Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
  • C12P7/56—Lactic acid
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
  • C12M47/10—Separation or concentration of fermentation products

Definitions

• This invention describes the production process of polylactic acid (PLA) obtained by sterification and polymerisation of purified lactic acid, produced through a fermentative process.
• PLA polylactic acid
• the PLA is a natural polymer composed by lactic acid chains.
• This polymer is a completely biodegradable aliphatic polyester when placed in a biologically rich environment, such as a composting plant.
• a biologically rich environment such as a composting plant.
• this polymer is a substitute for the common polymers obtained through petroleum is still quite small. This situation is a consequence of the fact that the industrial processes used nowadays to produce this polymer are not able to provide enough good quality product at competitive prices.
• This polymer is obtained by sterification and polymerisation of lactic acid dimmers.
• This acid has an asymmetrical carbon that can exist under the form of two enantiomers, L (+) - lactic acid and a D (-) - lactic acid.
• the obtain dimmer has two asymmetrical carbons and, consequently, can exist under the form of three enantiomers: dimmer L (two asymmetrical carbons have configuration L), the D dimmer (two asymmetrical carbons in configuration D) and the meso dimmer (an asymmetric carbon with configuration D and the other configuration L).
• the properties of the obtained polymer are subject to the stereochemistry of the lactic acid and its dimmer.
• Polylactic acid is synthesized mainly through isomer L.
• the lactic acid is obtained, mainly, in a biological way, through fermentative processes using lactic bacteria, in rich environments containing fermentable sugars.
• the lactic acid can be obtained by a great variety of bacteria of the Lactobacillus type, but according to the culture used in fermentation we can obtain any of the two isomers and their matching racemic mixtures.
• the lactic acid isolation and purification use techniques such as filtration, extraction, electrodialysis, reversed osmosis and chromatography, or, in some cases, the combination of two or more techniques at the same time. Some of these techniques are very expensive and difficult to put into practise or use organic solvents harmful to the environment.
• the lactic acid sterification and polymerisation into PLA is often carried out using chemical methods involving organic solvents, which originates effluents that are difficult to treat and to eliminate.
• the European patent nr. 230,021 describes a process through which glucose is continuously fermented into lactic acid and in which this acid is continuously removed by direct electrodialysis of the fermentation broth.
• Yeast extracts and inorganic salts are used in this process as supplements.
• the U.S. patent nr. 5,002,881 describes a lactic acid purification method in solutions, in which the lactic acid is produced as ammonia lactate, through a medium with glucose. The removal of the ammonia lactate is carried out using UF and in which the retained returns to the fermenter, reducing the energetic consumption. The fermentation is carried out by Bacillus coagulans. The permeable concentration is made by reverse osmosis (RO) and the lactic acid recovery is made by ED.
• RO reverse osmosis
• the main disadvantage of this process is in the use of a bacterial strain that requires the addition of amino acids as a nutrient, which increases the costs of the process. 4.
• the US patent 6,319,382 B1 describes the use of enzymes directly in the fermenter in order to hydrolyse the lactic proteins supplied as a nutritional supplement for the fermentation of the lactic acid.
• Commercial proteases are also used according to the fermentation pH to hydrolyse the existing proteins in the environment.
• a combination of ion-exchange resin ED is used in the recovery of lactic acid.
• the US patent 5,892,109 B1 describes a process for the production of lactic acid and its separation and recuperation, using, to this effect, an extraction with water-soluble trialquilamine in the presence of carbon dioxide.
• the lactic acid is recovered in the resulting organic phase.
• the main disadvantage of this process is precisely the use of organic solvents to purify the lactic acid.
• the US patent 6,187,951 describes a very similar process to the one in the previous patent. It describes the lactic acid production through the fermentation of a sugar solution carried out by Lactobacillus. The lactic acid produced is precipitated by the addition of ammonia carbonate and then recovered using long chain trialquilamine.
• a sugar rich growth medium is used, specifically, sugar-beet molasses.
