Jourrid
of
Gtwrril Micwhiologj, ( 1985), 131. 39-45.
Printed irr Great Brituiri
39
Mixed Substrate Utilization in Micro-organisms: Biochemical Aspects and
Energetics
B y W O L F G A N G BABEL* A N D R O L A N D H . M U L L E R
Academj, o j Sciences of the GDR, Institute of Technical Chernistrj,, 7050 Leipzig,
Permoserstr. 15, German Democratic Republic
(Received 24 February 1984; reuised 14 August 1984)
~
~
~~
The energy-based classification of heterotrophic substrates requires biochemical evaluation
because some substrates can be assimilated by a variety of different metabolic pathways. By
using the Y,,,-concept it was shown that the classification depends on the yield of ATP and
reducing equivalents already generated on the way to the precursor (phosphoglycerate). With
carbon-excess substrates a part of the total substrate consumed must be oxidized to completion
merely for energy production, whereas with energy-excess substrates more energy is provided
on the route to the precursor than is needed for assimilation of the precursor carbon. By means of
this approach it was possible to assess experimental growth yields obtained on mixed substrates
and to predict the optimum mixing proportion in order to attain the maximum carbon conversion
efficiency. The validity of this method was shown for some examples.
INTRODUCTION
Substrates for chemo-organotrophic nutrition of micro-organisms differ from each other with
regard to their carbon/energy ratio (Babel, 1979). Methane, methanol, ethanol and n-alkanes are
energy-excess substrates compared with the combustion enthalpy of the microbial cell
substance. Glucose, acetate and formate are energy-deficit substrates if they are used as sole
carbon and energy source.
The macromolecular composition (i.e. the content of proteins, lipids, carbohydrates and
nucleic acids; Stouthamer, 1973),the elementary composition (Sukatsch & Faust, 1977; Lebeault,
1979), the combustion enthalpy (Babel, 1979, 1980) and the available electrons per carbon atom
(Lebeault, 1979) of the microbial biomass are nearly independent of the carbon and energy
source used for growth. It is possible, therefore, that by combining substrates of both categories,
and hence by balancing the carbon/energy ratio, the loss of energy or carbon, respectively,
which takes place during growth on a single substrate, can be avoided.
With energy-excess substrates, a 100% carbon conversion should be possible (Babel, 1979,
1980, 1982), but there generally seems to be a measured limit at about 67% (Linton &
Stephenson, 1978; Babel, 1979). On the other hand, experimental values for some so-called
energy-excess substrates have been found which fall short of this apparent limit or which exceed
it. This is the case, for instance, with methanol (Goldberg, 1981) and ethanol (Suomalainen &
Oura, 1979), respectively. Thus the energy-based classification of substrates, which was
heuristically valuable for developing the auxiliary-substrate concept (Babel, 1979), requires a
biochemical evaluation. This is possible by means of the Y,Tp-concept and by determining the
carbon conversion efficiency using substrate mixtures.
METHODS
Growth yields were calculated on the basis of the Y,4,,-concept (Stouthamer, 1973, 1979; Van Dijken &
Harder, 1975; Anthony, 1978, 1980; Papoutsakis & Lim, 1981). This method requires knowledge of the metabolic
pathway for conversion of carbon substrate to the central carbon precursor (3-phosphoglycerate; PGA) from
Abbretliation: PGA phosphoglycerate.
0001-1811
0 1985 SGM
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40
BABEL AND R . H. MULLER
which all cell components are synthesized, as well as knowledge about the P/O-quotient and the elementary
cell composition. The energy requirement for the synthesis of cell substance from this precursor can he assumed
to be constant (Y:q$’ = 10.5 g dry wt per mol ATP; Stouthamer & Bettenhausen, 1973). Then, by using a cell
formula of C , H , 0 2 N , , with N H 3 as the nitrogen source, the equation for the synthesis of biomass rcads 3s:
4 PGA
+ NH, + 29 ATP + 5.5 NAD(P)H
+
(CjHsOZN,)3
The energy and reducing equivalents necessary for the synthesis of biomass are generated by metabolism of substrate to the central precursor (PGA) or by oxidation to COz (or both).
