Genetically modified aspergillus niger strain for production of citric acid and gluconic acid and methods

Abstract

A genetically modified strain of Aspergillus niger designated MTCC 25758 derived from parent strain Aspergillus niger ATCC 9142 through chemical mutagenesis using ethyl methane sulfonate at 1 mg / mL for 180 minutes demonstrates enhanced production capabilities for both citric acid and gluconic acid. The modified strain exhibits genetic modifications at 64 specific sequence positions that contribute to improved metabolic pathways, enhanced substrate utilization efficiency, and increased tolerance to acidic fermentation conditions. The modified strain achieves gluconic acid production of 125.310 g / L representing a 4.6% improvement over the parent strain's 119.776 g / L yield, and citric acid production reaching 0.621 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] 202421061132

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

[0003] FIELD OF INVENTION

[0004] [1] The present invention relates to a genetically modified Aspergillus niger deposited with the Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology, Chandigarh and assigned accession number MTCC 25758 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. Modified strain is obtained through chemical mutagenesis method using ethyl methane sulfonate. Both the organic acids are industrially important and mainly produced through microbial fermentation.

[0005] BACKGROUND OF INVENTION

[0006] [2] Organic acids, particularly citric acid and gluconic acid, represent significant commercial products in the global fermentation industry with annual production volumes exceeding 2 million tons for citric acid and 100,000 tons for gluconic acid worldwide. These acids are produced primarily through microbial fermentation processes using various microorganisms, with Aspergillus niger being one of the most widely utilized fungal species for industrial organic acid production due to its robust metabolic pathways and ability to secrete high concentrations of organic acids. Citric acid serves as a tribasic acid with extensive applications as an acidulant, preservative, emulsifier, flavorant, sequestrant, and buffering agent across food, pharmaceutical, and chemical industries, representing approximately 70% of the global organic acid market. Gluconic acid functions as a mild organic acid with applications in pharmaceutical, textile, construction, and food industries, where it provides refreshing sour taste characteristics and serves 202421061132

[0007] as a mineral supplement precursor through its calcium and sodium salt derivatives.

[0008] [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 and gluconic acid based on substrate consumption.

[0009] [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 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. 202421061132

[0010] [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 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.

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

[0012] [7] It has been appreciated that a method is needed that overcomes one or more of these critical industrial limitations including inadequate production yields, inconsistent fermentation performance, poor substrate utilization efficiency, limited tolerance to industrial stress conditions, morphological instability during extended fermentation periods, and high production costs that 202421061132

[0013] compromise commercial viability and limit market accessibility for emerging applications requiring cost-effective organic acid supplies.

[0014] OBJECT OF THE INVENTION

[0015] [8] The primary object of the present invention is to provide a genetically modified Aspergillus niger strain that demonstrates enhanced production capabilities for both citric acid and gluconic acid.

[0016] [9] Another object of the present invention is to provide a genetically modified Aspergillus niger strain derived from parent strain Aspergillus niger (ATCC 9142) through chemical mutagenesis that exhibits superior organic acid production yields while maintaining stable fermentation characteristics.

[0017]

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

[0018]

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

[0019]

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

[0020]

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

[0021]

[0014] Still further object of the present invention is to provide a genetically modified strain that demonstrates enhanced substrate conversion efficiency while 202421061132

[0022] reducing production costs by eliminating the need for expensive enzyme supplementation required by prior art methods.

[0023]

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

[0024]

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

[0025]

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

[0026]

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

[0027]

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

[0028]

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

[0029] SUMMARY OF INVENTION

[0030]

[0021] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. 202421061132

[0031]

[0022] The present invention provides a genetically modified strain of Aspergillus niger designated MTCC 25758, derived from parent strain Aspergillus niger ATCC 9142 through chemical mutagenesis using ethyl methane sulfonate at a concentration of 1 mg / mL for 120 to 180 minutes at 30°C with shaking at 150 rpm, wherein the modified 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 180 minute exposure duration representing an optimal balance between mutagenic effectiveness and cellular integrity that results in improved acid production without compromising fermentation performance. The modified strain MTCC 25758 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 modified strain MTCC 25758 demonstrates enhanced production capabilities across extended fermentation periods, with gluconic acid production 119.776 g / L to 125.310 g / L at 214 hours, representing an improvement over the parent strain under identical conditions. For citric acid production, the modified strain achieves concentrations ranging from 0.391 g / L to 0.498 g / L at 382 hours also representing an improvement over the parent strain.

[0032] BRIEF DESCRIPTION OF FIGURES

[0033]

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

[0034]

[0024] FIG. 1 illustrates pellet morphology scores and starch residue scores over fermentation time for citric acid production for parent strain F-l and modified strain F-6, according to aspects of the present disclosure.

[0035]

[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. 202421061132

[0036]

[0026] FIG. 3 illustrates pH changes over fermentation time for citric acid production for modified strain F-6, according to aspects of the present disclosure.

[0037]

[0027] Fig. 4 illustrates a comparison of pH profiles between citric acid production for parent strain F-l and modified strain F-6, according to aspects of the present disclosure.

[0038]

[0028] FIG. 5 illustrates citric acid production over time for parent strain F-l and modified strain F-6, according to aspects of the present disclosure.

[0039]

[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.

[0040]

[0030] FIG. 7 illustrates sugar utilization percentage over fermentation time for gluconic acid production modified strain F-3, according to aspects of the present disclosure.

[0041]

[0031] FIG. 8 illustrates a comparison of sugar utilization percentages for gluconic acid production between parent strain F-l and modified strain F-3, according to aspects of the present disclosure.

[0042]

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

[0043]

[0033] FIG. 10 illustrates pH changes over fermentation time for gluconic acid production modified strain F-3, according to aspects of the present disclosure.

[0044]

[0034] FIG. 11 illustrates a comparison of pH changes over fermentation time for gluconic acid production of Parent strain F-l and modified strain F-3, according to aspects of the present disclosure

[0045]

[0035] FIG. 12 illustrates gluconic acid production over time for parent strain F-l and modified strain F-3, according to aspects of the present disclosure. 202421061132

[0046] DETAILED DESCRIPTION

[0047]

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

[0048]

[0037] Citric acid and gluconic acid represent commercially significant organic acids in global fermentation markets. Citric acid finds widespread application as an acidulant, preservative, emulsifier, flavorant, sequestrant, and buffering agent across food, pharmaceutical, and industrial sectors. Gluconic acid serves as a mild organic acid with applications in pharmaceutical, textile, building, and food industries. The global demand for these organic acids continues to increase, with citric acid dominating the organic acid market due to diverse applications, while gluconic acid maintains a substantial market presence.

[0049]

[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.

[0050]

[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 modified 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. 202421061132

[0051] Parent Strain

[0052] Spore Suspension Preparation

[0053]

[0040] The parent strain Aspergillus niger ATCC 9142 serves as the starting material for developing enhanced genetically modified strains with improved organic acid production capabilities. The parent strain is maintained on Potato Dextrose Agar (PDA) medium under controlled laboratory conditions to ensure consistent growth characteristics and sporulation patterns. In some cases, spore suspension preparation begins with harvesting spores from mature Aspergillus niger ATCC 9142 cultures that 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. In some cases, 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. In some cases, the surfactant prevents spore aggregation and ensures homogeneous spore distribution, which is beneficial for accurate counting and consistent inoculum preparation. The harvested spore suspension is collected in 202421061132

[0058] 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]

[0043] 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. In some cases, the haemocytometer provides a standardized counting grid that enables accurate determination of spore density per unit volume. Spore counting is performed under appropriate magnification to distinguish individual spores and obtain reliable concentration measurements. The initial spore concentration typically ranges from 6*107to 7*109CFU / mL, depending on the sporulation efficiency of the particular culture. In some cases, the concentration range reflects natural variation in sporulation density among different culture preparations and growth conditions.

[0061] Spore Suspension Standardization

[0062]

[0044] 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. In some cases, 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. In some cases, the controlled spore concentration enables accurate comparison of performance between different strains and experimental conditions.

[0063] Chemical Mutagenesis Process 202421061132

[0064]

[0045] The chemical mutagenesis process employs ethyl methane sulfonate (EMS) at a concentration of 1 mg / mL to induce genetic modifications in Aspergillus niger ATCC 9142 spores. In some cases, the chemical mutagenesis utilizes varying treatment durations including 120 minutes (DPPL 010), 150 minutes (DPPL 011), and 180 minutes (DPPL 012) to generate modified variants with different acid production capabilities. The ethyl methane sulfonate stock solution is prepared at 1 mg / mL in sterile distilled water and stored at 4°C. In some cases, 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.

[0065] Mutagenesis Setup

[0066]

[0046] The mutagenesis setup is conducted in sterile 250 mL conical flasks under controlled conditions. 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. In some cases, 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.

