BACrERIOLOGICAL REVIEWS, June 1975, p. 144-167 Copyright 0 1975 American Society for Microbiology
Vol. 39, No. 2 Printed in U.S.A.
Psychrophilic Bacteria1 RICHARD Y. MORITA
Department of Microbiology and School of Oceanography, Oregon State University, Corvallis, Oregon 97331 INTRODUCTION
.......................................................
BACKGROUND INFORMATION
Definition used to describe psychrophiles
........................................
ISOLATION OF PSYCHROPHILIC BACTERIA
ECOLOGICAL DISTRIBUTION OF PSYCHROPHILES
OF
PSYCHROPHILIC
.........................
149
Temperature characteristic of growth
Proteins
on
148
BACTERIA.150
PHYSIOLOGY OF PSYCHROPHILES Effect of temperature
146 147
ECOLOGICAL SIGNIFICANCE OF PSYCHROPHILES
GROWTH
144
................................................
153 ...........................................
respiration and viability
153
.154
.......................................................
155
156 Thermally induced leakage 157 Thermally induced lysis .158 Temperature effects of permeability 158 Temperature effects on macromolecular synthesis Ecological factors affecting psychrophiles.159 .....................................................
.......................................................
...............................
CONCLUSION
LITERATURE CITED
161
..............................................
.
INTRODUCTION For the purposes of this review, psychrophiles are defined as organisms having an optimal temperature for growth at about 15 C or lower, a maximal temperature for growth at about 20 C, and a minimal temperature for growth at 0 C or below. In order to avoid as much confusion as possible concerning the term "psychrophile" in this review, the term "psychrotrophic" will be used for organisms that do not meet the definition as stated above, even though the cited author(s) may have referred to them as psychrophiles. Likewise, the term "psychrophile" will include those organisms previously referred to as obligate psychrophiles. Yeasts that meet the above definition are known to exist but they will not be discussed in this review. The main reasons for employing the above definition are to eliminate future confusion and to adhere to the etymological significance of the term psychrophile. Data are rapidly accumulating on the existence of bacteria that meet the above definition. The optimal temperature listed in this definition adheres to that given in the Dictionary for Microbiology (88). Because of the recent isolation of psychrophiles and the small number of investigators dealing with this unique group of bacteria, there are many areas not yet investigated. Also, because of the restrictive definition employed in this review there is a paucity of data from which
162
to draw. Nevertheless the major objectives of this review are to make microbiologists aware that "psychrophiles are psychrophiles" and to eliminate confusion in the literature concerning the term. The general effects of temperature on microbial physiology and growth are well discussed in many textbooks on microbiology and will not be repeated in this review. Hopefully this review will stimulate others to use the terms psychrophile and psychrotrophs as defined in this review. Although there may never be a complete agreement on these terms, a start must be made to end the confusion about the lower end of the thermal spectrum of microbial life. The terms applied to the various thermal groups are useful in microbiology, but not when confusion about them exists.
' Published as technical paper no. 4006, Oregon Agricultural Experiment Station. 144
BACKGROUND INFORMATION Textbooks in general microbiology for many years have classified microorganisms into various thermal groups based on their cardinal temperatures. Of these groups, the cold-loving bacteria are usually referred to as facultative psychrophiles and psychrophiles. However, the terms used most often in the published papers to describe these groups are psychrophile and obligate psychrophile. Furthermore, textbooks generally list the optimum growth temperature for psychrophiles as 15 C. Yet, to most microbiologists, the term "psychrophile" does not refer to the truly cold-loving bacteria but those that have the ability to grow at low temperatures.
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(The early literature would say "grow rapidly," meaning growth in at least a week.) The term psychrophile today is still misused, and the misnomer continues to be perpetuated in the literature. That certain organisms can overlap any two adjacent thermal groups is well recognized. For those mesophilic organisms that have the capability to grow at 0 C, the term "psychrotolerant" (98) and "psychrotrophic" are perhaps the best terms. The latter appears to be used more frequently in the literature today. There are many review articles dealing with "psychrophilic" bacteria (30, 83, 84, 85, 86, 159, 173, 187); however, they cover the psychrotrophic bacteria, not the psychrophilic bacteria. There is a common misconception concerning the maximum, minimum, and optimum growth temperatures for psychrophilic bacteria. The reports in the literature dealing with psychrophilic bacteria refer to organisms that are coldtolerant rather than cold-loving. This was especially true before the 1960's. Much of the confusion in the early literature arose from the lack of adequate data to support the concept of psychrophily. The early investigators concentrated on the ability to grow at or near 0 C. This ability to grow at 0 C was first reported by Forster (59) in 1887. The organisms were isolated from fish preserved by cold temperatures and were phosphorescent (bioluminetent). Later Forster (60)
145
reported the isolation of other bacteria from various environments (natural water, foods, surface and in intestines of freshwater fish, and especially in sea water and ocean fish) that were capable of growth at 0 C. From his studies in the Antarctic, Ekelof (51) proposed the existence of psychrophiles. The main deterrent to obtaining adequate data in the early investigations was probably the lack of refrigerated incubators. In addition, the investigators were unaware of the thermolability of psychrophilic organisms at laboratory temperatures. Because of this, early investigators (as listed in Table 1) did not determine the cardinal temperatures for their isolates. This was the beginning of the confusion concerning the cardinal temperatures for psychrophilic bacteria. In 1902 Schmidt-Nielsen (161) grew microorganisms at 0 C which he termed "psychrophiles." A year later Muller (138) objected to the term since the organisms described actually grew well at higher temperatures. The cultures isolated by early microbiologists actually grew better in the mesophilic temperature range (Table 1). As'a result of this inconsistency, various terms were coined by early investigators to describe organisms which had the ability to grow at 0 C (Table 2). A survey of the literature for the cardinal temperatures of psychrophiles was made by Morita (121). Unfortunately, the psychrophiles described before 1960's were not
TABLE 1. Early investigators reporting bacterial growth at 0 C or lower Source material or organism
Investigator and year
Forster (1887, 1892)
Fischer (1888, 1893) Conradi and Vogt (1901) Baur (1902) Brandt (1902) Schmidt-Nielsen (1902)
Miller (1903) Feitel (1903) Tsiklinsky (1908) Richardson (1908) Pennington (1908) Ravenel et al. (1910) Conn (1910,1912, and 1914) Brown and Smith (1912) Reed and Reynolds (1916) Vanderleck (1917 and 1918) Vass (1919) Fay and Olson (1924) Bardach (1924) Rubentschik (1925)
Fish, natural waters, foods, wastes, rubbish, soil, surface and intestines of fish Harbor water and soils, certain pathogenic bacteria "proteus bacillus" Bacterium lobatum Bacterium lobatum Bacillus aquatile fluorescense non-liquefaciens, B. granulosum, B. paracoli gasoformans anindolicum, B. radiatum, B. tarde fluorescens, B. pestis, and B. proteus fluorescens. Sausage meat, fish, intestinal contents of fish, milk, vegetables, meal, garden soil and muck Dentrifying bacteria from deep sea water Antartic fish Nonproteolytic bacteria on frozen meat Milk Milk Frozen soils Frozen soils Milk Frozen soils Frozen soils Ice cream
Sewage, sludge and salt water Urbacillus psychrocartericus, Urosarcina psychrocartericus
146
MORITA
BACTERIOL. REV.
TABLE 2. Terms used to describe bacteria capable of at 0 C but not at 32 C, whereas the latter could growing at or below 0 C grow at 0 C and 32 C. Many microgiologists Term
Reference
Glaciale Bakterien Rhigophile Psychrophile Psychrotolerant Psychrocartericus Psychrobe
Forster (1892) Forster (1892) Schmidt-Nielsen (1902) Kruse (1910) Rubentschik (1925) Horowitz-Wlassowa and
Thermophobic bacteria
Edsall and Wetterlow
Cryophile
Edsall and Wetterlow
Facultative psychrophile Obligate psychrophile Psychrotrophic
Hucker (1954) Hucker (1954) Eddy (1960)
Grinberg (1933) (1947)
(1947)
truly psychrophilic (with one exception [177]). After surveying the literature, Berry and Magoon (13) stated that "the contention that a truly cold-loving or psychrophilic group of microorganisms exists, appears unwarranted by available evidence. While organisms recorded as growing at 0 C or below may with propriety be regarded as forming a cold-tolerant group, their temperature requirements for best growth definitely place them in the mesophilic class." Other workers (69, 82, 83, 84, 85, 104, 105) also concluded that there was a lack of adequate data to substantiate the existence of psychrophiles. In 1963, Ingram (86) stated that the term psychrophile was a misnomer. Since SchmidtNielsen (161) first used the term "psychrophile," it has been employed indiscriminately so that there is much confusion in the literature. Hagen et al. (71) point out that the term psychrophile has been used and defined in many ways and most of the definitions ignore the etymological significance of the word. Definitions used to describe psychrophiles. Schmidt-Nielsen (161) defined psychrophiles as organisms which are not only able to survive but also multiply at 0 C. The term "psychrophile," according to Horowitz-Wlassowa and Grinberg (81), should be used for the commonly isolated bacteria that could grow at 0 C but actually grow better at higher temperatures; and the term "psychrobe" should be used for the true psychrophile if they existed. Without adequate data in the literature for the existence of psychrophiles, the Dictionary for Microbiology (88), published in 1957, defined psychrophiles as those bacteria having an optimum growth temperature of 15 C or lower. Organisms that grew at 0 C were separated into two categories, obligate and facultative, by Hucker (82). The former were capable of growth
today consider psychrophiles to be organisms that more or less fit the facultative category. Ingraham (83) suggested that psychrophiles be defined not by their maximum or optimum growth temperature but whether they grow rapidly at 0 C. The term "rapidly" generally meant 1 or 2 weeks, a period much too long for psychrophiles. Later Ingraham and Stokes (85) redefined the term to refer to bacteria that grow well at 0 C within 2 weeks, disregarding the cardinal temperatures. The term "grow well" referred to a visable colony or turbidity to the naked eye. They also suggested that the obligate psychrophile be defined as those bacteria that grew rapidly at 0 C but also grew most rapidly at temperatures below 20 C. The facultative psychrophile was capable of growth at 0 C. Microorganisms capable of growing at 5 C and below, according to Eddy (48), should be classified as psychrotrophic regardless of the optimum temperature and that the term psychrophile should be used only when a low optimum temperature is implied. There are many microbiologists who employ the term psychrotrophic (e.g., 47, 102, 114, 137, 186, 189), and this term is gaining wider acceptance among food and dairy microbiologists. Ingram (86) gives the best reasons published in the literature as to why the term psychrotroph should be employed for most of the organisms listed in the literature as psychrophiles. Baxter and Gibbons (10) defined psychrophiles as organisms with a temperature optimum of 20 C or below. Rose (158) applied the term to organisms having an optimum for growth at 25 C or below without reference to growth at or near 0 C and defined obligate psychrophiles as having a maximum growth temperature of around 30 C. The definition employed most often in the literature is the one proposed by Stokes (173) in which he defined psychrophiles as organisms which grow at 0 C so that they can be detected visually within 1 week; facultative psychrophiles have an optimum temperature of 20 C or higher, whereas the optimum growth temperature of obligate psychrophiles is 20 C or lower. The more recent definitions for psychrophile by Stokes (173) and Rose (158) are discussed by Ingram (87), who points out the inadequacies of their definitions. Ingram (87) also states that the term psychrophile has been misapplied to organisms which grow at 20 but not at 30 C and that there is a preponderant group of organisms, with optimum temperatures between 20 and 30 C, that are not "facultative psychrophiles" in the true meaning of the term. This latter group
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cannot be named satisfactorily (87). For the best discussion of the inconsistency in the logic of nomenclature dealing with psychrophiles, Ingram (87) should be consulted. Optimum growth temperature, ability to grow at low temperature, method of enumeration, and criteria which are independent of the temperature have been used to define psychrophiles (187). Different definitions are held by various areas of microbiology (e.g., dairy and food microbiology). All this adds to the confusion in the literature and to the definition of the term "psychrophile." For more discussion on the subject, Berry and Magoon (13), Hucker (82), Ingraham and Stokes (85), Witter (187), Ingraham (84), Ingram (87), and Morita (121) should be consulted. If the definition of psychrophile is to be changed in the future, then it is my opinion that the cardinal temperatures will be lower than those that I have given in my definition.
ISOLATION OF PSYCHROPHILIC BACTERIA Tsiklinsky in 1908 (177) reported the existence of a psychrophile. The psychrophile was isolated from the excrement of a fish (environmental temperature was - 1.5 C) and had a growth temperature range from 0 to 20 C, with an optimal temperature for growth at 16 C. It appears that the isolate went into limbo and the significance of this isolate (probably a vibrio according to her description) has been neglected by microbiologists. In 1948, Borg (15) isolated the etiological agent for "cold water" disease of silver salmon. The organism was assigned the name Cytophaga psychrophila and had an optimum temperature between 15 and 20 C. This organism, the first psychrophile to be well characterized, fits the definition of psychrophile better than any organisms described prior to 1963, but unfortunately Borg's thesis went unnoticed. In 1964 Eimjhellen (71), Morita and Haight (135), and Sieburth (164) reported the existence of psychrophilic bacteria. Since these reports, others have reported the existence of psychrophilic bacteria. All the isolates were from the marine environment. Eimjhellen isolated his organism from flounder eggs. Because the organism produces a red pigment, it was tentatively designated as a Serratia sp. but was later classified as Vibrio psychroerythrus (41). The red pigment of V. psychroerythrus is different from five other prodigiosin-like compounds found in other organisms (39). Morita and Haight (135) isolated their organism from oceanic waters 125 miles off the Oregon coast (1,200 meters, 3.24 C) and was identified as
147
Vibrio marinus, MP-1 (32). Sieburth's isolates from Narragansett Bay, R.I., were Arthrobacter spp.
The two main reasons why microbiologists in the past were not successful in isolating psychrophilic bacteria are probably the improper selection of source material and the laboratory procedure for handling material. The source material must be selected carefully. The past history of the source material is critical since it must never reach warm temperature for any extended period of time. For example, if sufficient radiant energy should fall on the source material it might be adequate to thermally inactivate the psychrophilic bacteria, if present. Hence the environment from which the source material is taken must remain cold constantly. Llano (103) recorded a change from -15 to 27.8 C on rock surfaces in the Horlick Mountains (40 from the geographical South Pole) within 3 h due to solar radiation even though the air temperature was around 0 C. Boylen and Brock (17) were not able to demonstrate the development of a true psychrophilic flora in Lake Wingra sediments which had a temperature of 4 C for only 3 months of the year. Brock et al. (21), using proper techniques for the isolation of psychrophiles, were unable to obtain a psychrophile from the cold springs in caves located in southern Indiana. The above only indicates that the past history of the environment from which sample material is taken is very important. Oceanic water has been cold for eons of time, and as a result it is easy to isolate psychrophiles in waters below the thermocline in the ocean as well as in arctic and antarctic waters. The isolation procedure for psychrophilic bacterial cells calls for the usual microbial methods except that the source material, pipettes, media, etc., are kept cold at all times (22, 121, 185). The above emphasizes the abnormal thermal lability of the psychrophilic bacteria. The pour plate technique employing agar medium cannot be recommended. It was shown that most marine forms are thermosensitive to the plating temperature of agar (192). The psychrophile, V. marinus MP-1, can be rendered nonviable when exposed to melted agar at 42 C for 1 min (135). The enrichment technique, with the desired medium, is recommended before trying to isolate pure cultures. The inoculated enrichment medium should be incubated between 0 and 5 C before proceeding with the isolation procedure. Stock cultures of psychrophiles should be transferred frequently, and at no time should the cultures be exposed to elevated temperatures for any extended period of time. Frequent transfer is necessary since the temperature of
148
MORITA
the laboratory refrigerator is nearly optimal for the growth of some psychrophiles. Psychrophiles are not rare in permanently cold environments and can be isolated easily if the correct application of the above procedures is followed.
ECOLOGICAL DISTRIBUTION OF PSYCHROPHILES Most biologists tend to neglect the fact that the dominant environment of the biosphere is cold. The polar regions and the oceans are 14 and 71% of the earth's surface, respectively. More than 90% (by volume) of the oceans is 5 C or colder. The oceans are a natural habitat for psychrophilic bacteria. Because of this situation it is relatively easy to isolate psychrophilic bacteria from below the thermocline of the oceans, in salt wedges (cold oceanic water intrusion) of bays as well as both polar regions. Psychrophilic bacteria have been isolated off the Oregon coast as well as the Arctic and northern Pacific waters (130). For instance, 65 miles off the Oregon coast 79 and 27 psychrophiles were found per 100 ml of sea water obtained from depths of 350 and 2,800 meters, respectively. Freshwater psychrophilic bacteria are readily isolated adjacent to melting glaciers in Alaska (Burton, personal communication). Sieburth (164, 166) examined various organisms isolated at 0, 18, and 36 C from waters of Narragansett Bay, R. I., and found that there definitely were microorganisms belonging to the psychrophilic group. These psychrophiles were only obtained on plates incubated at 0 C upon initial isolation and only when the water temperature was below 10 C. Many of the isolates had optimum temperatures for growth around 10 C. They were tentatively identified as Arthrobacter spp., Achromobacter spp., Flavobacterium spp., Vibrio spp., and Pseudomonas spp. A seasonal selection for the various thermal types was also observed so that the minimal, optimal, and maximal growth temperature of the dominant microbial flora continuously change as a result of the continual seasonal changes in water temperature. For example, certain isolates made at 0 C in the spring failed to grow above 16 C whereas isolates made during the summer could grow above 16 C. According to Sieburth (164), the life cycle of Arthrobacter spp. appear to be associated with different temperatures (i.e., life cycle was a function of water temperature). From the North Sea, Harder and Veldkamp (75) isolated 69 strains of bacteria capable of growth at low temperatures. Ten of these orga-
BACTERIOL. REV.
