Revista de Microbiologia (1999) 30:287-298 Extremely thermophilic microorganisms ISSN 0001-3714 EXTREMELY THERMOPHILIC MICROORGANISMS AND THEIR POLYMER- HYDROLYTIC ENZYMES Carolina M.M.C. Andrade1*; Nei Pereira Jr.1; Garo Antranikian2 1Departamento de Engenharia Bioquímica, Escola de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ, Brasil. 2Department of Technical Microbiology, Technical University Hamburg- Harburg, Hamburg, Germany Submitted: May 25, 1999; Returned to authors for corrections: July 29, 1999; Approved: August 26, 1999 MINI-REVIEW ABSTRACT Thermophilic and hyperthermophilic microorganisms are found as normal inhabitants of continental and submarine volcanic areas, geothermally heated sea-sediments and hydrothermal vents and thus are considered extremophiles. Several present or potential applications of extremophilic enzymes are reviewed, especially polymer-hydrolysing enzymes, such as amylolytic and hemicellulolytic enzymes. The purpose of this review is to present the range of morphological and metabolic features among those microorganisms growing from 70oC to 100°C and to indicate potential opportunities for useful applications derived from these features. Key words: Archaea, extremophiles, amylases, xylanases, pullulanases, thermostability INTRODUCTION especially the thermophiles, is an area that is receiving increasing attention. In general, moderate In recent years it became obvious that thermophiles are primarily bacteria and display extremophilic microorganisms differ from eucaryotic optimal growth temperature between 60°C and 80°C. cells because they have adapted to grow under Hyperthermophiles are primarily archaea and growth extreme conditions such as high temperature optimally at 80°C or above, being unable to grow (>100°C), high salinity (saturated NaCl), extremes below 60°C (47). of pH (<2.0, >10.0), and substrate stress. These kinds The hyperthermophiles are now well characterised of extreme microbial growth conditions are found taxonomically at the DNA-DNA hybridisation level, in exotic environments which were more widespread and their evolutionary relatedness has been examined. on primitive Earth. Extreme environments include By using 16S rRNA sequence comparison, an archaeal also high pressure (> 50 MPa) and the presence of phylogenetic tree has been proposed (54), with a organic solvents (e.g. > 1% toluene) or heavy metals. tripartite division of the living world consisting of the The evolution and taxonomy of extremophiles, domains Eucarya, Bacteria, and Archaea. In this * Corresponding author. Mailing address: Universidade Federal do Rio de Janeiro, Ilha do Fundão Centro de Tecnologia, Bloco E, Escola de Química, Departamento de Engenharia Bioquímica, Laboratório de Engenharia Bioquímica, CEP 21949-900, Rio de Janeiro, RJ, Brasil. Fax: (+5521) 590-4991, E-mail: [email protected] 287 C.M.M.C. Andrade et al. division Sulfolobales and Thermoproteales form one Thermophiles and hyperthermophiles: branch (the Crenarchaeota) and the remaining physiological and morphological aspects. Most of thermophiles form another branch containing the anaerobic thermophilic bacteria are methanogens, and extreme halophiles (the chemoorganotrophic in their metabolism. The Euryarchaeota). Currently, the only hyperthermophilic bacterial thermophilic thermoanaerobes, for organisms within the Bacterial domain are members example, belong to nearly the same range of of the genus Thermotoga and Aquifex (47). Until now, nutritional categories as do mesophilic bacteria. The no hyperthermophilic microorganisms in the domain hyperthermophilic bacteria Thermotoga are able to Eucarya have been reported. ferment various carbohydrates like glucose, starch Hyperthermophiles are represented at the deepest and xylans, forming acetate, L-lactate, H2 and CO2 and shortest lineages, including both genera of as end product (47), while the hyperthermophilic hyperthermophilic bacteria and the genus Aquifex is strictly chemolithoautotrophic, using Pyrodictium, Pyrobaculum, Desulfurococcus, molecular hydrogen, thiosulfate and elemental Sulfolobus, Methanopyrus, Thermococcus, sulphur as electron donors and oxygen (at low Methanothermus, Archaeoglobus within the concentrations) and nitrate as electron acceptors (22). Archaea. Recently, genetic elements, e.g. viruses and In general, the physiological processes for plasmids (excluding IS elements and transposons) adaptation to environmental stress in anaerobic have been described in the kingdom Crenarcheota bacteria seem to have involved different factors from (Thermoproteales and Sulfolobales) and in the those in aerobic bacteria. First, anaerobes are energy kingdom Euryarchaeota (Thermococcales and limited during the chemoorganotrophic growth Thermoplasmales) of the