Purification of Bacillus amyloliquefaciens lipopeptides for postharvest disease control

Mazibuko, Sebenzile (2018-03)

Thesis (MEng)--Stellenbosch University, 2018.


ENGLISH SUMMARY: Africa is the largest exporter of fresh fruit (by volume) in the southern hemisphere and as such, the quality of fruit must be maintained throughout the global supply chain. Several fungal pathogens contribute to significant fruit loss and wastage due to disease during postharvest storage. The use of chemical fungicides as a postharvest disease control strategy has become limiting due to negative environmental concerns (toxicity, non-specificity and non-biodegradability) while the efficient use of bacterial cells/spores as biocontrol agents is restrictive as the viability is dependent on suitable postharvest environments. An alternative and novel biocontrol strategy proposed in this study is to use antifungal lipopeptides, namely fengycin and iturin, produced by Bacillus amyloliquefaciens DSM 23117, as second-generation biocontrol agents for postharvest disease control. However, to be sufficiently effective, concentration and purification of the lipopeptides is necessary due to the low yields produced and the presence of metabolic products such as lipid and protein impurities that are not desired in the final product. The aim of the study was to develop an appropriate downstream concentration and purification programme for antifungal lipopeptides using acid precipitation, solvent extraction and macroporous adsorption as downstream unit operations. To concentrate the lipopeptides, while effecting some degree of purification, acid precipitation studies were conducted by acidification of the cell-free supernatant to pH values 1 – 4 to determine the effect of pH on the recovery and purity of fengycin and iturin. Reverse-phase high performance liquid chromatography (RP-HPLC) quantification of acid precipitate showed high recoveries of 78% and 62% for fengycin and iturin respectively at pH 2 with an optimal fengycin purity of 64% obtained at pH 3. Acid precipitation of the cell-free supernatant at pH 3 would be suitable as the initial concentration step, due to the percentage purity of fengycin and iturin. To improve the purity of fengycin and iturin above that obtained from acid precipitation, solvent extraction was the next unit operation in the purification programme. Organic solvents of varying polarity indices were screened for their efficiency in extracting fengycin and iturin. Following screening experiments, a three-stage methanol extraction and a three-stage diethyl ether-methanol extraction were conducted to further improve the purity. Lipopeptides in solvent extracts were quantified by thin layer chromatography (TLC) which showed methanol to be the best solvent which extracted 100% of lipopeptides with a purity of 74% for fengycin. A final purity of 89% and 16% for fengycin and surfactin respectively was obtained after the 3-stage methanol extraction, while final purities of 74% and 13% were obtained for fengycin and surfactin respectively, with the diethyl ether-methanol extraction. The extraction efficiency of fengycin by the organic solvents was found to be related the functional groups of the solvents, in addition to the polarity. Further analysis of the lipopeptide solvent extract by liquid chromatography electrospray ionisation mass spectroscopy (LC-ESI-MS) was conducted to identify the two bands that appeared on the plate when the solvent extracts were analysed by TLC, and which were assumed to be fengycin. LC-ESI-MS indicated inconclusive results and it could not be confirmed with certainty that both bands were indeed fengycin. Adsorption was the final unit operation used to improve the purity of fengycin and iturin. Batch adsorption experiments were designed through a central composite design (CCD) to elucidate the optimal adsorption conditions for fengycin and iturin on a polymeric macroporous resin HP-20. Batch adsorption experiments showed the lipopeptide to resin (LP/R) ratio and pH to be significant (α = 0.05) parameters in the adsorption of fengycin with an optimal LP/R of 0.5 and optimal pH of 10. Although the LP/R