Wei Wen Su

Bioprocessing technology for plant cell suspension cultures

Considering various forms of in vitro plant tissue cultures, cell suspension culture is most amenable to large-scale production of natural compounds, owing primarily to its superior culture homogeneity. This fact has already been demonstrated in several largescale applications, including the commercial shikonin process. The scope of this work is to review the state of the art in bioprocessing technologies pertinent to the secondary metabolite production from suspension cultures of callus cells. In the first part of the review, plant cell physiology relevant to bioprocess design is considered. This is followed by an in-depth discussion on the bioreactor design and operation and its effect on plant cell suspension cultures. Finally, recent commercial exploitation and development are summarized. Following the review, related patents and literature are listed.

Online estimation of stirred-tank microalgal photobioreactor cultures based on dissolved oxygen measurement

Abstract Photobioreactor sensing presents unique challenges not met in conventional fermentors. First and foremost, photobioreactor processes are governed by photosynthesis, and hence many parameters important to photobioreactors are not considered in conventional fermentors. Furthermore, photobioreactor processes are typically associated with stringent cost constraints, and thus the use of complex sensing hardware is precluded. This calls for innovative approaches to address sensing problems in photobioreactors. Here, we report the development of an effective model-based estimator that is capable of tracking key culture states in stirred-tank microalgal photobioreactor systems. A marine micro-alga Dunaliella salina was used as a model organism in this study. Extended Kalman filter (EKF) was applied here to provide optimal estimates of photobioreactor states, based on a dynamic process model in conjunction with online dissolved oxygen measurement. The process model consists of a growth model and a light transport model. The former associates growth to average light intensity in the reactor, while taking into account both photoinhibition and oxygen inhibition. The latter is based on a modified radial model to estimate the average light intensity. The estimator is capable of estimating biomass density, specific growth rate, dissolved oxygen concentration, photosynthetic efficiency, and average light intensity in the photobioreactor illuminated either with constant incident light at different intensity levels or with time-varying incident lights. For the latter, an auxiliary internal model EKF was used to accurately track the variation rate of the incident lights. This paper also presents a detailed analysis on the tuning of EKF for optimal estimation. This state estimation system offers a cost-effective means for monitoring the process dynamics of microalgal photobioreactor cultures online, through which the productivity of such a process could be optimized.

Green fluorescent protein as a secretory reporter and a tool for process optimization in transgenic plant cell cultures

Green fluorescent protein (GFP) is an attractive reporter for bioprocess monitoring. Although expression of GFP in plants has been widely reported, research on the use of GFP in plant cell cultures for bioprocess applications has been limited. In this study, the suitability of GFP as a secretory reporter and a useful tool in plant cell bioprocess optimization was demonstrated. GFP was produced and secreted from suspension cells derived from tobacco that was transformed with a binary vector containing mgfp5-ER cDNA, a modified GFP for efficient sorting to the endoplasmic reticulum, under control of the CaMV 35S promoter. For cell line gfp-13, extracellular and intracellular GFP accumulated to 15.4 and 29.4 mg x 1(-1), respectively. Extracellular GFP accounted for 30.9% of the total extracellular protein. The molecular mass of extracellular GFP was nearly identical to that of a recombinant GFP standard, indicating cleavage of the signal sequence. Neomycin phosphotransferase II, a cytosolic selection marker, was found almost exclusively in cellular extracts with less than 2% in the extracellular medium. These results suggest that extracellular GFP is most likely the result of secretion rather than nonspecific leakage from cells. Furthermore, medium fluorescence intensity correlated nicely with extracellular GFP concentration supporting the use of GFP as a quantitative secretory reporter. During the batch cultivation, culture GFP fluorescence also followed closely with cell growth. A medium feeding strategy was then developed based on culture GFP fluorescence that resulted in improved biomass as well as GFP production in a fed-batch culture.

