Enhancement of L-Asparaginase Production by Isolated <Emphasis Type="Italic">Bacillus circulans</Emphasis> (MTCC 8574) Using Response Surface Methodology

L-asparaginase production was optimized using isolated Bacillus circulans (MTCC 8574) under solid-state fermentation (SSF) using locally available agricultural waste materials. Among different agricultural materials (red gram husk, bengal gram husk, coconut, and groundnut cake), red gram husk gave the maximum enzyme production. A wide range of SSF parameters were optimized for maximize the production of L-asparaginase. Preliminary studies revealed that incubation temperature, moisture content, inoculum level, glucose, and L-asparagine play a vital role in enzyme yield. The interactive behavior of each of these parameters along with their significance on enzyme yield was analyzed using fractional factorial central composite design (FFCCD). The observed correlation coefficient (R ²) was 0.9714. Only L-asparagine and incubation temperature, were significant in linear and quadratic terms. L-asparaginase yield improved from 780 to 2,322 U/gds which is more than 300% using FFCCD as a means of optimizing conditions.     

Kinetics of Growth and Enhanced Sophorolipids Production by Candida bombicola Using a Low-Cost Fermentative Medium

In this study, effect of various parameters on sophorolipid (SL) production by the yeast Candida bombicola was investigated for the enhancing of its production by employing L18 orthogonal array design of experiments. At optimum conditions of sugarcane molasses 50 g l⁻¹, soybean oil 50 g l⁻¹, inoculum size 5% (v/v), temperature 30 °C, inoculum age 2 days, and agitation 200 rpm, the yeast produced almost equal amounts of the product in batch shake flasks and in a 3-l fermentor without any pH control (45 and 47 g l⁻¹, respectively). However, the yield increased to 60 g l⁻¹ in the fermentor under controlled pH environment. Time course of SL production, yeast biomass growth, and utilization of sugarcane molasses and soybean oil at these optimized conditions were fitted to existing kinetic models reported in the literature. Estimated kinetic parameters from these models suggested that conventional medium containing glucose can very well be replaced with the present low-cost fermentative medium.     

Optimization of Influential Parameters for Extracellular Keratinase Production by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> (MTCC9102) in Solid State Fermentation Using Horn Meal—A Biowaste Management

A Bacillus subtilis (MTCC9102) isolate was shown to produce significant amount of keratinase under optimized conditions in solid-state fermentation using Horn meal as a substrate. Optimized value for moisture, inoculum, and aeration were found to be 100% (v/w), 50% (v/w), and 150% (w/w), respectively, and the optimum nitrogen source was peptone and carbon source was dextrose. Maximum keratinolytic activity was observed at 48 h after incubation, and the optimum age (24 h) of inoculum was significant. The influence of cultivation temperature and initial pH of the medium on keratinase production revealed the optimum values for the temperature and pH as 37 °C and 7, respectively. Maximum keratinase activity of the crude extract was 15,972 U/mg/ml. These results indicate that this bacterial strain shows a high biotechnological potential for keratinase production in solid-state fermentation, and use of the horn meal as the substrate can be implemented for keratinous solid wastes management.     

Compactin Production Studies Using <Emphasis Type="Italic">Penicillium brevicompactum</Emphasis> Under Solid-State Fermentation Conditions

In the present study, compactin production by Penicillium brevicompactum WA 2315 was optimized using solid-state fermentation. The initial one factor at a time approach resulted in improved compactin production of 905 μg gds⁻¹ compared to initial 450 μg gds⁻¹. Subsequently, nutritional, physiological, and biological parameters were screened using fractional factorial and Box–Behnken design. The fractional factorial design studied inoculum age, inoculum volume, pH, NaCl, NH₄NO₃, MgSO₄, and KH₂PO₄. All parameters were found to be significant except pH and KH₂PO₄. The Box–Behnken design studied inoculum volume, inoculum age, glycerol, and NH₄NO₃ at three different levels. Inoculum volume (p = 0.0013) and glycerol (p = 0.0001) were significant factors with greater effect on response. The interaction effects were not significant. The validation study using model-defined conditions resulted in an improved yield of 1,250 μg gds⁻¹ compactin. Further improvement in yield was obtained using fed batch mode of carbon supplementation. The feeding of glycerol (20% v/v) on day 3 resulted in further improved compactin yield of 1,406 μg gds⁻¹. The present study demonstrates that agro-industrial residues can be successfully used for compactin production, and statistical experiment designs provide an easy tool to improve the process conditions for secondary metabolite production.     

