Field of technology

The invention relates to a method of activation and cultivation of production

microorganisms, the fermentation of fermentation products by these microorganisms and the use of this method. The solution also relates to a method of discontinuous and continuous fermentation.

Background of the invention

With standing utilized or proposed methods of activation of non-vegetative cells of primary metabolites production microorganisms for obtaining of active vegetative forms and their cultivation the practices based on the sterilization of suitable substratum or cultivating or fermentation medium are used in order to prevent or limit the activation and development of competing undesirable microorganisms. Typical primary metabolites of production microorganisms include, for example, ethanol, acetone, n-butanol, butanediol, isopropanol, butyric acid, citric acid, fumaric acid, lactic acid, propionic acid, succinic acid, itaconic acid, acetic acid, acetaldehyde, 3-hydroxy-propionic acid, gluconic acid, tartaric acid, or salts of these acids. The most used sterilizations for solid and liguid substrates and cultivating media are thermal, chemical and/or the sterilization with the use of UV radiation. For example, in the U.S. Patent 5868997 for sterilization of the fermentation eguipment a direct washing with warm alcohol is used. Similarly, in the U.S. 61149949 document pasteurization during the production of an alcoholic beverage is described, based on admixing to the fermentation broth an extract containing ethanol. Another example for chemical sterilization is the use of iontophoretic antibiotics for inhibition of bacterial growth during alcoholic fermentation (U.S. 5888788).

For activation of desirable microorganisms the pure cultures or the inoculum that does not contain unwanted cultures are introduced in the sterilized substratum or cultivating medium or fermentation broth. Also during the preparation of the inoculum of the desired culture the cultivation with high demands not only on sterility of cultivation medium, the substrate and the purity of the inoculum culture, but also the purity of air and all parts of the cultivating eguipment, which may come into contact with the cultivating medium. High sterility, achieved mainly by chemical sterilization, does not ensure desirable course of development of demanded microorganisms, cultivation or fermentation process. The most commonly used substrates for cultivation and fermentation are based on sugar-yielding substances contained in the raw materials such as beet or cane juice; diluted molasses of the same, hydrolyzed cereals (e.g. maize or wheat), hydrolyzed starch tubers (e.g. potatoes or artichokes), grape or fruit must, but also various hydrolysates of cellulose and hemicellulose (hydrolysates of wood and. straw). During fermentation or cultivation utilizing these substrates, often prepared from waste materials, the activation and development of undesirable competing stems is caused, in addition to physical, chemical and biological conditions (temperature, pressure, pH, presence of macro-and microelements, etc.), also by the purity of raw materials and sufficient number of cells of desired microorganisms. Therefore demands for substrate sterility are high, and even small imperfections in the sterilization process and a minority in presence of desirable microorganisms often lead to unwanted negative cultivation or fermentation development, or perhaps even to its reversion. This greatly limits- or even prevents keeping of cultivation or fermentation processes continuous. Furthermore, the sterilization affects the quality of the substrate or, during the chemical sterilization, it also acts as an inhibitor for desired production cultures and the affects quality of obtained products, or increase demands for their separation and purification. The activation of microbiological inoculum, especially spores and conidia into a vegetative state (initiation of cells' development, start of lag phase)up to now has been carried out by contacting the inoculum with cultivating medium under suitable conditions, with a possible doping of the medium with growth additives, for example yeast extract, vitamin complexes, etc. Similarly, during the isolation of microorganisms' cultures from mixed inoculative cultures, such as compost, soil, air or water, relatively high dilution of the inoculum for obtaining sufficiently developed colonies of desirable microorganism is necessary, which at the same time reduces the percentage of isolation. Isolated colonies, or cells from these colonies, are subsequently used for inoculation of suitable cultivating media to form a pure culture and for its multiplication to an inoculum necessary for the production fermentation. However, the risk of unwanted contamination with competing cultures is not reduced.

