Colloids and Surfaces B: Biointerfaces 122 (2014) 583–590
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Production of a value added compound from the H-acid waste water—Bioflocculants by Klebsiella pneumoniae Chunying Zhong a,b,1 , Aihua Xu a,1 , Buyun Wang a , Xianghui Yang a , Wentao Hong a , Baokun Yang a , Changhong Chen a , Hongtao Liu c , Jiangang Zhou a,∗ a
School of Environmental Engineering, Wuhan Textile University, Wuhan 430073, China School of Chemistry and Life Science, Hubei University of Education, Wuhan 430205, China c School of Materials Science and Engineering, Wuhan Textile University, Wuhan 430073, China b
a r t i c l e
i n f o
Article history: Received 31 March 2014 Received in revised form 20 July 2014 Accepted 22 July 2014 Available online 31 July 2014 Keywords: Bioflocculant H-acid Klebsiella pneumoniae Response surface methodology Bioflocculant stability
a b s t r a c t A novel strain (designated as ZCY-7) which could convert H-acid into bioflocculants was isolated from H-acid wastewater sludge. Conditions for bioflocculants production were optimized by response surface methodology (RSM) and determined to be inoculum size 9.65%, initial pH 7.0, and CODCr of the H-acid wastewater 520 mg/L. The highest flocculating efficiency achieved for kaolin suspension was 95.1%, after 60 h cultivation. The yielded bioflocculant was mainly composed of polysaccharide (82.4%) and protein (14.2%), and maintained its flocculating activity in 0.4% (w/w) kaolin suspensions over pH 2–8 and 20–80 ◦ C. Fourier transform infrared (FTIR) spectra showed that amino, amide and hydroxyl groups were present in the bioflocculant molecules. A viable alternative treatment technology of H-acid wastewater using this novel strain is suggested, which could largely reduce bioflocculants costs. In addition, flocculating mechanism investigation reveals that the bioflocculant could cause kaolin suspension instability by means of charge neutralization firstly and then promoted the aggregation of suspension particles by adsorption and bridge. It is evident from the results that H-acid wastewater could be used as a source to manufacture bioflocculants. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In view of environmental concern, there are renewed interests on microbial flocculants in the recent past due to their low levels of toxicity, high biodegradable nature, high selectivity, and specificity even under extreme conditions like pH, temperature, and salinity, considering them as potential alternatives to synthetic flocculants [1,2]. These salient features make them potential candidates for a wide range of applications in diverse areas including water supply, wastewater treatment and downstream processing for fermentation industries [3,4]. The high-cost and low yield of production, however, is the major limitation of bioflocculants development for commercial use in wastewater treatment. Although several studies using
∗ Corresponding author at: School of Environmental Engineering, Wuhan Textile University, Wuhan 430073, China. Tel.: +86 27 87611607; fax: +86 27 87611623. E-mail address: [email protected] (J. Zhou). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.colsurfb.2014.07.036 0927-7765/© 2014 Elsevier B.V. All rights reserved.
synthetic and low-cost substrates for bioflocculant production have been reported [5,6], there has been no report on the production of bioflocculants using H-acid wastewater as a substrate. H-acid (amino-8-naphthol-3,6-disulfonic acid) is a xenobiotic compound (PAH) used in the production of several direct, acid, reactive dyes and medicine [7–9]. The H-acid wastewater from the manufacturing processes is rich in various substituted derivatives of Naphthalene compound, which are not readily degradable compared with glucose or sucrose [7–9]. Additionally, wastewater from the manufacturing processes is released from the acidulation precipitation process and exhibits very high COD (30,000–50,000 mg/L), acidity (pH 1.5–3.5) and chroma, which can inhibit microbial activities. Strains that can effectively utilize Hacid wastewater to produce bioflocculants are of academic and practical interests. Therefore, specific objectives of this study were focused on the following factors: (1) isolation and identification of bioflocculant-producing strains from H-acid wastewater sludge; (2) production of bioflocculant using isolated strains from H-acid wastewater; (3) the performance of this bioflocculant and its flocculating mechanisms.
