345
FEMS Microbiology Ecology 74 (1990) 345-356
Published by Elsevier
FEMSEC 00305
Effects of substrate concentration, growth state,
and oxygen availability on relationships among bacterial carbon,
nitrogen and phospholipid phosphorus content *
J. Brinch-Iversen and G.M. King
Institute of Ecologv and Genetics, University of irhus, Arhus, Denmark
Received 12 June 1990
Revision received and accepted 13 September 1990
Key words: Phospholipid; Biomass; Bacteria, anaerobic; Growth state
1. SUMMARY
The utility of phospholipid phosphorus (Plip-P)
concentrations as a measure of microbial biomass
is dependent upon the accuracy of the conversion
factors used for relating Plip-P to cell carbon,
nitrogen or weight. Mixed cultures enriched from
marine sediments have been used to evaluate the
reliablity of these relationships as a function of
substrate concentration, growth state and the presence of oxygen. Ratios of Plip-P to carbon and
nitrogen were independent of growth state and
substrate level but were sensitive to oxygen status.
For aerobic incubations, average ratios of 190 and
730 pmol Plip-P/g carbon and nitrogen were obtained, respectively. Average ratios of 100 and 350
pmol Plip-P/g carbon and nitrogen were obtained
from anaerobic incubations. As a result, it is not
likely that a single conversion factor can be applied to samples from both aerobic and anaerobic
systems. In addition, a limited survey of algal
Correspondence lo (Present address): G.M. King, Darling
Marine Center, University of Maine, Walpole, ME 04573,
U.S.A.
Contribution 220 from the Darling Marine Center.
0168-6496/90/$03.50
Q
Plip-P, carbon and nitrogen concentrations indicates that the presence of a significant algal biomass can result in erroneous estimates of bacterial
abundance.
2. INTRODUCTION
Phospholipids are components of all cellular
membranes, accounting for up to 90% of the total
extractable lipids in bacteria [ 1,2]. Phospholipids
have a relatively fast turnover and are not thought
to accumulate outside living cells [3,4]. As a result,
phospholipid phosphorus has been proposed as a
convenient tool for estimating microbial biomass.
The use of phospholipid phosphorus (Plip-P)
concentrations as an estimate of total microbial
biomass in sediments was first described by White
et al. [3], who also introduced a conversion factor
for Plip-P to bacterial dry weight [ 5 ] . They based
their conversion factor on monocultures of common laboratory and clinical isolates which had
been grown at high substrate concentrations or
sampled during logarithmic growth. A general
conversion factor of 50 pmol Plip-P/g dry weight
for natural populations of bacteria was suggested.
1990 Federation of European Microbiological Societies
346
Since bacteria typically exist in complex communities in situ with varying growth states, a
conversion factor derived from pure cultures may
be inappropriate. In order to further evaluate the
relationship between Plip-P concentrations and
cellular carbon and nitrogen, we have used mixed
enrichment cultures from marine sediments. These
cultures, which contained a diversity of organisms,
were grown at different substrate concentrations
and sampled at different growth states. Since most
marine and freshwater sediments become anoxic
within the first centimeter, we also determined the
sensitivity of the Plip-P conversion factors to
anoxia.
3. MATERIALS AND METHODS
3.I . Enrichment cultures
Enrichment cultures were obtained from sediments collected at two different sites: an intertidal
estuarine sediment (Norsminde Fjord) and a subtidal coastal sediment (Aarhus Bay). These sites
have been previously described [6,7]. Sediment
was sampled from the estuarine site by hand using
acrylic cores (diameter 5 cm) and from the subtidal sediment using a Haps grab. Approximately
1 cm3 of sediment was inoculated into 100 ml of a
modified Widdel & Pfennig medium [8] containing the following salts (g I-’ distilled water): NaCI,
20.0; anhydrous Na,SO,, 2.84; MgCl, . 6H,O,
1.14; CaC1, . 2H,O. 0.14; KH,PO,, 0.068;
Na,EDTA, 0.01 and 10 ml of a trace element
solution containing (mg I - ’ distilled water):
FeSO,, 100.0; ZnSO, 7H,O. 10.0; MnCl .4H,O,
3.0; CoCl, .6H,O, 20.0; CuCl, 2H,O, 1.0; NiCI,
6H,O, 2.0; Na,MoO, * 2H,O, 3.0. The medium
was adjusted to pH 7.0 using either dilute HC1 or
NaOH as necessary, sterilized by autoclaving and
amended with 0.1% peptone and 0.005% yeast
extract. Both the peptone and the yeast extract
were made up as concentrated stock solutions
adjusted to p H 7 and sterilized by autoclaving.
