A subset of industrial wastewaters including pulp & paper have lower than optimal C:N:P ratios. To ensure proper bacterial growth, these facilities often add various nutrient blends to supplement both N & P. What happens when they do not supplement macronutrients at optimal levels or even add not nutrients at all?

We have long known that deficient macronutrients often impacts floc formation and the filament/floc forming organism balance. But what happens with the actual microbial makeup in the biomass. Molecular testing allows for conducting a total microbial census and we can compare similar systems with and without added macronutrients.

Diazatrophs are microbial organisms that use N2 gas - present at approximately 80% of our atmosphere - to provide nitrogen needed for cell growth. This reaction is most described in agriculture with legume crops where the microbes grow in root nodules. In wastewater, diazotrophs include a diverse mix of organisms including Rhizobia, Rhodopseudomonas, Frankia, Klebsiella, Azotobacter, Paenibacillus, and many cyanobacteria genera. Some of these organisms are floc forming and desirable in wastewater but others like cyanobacteria can create water quality issues.

In systems adding macronutrients, operators want to add sufficient N & P to keep a healthy, floc forming microbial population but not overdose nutrients. In the past, nutrient dose was based on finding N & P residuals in the effluent and maintaining these at target residuals. With molecular testing, we are now able to monitor for the presence of AOB/NOB (feeding too much nitrogen) and diazotroph populations (if in excess, you have insufficient nitrogen).

Aster Bio has been testing an increasing number of BNR plants with excellent phosphorous removal efficiencies. Our testing - Environmental Genomics™ MCA - is a total microbial census of MLSS with relative % of each genera. Using this molecular sequencing technology, you can see key differences among microbial populations based on system design and operation. 

Data from MCA Test
We use high-throughput sequencing of the 16s region to conduct the microbial census. The sequencing produces over 100 MB of data per run, so making it useful also means summarizing the data in a convenient table. Below are two different runs from a healthy BNR system with biological phosphorus removal and a conventional activated sludge system. The table presentation gives % of total reads for common functional groups - of course there is a lot of data behind this summary that is also interesting.

Key Differences
The Non-BNR systems of course have fewer PAO/GAO populations. The "Bulking" column is composed of Zooglea/Thauera genera. These genera are common bacteria that can form good floc, denitrify, and degrade a wide range of organic compounds. Under certain influent conditions, the bulking group can produce excess EPS that causes non-filamentous bulking.

BNR system testing reveals that the PAO/GAO cultures are inversely related to the Bulking genera. I have also noted that the BNR systems also tend to have more known filamentous bacteria genera present. 

With a complex community of inter-related microbes, anaerobic require balanced populations of fermentative bacteria and methanogenic archaea. Most textbooks breakdown the microbial communities into three groups:

• ​Hydrolyzing & Acidogenic bacteria are bacteria with anoxic and fermentative biological pathways. These organisms degrade complex organics into forms used by other anaerobic organisms. These microbes utilize any remaining alternative electron acceptors and eventually use organic compounds as the terminal electron acceptor.  This stage can also product nuisance H2S gas
• Acetogenic bacteria are anaerobic cultures that continue the fermentation process and produce the acetate/acetic acids that are the preferred feedstock for developing methanogenic archaeal populations. In both the Hydrolyzing and Acetogenic processes, H2 and CO2 gases are produced.
• Methanogenic archaea - note this is a separate kingdom from bacteria with unique enzyme cofactor micronutrients - are obligate anaerobic cultures responsible for producing valuable methane gas (CH4). Two varieties of methanogens exist based on substrates utilized:
  Acetic Acid Cleavage - CH3COOH --> CH4 + CO2
  Carbon Dioxide Reduction - CO2 + 4H2 --> CH4 + 2H2O
  
  

Traditional Monitoring

• Methane and CO2 percentages in produced gas
• pH, Temperature, ORP
• Alkalinity
• Organic acids
• H2S
• Trace micronutrients used by methanogens that may not be present in the influent
  
  

Adding Environmental Genomic testing to monitoring
Aster Bio uses Microbial Community Analysis (MCA) as a part of our Environmental Genomics testing suite.  The MCA test is a full microbial census for both bacteria and archaeal populations using high throughput sequencing technology. The resulting census gives relative % of the genera present. For monitoring, this shows drifts and changes in populations in response any operational change. MCA also proves useful in discovering problem sources in digesters not achieving desired methane conversion. While not a daily test, routine monitoring with MCA supplements traditional monitoring and an help improve anaerobic digester performance while reducing operating costs.

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.