Antonie van Leeuwenhoek 50 (1984) 625 639

Penicillin p r o d u c t i o n : b i o t e c h n o l o g y a t its best

C. P. VAN DER BEEK a n d J. A. ROELS

Gist-brocades N. V., Research and Development, P.O. Box 1, 2600 M A Delft,

The Netherlands

VAN DER BEEK, C. P. and ROELS,J. A. Penicillin production: biotechnology at

its best. Antonie van Leeuwenhoek 50: 625-639.
A brief description is given of the history of penicillin production in the Neth-
erlands.
The development of today's penicillin production technology is analysed in
terms of changes in the quality and intensity of the production process. Techno-
logical as well as genetical developments are shown to be of influence on the
quality and the intensity of the production process.
The analysis is illustrated by a brief description of the productivity improve-
ment of the penicillin fermentation as it occurred at Gist-brocades during the
past 20 years.

INTRODUCTION

It is well known that Alexander Fleming discovered, more or less by chance,

the lysogenic action of a compound, secreted by the mould Penicillium notatum,
towards a number of bacteria recognized as pathogenic (Fleming, 1929). He
coined the term penicillin for the substance responsible for the action. It was
to take another ten years before the discovery of Fleming initiated the develop-
ment of penicillin production technology as we witness it today, World War
II being the main factor in the stimulation of the early phase of its development.
In 1938 Chain took up Fleming's original work, which had been discontinued
after Clutterbucks failure to isolate the antibacterial substance (Clutterbuck et
al., 1932).
After successful animal tests (Chain et al., 1940) and clinical trials (Abraham
et al., 1941) during the first year of the war an arrest of the scale-up work in
England resulted as a consequence of the priority of war-related efforts. Still,
the possible value of penicillin was recognized and Florey, Chain's superior,
626 C. P. V A N D E R BEEK A N D J. A. R O E L S

was sent to the United States to discuss the possibility of a joint project. This
grew into an Anglo-American war project with top priority. Major problems
in fermentation, fermentor design and recovery were solved in a co-operation
between governmental, university and industrial laboratories.
From the Department of Agriculture in Peoria in the U.S.A. the industry
was supplied with fermentation advice and in line with the discovery of Kluyver
and Perquin (1933) that moulds can be cultivated in submerged culture, this
latter technique was adopted to apply to Penicilliurn. In the course of the devel-
opment programme the old production strain, Penicillium notatum, was replaced
by Penicillium chrysogenum N R R L 1951. This strain, after mutation and selec-
tion, led to the ancestor of all the major industrial strains: Wisconsin Q-176.
The latter relatively highly productive strain did, however, in the early corn-steep
liquor-containing media, produce penicillin K instead of penicillin G. This led
to the development of the so-called precursor fermentation in which the side-
chain phenylacetic acid, the 6-position substituent characterizing penicillin G,
was added.
Industrial companies like Pfizer, Merck, Eli Lilly in the U.S.A. and Glaxo
in Great Britain, reaped the fruits of the wartime-induced Anglo-American ef-
fort.
In the Netherlands penicillin research started secretly in t944, under German
occupation. By means of radio broadcasts from England researchers from the
Nedertandsche Gist en Spiritus fabriek (now known as Gist-brocades N.V.) be-
came aware of the discovery ofa wonderdrug called penicillin. This was correlat-
ed to the mould Penicillium, and soon the pre-war publication of Fleming was
found. An article of Kiese (1943) confirmed this correlation and research was
initiated by evaluating the antibiotic production capacity of some twenty Penicil-
lium species from the culture collection of the Centraalbureau voor Schimmel-
cultures at Baarn. Agar pieces grown with Penicillium baculatum produced the
largest inhibition zones on plates seeded with staphylococci, and so this species
was chosen for the production of Bacinol, which was the code name given to
the antibacterial substance.
When the contents of ampoules of American penicillin, which were contained
in the allied food and medicine droppings after the liberation of the Netherlands
in 1945, were compared with the product which had been independently devel-
oped by the Dutch researchers, both samples proved to be identical and compa-
rable in purity. A study trip to the U.S.A. at the end of 1945 incorporated the
knowledge, which had been developed in the Anglo-American war project, in
the Dutch penicillin production process. As a result the total Dutch requirement
for penicillin could be supplied by in 1948.
In 1949 penicillin became available for export. Fig. 1 (Hersbach et al., 1984)
shows the development of total penicillin production of Gist-brocades from 1949
to 1981. The increase in the world production of penicillin shows a similar pat-
tern. In 1945 world production amounted to about 5 tons (Sylvester and Coghill,
PENICILLIN PRODUCTION 627

