Effect of inorganic salt stress on the thermotolerance and ethanol production at high temperature of <Emphasis Type="Italic">Pichia kudriavzevii</Emphasis>

Application of cross-protection is expected to improve the thermotolerance of yeasts to enhance their ethanol production at high temperature. In this study, the effects of eight kinds of inorganic salts on the thermotolerance and ethanol production at high temperature in Pichia kudriavzevii were investigated. P. kudriavzevii showed strong thermotolerance and the ability to produce ethanol at high temperature, and higher ethanol production of P. kudriavzevii was observed at high temperature (37–42 °C) compared with that at 30 °C. Inorganic salt stresses induced obvious cross-protection of thermotolerance in P. kudriavzevii. The presence of 0.1 mol/L KNO₃ or Na₂SO₄ or 0.2 mol/L NaCl, KCl, NaNO₃, K₂SO₄ or MgCl₂ increased the yeast biomass in YEPD medium at 44 °C to 2.72–3.46 g/L, obviously higher than that in the absence of salt stress (2.17 g/L). The addition of NaCl, KCl, NaNO₃, KNO₃, Na₂SO₄, K₂SO₄, CaCl₂ and MgCl₂ significantly increased the ethanol production of P. kudriavzevii in YEPD fermentation medium at 44 °C by 37–58%. KCl and MgCl₂ exhibited the best performance on improving the thermotolerance and ethanol production, respectively, of P. kudriavzevii. A highly significant correlation (P?<?0.01) was obtained among ethanol production, biomass and glucose consumption, suggesting the important role of thermotolerance and glucose consumption in enhanced ethanol production. The combination of NaCl, KCl and MgCl₂ had a synergistic effect on the improvement of thermotolerance and ethanol production at high temperature in P. kudriavzevii. This study provides some important clues for improving ethanol production of thermotolerant yeasts at high temperature.     

Medium optimization for keratinase production in hair substrate by a new <Emphasis Type="Italic">Bacillus subtilis</Emphasis> KD-N2 using response surface methodology

Culture medium for keratinase production from hair substrate by a new Bacillus subtilis strain, KD-N2, was optimized. Effects of culture conditions on keratinase production were tested, and optimal results were obtained with 10% inocula (v/v), 16 g/L hair substrate, an initial pH value of 6.5 and a culture volume of 20 mL. Several carbon sources (sucrose, cornflour) and nitrogen sources (yeast extract, tryptone and peptone) had positive effects on keratinase production, with sucrose giving optimal results. To improve keratinase yield, statistically based experimental designs were applied to optimize the culture medium. Fractional factorial design (FFD) experiments showed that MgSO₄ and K₂HPO₄ were the most significant factors affecting keratinase production. Further central composite design (CCD) experiments indicated that the optimal MgSO₄ and K₂HPO₄ concentrations were 0.91 and 2.38 g/L, respectively. Using an optimized fermentation medium (g/L: NaCl 1.0, CaCl₂ 0.05, KH₂PO₄ 0.7, sucrose 3, MgSO₄ 0.91, K₂HPO₄ 2.38), keratinase activity increased to 125 U/mL, an approximate 1.7-fold increase over the previous activity (75 U/mL). Human hair was degraded during the submerged cultivation.     

Optimal conditions for the production of exopolysaccharide by marine microorganismHahella chejuensis

A marine microorganism, strain 96CJ10356 produced exopolysaccharide, designated as EPS-R. To optimize culture conditions for the production of EPS-R, carbon and nitrogen sources, mineral salts, temperature, and pH were examined. From this study, STN medium for the production of EPS-R was suggested as follows; sucrose 20 g, tryptone 10 g, NaCl 10 g, MgSO₄ 5 g, CaCl₂ 1 g, KH₂PO₄ 76 mg, K₂HPO₄ 83 mg, FeCl₂ 5 mg, MnCl₂ 1 mg, NaMoO₄ 1 mg, and ZnCl₂ 1 mg per liter at pH 7.0. About 9.23 g/L of EPS-R was obtained from STN medium after cultivation for 120 h at 25°C in a 5-liter jar fermentor with an aeration rate of 0.17 vvm. Apparent viscosity and flocculation activity of the culture broth were increased with the production of EPS-R and the maximal values were 415 cP and 1400 unit/mL against 0.5% activated carbon, respectively.     

