The Chemical Engmeermg Journal, 41 (1989) B27 - B35 B27

A Detailed Analysis of Saccharomyces Cerevkiae Growth Kinetics in Batch, Fed-batch, and Hollow-fiber Bioreactors

STEVEN J COPPELLA* and PRASAD DHURJATI

Department of Chemical Engmeenng, Unwerszty of Delaware, Newark, DE 19716 (US A )

(Received December 31, 1988)

ABSTRACT

The use of the yeast Saccharomyces cerevz- szae has zncreased greatly over the past few years for the productzon of pharmaceutzcals, specialty chemzcals, and other commodztzes One reason for thzs zs the many advantages zt offers for synthesis and secretion of recombinant DNA products However, the growth characterzstzcs of thzs yeast are quite complex and only recently has detazled analy- szs been available to provide addztzonal znszght zn to such phenomena as bzphaszc growth and catabolzc repression

mductlon Cell growth occurs m the Gl phase until start 1s reached, when a crltlcal mass is obtamed by the mother cell At this point m the Gl phase, cell rephcatlon, segregation, and dlvlslon begins Length of the Gl phase 1s strongly dependent on the specific growth rate of the cell, while the size of the mother cell is approximately independent of the growth rate Gl 1s the dommant phase at slow specific rates and 1s negligible at high specific growth rates Conversely the times of the S (DNA synthesis), G2 (gap 2), and M (mltosls) phases are nearly independent of growth rate

Thzs paper summarizes and organizes cur- rent literature and also details the experzmen- tal behavior of on-line and off-lzne varzables of S cerevzszae zn glucose-lzmzted-batch, fed- batch, and hollow-fiber bzoreactors The observed behavzor zs interpreted using knowl- edge of yeast catabolzsm bzochemzstry and physzology Speczal focus zs placed on the interpretation of off-gas behavzor wzth respect to TCA enzyme actzvzty and the saturatzon level of oxygen utzlzzatzon capaczty

Reproduction via buddmg for the haplold 1s highly asymmetrlc (volume of the daughter 4 volume of the mother) at high growth rates and approaches binary fission at slow growth

Two catabohc shifts predommate m yeast The Pasteur effect 1s the shift from oxlda- tlve to fermentatlve metabohsm when the oxygen supply 1s interrupted The second shift 1s dependent upon the glucose concen- tration and 1s referred to as the Crabtree effect

1 LITERATURE REVIEW

The growth behavior of Saccharomyces cerevzszae IS well documented and has been summarized by Llevense and Llm [ 1 ] There are three important metabolic features to consider when interpreting the behavior of this yeast cell dlvlslon mltlatlon, highly asymmetric cell cycle and dlvlslon, and cata- bolic shifts due to glucose represslon and

For glucose concentrations below 50 - 130 mg l-l, S cerevzszae oxldlzes glucose (eqn 1) with a maximum speclflc growth rate of 0 25 - 0 30 h-l, a substrate yield of 0 5 g dry cell wt g glucose consumed, a respiratory quotient (RQ) of 0 9 - 1 0, and an ATP yield of 16 - 28 per mole of glucose oxidized Erratic growth 1s observed for glucose below 10 mg 1-l

C606H12 + 60, - 6H,O + 6C0, (1)

At larger glucose concentrations, m an

*Author to whom all correspondence should be addressed Current addresses Department of Cheml- Cal Engineering, Umverslty of Maryland, Baltimore County, Baltimore, MD 21228, U S A , Medxal Blo- technology Center of the Maryland Biotechnology Institute, Umverslty of Maryland, Baltimore, MD 21201, U S A

aerobic environment, glucose is predoml- nantly fermented (eqn 2) despite the avall- ability of oxygen, and ethanol 1s not oxidized (the Crabtree effect) Maximum specific growth rate 1s 0 4 - 0 45 h-l, substrate yield 0 15, RQ 1s greater than 1, ATP yield is 2 per mole of glucose consumed, and ethanol 1s

0300-9467/89/$3 50 0 Elsevler Sequola/Prmted m The Netherlands

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the glucose to be fed Medium and fermentor equipment were autoclaved for 40 mm at 121 C Glucose stock solution was 300 g 1-l and was autoclaved separately Water used for all media and solutions was dlstllled then purified with a Barnstead Nanopure water purifier with a resulting resistance of about 10 rnfi cm-

