Biotechnology

Engineering Considerations for the Application of Extremophiles in Biotechnology

to those characteristic of the major extremophiles (psychrophiles, thermophiles, barophiles, alkalophiles, acidophiles, and halophiles), Heavy-metal contamination, high irradiation, and low nutrient concentration are conditions not usually included in the well-studied moderate world. Organisms from these environs will not be included in this review; for more information about these species, the reader is referred to reviews by Ehrlich,' Nasim and James,' Gadd,3 Whitelam and C ~ d d and ,~ Morgan and D o w . ~

Jan M. Ludlow and Douglas S. Clark

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ABSTRACT 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.

II. DEFINITION, HABITAT, AND CHARACTERISTICS OF EXTREMOPHILES A. Psychrophiles A number of definitions of the term psychrophile have been proposed. In early nomenclature, psychrophiles are organisms which grow at O°C,6 whereas psychrotrophs are capable of growth at 5°C.' While this definition of psychrotrophy is still commonly accepted, the most widely used definition of psychrophily is based on cardinal growth temperatures. A psychrophile exhibits an optimum growth temperature of 15°C or below, a maximum growth temperature of 20°C or below, and a minimum growth temperature of 0°C or below.8 However, since many organisms can grow at temperatures 10 to 20°C higher than that of their natural en~ironment,~ organisms isolated from cold habitats do not necessarily fit the definition of a psychrophile. Bacteria are found in a variety of natural and man-made cold environments. The largest such environment is the sea. More than 90% of marine water is below 5°C. Psychrophiles have been isolated from the Pacific Ocean, Puget Sound, the North Sea, and the Antarctic Ocean.'O.'' Organisms are found in solution, attached to debris, within higher organisms, and in sediment. In addition to cold seawater, many organisms are found in arctic and antarctic soil, deserts, and glaciers. The major man-made cold environment is far less exotic - the refrigerators and freezers of industrial and domestic food storage. The ecology of psychrophiles has been previously reviewed. I' In general, the low maximum growth temperature of psycrophiles is attributable to extreme thermosensitivity of one or more cellular constituents. Specifically, protein synthesis, enzyme activity, and cell membrane and wall structure are all negatively affected at temperatures generally considered mod-

1. INTRODUCTION Microorganisms have served mankind for centuries -from the traditional fermentation of foods and beverages to the modem production of pharmaceuticals and specialty chemicals. Most of these processes employ organisms (or their components) which grow in moderate conditions: moderate temperature, neutral pH, low salinity, and atmospheric pressure. However, some biological habitats differ significantly from the above-described "moderate world. " These extreme environments can be characterizedby high and low temperature, strong alkalinity and acidity, high ionic strength, and elevated pressure. Extremophiles are organisms that thrive under immoderate conditions, exhibiting optimal growth in one or more of the extremes listed, and sometimes finding moderate conditions fatal. In contrast, extremotolerant organisms are capable of growing in or surviving exposure to extreme conditions, but exhibit optimal growth rates under moderate conditions. Similar definitions can be applied to proteins and their biological functions. The focus of this review is the application of extremophilic and extremotolerant organisms and proteins in novel biotechnological processes. Specifically, equipment and materials employed in the study of extremophiles are described, as well as existing and potential industrial applications. This review is not intended as a comprehensive overview of all aspects of extremophily; therefore, sources of detailed taxonomic, physiological, and ecological information are noted in the text. Several other extreme conditions are recognized in addition

J. M. Ludlow is a graduate student in the Department of Chemical Engineering, 201 Gilrnan Hall, University of California, Berkeley 94720. D. S. Clark earned a Ph.D. at the California Institute of Technology, Pasadena. Presently, Dr. Clark is Associate Professor, Department of Chemical Engineering, University of California, Berkelev.

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erate.I3 Also of interest, particularly in the context of novel biochemical technology, are the properties of psychrophiles that permit growth below the limits of other organisms. Studies of mesophiles indicate that reduced feedback inhibition may be significant. For example, phosphoribosyl ATP pyrophosphorylases from cold-sensitive E. coli mutants retained near normal levels of activity at 37"C, but were 100 to lo00 times more sensitive to feedback inhibition by histidine than the enzyme in the native strain.I4 Further, a number of mesophilic enzymes, e.g., fructose 1,6 diphosphatase from rat liver,I5 and aspartic transcarbamylase from S. cerevisiae,I6 are more sensitive to feedback inhibition at lower temperatures. In addition to increased regulatory control, some cold-sensitive mutants are unable to assemble ribosomal subunits at low temperatures. l 7 However, the low-temperature limit for native strains is more likely a result of cessation of polysome formation. Finally, although many researchers have studied the effect of temperature on lipid composition and membrane permeability, limitations in membrane fluidity and transport do not seem to be responsible for low temperature limits of growth. These and other aspects of psychrophile physiology are discussed in detail in the review by Innis and 1ng~aham.l~

temperature of >50°C; a caldoactive organism (sometimes termed "extreme thermophile") has a maximum growth temperature >90"C and an optimum growth temperature >65"C.I9 Hyperthermophiles are capable of growth at or above I00"C. It should also be noted that many species are called thermophilic if they grow at a temperature higher than the optimum or maximum typical of most strains of the same species. Thus, not all species designated in the literature as thermophilic will exhibit true thermophilic growth characteristics. Thermophiles have been found in diverse high temperature environments: geothermally heated sources such as volcanic regions, superheated and hot springs, and deep-sea hydrothermal vents; solar-heated sources such as soil, rock surfaces, and ground litter; metabolically or self-heated sources such as compost, seaweed, hay, straw, sawdust, or coal refuse piles; and artificially heated sources such as water heaters and industrial steam and condensate lines. In addition to their thermal tolerance, some thermophilesare also acidophilicor akalophilic. Of all the extrernophiles, themophiles have been, and continue to be, the most widely studied. Many studies have focused on proteins from thermophiles with the aim of determining the limits and/or elucidating the mechanisms of their thermostability.20*21 In general, enzymes from thermophiles are thermostable; however, thermostable enzymes can also be derived from mesophiles,22and highly purified enzymes from thermophiles are sometimes heat-labile.I ' Thus, stability can either be an inherent property of the protein, or be conferred by the intracellular environment. Studies of homologous mesophilic and thermophilic enzymes indicate that relatively small changes in amino acid content can significantly improve thermostabil-

8. Thermophiles As with psychrophiles, a number of definitions have been proposed for thermophily and thermotolerance. Wiegel and Lungdahll8 have proposed a classification system that spans a range of more than 75°C (see Table 1). According to another useful definition, a thermophile is an organism with a maximum growth temperature of >60T and an optimum growth

Table 1 Definitions of Thermophily Term Thermotolerant Thermophiles Temperature-tolerant extreme thermophiles Extreme thermophiles (caldoactive) Barothermotolerants Barothermophiles

>25

545 >45

>45 >50

235

265

210

350

3-65

270

Methanococcus maripaludis Bacillus stearothermophilus Thermus aquaticus

Methunorhermus fervidus Thermothris thiopara Methanococcus jannaschii > 100 Bacillus caldolyticus Pyrococcus furiosus > 100 Pyrodictium occulrum Pyrodictium brockii

Nore: Wiegel and Ljungdahl coined the words "barothermotolerant" and "bar-

othermophilic" to represent bacteria that can grow or grow best, respectively, above 100°C if sufficient pressure is applied to keep liquid media from boiling. Organisms capable of growth at or above 100°C are generally referred to as hyperthermophiles. Additional examples can be found in the original reference.I s From Wiegel, J . and Ljungdahl, L. G . , CRC Crir.Rev. Biotech., 3 , 39, 1986.

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ity .23 Increased hydrogen, hydrophobic, and ionic bonding appear to enhance therm~stability.~~

itself, pressure can influence binding of substrate to enzyme, release of product, medium pH, and solute solubility, all of which may cause apparent changes in reaction and growth rates.

C. Barophiles Interest in the effect of pressure on microbial growth originated with marine biologists who wished to study organisms under the conditions found in the sea, from 1 to approximately 1150 bar of hydrostatic pre~sure.'~ Barotolerant or baroduric organisms are capable of growth over the range 1 to 600 atm, whereas barophiles grow faster at increased pressure. Obligate barophiles require elevated pressure for growth, and barosensitive organisms find any pressure over 1 atm fatal.26Barophilic organisms are typically found in the deep-sea and deep-oil or sulfur wells. Since barophiles exhibit enhanced growth with pressure, they must contain cellular components which are either inherently or conditionally pressure-stable. Moreover, pressure may enhance metabolic, genetic, and/or transport processes. Studies indicate that pressure stabilizes native DNA up to 10,OOO bar and condenses membrane lipids up to 3500 bar.27 Generalizations cannot be made, however, about the effect of pressure on protein denaturation (i.e., unfolding), which is governed by the sign and magnitude of the volume change of denaturation, AV. From Le Chatelier's principle, an increase in pressure will favor denaturation if it results in a negative AV and suppress denaturation if it results in a positive AV. Model systems provide evidence that pressure favors some denaturation processes and inhibits others. When a protein unfolds, buried intramolecular hydrogen bonds and charged groups may be transferred to aqueous surroundings, resulting in stronger hydrogen bonds and greater electrostriction, respectively. Both of these processes are promoted by pressure. On the other hand, studies of compounds believed to mimic hydrophobic groups of proteins (low molecular weight alcohols, ethers, ketones, and amides) suggest that volume changes for the exposure to water of buried nonpolar groups are positive.28Thus, increased pressure may stabilize the folded conformation if hydrophobic interactions dominate the free energy of the native state. Experimentally, pressure has been both a stabilizer and a denaturant of various proteins up to a limit of 4000 bar, above which proteins Several additional reviews of the effect of pressure on biological components have been ~ritten.~""~ Pressure can also affect enzymatic reaction rates similarly. According to the transition-state theory of kinetics, reactions proceed from an activated complex. Formation of the activated complex may cause the exposure of hydrophobic groups, intramolecular hydrogen bonds, and charges to water, resulting in a volume change called the activation volume, AV". If AV" is negative, pressure will enhance the formation of the activated complex, and the reaction will proceed more rapidly. Conversely, pressure retards reactions exhibiting a positive AV". In addition to the effect that pressure has on the reaction rate

D. Alkalophiles Organisms growing in alkaline environments were first reported in the 1 9 2 0 ~but , ~ have ~ only been studied in depth for the last 30 to 40 years.33Three classifications of alkalophiles have been proposed. Alkalophiles have a pH optimum for growth between 10 to 12, and grow very slowly at neutral pH. Obligate alkalophiles show similar pH optima, but do not grow at all in neutral solutions. Alkalotolerantbacteria have a slightly elevated pH optimum of 8.5 to 9 and grow well at neutral pH. Highly alkaline natural environments which contain organisms include desert soils and soda lakes. Man-made alkaline environments are most typically industrial waste streams. Alkalophiles have also been isolated from nonalkaline habitats such as neutral and acidic soils and thus appear to be fairly widespread. One of the most remarkable characteristics of many allcalophilic organisms is their ability to modify their environment. Many alkalophiles are able to alkalinize neutral media or acidify highly alkaline media to optimize the external pH for growth. On the other hand, the internal pH of most alkalophiles is lower (7 to 9) than the external medium. Thus, alkalophilicity can be achieved through membrane properties and transport systems and does not necessarily rely on alkali-resistant intracellular enzymes.33 E. Acidophiles Naturally acidic biological environments, often the result of aerobic oxidation of sulfide, are more common than alkaline environments. Organisms which can grow at pH < 4,but have optima in the neutral range, are acid tolerant. Acidophiles generally have a pH optimum of 2 to 4;such organisms unable to grow at neutral pH are obligate acidophiles." Very few organisms survive below pH 1.34 Two types of naturally acidic environments can be distinguished: the moderately acidic (pH 3 to 4) and the strongly acidic (pH 1 to 3). Moderately acidic habitats include some lakes, soils, and bogs. Strongly acidic habitats are usually characterized by both high levels of sulfide and high temperature. They include volcanic soils and lakes, and some springs, pools, and solfataras (geothermally produced acidic soil regions). In addition, mining and other industrial waste is often acidic or acidified by oxidation and may also be a source of organisms.I ' Acidophiles are similar to alkalophiles in that they can metabolically adjust the pH of neutral media to one more suitable for growth. It is less common, however, for acidophiles to neutralize media more acidic than the optimum. Internal pH is again observed to be nearly neutral (5.5 to 7), indicating

