Characterization and flocculation properties of biopolymeric flocculant (glycosaminoglycan) produced by Cellulomonas sp. Okoh

Authors

  • U.U. Nwodo,

    Corresponding author
    • Department of Biochemistry and Microbiology, Applied and Environmental Microbiology Research Group (AEMREG), University of Fort Hare, Alice, South Africa
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  • A.I. Okoh

    1. Department of Biochemistry and Microbiology, Applied and Environmental Microbiology Research Group (AEMREG), University of Fort Hare, Alice, South Africa
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Correspondence

U.U. Nwodo, Department of Biochemistry and Microbiology, Applied and Environmental Microbiology Research Group (AEMREG), University of Fort Hare, Private Bag X1314, Alice 5700, South Africa. E-mail: UNwodo@ufh.ac.za

Abstract

Aims

Bioflocculant production potential of an actinobacteria isolated from a freshwater environment was evaluated and the bioflocculant characterized.

Methods and Results

16S rDNA nucleotide sequence and BLAST analysis was used to identify the actinobacteria and fermentation conditions, and nutritional requirements were evaluated for optimal bioflocculant production. Chemical analyses, FTIR, 1H NMR spectrometry and SEM imaging of the purified bioflocculant were carried out. The 16S rDNA nucleotide sequences showed 93% similarities to three Cellulomonas species (strain 794, Cellulomonas flavigena DSM 20109 and Cellulomonas flavigena NCIMB 8073), and the sequences was deposited in GenBank as Cellulomonas sp. Okoh (accession number HQ537132). Bioflocculant was optimally produced at an initial pH 7, incubation temperature 30°C, agitation speed of 160 rpm and an inoculum size of 2% (vol/vol) of cell density 1·5 × 10cfu ml−1. Glucose (88·09% flocculating activity; yield: 4·04 ± 0·33 g l−1), (NH4)2NO3 (82·74% flocculating activity; yield: 4·47 ± 0·55 g l−1) and MgCl2 (90·40% flocculating activity; yield: 4·41 g l−1) were the preferred nutritional source. Bioflocculant chemical analyses showed carbohydrate, protein and uronic acids in the proportion of 28·9, 19·3 and 18·7% in CPB and 31·4, 18·7 and 32·1% in PPB, respectively. FTIR and 1H NMR indicated the presence of carboxyl, hydroxyl and amino groups amongst others typical of glycosaminoglycan. SEM imaging revealed horizontal pleats of membranous sheets closely packed.

Conclusion

Cellulomonas sp. produces bioflocculant predominantly composed of glycosaminoglycan polysaccharides with high flocculation activity.

Significance and Impact of the Study

High flocculation activity suggests suitability for industrial applications; hence, it may serve to replace the hazardous flocculant used in water treatment.

Introduction

Flocculation is a pertinent process in municipal and waste water treatment, bioprocessing of coal, dredging and other industrial processes including downstream processing in fermentation processes (Fujita et al. 2000; Vijayalakshmi and Raichur 2002; Qiang et al. 2010; Mabinya et al. 2012). Traditional flocculants, such as salts of aluminium (aluminium sulfate and polyaluminium chloride) and the derivatives of polyacrylamide and polyethylene imines (organic synthetic polymeric flocculants), have played dominant role in flocculation processes due to their low cost and good flocculation efficiency (He et al. 2010; Cosa et al. 2011; Mabinya et al. 2012). However, the association of debilitating deleterious health problems including cancer, neurotoxicity and the neurodegenerative disease termed Alzheimer's disease (Rudén 2004; Matthys et al. 2005; Piyo et al. 2011), has led to the search for alternatives. In addition, it has been reported that many countries have placed ban on the use of these hazardous flocculants (Xiong et al. 2010); consequently, an alternative becomes imperative.

Some microbial species produce extracellular biopolymers, which have been reported to mediate flocculation (Yokoi et al. 1995; Jang et al. 2001; Haijun et al. 2010; Mabinya et al. 2012). These biopolymers are designated bioflocculants, and the vast majority of the bioflocculant-producing microbes, reported, has been bacteria (Salehizadeh and Shojaosadati 2001; Zhang et al. 2010). Bioflocculants are attributed with good flocculation efficiency, however, innocuousness and biodegradability are the most favourable advantage it has over other forms of flocculants (Zhang et al. 2007; Piyo et al. 2011). Hence, bioprospecting for microbial strains with bioflocculant-producing potentials has been topical. Nonetheless, high production cost has been a limitation to industrial scale production and application of bioflocculants. Furthermore, the low flocculation efficiency reported for some of the bioflocculant (Li et al. 2008) necessitates a continuum in the exploration of microbes for high bioflocculant yield and high flocculation efficiency at minimal production cost via the utilization of cost-effective materials.

