Earth’s Cryosphere, 2024, Vol. XXVIII, No. 2, p. 12-19.
PERMAFROST MICROBIOLOGY
MICROBIAL REDUCTION OF Fe(III) IN TUNDRA SOIL SAMPLES FROM THE EAST SIBERIAN ARCTIC
A.G. Zakharyuk1,*, V.E. Trubitsyn1, T.A. Vishnivetskaya2, E.M. Rivkina2, V.A. Shcherbakova1
1 Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, prosp. Nauki 5, Pushchino, Moscow region, 142290 Russia
2 Institute of Physicochemical and Biological Problems of Soil Science, Russian Academy of Sciences, prosp. Nauki 2, Pushchino, Moscow region, 142290 Russia
*Corresponding author; e-mail: kuran82@mail.ru
Enrichment cultures of psychrophilic and psychrotolerant bacteria capable of reducing ferric iron at temperatures of 6 to 15°C were obtained from samples of two tundra soils from the Bykovsky Peninsula (Northern Yakutia, eastern sector of Russian Arctic). The highest concentrations of Fe(II) were observed in enrichment cultures grown with the use of a soluble ferric salt in the form of Fe(III) citrate. Furthermore, anaerobic communities from two enrichment cultures derived from permafrost soil samples of the Mammoth Khayata tract and cultivated at 15°C demonstrated a preference for insoluble Fe(III) oxide as an electron acceptor while utilizing acetate and formate as electron donors. Experimental data on the composition of microbial communities inhabiting permafrost soils were obtained through molecular biology and bioinformatics methods. Notably, this study presents a novel comparison between the composition of a naturally occurring microbial community that developed over an extended period under natural conditions at low temperatures, and a laboratory-cultivated microbial community. The results demonstrate that prokaryotic communities of the soils of Arctic ecosystems of Yakutia are phylogenetically diverse despite the cold and oligotrophic (nutrient-poor) conditions. While representatives of Proteobacteria and Actinobacteria phyla dominate in natural samples of tundra soil (~30–50%), the cultivated microbial community obtained in vitro from natural samples was dominated by Firmicutes (38%).
Keywords: tundra zone soils, Arctic, iron reduction, anaerobic communities, metagenome.
Recommended citation: Zakharyuk A.G., Trubitsyn V.E., Vishnivetskaya T.A., Rivkina E.M., Shcherbakova V.A., 2024. Microbial reduction of Fe(III) in tundra soil samples from the East Siberian Arctic. Earth’s Cryosphere XXVIII (2), 12–19.
Full text.
INTRODUCTION
For at least the last million years, climatic conditions in the Northern Hemisphere have been favorable for the formation of permafrost [Abramov et al., 2021]. Bacteria and archaea are discovered in Arctic tundra soils and permafrost of various ages. In permafrost, prokaryotes are forced to survive at subzero temperatures, low nutrient supply, and low free water content for long periods.
Biogeochemical studies of Arctic soils provide a comprehensive view of the interactions between the environment and soil communities and their contribution to ecosystem functioning and global climate regulation. The iron cycle carried out by microorganisms is one of the most important processes of biogeochemical cycles. For a long time, redox reactions involving iron were considered predominantly abiotic processes [Kappler et al., 2021]. Iron reduction by microorganisms attracted the attention of scientists in the 1950s. At that time, the anaerobic iron cycle was considered mainly in the works of soil microbiologists and concerned the reduction of iron in soils and silts by heterotrophic microorganisms, which most often reduce iron through the assimilation pathway [Bromfield, 1954; Kalakutsky, Duda, 1964]. In this case, iron reduction is not accompanied by the release of energy, but it can stimulate growth and change the composition of enzymatic products. Microorganisms typically use trivalent iron as an acceptor for dumping excess electrons. The question of whether prokaryotes are able to obtain energy from iron reduction remained open for a long time. However, in 1980, the possibility of iron reduction via using molecular hydrogen as an electron donor was demonstrated [Balashova, Zavarzin, 1980]. Soon after, within a very short time, a physiological group of dissimilatory iron reducers using various iron compounds for respiration was discovered [Slobodkin, Wiegel, 1997; Holmes et al., 2004; Nixon et al., 2017; Zhang et al., 2022]. To date, a number of studies have been published showing that the reduction of Fe(III) plays an important role at the terminal stages of organic carbon oxidation under anaerobic conditions in Arctic soils and sediments [Kostka et al., 1999; Glud et al., 2000; Rivkina et al., 2020].
