Initial Medium pH Research Articles

Optimization of Citrus Pulp Waste-Based Medium for Improved Bacterial Nanocellulose Production.

Bacterial nanocellulose (BC) has attracted significant attention across a wide array of applications due to its distinctive characteristics. Recently, there has been increasing interest in leveraging waste biomass to improve sustainability in BC biogenesis processes. This study focuses on optimizing the citrus pulp waste (CPW) medium to enhance BC production using Komagataeibacter sucrofermentans. The screening of initial medium pH, yeast extract, CPW sugar and inoculum concentrations was conducted using the Plackett-Burman design, with BC yield (mgDW/gCPW) as the model response. The significant parameters, i.e., CPW sugars and yeast extract concentrations, were optimized using response surface methodology, employing a five-level, two-factor central composite design. The optimized CPW-based growth medium resulted in a final yield of 66.7 ± 5.1 mgDW/gCPW, representing a 14-fold increase compared to non-optimized conditions (4.3 ± 0.4 mgBC/gCPW). Material characterization analysis indicated that the produced BC showed high thermal stability (30% mass retained at 600 °C) and a crystallinity index value of 71%. Additionally, to enhance process sustainability, spent baker's yeast hydrolysate (BYH) was assessed as a substitute for yeast extract, leading to a final BC titer of 9.3 ± 0.6 g/L.

Reusable Immobilized Lactobacillus futsaii CS3 for Enhanced GABA Synthesis using Low-Cost Substrates in Fermenter-Scale Batch and Fed-Batch Fermentations

On the industrial scale, the cost of commercial culture media for the production of gamma-aminobutyric acid (GABA) is a very important factor. This study employed a low-cost substrates and by-product from agri-food industry for GABA synthesis by Lact. futsaii CS3 at the fermenter-scale. Lact. futsaii CS3 cells were immobilized in 3 % (w/v) sodium alginate and employed for GABA synthesis in the optimal modified MRS medium (3.48 % (w/v) cane sugar, 3.84 % (w/v) tuna condensate waste and 10.77 % (w/v) monosodium glutamate (MSG)) with the initial pH medium of 5, fermentation temperature at 37 °C and agitation speed at 30 rpm. During the 60th h of batch fermentation without pH control, immobilized Lact. futsaii CS3 efficiently transformed MSG into the highest GABA of 19.05 g/L, achieving the volumetric productivity of 0.32 g/L/h with a bioconversion rate of 29.01 %. To further enhance GABA production, MSG (7.5 % w/v) was fed into the fermenter during the 48th h of fed-batch fermentation, aiming to amplify GABA synthesis. The maximum GABA (26.28 g/L) was synthesized at 84 h of fed-batch fermentation, and the volumetric productivity and a bioconversion rate of 0.31 g/L/h and 23.59 %, respectively, were obtained. Fed-batch fermentation significantly outperformed batch fermentation, resulting in a 37.95 % increase in GABA production. Moreover, immobilized Lact. futsaii CS3 cells could be reused in 2 batch cycles. In the 1st reusability, the maximum GABA synthesis reached 25.94 g/L, and the volumetric productivity and a bioconversion rate of 0.31 g/L/h and 23.29 %, respectively, were achieved at 84 h of fed-batch fermentation. While in the 2nd reusability, GABA synthesis decreased to 23.29 g/L, with the volumetric productivity and a bioconversion rate of 0.28 g/L/h and 20.91 %, respectively, at 84 h of fed-batch fermentation. HIGHLIGHTS GABA production utilizing agri-food industry by-product. GABA production was enhanced by cane sugar and tuna condensate waste. Immobilized futsaii CS3 produced GABA 19.05 g/L (batch). Immobilized futsaii CS3 produced GABA 26.28 g/L (fed-batch). Immobilized futsaii CS3 could be reused in 2 batch cycles on GABA production. GRAPHICAL ABSTRACT

Characterization of newly isolated Bacillus cereus D3 Co-producing α-amylase and protease and its application in food raw materials

