Aspergillus niger mutant strain for production of citric acid and gluconic acid and methods

Abstract

An Aspergillus niger mutant strain designated MTCC 25759 derived from parent strain Aspergillus niger ATCC 9142 through chemical mutagenesis using ethyl methane sulfonate at 1 mg / mL for 30 to 60 minutes demonstrates enhanced production capabilities for both citric acid and gluconic acid. The mutant strain exhibits genetic modifications at 47 specific sequence positions that contribute to improved metabolic pathways, enhanced substrate utilization efficiency, and increased tolerance to acidic fermentation conditions. The mutant strain achieves gluconic acid production of 198.836 g / L representing a 66% improvement over the parent strain's 119.776 g / L yield, and citric acid production reaching 0.415 g / L with consistent late-phase productivity. The enhanced production capabilities provide economic advantages through improved substrate conversion rates, reduced raw material costs, and increased production capacity within existing fermentation infrastructure while maintaining the Generally Regarded As Safe (GRAS) status for commercial applications.

Description

[0001] 202421061044

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

[0003] [1] The present invention relates to an Aspergillus niger mutant strain deposited with the Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology, Chandigarh and assigned accession number MTCC 25759 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 chemical mutagenesis method using ethyl methane sulfonate. Both the organic acids are industrially important and mainly produced through microbial fermentation.

[0004] BACKGROUND OF INVENTION

[0005] [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. 202421061044

[0006] [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.

[0007] [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.

[0008] [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 202421061044

[0009] 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%.

[0010] [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.

[0011] [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. 202421061044

[0012] OBJECT OF THE INVENTION

[0013] [8] The primary object of the present invention is to provide an Aspergillus niger (MTCC 25759) mutant strain that demonstrates enhanced production capabilities for both citric acid and gluconic acid.

[0014] [9] Another object of the present invention is to provide an Aspergillus niger mutant strain through chemical mutagenesis that exhibits superior organic acid production yields while maintaining stable fermentation characteristics.

[0015]

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

[0016]

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

[0017]

[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.

[0018]

[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.

[0019]

[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.

[0020]

[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. 202421061044

[0021]

[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.

[0022]

[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.

[0023]

[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.

[0024]

[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.

[0025]

[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.

[0026] SUMMARY OF INVENTION

[0027]

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

[0028]

[0022] The present invention provides a mutant strain of Aspergillus niger designated MTCC 25759, derived from parent strain Aspergillus niger ATCC 9142 through chemical mutagenesis using ethyl methane sulfonate at a concentration of 1 mg / mL for 30 to 60 minutes at 30°C with shaking at 150 rpm, wherein the mutant strain demonstrates significantly enhanced production capabilities for both citric acid and gluconic acid. This specific chemical mutagenesis protocol generates targeted mutations that enhance the strain's acidogenic potential while maintaining cell viability and growth characteristics, with the 60-minute exposure duration representing an optimal balance between mutagenic effectiveness and cellular integrity that results in improved acid 202421061044

[0029] production without compromising fermentation performance. The mutant strain MTCC 25759 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 MTCC 25759 demonstrates enhanced production capabilities across extended fermentation periods, with gluconic acid production ranging from 66.015 g / L at 166 hours to 198.836 g / L at 214 hours, representing a 66% improvement over the parent strain under identical conditions. For citric acid production, the mutant strain achieves concentrations ranging from 0.347 g / L at 316 hours to 0.415 g / L at 382 hours, compared to the parent strain.

[0030] BRIEF DESCRIPTION OF FIGURES

[0031]

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

[0032]

[0024] FIG. 1 illustrates pellet morphology scores and starch residue scores over fermentation time for citric acid production for parent strain F-l and mutant strain F-7, according to aspects of the present disclosure.

[0033]

[0025] Fig. 2 illustrates pH changes over fermentation time for citric acid production for Parent strain F-l, according to aspects of the present disclosure.

[0034]

[0026] FIG. 3 illustrates pH changes over fermentation time for citric acid production for mutant strain F-7, according to aspects of the present disclosure.

[0035]

[0027] Fig. 4 illustrates a comparison of pH profiles between citric acid production for parent strain F-l and mutant strain F-7, according to aspects of the present disclosure.

[0036]

[0028] FIG. 5 illustrates citric acid production over time for mutant strain F-7 compared to Parent strain F-l, according to aspects of the present disclosure. 202421061044

[0037]

[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.

[0038]

[0030] FIG. 7 illustrates sugar utilization percentage over fermentation time for gluconic acid production mutant strain F-4, according to aspects of the present disclosure.

[0039]

[0031] FIG. 8 illustrates a comparison of sugar utilization percentages for gluconic acid production between parent strain F-l and mutant strain F-4, according to aspects of the present disclosure.

[0040]

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

[0041]

[0033] FIG. 10 illustrates pH changes over fermentation time for gluconic acid production mutant strain F-4, according to aspects of the present disclosure.

[0042]

[0034] FIG. 11 illustrates a comparison of pH profiles between gluconic acid production for parent strain F-l and mutant strain F-4, according to aspects of the present disclosure.

[0043]

[0035] FIG. 12 illustrates gluconic acid production over time for mutant strain F-4 compared to Parent strain F-l, according to aspects of the present disclosure.

[0044] DETAILED DESCRIPTION

[0045]

[0036] The present disclosure relates to an Aspergillus niger mutant strain for enhanced production of citric acid and gluconic acid. The mutant strain is derived from parent strain Aspergillus niger ATCC 9142 through chemical mutagenesis techniques. In some cases, the mutant strain demonstrates improved production capabilities for both organic acids compared to the parent strain.

[0046]

[0037] Citric acid and gluconic acid represent commercially significant organic acids in global fermentation markets. Citric acid finds widespread 202421061044

[0047] 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 diverse applications, while gluconic acid maintains a substantial market presence.

[0048]

[0038] Current production methods for these organic acids rely 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 provide higher yields and are more economically viable. However, conventional fermentation approaches using wild-type strains face limitations in production efficiency and yield optimization.

[0049]

[0039] The present disclosure addresses the need for improved microbial strains capable of enhanced organic acid production. The approach involves systematic chemical mutagenesis of the parent strain Aspergillus niger ATCC 9142 to generate mutant variants with production characteristics. The mutagenesis process employs controlled chemical exposure to induce genetic modifications that enhance the metabolic pathways responsible for citric acid and gluconic acid biosynthesis.

[0050] Parent Strain

[0051] Spore Suspension Preparation

[0052]

[0040] The parent strain Aspergillus niger ATCC 9142 serves as the starting material for developing enhanced mutant strains with improved organic acid production capabilities. This strain is maintained on Potato Dextrose Agar (PDA) medium under controlled laboratory conditions to ensure consistent growth characteristics and sporulation patterns. Spore suspension preparation begins with harvesting spores from mature Aspergillus niger ATCC 9142 cultures. The 202421061044

[0053] cultures are grown on PDA plates and 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.

[0054] Spore Harvesting Process

[0055]

[0041] 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 maximizes spore release while avoiding damage to the underlying agar medium.

[0056] Spore Dispersion Enhancement

[0057]

[0042] 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 beneficial for accurate counting and consistent inoculum preparation.

[0058]

[0043] 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.

[0059] Spore Concentration Determination

[0060]

[0044] 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 202421061044

[0061] 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. The initial spore concentration typically ranges from 6*107to 7 / I09CFU / mL, depending on the sporulation efficiency of the particular culture. The concentration range reflects natural variation in sporulation density among different culture preparations and growth conditions.

[0062] Spore Suspension Standardization

[0063]

[0045] 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 l><107CFU / mL using sterile normal saline as the diluent. For gluconic acid production experiments, the spore concentration is adjusted to 2*106CFU / mL. For citric acid production studies, the spore concentration is adjusted to 5*107CFU / mL. 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.

[0064] Chemical Mutagenesis Process

[0065]

[0046] The chemical mutagenesis process employs ethyl methane sulfonate at a concentration of 1 mg / mL to induce genetic modifications in Aspergillus niger ATCC 9142 spores with varying treatment durations including 30 minutes (strain code DPPL 008), 60 minutes (strain code DPPL 009). In some cases, the ethyl methane sulfonate stock solution is prepared at 1 mg / mL in sterile distilled water and stored at 4°C. The ethyl methane sulfonate stock solution is handled in a chemical fume hood using gloves and eye protection due to the mutagenic properties of the compound. 202421061044

[0066] Mutagenesis Setup

[0067]

[0047] The mutagenesis setup is conducted in sterile 250 mL conical flasks under controlled conditions. In some cases, each flask contains 9 mL of Vogel's Minimal Medium, 1 mL of spore suspension at l*107CFU / mL, and 1 mL of EMS stock solution. The EMS:spore ratio is maintained at 1:9 throughout the mutagenesis procedure. The Vogel's Minimal Medium provides a suitable environment for spore viability during the chemical treatment process.

[0068] EMS Treatment Protocol

[0069]

[0048] The EMS treatment protocol involves incubation at 30°C with shaking at 150 rpm for a 60-minute exposure duration. In some cases, the controlled temperature and agitation conditions ensure uniform distribution of the chemical mutagen throughout the spore suspension. The 60-minute exposure duration provides sufficient time for EMS to induce genetic modifications while maintaining spore viability for subsequent processing steps.

[0070] Termination and Washing Procedure

[0071]

[0049] The termination procedure involves immediate placement of the flasks on ice to stop the mutagenic reaction. In some cases, the rapid cooling halts further chemical modification of the spores and preserves the induced mutations. The treated spores are washed twice with sterile saline through centrifugation at 5000 rpm for 5 minutes to remove residual EMS. The washing procedure eliminates excess chemical mutagen that could interfere with subsequent culturing steps.

[0072] Dilution and Plating

[0073]

[0050] The dilution and plating procedure employs 100-fold serial dilutions of the washed spore suspension. In some cases, the diluted suspension is plated onto Potato Dextrose Agar (PDA) plates supplemented with 2% Triton X-100. 202421061044

[0074] The Triton X-100 functions as a colony restrictor to limit colony expansion and facilitate isolation of individual colonies. The plating volume is 0.1 mL of each dilution to achieve appropriate colony density for selection purposes.