Shake flask experiments were performed m 150 ml of medium m a 250 ml Erlenmeyer flask within an air bath heated to 30 C and rotated at 260 rpm with an recn-culatlon Dry cell weight was measured using a Bausch and Lomb Spectromc 2000 spectrophotometer at 600 nm using the off-line correlation pre- sented elsewhere [ 1 l] Samples were diluted forOD>l65

The batch, fed-batch, and hollow-fiber expenments used a 20 series 500 LH Fermen- tation System (LH Fermentation Inc , Hayward, CA) Agitation was controlled at 750 rpm by a direct drive 502D agitator, pH at 6 0 + 0 1 with a 505 controller using a 465-35-K9 Ingold probe (Ingold Electrodes Inc , Wllmmgton, MA), and temperature at 30 C with a 503 controller. Mass transfer limitations were found to be mslgmflcant by comparing batch results at 500 and 750 rpm Fermentor schematics, dissolved oxygen, optical density/dry cell weight correlation, sterlhzatlon procedure, and off-line analysis have been described elsewhere [ 111 A solu- tion of 60 g 1-l glucose was fed by a 7520-35 masterflex pump with no 13 silicon tubing for the fed batch fermentation Flow rate error was approxunately 0 6 ml h-l

The hollow-fiber bloreactor schematics are shown m Fig 2, with the cells on the shell side and medium on the tube side The debubbler with vent on the cell side ehml- nated an bubbles, alleviated pressure resulting from moculatlon, and allowed air to fill the vacuum resultmg from sample withdrawal The Hybrmet Hollow Fiber Bloreactor (Que/Monsanto) used was made of a 160 pm thick polysulphone fiber m a polycarbonate housing with a surface area of 1850 cm?, pore size of 300 ii, and a lo6 molecular weight cut off The total volume of the cell side was 85 ml including 45 ml wlthm the hollow fiber The shell side recycle flow of 55 ml mu--l gave a residence tnne of 0 026 h, much below the yeast mmlmum doubling tnne of 1 6 h and thus mmlmlzmg bulk mass transfer

CELL SIDE (SHELL) MEDIUM SIDE CTU%E)

1 1 FERMENTOR ]

Fig 2 Hollow-fiber bloreactor schematxs

hmltatlons on the shell side Results showed that this high recycle had no adverse effect on the cells The tube side volume was 90 ml with a recycle flow also of 55 ml mm- giving a residence time of 0 01 h which was much below the on-line sampling time of 0 13 h

Samples were spun for 20 mm at 5000 g in a Sorvall RC-5B Super Centrifuge at 4 C Off- lme analysis included glucose, ethanol, and dry cell weight for the hollow fiber experi- ment

3 RESULTS

3 1 Batch fermentatzon Results of the aerated batch fermentation

are presented m Fig 3 The dry cell weight and dissolved oxygen m Fig 3(a) shows the dlauxlc growth of yeast on glucose as described earlier DO did not rise back to 100% saturation despite the OUR falling to 0 This growth 1s also seen from the off-gas plots m Figs 3(b) and 3(c) where a high CPR and low OUR are seen m the first phase of growth when the glucose 1s fermented The RQ rises from one to a value greater than 10 as the cells switch from the oxldatlve enzyme pools last used m the moculum to the fermentatlve pools required for the batch mltlal condl- tlons An abrupt change m the RQ is seen at glucose exhaustion as the cells quickly adapt

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W :ti.-,..oo l- 02 0 10 20 30 40 50

(a) TIME (hr)

2

0 55

0 10 20 30 40 50

(cl TIME (hr)

10 -

0 --

2 --

01 : I : I 1 l 00 05 10 15 20

(e) DRY CELL WT (g/l)

25

i

@I

20 30

TIME (hrj

6 ?

?

