Bioresource Technology 171 (2014) 336–342

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Production and characterization of high efficiency bioflocculant isolated from Klebsiella sp. ZZ-3 Ya-Jie Yin a, Zun-Ming Tian b, Wei Tang a, Lei Li a, Li-Yan Song a,c,⇑, Shawn P. McElmurry d a

Environmental Microbiology and Ecology Research Center, Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences, Chongqing 400714, PR China Shanghai Information Center for Life Sciences, Shanghai Institute of Biological Sciences, Chinese Academy of Science, Shanghai 200031, PR China c Key Laboratory of Reservoir Aquatic Environment, Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences, Chongqing 400714, PR China d Department of Civil & Environmental Engineering, Wayne State University, Detroit, MI 48202, USA b

h i g h l i g h t s  The high efficient bioflocculant was produced by isolated Klebsiella sp. ZZ-3.  The bioflocculant is pH tolerant and thermal stable.  The bioflocculant is a b-type heteropolysaccharide containing some protein.  High molecular weight and multi-functional groups contributed to the flocculation.  Bridging is the main flocculation mechanism of ZZ-3.

a r t i c l e

i n f o

Article history: Received 23 June 2014 Received in revised form 21 August 2014 Accepted 22 August 2014 Available online 30 August 2014 Keywords: Klebsiella sp. ZZ-3 Bioflocculant Thermal stability pH tolerant Industrial

a b s t r a c t In this study, a new bioflocculant (ZZ-3) is isolated and evaluated. This novel flocculant was derived Klebsiella, which was identified by 16S rDNA sequencing as well as biochemical and physiological analyses. The composition of ZZ-3 was found to be 84.6% polysaccharides and 6.1% protein. More specifically, the amount (moles) of the polysaccharides rhamnose, mannose, and galactose were found to be 6.48, 2.47, and 1.74 greater than glucose, respectively. Results show ZZ-3 has a relatively high molecular weight (603–1820 kDa) and contains many functional groups (hydroxyl, amide, carboxyl, and methoxyl) that likely contribute to flocculation. The results of microscopic observation, zeta potential measurements, and ZZ-3 bioflocculant structure suggested that bridging was the main mechanism for flocculation with kaolin. Based on a high flocculation efficiency, thermal stability, pH tolerance and the ability to flocculate without additional cations, ZZ-3 shows potential for industrial application. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Chemical flocculants (e.g., inorganic aluminum, ferric salts and synthetic organic polymers) are widely used for wastewater and drinking water treatment as well as in the food industry (Salehizadeh and Shojaosadati, 2001). Due to their biochemical stability, residues and derivatives of chemical flocculants are found in the environment (Rudén, 2004). New evidence suggests some traditional flocculants may be toxic to humans. For example, aluminum salts (e.g. alum) are suspected of inducing Alzheimer’s ⇑ Corresponding author at: Environmental Microbiology and Ecology Research Center, Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences, Chongqing 400714, PR China. E-mail address: [email protected] (L.-Y. Song). http://dx.doi.org/10.1016/j.biortech.2014.08.094 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.

disease (Crapper et al., 1973). Polyacrylamides, a common type of anionic flocculant, are found to be both neurotoxic and strong human carcinogens (Salehizadeh and Shojaosadati, 2001). In contrast, so called bioflocculants – flocculants produced by microorganisms – are typically biodegradable and considered relatively safe for the environment and humans (Salehizadeh and Shojaosadati, 2001; Salehizadeh et al., 2000). Due to their benign characteristics, bioflocculants show great potential to replace the traditional chemical flocculants. In recent years, the identification and characterization of bioflocculants has increased. Strains of highly efficient bioflocculants isolated from an array of environments include Proteus mirabilis TJ-1 (Xia et al., 2008) and Bacillus licheniformis X14 (Li et al., 2009) from water treatment process, Bacillus sp. (Zheng et al., 2008) and Bacillus mojavensis strain 32A (Elkady et al., 2011) from typical soils, and Nannocystis sp. NU-2

