Wael Sabra

Microbial production of diols as platform chemicals: Recent progresses

Diols are chemicals with two hydroxyl groups which have a wide range of appealing applications as chemicals and fuels. In particular, four diol compounds, namely 1,3-propanediol (1,3-PDO), 1,2-propanediol (1,2-PDO), 2,3-butanediol (2,3-BDO) and 1,4-butanediol (1,4-BDO) can be biotechnologically produced by direct microbial bioconversion of renewable materials. These diols are considered as platform green chemicals. We review and discuss here the recent development in the microbial production of these diols, especially regarding the engineering of production strains and optimization of the fermentation processes.

Bacterial alginate: physiology, product quality and process aspects.

Abstract. Alginate, a copolymer of β-D-mannuronic acid and α-L-guluronic acid and currently commercially produced from the marine brown algae, can also be biologically produced by bacteria such as Azotobacter vinelandii, A. chroococcum and several species of Pseudomonas. The ever-increasing applications of this polymer in the food and pharmaceutical sectors have led to continuing research interest aimed at better understanding the metabolic pathways, the physiological or biological function of this polymer, the regulation of its formation and composition, and optimising the microbial production process. These aspects are reviewed here, with particular attention to alginate formation in the soil bacterium A. vinelandii. In addition, the biotechnological and industrial applications of alginate are summarised.

Effect of oxygen on formation and structure of Azotobacter vinelandii alginate and its role in protecting nitrogenase

ABSTRACT The activity of nitrogenase in the nitrogen-fixing bacteriumAzotobacter vinelandii grown diazotrophically under aerobic conditions is generally considered to be protected against O2 by a high respiration rate. In this work, we have shown that a high rate of respiration is not the prevailing mechanism for nitrogenase protection in A. vinelandii grown in phosphate-limited nitrogen-free chemostat culture. Instead, the formation of alginate appeared to play a decisive role in protecting the nitrogenase that is required for cell growth in this culture. Depending on the O2 tension and cell growth rate, the formation rate and composition of alginate released into the culture broth varied significantly. Furthermore, transmission electron microscopic analysis of cell morphology and the cell surface revealed the existence of an alginate capsule on the surface of A. vinelandii. The composition, thickness, and compactness of this alginate capsule also varied significantly. In general, increasing O2 tension led to the formation of alginate with a higher molecular weight and a greater l-guluronic acid content. The alginate capsule was accordingly thicker and more compact. In addition, the formation of the alginate capsule was found to be strongly affected by the shear rate in a bioreactor. Based on these experimental results, it is suggested that the production of alginate, especially the formation of an alginate capsule on the cell surface, forms an effective barrier for O2 transfer into the cell. It is obviously the quality, not the quantity, of alginate that is decisive for the protection of nitrogenase.

Biosystems analysis and engineering of microbial consortia for industrial biotechnology.

The development of industrial biotechnology for an economical and ecological conversion of renewable materials into chemicals and fuels requires new strategies and concepts for bioprocessing. Biorefinery has been proposed as one of the key concepts with the aim of completely utilizing the substrate(s) and producing multiple products in one process or at one production site. In this article, we argue that microbial consortia can play an essential role to this end. To illustrate this, we first briefly describe some examples of existing industrial bioprocesses involving microbial consortia. New bioprocesses under development which make use of the advantages of microbial consortia are then introduced. Finally, we address some of the key issues and challenges for the analysis and engineering of bioprocesses involving microbial consortia from a perspective of biosystems engineering.

Metabolic network analysis and experimental study of lipid production in Rhodosporidium toruloides grown on single and mixed substrates

