Calorimetric versus Growth Microbial Analysis of CellulaseEnzymes Acting on Cellulose
REX E. LOVRIEN,* KARL K. WILLIAMS,t MARK L. FERREY, AND DAVID A. AMMEND
Biochemistry Department, University of Minnesota, St. Paul, Minnesota 55108
Received 9 September 1987/Accepted 15 September 1987
Assay of cellulase enzymology on cellulose was investigated by two methods: (i) plate colony counting todetermine microbial growth and (ii) microbial calorimetry. These methods were chosen because they acceptraw samples and have the potential to be far more specific than spectrophotometric reducing sugar assays.
Microbial calorimetry requires ca. 0.5 to 1 h and 10 to 100 ,uM concentrations of cellulolytic lower sugars
(glucose and cellobiose). Growth assay (liquid culture, plating, colony counting) requires 15 to 20 h and ca. 0.5mM sugars. Microbial calorimetry requires simply aerobic metabolism, whereas growth assay requirescompletion of the cell cycle. A stripping technique is described for use in conjunction with the calorimetricmethod to enable separate analysis of the two sugars. Mixtures of glucose and cellobiose are equilibrated withEscherichia coli and spun out to remove glucose. The supernatant is calorimetrically combusted with Klebsiellasp. to quantitate cellobiose, and the same organism combusting the nonstripped mixture gives heatproportional to the sum of the two sugars. Calorimetry of cellulolysis products from individual exo- andendocellulases, and from their reconstituted mixture, was carried out to develop a microbial calorimetricmeans for demonstrating enzyme synergism.
Two methods are available for analysis of sugars in raw or
turbid samples by microbial means: microbial growth on
sugars and microbial calorimetry. Both methods can acceptraw samples otherwise not suitable for direct spectro-photometric assays when the samples are turbid (e.g., soil).However, focus on what sugar concentrations microbialgrowth versus microbial calorimetry can deal with is needed.The time needed to perform assays is of increasing concern
and so is the question of specificities for individual sugars inmixtures of sugars. We investigated the two principal lowersugars from enzymic cellulolysis in regard to these ques-tions: glucose and cellobiose. Cellulase enzyme concentra-tions and specific activities in animal digestion set many ofthe ranges or conditions within which glucose and cellobioseassays are needed to work.
In microbial calorimetry, one may use any microorganismthat can aerobically metabolize the sugars in question.(Microbial calorimetry can also be used under anaerobic,nitrogen-purged conditions; however, aerobic metabolicheat is generally much larger than anaerobic heat and is morerapidly generated [9]. Hence, for convenience, speed ofanalysis, and sensitivity, aerobic conditions are chosen forcalorimetric analysis.) For growth assays, any cell able togrow on the sugar in question and form distinct colonies onplates might be adequate. We used Escherichia coli B/5 forboth calorimetric and growth assays of glucose. A Klebsiellastrain for microbial calorimetry of cellobiose was also used,together with a technique we call stripping, for distinguishingglucose and cellobiose from cellulolytic production. Strip-ping is very simple. It is treatment of a mixed sample ofglucose and cellobiose with an organism that binds glucosebut not cellobiose, namely, E. coli. On centrifuging, glucoseis carried down with the E. coli. The supernatant, with itscellobiose but stripped of glucose, is injected into a heat
* Corresponding author.t Present address: Oral Roberts University School of Medicine,
Tulsa, OK 74137.
conduction calorimeter to burn the cellobiose by means ofKlebsiella sp. The three methods are summarized in Fig. 1.
MATERIALS AND METHODSE. coli B/5. E. coli was grown on Davis-Mingioli minimal
medium (3) and trace elements (1) in shake flasks at 37°C for6 h. The cells were harvested, washed three times bycentrifugation and suspension in minimal medium, and thenstarved for 1 to 2 h before use in calorimetry or for stripping.The carbon source was 0.1% glucose for raising initialcultures. Cell concentrations in culture were determined viaspectrophotometry using the conversion factor 2.0 x 109 +
0.2 x 109 x A66o units (1-cm cuvette) = cells per milliliter.Kkbsiella sp. KAY2026. Klebsiella sp. strain KAY2026
was donated by Georg Sprenger (20). It was grown on thesame minimal salts used for E. coli except that it was
supplemented with 4 x 10-4 M arginine and guanine (21).For microbial calorimetry of cellobiose, the cells were grownon 0.1% cellobiose and centrifugally washed in minimal saltsthree times.
