D.O. meters report dissolved oxygen in the water phase of the aeration basin.  We know that in a flask, obligate aerobes such as AOB do very well at 2.0 mg/L DO.  Why is it that in many aeration basins the DO must be at least 3.5 mg/L for proper obligate aerobic organism growth?

I have seen two reasons why you may need to run higher than textbook D.O. residuals.

• DO probe placement – a single probe can have sampling error and can easily be checked by testing other points in the basin with a portable meter.
  
  
• DO in floc is different than in the water phase (where the probe is located) - floc is composed of microbes, EPS, non-soluble organics, and inorganics.  DO must penetrate the floc to reach microbes deep inside the floc.  Larger, higher EPS flocs can have anoxic/anaerobic zones inside.
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Do you have any other ideas why many systems require operation above 2.0 mg/L D.O. residuals in the aeration basin?

Aerobic microbes rely on oxygen to carry out their life functions.  Oxygen, being only moderately soluble in water, must be constantly added to aerobic wastewater treatment systems to ensure proper waste treatment.  Aeration energy use is one of the largest costs in running a treatment system, so finding the best aeration type is a key part of running and maintaining a system.

How oxygen is transferred into water
Oxygen dissolves into water at the air/water interface.  Oxygen from the atmosphere (20% O2) naturally enters water at the surface.  In most aerobic treatment systems oxygen transfer is done via air bubbles passing through the water (diffuser or jet type aeration) or through the mechanical action of surface aerators.  Key is the surface to volume of the bubble - the smaller the bubble the higher ratio of surface area for diffusion compared to larger or coarse bubbles.  

What we want aeration systems to do

• Provide efficient DO transfer
• Keep biological solids suspended
• Provide sufficient mixing to avoid short circuiting or dead zones
• Problem is you cannot have everything!
• Best solution may be combining multiple types into a hybrid system

Aster Bio uses molecular testing including Next Generation Sequencing (NGS) and qPCR to monitor the microbial populations in wastewater and other environmental samples.  With both technologies, samples require DNA extraction and purification before the actual testing starts.  To speed up sample processing times, Aster Bio has invested in a new robot to process samples in what was a time-consuming, labor-intensive step. 

With increased automation, we will be able to increase our sample throughput and give rapid results on microbial population composition to our clients.  The most common applications of Enviromental Genomics™ testing include:
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• Microbial Community Analysis (MCA) - a total microbial census of all bacteria present with information on functions or physical characteristics such as Ammonia Oxidation, Phosphate Removal, Bulking, Foaming, or Sulfur Reducing/oxidizing.
  
  
• AOB/NOB qPCR - with in-house developed primers, Aster Bio evaluates ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) in wastewater samples.  The qPCR results provide information well before effluent ammonia or nitrite residuals change.
  
  
• Bulking & specific filament - often customers find that key bulking or filamentous organisms appear during MCA testing. Aster Bio is now able to build rapid, accurate qPCR tests for monitoring these specific organisms with results being available in hours versus days.

 We often run OUR tests and record the oxygen uptake of a sample as mg O2/L/hr.  The key is to make sense of the respiration rate.  First, unless you run a constant MLSS or MLVSS, you should divide the OUR by MLSS in grams to get the SOUR which helps account for background solids impact on respiration.  Now with the OUR or SOUR numbers, you need to think about what the test is telling you about microbial health.
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• Sudden increase in OUR is normally see with high soluble BOD (organic) loadings.  If you run ATP tests, you should see an increase in free ATP (ATP in solution) which indicates rapid cell division or a move  to the left on the growth curve.
  
  
• A slow increase in OUR numbers can be from changes in total biological solids which is why SOUR calculation is important.  I have also seen increases in VSS relative to MLSS create a change in OUR numbers.  Other factors can be "fat slduge" which is sludge with adsorbed insoluble organics or high EPS which is simply food in storage. 
  
  
• What about a sudden drop in OUR numbers.  Not often seen in domestic wastewater, shock or toxic loadings can cause a sudden drop in biological activity or respiration rates.  In this case, you will see a drop in OUR and other signs of stress such as deflocculation, increased turbidity, loss of indicator protozoa, and loss of nitrification.  If the shock was due to a short term spill, you will see the low OUR replaced by a rapid increase in OUR as the surviving bacteria begin to multiply in log growth.

