Mierob Eeol (1986) 12:111-119
MICROBIAL ECOLOGY 9 1986 Springer-Verlag
The Biotechnological Future for Newly Described, Extremely Thermophilic Bacteria Jody W. Deming Chesapeake Bay Institute, The Johns Hopkins University, 4800 Atwell Road, Shady Side, Maryland 20764 USA
Abstract. Recent explorations of aquatic volcanic environments have led to the isolation of novel microorganisms with optimal growth temperatures of 80~ or higher. Expectations of equally novel, highly thermostable biocatalysts and specialty chemicals from such organisms remain high but must be tempered with the laboratory realities of manipulating unusual bacteria whose growth characteristics are as yet poorly defined. Advancing the biotechnological future of "'super-thermophiles" will require new cultivation methods, including the use of highly thermostable gels and pressurized bioreactors.
Introduction One of the greatest attractions of the rapidly emerging field of biotechnology is the promise of "doing something new, not just something better: opening new markets or sources of supply, not just refining or improving old ones" [12]. The key to this "something new" rests, inevitably, with the pool of genes available to the biotechnologist for directed manipulation, whether those genes are constructed in vitro or extant in living organisms. In the latter case, exPansion of the available genetic pool seems assured by continued searches for microbial life in novel and extreme environments. In the last 4 years alone, at least a dozen new microorganisms, containing genetic material as yet untapped by the biotechnologist, have been discovered in environments characterized by superheated temperatures (> 100~ and Perhaps most vividly described as primeval [4, 11, 35]. These are the aquatic environments influenced by magmatic or volcanic activity: fumaroles in the craters of active volcanoes like Mt. St. Helens of Washington State; marine Solfatara fields of volcanic islands like Iceland and Vulcano Island, Italy; and remote submarine hydrothermal vents at tectonically active and deep (1,5004,500 m) sites along the East Pacific seafloor. These seemingly hostile environments have more in common than the influence of geothermal activity; they each serve as habitats for thermophilic bacteria that grow optimally, at least to the extent tested in the laboratory, at temperatures above 80~ (Table I).
112
J.W. Deming
Table 1. Newly described, extremely thermophilic bacteria with optimal growth temperatures exceeding 80~ Cardinal temperatures (*C) at < 2 arm Organism
Source
Methanothermus Iceland, hot spring fervidus Methanococcus EastPacific Rise, jannaschii "smoker" sedi-
ment Vulcano,Italy, hot seawater Iceland and Mt. spp. Lassen, Cat, hot springs Sulfur-respiring, East Pacific Rise, heterohot "'smoker" eftrophic atfluent chaebacterium ChemolithoMt. St. Helens, trophic arWash, fumaroles chaebacteria Pyrodictium oc- Vulcano, Italy, hot culture seafloor "Black smoker" East Pacific Rise, bacteria Juan de Fuca Ridge, hot "'smoker" effluents Thermodiscus maritimus Thermoproteus
MiniMaximal Optimal real
Reference
65
83
97
[36]
?
100
120?
