Lanthanide-Dependent Methanol Metabolism of a Proteobacteria-Dominated Community in a Light Lanthanide-Rich Deep Environment.

This study investigated the occurrence and diversity of proteobacterial XoxF-type methanol dehydrogenases (MDHs) in the microbial community that inhabits a fossil organic matter- and sedimentary lanthanide (Ln3+)-rich underground mine environment using a metagenomic and metaproteomic approach. A total of 8 XoxF-encoding genes (XoxF-EGs) and 14 protein sequences matching XoxF were identified. XoxF-type MDHs were produced by Alpha-, Beta-, and Gammaproteobacteria represented by the four orders Methylococcales, Nitrosomonadales, Rhizobiales, and Xanthomonadales. The highest number of XoxF-EG- and XoxF-matching protein sequences were affiliated with Nitrosomonadales and Rhizobiales, respectively. Among the identified XoxF-EGs, two belonged to the XoxF1 clade, five to the XoxF4 clade, and one to the XoxF5 clade, while seven of the identified XoxF proteins belonged to the XoxF1 clade, four to the XoxF4 clade, and three to the XoxF5 clade. Moreover, the accumulation of light lanthanides and the presence of methanol in the microbial mat were confirmed. This study is the first to show the occurrence of XoxF in the metagenome and metaproteome of a deep microbial community colonizing a fossil organic matter- and light lanthanide-rich sedimentary environment. The presented results broaden our knowledge of the ecology of XoxF-producing bacteria as well as of the distribution and diversity of these enzymes in the natural environment.

Postdiagenetic Changes in Kerogen Properties and Type by Bacterial Oxidation and Dehydrogenation.

A significant part of organic carbon found on the earth is deposited as fossil organic matter in the lithosphere. The most important reservoir of carbon is shale rocks enriched with organic matter in the form of kerogen created during diagenesis. The purpose of this study was to analyze whether the bacterial communities currently inhabiting the shale rocks have had any impact on the properties and type of kerogen. We used the shale rock located on the Fore-Sudetic Monocline, which is characterized by oil-prone kerogen type II. We were able to show that shale rock inhabited by bacterial communities are characterized by oxidized and dehydrated kerogen type III (gas-prone) and type IV (nonproductive, residual, and hydrogen-free). Bacterial communities inhabiting shale rock were dominated by heterotrophs of the Proteobacteria, Firmicutes, and Actinobacteria phyla. Additionally, we detected a number of protein sequences in the metaproteomes of bacterial communities matched with enzymes involved in the oxidative metabolism of aliphatic and aromatic hydrocarbons, which may potentially contribute to the postdiagenetic oxidation and dehydrogenation of kerogen. The kerogen transformation contributes to the mobilization of fossil carbon in the form of extractable bitumen dominated by oxidized organic compounds.

Biosynthesis of Tetrapyrrole Cofactors by Bacterial Community Inhabiting Porphyrine-Containing Shale Rock (Fore-Sudetic Monocline).

This study describes for the first time the comprehensive characterization of tetrapyrrole cofactor biosynthetic pathways developed for bacterial community (BC) inhabiting shale rock. Based on the genomic and proteomic metadata, we have detailed the biosynthesis of siroheme, heme, cobalamin, and the major precursor uroporphyrinogen III by a deep BC living on a rock containing sedimentary tetrapyrrole compounds. The obtained results showed the presence of incomplete heme and cobalamin biosynthesis pathways in the studied BC. At the same time, the production of proteins containing these cofactors, such as cytochromes, catalases and sulfite reductase, was observed. The results obtained are crucial for understanding the ecology of bacteria inhabiting shale rock, as well as their metabolism and potential impact on the biogeochemistry of these rocks. Based on the findings, we hypothesize that the bacteria may use primary or modified sedimentary porphyrins and their degradation products as precursors for synthesizing tetrapyrrole cofactors. Experimental testing of this hypothesis is of course necessary, but its evidence would point to an important and unique phenomenon of the tetrapyrrole ring cycle on Earth involving bacteria.

Characterization of Halomonas sp. ZM3 isolated from the Zelazny Most post-flotation waste reservoir, with a special focus on its mobile DNA.

