Kinetic evaluation of the production of Bacillus lipopeptides effective against Tuberculosis

Johannes, Emile (2019-04)

Thesis (MEng)--Stellenbosch University, 2019.

Thesis

ENGLISH ABSTRACT:Tuberculosis (TB), caused by Mycobacterium Tuberculosis, is the second largest cause of death resulting from a single infectious agent globally. South Africa has one of the highest number of active TB cases globally and it was estimated that approximately 1% of South Africans develops active TB each year. Multi-drug resistant TB (MDR-TB) is of even greater concern due its low cure rate of only 50% for treated MDR-TB patients. The lipopeptide biosurfactant, surfactin, produced by various Bacillus species, offers a promising alternative antimicrobial agent against TB causing organisms due to its ability to lyse cell membranes and alter membrane permeability. The haemolytic activity of surfactin, however, limits its use as a medical drug to be ingested by humans, but does not limit its use in other applications such as detergents and disinfectants in the fight against TB. The large-scale production of surfactin is limited by low yields and high purification costs, hence economically attractive approaches needs to be developed to realise the commercial production of surfactin as an antimicrobial agent to be used in the fight against TB. Lipopeptide production greatly relies on factors such as medium composition, process conditions and environmental factors, thus by optimising these conditions the cost of both upstream processing, and downstream purification, can be reduced significantly. The overall aim of this study was to investigate the effect of medium composition and process conditions on the growth and lipopeptide production kinetics of B. subtilis in batch culture and advise on the conditions that will improve the upstream production of surfactin, for possible use as an antimicrobial agent against M. tuberculosis. Shake flasks were used to study the effect of distinct nitrogen sources (NH4+ and NO3-) on the process kinetics by supplying ammonium and nitrate at discrete nitrogen ratios (NH4-N:NO3-N). A rigorous kinetic analysis yielded the optimum nitrogen source ratio for surfactin production by B. subtilis to be 0.5:0.5. An NH4-N:NO3-N ratio of 0.5:0.5 yielded the highest surfactin concentration (1084 mg/L), the highest surfactin productivity (36.1 mg/L/h), the second highest specific surfactin production (Yp/x = 0.078), the highest surfactin yield on glucose (Yp/s = 0.031) and the third highest surfactin selectivity (5.11 gsurfactin / gfengycin). The effect of manganese concentration on the process kinetics were also studied in shake flasks and rigorous kinetic evaluation yielded the optimal manganese concentration for surfactin production to be 0.1 mM, however increasing the manganese concentration from 0.05 to 0.1 mM did not significantly improve the surfactin production kinetics. 0.1 mM manganese yielded the highest surfactin concentration (884 mg/L), the highest surfactin yield on glucose (Yp/s = 0.022), the highest surfactin productivity (46.5 mg/L/h), and the second highest the highest specific surfactin productivity (Yp/x = 0.089) and surfactin selectivity (5.9 g surfactin / g fengycin). The optimal nitrogen source ratio and manganese concentration from the shake flask studies were used to evaluate the process kinetics under controlled conditions in a batch bioreactor and were compared to the process kinetics obtained in the shake flasks. All bioreactor kinetic parameters (surfactin concentration – 891 mg/L; Yp/x – 0.113; Yp/s – 0.021) were almost identical to those in shake flasks (surfactin concentration – 854 mg/L; Yp/x – 0.087; Yp/s – 0.022), except for μmax (0.39 h-1 in the bioreactor and 0.5 h-1 in the shake flask culture) and surfactin productivity (18.56 mg/L/h in the bioreactor culture and 44.95 mg/L/h the shake flask culture). The differences were attributed to interference caused by antifoam addition in the bioreactor culture due to vigorous foaming, however further investigation is required. It was also recommended that alternative methods to handle foaming, such as foam fractionation, should be investigated in future work. A response surface methodology (CCD) design of shake flask experiments yielded a nitrogen source ratio (NH4-N:NO3-N) of 0.35:075, manganese concentration of 0.06 mM, and a relative filling volume (RFV) of 0.5 as optimal to achieve maximum surfactin production by B. subtilis. NH4-N:NO3-N ratio and oxygen availability (relative filling volume) were significant parameters (α = 0.05) affecting surfactin concentration, Yp/x, and surfactin selectivity, whilst manganese concentration did not have a significant effect on any of the responses. It was recommended that nitrogen source ratio and oxygen availability should be optimised under controlled conditions in a batch bioreactor as shake flasks offer limited control over oxygen availability Finally, the cell-free supernatant was used to test for antimicrobial activity against Mycobacterium aurum. The antimicrobial cell-free supernatant did not show any antimicrobial activity against M. aurum. It was recommended that the supernatant undergo further processing such as acid precipitation, solvent extraction and/or adsorption followed by antimicrobial testing against M. aurum after each purification step. Different methods for antimicrobial testing should also be investigated.

