Professional Documents
Culture Documents
Citric Acid
Biotechnology
BJRN KRISTIANSEN
Borregaard Industries Ltd, Norway
MICHAEL MATTEY
Department of Bioscience and Biotechnology, University of Strathclyde, UK
JOAN LINDEN
Gluppevelen 15, 1614 Fredikstad, Norway
Contents
page 1
1
2
2
3
4
5
6
7
7
8
8
9
11
11
12
19
21
24
25
33
33
35
46
50
50
4 Strain Improvement
4.1 Introduction
4.2 General aspects of strain improvement
55
55
55
v
Contents
vi
4.3
4.4
4.5
4.6
4.7
60
61
64
65
65
5 Fungal Morphology
5.1 Introduction
5.2 Factors affecting Aspergillus niger morphology in submerged culture
5.3 Effect of agitation
5.4 Effect of nutritional factors
5.5 Effect of inoculum
5.6 Conclusions and perspectives
5.7 References
69
69
69
70
74
82
82
82
85
85
85
86
87
88
89
91
95
95
101
102
103
105
105
107
113
119
119
121
121
122
123
125
128
130
131
132
135
135
136
Contents
9.3
9.4
9.5
9.6
9.7
9.8
9.9
vii
Solvent extraction
Adsorption, absorption and ion exchange
Liquid membranes
Electrodialysis
Ultrafiltration
Immobilization of micro-organisms
References
139
142
143
144
145
146
146
10 Fermentation Substrates
10.1
Introduction
10.2
Molasses
10.3
Refined or raw sucrose
10.4
Syrups
10.5
Starch
10.6
Hydrol
10.7
Alkanes
10.8
Oils and fats
10.9
Cellulose
10.10 Other medium redients
10.11 Conclusion
10.12 References
149
149
150
156
156
157
157
157
158
158
158
159
159
161
161
163
163
165
167
167
169
169
170
171
174
176
177
178
182
183
183
183
184
Index
187
Contributors
Ho Ai Meng Amy
Blk 135 Pasir Ris Street 11, # 06-239, Singapore 510135
Marin Berovic
Department of Chemistry and Biochemical Engineering, National Chemistry Laboratory
for Biotechnology and Industrial Mycology, 1115 Slo, Ljubljana, Hajdrihova 19 POB 30,
Slovenia
Pawel Gluszca
Department of Bioprocess Engineering, Technical University of Lodz, Wolczanska 175
90-924 Lodz, Poland
Bjrn Kristiansen
Borregaard Industries Ltd, PO Box 162, 1701 Sarpsborg, Norway
Liliana Krzystek
Department of Bioprocess Engineering, Technical University of Lodz, Wolczanska 175
90-924 Lodz, Poland
Christian Kubicek
Institute for Biochemical Technology and Microbiology, University of Technology
Getreidemarkt 9/1725, A-1060 Wien, Austria
Contributors
Staniskaw Ledakowicz
Department of Bioprocess Engineering, Technical University of Lodzul, Wolczanska 175
90-924 Lodz, Poland
Wladyslaw Lesniak
Food Biotechnology Department, Academy of Economics, Komandorska 118/120 PL 53345 Wroclaw, Poland
Michael Mattey
Department of Bioscience and Biotechnology, University of Strathclyde, Todd Centre 33
Taylor Street, Glasgow G4 0NR
Maria Papagianni
8 Kamvounion Street, 54 621 Thessaloniki, Greece
George Ruijter
Section of Molecular Genetics of Industrial Microorganisms, Wageningen Agricultural
University, Dreijentlaan 2, 6703 HA Wageningen, The Netherlands
Jaap Visser
Section of Molecular Genetics of Industrial Microorganisms, Wageningen Agricultural
University, Dreijentlaan 2, 6703 HA Wageningen, The Netherlands
Frank Wayman
Department of Bioscience and Biotechnology, University of Strathclyde, Todd Centre 33
Taylor Street, Glasgow G4 0NR
Markus Wolschek
Institute for Biochemical Technology and Microbiology, University of Technology
Getreidemarkt 9/1725, A-1060 Wien, Austria
It is probably no more than a coincidence that Sir Eric Geddes uttered his now famous
phrase at the time that the industrial production of citric acid by fungal fermentation was
being developed to circumvent the high price and lack of availability of lemon juice. However,
the association of taxation and squeezing lemons is appropriate, as the history of citric acid
reflects the politics and economics of the era as well as the science. Indeed the production
of citric acid is a classical biotechnology phenomenon, where the science, though important,
is secondary to the economics and politics of production. This book seeks to reflect that
balance between practical science, fundamental understanding and economics.
Citric acid derives its name from the Latin citrus, the citron tree, the fruit of which
resembles a lemon. The acid was first isolated from lemon juice in 1784 by Carl Scheele, a
Swedish chemist (17421786), who made a number of discoveries important to the advance
of chemistry, amongst them hydrofluoric, tartaric, benzoic, arsenious, molybdic, lactic, citric,
malic, oxalic, gallic and other acids as well as chlorine, oxygen (1772, published in English
in 1780, predating the discovery by Priestly in 1774), glycerine and hydrogen sulphide.
Citric acid was thus one amongst many natural organic acids.
Citric acid was produced commercially from Italian lemons from about 1826 in England
by John and Edmund Sturge, but with the increasing importance of citric acid as an item of
commerce, production was started in Italy by the lemon growers, who established a virtual
monopoly during the rest of the nineteenth century. Lemon juice remained the commercial
source of citric acid until 1919 when the first industrial process using Aspergillus niger
began in Belgium.
Lemon juice itself remains an important product. World lemon production averages about
3.3 million metric tonnes (US Foreign Agricultural statistics); about 75 per cent comes
from the United States, Italy, Spain and Argentina, with the rest from some 15 other producer
countries.
1
Marketing of lemons is the subject of political control both in Europe and the USA. In
Europe the processing of lemons to juice carries a processing subsidy which makes it attractive
to process the lemons rather than sell them as fresh produce; additionally the EU intervention
mechanism results in significant quantities of lemons being destroyed. In the USA marketing
is controlled by the United States Department of Agriculture (USDA) Lemon Administrative
Committee which determines how many lemons will be sold into the fresh market and what
growing areas will be allowed to sell them.
The economic result of any monopoly tends to be to make the product expensive; without
the spur of competition the control of costs, the development of the process and the efficiency
of production are neglected. Citric acid in the nineteenth century was no exception; the
Italian monopoly resulted in high prices that tempted the entrepreneurs of the era to seek
alternative sources of the increasingly useful product. Unable to find an alternative botanical
source of citric acid, the nineteenth century advances in chemistry and microbiology were
examined. By the turn of the century both possibilities existed.
sp., Botrytis sp., Eupenicillium sp., Mucor piriformis, Penicillium janthinellum, P. restrictum,
Talaromyces sp., Trichoderma viride and Ustulina vulgaris.
Currie (1917) found strains of A. niger that produced citric acid when cultured in media
with low pH values, high sugar levels and mineral salts. Prior to this A. niger was known to
produce oxalic acid; the key difference was the low pH which, as we now know, suppressed
both the production of oxalic acid, which would be toxic, and gluconic acid, which has a
significantly higher production rate from sugar than citric acid. Currie subsequently joined
Chas. Pfizer & Co. Inc. and his discovery formed the basis of the citric acid plant established
in the USA by the firm in 1923. This plant and the other similar processes established first
in Belgium then in England by J.E.Sturge, in Czechoslovakia and in Germany in the next
few years used the surface process. The details of this process are not well documented
despite its long history, due in part to the restriction of information by manufacturers. In
biotechnological terms, citric acid is known as a bulk, or low value, product. The market is,
and always has been, very competitive, so the profit margins are small. Improvements in
productivity depend on the detail of the various processes, many of which are not easily
protected by patents, so that secrecy is important and understandable.
salt of citric acid. It is not possible to crystallize the acid directly from the crude molasses
medium although this can be done if pure sucrose is used as the carbon source. The
precipitate of calcium citrate is washed and suspended in enough sulphuric acid to
precipitate the calcium as calcium sulphate. This releases the citric acid into solution
from where it can be treated further as required.
The surface process, though commercially profitable for many years, is labour intensive
and inefficient in its use of space; there is a limit as to how high a large tray can be lifted!
The production of citric acid by surface culture was challenged at the beginning of the
1940s by the development of submerged fermentation processes. When Shu and Johnson
published their work on the effect of medium ingredients and their concentrations on
citric acid production in submerged culture, the fundamental technology for submerged
production was ready to be exploited on an industrial scale (Shu and Johnson, 1948a,
1948b).
Many reports suggest that the morphology of the mycelium is crucial to the ultimate
yield; not only with respect to the shape of hyphae, but also their aggregation. Several
studies suggest that hyphae should be abnormally short, bulbous and heavily branched. It is
recognized that this condition is brought about by manganese deficiency or related to the
addition of ferrocyanide, which is probably the same thing. The mycelium should also form
small (less than 0.5 mm) pellets with a smooth, hard surface. Such pellets are produced
when a number of factors are controlled, such as ferrocyanide levels, manganese levels, low
iron (less than 1 ppm), low pH, control of aeration and agitation or the amount of spore
inoculum.
It is clear that this morphological appearance is not in itself necessary for a successful
yield, but is a result of the correct process parameters. Pellet formation is not necessary, but
does give a broth with a lower energy requirement for mixing. When a change to a filamentous
growth type occurs, the dissolved oxygen level may fall by 50 per cent for a fixed input.
That filamentous growth can give satisfactory yields has been demonstrated and consideration
of the diffusion characteristics of pellets versus filamentous mycelium would suggest that
while yields may be similar, productivity should be greater without the additional diffusional
constraint of pellets.
Aeration is a significant factor in the cost of the process, and although a constant aeration
rate is used in many laboratory scale studies, the industrial practice is to use relatively low
aeration rates initially (0.1 vvm) rising to 0.51 vvm as growth proceeds. Such aeration
rates will lead to foaming and various devices and agents are available to minimize the
problem. Although very high yields are possible, the productivity is a more important
consideration on an industrial basis, and it is rare that the process is allowed to continue to
the maximum yield.
The processes run today owes much to the pioneering work carried out by D.S.Clark
and his co-workers at the Northern Regional Research Laboratories in Canada during the
1950s and early 1960s. Here, the technology for large-scale production of citric acid with
A. niger using molasses was established. After the fermentation characteristics were worked
out, attention was given to the controlling mechanisms of the fermentation. Numerous
reports have been published on the role of metal ions on the citric acid cycle, in particular.
After decades of academic discussion, there is general agreement about the factors that
regulate the fermentation and give rise to the high yields obtained in industry (Mattey,
1992).
mycelium in alginate beads or collagen proved possible, but with very low production
rates. The difficulties of avoiding oxygen limitation when preparing beads, and
preventing further growth, which reduces oxygen transfer rates, have led to the
immobilization of conidia which are then grown under nitrogen limitation to the desired
compact pellet. While giving a manageable system, the productivity was still too low to
be of industrial interest.
Other constructs for immobilization that have been more successful are the use of exchange
filtration, and a rotating disc with an adhering mycelial film, reminiscent of sewage treatment
techniques. These radical methods are unlikely to gain acceptance, even were they to give
economic productivity gains, unless the engineering problems of scale-up can be overcome
without making the capital costs too large.
on the process developed for the yeast strain that had a substrate concentration of 10 per
cent n-decane, although n-alkanes from 9 to 20 carbons could be used. The availability and
cost of Libyan n-alkanes, which lead to the development of this and other plants, including
the dual substrate plants, has changed over the last three decades. One unique feature of the
n-alkane process is the insolubility of the substrate. To ensure a rapid conversion the nalkane has to be thoroughly dispersed, so additives such as polyoxypropylene glycol ether,
at concentrations from 20 to 200 ppm, are used to enhance this.
1.11 Conclusions
Books must follow science, not science books.
(Francis Bacon, Propositions touching Amendment of Laws)
For the last 80 years citric acid has been produced on an industrial scale by the fermentation
of carbohydrates, initially exclusively by Aspergillus niger, but in recent times by Candida
yeasts as well, with the proportion derived from the Candida process increasing. The higher
productivity of the yeast-based process suggests it will be the method of choice for any new
plants that may be built.
The intimate knowledge about the large-scale fermentation and subsequent recovery
processes are still regarded as industrial property. Nevertheless, the citric acid process is
one of the rare examples of industrial fermentation technology where academic discoveries
have worked in tandem with industrial know-how, in spite of an apparent lack of collaboration,
to give rise to a very efficient fermentation process.
The current world market for citric acid and its derivatives is difficult to estimate
accurately; no international statistics are collected, but industry estimates suggest that upwards
of 400 000 tonnes per year may be produced. Citric acid is a mature product but the
upward trend in its use seen over many years is an annual 23 per cent increase.
The price is such that profit margins are low, and with significant, but erratic, quantities
appearing on the world market from countries such as China the situation is unlikely to
improve.
The lemon, which started it all, is doing well, with an estimated world production of 3 to
4 million tonnes per year. Commercial varieties such as Eureka are all high acid lemons,
with the acid content exceeding 4.5 per cent by weight, so that some 140 000 tonnes of
citric acid are still produced by lemons!
The various themes touched on in this introduction are dealt with in greater depth in the
following chapters.
1.12 References
COOPER, W C and CHAPOT, H, 1977. Fruit Productionwith special emphasis on fruit for processing.
In Citrus Science and Technology, Vol. 2. Eds S Nagy, P E Shaw and M K Veldhuis (AVI Publishing
Co., Westport, CT, USA).
CURRIE, J N, 1917. The citric acid fermentation of A. niger, Journal of Biological Chemistry, 31, 5.
GRIMOUX, E and ADAMS, P, 1880. Synthese de lacide citrique, C.R.Hebd. Seances Acad. Sci., 90,
1252.
KRISTIANSEN, B and CHARLEY, R C, 1981. The effect of medium composition on citric acid
production in continuous culture, Presented at 2nd European Congress of Biotechnology, UK.
KRISTIANSEN, B and SINCLAIR, C G, 1979. Production of citric acid in continuous culture,
Biotechnology and Bioengineering, 21, 297.
MATTEY, M, 1992. The production of organic acids, CRC Critical Reviews in Biotechnology, 12, 81.
PASTEUR, L., 1875. Nouvelle observations sur la nature de la fermentation alcoolique, C.R. Acad.
Sci., 80, 452.
REUTHER, W, CALAVAN, E C and CARMAN, G E, 1967. The Citrus Industry, Vol. 1. History,
World Distribution, Botany and Varieties. Univ. Calif. Div. Agric. Nat. Res., San Pablo, California.
ROSENBAUM, J B, MCKINNEY, W A, BEARD, H L, CROCKER, L and NISSEN, W I, 1973.
Sulphur Dioxide Emission Control by Hydrogen Sulphide Reaction in Aqueous Solution. The
Citrate System. US Bureau of Mines, Report 1774.
SCHEELE, C, 1793. Crells Ann. 2, 1 1784, from Smmtliche Physische und Chemische Werke.
Hermbstdt (Berlin).
SHU, P and JOHNSON, M J, 1948a. Citric acid production submerged fermentation with Aspergillus
niger, Industrial and Engineering Chemistry, 40, 1202.
SHU, P and JOHNSON, M J, 1948b. The interdependence of medium constituents in citric acid
production by submerged fermentation, Journal of Bacteriology, 54, 161.
WEHMER, C, 1893. Note sur la fermentation Citrique, Bull. Soc. Chem. Fr, 9, 728.
2.1 Introduction
The biochemical mechanism by which Aspergillus niger accumulates citric acid has
attracted the interest of researchers since the late 1930s when the optimization of this
accumulation to give a commercial process began. In this sense, the various theories
which have been proposed to explain the accumulation of citric acid in such high yields
also reflect the general biochemical knowledge at the time the respective research was
done. In view of the high input into this research through more than 50 years it is therefore
rather disappointing that there is still no explanation of the biochemical basis of this
process which would consistently explain all the observed factors influencing this
fermentation. Reasons for this are manifold. First, citric acid is only accumulated when
several nutrient factors are present, either in excess (i.e. sugar concentration, H+, dissolved
oxygen), or at suboptimal levels (trace metals, nitrogen and phosphate), and thus is subject
to multifactorial influence. Hence it is unlikely that single biochemical events are solely
responsible for citric acid overflow.
Secondly, an appreciable part of the literature consists of work which has been performed
using low or only moderately producing strains or by applying nutrient conditions not optimal
for citric acid production, and while this may be justified for special reasons in individual
cases, the respective results are not comparable to those obtained by others. Moreover, their
significance for the understanding of the commercial citric acid fermentation is questionable.
Thirdly, the biochemical knowledge of filamentous fungi is still significantly inferior to
that of, for example, Saccharomyces cerevisiae or higher eukaryotes and, moreover, results
from these sources cannot be uncritically transformed to filamentous fungi, which impedes
a biochemically correct interpretation of results in several areas. Hence, although a
considerable amount of basic biochemical research has been carried out with A. niger, the
present state of understanding of the events relevant for citric acid accumulation (not to say
production) is still a poorly resolved puzzle.
This chapter attempts to draw the currently recognizable picture and to aid in the further
fitting together of the other scattered bits and pieces.
11
12
13
Figure 2.1 Metabolic pathways from glucose to citric acid by (a) involvement of an
anaplerotic carbon dioxide fixation (Cleland and Johnson, 1954), and (b) sole involvement of
the citric acid cycle. Only relevant intermediates are given, and arrows may indicate more
than a single enzymatic step. Note that in (b), each of the two acetyl-CoA molecules is
subject to one turn of the tricarboxylic acid cycle.
dioxide and oxygen in the exit air of a pilot plant citric acid fermentation, observed that
during the first 70 hours of fermentation the respiratory coefficient (i.e. CO2 released/O2
taken up) is close to 1; it starts to decrease thereafter and reaches the level predicted from
the operation of the pyruvate carboxylase reaction (0.66) only at stages where citrate
accumulation is already taking place at a constant rate (e.g. <120 hours). The Cleland and
Johnson reaction may therefore only be important at later stages of fermentation, whereas
the initial phase of citric acid accumulation takes place without anaplerotic carbon dioxide
supply.
Although not directly within the topic of this chapter, it should be noted that A. niger is
also capable of accumulating another organic acidoxalic acidas a (toxic) byproduct of
citric acid fermentation. The biosynthesis of this compound is controversial (Mller, 1975;
Mller and Frosch, 1975; Kubicek et al., 1988), and appears to depend on whether glucose
or citric acid is used as the carbon source. In the latter case, the glyoxalate cycle has been
implicated in its biosynthesis (Mller, 1975). Its biosynthesis on glucose as a carbon source
occurs by the hydrolysis of oxaloacetate catalyzed by oxaloacetate hydrolase, which is
cytosolically located and appears to act as a valve by which the carbon overflow can be
chanelled into an energetically neutral pathway (Figure 2.3) and so compete with citrate
overproduction (Kubicek, 1988). Although production of oxalate is, because of its toxicity,
of considerable interest to citric acid fermentation, the regulation of its biosynthesis is
controversial (Kubicek et al., 1988; Strasser et al., 1994).
Figure 2.2 Metabolic and regulatory network of citric acid biosynthesis from sucrose in A.
niger. For convenience, sucrose is assumed to be split into glucose and fructose by invertase
extracellularly (Boddy et al., 1993; Rubio and Maldonado, 1995) and only the
monosaccharides are taken up. The double line indicates the plasma membrane, the hatched
double line the mitochondrial membrane. Circles inserted into the membranes indicate
known or assumed transport steps (hatched: characterized in A. niger; full: assumed, but not
yet characterized; empty: countertransport, to be verified). Thick lines and arrows indicate
metabolic reactions; thin lines and arrows indicate regulatory interactions (*activation: //
inhibition). Intermediates of regulatory importance are boxed.
15
Figure 2.3 Pathway of oxalate biosynthesis by Aspergillus niger. Note that concentrations of
acetate corresponding to those of oxalate have not been detected in culture filtrates of A.
niger, and the metabolism of acetate therefore requires further study
16
Table 2.1 Genes encoding enzymes involved in the biosynthesis of citric acid by A. niger,
which have already been cloned from A. niger or other Aspergillus spp.
It is intriguing that the above named binding sites have so far not been detected in the 5'noncoding sequences of the few glycolytic genes studied in A. niger or the close relative
Aspergillus nidulans (Table 2.1, Figure 2.4). Transcriptional regulation of glyceraldehyde3-phosphate dehydrogenase (Punt et al., 1988, 1990, 1992) and of 3-phosphoglycerate kinase
(Clements and Roberts, 1986; Streatfield et al., 1992) has been studied in some detail in the
closely related fungus A. nidulans. Its transcription depends on positive control by several
co-operating DNA-binding proteins since a truncated core promoter of the pgkA gene only
containing the CAAT, TATA and CT-rich elements could not trigger transcription. Punt et
al. (1988) identified a glycolytic box as responsible for transcription. No differences in
expression of gpdA were observed on 1% glucose or 0.1% fructose (Punt et al., 1990).
A 24-bp region, which shares 60 per cent similarity with the glycolytic box, is also
present at -638 and -488 of the pgkA promoter (Figure 2.4). However, another sequence,
located between -161 and -120, in the pgkA promoter was shown to be essential for expression
of the respective gene. It consists of two non-overlapping octameric sequences that match
in seven out of eight nucleotides to the higher eukaryotic consensus ATGCAAAT (Falkner
et al., 1986).
A 17-base pair sequence was found in the 5'-regions of the A. nidulans and A. niger pkiA
genes that may act as an upstream regulating sequence (de Graaff et al., 1992). This sequence
was shown to be distinct from the proposed cis-acting element mediating increased
transcription of pyruvate kinase on glycolytic carbon sources (de Graaff et al., 1988).
Figure 2.4 Regulatory nucleotide motifs present in the 5'-non transcribed sequences of three glycolytic genes of A. nidulans (gpdA,
pgkA) and A. niger (pkiA). Only characterized nucleotide sequences are given. The boxed bar marker indicates 100 bp, and all
genes are drawn to scale.
Table 2.2 Enzymes of A. niger, involved in citric acid biosynthesis and catabolism, with noteworthy regulatory properties
19
20
recombinant strain of A. niger, which carries a disrupted copy of the constitutively expressed
trehalose-6-phosphate synthase gene tpsA (Wolschek and Kubicek, 1997), produces citric
acid at increased rates (Arisan-Atac et al., 1996). Similarly, a strain bearing multiple copies
of tpsA and hence overproducing trehalose-6-phosphate synthase exhibited a reduced rate
of citrate production. These data indicate that the cellular level of trehalose-6-phosphate
regulates the flux from glucose to citric acid and are thus in accordance with the conclusions
of Torres et al. (1996a) that hexokinase most likely accounts for the major part of regulation
at the early steps of glycolysis, thereby supplying an increased concentration of substrate
for PFK2.
However, most recently Panneman et al. (1996) reported on the isolation and
characterization of a glucokinase from A. niger N400, a strain producing only low levels of
citric acid, which has properties different from the hexo/glucokinase purified by Steinbck
et al. (1994). They also concluded that by analogy with A. nidulans (Ruijter et al., 1996a)
there may be also at least one separate hexokinase as well. The difference between the
results of Steinbck et al. (1994) and Panneman et al. (1996) are currently unresolved.
Hybridization of an A. niger ATCC 11414 DNA with a Kluyveromyces lactis hexokinaseencoding gene as a probe showed hybridization to a single fragment only (F.Narendja and
C.P.Kubicek, unpublished data). The gene from strain ATCC 11414 has recently been cloned
in our laboratory, and its characterization has to be awaited for clarification of this situation.
Whatever the results of this investigation, the results by Arisan-Atac et al. (1996) clearly
show that a relief from trehalose-6-phosphate inhibition positively influences the glycolytic
flux at high sugar concentrations, and the hexose-phosphorylating step is therefore a major
regulatory point in this fermentation.
Glucose uptake by A. niger was investigated by Torres et al. (1996a). A. niger ATCC
11414 contains two transporters with different Km and Vmax. However, the high-affinity
permease can only be detected during growth on low glucose concentration (1% w/v),
whereas the low-affinity permease is detectable in the presence of high glucose
concentrations. The latter may therefore contribute to the increased glycolytic flux during
growth on high glucose concentrations.
Several lines of evidence suggest that the regulation of PFK1 by Fru-2,6-P2 may not be
the only parameter regulating citrate accumulation. Citrate inhibition of PFK1 also seems
in vivo to be antagonized by ammonium ions (Habison et al., 1979). This antagonism is
functionally linked to the well known effect of trace metal ions (particularly manganese
ions) on citric acid accumulation (Shu and Johnson, 1948b; Tomlinson et al., 1950; Trumpy
and Millis, 1963), as one of the effects caused by manganese deficiency is an impairment of
macromolecular synthesis in A. niger (Kubicek et al., 1979a; Hockertz et al., 1987), which
causes increased protein degradation (Kubicek et al., 1979a; Ma et al., 1985). As a
consequence, mycelia accumulate elevated concentrations of NH4+ (Kubicek et al., 1979a).
Proof for the role of manganese ions in this process has been obtained by the isolation of
mutants of A. niger whose PFK1 was partially citrate-insensitive and whose citric acid
accumulation was simultaneously more tolerant to the presence of Mn2+ (Schreferl et al.,
1986). Furthermore, several authors have reported that the exogenous addition of NH4+
during citric acid fermentation even stimulates the rate of citrate production (Shepard, 1963;
Choe and Yoo, 1991; Yigitoglu and McNeil, 1992), which is consistent with this effect of
NH4+ on PFK1. The latter authors documented that both the time of addition as well as the
concentration of NH4+ were important, and its addition during inappropriate fermentation
phases even decreased acid accumulation.
The reason for the impairment of macromolecular synthesis under manganese deficient
cultivation conditions had originally been assumed to be at the translational level (Ma et
21
al., 1985). However, Hockertz et al. (1987) have demonstrated that the absence of
manganese ions from the nutrient medium of A. niger causes a reversible inhibition of
DNA, but not RNA biosynthesis. This is supported by the findings that the effect of
manganese deficiency can be mimicked by addition of hydroxyurea, an inhibitor of
ribonucleotide reductase (Hockertz et al., 1987). They proposed that manganese deficiency
may primarily impair DNA synthesis by causing a shortage of desoxyribonucleotides
required for DNA replication.
A further mechanism of regulation of PFK1 was proposed by Legisa and co-workers,
who postulated that PFK1 is regulated by phosphorylation by cyclic-AMP dependent protein
kinase A (Legisa and Bencina, 1994). They speculate that a high concentration of sucrose
causes an increase in mycelial cyclic-AMP levels which trigger the phosphorylation of
PFK1, thereby converting an inactive (non-phosphorylated) form into an active
(phosphorylated) form (Legisa and Gradisnik-Grapulin, 1995). The support for their model
is their observation that PFK1 was inactivated by treatment with alkaline phosphatase (Legisa
and Bencina, 1994). However, this model, while intriguing, has to be treated cautiously
until solid evidence for it has been obtained, as the molecular weight of the PFK1 purified
by Legisa and Bencina (1994) and used for their studies was 48 kDa which is not that of
native PFK1 (84 kDa). Moreover, the method section of their paper does not indicate whether
(and how) the alkaline phosphatase had been removed or inactivated prior to the PFK1
assay. If this was not done, the inactivation of PFK1 may have been due to a removal of
Fru-6-P from the assay and thus be an artefact. Proof for a regulation of PFK1 by
phosphorylation is therefore still needed.
A stimulation of citric acid accumulation by increased cyclic-AMP levels had also been
postulated earlier (Wold and Suzuki, 1973, 1976a, 1976b). They showed that the stimulatory
effect was dependent on the zinc concentration of the medium. Adenylate cyclase from A.
niger has been described as Zn2+ dependent (Wold and Suzuki, 1974). A bottleneck of their
investigations, however, is that they were using 1% (w/v) sucrose throughout, and hence the
relevance of their findings to the effect of zinc under citric acid fermentation conditions is
unclear. Xu et al. (1989b) studied the intracellular concentration of cyclic-AMP in A. niger
during citric acid biosynthesis on media with and without Mn2+ ions added, and with high
(14%) and low (1%) sucrose concentrations. They reported that the cyclic-AMP levels were
growth rate dependent, and comparable if phases of similar growth rates were compared.
Whether or not cyclic-AMP is in fact involved in the regulation of citrate overproduction
remains to be assessed.
22
1986). While this view has an extraordinary long half-life in the review literature, solid
evidence for the presence of an intact citric acid cycle during citric acid fermentation was
presented 25 years ago (Ahmed et al., 1972), and explanations based on this view are
therefore simply incorrect.
The requirement of citric acid accumulation of a deficiency in some metal ions (e.g.
Mn2+, Fe3+) has frequently been used to explain an inhibition of some enzymes of the TCA
cycle (for review see Kubicek and Roehr, 1986). Thus, iron deficiency has been claimed to
inhibit aconitase (Szczodrak and Ilczuk, 1985). However, the activity of this enzyme during
citric acid accumulation has been demonstrated clearly by others both in vitro (La Nauze,
1966; Ahmed et al., 1972; Mattey, 1977) as well as in vivo (Kubicek and Roehr, 1985). It
should be kept in mind that the enzymes of the respiratory chain, which also require iron,
are highly active during citric acid accumulation (Ahmed et al., 1972; Hussain et al., 1978).
By a similar rationale, the necessity for Mn2+ deficiency has been used to claim an inhibition
of either of the two isocitrate dehydrogenases which require divalent metal ions for activity
(cf. Gupta and Sharma, 1995). However, this requirement is for chelation of the substrate
(i.e. isocitrate; cf. Bowes and Mattey, 1979; Meixner-Monori et al., 1986). In view of the
fact that Mg2+ (which is present in excess) can take over the chelating role of Mn2+ efficiently,
this interpretation is unlikely to explain the effect of Mn2+.