BACKGROUND: Halomonas sp. ZM3 was isolated from Zelazny Most post-flotation mineral waste repository (Poland), which is highly contaminated with heavy metals and various organic compounds. Mobile DNA of the strain (i.e. plasmids and transposons) were analyzed in order to identify genetic information enabling adaptation of the bacterium to the harsh environmental conditions. RESULTS: The analysis revealed that ZM3 carries plasmid pZM3H1 (31,370 bp), whose replication system may be considered as an archetype of a novel subgroup of IncU-like replicons. pZM3H1 is a narrow host range, mobilizable plasmid (encodes a relaxase of the MOBV family) containing mercury resistance operon (mer) and czcD genes (mediate resistance to zinc and cobalt), which are part of a large truncated Tn3 family transposon. Further analysis demonstrated that the phenotypes determined by the pZM3H1 resistance cassette are highly dependent on the host strain. In another strand of the study, the trap plasmid pMAT1 was employed to identify functional transposable elements of Halomonas sp. ZM3. Using the sacB positive selection strategy two insertion sequences were identified: ISHsp1 - representing IS5 group of IS5 family and ISHsp2 - a distinct member of the IS630 family. CONCLUSIONS: This study provides the first detailed description of mobile DNA in a member of the family Halomonadaceae. The identified IncU plasmid pZM3H1 confers resistance phenotypes enabling adaptation of the host strain to the Zelazny Most environment. The extended comparative analysis has shed light on the distribution of related IncU plasmids among bacteria, which, in many cases, reflects the frequency and direction of horizontal gene transfer events. Our results also identify plasmid-encoded modules, which may form the basis of novel shuttle vectors, specific for this group of halophilic bacteria.

Postdiagenetic Bacterial Transformation of Nickel and Vanadyl Sedimentary Porphyrins of Organic-Rich Shale Rock (Fore-Sudetic Monocline, Poland).

Nickel and vanadyl porphyrins belong to the so-called fossil geo- or sedimentary porphyrins. They occur in different types of organic matter-rich sediments but mostly occur in crude oils and their source rocks, oil shales, coals, and oil sands. In this study, we aimed to understand the process of bacterial transformation of geoporphyrins occurring in the subsurface shale rock (Fore-Sudetic Monocline, SW Poland). We studied these transformations in rock samples directly obtained from the field; in rock samples treated with bacterial strain isolated from shale rock (strain LM27) in the laboratory; and using synthetic nickel and vanadyl porphyrins treated with LM27. Our results demonstrate the following: (i) cleavage and/or degradation of aliphatic and aromatic substituents of porphyrins; (ii) degradation of porphyrin (tetrapyrrole) ring; (iii) formation of organic compounds containing 1, 2, or 3 pyrrole rings; (iv) formation of nickel- or vanadium-containing organic compounds; and (v) mobilization of nickel and vanadium. Our results also showed that the described bacterial processes change the composition and content of geoporphyrins, composition of extractable organic matter, as well as nickel and vanadium content in shale rock.

Sedimentary Cobalt Protoporphyrin as a Potential Precursor of Prosthetic Heme Group for Bacteria Inhabiting Fossil Organic Matter-Rich Shale Rock.

This study hypothesizes that bacteria inhabiting shale rock affect the content of the sedimentary cobalt protoporphyrin present in it and can use it as a precursor for heme synthesis. To verify this hypothesis, we conducted qualitative and quantitative comparative analyses of cobalt protoporphyrin as well as heme, and heme iron in shale rock that were (i) inhabited by bacteria in the field, (ii) treated with bacteria in the laboratory, and with (iii) bacterial culture on synthetic cobalt protoporphyrin. Additionally, we examined the above-mentioned samples for the presence of enzymes involved in the heme biosynthesis and uptake as well as hemoproteins. We found depletion of cobalt protoporphyrin and a much higher heme concentration in the shale rock inhabited by bacteria in the field as well as the shale rock treated with bacteria in the laboratory. Similarly, we observed the accumulation of protoporphyrin in bacterial cells grown on synthetic cobalt protoporphyrin. We detected numerous hemoproteins in metaproteome of bacteria inhabited shale rock in the field and in proteomes of bacteria inhabited shale rock and synthetic cobalt protoporhyrin in the laboratory, but none of them had all the enzymes involved in the heme biosynthesis. However, proteins responsible for heme uptake, ferrochelatase and sirohydrochlorin cobaltochelatase/sirohydrochlorin cobalt-lyase were detected in all studied samples.

