Extremophiles in a changing world.

Extremophiles and their products have been a major focus of research interest for over 40 years. Through this period, studies of these organisms have contributed hugely to many aspects of the fundamental and applied sciences, and to wider and more philosophical issues such as the origins of life and astrobiology. Our understanding of the cellular adaptations to extreme conditions (such as acid, temperature, pressure and more), of the mechanisms underpinning the stability of macromolecules, and of the subtleties, complexities and limits of fundamental biochemical processes has been informed by research on extremophiles. Extremophiles have also contributed numerous products and processes to the many fields of biotechnology, from diagnostics to bioremediation. Yet, after 40 years of dedicated research, there remains much to be discovered in this field. Fortunately, extremophiles remain an active and vibrant area of research. In the third decade of the twenty-first century, with decreasing global resources and a steadily increasing human population, the world's attention has turned with increasing urgency to issues of sustainability. These global concerns were encapsulated and formalized by the United Nations with the adoption of the 2030 Agenda for Sustainable Development and the presentation of the seventeen Sustainable Development Goals (SDGs) in 2015. In the run-up to 2030, we consider the contributions that extremophiles have made, and will in the future make, to the SDGs.

Thermostable lipases from the extreme thermophilic anaerobic bacteria Thermoanaerobacter thermohydrosulfuricus SOL1 and Caldanaerobacter subterraneus subsp. tengcongensis.

Two novel genes encoding for heat and solvent stable lipases from strictly anaerobic extreme thermophilic bacteria Thermoanaerobacter thermohydrosulfuricus (LipTth) and Caldanaerobacter subterraneus subsp. tengcongensis (LipCst) were successfully cloned and expressed in E. coli. Recombinant proteins were purified to homogeneity by heat precipitation, hydrophobic interaction, and gel filtration chromatography. Unlike the enzymes from mesophile counterparts, enzymatic activity was measured at a broad temperature and pH range, between 40 and 90 degrees C and between pH 6.5 and 10; the half-life of the enzymes at 75 degrees C and pH 8.0 was 48 h. Inhibition was observed with 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride and phenylmethylsulfonylfluorid indicating that serine and thiol groups play a role in the active site of the enzymes. Gene sequence comparisons indicated very low identity to already described lipases from mesophilic and psychrophilic microorganisms. By optimal cultivation of E. coli Tuner (DE3) cells in 2-l bioreactors, a massive production of the recombinant lipases was achieved (53-2200 U/l) Unlike known lipases, the purified robust proteins are resistant against a large number of organic solvents (up to 99%) and detergents, and show activity toward a broad range of substrates, including triacylglycerols, monoacylglycerols, esters of secondary alcohols, and p-nitrophenyl esters. Furthermore, the enzyme from T. thermohydrosulfuricus is suitable for the production of optically pure compounds since it is highly S-stereoselective toward esters of secondary alcohols. The observed E values for but-3-yn-2-ol butyrate and but-3-yn-2-ol acetate of 21 and 16, respectively, make these enzymes ideal candidates for kinetic resolution of synthetically useful compounds.

Rapid access to genes of biotechnologically useful enzymes by partial genome sequencing: the thermoalkaliphile Anaerobranca gottschalkii.

Anaerobranca gottschalkii strain LBS3 T is an extremophile living at high temperature (up to 65 degrees C) and in alkaline environments (up to pH 10.5). An assembly of 696 DNA contigs representing about 96% of the 2.26-Mbp genome of A. gottschalkii has been generated with a low-sequence-coverage shotgun-sequencing strategy. The chosen sequencing strategy provided rapid and economical access to genes encoding key enzymes of the mono- and polysaccharide metabolism, without dilution of spare resources for extensive sequencing of genes lacking potential economical value. Five of these amylolytic enzymes of considerable commercial interest for biotechnological applications have been expressed and characterized in more detail after identification of their genes in the partial genome sequence: type I pullulanase, cyclodextrin glycosyltransferase (CGTase), two alpha-amylases (AmyA and AmyB), and an alpha-1,4-glucan-branching enzyme.

Oligonucleotide microarrays for the detection and identification of viable beer spoilage bacteria.

