Utilization of a Computer in On-Line Control and Optimization of a

20 Utilization of a Computer in On-Line Control and Optimization of a Batch Process for Microbial

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Conversion of Ethanol to Protein ARTHUR E. HUMPHREY, DANE W. ZABRISKIE, WILLIAM B. ARMIGER, and WILLIAM M. ZIEGLER Department of Chemical and Biochemical Engineering, University of Pennsylvania, Philadelphia, PA 19104 The

first

persons to point out

monitoring of Hisashi, U.S.

the p o s s i b i l i t y of computer

i n d i r e c t l y measured parameters were Yamashito,

and Inagaki

Patent in 1975

in

1969

(3).

(2).

The method was described

Examples

of

the

-aided i n d i r e c t l y measured parameters to t i o n of batch-fed Jefferis in

1977

in

this

in

Most of (SCP)

the

to

ethanol,

SCP.

increasing

early

the

be

disin-

use of

this

substrate,

a yeast

single

cell

cell

In recent years oxygenated compounds such as Key papers discussing the by microorganisms

Mor and F i e c h t e r ,

observation

aceticus

single

this

in

the

work.

pro-

conversion of petroleum-derived mate-

(7,8),

kinetics

of

(6)

aero-

include those of and Humphrey (10).

Laskin report which s p e c i f i c a l l y

Laskin

meth-

received

Prokop, Votruba, Sobotka, and

and Ristroph, Watteeuw, Armiger,

undertake

stated that

that an SCP fermentation

"laboratory utilizing

A.

amount

ethanol." coefficient

(yield)

The problem is

strongly

of protein is

and/or

two-fold.

dependent

content of

expercalcoat

product

biomass produced from

Firstly,

on the

the

It

led us

should be operated under e t h a n o l - l i m i t i n g conditions

high growth rates to maximize the protein the

carbon

acetate and c e l l u l o s i c materials have

iments have indicated

and

application

computer-aided

work on the production of

attention.

(6), (9),

was the to

of

the production of

u t i l i z a t i o n of ethanol

Laskin

this

and growth rate and the

feed back control

was based on the

anol,

Panos

of

The work to

SCP from Ethanol

rials

bic

and optimiza-

and by Wang, Cooney, and Wang

another example of

a process for

a

(SCP).

Production of

tein

is

c e l l biomass

information in the ethanol,

(4)

in

computer-

were described by

review by Humphrey (1).

report

d i r e c t l y measured

protein

control

Background and detailed history

can be found in the cussed

the

Baker's Yeast fermentation

and Humphrey in 1973 (5).

application of

the

substrate

growth r a t e .

This

0-8412-0549-3/80/47-124-355$05.00/0 © 1980 American Chemical Society In Computer Applications to Chemical Engineering; Squires, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

yield is

so

C O M P U T E R APPLICATIONS T O C H E M I C A L ENGINEERING

356 because the maintenance

coefficient

for ethanol u t i l i z i n g micro-

organisms has been observed to be quite high, 0.1

g ethanol/g c e l l

biomass/hr.

reasonably high growth r a t e s , appreciable tenance. ance 0.1

at

For example

17% of

a growth rate of

hr"-*-.

i.e.

f r a c t i o n of energy

will

and 33%

1

Secondly, both acetaldehyde


(9)

report

inhibited

is

in

excess.

at

acetaldehyde

inhibitory

to

Prokop, Votruba, concentrations

acetate concentrations

of

that

for Acinetobacter

inhibitory

than 0.1

g/liter

However, ing

acid,

growth.

for

acetaldehyde

acetaldehyde

however,

problem.

it

is

concentration Armiger,

batch-fed yeast grown on ethanol concentration

g/liter

saturation

constant at

Sobotka,

(point

the growth rate

This means that

was 0.03

to

at

for

should be 74% of

stopped grow-

that

is

In

the

(10)

in

their

control

of

ethanol

acetate started accumulating and Panos

which the

starvation. (9)

ethanol

the

concentration

is

0.35

ethanol concentration

no i n h i b i t i o n occurs.

