Studies on bioflocculant production by a mixed culture of Methylobacterium sp. Obi and Actinobacteriumsp. Mayor
© Luvuyo et al.; licensee BioMed Central Ltd. 2013
Received: 3 May 2013
Accepted: 25 July 2013
Published: 1 August 2013
Bioflocculants effect the aggregation of suspended solutes in solutions thus, a viable alternative to inorganic poly-ionic and synthetic organic flocculants which are associated with deleterious health problems. Consequently, a consortium of two bacteria species were evaluated for optimized bioflocculant yield following the inadequacies of axenic cultures.
16S rDNA nucleotide sequencing and BLAST analysis of nucleotide sequences were used to identify the bacterial species, carbon and nitrogen sources optimally supporting bioflocculant production were assessed and the purified bioflocculant characterized.
Nucleotide sequences showed 97% and 96% similarity to Methylobacterium sp. AKB-2008-KU9 and Methylobacterium sp. strain 440. The second isolate, likewise, showed 98% similarity to Actinobacterium OR-221. The sequences were deposited in GenBank as Methylobacterium sp. Obi [accession number HQ537130] and Actinobacterium sp. Mayor [accession number JF799090]. Flocculating activity of 95% was obtained in the presence of Ca2+ and heat-stability was exhibited with retention of above 70% activity at 100°C in 30 min. In addition, bioflocculant yield was about 8.203 g/l. A dose of 1 mg/ml of purified bioflocculant was optimal for the clarification of Kaolin suspension (100 ml) following Jar test. FTIR spectrum revealed the presence of carboxyl and hydroxyl functional groups amongst others.
The mixed culture produced bioflocculant with high flocculating activity and an improved yield. The efficiency observed with jar test may imply industrial applicability.
Flocculants may be synthetic or natural in origin. However, they lead to the fluffy mass formation of suspended particles . Flocculants are extensively applied in the treatment of wastewaters and other industrial effluents [2, 3]. Other applications have included the recovery of suspended solutes from solutions . Nonetheless, inorganic flocculants which includes the salts of poly-aluminium chloride and aluminium sulphate as well as the organic synthetic flocculants (poly-acryl amide and polyethylene amine) have been implicated in various human health problems such as nuerotoxicity, cancer and a medical disorder leading to dementia (Alzheimer’s disease). The organic synthetic flocculants are also known to be non biodegradable hence, not environmentally friendly . In contrast, bioflocculants have not been associated with any medical problem and are biodegradable; as such, are considered environmentally friendly [6, 7].
Considerable attention has been directed towards studying bioflocculant producing bacteria in axenic culture and yield optimization has been attempted through the manipulation of fermentation and nutritional conditions. Following the aforementioned techniques, high flocculation activities have been documented. However, low bioflocculant yield and lack of cost effectiveness in the production of bioflocculant militates against the application of these bioflocculants in industrial processes, such as in wastewater treatment [8, 9].
Consequently, it has become imperative to explore alternative means of bioflocculant yield optimization [10–12]. The application of mixed culture in the production of bioflocculant has been attempted by Kurane and Matsuyama  as well as Zhang et al.  and bioflocculant yield was reported to have improved. Following these findings, we evaluated the bioflocculant production potentials of a consortium of two fresh water bacteria belonging to Methylobacterium and Actinobacterium genera and the bioflocculant was characterized for novelty.
Bacterial strains were previous isolates from the Tyume River in the Eastern Cape Province of South Africa. Isolates were preserved in glycerol at −80°C as part of the culture collection of the Applied and Environmental Microbiology Research Group (AEMREG), University of Fort Hare, South Africa. However, prior to storage, the test bacteria were identified as Actinobacterium sp. Mayor and Methylobacterium sp. Obi through partial nucleotide sequencing of their 16S rRNA genes with subsequent BLAST analyses. Nucleotide sequences were deposited in GenBank and the repository accession numbers were JF799090 and HQ537130 respectively.
