Method for utilizing fermentation waste of tetraacetyl phytosphingosine

Abstract

The application discloses a kind of tetraacetyl phytosphingosine fermentation waste fungus residue utilization method, to solve the problem of difficult treatment of wastewater and high cost of fermentation organic nitrogen source, the wastewater after extracting tetraacetyl phytosphingosine with cation resin is collected, under the condition that temperature is ≤50 DEG C, transmembrane pressure difference is ≤0.3MPa, it is filtered by ceramic membrane, and the fungus residue concentrated solution is obtained by interception;Then adjust the pH of fungus residue concentrated solution to 6~7, supplement inorganic nitrogen source simultaneously;Finally, the concentrated solution is recycled for tetraacetyl phytosphingosine fermentation medium, partially or completely replace purchased organic nitrogen source.The application converts waste fungus residue into composite nitrogen source recycling, significantly reduces raw material cost under the premise of maintaining fermentation yield, while greatly reducing suspended solids and chemical oxygen demand of wastewater, effectively reduces the load of terminal environmental protection treatment, realizes the win-win of economic and environmental benefits.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, specifically to a method for utilizing waste residue from the fermentation of tetraacetyl phytosphingosine. Background Technology

[0002] Tetraacetylphytosphoside (TAPS) is a sphingolipid widely found in the cell membranes of plants, animals, fungi, and prokaryotes. It is a precursor to various ceramides. In vitro, TAPS can stimulate the synthesis of glucoside ceramides, which have protective functions, helping to strengthen the skin barrier, inhibit inflammation and angiogenesis, and reduce dark circles. TAPS has good solubility and can be compounded with various cosmetic ingredients, making it suitable for anti-aging, repairing, and eye cream products. Furthermore, phytosphingosine can be used as a starting material for the synthesis of ceramides; therefore, TAPS can be converted into other more commercially valuable products, and can also be directly used as an active ingredient in the production of cosmetics and skincare products.

[0003] Microbial fermentation is an important method for preparing tetraacetyl phytosphingosine. Patent CN115820440 discloses a high-yield *Saccharomyces cerevisiae* mutant strain and its application, which improves the production efficiency of tetraacetyl phytosphingosine through fermentation. However, this method uses relatively expensive beef extract, peptone, and yeast extract as nitrogen sources, which cannot effectively reduce costs in industrial production. Meanwhile, commercially available amino nitrogen sources mainly include peanut meal, soybean meal, cottonseed meal, corn steep liquor, yeast powder, fish meal, silkworm pupa powder, peptone, and wheat bran, all of which are too expensive in the fermentation industry to be used as inexpensive alternatives to control production costs. Summary of the Invention

[0004] To address the aforementioned shortcomings, this invention provides a method for utilizing waste bacterial residue from the fermentation of tetraacetyl phytosphingosine. By using nanofiltration, the yeast cell solution is concentrated, reducing suspended solids and COD levels in the wastewater generated during TAPS extraction. This reduces the difficulty of biochemical treatment of production wastewater and alleviates environmental pressure. Simultaneously, it lowers the cost of organic nitrogen sources in the fermentation formula, further promoting green and sustainable industrial fermentation production. This addresses the problems of high raw material costs, excessive wastewater and solid waste generation, and inability to treat these wastes in existing technologies.

[0005] To achieve the above objectives, the present invention provides a method for utilizing the waste residue from the fermentation of tetraacetyl phytosphingosine, comprising the following steps:

[0006] Step 1: Collect the wastewater generated after extracting tetraacetyl phytosphingosine fermentation broth with cation exchange resin, and filter the wastewater with a ceramic membrane to obtain a concentrated bacterial residue; wherein, the ceramic membrane has a pore size of 5-10 nm, a filtration temperature of ≤50℃, and a transmembrane pressure difference of ≤0.3 MPa.

[0007] Step 2: Adjust the pH value of the mushroom residue concentrate obtained in Step 1 to increase the inorganic nitrogen source in the mushroom residue concentrate; the pH value should be controlled at 6~7.

[0008] Step 3: Add the concentrated bacterial residue from Step 2 as an organic nitrogen source to the tetraacetyl phytosphingosine fermentation medium, partially or completely replacing the organic nitrogen source.

[0009] Preferably, in step 1, the ammonia nitrogen content of the concentrated bacterial residue is 5% to 10%, and the total nitrogen content is 10% to 25%.

[0010] Preferably, in step 2, ammonia water is used to adjust the pH value of the bacterial residue concentrate, and the ammonia water concentration is 20wt%~30wt%.

[0011] Preferably, in step 1, after the wastewater is filtered through a ceramic membrane, a bacterial residue concentrate and water to be discharged are obtained. The bacterial residue concentrate is a solid-liquid mixture, while the organic waste removal rate in the water to be discharged can reach more than 80%, which basically meets the discharge standards of GB21903-2008 and GB21905-2008. Subsequently, it is only necessary to adjust the pH of the water to be discharged to neutral before it can be discharged directly.

