PRESENTER: IP MOLOBELA MAY 10,2011

Microbial biofilms and their impact in the water industry College of Science, Engineering and Technology Civil and Chemical Engineering Department University of South Africa

Objectives  To introduce you to the concept of biofilms  To let you know why the study of biofilms is important in the water industry  To help you see how biofilms are radically changing the way we understand and deal with them as they change  To excite you about the prospects of the continued study of biofilms and the technologies on biofilm control and removal

Outline  Biofilms What are biofilms?  What are the parameters contributing to their formation and development?  Why is it important to study biofilms in the water industry – is it really necessary?  What are the disadvantages of chemical, physical and mechanical strategies in controlling but most importantly removing microbial biofilm?

 Are the above mentioned strategies successful in removing microbial biofilms? The answer is “ ”  “Enzyme Technology”- An emerged technology for microbial biofilm control and removal. Is this technology effective? The answer is “ ”

What are biofilms?  Prof Flemming simply defines biofilms city of microbes; EPS houseof microbes  Group of microorganisms (bacteria, fungi etc) ( attached to biotic or abiotic surfaces immersed in an aqueous environment )  Embedded in extracellular polymeric substances (EPS).  Biofilms have complex social structures that scientists and engineers are still trying to unravel Cont

 EPS is made up of carbohydrates, proteins, amino acid, lipids including humic substances  Biofilms produce different EPS structures (depending on growth conditions)

What is the role of EPS during biofilm formation?  Facilitates attachment (protects biofilm cells)  Maintains micro colonies (structural integrity)  Protects the biofilm cells from harsh conditions and predation  Enables the biofilms to capture nutrients (surrounding fluid medium)

 Enhances biofilm resistance to environmental stress, antimicrobial agents (bactericidal, fungicidal etc)

Pseudomonas fluorescens biofilms (Picture, Molobela IP, PhD studies ) EPS structures

Pseudomonas fluorescens and Staphylococcus lentus biofilms (Pictures, threat like structures EPS

Biofilm developmental cycle (stages)  Attachment (Protein molecules, flagella, fimbria etc) (conditioned surface) Primary stage (reversible) Secondary stage (irreversible)  Formation of micro colonies  Biofilm maturation  Detachment and dispersal of biofilm cells

Picture of Liang Research group (LL)

What are parameters influencing biofilm attachment and formation?  Nutrients  Temperature effects  Surface condition  Hydrodynamics  Quorum sensing (QS) (Gene expression and regulation)

Why is it important to study biofilms in the water industry?  They are the mainly reason water infrastructures and pipe materials are corroded (causative agent of biofouling and biocorrosion  Corrosion is the mainly problem in the water industry  Biofilms are also the biological contaminants of drinking water

Developed methods for biofilm control and removal 1.Chemical method (Bactericidal, Fungicidal, antimicrobials etc) Disadvantages  Partially effective  Fails to penetrate the EPS  Cannot destroy the biofilm cells  Some toxic to the surface materials

 Environmental unfriendly (produce odour) (their use-regulated by the EPA) 2. Mechanical method (machines for cleaning) Disadvantages  Cannot penetrate to biofilm contaminated sites/ areas  Costly- involves equipments down site - also labour expenditure

Enzyme Technology for microbial biofilm removal  Effective and robusting Enzymes are specific - target specific components Therefore: Enzyme can also target biofilm EPS (EPS - proteins, carbohydrates, lipid, amino acid) Note: It is important to understand the nature of EPS so that relevant enzymes are used

Enzymatic degradation of biofilm EPS (Molobela et al., 2010) Proteases (Savinase, Evelase, Esperase, polarzyme ) Single Species multi species Single Species multi species Mechanism of removal - Degradation of EPS single multi species  Savinase >80%>80%  Evelase >80%>80%  Esperase >80%>80%  Combination >90%>80 Mechanism of removal - Degradation of EPS single multi species  Savinase >80%>80%  Evelase >80%>80%  Esperase >80%>80%  Combination >90%>80 Tested biofilms Single multi species Tested biofilms Single multi species Tested enzymes Individually Combination Tested enzymes Individually Combination Highly effective IndividuallyCombination

Amylases (Amyloglucosidase (AMG), Bacterial Amylase Novo (BAN) Amylases (Amyloglucosidase (AMG), Bacterial Amylase Novo (BAN) Tested enzymes Individually Combination Tested enzymes Individually Combination Tested biofilms Single multi species Tested biofilms Single multi species Single Species multi species Single Species multi species Mechanism of removal - Degradation of EPS single multi species  AMG >50%>50%  BAN <50%<40%  Combination >50%>50% Mechanism of removal - Degradation of EPS single multi species  AMG >50%>50%  BAN <50%<40%  Combination >50%>50% IndividuallyCombination Partially effectiveNot effective

Combination of protease and amylase enzymes Combination of protease and amylase enzymes Tested biofilms Single multi species Tested biofilms Single multi species Single Species multi species Single Species multi species Combination Highly effectiveeffective Mechanism of removal - Degradation of EPS single multi species >80% <80% Mechanism of removal - Degradation of EPS single multi species >80% <80%

Methods used to study biofilm EPS  Protein assay- Froelund et al., 1996  Carbohydrate assay - Gaudy’s, 1962  Biofilm growth Assay – Molobela et al., 2010  Enzymatic degradation of biofilm EPS – Molobela et al., 2010

Conclusion  Study the nature of the biofilm EPS  Study the type of microbes within a biofilm  Thorough study on the impcat of parameters influencing biofilm formation and structures Cont

 Design of models for monitoring of biofilm attachment, formation and development and study the structures of biofilm EPS  Enzymatic technology seems promising on biofilm removal. However, more research still need to be done on this study  Collaborative research on biofilm study (involvement of expertise – different disciplines

Note to take home!  We have talked about biofilms being bad! Yes many of them are quite harmful and must be controlled and most importantly removed.  However Other biofilms are beneficial, and can be used to help fix serious problems (such as ground contamination from an oil spill, bioremediation, nitrogen fixers, rhizobial biofilms etc)

I thank you!!!!!!!!!

I am a strong biofilm, I destroy and destroy and destroy!!!!!!!! Hahahahahaha!!!!!!!!!!!!!!!!!!!