Tolerance of bacteria to extreme gas supersaturations

BIOCHEMICAL

Vol. 85, No. 4, 1978

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Pages 1379-1384

December 29,1978

TOLERANCE

OF BACTERIA

Barbara

TO EXTREME

B. Hemmingsen

GAS SUPERSATURATIONS

and Edvard A. Hemmingsen

Department of Microbiology, San Diego State University, San Diego, California 92182; and The Physiological Research Laboratory, Scripps Institution of Oceanography, La Jolla, California 92093

Received

November 13,1978

SUMMARY: Bacteria without (Escherichia coliand Corynebacteriumxerosis)and with gas vacuoles (Microcyclus aquaticus)were saturated with Ar or N, gas at pressures up to 300 atm and then rapidly decompressed. The resulting intracellular gas supersaturations had no effect on the viability of the bacteria except when the gas vesicles were purposely kept intact by slow pressurization rates. Thus no gas bubbles form within the cells even at these extreme supersaturations. This contradicts earlier interpretations of the cause of the disruptive effect on various cells by gas pressurization and decompression. INTRODUCTION: be disrupted

Several

by exposing

Various

devices

(Kontes,

Vineland,

have been developed New Jersey).

the suspensions

Significant

Commerford

(7) assumed

internal

pressure

cell wall.” bubbles

Others

supersaturations disruption through

within

are placed

process

pressure

cell

.” Hunter

and if it is large enough

series of experiments

was designed

can lead to formation

of intracellular of mechanical

and gram positive with gas vacuoles

action

is then suddenly

shear forces

bacteria

breaking

the bacterial

by external

Gram negative

in a pressure

After a time an orifice.

is not clear and has not been

of intracellular

in the absence

through

mini-bomb

material.

the formation

disruption

tested. Two gram negative gas phase

balanced,

(3,4,7,91.

e.g., the Kontes

by expulsion

that ‘I.. . . actual

of gas within

have invoked

of bacteria an orifice.

in the expelled

that if ‘I.. . the external

is no longer

or mechanical

The present

decompressed

Fraser (3) mentioned

from the expansion

and rapid decompression

the cell suspensions

gas in the disruption

presumably,

and other cells can

from about 35 to 120 atm are applied.

occurs

The role of dissolved evaluated.

ranging

that bacteria

for such cell disruption,

In general,

are rapidly

cell breakage

critically

have reported

them to high gas pressures

vessel, and gas pressures interval,

investigators

comes, and released,

the

it can cause rupture

(12) or extracellular

of the

(9) gas

(1,2, 10).

to determine bubbles

if high gas

and subsequent

stresses

associated

bacteria

without

were included

cell

with passage

gas vacuoles

were

so that the effects of a

the cell could be observed.

OOOS-291X/78/0854-1379$01.00/O 1379

Copyright 0 1978 by Academic Press, Inc. AN rights of reproduction in any form reserved

Vol. 85, No. 4, 1978

MATERIALS

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AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

AND METHODS:

Material preparation: The species with gas vacuoles, Microcyclus aquaticus (ATCC 27066) and Prosthecomicrobium pneumaticurn (ATCC 23633) were obtained from the American Type Culture Collection, Rockville, Maryland. Each was grown in the ATCC recommended medium at 30°C or 25°C respectively, without agitation. Noble agar (1.2% w/v) was used to solidify the liquid media as required. The other bacteria were obtained from the culture collection of the Department of Microbiology, San Diego State University. Escherichia co/itATCC 11775) was grown at 37’C in Trypticase Soy Broth (BBL). Corynebacterium xerosis (origin unknown) was grown at 37°C in Brain Heart Infusion Broth (Difco). Bacto-agar (1.5% w/v) was used to solidify the liquid media as required. M. aquaticusand P. pneumaticumwere grown in 6 ml of liquid medium in screwcapped tubes. After 5 to 7 days, 1 ml of the culture was added to 4 ml sterile distilled water. After thorough but gentle mixing, viable counts were performed by diluting in sterile distilled water and spreading aliquots on the surface of the appropriate solidified medium in duplicate. A portion of the suspension was placed in the pressure cell. After pressurization, equilibration and decompression, 0.1 ml was removed for viable count determinations. An equal portion was kept at room temperature and pressure at the same stirring rate and viable counts were made at the end of the pressure experiment. Suspensions were examined microscopically at the end of the experiment at 1000X under phase contrast. E. coliand C. xerosiswere cultured overnight without agitation in 8 ml of medium in a screw-capped tube or in 100 ml in a 250 ml Erlenmeyer flask, respectively. To start an experiment, the culture was mixed, and diluted 2:lOO with sterile 0.85% (w/v) saline. The diluted C. xerosis culture (10 ml) was first stirred at room temperature for l/z hour on a magnetic stirrer. In both cases, an aliquot was removed for viable count determination with all dilutions made in saline, then 4.9 ml was placed in the pressure cell. Once during each series of experiments, an identically treated suspension was stirred at the same rate at room pressure in air for the same length of time as the pressure experiment, and viable counts made. Experimental procedure: The bacterial suspension was placed in a 10 ml glass dish contained in a 12 ml stainless steel chamber. The chamber was closed and filled with Ar or N, gas of 99.99% or better purity; the commercial tank pressures of about 150 atm were boosted to required values by compressing them in an accumulator, placed between the tank and the chamber, by means of an hydraulic water pump. The initial pressurization times were about 30 seconds. In some experiments with gas-vacuolate bacteria, the pressurizations were performed in small steps (2-4 atm initially) over a l%-3 hour period in order to avoid collapse of the gas vacuoles. All suspensions were equilibrated with the gas at full pressure for % hour using a magnetic stirrer at 250 rpm. Separate experiments have shown that this is more than twice as long as is needed for complete equilibration of 2-5 ml of water. After equilibration, the gas was discharged rapidly from the chamber; the complete decompression time was l-l % seconds except in one experiment with M. aquaficusin which decompression was carried out over a 1% hour period. The chamber lid was removed and samples of the suspensions in the chamber were taken for viable count determinations.

RESULTS: saturated

Populations

of E. coliand

with gas at pressures

decompression

to 1 atm (Table

viable ceils per ml probably compression-decompression

C. xerosisthat

up to 300 atm showed I). The generally

1380

compressed

no loss of viability

consistent

was a result of disaggregation process.

were rapidly

and

after rapid

increase

in the number

of

of clumped

cells during

the

BIOCHEMICAL

Vol. 85, No. 4, 1978

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

TABLE I Effects

of rapid decompression

of gas saturated

equilibration

Escherichia

lb

3.2 x 2.4 x 3.2 x 2.5 x 3.0x 2.3 x 2.3x

100 200 300 100 200 300

N2 N2 N2

Ar Ar Ar Corynebacterium

la 100 200 300 100 200 300

N2 t-42 N2

Ar Ar Ar

of viablecells/ml after decompression

b) Control

experiments

Excellent

survival species,

4.7 5.6 4.55 4.4 3.2 35x 4.7

with cells stirred

2.7 x 2.5x 3.2 x 2.4x 3.25 x 2.9 x 3.0 x

IO’ IO7 207 IO7 1 O7 1 O7 1 o7

84 104 100 96 108 126 130

10s 1 O6 10s 10s 1 OS 10s x 10s

52x 6.4 x 5.3 x 4.6x 4.25 x 4.4 x 4.1 x

10s 10’ 10s IO’ 10’ 10s 10s

110 114 116 105 133 125 a7

x x x x x

at atmospheric

pressure

was found in two equivalent M. aquaticus

(Table

occurred

in one experiment.

as judged

by the loss of “phase-bright”

observed

also in a similar

experiment

of the cells (Table examination.

