Microbial response to oxidising biocides

Jackson, Vanessa A. (Vanessa Angela) (2003-03)

Thesis (MSc)--University of Stellenbosch, 2003.

Thesis

ENGLISH ABSTRACT: Biofouling of water systems is a problem extensively experienced in industry. Although this subject is the focus of many studies, the ability of microorganisms to survive exposure to biocides is still poorly understood. This study aimed to assess the biocidal effect of ozone on planktonic cells and biofilm communities, to evaluate different ozone generation techniques, and to follow population shifts within the biofilm community. Specific objectives included determining the effect of different ozone concentrations, the effect of different exposure times, and an assessment of microbial responses after exposure to sub-lethal ozone concentrations. Typically, 300 ml of an ovemight bacterial culture was exposed to ozone that was generated by anodic oxidation (0.3% wt or 18- 20% wt, respectively) or silent electric discharge (3.5% wt 03). The ozone was purged into the culture for 5-, 7-, 10- and 15 min., respectively. Enumeration of cells following ~10 min. exposure to 18-20% wt ozone showed a significant reduction in viable cell numbers. In contrast, when exposed to the two lower 03 concentrations, there was little change in the viable cell numbers even after prolonged exposure (30- and 60 min.). To evaluate biofilms, ozone was bubbled into the irrigation that was pumped through replicate flow cell channels. Response to ozone exposure was evaluated after staining the biofilms with the Baclight Viability probe, observation with fluorescence microscopy, and image analysis. The higher ozone concentration (18-20% wt 03) more effectively disrupted the biofilm structure of denser biofilms than the lower concentration, especially after 90 min. exposure. When compared to the controls, the 90 min. exposure resulted in a notable reduction in viable cells from 69% to 38% and a corresponding increase in nonviable cells from 29% to 62%. The lower concentration ozone (3.5% wt 03) was effective against the less dense, thinner biofilms evaluated, but not effective against the thicker biofilm. An analysis of the differences between continuous culture biofilms and batch culture biofilms showed that the biofilms in the batch system were less rigid. To evaluate microbial response to biocides, techniques such as Biolog whole-community metabolic profiles and terminal restriction fragment length polymorphisms (T-RFLP) were used. Biolog analysis of planktonic cells revealed changes following exposure to sub-lethal biocide concentrations, however carbon utilisation profiles resembled that of the controls after 24-48 hours. For biofilm communities, no carbon utilization differences could be detected under these conditions. There was, however differences in T-RFLP patterns between treated and untreated biofilm communities.

AFRIKAANSE OPSOMMING: Biobevuiling van watersisteme is 'n probleem wat algemeen in industriëe ervaar word. Alhoewel hierdie onderwerp die fokus van vele studies is, word die vermoëns van mikroorganismes om blootstelling aan biosiede te weerstaan steeds swak verstaan. Die doel van hierdie studie was om die biosidiese effek van osoon op planktoniese selle en biofilm gemeenskappe waar te neem, om die verskillende osoon generasie tegnieke te evalueer, asook om verskuiwings in die samestelling van die biofilm gemeenskap waar te neem. Spesifieke doelwitte sluit in die bepaling van die effek van verskillende osoon konsentrasies, die blootstellingtye, en 'n waarneming van mikrobiese reaksies na blootstelling aan sub-dodings osoon konsentrasies. Drie honderd ml van 'n oornag bakteriese kultuur was aan osoon, wat deur anodiese oksidasie (0.3% wt of 18% - 20% wt) of geluidlose elektriese ontlading (3.5% wt), gegenereer is, blootgestel. Tye van blootstelling was 5-, 7-, 10-, of 15 min., onderskeidelik. Bepaling van selgetalle na :2:10 min. blootstelling aan 18 - 20% wt osoon, het 'n betekenisvolle verlaging in die getal lewensvatbare mikrobeselle getoon. In teenstelling hiermee, het blootstelling aan twee laer osoon konsentrasies min verskil in die lewensvatbare selgetalle, selfs na verlengde blootstellingstye (30- en 60 min.), getoon. Om biofilms te evalueer is osoon in die medium geborrel wat deur replikaat vloeisel kanale gepomp is. Na osoon blootstelling, was die vloeisel onderwerp aan beeld analise deur gebruik te maak van die Baclight lewensvatbare peiler en fluoressensie mikroskopie. Die hoër osoon konsentrasie (18 - 20% wt 03) het die struktuur van dikker biofilms meer effektiefuiteengeskeur as die laer konsentrasie, veral na 90 min. blootstelling. In vergelyking met die onderskeie kontroles, het die getalle van lewensvatbare selle na 90 min. blootstelling drasties verlaag vanaf 69% tot 38% en 'n ooreenstemmende toename in die nie-lewensvatbare selgetalle vanaf 29% tot 62%. Die laer osoon konsentrasie (3.5% wt 03) was meer effektief teenoor die minder digte en dunner biofilms wat ge-evalueer was, maar nie so effektief teenoor die dikker biofilms nie. 'n Analise van die verskille tussen kontinue-kultuur biofilms en lotkultuur biofilms het getoon dat die lot-kultuur biofilms minder rigied is. Vir die evaluering van mikrobiese reaksies na biosied blootstelling, is tegnieke soos Biolog gemeenskap metaboliese profiele en eind-restriksie-fragment-lengte polimorfisme (TRFLP) gebruik. Biolog analise van planktoniese selle het verskille getoon na blootstelling aan sub-dodelike biosied konsentrasies. Koolstof benutting het wel na 24 - 48 ure met dit van die kontrole ooreengestem. Vir biofilm gemeenskappe was daar geen noemenswaardige verskille in koolstof benutting nie. Daar was wel verskille in T-RFLP patrone tussen die onbehandelde en biosied-behandelde biofilm gemeenskappe.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/53499
This item appears in the following collections: