Effect Of Uv C On Nutrient Limited Bacteria Biology Essay

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Recent studies have shown that a range of spore and non-spore forming bacteria can be isolated from the stratosphere. The aim of the work reported here was to determine some of the factors which may increase the rate of survival of Bacillus simplex and Staphyloccus pasteuri in this UV-rich environment. The results show that, while both UV-B and C are lethal to both B.simplex and S.pasteuri,"shading" by particulates, similar to those likely to present in the stratosphere, as well as the presence of low temperature and nutrient limitation are likely to increase the ability of bacteria to survive in the UV-rich stratosphere. Although the degree of protection, to UV, by the factors tested here afforded is relatively small, any degree of increased survival is likely to provide a selective advantage, allowing bacteria to survive in the stratosphere or perhaps deeper into space.

Introduction

Recent studies have shown that bacteria can be isolated from the stratosphere at heights of 20 1-2 and 41km 3-5, thereby confirming earlier reports of the presence of a stratospheric bacterial flora 6. The stratosphere is characterised as a region of low temperature and high degree of exposure to UV light, notably highly biologically damaging UV-C 7-8. The aim of the work reported here was to determine the effects of UV-B and UV-C on bacteria similar to those found in the stratosphere and to determine if growth under low nutrient and low temperature influences the survival in the presence of UV of two bacteria, the spore-forming Bacillus simplex and the non-spore forming Staphylococcus pasteuri. Additionally, the effects of UV-shading by a range of particulates, likely to found in the stratosphere were determined.

Materials and Methods

The bacteria used in this study were laboratory strains of Bacillus and Staphylococcus which have been isolated from the stratosphere and E.coli, and non-stratospheric organism; the cultures were obtained from DSMZ, Germany. Bacteria were grown in Nutrient Broth (Oxoid) for 48h at 37°C; B.simplex with shaking, and S.pastueri without shaking. Following incubation, the cells were harvested by centrifugation at 5000 rpm for 10 mins. and then stored in phosphate buffered saline (PBS) at 4°C. In order to obtain spores, B.simplex was grown on the complex medium recommended by Munakata and Rupert (1972) 9 at 30°C for 7days and the spores were harvested using the method recommended by Nicholson and Law (1999) 10. The spore suspensions were adjusted to a spore concentration of between 107 and 108 spores ml-1 and stored in buffed PBS at 4°C.

Effect of UV-C on nutrient limited bacteria

For studies on the effect of UV-C on nutrient-limited bacteria, B.simplex and Staphylococcus pasteuri was grown in peptone glucose broth medium with carbon or nitrogen, or both carbon and nitrogen omitted.

Effect of low temperature exposure on UV-C on bacteria

The bacteria were grown in nutrient broth (Oxoid) and then centrifuged to from a pellet. The medium was removed and the pellet left at -7°C for 2 days; the pellet was then thawed at room temperature and exposed to UV-C (wavelength 254nm, from a UVP,18,UV source, Upland, California, USA).

RESULTS AND DISCUSSION

The effects of the three types of UV-radiation on the survival of sporulating (B.simplex) and a non-spore-forming bacteria (S.pasteuri) is shown in Table 1.

Table 1. Lethal effects of different forms of UV on a range of bacteria

C

B

A

13

21

-

B.amyloliquefaciens

10

8

-

B. cereus

11

24

-

B. pumilus

12

19

-

B. simplex

11

18

-

B. subtilus

7

12

-

E. coli

8

12

-

S. aureus

Killing point in minutes

Ultraviolet-A had no effect of either of the bacteria, while UV-B and C proved lethal to both, with UV-C proving to be approximately twice as lethal as UV-B. The killing point of UV-C was also approximately twice that of UV-B. These results confirm the generalisations that UV-A is not lethal to bacteria, UV-C is particularly inhibitory, while UV-B is intermediate between the two in regard to its bacteriocidal effects. Similarly, it is well known that spore-forming bacilli are more resistant to UV than are non-spore formers, such as species of Staphylococcus 11. It is not surprising then that most of the bacteria which have, to date, been isolated from the stratosphere are spore forming members of the genus Bacillus 4. Despite this fact, both spore formers and non-sporulating bacteria have been isolated from this environment 2,12 .This apparent paradox can be explained by assuming that so-called "UV-shielding" of bacteria occurs due to the presence of particulates, such as cosmic dust, which are present in the stratosphere; these particulates are impervious to UV light and thereby protect bacteria from its lethal effects.

