We have used 16S rRNA phylogenetic analysis to investigate the microbial diversity of sulfur-rich black mud marine sediments. Two distinct environments were examined: mud from a hypersaline marsh and mud from a brackish marsh. Only two out of one hundred cloned 16S rDNA sequences were identical, indicating a very complex community of microbial species. Both environments were dominated by sulfate-reducing d-Proteobacteria, indicating that these organisms are likely the primary source of reduced forms of sulfate required by other species. Since seawater has abundant quantities of sulfate, micro-organisms that reduce this molecule are important for the cycling of sulfur in the global ecosystem. The 16S rDNA sequences for the majority of cultivable organisms were 98-99% identical to the 16S rDNAs of known organisms, whereas PCR of the 16S rDNAs extracted directly from the mud without culturing revealed that most of the population was less than 95% identical to known 16S rDNAs, and six of the one hundred sequences analyzed were only 80-85% identical to known sequences. Sequences from this more divergent group were identified as new candidate divisions. Although sulfate-reducing bacteria dominated both ecosystems, the hypersaline black mud also harbored organisms related to known halophiles. A search for Archaea uncovered a diverse collection of halophilic species that showed taxonomic affiliation with the Halobacteriales. From our sampling of 100 rDNA clones, we estimate that the diversity of organisms in these black mud samples is approximately 10,000 species – close to the number of Archaea and Bacteria currently cataloged by GenBank. Additionally, we note a lack of predominant organisms in our sample. This suggests a modestly ambitious DNA sequencing effort of rDNAs from black mud would yield thousands of new species and many new candidate divisions. 

Black Mud Tree

Figure 1.  The phylogenetic tree shown above contains 'hotmapped' links via the major taxonomic categories listed to the right of the brackets.   Although present, Eucarya were not examined from this environmnet.  Tetrahymena thermophila was used as an outgroup.  Only species labeled 'Kairos Black Mud Clone' were found in the samples studied here.  Other named species listed show the phylogenetic relationship of the newly isolated Black Mud Clones relative to known species or clones.  Hyperlinks from this tree will retrieve tables of each of the species with their accession numbers and a hyperlink to the actual sequence file at GenBank.  As we continue to add to this tree, more intricate html paging will be implemented to facilitate access to the thousands of species found in black mud.

This work is supported by the U.S. Department of Energy, Office of Basic Energy Sciences under grant 99ER20211.