Mikrobiol. Z. 2022; 84(3):17-28.
Features of the Synthesis of Extracellular Cytotoxic Lectin Bacillus subtilis IMV B-7724,
Depending on the Cultivation Conditions in the Laboratory Fermenter
O.G. Kisten1, K.I. Hetman1, E.V. Koval2,3, I.O. Hretskyi1, L.F. Zyryanova1,
L.M. Tyshchenko2,4, N.I. Fedosova2, N.L. Cheremshenko2, A.V. Chumak2
1Zabolotny Institute of Microbiology and Virology, NAS of Ukraine
154 Acad. Zabolotny Str., Kyiv, 03143, Ukraine
2Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NAS of Ukraine
45 Vasylkivska Str., Kyiv, 03022, Ukraine
3National University of Food Technologies
68 Volodymyrska Str., Kyiv, 01601, Ukraine
4National University of Life and Environmental Sciences of Ukraine
15 Heroiv Oborony Str., Kyiv, 03041, Ukraine
The level of oxygen mass transfer (KV) is an important parameter influencing the growth rate of aerobic microorganisms and the synthesis of metabolites. It is mainly determined by the agitation and the aeration rates in the fermenter. Aim. To study changes in pH, optical density (OD), and hemagglutinating (lectin) activity (HAA) of culture fluid (CF) of Bacillus subtilis strain IMV B-7724, a producer of extracellular cytotoxic lectin (ECL), during its cultivation in a laboratory fermenter at different agitation and aeration rates as well as to determine and compare the HAA, carbohydrate specifi city, and cytotoxic properties of the corresponding samples of the preparation isolated from CF. Methods. Batch antifoam-free fermentations were performed by culturing the strain in the modified Gause medium with galactose in two identical lab-scale fermenters with a working volume of 2.5 L at 37ºC for 48—72 h according to three fermentation variants. Variant 1: n — 400 rpm for the whole cultivation, the air supply to the CF — through a sparger at 0.1 vvm until the 39th h with further gradual decrease, KV — 4.2±0.3 g O2·L−1·h−1. Variant 2: n — 400 rpm for the first 24 h, then a gradual decrease to 200 rpm, air supply — through a sparger at 0.1 rpm for the first 12 h, followed by its switching into the fermenter free space, corresponding KV — from 4.2±0.3 to 0.3±0.1 g O2·L−1·h−1. Variant 3: n — 400 rpm and air supply to the fermenter free space during the whole cultivation, KV — 4.0±0.3 g O2·L−1·h−1. A number of biological properties of strain CF and isolated lectin samples were evaluated by biochemical, spectrophotometric, immunological, and culture methods. Statistical analysis was performed using Student’s t-test. Results. The maximum increase in the OD of CF relative to the initial values (28 and 21-fold) at the end of the period of the rapid growth of the strain (at 9th h), the μmax values of 0.33 and 0.41 h−1, and pH not lower than 6.7 and 6.3 units were observed for fermentation variants 1 and 2, respectively. In the case of variant 2, the HAA of CF reached 32 hemagglutinating units (HAU), and the samples isolated from it had a lectin activity of 512±64 HAU, whereas for variant 1 such values were lower:16 and 32±8 HAA, respectively; carbohydrate specificity of preparations to bovine submandibular gland mucin was the same, i.e. 0.2±0.1 mg/mL. In contrast to the above, a slower increase in the OD of the CF, a decrease in μmax, and significant acid formation (15-fold at the 9th h, 0.25 h−1, and pH decrease to 5.8 units, respectively) were observed for variant 3; in this case, the level of HAA of CF was minimal (2—4 HAU) and was absent in the corresponding isolated samples. The probable reason for such differences was the limited mass transfer in the CF due to the isolating effect of the foam layer on its surface formed as a result of intensive agitation. Conclusions. The rapid growth of the strain and an increase in the HAA of CF were observed during cultivation in a lab-scale fermenter by maintaining the maximum level of oxygen mass transfer with air supply into the CF through a sparger until the maximum OD was reached and the subsequent gradual decrease in the specifi ed level during further cultivation started.
Keywords: Bacillus subtilis IMV B-7724, laboratory fermenter, oxygen mass transfer, extracellular cytotoxic lectin, hemagglutinating activity, carbohydrate specifi city, cytotoxic properties.
- Kudryavtseva IG, Sharykina NI, Kovalenko EO, Khavych OO, Pavlovska GP. [Antitumor activity and toxicity of sialic acid-specifi cic lectins from bacteria]. Liky. 2006; (1—2):89—93. Ukrainian.
- Chekhun VF, Didenko GV, Cheremshenko NL, et al. [Strain of bacteria Bacillus subtilis IMB B-7724 — producer of cytotoxic substances with antitumor activity (Pat. N131824 UA)]. Publ. 25.01.2019. Bul. N2. Ukrainian.
