Immobilization of Clostridium perfringens type D in calcium alginate beads: toxin production mimics free cell culture
-
Hakimeh Rakhshandeh
,
Mehrdad Shamsaddini Bafti
,
Behnaz Familsatarian
,
Maryam Nooshadokht
,
Payam Khazaeli
,
Omid Raiesi
,
Bagher Amirheidari
Abstract
Background and Objectives: Cell-immobilization is used to maintain microbial culture to produce metabolites in repeated-batch or continuous fermentations, thereby reducing the time and resources spent on delivering mass production of microbe. The technique also enables shortening of the detoxification phase and the amount of formaldehyde required due to low incidence of viable bacteria in the extract.
Materials and Methods: A solution of sodium alginate containing Clostridium perfringens cells was dropped into stirring CaCl2 solution via a sterile syringe needle. Optimizations resulted in reasonably uniform beads containing C. perfringens. Beads were externally stabilized by poly L-lysine, followed by immersion in a solution of Na-alginate to coat them with a new layer of alginate forming an alginate-PLL-alginate cortex.
Results: This study proved successful in immobilizing C. perfringens cells inside uniform alginate microspheres. Cell loading and cell propagation inside the beads were measured. The cell loaded beads were cultivable in liquid media producing 550 minimum lethal doses per milliliter (MLD/ml) in a 72 h.
Conclusion: The research paved the way for further investigations to optimize and establish an efficient bacterial encapsulation method. Thus, it seems possible to produce toxins from beads engulfing C. perfringens on larger scales via repeated-batch or continuous fermentation processes.
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2. Kiu R, Hall LJ. An update on the human and animal enteric pathogen Clostridium perfringens. Emerg Microbes Infect 2018; 7: 141.
3. Uzal FA, Songer JG. Diagnosis of Clostridium perfringens intestinal infections in sheep and goats. J Vet Diagn Invest 2008; 20: 253-265.
4. Rood JI, Adams V, Lacey J, Lyras D, McClane BA, Melville SB, et al. Expansion of the Clostridium perfringens toxin-based typing scheme. Anaerobe 2018; 53: 5-10.
5. Seyed Sayyah P, Golestani B, Pilehchian Langroudi R. Cloning of Clostridium perfringens iota toxin gene in Escherichia coli. Arch Razi Inst 2018; 73: 107-111.
6. Smedley JG 3rd, Fisher DJ, Sayeed S, Chakrabarti G, McClane BA. The enteric toxins of Clostridium perfringens. Rev Physiol Biochem Pharmacol 2004; 152: 183-204.
7. Pluvinage B, Massel PM, Burak K, Boraston AB. Structural and functional analysis of four family 84 glycoside hydrolases from the opportunistic pathogen Clostridium perfringens. Glycobiology 2019; 30: 49-57.
8. Bakhshi F, Pilehchian Langroudi R, Eimani BG. Enhanced expression of recombinant beta toxin of Clostridium perfringens type B using a commercially available Escherichia coli strain. Onderstepoort J Vet Res 2016; 83(1):a1136.
9. Hussain R, Guangbin Z, Abbas RZ, Siddique AB, Mohiuddin M, Khan I, et al. Clostridium perfringens types A and D involved in peracute deaths in goats kept in Cholistan ecosystem during Winter season. Front Vet Sci 2022; 9: 849856.
10. Vieco-Saiz N, Belguesmia Y, VachÚe A, Le MarÚchal C, Salvat G, Drider D. Antibiotic resistance, genome analysis and further safe traits of Clostridium perfringens ICVB082; a strain capable of producing an inhibitory compound directed only against a closely related pathogenic strain. Anaerobe 2020; 62: 102177.
11. Cho YG, Rhee SK, Lee ST. Influence of phenol on biodegradation of p-nitrophenol by freely suspended and immobilized Nocardioides sp. NSP41. Biodegradation 2000; 11:21-28.
12. Kariminiaae-Hamedaani H-R, Kanda K, Kato F. Wastewater treatment with bacteria immobilized onto a ceramic carrier in an aerated system. J Biosci Bioeng 2003; 95: 128-132.
13. Bangrak P, Limtong S, Phisalaphong M. Continuous ethanol production using immobilized yeast cells entrapped in loofa-reinforced alginate carriers. Braz J Microbiol 2011; 42: 676-684.
