Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2015 Apr 5.
Published in final edited form as: J Appl Microbiol. 2011 Jul 6;111(3):547–558. doi: 10.1111/j.1365-2672.2011.05080.x

Production of a novel bioflocculant MNXY1 by Klebsiella pneumoniae strain NY1 and application in precipitation of cyanobacteria and municipal wastewater treatment

M Nie 1,2, X Yin 2, J Jia 1, Y Wang 2,3, S Liu 4, Q Shen 3, P Li 1, Z Wang 1
PMCID: PMC4385668  NIHMSID: NIHMS303682  PMID: 21679283

Abstract

Aims

To isolate and characterize the novel bioflocculant-producing bacteria, to optimize the bioflocculant production and evaluate its potential applications.

Methods and Results

Klebsiella pneumoniae strain NY1, a bacterium that produces a novel bioflocculant (MNXY1), was selected on the chemically defined media. It was classified according to the 16S rRNA gene sequence, morphological and microscopic characteristics. MNXY1 was characterized to contain 26% protein and 66% total sugar. The constituent sugar monomers of MNXY1, revealed by NMR analysis, are glucose, galactose and quinovose. Favorable culture conditions for MNXY1 production were determined. Strain NY1 produces a high level (14.9 g l−1) of MNXY1. MNXY1 is thermostable and tolerant to the extreme pH. It precipitated 54% of cyanobacteria from laboratory culture and 72% of the total suspended solids from raw wastewater.

Conclusions

Strain NY1 was identified to produce a novel bioflocculant MNXY1. The outstanding performance of MNXY1 in practical applications and its availability in copious amounts make it attractive for further investigation and development for industrial scale applications.

Keywords: Klebsiella pneumoniae strain NY1, bioflocculant MNXY1, hyper production, cyanobacteria precipitation, city wastewater treatment

Introduction

Bioflocculants are natural product metabolites produced by a wide variety of microorganisms including bacteria, fungi, yeast and algae (Nakamura et al. 1976b; Kurane et al. 1986a; Salehizadeh and Shojaosadati 2001). They are commonly identified as polysaccharides, proteins, lipids, glycolipids and glycoproteins (Kurane and Matsuyama 1994). The basic building blocks found in bioflocculants include sugar monomers (fructose, galactose, glucose, mannose, raffinose, rhamnose, trehalose, xylitol, xylose, galacturonic acid, glucuronic acid, guluronic acid, mannuronic acid, 2-ketogluconic acid, etc.), amino sugars (galactosamine and glucosamine), organic acids (acetic acid, formic acid, pyruvic acid, succinic acid, uronic acid, etc.), amino acids, nucleic acids, fatty acids, sulfate groups, and O-acetyl esters (Nakamura et al. 1976a; Takagi and Kadowaki 1985b; Toeda and Kurane 1991; Kurane and Matsuyama 1994; Kurane et al. 1995; Yokoi et al. 1995; Yokoi et al. 1997; Dermlim et al. 1999; Nakata and Kurane 1999; Salehizadeh et al. 2000; Shih et al. 2001; He et al. 2002; Kobayashi et al. 2002; Salehizadeh and Shojaosadati 2002; Zhang et al. 2002; Kumar et al. 2004; Yim et al. 2007; Li et al. 2009a; Patil et al. 2009; Liu et al. 2010b). Chemical flocculants are generally classified into two groups. Inorganic flocculants include aluminum sulfate, polyaluminum chloride (PAC) and ferric chloride. Synthetic organic flocculants are represented by polyacrylic acid, polyacrylamide (PAA) derivatives and polyethylene imine (Takagi and Kadowaki 1985a; Toeda and Kurane 1991). Chemical flocculants are widely employed in wastewater treatment, tap water production, and in the food and fermentation industries for downstream processing due to their low cost and high efficiency (Nakamura et al. 1976b; Takagi and Kadowaki 1985a; Kurane et al. 1986a). However, the wide use of these substances has raised serious environmental and health concerns. It has been reported that aluminum salts are associated with Alzheimer’s disease (Kowall et al. 1989; Pullen et al. 1990; Arezoo 2002) and also that polyacrylamides containing residual acrylamide monomers are neurotoxic and carcinogenic to humans (Vanhorick and Moens 1983; Dearfield et al. 1988). In addition, chemical flocculants are not easily degraded in nature. These unavoidable disadvantages of chemical flocculants make bioflocculants and their producing microorganisms important targets for investigation. Compared to chemical flocculants, bioflocculants have unrivaled benefits because they are environmentally friendly, biodegradable, nontoxic and harmless to human and animal health, and free of the risk of secondary pollution.

Like chemical flocculants, bioflocculants have been widely used to treat starch wastewater (Deng et al. 2003), river water, brewery waste, soy sauce brewing and meat processing wastewater, and the effluent from pulp and paper mills (Gong et al. 2008). Bioflocculants were studied to decolorize molasses wastewater (He et al. 2004) and dye solutions (Zhang et al. 2002; Gao et al. 2009; Liu et al. 2009), to eliminate heavy metal ions in wastewater (Gao et al. 2009), and to purify drinking water at low temperature (Li et al. 2009b). Bioflocculant p-KG03 was shown to have strong antiviral and immunostimulatory activities (Yim et al. 2007). In addition, the bioflocculant from Klebsiella terrigena was evaluated for the removal of high numbers of oocysts of the human pathogen Crytosporidium parvum from tap water and for the removal of Salmonella, a potent pathogen prevalent in poultry wastewater (Ghosh et al. 2009a; Ghosh et al. 2009b).

To date, more than 75 different microbial strains have been reported to produce bioflocculants (Salehizadeh and Shojaosadati 2001). Among them are five Klebsiella strains that include K. sp. strain S11 (Dermlim et al. 1999), K. pneumoniae H12 (Nakata and Kurane 1999; Kobayashi et al. 2002), K. sp. MYC (Yue et al. 2006), K. mobilis (Wang et al. 2007) and K. terrigena (Ghosh et al. 2009b). Except for K. sp. MYC whose bioflocculant product was not chemically characterized, the other four Klebsiella strains were all demonstrated to produce polysaccharide bioflocculants. The glycoside composition and sequence of the polysaccharide from K. pneumoniae H2 were partially elucidated by using NMR and MS methods with hydrolyzed samples (Kobayashi et al. 2002). In order to isolate and characterize the novel bioflocculant-producing bacteria, and to optimize the bioflocculant production and evaluate its potential applications, we set out to screen the samples collected from sediment of the wastewater treatment plants. In this report, we describe the identification and characterization of a novel bioflocculant MNXY1 and its hyper producing bacterium Klebsiella pneumoniae strain NY1, the determination of favorable culture conditions for MNXY1 production, the utilization of MNXY1 to efficiently precipitate cyanobacteria from laboratory culture, and to remove the suspended solids from municipal wastewater.

