A. Oxygen from the air diffused into the broth and the lack of enzymes capable of breaking down the superoxide anion lead to death by oxygen toxicity.
B. Fungal spores in the air landed in the broth and the subsequent fungal growth consumed the available nutrients.
C. The ambient temperature of the air cooled the broth below the optimal growing temperature of 37ºC, causing the Clostridium to die off.
D. The failure to cap the test tubes was irrelevant and the Clostridium must have died due to some unknown factor.
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B: The question makes no mention of fungal growth, and we have no reason to suspect that fungal spores in the air would be of a species capable of out-competing the bacteria for nutrients.
C: The ambient temperature in the room is unlikely to kill off bacteria. Cooler than optimal growing conditions may slow bacterial growth, but room temperature is unlikely to be fatal.
D: See explanation above for (A).
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