Optical sensor enabled rocking T-flasks as novel upstream bioprocessing tools

During the past decade, novel disposable cell culture vessels (generally referred to as Process Scouting Devices or PSDs) have become increasingly popular for laboratory scale studies and seed culture generation. However, the lack of engineering characterization and online monitoring tools for PSDs makes it difficult to elucidate their oxygen transfer capabilities. In this study, a mass transfer characterization (k_La) of sensor enabled static and rocking T‐flasks is presented and compared with other non‐instrumented PSDs such as CultiFlask 50®, spinner flasks, and SuperSpinner D 1000®. We have also developed a mass transfer empirical correlation that accounts for the contribution of convection and diffusion to the volumetric mass transfer coefficient (k_La) in rocking T‐flasks. We also carried out a scale‐down study at matched k_La between a rocking T75‐flask and a 10 L (2 L filling volume) wave bioreactor (Cultibag®) and we observed similar DO and pH profiles as well as maximum cell density and protein titer. However, in this scale‐down study, we also observed a negative correlation between cell growth and protein productivity between the rocking T‐flask and the wave bioreactor. We hypothesize that this negative correlation can be due to hydrodynamic stress difference between the rocking T‐flask and the Cultibag. As both cell culture devices share key similarities such as type of agitation (i.e., rocking), oxygen transfer capabilities (i.e., k_La) and disposability, we argue that rocking T‐flasks can be readily integrated with wave bioreactors, making the transition from research‐scale to manufacturing‐scale a seamless process. Biotechnol. Bioeng. 2012;109: 2295–2305. © 2012 Wiley Periodicals, Inc.     

Use of computational fluid dynamics as a tool for establishing process design space for mixing in a bioreactor

The concept of "design space" plays an integral part in implementation of quality by design for pharmaceutical products. ICH Q8 defines design space as "the multidimensional combination and interaction of input variables (e.g., material attributes) and process parameters that have been demonstrated to provide assurance of quality. Working within the design space is not considered as a change. Movement out of the design space is considered to be a change and would normally initiate a regulatory post-approval change process. Design space is proposed by the applicant and is subject to regulatory assessment and approval." Computational fluid dynamics (CFD) is increasingly being used as a tool for modeling of hydrodynamics and mass transfer. In this study, a laboratory-scale aerated bioreactor is modeled using CFD. Eulerian-Eulerian multiphase model is used along with dispersed k-ε turbulent model. Population balance model is incorporated to account for bubble breakage and coalescence. Multiple reference frame model is used for the rotating region. We demonstrate the usefulness of CFD modeling for evaluating the effects of typical process parameters like impeller speed, gas flow rate, and liquid height on the mass transfer coefficient (k(L)a). Design of experiments is utilized to establish a design space for the above mentioned parameters for a given permissible range of k(L)a.     

A comparative study of solid and liquid non‐aqueous phases for the biodegradation of hexane in two‐phase partitioning bioreactors

A comparative study of the performance of solid and liquid non‐aqueous phases (NAPs) to enhance the mass transfer and biodegradation of hexane by Pseudomonas aeruginosa in two‐phase partitioning bioreactors (TPPBs) was undertaken. A preliminary NAP screening was thus carried out among the most common solid and liquid NAPs used in pollutant biodegradation. The polymer Kraton G1657 (solid) and the liquid silicone oils SO20 and SO200 were selected from this screening based on their biocompatibility, resistance to microbial attack, non‐volatility and high affinity for hexane (low partition coefficient: K = C_g/C_NAP, where C_g and C_NAP represent the pollutant concentration in the gas phase and NAP, respectively). Despite the three NAPs exhibited a similar affinity for hexane (K ≈ 0.0058), SO200 and SO20 showed a superior performance to Kraton G1657 in terms of hexane mass transfer and biodegradation enhancement. The enhanced performance of SO200 and SO20 could be explained by both the low interfacial area of this solid polymer (as a result of the large size of commercial beads) and by the interference of water on hexane transfer (observed in this work). When Kraton G1657 (20%) was tested in a TPPB inoculated with P. aeruginosa, steady state elimination capacities (ECs) of 5.6 ± 0.6 g m^?3 h^?1 were achieved. These values were similar to those obtained in the absence of a NAP but lower compared to the ECs recorded in the presence of 20% of SO200 (10.6 ± 0.9 g m^?3 h^?1). Finally, this study showed that the enhancement in the transfer of hexane supported by SO200 was attenuated by limitations in microbial activity, as shown by the fact that the ECs in biotic systems were far lower than the maximum hexane transfer capacity recorded under abiotic conditions. Biotechnol. Bioeng. 2010;106: 731–740. © 2010 Wiley Periodicals, Inc.     

