WO2006103699A1

WO2006103699A1 - A process for the extraction of polyhydroxyalkanoates from bacteria

Info

Publication number: WO2006103699A1
Application number: PCT/IN2006/000084
Authority: WO
Inventors: Kshama Lakshman; Tumkur Ramachandriah Shamala
Original assignee: Council Of Scientific & Industrial Research
Priority date: 2005-03-31
Filing date: 2006-03-08
Publication date: 2006-10-05

Abstract

Polyhydroxyalkanoate (PHA) is gaining importance as an alternative source to synthetic polymers because of similar properties possessed by it compared to synthetic plastics and its biodegradability. The polymer is produced intracellularly and hence various methods are reported for its extraction. In order to minimize the changes in the polymer that occurs during chemical extraction and to avoid multiple enzyme usage, a biological method for the extraction of polyhydroxyalkanoates is proposed, which comprises of cultivation of PHA producing bacteria such as Sinorhizobium meliloti (MTCC 100) in nutrient medium, thermal inactivation of PHA containing cells along with the culture medium at 80° C, digestion of cell materials by growing any actinomycetes culture which can lyse the bacterial cells or by using its culture filtrate, and isolation of the released polymer by the use of water immiscible solvent or an admixture of a surfactant and a chelating agent. This method involves simple and easier extraction of PHA from the cells.

Description

A PROCESS FOR THE EXTRACTION OF POLYHYDROXYALKANOATES FROM

BACTERIA

Field of Invention

The present invention relates to a process for the extraction of polyhydroxyalkanoates from bacteria. The main usage of the invention is in the production and modified extraction of the biopolymer for various packaging applications.

Description of Prior Art

The synthetic polymers that are produced from petrochemical sources are not biodegradable; hence they are accumulating in the environment at an alarming rate and are causing pollution problems. Polyhydroxyalkanoate (PHA) is gaining importance as an alternative source to synthetic polymers because of similarities in it's properties as compared to synthetic plastics coupled to it's susceptibility towards biodegradation. Polyhydroxyalkanoate is a biodegradable polymer, which is produced intracellular^ in bacteria. Several bacteria produce PHA as carbon and energy reserves under certain nutrient depletion conditions. However, the bacterial cells should be hydrolyzed to recover the polymer. Therefore, the extraction of the polymer from the cell biomass is a critical step for its economical production. The main usage of the invention relates to a simplified method of extraction of PHA from bacterial cells.

Reference may be made to Baptist, J.N. (1962) Process for preparing poly-² - hydroxybutyric acid. U.S patent 3044942; Noda, Isao, Schechtman and Lee Arnold (1999), Solvent extraction of polyhydroxyalkanoates from biomass, U.S. patent No. 5942597; Herscovici E. J. (1980), Polymer recovery process, U.S. Patent 4205162; Vanlautem, N; Gilain, J. (1982), Process for separating poly-beta-hydroxybutyrates from a biomass, U.S. Patent 4310684; Senior, P.J., Wright, L. F. and Alderson, B. (1982), Extraction process, U.S. Patent 4324907; Walker, J., Whitton, J. S., (1982) Extraction of (beta-hydroxy butyric acid), U.S. Patent 4358583; Braham, PJ. , Selwood, A. (1983), Extraction of (beta-hydroxy butyric acid), U.S. Patent 4391766; Stageman, J. F. (1985), Extraction process, U.S. Patent 4562245; Vanlautem, N., Gilain, J. (1987), Process for extracting poly-beta-hydroxyalkanoates by means of a solvent from an aqueous suspension of microorganisms., U.S. Patent, 4705604; Traussnig, H., Kloimstein, E., Kroath, H., Estermann, S. (1990), Extracting agents for poly-D(-)-3hydroxybutyric acid., U.S. Patent, 4968611.; Blauhut, W., Gierlinger, W., Stremffl, F., (1993), Process for obtaining a polyhydroxyalkanoate from the cell material of a microorganism., U. S. Patent 5213976.; Ohleyer, E. (1995), Method for obtaining poly-beta-hydroxyoctanoic acid via solvent extraction., U.S. Patent, 5422257.; Kurdikar, D. L., Strauser, F. E., Solodar, A. J., Paster, M.D., Asrar, J. (2000), Methods of PHA extraction and recovery using non-halogenated solvents, U. S. Patent, 6043063., wherein extraction of PHA is achieved by solvents such as acetone, acetonitrile, benzene, butyl acetate, butyl propionate, beta- butyrolactone, gamma-butyrolactone, chloroform, diethyl carbonate, dichloromethane, 1 ,4-dioxane, diethyl formamide, dimethyl succinate, dimethyl sulfoxide, ethyl acetate, ethylene glycol diacetate, methyl acetate, methyl ethyl ketone, methylene chloride, propylene carbonate, tetrahydrofuran, toluene, xylene, individually or mixtures thereof. The drawbacks are that the solvent extraction is not desirable because of the harmful effects of solvents used, viscous nature of the cell material particularly where the proportion of non-HB polymer cell material to be separated is more, which renders filtration or centrifugation steps of such material difficult. Further problems encountered are utilization of large volumes of solvents involved in extraction and recovery of the solvents.

