Method for continuous cultivation of methane-oxidizing bacteria methylococcuscapsulatus for production of protein product from methane with controlled microbial contamination

Abstract

FIELD: biotechnology.SUBSTANCE: method is proposed for the continuous cultivation of methane-oxidizing bacteria Methylococcus capsulatus for obtaining a protein product from methane with controlled microbial contamination, including continuous cultivation of a Methylococcus capsulatus culture on an aqueous nutrient medium containing nitrogen and phosphorus sources, with the supply of methane as a carbon source and the simultaneous selection of a portion of the culture fluid, wherein methanol is introduced into the culture fluid stepwise and brought to 0.5–1.0 vol.%, followed by holding for 6–12 hours, with a decrease in the flow rate to a dilution factor D of 0.01–0.05 h-1, then a mixture of bacteriophages, bacterial contaminants of the genera Bacillus, Pseudomonas, Acinetobacter and / or the Enterobacteriaceae family are introduced into the culture fluid in a given amount, the antimycotic agent fluconazole at a concentration of 2–6 mg / l and / or terbinafine at a concentration of 2–5 mg / l and after recording a decrease in the bacterial load, the antibiotic vancomycin is introduced at a concentration of 25–50 mg / l and / or gentamicin at a concentration of 3–5 mg / l, maintained at the specified dilution factor for 6–12 hours, repeating the specified cycle if necessary until contamination is reduced to a level of less than 102 CFU / ml in the cultures, then the dilution factor D is gradually increased to 0.15–0.35 h-1 over 8–12 hours, after which the process is returned to the normal continuous cultivation mode without emptying the bioreactor.EFFECT: reduction in bacterial and fungal contamination of the Methylococcus capsulatus culture without stopping the fermentation process.3 cl, 4 ex

Description

[0001] The invention relates to the microbiological industry, namely: to the production of microbial protein mass from a strain of methane-oxidizing bacteria Methylococcus capsulatus, which can be used in agriculture for animal feed.

[0002] Methanotrophic bacteria (e.g. Methylococcus capsulatus) are known to be used to produce feed microbial protein from methane (natural gas).

[0003] From the field of technology, patent RU 2720121 published on 04 / 24 / 2020 “Method for producing microbial protein based on hydrocarbon raw materials” is known to be the closest in technical essence and the achieved result (Prototype).

[0004] The said method according to the patent discloses the continuous cultivation of Methylococcus capsulatus (strain GBS-15) in a 40-liter ejector-type loop fermenter with a constant supply of natural gas and air at 42°C and a pH of 5.6-5.8. Fermentation of bacterial cultures is carried out with a constant supply of culture liquid, ammonia solution and a gas mixture at a temperature of 40-45°C in a continuous flow of 0.2-0.3 of the fermenter volume per hour, separation, obtaining finished biomass and drying it,

[0005] A key feature is the return (recirculation) of a portion of the spent culture fluid instead of fresh water, which increases the yield of the finished product and fermentation productivity (increasing microbial protein yield by 10-30%) while simultaneously reducing the consumption of mineral salts and treated water. It is claimed that with this long-term continuous cultivation mode (using natural gas of varying compositions and with different water sources), no phagolysis of the culture is observed, indicating that the culture is resistant to bacteriophages. However, this method does not include special measures to protect against contamination by foreign bacteria. Its implementation requires maintaining strict aseptic conditions; if bacterial contamination occurs in a long-running fermenter, the process must be stopped for cleaning and restart, reducing the efficiency and cost-effectiveness of production.

[0006] Continuous methane cultivation provides high productivity; however, prolonged, uninterrupted fermentation carries the risk of microbial contamination. Foreign microorganisms can enter the culture from equipment, nutrients, water, or gas and develop within the culture. Contamination, typically caused by Gram-positive microflora, leads to a decrease in biomass yield, extraction efficiency, and process stability, and, if severe, can lead to complete loss of the culture. For example, in continuous ethanol fermentation, contamination by Lactobacillus species caused a decrease in the target product concentration by approximately 40%. In a continuous methane process, stopping fermentation due to contamination leads to significant technological and economic losses (equipment downtime, product loss, cleaning and restart costs).Thus, maintaining sterility and preventing contamination are critical tasks during long-term continuous cultivation of methanotrophs on a gaseous substrate.

