Performance of Roller Agitators for Cultivation of Microorganisms

BENEDICT MARK HALL

Department of Biology, Temple University, Philadelphia, Pennsylvania

Received for publication February 26, 1960

Rapidly growing aerobic organisms can deplete oxy-gen in the nutrient medium even when culture vesselsare open to the air. Agitation is beneficial under theseconditions because it tends to expose more liquid tothe air, and because the mixing tends to make condi-tions more homogeneous throughout the volume of themedium. Rotary and reciprocating shakers are capableof high rates of aeration when used with baffled Erlen-meyer and Fernbach flasks. However, the standardreciprocating or rotary shakers are not adapted toaerating test tube cultures.

Aeration by rolling has been used previously fortightly stoppered cultures of animal cells (White, 1954).A typical application of the roller tube method makesuse of standard 16- by 150-mm test tubes. These arecharged with a small quantity of liquid nutrient, thenplaced in a drum revolved about its longitudinal axis.The axis of revolution is inclined slightly from the hori-zontal so that the liquid which covers the lower sideof the tube wets it about halfway or two thirds of thedistance to the stopper. The drum turns from six to ashigh as 3000 revolutions per hr, depending on whetherit is desired that the cells grow on the surface of theglass or in suspension in the liquid medium (Paul,1959). In a somewhat different way Monod (1950)has used the principle of rolling to supply aeration in acontinuous flow device called the "Bactogen." A signifi-cant difference between the Bactogen and the rollertube system is that in the Bactogen the culture vesselrevolves about its own axis at approximately 400 rpm,a speed much higher than that of the roller drum. Inboth devices aeration is accomplished through themechanical action which spreads the liquid over theinner surface of the culture vessel. The fact that rapidgrowth of animal cells occurs in roller tubes does notindicate that this device is capable of very high rateof oxygen transfer, since total cell mass and growthrate remain low in contrast with aerobic cultures ofmolds, yeasts, or bacteria. However, the simplicity ofthe rolling principle of aeration encouraged me toattempt it with test tube and flask cultures of aerobicorganisms, and to make the quantitative tests whichare described below. My particular need was for a

method of measuring the growth of yeasts turbido-metrically in 13-mm special test tubes designed for theKlett-Summerson Colorimeter.1 A high rate of aeration

1 Klett Manufacturing Company, New York, New York.

was desirable. The mechanism and procedure describedhere secured a satisfactory aeration rate, preventedcontamination of cultures, and simplified growth meas-urements. The procedure made it possible to followthe whole growth cycle without removing the culturesfrom the test tubes.

DESCRIPTION OF ROLLER AERATOR

This machine is similar in operating principles tothe standard roller tube drum (White, 1954, page 117).The culture vessels are supported by a rotating cageor drum with their long axes parallel to the axis ofrotation, which is inclined sufficiently from the hori-zontal to retain the liquid within the culture vessel.The drive shaft, turning generally less than 60 rpm,rotates the cage about its axis, causing the culture ves-sels to orbit about the drive shaft. Since the rotationalspeed is slow, centrifugal force is negligible, and theliquid remains on the lower side of the culture vessels.The turning motion wets the inner surface of the ves-sels, exposing a large surface of liquid to the air. Themachine consists of a power source, capable of provid-ing a range of rotational speeds, coupled to a driveshaft within the incubator. The drive shaft is supportedby bearings which allow adjustment to maintain theshaft at the proper inclination. Two detachable test-tube baskets and an adjustable rack for larger vesselsare supported by the shaft (see figure 1).

External drive. Power is provided by a 20:1 wormgear reducer, V-belt driven at 860 rpm by a 1725 rpmelectric motor. A stub shaft, supported by the incu-bator wall, is belted to the driving end of the reducer.Through a combination of pulleys at the driving endof the reducer and the stub shaft a range of speeds from6.3 to 76 rpm was obtained.

Drive shaft. The 2-in. drive shaft inside the incu-bator is powered through a universal joint by the stubshaft through the incubator wall. A self-aligning, ad-justable shaft support regulates the height of the distalend of the drive shaft, maintaining the proper slopeof liquid in the test tubes and other vessels.

