Archaebacteria: ancient organisms with commercial potential

Letters in Applied Microbiology 1989,9, 33-39

GWGJ048

A Review Archaebacteria : ancient organisms with commercial potential D . W . HOUGH& M . J . DANSON* Department of Biochemistry, University of Bath, Bath BA2 7 A Y , U K Received 11 May 1989 and accepted 22 May 1989 The archaebacteria-their phylogeny, their potential From comparisons of rRNA sequences, it has been proposed that the archaebacteria are a phylogenetically distinct group of organisms and thus they constitute a third Kingdom, in addition to the eubacteria (the true bacteria) and the eukaryotes (reviewed in Woese & Wolfe 1985; Woese 1987; Matheson & Dennis 1989). The archaebacteria encompass three basic phenotypes, namely halophilic, thermophilic and methanogenic. Central to this review is the fact that they all live under extreme conditions, typical of the environment thought to exist during early life on earth (Table I), and thus the name archaebacteria was suggested. However, the question of their primitive nature is still a matter of debate, although the rRNA sequence analysis does indicate that the archaebacteria are indeed an ancient evolutionary lineage, the oldest branch of which is probably that of the thermoacidophiles. Many biochemical features of the archaebacteria reinforce this proposed distinct phylogeny. These characteristics span a broad range of cellular biochemistry, including the structures of proteins, enzymes, co-enzymes, lipids, ribosomes and cell walls, the pathways of metabolism and the processes of transcription and translation. Some of the features are unique to the archaebacteria, whereas others are typically eubacterial or eukaryotic. Recently, Lake (1988) has questioned the trichotomous division of living organisms. Using a newlydeveloped tree algorithm, but the same rRNA sequence data, Lake derives an evolutionary tree composed of two taxonomic divisions; these are termed the proto-eukaryotic group (giving rise to the eukaryotes and the eocytes-the sulphur-metabolizing ‘archaebacteria’) and the parkaryotic group (the eubacteria, the halophiles and the methanogens). This tree, whilst arguing against the concept of the archaebacteria as a monophyletic group, also predicts that the last common ancestor of extant life was a sulphur-metabolizing, extreme thermophile. Clearly, the construction of phylogenetic trees is still a contentious issue. However, for the purposes of this review, these arguments need not concern us; what is important here is the observation that the group of organisms commonly known as archaebacteria have unique biochemical features and that they all live under extreme conditions. The latter implies that their cellular components, whether individual macromolecules or macromolecular assemblies, are adapted to function in environments destructive to their mesophilic counterparts. The use of biological components in industrial technology demands robust molecules and demands unique capabilities : the archaebacteria and their constituents can help meet these demands.

The biotechnological potential of the archaebacteria This brief review is intended to highlight the contributions that the archaebacteria can make to the present and future needs of biotechnology. The brevity may often prevent detailed discussion but, wherever possible, comprehensive reviews will be referred to; indeed, our purpose is to stimulate the

* Corresponding author.

D . W . Hough and M . J . Danson

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reader to look further into this unique group of organisms, not only for their usefulness but also because they are biochemically and physiologically fascinating in themselves. We will discuss the organisms in terms of their phenotypes rather than their phylogenetic status because, as indicated above, it is the phenotypic characteristics that determine their biotechnological potential. HALOPHILIC ARCHAEBACTERIA

Halophiles are adapted to life in high salt concentrations (Table 1) and it is the consequent stability of their proteins and the unique nature of some of their membrane components that lend themselves to biotechnological uses. Halophilic enzymes The extremely halophilic archaebacteria live in very high concentrations of NaCl, with some members growing in saturated (5.2 mol/l) salt solutions. The intracellular concentration of salt is >3.5 mol/l, but is mainly KCl with lower concentrations of NaCl (Kushner 1985). Salts tend to withdraw water from proteins and make hydrophobic bonds strong, causing the polypeptides to aggregate and collapse; these effects have been overcome in halophilic proteins by an increased frequency of negatively-charged amino acids (glutamate and aspartate) and a decrease in non-polar residues (Eisenberg & Wachtel 1987; Danson 1988). Table 1. Archaebacterial phenotypes and their environments Phenotype

