Changes In Fermentation Conditions Research Articles

Influence of fermentation temperature and duration on survival and biocontrol efficacy of Pseudomonas fluorescens Pf153 freeze-dried cells.

The aim of this paper was to determine whether the quality of formulated Pseudomonas fluorescens Pf153 can be influenced by changes in fermentation conditions. In this study, the influence of the fermentation temperature on the growth and its consequence on shelf life, viability and biocontrol efficacy of freeze-dried cells of P. fluorescens Pf153 was investigated. Cells of P. fluorescens Pf153 were grown at 20 and 28°C in flasks and fermenter and harvested in the mid-log and the beginning of the stationary phase. The survival during storage of freeze-dried cells was tested at 25°C. Cells fermented at 20°C survived in storage better than those grown at 28°C, irrespective of the harvesting time. Compared to the untreated control, in in vitro tests Pf153 was in all production temperature/duration combinations significantly effective against all tested Botrytis cinerea strains. But no differences between temperature/duration combinations were found. In bioassay on detached Vicia faba leaves, it was found that young cells, when fermented at 28°C had a significant positive influence on the biocontrol efficacy. These results demonstrate that fermentation parameters have an influence on the performance and quality of a formulated product. Only limited numbers of biocontrol products based on antagonistic pseudomonads are on the market. This can be attributed to the lack of suitable formulated products with high numbers of viable cells and a good shelf life. Currently, only limited information on the influence of the fermentation on subsequent downstreaming process is available. Within this study, we focused on the influence of the two important parameters fermentation temperature and harvest time on survival, shelf life and biocontrol efficacy of P. fluorescens Pf153.

An automated microscale chromatographic purification of virus‐like particles as a strategy for process development

The development of fermentation processes for recombinant vaccines requires optimizing expression while maintaining high product quality. Changes to cell fermentation conditions are typically evaluated following cell disruption, with expression levels quantified by immunoassay, liquid chromatography or enzyme activity. However, assay titres do not always predict the effects that intracellular aggregation, proteolysis, post-translational modifications and differences in relative impurity levels can have on purification yield and product purity. Furthermore, heterogeneity in the size and surface properties inherent in viral particles makes unit operations such as chromatography less predictable. In these cases, the purification procedure (or a mimic thereof) must be carried out to give accurate information on the impact of changes in fermentation conditions on purification process performance. This was demonstrated for the development of a recombinant vaccine against human papillomavirus produced in Saccharomyces cerevisiae, where the most informative feedback on fermentation variables was obtained by completing a multistep chromatographic purification to evaluate process yield and product purity. To increase the purification throughput and reduce labour, the chromatography was miniaturized 1000-fold from the laboratory scale using microlitre volumes of adsorbent in a pipette tip and automated on a robotic workstation. The microscale purification is shown to be predictive of the laboratory-scale purification in terms of yield and purity, while providing over a 10-fold increase in throughput and allowing for increased monitoring of fermentation processes. In addition, by reducing the volume of cells needed for this assessment, the fermentation can be correspondingly reduced in scale and carried out in parallel for additional throughput gains.

Estimation of temperature dependent parameters of a batch alcoholic fermentation process

In this work, a procedure was established to develop a mathematical model considering the effect of temperature on reaction kinetics. Experiments were performed in batch mode in temperatures from 30 to 38 degrees C. The microorganism used was Saccharomyces cerevisiae and the culture media, sugarcane molasses. The objective is to assess the difficulty in updating the kinetic parameters when there are changes in fermentation conditions. We conclude that, although the re-estimation is a time-consuming task, it is possible to accurately describe the process when there are changes in raw material composition if a re-estimation of parameters is performed.

Expression of Sonic hedgehog-Fc fusion protein in Pichia pastoris. Identification and control of post-translational, chemical, and proteolytic modifications

We have investigated the suitability of Pichia pastoris as an expression system for the candidate therapeutic protein, Sonic hedgehog fused to an immunoglobulin Fc domain (Shh-Fc). Sonic hedgehog is a morphogen protein involved in the patterning of a wide range of tissues during animal embryogenesis. The presence of Sonic hedgehog and its receptor, Patched, in adult nervous tissue suggests possible applications for the protein in the treatment of neurodegenerative disease and injury. We have engineered the Shh-Fc fusion protein in order to improve binding affinity and increase systemic exposure in animals. N-terminal sequencing, peptide mapping, mass spectrometry, and other biochemical and biological methods were used to characterize the purified protein. These analyses revealed several unanticipated problems, including thiaproline modification of the N-terminal cysteine, cleavage by a Kex2-like protease at a site near the N-terminus, proteolysis at sites near the hinge, addition of a hexose in the CH3 domain of the Fc region, and several sites of methionine oxidation. Sequence modifications to the protein and changes in fermentation conditions resulted in increased potency and greater consistency of the product. The final product was shown to be biologically active in animal studies.

Influence of fermentation conditions and microfiltration processes on membrane fouling during recovery of glucuronane polysaccharides from fermentation broths.

