Assimilation Efficiency and Energy Contents of

Prototrophic Bacteria

K . P. HO and W. J . PAYNE, Department of Microbiology,

University of Georgia, Athens, Georgia 30602

Summary
When the assimilation efficiency in batch culture of a variety of heterotrophic
bacteria was tested in media containing progressively smaller concentrations of
carbon substrate, two groups emerged. Those in the first group assimilated carbon
progressively more efficiently as the initial concentration in the culture medium was
lowered below 2 mg C/ml: some displaying efficiencies exceeding 80%. Those in the
second group assimilated carbon at rates that changed little, if at all, with changes
in the initial concentrations of carbon substrate. Members of the second group
differed from the first in their metabolic versatility as evidenced by the ability of
each to catabolize hydrocarbons. The energy content (heat of combustion) of the
cells of the species tested vaned little with changes in cultured conditions, and
values for all’fell close to the average for bacteria (5411 cal/g ash-free dry wt). In
continuous aerobic culture on glucose-minimal medium, the carbon assimilation and
energy assimilation efficiencies were simultaneously maximal for Enrerobacter acvo-
genes when the input carbon concentration was 0.2004 mgiml and the dilution rate
was 0.57. An equation for predicting heat of combustion of cells from knowledge of
their C,H,N, and 0 ratios was devised and tested. Precision ranged from +6% to
- 16%.

INTRODUCTION

When required to utilize a single organic substrate (or a simple

mixture) as a sole source of carbon and energy, prototrophic het-
erotrophs reportedly assimilate 60(+5)% of the and the
energy content5p6of the molecule(s) and lose the remainder to dis-
similation. It would seem reasonable that growth in a complex
medium would result, in contrast, in an increased efficiency of
assimilation. Experimental examination of this suggestion7 revealed
that Enterobacter aerogenes and Pseudomonas perfectomarinus did
indeed incorporate 75 to 85% of the carbon taken from carbon-
limited cultures enriched by small additions of casamino acids or
yeast extract.* Yet, despite the increase in assimilation efficiency,
the cells from bacterial cultures on both minimal and nutritionally

Biotechnology and Bioengineering, Vol. XXI, Pp. 787-802 (1979)

@ 1979 John Wiley & Sons, Inc. OOO6-3592/79/0021-0787$01.OO
788 HO AND PAYNE

rich media were found to have energy-content values clustering

around the average of 5.41 1 kcal/g ash-free dry ~ t These . ~obser-
vations indicate that the richness of resources positively alters the
assimilation efficiency of the growing prototrophs; but, on a per cell
basis, the energy content remains unchanged.
Assimilation efficiency has historically received more attention
than cellular energy contents. Close estimates of both are essential,
however, to the development of yield predictions. So little is known
about cellular energy contents that they seem timely properties for
experimental exploitation. Energy values seem likely candidates to
replace dry weight figures in growth yield equations.
Having previously considered the effect of nutritional enrichment
on assimilation efficiency, the purposes of the current study were
then twofold: i) to determine if decreasing rather than increasing
the quantity of available carbon influences the assimilation effi-
ciency of bacteria growing prototrophically, and ii) to examine the
possible influences of varying growth conditions on the cellular
energy contents of various bacterial species. As an ancillary effort,
an equation for predicting caloric content of cells from analyses of
their elemental composition was developed and tested for applica-
bility.

MATERIALS AND METHODS

Microorganisms
We employed Bacillus stearothermophilus WT.2 provided by R.
J. Downey; Beneckea natriegens ATCC 4048, Pseudomonas per-
fectomarinus ATCC 14405, Halobacterium salinarium 34002, and
Sarcina litoralis 16006 supplied by N. E. Gibbons; Escherichia coli
K-12 supplied by G. J . Tritz; Acinetobacter HOI-N provided by W.
R. Finnerty; Candida lipolytica 4-1 obtained from V. Munk; My-
cobacterium vaccae JOB5 supplied by W. R. Finnerty ; Enterobac-
ter aerogenes ATCC 13048-3,Pseudomonas aeroginosa PA01 wild
type, and Serratia marcescens purchased from Midwest Culture
Service.

