A closed-circuit system for green bio-preparation of glucaric acid

Abstract

The present application discloses a closed loop system for green biological preparation of glucaric acid, which comprises a subcritical water pretreatment module, an integrated biological processing module, a filtration module, a glucaric acid separation module and an acetone distillation recovery module, and provides a highly integrated green preparation process of glucaric acid, which eliminates the steps of solid-liquid separation, sterilization, filtrate treatment, wastewater treatment and the like after pretreatment, maintains high yield, high output and high yield of glucaric acid, and greatly reduces the production cost of glucaric acid. The whole process adopts a new type of clean and environmentally friendly subcritical water pretreatment; the domesticated artificial microbial flora produces glucaric acid coupled with in-situ detoxification, realizing direct integrated biological processing of subcritical water pretreated plant straw to produce glucaric acid; a new type of anti-solvent crystallization method is used to separate glucaric acid, and acetone is distilled and recovered, thereby constructing a closed loop system from pretreatment to separation and purification, which is zero wastewater discharge, low cost and high yield.

Description

Technical Field

[0001] This field relates to biochemical engineering and biomass chemicals, specifically to a closed-loop system for the green biological preparation of gluconic acid. Background Technology

[0002] Against the backdrop of striving to develop a green economy, the green bio-manufacturing of glucaric acid based on microbial fermentation has attracted widespread attention in recent years due to its low cost and environmental friendliness. We have made significant progress in the bioprocessing of lignocellulose to produce glucaric acid using artificial microbial communities. First, it has been reported that the consolidated bioprocessing (CBP) of lignocellulose by engineered Trichoderma reesei and Saccharomyces cerevisiae has advantages over separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF), and is the optimal route for the biorefining of lignocellulose to produce glucaric acid (Li C., Lin X., Ling X. et al. Consolidated bioprocessing of lignocellulose for production of glucaric acid by an artificial microbial consortium. Biotechnol Biofuels, 14, 110 (2021).). Research has found that a collaborative push-pull strategy improves the efficiency of gluconic acid production from CBP lignocellulose, resulting in a highly efficient artificial microbial community composed of engineered Trichoderma reesei C10 and engineered Saccharomyces cerevisiae LGA-1C3S2, and a highly efficient CBP technology for gluconic acid production from lignocellulose. This technology can significantly improve the efficiency of CBP from artificial microbial communities. Moreover, this method is not only applicable to improving the efficiency of one-step gluconic acid production from artificial microbial communities, but also has broad applicability to other CBP processes using Trichoderma reesei and Saccharomyces cerevisiae communities, such as producing single-cell protein from baijiu lees. The significantly increased SCP yield after adopting the collaborative push-pull strategy demonstrates that this strategy is an effective and universally applicable method for improving the integrated bioprocessing efficiency of artificial microbial communities (see patent application number 202210044625.2 for details). However, the problems of degraded sugars, inhibitors, and waste liquid generated during the pretreatment of raw materials in this technology urgently need to be solved. The waste liquid has a low sugar concentration and contains inhibitors, and it is not cost-effective to establish an additional bioutilization pathway. We have established a technology for the production of gluconic acid from lignocellulose by domesticating artificial microbial communities, which solves the above problems and greatly simplifies the entire process (see patent application number 202211163383.5 for details).

[0003] However, there is currently no report on a highly integrated green bio-preparation process for gluconic acid that eliminates the steps of solid-liquid separation, sterilization, filtrate treatment, and wastewater treatment after pretreatment. Summary of the Invention

[0004] To address the aforementioned technical challenges, the applicant scaled up dCBP to a 10L fermenter and established a separation and purification process. They discovered that the domesticated artificial microbial flora possesses in-situ detoxification capabilities (undomesticated flora cannot grow normally in pretreated solutions containing inhibitors, nor do they exhibit any detoxification effect on dCBP). This capability can be fully utilized to establish a closed-loop system encompassing subcritical water pretreatment (SCWP), dCBP, and product separation, achieving a highly integrated green bio-production process for gluconic acid. This eliminates the need for wastewater treatment and allows for water recycling. Furthermore, this invention integrates the production process of gluconic acid from lignocellulose using artificial microbial flora dCBP, providing a more economically feasible new production technology for the green bio-production of gluconic acid.