Genera

Typical environments

Halobacterium Halococcus Natronobacterium Natronococcus

30"-40"C,

pH 6 7 ,

3-5 mold NaC1, aerobic

30"#C,

pH 9-11,

3-5 mol/l NaCI, aerobic

Thermophilic

Thermoplasma Sulfolobus Acidothermus Thermoproteus Thermojilum Desulfurococcus Thermococcus Thermodiscus Archaeoglobus Pyrobaculum Pyrococcus Pyrodictium

50"-60"C, 6Oo-90"C, 85"-93"C, 70°-l10"C, 55"-1WC, 8Oo-10O"C, 75"-90"C, 75"-98"C, 70°-92"C, 74"-102"C, 70"-103"C, 85"-llO"C,

pH pH pH pH pH pH pH pH pH pH pH pH

aerobic aerobic and anaerobic species facultatively aerobic anaerobic anaerobic anaerobic anaerobic anaerobic anaerobic anaerobic anaerobic anaerobic

Methanogenic

Methanococcus Methanothrix Methanosarcina Methanolobus Methanomicrobium Methanogenium Methanoplanus Methanospirillum Methanobacterium Methanobrevibacter Methanothermus

All species are anaerobic with pH optima pH 6 8 and most are mesophilic (30"-40"C) except:

Halomethanococcus MethanohaloDhilus

30"4"C, 30"45"C.

Halophilic

1-3, 1-5, 1-5, 2-7, 3-7, 5-7, 5-6, 5-7, 7, 5-7, 5-9, 5-7,

Extremely thermophilic (> 80°C) e.g. Methanothermus fervidus Methanococcus jannaschii Moderately thermophilic (65"-80°C) e.g. Methanococcus thermolithotrophilus Methanobacterium thermoautotrophicum pH 6 8 , uH 9,

anaerobic, 2-3 mold NaCl anaerobic. 0.2-2 molfl NaCl

The list of genera is not exhaustive but serves to illustrate the environments from which archaebacteria have been isolated. The order of genera is not based on phylogeny.

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Halophilic enzymes are therefore uniquely adapted to function in conditions of low water potential. In industrial organic syntheses, enzymes are becoming increasingly important on account of their high specificity, stereospecificity and efficiency, but one immediate problem is that the economics of such processes often demand that the enzymes operate in the presence of very high concentrations of substrate-that is, at low water activities. Enzymes from 'normal' organisms are often inactive in these situations, but clearly those from the extremely halophilic archaebacteria may be of use. That is, either they may be used in the process itself or, from a study of their structures, we may come to understand the features of these proteins that confer stability at low water activities and thus be able to engineer those features into a mesophilic enzyme of choice. The latter approach is obviously preferable as an enzyme can be tailored to the exact conditions of the organic synthesis in question. Bacteriorhodopsin

Bacteriorhodopsin of Halobacterium halobium is an integral membrane protein (purple membrane) consisting of a single polypeptide chain containing the chromophore, retinal (Kushner 1985). It effects the light-dependent translocation of protons from the inside to the outside of the cell and thereby generates a transmembrane electrochemical gradient which can subsequently be used to drive ATP synthesis. Biotechnologically, it is hoped to use artificial membranes incorporating bacteriorhodopsin to generate electricity from sunlight (Prentis 1981); in this situation, ATP synthesis would be prevented and the electrical potential arising from the proton gradient could be used as a source of electricity. Although the potential for low cost, low technology production of cheap electricity is considerable, the technical problems are formidable but not insuperable. For example, bacteriorhodopsin has to be prepared and fixed to a support which will permit the passage of protons but not other ions; furthermore, the protein molecules must be all in the same orientation so that protons are pumped in the same direction. Fortunately, purple membranes are remarkably resilient, undergoing complete renaturation even after extensive denaturation; also, the molecules can be correctly orientated by electric field application (Kungi et al. 1988). Laboratory-scale systems have been constructed and shown to be capable of generating electric currents, but much research and development is needed before commercial production and usage. The electrical response elicited by light through bacteriorhodopsin may also have applications in the development of biochips for new generations of computers (Hong 1986). The robust nature of the protein and its sensitivity to light are seen as distinct advantages in this area, plus the fact that it is a complex molecule which would permit manipulations by protein chemistry and genetic engineering. Other applications of purple membrane have been suggested (Prentis 1981). Co-immobilization of a H+/Na+ antiport with bacteriorhodopsin would pump Na' and C1- ions across a membrane and so might be used to desalinate salt water. In addition, the light-generated proton gradient could be used to produce large quantities of ATP cheaply. THERMOPHILIC ARCHAEBACTERIA

