Studies on Bioflocculant Production by Arthrobacter sp. Raats, a Freshwater Bacteria Isolated from Tyume River, South Africa

A bioflocculant-producing bacteria was isolated from Tyume River in the Eastern Cape Province, South Africa and identified by 16S rRNA gene nucleotide sequence to have 91% similarity to Arthrobacter sp. 5J12A, and the nucleotide sequence was deposited in GenBank as Arthrobacter sp. Raats (accession number HQ875723). The bacteria produced an extracellular bioflocculant when grown aerobically in a production medium containing glucose as sole carbon source and had an initial pH of 7.0. Influences of carbon, nitrogen and metal ions sources, as well as initial pH on flocculating activity were investigated. The bacteria optimally produced the bioflocullant when lactose and urea were used as sole sources of carbon and nitrogen respectively with flocculating activities of 75.4% and 83.4% respectively. Also, the bacteria produced the bioflocculant optimally when initial pH of the medium was 7.0 (flocculating activity 84%), and when Mg2+ was used as cation (flocculating activity 77%). Composition analyses indicated the bioflocculant to be principally a glycoprotein made up of about 56% protein and 25% total carbohydrate

Production and biochemical characterization of new bioflocculants from bacteria isolated from freshwater and marine environments of the Eastern Cape in South Africa

The production and characterization of bioflocculants produced by three bacteria belonging to Arthrobacter, Halomonas and Micrococcus genera and isolated from freshwater and marine environments were evaluated both as axenic cultures and as consortia. The influences of cultutre conditions such as carbon, nitrogen and metal ions sources, as well as initial pH on bioflocculant production by individual isolates were investigated. Both Arthrobacter sp. Raats and Halomonas sp. Okoh utilized urea as a nitrogen source of choice for optimal production of the bioflocculants with Micrococcus sp. Leo having a preference for peptone. All three strains differed in as far as the carbon source of choice was concerned with lactose, glucose and sucrose the preferred carbon sources respectively. Also, all three bacterial strains produced an extracellular bioflocculant aerobically but an intial pH 7.0 of the culture media was suitable for both Arthrobacter sp. Raats and Halomonas sp. Okoh with a slightly alkaline pH of 9.0 preferred by Micrococcus sp. Leo. The presence of Mg2+ cations stimulated bioflocculant production by both Arthrobacter sp. Raats and Micrococcus sp. Leo while Ca2+ resulted in more efficient bioflocculant production by Halomonas sp. Okoh. Chemical analyses revealed the bioflocculants produced by both Halomonas sp. Okoh and Micrococcus sp. Leo to be predominantly polysaccharides whereas Arthrobacter sp. Raats produced principally a glycoprotein composed of about 56percent protein and 25percent total carbohydrate. Response surface methodology (RSM) was used to optimize production medium for bioflocculant production by a consortium of Halomonas sp. Okoh and Micrococcus sp. Leo. Plackett-Burman experimental design showed that fructose, ammonium sulphate and MgCl2 were significant in the high yield of the bioflocculant. Furthermore, central composite design showed that optimal concentration of these critical nutritional sources were 16.14 g/L, 1.55 g/L and 1.88 g/L for fructose, ammonium sulphate and MgCl2 respectively. Quantification of the bioflocculant showed a yield of 6.43 g/L which was in close accord with the predicted value of 6.51 g/L. FTIR spectrometry of the bioflocculant indicated the presence of carboxyl, hydroxyl and amino groups, typical for heteropolysaccharide, while SEM imaging revealed a lattice-like structure. The efficiency of the nutrient optimization suggests suitability for industrial applicability.Thesis (PhD) -- Faculty of Science and Agriculture, 201

Production and characterization of a bioflocculant from a consortium of bacteria belonging to the halomonas and micrococcus genera.

