AU611869B2 - A method for improving recovery by centrifugation of microorganisms from suspension in aqueous solutions - Google Patents

Description

AU-.A-81507/87

PCT

WORLD INTELLECTUAL PROPERTY O?,GANIZATIONs Internauioal Bureau INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 1) International Patent Classification 4 11!) International Publication Number: WO 88/ 02662 B04B 11/00, C12N 1/ b Qnational Publication Date: 21 April 1988 (21.04.88) (21) International Application Number: PCT/AU87/00354 (22) International Filing Date: 16 October 1987 (16.1,9.87) (31) Priority Application Numbers: PH 8566 PI 0402 (32) Priority Dates: (33) Priority Country: 17 October 1986 (17.10,86) 17 February J 087 (17.02,87)

AU

(74) Agent: TAYLOR, Paul, Robert; Arthur S. Cave Company, Goldfields House, I Alfred Street, Sydney, NSW 2000 (AU).

(81) Designated States: AT (European patent), A U, BE (European patent), CH (European patent), DE (European patent), FR (European patent), GB (European patent), IT (European patent), LU (European patent), NL (European patent), SE (European patent), US.

Pulished With international search report.

Before the expiration of the time liit 'for amiending the claims and to be republished in the ev'ent of the receipt of amendments,

AUSTRALIAN

-6MAY 1988 PATENT OFFICE (71) Applicant (for all designated States except US); WEST- ERN BIOTECHNOLOGY LIMITED fAU/AUJ: Griffin Centre, 28 The Esplanade, Perth, W,A, 6000

(AU).

(72) Inventor; and Inventor/Applicant (for US onl0y: MACKAY, Bruce, John (AU/AU]; 216 Railway Parade, Leederville, 6077 (AU).

4 fl.'S'I'J I JII 1 (54)Title: A METHOD FOR IMPROVING RECOVERY BY FROM SUSPENSION IN AQUEOUS SOLUTIONS CENTRIFUGATION O~kfR-AizG4&tAZUALj (57) Abstract An apparatus and method for the separation of a fragile material from the liq~uid medium utilising centrif'ugation.

The apparatus is constructed to cause minimum shear during centrifugation to prevent rupture or the fragile material. A further improvement is provided by the dilution of the input media to improve separation efficiency, -1- The present invention relates to a method of obtaining microorganisms from suspension to be used, for example, in the harvesting of marine or salt tolerant species. In particular, the present invention relates to harvesting unicellular algae, such as, for example, of the genus Dunaliella, from fluids, such as, for example, brine, in order to recover materials of some economic value, such as, for example, carotenoids, particularly 3-carotene from the harvested material.

Although the present invention will be described with particular reference to the harvesting of algae of the genus Dunaliella, it is to be noted that the present invention is not so limited and extends to include other methods of obtaining other microorganisms.

Previous attempts to recover algae from brine have not entirely been successful for one or other reasons, One previous attempt is the subject of Australian Patent No. 486 999, which discloses a filtration process for the recovery of caroteniferous algae from brine suspensions in which a filter aid is added to a suspension of the algae prior to the recovery of 0:00 the combined filter aid and algae by filtration. However, one disadvantage of this process Includes the tendency of the microscopic algae to block the filter, even in the presence of the filter aid, which reduces the flow rate through the filter material. A further disadvantage is that large volumes of filter aid must be mixed with the algae in order to obtain reasonable filtration from which the desired algae is not readily separable after filtration which adds a further step to the overall process of obtaining the algae in a usable form.

V* Centrifugation is another cell harvesting technique which has been attempted, but has been found unsatisfactory because the action of most centrifuges will rupture the algal cell membrane, and because separation efficiency of the algal cells from the brine is poor.

In one broad form the invention comprises a method of separating microorganisms from a liquid medium comprising the steps of: 1, diluting the medium sufficient to Increase the density difference between the microorganisms and the medium; 2, transferring the diluted medium to a centrifuge before the microorganisms are able to equilibrate In density with the medium; and 3, separating the microorganisms from the diluted medium in the centrifuge. i The Invention also provides microorganisms separated from a liquid medium by means of the method of the invention, In one embodiment of the present Invention there is provided an GSA/1163C -2improved method for harvesting algae of the genus Dunaliella, which alleviates the problems of previously attempted methods related to this material.