Culture Media
Bacillus stearothermophilus was grown in a New Brunswick
shaker incubator on a medium described by Downey .lo Halobac-
terium salinarium and Sar. litoralis were grown on a medium de-
 ASSIMILATION EFFICIENCY AND ENERGY CONTENT 789

scribed by Gibbons and Payne. l1 The other organisms were grown

on a basal medium (pH 7.2) with the following composition (g/liter):
K2P0,,9.28; KH2P04,1.8 I ; (NH,),S0,,3.5; Na,SO,,O.5; MgS0,,0.2;
FeS0,-7H20,0.001. The basal salts were supplemented with one of
the following carbon and energy sources: glucose, succinate, as-
paragine, hexadecane, 0.5% tryptone and 0.15% yeast extract, 0.8%
nutrient broth, and 0.5% yeast extract in the quantities listed in
Tables I and I1 and Figures 1 and 2. For growth of the marine
bacteria, 2% NaCl was included in the medium, and 25% NaCl was
supplied in media used for growth of the two halophiles. For deni-
trifying cultures, 0.1% KNO, was added to the media. (Percentage
indicates a weight-to-volume basis.) Basal salts and complex carbon
sources were sterilized by autoclaving. Hexadecane was separately
autoclaved, then added aseptically to the basal salts. The other
carbon sources were filter sterilized and added aseptically to the
basal salts.

Batch Cultures
Cells were grown in 500 ml Erlenmeyer flasks containing 100 ml
basal medium supplemented with a carbon and energy source. The
cultures were incubated at 30°C on a New Brunswick gyrotory
shaker at 150 rpm with a stroke of 2.5 in. Turbidity resulting from
cell growth was monitored with a Bausch and Lomb Spectronic 20
set at 420 nm. Carbon incorporation efficiencies and heats of com-
bustion were determined for cells harvested late in the log phase of
growth.

Continuous Cultures
Chemostat cultures of Ent. uerogenes were grown in a specially
designed 2 liter Erlenmeyer flask enclosed in a waterjacket. Growth
temperature was controlled at 30( +5)"C 'by a Lauda K-2/R water-
circulator temperature control unit. The culture was agitated by a
spinning, 3 in. Teflon stirring bar driven at maximum speed by a
magnetic stirrer. The medium was sparged at 2 vol air/ vol growth
medium/ min. The oxygen absorption rate of the system (measured
by the sulfate oxidation method'? was 82 mmol 02/liter/hr. (Paca13
noted that the respiration rates of Klebsiella uerogenes never ex-
ceeded 40 mmol 02/g/hr during continuous growth at a density of
1.5 g cells/liter under different rates of oxygen supply. Hence, the
rate of absorption of oxygen we employed was considered ade-
quate.) Rate of inflow of nutrient medium was controlled with a
790 HO AND PAYNE

Buchler polystaltic pump. A constant working volume of 500 ml

was maintained by use of an external overflow tube. In these test
cultures, the carbon and energy sources were the limiting compo-
nents of the system. Starvation experiments were carried out by
suspending washed cells in the original culture volume of 0.066M
phosphate buffer (pH 7.2) and incubating at 30°C with shaking for
24 or 55 hr.

Dry- Weight Analyses

Direct dry weights were obtained by depositing the cells from 10-
40 ml suspension (depending on cell density) on predried Millipore
membrane filters (47 mm diam, 0.45 pm pore size). The deposits
were washed with a second volume of distilled water (0.052M
MgClz was used to wash the Ben. natriegens and Ps. perfectomar-
inirs cells). The filtered and washed cells were then dried at 105°C
for 15 hr and weighed. Several samples were washed with dilute
phosphoric acid to remove carbonates that might contribute to dry
weight, but the dry weight of the cells in these samples was not
different from that of cells washed with water. A filter through
which only uninoculated medium and water were filtered and
washed was dried and weighed as a control.