Materials and Methods

Screening and isolation of bioflocculant-producing bacterial strains

The samples collected from sediment of the wastewater treatment plants in Xi’an, China, were suspended in sterile water and a series of 10-fold dilutions from 10−1 to 10−6 were plated on agar plates containing the Bioflocculant Initial Screening Medium BISM (per liter): 5 g peptone and 3 g beef extract, pH 7.0. The inoculated plates were incubated at 30°C for 1 to 5 days during which the colony growth and morphologies were observed daily. The colonies which appeared to be mucoid and grew robustly were selected for further colony purification and confirmation on agar plates containing the Basal Bioflocculant Selection Medium (BBSM, per liter): 20 g glucose, 0.5 g yeast extract, 0.5 g urea, 0.2 g (NH4)2SO4, 0.2 g KH2PO4, 0.5 g K2HPO4, 0.2 g MgSO4.7H2O, 0.1 g NaCl, pH 7.0. To confirm bioflocculant production, a fresh single purified colony was inoculated in a 14 ml plastic test tube containing 5 ml LB broth. This seed culture was grown on a rotary shaker at 200 rpm at 30°C overnight, and 0.5 ml of this culture was then inoculated into a 250 ml Erlenmeyer flask containing 100 ml BBSM broth. The culture was incubated at 30°C with shaking. Culture samples at the time points from 6, 12, 24, 30, 36, 48, 54, 60, 72, 78, 84 and 96 h were collected and directly used for the preliminary flocculant activity assays with Kaolin clay suspension. One strain, NY1, exhibiting high bioflocculant activity was selected for detailed characterization.

Genomic DNA preparation, PCR amplification, DNA sequencing and analysis

Preparation of chromosomal DNA from strain NY1 was conducted according to a standard E. coli genomic DNA preparation protocol as described by Sambrook et al. (Sambrook 2001). To amplify the 16S rRNA gene from strain NY1, a pair of primers fD1 and rP2 were adopted from the literature (Weisburg et al. 1991). The PCR reaction conditions were as described previously (Yin et al. 2003), except for substitution of the forward and reverse primers with fD1 and rP2 as well as 1 μg genomic DNA of strain NY1. Primers used for PCR and DNA sequencing were obtained from Fisher. The PCR product from the agarose gel was purified using QIAquick Gel Extraction kits from Qiagen. DNA sequencing was performed at the Oregon State University Center for Genome Research and Biocomputing (CGRB) using the Amplitaq dye-terminator sequencing system (Perkin Elmer) and Applied Biosystems automated DNA sequencers (models 373 and 377). Nucleotide sequences were determined for both strands. Sequence analysis was carried out using Vector NTI (Invitrogen) software. Nucleotide sequence similarity comparisons were carried out in public databases using the BLAST program (Altschul et al. 1990). The 16S rRNA gene sequence of strain NY1 was deposited in GenBank under the accession number GU377208.

Culture conditions for growth of Klebsiella pneumoniae strain NY1 and production of MNXY1

Strain NY1 was permanently stocked in 20% glycerol solution at −70 °C and temporarily plated and maintained on Luria-Bertani (LB) agar for fresh inoculation of liquid culture. We evaluated the growth of strain NY1 in a series of liquid and solid media. They include the liquid media LB, yeast extract and tryptone (2 × YT), tryptic soy broth (TSB), yeast extract-malt extract (YM), glucose-yeast extract-peptone (YGP) and brain heart infusion (BHI), and the solid media LB, YM, and international Streptomyces project media (ISP2 and ISP4). Production of MNXY1 was affected by a number of factors including the inoculum size, initial pH, culture temperature, sources of carbon and nitrogen, shaking speed, metal ions and culture age. For MNXY1 production, strain NY1 was routinely grown in BBSS broth where the glucose in BBSM was replaced by sucrose.

Isolation and purification of MNXY1

Isolation and preparation of MNXY1 from a 72 h culture was conducted using a procedure described previously by Nakata et al. (Nakata and Kurane 1999). A 200 ml culture was centrifuged at 4000 g at 30°C for 20 min. The supernatant was treated by adding 2.5 volumes of 100% ethanol, and instantly white cotton-like flocs were formed. After gentle and thorough mixing, the precipitation of bioflocculant was completed at 4°C overnight. The precipitated bioflocculant was then recovered by centrifugation at 4000 g at 30°C for 5 min. The resulting pellet was re-dissolved in 20 ml deionized water and purified by three successive precipitations with 100% ethanol. The precipitated MNXY1 was re-dissolved in 10 ml deionized water and dialyzed in Milli-Q water with Slide-A-Lyzer® 3.5 K dialysis cassette (Pierce, Rockford, IL, USA). After lyophilizing the dialyzed solution, bioflocculant MNXY1 was recovered as white powder and used for bioflocculant activity assays, chemical analysis and application studies.

Flocculant activity assay

A series of concentrations of bioflocculant MNXY1 was prepared in deionized water. Kaolin clay (ACROS Organics, New Jersey, USA) suspension (2.5 g l−1) was freshly prepared, and after letting it stand for 5 min at room temperature to eliminate the self-precipitates, the upper phase solution was used for the flocculant activity assay. Unless stated otherwise, a typical standard flocculant activity assay was conducted in a 30 ml screw-cap glass test tube. 50 μl MNXY1 solution (25 g l−1) was added into 25 ml Kaolin suspension (2.5 g l−1) at pH 7.5, previously supplemented with 100 μl of 1 mol l−1 Ca2+. After vigorous vortex mixing for 1 min and letting the flocs grow for 2 min by gentle mixing, the tube was left to stand for 5 minutes at room temperature and a 1 ml top layer was then taken to read the absorbance at 550 nm (A) with a BIO-RAD SmartSpec Plus Spectrophotometer. As control, a 1 ml Kaolin suspension without addition of bioflocculant but with Ca2+ was used for measuring the absorbance at 550 nm (B). All assays including the control were performed in triplicate. The flocculant activity was calculated based on the formula (B-A/B) × 100% as previously described by Kurane et al. (Kurane et al. 1986b). Alternatively, to detect the flocculant activity of MNXY1 during growth of Klebsiella pneumoniae strain NY1, 10 μl bacterial suspensions from the actively growing culture were directly taken and added into the assay reaction without prior centrifugation to separate the supernatant from the cells.

Effects of initial pH, culture temperature, sources of carbon and nitrogen, and metal ions on MNXY1 production

Unless otherwise stated, all liquid cultures were grown in triplicate in 500 ml Erlenmeyer flasks containing 200 ml medium (BBSM or its derivatives) on a rotary shaker at 200 rpm at 30°C. The cultures were harvested at 72 h. An overnight culture of strain NY1 grown in 5 ml LB broth was used to inoculate the above 200 ml fermentation at ratio of 1:100 (v/v). To determine the effect of pH on MNXY1 production, the initial pH values in the cultures were adjusted to 2, 3, 4, 5, 6, 7, 8, 9, and 10. To monitor bacterial growth, evolution of culture pH, MNXY1 production and flocculant activity, strain NY1 was grown in BBSS medium with sucrose as the sole carbon source. The time point samples at 6, 12, 18, 24, 30, 36, 48, 54, 60, 72, 78, 96 h were taken to measure both the optical density at 600 nm and pH, determine the yield of MNXY1 and assay for flocculant activity. To evaluate the effect of the carbon source on MNXY1 production, the original carbon source (glucose) in BBSM was replaced with 20 g l−1 of one of the following carbohydrates: maltose, lactose, sucrose, malt extract, starch, dextrose or dextrin. In addition, organic acids (maleic acid, calcium lactate, sodium citrate, sodium acetate and lactic acid), and alcohols (ethanol, PEG1000 and glycerol) were tested as alternative carbon sources for MNXY1 production. To examine the effect of nitrogen source on the production of MNXY1, both single and complex nitrogen sources were investigated by omission and substitution of the original nitrogen sources (yeast extract, urea and (NH4)2SO4) in BBSM medium. The nitrogen sources, including urea, yeast extract, (NH4)2SO4, (NH4)2NO3 and KNO3, were added at 1.0 g l−1. To study the effect of metal ions on MNXY1 production, the original divalent metal ion Mg2+ in BBSM was replaced with one of the following metal ions: Fe2+/Fe3+, Ca2+, Mn2+, Ni2+, Ba2+, Cu2+ and Co2+, which corresponded to the salts: Fe (Kurane et al.)2/FeCl3 · 6H2O, CaCl2 · 2H2O, MnSO4 · H2O, NiCl2 · 6H2O, BaCl2, CuSO4, and CoCl2, respectively. The final concentration of each of these salts was 0.02 g l−1. To determine the effect of temperature on MNXY1 production, the cultures were incubated at 20, 25, 30, 35 and 40°C.

