Optimisation of Biofouling Control in Industrial Water Systems T.E. Cloete, V.S.Brozel, E.E de Bruyn and B. Pietersen WRC Report No.318/1/94 Optimisation of Biofouling Control in Industrial Water Systems by T.E. CLOETE, V.S. BROZEL, E.E. DE BRUYN AND B. PIETERSEN Environmental Biotechnology Programme Department of Microbiology and Plant Pathology University of Pretoria Pretoria, 0002, South Africa Report to the Water Research Commission on the Project "Optimisation of Biofouling Control in Industrial Water Systems" Head of Department: Prof. T.E. Cloete Project Leaden Prof. T.E. Cloete WRC Report No: 318 ISBN 1 86845 901 0 June 1994 EXECUTIVE SUMMARY Optimisation of Biofouling Control in Industrial Water Systems by T.E. CLOETE, V.S. BROZEL, E.E. DE BRUYN AND B. PIETERSEN Environmental Biotechnology Programme Department of Microbiology and Plant Pathology University of Pretoria Pretoria, 0002, South Africa Supplement to WRC Report No. 3*? which is the full report to the Water Research Commission on the Project "Optimisation of Biofouling Control in Industrial Water Systems" Head of Department: Prof. T.E. Cloete Project Leader: Prof. T.E. Cloete EXECUTIVE SUMMARY Both water consumption and discharge in industrial water systems are currently minimised. The circulation of such water results in concentration of dissolved and suspended substances, promoting the growth of waterbome microbes, biofouling and subsequent macrofouling of the system and concomitant microbially induced corrosion. A number of reviews have been published on the mechanisms of microbially induced corrosion and the organisms involved. The subject of biofilm formation has also been well covered in the literature. A lack of information on the community structure and physiology is, however, apparent. Many advances have, nonetheless, been made from planktonic bacterial monitoring to sessile bacterial monitoring. This led to the introduction of a variety of different sessile monitoring techniques. Much experience has since been obtained on the use and limitations of these techniques and, to date, one of the main problem areas remaining is the monitoring of biofouling. Research has also indicated the problem of microbial resistance to nonoxidising biocides. This has suggested that some of these compounds may be mutagens. From an environmental point of view, it has become very important to verify this. This has also indicated the need to develop biocides which do not induce resistance in micro-organisms, and to investigate whether oxidising biocides are also capable of inducing resistance in micro-organisms. Recent studies have indicated that biofilm ecosystems respond to stress (i.e. biocides) in ways similar to macro-ecosystems. Generally, there is a decline in species diversity and a selection of more tolerant isolates. These developments have placed the spotlight on alternative technologies, like biodispersants, which have shown potential as biofouling control agents, and which should be investigated further. Physical control measures are currently still limited to pigging, although a number of other technologies show promise. Although fluid dynamics and their effect on biofouling control programmes have been well reported in the literature, it remains an aspect which is neglected by industry in terms of practical applications. 11 The objectives of this study were therefore: * To develop an in situ biocide concentration monitoring technique which could be used by field personnel. This technique would be aimed at determining biocidal activity, rather than pure chemical analysis. This technique would also be used for determining the influence of other water treatment chemicals on the biocidal activity of biocides. * To determine the sessile microbiological tolerance level before biofouling sets in. This technique would in practice be used to monitor the efficacy of biocide programmes. * To determine whether bacteria develop a resistance to biocides and how this would influence biocide programmes. * To develop a rapid, easy to use technique for identifying an enumerating sulphate reducing bacteria (SRB) in situ and to determine their role in microbial induced corrosion (MIC). * To determine the effect of nutrients on biofilm activity in industrial water systems and * To investigate the development of cross resistance amongst different bactericides. DETERMINATION OF IN SITU BIOCIDE CONCENTRATIONS Biofouling in industrial water systems is normally prevented by the use of bactericides. However, bactericide programmes often fail owing to the lack of suitable techniques for determining the in situ bactericide concentration and this usually results in either inadequate or excessive bactericide concentrations. In this study, the Sterikon00 bioindicator was evaluated