Patent Application
20110014648
The field of biotechnology is currently marked by a strong trend of developing new technologies also for high-volume products, such as fuels, chemicals and bioplastics. Consequently, industrial biotechnology and microbiology are rapidly extending their domain not as before only mainly into enzymes, drugs and other products with a relatively low production volume but also into new focal areas. This development arises from such as concern over the availability and sufficiency of oil and other fossil fuels and their soaring prices, state of the environment and climate development. A positive impact on these issues can be achieved by a more wide-spread use and scope of industrial biotechnology. Biotechnology can also help us to avoid using many high-risk, dangerous or harmful chemicals.
In order to reach the objectives listed above, we will need new technologies for such as implementing biotechnical reactors, or bioreactors or fermentors, in a novel way, efficiently and optimally. Similarly, the post-treatment and recovery of products need to be developed. In order to reach optimal results in these tasks, or performing bioreactions in reactors and simultaneous or subsequent post-treatment processes, advanced measurement technology, process control and modelling are needed.
Biotechnology solutions typically build on biomass based raw materials, which often can be organic waste of various types. Plant biomass is another typical raw material. In this case, plants with a high sugar or starch content can be used. On the other hand, the use of plant materials, such as pulp, hemicellulose and lignins from wood materials has also been envisaged, and research and production projects of this type have already been realised. When using these macromolecules as sources of carbon and energy for microbial processes, it is usually necessary to hydrolyse them, or subject them to other types of physical, chemical or enzymatic treatment. This preliminary treatment enables an efficient use of the chemical energy bound in these materials for microbial growth and product formation. Lots of these plant-based macro-molecules or polymers are available in the nature, not to mention many waste materials. When using these, it may often be beneficial to add such as nitrogen components in the process or process solution as a source of nitrogen for the microbes, or various types of vitamins or other growth factors vital for the microbe being used.
Other factors important for microbial growth and function include regulation ensuring a favourable physicochemical process environment and compensating for environment changing factors caused by the microbe, eliminating any risks to the microbe growth, process or product formation. Various environmental factors that need be taken into consideration in optimisation include temperature, pH, partial pressures of oxygen and other gases, osmolarity, viscosity of the process solution etc. Many of these properties also play a role in the recovery and post-treatment of products.
Normally, a bio-reaction or fermentation takes place in a solution as a submerged cultivation: microbes take substances from the process solution, and the products are also formed in the solution. In case the product is the actual microbe cell material, this needs to be separated from the process solution and recovered using different types of post-treatment and purification methods. Microbe cells or enzymes, or other similar biocatalysts performing a reaction, may occur freely in the process solution. Frequently, they may also be attached to a carrier material or phase, i.e. immobilised. This often crucially extends the service life of the biocatalyst and considerably increases its tolerance of various environmental pressures and stresses compared to a non-immobilised biocatalyst. In some cases, immobilisation also facilitates post-treatment.
When using microbes, the bioprocess often has the advantage of avoiding complex reaction pathways. Especially, when using microbes that assimilate or are able to fix atmospheric nitrogen, these advantages are highlighted. In some cases, biocatalyst properties can be improved by means of genetic manipulation, but in this case, close attention should be paid to health-related and ecological considerations. At its best, the use of microbes considerably reduces the formation of harmful wastes.
Processes developed by the assistance of microbes are often called bio-refineries, especially when intended to complement or replace petrochemical industry. One benefit of these activities is that a number of products and processes can be run simultaneously. On the other hand, the same production and post-treatment equipment can be used for different production and post-treatment processes. This may reduce the investment costs in biotechnical processes compared to those of chemical or petrochemical industries. Additional costs in biotechnology may, however, be incurred by such as the requirement of asepticity, which places high demands as to the quality of materials, leadthroughs etc. Similarly, the heterogeneity required of the process solution may increase the costs significantly. In case waste is used as raw material, on the other hand, its negative price may have a positive effect on the total costs of the process, as do the multiplier effects of positive environmental impacts or emission reduction.
What also often is problematic in terms of the total yield of a bioprocess is the fact that the product formation or production does not take place at high enough a rate. This results in diluted process solutions or product mixtures, leading into a high expense of product recovery and purification. Similarly, in case the product is gaseous or vaporising and can be separated by such means as distillation, the concentrations and production rate often remain low. In these situations, an adequate product concentration cannot be obtained sufficiently fast, which has a negative impact on the cost-effectiveness of the whole process. The risks of contamination by undesirable microbes and consequently great losses are increased. In order to optimise the entire process, it is often sensible to model it, allowing the simulation of the effects of various regulations on the reactions in the process solution through the model. These regulations cannot, however, replace on-line measurements that yield accurate information on the process that is under way.
