Bioresource Technology 100 (2009) 4996–5001

Contents lists available at ScienceDirect

Bioresource Technology

journal homepage: www.elsevier .com/locate /bior tech

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

Qi Gao, Xiu-Hua Zhu *, Jun Mu *, Yi Zhang, Xue-Wei DongSchool of Environmental and Chemical Engineering, Dalian Jiaotong University, Dalian 116028, China

a r t i c l e i n f o a b s t r a c t

Article history:Received 1 February 2009Received in revised form 8 May 2009Accepted 17 May 2009Available online 16 June 2009

Keywords:Ruditapes philippinarumConglutination mudBioflocculantWastewater treatment

0960-8524/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.biortech.2009.05.035

* Corresponding authors. Tel.: +86 411 8410 933(X.-H. Zhu); tel.: +86 411 8410 6890; fax: +86 411 84

E-mail addresses: zhuxiuhua1@hotmail.com (X.-H(J. Mu).

A novel bioflocculant-producing bacterium, ZHT4-13, was isolated from Ruditapes philippinarum conglu-tination mud. By biomicroscope morphological observation, 16S rDNA sequence identification and phys-iological and biochemical characteristics, strain ZHT4-13 was identified as Rothia sp. The bioflocculantMBF4-13 produced by strain ZHT4-13 had a flocculating efficiency of 86.22% for 5 g L�1 Kaolin clay sus-pension when the initial pH was 9 and the temperature was 20 �C. It had flocculating effect in a widerange, pH 1–13 and temperature 4–100 �C. Analysis of MBF4-13 by UV–Vis spectrophotometer, Fou-rier-transform infrared spectrophotometer (FT-IR) and 1H nuclear magnetic resonance (NMR) indicatedthat the main component of MBF4-13 is polysaccharide. The culture conditions to produce strainZHT4-13 were optimized with orthogonal design of experiments. MBF4-13 had high efficiency indecolorizing dye solutions, had some abilities to remove heavy metal ions (Cr2O2�

7 , Ni2+) and improveperformance of activated sludge.

� 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 micro-bial flocculants (MBF) has attracted wide-attention in water treat-ment research field, and MBFs are widely used in wastewatertreatment, such as downstream processing, food and industrywastewater processing (Salehizadeh et al., 2000; Salehizadeh andShojaosadati, 2001; You et al., 2008).

MBF-producing bacteria were screened out from almost allkinds of environment of land or water, such as soil, activatedsludge, wastewater, and river (Deng et al., 2005; Gong et al.,2008; He et al., 2004; Kumar et al., 2004; Kurane et al., 1994; Sale-hizadeh et al., 2000; Salehizadeh and Shojaosadati 2001; Shih et al.,2001; Suh et al., 1997; Wang et al., 2007; Wu and Ye, 2007; Xiaet al., 2008; Yim et al., 2007;You et al., 2008; Zheng et al., 2008.),but to our knowledge, there is no MBF-producing bacteria isolatedfrom Ruditapes philippinarum conglutination mud. It is well knownthat R. philippinarum is a kind of delicious sea food, it lives in mudor sand at near seashore, and it was noticed that the water above itis very clear, it maybe deduced that there are probably some floc-culating effect from the secretion of R. philippinarum.

The aim of this study was to isolate bioflocculant-producingbacterium from R. philippinarum conglutination mud, optimizing

ll rights reserved.

5; fax: +86 411 8410 689010 6890 (J. Mu).. Zhu), mujun2003@263.net

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

2. Methods

2.1. Isolation and identification of bioflocculant-producing microor-ganism

Totally, 62 aerobic bacteria were isolated from R. philippinarumconglutination mud. Each isolated strain was inoculated in 250 mLErlenmeyer flasks containing 100 mL culture medium and incu-bated for 4 days in a rotary shaker at 150 rpm and 30 �C. The floc-culating efficiency of the strains was measured by Kaolin claysuspensions method (Kurane et al., 1986), and the strains whichshowed high flocculating efficiency were selected as biofloccu-lant-producing bacteria for further studies.

