Synergic effect of adsorption and biodegradation enhance cyanide removal by immobilized Alcaligenes sp. strain DN25

Accepted Manuscript Title: Synergic effect of adsorption and biodegradation enhance cyanide removal by immobilized Alcaligenes sp. strain DN25 Authors: Qingyun Li, Hui Lu, Yexing Yin, Yiming Qin, Aixing Tang, Haibo Liu, Youyan Liu PII: DOI: Reference:

S0304-3894(18)30897-5 https://doi.org/10.1016/j.jhazmat.2018.10.007 HAZMAT 19827

To appear in:

Journal of Hazardous Materials

Received date: Revised date: Accepted date:

21-5-2018 8-8-2018 2-10-2018

Please cite this article as: Li Q, Lu H, Yin Y, Qin Y, Tang A, Liu H, Liu Y, Synergic effect of adsorption and biodegradation enhance cyanide removal by immobilized Alcaligenes sp. strain DN25, Journal of Hazardous Materials (2018), https://doi.org/10.1016/j.jhazmat.2018.10.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synergic effect of adsorption and biodegradation enhance cyanide removal by immobilized Alcaligenes sp. strain DN25 Qingyun Lia,b, Hui Lua, Yexing Yina, Yiming Qina, Aixing Tanga, Haibo Liua, Youyan Liua,b,c*

(bGuangxi Key Laboratory of Biorefining, Nanning, 530003, Guangxi, PR China)

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(aCollege of Chemistry and Chemical Engineering, Guangxi University, Nanning, 530004, Guangxi, PR China)

(cGuangxi Colleges and Universities Key Laboratory of New Technology and Application in Resource Chemical Engineering, Guangxi University, Nanning, 530004, Guangxi, PR China)

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Graphical abstract:

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Highlights •A cyanide removal system with a synergic effect of adsorption and biodegradation. •The PUF-immobilized cells system showed higher removal efficiency and stability. •Removal capacity of the PUF-immobilized cells system could reach 1200 mg CN-/L.

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• The enhancement of the SAB immobilized cells system was discussed.

Abstract

A high efficiency and stability polyurethane-foam (PUF)-immobilized cell system was constructed to remove cyanide based on simultaneous adsorption and biodegradation (SAB). The performance of the PUF-

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immobilized system was evaluated by comparison with the freely suspended cell system. The SAB system

exhibited more effective and robust, and could still remain degradation activity even at 40°C or pH 11.0. The

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SAB system completely removed 500 mg CN-/L within 8 hours at 30°C, pH 8.0, and 120 rpm, whereas 12 hours

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were required for the free cells system. Moreover, the SAB system showed apparent superiority in removing

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higher cyanide concentrations up to 1200 mg /L. A continuously stirred tank bioreactor (CSTR) was successfully designed and steadily operated with approximately 85% of the total average removal efficiency for 52 days at

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an influent cyanide concentration of 100-200 mg/L, which demonstrated a favorable reliability. Cyanide removal

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process could be well described using a pseudo first-order model, and the higher apparent rate constants (k) of the immobilized cells showed the synergic effect of adsorption and biodegradation significantly enhanced cyanide removal. Preliminarily, it was found that the foam characteristic might play a not negligible role on the cyanide-degrading enzyme expression of strain DN25 in the SAB system.

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Keywords: Cyanide; Immobilized cells; Biodegradation; Synergy; Bioreactor

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*Corresponding author; Tel.: +86 771 323 6385; Fax: +86 771 323 3718; E-mail: [email protected]

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1. Introduction As a potent metabolic inhibitor to humans and aquatic organisms, cyanide is listed as a priority pollutant by the U.S. Environmental Protection Agency (EPA) [1] as well as in China. The permitted standard level of total cyanide is set to be 0.5 mg/L for integrated wastewater discharge by Ministry of Ecology and Environment of China. However, many industries, such as mining, dye production, synthetic fibers and plastics, use cyanide

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as raw material. The total worldwide discharge amount of cyanide is estimated to be nearly 14×106 kg per year

[2], and cyanide leakage occurs unpredictably all over the world. Biotreatment mediated by microorganisms or enzymes has become a common choice for the removal of such hazardous chemicals [3]. Many microorganisms,

including bacterial strains of Pseudomonas, Klebsiella and Alcaligenes as well as several fungi spices of

Fusarium solani and Trametes versicolor were thus isolated for the treatment of cyanide and its derivatives [5][7], and certain microorganisms have even been employed in practical application [8][9].

The technique of immobilization has many applications as a measure to improve the stability and efficiency

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in the bioremediation process [10][11]. More attractively, a proper and elaborate support would endow the

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immobilized cells with certain unique properties, such as the recently reported simultaneous adsorption and

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biodegradation (SAB) system, in which porous materials, active carbon [12], macroporous resin [13], and bamboo charcoal [14] were chosen to immobilize the cells. The observed synergistic effects were generally

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considered to be related to the dual-function of the support. The support would provide an interface for enhanced biomass accumulation, and the adsorption of the target pollutant on solid surface would promote sufficient

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contact with the cells, which results in the acceleration of the degradation process [15][16]. Dash et al. reported the removal of ferrocyanide by employing Pseudomonas fluorescens immobilized on granular activated carbon

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[17]. The removal efficiency of 300 mg/L ferrocyanide increased from 69.3% to 81.8% through the synergistic action of adsorption and biodegradation. Therefore, such immobilized systems appear an appealing for the establishment of this rapid treatment process.

