- Email: [email protected]
Isolating and characterizing deep-sea marine microorganisms
6
Isolating and characterizing deep-sea marine microorganisms Chiaki Kato, Akira lnoue and Koki Horikoshi We have isolated several microorganisms
that are adapted to living in the extremes
of the deep-sea environment. They include barophilic bacteria, which are able to grow at high hydrostatic pressure, but that are unable to grow at atmospheric pressure, and organic-solvent-tolerant
bacteria, which are able to grow in the
presence of toxic organic solvents such as toluene or benzene. In this review, we describe how to isolate such extremophiles,
and we outline the characteristics of
several strains that have been recovered from the deep-sea environment.
The deep-sea bed is a unique environment that experiences extremely high pressures and low temperatures. Microorganisms living there have developed particular characteristics that allow them to thrive at such extremes. In studies aimed at improving our understanding of microbial adaptation to the deep-sea e11vironment, we have isolated and characterized a number of microorganisms from samples of deep-sea mud obtained by the manned submerGble S&&i 6500. This vehicle, which is operated by the Japanese Marine Science and Technology Center (JAMSTEC), has the ability to submerge to a depth of 6300 m (Ref. 1). In this review, we describe two specific types of deep-sea microorganisms that have been isolated in our laboratory. The first group comprises barophilic microorganisms, which were isolated from the deep-sea samples at depths of greater than 25OC)m: they can grow only at high precsure, and not at normal atmospheric pressure. The second group comprises organicsolvent-tolerant microorganisms, which were mainly isolated from the samples taken at depths of less than 2000 m; thev can grow in the presence of high concentrations of toxic organic solvents such as toluene or benzene. It is not yet known whether these two groups are closely related, because ‘barophilic organicsolvent-tolerant bacteria’ have only just been isolated. However, some of the organic-solvent-tolerant bacteria can also grow at high pressure, so it is possible that such microorganisms will be found. Such microorganisms could prove to be useful for new biotechnology applications such as high-pressure bioreactor5 and two-phase (water/organic solvent) fermentation systems.
TIBTECH JANUARY
1996 IVOL
141
Deep-sea barophilic bacteria In 1957, Zobel and Morita’ were among the first researchers who attempted to isolate microorganisms that were specifically adapted to the high pressures associated with the deep-sea environment - they called them barophilic bacteria. However. it was only in 1979 that Yayanos et al.” were able to isolate barophilic bacteria, thanks to technlcal support and the development of procedures for investigating deep-sea environments. We are keen to isolate new deep-sea-adapted microorganisms and to characterize them for their possible application to high-preswre biotechnology. Several barophilic bacteria have been isolated from camples of deep-sea mud that were collected using sterilized mud samplers on the submersible Shkai f;5UO (KetS 3,5). The isolated barophilic bacteria (Table 1) were grown in pressure vessels under a range of hydrostatic pressures (0.1-80 Ml’s) and temperatures (;C15”C). The optinral pressure for growth of barophilic strains l~B5301, 11B6101, DB6705 and DB6906 was about 50MPa at IO”C, and 70 and hUMPa for strains 11B172F and l?B172R, respectively, at 10°C. None of these strains was able to grow at temperatures above 3VC under any pressure conditions. The growth-rate profiles (Fig. 1) of these barophilic strains indicate that their response to pressure is greatest near their upper temperature limit (15’C) for growth4,5. For example. the optimal growth pressures of L>B6101 and DB6705 at 15°C were about 70 and 60 MPa, respectively (Figs lb and 1 c), and the strains DB6705, 1)86906, DB172F and DB172R were cap,lble of growth only at pressures above 10 MPa (Figs lc-lf): however, the growth-rate profiles of these latter strains at 4’C were similar to the profiles of barotolerdnt (pressure-tolerant) strains. Yayanos” reported the effects of pressure and temperature on the growth rates of Feveral isolates of deep-sea 0 1996.
Elsewr
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Figure 1 Growth profiles of barophilic bacteria, strains (a) DB5501, (b) DB6101, (c) DB6705, (d) DB6906, (e) DB172F, and (f) DB172R at various pressures and temperatures; open circles denote growth at 15”C, filled circles denote growth at lO”C, and diamonds denote growth at 4°C (data from Refs 4 and 5). The growth rate is shown as l/td, where t, represents the doubling time (in hours). Barophllic bacteria were isolated keeping samples under high pressure and low temperature. Marine-broth 2216 medium, containing low-temperature-melting agar, was inoculated and incubated under high pressure. Subsequently, a test for barophily was conducted by comparing the growth rate of bacteria at 65 MPa and 0.1 MPa (Ref. 4).
