Usama Beshay

Phenol biodegradation by free and immobilized Acinetobacter

Acinetobacter sp. strain W-17, immobilized on porous sintered glass completely degraded 500 mg phenol l−1 in 40 h, but free cells required 120 h for this to be achieved. Immobilized cells can be used 7 times without losing their activity.

Effects of mixed nitrogen sources on biodegradation of phenol by immobilized Acinetobacter sp. strain W-17

Using Ca-alginate immobilized cells of Acinetobacter sp. strain W-17, the effects of ammonium-N and nitrate-N on the biodegradation of phenol were investigated. Degradation experiments in three different culture media; minimal salts medium (MSM), simulated (SW) and modified simulated wastewater (MSW) were performed. With the freely suspended cells (cell dry weight 0.2 g/l), complete phenol (500 mg/l) degradation was achieved after incubation for 120 h. Using the immobilized cells, the time was reduced to 24 h in MSM medium, and 15 h in the MSW. The results also indicate that strain W-17 can tolerate to high concentrations of NH4+-N (63 mg/l) and NO3--N (1000 mg/l) without a significant loss in the phenol biodegradation rate. Moreover, the presence of 500 mg/l phenol in the MSW had no considerable effect on the removal of both ammonium-N and nitrate-N. Repeated use of immobilized cells revealed that they could be used as much as five times without loss of activity. Our findings could be extended to enhance biotreatment of phenol contamination in a variety of biological treatment processes. (African Journal of Biotechnology: 2003 2(1): 8-12)

Cultivation of Dictyostelium discoideum in immobilized form by colonization of porous supports

Dictyostelium discoideum is a social amoeba. It shows promising characteristics as an expression system for the production of recombinant glycoproteins owing to its ability to perform a whole spectrum of post-translational modifications. However, its application is seriously affected by slow growth rates as well as low maximal cell densities. Since the technique of immobilization often is applied in order to achieve high cell concentrations, D. discoideum was cultivated in the presence of different porous supports with the aim of studying its growth in an immobilized state. On a small scale a shake flask cultivation system with external loop was applied for this purpose. On a larger scale a bioreactor was coupled with an external loop of a column containing a fixed bed of the porous support. During growth on standard axenic (soluble) media, cell densities in the pores of the supports were attained, which were more than one magnitude higher than those normally obtained during suspension culture. Electron micrograph images indicate how D. discoideum colonizes porous support particles

β-Glucanase production from genetically modified recombinant Escherichia coli: Effect of growth substrates and development of a culture medium in shake flasks and stirred tank bioreactor

Cultivation conditions for the extracellular production of a hybrid β-1,3-glucanase (EC 3.2.1.39) from Bacillus were established using a recombinant Escherichia coli BL21 (DE3) pET-bgl-hisactophillin-sec. Maximal β-1,3-glucanase production of 510 U ml−1 after 28 h, was obtained using an optimized medium which had an initial lactose level of 7 and 24 g l−1 yeast extract and 5 g l−1 NaCl. Lactose was a potent inducer of β-1,3-glucanase. It is a cheap and easily available substrate for large-scale cultivations. In addition, the kinetics of extracellular production of β-glucanase depended on salt concentration (NaCl). Batch fermentation in a 3-l laboratory fermentor using the optimized medium allowed the production of 495 U ml−1 β-glucanase after only 15 h. Final enzyme activity in the optimized medium can be increased up to 3.5 times (510 U ml−1) compared with the concentration obtained with the starting medium (150 U ml−1).

Applicability of Euglena gracilis for biorefineries demonstrated by the production of α-tocopherol and paramylon followed by anaerobic digestion.

In this study the use of Euglena gracilis biomass for α-tocopherol, paramylon and biogas production in a value-added chain was investigated. Therefore, we analyzed the dry cell weight and product concentrations at different growth phases during heterotrophic, photoheterotrophic and photoautotrophic cultivation in a low-cost minimal medium. Furthermore, the specific biogas yields for differently derived biomass with and without product recovery were investigated. We demonstrate that growth phase and cultivation mode not only have a significant impact on product formation, but also influence the yield of biogas obtained from anaerobic digestion of Euglena gracilis biomass. The maximum dry cell weight concentration ranged from 12.3±0.14gL(-1) for heterotrophically to 3.4±0.02gL(-1) for photoautotrophically grown Euglena gracilis cells. The heterotrophically grown biomass accumulated product concentrations of 5.3±0.12mgL(-1) of α-tocopherol and 9.3±0.1gL(-1) of paramylon or 805±10.9mL of biogasgvs(-1) (per gram volatile solids). The results for photoautotrophically grown cells were 8.6±0.22mgL(-1) of α-tocopherol and 0.78±0.01gL(-1) of paramylon or 648±7.2mL of biogasgvs(-1). For an energy-saving downstream procedure the extracting agent methanol does not have to be removed strictly. Samples with residual methanol showed a significantly increased biogas yield, because the solvent can be used as an additional substrate for methane production by archaebacteria.