[0005] The technical solution of the present invention is as follows:

[0006] This invention provides a closed-loop system for green biological preparation of gluconic acid, which is used to prepare gluconic acid from plant straw as raw material. The closed-loop system includes a subcritical water pretreatment module, an integrated biological processing module, a filtration module, a gluconic acid separation module, and an acetone distillation recovery module.

[0007] The subcritical water pretreatment module is used to pretreat plant straw with water to obtain subcritical water pretreated plant straw.

[0008] The integrated bioprocessing module is used to carry out microbial fermentation of plant straw after subcritical water pretreatment. The microbial fermentation uses Trichoderma reesei engineered strain C10 and Saccharomyces cerevisiae LGA-1C3S2.

[0009] The filtration module is used to separate the solid and liquid components of the fermentation mash obtained from microbial fermentation, wherein the liquid contains the target product gluconic acid.

[0010] The gluconic acid separation module is used to adjust the pH of the liquid obtained by the filtration module, and then add acetone to precipitate and obtain gluconic acid.

[0011] The acetone distillation and recovery module is used to distill and recover acetone from the remaining liquid after obtaining gluconic acid through acetone precipitation. The remainder is wastewater, which is used as water added during the next round of preparation after mixing with plant straw for subcritical water pretreatment.

[0012] Preferably, the plant straw is corn straw, wheat straw, rice straw, or willow sorghum.

[0013] This invention also provides a green biological method for preparing gluconic acid, comprising the following steps:

[0014] (1) Plant straw is mixed with water and then pretreated with subcritical water to obtain plant straw pretreated with subcritical water.

[0015] (2) The plant straw pretreated by subcritical water in step (1) is subjected to microbial fermentation. The microbial fermentation strains used are Trichoderma reesei engineered strain C10 and Saccharomyces cerevisiae LGA-1C3S2.

[0016] (3) The fermentation mash obtained from microbial fermentation in step (2) is subjected to solid-liquid separation, wherein the liquid contains the target product gluconic acid;

[0017] (4) Adjust the pH of the liquid obtained after solid-liquid separation in step (3), and then add acetone to precipitate and obtain gluconic acid;

[0018] (5) The remaining liquid after obtaining gluconic acid by acetone precipitation in step (4) is distilled to recover acetone. The remainder is wastewater, which is used as the water added in the subcritical water pretreatment after mixing with plant straw in step (1) during the next round of preparation.

[0019] The green biological method for preparing gluconic acid uses the closed-loop circulation system.

[0020] In step (1), the subcritical water pretreatment conditions are heating to 210°C and maintaining for 20 minutes.

[0021] Specifically, after subcritical water pretreatment in step (1), a filtrate with subcritical water pretreatment was obtained. The filtrate with subcritical water pretreatment was used to acclimate the Trichoderma reesei engineered strain C10 and Saccharomyces cerevisiae LGA-1C3S2 described in step (2). The plant straw with subcritical water pretreatment was added to the fermentation medium as a component of the fermentation medium for microbial fermentation in step (2).

[0022] The fermentation medium consisted of: a plant straw solution pretreated with 35.7 g / L subcritical water, 1 g / L peptone, 1 g / L yeast extract, 10% (v / v) Mandels nutrient solution, 0.1% (v / v) Mandels trace element solution, 5% (v / v) citrate buffer, and 0.1 g / L Tween-80.

[0023] Preferably, the concentration of the filtrate after subcritical water pretreatment is gradually increased to acclimate the engineered Trichoderma reesei C10 and Saccharomyces cerevisiae LGA-1C3S2.

[0024] Specifically, the steps for acclimating the engineered Trichoderma reesei C10 and Saccharomyces cerevisiae LGA-1C3S2 strains with the filtrate after subcritical water pretreatment are as follows:

[0025] (1) Inoculate Trichoderma reesei engineered strain C10 and Saccharomyces cerevisiae LGA-1C3S2 into fermentation medium and culture them.

[0026] (2) Add 10% of the filtrate to the fermentation medium and continue fermentation. Use the streak plate method to separate single colonies 1 that are tolerant to 10% of the filtrate.

[0027] (3) Inoculate the single colony 1 obtained in step (2) into the fermentation medium and culture it. Add 20% of the filtrate by volume to the fermentation medium and continue fermentation culture. Use the streak plate method to separate the single colony 2 that is tolerant to 20% of the filtrate by volume.