Actinobacteria have remained good sources of secondary metabolites of economic importance, yet information on their role in the production of bioflocculants is dearth. Consequently, we evaluated a freshwater actinobacteria previously isolated from Tyume River in the Eastern Cape Province of South Africa for bioflocculant production with subsequent yield optimization via manipulation of physicochemical parameters and subsequently characterizing it for novelty.

Materials and methods

Identification of the test actinobacteria

The actinobacteria was previously isolated from sediment samples of Tyume River in the Eastern Cape Province of South Africa and preserved in glycerol at −80°C as part of the culture collections of the Applied and Environmental Microbiology Research Group (AEMREG), University of Fort Hare, South Africa. It was identified by 16S rDNA sequence analyses following the description of Cook and Meyers (2003). The 16S rDNA gene of the actinobacteria was amplified by polymerase chain reaction (PCR) followed by sequence analysis of the amplified gene. DNA was extracted through the boiling method, and the PCR amplification was carried out in 50-μl reaction volume containing 2 mmol l−1 MgCl2, 2 U Supertherm Taq polymerase, 150 mmol l−1 of each dNTP, 0·5 mmol l−1 of each primer (F1: 59-AGAGTTTGATCITGGCTCAG-39; I = inosine and primer R5: 59-ACGGITACCTTGTTACGAC TT-39) and 2 ml of the template DNA. Primer F1 and R5 binds to base positions 7–26 and 1496–1476 of the 16S rRNA gene of Streptomyces ambofaciens ATCC 23877, respectively (Cook and Meyers 2003). The primers in this study were used to amplify nearly full-length 16S rDNA sequences. The PCR programme used was an initial denaturation (96°C for 2 min), 30 cycles of denaturation (96°C for 45 s), annealing (56°C for 30 s) and extension (72°C for 2 min), and a final extension (72°C for 5 min). Gel electrophoresis of PCR products were conducted on 1% agarose gels to confirm that a fragment of the correct size had been amplified. Automated sequencing of the 16S rRNA genes of the bacterial isolates was performed using the Spectrumedix SCE2410 (Spectrumedix ILL, State College, PA, USA) genetic analysis system with 24 capillaries. The sequencing reactions were performed according to the manufacturer's instructions, using the Big Dye version 3.1 dye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) and 27F primer. The sequences were edited manually based on the most similar sequences.

Screening of actinobacteria for bioflocculant production

The bacteria was screened for bioflocculant production in accordance with the methods we described elsewhere (Nwodo et al. 2012). The fermentation basal salt media (BSM) was composed of the follows (g l−1): glucose, 10; tryptone, 1; K2HPO4, 5; KH2PO4, 2 and MgSO4·7H2O, 0·3. A set of Erlenmeyer flasks, of 500 ml capacity, containing 200 ml fermentation medium (BSM) were aseptically inoculated with 2% (4 ml) of the activated culture adjusted to cell density of about 1·5 × 10cfu ml−1 (Gibbons and Westby 1986; NCCLS 1993) and incubated at a temperature of 28°C in a shaker incubator for 72 h at 140 g. After the incubation period, the broth was centrifuged at 3000 g for 30 min at 15°C, and the cell-free supernatant was assessed for flocculation activity. Inoculum cell density optimal for bioflocculant production was evaluated as described elsewhere (Nwodo et al. (2012).

Flocculation activity assay

Flocculation activity was measured as described by previous reports (Zhang et al. 2007; Nwodo et al. 2012). Briefly, 0·3 ml of 1% CaCl2 and 0·2 ml of cell-free broth (bioflocculant rich broth) were added into 10 ml of Kaolin suspension (4·0 g L−1) in a test tube. The mixture was vortexed using a vortex mixer (VM-1000; Digisystem, Keelung, Taiwan) for 30 s and kept still for 5 min afterwards, 2 ml of the upper layer was carefully withdrawn and its optical density (OD) read spectrophotometrically (Helios Epsilon, Madison, WI, USA) at 550 nm wavelength. Control included repeating same process but the bioflocculant broth was replaced with sterile (uninoculated) fermentation medium. All assays were in triplicates and flocculation activity calculated using the following equations:

display math

A and B are OD550 (optical density; 550 nm) of the control and sample, respectively.