Provided the energy and reducing equivalents generated on the route from the substrate to the central carbon
precursor (PGA) are sufficient (or more than sufficient) for the synthesis of the ‘cell molecule’ then a substrate
exhibits an excess of energy and hence is classified as such. In contrast, energy-deficit substrates are tho,se in which
a certain amount of the total substrate consumed must be oxidized to COz merely for production of energy or
reducing equivalents.
The balance equations of single substrates were used to calculate the carbon conversion efficiency of substrate
mixtures (Table 1). The substrates were mixed in such a proportion as to supplement carbon and energy. If one
substrate exhibits an excess of energy the loss of carbon is reduced to a level which is only determined by unavoidable oxidative decarboxylations during synthesis of cell constituents.
RESULTS A N D DISCUSSION
Biochemical evaluation o j heterotrophic substrates
The balance equations for the synthesis of the general carbon precursor (PGA), invcllving different pathways for the assimilation of certain substrates, are summarized in Table 1.
Methanol. During the synthesis of PGA from reduced C, compounds such as methanol, three
molecules (three carbon atoms) are used, regardless of the assimilation pathway involved.
Hence, up to the central carbon precursor (PGA) the carbon conversion efficiency is 100%.
However, the experimental growth yield on methanol is always less than 100% (Goldberg,
1981). The greatest growth yield should be possible with bacteria using the hexulose phosphate
pathway (Van Dijken & Harder, 1975; Anthony, 1978, 1980). Only if methanol is oxidized to
formaldehyde by a NAD+-linked enzyme, and not by the pyrrolo-quinoline-quinone-dependent
methanol dehydrogenase (which probably yields only 1 ATP; Anthony, 1982), and if‘ the P/Oquotient of NADH is 3, could 100% carbon conversion efficiency be imagined with bacteria
using the hexulose phosphate pathway (fructose bisphosphate variant plus transketolase/transaldolase/transketolase rearrangement of the C acceptor) and NH, as nitrogen source (Anthony,
1983). In all other cases, including methylotrophic yeasts with an NAD+-linked methanol dehydrogenase instead of methanol oxidase, additional methanol must be oxidized. Consequently,
methanol is practically always an energy-deficit substrate (Babel, 1980; Muller et al., 1983).
In the case of some bacteria using the serine pathway the growth yield or carbon conversion
efficiencyis NAD(P)H-limited, while in those using the hexulose phosphate pathway it is mainly
ATP-limited (Anthony, 1978). This distinction may be important if, for example, the growth
yield is to improve by genetic manipulations or if an auxiliary substrate is being looked for.
Glycerol. Glycerol is a balanced substrate in terms of its carbon/energy ratio (Babel, 1979).
From the experimental growth yield on this substrate (Payne, 1970) it is apparent that about 2
glycerol molecules must be oxidized exclusively for energy production at a carbon conversion
efficiency of about 66%. Again, the theoretical carbon conversion efficiency depends on the
route of carbon catabolism (see Table l), but in all cases more energy must be expended for the
incorporation of PGA than is made available on the route to this precursor. The sarne is true
for the carbon/energy balanced substrate mannitol (Babel, 1979).
Glucose. Glucose is an energy-deficit substrate when it is assimilated via the glycolytic
sequence as well as the Entner-Doudoroff pathway (Table 1). However, glucose can be
assimilated via the hexose monophosphate pathway, and in this case it becomes an energyexcess substrate, since on the way to the precursor carbon is lost by oxidative decarboxylation.
