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Effect of pH and stirring rate on itaconate production by Aspergillus terreus
Journal of Biotechnology 83 (2000) 219 – 230 www.elsevier.com/locate/jbiotec
Effect of pH and stirring rate on itaconate production by Aspergillus terreus Emanuele Riscaldati a, Mauro Moresi a,*,1, Federico Federici, Maurizio Petruccioli b b
a Istituto di Tecnologie Agroalimentari, Uni6ersity of Tuscia, Via S. Camillo de Lellis, 01100 Viterbo, Italy Dipartimento di Agrobiologia ed Agrochimica, Uni6ersity of Tuscia, Via S. Camillo de Lellis, 01100 Viterbo, Italy
Received 28 January 2000; received in revised form 5 June 2000; accepted 28 June 2000
Abstract The production of itaconic acid from glucose-based media by Aspergillus terreus NRRL 1960 was found to be controlled by stirring rate and pH. When the phosphorous (P) level in the production medium was reduced to less than 10 mg l − 1, the fungal mycelium exhausted its primary growth and started to excrete itaconic acid, while it continued its secondary growth at the expense of ammoniacal nitrogen. The fermentation exhibited a mixed-growthassociated product formation kinetics, the non-growth associated production term (mI) being practically zero only when the pH was left free to change from 3.4 down to 1.85. On the contrary, when the pH was kept reducing up to a constant value by automatic addition of KOH 4 mol l − 1, the itaconate yield coefficient on the initial glucose supplied (YI/So) and mI and were 0.53 g g − 1 and 0.028 h − 1 at pH 2.4 and 320 rev min − 1 and 0.5 g g − 1 and 0.036 h − 1 at pH 2.8 and 400 rev min − 1, respectively. Although the differences between mI and YI/So were statistically insignificant at the 95% confidence level, the net difference in the corresponding yield coefficients for itaconic acid on mycelial biomass resulted in a maximum itaconate production rate of 0.41 g l − 1 h − 1 at pH 2.8 and 400 rev min − 1, thus showing that this operating condition is no doubt optimal for the process under study. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Itaconic acid; Production; Aspergillus terreus; pH; Stirring rate; Mathematical modelling
1. Introduction
* Corresponding author. Tel.: + 39-0761-357494; fax: +390761-357498. E-mail address: [email protected] (M. Moresi). 1 Present address: Istituto di Tecnologie Agroalimentari, University of Tuscia, Via San Camillo de Lellis, 01100 Viterbo, Italy.
Owing to its conjugated double bonds and two carboxyl groups, itaconic acid is a very reactive compound and is used as monomer or comonomer for plastics, resins, synthetic fibers and elastomers (Tate, 1981; Milson and Meers, 1985). Laboratory — and pilot-plant production of itaconic acid by surface — and submerged-cul-
0168-1656/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 5 6 ( 0 0 ) 0 0 3 2 2 - 9
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ture fermentations was firstly reported by the Research Laboratories of the US Department of Agriculture (Lockwood and Ward, 1945; Nelson et al., 1952) and the strain Aspergillus terreus NRRL 1960 has been so far used as a target microorganism for optimising the fermentation process. Typical yields of 45 – 54 g of itaconic acid per 100 g of glucose supplied are generally obtained in 4–6 days of fermentation (Nelson et al., 1952). Nowadays, itaconic acid is produced by submerged culture fermentation with A. terreus in a medium containing molasses as the sugar source at 32–40°C and pH of 1.8 – 4.0 under 0.25 – 0.5 volumes of air per volume of medium per minute (l l − 1 min − 1) for 48 – 72 h (Milson and Meers, 1985; Roehr and Kubicek, 1996). The mycelial growth is sensitive to the ferrous ion content and limited by the phosphate concentration in the fermentation medium (Roehr and Kubicek, 1996). Several byproducts, such as erythritol and a-ketoglutaric, L-malic, succinic and itatartaric acids, have been reported to be produced during the itaconic acid fermentation (Larsen and Eimhjellen, 1955; Kobayashi, 1967). However, their formation can be suppressed by controlling the pH of the medium at 3.0 – 5.0 once the itaconate concentration reaches 4.6% (Batti, 1964). Although several studies have been so far carried out to optimise the fermentation process (Milson and Meers, 1985; Gyamerah, 1995; Roehr and Kubicek, 1996; Yahiro et al., 1997), some operating parameters are not fully defined yet. In fact, their so called optimal values vary in quite a large range and, sometimes, are even discordant (Roehr and Kubicek, 1996). As for the pH, for instance, good itaconic acid yields have been claimed on condition that the pH of the production medium be kept at 1.8 – 2.2 (Larsen and Eimhjellen, 1955; Elnaghy and Megalla, 1975) or 2.9 – 3.5 (van der Westhuizen et al., 1951; Rychtera and Wase, 1981). The effects of dissolved oxygen concentration (DO) and stirring speed at pH 2.0 were assessed by Park et al. (1993). A maximum yield coefficient of 0.55 g of itaconic acid per g of glucose consumed was obtained provided that DO was about 20% of the saturation value and the peripheral
speed (6I) (60-mm Rushton impellers) was about 0.94 m s − 1, equivalent to a stirring rate of 320 rev min − 1. Any increase or reduction in stirring rate would lower the itaconate production because of either mechanical damage of mycelial structure or insufficient oxygen transfer rate. On the contrary, Rychtera and Wase (1981) found that the best conditions for itaconate production were a pH between 2.9 and 3.1 and a peripheral impeller speed of 0.71 m s − 1. The main aim of this work was to maximise itaconic acid yield and productivity with respect to pH and stirring rate. Therefore, a series of fermentations was firstly carried out to assess the effect of pH on the itaconic fermentation under constant stirring rate. Secondly, another series of trials was designed to assess the effect of stirring speed at the optimal pH suggested by Rychtera and Wase (1981). Finally, mathematical modelling of itaconate formation kinetics was used to confirm the optimal operating conditions for this production.
2. Materials and methods
2.1. Microorganism A. terreus NRRL 1960 was obtained from the culture collection of the North Regional Research Laboratory (Peoria, IL). During this work, the culture was maintained on malt extract agar at 4–6°C and subcultured every month.
2.2. Apparatus A 15-l fermenter (Applikon, Schiedam, NL) with internal diameter 0.211 m and two turbines with six flat blades (diameter 75 mm) was used to study the fermentation process. The fermenter was equipped for the control of stirrer rate, temperature, pH, dissolved oxygen (DO) and air flow. Foaming or pH, when needed, was automatically controlled by adding a silicon antifoam (Antifoam A, Fluka) or an alkaline solution (4 mol l − 1 KOH), respectively.
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2.3. Culture medium and growth conditions The medium A for vegetative seed culture consisted of (per litre of de-ionised water): glucose, 20.0 g; (NH4)2SO4, 0.47 g; KH2PO4, 0.022 g; MgSO47H2O, 0.415 g; CaCl2 2H2O, 0.026 g; 5H2O, 0.04 mg; NaCl, 0.015 g; CuSO4 FeSO47H2O, 1.1 mg; MnCl24H2O, 0.14 mg; ZnSO47H2O, 0.25 mg. The production medium B contained (per litre of de-ionised water): glucose 100.0 g; (NH4)2SO4, 2.36 g; KH2PO4, 0.11 g; MgSO47H2O, 2.08 g; CaCl22H2O, 0.13 g; NaCl, 0.074 g; CuSO4 5H2O, 0.2 mg; FeSO4 7H2O, 5.5 mg; MnCl2 4H2O, 0.7 mg; ZnSO4 7H2O, 1.3 mg. The pH of medium A or B was adjusted to 4.2 or 3.4, respectively. All media were sterilised at 121°C for 20 min. For inoculation, 5 ml of spore suspensions containing about 4× 107 spores ml − 1 were transferred into 1-l Erlenmeyer flasks containing 200 ml of sterile medium A and incubated on a rotary shaker at 200 rev min − 1 and 35°C for 24 h. Then, 1 l of the vegetative seed culture were aseptically transferred to the fermentation vessel previously filled with 9 l of sterile medium B. The itaconate fermentation trials were performed batchwise under the following operating conditions: 35°C, initial pH 3.4, and 0.4 l l − 1 min − 1. During the itaconate production, the pH tended to reduce and was left free to change up to a final value of 1.85. Alternatively, as the pH was 1.95, 2.2, 2.4 or 2.89 0.05 pH units, it was kept constant by automatic addition of 4 mol l − 1 KOH. If not indicated otherwise, the stirring rate was 320 rev min − 1, this being equivalent to a peripheral impeller speed of 1.256 m s − 1. Any experimental trial was replicated twice, thus involving a coefficient of variation (i.e. the ratio between the standard deviation and sample mean) for all stoichiometric and kinetic parameters estimated here ranging from 0.05 to 0.1.