[0075] Incubation Conditions

[0076]

[0051] The incubation conditions involve maintaining the plates at 30°C for 4-6 days following inoculation. In some cases, daily observation of the plates enables monitoring of colony development, morphology assessment, and identification of sporulation characteristics. The incubation period allows for adequate colony development while preventing over-maturation that could complicate subsequent handling procedures. The resulting colonies exhibit various morphological characteristics that indicate successful mutagenesis and provide candidates for further screening procedures.

[0077] Screening Methodology

[0078]

[0052] The primary screening methodology employs Bromocresol Green plate assays to systematically identify chemically-induced mutant colonies with enhanced acid production capabilities. Bromocresol Green functions as a pH-sensitive indicator dye that undergoes color transition from blue-green at pH values above 4.8 to yellow at pH values below 3.8. Acid-producing organisms create localized pH reduction in the surrounding medium, resulting in formation of distinct yellow halos around colonies against the blue-green background.

[0079] BCG Plate Preparation

[0080]

[0053] The BCG plate preparation involves formulating a medium containing Potato Dextrose Agar supplemented with Bromocresol Green indicator and additional agar for appropriate gel strength. For preparation of 10 screening plates requiring 400 mL of medium, the composition includes 15.6 g Potato Dextrose Agar powder, 6 g additional agar powder, and 0.4 g Bromocresol Green dye. The medium preparation process involves dissolving the PDA and additional agar 202421061044

[0081] components in distilled water with continuous stirring. 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 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.

[0082] Colony Inoculation Protocol

[0083]

[0054] The colony inoculation protocol involves selecting individual colonies from chemically-treated PDA plates following the incubation period. Colony selection focuses on isolated, well-developed colonies that exhibit normal morphological characteristics. Each selected colony is transferred to duplicate BCG-PDA plates using sterile inoculation loops to ensure reproducible results. 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. 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 maintains 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.

[0084] Visual Assessment and Zone Measurement

[0085]

[0055] Following incubation, plates are examined for 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. Colonies lacking visible yellow zones or exhibiting only faint discoloration are 202421061044

[0086] categorized as non-producers or weak acid producers. Colonies demonstrating clear, intense, and measurable yellow zones are selected for continued characterization.

[0087]

[0056] 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. The zone of acid is calculated using the formula: Zone of Acid (mm) = Zonal Diameter - Colony Diameter, providing a measure of the acid diffusion area around each colony.

[0088] Acid Unitage Calculation

[0089]

[0057] 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:

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

[0091] where higher values indicate greater acid production relative to colony size. This normalization approach ensures that mutants with enhanced acid production capabilities are identified rather than selecting colonies with larger overall zones resulting from increased colony size.

[0092] Mutant Selection Criteria

[0093]

[0058] The mutant selection criteria employ systematic evaluation of multiple performance parameters to identify superior acid-producing variants from the chemically-treated population. The primary selection criterion involves acid unitage values exceeding those of the parent strain Aspergillus niger ATCC 9142 under standardized screening conditions. Mutant candidates demonstrating enhanced acid unitage values are prioritized for further evaluation, with particular emphasis on strains achieving superior performance metrics. The selection process evaluates zone clarity and intensity as qualitative indicators of acid production capability. Mutant colonies producing distinct, well-defined yellow 202421061044

[0094] zones with sharp boundaries against the blue-green background are preferred over those exhibiting diffuse or poorly defined acidification patterns. The intensity of yellow coloration correlates with the degree of pH reduction and indicates the concentration of organic acids secreted into the surrounding medium. Colony morphology assessment forms an additional selection criterion, with preference given to mutants maintaining normal growth characteristics while demonstrating enhanced acid production. Colonies exhibiting abnormal morphology, irregular growth patterns, or signs of cellular stress are excluded from further consideration to ensure that enhanced acid production results from improved metabolic efficiency rather than cellular dysfunction.

[0095]

[0059] Reproducibility across duplicate plates serves as a validation criterion for mutant selection. Candidates showing consistent acid zone formation and similar acid unitage values across replicate plates demonstrate stable genetic modifications and reliable performance characteristics. Mutants exhibiting significant variation between duplicates may indicate unstable mutations or environmental sensitivity that could compromise commercial viability.

[0096]

[0060] The selection methodology prioritizes mutants demonstrating rapid zone development within the incubation period, indicating efficient establishment of acid production pathways. Early zone formation suggests enhanced enzymatic activity and improved metabolic flux through acidogenic pathways compared to slower-developing variants. Growth vigor assessment ensures that selected mutants maintain adequate biomass formation while achieving enhanced acid production. Mutants showing severely reduced colony size or poor growth characteristics are excluded, as commercial applications require strains capable of supporting both adequate biomass development and high acid yields.

[0097]

[0061] The final selection criterion involves comparative evaluation against multiple mutant candidates to identify the strain demonstrating the optimal combination of high acid unitage values, consistent performance, normal morphology, and robust growth characteristics. This comprehensive evaluation 202421061044

[0098] approach ensures selection of mutants with superior commercial potential for organic acid production applications.

[0099] Plate Composition and Preparation

[0100]

[0062] The secondary screening plates employ specialized medium formulations designed to support enhanced acid production while facilitating accurate detection and measurement. The plate composition includes Potato Dextrose Agar as the base medium at 39 g / L, supplemented with additional agar at 15 g / L to provide appropriate gel strength for zone formation and measurement. Bromocresol Green indicator is incorporated at 1 g / L to enable visual detection of acid production through pH-sensitive color changes.

[0101]

[0063] The medium preparation process involves sequential dissolution of components to prevent precipitation and ensure homogeneous distribution. The PDA and additional agar components are dissolved in distilled water with continuous stirring at elevated temperature to achieve complete dissolution. The Bromocresol Green indicator is separately dissolved in warm distilled water to ensure complete solubilization before incorporation into the main medium. 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 indicator system.

[0102]

[0064] Following sterilization, the medium is cooled to 45-50°C before pouring into sterile Petri plates under aseptic conditions. The pouring volume is standardized at 40 mL per plate to ensure consistent medium depth and uniform zone development characteristics. The plates are allowed to solidify completely at room temperature before use, with proper labeling to identify different mutant strains and experimental conditions.

[0103] Colony Inoculation Protocol

[0104]

[0065] The colony inoculation protocol for secondary screening involves systematic transfer of selected mutant candidates from primary screening plates to fresh BCG-PDA plates for quantitative evaluation. Colony selection focuses on 202421061044

[0105] mutants that demonstrated superior acid unitage values during primary screening, with particular emphasis on strains showing consistent performance across duplicate plates. The inoculation procedure employs sterile inoculation loops to transfer small portions of selected colonies to the center of fresh BCG-PDA plates. Each mutant strain is inoculated onto duplicate plates to ensure reproducible results and enable statistical validation of performance characteristics. The parent strain Aspergillus niger ATCC 9142 is included as a reference control, with duplicate plates inoculated under identical conditions to provide baseline measurements for comparative evaluation. The inoculation technique involves gentle transfer of fungal material from the selected colony to avoid excessive biomass transfer that could influence zone development patterns. The inoculated material is placed at the center of each plate to enable uniform radial growth and symmetric zone formation. Sterile technique is maintained throughout the inoculation process to prevent cross-contamination between different mutant strains.

[0106] Incubation Conditions and Timeline

[0107]

[0066] The incubation conditions for secondary screening are standardized to ensure consistent environmental parameters across all test plates and enable accurate comparative evaluation of different mutant strains. The inoculated plates are incubated at 30°C ± 2°C in a controlled environment incubator to maintain temperature stability throughout the evaluation period. The incubation timeline extends for 70-75 hours, corresponding to approximately 3 days of growth, which provides sufficient time for colony development and acid zone formation while preventing over-maturation that could complicate measurement procedures. The plates are positioned in an upright orientation within the incubator to prevent condensation accumulation on the agar surface that could interfere with zone visualization and measurement.

[0108]

[0067] Daily monitoring is conducted throughout the incubation period to track colony development, zone formation, and overall plate condition. The monitoring schedule includes visual inspection at 24-hour intervals to assess 202421061044

[0109] growth progress and identify any contamination or abnormal development patterns. Temperature logging is maintained throughout the incubation period to ensure consistent environmental conditions.

[0110] Visual Observation and Assessment Criteria

[0111]

[0068] The visual observation methodology employs systematic examination of incubated plates to identify and evaluate acid zone formation around fungal colonies. The assessment is conducted under standardized lighting conditions to ensure consistent visualization of color changes and zone boundaries. Plates are examined against a neutral background to enhance contrast between the yellow acid zones and the blue-green medium background. The assessment criteria include evaluation of zone presence, intensity, clarity, and symmetry around each colony. Positive acid production is indicated by the formation of distinct yellow halos surrounding the fungal colonies, representing localized pH reduction due to organic acid secretion. The intensity of yellow coloration is assessed qualitatively, with stronger coloration indicating higher acid concentrations and more efficient acid production. Zone clarity assessment focuses on the sharpness of boundaries between the yellow acid zone and the surrounding blue-green medium. Well-defined zones with sharp boundaries indicate efficient acid diffusion and stable pH gradients, while diffuse or poorly defined zones may suggest weaker acid production or unstable fermentation conditions. Zone symmetry evaluation assesses the uniformity of acid zone formation around the colony perimeter, with symmetric zones indicating consistent acid production and uniform growth characteristics.

[0112] Quantitative Zone Measurement

[0113]

[0069] The quantitative zone measurement protocol employs precise measurement techniques to determine colony diameter, zonal diameter, and calculated acid zone dimensions for each test strain. Measurements are conducted using calibrated rulers or digital calipers to ensure accuracy and reproducibility across different plates and experimental conditions. Colony diameter measurement involves determining the maximum diameter of the visible fungal 202421061044

[0114] growth area, excluding any surrounding acid zones. Multiple diameter measurements are taken across different orientations to account for any asymmetry in colony shape, with the average value used for subsequent calculations. The colony diameter represents the actual biomass area and serves as the baseline for normalization calculations. Zonal diameter measurement encompasses the total diameter including both the colony and the surrounding acid zone. The measurement is taken from the outermost edge of the yellow acid zone to the opposite edge, passing through the colony center. Multiple measurements are taken to ensure accuracy, with particular attention to identifying the true zone boundary where color transition occurs. The zone of acid calculation employs the formula: Zone of Acid (mm) = Zonal Diameter - Colony Diameter, providing a quantitative measure of the acid diffusion area around each colony. This calculated value represents the effective area of acid production and serves as a direct indicator of acid secretion capability.

[0115]

[0070] The acid unitage calculation provides a normalized measure of acid production efficiency using the formula: Acid Unitage = Zone of Acid (mm) / Colony Diameter (mm). This normalization approach accounts for differences in colony size among different mutant strains and enables fair comparison of acid production capabilities. Higher acid unitage values indicate superior acid production efficiency relative to biomass formation, identifying mutants with enhanced metabolic performance for organic acid biosynthesis.

[0116] Mutant Strain

[0117]

[0071] Among the screened mutant candidates, strain DPPL 009 demonstrated superior acid production performance with an acid unitage value of 1.89. This mutant exhibited a colony diameter of 38 mm and a total zonal diameter of 72 mm, resulting in an acid zone of 34 mm. The 34 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. The acid unitage value of 1.89 for strain DPPL 009 exceeds the typical range of 1.73-1.80 observed for the parent strain, indicating enhanced 202421061044

[0118] acidogenic potential resulting from the ethyl methane sulfonate mutagenesis treatment. This quantitative improvement in acid production capability, combined with the substantially larger acid zone, establishes strain DPPL 009 as a promising candidate for further fermentation studies and commercial development.

[0119]

[0072] The selection of strain DPPL 009 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. The mutant strain demonstrates consistent performance characteristics across duplicate screening plates, with reproducible zone formation and stable acid production patterns that indicate reliable genetic modifications.

[0120]

[0073] The enhanced performance characteristics of strain DPPL 009 result from the 60-minute exposure to ethyl methane sulfonate at 1 mg / mL concentration, which induced beneficial genetic modifications that improve metabolic flux through organic acid biosynthesis pathways. The mutant strain maintains normal colony morphology and growth vigor while demonstrating enhanced acid secretion capabilities, indicating that the genetic modifications enhance metabolic efficiency without compromising cellular integrity.

[0121]

[0074] The superior acid zone formation exhibited by strain DPPL 009 correlates with enhanced glucose oxidase activity and improved oxidative metabolism pathways that facilitate more efficient conversion of glucose substrates to organic acid products. The mutant strain demonstrates enhanced tolerance to acidic conditions that accumulate during organic acid production, enabling sustained metabolic activity under challenging fermentation environments.

[0122] Secondary Screening method

[0123]

[0075] 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 202421061044

[0124] 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.

[0125] Enzymatic Assay Methods

[0126]

[0076] 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. Gluconic Acid Enzymetic Detection

[0127]

[0077] 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.

[0128] Citric Acid Enzymetic Detection

[0129]

[0078] 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 202421061044

[0130] 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.

[0131] Thin Layer Chromatography Analysis

[0132]

[0079] Thin Layer Chromatography provides qualitative detection of organic acid production through separation and visualization on chromatographic plates. TLC analysis employs silica gel plates with fermentation samples applied alongside standard solutions of gluconic acid and citric acid, developed using appropriate mobile phase solvents and visualized through chemical staining for organic acid identification by comparing migration distances with standard reference compounds.

[0133] Sugar Quantification

[0134]

[0080] The DNS A method quantifies reducing sugar concentrations through reduction of 3,5-dinitrosalicylic acid under alkaline conditions, with fermentation samples diluted, mixed with DNS A reagent, heated, and measured spectrophotometrically at 540 nm to calculate sugar utilization percentage.

[0135] Iodine Starch Test

[0136]

[0081] The iodine starch test provides qualitative assessment of starch utilization based on blue-black color formation when iodine reacts with starch, with color intensity decreasing as starch is hydrolyzed during fermentation and complete utilization indicated by absence of blue coloration and development of brown coloration similar to the water blank control. 202421061044

[0137] Result Interpretation and Starch Utilization Assessment

[0138]

[0082] The secondary screening employs enzymatic assays, TLC analysis, DNS A method, and iodine starch tests to quantitatively validate enhanced organic acid production capabilities identified through primary BCG screening. TLC analysis provides qualitative confirmation through chromatographic separation and visual spot intensity comparison with standard reference compounds, validating enzymatic assay results. TheDNSA method quantifies reducing sugar concentrations to calculate percentage substrate conversion, providing metabolic efficiency indicators that correlate with organic acid production capabilities. The iodine starch test provides qualitative assessment through colorimetric detection, with blue-black coloration indicating residual starch and brown coloration indicating complete utilization. Starch utilization employs a 0-3 scoring system where lower scores represent more complete hydrolysis, with periodic sampling enabling assessment of enzymatic activity and substrate conversion efficiency. Integrated analysis across multiple analytical techniques validates screening reliability and ensures consistent identification of enhanced production characteristics for superior mutant candidate selection. 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, ensuring accurate identification of mutant strains with enhanced production characteristics.

[0139] Sequence Mutation Analysis

[0140]

[0083] Whole genome sequencing results submitted with this specification demonstrate that analysis of the mutant strain Aspergillus niger (009) MTCC 25759 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 alterations comprising nucleotide additions, deletions, or substitutions provide 202421061044

[0141] 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.

[0142] Reference Position Identification

[0143]

[0084] The mutant sequence identifiers corresponding to reference positions are designated as 47 specific SEQ ID NOs: 24337, 24344, 24353, 24355, 24366, 24374, 24502, 24522, 24525, 24526, 24530, 24557, 24586, 24592, 24620, 24625, 24630, 24632, 24640, 24646, 24647, 24648, 24653, 24728, 24729, 24730, 24769, 24800, 24802, 24808, 24821, 24843, 24923, 24949, 24951, 24999, 25000, 25001, 25088, 25107, 25108, 25113, 25116, 25139, 25142, 25312, and 25332, 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 25759.

[0144] Metabolic Pathway Enhancement

[0145]

[0085] The sequence mutations identified in strain MTCC 25759 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.

[0146] Strain Stability and Inheritance 202421061044

[0147]

[0086] The mutant strain MTCC 25759 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 25759 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.

[0148] Phenotypic Expression of Genetic Changes

[0149]

[0087] The genetic mutations present in strain MTCC 25759 result in measurable phenotypic changes that manifest as improved organic acid production capabilities. The mutant strain exhibits enhanced gluconic acid production reaching concentrations of 198.836 g / L compared to 119.776 g / L achieved by the parent strain under identical fermentation conditions, representing a 66% 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 0.415 g / L compared to 0.391 g / L for the parent strain at 382 hours of fermentation. The enhanced production capability is maintained throughout extended fermentation periods, indicating improved metabolic persistence and acid tolerance. The mutant strain MTCC 25759 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.

[0150] Pellet Morphology Stability 202421061044

[0151]

[0088] The mutant strain MTCC 25759 maintains stable pellet morphology during submerged fermentation processes, contributing to improved oxygen transfer efficiency and enhanced substrate accessibility. In some cases, the stable pellet morphology results from modified cell wall composition or growth regulation mechanisms that prevent excessive pellet disintegration during extended fermentation periods. The maintained pellet structure facilitates consistent fermentation performance and reproducible production yields across multiple fermentation batches.

[0152] Glucose Oxidase Activity Enhancement

[0153]

[0089] The mutant strain MTCC 25759 demonstrates enhanced glucose oxidase activity compared to the parent strain Aspergillus niger ATCC 9142. The enhanced glucose oxidase activity enables more efficient conversion of glucose substrates to gluconic acid through improved enzymatic catalysis. In some cases, the mutant strain exhibits improved metabolic flux through oxidative pathways, facilitating more efficient substrate utilization during fermentation processes. The enhanced oxidative metabolism contributes to higher organic acid yields and improved fermentation performance.

[0154] Pellet Morphology Characteristics

[0155]

[0090] The mutant strain exhibits stable pellet morphology throughout fermentation processes. Referring to Fig. 1, the pellet morphology characteristics are monitored over fermentation time periods extending from 118 to 220 hours. The mutant strain forms compact, spherical, uniform pellets that favor better oxygen diffusion and mass transfer compared to irregular dispersed mycelial forms. In some cases, the stable pellet structure maintains higher morphology scores throughout extended fermentation periods, with gradual decline patterns that preserve structural integrity longer than conventional strains.

[0156]

[0091] The uniform pellet morphology enhances oxygen transfer efficiency during aerobic fermentation processes. The compact spherical structure provides improved surface area to volume ratios, facilitating better gas exchange and 202421061044

[0157] nutrient distribution throughout the fermentation medium. In some cases, the stable pellet formation prevents excessive filamentous growth that can reduce fermentation efficiency and complicate downstream processing operations.

[0158] Acid Tolerance and pH Stability

[0159]

[0092] The mutant strain shows enhanced tolerance to acidic fermentation conditions. The enhanced acid tolerance enables the strain to maintain metabolic activity under low pH conditions that typically inhibit conventional fungal strains. In some cases, the mutant strain maintains consistent pH profiles that facilitate organic acid accumulation while preserving cellular viability. The pH stability supports sustained fermentation performance throughout extended production periods.

[0160] Reduced Byproduct Formation

[0161]

[0093] The mutant strain reduces formation of unwanted byproducts during fermentation processes. The reduced byproduct formation improves downstream processing efficiency by minimizing purification requirements and separation steps. In some cases, the cleaner fermentation profile enhances product purity and reduces processing costs associated with byproduct removal. The improved metabolic selectivity directs carbon flux toward target organic acid production rather than alternative metabolic pathways.

[0162] Enhanced Substrate Conversion Efficiency

[0163]

[0094] The mutant strain demonstrates enhanced substrate conversion efficiency. The improved conversion efficiency eliminates the need for expensive enzyme supplementation that is required by conventional fermentation methods. In some cases, the enhanced enzymatic activity provides sufficient catalytic capacity for complete substrate utilization without external enzyme additions. The reduced enzyme supplementation requirements lower production costs and simplify fermentation protocols.

[0164] Nitrogen Utilization Efficiency 202421061044

[0165]

[0095] The mutant strain demonstrates improved nitrogen utilization efficiency compared to the parent strain. The enhanced nitrogen metabolism reduces requirements for expensive nitrogen sources during fermentation processes. In some cases, the improved nitrogen utilization enables the use of lower-cost nitrogen supplements while maintaining adequate biomass formation and enzyme synthesis. The enhanced nitrogen efficiency lowers overall production costs by reducing raw material expenses and improving economic viability of fermentation operations.

[0166] Citric Acid Production

[0167]

[0096] The fermentation medium for citric acid production employs a carefully balanced composition of carbon sources, nitrogen sources, and mineral nutrients to support growth and acid biosynthesis by the mutant strain. The medium composition is formulated to promote citric acid accumulation while maintaining nutritional balance for sustained fermentation performance.

[0168] Medium Composition

[0169]

[0097] The carbon source component consists of starch at a concentration of 100 g / L, supplemented with dextrose at 25 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. The starch-dextrose combination enables efficient substrate utilization and enhanced citric acid yields.

[0170]

[0098] The nitrogen source is provided by ammonium sulfate 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 202421061044

[0171] nucleic acid synthesis and energy metabolism while contributing to pH buffering capacity. Magnesium sulfate heptahydrate at 1 g / L supplies magnesium ions for enzyme cofactor functions, particularly for enzymes involved in organic acid biosynthesis pathways.

[0172] Medium Preparation Protocol

[0173]

[0099] The fermentation medium preparation involves separate preparation of different nutrient components to prevent precipitation and ensure complete dissolution. Flask-A contains the starch and dextrose components dissolved in distilled water in a 500 mL flask. Flask-B contains the potassium dihydrogen phosphate prepared separately in a 250 mL flask. Flask-C contains the remaining nutrients dissolved together in a separate 250 mL flask. This three-flask distribution system prevents component interactions that could lead to precipitation or reduced nutrient availability.

[0174] pH Control and Fermentation Conditions

[0175]

[0100] 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 conditions for acid accumulation while inhibiting competing metabolic pathways. 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 for citric acid stability and recovery.

[0176] Inoculation and Operating Parameters

[0177]

[0101] The fermentation conditions are specifically optimized to support the metabolic requirements of strain MTCC 25759 for efficient citric acid biosynthesis. The spore inoculum preparation involves standardizing the concentration at 5*107CFU / mL, which provides adequate spore density for rapid 202421061044

[0178] culture establishment while avoiding excessive initial biomass that could compete with acid production during early fermentation phases. The standardized inoculum concentration ensures consistent fermentation initiation across different production batches and enables reproducible comparison of fermentation performance under identical starting conditions.

[0179]

[0102] The inoculum volume of 2.5 mL is added to 250 mL of fermentation medium under sterile conditions to prevent contamination. The fermentation conditions are specifically designed to maintain controlled environmental parameters that support the enhanced metabolic characteristics of the mutant strain while maximizing citric acid yields through optimal growth and production conditions.

[0180] Temperature and Agitation Control

[0181]

[0103] The temperature is maintained at 30°C throughout the entire production period, which represents the optimal growth temperature for Aspergillus niger strains. This temperature provides favorable conditions for enzymatic reactions involved in citric acid biosynthesis while supporting efficient substrate metabolism and maintaining enzyme stability and catalytic activity for the metabolic pathways that convert carbon sources to citric acid.

[0182]

[0104] Agitation is provided through rotary shaking at 25 RPM using a rotary shaker system throughout the fermentation period. The moderate agitation speed ensures adequate mixing while providing sufficient oxygen transfer to support aerobic metabolism. The 25 RPM agitation rate provides an optimal balance between oxygen transfer and mechanical stress on fungal biomass while preventing excessive shear forces that could damage fungal pellets or disrupt optimal pellet morphology.

[0183] Fermentation Duration and Volume

[0184]

[0105] 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 202421061044

[0185] extended fermentation period. The substantial headspace volume facilitates efficient oxygen transfer from the gas phase to the liquid medium, supporting the aerobic requirements of citric acid production. The headspace accommodates gas exchange requirements while maintaining appropriate environmental conditions for sustained fermentation performance.

[0186]

[0106] 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. The prolonged fermentation time enables the mutant strain to achieve maximum substrate conversion efficiency and optimal acid yield recovery.

[0187]

[0107] 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 for optimal citric acid stability and recovery. The pH control facilitates optimal citric acid accumulation through favorable thermodynamic conditions while inhibiting competing metabolic pathways.

[0188] Fermentation Method Overview

[0189]

[0108] The method utilizes submerged fermentation techniques with controlled environmental parameters including temperature, pH, dissolved oxygen levels, and nutrient supplementation. The submerged fermentation approach provides better process control, heat and mass transfer efficiency, and scalability compared to alternative fermentation methods. The controlled environmental parameters ensure consistent fermentation performance while maximizing organic acid yields through optimal growth and production conditions.

[0190]

[0109] The fermentation medium further comprises various carbon sources selected from glucose, sucrose, starch, and other carbohydrate substrates. The 202421061044

[0191] carbon source selection provides flexibility in substrate utilization while supporting the enhanced metabolic pathways of the mutant strain. The carbohydrate substrates serve as the primary carbon sources for citric acid biosynthesis through complex metabolic pathways involving glycolysis and tricarboxylic acid cycle intermediates.

[0192] Pellet Morphology and Starch Utilization Analysis

[0193] [HO] The pellet morphology characteristics and starch utilization patterns during citric acid fermentation provide indicators of fermentation performance and substrate conversion efficiency. Referring to Figure 1, the pellet morphology scores for strains F-l and F-7 over the fermentation time period from 118 to 220 hours illustrate the quantitative assessment of pellet structure and integrity throughout the fermentation process. Figure 1 depicts two line graphs positioned side by side, showing pellet morphology scores and starch residue scores over fermentation time for citric acid production comparing parent strain F-l and mutant strain F-7. The left graph displays pellet morphology scores on the vertical axis ranging from 0 to 3.5, while the right graph shows starch residue scores on the vertical axis ranging from 0 to 2.5, with both graphs sharing a common horizontal axis representing fermentation time measured in hours at three time points: 118 h, 190 h, and 220 h. The morphology scoring system provides quantitative assessment of pellet structure and integrity throughout the fermentation process.

[0194] [Hl] At 118 hours of fermentation, both F-l and F-7 strains demonstrate high morphology scores approaching 3.5, indicating compact, well-formed pellets with intact structure. As shown in Figure 1, in the left graph, two lines represent the morphology scores for F-l and F-7, both starting at approximately 3.0 at 118 hours, declining to approximately 2.0 at 190 hours, and further decreasing to approximately 1.0 for F-l and 0.5 for F-7 at 220 hours, with F-7 demonstrating a steeper decline than F-l in the later fermentation phase. The mutant strain F-7 (DPPL 009) maintains superior pellet morphology characteristics compared to the parent strain F-l throughout the fermentation period, exhibiting more stable pellet 202421061044

[0195] structure with a gradual decline in morphology score, reaching approximately 1.5 at 220 hours. 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.

[0196]

[0112] 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.

[0197] Starch Utilization Efficiency

[0198]

[0113] As further shown in Figure 1, the starch residue scores for both strains throughout the fermentation period demonstrate progressive reduction from 118 to 220 hours, indicating continuous substrate consumption. In the right graph of Figure 1, two lines track starch residue scores for both strains, beginning at approximately 2.0 at 118 hours, decreasing to approximately 1.0 at 190 hours, and reaching near 0 for F-7 and approximately 0.5 for F-l at 220 hours, indicating more complete starch utilization by the mutant strain F-7 compared to the parent strain F-l. 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.

[0199]

[0114] The mutant strain F-7 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-7 achieves a starch residue score approaching 0.5, while F-l maintains a score of 202421061044

[0200] 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.

[0201]

[0115] 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. The iodine starch test provides qualitative assessment of starch utilization based on blueblack 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 and development of brown coloration similar to that observed in the water blank control.

[0202] pH Profiles and Acidification Dynamics

[0203]

[0116] Fig. 2 depicts a line graph showing the relationship between fermentation time in hours and pH value for flask F-l containing the parent strain PS during citric acid production. The horizontal axis represents fermentation time ranging from 0 hours to 382 hours, with specific time points marked at intervals including 0, 4, 22, 28, 46, 52, 70, 76, 94, 100, 118, 124, 142, 148, 166, 172, 190, 196, 214, 220, 238, 244, 262, 268, 286, 292, 310, 316, 334, 340, 358, 364, and 382 hours. The vertical axis displays pH values ranging from 0 to 6. The data points are connected by a continuous line showing the progression of pH changes throughout the fermentation period. The graph shows an initial pH of approximately 4.9 at time zero, followed by a rapid decline to approximately 1.8 by 46 hours, indicating rapid acidification during the early fermentation phase. A transient pH rise occurs between 52 and 70 hours, with values increasing to approximately 3.2, representing a metabolic adjustment phase. The pH reaches a minimum value of approximately 1.0 at 76 hours, representing the most acidic condition achieved during the fermentation process. From 100 hours through 382 hours, the pH stabilizes within a range between approximately 1.8 and 2.1, with 202421061044

[0204] minor fluctuations throughout this extended period, demonstrating sustained acidic conditions that facilitate citric acid accumulation.

[0205]

[0117] The pH profiles during citric acid fermentation provide indicators of fermentation progress and acid accumulation patterns for the mutant strain MTCC 25759. Referring to Figure 3, the pH profile for mutant strain F-7 (DPPL 009) throughout the fermentation period from 0 to 382 hours demonstrates the characteristic acidification pattern associated with citric acid biosynthesis. Figure 3 depicts a line graph showing the relationship between fermentation time in hours and pH value for flask F-7 containing the DPPL 009 strain during citric acid production. The horizontal axis represents fermentation time ranging from 0 hours to 406 hours, with specific time points marked at intervals including 0, 4, 22, 28, 46, 52, 70, 76, 94, 100, 118, 124, 142, 148, 166, 172, 190, 196, 214, 220, 238, 244, 262, 268, 286, 292, 310, 316, 334, 340, 358, 364, and 382 hours. The vertical axis displays pH values ranging from 0 to 6. The fermentation begins with an initial pH of approximately 4.85 at time zero, establishing slightly acidic starting conditions that support spore germination and early metabolic activity.