• the beet is a root, where high amounts of sucrose are stored, and it is becoming more and more a source of common sugar. Only half of the available sugars are extracted from beet, because the other 50 % would imply a highly expensive process to extract.
• Three main products are obtained from the sugar extraction from beet: the refined sugar, the pulp and the molasses.
• the sugar-beet molasses contain a high organic content, which can become an environmental problem when released in the environment without prior treatment.
• sugar-beet molasses are all fermentable. Molasses are comprised of monosaccharides like fructose, disaccharides like sucrose and other polysaccharides. Besides sugars, this growth medium contains mineral and protein supplements, which, therefore do not need to be added in large amounts. The composition of common sugar molasses can be observed in the following table.
• Vitamins (mg/100g dry weight)
• the composition of each lot may vary slightly, even if the lots originate from the same source.
• the sugar-beet molasses In order to increase the fermentation yield, the sugar-beet molasses must be submitted to a pre- treatment. This way, the undesired impurities and toxic substances, which sometimes exist in sugar molasses, are separated and/or inactivated.
• the pre-treatment consists firstly, of a dilution to 35° Brix, followed by an acidification with H 2 SO 4 2 M to a pH of 4.0, boiled for 5 min and finally centrifuged and filtered. After this treatment the growth medium is now pasteurised.
• This growth medium is then conducted to a fermenter where it will be fermented by a pure culture of lactic bacteria, from a strain known as Lactobacillus delbrueckii.
• Lactobacillus delbrueckii a strain known as Lactobacillus delbrueckii.
• These bacteria exist in many food products and can be also found in the normal flora of the mouth and intestines of many animal species, including humans and are rarely pathogenic.
• the bacteria cells have an average diameter of about 0.7 micrometers.
• the microorganism chosen for this fermentation process is an unicellular bacteria.
• the cells have a rod like shape and do not possess mobility. They are Gram Positive bacteria, due to the peptidoglycan that exists on the cell wall, are microaerophile and have a strictly sarcolastic metabolism.
• the strain is homofermentative, which means that sugars fermentation into lactic acid occurs exclusively by the Embden-Meyerhof way, or glicolisys, and therefore there are no other products resulting from the fermentation.
• This strain produces only the L form of lactic acid and the bacteria do not consume this product. It has an optimal growth temperature between 30 and 50 °C and an optimal pH between 6 and
• the strain used in this invention has complex nutritional needs. Therefore, the growth medium must contain between 12 and 13 % of glucose, 0.25% of ammonia and phosphate, and B complex Vitamins. In order to satisfy these needs, after the dilution of the molasses, a solution of (NH 4 ) 2 HPO 4 has to be added, to suppress the growth medium lack of these two components.
• the growth medium contains a wide variety of remaining cells and spores from its constituting elements. These must be eliminated prior to inoculation, in order to maximize the fermentation yield.
• the sterilization occurs continuously and takes place in heat exchangers, with the shape of concentric tubes, where steam or overheated water at 140 °C passes trough the external tube and the growth medium passes trough the internal tube.
• the hold-up is between 30 and 120 seconds depending on work rate.
• the medium must then pass trough a cooler, in order to reduce its temperature to match the fermentation temperature.
• un-sterilized medium (cold) is crossed with sterilized medium so that the last one can be cooled to the fermentation temperature.
• This type of sterilization has the advantage of reducing the sterilization cycle time, easy control and productivity increase, without the destruction of the existing nutrients in the solution.
• a tensioactive compound such as Tween 80 in a concentration of 0.001 % can minimize this effect.
• the sterilization of the reactor and of the filtration systems is made by direct steam injection; this is possible because the filtration membranes are thermoresistant.
• the fermentation takes place in a continuously agitated reactor, like a chimostat, with a high cell concentration.
• This system avoids, simultaneously, substrate inhibition - since the substrate is continuously supplied into the fermenter - and product inhibition - since the lactic acid is continuously removed from the fermenter.
• the high cell concentration is obtained trough cell immobilization.
• the immobilization is achieved by restraining the cells to a confined space using a physical barrier.
