LV15474B - Method for producing single cell oil from biodegredable by-products - Google Patents

Abstract

A process for obtaining a microorganism oil, comprising the following sequential steps: (i) fermenting the substrate by culturing the microorganisms in a bioreactor, (ii) filtering, (iii) pre-extracting the biomass, and (iv) extracting, wherein the substrate is biodegradable. industrial by-products and step (iv) is performed by supercritical CO2 extraction. Process according to claim 1, characterized in that in step (iv) the supercritical CO2 extraction is carried out at a pressure of 20-40 MPa, at a temperature of 31-80 ° C, with a CO2 content of 1-12 kg CO 2 / hour and an extraction time is from 45 minutes to 6 hours. Process according to claim 1, characterized in that in step (i) biodegradable production by-products selected from industrial glycerol, whey, sulphite waste streams, molasses, fruit and vegetable processing residues are used as substrate.

Description

DESCRIPTION OF THE INVENTION

The invention relates to the fishing industry, in particular to methods for obtaining feed materials for the aquaculture industry. The method of the invention is the production of microorganism oil, which is an analogue of fish oil, in the fermentation process from biodegradable production by-products (waste). Microorganism oil is intended to be used as a raw material in fish feed.

Prior art

The main components of traditional fish feed are fish oil and fish meal. Fish oil and fishmeal are the most nutritious and easily digestible feed materials for aquaculture fish, which is the main reason why more than 70% of the world's fish oil is used as feed in aquaculture [1, 2].

Nowadays, fish oil is obtained from various species of fish, fish residues and other by-products of fish processing. Oily fish of the genus Engraulis (anchovies) are most widely used for this purpose. These catches are significantly influenced by factors such as the El Nino phenomenon and the various catch quotas that legislators are introducing in order to avoid overfishing. Overall, global natural catches have been stagnant for the last 20 years [3]. The growing demand for fish oil, both for fish feed and for human consumption, is also causing significant damage to the environment, as current fishing methods threaten the already scarce resources available. Natural catches have significantly worsened the condition of several fish species, some of which are leading to extinction [4, 5]. Unsatisfied demand, unpredictable catches and instability in fish populations not only make natural catches an unattractive source of raw materials, but also raise serious concerns about the impact of overfishing on biodiversity in exploited habitats [6, 7]. For these reasons, oil from natural catches is unable to meet the growing demand for fish oil from aquaculture and it is therefore necessary to find new sources of oil that can be used in fish feed.

To address this problem, fish oil is increasingly being replaced by vegetable oils in aquaculture. The use of vegetable oils in the diet of captive fish shows acceptable ratios of live weight gain and feed conversion. However, the use of plant-based feed reduces the levels of omega-3 fatty acids cervonic acid (DHA) and eicosapentaenoic acid (EPA) in fish tissues.

When fish farmed in this way are eaten, the human body no longer absorbs enough DHA and EPA. These fatty acids play an extremely important role in the human body, and regular consumption of these fatty acids improves cell membranes, brain function, nerve impulses, oxygen transfer to blood plasma, hemoglobin synthesis, cell division processes, and promotes brain development in newborns and is generally considered vitally necessary because the human body is unable to synthesize these fatty acids [4, 8, 9]. Another negative effect of using plant-based feed in fish is an increase in the concentration of omega-6 fatty acids in the tissues of farmed fish, which breaks down the ratio of omega-3 to omega-6 [4]. Changing the ratio of omega-3 to omega-6 (increased omega-6 fatty acid concentration) is one of the main causes of various cardiovascular, neurodegenerative, inflammatory and oncological diseases [10, 11]. Consequently, the use of vegetable oils in the diet of captive fish can have a negative impact on human health if such fish are consumed for a long time, so it is necessary to find alternative sources of nutrients that could be used in aquaculture without risk to human health.

The closest method to the invention is the production of microorganism oil by culturing oily microorganisms using vegetable sugars [4]. This technique is accepted as a prototype. The prototype technique includes the following sequential steps, culturing oily microorganisms, filtering, pre-extraction treatment, and oil extraction. The prototype method uses inefficient extraction methods and expensive raw materials (vegetable sugars), so this method has a number of disadvantages: expensive raw materials [4]; raw material extraction competes over limited arable land areas [2, 4, 12, 13]; Extraction methods often use environmentally harmful solvents such as chloroform, methanol, hexane, isopropanol, etc. [4]; During extraction, substances that are undesirable to animal health (mycotoxins, heavy metals, etc.) may enter the final product (oil) [4, 26].

