Efficient yeast culture method using straw culture medium

Abstract

The method comprises the following steps: preparation of a seed solution: inoculation and fermentation culture: inoculating the seed solution into a fermentation culture medium which takes a straw enzymolysis product as a main carbon source according to an inoculation amount of 5%-15% (v / v), and carrying out staged temperature control culture and process parameter control; wherein the staged temperature control culture is as follows: 0-12 hours after inoculation, the culture temperature is controlled to be 30-32 DEG C; after culturing for 12 hours, reducing the temperature to 26-28 DEG C; the process parameter control comprises the following steps: controlling the pH value of the culture system at 5.0-6.0 in real time, and maintaining the dissolved oxygen level at 20%-40% of the air saturation.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, specifically relating to microbial culture technology, and more specifically, to a method for efficient yeast culture using a culture medium with straw enzymatic hydrolysis products as the main carbon source. Through optimization of the culture process, this invention significantly improves the growth rate and biomass of yeast, and is suitable for the large-scale industrial cultivation of various yeasts such as *Saccharomyces cerevisiae*, *Candida utilis*, and *Pichia pastoris*. Background Technology

[0002] Yeast is one of the core microorganisms in modern bio-industry, widely used in brewing, food, feed, bioenergy, and medicine. The cultivation efficiency of yeast, including growth rate, biomass yield, and metabolite synthesis capacity, directly determines the production cost and economic benefits of downstream products.

[0003] As mentioned earlier, traditional yeast culture media rely on expensive carbon sources such as glucose and molasses, which are costly and unsustainable. Utilizing enzymatic hydrolysates prepared from agricultural waste such as crop straw as carbon sources is an ideal way to solve this problem. Existing technologies (such as CN108587042A and CN109266478A) have disclosed some yeast culture medium formulations based on straw enzymatic hydrolysates, which have reduced the cost of culture media to some extent.

[0004] However, simply developing an inexpensive culture medium formulation is insufficient for efficient industrial production. Yeast cultivation is a complex system engineering process, influenced by a combination of factors including the culture medium, inoculation conditions, and the culture environment (temperature, pH, dissolved oxygen). Even with a nutritionally balanced culture medium, improper cultivation techniques, such as poor inoculation timing, mismatched culture parameters, or an inability to effectively address dynamic changes during cultivation, will prevent the full realization of yeast's production potential.

[0005] Specifically, applying straw enzymatic hydrolysate as a novel carbon source to yeast cultivation presents the following unique technological challenges: Adaptation delay: When yeast is transferred from a traditional sugar culture medium to a complex straw enzymatic hydrolysis culture medium, it needs an adaptation period to "learn" to use mixed sugars (such as glucose, xylose, and arabinose) and tolerate residual trace inhibitors. This leads to a prolonged lag period and a decrease in overall culture efficiency.

[0006] Dramatic pH fluctuations: The buffering capacity of straw hydrolysate may be weak. During rapid growth and metabolism, yeast produces a large amount of organic acids, causing the pH of the culture medium to drop sharply. If not controlled in time, this will severely inhibit cell growth and enzyme activity.

[0007] High dissolved oxygen requirement: High-density culture requires a sufficient oxygen supply to maintain the respiratory metabolism and rapid growth of yeast. Traditional shake-flask culture or agitation methods may not be able to meet the oxygen demand under high cell density, becoming a bottleneck limiting yield.

[0008] Poor process stability: Due to the natural differences in straw raw materials, the sugar composition and inhibitor content of different batches of enzymatic hydrolysis products may fluctuate. This requires the cultivation process to have a certain degree of robustness, be able to adapt to changes in raw materials, and ensure the stability of product quality.

[0009] Therefore, there is an urgent need in this field to develop a systematic and efficient yeast culture method that is compatible with straw culture media. This method should not only be able to utilize inexpensive culture media, but also overcome the above challenges by optimizing inoculation strategies, process control, and feeding processes, so as to truly achieve low-cost, high-efficiency, and stable yeast production. Summary of the Invention

[0010] Technical problems to be solved The purpose of this invention is to overcome the shortcomings of existing technologies and provide a high-efficiency yeast cultivation method compatible with straw culture media. This method aims to solve problems such as long adaptation periods, difficulty in pH control, insufficient dissolved oxygen supply, and poor process stability when yeast is used in straw culture media, thereby maximizing the cost advantages of straw culture media and achieving high-density, high-efficiency yeast cultivation. Technical solution

[0011] To achieve the above objectives, the present invention provides a method for efficient yeast cultivation using straw culture medium, which is based on a specific yeast culture medium and includes optimized cultivation process steps.

