1

Introduction

Plant cell and tissue culture emerged years ago as a breakthrough technology, and plant scientists envis-aged several applications for this new technique, ranging from clonal propagation to the large scale production of plant-derived pharmaceutical compounds (Georgiev et al., 2009). This technique was initially used to gener-ate genetic variability (somaclonal variation), to produce pathogen-free material, to study responses to abiotic stresses, to store germplasm, and in several basic studies aiming at understanding the control of metabolic path-ways and cell responses. The production of genetically

modified plants using Agrobacterium or particle bom-bardment also relies on tissue culture. Having been recognized as a promising and powerful technology, the literature pertaining to plant tissue culture and its derived techniques has grown massively during the last few years. Currently, plant tissue culture is widely employed as a common technique in various commer-cial activities. The simplest application of aseptically cultivating plants is the large scale production of several crops, especially those having high economic value, such as cut flowers and ornamentals. The woody plant indus-try utilizes clonal propagation as an important strategy

REVIEW ARTICLE

Living between two worlds: two-phase culture systems for producing plant secondary metabolites

Sonia Malik1, Mohammad Hossein Mirjalili2, Arthur Germano Fett-Neto3, Paulo Mazzafera1, and Mercedes Bonfill4

1Department of Plant Biology, Institute of Biology, State University of Campinas, Campinas, Brazil, 2Department of Agriculture, Medicinal Plants and Drugs Research Institute, Shahid Beheshti University, Tehran, Iran, 3Plant Physiology Laboratory, Center for Biotechnology – UFRGS, Porto Alegre, Brazil, and 4Plant Physiology Laboratory, Faculty of Pharmacy, University of Barcelona, Barcelona, Spain

AbstractThe two-phase culture system is an important in vitro strategy to increase the production of secondary metabolites (SMs) by providing an enhanced release of these compounds from plant cells. Whereas the first phase supports cell growth, the second phase provides an additional site or acts as a metabolic sink for the accumulation of SMs and also reduces feedback inhibition. This review is focused on several aspects of the two-phase culture system and aims to show the diverse possibilities of employing this technique for the in vitro production of SMs from plant cells. Depending on the material used in the secondary phase, two-phase culture systems can be broadly categorised as liquid–liquid or liquid–solid. The choice of material for the second phase depends on the type of compound to be recovered and the compatibility with the other phase. Different factors affecting the efficiency of two-phase culture systems include the choice of material for the secondary phase, its concentration, volume, and time of addition. Factors such as cell elicitation, immobilization, and permeabilization, have been suggested as important strategies to make the two-phase culture system practically reliable on a commercial scale. Since there are many possibilities for designing a two-phase system, more detailed studies are needed to broaden the range of secondary phases compatible with the various plant species producing SMs with potential applications, mainly in the food and pharmacology industries.Keywords: Adsorbent, activated charcoal, dimethylpolysiloxane, lichroprep RP-8, lipophilic, liquid–liquid system, liquid paraffin, liquid–solid system, miglyol, plant cell culture, silicon oil

Address for Correspondence: Mercedes Bonfill, Plant Physiology Laboratory, Faculty of Pharmacy, University of Barcelona, Avda Joan XXIII s/n 08028 Barcelona, Spain. E-mail: mbonfill@ub.edu

(Received 06 August 2011; revised 14 December 2011; accepted 17 January 2012)

Critical Reviews in Biotechnology, 2013; 33(1): 1–22© 2013 Informa Healthcare USA, Inc.ISSN 0738-8551 print/ISSN 1549-7801 onlineDOI: 10.3109/07388551.2012.659173

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for producing large numbers of genetically uniform or homogeneous plants.

While tissue culture became very important for plant propagation, one of the fields in which plant cell culture achieved great significance is in the production of second-ary metabolites (SMs). These compounds, also referred to as natural products since they are usually related to medicinal herbs (Holt & Chandra, 2002), have always received the attention of the pharmaceutical industry and a significant proportion of drugs are derived from molecules isolated from plants (Cordell, 2000). The rea-son is obvious; 75% of the approximately 100 substances used as important drugs in the world were isolated from plants and have been used in traditional medicine (Cragg & Newman, 2001). Estimates in the 1990s indicated that approximately 25% of modern drugs were derived from an edible product (Verpoorte et al., 1999). The interest in plant SMs originated from ethnobotanical studies, which provide information about plant species being used by people in their day-to-day activities, particularly metab-olites used to treat various diseases (Simmonds, 2009).

Researchers very quickly came to understand that cells growing under controlled conditions might produce large amounts of SMs in bioreactors. By the late eighties it was reported that 30 cell culture systems were able to produce larger quantities of SMs than the respective plants (Wink, 1987). Additionally, it was discovered that the production of some metabolites might be stimulated by different treatments, such as UV irradiation, heat, cold, changes in medium pH, heavy metals, and the addition of elicitors derived from plant or microbial origins (Thorpe, 1990). During the last few years, genetic manipulation has received a lot of attention for altering the content of SMs in plant cells, through the over-expression of genes involved in the biosynthesis of a target compound or by redirecting the metabolic flow in the cell (Verpoorte & Memelink, 2002). Molecular biology tools also enable us to identify the genes of plant natural product biosyn-thetic pathways (O’Connor, 2009). Despite the employ-ment of several approaches in genomic studies, there has been only limited success in increasing SM production in plants. This is likely due to the complexity of biosynthetic pathways (Itkin & Aharoni, 2009).

However, independently of the strategy used to increase SM productions in plant cell cultures, some important questions precede the design of such systems: the price of the compound, market size, production costs, and the existence of substitutes (Mohd et al., 2005; Sasson, 1991). Regarding production costs, it is already known that some plant cells in liquid culture have a higher productivity (yield-/-mass) than a tissue in the plant. In addition, instead of extracting the compound from the cell mass, which means increased costs for bioreactor disassembly and the maintenance of aseptic conditions, the release of metabolites into the medium might be crucial for economic success (Thorpe, 1990). To promote metabolite release, several procedures have been attempted to permeabilize the cells (i.e. the use of

detergents, organic solvents, and polymeric adsorbents) but with the drawback of affecting cell viability (Felix, 1982). Fortunately, there is an increasing knowledge of ABC protein membrane transporters (Verrier et al., 2008) and genetically modified cells might be produced to release SMs into the medium. However, even using genetically modified cells, and still related to costs, the SMs should be removed from the medium continuously because of their eventual degradation by chemical insta-bility or by extracellular enzymes, and a possible feed-back inhibition of SM production due to accumulation (Beiderbeck & Knoop, 1988).

Plant cell culture are known to offer a high potential for obtaining SMs of pharmaceutical interest (Rao & Ravishankar, 2002), but many technical problems must be solved before they can be used routinely for the industrial production of valuable SMs. Despite tremendous efforts in the last decades to make plant cell suspension cultures economically viable, only a few natural products have reached an industrial scale using cell culture technology (Mohd et al., 2005). This is partly due to a lack of knowl-edge of the mechanisms of SM excretion from plant cells. By using an additional (or second) phase in the medium, it may be possible to enhance the production and release of SMs, and to decrease production costs. This review is focused on the use of two-phase culture systems for SM production, particularly in cell suspension cultures. Different types of two-phase culture systems, the various solvents used, as well as the adsorbents employed as a second phase and their compatibility with the product of interest are addressed. The applications and future prospects of this system are also discussed by taking into account its feasibility for the large-scale production of SMs at an industrial level.

What is a two-phase culture system?

In most plant cells, the production of SMs is not associ-ated with cell growth. SMs are often produced under stress conditions or at the end of the growth cycle, i.e. during the stationary phase (Dixon, 2001; Verpoorte, 2000). Two-stage culture media are generally employed, in which the cells are first cultured in a nutrient-rich medium suitable for growth (stage I) and are then transferred to an SM production medium that contains elicitors, precursors or stress-inducing compounds (stage II). It has been observed that cells in suspension cultures often synthesize very small amounts of SMs. Various strategies, such as the selection of high-yielding cell lines, manipulation of media, culture conditions, immobilization, and elicitation have been employed to improve the production of SMs from plant cell culture for their industrial exploitation. However, to date there have been only a few successful examples (Dicosmo & Misawa, 1996; Fujita et al., 1982; Malik et al., 2011; Rao & Ravishankar, 2002; Venkat, 1999). Due to low produc-tivity, as well as the complicated and uneconomical methods used to separate the products from the culture,

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the process of obtaining SMs from cell culture is not fre-quently regarded as advantageous for industrial produc-tion compared with product extraction from cultivated intact plants. There are several factors that may limit the SM production from plant cells): biochemical instability of compounds in the presence of producing cells, meta-bolic inhibition phenomena, loss of compounds due to their volatile nature, and product degradation (Berger, 1995). In addition, some SMs at certain levels are toxic to the producing cells and are hydrophobic in nature. In all of these cases, a “two-phase culture system” may be employed for the continuous removal of the products from the vicinity of the producing cells or the site of pro-duction (Serrano-Carreon, 2003). As the name suggests, a two-phase culture system consists of two-phases: a nutrient or culture aqueous phase for cell growth and a second phase with liquid or solid material to retain the SMs. The second phase constitutes an additional site or acts as a sink for the accumulation of SMs. The concept of generating an artificial accumulation site is derived from intact plants, wherein SMs, being toxic to cell functions, are stored away from protoplasts or synthesizing cells, usually in the vacuole or in specialized cells or structures. (Luckner, 1980). Several SM are stored in vacuoles, such as glucosinolates, cyanogenic glycosides, and tannins; SMs are also stored in the liposomes of specialized oil cells and idioblasts or released from the plasmalemma into cell walls as monolignols, as well as into extracellular spaces such as resin ducts (Denffer et al., 1978). A simi-lar cell compartmentalization has been observed in the cell cultures of many plant species (Bisson, 1983; Bisson et al., 1983; Nagel & Reinhard, 1975).

Two-phase culture systems have been designed to increase SM accumulation without significant detrimen-tal effects on cell growth and are based on these effects: a) minimization of interference between product accu-mulation and cell growth, thus allowing for the continu-ous expression of product at optimum production levels, b) reduction of product loss resulting from interaction among producing cells, released enzymes, and environ-mental conditions, and c) minimization of down-stream processing (Freeman et al., 1993).

Types of two-phase culture system

In two-phase culture systems, one phase is the aqueous phase that contains the nutrient medium for cell growth and another (second or extractive) phase can be either liquid or solid. Table 1 shows the different liquid and solid phases used for the extraction of SMs from plant cell cultures. Various studies have shown that both solid and liquid phases could be used to extract SMs from cells depending on plant species. The choice of material for the second phase depends upon the type of substance to be recovered and the compatibility between the phase and the substance.