In general, microorganisms can be used to meet the high demand for fish feed oil. Synthesized microbial oils do not reduce the level of high-quality fatty acids in fish tissues and do not disrupt the ratio of omega-3 to omega-6, as is the case when using vegetable oils in fish diet [10, 11]. However, if agricultural raw materials grown on agricultural land are used as feed for microorganisms, cultivation is expensive. Arable land areas are limited at the global level [12, 13], so the cultivation of high-sugar plants for microorganisms is in direct competition with the areas that can be used to grow crops for human and animal consumption. Cultivation using such raw materials can only be worthwhile if the oil obtained is marketed with high added value (medicines, baby food, etc.). The oil thus obtained for use in aquaculture as fish feed is not cost-effective, so it is necessary to cultivate microorganisms using cheaper raw materials (by-products of other industries) and to adapt oil extraction methods to the waste products used.

Purpose and essence of the invention

The object of the invention is to develop a production method for obtaining a microorganism oil from biodegradable production by-products.

The object of the invention is achieved by a prototype method (production of microorganism oil using vegetable sugars) by replacing vegetable sugars with biodegradable industrial waste and by using supercritical CO2 extraction.

[009] In microbiological fermentation, the carbon source accounts for about 60% of the total production costs of the product [4], thus the use of biodegradable production by-products can significantly reduce production costs. The use of biodegradable waste products in oil production also reduces the negative impact on the environment, as microbiological fermentation of waste products significantly reduces the values of biological oxygen demand (BOD) and chemical oxygen demand (BOD) in the waste products concerned. After fermentation, residues of these waste products are much less harmful to the environment than if they were released untreated into the environment [4].

The supercritical CO2 extraction method is a green extraction method because it does not use strong chemical solvents such as chloroform, methanol, isopropanol, hexane, etc. Solvents are widely used in the production of traditional fish oil, so solvent extraction has a negative impact on the environment. In turn, the supercritical CO2 extraction method uses CO2 gas as a solvent, which ensures the separation of polar compounds from non-polar ones under high pressure and elevated or moderate temperatures, thus the oil in biomass is extracted completely cleaned of any impurities, because microorganism oils are non-polar compounds [14 -18]. The resulting oil is pure from water, heavy metals and other organic and inorganic compounds. Because it is possible to perform the extraction at moderate temperatures during the process, no active oxidation of the oils takes place, thus the obtained microorganism oil is of high quality [14, 15, 19, 20].

DETAILED DESCRIPTION OF THE INVENTION

The method of the invention for obtaining a microorganism oil comprises the following sequential steps: (i) fermentation of the substrate by culturing the microorganisms in a bioreactor, (ii) filtration, (iii) pre-extraction treatment of biomass, and (iv) supercritical CO2 extraction.

The first step (i) is the fermentation or culturing of the substrate. The cultivation process is divided into two stages: 1) biomass increase; 2) fat accumulation. In the first stage, the culture medium is rich in nutrients (carbon and nitrogen-rich compounds, trace elements) that promote cell proliferation; then, in the second stage of cultivation, unfavorable conditions for microorganisms are created in the medium, i. reduction of the concentration of nitrogenous compounds, which causes abiotic stress, which in turn promotes the accumulation of fat in the cells of microorganisms [4]. In both stages, the development of microorganisms and the accumulation of fat require a carbon source in the medium. The invention uses biodegradable industrial by-products selected from industrial glycerol, whey, sulphite waste, molasses, fruit and vegetable processing residues as carbon sources, but many other by-products can also be used for cultivation.

The second step (ii) is the separation of the cell mass from the liquid medium by filtration: the solids (microorganism biomass) are separated from the liquid fraction (medium) [4]. Flocculation using food grade products can improve the overall filtration efficiency.

The third step (iii) is the pre-extraction treatment of the biomass, where the biomass from the filtration step is washed with water to remove larger solids from the biomass that failed to be removed in the filtration step, as well as to treat the cell walls of the microorganisms and thus improve efficiency of the extraction process [4].