Polyurethane foam (PUF) is a type of porous material that is recommended to be an emerging water

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treatment medium by the U.S. EPA because of its good mechanical properties, larger contaminant capture capacity, environmentally friendly and competitive price [18]. Similarly, PUF is a good carrier for enzyme or cell immobilization. Many studies have reported the application of PUF-immobilized cells to remove toxic

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chemicals, such as naphthalene [20],2-nitrotoluene [21], and chlorpyrifos [22]. A higher removal efficiency and better stability have been observed in these cases. However, the biotreatment of cyanide with PUFs as the immobilized support has not been reported as far as is known. Moreover, a deeper understanding of the SAB process must be obtained for future engineering applications. Based on the goal of constructing a purposeful immobilized cell system adapted to cyanide removal, PUF was thus selected because it showed good adsorption abilities for cyanide in our previous work. An Alcaligenes 3

sp. strain DN25 was immobilized to investigate the cyanide removal capacity. As expected, the immobilized cell system exhibited a strong synergistic effect and achieved higher removal efficiency, especially at a high concentration. Data relevant to the immobilized and freely suspended cells were included and compared to elucidate the synergistic effect. A continuous reactor was also constructed to evaluate the system stability. This PUF-immobilized system would provide a new strategy for the rapid elimination of this type of dangerous and

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toxic pollutant.

2. Materials and methods 2.1 Bacterial strain and materials

The cyanide-degrading strain DN25, identified as Alcaligenes sp. by a 16S rDNA sequence analysis, was

previously isolated in our laboratory and preserved in the China General Microbiological Culture Collection Center (CGMCC 5734). Polyurethane foam was purchased from Hu zhou H＆T New Material Techonlogy Co.,

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Ltd (Huzhou, China). All other chemicals used in this study were of analytical reagent grade or high purity.

Cyanide stock solution was prepared by dissolving a certain amount of KCN in 0.1 mol/L sodium

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hydroxide solution to obtain a final cyanide concentration of 2000 mg/L. Unless otherwise noted, the

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concentration of cyanide in this study refer to CN- concentration.

2.2 Preparation of supports and immobilization of strain Alcaligenes sp. DN25

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The PUF was cut into cubes (approximately 5mm ×5 mm ×5 mm) and then successively eluted with 5% NaOH and HCl solution, washed with distilled water several times to pH 7.0, and finally oven dried at 60°C to

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constant weight for the following use as immobilized supports.

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A pure culture of Alcaligenes sp. DN25 was incubated in a rotary shaker at 30°C with 120 rpm for 24 hours and used as an inoculum. The composition of the strain growth medium was as follows (g/L): 2.0 K2HPO4, 0.5 NaCl, 2.0 (NH4)2SO4, 5.0 yeast extract, 5.0 peptone, 10.0 glucose and pH 8.0. The immobilization was conducted according to the procedure described by Manohar [20] and Ory [23] with certain modifications. In this work, 0.0500 g dried foam cubes were weighed and placed in a 250 mL cotton-plugged flask containing 20 mL of

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growth medium. Next, the flasks were autoclaved at 121°C for 20 minutes and 2 mL of the Alcaligenes sp. inoculums prepared above was added after the media was cooled. All flasks were incubated by shaking at 30°C and 120 rpm for 48 hours (during the stationary phase) until immobilized biofilms formed on the surface of the

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supports. Then the biocarriers were collected and gently washed at least three times with 0.05 M phosphate buffer (pH 8.0) to remove any culture remnants. Consequently, a portion of immobilized cells were obtained for cyanide degradation experiments, or washed with deionized water for biomass determination. To prevent inactivation, the prepared immobilized cells can be stored at 4oC. 2.3 Cyanide removal experiments To better study the synergic effect of the immobilized cell system, the biomass of the system was set equally 4

as much as the freely suspended cell system. Cyanide removal experiments were performed as follows: a share of immobilized cells (0.0100 g dry weight) was added into a sterile 250 mL conical flask containing 20 mL phosphate buffer (pH 8.0). The KCN stock solution was sterilized with a 0.22μ filter and added to the conical flask to achieve a given concentration. All the flasks were tightly sealed with stoppers to prevent cyanide volatilization and then placed in a shaker at 30°C with 120 rpm for degradation. At regular intervals, samples

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were collected, and the concentration of residual cyanide was measured. Control experiments using freely suspended cells (0.0100 g dry weight) were simultaneously conducted for comparison.