There are different ways of classifying two-phase cul-ture systems depending on the nature or properties of the

second phase. However, in this review we will divide the two-phase culture systems as follows:

1. Liquid-liquid systemsa) Aqueous organic systemsb) Aqueous-aqueous or aqueous two-phase systems

(ATPs)2. Liquid-solid systems

Liquid–liquid systemsA liquid–liquid system is also known as a two-liquid-phase system or an in situ extraction. It consists of two immiscible phases obtained either by the mixture of water insoluble compounds (such as organic solvents or lipophilic compounds in liquid form) or soluble compounds (such as polymers or salts). The former is known as aqueous-organic whereas the latter is called an aqueous–aqueous or a biphasic system (Aguilar & Rito-Palomares, 2010; Choi et al., 2001).

Aqueous-organic systemsAn aqueous-organic system often contains an aqueous phase and an immiscible organic phase that contains organic solvents or lipophilic material in liquid form (Choi et al., 2001; Weatherley, 1996). This system has been successfully employed to accumulate and enhance the production of taxol from Taxus cuspidata (Xu et al., 2004), T. chinensis (Wang et al., 2001; Zuojun et al., 2003), and T. brevifolia (Collins-Pavao et al., 1996); shi-konin derivatives from Lithospermum erythrorhizon (Deno et al., 1987), Arnebia euchroma (Fu & Lu, 1998), and Echium italicum (Zare et al., 2010);and artemisinin from Artemisia annua (Newman et al., 2006). The prin-ciple of aqueous-organic systems involves the efficient contact of two liquid phases: a feed phase (aqueous phase) and an extracting solvent. The phases are brought into contact by dispersing one liquid by drop dispersion into the second liquid, which allows a continuous phase between the two liquids. The selection of an appropriate second organic phase is a critical step for the success of an organic-aqueous two-phase plant cell culture system. Different organic solvents used for the extraction of SMs from plant cells in aqueous-organic system include butyl acetate, terpineol, cyclohexane, n-hexadecane, octane, decanol, dibuthyl phthalate (DBP), oleic acid, oleic alco-hol, and castor oil. The solvent is chosen according to its chemical properties for facilitating the selective migra-tion of the desired product from the feed phase and for ensuring the rapid disengagement of the two phases after contact (Weatherley, 1996).

DBP is a phthalate ester that has been used as a sec-ond phase for the accumulation of taxol in T. chinensis cell cultures (Wu & Lin, 2003). According to Zuojun et al. (2003), oleic acid (monounsaturated omega-9 fatty acid) and DBP were found to be suitable solvents to improve the production of paclitaxel up to three-fold in T. chinen-sis cell cultures. The optimal volumetric percentage of these organic solvents in the culture medium was found

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Table 1. Secondary accumulation/extraction phases used in plant cell culture.

Secondary phaseMaterial in secondary phase

Plant species Secondary product(s) Reference(s)Nature PhaseLiquid Lipophilic Miglyol T. occidentalis Monoterpenes Berlin et al., 1984

V. vinifera Geraniol Cormier and Ambib, 1987C. glabra Anthraquinones Dörnenburg and Knorr, 1996

n-hexadecane T. occidentalis Monoterpenes Berlin and Witte, 1988L. erythrorhizon Shikonin Kim and Chang, 1990bM.citrifolia Anthraquinones Bassetti and Tramper, 1995A. euchroma Shikonin Fu and Lu, 1999

Decanol T. minuta Thiophenes Buitelaar et al., 1990Silicon fluid E. californica Benzophenanthridine

alkaloidsByun et al., 1990b

F. alnus Mill. Anthraquinones Sajc et al., 1995C. roseus Indole alkaloids Tikhomiroff et al., 2002

Tricaprilyn T. brevifolia Taxol Collins-Pavao et al., 1996Dimethylpolysiloxane P. tinctorium Indirubin Bae et al., 1998Dibutylphathalate T. chinensis Taxol Wang et al., 2001; Wu and Lin,

2003; Zuojun et al., 2003Paraffin oil L. erythrorhizon,

E. italicumShikonin Lin and Wu, 2002; Zare et al.,

2010Oleic acid T. chinensis Taxol Zuojun et al., 2003Coconut oil N. tabacum Scopoletin Iizuka et al., 2005

Aqueous Polyethylene glycol + dextran

L. vera Rosmarinic acid Pavlov et al., 2001

Solid Lipophilic LiChroprep RP-8 V. wallichii Valepotriates Becker and Herold, 1983M. piperita Essential oils Kim et al., 1995; 1996;

Chakraborty and Chattopadhyay, 2008

Adsorbent XAD-4 N. rustica Phenolics, Nicotine Maisch et al., 1986P. somniferum Sanguinarine Kurz et al., 1990; Williams et al.,

1992V. fragrans Vanilla flavour Knuth and Sahai, 1991C. Arabica Caffeine, Theobromine Kurata et al., 1994M. piperita Essential oils Kim et al., 1995; 1996T. cuspidate Taxol Kwon et al., 1998R. damascene Volatiles and polar

compoundsPavlov et al., 2005b

M. elliptica Anthraquinones Chiang and Abdullah, 2007L. vera, N. tabaum, H. annuus

Volatile compounds Georgiev et al., 2010

XAD-7 C. ledgeriana Anthraquinones Robins and Rhodes, 1986C. roseus Indole alkaloids Asada and Shuler, 1989; Kim et al.,

1995; 1996; Wong et al., 2004P. somniferum Sanguinarine Kurz et al., 1990T. rugosum Berberine Choi, 1992P. somniferum Sanguinarine Williams et al., 1992T. patula Thiophenes Buitelaar et al., 1993C. roseus Ajmalicine, Serpentine Lee-Parsons and Shuler, 2002P. rosea Plumbagin Komaraiah et al., 2003 E. californica Alkaloid Klvana et al., 2005Taxus chinensis Taxuyunnanine C Gao et al., 2011

XAD-2 G. vernum Anthraquinones Strobel et al., 1991Wofatit ES G. vernum Anthraquinones Strobel et al., 1991IR 120 V. vinifera Anthocyanins Cormier et al., 1992DMSO C. blumei Rosmarinic acid Park and Martinez, 1994Activated charcoal M. chamomilla Coniferyl alcohol Knoop and Beiderbeck, 1983

V. vinifera Anthocyanins, Phenolic acids

Cai et al., 2011

HP2MGL V. vinifera Trans-resveratrol Yue et al., 2011

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to be approximately 8%, and the favourable time for their introduction was at the exponential phase of cell growth.

In an aqueous-organic system, the presence of organic solvents allows for increased yields as well as higher rates of productivity when compared to a single-phase system, but there are several drawbacks to this system. For exam-ple, organic solvents, especially those with high carbon content, are difficult to remove by evaporation, and residual contamination may affect the quality of the final product. Second, organic solvents may exhibit cytotoxic-ity and affect the viability and/or metabolic activity of the plant cells, resulting in lengthy lag phases as well as the premature termination of the bioprocess, possibly due to the toxic effect of both the product and the solvent (Bruce & Daugulis, 1991; Hüsken et al., 2001). Thirdly, although some of the solvents have a high efficiency (partition coefficient) for extracting the compound of interest from the aqueous phase, the solvents may form emulsions with the aqueous phase upon stirring (Etschmann & Schrader, 2006), thus causing problems for optical den-sity measurements as well as separation of the phases. Attempts to avoid solvent toxicity have included the identification and design of solvent-tolerant biocatalysts (Rojas et al., 2004; Wery et al., 2000). Additionally, physi-cal barriers have been implemented in an attempt to separate organic solvents from the biocatalysts.

It remains unclear how extractive phases increase SM production. Wang et al. (2001) suggested that the increase in SM production is due to the interaction of the extractive phase, in this case liquid solvents, with the cell membrane. Perhaps the extractive phase influences the secretion of metabolites by modulating their transport across the external membrane, which has been shown to be active (Villegas et al., 2000).

Aqueous-organic systems with lipophilic materials include Miglyol, liquid paraffin, polysiloxanes, tricaprylin, or silicon fluid systems. A lipophilic second phase is com-patible with SMs that are hydrophobic in nature. Plant cell cultures with a lipophilic second phase have been reported to be helpful for the accumulation and detection of vari-ous SMs (Becker et al., 1984). Several studies have shown that plant cell cultures release lipophilic and volatile substances, such as ethylene, ethanol, and acetaldehyde. Hydrocarbons have also been detected in the spent air of a suspension culture of Matricaria chamomilla crown gall cells passing through ethanol (Byun et al., 1990a). The addition of a lipophilic phase to the culture medium can be used as a means to accumulate and detect these substances. Cell cultures with lipophilic second phases contain the nutrient medium (aqueous phase) and a small amount of lipophilic material in a liquid form. A limited number of second organic phases with high affinity for SMs have been previously employed for the in situ extrac-tion of the product from the cellular environment.

Miglyol, a neutral oil, is a water-insoluble triglyceride of low viscosity composed of fatty acids with 8–10 carbons that has been used as a liquid lipophilic phase for the in situ extraction of plant SMs (Beiderbeck & Knoop, 1987;

Dörnenburg & Knorr, 1996). Triacylgylcerols such as Miglyol, were first used to retain volatile hydrocarbons in Thuja accidentals (Berlin et al., 1984). A medium that con-tained 10–12% of Miglyol, used with transformed and non-transformed cell suspension cultures of M. chamomilla (Beiderbeck & Knoop, 1987), Nicotinia tabacum, and T. occidentalis (Berlin et al., 1984), showed unimpaired growth in comparison with a conventional single-phase culture. Simultaneously, a Miglyol phase has been shown to accumulate various SMs from cultured cells, depending on the species. For example, M. chamomilla cell suspen-sions yielded a mixture of UV-absorbing products that were synthesized and accumulated mostly during the log phase of cell growth (Beiderbeck & Knoop, 1987). A detailed analysis showed that during the first, second, or third week of the culture with Miglyol, a great variety of different compounds appeared in the cells, the aque-ous nutrient medium, and in the Miglyol phase. At any time during the culture period, a large number of differ-ent substances could be isolated from the Miglyol phase, but the pattern of substances varied in all three fractions (cells, medium, and Miglyol) from week to week. One such product was identified as the sesquiterpene alcohol α-bisabolole, a therapeutically important compound of the Matricaria flower, which has not been detected in con-ventional cell suspensions. Similarly, a suspension culture of T. occidentalis produced up to 3 mg g–1 DW day–1 of a mixture of monoterpenes (α-pinene, β-pinene, myrcene, lomonene, and terpinolene) in a two-phase culture with Miglyol compared with 0.8 mg g–1 DW day–1 obtained in a single-phase system. Additionally, labile iron-tropolonate complexes could be detected (Berlin et al., 1984). The addition of the Miglyol as a second phase to shoot cultures of Mentha canadensis during the last day of an incubation cycle of 4 weeks resulted in a monoterpene production of more than twice (1.1 mg g–1 fresh weight) the amount found in control cultures (0.5 mg g–1 fresh weight).

