APPL MICROBIOLOGY, Mar., 1967, p. 319-324 Vol. 15, No. 2Copyright 0 1967 American Society for Microbiology Printed in U.S.A.

Pilot-Plant Production of Protease byAlternaria tenuissima

ARNE G. JONSSONDepartment of Microbiology, Agricultural College, Uppsala, Sweden

Received for publication 6 October 1966

Agar slants of a selected Alternaria tenuissima strain were illuminated to giveconidia suitable for further propagation. For production of protease with an optimalcaseolytic activity in the region of pH 8 to 10, the fungus was cultivated in steelfermentors with 6- and 60-liter working capacity. Maximal activity, 1.5 enzymeunits per liter, was attained in a medium based on liver after about 60 hr of cultiva-tion. The protease was secreted parallel with, or slightly after, the main growthphase. The process could be run favorably with a relatively low aeration rate. ThepH of the culture decreased during the process from 7.0 to about 6.3. This was theoptimal region also when pH was kept constant by automatic pH control. Optimaltemperature was about 28 C, which resulted in a maximal productivity of 0.057enzyme units per liter per hr during the protease secretion phase. Replacement ofthe liver in the medium with skim milk, meat scrap, or rapeseed oil meal resultedin a decrease of the protease yield.

Some fungal proteolytic enzymes have beenproduced from submerged cultures in industrialor pilot-plant scale: the acid-type protease ofblack Aspergillus species such as A. saitoi and A.usamii (5), the acid-type protease of Trametessanguinea (13), the alkaline-type protease of thephycomycete Mortierella renispora (14), threeproteases of A. oryzae (2), and others. In previousstudies (9), protease production by Alternariatenuissima in the laboratory scale (shake flasks)appeared so attractive that studies were continuedon a larger scale in steel fermentors. This wasdone in the present study to investigate some ofthe factors that influence the enzyme secretion bythis fungus and also to secure an appropriateprocess for protease supply for studies on themajor proteolytic enzyme, which has a maximalcaseolytic activity in the region of pH 8 to 10.

MATERIALS AND METHODS

Pilot plant. The pilot plant has been previouslyused for various microbial processes, e.g., in studieson utilization of agricultural wastes (7, 12) and forproduction of steroid-induced enzymes (3) and p-diphenoloxidase (G. Fahraeus, in preparation). Thefermentation equipment used consists of eight 10-literfermentors and one 100-liter fermentor. The 10-literfermentors have been described previously (11). Theywere used for 6-liter culture. The 100-liter fermentor,the main parts of which were supplied by Getinge-verken, Getinge, Sweden, is shown in Fig. 1. It isnormally used for 60-liter culture. It is designed andequipped principally like the smaller ones in order to

allow a close translation to the larger scale. Thejacketed fermentor is made of stainless steel, with aheight of about 800 mm and an inner diameter of 410mm. The container can be lolwered from the lid, whichis fixed to the wall. The entire equipment of thefermentor is connected to the lid. This includes theaxis for agitation at various agitation rates with a1.4-hp motor, illumination and viewing windows,and an assembly for pH, temperature, and antifoamcontrol. Seven ports are designed for aeration, inocu-lation, sampling, and inlet of antifoam, acid, andalkali. Air from two pressure blowers (Fabriks A.-B.Osmund, Uppsala, Sweden), each with a capacity of150 liters per min, is sterilized by passing jacketedstainless-steel filters with glass wool and fed throughtwo tubes fixed to the lid and ending with half-circularring spargers just under the impeller. These air tubesare connected with two steel rings bearing the three(50 by 600 mm) baffles. The fermentor is sterilized bypassing steam at 110 C through the air inlets for thenecessary time. The recording and control units, aswell as equipment for air and steam supply, are soconstructed as to be able to serve the similarly de-signed 1,200-liter fermentor of the pilot plant.