Intermediate liquid cultures and growth assays. Glucoseconcentrations from 20 to 250 jig/ml were used with an
inoculate concentration of 9 x 106 cells per ml and incuba-tion times from 19 to 30 h. To decrease lag (13), 10 mMbicarbonate was incorporated in attempts to get cell growthproportional to glucose in submillimolar glucose concentra-tions. Serial dilutions were made from liquid cultures forplating out to obtain cell growth counts in culture from thenumber of colonies and the dilution factor (2).
Plate growth assays. Starting with diluted E. coli B/5 cells,100 to 1,200 cells per ml and 0.2 ml of inoculate were spreadon nutrient broth (Difco Laboratories) plates containing 9 gof NaCl, 9 g of nutrient broth, and 18 g of agar per liter.Incubation was for 18 h at 37°C. Triplicate plates were madefor each inoculate and averaged after counting.
Nonmetabolizable buffer for pH control in cellulose diges-tion. A buffer completely unusable as a carbon source forbacteria was used for pH control. Certain common buffersused in enzymatic cellulolysis, notably acetate, are readily
strip out glucose Calorimetry; cellswith E. coli; grown on cellobiosecentrifuge (Klebsiella)
FIG. 1. Summary of the growth method, the general calorimetricmethod for metabolically combusting both sugars, and the strippingcalorimetric method capable of resolving the two sugars.
metabolized by E. coli. When so metabolized, millimolarconcentrations of acetate and similar buffers produce heatthat is difficult to blank or cancel out in calorimeters.Accordingly, 30 mM cis-5-norbornene-endo-2,3-dicarbox-ylate buffer was used to prepare raw samples for calorime-try. This compound is not metabolized by bacteria (10). Itbuffers at about pH 5, which is optimal for cellulolysis. Itsuse ensures that heat production comes from cellulolyticproducts, not from metabolism of buffer carried in by raw orundiluted samples. The Aldrich Co. anhydride was con-verted to the buffering salt via hydrolysis with NaOH.
Cellulase enzymes produced here. Complete Trichodermareesei QM9414 cellulases, as well as the individual exo- andendoenzymes, were produced and assayed as in previouswork (14). Cellulolysis using the complete multienzymecomplex was performed at pH 5 and 50°C on Whatman no. 1filter paper substrate under Mandels and Sternberg condi-tions (11). Individual exo- and endoenzymes and reconsti-tuted mixtures of these two enzymes (0.5 mg [dry weight] ofenzyme per ml of digest on filter paper) were interacted withfilter paper for 2 h to produce cellulolysis digests.Commercial cellulase. Cellulysin brand cellulase was pur-
chased from Calbiochem-Boehring. Cellulysin was recentlycharacterized in several respects here (8). It contains ca.40% (dry weight) reducing sugars and ca. 20% dialyzablesugars and salts (8).
