Horia Leonard Banciu

Living with salt: metabolic and phylogenetic diversity of archaea inhabiting saline ecosystems

Archaea that live at high salt concentrations are a phylogenetically diverse group of microorganisms. They include the heterotrophic haloarchaea (class Halobacteria) and some methanogenic Archaea, and they inhabit both oxic and anoxic environments. In spite of their common hypersaline environment, halophilic archaea are surprisingly diverse in their nutritional demands, range of carbon sources degraded (including hydrocarbons and aromatic compounds) and metabolic pathways. The recent discovery of a new group of extremely halophilic Euryarchaeota, the yet uncultured Nanohaloarchaea, shows that the archaeal diversity and metabolic variability in hypersaline environments is higher than hitherto estimated.

Functional microbiology of soda lakes

Soda lakes represent unique permanently haloalkaline system. Despite the harsh conditions, they are inhabited by abundant, mostly prokaryotic, microbial communities. This review summarizes results of studies of main functional groups of the soda lake prokaryotes responsible for carbon, nitrogen and sulfur cycling, including oxygenic and anoxygenic phototrophs, aerobic chemolithotrophs, fermenting and respiring anaerobes. The main conclusion from this work is that the soda lakes are very different from other high-salt systems in respect to microbial richness and activity. The reason for this difference is determined by the major physico-chemical features of two dominant salts - NaCl in neutral saline systems and sodium carbonates in soda lakes, that are influencing the amount of energy required for osmotic adaptation.

Microbial sulfide oxidation in the oxic-anoxic transition zone of freshwater sediment: involvement of lithoautotrophic Magnetospirillum strain J10.

The oxic-anoxic transition zone (OATZ) of freshwater sediments, where opposing gradients exist of reduced iron and sulfide with oxygen, creates a suitable environment for microorganisms that derive energy from the oxidation of iron or sulfide. Gradient microcosms incubated with freshwater sediment showed rapid microbial turnover of sulfide and oxygen compared with sterile systems. Microcosms with FeS as a substrate also showed growth at the OATZ and subsequent dilution series resulted in the isolation of three novel strains, of which strain J10 grows chemolithoautotrophically with reduced sulfur compounds under microaerobic conditions. All three strains are motile spirilla with bipolar flagella, related to the genera Magnetospirillum and Dechlorospirillum within the Alphaproteobacteria. Strain J10 is closely related to Magnetospirillum gryphiswaldense and is the first strain in this genus found to be capable of autotrophic growth. Thiosulfate was oxidized completely to sulfate, with a yield of 4 g protein mol(-1) thiosulfate, and autotrophic growth was evidenced by incorporation of (13)C derived from bicarbonate into biomass. A putative gene encoding ribulose 1,5-bisphosphate carboxylase/oxygenase type II was identified in strain J10, suggesting that the Calvin-Benson-Bassham cycle is used for autotrophic growth. Analogous genes are also present in other magnetospirilla, and in the autotrophically growing alphaproteobacterium magnetic vibrio MV-1.

Halophilic and haloalkaliphilic sulfur-oxidizing bacteria

Chemotrophic sulfur-oxidizing bacteria (SOB) represent an important functional group of microorganisms responsible for the dark oxidation of reduced sulfur compounds generated by sulfidogens. Until recently, only a single genus of halophilic SOB (Halothiobacillus) has been described, and nothing was known about the ability of this group to grow at high pH. Investigation of soda lakes, unique extremely alkaline and saline habitats, led to the discovery of a novel ecotype of natronophilic SOB. In contrast to the previously known neutrophilic ecotype, this group cannot grow at neutral pH, but grows optimally in soda brines at pH values around 10. They were the first chemolithoautotrophs among the described alkaliphiles. The group, so far, includes four novel genera within the Gammaproteobacteria. The genera Thioalkalimicrobium and Thioalkalispira represent low salt-tolerant alkaliphiles tolerating up to 1.5 M Na+. The genus Thioalkalibacter is a high salt-tolerant facultative alkaliphile. The genus Thioalkalivibrio is the most diverse and includes aerobic extremely salt-tolerant members and moderately salt-tolerant thiocyanate-utilizing and facultatively anaerobic denitrifying strains. The genome sequence of two Thioalkalivibrio strains revealed the presence of a truncated Sox system lacking the SoxCD component which is typical for gammaproteobacterial SOB. Bioenergetic studies of high salt-tolerant Thioalkalivibrio strains showed an obligate sodium dependence for respiratory activity implying the presence of sodium-dependent elements. Investigation of hypersaline inland chloride-sulfate lakes and hypersaline brines of marine origin with neutral pH revealed an unexpectedly high culturable diversity of halophilic obligately chemolithoautotrophic SOB comprising seven different groups within the Gammaproteobacteria. Two groups of strictly aerobic moderate halophiles were represented by the known genera Halothiobacillus and Thiomicrospira. Under denitrifying conditions and with thiocyanate as e-donor, three novel groups of moderately halophilic SOB were represented by the genera Thiohalomonas, Thiohalophilus, and Thiohalobacter. At 4 M NaCl, two groups of extremely halophilic SOB (a type not known before among the SOB) had been discovered. The obligately aerobic extreme halophiles comprised a novel genus Thiohalospira, and the facultatively anaerobic nitrate-reducing extreme halophiles—a novel deep-lineage genus Thiohalorhabdus. Overall, the investigation of hypersaline and (halo)alkaline habitats uncovered a novel and diverse world of extremophilic SOB with properties previously unknown for chemolithoautotrophic bacteria.