[0007] Other technical solutions aimed at reducing the risk of contamination or its consequences during the cultivation of methanotrophs are also known. For example, international application WO 2022008478 proposes an improved method for producing single-cell protein using methane, involving the use of a loop reactor. The description of this application notes that the fermentation of natural gas is associated with the fermentation of various microorganisms, since minor hydrocarbons present in the gas must be processed by accompanying bacteria; otherwise, these impurities accumulate, causing a decrease in efficiency or a complete halt in the process. In fact, this source points to the inevitable presence of undesirable associated microorganisms in the culture during long-term continuous fermentation using impure methane. Similar data are cited in the scientific literature: in a study by Bothe et al.(2002) demonstrated that during industrial cultivation of Methylococcus capsulatus (Bath strain) on natural gas under conditions of partial sterility, the culture transforms into a consortium state, where, in addition to the target methanotroph, at least three foreign heterotrophic bacteria are constantly present. The isolated contaminants were identified (representatives of Aneurinibacillus, Brevibacillus agri, and Ralstonia) and were found to lack hemolysin or enterotoxin genes, meaning they did not produce dangerous toxins. Their persistent presence in M. capsulatus cultures demonstrates the difficulty of maintaining a methanotroph monoculture during long-term continuous cultivation and the risk of developing other, potentially more dangerous contaminants.

[0008] To prevent infection, solutions involving the introduction of antimicrobial additives into the culture are also known. For example, Australian patent application AU2023311362 proposes the use of hop acids to suppress bacterial contaminants during fermentation of methylotrophic and methanotrophic microorganisms. Hop α- and β-acids are known as natural antibacterial agents against gram-positive bacteria and are widely used in brewing—they inhibit the growth of lactic acid bacteria without inhibiting yeast culture at pH 5.2-5.6. In the context of methanotrophic cultivation, the addition of hop acids to the medium suppresses the growth of extraneous gram-positive microflora and prevents a decrease in the target biomass titer. The disadvantage of this approach is the need for additional chemical reagents and maintaining a specific pH range.The effectiveness of hop acids decreases significantly with increasing pH (above 6), so in industrial conditions it may be necessary to add high concentrations of these compounds, which increases costs and can affect the properties of the final product.

[0009] Another well-known approach to reducing contamination risks is improving the bioreactor design. Patent RU2777059 describes a loop fermenter for the continuous cultivation of microorganisms (specifically, methanotrophic bacteria), which eliminates long horizontal flow sections and improves gas exchange. It is known that in classic U-shaped reactors, the presence of long horizontal tubes and a large separation zone (the so-called "barrel") leads to the formation of stagnant zones and gas pockets. In such areas, mass transfer is reduced, deposits can accumulate, and favorable conditions for the development of harmful secondary microflora are created. In the solution described in RU2777059, thanks to a special loop configuration (alternating vertical and horizontal sections of optimal length, multi-point gas injection, enhanced circulation, etc.), uniform gas-liquid mixing is achieved without large stagnant volumes.This reduces the likelihood of localized accumulation of nutrient substrates and the development of infection foci within the apparatus. However, even with optimized hydrodynamics, it is impossible to completely eliminate the possibility of external contamination—the fermenter design does not eliminate the need for sterile nutrient media, gas, and inlet equipment. Therefore, although technical improvements such as RU 2777059 improve process stability, the risk of contamination during long-term operation remains a factor requiring attention.