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(Zhang et al., 2002) and Sorangium cellulosum NUST06 (Zhang et al., 2002) from saline soils. However, high yield costs and lower flocculation efficiency, relative to synthetic organic flocculants, currently limit the application of bioflocculants. Hence, identifying new bioflocculants with high flocculation efficiency and low yield costs is urgently needed. After this initial identification, further characterize is also necessary to better understand the mechanisms responsible for flocculation so that their efficiency can be optimized. In the present study, six strains of bioflocculant producing organisms were isolated from paper mill wastewater. Flocculation activity was then tested using a kaolin suspension without the aid of additional cations. The strain with highest flocculation efficiency was identified as Klebsiella sp. ZZ-3 by 16S rDNA sequences and subjected to further biochemical and physiological characterization. 2. Methods 2.1. Isolation bioflocculant producing microorganism An activated sludge sample was obtained from the primary settling tank of a paper mill wastewater treatment system in Chongqing, China. One gram of the active sludge was dispersed in 10 ml of sterile distilled water. Serial dilutions (1:10) of this sample were then made until a maximum dilution of 106 was achieved. From the last 3 dilution aliquots, 500 ll was planted on Luria–Bertani (LB) agar plates (tryptone, 10 g; yeast extract, 5 g; and NaCl, 10 g; agar, 15 g; per liter) and cultured at 30 °C overnight. Strains with unique colony morphologies were picked and inoculated into 250 ml flasks containing 100 ml flocculant selecting medium (glucose, 10 g; KH2PO4, 2 g; K2HPO4, 5 g; MgSO47H2O, 0.2 g; NaCl, 0.1 g; urea, 0.5 g; yeast extract, 0.5 g; per liter) for 3 days at 30 °C on a rotary shaker at 180 rpm. From this culture, 1 ml was used for testing flocculation activity. 2.2. Identification of bioflocculant producing microorganisms Extraction of genomic DNA was performed with TIA Namp Bacteria DNA Kit (TianGen, China) according to the vendor’s protocol. The 16S rRNA gene was amplified using bacterial universal primers 27F (50 -AGAGTTTGATCCTGGCTCAG-30 ) and 1492R (50 -GGTTACCTTGTTACGACTT-30 ). The conditions for PCR were as follows: 5 min of denaturation at 94 °C, followed by 30 cycles of 94 °C for 1 min, 55 °C for 1 min, 72 °C for 1 min and a final extension at 72 °C for 10 min. PCR products were visualized by 0.8% agarose gel (w/v) electrophoresis and purified by TIANgel Midi Purification Kit (TianGen, China). The purified DNA fragments were ligated into pMD19-T vectors (TaKaRa, China) according to the manufacturer’s instructions. Approximately 200 clones were grown in a LB plate supplemented with 50 lg/ml ampicillin. Three clones were picked and cultured overnight in a LB broth with same concentration ampicilin. The plasmids were then extracted by TIANprep Mini Plasmid Kit (TianGen, China), purified by TIANpure Mini Plasmid Kit (TianGen, China), and sequenced by BGI Corp. (Beijing, China) using an Applied Biosystems 3730XL DNA analyzer based on the 27F primer. Resultant 16S rRNA gene sequences were assembled using Seqman II 5.0 at DNASTAR and analyzed using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Alignment of related sequences was carried out via CLUSTAL_X (version 1.83). Gaps at the 50 and 30 ends and ambiguous bases were removed. Phylogenetic trees were constructed by the neighbor-joining and maximum-parsimony algorithms using MEGA (version 5.0). Evolutionary distances were calculated using Kimura’s two-parameter model and bootstrap values were based on 1000 replications. The GenBank accession number of the 16S rRNA gene sequence is KJ681370.

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Escherichia coli strains were used to clone the 16S rRNA gene and cultured in LB broth aerobically on a rotary shaker (200 rpm) or on LB plates at 37 °C with 100 lg/ml ampicillin. Physiological and biochemical characteristics of the strain were identified according to the Manual of Systematic Bacteriology (Dong and Cai, 2001). 2.3. Determination of flocculating efficiency The flocculating efficiency was determined based on the standard kaolin suspension method (Salehizadeh et al., 2000). Briefly, 1 ml of the flocculant was mixed into a 50 ml kaolin suspension (4 g/L) in a 50 ml graduated cylinder and covered. The test cylinder was then gently shaken and allowed to sit for 5 min at room temperature to allow settling. Three milliliters of supernatant was carefully removed from the upper layer of solution and the amount of absorbance at 550 nm was measured using a TU-1901 spectrophotometer (PERSEE, China). A control sample that did not receive a flocculation agent was also completed. Flocculation efficiency was calculated according to the following equation:

Flocculation efficiency ð%Þ ¼ ðB  AÞ=B  100% where A is the absorbance at 550 nm of a sample and B is the absorbance at 550 nm of the control. 2.4. Optimization of Klebsiella sp. ZZ-3 culture conditions Culture time and the carbon and nitrogen sources were the most important factors influencing the yield cost of bioflocculant production. Culture times between 0 and 24 h were investigated using the flocculant selecting medium. To minimize the yield cost, the composition of the flocculant selecting medium was also optimized. The use of glucose as the primary carbon source was evaluated by assessing the impact of replacing it with sucrose, lactose, starch, maltose, mannitol, and citric acid. As for the source of nitrogen, peptone and urea were replaced with NaNO3, NH4Cl, yeast extract, and beef extract and results were compared. 2.5. Production, extraction, and purification of bioflocculant ZZ-3 Strain Klebsiella sp. ZZ-3 was inoculated into a 250 ml flask containing 100 ml optimized production mediums and incubated on a shaker (200 rpm) for 15 h at 30 °C. The flocculation efficiency of extracted precipitates (cells) and supernatant (bioflocculant) were compared using different extraction methods (centrifugation, 80 °C heating, and 32% sulfuric acid) (Sun et al., 2012). Extraction via centrifugation was performed by centrifuging broth that had been incubated over night at 12,000 rpm for 5 min and washed three times with 0.9% NaCl. Heated extraction consisted of dissolving centrifuged pellets into 0.9% NaCl and heating the sample to 80 °C for 15 min (Sun et al., 2012). After ambient cooling, the cell-free supernatant was obtained by centrifugation at 12,000 rpm for 10 min and filtered using a 0.22 lm membrane (JinTeng, China). Cold ethanol was then added to the supernatant and left overnight at 4 °C. The precipitate was collected by centrifugation at 12,000 rpm for 10 min and dissolved in ultrapure water. Sulfuric acid extraction was performed by suspending the centrifuged precipitate in 40 ml deionized water and adding 1.6 ml of 32% (w/v) sulfuric acid to the solution. After 15 min, the solution was centrifuged at 12,000 rpm for 5 min, and the supernatant was filtered using a 0.22 lm membrane (JinTeng, China). For both the heating and sulfuric acid extraction methods, the final supernatant was dialyzed overnight against deionized water with a molecular weight cutoff of 8–14.4 kDa (36 MM, Biosharp, USA). The purified bioflocculant (ZZ-3) was finally lyophilized by a freeze dryer (Virtis BT4KXL, USA).

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Centrifugation, heating, and sulfuric acid extraction produced precipitates with flocculation efficiency of 94.5%, 76.4%, and 80.1%, respectively. Correspondingly, the flocculation efficiency of extracted supernatant was 36.9%, 84.0%, and 89.0%, respectively. These tests demonstrated that the 32% sulfuric acid extraction had the highest extraction efficiency and were therefore used to extract ZZ-3. 2.6. Evaluating flocculation activity of bioflocculant ZZ-3 To evaluate the flocculation activity of ZZ-3, a total of 12.6 mg lyophilized sample was dissolved in 100 ml of deionized water. Doses of 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, and 10.0 ml were then applied to kaolin suspensions to test the effect on flocculation efficiency. To examine the impact of pH on flocculation efficiency, 1 ml of the ZZ-3 solution was mixed into the kaolin suspensions made in solutions with a range of pH (3, 4, 5, 6, 7, 8, 9 and 10). To test the effects of cations on flocculation efficiency, 1 ml solutions containing 0.09 M of different chloride salts (CaCl2, NaCl, KCl, MgCl2, AlCl3 and FeCl3) were mixed into the kaolin suspension at pH 7. Thermal stability was examined by measuring the flocculation activity of ZZ-3 after 20 min of heating at 115 °C.

2.7.5. GPC analysis Molecular weights (MWs) of ZZ-3 were evaluated by GelPermeation Chromatography (GPC) using a LC-10ADVP GPC system with a RID-10A refractive index (RI) detector (Shimadzu, Japan) with a TSK G4000PWxl column operated at 40 °C. The column was calibrated by using dextran standards. The mobile phase adopted for this study was deionized-distilled (DDI) water at flow rate of 0.5 ml/min. Before injection, the sample was filtrated though 0.45 lm filter. The weight-averaged molecular weight was used to characterize MWs. 2.8. Flocculation mechanism To test the flocculation mechanism (bridging or charge neutralization), the zeta potentials of the bioflocculant, the kaolin clay suspension, and the mixture of the bioflocculant and kaolin were measured using a NanoZ zeta potential analyzer (Malvern, UK). 3. Results and discussion 3.1. Isolation and identification of bioflocculant producing microorganisms