[0028] (4) Gradually increase the volume percentage of the filtrate by 10%, repeat steps (2) and (3) until the volume percentage of the filtrate reaches 100%, and obtain a single colony a that is tolerant to the filtrate with a volume percentage of 100%.

[0029] (5) Inoculate single colony a, which is tolerant to 100% filtrate by volume, into fermentation medium and culture. Add 100% filtrate by volume to the fermentation medium and continue fermentation to obtain single colony b, which is tolerant to 100% filtrate by volume. This yields the domesticated Trichoderma reesei engineered strain C10 and Saccharomyces cerevisiae LGA-1C3S2. To consolidate the inhibitor resistance of the artificial microbial community, domestication can be repeated at least 30 times.

[0030] Preferably, the conditions for microbial fermentation are an aeration rate of 3 lpm, dissolved oxygen content maintained above 20%, and a culture temperature of 30°C.

[0031] In step (3), the fermentation mash obtained from microbial fermentation in step (2) is subjected to solid-liquid separation, and the solids therein can be used as organic fertilizer.

[0032] Preferably, the process used in step (4) is an anti-solvent crystallization separation process, the specific steps of which are as follows:

[0033] After the solid-liquid separation in step (3), the liquid obtained is passed through a 0.2μm ceramic filter, and the pH of the fermentation broth is adjusted to 3.5 with hydrochloric acid solution to form potassium gluconate. Acetone is added while stirring until potassium gluconate precipitates. After standing, it is separated and obtained by vacuum filtration.

[0034] Compared with existing technologies, this invention establishes a closed-loop system consisting of pretreatment, direct biointegrated processing, gluconic acid separation, and acetone distillation recovery. It provides a highly integrated green gluconic acid preparation process, eliminating the need for post-pretreatment solid-liquid separation, sterilization, filtrate treatment, and wastewater treatment. This maintains high yield and high production rate of gluconic acid while significantly reducing production costs. The entire process utilizes novel, clean, and environmentally friendly subcritical water pretreatment; the production of gluconic acid from domesticated artificial microbial flora is coupled with in-situ detoxification, achieving direct integrated bioprocessing of plant straw to produce gluconic acid; a novel antisolvent crystallization method separates gluconic acid, and acetone is recovered by distillation, constructing a closed-loop system from pretreatment to separation and purification. This system features zero wastewater discharge, low cost, and high yield. This invention provides a new, production-friendly technology for the green bio-production of gluconic acid. Attached Figure Description

[0035] Figure 1 Diagram of a closed-loop system for the green biological preparation of gluconic acid.

[0036] Figure 2 The graph shows the changes in gluconic acid concentration and filter paper enzyme activity during 30 cycles of operation of the closed-loop system. Detailed Implementation

[0037] Example 1

[0038] Figure 1 What is shown is a closed-loop system for green biological preparation of gluconic acid, which is used to prepare gluconic acid from plant straw. The closed-loop system includes a subcritical water pretreatment module, an integrated biological processing module, a filtration module, a gluconic acid separation module, and an acetone distillation recovery module.

[0039] The subcritical water pretreatment module is used to pretreat plant straw mixed with water using subcritical water to obtain subcritical water pretreated plant straw.

[0040] The integrated bioprocessing module is used for microbial fermentation of plant straw after subcritical water pretreatment. The microbial fermentation device is a fermentation tank, and the microbial strains used for microbial fermentation are Trichoderma reesei engineered strain C10 and Saccharomyces cerevisiae LGA-1C3S2, respectively. After microbial fermentation, fermented mash is obtained.

[0041] The filtration module is used to separate the solid and liquid components of the fermentation mash obtained from microbial fermentation. The liquid contains the target product gluconic acid, while the remaining solids can be used as organic fertilizer.

[0042] The gluconic acid separation module is used to adjust the pH of the liquid containing the target product gluconic acid obtained by the filtration module, add acetone, and then obtain the precipitate and the remaining liquid through the separation device. The precipitate is then purified to obtain gluconic acid.

[0043] The acetone distillation and recovery module is used to distill the remaining liquid after acetone precipitation to obtain gluconic acid from the separation device, and recover the acetone. The remaining wastewater is used as water added during the subcritical water pretreatment after mixing with plant straw in the next round of preparation. This is a closed-loop system for green biological preparation of gluconic acid, eliminating the need for solid-liquid separation, sterilization, filtrate treatment, and wastewater treatment after pretreatment.