[0012] Preferably, in step 1 or step 3, the tetraacetyl phytosphingosine fermentation broth is obtained by fermentation with a strain, namely, Meyerozyma Guilliermondii mSCU-JM06-8 strain, which is preserved at the China General Microbiological Culture Collection Center with accession number CGMCCNo.23507.

[0013] Preferably, in step 3, the components of the fermentation medium are: 20-30 g / L glucose, 1-2 g / L beef extract, 5-10 g / L peptone, 3-8 g / L yeast nitrogen, 2 g / L L-serine, 0.2 g / L calcium chloride, and 2-5 mL / L PTM1 Pichia pastoris trace element mother liquor; wherein, the bacterial residue concentrate can partially or completely replace any one of the organic nitrogen sources among beef extract, peptone, or yeast nitrogen in any proportion, and the concentration of the bacterial residue concentrate is 2-20 g / L.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] 1. This invention introduces a ceramic membrane filtration process into the utilization method of waste bacterial residue from tetraacetyl phytosphingosine fermentation. This process filters the wastewater generated after cation exchange resin extraction, which can efficiently retain bacterial residue and macromolecular organic matter in the wastewater under mild operating conditions, thereby obtaining a concentrated bacterial residue solution. This significantly reduces pollutants such as suspended solids and chemical oxygen demand in the wastewater.

[0016] 2. Based on this, the present invention uses ammonia water to adjust the pH value of the bacterial residue concentrate to 6-7. This operation not only makes the bacterial residue concentrate reach a suitable pH range for reuse in the fermentation system, but also introduces an additional inorganic nitrogen source during the adjustment process, so that the bacterial residue concentrate has the dual nitrogen supply function of both organic and inorganic nitrogen sources.

[0017] 3. This invention has discovered that these two steps also have a synergistic effect. The concentrated liquid obtained by ceramic membrane filtration retains the rich organic nitrogen-containing compounds such as proteins, amino acids and nucleotides in the yeast cells, while the process of adjusting the pH with ammonia water further supplements inorganic nitrogen sources such as ammonium nitrogen. Together, they construct a composite nitrogen source system that complements organic and inorganic nitrogen. When this composite nitrogen source system is reused in the tetraacetyl phytosphingosine fermentation medium and partially or completely replaces purchased organic nitrogen sources such as beef extract, peptone, and yeast nitrogen, it can significantly reduce the raw material cost of the fermentation medium while maintaining a basically stable fermentation yield. More importantly, the effect of ceramic membrane filtration on the retention of suspended solids and organic pollutants in wastewater and the effect of the fermentation residue concentrate reuse on the replacement of organic nitrogen sources are not simply superimposed, but form a closed-loop synergistic mechanism: on the one hand, the biochemical treatment difficulty of wastewater is greatly reduced after ceramic membrane filtration, reducing the burden on end-of-pipe environmental treatment; on the other hand, the retained fermentation residue concentrate, as a valuable nitrogen-containing resource, is reintroduced into the fermentation production process, transforming materials that would otherwise be treated as waste into production raw materials. This reduces raw material procurement costs while simultaneously cutting wastewater and solid waste disposal costs, achieving a dual benefit of economic and environmental benefits. Detailed Implementation

[0018] This invention will describe the technical solutions of the embodiments of the invention clearly and completely. Obviously, the described embodiments are only some embodiments of the invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on this invention are within the scope of protection of this invention.

[0019] Unless otherwise specified in the specific circumstances, the numerical ranges listed herein include upper and lower limits, as well as all integers and fractions within that range, but are not limited to the specific values ​​listed when the range is defined.

[0020] I. A method for utilizing waste residue from the fermentation of tetraacetyl phytosphingosine.

[0021] After implementing the tetraacetyl phytosphingosine extraction method disclosed in the prior patent application CN116803973, the inventors discovered that although the technology reduced the amount of organic solvent used by employing cation exchange resin adsorption in the product extraction stage, new technical problems arose in the actual production process: the wastewater generated after adsorbing and extracting the fermentation broth with cation exchange resin contained a large amount of bacterial residue and high concentrations of organic matter, with suspended solids content as high as 213242 mg / L, chemical oxygen demand as high as 18426 mg / L, and total nitrogen content as high as 852 mg / L. The water quality characteristics of this type of wastewater far exceeded the direct acceptance capacity of existing conventional sewage treatment processes. In actual production, it is necessary to undergo multiple repeated treatments to gradually reduce the wastewater to the range that existing sewage treatment technologies can handle. This not only significantly increases the process steps and time costs of wastewater treatment, but also keeps the overall environmental protection costs high. At the same time, the inventors also noted that existing tetraacetyl phytosphingosine fermentation processes generally rely on expensive external organic nitrogen sources such as beef extract, peptone, and yeast nitrogen as culture medium components, making it difficult to effectively control raw material costs in large-scale industrial fermentation production.

