Many of us in industrial wastewater have experienced a toxic shock event. In most cases, toxic shock is noted by loss of nitrification and deflocculation (turbidity/floating solids). Today, I will work thorough my process for identifying what caused or is causing the upset. 

1. Remember "toxicity" is not a thing - you must identify what caused loss of biological activity and steps to prevent it in the future.
2. Do not accept that "nothing changed" - something did change as evidenced by change in biomass activity.
3. Look into influent and process flows - instead of running random outside lab tests, identify most likely candidates for upset. This is a thinking exercise - look at past events or operator notes.
4. After identifying potential bad actors, you can start analytical testing. We have options tailored to specific industries. For example, Aster Bio uses a battery of petrochemical wastewater tests to identify levels of common bad actors in refinery wastewater. Using GC/MS and analytical tests, we can identify and quantify many "bad actors".
5. Fixing the problem - since we cannot hold back water for extended periods, we usually use slowing forward flows, diversion of high strength wastes, additional oxygen, and bioaugmentation (or bugs). Note that the response includes multiple activities - this is where consultants can help bring new technologies or thought processes.
6. After fixing and identifying the cause - develop monitoring and early warning abilities to meet the problem flows before acute toxicity. We can use many tools from step 5 above to prevent a major biological unit upset.

I like it when a facility looks under the microscope daily. Even if you do not have a high dollar phase contrast microscope, you can make valuable observations with they standard light microscope that is common to many high school classrooms. But what are you looking at when you observe samples at 100x, 400x, and even 1000x magnification? Today, I'll cover what you see when using a microscope.

First, the "bugs" we note using the microscope are mostly single celled protozoa and a few multicellular lifeforms. The bacteria - or the actual workers - can be seen as part of the floc or as very, very small particles in the water. Some bacteria can become filamentous in form or are large enough to be seen with microscope, but most even at 1000x appear as small rods or spheres. 

We rely on the protozoa and higher life forms as indicator organisms for the underlying bacteria populations. For example, stalk ciliates are only present and active when there is sufficient dissolved oxygen and low levels of inhibitory or toxic compounds. This just happens to be where we are in decline phase growth or target F/M for most wastewater treatment plants. Multicellular forms such as rotifers, worms, or tardigrades appear even further along the F/M curve and can indicate old sludge or too low an F/M where you are carrying too much dead or inactive biomass.

I recommend looking under the microscope daily for the following:

• Floc size & density
• Filaments if present and estimate their relative abundance
• Protozoa present and in what abundance
• Multicellular life forms and abundance
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Next Generation Technology for Biomass Monitoring
While I use the microscopic exam daily, I also like to run newer molecular testing that looks directly into the floc's microbial community. With high throughput sequencing, we look at DNA in the system and find our which microbes are present and at what % of total biomass reads (identifying DNA segments). This is a total microbial census of the MLSS and is good to run when establishing a baseline population database, making operational changes or on a quarterly basis for tracking long term changes. Following Microbial Community Analysis (MCA), we identify key microbes that are most important for good treatment. We can then use qPCR technology to track these specific microbes. qPCR is faster, highly quantitative, and cheaper than the full microbial census (MCA). 

Microbes consume dissolved oxygen (DO) when growing on wastewater pollutants.  The rate at which the microbes consumer oxygen is the respiration rate. During periods of high loadings the respiration rates increases as the microbes become more active and cellular division rates increase.

OUR rates increase in response to higher soluble organic loadings  (BOD5), faster microbial division (low F/M), and following upset conditions when microbes are moving back towards stable population - this is log phase growth.

Another facet of OUR testing includes a potential for a drop in respiration rates due to acute toxicity where previously respiring microbes are inactivated (killed). I have seen this with phenol, cyanide, and tall oil releases in industrial wastewater. 

So any large change in OUR rates should be investigated. If you are changing MLSS concentrations, you should standardize the OUR by using the SOUR calculation (divide OUR by MLSS or MLVSS in grams).

Here is a link to the OUR test protocol that I have used for the past 25 years. 
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Being both deadly in enclosed spaces and a nuisance at even low levels, hydrogen sulfide is among the most problematic of compounds in wastewater treatment. Instead of covering the whole geochemical sulfur cycle, I want to look into the wastewater specific cycle that converts benign sulfates and sulfur into the problematic reduced sulfide species. Instead of graphics, I want to detail each form of sulfur found in wastewater.

• Sulfate - one of the most common forms of sulfur found in the environment. Sulfate is used by plants and is an important micronutrient for life.
• Sulfur - the yellow powder or rock form of sulfur. 
• Reduced Sulfides - often seen as S= or in analytical tests as Total Reduce Sulfides. This form of sulfide can bind with iron and give the dark gray to black color seen in septic wastewater. Sulfides are more soluble in water with a higher pH. Reduced sulfides in wastewater have a high chemical oxygen demand and are toxic to Ammonia Oxidizing Bacteria (AOB) that are required for ammonia removal.
• Hydrogen Sulfide - a corrosive, highly toxic gas, H2S is released from water into the atmosphere as pH drops in wastewater. (This is why one of the collection system odor control methods is to increase sewer line pH).
  