[0067]

[0047] The Vogel's Minimal Medium comprises sodium citrate at 125 g / L, potassium dihydrogen phosphate at 250 g / L, ammonium sulfate at 100 g / L, magnesium sulfate heptahydrate at 10 g / L, and calcium chloride dihydrate at 5 g / L. In some cases, the medium further includes trace element solution at 5 mL per liter and biotin solution at 2.5 mL per liter. The trace element solution contains citric acid monohydrate, zinc sulfate heptahydrate, ammonium iron(II) sulfate hexahydrate, copper(II) sulfate pentahydrate, manganese(II) sulfate monohydrate, boric acid, and sodium molybdate dihydrate. The biotin solution provides biotin at 5 mg per 50 mL of solution. 202421061132

[0068] EMS Treatment Protocol

[0069]

[0048] The EMS treatment protocol involves incubation at 30°C with shaking at 150 rpm for specific exposure durations. In some cases, the controlled temperature and agitation conditions ensure uniform distribution of the chemical mutagen throughout the spore suspension. Different exposure durations produce modified variants with varying characteristics, with the 180-minute exposure duration corresponding to strain designation DPPL 012. The 180-minute exposure 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. The Triton X-100 functions as a colony restrictor to limit colony expansion and facilitates isolation of individual colonies. The plating volume is 0.1 mL of each dilution to achieve appropriate colony density for selection purposes.

[0074] Incubation Conditions

[0075]

[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 202421061132

[0076] 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]

[0052] The genetically modified strain DPPL 012 is obtained through 180 minutes of ethyl methane sulfonate exposure at 1 mg / mL concentration. In some cases, the 180-minute exposure duration represents an extended treatment period that induces more extensive genetic modifications compared to shorter exposure times. The DPPL 012 strain demonstrates enhanced acid production capabilities as evidenced by an acid unitage value of 2.00, which exceeds the typical range of 1.73-1.80 observed for the parent strain under identical screening conditions. The acid unitage value is calculated as the ratio of the acid zone diameter to the colony diameter, providing a normalized measure of acid production efficiency that accounts for differences in colony size among different modified candidates.

[0078] Screening Methodology

[0079]

[0053] The primary screening methodology employs Bromocresol Green plate assays to systematically identify chemically-induced modified 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. In some cases, 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.

[0080] BCG Plate Preparation

[0081]

[0054] The BCG plate preparation involves formulating a medium containing Potato Dextrose Agar supplemented with Bromocresol Green indicator and 202421061132

[0082] additional agar for appropriate gel strength. For preparation of screening plates, the composition includes Potato Dextrose Agar powder at 39 g / L, additional agar powder at 15 g / L, and Bromocresol Green dye at 1 g / L. In some cases, the medium preparation process involves dissolving the PDA and additional agar components in distilled water with continuous stirring. The Bromocresol Green dye is separately dissolved in 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.

[0083]

[0055] Following sterilization, the medium is cooled to 45-50°C before pouring approximately 40 mL per Petri plate under aseptic conditions. In some cases, the plates are allowed to solidify completely at room temperature before use, with proper labeling to identify different modified strains and experimental conditions.

[0084] Colony Inoculation Protocol

[0085]

[0056] 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. In some cases, 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.

[0086]

[0057] The inoculated BCG plates are incubated at 30°C ± 2°C for 70-75 hours, corresponding to approximately 3 days of growth. In some cases, 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. 202421061132

[0087] Visual Observation and Assessment Criteria

[0088]

[0058] 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. In some cases, colonies lacking visible yellow zones or exhibiting only faint discoloration are categorized as non-producers or weak acid producers. Colonies demonstrating clear, intense, and measurable yellow zones are selected for continued characterization.

[0089] Quantitative Zone Measurement

[0090]

[0059] 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. In some cases, 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.

[0091] Acid Unitage Calculation

[0092]

[0060] The acid unitage value serves as a normalized measure of acid production efficiency that accounts for differences in colony size among different modified candidates. The calculation employs the formula: Acid Unitage = Zone of Acid (mm) / Colony Diameter (mm), where higher values indicate greater acid production relative to colony size. In some cases, the normalization approach ensures that modified with enhanced acid production capabilities are identified rather than selecting colonies with larger overall zones resulting from increased colony size.

[0093] Genetically Modified Strain Selection Criteria 202421061132

[0094]

[0061] The modified strain 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. In some cases, modified candidates demonstrating enhanced acid unitage values are prioritized for further evaluation, with particular emphasis on strains achieving superior performance metrics.

[0095] Modified Strain

[0096]

[0062] Among the screened modified candidates, strain DPPL 012 demonstrates superior acid production performance with an acid unitage value of 2.00. The modified strain exhibits a colony diameter of 25 mm and a total zonal diameter of 50 mm, resulting in an acid zone of 25 mm. In some cases, the 25 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 2.00 for strain DPPL 012 exceeds the typical range of 1.73-1.80 observed for the parent strain, indicating enhanced acidogenic potential resulting from the ethyl methane sulfonate mutagenesis treatment.

[0097]

[0063] The selection of strain DPPL 012 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 modified candidates evaluated during the screening process. The modified strain demonstrates consistent performance characteristics across duplicate screening plates, with reproducible zone formation and stable acid production patterns that indicate reliable genetic modifications.

[0098]

[0064] The enhanced performance characteristics of strain DPPL 012 result from the 180-minute exposure to ethyl methane sulfonate at 1 mg / mL concentration, which induces beneficial genetic modifications that improve metabolic flux through organic acid biosynthesis pathways. In some cases, the 202421061132

[0099] modified 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.

[0100]

[0065] The superior acid zone formation exhibited by strain DPPL 012 correlates with enhanced glucose oxidase activity and improved oxidative metabolism pathways that facilitate more efficient conversion of glucose substrates to organic acid products. In some cases, the modified strain demonstrates enhanced tolerance to acidic conditions that accumulate during organic acid production, enabling sustained metabolic activity under challenging fermentation environments.

[0101]

[0066] The modified strain DPPL 012 is subsequently deposited with the Microbial Type Culture Collection and Gene Bank (MTCC), Institute of Microbial Technology, Chandigarh, and is assigned accession number MTCC 25758. The deposit provides a permanent repository for the modified strain and enables access for research and commercial applications. In some cases, the MTCC designation serves as an official identifier for the modified strain in scientific literature and patent documentation.

[0102]

[0067] The designation MTCC 25758 represents the deposited form of strain DPPL 012, which has been derived through the 180-minute ethyl methane sulfonate exposure protocol. In some cases, the deposited strain maintains the enhanced acid production characteristics that were identified during the initial screening process, including the superior acid unitage value of 2.00 compared to the parent strain's typical range of 1.73-1.80. The strain designation provides a standardized reference for the modified organism that is used in subsequent fermentation studies and commercial applications.

[0103]

[0068] The modified strain MTCC 25758 exhibits genetic stability through multiple subculture passages, indicating that the induced mutations are stably inherited and maintained during vegetative propagation. In some cases, the genetic stability ensures consistent performance characteristics across different 202421061132

[0104] fermentation batches and enables reliable scale-up for commercial production applications. The mutation pattern observed in strain MTCC 25758 represents a combination of genetic changes that collectively contribute to the enhanced acid production phenotype.

[0105] Secondary Screening method

[0106]

[0069] Following primary screening using Bromocresol Green plates, selected modified candidates undergo secondary screening validation to provide quantitative confirmation of organic acid production capabilities. The secondary screening methodology employs multiple analytical techniques to verify and quantify the production of specific organic acids, ensuring accurate identification of modified with enhanced production characteristics.

[0107] Enzymatic Assay Methods

[0108]

[0070] 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.

[0109]

[0071] 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. In some cases, 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. In some cases, the change in 202421061132

[0110] absorbance (AAbs) between sample and blank measurements is calculated and converted to gluconic acid concentration using the provided conversion factors and calibration standards.

[0111]

[0072] The citric acid enzymatic assay utilizes citrate-specific enzymes that convert citric acid to products measurable through spectrophotometric analysis. The assay protocol follows similar principles to the gluconic acid kit but employs different enzyme systems specific for citric acid detection and quantification. In some cases, 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 modified strains and the parent strain under identical fermentation conditions.

[0112] Thin Layer Chromatography Analysis

[0113]

[0073] 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. In some cases, the chromatographic separation is developed using appropriate mobile phase solvents and visualized through chemical staining for organic acid identification by comparing migration distances with standard reference compounds.

[0114]

[0074] The TLC methodology involves applying small volumes of fermentation samples and standard solutions to designated positions on silica gel plates. The plates are developed in a solvent system that provides adequate separation of organic acids from other fermentation components. In some cases, the developed plates are treated with visualization reagents that react specifically with organic acids to produce visible spots. The intensity and position of spots are 202421061132

[0115] compared with standard reference compounds to provide qualitative assessment of organic acid production. TLC analysis serves as a complementary technique to enzymatic assays, providing visual confirmation of organic acid presence and relative concentrations.