Adaptive strategies in the double-extremophilic prokaryotes inhabiting soda lakes

Haloalkaliphiles are double extremophilic organisms thriving both at high salinity and alkaline pH. Although numerous haloalkaliphilic representatives have been identified among Archaea and Bacteria over the past 15 years, the adaptations underlying their prosperity at haloalkaline conditions are scarcely known. A multi-level adaptive strategy was proposed to occur in haloalkaliphilic organisms isolated from saline alkaline and soda environments including adjustments in the cell wall structure, plasma membrane lipid composition, membrane transport systems, bioenergetics, and osmoregulation. Isolation of chemolithoautotrophic sulfur-oxidizing γ-Proteobacteria from soda lakes allowed the elucidation of the structural and physiological differences between haloalkaliphilic (prefer NaCl) and natronophilic (prefer NaHCO3/Na2CO3, i.e. soda) microbes.

Adaptation in Haloalkaliphiles and Natronophilic Bacteria

Haloalkaliphiles differ from natronophiles by their requirement for chloride ions in addition to high alkalinity. Natronophilic bacteria grow optimally in soda medium buffered at alkaline pH by a combination of NaHCO3 and Na2CO3. The majority of known haloalkaliphilic and natronophilic prokaryotes are isolated from saline–alkaline ecosystems such as soda lakes and saline–alkaline soils. A great taxonomic and metabolic biodiversity is found in soda systems, enabling the functioning of all the cycles of the essential elements. In spite of the increasing number of haloalkaliphilic and natronophilic isolates, scarce biochemical and functional information on simultaneous adaptation at high salinity and alkalinity is reported. Most of the available data on haloalkaline adaptation can be inferred from the functional characterization of alkaliphilic and halophilic bacterial models as well as from a few haloalkaliphilic and natronophilic genome sequences deposited in databases. At the level of cell envelopes (cell wall and cytoplasmic membrane), the salt and alkaline adaptation strategies are different and relatively conserved between Gram-positive and Gram-negative bacteria. The cell wall of the former group is characterized by the excessive presence of acidic polymers, while cell membranes abound in phospholipids with branched fatty acids. Cell membranes of salt- and alkaline-adapted Gram-negatives contain a large variety of fatty acids as well as significant amounts of nonpolar lipids. Osmotic adaptation mostly depends on the accumulation of organic compatible solutes either by active solute uptake or by combined strategies of importing osmolytes or osmolyte precursors and de novo synthesis of organic compatible solutes. Aerobic and anaerobic haloalkaliphiles are distinguished from each other by very different bioenergetics. Energy conservation in aerobic alkaliphiles and haloalkaliphiles is mainly based on functioning of H+-driven F-type ATP synthase. In spite of the low transmembrane electrochemical proton gradient (equivalent to proton-motive force, pmf) encountered in the alkali-exposed membrane, the energy metabolism remains highly efficient, supporting high growth rate and yield in many aerobic alkaliphiles and haloalkaliphiles. The energetics of haloalkaliphilic anaerobes is less understood, but it seems to involve a greater deal of Na dependency than in their aerobic counterpart. Na+-dependent ATPase activity is reported in a few anaerobic haloalkaliphiles and its role probably deals with active Na+ ejection from the cytoplasm. In haloalkaliphiles and natronophiles, the sodium-motive force (smf) is mainly driving the flagellar movement and sodium/solute symport. Cytoplasmic pH and ion homeostasis in haloalkaliphiles and natronophiles are most probably achieved by a concerted activity of a constellation of alkaline-activated ion transporters, among which Na+/H+ and Mrp-like antiporters have a major contribution.

Heavy metal resistance in halophilic Bacteria and Archaea

Heavy metals are dense chemicals with dual biological role as micronutrients and intoxicants. A few hypersaline environmental systems are naturally enriched with heavy metals, while most metal-contaminated sites are a consequence of human activities. Numerous halotolerant and moderately halophilic Bacteria possess metal tolerance, whereas a few archaeal counterparts share similar features. The main mechanisms underlying heavy metal resistance in halophilic Bacteria and Archaea include extracellular metal sequestration by biopolymers, metal efflux mediated by specific transporters and enzymatic detoxification. Biotransformation of metals by halophiles has implications both for trace metal turnover in natural saline ecosystems and for development of novel bioremediation strategies.