Reference is also made to methods evolved by Berger, E., Ramsay, B.A., Ramsay, J.A., Chavarie, C, Braunegg, G. (1989), PHB recovery by hypochlorite digestion, Biotechnol. Tech. 3:227-232; Hahn, SK., Chang, Y.K.,

Kim, B.S., Lee, K.M., and Chang, H.N. (1993) The recovery of poly (3- hydroxybutyrate) by using dispersions of sodium hypochlorite solution and chloroform. Biotechnol. Technol. 7:209-212; Ramsay, B.A., Ramsay, J., Berger, E., Chavarie, C, Braunegg, G. (1992), Separation of poly-beta- hydroxyalkanoic acid from microbial biomass, U.S. Patent, 5110980,wherein sodium hypochlorite is used for the differential digestion of non-PHA cellular materials and chloroform assists in the dissolution and extraction of the polymer. The drawback however is the generation of chlorine containing solution at the end of extraction process and degradation of polymer during treatment.

Reference may be made to enzymatic digestion methods as an alternative to solvent extraction, developed by Holmes, P.A. and Lim G. B. (1990), Separation process. U.S. Patent 4910145 discloses a related method, wherein thermally treated biomass is solubilized using various proteolytic . enzymes and /or phospholipase, anionic surfactant to solubilize non PHA- cellular materials. The enzymes used are pepsin, trypsin, bromelin, papain etc. The draw back envisaged is that the biomass suspension obtained after heat treatment is centrifuged, cells are resuspended for digestion, buffered (pH 7-8.5), and enzymatic solubilization is performed either in stages using various proteolytic enzymes, phospholipase, and/or surfactant or alternatively enzyme mixtures are to be evolved for synergistic action to solubilize non PHA cellular materials to recover purified product.

Therefore, keeping in view the hitherto known processes for the extraction of polyhydroxyalkanoates from bacteria, the inventors realized that there exists a dire need for developing a simplified process for the extraction of PHA from bacterial cells.

Objects of the Invention

The main objective of the present invention is to provide a simplified process for the extraction of polyhydroxyalkanoates, which obviates the drawbacks as detailed above.

Another object of the present invention is to provide a process which minimizes the volume of solvent used for the extraction of the polymer.

Still another object of the present invention is to provide a biological method for the hydrolysis of bacterial cells, which prevents the use of hypochlorite for such process and the generation of chlorinated effluent as a result of that process.

A further object of the present invention is to provide a biological method of hydrolyzing PHA containing bacterial cells, wherein the lytic culture can be grown without additional nutrient supply directly in the PHA broth, without further buffering, centrifugation and re suspension of the cells.

Yet another object of the present invention is to provide a process wherein the culture filtrate of the cell lytic culture can also be used directly and effectively for hydrolysis of bacterial cells to obtain PHA.

