Development and optimization of a new synthetic process for lorcaserin

A two-step process to synthesize racemic lorcaserin was developed from 2-(4-chlorophenyl)ethanol via formation of bromide or tosylate derivatives. These derivatives were reacted with allylamine in neat conditions to provide pure N-(4-chlorophenethyl)allylammonium chloride. This compound was cyclized in neat conditions using aluminum or zinc chloride to give racemic lorcaserin. After resolution of enantiomers, the wrong enantiomer was racemized and recycled to give new R-lorcaserin.     

Bioprocess in-line monitoring using Raman spectroscopy and Indirect Hard Modeling (IHM): A simple calibration yields a robust model

To increase the process productivity and product quality of bioprocesses, the in-line monitoring of critical process parameters is highly important. For monitoring substrate, metabolite, and product concentrations, Raman spectroscopy is a commonly used Process Analytical Technology (PAT) tool that can be applied in-situ and non-invasively. However, evaluating bioprocess Raman spectra with a robust state-of-the-art statistical model requires effortful model calibration. In the present study, we in-line monitored a glucose to ethanol fermentation by Saccharomyces cerevisiae (S. cerevisiae) using Raman spectroscopy in combination with the physics-based Indirect Hard Modeling (IHM) and showed successfully that IHM is an alternative to statistical models with significantly lower calibration effort. The IHM prediction model was developed and calibrated with only 16 Raman spectra in total, which did not include any process spectra. Nevertheless, IHM's root mean square errors of prediction (RMSEPs) for glucose (3.68 g/L) and ethanol (1.69 g/L) were comparable to the prediction quality of similar studies that used statistical models calibrated with several calibration batches. Despite our simple calibration, we succeeded in developing a robust model for evaluating bioprocess Raman spectra.     

Elevated pCO2 affects the lactate metabolic shift in CHO cell culture processes

The shift from lactate production to consumption in CHO cell metabolism is a key event during cell culture cultivations and is connected to increased culture longevity and final product titers. However, the mechanisms controlling this metabolic shift are not yet fully understood. Variations in lactate metabolism have been mainly reported to be induced by process pH and availability of substrates like glucose and glutamine. The aim of this study was to investigate the effects of elevated pCO₂ concentrations on the lactate metabolic shift phenomena in CHO cell culture processes. In this publication, we show that at elevated pCO₂ in batch and fed‐batch cultures, the lactate metabolic shift was absent in comparison to control cultures at lower pCO₂ values. Furthermore, through metabolic flux analysis we found a link between the lactate metabolic shift and the ratio of NADH producing and regenerating intracellular pathways. This ratio was mainly affected by a reduced oxidative capacity of cultures at elevated pCO₂. The presented results are especially interesting for large‐scale and perfusion processes where increased pCO₂ concentrations are likely to occur. Our results suggest, that so far unexplained metabolic changes may be connected to increased pCO₂ accumulation in larger scale fermentations. Finally, we propose several mechanisms through which increased pCO₂ might affect the cell metabolism and briefly discuss methods to enable the lactate metabolic shift during cell cultivations.     

The development of sampling techniques for populations of the tarnished plant bug,Lygus lineolaris (hemiptera: Miridae)

Summary The relationship of ‘mean crowding’ to mean density showed that counts of nymphs and adults of the tarnished plant bug,Lygus lineolaris (Beauv.), on birdsfoot trefoil were aggregated and that the index of basic contagion approached zero for each developing stage. Aggregation increased gradually from the first to the fourth instar and then decreased through the adult stage. The mortality process from the first to the fifth instar was inversely density dependent. Inter-sample variance was the major source of population variance although significant variance was occasionally associated with blocks and plots owing to heterogeneity of the host stand. The most appropriate sample unit was a 3 sq. ft. area of foliage and substrate. This was divided into four quarters; application of the variance component technique indicated that in most cases one quarter drawn at random was optimal for sampling different stages. The number of samples required to attain a given level of precision varied inversely with population density. The cost function for sampling was determined for specified levels of precision.     

Maximizing the stability of metabolic engineering‐derived whole‐cell biocatalysts

A systematic and powerful knowledge‐based framework exists for improving the activity and stability of chemical catalysts and for empowering the commercialization of respective processes. In contrast, corresponding biotechnological processes are still scarce and characterized by case‐by‐case development strategies. A systematic understanding of parameters affecting biocatalyst efficiency, that is, biocatalyst activity and stability, is essential for a rational generation of improved biocatalysts. Today, systematic approaches only exist for increasing the activity of whole‐cell biocatalysts. They are still largely missing for whole‐cell biocatalyst stability. In this review, we structure factors affecting biocatalyst stability and summarize existing, yet not completely exploited strategies to overcome respective limitations. The factors and mechanisms related to biocatalyst destabilization are discussed and demonstrated inter alia based on two case studies. The factors are similar for processes with different objectives regarding target molecule or metabolic pathway complexity and process scale, but are in turn highly interdependent. This review provides a systematic for the stabilization of whole‐cell biocatalysts. In combination with our knowledge on strategies to improve biocatalyst activity, this paves the way for the rational design of superior recombinant whole‐cell biocatalysts, which can then be employed in economically and ecologically competitive and sustainable bioprocesses.     