14. Guo X, Zhou J, Xiao D. Improved ethanol production by mixed immobilized cells of Kluyveromyces marxianus and Saccharomyces cerevisiae from cheese whey powder solution fermentation. Appl Biochem Biotechnol 2010; 160: 532-538.
15. Wang X, Liu J, Du G, Zhou J, Chen J. Efficient production of L-sorbose from D-sorbitol by whole cell immobilization of Gluconobacter oxydans WSH-003. Biochem Eng J 2013; 77: 171-176.
16. Sekoai PT, Awosusi AA, Yoro KO, Singo M, Oloye O, Ayeni AO, et al. Microbial cell immobilization in biohydrogen production: a short overview. Crit Rev Biotechnol 2018; 38: 157-171.
17. Li Y-M, Gao J-Q, Pei X-Z, Du C, Fan C, Yuan W-J, et al. Production of l-alanyl-l-glutamine by immobilized Pichia pastoris GS115 expressing α-amino acid ester acyltransferase. Microb Cell Fact 2019; 18: 27.
18. Coelho-Rocha ND, De Castro CP, De Jesus LC, Leclercq SY, De Cicco Sandes SH, Nunes AC, et al. Microencapsulation of lactic acid bacteria improves the gastrointestinal delivery and in situ expression of recombinant fluorescent protein. Front Microbiol 2018; 9: 2398.
19. Raymond M-C, Neufeld RJ, Poncelet D. Encapsulation of brewers yeast in chitosan coated carrageenan microspheres by emulsification/thermal gelation. Artif Cells Blood Substit Immobil Biotechnol 2004; 32: 275-291.
20. Lu J, Peng W, Lv Y, Jiang Y, Xu B, Zhang W, et al. Application of cell immobilization technology in microbial cocultivation systems for biochemicals production. Ind Eng Chem Res 2020; 59: 17026-17034.
21. Orive G, Hernández RM, Rodriguez Gascón A, Calafiore R, Chang TM, De Vos P, et al. History, challenges and perspectives of cell microencapsulation. Trends Biotechnol 2004; 22: 87-92.
22. Behera S, Kar S, Mohanty RC, Ray RC. Comparative study of bio-ethanol production from mahula (Madhuca latifolia L.) flowers by Saccharomyces cerevisiae cells immobilized in agar agar and Ca-alginate matrices. Appl Energy 2010; 87: 96-100.
23. John RP, Tyagi RD, Brar SK, Surampalli RY, Prévost D. Bio-encapsulation of microbial cells for targeted agricultural delivery. Crit Rev Biotechnol 2011; 31: 211-226.
24. Ribeiro CC, Barrias CC, Barbosa MA. Calcium phosphate-alginate microspheres as enzyme delivery matrices. Biomaterials 2004; 25: 4363-4373.
25. Takka S, Gürel A. Evaluation of chitosan/alginate beads using experimental design: formulation and in vitro characterization. AAPS PharmSciTech 2010; 11: 460-466.
26. Constantinidis I, Grant SC, Celper S, Gauffin-Holmberg I, Agering K, Oca-Cossio JA, et al. Non-invasive evaluation of alginate/poly-l-lysine/alginate microcapsules by magnetic resonance microscopy. Biomaterials 2007; 28: 2438-2445.
27. Bishnoi NR, Kumar R, Bishnoi K. Biosorption of Cr (VI) with Trichoderma viride immobilized fungal biomass and cell free Ca-alginate beads. Indian J Exp Biol 2007; 45: 657-664.
28. Shu XZ, Zhu KJ. The release behavior of brilliant blue from calcium–alginate gel beads coated by chitosan: the preparation method effect. Eur J Pharm Biopharm 2002; 53: 193-201.
29. Ferrari LA, Giannuzzi L. Clinical parameters, postmortem analysis and estimation of lethal dose in victims of a massive intoxication with diethylene glycol. Forensic Sci Int 2005; 153: 45-51.30. Moreno-Garrido I. Microalgae immobilization:
current techniques and uses. Bioresour Technol 2008; 99: 3949-3464.
31. Wu D-Q, Zhang G-L, Shen C, Zhao Q, Li H, Meng Q. Evaluation of diffusion in gel entrapment cell culture within hollow fibers. World J Gastroenterol 2005; 11: 1599-604.