==
1000"

10(

10.

0-1"

l~so 156o 147o 1~'8o

Fig. 1. Total production of penicillin G by Gist-brocades (From Hersbach et al., 1984).

1954); in 1982 it was estimated at 12000 tons (Hersbach, 1983). The price
equalled 8.106 S/ton in 1945, whilst it had decreased to 3.5.104 S/ton in 1980
(De Flines, 1980). If this were to be corrected for inflation the dramatic decrease
of penicillin production costs would become even more clearly visible. Close
to 20~ of the present-day total world production of penicillin is supplied by
the Gist-brocades production facilities at Delft.

SYNTHESIS OF PENICILLINS

The biosynthesis of penicillin by fungi has been excellently reviewed several

times recently (Aberhart, 1977; O'Sullivan and Abraham, 1981; Queener and
Neuss, 1982) and consequently this topic will be dealt with only very briefly.
Penicillin is synthesized from the amino acid precursors c~,aminoadipic acid (an
628 C. P. VAN DER BEEK AND .1. A. ROELS

C~-AMINOADIPIC ACID CYSTEINE VALINE

~______~
ATP

MP+PPi
TM

MPtPPi
~ A ATP
MP*PPi

SH CH 3

O='-AAA - N H - C - C H2
I
C~NH .....
//
O

C~-AMINOADIPYL-CYSTEINYL-VALINE

(TRIPEPTIDE)

NADP÷
'FAD
; FADH2
S .~NADPH

~_A AA_NH_CH_CH~ NC -.-~ CH3

I I I~ c.3
C-- N CH
0~" ~ ~ 0
C
OH

ISOPENiC|LLtN N AMP+PPt A T P

OL'-AA A'~------~I " ~

~ - ~ - PHENY L A C E T Y L - S - C o A <

¢CoA-SH
PAA

o 4 s
CH2-C-NH-CH-CH C
I I I"" c..,
C--N ~ CH
o-,
OH

penicillin G

Fig. 2. Biosynthesisof penicillinG (From Hersbachet al., 1984).

intermediate in the fungal biosynthesis of lysine), cysteine and valine which are
coupled yielding the tripeptide 6-(~-aminoadipyl)-cysteinyl-vatine. Formation
of a fl-lactam ring fused to a thiazolidine ring is the n e x t e v e n t which results
in a structure called isopenicillin N. Exchange of the e-aminoadipyl moiety for
phenylacetic acid or phenoxyacetic acid, which are added as a precursor to the
fermentation, results in either penicillin G or V (Fig. 2).
PENICILLIN PRODUCTION 629

RCONH-T--IS'~
~
0~ L - N ~ COOH

R name R name

.._
~O-?H

C2Hs
_. p r o p i c i l l i n
~ ,~,~'-CH 3
oxacillin

CI
CI
.. cloxacillin

OCH3 CH3
CI
~ CH---
ampicillin
C I ~ C ~ ~ iclOxacillin
H2
Cl
._• amoxycillin
~ _. flucloxacillin

~1: ' ~ ' ~ H3

~ CH---
earbenicillin
OOH
Fig. 3. Chemical structures of some semisynthetic penicillins.

