Design of fermentation processes

Chapter 9 VNL
(2 lectures)

Gas inlet

f, cf Gas outlet

Liquid inlet
e,ce
Liquid outlet

V = liquid volume

Contents
• How to design a process?
• Reactor types
• Characteristics of processes
• Microbial (”fermentation”)
• Mammalian
• Algal
• Waste water treatment
• The Stirred Tank Reactor
• Batch /Continuous operation/Fed-batch
• Some design cases
• Fed-batch (Example 9.7)
• Recirculation (Example 9.3)
• Multiple reactors (Example 9.2)

1
 Designing the process
When we (believe that we) have our production organism and we know
(certain aspects) of the kinetics of the process, we can start the design of
our process. There are two fundamental questions:
• What are the best conditions?
• i.e. what substrate concentration(s), T, pH etc should we have..
• How do we obtain the best conditions?
• What reactor type is most suitable?
• Stirred tank reactor, airlift, plug flow reactor, reactors in series,
recirculation reactors..
• What mode of operation is best?
• Batch, fed-batch, continuous operation.. (from a kinetic point of
view? From a practical point of view? From an economic point of
view?)

..but what do we mean by ”best”?

The three main (and conflicting..) overall aims are to get:

High yield
• We want as much as possible of our substrate to be converted into our sellable (our
most highly priced) product. A high yield is crucial for any bulk product where the
substrate cost is the dominating large fraction of the OPEX (operating costs) .
High titer (concentration)
• A major part of OPEX in almost any bioprocess are the downstream cost (i.e. the costs
for separation & purification). These costs are normally product concentration
dependent.
High productivity
• If CAPEX (capital costs, i.e costs for equipment/factory) are high, it may be more
important to produce more product per time and reactor than to achieve a high yield.

In addition, there could be issues relating to product quality, legislative

requirements and environmental concerns which limits our choice of process
design.

2
 Reactor types
bed with e.g.
immobilized aeration
cells
medium
inlet

draft
stirrer tube

sparger aeration
sparger
pump
a b c d e
Stirred tank Column type reactors

Stirred tank reactors –

Common in labs and industry

Lab-scale bioreactor
Steel reactor (10 – 20L)
Cell culture – lab scale

3
 Air-lift reactors
For the really big scale

Airlift reactors (pilot-scale)

AL200 – Bioreactor Sciences, Georgia

Figure 9.17 Probably the largest air lift reactor ever constructed with a volume of
1900 m3 and a height of 60 m. The reactor was commissioned around 1970 and
used for production of SCP from methanol by the ICI company at Billingham, UK

4
Plug flow (packed bed) reactors
For immobilized systems

J. N. Warnock et al, Packed Bed Bioreactors, in Bioreactors for Tissue Engineering, Eds. Chaudhuri and El-
Rubeai, Springer, Dordrecht, 2005

Encapsulation
Surface attachment
(biofilm)

Hollow fiber
Entrapment

Immobilized system in stirred reactors -

"MBBR" - moving bed bioreactors
• Provides support for establishment of a
biofilm
• Retains biomass in the reactor, while still
permitting mixture /gas liquid mass
transfer

AnoxKaldnes biocarriers

5
Forced loop bioreactor
A hybrid reactor

Fig. 9.18 – Textbook

Microbial bioprocesses

Production of secreted proteins (enzymes, recombinant

proteins/peptides) or smaller molecules (metabolites) using
production organisms bacteria (E. coli, C. glutamicum, Bacillli..), Yeast
(S. cerevisiae, P. pastoris), Fungi (A. niger, T. reesei (H. jecorina)..)
Characteristics
• Fast growth
• This results in need of high oxygen transfer rates (if high cell density
cultures). High stirring rates – efficient gas-liquid mixing.
• Overflow metabolism
• Need to control substrate concentration, which favours fed-batch or
continuous cultivation
• High titers desired (when producing small molecules)
• Fungal cultivations may give mixing problems due to viscosity issues
(more about that later..)

6
 Mammalian cell cultures – it is all
about mAbs!

Top selling mAb product:

Humira (adalimumab)
Accumulated sales 2014-2017:
> 60 billion USD

This is a TNF inhibitor used for

treating autoimmune diseases, such
as rheumatoid arthritis

mAbs global annual sales value expressed as a percentage of

total biopharmaceutical global sales for the indicated years.
Financial data from La Merie Business Intelligence.