The morphological character of the best strain was observedwith Olympus CX31 biomicroscope (Japan), the physiological andbiochemical characteristics of that were identified according toBergey’s Manual of systematic bacteriology (Buchanan and Gib-bens, 1984). PCR amplification of 16S rDNA was identified by Taka-ra Biotechnology (Dalian, China) Co., Ltd.

2.2. The components of culture medium

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

Q. Gao et al. / Bioresource Technology 100 (2009) 4996–5001 4997

(Mu et al. 2008). The initial pH was 7.0–7.2. After autoclaving at115 �C for 30 min, it was made plate (9 cm) for use.

For the screen fermentation, the culture medium (1 L) consistedof 20 g of glucose, 0.2 g of (NH4)2SO4, 0.5 g of urea, 0.5 g of yeastextract, 0.2 g of MgSO4�7H2O, 2.0 g of KH2PO4, 5.0 g of K2HPO4,and 1000 mL of artificial seawater. The initial pH was 7.0–7.2. Itwas autoclaved at 115 �C for 30 min.

2.3. Optimization of culture conditions of ZHT4-13 for bioflocculantproduction

Experiments were designed according to Taguchi’s L9 orthogo-nal array (http://en.wikipedia.org/wiki/Taguchi_methods). Fourfactors including carbon source, nitrogen source, initial pH, andculture time were investigated in the optimization of the cultureconditions for ZHT4-13 strain. Three different levels for initial pHand culture time were chosen, 6.0, 7.0, and 8.0 for initial pH, and2, 3 and 4 days for culture time. Three different kinds of carbonsources and nitrogen sources were chosen also, they were glucose,D-fructose, and saccharose for carbon source, and urea, ammoniumsulfate ((NH4)2SO4), and peptone for nitrogen source. The othercomponents of the culture medium were 0.2 g of MgSO4�7H2O,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 Centri-fuge, Hermle LaborTechnik Gmbh) at 5000 rpm for 15 min to re-move the cell pellets. The supernatant liquor was poured intotwo volume of cold ethanol with stirring and kept for 12 h at4 �C to precipitate the bioflocculant. The resulting precipitate wascollected by centrifugation at 10000 rpm for 15 min and washedby redissolving in distilled water. After three times repeat suchtrial, the crude bioflocculant was evaporated 2 h for dryness to re-move all water and ethanol in vacuo by a rotary evaporator andvacuum drying overnight (12 h), and then the primarily purifiedbioflocculant was obtained.

2.5. Physical analysis of bioflocculant

The bioflocculant produced by ZHT4-13 was named as MBF4-13in this paper. MBF4-13 was characterized using a Fourier-trans-form infrared spectrophotometer (FT-IR, Bruker TENSOR 27, Ger-many). The dried sample was ground with KBr powder andpressed into pellets for FT-IR spectra measurement in the fre-quency range of 4000–500 cm�1. The 1H NMR spectra of MBF4-13 were recorded on a 500 MHz Bruker Avance500 NMR spectrom-eter (Switzerland). The UV spectra of MBF4-13 were analyzed onUV-2102PCS (Unico (Shanghai, China) Instruments Co., Ltd.).

2.6. Assay of flocculating efficiency

Flocculating efficiency of the samples was calculated as floccu-lating rate (FR) and measured using the Kaolin clay suspensionmethod (Kurane et al., 1986). Kaolin clay (0.5 g) was suspendedin 93 mL deionized water, 5 mL CaCl2 (10 g L�1), and 2 mL bioflocc-ulant of 2 g L�1 were added, and the pH value of that was adjustedto 7.5 with diluted hydrochloric acid and sodium hydroxide solu-tion. The above mixture solution was quickly stirred at 200 rpmfor 1 min, slowly stirred at 80 rpm for 2 min, and then it was keptstill for 5 min. The absorbance of the supernatant liquor of theabove mixture (B) and the blank control sample (A) (i.e. the deion-ized water was added into the Kaolin clay suspension instead ofthe bioflocculant solution) was measured at 550 nm with a spec-

trophotometer. The FR can be calculated according to the followingformula:

FRð%Þ ¼ 100� ðA� BÞ=A ð1Þ

The effects of pH (1–13) and temperature (4–100 �C) on theflocculation efficiency were also investigated.

