RU2118663C1 - Method of utilization of palm oil production liquid waste - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: method involves culturing the following organisms on liquid waste of palm oil production: cellulose-destructing fungi of genus Mucor and/or yeasts of genus Candida, and/or bacteria of genus Bacillus. Of fungi genus Mucor strains Mucor sp. MF-1 and/or Mucor sp. MF-2 can be used; of yeast Candida strains Candida foliarum M-07 and/or Candida utilis M-08 or M-09 can be used; of bacterium Bacillus strains Bacillus pumilis 7a or Bacillus cereus KP-4 can be used. Along with fungi Mucor lignin-destructing fungi Phanerochaete chrysosporum P-66 can be grown advisably. Yeast Candida should be grown with fungi simultaneously or successively. Before microorganisms culturing solid waste of this production or waste from agriculture can be added to liquid waste of palm oil production. After culturing yeasts or fungi the following strains of bacterium can be grown on residual substrate: Bacillus pumilis 7a, Bacillus cereus KP-4, Pseudomonas putida DB, Tiodictyon elegans HP or Sulfobacillus thermotolerans M. Liquid waste of palm oil production can be diluted or concentrated. EFFECT: improved method of waste utilization. 9 cl, 4 tbl, 11 ex

Description

 The invention relates to the field of biotechnology and relates to the disposal of liquid wastes from the production of palm oil by processing the latter into useful products using the microbiological method.

 A significant part of the global production of palm oil is concentrated in the countries of Southeast Asia with developed agriculture, and the industrial waste associated with this branch of production is one of the main environmental pollutants.

 On the other hand, this waste is a rich natural source of carbon and can be used as a substrate for growing microorganisms synthesizing useful products.

 This nature of the utilization of palm oil production waste seems to be the most promising and, starting from the 70s, a series of works on the use of microorganisms for the treatment of liquid waste and wastewater from the production of palm oil was published.

 In particular, in aerobic and anaerobic fermentation of liquid wastes of palm oil production by natural associations in purification ponds, the process takes 20 to 100 days (AN Ma, SC Cheah and MC Chow - Current status of palm oil processing wasters management, In book: Waste Management in Malaysia - Current Status and Prospects for Bioremediation. BG Yeoh et al. (Eds.), 1993, pp. 111-136). During purification, biogas is formed, and the resulting biomass is used as organic fertilizer.

 Sewage from ponds can be used as a substrate for growing Trichoderma viride ATEC 32086 to produce 1.37 - 1.42 g / l biomass containing 37.6 - 40.7% crude protein after 10-14 days of fermentation (MIA Karim and AQA Kamil - Biological Treatment of Palm Oil Mill Effluent using Trichoderma vivide, Biological Wastes, 27 (1989), pp. 143-152). The resulting product could serve as a source of protein for animal feed.

 Quite interesting were attempts to use liquid palm oil production waste, pre-treated for 4 days, as a substrate for growing photosynthetic bacteria Rhodotonacter sphaeroides (IFO 12203) to produce biodegradable polymers such as polyhydroxyalkonates. After 5 days of fermentation, 0.37 g / L biopolymer was obtained. (Hassan MA, Salam M., Ismail F., Abdul Karim MA and Shirai Y. - Bioplastics from palm oil mill effleuenr by photosynthetic bacteria, Challenges in Microbiology for the 21 s Century. Proceedings of the 17th. Malaysian Microbiology Symposium, Penang Malaysia , 29-30 Nov., 1994).

It was shown that liquid wastes from palm oil production with additional sources of nitrogen and phosphorus can serve as a substrate for growing Aspergillus oryzae IMI 44242 mushrooms. After 72 hours of growing mushrooms in flasks at a temperature of 30 ^° C, biomass containing 22–35% of crude protein was obtained. with a biomass yield of 52 - 59% of the organic substrate. During periodic cultivation in a fermenter after 48 to 72 hours, 4 g / l of crude protein was obtained with a biomass yield of 50% of the organic substrate. (TW Barker and JT Worgan - The Utilization of Palm Oil Processing Effieuents as Substrates for Microbial Protein Production by the Fungus Aspergilus oryzae, European Journal of Applied Microbiology and Biotechnology, 1981, 11, 234 - 240).

