Abstract
Shake-flask in vitro culture of Buddleja cordata cells produces large amounts of biomass and synthetizes verbascoside (VB), linarin and hydroxycinnamic acids, bioactive phenolic secondary metabolites (PSMs). In this work, we determined the effect of stirring speed on the growth of and production of PSMs [total phenolic, phenylethanoid glycoside and flavonoid contents (PeC, PeGC and FC, respectively)] by B. cordata cells cultured in two bioreactors. Two different stirring speeds (120 and 400 rpm) were tested in two stirred-tank bioreactors: a 2 L bioreactor equipped with a ring diffuser (B2RD) and a 3 L bioreactor with a sintered diffuser (B3SD). Growth kinetics of B. cordata cells were measured in the bioreactors and shake-flask systems. The stirring speed and type of bioreactor affected phases, parameters of growth and production of PSMs. The highest production of biomass (13.62 g L−1) and PSMs [PeC of 64.63 mg gallic acid equivalents g−1 (mg GAE g−1); PeGC of 119.24 mg VB equivalents g−1 (mg VBE g−1); and FC of 5.02 mg quercetin equivalents g−1 (mg QE g−1)] occurred in B2RD at 400 rpm. These values were similar to the found in shake-flasks system. This work establishes the basis for bioprocess advances of B. cordata focused on the development of a sustainable strategy for the management of natural resources and as a source of bioactive PSMs on a large scale.
Key message
Buddleja cordata cells cultured in a mechanically agitated bioreactor possess an outstanding biosynthetic potential that represents a suitable biotechnological alternative for the production of bioactive phenolic secondary metabolites.
Similar content being viewed by others
Abbreviations
- µ:
-
Specific growth rate
- ANOVA:
-
Analysis of variance
- B2RD:
-
2 L Bioreactor equipped with a ring diffuser
- B3SD:
-
3 L Bioreactor equipped with a sintered diffuser
- CSCBc:
-
Cell suspension culture of B. cordata
- CV:
-
Cellular viability
- DO:
-
Dissolved oxygen
- DW:
-
Dry weight
- F:
-
Flavonoid (s)
- FC:
-
Total flavonoid content
- GI:
-
Growth index
- GAE:
-
Gallic acid equivalent
- MB:
-
Maximum biomass
- MSDK:
-
Murashige and Skoog-modified culture medium
- Pe:
-
Phenolic compound (s)
- PeC:
-
Total phenolic content
- PeG:
-
Phenylethanoid glycoside (s)
- PeGC:
-
Total phenylethanoid glycoside content
- PSM:
-
Phenolic secondary metabolite (s)
- QE:
-
Quercetin equivalent
- qpF :
-
Specific production rate of total flavonoids
- qpPe :
-
Specific production rate of total phenolics
- qpPeG :
-
Specific production rate of total phenylethanoid glycosides
- RA:
-
Rosmarinic acid
- SM:
-
Secondary metabolite (s)
- td:
-
Doubling time
- TSC:
-
Total sugar content
- VB:
-
Verbascoside
- VBE:
-
Verbascoside equivalent
- Yx/s :
-
Yield of biomass from substrate
References
Ahmadi-Sakha S, Sharifi M, Niknam V (2015) Bioproduction of phenylethanoid glycosides by plant cell culture of Scrophularia striata Boiss: from shake-flasks to bioreactor. Plant Cell Tissue Organ Cult 124:275–281. https://doi.org/10.1007/s11240-015-0891-3
Ahmed S, Hahn EJ, Paek KY (2008) Aeration volume and photosynthetic photon flux affect cell growth and secondary metabolite contents in bioreactor cultures of Morinda citrifolia. J Plant Biol 51:209–212. https://doi.org/10.1007/BF03030700
Alamgir ANM (2017) Therapeutic use of medicinal plants and their extracts: volume 1. Progress in drug research, vol 73. Springer, Cham. https://doi.org/10.1007/978-3-319-63862-1_9
Alipieva K, Korkina L, Orhan IE, Georgiev MI (2014) Verbascoside—a review of its occurrence, (bio) synthesis and pharmacological significance. Biotechnol Adv 32:1065–1076. https://doi.org/10.1016/j.biotechadv.2014.07.001