archaeal domain (57). because they can not couple dehydrogenation Some similarities between the archaeal virus FH and reaction to oxygen reduction and gain a high level the bacterial phage P1 strongly indicate that this of chemical free energy. Second, growth of most temperate phage type already existed before the chemoorganotrophic anaerobes (except for separation of the Archaea from the Bacteria, which methanogens) is naturally associated with the was the first documented lineage diversion in cellular generation of toxic end products (e.g., organic acids evolution (57). Based on these observations, or alcohols, HS-), which requires that anaerobic hyperthermophiles may still be rather primitive and species develop some sort of dynamic adaptation the last common ancestor, the progenota, may have mechanism or tolerance to their catabolic end been a hyperthermophile (47, 54). products. In the last decades thermophilic and The most interesting group of thermophiles is the hyperthermophilic anaerobes have been isolated hyperthermophiles, since the isolation of these from continental and submarine volcanic areas, such organisms has caused a revaluation of the possible as solfatar fields, geothermal power plants, habitats for microorganisms and has increased the geothermally heated sea sediments and hydrothermal high-temperature limits at which life is known to vents (14, 18, 47, 50). Sites from which exist. The hyperthermophilic anaerobic archaea have hyperthermophilic organisms have been isolated almost the same size as one typical procaryotic cell, comprises solfataric fields; steam-heated soils, mud about 0.5 - 2.0µm, although some of them have holes, surface waters; deep hot springs; geothermal unusual morphological features (47). power plants as well as submarine hot springs and Hyperthermophiles are rather diverse with respect fumaroles; hot sediments and vents,black smokers to their metabolism, since they include methanogens, or chimneys; and active sea-mounts. sulphate-reducers, nitrate-reducers and also the It is interesting that some organisms have been aerobic respirers. However the majority of the species isolated from areas with temperatures much higher know at the present are strictly anaerobic than their maximum growth temperature, e.g., heterotrophic S0 reducers (24). Among the terrestrial Hyperthermus butilicus (56) and Pyrococcus abyssi Archaea, three groups can be distinguished. (18), which suggests that in these environments the Acidophilic extremethermophiles, which are found organisms may not be actively growing. The same exclusively within continental solfataric fields. The could be true for the organisms isolated from organisms are coccoid-shaped, strict and facultative temperatures much below their growth temperature aerobes, and require acidic pH (opt. approx. pH 3.0) optimum, such as Archaeoglobus profundus (7). to grow. Phylogenetically, they belong to the archaeal 288 Extremely thermophilic microorganisms genera Sulfolobus, Metallosphaera, Acidianus, and cells are connected by a network of ultra thin hollow Desulfurolobus (47). On the other hand, the slightly tubules (47). Strains of Pyrodictium are usually acidophilic and neutrophilic thermophiles are found chemolitoautotrophs gaining energy by reduction of 0 both in continental solfataric fields and in submarine S by H2. Although growth is stimulated by yeast hydrothermal systems. All of them are strict extract, both species of Pyrodictium are strictly anaerobes. Solfataric fields contain members of the dependent upon H2. genera Thermoproteus, Pyrobaculum, As an exception, Pyrodictium abyssi is a Thermophilum, Desulfurococcus, and heterotroph growing by fermentation of peptides and Methanothermus. Pyrobaculum islandicum is able is unable to grow chemolitotrophically on H2/CO2 0 -2 to grow autotrophically by anaerobic reduction of either in the presence of S or S2O3 . Similar to the 0 S with H2 as electron donor (35), but is also able to other members of the genus, the cells of Pyrodictium grow heterotrophically by sulphur respiration (47). abyssi are highly polymorphous, often disk-shaped, Strains of Thermophilum and Pyrobaculum and display ultra flat areas. The cell envelope consists organotrophum are obligate heterotrophs. They grow of the cytoplasmic membrane, a periplasmic space, by sulphur respiration using different organic and a surface layer protein. The ultra thin sections substrates. Interestingly, Thermophilum pendens also reveal a zigzag structure of the S-layer (40). shows an obligate requirement for a lipid fraction of Usually S-layer proteins are highly stable, maintain Thermoproteux tenax (117). the structural integrity of bacterial cells under The variety of hyperthermophilic archaea that are extreme environmental