Enhanced Fumaric Acid Production from Brewery Wastewater and Insight into the Morphology of Rhizopus oryzae 1526

The present work explores brewery wastewater as a novel substrate for fumaric acid production employing the filamentous fungal strain Rhizopus oryzae 1526 through submerged fermentation. The effects of different parameters such as substrate total solid concentrations, fermentation pH, incubation temperature, flask shaking speed, and inoculum size on the fungal morphologies were investigated. Different morphological forms (mycelium clumps, suspended mycelium, and solid/hairy pellets) of R. oryzae 1526 were obtained at different applied fermentation pH, incubation temperature, flask shaking speed, and inoculum size. Among all the obtained morphologies, pellet morphology was found to be the most favorable for enhanced production of fumaric acid for different studied parameters. Scanning electron microscopic investigation was done to reveal the detailed morphologies of the pellets formed under all optimized conditions. With all the optimized growth conditions (pH 6, 25 °C, 200 rpm, 5 % (v/v) inoculum size, 25 g/L total solid concentration, and pellet diameter of 0.465?±?0.04 mm), the highest concentration of fumaric acid achieved was 31.3?±?2.77 g/L. The results demonstrated that brewery wastewater could be used as a good substrate for the fungal strain R. oryzae 1526 in submerged fermentation for the production of fumaric acid.     

Optimisation of Batch Culture Conditions for Cell-Envelope-Associated Proteinase Production from Lactobacillus delbrueckii subsp. lactis ATCC? 7830?

Using a combination of conventional sequential techniques, the batch growth conditions for the production of cell-envelope-associated proteinases have for the first time been studied and optimised in Lactobacillus delbrueckii subsp. lactis 313 (ATCC 7830; LDL 313). Concentrations of inoculum (0.1?<?X?<?10?% vol/vol), agitation speed (0?<?S?<?200?rpm), varying incubation temperature (30?<?T?<?50?°C), starting pH (4.5?<?pH?<?7) and carbon/nitrogen ratio of production medium (0.2?<?r?<?5) had an individual effect on proteinase yield (p?<?0.01). Optimal conditions for proteinase production included an initial pH of 6.0, 45?°C incubation temperature, 2?% (v/v) inoculum size of OD₅₆₀?=?1, 150?rpm agitation speed, and growth medium carbon/nitrogen ratio of 1.0. Maximum proteinase activity obtained for whole cells was 0.99 U/ml after 8?h of incubation. The variables studied are very relevant due to their significance in improving the productivity of proteinase synthesis from LDL 313, under process and, likely, economic optimum conditions.     

Production and Characteristics of the Whole-Cell Lipase from Organic Solvent Tolerant <Emphasis Type="BoldItalic">Burkholderia</Emphasis> sp. ZYB002

The thermostable and organic solvent tolerant whole-cell lipase (WCL) was produced by Burkholderia sp. ZYB002 with broad spectrum organic solvent tolerance. The production medium of the WCL was primarily optimized, which resulted in the maximum activity of 22.8 U/mL and the 5.1-fold increase of the WCL yield. The optimized culture medium was as follows (% w/v or v/v): soybean meal 2, soybean oil 0.5, manganese sulfate 0.1, K₂HPO₄ 0.1, olive oil 0.5, initial pH 6.0, inoculum density 2, liquid volume 35 mL in 250-mL Erlenmeyer flask, and incubation time 24 h. The biochemical characterization of the WCL from Burkholderia sp. ZYB002 was determined, and the results showed that the optimal pH and temperature for lipolytic activity of the WCL was 8.0 and 65°C, respectively. The WCL was stable at temperature up to 70°C for 1 h and retained 79.2% of its original activity. The WCL was highly stable in the pH range from 3.0 to 8.5 for 6 h. Ca²⁺, K⁺, Na⁺, NO₃⁻, etc. ions stimulated its lipolytic activity, whereas Zn²⁺ ion caused inhibition effect. The WCL was also relatively stable in n-butanol at a final concentration of 50% (v/v) for 24 h. However, the WCL was strongly inhibited in Triton X-100 at a final concentration of 10% (v/v). The WCL with thermal resistance and organic solvent tolerance showed its great potential in various green industrial chemical processes.     