AIMS: The design and evaluation of an oligonucleotide microarray in order to detect and identify viable bacterial species that play a significant role in beer spoilage. These belong to the species of the genera Lactobacillus, Megasphaera, Pediococcus and Pectinatus. METHODS AND RESULTS: Oligonucleotide probes specific to beer spoilage bacteria were designed. In order to detect viable bacteria, the probes were designed to target the intergenic spacer regions (ISR) between 16S and 23S rRNA. Prior to hybridization the ISR were amplified by combining reverse transcriptase and polymerase chain reactions using a designed consenus primer. The developed oligonucleotide microarrays allows the detection of viable beer spoilage bacteria. CONCLUSIONS: This method allows the detection and discrimination of single bacterial species in a sample containing complex microbial community. Furthermore, microarrays using oligonucleotide probes targeting the ISR allow the distinction between viable bacteria with the potential to grow and non growing bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The results demonstrate the feasibility of oligonucleotide microarrays as a contamination control in food industry for the detection and identification of spoilage micro-organisms within a mixed population.

Practical application of different enzymes immobilized on sepabeads.

The immobilization of an endoglucanase, benzoylformate decarboxylase (BFD) from Pseudomonas putida, as well as of lipase B from Candida antarctica (CALB) onto the carrier supports Sepabeads EC-EP, Sepabeads EC-EA, and Sepabeads EC-BU was accomplished. It is shown that via these immobilized biocatalysts the synthesis of both fine and bulk chemicals is possible. This is illustrated by the syntheses of polyglycerol esters and (S)-hydroxy phenyl propanone. The benefit of immobilization is illustrated by repetitive use in a bubble column reactor as well as in a stirred tank reactor. High stability of two biocatalysts was achieved and reusability up to eight times was demonstrated. The comparison of CALB immobilized on Sepabeads EC-EP to Novozym 435 shows similar activity.

High yield of mannosylglycerate production by upshock fermentation and bacterial milking of trehalose-deficient mutant Thermus thermophilus RQ-1.

A production process, using upshock fermentation and osmotic downshock, for the effective production/excretion of mannosylglycerate (MG) by the trehalose-deficient mutant of the strain Thermus thermophilus RQ-1 has been developed. In the first phase of fed-batch fermentation, the knockout mutant was grown at 70 degrees C on a NaCl-free medium. After the culture reached the end of the exponential growth phase, upshift in temperature and NaCl concentration was applied. The temperature was increased to 77 degrees C, and NaCl was added up to 3.0% and kept constant during the second phase of fermentation. Although this shift in cultivation parameters caused a dramatic drop of cell density, a significant improvement in accumulation of MG up to 0.64 micromol/mg protein compared to batch fermentations (0.31 micromol/mg protein) was achieved. A total yield of 4.6 g MG/l of fermentation broth was obtained in the dialysis bioreactor with a productivity of 0.29 g MG l(-1) h(-1). The solute was released from the harvested biomass by osmotic downshock using demineralized water at 70 degrees C. More than 90% of the intracellularly accumulated solute was recovered from the water fraction. The process was very efficient, as hyperosmotic shock, release of the solute, and reiterative fed-batch fermentation could be repeated at least four times.

Genome sequence of Picrophilus torridus and its implications for life around pH 0.

The euryarchaea Picrophilus torridus and Picrophilus oshimae are able to grow around pH 0 at up to 65 degrees C, thus they represent the most thermoacidophilic organisms known. Several features that may contribute to the thermoacidophilic survival strategy of P. torridus were deduced from analysis of its 1.55-megabase genome. P. torridus has the smallest genome among nonparasitic aerobic microorganisms growing on organic substrates and simultaneously the highest coding density among thermoacidophiles. An exceptionally high ratio of secondary over ATP-consuming primary transport systems demonstrates that the high proton concentration in the surrounding medium is extensively used for transport processes. Certain genes that may be particularly supportive for the extreme lifestyle of P. torridus appear to have been internalized into the genome of the Picrophilus lineage by horizontal gene transfer from crenarchaea and bacteria. Finally, it is noteworthy that the thermoacidophiles from phylogenetically distant branches of the Archaea apparently share an unexpectedly large pool of genes.