Additionally, experiments of cyanide adsorption onto uninoculated and sterilized foam cubes (0.0500 g dried weight) were investigated with the same procedure mentioned above. Another control set of uninoculated flasks, i.e., without foam cubes and biomass, were also conducted concurrently to examine the possible removal of cyanide due to other causes. 2.4 Influence of different factors on cyanide removal

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To assess the effectiveness of the immobilized cell system, key factors, including temperature (20-40°C),

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pH value (7.0-11.0), and CN- concentration (10-800 mg/L), were examined according to the procedures mentioned above.

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The cyanide removal efficiency was determined by calculating the average specific biodegradation rate,

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which was expressed as mg/L•h•g CDW. The cyanide adsorption efficiency was calculated from the difference between the initial and residual cyanide concentration, following the equation (%) = (c0-ct)/c0 ×100, where c0

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and ct were the cyanide concentrations at time zero (0) and time t (h), respectively. 2.5 Kinetic analysis of cyanide removal

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Pseudo first-order kinetics is generally used to describe the biodegradation of different pollutants. The kinetics of cyanide removal process was evaluated based on data obtained from experiments using a pseudo firstorder model. The pseudo first-order rate equation is as follows: ct = c0e−kt

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where ct is the concentration of cyanide after treatment, c0 is the initial concentration of cyanide, k is the degradation constant, and t is time. 2.6 Construction and operation of immobilized cell bioreactors

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The long-term stability of the PUF-immobilized cell system for cyanide removal was tested in the

sequencing batch reactor (SBR) and continuously stirred tank reactor (CSTR), respectively. The 250 mL conical flasks were used as the SBRs, and cyanide loading was 100 mg CN-/L. The operating conditions of the SBRs were the same as those described in Section 2.3. After every 24 hours, the immobilized cells were filtered and washed slightly with phosphate buffer (pH 8.0). Then, 20 mL of fresh buffer containing CN- was added to start a new cycle. The residual cyanide concentration was analyzed at the end of each batch. 5

A CSTR was basically composed of an influent tank, a stirred tank with water bath and magnetic mixer, two constant flow pumps and an effluent tank (Fig.1). The working volume of the reactor was approximately 1.0 L. To minimize cyanide leakage, the top of all the tanks were covered and sealed with plugs or tapes. At the bottom of CSTR, the previously prepared immobilized cells were evenly fixed on a stainless shelf and a magnetic stirring was used for the better mass transfer. The CSTR was started up with simulated wastewater, i.e.,

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phosphate buffer (pH 8.0) containing cyanide with a concentration of 100 mg/L at a flow rate of 70 mL/ h via a peristaltic pump. The temperature of the whole reactor was kept at 30±1°C through a constant temperature water

bath with hot water circulation. The residual cyanide concentration was measured by sampling the effluent from the outlet filter. 2.7Analytical methods

The cyanide concentrations were determined by pyridine-barbituric acid colorimetric methods at 580nm [24]. The biomass analysis was based on the cell dry weight method. Freely suspended biomass samples (cell

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dry weight, CDW) were dried at 80°C in a vacuum oven to a constant weight. The biomass amount on the foam

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cubes was calculated according to the weight difference before and after the immobilization by subtracting the

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weight of the blank control samples. The morphologies of PUF and immobilized cells were observed using scanning electron microscopy (Hitachi, SU8020), and a brief description of the preparation was provided in

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Supplementary Material. The methods of total RNA isolation, reverse-transcription PCR and Quantitative realtime PCR reaction were briefly described in Supplementary Material.

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The formate and formamide were analyzed by a high performance liquid chromatograph (HPLC Dionex Ultimate 3000, USA) according to the methods of Ingvorsen et al.[25] using a Rspak KC-811 (8 mm × 300 mm,

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6-μm particles; Shodex, Japan) and a mobile phase of H3PO4 (pH 2.0) in H2O (flow rate: 0.5 mL/min; 30°C). The UV detector was set at 200 nm. The ammonia concentrations were determined following the Nessler's reagent colorimetric methods described in Standards Methods [24]. In this work, all the single factor experiments were repeated at least in triplicate. The mean values and

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standard deviations were calculated by excel. Data are shown as the mean ± SD (error bars) of three replicates.

3. Results and discussion

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3.1 Cyanide removal experiments Cyanide is volatile and photolysis. In order to determine the role of adsorption, biodegradation and other

abiotic factors in cyanide removal, corresponding experiments were performed, respectively. Fig. 2 presented the time curve of cyanide adsorption and desorption by the foam cubes. The maximum cyanide adsorption of 50 mg CN-/L by the foam cubes was approximate 16.1%, and the adsorption equilibrium was reached after two hours. A similar trend as adsorption was observed for cyanide desorption, which was relatively easy with a maximum desorption of 77.7%. In the control groups with no foam cubes and the cells, only less than 3% 6

cyanide reduction was observed, thus showed that volatilization and photolysis was only a small proportion for cyanide removal. In contrast, cyanide degradation mediated by both immobilized and free cells was rather fast as shown in Fig. 3. For the cyanide solution of 50 mg CN-/L, more than half of the cyanide could be degraded by the freely suspended cells within 45 minutes, and 98.4% of cyanide was removed after 3 hours. The cyanide removal rate

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of the PUF-immobilized cells was even higher that half of cyanide degradation after only 20 minutes, and the cyanide content in the solution decreased to an undetectable level after 2 hours. Furthermore, limited cyanide was detected in the foam cubes when degradation finished (data not shown), indicating that cyanide was

ultimately completely degraded by the immobilized cells. In addition, we traced the products and found that formate, formamide and ammonia were detected during cyanide biodegradation compared to the control groups (Fig.S1). And moreover, the formation of these products occurred with the decreasing of cyanide, meaning a successful detoxification was achieved for the environment.

