ABSTRACT

The present report aims to shed light on the

optimization of the solid/liquid separation using centrifugation. This involves

both type of centrifuge used and operation characteristics, such as flow rate.

Moreover, it investigates the centrifuge operation upon integration with

following processing steps, such as filtration and chromatography.

For this reason, the disk stack centrifuge was used to

clarify yeast homogenate, in different flow rates. The clarification was

estimated based upon the optical density values obtained for the supernatant

after the centrifugation. Furthermore, evaluation of the centrifugation was

carried out through filtration experiments for each flow rate. This was done by

obtaining the flow rate of the supernatant when passing it through the filter.

The obtained results were compared to analogous experiments for yeast and E. coli.

The experiments showed better clarification for low

flow rates. The highest clarification achieved was 99.31% and was obtained when

the flow rate was the lowest studied, and equal to 0.6 L/min. The filtration

results showed decreasing filtration flow rate for increasing flow rate in

centrifugation.

In conclusion, this study showed decreasing

clarification for increasing flow rate, but showed that other aspects are

important as well, especially when it is integrated is a multistep process.

(200 words)

RESULTS

The optical

density of the supernatant for each flow rate is plotted versus time in Figure

1.

Figure 1: Optical density of the supernatant measured

each minute over 10 minutes time, for each flow rate studied. The optical

density values reach a steady state, different for each flow rate used.

The

steady state optical densities observed for each flow rate, as well as the time

needed to achieve that value are presented in table 1.

Table 1: Steady state optical densities and time

required to reach these values for all of the flow rates studied. The number of

bowl volumes of feed until steady state are also presented, showing higher

volume requirements for higher flow rates.

Flow rate (L/min)

Steady state OD value

Time required to achieve steady state (mins)

Number of bowl volumes of feed required to

reach steady state

0.6

0.105

6

3.6

0.875

0.116

7

6.1

1.2

0.146

8

9.6

1.5

0.147

8

12

2.2

0.171

7

15.4

Figure 2

shows the dependence of the steady state OD values of the supernatant on the

flow rate used.

Figure 2: Steady state ODs measured for each feed flow

rate. In the diagram it seems that the higher the flow rate, the higher the

steady state OD value.

The data

acquired suggest a quite good quality of the experiments, as they showed the

expected. That is the lower ODs observed were for the lower flow rates used.

The number of bowl volumes of feed required to reach the

steady state clarification are summarized in table 1, for every flow rate

studied. This number is indicative of the flow pattern in the centrifuge and

thus it can be used to predict the likely flow pattern in the centrifuge. One

centrifuge volume required to be processed until steady state is reached, is

indicative of plug flow, i.e. ideal flow without mixing. On the other side, for

the fluid to be well mixed the volume processed until steady state should be

five or more bowl volumes.

Since all flow rates investigated required more than 3.6 bowl volumes of feed to be processed to reach steady state, in none

of the cases there is plug flow. Instead, almost all of them are well mixed. When

the flow rate was 0.6 L/min 3.6 bowl volumes of feed were processed until

steady state was reached. In this case the flow pattern is neither plug flow

nor well mixed, but something in between. The higher flow rates, i.e. 0.875

L/min, 1.2 L/min, 1.5 L/min and 2.2 L/min led to the processing of 6.1, 9.6, 12

and 15.4 bowl volumes of feed until steady state, which is indicative of a well

mixed flow pattern in the centrifuge.

The clarifications achieved for each one of the flow

rates, after steady state was reached, are provided in table 2.

Table 2:

Clarification values observed for the different flow rates studied. The

increase in the flow rate leads to decrease in the clarification achieved. The

Q/?

(m/s) values are presented as well, following an increasing path when flow rate

is increased.

Flow rate (L/min)

Clarification %

Q/? (m/s)

0.6 (0.1 L/s)

99.31

0.00019

0.875 (0.15 L/s)

98.98

0.00028

1.2 (0.2 L/s)

98.07

0.00038

1.5 (0.25 L/s)

98.04

0.00048

2.2 (0.37 L/s)

97.31

0.00070

The ? factor

for the centrifuge used in the present experiments, which was the CSA-1 disk

stack centrifuge, was calculated to be equal to 525.0 m2,

and the Q/? ratios for each flow rate are presented in table 2.