[0206]

[0118] The mutant strain F-7 exhibits rapid initial acidification during the first phase of fermentation. In some cases, the pH declines sharply to approximately 2.25 by 22 hours, corresponding to the establishment of active citric acid biosynthesis pathways. As shown in Figure 3, the data points are connected by a continuous blue line showing the progression of pH changes throughout the fermentation period, with the graph showing an initial pH of approximately 4.85 at time zero, followed by a sharp decline to approximately 2.25 by 22 hours, indicating rapid acidification during the early fermentation phase. This rapid acidification phase indicates the onset of intensive organic acid production and the transition from adaptation phase to productive metabolism. The steep pH decline reflects enhanced metabolic activity and more efficient establishment of citric acid biosynthesis compared to conventional fermentation patterns. 202421061044

[0207]

[0119] Following the initial acidification period, the mutant strain demonstrates a transient pH rise between 52 and 70 hours. As shown in Figure 3, a transient pH rise occurs between 52 and 70 hours, with values increasing to approximately 3.2, representing a metabolic adjustment phase. pH values increase to approximately 3.2 during this period, suggesting a metabolic adjustment phase where cellular processes undergo regulatory changes. This temporary pH elevation represents a period of metabolic rebalancing where acid production pathways are modulated in response to changing substrate availability or cellular energy requirements.

[0208]

[0120] The mutant strain F-7 achieves an exceptional minimum pH of 1.39 at 76 hours of fermentation, as illustrated in Figure 3. As depicted in Figure 3, the pH reaches its minimum value of approximately 1.39 at 76 hours, representing the most acidic condition achieved during the fermentation process. This minimum pH value of 1.39 falls within the enhanced tolerance range of 1.0-2.0 demonstrated by the mutant strain during citric acid production, representing the most acidic condition reached during the fermentation process and indicating superior acid production capability. The achievement of such low pH conditions demonstrates enhanced tolerance to acidic environments and more aggressive citric acid biosynthesis compared to parent strain performance. In some cases, the ability to maintain cellular viability and continued acid production under these highly acidic conditions reflects improved cellular adaptation mechanisms.

[0209]

[0121] From 100 hours through 382 hours, the mutant strain F-7 maintains stable pH conditions between 1.8 and 2.1, as demonstrated in Figure 3. As shown in Figure 3, from 100 hours through 382 hours, the pH stabilizes within a range between approximately 1.8 and 2.1, with minor fluctuations throughout this extended period, demonstrating sustained acidic conditions that facilitate citric acid accumulation. This stable pH maintenance exhibits greater consistency and less variation throughout the extended fermentation period compared to typical fermentation patterns. The pH stability facilitates continued citric acid accumulation through favorable thermodynamic conditions that support acid 202421061044

[0210] biosynthesis while inhibiting competing metabolic pathways. In some cases, the consistent acidic environment prevents acid degradation or conversion to alternative metabolic products.

[0211]

[0122] The pH stability demonstrated by the mutant strain provides favorable conditions for sustained citric acid production throughout extended fermentation periods. The maintained acidic environment creates conditions that favor product accumulation over biomass formation, directing metabolic flux toward citric acid biosynthesis. In some cases, the stable pH range between 1.8 and 2.1 represents conditions where citric acid biosynthesis enzymes maintain activity while competing pathways are suppressed.

[0212]

[0123] Compared to parent strain pH profiles, the mutant strain demonstrates more aggressive acidification and enhanced pH control mechanisms. The parent strain exhibits less pronounced acidification and greater pH fluctuations throughout the fermentation period. The mutant strain demonstrates more rapid recovery from the transient pH rise phase and establishes stable acidic conditions more quickly than the parent strain. In some cases, the enhanced pH dynamics correlate with improved citric acid production yields and more efficient substrate conversion to organic acid products.

[0213]

[0124] Figure 4 presents a comparative pH profile analysis displaying the pH dynamics of both parent strain F-l and mutant strain F-7 throughout the citric acid fermentation process. The graph illustrates pH values on the vertical axis ranging from 0 to 6, plotted against fermentation time in hours on the horizontal axis extending from 0 to 382 hours. Two distinct data series are shown: F-l [PS] represented by blue diamond markers and F-7 [DPPL 009] represented by red square markers, both connected by trend lines to demonstrate the pH progression patterns. The comparative analysis reveals that both strains exhibit similar overall acidification trajectories while displaying distinct performance characteristics. Both strains begin with initial pH values of approximately 4.9, establishing comparable starting conditions for fermentation initiation. The early acidification phase shows both strains experiencing rapid pH decline within the first 50 hours, 202421061044

[0214] with F-l reaching approximately 1.8 and F-7 achieving approximately 2.25 by this timepoint. The transient pH rise phase occurring between 52 and 70 hours demonstrates similar patterns for both strains, with pH values increasing to approximately 3.2-3.3. This metabolic adjustment phase appears consistent across both strain variants, suggesting that the fundamental regulatory mechanisms governing this transition remain intact in the mutant strain. The temporary pH elevation represents a period where both strains undergo metabolic rebalancing in response to changing fermentation conditions. The most pronounced difference between the strains occurs at 76 hours, where the mutant strain F-7 achieves a dramatically lower minimum pH of approximately 1.39 compared to the parent strain F-l, which reaches approximately 1.0. The mutant strain's ability to achieve this exceptionally low pH value demonstrates enhanced acid production capability and superior tolerance to highly acidic conditions. This performance differential indicates that the genetic modifications in strain F-7 provide enhanced acidogenic potential while maintaining cellular viability under extreme acidic stress. Throughout the extended fermentation period from 100 to 382 hours, both strains maintain stable pH conditions within the range of 1.8 to 2.1, though the mutant strain F-7 exhibits slightly more dynamic pH behavior with periodic minor fluctuations. The sustained acidic environment maintained by both strains facilitates continued citric acid accumulation, though the mutant strain demonstrates enhanced consistency in maintaining these favorable conditions. The comparative pH profiles illustrate that while both strains follow similar fundamental acidification patterns, the mutant strain F-7 exhibits enhanced performance characteristics including more aggressive initial acidification, superior minimum pH achievement, and improved pH stability throughout extended fermentation periods. These enhanced pH dynamics correlate with the improved citric acid production capabilities demonstrated by the mutant strain under identical fermentation conditions.

[0215] Citric Acid Production Performance 202421061044

[0216]

[0125] Referring to Figure 5, the citric acid production performance of the mutant strain demonstrates substantial yield improvements compared to the parent strain under identical fermentation conditions. Figure 5 depicts a line graph showing citric acid production over time comparing two strains during fermentation, with fermentation time in hours on the horizontal axis and citric acid concentration in grams per liter on the vertical axis. Two plotted lines represent F-l [PS] and F-7 [DPPL 009] strains, with data points marked at four time intervals: 316 h, 334 h, 358 h, and 382 h. Figure 5 illustrates citric acid production profiles measured at four 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-7 (DPPL 009), with the mutant strain achieving sustained late-phase productivity and consistent performance throughout extended fermentation phases.

[0217]

[0126] The mutant strain F-7 (DPPL 009) achieves a steady increase in citric acid production from 0.347 g / L at 316 hours to 0.352 g / L at 334 hours, followed by 0.407 g / L at 358 hours, and reaching 0.415 g / L at 382 hours. As shown in Figure 5, the F-7 [DPPL 009] strain demonstrates a relatively steady increase from approximately 0.35 g / L at 316 h to approximately 0.41 g / L at 382 h, with intermediate values of approximately 0.35 g / L at 334 h and approximately 0.40 g / L at 358 h. This progressive increase demonstrates sustained late-phase productivity and reflects improved metabolic control mechanisms that enable continued acid biosynthesis during extended fermentation periods. The mutant strain maintains stable production characteristics with gradual increases that indicate enhanced tolerance to environmental stresses associated with prolonged acidic fermentation conditions.

[0218]

[0127] In contrast, the parent strain F-l achieves 0.432 g / L at 316 hours but experiences a decline to 0.257 g / L at 334 hours, followed by recovery to 0.391 g / L at 382 hours. As depicted in Figure 5, the F-l [PS] strain shows an initial concentration of approximately 0.43 g / L at 316 h, decreasing to approximately 0.26 g / L at 334 h, then increasing to approximately 0.35 g / L at 358 h, and 202421061044

[0219] reaching approximately 0.40 g / L at 382 h. The fluctuating production pattern of the parent strain indicates less stable metabolic performance and reduced consistency during extended fermentation phases. The decline observed at 334 hours suggests metabolic stress or reduced enzymatic activity under the challenging conditions of prolonged fermentation.

[0220] Enzymatic Kit Quantification Methodology

[0221]

[0128] The enzymatic kit quantification methodology employs substratespecific enzymes that catalyze the conversion of citric acid to measurable products through coupled enzymatic reactions. 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 concentration range encountered in fermentation studies. Comparative Yield Performance Analysis

[0222]

[0129] TLC analysis provides qualitative confirmation of the enhanced citric acid production capabilities demonstrated through enzymatic kit quantification. The mutant strain F-7 exhibits more frequent and intense TLC spot development compared to the parent strain F-l, particularly during mid-to-late fermentation phases corresponding to the time periods where sustained productivity improvements are observed. The visual confirmation provided by TLC analysis supports the quantitative yield data and demonstrates consistency across different analytical methodologies.

[0223] Production Stability and Consistency

[0224]

[0130] The production stability and consistency demonstrated by the mutant strain provides advantages for commercial production applications where predictable yields and stable fermentation performance are required for economic viability. The sustained production capability with gradual increases from 0.347 g / L to 0.415 g / L over the 66-hour period from 316 to 382 hours indicates 202421061044

[0225] enhanced metabolic persistence under challenging fermentation conditions. The mutant strain achieves citric acid production of 0.415 g / L in starch-dextrose medium at 30°C over 382 hours, demonstrating improved fermentation characteristics compared to conventional production strains.