2.7. Characterization of bioflocculant ZZ-3 2.7.1. Chemical analysis The sugar content of ZZ-3 was determined according to the phenol–sulfuric acid method using a glucose standard (Lu et al., 2005). The protein content was determined by the Bradford method using a bovine serum albumin standard (Bradford, 1976). 2.7.2. GC–MS analysis To analyze the monosaccharide composition, 10 mg of ZZ-3 was hydrolyzed in 2 ml of 1 M H2SO4 at 100 °C for 4 h in a sealed glass tube. The hydrolysate was neutralized with BaCO3 to pH 7.0, and then centrifuged at 8000 rpm for 5 min. The supernatant was then filtered using a 0.22 lm hydrophilic membrane filter (JinTeng, China) and the filtrate was collected and lyophilized (Virtis, BT4KXL, USA). The lyophilized powder was then reacted with 10 mg hydroxylamine hydrochloride and 0.5 ml pyridine a 90 °C for 30 min and then was acetylated with 0.5 ml acetic anhydride at 90 °C for another 30 min. Standard sugars were prepared using the same procedure. The acetylated aldononitrile of ZZ-3 was analyzed using an Agilent 7890A-5977 gas chromatograph–mass spectrometer (GC–MS) operated in the electron ionization mode. A HP5MS fused silica capillary column (30 m  0.32 mm  0.25 mm) was used with helium as the carrier gas at a flow rate of 1 ml/ min. The temperatures of injector and detector were set at 300 and 260 °C, respectively. A temperature gradient was employed consisting of an initial column temperature of 130 °C that was held for 5 min and increased at a rate of 4 °C min1 to a final temperature of 240 °C and then held for 5 min. Sugar was identified by comparison with reference sugars. The relative molar proportions were calculated by the area normalization method. 2.7.3. XPS analysis Elemental analysis of ZZ-3 was performed by XPS (XSAM800, Kratos, UK) under FAT mode with a 5  107 Pa vacuum, 12 kV power and 12 mA current. The laser source was an aluminum target and 284.8 eV was used for correction. 2.7.4. Infrared spectrometer analysis A Nicolet 6700 infrared spectrometer (ThermoFisher, USA) was used to identify functional groups of ZZ-3 by measuring from 400 to 4000 cm1.

Six strains of organisms which produced bioflocculants were isolated from the activated sludge. Among these strains, strain ZZ-3 showed the highest flocculating activity to kaolin suspension (93.9%) and was thus selected for the further study. The strain ZZ-3 formed small (1.5 mm in diameter), milk white, smooth and humid colonies on the agar culture media after 24 h of aerobic incubation. Scanning electron microscopy (SEM) images show the strain is a short rod-shaped organism that can be characterized as being 1.2–2.0 lm long and 0.6–0.8 lm wide. The strain ZZ-3 formed 50–200 lm flocs when flocculated in kaolin clay suspension. Biochemical and physiological characteristics of the strain were tested according to standard procedure. Strain ZZ-3 is gram-negative, aerobic, non-motile and rod-shaped. ZZ-3 is negative for oxidase, gelatinase, and H2S production, but over 50% positive for Voges–Proskauer and methyl red test. Substrates utilized as sole carbon sources showed that ZZ-3 can utilize D-glucose, D-glucose, inositol, mannitol, D-mannose, D-sorbitol, L-rhamnose, melitriose, D-cellobiose, L-arabinose, melibiose, D-xylose, maltose, sucrose, starch and lactose. Therefore, according to the manual of systematic bacteriology, the biochemical and physiological characteristics indicate that ZZ-3 belongs to the genus Klebsiella. The nearly complete 16S rRNA gene sequence (1462 bp) of strain ZZ-3 was compared with the corresponding sequences of other bacterial strains in GenBank database through BLAST. BLAST results indicated that strain ZZ-3 was most similar to Klebsiella pneumoniae (99% similarity). A phylogenetic tree was constructed according to the neighbor-joining algorithm (Fig. 1). The tree topology, supported by high bootstrap values, clearly showed that strain ZZ-3 is within the genus Klebsiella. Strains in the family Enterobacteriaceae have been reported to produce bioflocculants. In the family, most reported strains belong to the genera Citrobacter (Fujita et al., 2000), Enterobacter (Liu et al., 2012) and Klebsiella (Nie et al., 2011). To date, seven Klebsiella strains have been reported to produce bioflocculants: Klebsiella sp. strain S11 (Dermlim et al., 1999), K. pneumonia H12 (Nakata and Kurane, 1999), Klebsiella sp. MYC (Yue et al., 2006), Klebsiella mobilis (Wang et al., 2007), Klebsiella terrigena (Ghosh et al., 2009), K. pneumonia strain NY1 (Nie et al., 2011) and K. pneumonia strain MBF-5 (Zhao et al., 2013). However, with the exception of K. pneumonia MBF-5 isolated from sputum, these bioflocculants