G. Walsh. Biopharmaceutical benchmarks 2018, Nature biotechnology, 36: 1136-1145, 2018

Mammalian cell cultures

The dominating production host (> 70%) is CHO (Chinese hamster
ovary) cells*. Characteristics:
• Slow growth (0.01- 0.05 h-1) which gives long process times (10-12
days in fed-batch). This gives a poor productivity (but production
costs are not necessarily an issue..), and increases risks of infection.
Titers today are as high as 5 g/L.
• Media used are complex. Glucose and glutamine are the main
substrates, but various growth factors are needed. There is a
desired to go towards defined media and avoid serum for product
safety reasons.
• Lactate is produced as an undesired by-product and ammonia is
produced from glutamine.
• Shear sensitive cultures! Only gentile aeration methods are
possible (propeller type agitators operating at low stirrer rates,
roller bottles or ”skakbord”)

*Other hosts could be cell lines of human origin, such as the HeLa cell line (named after the cancer
patient Henrietta Lacks from 1951) or BHK – baby hamster kidney cells

7
 Reactors for Mammalian cell cultures

• The issues with sterility and legislative

requirements steers towards disposable (pre-
sterilized) reactors.
Corning Dow disposable reactor for
antibody&recombinant protein (about 450
USD)
• The desire for increasing productivity has led to
development of perfusion reactors/membrane
reactors
• Keep the cells at high concentrations, remove
product

Hollow fiber module Xcell ATF10

Photobioreactors (algal reactors)

Central design criteria:
• Illumination (or light penetration)
• Due to Lambert-Beers law, photobioreactors need to be thin!
Main designs are either flat bed or tubular reactors.
• Wall growth is a problem (ever had an aquarium?)
• Carbon dioxide addition/mass transfer

Flat bed photobioreactor advertised on India mart Tubular photobioreactor from bbi biotech

8
 Mode of operation

The stirred tank reactor –

Governing equations

 f , cf
Inflow
 e , ce
Outflow

V, c
d (Vc)
 V (q t  q)  v f c f  ve c e Note: A system of ODEs
dt
Volumetric transfer rate Volumetric production rate
(gaseous compounds)

9
 Considering only the liquid phase
Batch Continuous Fed-batch
(no inflow, no outflow of (inflow and outflow of liquid) (inflow but no outflow of
liquid) liquid)

 , cf  , cf
, c

at ss
dc dc v
 q(c) 0  q(c)  (c f  c)
dc
 q(c) 
v(t )
(c f  c)
dt dt V dt V (t )

Batch
Two (or more) coupled ODEs Simplest case - Monod
Monod kinetics
100
Substrate & biomass concentrations

Kinetics Initial conditions 80

60

dx
 x ; x(t  0)  x0
40

dt 20
ds
 Yxs x ; s(t  0)  s 0 0
dt
-20
0 2 4 6 8 10 12 14 16 18 20
Time (h)

Note: This is not a lag phase max = 0.5 h-1, Ks = 0.1 g L-1
A lag phase can only be seen in a plot of log x vs time

10
 Batch
Batch processes are simple, but (typically) have a
low average productivity
Final productivity
Monod kinetics
100
Substrate & biomass concentrations

80 Average productivity of biomass

(given by slope)
60

40 Decay of biomass?

20

-20
0 2 4 6 8 10 12 14 16 18 20
Time (h)

The average productivity can be clearly increased by pushing

towards higher final biomass concentrations, but that gives
problems in terms of e.g. volumetric oxygen transfer capacity

Continuous cultivation
How do the concentrations change with time?
Biomass

Steady-state operation
? Long term stability?

Start-up

time
No change in concentration with time!

11
 Continuous cultivation at SS
Concentrations vs D for a simple Monod case

Monod kinetics
120
s
q x   x   max x  Dx

Concentration (g/L)
s  Ks 100

80

 max s DK s
Ds 60 Biomass
Ks  s  max  D
40

max s f 20
Prerequisite: D Substrate
Ks  s f 0
0 0.1 0.2 0.3 0.4 0.5 0.6
D (1/h)
If this is not fulfilled, wash-out occurs max = 0.5 h-1, Ks = 0.1 g L-1
wash-out
The yield coefficients are constant, and for a reasonable affinity for the substrate, the
obtained concentrations are almost unchanged for a wide range of dilution rates.