[0010] Furthermore, methods for selectively suppressing accompanying microflora using antimicrobial substances are known. For example, patent RU 2765490 proposes introducing an ionophore antibiotic into the gas-phase fermentation process to control contamination. The ionophore (e.g., monensin) is selected to selectively inhibit the growth of undesirable competing microorganisms (e.g., acetogens / homoacetogens) while minimally affecting the target producer strain. This approach effectively suppresses the growth of contaminants in the culture and prevents the formation of their metabolic byproducts. However, the use of antibiotics has several disadvantages. Firstly, it entails additional costs and complication of the technological process (the need to dose the drug and control its concentration). Secondly, there is a risk of developing resistant pest strains with prolonged exposure to the antibiotic.Thirdly, the use of antibiotic additives in the production of feed protein is undesirable due to the possibility of residual drug content in the biomass, which can create risks when fed to animals and is not welcomed by regulators.

[0011] Thus, an analysis of the state of the art shows that the problem of contamination remains relevant during the continuous cultivation of Methylococcus capsulatus on methane. Known solutions either require the addition of special additives (acids, antibiotics) and the use of complex engineering measures to inhibit the growth of foreign microflora, or allow the presence of unwanted companion organisms in the culture, which in any case negatively impacts the stability, productivity, and economics of the process. All of these shortcomings confirm the need to develop a new method for the continuous cultivation of M. capsulatus that eliminates these limitations. The proposed method is aimed at ensuring long-term stable methane fermentation, producing a high-quality protein product, while simultaneously eliminating the risks of contamination and the associated consequences.

[0012] In the continuous submerged cultivation of methanotrophic microorganisms (e.g., Methylococcus capsulatus) in a large-volume industrial bioreactor (approximately 100 m³), ​​the risk of contamination by foreign microflora inevitably increases over time. Even with regular CIP / SIP procedures, micro-leakage through worn seals and gaskets, degradation of sterilizing filters, biofilm formation, and the introduction of single cells during sampling and sensor replacement are possible. In long-term continuous cycles (weeks and months), even minor initial contamination can lead to the adaptation and proliferation of contaminants, which have a higher specific growth rate and can eventually displace the producer culture.

[0013] The growth of foreign bacterial or fungal microflora in the apparatus leads to competition for the substrate (methane) and oxygen, changes in pH and medium composition, and the accumulation of toxins, mucus, and foam. As a result, synthesis of the target product is reduced or completely stopped, and the methanotrophic culture degrades and is displaced by contaminants. The resulting biomass no longer meets microbiological safety and quality requirements (feed value, uniformity, and the absence of pathogens). Since the obtained biomass is intended for use in feed / feed additives, it must comply with the mandatory requirements for microbiological safety established by the Unified Veterinary and Sanitary Requirements of the EAEU (decision of the Customs Union No. 317 of 18.06.2010) and veterinary and sanitary standards for the quality of feed (15.07.1997 No. 13 7 2 / 1010), which provide for the inadmissibility of dangerous / pathogenic microflora (including Salmonella in 25 g) and the standardization of the total microbial contamination.Additionally, for protein feed products of microbiological origin, requirements are specified by national standards, including GOST R 71301-2024 (gaprin) and GOST R 57683-2017 (IPCC 60), which regulate maximum levels of QMAFAnM, yeasts, and molds, and the prohibition of Salmonella. Consequently, contamination during the cultivation process directly creates the risk of non-compliance with mandatory standards and product rejection. Typically, such a scenario necessitates an emergency shutdown of the process: the culture must be completely drained and disposed of, the apparatus must undergo lengthy cleaning and sterilization, filters and seals must be replaced, after which the production cycle must be restarted through a series of inoculators. Thus, a single contamination event results in significant technological and economic losses and also jeopardizes the biosafety of the product.

[0014] The technical problem addressed by the invention is the development of a method for the continuous cultivation of methane-oxidizing bacteria Methylococcus capsulatus for the production of a protein product from methane with controlled microbial contamination in a continuous mode without interrupting the process or draining the working volume. This ensures the selective elimination of foreign microflora while maintaining the viability and productivity of the target product, which will prevent lengthy downtime and product disposal in the event of contamination.