  

Now for the microbes involved in wastewater sulfur cycles:

• Sulfate Reducing Bacteria - these bacteria under anoxic conditions use sulfate as electron acceptor to degrade organics. If you have oxygen or nitrate in the system, sulfate reducing bacteria are outcompeted by harmless bacteria that obtain more energy using oxygen or nitrate as an electron acceptor.
• Sulfur Reducing Bacteria - these chemotrophic bacteria obtain energy by reducing sulfur into sulfide. In most wastewater, these are less common than the Sulfate Reducing Bacteria that use sulfur as an alternative electron acceptor. You would find these organisms in mines, subsea volcanic vents, hot springs, and oil formations.
• Sulfur Oxidizing Bacteria - chemotrophic bacteria with the metabolic ability to convert reduced sulfur species into sulfur and sulfate. While some species can use nitrate as the electron acceptor, most sulfur oxidizers require oxyen as the terminal electron acceptor. If taken to conclusion, you produce sulfuric acid (H2SO4). These are the well documented sewer corrosion bacteria that can form if a vapor phase H2S concentration reaches sufficient levels. In wastewater, Sulfur Oxidizing Bacteria (SOB) are vital to removing reduce sulfides. This does require alkalinity to buffer the system from pH drop. Common genera of wastewater SOB include Thiobacillus, Anwoodia, Paracoccus, and Thiosphaera.
  
  

Solutions to sulfide generation in collection systems, equalization tanks, and headworks.

• Sulfides can be kept in water phase by increasing pH, adding ferric or sulfide scavengers to the collection system. This is a quick fix, but does not solve generation of sulfides in anoxic/anaerobic zones.
• Adding alternative electron acceptors to prevent growth of sulfate reducing bacteria - common additions include nitrate, liquid oxygen, and hydrogen peroxide. All work by giving a higher energy electron acceptor than sulfate/sulfide for bacteria in the system.
• Using a comprehensive control program to prevent sulfide formation. This uses monitoring/testing to find zones where redox potential favors the growth of SRB. To combat the SRB, you add the best alternative electron acceptor based on the location and environmental conditions. We have also found it beneficial to add microbes capable of both using the alternative electron acceptor and oxidizing reduced sulfides. In Aster Bio's products we favor Paracoccus and Thiosphaera as they grow well from 5.5 - 8.5 pH - unlike most Thiobacillus that thrive at pH <5.0 where you get significant H2S formation.

Sulfides are a problem in wastewater. Unless you have an influent source of reduced sulfides, most sulfide is formed in water under anoxic/anaerobic conditions that favor SRB growth. The best solution for sulfides forming in the system is to disfavor the growth of SRB. How to accomplish this requires a system survey and determination of which solution best fits your situation. But before selecting a solution or choosing a single solution for a complex system, do a full survey of Redox (ORP), pH, Total Reduced Sulfides, and H2S.

Activated sludge systems operate by moving a suspended biomass (MLSS) through an aerated basin and then separating solids for recycle back to the aeration basin.  This process was invented in the early 20th century for treating wastewater and many variations on the process exist including contact stabilization, extended aeration, batch reactors, oxidation ditches, and pure oxygen systems.   A key operating parameter has been the Food to Microorganisms (F/M) ratio.   F/M is traditionally calculated using MLSS or MLVSS as the M and BOD5 as the F.   Key is to be consistent and always know what you are using to calculate F/M and identify the best F/M ratio for operations. 

Now we move to biofilm type systems versus suspended growth.   Here we have tricking filters (old but still good), MBBR, and fixed film media.  All relay on a biofilm which is actually just floc grown in attached form.  Calculated F/M in this system is difficult since the biomass is attached to media.  So we operate any biofilm based system on loading per unit of surface area - this can be BOD5/square meter or Ammonia/square meter.   Again just be consistent in calculating.

The key benefit of a biofilm vs suspended growth system is the ability to hold more biomass than comparable activated sludge units which rely totally on solids separation to keep biomass in the aeration tank.  Of course this advantage is not present for all influents.  For example, systems with significant influent oil & grease can foul (coat) the media which can be a big problem.   

Nitrosomonas species also known as Ammonia Oxidizing Bacteria (AOB) have always been discussed as obligate aerobic chemoautotrophs. Under aerobic conditions, the Nitrosomonas convert ammonia  to nitrite while using carbon dioxide  and carbonate as a carbon source. 

I have recently read multiple peer reviewed papers that document the ability of Nitrosomonas to utilize various organic compounds as an energy source with nitrite as an alternative electron acceptor - producing N2 gas as a final product. This ability to use their waste product from aerobic reactions as an electron acceptor reveals how Nitrosomonas can survive in systems with substantial anoxic periods.

