Microscopic exam should be performed daily or every shift by operators. When I say microscopic exam, I am not referring to filamentous bacteria ID or using any stain procedure. Instead, use a decent quality microscope (can be phase contrast or more simple light microscope) and have the operator look at a sample at 100x and 400x magnification (10x & 40x objectives). Use a standard transfer pipette to place a small amount of fresh MLSS on a slide. If you have a lagoon system, you will need to use a centrifuge to collect sufficient biomass to observe. Here is what should be noted - I promise it takes less than 30 seconds:

• Note floc size, density, and filaments - compare to "normal" system levels (100x)
• Free bacteria and pin floc (best seen at 400x)
• Now look for indicator protozoa (amoeba, flagellates, ciliates, and stalk ciliates)
• Any higher life forms such as rotifers or worms
• Finally, anything unusual or big changes from previous exam

A good idea is to use a written exam sheet with indicator organisms and floc characteristics recorded. This can be used to establish baseline observations and detect when something is impacting a system's biomass. Early detection of shocks, bulking, and F/M changes can be seen using microscopic exam before changes happen in SV30, foaming, or effluent quality.

Plug-flow systems are normally represented by a pipe with flow going in one direction towards the effluent. Many of our common systems operate in plug-flow or close to plug-flow reactor design. The most prevalent being oxidation ditches and what I call tightly baffled activated sludge where the flow runs through narrow, long channels. 

Let's recap the benefits of plug-flow:

• Good for BOD5 removal followed by ammonia oxidation, denitrification, and even phosphorus removal. Much flexibility in changing aeration, anoxic, anaerobic zone sizes.
• Works well with low BOD5 or degradation of insoluble BOD5 including resistant organics 

Now the problems

• High soluble BOD5 loadings near the inlet can create low D.O., promoting filaments
• Susceptibility to shocks - pH, influent temperature, toxic shock loadings (this is why plug flow is much less common in industrial wastewater)

For most plug flow systems, the biggest problems are caused by high soluble organic loadings near the influent depressing DO and promoting the low DO filaments. If the loadings are consistent and the ability to add more oxygen to the first part of the system is not efficient, another solution is to step-feed the influent. By having multiple injection sites along the length of plug-flow system, you reduce the amount of soluble BOD5 entering any one section per unit of aeration. This prevents excessive anoxic or low DO conditions favoring filaments and odors. It can also be useful in increasing nutrient removal efficiency by altering DO, Redox, nutrient, or soluble organic concentrations in individual sections.

The SV30 test uses a dedicated settleometer or 2L graduated cylinder to measure settling velocity and compaction in wastewater treatment biomass. (Note - always use the same size cylinder or settleometer to run the tests). To account for changes in MLSS concentration, the SVI takes the SV30 number and divides by MLSS in grams (some facilities use MLVSS in grams) - just use the same divisor each time.

Now for the SVI or SV30 being too low. Usually compact sludge is a good thing - it means no bulking and clarifiers with not solids carryover. However, if sludge settles too quickly or compacts too much there can be problems including turbidity, recycle (RAS) system troubles, and occasionally floating fines causing effluent TSS problems. Usually the problem is "Old Sludge" where lower F/M conditions over the long term result in bacteria consuming the extracellular polymer substances (EPS). Once the EPS percentage drops:

• the floc size begins to decrease 
  
• floc density increases and settles rapidly
  
• "fines" or small solids chunks break off from the floc and enter solution
• Polymer at the secondary clarifier to reduce TSS or clear up turbidity

How do you investigate and fix the problem

• Perform microscopic exam - look for smaller floc sizes, high density of floc, presence of low F/M filaments, and increased numbers of multicellular indicator organisms such as rotifers, tardigrades (water bears), and even nematodes.
• See if SOUR or DOUR has decreased. Lower oxygen uptake rates along with very low SVI are indicative of "old sludge"
• Determine if low influent organic concentrations is going to be an ongoing phenomenon and adjust target MLSS/MLVSS concentrations
• Increase wasting in 10% steps while monitoring sludge quality to see if EPS increase is triggered by increasing F/M and lower sludge age (MCRT)

A "milky" water usually means that you have an emulsion forming. Emulsions are insolubles such as grease, oils, or fats suspended in the water. Surfactants with non-polar and polar ends attach to the oils and water creating micelles. Each micelle in the milky water case contains oil attached to the non-polar end of the surfactant. The polar end of the surfactant keeps the non-polar oils suspended or dispersed in the water phase. It is the dispersed oils in this case that cause the milky appearance - interestingly milk is a suspension of fats and proteins in water hence the appearance.

Foaming in this case is caused by the surfactants and is usually stable and light in color. Anti-foams tend to work well on surfactant foams.