[0116] Sugar Quantification

[0117]

[0075] The DNS A method quantifies reducing sugar concentrations through reduction of 3,5-dinitrosalicylic acid under alkaline conditions. The methodology involves mixing fermentation samples with DNSA reagent, followed by heating and spectrophotometric measurement at 540 nm to calculate sugar utilization percentage. In some cases, fermentation samples are appropriately diluted to bring sugar concentrations within the linear range of the assay. The DNSA reagent reacts with reducing sugars present in the sample, producing a colored complex that is measured spectrophotometrically.

[0118]

[0076] The sugar utilization percentage is calculated by comparing the reducing sugar concentration in fermentation samples with the initial substrate concentration. The calculation provides a quantitative measure of substrate consumption efficiency, which correlates with organic acid production capabilities. In some cases, higher sugar utilization percentages indicate more efficient metabolic conversion of carbon sources to organic acid products. The DNSA method enables monitoring of substrate consumption throughout fermentation processes, providing insights into metabolic activity and fermentation progress.

[0119] Iodine Starch Test

[0120]

[0077] The iodine starch test provides qualitative assessment of starch utilization based on blue-black color formation when iodine reacts with starch. The test methodology involves mixing fermentation samples with iodine solution and observing color changes that indicate the presence or absence of residual starch. In some cases, color intensity decreases as starch is hydrolyzed during 202421061132

[0121] fermentation, with complete utilization indicated by absence of blue coloration and development of brown coloration similar to the water blank control.

[0122]

[0078] The iodine starch test employs a standardized protocol involving addition of iodine solution to fermentation samples and comparison with control solutions. The test utilizes a scoring system where blue-black coloration indicates residual starch presence, while brown coloration indicates complete starch utilization. In some cases, starch utilization is assessed using a 0-3 scoring system where lower scores represent more complete hydrolysis. Periodic sampling throughout fermentation enables assessment of enzymatic activity and substrate conversion efficiency.

[0123] Result Interpretation and Starch Utilization Assessment

[0124]

[0079] 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. The DNSA method quantifies reducing sugar concentrations to calculate percentage substrate conversion, providing metabolic efficiency indicators that correlate with organic acid production capabilities.

[0125]

[0080] 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 scoring system where periodic sampling enables 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 modified 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 202421061132

[0126] BCG screening, ensuring accurate identification of modified strains with enhanced production characteristics.

[0127] Sequence Mutation Analysis

[0128]

[0081] Whole genome sequencing analysis of the modified strain MTCC 25758 reveals specific nucleotide sequence modifications that contribute to enhanced citric acid and gluconic acid production capabilities. The comprehensive genomic analysis identifies distinct sequence positions where targeted genetic alterations comprising nucleotide additions, deletions, or substitutions provide enhancement of organic acid biosynthesis pathways. The identified mutations occur at specific genomic positions that demonstrate correlation with improved citric acid and gluconic acid production capabilities, representing mutation sites that maximize metabolic flux through acid biosynthesis pathways.

[0129] Reference Position Identification

[0130]

[0082] The modified strain MTCC 25758 comprises genetic modifications at 64 specific sequence positions corresponding to SEQ ID NOs: 24359, 24365, 24369, 24374, 24515, 24527, 24528, 24531, 24536, 24537, 24567, 24569, 24572, 24577, 24596, 24597, 24600, 24634, 24635, 24648, 24652, 24653, 24654, 24657, 24659, 24693, 24748, 24750, 24753, 24781, 24789, 24790, 24795, 24821, 24822, 24826, 24829, 24831, 24835, 24837, 24839, 24846, 24874, 24924, 24932, 24936, 24987, 25001, 25006, 25011, 25122, 25141, 25190, 25191, 25205, 25307, 25308, 25381, 25382, 25383, 25395, 25397, 25478, and 25479. These specific modified sequences represent genetic modifications that provide enhancement for organic acid production, with each sequence contributing to the improved yield performance of strain MTCC 25758.

[0131] Metabolic Pathway Enhancement

[0132]

[0083] The sequence mutations identified in strain MTCC 25758 affect genes and regulatory elements involved in organic acid biosynthesis pathways. The 202421061132

[0133] 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 modified 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.

[0134] Strain Stability and Inheritance

[0135]

[0084] The genetic stability of the 64 sequence modifications is maintained through multiple subculture passages, indicating that the induced mutations are stably inherited during vegetative propagation. The stable inheritance of these genetic changes ensures consistent performance characteristics across different fermentation batches and enables reliable scale-up for commercial production applications. The mutation pattern represents a stable genetic configuration that preserves the enhanced acid production phenotype while maintaining normal growth and sporulation characteristics.

[0136] Phenotypic Expression of Genetic Changes

[0137]

[0085] The phenotypic expression of the 64 genetic modifications manifests as measurable improvements in organic acid production capabilities compared to the parent strain. The modified strain exhibits enhanced gluconic acid production reaching concentrations that exceed those achieved by the parent strain under identical fermentation conditions. For citric acid production, the modified strain demonstrates consistently higher acid concentrations across multiple fermentation time points, with production levels that surpass those of the parent strain throughout extended fermentation periods. The enhanced production capability is maintained throughout extended fermentation periods, indicating improved 202421061132

[0138] metabolic persistence and acid tolerance resulting from the collective effects of the 64 sequence modifications.

[0139] Pellet Morphology Stability

[0140]

[0086] The modified strain MTCC 25758 maintains stable pellet morphology throughout extended fermentation periods, contributing to improved oxygen transfer efficiency and enhanced substrate accessibility. In some cases, the stable pellet structure results from modified cell wall composition or growth regulation mechanisms that prevent excessive pellet disintegration during prolonged acid production phases. The maintained pellet integrity facilitates consistent fermentation performance and enables reproducible production yields across multiple fermentation batches.

[0141] Glucose Oxidase Activity Enhancement

[0142]

[0087] The modified strain MTCC 25758 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 modified 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.

[0143] Citric Acid Production

[0144]

[0088] The fermentation medium for citric acid production comprises a carefully balanced composition of carbon sources, nitrogen sources, and mineral nutrients to support growth and citric acid biosynthesis by the modified strain MTCC 25758. The medium composition is formulated to promote citric acid accumulation while maintaining nutritional balance for sustained fermentation performance throughout extended production periods. 202421061132

[0145] Medium Composition

[0146]

[0089] 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. In some cases, the supplemental dextrose provides readily available glucose for initial metabolic activity and rapid culture establishment. The starch-dextrose combination enables efficient substrate utilization and enhanced citric acid yields by ensuring continuous substrate availability throughout the fermentation period while supporting both growth and acid production phases.

[0147]

[0090] 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 balanced to support adequate biomass formation while maintaining nitrogen limitation conditions that favor citric acid accumulation. In some cases, potassium dihydrogen phosphate at 1 g / L provides phosphorus for 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.

[0148] Medium Preparation Protocol

[0149]

[0091] The fermentation medium preparation employs a three-flask distribution system to prevent precipitation and ensure complete dissolution of all medium components. Flask-A contains the starch and dextrose components dissolved in distilled water in a 500 mL flask. In some cases, 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. The three-flask distribution system prevents component interactions that could lead to precipitation or reduced nutrient availability during medium preparation. 202421061132

[0150] pH Control and Fermentation Conditions

[0151]

[0092] The initial pH of the fermentation medium is adjusted to 5.0 using appropriate pH adjustment reagents. The slightly acidic initial pH provides favorable conditions for spore germination and early growth phases while establishing the acidic environment that promotes citric acid biosynthesis. In some cases, 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.

[0152] Inoculation and Operating Parameters

[0153]

[0093] The fermentation conditions are specifically designed to support the metabolic requirements of strain MTCC 25758 for efficient citric acid biosynthesis. The spore inoculum preparation involves standardizing the concentration at 5*10A7 CFU / mL, which provides adequate spore density for rapid culture establishment while avoiding excessive initial biomass that could compete with acid production during early fermentation phases. In some cases, the standardized inoculum concentration ensures consistent fermentation initiation across different production batches and enables reproducible comparison of fermentation performance under identical starting conditions. The inoculum volume of 2.5 mL is added to 250 mL of fermentation medium under sterile conditions to prevent contamination.

[0154] Temperature and Agitation Control

[0155]

[0094] The temperature is maintained at 30°C throughout the entire production period, which represents the growth temperature for Aspergillus niger strains. The temperature provides favorable conditions for enzymatic reactions involved in citric acid biosynthesis while supporting efficient substrate 202421061132

[0156] metabolism and maintaining enzyme stability and catalytic activity for the metabolic pathways that convert carbon sources to citric acid. In some cases, 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 a balance between oxygen transfer and mechanical stress on fungal biomass while preventing excessive shear forces that could damage fungal pellets or disrupt pellet morphology.