The

suggests that

the maximum and at 2 g / l i t e r

of the maximum rate i f

less

acetate.

g/liter.

and Humphrey

50% of the maximum)

1 g/liter

suggested

monitoring on the

during periods of ethanol

data of Prokop, Votruba,

rate

(6)

half and

found that whenever the

exceeded 4 g / l i t e r ,

only decreased

limits

Laskin

acetate not acetaldehyde

Ristroph, Watteeuw,

is

calcoaceticus are

and 1.0

work using NADH-NADPH fluorometric

and

A-49

g/liter

1

the

aerobic

the broth

Sobotka, and Panos

of 0.12

0.0175 g / l i t e r .

intermediIn

in

Laskin s data indicated the microorganisms

when

general,

levels

h r " l , an

a growth rate of

and acetic

that the growth rate of Candida u t i l i s at

than

grown at

be diverted to main-

ethanol fermentation both compounds can accumulate when ethanol

is

ethanol can be used for mainten-

hr"

ates in alcohol metabolism, are

greater

greater than 0.30

substrate

the

0.25

i.e.

Unless the organism

g/liter.

the growth it

will

Obviously

be 85%

it

is

a

t h i n balance point between s t r i v i n g for maximum growth rate while preventing acetate accumulation which adversely

affects

the growth

rate. It

appears,

therefore,

that the

batch ethanol SCP fermentation mand by i n d i r e c t l y estimating then to

add the

is the

to

concept to determine

cell

egy should prevent Normally,

the

one would do

reasons may include a desire letting

the

This

used in Baker's

of

the

cells

satisfies

this

the

This the

de-

strat-

end of

by continuous culture

However, there are

reasons for p r e f e r r i n g fed-batch to cells

it

2 g/liter.

growth rate and ultimately the SCP

than by a fed-batch process.

is

of

acetate from accumulating towards

the batch run a f f e c t i n g yield.

substrate de-

density and growth rate and

substrate in such a way that

mand but never exceeds a concentration

follow in a f e d the

to

rather

various process

continuous operation.

finish

o f f the

cell

age without substrate but under f u l l Yeast production to

as well as make them better

enhance

suited to

These

culture by the

aeration. stability

further p r o -

cessing. This

is

the

background to

A fed-batch process for the

the

control

strategy of

conversion of ethanol to

this

work.

a yeast SCP

In Computer Applications to Chemical Engineering; Squires, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

20.

HUMPHREY E T

Microbial

AL.

was to be computer

357

Conversion

controlled by i n d i r e c t l y monitoring the

cell

density and growth rate and then using these i n d i r e c t measurements to determine

the

amount of ethanol to

add to

the

fermentation.

The ethanol was to be added at a rate as high as possible but not s u f f i c i e n t l y high so that acetate would accumulate allowing rather than alcohol to ultimately Indirect

limit

it

growth.

Measurement of C e l l Biomass and Growth Rate

The p o s s i b i l i t y of i n d i r e c t l y measuring c e l l biomass and


growth rate by computer-aided on-line substrate balancing was first

mentioned in a publication by Humphrey (11).

The method

discussed in d e t a i l in a review by Humphrey (1). operates by noting that the difference component in and out of the vessel

is

organism for growth and maintenance metabolites

are

formed.

in

amount of a

equal to

if

no

Mathematically

particular

that used by the

appreciable

this

is

The technique

secondary

can be stated

as

x/i where Qi

= specific

m^

= s p e c i f i c maintenance

consumption rate of

component i ,

requirement

for

g±/g

cell-hr

component

i ,

g±/

g cell-hr X

= biomass concentration,

t

= time,

Y

x

/ i

g

cell/liter

hr

= maximum growth y i e l d ,

g c e l l / g ^ used for growth

The Q^X is the measured value of the In the

case of oxygen

this

oxygen in and out in the

is

gas

streams.

in the broth can be neglected The constants m and Y / i are x

data.

Equation

(1)

By i n i t i a l i z i n g at

because of i t s

the

very

the oxygen

low s o l u b i l i t y .

obtained from y i e l d s v s .

growth rate

the

(Q.X - m.X) ι ι

(2)

χ

c e l l concentration,

X(0),

either

from an

some point of the oxygen uptake or carbon dioxide

evolution rate or by knowing the grating

The accumulation of

can be rearranged to give the growth r a t e ,

| ~ = Υ dt χ/ι

estimation

substrate u t i l i z a t i o n .

simply the difference between

inoculum size

estimated growth rate over

time,

growth rate can be estimated o n - l i n e u t i l i z i n g substrate

(ethanol)

equation,

i.e.

demand,

and then

inte

both c e l l biomass and a computer.