On one type of palm oil production liquid waste - on sterile condensate containing 0.75% cellulose, after growing various strains of the Trichoderma reesei fungus at a temperature of 28 ^o C, a biomass with a crude protein content of 27% was obtained, and as a result of growing thermophilic microorganisms CF -27 at a temperature of 45 ^o C after 4 days, a cellulase complex of enzymes and biomass containing 30% crude protein was also obtained (SC Cheah, AN Ma, LCL Ooi and ASH Ong Biotechnological Applications for the Utilization of Wastes from Palm Oil Mills, Fat Sci. Technol., 90, Jahrgang, Mai, 1988, 536-540).

 The mentioned methods are undoubtedly of practical interest, since they make it possible to recycle industrial waste polluting the environment into valuable and useful products. However, these technologies using natural microbial associations and monocultures are very time-consuming and not adapted to industrial production. For this reason, their use does not yet allow a radical solution to the problem of protecting the environment from pollution by industrial waste, primarily palm oil waste.

The present invention allows to solve several problems aimed at the disposal of waste palm oil production:
significantly intensify the process of processing liquid wastes of palm oil production, reduce the duration of the production cycle to 10-36 hours;
to involve solid waste of this production, as well as other agricultural and industrial waste polluting the environment, such as husks of cocoa fruit or coffee, sawdust and the like, into the process of liquid liquid utilization of palm oil production;
to find high-performance strains of microorganisms that vigorously degrade palm oil production waste and waste from other industries;
pick up combinations of fungi, yeast and bacteria that can comprehensively and more energetically utilize agricultural and industrial waste than individual representatives of such an association;
to develop an industrially applicable technology for the release of the environment from palm oil production wastes and waste from other agricultural industries that are environmental pollutants;
to obtain biological products that can be used as feedstock for the isolation of biologically valuable substances, high-protein additives in animal feed, birds and fish or organic fertilizer.

 The tasks were solved by finding highly active cultures of fungi, yeast and bacteria that actively destroy palm oil waste and other agricultural waste and turn these waste into valuable biological products.

 Such cultures are strains of fungi Mucor sp. MF-1, Mocor sp. MF-2 and Phanerochaete chrysosporum F-66, strains of the yeast Candida foliarum M-07, Candida utilis M-08 and Candida utilis M-09, strains of the bacterium Bacillus pumilis 7a, Bacillus cereus KP-4, Pseudomonas putida DB, Thiodictyon elegans HB and Sulfobacillus thermotolerans M.

 Individual crops, and preferably combinations thereof, when grown under optimal conditions on liquid waste products of palm oil production, as the only carbon source, form up to 40 g / l of biomass containing 30-40% protein.

 Cellulose-containing wastes from other industries, for example, husks of cocoa fruits, can be added to the fermentation medium.

 The fermentation medium should be enriched with sources of nitrogen and phosphorus, in which ammonia, ammonium salts, urea, nitrates and phosphates can be used.

 The microbial biomass accumulated in this way can be used as a fertilizer, feed additive, and also for other utilitarian purposes.

 The essence of the present invention lies in the fact that liquid wastes from palm oil production are used as a nutrient substrate for the cultivation of microorganisms, turning polluting industrial wastes into bio-products of independent value.

 In particular, the resulting microbial biomass can be used as a protein supplement in the feed of farm animals and birds, as a starting material for the production of protein hydrolysates and nutrient media, as well as an organic fertilizer.

 The liquid waste products of palm oil production remaining after extraction of palm oil from the feedstock are rather rich in nutrients that can be utilized by microorganisms, as can be seen from table 1.

 Liquid palm oil production waste (OPPM) serves as the basis for a culture medium for the cultivation of the corresponding microorganisms.