Corrective Actions

• Discover source of both oils and surfactants - with the goal of keeping problems out of the wastewater treatment plant
• Use anti-foams to control foaming
• Be prepared to increase wasting as oils (grease) will get bound into the floc
• If you notice a loss of biological activity (OUR, Microscopic exam, effluent turbidity) - this is a case where bioaugmentation is very effective in keeping the system healthy and degrading the offending surfactants/grease

And remember that removing grease in the WWTP is important because:

• It can favor growth of Nocardia forms with foaming
• Makes dewatering sludge difficult
• Solids settling rates can decrease and floating MLSS in the clarifiers

Limited water exchange, high stocking density, and high protein feed to maximize growth rates; modern intensive aquaculture ponds often have problems with ammonia, nitrite, and other pollutants that stress the animals and open the door to disease. With several requests for an immediate ammonia response in problem ponds, I wanted to detail how to prevent the problem by good maintenance and how "nitrifiers" or AOB with their short shelf life and refrigeration requirements are not a good thing to add in almost all ponds.

First, ammonia in aquaculture comes from problems in the pond nitrogen cycle. As in nature, decaying animal wastes and excess feed (found in the pond bottom sludge) convert from organic nitrogen into ammonia. Ammonia can kill an aquaculture pond outright while still at less than 10 mg/L. Even at "manageable" levels, ammonia stresses the organisms which lowers feed conversion rates and weight gain. 

Ammonia can be removed from water in several ways from greatest to least important

• Algae thrives when given ammonia as a nutrient 
• Heterotrophic microbes use ammonia to fuel growth and make proteins
• Chemotrophic ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) exist in low concentrations in biological floc

So how do I see ponds likely to have ammonia problems during the grow out? Look at bottom sludge. This sludge is reservoir that continues to produce ammonia as organic solids decay. Of course overfeeding high protein feeds can exacerbate the problem. Now for the solution:

• Check pond bottom sludge levels prior to stocking 
• Ensure aerators are in working order 
• Use the correct feed composition and feeding rates
• Stock at appropriate density
• Adding heterotrophic waste degrading bacteria from the start of the season helps (adding before you get highly polluted water is key).
  
  

Benefits of adding heterotrophic bacteria:

• Organic wastes, including sludge, are consumed by microbes. This happens naturally, but often with high stocking density & feeding, you have waste buildup and start of problem anaerobic conditions (often seen as black sludge with hydrogen sulfide). 
• Microbes under low oxygen conditions (pond bottom) use nitrate/nitrite as alternative electron acceptor - this prevents inhibition of AOB/NOB activity
• Microbes in quality products also help reduce pathogenic bacteria growth via competitive exclusion
• Beneficial immune stimulation by select probiotic cultures 
• Promotes green algae (eukaryotic) organisms growth. Remember these microbes remove more ammonia from pond water than any other organisms.

We have done studies on the microbiome of successful aquaculture systems and found which microbes are associated with healthy ponds. The bacteria we selected for aquaculture products are not "superbugs" or GMO - they are concentrates of the beneficial microbes found in healthy ponds. Sometimes, the natural balance seen in healthy ponds is upset by stocking densities and feed rates. We just help restore the balance.
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Key point is to monitor the pond for pH, Dissolved Oxygen, Sludge Layer frequently. If stocking at high density, adding microbes early on in the grow out season will prevent pollution problems later in the season.

Removing biological solids and insoluble compounds requires microbes to form floc or biofilms. Many desirable wastewater microbes prefer to exist in a biofilm matrix.  Extracellular Polymer Substances (EPS) are the glue in biofilm and floc. Consisting of polysaccharides, proteins, DNA, and humic substances, EPS can be either attached to the cell (capsular) or free in solution (non-capsular). In typical floc, EPS constitutes 50 - 90% of total organic matter.

The Zooglea family of microbes was one of the first wastewater organisms associated with floc formation. While not the only producer of floc forming EPS, the Zooglea family are very common in most wastewater plants and can be the cause of non-filamentous bulking, difficult to dewater biological solids, and difficult to settle floc.

Why biofilm and floc forms:

• More consistent environmental conditions - protection from toxic compounds, pH swings, and predatory protozoa
• Allows syntrophic organisms to function efficiently, or one microbes' waste is another microbe's food source
• Helps to bring insoluble compounds close to microbes where extracellular enzymes can function
• Promotes concentration of nutrients - nitrogen, phosphorus, and micronutrients

When EPS goes from good to bad
​For keeping biological solids under control, you need EPS to be present in quantities that allow for binding biological solids. It is also better to have capsular EPS - attached to the cells - over free, unattached EPS. In free solution, the EPS acts much like agar or gelatin. Increasing viscosity, entrapping water, and creating the gelatinous matrix we associate with non-filamentous bulking. So what triggers the excess non-capsular EPS production?

• Low nutrients - usually  nitrogen and phosphorous - when microbes face nutrient related stress, they attempt to concentrate/and capture vital nutrients in their EPS matrix. 
• High levels of soluble organics - microbes use the EPS to accumulate soluble organics which can lead to excess EPS over extended periods of high organic loading
• Insoluble compounds such as grease, oil, and long chain fatty acids - while usually associated with Nocardia form growth, grease can also result in non-capsular EPS production
• Environmental stress - extreme temperatures, pH, and inhibitory influent organics can stress microbes which resort to producing more EPS for protection.