2.4. Analytical methods Samples were withdrawn at intervals of 6 – 8 h, diluted 1:5 with boiling distilled water, and filtered on pre-weighed Whatman GF/D discs to recover the mycelial biomass. The solid fraction
221
was washed twice with distilled water, dried at 105°C for 24 h and weighed to yield the mycelial biomass (X) concentration. Residual glucose (S) in the supernatant was spectrophotometrically measured by the DNSA method (Miller, 1959), while residual ammoniacal nitrogen (N) and phosphorous (P) in the supernatant were spectrophotometrically assayed as described by Strickland and Parson (1972). Itaconic acid (I) in the supernatant was determined by using a Violet Instruments (Rome, Italy) high-performance liquid chromatograph. The apparatus was equipped with a high-pressure volumetric pump (type Clar 003, Violet Instruments, Rome, Italy), a UV detector (Violet SP 430) and a 20-cm reverse phase LiChrospher, 100 RP-8, 5 mm (Merck, Darmstadt, Germany) column at room temperature. A buffer, 0.05 mol l − 1 KH2PO4 at pH 3, was used as eluent at a flow rate of 1.2 ml min − 1 and itaconic acid detection was performed at 210 nm.
3. Results and discussion To assess the optimal operating conditions for the itaconate production by A. terreus NRRL 1960 on glucose-based media, two series of fermentation trials were performed under constant stirring rate and pH, respectively.
3.1. Effect of pH Several fermentation trials were performed by varying the pH in the range 1.85–2.8 under a constant stirring rate of 320 rev min − 1. Despite the corresponding peripheral impeller speed (1.256 m s − 1) was greater than that (0.942 m s − 1) suggested by Park et al. (1993), such agitation rate was chosen to enhance mixing and oxygen transfer, being the working volume of the stirred fermenter used here 5 times greater than that used by Park et al. (1993). The mean values of the main results [initial (ti) and final (tf) times of the itaconate production phase together with the corresponding concentrations of mycelial biomass (Xi, Xf), glucose (SiF, SfF) and itaconic acid (Ii, If), and estimated values of itaconate production (RI), glucose consumption
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(RS) and mycelial growth (RX) rates and of yield coefficients for itaconic acid on glucose consumed (YI/S) or supplied (YI/So) and for mycelial biomass on glucose consumed (YX/S)] of all these fermentation runs are shown in the upper section of Table 1. As reported in Section 2, the coefficient of
Fig. 1. Time course of itaconic acid production by Aspergillus terreus NRRL 1960 at 320 rev min − 1 when the pH was left free to change: (a) Concentrations of glucose (S : ), itaconic acid (I : ") and mycelial biomass (X : ) as a function of the fermentation time (t); (b) Concentrations of dissolved oxygen (DO : D), ammoniacal nitrogen (N : ) and phosphorous (P : ), and pH (2) versus t. The continuous lines were calculated by using Eqs. (6) and (7) together with the empirical coefficients listed in Table 2.
variation for all the estimated parameters in Table 1 fell within 0.05 and 0.1. Itaconic acid yield (YI/S) and productivity (RI) appeared to be function of the pH of the medium and varied in the ranges of 0.33–0.63 g per gram of glucose consumed and 0.12–0.25 g l − 1 h − 1, respectively. Of the two itaconate yield coefficients estimated here, that on the initial glucose supplied (YI/So) appeared to be the most suitable for assessing the optimal pH for the fermentation process. When the pH was uncontrolled and reached a final value of 1.85, the acidity of the medium markedly limited the fungal utilisation of the glucose initially supplied and YI/So was only 0.21 g g − 1. On the contrary, when the pH was controlled at 2.4 the glucose was practically exhausted at the end of the fermentation (Table 1) and YI/So was 0.53 g g − 1. Figs. 1 and 2 illustrate the typical time course of the itaconate fermentation under the operating conditions mentioned above. As shown in Figs. 1a and 2a, after about 24 h of fermentation, the phosphorous (P) concentration in the production medium fell to about 10 mg l − 1 and the fungus started to excrete itaconic acid. During the production phase, the nitrogen (N) concentration continued to decrease from about 20 to less than 1 mg l − 1, while mycelial biomass increased from 3 to 10–11 g l − 1. The rapid phosphorous consumption during the first 24 h was accompanied by a dramatic fall in dissolved oxygen (DO) from 100 to 10–15% of the saturation value. After that time, DO remained almost constant up to the overall exhaustion of the nitrogen source, then started to increase again, thus tending to an equilibrium value when all mycelial metabolic activity was completely exhausted (Figs. 1b and 2b). Despite the above optimal pH of 2.4 is in line with several other reports (Larsen and Eimhjellen, 1955; Elnaghy and Megalla, 1975), it is worth noting that the itaconate yield coefficient (YI/S = 0.33) determined here (when using a 75-mm Rushton-type turbine operating at 6I = 1.256 m s − 1) was half that (YI/S = 0.64) obtained by Rychtera and Wase (1981), who worked at a pH of 2.9–3.1 with a 85-mm single flat-blade impeller operating at 6I = 0.71 m s − 1.
a
24.5 22.0 26.5 23.0 19.0 23.0 15.0 22.0
1.85a 1.95 2.2 2.4 2.8 2.9 2.8 2.8 165.0 141.0 209.0 251.0 137.0 166.5 121.0 128.5
tf (h)
3.1 2.5 2.6 1.9 3.0 4.5 2.6 4.0
XI (g l−1) 11.2 11.8 8.6 10.6 8.0 10.7 9.3 9.7
Xf (g l−1) 78.0 86.7 73.6 107.4 75.0 85.0 86.0 86.0
SiI (g l−1) 36.8 48.1 17.0 1.5 24.5 3.0 5.5 13.0
SfI (g l−1) 0.00 0.04 1.50 0.00 1.04 0.50 0.00 0.80
Ii (g l−1) 16.5 21.0 36.9 57.2 17.7 25.2 43.1 33.1
If (g l−1)
0.12 0.18 0.19 0.25 0.14 0.17 0.41 0.30
RI (g l−1 h−1)
Referred to the final pH of the medium when the fermentation was carried out with no pH control.
320 320 320 320 320 240 400 480
tI (h)
pH (−) Stirring rate (rev min−1)
-0.29 -0.32 -0.31 -0.46 -0.43 -0.57 -0.76 -0.69
RS (g l−1 h−1)
0.06 0.08 0.03 0.04 0.04 0.04 0.06 0.05
RX (g l−1 h−1)
0.40 0.54 0.63 0.54 0.33 0.30 0.54 0.44
YI/S (g g−1)
0.21 0.24 0.48 0.53 0.22 0.29 0.50 0.38
YI/So (g g−1)
0.20 0.24 0.11 0.08 0.10 0.08 0.08 0.08
YX/S (g g−1)
Table 1 Mean values of the main results of several itaconic acid production by A. terreus NRRL 1960 replicated twice: Effect of pH and stirring rate on the initial (ti) and final (tf) times of the itaconate production phase together with the corresponding concentrations of mycelial biomass (Xi, Xf), glucose (Si, Sf) and itaconic acid (Ii, If), and estimated values of itaconate production (RI), glucose consumption (RS) and mycelial growth (RX) rates and yield coefficient for itaconic acid on glucose consumed (YI/S) or initially supplied (YI/So) and for mycelium on glucose consumed (YX/S). The coefficient of variation for all these stoichiometric and kinetic parameters ranged from 0.05 to 0.1a
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Fig. 2. Time course of itaconic acid production by A. terreus NRRL 1960 at 320 rev min − 1 when the pH was left free to change up to 2.4: (a) Concentrations of glucose (S : ), itaconic acid (I : ") and mycelial biomass (X : ) as a function of the fermentation time (t); (b) Concentrations of dissolved oxygen (DO : D), ammoniacal nitrogen (N : ) and phosphorous (P : ), and pH (2) versus t. The continuous lines were calculated by using Eqs. (6) and (7) together with the empirical coefficients listed in Table 2.