barophilic bacteria that were isolated fi-ouj different depths of ocean. He reported maximal rates of reproduction at X-10°C for all isolates at various pressures, and that optimal growth occurred at a prewu-e similar to that encountered at their capture depth. However, two distinct growth profiles have been demonstrated for the barophilic strains isolated in our laboratory: the growth rates of strains L1BS.501, DB6101, DB6705 and DB6906 are higher at 15°C than at lower tenperatures, unlike the data obtained by Yayanos for the isolates”; and maximum growth rates for strains DB172F and DB172R were observed at lO”C, which was similar to growth profiles described by Yayanos. The differences in response to environmental conditions observed among previously reported barophilic bacteria, and those that we have isolated, are worth considering, both for their possible evolutionary significance, and for practical exploitation. Although the temperatures and preswre5 at which a change in growth response is observed can vary widely, the overall growth profiles are very similar for all the barophilic bacteria. We have also found that psychrophilic (i.e. those that have a temperature optimum for growth ofbetween 0°C and 30°C) barotolcrant bacteria isolated from deep-sea samples display a similar response profile to the barophilic organism~i. Indeed, even E.ccherirhi~ co/i responds in a similar way to variations in temperature and pressure’, perhaps implying that the same responw mechanisms are
widely conserved. At present, there are not enough data to draw any general conclusions about bacterial growth under high pressure, but It 1spossible that bacterial growth rates may be stimulated by high pressure near their maximum temperature for growth. From a comparison of the DNA sequences (held in the GenBank and EMBL databases) encoding 16s ribosomes. it was shown that the barophilic strains we isolated belong to the Proteobactcria, gamma subgroup”. The G+C content of chromosomal DNA from V&O spp. that belong to the gamma Pr~tec~hadcvia is between 10-50% (Ref. 9), and is considered to be typical of this subgroup. The G+C content of chromosomal DNA from the isolated barophilic strains was found to be similar, at -K-46’%. As a result of a taxonomic study of the obligate barophilic bacterium Coh~clIia hndnlicnsis, Deming et al.“’ reported that, based on its 5s ribosomal RNA sequence, this barophilic bacterium belongs to the gamma Proteobactcria. de Long and Franks” have also documented the existence of barophilic and psychrophilic deep-sea bacteria that belong to this group, as indicated by 16s ribosomal DNA sequence data. It is interesting to note that the 1 GS ribosomal I1NA sequences of the barophilic strains DBh’fOh, L)B172F and I>81 72R. 2nd the psychrophilic barotolerant strain DSS12 (Ref. 1) show the highest homology of all, indicating that these strains are very closely related. The relationship between the isolated strains and some strains of the gamma Pr(lfrobac&a TlBTECHJANUARY1996WOL14l
E. coli
S. marcescens
WHB46-2
DB5501
DB$lOl
\
\
Sh, a,ga
\
A. hydroi’hi’a
DB6705
Figure 2 Unrooted phylogenetlc tree showing the relatlonships between isolated barophilic strains withln the gamma subgroup of the Proteobacteria, as determined by comparing 16s ribosomal DNA sequences using the neighbor-joining method’z. The accession numbers of 16s ribosomal DNA sequences used in this figure are as follows; Escherichia coli (JO1695), Serratra marcescens (M591601, Plesiomonas shigelbdes (M59159), Proteus vulgaris (X07652), Vibno anguillarum (X16895), Aeromonas hydrophila (M591481, Shewanella alga (X81621), WHB46-1 (X54744), WHB46-2 (X54745), DB5501 (D21229), DB6101 (D21221), DB6705 (D21222), DB6906 (D21223), DSKl (D212241, DSS12 (D21225), DB172F (D63488). DB172R (D63824). (These accession numbers are for the EMBL, GenBank and DDBJ DNA databases.) K,,,, is the nucleotide substitution ratel2.
are shown in Fig. 2 in the form of a phylogenetic tree that uses the neighbor-joining method’“. The barophilic and barotolerant strains isolated from the deep-sea environment have been separated into one of the sub-branches in the gamma subgroup; the barophilic microorganisms reported by Liesack et al.‘” (strains WHB 46-l and WHI) 46-2) are also included in the sub-branch containing the strains isolated in our laboratory. These data suggest that the high-pressureadapted bacteria may belong to a new bacterial genus in the gamma subgroup of the Protrobacferia. Molecular-genetic analysis of the high-pressureadaptation mechanisms of such microorganisms is being carried out. Bartlett ct al.‘” have shown that the expression of the gene encoding the outer-membrane protein OmpH from a deep-sea P~~ofobactcrilrMI. strain SS9, is controlled by pressure, and the high-pressure regulation of cwyH gene expression has also been analysed1S-‘7. A pressure-regulated promoter region from a deep-sea barophilic bacterium strain DB6705 has been cloned and analysed’“, and it has been suggested that this promoter may be common to many barophilic bacteria’,lx. We believe that the new field of high-pressure biotechnology will develop from such basic studies. Organic-solvent-tolerant Organic-solvent-tolerant to grow in the presence lBTECHJANUARY1996WOL141
microorganisms microorganisms are able of high concentrations of
organic solvent. Organic solvents are generally toxic and kill most microorganisms at low concentrations (-0.1%). Although some microorganisms. including Pse~dowonas, ,Irlwomobarter and l%rclrtlia spp., can assimilate organic solvents, their solvent tolerance is less than 0.3%. We have determined that organic-solventtolerant microorganisms can grow on solvents such as toluene or benzene at concentrations of greater than 10% (v/v). While looking for such microorganisms around the islands of Japan, we discovered a variant strain of Pscudomm~s putida (strain IH2OOU), in mud samples taken from the Kumamoto Prefecture on Kyushu Island”‘; this is the first organic-solvent-tolerant bacterium that has been isolated. Strain IH2000 is able to grow in culture media containing more than 50% (v/v) toluene. We have also isolated useful organic-solvent-tolerant microorganisms (Table 1) from the deep-sea bed, and have found that 100 times more microorganisms of this type can be isolated from deep-sea mud samples than from soil samples taken from land. Organic-solvent-tolerant bacteria that exhibit the ability to degrade crude oil, polyaromatic hydrocarbons, or cholesterol, or the ability to utilize sulfur compounds, were isolated using the fcjllowing procedure: benzene was added to artificial sea-water-containing samples of deep-sea sediment to a concentration of 50’%, (v/v), and the cultures were incubated at room temperature for one week on a shaker; after
9 f ecus Table 1. Deep-sea microorganisms Bacterial strain
that have been isolated by the DEEPSTAR Group
Properties
Source
Ref.
