Everyone in wastewater treatment has seen the growth curve graphic during training classes. I find using the growth curve a great way to discuss wastewater system changes and dynamics. And, with our newer genetic/molecular wastewater testing, I am seeing how the system's microbial species change as we move along the growth curve.

For those in need of refresh, the Growth Curve describes microbial populations by dividing growth into (1) Lag, (2) Log, (3) Stationary, and (4) Decline (endogenous) phases. Species of microbes differ based on influent composition and environmental factors, but we also have a changes in species based on waste concentrations relative to microbial populations - this is also known as F/M ratio.

The growth curve is easiest to see in aerated lagoon systems. Near the influent, we find lag and log phase growth as abundant food favors fast growing k-rate strategist microbes. As you increase Dissolved Oxygen (DO) and BOD/COD (food) declines, you see a change to r-rate strategists such as ammonia oxidizing bacteria (AOB), Thauera/Zooglea (biopolymer producing low F/M microbes), and other niche organisms. What I find interesting is how much change we see in our Microbial Community Analysis (a total census of all microbes in the system) with slight variations in F/M or influent makeup at inflection points along the growth curve.

We call MLSS healthy when we meet the following:

• Meet effluent treatment goals
• SV30 & SVI within easy to operate range
• D.O. is above 2 mg/L throughout the system
• OUR or SOUR is at baseline indicating low growth rates
• F/M in target range
• Indicator protozoa are present & active (often stalk ciliates)

But at a bacterial level, what is a healthy MLSS? Using Aster Bio's Environmental Genomics testing, we now have an understanding of what a healthy MLSS looks like with respect to microbial species. In this case we are looking at extended aeration activated sludge which is the most common system that we encounter.

Early in the system startup or following a major upset, we see a reversion to k-rate organisms such as Pseudomonas, Bacillus, and a number of less known fast growing organisms that thrive with high COD/BOD with associated low D.O. and perhaps quasi toxic components. As fast growers, these organisms have a high OUR (respiration rate) and grow rapidly to reduce COD/BOD. As the the MLSS OUR drops, the cells aggregate lowering free bacteria in solution. If an upset, they colonize on the existing floc mass which includes extracellular polymers, dead biomass, particulates, and a lower fraction than normal living biomass. 

As the system matures, the mix of bacteria begins to favor r-rate strategist microbes that thrive in low F/M conditions seen in activated sludge or when water quality reaches effluent targets. The r-rate strategists tend to form EPS to store food and vital nutrients. This builds and maintains floc. Common r-rate wastewater strategists include Thauera, Zooglea, and AOB/NOB bacteria (in systems with nitrogen removal)

All bacteria in wastewater treatment have DNA. With molecular testing, such as Aster Bio's Environmental Genomics, we use specific DNA to ID populations of specific microorganisms (qPCR) or do a total microbial census (Microbial Community Analysis MCA). Identification gives us information on the genotype or the specific DNA sequences present.

Now comes the twist that can lead to some confusion. When we look under a microscope, we look at microbial appearance. The growth pattern or appearance of the microbe is called phenotype. For example, all human DNA sequences as species Homo sapiens. However we appear quite different which is due to phenotype.

Bacteria like humans can have the same genetic makeup but appear very different. Some examples here include an organisms going from floc forming to filamentous when D.O. drops. Others such as Thauera and Zooglea are important floc forming microbes in activated sludge but under certain conditions their growth can cause non-filamentous bulking. Other bacteria such as Nocardia​ are not always the foaming mess that stresses many operators.

Molecular testing needs to be coupled with other monitoring techniques such as SV30, F/M, MCRT, Respiration Rates, and microscopic exam to fully system microbial dynamics and make predictions on system performance. 

Aquatic toxicity tests, often called bioassay or biomonitoring, were developed to estimate the effluent impact on receiving streams. Using living higher life-forms such as fat head minnow and daphnia (water flea), the test looks for both acute and chronic toxicity when subjected to various concentrations of wastewater effluent.

Often the chronic toxicity which is reproduction and weight gain are the harder part of the test to pass. But what causes failure?

Obvious causes of failure

• Ammonia (very toxic to aquatic organisms)
  
• Toxic compounds such as phenol, cyanides, surfactants, and pesticides/herbicides
  
  

But what happens when you are meeting all effluent permit limits on listed parameters, but are still failing parts of the bioassay test? Then it is time to looks at less obvious sources of toxicity and how multiple inhibitory compounds at low concentration can work concurrently to inhibit weight gain or reproduction.

Non-obvious causes of failure:

• Intermediate microbial transforms can actually be made more soluble than the influent compounds and therefore be more toxic to aquatic organisms
• Interaction by minor inhibiting compounds to create a larger amount of inhibition than any compound alone

How to handle test failure:

• Look at changes in influent
• Use upstream acute toxicity testing if influent makeup is unknown or questions exist
• GC/MS testing to look for most common toxic compounds
• Evaluate treating effluent with activated carbon, peroxide, or zeolite to improve toxicity profile