High-Pressure Microfluidics for Ultra-Fast Microbial Phenotyping.

Here, we present a novel methodology based on high-pressure microfluidics to rapidly perform temperature-based phenotyping of microbial strains from deep-sea environments. The main advantage concerns the multiple on-chip temperature conditions that can be achieved in a single experiment at pressures representative of the deep-sea, overcoming the conventional limitations of large-scale batch metal reactors to conduct fast screening investigations. We monitored the growth of the model strain Thermococcus barophilus over 40 temperature and pressure conditions, without any decompression, in only 1 week, whereas it takes weeks or months with conventional approaches. The results are later compared with data from the literature. An additional example is also shown for a hydrogenotrophic methanogen strain (Methanothermococcus thermolithotrophicus), demonstrating the robustness of the methodology. These microfluidic tools can be used in laboratories to accelerate characterizations of new isolated species, changing the widely accepted paradigm that high-pressure microbiology experiments are time-consuming.

Characterizing the Piezosphere: The Effects of Decompression on Microbial Growth Dynamics.

The extent to which the full diversity of the subsurface microbiome can be captured via cultivation is likely hindered by the inevitable loss of cellular viability from decompression during sampling, enrichment, and isolation. Furthermore, the pressure tolerance of previously isolated strains that span surface and subsurface ecosystems can shed light into microbial activity and pressure adaptation in these transition zones. However, assessments of the effects of elevated pressure on the physiology of piezotolerant and piezosensitive species may be biased by high-pressure enrichment techniques. Here, we compared two high-pressure cultivation techniques-one that requires decompression of the whole cultures during sampling and one that employs the previously described isobaric PUSH devices-to explore the effects of repeated decompression during incubations performed to characterize isolates from deep environments. Two model sulfate-reducing prokaryotes were used to test the effects of decompression/repressurization cycles on growth rates, cell yields, and pressure tolerance. The mesophilic bacterium Desulfovibrio salexigens was cultivated from 0.1 to 50 MPa, and the hyperthermophilic archaeon Archaeoglobus fulgidus was tested from 0.1 to 98 MPa. For both cultivation methods, D. salexigens showed exponential growth up to 20 MPa, but faster growth rates were observed for isobaric cultivation. Furthermore, at 30 MPa minor growth was observed in D. salexigens cultures only for isobaric conditions. Isobaric conditions also extended exponential growth of A. fulgidus to 60 MPa, compared to 50 MPa when cultures were decompressed during subsampling. For both strains, growth rates and cell yields decreased with increasing pressures, and the most pronounced effects of decompression were observed at the higher end of the pressure ranges. These results highlight that repeated decompression can have a significant negative impact on cell viability, suggesting that decompression tolerance may depend on habitat depth. Furthermore, sampling, enrichment, and cultivation in isobaric devices is critical not only to explore the portion of the deep biosphere that is sensitive to decompression, but also to better characterize the pressure limits and growth characteristics of piezotolerant and piezosensitive species that span surface and subsurface ecosystems.

Continuous Segmented-Flow Synthesis of Ag and Au Nanoparticles Using a Low Cost Microfluidic PTFE Tubing Reactor.

We present in here a simple and low cost continuous segmented-flow process for the synthesis of Ag and Au spherical-shaped nanoparticles. Different residence times (RT) were used to perform the nanoparticle synthesis, observing that at low RT, the Ag nanoparticles production, which uses a fast reduction reaction with NaBH4, is improved due to an enhancement in the mixing of the reactants. However, the flow conditions have an opposite effect in the case of Au nanoparticles synthesis. Indeed, since the chemical reduction process (Turkevich method) exhibit a much slower kinetics, high RT (low flowrates) improve the synthesis yield and the quality of the nanoparticles. The Ag and Au nanoparticles were characterized by UV-Vis spectrophotometry (UV-Vis) and Transmission Electron Microscopy (TEM). The Ag spherical-shaped nanoparticles presented a LSPR at 400 nm (size ≈ 4 nm), while the synthesized Au nanoparticles exhibit LSPR and sizes in the range 520 - 550 nm and 14 - 17 nm, respectively.

Rate and Extent of Growth of a Model Extremophile, Archaeoglobus fulgidus, Under High Hydrostatic Pressures.