[0015] The technical result consists of reducing the bacterial and fungal contamination of the culture, achieving the absence of contaminant growth on the crops, selectively suppressing unwanted microflora without stopping the fermentation process and without emptying the apparatus, maintaining the stability of the Methylococcus capsulatus population at an acceptable controlled level of contamination.

[0016] The finished protein product will comply with current sanitary, veterinary and feed regulations for safety and quality.

[0017] Sanitation is performed directly in the bioreactor's working volume, limited to one or two short processing cycles instead of the multi-day downtime required for a full production restart. Each sanitation cycle reduces bacterial and fungal contamination of the culture by at least 3-4 orders of magnitude (CFU / ml), achieving zero contaminant growth in the cultures after one or two processing cycles. At the same time, the viability and production potential of the target strain are maintained: the decline in biomass concentration does not exceed 20-30%, the specific growth rate is restored to at least 70-80% of the original, and after completion of the "wash" process, process productivity returns to its original level (at least 4 g / L h).The continuous washing stage also ensures the removal of introduced reagents to acceptable residual levels: after 1-3 culture volumes, their concentrations are reduced by approximately 63-95%, which allows for compliance with regulatory requirements for residues of veterinary drugs in feed products.

[0018] The claimed method is characterized by high cost-effectiveness. The use of approved feed antibiotics, antifungal agents, and bacteriophages in sanitization cycles requires relatively low costs, significantly lower than the losses incurred during a complete shutdown and restart of the bioreactor. Even multiple in-situ sanitizations are less expensive than a single emergency drain of 100,000 liters of culture and a subsequent full CIP / SIP cycle with re-inoculation and process ramp-up. Thus, the combined features of the invention ensure not only microbiological contamination control but also the maintenance of continuous production while minimizing economic risks.

[0019] The technical problem is solved and the technical result is achieved by developing a method for the continuous cultivation of methane-oxidizing bacteria Methylococcus capsulatus for obtaining a protein product from methane with controlled microbial contamination, including continuous aerobic cultivation of a culture of Methylococcus capsulatus on an aqueous nutrient medium containing sources of nitrogen and phosphorus, with the supply of methane as a carbon source and the simultaneous selection of a portion of the culture fluid, including the introduction of selective reagents and the performance of regime operations for the sanitation of the culture without stopping the process.

[0020] The method is implemented by introducing a strictly defined set of selective reagents into the culture fluid and performing routine operations to ensure the sanitization of the culture within the apparatus's working volume. If signs of contamination appear, manifested by the growth of foreign bacterial and / or fungal microflora, confirmed by microbiological monitoring and / or deviations in process parameters (changes in turbidity, oxygen consumption, foaming, pH), the process is switched to a sanitary mode, which includes the following steps.

[0021] In the first stage, methanol is sterilely added to the culture fluid to a sublethal concentration of 0.1-1.0% vol. for the target strain. At this concentration, Methylococcus capsulatus maintains growth and metabolic activity, while the accompanying microflora experiences a stressful environment, increasing its sensitivity to subsequent selective action. Methanol is dosed through a separate sterile circuit to maintain the specified concentration in the working volume of the bioreactor.

[0022] After the methanol concentration has stabilized, a complex of antimicrobial agents, including a bacteriophage mixture, an antimycotic agent, and an antibiotic, is introduced into the culture fluid.

[0023] The bacteriophage mixture is formed from bacteriophages active against typical bacterial contaminants, in particular bacteria of the genera Bacillus, Pseudomonas, Acinetobacter, as well as representatives of the Enterobacteriaceae family (including Escherichia coli), taking into account the strains actually circulating at a specific production site. The bacteriophage mixture is added to the culture fluid to a total titer of 10 7 -10 9 phage units per ml, providing an infection rate of MOI = 5-20 in relation to the determined number of bacterial contaminants.