3.2. Effect of stirring rate To establish if the above experimental result (YI/S =0.33) was due to insufficient oxygen transfer in the fermenter used, a second series of trials was carried out by setting the stirring rate to 240, 400 and 480 rev min − 1 (these values corresponding to peripheral impeller speeds of 0.94, 1.57 and 1.88 m s − 1) and pH to about the optimal value
suggested by Rychtera and Wase (1981). The mean values of their main results are given in the lower section of Table 1. By increasing the stirring rate from 240 to 480 rev min − 1, the yield coefficients YI/So and YI/S firstly increased reaching a maximum value of 0.50 and 0.54 g g − 1 at 400 rev min − 1, respectively, and then decreased. On the contrary, YX/S appeared to be independent of agitation rate and practically equal to 0.08 g g − 1. Fig. 3 shows the typical time course of the itaconate fermentation at pH 2.8 and 400 rev min − 1. By only increasing the stirring rate to 400 rev min − 1, it was possible to confirm previous findings by Rychtera and Wase (1981), thus revealing a significant dependence of the itaconate yields upon the aeration-agitation conditions within the fermenter used. Although there are a number of reports in the literature of hyphal damage due to high agitation intensity [see for general reference the well known paper by Dion et al. (1955) and more specifically for the fermentation under study that by Park et al. (1993)], these results clearly show that the mycelium was not damaged by the shear stress exerted by the impeller blades at peripheral impeller speeds up to 1.57 m s − 1. This is in strict agreement with van Suijdam and Metz (1981) results on the effect of agitation intensity on the length of mycelial hyphae and with Moresi et al. (1987) on the effect of 6I in the range 1.76–3.8 m s − 1 on the stoichiometry and kinetics of untreated orange peel utilisation by Fusarium a6enaceum. The fact that the strain used in this work at pH 2.8 exhibited a higher resistance to shearing forces than that used by Park et al. (1993) at pH 2.0 might be in all probability attributed to the higher pH of the fermenting media. To test whether the experimental mean values of the yield coefficients for itaconic acid on glucose supplied (YI/So), that were observed at pH 2.8 and 400 rev min − 1 and at pH 2.4 and 320 rev min − 1, were significantly different, their standardised normal difference [d= (Y1 − Y2)/ ((s 21/p1)+ (s 22/p2)) where Yj, sj, and pj are the mean value, standard deviation and overall number of trials for the variables tested] was compared with the classic inequality of the hypothesis test for means,
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[i.e. d5ta,f the one-sided Student t-test for a confidence level a and f ( =p1 +p2 −2) degrees of freedom] (Bennett and Franklin, 1954). In this way, for s1 :s2 =0.03, p1 =p2 =2 and t0.05,2 =4.3, it was possible to reject the hypothesis of inequality (d=1.0 B4.3) at a probability level of 0.05. Similarly, the same inequality test performed on the itaconate formation rate (RI) values obtained at 320 rev min − 1 and pH ranging from 1.95 to 2.8 (upper section of Table 1) yielded
225
that there was no statistically significant difference between the yields and rates examined, thus justifying the discordant literature indications about the optimal pH of the medium during the itaconate production phase (Larsen and Eimhjellen, 1955; Elnaghy and Megalla, 1975; Rychtera and Wase, 1981). However, when the stirring rate was increased from 320 to 400 rev min − 1 at pH 2.8, the itaconate formation rate (RI) was found to increase from 0.259 0.02 to 0.419 0.03 g l − 1 h − 1 (Table 1). Since the difference in RI (6.3\ 4.3) was statistically significant at the 95% confidence level, the kinetics of this fermentation is no doubt controlled by the effective oxygen transfer capability of the fermenter used. In conclusion, despite the operation at pH 2.4 and 320 rev min − 1 would seem to be more convenient because the smaller the stirring speed the smaller the power input per unit itaconate formed and the smaller the pH of the medium the smaller the alkaline reactant consumption and the microbial contamination risk, the operation at pH 2.8 and 400 rev min − 1 has to be regarded as the optimal one because of the greater itaconate formation rate.
3.3. Kinetics of itaconate production Among the numerous models reported in the literature to describe quantitatively the formation rate of a metabolic compound (Moser, 1985), the now classic Luedeking and Piret (1959), kinetics has so far proved extremely useful and versatile in fitting product formation data from many different fermentations: RI = dI/dt = YI/X(dX/dt)+mI(X)
Fig. 3. Time course of itaconic acid production by A. terreus NRRL 1960 at 400 rev min − 1 when the pH was left free to change up to 2.8: (a) Concentrations of glucose (S : ), itaconic acid (I : ") and mycelial biomass (X : ) as a function of the fermentation time (t); (b) Concentrations of dissolved oxygen (DO : D), ammoniacal nitrogen (N : ) and phosphorous (P : ), and pH (2) versus t. The continuous lines were calculated by using Eqs. (6) and (7) together with the empirical coefficients listed in Table 2.
(1)
where mI is the specific product formation rate at zero mycelial growth rate and YI/X the yield factor for itaconic acid on unit mycelial biomass. In particular, the first term indicates the product formation rate in association with mycelial growth rate; while the second one is the nongrowth associated product formation rate. To apply this two-parameter kinetic expression, it is necessary to know the mycelial growth rate (RX), which can be generally described as:
E. Riscaldati et al. / Journal of Biotechnology 83 (2000) 219–230
226
Fig. 4. Itaconic acid production by A. terreus NRRL 1960 at pH= 2.8 and 400 rev min − 1: logarithm of (X/Xo : ") and concentrations of phosphorous (P : ) and ammoniacal nitrogen (N : ) versus time. The continuous line was calculated by using Eq. (4) together with the empirical coefficients listed in Table 2.
RX = dX/dt =m(X)
(2)
where X is the instantaneous mycelial concentration and m is the actual specific cell growth rate which is a more or less complex function of several variables such as, for instance, the limiting substrate concentration in the case of the Monod equation or the maximum mycelial concentration in the case of the logistic equation (Moser, 1985). By assuming no explicit relationship for m, Eq. (2) can be integrated as ln
X = X0
&
t
m dt
(3)
0
If the mycelium exponentially duplicates, the specific mycelial growth rate will coincide with the maximum specific one and the plot of the natural logarithm of the actual to the initial mycelial concentrations against time will be a straight line passing through the origin of the plot. In this specific fermentation, the general plot of ln(X/X0) versus time had the pattern shown in Fig. 4, which may be approximated by using at least two different straight lines, the first and second ones fitting the experimental data at the beginning and at the end of the fermentation, respectively. This means that mycelial growth may
be roughly described as double-substrate growth (Moser, 1985), the first limiting substrate being the phosphorous and the second one the nitrogen (Fig. 4). In fact, the ln(X/X0) deviates from the first straight line having slope m1 as the P content in the medium is smaller than about 10 mg l − 1 (Fig. 4). When the mycelium has adapted to the new cultural condition, it reduces its instantaneous specific growth rate and starts to excrete itaconic acid. In the circumstance, being the medium still rich in carbon and nitrogen sources, the mycelial weight concentration X will continue to increase at the expense of another limiting substrate, that is the ammoniacal nitrogen. Such a secondary mycelial growth is characterised by a secondary specific growth rate m2, which was found to be about one tenth of the primary one (m1). This microbial behaviour is not specific of the A. terreus strain used, since it was already observed in Yarrowia lipolytica (Moresi, 1994) and A. niger (Verhoff and Spradlin, 1976; Briffaud and Engasser, 1979) during the citric acid formation and in Saccharomyces cere6isiae (Martini et al., 1976) during the aerobic growth, both on media rich in glucose and devoid of the nitrogen source. To describe such double-substrate growth, the following empirical regression was used: ln
X(t) q = %jbj (1−e − mj t) Xo 1
(4)
where q is the number of statistically significant limiting substrates, mj the j-th maximum specific mycelial growth rate and bj the j-th empirical regression coefficient. By using the same procedure developed by Nussinovitch et al. (1989) and modified by Mancini et al. (1999) to correlate stress relaxation data, it was possible to fit all the experimental data by assigning initially an arbitrary spectrum of reaction times: 1 tj = = a x10z − j mj
(5)