High hydrostatic pressure (HHP) batch cultivation of a model extremophile, Archaeoglobus fulgidus type strain VC-16, was performed to explore how elevated pressures might affect microbial growth and physiology in the deep marine biosphere. Though commonly identified in high-temperature and high-pressure marine environments (up to 2-5 km below sea level, 20-50 MPa pressures), A. fulgidus growth at elevated pressure has not been characterized previously. Here, exponential growth of A. fulgidus was observed up to 60 MPa when supported by the heterotrophic metabolism of lactate oxidation coupled to sulfate reduction, and up to 40 MPa for autotrophic CO2 fixation coupled to thiosulfate reduction via H2. Maximum growth rates for this heterotrophic metabolism were observed at 20 MPa, suggesting that A. fulgidus is a moderate piezophile under these conditions. However, only piezotolerance was observed for autotrophy, as growth rates remained nearly constant from 0.3 to 40 MPa. Experiments described below show that A. fulgidus continues both heterotrophic sulfate reduction and autotrophic thiosulfate reduction nearly unaffected by increasing pressure up to 30 MPa and 40 MPa, respectively. As these pressures encompass a variety of subsurface marine environments, A. fulgidus serves as a model extremophile for exploring the effects of elevated pressure on microbial metabolisms in the deep subsurface. Further, these results exemplify the need for high-pressure cultivation of deep-sea and subsurface microorganisms to better reflect in situ physiological conditions.

Novel Intact Polar and Core Lipid Compositions in the Pyrococcus Model Species, P. furiosus and P. yayanosii, Reveal the Largest Lipid Diversity Amongst Thermococcales.

Elucidating the lipidome of Archaea is essential to understand their tolerance to extreme environmental conditions. Previous characterizations of the lipid composition of Pyrococcus species, a model genus of hyperthermophilic archaea belonging to the Thermococcales order, led to conflicting results, which hindered the comprehension of their membrane structure and the putative adaptive role of their lipids. In an effort to clarify the lipid composition data of the Pyrococcus genus, we thoroughly investigated the distribution of both the core lipids (CL) and intact polar lipids (IPL) of the model Pyrococcus furiosus and, for the first time, of Pyrococcus yayanosii, the sole obligate piezophilic hyperthermophilic archaeon known to date. We showed a low diversity of IPL in the lipid extract of P. furiosus, which nonetheless allowed the first report of phosphatidyl inositol-based glycerol mono- and trialkyl glycerol tetraethers. With up to 13 different CL structures identified, the acid methanolysis of Pyrococcus furiosus revealed an unprecedented CL diversity and showed strong discrepancies with the IPL compositions reported here and in previous studies. By contrast, P. yayanosii displayed fewer CL structures but a much wider variety of polar heads. Our results showed severe inconsistencies between IPL and CL relative abundances. Such differences highlight the diversity and complexity of the Pyrococcus plasma membrane composition and demonstrate that a large part of its lipids remains uncharacterized. Reassessing the lipid composition of model archaea should lead to a better understanding of the structural diversity of their lipidome and of their physiological and adaptive functions.

Microfluidics and Surface-Enhanced Raman Spectroscopy: A Perfect Match for New Analytical Tools.

In this perspective article, we emphasize the combination of Surface-Enhanced Raman Spectroscopy (SERS) and Microfluidic devices. SERS approaches have been widely studied and used for multiple applications including trace molecules detection, in situ analysis of biological samples and monitoring or, all of them with good results, however still with limitations of the technique, for example regarding with improved precision and reproducibility. These implications can be overcome by microfluidic approaches. The resulting coupling Microfluidics - SERS (MF-SERS) has recently gained increasing attention by creating thundering opportunities for the analytical field. For this purpose, we introduce some of the strategies developed to implement SERS within microfluidic reactor along with a brief overview of the most recent MF-SERS applications for biology, health and environmental concerns. Eventually, we will discuss future research opportunities of such systems.

Molecular chaperone accumulation as a function of stress evidences adaptation to high hydrostatic pressure in the piezophilic archaeon Thermococcus barophilus.

The accumulation of mannosyl-glycerate (MG), the salinity stress response osmolyte of Thermococcales, was investigated as a function of hydrostatic pressure in Thermococcus barophilus strain MP, a hyperthermophilic, piezophilic archaeon isolated from the Snake Pit site (MAR), which grows optimally at 40 MPa. Strain MP accumulated MG primarily in response to salinity stress, but in contrast to other Thermococcales, MG was also accumulated in response to thermal stress. MG accumulation peaked for combined stresses. The accumulation of MG was drastically increased under sub-optimal hydrostatic pressure conditions, demonstrating that low pressure is perceived as a stress in this piezophile, and that the proteome of T. barophilus is low-pressure sensitive. MG accumulation was strongly reduced under supra-optimal pressure conditions clearly demonstrating the structural adaptation of this proteome to high hydrostatic pressure. The lack of MG synthesis only slightly altered the growth characteristics of two different MG synthesis deletion mutants. No shift to other osmolytes was observed. Altogether our observations suggest that the salinity stress response in T. barophilus is not essential and may be under negative selective pressure, similarly to what has been observed for its thermal stress response.

Membrane homeoviscous adaptation in the piezo-hyperthermophilic archaeon Thermococcus barophilus.