nisms had a maximum growth temperature below 20 C, six of which were Pseudomonas, two were Vibrio, and two were Spirillum. Gounot (64, 65, 66) and Moiround and Gounot (120) investigated the microbial population in sediments and water below arctic glaciers, in arctic caves, and other cold environments. In her studies, she isolated an Arthrobacter sp. which had an optimum temperature for growth at 13 C. It also grew well at -5 and 18 C. She assigned the name Arthrobacter glacialis to the organism. Approximately 800 isolates were obtained from Arctic sediment samples (119), of which two hundred fifty were found to grow well on agar at 0 but not at 25 C. Unfortunately, these organisms were never characterized thermally. Stanley and Rose (171) isolated psychrophilic bacteria from various lakes on Deception Island, Antarctica, and found 2, 9, and 18 bacteria with optimum growth temperatures of 5, 10, and 15 C, respectively. A preliminary examination of 488 cultures isolated from the Antarctic was made by Wiebe and Hendricks (185) who found that 37.5% of the organisms would not grow at 15 C and 65% did not survive at 20 C. One of the species, designated as E5-4-4, had a maximal growth temperature at 10 C (31). Matches and Liston (115) isolated 288 clostridia from marine sediment collected in the Puget Sound area, of which 7 would not grow above 20 C. Five out of 29 clostridia studied by Finne and Matches (56) were psychrophiles. Approximately 400 isolates have been made by my associates during Cruises 46 and 51 of the U.S.N.S. Eltanin in the Antarctic. One hundred fifty of the 400 isolates were selected for further characterization, and it was found that 148 would grow at 10 C, 106 would grow at 15 C and 98 would grow at 20 C; 144 were oxidase positive, seven were agar digestors. All isolates demonstrated lipolytic activity and approximately 50% were chitin digestors. Also present were several psychrophilic anaerobes. Vishniac and Mainzar (183) investigated the dry valleys in southern Victorialand, Antarctica, for microbes. This area represents one of the driest regions on earth and the relative humidity is about 10%. Although temperature was not mentioned in the paper, the in situ 1-h experiments demonstrated the presence and activity of bacteria inhabiting the dry soils. The reason for the 1-h experiments was dictated by the ability of the investigators to work comfortably with their hands at subfreezing temperatures coupled with a wind velocity of 60 km/h. Since miltiplication of the microorganisms were
149
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VOL. 39, 1975
observed when water was available, the organisms could have been psychrophiles or psychrotrophs. Their conclusion was that the bacteria underwent rapid division within a few hours of the day when water was available. This water was the result of periodic snowfalls which melted when the radiant energy was present in sufficient quantity. If water was not available, the bacteria laid dormant for a week or 10 days at a time. Psychrotrophic bacteria have been known to exist in the Antarctic region since the early investigations conducted by Ekelif (51) during the Swedish Antarctic Expedition of 1901-1903. Most bacteriological investigations (16, 26, 27, 29, 42, 62, 117, 172) focused mainly on the demonstration of various types of bacteria present and not the existence of psychrophiles. Generally no precautions were taken to insure that psychrophiles would not be inactivated. For instance Boyd and Boyd (16) used nutrient agar for dilution of their Antarctic samples as well as an incubation temperature of 20 to 22 C, whereas Darling and Siple (42) used an incubation temperature of 35 and 37 C. Cameron et al. (28) incubated their cultures at 2 and 20 C but in only two samples were there more bacteria growing at 2 than at 20 C. Starka and Stokes (172), using soil samples, demonstrated the presence of psychrotrophic bacteria. It is difficult to determine from their data whether or not a psychrophilic bacterium was isolated that meets the definition given in this review. Since soil samples were employed it is likely that the true psychrophiles may have expired in the source material. For further details concerning the early Antarctic microbiological literature Sieburth (165) should be consulted. Unfortunately, there has never been an extensive effort made to determine ecological distri-
bution of psychrophiles in permanently cold environments. In the marine environment it is easy to isolate psychrophilic bacteria as well as many psychrotrophic forms. The latter generally have a wide growth temperature range. ECOLOGICAL SIGNIFICANCE OF PSYCHROPHILES In the natural environment it is very difficult to assess the importance of psychrophiles in the cycle of matter. Generally within any sample both psychrophiles and psychrotrophs can be isolated. If sufficient substrates are present at low temperatures near 0 C, a psychrophile can outgrow the psychrotrophic forms (130). Even at low substrate concentration and low temperature the psychrophiles will outgrow the psychrotrophic bacteria in continuous culture (77). These laboratory findings suggest that there will be a continuous selection for bacteria whose activity is greatest near the environmental temperature, especially in environments showing seasonal temperature changes (140, 164, 166). In Antarctic field experiments employing the heterotrophic activity method at temperatures slightly above and below 0 C, the average Vmax for glutamate uptake in water samples taken at the bottom of the euphotic zone was 1.09 x 10-2 Ag/liter per h. A portion of the data obtained during Cruise 46 of the U.S.N.S. Eltanin is given in Table 3. The heterotrophic activity method (79, 148, 187) is a kinetic approach which is based on the principles of the Michaelis-Menton theory. By using the kinetic approach, the maximum velocity of uptake (Vmax), the turnover time (Ti), and the sum of a "transport constant" and the natural substrate concentration (K, + SO) can be calculated. This type of data can be obtained in field studies where the water samples are immediately ana-
TABLE 3. Heterotrophic activity on "4C-labeled substrates by the indigenous microflora in water samples taken from various depths at Stations 8 and 9 during Cruise 46 of the U.S.N.S. Eltanina Station
Depth (m)
Substrate
Vma,,x(/g/liter per h)
8
10
Glu Glut Glu Glut Glut Tyr Lys Glu Glut Glu Glut
1.8 x 10-2 8.7 x 10-2 3.0 x 10-3 1.3 x 10-2 1.1 x 10-3 4.0 x 10-3 7.3 x 10-4 3.1 x 10-3 6.7 x 10-3 5.3 x 10-4 1.6 x 10- 3
50
9
500 10
50 a The water
Tt(h) 570 112 818 382
12,350 5,000 3,704 4,800 1,544 1,800 1,298
temperature was 0 C ± 1. The percentage of respiration equals 14CO, x
(jsg/liter)
K, + S.
% Respiration
10.4 9.7 2.4 5.0 13.8 2.0
36 74 36 70 77 9 4 24 64 21 51
2.7 11.8 10.3 1.0 2.0
100/"4C uptake + 14CO2.
150
BACTERIOL. REV.
MORITA
lyzed at an in situ temperature. Other measurements with other amino acids were also made (134). Employing the best field results obtained during Cruise 46 of the U.S.N.S. Eltanin, one can extrapolate these figures to a longer period of time and for a larger volume of water. By extrapolation 2.47 x 107 g/nautical mile3 per
day of carbon dioxide can be produced by the indigenous microorganisms in the Antarctic water (-1.0 C) (131). If one could legitimately extrapolate this figure to the entire volume of the ocean, then about 1015 g of carbon dioxide would be produced per day. The foregoing also indicates that organisms can function very well in cold environments where the temperature is 0 C. There are approximately 129 x 1018 g of carbon dioxide in the ocean (160). Although the conditions necessary to produce the extrapolated amount of carbon dioxide by microbial activity may not be present, the potential to produce this quantity of carbon dioxide is present. In consideration of the importance of the entire carbon dioxide system on earth, the function of microbes in the environment should not be taken lightly (125). Many of our psychrophilic isolates in our laboratory are extremely good chitin digestors at temperatures slightly above and below 0 C. The supernatant culture fluid of psychrophile 18-500 (tentatively identified as a Vibrio sp.) displays chitinase activity at 0 C. Although the organism has a maximum temperature for growth at 13 C, its chitinase functions at a much higher temperature (Table 4). ZoBell and Rittenberg (193) estimated that the annual chitin production from copepods is several billion tons. This does not take into consideration the amount of chitin produced by other marine forms (e.g., shrimps, crabs, etc.). Since fish eat organisms having exoskeletons composed of chitin, the supernatant fluid of a sculpin's (Leptocottus armatus) stomach content was checked for the presence of chitinoclastic bacteria. Per milliliter of supernatant fluid, 1.5 x 1010 chitinoclasts were isolated. The sculpin was obtained in the winter where its environmental temperature was 6 C. When a portion of the supernatant material from the fish was treated with toluene and placed in the presence of chitin, 162 gg of N-acetylglucosamine/g (dry weight) per h at 5 C was produced. Other exoenzymes are also produced by psychrophiles so that the cycle of matter can continue in environments close to 0 C. Whether or not there are pathogenic psychrophilic bacteria in the aquatic environment has yet to be investigated. The organism Cytophaga
TABLE 4. Chitinase activity by supernatant culture fluid resulting growth of 18-500 at 5 C for 5 daysa (C) Temp Temp (C)
N-acetylglucosamine produced ml)
~~~(;&g/h per
0 3 6 10 13.5 18.5 20 27 35
2.0 2.4 3.5 3.5 5.0 6.6 8.0 9.5 6.8
40
1.1
a The culture medium contained: yeast extract (Difco), 1.2 g; Trypticase (BBL), 2.3 g; sodium citrate, 0.3 g; L-glutamic acid, 0.3 g; sodium nitrate, 0.05 g; ferrous sulfate, 0.005 g; sodium chloride, 24 g; magnesium sulfate, 7 g; magnesium chloride, 5.3 g; potassium chloride, 0.7 g; and purified chitin, 1 g.
psychrophila (psychrotroph) does have the ability to cause cold-water disease (15, 148). Shell disease, burned spot disease, and rust disease (necrotic lesions in the exoskeleton of crustaceans) may be directly or indirectly caused by chitinoclastic bacteria since these organisms are not always isolated from the necrotic lesions (37). Apparently no precautions have been taken to isolate psychrophilic bacteria from shell disease. The role that psychrophiles play as producers of growth factors (e.g., B12), as food for higher forms, or as producers of hormonal-like compounds has yet to be assessed. GROWTH OF PSYCHROPHILIC BACTERIA Growth. The psychrotrophic bacteria described in the literature are actually slow growing at low temperatures and therefore are not representative of the psychrophiles that meet the definition in this review. In the figures and tables presented by various authors (e.g., 52, 78, 79, 83, 168, 174), the growth of the psychrotrophic bacteria at low temperatures (0 to 10 C) may be in the order of many hours, days, or even weeks. Because of the past and current data in the literature, the commonly accepted concept is that low temperature is iminical to the growth of "psychrophilic" bacteria. This concept is not valid when applied to psychrophiles. In order to refute the common concept that low cell yields and slow grow rates are manifestations of low incubation temperatures for bacteria, Morita and Albright (126) demonstrated that the psychrophile V. marinus MP-1 grows
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well at low temperature. The best cell yields found in the literature for previously described psychrotrophic strains of Pseudomonas fluorescens are by Hess (79) and Sinclair and Stokes (168). The former demonstrated that 9 x 108 cells could be obtained at 20 C in 72 h, 9.0 x 104 at5 C in72h, and3.4 x 103at0 Cin72h.The latter was able to obtain 1.8 x 1010 cells/ml at 30 C in 30 h and 1.7 x 1010 cells/ml at 10 C in 72 h. These data can be contrasted with the cell yields of V. marinus (126) which were 1.3 x 1012 cells/ml at 15 C in 24 h and 9 x 109 cells/ml at 3 C in 24 h. It can be readily seen that low temperatures of incubation for psychrophiles are not detrimental to large cell yields. Table 5 lists some of the generation times for previously reported psychrotrophic bacteria. It can be noted that the generation times are very long, especially for temperatures close to 0 C. In contrast the psychrophiles grow very well at low temperatures (31, 71, 123, 154, 166). The generation times for V. marinus at 15 and 3 C, respectively, are 80.7 and 226 min (126). Unfortunately the growth characteristics of all psychrophilic bacteria in this laboratory have not been investigated, but observations indicated that growth is excellent when cultures are incubated overnight at 0 to 3 C. Figure 1 illustrates the time-temperature growth plot of a psychrophile isolated from Antarctic waters. For V. marinus MP-1 the maximum growth temperature is about 20 C, the optimum growth temperature is about 15 C, and growth occurs at 1 C (lowest temperature tested; 135). We currently grow many psychrophiles routinely at -2.5 C but have never tried to determine the minimum temperature for growth for the various psychrophiles due to the lack of properly refrigerated incubators. How-
growth does occur at -5.5 C, the lowest we have tested. V. psychroerythrus, isolated by Eimjhellen and first reported by Hagen et al. (71), has a growth temperature range of 0 to 19 C, with an optimum growth temperature of about 15 C. When this organism was subjected to a temperature shift-up experiment (5 to 19.5 C), there was a slight temporary increase in the growth rate which lasted only 5 h. This increase was followed by a decrease in turbidity (70). The Pseudomonas, studied by Harder and Veldkamp (75) possessed a maximum growth tem-
ever,
temperature
1.6
O
1.2
0 0
@ 0.8 0
0.4
0
-2
0
2
6
4
8
FIG. 1. Growth of an Antarctic psychrophile (AP-2-24, designated tentatively as a Vibrio sp.). Incubation period was 80 h in Lib-X medium employing a temperature gradient incubator (Scientific Industries, Inc.). If the culture was incubated for 197 h, growth was noted at 10.5 C. Lib-X medium contains: yeast extract (Difco), 1.2 g; Trypticase (BBL), 2.3 g; sodium citrate, 0.3 g; L-glutamic acid, 0.3 g; sodium nitrate, 0.05 g; ferrous sulfate, 0.005 g; Rila marine mix, 33 g; and distilled water, 1,000 ml. The pH was adjusted to 7.5.
TABLE 5. Examples of generation time for psychrotrophic bacteria Organism
Pseudomonas fluorescensa Pseudomonas fluorescens0
Pseudomonas sp.
Generation time
Reference
6.68 h at 0.5 C 30.21hatO C
Hess (1934b)
10.65 h at 5 C 26.41 h at 0 C 2.66 h at 10 C
Hess (1934b)
Ingraham (1958)
10.33hat0 C Strain 82
21.58 h at 5 C
21.23hatO C Pseudomonas fluorescensc Bacillus psychrophilus Micrococcus cryophilus Pseudomonas strain 92
10
Temperature (C)
4.17 to 8.20 h at 4 C 6.30 h at -5 to -7 C 28.33 h at 0 C 11.1 h at 2 C
Upadhyay and Stokes (1962) Olsen and Jezeski (1963) Larkin and Stokes (1968) Tai and Jackson (1969) Frank et al. (1972)
a Inoculum prepared at 20 C. b Inoculum prepared at 5 C. c Generation time depends on type of substrates, stationary or aerated cultures.
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perature between 19 and 20 C with an optimum growth temperature at 14.5 C. The inability to grow at temperatures above 20 C might be the price the psychrophiles must pay for the advantage they have in the very cold natural environment (75). An organism in our laboratory designated as Ant-300 (tentatively identified as a Vibrio sp.) has a maximum growth temperature of about 13 C and an optimum growth temperature of 7 C; however, the minimum growth temperature has not been determined (63). (An
BACTERIOL. REV.
electron micrograph of a section of Ant-300 is shown in Fig. 2). There are quite a few isolates in our laboratory that have maximum growth temperatures of 13 C, but the majority have maximum growth temperatures between 20 C and 13 C. Classification of our psychrophiles is currently underway. The growth and activity of marine microorganisms is generally greater at temperatures 10 to 20 C above the environmental temperatures from which the organisms are isolated (14, 191).
FIG. 2. Electron micrograph of a thin section of Ant-300, a marine psychrophile. Cells were grown in glucose medium, washed in artificial sea water, fixed in 5% glutaraldehyde, and post-fixed in 1% osmic acid. Thin sections were stained with uranyl acetate and lead acetate. Bar = 1 jAm.
VOL. 39, 1975
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153
Although the maximum growth rate is attained tively, at the lower end of the thermal spectrum at optimal growth temperature, the maximum of growth. Their data helps establish the fact cell crop is obtained when organisms are incu- that making an Arrhenius plot for a microorgabated below the optimum for growth for ex- nism's growth response to temperatures is extended periods of incubation, probably due to cessive work when simple growth curves of the the efficient use of substrates (167). This situa- organism at different temperatures will give tion holds true for Ant-300 (63). Generally the essentially the same results. ocean environment is low in substrates, and the low temperature of the environment allows the PHYSIOLOGY OF PSYCHROPHILES utilization of the available substrates efficiently. The genetic constitution of the cell deterto of psychrophiles ability Nevertheless the function well in a cold environment has been mines its cardinal temperatures for growth. noted (131, 132, 133, 134). Ten to 20 C above the Although this premise is generally accepted, the organism's original environmental temperature data in the literature are completely lacking in may be too high for many of the currently order to postulate the exact genetic basis of known psychrophiles. However, this general psychrophily. Most of the research emphasis concept probably does apply to the psychro- have been placed on physiological studies. trophic bacteria. Many laboratory studies on Nevertheless, there is genetic evidence for the marine bacteria do not generally employ envi- conversion of a mesophile to a psychrotroph by ronmental temperatures, and therefore the data transduction (145), ultraviolet light (5), and the mutation by ultraviolet light from a psychrois difficult to extrapolate to the environment. Temperature characteristic for growth. It troph to a mesophile (176). Since phage have was suggested that the temperature characteris- been isolated from psychrotrophic bacteria (43, tic for growth (Au) could be used to determine 44, 99) transduction between cold-loving orgawhether or not microorganisms were psychro- nisms is possible. Unfortunately none of the philic or mesophilic (83). The bacterial growth host cells for the phage are psychrophiles. It rate is substituted for the reaction rate in the appears unlikely that either mutagenesis by van't Hoff-Arrhenius equation to obtain the At ultraviolet irradiation or transduction would value; g is then analogous to the activation change the total enzymic make-up of cells energy. Hence psychrophiles should have a sufficiently to promote psychrophily, since lower AL value than mesophiles. This concept there appears to be more than one enzyme in a was found to be erroneous (73, 162). Ingraham psychrophilic cell that is abnormally thermola(83) and Shaw (162) used a mesophile and a bile. One of the hypotheses of microbial evolution psychrotroph to obtain their results, whereas Hanus and Morita (74) employed a psychro- is that thermophiles were the first to evolve phile, a psychrotroph, and a mesophile all (20), followed by the mesophiles and then the belonging to the same genus. Baig and Hopton psychrophiles. The evolution of psychrophilic (6) found that organisms isolated from natural bacteria is probably due to many genetic events water having the fastest growth rates at 5 and since a psychrophile has more than one enzyme 10 C had the lowest g values and that the that is abnormally thermolabile and has differcorrelation is best when temperatures from 15 to ences in the membrane. According to Allen (4) 25 C are employed. Nevertheless, they state organisms that have lived in a cold environment that a low A value and psychrophilic properties for generations may have experienced mutation may be unrelated attributes of the bacteria that caused some of their critical proteins or capable of persisting in natural waters. Working protein complexes to become relatively heat with a thermophile and comparing their data labile. There are probably many pathways leadwith Hanus and Morita (74), Ward and Cockson ing to psychrophily, since we have found that a (184) could find no correlation between g and specific abnormally thermolabile enzyme in one psychrophile may not be abnormally thermolathe optimal growth temperatures. Harder and Veldkamp (77) determined the bile in another psychrophile. In the eighth edition of Bergey (25), V. maximum specific growth rates of four psychrotrophic and four psychrophilic bacteria. Their marinus MP-1 is classified as V. fisherii. Biodata show that the difference is found in the chemical characteristics used in taxonomy do temperature range in which the Arrhenius plots not characterize the thermal properties of enare linear, rather than in different temperature zymes or membranes. There is at present no characteristics of growth. Deviation from linear- data in the literature that indicate how the ity was at -4.5 to -5.0 C and 4 to 5 C for the code (probably sequence of codons) for "mesopsychrophiles and the psychrotrophs, respec- philic" enzyme differs from the code for "psy-
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crophilic" enzyme. The thermal properties of psychrophiles should be taken into consideration in bacterial classification, if we accept the hypothesis that the psychrophiles evolved from mesophiles. Krajewska and Szer (97) conducted comparative studies of amino acid incorporation in a cell-free system of a psychrotrophic Pseudomonas and Escherichia coli and found that the ribosomes and supernatant fractions of the psychrotrophic Pseudomonas and E. coli are interchangeable at 30 to 45 C in the poly U-promoted phenylalanine incorporation. At 2 to 9 C only the psychrotrophic Pseudomonas ribosomes are active with both supernatant fractions, and therefore the ability to perform protein synthesis at low temperature is related to ribosomal specificity. These authors also point out that the psychrotrophic system operates at a relatively high fidelity compared to the mesophilic and thermophilic subcellular systems. Does this indicate that through evolution the psychrophiles are more stable in terms of their genetic fidelity compared to the mesophiles and thermophiles? A positive correlation was found between the thermal stability of the ribosomes and the maximal growth temperature of the organisms studied. The percentage of guanine plus cytosine content of ribosomal ribonucleic acid (RNA) for V. marinus MP-1 and Bacillus stearothermophilus was 52.1 and 59.3 mol%, respectively. The percentage of guanine plus cytosine content of the deoxyribonucleic acid (DNA) was reported for the two organisms just mentioned as 40 and 51.5 mol%, respectively. Pace and Campbell (147) state that the thermal stability of the ribosome lies in the interaction between the RNA and the ribosomal protein. Numerous reasons have been put forth to explain why microorganisms expire when exposed to temperatures above their maximal growth temperature, but most of the research has been done with mesophiles. These explanations include the accelerated use of the intracellular amino acid pool (70), enzyme inactivation by elevated temperature (50, 100), changes in extent of cellular lipid saturation (91), disruption of the intracellular organization (84), inactivation of an enzyme-forming system (138, 178), the loss of permeability control (129), and thermally induced leakage of ribonucleotides, ammonia, ninhydrin-positive material, potassium, and phosphate (173). Effect of temperature on respiration and viability. Psychrophilic bacteria function well in terms of growth and respiration at environmental temperatures from which they were
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isolated. V. marinus MP-1 (grown at 15 C) was shown to be able to respire very well (13 ,u of oxygen uptake/mg of cell protein per 30 min) at its optimal growth temperature (15 C) and near its environmental temperature (4 C; 6.5 qI of oxygen uptake/mg of cell protein per 30 min). Cells grown at 4 C utilized 7.25 ,ul of oxygen/mg of cell protein per 30 min at 15 C and 3.5 ,ul of oxygen/mg of cell protein per 30 min at 4 C (72). The temperature at which the cells were grown governs their oxygen uptake. This same pattern of results was also shown by Christian and Wiebe (31). This situation could be attributed to the fluidity of the membrane at different temperatures, which, in turn, is reflected in the ability of the cells to transport various substrates at different temperatures. The respiratory ability of psychrophiles increases during the initial heat treatment at temperatures near and slightly above the maximum temperature for growth (31, 63, 75). Harder and Veldkamp (75), working with a psychrophilic Pseudomonas in continuous culture, found that the maximum growth temperature for the organism was 20 C, yet the maximum rate for respiration was at 23 C. Although the actual Q02 increased towards the maximum and minimum growth temperatures, there was a concomitant decrease in the molar growth yields. This decrease in the molar growth yields suggested to Harder and Veldkamp (75) that the energy needed per unit weight per unit time to maintain a certain growth increased with increasing deviation from the optimum growth temperature. Christian and Wiebe (31) restate Harder and Veldkamp's (75) and Brown's (23) results by saying that the portion of energy capable of being produced via respiration, which is utilized for reproduction, decreases as temperature increases. At 13 C (maximum growth temperature for Ant-300) and at slightly higher temperatures, the amount of carbon dioxide respired decreased with exposure time in the presence of exogenous glucose (Table 6). This decrease in respired carbon dioxide appeared to be the result of heat-induced restriction on glucose uptake which, in turn, accelerates starvation of the cells (63). An exposure of 110 min to 20.4 C will greatly reduce the ability of V. marinus MP-1 to take up oxygen endogenously or in the presence of glucose, thereby indicating that a thermal lesion(s) in the cells has taken place (157). This thermal injury appears to be a sequence of events since V. marinus MP-1 goes from an uninjured state rapidly through a state of reversible injury and then to a condition of
155
PSYCHROPHILIC BACTERIA
VOL. 39, 1975
TABLE 6. Uptake and "CO2 respired in the presence of [U-14Clglucose by Ant 300 cells exposed at various temperatures for various periods of timea dpmb Period of heat shock (h)
0 10 20 30 40 50 60
5 cc
00o
Uptake
% Respd
CO,
5039 5426 4629 5878 5064 5969 5499
17,852 13,658 11,240 12,515 10,894 11,670 11,042
34 40 41 47 46 51 50
12,381 11,244 10,031 8,873 7,995 6,840 5,211
CO,2
Uptake % Resp 32,632 22,124 19,428 16,907 15,223 13,701 9,231
18 C
15 C
13 C
38 51 52 52
53 50 56
14,018 13,100 8,235 6,859 4,904 3,984 1,754
Uptake % Resp 36,555 24,528 17,156 12,295 8,054 5,902 2,746
38 53 48 56 61 68 64
CO,
Uptake % Resp
15,698 38,776 9,536 20,154 3,422 6,599 1,052 1,052 383 383 144 144 0 0
40 47 52 100 100 100
a The cells were starved in a menstruum without any carbon source for 24 h at 5 C prior to exposure at various temperatures for various periods of time. After exposure, [U- 14C ]glucose (final concentration was 0.9 Ag/ml with a specific activity of 180 mCi/mmol) was added, and the mixture was incubated at the same temperature of exposure for an additional 2 h. Cells from an exponential phase culture (OD = 0.25 at 600 nm) grown in a glucose medium for 28 h at 5 C were employed. b dpm, Disintegrations per minute. Temperature of exposure and subsequent incubation with [4C ]glucose. dPercentage of respiration = CO/uptake x 100.