Cultivation. The medium normally used was thatselected in the shake-flask studies (9), which contained(grams per liter): liver (Difco), 4.0; glucose, 30.0;CaCOs, 5.0; KH2PO4, 1.0; MgSO4.7H20, 0.3; dis-tilled water, to 1,000 ml. (The pH was 6.2 beforesterilization.) The origin of the agricultural by-products used as substitutes for liver, meat scrap, andrapeseed oil meal has been outlined in an earlier re-port (7). Skim-milk powder was from Semper AB,Stockholm, Sweden ("Famos" brand).The strain selected, Alternaria tenuissima NRC

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FIG. 1. A 100-liter fermentor. The vessel is loweredfrom the lid with the main equipment of the fermentor.

E-34, was maintained as soil stocks. About 3 mg ofdried soil culture was inoculated on 10-ml agar slants(Sabouraud Glucose Agar, Oxo Ltd., London, U.K.)with 10-cm2 area. The slants were illuminated with a

daylight-type lamp (type 57223, Philips) at a distanceof 50 cm. After 7 days of incubation at 20 C, thecultures were suspended with a small platinum spatulain 8 ml of distilled water; 200-ml Erlenmeyer flaskscontaining 50 ml of Sabouraud glucose-peptonemedium were then inoculated with 0.5 ml of the sus-

pension and cultivated on a rotary shaker (110rev/min, 35-mm amplitude) for 2 days at 24 C. Eachof these cultures was finally used as inoculum foreach 1-liter Erlenmeyer flask containing 250 ml of thesame medium. After 2 days of incubation in the sameway, these cultures were used for inoculation of thefermentors with 100 ml of culture per 6 liters ofmedium.The following conditions were applied to the

fermentor cultures, unless otherwise mentioned:temperature, 28 C; agitation, 260 rev/min; aeration,0.5 liter per iter of medium per min. Silicon antifoamemulsion RD (Midland Silicones Ltd, Reading,U.K.), 1 ml per liter, was added before sterilization.ThepH was recorded but not controlled automatically.

Assay. Samples (10 ml) were withdrawn from thefermentors at intervals for assay and were filtered bydrawing the culture fluid into a pipette, the tip ofwhich was wrapped with a bit of cotton wool.

Protease was estimated as caseolytic activity atpH 9.5 by a modification of Anson's procedure aspreviously described (8). Absorbance at 660 mp, afterreaction with phenol reagent (E. Merck AG, Darm-stadt, Germany), was in this study determined in aSpectronic-20 colorimeter (Bausch & Lomb, Inc.,Rochester, N.Y.) after 10 min of developing. Theresults are expressed as enzyme units (EU), 1 unitbeing defined as that amount of enzyme whichliberated split products equivalent to -1 meq oftyrosine per min, under the conditions specified.

Glucose was estimated according to Hagedom-Jensen (Handbook of Chemistry and Physics, 32nded., p. 1524) and nitrogen by the micro-Kjeldahlmethod. The pH values of the cultures were auto-matically recorded and in stated cases controlled bya Speedomax recorder (model S, type G, Leeds &Northrup Co., Philadelphia, Pa., with a special con-troller design) by addition of 0.5 M NaOH or HCl.The standard error, m, was calculated according tothe formula m2 = S(x -x)2/n(n - 1).

Microscopy. Samples from the cultures werephotographed with a Zeiss Photomicroscope andAdox KB14 film.

RESULTSEffect of light on inoculation culture. For prop-

agation of the organism from the air-dried cultureto a culture suitable for inoculation of the fer-mentors, illumination at the agar-slant stepappeared important. Incubation in the dark re-sulted in a white mycelium with no conidia. How-ever, when the bottles with the slants wereexposed to light during incubation, the culturebecame green to black and formed abundantconidia (Fig. 2).