Several other explanations for citric acid accumulation are based on the postulation of a
metabolic inhibition of the NADP-specific isocitrate dehydrogenase by citrate (Mattey, 1977)
or glycerol (Legisa and Mattey, 1986), which would create a bottleneck in the tricarboxylic
acid cycle andbecause of the Keq of aconitaselead to a spilling over of citrate.
Unfortunately, none of the explanations which are based on an inhibition of NAD-or NADPspecific isocitrate dehydrogenases have ever been supported by evidence from in vivo
experiments. The glycerol theory (Legisa and Mattey, 1986; Gradisnik-Grapulin and Legisa,
1996), has recently been reassessed by studying the effect of increased mycelial glycerol
concentrations on the oxidation of 1,514C-citrate by mycelia and isolated mitochondria of
A. niger (Arisan-Atac and Kubicek, 1996). The appearance of 14C-labelled CO2which
because of the labelling position applied can only be released during the metabolic conversion
of citrate to a-ketoglutaratewas virtually unaffected by the glycerol concentration, thereby
clearly disproving an effect of glycerol on the activity of isocitrate dehydrogenases and
consequently this theory. Also, in contrast to the enzyme from crude cell-free extracts (Legisa
and Mattey, 1986), the purified NADP-specific isocitrate dehydrogenase was not inhibited
by citrate (Arisan-Atac and Kubicek, 1996).
It is surprising that the question of whether the isocitrate dehydrogenase step of the TCA
cycle is active during citric acid fermentation or not has never been viewed from a theoretical
point of view: using the cellular concentration of free and protein-bound glutamic acid as
an indicator of metabolic flux from glucose to a-ketoglutarate, there is no indication for a
significant change in this flux unless at late stages of fermentation where the fungal growth
(and also the need for glutamic acid) has stopped, and this flux is only 17 per cent lower
than that occurring in a culture accumulating 78 per cent less citric acid, and hence may not
be of high relevance to the mechanism of citric acid accumulation (O.Zehentgruber and
C.P.Kubicek, unpublished data).
With regard to the mechanisms which trigger the initial accumulation of citrate from the
mitochondria, a fact completely overlooked so far is the activity of the tricarboxylate
transporter. This carrier competes directly with aconitase for citrate, and if its affinity for
citrate were much higher than that of aconitase, would pump citrate out of the mitochondria
without any necessity for inhibition of one of the TCA cycle enzymes. As the tricarboxylate
carrier of mammalian tissues and yeast occurs by countertransport with malate (Evans et
23
al., 1983), such a situation is conceivable when its counter-ion malate accumulates in the
cytosol. Malate accumulation has in fact been shown to precede citrate accumulation (Roehr
and Kubicek, 1981). However, the mitochondrial citrate carrier of A. niger has not yet been
investigated, and this hypothesis clearly needs thorough investigation before it can be used
to explain citrate accumulation. It is also not known to what extent changes in the flux
through the NAD-dependent-, NADP-dependent isocitrate dehydrogenases, a-ketoglutarate
dehydrogenase and succinate dehydrogenase, contribute to a rise in the intramitochondrial
citrate concentration. As these enzymes are known to be regulated by the mitochondrial
NADH/NAD and NADPH/NADP ratios, as well as by AMP, cis-aconitate and oxaloacetate
(Chan et al., 1965; Meixner-Monori et al., 1985, 1986), fluctuations in the level of
mitochondrial TCA metabolites are likely.
24
bound ATPase during maintenance of the pH gradient between the cytosol and the
extracellular medium. The involvement of this enzyme in the maintainance of the pH
gradient in citric acid producing A. niger has been shown by Mattey et al. (1988). Hence
the requirement of a low pH for citric acid accumulation may be, at least in part, related
to a high turnover of the ATP formed, which otherwise would lead to a metabolic imbalance
and so stop acidogenesis. However, this explanation still requires experimental verification.
Most recently, single-point mutagenesis of a plant ATPase and its expression in yeast
resulted in increased H+-pumping and increased growth rates at low pH (Morsomme et
al., 1996).
25
The reason for the reciprocal effect of Mn2+ ions on export and import of citric acid may
also be related to yet another effect of manganese deficiency, i.e. inhibition of triglyceride
and phospholipid synthesis as well as a shift in the ratio of saturated to unsaturated fatty
acids of whole mycelial lipids (Orthofer et al., 1979; Jernejc et al., 1989) and of isolated
plasma membranes (Meixner et al., 1985). The different behaviour of the citrate export and
import system of A. niger may also be seen in the light of earlier studies on the antagonism
of several membrane affecting compounds on the detrimental action of manganese ions,
e.g. lower alcohols (Moyer, 1953), lipids (Millis et al., 1963; Gold and Kieber, 1967), or
tertiary amines (Batti, 1969). Also the technically important ability of Cu2+ ions to antagonize
the deleterious effect of Mn2+ may be related to citrate excretion, as Cu2+ strongly inhibited
the uptake of citric acid from the medium (Netik et al., 1997). However, the effect of Cu2+
(Schweiger, 1959) may also reside in its inhibition of the uptake of Mn2+ by A. niger (Hockertz
et al., 1987), which occurs by a specific, high affinity transport system (Seehaus et al.,
1990). The properties of the uptake and the export system are otherwise similar (?pH driven
proton symport) and it may be speculated that they are catalyzed by the same enzyme
system.
2.6 References
AHMED, S A, SMITH, J E and ANDERSON, J G, 1972. Mitochondrial activity during citric acid
production by Aspergillus niger, Transactions of the British Mycological Society, 59, 5161.
ARISAN-ATAC, I and KUBICEK, C P, 1996. Glycerol is not an inhibitor of mitochondrial citrate
oxidation in Aspergillus niger, Microbiology UK, 142, 29372942.
ARISAN-ATAC, I, WOLSCHEK, M and KUBICEK, C P, 1996. Trehalose-6-phosphate synthase A
affects citrate accumulation by Aspergillus niger under conditions of high glycolytic flux,
FEMS Microbiology Letters, 140, 7783.
ARTS, E, KUBICEK, C and ROEHR, M, 1987. Regulation of phosphofructokinase from Aspergillus
niger: effect of fructose-2,6-bisphosphate on the action of citrate, ammonium ions and AMP,
Journal of General Microbiology, 133, 11951199.
BAKER, H V, 1986. Glycolytic gene expression in Saccharomyces cerevisiae: nucleotide sequence
of GCR1, null mutations, and evidence for expression, Molecular and Cellular Biology, 6,
37743784.
BAKER, H V, 1991. GCR1 of Saccharomyces cerevisiae encodes a DNA binding protein whose
binding is abolished by mutations in the CTTCC sequence motif, Proceedings of the National
Academy of Sciences of the USA, 88, 94439447.
BATTI, M A, 1969. US Patent 3,438.863.
BERCOVITZ, A, PELEG, Y, BATTAT, E, ROKEM, J S and GOLDBERG, I, 1990. Localisation of
pyruvate carboxylase in organic acid producing Aspergillus strains, Applied and Environmental
Microbiology, 56, 15941597.
BERRY, D R, CHMIEL, A and AL-OBAIDY, Z, 1977. In: Genetics and Physiology of Aspergillus.
Eds J E SMITH and J A PATEMAN (Academic Press, London), pp. 405423.
BLOOM, S J and JOHNSON, M J, 1962. The pyruvate carboxylase of Aspergillus niger, Journal of
Biological Chemistry, 237, 27182720.
BODDY, L M, BERGES, T, BARREAU, C, VAINSTAIN, M H, JOBSON, M J, BALLANCE, D J
and PEBERDY, J F, 1993. Purification and characterization of an Aspergillus niger invertase
and its DNA sequence, Current Genetics, 24, 6066.
BOWES, I and MATTEY, M, 1979. A study of mitochondrial NADP +-specific isocitrate
dehydrogenase from selected strains of Aspergillus niger, FEMS Microbiology Letters, 7, 323
325.
26
27
GOLD, W and KIEBER, R J, 1967. Process of making citric acid by fermentation, US Patent
337,2094.
GRADISNIK-GRAPULIN, M and LEGISA, M, 1996. Comparison of specific metabolic
characteristics playing a role in citric acid excretion between some strains of the genus
Aspergillus, Journal of Biotechnology, 45, 265270.
GUPTA, S, and SHARMA, C B, 1995. Citric acid fermentation by the mutant strain of the Aspergillus
niger resistant to manganese ions inhibition, Biotechnology Letters, 17, 269274.
HABISON, A, KUBICEK, C P and ROEHR, M, 1979. Phosphofructokinase as a regulatory enzyme
in citric acid accumulating Aspergillus niger, FEMS Microbiology Letters, 5, 3942.
HABISON, A, KUBICEK, C P and ROEHR, M, 1983. Partial purification and regulatory properties
of phosphofructokinase from Aspergillus niger, Biochemical Journal, 209, 669676.
HARMSEN, H, KUBICEK-PRANZ, E M, VISSER, J, ROEHR, M and KUBICEK, C P, 1992.
Regulation of 6-phosphofructo-2-kinase from the citric acid producing fungus Aspergillus niger,
Applied Microbiology and Biotechnology, 37, 784787.
HAYASHI, S and NAKAMURA, S, 1981. Multiple forms of glucose oxidase with different
carbohydrate compositions, Biochimica et Biophysica Acta, 657, 4051.
HOCKERTZ, S, PLNZIG, J and AULING, G, 1987. Impairment of DNA formation is an early
event in Aspergillus niger under manganese starvation, Applied Microbiology and Biotechnology,
25, 590593.
HOCKERTZ, S, SCHMID, J and AULING, G, 1987. A specific transport system for manganese in
the filamentous fungus Aspergillus niger, Journal of General Microbiology, 133, 35133519.
HONECKER, S, BISPING, B, YANG, Z and REHM, H-J, 1989. Influence of sucrose concentration
and phosphate limitation on citric acid production by immobilized cells of Aspergillus niger,
Applied Microbiology and Biotechnology, 31, 1724.
HOSSAIN, M, BROOKS, J D and MADDOX, I S, 1984. The effect of sugar source on citric acid
production by Aspergillus niger, Applied Microbiology and Biotechnology, 19, 383391.
HUIE, M A, SCOTT, E W, DRAZINIC, C M, LOPEZ, M C, HORNSTRA, I K, YANG, T P and
BAKER, H V, 1992. Characterization of the DNA-binding activity of GCR1: in vivo evidence
for two GCR1-binding sites in the upstream activating sequences of TPI of Saccharomyces
cerevisiae, Molecular and Cellular Biology, 12, 26902700.
HUSSAIN, M, RAHMAN, R, CHOUDHURY, N and HUSSAIN, I, 1978. Studies on mitochondrial
respiration of some high citric acid-yielding mutants of Aspergillus niger, Journal of
Fermentation Technology, 56, 253256.
IWAHORI, K, TATSUTA, S, FUJITA, M and YAMAKAWA, M, 1995. Substrate permeability in
pellets formed by Aspergillus niger, Journal of Fermentation and Bioengineering, 79, 387
390.
JAKLITSCH, W M, KUBICEK, C P and SCRUTTON, M C, 1991. Intracellular organisation of
citrate production in Aspergillus niger, Canadian Journal of Microbiology, 37, 823827.
JERNEJC, K and LEGISA, M, 1996. Purification and properties of carnitine acetyltransferase from
citric acid producing Aspergillus niger, Applied Biochemistry and Biotechnology, 60, 151
158.
JERNEJC, K, VENDRAMIN, M and CIMERMAN, A, 1989. Lipid composition of Aspergillus
niger in citric acid accumulating and nonaccumulating conditions, Enzyme and Microbial
Technology, 11, 452456.
JERNEJC, K, PERDIH, A and CIMERMAN, A, 1991. ATP:citrate lyase and carnitine
acetyltransferase activity in a citric-acid-producing Aspergillus niger strain, Applied
Microbiology and Biotechnology, 36, 9295.
KIRIMURA, K, HIROWATARI, Y and USAMI, S, 1987. Alterations of respiratory systems in
Aspergillus niger under the conditions of citric acid fermentation, Agricultural Biological
Chemistry, 51, 12991303.
KIRIMURA, K, SARANGBIN, S, RUGSASEEL, S and USAMI, S, 1992. Citric acid production
by 2-deoxyglucose-resistant mutant strains of Aspergillus niger, Applied Microbiology and
Biotechnology, 36, 573577.
28
29
30
31
32
3.1 Introduction
In terms of bulk production citric acid is widely regarded as one of the most important of
the organic acids produced by microbiological methods, although reliable estimates of world
production are not easily obtained. A widespread perception has been that most of the
production is achieved with Aspergillus niger, in what has come to be regarded as the
traditional fermentation process, although the first indications of a microbiological process
for the production of citric acid were from Wehmer (1893), who noted that Citromyces
(now Penicillium) could accumulate citric acid.
Indeed until around 1970 A. niger was almost exclusively the organism used for the
production of citric acid; the ability of other filamentous fungi to excrete citric acid was
known and has been reviewed (Rhr and Kubicek, 1992), but they are of limited importance.
A more important class of production organisms found within the Candida yeasts, and an
increasing proportion of the total production of citric acid is now manufactured using strains
of Yarrowia lipolytica (the asexual form is Candida lipolytica, syn. Saccaromycopsis).
The Candida genus (family Cryptococcaceae, subfamily Cryptococcoideae) contains
30 species, and six varieties, many of which are pathogenic to animals, including humans.
With increasing numbers of immunodeficient people, either through retroviral disease or
the anti-rejection drugs used in organ transplantation, the pathogenic species such as C.
albicans have assumed a new importance. Many Candida species have been isolated from
fruit, seeds, soil, and similar sources.
Vegetative growth consists of budding cells and pseudomycelium, or true mycelium
with blastospores. C. lipolytica was isolated from margarine (hence its name, Gr. Lipos, fat;
lysis, breaking). The cells are variable, long oval to almost cylindrical and short oval. A well
developed pseudomycelium is frequently formed with some true mycelium. The organism,
as well as hydrolysing fats, will liquify gelatine, but does not ferment sugars.
As well as the industrial importance of the organism, it is being developed as a cloning
vehicle for the expression of heterologous proteins. Some understanding of the pathways
involved in citric acid production has resulted from the cloning of particular genes as a
result of this development; in particular our knowledge of the peroxisomal pathways has
33
34
35
decarboxylation of oxoglutarate becomes the limiting reaction in the TCA cycle. This
leads to oxoglutarate accumulation within the cells and secretion into the culture medium.
The glyoxylate cycle is used as an alternative pathway when the TCA cycle is impaired in
this way.
Although growth is usually limited by nitrogen exhaustion, limitation of growth by
sulphur, magnesium or phosphorus gives a similar effect (McKay et al., 1994). Citric acid
levels between 50 and 220 mM were measured after 168 hours, with nitrogen and sulphur
limitation giving the highest specific production rates. Potassium limitation was ineffective
(6 mM), and the glucose uptake rate was only 50 per cent of that achieved when nitrogen or
sulphur was limiting.
Since the utilization of alkanes overlaps considerably with the metabolism of fatty acids the
oleaginous yeasts such as C. lipolytica were obvious targets for fermentation processes
with this type of feedstock.
36
cytoplasmic end of these channels. The endoplasmic reticulum is the site of the initial
oxidation of the alkanes.
Alkane emulsions adhere to the cell wall of Candida yeast by a non-enzymatic mechanism
(Einsele et al., 1975; Kppeli and Fiechter, 1976, 1977). The binding is due to a
lipopolysaccharide in the cell wall that is induced by alkanes. The lipopolysaccharide, which
is mannan with about 4 per cent covalently linked fatty acid, has been isolated and
characterized (Kppeli et al., 1978).
37
Table 3.1 Ratio of odd chain fatty acids and C17 acids to total cellular fatty acids in Candida
lipolytica cells grown on n-alkanes and glucose (Tanaka et al., 1976)
Table 3.1 shows the relationship for C. lipolytica grown on a variety of substrates.
The correlation between odd chain length alkane substrate and odd chain length fatty
acids in the cells is clear. The high activity of a mono-oxygenase system, with its oxygen
radical mechanism, suggests that protection against damage by free radicals might be
important when yeast grows on alkanes. Indeed a considerable increase in copper and
zinc superoxide dismutase (SOD) is seen during growth on n-alkanes as compared to
glucose (Kujumdzievasavova et al., 1991). A correlation between SOD and catalase
was noted and resistance to oxygen free radicals observed as a result of the high levels
of copper/zinc SOD, which also protected against deleterious effects of Cu2+ and Zn2+
in the medium.
38
localization of these enzymes within the cells appears generalized. Early reports are
confusing, possibly because until 1974 the occurrence of peroxisomes was not known.
Osumi et al. (1974) have detected both dehydrogenases in peroxisomes, mitochondria and
microsomes.
39
Table 3.3 Comparison of the properties of acyl CoA synthetases for C. lipolytica
40
the characteristic enzymes of the TCA cycle, are present in the mitochondrial compartment
as might be anticipated (Kawamoto et al., 1977). Fatty acid -oxidation is not present in the
mitochondria of C. lipolytica (Mishina et al., 1978) or C. tropicalis (Kawamoto et al., 1978)
but appears confined to the peroxisome. This implies that acetyl CoA required for citrate
synthesis must be transported to the mitochondria from the peroxisome, probably by the
carnitine acyltransferase system (Kawamoto et al., 1978).
Methyl isocitrate cycle
The propionyl CoA, derived from odd chain length n-alkanes, is metabolized by a cyclic
pathway analogous to the first steps in the TCA cycle (Tabuchi, 1975a, b). This pathway is
based on the accumulation of pyruvate and seven carbon tricarboxylic acids in C. lipolytica
grown on odd chain length alkanes. Methyl citrate, methylaconitate and methylisocitrate
were detected as were the key enzymes described in the methyl citrate cycle. In this pathway
propionyl CoA from the odd chain fatty acid -oxidation sequence reacts with oxaloacetate
41
42
43
with acetyl CoA and oxalacetate respectively. The enzyme activity observed in extracts is
greater than that of isocitrate lyase, aconitase or isocitrate dehydrogenase (Behrens et al.,
1977; Omar and Rehm, 1980). Candida lipolytica has both an NAD+ and an NADP+dependent isocitrate dehydrogenase. The NADP+-dependent enzyme had Michaelis
Menten type kinetics with respect to isocitrate, and a Km of 80 M for isocitrate at pH 7.0.
There was inhibition by oxalacetate at 5 mM of about 40 per cent. The energy metabolism
of C. lipolytica has been examined during growth on n-alkanes; in particular, the
concentration of adenine nucleotides during a batch fermentation was measured (Marchal
et al., 1977).
After 25 hours there was a sharp drop in the concentration of ADP and AMP while the
ATP level rose. The total adenine nucleotide levels fell slightly, then recovered. These changes
coincided with the exhaustion of nitrogen in the medium and the effective cessation of
growth. At this point citric acid excretion began, together with isocitric acid. The proportion
of isocitric to citric acid was high, about 40 per cent, although the intracellular citric to
isocitric ratio was close to that expected from the aconitase equilibrium at about 90 per cent
citric:10 per cent isocitric acid. The adenylate energy charge (Atkinson, 1970) reflected the
changes in adenine nucleotides, rising to approach 1. However, the dramatic changes are
seen in the ATP:AMP ratio, and the most common allosteric effectors amongst the adenine
nucleotides are AMP and ATP, so that the ATP:AMP ratio is a better indicator of regulatory
changes. The enzyme that is regarded as a significant target for allosteric regulation during
growth on alkanes is mitochondrial NAD+-specific isocitrate dehydrogenase (Marchal et
al., 1977). The activity of this enzyme with respect to isocitrate and AMP is sigmoidal,
consistent with its structure which has four co-operative binding sites. The enzyme is totally
dependent on AMP for activity, with maximal activity shown at 0.1 mM AMP and 50 per
cent activity at 0.05 mM. At values below 0.01 mM the enzyme is virtually inactive.
Magnesium also behaved as an allosteric activator of the enzyme, apparently with two
co-operative sites. Since the substrate for the enzyme is magnesium isocitrate this is perhaps
surprising. There was no correlation between the rate of n-alkane uptake and nitrogen
44
exhaustion or changes in adenine nucleotide levels. This is unexpected since the cessation
of growth should greatly reduce the energy demand and hence the substrate uptake. The
implication is that the coupling between electron transport and ATP synthesis becomes
loose, or that energy is used in, for example, a transport process.
The mitochondrial ATP synthase genes have been studied in Y. lipolytica (Matsuoka et
al., 1994) and a 6.6 kilobase region sequenced. This closely resembled the human
mitochondrial genome with ATP synthase subunits 8 and 6 being followed by the genes for
cytochrome c oxidase subunit 3, NADH-ubiquinone oxidoreductase subunit 4 and ATP
synthase subunit 9. All the genes were transcribed from the same strand of DNA into
multigenic RNAs starting from a nonanucleotide sequence, 5'-ATA-TAAATA-3', similar to
other yeast mitochondrial promoters. In addition to these apparently normal mitochondrial
genes there is a cyanide-resistant oxidase (Medentsev and Akimenko, 1994) located in the
inner membrane. Its activity is typically blocked by benzohydroxamic acid. This resembles
the situation found in A. niger and the circumstances of uncoupled electron transport are
also similar.
The activity of the NAD+ isocitrate dehydrogenase, which is already low compared to
the level found in cells grown on glucose, is almost totally inhibited by the drop in AMP,
and the evolution of carbon dioxide mirrors this, being sharply reduced to very low levels
when growth ceases. The metabolic production of carbon dioxide from acetate via the
TCA cycle during growth is stopped by the inhibition at the level of isocitrate
dehydrogenase.
Isocitrate lyase was high compared to cells grown on glucose so that the entire carbon
flux through the mitochondrial compartment is via the glyoxylate cycle. The activity of
citrate synthase in C. lipolytica has not been extensively studied, but it is reported to show
limited inhibition by ATP (40 per cent at 5 mM ATP). Since the concentration of ATP
reported in the whole cell during the citric acid accumulation phase varied from 0.6 mM at
the start to 0.8 mM at the end, it is unlikely to rise much above 5 mM in the vicinity of
citrate synthase, even if most of the ATP is in the mitochondrial compartment. Further, the
level of acetyl CoA, which will be high during growth on n-alkanes, will reduce the ATP
inhibition of citrate synthase.
The 3-phosphoglycerate kinase gene has been isolated from a genomic library, by probing
with a PCR fragment amplified with primers deduced from two highly conserved regions
of various pyruvate kinases (Ledall et al., 1996). It encodes a polypeptide of 417 residues
with extensive homology to other kinases. The expression of the gene is higher on
gluconeogenic substrates, such as alkanes, than on glycolytic ones. Pyruvate kinase has
also been cloned as part of the development of expression/secretion systems for heterologous
proteins (Buckholz and Gleeson, 1991; Strick et al., 1992). Genomic clones were selected
by their specific hybridization to synthetic oligodeoxyribonucleotide probes based on
conserved sequences. The gene predicts a protein that is highly homologous to the
corresponding Saccharomyces cerevisiae enzyme and the gene further transforms wild type
Y. lipolytica with a twofold increase in pyruvate kinase activity. The gene sequence contained
an intron, the first reported in a Y. lipolytica gene.
45
direct citrate excretion across the plasma membrane by some form of facilitated diffusion,
active transport, or vacuolar transport, possibly by accumulation in vacuoles and exocytosis.
Studies of the activities of enzymes in acetate mutants of Y. lipolytica (Rymowicz et al.,
1993) indicated that the excretion of isocitric and citric acids depended more on the transport
system than metabolite levels within the cell.
The vacuolar transport idea appeared promising when vacuoles from Y. lipolytica isolated
during exponential growth showed the ability to concentrate citric acid through a citrate
uniporter. The vacuoles showed high ATPase activity (1000 mU/mg protein at six hours
growth, falling to 270 mU/mg after 48 hours), which was not sensitive to orthovanadate,
nor was it inhibited by azide or oligomycin. The citrate transport rate was up to 12 nmol/mg
protein/min after 12 hours growth, and calcium was also transported (140 nmol/mg/min).
The vacuoles generated both a proton gradient and a membrane potential. However during
the stationary phase, after nitrogen exhaustion, the transport ability fell to zero for both
calcium and citrate. This observation was found to be true regardless of the growth limiting
substrate, the carbon source, or whether citrate was released from the cells or not. The
conclusion was that the citrate transporting system of the vacuolar membrane was not involved
in the citrate release into the medium, and that that process was associated with transport
systems in the plasma membrane.
The ratio of excreted isocitric to citric acid is higher than would be expected from the
thermodynamic equilibrium of aconitase, being as high as 40 per cent in wild-type yeasts,
rather than the 7 per cent expected on an equilibrium basis. Nonetheless it is apparent that
the strategies used to mitigate this unwanted production of isocitric acid all have a common
theme in that they inhibit or delete aconitase. By limiting the formation of isocitrate in the
first place the problem is resolved. The strategies reported include: the use of iron-free
medium, which resulted in impairment of aconitase activity (Kimura and Nakanishi, 1973);
the addition of sodium tetraborate, which may complex with iron to give a similar outcome
(Furakawa et al., 1982); and the selection of mutants with low aconitase activities
(Benckiser, 1974). These have been selected by their ability to grow on n-alkanes but not
citric acid and the low aconitase results in improved citrate to isocitrate ratios and decreased
biomass, but the complete absence of aconitase would presumably be lethal. Other
strategies are the use of inhibitors such as monofluoroacetate (Benckiser, 1974) which is
metabolized to monofluorocitrate and acts as a competitive inhibitor of aconitase (Akiyama
et al., 1972, 1973a, 1973b), and 2,4-dinitrophenol, an uncoupler of oxidative
phosphorylation; and the addition of alcohols, up to oleyl alcohol (Kimura and Nakanishi,
1973). The first two strategies are of use in selecting mutants but would be undesirable in
a commercial fermentation. The relative levels of isocitrate lyase and aconitase in
determining the ratio of isocitrate to citrate was underlined by Finogenova et al. (1986)
with a study of a series of C. lipolytica mutants. Mutants with a high isocitrate lyase
activity and a low aconitase level synthesized citric acid almost exclusively regardless of
whether the carbon source was glucose, alkane, ethanol, acetate or glycerol. The mutant
low in isocitrate lyase but with a high level of aconitase produced primarily isocitric acid
on alkanes, where the ratio of citrate to isocitrate was 1:3.6, while on glucose the ratio
was 1.8:1. Wild-type strains with high levels of both enzymes gave intermediate results.
In the wild-type strains the ratio could be shifted towards isocitrate synthesis by inhibiting
isocitrate lyase with aconitate, the reverse of the industrial strategy.
The explanation advanced by Marchal et al. (1980) is still valid. They suggested that
the high isocitrate ratio was a result of compartmentation within the cell. Whereas citrate
is mainly mitochondrial, isocitrate is in the mitochondrial, the cytoplasmic and the
peroxisomal compartments. Isocitrate will be exported from the mitochondrial
46
compartment to the cytosol and then to the peroxisome where it will be converted to
glyoxylate and succinate. The absence of aconitase from the cytoplasmic compartment
will result in higher isocitrate levels with lower citrate levels, and it is presumably from
the cytoplasm that the acids are exported. It is further possible that citrate and isocitrate
have differential transport, but no mutants have been reported to suggest that there is a
separate export mechanism for each acid.
47
Figure 3.4 The metabolic relationships of citrate metabolism in yeasts with n-alkanes or
glucose as substrate. 1, Alkane monooxygenase; alkane, reduced rubridoxin:oxygen 1oxidoreductase, 1.14.15.3. 2, Alcohol dehydrogenase; alcohol:NAD+ oxidoreductase,
1.1.1.1. 3, Aldehyde dehydrogenase; aldehyde:NAD+ oxidoreductase, 1.2.1.3. 4, oxidation. 5, Hexokinase; ATP:D-hexose 6-phosphotransferase, 2.7.1.1. 6, Glycolysis. 7,
Pyruvate carboxylase; pyruvate:carbon dioxide ligase (ADP), 6.4.1.1. 8, Pyruvate
dehydrogenase; pyruvate:lipoate oxidoreductase (acceptor acylating), 1.2.4.1. 9, Citrate
synthase; citrate:oxaloacetate lyase (CoA acylating), 4.1.3.7. 10, Aconitase; citrate (isocitrate)
hydrolyase, 4.2.1.3. 11, Isocitrate dehydrogenase; threo-DS-isocitrate:NAD oxidoreductase
(decarboxylating), 1.1.1.41(42). 12. Isocitrate lyase; threo-DS-isocitrate:glyoxylate-lyase,
4.1.3.1. 13, Malate synthase; 1-malate glyoxylate-lyase (CoA-acetylating), 4.1.3.2., 14,
Gluconeogenesis
48
The subcellular location of the various enzymes has been determined (Sokolov et al.,
1995a). Pyruvate carboxylase was found in the cytoplasmic compartment in C. lipolytica,
and many other yeasts. The NADP+-dependent isocitrate dehydrogenase was found to be
distributed in both cytoplasmic and mitochondrial compartments, while ATP-citrate lyase
was found in the cytoplasmic compartment. The presence of this latter enzyme would be
required for lipid synthesis, but is presumably regulated so that it does not degrade a
significant amount of the cytoplasmic citrate. The number of peroxisomes in yeasts grown
on glucose is very small.
The properties of the NAD+-dependent isocitrate dehydrogenase are thought to be central
to citrate accumulation when glucose is used as a substrate as well as n-alkanes. The AMP
requirement for activity means that the very low AMP levels found during the stationary
phase induced by nitrogen depletion will result in very low activity of the isocitrate
dehydrogenase (Bartels and Jensen, 1979). The enzyme was shown to be allosterically
regulated by AMP (Sokolov et al., 1995b) although with excess isocitrate the rate became
AMP independent. It is also inhibited by ATP which is high during citric acid accumulation.