Continuous plant cell perfusion culture: Bioreactor characterization and secreted enzyme production

Culture perfusion is widely practiced in mammalian cell processes to enhance secreted antibody production. Here, we report the development of an efficient continuous perfusion process for the cultivation of plant cell suspensions. The key to this process is a perfusion bioreactor that incorporates an annular settling zone into a stirred-tank bioreactor to achieve continuous cell/medium separation via gravitational sedimentation. From washout experiments, we found that under typical operating conditions (e.g., 200 rpm and 0.3 vvm) the liquid phase in the entire perfusion bioreactor was homogeneous despite the presence of the cylindrical baffle. Using secreted acid phosphatase (APase) produced in Anchusa officinalis cell culture as a model we have studied the perfusion cultures under complete or partial cell retention. The perfusion culture was operated under phosphate limitation to stimulate APase production. Successful operation of the perfusion process over four weeks has been achieved in this work. When A. officinalis cells were grown in the perfusion reactor and perfused at up to 0.4 vvd with complete cell retention, a cell dry weight exceeding 20 g/l could be achieved while secreted APase productivity leveled off at approximately 300 units/l/d. The culture became extremely dense with the maximum packed cell volume (PCV) surpassing 70%. In comparison, the maximum cell dry weight and overall secreted APase productivity in a typical batch culture were 10-12 g/l and 100-150 units/l/d, respectively. Operation of the perfusion culture under extremely high PCV for a prolonged period, however, led to declined oxygen uptake and reduced viability. Subsequently, cell removal via a bleed stream at up to 0.11 vvd was tested and shown to stabilize the culture at a PCV below 60%. With culture bleeding, both specific oxygen uptake rate and viability were shown to increase. This also led to a higher cell dry weight exceeding 25 g/l, and further improvement of secreted APase productivity that reached a plateau fluctuating around 490 units/l/d.

State and parameter estimation of microalgal photobioreactor cultures based on local irradiance measurement

Local photosynthetic photon flux fluence rate (PPFFR) determined by a submersible 4pi quantum micro-sensor was used in developing a versatile on-line state estimator for stirred-tank microalgal photobioreactor cultures. A marine micro-alga Dunaliella salina was used as a model organism in this study. On-line state estimation was realized using the extended Kalman filter (EKF), based on a state model of the photobioreactor and on-line local PPFFR measurement. The dynamic state model for the photobioreactor was derived based on mass-balance equations of the relevant states. The measurement equation was established based on an empirical correlation between the microalgal biomass concentration and the local PPFFR measured at a fixed point inside the photobioreactor. An internal model approach was used to estimate the specific growth rate without the need of state-based kinetic expression. The estimator was proven to be capable of estimating biomass concentration and specific growth rate, as well as phosphate and dissolved oxygen concentrations in a photobioreactor illuminated with either fixed or time-varying incident radiation. The quantum sensor was shown to be robust and able to quickly respond to dynamic changes in local PPFFR. In addition, the quantum sensor outputs were not affected by bubble aeration or agitation within the typical operating range. The strong filtering capacity of EKF gives the state estimator superior performance compared to direct calculation from the empirical biomass/local PPFFR correlation. This state estimation system makes use of inexpensive and reliable sensor hardware to report key process dynamics of microalgal photobioreactor cultures on-line, enabling improved operation of such a process.

Co-immobilized enzymes in magnetic chitosan beads for improved hydrolysis of macromolecular substrates under a time-varying magnetic field

Glucoamylase and alpha-amylase co-immobilized with gamma ferric oxide powders in chitosan beads for consecutive starch liquefaction and saccharification under different magnetic fields was investigated. The chitosan concentration in the beads was found to greatly affect the immobilized enzyme performance. Superior immobilization efficiency and enzyme stability were noted when 2% instead of 4% chitosan was utilized. Using confocal microscopy and scanning electron microscopy, the beads with 2% chitosan were seen to exhibit a more rugged surface topology with more macropores and accommodate more protein near the external surface than with the 4% chitosan beads. An optimum loading ratio between alpha-amylase and glucoamylase exists that gives the highest glucose production, and this ratio varies with the size of the beads. The inclusion of the gamma ferric oxide powders renders the beads magnetically anisotropic and causes them to tumble under a single-phase alternating magnetic field, resulted in increased overall reaction rates. When exposed to a three-phase alternating magnetic field, these beads were stirred vigorously, also leading to enhanced reaction rates. The use of multi-enzyme co-immobilization in magnetic anisotropic chitosan beads may be extended to other practical applications that involve coordinated enzymatic reactions of macromolecular substrates.

