Valorization of Crude Glycerol, Residue Deriving from Biodiesel- Production Process, with the Use of Wild-type New Isolated Yarrowia lipolytica Strains: Production of Metabolites with Pharmaceutical and Biotechnological Interest

Author(s): Dimitris Sarris*, Zoe Sampani, Anna Rapti, Seraphim Papanikolaou.

Journal Name: Current Pharmaceutical Biotechnology

Volume 20 , Issue 10 , 2019

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Graphical Abstract:


Background & Objective: Crude glycerol (Glol), used as substrate for screening eleven natural Yarrowia lipolytica strains in shake-flask experiments. Aim of this study was to assess the ability of the screened strains to produce biomass (dry cell weight; X), lipid (L), citric acid (Cit), mannitol (Man), arabitol (Ara) and erythritol (Ery), compounds presenting pharmaceutical and biotechnological interest, in glycerol-based nitrogen-limited media, in which initial glycerol concentration had been adjusted to 40 g/L.

Methods: Citric acid may find use in biomedical engineering (i.e. drug delivery, tissue engineering, bioimaging, orthopedics, medical device coating, wound dressings). Polyols are considered as compounds with non-cariogenic and less calorigenic properties as also with low insulin-mediated response. Microbial lipids containing polyunsaturated fatty acids (PUFA) are medically and dietetically important (selective pharmaceutical and anticancer properties, aid fetal brain development, the sight function of the eye, hormonal balance and the cardio-vascular system, prevent reasons leading to type-2 diabetes, present healing and anti-inflammatory effects).

Results: All strains presented satisfactory microbial growth (Xmax=5.34-6.26 g/L) and almost complete substrate uptake. The principal metabolic product was citric acid (Citmax=8.5-31.7 g/L). Production of cellular lipid reached the values of 0.33-0.84 g/L. Polyols were also synthesized as strain dependent compounds (Manmax=2.8-6.1 g/L, Aramax ~2.0 g/L, Erymax= 0.5-3.8 g/L). The selected Y. lipolytica strain ACA-DC 5029 presented satisfactory growth along with synthesis of citric acid and polyols, thus, was further grown on media presenting an increased concentration of Glol~75 g/L. Biomass, lipid and citric acid production presented significant enhancement (Xmax=11.80 g/L, Lmax=1.26 g/L, Citmax=30.8 g/L), but conversion yield of citric acid produced per glycerol consumed was decreased compared to screening trials. Erythritol secretion (Erymax=15.6 g/L) was highly favored, suggesting a shift of yeast metabolism from citric acid accumulation towards erythritol production. Maximum endopolysaccharides (IPS) concentration was 4.04 g/L with yield in dry weight 34.2 % w/w.

Conclusion: Y. lipolytica strain ACA-YC 5029 can be considered as a satisfactory candidate grown in high concentrations of crude glycerol to produce added-value compounds that interest pharmaceutical and biotechnology industries.

Keywords: Arabitol, citric acid, crude glycerol, erythritol, mannitol, Yarrowia lipolytica.