Life cycle assessment as a tool for improving process performance: A case study on boron products

This paper explores the use of LCA as a tool for process environmental management, thereby moving the focus from product to process oriented analysis. The emphasis is on Improvement Assessment in which the “hot spots” in the system are targeted for maximum environmental improvements. In this context, it is useful to use multiobjective optimisation which renders Valuation unnecessary. The approach is illustrated by the case study of the system processing boron ores to make five different products. The results of Inventory Analysis and Impact Assessment are presented and discussed. In Improvement Assessment, a number of improvement options are identified and evaluated, using system optimisation. It is shown that the site environmental performance can be improved over current operation by an average of 20% over the whole life cycle. Thus the study demonstrates that the optimisation approach to environmental process management may assist in identifying optimal ways to operate a process or plant from “cradle to grave”. This may help the process industries not only to comply with legislation but also provide a framework for taking a more proactive approach to environmental management leading to more sustainable industrial operations and practices.     

RADseq as a valuable tool for plants with large genomes—A case study in cycads

Full genome sequencing of organisms with large and complex genomes is intractable and cost ineffective under most research budgets. Cycads (Cycadales) represent one of the oldest lineages of the extant seed plants and, partly due to their age, have incredibly large genomes up to ~60 Gbp. Restriction site‐associated DNA sequencing (RADseq) offers an approach to find genome‐wide informative markers and has proven to be effective with both model and nonmodel organisms. We tested the application of RADseq using ezRAD across all 10 genera of the Cycadales including an example data set of Cycas calcicola representing 72 samples from natural populations. Using previously available plastid and mitochondrial genomes as references, reads were mapped recovering plastid and mitochondrial genome regions and nuclear markers for all of the genera. De novo assembly generated up to 138,407 high‐depth clusters and up to 1,705 phylogenetically informative loci for the genera, and 4,421 loci for the example assembly of C. calcicola. The number of loci recovered by de novo assembly was lower than previous RADseq studies, yet still sufficient for downstream analysis. However, the number of markers could be increased by relaxing our assembly parameters, especially for the C. calcicola data set. Our results demonstrate the successful application of RADseq across the Cycadales to generate a large number of markers for all genomic compartments, despite the large number of plastids present in a typical plant cell. Our modified protocol was adapted to be applied to cycads and other organisms with large genomes to yield many informative genome‐wide markers.     

DNA barcoding as an effective tool to complement wetland management: A case study of a protected area in Italy

In recent years, DNA barcoding has been suggested as a useful molecular technique to complement traditional taxonomic expertise for fast species identification and biodiversity inventories. In this study, in situ application of DNA barcodes was tested on the plant community of a wetland area in central Italy. Four cpDNA markers (trnH–psbA, rbcL, rpoC1, and matK) were tested on 40 plant species, 26 of which strictly connected to the aquatic habitat. Universality of the method, ease of data retrieval, and correct assignation of the genetic markers to each species were evaluated. The markers showed different prospects of reliable applicability. The obtained sequences were blasted against the NCBI database to verify the correct species identification. A score ranging between 32% and 67% was achieved. Overall, eight species remained unidentified with all the tested barcodes due to the absence of conspecific sequences in the available databases. This work demonstrates some limitations in the applicability of DNA barcoding to accomplish complete taxonomical surveys. Difficulties encountered in this study urge refinement of technical protocols and accessibility to wider databases. Future technological advances and larger sample sets will certainly reinforce DNA barcoding as a useful tool to address knowledge and conservation of wetlands.     

Validation of 2D‐animated pictures as an investigative tool in the behavioural sciences: A case study with a West African cichlid fish,Pelvicachromis pulcher

Virtual stimuli represent an increasingly popular tool in the study of animal behaviour. Modern techniques have the potential to simplify and improve traditional experiments using live stimuli. However, the increasing availability of diverse techniques is associated with problems and limitations. Although many new methods have been developed, their validation remains largely untested. In the present study, we therefore performed two experiments to test whether 2‐D animations of predators and conspecifics elicit biologically appropriate behavioural responses in male rainbow kribs, Pelvicachromis pulcher. Individual responses towards a sympatric natural fish predator, Parachanna obscura, were tested using live predators and still colour photographs, animated using PowerPoint©. Compared to control trials (empty aquarium and white computer screen, respectively), individuals decreased their activity in response to both live and animated predators. We found no difference in activity between live and animation trials. Further, we tested individual aggression (frequency of aggressive behaviours) exhibited towards live and animated conspecifics. Individual aggressive behaviours shown towards live and animated conspecifics were positively correlated. Moreover, an individual's mean distance towards the opponent was a suitable proxy for individual aggression permitting the facilitation and standardisation of an individual's aggression through the use of a tracking software compared with the more laborious, traditional manual assessment. Our results show that simple, inexpensive animation techniques have the potential to provide an easy‐to‐apply and useful technological advance in animal behaviour research.