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3.2 Effect of temperature on cyanide removal

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Since temperature is commonly thought to exert a stronger effect on chemical process than physical process,

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bioprocess rather than cyanide adsorption process was more affected by temperature as shown in Fig. 4. The cyanide adsorption was maintained between 11 and 17% in the range of 20-40°C, with the maximum observed

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at 35°C. In terms of the bioprocess mediated by the freely suspended cells or immobilized cells, the highest removal rate was close to 4 times higher than the lowest one.

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Meanwhile, certain differences were observed between the two forms of cells. In the case of the freely

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suspended cells, the removal rate reached a maximum of 2503.25 mg/L•h•g CDW at 30°C and dramatically reduced when the temperature diverged from 30°C. PUF-immobilized cells tended to perform better in which the maximal average removal rate increased to 4104.70 mg/L•h•g CDW, which was nearly doubled that of the freely suspended cells. Even the temperature rose to 40°C, the immobilized cells could still maintain more than 50 percent of the maximum removal rate, suggesting better adaptability to the change in temperature.

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3.3 Effect of initial pH value on cyanide removal The pH value was another important factor affecting the process. Figure 5 depicted the variation trend of the cyanide adsorption and average specific removal rate with an initial pH value in the range of 7.0 to 11.0. The

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influence of pH on cyanide adsorption was far less notable than its influence on biodegradation. Cyanide adsorption appeared to decline as the pH value increased, and the maximum was observed at pH 7.0, which could be due to the competitive adsorption between cyanide ion and hydroxyl ion at high pH value. Similar to temperature, pH showed a U-typed effect on cyanide average specific removal rate. The freely suspended cells and PUFs-immobilized cells both achieved a maximum removal rate at pH 8.0, corresponding to 2498.75 and 3891.87 mg/L•h•g CDW, respectively. When the pH value was higher than 8.0, the cyanide average specific 7

removal rate in both systems started to decrease. However, the removal rate of PUF-immobilized cells declined more slowly than that of the free cells. The cyanide average specific removal rate at pH 11.0 was 880.44 mg/L• h•g CDW for PUF-immobilized cells and only 454.72 mg/L•h•g CDW for freely suspended cells. The good tolerability of the PUF-immobilized cells would be a clear benefit for practice. 3.4 Removal performance at cyanide concentration of 10-800 mg/L

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The cyanide concentration is anticipated to play an important role in the microbial activity and adsorption

behavior. As illustrated in Fig. 6, the adsorption capacity of the foam cubes obtained a maximum (5.88 mg CN/g foam cubes) at a cyanide concentration of 200 mg/L and remained constant with increased cyanide

concentration, which mean that the foam cubes reached adsorption saturation. Regarding the cyanide average specific removal rate, minor differences were observed between the freely suspended cells and PUF-immobilized

cells at concentrations below 100 mg/L. For example, at a cyanide concentration of 10 mg/L, the cyanide average

specific removal rate was 920.79 mg/L•h•g CDW for the former and 960.40 mg/L•h•g CDW for the latter. As

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the cyanide concentration increased, the differences became so great that the cyanide removal by the immobilized

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cells was at least one time faster than that by the free cells at a cyanide concentration of 800 mg /L. Of even

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greater concern was little toxic effect on the PUF-immobilized cells when the cyanide concentration was below 1200 mg/L. As a result, the immobilized cells obtained a maximum removal rate of 32534.65 mg/L•h•g CDW

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at 1200 mg/L, while the freely suspended cells reached a maximum removal rate of 17673.27 mg/L•h•g CDW at 700 mg/L and decreased dramatically with increased cyanide concentration until 1200 mg/L, when no activity

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3.5 Cyanide removal kinetics study

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was detected.

