Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
  • B03D—FLOTATION; DIFFERENTIAL SEDIMENTATION
  • B03D1/00—Flotation
  • B03D1/02—Froth-flotation processes
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
  • B03D—FLOTATION; DIFFERENTIAL SEDIMENTATION
  • B03D1/00—Flotation
  • B03D1/14—Flotation machines
  • B03D1/16—Flotation machines with impellers; Subaeration machines
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
  • B03D—FLOTATION; DIFFERENTIAL SEDIMENTATION
  • B03D1/00—Flotation
  • B03D1/14—Flotation machines
  • B03D1/24—Pneumatic
• • 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; 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
  • C12N1/06—Lysis of microorganisms
  • C12N1/066—Lysis of microorganisms by physical processes

Definitions

• Dunaliella salina normally is harvested from cultures that are produced in specially constructed outdoor ponds.
• the outdoor ponds typically are constructed in regions with a hot and arid climate with little rainfall and few cloudy days to promote carotenoid production.
• Two distinct methods of aquaculture have been developed for growing algae. These are an intensive mode and an extensive mode. Both aquacultural techniques require the addition of fertilizers to tne medium to supply the necessary inorganic nutrients, phosphorous, nitrogen, iron, and trace metals, that are necessary for biomass production through photosynthesis .
• the salinity of the growth medium is controlled within a specified range, usually between about 18 to 27 percent sodium chloride by weight per unit volume of brine. This range of concentrations is thought to provide the maximum carotenoid production.
• the optimum growth range for Dunaliella salina is said to be between about 18 and 21 percent salinity.
• Maximum carotenoid production in the algal biomass is said to occur at salinities greater than about 27 percent.
• Maximum carotenoid production per unit volume of the brine medium has been reported to occur at about 24 percent salinity.
• Outdoor ponds for intensive aquaculture typically are somewhat expensive and are frequently constructed of concrete and lined with plastic. Brine depth generally is controlled at 20 centimeters, which has been considered to be the optimum depth for producing algal biomass.
• the culture may become infested with a predator that may rapidly increase in number and decimate the Dunaliella salina population.
• the primary predators are the ciliated protozoan Fajbrea salina and the brine shrimp Artemia salina .
• Salt concentrations below about 15 percent other algae tend to compete with Dunaliella salina for nutrients and additional predators may further reduce the Dunaliella salina population.
• the invention is capable of economically dewatering algae obtained from the dilute suspensions found in naturally occurring lakes and ponds.
• Cell concentrations in dilute suspensions sometimes are as low as 2,000 cells per milliliter of growth medium.
• Multiple adsorptive bubble separation units can be used to dewater the algae.
• the algae become more and more concentrated in subsequent adsorptive bubble separation steps.
• the invention includes dispersed gas flotation methods generally, including mechanical and pneumatic froth flotation methods, dissolved gas flotation methods, and electrolytic methods for dewatering algal suspensions of algae of the genus Dunaliella and extracting components from the algae in the absence of undesirable chemical additives or treatments.
• Food grade solvents may be used that result in high recoveries of mixed carotenoids from Dunaliella .
• Electrolytic and dissolved gas flotation are not necessarily equivalent to dispersed gas flotation.
• the aqueous medium is concentrated brine, then more current is needed for the electrolytic flotation technique because brine is more conductive than fresh water. Gases typically do not dissolve as readily in concentrated brine as in fresh water.
• Dunaliella salina can be dewatered by rupturing the membrane that encapsulates the algal bodies and then removing the water by an adsorptive bubble process in the absence of coagulents or flocculating agents. While not wishing to be bound by theory, it is believed that when the membrane encapsulating the algal body ruptures, then the algal body adsorbs on hydrophobic gas bubbles that are intimately contacted with the brine.
• High shear conditions can be used that typically could be expected to disrupt a floe of algal bodies and would be considered undesirable where the process was directed to floating flocculated bodies.
• the algae also appear to contain naturally occurring surface active agents of sufficient concentration and power to readily produce a stable froth. Several rupturing methods are discussed below in the Detailed Description.