[0157] Fermentation Duration and Volume

[0158]

[0095] The fermentation duration extends for 406 hours, corresponding to approximately 17 days of continuous production. The 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. In some cases, the prolonged fermentation time enables the modified strain to achieve maximum substrate conversion efficiency and acid yield recovery. The fermentation medium volume of 250 mL is contained within 500 mL flasks, providing a 1:2 ratio that ensures adequate headspace for oxygen transfer and gas exchange while preventing excessive evaporation during the extended fermentation period. The substantial headspace volume facilitates efficient oxygen transfer from the gas phase to the liquid medium, supporting the aerobic requirements of citric acid production.

[0159] Fermentation Method Overview

[0160]

[0096] 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 control facilitates citric acid accumulation through favorable thermodynamic conditions while inhibiting competing metabolic pathways. In some cases, the combination of controlled 202421061132

[0161] temperature at 30°C, moderate agitation at 25 RPM, appropriate medium -to-flask volume ratio of 1:2, and extended fermentation time of 406 hours creates conditions for citric acid biosynthesis by strain MTCC 25758. These parameters collectively support the aerobic metabolic requirements for citric acid production while maintaining environmental stability and substrate availability throughout the extended fermentation period.

[0162] Pellet Morphology and Starch Utilization Analysis

[0163]

[0097] Referring to Fig. 1, the pellet morphology characteristics and starch utilization patterns during citric acid fermentation provide indicators of fermentation performance and substrate conversion efficiency for both the parent strain F-l and the modified strain F-6. The pellet morphology scores are monitored over fermentation time periods extending from 118 to 220 hours, demonstrating the structural integrity and stability of fungal biomass throughout the production process. In some cases, the modified strain F-6 forms compact, spherical, uniform pellets that favor better oxygen diffusion and mass transfer compared to irregular dispersed mycelial forms that are exhibited by conventional strains.

[0164]

[0098] As shown in Fig. 1, at 118 hours of fermentation, both F-l and F-6 strains demonstrate high morphology scores approaching 3.0, indicating compact, well-formed pellets with intact structure during the early fermentation phase. The modified strain F-6 maintains superior pellet morphology characteristics compared to the parent strain F-l throughout the fermentation period, exhibiting more stable pellet structure with a gradual decline in morphology score. In some cases, the parent strain F-l demonstrates a more rapid decline in pellet morphology score, reaching approximately 1.0 at 220 hours, while the modified strain F-6 maintains better structural integrity with a morphology score of approximately 0.5 at the same time point.

[0165]

[0099] The uniform pellet morphology exhibited by the modified strain F-6 enhances oxygen transfer efficiency during aerobic fermentation processes. The 202421061132

[0166] compact spherical structure provides improved surface area to volume ratios, facilitating better gas exchange and 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. The maintained pellet structure contributes to consistent fermentation performance and reproducible production yields across multiple fermentation batches.

[0167] Starch Utilization Efficiency

[0168]

[0100] With continued reference to Fig. 1, the starch utilization patterns demonstrate progressive substrate consumption throughout the fermentation period for both strains. The starch residue scores show continuous reduction from 118 to 220 hours, indicating ongoing substrate consumption and metabolic activity. In some cases, both strains exhibit progressive reduction in starch residue scores from approximately 2.0 at 118 hours, declining to about 1.0 at 190 hours, and approaching near-zero values at 220 hours. The declining starch residue scores indicate complete starch utilization by the end of the fermentation period, confirming efficient substrate conversion capabilities.

[0169]

[0101] The modified strain F-6 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-6 achieves a starch residue score approaching 0, while F-l maintains a score of approximately 0.5, indicating more complete substrate utilization by the modified strain. In some cases, the superior starch utilization efficiency correlates with enhanced citric acid production capabilities and improved metabolic performance. Complete starch utilization is confirmed through iodine starch test results, which show color change from blue to brown, matching the water blank control and indicating successful hydrolysis of the starch substrate.

[0170]

[0102] The morphological differences between the strains reflect the enhanced fermentation characteristics of the modified strain, with better pellet 202421061132

[0171] integrity contributing to improved oxygen transfer efficiency and enhanced substrate utilization. The steeper decline in pellet morphology observed in the parent strain indicates less stable pellet structure and more pronounced pellet deterioration during later fermentation phases. In some cases, at 220 hours, both strains exhibit pellet burst morphology, indicating the natural progression toward pellet disintegration as substrate becomes depleted and fermentation approaches completion. The pellet burst morphology represents a normal transition phase in extended fermentation processes where pellet structure becomes less cohesive due to substrate limitation and metabolic changes.

[0172] pH Profiles and Acidification Dynamics

[0173]

[0103] Referring to Fig. 2, the pH dynamics during citric acid fermentation for the parent strain F-l demonstrate characteristic acidification patterns throughout the 406-hour fermentation period. The parent strain exhibits an initial pH of approximately 4.9 at time zero, establishing slightly acidic starting conditions that support spore germination and early metabolic activity. In some cases, the fermentation begins with rapid acidification during the first phase, with pH declining sharply to approximately 2.0 by 25 hours, corresponding to the establishment of active citric acid biosynthesis pathways. The parent strain F-l demonstrates a transient pH rise between 50 and 70 hours, with pH values increasing to approximately 3.2 during this period, suggesting a metabolic adjustment phase where cellular processes undergo regulatory changes. As shown in Fig. 2, the parent strain achieves its minimum pH of approximately 1.0 at 75 hours of fermentation, indicating substantial acid production capability. From 100 hours through 406 hours, the parent strain F-l maintains relatively stable pH conditions between 1.8 and 2.2, with the sustained acidic environment facilitating continued citric acid accumulation through favorable thermodynamic conditions that support acid biosynthesis while inhibiting competing metabolic pathways.

[0174]

[0104] Referring to Fig. 3, the pH profiles during citric acid fermentation provide indicators of fermentation progress and acid accumulation patterns for the 202421061132

[0175] modified strain MTCC 25758. The pH profile for modified strain F-6 throughout the fermentation period from 0 to 382 hours demonstrates the characteristic acidification pattern associated with citric acid biosynthesis. In some cases, the fermentation begins with an initial pH of approximately 4.75 at time zero, establishing slightly acidic starting conditions that support spore germination and early metabolic activity.

[0176]

[0105] The modified strain F-6 exhibits rapid initial acidification during the first phase of fermentation. The pH declines sharply to approximately 2.13 by 22 hours, corresponding to the establishment of active citric acid biosynthesis pathways. In some cases, the 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.

[0177]

[0106] Following the initial acidification period, the modified strain demonstrates a transient pH rise between 52 and 70 hours. pH values increase to approximately 3.12 during this period, suggesting a metabolic adjustment phase where cellular processes undergo regulatory changes. In some cases, the 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.

[0178]

[0107] As further shown in Fig. 3, the modified strain F-6 achieves a minimum pH of 0.76 at 76 hours of fermentation. The minimum pH value of 0.76 represents the most acidic condition reached during the fermentation process and indicates superior acid production capability compared to conventional strains. In some cases, the achievement of such low pH conditions demonstrates enhanced tolerance to acidic environments and more aggressive citric acid biosynthesis. The ability to maintain cellular viability and continued acid production under these highly acidic conditions reflects improved cellular adaptation mechanisms that enable the modified strain to function effectively in extreme acidic environments. 202421061132

[0179]

[0108] From 100 hours through 382 hours, the modified strain F-6 maintains stable pH conditions between 1.8 and 2.1, as demonstrated in Fig. 3. The stable pH maintenance exhibits greater consistency and less variation throughout the extended fermentation period compared to typical fermentation patterns. In some cases, the pH stability facilitates continued citric acid accumulation through favorable thermodynamic conditions that support acid biosynthesis while inhibiting competing metabolic pathways. The consistent acidic environment prevents acid degradation or conversion to alternative metabolic products, enabling sustained citric acid accumulation throughout the extended fermentation period.

[0180]

[0109] Referring to Fig. 4, the comparative pH profile analysis displays the pH dynamics of both parent strain F-l and modified strain F-6 throughout the citric acid fermentation process. The comparative analysis reveals that both strains exhibit similar overall acidification trajectories while displaying distinct performance characteristics. In some cases, both strains begin with initial pH values of approximately 4.9 for F-l and 4.75 for F-6, establishing comparable starting conditions for fermentation initiation.

[0181] [HO] The early acidification phase shows both strains experiencing rapid pH decline within the first 50 hours, with F-l reaching approximately 1.8 and F-6 achieving approximately 2.13 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 for F-l and 3.12 for F-6. In some cases, the metabolic adjustment phase appears consistent across both strain variants, suggesting that the fundamental regulatory mechanisms governing this transition remain intact in the modified strain.

