COXIMTJNIC ATIONS

Liquid- Metering Device .for Continuous Culture

Summary

A device is described for aseptically metering liquids at rat,ej as low as 10 ml./hr. The device delivers

The volrime added is controlled The rnedirim does not erit>er thii

The metering rate is constant, brit can be readily changed. a measiired increment of liqnid every 10 sec. by electrical contact-s in an electrolyte tribe. t iilw.

Introduction

1let.hods of corit.rollirig dilrition rates i n cont,innoiis ciilt.ures of microorganisms have been reviewed by MAek and Fencl.1 When laboratory cont,iririoris fermen- tors of small volume (100-600 ml.) are operat,ed at, low dillition rates (0.1-0.2 hr.-1), precise control of dillition rat,e over a long period of t,ime is often difficiilt. The device here described has been in iise in oiir laborat,ory for 2 years and has been foiind to be accrirate and reliable.

Description

The device does not add medium to the fermeiitor continuotisly; an increment' is added every 10 sec. This is, in most cases, eqiiivalent to cont,iriuous addition.

The measiiring device is shown in Figure 1, and the valve-actuating circuit. is shown in Figure 2. In Figure 1 , which is not drawn to scale, the cycle begins with the opeiiirig of V1 (at, 0 sec.), Vz being closed. RIedium flows from A t,hroiigh P and V , into B, causing t.he electrolyte in tube C to rise in the right arm of the tube iiritil it makes elect,rical contact, wit,h the platinum wire 4. This contact, causes V1 to close. At 6 sec., V2 opens, and medium flows from tube B through V z iiit,o the ferment,or, t,hns causing t,he electro1yt.e in kibe C t,o rise in the left arm of the tribe until it makes contact with the p1at)iniim wire 5. This contact, caiises V 2 to close. The volume of medium delivered during each 10-see. cycle is determined by the amount. of elect.rolyt,e in t,iibe C. This may be adjristed by temporarily opening t.he glass stopcock and adjust,ing the electrolyt,e level by means of the clamp at, E. This clamp has a large area and is similar t.0 clamps iised to adjust Warbwg manometers. A millimet,er scale behind one of the arms of tube C facilit,ates adjustment, to the desired delivery rate. The electrolyte is 0.01M Na2C0, containing 0.5 g./l. of Tri1,on X-100 or other nonionic detergent. A small glars wool phig is inserted in the t,iibe just above the stopcock t,o prevent occasional specks of st'opcock grease from entering tube C. Plat.inirm wires (28 gage, 0.32 mm. diam.) are sealed through tithe C at 4, 5, and 6. Wires 4 and 5 are carefiilly centered in the hibe. Tnbe C is made from 5 mm. (0.11.) glass tubing; tube B from 7 or 8 mm. tubing. The distance from t>he lower end of wires 4 and 5 to the bot1,om of the U-tube is aborit 10 cm. Tithe D, which contains a little water arid is lined witlh filter paper, prevents evaporation of e lechlyte from tube C. The three-way glass stopcock at the left of titbe C normally connects

UIOTECHNOLOGY A N D BIOENGINEERING, VOL. IX , ISSUE 4

At, 10 sec., V , opens again, arid t,he cycle is repeat,ed.

COhlhIl JNICATIONS 03 1

Fig. 1. Iliagram (not to scale) of metering apparatiis: ( A ) reservoir of sterile medium, with air inlet tube arranged for con.;tant head; ( B ) mediilm deliver? tube; (C) metering t h e ; ( D ) hiimidifier tnbe; ( E ) rubber tube with adjustable clamp; ( F ) capillary glass tube for flow restriction; (VI, V,) two-way, normally closed small solenoid valves. Nnmbers 1-6 designate electrical connectioni to for- respondingly nnmbered points in Fig. 2 .

only tnbei R and C. It, is tiirned diiring adjnstment of liquid levels in tubes I? arid C .

Titbe H, after steam sterilization with its associat,ed components, is connected to tribe C by means of n shoit piece of heavy-walled rubber tiibing. A glass wool pliig in an enlarged section at the top of tribe B prevents contamination of the medium. The solenoid valves, V 1 and V2, a1e stainless steel Skinner type B2DA9173, with 115-V. a.c. coil. These valves have a small internal volume, and, when operated on d.c., will operate a t d.c. voltages above 25 V. The coil resistance is about 360 ohms. The valves are mounted in an inverted position to minimize bnbble entrapment. Glass tubing is sealed into the valve inlet and oullet. The connection of tube B to the tnbe connecting the two valves is made as indicated, to prevent air bubbles from entering tube B. Before steam sterilization of the valve assembly, the valve coils are removed from the valves. This is easily done after removing one retaining nnt.

The leiigth and internal diameter of the capillary tribe F is such that the rate of flow from the reservoir A to tube B is such that one increment of mediiim

Self-ciiring silicone rubber adhesive is a good sealing agent.

