Five commonly used extraction methods - regular centrifugation, EDTA extraction, ultracentrifugation, steaming extraction and regular centrifugation with formaldehyde (RCF) - were selected to study their effectiveness and repeatability in extracting extracellular polymeric substances (EPS) from aerobic/sulfate reducing and nitrifying/denitrifying biofilm samples. Biofilm EPS extraction yields were represented by carbohydrate and protein concentrations; the amount of cell lysis during the extractions was indicated by DNA concentration. The results showed that analyzing wash waters is essential in quantifying biofilm EPS; the contribution of this step varied from 8-50% of the total carbohydrate yield, depending on the extraction method. Among the extraction methods, the RCF extraction gave the greatest carbohydrate yield, the steaming extraction gave the greatest protein yield, and the other three extraction methods gave approximately equivalent amounts of carbohydrate and proteins for both types of biofilm. DNA in the EPS was 27 times smaller than in the pellets, indicating no significant cell lysis occurred during the extractions.
This study discovered that biofilm extracellular polymeric substances (EPS) are biodegradable by their own producers and by other microorganisms when they are starved. The study was performed in a comparative fashion to examine the biodegradability of biofilm EPS by the microorganisms from the original biofilm (its own producers) and from activated sludge (other microorganisms). Four distinctive phases were observed during EPS biodegradation. In the first phase, instantaneous concentration increases of carbohydrate and protein in the test solutions were observed when EPS was added; in the second phase, easily biodegradable EPS from the added EPS was quickly utilized; in the third phase, microorganisms began to produce soluble EPS, using the minimally biodegradable EPS left from the previously added EPS; in the fourth phase, cells consumed the newly produced EPS and microbial activity gradually stopped. This study suggests that EPS can be used as a substrate, and that the EPS carbohydrate can be utilized faster than the EPS protein. The EPS utilization rates (including carbohydrate and protein) in the activated sludge suspension were greater than those in the biofilm suspension. It may take microorganisms longer to get acclimated to a new nutrient environment if they are in a starved state.
Biofilm in drinking water systems is undesirable. Free chlorine and monochloramine are commonly used as secondary drinking water disinfectants, but monochloramine is perceived to penetrate biofilm better than free chlorine. However, this hypothesis remains unconfirmed by direct biofilm monochloramine measurement. This study compared free chlorine and monochloramine biofilm penetration into an undefined mixed-culture nitrifying biofilm by use of microelectrodes and assessed the subsequent effect on biofilm activity and viability by use of dissolved oxygen (DO) microelectrodes and confocal laser scanning microscopy (CLSM) with LIVE/DEAD BacLight. For equivalent chlorine concentrations, monochloramine initially penetrated biofilm 170 times faster than free chlorine, and even after subsequent application to a monochloramine penetrated biofilm, free chlorine penetration was limited. DO profiles paralleled monochloramine profiles, providing evidence that either the biofilm was inactivated with monochloramine's penetration or its persistence reduced available substrate (free ammonia). While this research clearly demonstrated monochloramine's greater penetration, this penetration did not necessarily translate to immediate viability loss. Even though free chlorine's penetration was limited compared to that of monochloramine, it more effectively (on a cell membrane integrity basis) inactivated microorganisms near the biofilm surface. Limited free chlorine penetration has implications when converting to free chlorine in full-scale chloraminated systems in response to nitrification episodes.
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