• the physical barrier used in this invention is cell recycling trough ultrafiltration membranes. This method has several advantages, mainly in product productivity increase, and in high fermentation yield. It also presents low energy costs and advantages in operation safety and stability of the production system.
• this system prevents many of the mass transfer problems, caused by diffusional problems, from occurring and also prevents contamination of the fermentation broth, where the final product can be found, as well as contamination of the fermenter, where the fermentation is occurring.
• this system allows operating at high dilution rates, without system collapsing, phenomenon known has wash-out.
• the high dilution rates at which the system operates is another very important factor that contributes to the productivity of the system. All of these factors combined make this a viable and profitable system.
• a pre-inocule For the fermentation process to be initiated, a pre-inocule must be prepared, with 10 % of the reactor's work volume. This can be prepared in smaller reactors. Moreover, the fermentation process must begin with a discontinuous phase of about 8 hours in order to obtain biomass.
• the bioreactor is ready for full functioning.
• a general diagram of the fermentation and purification process can be observed in the process description diagram. Probes control the fermentation conditions continuously. The information is analysed so that the conditions are stable, especially regarding temperature, pH and cell concentration. The pH is controlled by addition of concentrated NaOH (recovered from following process steps).
• the purification of the Lactic acid, that exits the fermenter in the form of Sodium Lactate, due to the added NaOH, is initiated in the ultrafiltration membrane system used to maintain the high cell concentration. These ceramic membranes retain bacterial cells and other macromolecules, which are then reintroduced in the reactor.
• the fermentation broth containing the lactic acid is conducted to further purification.
• the next purification step takes place in a nanofiltration system.
• This system contains nanofiltration membranes with an exclusion limit between 0.5 and 5 nanometers. This way all of the contaminants that are bigger than the lactic acid are eliminated.
• Electrodialysis is an electrochemical process that allows the separation, purification and concentration of ionic substances, by applying an electrical potential difference.
• ⁇ Ve is the use of the bipolar membrane that allows the purification and concentration to occur simultaneously.
• the electrodialysis process result two fractions, one containing lactic acid and residual sodium lactate, and the other fraction, containing NaOH.
• the residual sodium lactate fraction can be reduced by increasing the number of bipolar membranes used.
• PLA polylactic acid
• the lactic acid, produced, purified and concentrated in the previous steps is transported to a CSTR (continuously stirred tank reactor), where it will be dehydrated and pre-condensed.
• the reactor is equipped with a stirrer, a condenser and temperature control devices, important to assure that the reaction occurs for 2 hours at temperatures between 160 and 200 °C, at atmospheric pressure.
• the effluent water contains only residual lactic acid, meaning that it is not considered as a dangerous effluent and therefore does not require any treatment.
• lactic acid dimmers and oligomers are obtained, which are then separated, purified and concentrated by distillation in a distillation column.
• the effluent resulting from this process is recirculated and reintroduced in the first polymerisation reactor (CSTR 1).
• the concentrated dimmers and oligomers are then transported to a second reactor (CSTR 2) where the polycondensation reactions will occur.
• the reaction time for these reactions is 20 hours at temperatures between 160 and 200 °C, and at reduced pressure around 0,2 - 20 mmHg, in the presence of a catalyst, iron lactate, used in a concentration of 0.005-0.5% (p/p), regarding the initial lactic acid.
• the polycondensation reactions should take place, maintaining the initial conditions, until the desired molecular weight is achieved, around 25000 - 30000 kDa.
• the condensation step can be performed in several steps, lowering the pressure gradually.
• the yield of the polycondensation reaction is around 80% (p/p), and the yield in dimmers and oligomers around 20%.
• the polylactic acid is collected and the resulting dimmers and oligomers are transported to a fractional distillation column, where these dimmers and oligomers are purified at temperatures between 144-170 °C and a pressure between 0.5-0.8 mmHg. From this fractional distillation, purified dimmers are obtained which can be recycled and reintroduced in the polycondensation reactor (CSTR 2), and pure lactic acid residues and water, which are recycled and reintroduced in the first polymerisation reactor (CSTR 1). Since the non- condensate fractions are recycled, the overall yield is close to 100 %.