• a gas is dispersed into fine bubbles.
• the gas can be air or a gas that does not contain oxygen or oxidizing agents to avoid oxidation of the carotenoids.
• the fine bubbles and the algal sus--:nsion are intimately contacted to adsorb the algae onto the surfaces of the bubbles and to form bubble and alga agglomerates and a brine that is depleted in the algae .
• the bubble and alga agglomerates are separated from the liquid phase as a concentrated froth of algal suspension.
• Flotation aids can be used to enhance recovery, if desired. Flocculating or coagulating agents are not required, at least for dewatering Dunaliella salina in brine, but may be used if desired.
• the ruptured algae are floated by attachment to the gas bubbles, not by flocculation.
• a high shear field can be employed to provide small bubbles and to provide intimate bubble and particle contact, whereas in flocculation and flotation processes, low shear fields typically are used to minimize floe breakage.
• the gas can be dispersed into fine bubbles by sparging the gas into the liquid phase, as in a column.
• a multi-stage loop flow flotation column sometimes called the "MSTLFLO" column, is one apparatus useful in practicing this aspect of the invention.
• Figure 1 represents a flow diagram of a process for obtaining a suspension of algae, dewatering the algae, and extracting components from the algae;
• Figure 1 is discussed below with reference to dewatering a brine containing the alga Dunaliella salina for the purpose of extracting mixed carotenoids from Dunaliella salina .
• a feed stream comprising a suspension of Dunaliella salina in brine is obtained from a source thereof in accordance with step 20.
• the feed stream typically will be obtained by pumping an algal suspension from the source to the equipment that is used for dewatering the algae.
• a centrifugal pump will be used to harvest the algae, although other pumps may be substituted.
• the centrifugal pump is one of the most widely used pumps in the chemical industry for transferring liquids of all types.
• the algal suspension is transported from a source 34 to a dewatering device 36 through a pump 38, which may be a centrifugal pump.
• a pump discharge line 40 supplies brine to the dewatering device.
• a throttling valve 42 in the pump discharge line is provided for adjusting the pressure drop.
• the brine enters the dewatering device through feed inlet line 44.
• a recirculating line 46 is provided for recirculating brine from the discharge side to the intake side of the pump. The flow rate through the recirculating line is varied as necessary to provide the desired percent recycle through valve 50 in the pump loop.
• the process of the invention optionally can include various filtration steps.
• a mechanical filtration step is useful both before and after adsorptive bubble separation of the algae from the brine, as shown in steps 24 and 28, respectively.
• the algal suspension can be concentrated prior to adsorptive bubble separation by using deep bed filtration.
• the algal suspension can be concentrated after adsorptive bubble separation by microfiltration.
• any of these filtration steps can be performed either before or after adsorptive bubble separation and that, in some cases, adsorptive bubble separation may not be necessary.
• adsorptive bubble separation typically provides the most economical means for sufficiently concentrating the algal suspension to obtain its components.
• Suitable deep bed filtration media include those typically used in commercial processes, such as quartz sand, garnet sand, anthracite, fiberglass, and mixtures thereof .
• the media can be washed with fresh water or brine to recover the algal cells for further concentration of valuable components by adsorptive bubble separation.
• adsorptive bubble separation processes have a practical upper limit for the maximum concentration of carotenoids of less than about 10,000 ppm.
• the carotenoid concentration in an algal suspension should be greater than about 10,000 ppm for some purification processes to be economically viable, including dense gas extraction.
• Microfiltration has been determined to increase the carotenoid concentration in a suspension of Dunal iella salina in brine beyond that normally obtainable by adsorptive bubble separation by an order of magnitude with no measurable loss of carotenoids in the permeate. Concentrations of up to about 20,000 ppm have been obtained by the practice of microfiltration in connection with the practice of the invention.
• a schematic of a cross flow microfiltration process is represented in Figure 3. Microfiltration using the apparatus of Figure 3 is shown in the Examples, at VII C.
