Five different cultural and two direct microscopic methods were employed for estimating the abundance of bacteria in samples of sea water collected from both oceanic and neritic areas. The cultural methods included macrocolony counts on nutrient agar, silica gel, and membrane filters, microcolony counts on membrane filters, and the extinction dilution method.Direct microscopic counts were made of microbes on membrane filters and of microbes transferred from membrane filters to glass slides. Direct counts showed the presence of from 13 to 9,700 times as many bacteria as cultural methods.The extinction dilution method and the microcolony membrane filter method gave counts 20 and 35 times higher, respectively, than did any of the macrocolony methods. Direct microscopic counts on membrane filters were approximately 150 times higher than plate counts, and the numbers of microbes transferred from membrane filters to glass slides were approximately 2,000 times higher than plate counts.

A Mycobacterium sp., designated strain BG1, able to utilize the polycyclic aromatic hydrocarbon phenanthrene as the sole carbon and energy source was isolated from estuarine sediment following enrichment with the hydrocarbon. Unlike other phenanthrene degraders, this bacterium degraded phenanthrene via 1-hydroxy-2-naphthoic acid without accumulating this or other aromatic intermediates, as shown by high-performance liquid chromatography. Degradation proceeded via meta cleavage of protocatechuic acid. Different nonionic surfactants (Tween compounds) solubilized the phenanthrene to different degrees and enhanced phenanthrene utilization. The order of enhancement, however, did not correlate perfectly with increased solubility, suggesting physiological as well as physicochemical effects of the surfactants. Plasmids of approximately 21, 58, and 77 megadaltons were detected in cells grown with phenanthrene but not in those which, after growth on nutrient media, lost the phenanthrene-degrading phenotype. Given that plasmid-mediated degradations of aromatic hydrocarbons generally occur via meta cleavages, it is of interest that the addition of pyruvate, a product of meta cleavage, supported rapid mineralization of phenanthrene in broth culture; succinate, a product of ortho cleavage, supported growth but completely repressed the utilization of phenanthrene. The involvement of plasmids may have given rise to the unusual degradation pattern that was observed.

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Microbial Fe reduction in acetate-and succinate-containing enrichment cultures initiated with an estuarine sediment inoculum was studied. Fe reduction was unaffected when S042 reduction was inhibited by MoO42, indicating that both processes could occur independently. Bacterially produced sulfide precipitated as FeS but was not completely responsible for Fe reduction. The separation of oxidized Fe particles from bacteria by dialysis tubing demonstrated that direct bacterial contact was necessary for Fe reduction. Fe reduction in cultures amended with N03was delayed until N03and N02were removed. However, bacterial attachment to oxidized Fe particles in N03-amended cultures occurred early during growth in a manner similar to N03-free cultures. During late stages of growth, bacteria not attached to Fe particles became pale and swollen, while attached cells remained bright blue when examined by 4',6-diamidine-2-phenylindole epifluorescence microscopy. The presence of added oxidized Mn had no effect on Fe reduction. The results suggested that enzymatic Fe reduction was responsible for reducing Fe in these cultures even in the presence of sulfide and that cells incapable of Fe reduction became unhealthy when Fe(III) was the only available electron acceptor.

The polycyclic aromatic hydrocarbon phenanthrene was mineralized in two stages by soil, estuarine water, and sediment microbial populations. At high concentrations, phenanthrene was degraded, with the concomitant production of biomass and accumulation of Folin-Ciocalteau-reactive aromatic intermediates. Subsequent consumption of these intermediates resulted in a secondary increase in biomass. Analysis of intermediates by high-performance liquid chromatography, thin-layer chromatography, and UV absorption spectrometry showed 1-hydroxy-2-naphthoic acid (1H2NA) to be the predominant product. A less pronounced two-stage mineralization pattern was also observed by monitoring 14Co2 production from low concentrations (0.5 mg liter-') of radiolabeled phenanthrene. Here, mineralization of 14C-labeled 1H2NA could explain the incremental '4Co2 produced during the later part of the incubations. Accumulation of 1H2NA by isolates obtained from enrichments was dependent on the initial phenanthrene concentration. The production of metabolites during polycyclic aromatic hydrocarbon biodegradation is discussed with regard to its possible adaptive significance and its methodological implications.