Bacillus cereus, which effectively secretes various extracellular enzymes, has been widely used in the food, feed, and fermentation industries. One strain co-producing α-amylase and protease was newly isolated from Daqu and identified as B. cereus D3. It was shown that the production of α-amylase and protease co-produced by B. cereus D3 reached 13.89 ± 0.07 and 2468.45 ± 38.96 U/mL through optimization of fermentation conditions (nutrients (g/L): 20 of glucose, 20 of urea, 0.7 of MnCl2; incubation conditions: 24 h of incubation, initial medium pH of 7.0, temperature of 37 °C, 8% inoculum size, and 30% working volume). Moreover, the enzymatic genetic co-expression exhibited a similar pattern to the changes in enzymatic activities (growth-phase-dependent pattern). The two enzymes performed excellent thermal stability and were stable over wide pH ranges of 6.0–11.0 and 2.0–8.0, respectively. Finally, the hydrolysis degrees of raw materials including rice, sorghum, corn, and soybeans by α-amylase from B. cereus D3 reached 66.59 ± 4.21, 57.99 ± 4.39, 57.44 ± 4.67, and 38.01% ± 1.73%, respectively, producing glucose and maltose as the main products. The hydrolysis degrees of proteinous materials including mackerel heads and soybeans by protease from B. cereus D3 were 39.93 ± 1.99 and 30.44% ± 1.39%. SDS-PAGE analysis displayed extensive breakdown of macromolecular proteins, forming peptides of varied sizes. This is a systematic study on the co-production of α-amylase and protease by B. cereus, which is crucial to widen the understanding of this species co-producing multi-enzymes and explore its potential application in the food fermentation industry.

Production of Ganodiesel from the biomass of Ganoderma lucidum in air-L-shaped bioreactor (ALSB)

The increasing need for alternative and sustainable energy sources, prompted by the depletion of fossil fuels and the rise in greenhouse gas emissions, has generated attention towards exploring fast-growing filamentous fungi as a potential bioenergy source. This study aimed to optimize Ganoderma lucidum production for elevated biomass and lipid yields in submerged liquid fermentation. The optimization involved varying initial pH, glucose concentration, and agitation rate using response surface methodology (RSM) with central composite design (CCD). Glucose concentration and initial pH significantly influenced biomass production, while agitation rate had an insignificant effect. For lipid production, glucose concentration, initial medium pH, and agitation rate were identified as significant factors. The optimized conditions (initial pH 6, 50 g/L glucose concentration, and 113.42 rpm) were validated in 500 mL shake flasks and a 3 L Air-L-Shaped Bioreactor (ALSB). Shake flask results showed 8.33 g/L of biomass and 2.17 % of lipid, while the ALSB system produced 5.32 g/L of biomass and 2.35 % lipid. The obtained Ganoderma lucidum mycelial lipid underwent acid-catalysed transesterification to produce biodiesel, which was subjected to several tests to comply ASTM and EN standards. This study serves as a valuable reference for future biodiesel applications through the optimization of Ganoderma lucidum biomass and lipid production.

Maximization of uricase production in a column bioreactor through response surface methodology-based optimization

Uricase (EC 1.7.3.3) is an oxidoreductase enzyme that is widely exploited for diagnostic and treatment purposes in medicine. This study focuses on producing recombinant uricase from E. coli BL21 in a bubble column bioreactor (BCB) and finding the optimal conditions for maximum uricase activity. The three most effective variables on uricase activity were selected through the Plackett–Burman design from eight different variables and were further optimized by the central composite design of the response surface methodology (RSM). The selected variables included the inoculum size (%v/v), isopropyl β-d-1-thiogalactopyranoside (IPTG) concentration (mM) and the initial pH of the culture medium. The activity of uricase, the final optical density at 600 nm wavelength (OD600) and the final pH were considered as the responses of this optimization and were modeled. As a result, activity of 5.84 U·ml−1 and a final OD600 of 3.42 were obtained at optimum conditions of 3% v/v inoculum size, an IPTG concentration of 0.54 mM and a pH of 6.0. By purifying the obtained enzyme using a Ni-NTA agarose affinity chromatography column, 165 ± 1.5 mg uricase was obtained from a 600 ml cell culture. The results of this study show that BCBs can be a highly effective option for large-scale uricase production.