0 10 20 30 40 50

(d) TIME (hr)

251

5, : i : 1 : : : /

1 2 3 4 5

(f-1 DRY CELL WT (g/l) Fig 3 Glucose-hmlted batch fermentation results of AB103 1 pYaEGF-25 In YEPD (See text )

a

a

to oxidize the accumulated ethanol and the medium increases with growth during glucose OUR and CPR rise together fermentation due to the utlhzatlon of nltro-

Figure 3(c) shows the pH changes to follow gen After the dlauxlc lag, growth on ethanol the yeast growth curve The acidity of the causes the medium to become more basic for

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approximately 5 h but then switches back to causing the medium to become more acidic When the stationary phase 1s reached the pH again rises A shift m catabolism (glucose or ethanol exhaustion) elicits a temporary shift m the dlrectlon of pH change from down- wards to upwards This phenomenon may indicate a shift m nitrogen source from inorganic to organic

Glucose and ethanol concentrations m Fig 3(d) show the preferential fermentation of glucose to ethanol, then after derepresslon of the oxldatlve enzymes, the utlhzatlon of ethanol at glucose exhaustion Substrate yield plots for the batch fermentation compare very favorably with results reported elsewhere [ 1, 111 Dry cell mass yield on glucose 1s 0 18 and ethanol on glucose 1s 0 43 Ethanol 0x1- datlon yields 0 53 dry cell weight per g sub- strate Substrate yields have a relative error of f 6%

The slope from the OUR us dry cell weight plot during glucose fermentation m Fig 3(e) 1s 1 5 mmol 0,/g dew/h (+O 3 mmol 0,/g dew/h) and corresponds to approximately 10% activity of the TCA cycle From an oxy- gen balance over this region, 7 5% of the glucose is oxidized, which adJusts the dry cell mass yield on fermented glucose to 0 15 and ethanol from glucose to 0 46, which 1s very close to the stolchlometrlc value of 0 50 The slope for the OUR us dry cell weight plot during ethanol oxldatlon m Fig 3(f) of 6 0 mmol 0,/g dew/h (+O 5 mmol 0,/g dew/h) matches the maximum specific oxygen capac- ity for yeast found by Rleger et al [2] and Llevense [ 131 During oxldatlon, the capacity to utlhze oxygen may hmlt maxunum growth under resplratlon as suggested by Rleger et al [2] and Barford [12] The total carbon bal- ance closes to 102% on glucose to dry cell weight and carbon dioxide For this calcula- tion the cell composltlon determined by Llevense [13] of 44 35 wt % carbon 1s used

A Lmeweaver-Burke plot for glucose fermentation yields a maximum specific growth rate of 0 44 h- and a Monod satura- tion constant of 2 1 g 1-l The correction of glucose fermentation kmetlc parameters for glucose oxldatlon 1s negligible The specific growth rate was calculated from the slope of three dry cell wt. pomts then smoothed with a first-order time delay constant of -0 75 Slmlla.rly values for ethanol oxldatlon are

calculated to be 0 10 h-l and 1 4 g 1-l respec- tively These values have a relative error of k15%

3 2 Shake flask ferment&on The dry cell weight from the shake flask

experiment follows the previous batch fer- mentation results, which indicates that rota- tion speed and air reclrculatlon are sufficient Glucose and ethanol specific utlhzatlon rates compare favorably with the previous batch results Agitation 1s sufficient to provide ade- quate oxygen mass transfer to support the oxldatlon of ethanol after glucose exhaustion Substrate yields for dry cell weight on fer- mented glucose (Yxo,), ethanol on fermented glucose ( YEG), and dry-cell weight on ethanol ( YXE) are 0 17, 0 46, and 0 60 g/g respec- tively Glucose fermentation yields are adJusted for oxldatlon to 0 14 g dew/g glucose and 0 5 g ethanol/g glucose These values also agree with the previous data

The medium pH fell from 6 6 at mocula- tlon to 5 43 at glucose exhaustion, but rose to 5 80 at ethanol exhaustion Again the pH increases m acidity until a shift m catabolism (glucose exhaustion) that causes a temporary increase m pH Despite the absence of aera- tion and pH control, the behavior of the sys- tem m the shake flask 1s representative of the batch fermentation m growth and substrate yields

3 3 Fed-batch ferment&on Figure 4 contains fed-batch fermentation

results for glucose oxldatlon Feeding schedule and resulting dry cell weight are shown m Fig 4(a) Growth rates adJust to feed rates after an mltlal period of cellular enzyme pool response from the shake flask moculum Glucose and ethanol are seen tem- porarily to accumulate early m the fermenta- tion while the enzyme pools adJust to accom- modate the mltlal feeding, after which both substrates remam near 0 g 1-l during addl- tlonal constant feedmg Glucose peaks at 0 62 g 1-l at 5 1 h, then decreases causing an increase m the fermentatlve metabolism and a small accumulation of ethanol which peaks at 0 55 g 1-l at 17 9 h Both glucose and ethanol decay to near zero levels by 20 h

The OUR and CPR m Fig 4(b) contain spikes at the exhaustion of residual glucose and temporary production of ethanol at 10 h,

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6c6 1.

