Applied Microbiology and Biotechnology

, Volume 103, Issue 13, pp 5215–5230 | Cite as

Production of cellulosic butyrate and 3-hydroxybutyrate in engineered Escherichia coli

  • Dragan Miscevic
  • Kajan Srirangan
  • Teshager Kefale
  • Daryoush Abedi
  • Murray Moo-Young
  • C. Perry ChouEmail author
Biotechnological products and process engineering


Being the most abundant renewable organic substance on Earth, lignocellulosic biomass has acted as an attractive and cost-effective feedstock for biobased production of value-added products. However, lignocellulosic biomass should be properly treated for its effective utilization during biotransformation. The current work aimed to demonstrate biobased production of butyrate and 3-hydroxybutyrate (3-HB) in engineered Escherichia coli using pretreated and detoxified aspen tree (Populus tremuloides) wood chips as the feedstock. Various bioprocessing and genetic/metabolic factors limiting the production of cellulosic butyrate and 3-HB were identified. With these developed bioprocessing strategies and strain engineering approaches, major carbons in the hydrolysate, including glucose, xylose, and even acetate, could be completely dissimilated during shake-flask cultivation with up to 1.68 g L−1 butyrate, 8.95 g L−1 3-HB, and minimal side metabolites (i.e., acetate and ethanol) being obtained. Our results highlight the importance of consolidating bioprocess and genetic engineering strategies for effective biobased production from lignocellulosic biomass.


Escherichia coli Lignocellulosic hydrolysate Detoxification Over-liming Butyrate 3-Hydroxybutyrate 



The authors’ research is supported by Natural Sciences and Engineering Research Council (NSERC) and Networks of Centres of Excellence of Canada (BioFuelNet).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2019_9815_MOESM1_ESM.pdf (296 kb)
ESM 1 (PDF 296 kb)


  1. Achinas S, Euverink GJW. (2016). Consolidated briefing of biochemical ethanol production from lignocellulosic biomass. EJB 23(Supplement C):44–53Google Scholar
  2. Akawi L, Srirangan K, Liu X, Moo-Young M, Perry CC (2015) Engineering Escherichia coli for high-level production of propionate. J Ind Microbiol Biotechnol 42(7):1057–1072Google Scholar
  3. Alexeeva S, Hellingwerf KJ, Teixeira de Mattos MJ (2002) Quantitative assessment of oxygen availability: perceived aerobiosis and its effect on flux distribution in the respiratory chain of Escherichia coli. J Bacteriol 184(5):1402–1406Google Scholar
  4. Alriksson B, Horvath IS, Sjöde A, Nilvebrant N-O, Jönsson LJ (2005a) Ammonium hydroxide detoxification of spruce acid hydrolysates. In: Davison BH, Evans BR, Finkelstein M, McMillan JD (eds) Twenty-sixth symposium on biotechnology for fuels and chemicals. Humana Press, Totowa, pp 911–922Google Scholar
  5. Alriksson B, Horváth IS, Sjöde A, Nilvebrant N-O, Jönsson LJ (2005b) Ammonium hydroxide detoxification of spruce acid hydrolysates. Appl Biochem Biotechnol 124(1):911–922Google Scholar
  6. Alriksson B, Sjöde A, Nilvebrant N-O, Jönsson LJ (2006) Optimal conditions for alkaline detoxification of dilute-acid lignocellulose hydrolysates. Appl Biochem Biotechnol 130(1):599–611Google Scholar
  7. Alriksson B, Cavka A, Jönsson LJ (2011) Improving the fermentability of enzymatic hydrolysates of lignocellulose through chemical in-situ detoxification with reducing agents. Bioresour Technol 102(2):1254–1263Google Scholar
  8. Amann E, Ochs B, Abel K-J (1988) Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene 69(2):301–315Google Scholar