for determining the minimum inhibitory concentrations of 5 industrial bactericides (dichlorophen, sulphone, thiocarbamate, isothiazolone and a quaternary ammonium compound) for the monitoring of the concentrations of these compounds in industrial water Ill systems. The results indicated that the Sterikon(R) bioindicator can be used for the determination of bactericide concentrations. RESISTANCE OF BACTERIA FROM COOLING WATERS TO BACTERICIDES Bacteria from cooling water systems developed resistance to three different bactericides, i.e. quarternary ammonium compounds (QAC), isothiazolone and thiocarbamate. Resistance was induced by exposing isolates to increasing sublethal concentrations for a period of 10 weeks. Bacillus subtilis became resistant to 1000 mg.t1 QAC. Cross resistance was also detected, e.g. isothiazolone induced resistance to QAC and thiocarbamate. The relationship between culture age and resistance was investigated, as well as the resistance of attached cells. Resistance of Pseudomonas snuzeri and of Bacillus cereus doubled to all water treatment bactericides evaluated. Mucoid mutants did not exhibit increased tolerance to bactericides, indicating that extracellular polysaccharide does not confer increased resistance to bacteria in biofilms. However, attached cells were more resistant than free- living cells within 15 min following attachment. Cell age also had a marked influence on resistance, where actively growing cells were most resistant and late stationary phase cells were least resistant. A culture of Pseudomonas aeruginosa isolated from a cooling water system was grown in the presence of sub-inhibitory concentrations of the water treatment bactericide 2,2'- Methylenebis(4-chlorophenol) (MBC). It grew in the presence of increasing concentrations by a mechanism of adaptation. The initial minimum inhibitory concentration was 36 /ig/mf'1 and the highest attained was 80 /tg.mi"1. Resistant cultures exhibited a higher survival rate when exposed to 320 /xg.mf'" than did the original strain. Lipopolysaccharide and outer membrane protein profiles were determined by SDS PAGE. No changes were detected in the LPS profile. The quantity of OprP, a phosphate uptake protein in the outer membrane decreased to a low level, correlating with decreased phosphate (P;) uptake during growth. It is proposed that OprP is the place of entry for MBC through the outer membrane of P. aeruginosa and that the cell can adapt to growth in its presence tv by decreasing the level of OprP in the outer membrane. Resistance and the development thereof in Pseiidomonas aeruginosa to the bactericide sodium dimethyl dithiocarbamate (SMT) was investigated. Ps. aeruginosa was cultured in nutrient- poor broth in the presence of sub-inhibitory concentrations of SMT. It adapted over 21 days of exposure from 250 jtg.mf1 to 490 /ig.mf"1. The initial high MIC was ascribed to exclusion of SMT by the lipopolysaccharide layer since removal thereof by EDTA rendered cells highly susceptible. The alginate-producing mutant PAO 579 was much more susceptible to SMT than was its parent PAO 381, indicating that extracellular polysaccharide does not act as an exclusion barrier to SMT. Following 24 h exposure to SMT, Ps. aeruginosa had an altered profile of outer membrane proteins as determined by SDS PAGE. Resistant cells had a further altered profile. Resistance of Ps. aeruginosa is ascribed to a change in the outer membrane protein profile, leading to improved exclusion of SMT. P. aeruginosa was cultured in nutrient limited broth in the presence of sub-inhibitory concentrations of isothiazolone (a mixture of 1.15% 5-chloro-N-methylisothiazolone (CMIT) and 0,35% N-methylisothiazolone (MIT)). Three cultures tested in parallel adapted gradually over 15 days of exposure from an initial minimum inhibitory concentration (MIC) of 300 nL(~l to 607 fil.l'K The three parallel cultures adapted at similar rates, therefore the adaptation was not ascribed to mutation but to a specific mechanism. Resistant cells did not produce any extracellular isothiazolone-quenching compounds nor undergo detectable alterations in their lipopolysaccharide layer. In wild cells a 35 kD outer membrane protein (protein S) was detectable, whereas resistant cells lacked this protein. Production of protein S was suppressed within 24 h of exposure to isothiazolone. It was still suppressed after 72 h of growth in isothiazolone-free medium. We propose that P. aeruginosa acquires resistance to isothiazolone by a process of adaptation where the outer membrane protein S is suppressed. Hypochlorous acid and hydrogen peroxide are employed as biofouling control agents and as surface disinfectants. Whereas the bacterial development of resistance to non-oxidising bactericides is well established,