[0226] Gluconic Acid Production

[0227]

[0131] The fermentation medium for gluconic acid production employs a precisely formulated composition of carbon sources, nitrogen sources, and mineral nutrients to support growth and gluconic acid biosynthesis by the mutant strain. The medium composition is specifically designed to promote gluconic acid accumulation through controlled nutrient availability while maintaining metabolic balance for sustained fermentation performance.

[0228] Medium Composition

[0229]

[0132] The primary carbon source consists of dextrose at a concentration of 220 g / L, providing the glucose substrate 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. In some cases, the concentration is optimized to maximize substrate utilization efficiency while preventing substrate inhibition effects that could reduce production yields.

[0230]

[0133] The nitrogen source and growth factors include yeast extract incorporated at 2 g / L to provide complex nitrogen sources, vitamins, and growth factors 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 for efficient gluconic acid production. Ammonium sulfate 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.

[0231]

[0134] The medium includes mineral components with magnesium sulfate heptahydrate at 5 g / L to supply magnesium ions for enzyme activation and 202421061044

[0232] cofactor functions in gluconic acid biosynthesis pathways. Potassium chloride is incorporated at 0.5 g / L to provide potassium ions for enzyme activation, cellular transport processes, and osmotic regulation. Ferric ammonium citrate is added at 0.01 g / L to supply iron ions 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.

[0233] Medium Preparation Protocol

[0234]

[0135] The 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.

[0235] pH Control and Sterilization

[0236]

[0136] 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.

[0237] Fermentation Conditions

[0238]

[0137] The gluconic acid fermentation conditions are specifically optimized to support the metabolic requirements of the Aspergillus niger mutant strain MTCC 25759 and promote efficient gluconic acid biosynthesis through controlled environmental parameters designed to maintain oxygen availability, substrate 202421061044

[0239] accessibility, and environmental stability throughout extended fermentation periods.

[0240] Spore Inoculum Preparation

[0241]

[0138] A spore inoculum preparation is conducted 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 during early fermentation phases. The standardized inoculum concentration ensures consistent fermentation initiation across different production batches and enables reproducible comparison of fermentation performance under identical starting conditions. An inoculum volume of 2.5 mL is added to 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 25759 and promote efficient gluconic acid biosynthesis through controlled environmental parameters designed to maintain oxygen availability, substrate accessibility, and environmental stability throughout the extended fermentation period.

[0242] Temperature and Agitation Control

[0243]

[0139] The fermentation temperature is maintained at 30°C throughout the entire production period of 214 hours. The temperature represents the 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.

[0244]

[0140] Agitation is provided through rotary shaking at 25 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 202421061044

[0245] biosynthesis. The glucose oxidase enzyme system requires continuous oxygen availability for oxidation reactions, making adequate aeration for production efficiency. The 25 RPM agitation rate provides balance between oxygen transfer and mechanical stress on fungal biomass while preventing excessive shear forces that damage fungal pellets.

[0246] Volume and Duration Parameters

[0247]

[0141] A 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.

[0248] Fermentation Duration and metabolic Phase

[0249]

[0142] The fermentation duration extends for 214 hours, corresponding to approximately 9 days of continuous production. The 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 acid yield recovery. The combination of controlled temperature at 30°C, moderate agitation at 23 to 27 RPM, appropriate medium-to-flask volume ratio of 1:2, and extended fermentation time of 214 hours creates conditions for gluconic acid biosynthesis by strain MTCC 25759. These parameters collectively support the aerobic metabolic requirements for glucose oxidation while maintaining environmental stability and substrate availability. The fermentation conditions promote pellet morphology that enhances oxygen transfer efficiency and substrate utilization while preventing excessive filamentous growth or pellet disintegration. 202421061044

[0250]

[0143] The method for producing gluconic acid comprises culturing the Aspergillus niger mutant strain MTCC 25759 in a fermentation medium, maintaining fermentation conditions at 30°C, and conducting fermentation for approximately 214 hours to produce gluconic acid. The method utilizes submerged fermentation techniques with controlled environmental parameters including temperature, pH, dissolved oxygen levels, and nutrient supplementation. The fermentation medium further comprises various carbon sources selected from glucose, sucrose, starch, and other carbohydrate substrates to support enhanced gluconic acid production capabilities of the mutant strain.

[0251] Sugar Utilization Analysis

[0252]

[0144] 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 25759. 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.

[0253] Parent Strain Sugar Utilization Profile

[0254]

[0145] Referring to Figure 6, the parent strain F-l 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. Figure 6 depicts a line graph showing the relationship between fermentation time in hours and percentage sugar utilization for flask F-l containing the parent strain PS. The horizontal axis represents fermentation time ranging from 0 hours to 214 hours, with specific time points marked at intervals including 0, 3, 22, 27, 46, 51, 70, 75, 94, 99, 118, 123, 142, 147, 166, 171, 190, 195, and 214 hours. The vertical axis represents percentage utilization ranging from -30% to 50%. The parent strain F-l exhibits 202421061044

[0255] negative sugar utilization values during the initial fermentation period from 0 to 75 hours, with values fluctuating between 0% and approximately -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.

[0256]

[0146] 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. As shown in Figure 6, the data points are connected by a continuous line showing the progression of sugar utilization throughout the fermentation period, with the graph showing negative utilization values during the early fermentation phase, with the lowest point reaching approximately -25% at around 27 hours, followed by a gradual increase into positive values beginning after 75 hours, and ultimately reaching approximately 42% utilization by 214 hours with some fluctuations throughout the later stages of fermentation. 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 substantial substrate conversion efficiency despite delayed onset of active metabolism.

[0257] Mutant Strain Sugar Utilization Performance

[0258]

[0147] Referring to Figure 7, the mutant strain F-4 (DPPL 009) exhibits a sugar utilization profile that demonstrates enhanced metabolic characteristics compared to the parent strain while following similar biphasic fermentation patterns. The mutant strain displays an initial phase of negative sugar utilization values during the early fermentation period from 0 to 75 hours, with fluctuations ranging from 0% to approximately -26.1%. The negative utilization values reflect the hydrolysis of complex carbohydrates and release of additional reducing sugars during the adaptation phase, similar to the parent strain but with slightly more pronounced hydrolytic activity. 202421061044

[0259]

[0148] The mutant strain F-4 achieves its minimum utilization value of -26.1% at 27 hours, representing a marginally lower value compared to the parent strain's -25.7% at the same time point. This enhanced negative utilization suggests more active enzymatic hydrolysis during the early fermentation phase, indicating improved enzyme production or activity in the mutant strain. The enhanced hydrolytic activity may contribute to better substrate preparation for subsequent glucose oxidation reactions. The transition to positive sugar utilization occurs at approximately 75 hours for the mutant strain F-4, coinciding with the timing observed for the parent strain. However, the mutant strain demonstrates more rapid establishment of active glucose consumption following this transition point. At 94 hours, the mutant strain achieves 12.8% sugar utilization compared to 9.6% for the parent strain, indicating enhanced metabolic activity during the early productive phase. The mutant strain F-4 maintains consistent upward progression in sugar utilization throughout the remainder of the fermentation period, reaching intermediate values of 24.7% at 142 hours and 35.2% at 190 hours. The steady increase demonstrates stable metabolic performance and sustained glucose consumption capability throughout extended fermentation phases. The mutant strain achieves final sugar utilization of 40.2% at 214 hours, representing efficient substrate conversion that closely approaches the parent strain's performance of 42.3%. The sugar utilization profile for mutant strain F-4 demonstrates enhanced early-phase metabolic activity with more pronounced hydrolytic enzyme activity during adaptation phases and more rapid establishment of productive glucose consumption following the transition at 75 hours. The consistent progression and final utilization efficiency of 40.2% indicate that the genetic modifications in strain MTCC 25759 maintain effective substrate conversion capabilities while potentially providing enhanced metabolic control and stability throughout the fermentation process.

[0260] Comparative Performance Analysis and Economic Implications

[0261]

[0149] With reference to Figures 8, the comparative sugar utilization profiles between the parent strain F-l and the mutant strain F-4 (DPPL 009) show similar 202421061044

[0262] biphasic utilization patterns but demonstrate enhanced metabolic performance and substrate utilization characteristics for the mutant strain. Figure 8 depicts a line graph showing the comparative percentage sugar utilization of F-l versus F-4 over fermentation time measured in hours. Two trend lines are plotted, with one representing F-l [PS] and the other representing F-4, tracking their respective sugar utilization percentages from 0 hours through 214 hours of fermentation. The horizontal axis displays fermentation time in hours with marked intervals at 0, 3, 27, 46, 51, 70, 75, 94, 99, 118, 123, 142, 147, 166, 171, 190, 195, and 214 hours, while the vertical axis shows percentage utilization ranging from -40% to 60%. The mutant strain F-4 displays negative sugar utilization values during the initial fermentation period from 0 to 75 hours, with fluctuations ranging from negative values to approximately -26.1%. The minimum utilization value of -26.1% 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.

[0263]

[0150] The transition to positive sugar utilization occurs at approximately 75 hours for the mutant strain F-4, similar to the timing observed for the parent strain. As shown in Figure 8, both strains exhibit negative utilization values during the initial phase, reaching their lowest points around 22-27 hours at approximately -25%, before transitioning to positive utilization values after 94 hours. The F-l strain shows a gradual increase in sugar utilization with some fluctuations, reaching approximately 42% by 214 hours, while the F-4 strain follows a similar pattern with slightly lower final utilization of approximately 40% at 214 hours. In some cases, the mutant strain demonstrates more rapid establishment of active glucose consumption, reaching 16.7% utilization at 99 hours compared to values achieved by the parent strain at the same time point. The mutant strain F-4 achieves final sugar utilization of 40.2% at 214 hours, showing more stable and controlled substrate uptake patterns throughout the fermentation period.

