JZUS - Journal of Zhejiang University SCIENCE

Journal of Zhejiang University SCIENCE B 2016 Vol.17 No.4 P.262-270

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

Author(s): Wei Hu, Ji-hong Chen, Shu-yang Wang, Jing Liu, Yuan Song, Qing-feng Wu, Wen-jian Li
Affiliation(s): Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000, China; more
Corresponding email(s): chjh@impcas.ac.cn, wjli@impcas.ac.cn
Key Words: Carbon ion irradiation, Physiological properties, Mutation, Citric acid accumulation

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Abstract: The objective of this work was to produce citric acid from corn starch using a newly isolated mutant of Aspergillus , and to analyze the relationship between changes in the physiological properties of A. 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.

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

目的：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：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：（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：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%。

关键词：碳离子束辐照；生理特性；突变；柠檬酸积累

Reference

[1]Angumeenal, A.R., Venkappayya, D., 2013. An overview of citric acid production. LWT-Food Sci. Technol., 50(2):367-370.

[2]Bai, D.M., Zhao, X.M., Li, X.G., et al., 2004. Strain improvement of Rhizopus oryzae for over-production of L(+)-lactic acid and metabolic flux analysis of mutants. Biochem. Eng. J., 18(1):41-48.

[3]Betiku, E., Adesina, O.A., 2013. Statistical approach to the optimization of citric acid production using filamentous fungus Aspergillus niger grown on sweet potato starch hydrolyzate. Biomass Bioenerg., 55:350-354.

[4]Borgia, P.T., Iartchouk, N., Riggle, P.J., et al., 1996. The chsB gene of Aspergillus nidulans is necessary for normal hyphal growth and development. Fungal Genet. Biol., 20(3):193-203.

[5]Butler, M.J., Day, A.W., 1998. Fungal melanins: a review. Can. J. Microbiol., 44(12):1115-1136.

[6]de Nicolas-Santiago, S., Regalado-Gonzalez, C., Garcia-Almendarez, B., et al., 2006. Physiological, morphological, and mannanase production studies on Aspergillus niger uam-gs1 mutants. Electron. J. Biotechnol., 9(1):50-60.

[7]Dhillon, G.S., Brar, S.K., Verma, M., et al., 2011a. Recent advances in citric acid bio-production and recovery. Food Bioprocess Technol., 4(4):505-529.

[8]Dhillon, G.S., Brar, S.K., Verma, M., et al., 2011b. Utilization of different agro-industrial wastes for sustainable bioproduction of citric acid by Aspergillus niger. Biochem. Eng. J., 54(2):83-92.

[9]Fukuda, K., Yamada, K., Deoka, K., et al., 2009. Class III chitin synthase chsB of Aspergillus nidulans localizes at the sites of polarized cell wall synthesis and is required for conidial development. Eukaryot. Cell, 8(7):945-956.

[10]Goodhead, D.T., 1999. Mechanisms for the biological effectiveness of high-LET radiations. J. Radiat. Res., 40:S1-S13.

[11]Grewal, H.S., Kalra, K.L., 1995. Fungal production of citric acid. Biotechnol. Adv., 13(2):209-234.

[12]Grimm, L.H., Kelly, S., Krull, R., et al., 2005. Morphology and productivity of filamentous fungi. Appl. Microbiol. Biotechnol., 69(4):375-384.

[13]Haq, I.U., Ali, S., Iqbal, J., 2003. Direct production of citric acid from raw starch by Aspergillus niger. Process Biochem., 38(6):921-924.

[14]Heerd, D., Tari, C., Fernandez-Lahore, M., 2014. Microbial strain improvement for enhanced polygalacturonase production by Aspergillus sojae. Appl. Microbiol. Biotechnol., 98(17):7471-7481.

[15]Hu, G.R., Fan, Y., Zhang, L., et al., 2013. Enhanced lipid productivity and photosynthesis efficiency in a Desmodesmus sp. mutant induced by heavy carbon ions. PLoS ONE, 8(4):e60700.

[16]Hu, W., Liu, J., Chen, J.H., et al., 2014a. A mutation of Aspergillus niger for hyper-production of citric acid from corn meal hydrolysate in a bioreactor. J. Zhejiang Univ.-Sci. B (Biomed. & Biotechnol.), 15(11):1006-1010.

[17]Hu, W., Chen, J.H., Li, W.J., et al., 2014b. Mutant breeding of Aspergillus niger irradiated by ¹²C⁶⁺ for hyper citric acid. Nucl. Sci. Tech., 25(2):020302.