Enzymatic production of cyclodextrins.

Cyclodextrins (CD) are enzymatically modified starches with a wide range of applications in food, pharmaceutical and chemical industries, agriculture and environmental engineering. They are produced from starch via enzymatic conversion using cyclodextrin glycosyl transferases (CGTases) and partly alpha-amylases. Due to its low solubility in water, separation and purification of beta-CD is relatively easy compared to alpha- and gamma-CD. In recent years more economic processes for gamma-CD and especially alpha-CD production have been developed using improved CGTases and downstream processing. New purification steps, e.g. affinity adsorption, may reduce the use of complexing agents. The implementation of thermostable CGTases can simplify the production process and increase the selectivity of the reaction. A tabular overview of alpha-CD production processes is presented.

Thermoanaerobacter yonseiensis sp. nov., a novel extremely thermophilic, xylose-utilizing bacterium that grows at up to 85 degrees C.

A novel strictly anaerobic, extremely thermophilic, spore-forming and xylose-utilizing bacterium, designated strain KB-1TP (type and patent strain), was isolated from a geothermal hot stream at Sileri on Java island, Indonesia. The cells were rod-shaped, motile and had terminal spores. The newly isolated strain stained gram-positive and the cells occurred singly or in pairs during the exponential growth phase. The temperature optimum for growth was 75 degrees C and growth occurred in the range 50-85 degrees C. The pH range for growth was 4.5-9.0, with an optimum at pH 6.5. Strain KB-1TP grew chemo-organotrophically by fermenting a wide range of substrates such as glucose, fructose, D-xylose, lactose, maltose, sucrose, mannose, galactose, cellobiose, pullulan and soluble starch. Arabinose, xylan, cellulose, olive oil and Tween 80 were not fermented. The predominant fermentation end products after growth on glucose were lactate, acetate, ethanol, CO2 and small amounts of isovaleric acid, butyric acid, propionic acid, 1-pentanol and 2-propanol. Thiosulfate was reduced to H2S. Strain KB-1TP was sensitive to tetracycline, chloramphenicol, penicillin G, neomycin, kanamycin, vancomycin and rifampicin at concentrations of 100 microg ml(-1). No effect was observed with chloramphenicol and neomycin at concentrations of 10 microg ml(-1). This indicates that strain KB-1TP belongs to the bacterial domain. The G+C content of the DNA was 37 mol%. The comparison of the 165 rDNA sequence to that of closely related strains revealed that strain KB-1TP belonged to clostridial cluster V, showing highest sequence identities (92.7%) to members of the genus Thermoanaerobacter. Taking into account the physiological and molecular properties of the new isolate, it is proposed that strain KB-1TP should be classified as a new species of the genus Thermoanaerobacter, designated Thermoanaerobacter yonseiensis. The type strain, KB-1TP, has been deposited in the Korean Federation of Culture Collections (KFCC 11116P) as a patent strain and in the Deutsche Sammlung von Mikroorganismen und Zellkulturen as a type strain (= DSM 13777T).

Anaerobranca gottschalkii sp. nov., a novel thermoalkaliphilic bacterium that grows anaerobically at high pH and temperature.

A novel thermoalkaliphilic, obligately anaerobic bacterium was isolated from a humid soil sample of a hot inlet of Lake Bogoria, Kenya. The newly isolated strain grows optimally at pH 9.5 and 50-55 degrees C and its growth range is pH 6.0-10.5 and 30-65 degrees C. Unlike the already known thermoalkaliphiles, the strain grows heterotrophically on a variety of mono- and polysaccharides (glucose, ribose, mannose, fructose, sucrose, maltose, starch, pullulan, xylan and cellulose) and on proteinaceous substrates such as yeast extract, peptone and tryptone. No dissimilatory sulfate reduction was observed, whereas thiosulfate was found to enhance growth when glucose or starch were used as substrates. Under optimal conditions, the doubling time is 48 min. Sodium ions are necessary for growth, with an optimal concentration of 230 mM (1% NaCl, w/v) at pH 9.5. The rod-shaped cells are motile in the exponential growth phase under optimal growth conditions. Despite the Gram-negative staining and negative KOH assay, the strain is a Gram-positive organism, having an atypically thin cell wall. A sheath-like structure occurs at the cell separation area and parts of a surface layer-like structure were also observed. Based on physiological properties and molecular biological analysis, the strain falls within the radiation of the clostridia and represents a new species of Anaerobranca within the Clostridium/Bacillus subphylum of the Gram-positive bacteria. Strain LBS3T (= DSM 13577T) is named Anaerobranca gottschalkii sp. nov. and is designated as the type strain.

