reveal the correlation between SMP and effluent soluble COD. When no Zn (II) was added, as shown in Fig. 2, the SMP content was 49.2 ± 2.5 and 51.1 ± 3.5 mg/L for CR and ER, respectively. In the whole process, the SMP in CR did not change much. As for the ER, after the addition of 50 mg/L Zn (II), its SMP content was also comparatively stable (52.3 ± 3.5 mg/L). Even if further increasing the Zn (II) concentrations to 100, 200 and 400 mg/L, the SMP amount
Fig. 2. Effect of Zn (II) on the SMP and effluent COD under different Zn (II) con) SMP in CR; ( ) SMP in ER; () COD in CR; () COD in centrations: ( ER.
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Fig. 3. Effect of Zn (II) on (a) DNA, (b) protein, (c) humic substances and (d) polysaccharide production under different Zn (II) concentrations: (
in ER only increased in a very slight degree. However, when the synthetic wastewater with 600 mg/L of Zn (II) was added, the SMP content in ER began to rise rapidly and reached 90.3 ± 5.2 mg/L. When the additive Zn (II) concentration finally reached 800 mg/L, the SMP in ER had far exceeded that in CR. It was reported that heavy metals in high concentrations can disturb cellular functions, damage cell membranes and DNA structure [11], and even bring about significant lysis of biomass cells, which would inevitably induce the notable increase of SMP content [23]. Therefore, this sharp increase in SMP quantity under 600 and 800 mg/L Zn (II) was likely result of the mass death of activated sludge and rupture of cells. This result was consistent with the drastic rises in effluent COD under the same Zn (II) concentrations as shown in Fig. 1. It was widely acknowledged that SMP was an important part of soluble organic matter in effluent [35]. In this study, the effluent soluble COD in the stable state under each Zn (II) concentration was also presented in Fig. 2. Apparently, there was a close correlation between SMP and effluent COD, in both ER and CR. In addition, under the addition of 600 and 800 mg/L Zn (II), the effluent COD was found to rise more quickly than the SMP content in ER. This is probably because under these concentrations of Zn (II), the microorganisms began to die in large numbers and the substrate CH3 COONa was not adequately utilized. This part of unconsumed organics did not reflect in SMP content but substantially contributed to the sharp rise of the effluent COD. 3.3. Change of constituents in SMP It is commonly accepted that SMP is made up of two main parts: biomass-associated products (BAP) and utilizationassociated products (UAP) [23]. The BAP is generated due to biomass decay while UAP is produced from substrate metabolism and biomass growth [23,36]. In the present study, as CH3 COONa was utilized as the sole carbon source and it did not belong to any of the SMP categories, all the SMP detected was considered to be
) CR; (
) ER.
BAP. Meanwhile, polysaccharides, proteins and lipids are the dominating constituents of biomass cell walls [25] while DNA substance mainly distributes in cellular nucleus. Considering polysaccharide, protein and DNA are also the central components of SMP [24], large-scale biomass decay and cell lysis would inevitably induce a notable increase in the concentrations of the above three SMP components [23]. In order to uncover the change and role of the four main constituents in SMP, i.e. DNA, protein, humic substances and polysaccharide in resistance to high concentrations of Zn (II), their change pattern was further investigated and shown in Fig. 3. In terms of DNA content (Fig. 3a), with the increase of Zn (II) concentration from 0, 50, 100, 200 to 400 mg/L, a comparatively smooth trend was maintained. This phenomenon suggested that the toxic effect of Zn (II) under these concentration levels on the activated sludge was unapparent and did not cause significant cell rupture. However, when continuing to increase the Zn (II) concentrations to 600 and 800 mg/L, the DNA levels in ER significantly exceeded that in CR. Under 800 mg/L of Zn (II), the DNA content in ER and