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WO2013066149A1 - A method for producing a biopolymer - Google Patents

A method for producing a biopolymer Download PDF

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Publication number
WO2013066149A1
WO2013066149A1 PCT/MY2012/000247 MY2012000247W WO2013066149A1 WO 2013066149 A1 WO2013066149 A1 WO 2013066149A1 MY 2012000247 W MY2012000247 W MY 2012000247W WO 2013066149 A1 WO2013066149 A1 WO 2013066149A1
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WO
WIPO (PCT)
Prior art keywords
biopolymer
culture medium
producing
bioflocculant
range
Prior art date
Application number
PCT/MY2012/000247
Other languages
French (fr)
Other versions
WO2013066149A4 (en
WO2013066149A9 (en
Inventor
Azni IDRIS
Ahmad H. RAJAB
Norhafizah Abdullah
Rosfarizan Mohamad
Original Assignee
Universiti Putra Malaysia
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Publication date
Application filed by Universiti Putra Malaysia filed Critical Universiti Putra Malaysia
Priority to JP2014539896A priority Critical patent/JP5975236B2/en
Priority to CN201280053572.5A priority patent/CN103998612A/en
Priority to GB1405815.0A priority patent/GB2508569A/en
Publication of WO2013066149A1 publication Critical patent/WO2013066149A1/en
Publication of WO2013066149A4 publication Critical patent/WO2013066149A4/en
Publication of WO2013066149A9 publication Critical patent/WO2013066149A9/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/66—Aspergillus
    • C12R2001/67—Aspergillus flavus

Definitions

  • This invention relates to a method for producing a biopolymer, and more particularly to a method for culturing Aspergillus flavus to produce biopolymer such as bioflocculant, biocoagulant , and the like.
  • U.S. Patent Application Publication Number 2002/0074295 A1 disclosed a process for treating contaminated liquid, in which the liquid to be treated is contacted with combination of two oppositely charged polymeric material, wherein at least one of said polymeric materials is branched, and wherein said polymeric materials are preferably derived from a variety of natural sources (for example, algae, bacterial, and just to name a few). Floe formation is allowed and the floes are separated from the liquid.
  • the first and second polymeric materials may be selected from the group consisting of polysaccharides, proteins, lipids and polyhydroxy alcohols.
  • the drawback of this cited patent includes two steps of water treatment which the contaminated liquid is first treated with the first polymeric material (anionic, branched polymeric material which derived from blue-green algae), and after an interval of time, contacted with the second polymeric material (cationic, unbranched polymer derived from chitosan) to eliminate contaminants from water. These two steps are making the process more cumbersome, time consuming and also require large dosage of polymeric materials to reduce the contaminant concentration in the water.
  • the first polymeric material anionic, branched polymeric material which derived from blue-green algae
  • the second polymeric material cationic, unbranched polymer derived from chitosan
  • bioflocculants have received increased scientific and technical attention because they are biodegradable and non-toxic and their degradation intermediates does not involve secondary pollutants.
  • Several microorganisms are known to produce bioflocculants, including Aspergillus sojae, Paecilomyces sp., Rhodococcus erythropolis, Serratia ficaria, and Bacillus mucilaginosus.
  • U.S. Patent Number 5,250,201 disclosed a method of separating cyanobacteria, particularly strain J-1 from a liquid, and a method for culturing cyanobacteria to produce a polymer useful as an emulsifying agent for forming emulsion of hydrocarbons and oils in water.
  • the cited patent further relates to the polymeric substance produced by purification and separation techniques, and to methods of affecting the secondary recovery of petroleum through the use of a material excreted by cyanobacteria.
  • the emulsification activity of the emulcyan is temperature dependent and pH dependent, which shown to be at 100 % of maximal value at a temperature of approximately 26 °C and pH in a range of 5 to 9.
  • the drawback of this cited patent is that the emulsifying activity of the emulcyan has maintained their optimum activity at limited temperature range, as well as at a limited pH range.
  • U.S. Patent Number 4,948,733 disclosed two new bacterial strains designated Zoogloea ramigera 1 15SL and Zoogloea ramigera 1 15SLR which derived from the wild type Zoogloea ramigera 1 15.
  • Zoogloea ramigera cultures are stored frozen at -70 °C in trypticase soy broth (TSB) medium containing 7 % DMSO and 15 % glycerol.
  • TTB trypticase soy broth
  • the Zoogloea ramigera strains are routinely cultured in either the TSB medium or a defined medium comprising 25 g glucose, 2 g K 2 HP0 4 , 1 g KH 2 P0 4 , 1 g NH 4 CI, 0.2 g MgS0 4 '7H 2 0, 0.01 g yeast extract in one litre distilled water, where the glucose, MgS0 4 « 7H 2 0, yeast extract and salts are autoclaved separately.
  • 100 ml cultures of Zoogloea ramigera are grown on a rotary shaker or a stirred bioreactor (200 rpm) at 30 °C for period up to two weeks.
  • the two new strains produce a novel exopolysaccharide (EPS) and have several desirable characteristics that are absent from the parent strain, including improved culture properties, since they do not produce an EPS capsule layer like that of the parent 1 15 strain.
  • the 1 15SL EPS is instead excreted as a slime layer which is not confined to the immediate area surrounding the cells. Since cells are not trapped within a floe where they grow at a reduced rate or die because of nutrient starvation, the new strains have more consistent and reproducible growth cycles and increased growth rates. As a consequence, exopolysaccharide production is more consistent and titers are higher. The separation of the EPS from the cells is also much easier and more economical.
  • the Zoogloea ramigera strains have to be bred in a high salinity medium, which is cost consuming.
  • the Zoogloea ramigera strains without capsule layer may decrease the metal-binding capacity during water treatment due to fewer electric charges appear at the surrounding of the uncapsulated cell.
  • biopolymer substance may help in reducing water pollution and in turn conserving the valuable biodiversity in aquaculture.