Juszczyk, P.; Tomaszewska, L.; Kita, A.; Rymowicz, W. Biomass production by novel strains of Yarrowia lipolytica using raw glycerol, derived from biodiesel production. Bioresour. Technol., 2013, 137, 124-131.
Garlapati, V.K.; Shankar, U.; Budhiraja, A. Bioconversion technologies of crude glycerol to value added industrial products. Biotechnol. Rep. , 2016, 9, 9-14.
André, A.; Diamantopoulou, P.; Philippoussis, A.; Sarris, D.; Komaitis, M.; Papanikolaou, S. Biotechnological conversions of bio-diesel derived waste glycerol into added-value compounds by higher fungi: production of biomass, single cell oil and oxalic acid. Ind. Crops Prod., 2010, 31(2), 407-416.
André, A.; Chatzifragkou, A.; Diamantopoulou, P.; Sarris, D.; Philippoussis, A.; Galiotou‐Panayotou, M.; Komaitis, M.; Papanikolaou, S. Biotechnological conversions of bio‐diesel‐derived crude glycerol by Yarrowia lipolytica strains. Eng. Life Sci., 2009, 9(6), 468-478.
Rywińska, A.; Juszczyk, P.; Wojtatowicz, M.; Robak, M.; Lazar, Z.; Tomaszewska, L.; Rymowicz, W. Glycerol as a promising substrate for Yarrowia lipolytica biotechnological applications. Biomass Bioenergy, 2013, 48, 148-166.
Morgunov, I.G.; Kamzolova, S.V.; Lunina, J.N. The citric acid production from raw glycerol by Yarrowia lipolytica yeast and its regulation. Appl. Microbiol. Biotechnol., 2013, 97(16), 7387-7397.
Santos, E.O.; Michelon, M.; Gallas, J.A.; Kalil, S.J.; Burkert, C.A.V. Raw glycerol as substrate for the production of yeast biomass. Int. J. Food Eng., 2013, 9(4), 413-420.
Morgunov, I.G.; Kamzolova, S.V.; Lunina, J.N. Citric acid production by Yarrowia lipolytica yeast on different renewable raw materials. Fermentation, 2018, 4(2), 36.
Chatzifragkou, A.; Papanikolaou, S.; Dietz, D.; Doulgeraki, A.I.; Nychas, G-J.E.; Zeng, A-P. Production of 1, 3-propanediol by Clostridium butyricum growing on biodiesel-derived crude glycerol through a non-sterilized fermentation process. Appl. Microbiol. Biotechnol., 2011, 91(1), 101-112.
Chatzifragkou, A.; Makri, A.; Belka, A.; Bellou, S.; Mavrou, M.; Mastoridou, M.; Mystrioti, P.; Onjaro, G.; Aggelis, G.; Papanikolaou, S. Biotechnological conversions of biodiesel derived waste glycerol by yeast and fungal species. Energy, 2011, 36(2), 1097-1108.
Papanikolaou, S. 1, 3-Propanediol and citric acid production from glycerol containing waste discharged after bio-diesel manufacturing process In: Current Topics on Bioprocesses in Food Industry; Koutinas, A; Pandey, A; C, L, Eds.; AsiaTech Publishers Inc: New Delhi, 2008; 2, pp. 381-399.
Papanikolaou, S. Microbial conversion of glycerol into 1, 3-propanediol: glycerol assimilation, biochemical events related with 1, 3-propanediol biosynthesis and biochemical engineering of the process. In: Microbial Conversions of Raw Glycerol; Aggelis, G., Ed.; Nova Science Publishers Inc: New York, 2009; pp. 137-168.
Papanikolaou, S.; Aggelis, G. Biotechnological valorization of biodiesel derived glycerol waste through production of single cell oil and citric acid by Yarrowia lipolytica. Lipid Technol., 2009, 21(4), 83-87.
Soccol, C.R.; Vandenberghe, L.P.; Rodrigues, C.; Pandey, A. New perspectives for citric acid production and application. Food Technol. Biotechnol., 2006, 44(2), 141-149.
Anastassiadis, S.; Morgunov, I.G.; Kamzolova, S.V.; Finogenova, T.V. Citric acid production patent review. Recent Pat. Biotechnol., 2008, 2(2), 107-123.
Kubicek, C.P.; Röhr, M.; Rehm, H. Citric acid fermentation. Crit. Rev. Biotechnol., 1985, 3(4), 331-373.
Tran, R.T.; Zhang, Y.; Gyawali, D.; Yang, J. Recent developments on citric acid derived biodegradable elastomers. Recent Pat. Biom. Eng., 2009, 2(3), 216-227.