  9. Amartey S, Jeffries T (1996) An improvement in Pichia stipitis fermentation of acid-hydrolysed hemicellulose achieved by overliming (calcium hydroxide treatment) and strain adaptation. World J Microbiol Biotechnol 12(3):281–283Google Scholar
  10. Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2Google Scholar
  11. Baek J-M, Mazumdar S, Lee S-W, Jung M-Y, Lim J-H, Seo S-W, Jung G-Y, Oh M-K (2013) Butyrate production in engineered Escherichia coli with synthetic scaffolds. Biotechnol Bioeng 110(10):2790–2794Google Scholar
  12. Balan V (2014) Current challenges in commercially producing biofuels from lignocellulosic biomass. ISRN Biotechnology 2014:31Google Scholar
  13. Banerjee N, Bhatnagar R, Viswanathan L (1981) Inhibition of glycolysis by furfural in Saccharomyces cerevisiae. Euro J Appl Microbiol Biotechnol 11(4):226–228Google Scholar
  14. Bond-Watts BB, Bellerose RJ, Chang MCY (2011) Enzyme mechanism as a kinetic control element for designing synthetic biofuel pathways. Nat Chem Biol 7:222–227Google Scholar
  15. Brodeur G, Yau E, Badal K, Collier J, Ramachandran KB, Ramakrishnan S (2011) Chemical and physicochemical pretreatment of lignocellulosic biomass: a review. J Enzym Res 2011:17Google Scholar
  16. Carriquiry MA, Du X, Timilsina GR (2011) Second generation biofuels: economics and policies. Energy Policy 39(7):4222–4234Google Scholar
  17. Cavka A, Jönsson LJ. 2013. Detoxification of lignocellulosic hydrolysates using sodium borohydride. Bioresour Technol 136 (Supplement C):368–376Google Scholar
  18. Cherepanov PP, Wackernagel W (1995) Gene disruption in Escherichia coli: TcR and KmR cassettes with the option of Flp-catalyzed excision of the antibiotic-resistance determinant. Gene 158(1):9–14Google Scholar
  19. Choi YJ, Lee J, Jang Y-S, Lee SY (2014) Metabolic engineering of microorganisms for the production of higher alcohols. mBio 5(5):e01524-14Google Scholar
  20. Coz A, Llano T, Cifrián E, Viguri J, Maican E, Sixta H (2016) Physico-chemical alternatives in lignocellulosic materials in relation to the kind of component for fermenting purposes. Materials 9(7):574Google Scholar
  21. Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci 97(12):6640–6645Google Scholar
  22. Delidovich I, Hausoul PJC, Deng L, Pfützenreuter R, Rose M, Palkovits R (2016) Alternative monomers based on lignocellulose and their use for polymer production. Chem Rev 116(3):1540–1599Google Scholar
  23. Dionisi D, Anderson JA, Aulenta F, McCue A, Paton G (2015) The potential of microbial processes for lignocellulosic biomass conversion to ethanol: a review. J Chem Technol Biotechnol 90(3):366–383Google Scholar
  24. Dwidar M, Park J-Y, Mitchell RJ, Sang B-I (2012) The future of butyric acid in industry. Sci World J 2012:471417Google Scholar
  25. Egoburo DE, Diaz Peña R, Alvarez DS, Godoy MS, Mezzina MP, Pettinari MJ (2018) Microbial cell factories à la carte: elimination of global regulators Cra and ArcA generates metabolic backgrounds suitable for the synthesis of bioproducts in Escherichia coli. Appl Environ Microbiol 84Google Scholar
  26. El-Shahawy Tarek Abd E-G. (2015) Chemicals with a natural reference for controlling water hyacinth, Eichhornia crassipes (Mart.) Solms. J Plant Protect Res. p 294Google Scholar
  27. Entin-Meer M, Rephaeli A, Yang X, Nudelman A, VandenBerg SR, Haas-Kogan DA (2005) Butyric acid prodrugs are histone deacetylase inhibitors that show antineoplastic activity and radiosensitizing capacity in the treatment of malignant gliomas. Mol Cancer Ther 4(12):1952–1961Google Scholar