Occurrence of XoxF-type methanol dehydrogenases in bacteria inhabiting light lanthanide-rich shale rock.

This study analyzed the occurrence of lanthanide-dependent (XoxF type) methanol dehydrogenases in the bacterial community dominated by Proteobacteria inhabiting shale rock. In total, 22 sequence matches of XoxF were identified in the metaproteome of the community. This enzyme was produced by bacteria represented by orders such as Rhizobiales, Rhodobacterales, Rhodospiralles, Burkholderiales and Nitrosomonadales. Among the identified XoxF proteins, seven belonged to XoxF1 clade and 15 to XoxF5 clade. This study is the first to show the occurrence of XoxF proteins in the metaproteome of environmental lithobiontic bacterial community colonizing an underground rock rich in light lanthanides. The presented results broaden our understanding of the ecology of XoxF producing bacteria as well as the distribution and diversity of these enzymes in the natural environment.

The Oxidative Metabolism of Fossil Hydrocarbons and Sulfide Minerals by the Lithobiontic Microbial Community Inhabiting Deep Subterrestrial Kupferschiefer Black Shale.

Black shales are one of the largest reservoirs of fossil organic carbon and inorganic reduced sulfur on Earth. It is assumed that microorganisms play an important role in the transformations of these sedimentary rocks and contribute to the return of organic carbon and inorganic sulfur to the global geochemical cycles. An outcrop of deep subterrestrial ~256-million-year-old Kupferschiefer black shale was studied to define the metabolic processes of the deep biosphere important in transformations of organic carbon and inorganic reduced sulfur compounds. This outcrop was created during mining activity 12 years ago and since then it has been exposed to the activity of oxygen and microorganisms. The microbial processes were described based on metagenome and metaproteome studies as well as on the geochemistry of the rock. The microorganisms inhabiting the subterrestrial black shale were dominated by bacterial genera such as Pseudomonas, Limnobacter, Yonghaparkia, Thiobacillus, Bradyrhizobium, and Sulfuricaulis. This study on black shale was the first to detect archaea and fungi, represented by Nitrososphaera and Aspergillus genera, respectively. The enzymatic oxidation of fossil aliphatic and aromatic hydrocarbons was mediated mostly by chemoorganotrophic bacteria, but also by archaea and fungi. The dissimilative enzymatic oxidation of primary reduced sulfur compounds was performed by chemolithotrophic bacteria. The geochemical consequences of microbial activity were the oxidation and dehydrogenation of kerogen, as well as oxidation of sulfide minerals.

Bacterial weathering of fossil organic matter and organic carbon mobilization from subterrestrial Kupferschiefer black shale: long-term laboratory studies.

A large part of the organic carbon present in the lithosphere is trapped in fossil organic matter deposited in sedimentary rocks. Only specialized microorganisms are able to degrade it contributing to the return of the carbon to the global cycle. The role of bacteria in this process is not yet completely understood. In the present laboratory studies, subterrestrial organic-rich ∼256-million-year-old Kupferschiefer black shale was exposed to the activity of an indigenous consortium of lithobiontic bacteria for 365 days under aerobic conditions. An interdisciplinary research approach was applied, consisting of a detailed comparison of the chemical composition of extractable bitumens as well as resistant to extraction kerogen of the unweathered black shale to that of the bioweathered and chemically weathered, identification of mobilized organic compounds and spectrometry-based determination of proteomic composition of the bacterial biofilm. The oxidative bioweathering of bitumens and kerogen was confirmed. The mobilization of organic carbon in the form of oxidized organic compounds, such as monohydroxy and dihydroxy alcohols, aldehydes, monocarboxylic and dicarboxylic acids and esters due to the microbial activity, was documented. The enzymes crucial for the aerobic metabolism of aliphatic and aromatic hydrocarbons such as monooxygenases and dehydrogenases were identified in the epilithic biofilm inhabiting the black shale.