The aim of this study was to identify microbial processes of iron reduction, obtain enrichment cultures of iron-reducing bacteria (IRB), and conduct a comparative analysis of the phylogenetic diversity of prokaryotes both in enrichment cultures and in the original soil samples from the Bykovsky Peninsula.
MATERIALS AND METHODS
Area and objects of the study. In this study, samples were collected from two soil profiles in the eastern Arctic sector, on the Bykovsky Peninsula (Buor-Khaya Gulf) near Mammoth Khayata (Section 139-19; 71°46’56.3″ N, 129°24’47.5″ E) and near Ivashkina Lagoon (Section 149-19; 71°44’43» N, 129°24’21» E) (Fig. 1).
Sampling was carried out in compliance with all sterility requirements, as described previously [Gilichinsky et al., 1989; Shi et al., 1997]. Soil samples were stored frozen at –20°C until microbiological studies began.
The Mammoth Khayata site is located in the middle part of the Bykovsky Peninsula. Section 139-19 was laid on the surface of a hill with an absolute height of 40 m. For microbiological studies, three samples of the permafrost-affected soddy gley soil were taken from horizons designated as BG, AP and DP. The BG horizon (0–8 cm) was represented by loose peaty loam of brownish gray color with dark brown tint in the upper part, The AP horizon (3-18 cm) was represented by moist, compacted, structureless silty loam of nonuniform color with alternating bluish gray, brownish gray, and ocherous-brown mottles. The DP horizon (36–44 cm) was represented by moist, compacted, structureless bluish gray silty loam.
Section 149-19 was laid on a ridge of a polygonal bog on an alas terrace in the western part of the Ivashkina Lagoon basin. For microbiological studies, three samples of the permafrost-affected peat gley soil were taken from horizons designated as TG, G2 and GC. The TG horizon (8–17 cm) was represented by moist, dense, bluish peat with the high content of evenly distributed loamy material. The G2 horizon ( 27–35 cm) was represented by moist, dense, structureless silty clay loam of nonuniform color, with black mottles unevenly distributed in the background bluish gray material. The G2 horizon (35–70 cm) was represented by bluish gray, compacted, structureless silty clay loam containing ice crystals.
The maximum concentration of HCO3⁻ in the samples reached 42.7 mg/L. The highest concentrations of Cl– ions (74.3 mg/L) and Na⁺ ions (70.8 mg/L) were determined in the water extract from the TG horizon (8–17 cm) sampled in the Ivashkina Lagoon. The concentration of SO4²– in water extracts from the studied samples was high and varied from 14.4 to 56.5 mg/L. The pH of all samples was slightly acidic (Table 1).
Table 1. Physicochemical characteristics of the studied soil samples
Conditions for cultivating microorganisms. To obtain enrichment cultures of IRB for their further study, 1 g of soil was added to 60 mL of cultural medium. We used a modified medium of the following composition (g/L): NaCl – 1; MgCl2×6H2O – 0.2; NaHCO3 – 2.5; KH2PO4 – 0.68; CaCl2×2H2O – 0.1; NH4Cl – 1; yeast extract (Difco) – 0.002; solution of microelements [Slobodkin, Wiegel, 1997] – 1.0 mL; vitamin solution [Wolin et al., 1963] – 10.0 mL. A mixture of sodium formate and sodium acetate at a final concentration of 20 millimoles (mM) was used as a source of carbon and electron donor. Fe(III) citrate (10 mM) and an analogue of the natural mineral ferrihydrite in the form of amorphous iron (III) hydroxide (10 mM), which was prepared by titrating a 10% NaOH (wt/vol) solution of FeCl3×6H2O, were added as terminal electron acceptors. The preparation of the mineral medium and the cultivation of microorganisms were carried out under strictly anaerobic conditions under N2 (100% in the gas phase). The pH of the medium was 7.0–7.2. Numerous studies of permafrost have shown that microorganisms isolated from permafrost most often turn out to be mesophiles with a maximum growth temperature of 20–30°С [Bai et al., 2006; Steven et al., 2006, 2008; Zhang et al., 2007, 2013]. At the same time, true psychrophiles [according to Morita, 1975], having an optimal temperature for growth of ≤15°С, constitute a minority in the samples [Steven et al., 2007; Rivkina et al., 2016]. In order to cover the greatest possible diversity of cultivated cold-resistant microorganisms inhabiting tundra soil, the authors chose two cultivation temperatures: 6°C and 15°C. Incubation was carried out in the dark for 30–60 days. A mineral medium without inoculation was used as a chemical control to monitor abiotic iron reduction.