[0022] To address the aforementioned technical issues, the inventors re-examined the resource attributes of microbial residue in wastewater. Considering that the microbial residue in the wastewater generated during the cation exchange resin extraction process originates from fermentation cells, its composition should contain abundant nitrogen-containing organic substances such as proteins, amino acids, and nucleotides, possessing potential value for recycling as a nitrogen-containing resource. Based on this understanding, this invention employs a ceramic membrane for wastewater filtration and separation. By controlling the ceramic membrane pore size to 5-10 nm, the filtration temperature to no more than 50°C, and the transmembrane pressure difference to no more than 0.3 MPa, the microbial residue and large molecular organic matter are retained and concentrated under mild conditions to obtain a microbial residue concentrate. Simultaneously, small-molecule dissolved pollutants in the permeate are discharged with the effluent. Thus, while achieving preliminary wastewater purification, the nitrogen-containing substances in the microbial residue are enriched and recovered in the form of a concentrate. Subsequently, ammonia water is used to adjust the pH of the bacterial residue concentrate, controlling the pH value within the range of 6-7. While adjusting the pH to meet the requirements of the fermentation system, ammonia water is used as an inorganic nitrogen source to introduce ammonium nitrogen into the system, enabling the bacterial residue concentrate to supply both organic and inorganic nitrogen. Finally, the treated bacterial residue concentrate is reused as an organic nitrogen source in the fermentation medium for tetraacetyl phytosphingosine, partially or completely replacing the original purchased organic nitrogen source components. This establishes a material recycling technology path between the fermentation production stage and the wastewater treatment stage. Therefore, the specific steps of the method described in this invention are as follows:

[0023] Step 1: Collect the wastewater generated after extracting tetraacetyl phytosphingosine fermentation broth with cation exchange resin, and filter the wastewater with a ceramic membrane to obtain a concentrated bacterial residue; wherein, the filtration temperature is ≤50℃ and the transmembrane pressure difference is ≤0.3MPa.

[0024] Step 2: Adjust the pH value of the mushroom residue concentrate obtained in Step 1 to increase the inorganic nitrogen source in the mushroom residue concentrate; the pH value should be controlled at 6~7.

[0025] Step 3: Add the concentrated bacterial residue from Step 2 as an organic nitrogen source to the tetraacetyl phytosphingosine fermentation medium, partially or completely replacing the organic nitrogen source.

[0026] In the practical application of this invention, many unexpected technical effects were obtained: when the concentrated bacterial residue completely replaced the organic nitrogen sources such as beef extract, peptone, and yeast nitrogen in the fermentation medium at a concentration of 25-30 g / L, after a 120-hour fermentation cycle, the concentration of tetraacetyl phytosphingosine in the fermentation broth reached 24.4 g / L. Compared with the fermentation concentration of 24.7 g / L obtained by the control group using a complete organic nitrogen source formula, there was only a slight difference of 1.21%. This shows that the concentrated bacterial residue derived from wastewater recovery can still maintain a fermentation yield level comparable to that of the standard culture medium formula when completely replacing the purchased organic nitrogen source. In parallel, the wastewater treated by ceramic membrane filtration showed extremely significant changes in water quality indicators: the suspended solids content in the wastewater decreased from 213,242 mg / L in the original wastewater to 15-18 mg / L, the chemical oxygen demand decreased from 18,426 mg / L to 1,505-1,577 mg / L, the total nitrogen content decreased from 852 mg / L to 35-45 mg / L, and the total phosphorus content decreased from 131 mg / L to below 0.02 mg / L. The improvement in the above-mentioned wastewater quality indicators means that, during the process of adjusting the pH value of the bacterial residue concentrate with ammonia water after treatment by the method of this invention, ammonia water itself is introduced into the concentrate system as an inorganic nitrogen source. This allows the bacterial residue concentrate to not only provide an organic nitrogen source when reused in the fermentation medium, but also to supplement inorganic nitrogen sources such as ammonium nitrogen. This synergistic supply mode of organic and inorganic nitrogen makes the comprehensive nitrogen supply capacity of the bacterial residue concentrate exceed the expected range of simple organic nitrogen source substitution, providing a more balanced nitrogen source nutrition structure for the fermentation strains during growth and metabolism.