  28. Fakhrudin J, Setyaningsih D, Rahayuningsih M (2014) Bioethanol production from seaweed Eucheuma cottonii by neutralization and detoxification of acidic catalyzed hydrolysate. 455–458 p.Google Scholar
  29. Fenske JJ, Griffin DA, Penner MH (1998) Comparison of aromatic monomers in lignocellulosic biomass prehydrolysates. J Ind Microbiol Biotechnol 20(6):364–368Google Scholar
  30. Förster AH, Gescher J (2014) Metabolic engineering of Escherichia coli for production of mixed-acid fermentation end products. Front Bioeng Biotechnol 2(16)Google Scholar
  31. Gibson DG, Young L, Chuang R-Y, Venter JC, Hutchison CA, Smith HO (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat Meth 6(5):343–345Google Scholar
  32. Guevara-Martínez M, Gällnö KS, Sjöberg G, Jarmander J, Perez-Zabaleta M, Quillaguamán J, Larsson G (2015) Regulating the production of (R)-3-hydroxybutyrate in Escherichia coli by N or P limitation. Front Microbiol 6(AUG):844Google Scholar
  33. Gulevich AY, Skorokhodova AY, Sukhozhenko AV, Debabov VG (2017) Biosynthesis of enantiopure (S)-3-hydroxybutyrate from glucose through the inverted fatty acid β-oxidation pathway by metabolically engineered Escherichia coli. J Biotechnol 244(Supplement C):16–24Google Scholar
  34. Gunsalus RP (1992) Control of electron flow in Escherichia coli: coordinated transcription of respiratory pathway genes. J Bacteriol 174(22):7069–7074Google Scholar
  35. Heer D, Sauer U (2008) Identification of furfural as a key toxin in lignocellulosic hydrolysates and evolution of a tolerant yeast strain. Microbial Biotechnol 1(6):497–506Google Scholar
  36. Holt RA, Stephens GM, Morris JG (1984) Production of solvents by Clostridium acetobutylicum cultures maintained at neutral pH. Appl Environ Microbiol 48(6):1166–1170Google Scholar
  37. Jang Y-S, Woo HM, Im JA, Kim IH, Lee SY (2013) Metabolic engineering of Clostridium acetobutylicum for enhanced production of butyric acid. Appl Microbiol Biotechnol 97(21):9355–9363Google Scholar
  38. Jarmander J, Belotserkovsky J, Sjöberg G, Guevara-Martínez M, Pérez-Zabaleta M, Quillaguamán J, Larsson G (2015) Cultivation strategies for production of (R)-3-hydroxybutyric acid from simultaneous consumption of glucose, xylose and arabinose by Escherichia coli. Microb Cell Factories 14(1):51Google Scholar
  39. Jawed K, Mattam AJ, Fatma Z, Wajid S, Abdin MZ, Yazdani SS (2016) Engineered production of short chain fatty acid in Escherichia coli using fatty acid synthesis pathway. PLoS One 11(7):e0160035Google Scholar
  40. Jha AK, Li J, Yuan Y, Baral N, Ai B (2014) A review on bio-butyric acid production and its optimization. 1019–1024 p.Google Scholar
  41. Jiang Y, Zeng X, Luque R, Tang X, Sun Y, Lei T, Liu S, Lin L (2017) Cooking with active oxygen and solid alkali: a promising alternative approach for lignocellulosic biorefineries. ChemSusChem 10(20):3982–3993Google Scholar
  42. Jobling MG, Holmes RK (1990) Construction of vectors with the p15a replicon, kanamycin resistance, inducible lacZ alpha and pUC18 or pUC19 multiple cloning sites. Nucleic Acids Res 18(17):5315–5316Google Scholar
  43. Jönsson LJ, Palmqvist E, Nilvebrant N-O, Hahn-Hägerdal B (1998) Detoxification of wood hydrolysates with laccase and peroxidase from the white-rot fungus Trametes versicolor. Appl Microbiol Biotechnol 49(6):691–697Google Scholar
  44. Jönsson LJ, Alriksson B, Nilvebrant N-O (2013) Bioconversion of lignocellulose: inhibitors and detoxification. Biotechnology for Biofuels 6:6–16Google Scholar