Arriola-Padilla VJ, Estrada-Martínez E, Ortega-Rubio A, Pérez-Miranda R, Gijón-Hernández AR (2014) Deterioro en áreas naturales protegidas del Centro de México y del Eje Neovolcánico Transversal. Investig y Cienc 22:37–49
Ayoola GA, Eze SO, Johnson OO, Adeyemi DK (2018) Phytochemical screening, antioxidant, antiulcer and toxicity studies on Desmodium adscendens (Sw) DC Fabaceae leaf and stem. Trop J Pharm Res 17:1301–1307. https://doi.org/10.4314/tjpr.v17i7.11
Bulle S, Reddyvari H, Nallanchakravarthula V, Vaddi DR (2016) Therapeutic potential of Pterocarpus santalinus L.: an update. Pharmacogn Rev 10:43–49. https://doi.org/10.4103/0973-7847.176575
Chattopadhyay S, Farkya S, Srivastava AK, Bisaria VS (2002) Bioprocess considerations for production of secondary metabolites by plant cell suspension cultures. Biotechnol Bioprocess Eng 7:138–149. https://doi.org/10.1007/BF02932911
Chen SL, Yu H, Luo HM, Wu Q, Li CF, Steinmetz A (2016) Conservation and sustainable use of medicinal plants: problems, progress, and prospects. Chin Med 11:37–47. https://doi.org/10.1186/s13020-016-0108-7
Cheng XY, Zhou HY, Cui X, Ni W, Liu CZ (2006) Improvement of phenylethanoid glycosides biosynthesis in Cistanche deserticola cell suspension cultures by chitosan elicitor. J Biotechnol 121:253–260. https://doi.org/10.1016/j.jbiotec.2005.07.012
Bayer Healthcare LLC Consumer Care Division (2009) Composition for treating skin, US 2009/0028969A1. https://patentimages.storage.googleapis.com/9e/04/53/33ac508ee219b9/US20090028969A1.pdf. Accesed 31 July 2019
Doran PM (1999) Design of mixing systems for plant cell suspensions in stirred reactors. Biotechnol Prog 15:319–335. https://doi.org/10.1021/bp990042v
Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356. https://doi.org/10.1021/ac60111a017
Eibl R, Eibl D (2002) Bioreactors for plant cell and tissue cultures. In: Oksman-Caldentey KM, Barz WH (eds) Plant biotechnology and transgenic plants. Marcel Decker, New York, pp 163–199
Espinosa-Leal CA, Puente-Garza CA, García-Lara S (2018) In vitro plant tissue culture: means for production of biological active compounds. Planta 248:1–18. https://doi.org/10.1007/s00425-018-2910-1
Estrada-Zúñiga ME, Cruz-Sosa F, Verde-Calvo R, Rodríguez-Monroy M, Vernon-Carter EJ (2009) Phenylpropanoid production in callus and cell suspension cultures of Buddleja cordata Kunth. Plant Cell Tissue Organ Cult 97:39–47. https://doi.org/10.1007/s11240-009-9496-z
Estrada-Zúñiga ME, Arano-Varela H, Buendía-González L, Orozco-Villafuerte J (2012) Fatty acids, phenols content, and antioxidant activity in Ibervillea sonorae callus cultures. Rev Mex Ing Quim 11:89–96
European Pharmacopoeia (2005) European Pharmacopoeia: Fraxini folium, 5th edn. EDQM, Strasbourg, France
Georgiev MI, Weber J (2014) Bioreactors for plant cells: hardware configuration and internal environment optimization as tools for wider commercialization. Biotechnol Lett 36:1359–1367. https://doi.org/10.1007/s10529-014-1498-1
Georgiev MI, Weber J, Maciuk A (2009) Bioprocessing of plant cell cultures for mass production of targeted compounds. Appl Microbiol Biotechnol 83:809–823. https://doi.org/10.1007/s00253-009-2049-x
Georgiev MI, Ludwig-Müller J, Weber J, Stancheva N, Bley T (2011) Bioactive metabolite production and stress-related hormones in Devil’s claw cell suspension cultures grown in bioreactors. Appl Microbiol Biotechnol 89:1683–1691. https://doi.org/10.1007/s00253-010-3008-2
Georgiev MI, Eibl R, Zhong JJ (2013) Hosting the plant cells in vitro: recent trends in bioreactors. Appl Microbiol Biotechnol 97:3787–3800. https://doi.org/10.1007/s00253-013-4817-x
Godoy-Hernández GC, Vázquez-Flota FA, Loyola-Vargas VM (2000) The exposure to trans-cinnamic acid of osmotically stressed Catharanthus roseus cells cultured in a 14-L bioreactor increases alkaloid accumulation. Biotechnol Lett 22:921–925. https://doi.org/10.1023/A:1005672400219