Culture conditions and nutritional requirements

Fermentation media were adjusted to pH values corresponding from 2 to 12 using 0·1 mol l−1 NaOH and 0·1 mol l−1 HCl, respectively. The flasks were respectively, aseptically, inoculated with standardized culture (1·5 × 10cfu ml−1) amounting to 2% (v/v) of fermentation medium. The cultures were incubated at 28°C in a shaker incubator for 72 h at 140 rpm and afterwards flocculation activity was measured. Further optimization of fermentation pH, incubation temperatures and agitation speed were carried out in accordance with our previous reports (Mabinya et al. 2012; Nwodo et al. 2012). Glucose, fructose, sucrose, lactose, maltose and starch were the carbon sources evaluated, while nitrogen sources included urea, ammonium sulfate, ammonium nitrate, ammonium chloride and peptone. Cations sources evaluated were monovalent salts (KCl and NaCl), divalent salt (MgSO4, CaSO4.H2O, MnCl.4H2O, and FeSO4) and trivalent salt (FeCl3).

Bioflocculant production kinetics assay

A 4-ml standardized culture (1·5 × 10cfu ml−1) was inoculated into 200-ml fermentation medium (pH 7·2) and incubated at 30°C and 160 rpm in a shaker incubator (based on optimum conditions in previous sections). Bioflocculant production kinetics was assessed by withdrawing 5 ml of the fermentation broth of which 4 ml was used for pH and flocculation activity measurement, while the remaining 1 ml was used to determine bacterial cell growth via viable cell count (cfu ml−1).This process was conducted at 8-h interval for a period of 7 days.

Bioflocculant purification

The methods described by Yokoi et al. (1995) and Wu and Ye (2007) were followed for the extraction and purification of the bioflocculant. Briefly, fermentation broth was centrifuged (3000 g, 30 min, 15°C), and cell pellets separated from the supernatant by decantation. The supernatant was mixed with ice-cold ethanol (95%), at volume to volume ratio of 1:4 and kept at 4°C in a cold cabinet for 16 h to precipitate the bioflocculant. The resulting precipitate was collected by centrifugation (10 000 g, 30 min, 15°C) and redissolved in distilled water at ratio 1:4 (ml). This procedure was repeated twice successively and afterwards the partially purified bioflocculant (PPB) obtained was lyophilized and vacuum-dried. Additional purification step involved treating the partially purified bioflocculant with 10% cetylpyridinium chloride (CPC) solution after dissolution with 0·05 mol l−1 NaCl (Kumar et al. 2004). The mixture was stirred until bioflocculant–CPC complex was completely solubilized. The mixture was left overnight at room temperature (c. 25°C) after which the precipitate was recovered by centrifugation (10 000 g, 30 min, 15°C). The process was repeated twice successively and afterwards, the bioflocculant was redissolved in distilled water and dialysed overnight against distilled water at 4°C and the dialysed polymer was reprecipitated with ice-cold ethanol, followed by washing with distilled water, lyophilization and vacuum-dried. The lyophilized fraction designated CPB was used for further studies.

The effects of pH and cations on flocculation activity of the purified bioflocculant

Flocculation activities of PPB and CPB were comparably assessed using kaolin clay suspension as described previously. Conversely, aqueous solution of 0·4 mg ml−1 purified bioflocculants (PPB and CPB) was used in place of the bioflocculant cell-free broth. The effect of pH on flocculation activity was determined through varying the pH of the kaolin clay suspension by a factor of 1 unit and starting from pH 2 to 11. Likewise, the cation sources were varied from monovalent (NaCl and KCl) to divalent (CaSO4.H2O; MgCl2; MnCl.4H2O and FeSO4) and trivalent (FeCl3) metal ions, respectively. Flocculation activity assay were as described previously.

Analyses of bioflocculant composition

The total sugar and protein contents of the purified bioflocculant (PPB and CPB) were assayed following phenol-sulfuric acid and Folin-phenol methods of Dubois et al. (1956) and Lowry et al. (1951), respectively, using glucose and bovine serum albumen (BSA) as standards. Neutral sugars, amino sugars and uronic acids contents were further determined in accordance with the methods of Elson-Morgan and Morgan-Elson (Chaplin and Kennedy 1994), and carbazole-sulfuric acid technique for uronic acids quantification as described by Li et al. (2007).