Figure 3 shows the

clarification plotted versus Q/?

Figure 3: Clarification plotted versus Q/?. Higher flow rate, Q leads to higher Q/?. From the diagram it seems that there is a

clear decrease of the clarification with decreasing Q/?.

In Figure 3 there is a clear correlation of the Q/? with the clarification.

Filtration experiments were also carried out in order

to evaluate the performance of the centrifugation for each flow rate. For the

evaluation, the volume of filtrate was measured after one minute of filtration.

Then, the flow rate during filtration was calculated. These values are

summarized in table 3.

Table 3: Filtration rates for the different flow

rates that were used in the centrifugation. The filtration rate of the

homogenate feed and the well spun were evaluated as well and used as

references. Higher centrifugation flow rates led to lower filtration rates,

meaning that higher throughput led to lower performance of the centrifuge.

Sample

Filtration

volume in 1 min (mL)

Filtration rate (L/hr)

Homogenate feed

9

0,54

Well spun

11

0,66

centrifugation

flow rate (L/h)

Filtration volume in 1 min (mL)

Filtration rate (L/hr)

36

12

0.72

52.5

12

0.72

72

11

0.66

90

9

0.54

132

10

0.6

Table 3

shows a quite decreasing filtration rate for increasing flow rate during centrifugation,

which is result of the poorer performance of the centrifuge for higher flow

rates.

DISCUSSION

i)

The flow sheet from fermentation to the first

chromatography column is shown in Figure 4.

Figure

4: Flowsheet for the extraction of intracellular protein from yeast. After the

fermentation of the yeast cells, the broth is centrifuged to separate the cells

from the liquid phase, cells are then resuspended. Cell disruption is achieved

through high pressure homogenization and the liquid is centrifuged for a second

time in order to remove yeast cells debris. The product is in the supernatant,

which is later filtrated in order to be prepared for the first chromatography

step.

ii)

Centrifugation and filtration are two major steps

before chromatography, as they remove the solids that would lead to fouling of

the chromatography column. In the case of the yeast cells homogenate, the feed

inserted into the centrifuge is a mixture of large and smaller cell debris, as

well as proteins in a range of sizes. In order for the product protein to be extracted,

it has to be removed out of a pool with solids/proteins which have a comparable

size with the target protein. Therefore larger solids, i.e. membranes, large

protein aggregates and organelles, have to be removed first. This occurs mostly

during centrifugation. These attributes render centrifugation prior

chromatography an essential step.

iii)

Some of the most important aspects of the disk stack,

multichamber bowl, tubular bowl, solid bowl and scroll decanter centrifuges are

presented in table 4.

Table 4: Advantages

and disadvantages of the disk stack,

multichamber bowl, tubular bowl, solid bowl and scroll decanter centrifuges. The

most important characteristics of a centrifuge are the settling area and the

operating rotatioal speed and flow rate, as these are the factors that

influence the quality of roduct, i.e. clarity of the supernatant, and the

throughput of the centrifuge as well. 1-7

Centrifuge

type

Advantages

Disadvantages

Disk stack

·

Large

equivalent settling area ?, meaning

large processing area

·High

centrifugation speed available

· Easy to

operate

· Small

footprint

· Large

processing volumes

·

Operation

at high flow rates feasible

· ?nly

for small solid content in feed

Multichamber

bowl

· High

centrifugation speed available

· Small

settling area

· Operates at

low flow rates

Tubular bowl

· Processing

both liquid/liquid and solid/liquid separations

· Possible

foaming

Solid bowl

centrifuge

· Higher

centrifugation speed available

· Operates at

low flow rates

Scroll

decanter centrifuge

· Continuous

operation available

· Short

cleaning time

· Complex

iv) Steady state optical

densities observed for the different flow rates studied.