- Fedosova NI, Cheremshenko NL, Hetman KI, Karaman OM, Symchych TV, Ivanchenko AV, Danyliuk OI, Voyeykova IM, Didenko GV. Bioactivity of the Bacillus subtilis IMV B-7724 extracellular lectin. Mikrobiol Z. 2019; 81(4):107—117. Ukrainian. https://doi.org/10.15407/microbiolj83.01.039
- Fedosova NI, Cheremshenko NL, Hetman KI, Symchych TV, Chumak AV, Shliahovenko VO, Voyeykova IM, Didenko GV. Physicochemical and cytotoxic properties of Bacillus subtilis IMV B-7724 extracellular lectin. Mikrobiol Z. 2021; 83(1):39—48.
- Pidhorskii VS, Kovalenko EO, Rybalko SL, et al. [Lectin-containing drug for treating influenza (Pat. N83578 UA)]. Publ. 25.07.2008. Bul. N14. Ukrainian.
- Pidhorskii VS, Kovalenko EO, Rybalko SL, et al. [Medication, containing the bacterial lectin for treatment of patients, affected with a herpesvirus of 1 and 2 types (Pat. N84928 UA)]. Publ. 10.12.2008. Bul. N23. Ukrainian.
- Pidhorskii VS, Kovalenko EO, Rybalko SL, et al. [Medicament, containing bacterial lectin for treatment of patients, affected by hepatitis C (Pat. N83305 UA)]. Publ. 25.06.2008. Bul. N12. Ukrainian.
- Pidhorskii VS, Rybalko SL, Kovalenko EO, et al. [An application of sialospecific lectin, isolated from strain Bacillus subtilis 668 IMB for ingibition of reproduction of human immunodefi ciency virus (Pat. N68373 UA)]. Publ. 16.08.2004. Bul. N8. Ukrainian.
- Kovalenko EO. [Extracellular lectins of Bacillus genus bacteria]. Abstract of dissertation doctor biol. science. Kyiv; 1999. Ukrainian. http://library.nuft.edu.ua/ebook/file/03.00.07kovalenko.pdf
- Podgorsky VS, Kovalenko EA, Simonenko IA. [Lectins of bacteria]. Kyiv: Naukova dumka; 1992. Russian.
- Netrusov AI, Egorova MA, Zakharchuk LM, et al. [Practical training in microbiology: textbook for students of higher education institutions]. AI Netrusov, editor. Moscow: Academia; 2005. p. 39—40. Russian.
- Lutsyk MD, Panasyuk EN, Antonyuk VA. [Methods of searching for lectins (phytohaemagglutinins) and determination of their immunochemical specifi city: Methodical recommendations for biochemists and immunologists]. Lviv; 1980. Russian.
- Lakin G.F. [Biometry]. Moskow:Vysshaia shkola; 1990. Russian.
- Su Y, Liu C, Fang H, Zhang D. Bacillus subtilis: a universal cell factory for industry, agriculture, biomaterials and medicine. Microb Cell Fact. 2020; 19:173. https://doi.org/10.1186/s12934-020-01436-8
- Shih I-L, Lin C-Y, Wu J-Y, Hsieh C. Production of antifungal lipopeptide from Bacillus subtilis in submerged fermentation using shake flask and fermentor. Korean J Chem Eng. 2009; 26(6):1652—1661. https://doi.org/10.1007/s11814-009-0237-0
- Ha S, Kim HM, Chun HH, Hwang IM, Lee J-H, Kim J-C, Kim IS, Park HW. Effect of oxygen supply on surfactin production and sporulation in submerged culture of Bacillus subtilis Y9. Appl Sci. 2018; 8:1660. https://doi.org/10.3390/app8091660
- Takesono S, Onodera M. Biosurfactant production in stirred-tank fermenter under mechanical foam control. Trends in Chemical Engineering. 2019; 17:51—57.
- Delvigne F, Lecomte J-P. Foam formation and control in bioreactors. In: Encyclopedia of Industrial Biotechnology: Bioprocess, Bioseparation, and Cell Technology; 2010. p. 1—34. https://doi.org/10.1002/9780470054581.eib326
- Kisten OG, Kovalenko EO, Getman KI, Sashchuk OV, Pidgorsky VS, Tyshchenko LM. Extracellular lectin produced by Bacillus subtilis strain IMV B- 7014 depending on the culture conditions. Mikrobiol Z. 2019; 81(4):3—14. https://doi.org/10.15407/microbiolj81.04.003
- Research project report “Development of technological bases of microbial antiherpes drug in the conditions of current production”. Kyiv: Zabolotny Institute of Microbiology and Virology, National Academy of Sciences of Ukraine; 2018. p. 37. Ukrainian.
- Viesturs U, Leite M. [Certain new biotechnological processes and the equipment for their implementation (Rewiew)]. Prikl Biokhim Mikrobiol. 1997; 33(3):243—256. Russian.