Changes in the physiological properties and kinetics of citric acid accumulation via carbon ion irradiation mutagenesis of Aspergillus niger

Abstract

The objective of this work was to produce citric acid from corn starch using a newly isolated mutant of Aspergillus niger, and to analyze the relationship between changes in the physiological properties of A. niger induced by carbon ion irradiation and citric acid accumulation. Our results showed that the physiological characteristics of conidia in A. niger were closely related to citric acid accumulation and that lower growth rate and viability of conidia may be beneficial to citric acid accumulation. Using corn starch as a raw material, a high-yielding citric acid mutant, named HW2, was obtained. In a 10-L bioreactor, HW2 can accumulate 118.9 g/L citric acid with a residual total sugar concentration of only 14.4 g/L. This represented an 18% increase in citric acid accumulation and a 12.5% decrease in sugar utilization compared with the original strain.

1. Introduction

Citric acid, as an important microbial fermentation product, is widely used in various industrial applications due to its physiological advantages (Mostafa and Alamri, 2012; Angumeenal and Venkappayya, 2013). Several microorganisms (Betiku and Adesina, 2013) can produce citric acid through fermentation, including fungi (Aspergillus niger, Penicillium janthinelum, and A. awamori), yeast (Yarrowia lipolytica, Candida oleophila, and Candida tropicalis), and bacteria (Bacillus licheniformis, Corynebacterium sp., and Arthrobacter paraffinens). However, A. niger, as an important microbial cell factory, remains the best choice for the production of citric acid due to its high yield of citric acid and its ability to ferment various cheap raw materials (Grewal and Kalra, 1995; Schuster et al., 2002; Pel et al., 2007; Wang et al., 2015). Different cheap raw materials have been employed to produce citric acid, including starch materials such as corn starch (Hu et al., 2014b), Yam bean starch (Sarangbin and Watanapokasin, 1999), and liquefied corn (Hu et al., 2014a), and cheap agricultural products such as orange peel (Torrado et al., 2011), apple pomace (Dhillon et al., 2011b), whey, and sweet potatoes (Betiku and Adesina, 2013).

Worldwide citric acid production by an industrial-scale process of fermentation is 1.7×10⁶ t/a (Dhillon et al., 2011a), and the demand for citric acid is continuously increasing. China accounts for 60%–70% of the citric acid market share and the raw material used in China is mainly corn starch. Both the continuous growing demand for citric acid and the economics of fermentation encourage the exploration of different technical approaches to obtain improved varieties of A. niger using cheap raw materials for citric acid production (Parekh et al., 2000; Haq et al., 2003). Although physical or chemical mutagenesis agents are not novel and seem to be uneconomic, some remarkable microbial mutants have been obtained through mutagenesis and high-throughput screening (Heerd et al., 2014). Traditional irradiation technologies, such as ultraviolet irradiation, γ-rays, and chemical mutagenesis have been widely and frequently applied to improve the citric acid yield of A. niger (Lotfy et al., 2007; Javed et al., 2010). Also, some new and powerful mutagenesis methods have been applied to the breeding of high-yielding industrial strains. These new methods have technological advantages (higher mutation rates and more abundant phenotypic mutations) (Hu et al., 2013; Zhang et al., 2015), and include atmospheric and room temperature plasma mutagenesis technology in Beijing (Li et al., 2008; Wang et al., 2010; Lu et al., 2011; Zhang et al., 2014), and medium or high-energy heavy ion irradiation technology in Lanzhou, China. Specifically, heavy-ion beams, such as ¹²C⁶⁺, He²⁺, Ar³⁺, Zn²⁺, C⁴⁺, and C⁵⁺ (Yang et al., 2013), as a type of high linear energy transfer (LET) irradiation, have a higher relative biological effect (RBE) compared with X- and γ-rays (Yang et al., 2007; Li S.W. et al., 2011; Ota et al., 2013; Zhou et al., 2013), and are expected to increase mutation frequency and have a wide mutation spectrum. They have been used effectively as a breeding method in plants and microbes (Zhou et al., 2006; Wang et al., 2009; Kazama et al., 2011; Liu Q.F. et al., 2013).