nonreversible injury or death (67). Recovery of the reversible injury of the cells depends on the recovery medium. The thermal tolerance of V. marinus MP-1 was tested, and it was found that the organism in growth medium would expire when exposed for 50 h to a temperature slightly above 20 C (organism's maximal temperature for growth; 135). A time-temperature relationship was also noted. An exposure to 28.8 C for 6.25 h or 32.7 C for 1.25 h would render the organism nonviable. If nutrients (energy source) were not present in the suspending menstruum, then viability was lost in 15 min at 31 C (72). An exposure to 20.5 C for 110 min in the absence of nutrient will reduce the viable count from 3.8 x 1011 to 1.6 x 109 cells/ml (157). Temperatures above the maximum for growth of psychrophiles, although they may be less than room temperature, are inimical to the organisms. Proteins. Koffler and Gale (96) reported that cytoplasmic proteins isolated from four thermophiles were much more thermo stable than similar preparations from mesophiles. Using identical procedures on E. coli and V. marinus MP-1, Steenbergen and Morita (unpublished data) found that the cytoplasmic proteins from the psychrophile were more heat labile than those from the mesophile. Furthermore, some of the cytoplasmic proteins were heat labile between the psychrophile's optimal and maximal growth temperatures (Fig. 3). According to Koffler (95) death by heat was postulated to be brought about through the injury of a few essential molecules, perhaps only one. Within
limits cell inactivation is reversible as long as there is an opportunity for repair. Malic dehydrogenase from V. marinus MP-1 was studied in relation to its abnormal thermolability (100). The enzyme present in washed cells was found to be stable between 0 C and the organism's optimal temperature for growth (15 C). Between the organism's optimal and maximal growth temperatures there was an inactivation of the malic dehydrogenase. Thermolability studies on cell-free extracts for malic dehydrogenase indicated that almost all the activity was lost in 10 min at 30 C. Even at 0 C, there was a considerable loss in activity. Partially purified malic dehydrogenase (20fold) was found to be optimally stable between 15 and 20 C, and the inactivation became very pronounced when exposed to temperature above 20 C. Oxygen uptake studies gave no indications of the denaturation of enzymes at or near the organism's maximal growth temperature
(76).
In cell-free extracts of V. marinus, Mathemeier (Ph.D. thesis, Oregon State Univ., Corvallis, Ore., 1968) found that lactic dehydrogenase, hexokinase, malic dehydrogenase, phosphofructokinase/glyceraldehyde 3-phosphate dehydrogenase complex, and aldolase lost nearly all their activity when incubated at 35 to 40 C for 1 h. However, there is a possibility that a protease could be present in the crude extracts since no protease inhibitor was included in the assay mixture. Aldolase was purified from the same organism and found to be similar in properties to the Type II aldolases isolated from
156
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proteinases gave lower temperature characteristics than did proteinases from a Cytophaga sp. Z 6 A-20 and P. fluorescens 612. Unfortunately, the cardinal temperatures for these organisms were not determined but they did grow at 0 C. CLi The supernatant fluid of V. psychroerythrus 0.a (118) was found to contain proteolytic enzymes z 20-2 that hydrolyzed casein, f3-lactoglobulin, and hemoglobin. Although the organism is a psy-2 4 20-40 chrophile, the activity of the caseinase was optimal at 40 to 45 C at pH 10. The enzyme is \ relatively thermostable in the presence of cal2 cium; hence it appears to be similar to the proteinase of mesophiles. The best activity was CLl found between 35 and 45 C in the presence of C: . 40- 60 calcium (118). This proteinase was separated into three fractions; two were stable at 55 C and 30 35 20 25 0 5 10 15 one was almost inactivated at 55 C (141). A TEMPERATURE (C) FIG. 3. Heat denaturation of various ammonium white variant of the organism produced only sulfate fractions of V. marinus MP-1 cell-free extract two fractions on Sephadex chromatography in tris(hydroxymethyl)aminomethane-hydrochloride (142). Purified tryptophan synthetase from a buffer, pH 7.4. (Ammonium sulfate fractions: 0 to 20, psychrotrophic organism, designated as B-160, 20 to 40, and 40 to 60% saturation. The cell-free ex- lost 50% of its activity at 30 C in 5 min, whereas tract was prepared from cells grown at 15 C for 24 h, the tryptophan synthetase from E. coli is stable treated with a Biosonik III probe, and centrifuged at 34,500 x g for 30 min to remove the cellular debris. for several hours at 50 C (C. S. Cheng, M.S. z
0
Ammonium sulfate fractions were placed at various temperatures in a polythermostat for 12 h, followed by removal of the precipitate by centrifugation at 34,500 x g for 30 min. The supernatant fluid of each fraction was diluted, and the protein concentration was determined by the Lowry method). (Unpublished data, Steenbergen and Morita.)
other bacteria (L. P. Jones, Ph.D. thesis, Oregon State Univ., Corvallis, Ore., 1973). The temperature optimum for the-aldolase was 25 C with no activity evident above 32 C, but activity could be observed at 4 C (lowest temperature tested). Within experimental error of amino acid analysis, the amino acid composition of the aldolase from the psychrophile was the same as that from thermophilic aldolase (174) and from rabbit aldolase (163). Glucose-6-phosphate dehydrogenase from V. marinus MP-1 is not as abnormally thermolabile like the malic dehydrogenase from the same organism (W. W. Miller, M.S. thesis, Oregon State Univ., Corvallis, Ore., 1968). Partially purified glucose-6-phosphate dehydrogenase was found to be stable between 5 and 26 C, but when it was exposed to 36 C for 1 h it lost 90% of its activity. Glyceraldehyde-3-phosphate dehydrogenase from the same organism was purified and found to be quite thermostable (Howard, Miller, and Becker, personal communication). Proteinases in culture supernatant fluids of various Arctic bacteria hydrolyzed casein most rapidly between pH 7.0 and 8.0 (119). These
thesis, Brigham Young University, Provo, Utah, 1970). Thermally induced leakage. Leakage of 260nm-absorbing material from V. marinus MP-1 was first noted by Robison and Morita (157) when the cells were exposed to moderate temperatures of 20 to 30 C. The leakage products due to exposure to moderate temperatures listed in decreasing rates of leakage were identified as protein, deoxyribonucleic acid, ribonucleic acid, and amino acids (72). Leakage of nucleic acid material can be seen in Fig. 4. When the cells are exposed to 22.3 C (2.3 C above its maximal growth temperature), these cellular leakage materials appear in the super-
natant fluid after exposure for 45 min. After a 60-min exposure to 22.3 C, 21.5 mg of protein, 52 Atmol of amino acid, 2,100 ,g of RNA and 207 jig of DNA appeared in the supernatant fluid from 10 ml of cell suspension (optical density [OD] of 0.40 at 600 nm). If the exposure temperature was increased, then the amount of leakage material increased rapidly. The leakage of intracellular material was thought to be one of the causes for loss of viability in V. marinus MP-1 at moderate temperatures. However, it was found that 95% of the cells must expire before noticeable amounts of leakage products occur (93). In addition to the above named leakage products, it was found that malate dehydrogenase and glucose-6-phosphate appears in the supernatant fluid after exposure of the cells to moderate temperatures. Older cells were found to be more resistant to
VOL. 39, 1975
PSYCHROPHILIC BACTERIA
157
FIG. 4. Electron micrograph of a thin section of Vibrio marinus MP-1 exposed to 26.5 C for I min. Note the separation of the cell wall and the leakage of nuclear material from the cell. (Courtesy of R. R. Colwell.)
thermal death, lysis, and leakage. Harder and Veldkamp (75) detected a very small amount of 260-nm-absorbing material leaking from a psychrophilic pseudomonad at 18 to 19 C, but the amount was so small that it does not offer an explanation for the decreased protein synthesis. Because the use of spectrophotometric measurements is not as sensitive as radioisotope measurements, the problem of heat-induced leakage was reinvestigated employing Ant-300 cells grown in [U-_4C]glucose, harvested, starved, and exposed to temperatures near or slightly above the maximum for growth of the organism (63). Leakage, as evidenced by the loss of "C-labeled cellular material, was extensive during the first 10 h of incubation. At 18 C, 31% of the radioactivity was released from the cells. At and near the organism's maximal growth temperature (13 and 15 C, respectively), 16% of the radioactivity was released while only 7% was released at 5 C. The percentage of protein and protein plus DNA at 5 and 18 C are nearly equal, but the main high-molecularweight cellular leakage component at 18 C is RNA (Table 7). Identification of the lowmolecular-weight components have not been
identified (G. G. Geesey, M.S. thesis, Oregon State Univ., Corvallis, Ore., 1973). The question as to how much leakage is necessary before growth ceases must still be answered. The degree of leakage depends on the presence or absence of substrates. Thermally induced lysis. Phase optics indicate that V. psychroerythrus lyses when exposed to 20 C or higher (71,108) which is preceeded by death. With V. marinus MP-1 leakage and lysis occurred simultaneously but after 95% of the cells had expired (93). In nongrowing cultures once lysis is initiated at 25 C it cannot be stopped by transferring the culture back to 5 C, which suggests that the process is enzymatic. Lysis is not affected by metabolic inhibitors. The autolytic enzymes remain active at lower temperatures once activated by higher temperature. After lysis hexosamine as well as 260-nmabsorbing material can be found in the menstruum, and the breakdown of lipid phosphorus occurs after the onset of measurable lysis (71, 94). At 25 C phosphatidases attacking cells reduce the lipid phosphorus by more than half (94). These phosphatidases require salt for maximum activity. A drop in the turbidity of a sus-
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158
BACTERIOL. REV.
TABLE 7. High-molecular-weight components released from Ant 300 cells at 5 and 18 C for 4 ha shock Heat Heatshock Protein (%)
Protein + DNA (%)
RNA (%)
material ~~~~~~~~~~~~~~~~~~~~~~Total released (dpm/ml)b
5 18
17 (397 dpm) 16 (848 dpm)
18 (409 dpm) 17 (886 dpm)
10 (218 dpm) 14 (744 dpm)
2,264 5,161
a Cells were grown at 5 C for 22 h in a medium containing [U- "4C ]glucose (final concentration was 6.0 ,g/ml with an activity of 180 mCi/ml). The cells were harvested by centrifugation and suspended in artificial sea water to give a final concentration of 2 x 107 cells/ml. b dpm, Disintegrations per minute.
pension of water-lysed cells or cell envelopes at 25 C was also salt dependent. The ultrastructure of V. marinus MP-1 and V. psychroerythrus appear typical of gram-negative bacteria and both possess a trilaminar membrane structure (40, 55). Degradation of the cell wall and liberation of wall fragments occurs when lysis of the cell takes place at 37 C, and the trilaminar structure is rarely observed (40). This destruction occurs in the presence of all salt solutions tested. Temperature effects of permeability. The ability of the psychrophile to function well at cold temperature depends to a great degree on the availability of substrates. When the temperature is raised above the maximum temperature for growth, the transport of substrates is impaired. An apparent increase in malic dehydrogenase activity is noted in a psychrotrophic bacterium when the temperature of the system is raised above the maximum growth temperatures (129). In other words, near-maximal activity occurred even when the intracellular enzyme was being inactivated. This apparent increase in activity with whole cells is due to the alteration in permeability which permits diffusion rather than active transport of the substrate into the cells. When the temperature is lowered below the optimal growth temperature, the uptake of substrates is decreased (9, 92, 128, 150, 152). The foregoing data definitely indicate that changes in transport systems occur when the temperature is altered. B616hradek (11, 12) suggested that thermal death may be due to the solution of certain lipids and there was a correlation with the melting points of the lipids of the cell and death. Since that time, much research has been concentrated on membrane lipids in relation to growth temperature. As the temperature of growth is lowered, the proportion of unsaturated fatty acids (hexadecenoic and octodecenoic) increases, and this occurs when cells are grown at different temperatures in both minimal and complex medium (111). This situ-
ation also occurs in a psychrotrophic strain of V. marinus PS 207 (143) and with yeasts (90). The fatty acid composition of V. psychroerythrus (thought to be a Serratia sp. at the time of publication) was analyzed and compared to Serratia marcescens (90). The psychrophile did have more unsaturated fatty acids than S. marcescens. The percentage of unsaturated fatty acids is lower in V. marinus PS 207 than in many forms that have higher cardinal temperatures (143). The relative percentage of unsaturated fatty acids in V. marinus MP-1 (145) is 59.8% when the organism is grown at its optimum temperature for growth. The percentage of unsaturated fatty acids for V. marinus PS 207 grown at 25 C (optimum growth temperature) and 15 C are 68.5 and 72.0, respectively. Growth of both strains of V. marinus at their temperature optima reveal similar fatty acid patterns, but not when PS 207 is grown at 15 C (143). It therefore appears that within a species there is a shift toward more unsaturated fatty acids with a decrease in the temperature under which the organism is grown. Since V. marinus MP-1 has a much lower percentage of unsaturated fatty acid composition (146) compared to many of the other marine bacteria that are not psychrophiles (143), the question still remains as to how psychrophiles can maintain their membrane fluidity so that transport systems are still operative at 0 C or below. Temperature effects on macromolecular synthesis. When a psychrophilic Pseudomonas was grown above its optimal temperature for growth, there was an increase in the synthesis of RNA in the cells (75). This increased RNA synthesis was noted until a temperature of 18 C was reached (maximal growth temperature was 19 C). Between 18 and 19 C a drop in RNA content was noted. If the continuous culture system was elevated to 20 C, protein synthesis ceased and the culture was washed out of the continuous culture apparatus. The increased RNA of cells at the higher temperature was
VOL. 39, 1975
PSYCHROPHILIC BACTERIA
mainly ribosomal RNA. The ability of V. marinus MP-1 to take up [U-14C ]proline, synthesize protein (measured by incorporation of [U-14C]proline), and synthesize RNA (incorporation of [U-5H]uracil) was found to be a function of salinity and temperature. The critical temperature of the lesion(s) in protein synthesis increased with increasing salinity of the growth medium. At a salinity of 25%o and 22 C, protein synthesis was significantly inhibited, but not at a salinity of 35%o (maximal growth temperature at a salinity of 35%o is 21.2 C). When cells were incubated at 24 C at a salinity of 35%o, protein synthesis continues at a linear rate for 20 minutes before it begins to decrease. Total RNA synthesis continues at a linear rate for 20 min before salinities between 15 and 35%o. A salinity-temperature relationship is noted for both RNA and protein synthesis (38). However, it should be noted that both RNA and protein synthesis continue in growth media when cells are incubated a few degrees above the maximum for growth of a psychrophilic vibrio (127). At 5 C above the maximal growth temperature, the cessation of DNA and protein synthesis occurs approximately 30 min after exposure to the elevated temperature. An increase in protein and DNA was noted at the maximal temperature for growth over the amount produced at the optimum temperature for growth (75). No increase in protein, RNA, and DNA could be noted by Harder and Veldkamp (76) when cells were exposed in the presence or absence of substrate at 2.5 to 3.5 C above the maximal temperature for growth. Since no RNA synthesis could be detected at 2.5 to 3.5 C above the maximum growth temperature, they concluded that this may be the basis for the cessation of growth. However, as they point out, this does not account for the decline in the growth of the organism between the optimum and maximum growth temperatures. When a cell-free extract prepared from the psychrotrophic bacterium Micrococcus cryophilus was subjected to 5 C above the maximal growth temperature for the organism, three aminoacyl-tRNA synthetases (glutamic acid, histidine, and proline) were found to be temperature sensitive (109, 110, 111, 112). Unfortunately, the experiments were not carried out 1 or 2 C above the maximal growth temperature for M. cryophilus. At temperatures of 1 or 2 C above the maximal for growth, the cessation of macromolecular synthesis does not appear to be the cause of thermal death. Most thermal death experiments are carried out at 5 to 10 C above the maximal temperature for growth which is much
159
too high to determine the subtle changes in the organism which results in the cessation of growth. Ecological factors affecting psychrophiles. As stated previously, psychrophiles function well at environmental temperatures even though their optimum activity and growth may be several degrees higher than the environmental temperature. The main environmental factors other than the amount of substrates present that affect the psychrophiles' ability to function are salinity, temperature, and hydrostatic pressure. The effect of salinity and the various cations making up the salinity of the ocean on marine bacteria are well reviewed by MacLeod (106, 107). Membrane permeability has been shown to be salt dependent in marine bacteria (45, 46, 107). After washing cells of a marine pseudomonad free of medium components with 0.05 M MgSO4, there was an immediate increase in OD when the cells were suspended in solutions containing 200 mM of various salts (116). This increase in OD was followed by a slow decrease in OD. The decrease in OD can be prevented by omitting K+ from the suspending solution. The OD changes were not attributed to osmotic effects. The OD of cell envelopes prepared from the organism also increased with increases in NaCl but not in the presence of Mg2+. This initial increase in OD was attributed to an interaction of the salts with the components of the cell envelope, thereby causing the contraction of the envelopes and shrinkage of the cells. .05 .04
.03 E
.02
oC
.01
S%O 151o100
0
< -.01 O -.02 4
S=
-.03
O
2
3
4
5
150
6
7
TIME (MIN) EXPOSED TO VARIOUS SALINITIES
FIG. 5. Time-salinity effects on the OD of V. marinus MP-1 cells. Cells from a 24-h culture grown at 15 C were centrifuged, washed with artificial sea water (35 Vo), and centrifuged again. The sedimented cells were then suspended at various salinities for various periods of time. The initial OD reading was set at 0.2 (equals 0 on the figure). (Unpublished data, Haight and Morita.)..
