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The principal aim of this project was to investigate the distribution of hopanoids in marine environments and in cultured cyanobacteria in order to understand the biogeochemical significance of hopanoids preserved in ancient marine sediments. Specifically we asked the question: do hopanoids record ecological or biogeochemical processes in the upper ocean? The answer to this question ultimately rests on several key pieces of information concerning the sources, function, and fate of hopanoids in the ocean. At the outset of the project there was a large body of evidence supporting the idea that hopanoids could potentially be useful markers for cyanobacterial productivity.  However the abundances and structural diversity of hopanoids in the ocean was entirely unknown, and the potential biological sources enigmatic.

We analyzed for bacteriohopanepolyols using reversed-phase HPLC-APCI-MS and set out to reveal the spatial and temporal distribution of hopanoids in a range of marine environments. One of the advantages of the method is that BHPs can be resolved and detected in picogram quantities from total lipid extracts, requiring minimal sample preparation. The use of an internal standard (pregnone diacetate) greatly reduced error and, by comparison with an authentic standard of bacteriohopanetetrol (BHT), we have been able to report quantitative concentrations for BHT, and semi-quantitative concentrations for other BHP compounds. Given our detection limits (~100 pg on column) and the volumes of seawater we have extracted, we have been able to detect BHPs in the pg/L range.

Distribution of hopanoids in marine cyanobacteria

We screened over 30 strains of marine cyanobacteria from John Waterbury’s culture collection at WHOI and an additional 30 strains of heterotrophic bacteria from Tracy Mincer’s collection at WHOI to investigate the taxonomic distribution of hopanoids in marine bacteria. None of the heterotrophic bacteria contained hopanoids. Among the cyanobacteria, we could detect hopanoids in only three genera including Trichodesmium, Crocosphaera, and Cyanothece, all of which are capable of nitrogen fixation. We also observed a very low diversity of BHP structures in the marine cyanobacteria. The cyclitol ether was present in all three organisms and was nearly 10% by mass of the total lipid extract of Crocosphaera watsonii. This result stands in stark contrast to freshwater cyanobacteria, in which hopanoid production is widespread, structures are diverse, and generally range from 0.1-1% of the lipid extract mass. Our work, representing the largest survey of hopanoids in marine bacteria to date, suggests that hopanoids are rare in open ocean marine bacteria, at least in cultured strains, and predominately associated with nitrogen fixing cyanobacteria.

Distribution of hopanoids in the oceans

Our sample selection strategy was aimed at addressing five facets of hopanoid distribution: 1) land-sea distributions 2) open ocean distribution over a large spatial transect, 3) temporal variability in the upper ocean 4) patterns in vertical distribution, and 5) potential for export to marine sediments. During the course of support from this ACS funding, we have analyzed over 500 samples from over 20 sites distributed globally. From this data set we have begun to construct an understanding of how hopanoids are distributed in the contemporary marine realm.  In turn, this might inform us of past conditions in marine environments via their legacy in the sedimentary record.

Our results indicate that hopanoids are widespread in the oceans, and may be particularly useful as markers for processes in the photic zone and in low oxygen marine environments. We are however, far from understanding the biological sources of hopanoids in the ocean. Our culture survey suggests that marine cyanobacteria producing the cyclitol ether BHP should be prominent hopanoid producers in the oceans. In the environment, we see much a greater diversity of BHP structures, and often do not even detect the cyclitol ether, suggesting that there are many as yet uncultured hopanoid producing organisms in the oceans.

Another dilemma surrounds the origin and biogeochemical significance of 2-methylhopanoids, which have been widely applied as markers for cyanobacteria in the geologic record. We often see abundant 2-methylhopanoids in marine sediments, but have yet to detect them in the water column, or in any cultured marine cyanobacteria. We are beginning to map out the temporal seasonal variability in hopanoid structural diversity and abundance. Our preliminary observations suggest that certain structures may be associated with short-lived communities that are only present under certain conditions or at certain times of the year. These observations may go a long way towards accounting for the apparent absence of 2-methylhopanoids in the water column. While we cannot rule out non-cyanobacterial sources for 2-methylhopanoids in the oceans, or perhaps even their complete absence in marine bacteria, it is clear from our work that we only begun to scratch the surface of the biogeochemical significance of hopanoids in the ocean.

Ancient sediments and petroleum

Wide fluctuations in the abundances of 2-methylhopanoids, relative to their non-methylated counterparts continue to be reported in the literature and observed in several on-going studies in our laboratory.  The pattern that has emerged is that values of the 2-methylhopane index (2-MeH/ (2-MeH  + H) x 100) in the range of 3-5% is common in rocks and oils of all ages and facies.  Values in the range 5-10% is typical for marine carbonates.  Exceptionally high values in the range 15-35% are seen in sediments laid down during oceanic anoxic events irrespective of lithology.  Given the above results from cultures of marine cyanobacteria and marine environmental samples, it seems possible that the 2-methylhopane fossil record is consistent with what is observed in contemporary marine sediments.  However, it is also clear that we have not yet captured the responsible organism or organisms in our laboratory surveys.  Nor have we captured a modern environmental setting with the exceptionally high amounts of 2-methylhopanoids that are seen at OAE’s.  This suggests that anoxic/euxinic ocean waters are host to a particular group of 2-methylhopanoid-producing organisms and their biological affinities remain to be determined.