Development of a Novel Biofilm Continuous Culture Method for Simultaneous Assessment of Architecture and Gaseous Metabolite Production

Abstract

The way that gaseous metabolite production changes along with biofilm architecture development is poorly understood. To address this question, we developed a novel flow reactor biofilm culture method that allows for simultaneous assessment of gaseous metabolite production and architecture visualization. In this report, we establish the utility of this method using denitrification by Pseudomonas aeruginosa biofilms as a model system. Using this method, we were able to collect and analyze gaseous metabolites produced by denitrification and also visualize biofilm architecture in a nondestructive manner. Thus, we propose that this novel method is a powerful tool to investigate potential relationships between biofilm architecture and the gas-producing metabolic activity of biofilms, providing new insights into biofilm ecology.It is known that environmental biofilms often produce gaseous metabolites. For example, natural lake reed biofilms are reported to produce N 2 or N 2 O (28). Furthermore, microbial biofilms are utilized in wastewater treatment and other industrial processes to convert soluble material into gaseous material, such as the conversion of nitrate into N 2 or N 2 O by denitrification (2,21,30). Previous studies of biofilm architecture have reported correlations between metabolic substrates and biofilm architecture (11,24,27), and another study showed the importance of biofilm structures in the distribution of dissolved gases (4). However, such correlations between biofilm architecture and gaseous metabolite production remain poorly understood.If one were able to show simultaneously occurring changes in architecture and gaseous metabolite production, the existence of a correlation between biofilm architecture and gaseous metabolite production would be indicated. To date, the analysis of gaseous metabolite production by biofilms has mainly been performed using test tubes or flasks sealed with butyl rubber stoppers (28). These airtight containers allow for collection of gaseous metabolites and are suitable for batch culture experiments. However, there are currently no nondestructive methods to simultaneously allow for both biofilm visualization and gaseous metabolite analysis. Such a method would provide information on how gaseous metabolite production activity changes along with biofilm development.The flow reactor method (15, 18), combined with confocal laser scanning microscopy (CLSM), is preferred in studies of biofilm architecture and development. Both electron microscopy and CLSM are often used in visualizing biofilms (12); however, electron microscopy requires dehydration (and therefore termination of biofilm growth), which in some cases distorts or damages bacterial cells (7). In contrast, CLSM can be used to visualize the three-dimensional architecture of living biofilms and can be applied to the flow reactor method while maintaining continuous culture (15,18). If collection of gaseous metabolites is achieved in a flow reactor system, both biofilm visualization and gaseous metabolite analysis can...

Low concentrations of ethanol stimulate biofilm and pellicle formation in Pseudomonas aeruginosa

“…Biofilm formation reached a peak at 12 h in this flask. When the P. aeruginosa biofilm incubated for 12 h was observed by a confocal reflection microscopy technique, as described previously, 12) the presence of 1 and 2% ethanol resulted in increased biofilm formation at a thickness greater than 20 μm, though it was less than 10 μm in the absence of ethanol ( Fig. 1(B)).…”

Visualizing the Effects of Biofilm Structures on the Influx of Fluorescent Material Using Combined Confocal Reflection and Fluorescent Microscopy

“…However, when visualizing substrate penetration into biofilms solely with FCLSM, it is necessary to label the entire biofilm with another fluorescent dye or fluorescent proteins. In contrast, CRM enables the visualization of three-dimensional samples without fixation and fluorescent labeling (14,15,20). Thus, CRFP uses a simple procedure to track the penetration of fluorescent compounds while visualizing the entire structure of biofilms.…”

“…3). In a previous study, we reported that P. aeruginosa forms a three-dimensional mesh-like structure also under denitrifying conditions (20). SYTO9 fluorescent dye was added to the P. aeruginosa biofilm, and confocal images were acquired with both CRM and FCLSM at 1-min intervals after adding the dye.…”

“…Here, we demonstrated the utility of a simple procedure that combines confocal reflection microscopy (CRM) (14) and fluorescent confocal laser scanning microscopy (FCLSM) (9), termed CRFP (the combined reflection and fluorescent confocal microscopy test for dye penetration), to visually explain the effects of biofilm structure on mass transport. CRM is a variation of reflection microscopy that employs a confocal laser scanning microscope, permitting the visualization of three-dimensional samples without fixation and fluorescent labeling (14,15,20). Denitrification sludge and Pseudomonas aeruginosa biofilm, each possessing different structural properties, were used as model biofilms.…”

A Great Leap forward in Microbial Ecology

“…Natural samples such as activated sludge were observed with this technique as were model organisms, resulting in a high resolution that is comparable to the utilization of fluorescent proteins and dyes (170,171). By combining this technique with other techniques, it is possible to measure gas metabolites under anaerobic conditions and observe the correlation of the biofilm structure and material diffusion in biofilms (170,172).…”