The physicochemical properties of two bioflocculant producing bacteria; Halomonas sp. Okoh and Micrococcus sp. Leo were investigated. The optimum culture conditions for the individual species were determined. All the growth conditions examined for the individual bacteria were similar. Glucose and ammonium sulphate as sole carbon and nitrogen sources respectively resulted in optimum production of bioflocculant. The flocculating activity of the bioflocculants was stimulated when Al3+ was used as the coagulating aid under acidic medium. The information obtained from individual strains was used to produce a bioflocculant from the consortium of the two bacteria. After purification, the bioflocculant yields from 1L fermentation broths were 1.213 g from Halomonas sp. Okoh, 0.738 g from Micrococcus sp. Leo and 3.51 g from the consortium. The chemical analyses of the purified bioflocculants showed that they were glycoproteins. The thermostability property of the bioflocculants was investigated between 50-100oC and the results revealed that they are heat-stable. Fourier transform infrared revealed the presence of hydroxyl, carboxyl and amino groups in the bioflocculant molecules. Scaning electron microscope (SEM) images showed the structure of each bioflocculant(s) and kaolin clay before and after flocculation. From the results obtained, the idea of using the two strains in consortium for bioflocculant production resulted in an improvement in terms of flocculating activity and yield. The bioflocculants appears to have promise as an alternative to chemical flocculants used in various industrial processes such as wastewater treatment and drinking water purification.Thesis (MA) -- Faculty Science and Agriculture, 201

Production and characterization of a bioflocculant from a consortium of bacteria belonging to the halomonas and micrococcus genera

The physicochemical properties of two bioflocculant producing bacteria; Halomonas sp. Okoh and Micrococcus sp. Leo were investigated. The optimum culture conditions for the individual species were determined. All the growth conditions examined for the individual bacteria were similar. Glucose and ammonium sulphate as sole carbon and nitrogen sources respectively resulted in optimum production of bioflocculant. The flocculating activity of the bioflocculants was stimulated when Al3+ was used as the coagulating aid under acidic medium. The information obtained from individual strains was used to produce a bioflocculant from the consortium of the two bacteria. After purification, the bioflocculant yields from 1L fermentation broths were 1.213 g from Halomonas sp. Okoh, 0.738 g from Micrococcus sp. Leo and 3.51 g from the consortium. The chemical analyses of the purified bioflocculants showed that they were glycoproteins. The thermostability property of the bioflocculants was investigated between 50-100oC and the results revealed that they are heat-stable. Fourier transform infrared revealed the presence of hydroxyl, carboxyl and amino groups in the bioflocculant molecules. Scaning electron microscope (SEM) images showed the structure of each bioflocculant(s) and kaolin clay before and after flocculation. From the results obtained, the idea of using the two strains in consortium for bioflocculant production resulted in an improvement in terms of flocculating activity and yield. The bioflocculants appears to have promise as an alternative to chemical flocculants used in various industrial processes such as wastewater treatment and drinking water purification.Thesis (MA) -- Science and Agriculture, 201

Production and characterization of a bioflocculant from a consortium of bacteria belonging to the halomonas and micrococcus genera

The physicochemical properties of two bioflocculant producing bacteria; Halomonas sp. Okoh and Micrococcus sp. Leo were investigated. The optimum culture conditions for the individual species were determined. All the growth conditions examined for the individual bacteria were similar. Glucose and ammonium sulphate as sole carbon and nitrogen sources respectively resulted in optimum production of bioflocculant. The flocculating activity of the bioflocculants was stimulated when Al3+ was used as the coagulating aid under acidic medium. The information obtained from individual strains was used to produce a bioflocculant from the consortium of the two bacteria. After purification, the bioflocculant yields from 1L fermentation broths were 1.213 g from Halomonas sp. Okoh, 0.738 g from Micrococcus sp. Leo and 3.51 g from the consortium. The chemical analyses of the purified bioflocculants showed that they were glycoproteins. The thermostability property of the bioflocculants was investigated between 50-100oC and the results revealed that they are heat-stable. Fourier transform infrared revealed the presence of hydroxyl, carboxyl and amino groups in the bioflocculant molecules. Scaning electron microscope (SEM) images showed the structure of each bioflocculant(s) and kaolin clay before and after flocculation. From the results obtained, the idea of using the two strains in consortium for bioflocculant production resulted in an improvement in terms of flocculating activity and yield. The bioflocculants appears to have promise as an alternative to chemical flocculants used in various industrial processes such as wastewater treatment and drinking water purification.Thesis (MA) -- Science and Agriculture, 201

Production and characterization of a bioflocculant from a consortium of bacteria belonging to the halomonas and micrococcus genera.

