The yellowing of Norris glucose nitrogen-free medium containing bromothymol blue is primarily due to biochemical changes caused by bacteria isolated from earthworm guts. This color change is mainly attributed to a drop in pH resulting from the production of organic acids (such as lactic and acetic acids) during glucose fermentation. Although the bacteria may produce low levels of ammonia (5–6 ppm), which can initially increase pH, ammonia can be converted to ammonium ions in an acidic environment, contributing further to acidification. Additionally, processes such as nitrogen fixation and high overall metabolic activity may indirectly influence pH by shifting the chemical balance of the medium. To confirm these mechanisms, it is recommended to monitor pH over time and analyze metabolic byproducts such as organic acids and ammonia.

The yellowing of Norris Glucose Nitrogen-Free (NGNF) media is due to acid production from glucose fermentation by bacteria, which lowers the pH and turns the pH indicator bromothymol blue from green to yellow.

The yellowing of Norris Glucose Nitrogen-Free (NGNF) media during the growth of bacteria isolated from the earthworm gut indicates nitrogen fixation. Since the media lacks nitrogen, only nitrogen-fixing bacteria can grow. As these bacteria metabolize glucose and fix atmospheric nitrogen, they produce acidic byproducts that lower the pH, leading to a color change (yellowing) of the pH indicator in the medium.