- Isolate candidate microbes from environmental isolates, waste samples, or culture collections.
- Screen to ensure the candidates are non-pathogenic and classified as Bio Safety Level 1 organisms.
- Determine range of environmental conditions in which the strains grow. For example - pH, temperature, anaerobic growth, and nitrate/nitrite utilization.
- Growth on target organics and production of beneficial metabolites – this is where we see if it degrades problem compounds and forms desirable biofilm/floc. We aim to select the strains with best degradation capabilities and shortest-doubling time.
- Investigate the strain ability to be grown in industrial fermentation processes.
- Can the strain be preserved in shelf-stable forms. And conduct long term storage shelf life testing.
- Testing to ensure compatibility with other strains in the consortia.
- Lab testing on waste samples and compare growth to existing strains.
- Field testing to ensure lab results holdup in the real world environment.
- Introduce the new product.
Creating an effective bioaugmentation product (bugs) for use in wastewater systems in a multiple step process that involves screening multiple candidates and creating a decision matrix before selecting the ideal candidate for inclusion into a formulation. We spend significant amounts of time characterizing strains for environmental growth conditions and degradation capabilities. Additionally, complexities arise when you make a product with both spore forming and vegetative strains. In fact, many companies limit their product to spore forming microbes even if that means losing an large amount of metabolic diversity. Below, I have outlined the R&D process we use at Aster Bio (www.asterbio.com).
Bacterial communication in biofilms is more advanced than though - and what this means to environmental biotechnology
Researchers at University of California San Diego have found that bacteria in biofilms use ion channels to communicate with each other using electrical signals. This explains how a biofilm's outer cells slow growth and allow nutrients to reach the film's interior cells. In a biofilm, the exterior cells have access to nutrients but are exposed to potential toxic compounds and environmental hazards. Following a toxic event, the exterior cells that are severely damaged or dead are sloughed off creating turbidity and in big events increased effluent TSS. The undamaged cells inside the film rapidly replace the biopolymer matrix and reproduce to restore needed protection.
The research found that cells on the interior send electrical signals that reach the exterior cells via cell to cell signaling. The exterior cells slow reproduction and allow for transfer of nutrients to nurture growth inside the film. Remember biofilm is both found on fixed film (media systems) and suspended growth (as floc).
Biological wastewater treatment relies on the underlying role of bacteria and other microbes as "nature's recycling system" as the microbes covert organics into ever smaller compounds and finally into carbon dioxide, water, and trace elements. It is this recycling role that we harness in waste treatment to rid wastewater of potentially toxic compounds that could impact downstream water health.
As discussed in earlier posts, the biological treatment unit provides an "ideal" environment to encourage microbial growth. The goal being to maintain a highly active, extremely concentrated population of microbes that can rapidly metabolize (treat) influent wastes. What can derail this treatment process?
This brings me to the most frustrating (to me at least) source of problems in waste treatment. Simply, the lack treatment time in the biological unit. No matter how much you coddle the microbes - providing excess D.O., nutrients, increasing recycle rates, etc. Biological treatment of organics, especially recalcitrant, toxic, quasi-toxic or xenobiotic compounds, takes prolonged exposure to microbes for the multiple steps in biological transformation to occur.
So if you have a high COD wastewater with only a 4 hour residence time in the biological unit, you will get a reducing in highly soluble, easily degradable compounds. The more complex and less bioavailable compounds simply pass through or are incompletely metabolized. Sometimes the metabolites from initial degradation can be more toxic than the initial compound.
How much time is needed? This is where you do lab based treatability testing before designing a waste treatment unit. If you must build a unit with 2 - 4 hour residence time for industrial wastes, (Yes I often see this) - do not expect real biological treatment no matter what you do. You can "contact stabilize" where microbes adsorb some organics for later biodegradation in a digester. But most likely, you will have a simple physical treatment unit that aerates/mixes the influent only.
Biological waste treatment units are designed to provide an environment that promotes rapid microbial growth and metabolism. Our job in running the system is to ensure we provide the best possible growth environment, but what constitutes the ideal growth environment? I have a standard list that includes the following:
I often get questions regarding the potential impact of new waste stream on the biological treatment unit. After looking at reference materials on the compounds, we often do direct toxicity testing. The first test looks for inhibition of heterotrophic microbes. I prefer to use PolyTox test kits here. A quick 20-minute test that requires only a BOD bottle, stir plate, and BOD probe/meter; the PolyTox test used a known standardized biomass with the data being reduced oxygen uptake due to immediate inhibition.
Often the concern is the potential impact on ammonia oxidizing bacteria (AOB), which are much more sensitive and slower growing than heterotrophic microbes. Many compounds are highly toxic to ammonia oxidizing cultures including:
In order to determine the potential impact on ammonia removal, we can do a simulation using a concentrated Nitrosomonas culture. There are three basic toxicity screening tests we can do using a syringe of Nitrosomonas cell paste.
Recently I have been participating in an online discussion about Food/Microorganism (F/M) targets for a dairy wastewater treatment plant. The engineer wanted to know the target F/M ratio for dairy wastewater. Often I get this question with little supporting information such as: original system design and treatment targets.
When asked about F/M most consultants and engineers open a standard engineering text and give an answer from standard design numbers. I often will give a number from Metccalf & Eddy but indicate that this is just a starting point and influent makeup, temperature, pH, treatment targets, and a big "other factors" can require modifications on the number. For your reference here are a few of the target numbers:
In the dairy case that I first mentioned, I suggested that they look at their current F/M and check SV30, microscopic exam, and oxygen uptake rates to get a full picture of the system's biological state.
The easiest to interpret may be the microscopic exam. Use the following chart to determine how indicator protozoa, floc formation, and F/M relate. Note extended aeration operated at the far right side of the ideal F/M ratio section - this uses a lower F/M and higher mean cell residence time (MCRT) to both reduce solids yield and nutrient (N,P) control in systems with nutrient permits.
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