In China, the Heavy Ion Research Facility in Lanzhou (HIRFL) has been founded as a national laboratory and was opened for world-wide use in 1992. It now is an important institute contributing to global microbe breeding. Great progress has been made in radiation breeding of microbes of Desmodesmus sp. (Hu et al., 2013), Nannochloropsis (Ma et al., 2013), A. terreus (Li S.W. et al., 2011), Dietzia strains (Zhou et al., 2013), and oleaginous yeast (Wang et al., 2009) via ¹²C⁶⁺ ion beam irradiation at the Department of Biophysics, Institute of Modern Physics, Chinese Academy of Sciences (IMP, CAS), China. In 2014, a promising A. niger mutant, named H4002 (Hu et al., 2014a), was obtained after ¹²C⁶⁺ ion beam irradiation by IMP, CAS. Based on this mutant, citric acid accumulation can reach up to (187.5±0.7) g/L with extremely high productivity of 3.13 g/(L·h). The mutant has been used widely in industrial scale citric acid production. Citric acid biotechnology of A. niger has been significantly advanced by ¹²C⁶⁺ ion beam irradiation, but there have been few studies of the relationship between changes induced in physiological properties of A. niger via ¹²C⁶⁺ ion beam irradiation and citric acid accumulation.

The purpose of this study was to obtain different phenotypic A. niger mutants via ¹²C⁶⁺ ion beam irradiation. To our knowledge, it was the first time to study the relationships between alterations in the physiological properties of A. niger induced by ¹²C⁶⁺ ion beams and its secondary metabolite accumulation. This study indicated that the physiological characteristics of conidia in A. niger were closely related to citric acid accumulation, which can provide a new alternative way for screening high-yield citric acid A. niger mutants in the future.

2. Materials and methods

2.1. Strains

The original strain, named H4002 (Hu et al., 2014a; 2014b), is preserved in IMP, CAS. Mutants HW2 and H4 were obtained after ¹²C⁶⁺ ion beam irradiation.

2.2. Conidial diameter

Conidia of mutants and the original strain cultivated on potato dextrose agar (PDA)-containing slopes for 6 d were harvested in sterile distilled water, and then filtered through sterile filter paper with an aperture size of 20–25 μm. The filtered liquids were injected into a flow cytometer (MACSQuant™, Germany or FlowSight, USA) for measurement of conidial diameters.

2.3. Conidial viability

Conidia of mutants and the original strain cultivated on PDA-containing slopes for 6 d were harvested in sterile PDA-containing lipid medium (without agar). The conidial concentrations of the mutants and the original strain were all 4.65×10⁶ conidia/ml. A volume of 400 μl of conidial suspensions of mutants and original strain were added to wells in 24-well plates, and were cultivated for 2 h in a shake flask at a speed of 200 r/min. Then, 100 μl 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) liquid (5 mg/ml) was added to each well for 2 h at 50 °C. After standing, 500 μl hydrochloric acid (1 mol/L) was added to each well in the shake flask which was then shaken at a speed of 200 r/min for 10 min, followed by centrifugation (12 000 r/min, 5 min) and supernatant removal. A volume of 400 μl of isopropanol was added to the Eppendorf (EP) tubes for 10 min. After standing, the extraction supernatants of the mutants and original strain were collected followed by centrifugation (12 000 r/min, 5 min). Finally, 200 μl of each extraction supernatant was added to each well in a 96-well plate, and analyzed using an Infinite M200 PRO (Switzerland) microplate reader. The absorbance of each extraction supernatant was read at 560 nm.

2.4. Scanning electron microscopy

Conidia of the mutants and the original strain cultivated on PDA-containing slopes for 6 d were harvested using a sterile bamboo stick, and were shifted to Eppendorf tubes with 200 μl sterile water, followed by centrifugation (12 000 r/min, 5 min). The supernatants were removed after two washing steps with absolute ethyl alcohol, and then viewed using a JSM-5600LV scanning electron microscope (Japan).

2.5. Colony growth rates

Conidia of the mutants and the original strain cultivated on PDA-containing slopes for 6 d were harvested in sterile distilled water. The conidial concentrations were all 3.7×10⁵ conidia/ml. Details of procedures were reported by Liu et al. (2014). The solid plate medium had the following composition: 4.5 g/L glucose or 4.5 g/L maltose or 10 g/L starch as different carbon sources, 3 g/L sodium nitrate, 1 g/L dipotassium phosphate, 0.5 g/L magnesium sulfate, 0.5 g/L potassium chloride, 0.01 g/L ferric sulfate, 20 g/L agar, and 0.2 g/L bromocresol green. The cultivation conditions were 37 °C for 76 h.

2.6. Citric acid accumulation

The fermentation medium contained 120 g/L corn starch and 10.6 g/L nitrogen source material, which were hydrolyzed by α-amylase at 95–98 °C for 30 min in a 10-L bioreactor. Then, the whole medium was autoclaved at 115–118 °C for 30 min. Finally, equiponderant bran seeds of mutant HW2 and the original strain were injected into the 10-L bioreactor. The fermentation temperature was 37 °C, and the rotation speed (450±30) r/min.