Figure 2 shows that the lower the flow rate used the

better the clarification achieved. This means fewer solids in the supernatant

and thus lower OD values. For example, for the flow rates 0.6 L/min and 0.875

L/min, which were the lowest flow rates studied, the steady state OD values

were 0.105 and 0.116 correspondingly. On the other hand, for the higher flow

rates 1.2 L/min, 1.5 L/min and 2.2 L/min these values were 0.146, 0.147 and

0.171, which are higher than those observed with the low flow rates. These

findings are in line with the expected pattern of increasing OD values with

increasing flow rate, as less yeast debris are removed and ODs is proportional

to the cell debris in the liquid.

Clarification values observed for the different flow

rates studied

For the lowest flow

rate 0.6 L/min, the clarification percentage was 99.31%, which was the highest

clarification observed. The 0.875 L/min, 1.2 L/min and 1.5 L/min followed with

98.98%, 98.07% and 98.04% clarification correspondingly, while the highest flow

rate, i.e. 2.2 L/min resulted in 97.31% clarification, the lowest among all. As

explained earlier in this report, this is due to the fact that the low flow

rates enable the centrifuge operate in the best way, from the aspect of solid

removal.

Clarification

plotted versus Q/?

Table 2 shows that higher flow rates exhibited higher

Q/? values, as ? is constant for a specific centrifuge. So for

example, when the flow rate was 0.6 L/min, Q/? was 19*10-8 m/s, while for the 0.875 L/min

flow rate, the same value was 28*10-8 m/s. For operation at higher flow rate the Q/? values were higher. In more

detail, these where 38*10-8

m/s, 48*10-8 m/s and

70*10-8 m/s for the 1.2

L/min, 1.5 L/min and 2.2 L/min correspondingly.

Effect of Q/? on clarification

The values obtained

for the clarification, as presented in figure 3, show that clarification is

inversely proportional to the Q/?. The highest clarification that was achieved was 99.31 %, and was

observed when Q/? was equal to 19*108 m/s. the lower clarification values

98.98 %, 98.07 %, and 98.04 % follow, for the Q/? values 28*108 m/s, 38*108 m/s and 48*108 m/s correspondingly. The lowest clarification

corresponded to the highest Q/? value, 70*108 m/s,

and was found to be 97.31%.

Filtration experiments

Table 3 shows that high flow rates for centrifugation

resulted in lower clarification levels and higher ODs values, both indicating

higher presence of solids in the liquid. These solids are the key to the lower

flow rates observed during filtration, as these tend to block the membrane.

Similarly, lower flow rates led to lower presence of cell debris n the material

and therefore the liquid was able to move through the filter pores with more

ease. Specifically, the 36 L/hr and 52.5 L/h flow rates of centrifugation

resulted in the same filtration rate 0.72 L/h. The 72 L/h flow rate gave a

liquid which passed through the membrane with 0.66 L/h, while the higher flow

rates 90 L/h and 132 L/h were found to correspond to 0.54 L/h and 0.6 L/h

correspondingly. These values are not totally in line with the expected

pattern, such as the 90 L/h and 132 L/h flow rates where the higher flow rate

in centrifugation gave higher filtration flow rate. This deviation may be

attributed to human errors in handling during filtration, such as membrane

placement or time measurement. Other affecting factors could the fact that

there was only one filtration experiment for each sample, as well as the approximate

measurement of the volume filtrated in 1 min.

In the

case of an integrated platform of centrifugation and filtration as a

preparation step for a chromatography column, the size of the filter would

depend on the flow rate of the incoming liquid in the centrifuge. In order for

a high flow rate to be used during centrifugation, a larger size of filter

would be required to ensure the solid removal without blockage of the membrane,

as there would be more remaining solids in the liquid for high flow rates. In

this way, the whole volume of liquid will be processed during filtration and be

well prepared for chromatography. It is essential that solid removal is

successful prior to the chromatography step, as in the opposite case the column

would block. In the same way, in the case of lower centrifugation flow rates

used, a smaller filter would be sufficient for the preparation of the feed for

the chromatography, as the solids would be less, as indicated by the previous

data presented.

v)

My clarification values are similar to the clarification

values obtained for different Q/?,

as reported by Bracewell et al (2008) for yeast homogenates. The clarifications

achieved ranged from low percentages to 99% for the studied operating

conditions.