  45. Kang Y, Weber KD, Qiu Y, Kiley PJ, Blattner FR (2005) Genome-wide expression analysis indicates that FNR of Escherichia coli K-12 regulates a large number of genes of unknown function. J Bacteriol 187(3):1135–1160Google Scholar
  46. Kim SB, Lee JH, Yang X, Lee J, Kim SW (2015) Furfural production from hydrolysate of barley straw after dilute sulfuric acid pretreatment. Korean J Chem Eng 32(11):2280–2284Google Scholar
  47. Kumar R, Shimizu K (2011) Transcriptional regulation of main metabolic pathways of cyoA, cydB, fnr, and fur gene knockout Escherichia coli in C-limited and N-limited aerobic continuous cultures. Microb Cell Factories 10:3–3Google Scholar
  48. Lamsen EN, Atsumi S (2012) Recent progress in synthetic biology for microbial production of C3–C10 alcohols. Front Microbiol 3:196Google Scholar
  49. Lan EI, Liao JC (2012) ATP drives direct photosynthetic production of 1-butanol in cyanobacteria. Proc Natl Acad Sci 109(16):6018–6023Google Scholar
  50. Larsson S, Reimann A, Nilvebrant N-O, Jönsson LJ (1999) Comparison of different methods for the detoxification of lignocellulose hydrolyzates of spruce. Appl Biochem Biotechnol 77(1):91–103Google Scholar
  51. Lee IY, Kim MK, Park YH, Lee SY (1996) Regulatory effects of cellular nicotinamide nucleotides and enzyme activities on poly (3-hydroxybutyrate) synthesis in recombinant Escherichia coli. Biotechnol Bioeng 52(6):707–712Google Scholar
  52. Li H, Chen X, Ren J, Deng H, Peng F, Sun R (2015) Functional relationship of furfural yields and the hemicellulose-derived sugars in the hydrolysates from corncob by microwave-assisted hydrothermal pretreatment. Biotechnol Biofuels 8:127Google Scholar
  53. López MJ, Nichols NN, Dien BS, Moreno J, Bothast RJ (2004) Isolation of microorganisms for biological detoxification of lignocellulosic hydrolysates. Appl Microbiol Biotechnol 64(1):125–131Google Scholar
  54. Martinez A, Rodriguez M, York S, Preston J, Ingram L (2000) Effect of Ca(OH)2 treatments on the composition and toxicity of bagasse hemicellulose hydrolysates. 526–536 p.Google Scholar
  55. Martinez A, Rodriguez ME, Wells ML, York SW, Preston JF, Ingram LO (2001) Detoxification of dilute acid hydrolysates of lignocellulose with lime. Biotechnol Prog 17(2):287–293Google Scholar
  56. Marzan LW, Siddiquee KAZ, Shimizu K (2011) Metabolic regulation of an fnr gene knockout Escherichia coli under oxygen limitation. Bioeng Bugs 2(6):331–337Google Scholar
  57. Miller JH (1992) A short course in bacterial genetics: a laboratory manual and handbook for Escherichia coli and related bacteria. Cold Spring Harbor Laboratory Press, NYGoogle Scholar
  58. Mohr A, Raman S (2013) Lessons from first generation biofuels and implications for the sustainability appraisal of second generation biofuels. Energy Policy 63(Supplement C):114–122.Google Scholar
  59. Nikel PI, Pettinari MJ, Galvagno MA, Méndez BS (2006) Poly(3-hydroxybutyrate) synthesis by recombinant Escherichia coli arcA mutants in microaerobiosis. Appl Microbiol Biotechnol 72(4):2614–2620Google Scholar
  60. Nikel PI, Pettinari MJ, Galvagno MA, Méndez BS (2008a) Poly(3-hydroxybutyrate) synthesis from glycerol by a recombinant Escherichia coli arcA mutant in fed-batch microaerobic cultures. Appl Microbiol Biotechnol 77(6):1337–1343Google Scholar
  61. Nikel PI, Pettinari MJ, Ramírez MC, Galvagno MA, Méndez BS (2008b) Escherichia coli arcA mutants: metabolic profile characterization of microaerobic cultures using glycerol as a carbon source. J Mol Microbiol Biotechnol 15(1):48–54Google Scholar