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2. Materials and methods 2.1. Wastewater sample collection and isolation of bioflocculant producing strains Wastewater samples were collected from industrial effluent of JiHua Chemical Plant in Jiangsu Province, China. With serial dilution techniques, different strains of bacteria were isolated onto agar plates containing the following sterilized medium at pH 7.5 (per liter): 0.2 g H-acid, 5 g K2 HPO4 , 2 g KH2 PO4 , 0.2 g MgSO4 , 0.1 g NaCl, 0.5 g urea, 0.5 g yeast extract and 20 g agar. Plates were incubated at 25 ◦ C for 48–72 h. Isolated strains were named as ZCY1–ZCY25. Kaolin suspensions were then used to evaluate the flocculating capability of these microorganisms at the concentration of 4 g/L, and then the strains with high flocculating efficiency would be selected as the bioflocculant-producing strains for further investigation. Morphological, physiological and biochemical characteristics of the bacteria were identified according to Bergey’s Manual of Systematic Bacteriology. PCR amplification of 16 S rDNA was identified by Takara Biotechnology Co., Ltd [10]. 2.2. Bioflocculant production and flocculating activity tests H-acid wastewaters (CODCr = 41,000 mg/L, pH 3.0, BOD5 = 90 mg/L, color = 3900, Na2 SO4 = 250 g/L) were blending black liquor from the primary sedimentation tank of JiHua Chemical Plant in Jianggsu, China. The culture medium consisted of: diluted H-acid wastewater (CODCr = 500 mg/L−1 ) 1 L, 5 g K2 HPO4 , 2 g KH2 PO4 , 0.2 g MgSO4 , 0.5 g urea and 0.5 g yeast extract. Prior to cultivation, H-acid wastewater was diluted to the desired CODCr . Initial pH of the H-acid wastewater medium was adjusted to the determined value. Batch fermentations were performed in a 5-L stirred tank fermenter (Model KF5L, KoBio-Tech Co., Inchon, Korea) with 3-L working volume at 37 ◦ C for 96 h and agitation of 200 rpm. Samples were drawn at appropriate time intervals and monitored for pH, biomass, COD, BOD5 and flocculation properties. The fermented medium was harvested (10,000 rpm; 4 ◦ C, 20 min) and the cell-free supernatant was used as the source of bioflocculant. Purification of the bioflocculant was performed as described by the researchers [5]. The flocculating activity (FA) was measured using jar testers [11]. 2.3. Response surface methodology experimental design The Design Expert Software (version 8.0) was used for the statistical design of experiments and data analysis. The central composite design (CCD), a standard RSM, was selected for the optimization of the factors which made sense on the bioflocculant production. In this design, three factors were the CODCr of the H-acid wastewater, inoculum size, and initial pH respectively. Experiments were initiated as a preliminary study to determine a narrower range of H-acid wastewater CODCr , inoculum size, and initial pH before designing the experimental runs. Accordingly, CODCr of the H-acid wastewater from 100 mg/L was tried and the increments continued until appreciable reductions were observed during cell growth. Likewise, inoculum size range 1%–20% and initial pH range 2–11 were examined to search for a narrower and more effective range. As a result, it was chosen as follows: CODCr of H-acid wastewater 400–600 mg/L, inoculum size 5%–15% and initial pH 6–8. These three independent factors with five different levels (−1.682, −1, 0, +1, +1.682) of bioflocculant production were investigated and the experimental designs are shown in Table 1. The response variable (y) that represented flocculating rate was fitted by a second-order model in the form of quadratic polynomial
equation: Y = ˇ0 +
ˇiXi +
ˇiXi2 +
ˇijXiXj, . . .i,
j = 1, 2, 3, . . ., k
(1)
where Y is the predicted response, Xi and Xj are independent factors, ˇ0 is the intercept, ˇi is the linear coefficient, ˇii is the quadratic coefficient and ˇij is the interaction coefficient. 2.4. Characteristics of the bioflocculant Purified bioflocculant was analyzed by a Fourier transform infrared (FTIR) spectrophotometer (Made in Germany Model EQUINOX55). Spectrum of the sample was recorded on the spectrophotometer over a wave number range 400–4000 cm−1 under ambient conditions. Total sugar content was determined by the phenol sulfuric acid method with glucose as a standard. Uronic acids were determined with carbazole sulfuric acid method [12] using galacturonic acid as a standard. Amino sugars were determined according to the Elson–Morgan method with glucosamine as a standard. Protein was determined by the Lowry-Folin method with bovine serum albumin as a standard[13,14]. Monosaccharide composition of the purified biopolymer flocculant was analyzed after hydrolysis with 3 M HCl at 100 ◦ C for 4 h using cellulose TLC with ethyl acetate, pyridine, acetic acid and water (5:5:1:3, v/v) as solvent. Monosaccharides were detected by spraying with aniline phthalic acid reagent and heating at 110 ◦ C for 5 min. The gel filtration chromatography (Sepharose gel column, Pharmacia) was packed in a glass Column (1.2 cm × 50 cm) and was performed to determine the molecular weight of bioflocculant. A sample solution (20 L) was injected, and the column was eluted with 0.05 M NaCl solution at a flow rate of 0.6 mL/min. Molecular weight of the tested sample was based on the standard curve calibrated and established by standard-molecular-weight dextran [15]. Degradation temperature of the partially purified bioflocculant was studied using Thermogravimetric (STA 449/C Jupiter Netz, Germany; Perkin Elmer TGA7 Thermogravimetric Analyzer, USA) instrument. The bioflocculant measured was heated from 35 to 600 ◦ C at a constant rate of 10 ◦ C min−1 under constant flow of nitrogen gas. Characterization of charge (zeta potential) was implemented using Zetaphoremeter (Zetaphoremeter IV, Zetacompact Z8000, CAD Instrumentation, France) with the application of the Smoluckowski equation. Zeta potential values were obtained from the average of around 24 measurements, the average values are presented with its half-width confidence interval at 95% confidence level. 2.5. Flocculating properties of the purified bioflocculant To obtain the optimal concentration of bioflocculant, 993 mL kaolin suspension (4.0 g/L) was added into a 1000 mL beaker supplemented with various amounts of bioflocculant solution (final concentration of 2–100 mg/L) and 9 mM CaCl2 solution (5 mL) at pH7.0. Flocculating activity was then measured and calculated using the procedure described above. Effects of temperature and solution pH on flocculating activity were examined by measuring flocculating activity of the reaction mixture containing the optimal bioflocculant concentration at specified ranges pH 2–10 and at different temperatures 0–80 ◦ C. PH stability of bioflocculant was determined by measuring the residual activity after 24 h of preincubation at various pH values (1–12) and compared with that of a normal bioflocculant solution at pH 7.0. Thermal stability of bioflocculant was determined by measuring the residual activity after 60 min of incubation at various temperatures (0–100 ◦ C). Furthermore, effect of various cations and cation concentrations (0, 0.01, 0.1, 1, 10 and 100 mM) on flocculant activity was investigated
