An investigation into the use of the spinning cone column for in situ ethanol removal from a yeast...

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Process Biochemistry, Vol. 31, No. 7, pp. 651-658, 1996 Copyright © 1996 Elsevier Science Ltd

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An Investigation into the Use of the Spinning Cone Column for in situ Ethanol Removal from a Yeast Broth

A. J. Wright & D. L. Pyle Biotechnology and Biochemical Engineering Group, Department of Food Science and Technology, The University of Reading, Whiteknights, PO Box 226, Reading RG6 6AP, UK

(Received 13 December 1995; accepted 10 February 1996)

The spinning cone column (SCC), previously employed in the food industry for volatile removal at low temperatures, is investigated with the aim of removing ethanol in situ from a yeast broth. A 100 litre fermenter was integrated into the SCC pilot plant and broth passed continuously through the column. Although the SCC effectively removed ethanol from the broth, cell viability declined significantly with time. Laboratory scale experiments suggest that this is a result of various factors, the main one being the effect of reduced pressure upon cell size and shape. It was proposed that passing broth through the SCC at intervals rather than continuously, with periods to allow cells to 'recover' could prove to be more effective whilst keeping the broth ethanol concentration low. This process was employed upon cells at both stationary and logarithmic growth phases, and the results compared for cell viability and extent of recovery. Copyright © 1996 Elsevier Science Ltd

INTRODUCTION

The concept of using alternate stationary and spinning cones in a vertical countercurrent gas- liquid contacting device was initially developed in the 1930s for the separation of oxygen isotopes. 1 Today, the main use of the spinning cone column (SCC) is in the food industry for the removal of volatile compounds such as flavours and sulphur dioxide. 2

The internal layout of the SCC is shown in Fig. 1. The SCC consists of a series of alternate stationary and spinning cones, the spinning cones being attached to a centrally rotating shift. The cones lie at an angle of 45 ° to the shaft.

Liquid flow Liquid is pumped into the top of the column and falls onto a stationary cone below where a thin film of liquid flows across the upper surface

Fig. 1. (SCC).

651

' x \ \"~

.N\',-

" ~ +'+ IN

The internal layout of the spinning cone column

1.Feedm 2.L~do.t

652 A . J . Wright, D. L. Pyle

stallo~,/cone

Rotltmg cone

Fig. 2. SCC.

Path of liquid on ., s p i n ~ ©on*

) c ~

4 Pump blades on Spkmi~ C, om~e

The spiral path of l iquid be tween cones in the

under gravity towards the column shaft. The liquid then falls onto a rotating cone and is forced upward and outward across the cone surface since each liquid element experiences a centrifugal force proportional to its distance from the axis of rotation and to the square of its angular velocity. The liquid particles follow a spiral path as shown in Fig. 2. The centrifugal forces imposed upon the liquid produce a thin film across the spinning cone surface, the thick- ness of which decreases with distance from the axis of rotation. This film is turbulent under the normal operating conditions.

The liquid leaves the lip of the spinning cone essentially as a horizontal sheet which quickly breaks up into a spray of droplets against the column housing. A ring of rotating liquid forms against the housing losing its angular momen- tum due to friction. The liquid subsequently falls onto a stationary cone below and the process is repeated along the length of the SCC until the liquid flows out at the base.

The pathlength of the liquid is large and a high degree of turbulence is created throughout the flow field due to the rotation and spraying of the liquid against the housing. Each spinning cone redistributes the liquid flow so that chan- nelling does not occur. The nature of the liquid flow allows the column to handle highly viscous fluids and slurries, a property of great import- ance for food processing.

V a p o u r f l o w Vapour (steam or an inert gas feed) is pumped upwards through the column, thus creating a countercurrent flow. The vapour comes into close contact with the liquid as it traverses the spaces between alternate cones and removes volatile compounds which are later recovered at the top of the column by condensers (Fig. 1).

Fig. 3.

1. Stationary Cone 2. Rotary Cone 3. Fin 4. Rotaly Shaft 5. Inner Throat 6. Outer Throat

An SCC cone set.

The addition of fins to the underside of the rotating cones 3 converts each cone set into a radial flow gas pump so that the stack of cones in the column becomes a multistage centrifugal fan (Fig. 3). The rotation of the cones thus induces a pressure drop across the column which is opposite in sign to the pressure drop resulting from the upward vapour flow. There- fore, depending on the rate of rotation and upward vapour flow, the pressure drop can be negative, positive or zero. In addition, the rotation of the fins creates a large pathlength and produce a high degree of turbulence in both the vapour and liquid phases resulting in high mass transfer rates. The gas particles follow a spiral path as in the case of the liquid particles (Fig. 2) due to the rotation of cones and fins. Low entrainment of liquid droplets in the vapour phase is achieved due to the centrifugal forces.