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that intracellular components are not necessarily very acid tolerant.34 F. Halophiles Although the concentration of NaCl in seawater is 0.5 M, lo00 times higher than in freshwater, marine organisms are usually not considered halophilic. In general, halophiles are organisms which grow optimally in 0.5 M NaCl (or other salts in addition to a minimum amount of NaCI). Extreme halophiles grow best in 2.5 M to 5.2 M (saturated) salt media. Halotolerant organisms have growth optima in less than 0.5 M saline media hut can grow in higher salinity.'' Another, more detailed, classification system is presented by Kushner (Table 2).3s The most well-known extremely saline environments are salterns (salt evaporating ponds), which often exhibit colorful red cultures of halobacteria. Organisms are also found in salt lakes such as the Great Salt Lake in Utah and the Dead Sea. Man-made environments for halophile contamination are saltpreserved foods such as soy sauce, miso, and cured meats. Most saline environments are high in Na' and low in K + ; however, the interior of halophilic cells often contains significantly more K' and less Na+ than their exterior environment.35This situation is a result of limited sodium permeability through the membrane and transport processes that expel Na' . However, internal Na' levels are still higher than those of fresh water or marine organisms. In addition, the total ionic concentration is lower inside the cell than The cytoplasm of halophiles is often high in organic solutes such as sugars, polyols, or nitrogenous compounds (betaines)

to maintain cellular integrity (i.e., to avoid dessication by outward diffusion of water or collapse by osmotic pressure). Furthermore, studies suggest that changes in external salinity cause similar changes in internal solute concentration. For example, an increase in interior solute concentration may result from an increase in external salinity.36 Additional information about halophily may he found in recent compilation^.^^

111. SPECIALIZED EQUIPMENT AND PROCEDURES A. Materials Experiments and applications involving microorganisms and enzymes may require equipment constructed of carefully selected materials. The growth of some bacteria is inhibited by contact with stainless steel or some plastics, as is the activity of some enzymes. Although corrosion can be caused by moderate microorganisms or their metabolic products as well as by extremophiles, the problem is often exacerbated by extreme conditions such as high temperature, high pressure, or extremes of pH or salinity. Concrete conduits and tanks are often used in sewage treatment. Corrosion of concrete in such cases i s frequently a result of biogenic sulfuric acid production by Thiobucilli,3x acidophiles capable of producing an acidic environment (about pH 2) on concrete surfaces.3gRecently, the Biotest system was devised for the rapid evaluation of concrete resistance to hioThe Biotest system consisted of a temperature-

Table 2 Classification of Salt Response of Different Organisms Category Non-halophile Slight-halophile Moderatehalophile

Borderline extreme halophile Extreme halophile Halotolerant

Reaction

Examples

Grows best in media containing less than 0 . 2 M salt Grows best in media containing 0 . 2 4 . 5 M salt Grows best in media containing 0.5-2.5 M salt. Organisms able to grow in less than 0. I M salt are considered facultative halophiles Grows best in media containing 1 . 5 4 . 0 M salt Grows best in media containing 2.5-5.2 M salt (saturated) Nonhalophile which can tolerate salt. If the growth range extends above 2.5 M salt, it may be considered extremely halotolerant

Most normal eubacteria and most freshwater microorganisms Many marine organisms Bacteria and some algae

Ectothorhodospiru halophila Aciinopolyspora halophila The "red halophiles", halohacteria and halococci Staphlococcus aureus and other staphylococci; solute-tolerant yeast and fungi

Nnre: "Salt" is usually NaCl, but it can be other salts in addition to a minimum amount of NaCI; see the original reference'' for greater detail. From Kushner, D. J., Microbial Life in Extreme Environments. 1978, 317 With permission.

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Biotechnology controlled stainless steel chamber containing a pH-controlled water phase and a humidity-controlled gas phase. A nozzle permitted the aerosol application of suspended mixed cultures or liquid sulfur-containing substrates to concrete blocks housed in the chamber. Gas sampling and subsequent on-line analysis by gas chromatography permitted controlled addition of gaseous substrates and monitoring of gaseous products. The concrete blocks to be tested were scored so that sections of the surface could be removed from the chamber for cell growth and concrete degradation determinations. Initial tests using hydrogen sulfide as a substrate indicated that high cell counts of T . thiooxidans correlated with high corrosion of concrete (as measured by weight loss of sample), whereas levels of T. intermedius, T . novellus, and T . neapolitanus fluctuated during the course of degradation. It was possible to reproducibly evaluate the accelerated corrosion of three grades of concrete in nine months, rather than the 5 years estimated for field testing. In subsequent experiments with the Biotest system, three possible Thiobacillus substrates found in sewage systems, hydrogen sulfide, thiosulfate, and methylmercaptan, were tested for their effect on the colonization and corrosion of concrete.4o Hydrogen sulfide was the most destructive substrate, resulting in a loss of 3.3% of material and reduction of surface pH from approximately 10 to 1.5. Thiosulfate caused a 1.8% loss of material and pH reduction to 2.5, and methylmercaptan was innocuous, resulting in no loss of material and pH reduction only to 8.5. The dominant organism in tests using hydrogen sulfide as the reduced sulfur source was T . rhioxiduns, with both T . intermedius and T . neapolitanus exhibiting an order of magnitude lower colonization. Conversely, with thiosulfate as substrate, T. intermedius and T . neapolitanus were the dominant organisms, and T . thiooxidans was present at tenfold lower concentration. No Thiobacilli were detected with methylmercaptan as substrate. A more detailed understanding of the mechanism of concrete biocorrosion may aid in the appropriate selection of materials for various applications, including buildings in sulfur-containing polluted atmospheres, and retaining tanks or bases for microbial ore leaching and fuel desulfurization. Microorganisms corrode tanks and piping made of copper, nickel, and aluminum as well as steel. The electrochemistry of corrosion by organisms isolated from corroding copper, nickel, and mild steel surfaces has been s t ~ d i e d .Experiments ~' were conducted in a measurement cell consisting of two identical chambers containing nickel 201 separated by a cellulose acetate/cellulose nitrate membrane. Living Thermus aquaticus (an organism that requires temperatures from 60 to 80°C for growth) inoculated in one compartment generated an anodic current of 3.8pA/cmZ at 60°C whereas heat-killed or coldinactivated organisms produced no current. Thus, it appeared that metabolically active organisms were required for corrosion of nickel by T . aquaticus.

1991

To further characterize the observed biocorrosion, experiments were conducted with organism-free electrochemicalcells to investigate three mechanisms of corrosion related to colonization. Bare and colonized areas of the electrode exhibit differences in aeration that can cause current flow, resulting in the formation of a differential aeration cell. Microbial production of acids and entrapment of metal ions by biopolymers can also affect the electrochemical cell. In abiotic experiments mimicking these three phenomena, differential aeration, organic acid addition, and Fe (111) addition produced currents of 0, 0.1 and 0.3pA/cmz, respectively, with nickel electrodes, suggesting that none of these mechanisms dominated the corrosion of nickel by T . aquaticus. A Thermus species isolated from a corroded brazed nickel tee which failed in service at 60°C was also used to test the corrosion of metals by a thermophilic organism.42Various mild steel alloys incubated in cultures growing in complex media at 70°C for 5 d showed twice the loss of material as compared to cell-free controls. Electrochemicaldeterminationsof the corrosion potential and protection potential of 3 16 stainless steel after incubation with and without Thermus for 21 d at 70°C indicated that the growth medium probably passivated the stainless steel surface, but that microbial growth may have weakened the passivation layer. Perfusion systems were used to study attachment of the organism to various metals. After 2 d of incubation, cells showed greatest attachment to stainless steel, followed in descending order by titanium, aluminum, aluminum bronze, and copper/nickel. After 10 d, colonization had decreased on stainless steel and aluminum, remained the same on titanium, and increased on aluminum bronze and copperhickel. The changes in colonization may have been a result of metal toxicity or adaptation to tolerate heavy metals. Another study of metal colonization focused on the effect of various surface finishes. Samples of stainless steel (see Table 3) were incubated in shaken flask cultures of a common yeast, Candida tropicalus, at 30°C and pH 6 for 30 h and of an

Table 3 Steel Treatment for Biofouling Tests Sample 1

2 3 4 5

6

Treatment Washing with P3, ultrasonic cleaning Dipping only Dipping, additional electrolyte polishing Grinded (corn 360), additional polishing Electrolyte polishing at maximum (high-polished) Electrolyte polishing, additional ultrasonic cleaning

Norer Sample 1 was the control. Treatment for samples 2-45 was in addition to the treatment of the control. General parameters were: ultrasonic head velocity, 20 d s ; dip time, 1 h; electrolyte polishing, 15 min.

From Sonnleitner, B., Fiechter, A,, and Woschitz, D., J . Biotechnol., 6, 41, 1987. With permission.

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Critical Reviews In extremophile, Bacillus acidocaldurius, at 60°C and pH 3.5 for 90 h.43 Electron microscopy revealed that both the yeast and the extremophile adhered to all surfaces tested. The yeast adhered primarily in cell clusters and preferred the most highly polished surfaces, whereas the extremophile adhered as single cells with little discrimination of surface type. Neither culture cxhibited growth inhibition by the stainless steel in any surface finish. The results suggested that costly surface treatments may be of little value in reducing biofouling of stainless steel surfaces. Although many researchers have used electrochemical and cell count methods for evaluating microbial corrosion, Fourier transform infrared spectroscopy (FT/IR) and “signature” phospholipid fatty acid analysis have also been applied to both moderate organisms and extremophiles.44FT/IR permitted the nondestructive examination of surface biofilms (microbes and their extracellular products) on a scale approaching the size of the microbes themselves. “Signature” phospholipid fatty acid analysis permitted the identification of species within a mixed population of bacteria without the time consuming most probable number (MPN) culture test. For example, the acidophilic Thiobacilli were found to contain unusual ester-linked fatty acids in their phospholipids, making Thiobacilli easy to distinguish from other organisms. Further, Thiobacillus thioxiduns contained 88% cyclopropane 19:O phospholipid esterlinked fatty acid among its phospholipid ester-linked fatty acids, which was sufficient to differentiate it from other Thiobacilli.

