CET 96 DOI: 10.3303/CET2296067 Paper Received: 27 January 2022; Revised: 29 June 2022; Accepted: 6 September 2022 Please cite this article as: Brink H., Geyer-Johnson M., Nicol W., 2022, Malic Acid Production by Aspergillus Oryzae: the Effect of Alkaline- earth Carbonate Buffer Identity, Chemical Engineering Transactions, 96, 397-402 DOI:10.3303/CET2296067 CHEMICAL ENGINEERING TRANSACTIONS VOL. 96, 2022 A publication of The Italian Association of Chemical Engineering Online at www.cetjournal.it Guest Editors: David Bogle, Flavio Manenti, Piero Salatino Copyright © 2022, AIDIC Servizi S.r.l. ISBN 978-88-95608-95-2; ISSN 2283-9216 Malic Acid Production by Aspergillus oryzae: The effect of Alkaline-Earth Carbonate Buffer Identity Hendrik Brink*, Monique Geyer-Johnson, Willie Nicol Department of Chemical Engineering, University of Pretoria, Lynnwood, South Africa deon.brink@up.ac.za Malic acid is a specialty chemical that is currently mainly used in the food and beverage industry (market value of $182 million) but has a potential market value of $3.5 billion if used to produce maleic anhydride. The results from the study indicated that the production of malic acid by A. oryzae requires the presence of the alkaline earth metals calcium or magnesium in significant quantities. It was observed that replacing an amount of CaCO3 (240 g.l-1 CaCO3), significantly over that required for pH buffering (21 g.l-1 CaCO3), with an equivalent amount of MgCO3 (192 g.l-1 MgCO3 based on CO32+) results in similar malic acid yields and final malic acid titers. In contrast, a marked reduction in glucose consumption and malic acid production rates were observed. These observations are likely due to an evolutionary response to calcareous soils. These soils tend to immobilize minerals in solid precipitates resulting in nutrient depletion, while the production of malic acid solubilizes these minerals making them bioavailable. The higher rates observed for the calcium vs magnesium runs were likely a result of the stimulatory effect of Ca2+ on the ATP generating pathways as well as several regulatory responses within the fungal physiology. In addition, it was found that A. oryzae was capable of assimilating malic acid from the environment, therefore, minimizing the loss of valuable carbon due to malic acid excretion. This study provides invaluable information required for economically viable malic acid production by A. oryzae which could markedly reduce reliance on the petrochemical industry. 1. Introduction Developing a sustainable society requires a significant industrial shift away from petroleum-based products and towards renewable and bio-based technologies. This shift has been supported by renewed interest by consumers in natural, biodegradable, and environmentally friendly products (Mondala, 2015). As part of its drive towards bio-based products, the US Department of Energy has identified 12 priority platform chemicals required for bio-based chemical production; malic acid – along with the other two four carbon diprotic organic acids succinic- and fumaric acids- being considered among these (Werpy and Petersen, 2004). The current worldwide demand for malate is reported to be 200 kt/a (Chi et al., 2016) while the current international supply of L-malic acid is estimated at 40 kt/a (Liu et al., 2018). Currently, malic acid is commercially produced by the catalytic hydration of maleic or fumaric acid, both derived from maleic anhydride. Maleic anhydride is, in turn, produced from vapor phase oxidation of hydrocarbons, most prominently butane (Hermann and Patel, 2007). Unfortunately, this synthetic pathway produces a racemic mixture of L– and D–malic acid which is unsuitable for the food and beverage industry where malic acid is utilized as an acidulant (Knuf et al., 2014). The biological production of malic acid provides stereo selectivity since L–malic acid is a key intermediate in the tricarboxylic acid cycle (TCA) present in most microorganisms (Liu et al., 2017). Filamentous fungi of the genus Aspergilli are superior producers of