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Table 1 The central composite design of RSM for optimization of MBF-7 production by Klebsiella pneumoniae ZCY-7. Run
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Factors
MBF-7 yield(g/L)
COD of H-acid wastewater (mg/L)
Initial pH
Inoculum size (%)
X1
A (mg/L)
X2
B
X3
C
−1.000 1.000 1.682 1.000 0.000 0.000 1.000 −1.000 −1.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 −1.000 0.000 −1.682 0.000
400.0 600.0 668.2 600.0 500.0 500.0 600.0 400.0 400.0 600.0 500.0 500.0 500.0 500.0 500.0 500.0 400.0 500.0 331.8 500.0
1.000 1.000 0.000 −1.000 0.000 0.000 −1.000 −1.000 −1.000 1.000 0.000 0.000 0.000 0.000 −1.682 1.682 1.000 0.000 0.000 0.000
8.0 8.0 7.0 6.0 7.0 7.0 6.0 6.0 6.0 8.0 7.0 7.0 7.0 7.0 5.3 8.7 8.0 7.0 7.0 7.0
1.000 −1.000 0.000 −1.000 0.000 1.682 1.000 1.000 −1.000 1.000 −1.682 0.000 0.000 0.000 0.000 0.000 −1.000 0.000 0.000 0.000
15.0 5.0 10.0 5.0 10.0 18.5 15.0 15.0 5.0 15.0 1.5 10.0 10.0 10.0 10.0 10.0 5.0 10.0 10.0 10.0
6.49 7.57 7.07 7.42 8.92 7.75 7.39 6.67 7.25 7.72 7.93 8.88 8.89 8.85 7.53 7.43 6.75 8.86 5.82 8.91
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.21 0.25 0.19 0.32 0.33 0.28 0.26 0.23 0.25 0.30 0.24 0.34 0.35 0.19 0.27 0.29 0.25 0.28 0.23 0.31
The data are represented as mean value ± standard deviation (n = 3).
using the method described above, except that the CaCl2 solution was replaced by various metal salt solutions. Solutions of AgNO3 , Cu (NO3 )2 , Pb(NO3 )2 , Cd(NO3 )2 , Zn(NO3 )2 , Ca(NO3 )2 , Mn(NO3 )2 , Co(NO3 )3 and Fe(NO3 )3 were used as cations sources.
sequence similarity). Thus, according to its morphological, physiological properties and 16S rDNA BLAST result, the isolated strain ZCY-7 was identified as a strain of K. pneumoniae. Bioflocculant produced by this strain was named as MBF-7.
2.6. Bioflocculant toxicity test
3.2. Optimization of culture conditions for fermentation
Twenty white mice (25 ± 2 g) were obtained at 1 month of age from Wuhan University. The mice were divided into two groups randomly without consideration of gender. The mice in one group were fed with bioflocculant in the food, and that in the other group were fed with food only. These animals were kept in a room with temperature maintained at 22 ± 2 ◦ C, a relative humidity of 55 ± 5% and illumination of 12 h light/dark cycle. Bioflocculant was dissolved in water and mixed with food, a dosage of 1 g bioflocculant (kg animal)−1 day−1 was used. These mice were raised for 15 days while their posture, bite and sup, movement and weight were monitored [16].
3.2.1. Effect of the H-acid wastewater strength on MBF-7 yield The effect of H-acid wastewater strength (with various CODCr concentrations) on MBF-7 yield were studied and shown in Fig. 1A. The optimal CODCr concentration was 500 mg/L with the MBF-7 yield of 8.9 g/L. MBF-7 yield increased significantly (P < 0.05) when increasing CODCr concentration up to 500 mg/L. However, MBF7 yield decreased when the CODCr concentration was increased above 600 mg/L. When it was above 900 mg/L, MBF-7 yield were scarce. So it is possible that higher CODCr concentration could restrain K. pneumoniae ZCY-7 growth. As a result, the CODCr concentration of H-acid wastewater at 500 mg/L was used for all subsequent culture. Zhao [17] reported similar results for bioflocculant prodution under the conditon that is above or below optimum CODCr concentration of waste fermenting liquor.
3. Results and discussion 3.1. Identification of bioflocculant-producing bacterium In total, 25 aerobic bacteria were isolated from H-acid wastewater sludge and 9 strains were selected as the bioflocculantproducing bacteria. The strain of ZCY-7, which showed the highest flocculating efficiency of 92.3% under 4 g/L Kaolin clay suspensions, was chosen as bioflocculant-producing strain for further studies. It was found to be short, rod-shaped and a facultative aerobe with a size of approximately 1.01 m × 1.47 m. Its colony was circular, milk white, smooth and papillary. Some biochemical and physiological characteristics of the strain were as follows: gram stain (−), motility (−), capsule (+), endospore (−), citrate, simmons (+), gelatin hydrolysis 22 ◦ C (+), catalase (+), oxidase (−), phenylalanine aminotransferase (−), ornithine decarboxylase (−), arginine dihydrolase (−), Voges-Proskauer (+), d-glucose, gas production (+), d-glucose, acid production (+), lactose, gas production (+), malonate utilization (−), H2 S production (−) and urea hydrolysis (+). The analysis of the 16S rRNA gene sequences showed that strain ZCY-7 was closely related to the strains of Klebsiella pneumoniae (99.4%
3.2.2. Effect of the inoculum size on MBF-7 yield Inoculum size is an important parameter in the production of MBF-7. As seen in Fig. 1B, MBF-7 yield were affected by inoculum size. The initial MBF-7 yield increased with inoculum size. The maximum MBF-7 yield was obtained at the inoculum size of 10.0%. However, any further increase in inoculum size did not result in any higher MBF-7 yield. These results are in accord with previous findings [18]. As a result, an inoculum size of 10.0% was used for all subsequent cultures. 3.2.3. Effect of pH on MBF-7 yield in fermentation The effect of pH in fermentation runs was examined (Fig. 1C). Over the pH range 6–8, the highest MBF-7 yield was 8.9 g/L. The optimal pH for bioflocculant production was 7.0 because initial pH of culture medium could affect electrification state of microorganism cell, redox potential, microorganism nutriment assimilation and enzyme reaction [19,20].