[0024] Along with the bacteriophages, an antimycotic agent selected from the group of azole and / or allylamine derivatives is introduced into the culture fluid; fluconazole at a concentration of 2-6 mg / l and / or terbinafine at a concentration of 2-5 mg / l are preferably used. , which suppresses the growth of yeast and mold fungi and does not have a significant inhibitory effect on bacteria.

[0025] After recording a decrease in the bacterial load based on microbiological control data and / or indirect technological signs, an antibiotic to which the Methylococcus capsulatus culture has natural or induced resistance, active primarily against gram-positive and / or gram-negative bacterial contaminants, is introduced into the culture fluid at a concentration of 1-60 mg / l.

[0026] At the stages of introducing methanol, bacteriophage mixture, antifungal agent and antibiotic, the flow rate of the culture fluid is reduced, the dilution coefficient D is maintained at a reduced level of 0.01-0.05 h -1 , ensuring the required exposure of selective reagents and preventing their premature washout from the apparatus. The exposure time is sufficient to suppress contaminating microflora (6-12 hours).

[0027] The final stage of the sanitary regime is washing the culture, during which the dilution coefficient D is gradually increased to 0.15-0.35 h -1 The process is carried out in high-flow mode for 8-12 hours, ensuring the removal of dead microflora, lysis (protein breakdown products), and residual amounts of introduced reagents. If necessary, the sanitation cycle, including exposure and washing stages, is repeated until contamination is reduced to background levels according to microbiological monitoring data.

[0028] The transition to sanitary mode and the dosing of methanol, bacteriophage mixture, antifungal agent, and antibiotic are controlled by an automated process control and monitoring system. This system uses signals from pH, dissolved oxygen, optical density (turbidity), gas phase flow rate, and other parameters that form the normal Methylococcus capsulatus cultivation profile. Deviations from this profile trigger the automatic activation of the sanitary algorithm.

[0029] Antibiotics and antifungal agents are selected from the list of substances approved for use in feed production and comply with veterinary and sanitary requirements. Their concentrations and administration schedules are selected based on the permissible residual amounts of active ingredients in the finished product, with a washing step ensuring their reduction to regulatory levels.

[0030] An essential feature of the claimed method is that all of the above stages are carried out without stopping aeration and mixing, without draining the culture liquid and without re-accelerating the culture, which allows maintaining the continuity of the technological process, avoiding emergency stops and ensuring stable production of microbial protein while maintaining the growth and dominance of Methylococcus capsulatus.

[0031] The method is carried out as follows.

[0032] 1. Switching to sanitizing mode.

[0033] The dilution coefficient (flow rate of the culture liquid) is reduced to D = 0.01-0.05 h -1 To reduce leaching of the administered agents and ensure the required exposure in the culture fluid. Temperature, pH, gas phase composition, stirring intensity, and aeration are maintained within the ranges optimal for Methylococcus capsulatus growth, without abrupt changes in pressure or gas supply mode.

[0034] 2. Introduction of methanol.

[0035] Methanol, preferably as a 30% vol / vol solution in demineralized water sterilized by filtration through a 0.22 μm membrane, is dosed into the culture fluid through a separate sterile circuit. It is fed by a pump into the circulation circuit or into an intensive mixing zone to prevent localized concentration overshoot. The methanol concentration is increased stepwise: to 0.2-0.3% vol / vol over 2-3 h, then to the target range of 0.5-1.0% vol / vol while monitoring optical density, oxygen consumption, and the absence of a significant decrease in the growth rate of M. capsulatus. The achieved concentration is maintained for 6-12 h at a reduced dilution factor D 0.01-0.05 h -1 .

[0036] 3. Introduction of bacteriophage mixture and antimycotic agent.

[0037] After incubation in methanol, a bacteriophage mixture specific to the main bacterial contaminants and an antifungal agent at the working concentration are added to the culture fluid. The antifungal agent ensures suppression of yeast and mold microflora during sanitation, while the bacteriophages ensure lysis of sensitive bacterial contaminants.

[0038] 4. Administration of antibiotic.

