In activated sludge wastewater treatment, secondary clarifiers are designed to prevent TSS carryover to the effluent and concentrate biological solids (MLSS) for return to the aeration basin. A balance must be maintained by giving solids time to settle and compact, yet not so much time that the microbes in the bed become anaerobic. What we mean by anaerobic clarifier beds is that all dissolved oxygen is depleted. If nitrate/nitrite is present from ammonia oxidation, the microbes first turn to nitrate creating small bubbles and floating solids. If there is no nitrate, the microbes will start fermentative respiration and sulfate reduction. Both of these can be a problem for odors and creating what we call septic sludge.  

Most clarifiers are designed for between 2 - 4 hours hydraulic residence time, solids are recycled at a rate to prevent long solids residence times. A rule-of-thumb is to keep bed depths at 2 feet. With most clarifiers, a 2 foot bed recycles sufficiently concentrated MLSS while also preventing anaerobic conditions in the clarifier. Systems with bulking or mechanical problems often start to have trouble with 2 foot bed depths. Another problem with running deep clarifier beds is the potential for rake problems, usually torque on the motor. 

Good process monitoring habits for secondary clarifiers:

• Run SV30 and SVI on the MLSS to understand changes in settling rates. This can show potential problems in the secondary clarifiers early on. It will also inform operators on the need to add coagulants or flocculants to improve settling rates.
• Use a manual bed depth measuring tool and/or sonic levels to monitor bed depth every shift
• Return sludge concentration (RAS) concentrations should be checked
• Monitor rake torque
• Visually check for solids carryover, billowing, floating solids, scum, and problems with equipment.

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Water is an interesting polar solvent. Remembering back to my high school chemistry class - it was often called the universal solvent. Water's hydrogen bonding can make a solution with a multitude of compounds. However, oxygen is not very soluble in water at atmospheric pressures. In wastewater systems, we use fine bubble diffusers, jet aerators, and any number of strategies - yet most air (roughly about 19% oxygen) pushed into the water does not become "dissolved oxygen". 

Lately, I have been able to work with some newer technology for enhancing gas transfers into wastewater. Developed by BlueInGreen, the systems uses advanced high pressure adsorption technology to supersaturate water. As I am interested in getting oxygen into wastewater, I wanted to see that if supersaturating water could improve oxygen transfer. In most cases adding oxygen to wastewater in pure form results in most of the oxygen flashing off to the atmosphere due to low solubility in the water. The oxygen bubble coalesce and rise to the surface - thereby leaving the water with minimal transfer efficiency into D.O. 

Using BlueInGreen technology, we were seeking to avoid this waste and transfer much high levels of D.O. into solution (and for the wastewater bacteria). Instead of using air, the SDOX system uses pure oxygen. And, this is a big difference maker. Instead of a gas with 19% O2, we have 100% O2. But without advanced injection/adsorption system, it would be similar to past pure oxygen systems where we could get only temporary high DO levels before the bubbles coalesced. The difference is the pressure system and other bits of advanced engineering allowing for the production of nano-bubbles. Moving from micro to nano bubbles takes advantage of Zeta Potentials - with nano sized pure oxygen bubbles, you can create a stable colloidal solution of oxygen in water. The electrical charge of the nano-bubbles repels the individual oxygen bubbles. This results in D.O. levels above 30 mg/L with stability over 24 hours in wastewater. 

To show the impact of zeta potential forces in water, we added India Ink to a normal tap water (control) and tap water after SDOX treatment (creating a oxygen colloidal suspension). In the Control the India Ink drops to the bottom as expected. In the SDOX beaker, the ink particles have to navigate through the matrix of oxygen bubbles and are diffused into the solution. This action reveals how Zeta Potentials keep the nano oxygen bubbles stable in solution.

We usually think of microbes as one species at a time. A good example of this is a single strain on a petri plate. On a petri dish, we do not see the interactions that occur among microbes in the wild environment including soils, waters, and wastewater treatment plants. Some organisms require interactions with other species for growth. This symbiotic relationship, or syntrophy, is where one organisms by-products or waste fuels the other organisms growth. The reduction in by-products allows the first organism to continue to grow as the byproducts are often inhibitory to initial metabolic steps. One well known example is among ammonia oxidizing, nitrite oxidizing, an denitrifying bacteria. 

Some additional notes:

• Ammonia oxidation by ammonia oxidizing bacteria (AOB) often called just Nitrosomonas. pH above 8.5 begins to create unionized ammonia toxicity - NH4+ conversion to NH3 gas. So their energy source can become toxic. At pH < 6.5 we start to see toxicity as ammonia species move out of AOB metabolic ranges. 
• Nitrite oxidizing bacteria (NOB) often called just Nitrobacter. Usually grow faster than AOB, and are not often the rate limiting step. However, we have seen nitrite buildup in industrial wastewater that causes bioassay problems this was due to NOB partial inhibition.
• If you suspect toxicity from sulfides, metals, or organics - test using Aster Bio's Tox-Bac & Tox-N bench tests which give toxicity to heterotrophic and AOB with 3 hours. 
• Adding nitrifier concentrates to systems with insufficient AOB/NOB activity - run toxicity tests! I also have best luck adding heterotrophic cultures along with or before adding the AOB/NOB. The heterotrophs can remove many inhibitory compounds and form biofilms that prevent nitrifier washout.