Liquid paraffin is another lipophilic material that has been used as a second phase for the in situ extraction of plant SMs. Liquid paraffin or mineral oil, is a mixture of heavier alkanes, and has many names, including nujol, adepsine oil, alboline, glymol, medicinal paraffin, and saxol. It has a density of approximately 0.8 g cm-3 (WHO, 1976). It has been reported that the presence of liquid paraffin (10% v/v) as a second phase in M. chamomilla cell cultures caused the accumulation of UV-absorbing SMs from the culture medium. Any interference with cell growth was hardly detectable (Beiderbeck & Knoop, 1987). Paraffin oil is a compatible solvent for the extrac-tion of shikonin derivatives from cell suspension cultures of L. erythrorhizon (Heide & Tabata, 1987; Heide et al., 1989). Deno et al. (1987) tested several types of organic solvents with cell suspension cultures of L. erythrorhi-zon growing in M-9 medium. Their results revealed that, when paraffin was used as the solvent, the yield of shiko-nin derivatives increased to more than 1g L–1, and 80–90% of the derivatives produced were retained in the oily layer. Cells from suspension cultures of Selinum candolii

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produced 4–5 times more somatic embryos per fresh weight of inoculum when they were covered with a layer of liquid paraffin oil (Mathur, 1991), presumably due to the reduced concentration of oxygen in the medium. Recently, Zare et al. (2010) reported that the addition of liquid paraffin as a second phase in cell suspension cultures of E. italicum growing in B5 medium (Gamborg et al., 1968) was highly effective for the synthesis of shi-konin derivatives in the cultured cells. It has been sug-gested that liquid paraffin has a high affinity for secreted compounds, thus leading E. italicum cell suspension cultures towards further production of these pigments. A commonly used cell culture media, such as B5, appears to lack this potential, indicating that the presence of a lipophilic extractant is a key factor for the improved production of hydrophobic metabolites (e.g. shikonin derivatives). It was proposed that liquid paraffin could act as an indirect inducer through the adsorption of the produced pigments, diminishing the negative feedback of accumulation by acting as a sink for these products.

Silicon oil is another lipophilic material that has been reported as an organic second phase in two-phase culture systems. Silicon oil (polymerized siloxane with organic side chains) is a silicon analogue of a carbon-based organic compound, and can form (relatively) long and complex molecules based on silicon rather than carbon (Martín-Gil et al., 1997). In Catharanthus roseus hairy root cultures, Tikhomiroff et al. (2002) showed that alkaloids that normally remained intracellular were recovered extracellularly using silicon oil. Their results revealed that the addition of silicon oil increased the specific yields of lochnericine and tabersonine. Further, affinity of tabersonine and lochnericine for silicon oil was found to be nine times higher than for the aqueous phase, whereas the growth of the hairy roots was not significantly modified in the presence of silicon oil. The overall specific yields of tabersonine and lochnericine were increased by 100–400% and 14–200%, respectively, with the use of silicon oil in non-elicited control cultures. Serpentine was never found in the silicon oil. The specific yields for all measured alkaloids were higher using silicon oil and elicitation, suggesting that silicon oil, while acting as a sink for tabersonine and lochnericine, was efficient at increasing the metabolic fluxes of the secondary metabo-lism pathways. The circulation of the culture medium in an external loop containing a nontoxic organic phase was also shown to be efficient for the extraction of SMs from Hyoscyasmus muticus hairy root cultures (Corry et al., 1993). Overall, it appears that the effect of the extrac-tive phases is species-specific, and cell line-specific, and the mechanism of action remains unclear. For instance, it has not been determined whether the contact between the cells and the extractive phase is necessary for an increased SM production. Byun et al. (1990b) found that the addition of silicon oil to Eschscholtzia californica cell cultures increased alkaloid production 3.4-fold in shake-flask cultures, and three-fold in airlift fermentor cultures. Sajc and Vunjak-Novakovic (2000) developed a simple

mathematical model of co-current liquid–liquid extrac-tion in the riser of an external-loop airlift bioreactor. This bioreactor system, integrating biosynthesis and sepa-ration in a single-unit, was used for the production of anthraquinones by immobilized plant cells of Frangula alnus. The production of anthraquinones was affected by flow conditions, solvent properties, solvent droplet size, and contactor length on the product extraction effi-ciency. Product concentration profiles in the continuous aqueous phase and dispersed organic phase (silicon oil and n-hexadecane) could change production of these phenolics (Sajc & Vunjak-Novakovic, 2000).

Dimethylpolysiloxane (DMPS) belongs to a group of polymeric organosilicon compounds that are commonly referred to as silicones, and it has been used as a second organic phase in two-phase culture systems (Lotters et al., 1997). DMPS is the most widely used silicon-based organic polymer, and is particularly known for its unusual rheological (or flow) properties. DMPS is optically clear, and is generally considered inert, non-toxic, and non-flammable. It is occasionally called dimethicone and is one of several types of silicone oil (Lotters et al., 1997). The effect of DMPS on the production of indirubin from cell cultures of Polygonum tinctorium was studied in shake flasks and air-lift bioreactors by Bae et al. (1998). They observed that the introduction of DMPS as a sec-ond phase increased indirubin production up to 259% in shake flasks and 16% in air-lift bioreactors. The increased production of indirubin was possibly due to the non-tox-icity of the solvent to cell growth and to the continuous removal of the product from the cell surface.

Aqueous-aqueous or ATPs ATPs, also known as aqueous biphasic systems (ABSs), are the clean alternative for traditional organic-water solvent extraction systems, and were first described by Albertsson in 1958 (Aguilar & Rito-Palomares, 2010). These systems were employed initially for the separa-tion of plant organelles and viruses. An ABS is formed when two polymers (Polyethylene glycol[PEG], dextran, starch, and polyvinylalcohol), one polymer and one kosmotropic salt, or two salts (one chaotropic and other kosmotropic) are mixed together at appropriate concen-trations or at a particular temperature (Xu et al., 2001). The two phases are composed mostly of water and non volatile components, thus eliminating volatile organic compounds (Dutta et al., 1994). The interfacial tension between the phases is very low (i.e. approximately 400-fold less than that between water and an immiscible organic solvent), allowing small droplet size, large inter-facial areas, efficient mixing under very gentle stirring, and rapid partition. The partition of a solute between the two aqueous phases depends on its physicochemi-cal properties as well as on those of the two polymers (or polymer and salt; Albertsson, 1971). These systems have been used for many years in biotechnological applica-tions as non-denaturing and benign separation media (Aguilar & Rito-Palomares, 2010; Dutta et al., 1994).

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The systems have also been used successfully used for the separation of different types of cell membranes and organelles, for the purification of enzymes and extractive bioconversions. A great variety of separations has been achieved, the most important being the separation of enzymes from broken crude cell material. The separation may be achieved in a few minutes, minimizing the harm-ful action of endogenous proteases. Continuous liquid two-phase separation is easier than continuous solid/liquid separation using equipment commonly employed with immiscible solvent systems, for example disc-stack centrifuges and counter-current separators. Such sys-tems are very amenable to being scaled-up and may be employed in continuous enzyme extraction processes involving some recycling of the phases. ATPSs have been used in a variety of applications including protein extraction (Shanbhag & Johansson, 1974), the recovery of low-molecular weight volatile products such as etha-nol and acetone–butanol (Malinowski, 2001), microbial cultivation (Wubbolts et al., 1996), recombinant proteins (Hellwig et al., 2004) and plant SM production (Brodelius & Pedersen, 1993; Zare et al., 2010). The bioconversion process in ATPs is facilitated by low interfacial tension, which leads to the formation of small phase droplets dur-ing the mixing of the phases, hence minimizing migration distances and promoting mass transfer. These systems also provide an outstanding medium for biological pro-cessing because of the high water content of each phase (Albertsson, 1971). More importantly, as mentioned above, an ATPS has the ability to partition components such as nutrients, metabolites, proteins, cell particles, and whole cells unevenly between the two phases. ATPs strategies for the recovery and characterization of biolog-ical products from plants have been reviewed by Aguilar and Rito-Palomares (2010).

Incompatibility in solutions of agar, a water-soluble polymer, with soluble starch or gelatine was documented for the first time in 1896 (Beijerinck, 1910). Upon mixing, these materials separated into two immiscible phases (Aguilar & Rito-Palomares, 2010; Albertsson, 1958). Since then, several immiscible biphasic aqueous systems have been introduced based on their mixing materials includ-ing polymer/polymer, polymer/salt, and salt/salt. For instance, a polymer–polymer two-phase system can be obtained by mixing PEG and dextran in a certain propor-tion (Dutta et al., 1994). By adding specific amounts of these polymers to an aqueous feed phase (containing the solute), two aqueous phases, one rich in PEG, and the other rich in dextran, can be obtained.

PEG is a polyether compound with many applications, ranging from industrial manufacturing to medicine and has been used extensively in ATPs (Choi et al., 2001). It may contain some inhibitory compounds that can be toxic to the cells in culture (Kaul & Mattiasson, 1991), but is a well-known fusogen for various types of cells. The direct interaction of PEG with the cell surface and the molecular weight of PEG may affect plant cell growth in ABSs. Many ABSs containing PEG such as PEG/dextran,

PEG/polyvinylalcohol, PEG/starch, PEG/sodium car-bonate or PEG/phosphates, and citrates or sulphates are used extensively during downstream processing, mainly in the biotechnological and chemical industries (Hooker & Lee, 1990). The most thoroughly investigated of these is the aqueous PEG/dextran system, where PEG forms the more hydrophobic, less dense, upper phase and dextran the more hydrophilic, denser, lower phase. Phases are formed when limiting concentrations of the polymers are exceeded. Both phases contain mainly water (typi-cally 70–90%, w/w) and are enriched in one of the poly-mers. The limiting concentrations depend on the type and molecular weight of the polymers and on the pH, ionic strength, and temperature of the solution (Aguilar & Rito-Palomares, 2010; Byun et al., 1990a; Dutta et al., 1994).