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Fig. 1. Effect of CODCr concentrations, inoculum size, H-acid wastewater pH on MBF-7 yield. Error bars represent SD, n = 3.
3.2.4. Optimization of MBF-7 production with RSM Since inoculum size, initial pH, and CODCr of the H-acid wastewater had highly significant effects (P < 0.01) on MBF-7 production, it was desirable to investigate the interaction among the three most significant factors and optimize them to obtain higher flocculation efficiencies. Results from optimization experiments were analyzed by standard ANOVA (Table 2) and the central composite design was fitted with the polynomial equations: YMBF-7 production = 8.88 + 0.37 × A − composite design was 0.0027 × B − 0.0075 × C + 0.15 × A × B + 0.12 × A × C + 0.063 × B × C − 0.86 × A2 − 0.50 × B2 − 0.37 × C 2
(2)
where A, B and C are CODCr of the H-acid wastewater, inoculum size, and initial pH (all for coded factors), respectively. The fit of the model was checked by the coefficients of determination R2 , which was calculated to be 0.99, implying that 99% of the variability in the response could be explained by Eq. (2). Statistical significance of the model equations was evaluated by the F-test for ANOVA (Table 2). The Model F-value of 1688.00 implies the model is significant. There is only 0.01% chance that a “Model F-Value” this large could occur due to noise. Values of “Prob > F” less than 0.0500 indicate model terms are significant. The “Lack of Fit F-value” of 1.87 implies the Lack of Fit is not significant relative to the pure error. There is a 25.50% chance that a “Lack of Fit F-value” this large could occur due to noise. These results indicated that the model was suitable to describe the relationships between flocculation activity and these significant factors. Based on the results of RSM, the optimal condition for MBF7 production calculated from the regression equations were 520 mg/L CODCr of the H-acid wastewater, initial pH 7.0 and inoculum size 9.65%. The maximum values predicted for the responses was 8.99 g/L for MBF-7 production and the actual production obtained with the optimized medium was 8.91 g/L, which is in close agreement to the model prediction.
3.3. Growth profiles of K. pneumoniae ZCY-7 Time courses of the growth, pH and flocculating activity of K. pneumoniae ZCY-7 culture broth in the optimized medium are given in Fig. 2A. Flocculating activity of the culture broth increased in parallel with cell growth during the exponential phase. A sharp decline in pH levels was observed from 7.0 to 5.5 in the first 36 h, which might have been due to the presence of organic acid components of the flocculant polymer [21]. Beyond that point, pH was nearly stable for the following 60 h. The same phenomenon was reported in the cultivation of B. licheniformis [22,23]. The biomass reached its maximum yield of 2.9 g/L in the first 48 h, then flocculating activity started to decrease after 72 h because of cell lysis and enzymatic activity [3,23]. A yield of 8.91 g/L purified MBF-7 was obtained when the flocculating activity reached maximum. Fig. 2B demonstrated the change of CODcr removal rate during fermentation process. During the first day of experiment, the remaining CODcr decreased to 60%. It was less than 35% at the end of the experiments. The determined BOD5 of H-acid solution (CODcr = 500 mg/L) was 20 mg/L. The BOD5 /COD ratio of H-acid solution measured was 0.04, showing the resistance of H-acid to biodegradation. Fig. 1B also presents the variations of BOD5 /COD ratios from 0 h to 96 h. The biodegradability of solutions as measured by BOD5 /COD values increased during fermentation process. 3.4. Characterization of purified bioflocculant MBF-7 3.4.1. Chemical composition analysis of MBF-7 The MBF-7 from K. pneumoniae ZCY-7 was a proteoglycan comprised of carbohydrate (82.4%) and protein (14.2%). Further analysis of the hydrolyzed bioflocculant revealed that the mass proportion of neutral sugar, amino sugar, and uronic acid was 5:2:4. The neutral sugar components of hydrolyzed MBF-7 included dGlc, d-Man, d-Suc, d-Gal and d-Lac in an approximate molar ratio of 7.6:10.5:21.2:21.9:28.6. MBF-7 had appropriate content
C. Zhong et al. / Colloids and Surfaces B: Biointerfaces 122 (2014) 583–590
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Table 2 ANOVA for the response surface models. Source
Sum of squares
Kaolin clay flocculation efficiencya Model 16.33 Residual 0.011 Lack of fit 6.998E−003 3.750E−003 Pure error 16.34 Total a
DF
Mean square