One milliliter MBF4-13 (2 g L�1) and 10 mL Kaolin clay suspen-sion (5 g L�1) were added into a 50 mL test tube, which was mixedwith vortex agitator. To investigate the effect of pH on flocculatingactivity, the initial pH of the above solution was adjusted using di-luted hydrochloric acid and sodium hydroxide solution in the pHrange of 1–13.

To investigate the effect of temperature on flocculating activity,the tubes which contained 1 mL MBF4-13 (2 g L�1) and 10 mL Kao-lin clay suspension (5 g L�1) were put into water bath and kept inwater bath for 30 min, the temperature of the water bath was con-trolled 4, 20, 80 and 100 �C, respectively.

The scanning electron microscopy (SEM) of Kaolin clay beforeand after flocculation were captured with JEOL JSM-6360LV SEM(Japan) operated at 24 kV.

2.7. Applications in wastewater treatment

2.7.1. Decolorization experimentsBioflocculant (1 mL, 2 g L�1) was added to dye solution (10 mL,

10 mg L�1), and the solution was well mixed with a shaker for1 min. Then the mixture solution was kept still for 30 min. Theabsorbance of the supernatant liquor was measured under themaximum wavelength of the dye (660, 580, and 620 nm for meth-ylene blue, crystal violet, and malachite green, respectively) with aspectrophotometer. The decolorizing efficiency (DC) of the dyesolution 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 vol-ume deionized water was added into the dye solution instead ofthe bioflocculant); A is the absorbance of supernatant liquor ofthe dye solution after disposed with the bioflocculant.

2.7.2. Removal of heavy metal ionsMetal ion solutions of chromium(VI) and nickel(II) were pre-

pared by dissolving their respective salts, namely potassiumdichromate (K2Cr2O7) and nickel chloride (NiCl2) in deionizedwater. Bioflocculant (1 mL of 2 g L�1) was added to 10 mL metalion solutions (Cr2O2�

7 : 1 mg L�1, Ni2+: 20 mg L�1), and each solutionwas well mixed with a shaker for 1 min. Then the mixture solu-tions were kept still for 30 min. The absorbance of the supernatantliquor was measured with diphenylcarbohydrazide spectropho-tometry for sexavalence chromium and diacetyldioxime spectro-photometry for Ni2+ with a spectrophotometer (Yu, 2002). Theremoval efficiency (RE) of the heavy mental ion can be calculatedaccording to the following formula:

REð%Þ ¼ 100� ðA0 � AÞ=A0 ð3Þ

where A0 is the absorbance of the blank sample (i.e. the same vol-ume deionized water was added into the metal ion solution insteadof the bioflocculant); A is the absorbance of supernatant liquor ofthe heavy metal ion solution disposed with the bioflocculant.

2.7.3. Improving the performance of active sludgeActive sludge was obtained from Lingshui municipal swage

treatment plant (Dalian, China). The active sludge was culturedfor 2 days without aeration in order to get the bulking sludge. Thenbioflocculant of 2 g L�1 was added to the bulking sludge and cul-tured. The microscopic images of the bulking sludge’s structure

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.

4998 Q. Gao et al. / Bioresource Technology 100 (2009) 4996–5001

and microbial constitution before and after disposed with the bio-flocculant were recorded.

3. Results and discussion

3.1. Isolation and identification of bioflocculant-producingmicroorganism

Totally, 62 aerobic bacteria were isolated from R. philippinarumconglutination mud. And 17 strains were selected as the biofloccu-lant-producing bacteria. The strain of ZHT4-13, which showed thehighest flocculating efficiency of 83.9% for 5 g L�1 Kaolin clay sus-pensions, was chosen as bioflocculant-producing strain for furtherstudies. The colony of ZHT4-13 is small, circular, milk white, trimedge, and smooth. It’s Gram-positive, obligate aerobic bacterium,non-endospore forming, having no flagellum and immotile, andthe diameter of cells was 0.5–1 lm (The scanning electron micro-photograph (SEM) image of ZHT4-13 is shown in supplementarymaterial Fig. S1.).

