Biofilms as multifunctional surface coatings and adaptive systems: a biomimetic approach

Thesis (PhD)--Stellenbosch University, 2016.ENGLISH ABSTRACT: Biomimicry is an emerging scientific discipline that promotes nature-inspired innovation for sustainable solutions. Several patterns and survival strategies are repeated in Nature and these have been extrapolated into a hierarchical set of biomimetic principles that can be used to investigate the complexity of natural systems. A biomimetic approach was used to review biofilm literature and create a novel framework based on these principles to describe microbial biofilms on a molecular, structural and systems level. By reinterpreting current biofilm knowledge within a biomimetic framework, this study demonstrates that microorganisms use life-friendly chemistry to integrate biofilm development with growth, giving rise to resource-efficient systems. Furthermore, these structured microbial communities are responsive to their local environment, adapt to changes and, ultimately, evolve to survive. Subsequently, the application of biomimetic principles to biofilms was investigated using various analytical techniques. Two gfp-labelled Pseudomonas strains and an environmental multi-species community were selected for this study. Microscopic and spectroscopic techniques were used for biochemical investigations of single-species biofilm composition and structure. The distribution of biomolecules in Pseudomonas biofilms was investigated using protein- and glycoconjugate-specific fluorescent stains and confocal laser scanning microscopy (CLSM). CLSM was also used to investigate structural adaptations of Pseudomonas biofilms to changes in nutrient availability and hydrodynamic conditions. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy was used to explore biochemical adaptations of single- and multi-species biofilms cultivated in different nutrient media. ATR-FTIR spectra, visual observations and the quantification of biofilm parameters by digital image analysis of CLSM images support the hypothesis that biofilms are resource-efficient, self-organised systems that are built from the bottom up using life-friendly chemical principles. Both Pseudomonas strains adapted to environmental conditions by changing the three-dimensional structure of their biofilms, specifically in terms of biomass, substratum area coverage, average thickness and the surface area of biofilms exposed to the bulk liquid. In order to study biofilms as a system and investigate the responsiveness of a biofilm community as a whole, a relatively new approach was used to monitor biofilm responses in real time by measuring CO2 production as an indication of whole-biofilm metabolism. A CO2 evolution measurement system (CEMS) was combined with metabolic assays and direct plate count methods to monitor biofilm metabolism and biofilm-derived planktonic cell yield in response to environmental changes, i.e. changes in nutrient source and concentration or exposure to antimicrobial compounds (either streptomycin or a solution containing isothiazolone). The metabolic responses of biofilms, measured as CO2 production rates, showed that both single- and multi-species biofilms are able to respond rapidly to changes in nutrient availability or exposure to biocides and antibiotics. Multi-species biofilms generally recover faster after environmental changes or antimicrobial exposures, indicating that diversity adds to biofilm resilience and adaptability. Regardless of the conditions, single- and multi-species biofilms are able to maintain some level of metabolic activity, as well as release high numbers of planktonic cells into the effluent. The maintenance of biofilm-derived planktonic cell yield supports the hypothesis that biofilms are active proliferation sites in order to ensure survival – a feature of biofilms that is often overlooked in biofilm research. This study contributes to the growing field of biomimicry by applying biomimetic principles in biofilm research for the first time. A biomimetic approach can inform novel anti-biofilm strategies, promote biofilm-inspired innovation and explain complex microbial ecological phenomena. Within a biomimetic framework, the increasing degrees of complexity in biofilms are organised in a new way, demonstrating that the biochemical, structural and functional complexity of microbial communities are interconnected and need to be