Placing different types of probes and sensors in the right areas in the bioreactor, or in an adequate number of locations in the various sections of the process solution, may emerge as a problem. In high-volume production plants, this may end up extremely expensive, and the sensor leadthroughs may cause contamination problems. Consequently, obtaining accurate data on the entire bioreactor or the situation of the whole process solution for modelling and regulations may be difficult. In order to resolve this technical issue, too, new types of bioreactors are needed. In the production of biofuels, biochemicals or other large-volume products, these problems are often emphasised, as is the fact that the high requirements placed on various materials in the bioprocess, such as steel quality, will increase the overall costs.
Many quantities in bioreactors are measured continuously. These may include monitoring not only the above-mentioned physicochemical values, such as temperature, pH and oxygen partial pressure, but also measurements of substrate and end product concentrations. These give important regulation data on the progress of the process, use of raw materials and product formation. With respect to these measurements, it is also essential to get a true picture of the whole process solution or bioreactor status.
Because bioreactor raw materials and end products usually are heterogeneous, a type of platform thinking in the biochemical, biotechnical and microbiological sense is useful. Certain organisms and their metabolic pathways offer an opportunity to vary the production, raw material use, production directions as well as the formation and recovery of different products. This way, the bioreactor or bioprocessor in a certain sense is an ecosystem subject to the laws of microbiology, which can also be utilised in a technological sense. The basis of all macroscale technological design and construction should be an understanding of microscopic phenomena and observation of microbial interaction. What we need are new types of technological entities that take into account phenomena at the interface between the living biological material and engineering technology. These include comprehensive measurement and regulation methods, production control in the whole reactor volume, more efficient movement of materials, and development of the recovery, and its integration into the production method. In this wider sense, developing bioreactors will be the most important task of bioprocess technology in the near future.
Various bioreactor and fermentor solutions include Stirred Tank Reactors with mechanical stirring, static fermentors, equipment for cultivation and production reactions on solid or semi-solid beds (e.g. techniques used for cultivating plant and animal cells), columns of various types (that may feature bubbles), air lift reactors, hollow fibre fermentors, basins and containers (with or without stirring), and bioreactors with immobilised biocatalysts (packed-bed or fluidized-bed types). In this application, the words reactor, bioreactor and fermentor refer to equipment in which biochemical production or a reaction is performed. The fermentation or bioreaction may be of a fed-batch type or continuous. The formers starts at a certain point, subsequently going through the whole process, mainly with the same raw materials; whereas in a continuous reaction, new raw materials are constantly fed in and materials removed. The problems inherent in the latter type of reactor, however, include a low utilization rate of the raw materials, even if speeding up the reaction usually is possible e.g. in a chemostat type fermentor. Currently, the majority of large-scale process solutions are of the so-called fed-batch type, in which the raw materials (nutrient medium for microbes or other cells) are added in a number of batches during the bioreaction into the original process solution (and the products thus formed often also are recovered in batches). This naturally does not apply to additions of an acid or base used to regulate the fermentor or additions of other additives or regulators, but the addition of the actual main process raw material.
The objective of industrial microbiology and process biotechnology is to form the desired product, such as a cell mass, enzyme, chemical, polymer or fuel, by an energetically advantageous reaction in a bioreactor, making use of the biocatalysis of microbes or other cells (or filamentous growths) or the enzymes of the above-mentioned organisms. The biocatalyst may also be developed or refined through genetic recombination methods, in which case various safety considerations and risks should be particularly noted in the development of the biocatalysts, for example as regards unintentional transfers of genetic material. A part of a cell or cells may also sometimes work as a biocatalyst. What is essential is producing the desired products with lower amounts of reaction energy than in a similar chemical or physical reaction. Bioreactors and biocatalysts may, in such as polymerase industry, contribute to the production of substances and structures that cannot be achieved chemically by means of traditional synthesis. Similarly, by combining different microbes or organisms together (or with various enzymes) in the same reactions, processes or technological platforms, such as difficult-to-degrade materials can be efficiently broken down. This phenomenon of degradation as a result of the combined action of e.g. microbes, which no microbe alone could manage, is referred to as commensalism.
Cells used as biocatalysts are usually cultivated in the reactor as the process or reaction progresses. The cultivation may take place in one part of the reactor, or in case of reactors connected in a series, in some of the reactions, or during a temporally limited reaction period. In any case, what often may become a problem is the fact that the growth of microbes or other cells requires different conditions from the formation of the actual product. Many antibiotics and enzymes, for example, are secondary metabolites, which can only be formed after an active growth phase. In this case, one must be able to prevent the formation—after the growth or other previous reaction or treatment phases—of components preventing or slowing down the actual product formation, also by the biocatalysts themselves. Contaminating microbes occurring in the raw material or emerging during the process can also in a more or less crucial manner impede the action of the biocatalyst, and also the growth of the actual process microbe and performance of production.
