3-hydroxypropionaldehyde detection and extraction

Abstract

The present invention relates to a method of extracting 3-hydroxypropionaldehyde (3-HPA) and/or derivatives thereof from an aqueous solution comprising 3-HPA, the method comprising: (a) contacting the aqueous solution with chitosan and/or chitosan comprising polymers; (b) separating the 3-HPA bound chitosan and/or chitosan comprising polymers; and (c) washing the 3-HPA bound chitosan and/or chitosan comprising polymers at least once with a washing medium;
wherein 3-HPA and/or derivatives thereof is in the washing medium.

Claims

1. A method of extracting 3-hydroxypropionaldehyde (3-HPA) and/or derivatives thereof from an aqueous solution comprising 3-HPA, the method comprising: (a) contacting the aqueous solution with chitosan and/or chitosan comprising polymers; (b) separating the 3-HPA bound chitosan and/or chitosan comprising polymers; and (c) washing the 3-HPA bound chitosan and/or chitosan comprising polymers at least once with a washing medium; wherein 3-HPA and/or derivatives thereof is in the washing medium.

2. The method according to claim 1, wherein the washing medium is selected from the group consisting of water, an acid and mixtures thereof.

3. The method according to claim 2, wherein the acid is selected from the group consisting of acetic acid, formic acid, citric acid, carbonic acid, trifluoroacetic acid, hydrochloric acid, sulphuric acid, phosphoric acid and mixtures thereof.

4. The method according to claim 1, wherein (c) the washing step is carried out at least twice consecutively in at least two different washing mediums.

5. The method according to claim 4, wherein the two different washing mediums are (i) water and (ii) at least one acid or mixtures thereof.

6. The method according to claim 1, wherein chitosan and/or chitosan comprising polymers are recyclable.

7. The method according to claim 1, wherein the aqueous solution comprises 1 g of chitosan and/or chitosan comprising polymers for binding 50-2000 mg of 3-HPA.

8. The method according to claim 1, wherein (b) the step of separating is selected from the group consisting of filtration, centrifugation, decantation and combination thereof.

9. The method according to claim 1, wherein the chitosan and/or chitosan comprising polymers are capable of adsorption and desorption of 3-HPA.

10. The method according to claim 1, wherein the 3-HPA in aqueous solution is produced from at least one carbon source by at least one microorganism.

11. The method according to claim 10, wherein the microorganism is selected from the group consisting of Lactobacillus reuteri, Klebsiella pneumoniae, Citrobacter freundii, Clostridium butyricum, Clostridium acetobutylicum, and Enterobacter agglomerans.

12. The method according to claim 10, wherein the microorganism is a recombinant microorganism expressing glycerol dehydratase.

13. The method according to claim 10, wherein the carbon source is selected from the group consisting of glycerol and glucose.

14. The method according to claim 10, wherein the microorganisms are removed prior to (a) of contacting the aqueous solution with chitosan and/or chitosan comprising polymers.

15. The method according to claim 1, wherein (c) the washing step is carried out at least twice consecutively in two different washing mediums of (i) water and (ii) citric acid.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 is a graph showing the adsorption of 3-HPA (at different pH using buffers) to medium molecular weight chitosan (0.01 g).

(2) FIG. 2 is a graph showing the effect of molecular weight and concentration of chitosan used for making beads on the binding capacity for 3-HPA.

(3) FIG. 3 is a graph showing the effect of adsorption time on elution of 3-HPA from wet chitosan beads (3% medium molecular weight chitosan.

(4) FIG. 4A is a graph showing the adsorption, washing and elution of high concentration of 3-HPA (113 mM) from cross-linked beads using different elution system where the concentration of the elution medium is 1 M.

(5) FIG. 4B is a graph showing the adsorption, washing and elution of high concentration of 3-HPA (113 mM) from cross-linked beads using different elution system where concentrated elution mediums are used.

(6) FIG. 5 is a graph showing the adsorption, washing and elution of low concentration of 3-HPA (18 mM) from cross-linked beads using different elution systems. From the left to the right: 1 M acetic acid; 1 M formic acid; 1 M citric acid; conc. acetic acid; conc. formic acid; conc. citric acid and carbonic acid.

EXAMPLES

(7) The foregoing describes preferred embodiments, which, as will be understood by those skilled in the art, may be subject to variations or modifications in design, construction or operation without departing from the scope of the claims. These variations, for instance, are intended to be covered by the scope of the claims.