The export of isocitrate from the mitochondial compartment is presumably reduced when
glucose is a substrate, as the glyoxylate cycle is non-functional because of the low level of
isocitrate lyase and the absence of malate synthase (Finogenova et al., 1986). This may be
the reason for the improved ratio of citrate to isocitrate produced when glucose is a substrate.
The presence of the NADP+-dependent isocitrate dehydrogenase in both cytoplasm and
mitochondria has been noted but its role, if any, is not known.
An important factor in the over-production of citric acid is the maintenance of the flux
through glycolysis when metabolite levels rise. In particular the inhibition of
phosphofructokinase by citrate might be expected to regulate the precursors for citrate
production when citrate levels are high. In citrate producing strains of Y. lipolytica the citrate
inhibition of phosphofructokinase appears to be weak (Sokolov et al., 1996), while AMP
has no effect. Ammonium suppressed the inhibitory effect of citrate and activated the enzyme,
a similar mechanism to that suggested for A. niger (Habison et al., 1983). However in Y.
lipolytica under conditions of nitrogen exhaustion, when growth has ceased it is less likely
that there is a significant pool of intracellular ammonium.
The entry of glucose into the cell is normally regulated, and under conditions of citrate
accumulation there is indeed a reduction in the glucose uptake rate (Aiba and Matsuoka,
1978), suggesting that the regulation is present to some extent. The regulation of hexokinase
has been shown to be sensitive to trehalose-6-phosphate, which occurs in yeasts at about
0.2 mM (Blazques et al., 1993). This is well above the apparent Ki for Y. lipolytica hexokinase
and it was concluded that this compound was physiologically significant. There was, however,
49
no activity against glucokinase up to 5 mM so that high levels of glucose might avoid the
regulatory step at hexokinase.
A related substrate, glycerol, has attracted some attention, and the activities of glycerol
kinase and the NAD+ and FAD-dependent glycerol-3-phosphate dehydrogenases, involved
in the glycerol phosphate shuttle between cytoplasm and mitochondria, were determined
(Morgunov et al., 1991). Glycerol kinase was localized in the cytoplasm but both glycerol
phosphate dehydrogenases were associated with the membrane fraction of the cells. The
glycerol kinase was purified and found to be inhibited by AMP, but insensitive to fructose1,6-bisphosphate.
50
C. lipolytica at the time of nitrogen exhaustion inhibited the production of citric acid (Trutko
et al., 1993). At the same time, dinitrophenol (an uncoupler of oxidative phosphorylation),
reumycin (respiratory chain shunting agent), or arsenate (which forms ADP-arsenyl instead
of ATP) all decreased the yield of citric acid in proportion to the concentration of the agent.
There was no significant effect on biomass yield. Since the over-production of citric acid
appears to involve some uncoupling of the electron transport chain from ATP synthesis,
with maximal levels of ATP resulting, the requirement for ATP shown here may be connected
with export rather than synthesis, as may be the requirement for protein synthesis.
On the other hand, Kulakovskaya et al. (1994) showed that the activity of the plasma
membrane ATPase of Y. lipolytica decreased by a factor of ten during the course of nitrogen
limited growth with glucose as a carbon source. Citric acid excretion was independent of
glucose concentration and resistant to diethylstilboestrol, an inhibitor of the plasma membrane
ATPase, for the first 30 minutes of the excretion process. They concluded that the process is
independent of energy provision.
3.4 Conclusions
The over-production of citric acid by yeasts from both alkane and carbohydrate sources is
now well established, both commercially and scientifically. The basic pathways and some
of the enzymology are understood, although many details remain to be resolved. With both
substrates the overproduction appears to represent a mechanism for recycling reducing
equivalents and energy produced by unbalanced growth conditions in the form of the absence
of, and subsequent intracellular restriction on, a primary substrate from the growth medium.
Further developments in enzymology may arise coincidentally from the use of the
organism as a cloning vehicle, but one of the main unresolved problems is the mechanism
of excretion, which is central to the problem of high productivity.
3.5 References
ABE, M, TABUCHI, T and TAHARA, Y, 1970. Studies on organic acid fermentation in yeasts: further
investigations on production of citric and d-isocitric acid by yeasts. Journal of the Agricultural
Chemistry Society of Japan, 44, 499504.
AIBA, S and MATSUOKA, M, 1978. Citrate production from n-alkane by Candida lipolytica in
reference to carbon fluxes in vivo. European Journal of Applied Microbiology and Biotechnology,
5, 247261.
AIBA, S and MATSUOKA, A, 1979. Identification of metabolic model: citrate production from glucose
by Candida lipolytica, Biotechnology and Bioengineering, 1, 13731386.
AKIYAMA, S, SUZUKI, T, SUMINO, Y, NAKAO, Y and FUKUDA, H, 1972. Production of citric
acid from n-paraffins by fluoroacetate-sensitive mutants of Candida lipolytica. In 4th Proceedings
IFS: Fermentation Technology Today, 613.
AKIYAMA, S, SUZUKI, T, SUMINO, Y, NAKAO, Y and FUKUDA, H, 1973a. Induction and citric
acid productivity of fluoroacetate-sensitive mutant strains of Candida lipolytica, Agricultural
and Biological Chemistry, 37, 879884.
AKIYAMA, S, SUZUKI, T, SUMINO, Y, NAKAO, Y and FUKUDA, H, 1973b. Agricultural and
Biological Chemistry, 37, 885888.
ATKINSON, D E, 1970. Enzymes as control elements in metabolic regulation. In The Enzymes, Vol.
1. Ed. P D BOYER (Academic Press, London), pp. 461489.
BARTELS, P D and JENSEN, P K, 1979. Role of AMP in regulation of the citric acid cycle in
mitochondria from bakers yeast, Biochemica et Biophysica Acta, 582, 246259.
51
BARTH, G and SCHEUBER, T, 1993. Cloning of the isocitrate lyase gene (ICVL1) from Yarrowia
lipolytica and characterisation of the deduced protein, Molecular and General Genetics, 241,
422430.
BEHRENS, U, HIRZEL, K and SCHULZE, E, 1977. Enzymatische Untersuchungen zur Citrat-Isocitrat
Akkumulation bei Hefen, Nahrung, 21, 525529.
BENCKISER, J A, 1974. GesmbH, United States Patent 3,843,465.
BLAZQUES, M A, LAGUNAS, R, GANCEDO, C and GANCEDO, J M, 1993. Trehalosephosphate,
a new regulator of yeast glycolysis that inhibits hexokinases, Federation of European Biochemical
Societies Letters, 329, 5154.
BUCKHOLZ, R G and GLEESON, M A G, 1991. Yeast systems for the commercial production of
herterologous proteins, Biotechnology, 9, 10671072.
COOPER, T G and BEEVERS, H, 1969. -oxidation in glyoxosomes from castor bean endosperm,
Journal of Biological Chemistry, 244, 35143520.
DUPPEL, W, LEBEAULT, J M and COON M J, 1973. Properties of a yeast cytochrome P-450
containing enzyme system which catalyses hydroxylation of fatty acids, alkanes and drugs.
European Journal of Biochemistry, 36, 583592.
EINSELE, A, SCHNEIDER, H and FEICHTER, A, 1975. Journal of Fermentation Technology, 53,
241.
EITZEN, G A, TITORENKO, V L, SMITH, J J, VEENHUIS, M, SZILARD, R K and RACHUBINSKI,
R A, 1996. The Yarrowia lipolytica gene pay5 encodes a peroxisomal integral membrane protein
homologous to the mammalian peroxisomal assembly factor, PAF1. Journal of Biological
Chemistry, 271, 2030020306.
FINOGENOVA, T V, SHISHKANOVA, N V, ERMAKOVA, I T and KATAEVA, I A, 1986. Properties
of Candida lipolytica mutants with the modified glyoxylate cycle and their ability to produce
citric and isocitric acid, Applied Microbiology and Biotechnology, 23, 378383.
FURAKAWA, T, OGINO, T and MATSUYOSHI, T, 1982. Fermentative production of citric acid
from n-paraffins by Saccharomyces lipolytica, Journal of Fermentation Technology, 60, 281
293.
GALLO, M, BERTRAND, J C and AZOULAY, E, 1971. Federation of European Biochemical Societies
Letters, 19, 45.
GALLO, M, BERTRAND, J C and ROCHE, B, 1973. Alkane oxidation in Candida tropicalis,
Biochemica et Biophysica Acta, 296, 624638.
GMUNDER, F K, 1979. Die Assimilation von Hexadecan durch Candida tropicalis, Dissertation
ETH Zurich.
HABISON, A, KUBICEK, C P and RHR, M, 1983. Partial purification and properties of
phosphofructoskinase from Aspergillus niger, Biochemical Journal, 209, 669676.
HILDEBRANDT, W and WEIDE, H, 1974. Allg. Mikrobiol, 14, 47.
HIRAI, M, SHIOTANI, T, TANAKA, A and FUKUI, S, 1976a. Effect of carbon and nitrogen sources
on the level of several NADP- and NAD-linked dehydrogenase activities of hydrocarbon utilisable
Candida yeasts, Agricultural Biological Chemistry, 40, 18191827.
HIRAI, M, TAKASHI, S, TANAKA, A and FUKUI, S, 1976b. Intracellular localization of several
enzymes in Candida tropicalis grown on different carbon sources, Agricultural Biological
Chemistry, 40, 19791986.
HONES, I, SIMON, M and WEBER, H, 1991. Characterisation of isocitrate lyase from the yeast
Yarrowia lipolytica, Journal of Basic Microbiology, 31, 251258.
HOSAKA, K, MISHINA, M, TANAKA, T, KAMIRGO, T and NUMA, S, 1979. Acyl-coenzyme-A
synthetase I from Candida lipolytica, European Journal of Biochemistry, 93, 197204.
ILCHENKO, A P, MORGUNOV, I G, HONEK, H, MAUERBERGER, S, VASILKOVA, N N and
MULLER, H G, 1994. Purification and properties of alcohol oxidase from the yeast Yarrowia
lipolytica, Biochemistry-Moscow, 59, 969974.
KAMZOLOVA, S V, SHISHKANOVA, N V, ILCHENKO, A P, DEDYUKHINA, E G and
FINOGENOVA, T V, 1996. Effects of iron ions on biosynthesis of citric and isocitric acids by
mutant Yarrowia lipolytica N-1 under conditions of continuous cultivation, Applied Biochemistry
and Microbiology, 32, 3538.
KPPELI, O and FIECHTER, A, 1976. The mode of interaction between the substrate and cell surface
of the hydrocarbon-utilzing yease Candida tropicalis, Biotechnology and Bioengineering, 18,
967974.
52
KPPELI, O and FIECHTER, A, 1977. Component of the cell surface of the hydrocarbon utilising
yeast Candida tropicalis with possible relationship to hydrocarbon transport, Journal of
Bacteriology, 131, 917921.
KPPELI, O, MULLER, M and FIECHTER, A, 1978. Chemical and structural alterations at the cell
surface of Candida tropicalis induced by hydrocarbon substrates, Journal of Bacteriology, 133,
952958.
KAWAMOTO, S, UEDA, M, NOZAKI, C, YAMAMURA, M, TANAKA, A and FUKUI, S, 1977.
Localization of carnitine acyl transferase in peroxisomes and in mitochondria of n-alkane grown
Candida tropicalis, Federation of European Biochemical Societies Letters, 96, 3740.
KAWAMOTO, S, NOZAKI, C, TANAKA, A and FUKUI, S, 1978. Fatty acid beta-oxidation system
in microbodies of n-alkane grown Candida tropicalis, European Journal of Biochemistry, 83,
609613.
KAWAMOTO, S, YAMADA, T, TANAKA, A and FUKUI, S, 1979. Distinct subcellular localization
of NAD-linked and FAD-linked glycerol-3-phosphate dehydrogenases in N-alkane-grown Candida
tropicalis, Federation of European Biochemical Societies Letters, 97, 253256.
KIMURA, K and NAKANISHI, T, 1973. British Patent 1,332,180.
KUJUMDZIEVASAVOVA, A V, SAVOV, V A and GEORGIEVA, E I, 1991. Role of superoxide
dismutase in the oxidation of n-alkanes by yeasts, Free Radical Biology and Medicine, 11, 263
268.
KULAKOVSKAYA, T V, MATYASHOVA, R N, PETROV, V V and KURANOVA, E V, 1994. ATPase
of the plasma membrane of the yeast Yarrowia lipolytica is not involved in citrate excretion,
Microbiology, 63, 1215.
LAZAROW, P B and DE DUVE, C, 1976. A fatty acid acyl Co-A oxidizing system in rat liver
peroxisomes enhancement by clofibrate, a hypolipidemic drug, Proceedings of the National
Academy of Science of the USA, 73, 20432046.
LEBAULT, J M and AZOULAY, E, 1971. Metabolism of alkane by yeast, Lipids, 6, 444447.
LEBAULT, J M, ROCHE, B and DUVNJAK, Z, 1970a. Alcool-et aldehyde
deshydrogenasesparticulaires de Candida tropicalis cultiv sur hydrocarbures, Biochemistry
Biophysics Acta, 220, 373385.
LEBAULT, J M, MEYER, F and ROCHE, B, 1970b. Oxidation des alcools suprieurs chez C. tropicalis
cultiv hydrocarbures, Biochemistry Biophysics Acta, 220, 386395.
LEBAULT, J M, LODE, E T and COON, M J, 1971. Fatty acid and hydrocarbon hydroxylation in
yeast: role of cytochrome P-450 containing enzyme system in Candida tropicalis, Biochemistry
Biophysics Research Communications, 42, 413419.
LEDALL, M T, NICAUD, J M, TRETON, B Y and GAILLARDIN, C M, 1996. The 3-phosphoglycerate
gene of the yeast Yarrowia lipolytica de-represses on gluconeogenic substrates, Current Genetics,
29, 446456.
LIU, C M and JOHNSON, M J, 1971. Alkane oxidation by a participate preparation of Candida,
Journal of Bacteriology, 106, 830834.
MARCHAL, R, VANDECASTEELE, J-P and METCHE, M, 1977. Regulation of the central
metabolism in relation to citric acid production in Saccharomycopsis lipolytica, Archives of
Microbiology, 113, 99104.
MARCHAL, R, METCHE, M and VANDECASTEELE, J-P, 1980. Intracellular concentration of
citric acid and isocitric acid in cultures of the citric acid excreting yeast Saccharomycopsis lipolytica
grown on alkanes, Journal of General Microbiology, 116, 535538.
MATSUOKA, M, UEDA, Y and AIBA, S, 1980. Role and control of isocitrate lyase from Candida
lipolytica, Journal of Bacteriology, 144, 692697.
MATSUOKA, M, HIMENO, T and AIBA, S, 1984. Characterisation of Saccharomyces lipolytica
mutants that express temperature sensitive synthesis of isocitrate lyase, Journal of Bacteriology,
157, 899908.
MATSUOKA, M, MATSUBARA, M, INQUE, J, KAKEHI, M and IMANAKA, T, 1994. Organisation
and transcription of the mitochondrial ATP synthase genes in the yeast Yarrowia lipolytica, Current
Genetics, 26, 382389.
MCKAY, I A, MADDOX, I S and BROOKS, J D, 1994. High specific rates of glucose utilisation
under conditions of restricted growth are required for citric acid accumulation by Yarrowialipolytica
IMK-2, Applied Microbiology and Biotechnology, 41, 7378.
MEDENTSEV, A G and AKIMENKO, V K, 1994. Localisation of cyanide-resistant oxidase in
mitochondria of the yeast Yarrowia lipolytica, Microbiology, 63, 233236.
53
54
TABUCHI, T and HARA, S, 1970. Conversion of citrate fermentation to polyol fermentation in Candida
lipolytica, Journal of Agricultural Chemical Society of Japan, 47, 485489.
TABUCHI, T and SATOH, T, 1976. Distinction between isocitrate lyase and methylisocitrate lyase in
Candida lipolytica, Agricultural Biological Chemistry, 40, 18631869.
TABUCHI, T and SATOH, T, 1977. Purification and properties of methylisocitrate lyases, a key enzyme
in propionate metabolism in Candida lipolytica, Agricultural Biological Chemistry, 41, 169
174.
TABUCHI, T and SERIZAWA, N, 1975a. The production of 2-methylisocitrate from odd carbon Nalkanes by a mutant of Candida lipolytica, Agricultural Biological Chemistry, 39, 10551062.
TABUCHI, T and UCHIYAMA, H, 1975b. Methylisocitrate condensing and methylisocitrate cleaving
enzymes: evidence for the pathway of oxidation of propionyl CoA to pyruvate, Agricultural
Biological Chemistry, 39, 10491054.
TABUCHI, T, TANAKA, M and ABE, M, 1969. Journal of Agricultural Chemical Society of Japan,
43, 154.
TABUCHI, T, TAHARA, Y, TANAKA, M and YANAGIUCHI, S, 1973. Journal of Agricultural
Chemical Society of Japan, 47, 617.
TANAKA, A, HAGIHARA T, NISHIKAWA, Y, MISHINA, M and FUKUI, S, 1976. Effect of the
anti-lipogenic antibiotic cerulenin on growth and fatty acid composition of the n-alkane utilizing
Candida lipolytica, European Journal of Applied Microbiology, 3, 115124.
TANAKA, A, NABESHIMA, S, TOKDUA, M and FUKUI, S, 1977. Partial purification of isocitrate
lyase from Candida tropicalis and some kinetic properties of the enzyme, Agricultural Biological
Chemistry, 41, 795.
TERANISHI, Y, KAWAMOTO, S, TANAKA, A, OSUMI, M and FUKUI, S, 1974. Agricultural and
Biological Chemistry, 38, 12131225.
TRUTKO, S M, MATYASHOVA, R N and AKIMENKO, V K, 1993. The effect of cell deenergization
and malate addition on over-synthesis of citric acid by the yeast Candida lipolytica, Microbiology,
62, 603606.
UCHIYAMA, H and TABUCHI, T, 1976. Agricultural and Biological Chemistry, 40, 14111418.
WEHMER, C, 1893. Note sur la fermentation citrique, Bullitin Societe Chemie Francaise, 9, 728.
YAMADA, K, TAKAHASHI, J and VKOBAYASHI, K, 1963. Agricultural and Biological Chemistry,
27, 773.
ZYAKUN, A M, MUSLAEVA, I N, ASHIN, V V, PESHENKO, V P, ADANIIN, V M, MASHKINA,
L P, MATYASHOVA, R N and FINOGENOVA, T V, 1992. Heterotrophic fixation of carbon
dioxide by Candida lipolytica and its role in citric acid biosynthesis. Microbiology, 61, 390397.
Strain Improvement
GEORGE J.G.RUIJTER AND JAAP VISSER
4.1 Introduction
Many factors need to be considered by citric acid producers to obtain the economically
most favourable process. Strain breeding is one of these factors. In this chapter we will
summarize ways to improve citric acid production genetically. Commercial production of
citric acid is performed mainly with Aspergillus niger and to some extent with Candida (or
Yarrowia) lipolytica. As the existing fermentation processes usually give high yields, the
main objective of strain breeding nowadays is shortening of fermentation time. However,
other factors may also be relevant for strain improvement. For example, accumulation of a
high concentration of citric acid by A. niger results from quite extreme culture conditions
and strain breeding may decrease the sensitivity of the process to these conditions.
The number of reports considering strain improvement that have appeared in literature
is limited. Rhr et al. (1983), Kubicek and Rhr (1986) and Mattey (1992) have reviewed
much of the older work. However, some research and screening activities are hidden,
i.e. performed by industry and not published for obvious reasons. We have structured this
chapter more or less on the basis of the methodology used for strain improvement of A.
niger:
1 mutagenesis and selection;
2 the use of the parasexual cycle; and
3 genetic engineering.
As strain breeding involves fungal genetics, some aspects of the genetic methodology used
have been included.
56
individual colonies obtained from single spores (single spore method). Such a method
requires automated screening procedures that enable testing of thousands of colonies and
is therefore usually done by plate tests. A pH indicator is commonly included in the
medium to estimate acid production, but since a pH indicator does not distinguish between
citric acid and other acids, improved methods have been developed, e.g. using p-dimethylaminobenzaldehyde, which specifically measures citric acid (Rhr et al., 1979).
Yields are evaluated by determining the ratio between acid zone and colony diameter.
Obviously, statistical analysis of screening results is quite important to evaluate the
significance of a difference in acid production between the parental strain and strains
derived from it. Liquid cultures, such as shake flasks, are not suitable in the initial stage
of screening, but can be used in later steps for a limited number of selected strains. An
alternative to screening by plates would be the use of high throughput screening
procedures, making use of microtitre plate technology. This has an even higher capacity
than plates as it can be automated to a large extent. Nowadays, microtitre plate technology
is commonly used by industry in all kinds of screening processes, but it is not clear whether
citric acid producers also employ it.
The second method comprises selection of mutants with a specific trait from a large
population using a suitable discriminative growth condition (passage method). Selection
may be on the basis of resistance against an antimetabolite (Kirimura et al., 1992) or failure
to grow on a particular carbon source (Akiyama et al., 1973a). Mutants can arise
spontaneously or be produced by mutagenic treatment. A variety of methods are used for
mutagenesis including exposure to chemicals, UV light, g- and X-ray radiation (see e.g.
Begum et al., 1990; Hamissa et al., 1991; Avchieva and Vinarov, 1993; Gupta and Sharma,
1995). A serious drawback of mutagenic treatment is that high doses increase the chances
of obtaining more than one mutation per genome at a time. Thus, in addition to a mutation
that results in improved citric acid production, an isolate may have other mutations that
might, for example, result in (slightly) reduced viability. To minimize the chances to introduce
such unwanted mutations, mutagenic treatment should be performed in such a way that a
high percentage of survival is obtained.
When a better producing mutant is isolated it should be maintained in a proper way to
prevent decay, i.e. lose its particular characteristics favourable for citric acid production.
Decay is most pronounced during the vegetative stage and therefore storage of spores is the
best way to preserve a strain. The optimal storage method depends on the organism. A.
niger conidiospores are usually stored on silica beads at 4C or suspended in a 20 to 30 per
cent glycerol solution and frozen. Apart from natural variation certain mutations may be
particularly unstable, i.e. losing such mutation may be advantageous for the fungus. For
example a certain mutation may result in improved citric acid production, but concomitantly
cause reduced vitality. This necessitates careful preservation of original strains and possibly
frequent re-isolation.
The biochemistry of citric acid biosynthesis has been reviewed before (Kubicek and
Rhr, 1986; Mattey, 1992) and will not be treated at length here. Some aspects will however
be discussed in order to understand the rationale behind some strategies. Biosynthesis of
citric acid from hexoses is depicted in Figure 4.1. Following uptake, hexoses are degraded
mainly via glycolysis yielding pyruvate. Part of the pyruvate is converted to acetyl
CoA, part to oxaloacetate. Finally, these two compounds are condensed to citric acid,
which is secreted and accumulated in the medium. Only in a few cases is the genetic
basis or biochemical mechanism of the improved performance by a mutant known.
Schreferl-Kunar et al. (1989) isolated several mutants that grew better than the parent
Strain improvement
57
Figure 4.1 Schematic representation of biosynthesis of organic acids and polyols with A.
niger. The following steps are depicted: 1, glucose oxidase; 2, lactonase; 3, glucose transport;
4, hexokinase or glucokinase; 5, phosphoglucose isomerase; 6, fructose transport; 7,
hexokinase; 8, mannitol1-phosphate dehydrogenase; 9, mannitol-1phosphate phosphatase;
10, mannitol transport; 11, phosphofructokinase; 12, aldolase; 13, triosephosphate
isomerase; 14, glyceraldehyde3-phosphate dehydrogenase; 15, phosphoglycerate kinase;
16, phosphoglycerate mutase; 17, enolase; 18, pyruvate kinase; 19, pyruvate dehydrogenase;
20, pyruvate carboxylase; 21, oxaloacetate hydrolase; 22, oxalate transport; 23, malate
dehydrogenase; 24, citrate synthase; 25, tricarboxylate carrier; 26, citrate transport. Dashed
arrows are used for multiple steps in biosynthesis of erythritol and glycerol. PPP, pentose
phosphate pathway
58
on 14 per cent sucrose. The rationale of this selection procedure is the notion that a high rate
of citric acid production requires the ability for fast sugar metabolism. Four mutants consumed
sucrose faster and gave higher citric acid yields than the parental strain. Unfortunately, it is
not clear whether the productivity or just the final yield is improved in these mutants, although
faster sucrose consumption suggests increased productivity. Interestingly, all four mutants
had about twofold higher activity of the glycolytic enzymes hexokinase and
phosphofructokinase, suggesting that the activity of these two enzymes is important in
controlling the rate of sugar consumption. In the following sections a few specific objectives
for strain improvement will be discussed.
Strain improvement
59
localized mainly in the cell wall (Witteveen et al., 1992) and induced by hydrogen peroxide
and high glucose concentration (Witteveen et al., 1993). The enzyme is not stable at pH
values below 2 to 3 and hence not induced since no H2O2 is formed. Oxalic acid is produced
by oxaloacetate hydrolase, which is a cytoplasmic enzyme (Kubicek et al., 1988).
Biosynthesis of oxaloacetate hydrolase is also regulated by external pH, but in this case the
mechanism is unclear. Induction of the enzyme is optimal at pH 5 to 6, whereas a very low
oxaloacetate hydrolase activity is observed at pH 2 (Kubicek et al., 1988). In pure sugar
fermentations, production of gluconic and oxalic acid can thus be kept to a minimum by
starting the fermentation at a relatively low pH. In fermentations using molasses as a substrate,
an initial pH of 5 to 6 is commonly employed, because conidiospores will not germinate at
lower pH values. Therefore, in processes using molasses, production of gluconic and oxalic
acid may be a problem favouring production strains lacking glucose oxidase and oxaloacetate
hydrolase. In our laboratory a number of gox mutants have been isolated. One of the
mutations, goxC, results in the absence of glucose oxidase activity and strains carrying
goxC do not produce gluconic acid from glucose (Witteveen et al., 1990). Interestingly, a
goxC mutant produces more oxalic acid from glucose than wild-type A. niger (Van de Merbel
et al., 1994).
The major problem with production of citric acid by C. lipolytica is the simultaneous
production of considerable amounts of isocitric acid. Wild-type C lipolytica strains produce
approximately equimolar amounts of citric acid and isocitric acid from n-alkanes, whereas
less isocitric acid is produced from sugar substrates (Finogenova et al., 1986). Akiyama et
al. (1973a) reasoned that a low activity of aconitase, the enzyme catalyzing the conversion
of citric acid to isocitric acid, was essential to reduce production of isocitric acid. They
selected a mutant that was more sensitive to fluoroacetate than the wild-type strain. This
mutant had approximately 1 per cent of the wild-type aconitase activity and produced virtually
no isocitric acid (Akiyama et al., 1973a, 1973b).
60
phosphofructokinase that was less sensitive to citrate than the one in the parental strain; this
mutant accumulated approximately threefold more citric acid compared to the parent on a
medium containing 20 mM manganese. However, the citric acid yield of the mutant in the
presence of manganese was only half that obtained with the parental strain on manganese
deficient medium, indicating that the effects of manganese cannot be attributed to
phosphofructokinase alone.
Strain improvement
61
62
An important feature of MCA is the summation theorem, which states that the sum of
the flux control coefficients of the enzymes in a pathway is equal to 1. As a consequence,
the flux control coefficient of any enzyme in a very long pathway is probably very small as
there are many enzymes contributing to control. Moreover, when an enzyme with some
flux control is overproduced, control readily shifts to another step in the pathway. In practice
this means that genetic engineering is not easy in complex pathways. The benefit of control
analysis in designing strategies to optimize biotechnological processes depends heavily on
the availability of enzyme kinetic data and on the reliability of these data. In the case of
citric acid biosynthesis by A. niger quite a few enzymes have been studied now but a few,
such as the transport steps, are less well or not at all investigated, hampering a precise
analysis.
Recently, a few attempts have been performed to analyse flux control in citric acid
biosynthesis by A. niger (Torres, 1994a, 1994b; Ruijter et al., 1996; Torres et al., 1996a).
Torres performed modelling of the first part of the pathway, i.e. up to pyruvate (see Figure
4.1) using BST formalism and suggested that sugar transport and phosphorylation, which
are lumped into one step in the model, form the most important step in controlling the flux
through the pathway. Thus, according to this model, the cellular amount of sugar transporter
and/or hexokinase should be increased to obtain a higher metabolic flux. To a certain extent
these findings correlate with experimental data. As described in Section 4.2 certain mutants
with improved citric acid production had increased activity of hexokinase and
phosphofructokinase (Schreferl-Kunar et al., 1989) and Steinbck et al. (1994) found that
some 2-deoxyglucose resistant mutants had lower hexokinase activity and produced less
citric acid than the parent. From an investigation of glucose transport in A. niger, Torres et
al. (1996b) concluded that hexokinase contributed more to flux control in glycolysis than
glucose transport.
In a subsequent study (Torres et al., 1996a) it was concluded from flux optimization
calculations that simultaneous overproduction of seven enzymes was required for a significant
increase in flux. For practical reasons this is not achievable at the moment. Firstly, most of
the A. niger genes required for this approach are not available and secondly, simultaneous
overexpression of seven enzymes in a controlled way is experimentally difficult to
accomplish. Notably, this model has not incorporated the metabolism from pyruvate to
extracellular citric acid and hexokinase might have flux control in the conversion of glucose
to pyruvate, but the control in the complete pathway (hexose to citric acid) might be in later
steps i.e. between pyruvate and citric acid. Nevertheless, a modelling approach is worthwhile.
It may not produce an exact solution to improve the process, but it provides a guideline for
genetic engineering of A. niger.