The combination of effects described above upon the liquid and vapour phases makes for a highly efficient separation of volatile compounds such as flavours from a liquid phase. A thin, turbulent film of liquid is brought into close contact wth a countercurrent turbulent vapour flow over a large surface area. Both phases follow a large spiral pathlength induced by centrifugal forces created by the rotation of the spinning cones. The fact that the two phases are turbulent and that the interface between them is large ensures that transfer of volatile compounds is rapid and efficient.

The ability to minimise the pressure drop across the column allows low and uniform operating temperatures to be implemented without

Spinning cone column ethanol removal 653

an adverse effect upon the efficiency of separation. This is an important advantage of the SCC over other volatile removing methods, as ther- mal damage can be kept to a minimum. The retention time in the SCC is low compared to .other devices (minutes compared to hours) also minimising damage to the product and feed. l'he unit can operate efficiently over a wide range of flowrates. Results on the flooding characteristics of the SCC will be published separately.

The efficient removal of volatiles from a slurry at low operating temperatures by the SCC suggests that it could be part of an integrated fermentation process producing a volatile product. For example, ethanol extraction from a yeast broth may be possible in order to relieve the inhibition the alcohol exerts upon cells. Various in situ ethanol removal processes which utilise the difference in volatility between ethanol and the fermentation medium, com- posed mainly of water, have previously been developed: These processes which include the Vacuferm, 5 Flashferm 6 and Biostil, 7 enable ethanol extraction by boiling or evaporation at fermentation temperature thus forming a con- centrated vapour which can be condensed. However, problems with these and alternative in situ ethanol recovery 8'9 methods, including specificity, maintenance of cell viability and cost, have hampered further development. The use of the SCC for this purpose has not previously been considered. The benefits of maintaining a low concentration of ethanol in a fermentation broth are significant, particularly in the case of the yeast Kluyveromyces marxianus where, in addition to the production of biomass, an extracellular pectinase of great value in the food industry is produced. 1° In this paper, pre- liminary studies into the possibility of this further use for the SCC are presented.

MATERIALS AND METHODS

Table 1. Pilot plant parameters

Parameter Value

Temperature of system (°C) Pressure of system (kPa) Liquid flow rate (litre h ~) Strip rate (%) Frequency of cone rotation (Hz)

30" -95 100"

9" 35.3

"Setpoint.

operating parameters for the pilot plant in this study are given in Table 1.

A liquid flow rate of 100 litre h -1 was set, thus giving a mean residence time of approximately 3-4 min in the SCC pilot plant. The temperature of the system was maintained at 30°C using the product heater, and monitored and controlled throughout. The column temperature was chosen to be the same as in the fermenter. The strip rate equals the ratio of vapour mass to liquid mass flow.

Fermentations were performed in an LH fermenter of working volume 1001itre. The fermenter was sterilised at 121°C for 15 min, whereas the SCC was sterilised by blowing steam through for 20 min. The yeast Kluyvero- myces marxianus (NCYC 587) was grown in a medium containing 100 g litre-1 glucose, 10 g litre -1 yeast extract, 1 g l i tre-I MgzSO4, 1 g litre -~ KH2PO4 and 0.5 g litre-~ NHaSO4 at pH 4.5 and 30°C. The majority of experiments were performed on yeast broth grown to early stationary growth phase, i.e. the death phase had not begun. Stationary phase occurred as a result of ethanol inhibition and not as a consequence of substrate depletion. Viability was determined by plate counts with malt extract agar. The integration of the fermenter and SCC pilot plant is shown in Fig. 4. Samples throughout the investigations were taken from two points in the system, namely directly from the fermenter or from the return stream as shown in Fig. 4.

All trials were conducted on the Flavourtech Europe Pilot Plant which incorporates a Model 1000 Spinning Cone Column. The SCC has a nominal maximum liquid throughput of 1000 litre h-1, however, the sizing of ancillary com- ponents such as pumps and heat exchangers, limits the maximum liquid throughput of the pilot system as a whole to 600 litre h-1. The

RESULTS AND DISCUSSION

A yeast broth was grown in a batch culture to stationary growth phase (i.e. ethanol inhibition of growth occurred, substrate depletion did not play a role) and pumped through the SCC continuously and returned to the fermenter for 8 h. The ethanol concentration and cell viability in

654 A.J . Wright, D. L. Pile

D

.k,

~ - -~ [

Fmlu~r

Fig. 4.