B. Equipment Most extremophiles (acidophiles, alkalophiles, halophiles and psychrophiles) can be studied adequately in common laboratory and pilot plant equipment constructed of appropriate materials. In contrast, a variety of interesting apparatuses have been built for the study of organisms and enzymes at elevated temperatures and/or pressures. Descriptions of such equipment, along with other engineering considerations for the culture of extreme thermophiles, have been presented in recent rev i e w ~ . ~ ~This . ~ ’ review presents a general overview of the equipment used in high temperature-pressure experiments and specific descriptions of some recent developments. Moderate thermophiles have been studied in fairly typical laboratory equipment. Water baths have been used easily up to about 75”C, and sand or oil baths and heating blocks have been used at higher temperatures. For example, the hypertherniophile Pyrococcus furiosus has recently been cultivated at 98°C in a continuous culture apparatus consisting of a roundbottom flask heated by a controllable mantle.48Hot air incubators have also been used over a wide range of temperatures; the ovens commonly used in our own laboratory are rated up to 260°C. The discovery of organisms that grow at or above the boiling point of water necessitated methods of maintaining culture media in liquid form, and elevated pressure has been used to achieve this goal. Some of the simplest systems for studies at elevated tem-

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perature and hydrostatic pressure are modified autoclaves, pressurized syringes, and compressible metal or teflon bags. In general, these systems have been used for endpoint measurements, or samples can be removed for time-course analysis. One syringe system was made of a cylindrical sample chamber (the syringe) contained inside a pressure A piston was fitted inside the cylinder to separate the sample from the pressurizing fluid, and to transmit pressure from the pressurizing fluid to the sample during pressurization and sampling. Sampling was achieved by pumping preheated hydraulic fluid into the pressure vessel at the same rate sample liquid was removed from the syringe, allowing constant temperature and pressure to be maintained during sampling. Compressible bags have been sampled in a similar fashion, but the total amount of sample removed was limited by the minimum volume at which the bag ruptured, unless fresh medium replaced the samples and rate data were adjusted a~cordingly.~’ A liquid phase high pressure system suitable for continuous culture has also been described.s” The apparatus consisted of a sealed reservoir from which medium was hydraulically pumped at desired pressure into a heated 1 1 stainless steel reaction vessel. Effluent was removed through an overflow port at desired rates controlled by a micrometering valve. The inlet pump and outlet valve maintained the elevated pressure in the vessel. No gas phase was present. A liquid recirculation pump kept the culture well mixed. This apparatus was used to culture a facultatively anaerobic, thermophilic Bacillus species at 65°C and 200 bar for 72 h at a residence time of 18 h. The cells grown at high pressure were coccoidal rather than rod-shaped, and they lysed rapidly on exposure to atmospheric pressure. One especially ingenious hydrostatic pressure reactor was the high pressure-temperature gradient instrument described by Yayanos et al.51An aluminum block was fitted with nine cylindrical holes, eight to hold pressure vessels and the ninth to hold a thermistor for temperature measurement. The thermal gradient across the block (potentially ranging from -20 to 100°C) was established by circulating two thermostated fluids of different temperatures on either end of the block. The system was insulated to prevent heat losses; thus the gradient was linear and identical in each of the nine slots. Capped glass or polyethylene tubes containing inoculated liquid or solid media were placed in the pressure vessels and each vessel pressurized to a different pressure between 1 and 1100bar. This apparatus was used successfully to demonstrate the temperature-pressure boundaries of growth for the deep sea isolate CNPT-3, and may see future application in enzyme activity studies, microbial characterization, and culture isolation. In addition to high pressure systems employing only liquid or solid media, hyperbaric systems that use gases as pressurizing agents in direct contact with the liquid media have also been developed. One significant advantage of hyperbaric reactors is the availability of greater quantities of gaseous substrates. Caution must be exercised, however, in selecting the

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Biotechnology gas used to pressurize the system. Some inert gases have inhibited and oxygen sensitivity has been heightened at increased pressure.s3 The traditionally hydrostatic system of compressible vessels has been adapted for use with gaseous substrate^.^^ Elastic hydraweld nickel tubes were inoculated with liquid culture and the head space was repeatedly evacuated and filled to provide accurate concentrations of gaseous substrate. Nickel was chosen over silicon or teflon because of its low hydrogen permeability. Furthermore, neither Ni2 nor Cu' -Cuz leached into the sample solution at inhibitory levels. The tubes were loaded into one of four autoclaves, which were pressurized independently using hydraulic fluid. Heating up to 400°C was provided by ceramic insulated heaters encircling the autoclaves. Increases in temperature (90to 250°C)and pressure (up to 4000 bar) were completed within 10 and 2 min, respectively. Twentyfour hour endpoint growth studies of Methanococcus therrnolirhorrophicus were conducted in this apparatus over the pressure range 1 to 1500 bar at 56,65,70,and 75°C.At the optimum growth temperature of 65"C,pressures up to 500 bar enhanced the growth yield, whereas pressures above 750 bar resulted in cell lysis. Sturm et al. described a hyperbaric reactor built from a 500 ml teflon-lined autoclave.55The reactor was rated to 250°C and 200 bar, and was equipped with a magnetically driven stirrer and liquid and gas sampling ports. Temperature was measured by a thermocouple that extended into the liquid phase and was regulated within 2°C by the heating jacket. Pressurized gas was supplied from a compressed gas tank. Pressure was regulated to within 1 bar. In this apparatus, growth rates and the maximum growth temperature of Solfolobus acidocaldarius were seen to be approximately constant over the range 67 to 85°C and 1 to 120 bar. In contrast, no growth of S. acidocaldarius was observed at 70°C and 1 to 293 bar in hydrostatic syringe reactors, perhaps due to insufficient concentration of gaseous substrates. In our laboratory, two reactors have been built to study microbial growth and enzyme activity. The first reactor, suitable for precise studies of microbial growth and productivity at temperatures up to 260°C and pressures up to lo00 bar, consists of a stainless steel vessel housed in a thermostated oven (Figure 1).s6A synthetic sapphire vessel (rated to 350 bar) was originally employed, but it suffered structural failure after several years of use. A gas compressor permits the introduction of gases above cylinder pressure, and a liquid pump facilitates medium addition. A magnetically driven pump recirculates the gaseous substrate through the liquid to improve interphase mass transfer. Liquid samples are taken for off-line analysis, while gas samples are analyzed by an on-line gas chromatograph. This reactor was used to demonstrate that increasing the total pressure from 7.8 to 100 bar at a constant partial pressure (7.8bar) of gaseous substrate accelerated the production of methane and cellular protein by Methanococcus

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+

+

M

OT

ATM

1

utility

C 4 He

Y,

+

GB

Iiauid

Sample

I I

PP

FIGURE 1. Schematic diagram of the hyperbaric reactor used in our laboratory. Components are: (A) stainless steeI pressure vessel; (B) magnetically driven pump; (C) check valve; (D) oven; (DP) digital pressure gauge; (F)filter; (GB)anaerobic glove box; (GC) gas chromatograph; (M) motor; (OT) oxygen trap; (P) pressure gauge; (PC) pneumatic compressor; (PP) pneumatic pump; (T) thermocouple; and (TR) pressure transducer. ATM and VAC represent lines to amosphere and vacuum, respectively. (Adapted from Miller, J . F . , Almond, E. L., Shah, N. N . , Ludlow, J . M., Zollweg, J . A . , Srreett, W. B . , Zinder, S. H . , and Clark, D. S . , Biorechnol. Bioeng., 31, 407, 1988.)

jannaschii at 90°C and raised the maximum growth temperature from 90 to 92°C as measured by methane production. In further studies of the same organism, it was found that methanogenesis and growth at both 86 and 90°C were accelerated by pressure up to 750 atm, but growth was not observed above 90°C at either 7.8or 250 atm. Moreover, growth and methane production were decoupled above 90"C,and the high temperature limit for methanogenesis was increased by pressure from less than 94°C at 7.8 atm to 98°C at 250 atm.57Furthermore, the composition of the pressurizing gas had a strong effect on methanogenesis. When argon or substrate gas (4:lCO, and H,) was substituted for helium, no methanogenesis occurred at 86°Cand 250 atm, whereas limited methanogenesis occurred when hydrogen alone was substituted for helium. A similar reactor has been constructed for the spectrophotometric assay of enzyme activity in cell-free extracts of Methanococcusjannaschii. The salient modifications to the original design were the addition of a sample valve for the rapid highpressure gas injection of substrate into equilibrated enzyme solution, and a fiber optic probe for colorimetric measurements. The fiber optic probe consists of two optical fibers epoxied in a stainless steel tube fitted with a polished stainless steel mirror at a distance of a few millimeters from the fiber ends (Figure 2). The transmitting fiber is coupled to an LED source of 10 nm bandwidth, and the receiving fiber is coupled to a photomultiplier tube and photometer. The substrate injection valve and photometer are interfaced to a computer which actuates the injection valve and collects the data. In addition, the fiber optic probe can be removed and replaced by a capillary line to a gas chromatograph for alternate rate measurements.s8

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+I

7

1"

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9 20 bar

FIGURE 2. Vessel and probe used for high temperature-pressure fiber optic enzyme assays. Components are: (A) fiber optic probe; (B,C) taper seal fitting; (D) diilled taper seal to NPT coupling; (E,F.G) custom built high pressure fitting with NPT connector; (H)316 stainless steel vessel; (I) high pressure fitting opening; and (J) high pressure fitting. (Reproduced from Miller, J . F., Nelson, C. M., Ludlow, J. M., Shah, N. N.,and Clark, D. S., Biorechnol. Bioeng., 34, 1015, 1989.)

Initial studies in this reactor indicated that the methyl viologenreducing activity in cell free extracts of Methanococcus junnaschii was more than tripled at 86°C by an increase in pressure from about 7.5 to 260 atm.s9 One important aspect of work at elevated temperatures and pressures is the maintenance of constant pH at all conditions studied. The ionization of buffers (and therefore the pH solution) varies with both temperature and pressure. Thus, without proper pH measurement, results caused by pH changes may be attributed to temperature and/or pressure change. This is particularly significant in cases where CO, is present in the hyperbaric headspace and acidification of the medium may occur. Bernhardt et al. have recently adapted an electrochemical cell for the measurement of pH under culture conditions for Methanococcus thermolithotrophicus (Figure 3 ) The de-

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FIGURE 3. Device used for high pressure pH measurement in the presence of gases. Components are (1) Ag/AgC1, (2) pH glass electrode, (3) reference electrode, (4) silicon oil, (5) sample, (6) gas supply, (7) valve, (8) connecting tube, (9) pressure supply, (10) heating system, (11) O-ring, (12) pressure balancing inlet. (Adapted from Bernhardt, G., Distecke, A,, Jaenicke, R., and Stetter, K.-O., Appl. Microbial.Biotechnol., Koch, B . , Ludemann, H.-D., 28, 176, 1988. With permission.)

vice consisted of a Ag/AgCl-containingpH glass electrode and a Ag/AgCl-containingreference electrode submerged in sample medium covered by silicon oil as the pressurizing fluid. A valved tube extended into the sample fluid for the addition of CO, to the medium. The entire cell was jacketed by a heating system. Measurements of pH in the absence and presence of CO, were made from 1 to lo00 bar at 65°C in a mineral salts medium, supplemented mineral salts medium and HEPES- or PIPES-buffered supplemented mineral salts medium. Although the ApWlOOO bar of the supplemented medium in the absence of CO, was approximately the same with and without buffer (about +0.05), the ApWlOOO bar in the presence of CO, was significantly reduced by buffering (-0.07 vs. -0.26). Experiments were performed to evaluate the effect of buffering on growth at 65°C. Cell lysis occurred above approximately 10 bar in unbuffered medium and above approximately 50 bar in PIPES-buffered medium. Normal cell growth was enhanced

Volume 10, Issue 4

Biotechnology 2. Food Industry Processing of milk and cheese products may be facilitated by psychrophiles and their enzymes. Enzymes active at low temperatures sweeten milk, form curds, and ripen cheese with minimal mesophilic contamination. In some cases, psychrophilic enzymes required shorter reaction times as compared to mesophilic enzymes used near their low temperature limit. Furthermore, residual activities could be removed by pasteurization if the enzyme is sufficiently heat-labile. Use of psychrophiles in the dairy industry, as well as the negative effects of psychrophilic contamination in dairy products, meat, eggs, and produce were reviewed by Sharp and Munster."

in HEPES-buffered medium by pressures up to 400 bar; between 400 and lo00 bar, cells appeared elongated, and between lo00 and 1500 bar, mini-cells were formed.

