Bacteria require nutrients for building new cellular material and act as co-factors for various enzymes. The macronutrients are needed by most cells and at higher concentrations than micronutrients.

First the Macronutrients

• Carbon, Hydrogen, & Oxygen - the backbone of organic chemistry
• Nitrogen - used for proteins, enzymes, and nucleic acids
• Phosphorus - used in nucleic acids, phospholipids, and ATP
• Sulfur - for several amino acids and vitamins
• Potassium - in multiple enzymes
• Magnesium - cell stabilization of ribosomes and membranes
• Iron (Fe) 
• Calcium 

Micronutrients
Function as enzyme cofactors and are not required by all bacteria - concentrations required are in the ppb - or nanograms per Liter.  Micronutrients required by anaerobic methanogen archaea are noted.

• Manganese
• Copper
• Zinc
• Nickel - vital for methanogens
• Cobalt - vital for methanogens
• Selenium
• Molybdenum - vital for methanogens
  
  

Does adding micronutrients make a difference in microbial activity?
The short answer is "it depends" - in the case of domestic wastewater, you have an abundance of all nutrients. And, in industrial wastewater, you usually lack mostly nitrogen or phosphorus. All can be checked with laboratory analysis of the influent and if you are deficient in specific micronutrients, then can be added at low cost.

The "it depends" part comes from some pretreatment steps and other chemical additives having an ability to bind to trace metals making them not available to support bacterial metabolism. I have seen this with AOB/NOB cultures and in anaerobic digesters. 

One interesting paper found anaerobic digesters receiving alum or ferric pretreated domestic primary sludge benefitted from added Bacillus bioaugmentation and micronutrients. Here is the abstract:

Water Sci Technology 2005;52(1-2):275-81.
Effect of biological additives and micronutrients on the anaerobic digestion of physicochemical sludge
A Noyola 1, A Tinajero
Affiliations expand
PMID: 16180439
 
Abstract
Two additives (lyophilized bacilli and enzymes) and a solution of micronutrients (Fe, Co, Ni and Mo) were tried separately and in combination, in order to assess their effect on the anaerobic digestion of waste sludge from an enhanced primary treatment (EPT) of municipal wastewater. Three batch tests were carried out in serological bottles. In the first test, addition of bacilli increased production of methane from day 11 and at day 17 the production was 95% greater than the control. In that experiment, the concentration of volatile fatty acids (VFAs) was 1,391 mg/L, 40% lower than the control. In the second test, the combination of micronutrients with bacilli, reached from the first days a better methane production than the control, 167% higher in day 17. At the end of the experiment, this combination achieved a lower concentration of VFAs and a greater percentage of volatile solid removal than the rest of the treatments. The third test was based on an experimental design in order to statistically determine the best doses of bacilli additive and micronutrients. The anaerobic thermophilic digestion of sludge from aluminum sulfate EPT will be improved with the addition of Fe: 4.5 mg/g VS, Ni: 0.75 mg/g VS, Co: 0.45 mg/g VS, Mo: 0.09 mg/g VS and bacilli additive: 12 mg/g VS.

We have all read that for every 10 Deg C drop in water temperature, microbial activity drops by 50%. This is true in the mesophilic temperature range but there are some “hard” inflection points where microbial activity exhibits significant changes. With low wastewater temperatures, we often have trouble maintaining Ammonia Oxidizing Bacteria (AOB) and Nitrite Oxidizing Bacteria (NOB) populations. The normal slow growth rate of AOB & NOB is compounded by reduced activity brought on by low temperatures.
 
Before water temperatures drop, you should increase MLSS concentrations. More biomass means lower F/M for when organism metabolism slows. This is also the same as increasing MCRT.
 
Other engineering solutions in areas with prolonged low temperatures is to use deeper basins with less surface area. Aeration via diffusers (no surface aerators). Using attached growth (MBBR or RBC) and MBR systems also allow for higher biological solids concentrations and can be used to retrofit existing systems.
 
For occasions where biomass has not had time to increase/adapt and low temperatures slow growth, you can use bioaugmentation. This works by increasing the number of active microorganisms, especially those organisms that function well below 15 Deg C. Over the years, we have found wastewater heterotrophic organisms with growth down to 3 – 5 Deg C. 

What can cause sudden increase in effluent ammonia

• Rapid increase in loading – Ammonia & TKN
  • Look at influent loadings – total nitrogen loading is needed
  • As both AOB/NOB are slow growing it can take time to catch up to influent increase
• Inhibition – slowing of AOB & NOB metabolism
  • D.O. being < 2 mg/L
  • COD/BOD inhibition – Rule of Thumb – 80% COD needs to be removed before AOB/NOB tend to fully function in WW
  • pH < 7.0 or >8.5
  • Alkalinity
  • Temperature <12 or >38 Deg C
  • Enzyme cofactors – trace metals lacking or being bound
  • UV radiation (sunlight)
• Washout
  • Slow growth and short MCRT – can lead to AOB/NOB washout (population drop as reproduction is not fast enough)
  • This often goes along with inhibitory factors slowing growth rates
• Acute toxicity – kill of AOB/NOB
  • Some compounds have acute toxicity – thiourea, carbon disulfide, sulfide, cyanide, phenol
  • Total loss of aeration for extended periods with septicity 
  • Not that common in most WWTP 