 The organic substances contained in this waste can be the only and sufficient carbon source for developing microorganisms. Liquid palm oil wastes can be diluted with water or concentrated depending on the conditions of the growing technology.

 In addition, they can be supplemented with other carbon substrates, in particular solid waste from the production of palm oil - the inside of oil walnut (HMO), empty palm fruits (SPP) and others, as well as agricultural and food waste, such as sawdust (DO ) or husk of cocoa fruit (BOD). In this case, it is possible to simultaneously utilize the waste of one or more industries.

 Nutrient medium for microorganisms, the basis of which is the OPPM, should be enriched with sources of nitrogen and phosphorus, such as ammonium sulfate and ammonium phosphate. The content in the medium of the nitrogen source should be 1-5 g of nitrogen / l, and the phosphorus content of 0.3 - 2.0 g of phosphorus / l.

 The culture medium thus prepared is inoculated with an appropriate microorganism or microorganism association.

 Biological objects, partially destroying and disposing of liquid waste products from palm oil production, are cellulose-destroying fungi of the genus Mucor, in particular Mucor sp. MF-1 and MF-2; Candida utilis and Candida foliarum yeast; and Bacilus roll bacteria, in particular Bacillus pumilis, Bacillus cereus. They are able to independently and vigorously process plant waste from the food industry and agricultural production.

 The process of biodegradation of liquid wastes from the production of palm oil and similar products can be intensified as a result of the joint fermentation of cellulose-depleting fungi and yeast of the genus Candida. In particular, strains of Candida foliarum M-07, Candida utilis M-08, Candida utilis M-09 were found to be suitable for use with these fungi.

 In the case of sharing, cultures of fungi and yeast can be introduced into the fermentation medium simultaneously or sequentially, for example, yeast can be introduced 10-12 hours after the start of fermentation. In addition, the process can be multi-stage - at the first stage, mushrooms are grown, then after removal of the obtained biomass, yeast is sown. After removal of the yeast biomass, the medium can be seeded with bacteria, in particular Bacillus pumilis, Bacillus cereus, Pseudomonas putida, Thiodictyon elegans or Sulfobacillus thermotolerans. In this case, the most complete utilization of the substrate is obtained.

 Some results of the disposal of OPPM or its mixture with solid agricultural wastes from individual or mixed cultures of microorganisms are given in table 2.

 From the presented data it can be seen that in all cases, i.e. when using all substrates, there is an active growth of microbial cultures and utilization of substrates, but the use of mixed cultures gives better results - it accelerates the process and increases the protein yield.

 An even higher result can be achieved when, in addition to the above cultures, the Phanerochaete chrysosporum F-66 fungus, which decomposes lignin, is included in the microorganism association, especially in cases where the utilized substrate contains sawdust, which follows from the data in Table 3.

 In addition, it was shown that participation in the degradation of waste palm oil production and agricultural production of bacterial crops in the final stages is very useful.

 Subsequent treatment of the mixture of OPPM and cocoa husk first by the association of fungi Mucor sp. MF-2 and Phanerochaete chrysosporum FF-66, then with Candida utilis M-08 yeast, and in the end the bacterial culture of Bacillus pumilis 7a yielded the highest result - the biomass yield was 68% of the weight of the original substrate, and the protein content in the biomass was 42%.

 A similar treatment of the same substrate, but without the participation of a bacterial culture, led to a biomass yield of 60%, and the protein content in the biomass was 38%.

 Specific, mentioned above, representatives of those genera and types of microorganisms that are recommended for use within the scope of the present invention, are deposited in the collection of the Institute of State Research Institute of Synthesis Proteins (Russia).

 In more detail the essence of the invention is illustrated by the following examples, not limiting, however, the essence of the proposal.

 Example 1. In 3.5 l of liquid waste products of palm oil production (OPPM) containing 5% solids and 1.6% t of nitrogen, 45.0 g of ammonium sulfate and 16.0 g of disubstituted ammonium phosphate were added as a source of nitrogen, thereby preparing a nutrient medium in which the sole source of carbon was OPPM.