Increasing the secretion ability of the kil gene for recombinant proteins in Escherichia coli by using a strong stationary-phase promoter

By using a β-glucanase from Bacillus as a model protein, we investigated whether the secretion competence based on the action of the kil gene can be improved using stronger promoters for the expression of the kil gene. Since the production of extracellular target proteins also depends on the promoter strengths of the target gene, we constructed four expression vectors with all possible combinations of a weak and a strong stationary-phase promoter for the kil gene, and a weak and a strong constitutive promoter, respectively, for the β-glucanase gene. The results of batch fermentations showed that the use of stronger promoters generally decreased the cell density. However, a drastic increase of productivity of the cells to produce and secrete β-glucanase resulted in a significantly higher activity of extracellular β-glucanase. The yield of extracellular β-glucanase can be increased (to 168 %) by using a strong promoter for the β-glucanase alone. However, the increase was much higher when the weak promoter of the kil gene was replaced by a strong stationary-phase promoter (to 221 %). An even higher yield of extracellular β-glucanase was reached when β-glucanase was expressed by a strong promoter in addition indicating a combinatorial effect. This shows that the extracellular production of a recombinant target gene can be optimized by tuning the promoter strengths of components, the kil gene and the target gene.

Repeated batch production of alkaline protease using porous sintered glass as carriers

Abstract Whole cells of Teredinobacter turnirae were immobilized onto different sizes of porous sintered glass SIRAN. Production of alkaline protease by the immobilized whole cells was attempted in a batch system. Alkaline protease activity produced by immobilized whole cells was about 2.3 times higher than that produced by freely suspended cells under the same cultivation conditions. The reusability of the immobilized and freely suspended cells was examined. The activity of alkaline protease produced by freely suspended cells decreased with increasing use cycles while the activity of immobilized cells increased gradually and reached a steady state after 4 cycles. T. turnirae cells immobilized on different matrices were observed by scanning electron microscopy and the distribution of the cells on each matrix used either in and/or on the internal pores observed.

Dependency of the fatty acid composition of Euglena gracilis on growth phase and culture conditions

Usually, the fatty acid (FA) composition of lipids from microalgae is determined using samples taken at a single time point only, often without considering the medium composition and cultivation conditions. Therefore, the results only represent the FA composition of cells in a certain growth phase. Furthermore, they may misrepresent the capability of the organism to produce certain FA or lipid mixtures. In this study, 22 FA were analysed quantitatively during the cultivation of Euglena gracilis under different cultivation modes and conditions. For cell growth, various media compositions for heterotrophic and photoheterotrophic conditions were used. Results of extensive FA analysis allowed the determination of appropriate cultivation conditions and durations for yielding lipid mixtures with optimal composition, e.g. for the production of biodiesel or functional food. Drastic differences in the ratio between n3- and n6-polyunsaturated fatty acid (PUFA) ranging from 0.04 to 1.81 were detected. This effect was strongly influenced by the cultivation mode. In addition, media with higher nitrogen concentration resulted in a higher n3/n6-PUFA-ratio as well as improved specific growth rates for all analysed combinations of glucose and nitrogen concentrations. Furthermore, it was demonstrated that the inexpensive proteose peptone medium is ideal for lipid production by E. gracilis. This work provides valuable tools to optimise yield, productivity and n3/n6-PUFA ratio concerning the cultivation of E. gracilis as well as potentially other microalgae in general.

Influence of medium composition on growth behaviour of Dictyostelium discoideum for cultivation on axenic media

The social amoeba Dictyostelium discoideum represents an attractive host organism for the production of heterologous proteins. However, its application is seriously affected by slow growth rates as well as low maximal cell densities in the presence of axenic (liquid) media. Starting with standard complex media the influence of certain medium components was investigated. The kind and concentration of carbohydrates, the concentration of salt and ammonia and the supplementation of conditioned media were varied. These studies were performed by following cell growth in batch experiments over the whole growth cycle into the decline phase to obtain information about growth rates as well as maximal cell densities. Only maltose, glucose and α-trehalose were metabolised at significant rates. The concentration of ammonia produced correlated inversely with the concentration of carbohydrates metabolised. Under standard conditions ammonia reached concentrations of about 50 mM, most being produced during stationary phase. Ionic strength and the addition of ammonia affected the growth rate as well as the maximal cell density. Ammonia did not limit maximal cell density. D. discoideum can be cultivated in normal stirred bioreactors. A semi-empirical model is discussed for the description of the growth behaviour.