Mutant strain of aspergillus niger for production of citric acid and gluconic acid and methods

Abstract

The present disclosure provides a mutant strain of Aspergillus niger designated MTCC 25761 on deposit with the Microbial Type Culture Collection and Gene Bank, Institute of Microbial Technology, Chandigarh, derived from parent strain Aspergillus niger ATCC 9142 through UV mutagenesis at 254 nm wavelength for 19 minutes with constant stirring The mutant strain demonstrates enhanced production capabilities for both citric acid and gluconic acid compared to the parent strain. The mutant strain exhibits enhanced metabolic performance through improved enzyme expression, substrate utilization efficiency, and regulatory control mechanisms. Gluconic acid production is 1.37-fold higher (164.045 g / L) and citric acid production is 1.29-fold higher (0.506 g / L) than the parent strain.

Description

[0001] 202421061133

[0002] MUTANT STRAIN OF ASPERGILLUS NIGER FOR PRODUCTION OF CITRIC ACID AND GLUCONIC ACID AND METHODS

[0003] FIELD OF INVENTION

[0004] [1] The present invention relates to a mutant strain of Aspergillus niger deposited with the Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology, Chandigarh and assigned accession number MTCC 25761 for production of citric acid and gluconic acid. More specifically, the current invention belongs to the improvement of parent strain Aspergillus niger (ATCC 9142) for the better and efficient production of citric and gluconic acid. The mutant strain is obtained through UV mutation method. Both the organic acids are industrially important and mainly produced through microbial fermentation.

[0005] BACKGROUND OF INVENTION

[0006] [2] Organic acids, particularly citric acid and gluconic acid, represent significant commercial products in the global fermentation industry with annual production volumes exceeding 2 million tons for citric acid and 100,000 tons for gluconic acid worldwide. These acids are produced primarily through microbial fermentation processes using various microorganisms, with Aspergillus niger being one of the most widely utilized fungal species for industrial organic acid production due to its robust metabolic pathways and ability to secrete high concentrations of organic acids. Citric acid serves as a tribasic acid with extensive applications as an acidulant, preservative, emulsifier, flavorant, sequestrant, and buffering agent across food, pharmaceutical, and chemical industries, representing approximately 70% of the global organic acid market. Gluconic acid functions as a mild organic acid with applications in pharmaceutical, textile, construction, and food industries, where it provides refreshing sour taste characteristics and serves as a mineral supplement precursor through its calcium and sodium salt derivatives. 202421061133

[0007] [3] Traditional fermentation processes for organic acid production rely on wild-type strains of Aspergillus niger, which utilize various carbon sources including crude sugars, molasses, corn starch, and other carbohydrate substrates through complex metabolic pathways involving glycolysis, tricarboxylic acid cycle, and pentose phosphate pathway. The fermentation process typically involves aerobic conditions with controlled pH, temperature, dissolved oxygen levels (>30% saturation), and nutrient supplementation including nitrogen sources, phosphorus, trace metals, and growth factors to optimize acid production yields. Current industrial methods employ both submerged fermentation (SmF) and solid-state fermentation (SSF) techniques, with submerged fermentation being more commonly used for large-scale production due to better process control, heat and mass transfer efficiency, and scalability, typically achieving citric acid yields of 70-85% and gluconic acid yields of 85-95% based on substrate consumption.

[0008] [4] European Patent No. EP0745677B1 describes gluconic acid production using Aspergillus niger strains through submerged fermentation processes, achieving gluconic acid concentrations of 90-130 g / L through controlled glucose oxidation. However, this prior art is limited by inconsistent fermentation performance across different production batches, requiring complex medium sterilization procedures that increase energy consumption by 25-35%, demonstrates suboptimal substrate utilization efficiency leading to increased raw material costs, and shows poor tolerance to industrial stress conditions that affect production stability. The disclosed process requires expensive nitrogen source supplementation that complicates medium preparation and increases operational costs, exhibits lower production yields during extended fermentation periods compared to improved strain variants, and produces variable pellet morphology that reduces mass transfer efficiency, further decreasing overall process productivity and commercial viability.

[0009] [5] European Patent Publication EP 1096020 describes fermentation processes for organic acid production using Aspergillus niger strains with emphasis on substrate utilization and process optimization, achieving citric acid 202421061133

[0010] yields of 70-80% through controlled aeration rates of 0.5-1.5 vvm and agitation speeds of 200-400 rpm. However, this prior art focuses primarily on process parameters rather than strain improvement. The disclosed method requires specific substrate preparations including acid hydrolysis of starch that is not economically viable for all applications, increasing raw material costs by 10-20%, and demonstrates production capabilities that are insufficient for meeting growing industrial demand. The disclosed processes are limited to conventional wild-type strains without addressing the potential benefits of genetically improved variants, show reduced performance with alternative carbon sources, and require expensive medium sterilization procedures that increase energy consumption by 15-25%.

[0011] [6] Current microbial strain development approaches face significant technical limitations including suboptimal production yields, inconsistent fermentation performance with batch-to-batch variations and inadequate substrate utilization efficiency resulting in increased raw material costs. Existing wild-type strains demonstrate poor tolerance to industrial stress conditions including high product concentrations , elevated temperatures, and varying pH conditions, which limit their commercial scalability and require expensive process control systems. Furthermore, conventional strains exhibit morphological instability during extended fermentation periods, leading to reduced mass transfer efficiency, increased downstream processing complexity, and higher separation costs that negatively impact overall process economics.

[0012] [7] It has been appreciated that a method is needed that overcomes one or more of these critical industrial limitations including inadequate production yields, inconsistent fermentation performance, poor substrate utilization efficiency, limited tolerance to industrial stress conditions, morphological instability during extended fermentation periods, and high production costs that compromise commercial viability and limit market accessibility for emerging applications requiring cost-effective organic acid supplies.

[0013] OBJECT OF THE INVENTION 202421061133

[0014] [8] The primary object of the present invention is to provide a mutant strain of Aspergillus niger (MTCC 25761) that demonstrates enhanced production capabilities for both citric acid and gluconic acid.

[0015] [9] Another object of the present invention is to provide a mutant strain derived from parent strain Aspergillus niger (ATCC 9142) through UV mutagenesis that exhibits superior organic acid production yields while maintaining stable fermentation characteristics.

[0016]

[0010] Yet another object of the present invention is to provide a mutant strain that exhibits enhanced glucose oxidase activity and improved metabolic flux through oxidative pathways for more efficient substrate utilization.

[0017]

[0011] Another object of the present invention is to provide a mutant strain that demonstrates improved sugar utilization efficiency of 42-44% compared to conventional strains while maintaining stable pellet morphology throughout fermentation processes.

[0018]

[0012] Further object of the present invention is to provide a mutant strain that shows enhanced tolerance to acidic fermentation conditions and maintains consistent pH profiles that facilitate optimal organic acid accumulation.

[0019]

[0013] Yet another object of the present invention is to provide a mutant strain that reduces formation of unwanted byproducts during fermentation, thereby improving downstream processing efficiency and product purity.

[0020]

[0014] Still further object of the present invention is to provide a mutant strain that demonstrates enhanced substrate conversion efficiency while reducing production costs by eliminating the need for expensive enzyme supplementation required by prior art methods.

[0021]

[0015] Another object of the present invention is to provide a mutant strain that maintains consistent batch-to-batch performance with reduced contamination risks and improved process reliability for industrial applications. 202421061133

[0022]

[0016] Further object of the present invention is to provide a mutant strain that operates effectively under industrial stress conditions including high product concentrations, elevated temperatures, and varying substrate compositions.

[0023]

[0017] Still further object of the present invention is to provide a mutant strain that exhibits enhanced resistance to pH fluctuations and maintains optimal acid production even under varying fermentation conditions.

[0024]

[0018] Another object of the present invention is to provide a mutant strain that demonstrates improved nitrogen utilization efficiency, reducing the requirement for expensive nitrogen sources and lowering overall production costs.

[0025]

[0019] Yet another object of the present invention is to provide a mutant strain that achieves simultaneous production of both citric acid and gluconic acid in a single fermentation process, maximizing resource utilization and process efficiency.

[0026]

[0020] A still further object of the present invention is to provide a mutant strain that demonstrates scalability from laboratory to industrial production levels while maintaining consistent performance characteristics.

[0027] SUMMARY OF INVENTION

[0028]

[0021] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description.

[0029]

[0022] The present invention provides a mutant strain of Aspergillus niger designated MTCC 25761, derived from parent strain Aspergillus niger ATCC 9142 through UV mutagenesis at 254 nm wavelength for 19 minutes at a distance of 20 cm with constant stirring, wherein the mutant strain demonstrates significantly enhanced production capabilities for both citric acid and gluconic acid. This specific UV exposure protocol generates targeted mutations that enhance the strain's acidogenic potential while maintaining cell viability and growth characteristics, with the 19-minute exposure duration representing an optimal balance between mutagenic effectiveness and cellular integrity that results 202421061133

[0030] in improved acid production without compromising fermentation performance. The mutant strain MTCC 25761 exhibits superior organic acid production capabilities through improved metabolic pathways, enhanced substrate utilization efficiency, stable pellet morphology during fermentation, consistent pH profiles conducive to acid accumulation, and increased tolerance to acidic conditions that typically inhibit wild-type strains. The mutant strain produces citric acid and gluconic acid demonstrating substantial improvements in both citric acid and gluconic acid production yields. The mutant strain exhibits enhanced glucose oxidase activity and improved metabolic flux through oxidative pathways, enabling more efficient utilization of glucose substrates and providing substantially higher organic acid yields per fermentation batch, while demonstrating improved sugar utilization efficiency, maintaining stable pellet morphology throughout fermentation processes, showing enhanced tolerance to acidic fermentation conditions, and maintaining consistent pH profiles that facilitate optimal organic acid accumulation while reducing formation of unwanted byproducts.

[0031] BRIEF DESCRIPTION OF FIGURES

[0032]

[0023] Embodiments of the invention will be described, by way of example, with reference to the following drawings, in which:

[0033]

[0024] FIG. 1 illustrates pellet morphology scores over fermentation time for citric acid production for parent strain F-l and mutant strain F-5, according to aspects of the present disclosure.