[0039] After a reduction in bacterial load is detected (based on culture results, microscopy, and / or indirect technological indicators), an antibiotic to which the Methylococcus capsulatus strain is resistant is added to the culture fluid. The antibiotic is used to suppress residual and / or phage-resistant populations of bacterial contaminants while maintaining antifungal control of the fungal microflora.

[0040] 5. Washing and returning to normal operation.

[0041] When the required dynamics are achieved according to microbiological analysis, the dilution coefficient D is gradually increased to the working value of 0.15-0.35 h -1 , and the process is carried out in high-flow mode to remove (wash out) substances from protein breakdown products, suppressed microflora, and residual amounts of administered agents. The process is then returned to the standard continuous cultivation mode, maintaining the specified microbiological characteristics of the culture without emptying the apparatus or draining the working volume.

[0042] EXAMPLES OF IMPLEMENTING THE METHOD

[0043] Example 1 (240 L, severe mixed bacterial and fungal contamination)

[0044] In a bioreactor with a filling volume of 240 L, during continuous cultivation of M. capsulatus at a high density of 15-25 g a.d.w. / L, pronounced mixed contamination with bacteria (including Bacillus spp., representatives of Enterobacteriaceae) and yeast at a level of approximately 108 CFU / ml (according to the results of inoculation) was detected. The process was switched to sanitizing mode: the dilution factor was reduced to D = 0.01 h -1 Temperature, pH, and gas phase parameters were maintained at the standard operating level; the piping units were inspected, with the sterilizing filters for the gas and liquid reagent supply, as well as the sampling line and seals, being replaced.

[0045] Methanol was sterilely added to the culture liquid up to 1.0% vol., which amounted to 2.4 L of methanol per 240 L. Then, a bacteriophage mixture was simultaneously added, for example, SPO1, SP10, φ29, T4, T7 with a concentrate titer of 10 11 FO / ml. The introduction was carried out based on the calculation of MOI = 10, that is, until reaching about 10 9FO / ml in the reactor, which required 2.4 l of bacteriophage mixture (10 11 FO / ml). Then, antibiotics were dosed sequentially: vancomycin up to 50 mg / l (total 12 g) and gentamicin up to 5 mg / l (total 1.2 g). Additionally, an antifungal agent was introduced: fluconazole up to 5 mg / l (total 1.2 g) and terbinafine up to 5 mg / l (total 1.2 g). The exposure was carried out for 6-12 hours at D = 0.01 h. -1 , then “washing” was carried out by increasing D to 0.20-0.25 h -1 within 8-12 hours.

[0046] Based on microbiological monitoring after the first cycle, a reduction in the total bacterial and fungal load by at least four orders of magnitude was noted; however, contaminant growth was still detectable. Second and third sanitation cycles were conducted at the same dosages and time periods. After three cycles, no growth of foreign bacterial or fungal microflora was detected in the cultures. The producer biomass concentration remained at 15-18 g / L, and within 20 hours after completion of sanitation, the initial productivity of the continuous process was restored to a level of at least 4 g / L h without interruption or draining the unit.

[0047] The absence of contaminants according to microbiological control was maintained for at least 7 days.

[0048] Example 2 (240L, moderate contamination at early stage)

[0049] In a bioreactor with a filling volume of 240 l, during continuous cultivation of M. capsulatus with an average density of 10 g a.d.w. / l, moderate mixed contamination with spore-forming Bacillus spp. and yeast microflora at a level of about 10 was detected at an early stage. 6 CFU / ml. The process is switched to sanitizing mode: D is reduced to 0.03 h -1 , temperature / pH / gas phase parameters are maintained; liquid filters and sampling unit are replaced.

[0050] Methanol was added to the culture liquid up to 0.5% vol., which amounted to 1.2 liters per 240 liters. At the same time, a bacteriophage mixture was added against the identified bacterial contaminants with a titer of 10 11 FO / ml based on MOI = 10, until reaching about 10 7 FO / ml in the reactor, which amounted to 24 ml of bacteriophage mixture. Next, the antibiotic vancomycin was added to 30 mg / l (7.2 g total) and the antifungal fluconazole to 4 mg / l (0.96 g total).

