Aeration strategy: a need for very high ethanol performance in Saccharomyces cerevisiae fed-batch process.

In order to identify an optimal aeration strategy for intensifying bio-fuel ethanol production in fermentation processes where growth and production have to be managed simultaneously, we quantified the effect of aeration conditions--oxygen limited vs non limited culture (micro-aerobic vs aerobic culture)--on the dynamic behaviour of Saccharomyces cerevisiae cultivated in very high ethanol performance fed-batch cultures. Fermentation parameters and kinetics were established within a range of ethanol concentrations (up to 147 g l(-1)), which very few studies have addressed. Higher ethanol titres (147 vs 131 g l(-1) in 45 h) and average productivity (3.3 vs 2.6 g l(-1) h(-1)) were obtained in cultures without oxygen limitation. Compared to micro-aerobic culture, full aeration led to a 23% increase in the viable cell mass as a result of the concomitant increase in growth rate and yield, with lower ethanol inhibition. The second beneficial effect of aeration was better management of by-product production, with production of glycerol, the main by-product, being strongly reduced from 12 to 4 g l(-1). We demonstrate that aeration strategy is as much a determining factor as vitamin feeding (Alfenore et al. 2002) in very high ethanol performance (147 g l(-1) in 45 h) in order to achieve a highly competitive dynamic process.

Improving ethanol production and viability of Saccharomyces cerevisiae by a vitamin feeding strategy during fed-batch process.

Several bottlenecks in the alcoholic fermentation process must be overcome to reach a very high and competitive performance of bioethanol production by the yeast Saccharomyces cerevisiae. In this paper, a nutritional strategy is described that allowed S. cerevisiae to produce a final ethanol titre of 19% (v/v) ethanol in 45 h in a fed-batch culture at 30 degrees C. This performance was achieved by implementing exponential feeding of vitamins throughout the fermentation process. In comparison to an initial addition of a vitamin cocktail, an increase in the amount of vitamins and an exponential vitamin feeding strategy improved the final ethanol titre from 126 g l(-1) to 135 g l(-1) and 147 g l(-1), respectively. A maximum instantaneous productivity of 9.5 g l(-1) h(-1) was reached in the best fermentation. These performances resulted from improvements in growth, the specific ethanol production rate, and the concentration of viable cells in response to the nutritional strategy.

Mur1, a Streptococcus thermophilus peptidoglycan hydrolase devoid of a specific cell wall binding domain.

The gene encoding Mur1, a Streptococcus thermophilus peptidoglycan hydrolase, was cloned by homology with acmA, the Lactococcus lactis major autolysin gene. Mur1 is a 24.7-kDa protein endowed with a putative signal peptide. Sequence analysis evidenced that Mur1 encompasses exactly the AcmA region containing the catalytic domain, but lacks the one containing amino acid repeats involved in cell wall binding. Mur1 appears to be expressed and cell-associated in S. thermophilus, as revealed by immunoblot analysis. These results suggest that the cell wall attachment mode of Mur1 differs from that of most peptidoglycan hydrolases described so far.

Characterization of Streptococcus thermophilus strains that undergo lysis under unfavourable environmental conditions.

The autolysis of starter lactic acid bacteria appears as a promising way to enhance the flavour of fermented dairy products. The present work was aimed at investigating the autolysis phenomenon in Streptococcus thermophilus, a thermophilic lactic acid bacteria involved in the starters used for the production of yoghurts, Italian and Swiss-type cheeses. Out of 146 strains screened for their aptitude to spontaneously lyse at the end of growth in M17 medium containing lactose in limited concentration, six strains, among which is the type strain CNRZ 1358, were found to be highly autolytic. These autolytic strains are characterized by a typical bell-shaped growth curve. Lysis of the type strain, which was studied as the model, was triggered under unfavourable environmental conditions, such as lactose depletion and NaCl or organic solvents addition. The lysogenic character of this strain was evidenced. Taken together, our results indicate that the autolytic phenotype in S. thermophilus is linked to the lysogenic character but does not result from the massive prophage induction under stressing conditions.

The Streptococcus thermophilus autolytic phenotype results from a leaky prophage.

Streptococcus thermophilus autolytic strains are characterized by a typical bell-shaped growth curve when grown under appropriate conditions. The cellular mechanisms involved in the triggering of lysis and the bacteriolytic activities of these strains were investigated in this study. Lactose depletion and organic solvents (ethanol, methanol, and chloroform) were shown to trigger a premature and immediate lysis of M17 exponentially growing cells. These factors and compounds are suspected to act by altering the cell envelope properties, causing either the permeabilization (organic solvents) or the depolarization (lactose depletion) of the cytoplasmic membrane. The autolytic character was shown to be associated with lysogeny. Phage particles, most of which were defective, were observed in the culture supernatants after both mitomycin C-induced and spontaneous lysis. By renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a bacteriolytic activity was detected at 31 kDa exclusively in the autolytic strains. This enzyme was detected during both growth and spontaneous lysis with the same intensity. We have shown that it was prophage encoded and homologous to the endolysin Lyt51 of the streptococcal temperate bacteriophage phi01205 (M. Sheehan, E. Stanley, G. F. Fitzgerald, and D. van Sinderen, Appl. Environ. Microbiol. 65:569-577, 1999). It appears from our results that the autolytic properties are conferred to the S. thermophilus strains by a leaky prophage but do not result from massive prophage induction. More specifically, we propose that phagic genes are constitutively expressed in almost all the cells at a low and nonlethal level and that lysis is controlled and achieved by the prophage-encoded lysis proteins.

Purification, properties, and sequence specificity of SslI, a new type II restriction endonuclease from Streptococcus salivarius subsp. thermophilus.

SslI, a type II restriction endonuclease, was purified from Streptococcus salivarius subsp. thermophilus strain BSN 45. SslI is an isoschizomer of BstNI. SslI activity was maximum at pH 8.8, 0 to 50 mM NaCl, 2 to 8 mM Mg2+, and 42 degrees C. Activity against phage DNA in vitro was demonstrated.

Characterization and comparison of virulent bacteriophages of Streptococcus thermophilus isolated from yogurt.

Seven virulent bacteriophages of Streptococcus thermophilus were characterized at the molecular level and classified into 2 subgroups (A and B) by DNA/DNA hybridization experiments and analysis of their structural proteins. Two representatives of subgroups A and B were compared to 3 representatives of Neve's subgroups I, II and III (Neve et al, 1989) by Southern blot experiments. These isometric-headed phages possess a double-stranded DNA genome varying between 30-44 kilobase (kb) pairs. Subgroup A is composed of 3 phages (phi 57 as representative) with similar structural proteins as determined by sodium dodecyl sulfate-poly-acrylamide gel (SDS-PAGE) electrophoresis (estimated molecular weights of 31,000 and 27,500 for phage phi 57 and 32,000 and 27,000 for the 2 others). A common structural protein of 43,000 was found for phages of subgroup B. Phages phi 57 (subgroup A) and a10/J9 or PO (Neve's subgroups I or II, respectively) belonged to the same subgroup as determined by DNA/DNA hybridization experiments. Partial DNA homology was detected among all the phages tested except for phage phi ST27 of AW Jarvis. Phage-host interactions were also investigated by cross-propagation of the 7 studied phages on different indicator strains. A complete lack of correlation existed between the DNA homology grouping of the phages and their host range. Various restriction-modification systems were detected in some of the Streptococcus thermophilus strains.