Using Ruditapes philippinarum conglutination mud to produce bioflocculant and its applications in wastewater treatment

Bioresource Technology 100 (2009) 4996–5001

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Using Ruditapes philippinarum conglutination mud to produce bioflocculant and its applications in wastewater treatment Qi Gao, Xiu-Hua Zhu *, Jun Mu *, Yi Zhang, Xue-Wei Dong School of Environmental and Chemical Engineering, Dalian Jiaotong University, Dalian 116028, China

a r t i c l e

i n f o

Article history: Received 1 February 2009 Received in revised form 8 May 2009 Accepted 17 May 2009 Available online 16 June 2009 Keywords: Ruditapes philippinarum Conglutination mud Bioflocculant Wastewater treatment

a b s t r a c t A novel bioflocculant-producing bacterium, ZHT4-13, was isolated from Ruditapes philippinarum conglutination mud. By biomicroscope morphological observation, 16S rDNA sequence identification and physiological and biochemical characteristics, strain ZHT4-13 was identified as Rothia sp. The bioflocculant MBF4-13 produced by strain ZHT4-13 had a flocculating efficiency of 86.22% for 5 g L1 Kaolin clay suspension when the initial pH was 9 and the temperature was 20 °C. It had flocculating effect in a wide range, pH 1–13 and temperature 4–100 °C. Analysis of MBF4-13 by UV–Vis spectrophotometer, Fourier-transform infrared spectrophotometer (FT-IR) and 1H nuclear magnetic resonance (NMR) indicated that the main component of MBF4-13 is polysaccharide. The culture conditions to produce strain ZHT4-13 were optimized with orthogonal design of experiments. MBF4-13 had high efficiency in 2+ decolorizing dye solutions, had some abilities to remove heavy metal ions (Cr2 O2 7 , Ni ) and improve performance of activated sludge. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction For the remarkable advantages, such as safety to human health, biodegradability and cheapness on producing, the study of microbial flocculants (MBF) has attracted wide-attention in water treatment research field, and MBFs are widely used in wastewater treatment, such as downstream processing, food and industry wastewater processing (Salehizadeh et al., 2000; Salehizadeh and Shojaosadati, 2001; You et al., 2008). MBF-producing bacteria were screened out from almost all kinds of environment of land or water, such as soil, activated sludge, wastewater, and river (Deng et al., 2005; Gong et al., 2008; He et al., 2004; Kumar et al., 2004; Kurane et al., 1994; Salehizadeh et al., 2000; Salehizadeh and Shojaosadati 2001; Shih et al., 2001; Suh et al., 1997; Wang et al., 2007; Wu and Ye, 2007; Xia et al., 2008; Yim et al., 2007;You et al., 2008; Zheng et al., 2008.), but to our knowledge, there is no MBF-producing bacteria isolated from Ruditapes philippinarum conglutination mud. It is well known that R. philippinarum is a kind of delicious sea food, it lives in mud or sand at near seashore, and it was noticed that the water above it is very clear, it maybe deduced that there are probably some flocculating effect from the secretion of R. philippinarum. The aim of this study was to isolate bioflocculant-producing bacterium from R. philippinarum conglutination mud, optimizing

the culture conditions for the strain, producing new bioflocculant and applying it in wastewater treatment.

2. Methods 2.1. Isolation and identification of bioflocculant-producing microorganism Totally, 62 aerobic bacteria were isolated from R. philippinarum conglutination mud. Each isolated strain was inoculated in 250 mL Erlenmeyer flasks containing 100 mL culture medium and incubated for 4 days in a rotary shaker at 150 rpm and 30 °C. The flocculating efficiency of the strains was measured by Kaolin clay suspensions method (Kurane et al., 1986), and the strains which showed high flocculating efficiency were selected as bioflocculant-producing bacteria for further studies. The morphological character of the best strain was observed with Olympus CX31 biomicroscope (Japan), the physiological and biochemical characteristics of that were identified according to Bergey’s Manual of systematic bacteriology (Buchanan and Gibbens, 1984). PCR amplification of 16S rDNA was identified by Takara Biotechnology (Dalian, China) Co., Ltd. 2.2. The components of culture medium

* Corresponding authors. Tel.: +86 411 8410 9335; fax: +86 411 8410 6890 (X.-H. Zhu); tel.: +86 411 8410 6890; fax: +86 411 8410 6890 (J. Mu). E-mail addresses: [email protected] (X.-H. Zhu), [email protected] (J. Mu). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.05.035