160
Salinity changes can also do the same for V. marinus MP-1 (Fig. 5). Salt and low temperature are necessary for the stability of the cellular structure of V. psychroerythrus (40, 94), and lysis can occur at low ionic strength (108). V. psychroerythrus is stable in cold sea water or in a solution containing 0.1 M Mg2+ and 0.5 M NaCl. When subjected to water and in various monovalent salts (including 3.0 M NaCl), the turbidity of the cell suspension fell and some intracellular materials were released into the menstruum. Phase contrast microscopy revealed that the cell disintegrated into amorphous aggregates after first becoming swollen. The effectiveness of divalent salts in maintaining cell structure was in the order Cu2+ > Ni2+ > Ca2+ > Mn2+ > Mg2+
(94).
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Depending on the salt concentration in the medium, V. marinus MP-1 can possess a maximum growth temperature from 10.5 to 21.2 C (170). The lower the salt concentration, the lower the maximum growth temperature of the organism. This situation holds true for all the salt-requiring bacteria we have tested in our laboratory. In ecological situations where the salinity and temperature can change due to freshwater intrusions, evaporation, and diurnal insolation, the temperature-salinity regime may be an important factor in the enumeration of the microbial flora; hence an important thermal group may have been overlooked in microbial ecological studies (136). Figure 6 illustrates the co-effect of salinity and temperature variations on the growth of Vibrio marinus MP-1. The change in the maximum growth temperature was shown to be dependent upon the various ions present in the growth medium. The order of effectiveness in restoring the normal growth temperature (21.2 C), when added to dilute sea water, was Na+, Li+, Mg2+, K+, Rb+, NH,+. In a completely defined medium, the highest maximal growth temperature was 20.0 C at 0.40 M NaCl. A decrease in the maximal growth temperature was observed at both low and high concentrations of NaCl. In order to try to explain the change in the maximal growth temperature in relation to the salt concentration, studies were initiated to determine the effect of a salinity-temperature regime on macromolecular (see section on temperature effects on macromolecular synthesis). Rhodes and Payne (155) have found that the induction of washed cells of Pseudomonas natriegens (marine bacterium) to the oxidation of lactose and mannitol was dependent on the presence of Na+ with marine levels of Mg2+ and K+ in the suspending medium. A temperaturesalinity relationship was shown for the induc-
50 40 I
30 Co
20 10
0 0
4
8
12
16
20
TEMPERATURE (C)
FIG. 6. Contour surface diagram showing the coeffect of temperature and salinity on the growth rate as a number of generations/h of Vibrio marinus MP-1 in Lib-X broth. Symbols: 0, 0.28 generation/h, (maximum); A, 0.25 generations/h; 0, 0.20 generations/h; 0, 0.14 generations/h; and A, 0.06 generations/h.
tion of glutamic dehydrogenase in V. marinus MP-1. This interaction of salinity and temperature may be very important in the nearshore environment since it will determine whether or not a microorganism will have the ability to synthesize adaptive enzymes to cope with any foreign substrates that might enter the water. The pattern of carbon dioxide release from specific labeled carbons of glucose taken up by V. marinus MP-1 was found to change with salinity (68). Within the growth temperature range of the organism, temperature had no effect on this pattern. However, when the salinity is increased from 0.30 to 1.0 M NaCl, there is a decrease in the C6/C, (carbon dioxide) ratio. The changes observed in this study were not specific for Na+ or Cl-. The apparent shutdown of the hexose monophosphate pathway, as determined by radiorespirometric carbon dioxide release patterns, coincides with the minimum salinity of growth of V. marinus MP-1. This may in turn be related to the growth limitation at lower salinities. As the salinity was increased from 0.15 to 0.30 M NaCl, there was a shift in the total uptake patterns which suggests the formation and loss of metabolic by-products derived from the first, second, sixth, and presumably fifth carbons of glucose. The effects of hydrostatic pressure on psychrophiles in the marine environment takes on more importance when one takes into consideration the volume below the thermocline. In this region the pressure may be as high as 1,100 atm (average pressure is 380 atm) and the temperature is 5 C or colder. When pressure and temperature are considered as variables, a pressure-
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temperature interaction is found as would be predicted by the Ideal Gas Law. The pressure increases approximately 1 atm for every 10 m in depth. There have been many reviews dealing with hydrostatic pressure effects on microorganisms (19, 89, 122, 124, 169, 190). However, the data on pressure effects on psychrophiles is limited. V. marinus MP-1 was isolated from water taken from a depth of 1,200 m (135) where the temperature was 3.24 C. It has the ability to grow slightly better at 100 and 200 atm at 3 C than at 1 atm at 3 C. With increasing pressure above 300 atm, growth decreases and at 1,000 atm the organism expires. Death ensues at 800 atm when the temperature is 15 C (127). The response to pressure depends upon the environmental temperature from which the organisms were obtained. Hydrostatic pressure stimulated or supressed L-serine deamination by washed cells of V. marinus MP-1 depending upon the temperature at which the cells were grown and the incubation temperature of the reaction mixture (3). Cells grown at 15 or 4 C had deamination stimulated under pressure in the following cases: (i) cells grown at 15 C and tested for deamination at 15 C; (ii) cells grown at 4 C and tested at 4 C; and (iii) cells grown at 4 C at tested and 15 C. When cells were grown at 15 C and tested at 4 C, no stimulation of deamination activity due to pressure was observed. Unfortunately there is a tendency to neglect this fact when working on the physiological and biochemical reactions under pressure. The ability of a psychrophile to synthesize protein, RNA, and DNA under pressure was tested, and it was found that at 200 atm macromolecular synthesis was not affected. At 400 atm protein and RNA but not DNA synthesis was adversely affected, whereas at 500 atm there was a rather sharp decline in the synthetic rates. At 1,000 atm virtually no synthesis occurred (2). The effect of alternate pressurization-depressurization effects on growth and net synthesis of protein, RNA, and DNA was studied by Albright (1). He found that the application of 544 atm caused cell division and synthesis of RNA and protein to cease immediately. Upon release of the pressure cell division and synthesis of RNA and protein resumed the original rate. The second application of pressure had a less drastic effect on growth, and synthesis of RNA and protein. There was no immediate effect on DNA synthesis with the application of pressure, but this synthetic process slowed and ceased with time under pressure. Pressure was found to inhibit the transport of amino acids into a psychrotrophic bacterium (150). Once the amino acids are transported
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into the cells, the respiratory mechanism needed to produce carbon dioxide from the amino acids does not appear to be inhibited. Immediate pressurization of cells in the presence of an amino acid brings about inhibition of transport, and this transport inhibition is reversible when the pressure is released. If the cells were incubated in the presence of an amino acid, 3 h prior to pressurization, the ability of the transport mechanism for the amino acids is not impaired. The uptake of uracil was followed in two antarctic psychrophiles, V. marinus MP-1 and V. alginolyticus, and E. coli under pressure (8). Between 100 to 300 atm at 15 C, the ability of E. coli and V. alginolyticus to take up uracil was reduced approximately 70%. The same reduction was noted for V. marinus MP-1 at 5 C. The two antarctic psychrophiles were found to be more barotolerant since they had the ability to take up considerable uracil at 500 atm. It was shown that the mechanism for uracil uptake is more pressure sensitive than the RNA-synthetic mechanism. Growth studies were also conducted and correlated with uracil uptake at the same pressures and temperatures. In each organism there was a correlation between the organism's ability to divide and the rate of uracil uptake. The growth patterns, determined under pressures, of each psychrophile differed markedly. Morphological changes (spheroplast-like bodies and elongated forms) were commonly observed in cells obtained from cultures subjected to pressures limiting their growth. These morphological changes indicate that pressure profoundly influences the physiology of the cells. The above data on the pressure effects on psychrophiles strongly implicates the membrane as the primary site of pressure damage. CONCLUSION The data available concerning psychrophiles are too fragmentary to explain the biological basis of psychrophily. Nevertheless, there are sufficient data accumulated to use the term "psychrophile" in a meaningful way. By use of the definition employed in this review, a start can be made to end the confusion concerning the term. Furthermore, I believe that future scientific data on psychrophilic bacteria will more than justify this definition. ACKNOWLEDGMENTS I wish to thank my associates (J. A. Baross, P. A. Gillespie, R. P. Griffiths, G. G. Geesey, T. D. Goodrich, F. J. Hanus, S. S. Hayasaka, and L. P. Jones) for use of unpublished scientific data. This work was supported by National Science Foundation research grant GA 38583X.
MORITA
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269:2150-2152. 121. Morita, R. Y. 1966. Marine psychrophilic bacteria. Oceanogr. Mar. Biol. Annu. Rev. 4: 105-121. 122. Morita, R. Y. 1967. Effects of hydrostatic pressure on marine bacteria. Oceanogr. Mar. Biol. Annu. Rev. 5:187-203. 123. Morita, R. Y. 1968. The basic nature of marine psychrophilic bacteria. Bull. Misaki Mar. Biol. Inst. Kyoto Univ. 12:163-168. 124. Morita, R. Y. 1972. Pressure-bacteria, fungi, and blue-green algae, p. 1381-1388. In 0. Kinne (ed.), Marine ecology, vol. 1, pt. 3. Wiley Interscience Publishing Co., London. 125. Morita, R. Y. 1975. Microbial contribution to the evolution of the "steady state" carbon dioxide system. Origins Life 6:37-44. 126. Morita, R. Y., and L. J. Albright. 1965. Cell yields of Vibrio marinus, an obligate psychrophile, at low temperature. Can. J. Microbiol. 11:221-227. 127. Morita, R. Y., and L. J. Albright. 1968. Moderate temperature effects on protein, ribonucleic acid and deoxyribonucleic acid synthesis by Vibrio marinus, an obligately psychrophilic marine bacterium. Z. Allg. Mikrobiol. 8:269-273. 128. Morita, R. Y., and G. E. Buck. 1974. Low temperature inhibition of substrate uptake, p. 124-129. Inn R R. Colwell and R. Y. Morita (ed.), Effect of ocean environment on microbial activities. University Park Press, Baltimore. 129. Morita, R. Y., and S. D. Burton. 1963. Influence of moderate temperature on the growth and malic dehydrogenase of a marine psychrophile. J. Bacteriol. 86:1025-1029. 130. Morita, R. Y., and S. D. Burton. 1970. Occurrence, possible significance, and metabolism of obligate psychrophiles in marine waters, p. 275-285. In D. W. Hood (ed.), Organic matter in natural waters. Publication no. 1, Institute of Marine Science, University of Alaska, Fairbanks. 131. Morita, R. Y., G. G. Geesey, and T. D. Goodrich. 1974. Potential microbial contribution to the carbon dioxide system in the sea, p. 386-391. In R. R. Colwell and R. Y. Morita (ed.), Effect of the ocean environment on microbial activities. University Park Press, Baltimore. 132. Morita, R. Y., P. A. Gillespie, and L. P. Jones. 1971. Microbiology of antarctic sea water. Antarct. J. U.S. 6:157. 133. Morita, R. Y., R. P. Griffiths, and S. S. Hayasaka. 1972. Heterotrophic potential of antarctic marine microorganisms. Antarct. J. U.S. 7: 180-181. 134. Morita, R. Y., R. P. Griffiths, and S. S. Hayasaka. 1975. Heterotrophic potential of microorganisms in Antarctic waters. Third SCAR/ IUBS Symposium on Antarctic Biology N.A.S. (In press). 135. Morita, R. Y., and R. D. Haight. 1964. Temperature effects on the growth of an obligate
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MORITA psychrophilic marine bacterium. Limnol. Oceanogr. 9:102-106. Morita, R. Y., L. P. Jones, R. P. Griffiths, and T. E. Staley. 1973. Salinity and temperature interactions and their relationship to the microbiology of the estuarine environment, p. 221-232. In L. H. Stevenson and R. R. Colwell (ed.). Estuarine microbial ecology. University of South S. Carolina Press, Columbia. Mossel, D. A. A., and A. van de Mooskijk. 1964. The practical significance in the microbiological examination of cold stores foods of the allegedly low heat resistance among psychrotrophic organisms. J. Appl. Bacteriol. 27:221-223. Muller, M. 1903. Ueber das Wachstum und die Lebenstatigkeit von Bakterien sowie den Ablauf fermentativer Prozesse bei niederer Temperatur unter spezieller Beruicksichtigung des Fleisches als Nahrungsmittel. Arch. Hyg. 47:127-193. Nashif, S. A., and F. E. Nelson. 1953. The lipase of Pseudomonas fragi. II. Fractors affecting lipase production. J. Dairy Sci. 36:471-480. Nedwell, D. B., and G. D. Floodgate. 1971. The seasonal selection by temperature of heterotrophic bacteria in an intertidal sediment. Mar. Biol. 11:306-310. Nunokawa, Y., and I. J. McDonald. 1968a. Extracellular proteolytic enzymes of psychrophilic bacteria. I. Purification and some properties of enzymes of an obligately psychrophilic red-pigmented bacterium. Can. J. Microbiol. 14:215-231. Nunokawa, Y., and I. J. McDonald. 1968b. Extracellular proteolytic enzymes of psychrophilic bacteria. II. Production and heterogeneity of enzymes of an obligately psychrophilic red-pigmented bacterium and of a white variant. Can. J. Microbiol. 14:225-231. Oliver, J. D., and R. R. Colwell. 1973. Extractable lipids of gram-negative marine bacteria: fatty-acid composition. Int. J. Syst. Bacteriol. 23:442-458. Olsen, R. H., and J. J. Jezeski. 1963. Some effects of carbon source, aeration, and temperature on growth of a psychrophilic strain of Pseudomonas fluorescens. J. Bacteriol. 86:429-433. Olsen, R. H., and E. S. Metcalf. 1968. Conversion of mesophilic to psychrophilic bacteria. Science 162:1288-1289. Or6, J., T. G. Tornabene, D. W. Nooner, and E. Gelpi. 1967. Aliphatic hydrocarbons and fatty acids of some marine and freshwater microorganisms. J. Bacteriol. 93:1811-1818. Pace, B., and L. L. Campbell. 1967. Correlation of maximal growth temperature and ribosome heat stability. Proc. Natl. Acad. Sci. U.S.A. 57:1110-1116. Pacha, R. E. 1968. Characteristics of Cytophaga psychrophila (Borg) isolated during outbreaks of bacteria cold-water disease. Appl. Microbiol. 16:97-101. Parsons, R. R., and J. D. H. Strickland. 1962. On
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BACTERIOL. REV. the production of particulate organic carbon by heterotrophic processes in sea water. DeepSea Res. 8:211-222. Paul, K. L., and R. Y. Morita. 1971. The effect of hydrostatic pressure on the uptake and respiration of amino acids by a facultatively psychrophilic marine bacterium. J. Bacteriol. 108:835-843. Pennington, M. E. 1908. Bacterial growth and chemical changes in milk kept at low temperature. J. Biol. Chem. 4:353-393. Piperno, J. R., and D. L. Oxender. 1968. Amino acid transport systems in Escherichia coli K12. J. Biol. Chem. 243:5914-5920. Ravenel, M. P., E. G. Hastings, and B. W. Hammer. 1910. The bacterial flora of milk held at low temperatures. J. Infect. Dis. 7:38-46. Reed, H. S., and R. R. Reynolds. 1916. Some effects of temperature upon the growth and activity of bacteria in milk. Virginia Agricultural Experimental Station Technical Bulletin 10. Rhodes, M. E., and W. J. Payne. 1962. Further observations on effects of cations on enzyme induction in marine bacteria. Antonie van Leeuwenhoek J. Microbiol. Serol. 28:302-314. Richardson, W. D. 1908. The cold storage of beef and poultry, p. 261-316. In Premier Congress International du Froid, Paris, 1908. Tome 2. Rapports et Communications des Sections I, II, et III. Robison, S. M., and R. Y. Morita. 1966. The effect of moderate temperature on the respiration and viability of Vibrio marinus. Z. Allg. Mikrobiol. 6:181-187. Rose, A. H. 1962. Temperature relationships among microorganisms. Wallerstein Lab. Commun. 25:5-18. Rubentschik, L. 1925. Ueber die LebenstAtigkeit der Urobakterien bie einer Temperatur unter 0°. Zentr. Bakteriol. Parasitenkd. Infektionsk. Hyg. Abt. II. 64:166-174. Rubey, W. W. 1951. Geological history of sea water. Bull. Geol. Soc. Am. 62:1111-1147. Schmidt-Nielsen, S. 1902. Ueber einige psychrophile Mikroorganismen und ihr Vorkommen. Zentr. Bakteriol. Parasitenkd. Infektionsk. Hyg. Abt. II. 9:145-147. Shaw, M. K. 1967. Effect of abrupt temperature shifts on the growth of mesophilic and psychrophilic yeasts. J. Bacteriol. 93:1332-1336. Shimizu, H., and H. Ozawa. 1967. Studies on aldolases. I. Amino acid composition and subunit structure of rabbit-muscle aldolase. Biochim. Biophys. Acta 133:195-205. Sieburth, J. McN. 1964. Polymorphism of a marine bacterium (Arthrobacter) as a function of multiple temperature optima and nutrition, p. 11-16. Symposium on Experimental Marine Ecology, University of Rhode Island Occasional Publication no. 2. University of Rhode Island, Kingston. Sieburth, J. McN. 1965. Microbiology of the Antarctic, p. 267-295. In P. V. Oye and J. V.