F-value
p-value
9 10 5 5 19
1.81 1.075E−003 1.400E−003 7.500E−004
1688.00
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Production of a value added compound from the H-acid waste water—Bioflocculants by Klebsiella pneumoniae Chunying Zhong a,b,1 , Aihua Xu a,1 , Buyun Wang a , Xianghui Yang a , Wentao Hong a , Baokun Yang a , Changhong Chen a , Hongtao Liu c , Jiangang Zhou a,∗ a
School of Environmental Engineering, Wuhan Textile University, Wuhan 430073, China School of Chemistry and Life Science, Hubei University of Education, Wuhan 430205, China c School of Materials Science and Engineering, Wuhan Textile University, Wuhan 430073, China b
a r t i c l e
i n f o
Article history: Received 31 March 2014 Received in revised form 20 July 2014 Accepted 22 July 2014 Available online 31 July 2014 Keywords: Bioflocculant H-acid Klebsiella pneumoniae Response surface methodology Bioflocculant stability
a b s t r a c t A novel strain (designated as ZCY-7) which could convert H-acid into bioflocculants was isolated from H-acid wastewater sludge. Conditions for bioflocculants production were optimized by response surface methodology (RSM) and determined to be inoculum size 9.65%, initial pH 7.0, and CODCr of the H-acid wastewater 520 mg/L. The highest flocculating efficiency achieved for kaolin suspension was 95.1%, after 60 h cultivation. The yielded bioflocculant was mainly composed of polysaccharide (82.4%) and protein (14.2%), and maintained its flocculating activity in 0.4% (w/w) kaolin suspensions over pH 2–8 and 20–80 ◦ C. Fourier transform infrared (FTIR) spectra showed that amino, amide and hydroxyl groups were present in the bioflocculant molecules. A viable alternative treatment technology of H-acid wastewater using this novel strain is suggested, which could largely reduce bioflocculants costs. In addition, flocculating mechanism investigation reveals that the bioflocculant could cause kaolin suspension instability by means of charge neutralization firstly and then promoted the aggregation of suspension particles by adsorption and bridge. It is evident from the results that H-acid wastewater could be used as a source to manufacture bioflocculants. © 2014 Elsevier B.V. All rights reserved.
1. Introduction In view of environmental concern, there are renewed interests on microbial flocculants in the recent past due to their low levels of toxicity, high biodegradable nature, high selectivity, and specificity even under extreme conditions like pH, temperature, and salinity, considering them as potential alternatives to synthetic flocculants [1,2]. These salient features make them potential candidates for a wide range of applications in diverse areas including water supply, wastewater treatment and downstream processing for fermentation industries [3,4]. The high-cost and low yield of production, however, is the major limitation of bioflocculants development for commercial use in wastewater treatment. Although several studies using
∗ Corresponding author at: School of Environmental Engineering, Wuhan Textile University, Wuhan 430073, China. Tel.: +86 27 87611607; fax: +86 27 87611623. E-mail address: [email protected] (J. Zhou). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.colsurfb.2014.07.036 0927-7765/© 2014 Elsevier B.V. All rights reserved.
synthetic and low-cost substrates for bioflocculant production have been reported [5,6], there has been no report on the production of bioflocculants using H-acid wastewater as a substrate. H-acid (amino-8-naphthol-3,6-disulfonic acid) is a xenobiotic compound (PAH) used in the production of several direct, acid, reactive dyes and medicine [7–9]. The H-acid wastewater from the manufacturing processes is rich in various substituted derivatives of Naphthalene compound, which are not readily degradable compared with glucose or sucrose [7–9]. Additionally, wastewater from the manufacturing processes is released from the acidulation precipitation process and exhibits very high COD (30,000–50,000 mg/L), acidity (pH 1.5–3.5) and chroma, which can inhibit microbial activities. Strains that can effectively utilize Hacid wastewater to produce bioflocculants are of academic and practical interests. Therefore, specific objectives of this study were focused on the following factors: (1) isolation and identification of bioflocculant-producing strains from H-acid wastewater sludge; (2) production of bioflocculant using isolated strains from H-acid wastewater; (3) the performance of this bioflocculant and its flocculating mechanisms.
