Keeping a stable, healthy nitrifier population in wastewater treatment challenges many operators. As slow growing organisms, both Ammonia Oxidizing Bacteria (AOB) and Nitrite Oxidizing Bacteria (NOB) prove susceptible to washout when environmental conditions slow their growth.  From experience, it is more often a slow decline in AOB/NOB populations rather than an acute toxicity event (kill) that increases effluent ammonia. When you start to see effluent ammonia increasing, head off the problem and perform the following steps.

Step 1 - Determine what caused the problem by looking at influent & system data

• Acute toxicity event
• Gradual washout due to low DO, pH, or temperature slowing growth.
• Inhibition - temporary condition stops AOB/NOB metabolism - if it continues you will have washout.

Step 2 - Use qPCR test to confirm biomass AOB& NOB populations
Send a sample for a fast qPCR test for AOB & NOB checks. This gives information on what is causing loss of nitrification and the best corrective action. Given that a nitrifier panel using qPCR with wastewater specific primers is a rapid test and relatively low cost ($150 standard 2 day TNT or $250 rushed same day), this step can save money when compared to trucking in sludge or adding commercial nitrifiers. Based on quantitative results given by qPCR, you can chose the most efficient corrective action.

Step 3 - Corrective Action

• Make the environment favorable for AOB & NOB growth - pH, DO, Alkalinity, MCRT. Things that you can change.
• If part of the problem is inhibitory organics, adding heterotrophic cultures can help reduce some of the inhibition and get existing nitrifiers back to rapid growth.
• If qPCR finds insufficient AOB & NOB populations for ammonia (TKN) loadings, you can use either trucked in sludge or bioaugmentation with commercial nitrifiers. Dosing of commercial nitrifiers is done based on:
  • Amount of ammonia and nitrite to be oxidized
  • Environmental factors in the system - temp, pH, DO, etc
  • How much time do you have for full recovery - catching problems early is very important

MBR systems replace traditional secondary clarifiers (gravity separation) with a membrane containing pores. The small size of the pores - microfiltration or ultrafiltration - separates solids from the discharged water. MBR technology has come a long way in the past 20 years and is now a viable alternative to clarifiers. For those not familiar with MBR systems here is a quick summary of strengths and weaknesses

Strengths

• Allows for higher MLSS concentrations than conventional activated sludge (CAS)
• Total separation of solids and ultrafiltration improves effluent quality - ability to meet stringent permits
• Smaller system size than CAS

Weaknesses

• More expensive to operate
• Need to have pretreatment to remove grit and other particulates that can damage membrane

The ability of membrane to physically separate solids from the effluent leads many operators to assume that filamentous bacteria and non-filamentous bulking that cause problems in secondary clarifiers are not important to MBR operation. While not as susceptible to problems with bulking as CAS, MBR systems can develop problems related to bulking organisms.

Membrane systems are monitored on:

• Transmembrane Pressure (TMP)- force required to drive water through the membrane pores
• Flux Rate - flow per unit area of membrane
• Permeability = Flux Rate / TMP

Both filamentous and excessive EPS (non-filamentous bulking) organisms can limit membrane effectiveness. By clogging pores and fouling membranes, bulking results in decreased flux rates and higher TMP. This fouling can be countered by increased membrane backflushing and cleaning. Both of which reduce membrane operational time - less effluent per surface area of membrane. In practice, we have encountered MBR systems where discharge limits due to bulking create expensive bottlenecks in the treatment process. 

Below is an example of non-filamentous bulking by a Thauera sp. was limiting flux rates in a MBR system. We helped this client develop a qPCR test for key problem organisms and worked on operational methods to reduce likelihood of diminshed flux rates. Note how one strain was responsible for reduced flux rates and monitoring this population was a key performance indicator.

Aster Bio’s molecular testing capabilities include running 16s rRNA to conduct a total bacterial and archaeal (when needed) census for wastewater systems. The use of high throughput sequencing allows us do dive into what is doing the work in a biological treatment system. It identifies both good and bad actors and gives early warning of potential problems. When we first introduce this technology in 2017, the challenge was speeding up test turn-around-time (you don’t want to wait 4 – 6 weeks for operational test data) and reducing test cost which was much higher than standard operational tests. We have improved our equipment and testing methods to get a 4 – 7 business day TNT and have reduced costs by over 50% since we started testing. Another challenge was making the MCA and other molecular tests useful to operations. Early reports included 30 pages of pie charts and multiple spreadsheets that took hours to look up microbes and interpret the results. We have also improved this by creating a single spreadsheet for summarizing the results and giving users access to a secure online database for tracking trends in their system.
 
For a bit of background, Microbial Community Analysis (MCA) uses genetic information found in all bacteria and archaea as a “bar code”. The bar code is then processed through wastewater specific microbial databases for identification down to the genus level. At first, this was admittedly an academic exercise as the data generated from a single test often includes 70,000+ reads and over 100 MB of data. Making sense of the data was one of our earlier challenges.

The MCA report
 
Functional Groups Summary
This is the quick glance portion of the report. Genera with known ecological niches and biochemical pathways are classified into the groups of most concern to WW operations. The functional group summary allows for easy time series tracking of population drift associated with time and any operational changes. Below is the Function Groups Summary table - with % of reads in each functional group.

Diving Deeper into the report
After the Function Group Summary, top genera at >0.1% of total reads are listed in a table with their known functions. This gives information on system microbial diversity. This list can be over 100 genera in length, but below is a short example of the table.