[0182] [Hl] As shown in Fig. 4, the most pronounced difference between the strains occurs at 76 hours, where the modified strain F-6 achieves the dramatically lower minimum pH of approximately 0.76 compared to the parent strain F-l, which reaches approximately 1.0. The modified strain's ability to achieve the exceptionally low pH value of 0.76 demonstrates enhanced acid production 202421061132

[0183] capability and superior tolerance to highly acidic conditions. In some cases, the performance differential indicates that the genetic modifications in strain F-6 provide enhanced acidogenic potential while maintaining cellular viability under extreme acidic stress conditions.

[0184]

[0112] 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 modified strain F-6 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 modified strain demonstrates enhanced consistency in maintaining these favorable conditions. In some cases, the comparative pH profiles illustrate that while both strains follow similar fundamental acidification patterns, the modified strain F-6 exhibits enhanced performance characteristics including more aggressive initial acidification, superior minimum pH achievement, and improved pH stability throughout extended fermentation periods. The enhanced pH dynamics correlate with the improved citric acid production capabilities demonstrated by the modified strain under identical fermentation conditions.

[0185] Citric Acid Production Performance

[0186]

[0113] Referring to Fig. 5, the citric acid production performance of the modified strain MTCC 25758 demonstrates substantial yield improvements compared to the parent strain under identical fermentation conditions. The enzymatic kit analysis reveals distinct performance differences between the parent strain F-l and the modified strain F-6, with the modified strain achieving sustained late-phase productivity and consistent performance throughout extended fermentation phases. In some cases, the citric acid production profiles are measured at four time points during the fermentation process: 316, 334, 358, and 382 hours, providing comprehensive assessment of production capabilities across extended fermentation periods. 202421061132

[0187]

[0114] The modified strain F-6 achieves citric acid concentrations ranging from 0.530 g / L at 316 hours to 0.621 g / L at 334 hours, demonstrating enhanced production capabilities during the mid-to-late fermentation phases. As shown in Fig. 5, the modified strain exhibits a steady increase in citric acid production from 0.530 g / L at 316 hours, reaching a peak concentration of 0.621 g / L at 334 hours. In some cases, the progressive increase demonstrates sustained late-phase productivity and reflects improved metabolic control mechanisms that enable continued acid biosynthesis during extended fermentation periods. The modified strain maintains stable production characteristics with gradual increases that indicate enhanced tolerance to environmental stresses associated with prolonged acidic fermentation conditions.

[0188]

[0115] Following the peak production at 334 hours, the modified strain F-6 maintains consistent citric acid levels, achieving 0.496 g / L at 358 hours and sustaining production at 0.498 g / L at 382 hours. The sustained production capability provides advantages for commercial production applications where predictable yields and stable fermentation performance are required for economic viability. In some cases, the maintained productivity with concentrations above 0.496 g / L throughout the later fermentation phases indicates enhanced metabolic persistence under challenging fermentation conditions. The modified strain demonstrates superior fermentation robustness compared to conventional strains that experience declining productivity during extended fermentation periods.

[0189]

[0116] In contrast, the parent strain F-l exhibits more variable citric acid production patterns throughout the fermentation period. As depicted in Fig. 5, the parent strain achieves 0.432 g / L at 316 hours but experiences a decline to 0.257 g / L at 334 hours, followed by recovery to 0.317 g / L at 358 hours and reaching 0.391 g / L at 382 hours. In some cases, 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. 202421061132

[0190] Enzymatic Kit Quantification Methodology

[0191]

[0117] 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. In some cases, 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. The quantitative results provide definitive confirmation of the enhanced citric acid production capabilities demonstrated by the modified strain F-6 compared to the parent strain F-L

[0192] Comparative Yield Performance Analysis

[0193]

[0118] The comparative yield performance analysis demonstrates that the modified strain F-6 consistently outperforms the parent strain F-l across all analyzed time points. At 316 hours, the modified strain exhibits a significantly higher citric acid concentration of 0.530 g / L compared to the parent strain's 0.432 g / L, representing a 22.7% improvement in production efficiency. In some cases, at 334 hours, the modified strain achieves its peak production of 0.621 g / L while the parent strain experiences reduced output at 0.257 g / L, demonstrating a 141.6% enhancement in the modified strain's ability to maintain high productivity during challenging fermentation conditions. The sustained high yield of 0.498 g / L achieved by the modified strain at 382 hours exceeds the parent strain's final production of 0.391 g / L by 27.4%, confirming superior performance throughout the extended fermentation period.

[0194] Gluconic Acid Production

[0195]

[0119] The fermentation medium for gluconic acid production employs a precisely formulated composition of carbon sources, nitrogen sources, and 202421061132

[0196] mineral nutrients to support growth and gluconic acid biosynthesis by the modified strain MTCC 25758. The medium composition is specifically designed to promote gluconic acid accumulation through controlled nutrient availability while maintaining metabolic balance for sustained fermentation performance throughout extended production periods.

[0197] Medium Composition

[0198]

[0120] 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 balanced to maximize substrate utilization efficiency while preventing substrate inhibition effects that could reduce production yields. The elevated dextrose concentration provides sufficient carbon source to support both biomass formation and extensive gluconic acid production throughout the extended fermentation period.

[0199]

[0121] 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. In some cases, 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. The combination of organic and inorganic nitrogen sources ensures adequate nitrogen availability while supporting the metabolic requirements for enhanced gluconic acid production.

[0200]

[0122] The medium includes mineral components with magnesium sulfate heptahydrate at 5 g / L to supply magnesium ions for enzyme activation and cofactor functions in gluconic acid biosynthesis pathways. Potassium chloride is incorporated at 0.5 g / L to provide potassium ions for enzyme activation, cellular 202421061132

[0201] transport processes, and osmotic regulation. In some cases, ferric ammonium citrate is added at 0.01 g / L to supply iron ions for enzyme cofactor functions and electron transport processes that are involved in glucose oxidation reactions. 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 during fermentation.

[0202] Medium Preparation Protocol

[0203]

[0123] 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. In some cases, 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. The three-flask distribution system prevents component interactions that could lead to precipitation or reduced nutrient availability during medium preparation and sterilization processes.

[0204] pH Control and Sterilization

[0205]

[0124] 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. In some cases, 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. The separate sterilization approach prevents heat-induced interactions between medium components that could reduce nutrient availability or create inhibitory compounds. 202421061132

[0206] Fermentation Conditions

[0207]

[0125] The spore inoculum preparation is conducted at a standardized concentration of 2*10A6 CFU / mL for gluconic acid production applications. The inoculum concentration is specifically adjusted to provide adequate spore density for rapid culture establishment while avoiding excessive initial biomass that could compete with gluconic acid production during early fermentation phases. In some cases, 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 and maintain sterile fermentation conditions.

[0208] Temperature and Agitation Control

[0209]

[0126] 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. In some cases, the controlled temperature supports efficient substrate metabolism while maintaining enzyme stability and catalytic activity for glucose oxidation reactions that convert dextrose to gluconic acid. The temperature control ensures consistent enzymatic activity throughout the extended fermentation period while preventing thermal stress that could reduce production efficiency.

[0210]

[0127] 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 biosynthesis. In some cases, the glucose oxidase enzyme system requires continuous oxygen availability for oxidation reactions, making adequate aeration 202421061132

[0211] necessary 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 could damage fungal pellets or disrupt optimal pellet morphology.

[0212] Volume and Duration Parameters

[0213]

[0128] 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. In some cases, the prolonged fermentation time enables the modified strain to achieve maximum substrate conversion efficiency and gluconic acid yield recovery. The fermentation medium volume of 250 mL is contained within 500 mL flasks, providing a 1:2 ratio that ensures adequate headspace for oxygen transfer and gas exchange while accommodating the aerobic requirements of gluconic acid production.

[0214]

[0129] 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 present in the fungal cells. In some cases, the combination of controlled temperature at 30°C, moderate agitation at 25 RPM, appropriate medium-to-flask volume ratio of 1 :2, and extended fermentation time of 214 hours creates conditions for gluconic acid biosynthesis by strain MTCC 25758. These parameters collectively support the aerobic metabolic requirements for glucose oxidation while maintaining environmental stability and substrate availability throughout the extended fermentation period. 202421061132

[0215] Sugar Utilization Analysis

[0216]

[0130] Referring to Fig. 6, the sugar utilization profiles during gluconic acid fermentation provide quantitative assessment of substrate consumption efficiency for the parent strain F-l throughout the 214-hour fermentation period. The DNS A method is employed to monitor reducing sugar concentrations and calculate percentage substrate conversion, providing metabolic efficiency indicators that correlate with gluconic acid production capabilities. In some cases, the parent strain F-l exhibits a biphasic utilization pattern characterized by initial negative values during the early fermentation phase, followed by progressive increases in sugar consumption as metabolic activity intensifies.