Physiological aspects involved in production of xylanolytic enzymes by deep-sea hyperthermophilic archaeon Pyrodictium abyssi.

Xylanases (EC 3.2.1.8) catalyze the hydrolysis of xylan, the major constituent of hemicellulose. The use of these enzymes could greatly improve the overall economics of processing lignocellulosic materials for the generation of liquid fuels and chemicals. The hyperthermophilic archaeon Pyrodictium abyssi, which was originally isolated from marine hot abyssal sites, grows optimally at 97 degrees C and is a prospective source of highly thermostable xylanase. Its endoxylanase was shown to be highly thermostable (over 100 min at 105 degrees C) and active even at 110 degrees C. The growth of the deep-sea archaeon P. abyssi was investigated using different culture techniques. Among the carbohydrates used, beech wood xylan, birch wood glucuronoxylan and the arabinoxylan from oats pelt appeared to be good inducers for endoxylanase and beta-xylosidase production. The highest production of arabinofuranosidase, however, was detected in the cell extracts after growth on xylose and pyruvate, indicating that the intermediate of the tricarboxylic acid cycle acted as a nonrepressing carbon source for the production of this enzyme. Electron microscopic studies did not show a significant difference in the cell surface (e.g., xylanosomes) when P. abyssi cells were grown on different carbohydrates. The main kinetic parameters of the organism have been determined. The cell yield was shown to be very low owing to incomplete substrate utilization, but a very high maximal specific growth rate was determined (micromax = 0.0195) at 90 degrees C and pH 6.0. We also give information on the problems that arise during the fermentation of this hyperthermophilic archaeon at elevated temperatures.

Isolation of Thermoanaerobacter keratinophilus sp. nov., a novel thermophilic, anaerobic bacterium with keratinolytic activity.

Several thermophilic anaerobic bacteria with keratinolytic activity growing at temperatures between 50 degrees C and 90 degrees C were isolated from samples collected on the island of São Miguel in the Azores (Portugal). On the basis of morphological, physiological, and 16S rDNA studies, the isolate 2KXI was identified as a new species of the genus Thermoanaerobacter, designated Thermoanaerobacter keratinophilus. This strain, which grows optimally at 70 degrees C, pH 7.0, and 0.5% NaCl, is the first member of the genus Thermoanaerobacter that has been described for its ability to degrade native keratin. Around 70% of native wool was solubilized after 10 days of incubation under anaerobic conditions. The strain was shown to possess intracellular and extracellular proteases optimally active at 60 degrees C, pH 7.0, and 85 degrees C, pH 8.0, respectively. Keratin hydrolysis was demonstrated in vitro using a sodium dodecyl sulfate gel containing feather meal. The extracellular protease responsible for breaking down keratin fibers was purified to homogeneity in only one step by applying hydroxyapatite column chromatography. The enzyme belongs to the serine-type proteases and has a molecular mass of 135 kDa.

PCR performance of the B-type DNA polymerase from the thermophilic euryarchaeon Thermococcus aggregans improved by mutations in the Y-GG/A motif.