Tools to directly monitor AOB/NOB populations

• Aster Bio uses molecular tools to track for potential inhibition & washout
• qPCR for wastewater organisms – use primers based on actual WWTP data can directly enumerate populations
• MCA – total microbial census gives data on both AOB & NOB as % of total reads
• Answers questions – total toxicity, inhibition, or insufficient populations for current loadings

 
What to do if you suddenly see an increase in effluent ammonia

• Run qPCR to identify the root of the problem
• If influent ammonia/TKN increase is the issue
  • Slow influent flow if possible, this will give time for AOB/NOB growth
  • Adjust environmental conditions in the biological unit to maximize AOB/NOB growth
  • Add nitrifiers – decrease time required to adjust to full AOB/NOB population
• Toxicity – loss of all AOB/NOB populations
  • Find out what happened!
  • Remove problems from influent & increase wasting if it builds up in the MLSS
  • Adding nitrifiers or outside MLSS can help but only if inhibitors/toxic agents are removed
• Washout
  • Usually not a sudden increase – this is a gradual process, and you have warning
  • Reduce wasting rates
  • Enhance environmental conditions for AOB/NOB

 Key points
Check as to why you are seeing increased effluent ammonia

• Something had to change!
• Was it gradual or sudden
• Influent TKN is important - as much as influent ammonia
• If toxicity or inhibition is found, you must eliminate it as much as possible! Added nitrifiers from commercial blends are just as susceptible to inhibition as indigenous.
• qPCR is best tool to find out direct information on the AOB & NOB populations
• If adding nitrifiers, the dose depends upon growth rates and how much ammonia needs to be oxidized. We want to give the best growth environment possible.

 

We often use the terms “young sludge” and “old sludge” to describe problem biomass. What do these terms mean when it comes to the actual bacteria and biomass? 
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Components of floc

• Living bacteria – in Activated Sludge this is often only 5 – 15% of the solids.
• Dead bacteria
• Insoluble organics & inoraganics
• Extracellular Polymers (EPS) – made by bacteria to store organics, protect cells, and for extracellular enzyme control

 
The ideal floc contains a mix of the above at ratios where the solids are bound in a dense mass that settles well in clarification. In MBR systems, floc should readily separate from effluent and not blind pores in the membrane.
 
Young Sludge
Bacteria are rapidly dividing giving a high respiration rate (OUR) and free bacteria cells in solution. Rapidly dividing cells have yet to form sufficient dense EPS for target floc density. Color is often light. With respect to the growth curve, young sludge is seen where you have higher than design F/M (excess food).
 
Old Sludge
This is a system with low rates of bacterial division, and you get higher % dead bacterial cells and inorganics in the floc. With low division rates and fewer living bacteria, you also see lower respiration rates. The EPS that holds the floc becomes a source of “food” along with slower to degrade particulates. As the EPS is consumed, you end up with smaller pin floc and “fines” increasing turbidity in the supernatant. With the growth curve chart, you are in the decline/death phase.
 
Activated Sludge systems were designed to operate in the stationery to early decline phase of growth. This is where you maintain good levels of EPS with removal of soluble BOD (organics).

In wastewater treatment systems the referenced organisms responsible for ammonia and nitrite oxidation are Nitrosomonas sp.(AOB) and Nitrospira sp. (NOB). Both genera are chemolithotrophic organisms – they obtain energy from oxidation of ammonia or nitrite while using CO2 as carbon source. These and are other genera with similar chemolithotrophic nitrogen pathways responsible for ANAMMOX, COMAMMOX, and Ammonia Oxidizing Archaea (AOA). There are additional pathways for ammonia and nitrite oxidation via metabolic activities by heterotrophic organisms (heterotrophic organisms grown on organic compounds in wastewater and use this carbon to build cellular materials).
 
Heterotrophic Organisms with Ammonia Oxidation pathways
 
Methane Oxidizing organisms (Methanotrophs) are close cousins of Nitrosomonas and the enzymes produced for C1 oxidation can also oxidize ammonia. The bacteria do not gain any energy from this reaction and high ammonia concentrations can actually block methane degradation.
 
Paracoccus denitrificans is an interesting common wastewater organism that is capable of both sulfide and ammonia oxidation while also using nitrate/nitrite as alternative electron acceptors. P. denitrificans metabolism on ammonia was first investigated in the late 1980s and the ability to oxidize ammonia to nitrite coupled with simultaneous denitrification was noted in lab cultures. In real wastewater systems, this organism does aggressively denitrify, but often pursues higher energy yields using conventional heterotrophic pathways.
 
Similar to P. denitrificans, several Pseudomonas sp. have similar ammonia oxidation and denitrification pathways. To what extent these are responsible for ammonia oxidation in working wastewater systems is unknown. Other genera with potentially similar pathways include Alcaligenes and Bacillus.
 
What does this mean for wastewater operations
 

• Most ammonia and nitrite oxidation is accomplished via chemolithotrophic pathways by AOB & NOB cultures.

 

• When it comes to heterotrophic nitrification, just because an organism has the genes for ammonia oxidation pathways does not mean that the genes are turned on and the organism is oxidizing ammonia.
  
  
• Paracoccus denitrificans was one of the first heterotrophic organisms noted to have ammonia oxidation and denitrification pathways in a single organism. Paracoccus bacteria are commonly found in wastewater treatment systems – and are especially useful in improving nitrate/nitrite removal rates.

• Pseudomonas tend to also be aggressive denitrifiers and have been found to possess ammonia and nitrite oxidation genes. What we don’t know is how much Pseudomonas and other similar genera are responsible for primary ammonia oxidation in wastewater systems.