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339

ZZ-3 (KJ681370) 71 93

K. pneumoniaeT (X87276) K. variicolaT (AJ783916)

69

K. singaporensisT (AF250285)

95

K. pneumoniae subsp. rhinoscleromatisT (Y17657) K. pneumoniae subsp. ozaenaeT (AF130982) K. michiganensisT (JQ070300)

99

K. oxytocaT (AF129440)

99

Escherichia coliT (X80725)

85

Salmonella bongoriT (AF029227)

82

Serratia ficariaT (AJ233428) Yersinia pestisT (AF366383) Xenorhabdus bovieniiT (AY278673)

0.005 Fig. 1. Neighbor-joining phylogeny of Klebsiella sp. ZZ-3 and closely related strains. Numbers along branches represent bootstrap values; only values >50% are shown. GenBank accession numbers for the16S rRNA sequences used in the tree reconstruction are given in parentheses. Bar, 0.005 substitutions per nucleotide.

require cations to improve their flocculation efficiency. Like K. pneumonia MBF-5, Klebsiella ZZ-3 isolated in the present study does not require a paired cation. In the kaolin suspension test, 54.38 mg/L K. pneumonia MBF-5 was found to be the optimal dosage for maximum flocculation activity (over 80%). In contrast, 5.0 mg/L of bioflocculant ZZ-3 obtained a flocculation activity of 92.0%. The bioflocculant produced by B. licheniformis was found to have similar flocculation activity (>90% with 5.8 mg/L) as ZZ-3, but it also required 5.14 mM CaCl2 to achieve this level of flocculation (Xiong et al., 2010).

3.2. Optimization of culture conditions of Klebsiella sp. ZZ-3 Fig. 2a shows an expected increasing of both cell growth and flocculation efficiency for Klebsiella sp. ZZ-3 in the first 12 h. During exponential growth, production of the bioflocculant almost paralleled cell growth, indicating that the bioflocculant was produced by biosynthesis during growth and not by cell autolysis, as is the case for Enterobacter aerogenes (Lu et al., 2005). Cell concentrations were maximized at 14 h (the stationary phase) and the bioflocculant reached its maximum flocculating activity at 10 h (during late exponential growth). During the stationary phase, there was little variation in the cell concentration and the flocculating activity of the bioflocculant. Thus, 14 h is the optimal culture time for bioflocculant harvest with high biomass and flocculating activity. Fig. 2b showed the flocculating activity of Klebsiella sp. ZZ-3 after 14 h of cultivation in media containing different sources of carbon (sucrose, starch, mannitol, citric acid, lactose, maltose, and glucose). Glucose, starch and mannitol were more favorable for bioflocculant ZZ-3 production. Within them, glucose was the most preferred source. Given that glucose is also inexpensive, glucose was used as the carbon source during additional experiments. Fig. 2c shows the effect different nitrogen sources have on flocculation activity, with glucose as the carbon source after 14 h of cultivation. Peptone, yeast extract, and beef extract were found to result in poor cell growth and low flocculation activity, while NaNO3, NH4Cl and urea were good nitrogen sources for both cell growth of Klebsiella sp. ZZ-3 and bioflocculant production. Among them, the low cost of NaNO3 showed the highest flocculation efficiency (94.0%) and it was therefore selected as the nitrogen source.

