научная статья по теме BIOFLOCCULANT PRODUCTION BY BACILLUS SP. GILBERT ISOLATED FROM A MARINE ENVIRONMENT IN SOUTH AFRICA Химия

Текст научной статьи на тему «BIOFLOCCULANT PRODUCTION BY BACILLUS SP. GILBERT ISOLATED FROM A MARINE ENVIRONMENT IN SOUTH AFRICA»

ПРИКЛАДНАЯ БИОХИМИЯ И МИКРОБИОЛОГИЯ, 2014, том 50, № 1, с. 59-64

UDC: 576.80:541.18.041.2

BIOFLOCCULANT PRODUCTION BY Bacillus sp. Gilbert ISOLATED FROM A MARINE ENVIRONMENT IN SOUTH AFRICA

© 2014 A. M. Ugbenyen, S. Cosa, L. V. Mabinya, A. I. Okoh

Applied and Environmental Microbiology Research Group, Department of Biochemistry and Microbiology, University of Fort Hare, Private Bag X1314, Alice 5700, South Africa e-mail: aokoh@ufh.ac.za; ugbenyenanthony@gmail.com; sekco@webmail.co.za; lmabinya@ufh.ac.za

Received March 23, 2013

In our previous study we reported on the bioflocculant production by a Bacillus species isolated from sediment samples of Algoa Bay in the Eastern Cape Province of South Africa. In current study we carried out further evaluation on the effect of different culture conditions on the bioflocculant production, as well as characterised the bioflocculant produced in detail. The bacteria produced bioflocculant optimally under the following conditions: using sodium carbonate (95.2% flocculating activity) and potassium nitrate (76.6% flocculating activity) as carbon and nitrogen sources, respectively; inoculum size of 3% (v/v); initial pH 9.0; and Al3+ as coagulant aid. The crude bioflocculant retained 44.2% residual flocculating activity after heating at 100°C for 15 min. Chemical analysis of the Bacillus sp. Gilbert purified bioflocculant demonstrated that it was composed mainly of polysaccharide. Fourier transform infrared spectroscopy analysis revealed the presence of hydroxyl, carboxyl and methylene groups in the bioflocculant and energy-dispersive X-ray analysis detected the elemental composition in mass proportion (% w/w) of C, N, O, S and P as 4.12 : 7.40 : 39.92 : 3.00 : 13.91. Scanning electron micrograph image of the bioflocculant revealed an amorphous compound.

DOI: 10.7868/S0555109914010115

Bioflocculants are readily degradable metabolite compounds of microorganisms produced during their growth. Usually they are high molecular weight biopolymers released outside the cell [1]. Many biof-locculants have been reported to be polysaccharides, proteins, glycoproteins and nucleic acids [2, 3].

In recent years, bioflocculants have aroused the interest of many environmental researchers because of their biodegradability and the harmlessness of their degradative intermediates [4] compared to synthetic flocculant like polyacrylamide whose degradative intermediate, acrylamide has been implicated in cancer [5]. Screening new microorganisms for bioflocculant with high flocculating activity has become a subject of intensive investigations globally.

The genus Bacillus has been well reported for bioflocculant production [1, 4, 6—9] but isolates from the marine environment are scarce in literature with respect to bioflocculant production. Marine bacteria are amongst the most economically and biotechnological-ly valuable prokaryotes. They are responsible for the production of about 50% of all discovered bioactive secondary metabolites [10]. In our previous study [11] we reported a polysaccharide bioflocculant production by Bacillus sp. Gilbert. In current study we carried out further studies on effect of different culture conditions on the bioflocculant production, as well as characterised the purified bioflocculant produced in detail.

MATERIALS AND METHODS

Microorganism. The test bacterium was isolated from the sediment sample from Algoa Bay in the Eastern Cape Province of South Africa and reported in our previous study [11] as a bioflocculant producing bacterium. The culture of Bacillus sp. Gilbert was kept in 20% glycerol at -80°C.

Media and cultivation conditions. A medium described by Zhang et al. [12] with slight modifications was used for the bacterial growth. The medium contained (g/L offiltered natural sea water): glucose — 20; urea — 0.5; yeast extract - 0.5; (NH4)2SO4 - 0.2; KH2PO4 - 2; K2HPO4 - 5; NaCl - 0.1 and MgSO4 • 7H2O - 0.2. Two loopfuls ofbacterial colonies were inoculated in 50 mL of the medium and incubated with shaking at 28°C and 160 rpm for 72 h. At the end of incubation period,

2 mL of the fermentation broth was centrifuged (8000 g, 30 min) to separate the cells, and the cell free culture supernatant was analyzed for flocculating activity. The pre-culture was stored at 4°C and used for subsequent inoculations. Production medium contained the same components as the pre-culture medium.