SEM imaging, elemental analysis, FTIR and 1H NMR spectroscopy

Samples of the purified bioflocculant (PPB and CPB) were placed on carbon-coated stub and gold coated in a gold coating chamber, using Eiko IB.3 ION coater (Middleton, WI, USA). Scanning electron microscopic (SEM) image of the gold-coated bioflocculant was obtained using JEOL JSM-6390LV FEI XL30 (JEOL, Middleton, WI, USA) scanning electron microscope. The SEM was equipped with Noran Six 200 Energy Dispersive X-ray (EDX, Middleton, WI, USA) Analyzer, which was used to obtain bioflocculant elemental composition. Similarly, the functional groups present in the bioflocculants were determined using a Fourier transform infrared (FT-IR) spectrophotometer (2000 FTIRS Spectrometer; Perkin Elma System, Middleton, WI, USA) over a wave number range of 4000–500 cm−1. Proton nuclear magnetic resonance (1H-NMR) spectra of the purified bioflocculants were recorded in chloroform (CDCl3) at 20°C on a Varian inova 300 MHz spectrometer. Approximately 10 mg of the compounds were dissolved in 1 ml of chloroform and shaken overnight at 50 g. The solution was transferred into the 5-ml NMR tube, caped and all experiments were performed with the spectrometer operating at 299·949 MHz and equipped with a 5-mm 300AutoSw PFG probe at 293 K, and the residual chloroform peak was irradiated during the relaxation delay of 1·5 s and a total of 128 scans were collected. A line broadening of 0·5 Hz was applied to the spectra.

Thermal studies of the purified bioflocculant

Thermogravimetric analysis (pyrolysis studies) of the purified bioflocculants was carried out using a thermogravimetric analyzer (TGA 7; Perkin Elmer) fitted with thermal analysis controller (TAC 7/DX). About 2–3 mg of PPB and CPB each was loaded into an alumina cup and weight changes recorded as a function of temperature for a 10°C min−1 temperature gradient between 20 and 900°C. A purge gas of flowing nitrogen at a rate of 20 ml min−1 was used. Likewise, melting point/decomposition temperature of the bioflocculant was determined using Gallenkamp melting point apparatus.

Results

Actinobacteria identification

Polymerase chain reaction amplification of the actinobacterial 16S rRNA gene yielded the expected amplicon of 1·5 kb size, while Basic Local Alignment Search Tool (BLAST) analyses of the nucleotide sequences revealed 93% similarity to three Cellulomonas species (strain 794, Cellulomonas flavigena DSM 20109 and Cellulomonas flavigena NCIMB 8073), and the sequences was deposited in GenBank as Cellulomonas sp. Okoh (accession number HQ537132).

Effect of inoculum size on bioflocculant production

Inocula culture density of 1·0, 1·5, 3·0 and 5·0 (×108 cfu ml−1) used as 2% (v/v) of the fermentation media resulted in flocculation activities of 38, 54, 41 and 25%, respectively. Corresponding bacterial viable count varied from 27 × 1017 to 41 × 1026 (cfu ml−1) and 0·57 to 0·91 (Fig. 1).

Figure 1.

Effect of inoculum cell density on bioflocculant production by Cellulomonas sp. Okoh. (image_n/jam12095-gra-0001.png) Flocculation activity (%); (image_n/jam12095-gra-0002.png) Final cell density (cfu ml−1); (image_n/jam12095-gra-0003.png) Final cell density (OD600).

The effect of culture conditions on bioflocculant production

Evaluation of effect initial medium pH on bioflocculant production is summarized in Fig. 2. The highest flocculating activity (80%) was achieved with initial medium pH of 7. When the pH range was further spread between pH 6 and 8, optimum flocculation activity (81%) was again obtained at pH 7. Fermentation temperatures and agitation speeds evaluated showed 30°C and 160 g as optimum with corresponding flocculation activities as 82 and 73%, respectively. Higher values of agitation speed (200–400 rpm) and fermentation temperature (>30°C) resulted in decreases in flocculation activities (Fig. 2).

Figure 2.

