Kinetic evaluation of lipopeptide production by Bacillus amyloliquefaciens

Herbst, Willem Jacobus (2017-03)

Thesis (MEng)--Stellenbosch University, 2017.

Thesis

ENGLISH SUMMARY: Crop losses, caused by phytopathogens and pests, are estimated to be as high as 83% worldwide. These losses along with the world's growing population put additional strain on food production and security which emphasise the need for improved crop protection strategies. Chemically derived pesticides and fungicides are the preferred control strategy against postharvest diseases, however their detrimental effect on the environment and human life have directed research towards alternative strategies. Biocontrol have been identified as an alternative since they are environmentally safe, biodegradable and show antagonistic behaviour against fungi, bacteria and even viruses. Bacillus spp. have been shown to be effective as they produce the lipopeptides surfactin, iturin and fengycin. Direct application of the organism as cells and spores have been well documented and is the focus of commercially developed products. However, cell free lipopeptides have achieved greater inhibition against phytopathogens and are less sensitive to environmental factors. The study optimised upstream production of antifungal lipopeptides, iturin and fengycin by Bacillus amyloliquefaciens in controlled batch cultures. The effect of nitrogen source, concentration and dissolved oxygen availability were quantified through rigorous kinetic [NEXT THREE PARAGRAPHS DID NOT COPY. PLEASE REFER TO ORIGINAL DOCUMENT] production (158.15 mAU*min/g/l), highest maximum productivity (26.92 mAU*min/h) and competitively high selectivity (86.7%). A decrease in dissolved oxygen availability, decreased antifungal lipopeptide production kinetics. Low oxygen conditions forced nitrate to be used as an alternative electron acceptor, decreasing the amount of nitrate available for lipopeptide production. Optimum conditions cultured in the bioreactor performed better with respect to antifungal kinetics (maximum concentration, specific production and productivity) except µmax and CDW compared to the optimum reported in a previous study (8 g/l NH4NO3). Maximum concentration increased from 285.66 to 290.17 mAU*min, specific antifungal production from 51.85 to 58.1 mAU*min/g/l and productivity from 5.67 to 22.32 mAU*min/h. Culture supernatant, concentrated by acid precipitation, were used for antifungal efficacy tests. Fungal inhibition was observed against Botrytis cinerea, Alternaria brassicicola, Monilinia fructigena, Penicillium expansum and Rhizopus stolonifer while no inhibition was observed against Aspergillus sclerotiorum. The high effectiveness of antifungal lipopeptides in combination with kinetic data from this study indicate the potential to develop a standardised antifungal product for use against phytopathogens affecting post-harvest fruit. The effect of the process parameters on homologue production and ratio should be investigated, which could allow antifungal products to be tailored to contain specific homologues effective against specific phytopathogens. The use of continuous cultures for further kinetic evaluation and optimisation should be considered as it’s been shown, in this study, to be possible.