Figure 3 shows the result of such cultivation.After various incubation times, the culture wassuspended. Plate counts were made after 2 daysof incubation at 24 C, with 0.1 ml of suspensiondistributed on the surface of the same agar in a9-cm petri dish. Each point of the diagram in Fig.3 represents the mean from four independent

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PROTEASE PRODUCTION BY A. TENUISSIMA

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FIG. 2. Conidia of Alternaria tenuissimaE-34.

cultures, calculated per square centimeter oagar slant. Corresponding values at the tininoculation were 100 per agar slant. Cultiv;in light resulted in a 10- to 40-fold increase iplate count as compared with cultures kept idark. The maximal number of conidia and nlium fragments was reached in about 6 dayscorresponded to almost 100,000 conidia perof the illuminated slant. This level was ntained for at least an additional 8 daysmicroscopic examinations of suspensionsilluminated slants, a relation was observectween conidia and mycelial fragments Mcorresponded to the 10- to 40-fold increase fiin the plate counts. Conidia formation of (strains of A. tenuissima tested (NRC E-32, lE-33, and NRC E-36) was also strongly stlated by light, but one of these (NRC Iformed abundant conidia (70,000 per cm2)in the dark.When an agar slant with this high numb(

conidia was used as the starting material itpropagation work, shake cultures were obtawith a higher number of small-sized filamenpellets, a few millimeters in diameter, whichmore suitable as fermentor inoculum. Thependency on a heavy primary inoculum foiflasks was particularly evident when the xErlenmeyer flasks without baffles were used

Course of the process. The protease produ(process is illustrated in Fig. 4, which showsults from two representative cultures. Aftlag period of about 25 hr, the protease acdof the culture solution increased during the30 hr to 1.5 EU per liter. That is a productduring this latter phase of 0.05 EU per liteihr. As seen in Fig. 4, this phase correspondproximately to the growth phase of the fungreflected in the glucose utilization in the solu

co This was also observed visually, though a precisemeasure of the mycelium growth could not be ob-tained in this medium originally containing sus-pended liver and calcium carbonate. The main netutilization of dissolved nitrogen by the fungusoccurred, however, earlier in the process, afterwhich the nitrogen of the solution was constant asdetermined by the Kjeldahl method. The pHgenerally decreased slowly during the process, if

<2 no external pH control was applied.Examples of the microscopic appearance of the

mold from one of the cultures in Fig. 4 (0.5 liter0y of air per liter per min) is given in Fig. 5. No

general autolysis or other evident microscopicchanges of the mycelium seem to occur between

NRC incubation times of 41 hr (upper part) and 66 hr(lower part). At harvest, the heavy growth con-sisted of filamentous pellets (about 3 mm) in ausually dark-brown culture liquid. A certain

f the amount of growth on the surfaces of the fer-e. of mentor above the submerged culture alwaysation occurred.n the Effect of aeration. In preliminary experimentsn the it was noticed that aeration at the same level asnyce- has been frequently used in other processes, 1.0 to; and 1.5 liters of air per liter of culture per min, causedr cm2 a drastic decrease in the protease yield, as com-nain- pared with lower aeration rates. Figure 6 sum-s. In marizes the result of a closer study of the effect offrom various aeration rates from a total of 25 6-literI be- fermentor cultures. Aeration of 0.1 to 0.5 liter ofvhich air per liter of culture per min seems to be theound optimal region for production. This correspondsDther in the equipment used to an oxygen absorptionN.RC rate of 0.1 to 0.3 mmoles of oxygen per liter pertimu- min, measured by the sulfite method (1). Aera-E-33) tion rates within this region were therefore usedalso in the following work.er ofi thelineditouswere> de-r theasual

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FIG. 3. Plate counts ofsuspensionsfrom agar slants,with Alternaria tenuissima NRC E-34 kept in light(0) and in the dark (a).

VOL. 15, 1967 321

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JONSSON APPL. MICROBIOL.

temperature optimum of the process as performedwas indeed about 28 C (Fig. 8). In Fig. 8, themeans of four 6-liter fermentor cultures are

plotted for each incubation temperature. The

0 20 40 60 80

INCUBATION TIME (hr)

FIG. 4. Protease production by Alternaria tenuissimain 6-liter batches. Aeration, 0.1 (-) and 0.5 (0) literofair per liter per min.

