Plant Cell. Tissueand Organ Culture 43: 93-95, 1995. © 1995KluwerAcademicPublishers. Printedin the Netherlands.
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Use of non-conventional media in Morinda citrifolia cell cultures Lucilla Bassetti & Johannes Tramper Department of Food Science and Technology, Food and Bioprocess Engineering Group, Wageningen Agricultural University, P.O. Box 8129, 6700 EV Wageningen, The Netherlands
Key words: critical solvent concentration, log P, plant cells, solvent biocompatibility
Abstract Non-conventional media, containing organic solvents as supplement, were exploited to obtain the non-lethal product release of plant cell secondary metabolites, using suspension cultures of Morinda citrifolia as model system. The results of our preliminary studies about solvent biocompatibility show that the discrimination between biocompatible and toxic solvents can be achieved by means of two parameters: log P and critical solvent concentration. The last one seems to be a better indicator of solvent toxicity for living cells. Abbreviations: log P - logarithm of P; P - solvent partition coefficient in a standard system n-octanol/water
Introduction In the last decade, the interest in the use of nonconventional media in biocatalysis has been increasing. Non-conventional components, like water-immiscible organic solvents, have been introduced in reaction environments and growth media, because of the advantages an organic compound may bring to the system. Among these advantages, the selective extraction into an organic phase and the resulting reduction of product inhibition appear particularly attractive for use in plant cell biotechnology. A number of plant cell secondary metabolites have great economic value, and therefore are of commercial interest. Many secondary metabolites are, however, not spontaneously excreted and since the retention of intracellular products may affect the accomplishment of a continuous industrial process, cells need to be forced to release. Several procedures to permeabilize cells have been described (Felix 1982). In most cases product release was achieved, but cell viability was not preserved. It is important, in this respect, to develop a system in which a non-lethal product release can be accomplished. The use of organic solvents is one of the possible alternatives, which has been extensively investigated in our department using two model systems: hairy roots and suspensions of Tagetes
spp., producing thiophenes, and cell suspension cultures of Morinda citrifolia, producing anthraquinones as secondary metabolites.
Solvent biocompatibility Some general rules of thumb for the prediction of a biocatalytic activity in the presence of a given solvent were formulated for enzymes and microorganisms. Solvent hydrophobicity and activity retention were related by using the parameter log P. Log P is the logarithm of the solvent partition coefficient over a standard twophase system n-octanol/water. If log P is higher than 4, activity retention is full; if log P is lower than 2, there will be no activity left; between 2 and 4 no prediction is possible, as the activity retention is variable (Laane et al. 1987). Studies performed on our model systems showed that the previous relationships hold for plant cells as well, except that the break point shifted to a value of 5 instead of 4 (Buitelaar et al. 1990; Buitelaar et al. 1991; Bassetti & Tramper 1992). By using a two-phase system with n-hexadecane as second phase component, a significant enhancement of thiophenes excretion (+ 50%) was obtained in hairy roots cultures of Tagetes patula (Buitelaar et al. 1991). Molecular
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Fig. 1. Specificgrowth rate (e) and anthraquinoneaccumulation rate (o) as a functionof the solventlog P. 100%representsthe rates of the controlcells, grownin a solvent-freemedium(fromBassetti & Tramper1994)
toxicity (due to the solvent molecules dissolved into the aqueous phase; Bar 1987) was investigated in Morinda citrifolia suspensions cultures. Cells were grown over a period of 18 days in Gamborg's B5 media, saturated each with one of 33 solvents, which were selected according to their hydrophobicity (log P range:0.8/11.6). Two biological activities were considered as indicators of the solvent toxic effect: cell growth *and secondary metabolite accumulation. Results are reported in Fig. 1. The above-mentioned bioactivities were not affected by only 8 among the 33 tested solvents, those with log P higher than 5. At log P < 5, no growth and anthraquinone production were observed. Since a second, organic phase was not present, the toxic effects are exerted only at a molecular level. The observed shift of the break-point to a value of 5 indicates that plant cells are more sensitive than enzymes or microorganisms to organic solvents, with only the rather hydrophobic ones being detrimental (Bassetti & Tramper 1994). Despite of several studies, the mechanism of solvent toxicity has not yet been elucidated. If enzyme inactivation by solvents is generally explained by considering the distortion of the essential water layer surrounding the protein, cell toxicity is mainly attributed to a distorting action on cell membranes (Laane et al. 1987; Osborne et al. 1990). A deeper insight into the interaction of solvent /plant cell at molecular level was achieved by determining the values of the 'critical solvent concentrations' (Bassetti & Tramper 1994). The series of 1-alkanols and esters of acetic acid were tested. The critical concentration for a given solvent was