  • the present invention relates to a method for producing a biopolymer, comprising the steps of providing a culture medium comprising a carbon source, a nitrogen source, and a plurality of mineral sources; inoculating the culture medium with a Aspergillus flavus with an inoculum size in a range of 0.2 to 10 % by volume; incubating the culture medium; filtering the culture medium, thereby separating the Aspergillus flavus and the biopolymer; purifying the biopolymer; wherein the carbon source to nitrogen source is in a molar ratio of 5:1 , pH of the culture medium is 5 to 9, the temperature is 25 to 40 °C, inoculum size is 0.2 to 2 % by volume, shaking speed is 50 to 200 rpm, and culture time is 12 to 60 hours.
  • Figure 1 a shows a flocculating efficiency of bioflocculant in a kaolin suspension after 72 hours cultivation with a plurality of carbon sources (sucrose, glucose, fructose, lactose, starch, and glycerol);
  • Figure 1 b shows the flocculating efficiency of bioflocculant in the kaolin suspension after 72 hours cultivation with a plurality of nitrogen sources (peptone, yeast extract, glutamic acid, urea, ammonium sulphate, ammonium nitrate, and sodium nitrate);
  • nitrogen sources peptone, yeast extract, glutamic acid, urea, ammonium sulphate, ammonium nitrate, and sodium nitrate
  • Figure 1 c shows an effect of carbon source to nitrogen source molar ratio on the production of bioflocculant
  • Figure 2 shows the effect of pH in a culture medium on the production of bioflocculant
  • Figure 3 shows the effect of temperature in the culture medium on the production of bioflocculant
  • Figure 4 shows the effect of inoculum size of Aspergillus flavus in the culture medium on the production of bioflocculant
  • Figure 5 shows the effect of shaking speed of the culture medium on the production of bioflocculant
  • Figure 6 shows the effect of mineral sources present in the culture medium on the production of bioflocculant
  • Figure 7 shows a variation of bioflocculant production over a growth curve of Aspergillus flavus
  • Figure 8 shows the pH and thermal stability of the bioflocculant
  • Figure 9a shows the surface of bioflocculant
  • Figure 9b shows the surface of kaolin suspension
  • Figure 9c shows the formation of floes by bioflocculant.
  • the present invention relates to a method for producing a biopolymer, comprising the steps of:
  • a culture medium comprising a carbon source, a nitrogen source, and a plurality of mineral sources
  • said fungus is Aspergillus flavus
  • the carbon source is selected from the group consisting of sucrose, glucose, fructose, lactose, starch, and glycerol;
  • the nitrogen source is selected from the group consisting of peptone, yeast extract, glutamic acid, urea, ammonium sulphate, ammonium nitrate, and sodium nitrate;
  • the plurality of mineral sources is selected from the group consisting of magnesium, potassium, iron, chloride, or any combination thereof;
  • the carbon source to nitrogen source is in a molar ratio of 5: 1 , pH of the culture medium is 5 to 9, the temperature is 25 to 40 °C, inoculum size is 0.2 to 2 % by volume, shaking speed is 50 to 200 rpm, and culture time is 12 to 60 hours.
  • the culture medium is incubated in a rotary shaker or a stirred bioreactor.
  • the carbon source is sucrose.
  • the nitrogen source is peptone.
  • the mineral source is potassium.
  • the culture medium is in pH 7.
  • the culture medium is in 40 °C.
  • the inoculums size is 2 % by volume.
  • the shaking speed is 200 rpm.
  • the culture time is 60 hours.
  • the present invention further relates to a biopolymer produced from the method having a molecular weight in a range of 2.466x10" to 2.68 x10 4 Dalton, a plurality of functional groups, a plurality of chemical elements, a sugar, a protein, a minimum flocculating efficiency of 90 % in a pH range of 3 to 7 and a temperature range of 10 to 100 °C.
  • the plurality of functional group is selected from a group consisting of hydroxyl groups, hydrocarbons, amide groups, carboxyl groups, amines, methoxyl group, and any combination thereof.
  • the plurality of chemical elements comprises carbon, hydrogen, oxygen, nitrogen, and sulphur.
  • a fungus, Aspergillus flavus strain 44-1 was isolated by Department of Biotechnology and preserved at the Microbial Culture Collection Unit (UNiCC), Laboratory of Industrial Biotechnology, Institute of Bioscience, University Putra Malaysia, Kuala Lumpur, Malaysia.
  • Aspergillus flavus stock culture was maintained at 4 °C on a agar slant media and subcultured every 30 to 40 days, wherein the agar slant media comprises 4 g/L potato extract, 20 g/L glucose, 15 g/L agar and the initial pH was 5.6 ⁇ 0.2.
  • a culture medium was provided with a carbon source, a nitrogen source, and a plurality of mineral sources, wherein the carbon source is selected from the group consisting of sucrose, glucose, fructose, lactose, starch, and glycerol; the nitrogen source is selected from the group consisting of peptone, yeast extract, glutamic acid, urea, ammonium sulphate, ammonium nitrate, and sodium nitrate; and the plurality of mineral sources is selected from the group consisting of magnesium, potassium, iron, chloride, or any combination thereof.
  • the carbon source is selected from the group consisting of sucrose, glucose, fructose, lactose, starch, and glycerol
  • the nitrogen source is selected from the group consisting of peptone, yeast extract, glutamic acid, urea, ammonium sulphate, ammonium nitrate, and sodium nitrate
  • the plurality of mineral sources is selected from the group consisting of magnesium, potassium, iron, chloride
  • the culture medium comprises 30 g/L sucrose as carbon source, 3.0 g/L peptone as nitrogen source, 0.5 g/L hydrated magnesium sulphate (MgS0 4 *7H20), 0.5 g/L potassium chloride (KCI), 0.01 g/L iron (II) sulphate (FeS0 4 ), 1 .0 g/L dipotassium sulphate (K 2 HPO 4 ), and the initial pH was adjusted to 6.0.
  • the potassium chloride can be supplied in a form of sodium chloride, calcium chloride, magnesium chloride, manganese (I I) chloride and iron (I II) chloride.
  • the molar ratio of carbon source to nitrogen source is tested with 0: 1 , 1 : 1 , 2: 1 , 3: 1 , 4: 1 , 5: 1 , 10: 1 , 20: 1 , 30: 1 , 40: 1 and 50: 1.