Strain improvement
63
Figure 4.2 Schematic representation of the normal and alternative respiratory chains. The
normal respiratory chain (lower part) contains three complexes: NADH:ubiquinone
oxidoreductase (complex I), ubiquinol:cytochrome c oxidoreductase (complex III) and
cytochrome c oxidase (complex IV). In the alternative respiratory chain (top part) electrons
are tranferred directly from ubiquinol to oxygen
there is no growth in the stage of citric acid accumulation, the cells probably do not require
much ATP, and a switch from normal respiration to alternative oxidases would enable the
fungus to reoxidize its NADH without concomitant ATP production.
A very attractive hypothesis has been put forward by the group of Weiss. They found
that the proton-pumping NADH:ubiquinone oxidoreductase (complex I) is very fragile in
A. niger B-60, which is a good citric acid producer, compared to a wild-type A. niger strain
(Schmidt et al., 1992; Wallrath et al., 1992). The selective loss of complex I might result in
an increased NADH/NAD+ ratio in the cell, because the affinity of the alternative NADH
oxidase for NADH is approximately one order of magnitude lower than that of complex I.
Excretion of citric acid is a possibility in order to get rid of the excess reducing equivalents.
As such, the switch to alternative oxidases is not a reaction of the fungus to citric acid
production, but the loss of complex I results in initiation of citric acid accumulation. To test
this hypothesis one of the subunits of complex I was inactivated in a wild-type (bad
producing) A. niger strain by disruption of the corresponding gene, nuo51, by molecular
genetic techniques (Prmper et al., 1993). The mutant was unable to form a functional
complex I and should accordingly accumulate citric acid as B-60 does. Unexpectedly, the
mutant excreted virtually no citric acid, whereas the wild-type A. niger strain produced
approximately 30 per cent of the yield obtained with B-60. However, the mutant accumulated
high intracellular levels of TCA cycle intermediates, including citrate. Apparently, the mutant
is indeed unable to reoxidize NADH under these conditions, resulting in accumulation of
TCA cycle intermediates. Prmper et al. propose that, in contrast to wild-type A. niger and
strain B-60, the mutant is unable to excrete citric acid (or other TCA cycle intermediates).
This postulate, i.e. the presence of a citrate carrier, may explain the differences in citric acid
production between wild-type A. niger and B-60, but does not resolve the discrepancy
between wild-type A. niger and the mutant lacking complex I. It would be interesting to test
the effect of disruption of nuo51 in strain B-60. In addition to the effect it might have on
initiation of citric acid accumulation, it might also bring about an increase in the rate of acid
production. Assuming an excess of ATP during citric acid production, inactivation of complex
I would be a way to decrease such an excess, since less ATP is produced per NADH.
64
citric acid accumulation by a mutant of A. niger strain B-60 in which the gene encoding a
subunit of trehalose-6-phosphate synthase, ggsA, was disrupted. This mutant lacks trehalose6-phosphate synthase activity and the rationale for construction of this strain was the
following. Trehalose-6-phosphate and the enzyme catalyzing its biosynthesis have recently
been shown to play a role in regulation of glycolytic flux in the yeast Saccharomyces
cerevisiae (Thevelein and Hohmann, 1995). Trehalose-6-phosphate inhibits hexokinase
activity in S. cerevisiae in vitro (Blzquez et al., 1993) and this was found to be also the case
in A. niger (Arisan-Atac et al., 1996; Panneman et al., 1996). Inactivation of trehalose-6phosphate synthase would result in the inability to synthesize trehalose-6-phosphate and if,
under citric acid producing conditions, trehalose-6-phosphate inhibits glycolysis in A. niger,
the absence of trehalose-6-phosphate synthase might result in an increased glycolytic flux
and increased citric acid production. This was indeed found to be the case. The ggsA
disruption strain produced the same final yield of citric acid as the wild-type strain, but
reached this yield in a shorter fermentation time. This is the only case where genetic
engineering of A. niger results in improved citric acid production.
Recently we have studied in our laboratory, the effects of overproduction of two glycolytic
enzymes, phosphofructokinase and pyruvate kinase (Figure 4.1) on citric acid production
by A. niger (Ruijter et al., 1997). A few experimental studies have suggested that
phosphofructokinase might be an important step in control of the glycolytic flux. Firstly,
cultivation on a high concentration of sucrose, glucose or fructose stimulated citric acid
accumulation by A. niger and these conditions also led to increased intracellular levels of
fructose-2,6-bisphosphate, a potent activator of phosphofructokinase (Kubicek-Pranz et al.,
1990). Secondly, as already addressed in Section 4.2, mutants selected for the ability to
grow fast on high concentrations of sucrose exhibited increased citric acid production and
in these strains the activities of hexokinase and phosphofructokinase were twofold higher
than in the parental strain (Schreferl-Kunar et al., 1989). We have overexpressed
phosphofructokinase and pyruvate kinase, both individually and simultaneously, in A. niger
N400 (Ruijter et al., 1997). Unfortunately, moderate overexpression of these enzymes (three
to five times the wild-type level) did not enhance citric acid production by the fungus
significantly (Figure 4.3). Overexpression of pyruvate kinase even appeared to have a negative
effect on citric acid production. Thus, phosphofructokinase and pyruvate kinase do not
seem to contribute in a major way to flux control of the metabolism involved in the conversion
of glucose to citric acid. However, it must be noted that in cells overproducing
phosphofructokinase, the concentration of fructose-2,6-bisphosphate was decreased
approximately twofold compared to the wild-type. Hence, the fungus appears to adapt to
overexpression of phosphofructokinase by decreasing the specific activity of the enzyme
through a reduction in the level of fructose-2,6-bisphosphate. From his modelling studies
Torres (1994b) also concluded that phosphofructokinase and pyruvate kinase did not have
flux control. In the model of Torres, however, regulation of phosphofructokinase by fructose2,6-bisphosphate was not included. Our data suggest that overproduction of
phosphofructokinase, while maintaining or increasing fructose-2,6-bisphosphate levels, may
still increase glycolytic flux in A. niger.
Strain improvement
65
Figure 4.3 Citric acid fermentation from glucose by an A. niger N400 wild-type strain (+) and
transformants overproducing phosphofructokinase (O), pyruvate kinase (D) or
phosphofructokinase and pyruvate kinase (). Citric acid, glucose and dry weight are
indicated (data taken from Ruijter et al., 1997)
analysis of metabolism and metabolic pathway engineering are only just being implemented,
but in our view this is a promising approach, not so much as an alternative to the traditional
strain breeding methods, but complementary to it.
4.6 Acknowledgements
GR is financially supported by the Dutch Ministry of Economic Affairs, the Ministry of
Education, Culture and Science, The Ministry of Agriculture, Nature Management and
Fishery in the framework of an industrial relevant research programme of The Netherlands
Association of Biotechnology Centres (ABON).
4.7 Reference
AKIYAMA, S, SUZUKI, T, SUMINO, Y, NAKAO, Y and FUKUDA, H, 1973a. Induction and citric
acid productivity of fluoroacetate-sensitive mutant strains of Candida lipolytica, Agricultural
and Biological Chemistry, 37, 879884.
AKIYAMA, S, SUZUKI, T, SUMINO, Y, NAKAO, Y and FUKUDA, H, 1973b. Relationship between
aconitate hydratase activity and citric acid productivity in fluoroacetate-sensitive mutant strain
of Candida lipolytica, Agricultural and Biological Chemistry, 37, 885888.
66
Strain improvement
67
68
TORRES, N V, 1994b. Modelling approach to control of carbohydrate metabolism during citric acid
accumulation by Aspergillus niger: II. Sensitivity analysis, Biotechnology and Bioengineering,
44, 112118.
TORRES, N V, VOIT, E O and GONZLEZ-ALCN, C, 1996a. Optimisation of non-linear
biotechnological processes with linear programming: application to citric acid production by
Aspergillus niger, Biotechnology and Bioengineering, 49, 247258.
TORRES, N V, RIOL-CIMAS, J M, WOLSCHEK, M and KUBICEK, C P, 1996b. Glucose transport
by Aspergillus niger: the low-affinity carrier is only formed during growth on high glucose
concentrations. Applied Microbiology and Biotechnology, 44, 790794.
VAN DE MERBEL, N C, RUIJTER, G J G, LINGEMAN, H, BRINKMAN, U A TH and VISSER, J,
1994. An automated monitoring system using on-line ultrafiltration and column liquid
chromatography for Aspergillus niger fermentations, Applied Microbiology and Biotechnology,
41, 658663.
WALLRATH, J, SCHMIDT, M and WEISS, H, 1991. Concomitant loss of respiratory chain
NADH:ubiquinone reductase (complex I) and citric acid accumulation of Aspergillus niger, Applied
Microbiology and Biotechnology, 36, 7681.
WALLRATH, J, SCHMIDT, M and WEISS, H, 1992. Correlation between manganese-deficiency,
loss of respiratory chain complex I activity and citric acid production in Aspergillus niger, Archives
in Microbiology, 158, 435438.
WIEBE, M G, ROBSON, G D and TRINCI, A P J, 1989. Effect of choline on the morphology, growth
and phospholipid composition of Fusarium graminearum, Journal of General Microbiology,
135, 21552162.
WITTEVEEN, C F B and VISSER, J, 1995. Polyol pools in Aspergillus niger, FEMS Microbiology
Letters, 134, 5762.
WITTEVEEN, C F B, VAN DE VONDERVOORT, P J I, SWART, K and VISSER, J, 1990. Glucose
oxidase overproducing and negative mutants of Aspergillus niger, Applied Microbiology and
Biotechnology, 33, 683686.
WITTEVEEN, C F B, VEENHUIS, M and VISSER, J, 1992. Localisation of glucose oxidase and
catalase activities in Aspergillus niger, Applied Environmental Microbiology, 58, 11901194.
WITTEVEEN, C F B, VAN DE VONDERVOORT, P J I, VAN DEN BROECK, H C, VAN
ENGELENBURG, F A C, DE GRAAFF, L H, HILLEBRAND, M H B C, et al., 1993. Induction
of glucose oxidase, catalase, and lactonase in Aspergillus niger, Current Genetics, 24, 408416.
ZEHENTGRUBER, O, KUBICEK, C P and RHR, M, 1980. Alternative respiration of Aspergillus
niger, FEMS Microbiology Letters, 8, 7174.
Fungal Morphology
MARIA PAPAGIANNI
5.1 Introduction
In submerged culture the morphology of filamentous micro-organisms varies between pellets
and free filaments depending on culture conditions and the genotype of the strain. All the
growth forms have their own characteristics concerning growth kinetics, nutrient consumption
and broth rheology. Of the two extremes, pellet suspensions exhibit Newtonian rheological
behaviour, while the filamentous form produces more viscous media with consequent effects
of poor mixing and mass transfer. This is unfortunate, as it is very often the case that the
disperse filamentous form is the productive form. Another drawback with the pelleted
suspension is that cell growth occurs only at the surface of the pellets where contact with
oxygen and other nutrients is adequate, and the cells growing within a pellet respond to a very
different environment. Further into the pellet, mass transfer limitation will gradually occur
and cells could autolyse.
This chapter deals with the factors that affect Aspergillus niger morphology in submerged
culture and the influence of morphology on productivity in citric acid fermentation.
70
Since the morphological form can strongly influence the overall process productivity,
research on various aspects of morphological development has attracted the interest of
academia and industry and attempts have been made to induce a particular form of growth
and to relate morphology to product synthesis.
Initial investigations of mycelial morphology relied on manual measurements from
photographs and little quantitative work was presented until the 1970s. Detailed
morphological characterization of the free filamentous form was first presented by Metz et
al. (1981). Their method, which made use of an electronic digitizer to make measurements
from microphotographs, was time consuming, inaccurate and also difficult to automate. In
1988, Adams and Thomas presented the first image analysis method for morphological
measurements of a filamentous fungus, using images taken directly from the microscope to
an image analyzer. Since then, highly automated methods have been developed which have
many applications and allow detailed characterization of growth and simple differentiation
of filamentous micro-organisms (Thomas, 1992). With the large variety of products produced
by filamentous organisms and their complex physiology, a method that provides accurate
and reproducible quantitative morphological characterization is invaluable in studies of
process optimization and modelling.
In this chapter, the effects of agitation, nutritional factors (type and concentration of
carbon source, nitrogen and phosphate limitation, pH, dissolved oxygen tension, trace metals
levels) and inoculum size will be discussed with respect to the micro-morphology of A.
niger.
Fungal morphology
71
Figure 5.1 Effect of agitation on citric acid production and the relation between production
and morphology parameters in the tubular loop reactor
consecutive release of intracellular material may account for the decreased productivities
reported in many cases under intensive agitation conditions. This has been proven for
Penicillium chrysogenum. Smith et al. (1990) and Makagiansar et al. (1993) observed that
the lower rates of penicillin synthesis at high agitation speeds were due to increased damage
of hyphae, since it involved a greater frequency of circulation of mycelia through the high
energy dissipation zone around the impeller.
Case study 1
The effect of agitation on A. niger morphology and citric acid production has been studied
in a tubular loop (TLR) and a stirred tank reactor (STR), through a series of batch experiments
carried out at different circulation times (4 to 18 s) in the case of the TLR and at stirrer
speeds from 100 to 600 rpm for the STR (Papagianni, 1995). Both reactors were inoculated
with a vegetative inoculum. The inoculum filaments started to clump within 24 hours of
fermentation in all experiments.
Morphological measurements using image analysis showed that by increasing the intensity
of agitation, the size of clumps (P1) decreased, as did the length of the filaments (L) that
arose from the cores of clumps, while the diameter of filaments (d) increased. The perimeter
of the clumps was measured by joining the tips of the filaments that arose from the core of
the clumps. For the estimation of the core of clumps (P2), lines were drawn around the core
and their combined length was measured. For the estimation of L, the length of the filaments
and branches that arose from the core of the clump was measured.
In both fermenters, specific rates of citric acid formation, sp.rp, increased with agitation;
the amount of citric acid produced at the end of the runs (at 168 h) was dependent on the
circulation time and the stirrer speed. In the STR, as the stirrer speed increased citric acid
production increased up to a point (300 rpm) beyond which it remained constant. In Figures
5.1 and 5.2 the effect of agitation on citric acid production and the relation between production
and morphology is shown. In both reactors low dissolved oxygen levels were observed
under conditions of low agitation intensities.
If the intensity of agitation was changed, different patterns of morphological
development were observed in both fermenters. The effect of intensive agitation was
more pronounced in the stirred tank reactor, since the length of filaments was reduced by
a factor of three, while in the loop rector the reduction was much smaller. Figure 5.3 shows the
72
Figure 5.2 Effect of agitation on citric acid production and the relation between production
and morphology in the stirred tank reactor
Figure 5.3 Time course for the clump perimeter, P1, at different stirrer speeds in the stirred
tank fermentations
time course of P1 in the STR fermentations whilst the time course for the hyphal length in
the TLR is shown in Figure 5.4.
The mean diameters of filaments also changed during fermentation; filaments became
thinner with time in all experiments performed in both fermenters. The reduction of hyphal
diameter was found to be dependent on agitation: the faster the broth circulation and the
higher the stirrer speed, the more rapid was the reduction.
These differences in morphological development during fermentation could not be
explained by the assumption of increased branching alone (newly formed branches would
Fungal morphology
73
Figure 5.4 Time course for the specific growth rate and hyphal length, L, as a function of
circulation time in the tubular loop reactor
lower the mean values of L and P1) under conditions of intensive agitation. It is known that
increased branching frequency is associated with increased specific growth rates (Katz et
al., 1971; Morrison and Righelato, 1974). This is not the case for these experiments. For the
runs in which values for the specific growth were comparable rate (e.g. at circulation times
4s and 10s in the TLR), very different time courses of L as well as of P1 and P2 were
observed. This also applied to stirred tank fermentations; at 500 rpm, L decreased rapidly
for the first 100 hours and for the rest of the run decrease was slower, while m values for the
period 30 to 72 hours were lower than those noted at 300 rpm. Hyphal fragmentation as a
result of increased agitation intensity could explain the different patterns of time courses
observed in these studies. Thus, under intensive agitation conditions a cycle of fragmentation
and regrowth takes place while at low agitation intensities a gradual ageing process
predominates and the filaments grow long, with few branches remaining; mean values of L
and P1 are higher.
Mitard and Riba (1988), studying the effect of stirrer speed on A. niger growth and
morphology, observed that there was a relationship between the specific growth rate of the
organism and the rupture of mycelial aggregates. As the aggregates were broken, the specific
growth rate reduced; it increased again as the liberated filaments went on growing. This
could explain the lower specific growth rates observed at 500 rpm during the period between
30 and 72 hours and the rapid reduction of the length of filaments. At this stirrer speed P1
and P2 also decreased for 100 hours from inoculation; after this period their mean values
increased again towards the end of the run. This indicates that fragmentation and regrowth
took place.
From Figures 5.1 and 5.2, it can be observed that beyond a stirrer speed of 300 rpm,
changes in morphology were not followed by changes in citric acid production. However,
the specific rates of citric acid formation were stirrer speed dependent as was the
morphology. As the specific production rate increased with the stirrer speed, the parameters
74
Figure 5.5 Plots of the final values for citric acid concentration and the morphology
parameter P1 against the relative mixing time in the loop and stirred reactors
P1 and L reduced. The core perimeter, P2, appeared to be not affected by agitation and it
cannot be directly linked to citric acid production.
As Figures 5.1 and 5.2 show, the morphological characteristics of the broth are similar in
the two reactor systems and there appear to be no fundamental differences between the results
obtained from the loop and the stirred tank reactor. To compare the two different systems the
circulation times in the STR were estimated and they were found to be 1.1, 1.8, 2.2, 3, 3.8 and
6 seconds, while the mixing times were 8, 12, 14, 15, 16 and 22 seconds for the stirrer speed
range of 600 to 100 rpm. Compared to those of the loop reactor, both circulation and mixing
times in the STR were smaller. As a means of comparing the mixing characteristics of the two
ferments, the dimensionless parameter relative mixing time, tm, can be used. The relative
mixing time is equal to the mixing time divided by the circulation time (tm = tm/tc). It was
calculated and found to be within the range of 4 to 8 in both reactors.
In Figure 5.5, the final values for citric acid concentration and P1 have been plotted
against tm for both reactors. As shown in this figure, by increasing tm, citric acid production
increased, while the perimeter of clumps decreased, with good agreement between the two
reactor systems. It appears that the amount of product and morphology is a function of tm
and for this fermentation, production and morphology are strongly linked.
Fungal morphology
75
76
Figure 5.6 Effect of initial glucose concentration on length of filaments in batch fermentations
Figure 5.7 Effect of glucose levels on the specific rate of citric acid production in fed-batch
fermentations
Fungal morphology
77
Figure 5.8 Time course for the mean length of filaments under different glucose levels in fedbatch fermentations
the early stages of fermentation (48 hours), the highest specific growth rate values were
observed when glucose was maintained at the lowest glucose concentration of 5 g l-1. Later
in the fermentation, glucose level had little influence on the specific growth rate. The mean
length of filaments at 24 hours in fed-batch runs with glucose levels in the range from 130
g l-1 to 17 g l-1, decreased with decreasing glucose levels (Figure 5.8). Mean values of P1
(Figure 5.9) and L were found to be smaller in fed-batch runs with glucose levels between
130 g l-1 and 17 g l-1 after 24 hours of fermentation, than those observed in the batch run
with initial glucose at 150 g l-1. The values of the specific growth rate were significantly
higher in fed-batch experiments for the first two days of fermentation than those obtained in
the batch run at 150 g l-1 glucose. Since it was observed in both cultures that the specific
growth rate values for the first 48 hours were increased with decreasing glucose levels, the
reduction in L could be a result of increased branching.
Fermentation rates and morphology developed in a different way when glucose was
kept at 5 g l-1 throughout fermentation. The very low glucose levels in this run affected
metabolism, since citric acid formation was reduced in favour of cell growth and the
changes in metabolism were accompanied by changes in morphology, as shown in Figures
5.8 and 5.9. Morphological changes also included the appearance of pellets after three
days of fermentation, although the bulk of the mycelium was in the form of clumps. It
has been suggested that factors favouring increased growth rates may reduce pellet
formation in fungi (Hemmersdorfer et al., 1987). This is in contrast to our observations
78
Figure 5.9 Time course for the perimeter of the clumps under different glucose levels in fedbatch fermentations
of the values for the specific growth rate which were the highest noted and for the first time
pellets appeared in the broth.
It seems clear that the carbon source concentration affects citric acid production rates
and A. niger growth and morphology. For this system morphology and product formation
were closely related, since the reduction of the length of filaments and size of clumps was
associated with increased specific production rates.
Fungal morphology
79
Figure 5.10 Effect of phosphate level on A. niger morphology in the loop reactor at a
circulation time of 18 s
Case study 4
Figure 5.6 shows the time courses of the morphological parameters P1, P2 and L, of A. niger
clumps, for two experiments with KH2PO4 concentration in the fermentation medium of 0.1 g
l-1 and 0.5 g l-1. Both experiments were carried out in the loop fermenter of case study 1, at the
circulation time of 18 s. As Figure 5.10 shows, a small increase in phosphate level led to
drastic changes in morphology. The clump perimeter became almost three times larger, while
the perimeter of the core remained small. The clumps lost their compact structure and the
form of an extended growth around a small core predominated. The length of filaments increased
during fermentation, while at the lower phosphate level, after an early growth phase, L remained
at the same levels until the end of the run. The fermentation itself was also drastically affected,
since the yield of citric acid on glucose consumed fell from 70 per cent to 39 per cent on
increasing the phosphate level, while biomass concentration increased from 6.1 g l-1 at the
lower to 11.5 g l-1 at the higher phosphate level. The differences in specific growth rates and
biomass concentrations could explain the different time course for the hyphal length L in the
two runs. In contrast to the very high increase of P1, L remained comparatively small. This
could be an indication of increased branching with increasing phosphate levels.
Although the strain used in this work did not form macroscopic pellets but microscopic
clumps, factors such as increased phosphate concentrations seem to exert similar controls
on fundamentally different morphological types.
5.4.3 pH
Culture pH can have a profound effect on citric acid production by A. niger, since certain
enzymes within the TCA cycle are pH sensitive. The maintenance of a low pH during
fermentation is vital for a good yield and it is generally considered necessary for the pH
80
Figure 5.11 Effect of culture pH on citric acid production in the stirred tank reactor
to fall to around pH 2 within a few hours of the initiation of the process, otherwise the yields
are reduced (Mattey, 1992). Information concerning the effect of pH upon the morphology
of citric acid producing A. niger is very limited. Reports on the effect of pH on morphology
for other fungi are contradictory; either it influences morphology greatly or it has no effect
at all (Pirt and Callow, 1959; Van Suijdam and Metz, 1981; Miles and Trinci, 1983). The
following case study examines the effect of culture pH on citric acid production and A.
niger morphology in the stirred tank reactor.
Case study 5
Fermentations were carried out at 500 rpm and uncontrolled pH (resulting in a final pH of
1.6) and controlled pH (by addition of titrants) at 2.1 and 3 (Papagianni, 1995). Citric acid
production was highest when pH was maintained at 2.1, with 122 g l-1 at the end of the run
(168 hours of fermentation), compared to 75 g l-1 at pH 3 and 65 g l-1 at pH 1.6, as shown in
Figure 5.11. Biomass levels were slightly increased with increasing pH: 5.65 g l-1 at pH 1.6,
6.21 g l-1 at 2.1 and 7.40 g l-1 at pH 3. The highest values of specific rates of citric acid
formation were obtained at pH 2.1, with a maximum value 0.35 h-1 while it reached 0.18 l1
in the other two runs.
Mean values of the morphological parameters P1, P2, L and d at the end of the runs
performed at 500 rpm and pH 2.1, 3 and uncontrolled are shown in Table 5.1. P1, P2 and
L increased with pH whilst there was no unidirectional response for the diameter of
filaments. In addition to the small size of clumps and small length of filaments at final pH
1.6, there was an unusually high number of swollen cells and tips in the mycelium in this
run, as shown in Figure 5.11. A number of swollen cells were always present at stirrer
speeds above 400 rpm in the STR. The low pH in this experiment seemed to aid the
development of this morphological form which also gave low citric acid concentrations.
Fungal morphology
81
These experiments also indicated that productivity and morphology are linked. As with
agitation, the conditions which promote a certain morphological type, i.e. that of small
clumps and short filaments, favour citric acid production, much as observed with the
relationship between macro-morphology and citric acid production.
82
medium. Omission of Mn ions (less than 10-7) from the nutrient medium resulted in abnormal
morphological development that was characterized by increased spore swelling and squat,
bulbous hyphae. The inhibition of glucoprotein turnover caused by the presence of Mn ions
led to a possible loss of hyphal polarity and increased branching and chitin synthesis. Clark
et al. (1966) also discussed changes in A. niger morphology following the addition of Mn.
The authors noticed an undesirable change in morphology from the pellet like form to
filamentous form with the addition of 2 ppb Mn to ferrocyanide-treated molasses.
Morphological changes, which included prevention of clumping, absence of swollen cells
and reduced diameters of filaments, accompanied by a 20 per cent reduction in citric acid
yield, following the addition of 30 mg l-1 Mn to a Mn-free medium, were also reported by
Papagianni (1995).
5.7 References
ADAMS, H L, and THOMAS, C R, 1988. The use of image analysis for morphological measurements
on filamentous micro-organisms, Biotechnology and Bioengineering, 32, 707712.
Fungal morphology
83
84
MITARD, A and RIBA, A, 1988. Morphology and growth of Aspergillus niger ATCC 26036 cultivated
at several shear rates, Biotechnology and Bioengineering, 32, 835840.
MORRISON, K B and RIGHELATO, R C, 1974. The relationship between hyphal branching, specific
growth rate and colony radial growth in Penicillium chrysogenum, Journal of General
Microbiology, 81, 517520.
PAPAGIANNI, M, 1995. Morphology and citric acid production of Aspergillus niger in submerged
culture, PhD Thesis, University of Strathclyde, Glasgow, Scotland.
PAPAGIANNI, M, MATTEY, M and KRISTIANSEN, B, 1994. Morphology and citric acid production
of Aspergillus niger PM1, Biotechnology Letters, 9, 929934.
PIRT, S G and CALLOW, D S, 1959. Continuous-flow culture of the filamentous mould Penicillium
chrysogenum and the control of its morphology, Nature, 184, 307310.
RHR, M and KUBICEK, C P, 1981. Regulatory aspects of citric acid fermentation by Aspergillus
niger, Process Biochemistry, 16, 3444.
SHU, P and JOHNSON, M J, 1948. The interdependence of medium constituents in citric acid
production by submerged fermentation, Journal of Bacteriology, 56, 577585.
SMITH, M G and CALAM, C T, 1980. Variations in inocula and their influence on the productivity of
antibiotic fermentations, Biotechnology Letters, 2, 261266.
SMITH, J J, LILLY, M D and FOX, R I, 1990. The effect of agitation on the morphology and penicillin
production of Penicillium chrysogenum, Biotechnology and Bioengineering, 35, 10111023.
SNELL, R L and SCHWEIGER, L B, 1951. Citric acid by fermentation, British Patent No. 653 808,
Chemical Abstracts, 45, 8719a.
STEEL, R, MARTIN, S M and LENTZ, C P, 1955. A standard inoculum for citric acid production in
submerged culture, Canadian Journal of Microbiology, 1, 150157.
SVENSKA SOCKERFABRIK, A B, 1964. A method for producing citric acid, British Patent No. 951
629, Chemical Abstracts, 60, 2304a.
THOMAS, C R, 1992. Image analysis: putting the filamentous micro-organisms in the picture, Trends
in Biotechnology, 10, 343348.
TUCKER, K G and THOMAS, C R, 1992. Mycelial morphology: the effect of spore inoculum level,
Biotechnology Letters, 14, 10711074.
TUCKER, K G, KELLY, T, DELGRAZIA, P and THOMAS, C R, 1992. Fully automatic measurement
of mycelial morphology by image analysis, Biotechnology Progress, 8, 353359.
UJCOVA, E, FENCL, Z, MUSILCOVA, M and SEICHERT, L, 1980. Dependence of release of
nucleotides from fungi on fermentor turbine speed, Biotechnology and Bioengineering, 22, 237
241.
VAN SUIJDAM, J C and METZ, B, 1981. Influence of engineering variables upon the morphology of
filamentous molds, Biotechnology and Bioengineering, 23, 111148.
VAN SUIJDAM, J C, KOSSEN, N W F and PAUL, P G, 1980. An inoculum technique for the production
of fungal pellets, European Journal of Applied Microbiology, 8, 353359.
VECHT-LIFSHITZ, S E, MAGDASI, S and BRAUN, S, 1990. Pellet formation and cellular aggregation
in Streptomyces tendae, Biotechnology and Bioengineering, 35, 890896.
XU, D B, KUBICEK, C P and RHR, M, 1989. A comparison of factors influencing citric acid
production by Aspergillus niger grown in submerged culture and on filter paper, Applied
Microbiology and Biotechnology, 30, 444449.
Nomenclature
a,b
ae
aox
ared
k
n
EO2/H2O
Eh
Eo
F
N
Qg
pO2
pO2crit
P
R
rH
S
T
X
t
constants
electron activity
activity of oxidized form
activity of reduced form
redox reaction balance constant
number of electrons in redox reaction
standard potential1223 mV
potential measured in a solution,
based on standard hydrogen electrode
standard redox potential of a 50 per cent reduced substance
based on standard hydrogen electrode
Faraday constant
stirred speed
volumetric gas flow rate
dissolved oxygen partial pressure
critical dissolved oxygen partial pressure
citric acid concentration
gas constant
negative log of partial pressure of gaseous hydrogen
sugar concentration
temperature
biomass concentration
residence time
yield factors
(-)
(-)
(-)
(-)
(-)
(-)
(mv)
(mV)
(mV)
(s-1)
(vvm)
(atm)
(atm)
(g/l)
(cal/C mol)
(-)
(g/l)
(C)
(g/l)
(s)
(-)
6.1 Introduction
In living organisms oxidationreduction systems play such an intimate and essential a part,
that life itself might be defined as a continuous oxidationreduction reaction. It is not
85
86
6.2 Overview
Redox potential is, however, a parameter that can give valuable information about metabolism
taking place in various aerobic and anaerobic microbial cultures (Kjrgaard, 1977). The
significance of redox potential levels for high yielding citric acid biosynthesis has been
demonstrated in submerged citric acid fermentation (Berovic, 1996; Berovic and Cimerman,
1993).