Subject matter of the invention

Deficiencies of existing methods of activation and/or cultivation of production microorganisms producing primary metabolites, as fermentation products of sugar-yielding materials and/or glycerol, and/or deficiencies of methods of fermentations of these products by these microorganisms under set conditions, formed by the composition and possible limitation of the source substratum or cultivating medium or the fermentation broth, the temperature and pressure interval, pH, access or exclusion of air, and the mixing conditions, is eliminated by the invented solution, according to which the source substratum and/or the cultivating medium and/or the fermentation mixture, prepared for activation and/or cultivation of the desirable culture of production microorganisms producing the fermentation product, and/or prepared for fermentation by this culture, before and/or together with and/or after the inoculation with the material containing cells of the desirable production microorganisms, is added the fermentation product produced by the culture of the desirable production microorganisms so that the source substrate or the cultivating mixture or the fermentation broth contains a concentration of the fermentation product representing at least one- hundredth, preferably 3 to 15 hundredths of the value of the maximum concentration of the fermentation product, which is produced by the desirable culture of production microorganisms under the set conditions.

During the continuous, or semi-continuous cultivation and/or the fermentation the fresh cultivating and/or fermentation mixture containing concentration of the fermentation product, containing at least one hundredth of the maximal concentration of the fermentation product, which is produced by the desirable culture of production microorganisms under setup conditions, or the portion of the output mixture from the cultivation and/or fermentation and/or the pure form of the fermentation product and/or in a solution together with fresh cultivation and/or fermentation mixture in a such amount that the concentration of the fermentation product in added mixture will be at least one hundredth, preferably 3 to 7 hundredths of the maximal concentration of the fermentation product produced in the output metabolized cultivation and/or fermentation mixture, is added to the metabolized cultivation or fermentation mixture, containing the culture of the production microorganisms.

The activation and/or cultivation of the production microorganisms producing primary metabolite as a fermentation product of sugar-yielding raw materials and/or glycerol by adding this fermentation product to the cultivation medium and/or the cultivation mixture also may be used for the isolation of production microorganisms' cultures producing and/or metabolizing fermentation product.

The key advantage of the invented solution is the fact that the activating, or protective supplement is a natural metabolite of desirable microorganisms, occurrence of which in the substrate, or the cultivation or fermentation medium activates the start and development, or fermentation activity of desirable cultures, but inhibits the start and development of undesirable competing and ballast microorganisms. At the same time demands on sterility of substrate, or cultivation or fermentation medium, are reduced. This effect of the presence and concentration of the metabolite in the substrate or medium is caused by different sensitivity of the presented microorganisms' cells and different effects of the metabolite on the cellular activity. With the setup of favorable physico-chemical and vegetative conditions, the presence of the product in the cultivation or fermentation medium during the initiation and development of cells blocks the activity of those cells, microorganisms, for which the occurrence of the metabolite in the environment indicates unsuitability for their development. On the other hand, the presence of metabolites, with lower concentrations than the maximum concentration achieved under the given conditions, in the environment, simulates for the spores and vegetative cells of desirable production microorganisms the environment suitable for a development or the environment in which development is already in progress. This way is the sterilizing effect of the metabolite addition intensified and directed in favor of the desirable production of microorganisms, what controls the course of cultivation, or fermentation. By varying the supplemental fermentation product (metabolite) concentration it is possible selectively determine the development of multiple cultures or only the one culture of microorganisms inoculated into the substrate or cultivation media in a mixed form, for example in the form of substrate sample, compost, or water, or by exposition to the air. The activation of spores and conidian, or growth of vegetative cells will take place in the cultures for which the set conditions, including the presence of the given metabolite, are optimal. For example, for the identical physico-chemical conditions and the same composition of the cultivating medium, differing only in the metabolite concentration, the selective metabolite producers may gradually develop and possibly cultures metabolizing this product from individual groups according to phylogenetic relationships. This selection may be modified by adjusting of the substrate composition and setup conditions (straiten, expand or shift) as required.