32. Ching SH, Bansal N, Bhandari B. Alginate gel particles–A review of production techniques and physical properties. Crit Rev Food Sci Nutr 2017; 57: 1133-1152.
33. Chaikham P, Apichartsrangkoon A, George T, Jirarattanarangsri W. Efficacy of polymer coating of probiotic beads suspended in pressurized and pasteurized longan juices on the exposure to simulated gastrointestinal environment. Int J Food Sci Nutr 2013; 64: 862-869.
34. Mandal S, Hati S, Puniya AK, Khamrui K, Singh K. Enhancement of survival of alginate‐encapsulated Lactobacillus casei NCDC 298. J Sci Food Agric 2014; 94: 1994-2001.
35. Leung A, Trau M, Nielsen LK. Assembly of multilayer PSS/PAH membrane on coherent alginate/PLO microcapsule for long‐term graft transplantation. J Biomed Mater Res A 2009; 88: 226-237.36. De Prisco A, Maresca D, Ongeng D,
Mauriello G. Microencapsulation by vibrating technology of the probiotic strain Lactobacillus reuteri DSM 17938 to enhance its survival in foods and in gastrointestinal environment. LWT-Food SCI Technol 2015; 61: 452-462.37. Kanmani
P, Kumar RS, Yuvaraj N, Paari KA, Pattukumar V, Arul V. Cryopreservation and microencapsulation of a probiotic in alginate-chitosan capsules improves survival in simulated gastrointestinal conditions. Biotechnol Bioprocess Eng 2011; 16: 1106-1114.
38. Trabelsi I, Ayadi D, Bejar W, Bejar S, Chouayekh H, Ben Salah R. Effects of Lactobacillus plantarum immobilization in alginate coated with chitosan and gelatin on antibacterial activity. Int J Biol Macromol 2014; 64: 84-89.
39. Simó G, Fernández‐Fernández E, Vila‐Crespo J, Ruipérez V, Rodríguez‐Nogales JM. Research progress in coating techniques of alginate gel polymer for cell encapsulation. Carbohydr Polym 2017; 170: 1-14.
40. Hussack G, Grohs BM, Almquist KC, McLean MD, Ghosh R, Hall JC, et al. Purification of plant-derived antibodies through direct immobilization of affinity ligands on cellulose. J Agric Food Chem 2010; 58: 3451-3459.
41. Yu D, Wang N, Gong Y, Wu Z, Wang W, Wang L, et al. Screening of active sites and study on immobilization of Bacillus cereus phospholipase C. LWT 2022; 159: 113245.
42. Whelehan M, Marison IW. Microencapsulation using vibrating technology. J Microencapsul 2011; 28: 669-688.
2. Kiu R, Hall LJ. An update on the human and animal enteric pathogen Clostridium perfringens. Emerg Microbes Infect 2018; 7: 141.
3. Uzal FA, Songer JG. Diagnosis of Clostridium perfringens intestinal infections in sheep and goats. J Vet Diagn Invest 2008; 20: 253-265.
4. Rood JI, Adams V, Lacey J, Lyras D, McClane BA, Melville SB, et al. Expansion of the Clostridium perfringens toxin-based typing scheme. Anaerobe 2018; 53: 5-10.
5. Seyed Sayyah P, Golestani B, Pilehchian Langroudi R. Cloning of Clostridium perfringens iota toxin gene in Escherichia coli. Arch Razi Inst 2018; 73: 107-111.
6. Smedley JG 3rd, Fisher DJ, Sayeed S, Chakrabarti G, McClane BA. The enteric toxins of Clostridium perfringens. Rev Physiol Biochem Pharmacol 2004; 152: 183-204.
7. Pluvinage B, Massel PM, Burak K, Boraston AB. Structural and functional analysis of four family 84 glycoside hydrolases from the opportunistic pathogen Clostridium perfringens. Glycobiology 2019; 30: 49-57.
8. Bakhshi F, Pilehchian Langroudi R, Eimani BG. Enhanced expression of recombinant beta toxin of Clostridium perfringens type B using a commercially available Escherichia coli strain. Onderstepoort J Vet Res 2016; 83(1):a1136.
9. Hussain R, Guangbin Z, Abbas RZ, Siddique AB, Mohiuddin M, Khan I, et al. Clostridium perfringens types A and D involved in peracute deaths in goats kept in Cholistan ecosystem during Winter season. Front Vet Sci 2022; 9: 849856.