Accordingly, the present invention provides a process for the extraction of polyhydroxyalkanoates from commercially available bacterial strains characterized in that the hydrolysis of the polyhydroxyalkanoate producing bacteria is carried out using any commercially available cell lytic actinomycetes culture and the said process comprising the steps of:

[a] culturing the polyhydroxyalkanoate [PHA] producing bacteria in nutrient medium for a period of 2 to 3 days at a temperature ranging from 25 to 30 degree C under shaking at 150 to 300 rpm;

[b] heating the culture medium obtained in step [a] at a temperature ranging from 50 to 80 degree C;

[c] cooling the heated culture medium of step [b] at ambient temperature; [d] inoculating about 40 to 160 weight % of an actinomycetes culture in the culture medium as obtained from step [c] and culturing the same at a temperature ranging from 28 to 30 degree C under shaking at 150 to 250 rpm for a period of 1 to 2 days;

[e] optionally adding 10.0 to 50.0 % of the culture filtrate of said actinomycetes culture to thermally inactivated biomass of S. meliloti as obtained from step [b];

[f] separating the actinomycetes from the culture medium obtained in step [d] to obtain a hydrolysate;

[g] isolating the polyhydroxyalkanoates from the hydrolysate obtained in step [e] either by using a water immiscible solvent in the ratio of hydrolysate : solvent :: 1:1 to 4:1 or an admixture of a surfactant

[1.0 to 0.6%] and a chelating agent [0.1 to 0.06%].

In an embodiment of the present invention bacterium such as S. meliloti (MTCC 100), capable of producing PHA is cultivated under submerged fermentation conditions at ambient temperature ranging from about 25 to 30 degree C upto a period of about 72 hours.

In another embodiment of the present invention the culture broth having PHA containing cells are thermally processed at 60-80° C to inactivate the cells.

In yet another embodiment of the present invention actinomycetes culture such as Microbispora sp. [MTCC 964] is cultivated under submerged conditions and ambient temperature for 48 h - 72 h. In still another embodiment of the present invention the pelleted biomass of actinomycetes which can lyse bacterial cells is transferred to thermally treated S. meliloti culture broth and the inoculated broth is placed under submerged conditions and ambient temperatures up to 24 h in order to release PHA from the cells.

In a further embodiment of the present invention thermally inactivated S. meliloti is hydrolysed by the supernatant of actinomycetes culture to release PHA from the bacterial cells.

In yet another embodiment of present invention the PHA released from the bacterial cells by growth associated lytic activity of actinomycetes and its cell free lytic activity is separated from other cellular materials by using anionic surfactant in the range of 1.0 to 0.6% and a chelating agent in the range of 0.1 to 0.06% followed by drying the resultant sedimented PHA.

In another embodiment of the present invention the PHA released after hydrolysis of bacterial cell is solubilised in chloroform and separated from the non-PHA components and dried.

Detailed Description of the Invention

Inoculum of Sinorhizobium meliloti (MTCC 100) was prepared in nutrient medium at 25°-30 ⁰C and 200-300 rpm for 18-24 h. PHA production can be carried out in liquid medium which contained a source of nitrogen, phosphate, magnesium, carbon and buffering salts. Inoculated culture could be grown at 200-300 rpm and 25°-30⁰C for 48-72 h. At the end of the cultivation period the bacterial cells could be inactivated by heating the broth at 60° -80 ° C for 5-10 min. PHA was estimated by qualitative and quantitative methods.

Inoculum of the cell lytic culture as exemplified by Microbispora sp. [MTCC 964] was prepared by growing the culture in a nutrient medium at 25-30 ⁰C and 200-300 rpm for 48-72 h. After growth the pelleted cells were transferred at 30-40 mg level (dry weight basis) to every 200-300 mg % (dry wt.) of PHA containing cells in the form of inactivated culture broth of Sinorhizobium meliloti (MTCC 100) under sterile conditions and aerobic cultivation was carried further without additional supplementation of nutrients at 25-30° C, 100-150 rpm for 24-48 h. After growth Microbispora sp. cells that showed pelleted growth, were separated by simple filtration through glass wool. PHA could be isolated alternatively by:-

[1] the addition of Triton X 100 - a surfactant (1-0.6%) and EDTA (0.1-

0.06%) to the filtrate (pH 6-7) containing PHA. The broth was then incubated at 45 -50° C for 8-10 min to hydrolyse the non-PHA components and to chelate the polymer. The hydrolysed broth was centrifuged at 6000-7000 rpm for 15-20 min to isolate the polymer. The sedimented PHA was dewatered at low temperature (40-50° C). [2] As an alternate to this, 4 volumes of hydrolysed broth was mixed with one volume of chloroform and the chloroform layer containing PHA was separated and dried at 40-50° C to obtain PHA. The clear broth obtained after the growth of actinomycetes culture, which contained 175-200 U of lytic activity, was used at 0.5-1 volumes to hydrolyse 2-5 g/l of thermally inactivated biomass of S. meliloti. The hydrolysis was carried out at 40-50° C for 1-18 h and PHA was isolated using Triton X 100 and EDTA or with chloroform or by sedimentation as described above. Purity of the isolated product was determined by comparison with standard PHA samples.

