Review Article 1

2

A Review on Bioreactor Technology Assisted 3

Plant Suspension Culture 4

5 6

.7

ABSTRACT 8

This review is related to bioreactors for plant suspension culture and its products. Bioreactor plays an important role in bioprocess engineering. The core of bioprocessing technology is the bioreactor. A bioreactor is basically a device in which the organisms are cultivated and helps in production of desired products in a contained environment. Bioreactors are usually a containment which provides optimal condition for microorganisms in order to produce desired products. In this review, the bioreactor’s principle, working and its types are discussed. Enclosed by unit operations that carry out physical changes for medium preparation and recovery of products, the reactor is where the major chemical and biochemical transformations occur. In many bioprocess, characteristic of the reaction determined to a large extent the economic feasibility of the project. The integration of biosynthesis and separation is considered as a possible approach towards more efficient plant cell and tissue culture. In this review article, the aspects of bioprocess engineering for plant suspension culture and its products, bioreactor types, optimized strategies for production of secondary metabolites also and its industrial applications.

9 Keywords: Bioreactor, Secondary metabolite production, Industrial applications 10 11

1. INTRODUCTION 12 Plant tissue culture is generally refers to the in vitro cultivation of any plant segment, that 13 may be a single cell, a tissue or an organ. They are five main types of plant tissue 14 cultures: 1) seed lines of plants, 2) Isolated embryo,3) Isolated plant organs,4) Explant 15 cultures, 5) Isolate cells or small aggregates dispersed in liquid media. Generally, 16 commercial interests are secondary metabolites, which is categorized into essential oils, 17 glycosides alkaloids. These are synthesized by means of bioprocess engineering using 18 bioreactors [1]. Plants have evolved a vast chemical copiousness determined primarily 19 by the requirement to protect themselves from microbial pathogens, Insects and pests, 20 grazing animals. Special bioreactors are developed for some application. [2] Typical 21 bioreactors for plant cell and tissue cultures have been made of glass or stainless steel 22 for more than 40 years. In this area, stirred reactors, rotating drum reactors, airlift 23 reactors, bubble columns, fluidized bed reactors, packed bed reactors and trickle bed 24 reactors with culture volumes up to 75 m

3 as well as their modifications are the most 25

commonly used bioreactor types in research and commercial production processes [3]. 26 Bioreactors have been developed with disposable manner. These bioreactors consist of 27 a sterile plastic chamber that is partially filled with media (10% to 50%), inoculated with 28 cells and discarded after harvest. The single use chamber eliminating any need for 29 cleaning or sterilization is made of FDA approved biocompatible plastics such as 30 polyethylene, polystyrene and polypropylene. Generally, the disposable bioreactors are 31 low cost, simple to operate and undertaking high process security. It is suggested that 32 their use could improve process efficiency and results by reducing the time to time 33 market of new products. The objective of this review is to critically outline the potential of 34 the bioreactors for secondary metabolite production from suspension cultures, hairy 35

roots and embryogenic cultures [4]. With respect to the types of bioreactors reported in 36 the literature, their classification, application and characterization, here we describe the 37 features of bioreactors as well as summaries the results of hydrodynamic studies 38 (characterization of fluid flow, estimation of mixing time, distribution time, energy input) 39 and investigations of oxygen transport efficiency. [5] 40

41 2. PLANT CELL CULTURE 42

There are different plant cell culture techniques, used in industries and research purpose. 43 Majorly, there are different culture types of plant cell culture. They are, Callus, plant cell 44 suspension, Hairy root cultures, Embryogenic and shoot cultures [6]. 45

