DOT I DOT I rnbarl
C. Application of Byproducts
The procedures for the recovery of citric acid lead to an appreciable accumulation of byproducts, which are mainly mycelium, gypsum, and the waste fluid. We have previously mentioned that the mycelium can be dried and used as an animal feedstock or as an additive to fertilizers, provided digestible filter aids have been used. Recently an attractive proposal has been made for using the waste mycelium as a source of chitin for use as a biosorbent.'93 A chitosan-glucan complex was obtained by boiling mycelia in 30 to 40% NaOH for 4 to 6 hr; the complex was reported to display better chelating properties than animal chitosan.
The A . niger chitosan also has film-forming and coagulating properties of potential interest.
Little use has hitherto been made of the concentrated waste fluid. Recently several hy- drolytic enzymes have been reported to be formed and excreted into the fermentation medium during citric acid fermentation (e.g., pectinase, protease, cellulase, P-glucosidase). 194-1y6
From a biochemical point of view, this is surprising since the low pH and the high carbo- hydrate content of the medium would be expected to lead only to low levels of such enzymes
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364 CRC Critical Reviews in Biotechnology Table 13
APPLICATIONS OF CITRIC ACIDim
Food uses
Beverages, jellies, jams, preserves,
frozen food, fats and oils (as an antioxidant), anticoagulants
Pharmaceuticals
Solubilizing action for cathartics and analgesics, agent of effervescence, medicinal flavor, anticoagulant Cosmetics and toiletries
Industrial application
pH-adjustment, antioxidant, buffer
Metal cleaning purposes, detergents (phosphate replacement), agricultural remedy of metal ion deficiencies, dispersing agent in preparation of mineral or pigment slurries, electrolytic and nonelectrolytic deposition of metals, sulfur dioxide absorption and flue gas desulfurication, buffering agent in photography, oil well treatments, cements, textiles, lengthening setting times in concrete preparation, binding agent for refractory cements, primer for polyamide adhesives, preventing precoupling of diazocouplers, starter or chelator in polymerizations,
cieaning agent in waste water treatment by reverse osmosis
due to denaturation; enzymes formed under these conditions should thus dispiay unusuaI and interesting properties and be suitable for special purposes. However, calculations have to be made whether their separation from the culture broth before citrate precipitation is economically reasonable.
The gypsum obtained is usually heavily contaminated with impurities (HCF, charcoal, organic compounds from the molasses) and thus unsuitable for commercial purposes, there- fore, it is usually collected €or waste deposit.
VII. PROCESS KINETICS A. Kinetics of Citric Acid Fermentation by A. niger
The knowledge of process kinetics is an indispensable prerequisite both in the attempt to understand it, as well as for trials in process control and optimization. Citric acid production has long been the classical example of Gaden’s “Type 11” fermentati~n,’~’ which is char- acterized by the fact that both growth rate as well as product formation rate exhibit two maxima, one during growth (‘ ‘tropho”-) phase with active biomass production and little product formation, and one during stationary (“idio”-) phase with little growth and maximal product formation. Gaden’s classification was originaIIy based on Shu and Johnson’sB pioneering shake flask studies, which contained relativeIy few data points (in total, eight points for dry weight and eight points for citric acid concentration over a period of 240 hr).
Within this hiphasic behavior of biomass and product formation rates, claimed by CadenIg8 as typical for Type I1 fermentation, a significant ”second” growth rate between 80 and 150 hr of cultivation was apparent in Shu and Johnson’s data. Other workers also confirmed this unusuaI growth pattern. ‘99 During the authors’ investigations on pilot plant citric acid fer- mentation,*’” however, the second increase in dry weight could be attributed to considerabIe adhesion of extracellular material to the hyphae, which is not released by unusual washing procedures during dry weight determinations, but which can conveniently be removed by washing steps employing solutions with higher ionic strength- If this is done, a typical conventional growth curve is obtained (Figure 16), which exhibits o d y one fast-growth phase and one stationary phase. The growth curve so shown resembles more ctosely the
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Volume 3, Issue 4 365
0
2
100
c
U50 100 150
hrs
FIGURE 16.
biomass concentration; (A) citric acid concentration.””“
Typical example of growth and citric acid accumulation by A. niger in submerged culture; (0)
physiological state of the mycelium, as has been shown by RNA or soluble protein meas- urements.20k.201a A reexamination of the kinetics of citric acid fermentation has thus been peformed by Rohr et al. ,’O” using a pilot plant process with a high data point frequency (At of two data points maximally 2 hr). Thereby it was found that the process is characterized by the following features: in the phase of fast growth (trophophase), mycelial growth can conveniently be described by a sequence of logarithmic, cube root, and linear growth models, but there is also significant product formation directly depending on the growth rate; during stationary phase (idiophase), product formation is maximized, depending on the biomass concentration, but hardly any growth occurs. This general description is reminiscent of the Luedeking-Piret modelzo2 of microbial product formation, which has also been applied by Kristiansen and Sinclair’” in the description of citric acid production by A. phmnicis. In the course of our work as described above, however, some modifications were found nec- essary. The Luedeking-Piret model was only applicable to citric acid fmnentation when a certain “lag time” was taken into account for the hyphae to enter the physiological state to produce citric acid. (This “lag time” was originally introduced as “maturation time” by Brown and VassZo3 to describe antibiotic fermentations.) The folIowing equation thus de- sm*bes the time course of citric acid fermentation:
where P = citric acid concentration, g/t; X = biomass concentration, g / t ; t, is the lag time needed for a cell to enter the physiological state of citric acid production; and k, and k, are constants, 1.9 and 0.09, respectively. For determination oft,, see Reference 203.
Values of 15 hr have been obtained for A. niger citric acid fermentation, which can be interpreted in terms that A. niger hyphal cells must become subapical to produce citric acid (15 hr is approximately the generation time of A. niger under these conditions). By consid- ering the multicellular nature of a filamentous fungus like A. niger, it can also be explained why the simple Luedeking-Piret model, originaIIy devised for unicelluhr bacterial fermen- tations, is only roughly applicable to A. niger. Considering this, fdP/dt) may be expressed as
I-
-
&
0
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366 CRC Critical Reviews in Biotechnology
(dP/dt) * (dP/dS)