Total Carbon Determination

To estimate carbon incorporation efficiencies, the total amount
of dissolved carbon in a medium was measured, before and after
cell growth, with an F & M 185 carbon-hydrogen-nitrogen ana-
lyzer. The procedure outlined in the manufacturer’s handbook was
slightly modified to permit the measurement of total carbon in liquid
samples. Samples to be measured were acidified with concentrated
HCI and subjected to bubbling for 3 min with helium to remove
carbon dioxide before samples of precisely 0.05 ml were introduced
into the instrument for analysis. As a control, uninoculated media
samples were sparged with carbon dioxide for 20 min to saturate
with carbonate and then acidified and bubbled with helium for 3
min to ensure that the method used effectively stripped the fluids
of carbon dioxide.
For cells grown on hexadecane, the total carbon in the medium
before cell growth was calculated from the known amount of hex-
adecane added. Hexadecane not utilized by the cells can absorb to
cell surfaces and filters, presenting a problem in carbon measure-
 ASSIMILATION EFFICIENCY A N D ENERGY CONTENT 79 I

ments. To measure the total carbon in the spent medium after cell
growth, the cell suspension was acidified, sparged for 3 min with
helium to drive off carbon dioxide, and homogenized for 10 min
with a sonifier cell disruptor (Ultrasonic, Inc.). The total carbon of
the spent medium was calculated from the value of the total carbon
of the sonified cell suspension minus the amount of cell carbon in
the suspension.
Heuts cf Combustion
Cell suspensions were centrifuged at 8,000 g for IS min and
washed twice with distilled water (as before, 0.052M MgCI, was
used for washing the marine bacteria and 25% NaCl for washing
the halophiles). The washed cells were freeze dried and assayed for
heat of combustion with a Phillipson Micro Bomb Calorimeter.
Each value presented is the average derived from at least four
determinations. Samples of freeze-dried cells were held at 500°C for
10 hr and the residue weighed to provide a measure of ash content.

Elementul Composition
The carbon, nitrogen, and hydrogen contents of the freeze-dried
cells were measured with the F & M CHN analyzer.
Glucose was determined with enzymatic reagents purchased from
the Sigma Chemical Co., St. Louis.

Cell Yields
The cell yields were calculated as follows:
dry wt of cells produced (g/rnl)
molar growth yield (g/mol) =
substrate used (mol/ml)
carbon incorporation efficiency (%) =

wt of cells (g/ml) x C content of cells (%)

total C in medium before growth - total C in spent medium (g/ml)
x 100
energy assimilation efficiency (%) =

wt of cells (g/ml) x heat of combustion of cells (cal/g)

wt of substrate used (g/ml) x heat of combustion of substrate (calig)
x 100
192 HO AND PAYNE

RESULTS AND DISCUSSION

Batch Cultures
In minimal media, the polar flagellates, P s . aeruginosa and Ben.
natriegens, assimilated the carbon of glucose, and Ps. perfecto-
marinus the carbon of asparagine, increasingly more effectively in
media initially containing less than 1 mg Ciml than in media con-
taining greater concentrations of carbon (Fig. l(a)). Growing Ps.
perfectomarinus cells made more efficient use of carbon over a
longer range of concentration decrease than did P s . aeruginosa and
Ben. natriegens. During growth in a complex medium, carbon as-
similation efficiency in ps. perfectomarinus increased in a manner
different from that seen in minimal media. The increase proceeded
approximately linearly as initial carbon concentrations were de-
creased.
During growth of enteric bacteria in minimal media, the efficiency
of assimilation of decreasing initial quantities of glucose carbon
increased even more sharply. In Ent. aerogenes the steepest in-
crease in assimilation efficiency occurred when the initial concen-
trations were less than 2 mg C/ml as succinate (Fig. I(b)). For both

z 80
0
t
2 70
0
a
60
0
z 50
Z
0
m
[z 4 0
Q
0
8 30

o i z 3 4 5 0 1 2 3 4
ORIGINAL CARBON CONCENTRATION (mg/ml)

Fig. I . Demonstration in several bacterial species of increased efficiency of

carbon assimilation during growth o n culture media containing decreasing initial
quantities of organic substrate. (a) Polar flagellates: ( 0 )Ps. aeruginosa on glucose;
(0) Ben. narriegens on glucose; ( A) Ps. perfecrornarinus on trytone-yeast extract;
(0) Ps. perfectomarinus on asparagine. (b) Enteric bacteria: (m) Enr. aerogenes on
succinate; (A:0 ) Ent. uerogenrs and Serr. marcescens on glucose.
 ASSIMILATION EFFICIENCY AND ENERGY CONTENT 793