Exploitation of Selected Fungal Endophytes of Andrographis paniculata (Burm. f.) Wall. ex Nees for the Production and Optimisation of Tannase and Screening for their Associated Hydrolysing Enzymes; Amylase, Protease, Lipase, and Laccase

Endophytes represent a diverse domain of microorganisms with immense biotechnological potentialities. Metabolites from endophytes, especially fungi, are useful in industrial as well as pharmaceutical aspects. Here, endophytic fungal isolates of Andrographis paniculata collected from the Tapobon, Junglemahal region of West Bengal, India, have been studied for their hydrolyzing enzyme production abilities. Out of eighty-one isolates, twenty-one were positive for amylase, protease, lipase, laccase, and tannase action. Microscopic features of positive isolates revealed that the enzyme producers were Aspergillus sp., Fusarium sp., Alternaria sp., Trichoderma sp., Exerohilum sp., Nigrospora sp., Curvularia sp., Cladosporium sp., Cochliobolus sp., Tricothecium sp., Penicillium sp., Verticillium sp., and Cephalosporium sp. The amylolytic activity was remarkable in the case of endophytic Aspergillus sp. and Fusarium sp. Also, Aspergillus sp. and Alternaria sp. had proteolytic activity. Aspergillus sp., Mycelia sterilia-2, and Trichoderma sp. were potent lipase producers. Along with Exerohilum sp. endophytic Aspergillus sp. had positive laccase activity. The tannic acid degrading activity was highest for Aspergillus sp. APL11 followed by Helicosporium sp. and Fusarium sp. Tannase producing ability of Aspergillus sp. was optimized and an incubation time- 96h, incubation temperature- 29°C, initial medium pH- 6.5, carbon source- glucose, and nitrogen source- NaNO3 exhibited a two-fold scale-up of (2.83±0.05 u mL-1) in tannase action. These enzymes offer eco-friendly and efficient solutions across a wide range of industrial processes including food production, textile processing, and pharmaceutical synthesis. Fungal endophytes of medicinal plants act as an alternative source for hydrolysing enzymes.

Improving the citric acid production by mutant strains Aspergillus niger using carbohydrate-containing raw materials as a carbon source

The demand for citric acid (CA) as a component of food products, pharmaceuticals, and cosmetics is increasing yearly. The use of adapted micro-organisms that convert naturally occurring carbohydrates into organic acids makes it possible to increase annual CA production significantly. The research aim was to study CA production by the Aspergillus niger strain in the medium based on carbohydrate-containing raw materials as a carbon source. We used a fermentation by A. niger. Starch hydrolysates were chosen as a nutrient medium. To improve the CA production of A. niger, multi-step mutagenesis was performed. This resulted in mutant strain A. niger R5/4, which had the highest acidogenic activity among the samples. The study evaluated the effect of temperature on the productivity of the mutant strains. The quantitative content of citric acid was analyzed at different incubation times (144, 168, and 192 h). The effect of the initial medium pH (4.5, 5.0, and 5.5) on acid formation was also investigated. The strain's optimum temperature, pH, and cultivation time parameters were determined. A three-factor, three-level Box-Benken design (BBD) was used to optimize CA production by A. niger strain R5/4 on a starch-containing medium. When assessing the impact of temperature on CA production, the ideal range was between 29 and 31 °C.

Effect of the initial pH of the culture medium on the nutrient consumption pattern of Bifidobacterium animalis subsp. lactis Bb12 and the improvement of acid resistance by purine and pyrimidine compounds.

During fermentation, the accumulation of acidic products can induce media acidification, which restrains the growth of Bifidobacterium animalis subsp. lactis Bb12 (Bb12). This study investigated the nutrient consumption patterns of Bb12 under acid stress and effects of specific nutrients on the acid resistance of Bb12. Bb12 was cultured in chemically defined medium (CDM) at different initial pH values. Nutrient consumption patterns were analyzed in CDM at pH 5.3, 5.7, and 6.7. The patterns varied with pH: Asp+Asn had the highest consumption rate at pH 5.3 and 5.7, while Ala was predominant at pH 6.7. Regardless of the pH levels (5.3, 5.7, or 6.7), ascorbic acid, adenine, and Fe2+ were vitamins, nucleobases, and metal ions with the highest consumption rates, respectively. Nutrients whose consumption rates exceeded 50% were added individually in CDM at pH 5.3, 5.7, and 6.7. It was demonstrated that only some of them could promote the growth of Bb12. Mixed nutrients that could promote the growth of Bb12 were added to three different CDM. In CDM at pH 5.3, 5.7, and 6.7, it was found that the viable cell count of Bb12 was the highest after adding mixed nutrients, which were 8.87, 9.02, and 9.10 log CFU ml-1, respectively. The findings suggest that the initial pH of the culture medium affects the nutrient consumption patterns of Bb12. Specific nutrients can enhance the growth of Bb12 under acidic conditions and increase its acid resistance.