Keratinase Production by Endophytic <Emphasis Type="Italic">Penicillium</Emphasis> spp. Morsy1 Under Solid-State Fermentation Using Rice Straw

Among all endophytic keratinolytic fungal isolates recovered from marine soft coral Dendronephthya hemprichii, Penicillium spp. Morsy1 was selected as the hyperactive keratinolytic strain under solid substrate fermentation of different agriculture and poultry wastes. The optimization of extraction process, physicochemical parameters affecting the keratinase production in solid-state fermentation, and the purified keratinase parameters were studied. Maximum keratinase activity (1,600 U g⁻¹, initial dry substrate) was recovered from moldy bran with 0.1% Tween 80. The optimized production conditions were rice straw as carbon source, pH of medium 6, growth temperature 26 °C, initial moisture content of 80% (v/w), inoculum size of 10⁵ spores ml⁻¹, and an average particle size of the substrate 0.6 mm (3,560 U g⁻¹, initial dry substrate after 5 days of fermentation). Two types of keratinase (Ahm1 and Ahm2) were purified from the culture supernatant through ammonium sulfate precipitation, DEAE-Sepharose, and gel filtration chromatography. Enzyme molecular weights were 19 kDa (Ahm1) and 40 kDa (Ahm2). The kinetic parameters of purified keratinases were optimized for the hydrolysis of azokeratin by Ahm1 (pH 7.0–8.0, stable in pH range of 6.0 to 8.0 at 50 °C) and Ahm2 enzymes (pH 10.0–11.0, stable in pH range of 6.0 to 11.0 at 60–65 °C). Whereas inhibitors of serine (phenylmethylsulfonyl fluoride) and cysteine (iodoacetamide) proteases had minor effects on both Ahm1 and Ahm2 activity, both keratinases were strongly inhibited by chelating agents EDTA and EGTA. These findings suggest that serine and cysteine residues are not involved in the catalytic mechanisms, and they are metalloproteases.     

Synthesis,Characterization, and Oil Recovery Application of Biosurfactant Produced by Indigenous <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> WJ-1 Using Waste Vegetable Oils

A bacterial strain was isolated and cultured from the oil excavation areas in tropical zone in northern China. The biochemical characteristics and partial sequenced 16S rRNA gene of isolate, WJ-1, was identical to those of cultured representatives of the species Pseudomonas aeruginosa. This bacterium was able to produce a type of biosurfactant. Compositional analysis revealed that the extracted biosurfactant was composed of high percentage lipid (∼74%, w/w) and carbohydrate (∼20%, w/w) in addition to a minor fraction of protein (∼6%, w/w). The best production of 50.2 g/l was obtained when the cells were grown on minimal salt medium containing 6.0% (w/v) glucose and 0.75% (w/v) sodium nitrate supplemented with 0.1% (v/v) element solution at 37 °C and 180 rpm after 96 h. The optimum biosurfactant production pH value was found to be 6.0–8.0. The biosurfactant of WJ-1, with the critical micelle concentration of 0.014 g/L, could reduce surface tension to 24.5 mN/m and emulsified kerosene up to EI₂₄ ≈95. The results obtained from time course study indicated that the surface tension reduction and emulsification potential was increased in the same way to cell growth. However, maximum biosurfactant production occurred and established in the stationary growth phase (after 90 h). Thin layer chromatography, Fourier transform infrared spectrum, and mass spectrum analysis indicate the extracted biosurfactant was affiliated with rhamnolipid. The core holder flooding experiments demonstrated that the oil recovery efficiency of strain and its biosurfactant was 23.02% residual oil.     

Green Pigment from <Emphasis Type="Italic">Bacillus cereus</Emphasis> M<Superscript>1</Superscript><Subscript>16</Subscript> (MTCC 5521): Production Parameters and Antibacterial Activity

A bacterial strain, Bacillus cereus M¹ ₁₆ (MTCC 5521), isolated and identified in our laboratory produces a green pigment when grown in nutrient broth at stationary condition. Optimum fermentation parameters for maximum pigment production are pH 7.0, temperature 30°C, time of incubation 72 h and inoculum volume 1% from 20 h grown cell suspension. Magnesium ion enhances pigment production whereas calcium and zinc ions inhibit the process. The pigment is better extracted from the fermented broth with chloroform in comparison with diethyl ether, ethyl acetate, and butanol. The extracted crude pigment consists of three fractions as revealed from thin layer chromatogram on silica gel GF254 using ethyl acetate and hexane (1:1) solvent system. The major fraction C₃ shows antibacterial activity against different gram positive bacteria. The proposed structure of C₃ is 9-methyl-1,4,5,8-tetra-azaphenanthrene obtained by elemental analysis, GC-MS, and NMR spectra studies.     