For the isolation and purification, the culture medium (1 L) consisted of 45 g of nutrient agar and 1000 mL of artificial seawater

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(Mu et al. 2008). The initial pH was 7.0–7.2. After autoclaving at 115 °C for 30 min, it was made plate (9 cm) for use. For the screen fermentation, the culture medium (1 L) consisted of 20 g of glucose, 0.2 g of (NH4)2SO4, 0.5 g of urea, 0.5 g of yeast extract, 0.2 g of MgSO47H2O, 2.0 g of KH2PO4, 5.0 g of K2HPO4, and 1000 mL of artificial seawater. The initial pH was 7.0–7.2. It was autoclaved at 115 °C for 30 min. 2.3. Optimization of culture conditions of ZHT4-13 for bioflocculant production Experiments were designed according to Taguchi’s L9 orthogonal array (http://en.wikipedia.org/wiki/Taguchi_methods). Four factors including carbon source, nitrogen source, initial pH, and culture time were investigated in the optimization of the culture conditions for ZHT4-13 strain. Three different levels for initial pH and culture time were chosen, 6.0, 7.0, and 8.0 for initial pH, and 2, 3 and 4 days for culture time. Three different kinds of carbon sources and nitrogen sources were chosen also, they were glucose, D-fructose, and saccharose for carbon source, and urea, ammonium sulfate ((NH4)2SO4), and peptone for nitrogen source. The other components of the culture medium were 0.2 g of MgSO47H2O, 2.0 g of KH2PO4, 5.0 g of K2HPO4, 1000 mL of artificial seawater, and the inoculation amount was 108 CFU per 100 mL. 2.4. Bioflocculant purification The culture broth was centrifuged (Z36HK Refrigerated Centrifuge, Hermle LaborTechnik Gmbh) at 5000 rpm for 15 min to remove the cell pellets. The supernatant liquor was poured into two volume of cold ethanol with stirring and kept for 12 h at 4 °C to precipitate the bioflocculant. The resulting precipitate was collected by centrifugation at 10000 rpm for 15 min and washed by redissolving in distilled water. After three times repeat such trial, the crude bioflocculant was evaporated 2 h for dryness to remove all water and ethanol in vacuo by a rotary evaporator and vacuum drying overnight (12 h), and then the primarily purified bioflocculant was obtained. 2.5. Physical analysis of bioflocculant The bioflocculant produced by ZHT4-13 was named as MBF4-13 in this paper. MBF4-13 was characterized using a Fourier-transform infrared spectrophotometer (FT-IR, Bruker TENSOR 27, Germany). The dried sample was ground with KBr powder and pressed into pellets for FT-IR spectra measurement in the frequency range of 4000–500 cm1. The 1H NMR spectra of MBF413 were recorded on a 500 MHz Bruker Avance500 NMR spectrometer (Switzerland). The UV spectra of MBF4-13 were analyzed on UV-2102PCS (Unico (Shanghai, China) Instruments Co., Ltd.). 2.6. Assay of flocculating efficiency

trophotometer. The FR can be calculated according to the following formula:

FRð%Þ ¼ 100  ðA  BÞ=A

ð1Þ

The effects of pH (1–13) and temperature (4–100 °C) on the flocculation efficiency were also investigated. One milliliter MBF4-13 (2 g L1) and 10 mL Kaolin clay suspension (5 g L1) were added into a 50 mL test tube, which was mixed with vortex agitator. To investigate the effect of pH on flocculating activity, the initial pH of the above solution was adjusted using diluted hydrochloric acid and sodium hydroxide solution in the pH range of 1–13. To investigate the effect of temperature on flocculating activity, the tubes which contained 1 mL MBF4-13 (2 g L1) and 10 mL Kaolin clay suspension (5 g L1) were put into water bath and kept in water bath for 30 min, the temperature of the water bath was controlled 4, 20, 80 and 100 °C, respectively. The scanning electron microscopy (SEM) of Kaolin clay before and after flocculation were captured with JEOL JSM-6360LV SEM (Japan) operated at 24 kV. 2.7. Applications in wastewater treatment 2.7.1. Decolorization experiments Bioflocculant (1 mL, 2 g L1) was added to dye solution (10 mL, 10 mg L1), and the solution was well mixed with a shaker for 1 min. Then the mixture solution was kept still for 30 min. The absorbance of the supernatant liquor was measured under the maximum wavelength of the dye (660, 580, and 620 nm for methylene blue, crystal violet, and malachite green, respectively) with a spectrophotometer. The decolorizing efficiency (DC) of the dye solution can be calculated according to the following formula:

DCð%Þ ¼ 100  ðA0  AÞ=A0

ð2Þ

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 disposed with the bioflocculant. 2.7.2. Removal of heavy metal ions Metal ion solutions of chromium(VI) and nickel(II) were prepared by dissolving their respective salts, namely potassium dichromate (K2Cr2O7) and nickel chloride (NiCl2) in deionized water. Bioflocculant (1 mL of 2 g L1) was added to 10 mL metal 1 , Ni2+: 20 mg L1), and each solution ion solutions (Cr2 O2 7 : 1 mg L was well mixed with a shaker for 1 min. Then the mixture solutions were kept still for 30 min. The absorbance of the supernatant liquor was measured with diphenylcarbohydrazide spectrophotometry for sexavalence chromium and diacetyldioxime spectrophotometry for Ni2+ with a spectrophotometer (Yu, 2002). The removal efficiency (RE) of the heavy mental ion can be calculated according to the following formula:

REð%Þ ¼ 100  ðA0  AÞ=A0 Flocculating efficiency of the samples was calculated as flocculating rate (FR) and measured using the Kaolin clay suspension method (Kurane et al., 1986). Kaolin clay (0.5 g) was suspended in 93 mL deionized water, 5 mL CaCl2 (10 g L1), and 2 mL bioflocculant of 2 g L1 were added, and the pH value of that was adjusted to 7.5 with diluted hydrochloric acid and sodium hydroxide solution. The above mixture solution was quickly stirred at 200 rpm for 1 min, slowly stirred at 80 rpm for 2 min, and then it was kept still for 5 min. The absorbance of the supernatant liquor of the above mixture (B) and the blank control sample (A) (i.e. the deionized water was added into the Kaolin clay suspension instead of the bioflocculant solution) was measured at 550 nm with a spec-

4997

ð3Þ

where A0 is the absorbance of the blank sample (i.e. the same volume deionized water was added into the metal ion solution instead of the bioflocculant); A is the absorbance of supernatant liquor of the heavy metal ion solution disposed with the bioflocculant. 2.7.3. Improving the performance of active sludge Active sludge was obtained from Lingshui municipal swage treatment plant (Dalian, China). The active sludge was cultured for 2 days without aeration in order to get the bulking sludge. Then bioflocculant of 2 g L1 was added to the bulking sludge and cultured. The microscopic images of the bulking sludge’s structure

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3.2. Optimization of culture conditions of ZHT4-13 for bioflocculant producing

Fig. 1. Result of PCR product in 1% agarose gel electrophoresis of ZHT4-13. Lane M: DNA mark DL2000; Lane 1:ZHT4-13 PCR product; Lane +: positive control; Lane : negative control.

and microbial constitution before and after disposed with the bioflocculant were recorded.

3. Results and discussion 3.1. Isolation and identification of bioflocculant-producing microorganism Totally, 62 aerobic bacteria were isolated from R. philippinarum conglutination mud. And 17 strains were selected as the bioflocculant-producing bacteria. The strain of ZHT4-13, which showed the highest flocculating efficiency of 83.9% for 5 g L1 Kaolin clay suspensions, was chosen as bioflocculant-producing strain for further studies. The colony of ZHT4-13 is small, circular, milk white, trim edge, and smooth. It’s Gram-positive, obligate aerobic bacterium, non-endospore forming, having no flagellum and immotile, and the diameter of cells was 0.5–1 lm (The scanning electron microphotograph (SEM) image of ZHT4-13 is shown in supplementary material Fig. S1.). Some of the physiological and biochemical characteristics of the bacterium were as follows: Glycolysis, methyl-red, nitrate reduction and catalase test were all positive; indole, gelatin liquefaction, starch hydrolysis, Voges–Prokauer, H2S, oxidase and citrate test were all negative. The 16S rDNA of strain ZHT4-13 was sequenced and analyzed by Takara Biotechnology (Dalian, China) Co., Ltd. The result of PCR product in 1% agarose gel electrophoresis is shown in Fig. 1. The 16S rDNA sequences of strain ZHT4-13 was registered in GenBank and the accession number of strain ZHT4-13 is EU873349. According to the 16S rDNA sequence and the physiological and biochemica characteristic, strain ZHT4-13 could be identified as Rothia sp.