3.3. Factors influencing the flocculation activity of bioflocculant ZZ-3 The influence of dose on flocculating activity is described in Fig. 2d. Of the doses tested, 0.063, 0.126, and 0.252 mg of ZZ-3 in 50 ml suspensions resulted in over 80% flocculation efficiency with a dose of 0.252 mg having the highest efficiency (92%). These findings are consistent with previous studies that indicated there is an optimal bioflocculant dose (Salehizadeh and Shojaosadati, 2002). Lower doses do not offer sufficient amounts of the bioflocculant to adsorb the suspended kaolin clay particles. On the contrary, higher doses inhibited flocs from forming due to stronger repulsive forces between them (Yuan et al., 2011). Fig. 2e characterizes the effect of pH on flocculation activity. The flocculation activity of ZZ-3 was stable across a pH range of 3–7 (90.4–94.5%) but decreased at higher pH values (79.1% for pH 8, 60.6% for pH 10). These results demonstrate that ZZ-3 has a high flocculating efficiency across a wide pH range and suggests that it is tolerant to acidic (pH 3) and basic (pH 10) conditions. The deterioration of flocculation activity above pH 8 is probably due to the degradation and rearrangement of the molecular structure of polysaccharides (Aspinall, 1982). Like ZZ-3, the bioflocculant produced by Enterobacter cloacae WD7 has been found to be tolerant of the same pH range (3–9) (Prasertsan et al., 2006). Fig. 2f characterizes the effects various cations have on the flocculation activity of ZZ-3. Flocculation was not impacted by Na+, K+, Ca2+, and Mg2+; slightly impacted by Al3+; and dramatically impacted by Fe3+. Cations increase the initial adsorption of biopolymers on suspended particles by decreasing the negative charge on both the biopolymers and the particle and thus improve flocculation. For example, the bioflocculant produced by Enterobacter sp. required Al3+, Fe3+ and Ca2+ to achieve high flocculation activity (Yokoi et al., 1997). Prior to this study, very few bioflocculants have been reported to have high flocculation ability without a cation aid (e.g., K. pneumonia (Zhao et al., 2013)). The addition of Fe3+ into solution was found to reduce flocculation for both ZZ-3 and MBFF19 produced by Bacillus sp. F19 (Zheng et al., 2008). The mechanism for this interference is likely due to the high charge density of trivalent cations altering the surface charge of kaolin particles. The presence of high amounts of positively charged ions can induce charge reversal or inhibit ion exchange. Both of these mechanisms could reduce flocculation activity and may explain the observed decrease in flocculation.

Y.-J. Yin et al. / Bioresource Technology 171 (2014) 336–342 100

0.3 60 0.2

40 OD 600 (Ten times dilution)

0.1

20

Flocculant efficiency (%)

0.0

0 0

4

8

12

16

20

Flocculant efficiency (%)

80

Flocculant efficiency (%)

OD 600 (Ten times dilution)

0.8

100

a

0.4

Flocculant efficiency (%) OD 600 (Ten times dilution)

90

0.5

80

0.4 70 0.3 0.2

60

0.1 50

24

Glucose Sucrose

0.4 90 85

0.3

80 0.2

75 70 65

0.1 NaNO 3

NH 4 Cl

Urea

Peptone

d 60

40

20

0

Beef extract Yeast powder

0.013 0.025 0.063 0.126 0.252 0.631 1.262

100

90

Flocculant efficiency (%)

Flocculant efficiency (%)

Dosage (mg)

e

80

70

60

50

4

5

6

7

8

9

10

pH of reaction system

f

90 80 70 60 50 40

3

Maltose

80

Nitrogen source 100

Mannitol Citric acid Lactose

100

Flocculant efficiency (%)

Flocculant efficiency (%) OD 600 (Ten times dilution)

OD 600 (Ten times dilution)

Flocculant efficiency (%)

95

Starch

Carbon source 0.5

c

0.7 0.6

Time (h) 100

b

OD 600 (Ten times dilution)

340

None

NaCl

KCl

CaCl2 MgCl 2 AlCl 3

FeCl3

Different cations

Fig. 2. Optimization of culture time (a), carbon source (b), and nitrogen source (c) for Klebsiella sp. ZZ-3 growth. Impact of dosage (d), pH (e), and cations (f) on the flocculating efficiency of ZZ-3.

While Fe3+ was found to hinder flocculation for both ZZ-3 and MBFF19, flocculation with MBFF19 was completely inhibited by Fe3+ while flocculation with ZZ-3 still occurred, just at a lower a lower level. After being subjected to 115 °C for 20 min, the flocculation activity of ZZ-3 decreased from 92.6% to 53.5%. This modest reduction in flocculation activity at high temperature is to be expected and demonstrates thermal stability. Similar to ZZ-3, bioflocculants from B. licheniformis (Xiong et al., 2010) and E. cloacae WD7 (Prasertsan et al., 2006) have been found to maintain their flocculation ability below 80 and 70 °C, respectively.