ratio and temperature were found to be equally significant (α = 0.05) in the adsorption of iturin, no optimal conditions could be deduced as the LP/R ratio ranges may have existed outside the experimental region and thus further optimisation studies for iturin are required. To better understand the mechanism of fengycin and iturin adsorption, adsorption kinetics and adsorption isotherm studies were conducted, and the experimental adsorption data modelled through pseudo-kinetic-order rate models and the Langmuir and Freundlich isotherm models. Equilibrium times of 22 h and 24 h were obtained for fengycin and iturin respectively, at the optimal fengycin adsorption conditions. The experimental data was individually fitted to the pseudo-first-order rate model and pseudo-second-order rate model to determine whether the rate limiting step of fengycin and iturin adsorption could be explained by physisorption or chemisorption. Neither of the pseudo-order rate models could explain the kinetics of antifungal LP adsorption on HP-20 due to poor fit, suggesting that the kinetic data obtained in this study is inconclusive and further experimental work is required. After the kinetics of fengycin and iturin was studied, adsorption isotherms were conducted to determine the saturation concentration of fengycin and iturin at a constant temperature of 43°C and pH 10. A saturation concentration of 3 g/L and adsorption percentage of 76.5 ± 0.37% was obtained for fengycin while 0.5 g/L was determined to be the saturation concentration of iturin, with a % adsorption of 54.5 ± 3.99%. The experimental data was individually fitted to the Langmuir and Freundlich isotherm models to determine which model best represented the data and thus characterised the adsorption behaviour of the lipopeptides. As with the adsorption kinetic modelling, neither of the isotherm models could explain the adsorption mechanism of fengycin and iturin on HP-20, implying that other isotherm models such as the Redlich–Peterson model may be used in further experimental work to explain the adsorptive behaviour of the antifungal lipopeptides. To determine the effectiveness of the partially purified lipopeptides and purified lipopeptide mixtures, in vitro efficacy studies were conducted on six common fungal phytopathogens that cause diseases of fruit during postharvest storage. The fungal phytopathogens were grown separately on PDA plates containing the cell-free supernatant, resolubilised acid precipitate, methanol extract and diethyl ether-methanol extract. The growth inhibition was determined after a 5-day incubation period. 12 g/L fengycin resolubilised acid precipitate was the most effective treatment with fungal growth inhibition ranging between 30 – 100%, depending on the target phytopathogen. It was discovered that A. brassicicola was the most susceptible phytopathogen to all the treatments used while A. sclerotiorum was found to be the most resistant phytopathogen of the six phytopathogens studied. It was concluded that the effectiveness of the lipopeptides in fungal growth inhibition was the lipopeptide concentration and phytopathogen species dependent. It can be concluded from this study that an effective purification programme for fengycin and iturin would entail acid precipitation at pH 3, followed by a 3-stage extraction procedure involving methanol as the solvent, a programme which would potentially produce purities of 89% and 17% for fengycin and iturin respectively, with recoveries of 100% and 53% for fengycin and iturin respectively. To date, no study has systematically investigated existing downstream unit operations for the development of an appropriate concentration and purification programme for fengycin and iturin lipopeptides produced by B. amyloliquefaciens. Recommendations for further work include the following. While the optimal batch equilibrium adsorption conditions of 3 g/L fengycin at pH 10 and 43°C would ensure maximum fengycin adsorption on HP-20, further optimisation studies for iturin adsorption are required. Refinement in the adsorption kinetic and isotherm modelling studies is also required to elucidate the mechanism of fengycin adsorption.