For the aerobic experiments the cultures were
incubated on a shaker table at 25°C. For the
anaerobic experiments the shaker was placed in a
glovebag (Coy, Inc.) with an atmosphere of 90%
nitrogen and 10%hydrogen. After 24 h, both the
-
aerobic and the anaerobic cultures were diluted
1 : 100 and 1 : 10 respectively in fresh media, They
were incubated for 24 h and transferred similarly
to the final set of culture vessels. The medium
used for the anaerobic cultures was equilibrated
overnight in the glove bag. For the aerobic experiments, growth was followed for up to seven cultures amended with different amounts of peptone
and yeast extract respectively (% w/v): 0.01,
0.00125; 0.05, 0.0025; 0.1, 0.005; 0.2, 0.01; 0.4,
0.02; 0.8, 0.04; 1.6, 0.08. For the anaerobic experiments growth was followed for up to five cultures
with the following media composition (5% w/v):
0.1, 0.005; 0.2, 0.01; 0.4, 0.02; 0.8, 0.04; 1.6, 0.08.
Subsequently, these enrichment cultures will be
referred to by their peptone content only. Growth
was measured at intervals as optical density at 500
nm. At appropriate intervals, 1 to 20 ml of culture
were filtered by vacuum (15 kPa) through Whatman GF/F filters. The filters were washed with
one ml deionized water. Filters for Plip-P analysis
were immediately placed in an extraction mixture
(see below). Filters for C : N determinations were
rapidly heat fixed and stored at - 20°C until later
analysis. Filters for total cellular phosphate were
immediately placed in tubes containing 5 ml
saturated potassium persulfate (see below). All
filters were ‘fired’ at 450°C for 3 h prior to use.
For cell counts, one ml of the medium was fixed
in a 4%formalin solution and stored at 5°C.
Subsurface sediments from a location in
Norsminde Fjord reported to contain high numbers of methane-producing bacteria (K. Finster,
personal communication) were sampled with
acrylic cores (length 1 m, diameter 5 cm). A subsample from 50 cm depth was inoculated into a
reducing medium at 30°C (see below) with
methanol (20 mM) as the sole carbon source to
enrich for methanogenic bacteria. After several
transfers, the culture was grown to maximum optical density, harvested and sampled for Plip-P and
C :N analysis. The mixed culture produced copious amounts of methane.
3.2. Pure cultures
Sulfate reducers were obtained as highly enriched cultures that were grown and maintained
on a marine medium (91. The medium was mod-
341
ified by adding vitamins and trace elements [lo].
The purity of the cultures was monitored by microscopic inspection. The following three strains
were used: Desulfobacter sp., grown on 20 mM
acetate; Desulfouibrio sp., grown on 20 mM
lactate; Desulfobulbus sp., grown on 20 mM propionate. The cultures were incubated at 30°C and
sampled for Plip-P and C : N analysis after maximum optical density was achieved. Escherichia
coli (strain AS 19) was grown on a medium
amended with 2% glucose [ll] and incubated at
25°C. Growth was measured as before with Samples taken in mid-log and early stationary phase
for Plip-P, total carbon, total nitrogen and cell
numbers. Paracoccus denitrificans (strain DSM
413) was grown on a medium amended with 0.2%
peptone [12]. The culture was incubated aerobically 25°C and sampled as described above for E.
coli.
Two axenic algal monocultures, Synechococcus
sp. and Skeletonema sp. were obtained from the
Provasoli-Guillard Culture Collection for Marine
Phytoplankton (Bigelow Laboratory for Ocean
Science) and were grown and maintained on f/2
medium as described by Guillard [13]. The cultures were incubated in a shaking waterbath at
23°C with illumination by a mercury vapor lamp;
light intensities were not measured during growth.
The cultures were harvested for Plip-P, C :N and
total phosphate analyses at maximum density. An
additional axenic algal culture, Ditylum brightwelli
was grown on IMR/2 medium [14] and analyzed
as described above.