Effect of temperature and pH on flocculant activity of MNXY1

Two groups of treatments were performed to determine the temperature tolerance of MNXY1. The bioflocculant solution was prepared in deionized water at 25 g l−1. In group I, 30 ml of MNXY1 solution was heated at 100°C in a water bath. Samples were taken at 30, 60 and 120 min and used in the flocculant activity assay, after cooling down the bioflocculant solutions to room temperature. In group II, the bioflocculant solution was autoclaved at 121°C for 30 min. To determine the effect of pH on flocculant activity of MNXY1, pH values in the Kaolin suspensions were adjusted from 2 to 12 prior to addition of bioflocculant solution into the activity assay.

Chemical characterization of MNXY1

To reveal the composition of MNXY1, total sugar was determined by the phenol sulfuric acid method according to Dubois et al. (Dubois et al. 1956). The standard curve was prepared with D-glucose and drawn according to the absorbance measured at 490 nm. A linear regression equation was obtained as Y = 0.1918 X + 0.0182, R2 = 0.9957, where Y = glucose concentration and X = absorbance at 490 nm. MNXY1 solution was prepared by dissolving 30 mg of powder in 60 ml deionized water. The percentage of total sugar was calculated according to the above equation and the formula: v/w × 100% where V (mg ml−1) is the amount of total sugar measured and W (mg ml−1) is the weight of the original MNXY1 sample. Total protein was measured by the Bradford method (Bradford 1976), standardized with bovine serum albumin. To determine the functional groups of MNXY1, Fourier transform infrared spectra were measured in a pellet of potassium bromide using an IR Prestige-21-Shimadzu spectrometer. To elucidate the constituent sugar monomers of MNXY1, purified MNXY1 was hydrolyzed under mild acidic conditions (Kobayashi et al. 2002). The freeze-dried sample was then dissolved in deuterium oxide (D2O) at 60 mg ml−1. The proton, carbon, HSQC and TOCSY NMR analyses were performed on 600 MHz spectrometer. Chemical shifts are shown as δ. Reference is based on an internal standard trimethylsilyl propionate (TSP).

Determination of the molecular weight of MNXY1

Sepharose CL-4B (Sigma) packed in a glass Econo-column (1.5 × 50 cm) was used to estimate the molecular weight of MNXY1. Blue dextran (2 × 106 Daltons, Sigma) was used as molecular weight marker and distilled water as an eluant. The flow rate is 2 ml/min. Fractions (2 ml/tube) were collected and used for flocculant activity assay.

Precipitation of cyanobacteria with bioflocculant MNXY1

Cyanobacteria strain Synechocystis sp. UTEX 2470 (provided by Professor Philip J. Proteau, Oregon State University) was statically cultivated in BG11 medium at 28°C with a 16 h light/8 h dark cycle for two weeks (Proteau 1998). A culture of 500 ml was freshly harvested and aliquoted into 50 ml falcon tubes at 20 ml culture per tube. After the cultures had settled for 30 min at room temperature, the OD at 600 nm and pH were measured. Experiments for further precipitation of the algae from the intact culture were designed to include one untreated control, two treated controls and one treated sample. In the untreated control, the assay mixture only contained 20 ml original culture with no addititives. In the treated control 1, the assay mixture contained 20 ml original culture with the addition of 100 μl deionized water and 500 μl of 1 mol l−1 CaCl2. In the treated control 2, the assay mixture contained 20 ml original culture with the sequential addition of 100 μl of 1 mol l−1 NaOH and 500 μl 1 of mol l−1 CaCl2. In the treated sample, the assay mixture contained 20 ml original culture with the sequential addition of 100 μl of 1 mol l−1 NaOH supplemented with 2.5 mg MNXY1 and 500 μl of 1 mol l−1 CaCl2. Upon addition of the solutions, the assay mixtures were mixed gently and then left to settle at room temperature for 10 min in order to visually monitor the progress and completion of the cyanotacterial precipitation. After 10 min of precipitation, 1 ml, 3 ml and 10 ml supernatants taken from individual samples were used to measure the OD at 600 nm, the pH, and to centrifuge at 4000 g for 20 min, respectively. After centrifugation, portions of the supernatant were used to measure the OD at 600 nm and the pH, and the cell wet weight from 10 ml culture was determined. The differences in OD, cell wet weight and pH between the untreated control and the treated controls or the untreated control and treated samples were used to calculate the removal rates for cyanobacterial cells from the culture. The relative precipitation efficiency was calculated according to the formula: (Wo-W1 or W2 or Wt)/Wo × 100% where Wo represents the total cell wet weight or OD values from the 10 ml original culture without treatment, W1 or W2 or Wt represents the total cell wet weight or OD values from the 10 ml supernatant of the treated control 1, control 2 or treated sample, respectively.

Treatment of municipal wastewater

The raw wastewater samples (2 × 2 liters) were freshly collected from the city wastewater treatment facilities in Corvallis, Oregon, USA. The samples were collected from the pump station before passage through the bar screen and primary clarifiers. The control sample was an equal volume of untreated raw wastewater. After vigorously mixing the sample-containing bottles and leaving them stand for 30 min, an appropriate volume of the bioflocculant MNXY1 solution was added into the sample to achieve a final concentration of 44 mg/L. After thorough mixing, the bottle was left to settle on the bench at room temperature for three hours. Carbonaceous biochemical oxygen demand (CBOD), chemical oxygen demand (COD), ammonia as NH3 and total suspended solids were then measured by the City of Corvallis Water Quality Laboratory.

Results

Screening and isolation of the bioflocculant-producing bacterium strain NY1

We used a chemically defined medium BISM for initial screening of the bioflocculant-producing colonies from the sediment suspension. Fast-growing mucoid colonies were divided into phenotypic groups and purified by repeated dilution onto BBSM agar. 29 pure colony strains were further evaluated for their production of bioflocculants in BBSM liquid culture. One strain, NY1, which showed robust growth, higher bioflocculant production (an average of 8 g l−1) and higher flocculating ability (an average of removal rate of 85%) was selected for further study.

Classification of strain NY1

Using published eubacterial primers fD1 and rP2 (Weisburg et al. 1991) and genomic DNA prepared from strain NY1 as the PCR template, we amplified the expected 1.5 kb fragment. BLAST analysis of the 1498 bp sequence against the GenBank database (Altschul et al. 1990) showed 99% identity at the nucleotide level with 16S rRNA genes from Klebsiella pneumoniae strains (e.g. Accession Nos.: AP006725 and CP000647). Based on the BLAST results, morphological and microscopic characteristics, the pure isolate was classified as K. pneumoniae strain NY1.

