Pure-culture based studies are useful and especially have been in the past as they are the basis of most microbiological understanding, thus providing a wide range of references to use when investigating data obtained from an unculturable microorganism. These studies are also useful for physiological studies in simplified and controlled environment (Fererra and Sanchez, 2016). However, since the introduction of culture independent techniques, there has been a shift away from pure-culture based studies. This is because culture independent techniques have proven to be more accurate and sensitive.
Diversity in Wastewater
As many microbes involved in wastewater treatment cannot be cultured, the application of culture-independent techniques has become essential for identifying and tracking the diversity of microorganisms in wastewater. Techniques such as denaturing gradient gel electrophoresis (DGGE), terminal restriction fragment length polymorphism (RFLP), cloning and FISH have been used to identify the dominant phyla in activated sludge systems, such as Proteobacteria, Bacteroidetes and Actinobacteria (Ferrera and Sanchez, 2016).
Sequencing techniques such as high throughput sequencing (HTS) has identified groups previously unable to be identified with the above methods (Ferrera and Sanchez, 2016) as it has greater ability to identify a larger spectrum of genetic variations. Understanding the diversity of wastewater treatment is important as it can direct studies to explore the function and relevance of a certain group of bacteria or archaea in wastewater treatment. Additionally, diversity can be tracked over time to understand temporal stability of wastewater treatment plants and to detect how changing conditions affect the microbiome as a whole.
Phosphorus Removal in Wastewater
When using culture based methods, polyphosphate accumulating organisms (PAOs) were first identified as members of the genus Acinetobacter (Fererra and Sanchez, 2016). It was later found to be incorrect upon the usage of FISH to correctly identify a major PAO related to Rhodocyclus related beta-proteobacterium ‘Candidatus Accumulibacter phosphatis’ (Fererra and Sanchez, 2016). This example demonstrates how moving away from pure-culture based studies has increased the accuracy of research findings.
PAOs from sludge samples are very difficult to study in isolation. For example, a study for identification of PAOs by Crocetti et al. (1999) studied mixed cultures from sludge samples via FISH, staining and sequencing methods instead of using pure-culture methods because the organisms in question cannot be isolated from the mixed culture. A transition away from pure-culture studies allows for the identification of which bacteria, in a mixed culture, performs what function.
Filamentous Bacteria in Wastewater
Filamentous bacteria can cause bulking which reduces the settleability of bioflocs and solid separation from the final effluent (Fererra and Sanchez, 2016). A study run by Guo and Zhang (2012) created a 16S rRNA database of the most abundant groups of filamentous bacteria in wastewater treatment plants by utilising the 454-pyrosequencing method (Fererra and Sanchez, 2016). Further studies utilising metagenomics are able to identify the physiological traits that are key to the survival of filamentous bacteria, thus making it possible to experimentally adjust the environmental conditions of the microbiome in order to control the abundance of filamentous bacteria. This information can be useful for improving the operational efficiency of wastewater treatment plants, such a thing would be incredibly difficult to do so by only utilising pure-culture methods.
References
Crocetti, G., Hugenholtz, P., Bond, P., Schuler, A., Keller, J., Jenkins, D., Blackall, L. (1999). Identification of Polyphosphate-Accumulating Organisms and Design of 16S rRNA-Directed Probes for Their Detection and Quantitation. American Society for Microbiology, 66 (3), 1175-1182. doi: 10.1128/AEM.66.3.1175-1182.2000
Ferrera, I., Sanchez, S. (2016). Insights into microbial diversity in wastewater treatment systems: How far have we come?. Biotechnology Advances, 34, 790-802. doi: 10.1016/j.biotechadv.2016.04.003
Microbiomes in Aerobic Watewater Treatment 