RU2688315C1 - Method of producing protein-lipid biomass by growing hermetia illucens larvae on cellulose-containing wastes - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and can be used for cultivation of Hermetia illucens flies. Method of protein-lipid fodder biomass production envisages cellulose-containing wastes with moisture content of 50–70 %, addition of beet molasses in amount of 3–5 % of total weight of moist substrate with subsequent population of substrate with Hermetia illucens larvae of three-day age, their cultivation and inoculation of wastes with culture of thermophilic fungi Thielavia terrestris or Myceliophthora thermophile. Culture of thermophilic fungi is introduced into the substrate in the form of a liquid suspension with a CFU value of not less than 10⁷/ml in amount of 3–8 % of wet substrate weight during growth of larval biomass after substrate heating to 36–48 °C. After 10–14 days from the beginning of growth, larvae are separated, dried to moisture content of 5 % and milled.

EFFECT: invention increases yield of biomass of larval Hermetia illucens.

1 cl, 2 ex

Description

The invention relates to the field of biotechnology, can be used in the field of cultivation of the larvae of the fly Hermetia illucens, and consists of a method for preparing a substrate based on cellulose-containing waste (used poultry litter material), which involves the introduction of a mycelium of thermophilic mycelial fungi with high cellulose-lytic activity.

A known method of preparing a substrate for growing larvae of houseflies, for example, a mixture, which includes fish. A starter mixture is introduced into the mixture in the form of a water extract from horse manure. Eggs of flies are introduced into the prepared mixture so that there is enough food for the full growth and development of larvae (Proceedings of the Novosibirsk Agricultural Institute, Novosibirsk, 1986, v. 98, p. 93 [1]). The disadvantage of this environment is a small yield of larvae, the impossibility of extrapolating the results obtained on the technology, where it is supposed to use another species - Hermetia illucens, as well as other processed raw materials.

There is a method of processing organic waste, which includes a number of technological methods: first, egg-laying of synatropic flies is applied to a nutrient substrate consisting of milk and yeast, the larvae are cultivated under optimal hydrothermal conditions, after which they are transferred to organic waste and cultured to the state of seed chips (RU 2049389 [2 ]). The disadvantage of this method is the use of species of synanthropic flies, and, consequently, the irreproducibility of results in the case of replacing bioobjects with another species - Hermetia illucens, the absence of the effect of microbiological activation of the utilized substrate.

Also known medium for growing larvae of synanthropic flies (SU 954079 [3]). The medium consists of the following components, wt. %: manure 70-95, the contents of the scar 5-30. However, the inconstancy of the microflora of the scar, as well as the use of another type of fly, may not provide the necessary effect for Hermetia illucens.

There are ways to increase the efficiency of waste disposal in the system of microorganisms - earthworms, compared with the action of these organisms separately (Tereshchenko NN Ecological and microbiological aspects of vermicultivation / NN Tereshchenko. - Novosibirsk: Nauka, 2003. - 113 p. [4]; Stom DI Transformation of Oilin Soilbya Microbial Preparationand Earthworms / DI Stom, DS Potapov, AE Balayan, ON Matveeva // Eurasian Soil Science. - 2003. - Vol.36, No.3. - P. 329-331 / [five]).

However, these techniques are intended for the field of vermicomposting and have not been tested for the purpose of their inclusion in the technology of waste processing by insect larvae.

Known is a method of recycling cellulosic waste using vermiculture with earthworm Eisenia fetida and culture fungi Trichoderma viride, which envisages preliminary oral inoculation earthworm Eisenia fetida insulin syringe with felled needle point suspension of mycelia and spores cellulolytic strains week culture fungi Trichoderma viride with the contents therein colony forming units in an amount of 10 ³ / ml in an amount of 0.05 ml or daily exposing the worms in the substrate impregnated with a slurry (RU 2488997 C1, op b. 8/10/2013 [6]). The implementation of the invention allows to intensify the utilization of cellulosic waste by obtaining a population of earthworms with increased cellulase activity. The disadvantages of the method include the complexity of the procedures and the unsuitability of use for insect larvae.