Test tube baskets. The baskets are open sided boxesof }4-in. masonite. Each box is drilled to fit 18 tubes,one box being drilled for the 13-mm outside diameterspecial test tubes for the Klett-Summerson Colorimeter,the other for standard 16-mm test tubes. Semicircularrecesses of <4-in. radius are cut into the end and center

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1PERFORMIANCE OF ROLLER AGITATORS

plates of the baskets in such a way that the center linesof the recesses are parallel to the centers of the testtube apertures. The recesses fit against the drive shaft,insuring parallelism between the shaft and the tubes.I)uring operation the baskets are secured to the shaftly wiing nuts as shown in figure 2. The detachable bas-kets fa(ilitate handling of test tubes in the course ofturbidity measurements.

Figurle 1. Layout of machinery showing motor, 20:1 worm-

gear reducer, and pulley driving stub shaft in incubator wall.

The drive shaft, carrying a pair of detachable test tube basketsand a rack for cylindrical vessels, is held by a universal jointat one end and by an adjutstable shaft support at the other.

Figure 2. Detail of drive shaft and adjustable shaft supportwith one test tube basket removed. The basket inl place conI-

tains a partial load of Klett Colorimeter tuibes, colored liquiidhaving been added to indicate slope of vessels: 5 ml in Kletttubes and 200 ml in the cylindrical bottles.

A ccomodations for other vessels. A limited number ofErlenmeyer flasks may be hung from the ½-in. driveshaft by means of conventional flask clamps. Because ofthe moderate rotational speeds, this arranigement hasade(uate strength. For handling cylindrical bottles,a cage-type holder was constructed from light steelangles and spring clamps (see figure 2).

METHODS

The efficacy of the roller aerator was determinediodometrically by measuring the oxidation of sodiumsulfite solution catalyzed by copper sulfate (Cooperet al., 1944). All test tube runs were made with 5 mlsodium sulfite solution in the tubes, with the slope ad-justed so that the tube wall was wet to within 1.5 cmof the mouth. Oxygen absorption rate was computedas ml of oxygen per 100 ml liquid per hr. Biologicaltests were made with Torulopsis utilis strain ATCC99502 in Wickerham's medium (yeast nitrogen base3),a liquid nutrient providing mineral and organic re-quirements for yeasts but lacking a source of energyfor growth. Glucose or an organic acid was added as anenergy source. Inocula were prepared by serial dailytransfers through this medium. Measurements ofgrowth rate were made turbidometrically, and deter-mination of yield was made turbidometrically as wellas by weight.

RtESULTS

Effect of rolling speed. Variation of rolling speedbetween 6.3 and 60 rpm had no appreciable effect onrate of oxidation in test tubes (see table 1). Higherrates of rotation were not practical with test tubesbecause the surging of the liquid, caused by centrifugalforce, tended to wet the plugs. High speed rotationwith larger vessels did increase the aeration rate, butonly when critical speed (i.e., the point at which centrif-ugal force equals gravitational force) was approached.

2 American Type Culture Collection, Washington, D. C.3 1)ifco Laboratories, Detroit, Michigan.

TABLE 1

Effect of otlling spee(d on oxygen absorption rate in uinpluggedtest tubes*

Rolling Speed Oxidation Rate (02,/100 ml/hr)

rpmpnl60.0 94.056.0 97.030.0 96.013.0 103.06.3 95.0

Mean 97.0

* Test tubes 13-mm inside diameter bv 12.8 cm long (Kletttubes), containing 5 ml liquid, and slanted to wet glass within1.5 cm of tube mouth; temperature, 30 C.

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B. M. HALL

Near this point the fluid was lifted on the upswingof the vessel, then fell free on the downswing, splashingagainst the walls with considerable turbulence. Bafflingimproved this action and increased aeration corre-spondingly (see table 2).

Effect of closures. Cotton plugs did not greatly re-strict the rate of oxidation in test tubes in my own

TABLE 2

Comparative efficiency of rolling motion vs rotating shakingmotion with respect to oxygen absorption rate

OxygenAbsorp-

Method and Vessel Load Operating Conditions tion Rate(02/100ml/hr)

ml ml

Rolling motion*Tubes, 16 mm di- 5 53 rpm 82ameter

1000-ml Erlenmeyer 150 60 rpm 301-gal jug 100 57 rpm 791-gal jug, 1 baffle 100 76 rpm 150

Rotary shaking mo-tiont

Tubes, 16 mm di- 5 210 rpm, 34-in. throw 47ameter diameter (tubes

slanted)500-ml Erlenmeyer$ 100 220 rpm, 50-mm throw 37

diameter500-ml Erlenmeyer, 100 220 rpm, 50-mm throw 716

1 bafflet diameter1000-ml Erlenmeyer 150 210 rpm, Y4-in. throw 5

diameter1000-ml Erlenmeyert 200 285 rpm, 50-mm throw 56

diameter1000-ml Erlenmeyer§ 100 200 rpm, 4½-in. throw 34

diameter

* Axis of rotation horizontal, or nearly so.t Axis vertical.I Chain and Gualandi (1954).§ Corman et al. (1957).