  • the culture medium was then inoculated with Aspergillus flavus to produce Aspergillus flavus inoculums with an inoculums size in a range of 0.2 to 10 % by volume.
  • Aspergillus flavus was cultured in the agar slant media.
  • the agar slant media which carry Aspergillus flavus was cut into several pieces and soaked in 100 mL of distilled water.
  • Aspergillus flavus were suspended in the distilled water and then incubated by rotary shaker or stirred bioreactor at 30 °C for 24 hours.
  • the suspended Aspergillus flavus were then filtered to obtain Aspergillus flavus inoculum.
  • the culture process was optimized by varying a plurality of culture parameters such as carbon sources, nitrogen sources, molar ratio of carbon sources to nitrogen sources, pH, temperature, shaking speed, and culture time to obtain a biopolymer such as bioflocculant, biocoagulant, and the like.
  • bioflocculants are extracellular or intracellular biopolymeric substances secreted by bacteria, fungi, algae and yeast.
  • the composition and properties of bioflocculant depend on bioflocculant-producing microorgamisms (BPMs), medium culture composition and environmental conditions.
  • the culture medium was incubated in a rotary shaker or a stirred bioreactor with a shaking speed at a range of 0 to 250 rpm and a culture time for 12 to 96 hours with temperature 15 to 45 °C.
  • the culture medium was then filtered, thereby separating the Aspergillus flavus was obtained in a form of residue, while the bioflocculant in a form of filtrate.
  • the biomass of Aspergillus flavus was dried at 80 °C in an oven for 4 hours.
  • a kaolin suspension was used to measure the flocculating efficiency of the filtrate containing bioflocculant.
  • a 2 gram amount of Kaolin clay (Merck, Germany) was suspended in 1 Litre of deionized water.
  • 1 mL of filtrate containing bioflocculant was added into 99 ml of kaolin suspension in a 400 ml beaker, and the pH value was adjusted to 7.0 using 1 M sodium hydroxide or hydrochloric acid.
  • the mixture was vigorously stirred at 200 rpm for 1 minute and slowly stirred at 80 rpm for 5 minutes, and then allowed to stand for 5 minutes using 6-breaker jar tester (JLT6, VELP SCIENTIFICA, Italy).
  • the optical density (OD) of the clarifying solution was measured with a spectrophotometer (GENESYS 10 UV, Thermo Scientific, USA) at 550 nm.
  • a control experiment was conducted in the same manner by replacing filtrate containing bioflocculant with fresh culture medium (as blank).
  • the flocculating activity was calculated according to the following equation:
  • Flocculating efficiency (A-B)/A X 100 % where A and B were OD550 (optical density at 550 nm) of the control and sample supernatant, respectively.
  • Figure 1 a shows the flocculating efficiency in the kaolin suspension after 72 hours cultivation with various carbon sources (glucose, fructose, lactose, starch and glycerol) replacing sucrose at the same concentration.
  • Sucrose, starch and glucose were carbon sources favourable for bioflocculant production, while the production of bioflocculant was relatively low when fructose and glycerol were used as carbon sources in the culture medium.
  • sucrose was chosen as the sole carbon source due to the highest bioflocculant production shown in figure 1 a.
  • the effect of nitrogen sources was investigated by cultivating the Aspergillus flavus in the same culture medium, except that the nitrogen source was changed as shown in figure 1 b.
  • organic nitrogen sources (peptone, yeast extract and urea) were effectively used to produce bioflocculant by Aspergillus flavus, while inorganic nitrogen sources ammonium sulphate, and sodium nitrate led to poor bioflocculant production except ammonium nitrate was influenced the production of bioflocculant on the starch medium.
  • Peptone was the most favourable nitrogen source for bioflocculant production as shown in figure 1 b.
  • the pH of the culture medium can affect the nutrient absorption and enzymatic reaction of bioflocculant-producing microorgamisms. Over the pH range of 2 to 9, the lowest flocculating efficiency was at the acidic pH range of 2 to 4, while pH in the range of 5 to 9 was favourable for the bioflocculant production. However, pH 7 was optimum for bioflocculant production as shown in figure 2.
  • Figure 3 shows the effect of temperature in the culture medium on the production of bioflocculant.
  • the flocculating efficiency was increased dramatically after 25 °C of culture temperature and reached up to 86.2% at 40°C.
  • Maximum enzymatic activation can only be obtained at an optimum temperature.
  • a lower culture temperature might make Aspergillus flavus hibernate partially, and its enzyme system for bioflocculant production could not be activated completely.
  • the optimal temperature for bioflocculant production was 40 °C as shown in figure 3.
  • Figure 4 shows the effect of inoculum size of Aspergillus flavus on bioflocculant production.
  • the inoculums size of Aspergillus flavus is gradually increased from 0.2 to 2 % by volume.
  • the flocculating efficiency of bioflocculant reached to 86.6%.
  • any further increase in inoculum size did not result in any higher flocculating efficiency.
  • 2% by volume of inoculum was found to be the optimal inoculum size for Aspergillus flavus, which could acclimatize Aspergillus flavus to the culture medium and promote the productivity of bioflocculant.
  • FIG 5 shows the production of bioflocculant was high in shaking speed between 50 to 200 rpm. Increasing the mixing speed to 250 rpm decreased the production of bioflocculant. The shaking speed determines the level of dissolved oxygen in the culture medium which can also affect nutrient absorption and enzymatic reaction of Aspergillus flavus. The shaking speed of 200 rpm was the optimum speed for the production of bioflocculant.
  • Figure 6 shows the effect of mineral sources present in the culture medium on the production of bioflocculant. The bioflocculant production of Aspergillus flavus was stimulated in the presence of sodium, potassium, calcium, magnesium and manganese, or any combination thereof but was inhibited by iron as shown in figure 6. Referring to figure 6, potassium was the most favourable mineral source and used in the production of bioflocculant.