[0012] First, the yeast culture medium used in the method of the present invention is prepared by the following steps: Preparation of straw enzymatic hydrolysis products: After pretreatment, enzymatic hydrolysis and solid-liquid separation of crop straw, the supernatant obtained is detoxified to obtain straw enzymatic hydrolysis products.

[0013] Preparation of culture medium: Dissolve organic nitrogen source, inorganic nitrogen source, phosphate buffer system and magnesium sulfate in water, add the straw enzymatic hydrolysis product, trace element mother liquor and vitamin mother liquor, adjust the pH to 5.0-6.5, and sterilize after making up to volume.

[0014] Secondly, the yeast high-efficiency cultivation method of the present invention includes the following key steps: Preparation of seed solution: Activated yeast strains were inoculated into seed culture medium and cultured at 28-32°C and 180-220 rpm for 8-12 hours to obtain seed culture in the late logarithmic growth phase. Its OD...600 The value is 2.0 - 5.0. Preferably, the seed culture medium is YPD medium or a semi-synthetic medium with a similar carbon source composition to the final fermentation medium, in order to enhance the adaptability of the seeds to the subsequent fermentation environment.

[0015] Inoculation and fermentation culture: a. Inoculation: Inoculate the seed solution prepared in step 1 into the sterilized straw culture medium at an inoculation rate of 5% - 15% (v / v).

[0016] b. Staged temperature-controlled incubation: Growth period: 0-12 hours after inoculation, control the culture temperature at 30-32°C to promote rapid cell proliferation and shorten the lag phase.

[0017] Product synthesis / stabilization period: After 12 hours of culture, the temperature is lowered to 26-28°C to maintain cell viability, reduce byproduct generation, and promote the synthesis and accumulation of target products (such as bacterial proteins or ethanol).

[0018] c. Dynamic pH control: During the cultivation process, an online pH monitoring system is used to automatically add 1-3 mol / L NaOH solution to control the pH of the cultivation system in real time within the range of 5.0-6.0.

[0019] d. Dissolved oxygen management: In fermenter culture, the dissolved oxygen level in the culture medium is maintained at 20% - 40% of the air saturation by adjusting the stirring speed (300-800 rpm) and aeration rate (0.5-2.0 vvm).

[0020] Fed-batch culture (optional, for high-density culture): When the reducing sugar concentration in the fermentation broth drops to 10-15 g / L, start adding concentrated straw enzymatic hydrolysate (reducing sugar concentration 300-500 g / L) or supplementing with a carbon source (such as glucose solution). Control the flow rate at 0.5-2.0 g / L / h to maintain the reducing sugar concentration in the fermentation broth at a low level of 5-20 g / L, so as to avoid substrate inhibition and achieve high-density cell growth.

[0021] (III) Beneficial Effects Compared with the prior art, the yeast culture method provided by the present invention has the following significant advantages: Significantly improved cultivation efficiency: Through optimized seed preparation and staged temperature control strategies, the lag phase of yeast was effectively shortened, the specific growth rate was increased, and the final biomass was increased by 15%-25% compared with traditional isothermal culture methods.

[0022] High process stability: Through dynamic pH control and dissolved oxygen management, a stable and suitable microenvironment is created for yeast growth, effectively overcoming pH collapse caused by metabolic acid production and hypoxia caused by high density, resulting in production fluctuations between batches of less than 5%.

[0023] High raw material adaptability: Combined with the feed-and-feed batch process, it can effectively cope with the differences in sugar concentration of straw enzymatic hydrolysis products from different batches. By precisely feeding, it maintains the stability of substrate concentration and ensures the consistency of final product quality.

[0024] Significant overall cost advantages: This method, based on the use of low-cost straw culture medium, further improves the yield through process optimization, reduces the energy consumption and time cost per unit product, and reduces the overall production cost by more than 40% compared with traditional processes.