various bio-based chemicals including lipases, (Melo et al., 2011) xylanase, (Park et al., 2002), and various organic acids; Aspergillus flavus and A. oryzae widely considered the best biological producers of malic acid (Ochsenreither et al., 2014). However, A. flavus is known to produce hazardous amounts of carcinogenic aflatoxin, making the malic acid produced unsuitable for the food industry. In contrast, A. oryzae is a GRAS (Generally Regarded as Safe) organism that does not produce mycotoxins and has therefore been used in the production of sake, shochu, soy sauce, and miso for centuries (Payne et al., 2006). 397 Currently, laboratory-scale methods for malic acid production are limited due to the use of pellet morphology and CaCO3 as the go-to buffering agent which complicates downstream processing. Geyer et al. (2018) observed a maximum malic acid production rate of 0.09 g.l-1.h-1 for 20 g.l-1 CaCO3 was initially dosed as compared to a maximum malic acid production rate of 0.23 g.l-1.h-1 for 100 g.l-1 CaCO3. This compares well with the results obtained by Kövilein et al. (2021) who measured a maximum malic acid production rate of 0.052 g.l- 1.h-1 for 10 g.l-1 CaCO3 and 0.121 g.l-1.h-1 for 90 g.l-1 CaCO3. These results indicate a likely synergistic effect of excess CaCO3 on the excretion of malic acid in addition to its role as a pH buffer. To assess the effect of exchanging Ca2+ for Mg2+ on A. oryzae malic acid fermentation, while maintaining the buffering capacity and bicarbonate source in the medium, two different carbonate buffers were compared by adding the same amount of CO32- and changing only the cation (Ca2+ vs Mg2+). The resulting buffer concentrations used were 240 g/l CaCO3 and 192 g/l MgCO3 (impurities of each taken into consideration). 2. Materials and Methods 2.1 Micro-organisms and fermentation in shake flasks A. oryzae NRRL 3488 was obtained from the Agricultural Research Service Culture Collection in Illinois, USA. The stock cultures were stored at -40 °C in a 50 % w/w glycerol solution. Potato dextrose agar (PDA) (Merck KgaA, Darmstadt, Germany) plates were inoculated with the stock solution and incubated at 30 °C for 7 days. Approximately 60 mg of spores (circa 6.7 × 108 spores (Smith et al., 1988)) were harvested from two agar plates with sterilized distilled water. The inoculum was prepared by adding the spore solution to a 10 % w/w glycerol solution which was subsequently stored at -40 °C. Production was done using a one-step method and fermentation media adapted from Shigeo et al (1962) consisting of (in g/l): 60 glucose, 1.2 (NH4)2SO4, 0.75 KH2PO4, 0.75 K2HPO4, 0.1 MgSO4·7H2O, 0.1 CaCl2·2H2O, 0.005 FeSO4·7H2O and 0.005 NaCl. A 250 ml unbaffled Erlenmeyer flask was loaded with the medium, sterilized mixed, inoculated with the spore glycerol solution, and stirred (final spore concentration of 240 mg/l or 2.7 × 106 ml-1) and cultivated at 35 °C and 150 rpm for ~24 hours in an incubator shaker. There were 2 batches of shake flasks: one set used 240 g/l CaCO3, and one set used 192 g/l MgCO3 for pH buffering, each added at the start of the fermentation to the relevant flask. Both buffers were tested in triplicate. The choice of buffer amounts was 1) to ensure a complete excess of buffer at all times in the system, and 2) to ensure that equivalent amounts of CO32- (~140 g/l) were loaded in both. 2.2 Sample preparation It was hypothesized that there was a synergistic effect between pH control by CaCO3/MgCO3 dissolution and the precipitation of calcium salts (calcium–malate, –citrate, –fumarate). Due to this, the concentration of acids in the fermentation broth would be lower which in turn would allow A. oryzae to continue producing acid without possible adverse effects (pH, osmotic pressure, etc.). To determine how much acid, if any, had precipitated out of solution in the form of calcium/magnesium salts, 2 sets of HPLC and ICP-OES analyses were run on each sample: before acidification and after acid treatment. For this, a fraction of the supernatant was extracted and used for metabolite and cation analyses before acidification. Each sample remnant was then treated with 1 M HCl and incubated in the oven at 90 °C for 30 min (vortexed every 5 min) and then centrifuged. The subsequent supernatant was used for metabolite and ICP-OES analyses. 