Actinomycetes culture used in the invention produced lytic activity, which could solubilize S. meliloti cell wall materials and hence release the intracellular components such as PHA. The released PHA was separated from other non-PHA cellular materials either by selective solubilization using a solvent such as chloroform or by using a selective chelating agent.

Novelty:

The novelty of the present invention resides in the step of cell hydrolysis wherein the actinomycetes which can produce cell lytic enzyme can be directly grown for a shorter period without any additional nutrient supply in the residual fermentation broth of S. meliloti cultivated for polyhydroxyalkanoate production. The actinomycetes biomass obtained after growth is filamentous and can be easily separated from S. meliloti cells by filtration and PHA is recovered from the resulting hydrolyzed broth. The lytic enzyme obtained by the cultivation of actinomycetes alone is also used for the hydrolysis of S. meliloti cells to liberate the PHA. The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.

EXAMPLE 1

For PHA production using S. meliloti, inoculum was prepared in 100 ml medium (g I^"1): Na₂ HPO₄ 2H₂O, 4.4; KH₂PO₄ , 1.5; (NH₄) ₂SO₄, 1.0; MgSO₄ 7H₂O, 0.2; sucrose, 10; yeast extract, 0.5; pH 7.0. The inoculum was prepared at 30 ⁰C and 250 rpm for 24 h. PHA production was carried out in triplicate in 500 ml Erlenmeyer flasks in liquid medium (100 ml), which contained 20 g/l of sucrose. Incubation was performed at 250 rpm and 30 ⁰C for 72 h. At the end of the cultivation period the culture broth was heated to 80 ⁰ C for 10 min and cooled. PHA content of bacterial cells was determined gravimetrically by sodium hypochlorite hydrolysis (Williamson DH, Wilkinson

JF 1958, The isolation and estimation of poly-β-hydroxybutyrate inclusions of

Bacillus species. J. Gen. Microbiol. 19: 198-209; Law JH, Slepecky RA1961 ; Assay of polyhydroxybutyric acid. J. Bacteriol. 82: 33 -36). GC analysis of powdered PHA was carried out by the method described by Brandl et al. (Brandl H, Gross RA, Lenz RW, Fuller RC, 1988; Pseudomonas oleovorans

as a source of poly-β-(HA) for potential application as biodegradable

polyesters. Appl. Environ. Microbiol. 154: 1977-1982). For GC analysis 2 mg of PHA sample was subjected to methanolysis with a solution consisting of 1 ml chloroform, 0.85 ml methanol and 0.15 ml cone. H₂SO₄ at 100 ⁰C for 140 min. Deionized water was added to the cooled samples, the contents were homogenized and the bottom phase was used for GC analysis. The methyl esters were analyzed by GC with flame ionization detector, in a 30 m DB- 1 (fused silica gel - polymethyl siloxane) capillary column (internal diameter 0.25 mm and film thickness 0.25 microns). N₂ (1 ml/min) was used as a carrier gas. The injector and the detector were at 17O⁰C and 22O⁰C, respectively. The program used was: 55⁰C for 7 min; ramp of 4° C per min up to 100⁰C; 1O⁰C per min rise up to 200⁰C and hold at 20O⁰C for 10 min. Calibration was performed using standard P(HB-co-HV) containing 5 mol% of hydroxy valerate (Sigma) with benzoic acid as internal standard.