Kinetic and Stoichiometric Parameters in the Production of Carotenoids by <Emphasis Type="Italic">Sporidiobolus salmonicolor</Emphasis> (CBS 2636) in Synthetic and Agroindustrial Media

With the objective of determining the kinetic behavior (growth, substrate, pH, and carotenoid production) and obtain the stoichiometric parameters of the fermentative process by Sporidiobolus salmonicolor in synthetic and agroindustrial media, fermentations were carried out in shaken flasks at 25°C, 180 rpm, and initial pH of 4.0 for 120 h in the dark, sampling every 6 h. The maximum concentrations of total carotenoids in synthetic (913 μg/L) and agroindustrial (502 μg/L) media were attained approximately 100 h after the start of the fermentative process. Carotenoid bioproduction is associated with cell growth and the ratio between carotenoid production and cell growth (Y _P/X) is 176 and 163 μg/g in the synthetic and agroindustrial media, respectively. The pH of the agroindustrial fermentation medium varied from 4.2 to 8.5 during the fermentation. The specific growth rate (μ _X) for S. salmonicolor in synthetic and agroindustrial media was 0.07 and 0.04 h⁻¹, respectively. The synthetic medium allowed for greater productivity, obtaining maximum cell productivity (P _x) of 0.08 g L⁻¹ h⁻¹ and maximum total carotenoid productivity (P _car) of 14.2 μg L⁻¹ h⁻¹. Knowledge of the kinetics of a fermentative process is of extreme importance when transposing a laboratory experiment to an industrial scale, as well as making a quantitative comparison between different culture conditions.     

Alkaline Protease Production from <Emphasis Type="Italic">Brevibacterium luteolum</Emphasis> (MTCC 5982) Under Solid-State Fermentation and Its Application for Sulfide-Free Unhairing of Cowhides

Enzyme-based unhairing in replacement of conventional lime sulfide system has been attempted as an alternative for tackling pollution. The exorbitant cost of enzyme and the need for stringent process control need to be addressed yet. This study developed a mechanism for regulated release of protease from cheaper agro-wastes, which overcomes the necessity for stringent process control along with total cost reduction. The maximum protease activity of 1193.77 U/g was obtained after 96 h of incubation with 15% inoculum of the actinomycete strain Brevibacterium luteolum (MTCC 5982) under solid-state fermentation (SSF). The medium after SSF was used for unhairing without the downstream processing to avoid the cost involved in enzyme extraction. This also helped in the regulated release of enzyme from bran to the process liquor for controlled unhairing and avoided the problem of grain-pitting. Unhairing process parameters were standardized as 20% enzyme offer, 40% Hide-Float ratio at 5 ± 1 rpm, and process pH of 9.0. The cost of production of 1000 kU of the protease was calculated as 0.44 USD. The techno-economic feasibility studies for setting up an SSF enzyme production plant showed a high return on investment of 15.58% with a payback period of 6.4 years.     

Effects of Pluronic F68 on Manganese Peroxidase Production by Pelletized <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

In this study, a new process was developed for manganese peroxidase (MnP) production by Phanerochaete chrysosporium under an agitated and aerated cultivation condition. It was found that change of the inoculum from spore suspension to pellets resulted in enhanced MnP production of 200 U/L in rotated shake flasks. Several additives, including Pluronic F68, Tween 80, and PEG8000, significantly increased the enzyme production. With an optimal concentration in 125 mL flasks, Pluronic F68 increased MnP productivity by 180%. Moreover, successful enzyme production was achieved in a 5-L fermentor at an agitation speed of 300 rpm with the addition of 0.1% Pluronic F68.     