E nt . aerogenes and Serr. marcescens, the assimilation efficiency

response to glucose was fairly smoothly curvilinear over the range
between 0 and 2 mg C/ml.
The bacteria in this group are as efficient, therefore, as the most
effective animals (i.e., sedentary, growing, or poikilothermic me-
tazoa). l4
Another group of phototrophic bacteria and a yeast responded
less abruptly to lessened concentrations of carbon substrate (Fig.
2). Candida lipolytica assimilated between 65% and 55% of the
carbon of glucose removed from minimal medium over a relatively
broad range of initial carbon concentrations. The carbon assimila-
tion efficiency of M y c o . vaccae stayed near 50% over the entire
range of initial carbon concentrations, whereas that for Acineto-
bacter HOl-N ranged around 40% in both complex and aspargine
minimal medium. The lowest but reasonably constant assimilation
efficiency was exhibited by M y c o . vaccae growing on hexadecane
minimal medium. (An extensive study of the efficiency of assimi-
lation of the carbon of linear hydrocarbons will be published sepa-
rately.)
To rationalize these observations, we may suppose that the con-

ORIGHAL CARBON CONCENTRATON (mq/ml)

Fig. 2. Relatively unchanging efficiency of carbon assimilation in certain species
of microorganisms during growth on culture media containing decreasing initial
quantities of substrate. 1: (0) C. liporytica on glucose. 2: (A) M y c o . vaccae on
nutrient broth-yeast extract. 3: Acinetobacter HO1-N on (A) nutrient broth-yeast
extract and ( 0 )asparagine. 4: (0)
M y c o . vaccae on hexadecane.
794 HO A N D P A Y N E

nection between increased assimilation efficiency and lowered sub-

strate concentrations provides a competitive survival value for the
bacteria in the first group. In contrast, microorganisms in the second
group may depend less for competitive edge on assimilation effi-
ciency than upon versatility. These latter organisms all grow pro-
totrophically, for example, at the expense of a variety of complex
substrates such as hydrocarbons. The groups are not mutually ex-
clusive, for the impressively successful P s . aeruginosa is so ver-
satile as to fit into both.

CIi emos ttr t Culture

We established, empirically, a glucose concentration range that
permitted Ent. aerogenes to strip a minimal medium of all meas-
urable glucose and ail but minimal amounts of carbon while growing
at several dilution rates. Using the relationship described in the
Materials and Methods section to calculate energy assimilation ef-
ficiency (with heat of combustion of glucose = 3720 cal/g), it was
found that efficiency increased during growth in those cultures that
contained the lower substrate concentrations and were incubated at
the faster dilution rates (Table I ) . Ideal carbon concentrations
ranged from 0.101 to 0.2004 mg/ml and dilution rates from 0.57 to
0.75. It is significant to note that both the elemental composition
(C,N,H) and the heat'of combustion of the cells varied only slightly
during growth under these regimes.
From the molar growth ( Ysub) values as great as 90- 102 seen here,
we calculated Yay,- values6 of 3.75-4.25. Previous estimates of
rave-for glucose that averaged 3.33 were derived from the work of
a number of investigators,6 who used higher concentrations of sub-
strate than we employed in the current work and thus inadvertantly
failed to obtain maximal yields. Consistent with such a presumption
is the observation of similarly elevated Ysub values for growth of
Pserrdomonas j7uorescens and E. coli in dialysis cultures. 15,16

Energy Content of Cells

In a previous study,gthe average caloric content of bacteria grown
both aerobically and anaerobically in minimal and complex media
was 541 1 cal/g ash-free dry wt. The value for Ent. aerogenes in the
current investigation (5546 callg ash-free wt) lies close to that av-
erage, was not widely divergent for any of the cultures, and was
 ASSIMILATION EFFICIENCY AND ENERGY CONTENT 795

unaffected by growth rate or initial carbon concentration (Table I).

