Biopolymer extraction

Abstract

In a prior art reactor set up dense aggregates of microorganisms are formed, typically in or embedded in an extracellular matrix. Such may relate to granules, to sphere like entities having a higher viscosity than water, globules, a biofilm, etc. The dense aggregates comprise extracellular polymeric substances, or biopolymers, in particular linear polysaccharides. The present invention is in the field of extraction of a biopolymer from a granular sludge, a biopolymer obtained by such method, and a use of such method.

Claims

1. A method for extracting biopolymers from dense aggregates formed by microbial organisms, comprising the steps of: (i) producing dense aggregates comprising anionic biopolymers by bacteria, wherein the anionic biopolymer dense aggregate produced is a bacterial aerobic sludge or an anammox sludge of microbial cells self-immobilized through extracellular polymeric substances formed by microbial unicellular bacterial organisms, and providing the anionic biopolymer in dense aggregate aerobic sludge or anammox sludge, wherein the aerobic sludge or the anammox sludge comprises the extracellular polymeric substances, (iic) increasing the pH of the aerobic sludge or anammox sludge to 8.0-14.0 under addition to the aerobic sludge or anammox sludge of 1-20% v/v of at least one of Cl.sub.2, OCL.sup.− and H.sub.2O.sub.2, (a) wherein the temperature is increased to 50-100° C., during a period of 10-60 min, or (b) wherein the temperature is maintained at 10-30° C., during a period of 60-2880 min, and discoloring the anionic biopolymer, and (iii) extracting the discoloured anionic biopolymer, the discoloured anionic biopolymer comprising exopolysaccharides.

2. The method according to claim 1, further comprising the step of (iia) after providing the aerobic sludge or anammox sludge removing dense aggregate particles with a diameter larger than 500 μm.

3. The method according to claim 1, further comprising the step of (iib) after providing the aerobic sludge or anammox sludge in step (i) removing water to a 1-40% w/v content of the wet aerobic sludge or anammox sludge.

4. The method according to claim 1, further comprising the step of (iiia) reducing a pH by addition of an acid to produce an acidic gel.

5. The method according to claim 4, further comprising (iiib) reducing the pH and thereafter removing the aerobic sludge or anammox sludge by at least one of physical separation, settling, centrifugation, cyclonic separation, decantation, filtration, sieving, and flotation, under suitable conditions.

6. The method according to claim 1, wherein the anionic biopolymer dense aggregate is selected from exopolysaccharide, block-copolymers comprising uronic acid residues, lipids, and combinations thereof.

7. The method according to claim 6, wherein the aerobic sludge or anammox sludge has been substantially produced by bacteria belonging to the order Pseudomonadaceae; or, by bacteria belonging to the order Plancto-mycetales; or combinations thereof.

8. The method according to claim 1, wherein step (iic) further comprises addition of at least one of a salt comprising a base, and an oxidant.

9. The method according to claim 1, wherein after step (iic) a suspension of the aerobic sludge or anammox sludge is centrifuged, during 10-45 minutes, and a supernatant is collected for further processing.

10. The method according to claim 4, wherein after step (iiia) the acidic gel is centrifuged, during 10-45 minutes, and a supernatant is collected for further processing.

11. The method according to claim 1, wherein the extracted discolored anionic biopolymer is further treated.

12. The method according to claim 1, wherein the extracted discolored anionic biopolymer has a number averaged weight of >10,000 Dalton.

13. The method according to claim 1, wherein the extracted discolored anionic biopolymer has >30% with a molecular weight of >300,000 Da, >10% with a molecular weight of >100,000 Da, >15% with a molecular weight of >5,000 Da, and <10% with a molecular weight of <5,000 Da.

Description

SUMMARY OF FIGURES

(1) FIG. 1a-1c shows the effect of shear rate on viscosity of ALE and commercial alginate.

DETAILED DESCRIPTION OF FIGURES

(2) The figures are further detailed in the description of the experiments below.

Examples/Experiments

(3) The invention although described in detailed explanatory context may be best understood in conjunction with the accompanying examples and figures.