FEED 5-- - -- 5

? z

4 -- --4 I

: 5 9 --

2 3__ 3 k

& 2-- D

1 -.. -- 1

0 5 10 15 20 25 30 35

(a) TIME (hr)

02

-t ! RQ (02 t t

::::m:::;::::;:::: 00 0 5 10 15 20 25 30 35

(c) TIME (hr)

00 I 00

0 5 10 15 20 25 30 35

(b) TIME (hr)

65

I a 60

55- 0 5 10 15 20 25 30 35

(d) TIME (hr)

Fg 4 Glucose-hmlted fed-batch fermentation results of AB103 1 ~YcYEGF-25 m YEPD (See text )

and at the flow rate change of glucose and ethanol exhaustion at 22 h, demonstratmg a temporary period of cellular adJustment to the new growth rate Before the utlhzatlon of ethanol, the OUR remains constant at 3 mmol 1-l h-l for the feed rate of 2 4 ml h- glucose, and then after the exhaustion of accumulated substrates and the shift m feedmg to 5 ml h-l, the OUR remains constant at 6 mmol 1-l h- Both these values correspond to the stolchlo- metric requirement to oxldlze the fed glucose at 2 4 ml h- and 5 0 ml h- respectively Spikes are also seen m the RQ m Fig 4(c) However the RQ remains below or equal to 1 resulting from the oxldatlon of glucose and residual ethanol Spikes due to early changes m feeding (

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which causes a temporary change (lastmg approximately 5 h) m the pH dlrectlon The carbon balance closes to wlthm 110%

3 4 Hollow-fiber bloreactor fermentation Hollow-fiber bloreactor results are pre-

sented m Fig 5 Dry cell weight m Fig. 5(a) shows the exponential growth of the yeast up to a density of 35 g 1-l at which time msuffl- clent volume on the cell side remained to allow for addltlonal sampling The OUR m Fig 5(b) slightly increases as the fermentation progresses, but this low level could result from the high drift m the OUR measurement (>l mmol 1-l h-l) The CPR increases slgndi- cantly and peaks at 15 h owing to a decreas- mg growth rate resulting from a lower glucose concentration The RQ m Fig 5(a) slowly increases to nearly 3, mdlcatmg a predoml- nantly fermentatlve metabohsm

5oij 0

4.

40 --

0 0

(a) TIME (hr)

115

I 0 5 20 Cd) TIME (hr)

Fig 5 Glucose-hmlted hollow-fiber bloreactor fermentation results of AB103 1 pYaEGF-25 m YEPD (See text )

a

From the glucose concentration on both the cell and medmm sides shown m Fig 5(c) and from the calculated rate of glucose flux, an average mass transfer coefficient of 45 X lop5 cm s-l 1s calculated using Flcks law assuming no glucose loss on the medium side This value decreases for the last sample because of a reduced surface area due to decreased liquid volume on the cell side from sampling The decreased volume 1s responsible for the very small glucose concentration on the cell side at the end of the fermentation which limits cell growth despite the large concentration of glucose on the medium side The relative concentrations are reversed for ethanol, from which a mass transfer coefh- clent of 68 X 10m6 cm s-l IS found using Flcks law and assummg no loss of ethanol on the medium side This value IS nearly equal the predicted value from the Stokes-Emstem

O1

o/::::;::::;::::;::::co 0 5 10 15 20

(b) TIME (hr)

65r I

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equation using the mass transfer coefflclent for glucose Dry cell mass and ethanol yield on glucose are 0 18 and 0 53 respecclvely, nearly equal to the values found earlier for the batch fermentation Speclflc oxygen uptake rate 1s 1 7 mmol g- h-l for the early part of the fermentation, very near the value found for the batch experiment TCA enzyme actlvtty 1s again approximately lo%, which adJusts the fermented glucose yield to dry cell weight to 0 15 g/g and fermented glucose to ethanol to 0 49 g/g