A New <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis> Strain Producing Laccase. Use in Decolorization of Synthetics Dyes

Laccase activity was detected in a soil bacterium Stenotrophomonas maltophilia AAP56 identified by biochemical and molecular methods. It was produced in cells at the stationary growth phase in Luria Bertani (LB) medium added by 0.4 mM copper sulfate. The addition of CuSO₄ in culture medium improved production of laccase activity. However, one laccase enzyme was detected by native polyacrylamide gel electrophoresis. The enzyme showed syringaldazine (K _m = 53 μM), 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (K _m = 700 μM), and pyrocatechol (K _m = 25 μM) oxidase activity and was activated by addition of 0.1% (v/v) Triton-X-100 in the reaction mixture. Moreover, the laccase activity was increased 2.6-fold by the addition of 10 mM copper sulfate; the enzyme was totally inhibited by ethylenediaminetetraacetic acid (5 mM), suggesting that this laccase is a metal-dependant one. Decolorization activity of some synthetic dyes (methylene blue, methyl green, toluidine blue, Congo red, methyl orange, and pink) and the industrial effluent (SITEX Black) was achieved by the bacteria S. maltophilia AAP56 in the LB growth medium under shaking conditions.     

Optimization of fermentation conditions for production of glycopeptide antibiotic vancomycin by Amycolatopsis orientalis

Glycopeptides produced by Streptomyces species are the drugs used against β-lactam drug-resistant staphylococcal infections, and vancomycin is important among them. Increased prevalence of resistant strains increased the usage of vancomycin worldwide and also promoted attempts for indigenous production. The optimum process conditions pH, temperature, inoculum size, agitation, and aeration for vancomycin production by Amycolatopsis orientalis were evaluated, statistically analyzed, and the response surface curves were constructed. The optimum process conditions were a pH of 7.6, a temperature of 29°C, an inoculum size of 4.5%, an agitation of 255 rpm, and an aeration of less than 1:10 medium-to-air ratio.     

Phytase Production by a Marine Yeast <Emphasis Type="Italic">Kodamea ohmeri</Emphasis> BG3

The marine yeast strain Kodamea ohmeri BG3 isolated from the gut of a marine fish (Hexagrammes otakii) was found to secrete a large amount of phytase into the medium. The crude phytase produced by this marine yeast showed the highest activity at pH 5.0 and 65 °C. The optimal medium for phytase production contained oat 10.0 g/l, ammonium sulfate 15.0 g/l, glucose 30 g/l, and NaCl 20.0 g/l, while the optimal cultivation conditions for phytase production were pH 5.0, a temperature of 28 °C, and a shaking speed of 170 rpm. Under the optimal conditions, over 557.9 mU/ml of phytase activity was produced within 72 h of fermentation at the shake flask level. This is a very high level of phytase activity produced by yeasts. We think that the medium and process for phytase production by the marine yeast strain were very simple, and such marine yeast from the gut of natural marine fish may have a potential application in the maricultural industry and marine environmental protection. The results demonstrate that phytate was actively degraded by the crude phytase within a short period.     

Enhanced Production of an Extracellular β-<Emphasis Type="SmallCaps">d</Emphasis>-Fructofuranosidase Fructohydrolase from a 2-Deoxy-<Emphasis Type="SmallCaps">d</Emphasis>-glucose Stabilized Mutant of <Emphasis Type="Italic">Candida utilis</Emphasis>

The enzyme β-d-fructofuranosidase fructohydrolase (FFH) cleaves the α-1,4 glycosidic linkage between α-d-glucose and β-d-fructose molecules of sucrose, releasing monosaccharides by hydrolysis. In the present study, FFH production in Candida utilis GC-46, a lipolytic wild yeast strain was improved by exposure to N-methyl N-nitro N-nitroso guanidine (NG) and 2-deoxy-d-glucose (2dg) at various levels. The mutant strain NG-5 was obtained after exposure to 0.06 mg/ml of NG for 20 min. NG-5 offers improved extracellular FFH production (34 ± 2.6 U/ml/min) when compared to the wild strain (1.15 ± 0.01 U/ml/min). A 40-fold increase of FFH (45.65 ± 2.0 U/ml/min) was achieved when the process parameters, including incubation period (48 h), sucrose concentration (5.0 g/l), initial pH (6.0), inoculum size (2.0% v/v, 16 h old), and urea concentration (0.2%, w/v) were identified using Plackett–Burman design. The kinetic parameters viz. Q _p (0.723 U/g/h), Y _p/s (2.036 U/g), and q _p (0.091 U/g yeast cells/h) indicate that NG-5 is a hyperproducer of extracellular FFH with a concomitant increase in growth rate. The volumetric productivity of NG-5 was over sixfold improved over the parental strain. The enzyme production improvement is highly significant (HS, LSD 0.042, p ≤ 0.05), indicating commercial utility.     

