With the increasing numbers of warnings of pharmaceutical and personal care products (PPCP) passing through conventional wastewater treatment plants and entering the environment it is interesting to see a study on how constructed wetlands for polishing treated wastewater impact these residuals.
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Back when I started working in bioremediation and wastewater treatment, much research was conducted on using various white-rot fungi to biologically treat resistant xenobiotic compounds and decolorize paper mill and textile wastewater. As I have never seen large scale application of this technology, I wanted to see if there was any new novel research and ways to improve on early methods.

Background on P. chrysosporium
Like other white-rot fungi, P. chrysosporium is a basidiomycete fungi which produces a number of complex extracellular enzymes that breakdown lignin. In nature, the white-rot fungi are one of the primary decomposers of wood. What is most interesting for waste treatment is the ability of the lignin peroxidase and glyoxal oxidase to act on other pollutants (non-specific enzyme activity). 

Early test involved using white-rot fungi to decolorize pulp mill effluent where lignin creates highly colored water. In doing bench tests, researchers found the fungal enzymes also dechlorinated some of the halogenated organics found in chlorine  bleached pulp wastewater. This started a whole new area of research into using white-rot fungi to bioremediate such  compounds as:

• DDT & Lindane
• PCP (Pentachlorophenol) & Creosote - wood preserving wastes
• PCBs (Polychlorinated biphenyls)
• Dioxins

While the white-rot enzymes did initiate decomposition of many recalcitrant and chlorinated compounds, the challenge was how to get the enzyme to the substrate. Work on producing enzymes off-site and adding to contaminated soils and water proved expensive and has not been commercialized to any extent. The other option was to grow the white-rot fungi on wood chips and mix with the soil or water to be treated. While some sites were treated in this manner, it has never seemed to enter wide usage.

Revisiting the use of white-rot
With increased focus on decoloriziation and degrading many trace pharmaceutical products in wastewater, it may be time to reevaluate white-rot. Since target waste treatment is via a cometabolic pathway, we can use a relative inert carrier to hold/feed the white-rot cultures. The enzymes produced by the white-rot organisms can then cometabolize many of the problem trace xenobiotics.

Key items for research are:

• Finding an ideal immobilizing matrix for the fungi - alternative to wood chips
• Discover ways to optimize extracelluar enzyme production through carrier, nutrient, or environmental changes
• Evaluate how to put a biofilter with white-rot fungi to polish biologically treated waters to remove trae compounds

​Anammox (anaerobic ammonia oxidation) has received much attention in the past 10 years as it allows for lower utility costs over the more common practice of aerobic ammonia removal (nitrification). The reactions are as follows:

While the first step in ammonia removal is the same in both, the second step is biological short-cut that simplifies by converting nitrite and ammonia directly into nitrogen gas – thereby avoiding the denitrification step needed to meet modern total nitrogen permits.

While very successful anammox based systems have been constructed, we still do not really know much about the organisms that power the anammox process. So far what we know is:

• The microbes have a long generation time (slow growing)
• Grow in mixed culture with other organisms
• Form granules similar to that found in anaerobic digesters
• Possess an anammoxosome (membrane structure that actually does the conversion)

Data on the organisms is difficult to obtain as they are not readily cultured on standard microbiological media and the slow rate of growth. Over the past few years, researchers have evaluated anammox unit samples under various enrichment scenarios that make variation in the microbial populations. This involves varying any one or more environmental conditions or inputs to see what happens to the population as a whole. The testing then involves taking samples for metagenomics testing to determine what DNA is present in the biomass – thereby determining the organisms present and what relative frequency.
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The results of the results of the DNA sequencing have made the anammox puzzle even more interesting. Instead of typical bacteria, the anammox granules have a very unusual genome. They do not have many genes considered essential for bacteria. The researchers have called anammox populations the equivalent of “Microbial Dark Matter” – where much is yet to be learned.

​Last week, I was driving east of Houston and saw a survey crew examining the cap on the San Jacinto Waste Pits. These pits were used in the 1960s to dispose of paper mill bleaching waste which contained dioxin and other chlorinated organics. Over time, the pits were abandoned and the former barriers between the river and the landfill (pits) subsided and contaminants were being released from the site into the San Jacinto River and nearby Galveston Bay. In 2008, the site was give Superfund status by the EPA and an emergency cap was installed to contain the waste. As you can a see in the photo below, containing the waste in a pit located in a flood plain by the river is not a long run solution. So far the most proposed option is to excavate the waste and transport to incineration. Besides being expensive, excavation and transport could lead to more risks of exposure. So, this brings me back to thinking about the use of Zero Valent Iron (ZVI) or other metal and the ability of metals to dechlorinate many recalcitrant hydrocarbons.

Yesterday, I was looking under the microscope at a wastewater sample an noticed several suctorians on my slide. Not mentioned nearly as often as stalked or crawling ciliates, suctorians indicate generally good water quality with floc formation. If you don't recall much about a suctorian, they look like a pin-cushion on a stalk (at least that is the first call about one that I got from a customer years ago).

Anyway, suctorians anchor themselves to floc much like a stalk cilaite. "Spikes" extend from the organism into the water instead of the cilia seen on stalk ciliates. Suctorian feeding makes them one of the most interesting wastewater protozoa. Say a free swimming ciliate or flagellate happens to come  into contact with the suctorian's spikes. It is immediately immobilized by the spike and the ciliates cytoplasm is consumed by the suctorian. If you see this happening under the microscope, it makes excellent video.  

So what does having suctorians present in wastewater mean for biomass health?

• Sufficient D.O.
• Floc is forming as needed in Activated Slduge
• Toxic substances or other shock loadings are not present
• Soluble BOD5 has decreased leading to a mature biomass in decline phase growth

EPS are the complex high molecular weight compounds that are excreted by bacteria building floc or biofilms. The aggregation and adhesion of cells occurs once cellular division slows and the EPS builds on the cell surface. The mixture consists of polysaccharides, proteins, humic substances, and nucleic acids, with the majority being made up of polysaccharides and proteins. Key point here is you need an appropriate amount of EPS to get floc or biofilm yet too much EPS with entrained water can lead to viscous or zoogleal bulking.

​Research has found EPS composition varies with:

• ​​Carbon sources (what chemicals are present in BOD or COD)
• C:N:P ratio (key cause many bulking events)
• D.O. - higher DO makes for better adhesion & capsular EPS
• Temperature
  
• MCRT & HRT
• Growth stage (F/M ratio)
• Solution chemistry (pH, ionic strength, divalent cation)
• Toxic (biocides, heavy metals, CN, Phenol)

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​The study presented in Nature, evaluated the impact of salinity on cell adhesion and floc/biofilm formation. High Na+ can be a problem. While Na+ under 10 grams per liter has a negative impact on EPS formation, when you move from 10 g/L Na+ to 20 g/L Na+ that amount of EPS drops by 50%.

​The study then went into new territory by extracting the EPS from municipal and hypersaline wastewater biomass. What they found was the biomass manufactured more total EPS in the hyper saline environments where the EPS help protect against osmotic pressures caused by high Na+ concentrations. While the hypersaline had more total EPS, it has a different composition than municipal. The diversity of compounds in the EPS was lower in the hypersaline wastewater and the EPS was not as closely associated with cell aggregation/adhesion.

​Also both wastewaters had anerobic/anoxic tanks. What is interesting here is the EPS was not associated with aggregation. Apparently, DO is very important to give us the amount of aggregation needed for bioflm/floc formation.

​Here is a link to the Nature article if you would like to go deeper into the research. 

http://www.nature.com/articles/srep26721