AFRIKAANSE OPSOMMING: Suid-Afrika is die grootste uitvoerder van vars vrugte (per volume basis) in die Suidelike Halfrond en as gevolg hiervan moet die gehalte van vrugte regdeur die globale aanbod ketting gehandhaaf word. Verskeie swam patogene dra by tot aansienlike vrugte verlies en vermorsing as gevolg van siektes gedurende na-oes berging. Die gebruik van chemiese swamdoders as 'n na-oes siekte beheer strategie word beperk as gevolg van negatiewe omgewings bekommernisse (onder andere toksisiteit en nie-bioafbreekbaarheid) terwyl die doeltreffende gebruik van bakteriële selle/spore ʼn beperkende lewensvatbaarheid toon en grootliks afhanklik is van die geskikte na-oes omgewing. 'n Alternatiewe en nuwe strategie word voorgestel in hierdie studie aanrakend swamdodende lipopeptiedes, naamlik fengycin en iturin, geproduseer deur Bacillus amyloliquefaciens DSM 23117, as ‘n tweede generasie bio-beheer agent vir na-oes siekte beheer. Om egter voldoende doeltreffendheid te toon, is die suiwering van lipopeptiedes nodig as gevolg van die lae opbrengste geproduseer en die teenwoordigheid van metaboliese produkte soos lipied- en proteïen onsuiwerhede wat nie in die finale produk verlang word nie. Die doel van hierdie studie was die ontwikkeling van 'n toepaslike stroomaf konsentrasie en suiwerings program vir swamdodende lipopeptiedes deur gebruik te maak van suur neerslag, ekstraksie metodes en makro poreuse adsorpsie as stroomaf eenheid bedrywighede. Om die swamdodende lipopetides te konsentreer, terwyl ook 'n mate van suiwering aan te bring, was suur neerslag studies uitgevoer op die sel-vrye oplossing tussen pH waardes van 1 tot 4 om sodoende die suiwerheid van fengycin en iturin te bepaal. Suur neerslag omkeerbare-fase hoëverhittingvloeistofchromatografie het ‘n hoë opbrengs van 78% en 62% vir fengycin en iturin onderskeidelik getoon by ‘n pH waarde van 2 en ‘n optimale fengycin suiwerheid van 64% by pH 3 bekom. Sel-vrye suur neerslag by pH 3 is dus geskik vir die aanvanklike konsentrasie stap, as gevolg van die persentasie suiwerheid van fengycin en iturin bereik. Die suiwerheid van fengycin en iturin, bo wat reeds verkry is weens suur neerslagvorming, was verbeter deur gebruik te maak van ekstraksie metodes as die volgende eenheid bewerking in die suiwerings program. Organiese oplosmiddels van wisselende polariteit indekse was ondersoek vir hul doeltreffendheid in fengycin en iturin ekstraksie. Na aanleiding van die ondersoek was 'n drie-fase metanol ekstraksie en 'n drie-fase diëtieleter-metanol ekstraksie uitgevoer om verder die suiwerheid te verhoog. Die swamdodende lipopeptiede uittreksel was gekwantifiseer deur middel van dun laag Chromatografie en het getoon dat metanol die beste oplosmiddel is met ‘n 100% ekstraksie en ‘n 74% fengycin suiwerheid. ‘n Finale suiwerheid van 89% en 16% vir fengycin en surfactin is onderskeidelik verkry na die drie-fase ekstraksie met metanol terwyl diëtieleter-metanol ‘n finale suiwerheid van 74% en 13% onderskeidelik verkry het vir fengycin en surfactin. Tydens die studie was doeltreffende ekstraksie van fengycin gekoppel aan die oplosmiddel funksionele groepe, benewens die polariteit. ‘n Verdere ondersoek van die swamdodende lipopeptiede ekstraksie metodes deur middel van vloeibare Chromatografie elektro-sproei ionisasie massa spektroskopie was ingespan om die twee plaat bande te identifiseer wat of die bord verskyn het tydens die dun laag Chromatografie metode. Dit was aanvaar dat die plaat bande fengycin voorstel. Weens onvoldoende resultate was dit nie moontlik of vas te stel of beide plaat bande fengycin was nie. Adsorpsie was die laaste eenheid bewerking gebruik om die suiwerheid van fengycin en iturin te verbeter. Enkellading adsorpsie eksperimente was opgestel deur middel van ‘n sentrale saamgestelde ontwerp om sodoende die optimale voorwaardes uit te lig vir fengycin en iturin polimeriese makro poreuse HP-20 hars adsorpsie. Enkellading adsorpsie eksperimente het getoon dat die lipopeptiede tot hars verhouding en pH belangrike (α = 0.05) parameters vir fengycin adsorpsie is met 'n optimale fengycin lipopeptiede tot hars verhouding van 0.5 en ‘n optimale pH waarde van 10. Alhoewel die