[3, 5, 6, 14, 15]
:
[17,43] Belkin & Jannasch (Abstr, Annu Meet Am Soe Microbiol 1985, N71, p 224) [7] Baross (unpub data)
Each updated review o f bacteria from thermal e n v i r o n m e n t s includes, invariably, new isolates with cardinal growth temperatures exceeding the maxi m u m limits published, in some cases only m o n t h s earlier (compare lists c o m piled by Ljungdahl [23], Sonnleitner [29], and Sonnleitner and Fiechter [30]). This pace o f discovery has forced a constant reevaluation o f such terms as thermophilic, extremely thermophilic, and caldoactive. Perhaps inevitably, this pace has also exceeded the rate at which individual strains can be evaluated, either physiologically, genetically, or industrially. Thus, a review o f the newest " s u p e r - t h e r m o p h i l e s " m u s t necessarily be brief and speculative. Nevertheless, by drawing on existing knowledge o f m o r e c o n v e n t i o n a l thermophiles, as well as general characteristics c o m m o n to the organisms and their e n v i r o n m e n t a l sources o f origin, it is possible to predict specific areas o f research that m a y advance the biotechnological application o f the m o s t recently discovered, extremely thermophilic bacteria. A n y speculation a b o u t industrial returns from the isolation o f highly thermostable bacteria must be tempered by the realities o f manipulating new a n d unusual microorganisms in the laboratory [ 12, 30]. Intensive efforts to produce relatively well-described thermophiles or their b y - p r o d u c t s at industrially significant levels have repeatedly met with unexpected biological and technical
Future for Extreme Thermophiles
1 13
roadblocks [29-33]. Some of the disappointments, especially for bacteria having a maximal growth temperature above 75~ or higher [23], have included observations of low growth rate, low cell or product yield, incomplete substrate utilization, product inhibition, strain instability, culture collapse, contamination problems, biological inhibition by reactor materials, and reactor wear [ 10, 19, 25, 29-33]. Fortunately, investigators have remained undaunted by these problems and continue to find ingenious solutions, many of which are rooted in an appreciation for microbial interactions and ecology. For example, Ljungdahl and colleagues succeeded in obtaining significantly higher yields of ethanol from cellulose by combining the complementary metabolic functions of two extremely thermophilic microorganisms, Clostridium thermocellum and Therrnoanaerobacter ethanolicus, in mixed culture [24, 25]. At this time, the overall balance of laboratory realities, scientific ingenuity, and the ever-present promise of discovering novel, highly thermostable biocatalysts and unusual biochemical transformations still favors an important industrial future for microorganisms derived from superheated aquatic environments.
Some Characteristics of the Organisms and Their Environments Approximately 10-20 new strains and mixed cultures of extremely thermophilic bacteria, defined for purposes of this discussion as having optimal growth temperatures exceeding 80~ have been reported in the last 4 years. A partial listing of these organisms, along with known habitats and minimal, optimal, and maximal growth temperatures, is provided in Table 1. These microorganisms share a number of characteristics, in addition to their volcanic environments of origin and extreme cardinal temperatures, that will bear significantly on future industrial applications: 1) they are aquatic, nonsporulating anaerobes; 2) with one exception, they are facultative, if not obligate, chemolithotrophs that derive their carbon, nitrogen, and energy from inorganic compounds and gases (e.g., CO2, NH4, and H2 or reduced forms of sulfur and metals); 3) they are confirmed or, in some cases, inferred members of the archaebacteria [40] with representative methanogens (Methanothermusfervidus and Methanococcusjannaschii) and sulfur-metabolizing species (all others listed); and 4) where tested, they have been cultured at superheated temperatures (> 100~ using hyperbaric or hydrostatic pressures to prevent boiling [2, 3, 14, 15, 17, 19, 35]. Features of the environments from which these extremely thermophilic bacteria were obtained parallel the above-listed microbial characteristics: 1) they are aquatic and, where temperatures approach or exceed 100~ anaerobic; 2) they are rich in the inorganic compounds, gases, and metals of biological relevance; 3) methane and hydrogen sulfide are often found in saturating concentrations; and 4) as aquatic environments, they are subject to hydrostatic pressures (which increase 1 arm per 10-m depth increment) and, therefore, to Zones of superheated liquid water. In fact, these environmental parameters Could have served (and in most cases did serve) to predict the types of microOrganisms subsequently cultured from the samples collected. From this perSpective, it may be useful to consider other features of these environments, Particulady submarine hydrothermal vents, that may portend the physiological
114
J.W. Deming
nature ofthermophiles yet to be isolated and direct the development of methods for culturing them. Submarine hydrothermal vents, emitting superheated fluids from sulfide mounds or chimneys at depths of 1,500-4,500 m in the Pacific, have been named "smokers" because of the heavy and often black precipitates (thus, "black smokers") that form when hot (up to 350~ highly-reduced hydrothermal effluents mix with cold (
MICROBIAL ECOLOGY 9 1986 Springer-Verlag
The Biotechnological Future for Newly Described, Extremely Thermophilic Bacteria Jody W. Deming Chesapeake Bay Institute, The Johns Hopkins University, 4800 Atwell Road, Shady Side, Maryland 20764 USA
Abstract. Recent explorations of aquatic volcanic environments have led to the isolation of novel microorganisms with optimal growth temperatures of 80~ or higher. Expectations of equally novel, highly thermostable biocatalysts and specialty chemicals from such organisms remain high but must be tempered with the laboratory realities of manipulating unusual bacteria whose growth characteristics are as yet poorly defined. Advancing the biotechnological future of "'super-thermophiles" will require new cultivation methods, including the use of highly thermostable gels and pressurized bioreactors.
