Date of Award


Degree Type


Degree Name

Doctor of Philosophy (PhD)


Biological Science

First Advisor

Marta Gomez-Chiarri


In the United States of America, oyster production is an important component of the seafood economy in many communities in coastal states. The severe impact of disease outbreaks and mass mortality of oyster larvae in hatcheries impacts production, since the oyster industry is largely dependent on hatchery and nursery production. The use of probiotics has been proposed as a potential preventative measure to limit the impact of bacterial diseases in shellfish hatcheries. In previous laboratory studies, the probiotic bacteria Phaeobacter inhibens S4 and Bacillus pumillus RI06-95 improved the survival of eastern oyster (Crassostrea virginica) larvae against the pathogens Vibrio tubiashii RE22 (now V. coralliilyticus) and Roseovarius crassostreae CV919-312T (now Alliroseovarius crassostreae). The aim of this study is to evaluate the efficacy of candidate probiotics P. inhibens S4 and B. pumillus RI06-95 for disease prevention in shellfish hatcheries.

Chapter 1 provides an overview of bacterial disease in marine bivalves and the use of probiotics for disease prevention in bivalve hatcheries. Chapter 2 describes that the daily application of P. inhibens S4 and B. pumillus RI06-95 mixed with algal feed to culture tanks in the hatchery increased survival of oyster larvae to experimental challenge with V. coralliilyticus RE22. The levels of total Vibrios in water and surfaces of tanks treated with probiotics were significantly decreased (p < 0.05) compared to non-treated tanks, whereas there were no significant differences between treatments in levels of Vibrios in oysters. These probiotic strains had no significant impact on oyster larvae growth and survival rate at the hatchery.

Chapter 3 evaluates the safety and efficacy of candidate probiotic bacteria strains, P. inhibens S4 and B. pumilus RI06-95, in four bivalve species, including hard clams Mercenaria mercenaria, bay scallops Argopecten irradians, blue mussels Mytilus edulis, and razor clams Ensis directus. Pre-exposure of larvae to 104 CFU/ml of probiotics for 24 h in the laboratory did not protect these bivalve species to challenge with V. coralliilyticus RE22, but pre-exposure to 106 CFU/ml probiotics S4 and a mixture of S4 and RI did confer some protection to bay scallop larvae (RPS; 69 ± 4 %). Daily application of 104 CFU/ml probiotics to tanks with bay scallop larvae at the hatchery offered partial protection against bacterial infection without impacting levels of Vibrios in tank surfaces, water, and larvae. However, although daily probiotic treatment of tanks containing hard clam larvae led to a decrease in the levels of Vibrio sp. in rearing water and larvae, it provided no consistent protection to bacterial challenge.

Chapter 4 evaluates the effects of formulation methods on the viability and efficacy of two formulations of B. pumilus RI06-95, including a granulated (43 μm in size) and a lyophilized (containing 100 mM sucrose as a cryoprotectant) formulation. Granulation led to a decrease in cell viability from 108 CFU/mg to 105 CFU/mg. This level of viability was maintained for up to 8 weeks of storage. Lyophilization in the presence of 100 mM sucrose did not significantly impact the cell viability of RI06-95, but exposure of oyster larvae to this lyophilized formulation resulted in decreased survival compared to non-treated controls in a small-scale experiment. Furthermore, pretreatment of oyster larvae with the lyophilized formulation did not increase larval survival to challenge with the pathogen V. coralliilyticus RE22. More work needs to be done to develop effective probiotic formulations for shellfish hatcheries.

Chapter 5 characterized the microbial community of rearing water, tank surface, and oyster larvae during a pilot-hatchery trial using 16S rDNA-based MiSeq sequencing. The impact of treatment with probiotic B. pumilus RI06-95 on the microbial community at the oyster hatchery also described. Proteobacteria was the most abundant phylum in all collected samples at the hatchery trials. The proportion of bacterial groups at the phylum level was different for sources of collected sample (water, tank surfaces, and larvae). No shift was detected in the composition of the microbiome within/between treatments (probiotic and control) and time points. Therefore, application of probiotic B. pumilus RI06-95 at the oyster hatchery may not significantly impact on bacterial community as detected by 16S rDNA sequencing.