According to literature (Deng et al., 2005; He et al., 2004; Shih et al. 2001), the effective factors for producing bioflocculant were chosen. For carbon source (2%): glucose with one crystalwater (monosaccharide), D-fructose (monosaccharide), and saccharose (disaccharide); for nitrogen source (0.05%): urea (lower molecular weight organic nitrogen source), (NH4)2SO4 (lower molecular weight inorganic nitrogen source), peptone (higher molecular weight organic nitrogen source); for initial pH: as most of strains are able to grow in neutral condition (pH = 7.0), pH 6.0 (subacidity), pH 7.0 (neutrality) and pH 8.0 (alkalescence) were chosen; for culture time: as the liquid fermentation culture time is usually around 3 days, 2, 3 and 4 days were chosen. The other components of the culture medium are 0.5% NaCl, 0.1% MgSO47H2O, 0.5% KH2PO4, and 0.2% K2HPO4. The culture temperature was 30 °C, shaking speed was 150 rpm, inoculation amount was 108 CFU per 100 mL. Cell production amount (per 100 mL), MBF production amount (per 100 mL), cell FR, and MBF FR were measured to assess the optimum culture conditions for microbial flocculant production by strain ZHT4-13. The orthogonal design of experiments and results of optimized culture condition for ZHT4-13 are shown in Table 1. The optimum culture condition for strain ZHT4-13 to produce high-performance MBF4-13 was selected as follows: 20 g L1 of saccharose, 0.2 g L1 of (NH4)2SO4, 0.5 g L1 of peptone, 0.2 g L1 of MgSO47H2O, 2.0 g L1 of KH2PO4, 5.0 g L1 of K2HPO4, artificial seawater, pH 8.0, and 4 days cultivation (The detailed orthogonal experiments results and analysis of which are given in supplementary material.). The inoculation amount was 108 CFU per 100 mL. With the above culture medium, the FR of MBF4-13 for 5 g L1 Kaolin suspension was 86.01%. 3.3. Characteristics of the bioflocculant MBF4-13 It can be seen from the UV spectrum of MBF4-13 (The UV spectrum of MBF4-13 is shown in supplementary material Fig. S2.) that there are no nucleic acid (260 nm) and protein (280 nm) absorption peak, instead, there is an absorption peak at 200 nm characteristic for polysaccharide (Lu et al., 2005). From the FT-IR spectrum (The FT-IR spectrum of MBF4-13 is shown in supplementary material Fig. S2.), the characteristic chemical groups of MBF4-13 were observed as followed. The absorption peak at 3415 cm1 is characteristic of –OH stretching vibration. The single peak at 1660 cm1 is the absorptive band of –OH deformation vibration or C@O stretching vibration from conjugate aldehydes or a,b-unsaturated aldehydes. The absorptive peaks at 1401 cm1 is probably caused by tertiary alcohols and phenol. The absorptive peak at 1118 cm1 is characteristics of C–O–C stretching vibration. As there was no peak

Table 1 Orthogonal test design and results of optimized culture conditions for strain ZHT4-13. Test Test Test Test Test Test Test Test Test Test a

1 2 3 4 5 6 7 8 9

Carbon source

Nitrogen source

Initial pH

Culture time (d)

Cell production amount (g)

MBF production amount (g)

Cell FR (%)

MBF FR (%)

Glucose Glucose Glucose D-Fructose D-Fructose D-Fructose Saccharose Saccharose Saccharose

Urea (NH4)2SO4 Peptone Urea (NH4)2SO4 Peptone Urea (NH4)2SO4 Peptone

6.0 7.0 8.0 7.0 8.0 6.0 8.0 6.0 7.0

2 3 4 4 2 3 3 4 2

0.0013 0.0058 0.0055 0.0056 0.0268 0.0012 0.0102 0.0058 0.0257

0.1264 0.2385 0.1223 0.2394 0.2045 0.1433 0.0514 0.1497 0.0402

4.2 –a 51.1 36.0 28.2 37.7 64.5 72.8 43.7

11.2 42.5 64.3 46.0 30.9 58.6 75.7 70.0 50.9

The cell had no flocculability.