The growth, ferrous iron oxidation and ultrastructure of Acidithiobacillus ferrooxidans in the presence of dibutyl phthalate.

The iron-oxidizing bacteria Acidithiobacillus ferrooxidans is an example of strictly chemolitotrophic extremophile occurring in acidic environments. The prime niche of these microorganisms is an environment with low pH and high concentrations of iron, sulfide minerals or sulfur. Besides these environments, A. ferrooxidans is also isolated from heavy metal contaminated environments such as soil and sewage sludge and is known to be useful in bioremediation processes of these environments. In the current study, the influence of dibutyl phthalate on the growth, activity and ultrastructure of A. ferrooxidans ATCC19859 was shown. The presence of dibutyl phthalate in 9K medium did not influence A. ferrooxidans growth or ability to oxidize ferrous iron although changes in growth medium were accompanied by changes in the protein expression profiles of periplasmic fractions and remarkable changes in ultrastructure of the cell.

Determination of factors responsible for the bioweathering of copper minerals from organic-rich copper-bearing Kupferschiefer black shale.

The aim of this study was to investigate the bioweathering of copper minerals present in the alkaline, copper-bearing and organic-rich Kupferschiefer black shale through the action of a consortium of indigenous lithobiontic, heterotrophic, neutrophilic bacteria isolated from this sedimentary rock. The involvement of microorganisms in the direct/enzymatic bioweathering of fossil organic matter of the rock was confirmed. As a result of bacterial activity, a spectrum of various organic compounds such as urea and phosphoric acid tributyl ester were released from the rock. These compounds indirectly act on the copper minerals occurring in the rock and cause them to weather. This process was reflected in the mobilization of copper, iron and sulfur and in changes in the appearance of copper minerals observed under reflected light. The potential role of identified enzymes in biodegradation of fossil organic matter and role of organic compounds released from black shale as a result of this process in copper minerals weathering was discussed. The presented results provide a new insight into the role of chemical compounds released by bacteria during fossil organic matter bioweathering potentially important in the cycling of copper and iron deposited in the sedimentary rock. The originality of the described phenomenon lies in the fact that the bioweathering of fossil organic matter and, consequently, of copper minerals occur simultaneously in the same environment, without any additional sources of energy, electrons and carbon.

Extracellular compounds produced by bacterial consortium promoting elements mobilization from polymetallic Kupferschiefer black shale (Fore-Sudetic Monocline, Poland).

Culture experiments employing Fe-deficient medium showed that a consortium of indigenous microorganisms isolated from Kupferschiefer black shale produced a mixture of extracellular compounds containing siderophores which could form complexes with a wide range of elements and were able to mediate element mobilization from polymetallic black shale. The mobilization of a diverse array of elements including a number of essential trace elements (Co, Cu, Mn, Mo, Zn) and toxic species (As) was shown. Since the bacteria used in this study were originally obtained from a subsurface copper deposit, these results highlight the potential importance of extracellular compounds in biogeochemical cycles of elements in underground environment and their ecological significance in promoting the uptake of essential trace metals and resistance to toxic elements.

Diversity and role of plasmids in adaptation of bacteria inhabiting the Lubin copper mine in Poland, an environment rich in heavy metals.

The Lubin underground mine, is one of three mining divisions in the Lubin-Glogow Copper District in Lower Silesia province (Poland). It is the source of polymetallic ore that is rich in copper, silver and several heavy metals. Black shale is also significantly enriched in fossil organic matter in the form of long-chain hydrocarbons, polycyclic aromatic hydrocarbons, organic acids, esters, thiophenes and metalloporphyrins. Biological analyses have revealed that this environment is inhabited by extremophilic bacteria and fungi. Kupfershiefer black shale and samples of water, bottom and mineral sediments from the underground (below 600 m) Lubin mine were taken and 20 bacterial strains were isolated and characterized. All exhibited multi-resistant and hypertolerant phenotypes to heavy metals. We analyzed the plasmidome of these strains in order to evaluate the diversity and role of mobile DNA in adaptation to the harsh conditions of the mine environment. Experimental and bioinformatic analyses of 11 extrachromosomal replicons were performed. Three plasmids, including a broad-host-range replicon containing a Tn3 family transposon, carried genes conferring resistance to arsenic, cadmium, cobalt, mercury and zinc. Functional analysis revealed that the resistance modules exhibit host specificity, i.e., they may increase or decrease tolerance to toxic ions depending on the host strain. The other identified replicons showed diverse features. Among them we identified a catabolic plasmid encoding enzymes involved in the utilization of histidine and vanillate, a putative plasmid-like prophage carrying genes responsible for NAD biosynthesis, and two repABC-type plasmids containing virulence-associated genes. These findings provide an unique molecular insight into the pool of extrachromosomal replicons and highlight their role in the biology and adaptation of extremophilic bacteria inhabiting terrestrial deep subsurface.