Initial acidic media promotes quiescence entry in Saccharomyces cerevisiae.

Quiescence is a conserved cellular state wherein cells cease proliferation and remain poised to re-enter the cell cycle when conditions are appropriate. Budding yeast is a powerful model for studying cellular quiescence. In this work, we demonstrate that the pH of the YPD media strongly affects quiescence entry efficiency in Saccharomyces cerevisiae. Adjusting the initial media pH to 5.5 significantly improves quiescence entry efficiency compared to unadjusted YPD media. Thermotolerance of the produced quiescence yeast are similar, suggesting the media pH influences the quantity of quiescent cells more than quality of quiescence reached.

Using Static Culture Method to Increase the Production of Acetobacter Xylinum Bacterial Cellulose

ABSTRACT Bacterial cellulose is a natural polymer, usually produced through synthetic (chemically defined) or natural media. However, the current methods of bacterial cellulose production are not ideal because of their low productivity and large number of byproducts. In order to improve the yield of bacterial cellulose, the influence of medium composition, culture time, culture temperature and culture pH on the production of bacterial cellulose by Acetobacter xylinum ATCC 23,767 was studied by single factor method. The optimal medium composition for Acetobacter xylinum can be analyzed by Thermal field emission scanning electron microscopy (TF-SEM) as glucose (20 g/L), yeast extract (5 g/L), peptone (5 g/L), citric acid (2 g/L). Using the logarithmic calculation method, we can determine that the optimal culture conditions are controlled by initial pH of the culture medium of 6 and constant static culture at 28°C for 9 days. Using TF-SEM, Fourier transform infrared (FT-IR) spectroscopy and water contact angle test, the best post-treatment solution can be selected as sodium hydroxide (1%). The results showed that the yield of bacterial cellulose could reach up to 8.5 g/L under the above conditions, and the obtained product exhibited a dense three-dimensional network structure.

Optimization of polyhydroxybutyrate (PHB) production by Priestia megaterium ASL11 and glycerol and thermoplastic properties of PHB-based films

The production of polyhydroxybutyrate (PHB) by Priestia megaterium ASL11 was elucidated with various carbon sources. The optimized conditions for PHB production were as follows: C/N ratio of 6.99; mixture of 3% (v/v) glycerol, 0.079% (w/v) malt extract and 0.35% (w/v) sodium ammonium phosphate; initial medium pH of 8.0; and incubation time of 120 h. These optimal conditions increased the PHB concentration (g/L), PHB yield, and PHB content produced by ASL11 from 0.32, 0.13, and 15.8% (using glucose) to 0.92, 0.78 and 49.6% (using glycerol), accounting for 2.9-, 6.0- and 3.1-fold increases, respectively. FTIR analysis of the ASL11-PHB film revealed carbonyl (1720 cm−1) and alkane (1453–1379 cm−1) groups similar to those of standard PHB, and GC/MS analysis confirmed that the characteristics of PHB obtained with ASL11 were consistent with those of standard PHB. The ASL11-PHB film was resistant to heat (90–175 °C) and pH values from 5 to 10 and had a melting temperature of 165.1 °C, indicating typical thermobioplastic properties. The ASL11-PHB film could be completely dissolved in sulfuric acid, which is less toxic than chloroform. The mechanical properties of the ASL11-PHB film showed a low Young's modulus with an elongation at break of 5.27%, indicating brittle behavior with low mechanical strength. Thus, the ASL11-PHB film should be blended with other materials to reinforce the film strength and reduce its brittleness for future applications. Moreover, the studies of PHB production under optimal condition by ASL11 needs to be scaled up from the laboratory scale to the pilot scale.

