- Email: [email protected]
Decolorization of dye solutions with Ruditapes philippinarum conglutination mud and the isolated bacteria
Journal of Environmental Sciences 2011, 23(Supplement) S142–S145
Decolorization of dye solutions with Ruditapes philippinarum conglutination mud and the isolated bacteria Yinping Wei, Jun Mu ∗, Xiuhua Zhu, Qi Gao, Yi Zhang Key Laboratory of Environmental Science and Technology, Education Department of Liaoning Province; School of Environmental and Chemical Engineering, Dalian Jiaotong University, Dalian 116028, China. E-mail: [email protected]
Abstract Application of Ruditapes Philippinarum conglutination mud (RPM) for decolorizing synthetic dye solutions was studied. RPM showed good activity for decolorization of Methylene Blue, Crystal Violet, Malachite Green, and Ink Blue. The amount of the RPM had great effect on the decoloration rate of the dye solutions. However, the decoloration rate did not continue to increase when the amount of mud exceeded the optimum dose. The temperature of the dye solution had a remarkable effect on the decolorization rate of Ink Blue solution, but had little effect on the other three dye solutions. The initial pH of the dye solutions evidently affected the decolorization rate of Malachite Green solution, but had less effect on the other three. The decolorization rate of the dye solutions increased significantly with treatment time within 8 hr, but tended to be steady after 8 hr for Methylene Blue, Crystal Violet and Malachite Green solutions, and after 12 hr for Ink Blue solution. The decolorization efficiencies for the four dye solutions under the optimum conditions were all above 90%. Seventeen strains screened from RPM showed flocculation ability for kaolin clay suspension. Out of them, the flocculation rate of strain ZHT3-9 and strain ZHT4-13 were up to 88.14% and 86.01%, respectively. ZHT3-9 was studied, and its decolorization rate for Methylene Blue, Crystal Violet, and Malachite Green reached 90.02%, 89.21%, and 80.29%, respectively. By morphological, physiological and biochemical characteristics analysis and 16S rRNA sequencing, the strain ZHT3-9 was identified as Arthrobacter sp. Key words: Ruditapes Philippinarum; conglutination mud; bioflocculant; dye decolorization; Arthrobacter sp.
Introduction Synthetic dyes have been widely used in printing and dyeing industry and other relevant fields since 1850’s. The amount of dyes produced in the world was over 10,000 tons per year, and it was estimated that a loss of 1%–2% happened in production and 1%–10% loss was within use, thus brought great pollution to our environment. Traditional physical and chemical treatment strategies often showed unsatisfactory effects and accompanied secondary pollution. Recently, bioflocculants for decolorization of dye wastewater have become the research focus in wastewater treatment field and many bacteria for producing bioflocculants were isolated from soil or sludge of wastewater treatment plant. Compared with the traditional chemical high-polymer flocculants, bioflocculants tend to be biodegradable thus environmentfriendly, not only possess excellent flocculation function, but also exhibit good decolorization activities. Ruditapes Philippinarum is a kind of delicious seafood, residing in mud or sand at near seashore. It was noticed that the water above it was very clear and conglutination mud from it showed better flocculation characteristics, suggesting a marine-derived secretion of bioflocculant from Ruditapes Philippinarum. * Corresponding author. E-mail: [email protected]
This study aims to explore decolorization properties of Ruditapes philippinarum conglutination mud, screen marine-derived bioflocculant-producing bacteria, eventually apply them in dye wastewater decolorization.
