ABSTRACT This was done by obtaining the

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.

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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.

 

 

 

 

x

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