[18]Ikram-Ul-Haq, Ali, S., Qadeer, M.A., et al., 2003. Control of Aspergillus niger morphology to enhance citric acid production under liquid culture. Pak. J. Bot., 35(4):533-539.

[19]Javed, S., Asgher, M., Sheikh, M.A., et al., 2010. Strain improvement through UV and chemical mutagenesis for enhanced citric acid production in molasses-based solid state fermentation. Food Biotechnol., 24(2):165-179.

[20]Kazama, Y., Hirano, T., Saito, H., et al., 2011. Characterization of highly efficient heavy-ion mutagenesis in Arabidopsis thaliana. BMC Plant Biol., 11(1):161.

[21]Kiefer, J., 1992. Heavy-ion effects on cells: chromosomal-aberrations, mutations and neoplastic transformations. Radiat. Environ. Bioph., 31(4):279-288.

[22]Li, G., Li, H.P., Wang, L.Y., et al., 2008. Genetic effects of radio-frequency, atmospheric-pressure glow discharges with helium. Appl. Phys. Lett., 92(22):221504.

[23]Li, S.C., Zhu, Z.Y., Gu, S.B., et al., 2011. Mutation-screening in L-(+)-lactic acid producing strains by ion implantation. Indian J. Microbiol., 51(2):138-143.

[24]Li, S.W., Li, M., Song, H.P., et al., 2011. Induction of a high-yield lovastatin mutant of Aspergillus terreus by ¹²C⁶⁺ heavy-ion beam irradiation and the influence of culture conditions on lovastatin production under submerged fermentation. Appl. Biochem. Biotechnol., 165(3-4):913-925.

[25]Liu, H., Zheng, Z.M., Wang, P., et al., 2013a. Morphological changes induced by class III chitin synthase gene silencing could enhance penicillin production of Penicillium chrysogenum. Appl. Microbiol. Biotechnol., 97(8):3363-3372.

[26]Liu, H., Wang, P., Gong, G.H., et al., 2013b. Morphology engineering of Penicillium chrysogenum by RNA silencing of chitin synthase gene. Biotechnol. Lett., 35(3):423-429.

[27]Liu, J., Zhou, L., Chen, J.H., et al., 2014. Role of ozone in UV-C disinfection, demonstrated by comparison between wild-type and mutant conidia of Aspergillus niger. Photochem. Photobiol., 90(3):615-621.

[28]Liu, Q.F., Wang, Z.Z., Zhou, L.B., et al., 2013. Relationship between plant growth and cytological effect in root apical meristem after exposure of wheat dry seeds to carbon ion beams. Nucl. Instrum. Meth. Phys. Res. B, 305:9-15.

[29]Lotfy, W.A., Ghanem, K.M., El-Helow, E.R., 2007. Citric acid production by a novel Aspergillus niger isolate: I. mutagenesis and cost reduction studies. Bioresource Technol., 98(18):3464-3469.

[30]Lu, Y., Wang, L.Y., Ma, K., et al., 2011. Characteristics of hydrogen production of an Enterobacter aerogenes mutant generated by a new atmospheric and room temperature plasma (ARTP). Biochem. Eng. J., 55(1):17-22.

[31]Ma, Y.B., Wang, Z.Y., Zhu, M., et al., 2013. Increased lipid productivity and TAG content in Nannochloropsis by heavy-ion irradiation mutagenesis. Bioresource Technol., 136:360-367.

[32]Mesquita, N., Portugal, A., Pinar, G., et al., 2013. Flow cytometry as a tool to assess the effects of gamma radiation on the viability, growth and metabolic activity of fungal spores. Int. Biodeter. Biodegr., 84:250-257.

[33]Mostafa, Y.S., Alamri, S.A., 2012. Optimization of date syrup for enhancement of the production of citric acid using immobilized cells of Aspergillus niger. Saudi J. Biol. Sci., 19(2):241-246.

[34]Murai, K., Nishiura, A., Kazama, Y., et al., 2013. A large-scale mutant panel in wheat developed using heavy-ion beam mutagenesis and its application to genetic research. Nucl. Instrum. Meth. Phys. Res. B, 314:59-62.

[35]Ota, S., Matsuda, T., Takeshita, T., et al., 2013. Phenotypic spectrum of Parachlorella kessleri (Chlorophyta) mutants produced by heavy-ion irradiation. Bioresource Technol., 149:432-438.

[36]Papagianni, M., Mattey, M., Berovic, M., et al., 1999. Aspergillus niger morphology and citric acid production in submerged batch fermentation: effects of culture pH, phosphate and manganese levels. Food Technol. Biotechnol., 37(3):165-171.

