I had a recent question on why a system was with a history of stable operations was seeing an increase in non-volatile solids which we view as a decrease in the "bug" portion of MLSS.  Effluent quality remains excellent, but the local engineers wanted to think about what was driving this change. To understand MLSS to MLVSS ratio changes, it helps to think about what is happening in the system.

MLSS is total dried solids and includes “everything” after removing the water. This includes living microorganisms, dead microorganisms, extracellular materials including biopolymers, adsorbed organics, organic particulates, inorganic particulates.

MLVSS is the portion of MLSS that is removed during a high heat (550 Deg C) muffle furnace step. The residual contains non-volatile solids - Often benchmarked at 75% of solids being volatile in domestic WW.
How much of the volatile portion is living microbes? Usually less than 15% - longer sludge ages with lower F/M drop the % downward even further.

If you see an increase in the non-volatile fraction – this can mean

• More influent non-volatiles!
• Insufficient wasting leading to buildup of non-biodegradable or inorganic solids. As sludge becomes “old”, you enter endogenous respiration where active living biomass as a % falls
• Influent biodegradable organics drops so a lower microbial population is supported
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What does this MLSS/MLVSS ratio change mean for operations:

• Don’t use the ratio in a vacuum – use settling tests, turbidity, microscopic exam, respiration rates and other available tests to determine operations.
• Monitor & measure
• Look upstream to see if anything has changed

Quaternary Ammonium Compounds (QACs), often called quats, are cationic surfactants (positive charge). There are many QACs in use, but among the most discussed are the disinfectant QACs. The positive charge makes them bind to negatively charged cell walls in microbes where the QAC damages the cell walls/membranes. At concentrations between 200 – 400 mg/L, QACs are used to disinfect surfaces in food processing operations.  As cationic surfactants, QACs are also used in fabric softeners and hair conditioners to neutralize anionic charges (static). These QACs typically are not inhibitory to microbial growth and readily degrade in wastewater treatment plants.

With the number of facilities expressing concerns about QAC toxicity issues, I have been reading many academic papers about QAC impacts on wastewater facilities and in lab testing. So you don't have to read all these papers, I summarized some of the observations:

QAC wastewater impact

• 2 options – biodegradation and adsorption to anionic surfaces (soil, anionic surfactants, etc)
• Biofilms are much more resistant than free bacterial cells
• Most domestic WWTP have influent QAC in the 1 – 5 mg/L range
• QACs are a big group of compounds - not uniform in toxicity
  • Short chains are antimicrobial
  • Longer chains are fabric softeners etc
  • SC are more biodegradable
• Anionic surfactants complex with QAC and inactivate with respect to biocidal action these are also present in influent and will react with the QACs present. Anionic surfactants are in most cleaning products, laundry detergents, and liquid "soaps"
• EPS response to QAC increased MBR fouling

Key findings

• QAC – not a uniform toxicity across the board – certain ones are more harmful
• Anionic surfactants and other compounds present usually bind with the QAC and change/reduce their ability to damage bacterial cell walls and associated enzymes
• A variety of QAC neutralizers can be used to bind QACs – useful when influent QAC concentrations are above normal ranges – 1 – 5 mg/L range in the influent
• QAC toxicity usually impacts AOB/NOB first – 2 mg/L can cause significant inhibition. However, biofilm/floc reduces the ability to QAC to inhibit AOB/NOB.
• QACs reduce microbial diversity & result in changes in EPS (big for MBR fouling)
• Many common heterotrophs an degrade QAC – including Pseudomonas.
• Systems adapt to QAC by changing organism mix and EPS for increased tolerance and neutralization capability

What to do if you suspect QAC Problem
 
Confirm that QAC is the problem

• Do you have AOB/NOB? – test with MCA or qPCR (as the most sensitive organisms, their continued presence in MLSS indicates that QAC is not the problem)
• Quick lab kit tests don’t distinguish among QACs and can have interferences with common wastewater components. For “complete” testing send to an outside lab with HPLC capabilities.
• MCA tests can show changes in microbial diversity – a strong indicator of QAC at sub lethal concentrations
  
  

If there is QAC what can be done

• Find sources of QACs and make sure the users are using the products correctly and if they have neutralization capabilities – make sure they are being used!
• Use QAC binding chemistry to lower toxicity – multiple options are available.

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Those of us working with industrial wastewater treatment often complain about textbooks and training courses focusing on municipal wastewater. Rick Marshall & Steve Leach - both highly experienced wastewater professionals - are offering a petrochemical focused wastewater course in December 2020. This course will be online using Zoom but have the same content as in-person training. I highly recommend this course for those of you working in petrochemical or refining wastewater treatment. 

Microbial metabolism generates energy by moving electrons along metabolic pathways to a terminal electron acceptor. If this sounds complex or brings back images of the biology class electron transport chains, I apologize but it is good to know the general metabolic options in wastewater and not get bogged down by the individual biochemistry steps.

We often use the term aerobic, facultative anaerobe, or anaerobe when talking about wastewater bacteria. These terms are great, but the lines are not as clean as you may think. Let's start with the general options.