ABSs have also been employed successfully to accu-mulate and enhance the production of plant SMs. Pavlov et al. (2005a) studied the release of rosmarinic acid by Lavandula vera cell suspensions in an aqueous two-phase culture system. When a L. vera cell suspension was cultivated in a system formed by 4% PEG (MW 20,000) and 7.5% dextran (MW 70,000), 11.8% of the total ros-marinic acid content was released into the PEG phase. Hooker and Lee (1990) studied suspension cultures of N. tabacum grown in an ATPs comprised of PEG and dextran in a modified LS medium, and showed that the growth rate and stationary phase cell concentration were decreased by addition of low molecular weight PEG. The highest growth rate occurred in 3% PEG 20,000/5% crude dextran and approached growth rates and cell densi-ties of cultures grown in standard LS medium (Hooker & Lee, 1990). Although dextran is much less inhibitory, its concentration and molecular weight can similarly affect cell growth. Choi et al. (1999) prepared a 4.5% PEG 20,000 and 2.8% crude dextran system by mixing 15% of PEG 20,000, and 4% of crude dextran solution in the mass ratio of 1:2.33, which was used for Digitalis lanata cell cultures on the basis of the distribution characteris-tics of cells and product. Their results revealed that cells were totally partitioned in the bottom phase and cell growth was significantly inhibited by adding only 15% of PEG 20,000. Crude dextran (4%) also lengthened the lag period, but the growth rate after the lag was much higher than with PEG. However, in an ATPs with both PEG and dextran, the cell growth showed a similar pattern to that of the control (Choi et al., 1999).

Aqueous two-phase systems can also be generated using a polymer (e.g. PEG or dextran) and a salt such as sodium or potassium phosphate. Aqueous two-phase separations take place only at certain compositions (Hooker & Lee, 1990). The formation of PEG/salt systems was first observed in the 1950s, but the theoretical funda-mentals have not been well understood. It was observed that for PEG solutions, the addition of some inorganic salts (sulphates and carbonates) is more effective than the addition of other inorganic salts in reducing the critical concentration of cloud point curves (Albertsson,

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1958). These inorganic salts dramatically reduced the PEG cloud point at high temperatures. PEG/salt sys-tems have been introduced for the practical application of large-scale protein separation because of their larger droplet size, greater difference in density between the phases, lower viscosity and lower costs, leading to a much faster separation than in PEG/dextran systems. The industrial application of PEG/salt systems was improved by the availability of commercial separators, which allowed for faster and continuous protein yield. The application of a PEG/phosphate ATPs for the separa-tion of anti-HIV monoclonal antibodies from unclarified transgenic tobacco crude extract has been reported by Platis and Labrou (2009).

Liquid–solid systemsA liquid–solid system (or in situ adsorption) consists of liquid (nutrient) medium in aqueous form and secondary phase that contains solid compounds such as adsorbents or lipophilic material. Different adsorbents used in liq-uid–solid system include XAD-2, XAD-4, XAD-7, Zeolith, Wofatit ES, IR120, and activated charcoal. LiChroprep RP-8 is used as a solid lipophilic material.

Adsorbent materials such as polymer beads are used for adsorbing and removing target compounds from the aqueous phase while maintaining equilibrium con-ditions. In such systems, the capability of a polymer to adsorb the compound of interest is an important char-acteristic (Albertsson, 1958; Choi et al., 2001). Solid adsorbent materials, such as polycarboxyl ester based resins (AmberliteTM XAD) or activated carbon, represent an alternative to the organic solvents in biphasic systems (Held et al., 1999). A limitation of these materials may be the non-specific adsorption of organic compounds from solution. This can result in the removal of both product and substrate alike, which lead to decreased product removal efficiencies as well as increased substrate load-ings to compensate for adsorbed materials (Brodelius & Pedersen, 1993). In addition, polymeric resins, due to their porous and highly cross linked nature, are fragile and therefore are often not able to withstand the vigor-ous agitation that accompanies mechanically agitated systems. Separating the adsorbent materials from the environment of the bioreactor by way of external extrac-tion columns has been shown to be an effective strategy (Held et al., 1999). The adsorbent should be pre-treated to activate its surface before addition to the culture vessel or bioreactors for SM collection.

Adsorbents are generally used to adsorb volatile com-pounds because they are superior to organic solvents for the extraction of SMs due to toxic nature of the latter. According to Lilly et al. (1987), the presence of organic solvent in the medium changes the membrane perme-ability of the cells, which may result in inhibition of enzyme activity, protein deactivation and the collapse of transport mechanisms. It has also been hypothesized that solvents may cover a cell surface and thus inhibit the flow of nutrients and disrupt the cell wall to facilitate

the extraction of inner cellular components (Bar, 1987). An alternative to both immiscible organic solvents and solid-phase adsorbents may be thermoplastic polymers. The uptake of small molecules into thermoplastic poly-mers is analogous to their uptake into organic solvents, which operate on an absorption mechanism. Solid adsorbents operate on the basis of a surface adsorption phenomenon. These polymers have been successfully used to partition and deliver toxic organic molecules in biphasic systems, specifically for the biodegradation of phenols (Amsden et al., 2003; Prpich & Daugulis, 2006).

With the exception of lipophilic compounds, many products of plant cells are expected to have a polar nature and to be scarcely bound by lipophilic phases (Beiderbeck & Knoop, 1987). Therefore, more polar adsorbents such as polymeric resins may serve as second phases.

Solid polar adsorbents. XAD-4 is an absorber resin on a polystyrene base and is reported to adsorb a great variety of diverse substances such as phenols, alcohols, and organic acids (Wang et al., 2005). It is available as beads of 0.3–1.0 mm in diameter and can be recovered from cell suspension by repeated decanting. The solvent acetone elutes a broad pattern of substances bound to XAD-4 (Dutta et al., 1994). The additions of varying amounts of XAD-4 (up to a concentration of 2% w/v) to different cell lines of N. tabacum did not impair the growth of the cells (Beiderbeck & Knoop 1987). However, with an increase in concentration (>2% w/v), the cell lines were observed to behave differently: the growth of one line was reduced to one-third with 4% XAD-4 in the medium, whereas another cell line showed enhanced growth as well as the production of several SMs as com-pared with the control (adsorbent-free). The addition of XAD-4 at a concentration of 12.5% increased the produc-tion of chlorogenic acid more than 20-fold in this cell line (Beiderbeck & Knoop, 1987).

XAD-7, which is a polycarboxyl ester resin, has been used for the separation and enhanced production of berberine from cell suspension cultures of Thalictrum rugosum (Choi, 1992) and of anthraquinones from Cinchona ledgeriana (Robins & Rhodes, 1986). Similarly, an improved production of sanguinarine in a cell culture of Papaver somniferum by using XAD-7 has been shown by Williams et al. (1992). In 1996, Archambault et al. (1996) also reported the enhanced production of san-guinarine from elicited cells of same plant species in the presence of XAD-7. Their results showed that produc-tion levels of the larger cultures under apparently non-limiting conditions were up to 80 mg L−1 or 0.52% DW after 600 h, of which 70–80% was bound to the resin. The addition of polymeric resins to C. roseus suspension cell cultures has also been shown to increase the produc-tion of catharanthine and ajmalicine (Payne et al., 1988; Sim, 1994). Moreover, this extractive phase allowed the harvest of indole alkaloids which are known to remain intracellular.

Activated charcoal (AC) has a high capacity to bind to a wide spectrum of substances; its binding mechanism

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is not completely understood. AC is widely used in labo-ratories and for technical processes (Miguel et al., 2001). Among the available variety of different AC qualities, beads of 2–3 mm in diameter are most suitable as addi-tions to cell cultures because these are easy to separate from cells (Beiderbeck & Knoop, 1987). In a single-phase culture of M. chamomilla that released coniferyl alde-hyde into the medium, the addition of 0.8–4% (w/v) AC reduced the coniferyl aldehyde content of the aqueous phase depending on the adsorbent concentration. In contrast, compared with control cultures, 20- to 60-fold greater amounts of coniferyl aldehyde could be recovered from the adsorbent. The production of several unidenti-fied substances also showed substantial enhancement. Such an increased production of SMs may be traced partly to product accumulation in the AC and partly to a reduction of the rate of cell multiplication. After 1 week of culture with 0.8% AC as a second phase, the fresh weight of Matricaria cells was comparable to that of adsorbent-free controls, whereas the output of coniferyl aldehyde was increased by a factor of 20. With 4% AC the final fresh weight was reduced to one-third that of the controls, and the coniferyl aldehyde output per fresh weight was fur-ther increased (Beiderbeck & Knoop, 1987).

Various polymeric adsorbents including XAD-2, IRA-400, Wofalites (EP60, EP61, ES, and L60), Dowex 2X4, charcoal, and glass beads have been investigated by Strobel et al. (1991) to induce the release of anthraqui-nones from cultivated cells of Galium vernum and for the removal of SMs from the culture medium. These authors reported that XAD-2 and Wofatite ES were best suited for anthraquinone productivity and for their release from cell cultures of this plant species.

From the above literature, it is clear that there are a number of adsorbents available to recover SMs from plant cells. Adsorbents are useful for extraction when the aim is to extract a range of secondary products; other-wise, they should be designed to adsorb selectively the compound of interest. A disadvantage associated with the use of adsorbents is that, in addition to their adsorp-tion of toxic substances from the medium, they may sometimes remove nutrients or essential components required for cell growth and alter the pH of the culture. This is particularly true for AC which has low selectivity as adsorbent.

Solid lipophilic materials, such as LiChroprep RP-8, are also used in liquid–solid systems. RP-8 is a silica gel (particle size 40–60 µm) with outer SiOH groups cova-lently bound to C8 hydrocarbons (Kocjan & Sowa, 2002). These hydrocarbons coat the silica gel particles as a monomolecular lipophilic layer. RP-8 has been used as a second phase in suspension cultures of Pimpinella anisum. Cells of P. anisum released small amounts of the phenylpropanoid anethol into the RP-8 phase, whereas this compound could not be detected in single-phase cultures of the same cell line (Beiderbeck & Knoop, 1987). Cormier and Do (1988) and Teresa et al. (1995) have demonstrated the potential of RP-8 as a second phase to

enhance the overall productivity of M. piperita cell cul-tures as well as the selection of monoterpene-producing cell lines. Kim et al. (1995) reported that the use of RP-8 in cell cultures of this plant species enhanced the formation of essential oils without interfering with cell growth. In cell suspension cultures of Valeriana wallichii, RP-8 has been successfully used as a solid lipophilic phase to col-lect valepotriates (Becker & Herold, 1983).