• the membrane is typically in the configuration of a cylinder and the suspension is pumped through the cylinder.
• the brine passes through the membrane and is removed as permeate through line 69.
• the algal bodies remain in suspension and pass through the cylinder as retentate through line 70.
• the retentate may be returned to the holding tank and circulated through the filter several times until a sufficient concentration has been obtained. Alternatively, the retentate may be sent to another stage of microfiltration or directly to an extractor through line 72.
• the carotenoid globules usually are less than one tenth of a micron in diameter and significant losses normally would be expected in the permeate.
• the algal suspension In the collection zone the algal suspension is contacted with fine bubbles under conditions that promote intimate contact.
• the bubbles collide with the algal bodies and form bubble and alga agglomerates. It is desirable to generate intense mixing in the collection zone to provide a high frequency of collisions .
• the underflow from the rougher which is stream 118, may be provided as the feed stream to the initial scavenger 114 for processing in the scavenging zone 98.
• the underflow from initial concentrator 106 may be recycled to the rougher 100, if desired, or withdrawn as a waste stream, or included in the feed to the initial scavenger. 1.
• Roughing The rougher 100 functions as the initial froth flotation stage in roughing zone 94 for separating the algae from the brine.
• the objective of the rougher is to produce high algae recovery with a moderate increase in concentration. Therefore, the rougher typically is operated at conditions that maximize recovery of valuable products with a modest concentration factor.
• Flotation devices functioning as roughers typically operate at higher superficial gas velocity and thinner froth depth than those functioning as concentrators.
• Performance of the froth flotation device is quantified in terms of the carotenoid concentration in the froth and carotenoid recovery.
• 1. Mechanical Flotation Cells The hydrodynamic characteristics of a mechanical flotation cell 134 are illustrated in Figure 7. Mechanical cells typically employ a rotor and stator mechanism 136 for gas induction, bubble generation, and liquid circulation providing for bubble and alga collision.
• the ratio of vessel height to diameter termed the "aspect ratio" usually varies from about 0.7 to 2.
• pneumatic flotation cells serving as concentrators may be operated in either the collection limited regime or in the carrying capacity limited regime.
• the particle collection rate is limited by the number of collisions between bubbles and algae.
• the carrying capacity limited regime the bubble surfaces are saturated with algal material. Therefore, the particle collection rate is limited by the rate at which bubble surface area is added to the column. It is advantageous to produce a froth whose surface approaches saturation with algal material because it is desirable to minimize the volume of brine sent to the recovery process .
• a long residence time which also means a low superficial velocity, promotes a high algae collection efficiency, and therefore high recovery of carotenoids in the froth.
• a short residence time and high superficial velocity increases column throughput.
• J g values range from about 0.1 to 1.0 cm/s for the recovery of carotenoids from Dunaliella salina .
• stages of draft tubes are used to reduce the axial mixing in the column and minimize short circuiting, thus improving performance of the column.
• the number of stages ranges from about 1 to 5 for recovery of carotenoids from Dunaliella salina . More than one should ordinarily be used.
• the algal suspension feed enters the column through line 212 about 1 to 2 meters below the froth and liquid interface 214 and flows downward.
• the gas enters the base of the column through line 216 and is dispersed into fine bubbles, typically by means of a sparger 218.
• An inert gas including carbon dioxide, nitrogen, helium, or a noble gas may be used to minimize carotenoid degradation.
• the gas typically air, may be injected directly into the base of the column as an internal sparge as shown at 218, or it may be first contacted with water, algal suspension, frother solution, or a combination thereof, before injection as an external sparge.
• Internal spargers typically are fabricated from perforated pipe covered with fabric, such as filter cloth, or from perforated rubber .
• the algal feed suspension 80 ( Figure 5), is pumped to the top of the column through a venturi device which functions as the bubble generation zone 84 ( Figure 5) .
• Gas is entrained into the algal suspension and the resulting mixture passes down through the feed pipe, where most of the bubble and alga collisions occur.
• This feed pipe serves as the collection zone 86 ( Figure 5) .