lipopeptiede tot hars verhouding en temperatuur ewe belangrik was vir iturin adsorpsie kon geen optimale toestande vasgestel word nie weens die moontlikheid dat die lipopeptiede tot hars verhouding buite die eksperimentele streek lê, dus is verdere optimalisering studies vir iturin nodig. Om die fengycin en iturin adsorpsie begrip te verbeter, was adsorpsie kinetika en adsorpsie isoterm studies uitgevoer om sodoende die eksperimentele adsorpsie data deur middel van pseudo-orde kinetika en Langmuir en Freudlich modelle voor te stel. Ewewig tye van 22 h en 24 h was behaal vir fengycin en iturin onderskeidelik, by die optimale fengycin adsorpsie toestande. Die eksperimentele data was toegepas met beide ‘n pseudo-eerste-orde reaksie model en pseudo-tweede-orde reaksie model om te bepaal of die beperkende stap vir fengycin en iturin adsorpsie verduidelik kon word deur fisiesoprsie of chemisorpsie. Nie een van die modelle kon die kinetika van die swamdodende lipopeptiede HP-20 adsorpsie verduidelik nie as gevolg van die swak model passing. Hierdie is ‘n moontlike aanduiding dat die resultate onvoldoende is en dat verdere eksperimentele werk vereis word. Na die kinetika ondersoek ten opsigte van fengycin en iturin, is ‘n adsorpsie isoterm ondersoek uitgevoer om die versadigde konsentrasie van fengycin en iturin by 'n konstante temperatuur van 43° C en pH waarde van 10 te evalueer. 'n Versadiging konsentrasie van 3 g/L en adsorpsie persentasie van 76.5 ± 0.37% was vir fengycin verkry terwyl 0.5 g/L verkry was vir iturin met 'n % adsorpsie van 54.5 ± 3.99%. Die eksperimentele data was toegepas met Langmuir en Freudlich isoterm modelle om te bepaal watter model die beste die swamdodende lipopeptied data verteenwoordig. Soos met die adsorpsie kinetika modellering, kon nie een van die isoterm modelle die adsorpsie meganisme van fengycin en iturin op HP-20 verduidelik nie wat impliseer dat ander isoterm modelle soos die Redlich – verdor model of die Dubinin-Radushkevich model gebruik kan word in verdere eksperimentele werk om die adsorpsie gedrag van die swamdodende lipopeptiedes te benader. Om die doeltreffendheid van die gedeeltelik gesuiwerde swamdodende lipopeptiedes asook die gesuiwerde lipopeptiede mengsel te bepaal, was in vitro effektiwiteit studies uitgevoer op 6 algemene fitopatogene wat na-oes berging siektes veroorsaak. Die swam fitopatogene was gegroei op afsonderlike PDA bakkies wat die sel-vrye oplossing, suur neerslag presipitaat, metanol ekstraksie en diëtieleter-metanol ekstraksie bevat. Groei vertraging was na ‘n vyf dae inkubasie tydperk bepaal. Die mees effektiewe behandeling was die suur neerslag presipitaat wat groei vertraag het tussen 30-100%, afhangende van die teiken fitopatogeen. Die studie het uitgelig dat A. brassicicola was die mees vatbare fitopatogeen vir al die toegepaste behandelinge terwyl A. sclerotiorum die mees weerstandige van die ses ondersoekte fitopatogene was. Die gevolgtrekking was dat die effektiwiteit van die swamdodende lipopeptiedes vir groei vertraging afhanklik is van die lipopeptiede konsentrasie en tipe fitopatogeen. Die studie het bepaal dat 'n effektiewe program vir fengycin en iturin suiwering behels suur neerslag by ‘n pH waarde van 3, gevolg deur 'n drie-fase ekstraksie metode waarby metanol as die oplosmiddel gebruik was om sodoende 89% en 17% fengycin en iturin suiwerheid onderskeidelik te behaal met ‘n 100% en 53% fengycin en iturin opbrengs onderskeidelik. Hierdie studie is die eerste van sy soort en behels ‘n stelselmatige ondersoek van bestaande stroomaf eenheid bedrywighede vir die ontwikkeling van 'n toepaslike konsentrasie en suiwering program vir fengycin en iturin swamdodende lipopeptiedes geproduseer deur B. amyloliquefaciens. Aanbevelings vir verdere ondersoeke sluit die volgende in. Die optimale ewewig voorwaardes van 3 g/L fengycin by ‘n pH waarde van 10 en temperatuur van 43°C sal maksimum fengycin HP-20 adsorpsie verseker, alhoewel verder optimaliserings studies benodig word vir iturin adsorpsie. Verdere verfyning van kinetika en isoterm modellering studies word ook vereis om die fengycin adsorpsie meganisme uit te lig.

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