Although only limited attention has been paid to this phenomenon in the past, some
interesting and informative research work has been presented. Some workers have advocated
the use of redox potential measurements for monitoring and controlling dissolved oxygen
(Shibai et al., 1975; Radjai et al., 1984). At constant pH the relation between redox potential
and dissolved oxygen partial pressure can be simplified by logarithmic relation (Jacob,
1970; Memmert and Wandrey, 1987).
During the last few years a great deal of the attention for redox potential measurement
and it uses, has been given to anaerobic bioprocesses (Beck and Schink, 1995). The
importance of redox potential measurements was referred to in articles on waste water
bioprocessing, as in the case of propionate degrading Methanospirillum and
Methanocorpusculum bacteria in a fluidized bed reactor, where degradation was inhibited
at redox potential below 300 mV (Heppner et al., 1992), and in anaerobic digestion in
methanogenic fermentation where volatile fatty acids were used as the substrate (Peck and
Chynoweth, 1992).
Redox potential measurements have also been found to be important in extremely
thermophilic Thermotoga sp. bioprocessing, where most thermodynamic problems were
associated with the relatively high redox potential (Janssen and Morgan, 1992). In various
aerobic processes the importance of the redox potential has been observed. In the case of
87
6.3 Theory
Oxidation is a process in which a substance, molecule or ion loses or gives up electrons.
Reduction, on the other hand, is a process in which a substance, molecule or ion, is involved
in the taking up of electrons. Whenever one substance in a system is oxidized, another
substance must be reduced. The relation between reduction and oxidation may be expressed
as:
Reduced form Oxidized form + electron(s)
However, since free electrons never exist in any noteworthy concentration, reduction
and oxidation reactions are always coupled together, so that one reaction releases just as
many electrons as the other one consumes. Thus a pair of reactions always takes part in such
a process. These simultaneous and complementary reduction and oxidation processes are
generally known as redox reactions (Bhler and Galster, 1980). The oxidation (or reduction)
capacity of a solution is characterized by the free electron activity in it. Despite the fact that
the lifetime of a free electron is extremely short (10-110-15 seconds) there is a statistical
possibility of free electron existence at the moment of transformation from electron-donor
systems to electron-acceptor systems. (Balakireva et al., 1974).
The thermodynamic probability of electron emergence under activated reaction-capable
conditions (Inczedy, 1970) is understood as electron activity in a solution:
ae = (1/k)1/n (ared/aox)
(6.1)
The oxidation potential is a quantitative measure of redox capacity of a solution. It is an
electrical unit of charge of free energy in a redox interaction of the given system with a
standard system. The system:
2H++ 2e- H2
is a standard one. The oxidation potential is related to the electron activity in solution:
Eh = -RT/F lnae
(6.2)
88
(6.3)
(6.4)
In the first part of equation (6.4), kRT is equal to Eo, the standard redox potential of a 50 per
cent reduced substance, based on a standard hydrogen electrode.
Eh = Eo + RT/nF ln(ared/aox)
(6.5)
(6.6)
The potential values measured are dependent on pH, so that in each case measurements of
redox potential should be accompanied by a statement of the pH value at which they were
taken. In general a pH variation of one unit causes a potential variation of 57.7 mV (Jacob,
1970).
(6.7)
(6.8)
(6.9)
89
Figure 6.1 Electrode potential versus pH. Continuous line, theoretical curves, broken line,
actual system (from Hewitt, 1950)
90
Figure 6.2 Relationship between oxygen tension and redox potential (from Shibai et al.,
1975)
was useful in determining the oxygen transfer requirements when dissolved oxygen was
practically zero (Shibai et al., 1974; Akashi et al., 1978).
At constant pH the relation between redox potential and dissolved oxygen partial pressure
can be simplified by the following equation (Shibai et al., 1975), demonstrated in Figure
6.2:
logpO2 = aEh + b
(6.10)
A similar relationship between pO2 and Eh has been observed in amino acid production by
Corynebacterium glutamicum (Radjai et al., 1984):
logpO2 = 0.0157Eh - 0.071
(6.11)
Shibai et al. (1975) carried this further for inosine production by Bacillus subtilis; pO2crit
was determined by measuring the dissolved oxygen, the redox potential and cell respiration
rate in pH and temperature controlled culture. When the dissolved oxygen partial pressure
was above 1.10-2 atm, the redox potential had a linear relationship with the logarithm of the
dissolved oxygen partial pressure. Therefore pO2 = 1.10-2 atm was estimated by determining
the redox potential, on the assumption that there was a linear relationship even at the pO2
level less than 1.10-2 atm. The redox potential was markedly lowered by the physiological
change in the cells, when cell respiration was inhibited at Eh = -180 mV, which corresponded
to pO2 = 2.10-4 atm.
pO2 in this culture was recorded as nearly zero when the cell rapidly biosynthesized the
product. It went up above 1.10-2 atm at the end of fermentation, when the substrate was
almost completely assimilated. The data showed that maximum production was obtained
under limited oxygen supply, where cell respiration was inhibited. When cell respiration
91
was not inhibited, as the pO2 level rose above 1.10-2 atm, the cell did not produce the maximum
amount of L-leucine.
The lowest values of pO2crit that have been reported were 4.10-3 atm for Saccharomyces
cerevisiae, 3 10-4 atm for inosine and 2 10-4 atm for the leucine producer (Akashi et al.,
1978).
92
Figure 6.4 Redox potential measurements and citric acid formation by Aspergillus niger.
Curve 1: aerated sterile sugar beet molasses substrate including potassium ferrocyanide;
curve 2: inoculated and aerated sterile sugar beet molasses substrate excluding potassium
ferrocyanide; curve 3: inoculated and aerated sterile sugar beet molasses substrate including
potassium ferrocyanide
causes the formation not only of metal ion complexes, but also the Fe3+/Fe2+ redox couple,
which regulates the ion balance of the substrate (Clark and Cohen, 1923). The balance of
various redox couples and especially metal ion in fermentation broth is of essential importance
for citric acid biosynthesis. Related to this the influence of various influent factors and
substances on redox potential levels of beet molasses substrate have been studied (Figure
6.4).
A reference redox potential profile was obtained for sterile medium only, with no
addition of K4Fe(CN)6 (curve 1). After 24 hours of aeration, the redox potential reached
a stationary phase that was unchanged until the end of the experiment. In experiments
where inoculated substrate was used in absence of any addition of K4Fe(CN)6 (curve 2),
and with an initial addition of this compound (curve 3), the redox potential profile exhibited
a typical single peak. Only in the case where inoculated substrate with primary and
secondary addition of K4Fe(CN)6 was used (results shown in Figure 6.3), was the twin
93
Figure 6.5 Process variables in a low yielding, abnormal citric acid fermentation
peak redox potential course observed. The different metabolic activities in the fermentation
process are summarized in all the redox reactions and detected in redox potential
measurement. From these experiments we concluded that only in the presence of Aspergillus
niger were the relevant changes detected. Similar observations were made by Kwong and
Rao (1991, 1992) in amino acid fermentation using Corynebacterium glutamicum.
Redox potential measurements in citric acid fermentation might also give valuable
indications of product biosynthesis in the fermentation. From evaluation of more than 200
batches, we concluded that a high yielding fermentation is directly related to levels and
time course of redox potential.
The yield of citric acid is reflected in the time course of the redox potential. This is
shown in Figures 6.5 and 6.6, where experiments with different histories are shown.
Figure 6.5 presents the characteristics of an unsuccessful fermentation with respect to
94
Figure 6.6 Effect of temperature shift on citric acid fermentation with Aspergillus niger and
suger beet molasses medium
citric acid production. Growth was diffused and the low citric acid production was therefore
expected. This is also reflected in the course of the redox potential. The second peak is low
and almost negligible (190 mV).
The effect of temperature change is well reflected in the course of redox potential. Figure
6.6 presents the data from an experiment started at an initial temperature of 20C. The
temperature was changed after 20 hours to 30C. The effect of this change can be clearly
seen from the redox curve. In the same experiment foaming caused a loss of the substrate
(89 hours), which was also indicated in a new peak of the redox potential.
In a high yielding fermentation on beet molasses substrate, the redox potential course
starts at a level of 0 to 20 mV, as shown in Figure 6.3. After 12 hours, the culture reaches a
95
level of oxygenation which significantly influences germination of conidia in the lag phase
and the subsequent development of bulbous cells that appears at the first peak of the redox
potential at 260 mV. After the first peak, a period of inhibition followed by the first redox
minimum at 180 mV occurs. In this phase it seems that microbial activity stops. Oxygen
partial pressure in fermentation broth increases and the carbon dioxide and redox potential
decreases, indicating a reduced level of activity for the micro-organism. This phase is a
progressive transition from glucose to fructose consumption. For this reorganization, a low
redox potential level is needed, resulting in the change in morphology (Smith, 1983).
After this phase, the microbial growth mode changes to spherical pellets. This was
indicated by the second redox peak at 280 mV. After a decrease in redox potential to 80 mV,
the second minimum, citric acid production starts. As reported by Tengerdy (1961), at the
lowest redox potential level, the peak oxygen demand and initiation of rapid excretion of
citric acid can be observed. The low redox potential reveals the reducing state of the complex
redox system of the fermentation broth, where the respiratory enzyme system signifies
strong metabolic activity. It seems that citric acid biosynthesis (Matkovicz and Kovacz,
1957), as well as some other microbiological reactions, proceeds favourably at the redox
potential near the minimum of the redox curve for the particular culture involved (Hewitt,
1950; Tengerdy, 1961). This was found to be true in riboflavin fermentation.
The redox potential time-course in a high citric acid yielding fermentation reaches a
final level of 180 mV. Interestingly if significant amounts of oxalic acid, up to 20 mg/l, are
produced, the redox potential will only reach levels of 100 to 120 mV at the end of
fermentation. It has also been found that oscillations in redox potential greater than 20 mV
have a strong influence on further development of fermentation (Berovic, 1996).
Figure 6.7 Process parameters of citric acid fermentation at chemical regulation of redox potential using 0.1% sodium
sulphite as reductant and 0.1% hydrogen peroxide as oxidant
97
oxidants and reductants, and a physical method, based on simultaneous agitation and aeration
control were tested (Berovic and Cimerman, 1993).
98
Figure 6.8 Process parameters of citric acid fermentation at chemical regulation of redox
potential using 20% glucose as reductant and 0.1% hydrogen peroxide as oxidant
the microbial cell. According to the Nernst equation (equation 6.5), increasing aox in a cell
could strongly influence the metabolic and enzyme activities in micro-organisms (Harrison,
1972). In submerged citric acid fermentation with Aspergillus niger beet molasses, in addition
to the maxima and minima redox levels, timing of these events is of essential importance
(Berovic and Cimerman, 1982, 1993).
The possibilities of regulating both redox maxima and the first redox minimum were
tested, using agitation and aeration as a physical means of regulating redox potential. The
first maximum of Eh = 220 mV appeared at 23 hours (Qg = 0.4 vvm speed of agitation = 400
rpm). As the level of 220 mV was too low for further process development, increasing the
aeration rate Qg to 1 vvm and agitation to 600 rpm gave a redox level of 260 mV. By further
reducing the aeration rate to 0.3 vvm and agitation to 200 rpm, at 30 hours the first redox
minimum Eh = 180 mV was obtained. After this step, aeration was increased to 1.2 vvm and
agitation to 700 rpm. The second redox maximum Eh = 280 mV at 36 hours appeared. The
fermentation then proceeded at constant conditions of Qg = 1 vvm and N = 600 rpm until
the end of the process. As the course of redox potential was not maintained by aeration and
agitation during the last phase, it started to deviate from the optimal course with a third
maximum (265 mV) occurring at 48 hours and a third minimum (120 mV) at 75 hours. This
gave final biomass and citric acid concentrations of 11.4 and 68.5 g/l respectively (see
Figure 6.9).
Figure 6.9 Regulation of the first redox minimum and both maxima by manipulating airflow rate and stirred speed
Figure 6.10 Regulation of both redox minima and both maxima by manipulating airflow rate and stirred speed
101
Finally, optimized redox level profiles were followed using simultaneous regulation of
aeration and agitation during the whole course of the fermentation. This resulted in 14.7 g/
l of biomass and 95 g/l of citric acid at the end of the fermentation. The results are given in
Figure 6.10.
6.9 Scale-up based on redox potential
The aim of scale-up is to develop a method based on the physiological needs of the microorganism that would give high yielding and reproducible results on various scales. Scale-up
is usually based on criteria such as: geometrical similarity, power input, volumetric oxygen
transfer coefficient, mixing time, etc. (Nienow, 1992; Dunn et al., 1992, Dubuis et al.,
1993). However, we decided on scale-up based on redox potential, being the most relevant
process parameter for our process. As redox potential indicates oxygen demand of the culture,
the basic idea was to use a physiological criterion of our bioprocess for scale-up. If redox
potential indicates a microbial demand for oxygen, it could also reflect information on the
appropriate aeration and agitation conditions needed to meet this demand.
6.9.1 Bioreactor dimensions
The experiments were performed in 10 l Bioengineering AG, and 100 and 1000 l Chemap
AG bioreactors. These were all equipped with Rusthon turbines, but were not geometrically
similar. The reactor dimensions are given in Table 6.1.
6.9.2 Media composition
The fermentation substrate consisted of diluted beet molasses with 12.5 per cent of total
reducing sugars. It was treated by addition of potassium hexacyanoferrate K4[Fe(CN)6],
which balanced the ratio of heavy metals ions by the formation of metal complexes (Clark
et al., 1965). K4[Fe(CN)6] was added in two stages, before sterilization (primary addition)
and after (secondary addition) (Cimerman et al., 1974; Berovic and Cimerman, 1982). The
fermentations were carried out at T = 30C.
6.9.3 Laboratory scale experiments
Basic research for scale-up was performed in a 10 l laboratory fermentor. The best redox
profile was determined from some 200 fermentations. The objective of the scale-up was to
obtain a similar redox profile in the larger reactors by regulating the agitation and aeration.
102
6.10 Conclusions
For high citric acid yielding submerged fermentation on beet molasses the optimal redox
potential time course and its typical redox levels, with two maxima, 260 and 280 mV, and
two minima, 180 and 80 mV, are essentially important (Berovic, 1996; Berovic and
Cimerman, 1993). It is possible to influence the fermentation by changing the redox potential
profile as well as the magnitude of the maxima and minima. Regulating the redox by using
hydrogen peroxide as oxidant and sodium sulphite or glucose as reductant, resulted in a
favourable redox profile for the whole process, but the fermentation was affected to such an
extent that poor growth and reduced citric acid yields were obtained.
A better method for regulating the redox potential during fermentation is through alteration
in aeration and agitation. The desired redox profile is attained by respectively increasing,
and decreasing the aeration and agitation to obtain the desired maximum and minimum
values. It is a simple practical approach based on changing the gradient of oxygen transfer
in the fermentation broth, which influences changes in intracelluar oxygen concentration
and therefore the microbial physiology of the cell.
103
This method of regulating the redox profile was used as a scale-up criterion with the
process successfully scaled up from 10 to 1000 l. Considering the results obtained, it is
evident that this new scale-up method leads to very reproducible results even in geometrically
non-similar bioreactors.
6.11 References
AKASHI, K, IKEDA, S, SHIBAI, H, KOBAYASHI, K and HIROSE, Y, 1978. Biotechnology and
Bioengineering, 20, 2734.
ANDREEVA, E A, 1964. Mikrobiologija, 43, 780.
BALAKIREVA, L M, KANTERE, V M and RABOTNOVA, I L, 1974. The redox potential in
microbiological media, Biotechnology and Bioengineering Symposium, No. 4, 769780.
BECK, S O and SCHINK, B, 1995. Acetate oxidation through a modified citric acid cycle in
Propionobacterium freudenreichii, Archives in Microbiology, 163, 182187.
BEROVIC, M, 1996. PhD thesis, University of Ljubljana, Ljubljana.
BEROVIC, M and CIMERMAN, A, 1982. Redox potential in submerged citric acid fermentation,
European Journal of Applied Microbiology, 16, 185.
BEROVIC, M and CIMERMAN, A, (1993). Redox potential an effective tool for scaling-up of citric
acid fermentation from laboratory to pilot scale, 3rd International Congress on Bioreactor &
Bioprocess Fluid Dynamics. Ed. A NIENOW (MEP Publications), pp. 533545.
BEROVIC, M and ROSELJ, M, 1997. Possibilities of redox potential regulation in submerged citric
acid fermentation on beet molasses substrate (unpublished).
BHLER, H and GALSTER, H, 1980. Redox MeasurementPrinciples and Problems (Dr Ingold
AG, Zurich).
CIMERMAN, A, SKAFAR, S and JOHANIDES, V, 1974. YU Patent P2481/74.
CLARK, W M and COHEN, B, 1923. Public Health Report Washington, 38, 666.
CLARK, O S, ITO, K and TYMCHUK, P, 1965. Effects of potassium ferrocyanide addition on the
chemical composition of molasses mash used in the citric acid fermentation, Biotechnology and
Bioengineering, 7, 269271.
DUBUIS, B, PLSS, R, ROMETTE, J L, KUT, O M and BORNE, J, 1993. Physical factors affecting
the design and scale-up of fluidized bed bioreactors for plant cell culture, 3rd International
Congress on Bioreactor & Bioprocess Fluid Dynamics. Ed. A.NIENOW (MEP Publications),
pp. 89100.
DUNN, I J, HEINZLE, E, INGHAM, J and PRENOSIL, J E, 1992. Biochemical Reaction Engineering
(VCH), pp. 121123.
ERLICH, P, 1965. Recommendations 1964 of the International Union of Biochemistry (Elsevier).
GILLESPIE, L J, 1920. Soil Science, 9, 199.
HARRISON, D E F, 1972. Physiological effects of dissolved oxygen tension and redox potential on
growing populations of micro-organisms, Journal of Applied Chemistry and Biotechnology, 22,
417440.
HELMHOLTZ, H, 1883. Archives of Anatomy and Physiology.
HEWITT, L F, 1950. Oxidationreduction potentials. In Potentials in Bacteriology and Biochemistry,
6th edition (Livingstone, Edinburgh).
HEPPNER, B, ZELLNER, G and DIEKMANN, H, 1992. Start-up and operation of a propionate
degrading fluidised bed reactor, Applied Microbiology and Biotechnology, 36, 810816.
HILL, R, 1973. Bioenergetics, 4, 229.
HUANG, S Y and WU, C S, 1974. Redox potential in yeast cultivation broth using n-paraffins as
carbon source, Journal of Fermentation Technology, 52, 818827.
INCZEDY, J, 1970. Period Polytechnic Chemical Engineering, 14, 2.
ISHIZAKI, A, SNIBAI, H and HIROSE, Y, 1974. Basic aspects of electrode potential change in
submerged fermentation, Agricultural and Biological Chemistry, 38, 2399.
JACOB, H E, 1970. Methods in Microbiology, Vol. 2. Eds J R NORRIS and D W RIBBONS (Academic
Press).
JANSSEN, P H and MORGAN, H W, (1992). Heterotrophic sulphur reduction; end product inhibition,
FEMS Microbiology Letters, 2, 213218.
KJRGAARD, L, 1976. European Journal of Applied Microbiology, 2, 215.
104
7.1 Introduction
This chapter will focus on the kinetic modelling of industrial citric acid production by
Aspergillus niger and Candida (= Saccharomycopsis = Yarrowia) lipolytica. A good working
definition of kinetic modelling is the mathematically expressed correlation between the
rates and concentrations of reactants and products. When applied to appropriate mass
balances, it is possible to predict the utilization of substrates and the yield of individual
products. A well constructed model can be used to express the course of a whole fermentation
experiment based on a small set of initial values for the fermentation variables. Such models
can then be used as a basis for simulations which are essential for the optimal design and
operation of a given process.
106
of individual enzymes is related to their effect upon the overall reaction rate. This second
process has been carried out for the production of citric acid through the glycolytic
pathway by A. niger (Torres 1994a, 1994b) and was successful in showing that the
activities of glycolytic enzymes have little influence on the rate of product formation in
this system. A shortage of information currently prohibits the creation of a mechanistic
model for biomass production.
7.1.2 Unstructured models based on the Monod equation and other equations
The 1942 Monod equation (equation 7.1) relies on the principle that even when there are
many substrates the rate of biomass production depends on the concentration of just one
limiting substrate. At low concentrations of this substrate (S), is proportional to S, but
for increasing values of S an upper value max for the specific growth rate is gradually
reached. The maximum specific growth rate (max) and the saturation coefficient (Ks)
must be determined experimentally. This model has been shown to correlate with
fermentation data for many different micro-organisms, but fits best with well mixed
unicellular systems.
(7.1)
(7.2)
(7.3
(7.4)
This model was used to explain that part of the pellets was either not growing, or was
growing sub-optimally, because it would otherwise be expected that the pellets would
continue to grow exponentially until exhaustion of the nutrients. These equations are useful
when attempting to describe hyphal growth in filamentous fungi, particularly in conditions
that lead to the formation of pellets.
The rates of formation and depletion within the fermentation are linked to the formation
of biomass by yield coefficients, and are described mathematically as follows:
(7.5)
(7.6)
107
(7.7)
where rX, rS and rN are the rates of biomass accumulation, carbon source consumption and
nitrogen source consumption, respectively.
The LuedekingPiret (1959) equation (equation 7.8) is used to relate the formation of
products to either the biomass concentration or the rate of biomass accumulation:
rP = (a rX) + ( X)
(7.8)
This equation is often simplified by substituting zero for one of the product formation
constants, a or , giving equations for growth specific and non-growth specific product
formation.
7.2.1 A simple struct ured model for growth and citrate production
in A. niger
A typical growth curve for an A. niger under citric acid producing conditions has
been presented by Kubicek and Rhr (1989) as shown in Figure 7.1. This shows a
fast-growth phase followed by a slow-growth phase. This change in growth rate is
due to a change in the physiological state of the mycelium from the normal growth
form to the citrate excreting form. An examination of the kinetics of citric acid
production by Aspergillus niger growing on sucrose was carried out in a pilot plant
(Rhr et al., 1981). Cell growth and product formation were subdivided into several
phases, each described by a simple deterministic model. The growth phases identified
were the hyphal growth phase (B x), pellet growth phase (Cx), restricted growth phase
(D x), transition period between trophophase and idiophase (E x) and idiophase growth
(Fx). The growth in each phase was described by logarithmic, cube root and linear
equations and the best fitting equation was identified by evaluating the degree of
linearity within a limit of 5 per cent maximum deviation. The three phases are
illustrated in Figure 7.2, where the cell growth during citric acid fermentation by
Aspergillus niger B60/B3 has been plotted.
Product formation was related to the growth rate by a modified LuedekingPiret
equation. However, although the same descriptions were applicable for both growth and
108
Figure 7.1 Typical example of growth and citric acid accumulation by A. niger in submerged
culture (from Kubicek and Rhr, 1989)
acid formation (Figure 7.3), the acid formation kinetics usually differentiated from the growth
kinetics by a term that represented the lag time. This lag time is also known as the maturation
time, when the culture has taken up all the ammonium ions but does not yet produce citric
acid. The respective rate of product formation was then said to be proportional to the rate of
cells entering this physiological state and was expressed as follows:
rPt = krX(ttm)
(7.9)
where rP, t, X have their usual meanings and k and tm are the product formation rate constant
and maturation time respectively.
One problem with this equation is that k was found not to be constant but increases in
value during the fermentation. It was assumed that there were at least two different types of
cells within the mycelium with different productivities, and a production term for each type
could be described by the LuedekingPiret equation. Therefore, equation 7.9 was modified
to become:
rPt = k1rX(ttm) + k2(X)t-tm
(7.10)
Figure 7.2 Growth during citric acid fermentation by A. niger B60/B3 (from Rhr et al., 1981).
Figure 7.3 Citric acid concentration during citric acid fermentation by A. niger B60/B3 (from Rhr et al., 1981).
111
Table 7.1 Calculated values for tm and k (from Rhr et al., 1981)
(7.11)
(7.12)
(7.13)
where D is the dilution rate and subscripts b, c and d refer to basic, citric acid-producing
(carbon storage) and deactivated cells respectively. The dilution rate (D) in these equations
can be substituted by the overall growth rate () if they are to be applied to a batch type
fermentation (Sinclair et al., 1987).
It was assumed that the rate constants of the above equations took the following forms:
(7.14)
(7.15)
(7.16)
where the first rate constant, b, was the Monod expression for growth on a limiting substrate,
the second constant, c, accounted for the increase in mass of bulbous citrate producing
cells and kt was the rate of transformation of basic to storage cells.
Figure 7.4 Citric acid concentration during citric acid fermentation, as determined experimentally and as calculated from biomass values and
Table 7.1 (from Rhr et al., 1981).
113
Figure 7.5 Comparison of A. niger model with experimental data. X, Biomass; S, Substrate; P, product (from Ho et al., 1994)
Figure 7.6 Typical batch glucose and ammonia consumption, and biomass and acid production profiles from S. lipolytica fermentation (from
Klasson et al., 1989)
Figure 7.7 Comparison of S. lipolytica model with experimental data. X, (biomass); S, (substrate); P, (product); (from Klasson et al., 1991)
117
Figure 7.8 Citric acid production in batch culture by Yarrowia lipolytica ATCC 20346.
Concentrations of biomass (X) and citric acid (P) and weight fraction of intracellular nitrogen
(ZN) as a function of the fermentation time (t). Continuous lines were calculated using the
equations and yield coefficients reported (from Moresi, 1994)
118
Figure 7.9 Repeated batch citric acid production by Yarrowia lipolytica ATCC 20346.
Concentrations of biomass (X) and citric acid (P) and weight fraction of intracellular nitrogen
(ZN) as a function of the fermentation time (t) (from Moresi, 1994)
A linear type growth equation for trophophase was found to give an acceptable
correlation to the experimental profiles of X, S and N. After the nitrogen source is depleted,
the exhaustion does not prevent further growth but growth is accompanied by a
simultaneous decrease in the intracellular nitrogen content (ZN) before the start of citric
acid excretion (Figure 7.8).
The cell growth rate of this citric acid lag phase was modelled by the following
expression:
rX = -X (d ln zN/dt)
(7.21)
119
The citric acid production phase or idiophase was modelled using LuedekingPiret kinetics:
rp = (YP/S/YX/S) (rX) + mPX
(7.22)
However, as citric acid is formed by resting yeast cells, rX approximates to 0, so the above
LuedekingPiret equation is reduced to:
rP mPX
(7.23)
Confirmation of this can be seen in Figure 7.9, where the citric acid concentration increases
linearly with time while the biomass concentration remains constant in a repeated-batch
experiment.
7.4 Conclusion
Kinetic modelling is a powerful tool in the design and optimization of all biotechnological
processes, and the citric acid process is no different. The Candida lipolytica process is quite
well understood, and so it is not surprising that the published models are accurate and
applicable to a wide range of fermentation conditions.
On the other hand, the physiological change that occurs in Aspergillus niger mycelium
during the citric acid process is far from being well defined, and models which concentrate
purely on the production phase are therefore less susceptible to error. The single published
model which covers this transition phase is only capable of predicting the course of the
fermentation within a very narrow range of starting parameters. Outside this range, errors
start to accumulate after the transition phase.
If Aspergillus models are to reach the same degree of predictive accuracy as the Candida
models, a greater knowledge is needed of the internal metabolic changes that occur during
the transition phase, when the organism appears to become stressed.
7.5 References
HO, S F, KRISTIANSEN, B and MATTEY, M, 1994. Phase-related mathematical model of the
production of citric acid by Aspergillus niger, European Federation of Biotechnology International
Conference on Modelling of Filamentous Fungi, Otocec, Slovenia, p. 57.
KLASSON, T K, CLAUSEN, E C and GADDY, J L, 1989. Continuous fermentation for the production
of citric acid from glucose, Applied Biochemistry and Biotechnology, 20, 491509.
KLASSON, T K, CLAUSEN, E C, GADDY, J L and ACKERSON, M D, 1991. Modelling lysine and
citric acid production in terms of initial limiting nutrient concentrations, Journal of Biotechnology,
21, 271282.
KRISTIANSEN, B and SINCLAIR, C G, 1979. Production of citric acid in continuous culture,
Biotechnology and Bioengineering, 21, 297315.
KUBICEK, C P and RHR, M, 1989. Citric acid fermentation, CRC Critical Reviews in Biotechnology,
4, 331373.
LUEDEKING, R and PIRET, E L, 1959. A kinetic study of the lactic acid fermentation, Journal of
Biochemistry, Microbiology, Technology and Engineering, 1, 393412.
MONOD, J, 1942. Recherches sur la croissance des cultures bacteriannes (Hermann and Cie, Paris).
MORESI, M, 1994. Effect of glucose concentration on citric acid production by Yarrowia lipolytica,
Journal of Chemical Technology and Biotechnology, 60, 387395.
RHR, M, ZEHENTGRUBER, O and KUBICEK, C P, 1981. Kinetics of biomass formation and
citric acid production by Aspergillus niger on pilot plant scale, Biotechnology and Bioengineering,
23, 24332445.
120
SINCLAIR, C G, KRISTIANSEN, B and BULOCK, J D, 1987. Kinetics and Modelling (Taylor and
Francis, Open University Press), p. 56.
