In situ measurement of bacterial growth rates in wastewater treatment

Abstract

The development of advanced wastewater management today relies extensively on bacterial growth to remove/transform pollutants. Our understanding, the design, control and operation of these biological treatment processes is built upon indirect measurements of bacterial growth rates. However, little is known about the behaviour the bacteria in situ. The wastewater industry has considered the direct (in situ) measurement of bacterial growth rate impossible in the treatment environment.However, in natural aquatic environments bacterial growth rate is quantitatively measured in situ with radioactively labelled thymidine ([methyl-3H] thymidine) - the 'thymidine assay'. This thesis research has transferred and expanded this biotechnology into a broad range of wastewater treatment processes ranging from conventional dispersed phases to fixed-films. These included aerobic, anaerobic and anoxic dispersed phase processes and fixed-films (biofilms) on inert solids and on the living tissue of emergent macrophytes.Because most bacteria cannot be cultured outside the treatment environment, a number of alternative methods were used to evaluate the thymidine assay in situ:n n 1) the kinetics and isotope dilution of [methyl-3H] thymidinenincorporation were examined in the treatment processes;n n 2) specific growth rate measurements were compared to alternativenindirect measurements;n n 3) the consistency of the specific growth rate measurements werencompared with the design values used for the treatment processThe kinetic studies of each treatment process demonstrated that the rate of isotope uptake was always rapid and efficient. There was no lag before radioactivity was seen in bacterial DNA. Isotope diffusion did not limit the growth rate measurement. Generally, the best incubation time of sample with isotope was between 5 and 15 min. Isotope dilution was usually eliminated at between 12 and 25 mM of thymidine using a specific activity of 2 Ci.mmol-1nof [methyl-3H] thymidine. This represented an order of magnitude difference between engineered wastewater treatment processes and natural environments.Specific growth rate measurements (based on the thymidine assay) were compared to alternative indirect measurements. These other methods determined specific growth rates based on respirometry (OUR), increases in cell numbers (AODC) and hydraulic residence time. The thymidine measurements of specific growth rates of between 0.2 to 5 d-1were generally similar to those determined with these alternative methods.The scope of the thymidine assay was also expanded from community to population level measurements of specific growth rates by combining it with other molecular methods.n n