Microcalorimetry of microorganism metabolism of monosaccharides and simple aromatic compounds

Microcalorimetry of Microorganism Metabolism of Monosaccharides and Simple Aromatic

Compounds

REX LOVRIEN, GREGG JORGENSON, MAGGIE K. MA, and WESLEY E. SUND, Biochemistry Department, Gortner Laboratory, College of Biological Sciences, University of

Minnesota, St. Paul, Minnesota 55108

Summary Heat conduction solution calorimeters enable rapid determination of the heats of

aerobic and anaerobic metabolism of substrates by microorganisms. Aliquots of 1 .0 ml cell suspension, 5 x 1Og cells/ml, were mixed with a few dozen nmol substrate contained in 0.5 ml, under a controlled atmosphere of air, 0,, or N,. At these substrate concentrations, with adapted microorganisms, metabolism and its heat generation are usually complete within 300 to 600 sec. The raw data yield AH,,, values. The AH,,, were determined in the range 0.001 to 0.010% substrate, and extrapolated (limit substrate concentration 4 O), to yield AoR, the limiting differential molar heat of metabolism. The AoH values express the heat generated when there is rapid metabolism but little new growth, minimal contribution by H+ transfer from metabolites, and maintenance of aerobicity or anaerobicity as specified. Escherichiu coli Bi5 was used for aerobic and anaerobic combustion of eight sugars. Pseudo- monas multivoruns, and an Acinetobacter, strain B- I , were used for aerobic metabolism of benzene, toluene, naphthalene, and a methylnaphthalene. The large heats of combustion of the hydrocarbons enable the use of aqueous solutions of hydrocarbons well below their solubility limits. The quotient AoH/n (n = atoms carbon! molecule substrate) varies from (-)36 to (-)67 kcaUmol carbon for the sugars. The most reduced sugars yield the largest exothermic heats. The quotient varies from ( - )27 to (-)81 kcalhol carbon for the aromatic hydrocarbons. Comparison of the calorimetric heats of metabolism to those from total aerobic combustion in ayuo (where available) give measures of the efficiencies with which the heat contents of the aqueous substrates are used by the bacteria.

INTRODUCTION

Microorganisms utilize carbon compounds in many ways. The overall direction of utilization intimately depends on the oxygen tension; on aerobic versus anaerobic conditions. The metabolic velocity is dependent on the presence of constitutive enzymes for some substrates, and on both inducible and constitutive enzymes

Biotechnology and Bioengineering, Vol. XXII, Pp. 1249- 1269 (1980) @ 1980 John Wiley & Sons, Inc. 0006-359218010022- 1249$02.10

1250 LOVRIEN ET AL.

for the other substrates. The course of utilization is usually followed by disappearance of substrate, formation of product, growth, or oxygen respirometry . It is generally understood that following the metabolic heat production is a valid, possibly advantageous means of monitoring metabolism; e.g., in the case of thick suspensions, even sludges and soil, which are optically very dirty. In addition, the enthalpies give important quantities in cell thermodynamic bal- ances, as noted in 1972.'

A review of calorimetric heats of microbial metabolism cannot be made here, but one point is striking. Namely, the ratio of the amount of oxygen present with respect to the amount of substrate started with, in some calorimetric measurements. Consider a 1% solution of a hexose, such as glucose. Ten g glucose/liter are about 0.06M glucose, a commonly used concentration in previous calorimetric measurements. However, the solubility of air in water is only about 1.3 x lOP3M at 25°C. The concentration of 0, in water, using Henry's Law and that air is 20% oxygen, is only about 0.3 x 10-3M. In aerobic metabolism, about 3 mol O$mol hexose are required. Under such conditions, aerobic metabolism may get started, but there is an oxygen deficiency of about tenfold. Unless major amounts of oxygen are quickly added, which generates appreciable heats of mixing, the reaction is likely to become anaerobic soon after it is initiated. It is shown below that for Escherichia coli the characteristic heats for the two kinds of metabolism-aerobic versus anaerobic metabolism-are very different by a factor of about 10. Hence the main problems are not calorimetric sensitivity, calorimetric response time, or sample size, in modern calorimeters. The main problem is maintenance of specified reaction conditions while the reactions take place. The most simple way of managing this is to use amounts of substrate that are considerably smaller than the amount of available oxygen, in the calorimetry of aerobic metabolism. An added advantage is that heat production becomes nearly independent of titration heats of acids produced. Also, product inhibition is minimized when small amounts of substrate are turned over.

The calorimetric measurements here used the range 0.000 to 0.010% by weight sugar; to 10-4M sugar were the maximum concentrations. The aromatic hydrocarbons were used in concentrations well below their solubility limits. The instruments supplied to us measure heats in the 0- 10 mcal range with a precision and an accuracy of 54% in a single run. For hexoses, generation of 10

MICROCALORIMETRY OF METABOLISM 1251

mcal heat requires about 30 nmol hexose. The volumes that are mixed are 0.5 and 1.0 ml, in the mixing vessels.