We have investigated the recovery of exopolysaccharides produced by Sinorhizobium meliloti M5N1 CS bacteria from fermentation broths using different membrane filtration processes: cross-flow filtration with a 7 mm i.d. tubular ceramic membrane of 0.5-microm pores under fixed transmembrane pressure or fixed permeate flux and dynamic filtration with a 0.2 microm nylon membrane using a 16-cm rotating disc filter. With the tubular membrane, the polysaccharide mass flux was mainly limited by polymer transmission that decayed to 10% after 90 min. The mass flux of polymer produced under standard fermentation conditions (70 h at 30 degrees C) stabilized after 70 min to 15 g/h/m(2). This mass flux rises to 36 g/h/m(2) when the mean stirring speed during fermentation is increased and to 123 g/h/m(2) when fermentation is extended to 120 h. In both cases, the mean molecular weight of polysaccharides drops from 4.0 10(5) g/mol under standard conditions to 2.7 10(5) g/mol. A similar reduction in molecular weight was observed when the fermentation temperature was raised to 36 degrees C without benefit to the mass flux. These changes in fermentation conditions have little effect on stabilized permeate flux, but raise significantly the sieving coefficient, due probably to molecular weight reduction and the filamentous aspect of the polymer as observed from SEM photographs. The polymer-mass flux was also increased by reducing transmembrane pressure (TMP) and raising the shear rate by inserting a rod in the membrane lumen. Operation under fixed permeate flux instead of constant TMP inhibited fouling during the first 4 h, resulting in higher sieving coefficients and polymer mass fluxes. The most interesting results were obtained with dynamic filtration because it allows operation at high-shear rates and low TMP. Sieving coefficients remained between 90 and 100%. With a smooth disc, the polysaccharide mass flux remained close to 180 g/h/m(2) at 1500 rpm and cell concentrations from 1 to 3 g/L. When radial rods were glued to the disc to increase wall shear stress and turbulence, the mass flux rose to 275 g/h/m(2) at the same speed and cell concentration.

Diagnosing lactic acid fermentation based on specific rates of growth, substrate consumption and product formation

The on-line calculated specific rates of growth, substrate consumption and product formation were used to diagnose microbial activities during a lactic acid fermentation. The specific rates were calculated from on-line measured cell mass, and substrate and product concentrations. The specific rates were more sensitive indicators of slight changes in fermentation conditions than such monitored data as cell mass or product concentrations.

Fermentation of Idli: Effects of changes in raw material and physico-chemical conditions

Idli is a popular traditional South Indian steamed pancake prepared from a fermented batter containing both rice ( Oryza sativa ) and black gram ( Phaseolus mungo ). Analysis of 35 different samples of idli batters, both commercial and laboratory-prepared, revealed the occurrence of bacteria belonging to six species in the range of 10 6 –10 9 /g; 68 % of the samples were also positive for yeasts, ranging up to 10 6 /g and yielding six types. Laboratory studies on ways to improve the nutritional and organoleptic properties of idli identified changes in fermentation conditions that led to appreciable increases in the microbial cell counts, total acids, volume, soluble solids, reducing sugars, non-protein nitrogen, free amino acids, amylases, proteinases and water-soluble vitamins, including B 2 , B 2 , B 12 and C. Novel idli batters, prepared by replacing the conventional black gram with other legumes, revealed comparable changes but with differences in the levels of some biochemical constituents. Incubation of idli batters at 28 °C with initial pH around 4·5 and supplemented with sucrose (1–2%) proved to be favourable for improving the nutritional constituents and organoleptic characteristics.

Molecular weight of scleroglucan and other extracellular microbial polysaccharides by size-exclusion chromatography and low angle laser light scattering

High-performance aqueous size-exclusion chromatography coupled to a low angle laser light scattering detector has been applied to the analysis of scleroglucan and various other extracellular microbial polysaccharides. Emphasis has been focused on three main findings. (1) The molecular weight of these macromolecules is not very sensitive to changes in fermentation conditions. This is specially true in the case of scleroglucan and related (1 → 3)-β- d-glucans including schizophyllan, which all exhibited a constant weight-average molecular weight of 5·7×10 6±5%. (2) In contrast to plant polysaccharides, polydispersity is very low, usually near unity. (3) The molecular weight levels off quickly during biosynthesis since the molecular weight is constant from the middle of fermentation, if not before.

Submerged citric acid fermentation of ferrocyanide-treated beet molasses: morphology of pellets of Aspergillus niger.

A study of the internal structure of Aspergillus niger pellets, grown during submerged citric acid fermentation of ferrocyanide-treated beet molasses, was made using botanical microtechniques. Under optimum fermentation conditions, each pellet developed as a round mass of mycelium of uniform consistency during the first 24 hours of the fermentation (mash sparged with air); subsequently (mash sparged with oxygen), a dense crust of growth formed at the periphery of the pellet and autolysis of cells at the center began. At the end of fermentation (140 hours), the pellet consisted of a shell of mycelium occupying less than 50% of the pellet volume. Changes in fermentation conditions were reflected in the density of peripheral growth and in the rate and extent of autolysis.