632 COMMUNICATIONS

4

2

5

-6 .27K : 2w -3

5.6 IW --IcJrJ.hh- ;loo 1:;

Fig. 2. Valve-actuating circuit,. The two power transistors are RCA 40422 or other silicon power transist'ors having a breakdown voltage of 200 V. or more. The other four transistors are 2NX242A or other silicon transist.ors having low leakage current and high current gain. Itesistors are = t ~ l O 7 ~ , 0 .5 W. unless other- wise specified. S is the switch on a cycling timer; it changes position every 5 sec. (CM-2 timer wit>h R-12 gear rack; Indiistrial Timer Corp., Parsippany, N. J.). Numbered terminals indicate connectioii t o corresponding terminals in Fig. 1. The diodes are 200 mA. silicon rectifier diodes, 400 V. PI!'. Two of them are for bypassing turn-off transients. Coririeclion to the a.c. line is by means of a th ree prong plrrg, the ground terminal being connected to the metal enclosing cwe. The groiinded side of the a.c. line is comiecled to the negative side of the power supply. Leads 4 and 5, between the amplifier and t#he metering apparatus, should be en- closed in a grounded shield t,o minimize hiim pickup. If it, is desirable to count the niimber of increments of medium added to the fermentor, the 1K resistor in series with V2 is replaced by a 2K resistor shiiiited by a counter (CESOOBSSOZ), ITT General Controls, Des Plaines, Ill. If necessary, the input signal to the coiit,roller may be adjusted by changing the concentration of the elect.rolyte. The coupling indicated between the first and second st,ages is used in place of the simpler Darling- tori coupling because a phase inversion is necessary to allow operation of terminal 6 in titbe C as a positive terminal. The negative terminals 4 and 5 are depolarized by traces of dissolved oxygen in the electrolyte. Dissolved oxygen can become ex- hausted in the neighborhood of terminal 6.

enters the tube in about 2-3 sec. A section of capillary tubing is included iri the line from V2 to the fermentor (which is positioned below the control unit,) to adjust similarly the rate of oiitflow from tube B. The average level of medium in tube B should be adjust)ed (by means of the three-way stopcock) tJo be about the same as the average level of the electrolyte in tube C. The air between the medium and the electrolyte is then at a pressure close to atmospheric, and tends neither to dissolve in, nor extract gas from, the liquid phase.

The 5-mm. glass tubing (about 3.5 mm. 1.n.) specified for tube C is the smallest we have been able to use witshotit trouble from siirface tension effects. Tube C can, however, be made as large as desired, within obvious limit,s. Tube B, of course, must be correspondingly enlarged, arid a leveling bulb, rather t,hari a clamp, is used for adjusting the electrolyte level. When 5 mm. tubing is used

BIOTECIINOLOGY AND BIOENGINEERING, VOL. IX, ISSUE 4

COMMUNICATIONS 633

for ttibe C , incremeiitv ranging roiighly from 0.01 to 0.7 nil. can be added at 10 sec. intervals.

The valve-actuating circuit shown in Figure 2 is, of cotwe, only one of many possible designs. We have iised several arrangements, biit that of Figure 2 has proved to be simple and convenient.

Performance

The performance of the device was checked over a period of about 4 days, a t a number of delivery rates. It can be seen that increments of 6 pl. were not delivered with good reproducibilit,y. The reproducibility with larger increments was good, with both 5 mm. and 11 mm. tubes.

At very low delivery rat,es, appreciable errors occur if the ambient temperature changes several degrees. The electrolyte in tube C expands, and this increase in volume is subtracted from the vohime of the increment delivered. If the volume of air between the mediinn in tnbe B and t,he electrolyte in tube C varies,

Results are summarized in Table I.

TABLE I Performance of Liquid Metering Device

Experiment la Experiment 2* Experiment 3" Experiment 4b -

Elapsed Elapsed Elapsed Elapsed time, Increment time, Increment time, Increment time, Increment, days wt.," g. days wt.,c g. days wt.,d g. days wt.,e g.

0 .0 0.04 0.12 1.06 1.13 1.29 2.00 2.08 2.37 3.00 3.21 3.30 4.00

Av. St. dev.

0.00652 0.00663 0.00651 0.00651 0.00627 0.00657 0.00640 0.00597 0.00620 0,00681 0.00564 0.006 15 0.00609

0.0062.5 6.0';7,

0 .0 0 .23 0.83 1.06 1.25 1.83 2 . 2 3 2.87 3.94

0.02352 0.02329 0.02312 0.023.57 0.02337 0.02306 0.02295 0.02324 0.02342

0 .0 0.68 1.01 1.21 1.68 2.07 2.86 3.08 3.67

0.20643 0.20614 0.20605 0.20608 0.20592 0.20640 0.20636 0.20653 0.20650

Av. 0.02328 Av. 0.20627 St. dev. 0.90% Rt. dev. 0.087,

0 .0 0.58 0.90 1.12 1.58 1.77 2.58 2.92 3.60 3.98

Av. R t . dev

5.972 5.983 5.981 5.967 5.989 5.966 5.981 5.943 5.960 5.948

5.969 0.22yc

-

a 1)ianieter of tube C = 5 nim. b Diameter of tube C = 11 mm. c Average of 200-c500 increments d Average of 100 increments. e Average of 4 increments.

ti34 COMMUNICATIONS

error is introduced. This air coirtmcts (due to pressure increase) when Lhe medium rises in tube 13. If the air volume increases, t,his contraction will be greater, arid t.he increment delivered will be smaller. The magnitude of tbe errors allribrit,able to the above caiises may be readily calcdated, and in most cases is small.

Slipported in part by NII-I Giatit No. A1 02967-08.

Reference

1. I. MBlek and Z. Fencl, Yheorelical and kfethodoloyicul llusis oJ Conlinuovs Culture, Academic Press, New York, 1966.

11 LRVIN J. JOHNSON

Department of Biochemistry University of Wisconsin Madison, Wisconsin 53706

Received June 21, 1967