CR reached 16.56 ± 1.49 and 6.01 ± 0.45 mg/L, respectively. And the difference between the two values was 10.6 ± 0.4 mg/L, which was the highest DNA gap between ER and CR in the present study. As DNA substance is mainly located in cellular nucleus, it is probable that at this time the cell membrane as well as DNA structure were severely damaged due to the toxicity of Zn (II). As a result, many cells started to rupture and substantive DNA content was released into SMP. Combined with the change in effluent COD of ER, it was estimated that the change in DNA values would to a certain degree reflect the living state of biomass under the Zn (II) toxicity. Comparatively stable DNA value below 400 mg/L Zn (II) indicated the Zn (II) concentration was within the tolerance limit of activated sludge and the metabolism of microorganisms was maintained in a steady state. On the contrary, a dramatic increase in DNA under 600 and 800 mg/L Zn (II) implied that the Zn (II) concentration was beyond the tolerance of the biomass, which would lead to the mass death of
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Table 1 The Zn (II) concentrations in influent, effluent and SMP extraction liquid of ER in the stable state. Target (mg/L)
Influent (mg/L)
50 100 200 400 600 800
51.4 101 218 407 603 814
± ± ± ± ± ±
7.6 8 14 18 22 29
Effluent (mg/L) 4.07 4.12 3.40 3.93 148 314
± ± ± ± ± ±
0.30 0.68 0.54 0.53 7 13
microorganisms, sufficient release of DNA, and distinct decrease in the organic degradation capability of the system as shown in Fig. 1. Meanwhile, the protein, humic substances and polysaccharide content all varied in a trend similar to that of the DNA content, exhibiting a slowly increasing trend under 50–400 mg/L Zn (II) and then rose sharply under 600 and 800 mg/L. These data further confirmed that after long-term domestication, the activated sludge could endure 400 mg/L Zn (II) while 600 and 800 mg/L Zn (II) may cause large numbers of cells to die and break up. However, it should be noted that under 600–800 mg/L of Zn (II), the fluctuation in protein concentrations was more pronounced than that of humic substances, polysaccharide and DNA. For instance, the protein content under 800 mg/L Zn (II) was about 3.7 times as high as that under 400 mg/L Zn (II). The corresponding ratios for humic substances, polysaccharide and DNA were around 2.2, 2.6 and 2.7, respectively, all much lower than that of protein. Moreover, it has been well documented that protein plays a significant role in microorganisms’ adaptation to the presence of heavy metals, the mechanisms of which include sequestrating the metal through binding and mitigating the toxicity of metals by enzymatic detoxification [11]. This result suggested that the protein production in the activated sludge was likely more active under the excessive Zn (II) addition than the other three SMP constituents, which suggested the important role which protein played in resistance to Zn (II) toxicity. 3.4. The function of SMP compositions in resistance to Zn (II) To further uncover the function which the SMP played in resistance to the toxicity of Zn (II), the Zn (II) concentrations in the influent, effluent and SMP extraction liquid of ER in the steady state were determined and presented in Table 1. When the added Zn (II) concentration was below 400 mg/L, the Zn (II) concentration in the effluent of ER was comparatively low (around 4 mg/L) and did not changed significantly. Meanwhile, removing 200-mL mixed liquor from the 4-L reactor daily could just discharge a fairly small portion (about 5%) of Zn (II) out of ER. As a result, most of the added Zn (II) was retained in the system. However, the Zn (II) in SMP extraction liquid only increased from 0.72 ± 0.18 to 1.62 ± 0.29 mg/L when the added Zn (II) concentrations were raised from 50 to 400 mg/L. As mentioned above, the SMP extraction liquid was obtained by filtering the mixed liquor of ER through 0.22-m filter papers. Therefore, most of the retained Zn (II) in the ER was believed to be attached to the activated sludge