Nelson-Somogyi reducing sugar assays. Nelson and So-mogyi spectrophotometric analysis was performed as out-lined by Spiro (19). Our calibration slope, based on glucosestandards, was 6.3 x 10-3 A520 units/[centimeters x (nano-moles of glucose per milliliter of final assay volume)].Heat conduction calorimetry. Two batch mixing instru-
ments, which were constructed here and described before indetail (4), were used. These calorimeters measure heat from1 to 100 mcal (1 cal = 4.184 J) with ±3% precision andaccuracy using 0.5- to 2-ml samples and cell suspensions. Airin the samples plus air in the mixing vessel headspacesprovided the ca. 100-fold excess oxygen necessary to main-tain an aerobic state after mixture of the samples and cells.The calorimeters are differential instruments that automati-cally subtract the heat signal of the reference sample (mix-ture of cells with buffer and air but not metabolite) from theheat signal of the main sample (mixture of cells with buffer,air, and metabolite). The two compartments of the referencewere loaded with 1.0 ml of buffer and 2.0 ml of cells (2 x 109
to 5 x 109 cells per ml), respectively. The two compartmentsof the sample vessels were loaded similarly, except that the1.0-ml sample placed in the small compartment containedthe compound to be analyzed, the sample to be combusted.Calorimetric initial response time is 3 s, full-response risetime is ca. 10 s, and heat out-conduction relaxation time is 40s. Because the velocities of microbial uptake of sugars andsubsequent metabolism are appreciably slower than the heatconduction rates of the calorimeter, the apparent velocitiesof heat generation seen on the recorder are set almost solelyby microbial uptake and metabolic velocity. The area undereach recorded output is the integral of power with respect totime, which is directly proportional to the overall heat, Q.Calibration of the instruments was performed under bothwet (Tris hydrochloride neutralization) and dry (electricresistance heating) conditions in the 0.2- to 50-mcal range. Amain precaution is sterilization of the interior of the mixingvessels before they are reloaded with new samples. Anybacteria left in the compartment containing the compound tobe analyzed quickly combust the compound in the 10- to20-min wait needed to establish the base line before mixing.Consequently, the vessels were rinsed with 70% ethanol andthen sterile water after each unloading.Temperature during calorimetry. The temperature inside
the calorimeter for all measurements was 25°C.Stripping procedures. Cells to be used as strippers were
washed three times with either minimal salts or, whensamples were to be used for the Nelson-Somogyi assay, 0.15M NaCl to avoid reducing compounds. After washing, cellswere pelleted by centrifugation and the supernatant wasdiscarded. Samples were added, vortexed, incubated at 37°Cfor 10 min, and then iced for 5 min. Stripper cells were spundown for 10 min at ca. 3,000 x g. Stripped supernatant waswithdrawn and kept on ice for calorimetric analysis. Forstripping samples with initial volumes of 2 to 5 ml, 10 x 109to 15 x 109 cells were normally used before calorimetriccombustion of a 0.5- or 1-ml sample from the 2- to 5-mlsupernatant.
RESULTS
Assays of glucose based on growth ofE. coli B/5. On variousglucose concentrations greater than about 0.5 mM, new cellsare produced in proportionally increasing amounts, giving aplot of the kind shown in Fig. 2. Inoculates of 106 to 107viable cells per ml were used for the intermediate liquidculture stage. However, in each set of data leading to such aplot, the concentration of cells in each inoculate was fixed.An apparently linear relation, log net new cells versus logglucose concentration in the intermediate culture, was ob-tained from these relatively large glucose concentrationsabove 0.5 mM.
Decreasing the glucose concentration to the 0.04 to 0.07mM range produced countable new growth in 20 h at 37°C.However, fluctuations and inconsistency in the number ofcounts from triplicate cultures became large. Below 0.05 mMglucose, failure (region F of Fig. 2) or extreme variabilitywas obtained. The range down to ca. 1 ,uM glucose wasinvestigated.
Failure to grow in 1 to 50 ,uM glucose under the foregoingconditions could be ascribed to excessive inoculate size(which would use up all available sugar before growth oreven maintenance could be supported). Therefore, a fixedglucose concentration of about 10 ,uM (ca. 20 ,ug/ml) wasused and the concentration of the inoculate was varied.(Each inoculate was evaluated by serial dilution-enriched
FIG. 2. Net production of E. coli B/5, from inoculates having 106to 107 viable cells per ml as determined by colony counting, as a
function of glucose concentration in the intermediate liquid culture.Area F, Region of glucose concentration below 6 p.g of glucose per
ml, giving failure or wide fluctuations in net new production. Thethree kinds of symbols represent three independent determinationsstarting from different inoculates.
plate colony counting in determination of the concentrationof cells as viable cells.) Inoculation of >107 viable cells per
ml largely led to cell death under these conditions (Fig. 3).The 10 ,uM glucose disappeared in a short time, and even
(A)
0
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2 4 6 8 10
INOCULATE INITIAL CONCENTRATION
log(colls/mI)
FIG. 3. Production of E. coli cells in 19 h from an inoculateof constant size in 20 ,ug of glucose per ml-10 mM bicar-bonate-minimal medium.