Growth characteristics of strain NY1

After two days’ incubation on solid media (LB, ISP2, ISP4, and YM) at 30°C, robust growth was observed. Strain NY1 also grew well in liquid media including LB, 2xYT, TSB, YM, YGP, and BHI. Figure 1 shows a representative growth curve of strain NY1. During the approximately 18-h exponential phase, the pH of the culture dropped from 7.05 to 4.35. The production of the bioflocculant MNXY1 increased most rapidly in late exponential phase and slowly thereafter.

Figure 1.

Figure 1

Open in a new tab

Growth curve of strain NY1, and time courses of pH and MNXY1 production. Strain NY1 was cultivated in BBSS medium with sucrose as the sole carbon source. Error bars indicate the standard deviation. Inline graphic, pH; Inline graphic, Yield; Inline graphic, Optical density.

Determination of the molecular weight of MNXY1

Molecular weight of MNXY1 was determined by size exclusion chromatography. The strong flocculant activity (>95% removal rate for Kaolin suspension) was found in the fraction eluted just before blue dextran. Weak flocculant activity (<5%) was observed in the fraction co-eluted with blue dextran. This behavior indicates that the molecular weight of the major components of MNXY1 is > 2 × 106 Daltons. We compared the flocculant activities of the major and minor components and unfractionated MNXY1 and found no significant difference among them. No further effort to purify the original preparation was then made for the purpose of MNXY1 characterization and application studies.

Effect of pH, temperature, carbon and nitrogen sources and metal ions on MNXY1 production

In order to optimize the culture conditions, we systematically investigated the effect of several critical factors on MNXY production. The effect of initial pH values in the culture medium on MNXY1 production is shown in Figure 2. A slightly acidic pH was favorable for MNXY1 production. The growth of strain NY1 was completely inhibited at pH 2.0. The optimum initial pH range for MNXY1 production was 5.0 to 7.0. More acidic (below 5) or basic (over 8) pH values in the initial cultures significantly reduced MNXY1 production.

Figure 2.

Figure 2

Open in a new tab

Effect of the initial culture pH on bioflocculant MNXY1 production. Strain NY1 was cultivated in BBSM with glucose as the sole carbon source. Error bars indicate the standard deviation.

The effects of carbohydrates, organic acids, alcohols and metal ions on MNXY1 production and activity are presented in Table 1. Favorable culture conditions for MNXY1 production were determined. MNXY1 yields varied from 1.67 to 14.9 g l−1. The highest yield (14.9 g l−1) of MNXY1 was achieved when dextrin was used as the carbon source, whereas the lowest yield (1.67 g l−1) occurred with glucose as the sole carbon source. Starch, maltose, malt extract and sucrose were also less favorable carbon sources for MNXY1 production. The highest flocculant activity (85.4%) was obtained with lactose as the sole carbon source. The flocculant activity for MNXY1 grown on maltose or malt extract was superior to that for NY1 grown with sucrose and glucose. As alternative carbon sources, we studied the effects of several organic acids and alcohols on MNXY1 production. As shown in Table 1, three alcohols and four organic acids were not good carbon sources for MNXY1 production. The yield obtained with glycerol was comparable to that with glucose.

Table 1.

Effect of carbon sources on bioflocculant MNXY1 production and activity. Yield and activity were determined from the cultures in BBSM medium or its derivatives, harvested at 72 h. The values were means of triplicate experiments (± SD indicates the standard deviation).

Carbon sources Flocculant activity (% removal rate) Yield (gl−1)
Carbohydrate Starch 56.60 ± 6.59 13.70
Dextrin 69.17 ± 2.01 14.90
Dextose 69.39 ± 6.04 1.80
Maltose 78.33 ± 2.37 8.40
Malto extract 79.48 ± 1.38 5.05
Glucose 70.45 ± 4.62 1.67
Lactose 85.35 ± 0.56 2.51
Sucrose 71.55 ± 2.74 2.56
Alcohol Ethyl alcohol 55.88 ± 10.82 0.23
Glycerol 70.80 ± 5.62 1.85
PEG 1000 72.49 ± 4.97 0.21
Organic acid Calcium lactate 75.70 ± 1.12 1.55
Sodium citrate 37.35 ± 4.58 Nd
Sodium acetate 55.77 ± 5.33 0.96
Maleic acid 82.22 ± 3.73 1.85
Open in a new tab

Higher yields were obtained with organic nitrogen over inorganic nitrogen sources. Of the inorganics, (NH4)2SO4 was better than KNO3 and NH4NO3. Urea was found to be the most favorable sole nitrogen source for MNXY1 production and flocculant activity (Fig. S1). We also investigated combined nitrogen sources on MNXY1 production and activity, and found a combination of yeast extract, urea and (NH4)2SO4 at a ratio of 2.5: 2.5: 1 was superior to any single nitrogen source tested (Fig. S1).

Evaluation of individual metal ions indicated that Mg2+ was the most favorable one for MNXY1 production. Significant inhibition of MNXY1 production and flocculant activity was observed for the following metal ions (presented in the order of reduced inhibitory effects): Ni2+, Mn2+, Ba2+, Cu2+, Fe2+/Fe3+ and Ca2+, when magnesium chloride was absent from the culture medium (Fig. S2). Finally, a study of the culture temperature indicated that strain NY1 fermented at 30°C was more favorable to MNXY1 production compared to all other temperatures tested (Fig. S3).

Effects of temperature and pH on MNXY1 activity

Tolerance to temperature was determined by treatment of MNXY1 solutions in a 100°C water bath and at 121°C in an autoclave. Figure 3A shows that MNXY1 is quite thermally stable. After boiling a MNXY1 solution at 100°C for 30 or 60 min, its flocculant activity towards the Kaolin suspension was only reduced approx. 30% compared to the unheated control. It still retained 46% flocculant activity after treatment at 100°C for 2 h. However, MNXY1 completely lost its flocculant activity after autoclaving at 121°C for 30 min (Fig. 3A). The effect of pH on MNXY1 activity is shown in Figure 3B. MNXY1 is tolerant to the extreme pH and showed excellent activity either in a strongly acidic solution (pH below 5) or in a strongly basic solution (pH above 8). More than 90% removal rate was observed for Kaolin suspension either at strong acidic or basic pH range (Fig. 3B). Flocculant activity of MNXY1 was slightly higher in acidic (pH below 5) than in basic solution (pH above 8).

Figure 3.

Figure 3

Open in a new tab

Effect of temperature (A) and pH (B) on bioflocculant MNXY1 activity. Reduced flocculant activity was relative to the activity of unheated bioflocculant MNXY1. Error bars indicate the standard deviation. Activity assay was conducted in 30 ml mixed solution containing 500 μl MNXY1 (25 g l−1), 150 μl 1 mol l−1 CaCl2 and 2.5 g l−1 Kaolin clay suspension. 100 °C was achieved in boiling water bath and 121 °C by autoclave.

Chemical characterization of bioflocculant MNXY1

In 0.5 g l−1 of MNXY1 solution, 0.33 mg of total sugar was detected by the phenol sulfuric acid method while 0.13 mg of protein was revealed by the Bradford method. Therefore, the major content of MNXY1 was determined as 66% total sugar and 26% protein.

The Fourier transform infrared spectrum of MNXY1 is shown in Fig. S4. Peaks in the IR spectrum of MNXY1 are indicative of the following: hydroxy groups with a broad stretching band at 3402 cm−1, a weak C-H stretching band at 2930 cm−1, C=O stretching vibration band at 1647 cm−1, uronate-indicating band at 1400 cm−1, S=O symmetrical stretching band at 1242 cm−1 and presence of sugar derivatives at 1011 cm−1. To shed light on the constituent sugar monomers, acid hydrolyzed MNXY1 was analyzed by NMR. The proton, carbon, HSQC and TOCSY NMR spectra revealed the presence of three monomer residues, including glucose, galactose and quinovose as the constituent sugars of MNXY1 (Fig. 4, Fig. S5 and Fig. S6).