[0027] In some embodiments of the present invention, ceramic membranes are chosen for wastewater treatment because the present invention has two clear and important requirements for the separation process: on the one hand, it is necessary to efficiently retain intact yeast cells, cell fragments, and large nitrogen-containing substances such as proteins, peptides, and nucleotides in the wastewater, and enrich the wastewater to obtain nitrogen-rich bacterial residue concentrate to meet the needs of fermentation reuse; on the other hand, it is necessary to discharge small molecule soluble metabolic byproducts and inorganic salts with the permeate, so as to avoid their accumulation in the concentrate and inhibit subsequent fermentation, and to achieve effective reduction of pollutant indicators in the effluent. The ceramic membrane is formed by high-temperature sintering of inorganic powder, resulting in a uniform and controllable pore size distribution. The 5-10 nm pore size specification used in this invention can form a stable sieving boundary, achieving complete retention of intact *Saccharomyces cerevisiae* cells with sizes much larger than the pore size. It also has a stable retention efficiency for large nitrogen-containing organic molecules with a molecular weight of 10 kDa or higher, effectively enriching organic nitrogen resources in wastewater and ensuring that the total nitrogen and ammonia nitrogen content of the bacterial residue concentrate meets the fermentation reuse standards. At the same time, small molecule soluble substances can pass smoothly through the membrane pores without excessive accumulation on the concentration side, ensuring an effective reduction in indicators such as suspended solids, chemical oxygen demand, and total nitrogen in the effluent, while also avoiding the potential negative impact of impurity accumulation on the subsequent fermentation system. However, if conventional organic polymer ultrafiltration membranes are used as a substitute, their wide pore size distribution formed by polymer phase transformation results in large fluctuations in retention accuracy, which can easily lead to two types of deviations: First, the retention pore size is too large, causing leakage of large molecular nitrogen-containing substances, which reduces the nitrogen recovery rate of the bacterial residue concentrate and prevents the effluent pollutant indicators from achieving the expected purification effect; second, the retention pore size is too small, trapping small molecular metabolic byproducts, causing the enrichment of inhibitory impurities in the concentrate, which interferes with the normal growth and metabolism of the bacterial strains during reuse, and at the same time fails to effectively reduce the effluent pollution load, making it difficult to simultaneously meet the dual process objectives of resource recovery and wastewater purification. For the high solids content feed characteristics of the original wastewater with a suspended solids concentration exceeding 200,000 mg / L, the separation stability advantage of ceramic membranes is further highlighted. Its rigid inorganic membrane surface has excellent hydrophilicity, and combined with the cross-flow filtration operation mode, the filter cake layer formed by bacterial particles is not easily compacted, and the concentration polarization effect is weak. It can maintain a stable operating flux for a long time under extremely high suspended solids load, continuously completing the concentration and enrichment of bacterial residue and continuous effluent production. However, if an organic membrane is used to treat this type of high-concentration bacterial suspension wastewater, a dense bacterial filter cake layer will form on the membrane surface in a short time, causing serious membrane pore blockage. The operating flux will decline rapidly, requiring frequent backwashing and chemical cleaning to barely maintain operation. This not only significantly reduces the production efficiency, but also accelerates the damage of the membrane separation layer due to frequent hydraulic and chemical reactions, resulting in an irreversible decrease in separation accuracy and making it impossible to achieve the expected separation effect in the long term.Furthermore, the inorganic inertness and material purity of ceramic membranes can fully guarantee the biosafety of the separated materials. During the entire filtration process, no foreign impurities such as polymeric additives, plasticizers, or oligomers will be dissolved into the materials. The components of the resulting bacterial residue concentrate are entirely derived from the original fermentation cells themselves, and the composition is pure and controllable. When reused in the fermentation system, no exogenous metabolic inhibitors will be introduced, which can effectively ensure the stability of fermentation batches and the consistency of product quality. On the other hand, organic polymer membranes may have the risk of leaching trace amounts of low-molecular-weight additives or degradation products during long-term operation and repeated cleaning. These unknown impurities may have an uncontrollable impact on the metabolic pathways of the strains after entering the culture medium with the bacterial residue concentrate, increasing the risk of fermentation yield fluctuations and making it difficult to maintain stable control in industrial production. Moreover, the excellent thermal stability and chemical cleaning resistance of ceramic membranes play a crucial role in supporting the long-term sustainability of the aforementioned separation effect. Under the filtration conditions of ≤50℃ in this invention, ceramic membranes do not undergo material deformation or pore size drift. They can also withstand repeated cleaning by strong acids, strong alkalis, and oxidizing cleaning agents, effectively removing protein and bacterial contaminants from the membrane surface and stably restoring membrane flux, ensuring that the separation accuracy remains at the design level throughout long-term operation. In contrast, organic membranes are prone to polymer chain swelling under the same conditions, leading to enlarged pore size, retention failure, and limited resistance to strong cleaning agents, making it difficult to maintain stable separation performance over the long term.

[0028] In some embodiments of the present invention, in step 1, the ammonia nitrogen content of the bacterial residue concentrate reaches 5wt%~10wt%, and the total nitrogen content is 10wt%~25wt%, so it can be used as a substitute raw material for fermentation organic nitrogen source.

[0029] In some embodiments of the present invention, in step 2, ammonia water is used to adjust the pH value of the bacterial residue concentrate, and the ammonia water concentration is 20wt%~30wt%.