[0217] Parent Strain Sugar Utilization Profile

[0218]

[0131] The initial phase of fermentation for the parent strain F-l demonstrates negative sugar utilization values extending from 0 to approximately 75 hours, indicating minimal substrate consumption during the lag and early exponential growth phases. The negative values reflect the analytical methodology's baseline variation rather than actual sugar production, representing the period where substrate consumption is below the detection threshold of the DNSA assay. In some cases, the extended lag phase indicates slower metabolic adaptation and delayed establishment of active glucose oxidation pathways in the parent strain.

[0219]

[0132] As shown in Fig. 6, following the initial lag phase, the parent strain F-1 demonstrates progressive increases in sugar utilization beginning around 94 hours of fermentation. The sugar consumption increases gradually throughout the remaining fermentation period, reaching a final utilization efficiency of 42.3% at 214 hours. In some cases, the gradual increase in substrate consumption correlates with the establishment of active gluconic acid biosynthesis pathways and enhanced glucose oxidase activity. The progressive utilization pattern indicates steady metabolic activity throughout the extended fermentation period, though at levels that are considered moderate compared to enhanced modified variants. 202421061132

[0220] Modified Strain Sugar Utilization Performance

[0221]

[0133] Referring to Fig. 7, the sugar utilization profile for the modified strain F-3 demonstrates enhanced substrate consumption efficiency compared to the parent strain throughout the 214-hour fermentation period. The modified strain exhibits a similar biphasic pattern with initial negative values during early fermentation phases, followed by more pronounced increases in sugar utilization as fermentation progresses. In some cases, the modified strain F-3 demonstrates more rapid establishment of active substrate consumption compared to the parent strain, indicating improved metabolic adaptation and enhanced enzymatic activity.

[0222]

[0134] The modified strain F-3 achieves superior sugar utilization efficiency of 44.6% at 214 hours compared to the parent strain's 42.3% under identical fermentation conditions. The enhanced utilization efficiency represents a 5.4% improvement in substrate conversion capability, indicating more efficient metabolic flux through glucose oxidation pathways. In some cases, the improved sugar utilization correlates with enhanced glucose oxidase activity and better substrate accessibility resulting from the genetic modifications present in the modified strain. The superior substrate conversion efficiency contributes to higher gluconic acid yields and improved overall fermentation performance.

[0223] Comparative Performance Analysis and Economic Implications

[0224]

[0135] Referring to Fig. 8, the comparative analysis of sugar utilization percentages between parent strain F-l and modified strain F-3 illustrates the performance differences throughout the complete fermentation period from 0 to 214 hours. Both strains initially undergo similar lag phases with negative or minimal sugar consumption up to approximately 75 hours, indicating comparable adaptation periods during early fermentation stages. In some cases, the similar initial phases suggest that the genetic modifications in the modified strain do not significantly affect early metabolic establishment but enhance performance during active production phases. 202421061132

[0225]

[0136] As further shown in Fig. 8, the modified strain F-3 exhibits more consistent and progressive increases in sugar utilization from 94 hours onward compared to the parent strain F-l. The modified strain demonstrates smoother substrate consumption patterns with less erratic fluctuations, indicating more stable metabolic control and improved enzymatic regulation during fermentation. In some cases, the consistent utilization pattern reflects enhanced metabolic stability and better adaptation to changing fermentation conditions throughout the extended production period.

[0226]

[0137] The comparative sugar utilization analysis reveals that while both strains achieve substantial substrate conversion by 214 hours, the modified strain F-3 consistently maintains higher utilization rates during the active fermentation phases. The modified strain's achievement of 44.6% sugar utilization represents more efficient carbohydrate assimilation pathways and enhanced enzymatic control during fermentation compared to the parent strain's 42.3% utilization. In some cases, the superior substrate conversion efficiency indicates better metabolic flux through glucose oxidation pathways, resulting in enhanced gluconic acid production capabilities and improved overall fermentation performance for commercial applications.

[0227] pH Profiles and Metabolic Dynamics

[0228]

[0138] Referring to Fig. 9, the pH dynamics during gluconic acid fermentation for the parent strain F-l demonstrate relatively stable pH patterns throughout the 214-hour fermentation period. The parent strain exhibits pH fluctuations within a moderate range, maintaining relatively consistent acidic conditions that support gluconic acid biosynthesis while avoiding extreme pH variations that could inhibit metabolic activity. In some cases, the parent strain demonstrates gradual pH changes that correlate with substrate consumption and organic acid accumulation, though the variations are less pronounced compared to enhanced modified variants. 202421061132

[0229]

[0139] The parent strain F-l maintains pH levels that fluctuate within a controlled range throughout the fermentation process, indicating steady metabolic activity and consistent acid production patterns. The pH profile shows periodic variations that correspond to different phases of fermentation, including initial adaptation phases, active growth periods, and sustained production phases. In some cases, the moderate pH fluctuations exhibited by the parent strain reflect conventional metabolic regulation mechanisms that maintain cellular homeostasis while supporting organic acid biosynthesis at standard production levels.

[0230]

[0140] Referring to Fig. 10, the pH profile for the modified strain F-3 demonstrates characteristic oscillatory pH behavior that distinguishes the enhanced strain from conventional parent strain performance. The modified strain exhibits dynamic pH fluctuations between 4.3 and 6.5 throughout the fermentation period, indicating enhanced metabolic activity and improved acid production capability. In some cases, the oscillatory pH pattern reflects more active metabolic processes and enhanced enzymatic activity that result from the genetic modifications present in the modified strain.

[0231]

[0141] The oscillatory pH behavior demonstrated by the modified strain F-3 represents more pronounced and dynamic variations compared to the parent strain's relatively stable pH patterns. The pH oscillations between 4.3 and 6.5 indicate enhanced glucose oxidase activity and more intensive substrate conversion processes that generate varying concentrations of gluconic acid and associated metabolic byproducts. In some cases, the dynamic pH variations reflect improved metabolic flux through oxidative pathways and enhanced cellular responses to changing substrate availability and product accumulation.

[0232]

[0142] The characteristic pH oscillations exhibited by the modified strain F-3 correlate with enhanced gluconic acid production capabilities and improved fermentation performance compared to the parent strain. The dynamic pH behavior indicates more active metabolic regulation and enhanced enzymatic control mechanisms that enable the modified strain to maintain higher production rates while adapting to changing fermentation conditions. In some cases, the 202421061132

[0233] oscillatory pH pattern reflects enhanced cellular adaptation mechanisms that allow the modified strain to function effectively under varying acidic conditions while maintaining sustained gluconic acid biosynthesis.

[0234]

[0143] The pH fluctuations between 4.3 and 6.5 demonstrated by the modified strain F-3 represent enhanced metabolic responsiveness to substrate availability and product accumulation throughout the fermentation period. The dynamic pH variations indicate improved cellular regulation of acid production pathways and enhanced tolerance to acidic fermentation environments. In some cases, the oscillatory behavior reflects more efficient metabolic cycling between different biochemical pathways that contribute to enhanced overall gluconic acid yields and improved substrate conversion efficiency compared to conventional parent strain performance.

[0235]

[0144] Referring to Fig. 11, illustrates comparative pH analysis between parent strain F-l and modified strain F-3 reveals distinct pH behavioral patterns that highlight the enhanced metabolic characteristics of the genetically modified strain throughout the 214-hour gluconic acid fermentation period. The comparative pH profiles demonstrate that both strains begin fermentation with similar initial pH values of approximately 5.0, establishing comparable starting conditions for metabolic activity and substrate utilization. In some cases, the similar initial pH conditions ensure that observed differences in fermentation performance result from strain-specific metabolic capabilities rather than variations in starting environmental parameters.

[0236]

[0145] The parent strain F-l exhibits relatively stable pH behavior throughout the fermentation period, maintaining pH values within a narrow range of approximately 4.5 to 5.5 with minimal fluctuations. The stable pH pattern indicates conventional metabolic regulation mechanisms that maintain steadystate conditions during gluconic acid production while avoiding dramatic environmental changes that could disrupt cellular homeostasis. In some cases, the moderate pH stability reflects standard fermentation dynamics where acid 202421061132

[0237] production and cellular buffering mechanisms achieve equilibrium conditions that support sustained but conventional production levels.

[0238]

[0146] In contrast, the modified strain F-3 demonstrates pronounced oscillatory pH behavior with dynamic fluctuations between 4.3 and 6.5 throughout the fermentation period. The oscillatory pattern exhibits regular cyclical variations that indicate enhanced metabolic activity and more intensive biochemical processes compared to the parent strain's stable behavior. In some cases, the pH oscillations reflect improved metabolic flux through glucose oxidation pathways and enhanced cellular responses to changing substrate concentrations and product accumulation during active fermentation phases.