[0034]

[0025] FIG. 2 illustrates starch residue scores over fermentation time for citric acid production for parent strain F-l and mutant strain F-5, according to aspects of the present disclosure.

[0035]

[0026] FIG. 3 illustrates pH changes over fermentation time for citric acid production for parent strain F-l, according to aspects of the present disclosure. 202421061133

[0036]

[0027] FIG. 4 illustrates a comparison of pH profiles between citric acid production for parent strain F-l and mutant strain F-5, according to aspects of the present disclosure.

[0037]

[0028] FIG. 5 illustrates citric acid production over time for parent strain F-l and mutant strain F-5, according to aspects of the present disclosure.

[0038]

[0029] FIG. 6 illustrates sugar utilization percentage over fermentation time for gluconic acid production parent strain F-l, according to aspects of the present disclosure.

[0039]

[0030] FIG. 7 illustrates sugar utilization percentage over fermentation time for gluconic acid production mutant strain F-2, according to aspects of the present disclosure.

[0040]

[0031] FIG. 8 illustrates a comparison of sugar utilization percentages for gluconic acid production between parent strain F-l and mutant strain F-2, according to aspects of the present disclosure.

[0041]

[0032] FIG. 9 illustrates pH changes over fermentation time for gluconic acid production parent strain F-l, according to aspects of the present disclosure.

[0042]

[0033] FIG. 11 illustrates a comparison of pH profiles for gluconic acid production between parent strain F-l and mutant strain F-2, according to aspects of the present disclosure.

[0043]

[0034] FIG. 12 illustrates gluconic acid production over time for parent strain F-l and mutant strain F-2, according to aspects of the present disclosure.

[0044] DETAILED DESCRIPTION

[0045]

[0035] The present disclosure relates to a mutant strain of Aspergillus niger for enhanced production of citric acid and gluconic acid. The mutant strain is derived from the parent strain Aspergillus niger ATCC 9142 through UV mutagenesis techniques and demonstrates improved production capabilities for both organic acids compared to the parent strain. 202421061133

[0046]

[0036] Citric acid and gluconic acid represent two of the most commercially significant organic acids in global fermentation markets. Citric acid finds widespread application as an acidulant, preservative, emulsifier, flavorant, sequestrant, and buffering agent across food, pharmaceutical, and industrial sectors. Gluconic acid serves as a mild organic acid with applications in pharmaceutical, textile, building, and food industries. The global demand for these organic acids continues to increase, with citric acid dominating the organic acid market due to its diverse applications, while gluconic acid maintains a substantial market presence with approximately 60,000 tonnes produced worldwide annually.

[0047]

[0037] Current production methods for these organic acids rely primarily on microbial fermentation processes using Aspergillus niger, which is recognized as a Generally Regarded As Safe (GRAS) microorganism. While chemical, electrochemical, and bio-electrochemical production routes exist, fermentation processes typically provide higher yields and are more economically viable. However, conventional fermentation approaches using wild-type strains often face limitations in production efficiency and yield optimization.

[0048]

[0038] The present disclosure addresses the need for improved microbial strains capable of enhanced organic acid production. The approach involves systematic UV mutagenesis of the parent strain Aspergillus niger ATCC 9142 to generate mutant variants with superior production characteristics. The mutagenesis process employs controlled UV exposure at 254 nm wavelength for varying durations to induce genetic modifications that enhance the metabolic pathways responsible for citric acid and gluconic acid biosynthesis.

[0049]

[0039] The mutant strain development process includes comprehensive screening methodologies to identify variants with enhanced acid production capabilities. Primary screening utilizes Bromocresol Green (BCG) plate assays to detect acid-producing colonies through pH-sensitive color changes. Secondary screening employs specific analytical methods including enzymatic assays, thin 202421061133

[0050] layer chromatography, and quantitative measurement techniques to confirm and quantify the production of target organic acids.

[0051]

[0040] The resulting mutant strain demonstrates enhanced fermentation characteristics including improved substrate utilization, stable pH dynamics during fermentation, and increased organic acid yields compared to the parent strain. The mutant strain maintains the safety profile of the parent organism while exhibiting enhanced metabolic efficiency for organic acid production under controlled fermentation conditions.

[0052] Parent Strain

[0053] Spore Suspension Preparation

[0054]

[0041] Spore suspension preparation begins with harvesting spores from mature Aspergillus niger ATCC 9142 cultures grown on Potato Dextrose Agar (PDA) plates. The cultures are incubated at 30°C for 5 days to achieve adequate sporulation and colony maturation. The 5-day incubation period allows for complete spore development and ensures sufficient spore density for subsequent processing steps.

[0055] Spore Harvesting Process

[0056]

[0042] The spore harvesting process involves adding 15 mL of sterile 0.9% normal saline to each 5-day-old PDA culture plate. The saline solution provides an isotonic medium that maintains spore viability during the harvesting process. A sterile glass slide is used to gently scrape the surface of the fungal colony, releasing the spores into the saline solution. The scraping technique requires careful execution to maximize spore release while avoiding damage to the underlying agar medium.

[0057] Spore Dispersion Enhancement 202421061133

[0058]

[0043] Spore dispersion is enhanced through the addition of 0.01% sterile Tween 80 to the saline solution. Tween 80 functions as a non-ionic surfactant that reduces surface tension and promotes uniform spore distribution throughout the suspension. The surfactant prevents spore aggregation and ensures homogeneous spore distribution, which is necessary for accurate counting and consistent inoculum preparation.

[0059]

[0044] The harvested spore suspension is collected in sterile Petri plates and mixed gently to ensure homogeneity throughout the suspension volume. Gentle mixing prevents spore damage while achieving uniform distribution of spores within the liquid medium.

[0060] Spore Concentration Determination

[0061]

[0045] Spore concentration determination employs haemocytometer counting methodology. A 10 pL aliquot of the spore suspension is loaded onto a haemocytometer chamber for microscopic examination and counting. The haemocytometer provides a standardized counting grid that enables accurate determination of spore density per unit volume. Spore counting is performed under appropriate magnification to distinguish individual spores and obtain reliable concentration measurements.

[0062] 7 9

[0063]

[0046] The initial spore concentration typically ranges from 6^10 to 7x10 CFU / mL, depending on the sporulation efficiency of the particular culture. The wide concentration range reflects natural variation in sporulation density among different culture preparations and growth conditions.

[0064] Spore Suspension Standardization

[0065]

[0047] Spore suspension standardization involves dilution with sterile normal saline to achieve the desired final concentration for specific applications. For mutagenesis procedures, the spore concentration is adjusted to IxlO7CFU / mL using sterile normal saline as the diluent. For fermentation studies, different target 202421061133

[0066] concentrations are employed, such as 2*106CFU / mL for gluconic acid production experiments or 5*107CFU / mL for citric acid production studies.

[0067]

[0048] The standardized spore inoculum provides consistent starting conditions for subsequent experimental procedures, ensuring reproducible results across different fermentation batches and mutagenesis treatments. The controlled spore concentration enables accurate comparison of performance between different strains and experimental conditions.

[0068] UV Mutagenesis Process

[0069]

[0049] The UV mutagenesis process employs controlled ultraviolet radiation to induce genetic modifications in Aspergillus niger ATCC 9142 spores, generating mutant variants with enhanced organic acid production capabilities. The mutagenesis protocol utilizes UV light at 254 nm wavelength, which corresponds to the peak absorption wavelength of DNA and provides effective mutagenic activity through the formation of pyrimidine dimers and other DNA lesions.

[0070] UV Exposure Protocol Setup

[0071] 7

[0072]

[0050] The standardized spore suspension at lx 10 CFU / mL is dispensed into sterile Petri plates, with 10-15 mL of suspension allocated to each plate. Multiple plates are prepared to accommodate different UV exposure durations, allowing for systematic evaluation of mutagenic effects across a range of treatment intensities. Each plate is labeled with a specific strain code corresponding to the intended UV exposure duration.

[0073]

[0051] The UV exposure protocol encompasses eight different treatment durations ranging from 13 to 27 minutes. The exposure durations are systematically varied in 2-minute increments, with treatments at 13, 15, 17, 19, 21, 23, 25, and 27 minutes corresponding to strain codes DPPL 014, DPPL 015, DPPL 016, DPPL 017, DPPL 018, DPPL 019, DPPL 020, and DPPL 021, 202421061133

[0074] respectively. This range of exposure durations provides a comprehensive evaluation of mutagenic effects from mild to more intensive treatments.

[0075] UV Exposure Chamber Configuration

[0076]

[0052] The UV exposure chamber is configured with the spore suspension plates positioned at a fixed distance of 20 cm from the UV light source. This standardized distance ensures consistent UV intensity across all treatment groups and provides reproducible mutagenic conditions. The 20 cm distance represents a balance between adequate UV exposure intensity and practical handling considerations within the exposure chamber.

[0077]

[0053] During UV exposure, the Petri plate lids are removed to allow direct UV radiation contact with the spore suspension. The open plate configuration eliminates any UV filtering or attenuation that occurs through plastic or glass barriers, ensuring maximum mutagenic effectiveness.

[0078] UV Exposure Conditions

[0079]

[0054] Continuous stirring of the spore suspension is maintained throughout the UV exposure period using sterile magnetic stirrers. The stirring action ensures uniform UV exposure of all spores within the suspension, preventing settling or stratification that results in uneven mutagenic treatment. The continuous agitation promotes homogeneous distribution of UV dose across the entire spore population. Following UV exposure, the plates are immediately covered to prevent further unintended UV exposure that results in excessive mutagenic damage or spore mortality. The prompt covering of plates maintains the controlled nature of the mutagenic treatment and preserves spore viability for subsequent processing steps.

[0080] Post-UV Processing and Colony Isolation

[0081]

[0055] Post-UV processing involves serial dilution of the treated spore suspension to achieve appropriate colony density for isolation and screening 202421061133

[0082] procedures. A 100-fold serial dilution is performed using sterile normal saline as the diluent. The dilution process reduces the spore concentration to levels suitable for individual colony isolation and subsequent characterization.