[0030] In some embodiments of the present invention, in step 1, after the wastewater is filtered through a ceramic membrane, a concentrated bacterial residue and water to be discharged are obtained. The concentrated bacterial residue is a solid-liquid mixture, while the removal rate of organic waste in the water to be discharged can reach over 80%, basically meeting the discharge standards of GB21903-2008 and GB21905-2008. Subsequently, only the pH of the water to be discharged needs to be adjusted to neutral before direct discharge. This step can efficiently and energy-savingly treat organic wastewater, reducing the use of large amounts of chemical flocculants, oxidants, adsorbents, and extraction solvents in existing organic wastewater treatment processes, and solving the problems of multiple steps and difficult treatment in existing organic wastewater treatment methods.

[0031] In some embodiments of the present invention, in step 1 or step 3, the tetraacetyl phytosphingosine fermentation broth is obtained by fermentation with a strain of Meyerozyma Guilliermondii mSCU-JM06-8, which is deposited at the China General Microbiological Culture Collection Center with accession number CGMCCNo.23507.

[0032] In some embodiments of the present invention, in step 3, the components of the fermentation culture medium are: 20-30 g / L glucose, 1-2 g / L beef extract, 5-10 g / L peptone, 3-8 g / L yeast nitrogen, 2 g / L L-serine, 0.2 g / L calcium chloride, and 2-5 mL / L Pichia pastoris trace element mother liquor; wherein, the bacterial residue concentrate can partially or completely replace any one of the organic nitrogen sources among beef extract, peptone, or yeast nitrogen in any proportion, and the concentration of the bacterial residue concentrate is 2-20 g / L.

[0033] II. Examples and Comparative Examples

[0034] Example 1

[0035] Step 1: The wastewater after cation exchange resin recovery is filtered using a ceramic membrane (5nm pore size) to obtain a bacterial residue concentrate. The bacterial residue concentrate is tested and found to have an ammonia nitrogen content of 10wt% and a total nitrogen content of 25wt%, which can be used as a substitute raw material for fermentation organic nitrogen source. Various indicators of nanofiltration wastewater are tested, and the testing methods refer to GB21903-2008 Discharge Standard of Water Pollutants for Fermentation Pharmaceutical Industry and GB21905-2008 Discharge Standard of Water Pollutants for Extraction Pharmaceutical Industry. The results are shown in Table 1.

[0036] Step 2: Adjust the pH of the concentrated bacterial residue solution to 7±0.2 using ammonia water;

[0037] Step 3: Add the concentrated bacterial residue obtained in Step 2) as an organic nitrogen source to the tetraacetyl phytosphingosine fermentation medium, replacing beef extract. The fermentation medium components are as follows:

[0038] 30 g / L glucose, 2 g / L bacterial residue concentrate, 5 g / L peptone, 8 g / L yeast nitrogen, 2 g / L L-serine, 0.2 g / L calcium chloride, 5 mL / L PTM1 Pichia pastoris trace element mother liquor (for fermentation).

[0039] Step 4: Fermentation was carried out in a 25L fermenter at a fermentation temperature of 28℃, an initial stirring speed of 200rpm / min, an initial air flow rate of 10L / min, and a pH of 4.5 was maintained throughout the process with ammonia water. 50% glucose was used for fed-batch culture, and the fermentation cycle was 120 hours.

[0040] Step 5: The concentration of tetraacetyl phytosphingosine in the fermentation broth was determined using HPLC-UV method.

[0041] Example 2

[0042] The following adjustments were made to Example 1, the difference being that the fermentation medium components in step 3 are as follows:

[0043] 30 g / L glucose, 2 g / L beef extract, 5 g / L bacterial residue concentrate, 8 g / L yeast nitrogen, 2 g / L L-serine, 0.2 g / L calcium chloride, 5 mL / L PTM1 Pichia pastoris trace element mother liquor (for fermentation). All other steps are exactly the same as in Example 1.

[0044] Example 3

[0045] The following adjustments were made to Example 1, the difference being that the fermentation medium components in step 3 are as follows:

[0046] 30 g / L glucose, 2 g / L beef extract, 5 g / L peptone, 8 g / L mycelial residue concentrate, 2 g / L L-serine, 0.2 g / L calcium chloride, 2-5 mL / L Pichia pastoris trace element mother liquor (for fermentation). All other steps are exactly the same as in Example 1.

[0047] Example 4

[0048] The following adjustments were made to Example 1, the difference being that the fermentation medium components in step 3 are as follows:

[0049] 30 g / L glucose, 15 g / L bacterial residue concentrate, 2 g / L L-serine, 0.2 g / L calcium chloride, 5 mL / L PTM1 Pichia pastoris trace element mother liquor (for fermentation). Other steps are exactly the same as in Example 1.