The heat of combustion of the moderately halophilic (marine) bac-
terium, Ben. natriegens, was nearly identical to that for Ent. aero-
genes (Table 11). The value for E. coli was close to the mean as
well, and starvation changed it little, if at all. The heat of combustion
of one extreme halophile, H . salinarium, was close to the average
value for bacteria. The value for the other, Sarc. fitoralis, was 11%
lower than the average; but these are fragile bacteria, and even a
small amount of lysis would lower values. Caloric contents of the
other marine bacterium, Ps. perfectomarinus, varied but little
whether cells were grown aerobically or anaerobically as denitri-
fiers. Growth of Buc. stearothermophilus at 45"C, well below its
reported growth optimum of approximately 60"C, yielded cells with
diminished energy contents. Coultate and S ~ d a r a m ' reported ~
greater yields at the lower than the higher temperature. Additional
information on differences in cells derived from cultures at both
temperature extremes is needed if these disparities are to be ration-
alized. Growth as either an aerobe or a denitrifier at 60°C provided
Bac. stearothermophilus cells whose energy contents were scarcely
distinguishable, one from the other.
It is possible that gaining an energy content close to 5400 kcal/g
ash-free dry wt represents the primary objective of growing bacte-
ria-within a narrow range, an invariant basis upon which one may
develop a theory of heterotrophic growth. Our previous prediction
of the existence of a "budget" gene6 is made thus more tenable by
experimental establishment of a targeted outcome for the original
budgeting "plans" of the inoculum cells.

Basing Prediction o j Caloric Contents upon Elemental

Composition o j Cells

For some time, we have attempted to develop a mathematical

means of predicting caloric content of cells knowing only their
elemental analyses. Having that capability would obviate the ne-
cessity for the laborious calorimetry method. The following is a
description of our approach. Assays of 75 samples provided a value
of 26.8 kcaliave- (on an ash-free dry wt basis) as an average heat
content for a variety of bacteria (a value in close agreement with
26.9 derived by Minkevich and Eroshin'* for Pseudomonas and
Candida cells). Taking figures from Table I1 for H . salinarium as
 -4
W
m

TABLE I

Influence of Dilution Rate on the Carbon and Energy Assimilation Efficiencies of Enr. aerogenes Growing in Minimal Mineral Media Z
Containing Various Initial Concentrations of Glucose (Nitrogen Content as Ammonium Ion = 0.75 mg Niml) 0
>
2
Elemental composition Cellular U
of cells heat of
(%) combustion Energy
2.e
Original Cellular Carbon Total (calig assimilation 2
m
C conc Dilution dry wt incorporation C re- ash-free efficiency
(mg C/ml) rate (rngiml) Y,,, (%I maining C N H Ash dry wt) %
0.101 0.2 0.1033 77 57 0.014 47.5 12.9 6.7 8.5 5600 61
0.38 0.12 90 62 0.009 47.4 12.9 6.8 11.3 5502 70
0.57 0.1266 95 66 0.01 - - - 11.4 5527 75
0.75 0.1366 I02 80 0.023 45.9 12.7 6.7 8.4 5525 80

0.1326 0.2 0.1466 78 61 0.018 47.7 12.9 7.5 9 5474 65

0.38 0.19 101 72 0.008 47.4 13 7.6 9.7 5577 86
0.57 0. I9 101 74 0.01 47.6 13 7.5 11.7 5587 86
0.75 0.18 96 70 0.01 47.4 13 7. I 11.9 5573 81
0.2004 0.2 0.2433 87 63 0.014 47.7 12.9 6.7 8.4 5537 72
0.38 0.2733 97 68 0.014 46.5 12.7 6.3 8.3 5552 81
0.57 0.2866 I02 77 0.025 46.9 13 6.5 10.2 558 I 86
0.75 0.275 98 71 0.02 46.7 12.9 6.5 8.7 5578 82
>
CA

0.302 0.2 0.3 70 51 0.03 46.5 12.7 5.9 7.9 5520 59 z

0.38 0.33 77 56 0.028 46.5 12.7 5.9 9. I 5580
66 5
0.57 0.34 79 59 0.034 46.5 12.8 6.3 9.5 5545 67
0.75 0.36 84 66 0.047 46.7 12.9 6.3 7.9 5569 71 5
0
z
0.447 0.2 0.385 61 47 0.053 47.4 12.9 6. I 8 5548 51 m
?I
0.57 0.48 76 63 0.09 46.7 12.9 6. I 9.7 5517 63 3
0.75 0.52 82 63 0.06 46.9 13.1 6.3 10.5 5479 68 ci
m
I .06 0. I 1.08 76 52 0.15 47.1 13.1 6.2 8.2 5618 61
56
5.e
0.57 0.98 69 55 0.23 46.9 13.1 6.5 8.2 5500
0.75 47 58 0.54 46.3 12.7 6.5 9 5530 37.5 P
0.65 z
av = 5546 U
m
z
rn
m
n
4
0
0
z
1
m
5
-4
-4
\D
798 HO AND PAYNE

