Life at Interfaces: Biocomplexity in Extreme Environments

Enrichment Culture and Isolation of Novel Organisms
Principal Investigator: Tim Magnuson (ISU)

Microorganisms involved in reductive biogeochemical processes.

Microbiological enrichments have resulted in the culturing of several organisms of biogeochemical significance. A summary of the pure culture isolates obtained is shown in Table 1. Three isolates of sulfate-reducing bacteria (SRB) were readily isolated from Mickey Hot Spring M0290 by means of direct inoculation of a hydrothermal fluid-based medium supplemented with acetate and sulfate. The initial enrichments were then transferred to and maintained on a standard mineral salts minimal medium. One isolate, YPMS, has a very close (99%) relatedness to Desulfotomaculumkuznetsovii. Functional gene analysis of these isolates, using the dsrA gene as a target, confirms the functional identity of the organisms. All of the SRB isolates are also capable of utilizing lactate, ethanol, and hydrogen/acetate as electron donors in sulfate respiration.

A novel arsenic-transforming bacterium, strain YeAs-1, was cultured from M0290 sediments. Growth conditions were at pH 7.5, temperature 55°C. Gelrite was used to isolate individual colonies, which were then subcultured onto the same medium. These colonies and subcultures produced an unusual bright yellow precipitate, beta-realgar. The significance of these findings is that they demonstrate that novel culturing techniques isolate microorganisms with novel metabolisms.

Microorganisms involved in oxidative biogeochemical processes — arsenic oxidation.

Using a synthetic hot spring medium that closely mimics the chemistries of the spring water, we have isolated an arsenic-oxidizing bacterium from the high-arsenic Alvord Hot Spring outflow channels. This organism, a Thermus spp. related to a known arsenic-oxidizing organism, has been named strain A03C and a similar sequence has been detected in clone libraries made from the same orange biofilm material from which this strain was isolated. Importantly, this culture was obtained in pure form in only one month. Another arsenic-oxidizing isolate, OPOC, was isolated in the same manner from orange mineral deposits at the Mickey Hot Springs site. Orange deposits were suspended in hot spring waters and serially diluted. These dilutions were used to inoculate hot springs waters solidified with gelrite. After one week of incubation at 70°C, orange colonies appeared in the solid medium. One colony was transferred to artificial hot spring medium containing As(III), and after several transfers, was analyzed and judged to be a pure culture. Phylogenetic analysis of the 16S rDNA of OPOC indicates that it is a member of the Deinococcus/Thermus clade and falls into a novel clade with one previously cultured but undescribed species. Similar to AO3C, OPOC was obtained in pure culture in about one month. The importance of these discoveries is that it shows the effectiveness of novel culturing strategies in the rapid isolation of organisms of biogeochemical interest. Additionally, it demonstrates the efficacy of our culturing methods in isolating organisms that are abundant, as judged by molecular culture-independent methods.

Enrichment techniques using mineral surfaces as selective enrichment medium.

As an alternative strategy to more traditional culturing methods, mineral coupons were deployed into selected hot spring features at both Mickey and Borax. Colonized mineral samples were then returned to the laboratory and used as inocula for fresh coupons in hydrothermal water medium. Coupons were also used for DNA extraction and identification, and surface spectroscopic mineral analysis. Preliminary bacterial diversity analyses of the 16S rRNA genes amplified from coupons incubated in situ indicates the presence of organisms belonging to novel deeply branching Betaproteobacterial clades, novel green non-sulfur (GNS) bacteria, and a previously cultured Synechococcus group. Additional analysis of DNAs extracted from coupons using functional sulfate-reduction gene primers (dsrAB) shows the presence of these genes in surface-attached microbiota, particularly with certain mineral types (pyrrhotite).

In situ microbial enzyme analysis.

We have developed methods for the extraction, purification, and characterization of metal-transforming enzyme activities directly from microbial mats found at the hot springs . Arsenite oxidase is readily detected by means of a gel electrophoresis assay using mat protein extract resolved on the gel. This method has already garnered interest from collaborators at NASA-JPL, and is the subject of two more grant submissions.

Mickey Hot Spring, M0290.

Table 1. Summary of physiologic properties of organisms cultured from the Alvord Basin.

Isolate	Electron Donor	Electron Acceptor	Growth Temp (°C)	% Similarity	Closest Relative
SRB (sulfate-reducing bacteria)
YPMS	Lactate	Sulfate	65	99	Desulfotomaculum
YPU	Lactate	Sulfate	65	98	Desulfotomaculum
ASYP	Lactate	Sulfate	65	98	Desulfotomaculum
Others
YeAs-1	Yeast Extract	As(V)	55	96	Thermobrachium
YPAs-1	Yeast Extract	As(V)	55	NA	??
A03C	As(III)?	Oxygen	60	98	Thermus sp. H8
OPOC	As(III)?	Oxygen	60	96	Thermus/Deinococcus
YPMO	Methanol	Oxygen	60	NA	??

Table 2. Summary of anaerobic enrichments obtained from Mickey Hot Springs.

Enrichment	Electron Donor	Electron Acceptor	Growth Temp (°C)	Growth pH
ASYP	Acetate	Sulfate	55	8.5
ASYPB	Acetate	Sulfate	55	8.5
YPU	Acetate	Sulfate	65	8.5
YPMS	Malate	Sulfate	65	7.5
GABS	Glycerol	AQDS*	55	7.5
YEAS	Yeast extract	Arsenate	55	7.0
NMPU	Glycerol	Iron oxide	65	7.5

*Anthraquinone-2,6-disulfonate; Used as alternate electron acceptor for iron-reducing bacteria.