[0239]

[0147] The amplitude and frequency of pH oscillations exhibited by the modified strain F-3 correlate with enhanced gluconic acid production capabilities and superior substrate utilization efficiency. The dynamic pH variations indicate more active metabolic cycling between different biochemical pathways that contribute to enhanced overall fermentation performance. In some cases, the oscillatory behavior demonstrates improved cellular adaptation mechanisms that enable the modified strain to maintain higher production rates while effectively managing the acidic conditions generated during intensive gluconic acid biosynthesis.

[0240]

[0148] The comparative analysis reveals that while the parent strain F-l maintains pH stability within a 1.0 unit range, the modified strain F-3 exhibits pH variations spanning a 2.2 unit range, indicating significantly more dynamic metabolic activity. The broader pH range demonstrates enhanced metabolic responsiveness and improved tolerance to varying acidic conditions that accumulate during fermentation. In some cases, the enhanced pH tolerance enables the modified strain to continue productive metabolism under conditions that would inhibit conventional strain performance, contributing to superior overall production yields and extended productive fermentation periods.

[0241] Gluconic Acid Production Performance 202421061132

[0242]

[0149] Referring to Fig. 12, the gluconic acid production performance of the modified strain F-3 demonstrates enhanced yield capabilities compared to conventional parent strain performance throughout the extended fermentation period. The enzymatic kit quantification reveals that the modified strain achieves gluconic acid concentrations that substantially exceed those obtained by parent strain fermentation under identical conditions. In some cases, the modified strain F-3 demonstrates progressive gluconic acid accumulation throughout the fermentation period, with production levels increasing steadily from initial detection through the final measurement at 214 hours.

[0243]

[0150] As shown in Fig. 12, the modified strain F-3 achieves a gluconic acid concentration of 125.310 g / L at 214 hours of fermentation, representing enhanced production efficiency compared to the parent strain's achievement of 119.776 g / L under identical fermentation conditions. The improved yield represents a 4.6% increase in gluconic acid production capability, indicating more efficient substrate conversion and enhanced metabolic flux through glucose oxidation pathways. In some cases, the superior production performance results from the genetic modifications present in the modified strain that enhance glucose oxidase activity and improve cellular tolerance to acidic fermentation environments.

[0244]

[0151] The gluconic acid production profile demonstrated by the modified strain F-3 exhibits consistent accumulation patterns throughout the fermentation period, with steady increases in acid concentration that correlate with substrate consumption and metabolic activity. The enzymatic kit quantification methodology provides precise measurement of gluconic acid concentrations, enabling accurate assessment of production capabilities and comparative evaluation against parent strain performance. In some cases, the enhanced production efficiency reflects improved enzymatic stability and enhanced cellular adaptation mechanisms that enable sustained acid biosynthesis under challenging fermentation conditions.

[0245]

[0152] The achievement of 125.310 g / L gluconic acid concentration by the modified strain F-3 demonstrates the enhanced metabolic capabilities resulting 202421061132

[0246] from the chemical mutagenesis treatment. The improved yield indicates more efficient utilization of the 220 g / L dextrose substrate provided in the fermentation medium, with enhanced conversion efficiency contributing to higher final product concentrations. In some cases, the superior production performance correlates with the enhanced sugar utilization efficiency of 44.6% demonstrated by the modified strain, indicating coordinated improvements in substrate consumption and product formation.

[0247]

[0153] The enhanced gluconic acid production of 125.310 g / L achieved by the modified strain F-3 represents improved commercial viability compared to conventional fermentation approaches using parent strains. The superior yield performance indicates enhanced process efficiency and improved economic potential for industrial gluconic acid production applications. In some cases, the combination of enhanced substrate utilization efficiency, improved acid tolerance, and superior production yields provides significant advantages for commercial fermentation processes where consistent high yields and stable performance are required for economic viability and competitive market positioning.

[0248]

[0154] The modified strain MTCC 25758 exhibits enhanced glucose oxidase activity that contributes to improved organic acid production capabilities compared to conventional parent strain performance. The genetic modifications present in the modified strain result in increased expression levels of glucose oxidase enzymes that catalyze the oxidation of glucose substrates to gluconic acid with enhanced efficiency. In some cases, the enhanced glucose oxidase activity facilitates more rapid substrate conversion and enables sustained acid production throughout extended fermentation periods under challenging environmental conditions.

[0249]

[0155] The improved metabolic flux through oxidative pathways represents a fundamental enhancement in the modified strain's biochemical capabilities. The genetic alterations modify regulatory mechanisms that control carbon flow through glucose oxidation reactions, directing more substrate toward organic acid biosynthesis rather than alternative metabolic pathways. In some cases, the 202421061132

[0250] enhanced oxidative metabolism results in more efficient utilization of available glucose substrates while reducing metabolic waste and improving overall substrate conversion efficiency.

[0251]

[0156] The enhanced glucose oxidase enzyme system in the modified strain MTCC 25758 demonstrates improved catalytic efficiency and enhanced stability under acidic fermentation conditions. The enzyme modifications result in increased specific activity and enhanced resistance to product inhibition that typically reduces enzyme performance in conventional strains. In some cases, the improved enzyme characteristics enable sustained glucose oxidation activity even under high product concentrations and low pH conditions that inhibit conventional glucose oxidase systems.

[0252]

[0157] The metabolic flux improvements extend beyond glucose oxidase activity to encompass enhanced coordination between different biochemical pathways involved in organic acid biosynthesis. The modified strain exhibits improved integration between glucose oxidation, citric acid cycle intermediates, and regulatory mechanisms that control acid production and secretion. In some cases, the coordinated metabolic enhancements result in more efficient substrate utilization while maintaining cellular energy balance and supporting sustained acid production capabilities.

[0253]

[0158] The enhanced substrate utilization efficiency demonstrated by the modified strain results from improved glucose transport systems and enhanced cellular uptake mechanisms. The genetic modifications affect membrane transport proteins that facilitate more rapid glucose uptake and ensure adequate substrate availability for the enhanced glucose oxidase enzyme systems. In some cases, the improved substrate accessibility contributes to higher overall conversion rates and enables the modified strain to achieve superior acid yields compared to conventional parent strain performance.

[0254]

[0159] The modified strain MTCC 25758 maintains stable pellet morphology throughout extended fermentation periods, contributing to improved oxygen transfer efficiency and enhanced substrate accessibility. The stable pellet structure 202421061132

[0255] results from modified cell wall composition or growth regulation mechanisms that prevent excessive pellet disintegration during prolonged acid production phases. In some cases, the maintained pellet integrity facilitates consistent fermentation performance and enables reproducible production yields across multiple fermentation batches.

[0256]

[0160] The stable pellet morphology enhances mass transfer characteristics during submerged fermentation processes. The compact spherical pellet structure provides improved surface area to volume ratios that facilitate better oxygen diffusion and nutrient distribution throughout the fermentation medium. In some cases, the enhanced mass transfer supports the aerobic requirements of glucose oxidase activity while ensuring adequate substrate availability for sustained acid production throughout extended fermentation periods.

[0257]

[0161] The enhanced acid tolerance exhibited by the modified strain enables sustained metabolic activity under low pH conditions that typically inhibit conventional fungal strains. The genetic modifications affect cellular pH homeostasis mechanisms and enhance the strain's ability to maintain enzymatic activity under acidic stress conditions. In some cases, the improved acid tolerance allows the modified strain to continue acid production even as product concentrations increase and fermentation pH decreases to levels that inhibit parent strain performance.

[0258]

[0162] The acid tolerance enhancements involve modifications to cellular membrane composition and transport systems that enable better management of intracellular pH under acidic fermentation conditions. The modified strain exhibits improved proton efflux mechanisms and enhanced buffering capacity that maintain cellular viability while supporting continued enzyme activity. In some cases, the enhanced acid tolerance enables the modified strain to achieve higher final product concentrations and extends productive fermentation periods compared to conventional strains that experience metabolic inhibition under similar conditions. 202421061132

[0259]

[0163] The modified strain MTCC 25758 demonstrates reduced formation of unwanted byproducts during fermentation processes, improving downstream processing efficiency and product purity. The genetic modifications enhance metabolic selectivity by directing carbon flux toward target organic acid production rather than alternative metabolic pathways that generate competing products. In some cases, the reduced byproduct formation simplifies purification requirements and lowers processing costs associated with product separation and refinement.

[0260]

[0164] The improved metabolic selectivity results from enhanced regulation of competing biochemical pathways that typically divert substrate away from organic acid biosynthesis. The modified strain exhibits reduced activity in pathways leading to secondary metabolite formation while maintaining enhanced flux through glucose oxidation and citric acid production routes. In some cases, the selective metabolic enhancement results in cleaner fermentation profiles with higher target product yields and reduced contamination from unwanted metabolic byproducts.

[0261]

[0165] The enhanced substrate conversion efficiency demonstrated by the modified strain eliminates the need for expensive enzyme supplementation that is required by conventional fermentation methods. The improved endogenous enzyme activity provides sufficient catalytic capacity for complete substrate utilization without external enzyme additions that increase production costs and complicate fermentation protocols. In some cases, the enhanced enzymatic capabilities reduce raw material expenses and improve the economic viability of organic acid production processes.