  • FIG. 7 shows the variation of bioflocculant production over a growth curve of Aspergillus flavus. Overall, the production of bioflocculant paralleled the growth of biomass and increased with time. The flocculating efficiency of the bioflocculant reached its maximum (87.2%) in early stationary phase at 60 hours, which indicated that the bioflocculant was produced by biosynthesis during its growth. The flocculating efficiency began to decrease during the late stationary phase due to the activity of deflocculation enzymes. After 72 hours, as the death rate of the Aspergillus flavus began to exceed its birth rate, the strain entered the decline phase, and the flocculating efficiency of the bioflocculant decreased gradually.
  • the pH profile showed that the pH decreased from 7.0 to 5.3 within 48 hours, followed by a slight drop and increased to 5.6 at 96 hours. Therefore, 60 hours was the optimum culture time for the production of bioflocculant.
  • bioflocculant was mainly a polysaccharides bioflocculant
  • purified bioflocculant was hydrolyzed by trifluoroacetic acid to determine other saccharides such as neutral sugar, uronic acid and amino sugar.
  • the elemental analysis of purified bioflocculant revealed that the mass proportion of carbon (C), hydrogen (H), oxygen (O), nitrogen (N), and sulphur (S) was 29.9 %, 4.8 %, 34.7 %, 3.3 %, and 2.0 % by weight, correspondently.
  • GPC Gel Permeation Chromatography
  • Figure 8 shows the pH and thermal stability of the bioflocculant where more than 90 % of flocculating efficiency was achieved at range of pH 3.0 to 7.0. While pH of higher than 7.0 decreases the flocculating efficiency dramatically.
  • the floe formation may float to the top of the liquid, settle to the bottom of the liquid, or can be readily filtered or removed from the liquid.
  • the zeta potential indicates the degree of repulsion between adjacent, similarly charged particles (the vitamins) in a dispersion. Zeta potential with 0 mV indicates optimum flocculation performance in the process of liquid clarification.
  • Table 1 Zeta potential for bioflocculant, kaolin suspension and treated kaolin suspension

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Abstract

The present invention relates to a method for producing a biopolymer, comprising the steps of providing a culture medium comprising a carbon source, a nitrogen source, and a plurality of mineral sources; inoculating the culture medium with a Aspergillus flavus with an inoculum size in a range of 0.2 to 10 % by volume; incubating the culture medium; filtering the culture medium, thereby separating the Aspergillus flavus and the biopolymer; purifying the biopolymer; wherein the carbon source to nitrogen source is in a molar ratio of 5:1, p H of the culture medium is 5 to 9, the temperature is 25 to 40 °C, inoculum size is 0.2 to 2 % by volume, shaking speed is 50 to 200 rpm, and culture time is 12 to 60 hours. A biopolymer produced having a molecular weight in a range of 2.466x104 to 2.68x104 Dalton, a plurality of functional groups, a plurality of chemical elements, a sugar, a protein, a minimum flocculating efficiency of 90 % in a p H range of 3 to 7 and a temperature range of 10 to 100 °C.

Description

A METHOD FOR PRODUCING A BIOPOLYMER
Background of the Invention
Field of the Invention
This invention relates to a method for producing a biopolymer, and more particularly to a method for culturing Aspergillus flavus to produce biopolymer such as bioflocculant, biocoagulant , and the like.
Description of Related Arts
Water pollution occurs when a body of water is adversely affected due to the addition of large amounts of harmful substances such as microorganisms, colloids and toxic materials to the water. Improper disposal of hazardous chemicals down the drain will introduce toxic materials into the ecosystem, and in turn contaminating the water supplies in a way that can harm aquatic organisms. Coagulation and flocculation steps are most common method to remove colloids or suspended materials find in water and wastewater. As is well known, colloid is found to be negatively charged (approximately - 30 mV), and hence cationic synthetic polymers are generally employed to neutralise the colloid charge, which facilitates the floe settling. These polymers are expensive, and pollute environment and safety precautions must be followed during their handling. To overcome these existing problems an alternative and suitable way is using biocoagulants or bioflocculants produced by bacterial strains derived from activated sludge. U.S. Patent Application Publication Number 2002/0074295 A1 disclosed a process for treating contaminated liquid, in which the liquid to be treated is contacted with combination of two oppositely charged polymeric material, wherein at least one of said polymeric materials is branched, and wherein said polymeric materials are preferably derived from a variety of natural sources (for example, algae, bacterial, and just to name a few). Floe formation is allowed and the floes are separated from the liquid. The first and second polymeric materials may be selected from the group consisting of polysaccharides, proteins, lipids and polyhydroxy alcohols. However, the drawback of this cited patent includes two steps of water treatment which the contaminated liquid is first treated with the first polymeric material (anionic, branched polymeric material which derived from blue-green algae), and after an interval of time, contacted with the second polymeric material (cationic, unbranched polymer derived from chitosan) to eliminate contaminants from water. These two steps are making the process more cumbersome, time consuming and also require large dosage of polymeric materials to reduce the contaminant concentration in the water.
Microbial-produced bioflocculants have received increased scientific and technical attention because they are biodegradable and non-toxic and their degradation intermediates does not involve secondary pollutants. Several microorganisms are known to produce bioflocculants, including Aspergillus sojae, Paecilomyces sp., Rhodococcus erythropolis, Serratia ficaria, and Bacillus mucilaginosus. For example, U.S. Patent Number 5,250,201 disclosed a method of separating cyanobacteria, particularly strain J-1 from a liquid, and a method for culturing cyanobacteria to produce a polymer useful as an emulsifying agent for forming emulsion of hydrocarbons and oils in water. The cited patent further relates to the polymeric substance produced by purification and separation techniques, and to methods of affecting the secondary recovery of petroleum through the use of a material excreted by cyanobacteria. The emulsification activity of the emulcyan is temperature dependent and pH dependent, which shown to be at 100 % of maximal value at a temperature of approximately 26 °C and pH in a range of 5 to 9. However, the drawback of this cited patent is that the emulsifying activity of the emulcyan has maintained their optimum activity at limited temperature range, as well as at a limited pH range.