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from 910 to 650 cm1, it can be inferred that there was no structure of aromatic ring in MBF4-13. Based on the above proofs of the carboxyl and hydroxyl groups’ presence, and the strong absorption peak presented in the range from 1000 to 1200 cm1 which were generally known to be typical characteristics of all sugar derivatives (Gong et al., 2008), it was deduced that the main component of MBF4-13 should be polysaccharide. Moreover, the 1H NMR spectrum of MBF4-13 (The NMR spectrum of MBF4-13 is shown in supplementary material Fig. S3.) (tested in D2O added with CD3COOD) showed the signals at d 5.42 ppm (J = 3.9 Hz) (anomeric proton for a-sugar residue), d 4.7–4.8 ppm (anomeric proton for b-sugar residue, partially overlapped with the HDO residue solvent peak of D2O), d 3.48–4.22 ppm (plenty of other protons on saccharide rings), also proved that the major component of MBF4-13 was polysaccharide. It’s needed to be supplemented that limited by the sample’s too low solubility in the solvents for NMR testing, e.g., DMSO-d6, D2O, D2O added with CD3COOD or other solvents, the above protons’ signals were a little weak compared with the internal standard TMS (d 0.02 ppm) and the solvent peak at d 2.04 ppm (CD3COOD) and d 4.76 ppm (D2O). For the same reason, the necessary 13C NMR spectrum was not possible to be recorded to give further detailed structure information. As for the strong signals at d 2.93, 1.78, and 0.65 ppm, because of their disproportion to the intensity of the signals of polysaccharide, they were deduced to be some impurities more soluble than the main component in the recent test solvent. So, its exact structure is to be elucidated by further chromatographic purification, spectral tests, and hydrolyzing analysis. 3.4. Flocculating characteristics of MBF4-13 for Kaolin clay SEM of MBF4-13 was measured to research the flocculability of it for Kaolin clay suspension (The SEM images of Kaolin clay and Kaolin clay flocculated by MBF4-13 is shown in supplementary material Fig. S4). Compared with the scattered particles without processing, the flocculated Kaolin clay particles were connected together by bridge-like MBF4-13 or even trapped by the pectic net formed by MBF4-13. So it was speculated that the flocculation mechanism of MBF4-13 was probably bridging and network capturing bridging action. The range of pH and temperature for flocculating Kaolin clay suspension with MBF4-13 were also measured (Tables 2 and 3). From which, it can be seen that MBF4-13 had a flocculation effi-

Table 2 The influence of initial pH of Kaolin clay suspensions on the flocculation efficiency. pH

FR (%)

1 3 5 7 9 11 13

21.98 15.49 32.39 85.14 86.22 62.61 57.04

Table 3 The influence of the temperature of Kaolin clay suspensions on the flocculation efficiency. Temperature (°C)

FR (%)