The effect of mutations in the highly conserved Y-GG/A motif of B-type DNA polymerases was studied in the DNA polymerase from the hyperthermophilic euryarchaeon Thermococcus aggregans. This motif plays a critical role in the balance between the synthesis and degradation of the DNA chain. Five different mutations of the tyrosine at position 387 (Tyr387-->Phe, Tyr387-->Trp, Tyr387-->His, Tyr387-->Asn and Tyr387-->Ser) revealed that an aromatic ring system is crucial for the synthetic activity of the enzyme. Amino acids at this position lacking the ring system (Ser and Asn) led to a significant decrease in polymerase activity and to enhanced exonuclease activity, which resulted in improved enzyme fidelity. Exchange of tyrosine to phenylalanine, tryptophan or histidine led to phenotypes with wild-type-like fidelity but enhanced PCR performance that could be related to a higher velocity of polymerisation. With the help of a modelled structure of T.aggregans DNA polymerase, the biochemical data were interpreted proposing that the conformation of the flexible loop containing the Y-GG/A motif is an important factor for the equilibrium between DNA polymerisation and exonucleolysis.

Cloning, expression and biochemical characterisation of a unique thermostable pullulan-hydrolysing enzyme from the hyperthermophilic archaeon Thermococcus aggregans.

The gene for a new type of pullulan hydrolase from the hyperthermophilic archaeon Thermococcus aggregans was cloned and expressed in Escherichia coli. The 2181-bp open reading frame encodes a protein of 727 amino acids. A hypothetical membrane linker region was found to be cleaved during processing in E. coli. The recombinant enzyme was purified 70-fold by heat treatment, affinity and anion exchange chromatography. Optimal activity was detected at 95 degrees C at a broad pH range from 3.5 to 8.5 with an optimum at pH 6.5. More than 35% of enzymatic activity was detected even at 120 degrees C. The enzyme was stable at 90 degrees C for several hours and exhibited a half-life of 2.5 h at 100 degrees C. Unlike all pullulan-hydrolysing enzymes described to date, the enzyme is able to attack alpha-1,6- as well as alpha-1,4-glycosidic linkages in pullulan leading to the formation of a mixture of maltotriose, panose, maltose and glucose. The enzyme is also able to degrade starch, amylose and amylopectin forming maltotriose and maltose as main products.

A new thermoactive pullulanase from Desulfurococcus mucosus: cloning, sequencing, purification, and characterization of the recombinant enzyme after expression in Bacillus subtilis.

The gene encoding a thermoactive pullulanase from the hyperthermophilic anaerobic archaeon Desulfurococcus mucosus (apuA) was cloned in Escherichia coli and sequenced. apuA from D. mucosus showed 45.4% pairwise amino acid identity with the pullulanase from Thermococcus aggregans and contained the four regions conserved among all amylolytic enzymes. apuA encodes a protein of 686 amino acids with a 28-residue signal peptide and has a predicted mass of 74 kDa after signal cleavage. The apuA gene was then expressed in Bacillus subtilis and secreted into the culture fluid. This is one of the first reports on the successful expression and purification of an archaeal amylopullulanase in a Bacillus strain. The purified recombinant enzyme (rapuDm) is composed of two subunits, each having an estimated molecular mass of 66 kDa. Optimal activity was measured at 85 degrees C within a broad pH range from 3.5 to 8.5, with an optimum at pH 5.0. Divalent cations have no influence on the stability or activity of the enzyme. RapuDm was stable at 80 degrees C for 4 h and exhibited a half-life of 50 min at 85 degrees C. By high-pressure liquid chromatography analysis it was observed that rapuDm hydrolyzed alpha-1,6 glycosidic linkages of pullulan, producing maltotriose, and also alpha-1,4 glycosidic linkages in starch, amylose, amylopectin, and cyclodextrins, with maltotriose and maltose as the main products. Since the thermoactive pullulanases known so far from Archaea are not active on cyclodextrins and are in fact inhibited by these cyclic oligosaccharides, the enzyme from D. mucosus should be considered an archaeal pullulanase type II with a wider substrate specificity.

Crystallization and preliminary X-ray crystallographic studies of the thermoactive pullulanase type I, hydrolyzing alpha-1,6 glycosidic linkages, from Fervidobacterium pennivorans Ven5.