Biodegradation of organophosphorus insecticide chlorpyrifos into a major fuel additive 2,4-bis(1,1 dimethylethyl) phenol using white-rot fungal strain Trametes hirsuta MTCC-1171

Biodegradation of chlorpyrifos, a widely used organophosphorus insecticide, was accomplished by using a white-rot fungal strain (Trametes hirsuta MTCC-1171). The experimental results showed that the fungal strain can effectively and rapidly degrade chlorpyrifos while using it as a sole source of carbon and energy when provided with mineral salt medium (MSM). The optimum experimental conditions for degradation of chlorpyrifos in liquid media can be summed as follows: initial pH 6.0; mycelial inoculum 0.18 ​± ​0.01 ​g ​L⁻¹ (dry weight); chlorpyrifos concentration 150 ​mg ​L⁻¹; pH 6.0; temperature 30 ​°C; and shaking speed 150 ​rpm. Under these optimal experimental parameters, T. hirsuta MTCC-1171 achieved ≥95% degradation of chlorpyrifos in 16 ​h of incubation. The degradation rate was quantified by employing HPLC followed by identification of degradation metabolites using gas chromatography–mass spectrometry (GC-MS). 2,4-Bis (1,1 dimethylethyl) phenol, a fuel additive, was found to be a major metabolite product of chlorpyrifos degradation. However, no metabolite bioaccumulation was observed in the process. Additionally, soil studies were carried out to investigate the degradation ability of the strain against chlorpyrifos, in a natural environment. During the assessment 37 ​± ​2.3% degradation was observed after 15 days of incubation. These results illustrate that T. hirsuta MTCC-1171 has a potential of using chlorpyrifos as a sole source of carbon. Besides, fundamental understanding gained through this work lays a foundation to investigate efficient and rapid bioremediation processes in agricultural and forest environments.     

Sorption efficiency of a new sorbent towards uranyl: phosphonic acid grafted Merrifield resin

A novel sorbent resin consisting of a Phosphonic Acid grafted on Merrifield Resin (PA-MR) for the extraction of uranyl from nitrate media is described. The sorption behaviour of uranyl cation on PA-MR was investigated using batch equilibrium technique. The effects of parameters such as shaking speed, pH levels, contact time, metal concentrations, ionic strength and temperature were reported. The results show that the sorption capacity increases with increasing both initial uranyl ion concentration and temperature and decreases with increasing ionic strength. Therefore, the optimum condition for the present study should be using 6.6 mg adsorbent per 1.0 mg uranyl in solution with pH 3.6 and shaking at 250 rpm for 180 min. The adsorption behavior of the system was also investigated and found to be in line with Langmuir isotherm. The kinetic data was well described by the pseudo second-order. Thermodynamics data leads to endothermic process ∆H = + 31.03 kJ⁻¹ mol⁻¹, ∆S = + 146.64 J mol⁻¹ K⁻¹ and ∆G = −11.96 kJ mol⁻¹ at 20 K. ∆G decreased to negatives values with increasing temperature indicating that the process was more favoured at high temperature.     

Statistical and evolutionary optimisation of operating conditions for enhanced production of fungal <Emphasis Type="SmallCaps">l</Emphasis>-asparaginase

A three-level central composite design of the Response Surface Methodology and the Artificial Neural Network-linked Genetic Algorithm were applied to find the optimum operating conditions for enhanced production of l-asparaginase by the submerged fermentation of Aspergillus terreus MTCC 1782. The various effects of the operating conditions, including temperature, pH, inoculum concentration, agitation rate, and fermentation time on the experimental production of l-asparaginase, were fitted to a second-order polynomial model and non-linear models using Response Surface Methodology and the Artificial Neural Network, respectively. The Artificial Neural Network model fitted well, achieving a higher coefficient of determination (R ² = 0.999) than the second-order polynomial model (R ² = 0.962). The l-asparaginase activity (38.57 IU s mL⁻¹) predicted under the optimum conditions of 32.08°C, pH of 5.85, inoculum concentration of 1 vol. %, agitation rate of 123.5 min⁻¹, and fermentation time of 55.1 h was obtained using the Artificial Neural Networklinked Genetic Algorithm in very close agreement with the activity of 37.84 IU mL⁻¹ achieved in confirmation experiments.