[0083] Colony Plating and Incubation

[0084]

[0056] The diluted spore suspension is plated onto Potato Dextrose Agar plates supplemented with 2% Triton X-100. Triton X-100 functions as a colony restrictor, limiting colony size and spread to facilitate the isolation of well-separated individual colonies. The colony restrictor prevents excessive colony growth that interferes with colony selection and isolation procedures. Plating involves dispensing 0.1 mL aliquots of the 100-fold diluted suspension onto the PDA-Triton X-100 plates. The plating volume and dilution factor are calculated to yield approximately 30 colonies or fewer per plate, providing optimal colony density for individual colony selection and isolation. The reduced colony density prevents overcrowding and enables clear visualization of colony morphology and characteristics. The inoculated plates are incubated at 30°C for 4-6 days under controlled environmental conditions. The incubation temperature of 30°C represents the optimal growth temperature for Aspergillus niger and promotes healthy colony development while maintaining consistent growth conditions across all treatment groups. The 4-6 day incubation period allows for adequate colony development and sporulation while preventing over-maturation that complicates subsequent handling procedures. Daily observation of the plates during incubation enables monitoring of colony development, morphology assessment, and identification of any unusual growth characteristics that indicate successful mutagenesis. The observation period allows for documentation of colony appearance, size, color, and sporulation patterns that differ from the parent strain characteristics

[0085] Screening Method 202421061133

[0086]

[0057] Following incubation, the resultant colonies are subjected to screening procedures using Bromocresol Green plate assays to identify mutants with enhanced acid production capabilities. The screening process involves transferring individual colonies to PDA plates supplemented with Bromocresol Green, a pH-sensitive indicator dye that changes color in response to acid production. Bromocresol Green exhibits a color transition from blue-green at pH values above 4.8 to yellow at pH values below 3.8. Acid-producing colonies create localized pH reduction in the surrounding medium, resulting in the formation of distinct yellow halos or zones around the colonies. The appearance of yellow zones against the blue-green background provides a visual indication of organic acid production capability.

[0087] Acid Unitage Calculation Method

[0088]

[0058] The screening methodology involves measuring both colony diameter and the diameter of the surrounding acid zone for each tested colony. These measurements enable calculation of the acid unitage value, which serves as a quantitative measure of acid production capability relative to colony size.

[0089] The acid unitage value is calculated using the mathematical formula:

[0090] Acid Unitage = Zone of Acid (mm) / Colony Diameter (mm),

[0091]

[0059] where the Zone of Acid is determined by subtracting the colony diameter from the total zonal diameter. This calculation provides a normalized measure of acid production that accounts for differences in colony size, enabling fair comparison between different mutant candidates.

[0092] Mutant Selection Criteria

[0093]

[0060] Colonies exhibiting the highest acid unitage values are selected for further characterization and fermentation studies. The quantitative screening approach ensures selection of mutants with genuinely enhanced acid production 202421061133

[0094] capabilities rather than simply larger colonies or those with more visible but proportionally smaller acid zones.

[0095]

[0061] The primary screening methodology employs Bromocresol Green plates to systematically evaluate UV-induced mutant colonies for enhanced acid production capabilities. The screening process provides a rapid and reliable method for identifying promising mutant candidates through visual detection of acid zones surrounding fungal colonies.

[0096] Plate Composition and Preparation

[0097]

[0062] The screening medium consists of Potato Dextrose Agar supplemented with Bromocresol Green indicator dye and additional agar to achieve appropriate gel strength. For preparation of 16 screening plates requiring 640 mL of medium, the composition includes 24.96 g Potato Dextrose Agar powder, 9.6 g additional agar powder, and 0.64 g Bromocresol Green dye. The medium preparation process involves dissolving the PDA and additional agar components in distilled water with continuous stirring in a 2-liter flask. The Bromocresol Green dye is separately dissolved in approximately 50 mL of warm distilled water to ensure complete dissolution before addition to the main medium. The BCG solution is then incorporated into the PDA mixture, and the final volume is adjusted to 640 mL with distilled water.

[0098]

[0063] The complete medium is sterilized by autoclaving at 121°C for 15-20 minutes to eliminate microbial contaminants while preserving the pH-sensitive properties of the Bromocresol Green indicator. Following sterilization, the medium is cooled to 45-50°C before pouring approximately 40 mL per Petri plate under aseptic conditions to prevent contamination.

[0099] Colony Inoculation Protocol

[0100]

[0064] Individual colonies are selected from the UV-treated PDA-Triton plates following the 4-6 day incubation period. Colony selection focuses on isolated, well-developed colonies that exhibit normal morphological 202421061133

[0101] characteristics and adequate sporulation. Each selected colony is transferred to duplicate BCG-PDA plates using sterile inoculation loops to ensure reproducible results and account for potential variation in acid production expression.

[0102]

[0065] The inoculation process involves transferring the colony center or spore patch to the center of each BCG plate using sterile technique. The parent strain Aspergillus niger ATCC 9142 is included as a reference control, with duplicate plates inoculated under identical conditions to provide baseline acid production measurements for comparative evaluation.

[0103] Incubation Conditions and Timeline

[0104]

[0066] The inoculated BCG plates are incubated at 30°C ± 2°C for 70-75 hours, corresponding to approximately 3 days of growth. The temperature range of 30°C ± 2°C maintains optimal growth conditions for Aspergillus niger while ensuring consistent environmental conditions across all test plates. The plates are incubated in an upright position within a clean incubator to prevent condensation accumulation and maintain proper air circulation. The 70-75 hour incubation period provides sufficient time for colony establishment, growth, and acid production while preventing over-maturation that complicates zone measurement or leads to secondary metabolic changes that affect acid production patterns.

[0105] Visual Observation and Assessment Criteria

[0106]

[0067] Following incubation, plates are examined for the development of yellow acid zones surrounding the fungal colonies. Acid-producing colonies create distinct yellow halos against the greenish-blue background of the Bromocresol Green medium, indicating localized pH reduction due to organic acid secretion. The contrast between the yellow acid zones and the blue-green background provides clear visual differentiation for assessment purposes. Colonies lacking visible yellow zones or exhibiting only faint discoloration are categorized as non-producers or weak acid producers and are excluded from 202421061133

[0107] further consideration. Only colonies demonstrating clear, intense, and measurable yellow zones are selected for continued characterization and evaluation.

[0108] Quantitative Zone Measurement

[0109]

[0068] The screening methodology involves precise measurement of both colony diameter and total zonal diameter for each acid-producing colony. Colony diameter represents the actual fungal growth area, while zonal diameter encompasses both the colony and the surrounding acid zone. These measurements are performed using appropriate measuring instruments to ensure accuracy and reproducibility.

[0110]

[0069] The zone of acid is calculated by subtracting the colony diameter from the total zonal diameter, providing a measure of the acid diffusion area around each colony. This calculation isolates the acid production effect from the colony growth effect, enabling accurate assessment of acid production capability independent of colony size variations.

[0111] Acid Unitage Value Determination

[0112]

[0070] The acid unitage value serves as a normalized measure of acid production efficiency that accounts for differences in colony size among different mutant candidates. The calculation employs the formula: Acid Unitage = Zone of Acid (mm) / Colony Diameter (mm), where higher values indicate greater acid production relative to colony size. This normalization approach ensures that mutants with genuinely enhanced acid production capabilities are identified rather than simply selecting colonies with larger overall zones that result from increased colony size rather than improved acid production efficiency.

[0113] Mutant Strain

[0114]

[0071] Among the screened mutant candidates, strain DPPL 017 demonstrated superior acid production performance with an acid unitage value of 202421061133

[0115] 2.18. This mutant exhibited a colony diameter of 28 mm and a total zonal diameter of 61 mm, resulting in an acid zone of 33 mm. The 33 mm acid zone represents a substantial increase compared to the parent strain Aspergillus niger ATCC 9142, which typically exhibits acid zones of 20-22 mm under identical screening conditions.

[0116]

[0072] The acid unitage value of 2.18 for strain DPPL 017 exceeds the typical range of 1.73-1.80 observed for the parent strain, indicating enhanced acidogenic potential resulting from the UV mutagenesis treatment. This quantitative improvement in acid production capability, combined with the substantially larger acid zone, establishes strain DPPL 017 as a promising candidate for further fermentation studies and commercial development.

[0117]

[0073] The selection of strain DPPL 017 is based on the combination of high acid unitage value and large absolute acid zone measurement, both of which indicate enhanced organic acid production capability compared to the parent strain and other mutant candidates evaluated during the screening process. Following primary screening using Bromocresol Green plates, selected mutant candidates undergo secondary screening validation to provide quantitative confirmation of organic acid production capabilities. The secondary screening methodology employs multiple analytical techniques to verify and quantify the production of specific organic acids, ensuring accurate identification of mutants with enhanced production characteristics.

[0118] Secondary Screening Methods

[0119]

[0074] Enzymatic assay kits provide highly specific quantitative measurement of gluconic acid and citric acid concentrations in fermentation samples. The enzymatic approach utilizes substrate-specific enzymes that catalyze reactions producing measurable changes in absorbance, enabling precise determination of organic acid concentrations through spectrophotometric analysis.

[0120] Gluconic Acid Enzymatic Detection 202421061133

[0121]

[0075] The gluconic acid enzymatic kit employs a coupled enzyme system that converts gluconic acid to measurable products through sequential enzymatic reactions. The assay methodology involves sample preparation, enzyme reaction incubation, and spectrophotometric measurement at specific wavelengths to determine gluconic acid concentration. Sample preparation requires appropriate dilution of fermentation broth to bring gluconic acid concentrations within the linear range of the assay. The enzymatic reaction is initiated by addition of the enzyme mixture to the prepared sample, followed by incubation under controlled temperature and time conditions to ensure complete substrate conversion. Absorbance measurements are performed using a spectrophotometer at the wavelength specified by the kit manufacturer. The change in absorbance (AAbs) between sample and blank measurements is calculated and converted to gluconic acid concentration using the provided conversion factors and calibration standards.