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IV. APPLICATIONS A. Psychrophiles 7. Waste Treatment Psychrophiles are particularly attractive candidates for waste digestion at ambient temperature in cold climates. Research seeking to minimize the lengthy start-up times for psychrophilic digestors has been conducted.61In uninoculated cattle manure batch digestors operated for 5.5 months, the first steps of anaerobic degradation (hydrolysis and acidification) proceeded at temperatures from 5 to 30"C, but methane production was seen only at 25 and 30°C. In a separate experiment using a 1:l ratio of uninoculated manure and manure precultured at 18°C gas production was observed over the entire temperature range in just 50 d. In contrast, a similar mixture containing 50% manure precultured at 35°C showed no methane production below 15°C after 30 d. Greater accumulation of volatile fatty acids at 10°C than at 22°C indicated that methane formation may be inhibited at low temperatures in this system. Studies of CSTR operation at 20°C showed that overloading can be remedied by increasing the temperature to 25°C until volatile fatty acid levels drop. Digestion of pig manure was more problematic, but pilot scale accumulation systems for both substrates showed similar results, indicating that psychrophilic digestion may be feasible on a large scale as long as appropriate inoculum, initial temperature, and retention profiles are used. Batch studies have also been conducted on the psychrophilic digestion of excess sludge from fish processing plants.62Sludge inoculated with a soil sample (a possible source of psychrotrophs) exhibited a 1.6-fold higher methane and carbon dioxide yield at 15°C than sludge inoculated with mesophilic digestion sludge. Addition of a second soil sample and subsequent enrichment at low temperature resulted in doubling of the total gas generated at 5"C, and doubling of the rate of methane production at 15°C. In addition, the increased rate of methanogenesis was maintained for 30 d at 15°C before leveling off, as opposed to only 20 d in the unenriched culture. Thus, methane production by psychrophilic digestion of fish-processing excess sludge appeared feasible. Six hundred and five strains of proteolytic, chitinolytic, and cellulolytic organisms were isolated at 0°C from the antarctic shelf.63In more than 90% of the 605 strains, the proteolytic activity was greater at 0°C than at 20°C whereas the chitinolytic and cellulolytic activities were greater at 20°C than at 0°C in more than 67% of the strains. Although the main goal of this research was to characterize the distribution and degree of cold adaptation of degradative organisms in the antarctic environment, it may also provide a stock of possible organisms to use in isolated or mixed culture psychrophilic waste digestion.

B. Thermophiles 7. Waste Treatment and Energy Production Microbial digestion of organic waste is a prevalent technique in sanitary engineering. In general, thermophilic processes have appeared desirable because reaction rates may be increased, sludge formation may be lower, influent waste stream viscosities may be reduced, organics may be better solubilized, more pathogens may be destroyed (especially significant in the case of sewage and manure treatment), and the need to precool warm waste streams may be eliminated.@ In addition, many digestion processes can be performed anaerobically in mixed cultures that produce methane, a valuable byproduct. One disadvantage of thennophilic anaerobic digestion, however, is that many such processes have proven to be ~ n s t a b l e Further.~~ more, although many advantages can be predicted for thermophilic digestion reactors, some have exhibited rates of chemical oxygen demand (COD) removal and methane production no higher than those of their mesophilic counterparka Digestion of many substrates has been examined at 55°C or above in laboratory scale reactors, and some results are summarized in Table 4. In addition, livestock waste has been fermented on a pilot scale (5.1 m3 working volume fermentor) at temperatures up to 55"C.67The maximum methane production rate of 4.7 m3 CH4/m3fermentor-day was reported to be approximately four times higher than other pilot or full scale systems fermenting livestock waste. In addition to the largely empirical laboratory and pilot scale systems described previously, a pilot scale palm oil mill effluent (POME) digestor has been operated at 45 to 60°C. The data obeyed Monod kinetics, and the design parameters derived were in good agreement with a conventional commercial POME digestor operating at 44 to 52°C. The parameters were used in a theoretical design that could improve the plant's efficiency by the incorporation of solids recycle.68 One disadvantage associated with thermophilic anaerobic digestors is their sensitivity to shock loadings (sudden increases in quantity or strength of influent). Lloyd and Whitmore investigated control of a 55°C pig slurry CSTR.69Without control, volumetric or concentration overloading caused a sharp increase followed by a decrease and/or fluctuation in CH, pro-

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Critical Reviews In Table 4 Examples of Thermophilic Anaerobic Waste Digestion Reactor type Down flow stationary fixed film

Bean blanching waste

Upflow sludge blanket

Defined medium plus volatile fatty acids and sucrose or yeast extract Synthetic meat waste Complex medium containing sucrose

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Substrate

Attached film expanded bed

lnoculum

%COD removal

Ref.

Unspecified organisms acclimated to substrate Cow manure andlor sludge

U G 9I

165

78-45

166

Thermally adapted sludge Thermophilic and mesophilic sludge

49-59

167

70

I68

Nofe: All reactors operated at 55°C.

duction. A concurrent increase in hydrogen production was observed. When the dissolved hydrogen level was monitored by membrane inlet mass spectrometry and the feed rate adjusted to maintain 1 p V f H,, sustained increased levels of methane production were observed. At present, anaerobic digestion is feasible up to about 65°C. This temperature is the upper known limit for rapid methanogenesis from acetate,70 a major intermediate in the bioconversion of complex organic matter to methane. However, the isolation of an aceticlastic methanogen (a methanogen that converts acetate to methane) capable of growth at higher temperatures might enable higher reaction rates and shorter retention times in anaerobic digestion processes. In addition to the recovery of methane from anaerobic waste digestion, the production of ethanol from cellulose by thermophiles is a promising source of renewable energy. Biological ethanol production is traditionally carried out by mesophilic yeasts utilizing either carbohydrate-rich substrates such as corn or hydrolysis products of less costly cellulosic substrates. The cellulosic substrates must be pretreated either with strong acids or with cellulases to degrade cellulose to sugars for fermentation. One particularly thermostable cellulase that may be used in this application is produced by the moderate thermophile Acidothermus cellulolyticus." Concentrated A. cellulolyticus growth supernatant showed carboxymethylcellulose-degrading activity with an optimum temperature of 80°C and half-lives of 60 and 12 min at 85 and 90"C, respectively. Compared to yeast, thermophilic bacteria are better suited for cellulose digestion because they produce cellulase and because they can utilize a greater range of substrates (including pentoses), although no known species does both.a One organism commonly used in cellulose digestion is Closfridium therrnocellum, a thermophile that grows optimally at 60°C. C . therrnocellum cellulase complexes, termed cellulosomes by Lamed and Bayer,'* were found in multicomplex protuberances

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(polycellulosomes) on the cell surface. The polycellulosomes attached to the insoluble cellulose, affixing the cell to the substrate, After extended growth, the cells desorbed from the cellulose, leaving behind cellulosome clusters coating the substrate, presumably continuing degradati~n.~' Although C . rherrnocellurn can degrade cellulose to ethanol and acetate, it cannot convert some other substrates found in biomass. For maximum utilization of available substrates, thermophiles such as C. thermohydrosulphuricum, C . thermosaccharolyticum, and Thermoanaerobacter ethanolicus can be grown in co-culture with C . thermoceffurn to ferment pentoses present in biomass to ethanol .64 One disadvantage of employing thermophiles for ethanol production is their relatively low ethanol tolerance and yields. The ethanol tolerance of wild-type thermophilic bacteria is typically < 1%, but selective growth has produced strains that tolerate as much as 10% ethanoLM Although tolerant strains have been found, high-producing strains are less common. LyndM cites a maximum production of 3 to 4% ethanol by thermophilic fermentation vs. a value of 9 wt% for yeast. Continuous ethanol removal by solvent extraction or removal of enriched vapor may alleviate ethanol sensitivity. Ethanol production by thermophiles from cellulose has recently been reviewed by Lynd.M

2. Polymerase Chain Reaction The polymerase chain reaction (PCR)is a recent advance in gene amplifi~ation.~~ In the original method, double stranded DNA was denatured at 95"C, the temperature was lowered to 25 to 37"C, and oligonucleotide primers specific to the 5' ends of the targeted gene were annealed to single stranded DNA. The Klenow fragment of E . coli DNA polymerase I and suitable 2'-deoxyribonucleoside 5'-triphosphates (dNTPs) were added; formation of double stranded DNA of the desired sequence proceeded. This cycle was repeated 20 to 30 times and one

Volume 10, Issue 4

Biotechnology Double-stranded DNA

annealingheaction step permitted the amplification of one gene alone or the simultaneous amplification of a number of genes with similar 5' region^.'^ Furthermore, the higher temperature minimized the self-annealing of single stranded DNA to form hairpin loops that block polymerization; thus, longer genes (2000+ base pairs) could be amplified with Taq Pol I than with E. coli Klenow fragment^.^' Taq Pol I is available commercially as GENEAMP (PerkinElmer Cetus Instruments) and has been expressed in E. coli for ease of production and p ~ r if ic a tio n .A~ ~similar enzyme isolated from the thermoacidophile Sulfolobus acidocaldarius can also be used for thermostable PCR." Applications of gene amplification by PCR include detection and characterization of disease related facilitated cloning and ~equencing,'~-'~ and detection of viral pathogens, including HIV.86 In a 3 to 4 h automated process, PCR can achieve results that were formerly obtainable only after days or weeks of growth and purification.

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Strand separation

1

Primer binding

3. Carbohydrate Conversion Enzymes One major industrial use of thermostable enzymes is the conversion of starch and sugars to product sugars. For example, amylases hydrolyze starch to maltodextrins; glucoamylase and pullulanase convert maltodextrins to glucose; and glucose isomerase converts glucose to fructose. Glucose isomerase (Dxylose isomerase) is one of today's most important commercial enzymes. Used in high fructose corn syrup production, glucose isomerase converts glucose to a mixture of glucose and its sweeter isomer, One major source of commercial glucose isomerase is Bacillus coagulans, a thermophile with an optimum growth temperature of 37 to 45°C and a maximum growth temperature of 60°C.88 The application of these and other saccharification enzymes has recently been Developments in a-amylase production from thermophiles include the selective growth of a deregulated mutant in continuous culture of Bacillus caldolyticus strain SP at 60°C.90 Whereas the parent strain required maltose to induce amylase formation, the mutant strain (Ml) did not require maltose and produced twice as much amylase as measured by starch hydrolysis per volume of culture at 60°C. Furthermore, MI was resistant to glucose repression. The lack of required inducer and alleviation of glucose repression made this strain better for enzyme production. a-Amylase production also seemed to be increased in Bacilli by the introduction of D-cycloserine resistancea9'Thermophiles B. acidocaldarius A-2 and B. stearorhermophifus B-70 were exposed to UV mutation and cultured at sequentially higher concentrations of D-cycloserine. Amylase yield was increased 100-fold for both species, although the resulting enzymes were not characterized for thermostability in this study. Other a-amylase production schemes include adsorption of B . stearothermophilus to a thermostable ion-exchange resin for use in an airlift fermentor at 55"C.92 Amylase for the production of malt syrup has been produced

I

Primer extension

4

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I

I

I

I

I

I

I

I

1

I

I

I

I

I

I

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Many cycles

Enrichment of target sequence FIGURE 4. Polymerase chain reaction. (Adapted from Guyer, R. L. and Koshland, D. E., Jr., Science, 246, 1543, 1989. Copyright0 1989 by AAAS. With permission.)