Continuous cultivation at SS
Productivity (xD) vs D

Chemostat – Monod kinetics

50
d ( Dx )
0
40 dD
Productivity (g/L h)

30

20
Maximum

10

-10
0 0.1 0.2 0.3 0.4 0.5 0.6
D (1/h)

12
 What is the exact value of D at optimum conditions?
d ( Dx )
We have that 0
dD
Somewhat algebraically easier is to find the optimal substrate concentration, sopt

 s 
d  max YSX ( s f  s) 
K s
0  s  0
d ( Dx )
ds ds

s 2  2K s s  K s s f  0 s opt   K s  K s2  K s s f

Which gives the desired optimal dilution rate as

𝑚𝑎𝑥 𝑠𝑜𝑝𝑡
𝐷𝑜𝑝𝑡 =
𝐾𝑠 + 𝑠𝑜𝑝𝑡

This can also be expressed in dimensionless form

(common in Chapter 9)

𝑠
𝑆= ”Dimensionless concentration”
𝑠𝑓  S

m a  S
𝐾𝑠 ”Dimensionless affinity”
𝑎=
𝑠𝑓
 1
Since 0  SFor
 1Swe
< 1get  i.e. if a is small  m as S  1
m a  1

 S 
 max YSX d  (1  S ) 
using S and a, we get: aS  0
dS

S 2  2aS  a  0 Sopt  a  a 2  a

Note that if a > 1, Sopt > 1. What is the implication of this?

13
 Continuous cultivation
Example 9.1 Monod kinetics with maintenance
(the ”Herbert experiment”)

The stoichiometry of the main (“true”) reaction is obtained using the procedure in Chapter 3:
CH8/3O + 0.4483 O2 + 0.1368 NH3 → 0.6842 CH1.8O0.5N0.2 + 0.3158 CO2 + 0.923 H2O (1)

Maintenance energy is supplied by oxidation of glycerol to CO2:

CH8/3O + 1.1667 O2 → CO2 + 1.3333 H2O (2)

Ysxtrue = 0.6842 · (24.6/30.67) = 0.549 g biomass/g glycerol

Ysptrue = 0.3158 · (44/30.67) = 0.4531 g CO2/g glycerol
Ypxtrue = Ysxtrue /Ysptrue = 1.212 g biomass/g CO2

The observed yield coefficients will vary as a

function of D if maintenance is substantial
Example 9.1
Concentration (g/L)

x (biomass)

s (glycerol)

Dilution rate (1/h)

14
 Whether or not the change in yield coefficient is positive
depends entirely on the process!
Example 9.1
Ysp (g CO2/g glycerol)

Dilution rate (1/h)

CO2 in this example represents a maintenance coupled product, the yield of

which decreases as the specific growth rate increases.

Chemostat with substrate inhibition

m s
A typical kinetic expression is  2
in this case s  Ks  s
Ki
Or in dimensionless form

 S a
Ks sf
 2 where b
 m bS  S  a sf Ki

a
It is easily shown that  has a maximum for S opt 
b

15
 Chemostat with substrate inhibition

Sopt < 1, i.e. sopt < sf Sopt > 1, i.e. sopt > sf

/m /m

1 S 1 S

Multiple steady-states possible!

What D gives maximum productivity?
Somewhat more elaborate algebra needed. see Eq 9.21.

When is fed-batch operation beneficial?

• ”Overflow” metabolism, i.e. when metabolic pathway changes

occur at critical nodes as a function of concentration
• Limitations due to oxygen or heat transfer
• Inhibition effects due directly to substrate or due to impurities
(inhibitors) present in the feed. These may be bioconverted to
less inhibitory compounds if fed at low enough rate.