In terms of the overall cost-effectiveness of a bioreactor, it is also vital to ensure that the various nutrients are efficiently used and that the microbe's own control mechanisms, such as end product inhibition or catabolite repression, do not significantly reduce reaction efficiency. This can be influenced by using mixed instead of pure microbial cultivations, or for example by removing substances potentially inhibiting end or other products from the process solution in the cultivation or reactor (such as wastes) during the entire reaction as soon as these are formed. They can for example be recovered by means of sieving, filtering, two-phase fermentation, chromatography or vaporisation as well as evaporation (e.g. distillation). In many techniques, vacuum suction can be used. Of great importance industrially are the Escherichia coli bacteria, in the cultivation of which the use of a fed-batch reactor reduces the inhibitory acetate formation in so-called overflow metabolism, which does not support bacterial growth. The use of this reactor type also reduces catabolite repression compared to ordinary fed-batch cultivation.
An attempt can also be made to actively influence the genetic regulation systems of microbes by adding certain extra nutrients or additives, or activating or inhibitory factors, to a cultivation or reaction in a particular phase. In a traditional bioreactor, this usually affects the whole reaction mixture, as stirring usually spreads the substance in question everywhere in the process solution. In fact, this may even be involved in such as the normal regulation of fermentation pH or temperature. The optimum temperature for growth, for example, may be different from the optimum one for product formation. Similarly, e.g. the bacteria performing the 2,3-butanediol fermentation may grow fast in aerobic conditions, but the best yield of butanediol is achieved in micro-aerobic conditions.
In a biotechnical reaction, the impact of products separating from the catalyst on regulation systems and product formation is emphasised, in case their removal from the vicinity of cells or another catalyst is prolonged, for example because of diffusion restrictions of the substrate solution or compound. Similarly, the impact of wastes that inhibit or slow down the action of the biocatalyst is often highlighted because of diffusion restrictions. They also have an influence on the cells' nutrients supply and/or substrate intake. The diffusion of various gases also plays an important role, such as availability of oxygen to aerobic organisms. In addition to oxygen, other gases may also be highly significant (Hakalehto et al. 2007).
Of various organisms that are useful in biotechnology, sporogeneous aerobic and anaerobic bacteria could be mentioned, such as representatives of the family Bacillus in the aerobic group, and in the anaerobic group most importantly the Clostridium bacteria, which include such as the bacteria performing acetone-butanol fermentation, or butyric acid bacteria. Of the species Bacillus, thermophilic bacteria are particularly important in many applications, as at high temperatures their metabolism, and thus also product formation, usually speeds up considerably. Facultative anaerobic bacteria, such as E. coli (mixed acid fermentation) and Klebsiella or enterobacteria (2,3-butanediol fermentation), methane producers and methylotrophic bacteria, various types of degraders, e.g. the family Pseudomonas (aerobic), and many others also are of interest to the chemical and polymer industries. Of yeasts, such as Candida and Saccharomyces cerevisiae species are commonly used for various types of fermentation, for example ethanol fermentation, and fodder production. Moulds and filamentous actinomycetes perform a number of special biocatalytic functions. They are, for instance, important producers of antibiotics, and they form many hydrolytic enzymes, which are used for such as the hydrolysis of biomass macro-molecules.
The various biotechnical modellings may focus on enzymatic kinetic reactions, as well as on mass transfer properties as regards substrates, products, biocatalysts and wastes in the bioreactor and process solution. In the perspective of regulation, in order to prevent the creation of inhibitory by-product flows, it is important that the raw material in the reactor is converted into the desired products in its entirety. As the properties of enzymes (and also cells) as biocatalysts can be listed specificity, selectivity, specific speed of product formation, nutrient intake rate and stability of the biocatalyst. The mass transfer properties of substances call for a study of the mechanics and hydraulic properties of liquids. The vaporisation temperatures and other physical and chemical properties of various substances must be established. It is also important to know the temperature limits and optimums for growth and product formation of microbes acting in the reaction, as well as these values for the pH, dissolved oxygen and other environmental properties. By means of fluorescence spectrums, we can measure the concentrations of sugars, various ions, growth factors or carbon dioxide (e.g. monitoring of breathing), which can be used to obtain data that is vital for the process progression. Extending all these measurements to the whole reactor and process solution, however, often becomes prohibitively expensive. It is also important to establish the heat transfer properties of the entire reaction e.g. by using a thermal camera. A study of heat transfer and gas diffusion may yield information on such as the possibilities of forming biofilms in various parts of the reactor. Liquid flows and movements can also be simulated by computer modelling. The importance of determining all these process parameters is nothing but increased as the process is scaled up. At great volumes, arranging heat regulation and even distribution of the substances is more difficult than on the lab scale. The MFA procedure (“Metabolic Flux Analysis”) based on knowledge of all biochemical pathways is used to determine all metabolite pathways and transformations through modelling.
