Engineering considerations for the application of extremophiles in biotechnology

Biotechnology may soon take greater advantage of extremophiles--microorganisms that grow in high salt or heavy metal concentrations, or at extremes of temperature, pressure, or pH. These organisms and their cellular components are attractive because they permit process operation over a wider range of conditions than their traditional counterparts. However, extremophiles also present a number of challenges for the development of bioprocesses, such as slow growth, low cell yield, and high shear sensitivity. Difficulties inherent in designing equipment suitable for extreme conditions are also encountered. This review describes both the advantages and disadvantages of extremophiles, as well as the specialized equipment required for their study and application in biotechnology.     

Biochemical and thermodynamic analyses of energy conversion in extremophiles

A variety of extreme environments, characterized by extreme values of various physicochemical parameters (temperature, pressure, salinity, pH, and so on), are found on Earth. Organisms that favorably live in such extreme environments are called extremophiles. All living organisms, including extremophiles, must acquire energy to maintain cellular homeostasis, including extremophiles. For energy conversion in harsh environments, thermodynamically useful reactions and stable biomolecules are essential. In this review, I briefly summarize recent studies of extreme environments and extremophiles living in these environments and describe energy conversion processes in various extremophiles based on my previous research. Furthermore, I discuss the correlation between the biological system of electrotrophy, a third biological energy acquisition system, and the mechanism underlying microbiologically influenced corrosion. These insights into energy conversion in extremophiles may improve our understanding of the “limits of life”.

Abbreviations: PPi: pyrophosphate; PPase: pyrophosphatase; ITC: isothermal titration microcalorimetry; SVNTase: Shewanella violacea 5?-nucleotidase; SANTase: Shewanella amazonensis 5?-nucleotidase     

Homology Modeling and Comparative Profiling of Superoxide Dismutase Among Extremophiles: Exiguobacterium as a Model Organism

Superoxide dismutase (SOD), a well known antioxidant enzyme, is known to exert its presence across bacteria to humans. Apart from their well-known antioxidant defense mechanisms, their association with various extremophiles in response to various stress conditions is poorly understood. Here, we have discussed the conservation and the prevalence of SODs among 21 representative extremophiles. A systematic investigation of aligned amino acid sequences of SOD from all the selected extremophiles revealed a consensus motif D-[VLE]-[FW]-E-H-[AS]-Y-[YM]. To computationally predict the correlation of SOD with the various stress conditions encountered by these extremophiles, Exiguobacterium was selected as a model organism which is known to survive under various adverse extremophilic conditions. Interestingly, our phylogenetic study based on SOD homology revealed that Exiguobacterium sibiricum was one of the closest neighbors of Deinococcus radiodurans and Thermus thermophilus. Next, we sought to predict 3-D model structure of SOD for E. sibiricum (PMDB ID: 0078260), which showed >95 % similarity with D. radiodurans R1 SOD. The reliability of the predicted SOD model was checked by using various validation metrics, including Ramachandran plot, Z-score and normalized qualitative model energy analysis score. Further, various physicochemical properties of E. sibiricum SOD were calculated using different prominent resources.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-014-0482-8) contains supplementary material, which is available to authorized users.     

抗辐射菌Deinococcus radiodurans的多样性

抗辐射菌Deinococcus radiodurans是一种对电离辐射和其他DNA损伤因子具有极强抵抗能力的细菌,是研究DNA损伤与修复的模式生物。综述了国内外在抗辐射菌研究上取得的最新研究成果,从生存环境、对DNA损伤因子的抗性、抗性机理及其损伤修复关联基因等方面报道了抗辐射菌的多样性,并探讨了该细菌高效正确的DNA损伤修复机理的相关研究成果在生命科学、农业、环境修复及医学等领域的应用前景。     

Extremophiles: radiation resistance microbial reserves and therapeutic implications

Micro‐organisms with the ability to survive in extreme environmental conditions are known as ‘extremophiles’. Currently, extremophiles have caused a sensation in the biotechnology/pharmaceutical industries with their novel compounds, known as ‘extremolytes’. The potential applications of extremolytes are being investigated for human therapeutics including anticancer drugs, antioxidants, cell cycle‐blocking agents, anticholesteric drugs, etc. It is hypothesized that the majority of ultraviolet radiation (UVR)‐resistant micro‐organisms can be used to develop anticancer drugs to prevent skin damage from UVR. The metabolites from UVR‐resistant microbes are a great source of potential therapeutic applications in humans. This article aims to discuss the potentials of extremolytes along with their therapeutic implications of UVR extremophiles. The major challenges of therapeutic development using extremophiles are also discussed.     