3.3. Plip-P analysis
Lipids were extracted using a modified BlighDyer procedure [15]. Filters from the growth experiments above were placed in 30 ml glass tubes
with teflon-lined screwcaps. A total of 2.5 ml
dichloromethane (DCM) and 5 ml methanol were
added from a pre-mixed solution. The tubes were
closed, mixed vigorously and then allowed to stand
for 2 to 24 h. Subsequently 2.5 ml of DCM and 10
ml of saturated sodium bromide solution (0.8 g
ml- I , heated to aid dissolution, then cooled) were
added to separate the lipid-containing DCM phase
from the aqueous methanolic phase. The optimal
final ratio of DCM : methanol : sodium bromide
was 1: 1 : 2. The tubes were shaken and the organic
and aqueous phases allowed to separate for 2 to
24 h. The tubes were then centrifuged for 10 min
at 300 X g before subsamples of DCM (0.5-2 ml)
were pipetted into 2 ml glass ampoules. An additional 250 pl of DCM were pipetted to wash the
samples completely into the ampoules. Subsequently, DCM was removed under a stream of
nitrogen and with gentle heating. One ml of
saturated potassium persulfate (5 g K2S,0, dissolved in 100 ml deionized water) was added to
the ampoules which were sealed and incubated a t
100°C overnight.
The inorganic phosphate formed by the persulfate digestion was assayed by the method of
Van Veldhoven and Mannaerts [16]. Briefly, a
1.75% (w/v) solution of ammonium heptamolybdate was prepared in 2.61 N H,SO,. Malachite
green, 0.035% (w/v), was added to a solution of
0.35% (w/v) polyvinyl alcohol (PVA) prepared by
dissolution in deionized water at 80°C. The ammonium heptamolybdate (0.2 ml) was added to
the ampoules which were then mixed vigorously.
After standing at ambient temperature for 10 min,
0.2 ml of the malachite green solution was added.
Absorbance at 610 nm was measured after incubation at ambient temperature for 30 min on a
Hitachi U 2000 spectrophotometer.
Recovery of Plip-P from cells using an earlier
version of this technique has been previously reported to be quantitative [17]. The present method,
modified as described above, also gives quantitative recoveries of Plip-P from cells and environmental samples (King and Brinch-Iversen, unpublished data).
3.4. Total phosphorus analysis
The filters for total phosphate analysis were
placed in screw-cap test tubes (Pyrex) to which 5
ml saturated potassium persulfate solution (from
the stock described above) were added. The tubes
were closed with teflon-lined screw caps and incubated at 100°C overnight. The tubes were
centrifuged at 300 x g for 10 min. Subsamples (0.3
ml) were tranferred to disposable plastic test tubes
containing 0.7 ml 1.78 N €I,SO,. Phosphate was
measured colorimetrically as above with the ex-
348
c
ception that the ammonium heptamolybdate solution was prepared in deionized water.
1.4
1.2
3.5. Standardization
A standard curve was prepared by digesting a
varied amount of dioleoyl phosphatidylcholine
(from 0 to 20 nmol). The phosphate concentrations were analyzed as above. The relationship
between phosphate concentration and absorbance
was described approximately by y = 0.093 +
0 . 1 0 2 ~ ;r 2 = 0.99.
-
0.2
0.0
0 :
400
0
3.6. Other analyses
Filters for C :N determinations were packed in
tin capsules. Analyses were performed with a Carlo
Erba model 1400 elemental analyzer. The formalin-fixed samples for cell counts were stained
with crystal violet. Cell numbers were estimated
by direct counts using a Burker-Turk hemacytometer and an Axioskop (Zeiss Instruments, Inc.)
with phase contrast optics. A total of 20 fields
were counted for each determination.
+
3.8. Materials
All solvents and most salts were obtained as
analytical grade reagents from E. Merck, Darmstadt, Germany. Malachite green and dioleoyl
phosphatidylcholine were obtained from Sigma
Chem. Co. PVA ( M , = 14000; 100%hydrolyzed)
was obtained from Aldrich Chemical Co. Peptone
and yeast extract were obtained from Difco. Glass
ampoules were obtained from Wheaton Scientific.
Glass centrifuge tubes (Pyrex glass) for the lipid
extraction were fabricated locally. All glassware
used for lipid extraction was washed with a phos-
1200
1600
2000
Time (min)
20 0
1
E
4
=-a.
0
I
I
I
=
-37
d
1
16.0
120
0
c
a
g
8.0
c
a
0
E
4 0
0.0
3.7. Statistical analyses
A total of ten aerobic and six anaerobic enrichments were carried out. Triplicate samples were
taken for Plip-P and C : N analyses at each time
point. Triplicate samples for total phosphate
analyses and duplicate samples for cell counts
were taken for selected cultures. The pure cultures
were harvested once and triplicate samples were
taken for all analyses. Statistical analyses, one-way
ANOVA ( P= 0.05); two tail t-tests ( P= 0.05)
were performed using the StatView SE TM
program on an Apple MacIntosh.