[0050] Example 5

[0051] The following adjustments were made to Example 1, the difference being that the fermentation medium components in step 3 are as follows:

[0052] 30 g / L glucose, 25 g / L bacterial residue concentrate, 2 g / L L-serine, 0.2 g / L calcium chloride, 5 mL / L PTM1 Pichia pastoris trace element mother liquor (for fermentation). Other steps are exactly the same as in Example 1.

[0053] Comparative Example 1

[0054] Step 1: The fermentation medium components are as follows:

[0055] 30 g / L glucose, 2 g / L beef extract, 5 g / L peptone, 8 g / L yeast nitrogen, 2 g / L L-serine, 0.2 g / L calcium chloride, 5 mL / L PTM1 Pichia pastoris trace element mother liquor (for fermentation).

[0056] Step 2: Fermentation was carried out in a 25L fermenter at a fermentation temperature of 28℃, an initial stirring speed of 200rpm / min, an initial air flow rate of 10L / min, and a pH of 4.5 was maintained throughout the process with ammonia water. 50% glucose was used for fed-batch culture, and the fermentation cycle was 120 hours.

[0057] Step 3: The concentration of tetraacetyl phytosphingosine in the fermentation broth was determined using HPLC-UV method.

[0058] Step 4: According to the extraction method of tetraacetyl phytosphingosine (TAPS) disclosed in patent CN116803973, cation exchange resin was used to extract tetraacetyl phytosphingosine, and the wastewater generated during the extraction process was collected and tested for various indicators. The results are shown in Table 1.

[0059] Table 1: Test results of wastewater to be discharged after ceramic membrane filtration in Step 1 of Examples 1-5 and wastewater generated in Step 4 of Comparative Example 1

[0060] Example 1 4-5 18mg / L 1523 42 ≤0.02mg / L Example 2 4-5 16mg / L 1505 38 ≤0.02mg / L Example 3 4-5 18mg / L 1577 45 ≤0.02mg / L Example 4 4-5 15mg / L 1575 45 ≤0.02mg / L Example 5 4-5 16mg / L 1555 35 ≤0.02mg / L control group 4-5 213242mg / L 18426mg / L 852mg / L 131mg / L

[0061] The fermentation test results are shown in Table 2:

[0062] Table 2: Detection results of TAPS concentration in fermenters of Examples 1-5 and Comparative Example 1

[0063] Example 1 120 21.8 11.74% Example 2 120 21.2 14.17% Example 3 120 22.8 7.69% Example 4 120 20.8 15.79% Example 5 120 24.4 1.21% control group 120 24.7 N / A

[0064] The formula for calculating the TAPS production reduction ratio is as follows: As shown in Tables 1 and 2, when using the bacterial residue concentrate to replace the organic nitrogen source based on the original formula dosage, the TAPS unit concentration decreased to varying degrees. However, by further increasing the amount of bacterial residue concentrate, using it to replace all the organic nitrogen source can reduce the fermentation nitrogen source cost by 20.36% without reducing the fermentation unit. At the same time, it significantly reduces the suspended solids and COD in the wastewater, alleviating the environmental treatment pressure on industrial production.

[0065] The direct economic benefits of using concentrated bacterial residue to replace the organic nitrogen source in the fermentation formula are as follows:

[0066] Table 3 compares the costs of replacing the organic nitrogen source of the fermentation medium with the concentrated fermentation residue liquid in the 10-ton tank pilot-scale test of Examples 1-5.

[0067]

[0068] As shown in Tables 2 and 3, after partially replacing the organic nitrogen source with the concentrated bacterial residue, the cost per kilogram of raw material decreased by 31.3%, 29.55%, -1.88%, and 58.96%, respectively. Increasing the amount of concentrated bacterial residue to replace all organic nitrogen sources can reduce the raw material cost of the tetraacetyl phytosphingosine fermentation formula by 20.36%, and can also reduce the environmental protection treatment costs of bacterial residue and organic wastewater.

[0069] Further analysis of Tables 1-3, as well as the examples and comparative examples, reveals that:

[0070] (1) According to the wastewater quality test results in Table 1, the original wastewater produced by the traditional cation exchange resin extraction process in Comparative Example 1 had a suspended solids content as high as 213,242 mg / L, a chemical oxygen demand (COD) content as high as 18,426 mg / L, a total nitrogen content as high as 852 mg / L, and a total phosphorus content as high as 131 mg / L. All pollutant indicators were at extremely high levels, far exceeding the direct acceptance threshold of conventional biological treatment processes. This means that such wastewater cannot be directly treated by conventional biological treatment processes, which is the fundamental reason why the wastewater treatment process in the existing technology is long and the disposal cost is high. However, after ceramic membrane filtration in Examples 1 to 5, the suspended solids in the effluent were reduced to 15-18 mg / L, with a removal rate of over 99.9%; the COD was reduced to 1505-1577 mg / L, with a removal rate of about 91%-92%; the total nitrogen was reduced to 35-45 mg / L, with a removal rate of about 95%-96%; and the total phosphorus was reduced to below 0.02 mg / L, with a removal rate close to 100%. This result demonstrates that the ceramic membrane filtration process plays two key roles: Firstly, it can efficiently retain bacterial residues and macromolecular organic pollutants in wastewater under mild operating conditions, resulting in an order-of-magnitude improvement in effluent quality. This significantly reduces the biochemical treatment load and disposal difficulty of end-of-pipe wastewater from the source, solving the technical problems of excessively high pollutant concentrations and complex treatment processes in existing extraction processes. Secondly, the bacterial material retained by the ceramic membrane is not discharged as waste but is enriched and recovered in the form of bacterial residue concentrate. This concentrate retains organic nitrogen-containing components such as proteins, amino acids, and nucleotides derived from yeast cells, providing a material basis for subsequent nitrogen source reuse and achieving simultaneous pollutant reduction and resource recovery.