where a is an integer index ranging from 1 to 9 and z is an empirical exponent assigned to make the maximum reaction time coincident with the
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overall duration of the fermentation process. For instance, if the process lasts 300 h, z is equal to 4 so as to make the first reaction time (t1) varying from 1000 and 9000 h. By using a linear regression method, it was possible to discard firstly the reaction times, the contribution of which to the ln(X/Xo)-t relationship was statistically insignificant at the confidence level of 95%, and then to determine the optimal a value by maximising the coefficient of determinations (r 2) of Eq. (4). By applying the above operating procedure, there was no statistically significant improvement in fitting the time course of any experimental [ln(X/Xo)−t] data by considering more than two maximum specific cell growth rates in agreement with the aforementioned verbal kinetic model. Under the assumption that all the stoichiometric and kinetic parameters were constant, the above set of differential equations can be integrated, thus yielding the following: Á ÃX0 q X= Í ÃX0 exp %jbj (1− e − uj t) Ä 1 I = Io + mI
&
n
for t 5t0 for t \t0
(6)
t
X dt +YI/X (X − Xi)
(7)
tI
where the second term at the right hand side of Eq. (7) can be numerically integrated once X(t) is
227
known. By applying the analytical expressions given above in conjunction with the least squares method, it was possible to fit all the fermentation data collected and evaluate the kinetic and stoichiometric parameters listed in Table 2. The primary maximum specific cell growth rate (m1) ranged from 0.025 to 0.045 h − 1, while the secondary one (m2) was one tenth of m1. Moreover, the itaconate production by A. terreus NRRL 1960 exhibited a mixed-growth-associated product formation kinetics, the contribution of growth associated production being related to the secondary mycelial growth only and that of nongrowth associated production depending on pH and stirring rate. More specifically, at constant stirring rate of 320 rev min − 1 the specific product formation rate at zero mycelial growth rate (mI) was practically zero when the pH was left reducing without any control. This revealed a growth-associated product formation only when the mycelium was severely stressed by as low a pH as 1.85. By controlling the pH of the medium during the fermentation, mI progressively increased from as low as 0.004 h − 1 at pH 1.95 to a maximum value of 0.028 h − 1 at pH 2.4. At pH 2.8, mI was found to be also a function of the stirring rate, its maximum value (0.036 h − 1) being associated with a stirring rate of 400 rev min − 1. However, the difference in the last two maximum values of mI (d=3.1 B 4.3) was found to be statistically in-
Table 2 Main kinetic and stoichiometric parameters of several itaconic acid production by A. terreus NRRL 1960 replicated twice: Effect of pH and stirring rate on the primary (m1) and secondary (m2) maximum specific mycelial growth rates and their corresponding dimensionless contribution (bj ), specific product formation rate at zero mycelial growth rate (mI) and yield factor for itaconic acid on unit mycelial biomass (YI/X) together with their standard deviations and or coefficients of determinations (r 2)a pH (−)
1.85a 1.95 2.2 2.4 2.8 2.9 2.8 2.8 a
Stirring rate (rev min−1) 320 320 320 320 320 240 400 480
b1 (−)
m1 (h−1) b2 (−)
m2 (h−1) r 2
mI (h−1)
YI/X (g g−1)
r2
0.01 90.3 0.59 0.6 1.1 9 0.3 0.3 90.2 0.49 0.2 0.1 90.2 0.1 90.1 0.1 90.5
0.04 0.03 0.04 0.02 0.04 0.04 0.04 0.04
0.004 0.003 0.004 0.003 0.005 0.004 0.005 0.004
−0.00290.001 0.004 9 0.002 0.012 9 0.003 0.028 90.002 0.003 90.003 0.013 9 0.001 0.036 90.003 0.023 90.003
2.4 90.1 2.4 9 0.2 3.9 9 0.6 1.0 90.2 2.4 90.3 1.6 9 0.1 2.2 90.3 1.5 90.3
0.97 0.99 0.97 0.98 0.90 0.99 0.99 0.97
3.4890.09 3.659 0.2 1.329 0.12 2.989 0.07 1.83 9 0.08 2.009 0.08 2.199 0.05 2.2890.15
0.99 0.96 0.94 0.99 0.99 0.99 0.99 0.97
Referred to the final pH of the medium when the fermentation was performed with no pH control.
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significant at the 95% confidence level. On the contrary, at the same probability level the difference in the corresponding YI/X values (Table 2) resulted to be statistically significant (d= 4.7\ 4.3). Therefore, the net improvement in the RI value observed at pH 2.8 and 400 rev min − 1 with respect to that obtained at pH 2.4 and 320 rev min − 1 (Table 1) is most likely due to the difference in the yield coefficient for itaconic acid on mycelial biomass formed (Table 2). The continuous lines plotted in Figs. 1 – 3 were calculated by using Eqs. (6) and (7) together with the empirical coefficients reported in Table 2 and show a remarkable agreement between calculated and experimental X and I data. Therefore, all these results are consistent with the present hypothesis of mixed kinetics for itaconate production.
progressively increased from 0.004 h − 1 at pH 1.95 to 0.028 h − 1 at pH 2.4, even if such a maximum value was also associated with higher values of pH (2.8) and stirring rate (400 rev min − 1). Of these two operating conditions, the latter has to be definitively regarded as the optimal one for the itaconic acid fermentation owing to the greater product formation rate.
5. Notation a d DO I mI N
4. Conclusions P The discordant literature indications about the optimal pH of the medium during the production of itaconic acid from glucose-based media by A. terreus NRRL 1960 may be attributed to the fact that this fermentation is controlled by stirring rate and pH. A maximum yield coefficient (YI/S) of about 0.54 g of itaconic acid per g of glucose consumed was obtained under different operating conditions, such as pH 2.4 and 320 rev min − 1 or pH 2.8 and 400 rev min − 1, while their corresponding itaconate formation rates (RI) were 0.25 and 0.41 g l − 1 h − 1, respectively. This fermentation exhibited a mixed-growth-associated product formation kinetics, even if the contribution of non-growth associated production was mainly affected by the pH during the itaconate production phase and secondly by stirring rate. When no control of the pH was assured, the mycelium resulted to be so severely stressed as to stop itaconate production at pH 1.85, thus involving a strict growth-associated production kinetics. On the contrary, under pH control and constant stirring rate (320 rev min − 1) the specific product formation rate at zero mycelial growth rate (mI)
pj q r2 RI RS RX S sj t ta,f 6I X YI/S YI/So YI/X
integer index of Eq. (5). standardised normal difference between means. dissolved oxygen concentration (%). itaconic acid concentration (g l−1). specific product formation rate at zero mycelial growth rate (h−1). ammonia nitrogen concentration (mg l−1). phosphorous concentration (mg l−1). overall number of trials for the jth parameter tested. number of statistically significant limiting substrates. coefficient of determinations. itaconate production rate (g l−1 h−1). glucose consumption rate (g l−1 h−1). mycelial growth rate (g l−1 h−1). glucose concentration (g l−1). standard deviation of the j-th parameter tested. fermentation time (h). one-sided Student t-test. peripheral impeller speed (m s−1). mycelial biomass concentration (g l−1). yield coefficients for itaconic acid on glucose consumed (g g−1). yield coefficients for itaconic acid on glucose supplied (g g−1). yield factor for itaconic acid on unit mycelial biomass (g g−1).
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Yj YX/S
mean value of the j-th parameter tested. yield coefficients for mycelial biomass on glucose consumed (g g−1).
Greek symbols a confidence level. bj generic j-th empirical regression coefficient of Eq. (4). f degrees of freedom. m specific cell growth rate (h−1). mj generic j-th maximum specific mycelial growth rate (h−1). tj generic j-th reaction time (h). z empirical exponent of Eq. (5). Subscript o I f
referred to the initial value. referred to the beginning of the itaconate production phase. referred the end of the itaconate production phase.
Acknowledgements This research work was supported by a grant from the National Research Council (CNR) of Italy: Target Project on Biotechnology.
References Batti, M.A., 1964. Process for the production of itaconic acid. US Patent number, 3,162,582, (Dec. 22, 1964). Bennett, C.A., Franklin, N.L., 1954. Statistical Analysis in Chemistry and the Chemical Industry. Wiley, New York, pp. 176 – 185. Briffaud, J., Engasser, M., 1979. Citric acid production from glucose. I. Growth and excretion kinetics in a stirred fermentor. Biotechnol. Bioeng. 21, 2083–2092. Dion, W.N., Carilli, A., Sermonti, G., Chain, E.B., 1955. The effect of mechanical agitation on the morphology of Penicillium chrysogenum Thom in stirred fermenters. In: Rendiconto dell’Istituto Superiore di Sanita`, English, 17, 187 – 205. Elnaghy, M.A., Megalla, S.E., 1975. Itaconic acid production by a local strain of Aspergillus terreus. Eur. J. Appl. Microbiol. 2, 159 – 172.