Introduction One of the greatest attractions of the rapidly emerging field of biotechnology is the promise of "doing something new, not just something better: opening new markets or sources of supply, not just refining or improving old ones" [12]. The key to this "something new" rests, inevitably, with the pool of genes available to the biotechnologist for directed manipulation, whether those genes are constructed in vitro or extant in living organisms. In the latter case, exPansion of the available genetic pool seems assured by continued searches for microbial life in novel and extreme environments. In the last 4 years alone, at least a dozen new microorganisms, containing genetic material as yet untapped by the biotechnologist, have been discovered in environments characterized by superheated temperatures (> 100~ and Perhaps most vividly described as primeval [4, 11, 35]. These are the aquatic environments influenced by magmatic or volcanic activity: fumaroles in the craters of active volcanoes like Mt. St. Helens of Washington State; marine Solfatara fields of volcanic islands like Iceland and Vulcano Island, Italy; and remote submarine hydrothermal vents at tectonically active and deep (1,5004,500 m) sites along the East Pacific seafloor. These seemingly hostile environments have more in common than the influence of geothermal activity; they each serve as habitats for thermophilic bacteria that grow optimally, at least to the extent tested in the laboratory, at temperatures above 80~ (Table I).
112
J.W. Deming
Table 1. Newly described, extremely thermophilic bacteria with optimal growth temperatures exceeding 80~ Cardinal temperatures (*C) at < 2 arm Organism
Source
Methanothermus Iceland, hot spring fervidus Methanococcus EastPacific Rise, jannaschii "smoker" sedi-
ment Vulcano,Italy, hot seawater Iceland and Mt. spp. Lassen, Cat, hot springs Sulfur-respiring, East Pacific Rise, heterohot "'smoker" eftrophic atfluent chaebacterium ChemolithoMt. St. Helens, trophic arWash, fumaroles chaebacteria Pyrodictium oc- Vulcano, Italy, hot culture seafloor "Black smoker" East Pacific Rise, bacteria Juan de Fuca Ridge, hot "'smoker" effluents Thermodiscus maritimus Thermoproteus
MiniMaximal Optimal real
Reference
65
83
97
[36]
?
100
120?