16
 Fed-batch
Most fed-batch processes are started with a batch phase
x
Often xf =0 (no biomass in feed,
only substrate) ?
For biomass, we thus get

d (Vx ) dx 1 dV
Vx    x  x
dt dt V dt

dx 
 (  ) x
dt V t
Batch Fed-batch

Depends on on s(t), p(t)…

This ratio is controlled by the feed rate

Mode of Advantages Disadvantages

operation
1. Continuous a. Large scale production of cheap products
STR, especially CSTR, is by far the best due to
low capital and labour costs.
b. CSTR (or Fed-Batch) needed
when production of the desired
product is catabolite repressed.
c. Due to autocatalytic nature of microbial
reactions, productivity is high.
d. Product quality is (or can be) constant
a. Infection is a risk, e.g.
caused by a short stop of the
continuous feed sterilization by steam.
b. The strain may mutate to a non-
producing strain after long production
time.
c. Down-stream equipment can be
difficult to operate in the continuous
mode.
d. Very inflexible

a. Easily switched between different a. High labour costs.

2.Batch production duties with low retrofitting costs. b. Much idle time for sterilization,
operation b. Can be properly sterilized. outgrowth of
Small risk of infection and mutation (short inoculum and cleaning.
production time) c. Safety problems when filling and
emptying reactor

3.Fed-Batch a. Same advantages as CSTR a. More labour cost than CSTR.

(a. and b.). b. Large volume to be down stream
operation b. The production time is limited processed between runs. Holding
with smaller risk of mutations. tanks used.

17
 The origin of fed-batch
Bakers’ yeast production
• Long time ago in Egypt
• Distributed small scale production (keeping of inoculum)
• Middle ages
• Surplus yeast obtained from breweries
• About 1860 Drawing by Turpin from 1840
• Dedicated process for production (Vienna process) –
aeration introduced
• WW I
• Change of substrate (from grain to molasses)
• 1919
• Fed-batch (Soren Sak process)
• By controlling the specific growth rate, aerobic ethanol
formation (overflow metabolism) was avoided

Overflow metabolism
A B
Glucose Glucose
Low specific glucose uptake rate – respiratory metabolism

Glucose + O2  Biomass + CO2

High specific glucose uptake rate – partly (or mainly)

fermentative metabolism Ethanol CO2

CO2 CO2

Glucose + O2  Biomass + Fermentative products +CO2

Fig 7.6. Illustration of the “bottle-
neck” of the oxidative metabolism
in S. cerevisiae.

Ethanol (some yeasts), acetate (E. coli, +..)

18
 E. coli - Overfeeding may result in either
overflow metabolism or oxygen limitation

Overflow metabolism Goodsell, BIOCHEMISTRY AND MOLECULAR

BIOLOGY EDUCATION
Vol. 37, No. 6, pp. 325–332, 2009
Oxygen limitation
PEP

Pyruvate

NADH
NADH
Acetyl-CoA PEP Fumarate Succinate
TCA-
NADH
Acetyl-P cycle
Pyruvate Lactate
ATP
Acetate
Formate CO2 + H2

Acetyl-CoA
NADH

Acetyl-P
Acetaldehyde
ATP NADH
Acetate
Ethanol

Basan et al, Nature, 528:99-105

How should the fed-batch reactor be operated?

The optimization is clearly a dynamic problem, i.e. the
solution is not a point but a time dependent trajectory
Intensive variable, e.g.
Control variable, e.g. feed rate
Volumetric rate

Ri   qi ( z (u (t ), x)) dV
V

Objective function Reactor volume

F (u )   R (u (t )) dt Time dependent variable, for which a dynamic

optimization is made
t
Very often the feed rate is ”optimized” by setting the specific growth rate constant. This means
that the feed rate is controlled to give a constant substrate concentration in the reactor. This is
done until the need for volumetric oxygen transfer becomes too high.

19
 The ”normal” fed-batch control problem

Feed rate

Oxygen transfer limitation

Overflow
metabolism
Allowed
operational range

q s  VX 0  e t Time
F t  
S in  S t 
Optimal (extremum) control

Fed-batch

What should be the rate of this pump?