Effect ofpH. When the pH of the culture wasallowed to vary freely, a steady, slow decreasefrom pH 7.0 to about 6.3 occurred during theprocess, with occasional exceptions during theearly period of incubation when a small increasesometimes was seen (Fig. 4). The effect ofpH wasstudied in 6-liter cultures kept at constant pHvalues throughout the process by automaticaddition of hydrochloric acid or sodium hy-droxide. Figure 7 shows the results from thesefermentors up to a process time of 50 hr, at whichtime the main part of the protease production wascompleted. These results suggest an optimal pHfor protease production of 6.3 to 7.0. At higherpH values, a larger volume of sodium hydroxidewas used to maintain the high pH, but also atlower pH values sodium hydroxide addition pre-dominated. As the region for maximal proteaseproduction coincided with the pH of the cultureliquor without external pH control, the processeswere generally run without such control.

Effect of temperature. The applied temperatureof the process, 28 C, had been relatively arbi-trarily chosen. It appeared, however, that the

FIG. 5. Culture of Alternaria tenuissima after 41 hr(top) and 66 hr (bottom) of cultivation.

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FIG. 6. Effect of aeration on protease yield fromAlternaria tenuissima. Standard error is indicated byvertical bars.

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PROTEASE PRODUCTION BY A. TENUISSIMA

standard error of the mean is about ±10%.Productivity was calculated as the net increase ofprotease during a phase (10 to 20 hr) of a cultureat each temperature at which this increase wasmaximal for the culture. The maximal proteaseyield had less meaning, as the proteolytic ac-tivity of the cultures at the two lowest tempera-tures still were increasing after 70 hr of incuba-tion. The maximal yield in 50 and 70 hr, however,gave the same result as the productivity, about28 C.

Use of agricultural by-products and translationto 60-liter scale. Before the translation of theprotease production process to 60-liter scale,some less-expensive agricultural products and by-products were studied as substitutes for liver as amedium component. Previously (9), a milkmedium in shake flasks resulted in almost as higha protease yield as did the liver medium, althoughthe enzyme was more rapidly inactivated. In the10-liter fermentors, however, the excessive foam-ing demanded a relatively large amount of anti-foam addition (4 g per liter) to a medium in whichthe liver had been replaced by 7.5 g of skim-milkpowder per liter. In this culture, the protease ac-

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FIG. 8. Effect of cultivation temperature on proteaseproduction by Alternaria tenuissima. Yields after 50(0) and 70 (A) hr of incubation, and maximal pro-ductivity (D.

tivity was low, maximally 0.1 EU per liter. Lessthan 0.1 EU per liter was obtained from a mediumin which the liver was replaced by 4.5 g of meatscrap per liter. In this culture, however, the sus-pended meat scrap tended to stick to the airsparger, thus reducing the air flow. The bestresult with substitutes tested was obtained withsuspended rapeseed oil meal (7.5 g per liter): 0.2EU per liter after 70 hr of incubation.Compared with these results, the liver medium

appeared advantageous and was used in processeson the 60-liter scale. These were aerated with only0.2 liter of air per liter per min, to compensatefor the higher aeration efficiency of this equip-ment. The general course of these processescoincided with values obtained in the smallerfermentors. Thus, 1.3 EU per liter was obtainedafter 48 hr (Fig. 4).

DISCUSSIONIn a monograph by Joly (6), Alternaria species

A* - have been distinguished as to their sporulationcapacity into two groups: those with a constant

f* \ sporulation (among others, A. tenuis) and thoseJ whose sporulation capacity declines during main-

tenance in pure cultures (among others, A.tenuissima). During less than 1 year in pure cul-

Il tures, most of the A. tenuissima strains studied6 7 8 by him lost their sporulation capacity. Directp H sunlight stimulated to a certain extent the sporu-

protease yield from Al- lation of one of his strains of this fungus. Othershr of cultivation (A). Net have found similar effects of light on some othery for maintaining pH at Alternaria species (4), though factors like mois-separate series of runs: ture and temperature have sometimes been

judged as more important (10). With the strain

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used in this study, the illumination effect on theformation of conidia was drastic and had to beconsidered to obtain submerged growth suitablefor subsequent propagation. The shape of theconidia seems to coincide with drawings byNeergaard (10) and Joly (6).The time used for the main process in the

fermentors, about 60 hr, is approximately thesame as used for production of protease fromblack Aspergillus species (5) and from Mortierellarenispora (14), but is less than half of that re-ported for Trametes sanguinea (13) and Asper-gillus fumigatus (8). Glucose utilization is rela-tively limited, and the concentration of glucosein the medium may be restricted without con-siderable decrease of activity, as judged fromprevious shake-flask experiments (9). The simplepicture of the nitrogen content in the culturesolution, showing a decrease only in the early partof the process, may be the complex result of ni-trogen utilization on one hand and enzymaticdigestion of the suspended liver plus enzymesecretion on the other. Course of productionand microscopic appearance suggest that the pro-tease secretion to a large extent parallels growthand is not associated with general autolysis of my-celium.