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C. Zhong et al. / Colloids and Surfaces B: Biointerfaces 122 (2014) 583–590
2. Materials and methods 2.1. Wastewater sample collection and isolation of bioflocculant producing strains Wastewater samples were collected from industrial effluent of JiHua Chemical Plant in Jiangsu Province, China. With serial dilution techniques, different strains of bacteria were isolated onto agar plates containing the following sterilized medium at pH 7.5 (per liter): 0.2 g H-acid, 5 g K2 HPO4 , 2 g KH2 PO4 , 0.2 g MgSO4 , 0.1 g NaCl, 0.5 g urea, 0.5 g yeast extract and 20 g agar. Plates were incubated at 25 ◦ C for 48–72 h. Isolated strains were named as ZCY1–ZCY25. Kaolin suspensions were then used to evaluate the flocculating capability of these microorganisms at the concentration of 4 g/L, and then the strains with high flocculating efficiency would be selected as the bioflocculant-producing strains for further investigation. Morphological, physiological and biochemical characteristics of the bacteria were identified according to Bergey’s Manual of Systematic Bacteriology. PCR amplification of 16 S rDNA was identified by Takara Biotechnology Co., Ltd [10]. 2.2. Bioflocculant production and flocculating activity tests H-acid wastewaters (CODCr = 41,000 mg/L, pH 3.0, BOD5 = 90 mg/L, color = 3900, Na2 SO4 = 250 g/L) were blending black liquor from the primary sedimentation tank of JiHua Chemical Plant in Jianggsu, China. The culture medium consisted of: diluted H-acid wastewater (CODCr = 500 mg/L−1 ) 1 L, 5 g K2 HPO4 , 2 g KH2 PO4 , 0.2 g MgSO4 , 0.5 g urea and 0.5 g yeast extract. Prior to cultivation, H-acid wastewater was diluted to the desired CODCr . Initial pH of the H-acid wastewater medium was adjusted to the determined value. Batch fermentations were performed in a 5-L stirred tank fermenter (Model KF5L, KoBio-Tech Co., Inchon, Korea) with 3-L working volume at 37 ◦ C for 96 h and agitation of 200 rpm. Samples were drawn at appropriate time intervals and monitored for pH, biomass, COD, BOD5 and flocculation properties. The fermented medium was harvested (10,000 rpm; 4 ◦ C, 20 min) and the cell-free supernatant was used as the source of bioflocculant. Purification of the bioflocculant was performed as described by the researchers [5]. The flocculating activity (FA) was measured using jar testers [11]. 2.3. Response surface methodology experimental design The Design Expert Software (version 8.0) was used for the statistical design of experiments and data analysis. The central composite design (CCD), a standard RSM, was selected for the optimization of the factors which made sense on the bioflocculant production. In this design, three factors were the CODCr of the H-acid wastewater, inoculum size, and initial pH respectively. Experiments were initiated as a preliminary study to determine a narrower range of H-acid wastewater CODCr , inoculum size, and initial pH before designing the experimental runs. Accordingly, CODCr of the H-acid wastewater from 100 mg/L was tried and the increments continued until appreciable reductions were observed during cell growth. Likewise, inoculum size range 1%–20% and initial pH range 2–11 were examined to search for a narrower and more effective range. As a result, it was chosen as follows: CODCr of H-acid wastewater 400–600 mg/L, inoculum size 5%–15% and initial pH 6–8. These three independent factors with five different levels (−1.682, −1, 0, +1, +1.682) of bioflocculant production were investigated and the experimental designs are shown in Table 1. The response variable (y) that represented flocculating rate was fitted by a second-order model in the form of quadratic polynomial
equation: Y = ˇ0 +
ˇiXi +
ˇiXi2 +
ˇijXiXj, . . .i,
j = 1, 2, 3, . . ., k
(1)
where Y is the predicted response, Xi and Xj are independent factors, ˇ0 is the intercept, ˇi is the linear coefficient, ˇii is the quadratic coefficient and ˇij is the interaction coefficient. 2.4. Characteristics of the bioflocculant Purified bioflocculant was analyzed by a Fourier transform infrared (FTIR) spectrophotometer (Made in Germany Model EQUINOX55). Spectrum of the sample was recorded on the spectrophotometer over a wave number range 400–4000 cm−1 under ambient conditions. Total sugar content was determined by the phenol sulfuric acid method with glucose as a standard. Uronic acids were determined with carbazole sulfuric acid method [12] using galacturonic acid as a standard. Amino sugars were determined according to the Elson–Morgan method with glucosamine as a standard. Protein was determined by the Lowry-Folin method with bovine serum albumin as a standard[13,14]. Monosaccharide composition of the purified biopolymer flocculant was analyzed after hydrolysis with 3 M HCl at 100 ◦ C for 4 h using cellulose TLC with ethyl acetate, pyridine, acetic acid and water (5:5:1:3, v/v) as solvent. Monosaccharides were detected by spraying with aniline phthalic acid reagent and heating at 110 ◦ C for 5 min. The gel filtration chromatography (Sepharose gel column, Pharmacia) was packed in a glass Column (1.2 cm × 50 cm) and was performed to determine the molecular weight of bioflocculant. A sample solution (20 L) was injected, and the column was eluted with 0.05 M NaCl solution at a flow rate of 0.6 mL/min. Molecular weight of the tested sample was based on the standard curve calibrated and established by standard-molecular-weight dextran [15]. Degradation temperature of the partially purified bioflocculant was studied using Thermogravimetric (STA 449/C Jupiter Netz, Germany; Perkin Elmer TGA7 Thermogravimetric Analyzer, USA) instrument. The bioflocculant measured was heated from 35 to 600 ◦ C at a constant rate of 10 ◦ C min−1 under constant flow of nitrogen gas. Characterization of charge (zeta potential) was implemented using Zetaphoremeter (Zetaphoremeter IV, Zetacompact Z8000, CAD Instrumentation, France) with the application of the Smoluckowski equation. Zeta potential values were obtained from the average of around 24 measurements, the average values are presented with its half-width confidence interval at 95% confidence level. 2.5. Flocculating properties of the purified bioflocculant To obtain the optimal concentration of bioflocculant, 993 mL kaolin suspension (4.0 g/L) was added into a 1000 mL beaker supplemented with various amounts of bioflocculant solution (final concentration of 2–100 mg/L) and 9 mM CaCl2 solution (5 mL) at pH7.0. Flocculating activity was then measured and calculated using the procedure described above. Effects of temperature and solution pH on flocculating activity were examined by measuring flocculating activity of the reaction mixture containing the optimal bioflocculant concentration at specified ranges pH 2–10 and at different temperatures 0–80 ◦ C. PH stability of bioflocculant was determined by measuring the residual activity after 24 h of preincubation at various pH values (1–12) and compared with that of a normal bioflocculant solution at pH 7.0. Thermal stability of bioflocculant was determined by measuring the residual activity after 60 min of incubation at various temperatures (0–100 ◦ C). Furthermore, effect of various cations and cation concentrations (0, 0.01, 0.1, 1, 10 and 100 mM) on flocculant activity was investigated
