BRPI1005896A2 - system for obtaining bioproducts - Google Patents

Abstract

SYSTEM FOR OBTAINING BIOPRODUCTS. The present invention is an integrated system for obtaining large scale bioproducts, particularly biosurfactants, sugars, biofuels and enzymes for biofuel production. Said system comprises a bioreactor that integrates with different components in which special membranes are employed, which serve, among other purposes, the separation and recycling of microbial cells, ensuring the sterilization of the culture media involved in the process (without prejudice to thermolabile components), reduce foaming (thus avoiding the addition of defoamers that could de-characterize the product), allow to concentrate the products of interest and allow to recycle streams containing unused nutrients as well as water to the bioreactor.

Description

BIOPRODUCTS SYSTEM

FIELD OF INVENTION

The present invention relates to a system for obtaining large-scale bioproducts, whereby a bioreactor is integrated into different processes in which special membranes are used with the aim, among others, of separating microbial cells, ensuring sterilization of the medium. cultivation, reduce foaming, concentrate the products of interest and provide oxygen to the microbial cells. Among these products of interest that can be obtained with the integrated system are biosurfactants, biofuels and enzymes for biofuel production. BACKGROUND OF THE INVENTION

Biosurfactants are compounds with biologically produced surfactant properties using microbial strains, which are capable of improving the removal efficiency of certain types of pollutants, such as oil spills and their derivatives. These hydrocarbons aggregate with soil components, making their removal difficult and causing serious environmental problems. In the last decade these biosurfactant compounds have become

industries are of interest because they allow a variety of applications and low cost production possibilities, depending on the productivity of the selected strain, the raw material employed and the purification processes involved. Most known producer microorganisms need to be

aerobic conditions for the efficient production of these surfactants. However, introducing aeration into the culture medium, usually using oxygen or air bubbling, can lead to the formation of large amounts of foam. The technique therefore requires the use of foam controllers, generally defoaming agents, which contribute to the cost of the final product and the quality of that product.

Another aspect to be considered is the ease of contamination of equipment and materials by exogenous microorganisms, given the volume of product to be treated when working on a large scale. All equipment must be sterilized before and at the end of each production campaign, as well as the bioreactor feed media.

In most situations thermal sterilization cannot be applied as it could cause damage to microorganisms or cause degradation of inputs. One of the solutions that has been employed is the use of special membranes, developed to serve different purposes.

RELATED TECHNIQUE

Permeable membranes have long been used in various areas of

application. Improvement of product separation technologies adapted for use in the microbiological field is in rapid development worldwide.

The specialized technical literature describes the use of separating membranes in several situations: cultivation and cell separation in fermentative processes, blood plasma separation, enzyme and protein separation, among others.

Brazilian patent application Pl 0418820 (Probiogen AG) describes a bioreactor and a method for culturing and growing cells, wherein the cells to be cultured are situated in hollow filament membranes and are alternately brought into contact with a liquid phase. nutrient medium and with a gaseous phase, obtaining high cell density.

US 7129082 (CSIR) teaches a method of culturing living cells using a bioreactor that supports a matrix formed of a porous foam material into which living cells are anchored; The support also contains an oxygen carrier liquid that saturates the pores of the porous matrix. Liquid is a synthetic or semi-synthetic constituent that permeates the matrix releases oxygen to cells and absorbs carbon dioxide from cells.

The use of microfiltration units involving hollow fiber membranes to perform the sterilization of bioproduction process lines has been proposed by Carneiro, L. et al. - Desalination 148 (2002) - Cold sterilization and clarification of pineapple juice by tangential microfiltration ) 93-98.

Brazilian patent application Pl 0405952 (Petrobras) describes the obtaining of a biosurfactant and its use for the remediation of oil-impacted soils. The biosurfactant is constituted of the culture medium free of microbial cells, resulting from the cultivation of a strain of Pseudomonas aeruginosa isolated from petroleum environment, producing ramnolipids by fermentation conducted under membrane oxygen diffusion aeration.

In Brazilian patent application Pl 0600449 (PoIymar) a bioreactor equipped with continuous extraction using semi-permeable chitosan membranes is described, allowing to circumvent relevant technical aspects regarding the separation and concentration of products resulting in biotransformations. The process involves three steps: microbiological transformation, osmotic diffusion separation and continuous extraction through liquid-liquid extractor coupled to the system.

