Patricia Kieran

Plant cell suspension cultures: some engineering considerations

Higher plants are the source of a vast array of biochemicals which are used as drugs, pesticides, flavourings and fragrances. For some of these compounds, plant cell culture can provide a potential production alternative to traditional cultivation methods or chemical synthesis routes. Many systems have been patented and the last 20 years have seen considerable industrial and academic interest in the development of large scale cultures to produce pharmaceutically active, high value substances. However, the industrial application of plant cell suspension cultures has, to date, been limited. Commercialisation has essentially been impeded by economic feasibility, arising from both biological and engineering considerations. This paper reviews the commercial development of the technology to date and focuses on the impact of specific engineering-related factors, in particular, the shear sensitivity of plant cell suspension cultures. Evidence of sensitivity to hydrodynamic shear in bioreactors has generally been attributed to the physical characteristics of the suspended cells. Recent studies indicate that shear sensitivity may not be as important, in some cases, as initially anticipated.

Effects of Hydrodynamic and Interfacial Forces on Plant Cell Suspension Systems

Plant cells are perceived to be sensitive to the hydrodynamic environment in conventional bioreactors. Heightened sensitivity, relative to most bacterial cultures, is frequently attributed to larger plant cell sizes, extensive vacuolization and aggregation patterns. Early studies of shear sensitivity focused on cell lysis and/or loss of viability. More recently, an extensive array of sub-lethal responses has been identified. A fuller understanding of these sub-lytic effects may assist in the optimization of large-scale cultivation conditions. This paper reviews recent work on the hydrodynamic shear sensitivity of plant cell systems, under cultivation conditions and in purpose-built shearing devices. The relevance of different approaches to the characterization of the intensity of a given hydrodynamic environment is discussed. Indicators of cell response to hydrodynamic stress are evaluated. The potential significance of cellular defense mechanisms, observed in response to mechanical stimulants, is identified.

The effects of turbulent jet flows on plant cell suspension cultures

Cell suspensions of Morinda citrifolia were subjected to turbulent flow conditions in a submerged jet apparatus, to investigate their hydrodynamic shear susceptibility. The suspensions were exposed to repeated, pressure-driven passages through a submerged jet. Two nozzles, of 1 mm and 2 mm diameter, were employed. Average energy dissipation rates were in the range 10(3)-10(5) W/kg and cumulative energy dissipation in the range 10(5)-10(7) J/m3. System response to the imposed conditions was evaluated in terms of suspension viability (determined using a dye exclusion technique) and variations in both chain length distribution and maximum chain length. Viability loss was well-described by a first-order model, and a linear relationship was identified between the specific death rate constant and the average energy dissipation rate. This relationship was consistent with results obtained using the same suspension cultures in a turbulent capillary flow device. Morphological measurements indicated that exposure to the hydrodynamic environment generated in the jet resulted in a significant reduction in both the average and maximum chain lengths, and the reduction in the maximum chain length was identified as an appropriate measure of sustained damage. Analysis of both viability and chain length in terms of cumulative energy dissipated revealed good agreement with results reported by other authors for morphologically different plant cell systems. Copyright 1998 John Wiley & Sons, Inc.

Comparison of Morphological Characteristics of Streptomyces natalensis by Image Analysis and Focused Beam Reflectance Measurement

A morphological interpretation is presented for data collected during growth of a filamentous organism, using a focused beam reflectance measurement (FBRM) system. The morphology of the organism was also obtained using conventional semiautomatic image analysis to support the interpretation of the FBRM data. The model organism employed is the filamentous soil‐borne actinomycete Streptomyces natalensis, which produces the antifungal agent pimaricin. The organism was cultivated both in shake flasks and in a bench‐scale stirred tank bioreactor. It was found that FBRM could be used to track changes in the morphology of the organism throughout the course of its growth on both scales. These changes were highlighted using both the median chord length and length‐weighted mean chord length obtained from the chord length distribution measured with the FBRM probe. The ability of the FBRM probe to respond to changes in both the size and morphology of mycelial aggregates was supported by standard image analysis parameters, including equivalent diameter, convex area, and compactness.

Generation of reactive oxygen and antioxidant species by hydrodynamically stressed suspensions of Morinda citrifolia.