The

AS is then estimated by a similar


COMPUTER APPLICATIONS TO CHEMICAL ENGINEERING

358

μΧ

VAS = V

+ m

At

Χ

(3)

x/s where S

= substrate concentration,

V

= fermenter volume,

= s p e c i f i c growth rate =

μ

(1/X)(dx/dt),

Y /s

= maximum growth y i e l d of c e l l s

m

= s p e c i f i c maintenance requirement for

x


g/liter

liter

s

hr"

1

on substrate,

g/g

substrate,

g/g/hr

Materials and Methods Organism. work.

Candida u t i l i s

s t r a i n ATCC 26387 was used

The organism was stored on agar slants

medium in Table I

and 1% dextrose.

The culture was

at

28°C and 100

culture were used to New

transferred

liter

computer

controlled detail

Four hundred ml of

inoculate 7 l i t e r s

Brunswick fermenter.

tically 70

rev/min.

Four l i t e r s

after

7.5

flask

of medium in a 14

liter

this

40

elsewhere

(12).

This

The d e t a i l s

Media Composition.

culture were asep-

liters

(PDP-11/34 with 3 discs

fermentation system.

gyrator

shake

of

hours to

this

initially

propagated in 50 ml of media in 250 ml shake f l a s k s on a shaker

in

containing the

of medium in a

and 96K words of

core)

-

system has been described in

are

shown in Figure

1.

A l l experiments were run with ethanol as

the main carbon source.

Table I gives a l i s t

of components

ex

cluding ethanol: Table

I

Media Components in 40 (NH ) S0 4

2

KH P0 2

KH 2

Liters

200 g

4

300 g

4

P0

25 g

4

MgS0 -7 H 0 4

100 g

2

Yeast Extract Sigma Salts

40 g

(half

1

liter

strength) Antifoam

10 ml

Fermentation Conditions. c a r r i e d out in the

70

liter

The control experiments were a l l

computer-coupled fermentation system.

Data were logged and feed-back control commands issued every 2 or 5 minutes.

The dissolved oxygen was maintained above 45%

t i o n by a combined a g i t a t i o n and aeration controlled above 4.3 initially

at

28.0°C.

using NH 0H. 4

The temperature

However, in the

latter

cooling water and r e f r i g e r a t i o n systems heat evolution rate

from the

control.

satura

The pH was was maintained

stages of the run

could not keep up with

fermentation.

This

caused the


the

tem-


1

A

'

i . . ù

AIR FLOW ι METER AND CONTROLLER !

rO-ί

CQNC. DILUTE BASE-LINE FEED

"""SJ

FEED ADDITION

CONTROL FEED

IR

j

PH

ANALYZER

AIR FLOW RATE

ANALYZER

co2

MULTIPLEXER

œ

-

- o -

Schematic of 70-liter computer-coupled fermentation system

!

- , - - Q ; : : : : :

FEED BACK GROWTH RATE CONTROL PROGRAM

OUT

DRY § FILTER

DISSOLVED OXYGEN

TEMPERATURE

Jl METER

BASE RATE COUNTER

ι AIR

It

BASE

Figure 1.

r f l

•

FEED RATE COUNTER

A/D CONVERTERS


CRT GRAPHICS TELETYPE

COMPUTER APPLICATIONS TO C H E M I C A L ENGINEERING

360

perature to increase to 32°C and higher.

I t was then no longer

possible to maintain the growth rate within the desired range of 80-95% of the maximum. Ethanol Feed. first

The ethanol was fed by a dual system.

was a two f l a s k feed system,

base l i n e

feed r a t e .

solution.

Each flask contained 4000 ml of ethanol

Flask one contained 125 ml of 190 proof ethanol

g) in 3875 ml of H2O. ethanol


r

feed

will

a linear

occur.

at

a constant r a t e ,

increase in alcohol concentration in the

See Figure 2 f o r d e t a i l s .

a c e l l mass increase in the fermenter time.

When the system is feeding

from flask one to the fermenter

liter/hr,

This base l i n e

feed rate

will

that

This is

will

second or control

volume feeder.

See Figure

20 ml of 171 proof alcohol puter.

This could be every

r e s u l t in

second order in

always be less than the

desired growth rate as long as the correct feed rate The

(93.7

Flask two contained 3500 ml of 190 proof

(2623 g) plus 500 ml of water.

solution

The

used to maintain an ethanol

is

selected.

system was comprised of a constant 3 for d e t a i l s . (13 g shots)

This system will add

on demand from the com-

2 or 5 minutes depending upon the

frequency at which the computer was set to monitor the system and update the c o n t r o l . liter,

When the c e l l

concentration

approached 20 g /

the system was demanding nearly 400 g of ethanol/hour.