Figure 4.

Figure 4

Open in a new tab

Partial 1H NMR spectra showing two protons of the sugars of MNXY1.

Precipitation of the cyanobacterium Synechocystis UTEX 2470 from laboratory cultures

To determine whether bioflocculant MNXY1 could be used to precipitate cyanobacteria, we cultivated Synechocystis UTEX 2470 at laboratory scale (100 to 500 ml), yielding 2.67 ± 0.24 g l−1 of cell wet weight. As shown in Figure 5A, MNXY1 precipitated more than 50% of the cyanobacteria from the culture within 10 minutes. After standing at room temperature for 1 h, 95% of the cyanobacteria were precipitated. Without addition of MNXY1, the precipitation rate conferred by sodium hydroxide and Ca2+ together varied from 8.66% to 20.11%. Figure 5B compares the time course of cyanobacteria precipitation among controls and the treated sample. As shown in the figure, MNXY1 greatly enhanced the precipitation of cyanobacteria.

Figure 5.

Figure 5

Open in a new tab

Precipitation efficiency of blue-green algae with bioflocculant MNXY1 (A) and Precipitation of blue-green algae recorded as various time points (B). C1-OD, C2-OD and T-OD: precipitation rates for controls 1, 2 and treated sample, respectively, obtained by an optical density method (see Materials and Methods for details). C1-WT, C2-WT and T-WT: precipitation rates for controls 1, 2 and treated sample, respectively, obtained by the weight method. Precipitation rates were obtained by comparison of the measured values with those from untreated controls/intact culture. The assay mixtures contained (1) deionized water + Ca2+ for control 1, (2) deionized water + Ca2+ + NaOH for control 2, and (3) deionized water + Ca2+ + NaOH + bioflocculant MNXY1 for treated sample. The cut-off time for precipitation was 10 minutes.

Treatment of municipal wastewater

To determine whether bioflocculant MNXY1 could be used to improve the efficiency of municipal wastewater treatment, raw wastewater samples from the local city (Corvallis, Oregon, USA) wastewater treatment plant were freshly collected and treated with MNXY1. Removal of 72% of the total suspended solids from the wastewater by MNXY1 was achieved at a dose of 44 mg/L. The treatments resulted in reduction of CBOD and COD in the raw wastewater from 100% to 89% and 84%, respectively.

Discussion

There is a global need for biodegradable and renewable flocculants to replace the chemical flocculants in widespread use. Although bioflocculants hold great potential for broad real world applications, replacing chemical flocculants with bioflocculants would require significant reduction of their production costs. To this end, we set out to screen and isolate novel bioflocculant producers from the sediment of wastewater treatment facilities. We have isolated a high-yield strain, NY1, which we have classified as Klebsiella pneumoniae by BLAST analysis of its 16S rRNA gene sequence and morphological and microscopic characteristics.

In this study, we have characterized strain NY1 for its growth and favorable conditions for production of its bioflocculant, MNXY1. Growth of strain NY1, upon inoculation, quickly entered into the log phase without undergoing an obvious lag phase when inoculated at ratio of 1:100 (v/v). Absence of the lag phase and arrival at the maximum yield of MNXY1 in the early stationary phase would be ideal for economic production of MNXY1. A similar lag phase-lacking growth curve was previously reported for a proteoglycan bioflocculant producer Bacillus licheniformis and for the bioflocculant MBF-W6 producer Chryseobacterium daeguense W6, respectively (Liu et al. 2010a; Xiong et al. 2010). Like B. licheniformis, a prolonged stationary phase was observed during fermentation of strain NY1. Unlike Chryseobacterium daeguense W6, a typical bacterial death phase was not observed for strain NY1 (Fig. 1). This might be an indicator of autoregulation/degradation of MNXY1 production by strain NY1. We hypothesize that strain NY1 might be able to control its own MNXY1 production at certain levels and degrade the excessive MNXY1 and utilize the degraded products as nutrition for its continuous growth. This hypothesis could be further supported by the observation that at the stationary phase MNXY1 production displayed an oscillating curve (Fig. 1). It was unlikely that the fluctuation of MNXY1 production by strain NY1 during the stationary phase was associated with cell lysis as reported previously because no obvious decrease of the optical density in the culture was detected (Salehizadeh and Shojaosadati 2002).

Our interests in characterization of strain NY1 stemmed from its capability to produce a high yield of bioflocculant MNXY1. Under optimal conditions, the yield of MNXY1 could reach ~15 gl−1. Thus far, over 75 bioflocculant-producing microorganisms including five Klebsiella spp were described in the literature. Previously reported bioflocculant producers of the genus Klebsiella gave low yields of their bioflocullant products: 0.973 g l−1 [K. sp. S11 (Dermlim et al. 1999)], 3.0 g l−1 [K. pneumoniae H12 (Nakata and Kurane 1999)] and 2.58 g l−1 [K. mobilis (Wang et al. 2007)]. The bioflocculant yields from K. sp. MYC (Yue et al. 2006) and K. terrigena (Ghosh et al. 2009b) were not determined. There are only a handful of reported strains shown to have hyper-production of bioflocculants. These include Agrobacterium sp. M-503 (14.9 g l−1) (Li et al. 2010), a marine myxobacterium Nannocystis sp. NU-2 (14.8 g l−1) (Zhang et al. 2002), Bacillus licheniformis CCRC 12826 (14 g l−1) (Shih et al. 2001), Halomonas sp. V3a’ (5.58 g l−1) (He et al. 2009), and Azotobacter indicus (6.1 g l−1) (Patil et al. 2009). Because the high costs and low production of bioflocculants have greatly compromised their practical uses, there is a pressing need to identify microorganisms capable of producing high yields of desirable bioflocculants, like strain NY1.

Chemical characterization of bioflocculant MNXY1 indicated it is a protein-polysaccharide complex with the polysaccharide as the dominant component. The peaks revealed by Fourier transform IR of purified MNXY1 are characteristic of a protein-heteropolysaccharide complex and are similar to those observed previously (Kobayashi et al. 2002; Salehizadeh and Shojaosadati 2002; Deng et al. 2003; Kumar et al. 2004; Deng et al. 2005; Wang et al. 2007). Quinovose is 6-deoxy-D-glucose. The presence of quinovose in bioflocculant MNXY1 is fully supported by the HSQC and TOCSY NMR spectra which reveal the correlation between the H5 proton and the characteristic methyl group on carbon 5 (Fig. S5 and Fig. S6). MNXY1 has quinovose as one of its three constituent sugar monomers. Quinovose is a rare sugar found in bacteria (Kocharova et al. 2005). To the best of our knowledge, MNXY1 is the first reported quinovose-containing bioflocculant. The polysaccharide nature of MNXY1 is consistent with its thermal stability because polysaccharide-dominant bioflocculants are typically resistant to relatively high temperature (Salehizadeh and Shojaosadati 2002; Li et al. 2010; Patil et al. 2010). It was previously reported that the strongly acidic polysaccharide bioflocculant MS-102 from Bacillus firmus was thermostable and retained 48% of its initial activity after heating at 100°C for 50 min (Salehizadeh and Shojaosadati 2002). As shown in Figure 3B, after heating at 100°C for 60 min and 120 min, the flocculant activity of MNXY1 only decreased by 33 % and 54% of its initial activity, respectively. However, the protein part of MNXY1 was indispensible for displaying its full flocculant activity because MNXY1 flocculant activity was completely lost after autoclaving at 121°C for 30 min. Many protein-polysaccharide complex bioflocculants have been reported (Nakamura et al. 1976a; Yokoi et al. 1997; He et al. 2002; Kobayashi et al. 2002; Zhang et al. 2002; Xia et al. 2008; Zheng et al. 2008; Li et al. 2009b; Patil et al. 2009; Liu et al. 2010b; Xiong et al. 2010). It appeares that the native protein portions were required for maximum flocculant activity (Kurane et al. 1986b; He et al. 2002; Zhang et al. 2002). Bioflocculant MNXY1 was not only tolerant to heating but also retained high flocculant activity (over 90% removal rate for the Kaolin suspension) at either strongly acidic or basic pH (Fig. 3B). These characteristics of MNXY1 are favorable for its use under extreme temperature and pH conditions.