A perfusion air-lift bioreactor for high density plant cell cultivation and secreted protein production

A new bioreactor design that allows continuous perfusion cultivation of plant cell suspensions is described in this paper. This design incorporates an internal cell settling zone with an external-loop air-lift bioreactor. The settling zone is created by inserting a baffle plate into the upper portion of the downcomer. Using this bioreactor, Anchusa officinalis suspension culture was cultivated to a cell density of 27.2 g l−1 DW in 14 days at a perfusion rate of 0.123 per day. The maximum total extracellular protein concentration attained 1.11 g l−1. Complete cell retention was achieved throughout the culture during which the maximum packed cell volume (PCV) exceeded 80%. In comparison, the maximum cell density and extracellular protein concentration in the batch culture were 12.6 g l−1 DW and 0.47 g l−1, respectively. SDS-PAGE of the extracellular protein samples revealed two major bands at 58 and 47 kDa, each accounted for approximately 45% of the total secreted proteins.

High density cultivation of Anchusa officinalis in a stirred-tank bioreactor with in situ filtration

Previously, Su et al. [Biotechnol Bioeng 42: 884–890 (1993)] reported improved production of rosmarinic acid by Anchusa officinalis in shake-flask cultures using a cultivation strategy that involved intermittent medium exchange. Implementation of this cultivation strategy in 2.5-1 stirred-tank bioreactor cultures is investigated in the present study. Intermittent cell/medium separation in the bioreactor was accomplished by means of automated in situ culture filtration. In the bioreactor culture, rosmarinic acid production was found very sensitive to agitation and aeration conditions as well as dissolved oxygen concentration. A maximum cell density of 35 g dry weight/l and a rosmarinic acid concentration of 3.7 g/l were obtained by maintaining the dissolved oxygen concentration above 30% air saturation, gradually raising the impeller tip speed from 34 cm/s to 72 cm/s, and keeping the aeration rate at 0.44 vvm while increasing the O2: air ratio in the gas feed stream to 4:1. This result is comparable with the data obtained from shake-flask cultures using the same culture strategy.

Fluorescent proteins as tools to aid protein production.

Fluorescent proteins are genetically encoded, highly versatile reporters useful for monitoring various aspects of recombinant protein production. In addition to the widely popular green fluorescent protein (GFP) from Aequorea victoria, a variety of other fluorescent proteins have been discovered that display a wide range of spectral properties. Synthetic variants have also been developed to overcome limitations associated with their wild-type counterparts. Having a large repertoire of fluorescent proteins with diverse traits opens new opportunities for rapid monitoring and optimization of recombinant protein production.

Bioreactor Engineering For Recombinant Protein Production Using Plant Cell Suspension Culture

Plant cell culture has long been considered as a potential system for large-scale production of secondary metabolites. In recent years, with the advances in plant molecular biology, plant cell culture has also attracted considerable interests as an expression platform for large-scale production of high-value recombinant proteins. Many plant species can now be genetically transformed. Callus cells derived from the transgenic plants can be grown in simple, chemically defined liquid media to establish transgenic cell suspension cultures for recombinant protein production. For certain plant species, such as tobacco, it is also possible to establish transgenic suspension cell cultures by directly transforming wild-type cultured cells. There are several notable benefits of using plant suspension cultures for recombinant protein production. Plant cells, unlike prokaryotic hosts, are capable of performing complex post-translational processing, such as propeptide processing, signal peptide cleavage, protein folding, disulfide bond formation and glycosylation, which are required for active biological functions of the expressed heterologous proteins [1]. Plant cells are also easier and less expensive to cultivate in liquid media than their mammalian or insect cell counterparts. The potential human pathogen contamination problem associated with mammalian cell culture does not exist in plant cell culture since simple, chemically defined media are used [2]. When compared with transgenic plants, cultured plant cells also possess a number of advantages. Cultured plant cells have a much shorter growth cycle than that of transgenic plants grown in the field. Plant cell cultures are grown in a confined environment (i.e. enclosed bioreactor) and hence devoid the GMO release problem. Furthermore, cell suspension cultures consist of dedifferentiated callus cells lacking fully functional plasmodesmata and hence there is minimum cell-to-cell communication. This may reduce systemic post-transcriptional gene silencing (PTGS) which is believed to be transmitted via plasmodesmata and the vascular system [3,4]. On the down side, plant cells generally have a longer doubling time than bacterial or yeast cells. Genetic instability associated with de-differentiated callus cells due to somaclonal variation is another potential drawback in using cultured plant cells for recombinant protein production. Due in part to their more evolved and more tightly controlled gene/protein