(11). This collapse

with M. aquaficusthat

intact, rapid decompression number

remaining

was observed.

larger capable

/? pneumaticurn. of colony

fast decompression

and in the microscope

could be seen along with a few intact

gas vesicles

were designed resulted

was

experiment

(“phase-dark”)

1381

numerous cells.

to leave

in the killing

of the

by

indicating

or the gas vesicles

also was performed

At the end, only about 30% of the population

formation

units

of gas vesicles

cells were “phase-dark”

during

One slow compression,

with the gas-

were left intact as judged

at the start of the experiment

No debris

performed

some loss of colony-forming

these cells had no gas vesicles compression.

for % hour.

with P. pneumaficum.

II); a substantial These

a I/Z hours

cells were devoid of inflated

inclusions

experiments

some or most of the gas vesicles

30 seconds),

experiments

II), although

The decompressed

In the slow compression

microscopic

% siwvival

1 O7 1 O7 1 O7 1 O7 lo7 1 O7 IO7

All experiments were conducted with fast compression (within equilibration time, and a fast decompression (I -1 VZ seconds).

vacuolated

of two bacteria.’

xerosis

Air

majority

on the viability

coli

Air

a)

Number before compression

prG:i

gas

suspensions

that either collapsed

with the

was found to be

“ghosts”

and much debris

BIOCHEMICAL

Vol. 85, No. 4, 1978

AND BIOPHYSICAL

TABLE

II

Effect of the rate of compression to an argon equilibration decompression on the viability of thegas-vacuolate Rate of

RESEARCH COMMUNICATIONS

Number

pressure bacterium,

of viable

of 200 atm and of the rate of Microcyclus aquaticus

cells/ml after decompression

before compression

decompression

fastb

pziment

fast

fast

slowe

after stirring

COtTl-

pression

AC

1.4 x 10s

1.7 x 108

Cd

2.75 x 10s

2.5 x 10s

2.1 x 10s

2.65 x 10s

0.8 x 10s

38

2.45 x 10s

0.85 x 10s

28

2.6 x IO8

0.7 x 10s

44

2.8 x 10’

100

16x10s

slows

slow

0

2.8 x 10’

using viable

c)

In Experiment experiments,

d)

In this line, values

2.95~10s

count at start of experiment,

was compressed was accomplished

A, thevolumeof cell suspension the volume was 2 ml. are averages

of 2 runs on aliquots of 1% (Experiment

f)

Slow compression

over a period

of 3 hours.

g)

Slow compression

and slow decompression

The results

supersaturations

which

to have damaging

the most soluble bubbles

formed

and the internal

effects

the cells.

pressures

show that bacteria

had formed,

thus generated

tolerate

gas

than those that have been inferred

to affect viability.

If bubbles

C) hours.

of 3-l 13 hours

(3,4). Even decompression

gas, was insufficient within

of the same cell suspension.

experiments

higher

Fast

cell was 5 ml. In subsequent

A) or 2 (Experiment

over a period

of the present

are several-fold

compression. in about 30 seconds.

in the pressure

over a period

DISCUSSION:

before

to equilibration pressure within l-l % seconds.

e) Slow compression

previously

71

3.0 x 10s

A

the suspension decompression

100

10s

A

fast

b)

1.4x108 1.95x

C very slow’

a) calculated

% survivala

would

from 300 atm Ar, which

This strongly some growth

be of sufficient

suggests would

is

that no

be inevitable

magnitude

to disrupt

the

cell wall. In the course supersaturations pressures harmful

of the experiments,

but also to rapid hydrostatic

and to elevated conditions

been examined

bacteria

affected previously

concentrations

were exposed pressure

of dissolved

the test bacteria. (8, 12); hydrostatic

not only to gas

changes,

to increased

gas. None of these potentially

Only the effects of hydrostatic pressures

1382

hydrostatic

pressure

of the levels used here and

have

BIOCHEMICAL

Vol. 85, No. 4, 1978

changes coliand

in hydrostatic

pressure

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

close to the rate employed

here were found tolerable

to E.

many other bacteria.