Semisynthetic penicillins can be prepared from purified penicillin G (or V)

by removing the phenyl (or phenoxy) acetic acid side-chain chemically or enzy-
matically and replacing it by a side-chain which gives the penidllin molecule
more desirable pharmaceutical properties (Fig. 3).
Cephalosporins, another major group of/Llactam antibiotics, can be prepared
from penicillin by chemical expansion of the thiazolidine ring. Although cepha-
losporins can also be obtained from fermentations with the fungus Cephalospor-
iurn acrernoniurn ( = Acremoniurn chrysogenurn) the chemical route starting with
penicillin G has proven to be less expensive for at least some of the cephalospor-
ins (the oral preparations).

PRODUCTIVITY IN PENICILLIN FERMENTATION

Today in industrial penicillin production technology the submerged fermenta-

tion is the technology of choise. For obvious reasons the surface culture tech-
630 C. P. V A N DER BEEK A N D J. A. ROELS

nique has become obsolete and cannot be considered an economically viable

production option. Of course, there are several important factors, which deter-
mine the efficacy of the process for the production of penicillin:
1. The average productivity per unit fermentor volume, and per unit time.
2. The average efficiency of the conversion of the substrates to the product.
3. The efficiency of the process of recovery of the product from the often dilute
and contaminated solution, which is obtained after fermentation. Although
recovery certainly is an aspect of great importance, it will not be discussed
in detail in this paper. The reader is referred to the literature for a recent
overview (Hersbach et al., 1984).
If items 1 and 2 indicated above are analysed in somewhat more detail, it
becomes obvious that two types of factors can be distinguished in their impact
on the total productivity in and the economic viability of a fermentation process:
1. A group of process quality variables. These are reflected in the specific pro-
ductivity per unit mycelial dry substance and the often very closely related
efficiency of the conversion of substrate to penicillin. The quality variables
are dependent on genetically determined penicillin production capacity of
the strain, on the one hand, and medium composition in terms of concentra-
tions of growth and penicillin production-limiting, -enhancing or -inhibiting
compounds (e.g. the dissolved oxygen concentration) and quantities like pH
and temperature, on the other hand.
2. A group of process intensity variables. At a given specific productivity per
unit mycelial substance the actual productivity per unit of equipment volume
can be calculated if the amount of productive biomass which can be sustained
in the fermentor is known for each moment in time. This process intensity
is directly related to oxygen consumption, heat production and CO2 produc-
tion, i.e. a given amount of biomass requires a sufficient amount of metabolic
energy in order to be maximally productive. Therefore, process intensity is
determined by oxygen transfer, heat removal and carbon dioxide removal
characteristics of the equipment.
In the following the history of the penicillin production process will be ana-
lysed in terms of changes in quality and intensity variables. Of course, it is well-
known that the first commercial quantities of penicillin were obtained on the
basis of a surface culture technique. In an early stage, however, the submerged
culture emerged and became the standard technology for commercial penicillin
production in a relatively short period of time. Therefore, our discussion will
be limited to the submerged process. An overall account of the development
of penicillin productivity per unit volume and time at Gist-brocades is given
in Fig. 4. It reflects the net effect of the improvements in process intensity and
process quality as they occurred at Gist-brocades.
In the following the process intensity and quality improvements will be put
in their historic perspective and the discussion will distinguish between the effects
PENICILLIN PRODUCTION 631

4000-
000-

/ / oo
3000-

t
2000-

1000-
o
I i ! I / / I
1945 '50 '55 '60 '95 '70 '75 '80
year
Fig. 4. Mean penicillin G productivity at Gist-brocades, productivity per unit of volume and per
unit of time (From Hersbach et al., 1984).

of improved strains on the one hand and production technology on the other
hand, these having effects on both process quality and process intensity. The
effect of strain improvement resulted in a productivity rise between 1950 and
1975 of roughly a factor 16, whilst the technology contributed a factor of roughly
3 in that same period (Pirt, 1983).