20
Fed-batch control

Control Modelling

Measurement
Substrate tank

Reactor

Fed-batch control

• Empirical optimization
• Simply try and see what happens for different feed
profiles. If it works – use that feed profile! (still not
uncommon in industry..)
• Physiologically motivated feed-back controls
• Find information on the physiological status of the
culture, e.g. by probing control
• Model-based optimization
• Base your control on a kinetic (and reactor) model. This control
can be open loop or closed loop

21
 ”Probing control”

Make a perturbation in the inlet feed rate and

monitor the response
20
(b)
15

CER (mmol h )
-1
(I)
Gas Analyzer
10

Feed 5

0
0 0.1 0.2 0.3 0.4 0.5
Time (h)

Nilsson et al., 2001

”Probing control”

No response!
DOT (%)

DOT (%)

30 30

10 10
0 4
0 4
Flow rate
Flow rate

0 4 time
0 4 time
(min)
(min)

M. Åkesson PhD thesis, Lund University, 1999

22
Plug flow reactor
The PFR requires that the inflow contains biomass (or that biomass is retained)
The PFR is therefore used primarily for immobilized systems, but may also be
used as a final ”polishing” reactor in a reactor system
 dx S
x
S
V

dV s f xS0 q S
, sf

s  (s  ds)  qS dV  0
ds 𝑠
  qS 𝑉
=න
𝑑𝑠
dV  𝑠𝑓 𝑞𝑠

The reactor volume is thus obtained from integrating 1/qs

Capacity comparisons

The capacity of a given reactor can be said to be given

by the residence time, , required to obtain a specified
degree of conversion of the substrate.
For a reactant, s, we obtain from mass balances:
Tank reactor Plug flow reactor

s f sout  qs (sout )V  0 s  (s  ds)  qs (s)dV  0

ds  qs (s)dV  0

V s f  sout sf
 V ds
  qs (s) 
 
so u t
 qs ( s )
V 1
 
 D

23
 1
For a first order reaction,  qs

we qualitatively get

Tank
-qs
sout sf s
1
 qs

s
i.e. for a first order
reaction, the PFR is to be PFR
prefered (since the
colored area is smaller) sout sf s

What does this look like with Monod

kinetics?
 max s  s  s
 qS  q xYxs  x Yxs  Ysx ( s f  s ) max Yxs  ( s f  s ) max
Ks  s Ks  s Ks  s
Monod kinetics

12

10
s opt   K s  K s2  K s s f  2.31
8 Integral  VPFR
-1/qs (g/L h)

2 Rectangle area  VCSTR

0
1 2 3 4 5 6 7 8 9 10
s (g/L)

max = 0.5 h-1, Ks = 1 g/l and sf = 10 g/l.

24
 Finding the optimal value for s (and D) for maximizing
biomass productivit
20 20

Ysxsf /qx A Ysxsf /qx B

15 15
Substrate
inhibition
10 10

Figure 9.3 Productivity of a steady state stirred

tank reactor.
5 5
Monod kinetics: μmax = 0.5 h-1, Ks = 0.4 gL-1. sf = 2
= D-1 gL-1, Ysx = 0.5 g g-1.
0 0
Substrate inhibition: As in A, and Ki = 0.25 gL-1,
0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0 i.e. b = 8 in Eq. (9.18).
S S Product inhibition (Eq. (7.20)): As in A, and
sfYsp/pmax = 2/3.
20 20
Maintenance (Eqs. (9.4), (9.5)): As in A, and with b
Ysxsf /qx C Ysxsf /qx D
= 0.2 in (9.8),
15 15

maintenance
10 10

Product
5 5
inhibition
0 0
0.0 0.2 0.4 0.6 0.8 1.0 0.0 0.2 0.4 0.6 0.8 1.0

S S

Some design cases

• Fed-batch
• Baker’s yeast production
• Recirculation
• Waste water treatment
• Multiple reactors
• Waste water treatment

25
 Fed-batch design for Baker’s yeast production

Jästbolagets fabrik i Rotebro

https://www.jastbolaget.se/

Fed-batch design for Baker’s yeast production

Yeast is typically produced from molasses in fed-batch. In this example we will
simplify the analysis by using glucose 100 g L-1 as substrate feed.
Simplified stoichiometry:
CH2O + 0.3944 O2 + 0.115 NH3 → 0.5768 X (CH1.8O0.5N0.2) + 0.4232 CO2 + 0.65 H2O

The specific growth rate can be assumed given 0.4(h 1 ) s


by the Monod expression: s  150 (mg L-1 )

The critical specific growth rate above which overflow metabolism is about 0.25 h-1 ,
i.e. the feed rate should be controlled so that s = 250 mg L -1

26
 Fed-batch design for Baker’s yeast production
So, how do we actually feed so that we avoid overflow metabolism (i.e. keep s < 250 mg /L)?