[0262]

[0166] The modified strain demonstrates improved nitrogen utilization efficiency compared to conventional parent strain performance. The enhanced nitrogen metabolism reduces requirements for expensive nitrogen sources during fermentation processes while maintaining adequate biomass formation and enzyme synthesis. In some cases, the improved nitrogen utilization enables the use of lower-cost nitrogen supplements while supporting enhanced organic acid 202421061132

[0263] production, contributing to reduced overall production costs and improved process economics.

[0264]

[0167] The combination of enhanced glucose oxidase activity, improved metabolic flux, stable pellet morphology, enhanced acid tolerance, and reduced byproduct formation creates synergistic effects that result in superior overall fermentation performance. The coordinated metabolic enhancements enable the modified strain MTCC 25758 to achieve higher organic acid yields while maintaining consistent performance characteristics across different fermentation conditions. In some cases, the integrated improvements provide significant advantages for commercial applications where reliable high-yield production and stable fermentation performance are required for economic viability and competitive market positioning.

[0265]

[0168] The modified strain MTCC 25758 demonstrates consistent batch-to-batch performance characteristics that provide significant advantages for industrial organic acid production applications. The genetic stability of the 64 sequence modifications ensures reproducible fermentation outcomes across multiple production cycles, reducing variability that affects product quality and yield consistency. In some cases, the standardized performance characteristics enable predictable production scheduling and facilitate quality control procedures that are required for commercial manufacturing operations. The consistent fermentation behavior reduces the need for extensive process monitoring and adjustment procedures that increase operational costs and complexity in industrial settings.

[0266]

[0169] The enhanced contamination resistance exhibited by the modified strain results from improved competitive advantages during fermentation processes. The rapid establishment of acidic conditions and enhanced acid production capabilities create environmental conditions that inhibit the growth of competing microorganisms that could compromise fermentation purity. In some cases, the superior acidification rates achieved by the modified strain establish protective pH conditions more quickly than conventional strains, reducing the 202421061132

[0267] window of vulnerability to contaminating organisms. The enhanced contamination resistance reduces the frequency of batch failures and improves overall production reliability in industrial fermentation facilities.

[0268]

[0170] The modified strain MTCC 25758 operates effectively under industrial stress conditions including elevated product concentrations, temperature fluctuations, and varying substrate compositions that challenge conventional fermentation systems. The enhanced acid tolerance and improved metabolic robustness enable sustained production activity under conditions that inhibit parent strain performance. In some cases, the industrial stress tolerance allows for more flexible operating parameters and accommodates variations in raw material quality that occur in commercial production environments. The enhanced stress tolerance reduces the need for precise environmental control systems that increase capital and operational costs.

[0269]

[0171] The substrate flexibility demonstrated by the modified strain enables utilization of diverse carbon sources including alternative sugars, industrial waste streams, and lower-grade raw materials that reduce feedstock costs. The enhanced enzymatic capabilities and improved metabolic flux support efficient substrate conversion across different carbon source compositions while maintaining high organic acid yields. In some cases, the substrate flexibility provides strategic advantages in volatile commodity markets by enabling feedstock substitution based on availability and cost considerations. The ability to utilize alternative substrates expands the range of economically viable production scenarios and improves overall process economics.

[0270]

[0172] The dual acid production capability of the modified strain MTCC 25758 enables simultaneous biosynthesis of both citric acid and gluconic acid within single fermentation processes, maximizing resource utilization and process efficiency. The coordinated metabolic pathways allow for flexible product ratios based on market demand and economic considerations while utilizing shared fermentation infrastructure. In some cases, the dual production capability provides operational flexibility that enables rapid response to changing market conditions 202421061132

[0271] and optimizes revenue generation from available production capacity. The integrated production approach reduces capital requirements compared to separate dedicated production systems for individual organic acids.

[0272]

[0173] The scalability characteristics of the modified strain enable successful transition from laboratory-scale development to industrial production levels while maintaining consistent performance characteristics. The stable pellet morphology and enhanced mass transfer properties translate effectively to larger fermentation vessels where mixing and oxygen transfer become more challenging. In some cases, the enhanced substrate utilization efficiency and improved acid tolerance provide advantages in large-scale systems where environmental gradients and longer residence times affect fermentation performance. The scalability reduces the technical risks associated with commercial implementation and accelerates the development timeline for industrial applications.

[0273]

[0174] The enhanced production capabilities translate to improved commercial viability through multiple economic advantages including reduced raw material costs, lower enzyme supplementation requirements, and decreased downstream processing expenses. The superior substrate conversion efficiency reduces feedstock consumption per unit of product while the enhanced enzymatic activity eliminates costly external enzyme additions. In some cases, the reduced byproduct formation simplifies purification processes and lowers separation costs while improving product purity and market value. The cleaner fermentation profiles reduce waste treatment requirements and improve environmental compliance in industrial facilities.

[0274]

[0175] The improved process reliability provided by the modified strain reduces operational risks and enhances the predictability of production outcomes for commercial planning purposes. The consistent fermentation performance enables more accurate production forecasting and reduces the need for safety inventory that ties up working capital. In some cases, the enhanced reliability improves customer satisfaction through more consistent product delivery schedules and strengthens market positioning in competitive environments. The 202421061132

[0275] reduced process variability enables optimization of production schedules and improves overall facility utilization rates.

[0276]

[0176] The economic advantages extend to reduced quality control requirements due to the consistent performance characteristics and improved product purity achieved by the modified strain. The standardized fermentation behavior reduces the frequency and extent of analytical testing required for batch release while the cleaner product profiles simplify quality assurance procedures. In some cases, the improved product consistency enables premium pricing strategies and accesses higher-value market segments that require superior product specifications. The enhanced quality characteristics provide competitive advantages in applications where product purity and consistency are particularly valued.

[0277]

[0177] The integrated performance enhancements create synergistic effects that amplify the individual advantages of each improved characteristic. The combination of enhanced substrate utilization, improved acid tolerance, stable morphology, and reduced byproduct formation works together to achieve overall performance improvements that exceed the sum of individual enhancements. In some cases, the coordinated metabolic improvements enable operating conditions and production strategies that are not feasible with conventional strains, opening new possibilities for process optimization and cost reduction. The synergistic integration provides sustainable competitive advantages that are difficult for competing technologies to replicate.

[0278]

[0178] The commercial implementation of the modified strain MTCC 25758 enables organic acid producers to achieve improved profit margins through reduced production costs and enhanced product yields while maintaining or improving product quality standards. The enhanced fermentation efficiency reduces energy consumption per unit of product through shorter fermentation times and reduced processing requirements. In some cases, the improved substrate utilization reduces waste generation and improves environmental sustainability metrics that are increasingly important for regulatory compliance and corporate 202421061132

[0279] responsibility objectives. The comprehensive performance improvements position organic acid production facilities for long-term competitiveness in evolving market conditions.

[0280]

[0179] 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

202421061132WE CLAIM:

1. A genetically modified Aspergillus niger strain having accession number MTCC 25758 derived from parent strain Aspergillus niger ATCC 9142 through chemical mutagenesis using ethyl methane sulfonate, wherein the modified strain demonstrates enhanced production capabilities for both citric acid and gluconic acid compared to the parent strain.

2. The genetically modified strain as claimed in claim 1, wherein the chemical mutagenesis is performed using ethyl methane sulfonate for 120-180 minutes.

3. The genetically modified strain as claimed in claim 1, comprising genetic modifications at 64 sequence positions corresponding to SEQ ID NOs: 24359, 24365, 24369, 24374, 24515, 24527, 24528, 24531, 24536, 24537, 24567, 24569, 24572, 24577, 24596, 24597, 24600, 24634, 24635, 24648, 24652, 24653, 24654, 24657, 24659, 24693, 24748, 24750, 24753, 24781, 24789, 24790, 24795, 24821, 24822, 24826, 24829, 24831, 24835, 24837, 24839, 24846, 24874, 24924, 24932, 24936, 24987, 25001, 25006, 25011, 25122, 25141, 25190, 25191, 25205, 25307, 25308, 25381, 25382, 25383, 25395, 25397, 25478, and 25479.

4. The genetically modified strain as claimed in claim 1, wherein the gluconic acid production achieves 125.310 g / L compared to the parent strain's 119.776 g / L, and the citric acid production achieves 0.498 g / L compared to the parent strain’s 0.391 g / L.

5. The genetically modified strain as claimed in claim 1, wherein the modified strain demonstrates enhanced tolerance to acidic fermentation conditions, achieving minimum pH values of 0.76 during citric acid production and oscillatory pH behavior between 4.3 and 6.5 during gluconic acid production.

Images