Yang, J.; Webb, A.R.; Ameer, G.A. Novel citric acid‐based biodegradable elastomers for tissue engineering. Adv. Mater., 2004, 16(6), 511-516.
Rymowicz, W.; Fatykhova, A.R.; Kamzolova, S.V.; Rywińska, A.; Morgunov, I.G. Citric acid production from glycerol-containing waste of biodiesel industry by Yarrowia lipolytica in batch, repeated batch, and cell recycle regimes. Appl. Microbiol. Biotechnol., 2010, 87(3), 971-979.
Kamzolova, S.V.; Morgunov, I.G.; Aurich, A.; Perevoznikova, O.A.; Shishkanova, N.V.; Stottmeister, U.; Finogenova, T.V. Lipase secretion and citric acid production in Yarrowia lipolytica yeast grown on animal and vegetable fat. Food Technol. Biotechnol., 2005, 43(2), 113-122.
Almeida, J.R.; Fávaro, L.C.; Quirino, B.F. Biodiesel biorefinery: Opportunities and challenges for microbial production of fuels and chemicals from glycerol waste. Biotechnol. Biofuels, 2012, 5(1), 48.
Rymowicz, W.; Rywińska, A.; Gładkowski, W. Simultaneous production of citric acid and erythritol from crude glycerol by Yarrowia lipolytica Wratislavia K1. Chem. Pap., 2008, 62(3), 239-246.
Rymowicz, W.; Rywińska, A.; Żarowska, B.; Juszczyk, P. Citric acid production from raw glycerol by acetate mutants of Yarrowia lipolytica. Chem. Pap., 2006, 60(5), 391-394.
Rywińska, A.; Juszczyk, P.; Wojtatowicz, M.; Rymowicz, W. Chemostat study of citric acid production from glycerol by Yarrowia lipolytica. J. Biotechnol., 2011, 152(1-2), 54-57.
Rywinska, A.; Rymowicz, W.; Marcinkiewicz, M. Valorization of raw glycerol for citric acid production by Yarrowia lipolytica yeast. Electron. J. Biotechnol., 2010, 13(4), 9-10.
Tomaszewska, L.; Rakicka, M.; Rymowicz, W.; Rywińska, A. A comparative study on glycerol metabolism to erythritol and citric acid in Yarrowia lipolytica yeast cells. FEMS Yeast Res., 2014, 14(6), 966-976.
Papanikolaou, S.; Fakas, S.; Fick, M.; Chevalot, I.; Galiotou-Panayotou, M.; Komaitis, M.; Marc, I.; Aggelis, G. Biotechnological valorisation of raw glycerol discharged after bio-diesel (fatty acid methyl esters) manufacturing process: Production of 1, 3-propanediol, citric acid and single cell oil. Biomass Bioenergy, 2008, 32(1), 60-71.
Rakicka, M.; Rukowicz, B.; Rywińska, A.; Lazar, Z.; Rymowicz, W. Technology of efficient continuous erythritol production from glycerol. J. Clean. Prod., 2016, 139, 905-913.
Rakicka, M.; Rywińska, A.; Cybulski, K.; Rymowicz, W. Enhanced production of erythritol and mannitol by Yarrowia lipolytica in media containing surfactants. Braz. J. Microbiol., 2016, 47(2), 417-423.
Park, Y-C.; Oh, E.J.; Jo, J-H.; Jin, Y-S.; Seo, J-H. Recent advances in biological production of sugar alcohols. Curr. Opin. Biotechnol., 2016, 37, 105-113.
Rywińska, A.; Tomaszewska, L.; Rymowicz, W. Erythritol biosynthesis by Yarrowia lipolytica yeast under various culture conditions. Afr. J. Microbiol. Res., 2013, 7(27), 3511-3516.
Tomaszewska, L.; Rywińska, A.; Gładkowski, W. Production of erythritol and mannitol by Yarrowia lipolytica yeast in media containing glycerol. J. Ind. Microbiol. Biotechnol., 2012, 39(9), 1333-1343.
Saha, B.C.; Racine, F.M. Biotechnological production of mannitol and its applications. Appl. Microbiol. Biotechnol., 2011, 89(4), 879-891.
Song, S.H.; Vieille, C. Recent advances in the biological production of mannitol. Appl. Microbiol. Biotechnol., 2009, 84(1), 55-62.
Kordowska-Wiater, M.; Kubik-Komar, A.; Targoński, Z. Application of response surface methodology for the optimization of arabinose biotransformation to arabitol by Candida parapsilosis. Open Life Sci., 2013, 8(9), 835-842.
Koganti, S.; Kuo, T.M.; Kurtzman, C.P.; Smith, N.; Ju, L-K. Production of arabitol from glycerol: Strain screening and study of factors affecting production yield. Appl. Microbiol. Biotechnol., 2011, 90(1), 257-267.