Some of the physiological and biochemical characteristics of thebacterium were as follows: Glycolysis, methyl-red, nitrate reduc-tion and catalase test were all positive; indole, gelatin liquefaction,starch hydrolysis, Voges–Prokauer, H2S, oxidase and citrate testwere all negative.

The 16S rDNA of strain ZHT4-13 was sequenced and analyzedby Takara Biotechnology (Dalian, China) Co., Ltd. The result ofPCR product in 1% agarose gel electrophoresis is shown in Fig. 1.The 16S rDNA sequences of strain ZHT4-13 was registered in Gen-Bank and the accession number of strain ZHT4-13 is EU873349.

According to the 16S rDNA sequence and the physiological andbiochemica characteristic, strain ZHT4-13 could be identified asRothia sp.

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

Test Carbon source Nitrogen source Initial pH Culture time (d) Cell p

Test 1 Glucose Urea 6.0 2 0.0013Test 2 Glucose (NH4)2SO4 7.0 3 0.0058Test 3 Glucose Peptone 8.0 4 0.0055Test 4 D-Fructose Urea 7.0 4 0.0056Test 5 D-Fructose (NH4)2SO4 8.0 2 0.0268Test 6 D-Fructose Peptone 6.0 3 0.0012Test 7 Saccharose Urea 8.0 3 0.0102Test 8 Saccharose (NH4)2SO4 6.0 4 0.0058Test 9 Saccharose Peptone 7.0 2 0.0257

a The cell had no flocculability.

3.2. Optimization of culture conditions of ZHT4-13 for bioflocculantproducing

According to literature (Deng et al., 2005; He et al., 2004; Shihet al. 2001), the effective factors for producing bioflocculant werechosen. For carbon source (2%): glucose with one crystalwater(monosaccharide), D-fructose (monosaccharide), and saccharose(disaccharide); for nitrogen source (0.05%): urea (lower molecularweight organic nitrogen source), (NH4)2SO4 (lower molecularweight inorganic nitrogen source), peptone (higher molecularweight organic nitrogen source); for initial pH: as most of strainsare able to grow in neutral condition (pH = 7.0), pH 6.0 (subacidi-ty), pH 7.0 (neutrality) and pH 8.0 (alkalescence) were chosen;for culture time: as the liquid fermentation culture time is usuallyaround 3 days, 2, 3 and 4 days were chosen. The other componentsof the culture medium are 0.5% NaCl, 0.1% MgSO4�7H2O, 0.5%KH2PO4, and 0.2% K2HPO4. The culture temperature was 30 �C,shaking speed was 150 rpm, inoculation amount was 108 CFU per100 mL.

Cell production amount (per 100 mL), MBF production amount(per 100 mL), cell FR, and MBF FR were measured to assess theoptimum culture conditions for microbial flocculant productionby strain ZHT4-13. The orthogonal design of experiments and re-sults of optimized culture condition for ZHT4-13 are shown in Ta-ble 1.

The optimum culture condition for strain ZHT4-13 to producehigh-performance MBF4-13 was selected as follows: 20 g L�1 ofsaccharose, 0.2 g L�1 of (NH4)2SO4, 0.5 g L�1 of peptone, 0.2 g L�1