Optimal growth at 50 MPa and 10°C. Optimal growth at 50 MPa and 10°C. Optimal growth at 50 MPa and 10°C. No growth at atmospheric pressure. Optimal growth at 50 MPa and 10°C. No growth at atmospheric pressure. Optimal growth at 70 MPa and 10°C. No growth at atmospheric pressure. Optimal growth at 60 MPa and 10°C. No growth at atmospheric pressure
Suruga Bay, at a depth of 2485 m Ryukyu Trench, at a depth of 5110 m Japan Trench, at a depth of 6356 m
4
Japan Trench, at a depth of 6269 m
4
Izu-Bonin Trench, at a depth of 6499 m
5
Izu-Bonin Trench, at a depth of 6499 m
5
Barophilic bacteria DB5501 DB6101 DB6705 DB6906 DB172F DB172R
Organic-solvent-tolerant DS-711 DS-944 DS-1906 ST-l
Y-40
bacteria
Degrades crude oil Utilizes sulfur Degrades polyaromatic hydrocarbons Degrades cholesterol
Organic-sovent-tolerant
t
Suruga Bay, at a depth of 1945 m Sagami Bay, at a depth of 1168 m Sagami Bay, at a depth of 1168 m Okushiri Ridge, at a depth of 1963 m
yeast
Hydrocarbon-degrading yeast
incubation, the benzene layers were carefully separated from the sea-water layers, and a portion ofeach benzene layer was spread onto M-II agar medium”‘; colonies that grew on the medium after incubation for two days at 25’C or 30°C were isolated and purified. The following bacteria are among the benzene-tolerant strains that have been isolated: IX-7 1 I degrades crude oiP, LX-994 utilizes sulfilr compounds”, DS1906 degrades polyaromatic hydrocarbons”, and ST-1 degrades cholesterol”. These strains were isolated fi-om samples of deep-sea mud collected at Suruga Bay. Sagami Bay or Okushiri Ridge near Japan. From their morphological and biochemical characteristics, strains DS-711. l)S-994. US-1906 and ST-1 have been identified as FlavoDacterirrm sp., Barilhs sp., Bacillus sp. and A~tlwobactcr sp., respectively. Strain US-71 1 (Ref. 20) was selected as a hydrocarbon degrader, and it showed halotolerant growth and tolerance to various kinds of organic solvents, including 5% (v/v) benzene, 10% (v/v) toluene and 10%~ (v/v) p-- Y yl ene. The ability of strain IX-7 11 to degrade aromatic hydrocarbons and the rz-alkanes in kerosene was compared with the control strains DS203 (an Ahwmr~as sp.) and I? putida strain IH-2000. As shown in Table 2, all of the strains tested could degrade the rl-alkanes in kerosene, but did not degrade aromatic hydrocarbons. However, strain 11S-711 was much better at degrading rl-alkanes than were the control strains. In particular, the percentage of uz-decane and rl-undecane degraded by the control strains was only about 10.0%~ in each instance, whereas strain IX-711 degraded 71.4% and 68.0%, respectively. Strain DS-711 showed the highest activity of
Sagami Bay, at a depth of 1200 m
rl-alkane degradation among the strains tested, and the extent of degradation reached approximately 85% after seven days. Strain 1X-991 (Kef. 2 1) was identified by the ‘clear zone’ that formed around colonies producing sulfuric acid from Na,S,O,. This strain showed halotolerant growth and tol&ance to various kinds of organic solvents, including 5% (v/v) benzene, 10%~ (v/v) toluene and 10% (v/v) I-~ I Y)lr ene. To investigate its ability to utilize organic sulfur compounds such as dibenzothiophene (IIBT), thiophene (T) and ethyhnethyl sulfide (EMS). which are present in petroleum. DS-994 was incubated in a water-petroleum two-liquid-phase system. As shown in Table 3, the initial total sulfur concentration of DBT, T and EMS in the diesel oil decreased to 0.175%, 0.180’% and 0.170’%, resprctively. The extent of utilization (‘~4~) of the organic sulfur compounds solubilized in the diesel oil was higher than that of control 2 (see Table 2). In this experiment, no difference in the percentage utilization of organic sulfur compounds solubilized in the diesel oil was observed either in the presence or absence of organic solvent. Strain US-1 906 (Ref. 22) showed tolerance to various kinds of organic solvents, including 10% (v/v) benzene, 2O’% (v/v) cyclohexanc and 20’% (v/v) n-hexane; it could also degrade 483% naphthalene in the presence of +hexane. Furthermore, DS-1906 could degrade various kinds of polyaronratic conpound, such as Buorene, phenanthrene. anthracene, pyrene, chrysene and 1,2-benzopyrene in an aqueousorganic two-liquid-phase system. Strain ST-l (Ref. 23) showed the ability to degrade cholesterol, and also displayed tolerance to organic TIBTECHJANUARY1996WOL14)
Table 2. Hydrocarbon
degradationa (%) by solvent-tolerant
Hydrocarbons
strains of bacteriab
Strains Pseudomonas putida strain IH2000
Alteromonas sp. strain DS-201
FJavobacterium sp. strain DS-711
28.0 30.9 35.7 10.6 11.8 40.2 46.2 39.9 35.7 40.2
30.5 30.3 32.4 9.7 10.8 41.4 45.3 44.6 39.0 38.1
97.0 82.1 82.1 71.4 68.0 86.7 85.8 88.0 92.9 80.5
N.G. N.G. N.G.
0
n-Alkanes n-C, n-C, n-C9 Go n-Cl1 n-Cl2 n-Cl3
n-Cl4 “-Cl, “-Cl,
Aromatic hydrocarbon Benzene Toluene pXylene
N.G.C i
i
aDegradation 1%)was calculated using the following equation: Residual concentration Residual concentration of of hydrocarbon in control ’ I( hydrocarbon in inoculated sample 1 x loo Degradation (%) = i Residual concentration l of hydrocarbon in control i The residual content of n-alkane (number of mg In 1Oml of kerosene) after treament with the control is: n-C,, 34; n-C,, 38; n-C,, 190; n-C,,, 245; n-C,,, 270; n-C,,, 653; n-C,,, 501; n-C,,, 332; n-C,,, 188; n-C,,, 39. “Data from Ref. 20. ‘N.G., no growth.
Table 3. Utilization of organic-sulfur compounds by strain DS-994” Organic sulfur
Total sulfur concentration (%) SampleC
Control Id
Control 2e
Dibenzothiophene
0.175 (12.5)
0.2 (OP
0.195 (2.5)
Thiophene
0.180 (10.0)
0.2 (0)
0.195 (2.5)
Ethylmethylsulfide
0.170 (15.0)
0.2 (0)
0.190 (5.0)
aData from Ref. 21. bFigures in brackets denote percentage utilization. ‘Sample, mixture of 0.5 ml concentrated cell suspension (109 cells ml-11 and 5 ml diesel oil with 5% (v/v) organic solvent. dControl 1, no inoculation. eControl 2, no diesel oil and organic solvent.