The time course of cyanide removal by the PUF-immobilized cells and freely suspended cells were presented in Fig. 7. In term of either initial removal rate or average rate, the PUF-immobilized cells manifested higher efficiency than the free cells within the tested concentration range. The immobilized cells completely remove 500 mg/L of cyanide within only 8 hours, while the free cells took 12 hours. Compared to

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the zero-order and second-order equations (data not shown), the cyanide removal process could be well described using a pseudo first-order model (R2>0.98). The kinetic parameters listed inTable1showed much higher apparent rate constants (k) of the PUF-immobilized cells than those of the free cells. The maximum k reached at a cyanide

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concentration of 200 mg/L might be associated with the saturation of adsorption by the foam. Moreover, both the apparent rate constants of the two types of cells presented a decline trend with the increase in cyanide concentration, implying that highly concentrated cyanide has an inhibitory effect on the enzyme activity. The Haldane model was consequently proposed to describe the inhibition kinetic. Figure 8 revealed that the experimental data had preferably coherence with the theoretical data (R2>0.99) using professional data analytics software Origin Pro. 8.6. According to the inhibition kinetics constants of the Haldane model, the predicted 8

maximum removal rates were reached at a cyanide concentration of CPUF,opt =(KmKi)1/2=(2295.44 × 552.67)1/2=1126.3 mg/L and CFreely,opt =(KmKi)1/2=(588.60×774.44)1/2=675.2 mg/L for PUF-immobilized cells and freely suspended cells, respectively, which were consistent with our experimental results. 3.6 Performance of the bioreactors SBRs fed with simulated synthetic wastewater containing 100 mg/L of cyanide were examined to evaluate

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the stability of the PUF-immobilized cells. As shown in Fig.9, the PUF-immobilized cells could maintain above

95% of cyanide removal efficiency after 10 recycles. Although a declining trend was observed in the following 5 recycles, continuous and steady operation of the immobilized system was still anticipated. Subsequently, a

CSTR was developed and operated in the influent range from 100 mg CN-/L to 200 mg CN-/L. Figure 10 demonstrated that the average cyanide removal efficiency of the CSTR was above 90% in the first 27 days at

startup of 100 mg CN-/L, thus exhibiting favorable reliability and activity. Although the cyanide concentration of the influent was increased to 200 mg/L in the following days, the cyanide removal efficiency did not

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dramatically decline. As a consequence, the total average removal efficiency was calculated to be approximately

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85% during 52 days of operation. Therefore, the system here showed a satisfactory adaptability and flexibility.

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Furthermore, given that the cyanide treatment in the CSTR was mediated by the resting cells, a long-time operation and robust system should be realized if regulating the cells proliferation in the CSTR.

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Discussion

The immobilized cells systems with synergic effect of adsorption and biodegradation exhibited

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broad application prospects in the field of contaminants elimination and thus prompted researchers to design and

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construct systems especially for the treatment of high-concentration wastewater [25][27]. The PUF-immobilized cells system reported here might be considered as a successful example for cyanide treatment. The higher efficiency in the SAB system than adsorption and biodegradation alone was consistent with those reported in the literatures [14][17]. For cyanide concentration of 50 mg/L, the removal efficiency of SAB system was about 72.7% within 30 minutes, whereas the adsorption and biodegradation were 9.41% and 43.9%, respectively (Fig.

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2 and Fig.3). The maximum average specific cyanide removal rate of the SAB system was 1.8 times higher than that of the freely suspended one. The immobilized system not only showed high activity up to a cyanide

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concentration of 1200 mg/L, but also kept a steady and continuous operation for more than 50 days in the CSTR. Few studies have reported a maximum cyanide tolerance concentration higher than 1000 mg/L in the literature [28]. Therefore, the PUF-immobilized cell system has excellent performance in terms of efficiency and stability compared with the previous results. There are many explanations for the synergistic effect of the SAB system. A widely accepted was the mechanism of adsorption-desorption and biodegradation [16][26][29]. Firstly, the concentration of pollutants in 9

the bulk liquid would be decreased sharply in a short time by the fast adsorption of supports. Then the biodegradation process would occur following the pollutants desorption. Hence, to achieve the strengthening effect, supports with high adsorptivity always become the preferred option. Zheng et al. [25]developed a SAB system to remove anionic dye acid orange 10 (AO10) using mesoporous high-surfacearea activated carbon. The initial AO10 concentration of 1000 mg/L could be removed completely within 60

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min by the support. Benefiting from the highly efficiency adsorption of the support, the immobilized P. putida cells completely removed 6000 mg/L of AO10 within 84 h，whereas the maximum concentration of AO10 biodegraded by P. putida cells was only 250 mg/L in 72 h. On the other hand, the enhancement was related to the motivational role of adsorption in the transfer-reaction process. Adsorption would theoretically accelerate

the mass transfer from the bulk liquid to the support, leading to a more sufficient contact and a quick start for

biodegradation. Wang et al. successfully constructed a SAB system with high diesel oil biodegradation efficiency,

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where a full and direct contact between immobilized cells and oil resulting from the adsorption by the expanded

graphite (EG) played a main role [16]. Dash et al. used granular activated carbon as the support and found that

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the ferrocyanide removal started at an earlier time in the SAB system than that in biodegradation process alone

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[17]. Though there was no obvious quick start removal observed in our case (Fig.3), the cyanide removal

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efficiency of the SAB system was in general significantly higher than that of the free cells under the different temperature, pH value, and initial cyanide concentration conditions (as shown in Fig.4, Fig.5, and Fig.6). The higher apparent rate constants (k) of the immobilized cells (Table 1) and the shorter removal time (Fig.7) also

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manifested that the support adsorption effectively accelerated cyanide biodegradation. Therefore, the results

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suggested that the strengthened contact between the cells and cyanide via adsorption is a first step for the enhancement of the biodegradation process.