I recently helped a customer purchase a microscope for use in wastewater examination. While I have an excellent local microscope store - Land, Sea & Sky here in Houston, TX. I had not shopped for a microscope online in several years. And, things get confusing with every option and configuration making for a tough purchasing decision. I thought that it would help if I give you a few guidelines for selecting a microscope.
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1. What magnification - or is more magnification better? 
   Most microscopes have 4x, 10x, 40x, and 100x objectives. Even in the cheapest microscopes, the lower objectives are good enough for wastewater. Where I have seen the big difference is in the 40x and 100x objectives. Remember the eyepiece magnifies an additional 10x or 15x depending upon the eyepiece. What is equally important as magnification is resolution - which is why high end lenses made by Olympus, Nikon, and Zeiss always seem to have more "crisp" images. For routine wastewater exam- such as looking at floc, protozoa and metazoa most microscopes will suffice. Filamentous ID benefits from better quality lenses. For wastewater exam purposes, objectives over 100x are not very useful. You should also know 100x objectives require the use of immersion oil with the objective. Immersion oil is low cost, but can be messy if users do not clean the objectives after use. Often I find the 40x objective (no immersion oil required) to be the most magnification needed for daily microscopic exam.
   
   
2. Brightfield (light), Darkfield, or Phase Contrast
   Wastewater microorganisms are relatively clear and without staining, we do not see all their features when using brightfield microscopy. Adding phase contrast objectives allows viewing of minor changes in light refraction - which improves contrast. This is why professional microscopic exam pictures look so much better than on lower cost light microscopic views. Is phase contrast necessary? No, but the price for phase contrast condensers & objectives has dropped enough that I would recommend any new purchase include phase contrast and light field objectives. For wastewater, darkfield is not nearly as important as phase contrast. To learn more about phase contrast look here
    www.microscopyu.com/techniques/phase-contrast/introduction-to-phase-contrast-microscopy.
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3. Light source & power
   This used to be a big deal when we used old style bulbs. Most manufacturers now use halogen or LED light sources. Both last longer, with less heat, and good brightness than older bulbs. If I had a choice, I would go with LED but halogen is an excellent option too.
   
   
4. Mechanical stage 
   A mechanical stage allows for movement of the slide under the objective without touching the slide itself. All but the lowest end microscopes have mechanical stages - and unless your budget prevents it, get the mechanical stage.
   
   
5. Cover & case
   Dust is a huge enemy! Even in relatively clean labs, dust accumulates on light sources, phase contrast "discs", and lenses. Cleaning with lens paper, compressed air, and cotton swabs are good practice, but my best hint is to get a good microscope cover for when the microscope is not being used. If you are going to travel or move the microscope often, get a case to protect the scope.
   
   
6. Maintenance
   Periodically maintenance can be done at microscope repair shops - like Land, Sea & Sky - this includes taking the microscope completely apart to remove all dust, cleaning any "things" that get on the objectives, and adjusting the mechanical focus and stage mechanisms. The frequency of cleaning and maintenance all depends upon where the microscope is used, stored, and handled.

Resources for learning about microscopes:

• Zeiss has an excellent resource for microscope education - zeiss-campus.magnet.fsu.edu/index.html
  While a commercial company, the FSU based Zeiss site gives good general information no matter which microscope you select. 
• Also many university microbiology departments have training resources for using microscopes - if you have been in an intro microbiology lab, you will remember the laboratory activities including cheap microscopes, dust covered lenses, and looking at various prepared slides. And, afterwards forgetting all the techniques that the instructors tried to teach.

Biofilms are important for both attached and suspended growth biological wastewater treatment systems. We have designed systems that take advantage of natural biofilm formation tendencies in many environmental microorganisms. Take a minute to consider why biofilms are important in wastewater treatment.

What are the biofilm functions in wastewater treatment
Biofilms act to increase microbial population density in flow through systems. In attached growth, surface biofilms prevent hydraulic washout and promote more efficient pollution removal than seen in lagoons or ponds. Suspended growth systems with secondary clarification use gravity settling to keep microbial floc (biofilms) in circulation via the RAS line. The stronger the biofilm, the more resistant the biomass to both hydraulic and environmental shocks (this includes accidental spills and mechanical failure). A strong biofilm also supports a diverse microbial population including slower-growing, niche organisms such as ammonia oxidizing bacteria including both AOB & NOB. Without the biofilm, both AOB & NOB are much more subject to hydraulic washout and toxic shocks. Finally, a strong biofilm controls effluent solids and turbidity with minimal use of chemical polymers.

Why microbes form biofilms
Having established the importance of biofilms in wastewater, we need to consider why microbes form biofilms. There are three often discussed pressures for biofilm formation. First, the biofilm protects microbes from the surrounding environment and predation by protozoa or metazoan. Organisms inside the floc interact with the external environment via gradual diffusion of nutrients and oxygen across the matrix. The gradual concentration changes enable the cells to adjust to environmental changes over time versus responses seen by planktonic microbes. Secondly, biofilms also help increase nutrient concentrations. With lower F/M conditions, the EPS matrix includes insoluble organics and extracellular enzymes that enable the nutrient to cross cell walls. The biofilm also improved microbial access to nutrients such as nitrogen, phosphorus, and trace metals by storing and concentration the nutrients found in the influent. Finally, a diverse biofilm allows for syntrophic or symbiotic relationships among the microbes. Often individual strains perform only one step in the entire metabolic detoxification/degradation process. Other neighboring organisms use the intermediates for their metabolism, preventing inhibition by waste products from the first step. We see syntrophic interactions often in degradation of xenobiotics, ammonia oxidation, and anaerobic digester populations.