1 Materials and methods Ruditapes Philippinarum was cultivated in fresh sea water at 10–15°C with a depth of 10 cm for 1–2 days to collect the conglutination mud (RPM). Then the precipitate was collected from the bottom of the culture container. In order to remove the salts from sea water, the RPM was washed for 3–5 times with deionized water, then stored at 4°C until use. 1.1 Determination of decolorization efficiency RPM and the strains isolated from it were used as bioflocculants to decolorize dye solutions. Bioflocculants (1 mL, 2 g/L) were added into the dye solutions (10 mL, 10 mg/L), and the dye solutions were well mixed with a shaker for 1 min. Then the mixture solutions were kept still for 30 min (Chang et al., 2001). The absorbances of the supernatant liquor were measured under the maximum absorbing wavelength of the dye solutions (660, 580, 620, and 584 nm for Methylene Blue, Crystal Violet, Malachite Green and Ink Blue, respectively) with a spectrophotome-
Supplement
Decolorization of dye solutions with Ruditapes philippinarum conglutination mud and the isolated bacteria
ter. The decolorization efficiency (DC) of the dye solutions can be calculated according to the following Eq. (1): DC =
A0 − A × 100% A0
(1)
where, A0 is the absorbance of the blank sample (i.e. the same volume deionized water was added into the dye solution instead of the bioflocculant); A is the absorbance of supernatant liquor of the dye solution after treated with bioflocculants (Deng et al., 2005). 1.2 Influential factors determination of the dye wastewater treatment with RPM Effects of RPM amount on DC were investigated by adding 1, 2, 3, 4, 5 mL of RPM (2 g/L) into 10 mL (10 mg/L) dye solutions respectively. Then the DC values were measured following the above procedures. Effects of temperature, initial pH and treatment time on DC were determined separately by adding 1 mL RPM (2 g/L) and 1 mL CaCl2 (10%) into dye solutions (10 mL, 10 mg/L). For evaluating the effect of temperature, test tubes containing the dye solutions were kept in water bath for 30 min at 10, 20, 30, 40, and 50°C. The initial pH values ranging from 3.0 to 11.0 were applied to test the effect on DC. Treatment time varying from 2 to 24 min were performed under pH 7.0 to determine the effect on DC (Fei and Wu, 2007). 1.3 Screening and culturing of bioflocculant-producing microorganisms 1.3.1 Components of culture medium For the isolation and purification, the culture medium (1 L) consisted of 45 g of nutrient agar and 1000 mL of artificial seawater with initial pH 7.0–7.2. After autoclave at 121°C for 20 min, it was poured plates (9 cm Petri dishes) for use. For the fermentation and screening culture, the medium (1 L) consisted of 20 g of glucose, 0.2 g of (NH4 )2 SO4 , 0.5 g of urea, 0.5 g of yeast extract, 0.2 g of MgSO4 ·7H2 O, 2.0 g of KH2 PO4 , 5.0 g of K2 HPO4 , and 1000 mL of artificial seawater with the initial pH 7.0–7.2. It was autoclaved at 115°C for 30 min. 1.3.2 Bacteria isolation One milliliter of RPM (2 g/L) was well mixed in 99 mL sterilized distilled water to be a 10−2 diluent. Then this diluent was further diluted into a series of diluents: 10−3 , 10−4 , 10−5 , and 10−6 . From each serial diluent, 100 μL the solution were equably coated on nutrient agar medium in Petri dishes. After cultivation at (35 ± 1)°C for 2 days, bacterial colonies were picked out and purified on slant medium by streak method. After streaking isolation for 3 to 4 times, the purified strains mostly can be obtained. 1.3.3 Screening for bioflocculant-producing bacteria To choose high-efficiency bioflocculant-producing bacteria, the flocculation activities of the strains were measured using the kaolin clay suspension method. Briefly, kaolin clay (0.5 g) was suspended in 93 mL deionized water, 5 mL CaCl2 (10 g/L), and 2 mL bioflocculant
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solution (2 g/L) were added, and the initial pH value of which was adjusted to 7.0 with diluted hydrochloric acid and sodium hydroxide solution. The above mixture solution was quickly stirred at 200 r/min for 1 min, slowly stirred at 80 r/min for 2 min, then kept still for 5 min. The absorbance of the supernatant liquor of the above mixture solution (B) and the blank control sample (A) (i.e. the deionized water was added into the kaolin clay suspension solution instead of the bioflocculant solution) were measured at 550 nm with a spectrophotometer. The flocculating rate (FR) can be calculated according to the following Eq. (2) (Fei and Wu, 2007): FR =
A−B × 100% A
(2)
1.3.4 Identification of bioflocculant-producing bacteria The morphological characteristics of the strains were observed with Olympus CX31 bio-microscope, and their physiological and biochemical characteristics were determined according to Bergey’s Manual of systematic bacteriology. DNA extracts were prepared from the strains using Tiangen bacterial genome extraction package (Takara Biocompany, USA). Bacterial 16S rRNA genes were amplified by PCR in a 50-μL total volume. Each PCR mixture contained Premix Ex Taq 25 μL, DNA template 1 μL, primer 27f (5 -AGAGTTTGATCCTGGCTCAG-3 ) 1 μL, primer 1492r (5 -TACCTTGTTACGACTT-3 ) 1 μL, double distilled H2 O up to 50 μL. Cycling parameters were 5 min at 94°C, followed by 30 cycles of 1 min at 94°C, 1 min at 50°C, and 1.5 min at 72°C, followed by 5 min at 72°C. The PCR products were separated on 0.8% agarose gels. The DNA bands were excised from the gel and purified using a Qiagen QIA quick gel extraction kit following the manufacturer’s recommended protocol. Both strands of the purified DNA were then sequenced by an automatic DNA sequencer (ABI) with the primers in Takara’s 16S rDNA bacterial identification PCR kit, including RV-M (5 -GAGCGGATAACAATTTCACA CAGG-3 ), M13-47 (5 -CGCCAGGGTTTTCCCAGTCACGAC-3 ), and the seq internal (5 -CAGCAGCCGCGGTAATAC-3 ). All sequencing reactions were carried out with an ABI 3700 automated DNA sequencer at the Takara Biotechnology Co., Ltd. (China). Finally, the sequences were submitted to GenBank for deposit and BLASTn search.