[3, 5, 6, 14, 15]
:
[17,43] Belkin & Jannasch (Abstr, Annu Meet Am Soe Microbiol 1985, N71, p 224) [7] Baross (unpub data)
Each updated review o f bacteria from thermal e n v i r o n m e n t s includes, invariably, new isolates with cardinal growth temperatures exceeding the maxi m u m limits published, in some cases only m o n t h s earlier (compare lists c o m piled by Ljungdahl [23], Sonnleitner [29], and Sonnleitner and Fiechter [30]). This pace o f discovery has forced a constant reevaluation o f such terms as thermophilic, extremely thermophilic, and caldoactive. Perhaps inevitably, this pace has also exceeded the rate at which individual strains can be evaluated, either physiologically, genetically, or industrially. Thus, a review o f the newest " s u p e r - t h e r m o p h i l e s " m u s t necessarily be brief and speculative. Nevertheless, by drawing on existing knowledge o f m o r e c o n v e n t i o n a l thermophiles, as well as general characteristics c o m m o n to the organisms and their e n v i r o n m e n t a l sources o f origin, it is possible to predict specific areas o f research that m a y advance the biotechnological application o f the m o s t recently discovered, extremely thermophilic bacteria. A n y speculation a b o u t industrial returns from the isolation o f highly thermostable bacteria must be tempered by the realities o f manipulating new a n d unusual microorganisms in the laboratory [ 12, 30]. Intensive efforts to produce relatively well-described thermophiles or their b y - p r o d u c t s at industrially significant levels have repeatedly met with unexpected biological and technical
Future for Extreme Thermophiles
1 13
roadblocks [29-33]. Some of the disappointments, especially for bacteria having a maximal growth temperature above 75~ or higher [23], have included observations of low growth rate, low cell or product yield, incomplete substrate utilization, product inhibition, strain instability, culture collapse, contamination problems, biological inhibition by reactor materials, and reactor wear [ 10, 19, 25, 29-33]. Fortunately, investigators have remained undaunted by these problems and continue to find ingenious solutions, many of which are rooted in an appreciation for microbial interactions and ecology. For example, Ljungdahl and colleagues succeeded in obtaining significantly higher yields of ethanol from cellulose by combining the complementary metabolic functions of two extremely thermophilic microorganisms, Clostridium thermocellum and Therrnoanaerobacter ethanolicus, in mixed culture [24, 25]. At this time, the overall balance of laboratory realities, scientific ingenuity, and the ever-present promise of discovering novel, highly thermostable biocatalysts and unusual biochemical transformations still favors an important industrial future for microorganisms derived from superheated aquatic environments.
Some Characteristics of the Organisms and Their Environments Approximately 10-20 new strains and mixed cultures of extremely thermophilic bacteria, defined for purposes of this discussion as having optimal growth temperatures exceeding 80~ have been reported in the last 4 years. A partial listing of these organisms, along with known habitats and minimal, optimal, and maximal growth temperatures, is provided in Table 1. These microorganisms share a number of characteristics, in addition to their volcanic environments of origin and extreme cardinal temperatures, that will bear significantly on future industrial applications: 1) they are aquatic, nonsporulating anaerobes; 2) with one exception, they are facultative, if not obligate, chemolithotrophs that derive their carbon, nitrogen, and energy from inorganic compounds and gases (e.g., CO2, NH4, and H2 or reduced forms of sulfur and metals); 3) they are confirmed or, in some cases, inferred members of the archaebacteria [40] with representative methanogens (Methanothermusfervidus and Methanococcusjannaschii) and sulfur-metabolizing species (all others listed); and 4) where tested, they have been cultured at superheated temperatures (> 100~ using hyperbaric or hydrostatic pressures to prevent boiling [2, 3, 14, 15, 17, 19, 35]. Features of the environments from which these extremely thermophilic bacteria were obtained parallel the above-listed microbial characteristics: 1) they are aquatic and, where temperatures approach or exceed 100~ anaerobic; 2) they are rich in the inorganic compounds, gases, and metals of biological relevance; 3) methane and hydrogen sulfide are often found in saturating concentrations; and 4) as aquatic environments, they are subject to hydrostatic pressures (which increase 1 arm per 10-m depth increment) and, therefore, to Zones of superheated liquid water. In fact, these environmental parameters Could have served (and in most cases did serve) to predict the types of microOrganisms subsequently cultured from the samples collected. From this perSpective, it may be useful to consider other features of these environments, Particulady submarine hydrothermal vents, that may portend the physiological
114
J.W. Deming
nature ofthermophiles yet to be isolated and direct the development of methods for culturing them. Submarine hydrothermal vents, emitting superheated fluids from sulfide mounds or chimneys at depths of 1,500-4,500 m in the Pacific, have been named "smokers" because of the heavy and often black precipitates (thus, "black smokers") that form when hot (up to 350~ highly-reduced hydrothermal effluents mix with cold (
