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Chapter: Environmental Biotechnology: Fundamentals of Biological Intervention

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Extremophiles

Making use of these extremophile organisms could provide a way of develop-ing alternative routes to many conventional chemicals or materials in such a way as to offer significant advantages over existing traditional processes.

Extremophiles

As has been previously mentioned, in general the use of biotechnology for envi-ronmental management relies on mesophilic micro-organisms which have roughly similar environmental requirements to ourselves, in terms of temperature, pres-sure, water requirement and relative oxygenation. However, often some of their abilities, which are directly instrumental in enabling their use in this context, arose in the first instance as a result of previous environmental pressures in the species (pre)history. Accordingly, ancient metabolic pathways can be very valuable tools for environmental biotechnology. Thus, the selective advantages honed in Car-boniferous coal measures and the Pleistocene tar pits have produced microbes which can treat spilled mineral oil products in the present and methanogenesis, a process developed by the Archae during the dawn of life on earth, remains rele-vant to currently commonplace biological interventions. Moreover, some species living today tolerate extreme environments, like high salinity, pressures and tem-peratures, which might be of use for biotech applications requiring tolerance to these conditions. The Archaea (the group formerly known as the archaebacte-ria and now recognised as forming a distinct evolutionary line) rank amongst their numbers extreme thermophiles and extreme halophiles in addition to the methanogens previously mentioned. Other species tolerate high levels of ionis-ing radiation, pH or high pressures as found in the deep ocean volcanic vents known as ‘black smokers’.

 Making use of these extremophile organisms could provide a way of develop-ing alternative routes to many conventional chemicals or materials in such a way as to offer significant advantages over existing traditional processes. Many current industrial procedures generate pollution in one form or another and the chal-lenge of such ‘green chemistry’ is to design production systems which avoid the potential for environmental contamination. The implementation of ‘clean man-ufacturing technologies’ demands considerable understanding, innovation and effort if biologically derived process engineering of this kind is to be made a reality. With environmental concerns placing ever growing emphasis on energy efficiency and low carbon usage, industrial applications of the life sciences in this way seem likely to be increasingly relevant. To date, however, there has been little commercial interest in the extremophiles, despite their very obvious potential for exploitation.

 The existence of microbes capable of surviving in extreme environments has been known since the 1960s, but the hunt for them has taken on added impetus in recent years as possible industrial applications for their unique biological capa-bilities have been recognised. As might be expected, much of the interest centres on the extremophile enzymes, the so-called ‘extremozymes’, which enable these species to function in their demanding natural habitats. The global market for enzymes amounts to around $3 billion (US) annually for biomedical and other industrial uses and yet the ‘standard’ enzymes typically employed cease working when exposed to heat or other extreme conditions. This often forces manufactur-ing processes that rely on them to introduce special steps to protect the proteins during either the active stage or storage. The promise of extremozymes lies in their ability to remain functional when other enzymes cannot. The potential for the mass use of enzymatic ‘clean production’ is discussed more fully in the following, but the major benefit of using extremophile enzymes in this role is that they offer a way to obviate the requirement for such additional procedures, which inevitably both increases process efficiency and reduces costs. In addition, their novel and distinct abilities in challenging environments allows them to be considered for use as the basis of entirely new enzyme-based approaches to pro-cessing. Such methods, if properly designed and implemented, have the potential to give rise to major environmental and economic benefits compared with tra-ditional energy-intensive chemical procedures. However, the widespread uptake and integration of biocatalytic systems as industrial production processes in their own right is not without obstacles which need to be overcome. In many conven-tional catalytic processes, chemical engineers are free to manipulate turbulence, pH, temperature and pressure for process intensification, often using a variety of reactor configurations and regimes to bring about the desired enhancement of pro-ductivity (Wright and Raper 1996). By contrast, in biological systems, the use of turbulence and other such conventional intensification methods is not appropriate as the microbial cells are typically too sensitive to be subjected to this treatment, as are the isolated enzymes. Such procedures often irreversibly denature proteins, destroying enzymatic activity.