TIBTECH JANUARY 1996 WOL 14)
solvents such as 5% (v/v) benzene, 10% (v/v) toluene, and 10% (v/v) p-sylene. The ability of ST-1 to degrade cholesterol in a water-organic solvent twoliquid-phase system (water:organic solvent = 1: 1) was examined by adding cholesterol at a concentration of 1 mg 1111-l to the organic solvent. The results of a batch experiment are shown in Table 1. The data show a high level of cholesterol degradation, with the production of a large amount of by-product, identified as 1 ,&androstadiene-3.17-dione - a steroid hormone which was obtained using strain ST-I and the control strains when tested in organic solvents, but not in an aqueous medium; ST-1 showed the highest percentage of cholesterol degradation of all the strains tested, both with and without organic solvent. An organic-solvent-tolerant yeast has also been isolated from samples ofdeep-sea mud obtained from the floor of Sagami Bay at a depth of 1200 m. Strain Y-N (Ref. 24) has been sho\\rn to degrade hydrocarbons, and has been identified as a Cadida sp. Strain Y-40 grows well in the presence of solvents, such as
11 f OCUS
Table 4. Percentage of cholesterol degradation in two-liquid-phase systema Solvent
Water Tween 80
Strain
ST-1
Arthrobacter symplex ATCC6946
Flavobacterium dehydrogenans ATCC13930
Mycobacterium sp. ATCC29472
Mycobacterium smegmatis ATCC12549
68 (22Jb 78 (40)
2
52 (12) 60 (18)
49 (10) 59 (19)
2;
Two-liquid-phase systemC Benzene
22 (9)
5
7 (N.D.P
5 (N.D.)
4
Toluene pxylene
22 (10) (9) 24
i
;
L
:
n-Hexane nDecane n-Dodecane
56 (70) (54) 88 92 (78)
:: 67
38 (60) (44) 66 74 (65)
32 (62) (40) 66 73 (661
2; 77
aData from Ref. 23. ‘Figures in brackets “Two-phase systems dN.D., not detected.
denote the percentage comprise
the solvent
conversion of cholesterol shown and water.
n-octane, isooctane, cyclooctane. or kerosene at a concentration of 50’S) (v/v). The ability of Y-40 to degrade the hydrocarbons found in kerosene dissolved in 20% (v/v) fz-octane was compared with that of hydrocarbon-assimilating strains: C&i& tmpira/is strain IF0 1400 and Yurrou& lipolyti~a strain IF0 15-18; only strain Y-40 could degrade +alkanes with a carbon number of greater than eight in the presence of n-octane. The extent of degradation of PZ-alkanes in the presence of organic solvent was greater than that without an organic solvent. Microorganisms that can grow in high concerltrations of organic solvent are very useful for processing products that cannot dissolve in water. or for use in two-phase (water-organic solvent) fermentation systems.
Future applications The deep-sea environment is a source of unique microorganisms with great potential for biotcchnological exploitation. Very few studies concerning the isolation and characterization of deep-sea microorganisms have been carried out, and we think that investigations in this field may lead to many new discoveries. It is possible that life may have originated in the deep-sea, because in the distant past (3.5 billion years ago) the ultraviolet light from the sun was probably too stron, u on the land or in shallow water to permit the development of living organisms. Microorganisms living in the deep-sea have special features that allow then1 to live in an extreme environment, and it seems likely that further studies of these organisms will provide an important insight into the origin of life and its evolution. We have reported that a high-pressure-regulated system for gene expression is found not only in the deep-sea-adapted
into 1,4-androstadiene-3,17dione.
bacteria, but also in atmospheric-pressure-adapted bacteria such as E. di (Refs 25-27). These results suggest that the systems developed in a high-pressure environment may be conserved in organisms adapted to atmospheric pressure, possibly indicating that life emerged from the deep-sea environment a long time ago. In this review, we have described the isolation and characterization of two kinds of unique deep-sea microorganisms that display barophily and organicsolvent tolerance. To date, our investigations have not indicated any correlation between barophily and organic-solvent tolerance in deep-se,1 bacteria, despite the fact that both types of bacteria are adapted to the deep-sea environment. In our experience, it was not possible to isolate both types of lnicroorgallisnu from the same deep-sea samples, so it is possible that they thrive in different environlnents. These microorganisms may be very useful in new applications of biotechnology. For example, the genes and proteins from deep-sea barophilic bacteria arc adapted to highso they could be used for the pressure conditions’“,‘X, development of high-pressure bioreactors, for example. Furthermore, the organic-solvent-tolerant bacteria described here appear to be useful for degrading crude oil, removing several toxic conipound~ from the environment, and for producing steroid hormones from cholesteroPz~. Based on these new discoveries, fLIrther work aimed at developing connnerclal applications Ear these deep-sea microorganisms is in progress. Acknowledgements We thank Wayne 1~. Bellamy for reading the manuscript and for many useful discussions. We also thank the Shinhi operation team, and the crews of MS. K-i,kos~~ka, and M.S. !Vntsu.&m~. TIBTECH JANUARY1996WOL14)
12
TIBTECH Editorial Policy Trends in Biotechnology is a news, reviews and commentary journal designed to keep its international readership up to date with the current developments in biotechnology. TI3TECH occupies a niche between primary research journals and conventional review journals - it is not a vehicle for the publication of original research data or methods. There is strong emphasis on an integrated approach to communicating significant new advances in biotechnology, and discussing their commercial potential. This necessitates combining essential background with state-of-the-art information to make the topics addressed accessible and valuable to newcomers and experts in the field alike. All articles are subject to peer-review, and are prepared to strict standards to ensure clarity, scientific accuracy and readability. Commissioning does not guarantee publication. Reviews: Balanced and concise presentations of specific areas of research. The scope of such reviews is more limited than those of conventional review journals, but provides sufficient information to place the latest developments in a particular field in perspective. Focus: Short minireview-style articles that focus on a detailed examination of a narrow topic, thus providing a quick publication response to recognition of significant new directions of research. Forum: A platform for debate and analytical discussion of newly reported advances - whether reported in the research literature or at conferences. This section includes Workshops, Meeting reports and Letters to the Editor. Biotopics: A column providing a lighter, more-personal treatment of topics ranging from serious science and novel techniques, to finance and ethical questions. Features: Review-length articles that discuss business issues relevant to all biotechnologists - patenting, commercial opportunities and regulatory aspects.