[0122] Citric Acid Enzymatic Detection

[0123]

[0076] The citric acid enzymatic assay utilizes citrate-specific enzymes that convert citric acid to products measurable through Spectrophotometric analysis. The assay protocol follows similar principles to the gluconic acid kit but employs different enzyme systems specific for citric acid detection and quantification. The enzymatic reaction system provides high specificity for citric acid, minimizing interference from other organic acids or fermentation byproducts that are present in the sample matrix. The assay sensitivity enables detection of citric acid concentrations across the range typically encountered in fermentation studies. Quantitative results from enzymatic assays provide definitive confirmation of organic acid production levels, enabling accurate comparison between mutant strains and the parent strain under identical fermentation conditions. The enzymatic approach serves as the reference method for validating acid production capabilities identified through primary screening procedures.

[0124] Thin Layer Chromatography Analysis 202421061133

[0125]

[0077] Thin Layer Chromatography provides qualitative detection of organic acid production through separation and visualization on chromatographic plates based on characteristic migration patterns.

[0126] TLC Analysis Protocol

[0127]

[0078] TLC analysis employs silica gel plates with fermentation samples applied alongside standard solutions of gluconic acid and citric acid. Plates are developed using appropriate mobile phase solvents and visualized through chemical staining or UV illumination. Organic acid identification is accomplished by comparing migration distances with standard reference compounds, with semi-quantitative assessment performed by comparing spot intensities with known concentrations.

[0128] Sugar Quantification by DNS A Method

[0129]

[0079] The DNS A method quantifies reducing sugar concentrations through reduction of 3,5-dinitrosalicylic acid under alkaline conditions. Fermentation samples are diluted appropriately, mixed with DNS A reagent, heated under controlled conditions, and measured spectrophotometrically at 540 nm. Sugar utilization percentage is calculated by comparing concentrations with initial medium values.

[0130] Iodine Starch Test

[0131]

[0080] The iodine starch test provides qualitative assessment of starch utilization based on blue-black color formation when iodine reacts with starch. Color intensity decreases as starch is hydrolyzed during fermentation, with complete utilization indicated by absence of blue coloration.

[0132] Result Interpretation and Starch Utilization Assessment

[0133]

[0081] Complete starch utilization is indicated by the absence of blue coloration and the development of brown coloration similar to that observed in the 202421061133

[0134] water blank control. Partial starch utilization results in intermediate color intensities between the initial blue-black color and the final brown color. The iodine starch test provides a rapid and simple method for monitoring starch consumption during fermentation processes utilizing starch-containing media. The test enables assessment of amylolytic enzyme activity and substrate utilization efficiency in starch-based fermentation systems. The combination of enzymatic assays, TLC analysis, DNSA method, and iodine starch test provides comprehensive secondary screening validation that confirms and quantifies the organic acid production capabilities identified through primary BCG screening. These analytical methods ensure accurate identification of mutant strains with enhanced production characteristics and provide the quantitative data necessary for strain selection and fermentation optimization.

[0135]

[0082] The mutant strain DPPL 017 (1) has been deposited with the Microbial Type Culture Collection (MTCC) under accession number MTCC 25761. The deposited strain represents a genetically stable mutant derived from the parent strain Aspergillus niger ATCC 9142 through UV mutagenesis at 254 nm wavelength for 19 minutes exposure duration. The mutant strain MTCC 25761 exhibits specific genetic modifications that distinguish the strain from the parent organism and contribute to enhanced organic acid production capabilities. The genetic characterization of the mutant strain has identified multiple sequence mutation positions that correlate with improved metabolic performance for both citric acid and gluconic acid biosynthesis.

[0136] Sequence Mutation Analysis

[0137]

[0083] Whole genome sequencing results submitted with this specification demonstrate that analysis of the mutant strain Aspergillus niger MTCC 25761 has identified specific nucleotide sequence modifications including additions, deletions, and substitutions that exhibit maximum mutation probability and directly contribute to enhanced acid production yields. The comprehensive genomic analysis revealed distinct sequence positions where targeted genetic 202421061133

[0138] alterations comprising nucleotide additions, deletions, or substitutions provide optimal enhancement of organic acid biosynthesis pathways. The identified mutations occur at specific genomic positions that demonstrate the highest correlation with improved citric acid and gluconic acid production capabilities, representing critical mutation sites that maximize metabolic flux through acid biosynthesis pathways.

[0139]

[0084] The mutant sequence identifiers corresponding to reference positions are designated as SEQ ID NOs: 26886, 26904, 26906, 26909, 26916, 26927, 26935, 27080, 27104, 27116, 27123, 27128, 27150, 27170, 27171, 27213, 27225, 27262, 27273, 27275, 27280, 27291, 27292, 27294, 27295, 27339, 27341, 27399, 27401, 27413, 27447, 27450, 27452, 27466, 27493, 27502, 27516, 27532, 27663, 27690, 27698, 27734, 27739, 27885, 27957, 28140, 28141, and 28147, respectively. These specific mutant sequences represent the optimal genetic modifications that provide maximum mutation probability for enhanced organic acid production, with each sequence contributing to the overall improved yield performance of strain MTCC 25761.

[0140] Metabolic Pathway Enhancement

[0141]

[0085] The sequence mutations identified in strain MTCC 25761 affect genes and regulatory elements involved in organic acid biosynthesis pathways. The mutations contribute to enhanced enzyme expression, improved substrate utilization efficiency, and modified regulatory control mechanisms that collectively result in increased citric acid and gluconic acid production capabilities. The genetic modifications present in the mutant strain influence multiple aspects of cellular metabolism including carbon source utilization, energy metabolism, and organic acid transport systems. These changes result in improved fermentation characteristics including enhanced substrate conversion efficiency, stable acid production under varying environmental conditions, and increased tolerance to acidic fermentation environments.

[0142] Strain Stability and Inheritance 202421061133

[0143]

[0086] The mutant strain MTCC 25761 demonstrates genetic stability through multiple subculture passages, indicating that the induced mutations are stably inherited and maintained during vegetative propagation. The genetic stability ensures consistent performance characteristics across different fermentation batches and enables reliable scale-up for commercial production applications. The mutation pattern observed in strain MTCC 25761 represents a unique combination of genetic changes that collectively contribute to the enhanced acid production phenotype. The specific sequence alterations at the identified positions create a distinct genetic profile that differentiates the mutant strain from both the parent strain and other potential mutant variants.

[0144] Phenotypic Expression of Genetic Changes

[0145]

[0087] The genetic mutations present in strain MTCC 25761 result in measurable phenotypic changes that manifest as improved organic acid production capabilities. The mutant strain exhibits enhanced gluconic acid production reaching concentrations of 164.05 g / L compared to 119.78 g / L achieved by the parent strain under identical fermentation conditions, representing a 1.37-fold improvement in production efficiency. For citric acid production, the mutant strain demonstrates consistently higher acid concentrations across multiple fermentation time points, with production levels reaching 0.506 g / L compared to 0.391 g / L for the parent strain at 382 hours of fermentation, representing a 1.29-fold improvement. The enhanced production capability is maintained throughout extended fermentation periods, indicating improved metabolic persistence and acid tolerance. The mutant strain MTCC 25761 exhibits improved fermentation characteristics including more stable pH dynamics, enhanced substrate utilization patterns, and superior pellet morphology during submerged fermentation. These phenotypic improvements correlate with the underlying genetic modifications and contribute to the overall enhanced performance of the mutant strain for organic acid production applications.

[0146] CITRIC ACID PRODUCTION 202421061133

[0147] Citric Acid Fermentation Medium Composition

[0148]

[0088] The fermentation medium for citric acid production employs a carefully balanced composition of carbon sources, nitrogen sources, and mineral nutrients to support optimal growth and acid biosynthesis by the mutant strain MTCC 25761. The medium composition is specifically formulated to promote citric acid accumulation while maintaining appropriate nutritional balance for sustained fermentation performance.

[0149] Carbon Source Components

[0150]

[0089] The carbon source component consists of starch at a concentration of 80-120 g / L, supplemented with dextrose at 20-28 g / L. The starch serves as the primary carbon source, providing a sustained substrate supply through enzymatic hydrolysis during fermentation. The supplemental dextrose provides readily available glucose for initial metabolic activity and rapid culture establishment. This combination ensures continuous substrate availability throughout the fermentation period while supporting both growth and acid production phases.

[0151] Nitrogen and Mineral Components

[0152]

[0090] The nitrogen source is provided by ammonium sulphate at 3 g / L, supplying both nitrogen for protein synthesis and sulfur for amino acid biosynthesis. The concentration is optimized to support adequate biomass formation while maintaining nitrogen limitation conditions that favor citric acid accumulation. Potassium dihydrogen phosphate at 1 g / L provides phosphorus for nucleic acid synthesis and energy metabolism while contributing to pH buffering capacity. Magnesium sulphate heptahydrate at 1 g / L supplies magnesium ions for enzyme cofactor functions, particularly important for enzymes involved in organic acid biosynthesis pathways.

[0153] Medium Preparation Protocol 202421061133

[0154]

[0091] The fermentation medium preparation involves separate preparation of different nutrient components to prevent precipitation and ensure complete dissolution. The starch and dextrose components are dissolved in distilled water in a 500 mL flask. The potassium dihydrogen phosphate is prepared separately in a 250 mL flask. The remaining nutrients are dissolved together in a separate 250 mL flask.

[0155] pH Control and Fermentation Conditions

[0156]

[0092] The initial pH of the fermentation medium is adjusted to 5.0 using appropriate pH adjustment reagents. The slightly acidic initial pH provides favorable conditions for spore germination and early growth phases while establishing the acidic environment that promotes citric acid biosynthesis. During fermentation, the production pH is maintained at 2.0 through natural acidification resulting from citric acid accumulation, creating optimal conditions for acid accumulation while inhibiting competing metabolic pathways.

[0157] Inoculation and Operating Parameters

[0158] 7

[0159]

[0093] The spore inoculum for citric acid fermentation is prepared at 5x10 CFU / mL. This concentration provides adequate spore density for rapid culture establishment while avoiding excessive initial biomass that competes with acid production. The inoculum volume of 2.5 mL is added to 250 mL of fermentation medium under sterile conditions. The fermentation is conducted at 30°C to 35°C with agitation at 23-27 RPM using a rotary shaker, providing optimal environmental parameters for citric acid biosynthesis while maintaining adequate oxygen transfer for aerobic metabolism.