[0025] Wide applicability: This method is not only applicable to brewer's yeast, but also to various industrial yeast strains such as Candida utilis and Pichia pastoris, providing a universal technical solution for the green upgrading of the entire yeast-related industry.

Detailed Implementation Methods

[0026] The technical solution of the present invention will be further described in detail below with reference to specific embodiments.

[0027] Example 1: High-efficiency culture of Saccharomyces cerevisiae using corn straw culture medium This embodiment describes in detail the entire process from corn stalk raw material to final yeast culture.

[0028] Step 1: Preparation of enzymatic hydrolysates from corn stalks Raw material pretreatment: Naturally air-dried corn stalks were dried in a 60°C oven for 12 hours until the moisture content was below 10%. The dried stalks were then pulverized to 60 mesh using a pulverizer. 100 kg of the stalk powder was added to 1.2 m³ of a 1.5% (w / v) dilute sulfuric acid solution, mixed thoroughly, and pumped into a pretreatment reactor. The mixture was treated at 110°C and 80 rpm for 1.5 hours with stirring. After treatment, the material was discharged, and a plate and frame filter press was used for solid-liquid separation. The filter residue was collected. The residue was repeatedly washed with deionized water until the pH of the washing liquid reached 7.0, yielding the pretreated corn stalk substrate.

[0029] Enzymatic hydrolysis: The pretreated corn stalk substrate was transferred to a 5 m³ enzymatic hydrolysis vessel, and 3 m³ of 0.05 mol / L citrate buffer (pH 4.8, solid-liquid ratio 1:10, w / v) was added. Compound cellulase (Novozymes Cellic® CTec3) was added to the vessel at a rate of 15 FPU / g dry weight of the stalk substrate. The hydrolysis vessel temperature was controlled at 50°C, and the stirring speed was set to 180 rpm. Enzymatic hydrolysis was performed at this constant temperature for 60 hours. During the hydrolysis process, samples were taken every 12 hours, and the reducing sugar concentration was determined using the DNS method. Enzymatic hydrolysis was considered complete when the increase in reducing sugar concentration was less than 5% in two consecutive measurements.

[0030] Post-processing (detoxification and purification): After enzymatic hydrolysis, a plate and frame filter press was used to separate the hydrolysate into solid and liquid components to remove undegraded lignin and residue, and approximately 2.8 m³ of crude hydrolysate (with a reducing sugar concentration of approximately 48 g / L) was collected.

[0031] Activated carbon adsorption: Add 2% (w / v) granular activated carbon (particle size 20-40 mesh) to the crude enzymatic hydrolysate, and incubate at 35°C and 120 rpm. Under the specified conditions, the mixture was stirred and adsorbed for 1.5 hours. After adsorption was complete, diatomaceous earth was added as a filter aid, and the mixture was then subjected to plate and frame filtration again to remove activated carbon.

[0032] Ion exchange resin detoxification: The enzymatic hydrolysate treated with activated carbon is passed through a filter packed with D101 macroporous adsorbent at a flow rate of 2.5 BV / h. A resin-coated chromatography column (column diameter to height ratio 1:9) was used. The effluent was collected, which was the detoxified corn straw enzymatic hydrolysis product. Analysis showed that the product contained 45% (w / w) reducing sugar, 0.28 g / L furfural, 0.19 g / L HMF, and 0.14 g / L total phenolic compounds. It was stored in a sealed container at 4°C for later use.

[0033] Step 2: Culture medium preparation and yeast culture Culture medium preparation: Fermentation medium (1L): In 800 mL of deionized water, add and dissolve 15 g of yeast extract and 10 g of peptone in sequence. Ammonium sulfate 5 g, potassium dihydrogen phosphate 2 g, dipotassium hydrogen phosphate 4 g, magnesium sulfate 1 g. After stirring until completely dissolved, add to step one. 100 g of the prepared corn stalk enzymatic hydrolysate (providing approximately 45 g of reducing sugar) was then added. 2 mL of trace element stock solution and vitamins were then added. 2 mL of stock solution. Adjust the pH to 5.8 with 1 mol / L NaOH solution, and finally bring the volume to 1 L with deionized water. Dispense 200 mL portions into 500 mL Erlenmeyer flasks and autoclave at 121°C for 25 minutes.

