Cyclic fermentation method for preparing single-cell protein using methanol rectification fusel oil

Abstract

This invention discloses a cyclic fermentation method for preparing single-cell protein from methanol distillation fusel oil, belonging to the field of coal chemical by-product resource utilization and single-cell protein production technology. This method addresses the problems of complex composition, inhibitory impurities, and low direct fermentation efficiency of crude fusel oil produced by methanol distillation. It achieves efficient pretreatment of the fusel oil through ammonia washing and layering followed by atmospheric pressure flash evaporation, removing volatile sulfur, high-boiling-point higher alcohols, and other antibacterial components. Aeration oxidation / stripping desulfurization stabilizes and reuses the ammonium-containing aqueous phase, transforming waste into a nitrogen source. A methanol-utilizing Pichia pastoris strain is used, employing a staged fed-batch fermentation strategy to ultimately produce high-purity Pichia pastoris cell protein. This invention significantly reduces the interference of fusel oil inhibitors on yeast growth, achieves full recycling of carbon and nitrogen sources, reduces wastewater discharge and external nutrient consumption, has a short process flow, is easy to scale up, and possesses significant value for the high-value conversion and industrial promotion of coal chemical by-products.

Description

Technical Field

[0001] This invention belongs to the field of coal chemical by-product resource utilization and single-cell protein production technology, specifically involving a cyclic fermentation method for preparing single-cell protein by distilling fusel oil from methanol. Background Technology

[0002] Methanol, a core product of coal chemical industry, produces a large amount of fusel oil as a byproduct during its distillation and purification process. This byproduct typically contains methanol, ethanol, propanol, butanol, a small amount of pentanol components, and a certain proportion of water, and may also contain small amounts of volatile sulfur components and other polar impurities. Current disposal methods mainly involve low-value fuel utilization or incineration, which suffers from low added value, a large environmental burden, and unstable disposal costs.

[0003] Pichia pastoris ( Pichia pastoris It possesses strong methanol utilization and high cell protein accumulation capacity, making it one of the important candidate strains for industrial production of single-cell protein. However, the sources of methanol-distilled fusel oil are complex and its composition fluctuates greatly. Among them, higher alcohols and volatile sulfur components may have an inhibitory effect on yeast. When used directly for fermentation, it can easily cause problems such as slow cell growth, unstable fermentation, increased residual alcohol, and fluctuations in product quality.

[0004] Existing research indicates that different methanol-assimilating yeasts have varying tolerance to treated light alcohol fractions. For example, Hansenula polymorpha can grow under purified methanol conditions, but its growth and residual alcohol consumption significantly deteriorate when using treated light alcohol fractions. This suggests that the small amount of higher alcohols and trace impurities remaining in the light alcohol fractions may still inhibit some yeasts.

[0005] Meanwhile, washing crude fusel oil with alkaline or ammonia water will produce an ammonium-containing aqueous phase. This aqueous phase has some nitrogen source value, but it may contain free sulfides, hydrosulfates, volatile organic sulfur, and a small amount of dissolved alcohols. If it is directly reused for fermentation without treatment, it may release inhibitory components under storage, transportation, or fermentation aeration conditions, affecting the growth and fermentation stability of Pichia pastoris.

[0006] In summary, it is necessary to develop a short-process pretreatment, nitrogen source liquid stabilization, production strain suitability confirmation, and staged fermentation coupling process suitable for methanol distillation fusel oil, so as to convert the available alcohol components in the fusel oil into microbial protein and reduce waste liquid discharge and external nutrient consumption. Summary of the Invention

[0007] Technical Problem Solved: To address the aforementioned technical problems, this invention provides a cyclic fermentation method for preparing single-cell protein using methanol-distilled fusel oil. This method combines ammonia washing and stratification, atmospheric flash evaporation or atmospheric evaporation, ammonium-containing aqueous phase stabilization treatment, selection of existing methanol-utilizing Pichia pastoris production strains, seed culture expansion, and staged fed-batch fermentation. This reduces the adverse effects of inhibitory components in the fusel oil on fermentation, enabling the Pichia pastoris production strains to adapt to the influence of small amounts of higher alcohols in the light alcohol fraction, improving the carbon source utilization efficiency of the mixed alcohols, and achieving ammonium-containing aqueous phase reuse, thereby reducing external nitrogen source consumption and waste liquid discharge.