[0051] The exposure was carried out for 6-8 hours at D = 0.03 h -1, then “washing” was performed at D = 0.20-0.25 h -1 within 8-12 hours.

[0052] Microbiological monitoring (cultures / microscopy) showed a reduction in bacterial and fungal load to undetectable levels after just one sanitation cycle. The producer biomass concentration decreased by no more than 10-20% (from approximately 10 to 8-9 g / L), and within 12-18 hours, the specific growth rate returned to 0.25-0.30 h / m. -1 Without stopping or draining the apparatus. The absence of contaminants, as determined by microbiological testing, was maintained for at least 7 days.

[0053] Example 3 (1 m 3 , predominantly gram-negative bacterial contamination and yeast)

[0054] In a bioreactor with a filling volume of 1.0 m 3 (1000 l) M. capsulatus was continuously cultivated at a biomass concentration of 12 g a.d.w. / l and a working dilution factor D = 0.18 h -1Against the background of increased foaming and changes in the dynamics of dissolved oxygen, microbiological monitoring revealed contamination with gram-negative bacteria (for example, Pseudomonas pp., Acinetobacter spp.) in total 5×10 5 CFU / ml and yeast 1×10 5 CFU / ml.

[0055] The process has been switched to sanitizing mode: D has been reduced to 0.03 h -1 The sterilizing filters for the gas and liquid feed were replaced, and the sampling line was inspected. Methanol was then sterilely added to a concentration of 0.5% vol. (a total of 5.0 L per 1000 L) and held for 2 hours to stabilize the parameters. A bacteriophage mixture active against Pseudomonas / Acinetobacter isolates, with a titer of 10, was simultaneously added. 11 FO / ml, until reaching 10 7FO / ml in the reactor (MOI ≥ 10 relative to the bacterial load), which required 100 ml of bacteriophage mixture. Antifungal agents were simultaneously administered: fluconazole up to 4 mg / l (4 g total) and terbinafine up to 2 mg / l (2 g total). Three to four hours after the administration of the bacteriophage mixture (based on signs of a decrease in the bacterial load according to express testing), an antibiotic to which M. capsulatus is resistant, gentamicin up to 5 mg / l (5 g total), was administered.

[0056] The exposure was carried out for 10 hours at D = 0.03 h -1 , then “washing” was performed by increasing D to 0.30 h -1 for 9 hours (3 volume times) followed by a return to working D = 0.18 hours -1 .

[0057] According to the results of microbiological control after one cycle, bacterial and fungal contamination was reduced to a level below the detection limit (conditionally <10 2CFU / ml by culture). The concentration of the producer biomass decreased briefly by 10-15% (to 10.2-10.8 g / l) with recovery in less than 16 hours. The productivity of the continuous process 24 hours after the start of sanitation was restored to the standard level of at least 2.16 g / l h without stopping or draining the culture fluid.

[0058] During the next 14 days, under normal growth conditions, no contaminants were detected in the crops.

[0059] Example 4 (1 m3, severe bacterial contamination with spore-forming fungi + mold fungi; two-cycle sanitation)

[0060] M. capsulatus was continuously cultivated in a bioreactor with a filling volume of 1.0 m3 at a biomass concentration of 18 g a.d.w. / L and a working dilution factor of D = 0.20 h-1. Microbiological monitoring revealed significant contamination with spore-forming bacteria (e.g., Bacillus spp.) 2×107 CFU / ml and mold microflora (e.g., Aspergillus spp.) 104-105 CFU / ml.