2 Results and discussions 2.1 Influence factors of RPM on the decolorization efficiency 2.1.1 Effects of RPM amount on DC The effects on the decolorization efficiency of the dye solutions are shown in Fig. 1. It can be seen that RPM was able to decolorize the studied dyes and 1 mL RPM was the optimum dose for Methylene Blue and Crystal Violet solution, the DC of them were 95.56% and 95.91%, respectively; for Malachite Green solution, 3 mL was the optimum dose with the DC of 94.53%; and for Ink Blue
Journal of Environmental Sciences 2011, 23(Supplement) S142–S145 / Yinping Wei et al.
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Methylene Blue
Cystal Violet
Malachite Green
Ink Blue
90
bioflocculants produced by the bacteria in RPM which had effect for Ink Blue may be protein, for protein is heatlabile. Considering the DC and energy consumption, the optimum temperature for decolorizing the dye solutions was 30°C for Methylene Blue, Crystal Violet and Malachite Green, while 20°C for Ink Blue. 2.1.3 Effects of initial pH on DC
80
70 0
1
2 3 4 5 RPM dosage (mL) Fig. 1 Influences of RPM dosage on the decolorization rate of dye solutions.
solution, 2 mL was the optimum dose with the DC of 81.66%. The optimum amounts of RPM are different for different dyes and the bioflocculants exhibited different flocculation rates. Deng et al. (2005) also obtained the similar results. 2.1.2 Effects of temperature on DC The influences of temperature on DC of the dye solutions were evaluated. From Fig. 2, it can be seen that the temperature of the dye solutions had remarkable effects on the decolorization of Ink Blue solution, but had little effects on the other three dye solutions. It can be inferred that there are different kinds of bioflocculant-producing bacteria in RPM and the components of the bioflocculants are different. The main backbone of bioflocculants are polysaccharide presumably, because the polysaccharide bioflocculants are thermally stable. The main backbone of Methylene Blue
Malachite Green
Crystal Violet
Ink Blue
100
The influences of initial pH on DC of the dye solutions were investigated. From Fig. 3, it can be seen that the pH value had remarkable effects on the DC of Malachite Green solution, but had little effects on the other three dye solutions. The optimum initial pH value was 7 for decolorizing Methylene Blue, Crystal Violet and Ink Blue. With the initial pH over 9, Malachite Green solution become colorless when the RPM was added. Chang et al. (2001) found that the dye reduction rate increased nearly 2.5-fold as the pH was raised from 5.0 to 7.0, with removal rate insensitive to pH in the range of 7.0–9.5, which is in accordance with the Malachite Green result obtained in this study. Contrarily, Deng et al. (2005) observed the decolorization efficiency decreased with an initial pH from 2 to 12 for the four dyes (AB45, DB1, AO8 and RO16), and the decolorization efficiency decreased rapidly beyond pH 9 or 12 for different dyes. 2.1.4 Effects of treatment time on DC The influences of treatment time on the decolorization efficiency of the dye solutions are shown in Fig. 4. It 100
Decolorization rate (%)
Decolorization rate (%)
100
80
60 Methylene Blue Crystal Violet Malachite Green Ink Blue
40
90 80
2
8 10 12 pH Fig. 3 Influences of the initial pH value of dye solutions on its decolorization rate.