AFRIKAANS OPSOMMING: Daar word beraam dat gewasverliese, wat veroorsaak word deur fitopatogene en peste, so hoog kan wees as 83% wêreldwyd. Hierdie verliese, saam met die groeiende wêreldbevolking, plaas addisionele druk op voedselproduksie en –sekuriteit, wat die behoefte aan verbeterde gewas beskermingstrategieë beklemtoon. Chemikalieë wat verkry word uit plaag- en swamdoders is die gekose beheerstrategie teen na-oes siektes, alhoewel hul nadelige effek op die omgewing en menslike welstand, navorsing gerig het op alternatiewe beheerstrategieë. Bio-beheer is geïdentifiseer as ʼn alternatief aangesien dit omgewingsvriendelik is, bio-afbreekbaar is en antagonistiese gedrag toon teenoor fungi, bakterieë en selfs virusse. Bacillus spp. het effektief getoon as biologiese beheermaatreël aangesien dit die lipopeptiede surfactin, iturin en fengycin produseer. Direkte toediening van die organisme as selle en spore, is goed gedokumenteer en is die fokus van kommersieel ontwikkelde produkte. Sel-vrye lipopeptiede het egter groter inhibisie teen fitopatogene bereik en is minder sensitief vir omgewingsfaktore. Die studie optimaliseer die produksie van anti-fungale lipopeptiede, iturin en fengycin, deur Bacillus amyloliquefaciens in gekontroleerde lottestande. Die effek van stikstof bron, konsentrasie en opgeloste suurstof beskikbaarheid, is gekwantifiseer deur streng kinetiese evaluering en bevestig deur anti-fungale effektiwiteitstoetse. Kinetiese evaluering het staatgemaak op skudflesse en volledige instrumentele bio-reaktor kulture, van waar lipopeptiede monsters geneem is vir analise, deur middel van hoë-vloeistof chromatografie. Verhoogde stikstof (4 tot 8 g/l NH4NO3) in bio-reaktor kulture het µmax laat afneem van 0.237 h-1 na 0.19 h-1. Die maksimum anti-fungale konsentrasie het toegeneem van 247.2 na 340.7 mAU*min, die maksimum spesifieke anti-fungale konsentrasie van 43.7 na 124.35 mAU*min/g/l, maksimum anti-fungale produktiwiteit van 19 na 26.21 mAU*min/h en antifungale selektiwiteit het toegeneem van 73.8% na 92.0%. Die gebruik van twee unieke stikstof bronne (NH4Cl en NaNO3), het ʼn optimum verhouding (NH4-N tot NO3-N) vir bio-massa en lipopeptiede produksie kinetika gehad. Anti-fungale produksie is gemaksimeer met die 0:1 of 0.5:0.5 stikstof verhouding. Die optimum µmax van 0.258 h-1 was behaal met die 0.75:0.25 verhouding. Die optimum vir anti-fungale produksie was die 0.5:0.5 verhouding, aangesien dit die tweede hoogste maksimum konsentrasie (888.3 mAU*min), hoogste maksimum spesifieke produksie (158.15 mAU*min/g/l), hoogste maksimum produktiwiteit (26.92 mAU*min/h) en kompeterend hoë selektiwiteit (86.7%) gehad het. ʼn Afname in opgeloste suurstof beskikbaarheid het anti-fungale lipopeptiede produksie kinetika verminder. Lae suurstof toestande noodsaak die gebruik van nitraat as alternatiewe elektron akseptor, wat gevolglik die hoeveelheid beskikbaar vir lipopeptiede produksie laat afneem. Optimum toestande gekweek in die bio-reaktor het beter presteer met betrekking tot antifungale kinetika (maksimum konsentrasie, spesifieke produksie en produktiwiteit) behalwe µmax en CDW met vergelyking tot die optimum van ʼn vorige studie (8 g/l NH4NO3). Maksimum konsentrasie het toegeneem van 285.66 na 290.17 mAU*min, spesifieke anti-fungale produksie van 51.85 na 58.1 mAU*min/g/l en produktiwiteit van 5.67 na 22.32 mAU*min/h. Kultuur supernatant, gekonsentreer deur suur neerslag, is gebruik vir anti-fungale effektiwiteitstoetse. Fungale inhibisie is waargeneem teen Botrytis cinerea, Alternaria brassicicola, Monilinia fructigena, Penicillium expansum en Rhizopus stolonifera, terwyl geen inhibisie waargeneem is teen Aspergillus sclerotiorum nie. Die hoogste effektiwiteit van anti-fungal lipopeptiede in kombinasie met kinetiese data uit hierdie studie toon die potensiaal om ʼn gestandaardiseerde anti-fungale produk te ontwikkel vir gebruik teen fitopatogene wat na-oes vrugte affekteer. Die effek van die parameters op homoloog produksie en verhoudings moet ondersoek word. Dit sal tot gevolg hê dat antifungale produkte ontwerp kan word om spesifieke homoloë te bevat wat effektief is teen spesifieke fitopatogene. Die gebruik van aaneenlopende kulture vir verdere kinetiese evaluering en optimalisering moet oorweeg word aangesien dit, soos aangedui in hierdie studie, moontlik is.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/101012
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