[0008] Technical solution: In a first aspect, the present invention provides a cyclic fermentation method for preparing single-cell protein using methanol distillation of fusel oil, comprising the following steps: S1. The crude fusel oil, a by-product of methanol distillation, is washed with ammonia water, stirred evenly, and then allowed to stand to separate into layers, resulting in an upper organic alcohol phase and a lower ammonium-containing aqueous phase. S2. Stabilize the lower ammonium-containing aqueous phase obtained in step S1 to remove or reduce free sulfides and volatile sulfur components, and obtain a recovered nitrogen source liquid that can be reused in the fermentation system. S3. The upper organic alcohol phase obtained in step S1 is cut by atmospheric pressure flash evaporation or atmospheric pressure evaporation to obtain a light alcohol fraction suitable for fermentation and a heavy fraction rich in high-boiling-point inhibitory components. S4. A methanol-utilizing Pichia pastoris production strain was selected, and a seed culture was obtained that could be inoculated into the main fermentation system. S5. The seed liquid obtained in step S4 is introduced into the main fermentation system. The light alcohol fraction obtained in step S3 is fed into the main fermentation system. The feed-in batch fermentation is carried out in stages with controlled flow rate. At the same time, the recovered nitrogen source liquid obtained in step S2 is used as part of the nitrogen source and / or pH adjustment liquid and reused in the main fermentation system. S6. After solid-liquid separation, wet cells are obtained from the fermentation broth. After washing, purification, drying and dehydration, Pichia pastoris cell protein product is obtained.

[0009] Preferably, the amount of ammonia added is 4% to 12% of the total mass of crude fusel oil, and the mass fraction of the ammonia is 8% to 20%.

[0010] Furthermore, the amount of ammonia added is 6% to 10% of the total mass of the crude fusel oil, and the mass fraction of the ammonia is 12% to 18%.

[0011] Preferably, in step S1, the washing temperature is 15–40°C, the stirring time is 20–45 min, and the settling time is 1–4 h.

[0012] Preferably, in step S2, the stabilization treatment is at least one of aeration oxidation and stripping desulfurization. The aeration oxidation conditions are: atmospheric pressure, temperature 20-45℃, and treatment time 0.5-4h; the stripping desulfurization conditions are: atmospheric pressure / slight negative pressure, temperature 30-80℃, and treatment time 0.5-3h; the free sulfide content in the recovered nitrogen source liquid after treatment is less than 50mg / L, and the volatile sulfur component is less than 20mg / L.

[0013] Preferably, in step S3, the atmospheric pressure flash evaporation or atmospheric pressure evaporation cutting temperature is 75-95°C, and the isoamyl alcohol content in the obtained light alcohol fraction is less than 0.5 wt%.

[0014] Furthermore, in step S3, the atmospheric pressure flash evaporation or atmospheric pressure evaporation cutting temperature is 78-88℃, and the isoamyl alcohol content in the obtained light alcohol fraction is less than 0.2wt%.

[0015] Preferably, in step S4, the methanol-utilizing Pichia pastoris is... Komagataella phaffii GG7 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 39081 and deposit date December 2, 2025.

[0016] Preferably, in step S5, the main fermentation is carried out in three stages with the addition of light alcohol fractions: 2-3 mL / (L·h) during the growth phase, 3-5 mL / (L·h) during the logarithmic growth phase, and 6-9 mL / (L·h) during the protein accumulation phase; the fermentation temperature is 28-32℃, the pH is 4.8-5.8, and the dissolved oxygen (DO) is not less than 20%.

[0017] Preferably, in step S5, the recovered nitrogen source liquid is used alone or in combination with ammonium sulfate, ammonium chloride, and ammonia to control the initial total nitrogen concentration of the fermentation system to 0.8–1.2 g / L.

[0018] Preferably, in step S6, after solid-liquid separation, the bacterial cells are washed with water 1-2 times and then spray-dried to obtain the finished product.

[0019] In a second aspect, the present invention provides a Pichia pastoris cell protein product, which is prepared by the cyclic fermentation method described in the first aspect; the product, on a dry basis, has a crude protein content ≥55wt%, an ash content of 8-13wt%, and a moisture content ≤6wt%.