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Mieghem (ed.), Biogeography and ecology in Antarctica. Dr. W. Junk, Pub., The Hague. 166. Sieburth, J. McN. 1967. Seasonal selection of estuarine bacteria by water temperature. J. Exp. Mar. Biol. Ecol. 1:98-121. 167. Sieburth, J. McN. 1968. Observations on bacteria planktonic in Narragansett Bay, Rhode Island; a resume. Bull. Misaki Mar. Biol. Inst. Kyoto Univ. 12:49-64. 168. Sinclair, N. A., and J. L. Stokes. 1963. Role of oxygen in the high yields of psychrophiles and mesophiles at low temperature. J. Bacteriol. 85:164-167. 169. Sleigh, M. A., and A. G. MacDonald (ed.). 1972. The effects of pressure on organisms. Symp. Soc. Exp. Biol., vol. 26. Academic Press Inc., New York. 170. Stanley, S. O., and R. Y. Morita. 1968. Salinity effect on the maximum growth temperature of some bacteria isolated from marine environments. J. Bacteriol. 95:169-173. 171. Stanley, S. O., and A. H. Rose. 1967. Bacteria and yeasts from lakes on Deception Island. R. Soc. (London) Phil. Trans. B. 252:199-207. 172. Starka, R. P., and J. L. Stokes. 1960. Psychrophilic bacteria from Antarctica. J. Bacteriol. 80:622-625. 173. Stokes, J. L. 1963. General biology and nomenclature of psychrophilic bacteria, p. 187-192. In N. E. Gibbons (ed.), Recent progress in microbiology. University of Toronto Press, Toronto. 174. Stokes, J. L. 1968. Nature of psychrophilic microorganisms, p. 221-233. In J. Hawthorne (ed.), Low temperature biology of foodstuffs. Pergamon Press, Oxford. 175. Sugimoto, S., and Y. Nosoh. 1971. Thermal properties of fructose 1,6-diphosphate aldolase from thermophilic bacteria. Biochim. Biophys. Acta 235:210-221. 176. Tai, P.-C., and H. Jackson. 1969. Mesophilic mutants of an obligate psychrophile, Micrococcus cryophilus. Can. J. Microbiol. 15:1145-1150. 177. Tsiklinsky, M. 1908. La flore microbienne dans les regions du Pole Sud. Expedition antarctique francaise, 1903-1905. Masson et cie, Paris. 178. Upadhyay, J., and J. L. Stokes. 1962. Anaerobic growth of psychrophilic bacteria. J. Bacteriol. 83:270-275.
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179. Upadhyay, J., and J. L. Stokes. 1963. Temperature-sensitive formic hydrogenlyase in a psychrophilic bacterium. J. Bacteriol. 85:177-185. 180. Vanderleck, J. 1917. Bacteria of frozen soils in Quebec. Trans. R. Soc. Can. Ser. III, Sect. IV. 11:15-39. 181. Vanderleck, J. 1918. Bacteria of frozen soils in Quebec. II. Trans. R. Soc. Can. Ser. III, Sect. IV. 12:15-39. 182. Vass, A. F. 1919. The influence of low temperature on soil bacteria. N.Y. Agric. Exp. Stn. (Ithaca) Mem. 27:1039-1074. 183. Vishniac, W. V., and S. E. Mainzer. 1973. Antarctica as a Martian model, p. 3-31. In P. H. A. Sneath (ed.), Cospar life sciences and space Research XI. Akademie-Verlag, Berlin. 184. Ward, J., and A. Cockson. 1972. Studies on a thermophilic bacillus: its isolation, properties, and temperature coefficient of growth. J. Bacteriol. 112:1040-1042. 185. Wiebe, W. J., and C. W. Hendricks. 1971. Simple, reliable cold tray for the recovery and examination of thermosensitive organisms. Appl. Microbiol. 22:734-735. 186. Wilkins, P. O., R. Bourgeois, and R. G. E. Murray. 1972. Psychrotrophic properties of Listeria monocytogenes. Can. J. Microbiol. 18:543-551. 187. Witter, L. D. 1961. Psychrophilic bacteria-a review. J. Dairy Sci. 44:983-1015. 188. Wright, R. T., and J. E. Hobbie. 1965. The uptake of organic solutes in lake water. Limnol. Oceanogr. 10:22-28. 189. Zachariah, P., and J. Liston. 1973. Temperature adaptability of psychrotrophic Pseudomonas. Appl. Microbiol. 26:437-438. 190. Zimmerman, A. M. (ed.). 1970. High pressure effects on cellular processes. Academic Press Inc., New York. 191. ZoBell, C. E. 1961. Importance of microorganisms in the sea, p. 107-132. In Proceedings of the low temperature microbiology symposium. Campbell Soup Co., Camden, N.J. 192. ZoBell, C. E., and J. E. Conn. 1940. Studies on the thermal sensitivity of marine bacteria. J. Bacteriol. 40:223-238. 193. ZoBell, C. E., and S. C. Rittenberg. 1938. The occurrence and characteristics of chitinoclastic bacteria in the sea. J. Bacteriol. 35:275-287.
Vol. 39, No. 2 Printed in U.S.A.
Psychrophilic Bacteria1 RICHARD Y. MORITA
Department of Microbiology and School of Oceanography, Oregon State University, Corvallis, Oregon 97331 INTRODUCTION
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BACKGROUND INFORMATION
Definition used to describe psychrophiles
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ISOLATION OF PSYCHROPHILIC BACTERIA
ECOLOGICAL DISTRIBUTION OF PSYCHROPHILES
OF
PSYCHROPHILIC
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149
Temperature characteristic of growth
Proteins
on
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BACTERIA.150
PHYSIOLOGY OF PSYCHROPHILES Effect of temperature
146 147
ECOLOGICAL SIGNIFICANCE OF PSYCHROPHILES
GROWTH
144
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153 ...........................................
respiration and viability
153
.154
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156 Thermally induced leakage 157 Thermally induced lysis .158 Temperature effects of permeability 158 Temperature effects on macromolecular synthesis Ecological factors affecting psychrophiles.159 .....................................................
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CONCLUSION
LITERATURE CITED
161
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.
INTRODUCTION For the purposes of this review, psychrophiles are defined as organisms having an optimal temperature for growth at about 15 C or lower, a maximal temperature for growth at about 20 C, and a minimal temperature for growth at 0 C or below. In order to avoid as much confusion as possible concerning the term "psychrophile" in this review, the term "psychrotrophic" will be used for organisms that do not meet the definition as stated above, even though the cited author(s) may have referred to them as psychrophiles. Likewise, the term "psychrophile" will include those organisms previously referred to as obligate psychrophiles. Yeasts that meet the above definition are known to exist but they will not be discussed in this review. The main reasons for employing the above definition are to eliminate future confusion and to adhere to the etymological significance of the term psychrophile. Data are rapidly accumulating on the existence of bacteria that meet the above definition. The optimal temperature listed in this definition adheres to that given in the Dictionary for Microbiology (88). Because of the recent isolation of psychrophiles and the small number of investigators dealing with this unique group of bacteria, there are many areas not yet investigated. Also, because of the restrictive definition employed in this review there is a paucity of data from which
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to draw. Nevertheless the major objectives of this review are to make microbiologists aware that "psychrophiles are psychrophiles" and to eliminate confusion in the literature concerning the term. The general effects of temperature on microbial physiology and growth are well discussed in many textbooks on microbiology and will not be repeated in this review. Hopefully this review will stimulate others to use the terms psychrophile and psychrotrophs as defined in this review. Although there may never be a complete agreement on these terms, a start must be made to end the confusion about the lower end of the thermal spectrum of microbial life. The terms applied to the various thermal groups are useful in microbiology, but not when confusion about them exists.
' Published as technical paper no. 4006, Oregon Agricultural Experiment Station. 144
BACKGROUND INFORMATION Textbooks in general microbiology for many years have classified microorganisms into various thermal groups based on their cardinal temperatures. Of these groups, the cold-loving bacteria are usually referred to as facultative psychrophiles and psychrophiles. However, the terms used most often in the published papers to describe these groups are psychrophile and obligate psychrophile. Furthermore, textbooks generally list the optimum growth temperature for psychrophiles as 15 C. Yet, to most microbiologists, the term "psychrophile" does not refer to the truly cold-loving bacteria but those that have the ability to grow at low temperatures.
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(The early literature would say "grow rapidly," meaning growth in at least a week.) The term psychrophile today is still misused, and the misnomer continues to be perpetuated in the literature. That certain organisms can overlap any two adjacent thermal groups is well recognized. For those mesophilic organisms that have the capability to grow at 0 C, the term "psychrotolerant" (98) and "psychrotrophic" are perhaps the best terms. The latter appears to be used more frequently in the literature today. There are many review articles dealing with "psychrophilic" bacteria (30, 83, 84, 85, 86, 159, 173, 187); however, they cover the psychrotrophic bacteria, not the psychrophilic bacteria. There is a common misconception concerning the maximum, minimum, and optimum growth temperatures for psychrophilic bacteria. The reports in the literature dealing with psychrophilic bacteria refer to organisms that are coldtolerant rather than cold-loving. This was especially true before the 1960's. Much of the confusion in the early literature arose from the lack of adequate data to support the concept of psychrophily. The early investigators concentrated on the ability to grow at or near 0 C. This ability to grow at 0 C was first reported by Forster (59) in 1887. The organisms were isolated from fish preserved by cold temperatures and were phosphorescent (bioluminetent). Later Forster (60)
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reported the isolation of other bacteria from various environments (natural water, foods, surface and in intestines of freshwater fish, and especially in sea water and ocean fish) that were capable of growth at 0 C. From his studies in the Antarctic, Ekelof (51) proposed the existence of psychrophiles. The main deterrent to obtaining adequate data in the early investigations was probably the lack of refrigerated incubators. In addition, the investigators were unaware of the thermolability of psychrophilic organisms at laboratory temperatures. Because of this, early investigators (as listed in Table 1) did not determine the cardinal temperatures for their isolates. This was the beginning of the confusion concerning the cardinal temperatures for psychrophilic bacteria. In 1902 Schmidt-Nielsen (161) grew microorganisms at 0 C which he termed "psychrophiles." A year later Muller (138) objected to the term since the organisms described actually grew well at higher temperatures. The cultures isolated by early microbiologists actually grew better in the mesophilic temperature range (Table 1). As'a result of this inconsistency, various terms were coined by early investigators to describe organisms which had the ability to grow at 0 C (Table 2). A survey of the literature for the cardinal temperatures of psychrophiles was made by Morita (121). Unfortunately, the psychrophiles described before 1960's were not
TABLE 1. Early investigators reporting bacterial growth at 0 C or lower Source material or organism
Investigator and year
Forster (1887, 1892)
Fischer (1888, 1893) Conradi and Vogt (1901) Baur (1902) Brandt (1902) Schmidt-Nielsen (1902)
Miller (1903) Feitel (1903) Tsiklinsky (1908) Richardson (1908) Pennington (1908) Ravenel et al. (1910) Conn (1910,1912, and 1914) Brown and Smith (1912) Reed and Reynolds (1916) Vanderleck (1917 and 1918) Vass (1919) Fay and Olson (1924) Bardach (1924) Rubentschik (1925)
Fish, natural waters, foods, wastes, rubbish, soil, surface and intestines of fish Harbor water and soils, certain pathogenic bacteria "proteus bacillus" Bacterium lobatum Bacterium lobatum Bacillus aquatile fluorescense non-liquefaciens, B. granulosum, B. paracoli gasoformans anindolicum, B. radiatum, B. tarde fluorescens, B. pestis, and B. proteus fluorescens. Sausage meat, fish, intestinal contents of fish, milk, vegetables, meal, garden soil and muck Dentrifying bacteria from deep sea water Antartic fish Nonproteolytic bacteria on frozen meat Milk Milk Frozen soils Frozen soils Milk Frozen soils Frozen soils Ice cream
Sewage, sludge and salt water Urbacillus psychrocartericus, Urosarcina psychrocartericus
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TABLE 2. Terms used to describe bacteria capable of at 0 C but not at 32 C, whereas the latter could growing at or below 0 C grow at 0 C and 32 C. Many microgiologists Term
Reference
Glaciale Bakterien Rhigophile Psychrophile Psychrotolerant Psychrocartericus Psychrobe
Forster (1892) Forster (1892) Schmidt-Nielsen (1902) Kruse (1910) Rubentschik (1925) Horowitz-Wlassowa and
Thermophobic bacteria
Edsall and Wetterlow
Cryophile
Edsall and Wetterlow
Facultative psychrophile Obligate psychrophile Psychrotrophic
Hucker (1954) Hucker (1954) Eddy (1960)
Grinberg (1933) (1947)
(1947)
truly psychrophilic (with one exception [177]). After surveying the literature, Berry and Magoon (13) stated that "the contention that a truly cold-loving or psychrophilic group of microorganisms exists, appears unwarranted by available evidence. While organisms recorded as growing at 0 C or below may with propriety be regarded as forming a cold-tolerant group, their temperature requirements for best growth definitely place them in the mesophilic class." Other workers (69, 82, 83, 84, 85, 104, 105) also concluded that there was a lack of adequate data to substantiate the existence of psychrophiles. In 1963, Ingram (86) stated that the term psychrophile was a misnomer. Since SchmidtNielsen (161) first used the term "psychrophile," it has been employed indiscriminately so that there is much confusion in the literature. Hagen et al. (71) point out that the term psychrophile has been used and defined in many ways and most of the definitions ignore the etymological significance of the word. Definitions used to describe psychrophiles. Schmidt-Nielsen (161) defined psychrophiles as organisms which are not only able to survive but also multiply at 0 C. The term "psychrophile," according to Horowitz-Wlassowa and Grinberg (81), should be used for the commonly isolated bacteria that could grow at 0 C but actually grow better at higher temperatures; and the term "psychrobe" should be used for the true psychrophile if they existed. Without adequate data in the literature for the existence of psychrophiles, the Dictionary for Microbiology (88), published in 1957, defined psychrophiles as those bacteria having an optimum growth temperature of 15 C or lower. Organisms that grew at 0 C were separated into two categories, obligate and facultative, by Hucker (82). The former were capable of growth
today consider psychrophiles to be organisms that more or less fit the facultative category. Ingraham (83) suggested that psychrophiles be defined not by their maximum or optimum growth temperature but whether they grow rapidly at 0 C. The term "rapidly" generally meant 1 or 2 weeks, a period much too long for psychrophiles. Later Ingraham and Stokes (85) redefined the term to refer to bacteria that grow well at 0 C within 2 weeks, disregarding the cardinal temperatures. The term "grow well" referred to a visable colony or turbidity to the naked eye. They also suggested that the obligate psychrophile be defined as those bacteria that grew rapidly at 0 C but also grew most rapidly at temperatures below 20 C. The facultative psychrophile was capable of growth at 0 C. Microorganisms capable of growing at 5 C and below, according to Eddy (48), should be classified as psychrotrophic regardless of the optimum temperature and that the term psychrophile should be used only when a low optimum temperature is implied. There are many microbiologists who employ the term psychrotrophic (e.g., 47, 102, 114, 137, 186, 189), and this term is gaining wider acceptance among food and dairy microbiologists. Ingram (86) gives the best reasons published in the literature as to why the term psychrotroph should be employed for most of the organisms listed in the literature as psychrophiles. Baxter and Gibbons (10) defined psychrophiles as organisms with a temperature optimum of 20 C or below. Rose (158) applied the term to organisms having an optimum for growth at 25 C or below without reference to growth at or near 0 C and defined obligate psychrophiles as having a maximum growth temperature of around 30 C. The definition employed most often in the literature is the one proposed by Stokes (173) in which he defined psychrophiles as organisms which grow at 0 C so that they can be detected visually within 1 week; facultative psychrophiles have an optimum temperature of 20 C or higher, whereas the optimum growth temperature of obligate psychrophiles is 20 C or lower. The more recent definitions for psychrophile by Stokes (173) and Rose (158) are discussed by Ingram (87), who points out the inadequacies of their definitions. Ingram (87) also states that the term psychrophile has been misapplied to organisms which grow at 20 but not at 30 C and that there is a preponderant group of organisms, with optimum temperatures between 20 and 30 C, that are not "facultative psychrophiles" in the true meaning of the term. This latter group
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cannot be named satisfactorily (87). For the best discussion of the inconsistency in the logic of nomenclature dealing with psychrophiles, Ingram (87) should be consulted. Optimum growth temperature, ability to grow at low temperature, method of enumeration, and criteria which are independent of the temperature have been used to define psychrophiles (187). Different definitions are held by various areas of microbiology (e.g., dairy and food microbiology). All this adds to the confusion in the literature and to the definition of the term "psychrophile." For more discussion on the subject, Berry and Magoon (13), Hucker (82), Ingraham and Stokes (85), Witter (187), Ingraham (84), Ingram (87), and Morita (121) should be consulted. If the definition of psychrophile is to be changed in the future, then it is my opinion that the cardinal temperatures will be lower than those that I have given in my definition.
ISOLATION OF PSYCHROPHILIC BACTERIA Tsiklinsky in 1908 (177) reported the existence of a psychrophile. The psychrophile was isolated from the excrement of a fish (environmental temperature was - 1.5 C) and had a growth temperature range from 0 to 20 C, with an optimal temperature for growth at 16 C. It appears that the isolate went into limbo and the significance of this isolate (probably a vibrio according to her description) has been neglected by microbiologists. In 1948, Borg (15) isolated the etiological agent for "cold water" disease of silver salmon. The organism was assigned the name Cytophaga psychrophila and had an optimum temperature between 15 and 20 C. This organism, the first psychrophile to be well characterized, fits the definition of psychrophile better than any organisms described prior to 1963, but unfortunately Borg's thesis went unnoticed. In 1964 Eimjhellen (71), Morita and Haight (135), and Sieburth (164) reported the existence of psychrophilic bacteria. Since these reports, others have reported the existence of psychrophilic bacteria. All the isolates were from the marine environment. Eimjhellen isolated his organism from flounder eggs. Because the organism produces a red pigment, it was tentatively designated as a Serratia sp. but was later classified as Vibrio psychroerythrus (41). The red pigment of V. psychroerythrus is different from five other prodigiosin-like compounds found in other organisms (39). Morita and Haight (135) isolated their organism from oceanic waters 125 miles off the Oregon coast (1,200 meters, 3.24 C) and was identified as
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Vibrio marinus, MP-1 (32). Sieburth's isolates from Narragansett Bay, R.I., were Arthrobacter spp.
The two main reasons why microbiologists in the past were not successful in isolating psychrophilic bacteria are probably the improper selection of source material and the laboratory procedure for handling material. The source material must be selected carefully. The past history of the source material is critical since it must never reach warm temperature for any extended period of time. For example, if sufficient radiant energy should fall on the source material it might be adequate to thermally inactivate the psychrophilic bacteria, if present. Hence the environment from which the source material is taken must remain cold constantly. Llano (103) recorded a change from -15 to 27.8 C on rock surfaces in the Horlick Mountains (40 from the geographical South Pole) within 3 h due to solar radiation even though the air temperature was around 0 C. Boylen and Brock (17) were not able to demonstrate the development of a true psychrophilic flora in Lake Wingra sediments which had a temperature of 4 C for only 3 months of the year. Brock et al. (21), using proper techniques for the isolation of psychrophiles, were unable to obtain a psychrophile from the cold springs in caves located in southern Indiana. The above only indicates that the past history of the environment from which sample material is taken is very important. Oceanic water has been cold for eons of time, and as a result it is easy to isolate psychrophiles in waters below the thermocline in the ocean as well as in arctic and antarctic waters. The isolation procedure for psychrophilic bacterial cells calls for the usual microbial methods except that the source material, pipettes, media, etc., are kept cold at all times (22, 121, 185). The above emphasizes the abnormal thermal lability of the psychrophilic bacteria. The pour plate technique employing agar medium cannot be recommended. It was shown that most marine forms are thermosensitive to the plating temperature of agar (192). The psychrophile, V. marinus MP-1, can be rendered nonviable when exposed to melted agar at 42 C for 1 min (135). The enrichment technique, with the desired medium, is recommended before trying to isolate pure cultures. The inoculated enrichment medium should be incubated between 0 and 5 C before proceeding with the isolation procedure. Stock cultures of psychrophiles should be transferred frequently, and at no time should the cultures be exposed to elevated temperatures for any extended period of time. Frequent transfer is necessary since the temperature of
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the laboratory refrigerator is nearly optimal for the growth of some psychrophiles. Psychrophiles are not rare in permanently cold environments and can be isolated easily if the correct application of the above procedures is followed.