In other example, U.S. Patent Number 4,948,733 disclosed two new bacterial strains designated Zoogloea ramigera 1 15SL and Zoogloea ramigera 1 15SLR which derived from the wild type Zoogloea ramigera 1 15. Zoogloea ramigera cultures are stored frozen at -70 °C in trypticase soy broth (TSB) medium containing 7 % DMSO and 15 % glycerol. The Zoogloea ramigera strains are routinely cultured in either the TSB medium or a defined medium comprising 25 g glucose, 2 g K2HP04, 1 g KH2P04, 1 g NH4CI, 0.2 g MgS04'7H20, 0.01 g yeast extract in one litre distilled water, where the glucose, MgS04 «7H20, yeast extract and salts are autoclaved separately. 100 ml cultures of Zoogloea ramigera are grown on a rotary shaker or a stirred bioreactor (200 rpm) at 30 °C for period up to two weeks. The two new strains produce a novel exopolysaccharide (EPS) and have several desirable characteristics that are absent from the parent strain, including improved culture properties, since they do not produce an EPS capsule layer like that of the parent 1 15 strain. The 1 15SL EPS is instead excreted as a slime layer which is not confined to the immediate area surrounding the cells. Since cells are not trapped within a floe where they grow at a reduced rate or die because of nutrient starvation, the new strains have more consistent and reproducible growth cycles and increased growth rates. As a consequence, exopolysaccharide production is more consistent and titers are higher. The separation of the EPS from the cells is also much easier and more economical. However, the Zoogloea ramigera strains have to be bred in a high salinity medium, which is cost consuming. In addition, the Zoogloea ramigera strains without capsule layer may decrease the metal-binding capacity during water treatment due to fewer electric charges appear at the surrounding of the uncapsulated cell.
Accordingly, there is a need of present invention for a simple, convenient and cost effective method for culturing microorganism strain in suitable culture medium and environmental conditions to produce an optimal amount of biopolymer substance. In addition, this biopolymer substance may help in reducing water pollution and in turn conserving the valuable biodiversity in aquaculture.
Summary of Invention
It is an objective of the present invention to provide a method for producing biopolymer without the use of synthetic polymer so as to considerably reduce the production cost. It is also an objective of the present invention to provide a method for culturing a fungus to produce biopolymer for liquid clarification and water treatment.
It is yet another objective of the present invention to provide a method for culturing a fungus to produce optimal amount of biopolymer.
Accordingly, these objectives may be achieved by following the teachings of the present invention. The present invention relates to a method for producing a biopolymer, comprising the steps of providing a culture medium comprising a carbon source, a nitrogen source, and a plurality of mineral sources; inoculating the culture medium with a Aspergillus flavus with an inoculum size in a range of 0.2 to 10 % by volume; incubating the culture medium; filtering the culture medium, thereby separating the Aspergillus flavus and the biopolymer; purifying the biopolymer; wherein the carbon source to nitrogen source is in a molar ratio of 5:1 , pH of the culture medium is 5 to 9, the temperature is 25 to 40 °C, inoculum size is 0.2 to 2 % by volume, shaking speed is 50 to 200 rpm, and culture time is 12 to 60 hours. A biopolymer produced having a molecular weight in a range of 2.466x104 to 2.68 x104 Dalton, a plurality of functional groups, a plurality of chemical elements, a sugar, a protein, a minimum flocculating efficiency of 90 % in a pH range of 3 to 7 and a temperature range of 10 to 100 °C.
Brief Description of the Drawings
The features of the invention will be more readily understood and appreciated from the following detailed description when read in conjunction with the accompanying drawings of the preferred embodiment of the present invention, in which:
Figure 1 a shows a flocculating efficiency of bioflocculant in a kaolin suspension after 72 hours cultivation with a plurality of carbon sources (sucrose, glucose, fructose, lactose, starch, and glycerol);
Figure 1 b shows the flocculating efficiency of bioflocculant in the kaolin suspension after 72 hours cultivation with a plurality of nitrogen sources (peptone, yeast extract, glutamic acid, urea, ammonium sulphate, ammonium nitrate, and sodium nitrate);
Figure 1 c shows an effect of carbon source to nitrogen source molar ratio on the production of bioflocculant;
Figure 2 shows the effect of pH in a culture medium on the production of bioflocculant; Figure 3 shows the effect of temperature in the culture medium on the production of bioflocculant;
Figure 4 shows the effect of inoculum size of Aspergillus flavus in the culture medium on the production of bioflocculant;
Figure 5 shows the effect of shaking speed of the culture medium on the production of bioflocculant;
Figure 6 shows the effect of mineral sources present in the culture medium on the production of bioflocculant;
Figure 7 shows a variation of bioflocculant production over a growth curve of Aspergillus flavus; Figure 8 shows the pH and thermal stability of the bioflocculant;
Figure 9a shows the surface of bioflocculant;
Figure 9b shows the surface of kaolin suspension; and
Figure 9c shows the formation of floes by bioflocculant. Detailed Description of the Invention
As required, detailed embodiments of the present invention are disclosed herein; however, it is to be understood that the disclosed embodiments are merely exemplary of the invention, which may be embodied in various forms. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting but merely as a basis for claims. It should be understood that the drawings and detailed description thereto are not intended to limit the invention to the particular form disclosed, but on the contrary, the invention is to cover all modification, equivalents and alternatives falling within the scope of the present invention as defined by the appended claims. As used throughout this application, the word "may" is used in a permissive sense (i.e., meaning having the potential to), rather than the mandatory sense (i.e., meaning must). Similarly, the words "include," "including," and "includes" mean including, but not limited to. Further, the words "a" or "an" mean "at least one" and the word "plurality" means one or more, unless otherwise mentioned. Where the abbreviations of technical terms are used, these indicate the commonly accepted meanings as known in the technical field. For ease of reference, common reference numerals will be used throughout the figures when referring to the same or similar features common to the figures. The present invention will now be described with reference to Figs. 1 a-9c.