8Ox107O
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0 100 200 300 400 500
GLUCOSE (ug/ml)
FIG. 4. Net growth of cells from glucose concentrations farabove those yielded by most practical cellulase enzyme assays.Percentages and open circles on the vertical axis, net growth fromglucose already present in commercial Cellulysin cellulase prepara-tions (8), not produced in cellulolysis.
maintenance likely was not supported. With inoculates ofbelow 103 to 104 viable cells per ml, the plot of total cellspresent after 19 h of liquid culturing time decreased to zeroand so, accordingly, did the number of net new cells. Thus,there is a "window" of conditions, designated the usefulassay range in Fig. 3, outlining the inoculate concentrationsof viable cells that are usable for glucose concentrationassays.Optimum viable cell concentrations in inoculate, there-
fore, are in the region of 6 x 106 cells per ml. The responseof such an inoculate to various glucose concentration is seenin Fig. 4. The plot can be varied and perhaps improvedslightly by using bicarbonate in the medium (13) to give moresensitive response to glucose below ca. 0.5 mM glucose, thatis, to shorten lag time. However, it was not possible tomaneuver the conditions to make E. coli growth assaysapproach microbial calorimetric capability in this regard.Namely, to assay far below 0.5 mM glucose. The opencircles marked on the vertical axis of Fig. 4 and the percent-ages noted in Fig. 4 give apparent glucose concentrations(dry weight percentage) of Cellulysin commercial cellulase.This commercial enzyme preparation contains considerablereducing sugars, as noted in previous work (8), that is, asseen by growing E. coli using simply the enzyme sample asa carbon source, the product of control experiments fromCellulysin-cellulose digestion minus the cellulose. Thesepercentages, averaging ca. 46% reducing sugars in Cellulysinas glucose, agree reasonably well with the 40% sugarsdetermined before (8) via reducing sugar assays on Cellu-lysin.The foregoing results were obtained from 19 to 20 h of
growth in liquid culture after inoculation. Lengthening timesor shortening them (to ensure log-phase growth) did notimprove growth assay capacity or range. Attempts at growthfor longer than 25 h, even with relatively large glucoseconcentrations, gave 50% or larger decreases in cell produc-tivity, indicating cell death during the longer interval. Withinthe 20-h time, our yields were close to 0.70 mg (dry weight)of cells per mg of glucose consumed, which compares wellwith the values of Roberts et al. (17).
Microbial calorimetry of glucose and cellobiose. Figure 5illustrates calorimeter recorder outputs for metabolism of aglucose-cellobiose mixture (80 nmol of each sugar) before
0 O 00?0ooo0oo000ooooqoo oooopoooooooqooooooopoooooqoooc
0 2 4 6 8 10 12
TIME (minutes)
FIG. 5. Calorimetry of metabolism of a mixture of cellobiose andglucose before and after stripping, producing 107 and 30 mcal ofheat, respectively. Klebsiella sp. grown on cellobiose was used incalorimetric combustion; E. coli B/S grown on glucose was used forstripping glucose from the same mixture. Tris-H+ calibration illus-trates the velocity with which the instrument responds to instanta-neously generated heat.