The method of contact angle measurements and estimation of work of adhesion in bioleaching of metals.

In this paper, we present our method for the measurement of contact angles on the surface of minerals during the bioleaching process because the standard deviation obtained in our measurements achieved unexpectedly low error. Construction of a goniometer connected with a specially prepared computer program allowed us to repeat measurements several times over a short time course, yielding excellent results.After defining points on the outline of the image of a drop and its baseline as well of the first approximation of the outline of the drop, an iterative process is initiated that is aimed at fitting the model of the drop and baseline. In turn, after defining the medium for which measurements were made, the work of adhesion is determined according to Young-Dupré equation. Calculations were made with the use of two methods named the L-M and L-Q methods.

The role of microorganisms in dispersion of thallium compounds in the environment.

Thallium is a highly toxic element and very rarely studied in the context of environmental hazards connected with zinc and non-ferrous metal industry. Microorganisms naturally existing in post-flotation and smelt wastes can participate in thallium release from waste deposits and can contribute to its dispersion in the environment. Twenty-one isolates were obtained from wastes of a non-ferrous smelter in Southern Poland characterised by high heavy metal contamination. Ten isolates showed high activity in thallium leaching from wastes (post-flotation and smelt wastes) as well as from pure thallous sulphide. Additionally, cadmium and lead were bioleached from wastes. The isolated bacteria indicated thallium resistance at a concentration up to 100 mg/l and some of them were able to survive in good condition at a concentration of up to 4 g/l. The same bacteria were isolated from rivers and wastewater in this region. A preliminary characterisation of isolates was performed. It was shown that some petroleum products i.e. asphalt-base crude occasionally used for waste immobilisation at the edge of pond or flotation surfactants partially stopped the activity of sulphide oxidising bacteria.

Biotransformation of copper from Kupferschiefer black shale (Fore-Sudetic Monocline, Poland) by yeast Rhodotorula mucilaginosa LM9.

This study describes the yeast Rhodotorula mucilaginosa strain LM9 isolated from copper-bearing, organic-rich Kupferschiefer black shale and its role in copper biotransformation. Strain LM9 exhibited great ability to simultaneously mobilize and immobilize copper from this sedimentary rock. In addition, it showed considerable resistance to copper and high uptake of this metal. Moreover, malic and oxalic acid as well as siderophore (rhodotorulic acid) produced by this strain enhanced its resistance by promoting the mobilization and complexation of copper from black shale. These processes, characterized here under laboratory conditions, are assumed to play a role in copper cycling in black shale as well as in the adaptation of strain LM9 to the conditions prevailing in its natural mine habitat. The findings of this study indicate that yeast strain LM9 might be used for the recovery of copper particularly from alkaline or slightly neutral ores in a non-chemical environmentally-friendly procedure.

Bioweathering of Kupferschiefer black shale (Fore-Sudetic Monocline, SW Poland) by indigenous bacteria: implication for dissolution and precipitation of minerals in deep underground mine.

The Upper Permian polymetallic, organic-rich Kupferschiefer black shale in the Fore-Sudetic Monocline is acknowledged to be one of the largest Cu-Ag deposits in the world. Here we report the results of the first study of bioweathering of this sedimentary rock by indigenous heterotrophic bacteria. Experiments were performed under laboratory conditions, employing both petrological and microbiological methods, which permitted the monitoring and visualization of geomicrobiological processes. The results demonstrate that bacteria play a prominent role in the weathering of black shale and in the biogeochemical cycles of elements occurring in this rock. It was shown that bacteria directly interact with black shale organic matter to produce a widespread biofilm on the Kupferschiefer shale surface. As a result of bacterial activity, the formation of pits, bioweathering of ore and rock-forming minerals, the mobilization of elements and secondary mineral precipitation were observed. The chemistry of the secondary minerals unequivocally demonstrates the mobilization of elements from minerals comprising Kupferschiefer. The redistribution of P, Al, Si, Ca, Mg, K, Fe, S, Cu and Pb was confirmed. The presence of bacterial outer membrane vesicles on the surface of black shale was observed for the first time. Biomineralization reactions occurred in both the membrane vesicles and the bacterial cells.