ECOLOGICAL DISTRIBUTION OF PSYCHROPHILES Most biologists tend to neglect the fact that the dominant environment of the biosphere is cold. The polar regions and the oceans are 14 and 71% of the earth's surface, respectively. More than 90% (by volume) of the oceans is 5 C or colder. The oceans are a natural habitat for psychrophilic bacteria. Because of this situation it is relatively easy to isolate psychrophilic bacteria from below the thermocline of the oceans, in salt wedges (cold oceanic water intrusion) of bays as well as both polar regions. Psychrophilic bacteria have been isolated off the Oregon coast as well as the Arctic and northern Pacific waters (130). For instance, 65 miles off the Oregon coast 79 and 27 psychrophiles were found per 100 ml of sea water obtained from depths of 350 and 2,800 meters, respectively. Freshwater psychrophilic bacteria are readily isolated adjacent to melting glaciers in Alaska (Burton, personal communication). Sieburth (164, 166) examined various organisms isolated at 0, 18, and 36 C from waters of Narragansett Bay, R. I., and found that there definitely were microorganisms belonging to the psychrophilic group. These psychrophiles were only obtained on plates incubated at 0 C upon initial isolation and only when the water temperature was below 10 C. Many of the isolates had optimum temperatures for growth around 10 C. They were tentatively identified as Arthrobacter spp., Achromobacter spp., Flavobacterium spp., Vibrio spp., and Pseudomonas spp. A seasonal selection for the various thermal types was also observed so that the minimal, optimal, and maximal growth temperature of the dominant microbial flora continuously change as a result of the continual seasonal changes in water temperature. For example, certain isolates made at 0 C in the spring failed to grow above 16 C whereas isolates made during the summer could grow above 16 C. According to Sieburth (164), the life cycle of Arthrobacter spp. appear to be associated with different temperatures (i.e., life cycle was a function of water temperature). From the North Sea, Harder and Veldkamp (75) isolated 69 strains of bacteria capable of growth at low temperatures. Ten of these orga-
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nisms had a maximum growth temperature below 20 C, six of which were Pseudomonas, two were Vibrio, and two were Spirillum. Gounot (64, 65, 66) and Moiround and Gounot (120) investigated the microbial population in sediments and water below arctic glaciers, in arctic caves, and other cold environments. In her studies, she isolated an Arthrobacter sp. which had an optimum temperature for growth at 13 C. It also grew well at -5 and 18 C. She assigned the name Arthrobacter glacialis to the organism. Approximately 800 isolates were obtained from Arctic sediment samples (119), of which two hundred fifty were found to grow well on agar at 0 but not at 25 C. Unfortunately, these organisms were never characterized thermally. Stanley and Rose (171) isolated psychrophilic bacteria from various lakes on Deception Island, Antarctica, and found 2, 9, and 18 bacteria with optimum growth temperatures of 5, 10, and 15 C, respectively. A preliminary examination of 488 cultures isolated from the Antarctic was made by Wiebe and Hendricks (185) who found that 37.5% of the organisms would not grow at 15 C and 65% did not survive at 20 C. One of the species, designated as E5-4-4, had a maximal growth temperature at 10 C (31). Matches and Liston (115) isolated 288 clostridia from marine sediment collected in the Puget Sound area, of which 7 would not grow above 20 C. Five out of 29 clostridia studied by Finne and Matches (56) were psychrophiles. Approximately 400 isolates have been made by my associates during Cruises 46 and 51 of the U.S.N.S. Eltanin in the Antarctic. One hundred fifty of the 400 isolates were selected for further characterization, and it was found that 148 would grow at 10 C, 106 would grow at 15 C and 98 would grow at 20 C; 144 were oxidase positive, seven were agar digestors. All isolates demonstrated lipolytic activity and approximately 50% were chitin digestors. Also present were several psychrophilic anaerobes. Vishniac and Mainzar (183) investigated the dry valleys in southern Victorialand, Antarctica, for microbes. This area represents one of the driest regions on earth and the relative humidity is about 10%. Although temperature was not mentioned in the paper, the in situ 1-h experiments demonstrated the presence and activity of bacteria inhabiting the dry soils. The reason for the 1-h experiments was dictated by the ability of the investigators to work comfortably with their hands at subfreezing temperatures coupled with a wind velocity of 60 km/h. Since miltiplication of the microorganisms were
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VOL. 39, 1975
observed when water was available, the organisms could have been psychrophiles or psychrotrophs. Their conclusion was that the bacteria underwent rapid division within a few hours of the day when water was available. This water was the result of periodic snowfalls which melted when the radiant energy was present in sufficient quantity. If water was not available, the bacteria laid dormant for a week or 10 days at a time. Psychrotrophic bacteria have been known to exist in the Antarctic region since the early investigations conducted by Ekelif (51) during the Swedish Antarctic Expedition of 1901-1903. Most bacteriological investigations (16, 26, 27, 29, 42, 62, 117, 172) focused mainly on the demonstration of various types of bacteria present and not the existence of psychrophiles. Generally no precautions were taken to insure that psychrophiles would not be inactivated. For instance Boyd and Boyd (16) used nutrient agar for dilution of their Antarctic samples as well as an incubation temperature of 20 to 22 C, whereas Darling and Siple (42) used an incubation temperature of 35 and 37 C. Cameron et al. (28) incubated their cultures at 2 and 20 C but in only two samples were there more bacteria growing at 2 than at 20 C. Starka and Stokes (172), using soil samples, demonstrated the presence of psychrotrophic bacteria. It is difficult to determine from their data whether or not a psychrophilic bacterium was isolated that meets the definition given in this review. Since soil samples were employed it is likely that the true psychrophiles may have expired in the source material. For further details concerning the early Antarctic microbiological literature Sieburth (165) should be consulted. Unfortunately, there has never been an extensive effort made to determine ecological distri-
bution of psychrophiles in permanently cold environments. In the marine environment it is easy to isolate psychrophilic bacteria as well as many psychrotrophic forms. The latter generally have a wide growth temperature range. ECOLOGICAL SIGNIFICANCE OF PSYCHROPHILES In the natural environment it is very difficult to assess the importance of psychrophiles in the cycle of matter. Generally within any sample both psychrophiles and psychrotrophs can be isolated. If sufficient substrates are present at low temperatures near 0 C, a psychrophile can outgrow the psychrotrophic forms (130). Even at low substrate concentration and low temperature the psychrophiles will outgrow the psychrotrophic bacteria in continuous culture (77). These laboratory findings suggest that there will be a continuous selection for bacteria whose activity is greatest near the environmental temperature, especially in environments showing seasonal temperature changes (140, 164, 166). In Antarctic field experiments employing the heterotrophic activity method at temperatures slightly above and below 0 C, the average Vmax for glutamate uptake in water samples taken at the bottom of the euphotic zone was 1.09 x 10-2 Ag/liter per h. A portion of the data obtained during Cruise 46 of the U.S.N.S. Eltanin is given in Table 3. The heterotrophic activity method (79, 148, 187) is a kinetic approach which is based on the principles of the Michaelis-Menton theory. By using the kinetic approach, the maximum velocity of uptake (Vmax), the turnover time (Ti), and the sum of a "transport constant" and the natural substrate concentration (K, + SO) can be calculated. This type of data can be obtained in field studies where the water samples are immediately ana-
TABLE 3. Heterotrophic activity on "4C-labeled substrates by the indigenous microflora in water samples taken from various depths at Stations 8 and 9 during Cruise 46 of the U.S.N.S. Eltanina Station
Depth (m)
Substrate
Vma,,x(/g/liter per h)
8
10
Glu Glut Glu Glut Glut Tyr Lys Glu Glut Glu Glut
1.8 x 10-2 8.7 x 10-2 3.0 x 10-3 1.3 x 10-2 1.1 x 10-3 4.0 x 10-3 7.3 x 10-4 3.1 x 10-3 6.7 x 10-3 5.3 x 10-4 1.6 x 10- 3
50
9
500 10
50 a The water
Tt(h) 570 112 818 382
12,350 5,000 3,704 4,800 1,544 1,800 1,298
temperature was 0 C ± 1. The percentage of respiration equals 14CO, x
(jsg/liter)
K, + S.
% Respiration
10.4 9.7 2.4 5.0 13.8 2.0
36 74 36 70 77 9 4 24 64 21 51
2.7 11.8 10.3 1.0 2.0
100/"4C uptake + 14CO2.
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lyzed at an in situ temperature. Other measurements with other amino acids were also made (134). Employing the best field results obtained during Cruise 46 of the U.S.N.S. Eltanin, one can extrapolate these figures to a longer period of time and for a larger volume of water. By extrapolation 2.47 x 107 g/nautical mile3 per
day of carbon dioxide can be produced by the indigenous microorganisms in the Antarctic water (-1.0 C) (131). If one could legitimately extrapolate this figure to the entire volume of the ocean, then about 1015 g of carbon dioxide would be produced per day. The foregoing also indicates that organisms can function very well in cold environments where the temperature is 0 C. There are approximately 129 x 1018 g of carbon dioxide in the ocean (160). Although the conditions necessary to produce the extrapolated amount of carbon dioxide by microbial activity may not be present, the potential to produce this quantity of carbon dioxide is present. In consideration of the importance of the entire carbon dioxide system on earth, the function of microbes in the environment should not be taken lightly (125). Many of our psychrophilic isolates in our laboratory are extremely good chitin digestors at temperatures slightly above and below 0 C. The supernatant culture fluid of psychrophile 18-500 (tentatively identified as a Vibrio sp.) displays chitinase activity at 0 C. Although the organism has a maximum temperature for growth at 13 C, its chitinase functions at a much higher temperature (Table 4). ZoBell and Rittenberg (193) estimated that the annual chitin production from copepods is several billion tons. This does not take into consideration the amount of chitin produced by other marine forms (e.g., shrimps, crabs, etc.). Since fish eat organisms having exoskeletons composed of chitin, the supernatant fluid of a sculpin's (Leptocottus armatus) stomach content was checked for the presence of chitinoclastic bacteria. Per milliliter of supernatant fluid, 1.5 x 1010 chitinoclasts were isolated. The sculpin was obtained in the winter where its environmental temperature was 6 C. When a portion of the supernatant material from the fish was treated with toluene and placed in the presence of chitin, 162 gg of N-acetylglucosamine/g (dry weight) per h at 5 C was produced. Other exoenzymes are also produced by psychrophiles so that the cycle of matter can continue in environments close to 0 C. Whether or not there are pathogenic psychrophilic bacteria in the aquatic environment has yet to be investigated. The organism Cytophaga
TABLE 4. Chitinase activity by supernatant culture fluid resulting growth of 18-500 at 5 C for 5 daysa (C) Temp Temp (C)
N-acetylglucosamine produced ml)
~~~(;&g/h per
0 3 6 10 13.5 18.5 20 27 35
2.0 2.4 3.5 3.5 5.0 6.6 8.0 9.5 6.8
40
1.1
a The culture medium contained: yeast extract (Difco), 1.2 g; Trypticase (BBL), 2.3 g; sodium citrate, 0.3 g; L-glutamic acid, 0.3 g; sodium nitrate, 0.05 g; ferrous sulfate, 0.005 g; sodium chloride, 24 g; magnesium sulfate, 7 g; magnesium chloride, 5.3 g; potassium chloride, 0.7 g; and purified chitin, 1 g.
psychrophila (psychrotroph) does have the ability to cause cold-water disease (15, 148). Shell disease, burned spot disease, and rust disease (necrotic lesions in the exoskeleton of crustaceans) may be directly or indirectly caused by chitinoclastic bacteria since these organisms are not always isolated from the necrotic lesions (37). Apparently no precautions have been taken to isolate psychrophilic bacteria from shell disease. The role that psychrophiles play as producers of growth factors (e.g., B12), as food for higher forms, or as producers of hormonal-like compounds has yet to be assessed. GROWTH OF PSYCHROPHILIC BACTERIA Growth. The psychrotrophic bacteria described in the literature are actually slow growing at low temperatures and therefore are not representative of the psychrophiles that meet the definition in this review. In the figures and tables presented by various authors (e.g., 52, 78, 79, 83, 168, 174), the growth of the psychrotrophic bacteria at low temperatures (0 to 10 C) may be in the order of many hours, days, or even weeks. Because of the past and current data in the literature, the commonly accepted concept is that low temperature is iminical to the growth of "psychrophilic" bacteria. This concept is not valid when applied to psychrophiles. In order to refute the common concept that low cell yields and slow grow rates are manifestations of low incubation temperatures for bacteria, Morita and Albright (126) demonstrated that the psychrophile V. marinus MP-1 grows
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PSYCHROPHILIC BACTERIA
well at low temperature. The best cell yields found in the literature for previously described psychrotrophic strains of Pseudomonas fluorescens are by Hess (79) and Sinclair and Stokes (168). The former demonstrated that 9 x 108 cells could be obtained at 20 C in 72 h, 9.0 x 104 at5 C in72h, and3.4 x 103at0 Cin72h.The latter was able to obtain 1.8 x 1010 cells/ml at 30 C in 30 h and 1.7 x 1010 cells/ml at 10 C in 72 h. These data can be contrasted with the cell yields of V. marinus (126) which were 1.3 x 1012 cells/ml at 15 C in 24 h and 9 x 109 cells/ml at 3 C in 24 h. It can be readily seen that low temperatures of incubation for psychrophiles are not detrimental to large cell yields. Table 5 lists some of the generation times for previously reported psychrotrophic bacteria. It can be noted that the generation times are very long, especially for temperatures close to 0 C. In contrast the psychrophiles grow very well at low temperatures (31, 71, 123, 154, 166). The generation times for V. marinus at 15 and 3 C, respectively, are 80.7 and 226 min (126). Unfortunately the growth characteristics of all psychrophilic bacteria in this laboratory have not been investigated, but observations indicated that growth is excellent when cultures are incubated overnight at 0 to 3 C. Figure 1 illustrates the time-temperature growth plot of a psychrophile isolated from Antarctic waters. For V. marinus MP-1 the maximum growth temperature is about 20 C, the optimum growth temperature is about 15 C, and growth occurs at 1 C (lowest temperature tested; 135). We currently grow many psychrophiles routinely at -2.5 C but have never tried to determine the minimum temperature for growth for the various psychrophiles due to the lack of properly refrigerated incubators. How-
growth does occur at -5.5 C, the lowest we have tested. V. psychroerythrus, isolated by Eimjhellen and first reported by Hagen et al. (71), has a growth temperature range of 0 to 19 C, with an optimum growth temperature of about 15 C. When this organism was subjected to a temperature shift-up experiment (5 to 19.5 C), there was a slight temporary increase in the growth rate which lasted only 5 h. This increase was followed by a decrease in turbidity (70). The Pseudomonas, studied by Harder and Veldkamp (75) possessed a maximum growth tem-
ever,
temperature
1.6
O
1.2
0 0
@ 0.8 0
0.4
0
-2
0
2
6
4
8
FIG. 1. Growth of an Antarctic psychrophile (AP-2-24, designated tentatively as a Vibrio sp.). Incubation period was 80 h in Lib-X medium employing a temperature gradient incubator (Scientific Industries, Inc.). If the culture was incubated for 197 h, growth was noted at 10.5 C. Lib-X medium contains: yeast extract (Difco), 1.2 g; Trypticase (BBL), 2.3 g; sodium citrate, 0.3 g; L-glutamic acid, 0.3 g; sodium nitrate, 0.05 g; ferrous sulfate, 0.005 g; Rila marine mix, 33 g; and distilled water, 1,000 ml. The pH was adjusted to 7.5.
TABLE 5. Examples of generation time for psychrotrophic bacteria Organism
Pseudomonas fluorescensa Pseudomonas fluorescens0
Pseudomonas sp.
Generation time
Reference
6.68 h at 0.5 C 30.21hatO C
Hess (1934b)
10.65 h at 5 C 26.41 h at 0 C 2.66 h at 10 C
Hess (1934b)
Ingraham (1958)
10.33hat0 C Strain 82
21.58 h at 5 C
21.23hatO C Pseudomonas fluorescensc Bacillus psychrophilus Micrococcus cryophilus Pseudomonas strain 92
10
Temperature (C)
4.17 to 8.20 h at 4 C 6.30 h at -5 to -7 C 28.33 h at 0 C 11.1 h at 2 C
Upadhyay and Stokes (1962) Olsen and Jezeski (1963) Larkin and Stokes (1968) Tai and Jackson (1969) Frank et al. (1972)
a Inoculum prepared at 20 C. b Inoculum prepared at 5 C. c Generation time depends on type of substrates, stationary or aerated cultures.
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perature between 19 and 20 C with an optimum growth temperature at 14.5 C. The inability to grow at temperatures above 20 C might be the price the psychrophiles must pay for the advantage they have in the very cold natural environment (75). An organism in our laboratory designated as Ant-300 (tentatively identified as a Vibrio sp.) has a maximum growth temperature of about 13 C and an optimum growth temperature of 7 C; however, the minimum growth temperature has not been determined (63). (An
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electron micrograph of a section of Ant-300 is shown in Fig. 2). There are quite a few isolates in our laboratory that have maximum growth temperatures of 13 C, but the majority have maximum growth temperatures between 20 C and 13 C. Classification of our psychrophiles is currently underway. The growth and activity of marine microorganisms is generally greater at temperatures 10 to 20 C above the environmental temperatures from which the organisms are isolated (14, 191).
FIG. 2. Electron micrograph of a thin section of Ant-300, a marine psychrophile. Cells were grown in glucose medium, washed in artificial sea water, fixed in 5% glutaraldehyde, and post-fixed in 1% osmic acid. Thin sections were stained with uranyl acetate and lead acetate. Bar = 1 jAm.
