Continuous bioprocessing: The real thing this time?: 10th Annual bioProcessUK Conference,December 3–4, 2013, London,UK

The Annual bioProcessUK Conference has acted as the key networking event for bioprocess scientists and engineers in the UK for the past 10 years. The following article is a report from the sessions that focused on continuous bioprocessing during the 10^th Annual bioProcessUK Conference (London, December 2013). These sessions were organized by the ‘EPSRC Centre for Innovative Manufacturing in Emergent Macromolecular Therapies’ hosted at University College London. A plenary lecture and workshop provided a forum for participants to debate topical issues in roundtable discussions with industry and academic experts from institutions such as Genzyme, Janssen, Novo Nordisk, Pfizer, Merck, GE Healthcare and University College London. The aim of these particular sessions was to understand better the challenges and opportunities for continuous bioprocessing in the bioprocessing sector.     

Ecology of heath communities dominated by Cassiope tetragona at Alexandra Fiord, Ellesmere Island, Canada

At a high arctic lowland on Ellesmere Island, heath communities dominated by Cassiope tetragona were widespread, and occurred in a variety of habitats that differred in time of snowmelt, relative site moisture, soil thaw depth, and air and soil temperature. Cover, standing crop, and production were dominated by woody plants, notably Cassiope tetragona and Dryas integrifolia . The Cassiope -dominated heaths were similar in composition to those at other Canadian high arctic localities, but were less similar to localities in Greenland and Spitzbergen. Compositional relationships among Cassiope -dominated heaths in different habitats at the study site may be largely determined by two interrelated environmental factors, time of snowmelt and site moisture.
Aboveground vascular plant biomass was concentrated near the ground surface, resulting in simple vertical structure that takes advantage of relatively warm soil and air temperatures in summer, and a protective snow cover in winter. Aboveground vascular plant standing crop was largely comprised of attached dead tissue, and there were equal proportions of above- and belowground biomass. Low community production was due to dominance by long-lived, slow-growing species, and to short growing seasons and cold temperatures. Overall character of Cassiope -dominated heaths reflects the conservative, stress tolerant growth strategy of the dominant species, Cassiope tetragona .     

Glucose excretion by the symbiotic Chlorella of Spongilla fluviatilis

Chlorella sorokiniana strain 211-40c, a symbiotic Chlorella isolated from a freshwater sponge, excreted between 3% and 5% of assimilated ¹⁴CO₂ as glucose in the light, with a pH optimum around 5. This percentage increased when the illuminance was lowered (to 15% at 20 lx). Release of [¹⁴C]glucose continued in the dark and could be inhibited by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Net efflux of glucose occurred even at a concentration ratio of extracellular/intracellular glucose of 4. This, together with the sensitivity to FCCP, is taken as evidence for active transport. Exogenous [¹⁴C]glucose was taken up by the cells under conditions of net glucose efflux, showing uptake and excretion to take place simultaneously.Abbreviations FCCP carbonyl cyanide p-trifluoromethoxyphenylhydrazone - p.c. packed cells     

Soluble and membrane-bound ferrisiderophore reductases of Escherichia coli K-12

After uptake of microbial ferrisiderophores, iron is assumed to be released by reduction. Two ferrisiderophore-reductase activities were identified in Escherichia coli K-12. They differed in cellular location, susceptibility to amytal, and competition between oxygen and ferrichrome-iron(III) reduction. The ferrisiderophore reductase associated with the 40,000×g sediment (membrane-bound enzyme) was inhibited by 10 mM amytal in contrast to the ferrisiderophore reductase present in the 100,000×g supernatant (soluble enzyme). Reduction by the membrane-bound enzyme followed sigmoid kinetics, but was biphasic in the case of the soluble enzyme. The soluble reductase could be assigned to a protein consisting of a single polypeptide of M _r 26000. Reduction of iron(III) by the purified enzyme depended on the addition of NADH or NADPH which were equally active reductants. The cofactor FMN and to a lesser degree FAD stimulated the reaction. Substrate specificity of the soluble reductase was low. In addition to the hydroxamate siderophores arthrobactin, schizokinen, fusigen, aerobactin, ferrichrome, ferrioxamine B, coprogen, and ferrichrome A, the iron(III) complexes of synthetic catecholates, dihydroxy benzoic acid, and dicitrate, as well as carrier-free iron(III) were accepted as substrates. Both ferrisiderophore reductases were not controlled by the fur regulatory system and were not suppressed by anaerobic growth.Abbreviations DHB dihydroxybenzoic acid - MECAM 1,3,5-N,N,N-tris-(2,3-dihydroxybenzoyl)-triamino-methylbenzene - MECAMS 2,3-dihydroxy-5-sulfonyl-derivative of MECAM     

Nickel requirement for the formation of active urease in purple sulfur bacteria (Chromatiaceae)

Nickel was found to be required for expression of urease activity in batch cultures of Thiocapsa roseopersicina strain 6311, Chromatium vinosum strain 1611 and Thiocystis violacea strain 2311, grown photolithotrophically with NH₄Cl as nitrogen source. In a growth medium originally free of added nickel and EDTA, the addition of 0.1–10 M nickel chloride caused an increase in urease activity, while addition of EDTA (0.01–2 mM) caused a strong reduction. Variation of the nitrogen source had no pronounced influence on the level of urease activity in T. roseopersicina grown with 0.1 M nickel in the absence of EDTA. Only nickel, of several heavy metal ions tested, could reverse suppression of urease activity by EDTA. Nickel, however, did not stimulate and EDTA did not inhibit the enzyme in vitro. When nickel was added to cultures already growing in a nickel-deficient, EDTA-containing medium, urease activity showed a rapid increase which was not inhibited by chloramphenicol. It is concluded that the (inactive) urease apoprotein may be synthesized in the absence of nickel and can be activated in vivo without de novo protein synthesis by insertion of nickel into the pre-formed enzyme protein.     

Ferricrocin functions as the main intracellular iron-storage compound in mycelia ofNeurospora crassa

Summary Neurospora crassa produces several structurally distinct siderophores: coprogen, ferricrocin, ferrichrome C and some minor unknown compounds. Under conditions of iron starvation, desferricoprogen is the major extracellular siderophore whereas desferriferricrocin and desferriferrichrome C are predominantly found intracellularly. Mössbauer spectroscopic analyses revealed that coprogen-bound iron is rapidly released after uptake in mycelia of the wild-typeN.crassa 74A. The major intracellular target of iron distribution is desferriferricrocin. No ferritin-like iron pools could be detected. Ferricrocin functions as the main intracellular iron-storage peptide in mycelia ofN. crassa. After uptake of ferricrocin in both the wild-typeN. crassa 74A and the siderophore-free mutantN. crassa arg-5 ota aga, surprisingly little metabolization (11%) could be observed. Since ferricrocin is the main iron-storage compound in spores ofN. crassa, we suggest that ferricrocin is stored in mycelia for inclusion into conidiospores.