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Table 1 The central composite design of RSM for optimization of MBF-7 production by Klebsiella pneumoniae ZCY-7. Run
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
Factors
MBF-7 yield(g/L)
COD of H-acid wastewater (mg/L)
Initial pH
Inoculum size (%)
X1
A (mg/L)
X2
B
X3
C
−1.000 1.000 1.682 1.000 0.000 0.000 1.000 −1.000 −1.000 1.000 0.000 0.000 0.000 0.000 0.000 0.000 −1.000 0.000 −1.682 0.000
400.0 600.0 668.2 600.0 500.0 500.0 600.0 400.0 400.0 600.0 500.0 500.0 500.0 500.0 500.0 500.0 400.0 500.0 331.8 500.0
1.000 1.000 0.000 −1.000 0.000 0.000 −1.000 −1.000 −1.000 1.000 0.000 0.000 0.000 0.000 −1.682 1.682 1.000 0.000 0.000 0.000
8.0 8.0 7.0 6.0 7.0 7.0 6.0 6.0 6.0 8.0 7.0 7.0 7.0 7.0 5.3 8.7 8.0 7.0 7.0 7.0
1.000 −1.000 0.000 −1.000 0.000 1.682 1.000 1.000 −1.000 1.000 −1.682 0.000 0.000 0.000 0.000 0.000 −1.000 0.000 0.000 0.000
15.0 5.0 10.0 5.0 10.0 18.5 15.0 15.0 5.0 15.0 1.5 10.0 10.0 10.0 10.0 10.0 5.0 10.0 10.0 10.0
6.49 7.57 7.07 7.42 8.92 7.75 7.39 6.67 7.25 7.72 7.93 8.88 8.89 8.85 7.53 7.43 6.75 8.86 5.82 8.91
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.21 0.25 0.19 0.32 0.33 0.28 0.26 0.23 0.25 0.30 0.24 0.34 0.35 0.19 0.27 0.29 0.25 0.28 0.23 0.31
The data are represented as mean value ± standard deviation (n = 3).
using the method described above, except that the CaCl2 solution was replaced by various metal salt solutions. Solutions of AgNO3 , Cu (NO3 )2 , Pb(NO3 )2 , Cd(NO3 )2 , Zn(NO3 )2 , Ca(NO3 )2 , Mn(NO3 )2 , Co(NO3 )3 and Fe(NO3 )3 were used as cations sources.
sequence similarity). Thus, according to its morphological, physiological properties and 16S rDNA BLAST result, the isolated strain ZCY-7 was identified as a strain of K. pneumoniae. Bioflocculant produced by this strain was named as MBF-7.
2.6. Bioflocculant toxicity test
3.2. Optimization of culture conditions for fermentation
Twenty white mice (25 ± 2 g) were obtained at 1 month of age from Wuhan University. The mice were divided into two groups randomly without consideration of gender. The mice in one group were fed with bioflocculant in the food, and that in the other group were fed with food only. These animals were kept in a room with temperature maintained at 22 ± 2 ◦ C, a relative humidity of 55 ± 5% and illumination of 12 h light/dark cycle. Bioflocculant was dissolved in water and mixed with food, a dosage of 1 g bioflocculant (kg animal)−1 day−1 was used. These mice were raised for 15 days while their posture, bite and sup, movement and weight were monitored [16].
3.2.1. Effect of the H-acid wastewater strength on MBF-7 yield The effect of H-acid wastewater strength (with various CODCr concentrations) on MBF-7 yield were studied and shown in Fig. 1A. The optimal CODCr concentration was 500 mg/L with the MBF-7 yield of 8.9 g/L. MBF-7 yield increased significantly (P < 0.05) when increasing CODCr concentration up to 500 mg/L. However, MBF7 yield decreased when the CODCr concentration was increased above 600 mg/L. When it was above 900 mg/L, MBF-7 yield were scarce. So it is possible that higher CODCr concentration could restrain K. pneumoniae ZCY-7 growth. As a result, the CODCr concentration of H-acid wastewater at 500 mg/L was used for all subsequent culture. Zhao [17] reported similar results for bioflocculant prodution under the conditon that is above or below optimum CODCr concentration of waste fermenting liquor.
3. Results and discussion 3.1. Identification of bioflocculant-producing bacterium In total, 25 aerobic bacteria were isolated from H-acid wastewater sludge and 9 strains were selected as the bioflocculantproducing bacteria. The strain of ZCY-7, which showed the highest flocculating efficiency of 92.3% under 4 g/L Kaolin clay suspensions, was chosen as bioflocculant-producing strain for further studies. It was found to be short, rod-shaped and a facultative aerobe with a size of approximately 1.01 m × 1.47 m. Its colony was circular, milk white, smooth and papillary. Some biochemical and physiological characteristics of the strain were as follows: gram stain (−), motility (−), capsule (+), endospore (−), citrate, simmons (+), gelatin hydrolysis 22 ◦ C (+), catalase (+), oxidase (−), phenylalanine aminotransferase (−), ornithine decarboxylase (−), arginine dihydrolase (−), Voges-Proskauer (+), d-glucose, gas production (+), d-glucose, acid production (+), lactose, gas production (+), malonate utilization (−), H2 S production (−) and urea hydrolysis (+). The analysis of the 16S rRNA gene sequences showed that strain ZCY-7 was closely related to the strains of Klebsiella pneumoniae (99.4%
3.2.2. Effect of the inoculum size on MBF-7 yield Inoculum size is an important parameter in the production of MBF-7. As seen in Fig. 1B, MBF-7 yield were affected by inoculum size. The initial MBF-7 yield increased with inoculum size. The maximum MBF-7 yield was obtained at the inoculum size of 10.0%. However, any further increase in inoculum size did not result in any higher MBF-7 yield. These results are in accord with previous findings [18]. As a result, an inoculum size of 10.0% was used for all subsequent cultures. 3.2.3. Effect of pH on MBF-7 yield in fermentation The effect of pH in fermentation runs was examined (Fig. 1C). Over the pH range 6–8, the highest MBF-7 yield was 8.9 g/L. The optimal pH for bioflocculant production was 7.0 because initial pH of culture medium could affect electrification state of microorganism cell, redox potential, microorganism nutriment assimilation and enzyme reaction [19,20].