Only when the P/O-quotient falls below 1.3 must additional glucose be oxidized via the (oxidative
hexose monophosphate cycle (PGA
1 ATP + 3 C 0 2 5 NAD(P)H) or via the TCA cycle
+
+
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Table 1 . Balance equations and energetic classification of 'some substrates .fbr growth of' micro-organisms
-
-
EMP
ED
HMP
.-
-
-
-
-
Ser
Glyc
-
-
HuP
HuP
HuP
HuP
Ser
Ser
RBP
DHA
Ser
RBP
3
3
3
3
3
3
3
3
3
3
2
2
2
2
Methanol
Methanol
Methanol
((1)
Methanol
(P)
Methanol
(,f)
Methanol
Methanol
Methanol
(1)
Formate
Formate
Ethanol
Acetate
(g)
Oxalate
Oxalate
(h)
1 Glycerol
(i)
1 Glycerol
1 Succinate
6) 1 Xylose
(k)
3 Xylose
1 Glucose
1 Glucose
1 Glucose
(I)
1 Mannitol
( m ) 1 Mannitol
1 Phenol
(ti)
1 Hexadecane
(o)
1 Hexadecane
(h)
(c)
(a)
Pathway
+ 5 ATP
+ 5 ATP
+ 2 ATP
+ 1 ATP
+ 2 ATP
+ 5 ATP
+ 2 ATP
+ 5 ATP + 3 NAD(P)H
+ 8 ATP + 2 NAD(P)H
+ 3 ATP
+ 3 ATP
+ 3 ATP + 3 NAD(P)H
+ 3 ATP + 3 NAD(P)H
+ 1 ATP
+ 1 ATP
1 PGA
1 PGA
-+
-+
-+
-+
-+
-+
-+
-+
-+
-+
-+
-+
-+
-+
+
-+
1 PGA
1 PGA
1 PGA
1 PGA
I PGA
I PGA
+ 1 PGA
1 PGA
1 PGA
1 PGA
-+ 1 PGA
1 PGA
1 PGA
+ 1 PGA
+ 1 PGA
4 1 PGA
5 PGA
-+ 2 PGA
2 PGA
1 PGA
2 PGA
2 PGA
-+ 1 PGA
+ 4 PGA
-+ 4 PGA
-+
-+
1 NAD(P)H
1 NAD(P)H
+
+
+
+
I FADH?
1 FADH:
1 FADH?
+ 3 ATP
+ 1 ATP
+ 2 ATP
+ 2 NAD(P)H
+ 2 NAD(P)H
+ 1 NAD(P)H
+ 5 NAD(P)H
+ 5 NAD(P)H
+ 2 NAD(P)H
+ 2 NAD(P)H
+ 7 NAD(P)H
+ 3 NAD(P)H
+ 3 NAD(P)H
+ 3 NAD(P)H
+ 16 NAD(P)H
+ 15 NAD(P)H
1 FADH?
+ 2 FADH,
+ I I FADH,
+ 12 FADH?
+
+ 6 NAD(P)H 1 FADH2
+ 2 NAD(P)H + 1 FADH,
+
+
+ 1 NAD(P)H
+ 1 NAD(P)H
+ 1 NAD(P)H
+ 1 NAD(P)H
Balance equation
+ 3 coz
+ 4coz
+ 4 CO?
+ 3 CO?
+ 1 co2
+ 2 CO?
+ 1 CO,
+ 1 co,
+ 1 co,
+ 1 co,
Deficit
Deficit
Deficit
Deficit
Deficit
Deficit
Deficit
Deficit
Deficit
Deficit
Excess
Deficit
Deficit
Deficit
Deficit
Deficit
Deficit
Excess
Deficit
Deficit
Deficit
Excess
Deficit
Deficit
Excess
Excess
Excess
Energetic
classification
Ahhruciations: Ald. aldolase; DHA, dihydroxyacetone; ED, Entner-Doudoroff; EMP. Embden- Meyerhof-Parnas; FBP, fructose bisphosphate; Glyc,
glycerate; HMP, hexose monophosphate; HuP, hexulose 6-phosphate; ICL+, isocitrate lyase positive; K D P G , 2-keto-3-deoxy-6-phosphogluconate;
RBP, ribulose bisphosphate; Ser, serine ; TA, transaldolase; TK, transketolase. -, Not designated.