[0160] Citric Acid Fermentation Conditions

[0161]

[0094] The fermentation conditions are specifically optimized to support the metabolic requirements of strain MTCC 25761 and promote efficient citric acid biosynthesis. The controlled environmental parameters ensure consistent 202421061133

[0162] fermentation performance while maximizing organic acid yields through optimal growth and production conditions. Temperature is maintained at 30°C throughout the production period, representing the optimal growth temperature for Aspergillus niger strains and providing favorable conditions for enzymatic reactions involved in citric acid biosynthesis.

[0163]

[0095] Agitation is provided through rotary shaking at 23 to 27 RPM, ensuring adequate mixing while providing sufficient oxygen transfer to support aerobic metabolism. The moderate agitation speed prevents excessive shear stress that damages fungal pellets or disrupts optimal pellet morphology. The fermentation medium volume of 250 mL is contained within 500 mL flasks, providing a 1:2 ratio that ensures adequate headspace for oxygen transfer and gas exchange while preventing excessive evaporation during the extended fermentation period.

[0164]

[0096] The fermentation duration extends for 382 hours, corresponding to approximately 16 days of continuous production. This extended period allows for complete substrate utilization and maximum citric acid accumulation, accommodating the biphasic nature of citric acid production including initial growth phases followed by extended acid production and accumulation phases.

[0165]

[0097] The fermentation conditions promote the formation of compact, spherical pellet morphology that enhances oxygen transfer efficiency and substrate utilization. The optimal pellet formation contributes to improved fermentation performance and consistent acid production yields while preventing pellet disintegration or excessive filamentous growth that reduces fermentation efficiency.

[0166]

[0098] The controlled fermentation parameters maintain stable pH dynamics throughout the production period, with natural acidification occurring as citric acid accumulates in the medium. The pH progression from the initial value of 5.0 to the production pH of 2.0 indicates successful acid biosynthesis and provides the acidic environment necessary for optimal citric acid stability and recovery. 202421061133

[0167] Pellet Morphology and Starch Utilization Analysis

[0168]

[0099] The pellet morphology characteristics and starch utilization patterns during citric acid fermentation provide important indicators of fermentation performance and substrate conversion efficiency. Figure 1 illustrates the pellet morphology scores for strains F-l and F-5 over the fermentation time period from 118 to 220 hours. The morphology scoring system provides quantitative assessment of pellet structure and integrity throughout the fermentation process.

[0169] Pellet Morphology Performance Comparison

[0170]

[0100] At 118 hours of fermentation, both F-l and F-5 strains demonstrate high morphology scores approaching 3.5, indicating compact, well-formed pellets with intact structure. The mutant strain F-5 (DPPL 017 (1)) maintains superior pellet morphology characteristics compared to the parent strain F-l throughout the fermentation period, exhibiting more stable pellet structure with a gradual decline in morphology score, reaching approximately 1.5 at 220 hours.

[0171]

[0101] The parent strain F-l demonstrates a more rapid decline in pellet morphology score, reaching approximately 1.0 at 220 hours. The steeper decline indicates less stable pellet structure and more pronounced pellet deterioration during later fermentation phases. The morphological differences reflect the enhanced fermentation characteristics of the mutant strain, with better pellet integrity contributing to improved oxygen transfer efficiency and enhanced substrate utilization. At 220 hours, both strains exhibit pellet burst morphology, indicating the natural progression toward pellet disintegration as substrate becomes depleted and fermentation approaches completion. This represents a normal transition phase in extended fermentation processes where pellet structure becomes less cohesive due to substrate limitation and metabolic changes.

[0172] Starch Utilization Efficiency Assessment 202421061133

[0173]

[0102] Figure 2 demonstrates the starch residue scores for both strains throughout the fermentation period, providing quantitative assessment of substrate utilization efficiency. Both strains exhibit progressive reduction in starch residue scores from 118 to 220 hours, indicating continuous substrate consumption. At 118 hours, both strains show starch residue scores of approximately 2.0, indicating substantial remaining starch substrate in the fermentation medium.

[0174]

[0103] The mutant strain F-5 demonstrates more efficient starch utilization compared to the parent strain F-l, as evidenced by consistently lower starch residue scores throughout the fermentation period. At 220 hours, F-5 achieves a starch residue score approaching 0.5, while F-l maintains a score of approximately 1.0, indicating more complete substrate utilization by the mutant strain. The superior starch utilization efficiency correlates with enhanced citric acid production capabilities and improved metabolic performance. Complete starch utilization is confirmed through iodine starch test results, which show color change from blue to brown, matching the water blank control and indicating successful hydrolysis of the starch substrate.

[0175] pH Profiles and Acidification Dynamics

[0176]

[0104] The pH profiles during citric acid fermentation provide critical indicators of fermentation progress and acid accumulation patterns for both the parent strain and the mutant strain MTCC 25761. Figure 3 illustrates the pH profile for the parent strain F-l throughout the citric acid fermentation period from 0 to 382 hours, demonstrating the characteristic acidification pattern associated with citric acid biosynthesis, beginning with an initial pH of approximately 4.9.

[0177]

[0105] The parent strain F-l exhibits rapid initial acidification during the first 50 hours, with pH declining from 4.9 to approximately 1.8 by 46 hours. This rapid acidification phase corresponds to the establishment of active citric acid biosynthesis pathways and initial accumulation of organic acids. Following the initial acidification period, the parent strain demonstrates a transient pH rise 202421061133

[0178] between 52 and 70 hours, with pH values increasing to approximately 3.2, suggesting a metabolic adjustment phase. From 100 hours onward, the parent strain maintains a relatively stable pH range between 1.8 and 2.1 throughout the remainder of the fermentation period.

[0179]

[0106] Figure 4 presents the comparative pH profiles between the parent strain F-l and the mutant strain F-5 (DPPL 017 (1)) throughout the complete fermentation period. The mutant strain F-5 demonstrates more aggressive initial acidification compared to the parent strain, achieving a minimum pH of 0.5 to 1.0 at 75-76 hours of fermentation. This exceptionally low pH value represents the most acidic condition reached during the fermentation process and indicates superior acid production capability compared to the parent strain, which achieves a minimum pH of approximately 1.0 during the same time period.

[0180]

[0107] Following the initial acidification phase, the mutant strain F-5 exhibits similar pH fluctuations to the parent strain between 52 and 70 hours, with temporary pH elevation to approximately 3.3. However, the mutant strain demonstrates more rapid recovery from this transient pH rise and establishes stable acidic conditions more quickly. From 100 to 382 hours, the mutant strain F-5 maintains stable low pH conditions between 1.8 and 2.1, with greater consistency and less variation throughout the extended fermentation period. The pH stability facilitates optimal citric acid accumulation through favorable thermodynamic conditions while inhibiting competing metabolic pathways and preventing contamination.

[0181]

[0108] The citric acid production performance of mutant strain MTCC 25761 demonstrates substantial yield improvements compared to the parent strain Aspergillus niger ATCC 9142 under identical fermentation conditions. The quantitative assessment employs enzymatic assay kit methodology to provide precise measurement of acid concentrations throughout extended fermentation periods, enabling accurate comparison of production capabilities between the parent and mutant strains. 202421061133

[0182]

[0109] Figure 5 illustrates the citric acid production profiles for both strains measured at four critical time points during the fermentation process: 316, 334, 358, and 382 hours. The enzymatic kit analysis reveals distinct performance differences between the parent strain F-l and the mutant strain F-5 (DPPL 017 (1)), with the mutant strain consistently achieving higher citric acid concentrations throughout the measured fermentation period.

[0183] Enzymatic Kit Quantification Methodology

[0184]

[0110] The citric acid enzymatic assay kit employs substrate-specific enzymes that catalyze the conversion of citric acid to measurable products through coupled enzymatic reactions. The assay methodology involves precise sample preparation, controlled enzymatic reaction conditions, and spectrophotometric measurement at specific wavelengths to determine citric acid concentrations with high accuracy and specificity.

[0185]

[0111] The enzymatic reaction system provides high specificity for citric acid detection, minimizing interference from other organic acids, fermentation byproducts, or medium components present in the complex fermentation matrix. The enzyme-based detection method offers superior accuracy compared to chemical detection methods and enables reliable quantification across the range encountered in fermentation studies.

[0186] Comparative Yield Performance Analysis

[0187]

[0112] The citric acid production data presented in Figure 5 demonstrates the superior performance of mutant strain F-5 across all measured time points. At 316 hours, the mutant strain F-5 achieves 0.463 g / L compared to 0.432 g / L produced by the parent strain F-l, representing a 7.2% improvement. This early performance advantage indicates enhanced metabolic efficiency and more rapid establishment of citric acid biosynthesis pathways.

[0188]

[0113] The performance differential becomes more pronounced at 334 hours, where the mutant strain F-5 reaches 0.498 g / L while the parent strain F-l 202421061133

[0189] produces only 0.257 g / L, representing a 93.8% improvement. This dramatic yield difference indicates that the mutant strain maintains active citric acid biosynthesis during fermentation phases where the parent strain experiences reduced metabolic activity.

[0190]

[0114] At 358 hours, the mutant strain F-5 sustains high production at 0.497 g / L, while the parent strain F-l achieves 0.317 g / L, representing a 56.8% yield improvement. The sustained high production level demonstrates improved metabolic persistence and enhanced tolerance to the acidic fermentation environment that accumulates during extended production periods.

[0191]

[0115] The final measurement at 382 hours reveals the mutant strain F-5 achieving 0.506 g / L compared to 0.391 g / L for the parent strain F-l, representing a 29.4% improvement in final citric acid yield. The sustained production capability indicates enhanced acid tolerance and improved metabolic efficiency under the challenging conditions of prolonged acidic fermentation.

[0192] Enhanced Metabolic Efficiency Correlation

[0193]

[0116] The citric acid yield improvements demonstrated by mutant strain F-5 correlate directly with the enhanced pH dynamics and acidification patterns observed throughout the fermentation period. The superior acidification capability, including achievement of minimum pH values as low as 0.5, creates optimal thermodynamic conditions for citric acid accumulation while inhibiting competing metabolic pathways.