To summarise, solid phases perform well where the total product is required. However, the cumbersome means of their addition to and removal from the medium and their adsorption of nutrients along with SMs, have limited their use in a wide range of plant cell cultures.

Ionic liquids and two-phase culture systems

General features of ionic liquidsIonic liquids (ILs) may be defined as liquids composed completely of ions (Han & Armstrong, 2007), i.e. salts in which ions are not strongly coordinated, resulting in a liq-uid state below 100ºC or even at room temperature. Ionic liquids have at least one of the ions with a delocalised charge and one organic component, therefore hindering the formation of a stable crystal structure. Ionic liquids that have melting points below ambient temperature are referred to as Room Temperature Ionic Liquids (RTILs). Compared with classical organic solvents, ILs generally consist of bulky, nonsymmetrical organic cations and different inorganic or organic anions (Choi et al., 2011; Han & Row, 2010). In general, the cationic portions of ILs are organic-based molecules, such as imidazolium, N-alkylpyridinium, and tetraalkylammonium ions. The anion moieties can be organic or inorganic and include halides, acetate, nitrate, tetrafluoroborate, trifluorometh-ylsulfonate, and hexafluorophosphate.

Ionic liquids display unique physicochemical prop-erties, including low or negligible vapour pressure, a broad liquid range for temperature, good thermal sta-bility, tunable viscosity and miscibility with water and organic solvents, and good extractability for various organic compounds and metal ions. These versatile features vary mainly with their structures. Compared with low boiling point solvents, ILs are often signifi-cantly less toxic to biological molecules. Because of their unique properties, these liquids can have interactions with solutes in a variety of ways, solubilise organic and inorganic compounds, and be miscible or immiscible with water and several organic solvents. By combining different possible cations and anions, various ILs can be prepared to present specific properties. Because of this versatility, RTILs have been referred to as “tailor-made or designer solvents” (Han & Armstrong, 2007). RTILs have found a large array of applications in separation and extraction procedures, organic synthesis (particu-larly microwave-assisted), biotransformation processes, electrochemistry, liquid phase extraction, catalysis for clean technology and polymerization processes (Han & Row, 2010).

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Applications of ILs in whole cell cultures and their potential utility in two-phase culture systemsMost biological applications of ILs have focused on biotransformation studies using immobilized enzymes; however, whole cell biotransformation systems have also been reported. Use of ILs to improve the oxygenase-catalysed biotransformation of toluene by whole cells of Escherichia coli was able to protect the bacterial biocata-lyst from substrate toxicity by changing the availability of toluene in the aqueous phase (Cornmell et al., 2008). This property allowed the formation of a higher product con-centration than in aqueous reaction systems or in con-ventional two-phase systems. In a biocompatibility study involving E. coli and Saccharomyces cerevisiae, the use of 1-n-butyl-3-methylimidazolium hexafluorophosphate (BMIM[PF6]), BMIM-bis(trifluoromethanesulfonyl) imide (BMIM[Tf2N]) and methyltrioctylammonium-[Tf2N] (OMA[Tf2N]), all of them water immiscible ILs, did not damage microbial cells. These ILs were reported to be adequate substrate reservoir and in situ product-extracting agents for biphasic whole cell biocatalytic processes, replacing organic solvents and increasing the process efficiency (Pfruender et al., 2006).

An example of the successful application of ILs in two-phase culture systems that resembles the situation often found in plant cell cultures is the production of 2-phenyl-ethanol (PEA), a flavour compound with a rose-like odour, by S. cerevisiae in two-phase cell cultures (Sendovski et al., 2010). Phenylethanol is produced by yeast in a growth related manner, but as a result of strong feedback inhibi-tion, yields are quite low. A series of immiscible ILs were tested for biocompatibility and 5 out of 9 appeared to be biocompatible. Biocompatibility was dependent on the structure of the IL. For example, in imidazolium cation rings, the longer the side alkyl chain on the ring, the lower the biocompatibility. A second phase composed of 20% of one of the most biocompatible ILs allowed a three- to five-fold increase in PEA concentration. The ILs were able to extract effectively the inhibitory PEA from the aqueous phase, improving yields. BMIM[Tf2N] gave the best results in terms of biocompatibility and process performance (Sendovski et al., 2010). This was one of the first reports on the use of ILs in a two-phase system for the continuous extraction of a metabolite produced by growing and functional yeast cells. The marine diatoms Skeletonema marinoi and Phaeodactylum tricornutum experience a differential inhibition of growth and pho-tosynthetic efficiency in the presence of imidazolium ILs, the latter being much more tolerant. Less toxicity towards these processes was observed for monoethoxy and diethoxy side chains, compared with the alkyl coun-terparts for both organisms (Samorì et al., 2011).

Clearly, considering the versatility and the unique properties of “tailor-made” ILs, there is a great deal of potential for plant biotechnologists to fully explore the possible applications of molten salts in two-phase plant cell cultures directed at SM production. A large array of process optimization parameters is possible, including,

different types of ILs, times of addition, concentrations of second phases, and medium re-circulation designs for indirect cell-IL interactions.

Factors affecting system efficiency

Plant cell cultures are characterized by relatively high growth rates and increases in biomass are supported by rich media formulations often complemented with phy-tohormones involved in cell growth and division, such as auxins and cytokinins. However, the fast growth that warrants a sufficient biomass generation for extracting target metabolites also affects the metabolite production profile and yield, particularly for products that prefer-entially accumulate in the stationary phase (known as non growth related). The ultrastructure of cells in culture resembles that of meristematic cells, presenting rela-tively small vacuoles that limit the capacity of metabolite storage, increasing the feedback inhibition of metabolic pathways (Pasquali et al., 2006). In this situation, the culture medium becomes the storage compartment, but its role as a lytic compartment can substantially limit the yield of target metabolites. Product degradation is gener-ally caused by a number of released enzymes, re-uptake followed by intracellular degradation, and/or the action of aeration or light exposure. Therefore, the second phase may have a role of as a reservoir or storage compartment for metabolites released into the culture medium, pre-venting degradation and feedback inhibition. Not less important is the practical effect of a second phase, solid or liquid; the product is concentrated, which often sim-plifies extraction and recovery (Payne et al., 1991).

Several aspects must be considered in choosing an ideal material for the second phase, such as chemical stability under autoclaving, toxicity, interaction with the medium (aqueous phase) components, solubility, ability to dissolve, bind and stabilize SMs released by the cells, large accumulation capacity, easy recovery of retained SMs during simple extraction procedures, and specificity for binding the desired SMs (Beiderbeck & Knoop, 1987). With such requirements, it is understandable that an appropriate second phase for the accumulation of a cer-tain SMs must be tailored to each substance. Currently known second phases have shown the ability to bind a vast number of cell products in a more or less nonspecific manner (Choi et al., 2001; Dörnenburg & Knorr, 1996).

Factors such as the selection of second phase type, time of addition, concentration of the second phase, toxicity to cells (biocompatibility), ratio between phases, and pH changes are key elements in the design of suc-cessful culture systems that take advantage of different phases to optimize product yield, and will be discussed in this section.

Selection of second phaseThe second phase must have several features to be the optimum reservoir for the metabolites of interest. One aspect of major concern is a low to moderate degree

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of toxicity to cells, which allows a maximum use of the biosynthetic potential of plant cells without frequent changes in the productive biomass. The second phase property of not changing the components of the culture media (inertness) is another relevant point, for a modi-fied medium also demands frequent replacement. From an operational and commercial point of view, these manipulations reduce economic viability and, increase production costs and the chance of contamination. In addition, second phases should preferably be easy to sterilize, showing autoclaving stability and the possibility of disinfecting both phases in a single step.

The capacity of binding or solubilising and contain-ing metabolites of interest in a stable form, with a certain degree of specificity, is another feature relevant for sec-ond phase materials. For maximum interaction with the first phase of the culture and metabolite retention capac-ity, it is important to use a second phase that offers the most surface with minimum weight. A high specificity of the second phase for binding the products of interest is a bonus, but it must allow for relatively simple removal upon the application of extraction procedures. An expen-sive extraction procedure and/or a process that results in high quantity of effluent accumulation may hinder the overall viability of the target metabolite production. The extraction of shikonin, alkannin and derivatives from liq-uid paraffin phase used in two-phase cell cultures of E. italicum has been successfully achieved in a simple step using a Sep-Pack reverse phase column cartridge (Zare et al., 2010).

Choice of solvent/adsorbentThe choice between solid and liquid second phases depends on their physical properties, toxicity to cells and affinity for the metabolite of interest. In general, solid adsorbents are better suited for use with cell cultures, because they comprise a large variety of chemical com-positions, with different degrees of hydrophobicity and hydrophilicity. The problem of biocompatibility is gener-ally less pronounced with solid second phases because of their lack of solubility and the easy physical separation from cells (Payne et al., 1991). Liquid extractants, often organic in nature, must be immiscible with the aqueous media, restricting the choice of solvents to highly hydro-phobic ones. This can limit the capacity of liquid second phases for extracting more polar metabolites of interest and also tends to increase problems of biocompatibility, mostly by affecting the proper functioning of membranes and enzymes. The concept of biocompatibility is relatively intuitive, but a more exact definition has been proposed that establishes a threshold of metabolic activity reduc-tion of more than 50% than that observed in absence of the solvent (Vermuë et al., 1993). This definition, how-ever, is strongly dependent on the parameter(s) chosen to access metabolic activity. By knowing the aqueous solubility of a solvent and calculating its log p, it is pos-sible to determine the critical aqueous concentration of a solvent and the maximum concentration of a solvent

that can get into the membrane. It is generally accepted that the toxicity of organic solvents increases with decreasing log p (Harrop et al., 1992; Nikolova & Ward, 1993), where p is the partition coefficient of a compound over a standard i-octanol/water two-phase system. The log p of organic solvents can be found in the literature (Laane et al., 1987). In general, organic solvents with a log p > 4 have been found to be compatible with cells (Wu et al., 2000). For example, the log p values of oleic acid and DBP are 7.7 and 5.4, respectively, and the parti-tion coefficients of taxol in these solvents are 154 and 236, respectively. Zhang and Xu (2001) observed a four-fold increased production of taxol by using a mixture of oleic acid and terpineol, and elicitation with methyljasmonic acid and silver in cell suspension cultures of T. chinensis. Xu et al. (2004) have also investigated the effects of oleic acid and DBP on the viability and membrane integrity of T. cuspidata cells in two-liquid-phase suspension cultures. They observed that the cell-viability, electrical conductivity and concentration of malonyl dialdehyde did not change when the content of oleic acid or DBP was 2% (v/v), but varied markedly when the contents of these solvents were raised to 6% (v/v) or more, indicating that the organic solvents at higher concentrations severely affected the cell membrane permeability. Different sol-vents such as n-hexadecane, octane, cyclohexane, oleic acid, oleic alcohol, butyl acetate and castor oil have been employed to enhance the shikonin derivatives produc-tion from cell suspension cultures of A. euchroma (Fu & Lu, 1998). On the solvents tested, n-hexadecane and oleic alcohol were found to significantly improve the production of shikonin derivatives (2.29 and 2.45 times, respectively). In conclusion, a solvent will be biocompat-ible when its critical membrane concentration cannot be reached (Mojaat et al., 2008).

