Isolation and characterisation of lipolytic bacteria and investigation of their ability to degrade fats, oils and grease in grain distillery wastewater

Hendricks, Ashley Alfred (2015-04)

Thesis (MSc Food Sc)--Stellenbosch University, 2015.

Thesis

ENGLISH ABSTRACT: The large volumes of effluent water generated by distillery industries is an issue of great concern as it contains pollutants that must be treated according to environmental legislation. It has been reported that grain distillery wastewater (GDWW) is high in fats, oils and greases (FOG) that can be reduced by treating with suitable microorganisms. The objective of this study was to investigate the biodegradability of FOG in GDWW. This was done by isolating lipolytic bacteria from soil, which was situated close to the GDWW treatment plant at a distillery in Wellington, South Africa. These isolates were screened for lipolytic activity on various fat substrates. Secondly, the most desirable isolates were subjected to batch biodegradation trials using GDWW as substrate and tested for their ability to biodegrade FOG. Each of the four isolates, Pseudomonas fluorescens (1), Pseudomonas luteola (2), Stenotrophomonas maltophilia (3) and Bacillus licheniformis (4) were screened on three types of media: DifcoTM Spirit Blue Agar with Tributyrin (SBA-Tri); Victoria Blue B Agar with Cotton Seed Oil (VBB-CSO); and Victoria Blue B Agar with GDWW (VBB-GDWW) at different temperatures (25°C, 30°C, 37°C and 50°C) to determine optimal enzyme activity for lipolysis. Lipolysis was taken as positive when growth of dark blue colonies was formed or by the formation of a clear zone around the colony. Lipolysis was observed at all the aforementioned temperatures for P. fluorescens, P. luteola and S. maltophilia. Bacillus licheniformis failed to show any lipolytic activity at 50°C on the SBA-Tri. A decrease in lipolytic (clear) zone was observed at an increase in temperature from 25°C to 37°C for P. fluorescens. When VBB-GDWW was used as lipid substrate, isolates failed to indicate any clear zone of lipolysis, however, growth was present for all isolates in the form of a dark blue zone around colonies, which were also positive for lipolytic activity. Three lipolytic bacteria (P. luteola, S. maltophilia, and B. licheniformis) isolated from the above study were subjected to GDWW of various FOG concentrations (70 – 211 mg.L-1). These isolates were allowed to acclimatise to GDWW during a batch biodegradation period (18 – 21 d) at 37°C. Bacillus licheniformis showed the highest FOG reduction of 83% after 18 d exposure. All the strains showed that an initial acclimatisation phase improved the biodegradation of the FOG. A fatty acid profile was obtained for each batch biodegradation trial after the acclimatisation phase. It was found that these strains either biodegraded the fatty acids (FAs) or, as in the case of P. luteola, formed myristic and pentadecyclic acids from free FAs. The formation of FAs may have occurred through a process of inter-esterification. It was also found that certain precursors such as palmitoleic acid might be formed under aerobic or anaerobic conditions. In this study it was shown that biodegradation of FOG can be improved by an initial acclimatisation period. Single cultures with the desirable properties can be used to lower the FOG in GDWW and need not be used in mixed cultures that could produce inhibitory components that would otherwise upset the biodegradation activity of isolates present. Bacillus licheniformis could be used as a FOG-degrading isolate during the treatment of wastewaters high in FOG. However, future studies should focus on bioaugmenting the FOG degrading bacteria from this study with other strains to monitor its activity and ensure survival and activity in larger scale studies.

