טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentFried Tal
SubjectThe Effect of Surfactant on Biofilm Formation at an
Oil-Water Interface
DepartmentDepartment of Civil and Environmental Engineering
Supervisor Professor Guy Ramon
Full Thesis textFull thesis text - English Version


Abstract

Marine oil spills, natural or anthropogenic, can have disastrous environmental, societal and economic consequences, while cleaning up oil spills may require extended periods of time, ranging from weeks to years.

Microorganisms can colonize and degrade oil, at the sea surface and within the water column, resulting in effective and environmentally friendly oil-spill treatment - bioremediation.

However, there is a need to increase the rate of bacterial oil degradation, for which a better understanding of the mechanisms at play is required.

To date, very few studies have examined the formation of biofilms at the oil-water interface regarding bioremediation; moreover, biosurfactants are only indirectly mentioned.

In the present study, bacterial biofilm formation at the oil-water interface is examined, primarily in terms of its dependence on the presence of surfactants.

We used several methods in this study, including confocal microscopy, pendant-drop tensiometry, and an optical centrifuge, with which the differences between several bacterial strains were examined. These methods provide the interfacial tension over time, biofilm images, turbidity profiles, as well as flotation and sedimentation trends.

We compared two strains of Bacillus subtilis, an oil-degrading bacterial species: the 3610 (wild type - WT) and 3610 ΔsrfAA (MUT), which cannot secrete surfactin, providing a model system of surfactin influence, which is then further compared with another oil-degrading bacterial species - Pseudomonas putida.

Experimental results are presented in terms of biofilm formation rate, quantity, 3D structure, and general morphological characteristics, emulsion dynamics, and aggregation over time. Results indicated that the most stable emulsion (as defined in the research), both with and without bacteria, was formed with added TriX-100, a nonionic surfactant. WT, P. putida and MUT with added surfactin exhibited highly similar stability, lower than emulsions stabilized with TriX-100. MUT bacteria with no added surfactant showed low stability, while surfactin without bacteria did not stabilize the emulsion at all. MUT with added TriX-100 showed the poorest biofilm formation abilities. WT bacteria and MUT with added surfactin show high biofilm formation abilities, but with different morphologies, as observed through microscopy. 

Results indicate that the role of surfactin is broad and significant in biofilm formation, but is not exclusively a biosurfactant; if it were, WT and MUT with added surfactin would share a greater resemblance.


Comparing the results obtained for B. subtilis to those of P. putida sheds light on the different ways these bacteria achieve a similar goal. They are mostly similar, the main difference being morphology. B. subtilis displays complex aggregates composed of bacteria and oil droplets, while P. putida displays a biofilm that perfectly wrapped the oil droplets. The two species appear to have different adhesion and biofilm formation methods, making use of biosurfactants in different ways. For B. subtilis surfactin appears to be a major element in facilitating adhesion, while most of its other features are still unexplained in oil-water interface. It seems that different bacteria possess a different adhesion strategy, possibly involving biosurfactants.

In conclusion, while there is still much to be done on the subject, these results offer a step towards understanding the mechanics of bacterial adhesion on oil-water interfaces.