Crystals of the thermoactive recombinant F. pennivorans type I pullulanase, purified from the supernatant of a Bacillus subtilis culture, have been obtained by the vapour-diffusion method in the presence of the inhibitor beta-cyclodextrin (2 mM) by mixing protein (15 mg ml(-1)) with an equal volume of crystallization solution containing 0.1 M bis-tris propane pH 6.5, 50 mM MgCl(2) and 15% polyethylene glycol 3350. Crystals diffracted to 3.0 A using conventional Cu Kalpha radiation and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 76.8, b = 96.2, c = 98. 5 A. The asymmetric unit contains one monomer. A preliminary 26% complete data set has been collected at 2.2 A resolution using synchrotron radiation.

Alicyclobacillus hesperidum sp. nov. and a related genomic species from solfataric soils of São Miguel in the Azores.

Several acidophilic, slightly thermophilic or thermophilic Gram-positive isolates were recovered from solfataric soil at Furnas on the Island of São Miguel in the Azores. Phylogenetic analysis of the 16S rRNA gene sequence showed that these organisms represented two novel species of the genus Alicyclobacillus. Strains FR-11T and FR-1b had an optimum growth temperature of about 50 degrees C, whereas strains FR-3 and FR-6T had an optimum growth temperature of about 60 degrees C. Biochemical, physiological and chemotaxonomic characteristics did not distinguish isolates FR-3 and FR-6T from the type strain of Alicyclobacillus acidocaldarius; however, strains FR-11T and FR-1b could be easily distinguished from the type strain of Alicyclobacillus acidoterrestris by the carbon source assimilation pattern and the fatty acid composition. On the basis of the phylogenetic analysis, physiological and biochemical characteristics, and fatty acid composition the name Alicyclobacillus hesperidum is proposed for the species represented by strains FR-11T and FR-1b; a formal name for the new genomic species represented by strains FR-3 and FR-6T is not proposed at this time.

Cloning and expression of alpha-amylase from the hyperthermophilic archaeon Pyrococcus woesei in the moderately halophilic bacterium Halomonas elongata.

An extracellular alpha-amylase gene from the hyperthermophilic archaeon Pyrococcus woesei has been cloned and sequenced. The 1.4-kb protein-coding sequence is identical to that of the corresponding alpha-amylase gene of the closely related species P. furiosus. By using a shuttle cloning vector for halophilic bacteria, the P. woesei alpha-amylase was expressed in the moderate halophile Halomonas elongata, under the control of a native H. elongata promoter. The hyperthermophilic amylase activity expressed in the halophilic host was recovered completely in the crude membrane fraction of cell homogenates, suggesting the formation of inclusion bodies or that the secretion machinery of H. elongata may fail to recognize and release the pyrococcal alpha-amylase to the extracellular medium. However, thermal stability, metal ion interactions, optimal temperature and pH values for the crude and purified recombinant alpha-amylase were comparable with those of the native pyrococcal enzyme. The P. woesei amylase activity expressed in H. elongata was consistently detected in the cells upon growth on a wide range of NaCl concentrations (0.7-2.5 mol l-1). To our knowledge, this is the first report on the expression of an archaeal gene (P. woesei alpha-amylase) in a moderate halophilic host which serves as a cell factory able to grow under extreme salt conditions and with very simple nutritional requirements.

Naphthalene degradation and incorporation of naphthalene-derived carbon into biomass by the thermophile Bacillus thermoleovorans.

The thermophilic aerobic bacterium Bacillus thermoleovorans Hamburg 2 grows at 60 degrees C on naphthalene as the sole source of carbon and energy. In batch cultures, an effective substrate degradation was observed. The carbon balance, including naphthalene, metabolites, biomass, and CO(2), was determined by the application of [1-(13)C]naphthalene. The incorporation of naphthalene-derived carbon into the bulk biomass as well as into specified biomass fractions such as fatty acids and amino acids was confirmed by coupled gas chromatography-mass spectrometry (GC-MS) and isotope analyses. Metabolites were characterized by GC-MS; the established structures allow tracing the degradation pathway under thermophilic conditions. Apart from typical metabolites of naphthalene degradation known from mesophiles, intermediates such as 2, 3-dihydroxynaphthalene, 2-carboxycinnamic acid, and phthalic and benzoic acid were identified for the pathway of this bacterium. These compounds indicate that naphthalene degradation by the thermophilic B. thermoleovorans differs from the known pathways found for mesophilic bacteria.