Fig. 2. Logarithmof the criticalsolventconcentrationvalues as a functionof the solventhydrophobicity(log P) (fromB~setti & Tramper1994)
defined as the smallest amount of solvent needed to kill, within 48 hours, a fixed amount of Morinda citrifolia cells. Values were expressed in molality (moles of solvent / kg dry weight of biomass), since the amount of membranes (target of the solvent action) is dependant on the amount of biomass. In Fig. 2 the results are shown. It appears that hydrophobic solvents (high log P) are more toxic than hydrophilic ones (low log P): the longer the carbon-chain length, the smaller the value of the critical solvent concentration. Trends shown in Fig. 1 and 2 indicate that a careful choice of the biomass amount has to he made and that the parameter 'critical solvent concentration' is a better indicator than a percentage activity retention of solvent toxicity for living cells. Toxicity data found for Morinda citrifolia plant cells are consistent with investigations with bacterial cells (Vermu~ et al. 1993). In both cases the same type of mathematical equation can be used to represent the data, i.e. the linear correlation in Fig. 2. Critical solvent concentration data might imply that hydrophilic solvents, considered so far too toxic for use in extractive biocatalysis, may be utilized as well. A recent paper, in fact, reports on the use of hexane and pentane, 'toxic' and volatile solvents, for the integrated recovery of secondary metabolites from plant cells (Carry etal. 1993).
Conclusions
Though the mechanism of solvent toxicity has not yet been fully explained, some valid criteria for the
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use of organic solvents in extractive biocatalysis have been formulated and experimentally validated. Among them, the most important are: biocompatibility, nonbiodegradability, favourable distribution coefficient for the product, chemical and thermal stability (Bruce & Daugulis 1991). Since the introduction of an organic solvent leads to increasing complexity of the reaction system, novel bioreactors concepts are needed. An example is given by the Liquid-impelled Loop Reactor (Tramper et al. 1987), which was especially developed for fermentations with liquid-liquid two-phase systems.
References Bar R (1987) Phase toxicity in a water-solvent two-liquid phase microbial system. In: Laane C, Tramper J & Lilly MD (Eds) Biocatalysis in Organic Media (pp 147-153). Elsevier Science Publisher, The Netherlands Bassetti L & Tramper J (1992) Effect of organic solvents on growth and anthraquinone production in Morinda citrifolia cell cultures. In: Tramper J, Vermu~ MH, Beeftink HH & yon Stockar U (F_As) Biocatalysis in Non-Conventional Media, Progress in Biotechnology, Vol 8 (pp 617--622). Elsevier Science Publisher, The Netherlands Bassetti L & Tramper J (1994) Organic solvent toxicity in Morinda citrifolia cell cultures. Enzyme Microb. Technol. 8:642--648
Bruce LJ & Daugulis AJ (1991) Solvent selection strategies for extractive bioeatalysis. Biotechnol. Prog. 7:116-124 Buitelaar RM, Vermu~ MH, Schlatmann JE & Tramper J (1990) The influence of various organic solvents on the respiration of free and immobilized cells of Tagetes rninuta. Biotechnol. Techn. 6: 415--418 Buitelaar RM, Langenhoff AAM, Heidstra R & Tramper J (1991) Growth and thiophenes production by hairy root cultures of Tagetes patula in various two-liquid phase bioreactors. Enzyme Microb. Technol. 13:487---494 Corry JP, Reed WI & Curtis WR (1993) Enhanced recovery of solavetivone from Agrobacterium transformed root cultures of Hyoscyamus muticus using integrated product extraction. Biotechnol. Bioengin. 42:503-508 Felix H (1982) Permeabilized cells. Anal. Biochem. 120:211-234 Laane C, Boeren S, Vos K & Veeger C (1987) Rules for the optimization of biocatalysis in organic solvents. Biotechnol. Bioengin. 30: 81-87 Osborne SJ, Leaver J, Turner MK & Dunnill P (1990) Correlation of biocatalytic activity in an organic aqueous two-phase system with solvent concentration in the cell membrane. Enzyme Microb. Technol. 12:281-291 Tramper J, Wolters I & Vedaan P (1987) The Liquid-impelled Loop Reactor: a new type of density-difference mixed bioreactor. In: Laane C, Tramper J & Lilly MD (Eds) Biocatalysis in Organic Media (pp 311-316). Elsevier Science Publisher, Amsterdam Vermu~ MH, Sikkema J, Verheul A, Bakker R & Tramper J (1993) Toxicity of homologous series of organic solvents for Grampositive and Gram-negative bacteria. Biotechnol. Bioengin. 42: 747-758