The fourth step (iv) is the extraction of the oil, where the oil is separated from the biomass obtained by filtration and then washed. Supercritical CO2 extraction in the range of the following parameters is used for oil extraction: pressure 20-40 MPa, temperature 31-80 ° C, CO2 amount 1-12 kg СОг / hour and extraction time is from 45 minutes to 6 hours [15-20].

[016] Microorganism oil is lipids derived from oily microorganisms. Microorganism oil is a mixture of fatty substances in which the molecules consist mainly of aliphatic hydrocarbons, which are soluble in non-polar organic compounds but poorly soluble in water. Lipids are fats, oils, waxes, wax esters, sterols, terpenoids, isoprenoids, carotenoids, polyhydroxyalkanoates, nucleic acids, fatty acids, fatty alcohols, fatty aldehydes, fatty acid esters, phospholipids, glycolipids, sphingolipids, sphingolipids, sphingolipids. The most important lipids in the present invention are saturated fatty acids, monounsaturated fatty acids and polyunsaturated fatty acids.

[017] Microorganism oil can be obtained from bacteria, cyanobacteria, fungi (yeasts, microscopic mycelium), archaea and microscopic algae. Microorganisms can be both naturally and genetically modified to improve their lipid accumulation capacity. Oily micro-organisms are all micro-organisms that are able to accumulate in their cells at least 15% fat from the total dry matter of the cell biomass. As oily microorganisms, microorganisms from the genera Achlya, Acinetobacter, Apiotrichum, Aspergillus, Brachiomonas, Candida, Chaetomium, Chlorella, Cladosphora, Cladosporium, Cladosposridium, Claviceps, Crypthecodinium, Chamenococcus, Chamenococcus, Chamenococcus, Geotrichum, Glomus, Gordonia, Hansenula, Hantzschia, Humicola, Yarrowia, Leucosporidium, Lipomyces, Malbranchea, Mortierella, Mucor, Mycobacterium, Nannochloropsis, Nanochloris, Neochloris, Nitzschia, Nocardia, Pachysolen, Pietochlor, Parietochlor Rhodosporidium, Rhodotorula, Scenedesmus, Schizochytrium, Sirodotia Sporidiobolus, Sporobolomyces, Streptomyces, Traustrochytrium, Tremella, Trichosporon, Ulkenia, Umbelopsis, Waltomyces, Zygorhychus, etc. [4, 21].

[018] Biodegradable production by-products are any agricultural or industrial waste that is biodegradable as a result of the fermentation of microorganisms. The following can be used as biodegradable production by-products in the production of microorganism oil: sugar beet, sugar cane and soybean molasses and other sugar production by-products; milk processing residues (whey, juices, etc.); fruits, vegetables and their processing residues; cereal, soya and cotton processing residues (bran, stalks, straw, grains, beans, etc.) and their hydrolyses; by-products of starch manufacture and their hydrolysed products; poultry residues; by-products of beverage production (brewery by-products (crumbs, malt, etc.), fruit and vegetable pomace, etc.); crustacean processing residues; fish processing effluents; wastes and residues from glutamic acid production; Wastes from Capsicum oleoresin production (Capsicum powder, etc.); slaughterhouse waste (horns, hooves, feathers, blood, hair, etc.); paper and paper by-products (sulphite waste streams, lignin-containing residues, etc.); wastes and other residues from the manufacture of latex rubber; methane, methanol, acetic acid, formic acid, flue gases, etc. carbon-containing compounds and their industrial residues with impurities; glycerin; nparaffins, etc. petroleum by-products; biogas plant residues; municipal wastewater, etc. [22, 23]. Potentially the most practical biodegradable production by-products within the scope of this invention are glycerol, milk processing residues, sulfite waste streams, sugar production by-products and fruit and vegetable processing residues.

[019] Cultivation of microorganisms is a process during which the growth and development of oily microorganisms is promoted or allowed. During cultivation, the microorganisms contain the nutrients in the enzyme medium and accumulate fat inside the cell or in the medium, which can be extracted or collected. In the context of the present invention, the microorganisms are cultured under aerobic conditions, which means that the microorganisms use oxygen as an electron acceptor in energy production processes during cultivation. Generally, during cultivation, aerobic cultivation is provided by actively aerating the medium by stirring or adding oxygen or a gas mixture. The culturing process can be performed using any suitable culture method for the microorganisms, including batch (fed or batch), fed batch and continuous (English or. Continuous) cultivation. Cultivation for the production of microbial oil can take place under both sterile and non-sterile conditions. Sterile conditions in the context of cultivation of micro-organisms are conditions in which culture vessels, equipment, medium and other tools that come into contact with the micro-organism during cultivation are previously sterilized to ensure that no other undesirable cultures of micro-organisms develop in the medium during cultivation [24].