Besides measurement of reaction heats with microorganisms, part of the strategy is to observe the response when the microorganisms have adapted to certain metabolites. For example, all E . cofi will metabolize glucose. But glucose-grown E . coli frequently will not readily metabolize xylose. However, some strains will grow on and adapt to xylose, and generate appreciable heat, when mixed anew with xylose. Therefore by growing a strain of bacteria on various carbon sources, one may obtain a series of "reagents," that are adapted microorganisms, to work out the mosaic of reactivities toward various organic compounds, using calorimetric heat generation to monitor adaptation. The same methods work well for a-o- matic hydrocarbon-utilizing microorganisms.

EXPERIMENTAL METHODS

General Nature of the Experiments

An outline of the general protocol is illustrated in Figure 1. The cells were adjusted in their concentration by adding solvent (buffer

HARVEST, WASH, + STARVE, OBTAIN

CELL DENSITY

Grow cells on carbon source of interest

LOAD BOTH REACTION ~

VESSELS OF THE CALORIMETER

Cells in buffer, Carbon swrce in buffer

Reference vessel : Cells in buffer, Buffer with no

- - - - -

CONTROLGAS COMPOSITION

mix - Q m t +PROWCTS

heat 1 I

Retrieve for futher m l y s i s

IN THE HEADSRICE

(Air, 02 .N2 .CH4 .&I

Fig. I . General protocol for obtaining calorimetric heats of metabolism, starting from culture.

I252 LOVRIEN ET AL

plus supporting electrolyte) to dilute, or concentrated by centrifug- ing and removing excess solvent. Each calorimetric measurement involves adding four aliquots to the instrument, which is illustrated in Figure 2. After the run is finished, the products may be analyzed further for completeness of carbon utilization, pH shifts if any occurred.

Bacteria and Growth Media

Pseudornonas putida

This bacteria was originally isolated by Gibson and coworkers,2 and can grow on crystalline naphthalene as the sole carbon source.

M 3 cm

Fig. 2. Twin reaction vessel heat conduction microcalorimeter. One reaction vessel (the sample vessel) mixes bacteria plus carbon source in buffer. Second vessel (the reference vessel) mixes bacteria plus buffer containing no added carbon. Each vessel mixes 0.50 ml with 1.0 ml; mixing occurs upon simply inverting the whole cylinder. Control gases are humidified and thermally equilibrated with the heat sinks before entering the head spaces. Each reaction vessel transfers its power through the two Seebeck units that interface each vessel. Equilibration time after loading, to the point of mixing, is 10-15 min. Calorimetric figure of merit is 6.8 pWIpV net signal. Rise time for rapidly generated heat is 8 sec.

MICROCALORIMETRY OF METABOLISM I253

A culture of this organism was given to us by Dr. P. Chapman of this department. The base media contained the following salts (per liter): 4.2 g K,HP04.3H,0; 1 .O g NaH,PO,.H,O; 2.0 g NH,CI; 0.10 g nitriloacetic acid; 0.20 g MgSO4.7H,O; 0.012 g FeS04.7H,0; 0.003 g MnSO,.H,O; 0.003 g ZnS04.7H,0; 0.001 g CoCI2.6H20. Enough 1.ON NaOH was added, usually about 2.0 ml, to adjust the final pH to 7.0. The nitriloacetate allows the media to be autoclaved as a single solution.

Acinetobacter strain B-1 (Ac B-1)

This was provided by D. A. Kunz and P. J . Chapman of this department, an organism reported by them3 as an aromatic hydrocarbon-utilizing bacteria. It was handled in the same basic media plus trace metals, as that used for Ps. purida. The lower aromatic hydrocarbons (benzene and toluene) must be introduced to the liquid culture via the gas phase, from a separate cavity in the shake flask. Liquid hydrocarbon is toxic to these bacteria. For growth on benzene vapor, the population was increased first by growth on toluene vapor and then subculturing.

Escherichia coli BI5

This bacteria was the same as that utilized in our previous work. The solvent media was the Davis-Mingioli A buffer,, mixed with 0.8% NaCl, in a ratio of 1 vol A buffed9 vol NaCl. This was used in order to maintain the osmolality of the solvent, while providing a low enough buffer capacity during the calorimetric run to amplify any pH shifts of the solvent during combustion.

Cell suspension concentrations

Cell concentrations were determined by measurement of the op- tical density (OD) at 660 nm, based on independently determined viable cell concentrations from plate counting, as bef01-e.~ These two kinds of determinations gave the following relationships: E. coli, (B) = 2.00 k 0.05 x lo9 x OD660, where (B) = number of cells/ ml suspension, in a 1.00 cm path. For Ps. putida cells, the conversion factor was 1.7 k 0.2 x lo9.