[0194]

[0117] The stable pH maintenance between 1.8 and 2.1 demonstrated by the mutant strain supports sustained citric acid biosynthesis and prevents acid degradation or conversion to alternative metabolic products. The consistent acidic environment facilitates optimal enzyme activity while creating conditions that favor product accumulation over biomass formation.

[0195]

[0118] The TLC analysis results provide qualitative confirmation of the enhanced citric acid production capabilities demonstrated through enzymatic kit quantification. The mutant strain F-5 exhibits more frequent and intense TLC spot 202421061133

[0196] development compared to the parent strain F-l, particularly during mid-to-late fermentation phases corresponding to the time periods where the greatest yield improvements are observed.

[0197]

[0119] The correlation between TLC spot intensity and enzymatic kit measurements validates the enhanced citric acid production performance of the mutant strain. The visual confirmation provided by TLC analysis supports the quantitative yield data and demonstrates consistency across different analytical methodologies.

[0198] Production Stability and Consistency

[0199]

[0120] The citric acid production profile of mutant strain F-5 demonstrates remarkable stability and consistency throughout the extended fermentation period. Unlike the parent strain F-l, which exhibits significant yield fluctuations between measurement time points, the mutant strain maintains relatively stable production levels with a gradual increase from 0.463 g / L at 316 hours to 0.506 g / L at 382 hours.

[0200]

[0121] The production stability exhibited by the mutant strain indicates improved metabolic control mechanisms and enhanced tolerance to environmental stresses associated with prolonged acidic fermentation. The consistent performance characteristics provide advantages for commercial production applications where predictable yields and stable fermentation performance are required for economic viability.

[0201]

[0122] The approximately 40% overall improvement in citric acid yield achieved by mutant strain MTCC 25761 represents a substantial enhancement in production efficiency that directly translates to improved fermentation economics and commercial viability. The yield improvement, combined with enhanced fermentation stability and consistent performance characteristics, establishes the mutant strain as a superior production organism for citric acid biosynthesis applications. 202421061133

[0202] GLUCONIC ACID PRODUCTION

[0203] Gluconic Acid Fermentation Medium Composition

[0204]

[0123] The fermentation medium for gluconic acid production employs a precisely formulated composition of carbon sources, nitrogen sources, and essential mineral nutrients to support optimal growth and gluconic acid biosynthesis by the mutant strain MTCC 25761. The medium composition is specifically designed to promote gluconic acid accumulation through controlled nutrient availability while maintaining appropriate metabolic balance for sustained fermentation performance.

[0205] Carbon Source and Primary Substrate

[0206]

[0124] The primary carbon source consists of dextrose at 180-220 g / L, providing the glucose substrate required for gluconic acid biosynthesis through oxidative metabolism. The high dextrose concentration ensures abundant substrate availability while supporting the glucose oxidase enzyme system responsible for converting glucose to gluconic acid. The concentration is optimized to maximize substrate utilization efficiency while preventing substrate inhibition effects.

[0207] Nitrogen Source and Growth Factors

[0208]

[0125] Yeast extract is incorporated at 2 g / L to provide complex nitrogen sources, vitamins, and growth factors necessary for fungal metabolism and enzyme synthesis. The yeast extract supplies amino acids, peptides, and B-vitamins that support cellular growth and maintain the enzymatic machinery required for efficient gluconic acid production. Ammonium sulphate serves as the primary inorganic nitrogen source at 0.5 g / L, providing readily available nitrogen for protein synthesis while supplying sulfur for amino acid biosynthesis.

[0209] Essential Mineral Components

[0210]

[0126] Magnesium sulphate heptahydrate is included at 5 g / L to supply magnesium ions required for enzyme activation and cofactor functions in gluconic acid biosynthesis pathways. Potassium chloride is incorporated at 0.5 g / L to 202421061133

[0211] provide potassium ions necessary for enzyme activation, cellular transport processes, and osmotic regulation. Ferric ammonium citrate is added at 0.01 g / L to supply iron ions required for enzyme cofactor functions and electron transport processes. Potassium dihydrogen phosphate is included at 7 g / L to provide phosphorus for nucleic acid synthesis, energy metabolism, and phosphorylation reactions while serving as a pH buffering system.

[0212] Medium Distribution Protocol

[0213]

[0127] The gluconic acid fermentation medium preparation employs a three-flask distribution system to prevent precipitation and ensure complete dissolution of all medium components. Flask A contains 55 g of dextrose dissolved in 100 mL of distilled water and adjusted to 170 mL in a 500 mL flask. Flask B contains 1.75 g of potassium dihydrogen phosphate dissolved in 20 mL of distilled water and adjusted to 30 mL in a 250 mL flask. Flask C contains all remaining nutrient components dissolved in 30 mL of distilled water and adjusted to 50 mL in a 250 mL flask.

[0214] pH Control and Sterilization

[0215]

[0128] The initial pH of all medium components is adjusted to 7.0 to provide neutral conditions that prevent component degradation during sterilization while establishing appropriate starting conditions for spore germination and early growth phases. Each flask is sterilized separately by autoclaving to ensure complete sterilization while preserving component integrity. Following sterilization and cooling, the contents of all three flasks are aseptically combined to create the complete fermentation medium with a total volume of 250 mL.

[0216] Spore Inoculum Preparation and Concentration

[0217]

[0129] The spore inoculum for gluconic acid fermentation is prepared at a standardized concentration of 2*106CFU / mL. The inoculum concentration is optimized to provide adequate spore density for rapid culture establishment while avoiding excessive initial biomass that competes with gluconic acid production 202421061133

[0218] during early fermentation phases. The standardized inoculum concentration ensures consistent fermentation initiation across different production batches and enables reproducible comparison of fermentation performance between the parent strain and mutant strain under identical starting conditions.

[0219] Gluconic Acid Fermentation Conditions and Operating Parameters

[0220]

[0130] The inoculum volume of 2.5 mL is added to the 250 mL of complete fermentation medium under strict aseptic conditions to prevent contamination. The fermentation conditions are specifically optimized to support the metabolic requirements of strain MTCC 25761 and promote efficient gluconic acid biosynthesis through controlled environmental parameters designed to maintain optimal oxygen availability, substrate accessibility, and environmental stability throughout the extended fermentation period.

[0221] Temperature Control and Enzyme Optimization

[0222]

[0131] The fermentation temperature is maintained at 30°C throughout the entire production period of 214 hours. This temperature represents the optimal growth and metabolic temperature for Aspergillus niger strains and provides favorable thermodynamic conditions for the glucose oxidase enzyme system responsible for gluconic acid biosynthesis. The controlled temperature supports efficient substrate metabolism while maintaining enzyme stability and catalytic activity for glucose oxidation reactions that convert dextrose to gluconic acid.

[0223] Agitation and Oxygen Transfer Requirements

[0224]

[0132] Agitation is provided through rotary shaking at 23 to 27 RPM using a rotary shaker system throughout the 214-hour fermentation period. The moderate agitation speed ensures adequate mixing while providing sufficient oxygen transfer to support the aerobic metabolic requirements of gluconic acid biosynthesis. The glucose oxidase enzyme system requires continuous oxygen availability for oxidation reactions, making adequate aeration critical for 202421061133

[0225] production efficiency. The 25 RPM agitation rate provides optimal balance between oxygen transfer and mechanical stress on fungal biomass while preventing excessive shear forces that damage fungal pellets.

[0226] Medium Volume and Headspace Configuration

[0227]

[0133] The fermentation medium volume of 250 mL is contained within 500 mL flasks, providing a 1:2 ratio that ensures adequate headspace for oxygen transfer and gas exchange while accommodating the aerobic requirements of gluconic acid production. The substantial headspace volume facilitates efficient oxygen transfer from the gas phase to the liquid medium, supporting the high oxygen demand associated with glucose oxidase activity. The headspace accommodates hydrogen peroxide production as a byproduct of glucose oxidation, which is subsequently decomposed by catalase enzymes.

[0228] Fermentation Duration and Metabolic Phases

[0229]

[0134] The fermentation duration extends for 214 hours, corresponding to approximately 9 days of continuous production. This extended period allows for complete substrate utilization and maximum gluconic acid accumulation while accommodating the biphasic nature of fungal fermentation, including initial growth phases followed by extended acid production phases. The prolonged time enables the mutant strain to achieve maximum substrate conversion efficiency and optimal acid yield recovery.

[0230] Integrated Process Optimization and Performance

[0231]

[0135] The combination of controlled temperature at 30°C, moderate agitation at 25 RPM, appropriate medium-to-flask volume ratio of 1:2, and extended fermentation time of 214 hours creates optimal conditions for gluconic acid biosynthesis by strain MTCC 25761. These parameters collectively support the aerobic metabolic requirements for glucose oxidation while maintaining environmental stability and substrate availability. The fermentation conditions 202421061133

[0232] promote optimal pellet morphology that enhances oxygen transfer efficiency and substrate utilization while preventing excessive filamentous growth or pellet disintegration.

[0233] Sugar Utilization Analysis and Metabolic Performance Assessment

[0234]

[0136] The sugar utilization profiles during gluconic acid fermentation provide quantitative assessment of substrate consumption efficiency and metabolic activity patterns for both the parent strain and the mutant strain MTCC 25761. The substrate utilization analysis employs the DNSA (3,5-dinitrosalicylic acid) method to measure reducing sugar concentrations throughout the fermentation period, enabling calculation of percentage sugar utilization and evaluation of metabolic performance under controlled fermentation conditions.

[0235] Parent Strain F-l Sugar Utilization Profile

[0236]

[0137] Figure 6 illustrates the sugar utilization profile for the parent strain F-1 throughout the 214-hour gluconic acid fermentation period. The parent strain demonstrates a distinctive biphasic utilization pattern characterized by an initial phase of negative or minimal sugar utilization followed by progressive increase in substrate consumption during later fermentation phases. The parent strain F-l exhibits negative sugar utilization values during the initial fermentation period from 0 to 75 hours, with values fluctuating between 0% and -25.7%. The negative utilization values indicate apparent increases in reducing sugar concentration relative to initial measurement, reflecting hydrolysis of complex carbohydrates that release additional reducing sugars during early fermentation phases.