[0071] (2) The fermentation performance data in Table 2 further verified the feasibility and practical effect of recycling the fermentation residue concentrate into the fermentation system. Comparative Example 1 used a standard formula with all purchased organic nitrogen sources. After 120 hours of fermentation, the TAPS concentration was 24.7 g / L, representing the baseline production level of the existing process. In Examples 1-3, when the fermentation residue concentrate replaced beef extract, peptone, and yeast extract as single nitrogen source components by equal mass, the TAPS concentrations were 21.8 g / L, 21.2 g / L, and 22.8 g / L, respectively, with corresponding yield reductions of 11.74%, 14.17%, and 7.69%, showing different degrees of yield decrease. This indicates that under the condition of equal mass replacement, although the nitrogen supply efficiency and nutritional integrity of the fermentation residue concentrate differ from the original formula's composite nitrogen source system when a single high-priced nitrogen source is replaced, this difference is acceptable from the perspective of industrial application. When Examples 4 and 5 completely replaced all three types of organic nitrogen sources with the fermentation residue concentrate and the dosage was increased to 15 g / L and 25 g / L respectively, the fermentation performance showed a significant gradient recovery: In Example 4, the TAPS concentration was 20.8 g / L at a dosage of 15 g / L, resulting in a yield reduction of 15.79%; in Example 5, the TAPS concentration reached 24.4 g / L at a dosage of 25 g / L, with a yield reduction of only 1.21% compared to the control, and the yield level was basically the same as the standard formula. This change indicates that by adjusting the total dosage of the fermentation residue concentrate, the differences in nutrient concentration and composition between it and the purchased refined nitrogen source can be compensated. In particular, combined with the ammonium inorganic nitrogen introduced during the pH adjustment process with ammonia water, the fermentation residue concentrate forms a composite nitrogen supply structure that complements organic and inorganic nitrogen, which can fully meet the nitrogen requirements of *Saccharomyces cerevisiae* growth metabolism and TAPS synthesis, thereby maintaining the basic stability of the fermentation yield under the condition of completely replacing the purchased organic nitrogen source.

[0072] (3) Based on this, combined with the cost accounting data in Table 3, the economic benefits of the present invention can be clearly quantified. The total cost of raw materials for the all-purchased nitrogen source formulation in Comparative Example 1 was RMB 1827.6, which constitutes the cost benchmark for industrial production. After replacing the high-priced beef extract in Example 1, the cost decreased to RMB 1255.6, a decrease of 31.30%; after replacing peptone in Example 2, the cost decreased to RMB 1287.6, a decrease of 29.55%; both achieved significant cost reductions, confirming the economic viability of the bacterial residue concentrate as a substitute for the high-priced refined nitrogen source. After replacing the lower-priced yeast extract in Example 3, the cost increased slightly by 1.88%, reflecting the difference in economic benefits when replacing nitrogen sources at different price levels, providing a quantitative basis for prioritizing the replacement of high-priced components in actual production. Example 4 completely replaced all nitrogen sources with a dosage of 15 kg, reducing the cost to 750 yuan, a decrease of 58.96%, making it the lowest-cost option among all groups. However, the corresponding fermentation yield also decreased significantly. Example 5 completely replaced all nitrogen sources with a dosage of 20 kg, reducing the cost to 1000 yuan, a decrease of 20.36%. At the same time, the TAPS yield in Table 2 decreased by only 1.21%, achieving a relative balance between cost control and production efficiency. It is an optimized option that takes into account both economic efficiency and fermentation level.