The effects of initial fermentation pH, incubation temperature and agitation speed on bioflocculant production by Cellulomonas sp. Okoh. (image_n/jam12095-gra-0004.png) pH (a); (image_n/jam12095-gra-0005.png) pH (b); (image_n/jam12095-gra-0006.png) Incubation temperature (°C); (image_n/jam12095-gra-0007.png) Agitation speed (rpm).

The effect of carbon, nitrogen and cation sources on bioflocculant production

Of all the sole carbon sources assessed, glucose resulted in the highest flocculation activity of 88% and bioflocculant yield of 4·04 ± 0·33 g l−1. Conversely, other carbon sources showed lower flocculation activities as well as lower bioflocculant yield (Table 1). In the same vein, (NH4)2NO3 was the preferred nitrogen source as it showed optimum flocculation activity of about 82·74% and bioflocculant yield of 4·47 ± 0·55 g l−1. Urea and peptone followed suit with flocculation activities of 74·49 and 74·12% with bioflocculant yields of 3·81 ± 0·33 and 3·09 ± 0·15 (g l−1), respectively. Furthermore, the actinobacteria optimally utilized magnesium chloride as cation source for bioflocculant production resulting in flocculation activity of 90·4% and bioflocculant yield of 4·41 g l−1 (Table 1).

Table 1. The effects of nutritional factors on bioflocculant production by Cellulomonas sp. (Okoh) HQ537132
Carbon sourceGlucoseLactoseFructoseSucroseMaltoseStarch
Max. Flocculation activity (%)88·09 ± 1·2260·62 ± 2·6378·22 ± 4·0355·94 ± 2·6963·13 ± 1·4151·50 ± 1·08
Bioflocculant yield (g l−1)4·04 ± 0·333·35 ± 0·633·1 ± 0·122·78 ± 0·413·41 ± 0·362·9 ± 0·21
Nitrogen sourceUrea(NH4)2SO4(NH4)2NO3(NH4)2Cl4Peptone
Max. Flocculation activity (%)74·49 ± 1·1730·82 ± 2·5182·74 ± 0·9858·76 ± 1·1474·12 ± 2·50
Bioflocculant yield (g l−1)3·81 ± 0·332·31 ± 0·464·47 ± 0·552·16 ± 0·973·09 ± 0·15
Cation sourceKClNaClMgCl2CaSO4.H2OMnCl.4H2OFeSO4FeCl3
Max. Flocculation activity (%)52·0 ± 2·3131·47 ± 1·6390·40 ± 0·9178·29 ± 2·5738·97 ± 1·2641·26 ± 1·4825·25 ± 1·0
Bioflocculant yield (g l−1)2·39 ± 0·571·79 ± 0·594·41 ± 0·613·06 ± 0·191·85 ± 0·192·24 ± 0·281·77 ± 0·36

Kinetics of bioflocculant production

The kinetics of bioflocculant production using the optimum culture conditions determined above is summarized in Fig. 3. There was a steady increase in flocculation activity from 3·7% at 8 h of fermentation to a plateau of 86·3% after 88 h of fermentation. The gradient representing steep bioflocculant production coincides with the logarithmic growth phase of the actinobacteria as cell densities ranged from 1·36 × 1017 to 1·4 × 1020 (cfu ml−1), respectively. The optimal flocculation activity observed at the logarithmic phase of growth of the bacteria suggests that the bioflocculant is produced during active cell growth, thus the bacteria synthesizes bioflocculant in the presence of abundant nutrient.

Figure 3.

Bioflocculant prodution time course of Cellulomonas sp. Okoh using optimum fermentation conditions and nutritional sources. (image_n/jam12095-gra-0008.png) pH; (image_n/jam12095-gra-0001.png) Flocculation activity (%); (image_n/jam12095-gra-0009.png) Final cell density (cfu ml−1).

The effects of cations and pH on flocculation activity of purified bioflocculant

Divalent cations (Ca2+ and Mg2+) mediated flocculation better than the monovalent and trivalent counterpart (Fig. 4). Flocculation activities of 84% (Ca2+) and 82% (Mg2+) were achieved against PPB, while flocculation activities of 78% (Ca2+) and 73% (Mg2+) were similarly achieved against CPB. With regard to the monovalent and trivalent cations, flocculation activities ranged from 47% (Na+) to 61% (Fe3+) against PPB and from 39% (Fe3+) to 48% (Fe2+) against CPB, respectively. Additionally, optimal flocculation activities of 84 and 68% were correspondingly achieved against PPB and CPB at a neutral pH.