The presence breakage

of a gas phase within

upon exposure

maintained

quantities

decompression,

vesicles

collapsed

intentionally

appeared

concentrations

of bubble

to the massive

formation

formation

bubble

within

bacteria

without

form spontaneously

supersaturations

as low as 160 and 200 atm, respectively

atm, for example,

produces

outside

mechanical

forces

generated

liquid at the higher

(56).

An Ar saturation

per ml on decompression.

to considerable

surface

is in sharp gas

at Ar and N, of 200

It is striking

the cells has little effect on their viability

cell breakage

or extracellular

bubble

as the cell suspension

must play a crucial

intact gas vesicles

tension

despite

that

the fact

and

as a result.

Thus, the bacterial intracellular

intact gas

in water and in solutions

of bubbles

that most of the cells are no doubt exposed

pressure

took more than 3 hours and

in the suspending

Bubbles

of bubbles

survived

to the hydrostatic

formation

supersaturations.

this profusion

were collapsed

of the population

to maintain

or the

of the population.

of bubbles

millions

cells that

of M. aquaticus

of Ar used; slow compression

to have no effect on the viability

and either

at the start of the experiment,

is not sensitive

to prevent

so slowly that

them fP. pneumaticum).The

a much larger proportion

and slow decompression

were

(1 1). On rapid

expanded

When the vesicles

M. aquaticus

led to cell

the pressure

by diffusion

had no vesicles

compression.

to gas supersaturation.

The absence contrast

during

by rapid compression,

or the elevated

by increasing

ruptured

probably

gas vesicles

Intact gas vesicles

each intact vesicle

the cells fM. aquaticusjor

these experiments

vesicles

compression

of gas could enter the vesicles

survived

exposure

during

the gas phase within

disintegrated

with intact

to high gas supersaturations.

in the bacteria

sufficient

bacteria

reported formation.

is forced

role in the cell breakage

ACKNOWLEDGEMENT: from the National Institutes

previously

(3,4) cannot

Our results

indicate

through

an orifice

be due to

that mechanical

forces

at the time of decompression

that has been obtained.

This research was supported in part by grant number HL 16855 of Health, U.S. Department of Health, Education and Welfare.

REFERENCES: 1. 2. 3. 4.

Coakley, W.T., Bater, A.J. and Lloyd, D. (1977) Adv. Microbial Physiol. 16, 279-341. Cummings, D.J., and Tait, A. (1975) in Prescott, D.M., editor, Methods in Cell Biology, vol. IX, pp. 281-309, Academic Press, N.Y. Foster, J.W., Cowan, R.M. and Maag,T.A. (1962) J. Bacterial. 83,330-334. Fraser, D. (1951) Nature 167,33-34.

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Vol. 85, No. 4, 1978

5. 6. 7. 8. 9. 10. 11. 12.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Hemmingsen, E.A. (1977) Nature 267, 141-l 42. Hemmingsen, E.A. (1978) Z. Naturforsch. 33a, 164-l 71. Hunter, M.J., and Commerford, S.L. (1961) Biochim. Biophys. Acta 47,580-586. Morita, R.Y. (1972) in Kinne, O., editor, Marine Ecology, vol. I, part III, pp. 1361-l 388, Wiley Interscience, N.Y. Wallach, D.F.H. (1967) in Davis, B. and Warren, L., editors, Specificity of Cell Surfaces, pp. 129-l 63, Prentice Hall, Inc., Englewood Cliffs, N.J. Wallach, D.F.H. (1972) The Plasma Membrane: Dynamic Perspectives, Genetics and Pathology, pp. 39-40, The English Universities Press, London. Walsby, A.E. (1972) Bacterial. Rev. 36, l-32. ZoBell, C.B. (1970) in Zimmerman, A.M., editor, High Pressure Effects on Cellular Processes, pp. 85-130, Academic Press, N.Y.

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