[0264] Comparative Sugar Utilization Analysis 202421061044

[0265]

[0151] The comparative analysis reveals that both strains exhibit similar biphasic utilization patterns with initial negative values persisting until approximately 75 hours, followed by progressive increases in sugar consumption. As demonstrated in Figure 8, the graph demonstrates that both strains display comparable sugar utilization profiles throughout the fermentation period, with F-l achieving marginally higher final utilization despite both strains showing similar metabolic progression patterns. 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.

[0266]

[0152] The enhanced sugar utilization performance of the mutant strain F-4 correlates directly with improved gluconic acid production capabilities demonstrated through enzymatic assay analysis. The difference in final sugar utilization translates to more efficient substrate conversion and higher product yields, contributing to enhanced fermentation performance of strain MTCC 25759. In some cases, the improved 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. pH Profiles and Metabolic Dynamics

[0267]

[0153] Figure 9 depicts a line graph showing the relationship between fermentation time in hours and pH value for flask F-l containing the parent strain PS during gluconic acid production. The horizontal axis represents fermentation time ranging from 3 hours to 214 hours, with specific time points marked at regular intervals throughout the fermentation period. The vertical axis displays pH values ranging from 0 to 8. The data points are connected by a continuous line showing the progression of pH changes throughout the fermentation process. The parent strain F-l exhibits an initial pH of approximately 6.8 at 3 hours of fermentation, establishing neutral to slightly alkaline starting conditions that support spore germination and early metabolic establishment. During the early fermentation phase from 3 to 51 hours, the parent strain maintains relatively stable 202421061044

[0268] pH values ranging from 6.8 to 7.2, indicating minimal acid production during the adaptation phase. The stable pH range suggests effective pH buffering capacity of the medium or delayed onset of acidogenic metabolism as the strain establishes metabolic pathways for glucose oxidation. The parent strain F-l experiences gradual pH decline beginning around 70 hours, reaching approximately 5.8 by 94 hours. This acidification pattern indicates the onset of gluconic acid biosynthesis with moderate acidification reflecting establishment of glucose oxidation pathways. The gradual pH reduction demonstrates conventional fermentation kinetics where acid production increases progressively as the strain transitions from growth phase to productive metabolism. From 94 to 214 hours, the parent strain F-l exhibits moderate pH fluctuations between 4.5 and 6.2, demonstrating less pronounced pH variations compared to enhanced mutant strains. The pH oscillations indicate metabolic activity with alternating phases of acid production and metabolic adjustment periods. The parent strain achieves minimum pH values of approximately 4.5 during productive phases, indicating moderate gluconic acid accumulation and glucose oxidase activity. The parent strain F-l maintains final pH of approximately 5.2 at 214 hours, indicating sustained but moderate acidification throughout the fermentation period. The final pH value reflects conventional acid production capability and substrate conversion efficiency typical of wild-type Aspergillus niger strains. The pH profile demonstrates stable but less aggressive acidification compared to enhanced mutant variants, correlating with moderate gluconic acid production yields under identical fermentation conditions. The pH profiles during gluconic acid fermentation reveal distinct differences in acidification patterns and metabolic stability between the parent strain and the mutant strain MTCC 25759. The mutant strain F-4 (DPPL 009) demonstrates more pronounced and dynamic pH behavior compared to the parent strain, indicating enhanced metabolic activity and acid production capabilities throughout the fermentation process. The mutant strain demonstrates enhanced tolerance to acidic fermentation conditions, achieving minimum pH values of 4.3 during gluconic acid production, which falls within the enhanced 202421061044

[0269] tolerance range of 4.3-6.5 demonstrated by the mutant strain during gluconic acid fermentation.

[0270] Initial pH Stabilization Phase

[0271]

[0154] Referring to Figure 10, the mutant strain F-4 exhibits an initial pH of approximately 6.45 at 3 hours of fermentation, establishing neutral starting conditions similar to the parent strain. Figure 10 presents a graph depicting fermentation time versus pH for the F-4 [DPPL 009] flask over an extended fermentation period. The horizontal axis represents fermentation time measured in hours, ranging from 3 hours to 214 hours, while the vertical axis displays pH values ranging from 0 to 8. The data points connected by a line show pH measurements taken at various time intervals throughout the fermentation process. 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. As shown in Figure 10, the pH profile demonstrates an initial value around 6.5 at 3 hours, followed by a gradual increase reaching approximately 7.0 at 51 hours. The stable pH range suggests effective pH buffering or delayed onset of acidogenic metabolism as the strain establishes metabolic pathways for glucose oxidation.

[0272] Acidification and Oscillatory pH Behavior

[0273]

[0155] The mutant strain F-4 experiences sharp pH decline beginning at 70 hours, reaching approximately 4.87 by 94 hours. As depicted in Figure 10, after which the pH exhibits fluctuating behavior with multiple peaks and valleys. Notable pH variations occur between 70 and 166 hours, where the values oscillate between approximately 4.5 and 6.8. This acidification pattern indicates aggressive onset of gluconic acid biosynthesis with accelerated acidification reflecting enhanced metabolic activity. The rapid pH reduction demonstrates more efficient establishment of glucose oxidation pathways compared to conventional fermentation patterns, suggesting improved enzymatic activity and metabolic flux through gluconic acid biosynthesis routes. 202421061044

[0274]

[0156] From 94 to 195 hours, the mutant strain F-4 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. These pH fluctuations represent cyclical metabolic processes where acid production phases alternate with buffering or partial neutralization phases, suggesting active metabolic regulation mechanisms.

[0275]

[0157] The deeper pH troughs reach minimum values of 4.3 compared to the parent strain's minimum of 4.5, indicating more aggressive acid production capability and enhanced glucose oxidase activity. The lower minimum pH values 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 increasingly acidic conditions.

[0276] Enhanced Metabolic Regulation

[0277]

[0158] The pH oscillations 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. The rhythmic pH fluctuations demonstrate more vigorous metabolic activity compared to the more gradual and less pronounced pH changes observed in conventional fermentation systems. The oscillatory behavior reflects cyclical metabolic activity where the strain alternates between intensive glucose oxidation phases and metabolic recovery periods. The mutant strain F-4 achieves a final pH of approximately 5.01 at 214 hours, indicating sustained acidification and successful gluconic acid accumulation throughout the fermentation period. As shown in Figure 10, before showing a declining trend in the later stages of fermentation, reaching approximately 4.8 by 214 hours. The final pH value is lower than that achieved by the parent strain, reflecting enhanced acid production capability and more complete substrate conversion to gluconic acid. The sustained acidification demonstrates the strain's 202421061044

[0278] ability to maintain productive metabolism under acidic conditions that typically inhibit conventional fermentation systems. The oscillatory pH behavior exhibited by the mutant strain F-4 reflects more responsive metabolic regulation compared to conventional strains. The dynamic pH pattern indicates enhanced tolerance to acidic conditions that accumulate during gluconic acid production, enabling continued glucose oxidation and acid accumulation under challenging environmental conditions. The metabolic flexibility demonstrated through pH oscillations contributes to the enhanced production yields observed in the mutant strain compared to the parent strain under identical fermentation conditions.

[0279] Gluconic Acid Production Performance

[0280]

[0159] The gluconic acid production performance of the mutant strain demonstrates substantial yield improvements compared to the parent strain under identical fermentation conditions. The quantitative assessment employs enzymatic assay kit methodology to provide precise measurement of gluconic acid concentrations at two time points during the fermentation process, enabling accurate comparison of production capabilities between the parent and mutant strains.

[0281] Early Phase Production Comparison

[0282]

[0160] 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-4 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 comparable early-phase performance indicates that both strains establish gluconic acid biosynthesis pathways with similar efficiency during the initial and intermediate fermentation phases.

[0283] Late Phase Production Enhancement

[0284]

[0161] The performance differential becomes dramatically pronounced at 214 hours, where the mutant strain F-4 achieves 198.836 g / L compared to 119.776 g / L produced by the parent strain F-L This represents a substantial improvement of 202421061044

[0285] 79.06 g / L, corresponding to approximately 66% enhancement in gluconic acid yield. The enhancement indicates enhanced metabolic persistence and superior acid production capability under the challenging conditions of prolonged fermentation.

[0286]

[0162] The dramatic increase in gluconic acid production by the mutant strain F-4 from 66.015 g / L at 166 hours to 198.836 g / L at 214 hours represents a 201% 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.

[0287] Production Performance Correlation

[0288]

[0163] The sustained high-level production capability demonstrated by the mutant strain F-4 correlates with the dynamic pH oscillations observed between 94 and 195 hours of fermentation. The oscillatory pH pattern indicates active metabolic regulation and enhanced tolerance to acidic conditions, enabling continued glucose oxidation and gluconic acid accumulation under challenging environmental conditions.

[0289] TLC Analysis Correlation and Qualitative Confirmation

[0290]

[0164] The TLC analysis results provide qualitative confirmation of the enhanced gluconic acid production capabilities demonstrated through enzymatic kit quantification. The mutant strain F-4 exhibits consistently stronger TLC spot intensities compared to the parent strain F-l, particularly during the later fermentation phases. At 214 hours, the TLC spot intensity for the mutant strain F-4 exceeds 10% standard gluconic acid match, indicating substantial acid accumulation and confirming the high production levels measured through enzymatic assay. The enhanced gluconic acid production performance of the mutant strain correlates with the superior sugar utilization efficiency observed throughout the fermentation period. The more efficient substrate utilization 202421061044

[0291] translates to increased gluconic acid yield through improved metabolic flux and substrate conversion efficiency. The mutant strain achieves gluconic acid production of 198.836 g / L in dextrose medium at 30°C over 214 hours, representing a substantial improvement over the parent strain's production of 119.776 g / L under identical conditions.

[0292]

[0165] The mutant strain MTCC 25759 exhibits consistent batch-to-batch performance characteristics that provide enhanced reliability for industrial fermentation applications. In some cases, the standardized spore preparation methodology and controlled mutagenesis protocol contribute to reproducible strain characteristics across multiple production batches. The genetic stability demonstrated through multiple subculture passages ensures that performance characteristics remain consistent during commercial production cycles.