Life in blue: Copper resistance mechanisms of bacteria and Archaea used in industrial biomining of minerals

Industrial biomining processes to extract copper, gold and other metals involve the use of extremophiles such as the acidophilic Acidithiobacillus ferrooxidans (Bacteria), and the thermoacidophilic Sulfolobus metallicus (Archaea). Together with other extremophiles these microorganisms subsist in habitats where they are exposed to copper concentrations higher than 100 mM. Herein we review the current knowledge on the Cu-resistance mechanisms found in these microorganisms. Recent information suggests that biomining extremophiles respond to extremely high Cu concentrations by using simultaneously all or most of the following key elements: 1) a wide repertoire of Cu-resistance determinants; 2) duplication of some of these Cu-resistance determinants; 3) existence of novel Cu chaperones; 4) a polyP-based Cu-resistance system, and 5) an oxidative stress defense system. Further insight of the biomining community members and their individual response to copper is highly relevant, since this could provide key information to the mining industry. In turn, this information could be used to select the more fit members of the bioleaching community to attain more efficient industrial biomining processes.     

Searching for monooxygenases and hydrolases in bacteria from an extreme environment

Microbial oxidation potentials of extremophiles recovered from Pampo Sul oil field, Campos Basin, Brazil, in pure culture or in consortia, were investigated using high-throughput screening (HTS) and multibioreactions. Camphor (1), cis-jasmone (2), 2-methyl-cyclohexanone (3), 1,2-epoxyoctane (4), phenylethyl acetate (5), phenylethyl propionate (6), and phenylethyl octanoate (7) were used to perform multibioreaction assays. Eighty-two bacterial isolates were recovered from oil and formation water samples and those presenting outstanding activities in HTS assays were identified by sequencing their 16S rRNA genes. These results revealed that most microorganisms belonged to the genus Bacillus and presented alcohol dehydrogenase, monooxygenase, epoxide hydrolase, esterase, and lipase activities.     

An anti-HBV anthraquinone from aciduric fungus Penicillium sp. OUCMDZ-4736 under low pH stress

To obtain new bioactive natural products, the effect of acidic stress on the metabolites of an aciduric fungus was investigated. This fungus, Penicillium sp. OUCMDZ-4736, which was isolated from the sediment around roots of mangrove (Acanthus ilicifolius), produced different compounds and higher yields under pH 2.5 than under neutral conditions. Using spectroscopic analyses and calculations, three new anthraquinone derivatives (1–3) were isolated and identified from the acidic fermentation broth (pH 2.5) of OUCMDZ-4736. Compound 1 showed much stronger anti-hepatitis B virus activity than that of the positive control, lamivudine, strongly inhibiting HBsAg and HBeAg secretion from HepG2.2.15 cells. These results show that extremophiles are a valuable resource of bioactive compounds, and that pH regulation is an effective strategy to induce metabolite production in aciduric fungi.

     

Tardigrades in Space Research - Past and Future

To survive exposure to space conditions, organisms should have certain characteristics including a high tolerance for freezing, radiation and desiccation. The organisms with the best chance for survival under such conditions are extremophiles, like some species of Bacteria and Archea, Rotifera, several species of Nematoda, some of the arthropods and Tardigrada (water bears). There is no denying that tardigrades are one of the toughest animals on our planet and are the most unique in the extremophiles group. Tardigrada are very small animals (50 to 2,100 μm in length), and they inhabit great number of Earth environments. Ever since it was proven that tardigrades have high resistance to the different kinds of stress factors associated with cosmic journeys, combined with their relatively complex structure and their relative ease of observation, they have become a perfect model organism for space research. This taxon is now the focus of astrobiologists from around the world. Therefore, this paper presents a short review of the space research performed on tardigrades as well as some considerations for further studies.     