[0020] Beneficial effects: By washing and separating the layers with ammonia water and cutting with flash evaporation or evaporation at atmospheric pressure, the content of volatile sulfur components and high-boiling-point higher alcohols in fusel oil is reduced, thereby mitigating their inhibition on the growth and respiratory metabolism of Pichia pastoris. By stabilizing the ammonium-containing aqueous phase, the pretreatment by-product ammonium-containing aqueous phase is converted into a controllable and reusable recovered nitrogen source liquid, reducing the consumption of external inorganic nitrogen sources and waste liquid discharge; By comparing the carbon source suitability of different methanol assimilation yeasts, it was confirmed that Pichia pastoris GG7 has a stronger tolerance to small amounts of higher alcohols in the treated light alcohol fraction than Hansenula polymorpha, thus reducing the risk of fermentation instability caused by the direct use of by-product carbon sources. By using staged fed-batch fermentation, the carbon source supply is matched with the cell growth and protein accumulation process, which reduces residual alcohol accumulation and drastic fluctuations in dissolved oxygen, and improves fermentation stability and cell protein yield. The process flow is relatively short, making it easy to coordinate with methanol distillation units, fermentation units, and utility systems, and it has good value for industrial scale-up and resource utilization. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the overall process flow of the present invention. Detailed Implementation

[0022] The present invention will be described in detail below with reference to specific embodiments: Example 1

[0023] The raw material used was fusel oil, a byproduct of methanol distillation. Its main components were 35 wt% methanol, 8 wt% ethanol, 5 wt% isoamyl alcohol, and 52 wt% water, with a small amount of volatile sulfur components. Three batches of methanol-distilled fusel oil from different distillation times were selected for analysis. Specific compositional data are shown in Table 1 below. Table 1. Composition data of raw material fusel oil

[0024] Table 1 shows that the composition of the raw materials fluctuates to some extent. The overall process flow for pretreatment and subsequent fermentation using the technology of this invention is as follows: Figure 1 As shown, the specific operation is as follows: Add 8% (by mass) of crude fusel oil to 15% (by mass) industrial ammonia solution, stir at room temperature for 30 min, and allow to stand for 2.5 h to separate into an upper organic alcohol phase and a lower ammonium-containing aqueous phase. The lower ammonium-containing aqueous phase is then subjected to aeration oxidation treatment for 2 h to reduce the free sulfide content, yielding a recovered nitrogen source solution. The upper organic alcohol phase is sent to an atmospheric pressure flash evaporator and cut at 82℃. The light alcohol fraction from the top of the column is collected as a fermentation carbon source, while the heavy fraction from the bottom of the vessel is collected separately. Key indicators after pretreatment are shown in Table 2, and data before and after stabilization of the recovered nitrogen source solution are shown in Table 3. Table 2. Indicators of three batches of raw materials after pretreatment

[0025] As shown in Table 2, the content of volatile sulfur components and isoamyl alcohol in the light alcohol fraction decreased significantly after pretreatment, indicating that ammonia washing combined with flash evaporation can effectively reduce the risk of inhibitory components entering the fermentation system.

[0026] Table 3 Data before and after stabilization of the recovered nitrogen source solution

[0027] As shown in Table 3, after stabilization treatment, the free sulfides and volatile sulfur components in the recovered nitrogen source liquid were significantly reduced. No obvious antibacterial phenomenon was observed in the fermentation pre-experiment, indicating that it can be used as a partial nitrogen source or pH adjustment liquid for reuse in the fermentation system.

[0028] Pichia pastoris GG7 (preserved) was selected Komagataella phaffii GG7 (deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC No. 39081, deposit date December 2, 2025) was used as the fermentation strain. This strain is an existing methanol-utilizing Pichia pastoris production strain. After primary and secondary seed cultures, this strain was inoculated into the main fermentation system.

[0029] The primary fermentation was validated using a 5L fermenter with a working volume of 3L, a filling coefficient of 0.6, and an inoculum size of 10%. Fermentation was conducted in stages at 30℃, pH 5.0–5.5, and DO not less than 20%: 0–24 h, the light alcohol fraction flow rate was 2 mL / (L·h); 24–36 h, the light alcohol fraction flow rate was 4 mL / (L·h); 36–108 h, the light alcohol fraction flow rate was 8 mL / (L·h). Specific data are shown in Table 4. Table 4 Data on key processes of primary fermentation

[0030] As shown in Table 4, the pH and dissolved oxygen were maintained within a controllable range during the main fermentation process, and no residual alcohol accumulation was observed during the feeding stage, indicating that the staged feed strategy can match the cell growth and carbon source consumption capacity.

[0031] After fermentation, the fermentation broth was centrifuged, the bacterial cells were washed once with water and then spray-dried to obtain bacterial protein.

[0032] Comparative example: Comparative Example 1 Without ammonia washing and flash evaporation cutting, the crude fusel oil was directly fermented, and the rest was the same as in Example 1.

[0033] Comparative Example 2 The strain was Pichia pastoris GG7, the carbon source was refined methanol, and the rest was the same as in Example 1.