[0237]

[0138] Positive sugar utilization by the parent strain F-l begins to appear after 75 hours, indicating transition from adaptation phase to active glucose consumption for gluconic acid biosynthesis. The utilization values show clear upward trend from 94 hours onward, reaching 9.6% at 94 hours and progressively increasing throughout the remainder of the fermentation period. The parent strain F-l achieves maximum sugar utilization of 42.3% at 214 hours, demonstrating 202421061133

[0238] substantial substrate conversion efficiency despite delayed onset of active metabolism.

[0239] Mutant Strain F-2 Enhanced Utilization Performance

[0240]

[0139] Figure 7 presents the sugar utilization profile for the mutant strain F-2 (DPPL 017 (1)) throughout the same 214-hour fermentation period. The mutant strain exhibits a similar biphasic utilization pattern but demonstrates enhanced metabolic performance and superior final substrate utilization compared to the parent strain. The mutant strain F-2 displays negative sugar utilization values during the initial fermentation period from 0 to 75 hours, with fluctuations ranging from -2.3% to -32.5%. The minimum utilization value of -32.5% occurs at 27 hours, representing more pronounced apparent sugar increase compared to the parent strain, suggesting more active hydrolytic enzyme activity during the adaptation phase.

[0241]

[0140] The transition to positive sugar utilization occurs at approximately 75 hours for the mutant strain F-2, similar to the timing observed for the parent strain. However, the mutant strain demonstrates more rapid establishment of active glucose consumption, reaching 24.6% utilization at 99 hours compared to 19.2% achieved by the parent strain at the same time point. The mutant strain F-2 achieves superior final sugar utilization of 44.0% at 214 hours, representing a 4.0% improvement over the parent strain performance.

[0242] Comparative Performance Analysis and Economic Implications

[0243]

[0141] Figure 8 provides comparative analysis of sugar utilization profiles between the parent strain F-l and the mutant strain F-2 throughout the complete fermentation period. The comparative presentation enables direct assessment of performance differences and identification of enhanced metabolic characteristics of the mutant strain. Both strains exhibit similar biphasic utilization patterns with initial negative values persisting until approximately 75 hours, followed by 202421061133

[0244] progressive increases in sugar consumption. The parallel timing of metabolic phases indicates that fundamental fermentation kinetics remain similar between strains, with primary differences occurring in magnitude of substrate utilization rather than timing of metabolic transitions.

[0245]

[0142] The enhanced sugar utilization performance of the mutant strain F-2 correlates directly with improved gluconic acid production capabilities demonstrated through enzymatic assay analysis. The 4.0% improvement in final sugar utilization translates to more efficient substrate conversion and higher product yields, contributing to overall enhanced fermentation performance of strain MTCC 25761. The superior substrate utilization efficiency provides economic advantages for commercial gluconic acid production through improved substrate conversion rates and reduced raw material costs per unit of product.

[0246]

[0143] The pH profiles during gluconic acid fermentation provide critical indicators of fermentation progress, metabolic activity patterns, and acid accumulation dynamics for both the parent strain and the mutant strain MTCC 25761. The pH monitoring throughout the 214-hour fermentation period reveals distinct differences in acidification patterns and metabolic stability between the two strains, with the mutant strain demonstrating enhanced dynamic metabolic activity through characteristic oscillatory pH behavior.

[0247] Parent Strain F-l pH Profile and Acidification Pattern

[0248]

[0144] Figure 10 illustrates the pH profile for the parent strain F-l throughout the gluconic acid fermentation period from 0 to 214 hours. The parent strain exhibits an initial pH of approximately 6.3, reflecting the neutral starting conditions of the fermentation medium. During the early fermentation phase from 3 to 51 hours, the parent strain demonstrates a gradual pH increase to approximately 6.8, indicating minimal acid production and limited metabolic activity during the adaptation phase. 202421061133

[0249]

[0145] The parent strain F-l experiences a significant 4.0 to 5.0 drop beginning at 70 to 72 hours, with values declining to approximately 4.5 by 94 hours. This acidification phase corresponds to the onset of active gluconic acid biosynthesis and represents the transition from adaptation phase to productive metabolism. Following the initial acidification, the parent strain exhibits periodic pH fluctuations between approximately 4.5 and 6.5 throughout the remainder of the fermentation period, with oscillations occurring at regular intervals.

[0250]

[0146] The pH oscillations observed in the parent strain F-l indicate alternating phases of acid production and partial neutralization or consumption, suggesting dynamic metabolic activity with periods of active gluconic acid synthesis followed by metabolic adjustments. The final pH of approximately 5.2 at 214 hours indicates moderate acidification and sustained acid accumulation throughout the fermentation period.

[0251] Mutant Strain F-2 Enhanced pH Dynamics and Metabolic Activity

[0252]

[0147] Figure 11 presents the comparative pH profiles between the parent strain F-l and the mutant strain F-2 (DPPL 017 (1)) throughout the complete 214-hour fermentation period. The mutant strain F-2 demonstrates more pronounced and dynamic pH behavior compared to the parent strain, indicating enhanced metabolic activity and superior acid production capabilities.

[0253]

[0148] The mutant strain F-2 exhibits an initial pH of approximately 6.45, similar to the parent strain starting conditions. During the early fermentation phase from 3 to 51 hours, the mutant strain maintains stable pH values ranging from 6.45 to 6.88, indicating minimal acid production during the adaptation phase. The slightly higher pH values compared to the parent strain suggest more effective pH buffering or delayed onset of acidogenic metabolism.

[0254]

[0149] Beginning at 70 hours, the mutant strain F-2 experiences sharp pH decline, reaching approximately 4.87 by 94 hours. This acidification pattern is 202421061133

[0255] similar to the parent strain but demonstrates more rapid pH reduction, indicating more aggressive onset of gluconic acid biosynthesis. The accelerated acidification reflects enhanced metabolic activity and more efficient establishment of glucose oxidation pathways.

[0256] Oscillatory pH Pattern and Dynamic Metabolic Activity

[0257]

[0150] From 94 to 195 hours, the mutant strain F-2 exhibits characteristic oscillatory pH fluctuations between 4.3 and 6.5, demonstrating more pronounced and frequent pH variations compared to the parent strain. The oscillatory pattern indicates dynamic metabolic activity with alternating phases of intensive acid production and metabolic adjustment periods. The pH oscillations reach lower minimum values of approximately 4.3 compared to 4.5 achieved by the parent strain, indicating more aggressive acid production capability.

[0258]

[0151] The deeper pH troughs observed in the mutant strain F-2 correlate with enhanced gluconic acid production and more efficient glucose oxidation metabolism. The regular oscillatory pattern suggests well-regulated metabolic control mechanisms that enable sustained acid production while maintaining cellular viability under acidic conditions. The pH fluctuations between 4.3 and 6.5 represent a broader dynamic range compared to the parent strain, indicating more active metabolic turnover and enhanced acid production capacity.

[0259]

[0152] The oscillatory pH behavior exhibited by the mutant strain F-2 reflects cyclical metabolic activity where acid production phases alternate with buffering or partial neutralization phases. This dynamic pattern indicates active metabolic regulation and suggests enhanced tolerance to acidic conditions that accumulate during gluconic acid production. The rhythmic pH fluctuations demonstrate more vigorous metabolic activity compared to the more gradual and less pronounced pH changes observed in the parent strain.

[0260] Final pH Conditions and Fermentation Completion 202421061133

[0261]

[0153] The mutant strain F-2 achieves a final pH of approximately 5.01 at 214 hours, indicating sustained acidification and successful gluconic acid accumulation throughout the fermentation period. The final pH value is slightly lower than that achieved by the parent strain, reflecting enhanced acid production capability and more complete substrate conversion to gluconic acid.

[0262]

[0154] The pH profiles demonstrate that the mutant strain F-2 maintains more dynamic and responsive metabolic activity throughout the fermentation period, with enhanced acidification capability and superior pH control mechanisms. The oscillatory pH pattern between 4.3 and 6.5 from 94 to 195 hours represents a distinctive characteristic of the mutant strain that correlates with enhanced gluconic acid production performance and improved metabolic efficiency under controlled fermentation conditions.

[0263] Gluconic Acid Yield Performance and Production Analysis

[0264]

[0155] The gluconic acid production performance of mutant strain MTCC 25761 demonstrates substantial yield improvements compared to the parent strain Aspergillus niger ATCC 9142 under identical fermentation conditions. The quantitative assessment employs enzymatic assay kit methodology to provide precise measurement of gluconic acid concentrations at critical time points during the fermentation process, enabling accurate comparison of production capabilities and metabolic efficiency between the parent and mutant strains.

[0265]

[0156] Figure 12 illustrates the gluconic acid production profiles for both strains measured at two critical time points during the fermentation process: 166 hours and 214 hours. The enzymatic kit analysis reveals distinct performance differences between the parent strain F-l and the mutant strain F-2 (DPPL 017 (1)), with the mutant strain achieving substantially higher gluconic acid concentrations, particularly during the later stages of fermentation. 202421061133

[0266] Early Production Phase Performance Comparison

[0267]

[0157] At 166 hours of fermentation, both strains demonstrate comparable gluconic acid production levels, with the parent strain F-l achieving 65.225 g / L and the mutant strain F-2 producing 66.015 g / L. The marginal difference of 0.79 g / L represents approximately 1.2% improvement by the mutant strain during this early production phase. The similar production levels at 166 hours indicate that both strains establish gluconic acid biosynthesis pathways with comparable efficiency during the initial and intermediate fermentation phases.

[0268]

[0158] The comparable early-phase performance suggests that fundamental metabolic pathways for glucose oxidation and gluconic acid formation are established at similar rates in both strains. The enzymatic machinery responsible for glucose oxidase activity and subsequent gluconic acid formation appears to reach similar activity levels by 166 hours, indicating that the primary advantages of the mutant strain become apparent during extended fermentation periods.