COMPARATIVE STUDIES ON RESPIRATION : VIII. THE RESPIRATION OF BACILLUS SUBTILIS IN RELATION TO ANTAGONISM.

1. In relatively low concentrations of NaCl, KCl, and CaCl₂ the rate of respiration of Bacillus subtilis remains fairly constant for a period of several hours, while in the higher concentrations, there is a gradual decrease in the rate. 2. NaCl and KCl increase the rate of respiration of Bacillus subtilis somewhat at concentrations of 0.15 M and 0.2 M respectively; in sufficiently high concentrations they decrease the rate. CaCl₂ increases the rate of respiration of Bacillus subtilis at a concentration of 0.05 M and decreases the rate at somewhat higher concentrations. 3. The effects of salts upon respiration show a well marked antagonism between NaCl and CaCl₂, and between KCl and CaCl₂. The antagonism between NaCl and KCl is slight and the antagonism curve shows two maxima.     

Medium Optimization for Improved Production of Dihydrolipohyl Dehydrogenase from <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> PAD-91 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Dihydrolipohyl dehydrogenase (DLD) is a FAD-dependent enzyme that catalyzes the reversible oxidation of dihydrolipoamide. Herein, we report medium optimization for the production of a recombinant DLD with NADH-dependent diaphorase activity from a strain of Bacillus sphaericus PAD-91. The DLD gene that consisted of 1413 bp was expressed in Escherichia coli BL21 (DE3), and its enzymatic properties were studied. The composition of production medium was optimized using one-variable-at-a-time method followed by response surface methodology (RSM). B. sphaericus DLD catalyzed the reduction of lipoamide by NAD⁺ and exhibited diaphorase activity. The molecular weight of enzyme was about 50 kDa and determined to be a monomeric protein. Recombinant diaphorase showed its optimal activity at temperature of 30 °C and pH 8.5. K _m and V _max values with NADH were estimated to be 0.025 mM and 275.8 U/mL, respectively. Recombinant enzyme was optimally produced in fermentation medium containing 10 g/L sucrose, 25 g/L yeast extract, 5 g/L NaCl and 0.25 g/L MgSO₄. At these concentrations, the actual diaphorase activity was calculated to be 345.0 ± 4.1 U/mL. By scaling up fermentation from flask to bioreactor, enzyme activity was increased to 486.3 ± 5.5 U/mL. Briefly, a DLD with diaphorase activity from a newly isolated B. sphaericus PAD-91 was characterized and the production of recombinant enzyme was optimized using RSM technique.     

Optimization of nutrient medium containing agricultural waste for xylanase production by Bacillus pumilus B20

We aimed to optimize a nutrient medium containing agricultural waste for xylanase production by Bacillus pumilus B20. Xylanase production with lignocellulosic material was optimized in two steps using DeMeo’s fractional factorial design. A 3.4-fold increase in xylanase production (313.3 U/mL) was achieved using the optimized culture medium consisting of (g/L): K₂HPO₄, 2; MgSO₄·7H₂O, 0.3; CaCl₂·2H₂O, 0.01; NaCl, 2; peptone, 5 yeast extract, 4; and wheat bran, 50. _{B. pumilus} B20 produced a high level of xylanase, which may have potential industrial application.     

Development of a New <Emphasis Type="Italic">Amycolatopsis orientalis</Emphasis> Strain for High-Yield Eremomycin Production

A multistep random UV mutagenesis of an eremomycin-producing strain Amycolatopsis orientalis VKPM Ac-1125 with rather low productivity (0.5 g/L) and the further selection of the most high-yield mutant strains have resulted in a highly productive E 13-26 strain. After optimization of the fermentation medium by multifactor analysis, the most efficient composition for the fermentation medium was established, %: glycerol, 8; soybean meals, 2.0; MgCl₂, 0.02; KH₂PO₄, 0.02; KNO₃, 0.6; CaCl₂, 0.3; skim milk powder, 0.46; and glucose, 0.8. The maximum productivity of this strain on the optimized medium reached 3.42 ± 0.17 g/L. Therefore, the obtained strain is considered to be very promising for further use in the selection and development of technology for the large-scale eremomycin production.     