The thermophilic archaebacteria can grow in the temperature range 55"-11OoC (Table 1) and are the most thermally resistant of all living organisms. The cellular components are therefore extremely thermostable and these, together with their unique metabolic capabilities, offer considerable promise for biotechnological applications. Thermostable enzymes

As already mentioned, enzymes are becoming increasingly useful in the biotechnological industries (Battersby 1985; Kennedy 1987). They are used in degradative reactions (e.g. in biological detergents, in food processing), in synthetic processes (e.g. manufacture of pharmaceuticals) and in immobilized forms as biosensors. A significant part of the cost of such biotransformations is the enzyme catalyst and the increased stability of proteins from thermophiles is generally advantageous. Also, ther-

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D. W . Hough and M . J . Danson

mostable enzymes permit reactions to be carried out at higher temperatures than do their mesophilic counterparts, thereby increasing diffusion rates and solubility and decreasing viscosity, surface tension and the risk of microbial contamination. Furthermore, thermophilic enzymes often show an increased stability to organic solvents and denaturants, widening the scope of reaction conditions. For these reasons, enzymes from thermophilic organisms are generally favoured in biotechnological processes (Ng & Kenealy 1986;Wiegel & Ljungdahl 1986).In this sense, the advantages of enzymes from the thermophilic archaebacteria will undoubtedly be the same as those from thermophilic eubacteria. However, one further advantage offered by the archaebacteria is that many members can grow at temperatures much higher than the eubacteria. Generally, most thermophilic archaebacteria grow optimally at 80°C or above, with Pyrodictium, the most extreme thermophile in pure culture, having a growth optimum of 105°C (Stetter 1986). Therefore, the archaebacteria are a potential source of extremely thermostable enzymes; further research will determine if this potential can be realized and may give further insights into how these proteins can retain their activity at such extremes. Enzymes of unique specijications

Archaebacterial metabolic pathways are not well characterized, although investigations of their routes of central metabolism have already revealed pathways, and hence enzymes, not found in eubacteria or eukaryotes (Danson 1988).Therefore, in addition to highly stable enzymes, archaebacteria may provide enzymes of unique or unusual specificities. For example, while the nicotinamide nucleotide-dependent dehydrogenases of non-archaebacterial species are usually specific for either NAD' or NADP', a number of dehydrogenases from the thermoacidophilic archaebacteria accept both NAD' and NADP' on the same protein molecule. Such thermostable, dual cofactor-specific enzymes have obvious potential in industrial cofactor-regeneration systems and it is highly likely that many more archaebacterial enzymes will have unusual and useful properties. Biomining-the microbial leaching of mineral ores