46 2.1 Callus Culture 47 Callus is defined as a developing mass of unorganized parenchyma cells, the formation 48 of callus normally is achieved by placing the explant on an appropriate solid growth medium 49 with the necessary components such as plant growth regulators in a Petri dish and 50 incubated. Callus cultures have a developmental stage of cells described as dedifferentiated 51 cells. 52 2.2 Hairy Roots 53 54 Hairy roots are generated by the transformation of plants or explants with agro pine and 55 Mann opine type strains of Agrobacterium rhizogenic [7]. 56 57 2.3 Embryogenic and Shoot Culture 58 59 It is used for micro propagation and plant breeding and like hairy roots. They belong to the 60 group of differentiated organ culture. Meristem, seed germination or embryonic culture is 61 used to stable this kind of cultures. 62 63 2.4 Plant Cell Suspension 64 65 Plant cell suspension are highly variable in terms of morphology, rheological characteristics 66 ,growth and metabolic pattern .Rheological characters are important in the process of scale 67 up ,Plant cell suspensions are non-Newtonian fluids ,so the density of this cultures rises 68 proportional to cell density. As a result, plant suspension cells very rarely grow as single 69 cells, they form aggregates based on cell adhesion and results from the segregation of wall 70 extra cellular polysaccharides, this prevents cell separation [8]. 71 72 3. CULTURE CONDITIONS 73 74 Mostly of the plant cell cultures requires similar culture conditions as temperature, between 75 25 and 27°C, a medium pH of 5.0 and 6.0. Also, culture cells need some aeration; this 76 aeration is extremely lower than microbial system. Some cultures have periodic dark/light 77 cycle of 8 hr and 16 hr or continuous introduction of light (0.6 -10 Klux), mammalian cells 78 have to be cultivated in dark. Long lag phases (nearly 120 hours) can be eliminated by plant 79 cell cultures can be initiated with high cell concentrations (10% of culture volume) [9]. 80 81 3.1 Media 82 83 Culturing plant cells are based on nutrient supply. Doubled distilled and deionized water 84 which represents 95% of the media are the main substances used for culture. All culture 85 media have a basal medium with carbon source. Carbon sources include fructose, sucrose, 86 glucose and sorbitol, with organic and inorganic supplements. Supplements such as amino 87 acids, vitamins and cofactors as tocopherol are used for BY-2 sigmoid growth source. The 88

inorganic supplements are macro and microelements, the microelements are in µM 89 concentrations and microelements in mm 90 91 In addition to this nutrient supply needs some phytohormones as growth regulators like 92 auxins, cytokines and gibberellins. Growth regulators affect the growth process, cytokinin 93 which promotes cell division and auxins, like indole-3-acetic acid (IAA), or 2, 4-94 dichlorophenoxyacetic acid (2, 4-D) is used as a dedifferentiating hormone for rapid callus 95 induction. Under low auxin concentration and a high cytokinin concentration the cell growth 96 are stimulated and switched concentrations cell division are promoted [10]. 97 98 4. PROCESS DESIGN CONSIDERATIONS 99 100 Plant biotechnology requires a interdisciplinary research in plant physiology, cell and 101 molecular biology, pharmacology, toxicology, chemistry, and chemical engineering to 102 assess: 103 (1) Tissue morphology and configuration. 104 (2) Flow and mass transfer environments in the bioreactor. 105 (3) Kinetics of cell growth and product formation. 106 (4) Genetic stability of high-producing cell lines. 107 (5) Control of bioreactor micro and macro environs. 108 (6) Implications of bioreactor design on downstream processes. 109 (7) The potential for process scale up. 110 The goals of plant tissue process development is to achieve high productivity, high product 111 yield and high product concentration by bioreactor operating conditions. Insitu removal of 112 product by extracts of plant cell culture, the use of cell polymer constructs, application of 113 different precursors, elicitors and medium exchange strategies can improve the plant 114 metabolite production [11]. 115 116 5. BIOREACTORS 117 118 A bioreactor is defined as a closed system (vessel/bag or apparatus) in which a biochemical 119 reaction takes place with help of biocatalyst. The biocatalysts is converted into an expressed 120 protein which is biomass or expressed proteins in the process. The term “fermenter” is used 121 only for bioreactors which involves fast growing microorganisms, but in American English 122 this term is used in both bioreactors. The primary role of a bioreactor is to provide 123 containment with optimal conditions for cell growth and formation of products. 124 125 5.1 TYPES OF BIOREACTORS 126