2.3 Analytical methods Samples from the flasks were collected in pre-weighed CELLSTAR® tubes (Greiner Bio-One, Sigma Aldrich, St. Louis, MO, USA). The concentrations of glucose, glycerol, ethanol, and organic acids in the samples were determined with an Agilent 1260 Infinity HPLC (Agilent Technologies, Santa Clara, CA, USA) fitted with a refractive index detector. Samples (± 1 ml) were centrifuged at 16 600 × g for 90 s and filtered with 0.45 µm Nylon (Ministart®, Sigma Aldrich, St. Louis, MO, USA) syringe filters into HPLC vials and loaded into the autosampler tray of the HPLC. Samples (5 µl) were injected into a Micro-Guard® Cartridge (30 m × 4.6 m) that was attached to a 300 mm × 7.8 mm Aminex HPX-87H ion-exchange column (Bio-Rad Laboratories, Hercules, CA, USA) maintained at 60 °C. Organic acids and glycerol were measured with mobile phase A (0.02 M H2SO4) and glucose, lactic acid, and ethanol with mobile phase C (0.002 M H2SO4) both at a flow rate of 0.6 ml/min. Each sample was analyzed twice: the control supernatant and the pre-treated supernatant. The concentration of calcium and magnesium in the solution was measured using inductively coupled plasma mass spectrometry (ICP-OES, Perkin-Elmar, Waltham, MA, USA) using an Argon plasma to ionize the samples and measure the relevant concentrations. This was repeated for both control and pre-treated samples. 398 2.4 Curve fitting Time-dependent concentration profiles were approximated in the GraphPad Prism 7.0 (GraphPad Prism Software, San Diego, CA, USA) environment using a four-parameter logistic function 𝐶𝐶𝑖𝑖(𝑡𝑡) = 𝐶𝐶𝑖𝑖,𝑚𝑚𝑚𝑚𝑚𝑚 + (𝐶𝐶𝑖𝑖,𝑚𝑚𝑖𝑖𝑚𝑚 − 𝐶𝐶𝑖𝑖,𝑚𝑚𝑚𝑚𝑚𝑚 ) 1 + � 𝑡𝑡𝑡𝑡50 � 𝑘𝑘 (1) Consumption/production rates were determined by differentiating the fitted functions. 3. Results & Discussion The results for the malic acid concentration curves can be seen in Figure 1(a) for the CaCO3 experiment and in Figure 1(b) for the MgCO3 experiment. According to the proposed hypothesis, it was anticipated that the measured malic acid concentration would change significantly between the untreated and acid-treated samples. However, a Wilcoxon matched-pairs signed-rank test (α = 0.05) showed that there was not a statistically significant difference for the CaCO3 (Z = 0.257; p = 0.7969) or MgCO3 (Z = 0.873; p = 0.383) sets. This suggested that calcium-malate and magnesium-malate were not present in as significant concentrations as previously predicted. This contradicted the hypothesis that the precipitation of insoluble acid salts decreased the acid concentration in the fermentation broth which would decrease the inhibition of further acid production and therefore the effect of the CaCO3 or MgCO3 was likely related to either the supply of CO2 for anaplerotic reactions required for malate synthesis (Kövilein et al., 2021) or the effect of the alkaline earth cation identity itself. 0 100 200 300 400 500 600 15 30 45 60 Time (h) M al ic a ci d (g /L ) CaCO3 untreated CaCO3 acid-treated (a) 0 100 200 300 400 500 600 15 30 45 60 Time (h) M al ic a ci d (g /L ) MgCO3 untreated MgCO3 acid-treated (b) 0 49 82 101 127 148 194 242 290 361 505 0 25 50 75 100 125 150 Time (h) Ca lc iu m (g /L ) CaCO3 acid-treatedCaCO3 untreated (c) 0 48 96 146 192 241 290 353 505 0 20 40 60 80 100 Time (h) M ag ne si um (g /L ) MgCO3 acid-treatedMgCO3 untreated (d) Figure 1: Malic acid concentration curves for (a) CaCO3 and (b) MgCO3 shake flask experiments showing untreated and acid-treated measurements. The ICP-MS measured concentrations of each buffer cation in the solution for (c) CaCO3 and (d) MgCO3, including the untreated (aqueous cation) and acid-treated (total cation) measurements. Error bars indicated the standard deviation of triplicates. The calcium and magnesium concentrations measured with ICP-OES can be seen in Figures(c) and (d) below for the untreated and acid-treated samples. As more of the buffer dissolved to neutralize the pH, the concentration of the relevant cation in the solution increased, corresponding to a continuously increasing amount of buffer dissolution as a result of either the neutralization of the acids produced and/or the replacement of CO2 (in the form of HCO3-) consumed during the biosynthesis of malate (and minor amounts of succinate) during fermentation. The pH for both fermentations had average values of 6.9±0.5 and 8.0±0.5 for the CaCO3 and MgCO3, respectively, indicating that the predominant carbonic for was HCO3-. Therefore, the neutralization of 399 the produced organic acids required one carbonate ion for every proton neutralized. In addition, all produced acids would be fully dissociated for the pH range. From the results, it was observed that 0.88±0.22 M and 1.08±0.30 M of CO3- were liberated in the CaCO3 and MgCO3 runs, respectively. In comparison, total proton production of 0.79±15 M and 0.80±0.05 M were measured for the CaCO3 and MgCO3 runs, respectively. While CO2 demands of 0.40±0.08 M and 0.40±0.03 M for the synthesis of malate and succinate were determined. From the known equilibrium reactions for CaCO3 and MgCO3 in water (Aina et al., 2020), the neutralization of proton results in an equivalent molar formation of HCO3- from CO32- resulting in the dissolution of CaCO3/MgCO3. In addition, the assimilation of CO2 (as HCO3-) by the fungi results in an equivalent decrease in HCO3- in solution which results in the conversion of CO32- to HCO3- with the concomitant dissolution of CaCO3/MgCO3 to satisfy the prevailing equilibrium conditions. Therefore, it can be seen that nearly all the CO2 for anaplerotic reactions was obtained from MgCO3, while a very small fraction of CO2 for these reactions came from CaCO3. This implies that the main source of CO2 for malate synthesis in the CaCO3 was respiration, while in the MgCO3 system this was satisfied by the buffer. The results further demonstrate that a nearly constant concentration of calcium (as Ca2+ and CaCO3) and magnesium (as Mg2+ and MgCO3) were continuously present in the system, corresponding to an average of 96.2±20.1 g/l and 55.4±13.2 g/l for Ca2+ and Mg2+, respectively. These values correspond well with the originally loaded amounts of these ions. The fitted malic acid concentration curves with their corresponding malic acid production rates can be seen in Figures(a) and Figure 2(b). The glucose concentrations for control samples can be seen in Figure 2(c) with the consumption rate curves in Figure 2(d). The fitted parameters were summarised in Table 1. The CaCO3 set had significantly higher rates of glucose consumption and malic acid production compared to the MgCO3 set. Despite this, the measured malic acid concentrations were similar between the MgCO3 and CaCO3 experiments and the distributions in the group did not differ significantly (Mann-Whitney U = 32.5; n1 = 9; n2 = 8; p = 0.765 two-tailed). This indicated that the identity of the corresponding metal cation (Ca2+ or Mg2+) influenced the production/consumption rates. The glucose was completely consumed for the CaCO3 set after 250 hours with a decrease in malic acid seen after the same time. 0 100 200 300 400 500 600 15 30 45 60 Duration (hrs) M al ic a ci d (g /L ) MgCO3 CaCO3 (a) 0 100 200 300 400 500 600 0.1 0.2 0.3 0.4 Duration (hrs) M al ic a ci d pr od uc tio n ra te (g /L .h ) MgCO3 (b) CaCO3 100 200 300 400 500 600 0 20 40 60 80 100 120 Duration (hrs) G lu co se (g /L ) CaCO3 MgCO3 (c) 100 200 300 400 500 600 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Duration (hrs) G lu co se c on su m pt io n ra te (g /L .h ) CaCO3 MgCO3 (d) Figure 2: Malic acid (c) concentration curves and (d) production rate curves, glucose (e) concentration curves, and (f) consumption rate curves for the MgCO3 and CaCO3 control shake flask experiments. Curves were fitted using the logistic model (Equation 1) with the standard deviation of triplicates shown by the error bars. 400 Table 1: Curve fitting