Another useful tool in the output is the Taxonomic Chart
As we run the raw sequences through the databases, the reads are moved to increasingly specific classification. We move from Kingdom in steps down to Genus ID. Some reads do not fit and get stuck at higher levels. This data is not discarded, we know the reads and can make inferences based on information gathered from other systems. Below is the Taxonomic Chart presenting how the sequences are classified from less to more specific.

Experience in WW application of molecular testing
As a wastewater technology company with an advanced molecular testing lab, Aster Bio specializes in interpreting and working with our customers to deliver benefits from these new monitoring tools. Every facility has different needs and objectives, we look at each application as a way to further explore the wastewater microbiome and improve test usefulness. If you are interested in learning more about MCA and other molecular testing options, visit our website at www.environmentalgenomics.com, call us at 713.724.0082 or drop us an email at info@asterbio.com. 

Biological Oxygen Demand (BOD) tests measure oxygen use by aerobic microorganisms growing on samples. In a sealed bottle, you measure oxygen consumption by a seed over usually 5 days under strict temperature control and darkness (to control for photosynthesis by algae). 

Things that can confuse test results

• Insoluble organics – COD and TOC tests pick this up. But insoluble organics do not usually cause BOD test problems.
• Recalcitrant compounds – bacteria don’t grow on these. This means that the test misses these compounds and they do not show up in the test.
• .In bottle nitrification by AOB/NOB cultures – ammonia and nitrite oxidation consume high levels of DO. So any nitrification can compromise test accuracy.
• Seed variation – using MLSS or primary solids can have variation from operations and environmental factors.

Tests used to check for variation in seed

• Seed Control Factor (SCF)
  • Too low and seed may not have enough microbes.
  • Too high and seed may have too much background organic
  • Target is 0.6 – 1.0 mg/L
• GGA – Glucose Glutamic Acid standard
  • Well known soluble organic compound for standard. Used by most heterotrophic organisms.
  • Uniform QA/QC check - we know what the end result should be
  • Target is 198 + 30.5 mg/L

Options on BOD Seed
Goal of all procedures is to reduce variation from test to test. One way to help with this is to use a consistent seed. While MLSS is often used, it can vary in composition due to influent changes and seasonal variation. Additionally, MLSS often contains AOB/NOB cultures – which may require a nitrification inhibitor to get true BOD5 results.

What about primary effluent as the seed? This can contain high levels of organic compounds and a blend of true anaerobic cultures and other enteric organisms that can be highly variable.

Commercial seed – oldest and most used being PolySeed which was approved by EPA in 1982. Advantages related to the PolySeed:

• Contains a fixed blend of microorganisms with each batch tested for SCF and GGA standards.
• Does not contain AOB/NOB cultures but there is a PolySeed NX with nitrification inhibitor for use if you suspect your sample has high levels of AOB/NOB.
• By having a shelf-stable, fixed blend of seed, you have reduced variation in the test procedure.

The methanogens found in anaerobic digesters are actually not bacteria but from the kingdom archaea. Archaea are very different from bacteria and are their own unique life forms found in a variety of environments. In anaerobic systems, it is the methanogens that produce methane and maintaining a stable population of methanogens is key to successful digester operation. But, methanogens also have multiple metabolic pathways that impact digester performance. 

Archaea Methanogenic Pathways:
Acetoclastic                   CH3COOH  CH4 + CO2
Hydrogenotrophic       CO2 + 4H2  CH4 + 2H2O
Methylotrophic             4CH3OH  3CH4 + CO2 + 2H2O
 
Bacterial Pathways in Digesters:
            Fermentation              Produces organic acids from complex biological polymers
            Acetogenesis               Continued fermentation to CH3COOH
            Homoacetogeneis       4H2 + 2CO2  CH3COOH + 2H2O
                                                   Metabolic rate at 3 to 10x faster than HM Archaea
             SAO                             CH3COOH  4H2 + 2CO2
                                                  Syntrophic with Hydrogenotrophic methanogens
 
Causes of increases CO2 (lower methane %) in produced gas

• Acetoclastic and methylotrophic methanogens both produce CO2 along with methane. With the acetoclastic making equal parts CO2 and methane.  In digesters dominated by acetoclastic organisms (especially Methanothrix from Aster Bio's data), we see lower rates of methane production.
• Overgrowth by homoacetogenic bacteria - these organisms convert H2 and CO2 back to acetate which favors acetoclastic methanogen growth. These organisms deprive desirable hydrogenotrophic methanogens of needed inputs for growth.
• Need for SAO bacterial populations that work directly with hydrogenotrophic methanogens in a syntrophic relationship where the bacteria provide the archaea with H2 and CO2 - the result is improved methane yields. 

Good articles on the SAO & Homoacetogenesis bacteria in digester performance.

• Deep insights into the network of acetate metabolism in anaerobic digestion: focusing on syntrophic acetate oxidation and homoacetogenesis​ - ​www.sciencedirect.com/science/article/abs/pii/S0043135420313075
• Syntrophy mechanism, microbial population, and process optimization for volatile fatty acids metabolism in anaerobic digestion - www.sciencedirect.com/science/article/abs/pii/S1385894722046162
• Novel syntrophic bacteria in full-scale anaerobic digesters revealed by genome-centric metatranscriptomics   www.nature.com/articles/s41396-019-0571-0/