Here is a link to a 2009 paper with more details:
 https://www.ncbi.nlm.nih.gov/pubmed/19452213

Easily seen using a phase contrast microscope, filamentous bacteria provide the structural support on which floc forming organisms grow.  Without filaments, floc tends to shrink in size and be more susceptible to sheer.   Problems arise when filaments become overabundant and extend from the floc.  Once extended from floc and reducing floc density, filaments begin inhibiting settling rates.  So like many things, filaments are good in moderation but bad when overabundant.  What conditions promote filamentous growth?

• Low Dissolved Oxygen (D.O.)
• High influent soluble BOD & organic acids (causing a zone of highly depressed DO near the influent)
• Sulfides - several filamentous forms can also obtain energy from sulfide oxidation
• Low nitrogen or phosphorus - unbalanced C:N:P ratio
• Grease & Long chain fatty acids - this encourages Nocardia type foaming 

Some of the above problems can be fixed which reduces growth conditions favorable for filaments.  Consider corrective measures such as:

• Moving more aeration to the inlet area to avoid the DO depression often seen near the influent
• A long term solution is to step-feed influent which prevents DO drop and high concentrations of organic acids (also known as septicity in influent)
• Add nutrients if C:N:P ratio is triggering filaments (they have more surface area to uptake scarce nutrients)
• Adjust F/M or MCRT 
• Using hypochlorite, peroxide, or other oxidant to disrupt the filaments.  This is only for severe problems.  With more surface area, filaments are more susceptible to disinfection than floc forming bacteria.  Dosing can be determined by jar testing and looking for filament disruption prior to disinfection of the main system.

We sure love to use acronyms in environmental science and wastewater treatment. I have had full discussions with people mistaking "the nitrifiers" for "denitrifiers" when speaking - think about how close the words sound in normal speech. So, I thought a list of terms would help get everyone on the same page.

• Ammonia Oxidizing Bacteria (AOB) - these organisms convert ammonia (NH3) into nitrite (NO2)
• Nitrite Oxidizing Bacteria (NOB) - organisms converting nitrite (NO2) into nitrate (NO3)
  
• Denitrifying Organisms - many species can use nitrate (fewer nitrite) under anoxic conditions
  
• Anammox - or Anaerobic Ammonia Oxidation is biochemical pathway that has NO2 + NH4 --> N2 (gas) + H2O. This is an anaerobic process that requires a long MCRT & low F/M conditions. Often a fixed film with anammox cultures is required for significant development.
  
• Comammox - abbreviation for Complete Ammonia Oxidation - some organisms such as Nitrospira have the ability to completely convert ammonia into nitrate. Instead of two distinct microbial populations, Comammox have a single population completely removing problem nitrogen forms.
  
• Feammox - (the latest identified reactions) - iron reducing organisms work at lower pH without oxygen. Ammonia oxidation with concurrent Iron III reduction produces energy for this class of organisms. Research is still in the early phase regarding these organisms and their potential in waste treatment.
  

MBR systems use membrane separation instead of a DAF or clarifier to remove biological solids from effluent wastewater. MBR systems are becoming more common as membrane technology has improved and overall costs are lower when compared with larger foot print activated sludge systems. When compared with extended aeration activated sludge units, MBR systems typically have the following differences:

• Higher MLSS/MLVSS 
• Smaller aeration basin & overall foot print
• Better control of F/M & MCRT
• Fewer problems with solids in effluent

The MBR is technologically superior to older clarifier systems, but they still have issues that require operator attention. First since a membrane separates solids from discharged water, settling rates and floc formation would not seem as critical - however biological polymers are just as important for good MBR function as they are in systems with conventional clarification. Why do I say that floc formation is just as important in an MBR with advanced membrane technology?

Pores in the membrane allow for water to pass with solids being retained. As pores become clogged with solids - both biological & particulate - the system goes into a backwash cycle to clear the pores and restore membrane function. The problem is that backwashing is not perfect, overtime pores deteriorate with accumulated solids. Even with cleaning technologies that include oxidants, acids, or enzymes - the pores will eventually become plugged. 

A leading cause of plugged pores in biological waste treatment units is the accumulation of high molecular weight and "sticky" biological polymers. Organisms that thrive in the low F/M conditions found in MBR systems also tend to excrete copious amounts of extracellular polymeric substances (EPS). While some EPS is good and improves separation potential, the wrong EPS and overabundance of EPS can both create blinding conditions in membrane pores. 

The easiest ways to monitor EPS in a biological treatment unit is to run SV30, SVI, and microscopic exam on at least a daily basis. Additional monitoring with molecular testing is equally important - including both Microbial Community Analysis (MCA) and qPCR for specific problem or good organisms which gives earlier warnings for EPS blinding potential. When you have a buildup of problematic EPS, do not just assume that it cannot be corrected. Multiple control methods exist, it is just best to address the EPS problem before membrane issues start to compromise unit performance.