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.

Did you know that microbial populations in wastewater are constantly changing? F/M or influent organic makeup and concentrations are a major influence as to what microbes are present. Other factors influencing microbial populations include temperature, pH, D.O., inhibitory compounds, and macronutrients.

An underused tool in wastewater treatment is to check your system against the growth curve. A perfect way to watch the growth curve is in an aerated lagoon system since you move from lag or log phase growth all the way to endogenous respiration. 

Typical Aerated Lagoon Microbial Populations
Near the influent, you have high concentrations of organics with low microbial populations. This favors r-strategists - microbes with high growth rates. Other growth pressures include the presence of inhibitory compounds or tough environmental conditions. Using Microbial Community Analysis testing, we have found that many of these organisms are the most evaluated genera used in laboratory experiments.

By the mid-point of the example lagoon, soluble BOD5 is 80% removed and organisms that have new growth strategies become the dominant organisms. You notice an increase in EPS and cells forming floc. Indicator protozoa begin to favor ciliates and other more advanced forms. This is also where you have AOB & NOB populations becoming more active.

Moving through the system to the polishing pond, almost all soluble BOD5 is removed and floc is settling into a sludge layer where endogenous respiration occurs. 

Comparing a Lagoon to Activated Sludge
While you can move along the growth curve as water moves through a lagoon system, Activated Sludge with recycle MLSS tend to operate in a section of the growth curve. In conventional ASU systems, you target Decline Phase growth for maximum floc formation, BOD5 removal, and nutrient removal. Extended aeration systems and modern high MLSS (low F/M) systems also have endogenous respiration where cells lyse and solids yield decreases.

Using the MCA testing on ASU systems, we can identify the ideal mix of microbes for a given system. The organisms mix changes with influent and operational changes, but knowing the microbial mix allows for you to better identify your location on the growth curve and can help improve operational decisions.

My next blog posts will be from example MCA testing and show how microbial populations are impacted by system type and operations.

From an operational and microbial ecology perspective, BNR wastewater treatment systems have complex requirements. Aerobic, anoxic, and anaerobic zones must be optimized for both ammonia oxidation, denitrification, and phosphorus uptake. In addition to monitoring redox potentials (ORP), D.O., OUR, and F/M, monitoring the slower growing but vital niche organisms helps with operations. Since the most population changes happen gradually in BNR systems, we decided to test microbial populations using Microbial Community Analysis (MCA) which is a total microbial census using 16s rRNA. With the 16s rRNA serving as a microbial barcode, full information on all microbes in the MLSS with relative frequency is produced to give information on microbial populations when the system is running well and when efficiency declines.

Key Organism Groups to Monitor

• Ammonia Oxidizing Bacteria (AOB) & Nitrite Oxidizing Bacteria (NOB)
• Denitrifying bacteria
• Phosphorus Accumulating Organisms (PAO) and Glycogen Accumulating Organisms (GAO)
• Filamentous bacteria - filaments are favored by many of the conditions in BNR operations and should be monitored for overgrowth.

 Tracking the Key Functional Groups
​For ease or reading, the MCA findings are reported in a summary table.

We also include more data in a Sankey type chart that gives information on the organisms in the sample as you move from Kingdom (bacteria) to the more specific Genus level of ID. This chart involves more interpretation but is a much deeper look into population changes over time. Below is a rotated chart for use in this post, it is much easier to read in the spreadsheet output!

While the output looks complex, it has proven easy to use by everyone in the facilities. By directly measuring populations, MCA identifies drifting populations and the genera associated with best operations. 

Biological wastewater treatment has been a hot bed of innovation in the past ten years. Much of the innovation is being driven by new permits, a desire to recycle water, lowering energy use, and making systems more efficient. Below are some of the interesting projects that I have been fortunate to see in application.

Molecular Testing​​ 
Since I started working with wastewater molecular testing about seven years ago, it has developed from a curious novelty to a powerful operational control tool. Molecular or DNA testing can see slight changes in important populations including AOB/NOB, PAO/GAO, Filaments, Nocardia, Methanogens, and SRB. An example with AOB/NOB is a warning of nitrifier stress before effluent ammonia starts to increase. This technology continues to develop and will become even more useful as our knowledge increases.

Nanobubble & Microbubble Aeration
Maintaining sufficient D.O. in aerobic treatment systems is always a challenge and expensive. The development of new extremely efficient systems for oxygen transfer coupled with resistance to fouling is going to lead to improved aerobic systems. In field testing, I have been amazed with how much D.O. can be maintained and transferred while using less power than standard diffuser systems. 

Granular Sludge
Current systems rely on suspended floc or biofilms for treatment. First seen in anaerobic digesters, granular microbial aggregates can also develop in aerobic treatment systems. Key benefits are:

• Compact footprint as you maintain much higher levels of biomass in the system. 
• Having Aerobic, Anoxic, and Anaerobic zones within the granule - allowing for simultaneous nitrification/denitrification in one treatment zone.
• Fast settling rates reduce likelihood of hydraulic washout.
• Appears to be adaptable to existing systems.

Nitrogen and phosphorus from non-point sources have major implications for hazardous algae blooms. In natural low nutrient waters, algae provide both oxygen and primary food source for aquatic life. As we increase nitrogen and phosphorus concentrations, the delicate balance between algae varieties, protozoa, metazoa, and higher aquatic life becomes disrupted.

An interesting solution that may have application to waste water treatment in locations with available land is detailed in an article from the University of Illinois College of Agricultural, Consumer and Environmental Sciences published in Science Daily. Researchers detail how using wood chips in flow channels can remove much runoff water nitrogen by converting it to nitrogen gas. 

https://www.sciencedaily.com/releases/2021/04/210415170716.htm​