Morphology of microorganisms. Living bacterial cells were examined using an Axiostar PLUS light microscope with phase contrast(Carl Zeiss, Germany) at magnification of 1000×.
Analytical methods. The reduction of ferric iron was determined by the colorimetric method by the formation of a stable colored complex of ferrous iron with ferrozine [Viollier et al., 2000]. The concentration of Fe(II) was measured on a Spekol 221 spectrophotometer (Germany) at a wave length of 562 nm.
Molecular methods. To isolate total DNA from soil samples and enrichment cultures, the PowerSoil® kit (MO BIO Laboratories Inc., USA) was used; the resulting DNA was purified and concentrated using the Genomic DNA Clean and Concentrator® Kit (Zymo Research Corporation, USA). Sequencing was carried out using Oxford Nanopore technology in a MinIONMk1B sequencer on an R9.4.1 cell running the MinKNOW v.5.1.0 program. The assembly of metagenome contigs was performed by the Flye v.2.9-b1774 program with filtering by the length of incoming reads of 500 base pairs (bp). The quality of reads and statistical parameters of the assembly were assessed using FastQC v. 0.11.9 and prinseq-lite v. 0.20.4.
Phylogenetic analysis. Metagenome sequences were classified using Kraken2 v. 2.1.2 classifier and NCBI databases “bacteria,” “archaea,” “viral,”“fungi,” and the “—max-db-size” limit of 16 GB. For ease of use, the data are presented in the form of an interactive diagram in the Krona v.2.8.1 program. Bayesian reestimation of phylogenetic diversity and filtering of false entries were carried out by the companion program Bracken v.2.8. with an optimal kmer length of 6884 base pairs.
RESULTS AND DISCUSSION
Obtaining enrichment cultures of IRB. Overall, twenty-four accumulative cultures of bacteria capable of reducing Fe(III) compounds with formate and acetate as a carbon source and electron donor were obtained from various horizons of the studied soils at cultivation temperatures of 6 and 15°C. In model experiments, such common forms of reduced iron as ferrihydrite and soluble iron salt in the form of Fe(III) citrate were used as electron acceptors. On the thirteenth day of incubation, the amount of reduced iron in enrichment cultures obtained from the samples collected in the area of Mammoth Khayata varied from 0.2 to 15.0 mM. The most active reduction of iron was observed at cultivation temperature of 15°C. Maximum amounts of ferrous iron (10.5 and 15.0 mM) were recorded in the DP enrichment culture obtained from the DP horizon (36–44 cm). The minimum content of ferrous iron (0.2 mM) was detected in the BG enrichment culture obtained from the upper (BG) horizon (0–8 cm) with ferrihydrite as an electron acceptor (Table 2).
In enrichment cultures obtained from soil samples from the Ivashkina Lagoon area, the concentration of Fe(II) varied from 2.9 to 12.5 mM. The maximum of reduced iron was determined in the TG enrichment culture (12.5 mM) obtained from the TG horizon and grown with Fe(III) citrate at a temperature of 15°C. The minimum was recorded in enrichment cultures obtained from G2 and GC horizons (2.9 mM) cultivated at 15°C with ferrihydrite as an electron acceptor (Table 3). The concentration of Fe(II) in the chemical control did not exceed 0.1 mM.