TABLE 3Effect of closures on oxygen absorption rate

Oxygen AbsorptionRate (02/100 ml/hr)

Vessel Load ClosureWith Without

closure closure

ml ml ml

13-mm tubes .5 Cotton plug 78 9716-mm tubes ....... 5 Cotton plug 77 821000-ml Erlenmeyer, 200 Cotton plug 0.6* 96*not baffled.

1000-ml Erlenmeyer,not baffled........ 200 Alum. foil cap 0.21* 96*

2800-ml Fernbachindented ......... 300 Cotton plug 215t 685t

2800-ml Fernbachindented ......... 300 3 filter disks 471t 685t

* Chain and Gualandi (1954).

t Corman et al. (1957).

experiment. The comparison of closure effects reportedin the literature is not of much quantitative valuedue to the lack of sufficient information about thedesign and packing of plugs. However, the experienceof Corman et al. (1957) indicates the possibility ofdesigning closures which will allow sufficient oxygendiffusion. The values are summarized in table 3.

Absorption as a function of wet area. The rate of oxy-gen absorption for three different vessels and severalconditions of loading is computed as a function of wetarea (liquid surface plus wet glass area) in table 4.Fairly close agreement was obtained among variousvessels, and a mean was computed.

Comparative efficacy of the rolling vs. the rotary shakingmethod of aeration. Table 2 summarizes the results ofmy tests of the roller aerator with test tubes, flasks,and cylindrical vessels. For purposes of comparison,results which indicate the performance of typical rotat-ing shakers were selected from the literature.

TABLE 4Oxygen absorption in relation to wet area

Vessel Load Wet O0/100 ml/hr Oz/cm'/hr

ml cm2 ml ml

Milk dilution bottle* ......... 25 160 87 0.101000-ml Erlenmeyer. 100 436 38 0.0871000-ml Erlenmeyer. 150 484 30 0.0931000-ml Erlenmeyer. 200 541 28 0.1031-gal jug ..................... 100 874 79 0.0904

Weighed mean .......... .... 0.0936

* Compact square cross-sectional bottles (180 ml) (ArthurH. Thomas Co., Philadelphia, Pennsylvania).

TABLE 5Growth of Torulopsis utilis in rolled Klett Colorimeter tubes

and one-gallon bottles*

No. Previous Avg Cell Yield: g DryESt Concn of ES Passages, Division Yeast/g ES§Same ES Timne

% hr

Acetic acid .... 0.1 0 2.1Acetic acid ..... . .0.15 1.4Butyric acid . 0.05 46 6.0Glucose ....... 0.1 15 1.2Glucose.. 0.1 54 1.1Formic acid 0.05 21 1.5Lactic acid . 0.1 34 2.6Acetic acid 0.1 60 0.36Acetic acid 0.2 60 2.6 0.32Acetic acid 0.5 61 2.1 0.32¶Acetic acid 1.0 61 2.2 0. 32¶

* In Wickerham's medium (yeast nitrogen base); source ofenergy for growth indicated.

t ES = source of energy.$ In Klett tubes, 5 ml medium.§ One-gallon bottles, 100 ml medium.¶ Average of two samples, one brought to pH 5.0 with KOH

before inoculation; the other, with CaCO3.

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10PERIFORMANCE OF ROLLER AGITATORS

Growth of yeast on the roller. Table 5 indicates thegrowth characteristics of Torulopsis utilis in rolledvessels. Average cell division time (the time requiredfor doubling of cell mass during the period of logarith-mic growth) was lowest with glucose as an energysource, high with butyric acid, and intermediate withacetic, formic, and lactic acids. There was, in addition,a tendency for cell division time to decrease after sev-eral passages through Wickerham's medium and thesame energy source. Yield in g yeast per g of energysource was measured in one-gallon bottles with 100ml of growth medium. Yield remained constant, orpractically so (32 to 36 per cent), over the 10-foldconceintration ranige of acetic acid which was tested.