Enhancing ethanol production from cellulosic sugars using <Emphasis Type="Italic">Scheffersomyces (Pichia) stipitis</Emphasis>

Studies were performed on the effect of CaCO₃ and CaCl₂ supplementation to fermentation medium for ethanol production from xylose, glucose, or their mixtures using Scheffersomyces (Pichia) stipitis. Both of these chemicals were found to improve maximum ethanol concentration and ethanol productivity. Use of xylose alone resulted in the production of 20.68 ± 0.44 g L^?1 ethanol with a productivity of 0.17 ± 0.00 g L^?1 h^?1, while xylose plus 3 g L^?1 CaCO₃ resulted in the production of 24.68 ± 0.75 g L^?1 ethanol with a productivity of 0.21 ± 0.01 g L^?1 h^?1. Use of xylose plus glucose in combination with 3 g L^?1 CaCO₃ resulted in the production of 47.37 ± 0.55 g L^?1 ethanol (aerobic culture), thus resulting in an ethanol productivity of 0.39 ± 0.00 g L^?1 h^?1. These values are 229 % of that achieved in xylose medium. Supplementation of xylose and glucose medium with 0.40 g L^?1 CaCl₂ resulted in the production of 44.84 ± 0.28 g L^?1 ethanol with a productivity of 0.37 ± 0.02 g L^?1 h^?1. Use of glucose plus 3 g L^?1 CaCO₃ resulted in the production of 57.39 ± 1.41 g L^?1 ethanol under micro-aerophilic conditions. These results indicate that supplementation of cellulosic sugars in the fermentation medium with CaCO₃ and CaCl₂ would improve economics of ethanol production from agricultural residues.     

OPTIMIZATION OF FERMENTATION MEDIUM FOR ACETOIN PRODUCTION BY Bacillus subtilis SF4-3 USING STATISTICAL METHODS

To improve the acetoin-producing ability of Bacillus subtilis SF4-3, isolated from “natto,” a Japanese traditional food, the fermentation medium was optimized in shake-flask fermentation by statistically designed methods. Based on results of the single-factor experiment, orthogonal experiment, and Plackett–Burman design, yeast extract, corn steep liquor, and urea were identified as showing significant influence on the acetoin production. Subsequently, the optimum combination of the three factors was investigated by the Box–Behnken design (BBD) of response surface methodology (RSM) in order to further enhance the acetoin production. The maximum acetoin yield of 45.4 g/L was predicted when the concentrations of yeast extract, corn steep liquor, and urea were 8.5 g/L, 14.6 g/L, and 3.8 g/L, respectively. The results were further confirmed in triplicate experiments using the optimized medium (glucose 160 g/L, yeast extract 8.5 g/L, corn steep liquor 14.6 g/L, urea 3.8 g/L, manganese sulfate 0.05 g/L, ferrous sulfate 0.05 g/L), and an acetoin yield of 46.2 g/L was obtained in the validation experiment, which was in agreement with the prediction. After the optimization of medium components, an increase of 36.28% in acetoin production was achieved in comparison to that at the initial medium levels.     

Production,purification, and characterization of two extremely halotolerant,thermostable, and alkali-stable α-amylases from Chromohalobacter sp. TVSP 101

The halophilic bacterial strain Chromohalobacter sp. TVSP 101 was shown to produce extracellular, halotolerant, alkali-stable and moderately thermophilic α-amylase activity. The culture conditions for higher amylase production were optimized with respect to NaCl, pH, temperature and substrates. Maximum amylase production was achieved in a medium containing 20% NaCl or 15% KCl at pH 9.0 and 37 °C in the presence of 0.5% rice flour and tryptone. Addition of 50 mM CaCl₂ to the medium increased amylase production by 29%. Two kinds of amylase activity, designated amylase I and amylase II, were purified from culture filtrates to homogeneity with molecular masses of 72 and 62 kDa, respectively. Both enzymes had maximal activity at pH 9.0 and 65 °C in the presence of 0–20% (w/v) NaCl but amylase I was much more stable in the absence of NaCl than amylase II. The enzymes efficiently hydrolyzed carbohydrates to yield maltotetraose, maltotriose, maltose, and glucose as the end products.     

