Aerosols of the bacterium Serratia marcescens, and of uniformly labelled C-14 glucose, were created simultaneously and mixed in tubing leading to an aerosol chamber. During a subsequent period of about 5 hrs, C-14O2 was produced unequivocally within the chamber, and insoluble, labelled material within the suspended particles first increased, then decreased

Chatigny, M. A.

Dimmick, R. L.

Levin, G. V.

Schrot, J. R.

Straat, P. A.

Wolochow, H.

English

NASA Technical Reports Server

EVIDENCE FOR METABOLIC ACTIVITY
OF AIRBORNE BACTERIA
2nd Quarterly Report, 1973-74
NASA Contract W13450
Task No. 193-58-62-13-10
R. L. Dimmick, H. Wolochow, M. A. Chatigny, P. A. Straat,
J. R. Schrot and G. V. Levin
(NASA-CR-139620) EVIDENCE FOR METABOLIC N74-31
ACTIVITY OF AIRBORNE BACTERIA Quarterly
Report, 1973 - 1974 (Naval Biomedical Uncla
Research Lab., Oakland) 9 p HC .SCL 06M G30 731
NAVAL BIOMEDICAL RESEARCH LABORATORY
School of Public Health
University of California, Berkeley
Naval Supply Center
Oakland, California 94625
https://ntrs.nasa.gov/search.jsp?R=19740023439 2020-03-23T03:53:06+00:00Z
2nd Quarterly Report, 1973-74
NASA Contract W13450
Task No. 193-58-62-13-10
EVIDENCE FOR METABOLIC ACTIVITY OF AIRBORNE 
BACTERIA
R. L. Dimmick, H. Wolochow, M. A. Chatigny, P. A. Straat,
J. R. Schrot and G. V. Levin
Aerosols of the bacterium Serratia marcescens, I
and of uniformly labelled 14C glucose, were cre-
ated simultaneously and mixed in tubing leading
to an aerosol chamber. During a subsequent period
of about 5 hrs, 1 4 C002 was produced unequivocally
within the chamber, and "insoluble", labelled
material within the suspended particles first in-
creased, then decreased.
Current NASA planetary exploration projections include near fly-
by and probes of Jupiter, Saturn and possbily 
one or more of their
satellites. Planetary quarantine (PQ) constraints developed 
in in-
ternational agreements are applicable to such probes. Jupiter, 
in
particular, has a deep gaseous atmosphere which 
includes methane,
ammonia, and water, all in a dynamic convective 
mass in which there
is a zone about 80 Km thick where temperature 
and moisture conditions
do not preclude the possibility of microbial growth 
(1). Examination
of the probability of metabolism and growth (Pg) of spacecraft-borne
terrestrial microorganisms seeded into such 
clouds, and possibly main-
tained as aerosols, is an important factor in determining the 
degree
of control required to meet our international commitments.
There are numerous experimental aerobiology publications citing
presumptive evidence that airborne microorganisms 
support metabolic
functions (2), but direct evidence has been lacking. 
The question is
of importance not only in the approach to estimation of Pg in the
Jovian atmosphere, but also is implicated in mechanisms of microbial
survival in air (3), and is of fundamental, practical 
importance with
respect to the airborne biota of our own planet 
(4).
Groups in our laboratory are studying the 
problems in three
phases, listed here generally in order of ease of 
proof. They are:
(a) demonstration of the presence of some metabolic 
functions; (b)
demonstration of transcription or of gene replication 
and (c) demon-
stration of growth (Propagation) of selected microorganisms 
in atmos-
pheres providing organic matter potentially present 
or found in the
Jovian atmosphere (5). The work described is the demonstration 
of
the uptake of glucose and subsequent C02 production 
by airborne
Serratia marcescens (SM).
The strategy of the experiment was to mix two 
aerosols in a
closed chamber--one aerosol of semi-starved bacteria 
and the other
of 14C labeled glucose. If a "living" microbe collided with a glucose
2nd Quarterly Report, 1973-74
NASA Contract W13450
Task No. 193-58-62-13-10
particle, the glucose could be transported into the cell and incor-
porated into metabolic pathways for production of labeled C02.
Labeled materials within microbes would increase by incorporation,