In addition to biocompatibility, the maximum solu-bility for the product of interest and the extraction abil-ity must be considered. Unfortunately, biocompatible solvents are often poor extractants. An investigation of the effects of organic solvents on the membranes of T. cuspidata cell cultures showed little effect on electrical conductivity, concentration of malonyl dialdehyde, and cell viability when concentrations of oleic acid or dibu-tyl phthalate were 2% (v/v). However, these parameters were severely affected if concentrations increased three-fold or more, indicating damage to cell permeability control (Xu et al., 2004). Therefore, the main challenge is to identify solvents or solvent mixtures that have high extraction capacity but do not compromise cell metabo-lism and viability.

The polarity of the second phase is critical for its extrac-tion efficiency and is strongly dependent on the chemical properties of the metabolite of interest. An example is the production of lipophilic volatiles compounds that accu-mulate in the gas phase of culture vessels and bioreac-tors. Lipophilic second phases, such as RP-8, have been successfully used to increase the yields of menthol in M. piperita cell cultures by providing a steady-state recovery

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of this essential oil (Chakraborty & Chattopadhyay, 2008). As a general rule, the two most important aspects to consider in selecting organic solvents for two-phase liquid cultures are the biocompatibility of the solvents with the plant cells and a partition coefficient sufficiently high for the metabolite(s) of interest.

Considering the large number of solvents available and the diversity of production systems that can rely on two-phase culture, a method to predict the extraction capacity and biocompatibility of a solvent was developed for carotenoid production by the microalga Dunaliella salina (Mojaat et al., 2008). Initially, it was shown that log p and molecular mass were appropriate parameters for the selection of pure biocompatible solvents. The bio-compatible extraction of carotene from D. salina was only possible with a log p > 5 and a molecular mass greater than 150. In addition, a critical membrane concentration of a solvent, beyond which biocatalytic capacity ceases, has been considered, as proposed by Osborne et al. (1990). The use of Osborne´s model allowed the selection of biocompatible solvents from a series of chloro, ether, ketone and alcohol compounds in any given solvent in two-liquid-phase system using D. salina.

One approach to combine high extraction capacity and biocompatibility is to use mixtures that combine the good extractive and biocompatible properties of different solvents. In the case of D. salina carotenoid production, a mixture of dichloromethane – and decane was efficient at extracting carotene compared with pure decane. The increased extraction efficiency could result from the increased permeability caused by the action of dichlo-romethane on the cell membranes, because the concen-tration of solvent in the membrane increased by the use of a mixture compared with pure decane. The increased

carotene extraction capacity could be due to the uptake of solvent into the plasmatic or chloroplast membrane, leading to higher membrane activity, including exo- and endocytosis, with the release of carotene globules, or due to the solvent extraction of carotenoids (Mojaat et al., 2008). This important series of experiments clearly indicated that extraction capacity depends on the affin-ity of the solvent for the metabolite of interest and on the amount of solvent reaching the cell membrane (provided by an appropriate amount of toxic solvent). The overall practical significance of these observations is that the biocompatibility and efficient extraction capacity of solvent mixtures can be determined using the Osborne model and also depends on the adequate solubility of the metabolite of interest in the solvent mixture. Although developed for microalgae two-phase cultures aiming at the production of a highly apolar product, these guide-lines seem readily applicable to plant cells.

Time of second phase additionToxicity and extraction capacity can both be affected by the time of the second phase addition. The yield of metabolites that accumulate in a non-growth-related manner may often be increased by a late addition of the second phase during the growth cycle, a period in which metabolite accumulation is highest and toxicity will not compromise growth to a significant extent. Taxol production and release were promoted in T. chinensis cultures grown in 10% DBP with minimal cell growth inhibition by adding the solvent at the late log phase of the cell culture cycle (Wang et al., 2001). Taxane production in T. cuspidata cell cultures was enhanced by 45-fold in two-phase cultures with silicon cubes (Table 2). The time of addition was optimal 7 days after inoculation in a production medium (Kim et al., 1999).

Table 2. Increase in secondary metabolite production using a two-phase culture system.

Plant species Secondary compound

Culture type and Conditions

Material used in second phase

Increase in yield

Productivity/Production Reference(s)

M. chamomilla Coniferyl aldehyde C, Cr AC 20–60 times Knoop and Beiderbeck, 1983

M. chamomilla α-bisabolole C Migl detected Bisson et al., 1983

P. anisum Anethole C RP-8 detected Bisson, 1983V. wallichii Valepotriates C RP-8 46% Becker and Herold, 1983T. occidentalis Monoterpenoids C Migl 3.75 times 3.0 mg/

gDW/dBerlin et al., 1984

N. tabacum Chlorogenic acid C XAD-4 20% Maisch et al., 1986C. ledgeriana Anthraquinones C XAD-7 14.5 times 17.4 mg/L/d Robins and Rhodes, 1986L. erythrorhizon Shikonin C Hexd 1400 mg/L Deno et al., 1987C. roseus Ajmalicine,

SerpentineC XAD-7 6 times Payne et al., 1988

C. roseus Ajmalicine C XAD-7 5- to 6-fold 115.1mg/L Asada and Schuler, 1989L. erythrorhizon Shikonin I Hexd 23.96 times 71.9 mg/L Kim and Chang, 1990bE. californica Benzophenanthridine

alkaloidsC SF 3.1 times 6.2 mg/flask Byun et al., 1990b

G. vernum Anthraquinones C Wofatite ES 360% Strobel et al., 1991P. somniferum Sanguinarine C XAD-7 30–40% Williams et al., 1992

(Continued)

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Table 2. (Continued).

Plant species Secondary compound

Culture type and Conditions

Material used in second phase

Increase in yield

Productivity/Production Reference(s)

L. erythrorhizon Shikonin H, BC Hexd 3 times 572.6 mg/L Sim and Chang, 1993T. patula Thiophenes H XAD-7 40% Buitelaar et al., 1993M. canadensis Monoterpenes S Migl 2.2 times 1.1 mg/gFW Gräf and Knorr, 1993E. californica Sanguinarine C Trc 8.3 times Dutta et al., 1994V. planifolia Andrews

Vanillin A AC + FA 2 times 1.101μg/g DW Westcott et al., 1994

C. arabica Caffeine C XAD-4 1.4-fold Kurata et al., 1994M. piperita Essential oils

(monoterpenes)C, B RP-8 1.5-fold, 47% 6.9 μg/L/d Kim et al., 1995

M. citrifolia Anthraquinones C Hexd 2-fold 1.204/d Bassetti and Tramper, 1995

M. piperita Essential oils (monoterpenes)

C RP-8 1.5-fold 118 μg/L Kim et al., 1996

T. brevifolia Taxol C Trc 1.2 fold Collins-Pavao et al., 1996C. glabra Anthraquinones I Migl 5.5 fold Dörnenburg and Knorr,

1996T. cuspidata Taxol C XAD-7 40–70% Kwon et al., 1998P. tinctorium Indirubin C DMPS 259% and 2.6

fold248 mg/L Bae et al., 1998

P. tinctorium Indirubin C, B DMPS 16% 130 mg/L Bae et al., 1998T. cuspidata Taxol C XAD-4 40–70% - Kwon et al., 1998A. euchroma Shikonin C Hexd 2.29 times Fu and Lu, 1998

OA 2.45 timesA. euchroma Shikonin C, FE Hexd 6.15 fold 245.68 mg/L Fu and Lu, 1999T. cuspidata Paclitaxel C Sc 45 fold Kim et al., 1999C. roseus Alkaloids I, AL NKA-9 resin 4.5 times 380 mg/L Yuan et al., 1999R. akane Nakai Anthraquinones C + chitosan

+ 1% Tween 80

XAD-7 1.1 times 220 mg/L Shim et al., 1999

V. fragans (Salisbury) Ames

Vanilla C AC 54.3 times 16.3 mg/L Knuth and Sahai, 1999

V. fragans (Salisbury) Ames

Vanilla C XAD-4 9.1 times 0.91 g/mL Knuth and Sahai, 1999

T. chinensis Taxol C DBP 1.8 times 9.44 mg/L Wang et al., 2001C. roseus Ajmalicine I XAD-7 70% Lee-Parsons and Shuler,

2002L. erythrorhizon Shikonin C, U P 4.2 fold 354.8 mg/L Lin and Wu, 2002C. roseus Indole alkaloids H SF 100–400%

tabersonine, 14–200% löchnericine

- Tikhomiroff et al., 2002

P. rosea Plumbagin I + chitosan Amberlite XAD-7 2.54 times 92.13 mg/g DW

Komaraiah et al., 2003

T. chinensis Paclitaxel C DBP 3 fold 25.9 mg/L Zuojun et al., 2003OC 26.6 mg/L

T. chinensis Taxol C DBP 7–9 fold 19.2 mg/L Wu and Lin, 2003B. vulgaris Betalains H Silica/alumina

(1:1)71.39% Rudrappa et al., 2004

M. elliptica Anthraquinones C XAD-4 5.1 g/L Chiang and Abdullah, 2007

M. piperita Menthol C RP-8 + Menthone + -Cyclodextrin

148 mg/L Chakraborty and Chattopadhyay, 2008

V. vinifera Trans-resveratrol C HP2MGL + elicitors (SA + JA)

2666.7 mg/L Yue et al., 2011

A, Aerial roots; AL, Air-lift bioreactor; B, Bioreactor; BC, Bubble column bioreactor; C, Cell suspension; Cr, Crown-gall; FE, Fungal elicitor; H, Hairy roots; I, Immobilized cells; S, Shoot cultures; U, Ultrasound; AC, Activated charcoal; DBP, Dibutylphthalate; DMPS, Dimethylpolysiloxane; FA, Ferulic acid; Hexd, Hexadecane; JA, Jasmonic acid; Migl, Miglyol; OC, Oleic acid; OA, Oleic alcohol; P, Paraffin oil; Sc, Silicon cubes; SF, Silicon fluid; SA, Salicylic acid; Trc, Tricaprylin.