70 60
4
6
100
50
Decolorization rate (%)
Decolorization rate (%)
Vol. 23
40 30 20
90
80 Methylene Blue Crystal Violet Malachite Green Ink Blue
70 10
10
20
30 40 50 Temperature (ć) Fig. 2 Influences of temperature on the decolorization rate of dye solutions.
0
5
10
15 20 25 Standing time (hr) Fig. 4 Influences of standing time on the decolorization rate of dye solutions.
Decolorization of dye solutions with Ruditapes philippinarum conglutination mud and the isolated bacteria
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90 Flocculating rate(%)
80
Cell
Fermentation liquor
70 60 50 40 30 20 10 0
ZHT ZHT ZHT ZHT ZHT ZHT ZHT ZHT ZHT ZHT ZHT ZHT ZHT ZHT ZHT ZHT ZHT 1-1 1-3 1-4 1-5 2-5 2-6 2-7 3-3 3-5 3-6 3-7 3-8 3-9 4-1' 4-1" 4-2 4-13 Strain code Fig. 5 Flocculating activity of cell and fermentation liquor of bacteria.
identified as Arthrobacter sp. (Buchanan and Gibbens, 1984).
Fig. 6
SEM images of strains ZHT3-9 (a) and ZHT4-13 (b).
can be seen that the DC of the dye solutions increased significantly within 8 hr, but tended to be steady after 8 hr for Methylene Blue, Crystal Violet and Malachite Green solutions, while for Ink Blue solution was after 12 hr (Forgacs et al., 2004). 2.2 Screening and biological characteristics bioflocculant-producing microorganisms
of
From the above study, it can be concluded that RPM has the ability to decolorize the dye solutions. To investigate if the decolorizing ability was attributed to the microorganisms residing in the Ruditapes philippinarum conglutination mud, totally 62 aerobic bacteria were isolated from the RPM. Among them 17 strains which had flocculation activity were selected as bioflocculantproducing bacteria. From the Fig. 5 we can see that strains ZHT3-9 and ZHT4-13 had higher flocculation rates than others and thus they were chosen for further studies. Figure 6 shows the SEM images of strain ZHT3-9 and ZHT4-13. 2.2.1 Biological characteristics of ZHT3-9 The clone of ZHT3-9 is irregular, yellow, trim-edged, smooth, and humid. It belongs to Gram-positive, obligate aerobic bacterium. It forms endospore, with no flagellum and does not move. The cell is pear-shaped, with an average dimention of 1.0 μm in width and 1.2 μm in length. V. P test, gelatin hydrolysis test, and indole test showed negative results; while methyl-red test, nitrate reduction test, citrate test, starch test and catalase test were all positive. The 16S rRNA gene sequence of 1574 bp was determined and deposited in GenBank with the accession number HQ202575. Combining the BLASTn search and analysis of 16S rRNA gene sequence, strain ZHT3-9 was
2.2.2 Biological characteristics of ZHT4-13 The clone of ZHT4-13 is small, circular, milk white, trim-edged, and smooth. It is Gram-positive, obligate aerobic bacterium, non-endospore forming, without flagellum and not motile. The diameter of cell is 0.5–1.0 μm. Glycolysis test, methyl-red test, nitrate reduction test and catalase test showed positive reactions.
3 Conclusions The RPM had a strong ability to decolorize the four synthetic dye solutions. When the decolorizing reactions were performed at the conditions of the initial dye concentration 10 mg/L, initial pH 7.0, temperature 30°C, dosage of RPM (2 g/L) 1–3 mL and standing time 8 hr, the decolorizing rates were 97.73% for Methylene Blue solution, 95.91% for Crystal Violet solution, 94.85% for Malachite Green solution and 94.20% for Ink Blue solution. Totally 62 aerobic bacteria were isolated from the RPM, 17 strains among them showed flocculating ability to kaolin clay suspension. Strains ZHT3-9 and ZHT4-13 showed decolorizing effect for the investigated dye solutions. Based on morphological characteristics, physiological and biochemical properties and 16S rRNA gene sequencing analysis, the strain ZHT3-9 was identified as Arthrobacter sp.
Acknowledgments This work was supported by the Program for Liaoning Excellent Talents in University (No. 2009R08).