The letters in parentheses define variants of the respective pathways : ( a ) FBP/TK-Ald-TK ; ( h ) FBPITK-TA-TK ; (c) KDPG/TK-Ald-TK ; ( d )
KDPG/TK-TA-TK; (u) ICL+ via formaldehyde to activated C , , NAD+-linked formaldehyde and formate dehydrogenase; (g)via serine; ( h ) via glycerol
kinase or glycerol dehydrogenase plus di hydroxyacetone kinase or glycerol dehydrogenase plus glyceraldehyde kinase ; (i) via glycerol dehydrogenase plus
aldehyde dehydrogenase and glycerate kinase; 0')via TK-TA plus glucose 6-phosphate and 6-phosphogluconate dehydrogenase; ( k ) via TK-TA plus glycolytic sequence; (I) via fructose 1-phosphate; (m)via mannose 1-phosphate; ( n )by means of an NAD+-linked aldehyde dehydrogenase; (0)by means of
an FAD-linked aldehyde dehydrogenase.
r!
.-)
5.
2
s.
=:
z
Ts
E
b
&
s.
\
F.
.o
7
z
5
h
w.
42
+
(PGA --t 3 COz 4 NAD(P)H
an energy-deficit substrate.
BABEL AND R. H . M U L L E R
+ 1 FADH, + 2 ATP). Because of this, glucose becomes again
Xylose. Depending on the route for carbon catabolism, xylose, too, can be either an energydeficit or an energy-excess substrate (see Table 1).
Formate. Formate belongs to the class of energy-deficit substrates when used as the sole
growth substrate. However, if formate can only be oxidized, for example by means of an NAD+linked dehydrogenase, and its carbon cannot be assimilated (autotrophically or via the serineoxalate pathway), then it becomes an energy-excess substrate with an infinite energy/carbon
ratio (conditional energy-excess substrate) and it can help to increase the carbon conversion
efficiency of other substrates lacking energy up to the carbon-metabolism-determined upper
limit (Babel et a/., 1983).
Ethanol and n-alkanes. According to the energy-based classification, ethanol and n-alkanes are
energy-excess substrates, and this is also true from a biochemical point of view, because carbon
is lost on the way to the central precursor (Anthony, 1980), as is the case in the hexose monophosphate pathway (Table 1). But this is no longer true if the P/O-quotient of N A D H is smaller than
2 (Babel, 1983).
Acetate, oxalate and succinate. Acetate, oxalate and succinate are energy-deficit substrates and
they remain energy-deficient from a biochemical standpoint although carbon is lost as CO, on
the route to the precursor (Table 1).
Suitability of substrates as auxiliary substances
en erg^-escesslcarbon-excess substrates. From the above considerations it follows that ethanol
is an excellent substrate to use as an auxiliary substrate for improving growth yield. This is true
for n-alkanes (Heinritz et al., 1982) and possibly phenol, too. Experimentally this becomes
evident for ethanol if it is combined with sucrose, for example (which in this context is considered like hexoses; see glucose in Table 1). The carbon conversion efficiency of the substrate
mixture is greater than those of the single substrate, although the growth yield on ethanol is
smaller than expected (Table 2). This deviation is probably caused by the fact that NADPJH cannot
be used for ATP synthesis in yeasts. This might also be the reason for differences between the
calculated and the experimentally determined mixing proportions (Table 2).
Energ~,-de~cit/carbon-e.ucess
substrates. An auxiliary substrate effect can also occur if
substrates having different energy deficits are simultaneously assimilated, and some examples of
this are known; e.g. methanol/mannitol (Van Verseveld et al., 1979), methanol/glucose (Egli et
a/., 1982; Miiller et al., 1983), oxalate/formate and acetate/formate (Dijkhuizen & Harder,
1979a, b), methanol/formate (Van Verseveld & Stouthamer, 1978; Hazeu & Donker, 1983) and
methane/CO, and methanol/CO, (Malashenko et al., 1980). This phenomenon is surprising.