This is equivalent to a c e l l productivity of 5 g / l i t e r - h r . the

shots were being added every

f i v e minutes,

doses were required to meet the demand. ethanol were being added e s s e n t i a l l y

This meant that

instantaneously.

Since

control i n t e r v a l s

this

bordered on being undesirable,

were selected in the l a t t e r

runs.

that there would never be more than a double shot, alcohol/40

liters

added to the v e s s e l .

run

Control Program.

This

a c q u i s i t i o n and control by

than 0.1

control

9 instructions

ration.

Instructions

initialize

system is part of a fermentation data

(14).

It

it

The p a r t i c u -

is

for the control

for integration. file.

Next the necessary Then instructions

37 to

the proper density of ethanol being used, i . e . 190 or 200 proof.

Next the program i n i t i a l i z e s the

f o r the integration of the cumulative base l i n e

ethanol

feed and then calculates the amount of ethanol feed since the last

The

integ-

10-16 set up the permanent disc f i l e and

the variables

49 determine variables

(13).

consists of 129 i n s t r u c t i o n s .

read in the variables

parameters are read from the disc whether

At the end of the

g/liter.

scheme described here and detailed in Table II was

developed by Ziegler first

This meant i . e . 26 g

software package e n t i t l e d DATAN developed

Zabriskie and described in h i s Ph.D. thesis

lar

reached

2 minute

This was borne out in the

analysis of the broth.

the acetate was found to be less

39 g of

These conditions were

expected to avoid acetate accumulation. hourly gas-chromatograph

shot

This could

r e s u l t in a b r i e f period where the ethanol concentration 1 g/liter.

When

some t r i p l e

time i n t e r v a l .

Instructions

69 and 70 c a l l

for actual



20.

HUMPHREY E T AL.

Microbial

Conversion

361

FERMENTER

Figure 2.

Constant-volume-feed system control

ν* c

2

ETHANOL FEED CONCENTRATION C., = f ( t ) ? TO FERMENTER

FERMENTER CELL CONCENTRATION (XoVo)+Y,r(Cot+7re7rt ) V rt 0 +

Figure 3.

Mathematical representation of two-flask-feed system


C O M P U T E R APPLICATIONS

362

TABLE


0001 0002 0003 0004 0005 0006 0007 0008 0009 0010 0012 0013 0014 0015 0016 0017 0018 0019 0020 0021 0022 0023 0024 0025 0026 0027 0028 0029 0030 0031 0032 0033 0034 0035 0036 0037 0038 0039 0040 0043 0044 0045 0046 0047 0048 0049 0050 0051 0052 0053 0054 0055 0056 0058 0059

