Secondary Metabolites Production in Bioreactors (7 Production Types)

This article throws light upon the seven types of secondary metabolites production in bioreactors.

The seven production types are: (1) Shikonin Production (2) Berberine Production (3) Rosmarinic Acid Production (4) Indole Alkaloids Production (5) Organ Culture (6) Hairy Root Culture and (7) Commercialization.

Production Type # 1. Shikonin Production:

Shikonin is used as a remedy for various skin ailments and as a dye for skin and cosmetics. Shikonin is synthesized in cells of Lithospermum erythrorhizon from the shikimic acid and isoprenoid pathways as shown in figure. 28.6. Commercial production of shikonin was stimulated by its value and problems encountered in meeting the demand from the natural resources.

The first process for the commercial production of natural plant product by the cell suspension cultures was developed in Japan for the production of the naphthoquinone shikonin. Through visual inspection it was possible to identify the high-producing cultures.

Since the growth of cells was low in the medium (designated as M-9) which supports high levels of shikonin production, a two-stage culture process was developed. The cells are first grown in a growth promoting medium (designated as MG-5) for 9 days, filtered and pumped to a second vessel in which the M-9 production medium is added (Fig. 28.7).

The cells are cultured for 14 additional days, after which they are recovered by filtration, and the shikonin is extracted from the cells. Skikonin levels at that time were reported to be 4g 1^-1. They reported the effect of oxygen on shikonin production. Growth and production were enhanced by increased K_La for values up to 18 h^_1.

In a fermenter with a paddle-type impeller, shikonin production was enhanced by increased K_La up to 10 hr-¹. Paddle-type impeller favours cell growth however; shikonin production was reduced as compared to that in shake-flask cultured cells. High speed of the impeller further reduced shikonin production due to mechanical injury of the cells.

Efforts to use air-lift type bioreactor were frustrated by the ‘bubbling-up’ of the cells and the subsequent adherence of the cells to the tank wall. These authors then adopted a rotary drum type tank which revolved slowly, thus washing cells from the wall (Fig. 28.8). This system has been scaled-up to 1000L without loss in shikonin production.

Shikonin derivatives are a hydrophobic compound and exist on the surface of the cells as oil particles. Organic solvents such as ethanol or ether are commercially used for the extraction of the derivatives from the dried roots of L. erythrorhizon. The derivatives from the dried cultured cells are extracted with ethanol.

Production Type # 2. Berberine Production:

Berberine alkaloids are widely distributed in plants belonging to family’s berberidaceae, ranunculaceae, menispermaceae and rutaceae. Production of berberine has been scaled-up to the 400L bioreactor by Mitsui Petrochemicals Industries in Japan. Berberine is derived from two molecules of tyrosine as summarized in Fig. 28.9.

For the first time, Furuya et. al. (1972) succeeded in culturing the callus of Coptis japonica and isolated berberine, palmatine, coptisine, jatrorrhizin and magnoflorine. Nakagawa et. al. (1984) reported that time course of berberine production in cell suspension cultures of Thalictrum minus increased almost in parallel with cell growth.

Only small amount of berberine as the protoberberine-type alkaloids was found in T. minus original plant but, the callus cultures produced several protoberberine-type alkaloids, moreover, berberine was the main product. Berberine production in the cultured cells of Thalictrum was about 350 times higher than that in field grown plants.

In contrast with cell cultures of T. minus, Coptis japonica and Berberis aristata cells in culture accumulated most of the protoberberine alkaloid in the cells and released only a small amount of alkaloids into the medium, when partial autolysis of senescent cells occurred. Kobayashi et. al. (1988) developed an immobilized cell bioreactor for the production of berberine. Mitsui Petrochemical Industries has developed a technique for the commercial production of berberine using scaled-up system of 4000L vessel.

Production Type # 3. Rosmarinic Acid Production:

Rosmarinic acid (α-o-caffeoyl-3, 4-dihydroxypharyllacetic acid) has been most commonly found within the families lamiaceae and borigenaceae, and seems largely restricted to the tubiflorae. Rosmarinic acid is produced from the two distant precursors, phenylalanine and tyrosine (Fig. 28.10).

Although most of the rosmarinic acid was reported to be produced during the period of rapid growth, production only occurred in Coleus blumei cell cultures when detectable levels of (PAL) was present. Oxidized rosmarinic acid displayed anti-thyrotropic activities in tests with human thyroid membrane preperations and the pure compound has been shown to effectively suppress the complement-dependent components of endotoxin shock in rabbits.