* - ' t '-! I

The pilot plant SCC with integrated fermenter.

that in the SCC. After 20 min and 2 h, respectively, samples were taken and examined under an optical microscope. When compared to a control sample, a significant decrease in cell size was seen in both samples (Fig. 6). This suggests that the net result is cell implosion, most likely as a result of dissolved gases, less soluble at reduced pressure, leaking from the cell into the surrounding liquid and into the gas phase. Large vacuoles and protruding nuclei seen in the cells exposed to a vacuum support this hypothesis. Shadows around the cell perimeter

(i)

the fermenter broth as determined by plate counts are shown in Fig. 5. There was the expected decrease in the ethanol level (from 16 g li tre- 1 at time=0 to < 2 g l i tre- ~ after one pass through the column). However, cell viability fell continuously to around 25% of its initial value after 8 h operation (i.e. a turnover of 8 fermenter volumes). Two hypotheses for this decrease were proposed, namely damage to the membrane including the plasma membrane, or the removal of volatiles necessary for repro- duction and growth, e.g. oxygen (K. marxianus is a facultative anaerobe, however a tiny quan- tity of oxygen is always necessary for growth). Consequently, various laboratory scale experiments using a 1 litre fermenter were designed to determine the cause of the decrease in viability. The initial growth was carried out under sealed conditions so that there was no removal of ethanol from the system.

(1) A culture in the stationary growth phase was subjected to a vacuum of the same order of

(ii)

(iii)

16.00 ,

14.00 i [ * efu/ml 10A-7

12.00 /"-'41-- ethanol g/L

10.00

8.00

6.00 ~

4.00 - 0 -" -" e .~ 2.00 '.. = = = 0.00 I I I I

0 100 200 time/min 300 400 500

Fig. 5. Broth ethanol concentration and cell viability for broth pumped continuously through the SCC.

Fig. 6. The effect of reduced pressure upon cells in the stationary growth phase (i) control, (ii) after 20 min exposure to a vacuum, (iii) after 2 h exposure to a vacuum.


suggest that the alteration in the shape to that of a biconcave disc was possibly a result of cell wall damage.

(2) Three cultures at early stationary growth phase were exposed to a vacuum of the same order of that seen in the SCC. One culture was exposed to a continuous vacuum, one experienced pressure swings from atmospheric to vacuum and vice versa, whilst the third was exposed to a continuous vacuum while being sparged with air. The use of a series of pressure swings replicated more accurately the condi- )ions in the SCC as cells passed from the :ermenter at atmospheric pressure to a vacuum vessel (the column) and back to the fermenter continuously. Figure 7 shows the cell viability with time for each experiment.

In all cases a decrease in cell viability was observed on the application of a vacuum. A steady decline in cell viability was observed when a continuous vacuum was applied. How- ever, when a vacuum was applied at intervals, ':hus subjecting the cells to pressure swings, the 'all in viability was more pronounced. Despite :his, a recovery in viability was found when the .gressure was increased to atmospheric level. Blowing air through the broth did not appear to flleviate the decrease in cell viability when a vacuum was applied. The dramatic fall in cell viability observed with operation of the SCC ,vas not replicated, despite the fact that a simi- ,ar vacuum was applied. This is illustrated by ~he variation in the value of the death constant, KD, which was calculated to be 0.114 h -1 for a -'onstant vacuum compared to 0.15 h-1 for pas- rage through the SCC. This suggests that other factors combine with the adverse effect of the ~wings in pressure, to cause the large decrease in viability. These factors are likely to stem

from the passage of the cells through the whole pilot plant. The cells pass through powerful pumps and restrictions such as valves. The cells are also subjected to a large shear force created by the motion of the spinning cones thus increasing the likelihood of membrane damage.

The results above suggest that no advantage comes from the continuous passage of cells through the SCC pilot plant in order to remove ethanol, although the ethanol concentration is significantly lowered. However, it may be possible still to use the SCC for in situ ethanol removal if the cells were passed through the column for short intervals then being allowed to 'recover' before being pumped through the SCC again.