Sugars and aromatic hydrocarbons: Introduction into solvent

Crystalline, A.R. grade compounds were used throughout the work, from Sigma and Aldrich. The solubilities of a number of

1254 LOVRIEN ET AL.

monobenzene hydrocarbons in the media for handling bacteria were determined here. Naphthalene, benzene, and toluene solubilities in water (25°C) are known.6 We repeated the determinations for the case of the aqueous buffer and supporting salts systems used with the microorganisms. The solubilities of such hydrocarbons in the salts media generally are roughly half that in neat water, but the solubility of each compound has to be determined individually. This was carried out by determining the molar extinction coefficient, E ,

for the compound in hydroxylic solvent (alcohol-H,O), then using E for the spectrophotometric determination of the compound concentration for each aqueous hydrocarbon stock. It is necessary to determine the hydrocarbon concentration of aqueous stock solutions at least daily, preferably on a twice-daily basis. Each hydrocarbon saturate was prepared fresh each day, centrifuged, and stored for the next few hours use in a separatory funnel with excess hydrocarbon. Assiduous care and frequent monitoring of the concentrations must be done for aqueous hydrocarbons unsaturate very easily, losing hydrocarbon. Some improvement in stability can be made by adding aqueous solvent to the hydrocarbon saturate, but at the cost of decreasing hydrocarbon concentration.

Microcalorimeters

Two microcalorimeters were used, constructed in collaboration with Microcalorimeters, Inc., who supplied many of the parts of the amplifier. The instruments are nearly equivalent, having a figure of merit of 6.8 ? 0.2 pW/pV output. Each instrument is a twin batch mixing instrument. One of the instruments was modified as shown in Figure 2, for gas purging in anaerobic assays. Each calorimeter has two mixing vessels, a sample vessel, and a reference vessel. The sample vessels were usually loaded with 1.0 ml bacterial suspension in buffer, in the larger chamber, and 0.5 ml carbon source of the desired concentration in the same buffer, in the smaller chamber. The reference vessel was loaded similarly, with 1.0 and 0.5 ml aliquots, except that the smaller aliquot is only buffer, containing no added carbon source. The Seebeck sensors are connected such that the signal representing the heat from the reference mixing vessel subtracts from that of the sample mixing vessel. The instrument is a differential instrument, with a net signal output that measures the difference in heats between the sample and the reference, after mixing. Upon closing the ports, and purging when necessary, the instrument was left to reach internal thermal equilibrium before mixing. This requires a period of about 5 to 10 min. The cylinder


was rotated to mix the contents of both vessels, initiating the reactions. The net signal is led to an amplifier and thence to a strip- chart recorder with an integrator. The integral area under the tracing with respect to the baseline is directly proportional to the heat. Both chemical calibration [Tris amine buffer reaction with HCl; (-) 1 1.3 kcal/mol] and electrical calibration using a small heater, were carried out. The heat generated in the reference vessel from tumbling and mixing the bacterial suspension with buffer but no carbon source is quite small, generally less than 1 mcal. Therefore the reference or blank signal, while it is accounted for in the net signal, is not a large contribution in the amplification ranges generally employed. The 30 or 100 pV ranges on the amplifier were used for the 10 and 100 mcal ranges.

Control of the gas atmosphere and the dissolved gas was carried out by bubbling gases for about 30 sec through the solutions before loading them into the prepurged calorimeter reaction vessels. After loading, additional sweeping was carried out for several seconds, via the plumbing illustrated in Figure 2, and then stopped before establishing the calorimetric baseline. If too much purging is used, the system will be thermally shocked for some time because of the large heat of evaporation of water. If too little purging is used in the case of anaerobic systems, anomalously large heats may be generated if oxygen remains.

Monosaccharide Uptake Velocities

In order to measure rates of uptake of sugars by the cells, the same techniques used before for obtaining binding data5 were used, as a function of time of exposure, to obtain centrifuged supernatants with varying sugar concentrations. The cell uptake of sugar was stopped by a sudden thermal shock. The sugar concentrations were measured by means of a modification of the Nelson-Somogyi reducing sugar method.