then decrease through metabolism, and CO2 within the chamber would
increase to some maximal level; this sequence is essentially what was
observed.
Cultures of SM were grown in 0.01 M phosphate buffer with added
trace elements and ammonium ions, and with 0.5% glucose (as sole
carbon source) which was just sufficient to support growth of a popu-
lation of about 5 x 109 cells/ml.. Cultures were inoculated 16 hrs
prior to a given experiment. Cells were harvested by centrifugation1 0
within 10 minutes of the expected start time, and resuspended (ca 10
cells/ml) in growth medium without glucose.
Uniformly-labeled 14C-glucose (Schwarz/Mann) solutions were ad-
justed with unlabeled glucose to concentrations of 0.5% and a specific.
activities of 100 pCi/ml. Purity was established by thin-layer chroma-
tography on silica gel using n-butanol: acetone: water (30:50:20) as
the solvent system. Acidification of the glucose solutions released
less than 0.1% of the radioactivity as 14C02. Independent tests using
the labeled release technique (6) established that in vitro mixing of
these glucose solutions with SM resulted in the rapid evolution of
1 4C02
Measurements of radioactivity were performed on a Beckman liquid
scintillation counter equipped with a cesium external standard. The
counting "cocktail" consisted of 0.25 g of 1,4-bis(2-(5-phenyloxazoly))
benzene (POPOP) and 2 g of 2,5-diphenyloxazole (PPO) added to 500 ml
of tol ene and 500 ml of methanol. Counting efficiency was established
with 14C benzoic acid (New England Nuclear) as prime standard.
A cell suspension and a glucose solution were atomized separately
and simultaneously from two refluxing Wells atomizers having outputs
of 0.2 ml/min; efficiency of particulate emission was about 20% of
total mass loss from the atomizer (7). The two atomizers were posi-
tioned in a tee-joint, opposed, and separated by about 4 cm. The
side outlet of the tee led to a copper tube 0.9 cm in diameter and
33 cm long which was connected to the input tubing (0.3 cm in diameter,
44 cm long) of one or the other of a pair of rotating drums (1500
liters) (8). In independent experiments the coagulation efficiency
during the 5-second sojourn of particles in the tubing was found to
be about 15 percent; interaction of the two particulate species was
not dependent upon coagulation within the drum.
Drums were located in a temperature-controlled chamber (21 C)
and, prior to each run, were washed with clean air at 90-95% 
relative
humidity until the humidity of the exit air was identical to the in-
put air. Atomization then proceeded over a 5-minute spray time, after
2nd Quarterly Report, 1973-74
NASA Contract W13450
Tas!- No. 193-58-62-13-10
which each drum contained about 22 [Ci (30,000 dpm/liter) glucose with
(or without as appropriate) approximately 1010 (5 x 106 cells/liter)
viable bacterial cells suspended in the aerosol state. At various
times after aerosolization, AGI-30 (9) impinger samples of 12.5 liters
were collected into 20 ml of phosphate buffer during one minute. These
samples were analyzed for radioactive content and for viable bacterial
count by serial, ten-fold dilution in gelatin-phosphate medibm and
plating on trypticase soy agar.
Gas samples were also collected for 30-minute periods at various
times throughout a run. The gas sampler system consisted of a 5 ml
immunological pipette with the tip immersed into 2 ml of Hyamine hy-
droxide. The two were incorporated in a closed container such that
air entering the container was removed via the pipette at a rate of
about 60 ml/min. In independent experiments, we found essentially
100% collection of CO2 at this flow rate. During collection, samples
were first passed through a 0.1 pm membrane filter to remove radio-
active particles, and the volume of gas collected during the sampling
period was measured by water displacement. Samples were then assayed
for total radioactivity. Acidification of a Hyamine hydroxide aliquot
with entrapment of subsequently evolved volatiles, e.g. CO2 , on a
