Fed-batch process for the psychrotolerant marine bacterium Pseudoalteromonas haloplanktis

Abstract

BackgroundPseudoalteromonas haloplanktis is a cold-adapted γ-proteobacterium isolated from Antarctic sea ice. It is characterized by remarkably high growth rates at low temperatures. P. haloplanktis is one of the model organisms of cold-adapted bacteria and has been suggested as an alternative host for the soluble overproduction of heterologous proteins which tend to form inclusion bodies in established expression hosts. Despite the progress in establishing P. haloplanktis as an alternative expression host the cell densities obtained with this organism, which is unable to use glucose as a carbon source, are still low. Here we present the first fed-batch cultivation strategy for this auspicious alternative expression host.ResultsThe key for the fed-batch cultivation of P. haloplanktis was the replacement of peptone by casamino acids, which have a much higher solubility and allow a better growth control. In contrast to the peptone medium, on which P. haloplanktis showed different growth phases, on a casamino acids-containing, phosphate-buffered medium P. haloplanktis grew exponentially with a constant growth rate until the stationary phase. A fed-batch process was established by feeding of casamino acids with a constant rate resulting in a cell dry weight of about 11 g l-1 (OD540 = 28) which is a twofold increase of the highest densities which have been obtained with P. haloplanktis so far and an eightfold increase of the density obtained in standard shake flask cultures.The cell density was limited in the fed-batch cultivation by the relatively low solubility of casamino acids (about 100 g l-1), which was proven by pulse addition of casamino acid powder which increased the cell density to about 20 g l-1 (OD540 = 55).ConclusionThe growth of P. haloplanktis to higher cell densities on complex medium is possible. A first fed-batch fermentation strategy could be established which is feasible to be used in lab-scale or for industrial purposes. The substrate concentration of the feeding solution was found to influence the maximal biomass yield considerably. The bottleneck for growing P. haloplanktis to high cell densities still remains the availability of a highly concentrated substrate and the reduction of the substrate complexity. However, our results indicate glutamic acid as a major carbon source, which provides a good basis for further improvement of the fed-batch process.

Cytoplasmic and Periplasmic Proteomic Signatures of Exponentially Growing Cells of the Psychrophilic Bacterium Pseudoalteromonas haloplanktis TAC125

“…Recently, a P. haloplanktis expression system (12,17) was developed, which enables the use of this host for the low-temperature heterologous expression of otherwise "intractable" proteins. Furthermore, a first fed-batch cultivation strategy for this alternative expression host was established (50). However, the physiology and metabolic pathways of this bacterium, which facilitate remarkably high growth rates on complex substrates, are so far unknown.…”

Modelling microbial metabolic rewiring during growth in a complex medium

“…Here we used this model to investigate the metabolic rearrangements occurring during growth in a nutritionally rich medium. A detailed analysis of the data reported by Wilmes et al [14] allowed the identification of twelve distinct phases in the growth of Ph TAC125 on peptone medium, each of them corresponding to a time step of one hour. As during the last couple of phases almost no nutrients uptake was recorded, we limited our analyses to the first ten phases (P1 to P10).…”

“…The physiological data of Ph TAC125 growth in soy-peptone complex medium [14] was used to identify time steps corresponding to amino acid depletions. Overall, twelve different (one hour long) time steps were identified along the complete growth curve and, for each of these time steps, the uptake rates of each amino acid were estimated based on their relative decrease in concentration and on the growth rate.…”

“…To allow for growth simulations consistent with the experiment by Wilmes et al [14], we also had to compute the uptake fluxes (UF) for each amino acid at each time step. To do this, we used the following formula: …”

“…A genome-scale metabolic reconstruction of Ph TAC125 has been recently published by our group [17], allowing a system-level representation of its phenotypic landscape and boosting a holistic comprehension of its metabolic features. Interestingly, the growth curve of Ph TAC125 in a complex medium (peptone) is characterised by a number of metabolic switches among the amino acids available in the medium [14]. The progressive exhaustion of the different sets of amino acids (especially glutamate) defines a hierarchical usage of carbon sources and, consequently, multiple phases in its growth curve, characterized by a different set of metabolized substrates.…”