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Strickland, J.D.H., Parson, T.R., 1972. A Practical Handbook of Seawater Analysis. Fisheries Research Board of Canada, Canada. Tate, B.E., 1981. Itaconic acid and derivatives. Kirch–Othmer. In: Mark, H.F., Othmer, D.F., Overberger, C.G., Seaborg, G.T. (Eds.), Encycl. Chem. Technol, vol. 13, third. Wiley, New York, pp. 865 – 873. van der Westhuizen, G.C.A., Spruit, C.J.P., Sephton, H.H, 1951. Itaconic acid. I. Production by Aspergillus terreus from unrefined cane sugar. J. Appl. Chem. 1, 356–360.
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.
Effect of pH and stirring rate on itaconate production by Aspergillus terreus Emanuele Riscaldati a, Mauro Moresi a,*,1, Federico Federici, Maurizio Petruccioli b b
a Istituto di Tecnologie Agroalimentari, Uni6ersity of Tuscia, Via S. Camillo de Lellis, 01100 Viterbo, Italy Dipartimento di Agrobiologia ed Agrochimica, Uni6ersity of Tuscia, Via S. Camillo de Lellis, 01100 Viterbo, Italy
Received 28 January 2000; received in revised form 5 June 2000; accepted 28 June 2000
Abstract The production of itaconic acid from glucose-based media by Aspergillus terreus NRRL 1960 was found to be controlled by stirring rate and pH. When the phosphorous (P) level in the production medium was reduced to less than 10 mg l − 1, the fungal mycelium exhausted its primary growth and started to excrete itaconic acid, while it continued its secondary growth at the expense of ammoniacal nitrogen. The fermentation exhibited a mixed-growthassociated product formation kinetics, the non-growth associated production term (mI) being practically zero only when the pH was left free to change from 3.4 down to 1.85. On the contrary, when the pH was kept reducing up to a constant value by automatic addition of KOH 4 mol l − 1, the itaconate yield coefficient on the initial glucose supplied (YI/So) and mI and were 0.53 g g − 1 and 0.028 h − 1 at pH 2.4 and 320 rev min − 1 and 0.5 g g − 1 and 0.036 h − 1 at pH 2.8 and 400 rev min − 1, respectively. Although the differences between mI and YI/So were statistically insignificant at the 95% confidence level, the net difference in the corresponding yield coefficients for itaconic acid on mycelial biomass resulted in a maximum itaconate production rate of 0.41 g l − 1 h − 1 at pH 2.8 and 400 rev min − 1, thus showing that this operating condition is no doubt optimal for the process under study. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Itaconic acid; Production; Aspergillus terreus; pH; Stirring rate; Mathematical modelling
1. Introduction
* Corresponding author. Tel.: + 39-0761-357494; fax: +390761-357498. E-mail address: [email protected] (M. Moresi). 1 Present address: Istituto di Tecnologie Agroalimentari, University of Tuscia, Via San Camillo de Lellis, 01100 Viterbo, Italy.
Owing to its conjugated double bonds and two carboxyl groups, itaconic acid is a very reactive compound and is used as monomer or comonomer for plastics, resins, synthetic fibers and elastomers (Tate, 1981; Milson and Meers, 1985). Laboratory — and pilot-plant production of itaconic acid by surface — and submerged-cul-
0168-1656/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 5 6 ( 0 0 ) 0 0 3 2 2 - 9
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ture fermentations was firstly reported by the Research Laboratories of the US Department of Agriculture (Lockwood and Ward, 1945; Nelson et al., 1952) and the strain Aspergillus terreus NRRL 1960 has been so far used as a target microorganism for optimising the fermentation process. Typical yields of 45 – 54 g of itaconic acid per 100 g of glucose supplied are generally obtained in 4–6 days of fermentation (Nelson et al., 1952). Nowadays, itaconic acid is produced by submerged culture fermentation with A. terreus in a medium containing molasses as the sugar source at 32–40°C and pH of 1.8 – 4.0 under 0.25 – 0.5 volumes of air per volume of medium per minute (l l − 1 min − 1) for 48 – 72 h (Milson and Meers, 1985; Roehr and Kubicek, 1996). The mycelial growth is sensitive to the ferrous ion content and limited by the phosphate concentration in the fermentation medium (Roehr and Kubicek, 1996). Several byproducts, such as erythritol and a-ketoglutaric, L-malic, succinic and itatartaric acids, have been reported to be produced during the itaconic acid fermentation (Larsen and Eimhjellen, 1955; Kobayashi, 1967). However, their formation can be suppressed by controlling the pH of the medium at 3.0 – 5.0 once the itaconate concentration reaches 4.6% (Batti, 1964). Although several studies have been so far carried out to optimise the fermentation process (Milson and Meers, 1985; Gyamerah, 1995; Roehr and Kubicek, 1996; Yahiro et al., 1997), some operating parameters are not fully defined yet. In fact, their so called optimal values vary in quite a large range and, sometimes, are even discordant (Roehr and Kubicek, 1996). As for the pH, for instance, good itaconic acid yields have been claimed on condition that the pH of the production medium be kept at 1.8 – 2.2 (Larsen and Eimhjellen, 1955; Elnaghy and Megalla, 1975) or 2.9 – 3.5 (van der Westhuizen et al., 1951; Rychtera and Wase, 1981). The effects of dissolved oxygen concentration (DO) and stirring speed at pH 2.0 were assessed by Park et al. (1993). A maximum yield coefficient of 0.55 g of itaconic acid per g of glucose consumed was obtained provided that DO was about 20% of the saturation value and the peripheral
speed (6I) (60-mm Rushton impellers) was about 0.94 m s − 1, equivalent to a stirring rate of 320 rev min − 1. Any increase or reduction in stirring rate would lower the itaconate production because of either mechanical damage of mycelial structure or insufficient oxygen transfer rate. On the contrary, Rychtera and Wase (1981) found that the best conditions for itaconate production were a pH between 2.9 and 3.1 and a peripheral impeller speed of 0.71 m s − 1. The main aim of this work was to maximise itaconic acid yield and productivity with respect to pH and stirring rate. Therefore, a series of fermentations was firstly carried out to assess the effect of pH on the itaconic fermentation under constant stirring rate. Secondly, another series of trials was designed to assess the effect of stirring speed at the optimal pH suggested by Rychtera and Wase (1981). Finally, mathematical modelling of itaconate formation kinetics was used to confirm the optimal operating conditions for this production.
2. Materials and methods
2.1. Microorganism A. terreus NRRL 1960 was obtained from the culture collection of the North Regional Research Laboratory (Peoria, IL). During this work, the culture was maintained on malt extract agar at 4–6°C and subcultured every month.
2.2. Apparatus A 15-l fermenter (Applikon, Schiedam, NL) with internal diameter 0.211 m and two turbines with six flat blades (diameter 75 mm) was used to study the fermentation process. The fermenter was equipped for the control of stirrer rate, temperature, pH, dissolved oxygen (DO) and air flow. Foaming or pH, when needed, was automatically controlled by adding a silicon antifoam (Antifoam A, Fluka) or an alkaline solution (4 mol l − 1 KOH), respectively.