(4) Below exemplary embodiments of a method of extraction of a specific biopolymer (microbial alginate; ALE) is given. Note that various steps are optional.

(5) Hot Extraction of ALE from Granular Sludge

(6) 1) Sieve the granular sludge to collect granules diameter more than 200 μm, then wash with tap water.

(7) 2) Remove the excess water using tissue paper placed under the sieve.

(8) 3) Before starting the extraction, take ±1 gram of sludge for dry weight determination. Measure the empty cup, the filled cup and put in the oven (105° C.) to dry. Weigh again if dried.

(9) 4) Prepare granules suspension in tap water with a Total Solids TSS content of about 3%. This corresponds with 30-35 gram wet weight sludge in a total volume of 100 mL.

(10) Before adding the water, preheat it to save time in step 5.

(11) 5) Add thick bleach solution to reach 10% (v/v).

(12) 6) Mix thoroughly and place the suspension in a water bath on a hotplate stirrer set to 80° C. and 400 rpm, for 30 min. Add aluminium foil to the top to prevent evaporation.

(13) 7) Collect the suspension in 50 mL tubes and centrifuge the liquor at 4500 rpm for 20 min. Collect the supernatant in a glass beaker and discard the pellet.

(14) 8) Adjust the supernatant pH to 2.5 by adding 4 M HCl on a magnetic stirrer. Collect the acidic gel in 50 mL tubes.

(15) 9) Centrifuge the acidic gel at 4500 rpm for 10 min, and then collect the pellet.

(16) 10) The acidic gel can be stored at 4° C. or frozen.

(17) The gel can be further prepared according to purpose/client for example in the following ways:

(18) (a) To prepare Na-ALE.

(19) (b) To precipitate the Na-ALE, and

(20) (c) To prepare salt-free/low salt Na-ALE by desalination.

(21) (a) To prepare Na-ALE:

(22) Add 0.5 M NaOH to the acidic gel to reach the required concentration.

(23) (b) To precipitate the Na-ALE:

(24) Add ethanol 1:1 volume. Discard the supernatant and let the Na-ALE to dry in the oven at 85° C.

(25) (c) To desalinate:

(26) Put Na-ALE in SnakeSkin® dialysis tubing (3,500 MWCO*) with a volume capacity of (3.7 ml/cm); close the tubing ends by knotting or with tubing clips after edges folded over twice and leave overnight in a glass beaker with milliQ water while stirring.

(27) Cold Extraction of ALE from Granular Sludge

(28) 1) Sieve the granular sludge to collect granules diameter more than 200 μm, then wash with tap water.

(29) 2) Remove the excess water using tissue paper placed under the sieve.

(30) 3) Before starting the extraction, take ±1 gram of sludge for dry weight determination. Measure the empty cup, the filled cup and put in the oven (105° C.) to dry. Weigh again if dried.

(31) 4) Prepare granules suspension in tap water with TSS of about 3%. This corresponds with 30-35 gram wet weight sludge in a total volume of 100 mL.

(32) 5) Add thick bleach solution to reach 10% (v/v).

(33) 6) Mix thoroughly and place the suspension at room temp or 4 c for 24 hours. Add aluminium foil to the top to prevent evaporation.

(34) 7) Collect the suspension in 50 mL tubes and centrifuge the liquor at 4500 rpm for 20 min. Collect the supernatant in a glass beaker and discard the pellet.

(35) 8) Adjust the supernatant pH to 2.5 by adding 4 M HCl on a magnetic stirrer. Gel formation can be noticed with foam. If there is no foam, keep adding HCl.

(36) 9) Collect the acidic gel in 50 mL tubes and discard the foam from the top (containing some insoluble solid impurities).

(37) 10) Centrifuge the acidic gel at 4500 rpm for 10 min, and then collect the pellet.

(38) 11) The acidic gel can be stored at 4° C. or frozen.

(39) The gel can be further prepared according to purpose/client as indicated above in the hot extraction section.