800
I
I
I
I
I
I
0
20
40
60
80
100
pg Carbon rnl"
Fig. 1. (A) Optical density ( 0 ) .cell carbon (O), and Plip-P (m)
contents for an aerobic enrichment culture grown with 0.1%
peptone; each point represents the mean of triplicate determinations. (B) Plip-P vs. cellular carbon.
phate free detergent (Extran MA 3, from Merck),
rinsed at least 8 times in deionized water and
combusted at 450°C for 3 h. Ampoules were cornbusted at 450°C for 3 h, acid washed and rinsed in
deionized water. It should be noted that if appropriate precautionary steps are taken, it is possible to use disposable plastic pipet tips in handling
the organic solvents: pipet tips should be flushed
at least 3 times with DCM immediately prior to
use and tips should be calibrated with DCM.
4. RESULTS
4.I . Aerobic enrichments
During a typical time course experiment (Fig.
IA), growth in aerobic enrichment cultures ex-
349
hibited lag, logarithmic and stationary phases.
Cellular carbon and Plip-P concentrations paralleled optical density (Fig. 1A); total cellular
nitrogen and phosphorus also exhibited similar
trends (not shown). When enrichments were grown
with 0.1% peptone, values ranged from 6.87 to
21.39 pg ml-I (0.49-1.53 pmol m1-I) for nitrogen
and from 0.67 to 0.60 pg ml-I (21.03-18.67 nmol
ml-I) for total phosphorus during lag and stationary phases respectively. C : N ratios varied
from 4.2 to 4.9 over the growth cycle. Carbon,
nitrogen and total phosphorus were highly correlated with Plip-P. The relationships between these
parameters were described as follows for carbon,
y = - 2.187 + 0.200x, r 2 = 0.988 (Fig. 1B); for
nitrogen, y = - 3.966 + 0.921x, r 2 = 0.988; for
total phosphorus, y = -1.453
0.225x, r 2 =
+
1
0.25
-3
c
20:
0.2
0.15
'
200.0
E
*.--
1500
Q
2a
1000
0
f
0
50.0
E
0.0
ea
-.0
,-
I
,
100
150
I
I
250
300
200.0
E
n 1500
U
..-a
-
g
1000
ln
c
0
-
a
500
0
00
50
200
Fig. 3. (A) Regression analysis of Plip-P vs. cellular carbon
content for all time points sampled during the growth of the
aerobic enrichment cultures on the various peptone concentrations summarized in the text; each point represents the mean
of triplicate determinations for Plip-P and cellular carbon
concentrations. (B) As for A but Plip-P vs. cellular nitrogen.
0.1
0.05
0
60
40
20
80
100
120
Time (h)
2.4
2.0
$
16
'6
.=
2
12
2
a
08
B
A
pg Nitrogen ml.' culture
0"
-2
250.0
0
-a
s
.c
--8
E
(D
9
.$
aY
z
2
00
I B
I
5
2
Y
I
-
-
'
0
I
4
8
12
16
20
pg Carbon ml"
Fig. 2. (A) Optical density ( 0 ) .cell carbon ( 0 )and Plip-P (m)
contents for an anaerobic enrichment culture grown with 0.1%
peptone: each point represents the mean of triplicate determinations. (B) Plip-P vs. cellular carbon.
0.978. A conversion factor of 2.6 f 1.3 X 10' cells
per nmol Plip-P was derived from the enrichment
with 0.1% peptone. All slopes were significant at
P < 0.05.
4.2. Anaerobic enrichments
A typical growth cycle in anaerobic enrichment
cultures (Fig. 2A) exhibited the same growth
phases as seen in aerobic enrichment cultures.
Again cellular carbon and Plip-P concentrations
were congruent with optical density (Fig. 2A), and
nitrogen exhibited the same trend (not shown).
The concentrations of total phosphorus were not
measured for these particular cultures, but paralleled optical density in other anaerobic enrichments. Nitrogen ranged from 2.22 to 5.22 pg ml-'
(0.16-0.37 pmol ml-*) during the growth cycle for
350
an enrichment with 0.1% peptone. C : N ratios
decreased from 4.3 to 4.1 during the growth cycle.