Gutiérrez-Rebolledo GA, Estrada-Zúñiga ME, Nieto Trujillo A, Cruz-Sosa F, Jiménez-Arellanes MA (2018) In vivo anti-inflammatory activity and acute toxicity of methanolic extracts from wild plant leaves and cell suspension cultures of Buddleja cordata Kunth (Buddlejaceae). Rev Mex Ing Quim 17:317–330. https://doi.org/10.24275/uam/izt/dcbi/revmexingquim/2018v17n1/Gutierrez
Gutiérrez-Rebolledo GA, Estrada-Zúñiga ME, Garduño-Siciliano L, García-Gutiérrez GE, Reséndiz-Mora CA, Calderón-Amador J, Cruz-Sosa F (2019) In vivo anti-arthritic effect and repeated dose toxicity of standardized methanolic extracts of Buddleja cordata Kunth (Scrophulariaceae) wild plant leaves and cell culture. J Ethnopharmacol 240:111875. https://doi.org/10.1016/j.jep.2019.111875
Haas C, Weber J, Ludwig-Müller J, Deponte S, Bley T, Georgiev MI (2008) Flow cytometry and phytochemical analysis of a sunflower cell suspension culture in a 5-L bioreactor. Z Naturforsch C 63:699–705. https://doi.org/10.1515/znc-2008-9-1015
Isah T, Umar S, Mujib A, Sharma MP, Rajasekharan PE, Zafar N, Frukh A (2018) Secondary metabolism of pharmaceuticals in the plant in vitro cultures: strategies, approaches, and limitations to achieving higher yield. Plant Cell Tissue Organ Cult 132:239–265. https://doi.org/10.1007/s11240-017-1332-2
James E, Lee JM (2001) The production of foreign proteins from genetically modified plant cells. In: Zhong JJ et al (eds) Plant cells. Advances in biochemical engineering/biotechnology, vol 72. Springer, Berlin, pp 127–156. https://doi.org/10.1007/3-540-45302-4_5
Khanahmadi M, Paek KY (2017) Bioreactor technology for sustainable production of valuable plant metabolites: challenges and advances. In: Abdullah S, Chai-Ling H, Wagstaff C (eds) Crop improvement. Springer, Cham, pp 169–189. https://doi.org/10.1007/978-3-319-65079-1_8
Klöckner W, Gacem R, Anderlei T, Raven N, Schillberg S, Lattermann C, Büchs J (2013) Correlation between mass transfer coefficient KLa and relevant operating parameters in cylindrical disposable shaken bioreactors on a bench-to-pilot scale. J Biol Eng 7:28. https://doi.org/10.1186/1754-1611-7-28
Kolewe ME, Gaurav V, Roberts SC (2008) Pharmaceutically active natural product synthesis and supply via plant cell culture technology. Mol Pharm 5:243–256. https://doi.org/10.1021/mp7001494
Korkina LG, Mikhal’chik EV, Suprun MV, Pastore S, TosoR Dal (2007) Molecular mechanisms underlying wound healing and anti-inflammatory properties of naturally occurring biotechnologically produced phenylpropanoid glycosides. Cell Mol Biol 53:84–91. https://doi.org/10.1170/T822
Loza-Mejía MA, Salazar JR, Sánchez-Tejeda JF (2018) In silico studies on compounds derived from calceolaria: phenylethanoid glycosides as potential multitarget inhibitors for the development of pesticides. Biomolecules 8:121. https://doi.org/10.3390/biom8040121
Meijer JJ, Ten Hoopen HJG, Luyben KChA, Libbenga KR (1993) Effects of hydrodynamic stress on cultured plant cells: a literature survey. Enzyme Microb Technol 15:234–238. https://doi.org/10.1016/0141-0229(93)90143-P
Meijer JJ, Ten Hoopen HJG, Van Gameren YM, Luyben KChA, Libbenga KR (1994) Effects of hydrodynamic stress on the growth of plant cells in batch and continuous culture. Enzyme Microb Technol 16:467–477. https://doi.org/10.1016/0141-0229(94)90016-7
Mukta S, Ahmed SR, Afrin D (2017) Plant tissue culture—the alternative and efficient way to extract plant secondary metabolites. J Sylhet Agril Univ 4:1–13
Mulabagal V, Tsay HS (2004) Plant cell cultures—an alternative and efficient source for the production of biologically important secondary metabolites. Int J Appl Sci Eng 2:29–48