DIscussION AND CONCLUSIONS

The apparent independence of oxygen absorptionrate and speed of agitation is contrary to previous ex-perience. Chain aind Gualandi (1954) report a linearrelationship between rate of oxygen absorption andspeed of agitation, both in rotary shaken flasks and inpropeller agitated fermentors. Cooper, Fernstrom, andMiller (1944) agree with this in principle when theyconclude that aeration efficiency was proportional tothe power input per unit volume of liquid in stirredfermentors. It is conceivable that some of the peculiar-ities of my results may be due to the properties of thesulfite system; however, its general validity is not inquestion, and indeed, has been independently con-firmed. Chain and Gualandi (1954), in 24 pairs of deter-minations, obtained good correlation between the sul-fite and the amperometric method. Using a rotatingplatinum electrode, they found that the differenceswere usually within 10 per cent, and that over theexperiment as a whole, the differences cancelled out,indicating no appreciable bias.

Mly opinion is that the unexpected independence ofaeration rate and rotating speed is due to the differencein the mechanical action of the roller as opposed toother types of aeration systems. My experiments sup-port the theory that aeration in a slowly rolling vesselis a linear functioin of the wet surface area. In a rollingvessel the wetted area is constant within wide rangesof rotational speeds. Below this range, wet area de-creases because of drainage between wettings, andabove this range wet area increases somewhat becauseof splashing and the entrainment of bubbles. Rollingaeration rate is speed independent, in other words,because it does not depend on bubble entrainment asthe other systems do. An appropriate theory for thespeed dependence of aeration rate in shaken flasks andstirred fermentors is that higher speed increases thenumber of bubbles, decreases the average size of thebubbles, and therefore increases their total area, andthat the turbulence delays the escape of bubbles from

liquid film (Steel, 1958). It is obvious, furthermore,that in a shaken flask, baffled or not, increasing theshaking speed will increase the area of wet glass.The possibility that a film of 1 N sodium sulfite solu-

tion would not saturate with oxygen as quickly as

a film of culture solution suggests that the sulfitemethod might not be indicative of the true aerationrate in cultures rolled at a very low speed. Should theliquid film in the upper part of the rolled vessel becomesaturated with oxygen before being renewed by the turn-ing of the vessel, the average aeration rate would de-crease. I believe, however, that this bias, if it actuallyexists, would not be of any practical importance at

the rotatienal speeds likely to be employed for rollingvessels containing dense cultures of aerobic organisms.

Results indicate that the rolling machine is capableof inducing satisfactory growth in Torulopsis utilis, a

typical aerobic yeast. In this respect the biologicaltests corroborate the sulfite tests which had shown theroller to have as high an oxygenating rate as the con-

ventional unbaffled shaken flask systems commonlyused for growing aerobic organisms. Neither the growthrate nor the dry weight yield per g of energy source

differed significantly through the range of concentra-tions of energy source tested. If aeration rate had notbeen adequate, the effect would probably have beento depress growth rate and yield efficiency more in thedense cultures than in the dilute cultures.

Aeration of test tube cultures by rolling is more

effective than aeration by the rotary shaker tested.The rate of aeration in rolled test tubes is higher thanthat in shaken unbaffled flasks, but not so high as thatattainable in flasks with baffles.

For Erlenmeyer flasks without baffles, the rate ofaeration obtained by rolling is approximately equalto that obtained by shaking. Cylindrical vessels are

more efficient than Erlenmeyer flasks on the rollerbecause a cylindrical vessel of the same base and heightas an Erlenmeyer flask has considerably more internalarea, and because the inner surface of a cylindricalvessel is completely wetted even when the load is light.The one-gallon wide mouth jug used in my experimentshas a 6-in. base diameter, whereas the base diameterof a 1-L Erlenmeyer is 5 in.; the jug is /% in. taller.Although the two vessels occupy virtually the same

amount of space in an incubator, the jug aerates themedium twice as fast as the flask when both are rolled(see tables 2 and 4).