The generation of reactive oxygen species (ROS) by plant cell suspension cultures, in response to the imposition of both biotic and abiotic stress, is well‐documented. This study investigated the generation of hydrogen peroxide by hydrodynamically stressed cultures of Morinda citrifolia, over a 5‐h period post‐stress imposition. Suspensions were exposed to repeated passages through a syringe, under laminar flow conditions, corresponding to cumulative energy dissipation levels of approximately 3–6 J kg‐1. Extracellular hydrogen peroxide was detected using a luminol‐based chemiluminescence assay. The addition of exogenous hydrogen peroxide facilitated the detection of low levels of hydrogen peroxide in the presence of antioxidants. Immediately after shear exposure, there was evidence of significant antioxidative capacity in the sheared cell cultures, which potentially masked any oxidative burst (OB), but which decreased over the following 40 min. This antioxidative capacity was determined to derive from the shearing process. Trials in which ground cellular debris was added to control suspensions suggested that some of the antioxidative capacity observed in stressed suspensions was directly associated with debris generated by the shearing process. Using UV‐vis spectrophotometry and HPLC, stress‐related increases in the levels of phenolic compounds were detected in suspension filtrates. Under the stress conditions investigated, maximum hydrogen peroxide levels of 11.5 μM were observed, 5 h after shear exposure. This study emphasizes the importance of considering both oxidative and antioxidative capacities as part of a holistic approach to the determination of the OB in hydrodynamically stressed plant cell suspension cultures.

Teaching and learning across disciplines : student and staff experiences in a newly modularised system

Within modular degrees it is sometimes possible for students to broaden their education by taking modules from outside their main programme of study. This is one significant aspect of modular degrees which has not been studied. In an effort to better understand this issue, the research reported in this paper explored the experiences: (1) of students taking modules from outside their programme of study and; (2) of staff teaching modules with significant numbers of students from other programmes. In total, 820 undergraduate students responded to an on-line survey; 12 academic staff members participated in interviews. The survey focused on students’ reasons for choosing the module, their experiences of assessment and their perceptions of workload. Interviews with academic staff focused on the influence of non-programme students on teaching and assessment practices. The discussion addresses the implications of student choice and classroom diversity for teaching and assessment in modular systems.

Use of Fed-Batch Cultivation for Achieving High Cell Densities for the Pilot-Scale Production of a Recombinant Protein (Phenylalanine Dehydrogenase) in Escherichia coli

A fed-batch process for the high cell density cultivation of E. coli TG1 and the production of the recombinant protein phenylalanine dehydrogenase (PheDH) was developed. A model based on Monod kinetics with overflow metabolism and incorporating acetate utilization kinetics was used to generate simulations that describe cell growth, acetate production and reconsumption, and glucose consumption during fed-batch cultivation. Using these simulations a predetermined feeding profile was elaborated that would maintain carbon-limited growth at a growth rate below the critical growth rate for acetate formation (mu < mu(crit)). Two starvation periods are incorporated into the feed profile in order to induce acetate utilization. Cell concentrations of 53 g dry cell weight (DCW)/L were obtained with a final intracellular product concentration of recombinant protein corresponding to approximately 38% of the total cell protein. The yield of PheDH was 129 U/mL with a specific activity of 1.2 U/mg DCW and a maximum product formation rate of 0.41 U/mg DCW x h. The concentration of aectate was maintained below growth inhibitory levels until 3 h before the end of the fermentation when the concentration reached a maximum of 10.7 g/L due to IPTG induction of the recombinant protein.

Growth Behavior in Plant Cell Cultures Based on Emissions Detected by a Multisensor Array

The use of a multisensor array based on chemical gas sensors to monitor plant cell cultures is described. The multisensor array, also referred to as an electronic nose, consisted of 19 different metal oxide semiconductor sensors and one carbon dioxide sensor. The device was used to continuously monitor the off‐gas from two plant cell suspension cultures, Morinda citrifolia and Nicotiana tabacum, cultivated under batch conditions. By analyzing the multiarray responses using two pattern recognition methods, principal component analysis and artificial neural networks, it was possible to monitor the course of the cultivations and, in turn, to predict (1) the biomass concentration in both systems and (2) the formation of the secondary metabolite, antraquinone, by M. citrifolia. The results identify the multisensor array method as a potentially useful analytical tool for monitoring plant process variables that are otherwise difficult to analyze on‐line.

Optics education within engineering at University College Dublin

Undergraduate students within many of the departments which make up the Faculty of Engineering and Architecture at University College Dublin meet optics, optical inspection techniques and opto-electronics at various times during their standard four year undergraduate degree course. As well as optics fundamentals taught as part of their physics courses, engineering courses specifically concerning optics and opto-electronics are available. Furthermore many graduate students are involved in either projects, which are completely optics based, or carry out research projects involving the use or development of specific optical instruments. These projects include the development of image processing capture and processing software, high power laser machining of materials, design and testing of lasers for fiber optic telecommunication and sensing applications and photochemistry. In this paper we offer a brief review of some of the main optical educational themes covered within our faculty and present some details regarding a few optics based postgraduate research projects.