Another important factor underlying the enhancement could be the influence of support on the cells. Because porous materials promote the ability of microorganisms to attach and growth, a higher density of biomass is generally observed in immobilized cell systems, which is commonly considered to be the direct reason

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for the enhancement [29]30,[31]. Ory et al. reported that the strain of Acetobacter aceti was immobilized on the PUF with a large number of cells within the shortest time compared with Siran, and wood chips; thus it achieved

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the highest acetification rate [32]. In our study, a high density of Alcaligenes sp. strain DN25 was also observed on the foam cubes (Fig.S2), but the cyanide removal experiments were purposely try to conduct under the same biomass condition both in the immobilized and the free system, and the specific removal rates were compared as well. Therefore, we excluded the impact of increased biomass on the enhancement. Moreover, the support characteristics on the catalytic property of cells should not be overlooked because they play a more significant role in the biodegradation process. In fact, considerable research has shown that the special microenvironment around the supports is far different from that of the free cell system [33][34]. The effect of 10

the material properties on the enzyme production and activity has been reported in literatures. For example, Shim et al. found that the cells growth and lignin peroxidase production by Phanerochaete chrysosporium were effectively enhanced after immobilization and changed with different carriers [35]. Chen et al. suggested that the biodegradation of carbaryl may be dominantly controlled by the activity of the degrading microorganisms in the SAB system, based on the results that montmorillonite improved the activity of P. putida for biodegradation,

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whereas goethite displayed an inhibitory effect [36]. Ugochukwu et al. even identified the surface area and the local bridging effect of the clay mineral support as the important factors that determined the stimulatory role to

the cells [27]. According to the fine properties of PUF [37][38], we consequently speculated that the support might have a positive effect on the enzyme secretion and expression of the cell. Preliminarily, the q-PCR analysis

showed that the expression level of the cyanide-degrading enzyme of strain DN25 increased by 16% after immobilization (Fig.S3) However, further research is needed to determine which the feature parameters, such

as aperture, pore structure, surface area, and constituent, have a key effect on the enzyme secretion and

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expression, which would help to deeper understand the synergic effect in the SAB system.

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Conclusions

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In this study, PUF was purposely selected to immobilize Alcaligenes sp. for cyanide removal considering the synergic interaction between adsorption and biodegradation. Effects of important parameters on cyanide

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adsorption and degradation process were investigated. The PUF-immobilized cell system showed strong tolerance to high temperature, pH and cyanide concentration. The cyanide removal efficiency of the SAB system

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was greatly enhanced compared to the freely suspended cell system, which was thought to not only because of

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the PUF adsorption, but also the impact of support characteristics on the expression level of the cyanidedegrading enzyme. The good performance of the SAB system would provide a promising alternative for cyanidecontaining wastewater treatment. Conflict of interest

The authors of this study declare no conflict of interest.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (No.51108098) and the

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Scientific Research Foundation of GuangXi University (No.XJZ130360).

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mining and jewellery industries, Curr. Opin. in Biotechnol. 38 (2016) 9-13 [9] L.Mekuto, S.K.O.Ntwampe, A. Akcil, An integrated biological approach for treatment of cyanidation

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wastewater, Sci. Total Environ. 571(2016) 711-720 [10] B. Jiang, L. Tan, S.X. Ning, S.N. Shi, A novel integration system of magnetically immobilized cells

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and a pair of graphite plate-stainless iron mesh electrodes for the bioremediation of coking wastewater, Bioresource Technol. 216 (2016) 684-690. [11] J.J. Lin, L. Gan, Z. L. Chen, R. Naidu, Biodegradation of tetradecane using Acinetobacter venetianus immobilized on bagasse, Biochem. Eng. J. 100 (2015) 76-82.

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[12] M. S. Islam, Y.Y. Zhang, K. N. McPhedran, Y. Liu, M. Gamal Eldin, Granular activated carbon for simultaneous adsorption and biodegradation of toxic oil sands process-affected water organic compounds, J. Environ. Manage. 152 (2015) 49-57.

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[13] Y. Wang, H. Chen , Y. X. Liu, R. P. Ren, Y. K. Lv, An adsorption-release-biodegradation system for simultaneous biodegradation of phenol and ammonium in phenol-rich wastewater, Bioresource Technol. 211 (2016) 711-719. [14] Y. Chen, B. Yu, J.J Lin, R. Naidu, Z.L. Chen, Simultaneous adsorption and biodegradation (SAB) of diesel oil using immobilized Acinetobacter venetianus on porous material, Chem. Eng. J. 289 (2016) 463-470. 12

[15] H.F. Zhuang, H.J. Han, P. Xu, B.L. Hou, S.Y. Jia, D.X. Wang, K. Li, Biodegradation of quinoline by Streptomyces sp. N01 immobilized on bamboo carbon supported Fe3O4 nanoparticles, Biochem. Eng. J. 99 (2015) 44-47. [16] X. Wang, X. J. Wang, M. Liu, Y. J. Bu, J. Zhang, J. Chen, J. F. Zhao, Adsorption–synergic biodegradation of diesel oil in synthetic seawater by acclimated strains immobilized on multifunctional

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materials, Mar. Pollut. Bull. 92 (2015) 195-200. [17] R. R. Dash, C. Balomajumder, A. Kumar, Treatment of metal cyanide bearing wastewater by simultaneous adsorption and biodegradation (SAB), J. Hazard. Mater. 152 (2008) 387-396.