through bio-sorption, bioaccumulation and/or precipitation. To support this estimation, the MLSS and MLVSS values were also measured and presented. Under 50, 100, 200 and 400 mg/L of Zn (II), the MLSS achieved 2.27 ± 0.81, 3.52 ± 0.78, 4.77 ± 0.23 and 11.5 ± 4.5 g/L, respectively, which rose rapidly and far exceeded the comparatively steady MLSS value (1.19 ± 0.74 g/L) at the same time (Day 13–Day 208) in CR. Furthermore, the ratios between MLVSS and MLSS under 50, 100, 200 and 400 mg/L of Zn (II) continued to decline from 0.65 ± 0.10, 0.56 ± 0.04 and 0.45 ± 0.08 to 0.35 ± 0.02. Differently, the corresponding ratio in CR was relatively stable, with an average value of 0.85 ± 0.10. The sharp increase in MLSS and
SMP extraction liquid (mg/L) 0.72 0.86 1.24 1.62 76.0 182
± ± ± ± ± ±
0.18 0.21 0.23 0.29 10.5 15
SMP extraction liquid/effluent (%) 17.7 20.9 36.5 41.2 51.3 58.0
gradual decrease in [MLVSS]/[MLSS] ratio indicated that a large number of enthetic substances had entered into the MLSS. This result further confirmed that below 400 mg/L of Zn (II), most of the added Zn (II) had transferred into the activated sludge and this part of Zn (II) should be responsible for the significant increase in MLSS of ER. Nevertheless, when the Zn (II) concentrations in synthetic wastewater were raised to 600 and 800 mg/L, its concentrations in the effluent exhibited a large increase. The effluent Zn (II) concentrations accounted for approximately 25% and 39% of added Zn (II), respectively. It suggested that the current Zn (II) concentrations might have exceeded the adsorption and accumulation capabilities of the activated sludge. Meanwhile, the MLSS in ER under 600 and 800 mg/L Zn (II) were 13.7 ± 2.4 and 16.3 ± 2.1 g/L, respectively. Although the MLSS value still kept an upward trend, its growth rate had clearly slowed down. The corresponding [MLVSS]/[MLSS] ratios under 600 and 800 mg/L Zn (II) were 0.34 ± 0.02 and 0.33 ± 0.01, respectively, which also did not changed much compared to that under 400 mg/L. From these data it could be inferred that the growth–adsorption–growth equilibrium state in the activated sludge had been destroyed under 600 and 800 mg/L Zn (II), and the toxicity of Zn (II) became evident. This result was consistent with the conclusion that the activated sludge could stand 400 mg/L Zn (II) while 600 and 800 mg/L of Zn (II) exceeded the tolerance limit of the activated sludge in ER. Meanwhile, it should be noted that the ratio in Zn (II) concentrations between SMP extraction liquid and effluent gradually increased (Table 1), which demonstrated that more zinc ions were released into SMP extraction liquid. Meanwhile, a sharp rise in Zn (II) concentration of SMP extraction liquid was concomitant with the addition of 600 and 800 mg/L Zn (II). There were three possible sources for these emerging zinc ions: (1) after biomass decay and cell lysis, a portion of Zn (II) may still be attached to the SMP components such as polysaccharide, protein and humic substances through adsorption, ion exchange, and/or complexation [25,27,37]; (2) with the increase in SMP amount, some newly-released zinc ions by cell rupture and some newly-added zinc ions may be freshly bound and/or complexed by the chelating functional groups, such as carboxylates, hydroxyls and sulfydryls ( SH) in SMP constituents [23,38]; (3) some newly-added Zn (II), which had exceeded the adsorption and complexation ability of activated sludge and its SMP components, may still keep a unbound dissolved state and contribute to the sharp rise in Zn (II) concentration of SMP extraction liquid. It had been proven that the protein could bind heavy metals such as Zinc with different metal binding sites [11]. Moreover, the humic substances and polysaccharide were also reported to be able to bind Zn (II) through complexation and/or adsorption [39–42]. Therefore, the rise of Zn (II) concentrations in SMP extraction liquid may at least partly be attributed to the above three components of SMP. The preliminary results further indicated that protein, humic substances and polysaccharide might play an important role in the resistance to Zn (II) toxicity. Meanwhile, as the protein component increased in a larger degree than the humic substances and polysaccharide when the Zn (II) concentrations were raised from 400 to