Muñoz W, Vanegas OA, Guzmán A, Capataz J, Hoyos R, Orozco F (2006) Estimación de variables de operación de un biorreactor con células de Azadirachta indica A. Juss. Rev Fac Nac Agron Medellín 59:3467–3478
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
Ouyang J, Wang XD, Zhao B, Wang YC (2005a) Enhanced production of phenylethanoid glycosides by precursor feeding to cell culture of Cistanche deserticola. Process Biochem 40:3480–3484. https://doi.org/10.1016/j.procbio.2005.02.025
Ouyang J, Wang XD, Zhao B, Wang YC (2005b) Improved production of phenylethanoid glycosides by Cistanche deserticola cells cultured in an internal loop airlift bioreactor with sifter riser. Enzyme Microb Technol 36:982–988. https://doi.org/10.1016/j.enzmictec.2005.01.029
Pavlov AI, Georgiev MI, Ilieva MP (2005) Production of rosmarinic acid by Lavandula vera MM cell suspension in bioreactor: effect of dissolved oxygen concentration and agitation. World J Microbiol Biotechnol 21:389–392. https://doi.org/10.1007/s11274-004-3982-6
Pérez-Hernández J, Nicasio-Torres MP, Sarmiento-López LG, Rodríguez-Monroy M (2019) Production of anti-inflammatory compounds in Sphaeralcea angustifolia cell suspension cultivated in stirred tank bioreactor. Eng Life Sci 19:196–205. https://doi.org/10.1002/elsc.201800134
Piątczak E, Grąbkowska R, Wysokińska H (2018) Production of iridoid and phenylethanoid glycosides by in vitro systems of plants from the Buddlejaceae, Orobanchaceae, and Scrophulariaceae Families. In: Pavlov A, Bley T (eds) Bioprocessing of plant in vitro systems. Reference series in phytochemistry. Springer, Cham, pp 271–293. https://doi.org/10.1007/978-3-319-54600-1_3
Prakash G, Srivastava AK (2007) Azadirachtin production in stirred tank reactors by Azadirachta indica suspension culture. Process Biochem 42:93–97. https://doi.org/10.1016/j.procbio.2006.06.020
Ramos-Palacios R, Orozco-Segovia A, Sánchez-Coronado ME, Barradas VL (2012) Vegetative propagation of native species potentially useful in the restoration of Mexico City’s vegetation. Rev Mex Biodiv 83:809–816. https://doi.org/10.7550/rmb.21610
Rodríguez-Monroy M, Galindo E (1999) Broth rheology, growth and metabolite production of Beta vulgaris suspension culture: a comparative study between cultures grown in shake flasks and in a stirred tank. Enz Microbial Technol 24:687–693. https://doi.org/10.1016/S0141-0229(99)00002-2
Saimaru H, Orihara Y (2010) Biosynthesis of acteoside in cultured cells of Olea europaea. J Nat Med 64:139–145. https://doi.org/10.1007/s11418-009-0383-z
Smetanska I (2008) Production of secondary metabolites using plant cell cultures. Adv Biochem Eng Biotechnol 111:187–228. https://doi.org/10.1007/10_2008_103
Stancheva N, Weber J, Schulze J, Alipieva K, Ludwig-Müller J, Haas C, Georgiev V, Bley T, Georgiev M (2011) Phytochemical and flow cytometric analyses of Devil’s claw cell cultures. Plant Cell Tissue Organ Cult 105:79–84. https://doi.org/10.1007/s11240-010-9844-z
Thatoi H, Patra JK (2011) Biotechnology and pharmacological evaluation of medicinal plants: an overview. J Herbs Spices Med Plants 17:214–248. https://doi.org/10.1080/10496475.2011.602471
Trejo-Tapia G, Rodríguez-Monroy M (2007) La agregación celular en la producción de metabolitos secundarios en cultivos vegetales in vitro. Interciencia 32:669–674
Valdiani A, Hansen OK, Nielsen UB, Johannsen VK, Shariat M, Georgiev MI, Omidvar V, Ebrahimi M, Tavakoli Dinanani E, Abiri R (2018) Bioreactor-based advances in plant tissue and cell culture: challenges and prospects. Crit Rev Biotechnol 39:20–34. https://doi.org/10.1080/07388551.2018.1489778
