Microbial Ecology

The identification and in situ detection of microorganisms within activated sludge flocs using DNA probes have been reported for the subclasses of proteobacteria,115 Gram-negative filamentous bacteria,116 Acinetobacter spp.,117 and nitrifying bacteria, ammonia- and nitrite-oxidizing bacteria.118-121 Probes have been found to be effective in monitoring shifts in nutrient amended activated sludge samples and avoid biases associated with culture dependent methods.

Nucleotide probes are also being used in multimethod approaches to the analysis of marine snow. Grossart and Simon122 studied the bacterial colonization and microbial decomposition of lake snow with rRNA-targeted fluorescent oligonucleotide probes, used in conjunction with standard limnological parameters and measures of hydrolytic enzyme activities, bacterial production and growth rates, and changes in floc composition during aging and sinking. The flocs were shown to be hotspots of enhanced microbial activity, with the microbial community dominated by ^-Proteobacteria. The overall results suggested that lake snow and activated sludge flocs have similar functions in their aquatic ecosystems. A review and comparative analysis of the microbial ecology of flocs from many ecosystems have been published recently by Simon et al.,123 who consider marine, lacustrine, riverine, and estuarine environments. They describe methodology and reference it in considerable detail, and they conclude that the significance of bacteria in aggregation processes is much greater than previously estimated.

A variety of approaches permitting correlation of structure-function relationships with respect to specific metabolic and biogeochemical processes associated with flocs are available. Carbon-substrate oxidation patterns or phenotypic fingerprinting based on commercially available BIOLOG microplates allow for simultaneous testing of 95 separate carbon substrates producing patterns of metabolic response based on the reduction of tetrazolium dye as an indicator of sole carbon utilization.124 Brock and Brock125 introduced microautoradiography (MAR) as a tool for looking at the activities of individual microorganisms in their natural habitats. This technique has been used since to address many important ecological questions regarding bacterial activities in natural aquatic systems.126-129 MAR is a method that can be used to investigate the ability to assimilate organic or inorganic compounds by natural bacterial populations as well as assessing their abilities to be active under different operating conditions; for example, like the aerobic, anoxic, or anaerobic conditions encountered in some activated sludge systems.130-132 Radiolabeled (3H, 14C, 35S, or 33P) substrates are used and can become associated with cells or biological structures of interest either as a result of adsorption or active uptake. After coating cells with a photographic emulsion and following an appropriate time of exposure, uptake is indicated by deposition of silver grains on the surface of radioactive cells.

Recently, direct combination of fluorescent in situ hybridization (FISH) and MAR for simultaneous in situ identification and determination of substrate uptake patterns of individual microbial cells within complex microbial community has been applied to activated sludge.131-135 In the protocols used, an additional step of FISH prior to the 4',6-diamino-2-phenylindole (DAPI) stain, which is used to obtain total cell counts,115,130,136,137 and the MAR developing steps, is incorporated. This combination permits simultaneous in situ detection of DAPI signals, probe-conferred fluorescence, and silver grain formation (which indicates the presence of radioactive compounds within the fixed cells) at a single cell level. The technique has been used to examine the identity of unknown bacteria which carry out specific processes, including their roles in organic substrate uptake and biological phosphate removal.130,131 Some filamentous bacteria from activated sludge have been studied with these techniques, and their roles in the uptake of several organic substrates determined.133,134

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