Lawrence Berkeley National Laboratory

The important role of the normal anaerobic intestinal microbiota has been elucidated in humans and some domesticated animals; however, the presence of intestinal anaerobes, let alone their importance in invertebrates are largely unknown. The purpose of this research was to isolate and to identify obligate anaerobic members of the intestinal microbiota of the Pacific white shrimp, Litopenaeus vannamei. Intestinal samples for microbial isolation were taken from shrimp maintained at three salinities (3, 11, 31 ppt) and on a diet containing different protein concentrations. Members of the anaerobic intestinal microbiota were successfully isolated and cultivated in a Bactron II anaerobe chamber. Many of these isolates could not be identified using available biochemical and molecular-level techniques and databases, and consequently, may represent new, yet undescribed taxa. Of the isolates identified, the majority belonged to the genus Clostridium. Strains of the species, C. subterminale and C, beijerinckii were delineated by biochemical tests, while two species, C. butyricum GC subgroup A and C. bifermentans GC subgroup A were identified by whole-cell fatty acid methyl ester analysis. Isolates were tested for the expression of constitutive enzymes using the APIZYM system. The anaerobic bacteria demonstrated enzyme activities of acid and alkaline phosphatases, C4 and C8 esterases, C14 lipases, arylamidases, and glycosidases. Enzyme activities differed with isolates. Generally, strains identified as C. butyricum expressed enzymes for the breakdown of carbohydrates, while strains identified as C. bifermentans produced enzymes for the breakdown of proteins. Most isolates formed phosphatases possibly used for absorption of nutrients.