parameters for the concentration profiles in Figures (a) and (c) Dataset 240 g/l CaCO3 192 g/l MgCO3 Glucose Malic acid Glucose Malic acid 𝐶𝐶𝑖𝑖,𝑚𝑚𝑖𝑖𝑚𝑚 (g/l) 0 0 0 0 𝐶𝐶𝑖𝑖,𝑚𝑚𝑚𝑚𝑚𝑚 (g/l) 95.3 43.3 105 68.9 𝑘𝑘 -3.12 3.44 -2.07 1.75 𝑟𝑟𝑡𝑡50 (h) 142 108 299 311 𝑅𝑅2 0.956 0.876 0.952 0.958 The results presented in Figures 1 and 2 indicate that the effects of alkaline-earth buffer identity are extremely complicated. The results demonstrated that the hypothesis of immobilization of malate as Ca-malate or Mg- malate was not a significant factor in the system as negligible amounts of these complexes were detected. However, from the ionic dissolution measurements, it appeared that the type of buffer affected the source of the CO2 required for malate biosynthesis. This links strongly to the significantly increased rate of glucose consumption, with concomitant increased respiration rate, when comparing the CaCO3 and MgCO3 runs. The Ca2+ ion has been labeled the second messenger omnipresent in all fungi (Navazio and Mariani, 2008). This increased Ca2+ concentration within the external medium results in a coordinated range of intracellular signals to regulate the Ca2+ concentration (Roy et al., 2021), including stimulating the oxidative metabolism within the mitochondria to regulate ATP synthesis (Tarasov et al., 2012). This Ca2+ homeostasis and signaling have physiological implications for the growth, virulence, and stress responses in fungi (Lange and Peiter, 2020). These effects contributed to a significantly promoted rate of glucose transfer into the fungal biomass in Ca2+ stimulates fungi, while this same observation was not made for Mg2+ (Pitt and Ugalde, 1984). Applying the observed results in Figure 1 and Figure 2 to A. oryzae, the symbiotic relationship between CaCO3 and malic acid could be explained using a calcareous environment as a reference point. A. oryzae is part of a genus that is distinct from other microbes: they can utilize both a secondary and primary metabolism (Brown et al., 1996). In the primary metabolism, acidic compounds are secreted for nutrient acquisition from the soil (Wu et al., 2018). Specifically, for the extraction of phosphorous where Ca2+ sequesters phosphorus, malate improved the efficiency of phosphorus extraction from the soil (Ström et al., 2005). The secondary metabolism can utilize the acidic compounds from the primary metabolism. This allows A. oryzae to produce secondary metabolites that it can utilize to adapt to its current environment and inhibit the primary metabolic pathways (Brown et al., 1996). MgCO3 would act similarly when considering a dolomitic soil environment where phosphate would be sequestered by the Mg2+ and require a release for phosphorus uptake by the fungi. However, the reduced stimulation of the fungal physiology as compared to the CaCO3 system resulted in a less pronounced effect of Mg2+ on the system. This could be tested in the immobilized bio-reactor using NaOH/Na2CO3 to control the pH and spiking with CaCl2 or MgCl2. 4. Conclusions The study presents an investigation of the effect of alkaline earth buffer (CaCO3 and MgCO3) on the production of malic acid by A. oryzae in a batch bioreaction system. Based on the results, alkaline-earth metals play a key role in the production rate of malic acid with A. oryzae NRRL 3488. The glucose consumption and utilization were higher when CaCO3 was used as a buffer compared to MgCO3, however, similar amounts of malic acid were produced. This study provides invaluable insights into this potentially lucrative yet relatively simple avenue for the improvement of malic acid production by A. oryzae. However, it is clear that the production of malic acid by A. oryzae is a complex, multi-faceted problem requiring understanding of the physiological interactions of the biocatalyst with its environment. The current study is limited to a batch reaction system at laboratory scale and therefore larger scale study is an imperative before real-world application can be realised. 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