[37]Parekh, S., Vinci, V.A., Strobel, R.J., 2000. Improvement of microbial strains and fermentation processes. Appl. Microbiol. Biotechnol., 54(3):287-301.

[38]Paul, G.C., Priede, M.A., Thomas, C.R., 1999. Relationship between morphology and citric acid production in submerged Aspergillus niger fermentations. Biochem. Eng. J., 3(2):121-129.

[39]Pel, H.J., de Winde, J.H., Archer, D.B., et al., 2007. Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88. Nat. Biotechnol., 25(2):221-231.

[40]Sarangbin, S., Watanapokasin, Y., 1999. Yam bean starch: a novel substrate for citric acid production by the protease-negative mutant strain of Aspergillus niger. Carbohyd. Polym., 38(3):219-224.

[41]Schuster, E., Dunn-Coleman, N., Frisvad, J.C., et al., 2002. On the safety of Aspergillus niger—a review. Appl. Microbiol. Biot., 59(4-5):426-435.

[42]Shikazono, N., Suzuki, C., Kitamura, S., et al., 2005. Analysis of mutations induced by carbon ions in Arabidopsis thaliana. J. Exp. Bot., 56(412):587-596.

[43]Stentelaire, C., Antoine, N., Cabrol, C., et al., 2001. Development of a rapid and highly sensitive biochemical method for the measurement of fungal spore viability. An alternative to the CFU method. Enzyme Microb. Technol., 29(8-9):560-566.

[44]Suzuki, A., Sarangbin, S., Kirimura, K., et al., 1996. Direct production of citric acid from starch by a 2-deoxyglucose-resistant mutant strain of Aspergillus niger. J. Ferment. Bioeng., 81(4):320-323.

[45]Torrado, A.M., Cortes, S., Salgado, J.M., et al., 2011. Citric acid production from orange peel wastes by solid-state fermentation. Braz. J. Microbiol., 42(1):394-409.

[46]van Leeuwen, M.R., Krijgsheld, P., Bleichrodt, R., et al., 2013. Germination of conidia of Aspergillus niger is accompanied by major changes in RNA profiles. Stud. Mycol., 74(1):59-70.

[47]Wang, J.F., Li, R.M., Lu, D., et al., 2009. A quick isolation method for mutants with high lipid yield in oleaginous yeast. World J. Microb. Biot., 25(5):921-925.

[48]Wang, L., Zhang, J.H., Cao, Z.L., et al., 2015. Inhibition of oxidative phosphorylation for enhancing citric acid production by Aspergillus niger. Microb. Cell Fact., 14(1):7.

[49]Wang, L.Y., Huang, Z.L., Li, G., et al., 2010. Novel mutation breeding method for Streptomyces avermitilis using an atmospheric pressure glow discharge plasma. J. Appl. Microbiol., 108(3):851-858.

[50]Yang, Y.N., Ren, N., Xue, J.M., et al., 2007. Mutation effect of MeV protons on bioflocculant bacteria Bacillus cereus. Nucl. Instrum. Meth. Phys. Res. B, 262(2):220-224.

[51]Yang, Y.N., Liu, C.L., Wang, Y.K., et al., 2013. Mutation effects of C²⁺ ion irradiation on the greasy Nitzschia sp. Mut. Res. Fund. Mol. Mech. Mutagen., 751-752:24-28.

[52]Zhang, X., Zhang, X.F., Li, H.P., et al., 2014. Atmospheric and room temperature plasma (ARTP) as a new powerful mutagenesis tool. Appl. Microbiol. Biotechnol., 98(12):5387-5396.

[53]Zhang, X., Zhang, C., Zhou, Q.Q., et al., 2015. Quantitative evaluation of DNA damage and mutation rate by atmospheric and room-temperature plasma (ARTP) and conventional mutagenesis. Appl. Microbiol. Biotechnol., 99(13):5639-5646.

[54]Zhou, L.B., Li, W.J., Yu, L.X., et al., 2006. Linear energy transfer dependence of the effects of carbon ion beams on adventitious shoot regeneration from in vitro leaf explants of Saintpaulia ionahta. Int. J. Radiat. Biol., 82(7):473-481.

[55]Zhou, X., Xin, Z.J., Lu, X.H., et al., 2013. High efficiency degradation crude oil by a novel mutant irradiated from Dietzia strain by ¹²C⁶⁺ heavy ion using response surface methodology. Bioresource Technol., 137:386-393.

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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

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