The present invention relates to a method for producing a biopolymer, comprising the steps of:
providing a culture medium comprising a carbon source, a nitrogen source, and a plurality of mineral sources;
inoculating the culture medium with a fungus with an inoculum size in a range of 0.2 to 10 % by volume, wherein the fungus is cultured in a slant media; incubating the culture medium with a shaking speed at a range of 0 to 250 rpm and a culture time for 12 to 96 hours with temperature 15 to 45 °C;
filtering the culture medium, thereby separating the fungus and the biopolymer;
purifying the biopolymer;
characterized in that: said fungus is Aspergillus flavus;
the carbon source is selected from the group consisting of sucrose, glucose, fructose, lactose, starch, and glycerol;
the nitrogen source is selected from the group consisting of peptone, yeast extract, glutamic acid, urea, ammonium sulphate, ammonium nitrate, and sodium nitrate;
the plurality of mineral sources is selected from the group consisting of magnesium, potassium, iron, chloride, or any combination thereof;
the carbon source to nitrogen source is in a molar ratio of 5: 1 , pH of the culture medium is 5 to 9, the temperature is 25 to 40 °C, inoculum size is 0.2 to 2 % by volume, shaking speed is 50 to 200 rpm, and culture time is 12 to 60 hours.
In a preferred embodiment of the method for producing a biopolymer, the culture medium is incubated in a rotary shaker or a stirred bioreactor.
In a preferred embodiment of the method for producing a biopolymer, the carbon source is sucrose.
In a preferred embodiment of the method for producing a biopolymer, the nitrogen source is peptone.
In a preferred embodiment of the method for producing a biopolymer, the mineral source is potassium. In a preferred embodiment of the method for producing a biopolymer, the culture medium is in pH 7.
In a preferred embodiment of the method for producing a biopolymer, the culture medium is in 40 °C.
In a preferred embodiment of the method for producing a biopolymer, wherein the inoculums size is 2 % by volume. In a preferred embodiment of the method for producing a biopolymer, the shaking speed is 200 rpm.
In a preferred embodiment of the method for producing a biopolymer, the culture time is 60 hours.
The present invention further relates to a biopolymer produced from the method having a molecular weight in a range of 2.466x10" to 2.68 x104 Dalton, a plurality of functional groups, a plurality of chemical elements, a sugar, a protein, a minimum flocculating efficiency of 90 % in a pH range of 3 to 7 and a temperature range of 10 to 100 °C.
In a preferred embodiment of the biopolymer produced, the plurality of functional group is selected from a group consisting of hydroxyl groups, hydrocarbons, amide groups, carboxyl groups, amines, methoxyl group, and any combination thereof.
In a preferred embodiment of the biopolymer produced, the plurality of chemical elements comprises carbon, hydrogen, oxygen, nitrogen, and sulphur.
Below is an example of a method for producing biopolymer from which the advantages of the present invention may be more readily understood. It is to be understood that the following example is for illustrative purpose only and should not be construed to limit the present invention in any way.
Examples
A fungus, Aspergillus flavus strain 44-1 was isolated by Department of Biotechnology and preserved at the Microbial Culture Collection Unit (UNiCC), Laboratory of Industrial Biotechnology, Institute of Bioscience, University Putra Malaysia, Kuala Lumpur, Malaysia. Aspergillus flavus stock culture was maintained at 4 °C on a agar slant media and subcultured every 30 to 40 days, wherein the agar slant media comprises 4 g/L potato extract, 20 g/L glucose, 15 g/L agar and the initial pH was 5.6±0.2.
A culture medium was provided with a carbon source, a nitrogen source, and a plurality of mineral sources, wherein the carbon source is selected from the group consisting of sucrose, glucose, fructose, lactose, starch, and glycerol; the nitrogen source is selected from the group consisting of peptone, yeast extract, glutamic acid, urea, ammonium sulphate, ammonium nitrate, and sodium nitrate; and the plurality of mineral sources is selected from the group consisting of magnesium, potassium, iron, chloride, or any combination thereof. In a preferred embodiment, the culture medium comprises 30 g/L sucrose as carbon source, 3.0 g/L peptone as nitrogen source, 0.5 g/L hydrated magnesium sulphate (MgS04*7H20), 0.5 g/L potassium chloride (KCI), 0.01 g/L iron (II) sulphate (FeS04), 1 .0 g/L dipotassium sulphate (K2HPO4), and the initial pH was adjusted to 6.0. In other preferred embodiment, the potassium chloride can be supplied in a form of sodium chloride, calcium chloride, magnesium chloride, manganese (I I) chloride and iron (I II) chloride. In a preferred embodiment, the molar ratio of carbon source to nitrogen source is tested with 0: 1 , 1 : 1 , 2: 1 , 3: 1 , 4: 1 , 5: 1 , 10: 1 , 20: 1 , 30: 1 , 40: 1 and 50: 1.
The culture medium was then inoculated with Aspergillus flavus to produce Aspergillus flavus inoculums with an inoculums size in a range of 0.2 to 10 % by volume. In a preferred embodiment, Aspergillus flavus was cultured in the agar slant media. The agar slant media which carry Aspergillus flavus was cut into several pieces and soaked in 100 mL of distilled water. Aspergillus flavus were suspended in the distilled water and then incubated by rotary shaker or stirred bioreactor at 30 °C for 24 hours. The suspended Aspergillus flavus were then filtered to obtain Aspergillus flavus inoculum.
The culture process was optimized by varying a plurality of culture parameters such as carbon sources, nitrogen sources, molar ratio of carbon sources to nitrogen sources, pH, temperature, shaking speed, and culture time to obtain a biopolymer such as bioflocculant, biocoagulant, and the like. In a preferred embodiment, bioflocculants are extracellular or intracellular biopolymeric substances secreted by bacteria, fungi, algae and yeast. The composition and properties of bioflocculant depend on bioflocculant-producing microorgamisms (BPMs), medium culture composition and environmental conditions. In other preferred embodiment, the culture medium was incubated in a rotary shaker or a stirred bioreactor with a shaking speed at a range of 0 to 250 rpm and a culture time for 12 to 96 hours with temperature 15 to 45 °C. The culture medium was then filtered, thereby separating the Aspergillus flavus was obtained in a form of residue, while the bioflocculant in a form of filtrate. The biomass of Aspergillus flavus was dried at 80 °C in an oven for 4 hours.