IBTECH JANUARY 1996 (VOL 141
Isolating and characterizing deep-sea marine microorganisms Chiaki Kato, Akira lnoue and Koki Horikoshi We have isolated several microorganisms
that are adapted to living in the extremes
of the deep-sea environment. They include barophilic bacteria, which are able to grow at high hydrostatic pressure, but that are unable to grow at atmospheric pressure, and organic-solvent-tolerant
bacteria, which are able to grow in the
presence of toxic organic solvents such as toluene or benzene. In this review, we describe how to isolate such extremophiles,
and we outline the characteristics of
several strains that have been recovered from the deep-sea environment.
The deep-sea bed is a unique environment that experiences extremely high pressures and low temperatures. Microorganisms living there have developed particular characteristics that allow them to thrive at such extremes. In studies aimed at improving our understanding of microbial adaptation to the deep-sea e11vironment, we have isolated and characterized a number of microorganisms from samples of deep-sea mud obtained by the manned submerGble S&&i 6500. This vehicle, which is operated by the Japanese Marine Science and Technology Center (JAMSTEC), has the ability to submerge to a depth of 6300 m (Ref. 1). In this review, we describe two specific types of deep-sea microorganisms that have been isolated in our laboratory. The first group comprises barophilic microorganisms, which were isolated from the deep-sea samples at depths of greater than 25OC)m: they can grow only at high precsure, and not at normal atmospheric pressure. The second group comprises organicsolvent-tolerant microorganisms, which were mainly isolated from the samples taken at depths of less than 2000 m; thev can grow in the presence of high concentrations of toxic organic solvents such as toluene or benzene. It is not yet known whether these two groups are closely related, because ‘barophilic organicsolvent-tolerant bacteria’ have only just been isolated. However, some of the organic-solvent-tolerant bacteria can also grow at high pressure, so it is possible that such microorganisms will be found. Such microorganisms could prove to be useful for new biotechnology applications such as high-pressure bioreactor5 and two-phase (water/organic solvent) fermentation systems.
TIBTECH JANUARY
1996 IVOL
141
Deep-sea barophilic bacteria In 1957, Zobel and Morita’ were among the first researchers who attempted to isolate microorganisms that were specifically adapted to the high pressures associated with the deep-sea environment - they called them barophilic bacteria. However. it was only in 1979 that Yayanos et al.” were able to isolate barophilic bacteria, thanks to technlcal support and the development of procedures for investigating deep-sea environments. We are keen to isolate new deep-sea-adapted microorganisms and to characterize them for their possible application to high-preswre biotechnology. Several barophilic bacteria have been isolated from camples of deep-sea mud that were collected using sterilized mud samplers on the submersible Shkai f;5UO (KetS 3,5). The isolated barophilic bacteria (Table 1) were grown in pressure vessels under a range of hydrostatic pressures (0.1-80 Ml’s) and temperatures (;C15”C). The optinral pressure for growth of barophilic strains l~B5301, 11B6101, DB6705 and DB6906 was about 50MPa at IO”C, and 70 and hUMPa for strains 11B172F and l?B172R, respectively, at 10°C. None of these strains was able to grow at temperatures above 3VC under any pressure conditions. The growth-rate profiles (Fig. 1) of these barophilic strains indicate that their response to pressure is greatest near their upper temperature limit (15’C) for growth4,5. For example. the optimal growth pressures of L>B6101 and DB6705 at 15°C were about 70 and 60 MPa, respectively (Figs lb and 1 c), and the strains DB6705, 1)86906, DB172F and DB172R were cap,lble of growth only at pressures above 10 MPa (Figs lc-lf): however, the growth-rate profiles of these latter strains at 4’C were similar to the profiles of barotolerdnt (pressure-tolerant) strains. Yayanos” reported the effects of pressure and temperature on the growth rates of Feveral isolates of deep-sea 0 1996.
Elsewr
Science
Ltd 0167
- 7799/96/$15.00
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’ 90
(MPa)
Figure 1 Growth profiles of barophilic bacteria, strains (a) DB5501, (b) DB6101, (c) DB6705, (d) DB6906, (e) DB172F, and (f) DB172R at various pressures and temperatures; open circles denote growth at 15”C, filled circles denote growth at lO”C, and diamonds denote growth at 4°C (data from Refs 4 and 5). The growth rate is shown as l/td, where t, represents the doubling time (in hours). Barophllic bacteria were isolated keeping samples under high pressure and low temperature. Marine-broth 2216 medium, containing low-temperature-melting agar, was inoculated and incubated under high pressure. Subsequently, a test for barophily was conducted by comparing the growth rate of bacteria at 65 MPa and 0.1 MPa (Ref. 4).