T H E IMPACT OF TECHNOLOGICAL DEVELOPMENTS ON PENICILLIN PRODUCTIVITY

In the practical realisation of large-scale penicillin production a number of

aspects are of great importance and it is, within the scope of the present account,
impossible to treat all of them in detail (for a more detailed account see Hersbach
et al., 1984).
In present-day penicillin production technology the organism is grown in fer-
mentors of typically 150-200 m a in volume and an important aspect is to keep
the fermentor free from contamination by alien microorganisms. These both
compete with the production organism for nutrients and may degrade the/~-
lactam moiety of penicillin by the action of/Mactamases. The pH is controlled
632 C. P. V A N DER BEEK A N D J. A. ROELS

by the addition of alkali and acid or of the nitrogen source, the pH of choice
being around 6.2-6.8, which is dictated by both the rate of chemical degradation
of the ]3-1actammoiety at the high pH and an increasing toxicity of the precursor
(see later on) at low pH. The temperature of choice is in the region between
24 ° and 27 °C, the higher temperatures favouring growth, the lower ones being
more suited in view of both efficiency of penicillin synthesis and stability of
the product against chemical degradation.
Since the early days of penicillin production as a commercial activity the im-
portance of the dissolved oxygen level (D.O.) as a variable in the realization
of maximum efficiency of penicillin synthesis has been recognized. Typically,
the D.O. level must be higtier than 30~o o'f air saturation at 1 atmosphere pres-
sure. To sustain these oxygen concentration levels at the high oxygen demands
typical of the present-day state of the art, i.e. up to 30 moles.m-3 h- 1 or higher,
vigorous aeration and agitation are necessary. In spite of about 40 years of very
intensive research on alternative agitation and aeration systems the classical
Rushton turbine has kept its position as the stirrer of choice in the fermentation
industries. In general rather high rates of supply of compressed and sterile air
are necessary for both maintaining the above-mentioned high rates of oxygen
supply but, perhaps more importantly, also for ensuring sufficient ventilation
capacity, so that the carbon dioxide concentration in the air leaving the vessel
is kept well below 4 ~ (by volume), a level which has proven inhibitory to the
expression of the maximum biosynthetic activity with respect to penicillin.
Another important aspect of penicillin production technology, which will be
discussed in some depth is the mode of operation of the fermentation vessel.
In general at least three modes of operation can be distinguished: batch, fed-
batch and continuous culture. In a batch fermentation process all nutrients,
the carbon source, the nitrogen source and the like are presupplied at the begin-
ning of the fermentation, whilst oxygen, acid/alkali and/or the precursor may
be supplied during the fermentation. In the early days a batch process was em-
ployed for the synthesis of penicillin. The nutrient supply was based on a corn-
steep liquor-lactose medium, a mixture which allowed rather rapid growth in
the beginning of the fermentation process and slow, 13-galactosidase activity-
limited, growth during the later stage of the process. The rationale for using
this medium will become clearer later on.
A significant development resulted when it became common practice to add
the side-chain of the penicillin G molecule, the precursor phenylacetic acid, to
the production medium. This both greatly increased the productivity and re-
sulted in a higher selectivity towards penicillin G synthesis, i.e. the fomaation
ofpenicitlins with a side-chain other than phenylacetic acid was suppressed.
Of course, the batch process for the production of penicillin had an important
disadvantage: there was no direct way to regulate the energy flux through the
metabolism of the organism; in fact the organism more or less autonomously
regulated its initial period of a high growth rate and its subsequent period of
PENICILLIN PRODUCTION 633

slow, [3-galactosidase activity-limited, growth. As these activities are directly ref-