A NEWLY ANTI-Streptococcus suis BACTERIOCIN PRODUCING STRAIN FROM UNWEANED PIGLETS FECAL MATTER: ISOLATION,PRELIMINARY IDENTIFICATION,AND OPTIMIZATION OF MEDIUM COMPOSITION FOR ENHANCED BACTERIOCIN PRODUCTION

A newly isolated anti-Streptococcus suis bacteriocin-producing strain LPL1-5 was obtained from healthy unweaned piglets' fecal matter, and was designated as Lactobacillus pentosus LPL1-5 based on morphology, biochemical properties, and 16S rDNA sequencing analysis. The medium composition for enhanced bacteriocin production by L. pentosus LPL1-5 was optimized by statistical methodology. Yeast extract, K₂HPO₄ · 3H₂O, and MnSO₄ · H₂O were identified as significant components influencing pentocin LPL1-5 production using the Plackett–Burman method. Response surface methodology was applied for further optimization. The concentrations of medium components for enhanced pentocin LPL1-5 production were as follows (g/L): lactose 20.00, tryptone 10.00, beef extract 10.00, yeast extract 14.00, MnSO₄ · H₂O 0.84, K₂HPO₄ · 3H₂O 4.92, triammonium citrate 2.00, Na-acetate 5.00, MgSO₄ · 7H₂O 0.58, Tween 80 1.00. Under the optimized condition, a value of 3154.65 ± 27.93 IU/mL bacteriocin activity was achieved, which was 4.2-fold that of the original medium.     

Bioprocess optimization for pectinase production using <Emphasis Type="Italic">Aspergillus niger</Emphasis> in a submerged cultivation system

Background

Pectinase enzymes present a high priced category of microbial enzymes with many potential applications in various food and oil industries and an estimated market share of $ 41.4 billion by 2020.

Results

The production medium was first optimized using a statistical optimization approach to increase pectinase production. A maximal enzyme concentration of 76.35 U/mL (a 2.8-fold increase compared with the initial medium) was produced in a medium composed of (g/L): pectin, 32.22; (NH₄)₂SO₄, 4.33; K₂HPO₄, 1.36; MgSO₄.5H₂O, 0.05; KCl, 0.05; and FeSO₄.5H₂O, 0.10. The cultivations were then carried out in a 16-L stirred tank bioreactor in both batch and fed-batch modes to improve enzyme production, which is an important step for bioprocess industrialization. Controlling the pH at 5.5 during cultivation yielded a pectinase production of 109.63 U/mL, which was about 10% higher than the uncontrolled pH culture. Furthermore, fed-batch cultivation using sucrose as a feeding substrate with a rate of 2 g/L/h increased the enzyme production up to 450 U/mL after 126 h.

Conclusions

Statistical medium optimization improved volumetric pectinase productivity by about 2.8 folds. Scaling-up the production process in 16-L semi-industrial stirred tank bioreactor under controlled pH further enhanced pectinase production by about 4-folds. Finally, bioreactor fed-batch cultivation using constant carbon source feeding increased maximal volumetric enzyme production by about 16.5-folds from the initial starting conditions.

     

Development of optimal medium for production of commercially important monoclonal antibody 520C9 by hybridoma cell

Hybridoma HB-8696 produces monoclonal antibody (mAb) 520C9 (mouse IgG₁), which recognizes breast cancer oncoprotein c-erbB2. The objective of this study was to optimize the medium recipe of HB 8696 cell for production of mAb 520C9. The optimization consisted of two steps: (1) screening of significant nutrients to make subsequent experiments more efficient with less runs and (2) locating their optimal concentrations. 29 variables including essential and non-essential amino acids, glucose, serum and 6 salts, namely NaCl, KCl, CaCl₂, NaH₂PO₄, MgSO₄ and Na-pyruvate were chosen in screening phase. The Plackett–Burman method was used to screen the variables influencing mAb production. Seven factors namely glucose, serum, asparagine, threonine, serine, NaCl and NaH₂PO₄ were identified to have a positive influencing role on mAb production with a confidence level >90 % (p < 0.1). Finally, Response surface methodology revealed the optimal level of the variables. The mAb production and average specific mAb production rate were enhanced by 111.05 and 105 %, respectively, compared to control medium.     