The closest to the proposed method is a biologically active feed additive for farm animals and birds, which involves mixing 1 part by weight of the biomass of Hermetia illucens larvae grown on wheat grain and previously washed with tap water, with a protein content of at least 36 wt. %, with 1 mass part of the crude probiotic product obtained by solid-phase fermentation of the strain Bacillus subtilis VKPM B-8130 on a sterile carrier. The latter is a beet pulp, soaked in a nutrient medium. The resulting mixture is homogenized and carry out a combined process of solid-phase fermentation-drying at a temperature not higher than 55 ° C (RU 2576200 C1, 12.12.2014 [4]). The implementation of the invention provides increased productivity and safety of livestock of animals and birds, zootechnical indicators of animals by increasing the digestibility of feed, stimulation of metabolic and immune processes. The disadvantage of this method is the use of expensive substrate (wheat grain) and sterile media, involving the sterilization procedure associated with the use of additional equipment in the form of a steam sterilizer, and the cost of electricity for the implementation of sterilization cycles. In the present invention, the specified technological stage is missing.

The objective of the invention is the creation of a new method of obtaining protein-lipid feed biomass of high quality, increasing the productivity of farm animals and birds.

The task is solved by a method of obtaining protein-lipid forage biomass, consisting in the fact that the biomass of Hermetia illucens larvae is grown on a fermentable substrate of organic origin, and 50% -70% chopped cellulose wastes are used as the basis of organic origin in the amount of 3-5% of the total mass of the substrate, and the fermentation of waste is carried out by the culture of thermophilic fungi Thielavia terrestris or Myceliophthora thermophila, while the culture of the Ibs are introduced into the substrate in the form of a liquid suspension with a CFU value not lower than 10 ⁷ / ml in an amount of 3-8% of the weight of the wet substrate during the process of growing the biomass of the larvae after heating the substrate to a temperature of 36-48 ° C. After 10-14 days from the start of rearing, the larvae are separated, dried and ground.

The technical result achieved by the invention is to improve the bioconversion of cellulosic non-sterilized waste into the biomass of Hermetia illucens larvae, rich in protein and lipids, due to the use of a culture of thermophilic mycelial fungi Thielavia terrestris or Myceliophthora thermophila with pronounced cellulolyticus activity. The deposition of culture is carried out in the collection of the Penza State Agrarian University. The proposed method does not require the cost of sterilization of the substrate.

The proposed method is as follows. Organic waste with a high content of cellulose (for example, spent poultry manure litter) is mechanically crushed to a particle size of 1-5 mm and moistened to 50-70%. Then to the resulting material add beet molasses in the amount of 3-5% of the total final mass of the wet substrate. The resulting substrate is distributed in standard plastic containers with a layer of 150 mm. The next technological step is the colonization of the substrate by the larvae of the Diptera Hermetia illucens of the three-day age at the rate of 4 individuals per 1 cm ² . Mixed with waste beet molasses is rich in easily-assimilated metabolites and serves as a starting trophic factor designed to feed the insect larvae at the initial stage of bioconversion, which contributes to the manifestation of their active life activity. This fact, in turn, leads to the heating of the substrate to a temperature of 36-48 ° C in 24-48 hours from the moment of the process start. After the substrate has reached the specified temperature values, it is inoculated with a liquid seed suspension of cultures of the fungi Thielavia terrestris or Myceliophthora thermophila. The high temperature of the substrate provides the necessary growth intensity and cellulolytic activity of these fungal cultures. In turn, the accumulated mycelial biomass of the fungus, as well as low molecular weight products of the enzymatic degradation of the cellulosic components of the substrate are food sources for insect larvae in the final stages of bioconversion, when the main share of readily available components (mainly molasses sugars) is exhausted.

To obtain a liquid inoculum, the culture of mushrooms is grown on a liquid nutrient medium of the following composition (g / l):

Cellulose (powder) - 10

Glucose - 15

Corn extract - 10

Yeast Extract - 5

Glycerin - 3

Ammonium sulfate - 2

Sodium nitrate - 3

Magnesium sulfate - 0.25

Iron sulfate (II) - 0.01

Potassium phosphate monosubstituted - 2,5

Chemically precipitated chalk - 2.5

Water - up to 1 liter

pH of the medium - 6.8

The nutrient medium is poured into 100 ml Erlenmeyer flasks with a volume of 500 ml, sealed with cotton-gauze plugs and sterilized in an autoclave at a temperature of 115 ° C for 30 minutes. Then the medium is seeded with a test-tube culture of a mycelial fungus along with a fragment of the mown agar medium. Deep cultivation is carried out on a laboratory circular rocking chair with a rotation speed of 220 rpm, at a temperature of 40-44 ° C for 72-120 hours.

The amount of inoculum applied is 3-8% by weight of the wet substrate, while the CFU value is not lower than 10 ⁷ / ml. After 10-14 days of processing, the larvae are separated from the substrate using a vibrating screen, dried in a vacuum dryer to 5% moisture and crushed.