Mixed culture fermentation for bioflocculant production
Actinobacterium sp. and Methylobacterium sp. were activated by inoculation of 20 μL of the glycerol stock into a sterile 5 mL broth composed of (g/L); beef extract (3), tryptone (10) and NaCl (5) and each was incubated overnight at 28°C respectively. One percent (1%), each, of the activated culture was inoculated into 400 ml of bioflocculant production medium in 1000 ml conical flask. Bioflocculant production media was prepared in accordance with the methods of Zhang et al. . Briefly, glucose (20.0 g), KH2PO4 (2.0 g), K2HPO4 (5.0 g), (NH4)2SO4 (0.2 g), NaCl (0.1 g), MgSO4 · 7H2O urea (0.5 g) (0.2 g) and yeast extract (0.5 g) were dissolved in one litre of distilled water and the pH adjusted to 7. The incubation conditions for the mixed culture fermentation were an incubation temperature of 28°C, agitation speed of 160 rpm in a shaker incubator and fermentation time of 72 h. Thereafter, the fermentation broth was centrifuged at 3000 rpm for 30 min at 15°C and the cell-free supernatant was assayed for flocculation activity.
Effect of inoculum size and pH on bioflocculant production
Mixed culture inoculum volumes of 0.5%, 1%, 1.5% and 2% in proportion to the fermentation volume (400 ml) were respectively evaluated for bioflocculant production. The cultures were incubated at a temperature of 28°C for 72 h at 160 rpm. Thereafter, the fermentation broth was centrifuged (3000 rpm, 30 min, 15°C) and the supernatant was assessed for flocculation activity. Likewise, the initial fermentation pH regimes of 2 to 12 were evaluated for bioflocculant production while other conditions were kept constant.
Flocculation activity determination
A and B were optical densities at 550 nm of the sample and control respectively.
Purification of bioflocculant
The concentration and purification of bioflocculant from the bioflocculant-rich broth was in accordance with the methods of Chang et al. . One volume of distilled water was added to the cell-free-bioflocculant-rich broth and centrifuged at 10 000 rpm for 15 min at 15°C, the supernatant was decanted and the residue re-suspended with 20 ml of distilled water. Two volumes of cold ethanol were added to the bioflocculant solution and the mixture was left standing at 4°C for 12 h. after which the precipitate was collected through centrifugation (10 000 rpm; 15 min; 15°C). The residue was washed twice with distilled water, lyophilized and vacuum dried. The dried bioflocculant was used for subsequent assays.
Optimum bioflocculant concentration for flocculation activity – Jar test
In accordance with the methods of Wang et al. , Jar-test was employed, with some modification, to determine bioflocculant concentration optimally mediating flocculation of Kaolin clay suspension (4.0 g/L). Bioflocculant concentrations of (mg/ml); 0.5, 1.0, 1.5 and 2.0 were respectively added to 100 ml Kaolin clay suspension (4.0 g/L) containing 3 ml of 1% CaCl2 in 500 ml beakers. The mixture was rapidly stirred at 180 rpm for 3 min, followed by slow stirring at 40 rpm for 5 min. The solutions were then allowed to stand for 10 min. and afterwards, flocculating activity was measured and calculated as previously described.
Effect of temperature, pH and cations on flocculating activity
The effect of temperature regimes on the flocculating activity of purified bioflocculant were investigated; desired concentration of purified bioflocculant was reconstituted with 10 ml of distilled water and incubated in water bath at the respective temperatures; 50°C, 80°C and 100°C for a period of up to 30 min. Residual flocculating activity were measured afterwards . Similarly, the effect of pH on flocculation activity of bioflocculant was determined by adjusting the pH of Kaolin clay suspension from 3 to 12 using HCl or NaOH, before the addition of bioflocculant and CaCl2 as previously described. Furthermore, KCl, NaCl, LiCl, MgCl2, MnCl2, AlCl3 and FeCl3 were respectively assessed as cation sources in place of CaCl2, all conditions for flocculation activity assay were kept constant.
FT-IR spectroscopy and thermo-gravimetric analyses of purified bioflocculant
The functional groups of the bioflocculant were determined using Fourier transform infrared spectrophotometer (Perkin Elmer System 2000, FT-IR, England). The bioflocculant was ground with KBr at room temperature and pressed into a thin disc for FTIR spectroscopy over a wave number range of 4 000 - 370 cm-1. The thermo-gravimetric analysis of the purified bioflocculant was carried out at the temperature range of 20 to 900°C with a heating rate of 10°C/min under a constant flow of nitrogen gas, using a thermogravimetric analyzer (TGA 7; Perkin Elmer) fitted with thermal analysis controller (TAC 7/DX).