In a membrane contactor, the separation membrane is fully integrated with the extraction or absorption operation to fully exploit the benefits of both technologies. Applications that have been developed can be in both water and gas treatment. Some recent developments introduced in the industry teach its use from selective removal of heavy metals in galvanic process baths, through the recovery of organic water components in chemical industry processes and also ammonia recovery from gaseous streams, highlighting the wide scope of application. of these materials - Klaassen, R. and collaborators - "Membrane contactor applications" - Desalination 224 (2008) 81- 87.

One of the problems that needs to be overcome in a bioprocess is foaming. When using conventional air or oxygen bubbling technique, strict control of the process conditions is required so that stable foams are not formed. To avoid the use of defoaming agents that could de-characterize the product and compromise its quality, the most appropriate control can be done by means of a dispersive oxygenation system using membrane contactors (Kronemberger et al. "Oxygen-controlled biosurfactant"). production in a bench scale bioreactor "- Applied Biochemistry and Biotechnology 147 (2008) 33-45. SUMMARY OF THE INVENTION

The present invention is an integrated system for obtaining large scale bioproducts, particularly biosurfactants, biofuels and enzymes for biofuel production. This system uses a bioreactor integrated to different processes that employ special membranes, which serve, among other purposes, the separation of microbial cells, the sterilization of the culture medium and equipment involved in the process, the reduction of foam formation. , oxygen supply to the cells and concentration of the products of interest.

The system for obtaining large scale bioproducts comprises the following components: - a bioreactor (BR) integrated with the other components; - a culture medium preparation tank (TQ1), equipped with a mechanical stirrer, containing the nutrients necessary for the maintenance of the microorganisms;

- a first membrane microfiltration module (MF1) to ensure sterile culture medium is fed to

bioreactor;

- a second membrane microfiltration module (MF2) for separating microorganism cells after fermentation;

- a hollow fiber membrane contactor-type oxygenation module (OX);

- a reverse osmosis (OI) module for product concentration; depending on the product of interest, a filtering module (UF) may be used together or alternatively with that module;

- a product storage tank (TQ2);

- flow lines connecting the components, all provided with control means connected to a process control loop (C).

Membrane microfiltration modules (MF1 and MF2) ensure system sterilization. The control loop (C) monitors foam formation as well as the major operating conditions in the bioreactor such as pressure, temperature and pH. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows schematically the flowchart of the system object of the present invention.

Figure 2 schematically shows the operation of the oxygenation module of the system object of the invention.

Figure 3 graphically shows the production of single batch batch ramnolipid biosurfactants according to the system of the present invention. Figure 4 graphically shows the production of ramnolipid biosurfactants under fed batch regime according to the system of the present invention. DETAILED DESCRIPTION OF THE INVENTION In order that the integrated system for obtaining bioproducts can

better understood and evaluated, its detailed description will be based on and accompanying the figures accompanying this report.

The system of the present invention can be operated in distinct, single or combined regimes: single batch, fed batch, and continuous, introducing minor adaptations in the conduct of the process when required. Generally speaking, the system comprises an unprecedented arrangement of its components, as shown in Figure 1 and described below, in their main aspects. Initially, a culture medium (1) containing the nutrients

required for fermentation is prepared with purified water by means of membrane microfiltration or reverse osmosis before being introduced into a preparation tank (TQ1) fitted with a mechanical stirrer; only then is the mechanical stirrer triggered. This culture medium is then transferred to a bioreactor (BR) by means of a transfer pump (not shown in the figure), passing through a first membrane microfiltration module (MF1) to be sterilized.

The bioreactor (BR) is also equipped with a mechanical stirrer and has a double-walled outer jacket for the passage of thermal fluid. This thermal fluid allows the temperature to be kept constant during the fermentation process, and can also be used to thermally sterilize the bioreactor without the need for direct steam injection.