[0044] Example 2

[0045] This embodiment uses corn stalks as raw material to prepare gluconic acid.

[0046] I. Subcritical Water Pretreatment

[0047] In the pretreatment stage of SCWP, each batch of SCWP treats 20g of CS (corn stalks). 400mL of distilled water is added to a high-temperature, high-pressure reactor, heated to 210℃, allowing the water to remain in a liquid state and generating high pressure naturally for 20 minutes. The resulting SCWPCS (subcritical water pretreated corn stover) stock solution contains both solids and liquid. A 7-liter working volume requires 17.5 batches of SCWP, or 350g of CS.

[0048] II. Direct Integration of Bioprocessing

[0049] The artificial microbial community directly integrated into bioprocessing consists of *Trichoderma reesei* engineered strain C10 and *Saccharomyces cerevisiae* LGA-1C3S2. After overexpression of the exoglucanase II gene cbh2 from *Trichoderma reesei* C10 via the strong promoter Pcbh1, the activities of exoglucanase and the overall enzyme activity of cellulase were significantly increased, as detailed in the applicant's previous literature and patents (Fang et al., Bioresource Technology 2013, 144: 693-7; Patent Application No. 202210044625.2). *Saccharomyces cerevisiae* LGA-1C3S2 was derived from the high-glucosidic acid-producing engineered strain LGA-1; the specific modification steps are detailed in the literature (Li et al., Biotechnology for Biofuels 2021, 14: 110). Neurospora crassa was introduced into this strain. The crassa cellodextrin transport system, including membrane transport proteins CDT1 and CDT2 and intracellular β-glucosidase GH1-1, along with gluconic acid synthesis pathway-related enzymes Ino1, INM1, MIOX4, and Udh, aggregates via a protein scaffold to obtain Saccharomyces cerevisiae LGA-1C3S2. This system can efficiently ferment cellobiose and soluble cellooligosaccharides to produce gluconic acid. For specific steps, please refer to patent application number 202210044625.2.

[0050] Prepare seed culture of Trichoderma reesei C10 and Saccharomyces cerevisiae LGA-1C3S2.

[0051] Trichoderma reesei C10 seed culture medium (50 mL): 0.5 g / L glucose, 0.05 g / L peptone, 5 mL 10% (v / v) Mandels nutrient solution, 0.05 mL 0.1% (v / v) Mandels trace element solution, 2.5 mL 1M citrate buffer, 2 drops Tween-80, sterilized at 121℃ for 20 min. Trichoderma reesei C10 was cultured at 30℃ and 170 rpm for 36 h to obtain Trichoderma reesei seed culture. Saccharomyces cerevisiae LGA-1C3S2 seed culture medium (YPD): 10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose, sterilized at 121℃ for 20 min. Saccharomyces cerevisiae LGA-1C3S2 was cultured at 30℃ and 250 rpm until the OD600 value reached approximately 5 to obtain Saccharomyces cerevisiae seed culture. Transfer 2.5 mL of Trichoderma reesei seed solution and 2.5 mL of Saccharomyces cerevisiae seed solution to CBP medium and inoculate them into the medium. After inoculating with Trichoderma reesei and Saccharomyces cerevisiae seed solutions, culture at 30℃ and 180 rpm.