[0034] Comparative Example 3 The strain was replaced with Hansenula polymorpha (ATCC26012), the carbon source was light alcohol fraction, and the rest was the same as in Example 1.

[0035] Comparative Example 4 The strain was Hansenula polymorpha, the carbon source was refined methanol, and the rest was the same as in Example 1.

[0036] Comparative Example 5 The strain was *Hansenula polymorpha*, the carbon source was crude fusel oil, and the rest was the same as in Example 1.

[0037] Table 5 shows the evaluation data of the suitability of different yeasts for different carbon sources: Table 5. Evaluation data on the suitability of different yeasts for carbon sources

[0038] Table 5 shows that Pichia pastoris GG7 can maintain a growth level close to that under refined methanol conditions when using treated light alcohol fractions, while Hansenula polymorpha shows a lower OD value under treated light alcohol fractions. 600 The levels were significantly lower than when using refined methanol, indicating that not all methanol-assimilating yeasts are suitable for direct utilization of this light alcohol fraction. This result supports the strong tolerance of GG7 to small amounts of higher alcohols and trace impurities in light alcohol fractions.

[0039] The protein parameters obtained by fermenting different yeasts with different carbon sources are shown in Table 6: Table 6. Indicators of proteins obtained from fermentation in Example 1 and Comparative Examples 1-5

[0040] Microscopic observation showed that the bacterial cells obtained in the three batches of Example 1 were plump and uniform in morphology, with no contamination from other microorganisms. In Comparative Example 1, due to the lack of effective pretreatment, bacterial growth was inhibited, and the system failed. Comparative Example 2 demonstrated that GG7 could achieve a high bacterial yield under refined methanol conditions, providing a reference for light alcohol fraction fermentation. In Comparative Example 3, *Hansenula polymorpha* growth was inhibited under light alcohol fraction conditions, and residual alcohol was detected. In Comparative Example 4, *Hansenula polymorpha* could grow under refined methanol conditions, but the bacterial yield was lower than that of GG7. Comparative Example 5 showed that untreated crude fusel oil had a stronger inhibitory effect on *Hansenula polymorpha*. These results indicate that the fusel oil pretreatment, the selection of suitable *Pichia pastoris* GG7 production strain, and the staged fed-batch fermentation in this invention have a synergistic effect. Example

[0041] Scale-up verification was conducted using a 200L mechanically stirred and aerated fermenter. The fermenter had a working volume of 120L, a filling coefficient of 0.6, an inoculum size of 10%, and an inoculum seed liquid volume of 12L. The fermentation temperature was controlled at 30℃, the pH at 5.0–5.5, and the aeration rate at 11 m³ / s. 3The pressure in the tank was controlled at 0.03–0.06 MPa per hour, and the stirring speed was adjusted within the range of 120–260 r / min based on dissolved oxygen feedback, while the dissolved oxygen (DO) was controlled to be no less than 20%. The light alcohol fraction was added in stages using a metering pump. The recovered nitrogen source solution, after stabilization treatment, was reused as part of the nitrogen source and pH adjustment solution in the fermentation system. The main operating conditions for the 200L scale-up experiment are shown in Table 7 below: Table 7 Main operating conditions for 200L scale-up test

[0042] During the 200L scale-up experiment, the pH, DO, stirring speed, cell concentration, and residual alcohol content are shown in Table 8. Table 8. 200L Test Process Data

[0043] The endpoint of the 200L scale-up test and the product specifications are shown in Table 9: Table 9. 200L Test Endpoints and Product Specifications

[0044] Compared with Example 1, the fermentation cycle of the 200L scale-up experiment was the same, and the endpoint cell wet weight, cell dry weight, and crude protein index were at the same level. No obvious residual alcohol accumulation, uncontrollable foaming, or dissolved oxygen runaway was observed during the fermentation process. The above results indicate that the combined process of fusel oil pretreatment, nitrogen source recovery stabilization, selection of suitable Pichia pastoris production strains, and staged fed-batch fermentation described in this invention has good scale-up stability and industrial application feasibility.

[0045] In summary, this invention utilizes fusel oil, a byproduct of methanol distillation, as a carbon source for Pichia pastoris fermentation. After a short-process pretreatment, it produces cell protein products. Simultaneously, the ammonium-containing aqueous phase generated during pretreatment is stabilized and reused in the fermentation system. This process can be implemented in combination with methanol distillation, fermentation, solid-liquid separation, and drying units. It features clear raw material sources, easily achievable equipment conditions, clear process control parameters, low waste discharge, and feasibility for industrial scale-up, thus possessing industrial applicability.