Example 1

(8) Low and Medium Molecular Weight Chitosan Evaluated as Scavengers for 3-HPA

(9) Initially 0.1 g chitosan powder (low molecular weight component has a viscosity of 20-300 cP (1 wt. % in 1% acetic acid75 to 85% deacetylation) and the medium molecular weight component has viscosity of 200-800 cP (1 wt. % in 1% acetic acid75 to 85% deacetylation) as supplied commercially by Sigma) was added to 5 ml of 3-HPA solution (95.6 mM), pH 7. 3-Hydroxypropionaldehyde (3-HPA) was produced using Lactobacillus reuteri as described in Sardari et al. 2013 (Sardari, R. R., et al., Biotechnology and Bioengineering, 2013. 110(4): p. 1243-1248). The mixture was mixed for 30 min at room temperature, 22 C., after which the chitosan was separated by centrifugation at 3000g for 5 min. The concentration of residual 3-HPA in the supernatant 1 was measured by acrolein test. There was 60.1 mM of 3-HPA when low Mw chitosan was used and 53.1 mM of 3-HPA when medium Mw chitosan was used. The chitosan was then washed once by resuspension in 10 ml distilled water at room temperature for 10 min followed by centrifugation and determination of the 3-HPA concentration in the supernatant 2 using the acrolein test. Finally, 10 ml of 1 M organic acid (Acetic acid (Sigma Aldrich), Formic acid (Sigma Aldrich), Citric acid (Merck), Carbonic acid) was each separately added to the chitosan, mixed for 15 min, then centrifuged and the concentration of 3-HPA in the supernatant 3 was determined using the acrolein test.

(10) Acrolein test was used for quantitative analysis of 3-HPA. Two hundred microliter of a suitably diluted sample was mixed with 600 l HCl for the dehydration of 3-HPA to acrolein. DL-tryptophan (150 l) was added to the mixture, thereby obtaining an acrolein-chromophore complex (purple) which was quantified by absorbance at 560 nm on a spectrophotometer using acrolein as standard (Vollenweider, S., et al., Journal of Agricultural and Food Chemistry, 2003. 51(11): p. 3287-3293; Circle, S. Ind Eng Chem Anal Ed, 1945. 17: p. 259-262.).

(11) The results are shown in Table I below. Almost 50% of the bound 3-HPA was recovered in the water fraction (Elution 1) and the residual 50% was recovered in the organic acid fraction (Elution 2). The recovery (elution) of bound 3-HPA was quantitative.

(12) TABLE-US-00001 TABLE 1 Binding and elution of 3-HPA to/from low and medium molecular weight chitosan powder Chitosan Mwt Low Med Low Med Low Med Low Med Elution Svstem Acetic acid Formic acid Citric acid Carbonic acid Loading (mmol) 0.183 0.213 0.183 0.213 0.183 0.213 0.183 0.213 E1 (mmol) 0.093 0.111 0.091 0.113 0.095 0.110 0.098 0.122 E2 (mmol) 0.092 0.109 0.097 0.108 0.100 0.111 0.091 0.099 Total elution 0.185 0.220 0.188 0.220 0.195 0.220 0.189 0.221 % eluted 101.0% 103.4% 102.6% 103.4% 106.4% 103.4% 103.1% 103.8%

Example 2

(13) Effect of pH on Binding Capacity

(14) The effect of the solution pH on the binding capacity was also investigated using medium molecular weight chitosan as scavenger. Binding capacity was measured using the same method used in Example 1. The results are shown in FIG. 1. pH 7 was found optimum for 3-HPA binding.

Example 3

(15) Effect of Chitosan Molecular Weight and Concentration on 3-HPA Binding

(16) Different concentrations (2%, 4% and 6%, respectively) of low- and medium-molecular weight chitosan were dissolved overnight in 2%, 4% or 6% of acetic acid, respectively. Low molecular weight component has a viscosity of 20-300 cP (1 wt. % in 1% acetic acid75 to 85% deacetylation) and the medium molecular weight component has viscosity of 200-800 cP (1 wt. % in 1% acetic acid75 to 85% deacetylation) as supplied commercially by Sigma. The resulting viscous solutions were dropped in 2%, 4% and 6% of sodium hydroxide solution, respectively through a thick needle. The resulting beads were washed thoroughly with Milli-Q quality water and then with phosphate buffer (pH 7, 50 mM). One g of swollen beads was then placed in a 15-ml falcon tube and 3-HPA solution (5 ml of 46.6 mM, pH 7) was added to the beads and the tubes were mixed on a rocking table for 12 h. The concentration of residual 3-HPA in the solution was measured by acrolein test (as described under example 1) and the binding capacity was calculated for each chitosan molecular weight and concentration.