 This medium was seeded with native mycelium of the cellulose-depleting fungus Mucor sp. MF-1 based on a seed dose of 0.1 g / L.

Cultivation was carried out in a Biostat-E fermenter with a mechanical stirrer at a partial pressure of oxygen of 20, pH 5.0 and a temperature of 42 ^o for 34 hours.

 After completion of the fermentation, 84.0 g of biomass was obtained, which amounted to 58.7% of the original substrate, with a relative protein (crude protein) content of 33.2%.

 A total of 31.2 g of protein was obtained with a yield of 17.8%.

 Example 2. In 5 l of OPPM containing 6% solids was added 50 g of disubstituted ammonium phosphate.

 Inoculated with yeast Candida foliarum M-07 at the rate of 0.2 g / l.

They were grown on a Biostat E fermenter with a mechanical stirrer at a temperature of 43 ^o C, pH 5.0 for 24 hours.

 After fermentation was completed, 220 g of biomass containing 31.5% crude protein was obtained.

 Example 3. In 3.5 l of OPPM, 45.0 g of ammonium sulfate and 15.0 g of disubstituted ammonium phosphate were added and brought to 7.0 l with water. The solids content of the resulting nutrient medium was 165 g, and the nitrogen content was 2.97 g.

 The medium was inoculated with native mycelium of the fungus Mucor sp. MF-1 based on the inoculum dose of 0.14 g / L.

 The cultivation was carried out under the conditions described in example 1 for 11 hours, and then on Wednesday the seed culture of the yeast culture Candida utilis M-08 was introduced on the basis of a seed dose of 0.25 g / l and cultivation was continued for another 14 hours.

 After fermentation was completed, 116.0 g of biomass containing 40.4% protein was obtained.

 The biomass yield amounted to 70.3% of the initial substrate, and the protein yield of 28.4%.

 Example 4. In 6.0 l of OPPM, 45.0 g of ammonium sulfate and 15.0 g of disubstituted ammonium phosphate were added. The content in the resulting nutrient medium of the carbon substrate was 300 g, and nitrogen 18 g.

 The medium was inoculated simultaneously with native mycelium of the cellulose-depleting fungus Mucor sp. MF-1, native mycelium of the lignin-destructive fungus Phanerochaete chrysosporum F-66 and native yeast suspension Candida utilis M-08.

 Cultivation was carried out under the conditions described above for 24 hours.

 After the completion of the fermentation, 165.0 g of biomass containing 40.0% protein was obtained.

 The biomass yield was 55% of the initial substrate, and the protein yield, respectively, was 22.0%.

 Example 5. OPPM was concentrated twice, i.e. the solids content was 100 g / l. 45 g of ammonium sulfate and 15 g of disubstituted ammonium phosphate were added to 3.5 l of the concentrate.

 Inoculated with mushrooms Mucor sp. MF-1, and Phanerochaete chrysosporum F-66 and the yeast Candida utilis M-08 and Candida fuliarum M-07 at the same time.

 Cultivation was carried out for 24 hours.

 After fermentation was completed, 245 g of biomass containing 38.2% protein was obtained.

 The biomass yield was 70%, and the protein yield was 26.7%.

 Example 6. The nutrient medium was prepared by mixing OPPM and husk of cocoa fruit in a ratio of 1: 1 on a dry matter basis. The content of the obtained organic medium in the organic substrate was 420.0 g, and inorganic nitrogen - 28.8 g. The total volume of the nutrient medium was 5 l.

 The medium was seeded with the association of fungi Mucor sp. MF-2, Phanerochaete chrysosporum F-66 and yeast Candida utilis M-09.

 Cultivation was carried out in a Biostat-E fermenter at a partial pressure of oxygen of 10.0, pH 5.0 and a temperature of 42 degrees for 42 hours.

 After fermentation was completed, 245 g of biomass containing 40.8% protein was obtained.

 The biomass yield was 58%, and the protein yield was 23.8%.