A series of trials were performed where the broth was passed through the column for a specific length of time, T1, before being allowed to recover for 1 h, T2. The same values as above for the SCC parameters were employed. The cell viability was monitored throughout. At the start of the experiment, the broth was in the early stationary growth phase because of ethanol inhibition. Figures 8 and 9 illustrate the changes in viability of a broth returning to the fermenter and the broth in the fermenter, respectively, where T1 = 30 min and T2 = 1 h. The first two to three samples, i.e. 4-6 min after passage through the SCC was initiated, were diluted with liquid already in the system and therefore should be disregarded.

An overall decrease in cell viability with time during T1 was seen. However, unexpected and as yet unexplained peaks in the graphs were observed approximately 10-15min after the passage of cells through the SCC was initiated. The rapid fall in viability occurring due to passage through the SCC soon outweighs this

Fig. 7.

1.60E+I0

1.40E+I0 I ~ pressure. .swings ~ , , 1.20E+10 ~ an" spargmg [ ...." 1.00E+ 10

"~8 00E+09 ~ 6'00E+09 - 4.00E+09 2.00E+09 0.00E+00

0 50 I00 150 200 250 300 350 time/rain

Effects of continuous vacuum, pressure swings and continuous vacuum with air sparging upon cell viability.

656 A.J . Wright, D. L. Pile

Fig. 8.

2.50E+10

2.00E+I0

• "~ 1.50E+I0

~ 1.00E+I0

5.00E+09

0.00E+00

• ¢ O

OO •

I I I I

0 20 40 60 80 time/rain

I I T1 T2

Cell viability of broth in the return flow for T1---30 min and 7"2=1 h.

¢

100

I

m ~

(

120

I T1

140

effect. The experiment was also performed with a broth in the logarithmic growth phase for T1 =30 min and 15 min, respectively, and /'2=1 h (Figs 10 and 11).

The first passage through the SCC gave a similar graph to that for cells in the stationary

phase. However, while the latter cells recovered to a large degree, i.e. the viability increased to a value close to the initial value, this was not found with growing cells. After 1 h the viable cell count had risen by only a small fraction above that observed at the end of the period T1.

Fig. 9.

1.80E+I0

1.60E+I0

1.40E+I0

1.20E+I0

~I.00E+10

~8.00E+09

6.00E+09

4.00E+09

2.00E+09

0.00E+00

0

T1

I

20

i

• •

i I I I I

40 601me/miSO ~ . . . . ~ . 100 120

I I T2 T1

Cell viability of the broth in the fermenter for T1=30 min and Tt =1 h.

140

2.00E+I0

1.80E+I0

1 . 6 0 E + 1 0

1.40E+10

~ 1.20E+10 1.00E+10

S.00E+09 6.00E+09

4.00E+09

2.00E+09

0.00E+00

%

• • Has % %*** ~ "

I I I I I

20 40 60 80 100 timodmin

I T1 = 172h I T2 FT1=1/4~

120

Fig. 10. The effect of passing growing broth through the SCC - - return flow cell viability.


Fig. 11.

9.00E+09

8.00E+09

7.00E+09

6.00E+09

~ 5.00E+09

4.00E+09

3.00E+09

2.00E+09

1.00E+09

O.OOE+O0

I ~ e e % •

I I t I I

0 20 40 60 80 100 time/min

I I I I T1 = 1/2h T2 T1 = 1/4h

The effect of passing growing bro th th rough the SCC - - f e rmen te r cell viability.

120

Fhis suggests that the constantly dividing cells are more susceptible to membrane damage from the SCC than cells which have ceased growing.

CONCLUSIONS

Fhe spinning cone column was investigated with a view to employing it for the in situ removal of ethanol from a yeast broth. Results obtained from continuously pumping broth through the column indicate that although the ethanol removal efficiency after 8 h is high (> 85%), the yeast cells are adversely affected by the application of a vacuum and consequently viability falls. Cells observed under a microscope after the application of a vacuum show a rapid and significant decrease in size with an apparent change in shape to that of a biconcave disc. Implosion or collapse due to dissolved gases leaving the cells as a result of the reduced pressure is the most likely explanation for this observation.

Laboratory experiments replicating the changes in pressure experienced by the yeast passing through the pilot plant indicate that the decrease in cell viability as a result of pressure changes was more significant than as a consequence of vacuum alone. This may be alleviated by an increase in column temperature as a result of an increased pressure in the column. The difference in pressure between fermenter and SCC would then be considerably smaller. However, although a moderate increase in temperature would have little effect upon yeast cells, a large increase would result in tempera-

ture shock. Laboratory experiments indicate that a repeated momentary increase in temperature of above 50°C leads to a decline in biomass and ethanol production, the extent of which increases with increasing transient temperature. Therefore, any increased column temperature introduced to alleviate the effects of pressure swings cannot realistically be higher than 40-45°C if detrimental effects are to be avoided. Caution must be taken when increasing the temperature of cells due to the fact that the plasma membrane, the area most at risk from the effects of reduced pressure (hence the implosion observed in Fig. 6), increases in fluidity making it more vulnerable to damage. Sparging with air did not have any apparent advantage with respect to the decrease in viability observed when a vacuum was applied upon cells. This indicates that the decrease in dissolved oxygen concentration as a result of reduced pressure is not a major factor.