THOMAS, C R and PAUL, G C, 1994. Modelling of the penicillin fermentation, European Federation
of Biotechnology International Conference on Modelling of Filamentous Fungi Abstract, Otocec,
Slovenia, p. 19.
TORRES, N V, 1994a. Modelling approach to control of carbohydrate metabolism during citric acid
production by Aspergillus niger: I. Model definition and stability of the steady state, Biotechnology
and Bioengineering, 44, 104111.
TORRES, N V, 1994b. Modelling approach to control of carbohydrate metabolism during citric acid
production by Aspergillus niger: II. Sensitivity analysis, Biotechnology and Bioengineering, 44,
112118.
TRINCI, A P J, 1970. Kinetics of the growth of mycelial pellets of Aspergillus nidulans, Archiv fr
Mikrobiologie, 73, 353367.
Nomenclature
C
K
mATP
mS
mO
N
NP
concentration
ATP consumption for polymerization of
biomass precursors
specific maintenance requirements of ATP
specific maintenance requirements of
substrate
specific maintenance requirements of oxygen
moles ATP generated per 1 C-mol of
substrate by substrate level phosphorylation
moles ATP generated per 1 C-mol of
substrate before the biomass or product
formation diverges from the catabolic
pathway
rate of ATP consumption
rate of ATP consumption in maintenance
processes
rate of ATP conversion of compound j
maximum true yield of biomass on ATP
maximum true yield of product on ATP
yield on available electrons
true yield on available electrons
yield of ATP on substrate (moles ATP
generated per 1 C-mol of substrate from the
catabolic breakdown reaction)
yield of ATP on substrate (moles ATP
required for maintenance)
yield of oxygen on substrate
yield factor for compound j on compound i
(C-mol m-3)
(mol ATP (C-mol DM)-1)
(mol ATP (C-mol DM)-1 h-1)
(C-mol (C-mol DM)-1 h-1)
(mol O2 (C-mol DM)-1 h-1)
(mol ATP (C-mol)-1)
122
Yi,j
YC
YE
YH
d
gj
sj
h
xP
(C-mol (C-mol)-1)
(-)
(-)
(-)
(mol ATP (0.5 mol O2)-1)
(-)
(-)
(-)
(-)
(-)
Subscripts
O
P
X
S
max
pre
r
oxygen
product
biomass
substrate
maximum
biomass precursors
real
8.1 Introduction
For any bioprocess, rapid formation of an appropriate amount of biomass is of great
importance, both when it is the only expected product and when the purpose is to synthesize
definite chemical compounds. The simplest of the generally applied criteria of bioprocess
efficiency are mass yield coefficients. Precise determination of the amount of biomass
produced and substrate used is a starting point for the evaluation of all other process yields
and for mass and energy balances as well as elementary balances of carbon, oxygen, nitrogen,
etc. What makes the elementary balances much easier is the assumption of a constant average
weight fraction of carbon in the biomass (Erickson et al., 1979). Thus, it is specified how
much substrate an organism has used in the synthesis of its own mass and how much in the
production of energy. Furthermore, it is also important to understand the possibilities to
increase or decrease yields. This understanding is only possible by an extensive knowledge
of the biochemical intracellular reactions and processes that lead to biomass or products.
Such knowledge is best presented in the form of a metabolic model. The model is based on
the specification of a set of intracellular chemical reactions, which are derived from the
available biochemical information.
This chapter deals with mass and energy balances for Aspergillus niger growth kinetics.
The relations obeyed by observed and true yield coefficients resulting from balance equa
tions for carbon, reduction potential and energy during intensive cell growth and citric
123
Figure 8.1 Typical example of growth and citric acid accumulation by A. niger in submerged
culture (air-life bioreactor) (reprinted from Krzystek et al. (1996) with kind permission of
Elsevier Science)
acid overproduction are derived and discussed. Quantitative balances based on one macrochemical equation for checking the consistency of experimental data and evaluation of the
efficiency of conversion of organic substrates by A. niger are also presented.
124
Table 8.1 Stoichiometry of main metabolic pathways appearing in the citric acid production
by A. niger on sucrose (reprinted from Krzystek et al. (1996), with kind permission of Elsevier
Science)
listed in Table 8.1 such as synthesis of biomass precursors (I), production of citric acid (II),
catabolism of sucrose (VII), oxidative phosphorylation (VIII), polymerization of biomass
precursors (IX) and maintenance processes (X) are considered predominant. The formation
of any other by-products (i.e. polyhydric alcohols (IIIVI)) is assumed to be negligible,
since their total concentration is at a low level in comparison to citric acid (Rhr et al.,
1987). The stoichiometry determines the mass and energy balances of energy carriers (ATP,
GTP) and reducing equivalents (NADH2, NADPH2, FADH2):
125
The rate equations for substrate consumption and product formation could be derived after
applying a quasi-steady state approximation (QSSA) to biomass precursors, energy carriers
and reducing equivalents (Krzystek et al., 1996):
The equations obtained have the structure in which separate terms occur for substrate and
oxygen consumption associated with growth, product formation and maintenance. Formally,
they are identical to the most common assumption made on an a priori base, postulated by
Pirt (1975):
but now the yields are related to stoichiometric coefficients resulting from metabolic reactions
(Roels, 1983):
The above values were calculated taking the molecular mass MX of biomass 28.3 g DM
(C-mole DM)-1 assuming an ash content of 8 per cent DM. The P:O ratio was estimated as
-1
d = 2.17 mol ATP (0.5 mol O2)
(Roels, 1983; Garret and Grisham, 1995), while a mean
x
value of the true biomass yield on ATP, YAmTaP,X
, of 0.371 C-mol DM (mol ATP)-1, and the true
max
citric acid yield YAT P,X = -6 C-mol citric acid (mol ATP)-1 (Andrews, 1989; Roels, 1983).
The citric acid formation pathway generates 1/6 mol ATP from 1 C-mol of sucrose (Table
8.1), and, for simple products whose synthesis does not diverge from the catabolic pathway
YATP,P = .
8.3 Mass and energy balances
The yield coefficients are essentially thermodynamic quantities. They result from a balance
between the energy generated by the catabolic reactions and that consumed by the anabolic
reactions for the production of new cell mass. The following equations can be shown to
hold the mass and energy balances of ATP and reducing equivalents (Andrews, 1989):
126
Formation of mycelium of A. niger is an energy consuming process (Table 8.1, pathways (I)
and (IX)) and it implies that cell growth is energy limited. In turn, energy is generated in the
reaction of citric acid formation (Table 8.1, pathway (II)), so this is clearly a carbon limited
product. The yields YC, YH and YE represent their upper limits, referred to as carbon-limited,
reduction-limited and energy-limited, respectively. However, the mass and energy balances
on the reactions (equations (8.1)(8.3)) give the relations (8.9) (8.11) which are obeyed,
not only by the observed yields, but also the true yields.
In the case of theoretical yield all the substrate was used for production of a single
product and the cell maintenance is ignored. For the biomass the theoretical yield equals the
smallest of the following:
The value of YEX corresponds to the value of Yav,e = 0.11 C-mol DM (mol)-1. Similarly, the
calculations of theoretical citric acid yield give:
YCP = 1
YHP = 4/3 = 1.33
YEP = 1.33
and:
(YSP)max = YCP = 1 C-mol citric acid (C-mol sucrose)-1.
Simultaneous consideration of the mass and energy balances allows the calculation of the
highest biomass and product yields to be expected in practice (real values):
127
Figure 8.2 Possible biomass and product yields during citric acid production (reprinted from
Krzystek et al. (1996) with kind permission of Elsevier Science)
The graphical interpretation is given in Figure 8.2, where the citric acid yield is shown as
a function of dimensionless biomass. The highest theoretical biomass and citric acid yields
correspond to the point where the energy balance line for the value of YEX/YCX = 0.5,
crosses the diagonal line representing the mass balance (taking equality in relation (8.12)
and ignoring cell maintenance). The maximum real yield for citric acid is 0.8 C-mol citric
acid (C-mol sucrose)-1 and the corresponding maximum real yield of biomass is 0.18 Cmol DM (C-mol sucrose)-1 (i.e. 0.9 g citric acid (g sucrose)-1 and 0.18 g DM (g sucrose)1
, respectively.
The yield coefficient YE (equation (8.10)) can also be determined taking into account
the ATP requirement for maintenance. Calculated values for cells and product are as
follows:
The formation of biomass is also an energy consuming process as well as the production of
citric acid is a carbon-limited process. The calculated value of Yav,e now equals 0.16 C-mol
DM (mol)-1.
128
The form of equations (8.15) and (8.16) is identical to that of (8.4, 8.5) and (8.6, 8.7) if the
energy yield coefficients YE have the values of Y E (i.e. taking into account the ATP
requirement for maintenance). This makes the linear growth equations represent in fact the
overall energy balance where:
The energy and reduction limitation are the same in the case of formation of citric acid
since YATP,P = C-mol citric acid (mol ATP)-1 and the maintenance requirement for ATP is
met approximately by substrate-level phosphorylation. In addition, for carbon limited
products (YSP)max = YCP = YHP, thus from (8.14) and (8.15) the following equation can be
shown to hold:
129
Citric acid is formed by A. niger during the growth phase as well as in the stationary phase,
although mainly in the stationary phase when citric acid formation is maximized and hardly
any growth occurs (Kubicek and Rhr, 1986). It implies that in the intensive growth phase
the substrate consumption rates (sucrose and oxygen) can be described as:
On the basis of batch experimental data the yield coefficients in equations (8.37) and (8.38)
were verified (Krzystek et al., 1996). The equations are as follows:
The verification has been performed using the data from submerged citric acid processes in
sucrose mineral medium at the initial pH value about 2.5 carried out in a pilot plant air-lift
bioreactor (Gluszcz and Michalski, 1994). An agreement between the theory and
experimental results was observed, confirming the linear growth equation to be an energy
130
Table 8.2 Yield coefficients in citric acid production by A. niger. (reprinted from Krzystek et
al. (1996), with kind permission of Elsevier Science)
balance with the true yields coefficients. Experimentally obtained yield coefficients of citric
acid and biomass on sucrose and oxygen are presented in Table 8.2 and compared with
theoretical and true yield coefficients. The yield of citric acid on sucrose reached 83 per
cent of real maximum theoretical values. Yields of biomass on sucrose and oxygen were 96
per cent and 109 per cent, respectively.
On the basis of experimental data the maintenance coefficients were also estimated:
mS = mO = 0.026 C-mol sucrose (mol O2) (C-mol DM)-1(h)-1.
The resulting specific maintenance requirement of ATP was:
mATP = 0.015 mol ATP (C-mol DM)-1(h)-1,
which is of the order of common value for mATP (Solomon and Erickson, 1981).
The energy in the organic substrate is incorporated into biomass, evolved as heat (released as a
result of combustion or as electron equivalents that can be transferred to oxygen) or incorporated
into extracellular products. Available electron balance may be written in the form:
131
A balance analysis for sugar and oxygen uptake made by Rhr et al. (1983, 1987) showed
that during the first phase of citric acid accumulation (up to 130 hours) more sugar is taken
up than the production of biomass, CO2 and citric acid can account for. In contrast, during
later phases of fermentation, more citric acid, CO2 and mycelium are formed than sugar
uptake would theoretically allow.
A similar pattern was also reflected in a balance for oxygen uptake, where less uptake
occurs during the early phase of fermentation than needed for complete balance; the
reverse was observed during the late stage of fermentation. This was caused by the
intermediate accumulation and partial re-consumption of a number of polyhydric alcohols
such as glycerol, arabitol, erythritol and mannitol, up to almost 9 g l-1. This finding
explained earlier observations (Shu and Johnson, 1948) of accumulation of more citric
acid (9.9 g l-1) during the late stages of fermentation than sugar uptake can account for
(6.7 g l1), since the polyols become degraded during the late stages of fermentation. The
polyols as by-products of citric acid production account for 70 per cent of lacking material
(Rhr et al., 1983, 1987).
Nowakowska-Waszczuk and Sokolowski (1987), calculating the amounts of glucose
carbon utilized during the fermentation and its distribution to mycelium, citric acid and
CO2, also observed that the carbon content of consumed sugar and products did not balance.
During the first 24 hours of the process carried out in an air-lift bioreactor of 0.8 m3 working
volume (height about 11 m) only about 76 per cent sugar carbon was found in the products
(biomass, citric acid and CO2). From the second day a surplus of carbon was found in the
products. When the sugar content was very low or had been completely consumed, the
surplus carbon in the products was reduced to 0.95.3 per cent. In two different experiments
the conversion of glucose carbon to citric acid, mycelium and CO2 was 76.4 and 81 per
cent, 13.8 and 12.1 per cent and 13.54 and 11.8 per cent respectively. The pool of electrons
transferred to oxygen in the two runs was 4.89 and 4.55, corresponding to 1.22 and 1.14
mmol O2 dm-3. The carbon content in mycelium and the degree of reduction of its carbon
was accepted as 0.46 and 4.29, respectively.
8.6 Conclusion
Substrate and oxygen requirements as well as biomass and product yields, which are some
of the basic parameters that need to be considered in determining the feasibility of the
fermentation process, may only be estimated properly if material and energy balances can
be applied to the bioprocess. Available electron, ATP and carbon balances as well as the
comparison of estimated values of yields and maintenance parameters can be used to test
the consistency of the data in fermentations and to gain insight into the possibility of by-
132
product formation. Material balance for carbon is in particular widely examined during the
course of the process since it can be readily confirmed by actual proof on the basis of
experimental data.
The assumption that the micro-organisms are alike in their chemical and biochemical
properties leads to formulation of a macro-chemical equation. Obviously, this is a gross
simplification that only holds as a first rough approximation. However, for many biotechnological processes it is important to know accurately the yield values. This can be
realized by formulating the metabolic model.
A useful metabolic model should represent, in a concise way, the main biochemical
properties of a specific micro-organism specified as a limited number of reactions.
Such simple metabolic models give the proper formulation of the Pirt type relation
linking as well as more accurate yield values and the biochemical insight how these can
be manipulated. In linear relations following from restrictions out of the metabolic
network the yield values are a function of the biochemical ATP and decarboxylation
stoichiometry.
For citric acid production by A. niger the assumption of typical mechanistic
quantities such as and P:O enables the calculation of theoretical and true yields for
growth and citric acid production appearing in kinetic equations based on known
mechanism of the process. The analysis of the distribution of carbon and energy source
for biomass growth, product synthesis and maintenance processes stresses the
importance of maintenance requirements in the process. Thus, it is a useful way for
process design and optimization.
8.7 References
ANDREWS, G, 1989. Estimating cell and product yields, Biotechnology and Bioengineering, 33,
256265.
ERICKSON, L E, MINKEVICH, I G and EROSHIN, V K, 1979. Utilization of mass-energy balance
regularities in the analysis of continuous culture data, Biotechnology and Bioengineering, 21,
575591.
GARRET, R H and GRISHAM, Ch M, 1995. Biochemistry (Saunders).
GLUSZCZ, P and MICHALSKI, H, 1994. Cultivation of A. niger in a pilot plant external loop airlift
bioreactor, FEMS Microbiological Reviews, 14, 8388.
KRZYSTEK L, GLUSZCZ P and LEDAKOWICZ S, 1996. Determination of yield and maintenance
coefficients by A. niger, The Chemical Engineering Journal, 62, 215222.
KUBICEK, C P and RHR, M, 1986. Citric acid fermentation. CRC Critical Reviews of Biotechnology,
3, 331373.
MINKEVICH, I G and EROSHIN, V K, 1973. Productivity and heat generation of fermentation under
oxygen limitation, Folia Microbiologica, 18, 376385.
NOWAKOWSKA-WASZCZUK, A and SOKOLOWSKI, A, 1987. Application of carbon balance to
submerged citric acid production by A. niger, Applied Microbiology and Biotechnology, 26, 363
364.
PIRT, S J, 1975. Principles of Microbe and Cell Cultivation (Blackwell).
ROELS, J A, 1983. Energetics and Kinetics in Biotechnology (Elsevier).
RHR, M, KUBICEK, C P, ZEHENTGRUBER, O and ORTHOFER, R, 1983. A balance of carbon
and oxygen conversion rates during pilot plant citric acid fermentation by A. niger: identification
of polyols as major by-products, International Journal of Microbiology, 1, 1925.
RHR, M, KUBICEK, C P, ZEHENTGRUBER, O and ORTHOFER, R, 1987. Accumulation and
partial re-consumption of polyols during citric acid fermentation by A. niger, Applied Microbiology
and Biotechnology, 27, 235239.
SHU, P and JOHNSON, M J, 1948. Citric acid: production by submerged fermentation by A. niger,
Industrial Engineering Chemistry, 40, 12021204.
133
136
citrate and gypsum in the subsequent steps of downstream processing and reduces the yellow
hue of the citric acid solution.
Recovery of citric acid from pretreated fermentation broth may be accomplished by
several procedures: classical method of precipitation, solvent extraction, adsorption/
absorption on ion-exchange resins, and recently developed, more sophisticated methods
such as electrodialysis, ultra- and nanofiltration or application of liquid membranes.
9.2 Precipitation
The standard method of citric acid recovery has involved precipitating the insoluble tricalcium citrate by the addition of an equivalent amount of lime to the citric acid solution.
Successful operation of the precipitation depends on citric acid concentration, temperature,
pH and rate of lime addition. To obtain large crystals of high purity, milk of lime containing
calcium oxide (180250 kg/m3) is added gradually at a temperature of 90C or above and
pH below, but close to, 7. The concentration of citric acid in the solution should be above 15
per cent. The process of neutralization usually lasts about 120150 minutes. The minimum
loss of citric acid due to solubility of calcium citrate is 45 per cent.
If precipitation is properly done, most impurities remain in the solution and may be
removed by washing the filtered calcium citrate. Washing is performed with the smallest
amount possible of hot water (approx. 10 m3 of water per tonne of acid at the temperature
90C) until no saccharides, chlorides or coloured substances can be detected in the effluent.
The calcium citrate is then filtered off and subsequently treated with concentrated sulphuric
acid (6070 per cent) to obtain citric acid and the precipitate of calcium sulphate (gypsum).
After filtering off the gypsum a solution of 2530 per cent of citric acid is obtained. The
filtrate is treated with activated carbon to remove residual impurities or may be purified in
ion-exchange columns. The purified solution is then concentrated in vacuum evaporators at
temperature below 40C (to avoid caramelization), crystallized, centrifuged and dried to
obtain citric acid crystals. If crystallization is performed at temperatures below 36.5C, the
citric acid mono-hydrate is formed and above this transition temperature citric acid anhydrate may be obtained. The schematic flow-chart of the standard precipitation method is
shown in Figure 9.1.
The disadvantage of this technology is the large amount of lime required for citric acid
neutralization and of sulphuric acid for calcium citrate decomposition. Moreover, it results
in the formation of large amounts of liquid and solid wastes (solution after calcium citrate
filtration and gypsum). For one tonne of citric acid, 579 kg of calcium hydroxide, 765 kg of
sulphuric acid and 18m3 of water are consumed and approximately one tonne of waste
gypsum is produced.
With the aim of decreasing the amount of lime and sulphuric acid by about one third,
Ayers (1957) has proposed recovery of citric acid by precipitation of di-calcium acid citrate.
An additional advantage of this method is that di-calcium acid citrate has a definite crystalline
structure and washes cleaner than the amorphous tri-calcium citrate. Moreover, fewer
impurities are precipitated from a fermentation fluid with the di-calcium salt than with the
normal salt, when the reaction mixture is completely neutralized.
Di-calcium acid citrate precipitates from a citric acid solution that has been partially
neutralized by the addition of calcium hydroxide, calcium oxide or calcium carbonate at
an elevated temperature. It is believed that an equilibrium exists between tri-calcium
citrate and citric acid on the one side, and di-calcium acid citrate on the other. At room
temperature the rate of calcium hydrogen citrate formation is negligible, but if the
Figure 9.1 Flowsheet of the standard precipitation method of citric acid recovery from
fermentation broth
137
138
temperature is elevated above 40C the complete conversion of tri-calcium citrate mixed
with aqueous solution of citric acid occurs within a reasonable length of time (about 24
hours). According to this principle a new method of citric acid recovery has been
developed.
The citric acid solution, obtained from the fermentation broth, is divided in two parts.
The first part, about two thirds of the total volume, is completely neutralized with milk of
lime, and the tri-calcium citrate is filtered off and added to the remaining part of the
original citric acid solution. If the obtained mixture is heated above 40C, a precipitate of
di-calcium acid citrate will result. As an alternative method, an amount of calcium
hydroxide no greater than two thirds of that required for complete neutralization may be
added directly to a citric acid solution. This mixture of tri-calcium citrate and citric acid
may then be converted to di-calcium acid citrate by heating above 40C, preferably to
8095C (depending on the boiling point of the solution). It has been found that the
results of the process may be improved, both by shortening the time and by increasing the
yield, if the mixture is seeded with di-calcium acid citrate crystals (practically about 10 to
25 per cent of the expected yield).
As an alternative to the classical methods of precipitation, separation and purification of
citric acid from fermentation solutions, Schultz (1963) has suggested isolating the citric
acid from the fermentation solution in the form of its alkali metal salts and recovery of the
acid from such salts directly in one single operation. This process is based on the fact that
certain alkali metal salts of citric acid crystallize from a fermentation solution after
neutralization of the acid by the addition of alkaline alkali metal compounds (hydroxides,
bicarbonates or carbonates) in such a manner that the mono-, di- or tri-alkali metal citrates
are obtained.
The impurities contained in fermentation broth influence or even inhibit crystallization
of salts, so not all the theoretically possible alkali metal salts of citric acid can be produced
in crystalline form according to the process. Of the sodium salts, however, all three possible
salts can be recovered in the form of crystals.
Before neutralization the fermentation solution may be concentrated by vacuum
evaporation to a concentration of at least 40 per cent, calculated for free citric acid. After
neutralizing the alkali metal salts crystallize on standing or on slowly stirring the solution;
seed crystals may be added to enhance the rate of the process. Crystallization is ordinarily
completed within 24 hours. Separation of the crystals from the solution is performed by the
usual methods (filtration, centrifugation). After washing the crystals with a small amount of
water, an almost white or slightly yellowish-brown precipitate is obtained, depending upon
the type of alkali metal citrate recovered. Subsequent purification of citric acid may be
performed by ion exchange on cation exchange resins or by electrodialysis.
The yield of citric acid on recovering it in the form of its alkali metal salts is between 50
per cent and about 80 per cent depending on the salt used. Citric acid remaining in the
fermentation broth may be recovered by the classical method of precipitation in the form
of a calcium citrate and following treatment with the sulphuric acid. According to this process
considerable savings in chemicals are achieved and the amount of the spent gypsum produced
is reduced. Moreover, the obtained gypsum filters more rapidly, due to the presence of
alkali metal ions, than gypsum from the classical technology, produced in the absence of
the alkali metal ions.
The use of purer raw materials than molasses (e.g. sucrose or glucose) in citric acid
production leads to simplified methods for its recovery and purification. Crystalline or raw
sugar are the best raw materials in view of the high acid yield and relatively short fermentation
times attained. Crystalline sugar is also favoured by the reduced risk of infection with foreign
139
micro-organisms due to the low initial pH value of the nutrient medium (2.5 to 3), and by
the considerable reduction of the total amount of wastes and effluents.
Crystalline sugar based fermentation makes it possible to use a modified, citrate-free
method of citric acid recovery (Lsniak, 1989), applied in industrial practice in several
citric acid manufacturing plants in Poland and the Slovak Republic. This technology consists
of direct removal of impurities from the post-fermentation liquor, i.e. colloids (proteins),
mycelium derived substances, coloured substances formed on heating the fermentation
solution, and mineral salts introduced with the nutrient medium, substrate and water. These
impurities must be removed as they interfere with the subsequent crystallization process.
The first step of purification of the solution is achieved using suitably selected coagulating
agents and activated carbon and then filtering off the precipitates (Adamczyk et al., 1985).
Further treatment involves the removal of the remaining impurities by ultrafiltration and
retention of the mineral salts using ion-exchange resins. The purified citric acid solution is
concentrated, crystallized, centrifuged and dried according to the classical production process
flowsheet.
After the separation of citric acid crystals the supernatant liquid from the centrifuge is
recycled back to the concentration section where the so-called second crop and then a third
crop of crystals is obtained. The supernatant liquid obtained after removing the third crop
crystals by centrifugation contains a large amount of impurities and must be purified by the
classical method involving the precipitation of calcium citrate. Thus the citrate-free method
can be used for purifying only up to 80 per cent of the whole amount of citric acid. This
necessitates the construction of a separate process line in order to avoid plants using the
above technology to manufacture merely a 50 per cent solution of citric acid, making it
necessary to purify a part of the citric acid by the calcium citrate method.
It is also possible to produce half of the acid amount in crystalline form and the rest in
liquid form. In this case, citric acid solution purified by the citrate free method is thickened,
crystallized and centrifuged to obtain the first crop. The supernatant liquid from the centrifuge
(citric acid concentration of about 50 per cent) is purified by the described method so as to
meet the quality standard requirements in liquid form. The advantage of this technology
lies in the fact that about half of the product is obtained in crystalline form and the use of
lime and sulphuric acid is eliminated as well as the formation of large amounts of effluents
and solid wastes. The flowsheet of the simplified, non-citrate method of citric acid recovery
is shown in Figure 9.2.
140
Figure 9.2 Flowsheet of the simplified non-citrate method of citric acid separation and
purification
141
Extraction with organic solvents which are partly or wholly immiscible with water,
such as certain aliphatic alcohols, ketones, ethers or esters (Kasprzycka Guttman et
al., 1989).
Extraction with organophosphorus compounds, such as tri-n-butylphosphate (TBP) (Pagel
and Schwab, 1950) and alkylsulphoxides, e.g. trioctylphosphine oxide (TOPO) (Nikitin
and Egutkin, 1974; Grinstead, 1976).
Extraction with water-insoluble amines or a mixture of two or more of such amines, as a
rule dissolved in a substantially water-immiscible organic solvent, and extraction with
amine salts (Baniel, 1981; Bauer et al., 1988; Bizek et al., 1992; King, 1992; Prochazka
et al., 1994; Juang and Huang, 1995).
Each solvent used for extraction is characterized by its equilibrium distribution coefficient
which is defined as the ratio of the acid concentration of the extract to the acid concentration
of the aqueous phase. For low concentrations of citric acid in the raw fluid the distribution
coefficient depends strongly on the type of solvent; at higher acid concentrations differences
between solvents are much reduced.
Extraction with organic solvents (in practice ketones and alcohols are used) may be
useful in cases where the acid has a relatively high concentration in the aqueous system
from which it is to be extracted. These solvents have rather low distribution coefficients
(0.020.36), thus the extract is always more diluted than the raw liquor and multistage
extraction is necessary as a rule. Moreover, solvents with relatively higher distribution
coefficients (such as butanols) are too water-miscible, so they require energy-consuming
steps of subsequent solvent recovery. Thus, these extraction systems are relatively
inefficient for acid recovery from the dilute aqueous solutions found in most fermentation
streams.
Organophosphorus extractants have a significantly higher distribution ratio than carbonbonded solvents under comparable conditions, e.g. using undiluted TPB for citric acid
extraction a distribution ratio of about 2 may be obtained at a 0.1 mol initial acid
concentration at 25C (Pagel and Schwab, 1950). Alkylosulphoxides have been shown to
extract carboxylic acids with a distribution ratio even higher than that of TBP (Nikitin
and Egutkin, 1974). The value of the distribution coefficient is influenced not only by
acid concentration but also by temperature. In TBP the distribution ratio for citric acid
decreases by a factor of 4 in the 080C range. This property allows perfect control of the
process: extraction at low temperature (1030C) and re-extraction with water at higher
temperature (7095C).
For the extraction by means of amines, aliphatic, araliphatic or aromatic amines, or their
mixture, preferably with the average aggregate number of carbon atoms at least 20 for each
amino group, may be used. These reagents have the advantage of providing a favourable
coefficient of distribution of the citric acid between the aqueous and amine phases so the
acid may be extracted even from highly dilute solutions. On the other hand there is a problem
of decomposing the amine salt and recovering the acid and the amine separately, since the
amines are too expensive to be thrown out. Usually the amine is liberated by treatment of
the salt with an inorganic base (e.g. calcium hydroxide) or inorganic acid, and the salt is
thus obtained instead of free citric acid. In addition to the expenditure of chemicals, this
process has the disadvantage of requiring a number of processing steps.
The extraction by the amine salts may be considered as a variant of the extraction with
amines. In some cases the amount of acid that can be extracted with the water-immiscible
amine is stoichiometrically considerably in excess of the amine present in the amine solution.
142
The possible excess amount of extracted acid depends on several parameters, e.g.
concentration of the acid in the raw liquor, the nature of the amine and its solvent. In some
cases this phenomenon may be applied for extracting the acid from its concentrated aqueous
solution by means of salts of amines with the same acid. From the extract the excess acid
can be recovered by washing with water.
The organophosphorus and aliphatic amine extractants were developed initially for the
needs of inorganic extractive separation technologies. When these solvents are used for the
recovery of citric acid intended for the food industry, the question concerning their toxicity
should be settled. It is known that some of these compounds show teratogenic effects. On
the other hand the amine extractant patented by Baniel et al. (1981) and Baniel (1982) has
received approval by the US Food and Drug Administration for the use in food and drug
technology (Melsom, 1987; US Food and Drug Administration, 1975). Of the great amount
of patents concerning recovery of citric acid from the fermentation broth by extraction only
this one has been applied in large scale production.
143
decolorizing pulp mill bleaching effluent or removing pesticides from waste effluent.
Their effectiveness in the separation of citric acid from A. niger fermentation broth is
rather unexpected.