Figure 4.

The effects of cations and medium pH on the flocculating activity of purified bioflocculant produced by Cellulomonas sp. Okoh. (image_n/jam12095-gra-0011.png) Flocculation Rate of PPB (%); (image_n/jam12095-gra-0012.png) Flocculation Rate of CPB (%); (image_n/jam12095-gra-0010.png) Flocculation Rate of PPB (%); (image_n/jam12095-gra-0013.png) Flocculation Rate of CPB (%).

Purified bioflocculant compositional analyses

Compositional analyses of purified bioflocculant revealed the presence of carbohydrates, protein and uronic acids. About 100 mg of the purified bioflocculant yielded total sugar content of 28·9% in the CPB and 31·4% in the PPB, protein content of about 19·3% (CPB) and 18·7% (PPB) and uronic acid content of about 29·6% (CPB) and 32·1% (PPB), respectively. However, carbohydrates component further revealed the presence of neutral sugars (7·2 and 9·8%) and amino sugars (8·1 and 11·4%) for CPB and PPB, respectively. The proportion of total sugar, protein and uronic acid contents were 28·9, 19·3 and 18·7% in CPB and 31·4, 18·7 and 32·1% in PPB, respectively.

FTIR and NMR spectroscopy of the purified bioflocculant

Broad stretching peaks (Fig. 5 and Table 2) of 3589·78–3438·13 (cm−1), which is characteristic of hydroxyl groups from polymeric and dimeric OH stretch were obtained from PPB and CPB respectively. Peaks characteristics of other functional groups includes 1455·10 cm−1 (PPB) and 1455·04 cm−1 (CPB) typical of phenol and tertiary alcohol OH bend indicative of the presence of carboxylic groups, carboxylate ions, aromatic ring stretch and C–O and C–O–C from polysaccharides (Schmitt and Flemming 1998). Furthermore, wave numbers (2958·71–2317·30 cm−1) showing weak C–H stretching band from methylene group was similarly obtained, and an asymmetrical stretching bands at 1654·77–1544·57 (cm−1) indicative of aromatic ring presences (Schmitt and Flemming 1998; Coates 2000). The NMR spectra (Fig. 6) of the purified bioflocculant showed 1H chemical shift (in ppm) at 0·834–0·866 (R-CH3), 1·205–1·317 (R2-CH2 and R3-CH), 1·618 (Allylic grougs; C=C-CH3), 2·027 (Triple bond CC-H), 5·039–5·074 (C=C-H), 8·066–8·081 (ArOH), 8·404–8·441 (Ar-H) and 9·524–9·537 (R-(H)C=O and other aldehydes). Similar NMR spectra were demonstrated polyglutamic acid bioflocculant reported by Wu and Ye (2007).

Figure 5.

FTIR spectra of purified bioflocculant from Cellulomonas sp. Okoh. (a) PPB; (b) CPB.

Figure 6.

1H NMR Spectra of purified bioflocculant produced by Cellulomonas sp. Okoh.

Table 2. Position and characteristic bond obtained from Fourier Transform Infrared Spectroscopy of bioflocculants from Cellulomonas sp. (Okoh) HQ537132
CompoundOriginGroup frequency wave number (cm−1)Assignment/Functional group
AssignedPPBCPB
Hydroxy and ether compoundsO–H3570–3200 (broad)3589·783438·13Hydroxy group, H-bonded OH stretch
O–H3400–32003294·423309·91Normal ‘polymeric’ OH stretch
O–H3550–34503519·77 Dimeric OH stretch
O–H1410–13101455·101455·04Phenol or tertiary alcohol, OH bend
Amino compounds and PolysaccharidesN–H3400–33803414·623418·94Aliphatic primary amine, N–H stretch
N–H3510–34603439·13 Aromatic primary amine, N–H stretch

>N–H

>C=O

C–O

C–H

1650–1550

1654·77;

1605·23;

1539·01

1654·71;

1609·40;

1544·57

Secondary amine, NH bend Associated with proteins

>C=O stretch, Ether, Carboxylic groups

C–H bend from CH2, C–O bend from carboxylate ions

C–O and C–O–C from polysaccharides

Methyl (–CH3)–CH2935–2915/2865–2845

2958·70;

2924·27;

2854·39

2958·71;

2924·15;