million-fold amplificationresulted (see Figure 4). If one primer was used in substantial excess of the other, a single stranded product resulted which could be used for sequence analysis without further p ~ r i f i c a t i o n . ~ ~ Substitution of thermostable Polymerase I from Thermus aquaticus (Taq Pol I) has produced-several improvements to the original technique. Because Taq Pol I was highly thermostable, it survived the DNA denaturation step and was added only at the initiation of the reaction, not at the last step of each cycle; thus, the process can be automated.76The polymerization reaction was optimal at approximately 70°C; at this temperature, mismatched primer-template hybrids were unstable and target sensitivity and specificity were increased, with a resultant increase in product yield in cases where similar sequences exist in the genome. Varying the temperature of the

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Critical Reviews In by cloning a gene from B. stearothermophilus into B . subtilis, and the resulting recombinant amylase was found safe for use in the food industry.93 u-Amylase is also used to reduce wheat flour viscosity during wet milling. Malt amylase is sometimes used, but it imparts undesired odor, taste, and color. Thermophilic bacterial amylase causes no aesthetic problems, but because of its extreme thermostability, reduces viscosity to a value too low for bread manufacture. Recently, B. stearothermophilus a-amylase was acylated to reduce its temperature optimum from 70 to 60"C, and to reduce activity at 80 to 90°C to a negligible level. The maximum viscosity and the temperature of maximum viscosity of the resulting flour were similar to those of flour treated with malt a-amylase .94 In addition to the many thermostable a-amylases, a thermostable P-amylase has also been isolated. This enzyme, purified 98-fold from an overproducing strain of Clostridium therrnosulfurogene~,~~ hydrolyzed gelatinized starch at an optimum temperature of 75°C. The enzyme adsorbed tightly to raw starch and was active over a pH range of 4 to 7 , making it suitable for the commercial production of maltose-containing syrups. Another saccharification enzyme, lactase, was stabilized by polyethylenimine (PEI) in both free-cell and immobilized cultures of B. steurothemphilus." PEI-cured K-carrageenan beads containing B. stearothermophilus exhibited a lactase half-life of 39 d at 60°C in an operating plug flow reactor. In addition to its effect on the enzyme, PEI also stabilized the support. PEI-curing resulted in beads that were up to 4 . 7 times stronger than those subjected to KC1-curing. Moreover, the PEI-cured beads were resistant to thermal decay by agitated incubation at 60°C for 16 h and by autoclaving at 121°C for 20 min. 4. Proteases Proteases are used in a variety of commerciallindustrial applications, such as laundry detergents, leather preparation, protein recovery or solubilization, meat tenderization, and organic ~ynthesis.~' Proteases used in laundry detergents must be stable and active in the presence of surfactants, metal chelators, oxidizing agents, and moderately alkaline media. Some proteases recently isolated from t h e r m ~ p h i l e smay ~ ~ .thus ~ be unsuitable for use in detergents because they are dependent on calcium ions for thermostability. In contrast, the half-life at 95°C of a protease from Desulfurococcus was increased to more than 100 min by detergents such as 0.1% SDS and 1% Triton X-1O0.'Oo Moreover, stability was not effected by 10 mM EDTA, indicating possible applicability in detergents. Proteolytic activity in cell-free extracts of Pyrococcusfuriosus increased with temperature up to at least 105°C (above which no data are reported). lo'The enzyme was exceptionally stable as well: its half-life at 98°C was over 60 h, and nearly 50% activity remained after 12 h in 1% SDS at 98°C. Finally, a serine protease from Thermomonosporafuscu strain YX may also be of com-

332

mercial value, exhibiting temperature and pH optima of 80°C and 9, respectively, and good stability in the presence of 20 mM dithiothreitol, 0.2% SDS. or 0.1 A4 hydrogen peroxide."" Enzymatic protein processing such as soy, meat, fish, and gelatin hydrolysis can benefit from higher temperatures where catalytic efficiency may be increased, and mesophile contamination and feed viscosity may be red~ced.'~ In addition, proteins from thermophiles generally exhibit stability against chemical denaturants, pH, and organic solvents. I"' Thus, thermophile proteases may be applied to peptide synthesis, which is favored in nonaqueous environments. 'OM For example, thermolysin, a well-characterized protease isolated from B. ihrrmoproteolyticus, can be used in the synthesis of aspartame.".1o5 Another possible application of thermophilic proteases is the cleansing of fouled ultrafiltration or reverse osmosis membranes used in food processing industries. '"' Further reviews of proteases from thermophiles can be found in the literature."."'

5. Medical Analysis Ideally, enzymes used in clinical analysis should exhibit specificity, sensitivity, and stability. Recently, a glucokinase from B. stearothhrmophilus has been substituted for hexokinase from yeast for the detection of magnesium in serum.Lobglucose in serum and urine,"' and creatine kinase activity in serum."'" The resulting specificity and sensitivity were similar for both enzymes. The major advantage of the thermophilic glucokinase was its stability to storage, with reagent solutions remaining active for 1 to 3 months when stored refrigerated and 7 to 30 d at room temperature. In contrast, the traditional hexokinase systems were stable for only a few days, even when refrigerated. 'OR 6. Miscellaneous Uses Chirally pure organic compounds required for chemical synthesis can be produced by yeasts and other organisms, including thermophiles and their enzymes. For example, suspended whole cells of Thermounaerobium brockii converted 3-ketobutyrate and -valerate to (S)-3-hydroxyvalerate at 40% yield and 3 93% chiral purity at 72°C whereas baker's yeast did not perform this conversion. '" Immobilized alcohol dehydrogenase (ADH) from the same organism has been used for gram-scale production of R( ) 2-pentanol from 2-pentanone at approximately 80% optical purity at 23°C and 45% optical purity at 60°C. T. brockii ADH can also produce or recycle NADPH."" Enzymes from thermophiles hLve also been used in gasphase catalysis. Alcohol dehydrogenase from S. sulfaturicus was immobilized in cross-linked albumin glutaraldehyde particles and studied in batch and fixed bed reactors."' Immobilized S. sulfataricus ADH retained 75% of its alcohol oxidizing activity after 50 h at 60°C. In contrast, immobilized horse liver ADH lost 50% of its activity after 1.5 h at the same temperature. A fixed bed reactor operated at 60°C with immobilized S. solfutaricus ADH was 12 times more productive than the

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Biotechnology C. Barophiles 1. Applications of Barophiles There are relatively few practical applications of barophiles in the extremophile literature. Although the equipment used to study barophiles and barophilic behavior is well developed (see Section 111. B), most studies still focus on a better understanding of basic scientific principles. One exception is microbialenhanced oil recovery (MEOR). For example, microbes have been injected into oil wells 2000 to 3000-m deep at pressures of 200 to 450 bar or more (depending on overpressurization of the strata) to effect tertiary oil recovery.” At these depths, temperatures range from 60 to 123”C, and extremes of pH, salinity, and heavy metals may be encountered. Once in situ, the bacteria may produce gas and/or substances which reduce oil viscosity. Biosurfactants from nonbarophilic sources are also being developed with possible application in biologically enhanced oil recovery.

same system employing horse liver ADH. Immobilized ADH in gas-phase reactors may also be used to detect alcohol levels in the breath. I Support media can contain whole or disrupted cells as well as purified enzymes. Lyophilized S . solfataricus was incorporated in polysulfone capillary membrane fibers formed by phase inversion. I I 3 Although the cell-impregnated fibers were not as strong as those formed without cells, they could withstand a transmembrane pressure of 0.055 bar at 70”C, sufficient to observe the P-galactosidase activity of the entrapped cells. Thirty such fibers were assembled in a cross-flow bioreactor, and the performance was evaluated as a function of Peclet number (associated with transmembrane pressure) and a dimensionless Michaelis constant (associated with substrate feed concentration). High transmembrane pressure favored increased productivity but decreased product purity; high substrate concentration resulted in poor conversion. Thus, the choice of operating conditions may depend on the ease of product purification and/or substrate recycle. The immobilized cells were quite stable, retaining 50% of initial activity for approximately 100 d after an exponential decay in the first 20 d of operation at 70°C and 0.061 bar transmembrane pressure. Similarly, Calduriella acidophilu was immobilized in a variety of ultrafiltration membranes for observation of 9-galactosidase activity at 70 to 85°C. Entrapped cells showed higher enzyme activity than free cells, possibly due to changes in cytoplasmic membrane permeability caused by the entrapment procedure.115 Thermophiles and their enzymes were well suited to membrane immobilization because they were more resistant to the solvents and temperatures required for polymer formation and annealing. Because it has a two-dimensional crystalline structure, the cell wall surface layer (S layer) of thermophiles and other organisms can be used in applications requiring a regular array of molecules. Isoporous ultrafiltrationmembranes were formed by depositing S layers of B. stearothermophilus and C . thermohydrosulfuricum on large pore membrane supports and crosslinking the S layer fragments with glutaraldehyde.II6 The resulting membranes displayed sharp molecular weight rejection profiles with cut-off values between 30 and 45 kDa and were resistant to hydrochloric acid, sodium hydroxide, alcohols, ketones, chlorinated hydrocarbons, benzene, dimethyl sulfoxide, dimethylformamide, and acetonitrile. In another intriguing application, the S layer of S. acidocaldarius has been used as a template for metal lithography on the nanometer scale.11’ The regular protein array was bound to an amorphous carbon film, a metal film deposited nonuniformly, and the metal film ion-milled to produce a grid of oval holes whose centers were separated by approximately 19 nm. This technique was a preliminary result in the possible production of molecular electronic, chemical, and/or optical devices.

2. Applications of Pressure As mentioned in Section II.C, a number of theories and models have been proposed to explain the effect of pressure on protein structure. Depending on the system, pressure may stabilize proteins, enhance enzymatic rates, minimize competitive reactions, and/or regulate enzyme function. As an example of the potential use of pressure in applied enzymology, this review presents an overview of the effect of pressure on a number of proteases and describes two specific applications in which pressure is used to enhance or control protease action. Proteases are a well-studied class of enzymes capable of hydrolyzing a variety of substrates. Typically, proteases exhibit carboxypeptidase, amidase, and/or esterase activity. Because the rate limiting step of catalysis differs among substrates, pressure may affect these activities differently. Table 5 summarizes the results of kinetic studies conducted over the pressure range 1 to loo0 bar. The tabulated values, AVK, and AVka,, are parameters used to describe the effect of pressure on the Michaelis-Menten parameters, K, and kcat:

By analogy to the thermodynamic definitions of reaction and activation volumes, the dependence of K, on pressure can be described by the parameter AVK,:

AVKm= - RT-

d In K, dP

Likewise, the dependence of k,, on pressure can be described by AVkat:

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(3)

These values do not necessarily represent true volume changes because the parameters K, and k,,, can be combinations of rate and/or equilibrium constants. A negative value of AVK, indicates that K, increases with pressure, and a higher concentration of substrate will be required to achieve the maximum rate. Pressure enhances the maximum rate of reaction when AV,,,?is negative. Depending on the substrate concentration employed, one or both of these values will govern the effect of pressure on the observed reaction rate. At substrate concentrations significantly greater than K,, the rate of reaction, v , is equal to k,,,[E], and the effect of pressure on the overall reaction rate is governed by AVkcas.At the other extreme, if the substrate concentration is kept significantly below K,, v = k,,,/K,,[E][S], and the effect of pressure is governed by AVkeaJum, where

(

/I:

AVkcn,,Km = - RT d In - -

RT-

dP

dP

-

(4)

- RT

-)(d IndPK,

(5)

Table 5 Values of AVs of Protease Kinetics Determined From Pressure Experiments Enzyme

A V ~ C , ~ Ref.

