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Biofilm formation on materials leads to high costs in industrial processes, as well as in medical applications. This fact has stimulated interest in the development of new materials with improved surfaces to reduce bacterial colonization. Standardized tests relying on statistical evidence are indispensable to evaluate the quality and safety of these new materials. We describe here a flow chamber system for biofilm cultivation under controlled conditions with a total capacity for testing up to 32 samples in parallel. In order to quantify the surface colonization, bacterial cells were DAPI (4‘,6-diamidino-2-phenylindole)-stained and examined with epifluorescence microscopy. More than 100 images of each sample were automatically taken and the surface coverage was estimated using the free open source software g’mic, followed by a precise statistical evaluation. Overview images of all gathered pictures were generated to dissect the colonization characteristics of the selected model organism Escherichia coli W3310 on different materials (glass and implant steel). With our approach, differences in bacterial colonization on different materials can be quantified in a statistically validated manner. This reliable test procedure will support the design of improved materials for medical, industrial, and environmental (subaquatic or subaerial) applications.
The development of a test to evaluate the degradation of semi-volatile fuels as diesel by microorganisms is presented. This method is based on the principles described in the CEC-L-103 Standard procedure that is exclusively meant for testing the biodegradability of non-volatile lubricants. Therefore, significant modifications involve aseptic conditions for testing specific microorganisms and conducting the test in closed vessels avoiding evaporation losses, while fuel quantification using gas chromatography-flame ionization detection (GC-FID) is retained. It is suggested that the modified procedure should enable routine application for semi-volatile hydrocarbon-based fuels. GC-FID provides additionally valuable information on the alteration of fuel component patterns during biodegradation. The procedure was successfully tested using two bacteria (Pseudomonas aeruginosa and Sphingomonas sp.) and two yeasts (Moesziomyces sp. and Candida sp.) isolated from real diesel contamination cases. All tested microorganisms caused a significant degradation of diesel fuel achieving hydrocarbon degradation percentages ranging from 23% to 35%. Specific aspects on the test modification and prospects for further modification regarding targeted investigations in the field of fuel contamination by microorganisms are briefly discussed.
Quantification of microbial load in diesel storage tanks using culture- and qPCR-based approaches
(2016)
Microbial contamination of fuels, associated with a wide variety of bacteria and fungi, leads to decreased product quality and can compromise equipment performance by biofouling and microbiologically influenced corrosion of pipelines and storage tanks. Detection and quantification of biomass are critical in monitoring fuel systems for an early detection of microbial outbreaks. The aims of this study are (i) to quantify bacterial and fungal contamination in samples from diesel storage tanks of petrol stations, using both culture dependent- and culture independent (qPCR) approaches, and (ii) to analyse the diversity of cultivable diesel-contaminating microorganisms with the purpose to create a strain collection for further use in biodeterioration experiments. Both methodological approaches revealed a high microbial contamination in all studied samples, with the bacterial load being much higher than the fungal load. The diversity of cultivable microorganisms was rather low. Based on criteria of abundance and fuel degradation potential, the most relevant microorganisms were identified as bacteria of genera Bacillus, Citrobacter, Burkholderia and Acetobacter, the filamentous fungi Paecilomyces variotii and Pseudallescheria boydii, and a Dipodascaceae yeast. Furthermore the validity and utility of qPCR-based methods are discussed.