Results and discussion
Effect of inoculum size on bioflocculant production
Effect of pH on bioflocculant production
Bioflocculant yield and flocculation of kaolin clay
Mixed culture fermentation, following optimal conditions (starter culture density of 1%, initial fermentation pH of 9, agitation speed of 160 rpm and incubation temperature of 28°C), yielded bioflocculant to the tune of 8.203 g/l after purification. Similar account was documented by Zhang et al. . However, the yield with mixed cultures of Methylobacterium sp. Obi and Actinobacterium sp. Mayor reported in this work was lower than those from the consortium of Staphylococcus and Pseudomonas species .
Flocculation activity of purified bioflocculant - effects of physical-chemical factors
Compositional analysis of the purified bioflocculant
The bioflocculant thermogram revealed an initial weight loss between 20°C and 150°C and afterwards, other decompositions occurred at 590°C, 700°C and 850°C respectively. The thermogram profile indicates generic compounds present in the bioflocculant, with proteins and carbohydrates as an integral constituents.
The bioflocculant produced by the mixed cultures of Methylobacterium sp. Obi and Actinobacterium sp. Mayor is composed of proteins and polysaccharides and probably other constituents which have contributed to the high flocculation of Kaolin clay from the solution. In addition, the mixed culture of Methylobacterium sp. and Actinobacterium sp. have shown good bioflocculant producing potential, following high flocculation activity and bioflocculant yield obtained, in comparison to the yield and flocculation activity shown by the respective axenic cultures. Hence, bioflocculant produced by the consortium has good potentials for industrial applications.
We express our profound gratitude to the Govan Mbeki Research and Development Center (GMRDC), University of Fort Hare, for funding this research.
- Koizumi JI, Takeda M, Kurane R, Nakamura I: Synergetic flocculating of the bioflocculants FIX extracellularly produced by Nocardiaamare. J Gen Appl Microbiol. 1991, 37: 447-457. 10.2323/jgam.37.447.View ArticleGoogle Scholar
- Sanayei Y, Ismail N, Teng TT, Morad N: Studies on flocculating activity of bioflocculant from closed drainage system (CDS) and its application in reactive dye removal. International J Chem. 2010, 2 (1): 168-173.View ArticleGoogle Scholar
- Kurane R, Matsuyama H: Production of a bioflocculant by mixed culture. Biosci Biotechnol Biochem. 1994, 58: 1589-1594. 10.1271/bbb.58.1589.View ArticleGoogle Scholar
- Patil SV, Salunkhe RB, Patil CD, Patil DM: Bioflocculant exopolysaccharide production by Azotobacterinducus using flower extract of Madhucalatifolia L. Appl Biochem Biotechnol. 2011, 162: 1095-1108.View ArticleGoogle Scholar
- Matthys C, Bilau M, Govaert Y, Moons E, De HS, Willems JL: Risk assessment of dietary acrylamide intake in Flemish adolescents. Food Chem Toxicol. 2005, 43: 271-278. 10.1016/j.fct.2004.10.003.View ArticleGoogle Scholar
- He N, Li Y, Chen J: Production of a polygalacturonic acid bioflocculant REA-11 by Corynebacterium glutamicum. Bioresour Technol. 2004, 94: 99-105. 10.1016/j.biortech.2003.11.013.View ArticleGoogle Scholar
- Salehizadeh H, Shojaosadati SA: Extracellular biopolymeric flocculants recent trends and biotechnological importance. Biotechnol Adv. 2011, 19: 371-385.View ArticleGoogle Scholar
- Kurane R, Hatamochi K, Kakuno T, Kiyohara M, Hirono M, Taniguchi T: Production of a bioflocculant by Rhodococcus erythropolis S-1 grown on alcohols. Biosci Biotechnol Biochem. 1994, 58: 428-429. 10.1271/bbb.58.428.View ArticleGoogle Scholar
- Li Y, He N, Guan H, Du G, Chen J: A polygalacturonic acid bioflocculant REA-11 produced by Corynebacterium glutamicum: a proposed biosynthetic pathway and experimental confirmation. Appl Microbiol Biotechnol. 2003, 63: 200-206. 10.1007/s00253-003-1365-9.View ArticleGoogle Scholar