Suitable bioreactors for plant suspension culture are Stirred bioreactor, Bubble column 127 reactor, Airlift reactor, Bio Wave reactor, Wave & Undertow Bioreactor Slug Bubble 128 Bioreactor, Spray bioreactor, Mist bioreactor. The serious shear-related effects for plant cells 129 generally arise from the aeration and mixing system, the aeration rate or impeller tip speed 130 used. Through sparger rings, air (0.1 and 0.5 v m) is introduced directly, which is located in 131 the lower part of the bioreactor for plant cell cultures. In this way, air bubbles are generated 132 which can damage very sensitive plant suspension cell [12] . It causes bubble bursting. For 133 aerated airlift bioreactors and bubble columns it results in extensive foaming resulting from 134 extracellular polysaccharides, fatty acids and high sugar concentrations in the medium. This 135 can cause the dreaded wall growth phenomenon and blockage of the air exhaust filter, which 136 increases risk of contamination. Ceramic or sintering steel porous spargers, bubble free 137 aeration via tubes of silicone, external aeration via special devices, oxygen supplementation, 138 and changed geometry of the reactor and antifoam agents are suggested to overcome these 139 difficulties [13]. In the case of aeration in high biomass concentrations more than 30 g dry 140 weight, a further difficulty was reported for airlift bioreactors and bubble columns is poor 141

transfer of oxygen and heterogeneous biomass circulation. Therefore stirred bioreactors are 142 preferable for cell cultivation, with plants of high densities [14]. An impeller system and its 143 pumping mode for stirred bioreactors is chose on the basis of the aeration system type and 144 arrangement. The location of the sparger in the flow direction of the impeller guarantees 145 mass and temperature homogeneity as well as optimal gas dispersion. Hence, impellers 146 considered have included large slow-moving axial flow impellers with low tip speeds , such 147 as marine impellers or special pitched blade impellers till now, as well as impellers 148 positioned near the vessel wall, for example spiral stirrers, helical-ribbon impellers and 149 anchor impellers. Radial flow Rushton impellers with concave blades are occasionally used. 150 In addition, alternative impeller systems that includes cell-lift impeller, centrifugal-pump 151 impeller have been mounted in stirred vessels [15]. 152 153 The advantage of low-cost and disposable bioreactors having a gas-permeable cultivation 154 bag of plastic film was effectively proved in a number of plant cell cultivations. Whereas 155 Osmotek’s Life-Reactor and Curtis’ Plastic-lined Bioreactor represent pneumatically driven 156 bubble columns, the BioWave investigated by our team was the first mechanically driven bag 157 bioreactor [16]. Nowadays, further examples of such bag bioreactors is characterized by 158 wave-induced motion (where the mass and energy transfer is manually adjusted via rocking 159 angle, rocking rate, and filling level and culture bag geometry) are available, including, the 160 AppliFlex, Tsunami-Bioreactor, Optima and OrbiCell. A wave bag containing culture medium 161 keep in rocking their platform induces a wave in the bag. While mixing and oxygenation 162 occur, the surface of the medium is continuously renewed and bubble-free surface aeration 163 takes place. The BioWave has a leading position as a result of its scale-up capability to 300 l 164 culture volume and the availability of scale-up criteria based on its hydrodynamic 165 characterization [17]. Moreover, our studies reveal the potential of the BioWave for growing 166 tobacco, grape, apple and yew suspension cells up to 10 l culture volume. We achieved 167 maximum biomass productivities of 40 g fresh weight l-1 d-1 with excellent doubling times of 168 2 days. Furthermore, savings in time (no preparation, cleaning and sterilization are required), 169 reduced shearing (indicated by higher viabilities, biomass productivities and no significant 170 change in cell morphology), reduced foaming and a lower risk of contamination were found 171 in the Bio-Wave when compared to cultivations in dominating stirred bioreactor [18]. The 172 recent disposable bioreactor developments, namely the Wave & Undertow Bioreactor and 173 the Slug Bubble Bioreactor, were successfully used to grow tobacco and soya suspension 174 cells expression. There will be shear stress in negligible level [19]. 175 176 5.1.1 Disposable bioreactors for In-vitro culture 177 178 For cell culture, the Wave Bioreactor instrument is an effective, cost-efficient device. Culture 179 medium and cells made contact with each other by making contact with a presterile, 180 disposable chamber that is situated on a special rocking platform. Waves in the culture fluid 181 are created by rocking motion of the platform and offers continual mixing and oxygen 182 transfer, resulting in a healthy environment for cell growth. The Bioreactor requires no 183 sterilization, providing comfort of operation and protection against contamination. 184 185

