Isolation of obligately alkaliphilic magnetotactic bacteria from extremely alkaline environments

Abstract

Large numbers of magnetotactic bacteria were discovered in mud and water samples collected from a number of highly alkaline aquatic environments with pH values of ≈ 9.5. These bacteria were helical in morphology and biomineralized chains of bullet-shaped crystals of magnetite and were present in all the highly alkaline sites sampled. Three strains from different sites were isolated and cultured and grew optimally at pH 9.0-9.5 but not at 8.0 and below, demonstrating that these organisms truly require highly alkaline conditions and are not simply surviving/growing in neutral pH micro-niches in their natural habitats. All strains grew anaerobically through the reduction of sulfate as a terminal electron acceptor and phylogenetic analysis, based on 16S rRNA gene sequences, as well as some physiological features, showed that they could represent strains of Desulfonatronum thiodismutans, a known alkaliphilic bacterium that does not biomineralize magnetosomes. Our results show that some magnetotactic bacteria can be considered extremophilic and greatly extend the known ecology of magnetotactic bacteria and the conditions under which they can biomineralize magnetite. Moreover, our results show that this type of magnetotactic bacterium is common in highly alkaline environments. Our findings also greatly influence the interpretation of the presence of nanometer-sized magnetite crystals, so-called magnetofossils, in highly alkaline environments.

Ecology, Diversity, and Evolution of Magnetotactic Bacteria

“…Lefèvre et al (27) described an uncultured, moderately thermophilic, magnetotactic bacterium present in hot springs in northern Nevada with a probable upper growth/survival limit temperature of about 63°C. In addition, this same group isolated several strains of obligately alkaliphilic MTB from different aquatic habitats in California, including the hypersaline and extremely alkaline Mono Lake (28). The latter strains had an optimal growth pH of ϳ9.0.…”

“…The formation of sulfide in anaerobic cultures of sulfate-reducing MTB can interfere with iron availability for magnetosome formation (26,28). The strains of obligately alkaliphilic, sulfatereducing MTB, discussed above, initially displayed weak to no magnetotactic responses when first isolated, apparently due to scavenging of iron by sulfide produced during sulfate reduction, resulting in the precipitation of black iron sulfides.…”

“…Using either agar plates or shake tubes, colonies of MTB are usually brown or black due to the formation of magnetite (65,68). For those organisms that do not form colonies either on plates or in shake tubes, pure cultures can be obtained by a repeated series of dilutions to extinction in media as long as the dominant bacterium present in the original culture is the one targeted for isolation (26,28,49,53).…”

“…The strains of obligately alkaliphilic, sulfatereducing MTB, discussed above, initially displayed weak to no magnetotactic responses when first isolated, apparently due to scavenging of iron by sulfide produced during sulfate reduction, resulting in the precipitation of black iron sulfides. To obtain a stronger magnetotactic response, the iron concentration was increased from 20 to 200 M, and the headspace of the cultures was purged every other day with oxygen-free argon gas in order to decrease the concentration of hydrogen sulfide in the cultures (28). This issue of iron availability may be true for other sulfatereducing MTB such as Desulfovibrio magneticus, since this organism produces very few magnetosomes when grown anaerobically with sulfate compared to growth with fumarate (63).…”

“…Remarkably, the growth of cultivated Magnetospirillum species is inhibited at iron concentrations of Ͼ200 M (57), suggesting that intracellular magnetite biomineralization is not an adaptation specific to iron-rich environments. Ferric citrate, ferric quinate, ferric malate, and ferrous sulfate are the iron sources most often used for growth and magnetite or greigite biomineralization, as they can be prepared easily and autoclaved together with other medium components, usually without precipitation (28,(58)(59)(60). It is important to understand that Fe(II) and Fe(III) inverse concentration gradients form in the oxygen concentration gradient medium described in the paragraph above due to the presence of chemical reducing agents.…”