[18] C. Guimaraes, P. Porto, R. Oliveira, M. Mota, Continuous decolourization of a sugar refinery wastewater in a modified rotating biological contactor with Phanerochaete chrysosporium immobilized on polyurethane foam disks, Proc. Biochem. 40 (2005) 535-540.

[19] E. F. Gómez, X. Luo, C. Li, F.C.M. Jr, Y. Li, Biodegradability of crude glycerol-based polyurethane

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foams during composting, anaerobic digestion and soil incubation, Polymer Degradation & Stability

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[20] S. Manohar, C. K. Kim, T. B. Karegoudar, Enhanced degradation of naphthalene by immobilization

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of Pseudomonas sp. strain NGK1 in polyurethane foam, Appl. Microbiol. Biotechnol. 55 (2001) 311-

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[21] S. I. Mulla, M.P. Talwar, Z. K. Bagewadi, R. S. Hoskeri, H. Z. Ninnekar, Enhanced degradation of 2-

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EP TE

[22] M. Yadav, N. Srivastva, R. S. Singh, S. N. Upadhyay, S. K. Dubey, Biodegradation of chlorpyrifos by Pseudomonas sp. in a continuous packed bed bioreactor, Bioresource Technol. 165 (2014) 265269.

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wood chips and polyurethane foam as carriers for Acetobacter aceti, Process. Biochem. 39 (2004) 547-555.

[24] APHA, Standards Methods for the Examination of Water and Wastewater, American Public Health

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[25] K. Ingvorsen, B. Hojer-Pedersen, S.E. Godtfredsen, Novel cyanide-hydrolyzing enzyme from Alcaligenes xylosoxidans subsp. denitrificans. Appl. Environ. Microb. 57(1991) 1783-1789. [26] Y. Zheng, D. Y. Chen, N. J. Li, Q. F. Xu, H. Li, J. H. He, J. M. Lu, Highly efficient simultaneous adsorption and biodegradation of a highly-concentrated anionic dye by a high-surface-area carbonbased biocomposite, Chemosphere 179 (2017) 139-147. 13

[27] U. C. Ugochukwu , M. D. Jones, I. M. Head, D. A.C. Manning, C. I. Fialips, Biodegradation and adsorption of crude oil hydrocarbons supported on“homoionic” montmorillonite clay minerals, Applied Clay Science 87 (2014) 81-86. [28] Z. Javaheri Safa, S. Aminzadeh, M. Zamani, M. Motallebi, Signifcant increase in cyanide degradation by Bacillus sp. M01 PTCC 1908 with response surface methodology optimization, AMB Expr. 7

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cart before the horse. Biotechnology Advances 22 (2004) 633-658. [34] L. Kabaivanova ， G.E. Chernev ， P. Markov ， I.M.M. Salvado, Hybrid materials parameters

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influencing the enzyme activity of immobilized cells, Bulgarian Chemical Communications 46 (2014)

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Figure captions: Fig.1 Schematic diagram of the CSTR system Fig.2 Time curve of 50 mg CN-/ L percentage change at 30°C, pH 8.0, and 120 rpm. Solid and open up-pointedtriangles represent cyanide percentage of adsorption(▲) and desorption(△) by the foam cubes, respectively.

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Solid down-pointed-triangles represent cyanide percentage in the control groups(▼).Values are the mean ± SD (error bars) of three replicates.

Fig.3 Time curve of 50 mg CN- /L removal efficiency at 30°C, pH 8.0, and 120 rpm. Solid squares and circles represent cyanide removal efficiency of PUF-immobilized cells(■) and freely suspended cells(●), respectively. Values are the mean ± SD (error bars) of three replicates.

Fig.4 Effect of temperature on 50 mg CN-/L removal at pH 8.0, and 120 rpm. Solid squares and circles represent cyanide removal efficiency of PUF-immobilized cells(■) and freely suspended cells(●), respectively. Solid

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up-triangles represent the adsorption percentage of foam cubes(▲). Values are mean ± SD (error bars) of three

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replicates.

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Fig.5 Effect of pH on 50 mg CN- /L removal at 30°C, and 120 rpm. Solid squares and circles represent the cyanide removal efficiency of PUF-immobilized cells(■) and freely suspended cells(●), respectively. Solid up-triangles represent the adsorption percentage of foam cubes(▲). Values are mean ± SD (error bars) of three replicates. Fig.6 Specific removal rate at different initial CN- concentrations at pH 8.0, 30°C, and 120 rpm. Solid squares

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and circles represent cyanide removal efficiency of PUF-immobilized cells(■) and freely suspended cells(●),

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respectively. Solid up-triangles represent the adsorption capacity of foam cubes(▲). Values are the mean ± SD (error bars) of three replicates.