After culturing, the cell mass is separated from the liquid medium, which can be done by press, vacuum, centrifugation, screw press or other filtration methods, in which solid particles (microorganism biomass) are separated from the liquid fraction (medium). Flocculation using food grade products can improve the overall filtration efficiency.

[021] The biomass of microorganisms obtained before oil extraction can be rinsed with water and sonicated, microwave, mechanically ground (ball mills, etc.), frozen / thawed or lyophilized, as well as other pre-extraction treatments to destroy the cell walls of the microorganisms and skin. improve the efficiency of the extraction process.

[022] Extraction or oil extraction is a process during which oil is extracted from the obtained microorganism biomass. The oil is usually obtained mechanically (using press filters, screw presses, ball mills, etc. mechanical methods), chemically (using solvents and other lipid-soluble substances) or thermomechanically (combining mechanical methods with temperature change).

[023] The extraction methods widely used in the present invention are replaced by supercritical CO2 extraction, which has not been used so far in the extraction of microorganism oil from microorganism biomass. Supercritical fluid extraction is a process in which one component (extractant) is separated from another (matrix) using a supercritical fluid as the extraction solvent. The present invention uses carbon dioxide (CO2) as a supercritical fluid, which is recycled during the process, so that CO2 losses are minimal. A critical temperature of at least 31 ° C and a critical pressure of at least 7,4 MPa must be maintained in the extraction vessel to ensure a supercritical state of CO2 [25]. The extraction conditions mentioned in the literature and used in the laboratory for optimal oil production are as follows: pressure range from 20 to 40 MPa, temperature range from 31 to 80 ° C, CO2 amount from 1 to 12 kg СОг / hour and extraction time from 45 minutes to 6 hours [14-20]. Supercritical fluid extraction can be performed in a single extraction cell or in multiple extraction fractions. During fractionation, different pressure and temperature conditions are maintained in each cell, which allows some oil fractions to be extracted more efficiently than others [15].

Examples of implementation of the invention

Example 1: Method for obtaining microorganism oil from industrial glycerol Microorganism used: Yarrowia lipolytica (wild type).

Medium: 2 pg / L biotin; 400 pg / L calcium pantothenate; 2 pg / L folic acid; 400 pg / L niacin; 200 pg / L p-aminobenzoic acid; 400 pg / L pyridoxine hydrochloride; 200 pg / L riboflavin; 400 pg / L thiamine hydrochloride; 2 mg / L inositol; 500 pg / L boric acid; 40 pg / L copper sulphate; 100 pg / L potassium iodide; 200 pg / L ferric chloride; 400 pg / L manganese sulphate; 200 pg / L sodium molybdate; 400 pg / L zinc sulphate; 1 g / L potassium phosphate; 0.5 g / L magnesium sulphate; 0.1 g / L sodium chloride; 0.1 g / L calcium chloride; 50 g / L glycerol from biodiesel production; 0.26 g / L urea; distilled water.

(i) Cultivation conditions: ЗГС temperature; 250 rpm mixing speed; batch cultivation; pH 5.5 (adjusted with 1M NaOH); 120 hours of cultivation duration; aeration from mixing.

(ii) Filtration: filtration of the press through a 0.5 μm cellulose filter.

(iii) Pre-extraction treatment: drying at 38 ° C and mechanical grinding using a ball mill.

Amount of biomass obtained (grams per liter of medium): 11.27 ± 0.11 g / L (biomass was determined gravimetrically).

(iv) Supercritical CO2 extraction conditions: 25 MPa pressure; Temperature 39.85 ° C; 12 kg СОг / hour amount of CO2; 3 hours processing time.

Amount of oil obtained: 2.10 g / L (18.67% of biomass).

Example 2: Method for obtaining microbial oil from whey

[026] Microorganism used: Umbelopsis isabellina (wild type).