RESULTS

Microbial Reaction Conditions Affecting Heat Production

The need for care in adhering to anaerobic and aerobic conditions, depending on specifications as they are affected by sugar or hydrocarbon concentrations, was indicated above. Release of H+, from metabolic production of organic acids, generates characteristic heats, issuing from three main sources. They are represented in a

1256 LOVRIEN ET AL.

very general way, without stoichiometric coefficients, as:

substrate + O2 + RCOOH + C02 Q I (1)

RCOOH @ RCOO- + H+ Q 2 (2) H+ + titratable group * protonated acceptor Q 3 (3)

where Q is the heat generated. Under aerobic conditions, Q l is by far the larger quantity, commonly of the order of -300 kcal/mol (Table I). The heats of ionization of aliphatic carboxylic acids, Q2, usually are of the order of only + 1 to - 1 kcaVmol. Some carboxylic ionizations are athermal in the 25-37°C range. The value of Q 3 may be appreciable, depending on how the H+ are neutralized, if there is adequate buffering capacity. If the supporting buffer is phosphate, as in our work, the buffering capacity may be adjusted such that phosphate will almost certainly be the acceptor group. The mono- hydrogen phosphate ion HP042-, in its titration to H,PO,- has a Q of only -0.99 kcaVmol. But if H+ titrate imidazole side chains, on the cell's proteins, Q 3 will be -7.3 kcaVmol of H+. By measurement of buffer capacities and the pH shifts engendered by metabolism,

TABLE I Calorimetric Molar Enthalpies (Limit of Zero Concentration) for Microorganism

Metabolism of Compounds, 25°C

Monosaccharides (E. coli B/5) n ,

C atoms/ -AoH aerobic -AoH anaerobic Sugar molecule (kcal/mol) -AoH aerobicin (kcalimol)

Glycerol Sorbitol Mannitol Fructose Glucose Mannose Galactose Xylose

201 f 8 382 2 27 348 f 32 321 ? 15 3 0 4 2 18 245 2 18 230 f 27 178 ? 30

67 64 58 54 51 41 38 36

10 2 5 46 2 8 20 2 8 32 f 6 34 ? 3 25 ? 6 45 f 6

9 ? 4

Hydrocarbons (aerobic metabolism) n

carbon -A'H Compound Microorganism atoms (kcaUmol) -AoHln

Benzene Acinetobacter 6 191 f 32 32 Toluene Acinetobacter 7 188 ? 26 27 Naphthalene Ps. putida 10 810? 30 81 2-Methylnaphthalene Ps. putida 11 652 ? 15 59


the sum of Q2 and Q 3 can be semiquantitated. The correction on the overall measured heats, in aerobic conditions, was about 2 to 5% of Q ,.

Large corrections of the foregoing kind and the tendency of metabolic products to inhibit were avoided by use of small substrate concentrations. With the cell concentrations used, and with small amounts of substrate, anabolism and biosynthesis cannot proceed very far. The experimental heats were converted to the apparent molar heats, AHapp. The values of AHapp were determined as a function of c substrate, and the limit taken:

lim AHapp = AOIi Csubstrate-O

(4)

The quantity AOH is the limiting differential heat of metabolism. It gives the heat of metabolism of the substrate under conditions that require the least correction from heats of metabolite acid titration, etc.. described above.

Heat Generation from Adapted and Nonadapted Bacteria

One other important condition or parameter affects what is seen- namely, the velocity of uptake and metabolism of the substrate. In turn, such velocities are dependent on whether or not the cells are adapted to the substrate. The maximum velocity of the metabolism is expected when the substrate concentration is small relative to the number of cells, if uptake is not rate limiting. Achievement of maximum velocity of metabolism is another reason for determination of ha&, or of AH.,, at small substrate concentrations. Figure 3 is the record of heat generation from mixing 0.5 ml0.005% glucose with 1.0 ml glucose-grown E. coli B/5 cells. (After mixing, the sugar concentration is 0.005 x 0 3 1 . 5 = 0.0016%. The concentrations quoted are the concentrations before mixing.) Figure 3 includes a record of heat production from instantaneous heat generation upon mixing of a simple acid and base (H+ + Tris buffer) in a chemical calibration experiment. Such a measurement illustrates the rapidity of response of the instrument to a heat impulse. Comparison of the traces shows that in the case of glucose, there is no appreciable limitation by the instrument in responding to metabolic heats, within times of 10-50 sec. Rapid metabolism begins within about 5 sec after mixing. The maximum in glucose combustion occurs in about 300 sec, in contrast to the rise time of only about 10 sec to maximum signal from an instantaneously generated heat. Upon remixing, an-

1258

4 2

ln Y 80-

w I

LOVRIEN ET AL.

120r

‘ I i I J ( TRIS- HCl .CALIBRATION 1 HEAT DEVELOPED IMMEDIATELY UPON MIXING

I ’ I 1 I 1

I I I

0 200 400 600 800 1000 TIME AFTER MIXING, seconds

Development of heat as a function of time, upon aerobic mixing of glucose, with glucose-grown E . coli. I . O ml cells (2.5 x IOscells/ml) in each reaction vessel, 0.5 ml 0.002% glucose in the sample vessel before mixing. Lower zigzag trace represents mechanical integration to obtain the area under the curves.

Fig. 3 .

other “shot” of oxygen is made available, but with small amounts of sugar, most of the heat is out in 400 to 500 sec.