Certain characteristics of the rolling system of aera-

tion simplify the problem of experimental application.Since oxygen absorption is a linear function of wetarea which, in rolling, is determined by the shape ofthe vessel and is independent of other factors, anydesired aeration rate may be obtained simply by ad-justing the conditions of loading to the size of the vessel.

the liquid and reduces the thickness of the stagnant

1960] 381

A favorable characteristic of rolling aeration at

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P. A. HARTMAN AND D. V. HUNTSBERGER

moderate speeds in unbaffled vessels is that due to lowturbulence there is virtually no tendency to foam.Foaming is commonly observed in aeration systemswhich use baffled or indented flasks and depend fortheir efficacy on a high degree of turbulence and on theentrainment of air bubbles. Foam production interfereswith aeration and has other undesirable effects. It maybe reduced by chemical additives, but these additivesthemselves interfere with aeration. Corman et al. (1957)have shown that anti-foam agents may reduce theoxygen absorption rate drastically, in one case from455 to 54 ml oxygen per 100 ml per hr. It is likely,therefore, that rolling aeration is more efficient thanaeration by shaking with certain types of culture me-dia, since rolling aeration does not depend on bubbleentrainment, does not generate foam, and eliminatesthe need for anti-foam agents.

ACKNOWLEDGMENT

This work was facilitated by a grant from the TempleUniversity Committee on Research.

SUMMARY

For the aeration of liquid cultures of microorganismsin test tubes and larger vessels up to perhaps 1-L load,rolling has certain advantages. First, rolling of testtubes is more effective than shaking. For Erlenmeyer

flasks rolling and shaking are equally effective unlessbaffling is used. The installation of baffles increasesaeration about two-fold on the roller, but 10- to 20-fold on a high speed rotary shaker. Second, the absenceof foaming is a distinct advantage of rolling aeration.Third, because of the low rotation speed, centrifugalstresses are neglibible and power requirements aremoderate. Consequently, roller equipment is consider-ably simpler to design and construct than rotaryshakers.

R SFERENCES

CHAIN, E. B. AND GUALANDI, G. 1954 Aeration studies.Rend. inst. Super. sanitA (Eng. Ed.), 17, 5-60.

COOPER, C. M., FERNSTROM, G. A., AND MILLER, S. A. 1944Performance of agitated gas-liquid contactors. Ind.Eng. Chem., 36, 504-509.

CORMAN, J., ISUCHIYA, H. M., KOEPSELL, H. J., BENEDICT, R.G., KELLEY, S. E., FEGER, V. H., DWORSCHACK, R. G.,AND JACKSON, R. W. 1957 Oxygen absorption rates inlaboratory and pilot plant equipment. Appl. Microbiol.,5, 313-318.

MONOD, J. 1950 La technique de culture continue theorie etapplications. Ann. inst. Pasteur, 79, 390-410.

PAUL, J. 1959 Cell and tissue culture. The Williams &Wilkins Co., Baltimore, Maryland.

STEEL, R., ed. 1958 Biochemical engineering. The Mac-millan Co., New York, New York.

WHITE, P. R. 1954 The cultivation of animal and plant cells.Ronald Press Co., New York, New York.

Sampling Procedures for Bacterial Analysis of PreparedFrozen Foodsl

PAUL A. HARTMAN AND DAVID V. HUNTSBERGER

Departments of Bacteriology and Dairy and Food Industries and Department of Statistics, Iowa State University, Ames, Iowa

Received for publication March 7, 1960

Various investigators have reported that large differ-ences exist in the numbers of microorganisms associatedwith various types of frozen foods (cf. Borgstrom, 1955;Canale-Parola and Ordal, 1957; Huber et al., 1958;Ross and Thatcher, 1958). However, little practicalinformation is available regarding fluctuations of bac-terial numbers within a single brand and type of prod-uct. Sampling procedures which are of utmost importto processors and to others interested- in the microbi-

' Journal paper no. J-3733 of the Iowa Agricultural andHome Economics Experiment Station, Ames, Iowa. Projectno. 1379. This investigation was supported, in part, by researchgrant no. E-1141 from the Institute of Allergy and InfectiousDiseases of the National Institute of Health, U. S. PublicHealth Service.

ology of frozen foods have been in need of thoroughexamination. The following studies were made to deter-mine the impact of seasonal, day-to-day, diurnal, andsample-to-sample variations on the total counts tobe expected in a prepared frozen food.The data to be presented are exemplary of certain

sampling problems which are frequently encounteredin the microbiological enumeration of microorganismsin prepared frozen foods. Obviously, conditions ofproduction will vary between processing plants, be-tween processors, and even within the same plant fromyear to year as improvements are made. Bacterialcounts will be affected by processing technique, yetthe general considerations presented here should remainrelatively unchanged.

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