Substrate

BzArgOEt - 2 . 4 2 0.3 -1 1 622 BzArgNH, FuaGlyPhe -31 f 2 -4 2 2 CPase A -30 -t 3 FuaPhePhe -8 2 21 2 2 -23 2 2 ‘I‘hermolysin FuaGlyPheAla 13 FuaGlyLeuAla -26 2 2 23 2 2 -35 2 2 FuaGly PheNH, 10 It 4 -35 5 3 FuaGlyLeuNH, CPase Y FuaGlyPhe -16 2 2 27 2 2 - 22 FuaPhePhe 10 F I -6.0 2 1.0 - 1.5 5 0.5 FuaPheOEt -6.2 k 1.0 - 3 . 1 It 1.0 FuaPheOMe CPase P FuaPhePhe -34 5 3 -7 t 2 -8 2 2 BzGlyOPheLac - 3 s f 3 FuaPhePhe -2s 2 -8 2 CPase W -7 2 3 -21 2 2 FuaPheGly FuaPheOEt -3 2 2 -3 3 -2 k 2 -3 2 2 FuaPheOMe

Trypsin

*

*

*

*

169 170 171

172

173 174

*

Nore: The sign convention is described in the text and may differ

from the sign convention used in individual references.

334

-

Examination of the data in Table 5 leads to several conclusions which may be helpful in applying proteases to greatest advantage. At high substrate levels, pressure appeared to enhance carboxypeptidase (CPase) Y esterase activity while retarding its amidase activity, whereas both the esterase and peptidase activities of CPase P and CPase W were pressureenhanced. Furthermore, although the peptidase activity of CPase A, CPase P, and CPase W and the esterase activity of CPase Y, CPase P, and CPase W were promoted by pressure at high substrate concentrations, all these activities were retarded by pressure at low substrate concentrations. Although it is still necessary to know the actual values of the reaction constants in order to determine the feasibility of a particular reaction scheme, analysis of the effect of pressure on enzymatic reaction rates can be especially useful for those enzymes exhibiting multiple activities and substrate specificities. Practical application of pressure in protease-catalyzed reactions has been demonstrated. Digestion by thermolysin of several proteins important to the food and feed industry (e.g., soy protein, hemoglobin, and P-lactoglobulin) was enhanced by 2000 bar pressure.”’ Of particular interest in this study was the effect of pressure on digestion of a-lactalbumin and plactoglobulin in cow’s milk. or-Lactalbumin was not digested at either 1 or 2000 bar, and P-lactoglobulin was digested only at the elevated pressure. Removal of P-lactoglobulin while retaining a-lactalbumin made cow’s milk more like human milk. Although thermolysin has been shown to be activated by pressure, greater digestion may also be due to pressureinduced partial unfolding of the substrates, exposing sites for thermolysin attack. Pressure has also been used to enhance protease-catalyzed peptide formation by the mechanism shown in Figure 5. In initial experiments with CPase Y , peptide formation from an N-acyl amino acid ester (Fua-Phe-OEt) and an amino acid amide (either Gly-NH, or Phe-NH,) was increased as much as fivefold by the application of 1500 bar pressure.‘’’ Product peptide was not significantly hydrolyzed because CPase Y peptidase activity was strongly inhibited by pressure. In subsequent CPase Y studies, an N-acyldipeptide substrate (Fau-Gly-Phe) was substituted for the ester. Dipeptide formation with LeuNH, (i.e., amino acid substition to form Fua-Gly-Leu) was observed at 1000bar but not at 1bar.’21Furthermore, formation of the reaction intermediate Fua-Gly-Leu-NH, was at least doubled by application of 1000 bar at each pH and enzyme concentration tested, and hydrolysis of the substrate to byproduct Fua-Gly was decreased as much as 50%. In addition, dipeptide synthesis by thermolysin was studied. With the substrates Fua-Gly and Leu-NH,, the reaction proceeded very rapidly, and the product yield was governed by thermodynamic equilibrium rather than by kinetics. Pressurization to 2000 bar reduced the percent yield by 15 to 30% after 2 h incubation. Use of “nonspecific” substrates (those for which the enzyme

Volume 10, Issue 4

Biotechnology Fua AAIOEt

+ CPase Y

I1

Fua AAICPase Y (Acyl-enzyme)

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I11

~

cellulases active over the pH range 5 to 11 and alkalophilic Bacillus species No. 1139 produces a single cellulase of pH optimum 9. These enzymes have been isolated and their structural genes characterized. "' Conversion of xylan to reducing sugars may be more practically achieved at high pH because xylan is soluble in alkaline solution but insoluble in neutral water. Two xylanases were isolated from each of the alkalophilic Bacillus strains W1 and W2 grown at 45°C and pH 10. One xylanase from each strain had a pH optimum of 6.0 and a temperature optimum of 65"C, and produced xylobiose, xylotriose, xylotetraose, and xylopentaose from xylan. The other xylanase from each strain had a broad pH optimum of 7.0 to 9.0 and a temperature optimum of 70"C, and produced xylose, xylobiose, xylotriose, and xylopentaose from xylan. Similarly, two xylanases were isolated from the alkalophilic Bacillus strain C-125.'22One enzyme had a neutral pH optimum of 7, and the other had a broad maximum of pH 6 to 10, with activity remaining at pH 12. These three strains, therefore, each produce two extracellular xylanases, one of which has a neutral or near-neutral pH optimum and the other with a broad alkaline pH optimum. In contrast, the alkalophilic Bacillus strain AM-00 1 contained enzymes whose pH optima differed according to cell location. The cell-associated P-mannosidase activity showed a neutral pH optimum of 7, whereas the extracellular p-mannanase activity was optimal at pH 9.Iz5 In addition to the degradation of cellulose and its derivatives, alkalophiles can be used in the degradation of pectin. Pectinaceous citrus processing waste was assimilated by alkalophilic Bacillus species GIR 621-7, a mutant excreting 20 times the endopectate lyase activity of the parent. Uronic acid concentration (an indicator of treatment efficiency) decreased 93% in 20 h in supplemented waste at pH 9.5 and 27°C seeded with cells grown on pectic acid.Iz6

Fua AAWZ + CPase Y

+AA3NH,

VI

VII

t FuaAA1

-

formation. I: esterase FIGURE 5. Reactions in CPase Y-catalyzed dipeptide . . activity. 11: carboxypeptidase activity. 111: aminolysis. IV: amidase activity. V: carboxypeptidase activity. VI: carboxamidopeptidase activity. VII: hydrolysis. Acylated enzymes formed in IV, V, and VI are not explicitly shown. Fua = N-[3-(2-furayl)acryloyl]. AA = amino acid. (Adapted from Kunigi, S., Tanabe, K., Fukuda, M., Makimoto, S . , and Taniguchi, Y . , J. Chem. Soc. Chem. Commun., 1335, 1987.)

has low affinity) Cbz-Asp and Phe-OMe, resulted in slower reaction rates. After 4 h incubation, equilibrium had not been reached at either 1 or 1500 bar, but the yield at 1500 bar was up to six times the yield at 1 bar. Enhancement of the formation of Cbz-Asp-Phe-OMe may prove significant in sweetener production.

D. Alkalophiles

2. Amylase and Other Starch Conversion Enzymes Amylases degrade starch to oligosaccharides for use in food, pharmaceutical, and chemical products. Three alkaline amylases which produce maltohexaose as the main product from soluble starch have recently been isolated from alkalophilic Bacillus species H- 167 grown at 37°C and initial pH of 9.4. Iz7 These enzymes (mol wt 59, 73, and 80 kDa) showed the following similar properties: pH optimum of 10.5; stability in the pH range 7 to 12 as measured by retention of > 90% activity after incubation at 50°C for 30 min at each pH; temperature optimum of 60°C; and stability up to 55°C as measured by retention of > 90% activity after incubation for 30 rnin at pH 9 at each temperature. Maltohexaose-producing amylase activity was also observed in alkalophilic Bacillus species #707, and the gene was cloned for expression in B . subtilis and E. coli. Although the native strain produced five electrophoretically distinct bands of amylolytic activity capable of producing maltotetraose, maltopentaose, and maltohexaose, only

1. Cellulase and Other Waste Degrading Enzymes Cellulases active at elevated pH may be of greater utility than neutral or acidic cellulases because cellulose and hemicellulose are more soluble in alkaline solution. In addition, digestion of cellulosic waste found in human and animal excrement may occur at elevated pH as a result of ammonia production. A crude cellulase preparation from alkalophilic Aeromonas species No. 212 was used to decompose cellulosic material in human Five per cent of cellulose powder, 7.5% of microcrystalline cellulose, and 15% of available sewage (i.e., excrement and toilet paper) cellulose were enzymatically degraded at pH 8 in 20 h. Alkalophilic Bacillus strain C-1 1 was grown in hemicellulose-containing rayon waste supplemented with yeast extract and urea at pH 12 (initial) and 37"C.33Although hemicellulase activity was optimal at pH 7, approximately 20% of maximum activity remained at pH 12. In addition, alkalophilic Bacillus species N-4 produces several

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Critical Reviews In one maltohexaose producing band was seen with the plasmidcontaining host organisms. In culture, B. subtilis carrying plasmid pTUB8 12 produced approximately 70 times as much amylase activity per volume as the native strain. Production of amylase and other enzymes has also been stimulated by the addition of amino acids and their derivatives. The addition of 0.5 to 1.O% glycine, p-alanine, DL-norvaline (~~-a-amino-n-valeric acid), D-alanine, or D-methionine to alkaline starch medium increased the observed extracellular amylase activity of alkalophilic Bacillus No. A-40-2, while growth was maintained or inhibited. 12' Amylase activity was best enThe production and hanced (2.5-fold) by 0.5% ~~-norvaline. release into solution of P-galactosidase by alkalophilic Bacillus No. C-125 was enhanced by manganese and by glycine derivatives such as glycine methyl ester (GME), glycine ethyl ester (GEE). and glycinamide."" Addition of either 0.2 mM Mn2+ or 1.5% GEE alone more than doubled cell growth and more than quadrupled total P-galactosidase activity. GEE addition increased the ratio of extracellular to total activity from 1:6 to approximately 1:2. Addition of both factors nearly quadrupled growth and increased total P-galactosidase activity 20-fold, with more than 50% of the P-galactosidase activity excreted. Increase in the fraction of P-galactosidase activity excreted was attributed to an altered (leaky) cell membrane and to cell lysis.

5. Miscellaneous Alkalophilic organisms have been screened for antibiotic p r o d u ~ t i o n .One ~ ~ recent report described the production and isolation of phenazine antibiotics from the alkalophilic actinomycete, Nocardiopsis dassonvillei OPC-15. Incubation in complex media at pH 10 and 27°C for 6 to 8 days produced 1,6-dihydroxyphenazine and 1,6-dihydroxyphenazine 5-monooxide, whereas culturing as above for 6 d followed by a temperature shift to 4OC for 2 d resulted in production of 1.6dihydroxyphenazine 5-monooxide and 1,6dihydroxyphenazine 5,lO-dioxide. A novel engineering application of alkalophilic Bacillus species is the generation of electricity. '35 Stationary-phase cells were added to the anodic compartment of a fuel cell containing glucose and redox mediators (i.e., electron carriers) in pH 10 to 1 1 buffered solution. As the bacteria oxidized the glucose, the redox mediators took up electrons and transferred them across an ion-exchange membrane to a cathode compartment containing potassium fenicyanide in buffer. The optimal pH was 10.5 using methyl viologen (MV) and ferric EDTA (FE) as redox mediators. Fuel cells using MV and FE or MV and 2-hydroxy- 1,4-naphthoquinone (HNQ) as redox mediators at pH 10.5 and 35°C continuously produced currents on the order of milliamps for days at coulombic yields of 63 to 94%.