TABLE I1
Energy Contents, Elemental Composition, and Oxygen Combustion Demands

Cellular
caloric Elemental composition
content of cells (oxygen by difference)
(calig
(%)
Test bacteria ash-free
(and growth conditions) dry wt) Ash C N H 0

Ben. narriegens 5530 9.9 39.5 10.8 6.6 33.2

(aerobic)
Sar. litoralis 4797 46 26 5.3 3.3 20
(aerobic)
H . sulinarium 5204 48 25 6.9 3.1 16.7
(aerobic)
E . coli 5388 10.4 41 12 6.5 30
(aerobic)
E . coli 5227 12 40.8 12 6.3 28.9
(starved 24 hr)
E . coli 5317 12 40.3 12 6.2 29.5
(starved 55 hr)
Buc. stearorhermophilirs 4900 7.9 35.4 10 5.7 41
(45°C)
Bac. steurothermophilus 5286 9.6 38.6 10.8 5.8 30.5
(60°C)
Bac. steurothermophilus 5181 10 36.4 10 5.6 37.9
(denitrifying, 60°C)
P s . perjectomarinirs 5089 12 43 11.3 6.4 29
(aerobic)
Ps. perfectomnrinirs 5229 12.3 43 11.5 6.5 24
(denitrifying)
a Uo2 = g cell C burnedimol O2 consumed during bomb calorimetry.

an example, then:
heat of combustion (kcal/g)
= [mol 0, needed for burning I g ash-free dry wt cells]
x [4 av e-imolo, consumed]
 ASSIMILATION EFFICIENCY AND ENERGY CONTENT 799

of Various Types of Bacteria Grown under a Number of Conditions

Calculated values
0 2 Cellular Cellular Percent
required caloric caloric Cellular error
for com- content content caloric with
bustion (kcal/dv (kcaliav content respect
Empirical of 100g c - ash- c - ash- (calig to (g
formulas cells free free ash-free ash-free
for cells (moll c/o2" dry wt) dry wt) dry wt) dry wt)
-
3.9 10. I3 31.90 27.17 4704 -15

2.37 10.95 27.60 29.35 5092 + 6

2.32 10.77 28.90 28.78 5 148 - I

4.12 9.97 29.30 26.72 4929 - 8

4.07 10.02 28.30 26.85 4965 - 5

3.99 10. I I 29.30 27.10 4852 - 9

3.09 11.58 36.50 3 1 .OO 4159 - 15

3.56 10.84 33.50 29.05 458 I - I3

3.24 1 I .24 36.00 30.10 4339 - I6

4.29 10.1 26. I3 27.08 5 I44 + I

4.47 9.7 25.70 26.02 542 I + 4

Units for the first two bracketed terms are obvious. The last brack-
eted term is a "correction factor."
Step 1. Compute mol 0, required to burn one empirical formula
unit of cells:
+ 2.320, + 2.08C02 + 1 .53H20 + 0.245N2
C2.0aN0.4901.04H3.06
Use the term, 2.32 mol O,, in step 2.
Step 2. Compute mol 0, required to burn 1 g ash-free dry wt cells
so that, of 100 g
when the empirical formula is C,~oaNo,4901,04H3,06
800 HO AND PAYNE

cells (Table II), 52 g are the ash-free dry wt of cells:

- - - 4.46 x
2'32 mol 0,
52
Step 3. Compute V,, for H . salinarium:

25(% cell dry wt comprised of C)

uoz= 2.32 ( m o l 0 , to bum100 g cells)
= 10.77 (g cell C burnedimol 0, consumed)
Step 4. Estimate heat of combustion of cells:
heat of combustion = 4.46 x lo-* x 4

x p., (- 10 - 10.77 26.~)]

= 5149 cal/g ash-free dry wt cells

Step 5. Compare the calculated with the measured value:
5149
-x 100 = 9Wo error = -1%
5204
The calculated values given in Table I1 list the values obtained
for a variety of species by this method and indicate the relative
error for each prediction.