2854·20;

2352·91;

2317·30

Methylene C–H asym./sym. stretch
>CH -2900–2880  Methyne C–H stretch (Methyne)
Aromatic ring (aryl)C = C–C1510–14501455·101455·04Aromatic ring stretch

SEM micrography and elemental composition of the purified bioflocculant

Scanning electron micrographic imaging of PPB and CPB showed horizontal pleats of membranous sheets packed in such a manner that they are closely woven together for PPB and sparsely placed for CPB (Fig. 7). The interstice between horizontal-pleated sheets is less than 1 μm in PPB and more than 50 μm in CPB thereby making PPB to appear more like a mesh as against the flake-like structures seen with the sheets of CPB. Furthermore, the elemental composition of PPB and CPB is identical with exceptions in the quantity present as shown by spectral peaks, which was more in PPB, as well as the presence of aluminium in PPB, which was absent in CPB (Table 3). High ratio of carbon (C), nitrogen (N) and oxygen (O) was demonstrated in PPB and CPB, while other elements like sulfur and phosphorus were similarly present in varying concentration.

Figure 7.

SEM Micrograph of purified bioflocculant from Cellulomonas sp. Okoh. (a) PPB; (b) CPB.

Table 3. Elemental composition of purified bioflocculant produced by Cellulomonas sp. (Okoh) HQ537132
 Element lineElement wt. %Wt. % errorAtom %Atom % errorCompound formulaCompound wt. %
CPC purified BioflocculantC K3·26±0·246·07±0·45C3·26
O K39·95±0·4955·96±0·69O39·95
Na K1·05±0·101·02±0·10Na1·05
Mg K8·31±0·137·67±0·12Mg8·31
P K18·54±0·2213·41±0·16P18·54
Cl K0·62±0·060·39±0·04Cl0·62
K K1·40±0·070·80±0·04K1·40
Ca K25·12±0·3114·05±0·17Ca25·12
Cu K1·75±0·490·62±0·17Cu1·75
Partial Purified BioflocculantC K2·73±0·276·36±0·62C2·73
O K32·31±0·3356·50±0·58O32·31
Na K1·59±0·081·94±0·10Na1·59
Mg K6·84±0·107·88±0·11Mg6·84
Al K0·02±0·020·02±0·04Al0·02
P K13·47±0·1712·17±0·15P13·47
Cl K0·65±0·080·51±0·06Cl0·65
K K0·99±0·050·71±0·04K0·99
Ca K13·51±0·189·43±0·13Ca13·51
Cu K1·72±0·290·76±0·13Cu1·72
Au L26·17±2·933·72±0·42Au26·17

Thermal analyses of purified bioflocculant

Thermogravimetric analyses of the purified bioflocculant at temperature range of 20–900°C under nitrogen atmosphere showed mass loss of components starting from 54°C to 65°C against PPB and CPB, respectively (Fig. 8). PPB displayed a thermogram of about six decomposition steps at 65, 95, 256, 297, 395, 602 and 704°C, while CPB showed three distinct and several indistinct decomposition steps, respectively. The distinct decomposition pattern shown by CPB were at 65, 142 and 261°C, respectively, while the indistinct decomposition steps occurred at a temperature range of 356–486°C. PPB six decomposition steps corresponded to weight losses as follows: 0·22, 0·54, 0·98, 1·70, 4·01, 5·60 and 6·58 (mg), while CPB weight loss were similarly recorded as 0·04, 0·18 and 0·52 (mg), respectively, against the distinct decomposition steps. On a similar note, the thermal profile of bioflocculant produced by a marine dinoflagellate Gyrodinium impudicum indicated a complete degradation at 250°C (Yim et al. 2007).

Figure 8.

Thermogravimetric analysis curve showing the pyrolysis of purified bioflocculant (CPB and PPB) produced by Cellulomonas sp. Okoh. (image_n/jam12095-gra-0014.png) Weight of PPB; (image_n/jam12095-gra-0015.png) Weight of CPB.