[0269] Enhanced Late-Stage Production Performance

[0270]

[0159] The performance differential becomes dramatically pronounced at 214 hours, where the mutant strain F-2 achieves 164.045 g / L compared to 119.776 g / L produced by the parent strain F-L This represents a substantial improvement of 44.269 g / L, corresponding to approximately 37% enhancement in gluconic acid yield. The significant yield improvement during the later fermentation phase indicates enhanced metabolic persistence and superior acid production capability under the challenging conditions of prolonged fermentation.

[0271]

[0160] The 37% yield improvement achieved by the mutant strain F-2 represents a substantial enhancement in production efficiency that directly correlates with the enhanced sugar utilization performance observed throughout the fermentation period. The superior gluconic acid yield corresponds to the 4.0% 202421061133

[0272] improvement in final sugar utilization demonstrated by the mutant strain, indicating more efficient substrate conversion and enhanced metabolic flux through gluconic acid biosynthesis pathways.

[0273] Metabolic Persistence and Production Sustainability

[0274]

[0161] The dramatic increase in gluconic acid production by the mutant strain F-2 from 66.015 g / L at 166 hours to 164.045 g / L at 214 hours represents a 148.5% increase during the final 48-hour period. In contrast, the parent strain F-l achieves an 83.6% increase from 65.225 g / L to 119.776 g / L during the same period. The superior late-stage production performance indicates enhanced metabolic persistence and improved tolerance to the acidic fermentation environment that accumulates during extended gluconic acid production.

[0275]

[0162] The sustained high-level production capability demonstrated by the mutant strain F-2 correlates directly with the dynamic pH oscillations observed between 94 and 195 hours of fermentation. The oscillatory pH pattern between 4.3 and 6.5 indicates active metabolic regulation and enhanced tolerance to acidic conditions, enabling continued glucose oxidation and gluconic acid accumulation under challenging environmental conditions.

[0276] TLC Analysis Correlation and Qualitative Confirmation

[0277]

[0163] The TLC analysis results provide qualitative confirmation of the enhanced gluconic acid production capabilities demonstrated through enzymatic kit quantification. The mutant strain F-2 exhibits consistently stronger TLC spot intensities compared to the parent strain F-l, particularly during the later fermentation phases corresponding to the time periods where the greatest yield improvements are observed through enzymatic analysis. 202421061133

[0278]

[0164] At 214 hours, the TLC spot intensity for the mutant strain F-2 exceeds 10% standard gluconic acid match, indicating substantial acid accumulation and confirming the high production levels measured through enzymatic assay. The intense TLC spot development correlates directly with the 164.045 g / L gluconic acid concentration achieved by the mutant strain, providing visual confirmation of the enhanced production capability.

[0279]

[0165] The correlation between TLC spot intensity and enzymatic kit measurements validates the enhanced gluconic acid production performance of the mutant strain across different analytical methodologies. The visual confirmation provided by TLC analysis demonstrates consistency between qualitative and quantitative assessment methods, supporting the reliability of the yield improvement data.

[0280] Integrated Performance Analysis and Metabolic Correlation

[0281]

[0166] The enhanced gluconic acid production performance of mutant strain F-2 correlates directly with the superior sugar utilization efficiency and dynamic pH behavior observed throughout the fermentation period. The 44.0% final sugar utilization achieved by the mutant strain compared to 42.3% for the parent strain provides the substrate conversion foundation for enhanced acid production. The more efficient substrate utilization translates directly to increased gluconic acid yield through improved metabolic flux and substrate conversion efficiency.

[0282]

[0167] The oscillatory pH dynamics exhibited by the mutant strain F-2, with deeper pH troughs reaching 4.3 compared to 4.5 for the parent strain, indicate more aggressive acid production and enhanced glucose oxidase activity. The dynamic pH behavior reflects active metabolic regulation that enables sustained acid production while maintaining cellular viability under increasingly acidic conditions. 202421061133

[0283]

[0168] The combination of enhanced sugar utilization, dynamic pH regulation, and superior acid tolerance enables the mutant strain F-2 to achieve the substantial 37% improvement in gluconic acid yield. The integrated metabolic enhancements provide synergistic effects that collectively result in superior fermentation performance and establish strain MTCC 25761 as a significantly improved production organism for gluconic acid biosynthesis applications.

[0284]

[0169] The mutant strain DPPL 017 (1) development system combines UV mutagenesis, selective screening, and optimized fermentation to enhance gluconic and citric acid production while maintaining industrial safety standards. UV mutagenesis employs 254 nm wavelength for 19 minutes at 20 cm distance with 7

[0285] continuous stirring of lx 10 CFU / mL spore suspension to generate reproducible genetic modifications. Primary BCG screening identifies acid-producing mutants through pH-sensitive indicator zones, with acid unitage calculation normalizing production relative to colony size. Secondary validation uses enzymatic assays, TLC, and spectrophotometric analysis to confirm organic acid production capabilities and eliminate false positives. Optimized fermentation media employ 220 g / L dextrose for gluconic acid production and 25 g / L starch-dextrose combination for citric acid production with balanced nutrients. Fermentation conditions maintain 30°C temperature and 25 RPM agitation to support enhanced metabolic characteristics while preventing pellet damage. Extended fermentation periods of 214 hours for gluconic acid and 382 hours for citric acid enable complete substrate utilization and maximum yield recovery. The mutant strain demonstrates 44.0% sugar utilization efficiency, dynamic pH regulation, and enhanced acid tolerance compared to the parent strain. The genetic modifications present in strain DPPL 017 (1) at the identified sequence positions contribute to the enhanced metabolic performance through improved enzyme expression, substrate utilization efficiency, and regulatory control mechanisms. 202421061133

[0286]

[0170] The specific nucleotide alterations at positions corresponding to SEQ ID NOs: 26886, 26904, 26906, 26909, 26916, 26927, 26935, 27080, 27104, 27116, 27123, 27128, 27150, 27170, 27171, 27213, 27225, 27262, 27273, 27275, 27280, 27291, 27292, 27294, 27295, 27339, 27341, 27399, 27401, 27413, 27447, 27450, 27452, 27466, 27493, 27502, 27516, 27532, 27663, 27690, 27698, 27734, 27739, 27885, 27957, 28140, 28141, and 28147, collectively create the enhanced production phenotype while maintaining genetic stability through multiple subculture passages. Standardized protocols enable reproducible strain development addressing commercial requirements for enhanced organic acid production. Commercial viability is demonstrated through 1.37-fold gluconic acid yield improvement, achieving 164.045 g / L compared to 119.776 g / L for the parent strain (37% improvement), and 1.29-fold citric acid yield improvement, achieving 0.506 g / L compared to 0.391 g / L for the parent strain (29% improvement).

[0287]

[0171] Enhanced production provides economic advantages through improved substrate conversion, reduced raw material costs, and increased capacity within existing infrastructure. The reproducible methodology enables systematic generation of additional mutant variants while maintaining safety characteristics and regulatory compliance. The integrated system demonstrates synergistic interaction between mutagenesis, screening, and fermentation optimization to achieve superior organic acid production performance.

[0288]

[0172] Features of any of the examples or embodiments outlined above may be combined to create additional examples or embodiments without losing the intended effect. It should be understood that the description of an embodiment or example provided above is by way of example only, and various modifications could be made by one skilled in the art. Furthermore, one skilled in the art will recognise that numerous further modifications and combinations of various aspects are possible. Accordingly, the described aspects are intended to encompass all such alterations, modifications, and variations that fall within the scope of the appended claims.

Claims

202421061133We claim:

1. A mutant strain of Aspergillus niger having accession number MTCC 25761 derived from parent strain Aspergillus niger ATCC 9142 through UV mutagenesis, wherein the mutant strain demonstrates enhanced production capabilities for both citric acid and gluconic acid compared to the parent strain.

2. The mutant strain as claimed in claim 1, wherein theUV mutagenesis is performed at 254 nm wavelength for 13-27 minutes with constant stirring.

3. The mutant strain as claimed in claim 1, comprising genetic modifications at sequence positions corresponding to SEQ ID NOs: 26886, 26904, 26906, 26909, 26916, 26927, 26935, 27080, 27104, 27116, 27123, 27128, 27150, 27170, 27171, 27213, 27225, 27262, 27273, 27275, 27280, 27291, 27292, 27294, 27295, 27339, 27341, 27399, 27401, 27413, 27447, 27450, 27452, 27466, 27493, 27502, 27516, 27532, 27663, 27690, 27698, 27734, 27739, 27885, 27957, 28140, 28141, and 28147.

4. The mutant strain as claimed in claim 1, wherein the gluconic acid production yield is 1.37-fold higher than the parent strain, achieving 119.78 to 164.045 g / L, and the citric acid production yield is 1.29-fold higher than the parent strain, achieving 0.463 g / L 0.506 g / L.

5. The mutant strain as claimed in claim 1, wherein the mutant strain demonstrates enhanced tolerance to acidic fermentation conditions, achieving minimum pH values of 0.5-1.0 during citric acid production and minimum pH values of 4.0-5.0 during gluconic acid production.

6. A method for producing citric acid or gluconic acid with the mutant strain of claim 1, comprising:((a) subjecting the parent strain to UV mutagenesis to generate mutant variants; (b) preparing a spore suspension of the mutant strain for gluconic acid production or for citric acid production;202421061133(c) inoculating the spore suspension into a fermentation medium under sterile conditions;(d) fermenting the inoculated medium under controlled conditions to produce citric acid or gluconic acid; and(e) recovering the produced organic acid from the fermentation broth.

7. The method as claimed in claim 6, wherein the fermentation is conducted at a temperature of 30°C - 35°C with agitation at 23 - 27 RPM, and screening acidproducing mutants using Bromocresol Green plates to identify colonies with enhanced acid production capabilities.

8. The method as claimed in claim 6, wherein for gluconic acid production, the fermentation medium comprises dextrose at 180 - 220 g / L as a carbon source and the fermentation duration is 166 h to 214 hours.

9. The method as claimed in claim 6, wherein for citric acid production, the fermentation medium comprises starch at 80 - 120 g / L and dextrose at 20 - 28 g / L as carbon sources and the fermentation duration is 316 h to 382 hours.

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