An advantage of this solution is also the fact that, with help of available reagents and without high demands on sterility, it is possible to separate from mixed inoculate materials, such as soil substrates, compost, unfiltered air, bio-sludge, waste or natural waters, etc., cultures of production microorganisms and to cultivate their vegetative forms, simultaneously with significant acceleration of activation and cultivation processes. An advantage of this solution is also the fact that the method may be utilized in all methods of fermentation - batch, semi-continuous and continuous. Following the exhaustion of the substrate, or after reaching of the maximal product concentration in the fermentation mixture, it is possible to separate from the mixture a proportion of the product and by-products, replenish it with necessary components and reuse it for fermentation.

This process, according to the invention, may be used for separation, multiplication and cultivation of different cultures of microorganisms as well as the fermentative production of various materials in biotechnology processes, especially in the zymurgy and fermentation industry, in the production of bio-fuels and precursors for chemical products, such as polymers. The utilization of the process will reduce the cost ratio of the fermentative production, especially valuable bioproducts.

Also risks and direct effects of hazardous substances to the environment are reduced when natural metabolites are used in accordance with the solution, as well as the consumption of water, chemicals and energy is reduced.

Examples of an embodiment of the present invention

Example 1

The activation and isolation of the production mould strain, preparation of the inoculum and cultivation of mycelia with following fermentation of the itaconic acid. The activation of non-vegetative spore form for the preparation of the inoculum containing the vegetative form and for the isolation of Aspergillus terreus production mould mycelium is performed with the addition of 2 g of itaconic acid in 1 liter of source non- sterilized aqueous mixture containing 100 g of glucose, 2,5 g of ammonium sulfate, 0, 1 g of potassium dihydrophosphate, 2 g of heptahydrate of magnesium sulfate, 0, 1 g of sodium chloride, 0, 15 g of calcium chloride and mixture of microelements containing zinc, iron and manganese. Prepared cultivation mixture having pH value 3,0 is inoculated with spores of Aspergillus terreus, and it is cultivated in a stirred and aerated fermenter at 35 °C. After 80 hours of mycelium cultivation the pH reaches a value of 2.6 and the concentration of itaconic acid value of 4,6 g/L.

Cultivated mycelium of the production mould is introduced into the fermentation mixture, with a composition corresponding to a mixture containing 2 g/L of itaconic acid, and adjusted to the condition of the nitrogen and phosphorus limitation, when the ammonium sulfate is not present in the mixture and the potassium dihydrophosphate is replaced by potassium chloride (equimolar by potassium). Fermentation takes place in intensively aerated and stirred fermenter at the temperature of 35 to 38 °C. The fermenter is replenished with the amount of evaporated water in 24-hour intervals. After 200 hours of fermentation the concentration of itaconic acid will reach the level of 22 g/L.

The cultivation and fermentation is, under the same conditions as for the glucose substrate, repeated with use of the cultivation and fermentation mixture containing 100 g/L of glycerol and 4 g/L of itaconic acid. After 210 hours fermentation the concentration of itaconic acid will reach the value of 24,2 g/L.

Example 2

The Aspergillus niger cultivation and the itaconic acid fermentation with a change of the substrate.

50 mL of an inoculum mixture containing 20 wt % of dry Aspergillus niger mycelia and 4,4 g/L of itaconic acid is added to 0,95 liter of an aqueous mixture containing 10 wt % of sugar beet molasses, 0,25 wt % of NH ₄ CI, 0,095 wt% of MgS0 , 0,01 wt% of KH ₂ P0 ₄ , 0,0004 wt% of CuS0 ₄ , heated to 80 °C for 1 hour and then acidified to pH 4,0 with HCI. The content of itaconic acid (product) in such prepared production mixture, representing also the cultivation and fermentation mixture, is 0,22 g/L. The cultivation of mycelium and fermentation of the product runs for 120 hours at the temperature of 35 °C in an aerated fermenter equipped with a fine-meshed plastic sieve before the outlet. The maximum concentration of the product will reach 22 g/L.