 Example 7. A nutrient medium was prepared by suspending cocoa fruit husks in liquid waste at a ratio of 1: 1 (by dry weight). The total content of the substrate was 421 g.

 The medium was seeded in a Biostat E fermenter with a working volume of 5 l by the association of the cellulose-destroying fungus Mucoor sp. MF-2 and lignin-destructive fungus Phanerochaete chrysosporum F-66 and cultivation was carried out for 18 hours. Then, Candida utilis M-08 yeast culture was introduced into the fermentation medium and fermentation continued for another 12 hours.

 At the final stage, the medium was inoculated with the bacterial culture of Bacillus pumilis 7a and cultured for 6 hours.

 After completion of the growing process, 295 g of biomass was obtained with a protein content of 40%.

 The biomass yield was 70%, and the protein yield 28%. In the liquid medium, 4% of the starting organic substrate remained.

 Example 8. The culture medium obtained after cultivation of the mushroom association Mucor sp. MF-1, Mucor sp. MF-2, Phanerochaete chrysosporum F-66 and yeast Candida utilis M-08 on a mixture of OPPM and husk of cocoa fruit containing 20 g / l of residual organic matter.

Flasks containing 100 ml of the above medium without other additives were seeded with various strains of bacteria. The inoculum concentration was the same. The cultivation was carried out on a thermostatic shaker at a temperature of 42 ^o C and a pH of 7.0 for 10 hours. The results are shown in table 4.

 Example 9. The nutrient medium was prepared by suspension in 50 ml of OPPM 2.0 g of sawdust. On Wednesday, 0.15 g of disubstituted ammonium phosphate was added.

 The medium was inoculated with the association of the cellulose-depleting fungus Mucor sp. MF-2 and lignin-destructive fungus Phanerochaete chrysosporum F-66.

 Cultivation was carried out in flasks on a thermostatically controlled shaker at a temperature of 42 degrees for 48 hours.

 After completion of the fermentation process, 3 g of biomass with an organic nitrogen content of 4.5% was obtained.

 The biomass yield was 67%.

 Example 10. The nutrient medium was prepared by suspension in 50 ml of OPPM 2.0 g of empty palm fruits. On Wednesday, 0.15 g of disubstituted ammonium phosphate was added.

 The medium was inoculated with the association of the cellulose-depleting fungus Mucor sp. MF-2 and lignin-destructive fungus Phanerochaete chrysosporum F-66 and yeast Candida utilis M-09.

Cultivation was carried out in flasks on a thermostatically controlled shaker at a temperature of 42 ^o for 38 hours.

 Upon completion of the fermentation process, 3.1 g of biomass with an organic nitrogen content of 3.3% was obtained.

 The biomass yield was 69%.

 Example 11. The bacteria Bacillus pumilis 7a and Bacillus cereus KP-4 were grown in flasks containing 100 ml of liquid palm oil waste (OPPM) obtained by centrifugation for 20 minutes at 8000 G, with the addition of 2 g of monosubstituted ammonium phosphate. The cultivation temperature is 42 degrees, PH-6.8, the cultivation time is 16 hours. Biomass was isolated by centrifugation. The lyophilized dried biomass of Bacillus pumilis 7a contained 52% crude protein at a concentration of 12 g / L, and Bacillus cereus KP-4 contained 58% protein at a concentration of 10 g / L.

 Biomass accumulated as a result of the cultivation of fungi Mucor sp. and Phaenrochaete chrysosporum, yeast Candida utilis and Candida foliarum, bacteria Bacillus pumilis, Bacillus cereus and Pseudomonas putida on liquid waste products of palm oil is a dry, loose, heterogeneous mass from light to dark brown. The size of particles is up to 400 mesh, humidity is 5-6%. The ash content is not more than 8%, carbon - 42-45%, crude protein - not less than 35%, lipids - up to 6%, nitrates - not more than 200 mg / kg. The content of heavy metals is not more (mg / kg of product): lead - 5, arsenic - 2, mercury - 0.1, fluorine - 100, cadmium - 0.3.

