Abundance, distribution, and activity of Fe(II)-oxidizing and Fe(III)-reducing microorganisms in hypersaline sediments of Lake Kasin, southern Russia

Abstract

The extreme osmotic conditions prevailing in hypersaline environments result in decreasing metabolic diversity with increasing salinity. Various microbial metabolisms have been shown to occur even at high salinity, including photosynthesis as well as sulfate and nitrate reduction. However, information about anaerobic microbial iron metabolism in hypersaline environments is scarce. We studied the phylogenetic diversity, distribution, and metabolic activity of iron(II)-oxidizing and iron(III)-reducing Bacteria and Archaea in pH-neutral, iron-rich salt lake sediments (Lake Kasin, southern Russia; salinity, 348.6 g liter(-1)) using a combination of culture-dependent and -independent techniques. 16S rRNA gene clone libraries for Bacteria and Archaea revealed a microbial community composition typical for hypersaline sediments. Most-probable-number counts confirmed the presence of 4.26 × 10(2) to 8.32 × 10(3) iron(II)-oxidizing Bacteria and 4.16 × 10(2) to 2.13 × 10(3) iron(III)-reducing microorganisms per gram dry sediment. Microbial iron(III) reduction was detected in the presence of 5 M NaCl, extending the natural habitat boundaries for this important microbial process. Quantitative real-time PCR showed that 16S rRNA gene copy numbers of total Bacteria, total Archaea, and species dominating the iron(III)-reducing enrichment cultures (relatives of Halobaculum gomorrense, Desulfosporosinus lacus, and members of the Bacilli) were highest in an iron oxide-rich sediment layer. Combined with the presented geochemical and mineralogical data, our findings suggest the presence of an active microbial iron cycle at salt concentrations close to the solubility limit of NaCl.

Fig 1

Concentration of different iron fractions in a sediment profile of Lake Kasin. White bars, 0.5 M HCl-extractable (bioavailable) iron. Black bars, crystalline iron, extracted during incubation in 6 M HCl at 70°C for 24 h. Iron concentrations in the extracts were determined with the ferrozine assay (82). Error bars represent standard deviations calculated from duplicate samples.

Fig 2

Mössbauer spectra of the Fe-rich layer of Lake Kasin sediment (1.5 cm of depth) recorded at room temperature (RT) (top), 77 K (middle), and 5 K (bottom). Sextets (S) and doublets (D) are labeled as listed in Table S3 in the supplemental material.

Fig 3

Most-probable-number (MPN) counts of Fe(III)-reducing (FeRed) and anaerobic Fe(II)-oxidizing (anFeOx) microorganisms from the top 10 cm of Lake Kasin in mineral medium with 5 M (black bars) and 0.5 M NaCl (white bars), respectively. Medium for FeRed was supplemented with 0.5 M ferrihydrite as electron acceptor and 0.5 M lactate and acetate each as electron donors. For anFeOx, 10 mM FeCl₂ was added as the electron donor and 0.4 M NO₃⁻ as the electron acceptor. The Fe(II) oxidizer medium further contained 0.05 M acetate as a carbon source. Error bars denote 95% confidence intervals determined from seven replicate samples according to the work of Klee (34).

Fig 4

(A and B) Classification of 231 archaeal (A) and 299 bacterial (B) full-length 16S rRNA gene sequences retrieved from a 0- to 10-cm composite sample of Lake Kasin. (C and D) Rarefaction curves for the archaeal and bacterial sequences from the respective clone libraries for three different sequence identity cutoff values (99%, 97%, and 93%). The archaeal rarefaction curve for the 97% cutoff value in C is not visible because it exactly resembles the 93% curve. Rarefaction curves were calculated with the program MOTHUR (75).

Fig 5

Maximum likelihood trees of archaeal (A) and bacterial (B) 16S rRNA gene sequences directly amplified from Lake Kasin sediment or obtained from the Fe(III)-reducing enrichment cultures. (A) ML tree of the Halanaerobiaceae. (B) ML tree of the Bacilli and the Peptococcaceae family of the Clostridia. Groups for which at least one representative sequence was found in Lake Kasin sediment or enrichments are printed in black. Groups with no representatives from Lake Kasin sediment or enrichments are printed in gray. (Groups of) sequences that match both forward and reverse qPCR primers designed for Halobaculum gomorrense (A)- and Desulfosporosinus sp. or Bacilli (B)-related sequences are shown in boldface.

Fig 5

Maximum likelihood trees of archaeal (A) and bacterial (B) 16S rRNA gene sequences directly amplified from Lake Kasin sediment or obtained from the Fe(III)-reducing enrichment cultures. (A) ML tree of the Halanaerobiaceae. (B) ML tree of the Bacilli and the Peptococcaceae family of the Clostridia. Groups for which at least one representative sequence was found in Lake Kasin sediment or enrichments are printed in black. Groups with no representatives from Lake Kasin sediment or enrichments are printed in gray. (Groups of) sequences that match both forward and reverse qPCR primers designed for Halobaculum gomorrense (A)- and Desulfosporosinus sp. or Bacilli (B)-related sequences are shown in boldface.

Fig 6

Changes in relative abundance of potential Fe(III)-reducing taxa in a sediment profile of Lake Kasin. Bars indicate the abundance of Halobaculum gomorrense relatives (A), Desulfosporosinus sp. relatives (B), and Bacilli (C) relative to total Archaea (A) or total Bacteria (B and C) cell numbers for each sediment layer. Error bars refer to standard deviations of six individual qPCR measurements recorded in triplicate during two independent runs. The gray-shaded area in the background shows Fe (total) concentrations in % dry weight of the sediment.