The physicochemical properties of two bioflocculant producing bacteria; Halomonas sp. Okoh and Micrococcus sp. Leo were investigated. The optimum culture conditions for the individual species were determined. All the growth conditions examined for the individual bacteria were similar. Glucose and ammonium sulphate as sole carbon and nitrogen sources respectively resulted in optimum production of bioflocculant. The flocculating activity of the bioflocculants was stimulated when Al3+ was used as the coagulating aid under acidic medium. The information obtained from individual strains was used to produce a bioflocculant from the consortium of the two bacteria. After purification, the bioflocculant yields from 1L fermentation broths were 1.213 g from Halomonas sp. Okoh, 0.738 g from Micrococcus sp. Leo and 3.51 g from the consortium. The chemical analyses of the purified bioflocculants showed that they were glycoproteins. The thermostability property of the bioflocculants was investigated between 50-100oC and the results revealed that they are heat-stable. Fourier transform infrared revealed the presence of hydroxyl, carboxyl and amino groups in the bioflocculant molecules. Scaning electron microscope (SEM) images showed the structure of each bioflocculant(s) and kaolin clay before and after flocculation. From the results obtained, the idea of using the two strains in consortium for bioflocculant production resulted in an improvement in terms of flocculating activity and yield. The bioflocculants appears to have promise as an alternative to chemical flocculants used in various industrial processes such as wastewater treatment and drinking water purification.Thesis (MA) -- Faculty Science and Agriculture, 201

Evaluation of flocculating potentials and charecterization of bioflocculants produced by three bacterial isolates from Algoa Bay, South Africa

Flocculation has been widely adopted as one of the most effective methods to remove colloidal particles in water or wastewater treatment. Synthetic flocculants are conventionally used because of their high flocculating efficiency and cost-effectiveness. However, they have been reported to have hazardous properties and implicated in some serious health problems including senile dementia and neuro-toxicity, as well as being recalcitrant in the environment. Consequently, efforts are being geared away from the use of synthetic flocculants in water and wastewater treatment. Hence, the need for safe and eco-friendly flocculants has become imperative. Compared with synthetic flocculants, bioflocculants have special advantages such as safety, biodegradability and harmlessness to the environment and humans; attributes which make them potential alternatives in water treatment, downstream as well as fermentation processes. In the current study, the potentials of bacterial isolates recovered from Algoa Bay in the Eastern Cape Province of South Africa for bioflocculant production were investigated. The bacterial isolates were identified by polymerase chain reaction (PCR) as belonging to the Bacillus genus. The analysis of 16S ribosomal deoxyribonucleic acid (rDNA) nucleotide sequence of isolate M72 showed 99 percent similarity to Bacillus toyonensis strain BCT-7112 and was deposited in the GenBank as Bacillus toyonensis strain AEMREG6 with accession number KP406731. Likewise, the 16S rDNA nucleotide sequences of isolates M69 and M67 showed 98 percent sequence similarity to Bacillus licheniformis strain W7 and Bacillus algicola strain QD43 respectively; and M67 isolate was subsequently deposited in the GenBank as Bacillus sp. AEMREG7 with accession number KF933697.1. The results of the nutritional requirements and fermentation conditions revealed that optimum inoculum size for REG-6 production was 4 percent (v/v), while 5 percent (v/v) and 3 percent (v/v) were most favourable for MBF-W7 and MBF-UFH production respectively. Glucose was the best carbon source for the production of bioflocculants (REG-6 and MBF-UFH) by Bacillus toyonensis AEMREG6 and Bacillus sp. AEMREG7 respectively, while maltose supported optimum bioflocculant (MBF-W7) production by Bacillus specie. Inorganic nitrogen (NH4NO3) was the favoured nitrogen source for both REG-6 and MBF-W7 production, while mixed nitrogen sources [yeast extract + urea + (NH4)2SO4] supported the maximum production of MBF-UFH. The initial medium pH for REG-6 was 5, while MBF-W7 and MBF-UFH were both maximally produced at the initial pH of 6. After a 96 h cultivation period under optimal culture conditions, 3.2 g of purified REG-6 with a maximum flocculating activity of 77 percent was recovered from 1 L fermented broth of Bacillus toyonensis AEMREG6. Yields of 3.8 g and 1.6 g pure bioflocculants with the respective highest flocculating activities of 94.9 percent and 83.2 percent were also obtained from 1 L, 72 h-fermented broths of Bacillus licheniformis and Bacillus sp. AEMREG7 respectively. Furthermore, all the three bioflocculants (REG-6, MBF-W7 and MBF-UFH), displayed thermal stability within the temperature range of 50 to 100 oC, with strong flocculating activities of over 80 percent against kaolin suspension over a wide range of pH range (3–11) and relatively low dosage requirements of 0.1-03 mg/ml in the presence of divalent cations in the treatment of kaolin clay suspension and Thyme River waters. Chemical composition analyses of the bioflocculants showed them to be glycoproteins with a predominantly polysaccharide backbones as shown by the following carbohydrate/protein (w/w) ratios: 77.8 percent:11.5 percent (REG-6); 73.7 percent:6.2 percent (MBF-W7) and 76 percent:14 percent (MBF-UFH)