The degree of cell disruption caused by the passage through the SCC pilot plant could not be replicated in laboratory scale investigations, suggesting that other factors play a role. Likely candidates are the shear forces created by the rotation of the cones and the effect of high flow rate pumps and valves through the entire plant.

Although the continuous recycling of broth through the SCC could not be employed suc- cessfully as a consequence of the decline in cell viability, the option of intermittent recycling could in theory be used. However, this process depends upon the length of time of recycling (T1) and length of time allowed for recovery (T2) to a large degree. In the case of the stationary growth phase cells, recovery was

658 A.J. Wright, D. L. Pyle

achieved after a recovery period of 1 h. How- ever, no recovery was seen with actively growing cells, a result that leads to a greatly restricted process as biomass and ethanol productivities are greatest during the log phase of growth. More importantly, in the case of K. marxianus pectinase productivity is greatest towards the end of the log phase. Care must also be taken when using stationary phase cells to prevent the death phase of the growth process playing a significant role resulting in extensive loss of cells. The value of T1 must also be carefully chosen to prevent a widespread decrease in the viable cell count whilst allowing time for the ethanol concentration in the fermenter to be effectively lowered, i.e. a compromise between the two must be reached. Further research into this area is obviously necessary before any system can be developed and proposed as an efficient method of in situ ethanol removal.

We have not yet carried out a full economic analysis of the use of the spinning cone column as part of an integrated fermentation product removal process. However, this study (and other work as yet unreported) shows that high levels of ethanol recovery from the broth and high ethanol concentrations in the product stream can be achieved at very low strip rates, i.e. at low vapour/liquid ratios. From a thermo- dynamic perspective, then, the process is thus potentially extremely efficient, and this feature (which stems from the high stage efficiency and multistage nature of the SCC) has been recog- nised 12 as one which gives a distinct advantage over conventional evaporation processes.

ACKNOWLEDGEMENTS

We gratefully acknowledge Steve Sykes and Phil Riley for their assistance with experiments and discussions, and the financial support of Flavourtech.

REFERENCES

1. Pegram, G. B., Urey, H. C. & Huffman, J. R,. Distill- ing apparatus for separation of isotopes. The Physical Review, 49 (1936) 883.

2. Sykes, S. J., Casimir, D. J. & Prince, R. G. H., Recent advances in spinning cone column technology. Food Australia, 44 (10) (1992) 462-4.

3. Casimir, D. J. & Huntington, J. N., A distillation column for essence recovery. Int. Fruchtafttunion Tech. Komrn. Ber., 15 (1978) 115-24.

4. Shah, N., M.Sc. project dissertation. The University of Reading, UK, 1995.

5. Sundquist, J., Blanch, H. W. & Wilke, C. R., Vacuum fermentation. In Extractive Bioconversions, ed. B. Mattiasson & O. Hoist, Marcel Dekke r, New York, 1991, pp. 237-58.

5. Cysewski, G. R. & Wilke, C. R., Process design and economic studies of alternative fermentation methods for the production of ethanol. Biotech. Bioeng., 20 (1977) 1421-44.

7. Maiorella, B. L., Wilke, C. R. & Blanch, H. W., Alco- hol Production and Recovery. Adv. Biochem. Eng., 20 (1) (1981) 43-92.

8. Ehnstr6m, L., Frisenfelt, J. & Danielson, M., The Bio- stil process. In Extractive Bioconversions. ed. B. Mattiasson & O. Hoist, Marcel Dekker, New York, 1991, pp. 303-21.

9. Daugulis, A. J., Integrated reaction and product recovery in bioreactor systems. Biotechnol. Progress, 4 (3) (1988) 113-22.

10. Mattiasson, B. & Holst, O. (eds.), Extractive Biocon- versions, Marcel Dekker, New York, 1991.

11. Craig, A. J. McA., Flavour recovery by the Flavour- tech recovery system. Food Technol. Australia, 39 (1987) 102-4.

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