Measurement of flocculating activity. Flocculating activity was measured according to the method described elsewhere [11-15] with modifications. Briefly,

3 mL of 1% CaCl2 and 2 mL of cell free supernatant were added to 100 mL kaolin suspended solution (4 g/L) in 250 mL flask. The mixture was vigorously stirred,

poured into a 100 mL cylinder and allowed to stand for 5 min. The OD550 of the clarifying solution was measured spectrophotometrically. A control experiment was performed using the same method, but with fresh culture medium replacing the cell free supernatant. The flocculating activity was calculated according to the equation

Flocculating activity (%) = [(B - A)/A] x 100,

where A is the OD550 of the sample; B is the OD550 of the control experiment.

Effect of inoculum size. Inoculum size is an important parameter in the production of bioflocculants [12, 16]. Hence, we assessed the effect of different inoculum size on bioflocculant production by Bacillus sp. Gilbert. Flasks (150 mL size) containing 50 mL production medium were separately inoculated with 0.5, 1.0, 1.5 and 2.0 mL preculture of the test bacteria cultivated at 28°C and 160 rpm for 72 h. At the end of the incubation period, the fermentation broths were cen-trifuged (8000 g, 30 min) to separate the cells. The cell free culture supernatants were analyzed for flocculating activity.

Effect of carbon and nitrogen sources. The effects of organic and inorganic carbon sources on bioflocculant production of Bacillus sp. Gilbert were assessed. The organic carbon source candidates included glucose, sucrose, fructose, maltose, galactose and xylose; while the inorganic carbon sources included phthalate, sodium acetate and sodium carbonate. Also, the organic nitrogen sources such as peptone, tryptone, urea, yeast extract and casein, as well as inorganic nitrogen sources such as ammonium chloride, ammonium sulphate and potassium nitrate were assessed for their effect on bioflocculant production. In the production medium, urea, yeast extract and (NH4)2SO4 were replaced with one of the nitrogen source candidates in equivalent amounts.

Effect of initial pH and cations. The effects of initial pH and cations on bioflocculant production of Bacillus sp. Gilbert were evaluated in accordance with the method of Liu et al. [3]. The initial pH of the production medium was varied in the range of 3.0-12.0 using 0.1 M HCl and NaOH, while the cation candidates included Na+, K+, Li+, Mg2+, Mn2+, Al3+ and Fe3+. With regards to the effects of cations, flocculating activity assay was conducted as described above, with CaCl2 solution replaced by the solution of the cation candidates.

Thermal stabilty of bioflocculant. The effect of heat on the cell free bioflocculant produced by the test bacteria was assessed according to the method of Gong et al. [17]. Three different temperature regimes (50, 80 and 100°C) were used with the aid of a waterbath. The biof-locculant was placed in the waterbath and heated for 15 min. At 5 min intervals 2 mL of the bioflocculant was drawn and assessed for residual flocculating activity.

Time course assay. The composition of the medium for the time course assay is as described earlier [12]. The preculture was used to inoculate the production medium based on the previously determined optimal growth conditions. Samples (2 mL) were withdrawn at 12 h intervals over a period of 60 h, centrifuged (8000 g, 30 min) and the cell free supernatant was used to determine the flocculating activity. The pH of the broth was also recorded.

Extraction and purification of bioflocculant. Extraction and purification was done according to the method described by previous reports [11, 14, 18—20]. After 72 h of fermentation, the culture broth was cen-trifuged at 8000 g for 30 min to remove bacterial cells. One volume of distilled water was added to the supernatant and centrifuged at 8000 g for 15 min to remove insoluble substances. Two volumes of ethanol were added to the supernatant, stirred and left to stand for 12 h at 4°C. The precipitate was vacuum dried to obtain crude bioflocculant. One volume of the mixture of chloroform and n-butylalcohol (5 : 2, v/v) was added to the crude product dissolved in distilled water with stirring. The mixture was set aside for 12 h at room temperature. Then 2 volumes of ethanol were added to recover the precipitate, which was lyophilized.

Chemical analysis of purified bioflocculant. Total protein content of purified bioflocculant of Bacillus sp. Gilbert was determined by Lowry's method using BSA as a standard. The total sugar content of bioflocculant was determined by a phenol-sulphuric acid method using glucose as a standard solution [21], and uronic acid was quantified by the carbazole method [22].

Fourier transform infrared (FTIR) analysis of the purified bioflocculant was done using a Fouriertransform infrared spectrophotometer (Perkin Elmer System 2000, England) over a wave number range of 4000 to 370 cm-1.

Scanning electron microscopy (SEM) image of the purified bioflocculant was performed using JEOL (JSM-6390LV, Japan) and the energy dispersive X-ray analysis (EDX) was measured by a Thermo Super Dry II X-ray Detector (JEOL, Japan) using a Noran System Six Software package (Japan).

Statistical analysis. Data were analyzed by one-way analysis of variance (ANOVA) using MINITAB Student Release 12 statistical package (USA). The mean values were obtained from 3 replicates.

RESULTS AND DISCUSSION

Previous preliminary study on Bacillus sp. Gilbert had established the bacteria as a producer of b

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