ENHANCED PRODUCTION OF OXALIC ACID IN ASPERGILLUS NIGER BY THE ADDITION OF METHANOL

Waldemar Rymowicz, Dorota Lenart

ABSTRACT

The stimulating effects of methanol on the growth and production of oxalic acid from post-refining fatty acids (by-product of plant oil production containing free fatty acids) by a mutant of Aspergillus niger were investigated in submerged fermentation experiments. Both the final concentration and the yield of the product were highest when 1.5 % (w/v) methanol was added at the time of inoculation culture. With a medium containing of 50 g/l fatty acids, production reached a maximum of 75 g/l oxalic acid with yield on fatty acids consumed up to 1.57 g/g after 7 d. The concentration of oxalic acid and the product yield was 1.4 and 1.34 times higher, respectively, than the control (without methanol).

Key words: Aspergillus niger; biosynthesis; fatty acids; methanol; oxalic acid..

INTRODUCTION

Methanol as a stimulating agent is used in many fermentation processes. According to many investigators, supplementation of production medium with either methanol or ethanol enhances the production of various organic compounds, including organic acids and sugar alcohol [7, 14, 21, 22]. Methanol has a profound effect on the metabolism of sugars by A. niger. Maddox et al. [9] reported that the effect of methanol is at the cell permeability level, allowing metabolites to be excreted from the cell. Additionally, methanol has a direct effect on mycelial morphology and promotes pellet formation. It also increases the cell membrane permeability to provoke more citric acid excretion from the mycelial cells. Kumar et al. [8] and Raukas [13] reported that addition of 1- 6 % methanol resulted in a marked increase in the amount of citric acid formed by A. niger on fruits waste and the carob pod extracts, respectively. Raukas [14] found that addition of 6 % methanol affected the gluconic acid production from figs by A. niger in solid-state fermentation. Methanol is not assimilated by A. niger and its exact role in stimulating the production of citric or gluconic acid by A. niger is still not clear [7, 9]. Stimulatory effects of methanol on the xylitol production by Candida boidinni and Petromyces albertensis were also shown by Vongsuvanlert and Tani [21] and Dahiya [4], respectively. In contrast, Saha and Bothast [18] reported that methanol failed to increase the production of xylitol from xylose by Candida peltata.

A. niger produces oxalic acid as a by-product which causes problems with downstream processing of industrial production of citric acid. On the other hand, bio-hydrometallurgy groups are interested in the production of high amounts of oxalic acid on a large scale and at low pH. Due to its high reduction power, oxalic acid can be used for the removal of iron present in kaolin as impurity [2, 5]. Among the microorganisms accumulated in oxalic acid, A. niger is a very efficient oxalate producer, what has been reported by many authors [1, 3 , 6, 16]. According to Strasser et al. [20] sucrose and lactose permeate were suitable carbon sources for oxalic acid production. In sucrose medium A. niger produced high amounts of gluconic and oxalic acids, whereas in lactose permeate medium only oxalic acid was produced. Our previous work showed, that raw lipid materials can be regarded as efficient substrates for the biosynthesis of oxalic acid by A. niger at low pH rang ed from 4 to 5 [16, 17]. Production of oxalic acid from lipids by A. niger in the presence of methanol has not been investigated up till now.

The purpose of the present investigation was to determine the stimulating value of methanol on the production of oxalic acid from post-refining fatty acids by A. niger.

MATERIALS AND METHODS

Microorganism and Growth

The experiments were carried out with a high yielding strain of A. niger XP purchased from the Wroclaw University of Economics (Poland). The strain was maintained on potato dextrose agar. Spores from a slant agar were suspended in Tween 80 solution 0.1 %. Cultures were inoculated with spores solution to a final concentration of 10⁶/mL. The inoculation medium consisted (per liter) of 30 g post-refining fatty acids [by-product of plant oil production containing (% w/w) free fatty acids: 9.4 C_16:0, 3.3 C_18:0, 48.1 C_18:1, 31.2 C_18:2, 5.3 C_18:3, 0.8 C_20:1, 0.8 C_22:1], 1 g NH₄NO₃, 2.5 g KH₂PO₄, 0.3 g MgSO_{4 ·} 7H₂O, 0.1g FeSO₄ · 7H₂O, 0.3 g ZnSO₄ · 4H₂O and methanol from 0 to 20 g. The inoculation culture was grown in shaken flasks at 160 rpm at 30 ^oC for 48 h. The fermentation medium consisting (per liter) of 50 g post-refining fatty acids, 1 g NH₄NO₃, 2.5 g KH₂PO₄, 0.3 g MgSO₄ · 7H₂O, 0.1g FeSO₄ · 7H₂O, 0.3 g ZnSO₄ · 4H₂O and methanol from 0 to 30 g. Initial fatty acids concentration in the medium was 10 g/l. Additional acids were added between 48 h and 144 h at a constant feed rate of 1 g/h to final fatty acids concentration of 50 g/l. Methanol was added after sterilization of the medium. Fermentations were carried out in 5-l stirred tank reactor with a working volume of 2.5 l at 30 °C. 2.3 l of the fermentation medium was inoculated with 0.2 l of the inoculum culture. The aeration rate was fixed at 1 vvm. The agitation speed was adjusted to 500 rpm. The pH at 4 was automatically maintained by addition of 10 M KOH.

Analytical Methods

The biomass dry weight in the samples was determined by harvesting the mycelium by filtration through a pre-weighed membrane filter (cellulose nitrate filter, 1.2 µm pore size, Millipore) and drying at 80°C to a constant weight. Unconsumed lipids were extracted from the culture medium twice, using petroleum ether as solvent. The organic phase was dried at 50 °C to a constant weight. The concentrations of oxalic and citric acids were determined using HPLC on an Aminex HPX87H Organic Acids Column coupled to a UV detector at 210 nm. The column was eluted with 20 mM H₂SO₄ at room temperature and a flow rate of 0.6 ml/min.