of MgSO4�7H2O, 2.0 g L�1 of KH2PO4, 5.0 g L�1 of K2HPO4, artificialseawater, pH 8.0, and 4 days cultivation (The detailed orthogonalexperiments results and analysis of which are given in supplemen-tary material.). The inoculation amount was 108 CFU per 100 mL.With the above culture medium, the FR of MBF4-13 for 5 g L�1 Kao-lin 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 spec-trum of MBF4-13 is shown in supplementary material Fig. S2.) thatthere are no nucleic acid (260 nm) and protein (280 nm) absorp-tion peak, instead, there is an absorption peak at 200 nm character-istic for polysaccharide (Lu et al., 2005). From the FT-IR spectrum(The FT-IR spectrum of MBF4-13 is shown in supplementary mate-rial Fig. S2.), the characteristic chemical groups of MBF4-13 wereobserved as followed. The absorption peak at 3415 cm�1 is charac-teristic of –OH stretching vibration. The single peak at 1660 cm�1 isthe absorptive band of –OH deformation vibration or C@O stretch-ing vibration from conjugate aldehydes or a,b-unsaturated alde-hydes. The absorptive peaks at 1401 cm�1 is probably caused bytertiary alcohols and phenol. The absorptive peak at 1118 cm�1 ischaracteristics of C–O–C stretching vibration. As there was no peak

roduction amount (g) MBF production amount (g) Cell FR (%) MBF FR (%)

0.1264 4.2 11.20.2385 –a 42.50.1223 51.1 64.30.2394 36.0 46.00.2045 28.2 30.90.1433 37.7 58.60.0514 64.5 75.70.1497 72.8 70.00.0402 43.7 50.9

Q. Gao et al. / Bioresource Technology 100 (2009) 4996–5001 4999

from 910 to 650 cm�1, it can be inferred that there was no struc-ture of aromatic ring in MBF4-13. Based on the above proofs ofthe carboxyl and hydroxyl groups’ presence, and the strong absorp-tion peak presented in the range from 1000 to 1200 cm�1 whichwere generally known to be typical characteristics of all sugarderivatives (Gong et al., 2008), it was deduced that the main com-ponent of MBF4-13 should be polysaccharide. Moreover, the 1HNMR spectrum of MBF4-13 (The NMR spectrum of MBF4-13 isshown in supplementary material Fig. S3.) (tested in D2O addedwith CD3COOD) showed the signals at d 5.42 ppm (J = 3.9 Hz) (ano-meric proton for a-sugar residue), d 4.7–4.8 ppm (anomeric protonfor b-sugar residue, partially overlapped with the HDO residue sol-vent peak of D2O), d 3.48–4.22 ppm (plenty of other protons onsaccharide rings), also proved that the major component ofMBF4-13 was polysaccharide. It’s needed to be supplemented thatlimited by the sample’s too low solubility in the solvents for NMRtesting, e.g., DMSO-d6, D2O, D2O added with CD3COOD or other sol-vents, the above protons’ signals were a little weak compared withthe internal standard TMS (d 0.02 ppm) and the solvent peak at d2.04 ppm (CD3COOD) and d 4.76 ppm (D2O). For the same reason,the necessary 13C NMR spectrum was not possible to be recordedto give further detailed structure information. As for the strong sig-nals at d 2.93, 1.78, and 0.65 ppm, because of their disproportion tothe intensity of the signals of polysaccharide, they were deduced tobe some impurities more soluble than the main component in therecent test solvent. So, its exact structure is to be elucidated by fur-ther chromatographic purification, spectral tests, and hydrolyzinganalysis.

3.4. Flocculating characteristics of MBF4-13 for Kaolin clay

SEM of MBF4-13 was measured to research the flocculability ofit for Kaolin clay suspension (The SEM images of Kaolin clay andKaolin clay flocculated by MBF4-13 is shown in supplementarymaterial Fig. S4). Compared with the scattered particles withoutprocessing, the flocculated Kaolin clay particles were connected to-gether by bridge-like MBF4-13 or even trapped by the pectic netformed by MBF4-13. So it was speculated that the flocculationmechanism of MBF4-13 was probably bridging and network cap-turing bridging action.

The range of pH and temperature for flocculating Kaolin claysuspension with MBF4-13 were also measured (Tables 2 and 3).From which, it can be seen that MBF4-13 had a flocculation effi-

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

pH FR (%)

1 21.983 15.495 32.397 85.149 86.2211 62.6113 57.04

Table 3The influence of the temperature of Kaolin clay suspensions on the flocculationefficiency.