Photodegradation of antibacterial cefotaxime using Mn doped ZnO nanosphere

Elimination of pharmaceutical waste is essential for aquatic system preservation. In this report, the ZnO and Mn-ZnO nanospheres (MZNs) were effectively synthesized via facile co-precipitation method with different Mn doping concentrations as catalyst for photodegradation of cefotaxime. The structure features of the synthesized MZNs were successfully investigated. The obtained data were confirmed the successful fabrication of nanosphere ZnO and MZNs. The designed MZNs with various Mn concentrations were utilized in photodegradation of cefotaxime under optimum conditions. The photodegradation parameters such as antibiotic initial concentrations, catalyst dosage, and medium pH were carried out under UV irradiation. The superior photocatalytic activity was revealed when the MZNs molar ratio is 0.75 mol.%. The results show that the degradation efficiency of cefotaxime was 99% within 2 h at operating condition (initial concentration 30 mg/L, catalyst amount 0.5 g/ L and pH 9). The photodegradation mechanism of antibacterial cefotaxime drug in water is based on superoxide radicals which are considered the main reactive species. The developed MZNs exhibit high stability for removal of cefotaxime that reached to 98% after four times recycling. The MZNs demonstrated a simple and effective model for photocatalytic degradation of pharmaceutical waste.

Efficient pH and dissolved CO2 conditions for indoor and outdoor cultures of green alga Parachlorella.

Efficient pH and dissolved CO2 conditions for indoor (50-450mL scale) and outdoor (100-500L scale) culture of a green alga BX1.5 strain that can produce useful intracellular lipids and extracellular polysaccharides were investigated for the first time in Parachlorella sp. The cultures harvested under 26 different conditions were analysed for pH, dissolved CO2 concentration, and the biomass of extracellular polysaccharides. The BX1.5 strain could thrive in a wide range of initial medium pH ranging from 3 to 11 and produced valuable lipids such as C16:0, C18:2, and C18:3 under indoor and outdoor culture conditions when supplied with 2.0% dissolved CO2. Particularly, the acidic BG11 medium effectively increased the biomass of extracellular polysaccharides during short-term outdoor cultivation. The BG11 liquid medium also led to extracellular polysaccharide production, independent of acidity and alkalinity, proportional to the increase in total sugars derived from cells supplied with high CO2 concentrations. These results suggest Parachlorella as a promising strain for indoor and outdoor cultivation to produce valuable materials.

Optimization and reactor-scale production of plant growth regulators by Pleurotus eryngii.

The aims of the this study are to select the best cultivation type for plant growth regulator (PGR) production, to optimize PGR production with statistical experimental design, and to calculate bioprocess parameters and yield factors during PGR production by P. eryngii in flask and reactor scales. Submerged fermentation was the best cultivation type with 4438.67 ± 37.14, 436.95 ± 27.31, and 54.32 ± 3.21mg/L of GA3, ABA, and IAA production values, respectively. The Plackett-Burman and Box-Behnken designs were used to determine effective culture parameters and interactive effects of the selected culture parameters on PGR production by Pleurotus eryngii under submerged fermentation. The statistical model is valid for predicting PGR production by P. eryngii. After these studies, maximum PGR production (7926.17 ± 334.09, 634.92 ± 12.15, and 55.41 ± 4.38mg/L for GA3, ABA, and IAA, respectively) was reached on the 18th day of fermentation under optimized conditions. The optimum formula was 50g/L fructose, 3g/L NaNO3, and 1.5g/L KH2PO4, 1mg/L thiamine, incubation temperature 25°C, initial medium pH 7.0, and an agitation speed of 150rpm. The kinetics of PGR production was investigated in batch cultivation under 3-L stirred tank reactor conditions. Concentrations of GA3, ABA, and IAA of 10,545.00 ± 527.25, 872.32 ± 21.81, and 60.48 ± 3.48mg/L were obtained at the reactor scale which were 4.1, 3.4, and 2.3 times higher than the initial screening values. The specific growth rate (µ), the volumetric (rp) and specific (Qp) PGR production rates, 486.11mg/L/day and 107.43mg/g biomass/day for GA3, confirmed the successful transfer of optimized conditions to the reactor scale. In the presented study, PGR production of P. eryngii is reported for the first time.