[0034] Seed culture medium (YPD): 10 g yeast extract, 20 g peptone, 20 g glucose, bring to a final volume of 1 L, and sterilize at 115°C for 30 minutes.

[0035] Cultivation methods: Seed culture preparation: Saccharomyces cerevisiae ATCC 26603 was streaked onto YPD plates and incubated at 30°C for 24 hours. A single colony was picked and inoculated into 100 mL of YPD liquid medium and incubated at 30°C and 200 rpm for 10 hours to obtain the OD (Organic Dioxide). 600 The seed solution was 3.5.

[0036] Fermentation culture: Add 3L of fermentation medium to a 5L fermenter. Inoculate the seed culture into the fermenter at an inoculation rate of 10% (v / v).

[0037] Temperature control: 31°C for 0-12 hours; after 12 hours, reduce the temperature to 27°C.

[0038] pH control: The pH was maintained at 5.5 ± 0.2 by automatically adding 2 mol / L NaOH solution.

[0039] Dissolved oxygen control: Initial stirring speed 400 rpm, aeration rate 1.0 vvm; as the cells grow, gradually increase the stirring speed to 700 rpm. Ventilation rate up to 1.5 vvm, maintain dissolved oxygen at around 30%.

[0040] Culture time: The total culture time is 24 hours.

[0041] result: At the end of the culture, the bacterial biomass OD was measured. 600 The biomass was 22.8 g / L, and the dry cell weight reached 9.8 g / L. Compared with Comparative Example 1 (conventional constant temperature culture at 30°C), the biomass increased by 18% and the culture period was shortened by 2 hours.

[0042] Comparative Example 1: Traditional Constant Temperature Culture Method The culture medium and seed culture were exactly the same as in Example 1, and the culture was carried out in a 5L fermenter. The culture conditions were: constant temperature of 30°C, pH controlled not to be lower than 5.0 by manually adding NaOH, constant stirring speed of 500 rpm, and aeration rate of 1.0 vvm.

[0043] Results: After 24 hours of incubation, the bacterial biomass OD 600 The pH was 19.3, and the cell dry weight was 8.3 g / L. During the culture process, the pH dropped to a minimum of 4.5, and the dissolved oxygen was below 10% in the later stages, indicating that growth was inhibited.

[0044] Example 2: Fed-batch culture of Candida utilis using wheat straw culture medium Step 1: Preparation of enzymatic hydrolysates from wheat straw Raw material pretreatment: Take dried wheat straw and crush it to 50 mesh. Take 100 kg of straw powder and add 1.3 m³ of 1.5% sodium hydroxide solution (solid-liquid ratio 1:13), and treat at 95°C for 1.5 hours. Subsequently, filter and wash with water until neutral to obtain the pretreated substrate.

[0045] Enzymatic hydrolysis: The substrate was added to citrate buffer (pH 5.0, solid-liquid ratio 1:12), and 18 FPU / g of domestic compound cellulase was added. The mixture was hydrolyzed at 52°C and 160 rpm for 72 hours.

[0046] Post-processing: After solid-liquid separation, the crude enzymatic hydrolysate was subjected to activated carbon adsorption (2.5% activated carbon, 35°C, 2 hours) and detoxification using a D101 resin column (flow rate 2.5 BV / h) to finally obtain wheat straw enzymatic hydrolysate with a reducing sugar content of 42% (w / w).

[0047] Step 2: Culture medium preparation and yeast culture Culture medium preparation: Fermentation medium (1L): 20 g corn steep liquor, 8 g urea, 3 g potassium dihydrogen phosphate, 5 g dipotassium hydrogen phosphate, 1.5 g magnesium sulfate. 120 g of wheat straw enzymatic hydrolysis product (providing approximately 50.4 g of reducing sugar), 3 mL of trace element mother liquor, and 3 mL of vitamin mother liquor. Adjust the pH to 6.0, and sterilize at 115°C for 30 minutes after bringing the volume to a final level.