[0046] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A cyclic fermentation method for preparing single-cell protein using methanol distillation of fusel oil, characterized in that, Includes the following steps: S1. The crude fusel oil, a by-product of methanol distillation, is washed with ammonia water, stirred evenly, and then allowed to stand to separate into layers, resulting in an upper organic alcohol phase and a lower ammonium-containing aqueous phase. S2. Stabilize the lower ammonium-containing aqueous phase obtained in step S1 to remove or reduce free sulfides and volatile sulfur components, and obtain a recovered nitrogen source liquid that can be reused in the fermentation system. S3. The upper organic alcohol phase obtained in step S1 is cut by atmospheric pressure flash evaporation or atmospheric pressure evaporation to obtain a light alcohol fraction suitable for fermentation and a heavy fraction rich in high-boiling-point inhibitory components. S4. A methanol-utilizing Pichia pastoris production strain was selected, and a seed culture was obtained that could be inoculated into the main fermentation system. S5. The seed liquid obtained in step S4 is introduced into the main fermentation system. The light alcohol fraction obtained in step S3 is fed into the main fermentation system. The feed-in batch fermentation is carried out in stages with controlled flow rate. At the same time, the recovered nitrogen source liquid obtained in step S2 is used as part of the nitrogen source and / or pH adjustment liquid and reused in the main fermentation system. S6. After solid-liquid separation, wet cells are obtained from the fermentation broth. After washing, purification, drying and dehydration, Pichia pastoris cell protein product is obtained.

2. The cyclic fermentation method for preparing single-cell protein using methanol distillation of fusel oil according to claim 1, characterized in that: The amount of ammonia added is 4% to 12% of the total mass of crude fusel oil, preferably 6% to 10%; the mass fraction of the ammonia is 8% to 20%, preferably 12% to 18%.

3. The cyclic fermentation method for preparing single-cell protein using methanol distillation of fusel oil according to claim 1, characterized in that: In step S1, the washing temperature is 15-40℃, the stirring time is 20-45 min, and the settling time is 1-4 h.

4. The cyclic fermentation method for preparing single-cell protein using methanol distillation of fusel oil according to claim 1, characterized in that: In step S2, the stabilization treatment is at least one of aeration oxidation and stripping desulfurization. The aeration oxidation conditions are: atmospheric pressure, temperature 20-45℃, and treatment time 0.5-4h. The stripping desulfurization conditions are: atmospheric pressure / slight negative pressure, temperature 30-80℃, and treatment time 0.5-3h. After treatment, the free sulfide content in the recovered nitrogen source liquid is less than 50mg / L, and the volatile sulfur component is less than 20mg / L.

5. The cyclic fermentation method for preparing single-cell protein using methanol distillation of fusel oil according to claim 1, characterized in that: In step S3, the temperature for atmospheric flash evaporation or atmospheric evaporation is 75–95°C, preferably 78–88°C; the isoamyl alcohol content in the obtained light alcohol fraction is less than 0.5 wt%, preferably less than 0.2 wt%.

6. The cyclic fermentation method for preparing single-cell protein using methanol distillation of fusel oil according to claim 1, characterized in that: In step S4, the methanol-utilizing Pichia pastoris is... Komagataella phaffii GG7, accession number CGMCC No.39081, accession date December 2, 2025.

7. The cyclic fermentation method for preparing single-cell protein by distilling fusel oil from methanol according to claim 1, characterized in that: In step S5, the main fermentation is carried out in three stages with the addition of light alcohol fractions: 2-3 mL / (L·h) during the growth phase, 3-5 mL / (L·h) during the logarithmic growth phase, and 6-9 mL / (L·h) during the protein accumulation phase; the fermentation temperature is 28-32℃, the pH is 4.8-5.8, and the dissolved oxygen is not less than 20%.

8. The cyclic fermentation method for preparing single-cell protein using methanol distillation of fusel oil according to claim 1, characterized in that: In step S5, the recovered nitrogen source liquid is used alone or in combination with ammonium sulfate, ammonium chloride, and ammonia to control the initial total nitrogen concentration of the fermentation system to 0.8–1.2 g / L.

9. The cyclic fermentation method for preparing single-cell protein using methanol distillation of fusel oil according to claim 1, characterized in that: In step S6, after solid-liquid separation, the bacterial cells are washed with water 1-2 times and then spray-dried to obtain the finished product.

10. A Pichia pastoris cell protein product, characterized in that: It is prepared by the cyclic fermentation method according to any one of claims 1-9; the product, on a dry basis, has a crude protein content ≥55wt%, ash content 8-13wt%, and moisture content ≤6wt%.

Images