Cyanobacteria are found in lakes, ponds and rivers worldwide, and seasonal blooms of these organisms can severely affect fisheries, tourism, and human and animal health. For example, annual cyanobacterial blooms have been reported since 2007 in Taihu Lake (China’s third largest lake) (Guo 2007), adversely affecting more than two million people in Wuxi, Chian who use this Lake water as a drinking water source. The adverse effects of cyanobacterial blooms result from their production of a wide variety of toxins, including cyclic peptides (microsystins and nodularins) and alkaloids (van Apeldoorn et al. 2007). In addition, cyanobacterial die-off may deplete the limited oxygen supplies in water bodies of water, killing fish (Paerl and Huisman 2008).

The major function and application of bioflocculants are to precipitate suspended solids, which led us to speculate that cyanobacteria spread on the water surface could be precipitated by biodegradable bioflocculants. Effective precipitation of these bacteria from contaminated water may allow remediation of their outbreaks. Even if an outbreak of cyanobacteria has already occurred in a water body, they may be removed by precipitation and collection. These nutrition-rich bacterial cells may serve as fermentable substrates for manufacturing organic fertilizers. To this end, we evaluated the efficacy of MNXY1 in precipitating Synechocystis UTEX 2470 cultures. As demonstrated in Figure 5, MNXY1 could effectively precipitate the cyanobacteria at the concentration of 2.67 g l−1 cell wet weight with a dose of 200 mg l−1. Prior to our precipitation studies, there has been no report of methods for measuring this kind of removal process. We set out to develop our own methods for evaluating the efficiency of the process by optical density and biomass weight measurement. The evaluation of the precipitation efficiency by both methods led to quite similar results (Fig. 5A), which suggested they could be generalized for use in cyanobacteria precipitation experiments with other bioflocculants. Additionally, the precipitate obtained with addition of bioflocculant MNXY1 was much more tightly compressed than the control 2 and in no need of further dehydration, which would be simplify its downstream processing. MNXY1 also proved to be highly efficient in removal of suspended solids from raw municipal wastewater.

The mechanism of a bioflocculant is commonly deduced from the functional groups and chemical composition. It is generally accepted that the mechanism of the flocculation can be summarized as the interaction of bioflocculant with its target molecules, compounds, particles or cells via absorption, charge neutralization, bridging and aggregation (Deng et al. 2005; Li et al. 2009). In our case, the mechanism of flocculation with MNXY1 is likely similar to other protein-polysaccharide bioflocculant. Regarding precipitation of cyanobacteria, we can hypothesize that the interaction of the cyanobacterial cell surfaces with exposed functional groups of MNXY1 molecules must occur during the precipitation, and dose, pH and metal ions should play important roles in the observed precipitation. Most probably, these interactions are dependent on the electrical charges carried by overall or local bioflocculant molecules and the cyanobacterial cell surfaces.

Conclusion

We identified and characterized a novel quinovase-containing protein-polysaccharide complex bioflocculant MNXY1 and its hyper producing bacterium Klebsiella pneumoniae strain NY1. The outstanding performance of MNXY1 in practical applications, its availability in copious amounts from the producing organism, its high thermal stability and activity across a broad pH ranges, and its biodegradability, make MNXY1 an attractive candidate for further investigation and development for industrial scale applications.

Supplementary Material

Supp FigureS1-S5

Significance and Impact of the Study.

This is first report for the identification of a quinovose-containing bioflocculant and application of a protein-polysaccharide complex bioflocculant in precipitation of cyanobacteria. These findings suggest that MNXY1 holds great potential for use in management of harmful algae and city wastewater treatment.

Acknowledgments

This work was in part supported by Program for Changjiang Scholars and Innovative Research Team at Xi’an University of Architecture and Technology (PCSIRT, grant No. IRT0853), the Oregon State University and Nanjing Agricultural University collaborative grant 2010157 (XY), NIH Grants R01AI073784 (XY) and AI073784-03S1(XY). Professors Philip J. Proteau and T. Mark Zabriskie are thanked for helpful discussions and critical reading of this manuscript. David J. Kiemle (SUNY-ESF) is thanked for collecting 600 MHz NMR spectra. Daniel R. Hanthorn from Wastewater Operations and Denise Eason (Corvallis, Oregon, USA) and Shane Sinclair from the City of Corvallis Water Quality Laboratory are thanked for raw wastewater sample collection, analysis and excellent technical support. Yang Wang is a China-US joint training, visiting Ph.D. graduate student and supported by the State Scholarship Fund through the China Scholarship Council of the Ministry of Education of the P.R. China.

Footnotes

Support information

Supplementary materials with this manuscript include Fig. S1 Effects of nitrogen sources on MNXY1 activity. Fig. S2 Effect of metal ions in the culture on MNXY1 activity. Fig. S3 Effect of the culture temperatures on MNXY1 activity. Fig. S4 FT-IR spectra of MNXY1. Fig. S5 HSQC NMR spectrum of MNXY1. Fig. S6 TOCSY NMR spectrum of MNXY1.