The elementary composition of micro-organisms is nearly independent of growth substrate and
if no alternative, more efficient metabolism for precursor synthesis is involved the (auxiliary
effect can only be explained by an increase in the P/O-quotient due to the simultaneous assimilation of both substrates. This was shown to be the case with Paracoccus denitr$cans (Van Verseveld et d . , 1979). As the experimental growth yields of yeasts on methanol are around 0.38 g g-’
(Table 2) it follows that the P/O-quotient must be smaller than 2 during growth on this substrate
and, since the theoretical and experimental values of carbon conversion efficiency are similar
during growth on the mixture (Table 2) the P/O-quotient also seems to rise due to the presence of
glucose.
However, with Hansenula polymorpha no mixture of methanol and glucose could Ibe found
in which one substrate was assimilated and the other used exclusively for energy generation.
Obviously this metabolic configuration becomes possible with Paracoccus denitr$cans because
of the epigenetic regulation of the ribulosebisphosphate carboxylase. Probably, no ‘division of
labour’ can be induced in methylotrophic yeasts. This may be due to the fact that the enzymes
responsible for the assimilation of methanol (transketolase, Waites & Quayle, 1983, and
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Energetics of mixed substrate utilization
43
Table 2. E-uperimental and theoretical growth yields of single substrates and substrate mixtures
Organism
Candida utilis
Lodderomy ces
elongisporus
Hansenula
polymorpha
Paracoccus
denitrlficans
Paracoccus
denitr8cans
Beneckea
natriegens
Hansenula
polrmorpha
Molar
ratio
Substrate
Ethanol
Ethanol*
Sucrose
E t hanol/sucrose
E t hanol/sucrose *
Hexadecane
Hexadecane*
Sucrose
Hexadecane/sucrose
Hexadecane/sucrose*
Methanol
Methanol*
Glucose
Glucose*
Met hanol/glucose
Methanol/glucose*
Methanol
Methanol*
Manni to1
Mannitol*
Methanol/mannitol
Methanol/mannitol*
Methanol/mannitol*
Formate
Formate*
Mannitol
Mannitol*
Formate/mannitol
Formate/mannitol*
Formate
Forma te *
Glucose
Formate/glucose
Formate
Formate*
Glucose
Glucose*
Formate/glucose
Formate/glucose*
13.8: 1
10.8 : 1
0.27 : 1
0.33 : 1
38-8 : 1
39.0 : 1
2-1 :1
3.1 : 1
2.0 : 1
Y
[g (g
substrate)-']
Carbon
conversion
efficiency (%)
0.68
0.83
0.5
61.7
75.0
0.8
1.32
0.5
6.3 : 1
4.7 : 1
Feiler et al. (1980)
54.7
67.6
71-4
44.8
73.0
54.7
Heinritz et al. (1982)
ND
0.38
0.43
0.52
0-53
0.42
0.43
0.52
0.55
0.82
0.82
0.7
0.07
4.0 : 1
5.1 :1
Reference
0.12
0.52
0.55
0.7
0.7
0
0
0.45
0.52
0
0
0-52
0.53
0.7
0.7 1
56.0
47.7
54.5
61.1
61.9
58.0
55.0
53.0
53.6
62-2
65.8
98.2
98.2
83.8
12.7
21.8
62.2
65.8
83.8
83.8
0
0
52.9
61.1
0
0
61.1
61.9
82.2
83-3
Muller et al. (1983)
Van Verseveld
et al. (1979)
Van Verseveld &
Stouthamer (1980)
Linton et al. 1981)
Babel et al. ( 983)
* Calculated by Babel (1983): Yps;T+= 10.5, P/O-quotient = 2, cell molecule C4H,O2N ;glucose assimilation in
H . po/jwrorpha via EMP pathway; methanol and formate assimilation in P . denitrijicans via RBP pathway (cell
molecule C',,H, , O , N , , < ) : ND, nodata given variation of mixing proportions was aimed at increase of growth rate
only.