Carbon and nitrogen were significantly correlated
with Plip-P. The relationships for these parameters
were described as follows for carbon, y = -0.080
+ 0.117x, r 2 = 0.988 (Fig. 2B); nitrogen, y =
0.021 0.399x, r 2 = 0.978. A conversion factor of
4.3 1.1 x lo8 cells per nmol Hip-P was calculated. The relationship for total phosphorus for an
enrichment grown with 0.2% peptone was: y =
- 1.649 0.150x, r 2 = 0.924. All slopes were significant at P < 0.05.
When all data from the various aerobic enrichments were pooled, Plip-P concentrations were
highly correlated with total carbon and nitrogen
(Fig. 3A,B): for carbon, y = 2.323 + 0.190x, r 2 =
0.982; for nitrogen, y = 1.274 0.733x, r 2 = 0982.
*
+
+
+
Table 1
Summary of ratios of Plip-P to carbon and nitrogen as a
function of growth conditions in enrichment cultures
Numbers of time points sampled for each culture are given in
paren theses
Oxygen status
W Peptone
pmol Plip
(g C)-' a
pmol Plip
(g N)-' a
Aerobic (4)
Aerobic (4)
Aerobic (5)
Aerobic (4)
Aerobic ' (4)
Aerobic (3)
Aerobic (3)
Aerobic (3)
Aerobic (3)
Aerobic (3)
Anoxic (4)
Anoxic (2)
Anoxic (4)
Anoxic (4)
Anoxic (2)
Anoxic (3)
0.01
0.05
0.05
0.1
0.1
0.2
0.2
0.4
0.8
1.6
0.1
0.2
0.2
0.4
0.8
1.6
186.4+ 71.3
273 f 130.1
214.3 f 83.1
253.0 f 76.6
151.1 k 28.2
242.0f 95.0
205.1 f 14.1
194.9f 24.3
243.2 f 102.9
189.3 f 24.3
109.6k 8.5
96.7k 22.7
91.42 12 8
84.4f 23.6
123.6i 8.8
103.1k 16.7
690.5f264.7
1016.8f408.2
742.3 f253.3
869.2 f 294.3
587.1 It 146.0
918.4 f 345.8
679.6f 61.2
719.2f 98.7
832.8 f221 .O
695.7f 124.3
404.8f 35.0
378.3+ 79.8
274.4k 45.8
327.8+ 97.6
459.4f 24.4
371.2rt 69.2
MeankSD, calculated from the measured Plip-P, carbon
and nitrogen values.
Culture was enriched from an estuarine sediment (Norseminde Fjord).
' Culture was enriched from a subtidal sediment in Aarhus
Bay (water depth > 10 m).
a
00
0
20
40
80
60
100
pg Carbon mi" culture
c
10.0
I
0
5
I
10
,
15
Likewise, when the data for the anaerobic enrichment cultures were pooled, Plip-P concentrations
were also correlated with total carbon and nitrogen (Fig. 4A,B): for carbon, y = - 0.027 + 0.102x,
r z = 0.943; for nitrogen, y = 0.101 0.347x, r 2 =
0.916. Again, all slopes were significant at P <
0.05.
The ratios of Plip-P to carbon and nitrogen for
each individual enrichment culture were calculated (Table 1) and an analysis of variance
(ANOVA) performed to determine if there were
any significant differences among the cultures.
For the aerobic cultures, there were no significant
differences and the overall mean was 215.4 & 76.1
pmol Plip-P (g carbon)-' and 775.1 253.9 pmol
Plip-P (g nitrogen)-'. No significant differences
were observed for the anaerobic cultures either
and the overall mean was 99.6 & 18.8 pmol Plip-p
(g carbon)-' and 358.8 f 80.8 pmol Plip-P (g
nitrogen)-
+
20
25
30
pg Nitrogen ml-' culture
Fig. 4. (A) Regression analysis of Plip-P vs. cellular carbon
content for all time points sampled during the growth of the
anaerobic enrichment cultures on the various peptone concentrations summarized in the text; each point represents the
mean of triplicate determinations for Plip-P and cellular carbon
concentrations. (B) As for A but Plip-P vs. cellular nitrogen.
*
'.
351
Table 2
+
Summary of ratios of Plip-P to carbon and nitrogen (mean S.D.)in various pure cultures
Numbers of determinations are given in parentheses. NA, not available.