De-Eknamkul and Ellis (1985) examined the dynamics of growth and rosmarinic acid production by the cultures of Anchusa officinalis. They reported that the conditions which enhanced growth and also enhanced the accumulation of rosmarinic acid. Zenk et. al. (1977) cultured cells of C. blumei in a single-stage air-lift bioreactor but the production of rosmarinic acid was much low as compared to that in shake-flask cultures.

Latter, they reported serious mixing problems in a 32L air-lift bioreactor system. Therefore, they opted to use a mechanically agitated- type bioreactor system with helical impeller. They reported production of 5.5g of rosmarinic acid per liter in a two-stage batch culture system.

Firstly, cells were grown for 8 days in a glucose fed batch system and after suitable growth, cells were filtered out and pumped into a second culture vessel in which they were diluted with a sucrose solution to initiate production of rosmarinic acid. Incorporation of the precursors of rosmarinic acid biosynthesis inhibits both, growth and production.

Production Type # 4. Indole Alkaloids Production:

A number of indole alkaloids are produced by in vitro cultures of Catharanthus roseus including anti-tumour agents. Since C. roseus cells enlarge as they age, they may become more shear sensitive, and this increased shear sensitivity may explains low production of secondary metabolites.

Due to this shear sensitivity of cells, several workers have adopted pneumatically agitated type bioreactors for culturing C. roseus cells. Both, external loop and draft tube airlifts as well as bubble column systems have been used. In contrast, others used a mechanically agitated bioreactor with flat blade turbine impellers, and the alkaloid production in this type of bioreactor was less than that observed in shake flasks even when same inoculum was used in both the systems.

Since the medium which favours growth of cells do not favours production of the alkaloids in C. roseus cells, a two-stage culture system was used similar to that for the production of shikonin. By coupling filtration to aeration, it was possible to filter the cells from the medium without the need for operations outside the bioreactor.

This internal filtration operation reduces contamination risk and permits the use of a single bioreactor for both, growth and production phases. In a similar system, the spin filter-coupled agitation with filtration to achieve cell separation within a mechanically agitated bioreactor was used. It has been recorded that when NAA is used in place of 2,4-D, a single medium can be used for growth and production and medium exchange not required.

The relationships between the ajmalicine production rate by C. roseus and various constant glucose concentrations have been investigated. Highest ajmalicine production rate was greatly enhanced when glucose concentration was reduced from a constant 57 gl^-1 to a constant 32 gl^-1. A relationship between the age of cells in the inoculum and the ajmalicine production by C. roseus has been established.

The cells in late stationary phase produced 5 times more ajmalicine than cells in the early stationary phase. Young cells were not in appropriate state to withstand the osmotic shock of the high glucose concentration (80 gl^-1). Similarly, positive effect of 4% C0₂ in the inlet gas stream on the production of indole alkaloids has been recorded Schltmann et. al. 1993.

The importance of gaseous metabolites on ajmalicine production by feeding the exhaust air to the culture in an air circulation bioreactor has been established. With the use of such an experimental setup, the timing, amount and type of gaseous metabolite are under natural control of the cell suspension, in contrast to the addition of CO₂ or ethylene to the aeriation gas.

By removing CO₂, ethylene, or both from the air circulation stream, it was demonstrated that both gases play a negligible role in ajmalicine production. These investigations on the negative effect of high aeration rate on secondary metabolites were performed with relatively low biomass concentration (15 gl^-1).

Production Type # 5. Organ Culture:

Micro-propagation of a number of plant species by somatic embryogenesis, shoot culture and microbulbil formation is well established. Conventional micro-propagation methods require hundreds or even thousands of cultures to produce plantlets at commercial scale. The process is also labour intensive, time consuming and expensive.

Micro-propagation in suspension cultures are potential cost-effective as less material and labour may be required and shoot and root can be grown in the same vessel. But, the growth of organogenetic cultures using bioreactor system is not well established so far.

There are only a few reports on the production of somatic embryos, bulbils, or micro-tubers in bioreactor as compared to large number of reports on micro-propagation through organogenesis in callus culture. Timmis et. al. (1998) constructed a bioreactor comprising four independently controlled 1.5L vessels as a research tool for the improvement of somatic embryogenesis using several embryogenic lines of Douglas-fir (Pseudotsuga menziesii).

Bapat and colleagues at Bhabha Atomic Research Centre, Bombay have successfully produced large number of somatic embryos of Santalum album (sandal wood) using a stirred-type bioreactor system of one litre. Similarly, Bamboo shoots are produced in an air lift bioreactor at National Chemical Laboratory, Poona.

Production of Lillium longifolium bulblets, Gentiana shoot culture and Artemisia shoot culture are other examples of organ cultures raised in bioreactor.