lected in oxygen uptake rate, heat production rate and carbon dioxide evolution
rate profiles, also these latter quantities cannot be regulated in a direct way
in a batch process. Therefore, it becomes impossible to match the transfer capa-
bilities of the equipment to the demand of the microorganism for metabolic
energy. This was also the rationale behind the use of lactose, instead of the more
readily assimilated carbohydrates such as glucose and sucrose. The use of the
latter compound would have resulted in very' high oxygen demands, too low
oxygen levels and very little penicillin production already in the early stages
of the batch process.
The use of lactose has largely become a thing of the past and the so-called
fed-batch process has replaced the old technology. In a fed-batch process very
little of the carbon and energy source and often also the nitrogen source is sup-
plied at the beginning of the fermentation. By far the larger part is supplied
during the fermentation process and the carbon and energy source is generally
a rapidly metabolizable compound like glucose or sucrose. In general the carbon
and energy source is supplied at a growth-limiting rate after the initial 24 h
of autonomous batch growth. In this way the flux of metabolic energy through
the production organism can be closely regulated and the fermentation can be
designed in such a way that, given the transfer capabilities of the equipment,
a carbon source supply pattern is designed, which results in minimal production
costs. To this purpose mathematical modelling has been advocated as a powerful
tool and the importance of the profile of the growth-limiting rate of addition
of the carbon and energy source has been shown.
In this development it also became standard practice to use a continuous feed-
ing of the precursor phenylacetic acid in order to maintain its concentration
within a lower limit where itbecomes limiting to the rate of production of penicil-
lin G (0.1 kg- m 3) and a upper limit where it becomes toxic to the production
organism (1 kg.m-3).
Heijnen et al. (1979) presented extensive studies of the modelling of penicillin
fermentation. Some selected results of their modelling exercises are shown in
Fig. 5. As can be seen the final mycelial concentrations can be as high as 50
g of dry substance per kg in a fermentation process, which lasts 200 h. The
final penicillin concentrations are in the order of 25000 units, ml-1. One of the
important assumptions underlying the modelling exercises of Heijnen et al. is
related to the well-documented fate of the carbon and energy source in microbial
growth and product formation.
In general, the specific rate of carbon and energy source consumption, q~,
can, for the class of product formation processes to which penicillin production
belongs, be written as:
qs = I-t/Ysx+ qp/Y~p + ms in which
g is the specific growth rate of the mycelial dry matter (DM) (h- 1),
Ysxis the maximum yield of biomass dry matter on substrate (g DM .g- 1),
634 C. P. V A N D E R BEEK A N D J. A. R O E L S

MYCELIAL CONCENTRATION PENICILLIN CONCENTRATION

mole/kg g/kg mole/kg U/g

Cx cp

2.0- i(~ (B) 0.05

(A)
O.O4 24000 ~ (B)
(C)
0.03 - 18000
1.0 !5
/ 0.02 - 12ooo ~

0.01
~ (A)

6 ~ ~ (c)
/
i i i i

0 100 200 0 100 200

time (hr) time (hr~
Fig. 5. Penicillinand mycelialconcentrationcurvesin a representativefed-batchpenicillinfermenta-
tion (From Heijnenet al., 1979).

YspiS the maximum yield for product on substrate (g.g- ~),

m~is the specific maintenance substrate demand (g. g ~ DM h - 1),
q~is the specific rate of substrate consumption (g.g- 1 DM h - 1),
qpiS the specific rate of penicillin production (g,g-~ DM h - 1).
Furthermore, the specific rate of penicillin production is known to be depen-
dent on the specific growth rate g in the sense that qp increases with ~t up to
a specific growth rate of 0.01-0.015; if the specific growth rate increases further
no additional increase in % results. Therefore, the selectivity of the conversion
of substrate to biomass and the productivity per unit fermentor volume and
time become maximal in the above-indicated range of specific growth rates, i.e.
the specific growth rate must be rather low to result in maximal penicillin pro-
duction.
In their simulations Heijnen et al. (1979) present a rather detailed analysis
of the fate of the carbon and energy source in penicillin fermentation. In an
optimal process as far as productivity is concerned 65~o of the total carbon and
energy source consumption is directed towards maintenance metabolism, about
25~o is directed to biomass growth and 10~ results in the desired product, peni-
cillin. Indeed, these results clearly indicate the impact of microbial energetics
and more specifically of the specific maintenance substrate consumption in mod-
ern penicillin fermentation processes' state of the art.
An aspect of the fermentation process which merits attention and has not
been discussed up to now is related to broth viscosity, a phenomenon which
PENICILLIN PRODUCTION 635