2.7. Analytical methods

The concentrations of citric acid, total sugar, and biomass accumulation were measured using Fehling reagent (Hu et al., 2014a). The growth rates of the mutants and original strain were measure by colony diameters. The ratio of acid spot diameter to lawn diameter is defined to RALD. Each experiment was carried out three times.

3. Results

3.1. Conidial diameters and viability of mutants and the original strain

Mesquita et al. (2013) reported that flow cytometry can be an effective tool to assess the size and complexity of A. niger conidia after γ radiation, and that forward-scattered light (FSC) can provide information on spore size. Stentelaire et al. (2001) reported that MTT assay can be used to measure fungal conidial viability. In this study, we used flow cytometry and MTT assay to assess the average diameters and viabilities of conidia of the mutants and the original strain. The HW2 and H4 mutants had slightly larger conidia than the original strain (Table 1).

Table 1.

Comparison of conidial diameters among the original strain, mutant HW2, and mutant H4

Strain	FSC
Original strain	95.16±2.79
H4	103.76±8.22
HW2	96.72±10.90

FSC: forward-scattered light

Using MTT assay, we found that conidial vitality of the H4 strain was higher than that of the original strain. This implies that the metabolic activity of the H4 strain was higher than that of the original strain. The conidial vitality of the HW2 strain was not significantly different from that of the original strain (Fig. 1).

Fig. 1.

Comparison of the vitality of conidia among the original strain, HW2, and H4

Error bars indicate the standard deviation of the mean (n=3)

3.2. Differences in conidial morphologies and colony growth rates between mutants and the original strain

Scanning electron microscopy showed that the proportion of conidia with a wrinkled surface in the HW2 strain was higher than that of the original strain. The proportion in the H4 strain was not significantly different from that of the original strain (Fig. 2).

Fig. 2.

Conidial surface morphologies of the original strain, HW2, and H4

All photos were taken at the same magnification (×5000)

Growth rate experiments showed that when glucose, starch, or maltose was the carbon source, the lawn diameter of HW2 became smaller than that of the original strain, whereas the lawn diameter of the H4 strain was larger when starch or maltose was supplied as the carbon source (Figs. 3 and 4).

Fig. 3.

Effect of carbon source on the lawn diameters of the original strain, HW2, and H4

Error bars indicate the standard deviation of the mean (n=3). ^* P<0.05, ^** P<0.01, compared with the original strain

Fig. 4.

Variation in lawn diameter among the original strain, HW2 strain, and H4 strain in response to different carbon sources

The diameters of transparent halos are often used to screen mutants with a high yield of organic acids (Bai et al., 2004; Li S.C. et al., 2011). We analyzed differences in RALD between mutants and the original strain. When glucose, starch, or maltose was supplied as the carbon source, the RALD of HW2 was significantly larger than that of the original strain, whereas the RALD of the H4 strain was not significantly different from that of the original strain (Fig. 5). We conclude that mutant HW2 may exhibit enhanced citric acid accumulation.

Fig. 5.

Effects of different carbon sources on the RALD of the original strain, HW2, and H4

Error bars indicate the standard deviation of the mean (n=3). ^* P<0.05, compared with the original strain

3.3. Differences in citric acid accumulation between mutant HW2 and the original strain in the shake flask and bioreactor

Using corn starch as a carbon source, HW2 accumulated citric acid and subdued biomass accumulation under different fermentation time in the shake flask (Fig. 6). The genetic stability of the mutant HW2 was also investigated. Through four consecutive generations of citric acid production in the shake flask (48 h), mutant HW2 produced 37–38 g/L citric acid (Fig. 7). This suggests that mutant HW2 had a stable ability to produce citric acid.

Fig. 6.

Citric acid accumulation of the original strain and HW2 with starch as raw material under different fermentation time in a shaking flask

Fig. 7.

Genetic stability of the mutant HW2 (48 h)

Similar results were obtained using a 10-L bioreactor (Fig. 8). Under optimized culture conditions in the 10-L bioreactor, when the initial total sugar concentration was 120 g/L and fermentation time was 53 h, mutant HW2 could produce 118.9 g/L citric acid and the residual total sugar was only 14.4 g/L, whereas, the original strain produced 100.8 g/L citric acid and residual total sugar of 16.2 g/L. Mutant HW2 showed an 18.0% increase in citric acid accumulation and a 12.5% decrease in sugar utilization compared with the original strain. In other words, mutant HW2 own higher sugar-acid conversion rate than the original strain. Hence, the HW2 strain is a more promising strain than the original strain for industrial production of citric acid. Furthermore, during the whole metabolism process, HW2 showed more round and compact pellets than the original strain (Fig. 9).

Fig. 8.