VOL. 39, 1975
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153
Although the maximum growth rate is attained tively, at the lower end of the thermal spectrum at optimal growth temperature, the maximum of growth. Their data helps establish the fact cell crop is obtained when organisms are incu- that making an Arrhenius plot for a microorgabated below the optimum for growth for ex- nism's growth response to temperatures is extended periods of incubation, probably due to cessive work when simple growth curves of the the efficient use of substrates (167). This situa- organism at different temperatures will give tion holds true for Ant-300 (63). Generally the essentially the same results. ocean environment is low in substrates, and the low temperature of the environment allows the PHYSIOLOGY OF PSYCHROPHILES utilization of the available substrates efficiently. The genetic constitution of the cell deterto of psychrophiles ability Nevertheless the function well in a cold environment has been mines its cardinal temperatures for growth. noted (131, 132, 133, 134). Ten to 20 C above the Although this premise is generally accepted, the organism's original environmental temperature data in the literature are completely lacking in may be too high for many of the currently order to postulate the exact genetic basis of known psychrophiles. However, this general psychrophily. Most of the research emphasis concept probably does apply to the psychro- have been placed on physiological studies. trophic bacteria. Many laboratory studies on Nevertheless, there is genetic evidence for the marine bacteria do not generally employ envi- conversion of a mesophile to a psychrotroph by ronmental temperatures, and therefore the data transduction (145), ultraviolet light (5), and the mutation by ultraviolet light from a psychrois difficult to extrapolate to the environment. Temperature characteristic for growth. It troph to a mesophile (176). Since phage have was suggested that the temperature characteris- been isolated from psychrotrophic bacteria (43, tic for growth (Au) could be used to determine 44, 99) transduction between cold-loving orgawhether or not microorganisms were psychro- nisms is possible. Unfortunately none of the philic or mesophilic (83). The bacterial growth host cells for the phage are psychrophiles. It rate is substituted for the reaction rate in the appears unlikely that either mutagenesis by van't Hoff-Arrhenius equation to obtain the At ultraviolet irradiation or transduction would value; g is then analogous to the activation change the total enzymic make-up of cells energy. Hence psychrophiles should have a sufficiently to promote psychrophily, since lower AL value than mesophiles. This concept there appears to be more than one enzyme in a was found to be erroneous (73, 162). Ingraham psychrophilic cell that is abnormally thermola(83) and Shaw (162) used a mesophile and a bile. One of the hypotheses of microbial evolution psychrotroph to obtain their results, whereas Hanus and Morita (74) employed a psychro- is that thermophiles were the first to evolve phile, a psychrotroph, and a mesophile all (20), followed by the mesophiles and then the belonging to the same genus. Baig and Hopton psychrophiles. The evolution of psychrophilic (6) found that organisms isolated from natural bacteria is probably due to many genetic events water having the fastest growth rates at 5 and since a psychrophile has more than one enzyme 10 C had the lowest g values and that the that is abnormally thermolabile and has differcorrelation is best when temperatures from 15 to ences in the membrane. According to Allen (4) 25 C are employed. Nevertheless, they state organisms that have lived in a cold environment that a low A value and psychrophilic properties for generations may have experienced mutation may be unrelated attributes of the bacteria that caused some of their critical proteins or capable of persisting in natural waters. Working protein complexes to become relatively heat with a thermophile and comparing their data labile. There are probably many pathways leadwith Hanus and Morita (74), Ward and Cockson ing to psychrophily, since we have found that a (184) could find no correlation between g and specific abnormally thermolabile enzyme in one psychrophile may not be abnormally thermolathe optimal growth temperatures. Harder and Veldkamp (77) determined the bile in another psychrophile. In the eighth edition of Bergey (25), V. maximum specific growth rates of four psychrotrophic and four psychrophilic bacteria. Their marinus MP-1 is classified as V. fisherii. Biodata show that the difference is found in the chemical characteristics used in taxonomy do temperature range in which the Arrhenius plots not characterize the thermal properties of enare linear, rather than in different temperature zymes or membranes. There is at present no characteristics of growth. Deviation from linear- data in the literature that indicate how the ity was at -4.5 to -5.0 C and 4 to 5 C for the code (probably sequence of codons) for "mesopsychrophiles and the psychrotrophs, respec- philic" enzyme differs from the code for "psy-
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crophilic" enzyme. The thermal properties of psychrophiles should be taken into consideration in bacterial classification, if we accept the hypothesis that the psychrophiles evolved from mesophiles. Krajewska and Szer (97) conducted comparative studies of amino acid incorporation in a cell-free system of a psychrotrophic Pseudomonas and Escherichia coli and found that the ribosomes and supernatant fractions of the psychrotrophic Pseudomonas and E. coli are interchangeable at 30 to 45 C in the poly U-promoted phenylalanine incorporation. At 2 to 9 C only the psychrotrophic Pseudomonas ribosomes are active with both supernatant fractions, and therefore the ability to perform protein synthesis at low temperature is related to ribosomal specificity. These authors also point out that the psychrotrophic system operates at a relatively high fidelity compared to the mesophilic and thermophilic subcellular systems. Does this indicate that through evolution the psychrophiles are more stable in terms of their genetic fidelity compared to the mesophiles and thermophiles? A positive correlation was found between the thermal stability of the ribosomes and the maximal growth temperature of the organisms studied. The percentage of guanine plus cytosine content of ribosomal ribonucleic acid (RNA) for V. marinus MP-1 and Bacillus stearothermophilus was 52.1 and 59.3 mol%, respectively. The percentage of guanine plus cytosine content of the deoxyribonucleic acid (DNA) was reported for the two organisms just mentioned as 40 and 51.5 mol%, respectively. Pace and Campbell (147) state that the thermal stability of the ribosome lies in the interaction between the RNA and the ribosomal protein. Numerous reasons have been put forth to explain why microorganisms expire when exposed to temperatures above their maximal growth temperature, but most of the research has been done with mesophiles. These explanations include the accelerated use of the intracellular amino acid pool (70), enzyme inactivation by elevated temperature (50, 100), changes in extent of cellular lipid saturation (91), disruption of the intracellular organization (84), inactivation of an enzyme-forming system (138, 178), the loss of permeability control (129), and thermally induced leakage of ribonucleotides, ammonia, ninhydrin-positive material, potassium, and phosphate (173). Effect of temperature on respiration and viability. Psychrophilic bacteria function well in terms of growth and respiration at environmental temperatures from which they were
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isolated. V. marinus MP-1 (grown at 15 C) was shown to be able to respire very well (13 ,u of oxygen uptake/mg of cell protein per 30 min) at its optimal growth temperature (15 C) and near its environmental temperature (4 C; 6.5 qI of oxygen uptake/mg of cell protein per 30 min). Cells grown at 4 C utilized 7.25 ,ul of oxygen/mg of cell protein per 30 min at 15 C and 3.5 ,ul of oxygen/mg of cell protein per 30 min at 4 C (72). The temperature at which the cells were grown governs their oxygen uptake. This same pattern of results was also shown by Christian and Wiebe (31). This situation could be attributed to the fluidity of the membrane at different temperatures, which, in turn, is reflected in the ability of the cells to transport various substrates at different temperatures. The respiratory ability of psychrophiles increases during the initial heat treatment at temperatures near and slightly above the maximum temperature for growth (31, 63, 75). Harder and Veldkamp (75), working with a psychrophilic Pseudomonas in continuous culture, found that the maximum growth temperature for the organism was 20 C, yet the maximum rate for respiration was at 23 C. Although the actual Q02 increased towards the maximum and minimum growth temperatures, there was a concomitant decrease in the molar growth yields. This decrease in the molar growth yields suggested to Harder and Veldkamp (75) that the energy needed per unit weight per unit time to maintain a certain growth increased with increasing deviation from the optimum growth temperature. Christian and Wiebe (31) restate Harder and Veldkamp's (75) and Brown's (23) results by saying that the portion of energy capable of being produced via respiration, which is utilized for reproduction, decreases as temperature increases. At 13 C (maximum growth temperature for Ant-300) and at slightly higher temperatures, the amount of carbon dioxide respired decreased with exposure time in the presence of exogenous glucose (Table 6). This decrease in respired carbon dioxide appeared to be the result of heat-induced restriction on glucose uptake which, in turn, accelerates starvation of the cells (63). An exposure of 110 min to 20.4 C will greatly reduce the ability of V. marinus MP-1 to take up oxygen endogenously or in the presence of glucose, thereby indicating that a thermal lesion(s) in the cells has taken place (157). This thermal injury appears to be a sequence of events since V. marinus MP-1 goes from an uninjured state rapidly through a state of reversible injury and then to a condition of
155
PSYCHROPHILIC BACTERIA
VOL. 39, 1975
TABLE 6. Uptake and "CO2 respired in the presence of [U-14Clglucose by Ant 300 cells exposed at various temperatures for various periods of timea dpmb Period of heat shock (h)
0 10 20 30 40 50 60
5 cc
00o
Uptake
% Respd
CO,
5039 5426 4629 5878 5064 5969 5499
17,852 13,658 11,240 12,515 10,894 11,670 11,042
34 40 41 47 46 51 50
12,381 11,244 10,031 8,873 7,995 6,840 5,211
CO,2
Uptake % Resp 32,632 22,124 19,428 16,907 15,223 13,701 9,231
18 C
15 C
13 C
38 51 52 52
53 50 56
14,018 13,100 8,235 6,859 4,904 3,984 1,754
Uptake % Resp 36,555 24,528 17,156 12,295 8,054 5,902 2,746
38 53 48 56 61 68 64
CO,
Uptake % Resp
15,698 38,776 9,536 20,154 3,422 6,599 1,052 1,052 383 383 144 144 0 0
40 47 52 100 100 100
a The cells were starved in a menstruum without any carbon source for 24 h at 5 C prior to exposure at various temperatures for various periods of time. After exposure, [U- 14C ]glucose (final concentration was 0.9 Ag/ml with a specific activity of 180 mCi/mmol) was added, and the mixture was incubated at the same temperature of exposure for an additional 2 h. Cells from an exponential phase culture (OD = 0.25 at 600 nm) grown in a glucose medium for 28 h at 5 C were employed. b dpm, Disintegrations per minute. Temperature of exposure and subsequent incubation with [4C ]glucose. dPercentage of respiration = CO/uptake x 100.
nonreversible injury or death (67). Recovery of the reversible injury of the cells depends on the recovery medium. The thermal tolerance of V. marinus MP-1 was tested, and it was found that the organism in growth medium would expire when exposed for 50 h to a temperature slightly above 20 C (organism's maximal temperature for growth; 135). A time-temperature relationship was also noted. An exposure to 28.8 C for 6.25 h or 32.7 C for 1.25 h would render the organism nonviable. If nutrients (energy source) were not present in the suspending menstruum, then viability was lost in 15 min at 31 C (72). An exposure to 20.5 C for 110 min in the absence of nutrient will reduce the viable count from 3.8 x 1011 to 1.6 x 109 cells/ml (157). Temperatures above the maximum for growth of psychrophiles, although they may be less than room temperature, are inimical to the organisms. Proteins. Koffler and Gale (96) reported that cytoplasmic proteins isolated from four thermophiles were much more thermo stable than similar preparations from mesophiles. Using identical procedures on E. coli and V. marinus MP-1, Steenbergen and Morita (unpublished data) found that the cytoplasmic proteins from the psychrophile were more heat labile than those from the mesophile. Furthermore, some of the cytoplasmic proteins were heat labile between the psychrophile's optimal and maximal growth temperatures (Fig. 3). According to Koffler (95) death by heat was postulated to be brought about through the injury of a few essential molecules, perhaps only one. Within
limits cell inactivation is reversible as long as there is an opportunity for repair. Malic dehydrogenase from V. marinus MP-1 was studied in relation to its abnormal thermolability (100). The enzyme present in washed cells was found to be stable between 0 C and the organism's optimal temperature for growth (15 C). Between the organism's optimal and maximal growth temperatures there was an inactivation of the malic dehydrogenase. Thermolability studies on cell-free extracts for malic dehydrogenase indicated that almost all the activity was lost in 10 min at 30 C. Even at 0 C, there was a considerable loss in activity. Partially purified malic dehydrogenase (20fold) was found to be optimally stable between 15 and 20 C, and the inactivation became very pronounced when exposed to temperature above 20 C. Oxygen uptake studies gave no indications of the denaturation of enzymes at or near the organism's maximal growth temperature
(76).
In cell-free extracts of V. marinus, Mathemeier (Ph.D. thesis, Oregon State Univ., Corvallis, Ore., 1968) found that lactic dehydrogenase, hexokinase, malic dehydrogenase, phosphofructokinase/glyceraldehyde 3-phosphate dehydrogenase complex, and aldolase lost nearly all their activity when incubated at 35 to 40 C for 1 h. However, there is a possibility that a protease could be present in the crude extracts since no protease inhibitor was included in the assay mixture. Aldolase was purified from the same organism and found to be similar in properties to the Type II aldolases isolated from
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proteinases gave lower temperature characteristics than did proteinases from a Cytophaga sp. Z 6 A-20 and P. fluorescens 612. Unfortunately, the cardinal temperatures for these organisms were not determined but they did grow at 0 C. CLi The supernatant fluid of V. psychroerythrus 0.a (118) was found to contain proteolytic enzymes z 20-2 that hydrolyzed casein, f3-lactoglobulin, and hemoglobin. Although the organism is a psy-2 4 20-40 chrophile, the activity of the caseinase was optimal at 40 to 45 C at pH 10. The enzyme is \ relatively thermostable in the presence of cal2 cium; hence it appears to be similar to the proteinase of mesophiles. The best activity was CLl found between 35 and 45 C in the presence of C: . 40- 60 calcium (118). This proteinase was separated into three fractions; two were stable at 55 C and 30 35 20 25 0 5 10 15 one was almost inactivated at 55 C (141). A TEMPERATURE (C) FIG. 3. Heat denaturation of various ammonium white variant of the organism produced only sulfate fractions of V. marinus MP-1 cell-free extract two fractions on Sephadex chromatography in tris(hydroxymethyl)aminomethane-hydrochloride (142). Purified tryptophan synthetase from a buffer, pH 7.4. (Ammonium sulfate fractions: 0 to 20, psychrotrophic organism, designated as B-160, 20 to 40, and 40 to 60% saturation. The cell-free ex- lost 50% of its activity at 30 C in 5 min, whereas tract was prepared from cells grown at 15 C for 24 h, the tryptophan synthetase from E. coli is stable treated with a Biosonik III probe, and centrifuged at 34,500 x g for 30 min to remove the cellular debris. for several hours at 50 C (C. S. Cheng, M.S. z
0
Ammonium sulfate fractions were placed at various temperatures in a polythermostat for 12 h, followed by removal of the precipitate by centrifugation at 34,500 x g for 30 min. The supernatant fluid of each fraction was diluted, and the protein concentration was determined by the Lowry method). (Unpublished data, Steenbergen and Morita.)
other bacteria (L. P. Jones, Ph.D. thesis, Oregon State Univ., Corvallis, Ore., 1973). The temperature optimum for the-aldolase was 25 C with no activity evident above 32 C, but activity could be observed at 4 C (lowest temperature tested). Within experimental error of amino acid analysis, the amino acid composition of the aldolase from the psychrophile was the same as that from thermophilic aldolase (174) and from rabbit aldolase (163). Glucose-6-phosphate dehydrogenase from V. marinus MP-1 is not as abnormally thermolabile like the malic dehydrogenase from the same organism (W. W. Miller, M.S. thesis, Oregon State Univ., Corvallis, Ore., 1968). Partially purified glucose-6-phosphate dehydrogenase was found to be stable between 5 and 26 C, but when it was exposed to 36 C for 1 h it lost 90% of its activity. Glyceraldehyde-3-phosphate dehydrogenase from the same organism was purified and found to be quite thermostable (Howard, Miller, and Becker, personal communication). Proteinases in culture supernatant fluids of various Arctic bacteria hydrolyzed casein most rapidly between pH 7.0 and 8.0 (119). These
thesis, Brigham Young University, Provo, Utah, 1970). Thermally induced leakage. Leakage of 260nm-absorbing material from V. marinus MP-1 was first noted by Robison and Morita (157) when the cells were exposed to moderate temperatures of 20 to 30 C. The leakage products due to exposure to moderate temperatures listed in decreasing rates of leakage were identified as protein, deoxyribonucleic acid, ribonucleic acid, and amino acids (72). Leakage of nucleic acid material can be seen in Fig. 4. When the cells are exposed to 22.3 C (2.3 C above its maximal growth temperature), these cellular leakage materials appear in the super-
natant fluid after exposure for 45 min. After a 60-min exposure to 22.3 C, 21.5 mg of protein, 52 Atmol of amino acid, 2,100 ,g of RNA and 207 jig of DNA appeared in the supernatant fluid from 10 ml of cell suspension (optical density [OD] of 0.40 at 600 nm). If the exposure temperature was increased, then the amount of leakage material increased rapidly. The leakage of intracellular material was thought to be one of the causes for loss of viability in V. marinus MP-1 at moderate temperatures. However, it was found that 95% of the cells must expire before noticeable amounts of leakage products occur (93). In addition to the above named leakage products, it was found that malate dehydrogenase and glucose-6-phosphate appears in the supernatant fluid after exposure of the cells to moderate temperatures. Older cells were found to be more resistant to
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157
FIG. 4. Electron micrograph of a thin section of Vibrio marinus MP-1 exposed to 26.5 C for I min. Note the separation of the cell wall and the leakage of nuclear material from the cell. (Courtesy of R. R. Colwell.)
thermal death, lysis, and leakage. Harder and Veldkamp (75) detected a very small amount of 260-nm-absorbing material leaking from a psychrophilic pseudomonad at 18 to 19 C, but the amount was so small that it does not offer an explanation for the decreased protein synthesis. Because the use of spectrophotometric measurements is not as sensitive as radioisotope measurements, the problem of heat-induced leakage was reinvestigated employing Ant-300 cells grown in [U-_4C]glucose, harvested, starved, and exposed to temperatures near or slightly above the maximum for growth of the organism (63). Leakage, as evidenced by the loss of "C-labeled cellular material, was extensive during the first 10 h of incubation. At 18 C, 31% of the radioactivity was released from the cells. At and near the organism's maximal growth temperature (13 and 15 C, respectively), 16% of the radioactivity was released while only 7% was released at 5 C. The percentage of protein and protein plus DNA at 5 and 18 C are nearly equal, but the main high-molecularweight cellular leakage component at 18 C is RNA (Table 7). Identification of the lowmolecular-weight components have not been
identified (G. G. Geesey, M.S. thesis, Oregon State Univ., Corvallis, Ore., 1973). The question as to how much leakage is necessary before growth ceases must still be answered. The degree of leakage depends on the presence or absence of substrates. Thermally induced lysis. Phase optics indicate that V. psychroerythrus lyses when exposed to 20 C or higher (71,108) which is preceeded by death. With V. marinus MP-1 leakage and lysis occurred simultaneously but after 95% of the cells had expired (93). In nongrowing cultures once lysis is initiated at 25 C it cannot be stopped by transferring the culture back to 5 C, which suggests that the process is enzymatic. Lysis is not affected by metabolic inhibitors. The autolytic enzymes remain active at lower temperatures once activated by higher temperature. After lysis hexosamine as well as 260-nmabsorbing material can be found in the menstruum, and the breakdown of lipid phosphorus occurs after the onset of measurable lysis (71, 94). At 25 C phosphatidases attacking cells reduce the lipid phosphorus by more than half (94). These phosphatidases require salt for maximum activity. A drop in the turbidity of a sus-
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TABLE 7. High-molecular-weight components released from Ant 300 cells at 5 and 18 C for 4 ha shock Heat Heatshock Protein (%)
Protein + DNA (%)
RNA (%)
material ~~~~~~~~~~~~~~~~~~~~~~Total released (dpm/ml)b
5 18
17 (397 dpm) 16 (848 dpm)
18 (409 dpm) 17 (886 dpm)
10 (218 dpm) 14 (744 dpm)
2,264 5,161
a Cells were grown at 5 C for 22 h in a medium containing [U- "4C ]glucose (final concentration was 6.0 ,g/ml with an activity of 180 mCi/ml). The cells were harvested by centrifugation and suspended in artificial sea water to give a final concentration of 2 x 107 cells/ml. b dpm, Disintegrations per minute.