Inoculum of the actinomycetes culture such as Microbispora sp. [MTCC 964] was prepared by transferring the culture from 48 h old nutrient agar slant to 100 ml medium containing (g r¹): Na₂ HPO₄ 2H₂0, 4.4; KH₂PO₄ , 1.5; (NH₄) ₂SO₄, 1.0; MgSO₄ 7H₂O, 0.2; sucrose, 10; yeast extract, 1.5; pH 7.0. The inoculum was prepared at 30 ⁰C and 150 rpm up to 72 h. After growth the cells formed pellets and could be easily separated form the broth by decantation under sterile conditions. The pelleted cells were transferred at 40 mg level (dry weight basis) to every 100 ml of above described inactivated culture broth of Sinorhizobium meliloti under sterile conditions and aerobic cultivation was carried further without additional supplementation of nutrients at 30° C, 150 rpm for 24 h. After growth, Microbispora sp. [MTCC 964] cells that showed pelleted growth, was separated by simple filtration through glass wool. PHA was isolated from other cellular materials by 2 alternate methods: (1 ) A surfactant and a chelating agent were used for the isolation of the PHA (Chen Y, Yang H, Zhou Q, Chen J and Gu G., Cleaner recovery of poly -3- hydroxybutyric acid synthesized in Alcaligenes eutrophus; Process Biochemistry, 2001 , 36:501-516). Triton X 100, a surfactant (0.6%) and EDTA (0.06%), a chelating agent were added to the hydrolysate containing PHA. The broth was incubated at pH 6-7, 50° C for 10 min to hydrolyze the non- PHA components and to chelate the polymer. The hydrolyzed broth was centrifuged at 7000 rpm for 15 min to isolate the polymer. The PHA-sediment was dewatered at low temperature (50° C). (2) The hydrolyzed broth (4 volumes) was mixed with one volume of chloroform and stirred for 10 minutes at room temperature. The bottom phase of chloroform layer containing PHA was separated and dried at 50° C to obtain PHA.

Characterization of the Product

Purity of the isolated product was measured by crotanate assay. For this 5 mg of product was solubilized in 1 ml of chloroform and 10 Vi of this was mixed with 5 ml of concentrated H₂SO₄ and heated in a boiling water bath for 10 min and cooled. Crotoinic acid formed was measured at 235 nm against reagent blank. Concentration and purity of PHA was calculated by comparing with standard graph prepared using PHA.

The results in Table 1 indicate that extraction of PHA by chloroform or surfactant + chelating agent individually is poor. Microbispora sp. [MTCC 964] grew well on S. meliloti cells and the PHA released could be harvested after 24 h of growth. The polymer was recovered by using chloroform or surfactant + chelating agent. The purity of polymer was 90%. Table 1 : Growth associated extraction of polyhydroxyalkanoate (PHA) from S. meliloti cells using Microbispora sp. [MTCC 964]

Method of hydrolysis Extraction Dry wt. of PHA Purity of system S. meliloti recovered PHA (%) biomass (g); (g)

Control Chloroform 1 0.03 66

Control EDTA + Triton 1 0.16 68

Sod. hypochlorite Chloroform 1 0.47 96

Microbispora growth

Period Biomass

(mg %)

24 h 40 a. Chloroform 1 0.21 90 b. EDTA + Triton 1 0.49 90

48 h 110 a. Chloroform 1 0.17 94 b. EDTA + Triton 1 0.41 92

72 h 160 a. Chloroform 1 0.08 90 b .EDTA + Triton 1 0.07 90

EXAMPLE 2

The culture of Microbispora sp. [MTCC 964] was grown in mineral medium as described in Example 1 and clear broth obtained after the inoculation of pelleted cells was found to contain 175 -200 U/ml of lytic activity. Lytic activity was determined by hydrolyzing S. meliloti cells of known dry weight (3mg/5ml) with 48-72 h old clarified culture filtrate (0.5 ml) of Microbispora sp. [MTCC 964] at 50 ° C, pH 6-7 for 1 h. O.D. was measured at 620 nm and activity was calculated as 1000x (initial O.D. at 620 nm-final O.D. at 620 nm). The culture filtrate was used at 1 volume level to hydrolyze 3g/l of thermally inactivated biomass of S. meliloti. The hydrolysis was carried out at 50° C for 3 h and PHA was isolated using Triton X 100 and EDTA or with chloroform and purity of the polymer obtained was determined as described in Example 1.

