Characterization of Enrichment Cultures of Nitrate-Reducing Bacteria from Deep-Sea

Hydrothermal VentsCaroline Toney* and Costantino Vetriani

Deep-sea vents are one of the last frontiers of the unexplored world. These unique environments spew forth super-heated fluid that is laden with metals. The base of life which allow other organisms to live in this ecosystem are microorganisms which undergo chemosynthesis as a means of primary production.

Introduction

Figure 2. Sampling sites of deep-sea hydrothermal vents on the East Pacific Rise

Objectives• To grow cultures from samples taken at vent sites at 9°N and 13°N. •To isolate anaerobic, nitrate-reducing bacteria using different techniquesand media.•To compare the 16S rRNA gene sequences of these organisms to the databaseusing BLAST (Basic  Local Alignment Search Tool) to assess theirrelatedness to other species.

Media Carbon Source Electron Donor

Electron Acceptor

Temp (°C)

HB1 CO2 H2 NO3 50 & 75

Sulfurovum CO2 N2, thiosulfate NO3 35

Sulfurimonas CO2 H2, thiosulfate NO3 35, 50

ASW Organic N2 NO3 30

Caroline Toneycrt8347@uncw.edu(828) 429-9476

Figure 1. Illustration of deep-sea hydrothermal vent flow. (Brock et al., 2006).

Site Media Temp (°C) Identity (%) Description

Alvinella Tubes

HB1 50 99,97 Caminibacter mediatlanticus; Nautilia profundicula, abyssi

Sulfurimonas 35, 50 86 Sulfurospirillum carboxydovorans

Alvinella sulfide

HB1 50 97, 96 Caminibacter mediatlanticus

HB1 75 98 Desulfobacterium pacificum

Crab Spa Sulfurovum 35 93;96;93 Thioprofundum lithotrophica; Rhodobacter capsulatus; Marinobacter hydrocarbonoclasticus

Sulfurimonas 35, 50 97 Shewanella haliotisAnaerobic ASW 30 100 Klebsiella pneumoniae

Bottom Seawater

Anaerobic ASW (B)

30 99 Shewanella haliotis

Samples were taken during Nautile Dive (ND) #:ND 1726 Alvinella pompejana tubesND 1727 Alvinella pompejana encrusted sulfidesND 1729 Fluid from Crab Spa; Bottom Seawater

AcknowledgementsWe want to thank the crew of the R/V L’Atlante and the crew and pilots of the DSV/Nautile for sample collection We would like to offer our thanks to the members of the Deep Sea Microbiology Lab at Rutgers University their expertise, as well.This work was supported by an NSF grant.

Table 2. Results of sequencing and BLAST.

Conclusions•The molecular characterization of the organisms cultured in the samples taken from the dives reveals that there may be new species among them.•The sequences of the 16S rRNA gene from the organisms grown show that further isolation is needed for some samples in order to have a pure culture from which further investigation of the species can be done.• Future work would include characterization of isolated organisms. Morphological observation and studies of optimal growth in temperature, salinity and pH will be executed. Metabolism will be studied through growth under different conditions (i.e. electron donors and acceptors, pressure, etc.), in organic substrates, and resistance to antibiotics will also be tested.

Table 1. Characteristics of anaerobic media used.

Alvinella pompejana. (noc.soton.ac.uk)

Crab Spa (whoi.edu)