In wastewater and bioremediation, we usually deal with mesophilic environments. Mesophilic being defined as:

• Temperature 10 - 40 Deg C
• pH 6.0 - 8.5
• Salinity 0 - 33,000 ppm

When we move outside the above environments, the bacteria begins to change. For example, as temperatures increase over 43 Deg C, the bacteria populations change toward higher temperature mesophilic and finally to thermophilic organisms. Just how extreme can bacteria go? In the case of archaeal organisms it can get very extreme.

A recent paper found a new type of archaea living in a rift zone volcano with temperatures between 90 - 109 Deg C and a pH of 0. I don't think we would see this organism in most wastewater facilities, but it is a good read on life at the extremes.

https://www.nature.com/articles/s41598-019-44440-8

qPCR is an analytical test that can be used to estimate the amount of specific bacteria in a sample such as a wastewater MLSS sample. 

Different strains of bacteria each have unique genetic fingerprints encoded in DNA. qPCR (quantitative polymerase chain reaction) tests count these DNA fingerprints. Depending on how the test is run, a qPCR test can count all the bacteria, or just a specific subset, like the ammonia-oxidizing bacteria, present in a sample.
While qPCR can give absolute values (for example, 1.3 × 10^9 bacteria per milliliter of MLSS), the accuracy of the measurement depends on a lot of factors that are difficult to control. For this reason, we usually report relative numbers, which requires that we always run at least two tests on each sample.

The “Denominator” Test is for Total BacteriaFor every sample, we measure all the bacteria present in a sample using a standardized, reproducible protocol. This test measures a key gene, the 16S ribosomal RNA gene, that is found in all bacteria (and humans, too!). This number ends up being the denominator in all future calculations. Typical measurements of MLSS samples range from 1 × 10^6 to 5 × 10^7 copies per reaction.

The “Numerator” Test Varies
qPCR tests can be incredibly specific, able to detect and measure quantities of bacteria at the subspecies level! The test is also very sensitive: a properly designed test can measure 1 to 10 copies of the DNA fingerprint in a single reaction! And, samples can always be diluted to keep them within the upper limits of detection We routinely test for ammonia-oxidizing bacteria (AOBs), filaments, and zoogloeal bulking.
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The Results
​qPCR results are often reported as a fraction or percentage. For example, in a wastewater system with nitrification, the ammonia-oxidizing bacteria usually range from 0.1% to 1.0% of the total count. Lower numbers may indicate loss of nitrification, while higher numbers may indicate unusually heavy loadings of ammonia. Type 1701 filaments range from 0.002% to as high as 20%! 
While the relative numbers are useful, we find the trend in the numbers to be the real value. Each wastewater treatment plant is a little different, so the acceptable range for a particular microorganism differs from plant to plant. But, the trends within a single plant can reveal a slow increase in problematic filaments or foaming microorganisms to allow operators to take action before there is a crisis. Likewise, slow drops in nitrifiers can signal chronic toxicity before the entire population is lost.

Hyphomicrobium sp. form stalks with budding daughter cells during reproduction. It is the stalk extending from the biofilm that makes the Hyphomicrobium easy to see under a microscope. The daughter cells have flagella allowing for the Hyphomicrobium cells to swarm and colonize new areas. Hyphomicrobium in wastewater are associated with denitrification and are especially common in systems with methanol or other short chain alcohols.

So why do we commonly see Hyphomicrobium in systems without methanol in their influent? Stepping back and looking at the microbial community can give us the answer. Influent wastewater contains a big mix of organic and inorganic compounds. No one organism grows on all these compounds. Instead, we have an interaction among thousands of microbial forms. For example, a Pseudomonas may convert an insoluble fatty acid (grease) into soluble short-chain fatty acids which is a byproduct of their metabolism. Instead of short-chain fatty acids building up, bacteria such as Hyphomicrobium and Paracoccus begin to grow on this Pseudomonas waste product. This is an example of syntrophy among various organisms. Syntrohphy is also a reason why we cannot identify all microbes in wastewater MLSS using plate counts.

Here are some facts about Hyphomicrobium sp.:

• Aerobic and capable of using nitrate/nitrite as an alternative electron acceptor
• Uses methanol and other short-chain alcohols as a carbon source
• Under anoxic conditions Hyphomicrobium is a high rate denitrifier
• Biphasic life cycle – flagellated swarm then sessile where we see the stalk
• Studies have found the following pathways in various Hyphomicrobium isolates
  • Degrade PAH
  • Dichloromethane
  • Formaldehyde
  • Dimethyl sulfide & methanethiol oxidizing sulfide portion
  • Amines (MEA, DEA, TEA)

NITROSPIRA SP
Bergey’s Manual
Nitrospira make up the key nitrite-oxidizing bacteria in most wastewater treatment systems. While a lot of the literature highlights Nitrobacter, Nitrospira generally grow best on (very) low levels of nitrite found in WWTPs. Nitrobacter, on the other hand, likes higher nitrite concentrations and is easier to grow in the lab.

• Gram-negative
• Aerobic
• Some strains can use nitrate as a terminal electron acceptor

And, surprisingly, some strains of Nitrospira are Complete AMMonia OXidizers (COMAMMOX) capable of both ammonia and nitrite oxidation. In some WWTPs with significant ammonia nitrogen loadings, we have seen high levels of Nitrospira and extremely low levels of Nitrosomonas or other known ammonia-oxidizing bacteria. Using polymerase chain reaction assays (PCR), we can identify COMAMMOX strains present in some MLSS samples.

Short post continuing concepts with MLSS that does not compact in settling tests or in the secondary clarifier.

Poor settling and compacting sludge results in high SV30 and SVI. When sluddge does not settle or compact, the secondary clarifier bed depth increases. This results in less solids conentration in the RAS & WAS lines. What does this mean?

• Return line has less MLSS than is desirable. To maintain F/M ratio, you would have to return more flow via the RAS line.
• Hydraulics can become a problem if you increase return flows to maintain the F/M ratio.
• Waste sudge going for disposal has more water. Making dewatering more difficult or creating digester capacity issues.

While using our Microbial Community Analysis testing system on wastewater treatment system samples, Aster Bio has found that Nitrobacter (NOB) are not nearly as common as thought. Instead, the predominant NOB culture has been Nitrospira. Here is a bit more about Nitrospira (and forgive the filament photo - it was great for the blog header but is not Nitrospira)

Bergey’s Manual
Nitrospira make up the key nitrite-oxidizing bacteria in most wastewater treatment systems. While a lot of the literature highlights Nitrobacter, Nitrospira generally grow best on (very) low levels of nitrite found in WWTPs. Nitrobacter, on the other hand, likes higher nitrite concentrations and is easier to grow in the lab.

• Gram-negative
• Aerobic
• Some strains can use nitrate as a terminal electron acceptor

And, surprisingly, some strains of Nitrospira are Complete AMMonia OXidizers (COMAMMOX) capable of both ammonia and nitrite oxidation. In some WWTPs with significant ammonia nitrogen loadings, we have seen high levels of Nitrospira and extremely low levels of Nitrosomonas or other known ammonia-oxidizing bacteria. Using polymerase chain reaction assays (PCR), we can identify COMAMMOX strains present in some MLSS samples.

We have more on MCA identfied interesting microbes at https://blog.environmentalgenomics.com/