Biodegradation of Kupferschiefer black shale organic matter (Fore-Sudetic Monocline, Poland) by indigenous microorganisms.

This study provides the first evidence for the direct biodegradation of persistent organic matter extracted from the organic-rich polymetallic black shale ore Kupferschiefer, one of the most important sources of metals in the world. It was demonstrated that an enriched community of indigenous heterotrophic microorganisms isolated from black shale grown under aerobic conditions could utilize shale organic matter as the sole carbon and energy source. Colonization of shale organic matter was observed. The main biodegradation intermediates and products such as phosphonic acid dioctadecyl ester and isoindole-1,3 were detected in the aqueous phase of cultures. The bacterial community showed the ability to PAH biodegradation, assimilation of organic acids and esters as well as lipase activity. The intracellular accumulation of phosphorus by bacteria during growth on organic matter was confirmed. Strains within the genus Pseudomonas were found to dominate the bacterial population at the end of the experiment. The results of this study confirm that indigenous bacteria are likely to play a role in the biotransformation of black shale and can influence the geochemical cycles of ancient organic carbon in the deep terrestrial subsurface. This process may also occur in tailings ponds containing black shale, and cause the mobilization of potentially toxic compounds to the soil and groundwater.

Biotransformation of organic-rich copper-bearing black shale by indigenous microorganisms isolated from lubin copper mine (Poland).

The role of indigenous microorganisms in the biotransformation of refractory organic-rich copper-bearing black shale ore (Kupferschiefer) was confirmed in laboratory experiments. The persistent shale's organic matter was utilized by a mixture of bacterial strains as the sole carbon and energy source, and bacterial growth was accompanied by chemical and structural changes of black shale. The release of metallic elements and organic compounds into the aqueous phase was shown. Chemical analysis revealed the presence of long-chain aliphatic hydrocarbons and further biodegradation of these compounds by bacterial action. In this study, the release of metals from metalloorganic compounds present in organic-rich copper-bearing black shale was shown for the first time. The results have also confirmed the biotransformation of metalloporphyrins naturally occurring in black shale by indigenous microorganisms. Moreover, changes in the surface area and quantitative mineral composition of black shale were detected following bacterial treatment. This biotransformation activity is of potential use in biotechnological procedures for the recovery of copper and other valuable metals from tailings that contain up to 16% black shale. On the other hand, the release of organic carbon and heavy metals from black shale by biodegradation may significantly add to anthropogenic pollution.

Adaptive changes of chemolithoautotrophic acidophilic sulfur-oxidizing bacteria during growth in sewage sludge.

A chemolithoauthotrophic, acidophilic, sulfur-oxidizing strain was isolated from sewage sludge and identified as Acidithiobacillus thiooxidans. The morphology and physiology of the isolate grown in mineral medium or sterilized sewage sludge were investigated. Morphological and ultrastructural differences between cells grown in mineral medium and sewage sludge were clearly visible. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed some changes in the protein expression profiles in the periplasmic fraction as well as a lower level of cytochromes. Adaptation of A. thiooxidans to sewage sludge was not only a physiological process but also included genetic changes. Restriction fragment length polymorphism analysis using pulsed field gel electrophoresis showed structural changes in chromosomal DNA of such bacteria. Most of the restriction fragments were highly conserved and shared by strains grown under different conditions. Cultivation in mineral medium did, however, lead to the appearance of an additional restriction fragment. In combination, the obtained results provide evidence of adaptive responses by A. thioxidans during growth in sewage sludge and confirm that this bacteria can be useful in biotechnologies of heavy metal bioleaching from different environments polluted with hazardous compounds.