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2.3. Culture medium and growth conditions The medium A for vegetative seed culture consisted of (per litre of de-ionised water): glucose, 20.0 g; (NH4)2SO4, 0.47 g; KH2PO4, 0.022 g; MgSO47H2O, 0.415 g; CaCl2 2H2O, 0.026 g; 5H2O, 0.04 mg; NaCl, 0.015 g; CuSO4 FeSO47H2O, 1.1 mg; MnCl24H2O, 0.14 mg; ZnSO47H2O, 0.25 mg. The production medium B contained (per litre of de-ionised water): glucose 100.0 g; (NH4)2SO4, 2.36 g; KH2PO4, 0.11 g; MgSO47H2O, 2.08 g; CaCl22H2O, 0.13 g; NaCl, 0.074 g; CuSO4 5H2O, 0.2 mg; FeSO4 7H2O, 5.5 mg; MnCl2 4H2O, 0.7 mg; ZnSO4 7H2O, 1.3 mg. The pH of medium A or B was adjusted to 4.2 or 3.4, respectively. All media were sterilised at 121°C for 20 min. For inoculation, 5 ml of spore suspensions containing about 4× 107 spores ml − 1 were transferred into 1-l Erlenmeyer flasks containing 200 ml of sterile medium A and incubated on a rotary shaker at 200 rev min − 1 and 35°C for 24 h. Then, 1 l of the vegetative seed culture were aseptically transferred to the fermentation vessel previously filled with 9 l of sterile medium B. The itaconate fermentation trials were performed batchwise under the following operating conditions: 35°C, initial pH 3.4, and 0.4 l l − 1 min − 1. During the itaconate production, the pH tended to reduce and was left free to change up to a final value of 1.85. Alternatively, as the pH was 1.95, 2.2, 2.4 or 2.89 0.05 pH units, it was kept constant by automatic addition of 4 mol l − 1 KOH. If not indicated otherwise, the stirring rate was 320 rev min − 1, this being equivalent to a peripheral impeller speed of 1.256 m s − 1. Any experimental trial was replicated twice, thus involving a coefficient of variation (i.e. the ratio between the standard deviation and sample mean) for all stoichiometric and kinetic parameters estimated here ranging from 0.05 to 0.1.
2.4. Analytical methods Samples were withdrawn at intervals of 6 – 8 h, diluted 1:5 with boiling distilled water, and filtered on pre-weighed Whatman GF/D discs to recover the mycelial biomass. The solid fraction
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was washed twice with distilled water, dried at 105°C for 24 h and weighed to yield the mycelial biomass (X) concentration. Residual glucose (S) in the supernatant was spectrophotometrically measured by the DNSA method (Miller, 1959), while residual ammoniacal nitrogen (N) and phosphorous (P) in the supernatant were spectrophotometrically assayed as described by Strickland and Parson (1972). Itaconic acid (I) in the supernatant was determined by using a Violet Instruments (Rome, Italy) high-performance liquid chromatograph. The apparatus was equipped with a high-pressure volumetric pump (type Clar 003, Violet Instruments, Rome, Italy), a UV detector (Violet SP 430) and a 20-cm reverse phase LiChrospher, 100 RP-8, 5 mm (Merck, Darmstadt, Germany) column at room temperature. A buffer, 0.05 mol l − 1 KH2PO4 at pH 3, was used as eluent at a flow rate of 1.2 ml min − 1 and itaconic acid detection was performed at 210 nm.
3. Results and discussion To assess the optimal operating conditions for the itaconate production by A. terreus NRRL 1960 on glucose-based media, two series of fermentation trials were performed under constant stirring rate and pH, respectively.
3.1. Effect of pH Several fermentation trials were performed by varying the pH in the range 1.85–2.8 under a constant stirring rate of 320 rev min − 1. Despite the corresponding peripheral impeller speed (1.256 m s − 1) was greater than that (0.942 m s − 1) suggested by Park et al. (1993), such agitation rate was chosen to enhance mixing and oxygen transfer, being the working volume of the stirred fermenter used here 5 times greater than that used by Park et al. (1993). The mean values of the main results [initial (ti) and final (tf) times of the itaconate production phase together with the corresponding concentrations of mycelial biomass (Xi, Xf), glucose (SiF, SfF) and itaconic acid (Ii, If), and estimated values of itaconate production (RI), glucose consumption
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(RS) and mycelial growth (RX) rates and of yield coefficients for itaconic acid on glucose consumed (YI/S) or supplied (YI/So) and for mycelial biomass on glucose consumed (YX/S)] of all these fermentation runs are shown in the upper section of Table 1. As reported in Section 2, the coefficient of
Fig. 1. Time course of itaconic acid production by Aspergillus terreus NRRL 1960 at 320 rev min − 1 when the pH was left free to change: (a) Concentrations of glucose (S : ), itaconic acid (I : ") and mycelial biomass (X : ) as a function of the fermentation time (t); (b) Concentrations of dissolved oxygen (DO : D), ammoniacal nitrogen (N : ) and phosphorous (P : ), and pH (2) versus t. The continuous lines were calculated by using Eqs. (6) and (7) together with the empirical coefficients listed in Table 2.
variation for all the estimated parameters in Table 1 fell within 0.05 and 0.1. Itaconic acid yield (YI/S) and productivity (RI) appeared to be function of the pH of the medium and varied in the ranges of 0.33–0.63 g per gram of glucose consumed and 0.12–0.25 g l − 1 h − 1, respectively. Of the two itaconate yield coefficients estimated here, that on the initial glucose supplied (YI/So) appeared to be the most suitable for assessing the optimal pH for the fermentation process. When the pH was uncontrolled and reached a final value of 1.85, the acidity of the medium markedly limited the fungal utilisation of the glucose initially supplied and YI/So was only 0.21 g g − 1. On the contrary, when the pH was controlled at 2.4 the glucose was practically exhausted at the end of the fermentation (Table 1) and YI/So was 0.53 g g − 1. Figs. 1 and 2 illustrate the typical time course of the itaconate fermentation under the operating conditions mentioned above. As shown in Figs. 1a and 2a, after about 24 h of fermentation, the phosphorous (P) concentration in the production medium fell to about 10 mg l − 1 and the fungus started to excrete itaconic acid. During the production phase, the nitrogen (N) concentration continued to decrease from about 20 to less than 1 mg l − 1, while mycelial biomass increased from 3 to 10–11 g l − 1. The rapid phosphorous consumption during the first 24 h was accompanied by a dramatic fall in dissolved oxygen (DO) from 100 to 10–15% of the saturation value. After that time, DO remained almost constant up to the overall exhaustion of the nitrogen source, then started to increase again, thus tending to an equilibrium value when all mycelial metabolic activity was completely exhausted (Figs. 1b and 2b). Despite the above optimal pH of 2.4 is in line with several other reports (Larsen and Eimhjellen, 1955; Elnaghy and Megalla, 1975), it is worth noting that the itaconate yield coefficient (YI/S = 0.33) determined here (when using a 75-mm Rushton-type turbine operating at 6I = 1.256 m s − 1) was half that (YI/S = 0.64) obtained by Rychtera and Wase (1981), who worked at a pH of 2.9–3.1 with a 85-mm single flat-blade impeller operating at 6I = 0.71 m s − 1.
a
24.5 22.0 26.5 23.0 19.0 23.0 15.0 22.0
1.85a 1.95 2.2 2.4 2.8 2.9 2.8 2.8 165.0 141.0 209.0 251.0 137.0 166.5 121.0 128.5
tf (h)
3.1 2.5 2.6 1.9 3.0 4.5 2.6 4.0
XI (g l−1) 11.2 11.8 8.6 10.6 8.0 10.7 9.3 9.7
Xf (g l−1) 78.0 86.7 73.6 107.4 75.0 85.0 86.0 86.0
SiI (g l−1) 36.8 48.1 17.0 1.5 24.5 3.0 5.5 13.0
SfI (g l−1) 0.00 0.04 1.50 0.00 1.04 0.50 0.00 0.80
Ii (g l−1) 16.5 21.0 36.9 57.2 17.7 25.2 43.1 33.1
If (g l−1)
0.12 0.18 0.19 0.25 0.14 0.17 0.41 0.30
RI (g l−1 h−1)
Referred to the final pH of the medium when the fermentation was carried out with no pH control.
320 320 320 320 320 240 400 480
tI (h)
pH (−) Stirring rate (rev min−1)
-0.29 -0.32 -0.31 -0.46 -0.43 -0.57 -0.76 -0.69
RS (g l−1 h−1)
0.06 0.08 0.03 0.04 0.04 0.04 0.06 0.05
RX (g l−1 h−1)
0.40 0.54 0.63 0.54 0.33 0.30 0.54 0.44
YI/S (g g−1)
0.21 0.24 0.48 0.53 0.22 0.29 0.50 0.38
YI/So (g g−1)
0.20 0.24 0.11 0.08 0.10 0.08 0.08 0.08
YX/S (g g−1)
Table 1 Mean values of the main results of several itaconic acid production by A. terreus NRRL 1960 replicated twice: Effect of pH and stirring rate on the initial (ti) and final (tf) times of the itaconate production phase together with the corresponding concentrations of mycelial biomass (Xi, Xf), glucose (Si, Sf) and itaconic acid (Ii, If), and estimated values of itaconate production (RI), glucose consumption (RS) and mycelial growth (RX) rates and yield coefficient for itaconic acid on glucose consumed (YI/S) or initially supplied (YI/So) and for mycelium on glucose consumed (YX/S). The coefficient of variation for all these stoichiometric and kinetic parameters ranged from 0.05 to 0.1a
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Fig. 2. Time course of itaconic acid production by A. terreus NRRL 1960 at 320 rev min − 1 when the pH was left free to change up to 2.4: (a) Concentrations of glucose (S : ), itaconic acid (I : ") and mycelial biomass (X : ) as a function of the fermentation time (t); (b) Concentrations of dissolved oxygen (DO : D), ammoniacal nitrogen (N : ) and phosphorous (P : ), and pH (2) versus t. The continuous lines were calculated by using Eqs. (6) and (7) together with the empirical coefficients listed in Table 2.