Thermophiles

Of all the extremophiles, thermophiles are amongst the best studied, thriving in temperatures above 45 ◦ C, while some of their number, termed hyperther-mophiles, prefer temperatures in excess of 85 ◦C. Unsurprisingly, the majority of them have been isolated from environments which have some association with volcanic activity. The first extremophile capable of growth at temperatures greater than 70 ◦C was identified in the late 1960s as a result of a long-term study of life in the hot-springs of Yellowstone National Park, Wyoming, USA, headed by Thomas Brock of the University of Wisconsin-Madison. Now known as Thermus aquaticus, this bacterium would later make possible the widespread use of a revolutionary technology, the polymerase chain reaction (PCR). Shortly after this initial discovery, the first true hyperthermophile was found, this time an archaean which was subsequently named Sulfolobus acidocaldarius. Having been discovered in a hot acidic spring, this microbe thrives in temperatures up to 85 ◦ C. Hyperthermophiles have since been discovered from deep sea vent systems and related features such as geother-mal fluids, attached sulphide structures and hot sediments. Around 50 species are presently known. Some grow and reproduce in conditions hotter than 100 ◦C, the current record being held by Pyrolobus fumarii, which was found growing in oceanic ‘smokers’. Its optimum temperature for reproduction is around 105 ◦C but will continue to multiply up to 113 ◦C. It has been suggested that this rep-resents merely the maximum currently accepted for an isolated and culturable hyperthermophile and is probably not even close to the upper temperature limit for life which has been postulated at around 150 ◦ C, based on current understand-ing. Although no one knows for certain at this time, it is widely thought that, higher than this, the chemical integrity of essential molecules will be unlikely to escape being compromised.

 To set this in context, isolated samples of commonplace proteins, like egg albu-min, are irreversibly denatured well below 100 ◦C. The more familiar mesophilic bacteria enjoy optimum growth between 25 – 40 ◦ C; no known multicellular organ-ism can tolerate temperatures in excess of 50 ◦C and no eukaryotic microbe known can survive long-term exposure to temperatures greater than around 60 ◦ C. The potential for the industrial exploitation of the biochemical survival mech-anisms which enable thermo- and hyperthermophiles to thrive under such hot conditions is clear. In this respect, the inactivation of thermophiles at temper-atures which are still too hot for other organisms to tolerate may also have advantages in commercial processes. Though an extreme example in a world of extremes, the previously mentioned P. fumarii, stops growing below 90 ◦C; for many other species the cut-off comes at around 60 ◦ C.

 A good understanding of the way in which extremophile molecules are able to function under these conditions is essential for any future attempt at harnessing the extremozymes for industrial purposes. One area of interest in particular is how the structure of molecules in these organisms, which often very closely resemble their counterparts in mesophilic microbes, influences activity. In a number of heat-tolerant extremozymes, for example, the major difference appears to be no more than an increased prevalence of ionic bonds within the molecule.

 Though the industrial use of extremophiles in general has been limited to date, it has notably given rise to polymerase chain reaction (PCR), a major technique used in virtually every molecular biology laboratory worldwide. The application of PCR has, in addition, opened the flood gates for the application of genetic analyses in many other branches of life science, including forensics and medical diagnosis. Though this is a tool of genetic engineering rather than anything which could be argued as an ‘environmental’ application, it does illustrate the enormous potential of extremozymes. The process uses a DNA polymerase, called Taq poly-merase, derived from T. aquaticus, as mentioned earlier, and was invented by Kary Mullins in the mid-1980s. The original approach relied on mesophilic poly-merases and since the reaction mixture is alternately cycled between low and high temperatures, enzymatic denaturation took place, requiring their replenishment at the end of each hot phase. Samples of T. aquaticus had been deposited shortly after the organism’s discovery, some 20 years earlier, and the isolation of its highly heat-tolerant polymerase enabled totally automated PCR technology to be developed. In recent years, some PCR users have begun to substitute Pfu poly-merase, isolated from another hyperthermophile, Pyrococcus furiosus, which has an optimum temperature of 100 ◦C.


