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

Changes in the physiological properties and kinetics of citric acid accumulation via carbon ion irradiation mutagenesis of Aspergillus niger 碳离子束诱导黑曲霉生理特性的改变与其柠檬酸积累关系的研究 1 2 first-author 1 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:chjh@impcas.ac.cnwjli@impcas.ac.cn †E-mail:chjh@impcas.ac.cnwjli@impcas.ac.cn chjh@impcas.ac.cn 1 2 1 2 1 2 1 1 2 corresp ‡Corresponding Author ‡Corresponding Author †E-mail:chjh@impcas.ac.cnwjli@impcas.ac.cn †E-mail:chjh@impcas.ac.cnwjli@impcas.ac.cn chjh@impcas.ac.cn Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000, China Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000, China Key laboratory of Microbial Resources Exploition and Application, Lanzhou 730000, China Key laboratory of Microbial Resources Exploition and Application, Lanzhou 730000, China 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. 目的 2 探讨黑曲霉生理特性的改变对其柠檬酸积累的影响。 创新点 2 首次报道了碳离子束对黑曲霉生理特性的影响，提出黑曲霉生理特性的改变对其柠檬酸的积累有影响。 方法 2 （1）孢子直径和孢子活力的测定：利用流式细胞仪对两株黑曲霉突变菌株以及原始菌株进行了孢子直径测定。通过该实验说明碳离子束可以诱导黑曲霉孢子直径发生改变（表1）。利用MTT法，测定了突变菌株和原始菌株孢子活力。同时，通过该实验表明碳离子束可以诱导黑曲霉孢子活力发生改变（图1）。（2）生长速率的测定：利用不同糖分的平板生长法测定了突变体和原始菌株生长速度的差异。通过该实验说明碳离子束可以诱导黑曲霉生长速率发生改变（图3和图4）。（3）柠檬酸积累实验：利用摇瓶发酵实验和10 L发酵罐扩培实验测定了突变体和原始菌株柠檬酸积累的差异。通过该实验说明生理特性改变的突变体与原始菌株在柠檬酸积累上存在显著差异（图6~8）。 结论 2 通过两株黑曲霉突变体与原始菌株之间生理特性的差异研究，我们发现黑曲霉生理特性的改变对其柠檬酸积累有一定的影响，低的黑曲霉孢子活力以及生长速率对柠檬酸的积累是有益的。最终，获得了一株高产菌株HW2，其10 L发酵罐柠檬酸的积累能力比原始菌株提高了18%。 Carbon ion irradiation Physiological properties Mutation Citric acid accumulation 碳离子束辐照 生理特性 突变 柠檬酸积累 Introduction 1 Citric acid, as an important microbial fermentation product, is widely used in various industrial applications due to its physiological advantages (Mostafa and Alamri, 2012 2012 ; Angumeenal and Venkappayya, 2013 2013 ). Several microorganisms (Betiku and Adesina, 2013 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 1995 ; Schuster et al., 2002 2002 ; Pel et al., 2007 2007 ; Wang et al., 2015 2015 ). Different cheap raw materials have been employed to produce citric acid, including starch materials such as corn starch (Hu et al., 2014b 2014b ), Yam bean starch (Sarangbin and Watanapokasin, 1999 1999 ), and liquefied corn (Hu et al., 2014a 2014a ), and cheap agricultural products such as orange peel (Torrado et al., 2011 2011 ), apple pomace (Dhillon et al., 2011b 2011b ), whey, and sweet potatoes (Betiku and Adesina, 2013 2013 ). Worldwide citric acid production by an industrial-scale process of fermentation is 1.7×10 6 t/a (Dhillon et al., 2011a 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 2000 ; Haq et al., 2003 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 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 2007 ; Javed et al., 2010 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 2013 ; Zhang et al., 2015 2015 ), and include atmospheric and room temperature plasma mutagenesis technology in Beijing (Li et al., 2008 2008 ; Wang et al., 2010 2010 ; Lu et al., 2011 2011 ; Zhang et al., 2014 2014 ), and medium or high-energy heavy ion irradiation technology in Lanzhou, China. Specifically, heavy-ion beams, such as 12 C 6+ , He 2+ , Ar 3+ , Zn 2+ , C 4+ , and C 5+ (Yang et al., 2013 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 2007 ; Li S.W. et al., 2011 2011 ; Ota et al., 2013 2013 ; Zhou et al., 2013 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 2006 ; Wang et al., 2009 2009 ; Kazama et al., 2011 2011 ; Liu Q.F. et al., 2013 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 2013 ), Nannochloropsis (Ma et al., 2013 2013 ), A. terreus (Li S.W. et al., 2011 2011 ), Dietzia strains (Zhou et al., 2013 2013 ), and oleaginous yeast (Wang et al., 2009 2009 ) via 12 C 6+ 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 2014a ), was obtained after 12 C 6+ 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 12 C 6+ ion beam irradiation, but there have been few studies of the relationship between changes induced in physiological properties of A. niger via 12 C 6+ ion beam irradiation and citric acid accumulation. The purpose of this study was to obtain different phenotypic A. niger mutants via 12 C 6+ 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 12 C 6+ 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. Materials and methods 1 Strains 2 The original strain, named H4002 (Hu et al., 2014a 2014a ; 2014b 2014b ), is preserved in IMP, CAS. Mutants HW2 and H4 were obtained after 12 C 6+ ion beam irradiation. Conidial diameter 2 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. Conidial