filter pad moistened with barium hydroxide, established that the radio-
activity present in the Hyamine hydroxide samples from the drums was
CO2 (or possibly a weak volatile acid) and was devoid of glucose.
Results of a typical experiment in which separate preparations
of 14C-glucose and SM were mixed in the aerosol state are shown in
Table 1. These results are compared to a parallel experiment in which
glucose alone was aerosolized into a second drum in the absence of SM.
As shown, both glucose and the bacterial particles fell onto the walls
of the drum with time, decreasing the concentration in the aerosol
state. This fallout rate is a direct function of particle size and,
for glucose, the disappearance of radioactive-labeled particles from
the drum represents the actual fallout rate. For viable bacterial
cells, however, the decrease in concentration within the drum repre-
sents both fallout and the loss of cell viability. It should be
.noticed that the fallout rate of radioactive glucose is considerably
larger in the presence of SM than in the absence of bacterial cells
and that the humidity was the same in both drums. This indicates
that coagulation had occurred between glucose and SM forming larger
particles than in the absence of SM. As shown, in the drum containing
both glucose and SM, the amount of 14CO2 present gradually increased
until a plateau was reached after about hours. At the plateau, the
total amount of 14C02 represents approximately 7% of the original
amount of radioactivity added to the drum. We do not understand the
unusual, delayed disappearance of 1 4CO2 in this one test (compare
Table 3). Omission of SM from the drum resulted in production of less
than 15% of the carbon dioxide accumulated in the presence of SM during
the entire run. This indicates that most of the CO2 observed in the
presence of both SM and glucose is metabolic in origin and not a result
2nd Quarterly Report, 1973-74
NASA Contract W13450
Task No. 193-58-62-13-10
of some nonbiological interaction of glucose with residual material
possibly present on the walls of the drums. As for 
1 4C02 released
in the absence of SM, separate experiments have shown that, despite
scrupulous cleaning procedures between sequential experiments, some
residual radioactivity remained on the walls of the drum at the start
of the next experiment. Trace amounts were observed to be released
very slowly as CO2 under the experimental conditions. Sbmetimes,
after extended intervals, CO2 was comparable in magnitude to that
seen in Table 1 in the absence of SM.
The results shown in Table 1 do not allow a clear distinction to
be made between whether the source of CO2 production is from the
aerosol state or is a result of fallout and subsequent metabolism on
the wall. Indeed, the possibility of metabolism on the walls is
suggested in Table 1 because CO2 increased as the concentration of
airborne bacterial cells and glucose decreased by fallout; although
99% of the bacterial cells fell to the walls within 24 hours, the
amount of C02 in the drum was still increasing. The pair of experi-
ments in Table 2 show that, although cellular metabolism on the wall
did, indeed, occur, it did not account for the metabolic 
14C02 evolved
when SM and glucose were simultaneously aerosolized. In this experi-
ment, one drum was first preconditioned by aerosolizing with labeled
glucose alone and allowing all the glucose to fall out to the walls
of the drum during a 72-hr period. The drum was subsequently air-
washed and then filled with airborne SM. Any resulting 
1 4C0 in the
drum would then represent metabolism on the walls (plus any bleeding
effects). These results are compared to a parallel drum experiment
in which both SM and glucose were aerosolized simultaneously. The
results of this experiment (Table 2) show that metabolism on the walls
is not a significant contributor of CO2 until late in the time-course
of the reaction. The CO2 formed soon after aerosolization of both SM