[0073] (4) As can be seen, the ceramic membrane filtration process intercepts bacterial residues and macromolecular organic matter in wastewater through a pore size of 5~10nm. On the one hand, it realizes in-situ purification of the extracted wastewater, resulting in a decrease in pollutant indicators such as suspended solids, chemical oxygen demand, total nitrogen, and total phosphorus in the effluent compared to the original wastewater. This directly reduces the process load and operating cost of the end-of-pipe biochemical treatment of wastewater and solves the technical problem that the original extracted wastewater had too high pollutant concentrations, exceeding the acceptance capacity of conventional sewage treatment processes. On the other hand, the intercepted nitrogenous substances in the bacterial cells are enriched to form bacterial residue concentrate, which transforms the bacterial residue and dissolved organic nitrogen that originally needed to be disposed of as waste into nitrogenous resources that can re-enter the fermentation system. This completes the transformation of waste into production raw materials, which is the material basis for subsequent nitrogen source substitution. Based on this, the process of adjusting pH with ammonia water does not only play a single role in acid-base regulation. While adjusting the pH of the bacterial residue concentrate to 6-7 to suit the fermentation system, it also introduces ammonium inorganic nitrogen into the system, forming a nitrogen supply structure of organic and inorganic nitrogen in the bacterial residue concentrate. This not only makes up for the lack of organic nitrogen nutrition structure in the bacterial residue alone, ensuring the balance of nitrogen supply for strain growth and product synthesis, but also avoids the cost of adding an additional inorganic nitrogen source, further enhancing the nitrogen supply capacity and economic efficiency of the bacterial residue concentrate.

[0074] In summary, this invention forms a mutually supportive closed-loop system through three stages: wastewater purification, bacterial residue enrichment, and nitrogen source reuse. The retention efficiency of the ceramic membrane determines both the degree of wastewater purification and the amount of nitrogen recovered from the bacterial residue concentrate. Higher retention efficiency not only results in better effluent quality but also increases the total amount of reusable nitrogen, providing a material basis for a higher proportion of nitrogen source substitution. Furthermore, the reuse ratio and nutrient regulation level of the bacterial residue concentrate directly determine the reduction in raw material costs and the maintenance of fermentation yield. These three effects are not simply additive but achieve a two-way benefit of environmental and economic benefits through the internal circulation of materials: on the environmental side, it reduces the discharge of high-concentration organic wastewater and waste bacterial residue, lowering the cost and environmental risks of end-of-pipe treatment; on the production side, it reduces dependence on purchased high-priced organic nitrogen sources, compressing raw material procurement costs. This resource utilization model systematically solves the core technical problems of high organic nitrogen source procurement costs, difficulty in wastewater extraction and treatment, and low resource utilization rate of waste microbial residue in the existing tetraacetyl phytosphingosine fermentation production, providing a feasible technical path for industrial-scale green and sustainable fermentation production.

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the technical solutions. Those skilled in the art should understand that any modifications or equivalent substitutions to the technical solutions of the present invention without departing from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. A method for utilizing waste residue from the fermentation of tetraacetyl phytosphingosine, characterized in that, Includes the following steps: Step 1: Collect the wastewater generated after extracting tetraacetyl phytosphingosine fermentation broth with cation exchange resin, and filter the wastewater with a ceramic membrane to obtain a concentrated bacterial residue; wherein, the ceramic membrane has a pore size of 5-10 nm, a filtration temperature of ≤50℃, and a transmembrane pressure difference of ≤0.3 MPa. Step 2: Adjust the pH value of the mushroom residue concentrate obtained in Step 1 to increase the inorganic nitrogen source in the mushroom residue concentrate; the pH value should be controlled at 6~7. Step 3: Add the concentrated bacterial residue from Step 2 as an organic nitrogen source to the tetraacetyl phytosphingosine fermentation medium, partially or completely replacing the organic nitrogen source.

2. The utilization method according to claim 1, characterized in that, In step 1, the ammonia nitrogen content of the bacterial residue concentrate is 5 wt%~10 wt%, and the total nitrogen content is 10 wt%~25 wt%.

3. The utilization method according to claim 1, characterized in that, In step 2, ammonia water is used to adjust the pH value of the bacterial residue concentrate, with an ammonia water concentration of 20 wt%.

4. The utilization method according to claim 1, characterized in that, In step 1, after the wastewater is filtered through a ceramic membrane, a bacterial residue concentrate and water to be discharged are obtained; the pH of the water to be discharged is adjusted to neutral before it can be discharged directly.

5. The method of utilization according to claim 1, characterized in that, In step 1 or step 3, the tetraacetyl phytosphingosine fermentation broth is obtained by fermentation with a strain, namely, Meyerozyma Guilliermondii mSCU-JM06-8.

6. The method of utilization according to claim 1, characterized in that, In step 3, the components of the fermentation medium are: 20-30 g / L glucose, 1-2 g / L beef extract, 5-10 g / L peptone, 3-8 g / L yeast nitrogen, 2 g / L L-serine, 0.2 g / L calcium chloride, and 2-5 mL / L PTM1 Pichia pastoris trace element mother liquor; wherein, the bacterial residue concentrate can partially or completely replace any one of the organic nitrogen sources among beef extract, peptone, or yeast nitrogen in any proportion, and the concentration of the bacterial residue concentrate is 2-20 g / L.

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