Medium: 10 g / L yeast extract; 10 g / L peptone; 8 g / L sodium chloride; 0.12 g / L urea; autoclaved (2 bar pressure, 121 ° C temperature, 20 minutes treatment time) curd whey instead of water.

(i) Cultivation conditions: 28 ° C; 150 rpm mixing speed; batch cultivation; pH 6 (adjusted with 1M NaOH); 120 hours of cultivation duration; aeration from mixing.

(ii) Filtration: vacuum filtration through a 0.45 μm cellulose filter.

(iii) Pre-extraction treatment: rinsing with tap water, drying at 38 ° C and destruction of the cell walls using an ultrasonic bath.

Amount of biomass obtained (grams per liter of medium): 9.14 ± 0.08 g / L (biomass was determined gravimetrically).

(iv) Supercritical CO2 extraction conditions: 40 MPa pressure; 80 ° C temperature; 8 kg CO2 / hour amount of CO2; 45 minutes processing time.

Oil content obtained: 1.40 ± 0.02 g / L (15.27% of biomass).

Example 3: Method for obtaining microorganism oil from a sulphite waste stream [027] Microorganism used: Umbelopsis isabellina (wild type).

Medium: sulphite waste stream (obtained from wood in the laboratory) and distilled water in a ratio of 1: 1.

(i) Cultivation conditions: 30 ° C; 800 rpm mixing speed; batch cultivation; pH 6 (adjusted with 1M NaOH); 120 hours of cultivation duration; aeration was maintained at about 0.6 L minutes' ¹ .

(ii) Filtration: filtration of the press through a 0.5 μm cellulose filter.

(iii) Pre-extraction treatment: drying at 38 ° C and mechanical grinding using a ball mill.

Amount of biomass obtained (grams per liter of medium): 4.78 ± 0.12 g / L (biomass was determined gravimetrically).

(iv) Supercritical CO2 extraction conditions: 32 MPa pressure; 47 ° C temperature; 4 kg СОг / hour amount of CO2; 4 hours processing time.

Amount of oil obtained: 0.80 ± 0.04 g / L (16.76% of biomass).

Example 4: Method for obtaining micro-organism oil from molasses processing residues, fruit and vegetable processing residues

[028] Microorganism used: Rhodococcus opacus (wild type)

Medium: 10 g / L yeast extract; 10 g / L peptone; 8 g / L sodium chloride; 0.2 g / L urea; 70 g / L sugar cane molasses; autoclaved (2 bar pressure, 121 ° C temperature, 20 minutes treatment time) waste products: 10 g / L potato starch residues (fibers), 50 g / L apple squeezed.

(i) Cultivation conditions: 30 ° C; 120 rpm mixing speed; batch cultivation; pH 7 (adjusted with 1M NaOH); 72 hours cultivation duration; aeration from mixing.

(ii) Filtration: centrifugation (6000 rpm, 15 minutes).

(iii) Pre-extraction treatment: rinsing with tap water, drying at 38 ° C and destruction of the cell walls using an ultrasonic bath.

Amount of biomass obtained (grams per liter of medium): 2,02 ± 0,03 g / L (biomass was determined gravimetrically).

(iv) Supercritical CO2 extraction conditions (SFT-150): 20 MPa pressure; 35 ° C temperature; 1 kg СОг / hour amount of CO2; 6 hours processing time.

Oil content obtained: 0,66 ± 0,02 g / L (32,54% of biomass).

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Claims (3)

A process for obtaining a microorganism oil comprising the sequential steps of: (i) fermenting the substrate by culturing the microorganisms in a bioreactor, (ii) filtering, (iii) pre-extracting the biomass, and (iv) extracting, wherein the substrate is biodegradable. industrial by-products and step (iv) is performed by supercritical CO2 extraction. Process according to claim 1, characterized in that in step (iv) the supercritical CO2 extraction is carried out at a pressure of 20-40 MPa, at a temperature of 31-80 ° C, with a CO2 content of 1-12 kg CO 2 / hour and an extraction time is from 45 minutes to 6 hours. Process according to claim 1, characterized in that in step (i) biodegradable production by-products selected from industrial glycerol, whey, sulphite waste streams, molasses, fruit and vegetable processing residues are used as substrate.