The ores used as sources of many metals are often highly insoluble sulphides, and therefore it is necessary to degrade these compounds to release soluble metal ions, the recovery of which is then straightforward. If the concentration of metal in the ore is low, extraction by chemical means may be uneconomic, and microbial leaching is then preferred (Johnson 1985). For example, copper is currently commercially leached using bacteria. The eubacterium, Thiobacillus ferroxidans, is able to oxidize Cu,S to CuSO, directly; also sterile, acidic solutions of ferric sulphate are used to effect the same reaction and the T .ferroxidans then reoxidizes the ferrous ions produced back to ferric ions, to continue the sulphur oxidation. It has been recently recognized that the sulphur-metabolizing archaebacteria, Sulfolobus spp., are effective metal sulphide leachers (Brierly & Brierley 1986). They have the advantages of being extremely thermophilic (80"C), which speeds up the leaching process, and are able to attack the particularly refractory molybdenum-containing sulphide ores. In addition, their pH optima for growth are around pH 2 4 and these may be compatible with the acidic conditions generated in the leaching operations. These properties have aroused considerable interest in the archaebacteria for microbial leaching and several species are currently being investigated for bioleaching of refractory precious metal ores such as those containing gold and silver (Hutchins et al. 1988). In a process similar to microbial leaching, the thermoacidophilic archaebacteria are capable of the desulphurization of coal, with negligible loss of its energetic value (Kargi 1986).This is of considerable ecological importance; the sulphur content of coal varies from < 1% to > lo%, in the form of pyritic and organic sulphur, and on combustion this is released as SO, gas which is thought to be a major contributor to acid rain pollution. The archaebacterium, Sulfolobus acidocaldarius, has been successfully used for the removal of sulphur from coal (Kargi 1986), the high temperature of operation (70°C)improving the rate of chemical oxidation of pyritic sulphur by the ferric iron which is produced by microbial oxidation of pyritic iron in coal.

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Thermostable lipids and membranes A major objective in membrane technology is to create stable filtration membranes with selective porosity and low levels of surface fouling. Both natural and artificial membranes are under investigation. In the former case, the membranes of the archaebacteria may be advantageous to those from other species. In contrast to the straight-chain fatty acyl ester-linked glycerolipids (with sn-1,2-glycerol) of eubacterial and eukaryotic membranes, archaebacterial lipids are isopranyl ether-linked glycerolipids with an sn-2,3-glycerol configuration (Langworthy 1985). The thermophilic archaebacterial glycerolipids contain tetra-ethers, that is they possess two C,,-biphytanyl chains ether-linked simultaneously to two opposite glycerol molecules, allowing formation of lipid ‘monolayer’ membranes. In addition, the chains may contain one to four cyclopentyl rings which, along with the tetra-ethers, give considerable thermostability to these membranes. Moreover, the ether linkage is considerably more resistant to hydrolysis than the ester linkages found in the eubacteria and eukaryotes. With artificial membranes, selectivity of their sieving nature can be greatly enhanced through the surface deposition of crystalline bacterial cell envelope layers (S-layers) (Sleytr & Sara 1986). These S-layers are present in archaebacteria and the constituent protein and glycoprotein subunits have been characterized (Kandler & Konig 1985). Clearly, the thermostable S-layer proteins of the thermophilic archaebacteria may offer advantages in the field of ultrafiltration membranes. As for the fatty acid molecules of archaebacteria in general, there are no data to suggest whether or not their properties are of special importance as surfactants. Surfactants, of which fatty acids are one class, are amphiphilic molecules consisting of hydrophilic heads and hydrocarbon tails and therefore they have surface-active properties. Consequently, they have a wide range of industrial applications in both chemical and biochemical fields (Karsa 1987). In addition to the chemical characterization of archaebacterial fatty acids (Langworthy 1985), their physical properties now need to be documented.