I recommend daily running of OUR.  This test uses exisiting equipment with no reagents or excessive time requirements.  You can also use influente spiked OUR to predict toxicity or biomass inhibition.  Key thing is to run the test frequently so you know what the normal OUR is for the system and enable you to see any significant change in microbial activity.

Often called Zoogleal bulking, non-filamentous bulking causes floc to become more viscous.  By holding more water and having a loose structure, the non-filamentous bulking MLSS does not compact, dewater, or even pass efficiently through MBR system membranes.  Typically identified by microscopic exam and India Ink tests, non-filamentous bulking is often not detected until it becomes a severe problem.  In the photo above, Ryan Hennessey performed a microscopic exam identifying significant non-filamentous bulking using India Ink with DIC microscopy to show the high EPS surrounding the cells (https://rhwastewatermicrobiology.com/) 

Organisms responsible for non-filamentous bulking
Most non-filamentous bulking events are caused by either Zooglea or Thauera genera.  When at normal levels, both Zooglea and Thauera are valuable for removing organics and produce a good EPS that builds floc.  Problems happen when they start to over produce EPS which can be caused by high organic acids, limited macro-nutrients, or insoluble organics.  The EPS acts to store organics for later use by the bacteria.  If they do not consume the stored "food" before being "fed" again, you see an increase in EPS.  While the F/M ratio helps identify the feeding conditions, using molecular testing to monitor actual population of bulking genera can also predict the likelihood of having a non-filamentous bulking event.  

Applications of controlling non-filamentous bulking with molecular testing

1. ​A MBR system was having problems with high transmembrane pressures requiring limited forward flows.  Microscopic exam revealed high non-filamentous bulking but only once it became a problem.  To investigate the microbial population, the facility ran MCA tests to determine what bacteria were present during both good and bad operations.  From the MCA data, a qPCR test was developed to monitor for the exact organisms responsible for the worst cases on non-filamentous bulking.  The qPCR results give rapid information about increases in problem organisms and can direct increased wasting is needed to prevent a full bulking event.
   
   
2. An industrial wastewater system often had problems with clarifier solids not compacting sufficiently and WAS sludge requiring increased polymer dose to dewater.  Using MCA and qPCR testing developed specifically for the system, we were able to find the safe operating range for the Thauera populations.  When levels increased above ideal levels, the facility increased wasting rates to remove the excess EPS before it became a bulking event that caused operational problems.  

In both examples above, Aster Bio has utilized Environmental Genomics™ tools to create a monitoring program for facilities with a history of non-filamentous bulking.   Molecular testing proves valuable as it provides direct measures of microbes in the system.  Molecular tests are extremely sensitive to changes in populations and should be used with microscopic exam.   Additionally, Aster Bio with an in-house molecular lab, provides fast turn-around at an attractive price for routine monitoring purposes.

Microbial Community Analysis (MCA) relies on uses 16s rDNA to provide a microbial census of all organisms in the MLSS. The resulting data includes over 100 GB of individual sequences that are then run through databases to give us the ID and relative frequency of what is present. The challenge has been in interpreting this complex data. To help with interpretation, we include in our reports a "Taxonomy Chart", which we find a useful. In fact, it's usually the first place that we look when reviewing data. However, the chart layout does require some explanation. 

Bacteria are classified in a hierarchical taxonomy, from the broadest categories to the most specific: kingdom, phylum, class, order, family, genus, species, and in many cases, subspecies or strains. Some times, an MCA is unable to identify a population to the genus level, but the higher (more general) taxonomic levels can still provide some information: for example, the Firmicutes, which contain Bacillus and Clostridium, generally contain either facultative or obligate anaerobes. High levels of Firmicutes may suggest low dissolved oxygen in a system.

Our taxonomy chart is similar to stacking a bunch of bar charts side by side, showing the relative amounts of each taxon present at a taxonomic level, but with a twist. The first column covers the kingdom. In the example below, this kingdom is Bacteria, and it represents over 99% of the sequences. The next level shows the different phyla present in the sample, with Proteobacterium being the most-represented phylum. The chart repeats this all the way down to the genus level.