Werner S, Maschke RW, Eibl D, Eibl R (2018) Bioreactor technology for sustainable production of plant cell-derived products. In: Pavlov A, Bley T (eds) Bioprocessing of plant in vitro systems. Reference series in phytochemistry. Springer, Cham, pp 413–432. https://doi.org/10.1007/978-3-319-54600-1_6
Wilson SA, Roberts SC (2012) Recent advances towards development and commercialization of plant cell culture processes for synthesis of biomolecules. Plant Biotechnol J 10:249–268. https://doi.org/10.1111/j.1467-7652.2011.00664.x
Xue Z, Yang B (2016) Phenylethanoid glycosides: research advances in their phytochemistry, pharmacological activity and pharmacokinetics. Molecules 21:991. https://doi.org/10.3390/molecules21080991
Zhong JJ (2001) Biochemical engineering of the production of plant-specific secondary metabolites by cell suspension cultures. In: Zhong JJ et al (eds) Plant cells. Advances in biochemical engineering/biotechnology, vol 72. Springer, Berlin, pp 1–26. https://doi.org/10.1007/3-540-45302-4_1
Zhong JJ, Pan ZW, Wang ZY, Wu J, Chen F, Takagi M, Yoshida T (2002) Effect of mixing time on taxoid production using suspension cultures of Taxus chinensis in a centrifugal impeller bioreactor. J Biosci Bioeng 94:244–250. https://doi.org/10.1007/3-540-45302-4_1
Acknowledgements
The authors thank the Universidad Autónoma del Estado de México (UAEM) for financing this thesis project through the Programa de Investigación Científica, Innovación y Desarrollo UAEM 2014 (Project No. 3742/2014/CIB: Desarrollando Avances Biotecnológicos Sobre la Producción de Verbascósido por Cultivos de Células de Buddleja cordata) and to the Consejo Nacional de Ciencia y Tecnología (CONACyT) through grant number 620491 for the Master’s studies of AMV-M at the Posgrado en Ciencias Agropecuarias y Recursos Naturales from UAEM.
Author information
Authors and Affiliations
Contributions
AMV-M, as a Master student and for her thesis project, participated in all the experimental work, analysis and interpretation of data, and writing of this manuscript. CZ-G supervised the establishment of the experiments and provided chemical standards. CB-A participated in the standardization of analytical procedures to quantify the secondary metabolites. AB-A participated in the analysis of the growth kinetics and their statistical analysis. AN-T participated in the experimental work on phytochemical analysis. FC-S contributed to the design and execution of experiments on shake-flask cultures; participated in the analysis and interpretation of data. MR-M contributed to the thesis project of MAV-M as an assessor; supervised the establishment of bioreactor in vitro cultures; contributed to the planning and execution of this project, which led to this publication; and contributed to the preparation and writing of this manuscript. MEE-Z contributed to the design, execution and direction of all the experiments of this project, as she was the thesis project director of AMV-M; was the responsible for the design and direction of Project No. 3742/2014/CIB, which financially supported this thesis project; and critically contributed to the preparation of the manuscript until approving the final submitted version. All authors critically reviewed the manuscript and approved the final version.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Additional information
Communicated by Sergio J. Ochatt.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
About this article
Cite this article
Vazquez-Marquez, A.M., Zepeda-Gómez, C., Burrola-Aguilar, C. et al. Effect of stirring speed on the production of phenolic secondary metabolites and growth of Buddleja cordata cells cultured in mechanically agitated bioreactor. Plant Cell Tiss Organ Cult 139, 155–166 (2019). https://doi.org/10.1007/s11240-019-01673-9
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11240-019-01673-9