The archaeon Thermococcus barophilus, one of the most extreme members of hyperthermophilic piezophiles known thus far, is able to grow at temperatures up to 103°C and pressures up to 80 MPa. We analyzed the membrane lipids of T. barophilus by high performance liquid chromatography-mass spectrometry as a function of pressure and temperature. In contrast to previous reports, we show that under optimal growth conditions (40 MPa, 85°C) the membrane spanning tetraether lipid GDGT-0 (sometimes called caldarchaeol) is a major membrane lipid of T. barophilus together with archaeol. Increasing pressure and decreasing temperature lead to an increase of the proportion of archaeol. Reversely, a higher proportion of GDGT-0 is observed under low pressure and high temperature conditions. Noticeably, pressure and temperature fluctuations also impact the level of unsaturation of apolar lipids having an irregular polyisoprenoid carbon skeleton (unsaturated lycopane derivatives), suggesting a structural role for these neutral lipids in the membrane of T. barophilus. Whether these apolar lipids insert in the membrane or not remains to be addressed. However, our results raise questions about the structure of the membrane in this archaeon and other Archaea harboring a mixture of di- and tetraether lipids.

High hydrostatic pressure increases amino acid requirements in the piezo-hyperthermophilic archaeon Thermococcus barophilus.

We have established a defined growth medium for the piezophilic hyperthermophilic archaeon Thermococcus barophilus, which allows growth yields of ca. 10(8) cells/ml under both atmospheric and high hydrostatic pressure. Our results demonstrate a major impact of hydrostatic pressure on amino acid metabolism, with increases from 3 amino acids required at atmospheric pressure to 17 at 40 MPa. We observe in T. barophilus and other Thermococcales a similar discrepancy between the presence/absence of amino acid synthesis pathways and amino acid requirements, which supports the existence of alternate, but yet unknown, amino acid synthesis pathways, and may explain the low number of essential amino acids observed in T. barophilus and other Thermococcales. T. barophilus displays a strong metabolic preference for organic polymers such as polypeptides and chitin, which may constitute a more readily available resource of carbon and energy in situ in deep-sea hydrothermal vents. We hypothesize that the low energy yields of fermentation of organic polymers, together with energetic constraints imposed by high hydrostatic pressure, may render de novo synthesis of amino acids ecologically unfavorable. Induction of this metabolic switch to amino acid recycling can explain the requirement for non-essential amino acids by Thermococcales for efficient growth in defined medium.

Restoration of the di-myo-inositol-phosphate pathway in the piezo-hyperthermophilic archaeon Thermococcus barophilus.

Most Thermococcales accumulate di-myo-inositol-phosphate (DIP) as an organic solute as a response to heat stress. We have studied the accumulation of this osmolyte in the high-hydrostatic pressure adapted hyperthermophile Thermococcus barophilus. We found no accumulation of DIP under any of the stress conditions tested, although this archaeon harbors the 3 DIP synthesis genes. Lack of synthesis is due to the lack of expression of TERMP_01135 coding for the second step of DIP synthesis. In contrast to other species, the T. barophilus synthesis operon is interrupted by a four gene locus, in reverse orientation. Restoring an operon like structure at the DIP locus restored DIP synthesis, but did not have an impact on growth characteristics, suggesting that other mechanisms have evolved in this organism to cope with heat stress.

[The high pressure life of piezophiles]. / La vie sous pression des microorganismes piézophiles.

The deep biosphere is composed of very different biotopes located in the depth of the oceans, the ocean crust or the lithosphere. Although very different, deep biosphere biotopes share one common feature, high hydrostatic pressure. The deep biosphere is colonized by specific organisms, called piezophiles, that are able to grow under high hydrostatic pressure. Bacterial piezophiles are mainly psychrophiles belonging to five genera of γ-proteobacteria, Photobacterium, Shewanella, Colwellia, Psychromonas and Moritella, while piezophilic Archaea are mostly (hyper)thermophiles from the Thermococcales. None of these genera are specific for the deep biosphere. High pressure deeply impacts the activity of cells and cellular components, and reduces the activity of numerous key processes, eventually leading to cell death of piezosensitive organisms. Biochemical and genomic studies yield a fragmented view on the adaptive mechanisms in piezophiles. It is yet unclear whether piezophilic adaptation requires the modification of a few genes, or metabolic pathways, or a more profound reorganization of the genome, the fine tuning of gene expression to compensate the pressure-induced loss of activity of the proteins most affected by high pressure, or a stress-like physiological cell response. In contrast to what has been seen for thermophily or halophily, the adaptation to high pressure is diffuse in the genome and may concern only a small fraction of the genes.

Adaptation of the membrane in Archaea.

Microbes often face contrasted and fluctuating environmental conditions, to which they need to adapt or die. Because membranes play a central role in regulating fluxes inward and outward from the cells, maintaining the appropriate structure of the membrane is crucial to maintain cellular integrity and functions. This is achieved in bacteria and eucarya by a modification of the membrane lipid compositions, a strategy termed homeoviscous adaptation. We review here evidence for homeoviscous adaptation in Archaea, and discuss the limits of this strategy and our knowledge in this very peculiar domain of life.