Bioflocculant purification
2 Litre of 95 % cold ethanol (at 4 °C) were added to 1 Litre filtrate containing bioflocculant in order to obtain the precipitate. The precipitate obtained was redissolved in 100 mL deionized water and 50 mL of 2 % cetylpyridinium chloride solution (CPC) was added to the solution with stirring. After 3 hours, the precipitate was collected and dissolved in a 100 mL of 0.5 M sodium chloride solution. 2 Litre of 95 % cold ethanol were then added to obtain the precipitate. The precipitate was washed with ethanol and dissolved in 5 mL of deionized water and vacuum-dried. Approximately 0.402 g of pure bioflocculant was obtained in 1 L filtrate containing bioflocculant.
Determination of flocculating efficiency
A kaolin suspension was used to measure the flocculating efficiency of the filtrate containing bioflocculant. A 2 gram amount of Kaolin clay (Merck, Germany) was suspended in 1 Litre of deionized water. 1 mL of filtrate containing bioflocculant was added into 99 ml of kaolin suspension in a 400 ml beaker, and the pH value was adjusted to 7.0 using 1 M sodium hydroxide or hydrochloric acid. The mixture was vigorously stirred at 200 rpm for 1 minute and slowly stirred at 80 rpm for 5 minutes, and then allowed to stand for 5 minutes using 6-breaker jar tester (JLT6, VELP SCIENTIFICA, Italy). The optical density (OD) of the clarifying solution (supernatant obtained) was measured with a spectrophotometer (GENESYS 10 UV, Thermo Scientific, USA) at 550 nm. A control experiment was conducted in the same manner by replacing filtrate containing bioflocculant with fresh culture medium (as blank). The flocculating activity was calculated according to the following equation:
Flocculating efficiency = (A-B)/A X 100 % where A and B were OD550 (optical density at 550 nm) of the control and sample supernatant, respectively.
Figure 1 a shows the flocculating efficiency in the kaolin suspension after 72 hours cultivation with various carbon sources (glucose, fructose, lactose, starch and glycerol) replacing sucrose at the same concentration. Sucrose, starch and glucose were carbon sources favourable for bioflocculant production, while the production of bioflocculant was relatively low when fructose and glycerol were used as carbon sources in the culture medium. However, sucrose was chosen as the sole carbon source due to the highest bioflocculant production shown in figure 1 a. The effect of nitrogen sources was investigated by cultivating the Aspergillus flavus in the same culture medium, except that the nitrogen source was changed as shown in figure 1 b. Overall, organic nitrogen sources (peptone, yeast extract and urea) were effectively used to produce bioflocculant by Aspergillus flavus, while inorganic nitrogen sources ammonium sulphate, and sodium nitrate led to poor bioflocculant production except ammonium nitrate was influenced the production of bioflocculant on the starch medium. Peptone was the most favourable nitrogen source for bioflocculant production as shown in figure 1 b.
A dramatic increase on bioflocculant production was observed when the molar ratio of carbon source to nitrogen source (C:N) increased up to 5: 1 as shown in figure 1 c. Further increase on C:N molar ratio did not contribute to, and even led to a decrease on bioflocculant production. This indicates that the C: N in molar ratio of 5: 1 is favourable for bioflocculant production.
The pH of the culture medium can affect the nutrient absorption and enzymatic reaction of bioflocculant-producing microorgamisms. Over the pH range of 2 to 9, the lowest flocculating efficiency was at the acidic pH range of 2 to 4, while pH in the range of 5 to 9 was favourable for the bioflocculant production. However, pH 7 was optimum for bioflocculant production as shown in figure 2.
Figure 3 shows the effect of temperature in the culture medium on the production of bioflocculant. The flocculating efficiency was increased dramatically after 25 °C of culture temperature and reached up to 86.2% at 40°C. Maximum enzymatic activation can only be obtained at an optimum temperature. A lower culture temperature might make Aspergillus flavus hibernate partially, and its enzyme system for bioflocculant production could not be activated completely. Thus, the optimal temperature for bioflocculant production was 40 °C as shown in figure 3.
Figure 4 shows the effect of inoculum size of Aspergillus flavus on bioflocculant production. The inoculums size of Aspergillus flavus is gradually increased from 0.2 to 2 % by volume. When the inoculum size of the strain was 2% by volume, the flocculating efficiency of bioflocculant reached to 86.6%. However, any further increase in inoculum size did not result in any higher flocculating efficiency. 2% by volume of inoculum was found to be the optimal inoculum size for Aspergillus flavus, which could acclimatize Aspergillus flavus to the culture medium and promote the productivity of bioflocculant.
Figure 5 shows the production of bioflocculant was high in shaking speed between 50 to 200 rpm. Increasing the mixing speed to 250 rpm decreased the production of bioflocculant. The shaking speed determines the level of dissolved oxygen in the culture medium which can also affect nutrient absorption and enzymatic reaction of Aspergillus flavus. The shaking speed of 200 rpm was the optimum speed for the production of bioflocculant. Figure 6 shows the effect of mineral sources present in the culture medium on the production of bioflocculant. The bioflocculant production of Aspergillus flavus was stimulated in the presence of sodium, potassium, calcium, magnesium and manganese, or any combination thereof but was inhibited by iron as shown in figure 6. Referring to figure 6, potassium was the most favourable mineral source and used in the production of bioflocculant.
Figure 7 shows the variation of bioflocculant production over a growth curve of Aspergillus flavus. Overall, the production of bioflocculant paralleled the growth of biomass and increased with time. The flocculating efficiency of the bioflocculant reached its maximum (87.2%) in early stationary phase at 60 hours, which indicated that the bioflocculant was produced by biosynthesis during its growth. The flocculating efficiency began to decrease during the late stationary phase due to the activity of deflocculation enzymes. After 72 hours, as the death rate of the Aspergillus flavus began to exceed its birth rate, the strain entered the decline phase, and the flocculating efficiency of the bioflocculant decreased gradually. Corresponding to the profile of flocculating efficiency, the pH profile showed that the pH decreased from 7.0 to 5.3 within 48 hours, followed by a slight drop and increased to 5.6 at 96 hours. Therefore, 60 hours was the optimum culture time for the production of bioflocculant.