10. Vieco-Saiz N, Belguesmia Y, VachÚe A, Le MarÚchal C, Salvat G, Drider D. Antibiotic resistance, genome analysis and further safe traits of Clostridium perfringens ICVB082; a strain capable of producing an inhibitory compound directed only against a closely related pathogenic strain. Anaerobe 2020; 62: 102177.
11. Cho YG, Rhee SK, Lee ST. Influence of phenol on biodegradation of p-nitrophenol by freely suspended and immobilized Nocardioides sp. NSP41. Biodegradation 2000; 11:21-28.
12. Kariminiaae-Hamedaani H-R, Kanda K, Kato F. Wastewater treatment with bacteria immobilized onto a ceramic carrier in an aerated system. J Biosci Bioeng 2003; 95: 128-132.
13. Bangrak P, Limtong S, Phisalaphong M. Continuous ethanol production using immobilized yeast cells entrapped in loofa-reinforced alginate carriers. Braz J Microbiol 2011; 42: 676-684.
14. Guo X, Zhou J, Xiao D. Improved ethanol production by mixed immobilized cells of Kluyveromyces marxianus and Saccharomyces cerevisiae from cheese whey powder solution fermentation. Appl Biochem Biotechnol 2010; 160: 532-538.
15. Wang X, Liu J, Du G, Zhou J, Chen J. Efficient production of L-sorbose from D-sorbitol by whole cell immobilization of Gluconobacter oxydans WSH-003. Biochem Eng J 2013; 77: 171-176.
16. Sekoai PT, Awosusi AA, Yoro KO, Singo M, Oloye O, Ayeni AO, et al. Microbial cell immobilization in biohydrogen production: a short overview. Crit Rev Biotechnol 2018; 38: 157-171.
17. Li Y-M, Gao J-Q, Pei X-Z, Du C, Fan C, Yuan W-J, et al. Production of l-alanyl-l-glutamine by immobilized Pichia pastoris GS115 expressing α-amino acid ester acyltransferase. Microb Cell Fact 2019; 18: 27.
18. Coelho-Rocha ND, De Castro CP, De Jesus LC, Leclercq SY, De Cicco Sandes SH, Nunes AC, et al. Microencapsulation of lactic acid bacteria improves the gastrointestinal delivery and in situ expression of recombinant fluorescent protein. Front Microbiol 2018; 9: 2398.
19. Raymond M-C, Neufeld RJ, Poncelet D. Encapsulation of brewers yeast in chitosan coated carrageenan microspheres by emulsification/thermal gelation. Artif Cells Blood Substit Immobil Biotechnol 2004; 32: 275-291.
20. Lu J, Peng W, Lv Y, Jiang Y, Xu B, Zhang W, et al. Application of cell immobilization technology in microbial cocultivation systems for biochemicals production. Ind Eng Chem Res 2020; 59: 17026-17034.
21. Orive G, Hernández RM, Rodriguez Gascón A, Calafiore R, Chang TM, De Vos P, et al. History, challenges and perspectives of cell microencapsulation. Trends Biotechnol 2004; 22: 87-92.
22. Behera S, Kar S, Mohanty RC, Ray RC. Comparative study of bio-ethanol production from mahula (Madhuca latifolia L.) flowers by Saccharomyces cerevisiae cells immobilized in agar agar and Ca-alginate matrices. Appl Energy 2010; 87: 96-100.
23. John RP, Tyagi RD, Brar SK, Surampalli RY, Prévost D. Bio-encapsulation of microbial cells for targeted agricultural delivery. Crit Rev Biotechnol 2011; 31: 211-226.
24. Ribeiro CC, Barrias CC, Barbosa MA. Calcium phosphate-alginate microspheres as enzyme delivery matrices. Biomaterials 2004; 25: 4363-4373.
25. Takka S, Gürel A. Evaluation of chitosan/alginate beads using experimental design: formulation and in vitro characterization. AAPS PharmSciTech 2010; 11: 460-466.
26. Constantinidis I, Grant SC, Celper S, Gauffin-Holmberg I, Agering K, Oca-Cossio JA, et al. Non-invasive evaluation of alginate/poly-l-lysine/alginate microcapsules by magnetic resonance microscopy. Biomaterials 2007; 28: 2438-2445.