In addition to modelling, appropriate measurements are essential in order to control the events on real time and in real conditions. Predicting or monitoring different biological phenomena in particular through nothing but modelling may be an insurmountable obstacle, and even parallel measurements may produce widely different results due to the special features of biological material. In this connection, we must note that process solutions are not usually homogeneous at the microscopic level, but they contain cells and other particles, the behaviour of which is very challenging to model. In terms of practical reactor design, a great degree of homogeneity in the process solution is highly important, at least during a specific process phase. Implementing measurements at full reaction volume may involve difficulties, and the same applies to the arrangements of leadthroughs, which as such haven an impact on what happens in the process solution, as well as increase contamination risk and bring up the cost.
When biotechnology is needed to produce chemical components of a relatively low value on a large scale, new technical solutions are required. In addition to measurements, modellings and arrangements for mass transfers, the most important task is organising post-treatment and product recovery. This can often be integrated in the production process. The available methods include distillation, membrane filtration, sedimentation, sieving, centrifugation, evaporation, ultra-filtering and various chromatographic methods. These can be combined, and different products recovered in the same process.
The method and equipment in accordance with this invention typically allow for the implementation of a biotechnological process ensuring that the process solution travels in a desired direction. Various types of tanks, containers and reactors may also be built for preliminary treatments and intermediate phases, in which the process solution lingers for a time. As the process solution moves on, the desired gas or gas mixture, which may contain oxygen or be anaerobic, is simultaneously led into it. The movement of the process solution may be achieved for example through the action of a pump, and physical phenomena can be utilised in it, such as gravity, hydrostatic pressure, capillary action or diffusion. The movement of the process solution can also be affected by means of a conveyor, belt, wire or similar, causing the process solution to travel with it in a relatively thin layer, such as in a pulp and paper factory. The difference is, however, that the solution travels in a space limited in an optimal way, making it possible to create an environment that is aseptic or more hygienically controlled than in an open forest industry process. Similarly, when using the method and equipment in accordance with this invention, the partial pressures and gaseous states of gases in the process solution and in its immediate vicinity can be regulated. This also makes it possible to create desirable gas-liquid interfaces, and reactions may take place in different ways in a process solution where bubbles of various types of gaseous mixtures have been added, or in a similar liquid or suspension. The reactions may also take place in a foaming environment, in which an attempt is further made to speed up the mass transfers and the changes of phases from gas into liquid or vice versa.
A number of benefits can be achieved by leading gases into the process solution. They can be used to bring raw materials and growth factors for various microbes into the microbe nutrient medium. The diffusion of gases and nutrients is improved, wastes that inhibit microbe growth and product formation can be removed faster than before compared to such as a liquid phase, based on the pressure and temperature differences of various existing or verifiable gases. The absorption of gases and removal of volatile products also affect the temperature conditions of the process solutions and reactor, and these phenomena can be utilised in heat transfers to the various parts of the reactor and solution. A crucial difference to e.g. forest industry processes is that motive forces of such as gravity and hydrostatic pressure work in one direction, whereas forces associated with gas pressures caused by feeding in of bubbles and gases work in the other, opposite direction. Consequently, at the intersection of these different forces, conditions are formed where the mass transfers are strong and fast, facilitating biological, biochemical and biotechnical reactions. For one advantageous way of implementing equipment in line with this invention, see
By pumping or by—other methods, gas can be led into the process solution at points where the downward travelling direction is replaced by an upward one, and the recovery of gaseous fractions can be implemented in the opposite turning points where the process solution starts travelling downwards, through discharge outlets, pipe outlets or similar (7). In order to e.g. regulate pressure or prevent overflowing, these can conveniently be closed with semi-permeable membranes (8) or valves (9), or both. Remainder of the process solution is led to such as a separation line (10), where after-fermentation can take place, or merely the recovery of liquid fractions or, alternatively, solid fractions by using filtering, sieving, two-phase or centrifugal techniques or other methods. To separate out the various phases, precipitation can also be used, which can be implemented or intensified by adding the required substances in the remainder of the process solution. Structurally, the separation line may also be a moving wire or belt, or it can be or become a pipe (11), from which the liquid phase is e.g. led to chromatographic product recovery (12). The various fractions can subsequently be collected in product recovery containers or basins (13-15). These can be delivered for after-storage. Various fractions can also at least partially be recycled back to the process as necessary (16).
When using the method and equipment in accordance with the invention, such as the following benefits are achieved:
| Number | Date | Country | Kind |
|---|---|---|---|
| 20080249 | Mar 2008 | FI | national |
| PCT/FI2009/000040 | Mar 2009 | FI | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/FI09/00040 | 3/30/2009 | WO | 00 | 9/26/2010 |
