COMPARISON OF DIFFERENT STRAINS OF ASPERGILLUS NIGER FOR OXALIC ACID PRODUCTION FROM LIPID SUBSTRATES

Waldemar Rymowicz, Dorota Lenart

ABSTRACT

One wild-type strain and eight mutants of the fungi species of Aspergillus niger were screened for their ability to produce oxalic acid on lipids media using batch fermentation. All of the tested fungi strains were able to utilize crude rapeseed oil as energy and carbon source. A final oxalic acid concentration ranged from 8 to 66.1 g/L, depending on the strain used. A. niger XP (induced with UV irradiation) was found to be the most suitable for oxalic acid production from lipid substrates. The highest production of oxalic acid (66.1 g/L), the oxalate yield (1.39 g/g) and the overall oxalate productivity (9.4 g/L·d) were obtained when the fermentation medium contained 50 g/L of oil and at pH 5 maintained from the beginning of the fermentation process.

Key words: Aspergillus niger, oxalic acid, biosynthesis, lipids.

INTRODUCTION

Oxalic acid can be produced in a biotechnological process because several microorganisms are capable to produce it into the culture broth [5, 9, 13, 16]. Among the different microorganisms, the fungus A. niger is favored as the best oxalic acid production species. A. niger is capable of producing various organic acids (like citric, gluconic and oxalic) from carbohydrates [11, 12, 15]. Production of oxalic acid with filamentous fungi is largely influenced both by composition of the production medium and the culture conditions such as agitation and aeration [3, 9, 15]. Thus, oxalic acid production by A. niger can be favored maintaining a high pH, ranging from 6 to 7, when the culture medium is enriched with appropriate amounts of nitrogen and phosphorus. The production of high amounts of oxalic acid is of interest for bio-hydrometallurgical applications because oxalic acid can be used for the removal of the iron contained in kaolin as impurity [6, 17]. This allows for an increase in kaolin whiteness index, a very important factor for industrial application. Important for an economically competitive fermentation process is the ability of A. niger to grow and to produce oxalic acid on low cost carbon substrates as renewable products from agriculture and food industry. Up till now, the renewable products such as beet molasses, a green corn syrup and milk whey were used for oxalic acid production by A. niger [4, 8]. There are many data in literature describing biosynthesis of oxalic acid only on such carbohydrates as sucrose, lactose, maltodextrin and xylose by A. niger. Despite significant knowledge of oxalic acid fermentation in both synthetic lactose and sucrose media, little information is available about the production of this compound from crude lipid materials. Lipid substrates were successful used in various fermentations. In the previous investigations showed, that various types of vegetable oils (soya, rapeseed, sunflower, maize, olive and palm) c an be considered for biomass yeasts and citric acid production [7, 10]. According to Aurich et al. [2] citric acid was produced by Y.lipolytica from several refined and cold pressed plant oils and fatty acids with high citrate concentration and yield. According to Adham [1], olive oil at 4 % concentration added to beet molasses caused a considerable increase in citric acid yield from A. niger. In our previous work we showed that post-refining fatty acid (a by-product of plant oil production) was the efficient substrate for the biosynthesis of oxalic acid by a mutant of A. niger [14].

In this report, the results of the screening of 9 fungal cultures A. niger for oxalic acid production from crude rapeseed oil are discussed. The strains were compared for their product yields, specific acid production rates and ratios of oxalic acid to citric acid.

MATERIALS AND METHODS

Microorganisms. Two UV mutants of A. niger (strain 13/33, 13,32) obtained from the strain collection of the Agricultural University of Lublin (Poland), six UV mutants of A. niger (strain CH 11/21, IBR 6/2, SBJ, SBP, MB, XP) obtained from the Wroclaw University of Economics (Poland) and one wild strain F12 obtained from the Agricultural University of Wroclaw were employed.

Culture media. A solid medium contained (g/L): malt extract 30; peptone 3; yeast extract 2; agar 2; at pH 5,5 was used for the maintenance of the mold. The growth medium of the inoculum contained (g/L): crude rapeseed oil (low erucic) 30; KH₂PO₄ 2.5; NH₄NO₃ 1; MgSO₄ · 7H₂O 0.3; FeSO₄ · 7H₂O 0.1; ZnSO₄ · 7H₂O 0.25; in 1L tap water. Lipid fermentation medium contained (g/L): crude rapeseed oil from 50 to 60; KH₂PO₄ 2.5; NH₄NO₃ 1; MgSO₄ · 7H₂O 0.3; FeSO₄ · 7H₂O 0.1; ZnSO₄ · 7H₂O 0.25; in 1 L tap water.

Culture techniques. Spores suspension. Spores from the solid medium were suspended in Tween 80 0.1% and NaCl 0.9% solution and were recounted on a Thoma chamber.

Inoculum. Erlenmeyers of 250 mL with 50 mL of proliferation medium were inoculated with spores at the concentration of 10⁶ spores per mL. The culture temperature was 30 °C, shaking at 160 rpm. The fungus was grown for 2 days and the resulting biosuspension was used as the inoculum (100 mL per bioreactor) for stirred tank reactor experiments.