Diversity and Biomineralization Potential of the Epilithic Bacterial Communities Inhabiting the Oldest Public Stone Monument of Cluj-Napoca (Transylvania, Romania)

In this study, we investigated the biomineralization potential and diversity of the epilithic bacterial communities dwelling on the limestone statue of Saint Donatus, the oldest public monument of Cluj-Napoca city (Transylvania region, NW Romania). Their spatial distribution together with phylogenetic and metabolic diversity, as well as their capacity to precipitate calcium carbonate was evaluated by combining molecular and phenotypic fingerprinting methods with X-ray diffraction, Fourier transform infrared spectroscopy, and scanning electron-microscopy analyses. The results of real-time quantitative PCR, molecular fingerprinting and community-level physiological profiling showed that diverse and abundant bacterial assemblages that differ in relation to their collection site colonized the statue. The cultivation and molecular identification procedures allowed the characterization of 79 bacterial isolates belonging to Proteobacteria (73.4%), Firmicutes (19%), and Actinobacteria (7.6%). Amongst them, the 22 strains identified as being capable of calcium carbonate precipitation were found to belong mostly to Bacillus and Pseudomonas genera. We found that bacteria acted as nucleation sites, inducing the formation of nanoscale aggregates that were shown to be principally composed of vaterite. Furthermore, we expanded the current knowledge on culturable diversity of carbonatogenic bacteria by providing evidence for biogenic vaterite/calcite formation mediated by: Pseudomonas synxantha, P. graminis, Brevibacterium iodinum, Streptomyces albidoflavus, and Stenotrophomonas chelatiphaga. Overall, this study highlights the need to evaluate the carbonatogenetic potential of all the bacterial communities present on stone artwork prior to designing an efficient conservation treatment based on biomineralization.

Spatial Distribution and Molecular Diversity of Archaeal Communities in the Extreme Hypersaline Meromictic Brâncoveanu Lake (Transylvanian Basin, Romania)

ABSTRACT Dating from the Middle Miocene, the massive halite deposits lying beneath the Transylvanian Basin (Central Romania) have been valuable mineral resources quarried for millennia. Among the numerous hypersaline pit lakes that resulted from this mining, Brâncoveanu Lake is unique by its extreme salinity. Assessment of physicochemical variables, water chemistry and trophic status indicated that Brâncoveanu Lake is a permanently stratified, pH-neutral, NaCl-rich and eutrophied system. We investigated the abundance, molecular diversity and vertical distribution of archaeal community by culture-independent approaches. Additionally, the most relevant environmental parameters shaping the archaeal community composition were evaluated by statistical methods. Archaea appeared to largely outnumber Bacteria; altogether the great prevalence of Halobacteriaceae-related sequences could imply a major contribution of this group to the biogeochemical carbon turnover. The fairly distinct composition of archaeal communities reflects the lakes physicochemical stratification. Among the limnological factors, salinity and oxygen showed a significant impact on determining the composition and structure of archaeal assemblages. Furthermore, Brâncoveanu Lake might harbor novel microorganisms such as members of the recently described phylum Nanohaloarchaea. Overall, this study reported the occurrence of halophilic Archaea in a little explored hydrogeochemical system and provided a better insight into geomicrobiology of meromictic hypersaline pit lakes.

Novel approach to microbiological air monitoring in show caves

Air microbial pollution in touristic areas poses a risk for both the integrity of an ecosystem and human health. Microbiological monitoring together with environmental parameters monitoring allows for the assessment of the impacts and formulation of sound management decisions to protect humans and ecosystems. Four show caves from the Carpathian Mountains were selected for our study. The caves were sampled monthly to obtain an overview of the changes that occur over a yearly cycle. For the microbial monitoring, we used RIDA®COUNT test plates, while the environmental parameters were monitored with a variety of devices. Second and third generations of microbes extracted from the plates were grown on specific media for the purpose of 16S rRNA and 18S rRNA extraction and taxa identification. The bacterial communities identified in the air samples in the four investigated show caves were dominated by Staphylococcus, while regarding the fungi communities, Penicillium was more likely to occur in the touristic part of the caves and Cladosporium in the non-visited passages. Together with data on number of visitors, number of bats and radon levels we were able understand the impact of tourists on the cave environment and to generate microbiological risk maps for human health. This type of comprehensive study can be used not only to protect the integrity of a touristic area from the impacts caused by the introduction of allochthonous organic matter, but also for the protection of the tourists and guides from potential pathogenic taxa.