The technical result achieved by the use of fungal cultures is determined by a more efficient bioconversion of cellulose-containing non-sterilized wastes by Hermetia illucens larvae into biomass rich in proteins and lipids.

Example 1

For carrying out the production experiment in the conditions of Biogenesis LLC, two experimental and control options were formed, each of which included five boxes of substrate made from cellulosic waste (crushed litter bedding of poultry mixed with beetroot molasses). The density of settlement of the larvae was 4 individuals per 1 cm ² , i.e. 8976 individuals per container, and the mass of the substrate with a humidity of 60% was 15 kg. After 48 hours from the moment of colonization by the larvae, liquid inoculum of the Thielavia terrestris culture was introduced into the containers of the experimental group. In the containers of the control variant inoculum cellulose fungus was not made. In general, the process of bioconversion was carried out for 12 days

According to the results of cultivation, the accumulation of raw biomass of Hermetia illucens larvae, the degree of conversion of substrate material was evaluated. The introduction of the liquid inoculum of the thermophilic mushroom culture of Thielavia terrestris contributes to the activation of the process of bioprocessing waste containing a large proportion of cellulose. The total biomass of raw larvae in experimental variants is 1020 g per 15 kg of substrate 60% humidity. In the control variants, the same indicator is at the level of 865 g per 15 kg of substrate 60% humidity. Thus, the excess of the larvae biomass yield in the experimental variant is 17.9% relative to the control. Evaluation of the efficiency of the process of bioconversion of the processed substrate, carried out by comparing the residual amounts of non-recycled material, showed an excess in the experimental variant compared to the control one by 31%.

Example 2

For carrying out the production experiment in the conditions of Biogenesis LLC, two experimental and control options were formed, each of which included five boxes of substrate made from cellulosic waste (crushed litter bedding of poultry mixed with beetroot molasses). The process parameters (density of settlement of larvae in the container, humidity, the timing of the introduction of the thermophilic fungus inoculum, the duration of the cultivation of the larvae) were maintained at the same numerical values as the above example. The principal difference was in the use of the culture of Myceliophthora thermophila. In the containers of the control variant inoculum cellulose fungus was not made.

According to the results of cultivation, the accumulation of raw biomass of Hermetia illucens larvae, the degree of conversion of substrate material was evaluated. The introduction of liquid inoculum of the culture of the thermophilic myceliophthora thermophila fungus increases the efficiency of bio-processing of waste containing a significant proportion of which is cellulose. The total biomass of raw larvae in the experimental variants is 1230 g. In the control, the similar indicator is at the level of 865 g per 15 kg of substrate 60% humidity. Thus, the excess of the larvae biomass yield in the experimental variant is 42.2% relative to the control. Evaluation of the efficiency of the process of bioconversion of the processed substrate, carried out by comparing the residual amounts of non-recycled material, showed an excess in the experimental version compared to the control one by 54%.

The inclusion of beet molasses containing easily accessible carbohydrates into the substrate ensures a high intensity of the exchange and growth processes of the larvae at the initial stage of growth of the larvae and the processing of the substrate. Against this background, an increase in the motor activity of the larvae and the heating of the substrate to a sufficiently high temperature, which is optimal for the manifestation of the maximum growth potential, as well as the cellulolytic activity of the cultures of thermophilic mycelial fungi, are also noted. Thus, the increased biomass of fungi, as well as the resulting products of enzymatic hydrolysis of cellulosic components, are trophic factors in the late stages of the development of the larvae of Hermetia illucens on wastes with a high content of cellulosic materials. Bringing in the organic material at the same time beet molasses and liquid inoculum of a thermophilic fungus allows to obtain a substrate that provides higher rates of biomass accumulation of Hermetia illucens larvae compared with the control 1.2-1.4 times.

Claims (1)

The method of obtaining protein-lipid fodder biomass, consisting in the fact that the biomass of Hermetia illucens larvae is grown on a fermentable substrate of organic origin, characterized in that as the basis of the substrate they use crushed cellulose waste with a moisture content of 50-70%, in which they add beet pulp in the amount of 3 -5% of the total final weight of the wet substrate, and the fermentation of waste is carried out by the culture of thermophilic fungi Thielavia terrestris or Myceliophthora thermophile, while the specified culture of fungi contribute to the substrate atm in the liquid slurry with a value not less than 10 cfu ⁷ / ml in an amount of 3-8% by weight of the wet substrate during the growth of the larvae biomass after heating the substrate to a temperature of 36-48 ° C, and after 10-14 days from the beginning of cultivation the larvae are separated, dried and crushed.