Temperature (�C) FR (%)

4 65.120 86.0180 87.69100 86.01

ciency in a wide pH range of 1–13, the optimal pH for the floccu-lating was in the range of 7–9, and it kept a high flocculatingactivity in the temperature range 4–100 �C. The optimum pH ofMBF4-13 is quite different from the other polysaccharide bioflocc-ulants. The optimal pH of polysaccharide bioflocculants SF-1 was inthe weakly acidic or near neutral range of 5.0–7.0 and was notheatstable (Gong et al., 2008). The exopolysaccharide biofloccu-lants p-KG03 was an effective flocculant under acidic conditions(pH 3.0–6.0) and over a wide temperature range (4–90 �C) (Yimet 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 abilitiesin decolorization, removal of heavy mental ions, and improvingperformance of activated sludge.

3.5.1. Decolorization for dye solutionOne milliliter of MBF4-13 (2 g L�1) was added into 10 mL dye

solutions (10 mg L�1 methylene blue, 20 mg L�1 crystal violet,and 10 mg L�1 malachite green). After well mixing and standingstill for 30 min, the absorbance of the supernatant liquor was mea-sured by a spectrophotometer. The DC (%) for methylene blue, crys-tal violet and malachite green were 86.11%, 97.84% and 99.49%,respectively. It was found interestingly that MBF4-13 had a strongdecolorizing ability for blue and violet series of dyes, but had a rel-atively small decolorizing ability for red, pink and orange seriesdyes. The reason for this may be relevant to the composition andfunctional groups of MBF4-13 and dyes. Deng et al. (2005) foundthe bioflocculant produced by Aspergillus parasticus was moreeffective for Reactive Blue 4 and Acid Yellow 25 than for Basic BlueB. The results showed that depending on the dye used, the bioflocc-ulant exhibited different decolorzing efficiency.

3.5.2. Removal of heavy mental ionsOne milliliter of MBF4-13 (2 g L�1) was added into 10 mL heavy

metal ion solutions (1 mg L�1 Cr2O2�7 and 20 mg L�1 Ni2+, respec-

tively). After well mixing and standing still for 30 min, the absor-bance of that was determined. The RE (%) for Ni2+ and Cr2O2

7

were 19.2% and 69.3%, respectively. The removal efficiencies forNi2+ and Cr2O2

7 are quite different. According to Section 3.3,MBF4-13 has hydroxyl group, it can be inferred that maybe hydro-gen bonds were formed between MBF4-13 and Cr2O2�

7 , it had high-er removal rate for Cr2O2�

7 than for Ni2+.

3.5.3. Improving performance of activated sludgeOne milliliter of MBF4-13 (2 g L�1) was added into the bulking

activated sludge. After culturing for 48 h, the microscopic imagesof zoogloea and microorganisms were photographed. By compari-son of picture A and B in Fig. 2, it can be seen that the structure ofzoogloea between original sludge (picture A) and after flocculatedsludge (picture B) had a remarkable change. Flocculating by MBF4-13 made the zoogloea more tightly and firmly. Picture C and D inFig. 2 showed that the kinds of microorganisms in the activatedsludge were also quite different. After flocculation of sludge, the fil-amentous fungi which caused sludge bulking disappeared, andprotozoa increased, such as rotifers, vorticella present, which wereindicator organisms for good water quality.

3.5.4. Comparison with traditional flocculants and the otherbioflocculants

The traditional flocculants, sodium meta-aluminate, aluminumsulfate and polyaluminium chloride were chosen for comparingthe flocculating efficiency with MBF4-13. As the results shown inTable 4, the flocculating efficiency for Kaolin clay suspension using

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 inflocculated sludge.

5000 Q. Gao et al. / Bioresource Technology 100 (2009) 4996–5001

MBF4-13 was similar to or even better than some of the abovementioned traditional chemical synthetic flocculants.