METHANOGENIC A R C H A E B A C T E R I A

Of all the archaebacteria, it is the methanogens which have been exploited most successfully in terms of their biotechnological potential. They are obligate anaerobes that obtain their energy by the reduction of CO, ,or simple C1 compounds such as formate, methanol or acetate, to methane. Methanogenesis is obligatory and no secondary energy sources are known (Whitman 1985). They live in a variety of habitats such as waterlogged soils, sewage, sediments, hot springs and the gut of animals, and they encompass mesophilic, thermophilic and halophilic phenotypes (Table 1). Their biotechnological usefulness lies in the methane that they produce (methane is a good, clean fuel) and the digestion of waste materials from which it is generated (Daniels 1984; Kirsop 1984). Approximately 5 x lo” g of methane are released into the atmosphere each year, biogenic generation accounting for up to 70% of this, with the rest coming from emissions of natural gas, burning biomass and from coal mining. Clearly, biogas production could be an economic asset, although of at least equal importance is the anaerobic conversion of organic waste to volatile and harmless products. Methanogenesis is therefore an attractive alternative to aerobic methods of waste disposal, as little biomass needing itself to be disposed is produced and because the costly process of aeration is eliminated. In the anaerobic digestion of biomass to form methane, the microbiology is complex with the methanogens being responsible for the last step of the process. In all, three groups of bacteria act sequentially: first, the hydrolytic fermentative bacteria catabolize biopolymers to fermentative endproducts such as hydrogen, CO, , propionate, butyrate and ethanol; secondly, acetogenic bacteria convert the products of the first group to acetate which, along with H, and CO, , can be converted to methane by the third group, the methanogenic archaebacteria. To maximize biogas production it will be necessary to come to a fuller understanding of the thermodynamics, kinetics and metabolism of these interacting micro-organisms (Daniels 1984; Kirsop 1984). Daniels (1984) has considered the general waste types that can be used for biological methanogenesis. These include manure (especially from cattle and chickens), municipal and industrial solid wastes (currently much of this is buried), sewage and dilute industrial wastes (the disposal of which

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D . W . Hough and M . J . Danson

can be very expensive). Not only waste can be used; with the need for new sources of energy, it may be feasible and desirable to grow crops specifically for methanogenesis. A considerable research effort has been set up in the USA to look at the whole process of methane production from biomass, integrating land usage, climate, crops, transportation, economics, microbiology and engineering. This systems approach has been comprehensively reviewed by Smith & Frank (1988) and the outlook is highly optimistic. In terms of waste treatment for biogas production, the economics are often marginal and are dependent on factors such as the current prices of alternative fuels and of other means of waste disposal, capital costs, tax structures and incentives, operative costs and the production and sale of by-products. However, examples of biogas production can be quoted. In China, where 80% of the population live in rural areas, 5 million family-size methane digesters supply biogas for cooking and lighting, and many large biogas power plants are being built which are linked to industrial waste materials (Li 1987). Several landfills in the USA, Great Britain, Canada, Switzerland, Italy and West Germany currently produce methane and, in these cases, this is the cheapest way of disposing of solid wastes and therefore the gas generated is a further economic bonus.

Concluding remarks The literature on biotechnology is vast and is increasing at an ever-expanding rate. In this short manuscript, we have not attempted to review the subject of biotechnology; rather, we have tried to highlight some features of the archaebacteria which emphasize their commercial potential. The word ‘potential’ is perhaps the most significant because, with a few notable exceptions, their ‘useful’ features are truly latent-they have yet to be realized. However, the realization of that potential will only come about after a great deal more fundamental research into these unique organisms. We need to understand their metabolic pathways, how they are regulated and how they can be manipulated; we need to identify the structural features of archaebacterial proteins that confer extreme stability, in order to engineer similar stability into mesophilic proteins; we need to be able to clone the genes of ‘useful’ archaebacterial enzymes and to express them in an active form in organisms like Escherichia coli which have high growth yields ; we must understand the organization and expression of genes within the archaebacterial genomes as it may be necessary to introduce eubacterial and eukaryotic genes into them, thereby to manipulate the archaebacterial phenotypes. As investigations proceed, we are confident that the knowledge gained will not only allow us to capitalize on the potential we have discussed, but it will undoubtedly reveal far more of interest and use than we have learned of the archaebacteria since they were first recognized as a unique group of ancient organisms. Financial support for the work of the authors from SERC, The Royal Society, The British Council, NATO and ICI plc are gratefully acknowledged.

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