3.2. Effect of stirring rate To establish if the above experimental result (YI/S =0.33) was due to insufficient oxygen transfer in the fermenter used, a second series of trials was carried out by setting the stirring rate to 240, 400 and 480 rev min − 1 (these values corresponding to peripheral impeller speeds of 0.94, 1.57 and 1.88 m s − 1) and pH to about the optimal value
suggested by Rychtera and Wase (1981). The mean values of their main results are given in the lower section of Table 1. By increasing the stirring rate from 240 to 480 rev min − 1, the yield coefficients YI/So and YI/S firstly increased reaching a maximum value of 0.50 and 0.54 g g − 1 at 400 rev min − 1, respectively, and then decreased. On the contrary, YX/S appeared to be independent of agitation rate and practically equal to 0.08 g g − 1. Fig. 3 shows the typical time course of the itaconate fermentation at pH 2.8 and 400 rev min − 1. By only increasing the stirring rate to 400 rev min − 1, it was possible to confirm previous findings by Rychtera and Wase (1981), thus revealing a significant dependence of the itaconate yields upon the aeration-agitation conditions within the fermenter used. Although there are a number of reports in the literature of hyphal damage due to high agitation intensity [see for general reference the well known paper by Dion et al. (1955) and more specifically for the fermentation under study that by Park et al. (1993)], these results clearly show that the mycelium was not damaged by the shear stress exerted by the impeller blades at peripheral impeller speeds up to 1.57 m s − 1. This is in strict agreement with van Suijdam and Metz (1981) results on the effect of agitation intensity on the length of mycelial hyphae and with Moresi et al. (1987) on the effect of 6I in the range 1.76–3.8 m s − 1 on the stoichiometry and kinetics of untreated orange peel utilisation by Fusarium a6enaceum. The fact that the strain used in this work at pH 2.8 exhibited a higher resistance to shearing forces than that used by Park et al. (1993) at pH 2.0 might be in all probability attributed to the higher pH of the fermenting media. To test whether the experimental mean values of the yield coefficients for itaconic acid on glucose supplied (YI/So), that were observed at pH 2.8 and 400 rev min − 1 and at pH 2.4 and 320 rev min − 1, were significantly different, their standardised normal difference [d= (Y1 − Y2)/ ((s 21/p1)+ (s 22/p2)) where Yj, sj, and pj are the mean value, standard deviation and overall number of trials for the variables tested] was compared with the classic inequality of the hypothesis test for means,
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[i.e. d5ta,f the one-sided Student t-test for a confidence level a and f ( =p1 +p2 −2) degrees of freedom] (Bennett and Franklin, 1954). In this way, for s1 :s2 =0.03, p1 =p2 =2 and t0.05,2 =4.3, it was possible to reject the hypothesis of inequality (d=1.0 B4.3) at a probability level of 0.05. Similarly, the same inequality test performed on the itaconate formation rate (RI) values obtained at 320 rev min − 1 and pH ranging from 1.95 to 2.8 (upper section of Table 1) yielded
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that there was no statistically significant difference between the yields and rates examined, thus justifying the discordant literature indications about the optimal pH of the medium during the itaconate production phase (Larsen and Eimhjellen, 1955; Elnaghy and Megalla, 1975; Rychtera and Wase, 1981). However, when the stirring rate was increased from 320 to 400 rev min − 1 at pH 2.8, the itaconate formation rate (RI) was found to increase from 0.259 0.02 to 0.419 0.03 g l − 1 h − 1 (Table 1). Since the difference in RI (6.3\ 4.3) was statistically significant at the 95% confidence level, the kinetics of this fermentation is no doubt controlled by the effective oxygen transfer capability of the fermenter used. In conclusion, despite the operation at pH 2.4 and 320 rev min − 1 would seem to be more convenient because the smaller the stirring speed the smaller the power input per unit itaconate formed and the smaller the pH of the medium the smaller the alkaline reactant consumption and the microbial contamination risk, the operation at pH 2.8 and 400 rev min − 1 has to be regarded as the optimal one because of the greater itaconate formation rate.
3.3. Kinetics of itaconate production Among the numerous models reported in the literature to describe quantitatively the formation rate of a metabolic compound (Moser, 1985), the now classic Luedeking and Piret (1959), kinetics has so far proved extremely useful and versatile in fitting product formation data from many different fermentations: RI = dI/dt = YI/X(dX/dt)+mI(X)
Fig. 3. Time course of itaconic acid production by A. terreus NRRL 1960 at 400 rev min − 1 when the pH was left free to change up to 2.8: (a) Concentrations of glucose (S : ), itaconic acid (I : ") and mycelial biomass (X : ) as a function of the fermentation time (t); (b) Concentrations of dissolved oxygen (DO : D), ammoniacal nitrogen (N : ) and phosphorous (P : ), and pH (2) versus t. The continuous lines were calculated by using Eqs. (6) and (7) together with the empirical coefficients listed in Table 2.
(1)
where mI is the specific product formation rate at zero mycelial growth rate and YI/X the yield factor for itaconic acid on unit mycelial biomass. In particular, the first term indicates the product formation rate in association with mycelial growth rate; while the second one is the nongrowth associated product formation rate. To apply this two-parameter kinetic expression, it is necessary to know the mycelial growth rate (RX), which can be generally described as:
E. Riscaldati et al. / Journal of Biotechnology 83 (2000) 219–230
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Fig. 4. Itaconic acid production by A. terreus NRRL 1960 at pH= 2.8 and 400 rev min − 1: logarithm of (X/Xo : ") and concentrations of phosphorous (P : ) and ammoniacal nitrogen (N : ) versus time. The continuous line was calculated by using Eq. (4) together with the empirical coefficients listed in Table 2.