[0052] The artificial microbial community was gradually acclimated using the filtrate of SCWPCS. First, CS was pretreated with SCWP and then filtered through qualitative filter paper (pore size 30-50 mm). The resulting solids were SCWPCS, which were then used as substrates for consolidated bioprocessing (CBP). The CBP medium consisted of 35.7 g / L SCWPCS (equivalent to the SCWPCS concentration in 20 g CS stock solution, i.e., the filtered solids of SCWPCS plus 400 mL distilled water) (dry weight), 1 g / L peptone, 1 g / L yeast extract, 10% (v / v) Mandels nutrients, 0.1% (v / v) Mandels trace element solution, 5% (v / v) citrate buffer, and 0.1 g / L Tween-80. The medium was sterilized at 121°C for 30 min. Before CBP, the filtrate was slowly added to the CBP medium. 10% of the filtrate was added to the CBP medium (40 mL filtrate, 360 mL distilled water) to allow the artificial microbial community to slowly adapt (acclimatize). Microbial communities tolerant to inhibitors were isolated by streak plating, and the resulting single colonies of *Trichoderma reesei* and *Saccharomyces cerevisiae* were used for the next round of acclimatization. Then add 20% filtrate (80mL filtrate, 320mL distilled water), gradually increasing the ratio until the filtrate reaches 100% (SCWP stock solution, stock solution after SCWP of 10g or 20g raw material and 400mL distilled water). This eliminates the need for filtration and separation. SCWPCS, subcritical water pretreated wheat straw (SCWPWS), subcritical water pretreated rice straw (SCWPRS), and subcritical water pretreated switchgrass (SCWPSG) stock solutions can be directly integrated into direct consolidated bioprocessing (dCBP). (After SCWP, sterilization, separation, or detoxification is no longer required. After adding nutrients, Trichoderma reesei and Saccharomyces cerevisiae seed culture can be directly inoculated.) This yields Trichoderma reesei engineered strain C10a and Saccharomyces cerevisiae LGA-1C3S2a, which are tolerant to 100% filtrate by volume. After the final 100% stock solution was acclimatized for 30 additional rounds, the inhibitory resistance of the artificial microbial community was consolidated, resulting in the engineered Trichoderma reesei strain C10b and Saccharomyces cerevisiae LGA-1C3S2b.

[0053] The domesticated artificial microbial community was used to directly integrate the bioprocessing of SCWPCS stock solution to produce gluconic acid. dCBP was carried out in a 10L airlift fermenter with a working volume of 7L. The seed culture methods for *Trichoderma reesei* C10 and *Saccharomyces cerevisiae* LGA-1C3S2 were the same as above, with a total inoculum ratio of 10% (v / v), i.e., 350mL of *Trichoderma reesei* seed culture and 350mL of *Saccharomyces cerevisiae* seed culture. The dCBP medium directly used the SCWPCS stock solution, with the same concentration of peptone, yeast extract, and nutrients as the CBP medium added to a 7L volume. Water evaporation caused by pretreatment was compensated for by the seed culture, and volume deviation was negligible. The aeration rate was 3 lpm, dissolved oxygen was maintained above 20%, and the temperature was 30℃. After inoculation, dCBP was initiated, and samples were taken daily to measure gluconic acid content and filter paper enzyme activity.

[0054] III. Filtration, gluconic acid separation, and acetone distillation recovery

[0055] After dCBP is completed, the bacterial cells, residual substrate, and other solids are separated from the fermentation broth using plate and frame filtration. The former can be used as organic fertilizer. The fermentation broth is passed through a 0.2μm ceramic filter, and the pH is adjusted to 3.5 with HCl to promote the formation of monopotassium glucarate (KGA). Then, acetone is slowly added while stirring until the minimum solubility of KGA is reached. The mixed suspension is then placed at 4°C for 24 hours to promote crystal growth and improve the KGA crystal recovery rate. The KGA crystals are recovered by vacuum filtration. The residual acetone / fermentation broth is distilled to recover acetone, which is used in the next round of antisolvent crystallization to separate glucarate. The residual liquid is used in the next round of SCWP. Due to water loss caused by evaporation and adsorption, it is replenished with distilled water.

[0056] The above-mentioned closed-loop circulation system from SCWP to dCBP, and then to separation and purification, is as follows: Figure 1 As shown, the above cycle was run for 30 rounds. The experimental results for each round include the gluconic acid yield on day 7 and the filter paper activity (FPA) on day 5. Figure 2 As shown in the figure. The results showed that the 30-cycle operation was stable, with gluconic acid yield and FPA fluctuating slightly around 10 g / L and 35 IU / mL, respectively. This indicates that no inhibitors, toxic chemicals, or organic solvents accumulated during the closed-loop cycle, and no wastewater or waste was generated. The in-situ detoxification performance of the domesticated artificial microbial community was well utilized, and the entire process was clean, pollution-free, and environmentally friendly. This invention provides a new technology for the green bio-production of gluconic acid.