viability 2 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 4.65×10 6 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. Scanning electron microscopy 2 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). Colony growth rates 2 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 3.7×10 5 conidia/ml. Details of procedures were reported by Liu et al. ( 2014 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. Citric acid accumulation 2 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. Analytical methods 2 The concentrations of citric acid, total sugar, and biomass accumulation were measured using Fehling reagent (Hu et al., 2014a 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. Results 1 Conidial diameters and viability of mutants and the original strain 2 Mesquita et al. ( 2013 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 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 1 ). FSC: forward-scattered light Strain FSC Original strain 95.16±2.79 H4 103.76±8.22 HW2 96.72±10.90 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 1 ). Error bars indicate the standard deviation of the mean ( n =3) Differences in conidial morphologies and colony growth rates between mutants and the original strain 2 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 2 ). 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 3 and 4 4 ). Error bars indicate the standard deviation of the mean ( n =3). * P< 0.05, ** P< 0.01, compared with the original strain The diameters of transparent halos are often used to screen mutants with a high yield of organic acids (Bai et al., 2004 2004 ; Li S.C. et al., 2011 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 5 ). We conclude that mutant HW2 may exhibit enhanced citric acid accumulation. Error bars indicate the standard deviation of the mean ( n =3). * P< 0.05, compared with the original strain Differences in citric acid accumulation between mutant HW2 and the original strain in the shake flask and bioreactor 2 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 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 7 ). This suggests that mutant HW2 had a stable ability to produce citric acid. Similar results were obtained using a 10-L bioreactor (Fig. 8 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 9 ). All photos were taken at the same magnification (×10) 4 Discussion 2 Conidia are the main physiological structures in Aspergillus (van Leeuwen et al., 2013 2013 ), and have important physiological functions. The physiological properties of conidia have drawn the attention of biologists and breeding experts. Butler and Day ( 1998 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 2014 ). de Nicolas-Santiago et al. ( 2006 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 2014 ). Compared with traditional irradiation methods, LET heavy charged particles show denser ionization along their trajectories and higher RBE (Kiefer, 1992 1992 ; Goodhead, 1999 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 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 2005 ; Murai et al., 2013 2013 ). As a novel and efficient method for generating mutations, 12 C 6+ 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 1996 ; Haq et al., 2003 2003 ). Presently, China produces more than 60% of global citric acid (Hu et al., 2014a 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 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 1999 ; Grimm et al., 2005 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 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 1999 ; Ikram-Ul-Haq et al., 2003 2003 ). Paul et al. ( 1999 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 1996 ; Fukuda et al., 2009 2009 ). Liu et al. ( 2013b 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 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. 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High efficiency degradation crude oil by a novel mutant irradiated from Dietzia strain by 12C6+ heavy ion using response surface methodology Compliance with ethics guidelines Wei HU, Ji-hong CHEN, Shu-yang WANG, Jing LIU, Yuan SONG, Qing-feng WU, and Wen-jian LI declare that they have no conflict of interest. This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1500120 Project supported by the Agriculture Science Technology Achievement Transformation Fund (No. 2013GB24910680), China 2015-05-16 2016-03-15 2015-12-08 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2016 2016 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 04 17