(17) From the data as shown in FIG. 2 it is clear that there was no difference between the two molecular weights of chitosan for the binding capacity of 3-HPA when used at the same concentration (4%). However, by decreasing the concentration of the chitosan, the binding capacity was increased. In case of low molecular weight chitosan, the beads were extremely fragile at a concentration of 2% and did not solidify when dropped in sodium hydroxide solution. On the other hand, it gave highly stable beads when used at concentrations of 4% and 6%. The opposite was true for medium molecular weight chitosan where at a concentration of 2% and 4%, the beads was highly stable, however, for the 6% chitosan the solution was highly viscous. Hence, 2% medium molecular weight chitosan was found optimum for use in 3-HPA capture due to easy formulation and high binding capacity reaching 766 mg/g chitosan.

Example 4

(18) Effect of Duration of 3-HPA Binding to Chitosan on Elution

(19) To four 50 ml falcon tubes, each containing 1 g wet beads prepared from medium molecular weight chitosan (2%) in 2% acetic acid, 5 ml of 14 mM 3-HPA solution, pH 7 was added and shaken at room temperature for different time intervals (0.5, 1, 1.5 and 2 h, respectively). At each time period, one tube was removed, the solution separated and beads washed once with 5 ml MQ water and the amount of 3-HPA bound to the resin was calculated. Subsequently 3-HPA was eluted using 2.5 ml of elution system (ethanol/ammonium sulfate/HCl, 50%/6%/0.1 M) and the amount of 3-HPA in the eluent was measured.

(20) As can be seen in FIG. 3, the amount of 3-HPA bound during 0.5-2 h varied between 60 and 80 mg/g chitosan. The amount of 3-HPA eluted from the beads was on average 91.717%. Significant amount of 3-HPA was also recovered in the washing step that could be the aldehyde that was physically adsorbed to the beads.

Example 5

(21) Adsorption/Desorption of 3-HPA Using Cross-Linked Chitosan Beads: Using High Initial 3-HPA Concentration and Different Elution Systems

(22) In three 50 ml falcon tubes, 0.05 g cross-linked chitosan beads were mixed with 5 ml of 113 mM 3-HPA solution, pH 7 for 30 min on a rocking table.

(23) X-linked chitosan beads were prepared based on the method used by Wan Ngah etal. (2008). First, 1 g of medium molecular weight chitosan was dissolved in 35 ml of 5% (v/v) acetic acid. The mixture was shaken slowly overnight on a rocking table. The dissolved chitosan solution was filled in a 100 ml syringe and dropped through a needle into 250 ml sodium hydroxide solution (2% w/v). The formed wet beads were then washed by distilled water to remove any NaOH and air-dried (dry beads). For X-linking the chitosan beads, 0.1 M epichlorohydrin (ECH) solution was prepared and the pH was adjusted to 10 using 0.067 M NaOH. The dry chitosan beads were suspended in 62.5 ml of the 0.1 M ECH solution to obtain a ratio of 1:1 with chitosan (mol CH2O:mol CH2OH). The beads suspension was stirred continuously on a magnetic stirrer at 200 rpm for 2 h. Then the X-linked beads were washed with hot-followed by cold distilled water to remove any excess ECH solution, and then air-dried.

(24) The binding to the x-linked chitosan beads was followed by a washing step using 10 ml of distilled water and elution by 5 ml of 1 M acetic acid, 1 M formic acid, and 1 M citric acid, respectively, for 30 min (FIG. 4 A). A parallel experiment was performed in 4 falcon tubes using 0.1 g cross-linked chitosan and different concentrated acids (acetic acid, formic acid, citric acid, and carbonic acid) for elution (FIG. 4 B).

(25) As seen from FIG. 4, low concentration of acids, could release higher amount of 3-HPA, and 1 M acetic acid has the highest recovery.

Example 6

(26) Adsorption/Desorption of 3-HPA Using Cross-Linked Chitosan Beads: Using Low Initial 3-HPA Concentration and Different Elution Systems

(27) In seven 50 ml falcon tubes, 0.05 g of cross-linked beads were mixed with 5 ml of 18 mM 3-HPA solution, pH 7 for 30 min on a rocking table. The binding was followed by a washing step using 10 ml of distilled water and elution by 5 ml of different acids with different concentration as elution system for 30 min. The results are shown in FIG. 5.

(28) The recovery of 3-HPA was 89, 74, 67, and 73% using concentrated acetic acid, concentrated formic acid, and concentrated citric acid, respectively. Also, the recovery of 3-HPA was 74, 62, 72, and 73 using 1 M acetic acid, 1 M formic acid, 1 M citric acid, and carbonic acid.

(29) It can be concluded that adsorption and recovery are better at high initial 3-HPA concentration and 3-HPA is not stable in high concentration of acid.