An amount of inoculum (2) is fed to the bioreactor (BR), which operates under pressure up to 2 kgf / cm2 and which is controlled by means of a control loop (C) acting on two control valves (not shown). ), in split-range air addition and removal. The control loop (C) also acts on specific instruments such as pH and dissolved oxygen analyzers, temperature, pressure, media level and foam level transmitters installed on flow lines that are part of the system; Such instruments serve to monitor and control reaction conditions. The bioreactor output current (3) is fed to a second microfiltration module (MF2) or to an oxygenation module (OX). After filtration, the stream (5), rich in microorganism cells, is fed back to the bioreactor (BR). A portion of the cell-free permeate stream (4) leaves the second microfiltration module (MF2), is fed to the oxygenation module (OX) and then returned to the bioreactor (BR) as reaction medium (6). Another part (7) of this permeate stream (4) proceeds to a product storage tank (TQ2).

Membrane microfiltration of the reaction medium is of fundamental importance to the system, as is the concentration of microorganism cells by microfiltration and the concentration of the product by ultrafiltration or reverse osmosis, as it is a guarantee of product sterility and enables different operational configurations, such as continuous processing without bioreactor unloading or operation with cells at different metabolic stages. To ensure the good performance of the microfiltration modules (MF1 and MF2), a backwash system is provided, which is periodically operated to prevent clogging of the membranes.

The hollow fiber membrane contactor-type oxygenation module (OX) promotes oxygenation of the reaction medium stream (6). The two currents [liquid (L) and gaseous (G)] do not contact each other directly. This technique constitutes a non-dispersive oxygenation process, since the liquid current (L) is forced to circulate through the oxygenation module shell, being in contact only with the outside of the hollow fiber, while the gas stream (G) 1 selected from compressed air, oxygen enriched compressed air and pure oxygen, flows through the fiber interior. Figure 2 illustrates this oxygenation scheme in a simplified manner.

Depending on the type of crop being used and the oxygen demand required by the microorganism, different membrane types may be used. The combined use of this oxygenation module (OX) may also be required with conventional oxygenation techniques, such as bubbling of compressed air or oxygen, as is the case when using Pichia yeast strains, which have a high demand for oxygen. oxygen. Hydrophobic microporous membranes may also be used.

as contactors, provided that the culture medium has surface tension values close to those of pure water throughout the process. Microporous support composite membranes also coated with a thin layer of dense material such as silicone or Teflon may also be used. However, in most cases it is advisable to use a dense type silicone fiber membrane. The entire contact area between the liquid (L) and gas (G) phases is fixed and defined by the available membrane area. Oxygenation supplied to the system is controlled through the supply air pressure and purge flow. It is common for the product of interest to be at a concentration

lower than that desired at the end of the reaction. In this case, a part (8) of the product stream (9) leaves the product storage tank (TQ2) and is led to a reverse osmosis (OI) or ultrafiltration (UF) module for concentration, where particles of small dimensions, of the order of a few dozen Angstroms (in the case of OI), and may reach a few hundred Angstroms (in the case of UF), are retained. A pressure regulating valve installed downstream of the module (not shown in Figure 1) ensures a pressure differential between the concentrated product stream (10) and the permeate stream (11), which must be within a range of 15 kgf / cm2 and 25 kgf / cm2, preferably at 20 kgf / cm2.

The temperature of the concentrate is maintained by means of a heat exchanger so as not to affect product stability. The concentrated product stream (10) is fed to the product storage tank (TQ2). The permeate stream (11) is basically water which can be reused in the preparation tank (TQ1) for the composition of the culture medium together with the feedstock stream (12) forming the stream ( 13) which will be fed to the first microfiltration module (MF1) for sterilization, which is supplied to the bioreactor as a culture medium (1).

As mentioned above, the system of the invention can operate continuously in single batch and fed batch. As a result, product transfer valves are properly controlled and actuated due to the "modus operandi" of the process. Alternatively, depending on the product of interest, the

Reverse osmosis may be replaced by equipment more suited to the situation, such as filtering or nanofiltration modules, more compatible with the size of the molecules to be treated, ensuring the stability and quality of the final product, as well as recycling the unused substrate to start of the process.

The advantages gained from the integration of biotechnological processes become clear to those skilled in the art, especially when it comes to expanding the production of a particular bioproduct. These advantages include the quality assurance and standardization of the product, the possibility of using various types of raw materials, the possibility of employing various modes of conducting the process, obtaining more pure and concentrated products, greater control. In foam generation, the lowest energy expenditure for the sterilization of the inputs, facilitating their conservation and storage, and the integrated concentration of the product, allowing reuse of unused nutrients as well as water in the process.