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Fig. 1. Effect of CODCr concentrations, inoculum size, H-acid wastewater pH on MBF-7 yield. Error bars represent SD, n = 3.
3.2.4. Optimization of MBF-7 production with RSM Since inoculum size, initial pH, and CODCr of the H-acid wastewater had highly significant effects (P < 0.01) on MBF-7 production, it was desirable to investigate the interaction among the three most significant factors and optimize them to obtain higher flocculation efficiencies. Results from optimization experiments were analyzed by standard ANOVA (Table 2) and the central composite design was fitted with the polynomial equations: YMBF-7 production = 8.88 + 0.37 × A − composite design was 0.0027 × B − 0.0075 × C + 0.15 × A × B + 0.12 × A × C + 0.063 × B × C − 0.86 × A2 − 0.50 × B2 − 0.37 × C 2
(2)
where A, B and C are CODCr of the H-acid wastewater, inoculum size, and initial pH (all for coded factors), respectively. The fit of the model was checked by the coefficients of determination R2 , which was calculated to be 0.99, implying that 99% of the variability in the response could be explained by Eq. (2). Statistical significance of the model equations was evaluated by the F-test for ANOVA (Table 2). The Model F-value of 1688.00 implies the model is significant. There is only 0.01% chance that a “Model F-Value” this large could occur due to noise. Values of “Prob > F” less than 0.0500 indicate model terms are significant. The “Lack of Fit F-value” of 1.87 implies the Lack of Fit is not significant relative to the pure error. There is a 25.50% chance that a “Lack of Fit F-value” this large could occur due to noise. These results indicated that the model was suitable to describe the relationships between flocculation activity and these significant factors. Based on the results of RSM, the optimal condition for MBF7 production calculated from the regression equations were 520 mg/L CODCr of the H-acid wastewater, initial pH 7.0 and inoculum size 9.65%. The maximum values predicted for the responses was 8.99 g/L for MBF-7 production and the actual production obtained with the optimized medium was 8.91 g/L, which is in close agreement to the model prediction.
3.3. Growth profiles of K. pneumoniae ZCY-7 Time courses of the growth, pH and flocculating activity of K. pneumoniae ZCY-7 culture broth in the optimized medium are given in Fig. 2A. Flocculating activity of the culture broth increased in parallel with cell growth during the exponential phase. A sharp decline in pH levels was observed from 7.0 to 5.5 in the first 36 h, which might have been due to the presence of organic acid components of the flocculant polymer [21]. Beyond that point, pH was nearly stable for the following 60 h. The same phenomenon was reported in the cultivation of B. licheniformis [22,23]. The biomass reached its maximum yield of 2.9 g/L in the first 48 h, then flocculating activity started to decrease after 72 h because of cell lysis and enzymatic activity [3,23]. A yield of 8.91 g/L purified MBF-7 was obtained when the flocculating activity reached maximum. Fig. 2B demonstrated the change of CODcr removal rate during fermentation process. During the first day of experiment, the remaining CODcr decreased to 60%. It was less than 35% at the end of the experiments. The determined BOD5 of H-acid solution (CODcr = 500 mg/L) was 20 mg/L. The BOD5 /COD ratio of H-acid solution measured was 0.04, showing the resistance of H-acid to biodegradation. Fig. 1B also presents the variations of BOD5 /COD ratios from 0 h to 96 h. The biodegradability of solutions as measured by BOD5 /COD values increased during fermentation process. 3.4. Characterization of purified bioflocculant MBF-7 3.4.1. Chemical composition analysis of MBF-7 The MBF-7 from K. pneumoniae ZCY-7 was a proteoglycan comprised of carbohydrate (82.4%) and protein (14.2%). Further analysis of the hydrolyzed bioflocculant revealed that the mass proportion of neutral sugar, amino sugar, and uronic acid was 5:2:4. The neutral sugar components of hydrolyzed MBF-7 included dGlc, d-Man, d-Suc, d-Gal and d-Lac in an approximate molar ratio of 7.6:10.5:21.2:21.9:28.6. MBF-7 had appropriate content
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Table 2 ANOVA for the response surface models. Source
Sum of squares
Kaolin clay flocculation efficiencya Model 16.33 Residual 0.011 Lack of fit 6.998E−003 3.750E−003 Pure error 16.34 Total a
DF
Mean square
F-value
p-value
9 10 5 5 19
1.81 1.075E−003 1.400E−003 7.500E−004
1688.00
