Aster Bio has been working on adapting advanced molecular testing to wastewater treatment - a technology platform that we call Environmental Genomics. As with any new test, operators and engineers need information on what the tests actually measure and how the data can be applied to daily wastewater unit operations. First some background on the tests.

We currently are using two molecular testing technologies to examine and troubleshoot wastewater biomass. Both tests use the 16s region found in all bacterial cells and the best way to identify bacteria down to the species level. Each is detailed below:

Microbial Community Analysis 
Using high throughput sequencing, Aster Bio can take an MLSS sample and conduct a microbial census. This test gives the relative frequency of each organism with accuracy down to the genus level. Microbial community analysis picks up all genetic material present, even organisms that do not grow on traditional microbiological media plates. The results are often presented as a Sankey Chart with relative frequency and Observed Taxonomic Units (OTU) ~ which are akin to older plate count CFU numbers. The Microbial Community Analysis is a more complex test, but gives deep information never before available on what is in the system and doing the work. The test shows how changes in operations impact the total microbial populations and is also used to develop customized qPCR tests which are specific for "keystone" organisms.

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qPCR
After running Microbial Community Analysis tests, we can pick out key organisms related to good and bad operational efficiency. qPCR can test for AOB/NOB (nitrifiers), specific filamentous bulking organisms, SRB, and even non-filamentous bulking organisms. Once we select target organisms, Aster Bio's lab develops custom qPCR tests that can detect and quantify only matching 16s sequences in MLSS samples. qPCR testing can screen for multiple taxonomic units in each run. With its combination of speed, accuracy and lower cost, qPCR is designed for routine monitoring - where the Microbial Community Analysis is more of a high detail periodic monitoring test. Key to successful use of qPCR technology is having the right tests for your system's MLSS and the ability to adapt the output information into daily operations. As you can see from the output below, qPCR amplification real-time data requires some processing to make actionable reports.
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Nocardia is a very distinctive organisms in wastewater. Nocardia has true branching and stains gram positive. The photo above is a gram stained Nocardia filament from recent sample. While a filament, Nocardia forms do not cause bulking. The problem with Nocardia is the foaming caused by their production of hydrophobic extracellular polymeric substances (EPS). The foam is thick, resistant to anti-foams, and is very stable.

What causes Nocardia overgrowth?

• Grease (insoluble fatty acids) - this is why Nocardia is a huge problem in food processing and in high FOG municipal wastewater treatment systems
• Long sludge age - a slow grower, Nocardia requires longer sludge ages to develop. Once in a foam layer on the aeration basin, sludge age is less of a factor as the Nocardia are in the foam.

Control measures:

• Keep grease from entering the aeration basin. We have used collection system treatment to reduce long chain fatty acids (what we call grease) entering the WWTP. By depriving Nocardia of their ecological nice, you can reduce their advantages over "good" bacteria.
• If you have Nocardia foam, increase wasting. You may also need to consider adding bleach or other disinfectant to the RAS line. Although it will miss Nocardia suspended in the foam.
• Spray hypochlorite solution directly on the foam layer. This hits the nocardia directly.

Ammonia oxidation to nitrate is a two-step process in wastewater treatment. What we call nitrifiers are actually two groups of bacteria: (1) Ammonia Oxidizing Bacteria (AOB) and (2) Nitrite oxidizing bacteria (NOB). And some organisms can actually do both steps! Before we get to inhibition, here are some important facts on AOB & NOB:

• AOB & NOB have slower growth rates than heterotrophic microbes
• Obligate aerobic organisms
• Require pH between 6.5 - 8.5 best growth between 7.2 - 7.8
• Temperatures 10 - 40 Degrees C. (I have seen activity outside these ranges, but it is a stressor)
• Alkalinity for pH buffering 

The most important factor is the relatively slow growth of AOB/NOB cultures. This is why nitrifying systems require longer sludge ages and lower F/M than non-nitrifying biological treatment units. Now things that can harm an AOB/NOB community in the MLSS:

• Toxic compounds - sulfides, phenol, cyanide, solvents, and surfactants
• High COD - depression of dissolved oxygen inside the floc
• Effect of multiple stress factors - no one factor is completely inhibitory but creating conditions slowing AOB/NOB growth.
• Hydraulic overload (washout)

Most often AOB/NOB problems are related to longer term multiple stressors that lead to slower growth followed by a decrease in overall AOB/NOB populations as the organisms "washout". While this is not as easy to solve as a single compound or environmental condition, AOB/NOB populations can be cultivated by managing MCRT and optimizing the environment for their growth. In some systems with high stress, adding supplemental AOB/NOB cultures can help maintain needed populations for effective ammonia & nitrite oxidation.

qPCR (quantitative polymerase chain reaction) tools allow for real-time quantification of specific DNA strands. With qPCR we can look for specific DNA in a mixed sample and rapidly quantify the amount of that DNA present. So how can qPCR be used to monitor wastewater treatment systems?

The molecular (genetic testing) process normally starts with a full microbial census using advanced metagenomic sequencing. This test gives information on all organisms present in the wastewater and in what relative quantities. A more complex test, metagenomics gives us information on the microbial dynamics of the system. More importantly, we can identify the top species for "doing the work" and problematic organisms.

Once we have the key organisms both good and bad identified, we can create qPCR oligonucleotides to detect specific organisms in the system. Given advances in molecular testing, we can build customized testing kits without delay. This kit allows for rapid, real-time quantification and tracking of the key organisms. At my company, Aster Bio, we are using in-house metagenomic and qPCR testing to improve wastewater molecular testing. Environmental Genomics is our combination of tests and wastewater organisms databases that are cost-effective ways to use molecular testing in monitoring and controlling wastewater treatment units. Examples of qPCR applications include:

• System specific filamentous organisms (more quantitative than microscopic exam)
• Nutrient removal organisms such as AOB/NOB, Bio-P
• Specific degraders for resistant compounds
• Organisms associated with good and bad EPS production