It all starts with where the electron ends
Going back to the electron tower (pictured above), microbes generate energy for growth by moving the electron to the highest energy acceptor. If the terminal electron acceptor is oxygen, you have an extremely high energy yield. As you move to other electron acceptors, less energy is produced. 

Terms that we use in wastewater

• Obligate aerobe
  Only grows when oxygen is present. Often we say NItrosomonas (AOB) are obligate aerobes, but even Nitrosomonas has limited growth using alternative anoxic pathways. 
• Facultative anaerobe
  In wastewater, a facultative anaerobe is any culture having the ability to grow when dissolved oxygen is not present. They can use various alternative electron acceptors. If they do not have an appropriate electron acceptor they do not grow. These are the most common cultures in wastewater treatment systems - example genera include: Pseudomonas, Bacillus, Zooglea, Thauera, & Paracoccus.
• Obligate anaerobe
  Here we have organisms that are poisoned by free oxygen. They only grow in reducing (low redox) environments. Here you have organisms found in sludge layers, inside dense granular floc, or anaerobic digesters. Common examples include Clostridium (an acid forming bacteria) and Methanogens (archaeal cultures that produce methane from short-chain organic acids and hydrogen).
• Fermentative 
  This group includes some facultative anaerobes and obligate anaerobes! Fermentative respiration requires the terminal electron acceptor to be organic compounds. This is the acid forming step in anaerobic digesters. Fermentative respiration yields less energy than growth using oxygen, nitrate, nitrite, or sulfate as electron acceptors.
• Microaerophilic
  This includes cultures that thrive at low oxygen levels. They use oxygen as an electron acceptor but exploit a relatively low D.O. ecological niche.

Foam can tell you a lot about the biological state of your aeration basin. Some foams including the normal white foam and the greasy thick stable foams are produced from the byproducts of microbial growth. Here are the most common foams that you see on aeration basins:

Normal Biological Foam
As bacteria grow and divide, they produce extracellular materials including polysaccharides, proteins (enzymes), and even DNA/RNA. Also important for biofilm or floc adhesion, these materials trap air bubbles in a light color foam that is easily broken by waters spray if it even builds up more than 1 - 2" on the water surface. When the system is in log phase growth, you may notice both a lighter color and more foam than when the system is mature in decline phase growth.

Nocardia/Microthrix Parvicella (filaments)
Nocardia (often actually a Gordonia sp) is promoted by long chain fatty acids (FOG) and Microthrix is triggered often by low F/M, long chain fatty acids and lower temperatures. Both genera can exist in wastewater and not cause foaming, but under appropriate conditions, they start to produce hydrophobic extracellular materials. The hydrophobic biological polymers trap air and create the thick, stable, greasy foam. The best way to prevent foaming is to reduce grease and keep appropriate MCRT or F/M ratios in the system.

Surfactant Foams
Surfactants are used in many products not just cleaning agents. Firefighting foams, food product emulsion stabilizers, even many personal care products contain surfactant chemistries. Under normal concentrations, bacteria readily remove surfactants or they are diluted beyond their ability to reduce surface tension (creating foam). Periodically, you may see higher loadings hit the aeration basin. With surfactants the foam is usually stable and has a light color. While their presence is temporary and foam levels should drop, you can use commercial antifoam products to prevent excess foam during peak events.

Plant soaps, Starches, & Proteins
The above chemicals are natural polymers found in wastewater. Foaming occurrs when normal biological foam beomes stabilized by the natural polymers. In this case, antifoams and water sprays are often effective. We have also effectively used pretreatment with enzymes to pre-digest the polymers prior to the aeration basin - this can be very valuable in food processing wastewater treatment.

For years, I have always been told that Nitrosomonas sp are obligate aerobic bacteria. We all accepted that Nitrosomonas  used oxygen to convert ammonia into nitrite. That was the extent of their metabolic capabilities. However, Nitrosomonas have now been found to have a few other metabolic tricks available to survive in anoxic and anaerobic environments. How well does Nitrosomonas grow under D.O. limited environments, surprisingly well. In fact, Aster Bio's MCA tests on multiple anaerobic digesters has found significant Nitrosomonas populations. Here is what Nitrosomonas does in aerobic, anoxic, and anaerobic environments:

• Aerobic - this is where Nitrosomonas exhibits most rapid growth and is the best-known metabolic process. Using Ammonia + Oxygen to produce Nitrite.
  
  
• Anaerobic - Nitrosomonas can function as an ANAMMOX culture. In this pathway, Ammonium and Nitrite are the inputs with the final products being Nitrogen gas and H2O.  
  
  
• Anoxic - Nitrosomonas uses nitrite as the terminal electron acceptor and can use various cell intermediates and a few organic compounds to provide energy for growth. Technically, the anaerobic pathways listed above can be included with the anoxic systems as both require nitrite.

What does this mean for wastewater treatment? Well you may have noticed much emphasis on ANAMMOX technology where ammonia oxidation and denitrification happen in the same step. This saves on energy for aeration and makes denitrification more efficient. You do have to account for the slower growth under anoxic/anaerobic conditions, but Nitrosomonas may turn out to be one of the most important ANAMMOX cultures in wastewater systems as it also provides the NO2 used in ANAMOX during aerobic growth.