• the liquid and gas dispersion containing the bubble and alga agglomerates flows through a distributor into the separation zone 88 ( Figure 5) , which is in a separate vessel.
• the agglomerates float to the surface where they accumulate in a froth zone 90 ( Figure 5) .
• the brine depleted in algae underflows the vessel as stream 92 ( Figure 5) .
• the liquid used to generate the microbubbles exits the aeration zone depleted in bubbles through a port and typically is recycled to the microbubble generator.
• the microbubbles leave the aeration zone through the one way plate, where they enter the collection zone 86 ( Figure 5) which is the region below the feed distributor and above the aeration zone. Because of the small size of the bubbles, t -. flow in the collection zone is substantially quiescent, resulting in efficient countercurrent contact of the algal suspension and the microbubbles.
• the brine, depleted in algae leaves through a port in the one way plate as an underflow stream.
• the bubble and alga agglomerates rise through the collection zone where they accumulate in the froth zone.
• Extraction can be performed either batchwise or continuously.
• a batch extraction process has proved useful.
• the organic and aqueous phases are sufficiently agitated so that substantially all of the carotenoids are extracted into the organic phase. Agitation is then stopped.
• the dispersion is allowed to settle so that three distinct regions form, the raffinate, extract, and rag layers. The layers are separated by careful decantation for further processing as outlined below.
• the solvent can be dispersed in the algal suspension prior to one or more dewatering .eps.
• the solvent is said to be predispersed and the feed is said to be preconditioned for extraction.
• solvent can be predispersed in the algal suspension before initiating adsorptive bubble separation.
• the crude extract can be further concentrated in accordance with step 312 by one or more of several techniques, including evaporating the solvent via a flash, distillation, wiped film evaporation, short path distillation, and molecular distillation. Proper selection of a solvent will allow this concentration step to operate at low temperatures where the carotenoids do not degrade or reisomerize .
• the preferred method of processing the crude extract depends on the desired product.
• the column can be washed periodically to remove any polar carotenoids, lipids, and chlorophyll that are not eluted.
• the high purity natural 9-cis beta carotene is produced by dissolving the high purity natural beta carotene in a minimal amount of non-polar solvent warmed to 40° to 50°C to dissolve the beta carotene, chilling the solvent to -20°C to preferentially crystallize the all-trans isomer, and separating the solid and liquid phases.
• the crystallization step may be repeated to improve the purity of the crystals and the supernatant solution.
• the solvent is evaporated from the supernatant solution to yield a preparation enriched in 9-cis isomer to a concentration of at least 75% by weight.
• Beta carotene and other carotenoids derived by the process of the invention may be formulated for sale as any number of products. Spray dried Dunaliella salina powder can be incorporated into animal feeds.
• the rag layer can be contacted with ethanol to recover glycerol.
• the ethanol can then be evaporated and the resulting glycerol residue can be purified by distillation.
• the glycerol may be extracted, decolorized, and distilled for sale as a useful product.
• the cell mass remaining after the glycerol extraction is rich in proteins and can be dried to produce a protein enriched algal meal for use in animal feeds.
• the cell mass can be washed with water to remove residual salts before drying.
• Q 4 and Q 5 were determined using the procedure described previously and Q 2 is the flow rate through line 46.
• the pressure drop across throttling valve 42 ( ⁇ p t ) promotes cell rupture, and is calculated as follows : where p x and p 2 are the pressures measured at pressure indicator 56 and pressure indicator 58, respectively.
• the percent of algae ruptured in the process was determined from cell count data. The number of live cells per ml was measured for the feed intake to the pump through line 60, Q 0 , and the Jameson cell underflow stream 50, Q 7 .
• the percent of algae ruptured, F is calculated using the following equation:
• the cell count is defined as the number of whole cells per milliliter of brine.
• A is the cross sectional area of the filter available for flow
• M 2 and M x are the mass of permeate at times t 2 and t ⁇ , respectively.
• the feed flow rate was measured by a flow meter 78.