References Buchanan R E, Gibbens N E, 1984. Bergey’s Manual of Systematic Bacteriology (8th ed.). Science Press, Beijing. Chang J S, Chou C, Lin Y C, Lin P J, Ho J Y, Hu T L, 2001. Kinetic characteristics of bacterial azo-dye decolourization by Pseudomonas luteola. Water Research, 35: 2841–2850. Deng S, Yu G, Ting Y P, 2005. Production of a bioflocculant by Aspergillus parasiticus and its application in dye removal. Colloids and Surfaces B, 44: 179–186. Fei W Y, Wu J, 2007. Screeening of bioflocculant-producing strain and study on flocculating characteristics. Biotechnology, 17(3): 59–63. Forgacs E, Cserhati T, Oros G, 2004. Removal of synthetic dyes from wastewaters: A Review, Budapest. Environment International, 30: 953–971.
Decolorization of dye solutions with Ruditapes philippinarum conglutination mud and the isolated bacteria Yinping Wei, Jun Mu ∗, Xiuhua Zhu, Qi Gao, Yi Zhang Key Laboratory of Environmental Science and Technology, Education Department of Liaoning Province; School of Environmental and Chemical Engineering, Dalian Jiaotong University, Dalian 116028, China. E-mail: [email protected]
Abstract Application of Ruditapes Philippinarum conglutination mud (RPM) for decolorizing synthetic dye solutions was studied. RPM showed good activity for decolorization of Methylene Blue, Crystal Violet, Malachite Green, and Ink Blue. The amount of the RPM had great effect on the decoloration rate of the dye solutions. However, the decoloration rate did not continue to increase when the amount of mud exceeded the optimum dose. The temperature of the dye solution had a remarkable effect on the decolorization rate of Ink Blue solution, but had little effect on the other three dye solutions. The initial pH of the dye solutions evidently affected the decolorization rate of Malachite Green solution, but had less effect on the other three. The decolorization rate of the dye solutions increased significantly with treatment time within 8 hr, but tended to be steady after 8 hr for Methylene Blue, Crystal Violet and Malachite Green solutions, and after 12 hr for Ink Blue solution. The decolorization efficiencies for the four dye solutions under the optimum conditions were all above 90%. Seventeen strains screened from RPM showed flocculation ability for kaolin clay suspension. Out of them, the flocculation rate of strain ZHT3-9 and strain ZHT4-13 were up to 88.14% and 86.01%, respectively. ZHT3-9 was studied, and its decolorization rate for Methylene Blue, Crystal Violet, and Malachite Green reached 90.02%, 89.21%, and 80.29%, respectively. By morphological, physiological and biochemical characteristics analysis and 16S rRNA sequencing, the strain ZHT3-9 was identified as Arthrobacter sp. Key words: Ruditapes Philippinarum; conglutination mud; bioflocculant; dye decolorization; Arthrobacter sp.
Introduction Synthetic dyes have been widely used in printing and dyeing industry and other relevant fields since 1850’s. The amount of dyes produced in the world was over 10,000 tons per year, and it was estimated that a loss of 1%–2% happened in production and 1%–10% loss was within use, thus brought great pollution to our environment. Traditional physical and chemical treatment strategies often showed unsatisfactory effects and accompanied secondary pollution. Recently, bioflocculants for decolorization of dye wastewater have become the research focus in wastewater treatment field and many bacteria for producing bioflocculants were isolated from soil or sludge of wastewater treatment plant. Compared with the traditional chemical high-polymer flocculants, bioflocculants tend to be biodegradable thus environmentfriendly, not only possess excellent flocculation function, but also exhibit good decolorization activities. Ruditapes Philippinarum is a kind of delicious seafood, residing in mud or sand at near seashore. It was noticed that the water above it was very clear and conglutination mud from it showed better flocculation characteristics, suggesting a marine-derived secretion of bioflocculant from Ruditapes Philippinarum. * Corresponding author. E-mail: [email protected]
This study aims to explore decolorization properties of Ruditapes philippinarum conglutination mud, screen marine-derived bioflocculant-producing bacteria, eventually apply them in dye wastewater decolorization.