Halophilic and salts tolerant protoplast cultures of mangrove plants, Sonneratia alba and Avicennia alba

The effects of sea salts, NaCl, KCl, MgCl₂, MgSO₄, and CaCl₂, on the growth of protoplast cultures of two mangrove species, Sonneratia alba and Avicennia alba, were investigated using 96-well culture plates. Plants of these two species naturally grow at the seaward side of a mangrove forest. Cotyledon protoplasts of S. alba showed halophilic nature to NaCl, KCl, and MgCl₂ at low concentrations (10–50 mM) when cultured in Murashige and Skoog’s (MS) medium containing 0.6 M mannitol. CaCl₂ at a concentration higher than 25 mM was inhibitory to cell growth. On the other hand, in protoplast culture of A. alba suspension cells, which were induced from cotyledon tissues, in the modified amino acid (mAA) medium containing 1.2 M sorbitol, tolerance to NaCl, MgCl₂ and MgSO₄ were observed at a wide range of concentrations up to 400 mM. CaCl₂ was always inhibitory for cell divisions in A. alba, but stimulatory for spherical enlargement of cells. However, no difference in cell enlargement was observed among other salts. Similarity and difference in reactivity to salts between protoplasts and suspension cells from our previous studies were discussed in relation to the site of salt tolerance or halophilic adaptation within mangrove cells. For protoplast cultures, the site(s) for response of S. alba and A. alba are located in the cytoplasm and/or the cell membrane.     

红色诺卡菌固体培养基配方的优化

应用响应面优化设计法优化固体培养基配方,增大红色诺卡菌的固体培养细胞生物量。首先用Plackett-Burman法从现有培养基组分中找到影响红色诺卡菌细胞生物量的关键因素,再通过最陡爬坡法确定细胞生物量最大的配方,用作中心组合设计(Central Composite Design, CCD)实验的基础起始值,拟合数学模型方程,最后找到最优组分的组合。优化的配方转移至企业实施放大实验,对结果进行验证和比较。试验结果表明,培养基各组分中影响红色诺卡菌细胞生物量的关键因素为蛋白胨、NaCl、牛肉膏;最优固体培养基配方:蛋白胨42 g/L、牛肉膏8 g/L、NaCl 1.2 g/L、甘油10 mL/L、Na_2HPO_4·12H_2O 0.3 g/L、琼脂20 g/L。在细胞生物量方面最优固体培养基配方比原配方高104%。响应面优化设计可用于提高红色诺卡菌细胞生物量固体培养基的优化,也为红色诺卡菌培养条件、液体发酵的优化研究提供参考。     

蝉拟青霉液体发酵条件优化

采用液体发酵蝉拟青霉,对蝉拟青霉的发酵条件进行优化,以提高蝉拟青霉胞外多糖产量及生物量。摇瓶发酵条件下,在单因素基础上设计正交实验确定各因素的最佳组合。优化后得最佳发酵培养基:蔗糖8%,牛肉膏0.75%,酵母膏0.125%,MgSO_4·7H_2O 0.3%,KH_2PO_4 0.2%,麸皮0.5%。该条件下胞外多糖产量为5.96 g/L,生物量为42 g/L,较优化前提高了1倍。采用发酵罐进行扩大培养,对分批发酵时的初糖浓度进行了优化,并分析了补料分批发酵对发酵过程的影响。发酵罐培养时最适初糖浓度为5%,此时生物量最高为38 g/L,多糖含量最高为5.5 g/L;采用补料分批发酵时,多糖产量最高为5.89 g/L,生物量最高为40 g/L,效果优于分批发酵。     

Expression of Bacillus pumilus keratinase rK27 in Bacillus subtilis: enzyme application for developing renewable flocculants from bone meal

In this study thermostable keratinase rK₂₇ of Bacillus pumilus KS12 was expressed and secreted in Bacillus subtilis WB980 expression system under the control of xylose promoter (PxylA). The concentration of the recombinant keratinase rK₂₇ produced by B. subtilis reached 4,432 U/mL after 24 h of culture at 37 °C and 200 rpm with 0.5 % xylose at an initial concentration of 0.3 OD_600nm. Using the one-factor-at-a-time approach, we achieved an improvement in enzyme yield of up to 3.4-fold (15,390 U/mL) in the presence of 3 % yeast extract and 0.5 % tryptone. The enzyme was purified to homogenity using nickel affinity chromatography with a 3.63-fold purity and 80 % recovery. The purified enzyme rK₂₇ hydrolyzed 1 g bone meal after 12 h at 40 °C, pH 9, with a maximum protein release of 37.3 mg/g bone meal; in comparison subtilisin Carlsberg hydrolyzed 19.3 mg/g bone meal and proteinase K hydrolyzed 6.2 mg/g bone meal. The hydrolysate obtained after hydrolysis of bone by rK₂₇ was found to be effective as a flocculant at 0.1 mg in a 10 % (w/v) kaolin solution when compared with hydrolysates obtained from substilisin Carlsberg and proteinase K, which were effective at 0.5 mg and >2 mg, respectively.     