and glucose must, then, be attributable to metabolism in the aerosol
state.
An experiment (Table 3) was conducted in an effort to eliminate
the effects of metabolism on the walls. SM and glucose were simul-
taneously aerosolized into each of two drums, one of which was coated
with a 0.2N Na CO solution to trap any 14C02 formed by metabolism on
the walls. Initial values of CO2 were similar in both drums. How-
ever, as shown, CO2 gradually increased in the absence of carbonate,
whereas, in the presence of carbonate, CO2 remained constant or in-
creased only slightly after the initial evolution. These results
suggest that metabolism in air occurs soon after contact of the cell
with the glucose particle and that further increases in CO2 contain
a partial contribution of metabolism from cells on the walls. The
magnitude of the wall-effect seen in Table 3 is in general agreement
with the results shown in Table 2. The apparent magnitude of the CO2
increase (Table 3) is undoubtedly lower than the actual increase
because of the known "gettering" effect of Na2CO3.
2nd Quarterly Report, 1973-74
NASA Contract W13450
Task No. 193-58-62-13-10
As an additional indication of microbial metabolic activity in
the air, we collected samples for 1 minute on 0.45 pm membrane fil-
ters at flow rates of 5 liters of air/min. Filters were then rinsed
5 times with 2.0 ml quantities of unlabeled 0.5% glucose and the fil-
ters counted for 14C retention. Corrections were made for small
amounts of glucose which tends to be retained on the filter even with-
out cells. The remaining counts are referred to as insoluble, par-
ticulate label (IPL), and presumably represent an estimate of the
amount of incorporated glucose not yet metabolized to CO2 . Although
the dynamics of the IPL are complex, as is CO2 evolution, in all
tests the IPL first increased, then decreased to background levels
during the first 2 to 5 hr period of aerosol life. In one experiment
(Table 2), the IPL obtained with glucose plus SM appears to be the
same as glucose alone (Table 1). However, there was a gross difference
in physical fallout between the two experiments. When corrections are
made accordingly, the IPL with glucose plus SM is significantly greater
than that with glucose alone.
Together, these experiments indicate that metabolism can and does
occur in the aerosol state. Under experimental conditions, metabo-
lism starts soon after initial interaction between glucose and bac-
terial cells. The fact that CO2 evolution from the airborne cells
did not continue for an extended time after interaction with sub-
strate may reflect total utilization of all glucose available to the
bacterial cells, as the decline of IPL indicates. After the initial
aerosolization,.which optimizes coagulation, little additional coagu-
lation occurs between SM and glucose in the airborne state within the
drum. Alternatively, the outcome is similar to results of experiments
conducted at Biospherics (10) in which 1 4C-labeled substrates were
added to coliform organisms. An initial burst of 1 4CO2 evolution was
followed by a slower but continual rate of 14C02 release. Since coli-
form organisms were in continual contact with 1 4C-labeled sub-
strates, this kinetic phenomenon could not reflect lack of substrate
availability. The rate of 14C0 2 evolution in the "Biospherics" studies
increased again as growth and reproduction of the microorganisms began
to occur.
At present, we have no evidence that growth and/or reproduction
are occurring within the drum. It is possible that the kinetics ob-
served for airborne metabolism may, therefore, reflect the "early
burst" phenomenon and that, if the aerosol were maintained over
longer periods, until growth and reproduction could occur, the metab-
olic rate would be resumed. Thus, whether or not the kinetics of air-
borne metabolism in these experiments represent a substrate-limited
situation or reflect lack of growth is an issue to be resolved by
further experimentation.
2nd Quarterly Report, 1973-74
NASA Contract W13450