Table 2: Extraction of polyhydroxyalkanoate (PHA) from S. meliloti cells using lytic enzyme of Microbispora sp. [MTCC 964]

Method of Extraction Dry wt. of PHA Purity of hydrolysis system S. meliloti recovered PHA (%) biomass (g); (g) Control Chloroform 1 0.03 66

Control EDTA + Triton 1 0.16 68

Sodium hypochlorite Chloroform 1 0.47 96 Lytic enzyme a. Chloroform 1 0.22 86 b. EDTA + Triton 1 0.47 92

The results in Table 2 indicate that the culture filtrate of Microbispora sp. can be used effectively to solubilize S. meliloti cells and release PHA. PHA was further purified by using chloroform or by adding surfactant + chelating agent to obtain polymer of 86-92% purity.

Therefore, from the examples it can be concluded that Microbispora sp. [MTCC 964] culture used in the experiment produced lytic activity, which could solubilize the bacterial cell material and hence release the intracellular components such as PHA. The released PHA was separated from other non- PHA cellular materials either by selective solubilization using a solvent such as chloroform or by using a selective surfactant and a chelating agent to obtain 86-92% purity of the product. The novelty of the present invention with respect to prior art is that cell lytic culture is directly grown for a shorter period without any additional nutrient supply in the fermented broth containing S. meliloti cells having PHA and the lytic biomass obtained after growth was easily separated by filtration and PHA is recovered from the hydrolyzed broth. The enzyme obtained by the cultivation of Microbispora sp. can also be used directly for hydrolysis of S. meliloti cells to recover PHA.

Advantages:

The main advantages of the present invention are: 1 ) minimization of solvents used for the extraction of the polymer i.e., PHA;

2) biological method for the hydrolysis of bacterial cells employed, which prevents the use of hypochlorite for such hydrolysis thereby preventing the generation of chlorinated effluent;

3) biological method of cell hydrolysis of PHA containing bacterial cells where the lytic culture can be grown without additional nutrient supply directly in the PHA broth, without further buffering, centrifugation and re-suspension of the cells;

4) culture filtrate of cell lytic culture can also be used directly and effectively for hydrolysis of bacterial cells to obtain PHA.

Claims

1. A process for the extraction of polyhydroxyalkanoates from commercially available bacterial strains characterized in that the hydrolysis of the polyhydroxyalkanoate producing bacteria is carried out using any commercially available cell lytic actinomycetes culture and the said process comprising the steps of:

[g] isolating the polyhydroxyalkanoates from the hydrolysate obtained in step [e] either by using a water immiscible solvent in the ratio of hydrolysate : solvent :: 1 :1 to 4:1 or an admixture of a surfactant [1.0 to 0.6%] and a chelating agent [0.1 to 0.06%].

2. A process according to claim 1 , wherein the bacterial strain used for harvesting PHA is any commercially available PHA producing bacterial strain.

3. A process according to claim 2, wherein the PHA producing bacterial strain is Sinorhizobium meliloti MTCC 100.

4. A process according to claim 3, wherein the PHA producing bacteria is cultured for a period of about 3 days.

5. A process according to claim 3, wherein the PHA producing bacteria is cultured at a temperature of about 30 degree C.

6. A process according to claim 3, wherein the PHA producing bacteria is cultured preferably at 150 to 200 rpm.

7. A process according to claim 1 , wherein heating of the culture medium is carried out at a temperature of about 80 degree C.

8. A process according to claim 1 , wherein the actinomycetes used for the hydrolysis of PHA producing bacterial strain is any commercially available actinomycetes strain.

9. A process according to claim 8, wherein the actinomycetes strain is Microbisopra sp. MTCC 964.

10. A process according to claim 9, wherein the actinomycetes is inoculated in the culture medium at a concentration of about 40 weight %.

11. A process according to claim 9, wherein the actinomycetes is cultured preferably at a temperature of about 30 degree C for about 24 hours at around 150 rpm.

12. A process according to claim 1 , wherein separation of actinomycetes from the culture medium is carried out preferably by filtration or centrifugation.

13. A process according to claim 1 , wherein the water immiscible solvent used is preferably selected from the group consisting of chloroform, butylacetate, chloroform, diethyl carbonate, dichloromethane and the like.

14. A process according to claim 1 , wherein the surfactant triton X 100 is used at the concentration of about 0.06%.

15. A process according to claim 1 , wherein the chelating agent EDTA is used at the concentration of about 0.025%.

16. A process according to claim 1 , wherein the purity of isolated polyhydroxyalkanoate is up to 92 %.