Microbial diversity of deep-sea extremophiles--Piezophiles, Hyperthermophiles, and subsurface microorganisms]

Knowledge of our Planet's biosphere has increased tremendously during the last 10 to 20 years. In the field of Microbiology in particular, scientists have discovered novel "extremophiles", microorganisms capable of living in extreme environments such as highly acidic or alkaline conditions, at high salt concentration, with no oxygen, extreme temperatures (as low as -20 degrees C and as high as 300 degrees C), at high concentrations of heavy metals and in high pressure environments such as the deep-sea. It is apparent that microorganisms can exist in any extreme environment of the Earth, yet already scientists have started to look for life on other planets; the so-called "Exobiology" project. But as yet we have little knowledge of the deep-sea and subsurface biosphere of our own planet. We believe that we should elucidate the Biodiversity of Earth more thoroughly before exploring life on other planets, and these attempts would provide deeper insight into clarifying the existence of extraterrestrial life. We focused on two deep-sea extremophiles in this article; one is "Piezophiles", and another is "Hyperthermophiles". Piezophiles are typical microorganisms adapted to high-pressure and cold temperature environments, and located in deep-sea bottom. Otherwise, hyperthermophiles are living in high temperature environment, and located at around the hydrothermal vent systems in deep-sea. They are not typical deep-sea microorganisms, but they can grow well at high-pressure condition, just like piezophiles. Deming and Baross mentioned that most of the hyperthermophilic archaea isolated from deep-sea hydrothermal vents are able to grow under conditions of high temperature and pressure, and in most cases their optimal pressure for growth was greater than the environmental pressure they were isolated from. It is possible that originally their native environment may have been deeper than the sea floor and that there had to be a deeper biosphere. This implication suggests that the deep-sea hydrothermal vents are the windows to a deep subsurface biosphere. A vast array of chemoautotrophic deep-sea animal communities have been found to exist in cold seep environments, and most of these animals are common with those found in hydrothermal vent environments. Thus, it is possible to consider that the cold seeps are also one of slit windows to a deep subsurface biosphere. We conclude that the deep-sea extremophiles are very closely related into the unseen majority in subsurface biosphere, and the subsurface biosphere probably concerns to consider the "exobiology".     

深海极端微生物菌群及代谢产物多样性的研究进展

海洋覆盖了地球表面积的四分之三,它不仅是生命的起源,而且还孕育了各种极端微生物。它们存在于海洋极端环境中,如热液喷口、热泉、咸湖和深海层等,由于生境太过恶劣,一度被认为是生命的禁区。随着人类对深海极端环境微生物研究的不断深入,已经探索到那里具有丰富的菌群资源和具有潜在价值的天然生物活性产物。这些极端微生物能够适应极高温、极低温、高压、高盐、高放射性和极度酸碱性等极端环境,具有特殊的生物多样性、遗传背景和代谢途径,能够产生各种具有特殊功能的酶类及其他活性物质,展现出巨大的研究价值和应用潜力。研究海洋极端微生物对探索生物多样性、新资源开发利用及对地球生物学研究等都具有重要意义。     

嗜酸性硫杆菌群体感应系统研究进展

嗜酸性硫杆菌(Acidithiobacillus spp.)是一类重要的极端环境微生物与工业微生物。该类细菌通过氧化硫或亚铁获得电子以固定二氧化碳进行自养生长,是驱动矿山环境酸化和重金属溶出的关键菌群,也是生物冶金等微生物浸出技术中的核心菌群。群体感应(quorum sensing, QS)系统是细菌种内及种间信息交流的重要方式,广泛分布于嗜酸性硫杆菌等化能自养微生物中,比如类似于LuxI/R的AfeI/R系统。系统介绍近年来嗜酸性硫杆菌菌体感应系统研究成果,尤其是在AfeI/R种群分布、生物学功能、调节机制及其应用研究中的新发现与新理论。讨论今后嗜酸性硫杆菌群体感应系统研究的主要方向及需要解决的关键科学问题,以促进极端微生物群体感应系统理论研究的开展与产业应用技术的开发。     