4 20 80 100

65.1 86.01 87.69 86.01

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ciency in a wide pH range of 1–13, the optimal pH for the flocculating was in the range of 7–9, and it kept a high flocculating activity in the temperature range 4–100 °C. The optimum pH of MBF4-13 is quite different from the other polysaccharide bioflocculants. The optimal pH of polysaccharide bioflocculants SF-1 was in the weakly acidic or near neutral range of 5.0–7.0 and was not heatstable (Gong et al., 2008). The exopolysaccharide bioflocculants p-KG03 was an effective flocculant under acidic conditions (pH 3.0–6.0) and over a wide temperature range (4–90 °C) (Yim et al., 2007). 3.5. Applications of MBF4-13 in wastewater treatment MBF4-13 was added into dye solutions, heavy metal solutions, and bulking activated sludge to determine the flocculating abilities in decolorization, removal of heavy mental ions, and improving performance of activated sludge. 3.5.1. Decolorization for dye solution One milliliter of MBF4-13 (2 g L1) was added into 10 mL dye solutions (10 mg L1 methylene blue, 20 mg L1 crystal violet, and 10 mg L1 malachite green). After well mixing and standing still for 30 min, the absorbance of the supernatant liquor was measured by a spectrophotometer. The DC (%) for methylene blue, crystal violet and malachite green were 86.11%, 97.84% and 99.49%, respectively. It was found interestingly that MBF4-13 had a strong decolorizing ability for blue and violet series of dyes, but had a relatively small decolorizing ability for red, pink and orange series dyes. The reason for this may be relevant to the composition and functional groups of MBF4-13 and dyes. Deng et al. (2005) found the bioflocculant produced by Aspergillus parasticus was more effective for Reactive Blue 4 and Acid Yellow 25 than for Basic Blue B. The results showed that depending on the dye used, the bioflocculant exhibited different decolorzing efficiency. 3.5.2. Removal of heavy mental ions One milliliter of MBF4-13 (2 g L1) was added into 10 mL heavy and 20 mg L1 Ni2+, respecmetal ion solutions (1 mg L1 Cr2 O2 7 tively). After well mixing and standing still for 30 min, the absorbance of that was determined. The RE (%) for Ni2+ and Cr2 O27 were 19.2% and 69.3%, respectively. The removal efficiencies for Ni2+ and Cr2 O27 are quite different. According to Section 3.3, MBF4-13 has hydroxyl group, it can be inferred that maybe hydrogen bonds were formed between MBF4-13 and Cr2 O2 7 , it had high2+ than for Ni . er removal rate for Cr2 O2 7 3.5.3. Improving performance of activated sludge One milliliter of MBF4-13 (2 g L1) was added into the bulking activated sludge. After culturing for 48 h, the microscopic images of zoogloea and microorganisms were photographed. By comparison of picture A and B in Fig. 2, it can be seen that the structure of zoogloea between original sludge (picture A) and after flocculated sludge (picture B) had a remarkable change. Flocculating by MBF413 made the zoogloea more tightly and firmly. Picture C and D in Fig. 2 showed that the kinds of microorganisms in the activated sludge were also quite different. After flocculation of sludge, the filamentous fungi which caused sludge bulking disappeared, and protozoa increased, such as rotifers, vorticella present, which were indicator organisms for good water quality. 3.5.4. Comparison with traditional flocculants and the other bioflocculants The traditional flocculants, sodium meta-aluminate, aluminum sulfate and polyaluminium chloride were chosen for comparing the flocculating efficiency with MBF4-13. As the results shown in Table 4, the flocculating efficiency for Kaolin clay suspension using

5000

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Fig. 2. Micro-photos of activated sludge. (A) zoogloea in bulking sludge, (B) zoogloea in flocculated sludge, (C) filamentous fungus in bulking sludge, and (D) protozoan in flocculated sludge.

MBF4-13 was similar to or even better than some of the above mentioned traditional chemical synthetic flocculants. To date, many studies on the microbial production of flocculating substances have been reported from different viewpoints, most recently identified bioflocculants were polysaccharide-like substances, including poly-glutamic acids with molecular weight of 20–3000 kDa (Deng et al., 2003; Fujita et al., 2000; Toead and Kurane, 1991; Salehizadeh and Shojaosadati, 2001; Yokoi et al., 1996; You et al., 2008). The above mentioned study was mainly focused on the flocculating activity of bioflocculants for Kaolin suspension. Although the bioflocculant produced by A. parasticus could decolorize some kinds of dyes, the sludge produced after the dye molecules were absorbed on the flocculant did not compact well (Deng et al., 2005). In this study, it has been proved that bioflcculant MBF4-13 not only has the ability to flocculate Kaolin suspension effectively but also has the ability for decolorizing of dye solution, removal of some kinds of heavy metal ions in waste water and improving the performance of active sludge. The application range of bioflcculant MBF4-13 is much wider, and for it is produced from the strain ZHT4-13 which was isolated from R. philippinarum conglutination mud, maybe it can be deduced that bioflcculant MBF4-13 is harmless toward human. Bioflcculant MBF4-13 is anticipated like c-PGA to be utilized in the areas of wastewater treatment, drinking-water processing and downstream processing in food and fermentation industries as a new bioflocculant which is harmless towards humans and the environment (Yokoi et al. 1996). Table 4 Comparison of the flocculent activity of MBF4-13 with traditional flocculants. Flocculants

FR (%)

