AQUATIC MICROBIAL ECOLOGY Vol. 32: 85–93, 2003 Published May 12 Aquat Microb Ecol Distinct pigmentation and trophic modes in Beggiatoa from hydrocarbon seeps in the Gulf of Mexico Roxanne Nikolaus1, James W. Ammerman2, Ian R. MacDonald3,* 1US Commission on Ocean Policy, 1120 20th Street NW, Suite 200 North, Washington, DC 20036, USA 2Institute of Marine and Coastal Sciences, Rutgers University, 71 Dudley Road, New Brunswick, New Jersey 08901-8521, USA 3Department of Physical and Life Sciences, Texas A&M University–Corpus Christi, 6300 Ocean Drive, Corpus Christi, Texas 78412, USA ABSTRACT: Bacterial mats, which are comprised of spatially distinct, pigmented and non-pigmented filamentous Beggiatoa, are abundant at hydrocarbon seeps on the continental slope of the northern Gulf of Mexico. Samples of both filament types were collected, using the submarine ‘Johnson Sea Link’, from seeps at water depths of ~550 m. The water-soluble pigment of colored strains was inter- nal to the cells and had an absorbance peak of approximately 390 nm. Sulfur granules in both pig- mented and non-pigmented cells indicated that these Beggiatoa had the capability of oxidizing H2S. Non-pigmented filaments were capable of significant CO2 fixation based on incorporation of CO2 by whole, live cells and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) assays using cell-free extracts. RuBisCO activities for extracts from non-pigmented cells ranged from 9.92 to –1 –1 135.35 nmol CO2 fixed mg protein min . Activities varied significantly with temperature and pH. This ability to use CO2 as the primary carbon source, along with the ability to oxidize H2S for energy, suggests that these non-pigmented filaments were chemoautotrophic. Pigmented filaments, in contrast, had little CO2 incorporation ability. RuBisCO activities from pigmented mats ranged from –1 –1 0.17 to 0.92 nmol CO2 fixed mg protein min . These results suggest that geochemical processes at hydrocarbon seeps create an environment capable of supporting separate chemoautotrophic and heterotrophic (presumably organo-heterotrophic) Beggiatoa populations. KEY WORDS: Bacterial mat · Autotrophy · Heterotrophy · Chemosynthetic community · Digital camera array Resale or republication not permitted without written consent of the publisher INTRODUCTION of Mexico seeps include non-pigmented (white) and pigmented (yellow to orange) colonies, which are spa- Abundant bacterial mats, which consist of gliding, tially distinct as a general rule (MacDonald et al. 1989, filamentous, sulfide-oxidizing Beggiatoa, are preva- Sassen et al. 1994, Larkin & Henk 1996, Sen Gupta et lent at hydrocarbon seep sites in the Gulf of Mexico al. 1997). The mats occur on sedimentary settings near (Larkin et al. 1994). A distinctive characteristic of Beg- faults and fractures, covering shallow gas-hydrate giatoa filaments at hydrocarbon seeps is their large deposits, and peripheral to clusters of chemoauto- size. Wirsen et al. (1992) found 2 size-classes consisting trophic host organisms such as vestimentiferan tube of 30 to 50 µm and 85 to 108 µm wide filaments, while worms Lamellibrachia sp. and seep mussels Bathymo- Sassen et al. (1993) reported filaments as wide as diolus childressi (MacDonald et al. 1989, 1994, Fisher 200 µm. These widths greatly exceed those of B. gigan- 1990, Gustafson et al. 1998). The orange pigmentation tea, which had been the largest known species having of some of the mats is not typical of the cytochrome- filament widths greater than 25 µm. This size range is derived pink color that is commonly seen (Strohl & comparable to that at the Guaymas Basin hydrothermal Schmidt 1984), but its exact origin is still unclear. Mats vents, where Beggiatoa as wide as 122 µm have been of pigmented Beggiatoa have also been reported from collected (Nelson et al. 1989a). Beggiatoa mats in Gulf cold seeps in Monterey Canyon (Barry et al. 1996) and *Corresponding author. Email: [email protected] © Inter-Research 2003 · www.int-res.com 86 Aquat Microb Ecol 32: 85–93, 2003 from sediments adjacent to hydrothermal vents on MATERIALS AND METHODS Endeavour Ridge (Hedrick et al. 1992). The Beggiatoa at Gulf of Mexico seeps is unusual because one Setting. Samples were collected from 2 principal often observes distinct zonation of pigmented and non- hydrocarbon seeps on the northern continental slope pigmented mats over scales of a few cm. of the Gulf of Mexico (Fig. 1). The deeper site, desig- Sediment beneath Beggiatoa mats at hydrocarbon nated GC185 (27° 44’ N, 91° 30’ W), consists of an ellip- seeps contains high concentrations of hydrocarbon tical mound, approximately 750 m long and 500 m gases, C1 to C5, and oil that has undergone at least wide and elevated approximately 40 m above the sur- partial bacterial oxidation (Kennicutt & Sassen 1995, rounding seafloor. Its crest is at a water depth of 570 m. Sassen et al. 1998). Sassen et al. (1994) reported sub- The mound is aligned along a low-angle fault that stantially