Abstract

An increase in the number of culturable organisms and a decrease in the diversity of recoverable microbiota have been reported in deep subsurface materials after storage perturbation. The magnitude of the microbial community shift in stored samples was more pronounced at 4°C compared to −20°C. Phospholipid fatty acid analyses and acridine orange direct counts indicated that biomass did not increase significantly throughout storage. Changes in the types of fatty acid methyl esters determined over the time course indicated that some of the microbial community shift was due to bacterial proliferation. However, the recovery of new bacterial types only after the storage process suggested that some of the increase in culturable cell count was due to the resuscitation of dormant microorganisms, possibly activated by some aspect of sampling, sample handling, and/or storage. Comparison of acridine orange direct counts with phospholipid and diglyceride fatty acid content suggested that much of the biomass may have been non-living at early time points; however, after 30 days of storage most of the bacterial biomass was viable.

References

[1]
Hirsch
P.
Rades-Rohkohl
E.
(
1988
)
Some special problems in the determination of viable counts of groundwater microorganisms
Microb. Ecol.
 ,
16
,
99
113
.
[2]
Brockman
F.J.
Kieft
T.L.
Frederickson
J.K.
Bjornstad
B.N.
Li
S.W.
Spangenburg
W.
Long
P.E.
(
1992
)
Microbiology of vadose zone paleosols in south-central Washington state
Microb. Ecol.
 ,
23
,
279
301
.
[3]
Haldeman
D.L.
Amy
P.S.
White
D.C.
Ringelberg
D.B.
(
1994
)
Changes in bacteria recoverable from subsurface volcanic rock samples during storage at 4° C
Appl. Environ. Microbiol.
 ,
60
,
2697
2703
.
[4]
Fredrickson
J.K.
Li
S.W.
Brockman
F.J.
Haldeman
D.L.
Amy
P.S.
Balkwill
D.L.
(
1995
)
Time-dependent changes in viable count and activities of heterotrophic bacteria in subsurface samples
J. Microbiol. Meth.
 ,
21
,
253
265
.
[5]
Haldeman
D.L.
Amy
P.S.
Russell
C.E.
Jacobson
R.
(
1995
)
Comparison of drilling and mining as methods for obtaining microbiological samples from the deep subsurface
J. Microbiol. Meth.
 ,
21
,
305
316
.
[6]
Zobell
C.E.
(
1943
)
The effects of solid surfaces upon bacterial activity
J. Bacteriol.
 ,
46
,
39
56
.
[7]
Atlas
R.M.
Bartha
R.
(
1987
)
Microbial Ecology: Fundamentals and Applications
 
2nd Edn.
Benjamin/Cummings Publishing Co., Inc
,
Menlo Park, CA
.
[8]
Wollum
A.G.
(
1982
)
Cultural methods for soil microorganisms
In:
Methods of Soil Analysis, Part 2: Chemical and Microbiological Properties
 
Page
A.L.
, Ed) pp
781
802
American Society of Agronomy
,
Madison, WI
.
[9]
Ghiorse
W.C.
Balkwill
D.L.
(
1985
)
Microbiological characterization of subsurface environments
In:
Ground Water Quality
 
Ward
C.H.
Giger
W.
McCarty
P.L.
, Eds) pp
387
401
Wiley
,
New York
.
[10]
Rozak
D.B.
Colwell
R.R.
(
1987
)
Survival strategies of bacteria in the natural environment
Microbiol. Rev.
 ,
51
,
365
379
.
[11]
Kapreylants
A.S.
Kell
D.B.
(
1993
)
Dormancy in stationary-phase cultures of Micrococcus luteus: Flow cytometric analysis of starvation and resuscitation
Appl. Environ. Microbiol.
 ,
59
,
3187
3196
.
[12]
Bissonette
J.K.
Jezeski
J.H.
McFeters
G.A.
Stuart
D.G.
(
1975
)
Influence of environmental stress on enumeration of indicator bacteria from natural water
Appl. Environ. Microbiol.
 ,
29
,
186
194
.
[13]
Nilsson
L.L.
Oliver
J.D.
Kjelleberg
S.
(
1991
)
Resuscitation of Vibrio vulnificus from the viable but non culturable state
J. Bacteriol.
 ,
173
,
5054
5059
.
[14]
Caldwell
B.A.
Ye
D.
Griffiths
R.P.
Moyer
C.L.
Morita
R.Y.
(
1989
)
Plasmid expression and maintenance during long-term starvation-survival of bacteria in well water
Appl. Environ. Microbiol.
 ,
55
,
1860
1864
.
[15]
Henis
Y.
(
1987
)
Survival and dormancy of bacteria
In:
Survival and Dormancy of Microorganisms
 