After reaching the maximal product concentration, the aqueous phase of the production mixture will be discharged through a plastic sieve with preservation of the wet mycelium with a volume of 0, 1 liter in the fermenter. The quantity of product retained in wet mycelium volume is 0, 1 g. The mycelium in the fermenter is mixed with 0,9 liters of an aqueous mixture containing 200 mL of acid potato hydrolyzate (glucose content of 97 g/L, pH value of 2,0), 1 ,01 g of itaconic acid (product), 2 g of Na ₂ S0 ₄ , 0,2 g of MgS0 ₄ .7H ₂ 0, 0, 1 g of KCI, 0, 5 g of CaCI ₂ and a mixture of microelements containing zinc, iron, and manganese. The total concentration of added product in the fermentation mixture is 1 ,02 g/L. The fermentation is carried out at temperature of 35 °C with aeration, mixing and pouring water evaporated from the production mixture in about 20 hour intervals during 150 hours, when the product concentration in the output fermentation mixture reached 20,4 g/L.

After a discharge of 0,5 liter of water phase from the output fermentation mixture, the fermenter with the rest of the output fermentation mixture is replenished with 0,5 L of fresh aqueous mixture of the hydrolyzed potato substrate and the fermentation is repeated under the same conditions as in the previous fermentation. After 120 hours of fermentation, the concentration of the product in the fermentation mixture will reach 22,3 g/L. Example 3

Activation and cultivation of bacteria Actinobacillus succinogenes and fermentation of succinic acid.

With the tap water a suspension mixture is prepared, containing 20 g/l of glucose, 7,5 g/l of yeast extract, 1 ,5 g/L of K ₂ HP0 ₄ , 1 ,5 g/L of KH ₂ P0 ₄ , 1 ,5 g/L of NaCI, 0,07 g/L of MnCI ₂ , 0,4 g/L of CaCI ₂ and 22 g/L of MgC0 ₃ . Into the suspension mixture is admixed 3 vol. % of Actinobacillus succinogenes inoculum, and it is exposed to air for 0.5 hour. The mixture is homogeneously divided into two thermally insulated containers (production and comparative), heated to a temperature of 37 °C and equipped with the Pasteur cap filled with water. In the production container is added 1 volume unit of succinic acid solution with a concentration of 7 g/L to 9 volume units of the mixture. The total content of succinic acid, contained in the prepared cultivation/fermentation mixture in the form of succinate, is 0,7 g/L. To 9 volume units of suspension mixture in the comparative container is added to 1 volume unit of tap water. The pH of both such prepared cultivation /fermentation mixtures is 7,0. After 12 hours the content of succinic acid in the filtered sample from the production container is determined by the HPLC method, reaching value of 14.5 g/L. The sample from the comparative container after 12 hours is showing the multiplication of yeast cultures, while the content of succinic acid is not indicated.

Example 4

The cultivation of production bacteria and fermentation of butyrate, acetate, butanol, ethanol and acetone.

Into a fermenter, equipped with a pressure valve for gas bleeding, is added 1 liter of cultivation/fermentation mixture containing 1 g of magnesium chloride hexahydrate, 1 ,5 g of ammonium sulfate, 0,5 g of potassium dihydrophosphate, 10 mg of ferrous sulfate heptahydrate, 10 mg of manganese sulfate hydrate, 0, 15 g of calcium chloride dihydrate, 5 g of the yeast extract, 30 g of glucose, and 10 mL of the vitamin solution (thiamine chloride, nicotinic acid, riboflavine, vitamin B12, aminobenzoic acid, Capantothenate, lipoic acid). Into the mixture is added 50 mL of Clostridium acetobutylicum inoculum from a previous cultivation, containing 3 g/L of butyrate, 4 g/L of acetate, 7 g/L of butanol, 0,2 g/L of ethanol, and 2,7 g/L of acetone.

The mixture is warmed-up to a temperature of 35 °C under anaerobic conditions, with maintaining of the pH value between 4,5 and 5,5 by the addition of 0,5 M sodium hydroxide solution. After 50 hours of fermentation the mixture contains 3,2 g/L of butyrate, 3,7 g/L of acetate, 10 g/L of butanol, 0,3 g/L of ethanol and 3,5 g/L of acetone.

