Psychrophilic microorganisms from
the deep-sea environment

Tetsuo Hamamoto
Microbiology Laboratory

Abstract:

Psychrophilic microorganisms grow optimally at low temperatures, such as0-10 $^\circ$C. Isolation and characterization of psychrophilic microorganisms in the deep-sea environment, one of the most stable low-temperature environments, have been carried out. So far, more than 100 microorganisms of this sort have been isolated. Among them are 9 strains whose maximum growth temperature is lower than 20 $^\circ$C. Amylase activity was detected in two strains and lipase activity was detected in four. Enzymes produced by these microorganisms showed low thermostability and significant retention of activities at low temperatures, illustrating their physiological adaptation to

Bacteria capable of growing at low temperatures (0 $^\circ$C or lower) are referred to as psychrophiles and psychrotrophs. Psychrophiles are bacteria which have an optimal temperature for growth of less than 15 $^\circ$C, a maximal temperature for growth of less than 20 $^\circ$C, and a minimal temperature for growth at 0 $^\circ$C or less. Psychrotrophs can grow at low temperatures but their optimal growth temperature is higher than 15 $^\circ$C.$^{1)}$ Recent advances in the field of marine technology have made it possible to study in detail characteristics of deep sea and its floor.$^{2)}$ Since the temperature of deep-sea water is constant at about $-$1 to 3 $^\circ$C,$^{1,3,4)}$ we have begun to isolate psychrophilic and psychrotrophic bacteria from the deep-sea sediment samples. This paper describes characteristics of psychrophilic and psychrotrophic bacteria isolated from the deep sea and properties of their enzymes.

We have isolated more than 100 psychrotrophic and 9 psychrophilic bacteria from deep-sea sediment samples which were collected during a series of dives by the Shinkai 2000 and the Shinkai 6500 submersibles belonging to the Japan Marine

Table: Properties of Isolated Representative Psychrophiles and Psychrotrophs.

Science and Technology Center. The collected sediment samples were spread onto agar plates containing nutrients and 3% NaCl. Bacterial colonies on the plates incubated at 4 $^\circ$C were examined further. Taxonomic and growth properties of representative isolates, which tentatively indicate that all belong to the genus Vibrio, are summarized in Table 1. Among these bacteria, two strains (29-6 and 5710) possessed extremely low maximum (around 10 $^\circ$C) and optimum temperatures (around 7 $^\circ$C) for their growth (Fig. 1).

Figure: Effect of temperature on the growth of deep-sea psychrophilic bacteria, strains No. 29-6 and 5710. Growth is expressed as specific growth rate, ln 2 (=0.69)/generation time (hr).

In order to study cold adaptation of psychrophilic microorganisms, we have examined several extracellular enzymes which they produce.

Amylases were purified from the culture supernatants of two psychrotrophic isolates, Vibrio sp. strains No. 4-3$^{5)}$ and No. 814-4. Characteristics of these amylases are shown in Table 2. These amylases exhibited optimal temperatures for the activities at 35 and 25 $^\circ$C, respectively, and retained the activities at lower temperatures.

Table: Characteristics of amylases from deep-sea psychrotrophic bacteria.

Four strains produced $p$-nitrophenyl laurate-hydrolyzing enzymes in culture media. The four activities were completely lost after a 10 minute incubation at 40 $^\circ$C. As these activities could be observed in the presence of $n$-hexane, the effects of temperature on activities in aqueous and $n$-hexane-containing reaction mixtures were compared. Fig. 2 shows a temperature profile of the activity of strain 5710 as an example. In the $n$-hexane-containing reaction mixture at $-$10 $^\circ$C, enzyme reaction occurred at a rate of 28% of that at the optimal temperature. The optimal temperature for enzyme activity decreased by 10 $^\circ$C when $n$-hexane was present in the reaction mixture. However, only 6.9% of the activity observed in the aqueous reaction mixtures was observed in the presence of $n$-hexane.

The psychrophilic bacteria described in this paper are able to grow efficiently at low temperatures, illustrating their physiological adaptation to the permanently cold deep-sea environment. Exoenzymes produced by these bacteria displayed low optimal temperatures and low thermal stability, which are the main characteristics of cold adapted enzymes. In particular, lipase in reactions containing $n$-hexane, showed

activities at temperatures as low as $-$10 $^\circ$C. Although the low-temperature optima and the low thermostability reported here were observed with only six enzymatic activities, characterization of more enzymes from psychrophilic and psychrotrophic bacteria would provide useful tools for the biochemical study of psychrophily. It may be also possible to apply these enzymes in various chemical reactions at low temperatures.

Figure: Effect of temperature on enzyme activity of the culture supernatant of the strain No. 5710. Solid lines indicate the reactions without $n$-hexane, and broken lines indicate the reactions with $n$-hexane. Activity was expressed as the increase in absorbance at 405 nm by 1 ml of the sample in 1 minute.

References

1) R. Y. Morita: Bacteriol. Rev. 39, 144 (1975).

2) S. F. Myers and A. Anderson: Science 255, 28 (1992).

3) H. W. Jannasch and C. D. Taylor: Ann. Rev. Microbiol. 38, 487 (1984).

4) B. Austin: Marine microbiology. Cambridge University Press, Cambridge (1988).

5) T. Hamamoto and K. Horikoshi: FEMS Microbiol. Let. 84, 79 (1991).