Like most other submerged processes, proteaseproduction by A. tenuissima appeared to be verydependent on a suitable aeration rate. Excessiveaeration rates resulted in lower protease yields.Optimal pH for production, 6.3 to 7.0, was lowerthan that for enzyme activity. The temperaturewas also of considerable importance for the pro-ductivity of the process.The activity of the culture solution at harvest

corresponds to about 0.5 EU per g (dry weight),if the remaining glucose content is subtracted.This can be compared with the activity of a com-mercial preparation of a bacterial protease usedas supplement in detergents, 0.8 EU per g (dryweight), as estimated by the same method.

AcKNowiLEDGMENwrSI gratefully acknowledge the valuable assistance of

Christina Johnsson.This investigation was supported by the Swedish

Technical Research Council.

LITERATURE CITED1. AURO, M. A., H. M. HODGE, AND N. G. ROTH.

1957. Oxygen absorption rates in shaken flasks,Ind. Eng. Chem. 49:1237-1238.

2. BERGKVIST, R. 1963. The proteolytic enzymes ofAspergillus oryzae. I. Methods for the estima-tion and isolation of the proteolytic enzymes.Acta Chem. Scand. 17:1521-1540.

3. DELIN, S., P. G. SQUIRE, AND J. PORATH. 1964.Purification of steroid-induced enzymes fromPseudomonas testosteroni. Biochim. Biophys.Acta 89:398-408.

4. FAHIM, M. M. 1966. The effect of light and otherfactors on the sporulation of Alternaria porri.Brit. Mycol. Soc. Trans. 49:73-78.

5. ICHISHIMA, E., Y. GOMI, T. WATARAI, AND F.YOSHIDA. 1963. Studies on the proteolyticenzymes of black Aspergilli. Part IX. Effect ofagitation and aeration on submerged produc-tion of acid protease. Agr. Biol. Chem. (Tokyo)27:302-309.

6. JOLY, P. 1964. Le genre Alternaria, vol. 33. InEncyclopedie mycologique. Paul Lechevalier,Paris.

7. J6NSSON, A. G. 1962. Studies in the utilizationof some agricultural wastes and by-products byvarious microbial processes. Kgl. Lantbruks-Hogskol. Ann. 28:235-260.

8. JONSSON, A. G., IAN S. M. MARTIN. 1964.Protease production by Aspergillus fumigatus.Agr. Biol. Chem. (Tokyo) 28:734-739.

9. JONSSON, A. G., ANm S. M. MARTIN. 1965.Protease production by Alternaria tenuissima.Agr. Biol. Chem. (Tokyo) 29:787-791.

10. NEERGAARD, P. 1945. Danish species of Alternariaand Stemphylium. Einar Munksgaard, Copen-hagen.

11. NILSSON, P. E. 1957. The utilization of agricul-tural products by microbial fermentations. IVA28:117-144.

12. NILSSON, P. E., AND A. G. JONSSON. 1962. Culti-vation of azotobacters for production ofmicrobial proteins. Kgl. Lantbruks-Hogskol.Ann. 28:203-214.

13. TOMODA, K., AND H. SHIMAZONO. 1964. Acidprotease produced by Trametes sanguinea awood-destroying fungus. I. Purification andcrystallization of the enzyme. Agr. Biol. Chem.(Tokyo) 28:770-773.

14. WETTER, L. R. 1952. The proteolytic enzymes ofmicroorganisms. IV. Partial purification andsome properties of extracellular protease fromMortierella renispora Dixon-Stewart. Can. J.Botany 30:685-692.

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