Culture in a stirred tank reactor. A BIOFLO III (New Bruswick Scienfic) 3-L bioreactor with a working volume of 1.3 L was employed. Cultivation was carried out at 30 °C. The pH was automatically controlled either at 5 after 48 h or at 5 from the beginning of fermentation process by addition of 5N KOH. The aeration rate of 1 vvm and the agitation rate of 400 rpm were maintained. A sample of about 15 mL was taken daily.

Analytical methods. The dry weight of biomass 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 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 constant weight. The concentrations of oxalic and citric acids were determined by HPLC on an Aminex HPX87H Column Organic Acids 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 nine strains of A. niger (one wild-type and eight UV mutants) were screened for their ability to produce oxalic acid from crude rapeseed oil. In the first step of the screening procedure, the submerged fermentations were performed in 3-L stirred tank fermentor using pH-shift technique. This technique was recommended by Cameselle et al. [4] for the oxalic acid production by A. niger on lactose and sucrose media. After 2 days of fermentation, the pH was shifted from 2.5 to 5 and maintained at the same level to the end of fermentation process. The oxalic acid production from lipids by different strains of A. niger in the fermentor is summarized in Table 1. In 9 days submerged cultures, each process varied in the amount of the accumulated acids, the biomass formed, the dynamics of acids production, the oxalate yield coefficient, and the ratios of oxalic acid (OA) to citric acid (CA).

Even though the initial nitrogen concentration was equal in each process, the final biomass concentration varied from 11.7 g/L with SBJ strain to about 19.5 g/L with F12 strain. As can be seen, both the maximum volumetric oxalic acid production rate and the maximum yield were reached with XP strain, 6 g/L·d and 1.04 g/g, respectively. Additionally, the strain XP produced only small quantities of citric acid at oxalic to citric acid ratio of 4. With other strains, concentration of oxalic acid ranged from 4.9 to 51.8 g/L and ratios OA to CA were much lower (0.6 – 2.7). The two strains (CH 11/21 and IBR 6/2) behaved in prepared conditions differently from other ones. With these strains, the final concentration of oxalic acid was lower than citric acid. These A. niger strains were employed by Elimer [7] for citrate production from rapeseed oil. The oxalate yield (based on lipids consumed) ranged from 0.1 g/g with strain SBP to 0.86 and 1.04 g/g with strain 13/33 and XP, respective ly.

The six fungal strains selected from the first screening procedure, which produced the highest amount of oxalic acid, were tested for the production of oxalic acid in stirred culture, where pH was maintained at 5 from the beginning of the fermentation. According to our previous work this kind of cultivation was more efficient as compared to pH-shift technique, because higher oxalate productivity and yield were obtained [14]. Table 2 shows the final concentration of oxalic and citric acids and overall oxalic acid productivity and product yields. Based on the results obtained in these experiments, A. niger XP was selected as the best oxalate producer. In a shorter time of the fermentation (7 days), concentration of oxalic acid was 66.1 g/L. As a result, oxalate productivity of 9.4 g/L·d, specific oxalic acid production rate of 0.81 g/g·d and yield of 1.39 g/g were achieved. This corresponded to a 1.6-and 1.3 fold increase in productivity and yield, r espectively, as compared with the results obtained using pH-shift technique.

According to Bohlmann et. al [3] among various carbohydrates, only lactose can be considered for the oxalate fermentation by A. niger, since other by-products such as citric and gluconic acids are not produced. They showed, that a final concentration of oxalate (41.3 g/L) and yield (0.5 g/g) were reached with strain A. niger 1120 on day 6. In the present work, for all the strains of A. niger investigated, maintaining the pH at 5 from the beginning of fermentation increased oxalate but decreased citrate concentration in the culture medium compared with the results obtained using pH-shift technique. The ratio of oxalate to citrate depended on the particular strain used. The four strains produced very small quantities of citric acid (below 2.7 g/L). Figure 1 shows the variation of volumetric oxalic acid productivities during batch culture by different fungal strains on lipids under investigation. The maximum oxalate productivity (12.3-16.4 g/L& middot;d) was obtained for 2 to 3 days of fermentation with XP strain and (8.5-12 g/L·d) with other strains. The obtained results corresponded to a 1.6- and 2-fold increase in oxalate concentration and productivity, as compared with the results reported by other authors [3, 15].

Finally, it can be concluded that oxalate production with UV mutant strains XP, F12, 13/33 and 13/32 in this study are superior to previous reports in several respects [17]. Firstly, we obtained the highest oxalate concentration and yield among literature reported oxalate-producing microorganisms. Secondly, these strains produced only small quantities of undesired by-product i.e. citric acid, what is important for their potential application on an industrial scale. Our investigations on the production of oxalic acid by A. niger on raw lipid substrates of agro-industrial origin may be considered of interest because they represent the first approach to the utilization of these raw materials for biotechnological purposes.

Further optimization of both compositions of the production medium and the culture conditions will allow for even higher levels of oxalic acid production and consequently better industrial production of oxalic acid by means of microbiological process.

ACKNOWLEDGEMENTS

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

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