186 Fig. 1. Wave bioreactor 187

5.2. Continuous stirred tank bioreactor 188

A cylindrical vessel with motor driven central shaft that supports one or more agitators were 189 used in continuous stirred tank bioreactors. Usually, agitators also called as impellers. The 190 bottom of the bioreactor contains shaft (Fig. 2. A). According to, height to diameter ratio, 191 number of impellers vary. That height to diameter ratio is referred to as aspect ratio. The 192 aspect ratio of a stirred tank bioreactor is generally 3-5. The aspect ratio is less than 2, for 193 animal cell culture. Usually 1/3 rd. of the vessel diameter, gives the diameter of the 194 impellers. The distance between two impellers is approximately 1.2 impeller diameter. 195 Different types of impellers like Rustom disc, concave bladed, marine propeller etc., are in 196 use [20]. 197

In short stirred tank reactors (STRs), the air is added to the culture medium under pressure. 198 A device called sparger is used for aeration. Sparger may contains a ring with many holes or 199 a tube with a single orifice. For better gas division system throughout the vessel, spargers 200 along with impellers are used in this reactors. 201

Impellers broken down the bubbles generated and discrete throughout the medium. This 202 enables the formation of optimal environment throughout the bioreactor [21]. 203

204

(D) 205

Fig. 2. A) Continuous stirred bioreactor, B) Bubble column bioreactor, C) Internal loop 206 airlift bioreactor, D) External loop airlift bioreactor. 207

208 5.3 Bubble Column Bioreactors 209 210 The air or gas is introduced at the base of the column in bubble column reactor, all the way 211 through perforated pipes or plates, or metal micro porous spargers (Fig 2 B). The gas flow 212 rate influences the performance factors like O2 transfer and proper mixing. For better 213 performance the bubble column bioreactors may be fitted with perforated plates. In bubble 214 column reactors the vessel is cylindrical. The aspect ratio of this reactor is 4-6 with height to 215 diameter ratio. 216 217 5.4 Airlift Bioreactors 218 219 The medium of the vessel is divided into two interconnected zones by means of a baffle or 220 draft tube in air lift reactors. Among the two zones, one is stated as riser in which the air/gas 221 is pumped. The other zone is the down comer, which receives no gas. The dispersion flows 222 up the riser zone. The down flow occurs in the down comer. There are two types of airlift 223 bioreactors. They are : internal loop airlift reactor and external loop air lift reactor. 224

Internal-loop airlift bioreactor has a single container. Central draft tube that creates interior 225 liquid circulation channels are equipped in that single container. These bioreactors are 226 simple in design, with volume and circulation at a fixed rate for fermentation. 227

External loop airlift bioreactor (Fig. 2.D) possesses an external loop. This external loop 228 facilitates the liquid that circulates through separate independent channels. For different 229 fermentation process these reactors are suitably modified. In general, the airlift bioreactors 230 are more efficient than bubble columns. They are used particularly for denser suspensions of 231

