Identification of gastrointestinal microflora in Hawaiian green turtles (Chelonia mydas Linnaeus), and effects of glyphosate herbicide on their gastrointestinal bacteria













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Long-term conservation of the green turtle (Chelonia mydas Linnaeus), the largest marine herbivore in the Hawaiian archipelago, may depend on understanding their microflora. Using hindgut fermentation, green turtles rely on microbial degradation of cellulose and starch products from the seagrasses and macroalgae consumed. Few studies have examined bacterial communities of green turtles, and none has sampled in situ and identified hindgut microflora of green turtles. Fresh samples were taken from five locations along the gastrointestinal tracts of eight green turtles that had required euthanization. Bacteria were cultured, aerobically and anaerobically, on nutrient agar and four differential and selective media. Fecal samples at three sections along the gastrointestinal tracts of two green turtles were analyzed using 16S metagenomics on the Ion Torrent Personal Genome Machine (PGM). More than half of the 4,532,104 sequences belonged to the phylum Firmicutes, followed by Bacteroidetes and Proteobacteria, which are characteristic of herbivore gut microflora. The 16S sequence analysis provides a better representation of the total gastrointestinal bacterial community, much of which cannot be cultured using traditional microbial techniques. Accurate and precise enumeration and identification of green turtle microflora will help to clarify connections between diet and digestive bacteria, as well as provide new tools for assessing the health of green turtles grazing in different locales. In some foraging areas in the Hawaiian Islands, green turtles are growing at reduced rates. In the Hawaiian Islands, glyphosate-based herbicides are frequently sprayed to combat weeds, and may be affecting non-target marine species. Glyphosate inhibits a key enzyme in the shikimate pathway that is found in plants, fungi, and bacteria for the biosynthesis of aromatic amino acids. Studies have shown that glyphosate can persist in coastal environments. Glyphosate is toxic to beneficial gut bacteria in cattle and chickens, and can cause lowered digestive efficiency; however, no studies have assessed the impact of glyphosate on the bacteria in the GI tract of marine herbivorous turtles, which could be exposed to glyphosate via ingestion of glyphosate-contaminated seaweeds, seagrasses, or seawater during foraging. Four microfloral isolates obtained from freshly euthanized green turtles were exposed to fifteen different concentrations of glyphosate herbicide (2.2 x 10-4 to 3.6 g L-1 glyphosate) and a control of DI water, using a modified Kirby-Bauer disk diffusion assay. A response to glyphosate was observed in all taxa tested. In three out of the four taxa, at 0.028 g L -1 glyphosate, zones of growth inhibition were significantly different than the control. Proteus sp. was the least sensitive to glyphosate. Additionally, aliquots of mixed bacterial communities from turtle GI tracts, cultured in nutrient broth, were exposed to six different concentrations of glyphosate (2.2 x 10-4 to 3.6 g L-1 glyphosate) and a control of DI water. Transmittance at 600 nm wavelength was measured before and after 24 hours to assess bacterial density. Cultures with concentrations ≥ 2.2 x 10-4 g L-1 glyphosate showed significantly different greater transmittance than the control. Reduced growth or decreased survival of GI tract bacteria in green turtles exposed to glyphosate could have adverse effects on turtle digestion and overall health. This thesis brings together three chapters: 1) an overview of C. mydas digestion, microbiota, and glyphosate herbicide; 2) identification of gastrointestinal microflora collected in situ in C. mydas; and 3) glyphosate effects on gastrointestinal microflora of C. mydas. The objectives of this study were to collect in situ and identify the gastrointestinal microflora found in green turtles (Chelonia mydas) in Hawaii, and to quantify the sensitivity of these microbes to glyphosate-based herbicide (RoundUp®). The hypotheses were: 1) diverse bacteria occur in the GI tract of green turtles, including the following phyla: Actinobacteria, Bacteroidetes, and Firmicutes; 2) different species of bacteria from green turtles demonstrate different sensitivities to glyphosate; and 3) inhibition of bacterial growth is dependent on glyphosate concentration.



Microbiology, Environmental science, Toxicology, bacteria, Chelonia mydas, gastrointestinal microflora, glyphosate, herbicide toxicity, metagenomics



112 pages


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