Overview
When you test wastewater using the QG21W method, you measure two things:
Total ATP (tATP): all the ATP in a sample, including inside living cells and free outside them.
Dissolved ATP (dATP): only the ATP found outside of living cells (free ATP, bound extracellular ATP, or easily-extractable ATP).
From these, you calculate:
Cellular ATP (cATP): tATP – dATP → an estimate of the living, active biomass present.
Biomass Stress Index (BSI): (dATP ÷ tATP) × 100% → a measure of stress in the biomass.
Normally, dATP is only a small slice of tATP. But sometimes, you might get the surprising result that dATP is equal to or greater than tATP. On paper this looks impossible — but it does happen, and this article explains why, and what to do about it.
Why can dATP be higher than tATP?
There are a few possible reasons:
1. Biomass stress
When microbes experience nutrient deficiencies, toxins, or oxygen limitation, they begin leaking ATP.
This leakage raises the extracellular fraction (dATP) while lowering the intracellular fraction (cATP).
High dATP relative to tATP indicates that more ATP is outside the cells than inside, signaling stressed or dying biomass.
2. Process interferences
Certain conditions in wastewater can artificially alter test results:
High salinity (≥ 5% salts): suppresses light output in the tATP assay, reducing tATP values.
Heavy metals (e.g., copper, zinc): interfere with enzyme reactions, leading to artificially low tATP and inflated stress indices.
Very long solids retention times (SRT): lead to accumulation of non-biological solids, which stress the microbial community and skew results.
3. Normal variability
Wastewater processes are dynamic.
Microbial communities change hour by hour, so occasional spikes in BSI, even above 100%, can occur due to statistical variability.
What are acceptable BSI levels?
Think of BSI as a “stress meter” for your biomass:
BSI < 30%: Indicates a healthy, well-balanced biomass in most aerobic systems.
BSI > 75%: Biomass is under significant stress; investigate process conditions.
BSI ≥ 100%: A critical signal. Retest to confirm, then take action.
Remember: treat a confirmed BSI ≥ 100% as an urgent indicator of stress or interference. Report as 100% and begin investigating process conditions.
How do I check if interferences are the problem?
You can run a dilution-inhibition check. This test helps you figure out if chemicals in the sample are interfering:
After running a normal tATP test, save the diluted extract (the mix of sample and reagents).
In a new tube, mix 1 mL of that extract with 1 mL of fresh reagent (UltraLute).
Add Luminase, measure RLUs, and compare.
If the diluted sample gives about half the reading of the original, there’s no interference.
If the diluted sample gives much higher readings than expected, interference is present. Repeat with further dilutions until the interference disappears.
Once you find the right dilution that works, you can reduce your future sample size by that factor.
Example: If a 1/10 dilution removes inhibition, test future samples at 100 µL instead of 1 mL.
This method is simple, but very effective for uncovering hidden problems.
What should I do next if dATP > tATP?
If you confirm that dATP is equal to or higher than tATP:
Repeat the test to be sure it wasn’t a one-off variation.
Check process conditions: look at solids retention time, aeration, and nutrient dosing.
Consider stressors: possible toxins, high salt, or heavy metals.
Use trends, not single numbers. One result doesn’t tell the whole story. Compare across several days or weeks to see if it’s an ongoing issue.
Why this matters
High dATP relative to tATP is not just a quirk of the test, it’s an opportunity. It can alert operators to hidden stress in the system that, if corrected, can improve:
Microbial health and responsiveness
Energy efficiency (less over-aeration, fewer wasted nutrients)
Overall secondary treatment performance
Related articles
- What is the Biomass Stress Index (BSI) in Wastewater Treatment?
- What are relative light units (RLU) in ATP testing and how should I interpret them?
Need Assistance?
For additional support or to order kits, contact LuminUltra Support
By following these recommendations, your PhotonMaster luminometer will remain in excellent condition, delivering consistent, accurate results every time.