barophilic bacteria that were isolated fi-ouj different depths of ocean. He reported maximal rates of reproduction at X-10°C for all isolates at various pressures, and that optimal growth occurred at a prewu-e similar to that encountered at their capture depth. However, two distinct growth profiles have been demonstrated for the barophilic strains isolated in our laboratory: the growth rates of strains L1BS.501, DB6101, DB6705 and DB6906 are higher at 15°C than at lower tenperatures, unlike the data obtained by Yayanos for the isolates”; and maximum growth rates for strains DB172F and DB172R were observed at lO”C, which was similar to growth profiles described by Yayanos. The differences in response to environmental conditions observed among previously reported barophilic bacteria, and those that we have isolated, are worth considering, both for their possible evolutionary significance, and for practical exploitation. Although the temperatures and preswre5 at which a change in growth response is observed can vary widely, the overall growth profiles are very similar for all the barophilic bacteria. We have also found that psychrophilic (i.e. those that have a temperature optimum for growth ofbetween 0°C and 30°C) barotolcrant bacteria isolated from deep-sea samples display a similar response profile to the barophilic organism~i. Indeed, even E.ccherirhi~ co/i responds in a similar way to variations in temperature and pressure’, perhaps implying that the same responw mechanisms are
widely conserved. At present, there are not enough data to draw any general conclusions about bacterial growth under high pressure, but It 1spossible that bacterial growth rates may be stimulated by high pressure near their maximum temperature for growth. From a comparison of the DNA sequences (held in the GenBank and EMBL databases) encoding 16s ribosomes. it was shown that the barophilic strains we isolated belong to the Proteobactcria, gamma subgroup”. The G+C content of chromosomal DNA from V&O spp. that belong to the gamma Pr~tec~hadcvia is between 10-50% (Ref. 9), and is considered to be typical of this subgroup. The G+C content of chromosomal DNA from the isolated barophilic strains was found to be similar, at -K-46’%. As a result of a taxonomic study of the obligate barophilic bacterium Coh~clIia hndnlicnsis, Deming et al.“’ reported that, based on its 5s ribosomal RNA sequence, this barophilic bacterium belongs to the gamma Proteobactcria. de Long and Franks” have also documented the existence of barophilic and psychrophilic deep-sea bacteria that belong to this group, as indicated by 16s ribosomal DNA sequence data. It is interesting to note that the 1 GS ribosomal I1NA sequences of the barophilic strains DBh’fOh, L)B172F and I>81 72R. 2nd the psychrophilic barotolerant strain DSS12 (Ref. 1) show the highest homology of all, indicating that these strains are very closely related. The relationship between the isolated strains and some strains of the gamma Pr(lfrobac&a TlBTECHJANUARY1996WOL14l
E. coli
S. marcescens
WHB46-2
DB5501
DB$lOl
\
\
Sh, a,ga
\
A. hydroi’hi’a
DB6705
Figure 2 Unrooted phylogenetlc tree showing the relatlonships between isolated barophilic strains withln the gamma subgroup of the Proteobacteria, as determined by comparing 16s ribosomal DNA sequences using the neighbor-joining method’z. The accession numbers of 16s ribosomal DNA sequences used in this figure are as follows; Escherichia coli (JO1695), Serratra marcescens (M591601, Plesiomonas shigelbdes (M59159), Proteus vulgaris (X07652), Vibno anguillarum (X16895), Aeromonas hydrophila (M591481, Shewanella alga (X81621), WHB46-1 (X54744), WHB46-2 (X54745), DB5501 (D21229), DB6101 (D21221), DB6705 (D21222), DB6906 (D21223), DSKl (D212241, DSS12 (D21225), DB172F (D63488). DB172R (D63824). (These accession numbers are for the EMBL, GenBank and DDBJ DNA databases.) K,,,, is the nucleotide substitution ratel2.
are shown in Fig. 2 in the form of a phylogenetic tree that uses the neighbor-joining method’“. The barophilic and barotolerant strains isolated from the deep-sea environment have been separated into one of the sub-branches in the gamma subgroup; the barophilic microorganisms reported by Liesack et al.‘” (strains WHB 46-l and WHI) 46-2) are also included in the sub-branch containing the strains isolated in our laboratory. These data suggest that the high-pressureadapted bacteria may belong to a new bacterial genus in the gamma subgroup of the Protrobacferia. Molecular-genetic analysis of the high-pressureadaptation mechanisms of such microorganisms is being carried out. Bartlett ct al.‘” have shown that the expression of the gene encoding the outer-membrane protein OmpH from a deep-sea P~~ofobactcrilrMI. strain SS9, is controlled by pressure, and the high-pressure regulation of cwyH gene expression has also been analysed1S-‘7. A pressure-regulated promoter region from a deep-sea barophilic bacterium strain DB6705 has been cloned and analysed’“, and it has been suggested that this promoter may be common to many barophilic bacteria’,lx. We believe that the new field of high-pressure biotechnology will develop from such basic studies. Organic-solvent-tolerant Organic-solvent-tolerant to grow in the presence lBTECHJANUARY1996WOL141
microorganisms microorganisms are able of high concentrations of
organic solvent. Organic solvents are generally toxic and kill most microorganisms at low concentrations (-0.1%). Although some microorganisms. including Pse~dowonas, ,Irlwomobarter and l%rclrtlia spp., can assimilate organic solvents, their solvent tolerance is less than 0.3%. We have determined that organic-solventtolerant microorganisms can grow on solvents such as toluene or benzene at concentrations of greater than 10% (v/v). While looking for such microorganisms around the islands of Japan, we discovered a variant strain of Pscudomm~s putida (strain IH2OOU), in mud samples taken from the Kumamoto Prefecture on Kyushu Island”‘; this is the first organic-solvent-tolerant bacterium that has been isolated. Strain IH2000 is able to grow in culture media containing more than 50% (v/v) toluene. We have also isolated useful organic-solvent-tolerant microorganisms (Table 1) from the deep-sea bed, and have found that 100 times more microorganisms of this type can be isolated from deep-sea mud samples than from soil samples taken from land. Organic-solvent-tolerant bacteria that exhibit the ability to degrade crude oil, polyaromatic hydrocarbons, or cholesterol, or the ability to utilize sulfur compounds, were isolated using the fcjllowing procedure: benzene was added to artificial sea-water-containing samples of deep-sea sediment to a concentration of 50’%, (v/v), and the cultures were incubated at room temperature for one week on a shaker; after
9 f ecus Table 1. Deep-sea microorganisms Bacterial strain
that have been isolated by the DEEPSTAR Group
Properties
Source
Ref.