On the other hand the reported values for E.coli homogenates,

as published by both Li et al (2013) and Chatel et al (2014) are much different

than those of yeast homogenates. Both studies showed a lower clarification

achieved for E. coli homogenates. For

example, Chatel et al (2014) found that for Q/? 4*10-8 less than 60% of the solids were

removed. Same, Li et al (2013) found that only 30% of the solids were removed

for the same Q/?,

while the clarification achieved for yeast homogenates was more than 97% for

every Q/?.

Though, in all studies including the present report,

the clarification had a decreasing pattern for increasing Q/? values.

In fact, the above studies showed that the level of clarification of E. coli homogenates could reach the

clarification levels of the yeast homogenates for much lower operating Q/?, such as

less than10-9 value for Q/?.8-10

Solid removal

with combination of methods

The use of an integrated platform with a series of

steps enables further optimization of the process as a whole, to give better

results than each step alone would give. For example, the opportunity of

choosing a better throughput for the centrifugation step is given, as the

following filtration step will make up or the lower quality of separation

during the centrifugation step. To study this, I would conduct a series of

experiments using different centrifuge types operating at different flow rates

in combination with different filtration steps. The difference in the

filtration step could be the size of the filter, the size of the pores, as well

as the pump forcing the liquid to flow. The process could be evaluated on the basis

of output material quality and total operation time. Investigation of the final

material could be done through a following chromatography step, e.g. to measure

the fouling of the chromatography column, or measurement of its optical

density.

REFERENCES

1.

Letki,

Alan G. “Know When to Turn to Centrifugal Separation.” Chemical

Engineering Progress. September 1998: 29-44.

2.

McCabe,

Warren L., Julian C. Smith, and Peter Harriott. Unit Operations of Chemical

Engineering. 5th ed. McGraw-Hill, New York, 1993.

Print.

3.

Perry,

Robert H., and Don W. Green. Perry’s Chemical Engineers’ Handbook, 7th ed. New

York: McGraw Hill, 1997: 18-106 – 18-125. Print.

4.

Rousseau,

Ronald W. Handbook of Separation Technology New York: John Wiley & Sons,

1987: 163-167. Print.

5.

Schweitzer,

Philip A. Handbook of Separation Techniques for Chemical Engineers. 2nd ed.

New York: McGraw-Hill Inc., 1988: 4-59 – 4-88.

Print.

6.

Svarovsky,

Ladislav. Solid-Liquid Separation. 3rd ed. Boston: Butterworths & Co.,

1977: 260-273. Print.

7. Torzewski, Kate. “Facts at your

Fingerprints: Sedimentation Centrifuging”. Chemical

Engineering . January 2008: 27

8.

Li ,Q, Mannall, G, Ali,S, Hoare M (2013) An ultra

scale-down approach to study the interaction of fermentation, homogenisation

and centrifugation for antibody fragment recovery from rec E. coli ,

Biotechnology and Bioengineering (110): 2150-2160.

9.

Chatel, A, Kumpalaume, P, Hoare ,M. (2014) Ultra

scale-down characterisation of the impact of conditioning methods for harvested

cell broths on clarification by continuous centrifugation – recovery of domain

antibodies from rec E. coli , Biotechnology and Bioengineering (111):

913-924.

10.