Rakicka, M.; Mirończuk, A.M.; Tomaszewska-Hetman, L.; Rywińska, A.; Rymowicz, W. An Effective method of continuous production of erythritol from glycerol by Yarrowia lipolytica MK1. Food Technol. Biotechnol., 2017, 55(1), 125-130.
Rywińska, A.; Marcinkiewicz, M.; Cibis, E.; Rymowicz, W. Optimization of medium composition for erythritol production from glycerol by Yarrowia lipolytica using response surface methodology. Prep. Biochem. Biotechnol., 2015, 45(6), 515-529.
Chatzifragkou, A.; Petrou, I.; Gardeli, C.; Komaitis, M.; Papanikolaou, S. Effect of Origanum vulgare L. essential oil on growth and lipid profile of Yarrowia lipolytica cultivated on glycerol-based media. J. Am. Oil Chem. Soc., 2011, 88(12), 1955-1964.
Papanikolaou, S.; Aggelis, G. Yarrowia lipolytica: A model microorganism used for the production of tailor‐made lipids. Eur. J. Lipid Sci. Technol., 2010, 112(6), 639-654.
Papanikolaou, S.; Aggelis, G. Modeling lipid accumulation and degradation in Yarrowia lipolytica cultivated on industrial fats Curr. Microbiol, 2003, 46(6), 0398-0402.
Carsanba, E.; Papanikolaou, S.; Erten, H. Production of oils and fats by oleaginous microorganisms with an emphasis given to the potential of the nonconventional yeast Yarrowia lipolytica. Crit. Rev. Biotechnol., 2018, 1-14.
Zikou, E.; Chatzifragkou, A.; Koutinas, A.; Papanikolaou, S. Evaluating glucose and xylose as cosubstrates for lipid accumulation and γ‐linolenic acid biosynthesis of Thamnidium elegans. J. Appl. Microbiol., 2013, 114(4), 1020-1032.
Kolouchová, I.; Sigler, K.; Schreiberová, O.; Masák, J.; Řezanka, T. New yeast-based approaches in production of palmitoleic acid. Bioresour. Technol., 2015, 192, 726-734.
Papanikolaou, S.; Aggelis, G. Lipids of oleaginous yeasts. Part I: Biochemistry of single cell oil production. Eur. J. Lipid Sci. Technol., 2011, 113(8), 1031-1051.
Athenaki, M.; Gardeli, C.; Diamantopoulou, P.; Tchakouteu, S.S.; Sarris, D.; Philippoussis, A.; Papanikolaou, S. Lipids from yeasts and fungi: Physiology, production and analytical considerations. J. Appl. Microbiol., 2018, 124(2), 336-367.
Makri, A.; Fakas, S.; Aggelis, G. Metabolic activities of biotechnological interest in Yarrowia lipolytica grown on glycerol in repeated batch cultures. Bioresour. Technol., 2010, 101(7), 2351-2358.
Papanikolaou, S.; Beopoulos, A.; Koletti, A.; Thevenieau, F.; Koutinas, A.A.; Nicaud, J-M.; Aggelis, G. Importance of the methyl-citrate cycle on glycerol metabolism in the yeast Yarrowia lipolytica. J. Biotechnol., 2013, 168(4), 303-314.
Sarris, D.; Galiotou‐Panayotou, M.; Koutinas, A.A.; Komaitis, M.; Papanikolaou, S. Citric acid, biomass and cellular lipid production by Yarrowia lipolytica strains cultivated on olive mill wastewater‐based media. J. Chem. Technol. Biotechnol., 2011, 86(11), 1439-1448.
Papanikolaou, S.; Komaitis, M.; Aggelis, G. Single cell oil (SCO) production by Mortierella isabellina grown on high-sugar content media. Bioresour. Technol., 2004, 95(3), 287-291.
Fakas, S.; Papanikolaou, S.; Galiotou-Panayotou, M.; Komaitis, M.; Aggelis, G. Lipids of Cunninghamella echinulata with emphasis to γ-linolenic acid distribution among lipid classes. Appl. Microbiol. Biotechnol., 2006, 73(3), 676-683.
Tsakona, S.; Kopsahelis, N.; Chatzifragkou, A.; Papanikolaou, S.; Kookos, I.K.; Koutinas, A.A. Formulation of fermentation media from flour-rich waste streams for microbial lipid production by Lipomyces starkeyi. J. Biotechnol., 2014, 189, 36-45.
Miller, G.L. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem., 1959, 31(3), 426-428.
Rzechonek, D.A.; Dobrowolski, A.; Rymowicz, W.; Mirończuk, A.M. Recent advances in biological production of erythritol. Crit. Rev. Biotechnol., 2018, 38(4), 620-633.
