Unlocking the ‘black boxes’ of common biological wastewater treatment processes

Kevin Sheeran, Process Scientist at Aqua Enviro

Biological treatment plays a key role in wastewater treatment. Communities of bacteria facilitate sanitation, resource recovery and environmental compliance. These organisms would naturally play their part in the Nitrogen and Carbon cycles, but as we have harnessed their capabilities for wastewater treatment, their role in society and the circular economy has become critical.

Currently, these engineered systems are monitored using physiochemical parameters such as COD, suspended solids, ammonia and phosphorus concentrations. Some of these engineered processes reply on slow growing groups of microorganisms such as Phosphorus Accumulating Organisms (PAOs), Nitrifiers, and Anammox. These populations of bacteria can be vulnerable to competition and toxic shock events with the added detriment of having a slow recovery time. Changes in the population tend to occur before any tangible or noticeable changes on site, such as filamentous bulking that can be detected by the traditional physiochemical parameters. Therefore the increased monitoring of these populations could lead to further optimisation of assets to improve treatment and lower OPEX.

By using the latest sequencing technologies, it is possible to monitor bacterial populations in near real time. This technology has been around for a number of decades, being used already in a variety of industries such as Oil & Gas, Pharmaceuticals, and in food production, but only now has it advanced to a point of general usability and affordability. The price of sequencing has declined year-on-year and is now at point where it is accessible to areas outside of research.

DNA Sequencing Costs Data


The general principle is to exploit the 16S rRNA gene as a unique marker for each genus or species within the sample. This involves a typical DNA extraction step to separate the DNA from other cellular debris. This is followed by a library preparation using PCR which will amplify the extracted DNA, and more specifically, the target 16S rRNA gene. The library can then be sequenced to determine the individual DNA nitrogenous bases, A, T, G, or C and the order in which they are found. The sequence of the bases are compared to a database of known sequences for individual species and their corresponding 16S rRNA gene sequences. The relative abundances of each species present can then be determined by the frequency of the number of times its 16S rRNA gene was sequenced.

In the past, sequencing only a handful of genes—never mind an entire ecosystem—could take several days or weeks. Today, sequencing can be performed and completed in the same day with the latest sequencing platforms. To add to the advantageous nature of this new technology, it can also be used on site outside of a traditional laboratory setting.

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The information gained regarding the key species involved within nitrogen and phosphorus removal, and settlement characteristics, can lead to process optimisation in these areas through operational decisions in response to their changes. For instance, creating a baseline for the population within a system and measuring the variation away from that baseline to preempt operational issues. A simplistic approach would be to monitor the populations before and after major changes, such as the conversion to an Enhanced biological phosphorus removal (EBPR) system, had taken place. To go further would be to implement a continuous monitoring regime whereby, engineering and operational changes were implemented and the bacterial populations were monitored throughout these changes. You could then adjust the changes you were making based on the near real-time population data.

EBPR relies on the abundance of Phosphorus accumulating organisms (PAOs), yet Glycogen accumulating organisms (GAOs) can outcompete the PAOs which will hinder the EBPR process. Frequent monitoring of PAOs and GAOs can help inform on the parameters such as pH, temperature and nutrients required at a specific treatment works, as well as pushing the populations towards a more optimised system with greater biological nutrient removal.

Other areas in which sequencing could be advantageous are in pathogen detection and Antimicrobial resistance (AMR) monitoring. Genetic techniques have been used for decades to monitor Polio outbreaks and vaccination success in African countries and are now being used to monitor SARS-Cov-2 outbreaks and track variants. The general idea is that the pathogen, or at least the genetic material from the pathogen, is shed from humans into sewers and eventually into wastewater treatment plants. While qPCR is being used to detect the virus as a whole in local catchment areas, sequencing can be used to determine mutations and variants of the virus. This can lead to greater on the ground surveillance, restrictions and therapeutic approaches.

AMR is considered to be one of the greatest threats of the 21st century alongside climate change. Work is ongoing to understand how widespread AMR and AMR associated genes (ARGs) are in the environment. Wastewater epidemiology and pathogen tracking techniques could be applied to AMR, whereby we monitor catchments for large sources of AMR/ARGs to focus our attention on finding and addressing the hotspots.

Aqua Enviro are focused on unlocking the ‘black box’ of biological wastewater treatment technologies and believe that our knowledge and sequencing tools can be used to support the water utilities and others using these technologies. To learn more contact Aqua Enviro on 01924 242255 or email.


Posted 11th January 2022
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