1 Materials and methods Ruditapes Philippinarum was cultivated in fresh sea water at 10–15°C with a depth of 10 cm for 1–2 days to collect the conglutination mud (RPM). Then the precipitate was collected from the bottom of the culture container. In order to remove the salts from sea water, the RPM was washed for 3–5 times with deionized water, then stored at 4°C until use. 1.1 Determination of decolorization efficiency RPM and the strains isolated from it were used as bioflocculants to decolorize dye solutions. Bioflocculants (1 mL, 2 g/L) were added into the dye solutions (10 mL, 10 mg/L), and the dye solutions were well mixed with a shaker for 1 min. Then the mixture solutions were kept still for 30 min (Chang et al., 2001). The absorbances of the supernatant liquor were measured under the maximum absorbing wavelength of the dye solutions (660, 580, 620, and 584 nm for Methylene Blue, Crystal Violet, Malachite Green and Ink Blue, respectively) with a spectrophotome-
Supplement
Decolorization of dye solutions with Ruditapes philippinarum conglutination mud and the isolated bacteria
ter. The decolorization efficiency (DC) of the dye solutions can be calculated according to the following Eq. (1): DC =
A0 − A × 100% A0
(1)
where, A0 is the absorbance of the blank sample (i.e. the same volume deionized water was added into the dye solution instead of the bioflocculant); A is the absorbance of supernatant liquor of the dye solution after treated with bioflocculants (Deng et al., 2005). 1.2 Influential factors determination of the dye wastewater treatment with RPM Effects of RPM amount on DC were investigated by adding 1, 2, 3, 4, 5 mL of RPM (2 g/L) into 10 mL (10 mg/L) dye solutions respectively. Then the DC values were measured following the above procedures. Effects of temperature, initial pH and treatment time on DC were determined separately by adding 1 mL RPM (2 g/L) and 1 mL CaCl2 (10%) into dye solutions (10 mL, 10 mg/L). For evaluating the effect of temperature, test tubes containing the dye solutions were kept in water bath for 30 min at 10, 20, 30, 40, and 50°C. The initial pH values ranging from 3.0 to 11.0 were applied to test the effect on DC. Treatment time varying from 2 to 24 min were performed under pH 7.0 to determine the effect on DC (Fei and Wu, 2007). 1.3 Screening and culturing of bioflocculant-producing microorganisms 1.3.1 Components of culture medium For the isolation and purification, the culture medium (1 L) consisted of 45 g of nutrient agar and 1000 mL of artificial seawater with initial pH 7.0–7.2. After autoclave at 121°C for 20 min, it was poured plates (9 cm Petri dishes) for use. For the fermentation and screening culture, the medium (1 L) consisted of 20 g of glucose, 0.2 g of (NH4 )2 SO4 , 0.5 g of urea, 0.5 g of yeast extract, 0.2 g of MgSO4 ·7H2 O, 2.0 g of KH2 PO4 , 5.0 g of K2 HPO4 , and 1000 mL of artificial seawater with the initial pH 7.0–7.2. It was autoclaved at 115°C for 30 min. 1.3.2 Bacteria isolation One milliliter of RPM (2 g/L) was well mixed in 99 mL sterilized distilled water to be a 10−2 diluent. Then this diluent was further diluted into a series of diluents: 10−3 , 10−4 , 10−5 , and 10−6 . From each serial diluent, 100 μL the solution were equably coated on nutrient agar medium in Petri dishes. After cultivation at (35 ± 1)°C for 2 days, bacterial colonies were picked out and purified on slant medium by streak method. After streaking isolation for 3 to 4 times, the purified strains mostly can be obtained. 1.3.3 Screening for bioflocculant-producing bacteria To choose high-efficiency bioflocculant-producing bacteria, the flocculation activities of the strains were measured using the kaolin clay suspension method. Briefly, kaolin clay (0.5 g) was suspended in 93 mL deionized water, 5 mL CaCl2 (10 g/L), and 2 mL bioflocculant
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solution (2 g/L) were added, and the initial pH value of which was adjusted to 7.0 with diluted hydrochloric acid and sodium hydroxide solution. The above mixture solution was quickly stirred at 200 r/min for 1 min, slowly stirred at 80 r/min for 2 min, then kept still for 5 min. The absorbance of the supernatant liquor of the above mixture solution (B) and the blank control sample (A) (i.e. the deionized water was added into the kaolin clay suspension solution instead of the bioflocculant solution) were measured at 550 nm with a spectrophotometer. The flocculating rate (FR) can be calculated according to the following Eq. (2) (Fei and Wu, 2007): FR =
A−B × 100% A
(2)
1.3.4 Identification of bioflocculant-producing bacteria The morphological characteristics of the strains were observed with Olympus CX31 bio-microscope, and their physiological and biochemical characteristics were determined according to Bergey’s Manual of systematic bacteriology. DNA extracts were prepared from the strains using Tiangen bacterial genome extraction package (Takara Biocompany, USA). Bacterial 16S rRNA genes were amplified by PCR in a 50-μL total volume. Each PCR mixture contained Premix Ex Taq 25 μL, DNA template 1 μL, primer 27f (5 -AGAGTTTGATCCTGGCTCAG-3 ) 1 μL, primer 1492r (5 -TACCTTGTTACGACTT-3 ) 1 μL, double distilled H2 O up to 50 μL. Cycling parameters were 5 min at 94°C, followed by 30 cycles of 1 min at 94°C, 1 min at 50°C, and 1.5 min at 72°C, followed by 5 min at 72°C. The PCR products were separated on 0.8% agarose gels. The DNA bands were excised from the gel and purified using a Qiagen QIA quick gel extraction kit following the manufacturer’s recommended protocol. Both strands of the purified DNA were then sequenced by an automatic DNA sequencer (ABI) with the primers in Takara’s 16S rDNA bacterial identification PCR kit, including RV-M (5 -GAGCGGATAACAATTTCACA CAGG-3 ), M13-47 (5 -CGCCAGGGTTTTCCCAGTCACGAC-3 ), and the seq internal (5 -CAGCAGCCGCGGTAATAC-3 ). All sequencing reactions were carried out with an ABI 3700 automated DNA sequencer at the Takara Biotechnology Co., Ltd. (China). Finally, the sequences were submitted to GenBank for deposit and BLASTn search.