AFRIKAANSE OPSOMMING: Die groot volumes afloopwater wat opgelewer word deur die distilleer-industrie is ‘n kwessie wat groot kommer wek aangesien dit groot hoeveelhede besoedelende stowwe bevat. Daarom moet dit, volgens omgewingsverwante wetgewing, behandel word. Daar is voorheen gerapporteer dat graandistillerings-afloopwater (GDAW) hoog is in vette, olies en ghries (VOG) en dat hierdie VOG verminder kan word deur die GDAW te behandel met toepaslike mikroörganismes. Die oorhoofse doelstelling van hierdie studie was om die bioafbreekbaarheid van die VOG in GDAW te ondersoek. Dit is eerstens gedoen deur lipolitiese bakterieë uit grond wat naby ‘n graandistillerings-aanleg (Wellington, SuidAfrika) geleë is, te isoleer. Verskeie vetsubstrate is gebruik om hierdie isolate vir lipolitiese aktiwiteit te toets. Tweedens is die verkose isolate getoets vir lipolitiese aktiwiteit deur gebruik te maak van lot-bio-afbreekbaarheidsmetode. Tydens hierdie metode is GDAW as substraat gebruik en die verskillende bakterieë se vermoë om VOG af te breek is getoets. Om die optimale ensiemaktiwiteit vir lipolise van elk van die vier isolate nl. Pseudomonas fluorescens (1), Pseudomonas luteola (2), Stenotrophomonas maltophilia (3) en Bacillus licheniformis (4), vas te stel, is elk getoets op drie verkillende media: “DifcoTM Spirit Blue Agar” met Tributirien (SBA-Tri); “Victoria Blue B Agar” met Katoensaadolie (VBB-KSO); en “Victoria Blue B Agar” met GDAW (VBB-GDAW) teen verskillende temperature (25°C, 30°C, 37°C en 50°C). Indien donker-blou kolonies gevorm is of ‘n deursigbare sone rondom ‘n kolonie waargeneem is, is lipolise as “positief” beskou. Lipolise is waargeneem teen alle voorafgenoemde temperature vir P. fluorescens, P. luteola en S. maltophilia. Bacillus licheniformis het nie lipolitiese aktiwiteit getoon teen 50°C op SBA-Tri. ‘n Afname in die deursigbare sone is waargeneem teenoor ‘n toename in temperatuur vanaf 25°C tot 37°C vir P. fluorescens. In die geval van VBB-GDAW as lipiedsubstraat, het isolate geen deursigbare sone vir lipolise getoon nie. Daar was egter ‘n donker-blou sone rondom kolonies teenwoordig, wat ook positief is vir lipolitiese aktiwiteit. Drie lipolitiese bakterieë (P. luteola, S. maltophilia, and B. licheniformis) is geïsoleer uit bogenoemde studie en is aan inkubasie in GDAW teen verksillende VOGkonsentrasies (70 – 211 mg.L-1) blootgestel. Hierdie isolate is toegelaat om te akklimatiseer tot die GDAW tydens ‘n lot-bio-afbreekbaarheidstydperk (18 – 21 d) teen 37°C. Bacillus licheniformis het die hoogste VOG-afname van 83% na 18 d blootstelling getoon. Alle bakterieë het getoon dat ‘n aanvanklike akklimatiserings-tydperk die bioafbreekbaarheid van die VOG verbeter. ‘n Vetsuur-profiel is verkry vir elk van die lot-bio afbreekbaarheidstoetse na die akklimatiserings-fase. Daar is bevind dat hierdie bakterieë óf die vetsure afgebreek het óf, soos in die geval van P. luteola, miristiese en pentadesikliese sure, vanaf vry-vetsure, gevorm het. Die vorming van vetsure is moontlik as gevolg van die proses van inter-esterifikasie. Dit is verder bevind dat sekere voorlopers, soos palmitoë-oleïensuur, gevorm kan word onder aërobies of anaërobiese toestande. In hierdie studie is getoon dan die bio-afbreekbaarheid van VOG verbeter kan word deur ‘n aanvanklike akklimatiserings-tydperk toe te pas. Enkel-kulture met die verkose eienskappe kan gebruik word om die VOG in GDAW te verminder. Gemengde kulture, wat inhiberende komponente produseer wat moontlik die bio-afbreekbaarheids proses negatief kan beïnvloed, hoef dus nie gebruik te word nie. Bacillus licheniformis kan gebruik word as ‘n VOG-afbrekende isolaat tydens die behandeling van afloopwater wat hoog in VOG is. Verdere studies moet egter fokus op die samevoeging van VOGafbrekende bakterieë vanuit hierdie studie asook ander bakterieë om die aktiwiteit daarvan te monitor en sodoende oorlewing en aktiwteit op ‘n groter skaal te verseker.

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