To date, many studies on the microbial production of flocculat-ing substances have been reported from different viewpoints, mostrecently identified bioflocculants were polysaccharide-like sub-stances, including poly-glutamic acids with molecular weight of20–3000 kDa (Deng et al., 2003; Fujita et al., 2000; Toead and Kura-ne, 1991; Salehizadeh and Shojaosadati, 2001; Yokoi et al., 1996;You et al., 2008). The above mentioned study was mainly focusedon the flocculating activity of bioflocculants for Kaolin suspension.Although the bioflocculant produced by A. parasticus could decolor-ize some kinds of dyes, the sludge produced after the dye mole-cules were absorbed on the flocculant did not compact well(Deng et al., 2005). In this study, it has been proved that bioflccu-lant MBF4-13 not only has the ability to flocculate Kaolin suspen-sion effectively but also has the ability for decolorizing of dyesolution, removal of some kinds of heavy metal ions in waste waterand improving the performance of active sludge. The applicationrange of bioflcculant MBF4-13 is much wider, and for it is producedfrom the strain ZHT4-13 which was isolated from R. philippinarumconglutination mud, maybe it can be deduced that bioflcculantMBF4-13 is harmless toward human. Bioflcculant MBF4-13 isanticipated like c-PGA to be utilized in the areas of wastewatertreatment, drinking-water processing and downstream processingin food and fermentation industries as a new bioflocculant which isharmless towards humans and the environment (Yokoi et al.1996).

Table 4Comparison of the flocculent activity of MBF4-13 with traditional flocculants.

Flocculants FR (%)

Sodium meta-aluminate 50.28Aluminum sulfate 74.83Polyaluminum chloride 88.25MBF4-13 86.01

4. Conclusions

A novel bioflocculant-producing bacterium ZHT4-13 was iso-lated from the conglutination mud of R. philippinarum. It was iden-tified as Rothia sp. The bioflcculant MBF4-13 produced by ZHT4-13had 86.22% flocculating efficiency for Kaolin clay of 5 g L�1. Spec-tral analysis showed that the main constituent of MBF4-13 waspolysaccharide with complex composition. The results of experi-ments showed the optimal culture conditions for bioflocculantproduction were as follows: 20.0 g L�1 of saccharose as carbonsource, 0.2 g L�1 of peptone and 0.2 g L�1 of (NH4)2SO4 as complexnitrogen source, 2.0 g L�1 of KH2PO4, 5.0 g L�1 of K2HPO4, 1000 mLof artificial seawater, pH 8.0, and 4 days cultivation. The biofloccu-lant MBF4-13 was effective for flocculating Kaolin clay suspensionin the range of pH 1–13 and temperature 4–100 �C. MBF4-13 wasable to treat some kinds of dye wastewater and heavy metal waste-water, and also improve activated sludge property. MBF4-13 had astrong decolorizing ability for blue and violet series of dye. For10 mg L�1 malachite green solution, the DC of that was up to99.49%. The flocculating efficiency for Kaolin clay suspension ofMBF4-13 was similar to or even better than some of the traditionalchemical synthetic flocculants, such as sodium meta-aluminate,aluminum sulfate and polyaluminium chloride.

Acknowledgements

We are pleased to acknowledge the valuable comments madeby two anonymous reviewers. The study was supported by ChineseNational 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

Q. Gao et al. / Bioresource Technology 100 (2009) 4996–5001 5001

source and the comprehensive compare of the orthogonal experi-ments results for the yield of cells, the flocculating ability of cellsfor Kaolin clay suspension, the yield of MBF4-13 and the flocculat-ing ability of MBF4-13 for Kaolin clay suspension are listed. TheFigure of the SEM of Rothia sp. strain ZHT4-13, the UV (A) andFT-IR (B) spectra of MBF4-13, the 1H NMR spectra of MBF4-13and the SEM images of (A) Kaolin clay and (B) Kaolin clay floccu-lated by MBF4-13 are listed too. Supplementary data associatedwith this article can be found, in the online version, atdoi: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 bioflocculantproduced 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 Aspergillusparasiticus 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 fromacetic 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 inwastewater treatment. Bioresour. Technol. 99, 4668–4674.