(c)

microorganisms. Because in these bioreactors, the mixing of the contents is better compared 232 to bubble columns. 233

For aerobic bioprocessing technology, air lift bioreactors are used. By pumping, they ensure 234 a controlled liquid flow in a recycle system. Airlift bioreactors are sometimes preferred for 235 methanol production [22], waste water treatment [23], single-cell protein production [24] due 236 to its high efficiency. Airlift bioreactor’s performance is dependent on the pumping of air and 237 the liquid circulation. [25] 238

5.4.1Two-stage airlift bioreactors: 239 For the temperature dependent formation of products, two stage air lift bioreactors are used 240 [26]. Growing cells from one bioreactor which is maintained at 30°Care pumped into another 241 bioreactor [27] which is of temperature 42°C. It is very difficult to raise the temperature 242 quickly from 30°C to 42°C in the same vessel, therefore we use this reactor. Each 243 bioreactors is fitted with valves. The transfer tube and pump are used to connect the 244 bioreactors [28] (Fig. 3). Growing of cells takes place in the first bioreactor and the second 245 reactor is for bioprocessing. [29] 246

247

248 Fig. 3 Two stage airlift bioreactor. 249

250 5.5 Tower bioreactors 251 252 A tower bioreactor constitutes pressure cycle fermenter with large dimensions. At the bottom 253 of the reactor high hydrostatic stress is created that increases the solubility of O2 in the 254 medium [30]. At the top of the riser, reduces pressure and facilitates expulsion of CO2. The 255 down comer facilitates the back flow of the medium and completes the cycle. The tower 256 bioreactor has high aeration capacities without having moving parts. [31] 257

258 Fig 4 Tower bioreactor 259 260 5.6 Fluidized Bed Bioreactors 261 262 Fluidized bed bioreactor is similar to that of the bubble column bioreactor. But the top 263 position is expanded to reduce the velocity of the fluid in fluidized reactors. The design of the 264 fluidized bioreactors is mainly to facilitate the solids that are retained in the reactor. When 265 solids are retained, the liquid flows out (Fig.4). Fluid suspended biocatalysts such as 266 immobilized enzymes, immobilized cells, and microbial flocs reactions are carried out in 267 these reactors [32]. 268

269 Fig 5 Fluidized bed bioreactor. 270

271 Gas is spared to create a suitable gas-liquid-solid fluid bed, for efficient function of this 272 reactor. The suspended solid particles are not too light or too dense [33], and they are in a 273 fair suspended state. To maintain continuous contact between the reaction contents [35] and 274 biocatalysts, recycling is needed periodically. It ensures better efficiency of bioprocessing. 275 Fluidized bed reactors also facilitates the efficient transfer of heat to biomass particles. 276

Circulating fluidized beds are employed for biomass fast pyrolysis [36]. However, the scale-277 up process can be limited by overall heat transfer in this reactor. By adjusting the fluidization 278 gas flow rates, vapor residence time can be controlled; But in bubbling fluidized beds, the 279 undesired biomass entry of [37] particles can occur at high gas velocities. Small, inert sand 280 particles are provided in reactor bed to increase the heat transfer rate to the biomass 281 particles. The fluidized bed also contains catalytically active solid particles, which is 282 assimilated in the reactor. [38] 283 284 5.7 Packed Bed Bioreactors 285 286 In a packed bed reactors, a bed of solid particles, with biocatalysts on or within the matrix of 287 solids, packed in a column is provided (Fig 6). The solids used may be porous or non-porous 288 gels, compressible or rigid in nature. Over the immobilized biocatalyst, medium is 289 continuously flowed inside the reactors. In packed bed bioreactor, products are released into 290 the fluid and removed. The flow of the fluid can be upward or downward .Mostly down flow 291 under gravity is preferred [39] 292