The efficiency of the ion-exchange separation process may be greatly enhanced by
applying a so called simulated moving bed counter-current flow system. In this case the
apparatus consists of at least two static beds, connected with appropriate valving so that the
feed mixture is passed through one adsorbent bed while the desorbent material can be passed
through the other. Progressive changes in the function of each ion-exchange bed simulate
the counter-current movement of the adsorbent in relation to liquid flow. In such a system,
the adsorption and desorption operations are continuously taking place, which allows both
continuous production of an extract and a raffinate stream and the continual use of feed and
desorbent streams. The simulated moving bed system applied for citric acid recovery in a
pilot scale is proposed by Edlauer et al. (1990).
The disadvantage of the ion-exchange method may be seen in the fact that elution of
citric acid from the adsorption bed may require a large amount of desorbent, due to the
tailing effect known in chromatography, causing considerable dilution of the resulting citric
acid solution. The periodical regeneration of the ion-exchange resins by inorganic bases
may also be a source of unwanted effluent wastes.
144
of citric acid and the other the strip solution flowing in the lumen. The organic liquid
membrane is contained in the shell side between these two sets of hollow fibres. This
technique has been shown to be promising for citric acid separation even in the large scale,
as the extent of citric acid recovery of up to 99 per cent was linear with the membrane area,
suggesting easy scale-up.
The use of liquid membranes for the recovery of citric acid from fermentation broths
offers unique advantages over conventional techniques: lower energy consumption, higher
separation factors in a single stage, the ability to concentrate citric acid during separation
and smaller size of the complete separation apparatus. These advantages may result in a
reduction in overall recovery costs and in amount of wastes.
9.6 Electrodialysis
Another environmentally friendly alternative to the conventional methods of citric acid
recovery may be electrodialysis. This process enables separation of salts from a solution
and their simultaneous conversion into the corresponding acids and bases using electrical
potential and mono- or bipolar membranes. Bipolar membranes are special ion exchange
membranes which, in an electrical field, enable the splitting of water into H+ and OH- ions
(Strathmann et al., 1993). By integrating bipolar membranes with anionic and cationic
exchange membranes a three- or four-compartment cell may be arranged, in which
electrodialytic separation of salt ions and their conversion into base and acid takes place
(Voss, 1986; Sappino et al., 1996). According to Karklins et al. (1996), complete
transformation of sodium tri-citrate into citric acid in a four-compartment cell may be
achieved a little faster, but voltage on electrodes is higher than in a three-chamber cell.
Specific electroenergy consumption of the four-compartment cell was about 40 per cent
higher than that of a three-chamber apparatus.
When converting organic salts, high final acid concentrations may be achieved, as opposed
to mineral salts. It makes the process especially advantageous for citric acid recovery, as the
evaporation step normally required can be omitted. On the other hand organic salts such as
sodium citrate have a relatively large molecular weight and the solution also shows relatively
low conductivity. These properties make the separation more difficult and lead to higher
energy consumption, as in the case of inorganic compounds. The energy consumption
(excluding pumping) for the separation of 1 kg of citric acid using bipolar membranes is in
the range of 6.1 103 to 7.2 103 kWs (Novalic et al., 1995). Due to low mass transfer at
low pH values it is advantageous to adjust the pH of the feed acid stream to 7.5 (Moresi and
Sappino, 1996; Novalic et al., 1996).
Before the fermentation solution comes to the electrodialysis some pretreatment steps
are normally necessary: filtration of the broth, removal of ionogenic substances (especially
Ca++ and Mg++ ions) and neutralization by means of sodium hydroxide. In the subsequent
electrodialytic step the sodium citrate solution is converted into base and citric acid, which
is simultaneously concentrated and for the most part purified. The produced NaOH may be
reused for the neutralization (Novalic and Kulbe, 1996).
Although there have been several patents published concerning recovery and
purification of organic acids by electrodialysis (Gomez et al., 1991), this method is still
applied only in laboratory scale and requires optimization. The economics are mainly
influenced by the relatively high energy consumption, the membrane costs and the membrane
life time. However, due to the wider commercial availability of bipolar membranes in the
past few years and various advantages of the electrodialysis technique it is expected that
145
Figure 9.3 Scheme of citric acid separation by means of electrodialysis with bipolar
membranes (from Novalic and Kulbe, 1996)
this technology will soon be competitive with other processes (Novalic et al., 1996). Besides
the elimination of environmental problems, the use of electrodialysis enables continuous
separation of the citric acid from the broth during fermentation, leading to the decrease of
an inhibiting influence of the product. It is also possible to apply this technique for recovery
of the citric acid in continuous fermentation processes. The scheme of the proposed method
(Novalic and Kulbe, 1996) for citric acid separation by means of electrodialysis with bipolar
membranes is shown in Figure 9.3.
9.7 Ultrafiltration
Continuous separation and concentration of citric acid may be also achieved by ultra and/
or nanofiltration. Visacky (1996) verified in a laboratory scale a two-stage membrane process
for citric acid recovery from the broth obtained in A. niger cultivation on sucrose.
Polysulphone membrane with cut-off 10 000 used in the first stage allowed the product to
pass through to the permeate stream, while the retentate stream contained most of peptides
and proteins from the broth. The rejection coefficient for the product in this step was 3 per
cent, for the reducing sugars 14 per cent and for the proteins 100 per cent. Tighter
nanofiltration membrane with cut-off 200 in the second stage rejected approximately 90
per cent of citric acid and 60 per cent of reducing sugars (mono-saccharides). Concentration
of the product in the retentate stream was increased three times in comparison to the feed. A
similar two-stage membrane technique was adapted by Bohdziewicz and Bodzek (1994)
for simultaneous separation and concentration of pectinolytic enzymes and citric acid from
146
a fermentation broth. The dilute citric acid solution obtained as a permeate in the first step
of the post-fermentation fluid ultrafiltration was then concentrated up to 20 per cent using
reverse osmosis. Such membrane processes may give important benefits in industrial
technologies of citric acid recovery: low energy consumption, no wastes in comparison to
the conventional chemical methods, possibility of use in continuous processes. However,
they require practical verification and optimization in a pilot and industrial scale.
9.9 References
ADAMCZYK, E, LESNIAK, W, PIETKIEWICZ, J, PODGRSKI, W, ZIOBROWSKI, J and
KUTERMANKIEWICZ, M, 1985. Polish Patent 128,527.
ALBULESCU, C and GUZUN-STOICA, A, 1996. Emulsion liquid membrane extraction of citric
acid, Proceedings of the International Conference Advances in Citric Acid Technology, Bratislava,
October, p. 32.
ALTER, J E, BLUMBERG, R, 1981. US Patent 4,251,671.
AYERS, R, JR, 1957. US Patent 2,810,755.
BAKER, R W, TUTTLE, M E, KELLY, D J and LONSDALE, H K, 1977. Coupled-transport
membranes, Journal of Membrane Science, 2, 213221.
BANIEL, A M, 1981. Eur. Patent 0049,429.
BANIEL, A, 1982. US Patent 4,334,095.
BANIEL, A M and GONEN, D, 1991. US Patent 4,994,609.
BANIEL, A M, BLUMBERG, R and HAJDU, K, 1981. US Patent 4,275,234.
BASU, R and SIRKAR, K K, 1991. Hollow fibre contained liquid membrane separation of citric acid,
AIChE Journal, 37, 383393.
BASU, R and SIRKAR, K K, 1992. Citric acid extraction with microporous hollow fibres, Solvent
Extraction and Ion Exchange, 10, 119144.
BAUER, U, MARR, R, RUECKL, W and SIEBENHOFER, M, 1988. Extraction of citric acid from
aqueous solutions, Chemical and Biochemical Engineering, 2, 230232.
BIZEK, V, HORACEK, J, RERICHA, R and KOUSOVA, M, 1992. Amine extraction of
hydroxycarboxylic acids. 1. Extraction of citric acid with l-ocyanol/n-heptane solutions of
trialkalyamine, Industrial Engineering Chemistry Research, 31, 15541562.
147
148
MORESI, M and SAPPINO, F, 1996. Effect of temperature and pH on sodium citrate recovery from
aqueous solutions by electrodialysis, Proceedings of International Conference Advances in Citric
Acid Technology, Bratislava, October, p. 29.
NIKITIN, YU E and EGUTKIN, N L, 1974. Neftekhimiya, 14, 780785.
NOVALIC, S and KULBE, K D, 1996. Separation and concentration of citric acid by means of
electrodialytic bipolar membrane technology, Proceedings of International Conference Advances
in Citric Acid Technology, Bratislava, October, pp. 4144.
NOVALIC, S, JAGSCHITS, F, OKWOR, J and KULBE, K D, 1995. Behaviour of citric acid during
electrodialysis, Journal of Membrane Science, 108, 201205.
NOVALIC, S, OKWOR, J, and KULBE, K D, 1996. The characteristics of citric acid separation using
electrodialysis with bipolar membranes, Desalination, 105, 277282.
PAGEL, H A and SCHWAB, K D, 1950. Analytical Chemistry, 22, 1207.
PALLARES, J, RODRIGUEZ, S and SANROMAN, A, 1996. Citric acid production by immobilised
Aspergillus niger in a fluidised bed reactor, Biotechnology Techniques, 10, 5357.
PROCHAZKA, J, HEYBERGER, A, BIZEK, V, KOUSOVA, M and VOLAUFOVA, E, 1994. Amine
extraction of hydroxy-carboxylic acids. 2. Comparison of equilibria for lactic, malic and citric
acids, Industrial Engineering Chemistry Research, 33, 15651573.
SANROMAN, A, PINTADO, J and LEMA, J M, 1994. A comparison of two techniques for the
immobilisation of Aspergillus niger in polyurethane foam, Biotechnology Techniques, 8, 389
394.
SAPPINO, F, MANCINI, M and MORESI, M, 1996. Recovery of sodium citrate from aqueous solutions
by electrodialysis, Italian Journal of Food Science, 8, 239.
SCHGERL, K, 1994. Solvent Extraction in Biotechnology (Springer-Verlag).
SCHULTZ, G, 1963. US Patent 3,086,928.
STRATHMANN, H, RAPP, H J, BAUER, B and BELL, C H, 1993. Desalination, 90, 303310.
TSAY, S S and TO, K Y, 1987. Citric acid production using immobilized conidia of Aspergillus niger
TMB 2022, Biotechnology and Bioengineering, 29, 297304.
US FOOD and DRUG ADMINISTRATION, 1975. Federal Register, 40, 4908049082.
VISACKY, V, 1996. Membrane nanofiltration for citric acid isolation, Proceedings of the International
Conference Advances in Citric Acid Technology, Bratislava, October, p. 31.
VOSS, H, 1986. Deacidification of citric acid solutions by electrodialysis, Journal of Membrane Science,
27, 165172.
WANG, J L and LIU, P, 1996. Comparison of citric acid production by Aspergillus niger immobilised
in gels and cryogels of polyacrylamide, Journal of Industrial Microbiology, 16, 351353.
10
Fermentation Substrates
WLADYSLAW LESNIAK
10.1 Introduction
Fermentation industries have an advantage over some other manufacturing industries in
that their raw materials can be altered, within limits, allowing some buffering against
increasing world prices. However, the past 20 years have seen global changes in the prices
of all raw materials and consequently all fermentation substrates have suffered increases to
varying extents.
For processes where different substrates can be used, or both synthetic and biological
production routes exist, process economics is of paramount importance for survival. For
processes where the product is only obtainable through fermentation, profit margins can be
sustained by passing the price increases resulting from substrate cost increases on to the
consumer. Production of bulk products such as citric acid and antibiotics are obvious
examples.
These products therefore may have had less pressure on them than the others to search
for the cheapest possible substrate, but even here there is competition between rival companies
and ways to lower costs and increase profits are thus continually being sought. The choice
of substrate is therefore always under review (Ratledge, 1977).
There is always pressure to find a cheaper or better substrate, but the new substrate may
present storage problems, may be difficult to sterilize or have an unwanted variability in
composition. Increased productivity is not the only yardstick to be used. The substrate may
have a residue which poses product recovery and purification problems. The cheapest
substrate is therefore not often the best. In addition to these problems, any change in substrate
or amendment to the formulation of the medium will influence the characteristic of the
fermentation process, and has to be carefully evaluated.
A substrate must be readily available throughout most of the year. Seasonably produced
crops from which process wastes are used as fermentation feedstock are not suitable if the
harvest period is short and the material to be used is subject to contamination and spoilage.
Thus the industry must have substrates that are relatively stable and can be stored reasonably
easily for more than half a year.
A process, for example, citric acid production, can be changed to accommodate a new
substrate. The advent of cheap hydrocarbons in the 1960s led to many companies switching
149
150
Table 10.1 Relative carbon contents of fermentation substrates (from Ratledge, 1977)
over to this substrate. Aspergillus niger, the traditional producer, cannot grow on alkanes,
but a variety of yeast can and some will accumulate citric acid sufficiently enough for
industrial processes to be established (Shennan and Levi, 1974).
The price of the substrate is crucial. However, it is important to take into consideration
the amount of available carbon. This differs according to the type of substrate being used
(see Table 10.1). This suggests that if the choice of substrate is not limited, a carbohydrate
could be replaced by alkanes with no loss in process productivity (an important optimization
parameter for the citric acid process). However, others factors have to be taken into
consideration before this is acceptedincreased aeration or agitation rates may be necessary
with alkanes (being a more reduced substrate) and this factor must be met by the savings
from the change of substrate.
Transport costs for substrates from the collection or production point to the fermentation
plant have to be considered. These costs may become significant if too much water is present
and will mitigate against the use of some waste materials at sites removed from their point
of production. One substrate may be more attractive to use than another simply because it
poses fewer problems in the processes both before and after the fermentation.
Fermentation media for citric acid biosynthesis should consist of substrates necessary
for growth of the producer micro-organism and its citric acid biosynthesis, primarily the
carbon, nitrogen, phosphorus and microelements sources. Moreover process water and air
can be included as fermentation substrates.
The basic substrate for citric acid fermentation in plants using the surface method of
fermentation is beet or cane molasses. Plants using submerged fermentation can use not
only beet or cane molasses, but a substrate of higher purity such as hydrolysed starch,
technical and pure glucose, refined or raw sugar, purified and condensed beet or cane juice.
This is because use of a pure substrate may result in increases in yield, or reduction in
fermentation time.
10.2 Molasses
Molasses is a widely used substrate, coming in a variety of qualities. High quality molasses
is usually demanded for citric acid production while poorer quality molasses is used mainly
in the production of low value products such as alcohol, where the producer micro-organism
has a much greater tolerance to impurities in the medium.
Fermentation substrates
151
The composition of cane and beet molasses has recently been compared and the uses of
molasses as a fermentation feedstock have been discussed elsewhere (Hastings, 1971). Cane
and beet molasses are not identical in composition; often one type will be preferred to the
other. They are sometimes mixed to take advantage of the additional nutrients arising from
the differences in composition.
Besides substrate type (sugar beet, sugar cane), the chemical composition of molasses
depends on many factors such as soil and climate conditions, fertilization type, crop
method, time and conditions of storage, production technology, technical equipment of
plant, etc.
152
Table 10.2 Amino acid content of beet molasses (from Smirnow, 1983)
the fermentation. The amino acids content in molasses depends on the soil and climate
conditions and beet cultivation. Amino acid content of beet molasses is shown in Table
10.2.
The content of mineral substances in beet molasses amounts to 8.514.0 per cent. The
main ingredient of the mineral ash is K2O (6070 per cent of the total), CaO (4.57.0 per
cent) and MgO (about 1 per cent). The level of P2O5 in ash is normally very low (0.20.6
per cent), because over 90 per cent of phosphorus contained in beet is removed in the
sugar extraction process. If the method of juice alkalization by Na3PO4 (pH 8.38.5) is
used in the sugar production, the contents of P2O5 in molasses ash can reach 1.22.0 per
cent.
There are also many other elements, so-called microelements, which have a great
effect on the citric acid fermentation process. The amount of particular microelements
in different molasses can range widely as indicated in Table 10.3. Another important
Fermentation substrates
153
ingredient of molasses is vitamins, especially those that are known to stimulate microbial
activity. The content of vitamins (mg/100 g) in beet and cane molasses is shown in Tables
10.4 and 10.5 respectively.
The pH of molasses depends on the sugar extraction technology. It was considered that
a neutral, or slightly alkaline molasses gave the best citric acid yields. However, a fermentation
technology to tolerate the slightly acidic molasses produced in modern refineries has been
developed. Today, it is considered that for citric acid fermentation the buffering capacity of
the medium is more important than the pH value of the molasses. It is defined as the amount
of 1N solution of sulphuric acid (in cm3) used to reduce pH from 5.0 to 3.0 in 100 g molasses
solution diluted in 1:1 ratio with water and acidified to pH 5.0. The buffer capacity of beet
molasses usually ranges from 60 to 95 cm3. Citric acid production needs molasses with low
buffer ability, to make possible the required rapid fall of medium pH during fermentation.
154
Beet and cane molasses can also contain other substances which appear in small amounts,
but are often crucial in deciding whether the molasses are suitable for use in citric acid
biosynthesis. These are pesticides, fungicides and herbicides used in beet and cane cultivation
and also substances used for defoaming in sugar production process. All have mostly toxic
properties and negatively affect molasses usability. It is considered that the best molasses
for citric acid fermentation can be, as a rule of thumb, characterized as shown in Table 10.7.
According to all cited requirements, beet molasses is more suitable for citric acid fermentation
than cane molasses. It is especially relevant in submerged fermentation where the quality of
the substrate is more important for productivity and fermentation yield.
The microflora of molasses can be an agent of negative influence on yield and
productivity of fermentation. Molasses will always contain a certain number and type of
micro-organisms, sometimes the count can be higher than 10 000 per g of molasses. The
most common micro-organism in molasses is sporulating rods of Bacillus species (over
90 per cent of total molasses microflora), bacteria producing acids and gases (E. coli,
Pseudomonas and others), heterofermentative lactic acid bacteria (Leuconostoc
mesenteroides), sometimes yeasts of Candida species, and very rarely, moulds of
Penicillium, Aspergillus and other species.
Fermentation substrates
155
Bacteria of Bacillus species appear in molasses because their spores are present in
beet and are unaffected by high temperatures, even 125C (Bacillus subtilis). They are
destructive because some of them (B. megaterium, B. mesentericus) are able to reduce
nitrates to nitrites. Strains of Aspergillus niger can be very sensitive to nitrites (a NO2
concentration in medium of 0.05 per cent will retard growth and cut the citric acid
production by 50 per cent).
The greatest antagonists of Aspergillus niger among non-sporulating bacteria are E. coli
and Pseudomonas. They grow very quickly in many media over a wide temperature range,
decomposing sugar in solution to unwanted acids, alcohol and gases, and are able to reduce
nitrates to nitrites. Bacteria of Leuconostoc species convert sucrose to dextran. They also
produce unwanted volatile acids such as formic, acetic and propionic acid. Yeasts of Candida
species can propagate over a wide range of temperature (555C) and pH value of medium
(28). They can be very undesirable to Aspergillus niger strains, especially in submerged
fermentation, where they can stop citric acid biosynthesis.
156
of the medium may not be sufficient to ensure total sterility because some micro-organisms
can enter the fermentation broth via addition ports or from the air. Because of this, other
sterilizing agents such as formaline (at 0.0060.01 per cent) (in particular for the surface
fermentation) and furan derivatives are used.
Sulphamide preparations do not totally destroy the bacteria, but antibiotics, though they
do not have any negative influences, are too expensive (Karklinsh and Probok, 1972).
Applying chemical sterilizing agents enables softening of sharp thermal sterilization
conditions that have a negative effect on molasses quality. Other methods of sterilization
tested are UV and gamma radiation, ultrasound, and ultrafiltration. They are not used in
practice as they are cost-prohibitive compared with steam sterilization.
In tropical countries where date production is considerable, date syrup is a major product.
The chemical composition of this material differs from that of sugar beet molasses, but
when mixed with an equal volume of beet molasses it gives the same yield of citric acid as
for beet molasses based on the amount of sugar converted (Shadafza et al., 1976). Molasses
from the starch industry (hydrol molasses) is also widely used in citric acid fermentation.
10.4 Syrups
Syrups of beet or cane sugar can also be used as basic substrate for the submerged citric
acid fermentation. The great advantage with this substrate is its purity; however, the quality
of the syrups deteriorates rapidly during storage. Because of this they can only be used
during the sugar campaign season and only if the citric acid plant is not too far from the
sugar factory because of the large transport costs.
Preparation of the syrups for fermentation entails dilution with water to a sugar
concentration of 1520 per cent, addition of necessary nutrients (NH4NO3, KH2PO4, MgSO4,
(NH4)2C2O4), acidification with hydrochloric or sulphuric acid to pH 45 and sterilization
at 121C for 0.51 hours (Kutermankiewicz et al., 1980).
Fermentation substrates
157
10.5 Starch
Starch can be an attractive feed stock for many fermentation processes. It can be used
directly by many micro-organisms and is frequently incorporated into fermentation
media as a partial ingredient. Starch is widely used as the principal substrate for the
production of amylases and amyloses in the food and brewing industries. The production
of citric acid from sources of starch such as corn, wheat, tapioca and potato is widely
used.
The suitability of these substrates for citric acid fermentation depends on their purity
and method of hydrolysis. Acid hydrolysis, enzymatic hydrolysis, or a combination of the
two, are used. Preparation of starch substrates for fermentation is based on their enzymatic
liquefaction and saccharification to a defined hydrolysis level. Additional nutrients are added,
depending on which starch is used. The pH is adjusted to 34 using hydrochloric or sulphuric
acid and the medium is sterilized at 121C for 0.51 hour.
Good citric acid yields have been obtained using pure starch (potatoes, wheat or maize),
hydrolysed only to 1015 DE with a-amylase (Bolach et al., 1985). This was possible, as
the applied Aspergillus niger strain had the ability to produce its own amylolytic enzymes
which helped in the saccharification of the starch to available sugars. Dextrose syrup, obtained
by enzymatic hydrolysis of starch, is now employed as a basic substrate for citric acid
biosynthesis in laboratory and industrial scale. In this case it is especially important to
restrict the amount of heavy metals below critical levels; heavy metals should therefore be
removed by ion exchange.
When using an Aspergillus niger strain resistant to higher concentrations of heavy metals,
practically the same yield may be obtained on decationized and non-decationized dextrose
syrup (Pietkiewicz et al., 1996).
10.6 Hydrol
This is a paramolasses obtained as a by-product during crystalline glucose production from
starch. Because of the high glucose content (4045 per cent) and high purity coefficient it is
a very good substrate for citric acid production (Lesniak et al., 1986). Preparation of hydrol
for fermentation involves dilution to a sugar concentration of 15 18 per cent, addition of
necessary nutrients and adjustment of pH with hydrochloric or sulphuric acid to 3.04.0.
The solution is sterilized at 121C for 0.5 hour and cooled to 3235C.
10.7 Alkanes
The low price of alkanes, coupled with the ability of many organisms to utilize them, produced
major changes in the fermentation industry during the 1960s and 1970s. Citric acid
production, using Candida lipolytica, is a typical example and has been the subject of many
patents (Maldonado and Charpentier, 1975; Kimura and Nakanishi, 1985). However, there
are few industrial citric acid processes that are based on alkanes. There are two main reasons
for this. Firstly, in these processes isocitric acid would also be produced at concentrations
that would cause product recovery problems, as well as reduced citric acid yields
(Wojtatowicz and Sobieszczanski, 1981). Secondly, a fourfold increase in price since 1973
no longer makes alkanes a cheap substrate.
158
10.9 Cellulose
Cellulose is the major renewable form of carbohydrate in the world: about 1011 tonnes are
synthesized annually and much of this is waste. To use it as fermentation feedstock, it must
be first hydrolyzed to starch and then to sugar, either chemically or by cellulases. The
technology and economics of these processes are constantly being improved, but it is still
not apparent when the production of sugar syrups by this route is going to become profitable.
In the long term, cellulose could become a major resource of the fermentation industry in
general, including citric acid fermentation.
Fermentation substrates
159
is very different from academic research into citric acid fermentation. Here, a refined sugar
is invariably used as the carbon source and much work has been done on the level of nutrients,
in particular trace metals required for optimal acid yields and the role of individual metal
ions.
10.10.2 Water
Water used for diluting basic substrates should be at least of drinking water quality. There
should not be organic compounds and products of their decomposition (NH3, and H2S) and
the level of trace metals must be controlled. All the water must be sterilized to remove
contaminating micro-organisms.
10.11 Conclusion
Citric acid is a bulk product, with the substrate cost being a major part of the plant operating
cost. In terms of bulk, the carbon source is the most important substrate. The efficiency of
its conversion to citric acid will determine the profitability of the fermentation process.
For this reason, the carbon source is also the most important substrate for process
economics. This chapter has, therefore, concentrated on the various forms of carbon sources
used. Most processes are based on molasses, although the use of cleaner sources is gaining
ground. Whatever the source, its cost and preparation in order to permit optimal
fermentation conditions are two important aspects of the technology in citric acid
production.
10.12 References
BOLACH, E, LESNIAK, W and ZIOBROWSKI, J, 1985. Acta Aliment. Polonica, 11, 1.
ELIMER, E, 1994. Studies on Use of Plant Fats for Citric Acid Production by Aspergillus niger, PhD
thesis, University of Wroclaw, Poland.
GUTCHO, S J, 1973. Chemicals by Fermentation (Noyes Data Corporation, Park Ridge, NY, USA).
HASTING, J J H, 1971. Advances in Applied Microbiology, 14, 1.
IKENO, Y, MASUDA, M, TANNO, K, OOMORI, I and TAKAHASHI, N, 1975. Journal of
Fermentation Technology, 53, 752.
KARKLINSH, R J and PROBOK, A K, 1972. Organic acid biosynthesis (in Russian), Zinatne, Riga.
KIMURA, K and NAKANISHI, T, 1985. German Patent 2 065 206.
KUTERMANKIEWICZ, M, LESNIAK, W and BOLACH, E, 1980. Przem. Ferm. i Owoc.-Warzyw,
6, 27.
LEOPOLD, H and VALTR, Z, 1964. Die Nahrung 1, 37.
LESNIAK, W, 1972. Studies on Submerged Citric Acid Fermentation, PhD Thesis, University of
Wroclaw, Poland.
LESNIAK, W, 1976. Przem.Ferm. i Rolny, 6, 22.
LESNIAK, W, 1989. Polish Technical Review, 5, 185.
LESNIAK, W and KUTERMANKIEWICZ, M, 1990. Citric Acid ProductionBasic Review (in
Polish), STC, Warsaw.
LESNIAK, W, PODGORSKI, W and PIETKIEWICZ, J, 1986. Przem. Ferm. i Owoc.-Warzyw, 6, 22.
MALDONADO, P and CHARPENTIER, M, 1975. German Patent 2 551 469.
PIETKIEWICZ, J, PODGORSKI, W and LESNIAK, W, 1996. Proceedings of the International
Conference on Advances in Citric Acid Technology (Bratislava, Slovak Republic), p. 9.
RATLEDGE, C, 1977. Fermentation substrates, Annual Reports on Fermentation Processes, Vol. 1,
Chapter 3.
160
SHADAFZA, D, OGAWA, T and FAZELI, A, 1976. Journal of Fermentation Technology, 54, 67.
SHENNAN, L and LEVI, J D, 1974. Progress in Industrial Microbiology, 13, 3.
SMIRNOW, W A 1983. Food Acids (in Russian), Moscow, 105.
WOJTATOWICZ, M and SOBIESZCZANSKI, J, 1981. Acta Microbiologica Polonica, 30, 69.
11
Nomenclature
a
A
B
Cn
Cp
dhole
D
D
e
g
h
hf
HD
J
k
kL
K
K
lp
L
L
N
Np
P
DP
Q
r
R
specific area
area
permeability coefficient
(n = 1 5) constants
specific heat capacity
pore diameter
diameter
liquid diffusivity
fractional voidage
acceleration due to gravity
bed height
film heat transfer coefficient
dispersion height
flux
thermal conductivity
liquid side mass transfer coefficient
at the gasliquid interface
fluid consistency index
Kozeny constant
pore length
liquid height in the reactor
characteristic length in dimensionless numbers
impeller speed
power number
power input
pressure drop
volumetric gas flow rate at NTP
filtration resistance of the filter medium
universal gas constant
(m2 m-3)
(m2)
(m2)
(J kg-1 C-1)
(m)
(m)
(m2 s-1)
(-)
(m s-2)
(m)
(W m-2 C-1)
(m)
(m3 m-2 s-1)
(W m-1 C)
(m s-1)
(Pa s)
(-)
(m)
(m)
(m)
(s-1)
(-)
(kW)
(N m-2)
(m3 s-1)
(m-1)
161
162
T
u
V
temperature
superficial gas velocity
volume
(C)
(m s-1)
(m3)
Greek letters
(-)
(m-1)
(N m-1)
(Pa s)
(-)
(kg m-3)
(-)
(N s m-2)
(kg m-3)
Subscripts
a
d
D
eff
g
i
L
o
apparent
downcomer
total area
effective
gas
impeller
liquid
unaerated
(mg 1-1)
gDrr/s
ug(1+f)l/fds
Q/ND3
N2D/g
gDR3/heff
Cp/k
/rD
(min)
r
R
T
163
riser
reactor
top
164
165
Are there constraints imposed on the packaging material due to environmental legislation?
(see Livingstone and Sparks (1994) for a discussion on the effect of new packaging
laws).
Is the final product application known? For example, will it be used in dry formulations,
where a specific crystal size distribution is important, or will it be dissolved and used in
solution?
Are there any other specific customer requirements?
site selection;
a comparison of yields and productivity with different strains and substrates;
alternative processing routes; and
plant capacity and utilization.
