Thesis (PhD (Process Engineering))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: Biological agents offer several opportunities to address the many challenges faced in intensive
reticulated aquaculture. We therefore isolated and selected Bacillus spp. as potential biological
agents, because this group has demonstrated an array of biological activities of possible benefit to
aquaculture. They also display advantages in terms of robustness during bioprocessing and end
product application.
Natural isolates obtained from Cyprinus carpio, selected as a model high-value ornamental fish
species, and associated culture environments, were purified and assessed for in vitro efficacy
based on the inhibition of growth of pathogenic Aeromonas hydrophila and the decrease in
concentrations of ammonium, nitrite, nitrate and phosphate ions, typically found as waste
products in aquaculture systems. Based on suitability for aquaculture application, isolates B001,
B002 and B003 were selected and further evaluated in vitro and in an in vivo trial with C. carpio.
Inhibition of Aer. hydrophila growth and a decrease in concentrations of waste ions were
demonstrated in these studies. Based on 16S RNA sequence homology, the isolates were
identified as Bacillus subtilis, B. cereus and B. licheniformis, respectively. High sequence
homology between B. subtilis and B. anthracis necessitated further safety studies on the best
isolate, B. cereus NRRL100132 (B002). The isolate was shown not to contain the anthrax
virulence genes pOX1, pOX2 or the B. cereus enterotoxin.
Elucidation of the potential modes of action of a biological agent facilitates an understanding of
functionality and encourages technology uptake by end users. Competitive exclusion through
growth rate and competitive uptake of glucose and iron, the latter facilitated by siderophore
production, were shown to be key mechanisms at play in inhibition of Aer. hydrophila by the B.
cereus isolate.
As production cost is an important consideration in development of commercially relevant
biological products, we examined the optimization of nutrient supplementation, which has an
impact on high-density production of spores by fermentation. Corn steep liquor (CSL) was
identified as a lower cost and more effective nutrient source in comparison to conventional
nutrient substrates, in particular yeast extract and nutrient broth. The improved sporulation
performance of B. cereus could be related to the increased availability of free amino acids, carbohydrates, and minerals in CSL, which had a positive effect on organism growth and
sporulation efficiency. The impact of nutrient concentration on spore yield and productivity was
modelled to develop a tool for selection of optimal conditions. Excellent correlation with actual
laboratory fermentation data was demonstrated. A cost analysis revealed that production using
liquid phytase treated and ultra-filtered CSL was less expensive than spray dried CSL and
supported cultivation of B. cereus spores at densities higher than 1×1010 CFU ml 1.
Adoption of biological agents in commercial applications is lacking, due to limitations in process
and product development that address key end user product requirements such as cost, efficacy,
shelf life and convenience. The development of suitable spore recovery, drying, formulation and
tablet production process steps was thus performed. Key criteria used for downstream process
unit evaluation included spore viability, recovery, spore balance closure, spore re-germination,
product intermediate stability, end product stability and efficacy. A process flow sheet
comprising vertical tube centrifugation, fluidised bed agglomeration and tablet pressing yielded
an attractive product. The formulation included corn steep liquor and glucose to enhance
subsequent spore re-germination. Viable spore recovery and spore balance closure across each of
the process units was high (>70% and >99% respectively), with improvement in recovery
possible by adoption of continuous processing at large scale. Spore re-germination was 97%,
whilst a product half-life in excess of 5 years was estimated based on thermal resistance curves.
The process resulted in a commercially attractive product and affordable variable cost of
production.
Functionality of the product, incorporating the B. cereus isolate, was investigated across a range
of physiological conditions, including salinity, pH and temperature, based on rearing of C.
carpio. Temperature had a significant influence on germination, specific growth rate and increase
in cell number of B. cereus, whilst salinity and pH did not have any measurable effect on growth.
Controlled studies in bioreactors and modelling of the data to the Arrhenius function indicated
the existence of high and low growth temperature domains. The rates of pathogenic Aer.
hydrophila suppression and decrease in waste ion concentrations (ammonium, nitrite, nitrate and
phosphate) were translated into a linear predictive indicator of efficacy of the B. cereus isolate at
different temperatures. This study has resulted in development of an upstream and downstream process for production of
a new B. cereus isolate (NRRL 100132) which was shown to be safe, stable, functional, robust
and cost effective for application in aquaculture. / AFRIKAANSE OPSOMMING: Biologiese middels bied verskeie maniere om die veelvoudige uitdagings van intensiewe
netsgewyse akwakultuur aan te spreek. Gevolglik het ons uitgesoekte Bacillus spesies as
potensiële biologiese middels geïsoleer, omdat hierdie groep verskeie biologiese aktiwiteite
demonstreer wat van potensiële waarde kan wees in akwakultuur. Die groep toon ook voordele in
terme van robuustheid gedurende bioprosessering en eind-toepassings.
Natuurlike bakteriële isolate vanuit Cyprinus carpio geassosieerde kultuur omgewings,
geselekteer as 'n hoë-waarde model ornamentele spesie, is gesuiwer. Die in vitro
doeltreffendheid van die isolate is bepaal gebasseerd op die groei inhibisie van patogeniese
Aeromons hydrophila asook die afname in konsentrasies van ammonium, nitriete, nitrate en
fosfaat ione wat as tipiese afval produkte gevind word in akwakultuur sisteme. Isolate B001,
B002 en B003 is geselekteer op grond van geskiktheid en verder evalueer in in vitro en in vivo
proewe met C. carpio. Groei inhibisie van Aer. hydrophila asook 'n afname in konsentrasies van
afval ione was tydens die studies gedemonstreer is. Die isolate is identifiseer as Bacillus subtilis,
B. cereus en B. licheniformis, respektiewelik, op grond van 16S RNS volgorde homologie. Die
hoë volgorde homologie tussen B. subtilis en B. anthracis het verdere veiligheidstudies op die
beste isolaat, B. cereus NRRL100132 (B002) genoodsaak. Die isolaat het nie die antraks
virulensie plasmied pOX1, pOX2 of die B. cereus enterotoksien getoon nie.
