Concepts to determine geochemical heterogeneity within a multi component biofilm by microsensors

Bacteria in nature do not usually occur as single individual bacterial cells but rather in large communities of bacteria glued together by Extracellular Polymeric Substances (EPS). Such aggregates are called biofilms. They may significantly influence the transport of heavy metals in the geosphere by changing the geochemical conditions within the biofilm or simply by adsorption onto biological surfaces. For these biofilm studies we generate thick biofilms in a rotating annular bioreactor that provides a well mixed liquid phase, turbulent flow and constant shear fields [1]. For biofilm sampling, removable microscopic slides with affixed mineral platelets are placed in recessed slots on the outside of the inner cylinder. These slides can be removed via a port in the top of the reactor. The nutrient solution includes a bacteria cocktail (Pseudomonas stutzeri, Pseudomonas fluorescens, Flavobacterium aquatile, Bacillusus thuringiensis) was pumped through the reactor at 0.02 mL per minute into the base of the reactor, triggering a circulation from bottom to top at room temperature. After the experiment the mature confluent biofilm was removed from the reactor and the biofilm thickness of approximately 150 µm was determined by using Confocal Scanning Laser Microscopy (CLSM).

To measure the electrochemical conditions such as redox potential and/or pH in situ within the biofilm and in the surrounding bulk solution an experimental setup has been developed. Previous work has been carried out by means of several sensors, mainly focused on the interface basis material/biofilm to investigate microbial induced corrosion [2]. Because of low lateral dimensions for an analytical study of concentration profiles in biofilms only micro sensors with extremly small diameters are applicable. It is essential that the biofilm is not affected by the use of the grooved sensor and that the front surface exclusively acts sensitively. In our experiments an investigation concept is introduced, where the electrochemical micro sensor forms a unit with an integrated micrometer screw (advantage: not rotable spindle). In this way during the measurement the micro sensor can be moved in the biofilm in µm-steps vertically. The sensor systems to determine the redox potential and the pH value consist of indicator electrode and reference electrode, which are arranged sidewise and possess a ceramic diaphragm. The applied redox sensors have been developed in different constructions. On the one hand glass coated Pt sensors with an outer diameter of 300 µm have been constructed; on the other hand we applied micro sensors covered with wax and resin having an outer diameter of 120 µm. In both cases the diameter of the sensitive area amounts to 80 µm.
In our experiments we used the newly constructed micro sensors to determine the redox potentials and pH values within the biofilm generated in the rotating annular bioreactor. In the near future it is planned to develop micro sensors for measuring concentration gradients of other substances such as Cl- and NO3- [3], too.