Bracewell,

D.G., Boychyn, M., Baldascini, H., Storey, S.A., Bulmer, M., More, J. and

Hoare, M. (2008), Impact of clarification strategy on chromatographic

separations: Pre-processing of cell homogenates. Biotechnol. Bioeng.,

100: 941–949. doi:10.1002/bit.21823

APPENDIX

Estimation of the steady state OD values

These values

were estimated based on Figure 1. These correspond to the OD value where no changes

in OD are observed beyond that point. For example, for the 0.6 L/min flow rate,

the OD seems to be stabilized in the 0.105 value, as the next values are close

to 0.105 and 0.105 was the OD of the supernatant for a second time, after 9

minutes of centrifugation. The methodology used is clearer for the flow rate of

1.2 L/min. In this case, OD rises with time but stabilized at 0.146 which is

the steady state OD value.

Calculation of bowl volumes of feed

For the

calculation of this volume it is essential to know the time required to reach

steady state. This time is presented in table 4 as well, together with the OD

values for each time interval. By multiplying this time interval with the flow

rate, the calculated value is the total volume of feed needed to be processed

until steady state. The number of centrifuge bowls that this volume is equal

to, is (volume of feed processed)/(centrifuge volume). Therefore, the number of

bowl volumes of feed that are required to be processed until steady state OD is

equal to

(Q*ts)/VB,

Where Q

is the flow rate,

ts

is the time required until steady state and,

VB is

the total volume of the centrifuge.

For

example, for the calculation of the bowl volumes of feed required to reach ODs

for the 1.2 L/min it is:

Q=1.2

L/min

ts=8

min.

The total

centrifuge volume VB is 1.0 L for the Pathfinder disk stack

centrifuge and therefore it is:

Bowl

volumes of feed = (1.2 L/min)*(8 min)/(1.0 L) = 9.6

The exact

same methodology was used for all flow rates under investigation.

Calculation of clarification

The clarification

percentage for each flow rate was used via the formula

C =

(ODf-ODs)/(ODf-ODw)*100% (1),

Where ODf

is the OD value of the feed,

ODs the

OD value of the supernatant of the centrifuged feed under certain flow rate,

and

ODw is

the OD value of the supernatant after high speed centrifugation for long time-

this is practically the most clarification that can be achieved through

centrifugation.

The

clarification was calculated in the same way for every flow rate studied. An

example calculation is that for the 0.6 L/min flow rate.

The ODf

was measured to be equal to 3.377, while the ODw was 0.082. Also, the steady

state OD value for the 0.6 L/min was estimated to be 0.105. So, from (1) it is

C = (3.377-0.105)/(3.377-0.082)*100

= 99.312 % or 99.31%.

Calculation of the ? factor

The

equivalent settling area ?

of the Pathfinder disk stack centrifuge used, the CSA-1, is given by the

following equation:

?

= (2*?*F)/3*(z/g)*?2*cot?*(Ro3

– R13)*Cds (2),

Where

Ro is the

outer radius

R1 is the

inner disc radius

Z is the

number of discs in the stack

?

is the half disc angle

F, the

correction factor for area occupied by caulks, and

Cds is

the calibration factor for non-ideal flow.

For the centrfuge used these values

are known to be as:

Ro=0.055 m

R1=0.0261 m

z=45 discks

?=38.5 degrees

F=0.9

N=9800 rpm

Cds=0.4

The operating speed of the centrifuge

is 9800 rpm, which is equivalent to (9800 rpm)/(60 s*min-1) = 163.33

rps. Therefore ?, which is equal to 2*?*?, is

? = 2*?*? = 2*3.142*163.33 s-1 =

1026.387 s-1.

Since cot? = 1/tan? = 1/tan38.5 = 1/1.031

= 0,969 it is cot? = 0,969

Therefore (2) becomes

?

= (2*3.142*0.9)/3*45/(9.81m*s-2)*(1026.387s-1)2*cot(38.5)*( 0.055 m)3

– (0.0261 m)3*0.4

= 524.967 m2.

Calculation of Q/? values

Since

both the flow rate Q values and ?

are known, Q/?

values were calculated easily. The following is an example calculation.

For Q =

0.6 L/min = (0.6 L/min)/(60 sec*min-1) = 0.1 L/s or 0.1*10-3

m3s-1.

So, it is

Q/?

= 0.1*10-3 m3s-1/ 524.967 m2 = 0.00019*10-3

ms-1.