As a result of laboratory experiments, the authors did not identify a clear relationship between the cultivation temperature and the amount of reduced iron in the enrichment cultures. The form of the Fe(III)-containing compound had a greater influence on the process of microbial iron reduction. Thus, in all enrichment cultures obtained from soil samples of the Ivashkina Lagoon site and grown with Fe(III) citrate as an electron acceptor, the amount of Fe(II) was greater than in enrichment cultures grown under the same conditions, but using Fe (III) in the form of insoluble oxides. For 75% of enrichment cultures obtained from soil samples from the Mammoth Khayata site, soluble Fe(III) salt was also the preferred electron acceptor.
However, the anaerobic communities of two enrichment cultures obtained from the frozen soils of the Mammoth Khayata site upon cultivation at 15°С (Table 2) preferred insoluble Fe(III) oxide as an electron acceptor, and acetate and formate as electron donors. Both substrates are important carbon sources for anaerobic microbial iron reduction. As solid oxide forms of iron predominate in natural environments at a close to neutral reaction, microorganisms that reduce such substances are of particular interest from a biogeochemical point of view. Research has shown that iron-reducing microbial communities in various natural ecosystems subject to limited electron donors successfully compete with sulfate reducers or methanogens only in the presence of ferrihydrite or similar weakly crystallized forms of iron oxides [Roden, Wetzel, 2002].
As a result of microscopic studies of the obtained enrichment cultures of IRB, several morphotypes of bacterial cells were discovered. Each of the studied communities contained mobile and immobile rod-shaped cells of different sizes. During microscopy of enrichment cultures TG, BG and DP, rods with terminally located spores were observed. The presence of different cell morphotypes indicates the diversity of phylogenetic groups of psychrophilic and psychrotolerant microorganisms that formed in the studied microbial communities. The presence of spore-forming bacteria may be explained by their ability to survive as spores rather than as vegetative cells. The results obtained by the authors are consistent with the results previously published by Peterson with coauthors in a study of samples taken on the left bank of the Aldan River, at the Mamontova Mount site ( (Central Yakutia). A group of researchers demonstrated the presence of bacilli having relatively large rods with round spores in permafrost [Peterson et al., 2011]. It is known that spores of psychrophilic microorganisms are the most resistant [Nicholson et al., 2000] and probably aid survival at low temperatures.
Metagenome analysis. Analysis of annotated metagenomes of natural samples from the two sections showed that representatives of the phyla Proteobacteria and Actinobacteria dominated in the studied natural microbial communities (~30–50%). In addition, Firmicutes and Bacteroidetes were the major phyla (˃5%) present in all the samples. It should be noted that the number of representatives of the phylum Bacteroidetes increased with the depth in both soil profiles. Representatives of the phyla Chloroflexi and Acidobacteria were found almost everywhere, although in small quantities. The number of Acidobacteria decreased with the depth of the soil profiles. Minor components (˂5%) of natural microbial communities of Arctic soils were representatives of the phylum Cyanobacteria (Fig. 2).
At present, most of the dissimilatory described IRBs belong to the phylum Proteobacteria [Lovley et al., 2004; Sung et al., 2006; Weber et al., 2006]. In the upper horizon of the natural sample TG of section 149-19 (Fig. 2), representatives of Proteobacteria constituted more than 50% of the entire community. This suggests that Proteobacteria not only play an important role in the microbiocenosis of this region but are also likely to be directly involved in the processes of microbial iron reduction playing a key role in the biogenic transformation of iron minerals in Arctic soils.
To compare the diversity of the prokaryotic community inhabiting the tundra soils and the cultivated microbial community obtained in the laboratory, the TG enrichment culture from the upper (TG) soil horizon sampled at the Ivashkina Lagoon and cultivated with Fe(III) citrate at 6°C was selected.
As a result of sequencing the microbial community of the TG enrichment culture using Oxford Nanopore technology, after removing barcodes, a library was obtained with 542822 reads ranging in length from 1 to 178849 bp, assembled into 1688 contigs with a length of 548 to 387801 bp. The average contig length was 6884 bp. Using the Kraken 2 program, contigs belonging to the domains Bacteria (78.14%), Archaea (0.8%), and Basidiomycota (0.1%) were identified. Based on the above analysis, contigs from the Bacteria domain belonged to four phyla: Firmicutes (38%), Proteobacteria (14%), Actinobacteria (13%), and Bacteroidetes (7%) (Fig. 3).
