Sequential Batch Cultures Research Articles

Improved protein synthesis and secretion through medium enrichment in a stable recombinant yeast strain

Two Saccharomyces cerevisiae strains were employed to investigate the effects of medium enrichment on the expression and secretion of a recombinant protein. One was a stable autoselection strain with mutations in the ura3, fur1, and urid-k genes. The combination of these three mutations blocks both the pyrimidine nucleotide biosynthetic and salvage pathways and is lethal to the cells. Retention of the plasmid, which carries a URA3 gene, was essential for cell viability. Therefore, all media were selective, allowing cultivation of the strain in complex medium. The second strain was a nonautoselection (control) strain and is isogenic to the first except for the fur1 and urid-k mutations. The plasmid utilized contains the yeast invertase gene under the control of the MFalpha1 promoter and leader sequence. The expression and secretion of invertase for the autoselection strain were examined in batch culture for three media: a minimal medium (SD), a semidefined medium (SDC), and a rich complex medium (YPD). Biomass yields and invertase productivity (volumetric activity) increased with the complexity of the medium; total invertase volumetric activity in YPD was 100% higher than in SDC and 180% higher than in SD. Specific activity, however, was lowest in the SDC medium. Secretion efficiency was extremely high in all three media; for the majority of the culture, 80-90% of the invertase was secreted into the periplasmic space and/or culture medium. A glucose pulse at the end of batch culture in YPD facilitated the transport of residual cytoplasmic invertase. For the nonautoselection strain, invertase productivity did not improve as the medium was enriched from SDC to YPD, and plasmid stability in the complex YPD medium dropped from 54% to 34% during one batch fermentation. During long-term sequential batch culture in YPD, invertase activity decreased by 90% and the plasmid-containing fraction dropped from 56% to 8.8% over 44 generations of growth. The expression level for the autoselection strain, however, remained high and constant over this time period, and no reversion at the fur1 or urid-k locus was observed.

Physiological properties and plasma membrane composition of Saccharomyces cerevisiae grown in sequential batch culture and in the presence of surfactants

The effects of various polyoxyalkylene glycols (PAG) of different molecular mass, colza oil and oleic acid were studied on Saccharomyces cerevisiae grown without aeration in sequential batch culture on beet molasses wort. It was observed that PAG improve cell growth, viability and alcohol production with the best efficiency when their molecular mass was between 2000 and 3300 Da. Plasma membrane analysis revealed the presence of a higher percentage of ergosterol but a low percentage of lanosterol in cells showing the best viability. Colza oil had no significant action while oleic acid had a positive effect similar to that of the most efficient surfactant tested in this study. The kinetics of sphaeroplast production revealed that recycled cells were more resistant to lytic enzymes and that culture of the cells in the presence of the surfactants had no effect on cell wall lysis.

Evaluation of iron as a triggering factor for red tide blooms

We have examined the relationship between Fe and blooms of the toxic dinoflagellate Alexandrium tamarense (Balech) (formerly Gonyaulax tamarensis var. excavata (Lebour)) using a chemical method that estimates the biological avdability of Fe in seawater. The Fe requirement for optimal growth of A. tamarense in sequential batch culture (ca 3 nM 'available' Fe) was compared with Fe concentrations in waters of the Gulf of Maine, USA. Results indicated that Fe did not limit growth of the organism in nearshore coastal waters or over Georges Bank, but that the organism may have been Fe-limited in Gulf of Maine basin waters. The distribution of A. tamarense in the Gulf of Maine is consistent with these Fe data. Red tide outbreaks in the nearshore environment did not correlate with changes in total Fe or the estimated Fe availability. Although Fe did not appear to trigger outbreaks of A. tamarense in Maine coastal waters, the findings are consistent with suggestions that pulsed inputs of Fe may be important for the development of toxic dinoflagellate blooms in regions (e.g. Florida) where outbreaks are initiated offshore.

The production of secondary caries‐like lesions on cavity walls and the assessment of microleakage using an in vitro microbial caries system

The aim of this study was to assess microleakage along restored cavity walls using a new in vitro microbial technique. Extracted human teeth containing cavities restored with a microfine posterior composite were incubated in broth inoculated with a single strain of Streptococcus mutans for 10 days, using a sequential batch culture technique. Each margin of the cavities was finished in one of three ways: butt joint and etching; butt joint and no etching, or; bevel joint and etching. The assessment of microleakage was achieved by examining sections of the teeth histologically using polarized light for the presence or absence of caries-like cavity wall lesions. Outer (surface) lesions were also examined and displayed the characteristic zones of early natural caries lesions. The cavity wall lesions were observed as a translucent zone in 31% of butt and unetched margins, 16% of butt and etched margins, and 5% of bevelled and etched margins.

Stimulation by cysteine on growth of Clostridium thermoaceticum in minimal medium

An acetate-tolerant strain of Clostridium thermoaceticum (ATCC 31490), characterized as growing on a tryptone-yeast extract complex medium, was adapted to grow in a minimal medium containing glucose as sole carbon source. After five sequential batch cultures involving reducing and deleting organic materials from the original complex medium, the bacterium could grow on minimal medium with a supplement of nicotinic acid. However, growth and homoacetogenesis from glucose was remarkably retarded compared with that observed in the original complex medium. This could be strikingly improved up to the original level by replacing sodium thioglycolate (a reducing agent in the original medium) with L-cysteinλHCI supplemented to the minimal medium. Cysteine played an important role in stimulating growth and homoacetogenesis by not only supplying H2S as a sulphur source via cysteine desulphydrase (EC 4.4.1.1) but also maintaining a proper redox potential (—370 and —400 mV) in the growth phase during culture. Methionine could not serve as a sulphur source.

Demineralisation of Human Enamel by Streptococcus mutans NCTC 10832 Using a Sequential Batch Culture Technique

It has been recognised for some years that it is possible to produce caries-like areas of demineralisation in human enamel using relatively simple acidified-gel techniques [<i>Silverstone</i>, 1966]. Attempts to produce similar lesions by means of bacterial action have, in general, been thought to require complex apparatus in the form of ‘artificial mouths’ [<i>Pigman</i> et al., 1952; <i>Sidaway</i> et al., 1964; <i>Levine and Coulter</i>, 1976; <i>Yaari and Bibbby</i>, 1976]. Such apparatus is expensive as well as being difficult to sterilise and maintain sterile. In most instances only one specimen can be processed at a time. Artificial ‘white spot’ lesions have been produced using sequential batch culture techniques [<i>Jordan and Keγes</i>, 1966] but doubt has been cast on the comparability of these lesions to natural caries [<i>Hardie</i> et al., 1971]. The present technique uses simple inexpensive apparatus which is easily sterilised and which can be readily replicated.