3.4. Characterization of the bioflocculant ZZ-3 3.4.1. Chemical analysis of ZZ-3 A total of 126.2 mg of purified ZZ-3 was recovered from 1 L of fermentation broth. The purified ZZ-3 was found to form large flocs (20–100 lm) which are favorable for flocculation. The sugar and protein content of this flocculant was 84.6% and 6.1%, respectively, indicating that ZZ-3 is mainly composed of polysaccharides.

3.4.2. Molecular weight Two peaks identified by two retention times (9.95 and 12.72 min) were detected by RI during GPC. The corresponding weight-averaged MWs of the two constituents were 1969 and 281 kDa, respectively, accounting for 12.7% and 87.1% peak area. 3.4.3. Monosaccharide Alditol acetate derivatives from monosaccharides were examined by GC–MS and identified by comparing retention times observed with those of known standards. The results indicate that ZZ-3 polysaccharides consist primarily of rhamnose (17.69 min), mannose (23.82 min), glucose (24.08 min), and galactose (24.65 min). Based on observed concentrations, the molar ratios for these polysaccharides are 6.48:2.47:1:1.74. Furthermore, the monosaccharides detected were also confirmed by the corresponding mass spectra (data not shown). 3.4.4. Functional group analysis Infrared spectrometer spectrum of ZZ-3 displayed broad stretching peaks. The intense absorption peak at 3367 cm1

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Y.-J. Yin et al. / Bioresource Technology 171 (2014) 336–342 Table 1 Binding energies (eV) and assignment/quantization of XPS spectral bands of ZZ-3. Element

C

Percentage Peak (eV) Assignment Percentage

65.16 284.8 CA(C, H) 27.33

O 286.1 CA(O) 27.18

287.5 O@CO 7.86

288.9 C@O 2.79

indicated that ZZ-3 contained extensive hydroxyl groups which may be caused by the vibration of AOH or ANH bonds in the sugar ring of polysaccharides (Lian et al., 2008). A weak CAH stretching vibration band was observed at 2925 cm1 (Zheng et al., 2008). An asymmetrical stretching peak at 1635 cm1 and a week symmetrical stretching peak at 1403 cm1 showed the presence of carboxyl groups. Bands at 1074 and 1137 cm1 are related to methoxyl groups (Zheng et al., 2008). The peak at 1542 cm1 could be attributed to NH bending vibrations. The strong absorption peak presented at 1033 cm1 is related to CAO stretching and provides further evidence supporting the presence of methoxyl groups, typical group of sugar derivatives (Aljuboori et al., 2013). In all, the infrared spectrum showed characteristic peaks for carbohydrates and amides. Therefore, it can be inferred that the bioflocculant is a b-type heteropolysaccharide containing some proteins. 3.4.5. Elemental analysis The atomic composition and mass fraction of ZZ-3 was determined by XPS. Core peaks observed were C 1s, O 1s, N 1s and S 2p. Based on this analysis, the mass fractions of C, O, N and S were estimated to be 65.16%, 31.04%, 3.19% and 0.62%, respectively (Table 1). High resolution 1s XPS spectra showed the presence of four C (1s), two O (1s), two N (1s) and one S (2p). The C 1s peak was resolved into four component peaks. The peak at 284.8 eV (27.33%) is associated with CA(C, H) in lipids or amino acid side chains. The peak at 286.2 eV (27.18%) is associated with CA(O, N) in alcohols, ether amines or amides. The peak at 288.9 eV (2.79%) is associated with C@O. The peak at 287.5 eV (7.86%) is attributed to C@N groups. Within them, the peak at 284.8 and 286.2 eV constitute large percentages of the spectral band. The O 1s peak is decomposed into two peaks. The peak at 531.9 eV (11.68%) is attributed to C@O as in carboxylate, carbonyl, ester, or amide. The peak at 532.9 eV (19.36%) is attributed to CAO from the alcohols, hemiacetal, or acetal groups. The N 1s peak is also decomposed into two peaks. The peak at 399.7 eV (2.42%) is attributed to nonprotonated nitrogen from amines and amides, and the other peak at 401.1 eV (0.77%) is attributed to protonated amines, which are commonly found in amino acids and amino sugars. The peak at 168.8 eV (0.62%) was attributed to S from sulfate. Glycoproteins (Li et al., 2009; Mabinya et al., 2012) and lipids (Kurane et al., 1995), especially polysaccharides (Aljuboori et al., 2013; Xia et al., 2008; Zajic and Leduy, 1973) and proteins (Takeda et al., 1992; Yokoi et al., 1998) have been reported to be the key constituents of bioflocculants. For instance, P. mirabilis (Xia et al., 2008), B. licheniformis (Xiong et al., 2010), and Pullularia Pullulans (Zajic and Leduy, 1973) produced polysaccharides based bioflocculants, while Pseudomonas sp. A-99 was found to produce protein based bioflocculants (Yokoi et al., 1998). The key constituents of the bioflocculant isolated in this study are polysaccharides which consist primarily of rhamnose, mannose, glucose and galactose. Efficient bioflocculants tend to have a high MW, ranging from 102 to 103 kDa. For example, Bacillus megaterium TF10 was produced a bioflocculant 1037–2521 kDa (Yuan et al., 2011) and B. licheniformis yielded a bioflocculant 1800 kDa in size (Xiong et al., 2010). ZZ-3 also has high MWs, ranging from 280 to 2000 kDa. Higher MW bioflocculants can offer more binding sites for sorption and cation-bridging, resulting in higher flocculation activity and larger floc formations (Michaels and Morelos, 1955;