[0061] The process was switched to sanitizing mode: D was reduced to 0.015 h-1, components with an increased risk of contamination (gas filters, liquid filters, seals, sampling lines) were inspected and replaced. Methanol was added to the culture fluid up to 0.8% vol. (8.0 L in total). Then, a bacteriophage mixture of Bacillus spp. (and accompanying bacteria, if necessary) was added, with a titer of 1011 FO / ml, until 2×108 FO / ml was reached in the reactor (MOI = 10 relative to 2×107 CFU / ml), which required 2.0 L of the bacteriophage mixture. At the same time, an antifungal agent was added: fluconazole up to 6 mg / l (6 g in total) and terbinafine up to 4 mg / l (4 g in total). After 4-6 hours of exposure, an antibiotic to which the producer is resistant and which is active against the identified bacterial contaminants was introduced: vancomycin at 40 mg / L (40 g in total) and, additionally (to broaden the spectrum), gentamicin at 4 mg / L (4 g in total). Exposure was continued for a total of 12 hours at D = 0.015 h-1, followed by a "washout" at D = 0.30-0.35 h-1 for 10-12 hours.

[0062] Based on the results of monitoring after the first cycle, a decrease in the bacterial load by more than 3 orders of magnitude, and a decrease in the fungal load by more than 2 orders of magnitude was noted; however, isolated mold colonies remained. A second sanitation cycle was conducted at D = 0.02 h-1 while maintaining methanol at 0.5% vol. (additional addition to the target level), repeating the introduction of the bacteriophage mixture to an MOI ≥ 10 relative to the residual bacterial load and introducing an antifungal agent (fluconazole 4 mg / L, terbinafine 3 mg / L) and an antibiotic (vancomycin 25 mg / L). After the second cycle, no growth of foreign microflora was detected in the cultures.

[0063] The concentration of the producer biomass remained at a level of more than 16-17 g / l, the restoration of normal productivity occurred within 24-36 hours after the completion of sanitation, reaching 3.6 g / l h.

[0064] The entire range of sanitization measures was completed without stopping the unit and without draining the working volume, which prevented downtime associated with the full CIP / SIP cycle and re-acceleration of the culture.

Claims

1. A method for the continuous cultivation of methane-oxidizing bacteria Methylococcus capsulatus for obtaining a protein product from methane with controlled microbial contamination, comprising the continuous cultivation of a culture of Methylococcus capsulatus on an aqueous nutrient medium containing nitrogen and phosphorus sources, with the supply of methane as a carbon source and the simultaneous collection of a portion of the culture fluid, characterized in that methanol is introduced into the culture fluid stepwise, increasing its concentration in the culture fluid to 0.2-0.3 vol.% over 2-3 hours, then brought to 0.5-1.0 vol.%, followed by holding for 6-12 hours while reducing the flow rate to a dilution factor D of 0.01-0.05 h -1, then a mixture of bacteriophages, bacterial contaminants of the genera Bacillus, Pseudomonas, Acinetobacter and / or the family Enterobacteriaceae is introduced into the culture fluid in an amount that ensures an infection rate MOI of 5–20 and a total concentration of bacteriophages in the culture fluid of 10 7 -10 9 phage units / ml, the antifungal agent fluconazole at a concentration of 2-6 mg / l and / or terbinafine at a concentration of 2-5 mg / l, and after recording a decrease in the bacterial load, the antibiotic vancomycin is administered at a concentration of 25-50 mg / l and / or gentamicin at a concentration of 3-5 mg / l, maintained at the specified dilution factor for 6-12 hours, if necessary, repeating the specified cycle until contamination is reduced to a level of less than 10 2 CFU / ml by sowing, then a step-by-step increase in the dilution factor D to 0.15-0.35 h is carried out. -1 for 8-12 hours, after which the process is returned to the normal continuous cultivation mode without emptying the bioreactor.

2. The method according to claim 1, characterized in that the bacteriophage mixture contains at least one bacteriophage selected from the group: Bacillus phagephi29, Bacillus phageSPO1, Bacillus phageSP10, Escherichia phageT4, Escherichia phageT7.

3. The method according to paragraph 1, characterized in that the antibiotic is administered 3-6 hours after the administration of the bacteriophage mixture.