The culture of differentiated plant tissues is strongly affected by the four main factors in all bioreactors: moisture, temperature, soluble nutrients and gases. Light is also a critical factor required for some cultures. Assuring efficient gas exchange is a particular challenge, especially in densely packed reactors containing more than 50% biomass volume.

Nutrient mist technology, or aeroponics, is one approach to alleviate this problem. Inventions in the design of nutrient mist reactors, includes the use of acoustic windows, have made the technology simple and cost effective. Other then angiosperms, a few fern species has also been cultured in bioreactors like e.g., gametophytes of Pteridium aquilinum and Anemia phyllitidis in 8L air-lift type bioreactor.

Production Type # 6. Hairy Root Culture:

Hairy root cultures, as an alternative approach to produce secondary metabolites in vitro, have received attention during the last decade. It has been claimed that hairy root cultures have a stable and high biosynthetic capacity for the secondary metabolite production as compared to un-organized cultures.

Furthermore, hairy roots can be easily retained within the bioreactor, facilitating continuous operation. An additional feature of hairy roots is the low inoculum density required for growth, as compared to cell suspension cultures and immobilized cell systems where high density is required.

During incubation, the structured nature of hairy root cultures leads to formation of interconnected, non-homogeneous material unevenly distributed throughout the bioreactor, which made it necessary to develop novel process strategies and modified bioreactors.

Furthermore, the tendency of hairy roots to grow in a large cluster results in mass transfer limitations inside the cluster and in inefficient exploitation of the reactor volume. Scale-up of processes using hairy roots will be hampered by these technological drawbacks.

A large number of investigations have been carried out on the growth of roots and production of secondary metabolite using bioreactor systems. Catharanthus roseus roots were grown in been grown in 20L stirred-tank-reactor and the production of indole alkaloids in such cultures was reported.

The other works on root culture includes effect of reactor types on hairy root growth of C. roseus, cultivation of hairy roots of Datura stramonium in 14 L stirred tank bioreactor, the production of thiophene from hairy root cultures of Tagetes, the production of anthraquinone pigments by hairy roots of Rubia and Carrot hairy roots in 1.5L stirred tank bioreactor.

In 1990, a 500L bioreactor was developed, which could be operated as nutrient mist reactor as well as with submerged hairy root cultures. Unfortunately, the scale-up of hairy root culture is still troublesome. Despite of technological disadvantages, hairy roots can be grown at relatively high biomass densities in small bioreactors. Biomass yield of D. stramonium cultures grown in 1.5L bubble column reactor was achieved up to 40 gl^-1.

Production Type # 7. Commercialization:

Commercial-scale plant cell culture for the production of secondary metabolites has acquired much attention in Japan. Shikonin, a natural dye, is produced by Mitsui Petrochemcal Industries. Berberine is sold exclusively in Asia and can be produced commercially in a continuous-flow bioreactor in which cells are retained by a membrane and harvested periodically.

This system has been operated at a scale-up to 4000 L. Production of ginseng roots as organized tissues (tuberous- roots) is being exploited commercially. In Japan, Ushiyama (1989) successfully cultured ginseng roots in 20,000 L bioreactor and efforts are being made to exploit this system for the production of ginsenosides. The production of a natural vanilla from cell culture is being developed by Escagenetics in USA.

Elicitation is apparently a key component of the production strategy. A 300 L air-lift type bioreactor system has been developed by Vipont Research Laboratories, U.S.A. for the production of sanguinarine from Papaver somniferum. Colgate has signed a formal arrangement with Vipont, which suggests strong interest in completing the development of commercialization of this project.

A new start-up company, Phyton Catalytic, U.S.A. has been founded to make commercial scale levels of taxol and other plant products. In Europe, several processes have seriously considered for the commercialization of natural plant products but, ultimately put on hold or abandoned. However, a 75,000 L reactor facility has been established near Hamburg in Germany for use and exploitation of secondary metabolites production.

In addition to these efforts to produce secondary metabolites, whole plants are being produced. Levin et. al. (1988) reported that Plant Biotechnology Industries in Israel uses a bioreactor as an important component of a micro-propagation work that has already been successfully applied to several species. A collaborative venture between Kirin Brewery and the US Firm Plant Genetics has produced a large-scale lettuce and cereal crops from immobilization of somatic embryos.

Technology for the production of useful secondary metabolites has come to a threshold level. A final push is required to make this technology widely applicable for the production of pharmaceuticals, dyes and fragrances, demand for natural products for fragrances, dyes and pharmaceuticals (anti-AIDS, anti-aging, anti cancer etc.), technology should be ready to meet the demand in coming decades.