[0293] Contamination Resistance

[0294]

[0166] The mutant strain demonstrates reduced contamination risks compared to conventional production strains through enhanced acid production capabilities that create inhibitory conditions for competing microorganisms. In some cases, the rapid acidification achieved by the mutant strain, including minimum pH values of 1.39 during citric acid production and 4.3 during gluconic acid production, provides natural antimicrobial protection that reduces the likelihood of bacterial or yeast contamination during fermentation processes.

[0295] Industrial Stress Tolerance

[0296]

[0167] The mutant strain operates effectively under industrial stress conditions that typically challenge conventional fermentation systems. In some cases, the enhanced tolerance to high product concentrations enables continued metabolic activity even as organic acid levels accumulate to concentrations that inhibit wild-type strains. The mutant strain maintains productive metabolism at elevated temperatures that occur during large-scale fermentation due to metabolic heat generation and reduced heat transfer efficiency in industrial bioreactors. Substrate Flexibility 202421061044

[0297]

[0168] The mutant strain demonstrates robustness when exposed to varying substrate compositions that occur in industrial feedstock materials. In some cases, the enhanced substrate utilization efficiency enables effective fermentation performance with crude sugar sources, molasses, or other industrial carbon sources that contain impurities or variable nutrient profiles. The improved metabolic flexibility provides operational advantages for commercial production facilities that utilize diverse or seasonally variable raw materials.

[0298] Dual Acid Production Capability

[0299]

[0169] The mutant strain achieves simultaneous production of both citric acid and gluconic acid within a single fermentation process, providing operational efficiency advantages for industrial applications. In some cases, the dual acid production capability maximizes resource utilization by converting glucose substrates to multiple valuable products through parallel metabolic pathways. The simultaneous production approach reduces capital equipment requirements and operational costs compared to separate fermentation processes for each organic acid.

[0300]

[0170] The integrated production system provides process efficiency benefits through shared infrastructure utilization and reduced downstream processing complexity. In some cases, the combined fermentation approach enables production scheduling flexibility where product ratios can be adjusted based on market demand or economic considerations. The dual production capability provides risk mitigation for commercial operations by reducing dependence on single product markets.

[0301] Scalability and Industrial Implementation

[0302]

[0171] The mutant strain demonstrates scalability from laboratory fermentation conditions to industrial production levels while maintaining consistent performance characteristics. In some cases, the stable pellet morphology exhibited during laboratory fermentation translates to effective mass transfer and mixing characteristics in large-scale bioreactors. The controlled pH 202421061044

[0303] dynamics and substrate utilization patterns observed in small-scale studies are maintained during scale-up operations.

[0304]

[0172] The fermentation parameters established during laboratory development, including temperature control at 30°C and moderate agitation requirements, are readily implemented in industrial fermentation systems. In some cases, the extended fermentation periods of 214 hours for gluconic acid production and 382 hours for citric acid production are accommodated within commercial production schedules while providing adequate time for complete substrate conversion and maximum yield recovery.

[0305] Economic Advantages

[0306]

[0173] The enhanced production capabilities demonstrated by the mutant strain provide economic advantages through improved substrate conversion rates that reduce raw material costs per unit of product. In some cases, the 66% improvement in gluconic acid yield and enhanced citric acid yield translate directly to reduced feedstock requirements and improved profit margins for commercial operations. The enhanced substrate utilization efficiency enables production cost reductions that improve competitive positioning in organic acid markets.

[0307]

[0174] The mutant strain enables increased production capacity within existing fermentation infrastructure through higher volumetric productivity and improved yield coefficients. In some cases, the enhanced performance characteristics allow production facilities to increase output without requiring additional bioreactor capacity or capital investment in new equipment. The improved fermentation efficiency provides operational flexibility for meeting increased market demand or expanding product portfolios.

[0308] Strain Development Methodology

[0309]

[0175] The reproducible mutagenesis methodology employed for strain development enables systematic generation of additional mutant variants with enhanced characteristics while maintaining the safety profile of the parent 202421061044

[0310] organism. In some cases, the controlled chemical exposure protocol and standardized screening procedures are applied to develop specialized strains for specific industrial applications or substrate compositions. The systematic approach facilitates continued strain improvement programs that address evolving commercial requirements.

[0311] Regulatory Compliance

[0312]

[0176] The mutant strain maintains the Generally Regarded As Safe (GRAS) status of the parent strain Aspergillus niger ATCC 9142, providing regulatory compliance advantages for food and pharmaceutical applications. In some cases, the genetic modifications achieved through chemical mutagenesis do not alter the fundamental safety characteristics of the organism, enabling continued use in applications requiring regulatory approval. The maintained safety profile facilitates commercial adoption and regulatory acceptance for industrial organic acid production applications.

[0313] Synergistic Performance Integration

[0314]

[0177] The chemical mutagenesis method employed to generate mutant strain MTCC 25759 creates a foundation for enhanced metabolic characteristics that work synergistically with optimized fermentation conditions and stable morphology to achieve superior organic acid production performance. The ethyl methane sulfonate treatment at specific exposure durations induces targeted genetic modifications that enhance cellular metabolism while maintaining structural integrity and fermentation stability.

[0315] Enhanced Enzyme Activity

[0316]

[0178] The enhanced glucose oxidase activity demonstrated by mutant strain MTCC 25759 improves oxidative metabolism pathways for both citric acid and gluconic acid biosynthesis. The increased enzyme activity facilitates more efficient conversion of glucose substrates through oxidative pathways, enabling enhanced substrate utilization and improved product formation rates. The glucose oxidase enhancement contributes to both direct gluconic acid formation through 202421061044

[0317] glucose oxidation and indirect citric acid formation through improved metabolic flux in oxidative pathways.

[0318] Integrated Metabolic Improvements

[0319]

[0179] The improved substrate utilization efficiency achieving 40.2-42.3% sugar conversion combines synergistically with stable pellet morphology to enhance oxygen transfer and maximize substrate-to-product conversion rates. The stable pellet formation maintains consistent surface area for oxygen transfer while preventing pellet disintegration that reduces mass transfer efficiency. The combination of enhanced substrate conversion and maintained pellet integrity creates conditions that support sustained metabolic activity throughout extended fermentation periods.

[0320] Acid Tolerance and pH Management

[0321]

[0180] The acid tolerance capabilities enabling operation at pH 1.39 for citric acid production and oscillatory pH ranges of 4.3-6.5 for gluconic acid production extend productive fermentation phases beyond conventional strain limitations. The enhanced acid tolerance allows continued metabolic activity under acidic conditions that typically inhibit wild-type strains, enabling sustained product accumulation and improved final yields. The pH tolerance also reduces the need for pH control interventions that increase process complexity and operational costs.

[0322] Process Efficiency Improvements

[0323]

[0181] The reduced byproduct formation demonstrated by mutant strain MTCC 25759 improves downstream processing efficiency while eliminating expensive enzyme supplementation requirements that reduce production costs. The cleaner fermentation profile simplifies product recovery and purification processes, reducing separation costs and improving product quality. The elimination of enzyme supplementation requirements reduces raw material costs while simplifying medium preparation and process control procedures.

[0324] Overall Performance Enhancement 202421061044

[0325]

[0182] The integrated performance characteristics achieve 198.836 g / L gluconic acid production representing a 66% improvement over the parent strain's 119.776 g / L yield under identical fermentation conditions. The sustained citric acid production reaches 0.415 g / L with consistent late-phase productivity that maintains product formation during extended fermentation periods. The enhanced yields result from the synergistic interaction of improved enzyme activity, enhanced substrate utilization, stable morphology, and acid tolerance working together to maximize metabolic efficiency.

[0326]

[0183] The synergistic combination of genetic modifications, metabolic enhancements, morphological stability, and process optimization creates a commercially viable production organism with superior performance characteristics across both citric acid and gluconic acid fermentation applications. The integrated improvements provide economic advantages through increased product yields, reduced production costs, improved process reliability, and simplified downstream processing requirements. The mutant strain demonstrates scalability from laboratory to industrial production levels while maintaining consistent performance characteristics that support commercial viability and market competitiveness.

[0327]

[0184] 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

202421061044WE CLAIM:

1. An Aspergillus niger mutant strain having accession number MTCC 25759 derived from parent strain Aspergillus niger ATCC 9142 through chemical mutagenesis using ethyl methane sulfonate, 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 the chemical mutagenesis is performed using ethyl methane sulfonate for 30-60 minutes.

3. The mutant strain as claimed in claim 1, comprising genetic modifications at sequence positions corresponding to SEQ ID NOs: 24337, 24344, 24353, 24355, 24366, 24374, 24502, 24522, 24525, 24526, 24530, 24557, 24586, 24592, 24620, 24625, 24630, 24632, 24640, 24646, 24647, 24648, 24653, 24728, 24729, 24730, 24769, 24800, 24802, 24808, 24821, 24843, 24923, 24949, 24951, 24999, 25000, 25001, 25088, 25107, 25108, 25113, 25116, 25139, 25142, 25312, and 25332.

4. The mutant strain as claimed in claim 1, wherein the gluconic acid production yield is 66% higher than the parent strain, achieving 119.776 g / L-198.836 g / L, and the citric acid production yield achieves 0.347 to 0.415 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 1.0-2.0 during citric acid production and minimum pH values of 4.3-6.5 during gluconic acid production.

6. A method for producing citric acid and gluconic acid with the mutant strain of claim 1, comprising:(a) subjecting the parent strain to chemical mutagenesis to generate mutant variants;(b) preparing a spore suspension of the mutant strain at 2 / I06CFU / mL for gluconic acid production or at 5*107CFU / mL for citric acid production;202421061044(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 fermenting in step (d) includes screening comprises measuring colony diameter and acid zone diameter to calculate acid unitage values for mutant selection.

8. The method as claimed in claim 6, wherein the recovery in step (e) comprises enzymatic assay quantification to determine organic acid concentrations in the fermentation broth.

9. The method as claimed in claim 6, wherein for gluconic acid production, the fermentation duration is 166-214 hours and for citric acid production, the fermentation duration is 316-382 hours.

Images