Optimal growth at 50 MPa and 10°C. Optimal growth at 50 MPa and 10°C. Optimal growth at 50 MPa and 10°C. No growth at atmospheric pressure. Optimal growth at 50 MPa and 10°C. No growth at atmospheric pressure. Optimal growth at 70 MPa and 10°C. No growth at atmospheric pressure. Optimal growth at 60 MPa and 10°C. No growth at atmospheric pressure
Suruga Bay, at a depth of 2485 m Ryukyu Trench, at a depth of 5110 m Japan Trench, at a depth of 6356 m
4
Japan Trench, at a depth of 6269 m
4
Izu-Bonin Trench, at a depth of 6499 m
5
Izu-Bonin Trench, at a depth of 6499 m
5
Barophilic bacteria DB5501 DB6101 DB6705 DB6906 DB172F DB172R
Organic-solvent-tolerant DS-711 DS-944 DS-1906 ST-l
Y-40
bacteria
Degrades crude oil Utilizes sulfur Degrades polyaromatic hydrocarbons Degrades cholesterol
Organic-sovent-tolerant
t
Suruga Bay, at a depth of 1945 m Sagami Bay, at a depth of 1168 m Sagami Bay, at a depth of 1168 m Okushiri Ridge, at a depth of 1963 m
yeast
Hydrocarbon-degrading yeast
incubation, the benzene layers were carefully separated from the sea-water layers, and a portion ofeach benzene layer was spread onto M-II agar medium”‘; colonies that grew on the medium after incubation for two days at 25’C or 30°C were isolated and purified. The following bacteria are among the benzene-tolerant strains that have been isolated: IX-7 1 I degrades crude oiP, LX-994 utilizes sulfilr compounds”, DS1906 degrades polyaromatic hydrocarbons”, and ST-1 degrades cholesterol”. These strains were isolated fi-om samples of deep-sea mud collected at Suruga Bay. Sagami Bay or Okushiri Ridge near Japan. From their morphological and biochemical characteristics, strains DS-711. l)S-994. US-1906 and ST-1 have been identified as FlavoDacterirrm sp., Barilhs sp., Bacillus sp. and A~tlwobactcr sp., respectively. Strain US-71 1 (Ref. 20) was selected as a hydrocarbon degrader, and it showed halotolerant growth and tolerance to various kinds of organic solvents, including 5% (v/v) benzene, 10% (v/v) toluene and 10%~ (v/v) p-- Y yl ene. The ability of strain IX-7 11 to degrade aromatic hydrocarbons and the rz-alkanes in kerosene was compared with the control strains DS203 (an Ahwmr~as sp.) and I? putida strain IH-2000. As shown in Table 2, all of the strains tested could degrade the rl-alkanes in kerosene, but did not degrade aromatic hydrocarbons. However, strain 11S-711 was much better at degrading rl-alkanes than were the control strains. In particular, the percentage of uz-decane and rl-undecane degraded by the control strains was only about 10.0%~ in each instance, whereas strain IX-711 degraded 71.4% and 68.0%, respectively. Strain DS-711 showed the highest activity of
Sagami Bay, at a depth of 1200 m
rl-alkane degradation among the strains tested, and the extent of degradation reached approximately 85% after seven days. Strain 1X-991 (Kef. 2 1) was identified by the ‘clear zone’ that formed around colonies producing sulfuric acid from Na,S,O,. This strain showed halotolerant growth and tol&ance to various kinds of organic solvents, including 5% (v/v) benzene, 10%~ (v/v) toluene and 10% (v/v) I-~ I Y)lr ene. To investigate its ability to utilize organic sulfur compounds such as dibenzothiophene (IIBT), thiophene (T) and ethyhnethyl sulfide (EMS). which are present in petroleum. DS-994 was incubated in a water-petroleum two-liquid-phase system. As shown in Table 3, the initial total sulfur concentration of DBT, T and EMS in the diesel oil decreased to 0.175%, 0.180’% and 0.170’%, resprctively. The extent of utilization (‘~4~) of the organic sulfur compounds solubilized in the diesel oil was higher than that of control 2 (see Table 2). In this experiment, no difference in the percentage utilization of organic sulfur compounds solubilized in the diesel oil was observed either in the presence or absence of organic solvent. Strain US-1 906 (Ref. 22) showed tolerance to various kinds of organic solvents, including 10% (v/v) benzene, 2O’% (v/v) cyclohexanc and 20’% (v/v) n-hexane; it could also degrade 483% naphthalene in the presence of +hexane. Furthermore, DS-1906 could degrade various kinds of polyaronratic conpound, such as Buorene, phenanthrene. anthracene, pyrene, chrysene and 1,2-benzopyrene in an aqueousorganic two-liquid-phase system. Strain ST-l (Ref. 23) showed the ability to degrade cholesterol, and also displayed tolerance to organic TIBTECHJANUARY1996WOL14)
Table 2. Hydrocarbon
degradationa (%) by solvent-tolerant
Hydrocarbons
strains of bacteriab
Strains Pseudomonas putida strain IH2000
Alteromonas sp. strain DS-201
FJavobacterium sp. strain DS-711
28.0 30.9 35.7 10.6 11.8 40.2 46.2 39.9 35.7 40.2
30.5 30.3 32.4 9.7 10.8 41.4 45.3 44.6 39.0 38.1
97.0 82.1 82.1 71.4 68.0 86.7 85.8 88.0 92.9 80.5
N.G. N.G. N.G.
0
n-Alkanes n-C, n-C, n-C9 Go n-Cl1 n-Cl2 n-Cl3
n-Cl4 “-Cl, “-Cl,
Aromatic hydrocarbon Benzene Toluene pXylene
N.G.C i
i
aDegradation 1%)was calculated using the following equation: Residual concentration Residual concentration of of hydrocarbon in control ’ I( hydrocarbon in inoculated sample 1 x loo Degradation (%) = i Residual concentration l of hydrocarbon in control i The residual content of n-alkane (number of mg In 1Oml of kerosene) after treament with the control is: n-C,, 34; n-C,, 38; n-C,, 190; n-C,,, 245; n-C,,, 270; n-C,,, 653; n-C,,, 501; n-C,,, 332; n-C,,, 188; n-C,,, 39. “Data from Ref. 20. ‘N.G., no growth.
Table 3. Utilization of organic-sulfur compounds by strain DS-994” Organic sulfur
Total sulfur concentration (%) SampleC
Control Id
Control 2e
Dibenzothiophene
0.175 (12.5)
0.2 (OP
0.195 (2.5)
Thiophene
0.180 (10.0)
0.2 (0)
0.195 (2.5)
Ethylmethylsulfide
0.170 (15.0)
0.2 (0)
0.190 (5.0)
aData from Ref. 21. bFigures in brackets denote percentage utilization. ‘Sample, mixture of 0.5 ml concentrated cell suspension (109 cells ml-11 and 5 ml diesel oil with 5% (v/v) organic solvent. dControl 1, no inoculation. eControl 2, no diesel oil and organic solvent.