Example 5

Cultivation of yeast and fermentation of ethanolic federweisser.

20 liters of raw grape must (free-run containing 22 wt% of glucose), stored in the fermentation vessel with a volume of 30 I wash out by tap water, is admixed with a suspension of 5 g of vine yeasts in 50 mL of water, and then added 0,5 liter of 40 % ethanol. The fermentation vessel with such prepared cultivation/fermentation mixture is closed with help of the fermentation cap and placed in an area with a temperature between 25 and 28°C. After 28 hours of cultivation/fermentation it contains the federweisser with 10 vol. % of ethanol.

Example 6

Ethanol fermentation with yeast from sucrose.

Four containers (No.1-4), 500 ml_ each, are poured with 2 liters of non-sterilized fermentation mixtures inoculated with 5 mL of 10 wt% suspension of vegetative yeast Saccharomyces cerevisiae. The mixture contains sucrose (50 g/L), ammonium sulfate (2 g/L), potassium dihydrophosphate (0,15 g/L). The mixtures in containers 2, 3 and 4 are admixed with 99,7 % ethanol: 1 mL in the container No. 2; 2 mL in the container No. 3; and 4 mL in container No. 4. The containers are sealed by a Pasteur cap and stored at room temperature for 5 days. In the container No.1 (comparative fermentation) it is seen, besides a development of the yeast, a significant development of bacteria, while the content of ethanol is less than 0, 1 g/L. The content of ethanol in mixtures is as follows: 2,2 g/L in the container No.2; 6;8 g/L in No.3; and 10, 1 g/L in the mixture No.4.

Example 7

Activation, inoculum preparation and isolation of mould cultures producing the itaconic acid.

For preparation of the cultivating medium based on starch hydrolysate, 30 g of finely grated potato is suspended in 100 mL of water containing 10 mL of 20 % hydrochloric acid. The mixture is boiled at 100 °C for 30 minutes. For the adjustment of mixture pH the addition of sodium hydrogen carbonate is used. With the addition of ammonium hydroxide, the pH is increased to value of 8 and then by adding the phosphoric acid to pH 6,0. After the admixture of 0,5 g calcium carbonate powder, the pH of the mixture is settled between 6,3. The inoculum composition is prepared by mechanical homogenization of mature compost and topsoil samples. The cultivating medium is admixed with a homogenized inoculum mixture, the mixture is exposed to air and divided into six Petri dishes (A-F) with 20 g of the mixture. The inoculated soils in the individual dishes are admixed with components according to the Table 1 (itaconic acid, aluminum oxide, or calcium itaconate). The dishes are placed in the dark at ambient temperature of 20 to 25 °C. The results of visual and microscopic evaluations are listed in the Table 1. The comparative dish with solid cultivation soil (content of aluminum oxide) provides comparable results to that of the liquid soil in the B dish (without the addition of itaconic acid, or calcium itaconate) with a complex mixture of different competing cultures. In the F comparative dish (addition of the itaconate) the less developed of mould cultures is notable, but of different types than in comparative A and B dishes. From the C dish it is possible isolate only developed cultures of Aspergillum line, there are developed only 2 cultures of Aspergillum group in the D dish, and in the E dish there is only one type of developed mould (Aspergillum niger). W 201

Table 1

Industrial utilization

The invention is applicable to direct the activation of cultivation and production of microorganisms, their separation from mixed cultures, as well as to improve fermentation process stability carried out using these microorganisms, such as the biotechnological production of biopolymer precursors, antibiotics, foodstuffs and food ingredients, organic chemical materials, biofuel components etc.

The solution according to the invention may be utilized in production biotechnologies during the production of various fermentation products from sugar substrates, especially in the fermentation, pharmacological and food industries, as well as in industrial ecology in fermentation treatment of plant biomass and biowaste.