Calculation of the average velocity of aerobic glucose utilization, from the velocity of heat production, gave a rate of 1.2 x IO-l9 mol glucose/celYsec. Conventional uptake velocity experiments, not in- volving calorimetry, were performed using the reducing sugar assay to monitor uptake rates. They gave rates of uptake ranging from 0.7 X to 4 X mol glucose/cell/sec, depending on the extent to which the sugar was consumed. These values are in fairly good agreement with those of Hempfling et al. ,’ who obtained rates in the neighborhood of 3 x moVcelYsec, with E . coli B, under somewhat different conditions.

If bacteria were not previously adapted to the carbon source with which they are mixed, major changes in the rate of heat generation are seen in the expected direction. Figure 4 illustrates the course of heat generation when glucosamine-grown E. coli meet glycerol for the first time. Very little heat is produced for about 20 min. At least one, nearly two, doubling times elapse before generation of the appreciable heat which does eventually appear, as shown. The rate


of heat generation depends on adaptation, which in turn depends on the growth rate. Such reactions were not quantitated for this research, because unknown amount of glycerol were consumed in the various stages: adaptation and growth. However, the method is a convenient, frequently a vivid, means for monitoring adaptation.

For contrasting the behavior of adapted and nonadapted bacteria, E . coli B/5 were mixed with various sugars in a series of calorimetric measurements, employing the same cell concentration and sugar concentration in all cases; namely 5 x lo7 cells/ml (1.0 ml) and 0.002% by weight sugar (0.50 ml). Figure 5 shows the heats that were produced, as vertical bars with standard deviations, when various sugars are compared with respect to each other, and with respect to the substrate used for growth of the cells. The ordinates are the heats produced within 10 min after mixing. This period of time was selected because it is sufficient to generate practically all the heat from active metabolism by the cells if in fact they do transport the sugar, but it is too short for appreciable generation of newly adapted cells.

Figure 5 shows how for the metabolism of glucose, all E . cofi generate nearly the same amount of heat. For D-xylose and D- galactose metabolism, zero heat (within experimental error) is generated by glucose-grown cells. If the cells were adapted for xylose and galactose beforehand, rapid combustion occurs, according to the 10 min criteria. It is seen that the heat of xylose metabolism is lower than the other sugars at the same 0.002% concentration, and

Fig. 4. Adaptation of bacteria to a new carbon source illustrated by calorimetry. Cells grown on glucosarnine, but not previously exposed to glycerol, generate no heat on mixing with glycerol even at large concentrations (0.02% glycerol in 0.5 ml before mixing). After about one doubling time, the combustion of glycerol begins. Each remixing renews the oxygen in the solvent, to produce a burst of heat until the system lapses into aerobicity. Rate of heat generation depends on the rate of growth and the rate at which oxygen is made available at such large carbon source concentrations.

1260

measured. rnillical

Heat Of 12- metabolism d 55 n m - moles of carban sourfx

LOVRIEN ET AL.

-

-

2or r

d .t O L Cells

sum Mabolized

on -+ Glucose Glucose Xylose Xytose

4 Glucose Xylote Glucose Xylors

Glucose Uucose Galactose Galactose

Glucose Goloctosa Glucose Galactose

Fig. 5 . Raw experimental data for E. coli B/5, contrasting response to the sugar on which cells were grown vs. sugars for which the cells were not previously adapted. Bars represent Q, the amount of heat developed in 10 min, 0.5 ml0.002% sugar, 1 ml microorganism suspension, 25°C aerobic conditions. Brackets represent the standard deviation from five repeats.

so is its AoH value. The raw data from experiments represented by Figure 5 give a reasonably close approximation, with error between 5 to lo%, to the molar heats obtained from the more elaborate determination of AOH. In short, a molar heat of metabolism determined from the 0.002% sugar concentration conditions may often be adequate for certain practical purposes.

Molar Differential Heats of Metabolism of Monosaccharides by E . coli B15

The values of AoH were determined for aerobic and anaerobic conditions, at 25"C, from plots of the original data represented by Figure 6, according to the definition, eq. (4). The anaerobic or aerobic conditions, the cells, the substrate, and the temperature are specified. The AoH values are summarized in Table I. (The temperature increase in the reaction vessels at maximum power production after mixing is at most only a few thousandths of a degree, incon- sequentially small. Therefore the temporal temperature is assumed constant at 25°C for practical purposes.)

Occasionally in the anaerobic experiments (N, purged condi-


tions), at the lowest sugar concentrations, of the order of 0.0005 to 0.001% concentration, the heats are apparently high. Such a point is shown in Figure 6 for 0.001% glucose. Perhaps residual traces of oxygen remain, which obviously tends to cause some aerobic metabolism. Aerobicity generates a much larger characteristic heat than anaerobicity . Therefore for the anaerobic case, the AoH values are more desirable than individual AH,,, values, but more work is involved in obtaining AOH.