 This biomass does not have toxicity on the test - objects of the Tetrachimena pyrimiformis infusoria and white mice. Does not contain sanitary indicative microorganisms.

 It can be stored in a dry room in plastic bags that do not allow changes in the humidity of the product, at a temperature of 20 to 40 degrees for 2 years.

 This product can be used as a feed additive for farm animals.

 The biomass enriched with organic nitrogen, obtained by growing the association of mushrooms and yeast on a mixture of liquid waste with sawdust or empty palm fruits, can be used as an organic fertilizer.

Mushroom strain Mucor sp. MF-1 was isolated from palm oil production effluents, and strain MF-2 was isolated from the soil of Malaysia. Aerobes. They grow well in liquid aerated media, as well as on the surface of solid media. On solid nutrient media they form an abundant well-branched, unseparated substrate and aerial mycelium. Propagate sexually and vegetatively. They form large multispore sporangia of spherical shape, sitting on thick, vertical sporangios. Mature sporangia are dark gray, almost black. Disputes round with a shell, motionless, appear in the environment when a ripe sporangium ruptures. During the sexual process, zygotes are formed by the fusion of the two ends of the hyphae, which are separated by a septum from the rest of the mycelium. In a liquid well-aerated medium, many globules form. Assimilate various sugars, organic acids, starch and cellulose. Ammonium salts, nitrates and urea can be used as a source of nitrogen. Grow at a pH of 3 to 8 (optimum 5.0-5.5). They have a wide range of growth temperatures from 20 to 50 ^o . Temperature optimum 40-43 ^o . In laboratory conditions, the strains are maintained in test tubes on beveled wort agar. The frequency of reseeding once every 2-3 months. The strains are not toxic, not pathogenic, and also not phytopathogenic and not phytotoxic.

The strain of the fungus Phanerochaete chrysosporum F-66 was obtained from the collection of the Institute of State Research Institute of Synthesis and Synthesis, Russia. Aerobe. It grows well both on the surface of solids and in liquid synthetic aerated media. It forms an abundant branching substrate mycelium of white color. With prolonged cultivation, the mycelium disappears and microscopy shows that only spores (or conidia) of an egg-shaped white form remain. Propagated sexually and vegetatively. In liquid cultures, aeration can form globules. Assimilates sugars, organic acids and lignin. It does not have cellulose activity. The optimum pH is 5.0-5.5. Growth temperature up to 45 degrees, optimum 34-40 ^o . In laboratory conditions, the strain is maintained in test tubes on beveled wort agar. The frequency of reseeding once every 2-3 months. The strain is not toxic, not pathogenic, and also not phytopathogenic and not phytotoxic.

 Candida foliarum M-07 yeast strain isolated from palm oil production wastes in Malaysia. Cells in a glucose-peptone medium are single ellipsoidal in shape with kidneys located on the side, not along the center line. There can be 2-3 kidneys on one cell. It does not form ascospores. Pseudomycelia does not form. On malt wort forms rounded light cream almost white colonies, smooth with a low sheen, evenly rising with a smooth edge. The wort-agar line is flat, matte, light cream, slightly scalloped. Assimilates glucose, D-xylose, D-mannose, D (-) fructose, glycerin, adonite, succinic acid, citric acid. Non-assimilates sucrose, lactose, D-maltose, D (-) ribose, D (+) trehalose, D-galactose, D-cellobiose, D-arabinose, D-melibiosis, L-rhamnose, D-mannitol, starch, dextran, inulin , cellulose, hexadecane, inositol. Does not ferment glucose. Absorbs nitrate nitrogen. Needs B vitamins, especially thiamine. The need for thiamine is partially eliminated by biotin and nicotinic acid. The optimum growth temperature is 440C. It grows in a wide pH range from 4 to 8 at the same rate. Essential trace elements: Zn, Fe, Mn. Cu inhibits the growth of the strain. The strain is neither pathogenic nor toxic, nor phytopathogenic and phytotoxic.