has been extensively studied by Kossen and co-workers (Metz et al., 1979). One
of the important characteristics of filamentous broths are the rather high
viscosities which result if the mycelial dry matter concentration becomes high.
Viscosities of up to 2000 centipoises are by no means exceptional. The viscosities
have been shown to depend on the mycelial morphology and, very important,
on the fraction of the mycelium being present as so-called pellets, dense mycelial
aggregates. As the viscosity becomes a very important parameter in determining
the level of power input necessary to obtain a given level of the oxygen transfer
rate or, alternatively stated, as the level of the oxygen transfer rate which can
be realized at a given level of power input increases drastically with decreasing
viscosity, viscosity measurements and "viscosity engineering" are an important
tool in the optimization of the penicillin production process.

STRAIN SELECTION

In addition to process improvements, strain selection has played an important

role in the improvement of the productivity of penicillin fermentations. The main
target of strain selection is to increase the total output of penicillin per unit
of fermentor volume per unit of time. Not only strains with a higher specific
rate of penicillin synthesis, but also strains which can be grown to higher broth
densities without losing their production capacity, meet this requirement. That
means that both process quality and process intensity improvements can be a
target for strain selection. As the costs of the C-source are very important in
penicillin fermentation and the portion that is converted into penicillin is only
10-15~, improvement of the yield has become an additional selection criterium.
Other, more specialized properties such as improved filtration characteristics,
efficiency of side-chain incorporation or enhanced sporulation, may also be se-
lected for if l:heycontribute to an optimal performance of the process.
Strain selection programmes generally pass through five stages:
1. Induction of variation:
In principle three genetic techniques are available to Penicillium genetics: mu-
tation, recombination by means of the parasexual cycle and genetic engineering.
Mutation and recombination have already proven their value in the improve-
ment of penicillin production. Genetic engineering is being developed for this
purpose.
2. Preselection:
To reduce the number of strains to be tested in shake flasks after a mutagenic
treatment a preselection stage can be introduced. This preselection can either
be directed to a comparison of penicillin production capacity on solid media
like the potency index and the agar piece method, or to the selection of mutants
with properties that are suspected to have a beneficial effect on penicillin produc-
tion, the so-called rational selections. Sometimes the preselection stage is omit-
636 C. P. V A N D E R BEEK A N D J. A. R O E L S

ted and replaced by an increase in the capacity of (a miniaturized version of)

the shake flask selection.
3. Shake flask selection:
Several parameters may be evaluated during this stage and used for the selec-
tion of improved strains:
- penicillin production,
- yield on carbon source,
- efficiency of side-chain precursor incorporation.
This evaluation may be extended with several other parameters by performing
tests in laboratory fermentors.
4. Pilot plant evaluation:
During this stage the fermentation conditions of the production plant are
applied to the new strains. Some strain-specific adaptations in recipe and/or
fermentation conditions may emerge as a result of optimization experiments.
5. Introduction in the production plant:
Due to scale differences some optimization of the fermentation conditions
may still be necessary during this stage. Some adaptations may also be necessary
in the penicillin recovery process.

As an example, the improvement of penicillin productivity at Gist-brocades

over a period of 20 years is given in Fig. 6 (Van der Beek et al., 1984). From
a strain which was externally obtained in 1962 a series of strains has been devel-
oped of which the phylogeny is given on the same time scale as the productivity
improvement. Clearly it can be seen that some increases in productivity are due
to process improvements as they are accomplished without replacement of the
strain (1969-1973). Other productivity increases are obviously the result of the
introduction of improved strains (1978/1979, 1980/1981). Following the intro-
duction of a new strain optimization of fermentation conditions can provide
further productivity improvements for some years (1974-1978).