Infection Process of Plectosporium tabacinum on False Cleavers (Galium spurium)

A native fungus, Plectosporium tabacinum (van Beyma) M. E. Palm, W. Gams et Nirenberg, has potential as a bioherbicide for the control of both herbicide-resistant and herbicide-susceptible false cleavers. Limited information is available on the infection process of P. tabacinum. P. tabacinum spore distribution pattern, germination, penetration, and colonization on false cleavers leaves were examined using confocal, light, and scanning electron microscopy. The results demonstrated that conidia were distributed over the entire surface of leaves and cotyledons. More than 90% of the conidia germinated on the leaf surface 6-8 h after inoculation. Penetration of the leaf epidermis by conidia started 8-10 h after inoculation. Histological observation showed that no appressoria were formed by P. tabacinum, but its hyphae produced appressed club-like structures that penetrated the cuticle and epidermal layers. No stomata or other natural openings were observed on the upper leaf surface of false cleavers seedlings. Penetration occurs directly on epidermal cells with more frequent intercellular penetrations. Hyphal penetration was visualized at a depth of 30 and 40 üm after 8 and 16 h of incubation, respectively. Secondary hyphae colonized mesophyll cells 16 h after inoculation. Even spore distribution, short spore germination time, club-like infection structure formation, direct penetration, quick colonization, and mucous secretion on false cleavers leaves may contribute to the kill of false cleavers by P. tabacinum. Slow spore germination and germ tube growth, low spore germination numbers, and no infection structure formation on Brassica napus leaves may be factors affecting the host selectivity of P. tabacinum.     

Control of Polymorphs on the Crystallization of Glycine Using a WWDJ Batch Crystallizer

The control of crystal polymorphs was investigated using a WWDJ batch crystallizer and glycine as a model compound. The WWDJ batch crystallizer is a newly developed crystallizer, which is equipped with a slurry sprinkler named Wall Wetter fixed on the shaft of an impeller and a double‐deck jacket. When a conventional crystallizer was used, the unstable α‐form crystals were always obtained. However, when the WWDJ batch crystallizer was used, the stable γ‐form crystals were obtained. The appearance of different polymorphs depends on the cooling rate during the crystallization. The γ‐form crystals were obtained by slow cooling, while the α‐form was obtained by rapid cooling. It means that the solvent‐mediated transformation of glycine crystal polymorphs can be controlled by changing the cooling rate in the WWDJ crystallizer. These results were obtained due to the fact that the WWDJ batch crystallizer accelerates the dissolution of metastable crystals and the growth of stable crystals.     

The contribution of PAS 2050 to the evolution of international greenhouse gas emission standards

Background, aim, and scope  The assessment of greenhouse gas (GHG) emissions arising from products (goods and services) is emerging as a high profile application of life cycle assessment (LCA), with an increasing desire from retailers and other supply chain organizations to better understand, and in some cases communicate, the carbon footprint of products. Publicly Available Specification 2050:2008, Specification for the assessment of the life cycle greenhouse gas emissions of goods and services, addresses the single-impact category of global warming to provide a standardized and simplified implementation of process LCA methods for assessing GHG emissions from products. This paper briefly reviews the development process followed for PAS 2050, before examining the treatment of GHG-specific contribution of PAS 2050 to product carbon footprinting. Materials and methods  PAS 2050 was jointly sponsored by the Carbon Trust and the UK Department for Environment, Food and Rural Affairs and was published by the British Standards Institution on 29 October 2008. An independent steering group oversaw the development of the specification, including the establishment of an expert workgroup program, comprehensive international consultation, and expert input on the requirements of the specification. Results  The development process for PAS 2050 resulted in a specification that includes specific requirements that limit the interpretation of the underlying LCA approach to product carbon footprinting. These requirements, including goal setting and life cycle inventory assessment, aspects of system boundary identification and temporal aspects of GHG emissions, clarify the approach to be taken by organizations implementing product carbon footprinting, and simplify the application of LCA procedures in relation to product carbon footprinting. Discussion  Assessment of the emissions arising from the life cycle of products has a clear international component, and delivering consistent results across the supply chain requires the application of consistent methods. There is an emerging recognition that further standardization of methods for product carbon footprinting is needed, and the specific requirements resulting from the PAS 2050 development process make a valuable contribution across a range of GHG assessment issues. Conclusions  The widespread interest in PAS 2050 from individuals and organizations, together with the development of similar guidance by other organizations, confirmed that there is a need for clarification, certainty, and requirements in the field of product carbon footprint analysis. The use of PAS 2050 to refine, clarify, and simplify existing LCA methods and standards has resulted in specific approaches to key GHG assessment issues being developed; it is important that future standards development work considers the impact of these approaches and their further refinement. Recommendations and perspectives  It is the consumption of goods and services by individuals around the world that drives global GHG emission, and PAS 2050 is a first attempt to provide integrated, consistent approaches that directly address the role of consumption at the product level in contributing to GHG emissions. Climate science and GHG assessment techniques are both evolving areas and it will be necessary to review the approach taken by PAS 2050 in the future: a formal review process for PAS 2050 will commence towards the end of 2009 and practitioners are encouraged to participate in this review process.

Strategy for a protein purification design using C-phycocyanin extract

A variety of techniques have been developed for the separation and recovery of proteins. The cost of purifying the product is frequently determined by the desired quality of the final product, which is evaluated by measuring the purity. In this work the design of a protein purification process for C-phycocyanin, a phycobiliprotein that can be used in the food and medical industries, was established. The study evaluated the use of ammonium sulfate precipitation, ion exchange chromatography and gel filtration to purify C-phycocyanin in a variety of sequences. The final design included the C-phycocyanin extraction step, precipitation with ammonium sulfate and ion exchange chromatography. When the elution step was studied, the kind of elution and pH were considered in order to obtain a product with a final purity of 4.0 with a purification factor of 6.35, so that, at the end of the strategy, C-phycocyanin of analytical grade would be obtained.