2 Results and discussions 2.1 Influence factors of RPM on the decolorization efficiency 2.1.1 Effects of RPM amount on DC The effects on the decolorization efficiency of the dye solutions are shown in Fig. 1. It can be seen that RPM was able to decolorize the studied dyes and 1 mL RPM was the optimum dose for Methylene Blue and Crystal Violet solution, the DC of them were 95.56% and 95.91%, respectively; for Malachite Green solution, 3 mL was the optimum dose with the DC of 94.53%; and for Ink Blue
Journal of Environmental Sciences 2011, 23(Supplement) S142–S145 / Yinping Wei et al.
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Methylene Blue
Cystal Violet
Malachite Green
Ink Blue
90
bioflocculants produced by the bacteria in RPM which had effect for Ink Blue may be protein, for protein is heatlabile. Considering the DC and energy consumption, the optimum temperature for decolorizing the dye solutions was 30°C for Methylene Blue, Crystal Violet and Malachite Green, while 20°C for Ink Blue. 2.1.3 Effects of initial pH on DC
80
70 0
1
2 3 4 5 RPM dosage (mL) Fig. 1 Influences of RPM dosage on the decolorization rate of dye solutions.
solution, 2 mL was the optimum dose with the DC of 81.66%. The optimum amounts of RPM are different for different dyes and the bioflocculants exhibited different flocculation rates. Deng et al. (2005) also obtained the similar results. 2.1.2 Effects of temperature on DC The influences of temperature on DC of the dye solutions were evaluated. From Fig. 2, it can be seen that the temperature of the dye solutions had remarkable effects on the decolorization of Ink Blue solution, but had little effects on the other three dye solutions. It can be inferred that there are different kinds of bioflocculant-producing bacteria in RPM and the components of the bioflocculants are different. The main backbone of bioflocculants are polysaccharide presumably, because the polysaccharide bioflocculants are thermally stable. The main backbone of Methylene Blue
Malachite Green
Crystal Violet
Ink Blue
100
The influences of initial pH on DC of the dye solutions were investigated. From Fig. 3, it can be seen that the pH value had remarkable effects on the DC of Malachite Green solution, but had little effects on the other three dye solutions. The optimum initial pH value was 7 for decolorizing Methylene Blue, Crystal Violet and Ink Blue. With the initial pH over 9, Malachite Green solution become colorless when the RPM was added. Chang et al. (2001) found that the dye reduction rate increased nearly 2.5-fold as the pH was raised from 5.0 to 7.0, with removal rate insensitive to pH in the range of 7.0–9.5, which is in accordance with the Malachite Green result obtained in this study. Contrarily, Deng et al. (2005) observed the decolorization efficiency decreased with an initial pH from 2 to 12 for the four dyes (AB45, DB1, AO8 and RO16), and the decolorization efficiency decreased rapidly beyond pH 9 or 12 for different dyes. 2.1.4 Effects of treatment time on DC The influences of treatment time on the decolorization efficiency of the dye solutions are shown in Fig. 4. It 100
Decolorization rate (%)
Decolorization rate (%)
100
80
60 Methylene Blue Crystal Violet Malachite Green Ink Blue
40
90 80
2
8 10 12 pH Fig. 3 Influences of the initial pH value of dye solutions on its decolorization rate.