Papanikolaou, S.; Gortzi, O.; Margeli, E.; Chinou, I.; Galiotou‐Panayotou, M.; Lalas, S. Effect of Citrus essential oil addition upon growth and cellular lipids of Yarrowia lipolytica yeast. Eur. J. Lipid Sci. Technol., 2008, 110(11), 997-1006.
Kamzolova, S.V.; Shishkanova, N.V.; Morgunov, I.G.; Finogenova, T.V. Oxygen requirements for growth and citric acid production of Yarrowia lipolytica. FEMS Yeast Res., 2003, 3(2), 217-222.
Anastassiadis, S.; Rehm, H-J. Continuous citric acid secretion by a high specific pH dependent active transport system in yeast Candida oleophila ATCC 20177. Electron. J. Biotechnol., 2005, 8(2), 26-42.
Tchakouteu, S.S.; Kalantzi, O.; Gardeli, C.; Koutinas, A.A.; Aggelis, G.; Papanikolaou, S. Lipid production by yeasts growing on biodiesel‐derived crude glycerol: strain selection and impact of substrate concentration on the fermentation efficiency. J. Appl. Microbiol., 2015, 118(4), 911-927.
Papanikolaou, S.; Muniglia, L.; Chevalot, I.; Aggelis, G.; Marc, I. Yarrowia lipolytica as a potential producer of citric acid from raw glycerol. J. Appl. Microbiol., 2002, 92(4), 737-744.
Papanikolaou, S.; Rontou, M.; Belka, A.; Athenaki, M.; Gardeli, C.; Mallouchos, A.; Kalantzi, O.; Koutinas, A.A.; Kookos, I.K.; Zeng, A.P. Conversion of biodiesel‐derived glycerol into biotechnological products of industrial significance by yeast and fungal strains. Eng. Life Sci., 2017, 17(3), 262-281.
Rymowicz, W.; Rywińska, A.; Marcinkiewicz, M. High-yield production of erythritol from raw glycerol in fed-batch cultures of Yarrowia lipolytica. Biotechnol. Lett., 2009, 31(3), 377-380.
Rywinska, A.; Rymowicz, W. Citric acid production from raw glycerol by Yarrowia lipolytica Wratislavia 1.31. In: Microbial Conversions of Raw Glycerol; Aggelis, G., Ed.; Nova Science Publishers Inc: New York, 2009; pp. 19-30.
Rywińska, A.; Rymowicz, W. High-yield production of citric acid by Yarrowia lipolytica on glycerol in repeated-batch bioreactors. J. Ind. Microbiol. Biotechnol., 2010, 37(5), 431-435.
Kamzolova, S.V.; Fatykhova, A.R.; Dedyukhina, E.G.; Anastassiadis, S.G.; Golovchenko, N.P.; Morgunov, I.G. Citric acid production by yeast grown on glycerol-containing waste from biodiesel industry. Food Technol. Biotechnol., 2011, 49(1), 65-74.
Papanikolaou, S.; Kampisopoulou, E.; Blanchard, F.; Rondags, E.; Gardeli, C.; Koutinas, A.A.; Chevalot, I.; Aggelis, G. Production of secondary metabolites through glycerol fermentation under carbon‐excess conditions by the yeasts Yarrowia lipolytica and Rhodosporidium toruloides. Eur. J. Lipid Sci. Technol., 2017, 119(9)
Kamzolova, S.V.; Morgunov, I.G. Biosynthesis of pyruvic acid from glycerol-containing substrates and its regulation in the yeast Yarrowia lipolytica. Bioresour. Technol., 2018, 266, 125-133.
Aggelis, G.; Komaitis, M. Enhancement of single cell oil production by Yarrowia lipolytica growing in the presence of Teucrium polium L. aqueous extract. Biotechnol. Lett., 1999, 21(9), 747-749.
Finogenova, T.; Kamzolova, S.; Dedyukhina, E.; Shishkanova, N.; Il’chenko, A.; Morgunov, I.; Chernyavskaya, O.; Sokolov, A. Biosynthesis of citric and isocitric acids from ethanol by mutant Yarrowia lipolytica N 1 under continuous cultivation. Appl. Microbiol. Biotechnol., 2002, 59(4-5), 493-500.
Mirończuk, A.M.; Furgała, J.; Rakicka, M.; Rymowicz, W. Enhanced production of erythritol by Yarrowia lipolytica on glycerol in repeated batch cultures. J. Ind. Microbiol. Biotechnol., 2014, 41(1), 57-64.

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Year: 2019
Page: [881 - 894]
Pages: 14
DOI: 10.2174/1389201020666190211145215
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