Justification f o r Using tho Correction Fuctor and N o t Simply 26.8

The value 26.8 kcal/av e - is the average from 75 assays of cells
of 17 species grown on different substrates and under various con-
ditions. Most of the individual values are close to 26.8, but vary, of
course, on one side or the other. If this variance is not taken into
consideration, and 26.8 is used in all calculations, the estimates of
heat of combustion of those cells with a value higher or lower than
26.8 would diverge even more significantly from the experimental
value than do the figures given in Table 11. The term U,, was thus
derived to attempt to damp out these effects.
That not all cells have a kcal/av e - value of 26.8 might be due to
the slight difference in bond types and oxidation states. Since car-
bon is the predominant element in the cell, dividing the percentage
that is carbon by the mol 0, required to burn 100 g cells would give
a factor that might indicate relative oxidation states. Indeed, close
examination of the tables shows that cells with kcal/av e - values
 ASSIMILATION EFFICIENCY AND ENERGY CONTENT 80 1

close to 26.8 had U,, values near 10. Cells with a higher than 26.8
kcal/av e - value had a higher Uo2value than 10, and vice versa.
Since 26.8 kcaliav e - is imprecise but is used as a necessary but
correctable component of the equation, the value of Uo2 = 10 is
treated similarly.
What the complex correction factor does (or attempts) when the
Uo2value for a species is a part of the equation is to compare that
value with 10 and find the percent difference. This difference cor-
responds to a difference in kcaliav e - . If a value of Uo, = 10 is
plugged in, the whole correction factor will simply become 26.8. If
a U,,, is greater than 10, the correction factor becomes larger than
26.8, and vice versa. The error in the calculation should thus be
decreased.
The size of the error in the predictions derived by this equation
for several of the systems was unexpected. With no data other than
elemental composition in hand, we were able more accurately
( * 5%) to predict the cellular heat of combustion of bacteria grown
on hydrocarbons (unpublished observations). The usefulness of the
equation may thus be restricted to studies of hydrocarbon culture
systems.
CONCLUSIONS

Carbon assimilation efficiency of a group of prototrophic heter-

otrophic bacterial species growing in batch culture increased sharply
as the initial carbon concentration in chemically defined culture
media was decreased. Values as great as 7.545% were achieved.
The increases were most notable for polar flagellates in media con-
taining less than 1 mg C/ml, whereas the response in enteric bacteria
began when initial carbon concentration was less than 2 mg C/ml.
In contrast, the carbon assimilation efficiency of Ps. perfecromar-
inus in complex medium was approximately linear over a broader
range from 0.5 to 5.4 mg C/ml. A second group, comprising a yeast
and several bacterial species that can utilize hydrocarbons, assim-
ilated carbon less efficiently than the organisms in the first group.
The members of the second group did not respond sharply to dim-
inished concentrations of carbon substrate during growth in either
minimal or complex media.
Continuous cultures ofEnr. aerogenes (member of the first group)
revealed that the efficiency of carbon assimilation from glucose was
maximized when the dilution rate was 0.75 and the carbon concen-
tration was 0.101 mg C/ml. Efficiency of simultaneous assimilation
of carbon and energy of glucose was maximal at a dilution rate of
802 HO A N D PAYNE

0.57 and a carbon concentration of 0.2004 mg C/ml. The heat of

combustion of Ent. aerogenes cells varied but little from an average
of 5546 cal/g ash-free dry wt over a range of dilution rates and
glucose carbon concentrations. The heat of combustion of the ther-
mophile, Bac. stearothermophilus, was lower when cells were
grown at 45°C than when they were grown at 60°C either aerobically
or as denitrifiers. Cells grown at 60°C contained heat contents near
the average for bacteria (5411 cal/g ash-free dry wt). The heat of
combustion of the extreme halophile, Sar. litoralis, was lower than
expected (perhaps due to lysis), whereas that of H. salinarium was
nearer the average. Heats of combustion of two species of marine
bacteria, Ben. natriegens and P s . perfectomarinus (aerobic or den-
itrifying), and E. coli were also near the average.
An equation designed to aid in predicting heat of combustion of
cells knowing only the C,H,N and 0 ratios from elemental analysis
was tested and found to vary in precision from + 6% to - 16% from
the measured values for several species.

This study was supported by National Science Foundation under Grants No. DES
74-21338 and No. OCE 77-15854.

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Accepted for Publication July 18, 1978