Accumulation of citric acid by the original strain and HW2 in a 10-L bioreactor

Fig. 9.

Pellet morphologies of the original strain and HW2 after different fermentation periods in a 10-L bioreactor

All photos were taken at the same magnification (×10)

4 Discussion

Conidia are the main physiological structures in Aspergillus (van Leeuwen et al., 2013), and have important physiological functions. The physiological properties of conidia have drawn the attention of biologists and breeding experts. Butler and Day (1998) reported that conidia in A. niger showed strong tolerance to UV-rays due to the presence of melanin in conidia. Similar results were found by Liu et al. (2014). de Nicolas-Santiago et al. (2006) reported that differential mannanase production in original A. niger and mutants was associated with different conidial morphologies and diameters. These results proved that the physiological properties of fungal spores were related to their physiological metabolic functions. Changing in the physiological characteristics of microbes via mutagenesis indicated differential secondary metabolite accumulation (Zhang et al., 2014).

Compared with traditional irradiation methods, LET heavy charged particles show denser ionization along their trajectories and higher RBE (Kiefer, 1992; Goodhead, 1999), and result in complex DNA damage in the body, such as large deletions, rearrangements, or translocations, which can generate abundant mutants (Hu et al., 2013). The establishment of different high-throughput screening methods has also enabled some promising mutants to be obtained quickly. These mutants can be useful in functional gene research (Shikazono et al., 2005; Murai et al., 2013). As a novel and efficient method for generating mutations, ¹²C⁶⁺ ion beam irradiation has been widely applied in the breeding of industrial microorganisms in China due to its characteristics of high LET, high RBE, and high mutation rate.

In this study, we firstly showed that carbon ion irradiation may induce alterations in the physiological characteristics of conidia in A. niger. Our results suggested that changes in the physiological properties of A. niger, such as lower growth rate and viability of conidia, may provide a new strategy for screening high-yielding citric acid producing strains. To achieve economic production of citric acid in an industrial-scale process, a supply of cheap raw materials, such as starch materials, and high-yielding strains are highly desirable (Suzuki et al., 1996; Haq et al., 2003). Presently, China produces more than 60% of global citric acid (Hu et al., 2014a), and the raw material for citric acid production in China is mainly corn starch. Therefore, using corn starch as raw material, differences in citric acid accumulation between mutant HW2 and the original strain in shake flask and in a 10-L bioreactor were investigated. HW2 exhibited enhanced citric acid production and sugar-acid conversion rates compared with the original strain. Hu et al. (2014b) reported that, when using corn starch as a carbon source, the original strain used in this study could accumulate 187 g/L citric acid within 68 h. Therefore, we conclude that a more promising citric acid-producing strain was obtained via carbon ion irradiation. Morphological differences in pellets between mutant HW2 and the original strain during the fermentation process were also observed. It has been suggested that the morphology of filamentous fungi is strongly related to their productivity (Paul et al., 1999; Grimm et al., 2005). In this study, it was observed that, compared with the original strain, smaller pellets in fermentation broth in A. niger mutants may enhance citric acid accumulation (Fig. 8). Thus, heavy ion irradiation can be an effective tool for inducing significant alterations in physiological characteristics in microbes, including conidial growth rate, conidial vitality, and pellet morphology, which are related to secondary metabolite accumulation.

Studies of the relationship between morphology and citric acid production in A. niger have been reported (Papagianni et al., 1999; Ikram-Ul-Haq et al., 2003). Paul et al. (1999) discussed how morphology in A. niger affected citric acid accumulation and several other variables, such as oxygen uptake, glucose uptake, and carbon dioxide production. Chitin synthase genes are important in determining the hyphal morphology and conidial development of A. nidulans (Borgia et al., 1996; Fukuda et al., 2009). Liu et al. (2013b) reported that the chitin synthase gene can influence the morphology of P. chrysogenum, and morphological changes induced by class III chitin synthase gene silencing were related to penicillin production by P. chrysogenum (Liu et al., 2013a). In our study, the mutants HW2 and H4 had different colony and pellet morphologies from those of the original strain. Citric acid accumulation by A. niger may be associated with mutation in chitin synthase genes induced by carbon ion irradiation. We also found preliminary evidence that the expression levels of key genes involved in the pathway of starch bio-degradation, such as the glucoamylase gene and starch degradation regulation gene, showed obvious differences between the mutant and original strains. These findings suggest possible mechanisms to explain why the mutant strains HW2 and H4 showed differential citric acid accumulation compared with the original strain. This may be worthy of further study in the future.

Acknowledgements

The authors are grateful to all the staff of the Heavy Ion Research Facility in Lanzhou, China for providing the carbon beams.