Strain Candida utilis M-08 isolated from the soil of Malaysia. The yeast culture in a liquid medium is represented by single or assembled into short, branched chains cells. The shape of the cells is oval to cylindrical. Sizes 3.5 - 4.5 x 7.0 - 12.0 microns. May form primitive pseudomycelia. The wort agar colonies are round, smooth, grayish-cream with a low sheen. It does not form ascospores. Optional aerob. Assimilates glucose, sucrose, maltose, xylose, mannose, ethanol, mannitol, adonite, citric acid, a number of fatty acids, including high molecular weight ones. Ferment glucose and sucrose. Uses ammonium and nitrate nitrogen, as well as urea. Biotin-dependent. Calculation in a wide range of pH from 3 to 7 and a temperature of 20 - 47 ^o . In laboratory conditions, the strain is maintained in test tubes on beveled wort agar. It can also be maintained on synthetic media with sugars and additional growth factors or molasses. The strain is not toxic, not pathogenic, and also not phytopathogenic and not phytotoxic.

The strain Candida utilis M-09 is isolated from palm oil effluents. The yeast culture in a liquid medium is represented by single or assembled into short, branched chains cells. The shape of the cells is more rounded than that of strain M-08, and the stroke of the old culture on beveled wort agar is less rigid. Cell sizes 3.5 - 4.5 • 7.0 - 12.0 microns. May form primitive pseudomycelia. The wort-agar colonies are round, smooth, grayish-cream, with a low sheen. It does not form ascospores. Optional aerob. Assimilates glucose, sucrose, maltose, xylose, mannose, ethanol, mannitol, adonite, citric acid, a number of fatty acids, including high molecular weight ones. Ferment glucose and sucrose. Uses ammonium and nitrate nitrogen, as well as urea. Biotin-dependent. It grows in a wide range of pH from 3 to 7 and a temperature of 20 - 47 ^o . In laboratory conditions, the strain is maintained in test tubes on beveled wort agar. It can also be maintained on synthetic media with sugars and additional growth factors or molasses. The strain is not toxic, not pathogenic, and also not phytopathogenic and not phytotoxic.

The strain of the bacterium Bacillus pumilis 7a isolated from soils contaminated with oil. Aerobe. Thin straight sticks 0.6 - 0.8 microns wide, form filaments and clusters of filaments. Form round-oval endospores. Gram-positive or gram-variable. The colonies on the MPA are flat cream with a slightly raised darker middle and scalloped edges. It forms a gray-white film on the broth, which adheres to the edges of the tube. It dilutes gelatin, milk peptones. High catalase activity. It grows well in the temperature range 30 - 50 ^o C and pH 6.5 - 8.5. Uses organic and inorganic forms of nitrogen, including nitrates and urea. Uses a wide range of organic compounds, including heterocyclic and aromatic. Resistant to 7% NaCl. The strain is not toxic, not pathogenic, and also not phytopathogenic and not phytotoxic.

Strain Thiodictyon elegans HB - isolated from the bottom sediments of shrimp breeding farms in Malaysia. Long thin sticks 1-2 x 10-12 microns. Gram-positive, contain large gas vacuoles. Cells can form chains and clusters. Photoheterotroph, capable of active aerobic heterotrophic growth on complex organic and inorganic substrates, both in the light and in the dark. The culture grown in the dark under aerobic conditions in liquid media has a faint purple hue, which is significantly enhanced when the culture is exposed to light. It grows in a wide temperature range from 28 to 43 ^o C. The optimum pH is 6-7. The yield from the substrate is 50-60%.

Strain Sulaobacillus thermoaceticum M - isolated from the effluent of palm oil production in Malaysia. Sticks of 1-2 x 8-10 microns in size form clusters. Able to grow in complex organic environments. It is characterized by acid resistance and halophilicity. It can grow in the range of pH 4.0 - 7.0 and in the temperature range 30 - 45 ^o C.