Mass transfer coefficient for oxygen estl- mated from the Stokes-Em&em equation 1s 8 2 X 10 -5 cm s-l using the result for ethanol The calculated maximum oxygen transport capacity for the hollow fiber IS approximately 0 02 g O2 h- This level was only 2% of that required by the yeast after glucose exhaustion to oxldlze the ethanol These calculations are supported by the observed ethanol oxldatlon rate of less than 6% of the level expected for ethanol oxldatlon Thus transport capacity of oxygen IS inadequate m the hollow-fiber membrane to support oxldatlve growth

High cell density does not affect the sub- strate yields or cell growth The pH m Fig 5(d) decreases throughout the fermentation as expected, since no shifts m catabolism occur The carbon balance for this experi- ment closes to within 100%

4 CONCLUSIONS

Table 1 summarizes the yields of the prevl- ous experiments Substrate yields are both consistent throughout the experiments and with literature values

Glucose repression 1s shown by a reduced oxygen capacity of the TCA cycle of 1 5 mmol 0,/g dew/h for glucose concentrations >0 1 g 1-l This level represents approxl- mately 10% maximum TCA enzyme activity level When fully derepressed, the oxygen capacity IS 6 mmol 0,/g dew/h as reported earlier

With adequate agitation, shake flask kinetics are representative of batch fermenta- tion results Cell growth causes medium pH to decrease except followmg a shift m cata- bolism (substrate exhaustion or increase m feed rate) which causes a temporary increase m pH for approximately 5 h, after which the

TABLE 1

Substrate yields for AB103 1 ~YcYEGF-25 m YEPD

YXGF* yXGO YEG* YXE

gig gig g/g g/g

Batch 0 15 - 0 46 0 53

Shake 0 14 - 0 50 06 flask

Fed - 051 - -

batch

Hollow 0 15 - 049 - fiber

*Corrected for glucose oxldatlon

pH again decreases with cell growth This shift may indicate a switch m nitrogen source from the normal morgamc to organic caused by the unavallablhty of the carbon backbone of the preferred substrate Thus a control algorithm using pH control as a feedback variable must account for this shift m pH dlrectlon

The hollow-fiber bloreactor has no effect on glucose fermentation However, oxygen transport capacity of the membrane IS not adequate to support resplratlon of ethanol after glucose exhaustion

Growth kinetics and substrate yields are not affected by the different reactor conflg- uratlons, catabohsms, cell densities, or changing pH from 5 4 to 6 6

ACKNOWLEDGMENTS

We would like to thank Dr Jefferson C Llevense of the Eastman Kodak Co (Rochester, NY) and Dr Harold B White of the University of Delaware (Newark, DE) for their advice The authors wish to thank the Chlron Corp (Emeryvllle, CA) for their generous supply of the cultures used for the experimental study We especially acknowl- edge the help of Dr James P Merryweather and Dr Carlos George-Nasclmento for their continuous support

REFERENCES

1 J C Llevense and H C Llm In G T Tsao (ed ), The growth and dynamics of Saccharomyces cerevwae, Annual Reports on Fermentatron Pro

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cesses, Academic Press, New York, 1982, Vol 5, pp 211 - 262

2 M Rleger, 0 Kappeh and A Flechter, J Gen Mzcrobzol, 129 (1983) 653 - 661

3 T Yamane and S Shlmlzu, Adu Bzoch Eng /Bzo- technol, 30 (1984) 147 - 194

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5 E Oura, Bzotechnol Bzoeng 16 (1974) 1197 - , 1212

6 I Ohvero, C Rulz-Maclas, A Chord1 and J M Pelnado, Bzotechnol Bzoeng, 24 (1982) 2725 - 2729

7 G Parada and F Acevedo, Bzotechnol Bzoeng,

25 (1983) 2785 - 2788 8 K E Stevenson and T R Graumhch, Adv Appl

Mzcrobzol, 23 (1978) 203 - 217 9 H Y Wang, C L Cooney and D I C Wang,

Bzotechnol Bzoeng, 21 (1979) 975 - 995 10 A J Brake, J P Merryweather, D G Colt,

U A Herberlem, F R Maslarz, G T Mullenbach, M S Urdea, P Valenzuela and P J Barr, PNAS, USA, 81 (1984) 4642 - 4646

11 S J Coppella, Ph D Theszs, Umverslty of Delaware, 1987

12 J P Barford, Bzotechnol Bzoeng, 23 (1981) 1735 - 1762

13 J C Llevense, Ph D Theszs, Department of Chemical Engmeermg, Purdue Umverslty, 1984