Characterization of the bioflocculant
Composition analysis of bioflocculant
Chemical analysis of the purified bioflocculant showed that the proportions of total sugar and total protein were 28.3% and 5.7%, respectively. Since this result showed bioflocculant was mainly a polysaccharides bioflocculant, purified bioflocculant was hydrolyzed by trifluoroacetic acid to determine other saccharides such as neutral sugar, uronic acid and amino sugar. The elemental analysis of purified bioflocculant revealed that the mass proportion of carbon (C), hydrogen (H), oxygen (O), nitrogen (N), and sulphur (S) was 29.9 %, 4.8 %, 34.7 %, 3.3 %, and 2.0 % by weight, correspondently. By Gel Permeation Chromatography (GPC) system, the molecular weight of bioflocculant was determined to be 2.466x104 to 2.68 x104 Dalton.
Functional group analysis of bioflocculant
The infrared spectrum of purified bioflocculant displayed a broad stretching peak at approximately 3285 cm"1 characteristic of hydroxyl groups, and a weak C-H stretching band at 2927 cm"1. Both peaks at 1333 and 1633 cm"1 are the consequences of COO" bond asymmetric oscillation. The peak at 1538 cm"1 was attributed to the NH bending vibration and the strong absorption peak presented at 1007 cm"1 indicate the C-0 stretching vibration and the presence of methoxyl groups, which were generally known to be typical characteristics of all sugar derivatives. pH stability of bioflocculant
Figure 8 shows the pH and thermal stability of the bioflocculant where more than 90 % of flocculating efficiency was achieved at range of pH 3.0 to 7.0. While pH of higher than 7.0 decreases the flocculating efficiency dramatically.
Thermal stability of bioflocculant
An investigation of the thermal stability of the bioflocculant showed that the bioflocculant was extremely stable (Figure 8). Over 90 % of flocculating efficiency was maintained at a wide range of temperatures (10 to 100 °C), if the pH of the bioflocculant is in the range of 3.0 to 7.0, because the main component of this bioflocculant was a polysaccharide. Bioflocculant mechanism for clarifying liquid
Impurity either colloidal or particles in water body are negatively charged, thus using cationic bioflocculant will be most effective for flocculation. Electrostatic interaction between the surface of the bioflocculant (figure 9a) and colloidal will produce attraction and charge neutralization of the colloidal surface, leading to formation of floes (figure 9c) and decrease electrical repulsion between them. Table 1 shows the zeta potential for bioflocculant and kaolin suspension (synthetic turbid water), where treated kaolin suspension with zeta potential of -9.3 mV tends to flocculate in order to have a clearer liquid. The surface of the kaolin suspension can be seen in figure 9b. In a preferred embodiment, the floe formation may float to the top of the liquid, settle to the bottom of the liquid, or can be readily filtered or removed from the liquid. In other preferred embodiment, the zeta potential indicates the degree of repulsion between adjacent, similarly charged particles (the vitamins) in a dispersion. Zeta potential with 0 mV indicates optimum flocculation performance in the process of liquid clarification.
Table 1 : Zeta potential for bioflocculant, kaolin suspension and treated kaolin suspension
Figure imgf000016_0001
Although the present invention has been described with reference to specific embodiments, also shown in the appended figures, it will be apparent for those skilled in the art that many variations and modifications can be done within the scope of the invention as described in the specification and defined in the following claims.

Claims

Claims I/We claim:
1 . A method for producing a biopolymer, comprising the steps of:
providing a culture medium comprising a carbon source, a nitrogen source, and a plurality of mineral sources;
inoculating the culture medium with a fungus with an inoculum size in a range of 0.2 to 10 % by volume;
incubating the culture medium with a shaking speed at a range of 0 to 250 rpm and a culture time for 12 to 96 hours with temperature 15 to 45 °C;
filtering the culture medium, thereby separating the fungus and the biopolymer;
purifying the biopolymer;
characterized in that:
said fungus is Aspergillus flavus;
the carbon source is selected from the group consisting of sucrose, glucose, fructose, lactose, starch, and glycerol;
the nitrogen source is selected from the group consisting of peptone, yeast extract, glutamic acid, urea, ammonium sulphate, ammonium nitrate, and sodium nitrate;
the plurality of mineral sources is selected from the group consisting of magnesium, potassium, iron, chloride, or any combination thereof;
the carbon source to nitrogen source is in a molar ratio of 5: 1 , pH of the culture medium is 5 to 9, the temperature is 25 to 40 °C, inoculum size is 0.2 to 2 % by volume, shaking speed is 50 to 200 rpm, and culture time is 12 to 60 hours.
2. A method for producing a biopolymer according to claim 1 , wherein the culture medium is incubated in a rotary shaker or a stirred bioreactor.
3. A method for producing a biopolymer according to claim 1 , wherein the carbon source is sucrose.
4. A method for producing a biopolymer according to claim 1 , wherein the nitrogen source is peptone.
5. A method for producing a biopolymer according to claim 1 , wherein the culture medium is in pH 7.
6. A method for producing a biopolymer according to claim 1 , wherein the culture medium is in 40 °C.
7. A method for producing a biopolymer according to claim 1 , wherein the inoculums size is 2 % by volume.
8. A method for producing a biopolymer according to claim 1 , wherein the shaking speed is 200 rpm.
9. A method for producing a biopolymer according to claim 1 , wherein the culture time is 60 hours.
10. A biopolymer produced according to the method of claim 1 , wherein said biopolymer having a molecular weight in a range of 2.466x104 to 2.68 x104 Dalton, a plurality of functional groups, a plurality of chemical elements, a sugar, a protein, a minimum flocculating efficiency of 90 % in a pH range of 3 to 7 and a temperature range of 10 to 100 °C.
1 1. A biopolymer produced according to claim 1 1 , wherein the plurality of functional group is selected from a group consisting of hydroxyl groups, hydrocarbons, amide groups, carboxyl groups, amines, methoxyl group, and any combination thereof.
12. A biopolymer produced according to claim 1 1 , wherein the plurality of chemical elements comprises carbon, hydrogen, oxygen, nitrogen, and sulphur.
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