  62. Nikel PI, de Almeida A, Giordano AM, Pettinari MJ (2010) Redox driven metabolic tuning. Bioeng Bugs 1(4):293–297Google Scholar
  63. Okuda N, Soneura M, Ninomiya K, Katakura Y, Shioya S (2008) Biological detoxification of waste house wood hydrolysate using Ureibacillus thermosphaericus for bioethanol production. J Biosci Bioeng 106(2):128–133Google Scholar
  64. Palmqvist E, Hahn-Hägerdal B, Galbe M, Zacchi G (1996) The effect of water-soluble inhibitors from steam-pretreated willow on enzymatic hydrolysis and ethanol fermentation. Enzym Microb Technol 19(6):470–476Google Scholar
  65. Panagiotopoulos IA, Chandra RP, Saddler JN (2013) A two-stage pretreatment approach to maximise sugar yield and enhance reactive lignin recovery from poplar wood chips. Bioresour Technol 130:570–577Google Scholar
  66. Park DM, Akhtar MS, Ansari AZ, Landick R, Kiley PJ (2013) The bacterial response regulator ArcA uses a diverse binding site architecture to regulate carbon oxidation globally. PLoS Genet 9(10):e1003839Google Scholar
  67. Perez-Zabaleta M, Sjöberg G, Guevara-Martínez M, Jarmander J, Gustavsson M, Quillaguamán J, Larsson G (2016) Increasing the production of (R)-3-hydroxybutyrate in recombinant Escherichia coli by improved cofactor supply. Microb Cell Factories 15:91Google Scholar
  68. Perrenoud A, Sauer U (2005) Impact of global transcriptional regulation by ArcA, ArcB, Cra, Crp, Cya, Fnr, and Mlc on glucose catabolism in Escherichia coli. J Bacteriol 187(9):3171–3179Google Scholar
  69. Persson P, Andersson J, Gorton L, Larsson S, Nilvebrant N-O, Jönsson LJ (2002a) Effect of different forms of alkali treatment on specific fermentation inhibitors and on the fermentability of lignocellulose hydrolysates for production of fuel ethanol. J Agric Food Chem 50(19):5318–5325Google Scholar
  70. Persson P, Larsson S, Jönsson LJ, Nilvebrant N-O, Sivik B, Munteanu F, Thörneby L, Gorton L (2002b) Supercritical fluid extraction of a lignocellulosic hydrolysate of spruce for detoxification and to facilitate analysis of inhibitors. Biotechnol Bioeng 79(6):694–700Google Scholar
  71. Ranatunga TD, Jervis J, Helm RF, McMillan JD, Wooley RJ (2000) The effect of overliming on the toxicity of dilute acid pretreated lignocellulosics: the role of inorganics, uronic acids and ether-soluble organics. Enzym Microb Technol 27(3):240–247Google Scholar
  72. Ren Q, Ruth K, Thöny-Meyer L, Zinn M (2010) Enatiomerically pure hydroxycarboxylic acids: current approaches and future perspectives. Appl Microbiol Biotechnol 87(1):41–52Google Scholar
  73. Saini JK, Saini R, Tewari L (2015) Lignocellulosic agriculture wastes as biomass feedstocks for second-generation bioethanol production: concepts and recent developments. 3. Biotech 5(4):337–353Google Scholar
  74. Sárvári Horváth I, Franzén CJ, Taherzadeh MJ, Niklasson C, Lidén G (2003) Effects of furfural on the respiratory metabolism of Saccharomyces cerevisiae in glucose-limited chemostats. Appl Environ Microbiol 69(7):4076–4086Google Scholar
  75. Sateesh LR, Adivikatla V, Naseeruddin S, Yadav DS, Panda SH, Yenumula GP, Linga V (2011) Studies on different detoxification methods for the acid hydrolysate of lignocellulosic substrate Saccharum spontaneum. 53–57 p.Google Scholar
  76. Sawers G, Suppmann B (1992) Anaerobic induction of pyruvate formate-lyase gene expression is mediated by the ArcA and FNR proteins. J Bacteriol 174(11):3474–3478Google Scholar
  77. Shalel Levanon S, San K-Y, Bennett GN (2005) Effect of oxygen on the Escherichia coli ArcA and FNR regulation systems and metabolic responses. Biotechnol Bioeng 89(5):556–564Google Scholar
