The feasibility of using a live Escherichia coli population, which had been engineered to express the green fluorescent protein (GFP), coupled with fluorimetry, was tested as a means for determining protozoan ingestion rates. Its potential use was based on evidence that once cells are acidified, e.g. in a food vacuole, the fluorescence is lost. Of the 29 protozoa tested, over 85% ingested the GFP-expressing E. coli and a detailed experiment with the ciliate Tetrahymena pyriformis was carried out, principally to assess the performance of the live bacterium against two commonly used surrogate prey, i.e. fluorescently labelled bacteria (FLB) and fluorescently labelled microspheres (FLMs). A decrease in GFP-expressing E. coli fluorescence and, hence, concentration, was recorded by fluorimetry and epifluorescence microscopy, with calculated ingestion rates being equivalent. A higher ingestion rate was determined by counting the number of fluorescent E. coli within the ciliate over 120 s, but this was equivalent to that obtained for the stained E. coli using the same direct method of analysis. However, the ciliate was shown to process the stained and unstained E. coli cells differently, with only the latter resulting in an increase in ciliate abundance.

The digestion of heat-killed/stained Escherichia coli, Pseudomonas aeruginosa, Mesorhizobium sp. and Staphylococcus aureus was monitored within one food vacuole passage time in the ciliate Tetrahymena pyriformis using the pulse chase technique. Prey digestion proceeded in 2 phases: a digestive phase which lasted ~25 min and a defecation competent phase that showed limited digestive activity and was variable in length. The number of prey cells per food vacuole was found to influence the effectiveness of prey digestion. Complete digestion of the vacuole content was more likely to occur when the number of prey per vacuole averaged ~6 or less. At higher levels, only partial digestion of the vacuole content was recorded and some undigested prey were egested from the ciliate cell. A strain of Synechococcus sp. was never digested by this ciliate. Results suggest that bacteria do not necessarily require elaborate mechanisms to evade digestion by protozoa, as possessed by some pathogenic bacteria, but that inefficiency in the protozoan digestive system is all that is required to allow the release of undigested, apparently unharmed prey from their cells. Thus, models on carbon cycling which employ data on protistan ingestion rates alone should consider accounting for digestion efficiency and the subsequent effect of prey concentration, because prey carbon might not always be transferred efficiently to higher trophic levels.

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