The above described system is particularly interesting for obtaining large scale biosurfactants. These products are used for various applications, including cleaning of oil-impacted soils, advanced oil recovery due to reduced environmental impacts and replacement of chemically synthesized surfactants, which are widely used today. .

By providing a high oxygenation rate to the cells, the system is also interesting for the cultivation and production of bioproducts by Pichia yeast, particularly Pichia pastoris, in genetically modified strains, due to the fact that such cells commonly require high levels. oxygenation to achieve high cell density and produce bioproducts at higher rates. Bioproducts are obtained by biochemical process, which uses

microorganisms conditioned for this purpose, including bacteria of the genus Pseudomonas, which consume a substrate and produce ramnolipid biosurfactants. The process is strictly aerobic, so the use of conventional bubbling oxygenation leads to the formation of very stable foams. The use of oxygen by dispersion of air (or oxygen) in the liquid phase allows the bioproduct to be obtained on a large scale, since it allows to achieve a more complete control of foam formation. EXAMPLES

The integrated system object of the present invention will be illustrated below using glycerol as the carbon source supplied to microorganisms for obtaining large-scale ramnolipid biosurfactants.

The experiments were conducted in a bioreactor equipped with a mechanical stirrer working at a pressure of 2 kgf / cm2 at 30 ° C. However, the system can be scaled up without losing its characteristics and can operate in temperature ranges between 15 ° C and 60 ° C, under continuous, single batch and fed batch regime. According to a standard procedure adopted, the experiments were carried out under batch regime in a bioreactor equipped with mechanical stirrer, with capacity of 3.0 liters of culture medium, using non-dispersive oxygenation and dense silicone membrane contactors. hollow fiber form. The contact area between the liquid and gas phases is defined by the available membrane area, in this case equal to 32 m2. Four microfiltration modules were used in the microfiltration step to separate cells with hollow fiber membranes operating in parallel, totaling 100 m2 of area. These modules are powered by a circulation pump with a nominal flow rate of 7 m3 / h provided with a frequency converter operating in conjunction with the discharge pressure control loop. The pressure drop on these modules is about 0.6 kgf / cm2. The pressure differential between the module and the permeate is in the range 0.8 kgf / cm2 to 1.2 kgf / cm2, preferably 1 kgf / cm2, with a pressure drop of 0.1 kgf / cm2. , the control being made by means of the control loop that acts on the concentrate discharge valve. The permeate recovery was 80%. The backwash system was periodically activated to prevent clogging of the membranes. Example 1

The bioreactor containing culture medium for Pseudomonas aeruginosa PA1 was kept under agitation of 150 rpm and a temperature of 30 ° C, in a single batch regime, followed by graphing the consumption of raw material (glycerol) and the production of ramnolipids at the same time. over time. The resulting graph is shown in Figure 3.

36.6 mg / Lh of ramnolipids were produced after 7 days of production with a consumption rate of 119 mg / Lh of glycerol, resulting in a product concentration close to 7.0 g / L at the bioreactor exit before concentration by reverse osmosis. Example 2

The experiment was carried out under a batch fed regime in a bioreactor equipped with mechanical stirrer, with capacity for 3.0 liters of culture medium, using non-dispersive oxygenation and dense silicone membrane contactors in the form of hollow fibers. The bioreactor containing culture medium by Pseudomonas aeruginosa PA1 was kept under agitation of 150 rpm and temperature of 30 ° C, under long-term fermentation conditions with extra glycerol and sodium nitrate addition, followed by graphing the consumption of matter. (glycerol) and the production of ramnolipids over time. The resulting graph is shown in Figure 4.

The experiments indicated a maximum consumption of 150 mg / Lh of glycerol and 200 mg / Lh of oxygen, generating 44.3 mg / Lh of ramnolipids, resulting in a product concentration of 16.9 g / L at the bioreactor outlet, before concentration by reverse osmosis after 16 days of production.