  78. Shalel-Levanon S, San K-Y, Bennett GN (2005) Effect of ArcA and FNR on the expression of genes related to the oxygen regulation and the glycolysis pathway in Escherichia coli under microaerobic growth conditions. Biotechnol Bioeng 92(2):147–159Google Scholar
  79. Tian D, Chandra RP, Lee J-S, Lu C, Saddler JN (2017) A comparison of various lignin-extraction methods to enhance the accessibility and ease of enzymatic hydrolysis of the cellulosic component of steam-pretreated poplar. Biotechnol Biofuels 10(1):157Google Scholar
  80. Tokiwa Y, Ugwu CU (2007) Biotechnological production of (R)-3-hydroxybutyric acid monomer. J Biotechnol 132(3):264–272Google Scholar
  81. Tseng H-C, Martin CH, Nielsen DR, Prather KLJ (2009) Metabolic engineering of Escherichia coli for enhanced production of (R)- and (S)-3-hydroxybutyrate. Appl Environ Microbiol 75(10):3137–3145Google Scholar
  82. Ulbricht RJ, Northup SJ, Thomas JA (1984) A review of 5-hydroxymethylfurfural (HMF) in parenteral solutions. Fundam Appl Toxicol 4(5):843–53Google Scholar
  83. Valdivia M, Galan JL, Laffarga J, Ramos J-L (2016) Biofuels 2020: biorefineries based on lignocellulosic materials. Microb Biotechnol 9(5):585–594Google Scholar
  84. Valentine J, Clifton-Brown J, Hastings A, Robson P, Allison G, Smith P (2012) Food vs. fuel: the use of land for lignocellulosic ‘next generation’ energy crops that minimize competition with primary food production. GCB Bioenergy 4(1):1–19Google Scholar
  85. Van Zyl C, Prior BA, Du Preez JC (1988) Production of ethanol from sugar cane bagasse hemicellulose hydrolyzate by Pichia stipitis. Appl Biochem Biotechnol 17(1):357–369Google Scholar
  86. Volker AR, Gogerty DS, Bartholomay C, Hennen-Bierwagen T, Zhu H, Bobik TA (2014) Fermentative production of short-chain fatty acids in Escherichia coli. Microbiology 160(Pt_7):1513–1522Google Scholar
  87. Welton T (2015) Solvents and sustainable chemistry. Proc Math Phys Eng Sci 471(2183):20150502Google Scholar
  88. Wood BJB (1998) Microbiology of fermented foods. Springer, New YorkGoogle Scholar
  89. Xiros C, Olsson L (2014) Comparison of strategies to overcome the inhibitory effects in high-gravity fermentation of lignocellulosic hydrolysates. Biomass Bioenergy 65 (Supplement C):79–90Google Scholar
  90. Yim H, Haselbeck R, Niu W, Pujol-Baxley C, Burgard A, Boldt J, Khandurina J, Trawick JD, Osterhout RE, Stephen R and others. 2011. Metabolic engineering of Escherichia coli for direct production of 1,4-butanediol. Nat Chem Biol 7:445, 452Google Scholar
  91. Yu RJ, Van Scott EJ (2004) Alpha-hydroxyacids and carboxylic acids. J Cosmet Dermatol 3(2):76–87Google Scholar
  92. Zaldivar J, Martinez A, Ingram L (1999) Effect of selected aldehydes on the growth and fermentation of ethanologenic Escherichia coli. 24–33 p.Google Scholar
  93. Zheng Z, Gong Q, Liu T, Deng Y, Chen J-C, Chen G-Q (2004) Thioesterase II of Escherichia coli plays an important role in 3-hydroxydecanoic acid production. Appl Environ Microbiol 70(7):3807–3813Google Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Dragan Miscevic
    • 1
  • Kajan Srirangan
    • 2
  • Teshager Kefale
    • 1
  • Daryoush Abedi
    • 1
    • 3
  • Murray Moo-Young
    • 1
  • C. Perry Chou
    • 1
    Email author
  1. 1.Department of Chemical EngineeringUniversity of WaterlooWaterlooCanada
  2. 2.Biotechnology Research InstituteNational Research Council of CanadaMontrealCanada
  3. 3.Department of Drug & Food ControlTehran University of Medical SciencesTehranIran

Personalised recommendations