~
dihydroxyacetone kinase, Hofmann & Babel, 1980) and the dissimilation via formate are simultaneously regulated epigenetically.
We do not know why the upper exploitation limits of mannitol that were attained using
methanol and formate as energy donors were not identical. The growth yield obtained (0.82 g
dry wt per g mannitol) appears very high. Perhaps it is not justified to assume that methanol
cannot be assimilated. (The essential criterion is the measurability of ribulosebisphosphate carboxylase.) Thus it is better to take a certain portion of methanol into account when calculating
the growth yield. Assuming that the upper carbon conversion efficiency of mannitol is the same
as that of glucose (approximately 83%; Babel et a[., 1983), which is supported by the results
obtained with a formatelmannitol mixture (Van Verseveld & Stouthamer, 1980), then about
0.1 g dry wt of Paracoccus denitr$cans should be produced from methanol.
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44
w.
BABEL A N D R . H. M ~ J L L E R
The proportion of methanol calculated to be necessary for improving the carbon conversion
efficiency up to 98.2% was calculated to be higher than the experimental mixing proportion
(Table 2). This resulted from using a P/O-quotient of 2 in all calculations instead of the real
one, which was estimated to be 3 in this example (Van Verseveld et al., 1979).
Conditional energ~~-e.~~ess/carbon-e.ucess
substrates. Conditional energy-excess substrates are
characterized by the fact that they are used only to provide energy (i.e. no carbon is assimilated).
By using these it becomes possible firstly to detect the upper limit of carbon conversion
efficiency of any growth substrate, and secondly to determine whether and, if so, to what extent.
ii substrate exhibits an excess of' carbon or energy (cf. Table 1 ) .
Formate turns out to be such an energy donor. In Paracoccus denitri@cansand Hansenulm po1~'morpha the carbon conversion efficiency of growth on mannitol and glucose, respectively, could
be increased up to the maximum (Table 2). However, with Torulopsis sp. M H 26 a maximurn
growth yield on glucose of only 0-5 g g-I could be attained (data not shown). This indicates that
in this organism glucose is almost exclusively assimilated via the hexose monophosphate
pathway (Muller & Babel, 1984). We therefore suggest that with Beneckea natrieguns growing
under conditions such that the growth yield of 0.52 g g-I is the maximum value attainable on
glucose using formate as energy donor (Table 2), glucose assimilation does not take place via the
Embden-Meyerhof-Parnas pathway. Hence there is a lower upper limit of carbon conversion
efficiency.
Further aspects. Babel ( 1 979) predicted that the simultaneous utilization of heterotrophic substrates would be accompanied by an increase in the growth rate. This was confirmed even when
the auxiliary-substrate effect on the carbon conversion efficiency was not significant (Eggeling
& Sahm, 1981).
This increase in the growth rate cannot be explained definitively. A possible reason might be
to overcome a bottleneck. For example, when the growth rate is proportional to the energy production rate (Stouthamer, 1980) there must be a bottleneck in the energy-providing system.
An additional interesting phenomenon is that when two substrates are assimilated
simultaneously, such as methanol/glucose (Egli et al., 1983; Muller et al., 1983), the residual concentration of methanol during growth in continuous culture is lower than during growth on
methanol alone. This seems to be a universally valid but not yet fully understood phenomenon
(Harder & Dijkhuizen, 1982). Both the increase in the growth rate and the decrease of the
K , value (increase in substrate affinity), give micro-organisms an advantage in competing for
substrates in the course of evolution (Babel, 1982).
The authors are grateful to Dr Christopher Anthony for correction of the English text.
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