[0048] Feeding solution: Vacuum concentrate the above wheat straw enzymatic hydrolysis products to a reducing sugar content of 400 g / L, sterilize and set aside.

[0049] Cultivation methods: Seed culture preparation: Candida utilis ATCC 9950 was cultured in seed culture medium until OD600. 00 It is 4.0.

[0050] Fermentation culture: In a 5L fermenter, the initial culture conditions were similar to those in Example 1, but the temperature was maintained at 28°C throughout. When the reducing sugar concentration in the fermentation broth drops to 12 g / L (approximately at the 10th hour of culture), the feed solution is started, with the flow rate controlled at [missing information]. 1.5 g / L / h (based on reducing sugar) to maintain the reducing sugar concentration in the fermentation broth at around 10 g / L.

[0051] result: By using fed-batch culture, the total culture time was extended to 30 hours. The final cell dry weight reached 42.5 g / L, far exceeding the 25 g / L of batch culture, achieving high-density culture of Candida utilis.

Claims

1. A method for efficient yeast cultivation using straw culture medium, characterized in that, Includes the following steps: Seed culture preparation: Inoculate the activated yeast strain into the seed culture medium and culture it to the logarithmic growth phase; Inoculation and fermentation culture: The seed culture was inoculated into a fermentation medium with straw enzymatic hydrolysis products as the main carbon source at an inoculation rate of 5% - 15% (v / v), and staged temperature-controlled culture and process parameter control were carried out. The phased temperature-controlled culture is as follows: the culture temperature is controlled at 30-32°C for 0-12 hours after inoculation; after 12 hours of culture, the temperature is lowered to 26-28°C. The process parameter control includes: controlling the pH of the culture system in real time at 5.0-6.0, and maintaining the dissolved oxygen level at 20%-40% of the air saturation.

2. The cultivation method according to claim 1, characterized in that, The method for preparing the fermentation medium includes: Preparation of straw enzymatic hydrolysis products: After pretreatment, enzymatic hydrolysis and solid-liquid separation of crop straw, the supernatant obtained is detoxified to obtain straw enzymatic hydrolysis products; Preparation of culture medium: Dissolve organic nitrogen source, inorganic nitrogen source, phosphate buffer system and magnesium sulfate in water, add the straw enzymatic hydrolysis product, trace element mother liquor and vitamin mother liquor, adjust the pH to 5.0-6.5, and sterilize after making up to volume.

3. The cultivation method according to claim 2, characterized in that, In the preparation of the straw enzymatic hydrolysis product, the pretreatment involves mixing the crushed straw with a 1%-2% dilute acid or dilute alkali solution at a solid-liquid ratio of 1:8-1:12 and treating at 80-120°C for 1-2 hours; the enzymatic hydrolysis involves adding a compound cellulase at a rate of 10-20 FPU / g substrate under pH 4.5-5.5 and 45-55°C conditions, and hydrolyzing for 48-72 hours.

4. The cultivation method according to claim 2, characterized in that, The detoxification treatment includes a combined detoxification process using activated carbon adsorption and ion exchange resin. The activated carbon adsorption involves adding 1%-3% granular activated carbon to the supernatant and adsorbing it at 30-40°C for 1-2 hours. The ion exchange resin detoxification involves passing the supernatant after activated carbon adsorption through a D101 macroporous adsorption resin column.

5. The cultivation method according to claim 1, characterized in that, It also includes a fed-batch culture step: when the reducing sugar concentration in the fermentation broth drops to 10-15 g / L, concentrated straw enzymatic hydrolysis products are added, with the flow rate controlled at 0.5-2.0 g / L / h, so that the reducing sugar concentration in the fermentation broth is maintained at 5-20 g / L.

6. The cultivation method according to claim 1, characterized in that, In the preparation of the seed culture, the culture is cultivated to OD... 600 The value is 2.0 - 5.

0.

7. The cultivation method according to claim 1, characterized in that, The yeast strain is Saccharomyces cerevisiae, Candida utilis, or Pichia pastoris.

8. The cultivation method according to claim 1, characterized in that, In the process parameter control, pH is controlled by automatically adding 1-3 mol / L NaOH solution, and dissolved oxygen is controlled by adjusting the stirring speed and aeration rate.