31.04 531.9 C@O 11.68

N 532.9 CAO 19.36

3.19 399.7 NAH 2.42

S 401.1 NAO 0.77

0.62 168.8 SO4 0.62

Salehizadeh and Shojaosadati, 2001). The IR and XPS analysis demonstrated the presence of hydroxyl (AOH), amide (ACOANH), and carboxyl (ACOO) functional groups in ZZ-3. These structures are preferred for flocculation and also observed in other bioflocculants (Xia et al., 2008; Yuan et al., 2011). 3.5. Flocculation mechanism The precise mechanism for flocculation by ZZ-3 is not entirely clear. Bridging, charge neutralization and a combination of the two mechanisms have been identified as dominant mechanisms for bioflocculants (Salehizadeh and Shojaosadati, 2001; Xia et al., 2008). If charge neutralization was the main mechanism for the flocculation, flocculation should occur when the zeta potential of the particles is sufficiently low to eliminate repulsion between them (Salehizadeh and Shojaosadati, 2001). The zeta potentials of ZZ-3, Kaolin clay suspension, and ZZ-3/kaolin clay were 31.2 ± 1.56, 35.6 ± 1.66, and 48.4 ± 1.88 mV, respectively; suggesting that the charge neutralization is not the mechanism for the flocculation of bioflocculant ZZ-3. Bridging occurs when bioflocculants extend from the particles’ surface into the solution for a distance greater than the distance over which inter-particle repulsion occurs, consequently bioflocculant adsorb the particles to form flocs (Salehizadeh and Shojaosadati, 2001). The bridging mechanism is common for bioflocculants given the chemical composition, MW, and molecular structure of many bioflocculants, all properties which influence the effectiveness of the bridging mechanism (Xia et al., 2008; Yuan et al., 2011). B. megaterium TF10 (Yuan et al., 2011) and P. mirabilis TJ-1(Xia et al., 2008) both have been found to produce polysaccharides and bridging has been proposed as the primary mechanism for flocculation for these bioflocculants since they consist of large polysaccharides with a number of active sorption sites. In present study, high MW polysaccharides were identified as the main constitutes of ZZ-3. The polysaccharides have long backbones (composed of mannose, glucose, and galactose) with a large number of functional groups (hydroxyl, amide and carboxyl) that are likely to adsorb many particles to form large flocs. Therefore, bridging is suggested as the likely mechanism for ZZ-3 flocculation with kaolin but further investigations must be completed before this can be concluded. Future studies should evaluate ZZ-3’s ability to flocculate other particles and to economically scale up ZZ-3 production. Both of these aspects must be demonstrated before it can be determined ZZ-3 is appropriate for industrial applications. 4. Conclusions The bacterium Klebsiella sp. ZZ-3 was isolated from paper mill wastewater and found to produce the bioflocculant ZZ-3. This bioflocculant was found to be pH tolerant, have good thermal stability, not require additional cations and be highly efficient. The active constitutes of ZZ-3 were found to be polysaccharides consisting of rhamnose, mannose, glucose and galactose at a molar ratios of 6.48:2.47:1:1.74. High MWs and multiple functional groups present within ZZ-3 contributed to the high flocculation efficiency. Although further studies are required, the results of this study suggest ZZ-3 may be useful for industrial flocculation.

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