70 60
4
6
100
50
Decolorization rate (%)
Decolorization rate (%)
Vol. 23
40 30 20
90
80 Methylene Blue Crystal Violet Malachite Green Ink Blue
70 10
10
20
30 40 50 Temperature (ć) Fig. 2 Influences of temperature on the decolorization rate of dye solutions.
0
5
10
15 20 25 Standing time (hr) Fig. 4 Influences of standing time on the decolorization rate of dye solutions.
Decolorization of dye solutions with Ruditapes philippinarum conglutination mud and the isolated bacteria
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90 Flocculating rate(%)
80
Cell
Fermentation liquor
70 60 50 40 30 20 10 0
ZHT ZHT ZHT ZHT ZHT ZHT ZHT ZHT ZHT ZHT ZHT ZHT ZHT ZHT ZHT ZHT ZHT 1-1 1-3 1-4 1-5 2-5 2-6 2-7 3-3 3-5 3-6 3-7 3-8 3-9 4-1' 4-1" 4-2 4-13 Strain code Fig. 5 Flocculating activity of cell and fermentation liquor of bacteria.
identified as Arthrobacter sp. (Buchanan and Gibbens, 1984).
Fig. 6
SEM images of strains ZHT3-9 (a) and ZHT4-13 (b).
can be seen that the DC of the dye solutions increased significantly within 8 hr, but tended to be steady after 8 hr for Methylene Blue, Crystal Violet and Malachite Green solutions, while for Ink Blue solution was after 12 hr (Forgacs et al., 2004). 2.2 Screening and biological characteristics bioflocculant-producing microorganisms
of
From the above study, it can be concluded that RPM has the ability to decolorize the dye solutions. To investigate if the decolorizing ability was attributed to the microorganisms residing in the Ruditapes philippinarum conglutination mud, totally 62 aerobic bacteria were isolated from the RPM. Among them 17 strains which had flocculation activity were selected as bioflocculantproducing bacteria. From the Fig. 5 we can see that strains ZHT3-9 and ZHT4-13 had higher flocculation rates than others and thus they were chosen for further studies. Figure 6 shows the SEM images of strain ZHT3-9 and ZHT4-13. 2.2.1 Biological characteristics of ZHT3-9 The clone of ZHT3-9 is irregular, yellow, trim-edged, smooth, and humid. It belongs to Gram-positive, obligate aerobic bacterium. It forms endospore, with no flagellum and does not move. The cell is pear-shaped, with an average dimention of 1.0 μm in width and 1.2 μm in length. V. P test, gelatin hydrolysis test, and indole test showed negative results; while methyl-red test, nitrate reduction test, citrate test, starch test and catalase test were all positive. The 16S rRNA gene sequence of 1574 bp was determined and deposited in GenBank with the accession number HQ202575. Combining the BLASTn search and analysis of 16S rRNA gene sequence, strain ZHT3-9 was
2.2.2 Biological characteristics of ZHT4-13 The clone of ZHT4-13 is small, circular, milk white, trim-edged, and smooth. It is Gram-positive, obligate aerobic bacterium, non-endospore forming, without flagellum and not motile. The diameter of cell is 0.5–1.0 μm. Glycolysis test, methyl-red test, nitrate reduction test and catalase test showed positive reactions.
3 Conclusions The RPM had a strong ability to decolorize the four synthetic dye solutions. When the decolorizing reactions were performed at the conditions of the initial dye concentration 10 mg/L, initial pH 7.0, temperature 30°C, dosage of RPM (2 g/L) 1–3 mL and standing time 8 hr, the decolorizing rates were 97.73% for Methylene Blue solution, 95.91% for Crystal Violet solution, 94.85% for Malachite Green solution and 94.20% for Ink Blue solution. Totally 62 aerobic bacteria were isolated from the RPM, 17 strains among them showed flocculating ability to kaolin clay suspension. Strains ZHT3-9 and ZHT4-13 showed decolorizing effect for the investigated dye solutions. Based on morphological characteristics, physiological and biochemical properties and 16S rRNA gene sequencing analysis, the strain ZHT3-9 was identified as Arthrobacter sp.
Acknowledgments This work was supported by the Program for Liaoning Excellent Talents in University (No. 2009R08).
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