Engineering of bacterial exopolysaccharides: from synthesis to properties

Dissertação para obtenção do Grau de Doutor em Engenharia Química e BioquímicaFundação para a Ciência e Tecnologi

Characterization of bioflocculants produced by consortia of three marine bacteria belonging to the genera bacillus and cobetia previously isolated from the bottom sediment of Algoa Bay in the Eastern Cape Province of South Africa

The bioflocculant-producing potentials of three marine bacteria isolated from the sediment samples of Algoa Bay in the Eastern Cape Province of South Africa were assessed. Analysis of the partial nucleotide sequence of the 16S rDNA of the bacteria revealed 99 percent, 99 percent, and 98 percent similarity to Cobetia sp. L222, Bacillus sp. A-5A, and Bacillus sp. HXG-C1 respectively and the sequence was deposited in GenBank as Cobetia sp. OAUIFE, Bacillus sp. MAYA and Bacillus sp. Gilbert (accession number JF799092, JF799093, and HQ537128 respectively). Cultivation condition studies for Cobetia sp. OAUIFE revealed that bioflocculant production was optimal with an inoculum size of 2 percent (v/v), initial pH of 6.0, Mn2+ as the metal ion, and glucose as the carbon source. Metal ions, including Na+, K+, Li+, Ca2+and Mg2+ stimulated bioflocculant production resulting in flocculating activity of above 90 percent. This crude bioflocculant is thermally stable, with about 78 percent of its flocculating activity remaining after heating at 100 oC for 25 min. Analysis of the purified bioflocculant revealed it to be an acidic extracellular polysaccharide. FTIR analysis revealed the presence of methoxyl, hydroxyl, and carboxyl - groups in the compound bioflocculant and SEM micrograph of the bioflocculant revealed a crystal-linear structure. On the other hand, bioflocculant production by Bacillus sp. MAYA was optimal when glucose (95.6 percent flocculating activity) and ammonium nitrate (83.3 percent flocculating activity) were used as carbon and nitrogen sources respectively; inoculum size was 2 percent (v/v); initial pH 6; and Ca2+ as coagulant aid. Chemical analysis of the purified bioflocculant shows that it is composed of uronic acid, neutral sugar and protein. FTIR analysis also revealed the presence of methoxyl, hydroxyl, carboxyl and amino- groups in this bioflocculant. The bioflocculant is thermostable with about 65.6 percent residual flocculating activity retained after heating the bioflocculant at 100 oC for 25 min. However bioflocculant production by Bacillus sp. Gilbert was optimal when sodium carbonate (95.2 percent flocculating activity) and potassium nitrate (76.6 percent flocculating activity) were used as carbon and nitrogen sources respectively; inoculum size was 3 percent (v/v); initial pH 9; and Al3+ as cation. The crude bioflocculant retained 44.2 percent residual flocculating activity after heating at 100 oC for 15 min. FTIR analysis reveals the presence of hydroxyl, carboxyl and methylene - groups in the compound bioflocculant. SEM micrograph of the bioflocculant revealed an amorphous compound. The consortia of these bacteria strains also produced bioflocculants with high flocculating activities which were highly efficient in removing turbidity and chemical oxygen demand (COD) from brewery wastewater, diary wastewater and river water. The bioflocculants from the consortia seemed better than traditional flocculants such as alum . The characteristics of the bioflocculant produced by the consortium of Cobetia sp. OAUIFE and Bacillus sp. MAYA showed that this extracellular bioflocculant, composed of 66percent uronic acid and 31percent protein and an optimum flocculation (90 percent) of kaolin suspension, when the dosage concentration was 0.8 mg/ml, under weak alkaline pH of 8, and Ca2+ as a coagulant aid. The bioflocculant is thermally stable, with a high residual flocculating activity of 86.7 percent, 89.3 percent and 87.0 percent after heating at 50 oC, 80 oC and 100 oC for 25 min respectively. The FTIR analysis of the bioflocculant indicated the presence of hydroxyl, amino, carbonyl and carboxyl functional groups. Scanning electron microscopy (SEM) image revealed a crystal-linear spongy-like bioflocculant structure and EDX analysis of the purified bioflocculant showed that the elemental composition in mass proportion of C,N,O,S and P was 6.67:6.23:37.55:0.38:4.42 (percent w/w). However, the characteristics of the bioflocculant produced by the consortium of Cobetia sp OAUIFE and Bacillus sp. Gilbert showed an optimum flocculation (90 percent) of kaolin suspension when the dosage concentration was 0.2 mg/ml, under neutral pH of 7, and Ca2+ as a coagulant aid. The FTIR analysis of the bioflocculant indicated the presence of hydroxyl and carbonyl functional groups. Scanning electron microscopy (SEM) image revealed an amorphous morphology. On the other hand the bioflocculant produced by the consortium of Bacillus sp. MAYA and Bacillus sp. Gilbert showed similar characteristic with the bioflocculant from the consortium of Cobetia sp. OAUIFE and Bacillus sp. Gilbert except for Al3+ being the preferred coagulant aid. The characteristics of the bioflocculant produced by the consortium of Cobetia sp. OAUIFE, Bacillus sp. MAYA and Bacillus sp. Gilbert showed an optimum flocculation (87 percent) of kaolin suspension when the dosage concentration was 1.0 mg/ml. Under strong alkaline pH of 12, flocculating activity reached (95 percent) when Al3+ was the coagulant aid. The FTIR analysis of the bioflocculant indicated the presence of hydroxyl, amino, carbonyl and carboxyl and phosphoryl functional groups. Scanning electron microscopy (SEM) image revealed a flaky amorphous morphological structure. Due to the excellent COD and turbidity removal efficiencies of the bioflocculants produced by the consortia, these make those attractive candidates for use in water and wastewater treatment.Thesis (PhD) -- Faculty of Science and Agriculture, 201