He, N., Li, Y., Chen, J., 2004. Production of a novel polygalacturotic acid bioflocculantREA-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 andcharacterization of an extracellular polysaccharide from halolkalophilic Bacillussp.I-450. Enzyme Microb. Technol. 34, 673–681.

Kurane, R., Toeda, K., Takeda, K., 1986. Culture conditions for production ofmicrobial 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 LipidBioflocculant Produced by Phodococcus erythropolis. Biosci. Biotechnol.Biochem. 58, 1977–1982.

Lu, W.-Y., Zhang, T., Zhang, D.-Y., Li, C.-H., Wen, J.-P., Du, L.-X., 2005. A novelbioflocculant produced by Enterobacter aerogens and its use in defecating thetrona suspension. Biochem. Eng. J. 27, 1–7.

Mu, J., Jiao B., Liang Z., Lü H., Wei H., Zhou R., Yang D, 2008. The preparation andpurposes of the bioactive extracts from a strictly anaerobic bacterium. ChinaPatent No. ZL 2006100461785.

Salehizadeh, H., Shojaosadati, S.A., 2001. Extracellular biopolymeric flocculantsrecent trends and biotechnological importance. Biotechnol. Adv. 19, 371–385.

Salehizadeh, H., Vossoughi, M., Alemzadeh, I., 2000. Some investigations onbioflocculant producing bacteria. Biochem. Eng. J. 5, 39–44.

Shih, I.L., Van, Y.T., Yeh, L.C., Lin, H.G., Chang, Y.N., 2001. Production of a biopolymerflocculant from Bacillus licheniformis and its flocculation properties. Bioresour.Technol. 78, 267–272.

Suh, H.-H., Kwon, G.-S., Lee, C.-H., Kim, H.-S., Oh, H.-M., Yoon, B.-D., 1997.Characterization of Bioflocculant Produced by Bacillus sp. DP-152. J. Ferment.Bioeng. 84, 108–112.

Toead, K., Kurane, R., 1991. Microbial flocculant from Aalcaligenes cupidus KT201.Agric. Biol. Chem. 55, 2793–2799.

Wang, S.-G., Gong, W.-X., Liu, X.-W., Tian, L., Yue, Q.-Y., Gao, B.-Y., 2007. Productionof a novel bioflocculant by culture of Klebsiella mobilis using dairy wastewater.Biochem. Eng. J. 36, 81–86.

Wu, J.Y., Ye, H.F., 2007. Characterzation and flocculating properties of anextracellular biopolymer produced from a Bacillus subtillis DYU1 isolate.Process Biochem. 5, 1–10.

Xia, S., Zhang, Z., Wang, X., Yang, A., Chen, L., Zhao, J., Leonard, D., Jaffrezic-Renault,N., 2008. Production and characterization of a bioflocculant by Proteus mirabilisTJ-1. Bioresource Technol. 99, 6520–6527.

Yim, J.H., Kim, S.J., Ahn, S.H., Lee, H.K., 2007. Characterization of a novelbioflocculant p-KG03 from a marine dinoflagellate Gyrodinium impudicumKG03. Bioresour. Technol. 98, 361–367.

Yokoi, H., Arima, T., Hirose, J., Hayashi, S., Takasaki, Y., 1996. Flocculation propertiesof Poly (c-Glutamic Acid) produced by Bacillus subtilis. J. Ferment. Bioeng. 82,84–87.

You, Y., Ren, N., Wang, A., Ma, F., Gao, L., Peng, Y., Lee, D., 2008. Use wastefermenting liquor to produce bioflocculants with isolated strains. Int. J.Hydrogen energy 33, 3295–3301.

Yu, L., 2002. Standard Practice Manual of Water Quality Monitoring and AnalysisMethod. China environmental science publishing company, Beijing. pp. 89–166.

Zheng, Y., Ye, Z.-L., Fang, X.-L., Li, Y.-H., Cai, W.-M., 2008. Production andcharacteristics of a bioflocculant produced by Bacillus sp. F19. BioresourceTechnol 99, 7686–7691.