RX = dX/dt =m(X)
(2)
where X is the instantaneous mycelial concentration and m is the actual specific cell growth rate which is a more or less complex function of several variables such as, for instance, the limiting substrate concentration in the case of the Monod equation or the maximum mycelial concentration in the case of the logistic equation (Moser, 1985). By assuming no explicit relationship for m, Eq. (2) can be integrated as ln
X = X0
&
t
m dt
(3)
0
If the mycelium exponentially duplicates, the specific mycelial growth rate will coincide with the maximum specific one and the plot of the natural logarithm of the actual to the initial mycelial concentrations against time will be a straight line passing through the origin of the plot. In this specific fermentation, the general plot of ln(X/X0) versus time had the pattern shown in Fig. 4, which may be approximated by using at least two different straight lines, the first and second ones fitting the experimental data at the beginning and at the end of the fermentation, respectively. This means that mycelial growth may
be roughly described as double-substrate growth (Moser, 1985), the first limiting substrate being the phosphorous and the second one the nitrogen (Fig. 4). In fact, the ln(X/X0) deviates from the first straight line having slope m1 as the P content in the medium is smaller than about 10 mg l − 1 (Fig. 4). When the mycelium has adapted to the new cultural condition, it reduces its instantaneous specific growth rate and starts to excrete itaconic acid. In the circumstance, being the medium still rich in carbon and nitrogen sources, the mycelial weight concentration X will continue to increase at the expense of another limiting substrate, that is the ammoniacal nitrogen. Such a secondary mycelial growth is characterised by a secondary specific growth rate m2, which was found to be about one tenth of the primary one (m1). This microbial behaviour is not specific of the A. terreus strain used, since it was already observed in Yarrowia lipolytica (Moresi, 1994) and A. niger (Verhoff and Spradlin, 1976; Briffaud and Engasser, 1979) during the citric acid formation and in Saccharomyces cere6isiae (Martini et al., 1976) during the aerobic growth, both on media rich in glucose and devoid of the nitrogen source. To describe such double-substrate growth, the following empirical regression was used: ln
X(t) q = %jbj (1−e − mj t) Xo 1
(4)
where q is the number of statistically significant limiting substrates, mj the j-th maximum specific mycelial growth rate and bj the j-th empirical regression coefficient. By using the same procedure developed by Nussinovitch et al. (1989) and modified by Mancini et al. (1999) to correlate stress relaxation data, it was possible to fit all the experimental data by assigning initially an arbitrary spectrum of reaction times: 1 tj = = a x10z − j mj
(5)
where a is an integer index ranging from 1 to 9 and z is an empirical exponent assigned to make the maximum reaction time coincident with the
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overall duration of the fermentation process. For instance, if the process lasts 300 h, z is equal to 4 so as to make the first reaction time (t1) varying from 1000 and 9000 h. By using a linear regression method, it was possible to discard firstly the reaction times, the contribution of which to the ln(X/Xo)-t relationship was statistically insignificant at the confidence level of 95%, and then to determine the optimal a value by maximising the coefficient of determinations (r 2) of Eq. (4). By applying the above operating procedure, there was no statistically significant improvement in fitting the time course of any experimental [ln(X/Xo)−t] data by considering more than two maximum specific cell growth rates in agreement with the aforementioned verbal kinetic model. Under the assumption that all the stoichiometric and kinetic parameters were constant, the above set of differential equations can be integrated, thus yielding the following: Á ÃX0 q X= Í ÃX0 exp %jbj (1− e − uj t) Ä 1 I = Io + mI
&
n
for t 5t0 for t \t0
(6)
t
X dt +YI/X (X − Xi)
(7)
tI
where the second term at the right hand side of Eq. (7) can be numerically integrated once X(t) is
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known. By applying the analytical expressions given above in conjunction with the least squares method, it was possible to fit all the fermentation data collected and evaluate the kinetic and stoichiometric parameters listed in Table 2. The primary maximum specific cell growth rate (m1) ranged from 0.025 to 0.045 h − 1, while the secondary one (m2) was one tenth of m1. Moreover, the itaconate production by A. terreus NRRL 1960 exhibited a mixed-growth-associated product formation kinetics, the contribution of growth associated production being related to the secondary mycelial growth only and that of nongrowth associated production depending on pH and stirring rate. More specifically, at constant stirring rate of 320 rev min − 1 the specific product formation rate at zero mycelial growth rate (mI) was practically zero when the pH was left reducing without any control. This revealed a growth-associated product formation only when the mycelium was severely stressed by as low a pH as 1.85. By controlling the pH of the medium during the fermentation, mI progressively increased from as low as 0.004 h − 1 at pH 1.95 to a maximum value of 0.028 h − 1 at pH 2.4. At pH 2.8, mI was found to be also a function of the stirring rate, its maximum value (0.036 h − 1) being associated with a stirring rate of 400 rev min − 1. However, the difference in the last two maximum values of mI (d=3.1 B 4.3) was found to be statistically in-
Table 2 Main kinetic and stoichiometric parameters of several itaconic acid production by A. terreus NRRL 1960 replicated twice: Effect of pH and stirring rate on the primary (m1) and secondary (m2) maximum specific mycelial growth rates and their corresponding dimensionless contribution (bj ), specific product formation rate at zero mycelial growth rate (mI) and yield factor for itaconic acid on unit mycelial biomass (YI/X) together with their standard deviations and or coefficients of determinations (r 2)a pH (−)
1.85a 1.95 2.2 2.4 2.8 2.9 2.8 2.8 a
Stirring rate (rev min−1) 320 320 320 320 320 240 400 480
b1 (−)
m1 (h−1) b2 (−)
m2 (h−1) r 2
mI (h−1)
YI/X (g g−1)
r2
0.01 90.3 0.59 0.6 1.1 9 0.3 0.3 90.2 0.49 0.2 0.1 90.2 0.1 90.1 0.1 90.5
0.04 0.03 0.04 0.02 0.04 0.04 0.04 0.04
0.004 0.003 0.004 0.003 0.005 0.004 0.005 0.004
−0.00290.001 0.004 9 0.002 0.012 9 0.003 0.028 90.002 0.003 90.003 0.013 9 0.001 0.036 90.003 0.023 90.003
2.4 90.1 2.4 9 0.2 3.9 9 0.6 1.0 90.2 2.4 90.3 1.6 9 0.1 2.2 90.3 1.5 90.3
0.97 0.99 0.97 0.98 0.90 0.99 0.99 0.97
3.4890.09 3.659 0.2 1.329 0.12 2.989 0.07 1.83 9 0.08 2.009 0.08 2.199 0.05 2.2890.15
0.99 0.96 0.94 0.99 0.99 0.99 0.99 0.97
Referred to the final pH of the medium when the fermentation was performed with no pH control.
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significant at the 95% confidence level. On the contrary, at the same probability level the difference in the corresponding YI/X values (Table 2) resulted to be statistically significant (d= 4.7\ 4.3). Therefore, the net improvement in the RI value observed at pH 2.8 and 400 rev min − 1 with respect to that obtained at pH 2.4 and 320 rev min − 1 (Table 1) is most likely due to the difference in the yield coefficient for itaconic acid on mycelial biomass formed (Table 2). The continuous lines plotted in Figs. 1 – 3 were calculated by using Eqs. (6) and (7) together with the empirical coefficients reported in Table 2 and show a remarkable agreement between calculated and experimental X and I data. Therefore, all these results are consistent with the present hypothesis of mixed kinetics for itaconate production.
progressively increased from 0.004 h − 1 at pH 1.95 to 0.028 h − 1 at pH 2.4, even if such a maximum value was also associated with higher values of pH (2.8) and stirring rate (400 rev min − 1). Of these two operating conditions, the latter has to be definitively regarded as the optimal one for the itaconic acid fermentation owing to the greater product formation rate.
5. Notation a d DO I mI N
4. Conclusions P The discordant literature indications about the optimal pH of the medium during the production of itaconic acid from glucose-based media by A. terreus NRRL 1960 may be attributed to the fact that this fermentation is controlled by stirring rate and pH. A maximum yield coefficient (YI/S) of about 0.54 g of itaconic acid per g of glucose consumed was obtained under different operating conditions, such as pH 2.4 and 320 rev min − 1 or pH 2.8 and 400 rev min − 1, while their corresponding itaconate formation rates (RI) were 0.25 and 0.41 g l − 1 h − 1, respectively. This fermentation exhibited a mixed-growth-associated product formation kinetics, even if the contribution of non-growth associated production was mainly affected by the pH during the itaconate production phase and secondly by stirring rate. When no control of the pH was assured, the mycelium resulted to be so severely stressed as to stop itaconate production at pH 1.85, thus involving a strict growth-associated production kinetics. On the contrary, under pH control and constant stirring rate (320 rev min − 1) the specific product formation rate at zero mycelial growth rate (mI)
pj q r2 RI RS RX S sj t ta,f 6I X YI/S YI/So YI/X
integer index of Eq. (5). standardised normal difference between means. dissolved oxygen concentration (%). itaconic acid concentration (g l−1). specific product formation rate at zero mycelial growth rate (h−1). ammonia nitrogen concentration (mg l−1). phosphorous concentration (mg l−1). overall number of trials for the jth parameter tested. number of statistically significant limiting substrates. coefficient of determinations. itaconate production rate (g l−1 h−1). glucose consumption rate (g l−1 h−1). mycelial growth rate (g l−1 h−1). glucose concentration (g l−1). standard deviation of the j-th parameter tested. fermentation time (h). one-sided Student t-test. peripheral impeller speed (m s−1). mycelial biomass concentration (g l−1). yield coefficients for itaconic acid on glucose consumed (g g−1). yield coefficients for itaconic acid on glucose supplied (g g−1). yield factor for itaconic acid on unit mycelial biomass (g g−1).
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Yj YX/S
mean value of the j-th parameter tested. yield coefficients for mycelial biomass on glucose consumed (g g−1).
Greek symbols a confidence level. bj generic j-th empirical regression coefficient of Eq. (4). f degrees of freedom. m specific cell growth rate (h−1). mj generic j-th maximum specific mycelial growth rate (h−1). tj generic j-th reaction time (h). z empirical exponent of Eq. (5). Subscript o I f
referred to the initial value. referred to the beginning of the itaconate production phase. referred the end of the itaconate production phase.
Acknowledgements This research work was supported by a grant from the National Research Council (CNR) of Italy: Target Project on Biotechnology.
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