27. Bishnoi NR, Kumar R, Bishnoi K. Biosorption of Cr (VI) with Trichoderma viride immobilized fungal biomass and cell free Ca-alginate beads. Indian J Exp Biol 2007; 45: 657-664.
28. Shu XZ, Zhu KJ. The release behavior of brilliant blue from calcium–alginate gel beads coated by chitosan: the preparation method effect. Eur J Pharm Biopharm 2002; 53: 193-201.
29. Ferrari LA, Giannuzzi L. Clinical parameters, postmortem analysis and estimation of lethal dose in victims of a massive intoxication with diethylene glycol. Forensic Sci Int 2005; 153: 45-51.30. Moreno-Garrido I. Microalgae immobilization:
current techniques and uses. Bioresour Technol 2008; 99: 3949-3464.
31. Wu D-Q, Zhang G-L, Shen C, Zhao Q, Li H, Meng Q. Evaluation of diffusion in gel entrapment cell culture within hollow fibers. World J Gastroenterol 2005; 11: 1599-604.
32. Ching SH, Bansal N, Bhandari B. Alginate gel particles–A review of production techniques and physical properties. Crit Rev Food Sci Nutr 2017; 57: 1133-1152.
33. Chaikham P, Apichartsrangkoon A, George T, Jirarattanarangsri W. Efficacy of polymer coating of probiotic beads suspended in pressurized and pasteurized longan juices on the exposure to simulated gastrointestinal environment. Int J Food Sci Nutr 2013; 64: 862-869.
34. Mandal S, Hati S, Puniya AK, Khamrui K, Singh K. Enhancement of survival of alginate‐encapsulated Lactobacillus casei NCDC 298. J Sci Food Agric 2014; 94: 1994-2001.
35. Leung A, Trau M, Nielsen LK. Assembly of multilayer PSS/PAH membrane on coherent alginate/PLO microcapsule for long‐term graft transplantation. J Biomed Mater Res A 2009; 88: 226-237.36. De Prisco A, Maresca D, Ongeng D,
Mauriello G. Microencapsulation by vibrating technology of the probiotic strain Lactobacillus reuteri DSM 17938 to enhance its survival in foods and in gastrointestinal environment. LWT-Food SCI Technol 2015; 61: 452-462.37. Kanmani
P, Kumar RS, Yuvaraj N, Paari KA, Pattukumar V, Arul V. Cryopreservation and microencapsulation of a probiotic in alginate-chitosan capsules improves survival in simulated gastrointestinal conditions. Biotechnol Bioprocess Eng 2011; 16: 1106-1114.
38. Trabelsi I, Ayadi D, Bejar W, Bejar S, Chouayekh H, Ben Salah R. Effects of Lactobacillus plantarum immobilization in alginate coated with chitosan and gelatin on antibacterial activity. Int J Biol Macromol 2014; 64: 84-89.
39. Simó G, Fernández‐Fernández E, Vila‐Crespo J, Ruipérez V, Rodríguez‐Nogales JM. Research progress in coating techniques of alginate gel polymer for cell encapsulation. Carbohydr Polym 2017; 170: 1-14.
40. Hussack G, Grohs BM, Almquist KC, McLean MD, Ghosh R, Hall JC, et al. Purification of plant-derived antibodies through direct immobilization of affinity ligands on cellulose. J Agric Food Chem 2010; 58: 3451-3459.
41. Yu D, Wang N, Gong Y, Wu Z, Wang W, Wang L, et al. Screening of active sites and study on immobilization of Bacillus cereus phospholipase C. LWT 2022; 159: 113245.
42. Whelehan M, Marison IW. Microencapsulation using vibrating technology. J Microencapsul 2011; 28: 669-688.
| Files | ||
| Issue | Vol 14 No 4 (2022) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijm.v14i4.10236 | |
| Keywords | ||
| Clostridium perfringens; Cell-immobilization techniques; Encapsulation; Calcium alginate beads; Toxin production | ||
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This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. |
How to Cite
1.
Rakhshandeh H, Shamsaddini Bafti M, Familsatarian B, Nooshadokht M, Khazaeli P, Raiesi O, Amirheidari B. Immobilization of Clostridium perfringens type D in calcium alginate beads: toxin production mimics free cell culture. Iran J Microbiol. 2022;14(4):503-509.