pension of water-lysed cells or cell envelopes at 25 C was also salt dependent. The ultrastructure of V. marinus MP-1 and V. psychroerythrus appear typical of gram-negative bacteria and both possess a trilaminar membrane structure (40, 55). Degradation of the cell wall and liberation of wall fragments occurs when lysis of the cell takes place at 37 C, and the trilaminar structure is rarely observed (40). This destruction occurs in the presence of all salt solutions tested. Temperature effects of permeability. The ability of the psychrophile to function well at cold temperature depends to a great degree on the availability of substrates. When the temperature is raised above the maximum temperature for growth, the transport of substrates is impaired. An apparent increase in malic dehydrogenase activity is noted in a psychrotrophic bacterium when the temperature of the system is raised above the maximum growth temperatures (129). In other words, near-maximal activity occurred even when the intracellular enzyme was being inactivated. This apparent increase in activity with whole cells is due to the alteration in permeability which permits diffusion rather than active transport of the substrate into the cells. When the temperature is lowered below the optimal growth temperature, the uptake of substrates is decreased (9, 92, 128, 150, 152). The foregoing data definitely indicate that changes in transport systems occur when the temperature is altered. B616hradek (11, 12) suggested that thermal death may be due to the solution of certain lipids and there was a correlation with the melting points of the lipids of the cell and death. Since that time, much research has been concentrated on membrane lipids in relation to growth temperature. As the temperature of growth is lowered, the proportion of unsaturated fatty acids (hexadecenoic and octodecenoic) increases, and this occurs when cells are grown at different temperatures in both minimal and complex medium (111). This situ-
ation also occurs in a psychrotrophic strain of V. marinus PS 207 (143) and with yeasts (90). The fatty acid composition of V. psychroerythrus (thought to be a Serratia sp. at the time of publication) was analyzed and compared to Serratia marcescens (90). The psychrophile did have more unsaturated fatty acids than S. marcescens. The percentage of unsaturated fatty acids is lower in V. marinus PS 207 than in many forms that have higher cardinal temperatures (143). The relative percentage of unsaturated fatty acids in V. marinus MP-1 (145) is 59.8% when the organism is grown at its optimum temperature for growth. The percentage of unsaturated fatty acids for V. marinus PS 207 grown at 25 C (optimum growth temperature) and 15 C are 68.5 and 72.0, respectively. Growth of both strains of V. marinus at their temperature optima reveal similar fatty acid patterns, but not when PS 207 is grown at 15 C (143). It therefore appears that within a species there is a shift toward more unsaturated fatty acids with a decrease in the temperature under which the organism is grown. Since V. marinus MP-1 has a much lower percentage of unsaturated fatty acid composition (146) compared to many of the other marine bacteria that are not psychrophiles (143), the question still remains as to how psychrophiles can maintain their membrane fluidity so that transport systems are still operative at 0 C or below. Temperature effects on macromolecular synthesis. When a psychrophilic Pseudomonas was grown above its optimal temperature for growth, there was an increase in the synthesis of RNA in the cells (75). This increased RNA synthesis was noted until a temperature of 18 C was reached (maximal growth temperature was 19 C). Between 18 and 19 C a drop in RNA content was noted. If the continuous culture system was elevated to 20 C, protein synthesis ceased and the culture was washed out of the continuous culture apparatus. The increased RNA of cells at the higher temperature was
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PSYCHROPHILIC BACTERIA
mainly ribosomal RNA. The ability of V. marinus MP-1 to take up [U-14C ]proline, synthesize protein (measured by incorporation of [U-14C]proline), and synthesize RNA (incorporation of [U-5H]uracil) was found to be a function of salinity and temperature. The critical temperature of the lesion(s) in protein synthesis increased with increasing salinity of the growth medium. At a salinity of 25%o and 22 C, protein synthesis was significantly inhibited, but not at a salinity of 35%o (maximal growth temperature at a salinity of 35%o is 21.2 C). When cells were incubated at 24 C at a salinity of 35%o, protein synthesis continues at a linear rate for 20 minutes before it begins to decrease. Total RNA synthesis continues at a linear rate for 20 min before salinities between 15 and 35%o. A salinity-temperature relationship is noted for both RNA and protein synthesis (38). However, it should be noted that both RNA and protein synthesis continue in growth media when cells are incubated a few degrees above the maximum for growth of a psychrophilic vibrio (127). At 5 C above the maximal growth temperature, the cessation of DNA and protein synthesis occurs approximately 30 min after exposure to the elevated temperature. An increase in protein and DNA was noted at the maximal temperature for growth over the amount produced at the optimum temperature for growth (75). No increase in protein, RNA, and DNA could be noted by Harder and Veldkamp (76) when cells were exposed in the presence or absence of substrate at 2.5 to 3.5 C above the maximal temperature for growth. Since no RNA synthesis could be detected at 2.5 to 3.5 C above the maximum growth temperature, they concluded that this may be the basis for the cessation of growth. However, as they point out, this does not account for the decline in the growth of the organism between the optimum and maximum growth temperatures. When a cell-free extract prepared from the psychrotrophic bacterium Micrococcus cryophilus was subjected to 5 C above the maximal growth temperature for the organism, three aminoacyl-tRNA synthetases (glutamic acid, histidine, and proline) were found to be temperature sensitive (109, 110, 111, 112). Unfortunately, the experiments were not carried out 1 or 2 C above the maximal growth temperature for M. cryophilus. At temperatures of 1 or 2 C above the maximal for growth, the cessation of macromolecular synthesis does not appear to be the cause of thermal death. Most thermal death experiments are carried out at 5 to 10 C above the maximal temperature for growth which is much
159
too high to determine the subtle changes in the organism which results in the cessation of growth. Ecological factors affecting psychrophiles. As stated previously, psychrophiles function well at environmental temperatures even though their optimum activity and growth may be several degrees higher than the environmental temperature. The main environmental factors other than the amount of substrates present that affect the psychrophiles' ability to function are salinity, temperature, and hydrostatic pressure. The effect of salinity and the various cations making up the salinity of the ocean on marine bacteria are well reviewed by MacLeod (106, 107). Membrane permeability has been shown to be salt dependent in marine bacteria (45, 46, 107). After washing cells of a marine pseudomonad free of medium components with 0.05 M MgSO4, there was an immediate increase in OD when the cells were suspended in solutions containing 200 mM of various salts (116). This increase in OD was followed by a slow decrease in OD. The decrease in OD can be prevented by omitting K+ from the suspending solution. The OD changes were not attributed to osmotic effects. The OD of cell envelopes prepared from the organism also increased with increases in NaCl but not in the presence of Mg2+. This initial increase in OD was attributed to an interaction of the salts with the components of the cell envelope, thereby causing the contraction of the envelopes and shrinkage of the cells. .05 .04
.03 E
.02
oC
.01
S%O 151o100
0
< -.01 O -.02 4
S=
-.03
O
2
3
4
5
150
6
7
TIME (MIN) EXPOSED TO VARIOUS SALINITIES
FIG. 5. Time-salinity effects on the OD of V. marinus MP-1 cells. Cells from a 24-h culture grown at 15 C were centrifuged, washed with artificial sea water (35 Vo), and centrifuged again. The sedimented cells were then suspended at various salinities for various periods of time. The initial OD reading was set at 0.2 (equals 0 on the figure). (Unpublished data, Haight and Morita.)..
160
Salinity changes can also do the same for V. marinus MP-1 (Fig. 5). Salt and low temperature are necessary for the stability of the cellular structure of V. psychroerythrus (40, 94), and lysis can occur at low ionic strength (108). V. psychroerythrus is stable in cold sea water or in a solution containing 0.1 M Mg2+ and 0.5 M NaCl. When subjected to water and in various monovalent salts (including 3.0 M NaCl), the turbidity of the cell suspension fell and some intracellular materials were released into the menstruum. Phase contrast microscopy revealed that the cell disintegrated into amorphous aggregates after first becoming swollen. The effectiveness of divalent salts in maintaining cell structure was in the order Cu2+ > Ni2+ > Ca2+ > Mn2+ > Mg2+
(94).
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Depending on the salt concentration in the medium, V. marinus MP-1 can possess a maximum growth temperature from 10.5 to 21.2 C (170). The lower the salt concentration, the lower the maximum growth temperature of the organism. This situation holds true for all the salt-requiring bacteria we have tested in our laboratory. In ecological situations where the salinity and temperature can change due to freshwater intrusions, evaporation, and diurnal insolation, the temperature-salinity regime may be an important factor in the enumeration of the microbial flora; hence an important thermal group may have been overlooked in microbial ecological studies (136). Figure 6 illustrates the co-effect of salinity and temperature variations on the growth of Vibrio marinus MP-1. The change in the maximum growth temperature was shown to be dependent upon the various ions present in the growth medium. The order of effectiveness in restoring the normal growth temperature (21.2 C), when added to dilute sea water, was Na+, Li+, Mg2+, K+, Rb+, NH,+. In a completely defined medium, the highest maximal growth temperature was 20.0 C at 0.40 M NaCl. A decrease in the maximal growth temperature was observed at both low and high concentrations of NaCl. In order to try to explain the change in the maximal growth temperature in relation to the salt concentration, studies were initiated to determine the effect of a salinity-temperature regime on macromolecular (see section on temperature effects on macromolecular synthesis). Rhodes and Payne (155) have found that the induction of washed cells of Pseudomonas natriegens (marine bacterium) to the oxidation of lactose and mannitol was dependent on the presence of Na+ with marine levels of Mg2+ and K+ in the suspending medium. A temperaturesalinity relationship was shown for the induc-
50 40 I
30 Co
20 10
0 0
4
8
12
16
20
TEMPERATURE (C)
FIG. 6. Contour surface diagram showing the coeffect of temperature and salinity on the growth rate as a number of generations/h of Vibrio marinus MP-1 in Lib-X broth. Symbols: 0, 0.28 generation/h, (maximum); A, 0.25 generations/h; 0, 0.20 generations/h; 0, 0.14 generations/h; and A, 0.06 generations/h.
tion of glutamic dehydrogenase in V. marinus MP-1. This interaction of salinity and temperature may be very important in the nearshore environment since it will determine whether or not a microorganism will have the ability to synthesize adaptive enzymes to cope with any foreign substrates that might enter the water. The pattern of carbon dioxide release from specific labeled carbons of glucose taken up by V. marinus MP-1 was found to change with salinity (68). Within the growth temperature range of the organism, temperature had no effect on this pattern. However, when the salinity is increased from 0.30 to 1.0 M NaCl, there is a decrease in the C6/C, (carbon dioxide) ratio. The changes observed in this study were not specific for Na+ or Cl-. The apparent shutdown of the hexose monophosphate pathway, as determined by radiorespirometric carbon dioxide release patterns, coincides with the minimum salinity of growth of V. marinus MP-1. This may in turn be related to the growth limitation at lower salinities. As the salinity was increased from 0.15 to 0.30 M NaCl, there was a shift in the total uptake patterns which suggests the formation and loss of metabolic by-products derived from the first, second, sixth, and presumably fifth carbons of glucose. The effects of hydrostatic pressure on psychrophiles in the marine environment takes on more importance when one takes into consideration the volume below the thermocline. In this region the pressure may be as high as 1,100 atm (average pressure is 380 atm) and the temperature is 5 C or colder. When pressure and temperature are considered as variables, a pressure-
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temperature interaction is found as would be predicted by the Ideal Gas Law. The pressure increases approximately 1 atm for every 10 m in depth. There have been many reviews dealing with hydrostatic pressure effects on microorganisms (19, 89, 122, 124, 169, 190). However, the data on pressure effects on psychrophiles is limited. V. marinus MP-1 was isolated from water taken from a depth of 1,200 m (135) where the temperature was 3.24 C. It has the ability to grow slightly better at 100 and 200 atm at 3 C than at 1 atm at 3 C. With increasing pressure above 300 atm, growth decreases and at 1,000 atm the organism expires. Death ensues at 800 atm when the temperature is 15 C (127). The response to pressure depends upon the environmental temperature from which the organisms were obtained. Hydrostatic pressure stimulated or supressed L-serine deamination by washed cells of V. marinus MP-1 depending upon the temperature at which the cells were grown and the incubation temperature of the reaction mixture (3). Cells grown at 15 or 4 C had deamination stimulated under pressure in the following cases: (i) cells grown at 15 C and tested for deamination at 15 C; (ii) cells grown at 4 C and tested at 4 C; and (iii) cells grown at 4 C at tested and 15 C. When cells were grown at 15 C and tested at 4 C, no stimulation of deamination activity due to pressure was observed. Unfortunately there is a tendency to neglect this fact when working on the physiological and biochemical reactions under pressure. The ability of a psychrophile to synthesize protein, RNA, and DNA under pressure was tested, and it was found that at 200 atm macromolecular synthesis was not affected. At 400 atm protein and RNA but not DNA synthesis was adversely affected, whereas at 500 atm there was a rather sharp decline in the synthetic rates. At 1,000 atm virtually no synthesis occurred (2). The effect of alternate pressurization-depressurization effects on growth and net synthesis of protein, RNA, and DNA was studied by Albright (1). He found that the application of 544 atm caused cell division and synthesis of RNA and protein to cease immediately. Upon release of the pressure cell division and synthesis of RNA and protein resumed the original rate. The second application of pressure had a less drastic effect on growth, and synthesis of RNA and protein. There was no immediate effect on DNA synthesis with the application of pressure, but this synthetic process slowed and ceased with time under pressure. Pressure was found to inhibit the transport of amino acids into a psychrotrophic bacterium (150). Once the amino acids are transported
161
into the cells, the respiratory mechanism needed to produce carbon dioxide from the amino acids does not appear to be inhibited. Immediate pressurization of cells in the presence of an amino acid brings about inhibition of transport, and this transport inhibition is reversible when the pressure is released. If the cells were incubated in the presence of an amino acid, 3 h prior to pressurization, the ability of the transport mechanism for the amino acids is not impaired. The uptake of uracil was followed in two antarctic psychrophiles, V. marinus MP-1 and V. alginolyticus, and E. coli under pressure (8). Between 100 to 300 atm at 15 C, the ability of E. coli and V. alginolyticus to take up uracil was reduced approximately 70%. The same reduction was noted for V. marinus MP-1 at 5 C. The two antarctic psychrophiles were found to be more barotolerant since they had the ability to take up considerable uracil at 500 atm. It was shown that the mechanism for uracil uptake is more pressure sensitive than the RNA-synthetic mechanism. Growth studies were also conducted and correlated with uracil uptake at the same pressures and temperatures. In each organism there was a correlation between the organism's ability to divide and the rate of uracil uptake. The growth patterns, determined under pressures, of each psychrophile differed markedly. Morphological changes (spheroplast-like bodies and elongated forms) were commonly observed in cells obtained from cultures subjected to pressures limiting their growth. These morphological changes indicate that pressure profoundly influences the physiology of the cells. The above data on the pressure effects on psychrophiles strongly implicates the membrane as the primary site of pressure damage. CONCLUSION The data available concerning psychrophiles are too fragmentary to explain the biological basis of psychrophily. Nevertheless, there are sufficient data accumulated to use the term "psychrophile" in a meaningful way. By use of the definition employed in this review, a start can be made to end the confusion concerning the term. Furthermore, I believe that future scientific data on psychrophilic bacteria will more than justify this definition. ACKNOWLEDGMENTS I wish to thank my associates (J. A. Baross, P. A. Gillespie, R. P. Griffiths, G. G. Geesey, T. D. Goodrich, F. J. Hanus, S. S. Hayasaka, and L. P. Jones) for use of unpublished scientific data. This work was supported by National Science Foundation research grant GA 38583X.
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162 LITERATURE CITED
1. Albright, L. J. 1969. Alternate pressurizationdepressurization effects on growth and net protein, RNA and DNA synthesis by Escherichia coli and Vibrio marinus. Can. J. Microbiol. 15:1237-1240. 2. Albright, L. J., and R. Y. Morita. 1968. Effects of hydrostatic pressure on the synthesis of protein, ribonucleic acid and deoxyribonucleic acid by the marine psychrophilic bacterium, Vibrio marinus. Limnol. Oceanogr. 13:637-643. 3. Albright, L. J., and R. Y. Morita. 1972. Effect of environmental parameters of low temperature and hydrostatic pressure on L-serine deamination by Vibrio marinus. J. Oceanogr. Soc. Jpn. 28:63-70. 4. Allen, M. B. 1953. The thermophilic aerobic sporeforming bacteria. Bacteriol. Rev. 17:125-173. 5. Azuma, Y., S. B. Newton, and L. D. Witter. 1962. Production of psychrophilic mutants from mesophilic bacteria by ultraviolet irradiation. J. Dairy Sci. 45:1529-1530. 6. Baig, I. A., and J. W. Hopton. 1969. Psychrophilic properties and the temperature characteristic of growth of bacteria. J. Bacteriol. 100:552-553. 7. Bardach, I. 1924. Ueber die Lebensprozesse der Bakterien bei Temperaturen, die unter 0° stehen. Ber. Wiss. Forsch. Inst. Odessa 1:1-5. 8. Baross, J. A., F. J. Hanus, and R. Y. Morita. 1974. The effects of hydrostatic pressure on uracil uptake, ribonucleic acid synthesis, and growth of three obligately psychrophilic marine vibrios, V. alginolyticus and Escherichia coli. In R. R. Colwell and R. Y. Morita (ed.), Effect of the ocean environment on microbial activities. University Park Press, Baltimore. 9. Baur, E. 1902. Ueber zwei denitrifizierende Bakterien aus der Ostee. Wiss. Meeresuntersuchungen, Kiel, N. F. 6:9-22. 10. Baxter, R. M., and N. E. Gibbons. 1962. Observations on the physiology of psychrophilism in a yeast. Can. J. Microbiol. 8:511-517. 11. B6lehradek, J. 1931. Le mecanisme physicochimique de l'adaptation thermique. Protoplasma 12:406-434. 12. B6lhradek, J. 1935. Temperature and living matter. Gebruder Borntraeger, Berlin. 13. Berry, J. A., and C. A. Magoon. 1934. Growth of microorganisms at and below 0C. Phytopathology 24:780-796. 14. Braarud, T. 1961. Cultivation of marine organisms as a means of understanding environmental influences on populations, p. 271-298. Oceanography. American Association for the Advancement of Science Publication no. 67. American Association for the Advancement of Science, Washington, D.C. 15. Borg, A. F. 1960. Studies on myxobacteria associated with diseases in salmonid fishes. Wildl. Dis. 8:1-85. (2 Microcards.) 16. Boyd, W. L., and J. W. Boyd. 1962. Soil microorganisms of the McMurdo Sound Area, Antarc-
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tica. Appl. Microbiol. 11:116-121. 17. Boylen, C. W., and T. D. Brock. 1973. Bacterial decomposition processes in Lake Wingra sediments during winter. Limnol. Oceanogr. 18:628-634. 18. Brandt, K. 1902. Ueber den Stoffwechsel im Meere. 2. Abhandlung. Wiss. Meeresuntersuchungen, Kiel, N.F. 6:23-79. 19. Brauer, R. (ed.). 1972. Barobiology and the experimental biology of the deep sea. North Carolina Sea Grant Program, University of North Carolina, Chapel Hill. 20. Brock, T. D. 1967. Life at high temperatures. Science 158:1012-1019. 21. Brock, T. D., F. Passman, and I. Yoder. 1973. Absence of obligately psychrophilic bacteria in constantly cold springs associated with caves in southern Indiana. Am. Midl. Nat. 90:240-246. 22. Brock, T. D., and A. H. Rose. 1969. Psychrophiles and thermophiles, p. 161-168. In J. R. Norris and D. W. Ribbons (ed.), Methods in microbiology, vol. 3B. Academic Press Inc., London. 23. Brown, A. D. 1957. Some general properties of a psychrophilic pseudomonad: the effects of temperature on some of these properties and the utilization of glucose by this organism and Pseudomonas aeruginosa. J. Gen. Microbiol. 17:640-648. 24. Brown, P. E., and R. E. Smith. 1912. Bacterial activities in frozen soils. Iowa Agric. Exp. Stn. Res. Bull. 4:155-184. 25. Buchanan, R. E., and N. E. Gibbons. 1974. Bergey's manual of determinative bacteriology, 8th ed. Williams and Wilkins, Baltimore. 26. Bunt, J. S. 1971. Microbial productivity in polar regions, p. 333-354. In D. E. Hughes and A. H. Rose (ed.), Microbes and biological productivity. Symp. Soc. Gen. Microbiol. 21:333-354. 27. Bunt, J. S., and A. D. Rovira. 1955. Microbiological studies of some sub Antarctic soils. J. Soil Sci. 6:119-128. 28. Cameron, R. E., and R. E. Benoit. 1970. Microbial and ecological investigations of recent cinder cones. Deception Island. Antarctica-a preliminary report. Ecology 51:802-809. 29. Cameron, R. E., J. King, and C. N. David. 1970. Microbiology, ecology, and microclimatology of soil sites in dry valleys of Southern Victoria Land, Antarctica, p. 702-716. In M. W. Holdgate (ed.), Antarctic ecology, vol. 2. Academic Press Inc., London. 30. Campbell Soup Company. 1961. Proceedings of the low temperature microbiology symposium-1961. Campbell Soup Co., Camden, N.J. 31. Christian, R. R., and W. J. Wiebe. 1974. The effects of temperature upon the reproduction and respiration of a marine obligate psychrophile. Can. J. Microbiol. 20:1341-1345. 32. Colwell, R. R., and R. Y. Morita. 1964. Reisolation and emendation of description of Vibrio marinus (Russell) Ford. J. Bacteriol. 88:831-837. 33. Conn, H. J. 1910. Bacteria of frozen soil. Cent.
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