Culture
C :N (molar)
pmol P l i p - ~(g c)-'
pmol Plip-P (g N)-'
Aerobic enrichment
E. coli (2)
P. denirrificans (2)
Anaerobic enrichment (19)
Desulfouibrio sp. (4)
Desugobulbus sp. (4)
Desul/obacrer sp. (4)
Methanogenic bact. (4)
Skeletonema sp. (4)
Synechoococcus sp. (3)
D. brightwelli (5)
4.3
4.2
NA
4.1
4.7
5.2
4.5
5.6
9.9
6.2
6.2
215.4 f 76.1
168.1 f 10.9
63.4*21.8
99.6i18.8
127.3 f 15.4
253.4 f 35.8
118.1 f14.7
138.3f25.1
108.6k 7.8
13.5* 1.9
53.4f 7.9
775.1 f 253.9
498.2 f 137.9
NA
358.8k 80.8
509.4f 18.4
1096.1 30.4
496.0* 17.7
719.6k 66.9
917.6*121.7
71.6* 7.7
283.6f 35.8
In general, conversion factors from the enrichments were consistent with conversion factors from
monocultures (Table 2). A ratio of 168 pmol Plip-P
(g carbon)-' was calculated for E. coli and 63
pmol PIip-P (g carbon)-' for P. denitrificuns. The
ratio for the sulfate-reducing bacteria ranged from
118 to 253 pmol Plip-P (g carbon)-' for Desulfobacter and Desulfobulbus sp. respectively. The
methanogenic enrichment culture had a ratio of
138 pmol Plip-P (g carbon)-'. The Plip-P/carbon
ratio for the algal cultures ranged from 14 to 109
pmol g-' for Synechococcus and Skeletonema sp.
respectively. For these two algal cultures, the following C :N : P ratios were calculated: Skeletonernu sp.: 110 : 11 : 1; Synechococcus sp.:
16: 3 : 1.
5 . DISCUSSION
The use of DCM instead of chloroform and of
sodium bromide for phase separation are modifications of a technique described by Findlay et al.
[17].Sodium bromide facilitates sampling of the
DCM phase since DCM is less dense than the
final aqueous methanolic sodium bromide. This
'phase reversal' minimizes contamination and increases sample throughput relative to methods
used by others [3]; The use of DCM is also a
*
significant enhancement since it presents a much
lower health sisk than chloroform. DCM can be
recommended since there is no difference in the
extraction efficiency of DCM and chloroform
(Findlay, personal communication). In addition,
the use of sodium bromide to achieve phase separation does not influence the recovery of either
phosphatidylcholine or bacterial lipids from environmental samples (King and Brinch-Iversen,
unpublished data).
In this study, all time course experiments
showed a strong linear correlation between Plip-P
and carbon or nitrogen indicating that the growth
state of bacteria does not generally have any significant influence on these ratios (Figs. 1B and
2B). Exceptions occurred in old, senescent cultures
where the ratio of Plip-P to carbon and nitrogen
decreased. Similar observations have been reported by White et al. [ 5 ] .
Few others have considered the effect of growth
state on the ratio of Plip-P to carbon or nitrogen.
For purposes of comparison, previous literature
data have been recalculated on a carbon basis by
assuming that carbon is 50% of the cell dry weight.
Based on this conversion and a dry weight/wet
weight ratio of 0.4 [18], Randle et al. [19] have
found a relatively constant concentration of Plip-P
(g carbon)-' in an analysis of six aerobic cultures.
Estimated ratios range from 30 to 115 pmol Plip-P
352
(g carbon)-' among the cultures. These values are
all significantly lower than those observed during
this study with the exception of P. denitrijiicans.
For E. coli b growing on a glucose or glycerol
medium, Randle et al. have found from 267 k 75
and 243 f 71 pmol Plip-P (g carbon)-' (mean f
SD) respectively. The values obtained by Randle
et al. [I91 are somewhat higher than those reported
here. However, both sets of values are within the
range reported by others [ll]. Of course, it must
be noted that these comparisons should be viewed
with caution since the data from Randle et al.
were recalculated from cell wet weights based on
the assumptions given above. In other work, DeSiervo [ll] has observed that Plip-P concentrations increased from 110 to 240 pmol Plip-P (g
carbon)-' during a shift from early log to transition phase for E. coli b growing at 27°C. Card [20]
has also observed an increase in the Plip-P to
carbon ratio during growth of Bacillus
stearothermophillus, with values rising from 100
pmol to 150 pmol Plip-P (g carbon)-'. As a
result, it appears that Plip-P conversion factors are
generally insensitive to growth state but that exceptions do exist, especially in pure cultures. To
some extent, such changes may be masked within
diverse mixed cultures or in natural populations.
