Enumeration, Isolation, and Characterization of N 2 -Fixing Bacteria from Seawater

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Abstract
Marine pelagic N2-fixing bacteria have not, in general, been identified or quantified, since low or negligible rates of N2 fixation have been recorded for seawater when blue-green algae (cyanobacteria) are absent. In the study reported here, marine N2-fixing bacteria were found in all samples of seawater collected and were analyzed by using a most-probable-number (MPN) method. Two different media were used which allowed growth of microaerophiles, as well as that of aerobes and facultative anaerobes. MPN values obtained for N2-fixing bacteria ranged from 0.4 to 1 × 103 per liter for water collected off the coast of Puerto Rico and from 2 to 5.5 × 102 per liter for Chesapeake Bay water. Over 100 strains of N2-fixing bacteria were isolated from the MPN tubes and classified, yielding four major groups of NaCl-requiring bacteria based on biochemical characteristics. Results of differential filtration studies indicate that N2-fixing bacteria may be associated with phytoplankton. In addition, when N2-fixing bacteria were inoculated into unfiltered seawater and incubated in situ, nitrogenase activity could be detected within 1 h. However, no nitrogenase activity was detected in uninoculated seawater or when bacteria were incubated in 0.2-μm-filtered (phytoplankton-free) seawater. The ability of these isolates to fix N2 at ambient conditions in seawater and the large variety of N2-fixing bacteria isolated and identified lead to the conclusion that N2 fixation in the ocean may occur to a greater degree than previously believed.