3. Proteases Proteases active at alkaline pH can be used in detergent preparations as described in the section on proteases from thermophiles. A number of such enzymes has been isolated from alkalophilic organisms as reviewed by Horikoshi and Akiba33 and by Sharp and Munster. ' I Recently, a protease capable of hydrolyzing elastin has been isolated from culture broth of alkalophilic Bacillus strain Ya-B grown at 37°C and pH 10.1.l 3 I A protease of pH optimum 11.5 has also been isolated from Bacillus B21-2 grown at pH 10.2 and 30°C.132This strain is considered to have commercial potential because it produces large amounts of alkaline protease while growing on an inexpensive substrate mixture of glucose, soybean meal, and fish processing waste.

1. Microbial Leaching Microbial leaching by acidophiles is employed primarily in the recovery of copper and uranium from low grade ores. In the case of copper recovery, Thiobacillusferrooxidans oxidizes pyrite to ferrous sulfate,and sulfuric acid. The bacteria then further oxidize ferrous sulfate (FeSO,) to ferric sulfate (Fe,(SO,),), a strong oxidizing agent. 136

E. Acidophiles

4. Cyclodextrin Glucosyltransferases Cyclodextrins or Schardinger dextrins are cyclic molecules composed of 6 to 8 glucose units linked by 01-1,4 linkages. Cyclodextrins are good carrier molecules that may be used in drug formulations, flavor stabilizers, food and medicine deodorizers, pesticides, diagnostics, catalysis, phase transfer, and chromatography. Cyclodextrins are produced by the enzymatic degradation of starch by cyclodextrin glucosyltransferase (CG'T), which has been isolated from a variety of bacterial sources, including alkalophiles. CCTs derived from alkalophiles have recently been reviewed.

336

2FeS, (pyrite)

4FeS0,

+ 7 0 , + 2H20 +.

+ 0, + 2H,SO,

+-

2FeS0,

+ 2H,SO,

(7)

+ 2H,O

(8)

2Fe2(S0,),

The ferric sulfate then attacks copper sulfide minerals to generate cupric sulfate and elemental sulfur. CuFeS, (chalcopyrite)

Cu,S (chalcocite)

CuS (covellite)

Volume 10, Issue 4

+ 2Fe,(SO,),

+ 2Fe2(S0,),

+ Fe,(SO,),

+-

+ 2CuS0,

+ 5FeS0, + 2s'

CuSO, + 4FeS0,

(9)

+ So

(10)

+ 2FeS0, + So

( 1 1)

+ CuSO,

Biotechnology Cu,FeS, (bornite)

+ 6Fe,(SO,),

+ SCuSO,

+

13FeS0,

+ 4s'

(12)

In addition, the sulfuric acid generated converts copper oxide minerals to soluble cupric sulfate. Cu,(OH),(CO,), (azurite)

+ 3H,SO,

+ 3CuS0,

+

2C0,

+ 4H,O

(13)

pH ADJUSTMENT

IN-PLACE LEACH STOPES

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CuSiO, * 2H,O (chrysocolla)

CuO (tenorite)

+ H,SO, -+ CuSO, + SiO, + 3H,O

+ H,SO,

+ CuSO,

+ H,O

PREGNANT

(14)

LIQUOR RESERVOIR

or

(15)

+ 3 0 , + 2H,O + 2H,SO,

v

- SOLVENT EXTRACITON AND STRIPPING

Both Thiobacillusferrooxidans and T. thiooxidans can convert elemental sulfur to sulfuric acid to maintain low pH (1 to 3) and thereby facilitate acid solubilization reactions.

2s'

Fe SCRAP

either

SOLIDLIQUID

ELECTROWINNING

(16)

'

In addition to indirect leaching (described previously), direct leaching may occur in which bivalent metal sulfides are converted to metal sulfides without the production of ferric sulfate. Uranium, aluminum, gold, chromium, zinc, and vanadium can also be recovered microbially from low grade ores and industrial waste. A number of reviews on the chemistry and applications of metal recovery by microbial leaching have been published.136-'39 Ore leaching is commonly performed in dump, heap, or in situ processes; reactor systems may be used for high-value metal recovery. Limitations in dump and heap leaching include restricted oxygen transferI4' and heating up to 80'4'-90"C136in the interior of the pile. The tendency of dumps to self-heat may render thermophilic acidophiles, such as Sulfolobus species, more attractive in microbial leaching. A thought provoking review comparing the biotechnology of mining and metal processing to the biotechnology of pharmaceutical production addresses some of these problems.L42 In situ ore leaching is usually reserved for the lowest grade ores for which the cost of ore removal is prohibitive. Although the ore remains in place, fractionation of ore in situ and pumping dilute leachate from mines may also be Sanmugasunderam modeled an in situ stope leaching process using 5.4 m X 33 cm columns containing ore pellets 0.6 to 15 cm in diameter.143The columns were flooded with lixiviant (liquid that has trickled through a leaching dump) and then drained, sampled, and refilled each week. The design for the in situ process included a bioreactor to culture Thiobacilli to enrich the lixiviant before it is recirculated to the stope (see Figure 6). Copper extraction rates of 1 . 1 % per month obtained after 140 d were reported.

v

CEMENTATION

f

EFFLUENT TREATMENT

I

1

SETTLING POND

Cu CATHODE

CuCEMENT WASTE/SLUDGE

FIGURE 6. Proposed in situ stope leaching process. Bleed streams are indicted by (Reproduced from Sanmugasunderam, V . , Biotechnology for the Mining Metal-Refining and Fossil Fuel Processing Industries, 1986, 13.

In addition to ore leaching, acidophiles can be used to recover metal from industrial waste streams. Although chemical leaching may be more rapid, the gradual decrease in pH characteristic of microbial processes may cause different metals to be preferentially removed as the pH drops. Thus, products may be more easily separated when wastes containing more than one metal of interest are leached. For example, 100%vanadium extraction from filter press residue by T. thiooxidans was achieved in 8 d, whereas chromium extraction continued up to 23 d.I4 In the same study, a rough cost comparison of microbial and chemical (i.e., sulfuric acid) leaching of industrial waste showed that substrates for microbial acid production were less expensive than the sulfuric acid required for comparable recovery by chemical means. In contrast to the beneficial results of controlled microbial leaching, uncontrolled leaching may result in acid mine drainage, which pollutes soil and waterways. A method of controlling the effect of Thiobucilli on soil has been presented. 145 Anionic detergents such as sodium lauryl sulfate (SLS) were 1991

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inhibitory or toxic to T. ferruxidans in culture media and mine spoils. Moreover, SLS exhibited low mammalian toxicity and high biodegradability. Because SLS was both washed from the soil and/or degraded, repeated or timed application was required to maintain appropriate concentrations in the field. A combination of initial spray application followed by time release of SLS from cylindrical rubber pellets was proposed. SLS release from rubber pellets was demonstrated in the laboratory, and field tests were begun.

2. Desulfurization of Fossil Fuels The phenomenon of acid rain has become a significant environmental problem. Sulfur dioxide released from the burning of sulfur-containing fossil fuels combines with water and oxygen in the atmosphere to form sulfurous and sulfuric acid. Government regulations now limit the amount of SO, which may be released into the atmosphere, and most industries scrub or otherwise treat effluent gas streams before release. Sulfur can also be removed from the fuel before combustion. Conventional hydrodesulfurization of fuel oil is a catalytic process capable of reducing sulfur levels tenfold to provide low sulfur crude oil. Alternatively, microbial desulfurization, caused by reactions similar to those responsible for microbial leaching, can he employed. The microbial desulfurization of fossil fuels and an economic comparison with conventional hydrodesulfurization were described by Finnerty and Robinson. 14' The 1986 review concluded that microbial desulfurization was more than three times as costly as hydrodesulfurization, but that realistic improvements in the biological process design could make it competitive. In a related article, the cost of microbial desulfurization was compared to the market cost of naturally low-sulfur coal, and microbial desulfurization was found to be economically feasible.L47 Some recent experiments in microbial coal desulfurization attempted to build upon the understanding of the mesophilic acidophile Thiobacillus by studying the thermophilic acidophile Sulfulobus. Both species are known to attach to coal particles in general as well as to sulfur-containing pyrite, and both species oxidize Fe2+and So to Fe'+ and S04.148Sulfolobu~~ aridocaldarius was chosen to study the relationship between extent of attachment and sulfur leaching by thermophiles. 14* S. ai-idocaldarius reached equilibrium adsorption to coal particles after less than 5 min of exposure to finely divided coal samples at 72°C. On the coal sample with greater surface area and finer pyrite grains (KY #9), the culture showed greater cell attachment, half the lag time for leaching to begin, a slightly higher (< 20%) maximum iron removal rate, and twice the first order rate constant for sulfur leaching than on the coal samplc with less surface area and larger pyrite grains (OH mixture). Adsorptioddesorption experiments with both coals indicated that some cells became irreversibly attached to the surface. These results indicated that increased attachment and enhanced leaching were related, although more detailed ex-

338

periments were required to fully understand the mechanism of direct and indirect leaching in Sulfolobus. Knowledge of the action of thermophiles on coal was applied to a mathematical model of mesophilic leaching to develop a model for thermophilic leaching. 149 In addition to equations for (1) microbial oxidation of ferrous ions by suspended cells, (2) microbial oxidation of pyrite by attached cells, and (3) precipitation of sulfate and femc ions, it was necessary to add equations for (4) chemical oxidation of ferrous ions by dissolved oxygen and, ( 5 ) chemical oxidation of pyrite, reactions which became significant at the elevated temperature of 72°C but were negligible at 25 to 37°C. Mass balances for Fe(II), Fe(III), attached and free cells, and pyritic sulfur completed the model. The model adequately described the microbial oxidation of ferrous iron and the chemical leaching of coal samples, and provided a good description of the dynamics of the microbial coal leaching system. Adjustment of assumptions about cell attachment could alter the fit of the model to the data, and more experiments clarifying cell adsorption may make the model more generally applicable. In addition to systems employing thermophilic acidophiles, new coal desulfurization procedures employing Thiobacilli have been developed. One such process involved the containment of cell free extracts of T. ferrooxidans in reverse micelles for leaching of finely divided coal suspended in mineral oil.'5o Acidic aqueous solutions of sonicated cell suspensions were added at 1% (v/v) to slumes of crushed coal in mineral oil and 1.01% nonionic surfactant. Sonication and vigorous shaking produced reverse micelles which maintained an aqueous environment for the extracted enzymes. Spectrophotometric assays showed a decrease of almost 50% in Fe2+ in 2 h and analysis of washed coal removed from the system indicated removal in 1 d of more sulfur (> 37% of sulfur present in the raw coal) than the amount removed by whole cells in 3 d. Similar experiments were proposed for S. acidocaldurius but were not conducted.