The he flow rate of the nutrient broth with increase in concentration of the nutrients. It is 293 rather difficult to control the pH of packed bed bioreactors by the addition of acid or alkali, 294 because of poor mixing. Hence product-inhibited reactions use these reactors. These 295 bioreactors do not allow accumulation of the products to any significant level. [40] 296

297

Fig 6 Packed bed bioreactor. 298 5.8 Photo-Bioreactors 299 Photo bioreactors are closed systems. Photo bioreactor working either outdoors or indoors. 300 In photo bioreactors, single species is inoculated to keep a clean-culture operation. Closed 301 cultivation systems prevents contamination. It leads to higher growth and quality of the 302 product harvested. [41] Manufacturing costs increases for photo bioreactor’s products. Some 303 fermentation process can be carried out either by exposing to sunlight or artificial 304 illumination. Since artificial illumination is expensive, therefore outdoor photo-bioreactors are 305 preferred. Important compounds are produced by using photo-bioreactors are: p-carotene, 306 asthaxanthin. The different types of photo-bioreactors are depicted in Fig.7. Glass or more 307 commonly transparent plastic are used for making this reactors. The arrangement of tubes 308 or flat panels establish light receiving systems, which is commonly known as solar receivers. 309 Through the solar receivers culture can be circulated by methods such as using centrifugal 310 pumps or airlift pumps. It is necessary to keep cells in continuous circulation without forming 311

any sediments. Adequate penetration of sunlight should be maintained continuously. To 312 prevent rise in temperature, the tubes should be cooled periodically. Photo-bioreactors are 313 usually operated in a continuous mode and temperature should be maintained in the range 314 of 25-40°C. Microalgae and cyanobacteria are normally used to cultivate in photo bioreactor. 315 The products are produced in night time and the cell grows in daylight [42] 316

317

318 Fig 7 Photo bioreactor 319

320 5.9 Mist reactor 321 322 The cultivation of hairy roots with high potential is done by the gas phase reactors called 323 mist reactors. The roots have an optimal growth, if critical droplet size is between 1µm and 324 35 µm Liquid nutrients are homogeneously distributed in this case and the gas transfer into 325 the rots is does not experience oxygen stress. A glass vessel with stainless steel upper and 326 lower lids is equipped in mist reactors. The vessel stands vertically on four legs. In the 327 bioreactor’s lower lid thermos regulation system is located. Sensors for temperature and 328 levelling are located on the bioreactor’s upper lid. [43] It is supplied with a LED jacket, a 329 hydraulic spray nozzle, three sample holder plates and a ring sparger. Gas inlet/outlet ports 330 are located on the upper lid of the bioreactor. The exhaust gas condenser is fixed on the 331 upper part of the bioreactor. It prevent unwanted liquid particle flowing out from the vessels 332 environment. For easier autoclaving the design of the condenser provides an option for the 333 user to adjust its position. The condenser is provided with an adjustable gas valve for 334 pressure regulation [44]. 335 336 5.10 Low Cost Mist Bioreactor (LCMB) 337 338 The largest gas-phase bioreactor with a capacity of 60 L. Low cost mist bioreactors were 339 designed to grow Artemisia annul transformed roots and Dianthus caryophyllus shoots. The 340 reactors use similar mist generators but the culture chambers were modified to meet the 341 requirements of each application [45]. 342

343 Fig 8 Mist reactor 344 5.11 Wilson bioreactor 345 346 The largest hybrid bioreactor system with a capacity of 500 L. The spray reactor reminds a 347 gas-phase bioreactor with a cultivation container with horizontal meshes. This bioreactor has 348 some problems such as very laborious handling and often associated with contamination 349 issues, now is no longer in use [46]. 350 351 5.12 Balloon-type 352 353 It is a spherical-glass bioreactor, which is used for root culture due their advantages of easy 354 moisture and simplicity, although these assets are no-longer in use due to easy 355 contamination and difficulty of take out the roots useful for extracellular products [47]. 356 357 Table 1. Types of bioreactors 358