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The production of monoterpenes by M. piperita cell cultures was increased by almost 50% upon addition of Lichroprep RP-8 (Kim et al., 1995) (Table 2). The time of RP-8 addition, however, had different effects on cell growth. Exposure to RP-8 at 4 or 7 days of culture pro-moted cell growth, whereas at 0, 10, and 16 days, cell growth was inhibited. The ratio of extra to intracellular menthol and isomenthol increased after treatment with RP-8, indicating that the second phase treatment stimulated the secretion of metabolite into the medium. Menthyl acetate, which is often associated with the inhi-bition of oil production during flowering, accumulated in the RP-8 phase, which may also have contributed to the higher yields of essential oils (Kim et al., 1995).

The addition of a second phase may be coupled to an elicitor treatment to maximize biosynthetic responses. Shikonin production by cell cultures of A. euchroma was doubled by fungal elicitor treatment at the tenth day of culture. The simultaneous addition of elicitors and a second phase solvent, hexadecane, increased shiko-nin yield six-fold (Fu & Lu, 1999). A similar promotion was observed for paclitaxel yield in T. baccata cultures exposed to lauryl alcohol and methyl jasmonate; a dou-bling of paclitaxel yield was reported without significant growth depression (Yamamoto et al., 2006). However, this synergism is not always observed (Kim et al., 1995), as the interaction varies with plant species, type of elici-tor and second phase. More complex combinations of second phase (RP-8) time of addition, coupled with elicitation (γ-cyclodextrin for cell lysing) and precursor feeding (menthone), doubled menthol production and cell density and induced product secretion in M. piperita (Chakraborty & Chattopadhyay, 2008). Taxol yield in T. chinensis suspensions was promoted by a combina-tion of oleic acid: terpineol (1:1 v/v) at 3:50 (v/v) 4 days after a multifactorial elicitation (jasmonate, chitosan, and silver) applied to 10-day-old cultures (Zhang & Xu, 2001). This strategy increased the taxol yield two- and seven-fold compared with elicitation and in situ extrac-tion alone. Moreover, this study illustrates that separating the time of elicitation and the two-phase addition can be critical to avoid negative effects of the solvent phase on the action of elicitors, such as jasmonate.

A different approach to avoid the toxicity of organic compounds in two-phase plant cell cultures is to separate the adsorbing material from the cells. In two-phase sus-pension cultures of E. californica, a system was devised to extract benzophenanthridine alkaloids from the cell cul-ture, without cells contacting the extracting resin XAD-7, by recirculating the medium through a column filled with the organic phase (Klvana et al., 2005). With respect to the relative content of alkaloids of interest (sanguina-rine and chelerythrine) in the total alkaloids produced, this strategy compared very favourably with the con-ventional method of adding the resin directly to the cell suspensions. The recirculation simplified the alkaloid production process, enabling the continuous extraction of the products of interest without culture termination.

However, a limitation on flow rate through the recirculat-ing device makes the overall production equivalent to or slightly lower- than the conventional addition of resins in cell suspensions. Further improvements are needed for optimized performance.

Concentration and volume fraction of the second phaseThe main site of action of organic solvents is the plas-malemma, which participates in the selectivity of solute transport, energy maintenance, and homeostasis of the intracellular environment. The toxicity of second phases to cells is mainly the result of membrane damage due to the partitioning of the solvents into the lipid bilayer, which compromises cell viability (Sikkema et al., 1995). It is believed that the toxicity caused by solvents results from their incorporation into the membrane lipids, obliterating basic cell functions, such as transport, permeability, and enzyme activity, and eventually causing cell lysis at high concentration. It appears that the key factor in toxicity is the solvent concentration in the membranes, rather than a particular chemical structure (Sikkema et al., 1992). The use of solvents at subsaturating concentrations allows for the separation of the effects of phase (when there are clearly two separate phases, i.e. saturating concentrations of solvent) from those of molecular toxicity (caused by solvent molecules that dissolve in the aqueous phase; Mojaat et al., 2008). In phase toxicity, the close contact of cells with the solvent phase may lead to the partial disrup-tion of the cell wall, nutrient transport, and major leaking of inner cellular components due to membrane damage. In molecular toxicity, changes in membrane permeability, enzyme and transporter inactivation can take place due to dissolved solvent molecules in the lipid membranes.

For paclitaxel production in T. cuspidata cell cultures, the effect of different concentrations of a second phase made of silicone cubes was examined; a 10% (w/v) con-centration yielded the highest product concentration and specific productivity (Kim et al., 1999). An interest-ing feature of these experiments was that the addition of silicone cubes resulted in a major shift in the taxane composition of the culture from 10-deacetyl baccatin III and baccatin III towards paclitaxel (paclitaxel increased from 5 to 85% of the total taxanes). A strong preferential adsorption of paclitaxel by silicone cubes was observed compared with baccatin III and 10-deacetyl baccatin III, allowing for the effective removal of the final product without compromising its biosynthetic flux by precursor binding (Kim et al., 1999).

An investigation of various types of solvents (hydro-carbons, fatty alcohol, and triglyceride) on taxol pro-duction by T. baccata cell cultures evaluated the effect of the organic solvent volume fraction on production. A clear promotion of taxol productivity was observed as the organic solvent volume fraction increased from 10 to 50%, without a major impact on growth. The greater volume fraction likely allowed a more efficient removal of taxol from the extracellular compartment, relieving

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feedback inhibition. In addition, larger organic solvent volumes may have had a stronger impact on stress-induced taxol production (Yamamoto et al., 2007). The response was consistent for octadecane, lauryl alcohol, and tricaprylin. Only hexadene had the same effect at the volume fractions tested, presumably due to the adsorp-tion of metabolite intermediates or conditioning factors required for taxol production. A related approach was applied to taxol production by T. cuspidata cell cultures by the frequent refreshing of lauryl alcohol in a two-phase culture system (Yamamoto et al., 2009). The sequential replacement of the organic phase promoted taxol yield by increasing removal capacity and alleviating feedback inhibition, without negative effects on cell growth. The strategy was also successfully applied for combined two-phase and elicitation with methyl jasmonate.

A two-phase culture of T. cuspidata cells using 4% (v/v) oleic acid improved paclitaxel accumulation by 4.8-fold, compared with the control (Xu et al., 2005). Oleic acid may not only act as a solvent phase to partition taxol, but also as a trigger of defense responses. There is evidence that the activation of an extracellular signal-regulated kinase, such as MAPK depends on oleic acid stimulated G-protein and ion channel activity, but not on oxidative burst during apoptosis and taxol production (Cheng et al., 2008). A more detailed analysis of the cytological and cell cycle changes in the cells revealed a decreased mitotic activity and an increased cell apoptosis. Abnormal cells, chromosomal doubling, chromosomal bridges, bi-nucle-ated cells and the presence of micronuclei confirmed that oleic acid negatively affected normal mitosis of T. cuspidata cells in two-liquid-phase suspensions (Xu et al., 2005). Because abnormal mitoses were 3 to 4 times more frequent than apoptosis, it was suggested that apoptosis followed oleic acid-caused abnormal mitosis. There was a close relationship between the decreased mitotic index/increased apoptosis and improved taxol yield. However, this study did not present data on the metabolic changes associated with oleic acid use. A sec-ond phase concentration that promotes metabolite yield may cause genetic damage to cells, which could compro-mise the reutilization of cell biomass in the long term.

Hydrogen potentialPlant secondary metabolism can be affected strongly by pH. The protonation status of specific metabolites (e.g. anthocyanins), interferes with key aspects for ecochemi-cal interactions, such as colour, affecting interactions with pollinators and seed dispersers. The overall activ-ity of membrane transporters in tonoplasts, plastid and mitochondria envelopes and the plasmalemma itself can be modified by pH changes. Electrochemical gradients, on which certain transporters depend, can be enhanced or disrupted by pH changes of certain magnitude. These aspects alone could help to explain the relationship between intracellular pH and the SM profile.

Studies using fluorescent probes to monitor the acidi-fication of intracellular cytoplasm, coupled with the use

of pharmacological agents to modulate pH, revealed a positive correlation between cytoplasm acidification and the production of SMs. This response was observed independent of metabolite class or plant species. The authors suggested that cytoplasmic acidification may not be a necessary pre-requisite to metabolite production, but rather, it is part of a metabolic shift that accompanies metabolite production. In fact, the mechanism proposed to explain the results involves the fact that low cytoplas-mic pH may affect the transport of metabolites (for exam-ple using H+-antiporters) among cell compartments and from the cells to the culture media (Hagendoorn et al., 1994). Cytoplasmic acidification and the corresponding medium or apoplast alkalinization are also early events in elicitor-treated plant cells, leading to oxidative burst and the biosynthesis of defense-related SMs. These responses are related to the elicitor-induced depolarization of the plasma membrane, followed by exchanges of protons, potassium, calcium, and chloride ions (Zhao et al., 2005).

The control of pH can be challenging in some two-phase media cell cultures. Changes in pH can affect not only production of metabolites of interest and cell growth, but also the partitioning of metabolites between the phases. The alkaloid production profiles in C. roseus root cultures grown in the presence of silicon oil revealed the selective partitioning of the alkaloids tabersonine and löchnericine in the organic phase at pHs 6 and 7, which could be further increased at higher pH values (Tikhomiroff et al., 2002). Cell cultures of T. brevifolia in two-phase medium that contains tricaprylin had growth and extracellular taxol accumulation negatively affected above pH 7.5. However, taxol partitioning into the organic phase was highest at pHs 8 and 9 (Collins-Pavao et al., 1996). Therefore, despite the potential regulatory role of pH on metabolite yield and its impact on parti-tioning between phases, a compromise must be reached so that the maximum accumulation of metabolites in the extracellular fraction does not limit growth and/or biosynthesis.