TIBTECH JANUARY 1996 WOL 14)
solvents such as 5% (v/v) benzene, 10% (v/v) toluene, and 10% (v/v) p-sylene. The ability of ST-1 to degrade cholesterol in a water-organic solvent twoliquid-phase system (water:organic solvent = 1: 1) was examined by adding cholesterol at a concentration of 1 mg 1111-l to the organic solvent. The results of a batch experiment are shown in Table 1. The data show a high level of cholesterol degradation, with the production of a large amount of by-product, identified as 1 ,&androstadiene-3.17-dione - a steroid hormone which was obtained using strain ST-I and the control strains when tested in organic solvents, but not in an aqueous medium; ST-1 showed the highest percentage of cholesterol degradation of all the strains tested, both with and without organic solvent. An organic-solvent-tolerant yeast has also been isolated from samples ofdeep-sea mud obtained from the floor of Sagami Bay at a depth of 1200 m. Strain Y-N (Ref. 24) has been sho\\rn to degrade hydrocarbons, and has been identified as a Cadida sp. Strain Y-40 grows well in the presence of solvents, such as
11 f OCUS
Table 4. Percentage of cholesterol degradation in two-liquid-phase systema Solvent
Water Tween 80
Strain
ST-1
Arthrobacter symplex ATCC6946
Flavobacterium dehydrogenans ATCC13930
Mycobacterium sp. ATCC29472
Mycobacterium smegmatis ATCC12549
68 (22Jb 78 (40)
2
52 (12) 60 (18)
49 (10) 59 (19)
2;
Two-liquid-phase systemC Benzene
22 (9)
5
7 (N.D.P
5 (N.D.)
4
Toluene pxylene
22 (10) (9) 24
i
;
L
:
n-Hexane nDecane n-Dodecane
56 (70) (54) 88 92 (78)
:: 67
38 (60) (44) 66 74 (65)
32 (62) (40) 66 73 (661
2; 77
aData from Ref. 23. ‘Figures in brackets “Two-phase systems dN.D., not detected.
denote the percentage comprise
the solvent
conversion of cholesterol shown and water.
n-octane, isooctane, cyclooctane. or kerosene at a concentration of 50’S) (v/v). The ability of Y-40 to degrade the hydrocarbons found in kerosene dissolved in 20% (v/v) fz-octane was compared with that of hydrocarbon-assimilating strains: C&i& tmpira/is strain IF0 1400 and Yurrou& lipolyti~a strain IF0 15-18; only strain Y-40 could degrade +alkanes with a carbon number of greater than eight in the presence of n-octane. The extent of degradation of PZ-alkanes in the presence of organic solvent was greater than that without an organic solvent. Microorganisms that can grow in high concerltrations of organic solvent are very useful for processing products that cannot dissolve in water. or for use in two-phase (water-organic solvent) fermentation systems.
Future applications The deep-sea environment is a source of unique microorganisms with great potential for biotcchnological exploitation. Very few studies concerning the isolation and characterization of deep-sea microorganisms have been carried out, and we think that investigations in this field may lead to many new discoveries. It is possible that life may have originated in the deep-sea, because in the distant past (3.5 billion years ago) the ultraviolet light from the sun was probably too stron, u on the land or in shallow water to permit the development of living organisms. Microorganisms living in the deep-sea have special features that allow then1 to live in an extreme environment, and it seems likely that further studies of these organisms will provide an important insight into the origin of life and its evolution. We have reported that a high-pressure-regulated system for gene expression is found not only in the deep-sea-adapted
into 1,4-androstadiene-3,17dione.
bacteria, but also in atmospheric-pressure-adapted bacteria such as E. di (Refs 25-27). These results suggest that the systems developed in a high-pressure environment may be conserved in organisms adapted to atmospheric pressure, possibly indicating that life emerged from the deep-sea environment a long time ago. In this review, we have described the isolation and characterization of two kinds of unique deep-sea microorganisms that display barophily and organicsolvent tolerance. To date, our investigations have not indicated any correlation between barophily and organic-solvent tolerance in deep-se,1 bacteria, despite the fact that both types of bacteria are adapted to the deep-sea environment. In our experience, it was not possible to isolate both types of lnicroorgallisnu from the same deep-sea samples, so it is possible that they thrive in different environlnents. These microorganisms may be very useful in new applications of biotechnology. For example, the genes and proteins from deep-sea barophilic bacteria arc adapted to highso they could be used for the pressure conditions’“,‘X, development of high-pressure bioreactors, for example. Furthermore, the organic-solvent-tolerant bacteria described here appear to be useful for degrading crude oil, removing several toxic conipound~ from the environment, and for producing steroid hormones from cholesteroPz~. Based on these new discoveries, fLIrther work aimed at developing connnerclal applications Ear these deep-sea microorganisms is in progress. Acknowledgements We thank Wayne 1~. Bellamy for reading the manuscript and for many useful discussions. We also thank the Shinhi operation team, and the crews of MS. K-i,kos~~ka, and M.S. !Vntsu.&m~. TIBTECH JANUARY1996WOL14)
12
TIBTECH Editorial Policy Trends in Biotechnology is a news, reviews and commentary journal designed to keep its international readership up to date with the current developments in biotechnology. TI3TECH occupies a niche between primary research journals and conventional review journals - it is not a vehicle for the publication of original research data or methods. There is strong emphasis on an integrated approach to communicating significant new advances in biotechnology, and discussing their commercial potential. This necessitates combining essential background with state-of-the-art information to make the topics addressed accessible and valuable to newcomers and experts in the field alike. All articles are subject to peer-review, and are prepared to strict standards to ensure clarity, scientific accuracy and readability. Commissioning does not guarantee publication. Reviews: Balanced and concise presentations of specific areas of research. The scope of such reviews is more limited than those of conventional review journals, but provides sufficient information to place the latest developments in a particular field in perspective. Focus: Short minireview-style articles that focus on a detailed examination of a narrow topic, thus providing a quick publication response to recognition of significant new directions of research. Forum: A platform for debate and analytical discussion of newly reported advances - whether reported in the research literature or at conferences. This section includes Workshops, Meeting reports and Letters to the Editor. Biotopics: A column providing a lighter, more-personal treatment of topics ranging from serious science and novel techniques, to finance and ethical questions. Features: Review-length articles that discuss business issues relevant to all biotechnologists - patenting, commercial opportunities and regulatory aspects.
IBTECH JANUARY 1996 (VOL 141