and after stripping. Similar heat outputs for individual sugarsand other classes of compounds were reported earlier (9). Awet calibration trace is included in Fig. 5 to show theresponse of the instrument to heat generated instantaneouslyby mixing Tris buffer and a very dilute sample of HCI. Thedata in Fig. 5 pertain to Klebsiella sp. strain KAY2026,which can metabolize both glucose and cellobiose. Whenboth sugars were present, heat generation appeared bi-phasic. This occurred not because of competition but be-cause cellobiose was metabolized somewhat more slowlythan glucose. With the eight different species of bacteriawe've examined (4, 9), including Klebsiella sp., g'ucose wasaerobically metabolized to completion in 300 to 500 s when aconsiderable excess of cells was present to take it up.Disaccharides, in general, are metabolized more slowly (500to 800 s) under conditions of ca. 2 to 100 FtM disaccharideand 2 x 109 to 5 x 109 cells per ml. These relative rates wereapparent for the mixture of glucose and cellobiose (Fig. 5,upper trace). When the same mixture was stripped before-hand with E. coli cells, glucose was quantitatively removedfrom the mixture, giving the lower calorimetric trace ofpower versus time after mixing. Integrated over time (ab-scissa), the area under each envelope is the overall heat. Inthe example shown, 107 mcal of heat was obtained from themixture of 80 nmol each of cellobiose and glucose. Cello-biose alone, stripped of glucose, gave 30 mcal. Thus, Fig. 5illustrates the feasibility of the stripping (Fig. 1, method 3)via Klebsiella calorimetry of the glucose-cellobiose analytes.The measurement is completed in 15 to 20 min.
Efficiency of glucose stripping from glucose-cellobiose mix-tures by E. coli. The stripping step for removing glucose fromglucose-cellobiose mixtures needed optimization with re-spect to the ranges of cell concentrations required to removeglucose quantitatively. Figure 6 shows how E. coli func-tioned in two kinds of cases. On the left of Fig. 6 are data(bar graphs) obtained by varying cellobiose from 200 to 1,000
nmol with a fixed number (5 x 109) of E. coli cells used tostrip glucose from a 3-ml volume. The vertical bars show theamount of cellobiose recovered, measured by microbialcalorimetry using Klebsiella sp. to combust the samples.Stripped and nonstripped samples are compared. The threepairs of data on the left show that 5 x 109 E. coli cells maybind a modest fraction of cellobiose (ca. 5%) from the largerconcentrations of cellobiose. However, these concentra-tions, of ca. 1,000 nmol/3 ml (0.3 mM cellobiose), areconsiderably larger than that necessary for calorimetricanalysis of cellobiose. E. coli binds very little cellobiose atconcentrations required for calorimetric analysis.The set of bars on the right shows that, for 196 nmol of
glucose per ml, a range of E. coli cell concentrationsbetween 8 x 109 and 17 x 109 cells per ml is adequate forremoving such a concentration of glucose. These glucoseconcentrations also are larger than that needed for microbialcalorimetric analysis. Excess glucose loads and long incuba-tion times during stripping are undesirable. E. coli excretesmetabolites, depending on oxygen concentration, notablyacetate and ethanol. If these build up, considerable microbialcalorimetric heat may ensue from aerobic metabolism ofthem. Hence, stripping needs be limited to the times indi-cated above so that simply uptake, not complete metabo-lism, occurs.
Microbial calorimetry of cellulase-cellulose digestion. Cel-lulose was digested by complete cellulase enzymes for up to2 h. Half of the digests were stripped with E. coli cells andthe other half were not. Total heat production from Klebsi-ella sp. metabolism was plotted as a function of enzymaticdigestion time.
Since 3-glucosidase was present in complete cellulases,much of the cellulolysis product was glucose. Glucose wasrapidly, nearly quantitatively, removed by E. coli stripping.Klebsiella sp. combustion of stripped samples gave littleheat, which was expected if most of the lower sugars wereconverted to glucose. The slope of heat generated versusdigestion time for nonstripped samples was 16.6 mcal of heatper ml of original digest before dilution per min of cellu-lolytic digestion time. The slope for the same digests after E.coli stripping was 0.3 in the same units. Therefore, under thecellulolytic conditions outlined in Materials and Methods,
cc
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0
0
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aUIC
0
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0
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zz
1030 618 206 0 8 x 109 17x 109
NANOMOLES CELLOBIOSE NUMBER OF E. cog CELLS:ADDED, 5 x 109 E. coO CELLS 196 NANOMOLES
GLUCOSE/ml ADDED
FIG. 6. Efficiency of stripping. Left half, a fixed number of E.coli cells refrained from binding appreciable cellobiose untilcellobiose concentrations became 10 times as large as those nor-mally obtained in cellulolysis. Right half, ability of variable numbersof E. coli to strip a fixed amount of glucose from a 5-ml total volume.