AFRIKAANSE OPSOMMING: Tuberkulose (TB), veroorsaak deur Mycobacteriuim Tuberculosis, is die tweede grootste oorsaak van sterftes wat resulteer uit ʼn enkel aansteeklike agent wêreldwyd. Suid-Afrika het een van die hoogste aantal aktiewe TB-gevalle wêreldwyd en dit is beraam dat omtrent 1% van Suid-Afrikaners aktiewe TB elke jaar ontwikkel. Multimiddelweerstandige-TB (MDR-TB) is van groter kommer as gevolg van die lae genesingstempo van slegs 50% vir behandelde MDR-TB pasiënte. Die lipopeptiede biosurfaktant, surfactin, vervaardig deur verskeie Bacillus-spesies, lyk na ʼn belowende alternatiewe antimikrobiale agent teen organismes wat TB veroorsaak as gevolg van sy vermoë om selmembrane te liseer en membraan deurlatentheid te verander. Die hemolitiese aktiwiteit van surfactin beperk egter die gebruik daarvan as ʼn mediese geneesmiddel wat deur mense ingeneem kan word, maar beperk nie sy gebruik in ander toepassings, soos in waspoeiers en ontsmettingsmiddels, in die stryd teen TB nie. Die groot-skaal produksie van surfactin word beperk deur lae opbrengste en hoë suiweringskostes, daarom die behoefte om ekonomies aanloklike benaderings te onwikkel om die kommersiële produksie van surfactin as ʼn antimikrobiale agent te realiseer vir die gebruik in die stryd teen TB. Lipopeptiede produksie steun grootliks op faktore soos medium samestelling, proses toestande en omgewingsfaktore. Deur hierdie toestande te optimeer sal die kostes vir beide stroomop-prosessering en stroomaf-suiwering, beduidend verminder word. Die algehele doel van hierdie studie was om die effek van medium samestelling en proses toestande op die groei en lipopeptiede produksie kinetika van B. subtilis in lotkultuur te ondersoek, en aanbevelings te maak oor die toestande wat die stroomop-produksie van surfactin, vir moontlike gebruik as ʼn antimikrobiale agent teen M. tuberculosis, te verbeter. Skudflesse is gebruik om die effek van onderskeie stikstofbronne (NH4+ en NO3-) op die proses kinetika te bestudeer deur ammonium en nitraat by diskrete stikstofverhoudings (NH4-N:NO3-N) te verskaf. ʼn Streng kinetiese analise het die optimale stiksofbronverhouding vir surfactin produksie deur B. subtilis gelewer, naamlik 0.5:0.5. ʼn NH4-N:NO3-N verhouding van 0.5:0.5 het die hoogste surfactin konsentrasie (1084 mg/L), die hoogste surfactin produktiwiteit (36.1 mg/L/h), die tweede hoogste spesifieke surfactin produksie (Yp/x = 0.078), die hoogste surfactin opbrengs op glukose (Yp/s = 0.031) en die derde hoogste surfactin-selektiwiteit (5.11 g-surfactin / g-fengycin), gelewer. Die effek van mangaankonsentrasie op die proses kinetika is ook bestudeer in skudflesse. Streng kinetiese evaluasie het die optimale mangaankonsentrasie vir surfactin produksie gelewer, naamlik 0.1 mM, alhoewel die verhoging van mangaankonsentrasie van 0.05 mM tot 0.1 mM nie die surfactin produksie kinetika beduidend verbeter het nie. 0.1 mM mangaan het die hoogste surfactinkonsentrasie (884 mg/L), die hoogste surfactin opbrengs op glukose (Yp/s = 0.022), hoogste surfactin produktiwiteit (46.5 mg/L/h), die tweede hoogste spesifieke surfactin produktiwiteit (Yp/x = 0.089) en surfactin-selektiwiteit (5.9 g-surfactin / g-fengycin), verskaf. Die optimale stikstofbronverhouding en mangaankonsentrasie van die skudflesstudies is gebruik om die proses kinetika onder beheerde toestande in ʼn lotbioreaktor te evalueer en is vergelyk met die proses kinetika verkry in die skudflesse. Alle bioreaktor kinetiese parameters (surfactinkonsentrasie – 891 mg/L; Yp/x – 0.113; Yp/s – 0.021) is amper identies aan dié in die skudflesse (surfactinkonsentrasie – 854 mg/L; Yp/x – 0.087; Yp/s – 0.022), behalwe vir μmax (0.39 h-1 in die bioreaktor en 0.5 h-1 in die skudfleskultuur) en surfactin produktiwiteit (18.56 mg/L/h in die bioreaktorkultuur en 30.63 mg/L/h in die skudfleskultuur). Die verskille word toegeskryf aan steuring veroorsaak deur die byvoeging van skuimweerder in die bioreaktorkultuur as gevolg van kragtige skuiming, maar verdere ondersoek word benodig. Dit was ook voorgestel dat alternatiewe metodes in die toekoms ondersoek moet word om die skuiming te hanteer, soos skuim fraksionering. ʼn Respons oppervlak metodologie (CCD) ontwerp van die skudfleseksperimente het ʼn stikstofbronverhouding (NH4-N:NO3-N) van 0.35:0.75, mangaankonsentrasie van 0.06 mM, en ʼn relatiewe vulvolume (RFV) van 0.5 as optimaal gelewer om maksimum surfactin produksie by B. subtilis te bereik. NH4-N:NO3-N-verhouding en suurstof beskikbaarheid (RVF) was beduidende parameters (α = 0.05) wat surfactinkonsentrasie, Yp/x, en surfactin-selektiwiteit, affekteer, terwyl mangaankonsentrasie nie ʼn beduidende effek op enige van die response gehad het nie. Dit was aanbeveel dat stikstofbronverhouding en suurstof beskikbaarheid geoptimeer moet word onder beheerde toestande in ʼn lotreaktor, aangesien skudflesse beperkte beheer oor suurstof beskikbaarheid het. Laastens, die selvrye bo-drywende stof is gebruik vir toetse vir antimikrobiese aktiwiteit teen Mycobacterium aurum. Die antimikrobiese selvrye bo-drywende stof het nie enige aktiwiteit teen M. aurum gewys nie. Dit was aanbeveel dat die bo-drywende stof verdere prosessering soos suurpresipitasie, oplossing ekstraksie en/of adsorpsie ondergaan, gevolg deur antimikrobiale toetse teen M. aurum na elke suiweringstadium. Verskillende metodes vir antimikrobiale toetse moet ook ondersoek word.

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