In this study, increased nutrient concentrations
resulted in higher growth rates and biomass but
also proportionally higher Plip-P concentrations.
An ANOVA performed on the Plip-P to carbon
and nitrogen ratios from the individual enrichments (Table 1) indicated no significant differences between different nutrient amendments.
Pooling all data and performing a linear regression analysis gave similar estimates (Figs. 3A,B
and 4A,B). The differences between the means
calculated in Table 1 and the ratios obtained from
the regression data were not significant (two tail
t-test, P = 0.05). Thus, the Plip-P to carbon ratio
appears relatively constant as a function of substrate concentration, even though it is clear that
the specific fatty acid and sub-class composition
of PIip-P is influenced by the nature of growth
substrates (e.g. ref. 21). Further experiments are
necessary to determine if Plip-P conversion factors
vary as a function of substrate class, for example,
protein versus carbohydrate, or the availability of
inorganic nutrients, for example, nitrogen or phosphorus. Such information is necessary to further
validate the application of culture-based conversion factors to natural populations.
Some additional insights into the response of
Plip-P conversion factors to both growth state and
substrate concentration are available from studies
of bacterial starvation. Several studies report losses
of lipids during starvation [22,23]. However,
calculating the approximate lipid phosphate to
carbon ratio for cultures grown by Oliver and
Stringer [22] shows that the ratio increased for two
cultures and was constant for a third during the
first 14 days of starvation. Estimates of Plip-P to
carbon ratios from data of Thomas and Batt [23]
show a 4%increase during the first 24 h and a 23%
decrease over 55 h of starvation. During starvation
of Arthrobacter crystallopoietes for two weeks, the
losses of Plip-P were proportional to the losses of
cellular carbon [24]. The concentrations of Plip-P
and carbon did decrease with time for a culture in
a medium not supporting growth, however the
Plip-P to carbon ratio was not different (two tail
1-test, P = 0.05) from the ratio of the amended
cultures (Table 1).
Because of the good correlation between Plip-P
and carbon or nitrogen despite growth state or
nutrient load, it is possible to establish a general
Plip-P to carbon or nitrogen ratio. Based on the
results from the aerobic enrichment cultures, we
suggest 190 pmol Plip-P (g carbon)-' and 730
pmol Plip-P (g nitrogen)-' (2.28 and 10.22 pmol
mmol- carbon and nitrogen respectively). On the
basis of the anaerobic enrichment cultures, we
suggest 100 pmol Plip-P (g carbon)-' and 350
pmol Plip-P (g nitrogen)-' (1.20 and 4.90 pmol
mmol- carbon and nitrogen respectively). These
ratios are consistent with both the pure cultures
examined in this study (Table 2) as well as with
some of the values reported by others (e.g. ref. 5 ) .
The ratio observed here for P. denitrificans is
much lower than the other aerobic cultures examined. However, a very low growth rate was
observed for this culture so the measured ratio
may be due to sub-optimal conditions. Likewise,
Desulfobulbus sp. grown on propionate shows a
ratio significantly higher than the other anaerobic
cultures. However, it is not clear whether this
'
'
353
reflects the growth conditions that were used or a
consistent difference between Desulfobulbus sp.
and other anaerobes.
Differences among cultures or enrichments in
Plip-P conversion factors may depend in part on
membrane structure. For example, a shift from
anaerobic to aerobic growth conditions for a
monoculture of Staphylococcus aureus resulted in
a change in Plip-P from 80 to 140 pmol (g
carbon)-' [25].This change was apparently due to
the formation of a membrane-bound electron
transport system. The higher Plip-P conversion
factors found in the aerobic enrichments in the
study reported here may be explained similarily.
Differences between the aerobic and anaerobic
enrichments may also be due in part to differences
in the relative abundance of Gram-positive and
Gram-negative bacteria since the membranes of
Gram-negative organisms contain two phospholipid bilayers. However, there is no consistent evidence for such differences from data presently
available (Table 3). Thus, biomass estimates from
Plip-P concentrations may be less sensitive to
population structure than estimates from other
parameters, for example, lipopolysaccharide or
muramic acid concentrations.