The number of sample measurements varied from 9 for glycerol to 49 for glucose, in the aerobic systems. From 12 calorimetric runs for fructose to 28 runs for mannitol were performed for the anaerobic case. Between 21 and 24 combustions were determined for each hydrocarbon, in the case of all four hydrocarbons. For both glucose and mannitol, there appeared to be no real difference between data from use of glucose (or mannito1)-grown cells, and cells grown on other sugars, which were also used to combust glucose

t z W a d n a

0.000 0.004 0908 % BY WEIGHT SUGAR IN 0 . 5 0 m l ALIOUOT

Fig. 6. Apparent molar heats of metabolism for glucose under aerobic and anaerobic conditions, as a function of concentration of the sugar before mixing. Ex- trapolation to c -+ 0 gives AoH, the differential molar heat of metabolism under zero growth conditions.

1262 LOVRIEN ET AL.

and mannitol. In short, the same or very nearly the same heat was produced for glucose and mannitol combustion for all E. coli B/5, regardless of the carbon source used for growth. Table I also gives the quotient AoH/n, where n is the number of carbon atoms in the metabolite, for relating heat generation to molecular structure.

Anaerobic Metabolism of Monosaccharides b y E . coli

Anaerobic conditions were imposed by purging with humidified gases (N2) by means of the plumbing shown in Figure 2. Because of the general importance of glucose fermentation and the place of E. coli in microbial technology, we report some of the heats obtained under anaerobic conditions in extenso. Table I1 shows some of the data obtained at various glucose concentrations up to 0.010% glucose. The 0.001% glucose level gives an anomolously large value, for reasons described above. This tends to happen with other sugars as well. Under the conditions used, 30 nmol sugar are metabolized at the 0.001% level. We recommend using 0.002 to 0.005% concentrations and averaging. The final result (leaving out the 0.001% heat) yields a value of (-)34 2 6 kcaYmol for glucose, c = 0.002 to 0.010%. Anaerobic heats, the AoH values for other monosaccharides, are compiled in Table I.

There is a possibility, as in the case of glucose oxidase,16 that

TABLE I1 Anaerobic Molar Heats for E . coli B/5 Glucose Metabolism for

Individual Sugar Concentrationsa

D-Glucose concentration before mixing - AHa,,

(%I (kcaVmol)

0.001 0.002 0.003 0.004 0.005 0.006 0.007 0.008 0.009 0.010

67 f 12 3 3 2 3 442 4 4 0 + 8 32 f 12 3 6 2 5 3 3 2 5 2 6 2 1 2 8 2 7 3 2 ? 6

Average AH,,, = -34 f 6 kcaUmol

"AU values are exothermic. Final average does not include the 0.001% value.


there might be a preference for certain anomers. In an equilibrium mixture of glucose, the alp ratio of anomeric forms is nearly 2. Therefore the heat of mutarotation might be involved. However, for glucose in solution this enthalpy is small, only about 0.16 kcall mol,* and hence may be neglected.

Aqueous Aromatic Hydrocarbons in the 10-80 nmol Range: Metabolism and Calorimetric Combustion

The molar enthalpies of aerobic metabolism of benzene, toluene, naphthalene, and 2-methylnaphthalene were determined. At the concentrations that were used, combustion proceeds rapidly and can therefore be monitored nearly as conveniently as for sugars. Figure 7 reproduces one of the scans from toluene metabolism, by Acinetobacter which was grown on toluene. Benzene and toluene combustion rates by Acinetobacter are quite comparable, when the cells were grown (then washed and starved) on the carbon source of interest. Metabolism of naphthalene and 2-methylnaphthalene also proceeded rapidly in the specified concentration ranges. The microorganism Ps. putidu was used for the naphthalene hydrocarbons. With the naphthalenes, maximum power is produced in less than 1 min, and all the heat is evolved in 8- 10 min. At the substrate concentrations that were used, the cells probably were not saturated with hydr~carbon,~ in keeping with the aims here which are to determine AOH.

2 3 4 5 6 Time, minules

Fig. 7. Calorimetric response in production of exothermic heat from aerobic metabolism of toluene by Acinetobacter B-I; 5 x 108 cells washed and starved 2 hr before use; 40 nmol toluene, pH 7.0, buffer capacity 5 X 10" standard units, 25°C; 7.2 mcal of net heat. Reference: microorganisms mixed with buffer containing no toluene.

I264 LOVRIEN ET AL.