F. Halophiles 7. Medical and Biological Research Applications One recent area of applied halophile research is screening for antibiotic sensitivity. Antibiotics to be used in cancer therapy are sometimes screened by their effect on E . coli mutants. However, since most antibiotics active against eucaryotic proteins (e.g., tubulin, type I1 DNA topoisomerase) have no effect on similar eubacterial proteins, only antibiotics which interact directly with DNA will be detected by this method.'" On the other hand, the extreme halophile Halobacteriuim halobium was sensitive to antibiotics which target the proteins DNA topoisomerase TI, actomyosin, and tubulin in eucaryotic cells. Is2 For example, growth of H. haiobium was inhibited 50% in a complex medium containing 200 g/l NaCl and 20 gil MgS04-7H20at pH 7.5 by each of the following: 10kg/inl adriamycin, 7 pg/d daunorubicin, 25 pg/d etoposide (VP16),

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Biotechnology 20 pg/ml teniposide (VM26), 5 pg/ml cytochalasin B, 50 pg/ml cytochalasin D, 25 pg/ml vincristine, 8 pg/ml podophyllotoxin, and 35 pg/ml nocodazalone. Although neither 2methyl-9-hydroxy ellipticine nor m-4'-(9-acridinylamino) methane sulfon-m-aniside inhibited growth of H . halobium, at concentrations of 30 pg/ml and 3 pg/ml, respectively, each antibiotic inhibited by 50% the growth of the haloalkalophile Natrobacterium gregoryii in a medium containing 1 g/l MgS04.7H,0 (pH 10.5). In addition, antibodies against chicken actin and tubulin reacted with crude extracts of H . halobium, and a yeast actin DNA probe hybridized with H . halobium DNA digests, indicating similarities between the eucaryotic and archaebacterialproteins beyond the functional level. Thus, Halobacterium halobium, and possibly other halophiles and archaebacteria, may be more suitable for screening antitumor agents than eubacteria. In a broader study, eleven species of the genera Halobacterium, Halococcus, and Halaoarcula were screened for complete growth inhibition by twenty antibiotics at concentrations up to 1500 ~ g / m l .All '~~ species were resistant to ampicillin, carbenicillin, cefotaxime, and D-cycloserine. All species were sensitive to haloquinone and all but Haloarcula were sensitive to novobiocin and rifampicin. Moreover, although native H . mediterranei was sensitive to several antibiotics (D-cycloserine, bacitracin, erythromycin, troleandomycin, clindamycin, josamycin, rifampicin, novobiocin, kanamycin, chloramphenicol, and haloquinone), spontaneous mutants with resistance to bacitracin, chloramphenicol, and josamycin were obtained for this species. These mutants might prove useful as genetic markers. Transformation methods for halophiles have been described by Cline et Centrifuged cells of either H . halobium or H . volcanii were resuspended in a buffered monovalent salt and sucrose solution, and converted to spheroplasts by EDTA chelation of residual divalent cations essential to cell envelop integrity. Transforming DNA was introduced to the spheroplasts by 30% PEG. Cell lysis or a high concentration of DNA resulted in DNA precipitation and poor or failed transformation. The transformed spheroplasts were then diluted in a buffered solution containing Mg2+ and mixed with regeneration medium and plated. Halobacterial spheroplasts regenerated glycoprotein cell envelopes in the presence of sufficient magnesium, making the reestablishmentof normally dividing transformed cell lines relatively straightforward. In addition to transformation of both H . halobium and H . volcanii by linear genomic and plasmid DNA, H . halobium was transfected by a - H phage particles and H . volcanii was transformed by @H phage DNA. These techniques can be applied in strain construction, genetic fine mapping, and studies of genetic relatedness and recombination. Unilayer lipid vesicles are synthesized as model membranes to study mass and energy transport in living cells. Because the Halobacteria contain no zwitterionic or cationic lipids, a re-

cently reported vesicle formation method requiring anionic lipids may be especially applicable. ' 5 5 The procedure consisted of suspending the lipids in chloroform, washing with mild acid, rotoevaporation to remove solvent and form a lipid film, suspension of the film in vesicle forming medium, titration to pH 11.O, and subsequent reduction of pH to 7.55. The method was rapid and produced uniform vesicles of 150 to lo00 nm diameter with polydispersity indices ranging from 0.06 to 0.20. Size and its uniformity were found to depend on the lipids used, the ionic strength of the vesicle forming medium, and timing of the pH adjustment. This technique was shown to be effective with extracts of H . cutirubum as well as with pure phytanyl anionic lipids. Envelope vesicles of Halobacterium halobium were used to demonstrate the use of lipophilic ions (e.g., phosphonium cations such as triphenylmethylphosphonium) to monitor the membrane potential of cells and vesicles. 156 A known quantity of ions was added to a vesicle solution. The ions partitioned across the membrane according to its potential. The external ion concentration was measured, and the potential was determined by a mass balance. One drawback to this technique was the possible binding of ions to the membrane or cell components, adding a third population to the mass balance. H . halobium envelope vesicles were chosen to demonstrate this technique because they contain no internal components and because binding to the membrane in the absence of a lightinduced membrane potential could be measured, assuring the validity of the measurement. It has been noted that the Halococci are less studied than the Halobacteria because the Halococci have cell envelopes resistant to rupture. '51 A new species of Halobacterium, Halobacterium mediterranei, is more resistant to disruption than most Halobacteria, and its lysis has been ~ptimized.'~'The results obtained may be applicable to other halophiles, although no experiments were conducted to test the generality of the optimization. Release of three enzymes was maximized by treatment with 19,200 psi in a French Pressure Cell and by ultrasonic disintegration at 90 to 100 W for 4 min. Digestion with lysozyme (with added EDTA to increase substrate availability), reduction of medium ionic strength, and mechanical damage by shaking with 0.1-mm beads were also effective. However, use of EDTA and reduced ionic strength may not be useful for releasing enzymes which require ions for stability. Breakage by mortar and pestle and by freeze-thaw cycling were least effective. Permeabilization by 2 to 6% toluene and/or 0.6 to 1.2 mMEDTA were also attempted, but effects of the solvent and chelating agent on the enzymes observed made these techniques least desirable.

2. Nutritional Applications Pediococcus halophilus is a lactic acid-producing organism used in soy sauce fermentation. Although the native organism grows optimally in 5% NaC1, it must grow in 18% NaCl for

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Critical Reviews In industrial brewing. Adaptation of the cells to 18% NaCl prior to industrial use minimized the initial lag phase, which can be 10 h or more. A method has been developed for rapid production of salt tolerant cultures. A cross-flow filtration system was used to remove inhibitory levels of the lactate produced, and the salt level in the fresh medium supplied to the continuous system was increased linearly or stepwise from 5% to 18%. The result was approximately 30-fold higher cell concentrations of 8.48 to 9.86 g/l dry cells in approximately 50 h of fermentation as compared to 0.34 g/l dry cells produced by batch adaptation in 18% NaCl for 93 h. The flavoring agents 5'-GMP and 5'-IMP can be produced by the enzymatic degradation of RNA. Micrococcus varians halophilus produced extracellular nuclease H and 5'-nucleotidase in 12% NaCl culture medium. Nuclease H hydrolyzed RNA to produce the desired 5'-GMP with a temperature optimurn of 5 5 T , whereas the 5'-nucleotidase dephosphorylated 5'-GMP to the nucleoside base at an optimum temperature of 20°C. Thus, incubation of M . vuriuns halophilus culture supernatant with 5 g/l salt-extracted yeast RNA for 24 h at 60°C resulted in 72% degradation of the RNA with only 1.5% of the resulting nucleotides degraded to nucleosides. Although the process required specially extracted RNA rather than commercially available RNA to avoid salt precipitation of the substrate, the high (12%) salt content minimized the possibility of Bacillus contamination, which is common in the food industry.'60 Organisms capable of growing in brackish water can be a valuable source of nutrition for humans and animals, especially in areas where fresh water is less plentiful. Spirulina platensis, a multicellular cyanobacterium found growing in high-salt alkaline lakes in Africa, is traditionally harvested and dried for human consumption. Extracts of the halotolerant algae Dunuliellu grown in salty ponds have been tested as a protein supplement in bread.'62 Loaves with acceptable rise characteristics were obtained only with water-insoluble algae fractions. Limitations for practical use included the imparting of green color to the bread by chlorophyll and the high nucleic acid to protein ratio usually associated with unicellular organisms. The water-soluble algae fractions were not acceptable because they contained too much salt. Extracts of the halotolerant thermophilic blue-green alga Synechococcus elongutus can be used to promote growth of human cell lines in serum-free medium.'6' The extract was more effective than a mixture of insulin, transferrin, ethanolamine, and selenite (ITES) in promoting growth of a human myeloma line (RPMI 8226) and two human B-lymphoblastoid lines (HMY-2 and HO-323). Neither ITES nor S. elongatus extract significantly increased growth of human acute T-lymphatic leukemia line HSB-2, and ITES was slightly more effective in promoting growth of human acute T-lymphatic leukemia line Molt-4. The growth promoting activity of S. elongatus was very stable, losing less than 20% of its growth enhancing ca-

340

pacity after 8 months storage at 4°C and exhibiting no activity loss after incubation for 20 min at 100°C. Use of microalgae such as Synechococcus as a source of growth factors for cell culture was significant because large quantities of fresh algae can be produced more easily and inexpensively than tissues and organs of animals, the traditional source of growth factors.

3. Miscellaneous Uses Photochemically active purple membranes of Hulabacterium halobium have been dispersed in a polymer matrix to form biochrom films for the real time recording of optical information. The photocenters of the purple membranes, bacteriorodopsin, were asymmetric and absorbed light according to their orientation in the photon beam. Because the bacteriorodopsin molecules were randomly oriented in the film, the film was initially isotropic. When polarized light excited the film, those molecules aligned parallel to the light were bleached, and those perpendicular to the beam remained unexcited, creating an anisotropic film. This property can be exploited to utilize Biochrom films for real time holography, as demonstrated by Burykin et al. lh4 Bio-chrom film demonstrated a diffraction efficiency of 0 to 1.5%, sensitivity of 10 mW/cm2, spatial resolution greater than 5000 lines/mm, and a cycle lifetime of a few seconds.

V. CONCLUSION As demonstrated by the examples cited in this review, extremophiles can be used in a wide variety of interesting and useful applications. These hardy organisms and their components,which can withstand unusually harsh conditions, expand the range of biotechnology. Furthermore, as research in this dynamic field progresses, mechanisms of extremophily will be better understood and greater application of extremophiles will surely be seen.

ACKNOWLEDGMENTS The authors are grateful for the financial support of the Office of Naval Research (N00014-89-5-1884) and the National Science Foundation (CBT-8696159). J. M. Ludlow is a National Science Foundation Fellow.

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Biotechnology 167. Rudd, T., Hicks, S. J., and Lester, J. N., Comparison of the treatment of a synthetic meat waste by mesophilic and thermophilic anaerobic fluidized bed reactors, Environ. Technol. Lett., 6 , 209, 1985. 168. Schraa, G. and Jewell, W. J., High rate conversions of soluble organics with a thermophilic anaerobic attached film expanded bed, J. WPCF, 56, 226, 1984. 169. Kunugi, S., Fukuda, M., and Ise, N., Pressure dependence of trypsin-catalyzed hydrolyses of specific substrates, Biochim. Biophys. Acta, 704, 107, 1982. 170. Fukuda, M., Kunugi, S., and Ise, N., Pressure dependence of carboxypeptidase A action, Bull. Chem. SOC. Jpn., 56, 3303, 1983. 172. Fukuda, M. and Kunugi, S., Pressure dependence of thermolysin catalysis, Eur. J. Biochem.. 142, 565, 1984. 172. Fukuda, M. and Kunigi, S., Mechanism of carboxypeptidase-Y-catalyzed reaction deduced from a pressure-dependence study, Eur. J . Biochem., 149, 657, 1985. 173. Fukuda, M., Shima, H., and Kunugi, S., Kinetic study of carboxypeptidase P-catalyzed reaction: pressure and temperature dependence of kinetic parameters, J. Biochem, 98, 517, 1985. 174. Fukuda, M. and Kunugi, S., Kinetic studies of wheat carboxypeptidase-catalyzed reaction: differences in pressure and temperature dependence of peptidase and esterase activities, J. Biochem. 101, 233, 1987. 175. Guyer, R. L. and Koshland, D. E., Jr., The molecule of the year, Science, 246, 1543, 1989.

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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...
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