Sodium meta-aluminate Aluminum sulfate Polyaluminum chloride MBF4-13

50.28 74.83 88.25 86.01

4. Conclusions A novel bioflocculant-producing bacterium ZHT4-13 was isolated from the conglutination mud of R. philippinarum. It was identified as Rothia sp. The bioflcculant MBF4-13 produced by ZHT4-13 had 86.22% flocculating efficiency for Kaolin clay of 5 g L1. Spectral analysis showed that the main constituent of MBF4-13 was polysaccharide with complex composition. The results of experiments showed the optimal culture conditions for bioflocculant production were as follows: 20.0 g L1 of saccharose as carbon source, 0.2 g L1 of peptone and 0.2 g L1 of (NH4)2SO4 as complex nitrogen source, 2.0 g L1 of KH2PO4, 5.0 g L1 of K2HPO4, 1000 mL of artificial seawater, pH 8.0, and 4 days cultivation. The bioflocculant MBF4-13 was effective for flocculating Kaolin clay suspension in the range of pH 1–13 and temperature 4–100 °C. MBF4-13 was able to treat some kinds of dye wastewater and heavy metal wastewater, and also improve activated sludge property. MBF4-13 had a strong decolorizing ability for blue and violet series of dye. For 10 mg L1 malachite green solution, the DC of that was up to 99.49%. The flocculating efficiency for Kaolin clay suspension of MBF4-13 was similar to or even better than some of the traditional chemical synthetic flocculants, such as sodium meta-aluminate, aluminum sulfate and polyaluminium chloride. Acknowledgements We are pleased to acknowledge the valuable comments made by two anonymous reviewers. The study was supported by Chinese National Programs for High Technology Research and Development (No. 2006AA09Z426). Appendix A. Supplementary data Details of statistical analysis results about the intuitive analysis, variance analysis, interaction analysis of carbon source/nitrogen

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source and the comprehensive compare of the orthogonal experiments results for the yield of cells, the flocculating ability of cells for Kaolin clay suspension, the yield of MBF4-13 and the flocculating ability of MBF4-13 for Kaolin clay suspension are listed. The Figure of the SEM of Rothia sp. strain ZHT4-13, the UV (A) and FT-IR (B) spectra of MBF4-13, the 1H NMR spectra of MBF4-13 and the SEM images of (A) Kaolin clay and (B) Kaolin clay flocculated by MBF4-13 are listed too. Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biortech.2009.05.035. References Buchanan, R.E., Gibbens, N.E., 1984. Bergey’s Manual of Systematic Bacteriology, eighth ed. Science Press, Beijing. Deng, S.B., Bai, R.B., Xu, X.M., Luo, Q., 2003. Characteristics of a bioflocculant produced by Bacillus mucilaginosus and its use in starch wastewater treatment. Appl. Microbiol. Biotechnol. 60, 588–593. Deng, S.B., Yu, G., Ting, Y.P., 2005. Production of a bioflocculant by Aspergillus parasiticus and its application in dye removal. Colloids Surf. B 44, 179–186. Fujita, M., Ike, M., Tachibana, S., Kitada, G., Kim, S.M., Inoue, Z., 2000. Characterization of bioflocculant produced by Citrobacter sp. TKF04 from acetic and propionic acids. J. Biosci. Bioeng 89, 40–46. Gong, W.-X., Wang, S.-G., Sun, X.-F., Liu, X.-W., Yue, Q.-Y., Gao, B.-Y., 2008. Bioflocculant production by culture of Serratia ficaria and its application in wastewater treatment. Bioresour. Technol. 99, 4668–4674. He, N., Li, Y., Chen, J., 2004. Production of a novel polygalacturotic acid bioflocculant REA-11 by Corynebacterium ghutamicum. Bioresour. Technol. 94, 99–105. Kumar, C.G., Joo, H.-S., Choi, J.-W., Koo, Y.-M., Chang, C.-S., 2004. Purification and characterization of an extracellular polysaccharide from halolkalophilic Bacillus sp.I-450. Enzyme Microb. Technol. 34, 673–681. Kurane, R., Toeda, K., Takeda, K., 1986. Culture conditions for production of microbial flocculant by Phodococcus erythropols. Agric. Biol. Chem. 50, 2309– 2313. Kurane, R., Hatamochi, K., Kakuno, T., Kiyohara, M., Kawaguchi, K., Mizuno, Y., Hirano, M., Taniguchi, Y., 1994. Puification and Characterization of Lipid Bioflocculant Produced by Phodococcus erythropolis. Biosci. Biotechnol. Biochem. 58, 1977–1982.

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