Uitklaring van die potensiële meganismes van aksie van biologiese middels fasiliteer 'n begrip
van funksionaliteit en moedig tegnologie aanvaarding deur eind-gebruikers aan. Mededingende
uitsluiting deur groeitempo en mededingende opname van glukose asook die produksie van
siderofore is bewys as sleutel meganismes betrokke in die inhibisie van Aer. hydrophila deur die
B. cereus isolaat.
Aangesien koste 'n belangrike oorweging is in die ontwikkeling van kommersiële toepaslike
biologiese produkte, is die optimisering van voedingstof aanvullings wat 'n impak het op hoëdigtheid
produksie van spore deur fermentasie ondersoek. Week-vloeistof van mielie
prosessering (CSL) is identifiseer as 'n lae koste en effektiewe voedingsbron in vergelyking met
konvensionele voeding substrate, veral gisekstrak en voedingsboeljon. Die verbeterde sporulering
prestasie van B. cereus kon toegeskryf word aan die verhoogde beskikbaarheid van vrye aminosure, koolhidrate en minerale in CSL, wat 'n postitewe effek op organisme groei en
sporulerings effektiwiteit getoon het. Die impak van voedingstof konsentrasie op spoor opbrengs
en produktiwiteit is gemodelleer om 'n werktuig vir die selektering van optimale kondisies te
ontwikkel. Uitstekende korrelasie met werklike laboratorium data is gedemonstreer. Koste
analises het getoon dat produksie deur middel van vloeibare fitase-behandelde en ultra-filtreerde
CSL goedkoper is as sproei-gedroogde CSL en ondersteun verder die kultivering van B. cereus
spore teen digthede hoër as 1 x 1010 kolonie vormende eenhede.ml-1.
Die opname van biologiese middels in kommersiële toepassings skiet tekort as gevolg van
beperkinge in proses en produk ontwikkeling wat belangrike eind-gebruiker vereistes soos koste,
doeltreffendheid, rak leeftyd en gerieflikheid aanspreek. Die ontwikkeling van toepaslike
prosesse vir spoor herwinning, droging, formulering en tablet produksie is gevolglik uitgevoer.
Belangrike maatstawwe wat gebruik is vir stroomaf proseseenheid-ontwikkeling het
lewensvatbaarheid, herwinning, spoor balans sluiting, spoor her-ontkieming, intermediêre produk
stabiliteit, eindproduk stabiliteit en doeltreffendheid ingesluit. 'n Proses vloeidiagram bestaande
uit vertikale buis sentrifugasie, vloeibare bed agglomerasie en tablet persing het 'n aantreklike
produk voortgebring. Die formulering het ook CSL en glukose ingesluit om gevolglike spoor herontkieming
te verbeter. Lewensvatbare spoor herwinning en spoor balans sluiting oor elke proses
eenheid was hoog (>70% en 99% respektiewelik) met verbetering in herwinning wat moontlik
gemaak is deur die gebruik van aaneenlopende prosessering op groot skaal. Spoor her-ontkieming
was 97%, terwyl produk halfleeftyd langer as 5 jaar beraam is, gebasseer op termiese weerstand
grafieke. Die proses het gelei tot 'n kommersiële aantreklike produk asook bekostigbare
veranderbare produksie koste.
Die funksionaliteit van die tablet-produk met die ingeslote B. cereus isolaat is ondersoek oor 'n
reeks fisiologiese kondisies insluitend soutgehalte, pH en temperatuur, gebasseer op die
kultivering van C. carpio. Temperatuur het 'n betreklike invloed op ontkieming, spesifieke
groeitempo en toename in sel hoeveelheid van B. cereus gehad, terwyl soutgehalte en pH nie
enige meetbare effek op groei gehad het nie. Gekontrolleerde studies in bioreaktors en
modellering van die data op die Arrhenius funksie het hoë en lae groei temperatuur domeins
gewys. Die tempo van patogeniese Aer. hydrophila onderdrukking en afname in konsentrasies
van afval-ione (ammonium, nitriete, nitrate en fosfaat) is herlei na 'n liniêre voorspellende
aanwysing van effektiwiteit van B. cereus isolate by verskillende temperature. Die studie het gelei tot die ontwikkeling van stroomop- en stroomaf-prosesse vir die produksie
van 'n nuwe B. cereus isolaat (NRRL 100132) wat bewys is as veilig, stabiel, funksioneel,
robuust en koste effektiewe vir toepassing in akwakultuur.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/5300 |
| Date | 12 1900 |
| Creators | Lalloo, Rajesh |
| Contributors | Gorgens, J. F., University of Stellenbosch. Faculty of Engineering. Dept. of Process Engineering. |
| Publisher | Stellenbosch : University of Stellenbosch |
| Source Sets | South African National ETD Portal |
| Detected Language | English |
| Type | Thesis |
| Format | 132 p. : ill. |
| Rights | University of Stellenbosch |
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