Task No. 193-58-62-13-10
ACKNOWLEDGEMENTS
Work supported in part by NASA, (Office of Planetary Quarantine)
Contract W-13,450 and in part by the Office of Naval Research. We
acknowledge the excellent assistance of Bruce Conner, Al Boyd and
Steve Dunn.
REFERENCES
1. D. Neil. The Planet Jupiter, NASA SP-8069 (1971).
2. J. E. Benbough and P. Hambleton in Airborne Transmission and 
Air
Infection, J. F. Hers and K. C. Winkler, Eds. (Oosthoek Publish-
ing Co., Utrecht, The Netherlands, (1973) p. 135; R. L. Dimmick,
ibid. p. 100; P. Hambleton, J. Gen. Microbiol. 69:81 (1971);
C. S. Cox and F. Baldwin, ibid, 44:15 (1966).
3. M. T. Hatch and H. Wolochow in Intro. to Exptl. Aerobiol., R. L.
Dimmick and A. Akers, Eds. (J. Wiley, N. Y. 1969) p. 267.
4. P. C. Parker, Nat. History, Oct. 55 (1970).
5. F. Woeller and C. Ponnamperuma., ICARUS, 10:386 (1969).
6. G. V. Levin, A. H. Heim, J. R. Clendenning and M. F. Thompson,
Science, 138:114 (1962); G. V. Levin, ICARUS, 16:153 (1972).
7. K. B. DeOme et al., Am. J. Hyg. 40:239 (1944).
8. L. J. Goldberg et al., ibid, 68:85 (1958).
9. P. S. Brachman et al., Science, 144:1295 (1964).
10. G. V. Levin, V. R. Harrison and W. C. Hess, J. Am. Water Works
Assoc., 49:1069 (1957); G. V. Levin, Adv. Appl. Microbiol. 5:
95 (1963).
Table 1. Test of metabolism; glucose against glucose plus bacteria. E C
Glucose Glucose & SM
rtrt
dpm/l cells/1 dpm/1 dpm/1 cells/1 dpm/1 H
14 F W nAerosol time (Impinger) (Impinger) 1 4 0 IPL F  Aerosol time (Impinger) (Impinger) CO2  IP r LF2 __ U
BA 0 BA - 130 - 0
4 4I U6rt
1 min. 2.7 x 10 - 70 1 min. 3.8 x 10 3.7 x 106 - 560
32 min. - - 91 - 10 min. - - 92 - c
3.3 hrs 2.6 x 10 - - 250 3.5 hrs 3.0 x 10 3.0 x 106 - 1400
4.0 hrs - 34 - 3.6 hrs - 555 -
5.3 hra 2.3 x 10 - - 250 5.5 hrs 2.8 x 10 2.4 x 106 - 1700
5.6 hrs - - 137 - 5.6 hrs - - 798 -
21.5 hrs - - 0 - 22.5 hrs - - 2,630 -
23.0 hrs 2.1 x 104 - - 60 23.0 hrs 1.6 x 10 2.1 x 105 - 280
27.5 hrs 1.8 x 104 - - - 27.5 hrs 6.8 x 103 4.2 x 104 -
28.0 hrs 
- - 307 100 27.6 hrs 
- - 2,270 150
47.0 hrs - 0 - 46.5 hrs - - 566 -
14C-glucose was aerosolized into each of two drums with and without simultaneous aerosolization of Serratia
Marcescens, as indicated. At various times after aerosolization, impinger samples were withdrawn to determine
14the remaining suspended C-glucose and bacterial cell count. Gas samples were also withdrawn to determine
14the amount of CO2 in the drums.
*BA = Before Aerosolization
F = Uncorrected for fallout or background; see text.
Page intentionally left blank 
Table 3. Test of metabolism; with and without CO adsorbant. N)
oi
Glucose & SM (- Na2CO3) Glucose & SM (+ Na2CO3)
Aerosol time dpm/l cells/i d/1 F dpm/l cells/i d0/l H
(Impinger) (Impinger) 14CO2  IPLF  Aerosol time (Impinger) (Impinger) 14CO 2  IPLF
BA* - - 0 - BA*- 0 - 0
4 6 4 6 kn"1 min 3.6 x 10 4.5 x 10 - - 1 min 3.6 x 10 5.9 x 10 - - o r
4min - - - 742 3 min - - 354 H
o
20 min - - 349 - 8 min - 289 -
3 hrs 2.2 x 10 2.2 x 106  - 578 3.2 hrs 2.1 x 10 1.9 x 106 - 402
3.5 hrs 729 3.3 hrs - - 462 -
4.7 hrs 1.5 x 10 1.4 x 106 - 119 5.2 hrs 1.6 x 104 1.2 x 106 - 362
5.6 hrs - - 511 - 5.5 hrs - - 332 -
23.5 hrs - - 1,448 - 23.4 hrs - - 318 -
24.6 hrs 5.4 x 103 2.2 x 105  - 78 24.7 hrs 5.0 x 103  2.4 x 105 - 82
28.0 hrs 4 x 103 1.4 x 105 - 44 28.5 hrs 3.7 x 103  1.2 x 105 - -
28.6 hrs - - 1,218 - 28.6 hrs - - 374 28
48.7 hrs 1.8 x 103  3 x 10 - - 49.2 hrs 2.0 x 102 - -
49.5 hrs - - 1,348 - 51.5 hrs - 419 -
14
C-glucose.and Serratia marcescens (SM) were simultaneously aerosolized into each of two drums, one of which had
previously been lined with Na2C03, as indicated. At various times after aerosolization, impinger samples were
14
withdrawn to determine the remaining suspended C-glucose and bacterial cell count. Gas samples were also with-
drawn to determine the amount of 14CO 2 in the drums.
*BA = Before Aerosolization
F = uncorrected for fallout and background, see text.


Evidence for metabolic activity of airborne bacteria

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