Other extremophiles

As was stated earlier, the thermophiles are amongst the best investigated of the extremophiles, but there are many other species which survive under equally challenging environmental conditions and which may also have some potential as the starting point for future methods of reduced pollution manufacturing. For example, cold environments are more common on earth than hot ones. The aver-age oceanic temperature is around 1 – 3 ◦ C and vast areas of the global land mass are permanently or near-permanently frozen. In these seemingly inhospitable con-ditions, extremophiles, known as psychrophiles, flourish. A variety of organisms including a number of bacteria and photosynthetic eukaryotes can tolerate these circumstances, often with an optimum functional temperature as low as 4 ◦ C and stopping reproduction above 12 or 15 ◦C. Intensely saline environments, such as exist in natural salt lakes or within the artificial confines of constructed salt evaporation ponds are home to a group of extremophiles, termed the halophiles. Under normal circumstances, water flows from areas of low solute concentra-tion to areas where it is higher. Accordingly, in salty conditions, unprotected cells rapidly lose water from their cytoplasm and dehydrate. Halophilic microbes appear to deal with this problem by ensuring that their cytoplasm contains a higher solute concentration than is present in their surroundings. They seem to achieve this by two distinct mechanisms, either manufacturing large quantities of solutes for themselves or concentrating a solute extracted from external sources.

 A number of species, for example, accumulate potassium chloride (KCl) in their cytoplasm, with the concomitant result that extremozymes isolated from these organisms will only function properly in the presence of high KCl levels. By the same token, many surface structural proteins in halophiles require severely elevated concentrations of sodium salts.

 Acidophiles thrive in the conditions of low pH, typically below 5, which occur naturally as a result of sulphurous gas production in hydrothermal vents and may also exist in residual spoils from coal-mining activity. Though they can tolerate an externally low pH, an acidic intra-cellular environment is intolerable to acidophilic organisms, which rely on protective molecules in, or on, their cell walls, membranes or outer cell coatings to exclude acids. Extremozymes capable of functioning below pH1 have been isolated from these structures in some acidophile species.

 At the other end of the spectrum, alkaliphiles are naturally occurring species which flourish in soda lakes and heavily alkaline soils, typically enduring pH9 or more. Like the previous acidophiles, alkaliphiles require more typically neutral internal conditions, again relying on protective chemicals on or near their surfaces or in their secretions to ensure the external environment is held at bay.


Diverse degradative abilities

Bacteria possessing pathways involved in the degradation of a number of organic molecules of industrial importance, have been acknowledged for some time. One oft-quoted example is that for toluene degradation in Pseudomonas putida, which exhibits a fascinating interplay between the genes carried on the chromosome and the plasmids (Burlage, Hooper and Sayler 1989). Bacteria are constantly being discovered which exhibit pathways involved in the degradation and synthesis of chemicals of particular interest to environmental biotechnologists. For example, a new class of biopolymer produced by the bacterium, Ralstonia eutropha, contain-ing sulphur in its backbone, has recently been identified. (Lutke¨-Eversloh et al. 2001) It is possible that these and other novel biopolymers awaiting discovery, will have innovative and exciting applications in clean technology.

 In very recent years, bacteria representing very diverse degradative abilities have been discovered in a variety of niches adding almost daily, to the pool of organisms of potential use to environmental biotechnology. By illustration these include phenol-degrading Oceanomas baumannii isolated from estuarine mud from the mouth of the River Wear, UK (Brown, Sutcliffe and Cummings 2001), chloromethane utilising Hyphomicrobium and Methylobacterium from polluted soil near a petrochemical factory in Russia (McDonald et al. 2001) and a strain of Clostridium able to degrade cellulose, isolated from soil under wood chips or the forest floor in northeast USA. In addition to their cellulytic activity, these Clostridia were also found to be mesophilic, nitrogen-fixing, spore-forming andobligate anaerobes (Monserrate, Leschine and Canale-Parola 2001). Again, there is interest in this organism with regard to clean technology in the hope that it may be used to convert cellulose into industrially useful substances. A note of caution is that cellulose is a major product of photosynthesis and, being the most abundant biopolymer on this planet, has a vital role to play in the carbon cycle. Large-scale disturbance of this balance may have consequences to the environment even less welcome than the technologies they seek to replace. However, judicious use of this biotechnology could reap rewards at many levels.

Bacteria have also adapted to degrade man-made organics called xenobiotics

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