Henis
Y.
, Ed) pp
1
108
Wiley
,
New York
.
[16]
Murno
P.M.
Gauthier
M.J.
Greittmayer
V.A.
Bongiovanni
J.
(
1989
)
Influence of osmoregulation processes on starvation survival of Esherichia coli in seawater
Appl. Environ. Microbiol.
 ,
55
,
2017
2024
.
[17]
Haldeman
D.L.
Amy
P.S.
(
1993
)
Bacterial heterogeneity in deep subsurface tunnels at Rainier Mesa, Nevada Test Site.
Microb. Ecol.
 ,
25
,
183
194
.
[18]
Haldeman
D.L.
Amy
P.S.
Ringelberg
D.
White
D.C.
(
1993
)
Characterization of the microbiology within a 21 m3 section of rock from the deep subsurface
Microb. Ecol.
 ,
26
,
145
159
.
[19]
Kaplan
L.A.
Bott
T.L.
Bielicki
J.K.
(
1992
)
Assessment of [3H]thymidine incorporation into DNA as a method to determine bacterial productivity in stream bed sediments
Appl. Environ. Microbiol.
 ,
58
,
3614
3621
.
[20]
Thorn
P.M.
Ventullo
R.M.
(
1988
)
Measurement of bacterial growth rates in subsurface sediments using the incorporation of tritiated thymidine into DNA
Microb. Ecol.
 ,
16
,
3
16
.
[21]
Amy
P.S.
Haldeman
D.L.
Ringelberg
D.
Hall
D.H.
Russell
C.
(
1992
)
Comparison of identification systems for classification of bacteria isolated from water and endolithic habitats within the deep subsurface
Appl. Environ. Microbiol.
 ,
58
,
3367
3373
.
[22]
Beloin
R.M.
(
1987
)
2nd edition
, In:
Master's Thesis
 , pp
7
Cornell University
,
Ithaca, NY
Thesis.
[23]
Ghiorse
W.C.
Miller
D.N.
Sandoli
R.L.
Siering
P.L.
(
1995
)
Application of laser scanning microscopy for analysis of aquatic microhabitats
Dobbs
Fred
, Ed)
2nd edition
, In:
Special Issue on Microscopic Studies of Microorganisms
 (manuscript submitted).
[24]
Reference omitted.
[25]
Kieft
T.L.
Ringelberg
D.B.
White
D.C.
(
1994
)
Changes in ester-linked phospholipid fatty acid profiles of subsurface bacteria during starvation and dessication in a porous medium
Appl. Environ. Microbiol.
 , in press.
[26]
Bligh
E.G.
Dyer
W.M.
(
1959
)
A rapid method of lipid extraction and purification
Can. J. Biochem. Physiol.
 ,
35
,
911
917
.
[27]
White
D.C.
Davis
W.M.
Nickels
J.S.
King
J.D.
Bobbie
R.J.
(
1979
)
Determination of sedimentary microbial biomass by extractable lipid phosphate
Oecologia
 ,
40
,
51
62
.
[28]
O'Leary
W.M.
Wilkinson
S.G.
(
1988
) In:
Microbial Lipids
 
Ratledge
E.
Wilkinson
S.G.
, Eds)
2nd edition
, Vol.
1
, pp
122
Academic Press
,
London
.
[29]
Balkwill
D.L.
Leach
F.R.
Wilson
J.T.
McNabb
J.F.
White
D.C.
(
1988
)
Equivalence of microbial biomass measures based on membrane lipid and cell wall components, adenosine triphosphate, and direct counts in subsurface aquifer sediments
Microb. Ecol.
 ,
16
,
73
84
.
[30]
Amy
P.S.
Durham
C.D.
Hall
D.
Haldeman
D.L.
(
1993
)
Starvation survival of deep subsurface isolates
Curr. Microbiol.
 ,
26
,
345
352
.
[31]
Shivaji
S.
Shymala Rao
N.
Saisree
L.
Reddy
G.S.N.
Seshu Kumar
G.
Bhargava
P.M.
(
1989
)
Isolates of Arthrobacter from soils of Schirmacher Oasis, Antarctica
Polar Biol.
 ,
10
,
225
229
.
[32]
Nelson
L.M.
Parkinson
D.
(
1978
)
Effect of freezing and thawing on survival of three bacterial isolates from an antarctic soil
Can. J. Microbiol.
 ,
24
,
1468
1474
.