Figure 8 plots the completed data for heats of Ps. putida metabolism of naphthalene and 2-methylnaphthalene. The slopes of the plots give the AH,,, values. Since the plots are quite linear, in the ranges of less than 70 nmol hydrocarbon, the AH,,, values are considered to be good measures of AOH. The latter values are listed in Table I

The restrictions in metabolite molecular structure which pertain to the carbon source which the bacteria may utilize are rather narrow. This is expected from many previous growth studies in- volving aromatic hydrocarbon^.^.^^ Our naphthalene-grown Ps. putida cells readily utilized 2-methylnaphthalene. However, no heat was obtained when the same cells were mixed with 2-ethylnaphthal- ene, acenaphthene, or phenanthrene.

Very Slow Metabolism: L-Arabinose

If bacteria metabolize a substrate extremely slowly, power production will be very small. Consequently, little or no signal will be available from the calorimetric experiment, even if the overall heat, the power integrated over a very long time-were appreciable. In

Mdas HYDROCARBON X I 0 a

Fig. 8. Heats of aerobic metabolism of naphthalene and 2-methylnaphthalene, by naphthalene-grown Ps. putidu, 5 x 1oD cells in each determination. Slopes give the Aofi values, (-)810 k 30 kcal/mol for (0) naphthalene, (-)652 2 15 kcaYmol for ( x )

2-methylnaphthalene, 25T, pH 7.


Figure 4, the process of adaptation is shown, wherein power generation increases markedly some time after mixing. In the case of L-arabinose, metabolism is so slow that appreciable power production for calorimetric measurement is not produced even after adaptation. Escherichia coli B/5 were grown over a period of two days on L-arabinose as the sole carbon source. Of course the bacteria metabolized the L-arabinose, since they grew on it. Upon harvesting the cells and washing them, the L-arabinose-grown cells rapidly produced heat upon mixing with glucose and mannitol. The aerobic molar heats were -271 ? 25 and -327 ? 22 kcal/mol, respectively, for these two sugars. However, the L-arabinose-grown cells produced nearly zero heat (within experimental error) with L-arabinose. What is seen in heat generation is governed by rate processes with this particular sugar. Arabinose is regarded as an "unnatural car- bohydrate" in E . coli metabolism. Although certain strains can grow on this monosaccharide, they are likely to be deficient in enzymes involved in the early catabolic steps, perhaps a kinase or a phosphoarabinoisomerase."

DISCUSSION

There are two main categories for use of data from calorimetric combustion of aqueous substrates: 1) In fermentation engineering when scaling up involves production of heat which must be re- moved, and in applications such as the Vacuferm process1* whereby fermentation exothermic heat is conserved to help balance the en- dothermic heat of distilling off product. 2) As a method for monitoring microbial metabolic processes.

Calorimetry of anaerobic metabolism has particular attraction because of the difficulties in handling certain anaerobic systems.13 Initial velocities of metabolism and the response toward various carbon sources according to the state of the cells may be discerned rapidly, in the seconds to minutes ranges. We have carried out aqueous combustions of lower alcohols such as methanol and ethanol in submillimolar concentrations, and investigated the use of metabolic inhibitors and cell surface perturbing compounds5 by means of microcalorimetry. Mou and Cooney14 have discussed a number of other facets of the method as it applies to fermentation technology.

The heats from aqueous naphthalene and from aqueous glucose metabolism shall be compared with the heats of complete combustion to C02 and H 2 0 in bomb calorimetry, at 25" C. Since the

I266 LOVRIEN ET AL.

reference states of both reactants and products are different in the two methods, corrections have to be inserted. For naphthalene (CI&) according to Cox and Pilcher,I5 in bomb calorimetry:

C1,,H81x,al) + 120z (r) + 10C0, tr, + 4HD([, AHzg8, = -1230 kcaymol

The heats of formation of aqueous CO, and 0, are given by Wil- hoit . I 6 Conversion of this reaction to aqueous conditions requires calculation of the heat of transfer for naphthalene: C10H8 (x,a,) -+

CIJ-IslaQ,. This was done via use of the heat of fusion," A H m = +4.6 kcal (since AHm = T O , ) , and that transfer of liquid CloH8 to water is likely nearly athermal, on the basis of the values for a number of aromatic compounds.18 Accordingly,

claHIdq) + 1 2 0 ~ ~ ~ ~ ~ ) + ~ O C O ~ , ~ ~ , + 4 ~ , 0 ( ~ ,

AHzg8, = (-)1237.2 kcal/mol

This value is compared to the aerobic AoH value of (-)810 kcali mol for the Ps. putida metabolism of naphthalene. The fraction of naphthalene's heat content that is conserved is (1237 - 810)/1237 = 0.35. Judging by the average levels of coenzymes normally contained in many cells,1g it seems unlikely that the corresponding amount of carbon that is conserved can be accounted for as simply as "priming metabolism" for buildup of coenzymes, unless abnor- mally large amounts of coenzymes are initially made. Some carbon likely goes elsewhere, presumably into biosynthesis. However, enough CO, and heat are rapidly produced to allow monitoring of the response by starved cells.