As can be seen, the integrated system proposed here allows to circumvent problems commonly encountered in biotechnological processes, such as excessive foaming. By integrating different types of processing, microbial cells can be concomitantly microfiltrated and returned to the bioreactor, allowing for high product concentrations as well as the subsequent concentration of the product stream in reverse osmosis or filtration modules, depending on the product of interest. On the other hand, unconsumed nutrients can be recycled to the bioreactor, contributing to the economic viability of the processes.

In order to illustrate the inventive concept, the system has been described preferably with respect to the production of biosurfactants. However, it can be configured for other applications, such as for the production and concentration of enzyme-containing streams, which can be obtained using different microorganisms, such as Pichia sp. and from other strains (Candida sp., Rhizopus sp., Geotrichum sp., Penicillium sp., Aspergillus sp.) as well as other hydrolases such as cellulases, xylanases, amylases, from Penieillium sp., Aspergillus sp. and Triehoderma sp., for use in the production of biofuels, such as biodiesel, bioethanol and other chemicals of interest.

Claims (10)

1. Large-scale BIOPRODUCTS SYSTEM, such as biosurfactants, biofuels, enzymes and chemicals for biofuel production, characterized by the following components: - a bioreactor (BR) integrated with the other components; - a culture medium preparation tank (TQ1), equipped with a mechanical stirrer; - a membrane microfiltration module (MF1) to ensure that the bioreactor is fed with sterile culture medium; - a membrane microfiltration module (MF2) for separating microorganism cells after fermentation; - a hollow fiber membrane contactor-type oxygenation module (OX); - a reverse osmosis (OI) module for product concentration; - a product storage tank (TQ2); - flow lines connecting the components, all provided with control means connected to a process control loop (C). BIOPRODUCTS OBTAINING SYSTEM according to claim 1, characterized in that said system operates alternatively in single batch, fed batch or continuous batch mode. BIOPRODUCTS SYSTEM according to Claim 1, characterized in that said bioreactor (BR) operates under pressure, is equipped with a mechanical stirrer and has a double-walled external jacket for the passage of thermal fluid which maintains a constant temperature during the fermentation process in the range of 15 ° C to 60 ° C, which additionally thermally sterilizes the bioreactor without the need for direct steam injection. 4. A system for obtaining bio-products according to claims 1 and 3, characterized in that said bioreactor (BR) is integrated with the other components and its operation is carried out by means of a control loop (C). 5.- BIOPRODUCTS OBTAINING SYSTEM according to Claims 1, 3 and 4, characterized in that said control loop (C) acts on two split-range control valves of air addition and withdrawal and instruments that are part of the system installed on flow lines such as pH and dissolved oxygen analyzers, temperature transmitters, pressure transmitters, media level transmitters and foam level transmitters to track and monitor reaction conditions. . 6. A system for obtaining bio-products according to claim 1, characterized in that said membrane microfiltration modules (MF1 and MF2) enable feeding to cells at different metabolic stages, continuous processing without bioreactor discharge and sterility. of product. 7. A system for obtaining bio-products according to claim 1, characterized in that said oxygenation module (OX) is of the hollow-fiber membrane contactor type and promotes oxygenation of the reaction medium stream by means of a non-dispersive oxygenation process, whereby the liquid current (L) is forced to circulate through the oxygenation module shell, being in contact only with the outside of the hollow fiber, while the gaseous current (G) 1 selected from compressed air, compressed air enriched with oxygen and pure oxygen, flows into the fiber to maintain a pressure differential between the reaction medium and the gas stream (G) in the range 0.8 kgf / cm2 to 1, 2 kgf / cm2, preferably at 1 kgf / cm2. 8. A system for obtaining bio-products according to claim 1, characterized in that it employs membranes of different types, such as hydrophobic microporous membranes and microporous support composite membranes covered with a thin layer of dense material. A system for obtaining bio-products according to claim 1, characterized in that in said reverse osmosis module (OI) for product concentration a pressure differential between the concentrated product stream (10) and the permeate stream ( 11) be in the range of 15 kgf / cm2 to 25 kgf / cm2, preferably 20 kgf / cm2. A system for obtaining bio-products according to claim 1, characterized in that said reverse osmosis (OI) module in one embodiment of this invention is alternatively replaced by modules more compatible with the size of molecules to be treated, such as uFiltration and nanofiltration.