• the temperature was measured by a thermometer. Samples were taken of the retentate and permeate, and analyzed for carotenoids.
• Diafiltrations were performed to reduce the salt concentration in the retentate. Fresh water was added to feed tank 62. Additional filtration and water dilution stages were performed as required to achieve the desired salt concentration in the final retentate. At the end of the 6 hour run, the remaining retentate was diluted with an equal volume of fresh water and filtered for 13 minutes to return to the initial volume. A second diafiltration was performed by addition of another equal volume of fresh water followed by filtration for 13 minutes to return to the initial volume. The flux measured for these diafiltration experiments is presented in Figure 4.
• Brine containing a suspension of Dunaliella salina was processed in a mechanical pretreatment device to rupture the cells and then treated in a . Jameson cell.
• the Jameson cell had a ratio of downcomer diameter to orifice diameter of 8.6 and a ratio of riser diameter to downcomer diameter of 5.
• the cell J was 0.44 cm/s.
• the jet velocity was 21.5 m/s.
• the downcomer superficial velocity was 0.20 m/s.
• the downcomer residence time was 15.1 s.
• the air to feed ratio was 0.52.
• the solids fraction in the froth was 0.02% on a gas free basis.
• Example 38 Froth generated during the run described in Example 34 was collected and processed in a Jameson cell having the geometry described in Example 34 to further concentrate the carotenoids.
• the cell J g was 0.29 cm/s.
• the jet velocity was 11.9 m/s.
• the downcomer superficial velocity was 0.14 m/s .
• the downcomer residence time was 21.7 s.
• the feed pressure was 22 psi.
• the air to feed ratio was 0.49.
• Carotenoid recovery over the course of the run averaged 68%.
• the fraction of solids in the froth was 8.3% on a gas free basis.
• Example 48 The MSTLFLO column described above was operated with all the draft tubes removed. No frother was added. The pH of the brine ranged between 6 and 7. The same sparger was used. The feed distributor was located about 36 inches below the froth overflow weir. Example 48
• the flow of gas to the air sparged hydrocyclone was started at the desired rate be tore the feed flow commenced at the setpoint value.
• the ASH unit consisted of a plastic shell that contained a 2 inch diameter polyethylene membrane approximately 18 inches in length with an average pore size of 2C microns. The gas pressure on the pressurized side of the membrane was maintained between 15 and 10 psig. No surface active materials were added to facilitate the flotation. Samples were taken of the feed, froth, and underflow to quantitate ASH performance. The J g was 5.9 cm/s. The gas to feed ratio was held at 5.8. The liquid residence time was 1.3 s. The solids fraction in the froth was 0.09% on a gas free basis. The recovery of carotenoids was 68%. Examples 51 to 54
• the mixture was agitated at 600 rpm for 20 minutes. Samples of brine were periodically removed to determine mass—transfer kinetics. After 20 minutes, the mixer was stopped and the phase separation time was recorded. The oil phase was decanted, and the brine phase returned to the mixer. Four hundred milliliters of fresh solvent were charged to the extractor, and the multiphase mixture was agitated at 600 rpm for 20 minutes. The phases were again allowed to separate for 20 minutes. The solvent phases from both extraction stages were allowed to settle for an additional four hours to reduce the volume of the gelatinous algal residue. The solid phase was then centrifuged to separate the solvent and brine phases from the algal residue. The solvent was evaporated from the carotenoid extract and olive oil was added to produce a suspension of carotenoids in olive oil. Extraction and phase separation data are provided in the following examples.
• Example 66 Extraction of carotenoids from concentrated froth into limonene.
• Example 67 Extraction of carotenoids from concentrated froth into ethyl butyrate.

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• 1996
  • 1996-12-20 US US08/772,589 patent/US6000551A/en not_active Expired - Lifetime
• 1997
  • 1997-12-10 JP JP52883898A patent/JP2001506866A/ja not_active Ceased
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  • 1997-12-10 AU AU58978/98A patent/AU5897898A/en not_active Abandoned
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