Claims

1. A closed-loop recycling system for the green biological preparation of gluconic acid, used to prepare gluconic acid from plant straw as raw material, characterized in that, The closed-loop circulation system includes a subcritical water pretreatment module, an integrated biological processing module, a filtration module, a gluconic acid separation module, and an acetone distillation recovery module. The subcritical water pretreatment module is used to pretreat plant straw with water to obtain subcritical water pretreated plant straw. The integrated bioprocessing module is used for microbial fermentation of plant straw after subcritical water pretreatment. The microbial fermentation uses domesticated Trichoderma reesei engineered strain C10 and Saccharomyces cerevisiae LGA-1C3S2. The domestication is carried out by gradually increasing the concentration of the filtrate after subcritical water pretreatment, thereby domesticating Trichoderma reesei engineered strain C10 and Saccharomyces cerevisiae LGA-1C3S2. The specific domestication steps are as follows: (1) Inoculate Trichoderma reesei engineered strain C10 and Saccharomyces cerevisiae LGA-1C3S2 into fermentation medium and culture them; (2) Add 10% of the filtrate by volume to the fermentation medium, continue fermentation, and use the streak plate method to separate single colonies 1 that are tolerant to 10% of the filtrate by volume. (3) Inoculate the single colony 1 obtained in step (2) into the fermentation medium and add 20% of the filtrate by volume to the fermentation medium. Continue fermentation and culture, and use the streak plate method to separate the single colony 2 that is tolerant to 20% of the filtrate by volume. (4) Gradually increase the volume percentage of the filtrate by 10%, repeat steps (2) and (3) until the volume percentage of the filtrate reaches 100%, and obtain a single colony a that is tolerant to the filtrate with a volume percentage of 100%. (5) Inoculate a single colony a that is tolerant to 100% of the filtrate into the fermentation medium and culture it. Add 100% of the filtrate to the fermentation medium and continue fermentation to obtain a single colony b that is tolerant to 100% of the filtrate. This yields the domesticated Trichoderma reesei engineered strain C10 and Saccharomyces cerevisiae LGA-1C3S2. The filtration module is used to separate the solid and liquid components of the fermentation mash obtained from microbial fermentation, wherein the liquid contains the target product gluconic acid. The gluconic acid separation module is used to adjust the pH of the liquid obtained by the filtration module, and then add acetone to precipitate and obtain gluconic acid. The acetone distillation and recovery module is used to distill and recover acetone from the remaining liquid after obtaining gluconic acid through acetone precipitation. The remainder is wastewater, which is used as water added during the next round of preparation after mixing with plant straw for subcritical water pretreatment. Furthermore, the method for preparing gluconic acid using the closed-loop circulation system includes the following steps: (a) Plant straw was mixed with water and then subjected to subcritical water pretreatment. The conditions were heating to 210°C and maintaining for 20 min to obtain plant straw pretreated with subcritical water. (b) The plant straw pretreated with subcritical water in step (a) was subjected to microbial fermentation. The microbial fermentation strains used were Trichoderma reesei engineered strain C10 and Saccharomyces cerevisiae LGA-1C3S2. The fermentation medium consisted of: 35.7 g / L subcritical water pretreated plant straw solution, 1 g / L peptone, 1 g / L yeast extract, 10% (v / v) Mandels nutrient solution, 0.1% (v / v) Mandels trace element solution, 5% (v / v) citrate buffer, and 0.1 g / L Tween-80. (c) The fermentation mash obtained from microbial fermentation in step (b) is subjected to solid-liquid separation, wherein the liquid contains the target product gluconic acid; (d) After the solid-liquid separation in step (c), the liquid obtained is passed through a 0.2 µm ceramic filter, and the pH of the fermentation broth is adjusted to 3.5 with hydrochloric acid solution to form potassium gluconate; acetone is added while stirring until potassium gluconate precipitates; after standing, it is separated and obtained by vacuum filtration. (e) The remaining liquid after obtaining gluconic acid by acetone precipitation in step (d) is distilled to recover acetone. The remainder is wastewater, which is used as the water added in the subcritical water pretreatment after mixing with plant straw in step (a) during the next round of preparation.

2. The closed-loop recycling system for green bio-production of gluconic acid as described in claim 1, characterized in that, The plant straw is corn stalk, wheat straw, rice straw, or willow sorghum.

3. The closed-loop recycling system for green bio-production of gluconic acid as described in claim 1, characterized in that, The conditions for microbial fermentation in step (b) are an aeration rate of 3 lpm, dissolved oxygen content maintained above 20%, and a culture temperature of 30°C. In step (c), the fermentation mash obtained from microbial fermentation in step (b) is subjected to solid-liquid separation, and the solids are used as organic fertilizer.

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