considered together in biofilm studies. To this end, the usefulness of CEMS as a non-destructive technique to study real-time biofilm responses is demonstrated.AFRIKAANS OPSOMMING: Biomimiek is ’n ontluikende wetenskaplike dissipline wat natuur-geïnspireerde innovasie vir volhoubare oplossings bevorder. Verskeie patrone en oorlewingstrategieë word in die natuur herhaal. In biomimiek word dié strategieë veralgemeen in ’n hiërargiese stel beginsels wat gebruik kan word om die kompleksiteit van natuurlike stelsels te ondersoek. Biomimiekbeginsels en ’n literatuuroorsig van biofilmnavorsing word hier gebruik om ’n nuwe raamwerk te skep wat mikrobiese biofilms op ’n molekulêre, strukturele en sistemiese vlak beskryf. Deur huidige biofilmkennis binne ‘n biomimiekraamwerk te herinterpreer, demonstreer hierdie studie hoe mikroorganismes lewensvriendelike chemiese reaksies gebruik om hulpbron-doeltreffende stelsels te skep wat ontwikkeling met die groei van die stelsel integreer. Hierdie gestruktureerde mikrobiese gemeenskappe reageer op hul biotiese en abiotiese omgewing, pas aan by omgewingsveranderinge en evolueer om te oorleef. Gevolglik is die toepassing van biomimiekbeginsels op biofilms deur ‘n verskeidenheid analitiese tegnieke getoets. Twee gfp-gemerkte Pseudomonas-stamme asook ’n multi-spesie-omgewingsgemeenskap is gekies vir hierdie studie. Mikroskopiese en spektroskopiese tegnieke is gebruik in biochemiese ondersoeke na die samestelling en struktuur van enkelspesies biofilms. Konfokalelaseraftasmikroskopie (KLAM) en fluoreserende kleurstowwe met proteïen- en glikokonjugaat-spesifisiteit is gebruik om die verspreiding van biomolekule in Pseudomonas-biofilms te bestudeer. KLAM is ook gebruik om die strukturele aanpassings van Pseudomonas-biofilms wat blootgestel is aan verskillende voedingsmediumkonsentrasies en hidrodinamiese toestande te ondersoek. Die biochemiese samestellings en aanpassings van hierdie biofilms is deur verswakte totale-refleksie Fourier-transformasie-infrarooi (VTR-FTIR) spektroskopie ondersoek. VTR-FTIR-spektra, visuele waarnemings en die kwantifisering van biofilmparameters deur digitale analises van KLAM-beelde ondersteun die hipotese dat biofilms hulpbron-doeltreffende, self-georganiseerde stelsels is wat van onder af boontoe gebou word deur lewensvriendelike chemiese beginsels. Beide Pseudomonas-stamme kon aanpas by omgewingstoestande deur die drie-dimensionele struktuur van hul biofilms te verander, spesifiek ten opsigte van biomassa, oppervlakbedekking, gemiddelde biofilmdikte en die oppervlakarea wat blootgestel word aan die omgewing. ’n Relatief nuwe stelsel, wat biofilms se CO2-produksie meet as ’n aanduiding van hul algehele metabolisme, is gebruik om biofilms as sisteme te bestudeer. ’n CO2-evolusie-metingstelsel (CEMS), metaboliese toetse en direkte plaattellingmetodes is gebruik om te monitor hoe veranderinge in omgewingstoestande biofilmmetabolisme en planktoniese sel-opbrengs beïnvloed. Biofilms is blootgestel aan verskillende voedingsbronne en -konsentrasies of aan antimikrobiese middels (streptomisien of ’n oplossing wat isotiasoloon bevat). Die metaboliese reaksies van biofilms, gemeet as CO2-produksie tempo’s, wys dat beide enkel- en multi-spesiebiofilms in staat is om vinnig te reageer op veranderinge in voedingsbronbeskikbaarheid of op blootstelling aan antimikrobiese middels. Multi-spesiebiofilms het oor die algemeen vinniger herstel wat daarop dui dat diversiteit bydra tot die veerkragtigheid en aanpasbaarheid van biofilms. Die biofilms is in staat om ’n sekere metaboliese vlak te handhaaf en ’n hoë aantal planktoniese selle vry te stel ongeag die omgewingstoestande. Hierdie resultate het ook bevestig dat biofilms optree as aktiewe selvormingsetels. Hierdie studie dra by tot die ontwikkelende veld van biomimieknavorsing deur biomimiekbeginsels vir die eerste keer in biofilmnavorsing toe te pas. ’n Biomimiekbenadering kan gebruik word om nuwe anti-biofilmoplossings na te vors, biofilm-geïnspireerde innovasie te bevorder en om komplekse mikro-ekologiese verskynsels te verduidelik. Binne ’n biomimiekraamwerk word die toenemende kompleksiteit van biofilms op ’n nuwe manier gerangskik. Sodoende word die verbintenis tussen die biochemiese, strukturele en funksionele kompleksiteit van mikrobiese gemeenskappe gedemonstreer. Die waarde van CEMS as ’n tegniek vir die nie-destruktiewe bestudering van biofilms word uitgelig.Doctora