Extremolytes: natural compounds from extremophiles for versatile applications

Extremophilic microorganisms have adopted a variety of ingenious strategies for survival under high or low temperature, extreme pressure, and drastic salt concentrations. A novel application area for extremophiles is the use of “extremolytes,” organic osmolytes from extremophilic microorganisms, to protect biological macromolecules and cells from damage by external stresses. In extremophiles, these low molecular weight compounds are accumulated in response to increased extracellular salt concentrations, but also as a response to other environmental changes, e.g., increased temperature. Extremolytes minimize the denaturation of biopolymers that usually occurs under conditions of water stress and are compatible with the intracellular machinery at high (>1 M) concentrations. The ectoines, as the first extremolytes that are produced in a large scale, have already found application as cell protectants in skin care and as protein-free stabilizers of proteins and cells in life sciences. In addition to ectoines, a range of extremolytes with heterogenous chemical structures like the polyol phosphates di-myoinositol-1,1′-phosphate, cyclic 2,3-diphosphoglycerate, and α-diglycerol phosphate and the mannose derivatives mannosylglycerate (firoin) and mannosylglyceramide (firoin-A) were characterized and were shown to have protective properties toward proteins and cells. A range of new applications, all based on the adaptation to stress conditions conferred by extremolytes, is in development.     

Molecular biology of extremophiles: recent progress on the hyperthermophilic archaeon Sulfolobus

Extremophiles are microorganisms that flourish in habitats of extreme temperature, pH, salinity, or pressure. All extreme environments are dominated by microorganisms belonging to Archaea, the third domain of life, evolutionary distinct from Bacteria and Eucarya. Over the past few years the biology of extremophilic Archaea has stimulated a lot of interest, aimed at understanding at molecular level the adaptation to their life conditions, as well as their evolutionary relationships to other organisms. Here, we review recent insights in the molecular biology of thermoacidophilic Archaea of the genus Sulfolobus, which has been used as a model system for biochemical, structural, and genetic studies in Archaea and extremophiles in general. With the recent completion of the genome sequence of Sulfolobus solfataricus it is expected that these organisms will contribute new discoveries in the near future. This revised version was published online in August 2006 with corrections to the Cover Date.     

Extremophiles as sources of inorganic bio-nanoparticles

Industrial use of nanotechnology in daily life has produced an emphasis on the safe and efficient production of nanoparticles (NPs). Traditional chemical oxidation and reduction methods are seen as inefficient, environmentally unsound, and often dangerous to those exposed and involved in NP manufacturing. However, utilizing microorganisms for biosynthesis of NPs allows efficient green production of a range of inorganic NPs, while maintaining specific size, shape, stability, and dispersity. Microorganisms living under harsh environmental conditions, called “Extremophiles,” are one group of microorganisms being utilized for this biosynthesis. Extremophiles’ unique living conditions have endowed them with various processes that enable NP biosynthesis. This includes a range of extremophiles: thermophiles, acidophilus, halophiles, psychrophiles, anaerobes, and some others. Fungi, bacteria, yeasts, and archaea, i.e. Ureibacillus thermosphaericus, and Geobacillus stearothermophilus, among others, have been established for NP biosynthesis. This article highlights the extremophiles and methods found to be viable candidates for the production of varying types of NPs, as well as interpreting selective methods used by the organisms to synthesize NPs.     