Advantages and applications of two-phase culture system

A second phase in the culture medium provides an artificial site that can sequestrate metabolites and con-comitantly retard the loss of volatile compounds, prevent feedback inhibition, and eliminate unwanted secondary transformations that may occur by reactions with the constituents of the medium (Banthorpe, 1994). Apart from product removal, two-phase culture systems offer a number of key potential advantages, which are described below.

Increase in productivityA secondary phase in culture acts as a metabolic sink for the accumulation of SMs and directs the metabolic flux towards the desired end product or intermedi-ate within the pathway (Tikhomiroff et al., 2002). It has

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been observed in many plant species that in situ prod-uct removal by using a liquid or solid phase enhanced or stimulated the SM production and the products were selectively released from the cells and dissolved in the solvents or bound to adsorbents (Kim & Chang, 1990a, c; Shim et al., 1999). The removal of compounds from the cell surface enhances the biosynthetic capability of the cell, thus leading to a continuous production of the desired compounds, because the storage capacity of cells does not become the limiting factor (Strobel et al., 1991). There are a number of examples in which two-phase culture systems have been successfully employed for the enhanced production of SMs; a few of which are listed in Table 2.

Cost-effectiveness and ease in down-stream processesThe high cost and complex procedures associated with the separation of the compounds of interest and com-plicated downstream processes limit the industrial pro-duction of SMs from in vitro cultures. The cost-effective separation of SMs from the cell culture is of prime inter-est for making the process feasible on a commercial scale. Since the products are selectively released from the cells and dissolved in the solvents or adsorbents, a two-phase method reduces the cost and time required to harvest the product through complicated procedures. (Aguilar & Rito-Palomares, 2010; Shim et al., 1999). In a two-phase culture system, cells continuously grow in the culture medium while the excreted product is released in to the second phase where it is easily harvested without disrupting the cell or disturbing the culture process. Cells may be recycled by adding fresh solvent or adsorbent. This method also eliminates the labour and expenses involved in regenerating the biomass and make the downstream processes simple and economi-cal. The product can be concentrated by in situ recovery, and downstream purification may be reduced if product removal from the culture medium and cells is selective. Consequently, the recovery and purification are gener-ally simplified, thus reducing production costs.

Non-toxicity to cellsSubstances, including the SMs of interest, released from cells into the culture medium may be harmful to cell growth and consequently decrease SM production. Adsorbents, e.g. charcoal in the medium as a second phase, are able to adsorb not only the SMs of interest but also waste materials released from cells, thus alleviating their toxic effects (Thomas, 2008).

Relief of feedback inhibitionThe over-accumulation of SMs in cell cultures lead to feedback inhibition as observed in several plant species, including Taxus and Lithospermum (Collins-Pavao et al., 1996; Deno et al., 1987). By using a two-phase culture system, it is quite possible to eliminate the feedback inhi-bition of metabolic enzymes as well as the inhibition of membrane transport. This method can even eliminate

traces of products from the culture medium, which may up-regulate the biosynthesis pathways of SMs by alleviat-ing negative feedback. In T. chinensis, paclitaxel produc-tion was improved by reducing the feedback depression caused by the accumulation of this compound in the medium. More than 80% of the total paclitaxel was extracted from the organic phase (Zuojun et al., 2003). Similarly, in Galium vernum, the secretion and in situ removal stimulated anthraquinones production from cell suspension cultures (Strobel et al., 1991). According to Beiderbeck and Knoop (1987), in situ product separation can enhance the production of SMs by removing feed-back regulation mechanisms and non-specific inhibitors in plant cell cultures.

Many approaches have been developed to remove metabolites of interest in situ along their production. Two-phase culture systems such as liquid–liquid and solid–liquid systems, in which the products are extracted from the non-aqueous phase, have been introduced to remove the feedback regulatory mechanism in plant cells (Choi et al., 2001; Shim et al., 1999). By using a two-phase culture, the accumulation of SMs inside the cell, in the culture medium, and in the accumulation phase should arrive at an equilibrium depending on the affinity, stor-age capacity, and the amount of the second-phase mate-rial (Abdullah et al., 2005; Beiderbeck & Knoop, 1987).

Prevention of product degradation and lossVarious SMs produced by cells suspension cultures may be lost (e.g. volatile products) or degraded by excreted catabolic enzymes and acids in the culture medium. A two-phase culture system could efficiently limit the deprivation of SMs in cell culture and protect the SMs from degrading enzymes (Becker et al., 1984; Robins & Rhodes, 1986). Evaporation of the product into the gas phase can also be reduced by trapping flavour com-pounds in artificial accumulation sites. The desired plant products can then be removed selectively from the cul-ture systems (Dörnenburg & Knorr, 1996; Payne et al., 1988).

Synergistic effect of in situ product removal and stimulating strategiesIn situ product removal in combination with other meth-ods for enhancing SM production, such as elicitation, immobilization, and permeabilization has led to signifi-cant enhancements in SM production from plant cells. For example, a two-phase culture system in combination with immobilization improved the specific productivity of anthraquinones 5.5-fold in Cruciata glabra (Dörnenburg & Knorr, 1996). In another case with cell suspension cul-tures of Papaver somniferum, the overall yield of sanguin-arine was found to reach more than 39 mg g−1 DW when elicitation was combined with in situ adsorption which was only 21 mg g−1 DW for the elicited culture (Williams et al., 1992). The production of anthraquinones was enhanced two-fold in the presence of hexadecane and the surfactant Pluronic F-68 (Bassetti & Tramper, 1995). The

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simultaneous use of low-energy ultrasound and in situ extraction using paraffin oil resulted in two-to three-fold increases in shikonin yield in cell suspension cultures of Lithospermum erythrorhizon (Lin & Wu, 2002). The con-current use of Miglyol as a lipophilic accumulation phase and immobilization improved the specific productivity of anthraquinones 5.5-fold in C. glabra (Dörnenburg & Knorr, 1996). A synergistic effect of in situ product removal, elicitation, and immobilization has been shown in C. roseus cell cultures. The production of ajmalicine in cell suspension cultures of this species was increased (from 2 mg L–1 to 90 mg L–1) when subjected to all of these treatments simultaneously (Asada & Shuler, 1989). The yield of the naphthoquinone plumbagin, which is a pharmaceutically important compound, was enhanced 21 times (92.13 mg g−1DW) upon treatment with elicita-tion, immobilization and in situ adsorption (Komaraiah et al., 2003). The combined use of ultrasound, the elicitor methyl jasmonate and in situ solvent extraction resulted in 33–35 mg L–1 of taxol production, which was 17-fold higher than the control (1.9 mg L–1). This enhanced pro-duction of taxol occurred due to the stimulation of the enzyme activities in the biosynthetic pathway in response to all of these treatments (Wu & Lin, 2003).

Successful application of the two-phase culture system to produce plant SMs

Studies using a two-phase culture system have had vary-ing degrees of success although virtually all have resulted in an increased production of SMs. In the best of cases, production has been increased 10-fold or even more. In Table 2, we have gathered examples of the whole range of published results since the system began to be employed in the 1980s.

SM production increases of two to 60-fold using a sec-ond accumulation phase have been reported (Table 2). There are a few examples that show even higher increases in production. For example, an increase of 360% in anthraquinones yield was achieved using Wofatite resin in Galium vernum cell suspension cultures (Strobel et al., 1991), and a 100–400% increase in tabersonine yield was observed using silicon oil in C. roseus hairy roots (Tikhomiroff et al., 2002).

In general, the best results have been achieved using solid second phases (in situ adsorption), such as acti-vated charcoal and the resins XAD-4 and XAD-7, as compared with liquid secondary phases (in situ extrac-tion). AC has been useful for increasing the production of coniferyl aldehyde (20–60 times) and vanilla (54.3 times). XAD-4 increased chlorogenic acid (20%), caffeine, taxol (40–70%), and vanilla (9.1 times) production. XAD-7 has been the most commonly employed solid second-ary phase and its addition in culture medium improved the production of several SMs including anthraquinones (14.5 times), ajmalicine (70%), serpentine (6 times), san-guinarine (30–40%), thiophenes (40%), plumbagin (2.5 times), and taxol (40–70%).

A liquid second phase has been used more specifically for the accumulation of one or two SMs, whereas a second phase with solid materials is generally employed to retain a wide range of secondary products. In this way, n-hexadec-ane has been used successfully to increase the production of shikonin, dibutylphtalate for taxol and Miglyol for mono- and sesquiterpenoids. Examples of the successful yield improvements of various SMs with two-phase cultures exist for herbaceous and woody species, and span a wide array of metabolite classes and second phase types (Table 2).

A closer look at Table 2 reveals that most of the findings/reports on two-phase culture for SM production appeared in the 1990s. During this period, a large variety of liquid/liquid and liquid/solid second phases were assayed and, in general, the best product yields were achieved. Also during this period, a patent on vanilla production using activated charcoal and XAD-4 appeared (Knuth & Sahai, 1999). However, this method has not been sufficient to scale-up the system to an industrial level and more efforts should be made toward achieving this goal.

Conclusions and future perspectives

A two-phase culture system can be a powerful tool to stimulate plant cell cultures to produce SMs, simulat-ing at least some of the complex compartmentation and metabolic fluxes inherent to intact plants. The secondary accumulation phase has the important role of providing the cells in suspension cultures a surrogate storage and metabolic regulator compartment, thus allowing a con-tinuous production of SMs at a level comparable to that found in the whole plant. The combination of two-phase culture systems with elicitation strategies, precursor feeding and different bioreactor designs are key compo-nents of the full development of this technology.

Most of the reports of two-phase culture systems have achieved improvements in yield and productivity; how-ever, from its beginning in the 1980s, their use for in vitro culture has not been as widespread as might be expected. Perhaps this is because, ideally, the second phase should be optimized for each plant species and SMs of interest, and this would require previous studies of the range of possible secondary phases to minimize non-specific behaviour and achieve optimal results. Future studies should focus on finding solvents and adsorbents other than those that have already been tested. A more detailed knowledge of the properties of accumulation phases is required before the successful large scale production of SMs from plant cells using these systems becomes economically viable. The release mechanisms of SMs from the cells also requires a more in-depth study. Such insights would provide a solid base for improving indus-trial phytochemical production using in vitro cultures.

Acknowledgments

S. Malik acknowledges the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for a

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post-doctoral fellowship (process 2009/53450-8). A. G. Fett-Neto and P. Mazzafera acknowledge Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for research fellowships. M. Bonfill would like to thank Spanish MEC (grant BIO2008-01210) and Calatan Government (grant 2009SGR1217) for supporting this work.

Declaration of interest

All the authors have read the manuscript and there are no conflicts of interest.

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