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The in vitro liquid mass culture of entomopathogenic nematodes in shake flasks and bioreactors
Thesis (PhDConsEcol)--Stellenbosch University, 2023.
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| Format: | Thesis |
| Language: | English |
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Stellenbosch : Stellenbosch University
2023
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| _version_ | 1867614047680069632 |
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| access_status_str | Open Access |
| author | Dunn, Murray David |
| author2 | Malan, Antoinette Paula |
| author_browse | Dunn, Murray David Malan, Antoinette Paula |
| author_facet | Malan, Antoinette Paula Dunn, Murray David |
| author_sort | Dunn, Murray David |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (PhDConsEcol)--Stellenbosch University, 2023. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/128467 |
| institution | Stellenbosch University (South Africa) |
| language | English |
| last_indexed | 2026-06-10T12:45:50.231Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2023 |
| publishDateRange | 2023 |
| publishDateSort | 2023 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/128467 The in vitro liquid mass culture of entomopathogenic nematodes in shake flasks and bioreactors Dunn, Murray David Malan, Antoinette Paula Belur, Prasanna D. Stellenbosch University. Faculty of AgriSciences. Dept. of Conservation Ecology and Entomology. Entomopathogenic nematodes -- Biotechnology Insect nematodes -- Biological control Insect nematodes -- Classification Bioreactors -- Fluid dynamics Nematodes -- Life cycles Mass production UCTD Thesis (PhDConsEcol)--Stellenbosch University, 2023. ENGLISH ABSTRACT: The commercial use of entomopathogenic nematodes (EPNs) as a biopesticide has significant barriers in South Africa, including those in terms of production, market access, registration, and education. The in vitro liquid mass production of EPNs begins with the development of a standard protocol for production in shake flasks, with high yields in a short production time. This is followed by identifying optimisation factors that can improve the mass production process, which is coupled with scale-up from desktop to industrial-sized bioreactors. The steps involved were thus followed for the investigation and development of a standard protocol for the in vitro liquid mass production of Steinernema yirgalemense and Heterorhabditis zealandica. After the establishment of a monoxenic stock culture of S. yirgalemense, subsequent optimisation in the protein, lipid and nitrogen sources yielded unexpected results with a large increase in yields and a reduction in production costs, when replacing soy powder protein and olive oil with egg yolk and canola oil, respectively. This was followed by conducting investigations into the life cycle of S. yirgalemense, whose life cycle in the flask was compared to that in a 10-L custom-designed desktop bioreactor. The flask and bioreactor life cycle results indicate that the growth and development of S. yirgalemense in the bioreactor vessel is slower than that which it experiences in the flask. However, similar yields of up to 2.5-3.0 × 105 IJs/ml can be achieved, supporting the transfer of the scale-up process to larger bioreactors. Such optimisation factors as the IJ inoculum concentration and timing were used to further improve the mass culture process. The results indicate a larger IJ inoculum concentration (of up to 6000 IJs/ml) to be the optimum IJ concentration, as it results in a significantly higher number of adults, increased yield (up to 3.0 × 105) and reduced batch variation. The results showed the successful development of a standard protocol for the in vitro liquid mass production of S. yirgalemense in shake flasks and in a 10-L desktop bioreactor, which supported the next phase of research, being the development of a standard protocol for Heterorhabditis zealandica. In contrast, however, H. zealandica trials proved to be far more difficult than that of S. yirgalemense in terms of producing a monoxenic stock culture due to the reproductive strategies and copulation methods of Heterorhabditis species. The first trials with H. zealandica, and its symbiotic bacteria, Photorhabdus thracensis, indicated that the preferred protein is soy, and not egg yolk. The results of this report and the success attained in the development of the standard protocol of mass production for two South African EPN isolates greatly supports further research, as well as the commercialisation of a South African EPN-based biopesticide product. AFRIKAANSE