Discussion

Water milieu remains good source of microbes with novel metabolites and as such, the fresh water actinobacteria identified in this study as Cellulomonas sp. produced bioflocculant with high flocculation activity. Although accounts of bioflocculant production have been documented for several bacterial species (Kurane et al. 1994; Salehizadeh and Shojaosadati 2001; Zhang et al. 2010), however, bioflocculant production by Cellulomonas sp. is novel. Activation of actinobacteria culture and the optimization of cell density to nutrient ratio were necessary, as this process reduces fermentation time and increases bioflocculant productivity (Smith et al. 2004). Similarly, variation of fermentation conditions was crucial towards achieving conditions optimal for the maximization of the production of products of interest; hence, neutral pH, incubation at 30°C and agitation speed of 160 rpm were the optimal conditions for the production of bioflocculant. Conversely, glucose, ammonium nitrate and magnesium chloride, respectively, served as preferred nutritional sources by Cellulomonas sp. for the production of bioflocculant, however, saccharides such as glucose, fructose, sucrose as well as nitrogen and cation sources including urea, ammonium sulfate, calcium and magnesium have been, respectively, documented as components of suitable media for bioflocculant production (Kurane et al. 1994; Fujita et al. 2000).

The ability of Cellulomonas sp. to produce bioflocculant over time showed that peak production resided at the exponential growth phase; hence, it may be deduced that cell biosynthetic processes are responsible for production. This point was further buttressed as flocculation activity apparently stagnated at the stationary phase of the bacterial growth curve and declined with decline/death phase. These patterns of results are consistent with similar studies showing the link between bacterial growth curve and bioflocculant production (Lu et al. 2005; Gong et al. 2008; Wu et al. 2010).

Cations mediate flocculating activity, and this process has been reported for various kinds of bioflocculants produced by different microbial species, however, flocculation activity has optimally revolved around divalent and trivalent cations (Takeda et al. 1992; Watanabe et al. 1999). Nonetheless, the role of cations in mediating flocculation activity was demonstrated in this study. The divalent cations mediated flocculation activity better with both forms of purified bioflocculant (PPB and CPB) in comparison with the monovalent and trivalent cations. The reason behind this concept may lie on the binding capabilities of the cations to the bioflocculants or kaolin clay, which may lead to charge neutralization or anchorage of biomolecular flocculants in such a spatial arrangement as to enhance flocculation process. Nonetheless, Yokoi et al. (1995) and Nwodo et al. (2012) similarly reported divalent cations to better mediate flocculation activity. Furthermore, PPB and CPB showed optimum flocculation activity as neutral pH, however, flocculation was recorded at a wide pH range (3–11), which indicates good stability to both acidic and alkaline pH. In corroboration to these attributes, Corynebacterium glutamicum and Streptomyces sp. Gansen similarly produced bioflocculants with a wide range of stability to pH (Lu et al. 2005; Nwodo et al. 2012).

In a bid to improve upon the quality of the bioflocculant, treatment with CPC and subsequent dialysis proved to be an unnecessary step. This was so because flocculation activity declined, and this process was not cost effective if industrial applicability is to be anticipated. Moreover, decreased flocculation activity may be attributed to the lowered concentration of cations from CPB; hence, PPB contained more metal ions with consequent higher flocculation activity. However, on the contrary, the chemical composition of CPB and PPB remained fairly the same. Furthermore, the spectroscopic analyses of the purified bioflocculant via FTIR, 1H NMR and EDX revealed more details to account for compounds not assayed for in the quantitative analyses for chemical composition. This is supported by the multiple steps shown by the pyrolysis studies and at the same time, resilience of the bioflocculant to thermal degradation at a temperature above 500°C implies good thermal stability. Additionally, vinylic, aromatic, benzylic, allylic, hydroxyl, carboxyl, esters and amino groups amongst other functional groups demonstrated to be present in the bioflocculant yields charged moieties favourable for flocculation processes. However, the presence of uronic acids, carbohydrates and proteins is a strong indication that that the bioflocculant is glycosaminoglycan in nature (Esko and Lindahl 2001). The moieties and functional groups revealed by FTIR and 1H NMR further confirm this assertion. In conclusion, Cellulomonas sp. Okoh produced a unique bioflocculant, which is glycosaminoglycan in nature, stable at a wide range of pH and temperature and shows strong flocculation activity at moderate conditions. Equally, the presence of vinylic, aromatic, benzylic, allylic, hydroxyl, carboxyl, esters and amino moieties, which are usually charged, accounts for the uniqueness and novelty of the bioflocculant as well as the strong flocculation activity; hence, it shows an indication of potential industrial applicability.

Acknowledgments

We are grateful to the National Research Foundation (NRF) of South Africa and the University of Fort Hare for financial support.

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