(40) ALE Molecular Weight Analysis

(41) Size exclusion chromatography was performed with a Superdex 75 10/300 GL column (AKTA Purifier System, GE Healthcare). Elution was carried out at room temperature using Phosphate Buffer Saline (PBS) containing 10 mM (HPO42-, H2PO4-) with a pH of 7.4, and further having 2.7 mM KCl and 137 mM NaCl, at a constant 0.4 mL/min flow rate. The detection was monitored by following the absorbance of the eluted molecules at a wavelength of 210 nm.

(42) The Superdex 75 10/300 GL column is capable of separating molecules of 1,000 to 70,000 Daltons (Da). Measurement of the elution volume of dextran standards (i.e. 1000 Da, 5000 Da, 12000 Da, 25000 Da and 50000 Da) led to the calibration equation:
Log(MW)=6.212−0.1861 Ve;
Wherein MW: Molecular Weight of the molecule in Dalton (Da), and Ve: elution volume in mL (assayed at the top of the peak).

(43) Chromatogram profiles were recorded with UNICORN 5.1 software (GE Healthcare). Peak retention times and peak areas were directly calculated and delivered by the program.

(44) Results

(45) TABLE-US-00001 TABLE 1 Molecular weight of different fractions in alginate-like exopolysaccharides and their percentage. Elution volume Percentage of the of the peak Molecular weight fraction (ml) (kDa) (% peak area) 7.83 >70 29.74 13.48 14.4 18.82 15.57 5.79 45.15 17.58 2.15 4.42 20.13 0.656 1.87
Rheology Experiments

(46) Viscosity is considered to be an important parameter for biopolymers, such as alginate. Rheology studies the phenomena that appear during deformation and flow of fluids, solids and of solid systems under the influence of external forces. Newton's law is considered to apply for fluids such as to ideal elastic and viscous materials.

(47) Rheological experiments are performed to determine the viscosity versus the shear rate, the critical overlap concentration, the thermal stability and the salinity stability. The viscosity is measured as a function of shear rate using an AR-G2 Rheometer.

(48) Materials and Method

(49) The rheology experiments are performed in an AR-G2 Rheometer using Couette Geometry. The Rheometer is filled with 20 ml samples of the polymer solution Na-ALE in the desired concentrations and salinity's. The alginic acid is converted to the desired polymer solution (sodium alginate (Na-ALE)) by adding NaOH and deionized water.

(50) To prepare the polymer solution samples for the rheology experiments a stock solution of the highest concentration is prepared first. Thereafter the highest concentration stock solution is diluted to the desired (lower) concentrations. The stock solution is prepared as follows:

(51) The amount of alginic acid required is weighted with a mass balance.

(52) Subsequently NaOH (0.1 N) is added gently to the solution to avoid particle agglomeration.

(53) The solution is stirred and the pH is measured continuously.

(54) NaOH (0.1 N) is added up to a final pH of approximately 8.3 and the solution is supplemented to the required volume with deionized water.

(55) The beaker is stirred for 30 minutes at high speed and covered with aluminum foil to prevent contact with air.

(56) Subsequently, the stirrer is reduced to medium speed and the solution is stirred and degassed for at least one day to create a homogeneous polymer solution in equilibrium and to guarantee hydration.

(57) Finally the stock solution is diluted with deionized water to 20 ml of polymer solution to the desired concentrations.

(58) Results

(59) The viscosity of ALE and alginate solutions at various shear rates is shown in FIG. 1a-c). The viscosity (vertical axis, Pa*s) of ALE decreases as the shear rate increases (horizontal axis, 1/s). This is shown for four different solutions, from top to bottom, having 5%, 3%, 2%, and 1% alginate, respectively. Apparently the present solutions, comprising the present ALE, show non-Newtonian behavior in this respect.

(60) In comparison, in FIG. 1b the viscosity of algae alginate is affected less by changing shear rate. This is shown for five different solutions, from top to bottom, having 10%, 5%, 3%, 2%, and 1% algae alginate, respectively.

(61) Such is considered an indication that the solution of ALE is more pseudoplastic than that of comparable algae alginates. This property may provide advantages in processing, such as pumping and filling.