It was also found that in the cultivated IRB community, the dominance of representatives of the phylum Firmicutes was strongly expressed compared to the natural samples. It is known that many representatives of this phylum are capable of reducing Fe(III)and use it to drain electrons in the anaerobic microbial community [Zhilina et al., 2009; Moe et al., 2012]. Kappler et al. [2004] demonstrated that enzymatic bacteria represent the largest population in neutral natural environments and play an important role in the reduction of humus and humic acids. The enzymatic iron reducer Pelosinus baikalensis, previously isolated by the authors [Zakharyuk et al., 2023] from a cold freshwater lake, was capable of restoring an analogue of humic substances—anthraquinone-2,6-disulfotane (AQDS)—in the presence of a fermentable substrate with the formation of a reduced form of AHQDS. Additionally, in laboratory experiments, this strain oxidized lactate and reduced synthesized ferrihydrite (an insoluble electron acceptor) using humic quinones (in our case, AQDS) as shuttle electron carriers.
Analysis of the content of lower-order taxa using Bracken showed that the enrichment culture contained eight species belonging to the genera Acetobacterium (52.15%), Pseudomonas (23.54%), Pelosinus (16.36%), and Proteiniphilum (7.85%). The most widely represented were Acetobacterium woodii (29.28% of selected reads) and Pseudomonas fluorescens (23.54% of reads) (Table 4).
Currently, the genus Acetobacterium includes 11 species, including Acetobacterium tundrae isolated from the tundra soil of the Northern Urals [Simankova et al., 2000]. Studies on the ability to reduce iron compounds have not been conducted for any of the described species. However, acetobacteria produce acetate while growing on Н2/СО2 and formate, which is an electron donor for iron-reducing bacteria [Balch et al., 1977; Braun, Gottschalk, 1982]. In turn, it has been established that the strains of Pseudomonas fluorescens isolated from gley soil under anaerobic conditions use acetate as a carbon source and electron donor, thereby reducing ferric iron compounds [Pukhova, 2018]. Representatives of Pelosinus fermentans [Shelobolina et al., 2007] and Pelosinus sp. strain UFO1[Ray et al., 2018] found by the authors in the enrichment culture are also capable of reducing various compounds of FeIII) and humic acids in the presence of a fermentable substrate in the medium.
As a result, in laboratory conditions the authors obtained an accumulative culture, the microbial community of which was formed by representatives of different phylogenetic groups, both capable of forming acetate under anaerobic conditions, and capable of using acetate and other organic substances while reducing various compounds of Fe(III).
CONCLUSION
The results of laboratory experiments suggest that the highest concentrations of Fe(II) are recorded in enrichment cultures grown using soluble compounds of Fe(III). The process of iron reduction in such enrichment cultures occurs faster than in the case of using ferrihydrite.
As Fe(III) can be used by microorganisms not only as an electron acceptor during the respiratory type of metabolism but can also be reduced by many prokaryotes with a fermentative type of metabolism, carrying out the so-called facilitated fermentation [Slobodkin et al., 2006; Shelobolina et al., 2007; Pollock et al., 2007], it is possible that in the microbial communities obtained by the authors, iron is reduced in two ways: assimilation during facilitated fermentation and dissimilation using Fe(III) as an energy source.
For the first time, a comparison was made between the composition of a natural microbial community formed over a long period of time under natural conditions at low temperatures and a cultured microbial community reducing Fe (III) and obtained from a soil sample of the tundra zone (TG horizon, section 149-19). Data on the composition of microbial communities inhabiting soils of the Siberian sector of the Arctic and participating in iron reduction were obtained using the methods of molecular biology and bioinformatics.
Further studies of enrichment cultures and detailed analysis of metagenomes will allow a more detailed study of the pathways and mechanisms of biotransformation of iron minerals in the active layer, and will also provide an opportunity to assess the role of psychrophilic iron-containing bacteria in the destruction of organic matter and the development of redox conditions in the modern permafrost soils of northern Yakutia.
Acknowledgments. The work was carried out with financial support from the Russian Science Foundation grant No. 22-24-00518.
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Received June 26, 2023
Revised February 4, 2024
Accepted February 10, 2024
Translated by E. Shelekhova