Bioflocculant production by aspergillus flavus using chicken viscera as a substrate

The biodegradability and safety of the bioflocculants make them potential alternative to non-biodegradable chemical flocculants for wastewater treatment. However, low yield and production cost has been reported to be the limiting factor for large scale bioflocculant production. The sustainability and economics of bioflocculant production is dependent on the use of low-cost substrate at optimum culture conditions. This study focused on the optimization of culture conditions for Aspergillus flavus growth and its bioflocculant production using chicken viscera hydrolysate as substrate. The effects of culture conditions including pH, shaker speed, temperature and inoculum size on bioflocculant production were investigated and optimized via response surface method in accordance with the critical component design (CCD) package of design expert. The purified bioflocculant was characterized using physical and chemical analysis. The flocculation performance and effect of cations on the bioflocculant was investigated using jar testing and Kaolin clay suspension as wastewater model. Under optimized culture conditions, 6.75 g/L of crude bioflocculant was produced. The bioflocculation activity was mostly distributed in the cell free supernatant with optimum efficiency of 91.8% at dose of 4 mL/100 mL Kaolin suspension. The purified bioflocculant was an uneven, coarse EPS assemblage in netted texture consisting of 23.46% protein and 74.5% polysaccharide, including 46% neutral sugar and 2.01% uronic acid with zeta potential of -25.28 ± 2.7 mV at pH 6.2. The Fourier-transform infrared (FTIR) and X-ray photoelectron spectroscopy (XPS) analysis indicated the presence of carbonyl, amino, hydroxyl and amide functional groups and mass proportion of C, O and N at 63.46%, 27.87% and 8.86%, respectively. It had a minimum of 83.1% efficiency in flocculating 2-12 g/L Kaolin clay suspension over a wide temperature range (4 - 80°C) and function optimally at neutral pH. It effectively flocculated different suspended particles such as activated carbon (92%), soil solids (94.8%) and algae (69.4%) at varying concentrations. Addition of both Ca2+ and Mg2+ stimulated the efficiency of the bioflocculant at all the concentrations tested with optimum flocculation efficiency of 95% recorded with 5 mL 1% Ca2+. Lower concentrations (1-2 mL) of Al3+ also stimulated the bioflocculant to about 94%, K+ slightly enhanced the flocculation at 4 – 10 mL 1%, while Na+ and Fe3+ inhibited the flocculation. This study indicates high potential of cation dependent bioflocculant production from chicken viscera at appropriate culture conditions and stand as an attractive candidate for additional exploration and development for large-scale bioflocculant production and application