- He J, Zou J, Shao Z, Zhang J, Liu Z, Yu Z: Characteristics flocculating mechanism of a novel bioflocculant HBF-3 produced by deep-sea bacterium mutant Halomonas sp. V3a. J Microbiol Biotechnol. 2010, 26: 1135-1141. 10.1007/s11274-009-0281-2.View ArticleGoogle Scholar
- Xia S, Zhang Z, Wang X, Yang A, Chen L, Zhao J, Leonard D, Jaffrezic-Renault N: Production and characterization of bioflocculant by Proteus mirabilis TJ-1. Bioresour Technol. 2008, 99: 6520-6527. 10.1016/j.biortech.2007.11.031.View ArticleGoogle Scholar
- Ma F, Liu JL, Li SG, Yang JX, Zhang LQ, Wu B, Zhu YB: Development of complex microbial flocculant. China Water Wastewater. 2003, 19: 1-4.Google Scholar
- Zhang ZQ, Lin B, Xia SQ, Wang XJ, Yang AM: Production and application of a novel bioflocculant by multiple microorganism consortia using brewery wastewater as carbon source. J Environ Sci. 2007, 19: 667-673. 10.1016/S1001-0742(07)60112-0.View ArticleGoogle Scholar
- Wang Y, Gao BY, Yue QY, Wei JC, Zhou WZ, Gu R: Color removal from textile industry wastewater using composite flocculants. Environ Technol. 2010, 28 (6): 629-637.View ArticleGoogle Scholar
- Chang WC, Soon AY, In HO, Sang HP: Characterization of an extracellular flocculating substance produced by a planktonic cyanobacterium, Anabaena sp. Biotechnol Lett. 1998, 20 (12): 643-646.View ArticleGoogle Scholar
- Jie G, Hua-ying B, Ming-xia X, Qian L, Yan-fen : Characterization of a bioflocculant from a newly isolated Vagococcus sp. W31. J Zhejiang Univ Sci B. 2006, 7 (3): 186-192. 10.1631/jzus.2006.B0186.View ArticleGoogle Scholar
- Wang S, Gong W, Lui X, Tian L, Yue Q, Gao B: Production of novel bioflocculant by culture of Klebsiella mobilis using dairy wastewater. J Biochem Eng. 2007, 39: 81-86.View ArticleGoogle Scholar
- Nwodo UU, Agunbiade MO, Green E, Mabinya LV, Okoh AI: A Freshwater Streptomyces, isolated from Tyume River, produces a predominantly extracellular glycoprotein bioflocculant. Int J Mol Sci. 2012, 13: 8679-8695. 10.3390/ijms13078679.View ArticleGoogle Scholar
- Chan WC, Chiang CY: Flocculation of clay suspensions with water insoluble starch grafting acrylamide/sodium allylsulfonated copolymer powder. Appl Polymer Sci. 1995, 58: 1721-1726. 10.1002/app.1995.070581009.View ArticleGoogle Scholar
- Wang L, Ma F, Qu Y, Sun D, Li A, Guo J, Guo J, Yu B: Characterization of a compound bioflocculant produced by mixed culture of Rhizobium radiobacter F2 and Bacillus sphaeicus F6. World J Microbiol Biotechnol. 2011, 10: 1007-1012.Google Scholar
- Zheng Y, Ye Z, Fang X, Li Y, Cia W: Production and characteristics of a bioflocculant produced by Bacillus sp. F19. Biosour Technol. 2008, 99: 7686-7691. 10.1016/j.biortech.2008.01.068.View ArticleGoogle Scholar
- 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. 2010, 101: 1044-1048. 10.1016/j.biortech.2009.08.108.View ArticleGoogle Scholar
- Zhi L, Baoping H, Hong L: Optimum condition to high-concentration microparticle slime water with bioflocculants. Mining Sci Technol. 2010, 20: 0478-0484.Google Scholar
- Suh H, Kwon G, Lee C, Kim H, Yoon B: Characterization of bioflocculant produced by Bacillus sp. DP-152. J Ferment Bioeng. 1997, 84: 108-112. 10.1016/S0922-338X(97)82537-8.View ArticleGoogle Scholar
- Kumar CG, Joo HS, Kavali R, Choi JW, Chang CS: Characterization of an extracellular biopolymer flocculant from a haloakalophilic Bacillus isolate. World J Microbiol Biotechnol. 2004, 20 (8): 837-843. 10.1007/s11274-004-9008-6.View ArticleGoogle Scholar
- Comte S, Guibaud G, Baudu M: Bio sorption properties of extracellular polymeric substances (EPS) resulting from activated sludge according to their type: soluble or bound. Proces Biochem. 2006, 41: 815-823. 10.1016/j.procbio.2005.10.014.View ArticleGoogle Scholar
- Kumar CG, Anand SK: Significance of microbial biofilms in food industry: a review. Int J Food Microbiol. 1998, 42: 9-27. 10.1016/S0168-1605(98)00060-9.View ArticleGoogle Scholar
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