The most critical step in the application of
Plip-P for estimating bacterial biomass is the
choice of an appropriate conversion factor. Several
experiments where Plip-P concentrations have
been measured in monocultures have been summarized by White et al. [ 5 ] . Based on those data, a
general conversion factor of 100 pmol Plip-P (g
carbon)-' has been proposed. A higher conver-
Table 3
Summary of Plip-P to carbon ratios from the literature
~
Organism
Remarks
pmol Plip-P
(g C)-l
growth cycle
shift from 37 to 25 C
shift to aerobic conditions
growth cycle
logarithmic growth
glycerol limitation
growth cycle at 27OC
growth cycle at 37 C
growth cycle with glucose
growth cycle with glycerol
maximum density
maximum density
growth cycle
growth cycle
growth cycle
growth cycle
growth cycle
stationary phase
logarithmic growth
Aerobic enrichment
maximum density
maximum density
maximum density
maximum density
120
80- 120
80-140
100-140
160
80-100
110-240
125-170
223-380
148-340
168
63
55- 34
36- 95
45- 60
74-110
114-184
120
100
150
118
127
253
310
Aerobic enrichment
maximum density
520
Staphylococcus aureus
Bacillus sraerothermophillus
3. Iicheniformis
B. subrilis
E. coli
paracoccus denitrijicans
Agrobacterium tumefaciens
Azorobacter agilis
Chromobacter violaceium
Proreus vulgaris
pseudomonas aerugrnosa
Bacteroidesmelanmogenicus
Micrococcus denirriJcans
Haemophilus parainfluenzea
Desuyobacter sp.
Desul/ourbrio sp.
Desulfobulbus sp.
Gram
reaction
Ref.
+
+
1331
-
1191
~ 9 1
~ 9 1
-
+-
-
111
1361
121
This study
This study
This study
King et al.
(unpubl.)
1171
354
sion factor has been reported recently [17] for an
aerobic enrichment. The conversion factor suggested, 521 pmol Plip-P (g carbon)-', is 2.5- to
3-fold higher than that reported here for similar
enrichments. A ratio of 310 pmol Plip-P (g
carbon)-' has also been measured for a mixed
culture enriched from a coral reef sediment St.
Croix, U.S.V.I. (King and Brinch-Iversen, unpublished data). Both of these mixed culture conversion factors as well as the aerobic conversion
factor reported here are significantly higher than
the conversion factor originally proposed by White
et al. [5]. In contrast, the conversion factor reported here for anaerobic incubations is similar to
that of White et al. [5]. This factor may find the
widest application for sediments since oxygen
penetration is generally limited to a few millimeters in lacustrine as well as near-shore and even
deep pelagic systems [26-281. The variability observed for conversion factors derived from aerobic
incubations suggests that greater caution may be
required for estimation of biomass in oxic samples.
A final point concerns the algal Plip-P conversion factors. These ranged from 14 to 110 pmol
Plip-P (g carbon)-' for a cyanobacterium and a
diatom respectively. The low ratio observed for
the cyanobacterium may be due to the formation
of a typical mucus layer [29] which would increase
the apparent carbon content without requiring
proportional increases in phospholipids or membrane area. More important though, the large variability observed for the conversion factors could
pose significant problems when assessing bacterial
biomass in the presence of significant concentrations of algae. One possible solution may be to
establish a relationship between Chl-a and Plip-P
and to measure these two parameters simultaneously. Volkman et al. [30] have reported that the
ratio of total fatty acids to Chl-a in 10 monocultures of different microalgae varies from 1.8 to
8.9, with most values in the range of 4-6. A more
detailed survey may narrow this range to more
acceptable limits. Simultaneous measurement of
Chl-a and Plip-P concentrations could then allow
an appropiate correction for and estimate of algal
biomass. Such measurements are facilitated by the
fact that chlorophylls are extracted efficiently and
partitioned into the DCM phase along with phospholipids using the methods described here (King,
unpublished data).
In summary, the use of Plip-P concentrations
as an estimate of total microbial biomass is appealing because it is simple, rapid and accurate
relative to many other methods (e.g. refs. 3, 17, 31,
32). A new set of conversion factors has been
proposed here. These should be regarded with
some caution due to the variability observed for
monocultures (Table 3) and for mixed cultures
enriched from different sediments.
ACKNOWLEDGEMENTS
We thank P. Haecky for D. brigthwelli; T.
Wiegers for E. coli; T. Dalsgaard for P. denitrificans and F. Bak for the sulfate reducers. We are
also grateful to F. Bak and K. Finster for their
advice on growing sulfate-reducing bacteria and to
T.H. Blackburn for advice and encouragement
throughout. This work was supported in part by
NSF OCE-8700358 and OCE-8900358.
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