References

  1. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Arezoo C. The potential role of aluminium in Alzheimer’s disease. Nephrol Dial Transplant. 2002;17:17–20. doi: 10.1093/ndt/17.suppl_2.17. [DOI] [PubMed] [Google Scholar]
  3. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Dearfield KL, Abernathy CO, Ottley MS, Brantner JH, Hayes PF. Acrylamide: its metabolism, developmental and reproductive effects, genotoxicity, and carcinogenicity. Mutat Res. 1988;195:45–77. doi: 10.1016/0165-1110(88)90015-2. [DOI] [PubMed] [Google Scholar]
  5. Deng S, Yu G, Ting YP. Production of a bioflocculant by Aspergillus parasiticus and its application in dye removal. Colloids Surf B Biointerfaces. 2005;44:179–186. doi: 10.1016/j.colsurfb.2005.06.011. [DOI] [PubMed] [Google Scholar]
  6. Deng SB, Bai RB, Hu XM, Luo Q. Characteristics of a bioflocculant produced by Bacillus mucilaginosus and its use in starch wastewater treatment. Appl Microbiol Biotechnol. 2003;60:588–593. doi: 10.1007/s00253-002-1159-5. [DOI] [PubMed] [Google Scholar]
  7. Dermlim W, Prasertsan P, Doelle H. Screening and characterization of bioflocculant produced by isolated Klebsiella sp. Appl Microbiol Biotechnol. 1999;52:698–703. doi: 10.1007/s002530051581. [DOI] [PubMed] [Google Scholar]
  8. Dubois M, Gilles K, Hamilton J, Rebers P, Smith F. Colorimetric method for determination of sugars and related substances. Anal Chem. 1956;28:350–356. [Google Scholar]
  9. Gao Q, Zhu XH, Mu J, Zhang Y, Dong XW. Using Ruditapes philippinarum conglutination mud to produce bioflocculant and its applications in wastewater treatment. Bioresour Technol. 2009;100:4996–5001. doi: 10.1016/j.biortech.2009.05.035. [DOI] [PubMed] [Google Scholar]
  10. Ghosh M, Ganguli A, Pathak S. Application of a novel biopolymer for removal of Salmonella from poultry wastewater. Environ Technol. 2009a;30:337–344. doi: 10.1080/09593330902732093. [DOI] [PubMed] [Google Scholar]
  11. Ghosh M, Pathak S, Ganguli A. Effective removal of Cryptosporidium by a novel bioflocculant. Water Environ Res. 2009b;81:160–164. doi: 10.2175/106143008x325818. [DOI] [PubMed] [Google Scholar]
  12. Gong WX, Wang SG, Sun XF, Liu XW, Yue QY, Gao BY. Bioflocculant production by culture of Serratia ficaria and its application in wastewater treatment. Bioresour Technol. 2008;99:4668–4674. doi: 10.1016/j.biortech.2007.09.077. [DOI] [PubMed] [Google Scholar]
  13. Guo L. Ecology. Doing battle with the green monster of Taihu Lake. Science. 2007;317:1166. doi: 10.1126/science.317.5842.1166. [DOI] [PubMed] [Google Scholar]
  14. He J, Zhen Q, Qiu N, Liu Z, Wang B, Shao Z, Yu Z. Medium optimization for the production of a novel bioflocculant from Halomonas sp. V3a’ using response surface methodology. Bioresour Technol. 2009;100:5922–5927. doi: 10.1016/j.biortech.2009.06.087. [DOI] [PubMed] [Google Scholar]
  15. He N, Li Y, Chen J. Production of a novel polygalacturonic acid bioflocculant REA-11 by Corynebacterium glutamicum. Bioresour Technol. 2004;94:99–105. doi: 10.1016/j.biortech.2003.11.013. [DOI] [PubMed] [Google Scholar]
  16. He N, Li Y, Chen J, Lun SY. Identification of a novel bioflocculant from a newly isolated Corynebacterium glutamicum. Biochem Eng J. 2002;11:137–148. [Google Scholar]
  17. Kobayashi T, Takiguchi Y, Yazawa Y, Nakata K, Yamaguchi T, Kurane R. Structural analysis of an extracellular polysaccharide bioflocculant of Klebsiella pneumoniae. Biosci Biotechnol Biochem. 2002;66:1524–1530. doi: 10.1271/bbb.66.1524. [DOI] [PubMed] [Google Scholar]
  18. Kocharova NA, Ovchinnikova OG, Toukach FV, Torzewska A, Shashkov AS, Knirel YA, Rozalski A. The O-polysaccharide from the polysaccharide of Providencia stuartii O44 contains L-quinovose, a 6-deoxy sugar rarely occurring in bacterial polysaccharides. Carbohydr Res. 2005;340:1419–1423. doi: 10.1016/j.carres.2005.02.020. [DOI] [PubMed] [Google Scholar]
  19. Kowall NW, Pendlebury WW, Kessler JB, Perl DP, Beal MF. Aluminum-induced neurofibrillary degeneration affects a subset of neurons in rabbit cerebral cortex, basal forebrain and upper brainstem. Neuroscience. 1989;29:329–337. doi: 10.1016/0306-4522(89)90060-2. [DOI] [PubMed] [Google Scholar]
  20. Kumar CG, Joo HS, Choi JW, Koo YM, Chang CS. Purification and characterization of an extracellular polysaccharide from haloalkalophilic Bacillus sp. I-450. Enzyme Microb Technol. 2004;34:673–681. [Google Scholar]
  21. Kurane R, Hatamochi K, Kakuno T, Kiyohara M, Tajima T, Hirano M, Taniguchi Y. Chemical Structure of Lipid Bioflocculant Produced by Rhodococcus erythropolis. Biosci Biotechnol Biochem. 1995;59:1652–1656. [Google Scholar]
  22. Kurane R, Matsuyama H. Production of a bioflocculant by mixed culture. Biosci Biotechnol Biochem. 1994;58:1589–1594. doi: 10.1271/bbb.58.1589. [DOI] [PubMed] [Google Scholar]
  23. Kurane R, Takeda K, Suzuki T. Screening for and Characteristics of Microbial Flocculants. Agric Biol Chem. 1986a;50:2301–2307. [Google Scholar]
  24. Kurane R, Toeda K, Takeda K, Suzuki T. Culture Conditions for Production of Microbial Flocculant by Rhodococcus erythropolis. Agric Biol Chem. 1986b;50:2309–2313. [Google Scholar]
  25. Li Q, Liu HL, Qi QS, Wang FS, Zhang YZ. Isolation and characterization of temperature and alkaline stable bioflocculant from Agrobacterium sp. M-503. N Biotechnol. 2010 doi: 10.1016/j.nbt.2010.09.002. [DOI] [PubMed] [Google Scholar]
  26. Li Z, Chen RW, Lei HY, Shan Z, Bai T, Yu Q, Li HL. Characterization and flocculating properties of a novel bioflocculant produced by Bacillus circulans. World J Microbiol Biotechnol. 2009a;25:745–752. [Google Scholar]
  27. Li Z, Zhong S, Lei HY, Chen RW, Yu Q, Li HL. Production of a novel bioflocculant by Bacillus licheniformis X14 and its application to low temperature drinking water treatment. Bioresour Technol. 2009b;100:3650–3656. doi: 10.1016/j.biortech.2009.02.029. [DOI] [PubMed] [Google Scholar]
  28. Liu W, Wang K, Li B, Yuan H, Yang J. Production and characterization of an intracellular bioflocculant by Chryseobacterium daeguense W6 cultured in low nutrition medium. Bioresour Technol. 2010a;101:1044–1048. doi: 10.1016/j.biortech.2009.08.108. [DOI] [PubMed] [Google Scholar]
  29. Liu W, Yuan H, Yang J, Li B. Characterization of bioflocculants from biologically aerated filter backwashed sludge and its application in dying wastewater treatment. Bioresour Technol. 2009;100:2629–2632. doi: 10.1016/j.biortech.2008.12.017. [DOI] [PubMed] [Google Scholar]