T O C H E M I C A L ENGINEERING

II

SUBROUTINE CONTRL < ETHRS F 1 F L AG7 91FL AG8 » L I N F E D FCUMSUBf MU » MUACΤL9 2NUM79NUH8FCUM79CUM8F C U M F E D 9 6 R 0 2 F X02 F I R E C 9 F I L O U T F N S I Z E F V O L F E R ) R E A L 4 8 DELT 9LINFEDF L I N F D L 9 N B tNBO 9NBL F ETFDNBDT 9VBT» CB rDLINFD R E A L M MAINFMUFMUOFMUACTL DIMENSION I ( 1 0 ) F P < 3 8 ) F D ( S 7 ) F I N T < 1 4 ) F Z E R O ( 2 0 ) F I M S 6 ( 1 3 ) I N T E G E R * 2 SWITCH DATA DNAME/6RDKHAND/ IFLAG7«0 IFLAG8«0 I F ( I R E C . E Q . D GO TO 5 0 C A L L A S S I G N ( I F ' C O N T R O L · 0 0 0 ' F 0 F 'RDO'F 0 F 1 ) DEFINE F I L E 1(2F128FUFIVAR) READ ( I ' D ETHRSLFLINDFLFNBLFCUMSBL CALL CLOSE ( 1 ) GO TO 6 0 50 CONTINUE DO 40» I J = l r 2 0 ZERO(IJ)=0.0 40 CONTINUE C A L L A S S I G N ( I F CONTROL » 0 0 0 ' F 0 F 'NEW' F 0 F D DEFINE F I L E 1(2F128FUFIVAR) WRITE ( I ' D ZERO CALL CLOSE ( 1 ) CUMSBL=0.0 ETHRSL»0*0 LINFDL-0.0 60 CONTINUE C A L L A S S I G N (1F'PARAMS.OOO'FOF'OLD'FOF1) CALL FDBSET ( I F ' O L D ' ) DEFINE F I L E 1(2F256FUFIVAR) READ ( I ' D (I(N)FN 1F10)FNUM7FNUM8FSWITCH READ ( 1 ' 2 ) F I L N A M F ( P ( N ) F N = 1 F 3 8 ) F S I Z E 7 F S I Z E 8 F E T H 7 F E T H 8 F V 0 L 7 F V 0 L 8 F 1VOLTOT F V O L E T A F V O L E T B F FLOWRT F GROMAX F Y X S F M A I N » C 0 D E 9 CALL CLOSE(1) V0L0«P(3> XIN0«P(33) IPR00F*I(10) RH0190«0*7493 RH0200=0»7893 I F ( I P R 0 0 F . E Q . 2 0 0 ) GO TO 1 0 0 I F ( I P R 0 0 F » E Q » 1 9 0 ) GO TO 1 1 0 TYPE 120 120 FORMAT (' T H I S S Y S T E M I S ONLY C A P A B L E OF O P E R A T I N G WITH'F 1 / F ' 1 9 0 OR 2 0 0 PROOF ETHANOL.') RETURN RH0=RH0200 100 GO TO 111 110 RHO-RH0190 111 CONTINUE CONCA*RHO*VOLETA/VOLTOT NB=NBL DELT=0.001*(ETHRS-ETHRSL) LINFED=LINFDL NBO»RHO*VOLETB I F ( I R E C . E G . D NB-NBO VBO=VOLTOT ET=ETHRSL Q=FLOWRT 7

S


20.

HUMPHREY E T A L .

Microbial


TABLE II

0060 0061 0062 0063 0064 0065 0066 0067 0068 2000 0069 0070 0071 0072 0073 0075 0076 0078 800 0080 0081 0083 0085 0086 0087 0088 0089 900 0090 0091 0092 950 0093 0094 0096 0098 0099 300 0100 0101 0102 0103 0104 1008 0105 0106 0107 0108 0109 0110 0111 500 0112 0113 0114 0115 0116 0117 0118 0119 0120 0121 0122 0123 0124 0125 0126 0127 0128 0129

600

Conversion

363

Continued

2000 J = l f l 0 0 0 ET=ET+DELT DNBDT* < Q * C 0 N C A / 2 • 0 ) - ( Q * N B ) / < V B 0 - Q * E T / 2 • 0 ) NB=NB+DNBDT*DELT VBT»VB0-Q*ET/2*0 CB=NB/VBT DLINFD=CB*Q*DELT LINFED«LINFED+DLINFD CONTINUE MUACTL-GR02/X02 X=X02 MU0*0»9*GR0MAX MU=MU0 I F MU=0*8*GR0MAX I F (MU.GT•GROMAX) MU-GROMAX CONTINUE I F ( I R E C * L E » S W I T C H ) MU-MUO I F ( I R E C » L E • S W I T C H ) X*XINO*EXP MU=1.01*MU DELSUB*VOLFER*(MU*X/YXS+MAIN*X)* < ETHRS-ETHRSL) CUMSUB=CUMSBL+DELSUB GO TO 9 0 0 CONTINUE WTSH7*SIZE7*RH0*ETH7/V0L7 WTSH8»SIZE8*RH0*ETH8/V0L8 CONTINUE ETHXS*WTSH7*NUM7+WTSH8*NUM8+LINFED-CUMSUB I F ( E T H X S » G T » 0 » 0 ) GO TO 5 0 0 IF GO TO 3 0 0 GO TO 5 0 0 CONTINUE IMSG(1)=2 IMSG IMSG~28672 C A L L SEND (DNAMEF1MSG F IDSW > NUM8=NUH8+1 IFLAG8=1 GO TO 9 5 0 CONTINUE C A L L A S S I G N < 1 F 'PARAMS•000' F 0 F 'OLD' F 0 F 1 ) C A L L F D B S E T

Utilization of a Computer in On-Line Control and Optimization of a

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