Microbial Transformation of Pimavanserin by Cunninghamella blakesleeana AS 3.970

Pimavanserin is an approved selective 5-HT2A receptor inverse agonist for treating Parkinson’s disease psychosis. However, few studies on its metabolism in vitro have been investigated. In this research, eight strains of fungi are used to study the pimavanserin metabolism profiles in vitro and six of them demonstrated positive transformation results. Factors influencing the transformation rate, like substrate concentration, culture time, initial media pH value, culture temperature, and shaking speed, were evaluated and optimized. Cunninghamella blakesleeana AS3.970 provided the best transformation rate of 30.31%, and 10 unreported metabolites were screened by LC-MS/MS. Among these metabolites, M1 is the major one and identified as 1-(4-fluorobenzyl)-3-(4-(2-hydroxy-2-methylpropoxy)benzyl)-1-(1-methylpiperidin-4-yl)urea, which is a hydroxylation product of the pimavanserin. A preliminary molecular docking simulation was performed, which indicated that M1 exhibits similar binding properties with pimavanserin and may become a potential candidate for Parkinson’s disease treatment.

Screening of xylanase producing Bacillus species and optimization of xylanase process parameters in submerged fermentation

One of the main process inputs in the microbial enzyme, an environment friendly and an extraordinary catalyst, production is characteristics of the microorganism. This study aimed to screen newly isolated Bacillus species for the xylanase production potentiality and investigate crucial parameters that affect the xylanase producing process. Bacteria samples were isolated from potential agricultural and food sources such as soil, wheat bran, and sourdough. Eight xylanase producing isolates showed 99% homology to Bacillus species according to 16S rRNA sequence analysis. Isolate A67 has been found the best xylanase producer isolate and identified as Bacillus halotolerans DSM 8802. Incubation temperature, initial medium pH, and agitation speed parameters were optimized by RSM in the shake flask scale. The optimum xylanase production points were found as 30 °C, pH 8, and 127.27 rpm under submerged fermentation conditions. Xylanase activity was obtained experimentally as 23.47 U/mL resulting 4.21-fold increase with the statistical optimization. R2 and predicted R2 values that reflects the significance of the designed model were calculated as 0.9964 and 0.9674, respectively. Incubation temperature has been concluded as the most important parameter that affects the xylanase activity for isolate A67. As it has low incubation temperature demand, metabolizes xylan polymer as a carbon source and most of the Bacillus species do not produce any toxin, isolate A67 could have potential for the usage of the lignocellulosic biomass added media to make fermentation cost effective in food-grade xylanase production process.

EFFECTS OF MEDIA COMPOSITIONS ON AMYLASE AND D-LACTIC ACID PRODUCED BY A POTENTIAL STARCH-UTILIZING BACTERIUM

The high demand for amylase and lactic acid in various industries made enzyme and acid production from cheap substrates very attractive. This study investigated the effect of major medium constituents on amylase and D-lactic acid production by the potential starch-utilizing and optical pure D-lactic acid-producing Lactobacillus sp. SUTWR 73, to provide data for the efficient application of the lactic acid bacterium. Its amylase and D-lactic acid production was considerably affected by the addition of organic nitrogen tryptone to certain cassava starch concentrations as a carbon-source. While the other medium compositions: yeast extract and cheap nitrogen sources (soy protein, defatted rice bran, and spent brewery yeast) compared to tryptone as equal total nitrogen contents, did not have any effect on either metabolite production. The medium containing cassava starch (4.0%, w/v) and tryptone (4.5%, w/v) can be satisfactorily used for both products. The initial pH of the production medium was another factor affecting metabolite production. A suitable initial pH was 8.0 and 7.0-10.0 for amylase and D-lactic acid production, respectively. Amylase activity was detected at the early log growth phase while the D-lactic acid was produced at the late log phase. Understanding the relationship between amylase and D-lactic acid production from this study is important for designing an economical operating system for the direct production of D-lactic acid from cheap cassava starch without adding any commercial amylase as a substrate in pretreatment resulting in reduction costs of the production process.

Thrombolytic and anticoagulant efficiencies of purified fibrinolytic enzyme produced from Cochliobolus hawaiiensis under solid-state fermentation.