Strain Bacillus cereus KP-4 - isolated from plant agricultural waste. Sticks measuring 1-2 x 5-7 microns, form filaments. It grows on various organic media - mono-, di-, polysaccharides, including glucans and fructans. It is distinguished by its ability to decompose a wide range of nitrogen-containing compounds, including plant proteins. Thermotolerant. On agar media it grows in the form of folded colonies similar to fungal colonies. Gram-positive and spore-forming. Temperature range 25 - 43 ^o C, pH 6.0 - 7.0.

The strain Pseudomonas putida DB - isolated from the soil of Malaysia. Short sticks 2-3 x 4-5 microns. Aerobic, gram-negative, non-spore forming sticks. Able to oxidize a large number of organic compounds, such as monosaccharides, organic acids, hydrocarbons - octodecane, hexadecane, benzene, polysaccharides, dextran, xanthan, various proteins and their decomposition products. It is characterized by acid resistance and thermotolerance. The temperature range of growth is 30 - 43 ^o C and pH 4.0 - 7.0.

 Thus, which is confirmed by specific examples of the invention, the proposed method can dramatically increase the productivity of the process of recycling palm oil production waste and carry out this process on an industrial scale.

 The proposed method differs from the existing known methods of recycling palm oil production waste using microorganisms cultures not previously used for these purposes and using mixed microbial cultures, which was also not used previously to solve this problem.

 The microorganisms Mucor sp., Phanerochaete chrysosporum, Candida foliarum, Candida utilis, Bacillus pumilis, Bacillus cereus and Pseudoomonas putida microorganisms used by the applicant are not commonly used and common producers of cellulite enzymes and are not even mentioned in the latest bioconversion of plant materials A. , A.V. Gusakov, V. M. Chernoglazov, Bioconversion of lignocellulosic materials (Moscow: Publishing House of Moscow University, 1995, p. 220). Therefore, their use for solving the tasks set by the applicant is not obvious.

The following links provide information on determinants on the basis of which strains can be identified:
1) Key to lower plants (mushrooms). / Edited by L.I. Kursanov, Moscow: Soviet Science, 1954.

 2) Microrology. E. Muller et al. M .: Mir, 1995.

 3) A brief identifier of Bergi bacteria. Translation edited by G.A. Zavarzin. - M .: Mir, 1980.

Claims (9)

 1. The method of disposal of liquid waste products of palm oil production by growing microorganisms on them, characterized in that the microorganisms use cellulose-destroying fungi of the genus Mucor, yeast of the genus Candida and / or bacteria of the genus Bacillus.  2. The method according to claim 1, characterized in that strains of Mucor sp. Are used from cellulose-depleting fungi of the genus Mucor. MF-1 and / or Mucor.sp.MF-2.  3. The method according to claim 1, characterized in that strains of Candida foliarum M-07 and / or Candida utilis M-08 or Candida utilis M-09 are used from yeast of the genus Candida.  4. The method according to claim 1, characterized in that the bacteria of the genus Bacillus use strains of Bacillus pumilis 7a or Bacillus cereus KP-4.  5. The method according to PP. 1 and 2, characterized in that lignin-destroying fungi, in particular Phanerochaete chrysosporum F-66, are grown on liquid wastes of palm oil production simultaneously or sequentially with fungi of the Mucor genus.  6. The method according to claims 1, 2 and 5, characterized in that the liquid waste of palm oil production simultaneously or sequentially with fungi grow yeast of the genus Candida, in particular Candida foliarum M-07 and / or Candida utilis M-08 or Candida utilis M-09.  7. The method according to claims 1, 2, 3, 5 and 6, characterized in that after growing on liquid waste production of palm oil of mushrooms and yeast, bacteria Bacillus pumilis 7a, Bacillus cereus KP-4, Pseudomonas putida DV, are grown on a residual substrate Thiodictyon elegans HB or Sulfobacillus thermotolerans M.  8. The method according to PP. 1 to 7, characterized in that the solid waste from the production of palm oil or other agricultural industries is additionally added to the liquid waste from the production of palm oil.  9. The method according to claims 1 to 7, characterized in that before the cultivation of microorganisms, the liquid palm oil waste is diluted or concentrated.

Images