FIG. 7. Correlation of heat production (Klebsiella sp.) withNelson-Somogyi reducing sugar assays of cellulolysis digests offilter paper cellulose.
about 1 min of cellulolysis on crystalline cellulose with 0.5mg of moderately active complete cellulase produces abouttwo to five times as much saccharide as that needed for acomfortable range of measurement via microbial calorime-try. During shorter cellulolysis times, the dominant sugar isglucose, when ,-glucosidase is active and not product inhib-ited.Reducing sugar assays are much used for monitoring
cellulolysis. Therefore, heat production (nonstripped sam-ples) was plotted against estimates of reducing sugar asglucose in a Nelson-Somogyi assay of the same digests fromcellulolysis for up to 1 h (Fig. 7). The longer digestion timesneeded to produce adequate sugars for Nelson-Somogyiassays engender great calorimetric heat (Klebsiella metabo-lism) if not diluted i.e., several hundred millicalories of heatand far off scale. Accordingly, the digestion samples werediluted before microbial calorimetry to keep the values onscale. The dilutions were taken into account and incorpo-rated in the ordinate of Fig. 7. That is, each ordinal valuegives the heat content (microbial combustion) of the corre-sponding digest before dilution. Most of the dilution factorsfrom cellulolysis (0.5 mg of enzyme, 50 mg of filter paper, 0.2to 1 h) were diluted by 50-fold to give 1 ml of sample suitablefor batch calorimetry. Table 1 summarizes the data fromboth spectrophotometric reducing sugar assays and Klebsi-ella calorimetric combustion assays of six cellulolyses under
TABLE 1. Sugar apparently remaining in stripped versusnonstripped products from cellulase digests of filter paper
cellulose as analyzed by conventional reducingsugar and by microbial calorimetric assays
Glucose concn (pLg/ml) Heat production (mcal/ml)Digestion by Nelson-Somogyi Ha rdcin(cUiconditions reducing sugar assay by calorimetric assay(°C, min)
FIG. 8. Calorimetry of Klebsiella sp. combustion of digests fromindividual exo- and endocellulase enzymes and from recombinedmixtures of these two enzymes to reconstitute a cellulase lackingP-glucosidase activity.
various conditions. These data show how stripping per-formed. Stripping removed most, but not all, of the reducingsugars from the digests seen via spectrophotometry. Strip-ping removed nearly all glucose. Because almost no heat wasproduced after stripping, little cellobiose should have beenpresent, since cellobiose-grown Klebsiella sp. was used forcalorimetric combustion.
Individual exo- and endoenzyme cellulolysis. Synergismbetween the exo- and endocellulase enzymes is important incellulolysis when the substrate, such as filter paper, is verycrystalline. Well-purified individual exo- and endocellulaseenzymes from Trichoderma sp. were produced and exam-ined for purity via four criteria outlined before (14), namely,via slab gel electrophoresis, by the criterion of synergism oncotton (nearly perfectly crystalline), by identity assays de-signed here, and by their weight and molar absorptioncoefficients. Evidence of enzyme purity is important inreconstitution of cellulase mixtures in attempts to create asystem free or nearly free of ,B-glucosidase which producesmuch larger proportions of cellobiose than complete, -glucosidase-containing cellulase.
