Impact of aeration strategies on fed-batch cell culture kinetics in a single-use 24-well miniature bioreactor

The need to bring new biopharmaceutical products to market more quickly and to reduce final manufacturing costs is driving early stage, small scale bioprocess development. This work describes a comprehensive engineering characterisation of a novel, single-use 24-well parallel miniature bioreactor system. Cell culture performance is also investigated, with particular focus on the aeration strategies adopted at this small scale (7 mL) either by headspace sparging alone or by direct gas sparging into the culture medium. Apparent volumetric oxygen mass transfer coefficient (kLa) values ranged between 3–22 h−1 and 4–53 h−1 for headspace aeration and direct gas sparging respectively. The higher kLa values with direct gas sparging correlated directly with the increase in gas–liquid interfacial area per unit volume. Mixing times (tm) over a range of conditions were generally in the range 1–13 s and decreased with increasing shaking frequency (500–800 rpm). Direct gas sparging also served to reduce tm values by a factor of up to 19 fold. The impact of aeration strategies on cell culture kinetics of a model CHO cell line was also determined. Cultures performed with head space aeration alone showed the highest viable cell density (VCD) (15.2 × 106 cells mL−1), viability retention and antibody titre (1.58 g L−1). These were greater than in conventional shake flask cultures due to the improved control of the μ24 bioreactor system. In all cases the miniature bioreactor managed good control of process parameters such as pH 6.95 ± 0.4, temperature T°C 37 ± 0.4 and DO% 57 ± 32. Cultures performed with direct gas sparging showed a 25–45% reduction in VCD (depending on the aeration strategy used) and a similar reduction in antibody titre. Overall this work shows the successful application of the miniature bioreactor system for industrially relevant fed-batch cultures and highlights the impact of the dispersed gas phase on cell culture performance at the small scale

Environmental influences on akinete germination and development in Nodularia spumigena (Cyanobacteriaceae), isolated from the Gippsland Lakes, Victoria, Australia

This study was carried out to investigate the genesis of N. spumigena blooms by specifically studying the effects of environmental variables (salinity, nitrogen, phosphorus and light) on the germination of N. spumigena akinetes. Optimal conditions for maximum germination and germling growth were determined by exposing akinetes to a range of salinities and nutrient (nitrogen and phosphorus) concentrations under two different irradiances. At pre-determined time periods, treatments were sampled and the percent germination and length of germlings assessed. The results indicated that akinete germination and germling growth were optimal at salinities from 5 to 25 and significantly reduced outside this range. A positive correlation in germination was observed with increasing nutrient (phosphorus and nitrate) concentration. Similarly, germling growth increased with increasing concentrations of both nutrients. Irradiance significantly influenced both germination and growth during salinity experiments, whereas in nutrient addition experiments, irradiance had no effect on germination; however, growth was significantly influenced during phosphorus addition experiments. Consequently, salinity and light appeared to be most critical in the germination process for N. spumigena akinetes, with phosphorus most important for germling growth. The study showed that N. spumigena may be able to germinate under environmental conditions outside its optimal range, but the growth of the germling is significantly reduced, which in turn suggests that its ability to form a bloom outside its optimal environmental conditions would also be greatly reduced.<br /

Occurrence of glucosylsucrose [α-D-glucopyranosyl- (1→2)-α-D-glucopyranosyl-(1→2)-β-D-fructofuranoside] and glucosylated homologues in cyanobacteria: Structural properties, cellular contents and possible function as thermoprotectants

Little is known about the structure and function of oligosaccharides in cyanobacteria. In this study, a new class of saccharides from Nostoc was identified by MS and NMR techniques, consisting of α-d-glucopyranosyl-(1→2)-[α-d-glucopyranosyl-(1→2)]n-β-d-fructofuranosides ranging from the trisaccharide (n = 1) to decasaccharide (n = 8). In Nostoc ellipsosporum the cell content of saccharides increased 10–20-fold after heat stress (1 day, 40 °C) or during prolonged cultivation. Under these conditions the abundance of homologues of higher molecular mass (> pentasaccharide) increased and finally exceeded that of homologues of lower molecular mass including sucrose. Total intracellular content of the saccharides after heat stress was 5–10 mg·(g dry weight)-1 corresponding to intracellular concentrations of 0.25–0.5% (w/v). A possible role of the oligosaccharides identified is in the protection of enzymes against heat inactivation. Whereas amylase from Nostoc was only weakly protected by the decasaccharide, α-amylase from porcine pancreas was more efficiently stabilized by the octasaccharide and decasaccharide. Evidence is presented for the widespread occurrence of the newly identified saccharides in cyanobacteria. The results are discussed including previous reports on cyanobacterial oligosaccharides and with respect to possible functions of these compounds in the living cell