We could have a glucose sensor on-line and apply a PID controller (perhaps).

Alternatively we use a simple feed forward design based on the mass balance
equation.
The fed-batch operation is likely to start after a batch phase, which ends at a relatively
low biomass concentration, say 1 g/L.

We could try to set the feed rate to 0.25*V (i.e. a "dilution rate" of 0.25 h-1, which is
the desired value of 

If the initial concentration of s at the start of

the fed-batch is the desired, it is easy to decide 𝑐𝑟𝑖𝑡 𝑉𝑥𝑌𝑥𝑠
= ൘(𝑠 − 𝑠 )
on a strategy. We can set 𝑓 𝑐𝑟𝑖𝑡

Fed-batch design for Baker’s yeast production

Eventually, the feed rate will approach 0.25*V (when x approaches Ysx(sf-scrit).
However, starting with that rate will not be good.

Huge overfeeding!

27
 Fed-batch design for Baker’s yeast production
If the initial concentration of s at the start of the fed-batch is the desired, it is easy to
decide on a strategy. We can set  𝑉𝑥𝑌𝑥𝑠
 = 𝑐𝑟𝑖𝑡 ൘(𝑠 − 𝑠 )
𝑓 𝑐𝑟𝑖𝑡

Assume a start volume of 1 m3

Starting at x=1 g/L (low inoculum) Starting at x=30 g/L (very dense inoculum)

x V

V x

Apart from the over-flow metabolism, we also

need to worry about oxygen transfer rates!
𝑞𝑜,𝑚𝑎𝑥 = 𝑘𝑙 𝑎(𝐶𝑂∗ − 0.1𝐶𝑂∗ ) 𝑞𝑜,𝑚𝑎𝑥 = 𝑌𝑥𝑜 𝑥
Maximum transfer rate Metabolic oxygen demand

For a given value of kl a we can determine the maximum biomass concentration for
our feed strategy.

Assume that kl a = 650 h-1 (which is possible in a stirred reactor - but not in the type
of reactor used ), we get

(- q0)max = 650 · 0.244· (1 – 0.1) = 142.5 mmol L-1 h-1

Yxo = 0.3944/0.5768 = 0.684 mol O2/C-mol X

xmax = 142.5 10-3 /( 0.684* 0.25) = 0.833 C-mol L-1 = 20.47 g L-1.

28
 Waste water treatment (Example 9.3)
A hypothetical waste water stream treated in a CSTR
𝑚 = 1 ℎ−1
sf = 4 g L-1 𝑚 𝑠
= 𝐾𝑠 = 1 𝑔 𝐿−1
𝐾𝑠 + 𝑠
=1 m3 h -1
𝑌𝑠𝑥 = 0.5 𝑔 𝑔−1

V = 0.5 m3


Wash-out! No removal of s at all! 𝐷= = 2 ℎ−1
𝑉

Waste water treatment (Example 9.3)

sf = 4 g L-1
What is the best choice of 1?
 = 1 m3 h -1
1
(partly cleaned)

V = 0.5 m3
2
(uncleaned)
𝑠𝑜𝑝𝑡 = −𝐾𝑠 + 𝐾𝑠2 + 𝐾𝑠 𝑠𝑓 = 1.23 𝑔 𝐿-1 Dopt = 0.552 h-1

1 = 0.552*0.5=0.276 m3 h-1

The BEST you can do is thus to let 73.4 % of the waste simply by-pass the CSTR

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What if you do not want to do that?
Then you will need a different design– one possibility is cell
recirculation
Same volumetric flow rate into and out of system

Centrifuge/or other
v, sf v(R+1) separation method

x, s, p v xe, s, p

Higher flow rate after

the mixing point
vR xR, s, p

Alternative configurations

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 Centrifuge/or other
v, sf v(R+1) separation method
Recirculation
x, s, p v xe, s, p

Mass balance for the biomass over the entire

system gives
vR xR, s, p
0 − 𝑥𝑒 + 𝑉𝑥 = 0
𝑥𝑒
−𝐷𝑥𝑒 + 𝑥 = 0 Define 𝑓= −𝐷𝑓𝑥 + 𝑥 = 0  𝐷𝑓 = 
𝑥
Clearly, we can now maintain biomass at SS as long as we have Df < wash-out

x
xe s Biomass in reactor

xe s

end

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