OPSOMMING: Die kommersiële gebruik van entomopatogeniese nematodes (EPNs) as ʼn biologiese beheer agent van pes insekte, ondervind probleme in Suid-Afrika, in terme van onder andere marktoegang, registrasie en opleiding. Die in vitro massa-produksie van EPNs begin deur die ontwikkeling van ʼn standaard-protokol in skudflesse, met die doel om hoë opbrengste te handhaaf in ʼn kort produksietyd. Die proses word gevolg deur die identifisering van optimiserings faktore wat proses kan verbeter en wat gepaard gaan met opskaling tot industriële grootte bioreaktore. Die stappe is gevolg vir die ontwikkeling van ʼn standaard-protokol vir die in vitro massa-produksie van Steinernema yirgalemense en Heterorhabditis zealandica. Na die vestiging van ʼn suiwer inokulum-kultuur van S. yirgalemense, het daaropvolgende optimalisering van die proteïen-, lipied- en stikstofbronne onverwagte resultate gelewer met ʼn verhoogde toename in opbrengs en ʼn verlaging in produksiekoste, wanneer soja-poeier proteïen en olyfolie met eiergeel en Canola olie vervang is. Na afloop van die resultate is ʼn die lewensiklus van S. yirgalemense in ʼn 10-L pasgemaakte bioreaktor en in flesse met mekaar vergelyk. Die fles- en bioreaktor-lewensiklus resultate het daarop gedui dat die ontwikkeling en groei van S. yirgalemense stadiger plaasgevind het in bioreaktore, in vergelyking met skudflesse. Ongeag is daar steeds soortgelyke opbrengste te behaal van 2.5-3.0 × 10⁵ IJs/ml met opskaling. Optimiseringsfaktore soos van die inokulum-konsentrasie en tydsberekening is gebruik om die massa-produksie proses verder te verbeter. Die resultate dui aan dat ʼn verhoogde konsentrasie van 6000 IJs/ml die optimum-konsentrasie is, aangesien dit lei tot beduidende hoër aantal volwassenes, verhoogde opbrengste asook verminderde groepsvariasie. Die resultate het ook die suksesvolle daarstelling van ʼn standaardprotokol vir die in vitro massa-teling van S. yirgalemense in skudflesse en in ʼn 10L-bioreaktor tot gevolg gehad wat die volgende fase van navorsing ondersteun het, naamlik die ontwikkeling van ʼn standaard-protokol vir Heterorhabditis zealandica. In teenstelling hiermee was dit duidelik dat die ondersoek met H. zealandica veel meer uitdagend was as vir S. yirgalemense in terme van die produksie van ʼn suiwer kultuur as gevolg van die voorplanting strategieë en kopulasie proses van Heterorhabditis. Die eerste eksperimentele ondersoeke met H. zealandica en sy simbiotiese bakterieë Photorhabdus thracensis, het wel aangedui dat die voorkeurproteïen soja is en nie eiergeel nie. Die resultate van hierdie verslag asook die sukses wat behaal is in die ontwikkeling van ʼn standaardprotokol vir massaproduksie van twee Suid-Afrikaanse EPN-spesies ondersteun grootliks verdere navorsing, asook die kommersialisering van ʼn Suid- Afrikaanse EPN gebaseerde biologiese beheer produk. Doctoral 2023-03-05T16:11:39Z 2023-08-30T13:13:06Z 2023-03 2023-03-05T16:11:39Z 2023-08-31T09:18:48Z 2023-03-05T16:11:39Z 2023-08-31T09:18:48Z 2023-03 Thesis https://scholar.sun.ac.za/handle/10019.1/128467 en Stellenbosch University application/pdf xx, 164 pages : illustrations application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Entomopathogenic nematodes -- Biotechnology Insect nematodes -- Biological control Insect nematodes -- Classification Bioreactors -- Fluid dynamics Nematodes -- Life cycles Mass production UCTD Dunn, Murray David The in vitro liquid mass culture of entomopathogenic nematodes in shake flasks and bioreactors |
| title | The in vitro liquid mass culture of entomopathogenic nematodes in shake flasks and bioreactors |
| title_full | The in vitro liquid mass culture of entomopathogenic nematodes in shake flasks and bioreactors |
| title_fullStr | The in vitro liquid mass culture of entomopathogenic nematodes in shake flasks and bioreactors |
| title_full_unstemmed | The in vitro liquid mass culture of entomopathogenic nematodes in shake flasks and bioreactors |
| title_short | The in vitro liquid mass culture of entomopathogenic nematodes in shake flasks and bioreactors |
| title_sort | in vitro liquid mass culture of entomopathogenic nematodes in shake flasks and bioreactors |
| topic | Entomopathogenic nematodes -- Biotechnology Insect nematodes -- Biological control Insect nematodes -- Classification Bioreactors -- Fluid dynamics Nematodes -- Life cycles Mass production UCTD |
| url | https://scholar.sun.ac.za/handle/10019.1/128467 |
| work_keys_str_mv | AT dunnmurraydavid theinvitroliquidmasscultureofentomopathogenicnematodesinshakeflasksandbioreactors AT dunnmurraydavid invitroliquidmasscultureofentomopathogenicnematodesinshakeflasksandbioreactors |