Cochliobolus hawaiiensis Alcorn Assiut University Mycological Centre 8606 was chosen from the screened 20 fungal species as the potent producer of fibrinolytic enzyme on skimmed-milk agar plates. The greatest enzyme yield was attained when the submerged fermentation (SmF) conditions were optimized, and it was around (39.7U/mg protein). Moreover, upon optimization of fibrinolytic enzyme production under solid-state fermentation (SSF), the maximum productivity of fibrinolytic enzyme was greatly increased recorded a bout (405U/mg protein) on sugarcane bagasse, incubation period of 5 days, moisture level of 100%, initial pH of salt basal medium 7.8, incubation temperature at 35°C, and supplementation of the salt basal medium with corn steep liquor (80％, v/v). The yield of fibrinolytic enzyme by C. hawaiiensis under SSF was higher than that of SmF with about 10.20-fold. The purification procedures of fibrinolytic enzyme by ammonium sulfate (70%), gel filtration, and ion-exchange columns chromatography caused a great increase in its specific activity to 2581.6U/mg protein with an overall yield of 55.89%, 6.37 purification fold and molecular weight of 35kDa. Maximal activity was recorded at pH 7 and 37°C. Significant pH stability was recorded at pH 6.6-7.2, and thermal stability was recorded at 33-41°C. The enzyme showed the highest affinity toward fibrin, with Vmax of 240U/mL and an apparent Km value of 47.61mmol. Mg2+ and Ca2+ moderately induced fibrinolytic activity, whereas Cu2+ and Zn2+ greatly suppressed the enzyme activity. The produced enzyme is categorized as serine protease and non-metalloprotease. The purified fibrinolytic enzyme showed efficient thrombolytic and antiplatelet aggregation activities by completely prevention and dissolution of the blood clot which confirmed by microscopic examination and amelioration of blood coagulation assays. These findings suggested that the produced fibrinolytic enzyme is a promising agent in management of blood coagulation disorders.

Enhancement of alkaline protease production in recombinant Bacillus licheniformis by response surface methodology

Alkaline protease is widely used in the food, detergent, and pharmaceutical industries because of its comparatively great hydrolysis ability and alkali tolerance. To improve the ability of the recombinant Bacillus licheniformis to produce alkaline protease, single-factor experiments and response surface methodology (RSM) were utilized to determine and develop optimal culture conditions. The results showed that three factors (corn starch content, soybean meal content, and initial medium pH) had significant effects on alkaline protease production (P < 0.05), as determined through the Plackett‒Burman design. The maximum enzyme activity was observed with an optimal medium composition by central composite design (CCD): corn starch, 92.3 g/L; soybean meal, 35.8 g/L; and initial medium pH, 9.58. Under these optimum conditions, the alkaline protease activity of strain BL10::aprE was 15,435.1 U/mL, 82% higher than that in the initial fermentation medium. To further investigate the application of the optimum fermentation medium, the overexpressed strain BL10::aprE/pHYaprE was cultured using the optimized medium to achieve an enzyme activity of 39,233.6 U/mL. The present study achieved the highest enzyme activity of alkaline protease by B. licheniformis at the shake-flask fermentation level, which has important application value for large-scale production.Graphical

Valorization of shalgam juice plant waste for the production of carotenoids by Rhodotorula glutinis

Food waste is an emerging global problem that should not be underestimated. One of the most abundant wastes in Türkiye and neighbour countries is the waste of shalgam juice plants (WSJP). In this study, WSJP was used as a growth medium for Rhodotorula glutinis. The effects of initial medium pH (3.4-5.4), carbon (0-60 g/L glycerol) and nitrogen sources (0-1 g/L urea) were investigated on biomass, medium pH, reducing sugar concentration, total lipid and carotenoid contents, as well as carotenoid composition (torulene, torularhodin, β-carotene). When crude waste extract (no additional nutrient) was used as growth medium (initial medium pH 3.4, 6.14 g/L sugar), biomass was relatively low (1.47±0.055 g/L), due to acidic pH and insufficient nutrients. However, these stress conditions increased the production of total lipid and carotenoid contents by R. glutinis. The highest lipid and carotenoid contents were obtained as 0.14±0.0004 g/g dry cell weight (dcw) and 1221.57±0.59 µg/g dcw, respectively, under these circumstances. As additional stress factor fermentation medium -crude WSJP extract- was illuminated for 72 h. This increased lipid content by 1.7-fold, while showing low impact on carotenoid content. Interestingly, illumination changed the carotenoid composition by decreasing torulene and β-carotene percentages, but increasing torularhodin percentage. On the other hand, tuning the initial pH to an ambient value (5.4) and the addition of carbon and nitrogen sources stimulated cell growth (4.67±0.07 g/L). This study presents the first time use of WSJP extract as a growth medium, without any additional nutrition, moreover, the simultaneous production of high-value added carotenoids.