There is a lot of discussion on micropollutants such as pharmaceuticals, chlorinated organics, pesticides/herbicides, and even common things such as artificial sweeteners. The biggest issues are these xenobiotics (not naturally occurring compounds) are passing through biological treatment without degradation. So, a lot of effort is being directed at finding bacteria that can degrade these compounds. In traditional microbiology, we pften screen organisms by sole-carbon source testing. In sole-carbon source testing, you look for microbes that can initate biodegradation of the target compound. Often we can find strains that grow on the target compound. For more difficult compounds, we may need to a cometabolism component. If organisms exist that can degrade these compounds, why are the difficult to treat in wastewater, soils, and groundwater? Here are a few possible explanations:
​

• The most discussed reason is that ​target concentrations are too low for supporting the organisms. At concentrations in the ppb (parts per billion), there is often too little of the compound for direct degradation. In this case you need to evaluate cometabolism where a non-specific enzyme starts decomposition.
  
  
• Another cause could be that moving from a sole-carbon source environment to one with other available "energy" sources will change microbial behavior. In the presence of easy-to-degrade substrates (sugars, starches, etc), we often see microbes conusmer the "easy" compounds first. This leaves low energy yeilding compounds unaltered. Think of this as similar to giving kids a choice of candy or broccoli. Which one would most kids select... of course you know it is candy. 

Oxygen Uptake Rate (OUR or sometimes DOUR) uses a dissolved oxygen probe with a BOD bottle to measure oxygen uptake by biomass. While you could purchase a highly automated, advanced respirometer, for wastewater operations we can do just fine with a standard BOD probe and bottle setup. 

Aerobic wastewater treatment microbes consue oxygen when growing. When you saturate a sample with oxygen and then cap the bottle with a DO probe, you measure the oxygen consumed by the bacteria. The observed oxygen uptake per minute gives your the OUR number. Here is a OUR test protocol - https://www.biologicalwasteexpert.com/useful-information.html.

Few bullet points on OUR/SOUR and what the numbers mean for operations.

• OUR should be run frequently to get a baseline normal rate. This is very important, your normal OUR is specific to your system and MLSS concentrations. Using similar systems or rules-of-thumb are just not as good.
• Make sure you calibrate your DO meter & probe - sometimes we get in a rush and don't check. This is where mistakes happen.
• ​Higher respiration rates indicate more soluble organics (higher BOD5)
• Low OUR indicates soluble BOD has been substantially removed and biomass is entering endogenous respiration. 
• If the DO drops below 1 mg/L - the meter is not as accurate. Make sure you calculate based on DO from saturation down to a minimum of 1 mg/L.
• For industrial waters, if you see a very low OUR & have normal/high COD influent - you may have toxicity. Be sure to check for changes in influent. Be aware of suddenly low OUR numbers.
• If you change MLSS concentrations, convert OUR to SOUR which is OUR divided by MLSS (grams). Make sure you do the calculations the same every day - don't switch between MLSS and MLVSS for calculating SOUR.

Denitrifcation happens when bacteria begin to use nitrate and nitrite as an alternative electron acceptor. This a complex way to say that the bacteria are using the oxygen in nitrate and nitrite, leaving nitrogen gas. If you get denitrification in a treatment basin, it is a good sign and often required for facilities with nitrite/nitrate discharge limits. If you have denitrification in a secondary clarifier, you can have floating solids with TSS carryover into the effluent. So what can you do to prevent denitrification in secondary clarifiers:

• Control bed depths to between 2 - 3 feet by adjusting recycle rates
• Hydraulic residence time in clarifier 2 - 4 hours
• Warm temperatures increase denitrification (high metabolic activity) so problem is usually more pronounced in summer months
• Use a water spray to degas the floating solids - you will see the bubbles breakout and the sludge sink

I still like and am fascinated by trickling filter wastewater treatment systems. A simple trickling filter is a tank or tower packed with media on which biofilm grows. Trickling filter biofilm is just like the biofilms you find on Rotating Biological Contact (RBC), MBBR, and any type system where you have a support matrix for biofilm growth.

In an old tricking filter, you can see how biomass develops as the wastewater moves through the media. Early stages have heterotrophic bacteria that remove soluble BOD. Protozoa in highly loaded sections favors amoeba and flagellates. As souble BOD drops and oxgyen increases, you start to see develoment of higher indicator protozoa and appearance of slower growing bacteria such as nitrifiers and sulfur oxidizers. 

Pros of Biofilm Systems

• ​Can support more biomass than suspended growth systems in a given tank/clarifieSor configuration
• Effective for allowing slow growing organisms such as nitrifiers to develop
• Biofilm offers some protection against hydraulic stress (washout) and limited toxic shock (pH, inhibitory chemicals)
• Less operator action required than suspended growth
• Effective for pre-treatment of high strength waste (roughing filters)
  ​
  

Cons of Biofilms Systems​

• ​Can be fouled by Fats, Oils, Grease (FOG) - high FOG can "coat" the biofilm
• Less ability to change operations (MCRT, F/M) than suspended growth 
• Favors filmentout forms that can be difficult to remove with gravity solids separation

We don't often get back to an old test such as BOD very often. However, I have seen posts where people are seeing BOD5 removal drop and they are wondering why their treatment systems is running well, yet effluent BOD is high.