According to the preferred embodiment for harvesting fragile organisms such as Dunaliella, a number of alterations to conventional centrifugation practices minimise damage to the algae cells. The combined effect of dilution and these alterations make centrifugation cost effective for separating species of the genus Dunaliella and other materials.

In accordance with another embodiment of the invention there is provided a method of producing -carotene from the Dunaliella cells harvested by the method of the invention.

The majority of industrial centrifuges cause unacceptably high levels of cell rupture to Dunaliella species, or any other fragile materials.

Members of the genus Dunaliella do not have a cell wall, but are surrounded by a thin elastic membrane or periplast, this makes the cells particularly susceptible to rupture.

Shear and cavitation within the centrifuge are two major reasons for damage to fragile materials. The amount of damage can be minliised by alterations to centrifuges. One or several or all of the following features help reduce damage to fragile materials: 1. Hermetic centrifuge design which restricts the entry of air thereby limiting cavitation; 2. Feed and discharge pumps, whether they be an integral part of the centrifuge or separate to the centrifuge, should be of a design which minimises shear; 3. The liquid entering the machine should be gradually accelerated in VI angular velocity, to minimise shear as the liquid enters the bowl. Gradual acceleration in angular velocity can be achieved by introducing liquid into the bowl through a rotating hollow spindle as, for instance, found in S 30 certain hermetic milk separators.

Certain centrifuges can be altered to reduce cell rupture by removing feed and/or discharge paring discs. The paring discs are centripetal pumps in the centrifuge, they create shear which damages fragile materials. When the feed paring disc Is removed, it would be necessary to feed the machine using an external pump of a type which would cause minimal shear. Such pumps Include helical rotor pumps or peristaltic pumps.

The separation efficiency of Dunaliella and other materials from brine or other media using a centrifuge is improved dramatically by dilution of the media.

'"GSA/163C 7-

I

-3 Dilution of the media causes a number of changes to the physical characteristics of the algae and the continuous phase (media) which affect sedimentation velocity: 1. Dilution of the culture media causes lowering of the density of the media. The lowering of the density of the media leads to an increase in the difference in density between the algal cells and the media, thereby increasing sedimentation velocity; 2. Viscosity of the media will be reduced by dilution, this will increase sedimentation velocity of algal cells; 3. Dilution of algal culture media is reported to cause swelling of the algal cells. The swelling of the algal cells effectively increases particle diameter, therefore increasing sedimentation velocity.

The increased sedimentation velocity caused by the dilution will lead to an improvement in separation efficiency.

An application of the invention for commercial harvesting of Dunaliella speci es i s represented i n Fi gure 1 This manner of harvesting Dunaliella has a number of advantages over other harvesting methods, including filtration and flocculation methods. These advantages include'.

1. Minimisation of the number of unit steps in harvesting; 2. The Dunaliella cells not recovered during the harvesting operation are not damaged, hence, they can be immediately returned to the growing ponds as seed inoculum; 3. Dilution of the culture, as recommended in this invention, Is an advantage in most Dunaliella production situations. Duznaliella species are naturally occurring, and usually cultivated, in an environment where evaporation rate Is extremely high. Hence, dilution of the growing media Is necessary to maintain constant salinity. Dilution and mixing of the Dunaliella salina culture during the harvesting operation reduces the number of unit operations in algal production, and improves mixing of dilution water and brine before return into growing ponds.

Operating "Alfa Laval" Model 2181 centrifuge with and without feed parin~g disc.

A Dunaliella sal na culture, containing 6.9 x 10O4 cells. Mlwas pumped Into an "Alfa Laval" Model 2181 centrifuge. The pump was a "Mono" helical rotor pump which was coupled to a "Toshiba" Inverter to allow accurate variable flow rate.

