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Field Sensor for In-Situ Detection Of Marine Bacterial Biofilms
Novel Sensor Concept Enables Time-Resolved Detection of Bacteria

Matthias Fischer

Martin Wahl

Dr. Gernot Friedrichs

A photograph of the biofilm sensor without waterproof housing. (Bottom) Schematic longitudinal cross-sectional view of the cylindrical sensor head with the main components and a color-coded region illustrating the excitation of the UV-LED and fluorescence detection efficiency pattern.
In marine environments, nearly all surfaces become rapidly covered by microorganisms, forming a biofilm. Following a biochemical conditioning phase, the biofilm formation starts within the very first minutes when pioneer bacteria cells adhere to the submerged surface.

Marine biofilms cause serious technical problems by settling on ship hulls and their water conduits, navigational equipment, stationary port structures, industrial pipelines and tidal power plants. They cause severe damage by increasing the drag, roughness and friction resistance of submerged objects, and accelerate biocorrosion of metals.

In natural ecosystems, however, bacterial biofilms serve as a unique living habitat and enable or prevent additional bio'fouling by micro-organisms and macro-organisms. Biofilms on the surfaces of marine organisms can substantially change their ecology and well-being.

To gain insight into biofilm formation kinetics and dynamics, continuous and in-situ monitoring of biofilm establishment in the marine habitat is desirable, but the required temporal and spatial resolution is difficult to achieve.

Biofilm Sensor Concept
For marine applications, sensor requirements differ considerably from those for highly sophisticated laboratory instrumentation. The aim was to develop a robust and reliable ready-to-use sensor to detect biofilm formation dynamics in situ, online and nondestructively in the marine environment. The sensor concept should allow for autonomous operation over several months, as well as selective detection, i.e., distinguishing between organic and inorganic material.

A sufficient penetration depth is desired to account for the 3D structure of biofilm, which typically constitutes highly patchy cell clusters up to several-hundred micrometers in diameter. To ensure a representative sensor signal of the inhomogeneous biofilm, a large detection area of 1 square centimeter is required, while keeping a low detection limit and a wide dynamic range to quantify the entire growth range, from initially adsorbed bacteria cells up to a complex biofilm community. All organisms contain natural intracellular fluorophores, which can be utilized for fluorescence-based detection methods as they provide high sensitivity and selectivity, fast response time and the capability of monitoring large areas in situ without sample contact.

The natural protein fluorescence of bacteria, stemming, for instance, from amino and nucleic acids, has long been known to indicate biomass and metabolic activity.

At wavelengths in the ultraviolet (UV) range, intrinsic protein fluorescence originates mainly from the aromatic amino acids tyrosine, phenylalanine and tryptophan. Due to a very low quantum yield of phenylalanine and common quenching mechanisms of the emission of tyrosine, the native fluorescence in proteins is dominated by tryptophan. The indole chromophore of tryptophan can be selectively measured by optical excitation at a wavelength of 280 nanometers with detection of the corresponding peak fluorescence centered around 350 nanometers.

Field Sensor Layout
The portable fiber-optic probe is contained in a waterproof housing (12 centimeters in diameter and 40 centimeters long) that is deployable to 50 meters depth. The sensor head comprises a substrate for biofilm establishment, the light source with an excitation filter on top, the collecting fiber optics and a motor-driven cover plate.

A UV transparent quartz window, equipped with a gas permeable and low-fluorescent foil for optimized bacterial growth, is employed as a settling substrate. The biofilm is back-illuminated and excited through the substrate using a 280-nanometer UV-LED with a narrow bandwidth interference filter.

Eighteen bundles of 30 optical fibers each are arranged hemispherically around the LED to collect the emitted fluorescence light. The angle of the fibers is optimized to constrain the detected fluorescence volume to a layer close to the surface and for spatially uniform sensing of an effective area approximately 0.5 square centimeters. At the end of the combined fiber bundles, the collected fluorescence emission is spectrally separated by a combination of two interference filters centered at 350 nanometers prior to detection on a photomultiplier tube operating in single-photon counting mode.

The timing of the electronics, the readout of the detector and the data recording on a 1-gigabyte SD memory card is accomplished by a programmable microcontroller, making the sensor package ready for use as a field data logger. A National Instruments Corp. LabVIEW-based graphical user interface allows the user to control the sensor settings, including the timing and sampling interval, via USB. Either a universal AC adaptor or seven nickel-metal-hydride rechargeable batteries with charge capacities of 4,500 milliampere hours at 1.2 volts could power the biofilm sensor unit. To continue this article please click here.

Matthias Fischer is a researcher with a background in biomedical engineering. He is conducting his Ph.D. research at the Helmholtz Centre for Ocean Research (GEOMAR) in Kiel, Germany. He has developed an optical biofilm sensor for quasi-continuous, in-situ measurements of biofilm formation dynamics in marine environments.

Martin Wahl is a professor of marine benthic ecology at Helmholtz Centre for Ocean Research (GEOMAR). His research focuses on community structures and dynamics of life on living and nonliving surfaces, stress, chemical and global change ecology in marine habitats. He is the founder of an international training program that combines applied research with training for young scientists.

Gernot Friedrichs is a professor of physical chemistry at Kiel University with a focus on the application of laser spectroscopic methods in the field of chemical reaction kinetics and ocean surface chemistry. Within the framework of The Future Ocean Cluster of Excellence, he promotes the use of optical detection technology in marine environments.

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