Extremo-phage: In vitro selection of tolerance to a hostile environment

Extremophiles are microorganisms that thrive under extreme conditions such as temperatures above 65°C, pHs below 4 or above 10, salt concentrations above 0.5 m, or pressures of 600 atm. While studies of enzymes either isolated from extremophiles, or generated using site-specific mutagenesis, or adapted by in vivo or in vitro selection have established a precedent for the engineering and application of proteins at extreme conditions, generalization of the approaches to more complex multimolecular or multitask systems has remained elusive. Here we demonstrate that a significantly more complex system—a bacteriophage—can over a number of generations be adapted to tolerate a hostile and unnatural environment. An in vitro selection strategy was used to adapt phage to urea, a protein denaturing agent. As the concentration of urea employed in selections over 20 generations was gradually increased from 5 to 9 m, the surviving phages steadily improved their tolerance, finally achieving a greater than 350-fold stability enhancement over the original population.Correspondence to: J. Yin     

Structural adaptation of active center channels of octaheme nitrite reductases from the haloalkaliphilic bacteria <Emphasis Type="Italic">Thioalkalivibrio nitratireducens</Emphasis> to a proton deficit

Study of the adaptation mechanisms of proteins from extremophiles paves the way for the development of new biocatalysts that are resistant to extreme conditions. Here, we studied the structural adaptation of active center channels of octaheme nitrite reductase from the haloalkophilic bacterium Thioalkalivibrio nitratireducens (TvNiR) to high pH. Comparative analysis of the structures of octaheme nitrite reductases adapted to different environmental conditions revealed unique adaptation mechanisms for TvNiR, which play an important role in binding rare protons and substrate and product migration in the active-site channels.     

The impact of medicinal brines on microbial biofilm formation on inhalation equipment surfaces

Abstract

Materials such as polyvinyl chloride, polypropylene, and polyethylene are used for the construction of medical equipment, including inhalation equipment. Inhalation equipment, because of the wet conditions and good oxygenation, constitutes a perfect environment for microbial biofilm formation. Biofilms may affect microbiological cleanliness of inhalation facilities and installations and promote the development of pathogenic bacteria. Microbial biofilms can form even in saline environments. Therefore, the aim of this study was to evaluate the effect of medicinal brines on microbial biofilm formation on the surfaces of inhalation equipment. The study confirmed the high risk of biofilm formation on surfaces used in inhalation equipment. Isolated microorganisms belonged to potential pathogens of the respiratory system, which can pose a health threat to hospital patients. The introduction of additional contaminants increased the amount of bacterial biofilm. On the other hand, the presence of brines significantly limited the amount of biofilm, thus eliminating the risk of infections.     

High glucose induced endothelial cell growth inhibition is associated with an increase in TGFbeta1 secretion and inhibition of Ras prenylation via suppression of the mevalonate pathway

ObjectiveRas proteins are known to affect cellular growth and function. The influence of the prenylation status of Ras on the observed changes in endothelial cell growth under high glucose conditions has not previously been examined.MethodsHuman umbilical vein endothelial cells were exposed to normal or high glucose conditions for 72 h. They were then examined for proliferative and hypertrophic effects, transforming growth factor β₁ (TGFβ₁) release, and phosphorylated p38 expression. The importance of prenylation was explored by the addition of mevalonate, isoprenoids or farnesyltransferase inhibitors to control the high glucose media and by measuring changes induced by high glucose and exogenous TGFβ₁ in Ras prenylation and farnesyltransferase activity. Kidneys from diabetic rats treated with atorvastatin were also compared to specimens from untreated animals and the expression of the Ras effector p-Akt examined.ResultsHigh glucose conditions caused a reduction in cell number. This was reversed in the presence of mevalonate or farnesylpyrophosphate (FPP), suggesting that the cell growth abnormalities observed are due to high glucose induced inhibition of the mevalonate pathway and subsequent prenylation of proteins. Endothelial cells exposed to high glucose increased their secretion of TGFβ₁ and the phosphorylation of p38 both of which were reversed by concurrent exposure to FPP. A reduction in farnesyltransferase activity was observed after exposure to both high glucose and TGFβ₁. Exposure to a farnesyltransferase inhibitor in control conditions mimicked the growth response observed with high glucose exposure and prenylated Ras was reduced by exposure to both high glucose and TGFβ₁. Finally, interruption of the mevalonate pathway with a statin reduced the expression of p-Akt in diabetic rat kidneys.ConclusionThis study demonstrates that high glucose induced significant alterations in endothelial cell growth by inhibition of the mevalonate pathway, which subsequently mediates the increase in TGFβ₁ and inhibition of Ras prenylation.