The centrifuge was first tested with the paring discs on the feed and discharge removed. In the culture discharged from the centrifuge It was GSA/i1163C q -4found that there was negligible cell damage.

i The centrifuge was then tested with the feed paring disc inserted i andthe discharge paring disc removed. A feed nozzle designed for maximum ji throughput was inserted into the feed paring disc. Culture from the discharge was examined microscopically. Ruptured cells and active cells were counted, and it was noted that significant cell rupture was caused when the centrifuge was operated with the feed paring disc inserted. The results are shown in Figure 1.

EXAMPLE 2 Separation efficiencies at different dilution.

A Dunaliella salina culture, containing 7.2 x 10 4 cells, ml" was pumped into an "Alfa Laval" Model 2181 centrifuge with both feed and discharge paring discs removed. Before entering the centrifuge the algal culture was mixed with freshwater in a "Lightnin" static mixer. Flowrates of fresh water and culture were controlled by identical variable speed "Mono" helical rotor pumps. A diagram indicating the equipment used in this test is given in Figure 3.

i*9 The amount of -carotene in the discharge from the centrifuge was determined (the 8-carotene concentration being proportional to the concentration of Dunaliella salina cells). From these results the amount of 3-carotene recovered in the centrifuge was calculated. The results of this test are given in Figure 4. It can be seen that dilution of the culture increases separation efficiency markedly at flowrates over 500 litres, hr 1 (for example, at a culture flowrate of 3,000 litres.

hr with no dilution 3.6 grams of a-carotene. hr- I was recovered. At a culture flowrate of 3,000 litres. hr with dilution of 1 part culture to 1 part freshwater 11.7 grams of 3 -carotene. hr 1 was recovered).

It should therefore be obvious that the present invention provides an improved method of recovery by centrifugation of fragile materials from suspension in aqueous solutions of about 30 parts per thousand or greater, which comprises the steps of: Dilution of the culture prior to feeding into the centrifuge to improve separation efficiency; and Utilising a centrifuge In which the shear is minimised.

This can be caused by the removing from conventional centrifuges of the feed paring discs, the feed nozzle and the discharge paring disc.

Further, the feed to the centrifuge could be by way of a low shear pump as a distinct entity to that of the centrifuge, whereby the aqueous solution GSA/1163C

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7 is gradually accelerated in angular velocity to approximately match up to that of the centrifuge itself.

Those skilled in th art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is understood that the invention includes all such variations and modifications which fall into its spirit and scope.

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GSA/1163C

Claims (9)

1. A method for separating microorganisms from a liquid medium, which method comprises the steps of: diluting the medium sufficient to increase the density difference between the microorganisms and the medium; (11) transferring the diluted medium to a centrifuge before the microorganisms are able to equilibrate in density with the medium; and (iii) separating the microorganisms from the diluted medium in the centrifuge.

2. A method according to claim 1 wherein the microorganisms are algae.

3. A method according to claim 2 wherein the algae are species of S the genus Dunaiella.

4. A method according to any one of claims 1 to 3 wherein the medium is brine. A method according to claim 4 wherein the brine is diluted with fresh water.

6. A method according to any one of claims 1 to 5 wherein the centrifuge is modified su as to minimise damage to the microorganisms.

7. A method according to claim 6 wherein the centrifuge is modified by one or more of removing feed paring disc, removing feed nozzle, removing discharge paring disc, and feeding the centrifuge using a low shear pump distinct from the centrifuge.

8. A method for separating microorganisms from a liquid medium substantially as hereinbefore described with reference to any one of the Examples.

9. Microorganisms isolated by the method of any one of claims 1 to 8. A method of producing 3-carotene, which method comprises extracting 3-carotene from Dunaliella separated from brine according to the method of any one of claims 3 to 8.

11. s-carotene produced by the method of claim DATED this TWENTY-SECO D day of FEBRUARY 1991 Western Biotechnology Limited o Patent Attorneys for the Applicant SPRUSON FERGUSON GSA/1163C