Mixtures of the two isolated enzymes, exoenzyme plusendoenzyme, as well as the individual enzymes of equal-weight concentrations, were digested separately with cellu-lose under standard digestion conditions. Figure 8 and Table2 show heats of Klebsiella metabolism of digests and theeffect of stripping with E. coli versus no stripping. Digestsfrom these ,B-glucosidase-poor enzyme systems still pro-duced considerable heat from their stripped digests andtherefore appeared to have mainly cellobiose insofar asKlebsiella combustion was a criterion. This behavior was inmarked contrast to the ,3-glucosidase-containing completecellulases, which produced digests that were mostly strip-pable via E. coli cells which remove glucose (and therefore,as reported above, produce very little heat after stripping).Either individual enzyme alone, the exoenzyme or theendoenzyme, even in concentrations of up to 0.5 mg (dryweight) of enzyme per ml of digest, produced very littlemetabolic heat via Klebsiella calorimetry (Fig. 8, lowest
TABLE 2. Metabolic heat levels (Klebsiella sp.) of cellulolysisproducts from individual-enzyme treatment and from treatment
with reconstituted synergistic mixtures of the endo-and exocellulases of filter paper cellulose
with 500 p.g of the enzymes
Incubation Calorimetric heattime (min) (mcal/ml of digest)
30 + + 75860 + + 65890 + + 800120 + + 996
120 - + 70120 + - 0120 None None 8
plot; Table 2). This result needs to be gauged with respect totheir expected behavior pertaining to synergism, which isdiscussed below.
DISCUSSION
Although data on heat for aerobic metabolism are lackingfor most compounds, enough is known (9) to make estimatespossible regarding how microbial calorimetry should com-
pare in applications of this kind, that is, in carbohydrateanalysis. Under aerobic conditions, glucose is metabolizedby E. coli with a heat of (-)304 + 18 kcal/mol (9), withexcess cells, oxygen, and 5 to 500 ,M glucose. Close to thesame heat for aerobic glucose metabolism has been mea-sured here for numbers of other bacteria, Klebsiella sp.,Pseudomonas sp., and several soil isolates. Gordon et al. (6)measured (-)295 to (-)310 kcal/mol of glucose, using Vibrioalginolyticus. Constant heat for this metabolism, good for allconcentrations, is not expected. When there is sufficientmetabolite to supply the later or branched (22) sectors ofcarbon utilization, e.g., if much glucose is routed to glycogenor polyhydroxybutyrate, the overall aerobic heat can beexpected to be lower and, of course, far lower if an anaero-bic state sets in. However, under the conditions of interesthere, realistic, albeit rough, estimates are useful concerninghow much analyte (glucose) is required. Viz., if one has 0.2-to 1.0-ml samples and a useful calorimeter range of 2 to 50mcal of heat, (-)304 kcal heat per mol of glucose indicatesthat 5 to 200 ,uM glucose (5 to 200 nmol of glucose per ml) inthe sample is suitable. Comparison with average amounts ofreducing sugars in spectrophotometric assays may be made.If the initial sample is 1 ml and 1-cm cuvettes and generationof absorbancies from 0.10 to 0.70 are assumed, the followingamounts of carbohydrate are needed: dinitrosalicylate-basedmethod, 500 to 3,500 nmol; glucose oxidase, Nelson-Somogyi, and phenol-sulfuric acid methods, 30 to 200 nmolof hexose.Mountfort and Asher (12) and Pearce and Bauchop (16)
recently investigated rumen fungus cellulolysis in vitro onvarious substrates. It is useful to compare the microbialcalorimetric needs for lower sugars to get in measurementrange with the amounts of sugars provided by cellulolysis.Their cultures produced 0.004 to 0.006 U of overall cellulaseactivity per ml. (We converted their units quoted in micro-grams of glucose to the more standard micromoles of glu-cose.) On average, from their substrates, roughly 0.01 p.molof glucose per min per ml of culture was obtained. There-fore, in 10 min, roughly 100 nmol of glucose per ml can beexpected, an amount in the upper end of the range needed
for microbial calorimetry. These estimates, based on reduc-ing sugars reported as equivalents of glucose, do not de-scribe the proportion of cellobiose and glucose actuallypresent. Plainly, the combined microbial calorimetric andstripping methods can resolve these sugars simultaneously ina multichannel instrument of the kind commonly used now(4). Normally, the precision for determining sugars and otherlower-molecular-weight fermentation products, such as glyc-erol and acetate, via microbial calorimetry is about +3%.Depending on how well organisms can be confined to me-tabolism of particular substrates, comparable accuracies inanalysis can also be expected.The question of the specificity of microbial calorimetric