Fig.7 Time curve of cyanide removal by (A) PUF-immobilized cells and (B) freely suspended cells at 30°C, pH 8.0, and 120 rpm. Open down-pointed-triangles, squares, diamonds, circle, and up-pointed-triangles represent 50(▽), 100(□), 200(◇), 300(○), 500 mg/L(△), respectively. Values are the mean ± SD (error bars) of three replicates.

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Fig.8 Experimental and predicted initial rates of cyanide removal by Haldane’s model. Open squares and circles represent the experimental data of PUF-immobilized cells(□) and freely suspended cells(○), respectively. The red solid line represents the predicted data of Haldane’s model. Values are the mean ± SD (error bars) of three

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replicates.

Fig.9 Removal efficiency in the sequencing batch reactor(SBR) at 30°C, pH 8.0, and 120 rpm with 100 mg CN/ L. Fig.10 Removal efficiency in the continuous stirred tank reactor(CSTR) at 30°C, pH 8.0 with different CNconcentration. Open squares represent cyanide removal efficiency of the effluent(□), and stars represent the influent CN- concentration(＊). 15

Table legends :

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A

N

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SC RI PT

Table 1 Kinetic results of the cyanide removal by immobilized and freely suspended cells

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Figure list

16

D

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CC

A

SC RI PT

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N

A

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Fig.1

17

120

80 60

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Percentage (%)

100

Control groups Desorption Adsorption

40 20 0

0

1

2

3

4

5

Time (h)

A

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D

M

A

N

U

Fig.2

18

100 80 60 40

PUF-immobilized cells Freely suspended cells

20 0

SC RI PT

Removal efficiency (%)

120

0

30

60

90

120

150

180

Time (min)

A

CC

EP TE

D

M

A

N

U

Fig.3

210

19

240

80

SC RI PT

4000 3500

60

3000

40

2500 2000

20

1000

20

25

30

35 o

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Temperature ( C)

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1500

A

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D

M

A

Fig.4

20

40

0

Adsorption (%)

4500

100 PUF-immobilized cells Freely suspended cells Cyanide adsorption

-

Specific removal rate (mg CN / L·h·g CDW)

5000

80

4000

SC RI PT

3500 60

3000 2500

40

2000 1500

20

1000 500 7

8

9

10

11

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pH

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Fig.5

21

Adsorption (%)

4500

100

PUF-immobilized cells Freely suspended cells Cyanide adsorption

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-

Specific removal rate (mg CN /L·h·g CDW)

5000

0

25000

16

20000 15000

SC RI PT

12

8

10000

4

5000 0

0

200

400

600

800

-

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CN concentration (mg/L)

A

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D

M

A

N

Fig.6

22

Adsorption capacity (mg / g foam cube)

20

PUF-immobilized cells Freely suspended cells Cyanide adsorption

-

Specific removal rate (mg CN / L·h·g CDW)

30000

0

A

400 300 50 mg/L 100 mg/L 200 mg/L 300 mg/L 500 mg/L

200 100 0 0

1

2

3

4

5

6

7

8

N

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Time (h)

SC RI PT

-

CN concentration (mg / L)

500

A

B

M

400

50 mg/L 100 mg/L 200 mg/L 300 mg/L 500 mg/L

D

300 200 100 0

2

4 6 Time (h)

8

10

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0

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-

CN concentration (mg / L)

500

A

Fig.7

23

12

Experimental values of PUF-immobilized cells Experimental values of Freely suspended cells Theoretical values by Haldane model

250

-

Initial rate of removal (mg CN / L·h)

300

200

SC RI PT

150 100 50 0

0

100

200

300

400

500

600

700

800

-

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CN concentration (mg/L)

A

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D

M

A

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Fig.8

24

80 60

SC RI PT

Rmoval efficiency (%)

100

40 20 0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Time (d)

A

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D

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A

N

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Fig.9

25

80

150

60 40 50

20 0

Influent Effluent

0

4

8 12 16 20 24 28 32 36 40 44 48 52

-

100

0

Time (d)

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D

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A

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Fig.10

SC RI PT

200

CN concentration (mg/L)

Removal efficiency (%)

100

26

Table list

k（h-1） Immobilized cells

Free cells

Immobilized cells

Free cells

Immobilized cells 0.9889

0.9219

0.7634

0.75

0.91

100

0.9801

0.7380

0.71

0.94

200

1.3661

0.7527

0.51

0.92

300

0.7507

0.4444

0.92

1.56

500

0.5461

0.3665

1.27

1.89

A

CC

EP TE

D

M

A

N

U

50

27

Free cells 0.9864

SC RI PT

CN- concentration（mg/L）

R2

t1/2 (h)

0.9929

0.9901

0.9916

0.9941

0.9937

0.99

0.9964

0.9926