The bomb calorimetric heats of combustion of benzene and toluene are -781 and -935 kcalimol. The maximum heats of combustion in water are calculated to be -781 and -943 kcalimol, respectively. Acinetobacter aerobic combustion gave - 191 and - 188 kcali mol. Hence the fractions of hydrocarbon total heat content that were conserved appear to be 76 and 80% for benzene and toluene, respectively.

For glucose, the thermochemical values are:16

ca1 '206 + 60!2 * 6co!2 (d(l) + 6H20(/)

A Hzg8, = - 684 kcallmol

From a material balance cited by Roberts et al. ,20 45% of the glucose metabolizes to C02, and about 16% forms acetate:


Approximately 30% may be used in synthesis, and 9% forms other products. The acetate formation reaction has an enthalpy of (-)42.2 kcalimol glucose. According to an estimate by Morowitz,21 E . coli cell synthesis from glucose has a "theoretical" enthalpy (based on bond distribution values) of (-) 124 calig glucose or about (-)22 kcal/mol glucose. The other products cannot be precisely evaluated in their heat production since they are undesignated, but they probably do not produce large heats. (e.g., the glucose + ethanol + 2C0, reaction would normally produce about -33 kcal enthalpyi mol, and therefore about -3 kcal enthalpy in the present case).

Upon adding the foregoing values together, the aerobic utilization of glucose by E . coli should produce approximately (-)322 kcalimol with growth and (-)315 kcalimol without growth. In the limit for determination of AOH, presumably there is little growth. Therefore we compare the (-)3 15 kcal value with our observed AoH = (-)304 2 18 kcalimol glucose. The numbers may be fortuitously close. In any case, our 14C-labeled glucose results5 with E. coli aerobic utilization produced close to 3 CO,/glucose, consistent with the material balance just cited. The heat from that pathway is large relative to the other contributions, and dominates the final values.

For anaerobic fermentation, one of the simplest overall reactions is that outlined by Woodz2 (see also DoelleZ3):

2 glucose + H,O -+ 2 lactate + acetate + ethanol + 2C0, + 2H,

All the heats of formation (in q u o ) are also given by Wilhoit.lG These yield an estimated heat of -20.2 kcal/mol glucose, and may be compared with our value of -34 2 3 kcalimol for anaerobic metabolism.

The quotients AOHh for the aerobic metabolism of sugars and aromatic hydrocarbons are listed as part of Table I; n is the number of carbon atoms in the molecule. For the sugars, A 0 H h varies by a factor of nearly 2 , comparing D-xylose [(-)36 kcal/mol carbon atom] to the sugar alcohols [( -)58-( -)67 kcaVmol carbon atom]. The three sugar alcohols are of course the most reduced of the sugars. The aromatic hydrocarbons also exhibit a considerable spread in both AoH and AOHh values. Evidently, the microorganisms that were used for combusting naphthalene and 2-methylnaphthalene conserve less of the available energy than can the benzene- and toluene-utilizing bacteria.

Oxygen consumption rates for E . coli have been quoted by Har- risonZ4 for the respirometric growth rate, Qo,, and those needed simply for maintenance, Mo,. Generally, Qo, >Mo, by a factor of

I268 LOVRIEN E T AL.

about 10. These rates have been used as part of the basis for some of the current thinking about energy conservation in microbial metabolism. Directly measured heats are measures of waste enthalpy, and reflect oxygen consumption. Hence we compare the Qo, values, with what the AoH quantity would predict. For glucose, Qo, is 5 mmol O,/g dry cells/hr, at 35°C. Using 4 x 10l2 cells/dry g, we have 3 0, consumed/mol glucose, and a production of (-)18 mcal heat from 2.5 x lo9 cells in 400 sec. Hence we calculate Qo, = 2.6 mmol O,/dry g/hr, all at 25°C. It is likely this would be increased at 35°C to compare with the literature value. Thus the microcalorimetric heats monitor microbial metabolism in a manner comparable to respirometry. This was also illustrated by Mou and C ~ o n e y ' ~ in a Streptomyces niveus fermentation.

The concentration levels for the aromatic hydrocarbons in water with which one can deal in this way are comparable to those used in some gas chromatographic techniques, It remains to be seen whether selections of hydrocarbon-utilizing bacteria may be de- ployed as reagents to assay such systems on a broad scale, or for a large variety of aqueous hydrocarbons. Most of the thermochemical data for evaluating aqueous hydrocarbons in their function as growth substrates for protein production on the one hand (and as pollutants on the other hand), remain to be gathered.

This work was supported by the Minnesota Energy Agency and the University of Minnesota Graduate School. The authors are indebted to P. Chapman, D. Kunz, and J. Fuchs of this department for microbial cultures and much advice.

References

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Accepted for Publication October 15, 1979

Microcalorimetry of microorganism metabolism of monosaccharides and simple aromatic compounds

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