BOD5 is also called soluble BOD. This means that BOD5 represents oxygen demand by most common wastewater components such as sugars, fatty acids, starches, etc. What remains in the BOD5 bottle after 5 days tends to be insoluble or recalcitrant organic compounds. This could be particulates, FOG, long chain hydrocarbons, xenobiotics, pharmaceuticals, lignin, and other slow to degrade or insoluble organics. In theory, if we continued to run the BOD test for 20 days, the microbes would continue to consume oxygen (although at a slower rate) as they degraded these compounds. 

The BOD20 vs BOD5 can be related to BOD5 removal efficiency by considering how non-soluble or recalcitrant compounds are processed by  microbes. For example, consider grease (a very common insoluble in wastewater). If you do not remove grease in pretreatment, the following happens in the biological unit:

• Option 1 - the grease can float across treatment system straight to the effluent
• Option 2 - insoluble grease is adsorbed (fixes) into the MLSS - this can also lead too floating scum on the clarifier.

Now we will assume tht the grease is adsorbed into floc. As the soluble BOD decreases, the bacteria begin to use a combination of biosurfactants & exocellular enzymes to carve up the long chain hydrocarbon into forms capable of being transported across the cell wall. In the case of long chain fatty acids (FOG), biodegradation takes place via Beta-Oxidation where fatty acid length is decreased two carbons each cycle through the beta-oxidation pathway. Once the fatty acid drops to less than 10 carbons in length, solubility increases and degradation rates increase as you have converted the organic into soluble BOD. This is how microbes can increase the soluble BOD5 while degrading organics that were previously not detected by the 5 day test.

​Now back to case of BOD5 being possibly generated across a aeration basin. If you expect potential conversion of insoluble organics into soluble (BOD5) forms across your biological unit, you an check by running COD or filtered COD. Besides being a quick test, COD is effective in rapid detection of things contributing to ultimate BOD. 

Microscopic exam should be done often by wastewater system operators. While a high end, phase contrast scope is great, a simple high school biology level microscope will allow you to see indicator organisms, floc size & density. Now that you are performing a microscopic exam, do you have to ID each protozoa, metazoa, and filament? The answer is NO! Operators have enough to do without performing a microscopic exam that involves ID of every organisms and in the case of filaments, often using staining. I like to use a simple daily exam sheet to record the following groups and their relative frequency.

Amoeba
Amoeba are slow moving single celled protozoa. With streaming cytoplasm, amoebae envelop free bacteria, organic particles, and other "food" sources. In wastewater, we often have "free" amoeba which are jusAt the streaming cytoplasm inside a cell envelope. There is also the testate form which has a shell. The free amoeba are most common during early phases of growth, but you can see testate amoeba even with low F/M, mature systems.

Flagellates
A large and diverse group of protozoa, flagellates are grouped by how they move. Propelled by whip like flagella, these protozoa tend to "bounce" and move in what seems a somewhat random manner. With large flagellates or when using a phase contrast microscope, you can often see the flagella propelling the organism. Flagellates are seen early on the growth curve and tend to decrease in relative frequency as you move along the growth curve. 

Free Swimming & Crawling Ciliates
Ciliates move faster and in a more purposeful manner than flagellates. They also tend to be larger and easier to observe with 10x and 40x objectives (100x and 400x magnification). When ciliates are the dominate protozoa, you will have floc forming, Dissolved Oxygen increasing, and a more mature biomass. In the exam, look for:

• Diversity - free swimming vs crawling
• How activate the ciliates are (how fast they are moving)

Stalk Ciliates & Suctorians​
Stalk ciliates and suctorians are the ideal indicator protozoa for most wastewater systems. Present only in systems with good D.O., low soluble organics, and good floc forming conditions; stalk forms do not move and are easy to observe. These protozoa anchor to the floc and are only mobile in early phases and after shock events. Stalks are highly susceptible to shock events - pH swing and toxic compounds. So, note any change in their frequency. Note the following:

• Single stalk forms - most common
• Colonial stalks - if seen, you have great quality water
  
• Suctorians - similar to a stalk, but look like a pin cushion and stab/suck cytoplasm from passing flagellates and ciliates. These are very fun to watch if you have a few minutes.
  

Metazoa and higher forms
Everybody loves the rotifers and tardigrades (water bears). They are easy to see and interesting to watch. If they become the dominant indicators, you are probably tilting toward an old sludge. Also they are more resistant to shocks than stalk forms, so you can have some problems and still see rotifers. 

Floc Size & Density
It is also important to note the floc. How big are the floc particles (you don't have to get a precise measurement, just use relative size from day to day). Also not floc density and can you see filaments extending from the floc. Inside the floc, filaments are not bulking and function as a structure to improve floc strength. Also not if there are free bacteria and pin flocs in the sample.  Again, you need to note changes from day to day. And if you see something strange, look for a cause and definitely note the observation.