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600 and 800 mg/L (Fig. 3), protein was postulated to play a more important role in resistance to heavy metal toxicity. More in-depth research and analysis are still needed to confirm the speculation. 4. Conclusion (1) After long-term acclimatization, the activated sludge in SBR could tolerate 400 mg/L Zn (II) while 600 and 800 mg/L of Zn (II) would led to the mass death of activated sludge and cell rupture. (2) A close correlation was observed between SMP and effluent soluble COD, in both ER and CR. (3) Under increasing Zn (II) concentrations, the SMP content, together with its four components, first maintained a slight increasing trend under 50–400 mg/L and rose rapidly between 600 and 800 mg/L. The quick increase was probably due to the mass death of activated sludge and cell lysis. (4) The Zn (II) concentrations in the effluent and SMP extraction liquid varied in a similar way as SMP, further confirming that the domesticated activated sludge could stand as high as 400 mg/L Zn (II). Acknowledgments The authors wish to thank National Key Technologies for Water Pollution Control (no. 2009ZX07318-007-01), State Key Laboratory of Pollution Control and Resources Reuse Foundation (no. PCRRF10003) and National Natural Science Foundation of China (no. 51078091) for the financial support of this study. References ˜ [1] A. Hammaini, F. González, A. Ballester, M.L. Blázquez, J.A. Munoz, Biosorption of heavy metals by activated sludge and their desorption characteristics, J. Environ. Manage. 84 (2007) 419–426. [2] EPA, Local Limits Development Guidance, EPA/833/R-04/002A, U.S. Environmental Protection Agency, Washington, DC, 2004. [3] H. Yamagata, M. Yoshizawa, M. Minamiyama, Assessment of current status of zinc in wastewater treatment plants to set effluent standards for protecting aquatic organisms in Japan, Environ. Monit. Assess. 169 (2010) 67–73. [4] General Administration of Quality Supervision, Inspection and Quarantine of PR China, Integrated Wastewater Discharge Standard, GB 8978 1996, China Environmental Science Press, Beijing, 1996. [5] X.F. Zeng, X.M. Yu, Z.W. Wang, D.B. Wei, Research on removal and recovery of heavy metals from municipal sludge, China Water Wastewater 25 (2009) 81–84. [6] F.M. Ren, Y.S. Zhou, M.C. Niu, Z.Y. Xu, Characteristics analysis and environmental assessment on heavy metals in the sludge of sewage, J. Beijing Jiaotong Univ. 31 (2007) 102–105. [7] W. Liang, H.Y. Hu, H. Wang, Y.F. Gu, Y.D. Song, Y.L. Che, Effects of micronutrients on biological treatment efficiency of textile wastewater, Fresenius Environ. Bull. 16 (2007) 1578–1582. [8] T.A. Özbelge, H. Önder Özbelge, P. Altınten, Effect of acclimatization of microorganisms to heavy metals on the performance of activated sludge process, J. Hazard. Mater. 142 (2007) 332–339. [9] X. Bing, Analysis of the effect of zinc ions on activated sludge microbes by random amplified polymorphism DNA (RAPD), Int. J. Environ. Pollut. 21 (2004) 533–546. [10] X.H. Zhou, T. Yu, H.C. Shi, H.M. Shi, Temporal and spatial inhibitory effects of zinc and copper on wastewater biofilms from oxygen concentration profiles determined by microelectrodes, Water Res. 45 (2011) 953–959. [11] M.R. Bruins, S. Kapil, F.W. Oehme, Microbial resistance to metals in the environment, Ecotoxicol. Environ. Saf. 45 (2000) 198–207. [12] E. Peltier, J. Vincent, C. Finn, D.W. Graham, Zinc-induced antibiotic resistance in activated sludge bioreactors, Water Res. 44 (2010) 3829–3836. [13] J. Li, T. Zhang, L. Wang, Y. Liu, R.H. Dai, X. Liu, Characterization and quantification of the nickel resistant microbial community in activated sludge using 16S rDNA and nickel resistance genes, Environ. Technol. 32 (2011) 533–542.
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