FUTURE DEVELOPMENT

In the foregoing we have analysed the basic philosophy underlying modern

penicillin production technology and have indicated aspects of its development
to its present-day state of the art. The developments which have been discussed
have resulted in a quite low level of the production costs and, according to the
literature (De Flines, 1980), a high level of raw material costs in the total produc-
tion costs (i.e. up to 50~o). Furthermore, the costs of the Carbon source are an
important part of the total raw material costs and as only 10-15~ of the C-source
is directly used for the synthesis of penicillin, the improvement of the efficiency
of conversion of this C-source into penicillin can be understood to be an impor-
tant target for further research.
PENICILLIN PRODUCTION 637

PHYLOGENY AND PRODUCTIVITY OF PENICILUUM CHRYSOGENUM STRAINS AT GIST-BROCADES.

fermentation productivity
[abit~ary units per unit of volume and time]

~t962~ ~1964 * I19661 ~1968 ~ 11970' ~1972= I ~1974¢ I i~761 J lg78t t 1980~ ~ 1982~ '

--.--..-- EMS EMS EM EMS E M $ EMS EM

diepoxy UV V

Horizontal lines: mLdation f o l l o w e d b y selection

Vertical lines: selections without mutation
Mutagenic a g e n t s are indicated
P O d t on s t r a r~s a

Fig. 6. Phylogeny of Penieillium ehrysogenum strains and the development of penicillin productivity
at Gist-brocades (From Van der Beeket aL, 1984).
638 C. P. VAN DER BEEK AND J. A. ROLLS

I n o u r c o n v i c t i o n classical m u t a t i o n a n d s c r e e n i n g a n d , in a l a t e r stage, r a t i o -
nal g e n e t i c e n g i n e e r i n g - b a s e d a p p r o a c h e s will c o n t i n u e to c o n t r i b u t e t o w a r d s
the a b o v e - i n d i c a t e d goal. A l s o n e w t e c h n o l o g i c a l d e v e l o p m e n t s , s u c h as the use
o f i m m o b i l i z e d m y c e l i u m , m a y c o n t r i b u t e to f u r t h e r p r o c e s s i m p r o v e m e n t , b u t
at p r e s e n t this r e m a i n s s p e c u l a t i v e .
A s far as the t e c h n o l o g y o f the p r o c e s s is c o n c e r n e d a h i g h level o f m a t u r a t i o n
has b e e n r e a c h e d , i.e. p r o c e s s i m p r o v e m e n t b y m e a n s o f o t h e r t h a n s t r a i n i m - ,
p r o v e m e n t s t e n d s to b e c o m e i n c r e a s i n g l y difficult. N o d o u b t , p r o g r e s s in this
a r e a will be far less s p e c t a c u l a r t h a n it has b e e n in the past. H o w e v e r , t w o a r e a s
o f f r u i t f u l f u r t h e r r e s e a r c h m a y be i n d i c a t e d . F i r s t l y , t h e r e a r e t h e p r o s p e c t s
o f " v i s c o s i t y e n g i n e e r i n g " w h i c h m a y a l l o w h i g h e r b i o m a s s c o n c e n t r a t i o n s to
be m a i n t a i n e d in e x i s t i n g e q u i p m e n t , a n d s e c o n d l y , t h e r e is t h e p r o s p e c t o f p r o -
cess i m p r o v e m e n t t h r o u g h m a t h e m a t i c a l m o d e l l i n g a n d / o r i m p r o v e d ( c o m -
p u t e r ) c o n t r o l o f the f e r m e n t a t i o n process.

REFERENCES

ABERHART, D. J. 1977. Biosynthesis of 13-1actam antibiotics. - - Tetrahedron 33:1545-1559.

ABRAHAM,E. P., CHAIN, E., FLETCHER,C. M., FLOREY,H. W., GARDNER,A. D., HEATLEY,N.
G. andJENN1NGS,M.A. 1941. Further observations on penicillin. - - Lancet ii: 177 188.
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