RESULTS AND DISCUSSION

The stimulatory effects of methanol on the formation of oxalic acid by A. niger XP strain in post-refining fatty acids containing media (50 g/l) were investigated in submerged fermentation experiments. Methanol was added either throughout the fermentation period or at the time of inoculation culture. In the first part of the investigations, methanol was added throughout the fermentation period. Concentration of methanol in the fermentation medium ranged from 0 to 3 % (w/v). Table 1A shows the final oxalic acid concentration, as well as the overall volumetric oxalic acid productivity and yields during several fermentations in the presence of methanol concentrations varying from 0 to 3 % (w/v). As can be seen, addition of methanol into the fermentation medium resulted in a significant increase in oxalic acid production from fatty acids. The observed increase in oxalic acid concentrations and yields show, that methanol has a profound effect on the metabolism of lip ids by A. niger. The highest oxalate concentration and yield were 73 g/l and 1.56 g/g, respectively, and were obtained at 2 % (w/v) concentration of methanol after 7 d. According to Camessele et al. [3] and Santoro et al. [19], maximum oxalate yields of 0.4-0.5 g/g were reached for the strains of A. niger growing on lactose in batch cultures. Methanol at a level of 3 % (w/v) was an inhibitor, as it presence in the fermentation medium caused significantly lower oxalate concentration and yield by A. niger XP. Under this condition, concentration of citric acid, an undesired by-product, and fungal biomass weight (21.1 g/l), were the highest. In our experiments addition of methanol from 0 to 3 % increased amount of the fungal biomass from 12 to 21 g/l (Table 1A). Kumar et al. [8] and Haq et al. [7] reported that addition of methanol into medium containing fruits waste and cane molasses, reduced growth of the A. niger but increased citric acid yield. As can be seen, the excretion of citric acid by A. niger XP started after 2 days of fermentation process reached 13.2 g/l for 7 d (Fig. 1). Haq et al. [7] and Manonmani and Sreekantiah [10] demonstrated that methanol or ethanol addition to the production medium changed the activity of some enzymes of the tricarboxylic acid cycle or related with this cycle, rendering them suitable for citric acid accumulation. Addition of ethanol resulted in 2-fold increase in citrate synthase activity (enzyme responsible for citrate synthesis) and 75 % decrease in the aconitase activity. Previously, methanol was reported to enhance fungal production of citric acid from different raw materials [8, 13, 22]. These authors reported that methanol affects the permeability properties of the cell membrane and enables greater excretion of organic acids. Our study shows that methanol is also a good stimulant for oxalic acid production from fatty acids by A. niger in the stirred ferme ntor.

In other studies we examined oxalate production during submerged fermentation, when methanol was added at the time of inoculation. Concentration of methanol ranged from 0 to 2 % (w/v). The effects of methanol concentration added to inoculum culture on the kinetics of fatty acids fermentation is shown in Table 1B. All the fermentation parameters, including biomass dry weight, increased with the increase in methanol concentration from 0 to 2 % (w/v). This kind of addition of methanol was more efficient as compared to that described above, because A. niger XP did not produce citric acid. When 1.5 % (w/v) methanol was added into inoculum culture, the concentration of oxalic acid and the product yield increased up to 75 g/l and 1.57 g/g in 7 d, respectively, which was 1.4 and 1.34 times higher than without methanol. Our data are not in agreement with the results reported by Bohlmann et al. [1], which suggested that concentration of 45 g/l oxalic acid inhibits completely the fermentation process. These authors reported production of 41 g/l oxalic acid from 180g/l milk whey in 13 d. Production of 38 g/l oxalic acid, which is close to the border of sodium oxalate solubility, was reported by Strasser et al. [20], using a fed-batch process at pH 6 with sucrose as the carbon source.

Further increase in methanol concentration up to 2 % (w/v) did not enhance oxalic acid accumulation by XP strain. The addition of methanol not only increased oxalate production but also influenced pellet formation (data not shown). Paul et al. [11] demonstrated that palleted fermentation gave significantly higher oxalic acid concentration than the non-pelleted one. Figure 2 shows the variations of volumetric oxalic acid productivity during batch culture on fatty acids supplemented with methanol. The maximum oxalate productivity (14-18 g/lˇd) was maintained for 2 to 4 d of fermentation. This corresponded to a 1.8–fold increase in productivity, in comparison with the results reported by other authors [4, 20]. Bohlmann et al. [1] reported that the production of oxalic acid from milk whey started slowly but from sixth day onwards the maximum oxalate production rate of 6.5 g/lˇd was maintained for one week.

Of the oxalate producing microorganisms quoted in the literature, the mutant A. niger XP from renewable lipid resources such as post-refining fatty acids produced the highest oxalic acid concentration and yield when methanol was added to inoculum culture.

Our data show that methanol is favorable for oxalate biosynthesis by A. niger XP on lipids media and is an important stimulant in determining the overall performance of oxalic acid processes. The activity of OAH, the enzyme believed to be responsible for oxalate formation in A. niger, correlated with oxalate production [12, 15]. Further investigation concerning determination of OAH (OAH, EC.3.7.1.1) activity in A. niger XP cultured on the lipids media, containing different concentration of methanol can explain correlation between presence of methanol, activity of OAH and oxalic acid accumulation by this strain.

ACKNOWLEDGEMENTS

This work was financed by the State Committee for Scientific Research (KBN) within the project No 2P06T 113 26.

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