• Biodegradable which means that microorganisms are able to alter the plastic properties and modify its chemical structure.
• PLA can be produced with a wide variety of properties and characteristics, because the lactic acid molecule is a chiral molecule with two asymmetry centers and therefore three possible enantiomers derived from this dimmer. This way controlling the stechiometry of the lactic acid dimmer formation, it is possible to obtain different mixtures of lactic acid containing the L and D or meso-lactate forms.
• PLA is often compared to PET (Polyethylene thereftalate), due to the enormous similarity between the properties of the two compounds.
• PLA can be used for the same purposes as normal polyesters.
• the main applications are:
• PLA has many other different applications in the form of fibber, resin or thermoplastic. Future perspectives are very positive for PLA. The decrease in the price of production is increasing the range of applications and the level of substitution of petroleum-based plastics for biodegradable plastics such as PLA will increase significantly in the forecoming years.
• the diagram represents the continuous process for the production of polylactic acid from sugar molasses.
• the entrance flow (1) (constituted by fresh broth previously prepared), passes through a sterilizer and then through a cooler (2) (where it is sterilized and cooled to the fermentation temperature). It then enters in the fermentation tank (3) in witch sugars are fermented to lactic acid.
• the temperature inside the tank is held constant with circulating water.
• Into the fermentator enters a sodium hydroxide flow (4) from the electrodialysis system (5).
• the exit flow (6) feeds an ultrafiltration system (7) in witch cells are separated from the fermentation broth.
• Cells can or not (in case of purge), re-enter the fermentator through flow (8).
• the fermentation broth, containing the sodium lactate flows (9) to a nanofiltration system to separate macromolecules.
• the held can or not, be recirculated to dilute the entrance flow (1), through flow (10).
• Dimmers can be directly polymerised in the casting box (25) by adding a catalytic octanoate (between 180 and 195°C) and impurities are recirculated to flow (14) to a new polycondensation.
• Lactic acid fermentation is held in a 50L fermentator coupled to an ultrafiltration system with a size exclusion of 5 kDa and a total membrane area of 3.75 m 2 , with internal and external pressures of 4.4 e 2.9 bar, respectively.
• aqueous broth composed of whey, milk protein concentrate and additional nutrients such as inorganic salts and cystein
• additional nutrients such as inorganic salts and cystein
• 9 g of Lactobacillus helveticus and 26.5 g of Flavourzyme ® were added. Fermentation in batch mode was taken for 9 hours, after witch continuous mode was started. Added fresh broth contained only whey, lactose and Flavourzyme ® . pH was made constant at 5.75 by addition of ammonia gas.
• Biomass density was held constant between 7 and 8% by a continuous purge. Permeate flow was continuous during all fermentation at 1 Uminute.
• Dilution rate varied between 0.15 and 0.3 h "1 . This rate did not alter the yield that was constant during the 34 days of fermentation.
• the concentration of lactate in the flow-through was about 4% and productivity, at a dilution rate of 0.3 h-1 was 12 g/l.h.
• the lactic acid in the permeate was isolated using a combination of ion-exchange resins with quelating agents, followed by two consecutive electrodialysis steps.
• the recovery rate was about 85-90% depending on the starting sugar amount.
• the aqueous lactic acid solution from purification processes is led to a 25 dm 3 CSTR.
• This solution contains 85% in weight of L-lactic acid (17.6 kg).
• the reactor is equipped with a condenser, heating shirt and temperature control. Reaction lasts for 2 hours at 160-200 °C and at room pressure in order to obtain 4.38 kg of distillate.
• Condensation reactions should occur, in the previously described conditions, until the polymer has the desired molecular weight (25000-30000). Condensation step can be taken in several steps, with a progressive diminution of pressure.
• Polylactic acid is redraw and dimmers are redirected to a fractionated distillation column, in witch they are purified at temperatures between 144 - 170 °C, and a pressure between 0.5 - 0.8 mmHg.
• the purified dimmers are recirculated to the second CSTR and the water and monomers are recirculated to the first one.

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