Answer:
The correct answer is option C. "by applying heat and antimicrobial treatment to sewage samples and observing that nitrate was no longer formed".
Explanation:
The easiest way to demonstrate that an element is responsible for a reaction, is to remove the element and see if the reaction still occurs. In this case, early microbial ecologists removed microbes from sewage samples by applying heat. When ecologists observed that nitrate was no longer formed in the samples, they concluded that microbes are responsible for hydrogen gas oxidation in soil and ammonia oxidation in sewage. Nowadays, the bacteria that perform these ecological reactions are known as nitrifying bacteria, and the reactions that they perform are well characterized.