Production of mycelial biomass and exo-polymer by <Emphasis Type="Italic">Hericium erinaceus</Emphasis> CZ-2: Optimization of nutrients levels using response surface methodology

The Doehlert experimental design was used to optimize the production of mycelial biomass and exopolymer from Hericium erinaceus CZ-2 in this study. Statistical analysis showed that the linear and quadric terms of 3 variables: corn flour, yeast extract, and corn steep liquor had significant effects. The optimized combination of these 3 variables was confirmed through validation experiments. The optimal conditions for higher production of mycelial biomass (19.92 g/L) were estimated when the media composition concentrations were set as: 30.85 g/L, corn flour; 2.81 g/L, yeast extract; 16.9 mL/L, corn steep liquor; 10 g/L, glucose; 1 g/L, KH₂PO₄; and 0.5 g/L, MgSO₄·7H₂O; while a maximal exo-polymer yield (1.653 g/L) could be achieved when setting concentrations of: 32.71 g/L, corn flour; 2.35 g/L, Yeast extract; 14.42 mL/L, Corn steep liquor; 10 g/L, glucose; 1 g/L, KH₂PO₄; and 0.5 g/L, MgSO₄·7H₂O. The upscale production was also investigated using a 15 L fermentor using the optimized medium.     

Identification of Newly Isolated Talaromyces pinophilus and Statistical Optimization of β-Glucosidase Production Under Solid-State Fermentation

Fungi able to degrade agriculture wastes were isolated from different soil samples, rice straw, and compost; these isolates were screened for their ability to produce β-glucosidase. The most active fungal isolate was identified as Talaromyces pinophilus strain EMOO 13-3. The Plackett–Burman design is used for identifying the significant variables that influence β-glucosidase production under solid-state fermentation. Fifteen variables were examined for their significances on the production of β-glucosidase in 20 experimental runs. Among the variables screened, moisture content, Tween 80, and (NH₄)₂SO₄ had significant effects on β-glucosidase production with confidence levels above 90% (p < 0.1). The optimal levels of these variables were further optimized using Box–Behnken statical design. As a result, the maximal β-glucosidase activity is 3648.519 U g^?1, which is achieved at the following fermentation conditions: substrate amount 0.5 (g/250 mL flask), NaNO₃ 0.5 (%), KH₂PO₄ 0.3 (%), KCl 0.02 (%), MgSO₄ · 7H₂O 0.01 (%), CaCl₂ 0.01 (%), yeast extract 0.07 (%), FeSO₄ · 7H₂O 0.0002 (%), Tween 80 0.02 (%), (NH₄)₂SO₄ 0.3 (%), pH 6.5, temperature 25°C, moisture content 1 (mL/g dry substrate), inoculum size 0.5 (mL/g dry substrate), and incubation period 5 days.     

MEDIUM OPTIMIZATION OF ANTIFUNGAL ACTIVITY PRODUCTION BY Bacillus amyloliquefaciens USING STATISTICAL EXPERIMENTAL DESIGN

In order to overproduce biofungicides agents by Bacillus amyloliquefaciens BLB371, a suitable culture medium was optimized using response surface methodology. Plackett–Burman design and central composite design were employed for experimental design and analysis of the results. Peptone, sucrose, and yeast extract were found to significantly influence antifungal activity production and their optimal concentrations were, respectively, 20 g/L, 25 g/L, and 4.5 g/L. The corresponding biofungicide production was 250 AU/mL, corresponding to 56% improvement in antifungal components production over a previously used medium (160 AU/mL). Moreover, our results indicated that a deficiency of the minerals CuSO₄, FeCl₃ · 6H₂O, Na₂MoO₄, KI, ZnSO₄ · 7H₂O, H₃BO₃, and C₆H₈O₇ in the optimized culture medium was not crucial for biofungicides production by Bacillus amyloliquefaciens BLB371, which is interesting from a practical point of view, particularly for low-cost production and use of the biofungicide for the control of agricultural fungal pests.