higher concentrations of hydrocarbons in intersects the seafloor (Cook & D’Onfro 1991) and pro- sediments covered with pigmented mats than in those vides a conduit for hydrocarbons that migrate to the beneath adjacent non-pigmented mats. However, the seafloor from deeply buried reservoirs (Kennicutt et al. gradients they observed were hundreds of parts per 1987). An abundant benthic fauna, including vesti- million over lateral and vertical scales of a few cm. mentiferan tube worms, seep mussels, and associated So, it is not clear whether the different pigmentation heterotrophic animals, colonizes the fault axis along styles are associated with distinct geochemical envi- the crest of the mound (MacDonald et al. 1989). The ronments or with transient pockets and veins of oil second site, designated GC234 (27° 44’ N, 91° 33’ W), is and gas in an extremely high-flux setting. The bacter- similarly colonized by seep fauna, but is situated in a ial oxidation of this abundant organic carbon has an half-graben at water depths of 525 to 550 m (Fig. 1). important impact on the seep environment. Bacterial Thermogenic hydrocarbons, including gases (methane oxidation of hydrocarbons causes depletion in the sup- to pentane) and high molecular weight hydrocarbons ply of O2 in the sediment and leads to the bacterial (liquid oil), are prevalent in surface sediments of both reduction of sulfate and production of H2S (Aharon & sites (Sassen et al. 1994). Fu 2000). The sediment at hydrocarbon seeps there- Bacterial mats were widespread at both sites. Typi- fore contains an ample supply of both CO2 and H2S in cally, non-pigmented mats consist of reticulated addition to organic compounds in the form of the strands that extend across the surface sediments in hydrocarbons and bacterial metabolites derived from irregular arrays that can be 1 m or more in width. Pig- heterotrophic consumption of hydrocarbons. Such con- mented mats generally appear denser and more com- ditions would support microbial heterotrophy as well pact. It is not uncommon to find distinct zones of pig- as autotrophy. Nelson et al. (1986) demonstrated mented mat surrounded by a fringe of non-pigmented chemoautotrophic growth by Beggiatoa in oxygen- filaments (Fig. 2A). Pigmented mats were often asso- sulfide micro-gradients. The characterization of hydro- thermal vent Beggiatoa by Nelson, et al. (1989b) included measurements of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) activity and CO2 fixation. RuBisCO activities in filaments in the 24 to 32 µm and 116 to 122 µm width-classes were found to be at levels near those of known chemoautotrophs. Carbon isotope analysis reveals seep Beggiatoa bio- mass to have a light δ13C value of –27.9% PDB (Sassen et al. 1993). This is compatible with typical values seen for chemoautotrophic carbon and lends credence to the idea that seep Beggiatoa use CO2 generated by the bacterial oxidation of hydrocarbons as their carbon source, and oxidize H2S for energy. Results reported by Wirsen et al. (1992) indicated significant rates of CO2 fixation for non-pigmented Beggiatoa from Gulf of Mexico seeps, whereas pig- mented Beggiatoa had ‘virtually no fixation activity.’ This investigation was undertaken to examine evi- dence for multiple trophic modes in Beggiatoa from hydrocarbon seeps in the Gulf of Mexico and to deter- Fig. 1. Study sites in northern Gulf of Mexico (M); see mine whether distinct trophic modes are associated ‘Materials and methods; Setting’ for descriptions of sites. with filament size or pigmentation. Depth contours in m Nikolaus et al.: Trophic modes in Beggiatoa 87 A C B D Fig. 2. Beggiatoa. Bacterial mats at GC234 study site, northern Gulf of Mexico. (A) Mix of pigmented and non-pigmented mats. Scale bar ~10 cm. (B) Gradient between pigmented and non-pigmented mats. Scale bar ~ 2 cm. (C) Detail of gradient between pigmented and non-pigmented mats. Scale bar ~1 cm. (D) Detail of amorphous flocculent layer on sediments disturbed by sampling operations. The layer formed in a matter of days following disturbance and covered a total area >15 m2 ciated with shallow or outcropping deposits of gas mination. Camera function was controlled from the hydrate (MacDonald et al. 1994, Sassen et al. 1998). submarine by entering commands on a hand-held Field methods. Collections of Beggiatoa were made computer. The visual distinction between pigmented using the submarine ‘Johnson Sea Link’. High-resolu- and non-pigmented mats is generally evident and, tion photographs of mats were taken with a newly de- where the 2 types co-occur, the gradation from pig- signed digital camera array mounted on the sub- mented to non-pigmented filaments occurs over dis- mersible arm. The camera, a Nikon Coolpix 990®, was tances of <1 cm (Fig. 2A–C). It was therefore possible modified to fit in a titanium alloy housing with a flat to target collections of Beggiatoa mats based on their optical port. A pair of 250 W quartz lamps co-mounted in-situ appearance.