Table 2: Effect of particulate shedding on UV-C effect on bacteria cell surviving 10 mins colony forming units (1x103)

B. simplex

UV-B

UV-C

No particulates

7.8

1.4

Buckminsterfullerene

81.25*

352.4*

Charcoal

0.5*

190.0*

Clay

11.9*

140.0*

Silicic acid

0.0*

30.5*

Meteor dust

8.0

150.0*

S. pasteuri

No particulates

12

8

Buckminsterfullerene

27*

4.8*

Charcoal

20*

0.4

Clay

22.0*

0.3

Silicic acid

0.27

0.0

Meteor dust

21.0*

0.3

* Significantly different from control (p≤0.05)

The results shown in Table 2 confirm that a range of particulates, typical of those likely to be found in the stratosphere, or space in general, protect both B. simplex and S.pasteuri from the lethal effects of both UV a and UVB. Most of the particles reduced the lethal effects of UV B and C in both bacteria, but the effect was most pronounced in the presence of buckminsterfullerene, a finding which can be explained by the fact that this carbon allotrope, (which has been reported occurring in space) 13, contains numerous pores in which bacteria may "hide" and thereby be protected from lethal UV-radiation. The protective effect of buckminsterfullerene would presumably be particularly useful in the case of non-sporing bacteria, like S. pasteuri which, in the absence of any protection are extremely sensitive to both UV-B and UV-C. Such UV-protection provided by particulates, even if it occurs for only a short period, would provide a selective advantage for any bacteria being in this way protected from stratospheric-UV.

Since the stratosphere is extremely cold and likely to be lacking in bacteria-available nutrients, this study determined the effects of nutrient limitation and pre-exposure to low temperature on the lethal effects, on bacteria, of both UV-B and UV-C. Incubation of the bacteria under nutrient limitation prior to UV-B and C exposure generally had no significant effect on the killing point produced by both forms of UV. An exception occurred however, when B simplex was grown in a medium lacking both carbon and nitrogen prior to UV exposure (Table 3).

Table 3: Effect of low temperature (-70°C) on bacterial response to UV- C

25 °C

-70 °C

B

C

B

C

B. simplex (Complete medium)

17

9

25

18

B. simplex (C-deficient)

14

10

21

18

B. simplex (N-deficient)

17

10

26

16

B. simplex (C and N deficient)

31

22

36

30

S. aureus (Complete medium)

8

7

12

7

S. aureus (C-deficient)

8

8

12

9

S. aureus (N-deficient)

10

8

12

8

S. aureus (C and N deficient)

11

8

15

8

Killing point in minutes

In this case, a marked, and significant increase in the time required for both types of UV radiation to kill this bacterium occurred; no similar effect was seen with S.pasteuri. The ability of B.simplex to survive for longer periods in the absence of nutrient in the stratosphere is again likely to provide it with a selective advantage over other bacteria which are unable to do so. Table 4 shows that a period of low temperature incubation prior to UV exposure had a remarkable protective effect on the ability of both bacteria to survive both UV-B and C. Both carbon and nitrogen limitation individually had no significant effect on survival, but the removal of both carbon and nitrogen from the medium led to a significant increase in survival times for both B.simplex (UV-B and C) and S.pasteuri (UV-B). These results show that low temperature exposure, far from making bacteria more vulnerable to the lethal effects of UV provides some protection, which is most marked in the response of B.simplex to UV-C.

Table 4: Effect of nutrient limitation on the lethal effects of UV- C on B. simplex and S. aureus

B

C

B. simplex (Complete medium)

17

12

B. simplex (C-limited)

14

10

B. simplex (N-limited)

18

10

B .simplex (N and C-limited)

32*

24*

S. pasteuri (Complete medium)

8

7

S. pasteuri (C-limited)

8

8

S. pasteuri (N-limited)

10

8

S. pasteuri (N and C-limited)

10

8

Killing point in minutes

* Significantly different from control (p≤0.05)

The above results show that while both UV-B and C are lethal to both B.simplex and S.pasteuri "shading" by particulates, similar to those likely to present in the stratosphere, as well as the presence of low temperature and nutrient limitation are likely to increase the ability of bacteria to survive in the UV-rich stratosphere. Although the degree of protection, against UV, by the factors tested here is relatively small, any degree of increased survival is likely to provide a selective advantage, allowing bacteria to survive in the stratosphere or perhaps deeper into space 14.

Acknowledgement

Thanks are due to Prof. Milton Wainwright for her contribution to this study. The study was supported in part by the Centre for Excellence and Diversity, King Saud University; I also thank the College of Science Research Center, King Saud University, Saudi Arabia, for support.

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