A process description and material balance quantifies aspects such as effluents, by-products
and site storage requirements for raw materials and products. Usually such a mass balance
is calculated backwardsi.e. starting with the stated customer requirement. The approach
to process development has also been fixed at this stage. In other words: To
Figure 11.2 Schematic flow diagram for the production of citric acid
167
what extent will licensed technology be sought and which aspects might be developed fully
in-house. The process flow diagram (PFD) with the mass balance will now form the baseline
for more detail design.
The production of citric acid is a fairly mature technology, and it is unlikely to expect a
radical breakthrough. More likely would be incremental advances (see Roussel et al. (1991,
p. 54) for the context of the terms radical and incremental) in technology, as major producers
focus R&D efforts on staying competitive. Thus the processing of citric acid can be expected
to remain within the scheme as set out in Figure 11.2. Two areas where new technology
might impact, will be on fermenter design and direct crystallization routes. The former
refers to the probable phasing out of mechanically stirred vessels, while the latter includes
all processes aimed at recovering citric acid without a precipitation sequence. This would
include:
membrane applications;
novel ion exchange resins;
solvent extraction;
electrodialysis; and
chromatography.
168
169
approval. At this stage the design concepts and capacities are frozen, so that the focus is on
the detail design of the individual units. The plant battery limits have also been set, and the
various interfaces are clearly defined. In parallel to the detail unit design, the drawing office
can start on plant layout options.
170
can still be done in situ. One of the reasons to avoid batch sterilization of media on the
production scale, is the possible formation of complexes such as hydroxymethyl-furfural
(HMF). Formed from the reaction between glucose and ammonium and nitrogen compounds
at elevated temperatures, HMF is a strong respiratory inhibitor. Due to the long heating and
cooling cycle of large scale batch sterilization, HMF produced might inhibit the subsequent
fermentation to non-optimal productivity levels. In discussing fermentation design, Sderberg
(1983) lists the advantages of continuous sterilization and Wallhuser (1985) offers a
comprehensive treatment of media and vessel sterilization.
11.8 Fermentation
The mechanically stirred tank reactor (STR) has been the standard in bioprocessing for at
least 40 years. Although the picture is now changing, fewer scale-up studies (especially on
the larger scale) have been done on airlift reactors, while scale-up and mixing in the STR
has been extensively researched and several design correlations are available. The reader is
referred to a review article by Berovic (1991) where he discusses advances in reactor design.
rules of thumb;
scaling according to one specific parameter;
geometric similarity; and
scale-down method.
Rule of thumb guidelines, based on historical data and conventions followed with previous
successful designs, provide the starting point for further detail calculations. The following
set (Sections 11.8.211.8.3) is not intended as a complete list, but as a summary of typical
aspects which would be included.
171
the mixing power input into the vessel, but only to an exponent of 0.2 to 0.3 (Oldshue,
1985). As the mechanical energy input is dissipated as thermal energy, it does not
help to increase agitator power input in order to improve heat transfer. Indications are
that the presence of dispersed air bubbles at the heat transfer surfaces increases the
coefficient (De Maerteleire, 1982). This is possibly due to a scouring action on the
surface. Effective utilization of mixing energy input to ensure complete gas dispersion,
would thus be an important consideration.
Increase the available heat transfer surface.
In the laminar flow range the power number declines linearly from an initial maximum
value, while it is independent of the Reynolds number (and constant) in the turbulent flow
regime. The power number varies according to the impeller geometry, but shows the same
profile for a wide range of impellers (Mockel and Wollechensky, 1990).
172
which can be applied for non-Newtonian media (Taguchi and Miyamoto, 1966). In equation
(11.2), the constant, C1, is dependent on system geometry.
The constant C2 and exponents a, and d are system specific (i.e. scale dependent and
function of geometry) and must be determined experimentally. However, the following
generalizations can be stated:
For Newtonian fluids the value of d is usually small and in the region of 0.100.14.
Hence errors in the viscosity term do not drastically influence the accuracy of the result.
Applying the concept of an apparent viscosity, this term can also be used in correlating
data for non-Newtonian rheology. The wall viscosity, w, can be taken to be equal to the
viscosity at zero biomass concentration.
and are positive and generally in the range, 0.25 < a, < 0.9.
173
(11.5)
174
The purpose of doing the initial regime analysis is to establish which mechanisms are rate
determining. This is done by comparing the order of magnitude of the different characteristic
times. If the small scale study is to be representative, the relative time-constants of ratelimiting steps must remain in the same order. It should be noted that the method is only an
order of magnitude comparison and not an exact procedure. Thus if the time constants for
two processes (such as mixing time and oxygen transfer) are similar, the mechanisms should
be investigated further.
(11.7)
175
(11.8)
This is the sum of the gas kinetic energy and the compression energy to overcome the
pressure drop. The parameter, a, is the ratio of the maximum hydrostatic head, to the pressure
at the liquid surface:
a
= rL(1 - f)gL/PT.
(11.9)
Gas hold-up
The gas residence time in the reactor is determined primarily by the liquid circulation velocity
and the bubble swarm rise velocity. As the liquid circulation velocity increases, the degree
of back-mixing in an airlift reactor and the fractional hold-up increases. This means more
efficient utilization of the available oxygen than in an STR of similar geometry. Therefore
airlift reactors typically employ aspect ratios as high as 10, to develop high liquid circulation
velocity (Onken and Weiland, 1983). Hold-up has been correlated as being directly
proportional to ug, i.e.
f
= C3ug
(11.10)
= (ug/rL)/(30 + 2ug)(72/s)-1/3
(11.11)
-1
which is applicable for Newtonian fluids in the range 5 < ug < 12 cm s . For non-Newtonian
fluids, Barker and Worgan (1981) correlated hold-up data to the consistency index, K,
according to:
f
(11.12)
176
hf = 1380(ug)0.22/(Pr)0.5
(11.13)
(11.14)
and
hf = 13.34(1 + Ar/Ad)-0.7(ug)0.275
(11.15)
(11.16)
kLaa(ug)n/(a)b
(11.17)
or:
with the coefficient, n, typically in the range 0.70.8. For airlift fermenters, Popovic and
Robinson (1989) found:
(11.18)
which can be reduced to equation (11.16) or (11.17). In this case Al is proportionality constant,
and refers to the sparger hole diameter [m]. The exponents a1, b1, d1, e1, f1, g1 and h1 must
be determined experimentally.
Dimensional analysis approach
The general equation proposed for design purposes (Deckwer, 1985), incorporates several
dimensionless groups:
kLa = C4(D/DR2)Scb1 Bob2 Gab3 Fr(1 + C5Dem)-b4
(11.19)
Again C4 and C5 are constants and b14 must be determined experimentally. Depending on
the system under investigation, not all groups would necessarily be relevant. The Froude
number, Fr, is often omitted where vortex formation is not a factor, while the Deborah, De,
number accounts for the elastic properties of the broth. A number of correlations of the
format of equation (11.18) and (11.19) are summarized by Chisti (1989).
177
The design of the filter is based on the application of the Poiseuille equation (Boss, 1983).
The equation can be written in various forms (Coulson and Richardson, 1983, p. 323), but
always relates the rate of filtration, dV/dq, to the pressure drop across the filter, DP, filtration
area, A, liquid viscosity, , resistance to filtration, r, and cake compressibility, d:
(11.20)
where a is the average specific cake resistance, which is a function of the pressure applied and
the cake compressibility d: a = aDPd, where a is a constant related to the size of the particles
in the cake. A value of d = 0 corresponds to an incompressible filter cake, while d = 1 would
be a gelatinous protein sludge. From equation (11.20) it follows that for such cases, the rate of
filtration is independent of the applied pressure. The resistance of the filter medium, r, is often
expressed as an additional cake resistance, with a fictitious thickness, to simplify the handling
of experimental data. On a (batch) laboratory apparatus, the rate of filtration is measured
versus the applied pressure. The parameters of the equation are then determined through
curve fitting of the data. Scaling up the filter design, is in essence the specification of a required
filter area to achieve a certain rate of filtration, at the allowable pressure drop.
Equation (11.20) can be extended to account for applications such as a rotary drum
filter, where the total filter area is not continuously submerged in the slurry (Peters and
Timmerhaus, 1968, p. 487), by defining an effective area: ADyf. If AD is the total filter area
and yf the fraction immersed in the slurry, then:
(11.21)
where VR is the volume of filtrate per revolution of the filter.
Final points to consider in selecting a suitable filter include:
Will washing of the filter cake be necessary to recover the maximum citric acid?
What is the desired or acceptable moisture content of the cake?
Can a filter aid such as diatomaceous earth be used, or is this undesirable? If so, is this
from an economical point, or because it impacts on the fodder value of the biomass?
Can gypsum be used as a filter aid?
Is there an additional time constraint on the required rate of filtration? This applies in
cases where the operation must be completed within a short time (usually less than 16
hours) after the end of fermentation, before complete cell lysis occurs.
178
11.13 Purification
11.13.1 Membrane applications
Microfiltration, ultrafiltration and nanofiltration have been investigated for application in
citric acid processes. Microfiltration, as with lactic acid production, offers possibilities with
regard to cell retention in continuous citric acid fermentation systems (Enzminger and Asenjo,
1986; Daniel and Brauer, 1994; Rubbico et al., 1996).
Where the aim is to remove proteins or enzymes from the fermentation broth, ultrafiltration
(UF) or nanofiltration (NF) is employed (Bohdziewicz and Bodzek, 1994). With NF it is
also possible to remove residual sugars from the process liquor (Raman et al., 1994) at low
pH. This is due to the structure of the NF membrane, where the active membrane layer
typically consists of negatively charged groups. Thus salts are rejected due to electrostatic
interaction between the ions and the membrane while sugars are rejected on molecular size.
At low pH values, the citric acid is un-dissociated and permeates the membrane in spite of
sugars (with a similar molecular weight) being rejected. Conceivably it is therefore possible
to put together a process scheme which would eliminate the lime precipitation step by
removing, not only proteins, but also a significant percentage of residual sugars, through
NF.
Industrial exploitation of this concept has been hindered by:
Developing membrane material that offers long-term stability at low (2) pH.
Membrane fouling.
Cost of membranes.
Energy requirements due to high trans-membrane pressures.
These factors are being resolved with continued research in the field: developments such as
ceramic membranes offer mechanically rigid filters resistant to chemical attack. One
favourable aspect of the membrane applications is that scale-up can be done through modular
duplication of pilot plant units. Hence it is possible to predict accurately the performance of
a full-scale unit from a series of laboratory or pilot plant tests. As with standard filter
operations, it is however of prime importance to use a representative sample in doing
experiments.
The application areas of UF and NF overlap to some extent, but can be grouped according
to molecular cut-off point (Gyure, 1992). Mathematically, ultrafiltration can be modelled with
the HagenPoiseuille equation (Kula, 1985). Analogous to equation (11.20), the flux, J is
related to the trans-membrane pressure applied, dynamic viscosity and membrane properties:
(11.22)
179
Equation (11.22) applies in the ideal case, where the membrane pores are of uniform
distribution and size and fouling of the membrane or concentration polarization can be
neglected. The latter effect occurs due to an accumulation of the retained solute at the
membrane surface, resulting in a concentration higher than the bulk concentration. Such
concentration of the retained species means that the component will diffuse back to the
bulk flow conditions. At a sufficiently high concentration of the retained species,
saturation concentrations at the membrane surface lead to the formation of a gel layer,
which then offers an additional filtration resistance. Once such gel polarization is
established, the flux becomes independent of the pressure: increased pressure forms a
thicker gel layer, which in turn offers increased resistance and hence the flux does not
increase.
In practice, the UF process is better described by the mass transfer limited (i.e. diffusion
limited) models. For a comprehensive treatment of the topic, the reader is referred to the
Ultrafiltration Handbook (Cheryan, 1986); Reisman (1988) also presents some comparative
data on capital and operating costs of membrane units.
(11.23)
where:
B = [1/K][e3(S2(1 - e)2)]
(11.24)
B, is the bed permeability coefficient, while the Kozeny constant, K, is generally assumed
to be 5. This constant is a function of particle shape and porosity.
It should be noted that the equation was derived on the basis of the bed consisting of
uniformly sized, spherical particles. Where significant deviation occurs from this situation,
some corrections have to be taken into account (Coulson and Richardson, 1983). Equation
(11.24) can also be applied to calculate the pressure drop across an ion-exchange column
resin bed. In such a case it is to be expected that the particles will be of uniform size and
spherical.
180
11.13.3 Ion-exchange
Ion exchange involves the interchange of any ion between the process liquor and the polymer
resin. The essential points (Dechow, 1983) are that ion exchange reactions are:
stoichiometric;
reversible;
possible with any ionizable compound;
a function of the resin selectivity and reaction kinetics;
subject to the usual chemistry kinetic behaviour with regard to concentration and
temperature.
Typically, resins are polymers based on the cross-linking of polystyrene with divinylbenzene.
Other possibilities are the cross-linking of divinyl-benzene with an acrylate or acrylonitrile,
as well as phenol-formaldehyde and polyalkylamine resins. Three types of resin exchange
reaction can be found in the production of citric acid:
Demineralization.
Metathesis, which is the conversion of salts of citric acid to the acid.
Adsorptive purification.
The metathesis reaction can be represented as:
[resin]-H+ + Na+-[citrate]- [resin]-Na+ + H+-[citrate]with equilibrium constant, K, defined as the concentration of products divided by reagents
(at equilibrium). A large K-value indicates a high affinity of the resin for sodium ions and
means that an excess of strong acid will be required to regenerate the resin. The polymer
structure and composition determine the resin selectivity for a specific ion, but at ambient
temperatures, two generalizations apply to dilute aqueous solutions:
the exchange potential increases with increasing ion valence; and
at the same ion valence, the exchange potential increases with atomic number.
Thus, in increasing order of exchange potential:
Na+ < Ca++ < Al+++ and
Li+ < Na+ < K+ or Mg++ < Ca++ < Sr++ < Ba++
Similarly, for anions: F- < Cl- < BrThese principles are important in monitoring the ion exchange column effluent: it follows
that the monovalent ions would be expected to break through first as the resin reaches
capacity loading. As the resin reactions are stoichiometric, the quantity of resin material
required to remove a certain concentration of cations or anions, can easily be calculated.
The resin capacity is expressed as equivalents per kilogram (on a dry basis) or per litre on a
wet basis (eq/l). The equivalents number is simply an indication of the number of active
sites available for adsorption and can be obtained from the resin supplier. Analysis of the
process liquor will then determine the quantity of resin to be used for the required throughput.
Because the exchange process is an equilibrium reaction, the resin is utilized at a level well
below the theoretical capacity, thereby shifting the equilibrium in the desired direction (Le
Chteliers principle). This does not significantly increase costs, as the resin cost is typically
only 10 per cent of the unit cost (Dechow, 1983).
181
(11.25)
Thus the resin volume and hence column capacity more than doubles, but the capital cost to
fabricate and install the unit does not increase by the same factor. The calculation of the
pressure drop for a specific column geometry can be done with equation (11.23).
Adsorptive purification
Possibly a commercially viable direct crystallization route, this process is already employed
for the recovery of lysine. In some cases, adsorption is done directly from the fermentation broth
(Van Walsem et al., 1997) thereby simplifying the flowsheet considerably. A scheme proposed
for the recovery of citric acid (Ernst and McQuigg, 1992) uses temperature swing adsorption
(TSA) to purify the citric acid solution. In this case the regeneration is done by utilizing the
difference in resin capacity for citric acid as a function of temperature. Thus citric acid is adsorbed
at ambient temperatures and desorbed with hot water. Resin capacity in excess of 155 g citric
acid per litre resin, with a 96 per cent reduction in RCS values were reported.
While such a process eliminates the lime precipitation route and associated by-product
disposal dilemma, the environmental focus might shift to the actual resin in this case. The
adsorptive resins are structured from poly-vinylpyridine-co-divinylbenzene, the production
process of which generates some environmental concerns in itself. Although the effluents
(pyridine compounds) can be treated, it ultimately becomes a cost which is passed on to the
end user in the pricing of the resin.
11.13.4 Electrodialysis
Recovering lactic acid by electrodialysis has been researched and several processes patented
during the last 20 years (Nomura et al., 1987; Siebold, et al., 1995). Although it is actively
being researched (Karklins et al., 1996; Moresi and Sappino, 1996), it is this authors opinion
that employing electrodialysis for citric acid recovery is not at the point of commercial
exploitation yet. The technology is proven, but the energy consumption does not yet offer a
competitive advantage (Novalic and Kulbe, 1996). Membrane filtration routes, solvent
extraction and adsorptive ion exchange seem more likely to succeed on a cost competitive
basis.
182
substrates such as glucose syrups, to avoid the extraction of impurities in beet and cane
molasses. Once again, with increasing environmental pressure on reducing effluents, this
might become an economically viable alternative to the classical lime precipitation route.
Presenting a discussion of solvent extraction principles is beyond the scope of this text.
Suffice it to say that processes utilizing butan-2-ol tributyl phosphate plus kerosene and
tertiary amines have been published (Melsom and Meers, 1985). A further simplification of
the flow sheet would be direct extraction from the fermentation broth (Stuckey, 1997),
which is currently being researched.
11.14.1 Evaporation
At the evaporation stage, the process liquor will contain 15 to 20 per cent citric acid in
solution. It is an energy intensive operation and the efficient utilization of energy is an
important design consideration. The norm is to specify multiple effect evaporators, where
vapour from one effect is condensed in the subsequent unit re-boiler, with the process side
operated at a lower pressure. Mechanical vapour recompression can also be considered and
depending on the relative steam/electricity cost, is often economical at large capacities. The
types of evaporators employed vary, but forced circulation and falling film types have been
used successfully for a number of years. In planning the energy integration, care must be
taken to ensure that the citric acid will not be discoloured through exposure to high
temperature. Especially if a direct crystallization route is considered, trace amounts of residual
sugar are still present at the evaporation stage. This means that the temperature in the first
effect evaporator, where steam is used as the heating medium, must be limited to well below
100C. Such a constraint necessitates the use of low-pressure steam and also dictates the
vacuum required in subsequent stages.
Removing colour from the concentrated citric acid solution after the evaporator stage
presents practical problems and should be avoided where possible. As the solution is close
to saturation, the prevention of blockages due to crystals settling in a unit such as an activated
carbon column is cumbersome.
11.14.2 Crystallization
Typically this is a two-stage operation, where the first stage is the final purification step.
The second crystallization must yield the correct crystal size distribution, according to the
specified customer requirement. Usually continuous forced circulation crystallizers, in line
with pusher centrifuges will be used. The mother liquor produced from the crystallizers can
be recycled through an adsorptive ion exchange unit, or utilized for the production of sodium
citrate. Alternatively, the acid can be recovered with the lime precipitation route.
In discussing the unit specification with a vendor, it will be necessary to stipulate if the
option of anhydrous and monohydrate acid is required. As the crystallization temperature of
monohydrate citric acid is lower, this impacts on the capacity of the vacuum ejectors/pumps.
183
11.17 In conclusion
The preceding paragraphs bear out the fact that plant design is largely a discipline generic
to the chemical engineering industry. However, it should also be stressed that fermentation
plants require unambiguous communication between the chemical engineer and
microbiologist. Provided the process engineer can correctly interpret the sometimes unusual
requirements of a living system, the application of sound engineering practice will ensure
a successful design.
184
11.18 References
ALLEN, D G and ROBINSON, C W, 1990. Measurement of rheological properties of filamentous
fermentation broths, Chemical Engineering Science, 45, 3748.
BARKER, T W and WORGAN, J T, 1981. The application of airlift fermenters to the cultivation of
filamentous fungi, European Journal of Applied Microbiology and Biotechnology, 13, 7783.
BEROVIC, M, 1991. Advances in aerobic bioreactor design, Chemical Biochemical Engineering
Quarterly, 5, 189192.
BOHDZIEWICZ, J and BODZEK, M, 1994. Ultrafiltration preparation of pectinolytic enzymes from
citric acid fermentation broth, Process Biochemistry, 29, 99107.
BOSS, F C, 1983. Filtration. In Fermentation and Biochemical Engineering Handbook, ed. H C VOGEL
(Noyes Publications).
CHERYAN, M, 1986. Ultrafiltration Handbook (Technomic Publishing Company Inc.).
CHISTI, M Y, 1989. Airlift bioreactors (Elsevier Applied Science).
CHOI, P B, 1990. Designing airlift loop fermenters, Chemical Engineering Progress, December, 32
37.
COULSON, J M and RICHARDSON, J F, 1983. Chemical Engineering, Volume Two (Pergamon
Press).
DANIEL, ST and BRAUER, H, 1994. Continuous production of citric acid in the reciprocating-jetbioreactor, Bioprocess Engineering, 11, 123127.
DECHOW, F J, 1983. Ion exchange. In Fermentation and Biochemical Engineering Handbook, ed. H
C VOGEL (Noyes Publications).
DECKWER, W, 1985. Bubble column reactors. In Biotechnology, Volume 2, ed. H BRAUER, (VCH).
ENZMINGER, J D and ASENJO, J A, 1986. Use of cell recycle in the aerobic fermentative production
of citric acid by yeast, Biotechnology Letters, 8, 712.
ERNST, E E and MCQUIGG, D W, 1992. Adsorptive purification of carboxylic acids, presented at
AIChE meeting, Miami, November.
GYURE, D C, 1992. Set realistic goals for cross-flow filtration, Chemical Engineering Progress,
November.
HUDCOVA, V, MACHON, V and NIENOW, A W, 1989. Gas-liquid dispersion with dual Rushton
turbine impellers, Biotechnology and Bioengineering, 34, 617628.
KARKLINS, R, SKRASTINA, I and LEMBA, J, 1996. Electrodialysis method in citric acid and its
salts recovery process. Presented at Advances in Citric Acid Technology, Bratislava, Slovakia.
KULA, M, 1985. Recovery operations. In Biotechnology, Volume 2, ed. H BRAUER (VCH).
LIVINGSTONE, S and SPARKS, L, 1994. The new German packaging laws: effects on firms exporting
to Germany, International Journal of Physical Distribution & Logistics Management, 24, 15
25.
MASHELKAR, R A, 1970. Bubble columns, British Chemical Engineering, 15, 274281.
MCFARLANE, C M, ZHAO, X and NIENOW, A W, 1995. Studies of high solidity ratio hydrofoil
impellers for aerated bioreactors, Biotechnology Progress, 11, 608618.
MELSOM, P E and MEERS, J L, 1985. Citric Acid. In Comprehensive Biotechnology, Vol. 3, ed. M
MOO-YOUNG (Pergamon Press).
MITARD, A and RIBA, J P, 1988. Morphology and growth of Aspergillus niger ATCC 26036 cultivated
at several shear rates, Biotechnology and Bioengineering, 32, 835840.
MOCKEL, H O and WOLLECHENSKY, E, 1990. Modelling of the calculation of the power input for
aerated single- and multistage impellers with special respect to scale-up, Acta Biotechnology, 10,
215224.
MORESI, M and SAPPINO, F, 1996. Effect of temperature and pH on sodium citrate recovery from
aqueous solutions by electrodialysis, Presented at Advances in Citric Acid Technology, Bratislava,
Slovakia.
NOMURA, Y, IWAHARA, M and HONGO, M, 1987. Lactic acid production by electrodialysis
fermentation using immobilized growing cells, Biotechnology and Bioengineering, 30, 788
793.
NOVALIC, S and KULBE, K D, 1996, Separation and concentration of citric acid by means of
electrodialytic bipolar membrane technology, presented at Advances in Citric Acid Technology,
Bratislava, Slovakia.
OLDSHUE, J Y, 1985. Transport phenomena, reactor design and scale-up, Biotechnology Advances,
3, 219237.
185
Index
absorption 142
absorptive purification 181
activated carbon 139
acyl CoA synthetase 389
adenine nucleotides 43
adsorption 142
aerated systems 172
aeration 5
agitation effects 70
air-lift bioreactor 129, 131, 174
alcohol dehydrogenase 37
alcohols 37
aldehyde dehydrogenase 37
aliphatic alcohols 141
alkali metal salts 138
alkanes 6, 157 uptake 35
alkylsuphoxides 141
alternative oxidase 123
ammonia 20, 158
anion exchange resins 142
approaches to design 174
arabinose 151
aspergillus model 107
available electron balance 130
axial impellers 173
beet molasses 1513 microelements 152
vitamins 153 amino acid content 152
betaine 151
bipolar membranes 144
blackstrap 153
bubble column reactor 174
butanol 139
cane molasses 1535 vitamins 153
composition 154
caramel 151
carbon balance 130
carbon content of substrates 150
Carmen-Kozeny equation 179
188
downstream processing 13546
economics 1
effective viscosity 175
effluent 8, 183
electrodialysis 138, 144, 181 scheme 145
electron activity 87, 88
elemental composition 124
elementary balances 122
energy balance 121
energy consumption 144
energy yield coefficients 128
equilibrium distribution coefficient 141
esters 141
ethers 141
ethylenediamineacetic acid 155
evaporation 182
exchange potential 180
fats 158
fermentation aspects of design 170
filter types 177
fixed bed filter 142
formaline 156
four compartment cell 144
Froude number 173
fructose-2, 6-bisphosphate 19
gas hold-up 175
genes 1519, 44
geometric similarity 172
gluconic acid 12, 58
glucose oxidase 12
glucose uptake 20
glutamate 49
glycerol 22, 49
glycolytic pathway 12, 58
glyoxylate cycle 39
growth kinetics 127
Hagen-Poiseuille equation 178
heat removal 3
heat transfer 170, 175
hydrocarbons 149
hydrol 157
idiophase 117, 119, 123
immobilization 5, 145
initial conditions model 113
inoculum 4
ion exchange 1801, 1389, 142
iron 81
isocitric acid 59
isocitrate lyase 41
kestose 151
ketones 141
Index
kinetic modelling 105
Kjrgaard equation 87
K a 172
L
Kozeny
constant 179
lemon 1
lime 1367
linear growth model 1067, 118
liquid membranes 143
log growth model 1067
logistic growth equation 113
Ludeking-Piret equation 1078, 113, 119
magnesium 81
manganese 21, 22, 25, 5960, 81
mannose 151
market 2, 9
marketing information 164
mass balance 121, 126
mass transfer 171
mass transfer correlations 176
mass yield coefficient 122
mechanistic models 105
melanoidines 151
membrane applications 178
metabolic control analysis 612, 106
metabolic description of A. niger growth 123
metathesis reaction 180
methyl citrate 401
methyl isocitrate 401
microelements 158
microfiltration 178
microporous hollow fibres 143
mitochondria 22
mixed biomass 111
modelling 10519
molasses 1506 colour 151 microflora 154
non-volatile compounds 151 pH 153
volatile compounds 151 nitrogen
compounds 151, 153
Monod equation 106, 111
monopotassium phosphate 158
morphology 5, 23, 60 A. niger 71 dissolved
oxygen 81 effect of carbon source 75 effect
of inoculum 82 effect of nutritional factors
74 effect of pH 79 initial glucose
concentration 75 nitrogen limitation 78
phosphate level 78 trace metal levels 81
mutagenesis 56
mycelium formation 126
NADH oxidation 23, 623
NADH: ubiquinone oxidoreductase 63
nanofiltration 145, 178
neokestose 151
Nernst equation 87, 98
new technology in design 167
nitrogen metabolism 49
Index
nitrogen source 158
nomenclature 85, 121, 126, 1613
non-aerated power input 171
non-ionic resins 142
non-Newtonian behaviour 175
oils 158
organophosphorus compounds 141
oxalate biosynthesis 15
oxalic acid 13, 589 removal 135
oxidation potential 87, 8
oxidative phosphorylation 124
oxygen yield coefficient 128
oxygenation 3
parasexual cycle 60
pectinolytic enzymes 145
pellets 5, 69
pentose phosphate pathway 12
peroxisomes 3840
pH 3, 6, 23, 24
phase related model 117
phosphofructokinase overexpression 64
phosphorus source 158
Poiseuille equation 177
polyhydric alcohols 131
polysulphone 145
potassium ferricyanide 91
potassium ferrocyanide 155
power law 175
precipitation 136 flow sheet 137
pre-treatment 144 submerged fermentation
135 surface process 135
process economics 149
process flowsheet 169
process package 167
product drying 183 isolation 176 packaging
183
purification 178
pyruvate kinase overexpression 64
QSSA 125
raffinose 151, 153
raw materials 169
redox dyes 88
redox electrodes 88 calibration 88
redox potential 85 in citric acid fermentation
915 measurement 88 optimal 102
regulation 95 significance 89 theory 87
time course 923 dissolved oxygen
relationship 90 temperature change 94
redox regulation chemical 97 physical 97100
refined sucrose 156
regime analysis 173
regulatory enzymes 18
regulatory network 14
respiratory chain 62, 63
189
reverse osmosis 145
Rushton impeller 173
Rushton turbine 173
scale down 173
scale up 101, 170, 172
scope definition 1678
seeding 138
shear 70
shear rate 173, 175
simple structured model for A. niger 107
solvent extraction 13941, 181
starch 58, 157
sterilisation 155
stirred tank reactor 71, 170 design 171
stoichiometry 124
structured models 105
submerged method 150
substrate level phosphorylation 128
substrate preparation 169
substrates 149
sucrose 151, 153
sulphamide 156
superficial gas velocity 176
surface method 150
syrups 156
technical data in design 165
teratogenic effect 142
three-compartment cell 144
transcriptional regulation 1516
transport costs 150
treatment of molasses 155
therapies-6-phosphate 20, 64
tricalcium citrate 136, 137
tricarboxylate transporter 22
tri-n-butylphosphate 141
trioctylphosphine oxide 141
trophophase 11718, 123
tubular loop reactor 71
ultrafiltration 139, 1456, 178
unstructured models 105
vacuum evaporation 138
wastes 136
water 159
water-soluble amines 141
xylose 151
yeast based models 113
yeasts 6
yield coefficients 1067, 113, 1259
zinc 81