analysis hinges largely on how specific organisms can bemade for use on particular test compounds and on howsimple techniques like stripping can be developed. A fewcompounds, such as methanol and several substitutedphenols, can be expected to yield to calorimetric analysis byorganisms narrowly specific in utilizing them. Obviously,glucose is at the other end of the spectrum. Most organismscan use glucose, so its heat of metabolism will add to that ofany other analyte. At the same time, glucose is easy toremove via the stripping technique, leaving the remnantsugars more clearly quantitated upon use of the calorimetricstep.Two other aspects of microbial behavior bear on analysis
of metabolities and are part of the work reported above.Rather rapidly generated heat outputs of the kind shown inFig. 5 can be expected. Glucose uptake rates (5), shifts inAtkinson energy charge (18), and H+ extrusion-K+ transportoccur in the same time ranges, 300 to 600 s, with mostbacteria. Therefore, if a bacterium is adapted to a particularcarbon source, cellobiose, lactose, a phenol, it is not sur-prising that it generates its principal metabolic heat envelopein comparable times (in 200 to 500 s at 25 to 37°C). Since thistime frame holds for many bacteria, microbial calorimetryusing such bacteria can be expected to finish and give backthe completed data in a few hundred seconds. Bacteria notadapted to an analyte take much longer to combust calori-metrically, as in a case described before (9). E. coli wasgrown on glucosamine, isolated, and then mixed with glyc-erol. Lags of ca. 30 min elapsed before heat started togenerate because new growth actually had to occur insidethe mixing vessel before glycerol could be utilized. On theother hand, glycerol-grown E. coli completely combustedmicromolar glycerol in a few minutes after mixing.Aerobic heats of oxidation usually are much larger than
the characteristic heats of most other classes of reactions inwater. A referee asked why heats of hydrolysis of glycosidicbonds should not serve as well as metabolic heats for ananalytic basis. There are four main reasons why approachesbased on hydrolysis heat compare very poorly with aerobicmetabolic heat in this kind of application. Only one reasoncan be described here. The hydrolytic heats of organiccompounds in water are actually heat levels of group transferto solvent molecules and do not reflect chemical bondenergies (7). These heats of transfer, cum hydrolysis, arealmost always relatively small, if not athermal. The same istrue for esterification, alcoholysis, etc. in hydroxylic sol-vents (23). From zero to only perhaps 5 kcal of heat per molcan be expected from glycoside hydrolysis, as seen previ-ously (15). But heat levels caused by aerobic oxidation aremuch larger than those due to hydrolysis, at least 10 andmostly close to 100 times larger, both on a molar and on a percarbon atom basis (9). In sum, adequate sensitivity toglycosidic conversion for analysis based on heat production
is hardly expected from hydrolysis but is expected, andobserved, with aerobic metabolism.The results of Fig. 5 and 8 and Table 2 fit well with how the
individual depolymerizing enzymes of cellulases normallybehave when pure. When pure, the individual enzymes,even when used in 500-,ug amounts in volumes of a fewmilliliters, should produce very little metabolizable sugarfrom 50 to 100 mg of cellulose. On reconstitution of themixture of exo- and endoenzymes, a 500-,ug mixture (250 ,ugof each enzyme) produces relatively huge amounts ofmicrobially combustible sugar, and therefore heat, far offscale if not diluted. If either individual enzyme were cross-contaminated, calorimetrically combustible sugars certainlywould be seen, in parallel to the identity assays developedrecently (14). In sum, microbial calorimetric assay ofcellulolysis products from these enzymes provides a rapidmeans for assaying enzyme purity, capacity to exert syner-gism and quantitates the ratio of glucose to cellobiose.
ACKNOWLEDGMENTS
This work was supported by the Bioprocess Technology Instituteand by the Agricultural Experiment Station of the University ofMinnesota.
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