S.no

Types Driven Aerati

on Product

Starter Culture

Advantages Disadvantages Uses

1

Stirred bioreactor

Mechanic

Bubbles/

airlift - -

Easy scale up Simple

Well known

Shear stress Mechanic stress

Foam Suspensions

2 Bubble Column

Airlift

Pneumatically

Bubbles/

airlift - -

High aeration Low cost

High efficient mass transfer

Viscosity Foam

Shear stress

Immobilized Suspensions.

3

Hollow fiber

pneumatically

Perfusion

pumps - -

Easy downstream Fast, small

Scale, flexible

Contamination. Mass transfer

limitation. Low culture of

volume.

Adherent cells.

Proteins.

359 CONCLUSION 360 361 A variety of bioreactor types providing growth and expression of bioactive substances are 362 developed for plant suspension culture. Plant cell suspension culture as a bio production 363 platform provides a number of unique advantages for recombinant protein production [49]. 364 These bioreactors types used for synthesis of complex chemicals whereas low biomass and 365 product level from plants can be achieved. 366 In the early past, industries use stirred bioreactors, hollow fibers and bubble columns for 367 their production as it’s possible to see in Phyton Biotech [50], which uses bioreactors of 368 stainless steel. But now, single-use bioreactors are gone to set aside this last device, 369 Phytocelltec [51] uses Malus Domestica for obtaining a component for a liposomal active 370 ingredient based on stem cells from the Tutwiler Spat Lauper apple, this cosmetic industry 371 uses bio wave bioreactors in sequence instead of huge stainless-steel bioreactors [52]. 372 Single-use bioreactors are a reality in R+D laboratories due to their high amount of data in 373 less time, low cost and safety but in industry stainless steel bioreactors are yet implemented; 374 however low-cost safety, short time of production (it’s not necessary to get clean and 375 disinfected), high flexibility and simplicity are going to change this trend. Products as their 376 culture types are different, consequently the disposable bioreactors are lots, it seems that 377 stirred bioreactors with different impellers are the most used kind of device, it has plenty of 378 advantages and low cons, so the combination of stirred devices with bubble columns or 379 Airlifts, suggests a great solution [54]. Orbital bioreactors have more advantages than stirred 380 but they only can be used for low-oxygen cell lines. Hydraulic and hollow fibers are only 381 focused in Immobilized and attached cells. Root culture, due their difficulty and own 382 characteristics has specific bioreactors, now the most used is the Mist bioreactor [55], 383 Wilson bioreactor have a great capacity and product titer but it has plenty of contamination. 384 In the other hand, single use bioreactors, stirred ones have importance, but their lack in no 385 foam formation and shear stress improves the acceptance of wave-mixing bioreactors, the 386 only problem of this kind of device is the rheological issues of plant cultures, but this could 387 be avoided by rise the rocking angle and velocity. 388 389 REFERENCES 390 391 1. Sajc L, Grubisic D, and Novakovic GV. Bioreactors for plant engineering: an outlook for 392 further research. Biochem. Eng. 2000, 4, 89999. 393

4

Orbitally Mechanic

Bubbles

Airlift absen

ce

18mg/l

Shaken:

20mg/l

5L 50Ml

Simple. Well cost efficiency.

Easy to scale up

Only suitable for certain cell lines.

low oxygen cell

Lines.

5 Life

reactor Pneumatic

ally Bubbl

es - -

Easy scale up Simple.

Well known.

Low culture Volume

embriocrops

6

Wave Mechanic bubble

s

Non detect

ed

5L for 15%

inoculum

Bubble free surface

aeration. Well

investigated Uniformity of

energy negligible foaming.

Rheological Issues of plant

culture

Suspension and

Immobilized cells

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