  30. Liu WJ, Wang K, Li BZ, Yuan HL, Yang JS. Production and characterization of an intracellular bioflocculant by Chryseobacterium daeguense W6 cultured in low nutrition medium. Bioresour Technol. 2010b;101:1044–1048. doi: 10.1016/j.biortech.2009.08.108. [DOI] [PubMed] [Google Scholar]
  31. Nakamura J, Miyashiro S, Hirose Y. Purification and Chemical Analysis of Microbial Cell Flocculant Produced by Aspergillus sojae AJ7002. Agric Biol Chem. 1976a;40:619–624. [Google Scholar]
  32. Nakamura J, Miyashiro S, Hirose Y. Screening, Isolation, and Some Properties of Microbial Cell Flocculants. Agric Biol Chem. 1976b;40:377–383. [Google Scholar]
  33. Nakata K, Kurane R. Production of an extracellular polysaccharide bioflocculant by Klebsiella pneumoniae. Biosci Biotechnol Biochem. 1999;63:2064–2068. doi: 10.1271/bbb.63.2064. [DOI] [PubMed] [Google Scholar]
  34. Paerl HW, Huisman J. Climate. Blooms like it hot. Science. 2008;320:57–58. doi: 10.1126/science.1155398. [DOI] [PubMed] [Google Scholar]
  35. Patil SV, Patil CD, Salunke BK, Salunkhe RB, Bathe GA, Patil DM. Studies on Characterization of Bioflocculant Exopolysaccharide of Azotobacter indicus and Its Potential for Wastewater Treatment. Appl Biochem Biotechnol. 2010 doi: 10.1007/s12010-010-9054-5. [DOI] [PubMed] [Google Scholar]
  36. Patil SV, Salunkhe RB, Patil CD, Patil DM, Salunke BK. Bioflocculant Exopolysaccharide Production by Azotobacter indicus Using Flower Extract of Madhuca latifolia L. Appl Biochem Biotechnol. 2009;164:1095–1108. doi: 10.1007/s12010-009-8820-8. [DOI] [PubMed] [Google Scholar]
  37. Proteau PJ. Biosynthesis of phytol in the cyanobacterium synechocystis sp. UTEX 2470: utilization of the non-mevalonate pathway. J Nat Prod. 1998;61:841–843. doi: 10.1021/np980006q. [DOI] [PubMed] [Google Scholar]
  38. Pullen RG, Candy JM, Morris CM, Taylor G, Keith AB, Edwardson JA. Gallium-67 as a potential marker for aluminium transport in rat brain: implications for Alzheimer’s disease. J Neurochem. 1990;55:251–259. doi: 10.1111/j.1471-4159.1990.tb08846.x. [DOI] [PubMed] [Google Scholar]
  39. Salehizadeh H, Shojaosadati SA. Extracellular biopolymeric flocculants. Recent trends and biotechnological importance. Biotechnol Adv. 2001;19:371–385. doi: 10.1016/s0734-9750(01)00071-4. [DOI] [PubMed] [Google Scholar]
  40. Salehizadeh H, Shojaosadati SA. Isolation and characterisation of a bioflocculant produced by Bacillus firmus. Biotechnol Lett. 2002;24:35–40. [Google Scholar]
  41. Salehizadeh H, Vossoughi M, Alemzadeh I. Some investigations on bioflocculant producing bacteria. Biochem Eng J. 2000;5:39–44. [Google Scholar]
  42. Sambrook J, Russell DV. Molecular cloning A laboratory manual. Third. Cold Spring Harbor Laboratory Press, Cold Spring Harbor; New York: 2001. [Google Scholar]
  43. Shih IL, Van YT, Yeh LC, Lin HG, Chang YN. Production of a biopolymer flocculant from Bacillus licheniformis and its flocculation properties. Bioresour Technol. 2001;78:267–272. doi: 10.1016/s0960-8524(01)00027-x. [DOI] [PubMed] [Google Scholar]
  44. Takagi H, Kadowaki K. Flocculant Production by Paecilomyces sp. Taxonomic Studies and Culture Conditions for Production. Agric Biol Chem. 1985a;49:3151–3157. [Google Scholar]
  45. Takagi H, Kadowaki K. Purification and Chemical Properties of a Flocculant Produced by Paecilomyces. Agric Biol Chem. 1985b;49:3159–3164. [Google Scholar]
  46. Toeda K, Kurane R. Microbial Flocculant from Alcaligenes cupidus KT201. Agric Biol Chem. 1991;55:2793–2799. [Google Scholar]
  47. van Apeldoorn ME, van Egmond HP, Speijers GJ, Bakker GJ. Toxins of cyanobacteria. Mol Nutr Food Res. 2007;51:7–60. doi: 10.1002/mnfr.200600185. [DOI] [PubMed] [Google Scholar]
  48. Vanhorick M, Moens W. Carcinogen-mediated induction of SV40 DNA amplification is enhanced by acrylamide in Chinese hamster CO60 cells. Carcinogenesis. 1983;4:1459–1463. doi: 10.1093/carcin/4.11.1459. [DOI] [PubMed] [Google Scholar]
  49. Wang SG, Gong WX, Liu XW, Tian L, Yue QY, Gao BY. Production of a novel bioflocculant by culture of Klebsiella mobilis using dairy wastewater. Biochem Eng J. 2007;36:81–86. [Google Scholar]
  50. Weisburg WG, Barns SM, Pelletier DA, Lane DJ. 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol. 1991;173:697–703. doi: 10.1128/jb.173.2.697-703.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Xia S, Zhang Z, Wang X, Yang A, Chen L, Zhao J, Leonard D, Jaffrezic-Renault N. Production and characterization of a bioflocculant by Proteus mirabilis TJ-1. Bioresour Technol. 2008;99:6520–6527. doi: 10.1016/j.biortech.2007.11.031. [DOI] [PubMed] [Google Scholar]
  52. Xiong Y, Wang Y, Yu Y, Li Q, Wang H, Chen R, He N. Production and characterization of a novel bioflocculant from Bacillus licheniformis. Appl Environ Microbiol. 2010 doi: 10.1128/AEM.02558-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Yim JH, Kim SJ, Ahn SH, Lee HK. Characterization of a novel bioflocculant, p-KG03, from a marine dinoflagellate, Gyrodinium impudicum KG03. Bioresour Technol. 2007;98:361–367. doi: 10.1016/j.biortech.2005.12.021. [DOI] [PubMed] [Google Scholar]
  54. Yin X, O’Hare T, Gould SJ, Zabriskie TM. Identification and cloning of genes encoding viomycin biosynthesis from Streptomyces vinaceus and evidence for involvement of a rare oxygenase. Gene. 2003;312:215–224. doi: 10.1016/s0378-1119(03)00617-6. [DOI] [PubMed] [Google Scholar]
  55. Yokoi H, Natsuda O, Hirose J, Hayashi S, Takasaki Y. Characteristics of a Biopolymer Flocculant Produced by Bacillus sp. PY-90. J Ferm Bioeng. 1995;79:378–380. [Google Scholar]
  56. Yokoi H, Yoshida T, Mori S, Hirose J, Hayashi S, Takasaki Y. Biopolymer flocculant produced by an Enterobacter sp. Biotechnol Lett. 1997;19:569–573. [Google Scholar]
  57. Yue LX, Ma CL, Chi ZM. Bioflocculant Produced by Klebsiella sp. MYC and Its Application in the Treatment of Oil-field Produced Water. J Ocean Univ China. 2006;5:333–338. [Google Scholar]
  58. Zhang J, Liu Z, Wang S, Jiang P. Characterization of a bioflocculant produced by the marine myxobacterium Nannocystis sp. NU-2. Appl Microbiol Biotechnol. 2002;59:517–522. doi: 10.1007/s00253-002-1023-7. [DOI] [PubMed] [Google Scholar]
  59. Zheng Y, Ye ZL, Fang XL, Li YH, Cai WM. Production and characteristics of a bioflocculant produced by Bacillus sp. F19. Bioresour Technol. 2008;99:7686–7691. doi: 10.1016/j.biortech.2008.01.068. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp FigureS1-S5
NIHMS303682-supplement-Supp_FigureS1-S5.pdf (82.3KB, pdf)

RESOURCES