Most wastewater treatment theory sees each section or segment as having homogenous ecological environments. Instead, you find that environmental variables change with physical distance from the floc surface or depth in the biofilm. For microbes in activated sludge floc, distance from the floc surface also changes D.O., pH, nutrients, exposure to both organics and potentially toxic organics. Organisms deeper in the floc have a more uniform environment, but have less D.O. and access to soluble organics than floc surface organisms.

In addition to physical location in the biofilm/floc, you also need to consider how biological unit conditions can impact floc composition. Higher stress levels (pH, temperatures, toxic compounds, etc) encourage the production of protective extracellular polymer substances (EPS). The EPS acts as a permeable barrier between the microbial cell walls and the external environment. While necessary for building the floc aggregates, EPS can also cause non-filamentous bulking, problems with dewatering, or weak floc structure. Therefore, changes in EPS composition or quantity also changes the ecological conditions for organisms inside the floc.
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While a system may be aerobic with D.O. levels measured at above 3.0 mg/L throughout a basin, the underlying biomass can have organisms experiencing aerobic, anoxic, and anaerobic environments. This variation in environmental conditions within biofilms requires us to think about environmental micro-niches. This explains why 16sRNA genomic testing often finds obligate anaerobic cultures in “aerobic systems”. Or how long sludge ages in industrial wastewater treatment can have anammox cultures. If you observe unusual behavior in biological waste treatment systems, you may want to look at potential micro-niches for the explanation.

  Over the past several years, Aster Bio has used advanced DNA testing including metagenomics, 16s rRNA and qPCR. We have found the technologies have given better information than traditional microbiological testing (plate counts, stains, etc) for many of our daily activities. That left us wondering, could Aster Bio reduce both the time and cost of running molecular (DNA) tests. Through over three years of work, we have reached a point where molecular testing is now a practical monitoring and troubleshooting tool for wastewater operators. Here is how we have used Environmental Genomics™ testing results:

• Identify changes in the MLSS microbial mix following any change in a system
• Find key organisms associated with "good" operational efficiency
• Evaluate what is actually going on in the biomass - for example, how low DO and anaerobic organisms were found and growing in an "aerobic" basin
• Discover microbial candidates for addition following shock loadings or washout (custom bioaugmentation products)
• In biological nutrient removal (Phosphorus, Ammonia, Nitrite/Nitrate, and Anammox processes) - we can look into the microbial mix to ensure the correct cultures are thriving.

For more information on Environmental Genomics testing 
http://www.asterbio.com/environmentalgenomics/
or email us at
info@asterbio.com

We do F/M calculations with either MLSS or MLVSS standing in as "microbes". However, both MLSS & MLVSS contain more "other" than actual living microbes. Depending upon the system, the living microbe portion of MLVSS can have a range from <10 to over 20% with younger sludge ages having a greater percentage as living, active microbes.

As ammonia has a high oxygen demand and potential toxicity to animals in receiving streams, ammonia has long been permitted in wastewater treatment plants Additional concerns about nitrite and nitrate creating algae blooms has created a need for completing the nitrogen cycle by reducing nitrate/nitrite concentrations via denitrification (producing nitrogen gas. Research over the past few decades has increased our understanding of the various bacteria and archaea associated with the nitrogen cycle. For reference, I will break out the organisms into their kingdom and ecological niche:

• Ammonia Oxidizing Bacteria (AOB) convert Ammonia --> Nitrite
• Ammonia Oxidizing Archaea (AOA) convert Ammonia --> Nitrite
• Nitrite Oxidizing Bacteria (NOB) convert Nitrite --> Nitrate
• Anammox - Nitrite + Ammonia --> Nitrogen Gas
• Heterotrophic (Denitrification & Nitrification) 
  BOD5 + Nitrate/Nitrite --> Nitrogen gas (oxidized nitrogen as alternative electron acceptor)
  Ammonia/Nitrite --> Proteins, oxidized nitrogen (not primary energy source for microbes)
  

Wastewater treatment plants face increasingly stringent permits requiring removal of nitrate, nitrite, and soluble phosphate from effluent flows. Often biological nutrient removal systems are extensions on the conventional activated sludge system. Nitrate/nitrite removal is accomplished by adding an anoxic zone following the aerobic treatment section. For proper nitrate/nitrite removal, the anoxic basin much have sufficiently low redox potentials and soluble BOD5 (for the microbes to consume while using nitrate/nitrite as an electron acceptor). If biological phosphate removal is required, the system must have an anaerobic influent section with sufficient organic acids to encourage PAO growth. The anaerobic section must have sufficient organic acids and no nitrate which can favor non-PAO organisms. Besides the need for soluble organics in the anaerobic and anoxic sections, operators must balance flows to maintain residence times and monitor for the growth of problematic microbes.

Given the complexities of operating a biological nutrient removal system, research on creating alternative treatment technologies has been a priority. One of the promising technologies is aerobic granular sludge(AGS). While granular anaerobic systems have existed for years, aerobic granules are new to wastewater treatment. In nature, microbes form diverse communities in the form of biofilms (or floc in activated sludge). As the microbial community matures, distinct ecological zones form inside the film. The film surface has free oxygen present, favoring aerobic organisms. In the aerobic zone you have a highly diverse collection of microbes including ammonia oxidizing bacteria (AOB). Moving deeper into the biofilm, an anoxic zone exists. In the anoxic zone, free oxygen is not present and bacteria work by using alternative electron acceptors such as nitrate/nitrite. Given ammonia oxidation in the aerobic surface layer, the anoxic zone bacteria make use of nitrate/nitrite produced in the aerobic layer. In mature granules, we also see the presence of anammox bacteria that utilize ammonium (NH4+) and nitrite (NO2) to make dinitrogen gas (N2). Anammox microbes are interesting in that they reduce oxygen requirements for ammonia removal while also avoiding the need to remove nitrate/nitrite in a separate step. At the core of the granule, or deepest section of a biofilm, exists a true anaerobic zone. This section utilizes soluble organics and contains both PAO that instead of using aerobic conditions to uptake phosphorus utilize nitrate/nitrite present in low concentrations at the deepest part of the granule/biofilm.

The key part of aerobic granule systems is the ability to build a fast settling granular biomass. This has been accomplished in SBR (batch reactors) where operators can control operational parameters including mixing, aeration, and residence time with maximum flexibility. In practice, granular systems have performed will for treatment, but may require a tertiary clarifier to remove residual solids that come from sloughing biomass.

.Environmental Genomics™ technology combines molecular testing using advanced metagenomics technologies including 16s rDNA, qPCR, and full metagenomic sequencing. This technology looks directly into the genetic makeup of the biomass and gives direct insight into what is happening at a genetic level. Recent improvements in sequencing technologies allow for cost-effective and timely analysis that proves valuable for monitoring wastewater treatment systems in achieving effluent permits while controlling treatment costs. 

At Aster Bio we have been using metagenomic testing for several years and building a database on environmental cultures found in wastewater and soil remediation sites. We are now ready to work with our customers to implement metagenomics testing as a routine monitoring technology. For more information on Environmental Genomics, contact Aster Bio by email (info@asterbio.com) or phone 1 (713) 724-0082