BackgroundMicrobial lipids (triacylglycerols, TAG) have received large attention for a sustainable production of oleochemicals and biofuels. Rhodosporidium toruloides can accumulate lipids up to 70% of its cell mass under certain conditions. However, our understanding of lipid production in this yeast is still much limited, especially for growth with mixed substrates at the level of metabolic network. In this work, the potentials of several important carbon sources for TAG production in R.toruloides are first comparatively studied in silico by means of elementary mode analysis followed by experimental validation.ResultsA simplified metabolic network of R.toruloides was reconstructed based on a combination of genome and proteome annotations. Optimal metabolic space was studied using elementary mode analysis for growth on glycerol, glucose, xylose and arabinose or in mixtures. The in silico model predictions of growth and lipid production are in agreement with experimental results. Both the in silico and experimental studies revealed that glycerol is an attractive substrate for lipid synthesis in R. toruloides either alone or in blend with sugars. A lipid yield as high as 0.53 (C-mol TAG/C-mol) has been experimentally obtained for growth on glycerol, compared to a theoretical maximum of 0.63 (C-mol TAG/C-mol). The lipid yield on glucose is much lower (0.29 (experimental) vs. 0.58 (predicted) C-mol TAG/C-mol). The blend of glucose with glycerol decreased the lipid yield on substrate but can significantly increase the overall volumetric productivity. Experimental studies revealed catabolite repression of glycerol by the presence of glucose for the first time. Significant influence of oxygen concentration on the yield and composition of lipids were observed which have not been quantitatively studied before.ConclusionsThis study provides for the first time a simplified metabolic model of R.toruloides and its detailed in silico analysis for growth on different carbon sources for their potential of TAG synthesis. Experimental studies revealed the phenomenon of catabolite repression of glycerol by glucose and the importance of oxygen supply on the yield and composition of lipids. More systematic studies are needed to understand the mechanisms which should help to further optimize the lipid production in this strain of industrial interest.

Effect of phosphate and oxygen concentrations on alginate production and stoichiometry of metabolism of Azotobacter vinelandii under microaerobic conditions

Abstract Alginate production by Azotobacter vinelandii was studied in batch and continuous cultures under microaerobic conditions. In batch culture at a pO2 of 2–3% (air saturation) alginate production was enhanced by decreasing the PO3−4 level in the medium. Alginate yield from biomass (YP/X) reached the highest value of 0.66 g/g at the lowest phosphate level (100 mg/l), compared to 0.40 g/g and 0.25 g/g at higher phosphate levels (200 mg/l and 400 mg/l, respectively). In contrast, biomass formation behaved differently and the growth yield (YX/S) decreased with decreasing PO43− concentrations. Moreover, the respiratory quotient (RQ) of the culture was dependent on the initial phosphate concentration, especially in the phosphate-limited phase of growth. As the initial phosphate level decreased from 400 mg/l to 100 mg/l, the average RQ value of the culture declined from 1.46 to 0.89. The low RQ value is very close to the theoretical optimum RQ, calculated to be 0.8 on the basis of the stoichiometry of the metabolic pathways for alginate formation from sucrose. This optimum RQ was also confirmed in continuous culture at different dilution rates. Independent of the dilution rate, a pO2 value of 2–5% (air saturation) was found to be optimal for alginate production, the corresponding RQ values being 0.80–0.84. In addition, the molecular mass and composition of alginate were also found to be affected by both phosphate and oxygen concentrations. In conclusion, the RQ appears to be a useful parameter for optimum control of alginate production with this microorganism.

Draft Genome Sequence of Type Strain Clostridium pasteurianum DSM 525 (ATCC 6013), a Promising Producer of Chemicals and Fuels

ABSTRACT Clostridium pasteurianum, an anaerobic bacterium able to utilize atmospheric free nitrogen for biosynthesis, has recently been proven to be a promising producer of chemicals and fuels, such as 1,3-propanediol and n-butanol. Here, we report the high-quality draft genome sequence of DSM 525, a type strain of C. pasteurianum.

Microbial Cell Factories for Diol Production.

Diols are compounds with two hydroxyl groups and have a wide range of appealing applications as chemicals and fuels. In particular, five low molecular diol compounds, namely 1,3-propanediol (1,3-PDO), 1,2-propanediol (1,2-PDO), 2,3-butanediol (2,3-BDO), 1,3-butanediol (1,3-BDO), and 1,4-butanediol (1,4-BDO), can be biotechnologically produced by direct microbial bioconversion of renewable materials. In this review, we summarize recent developments in the microbial production of diols, especially regarding the engineering of typical microbial strains as cell factory and the development of corresponding bioconversion processes.

Fermentation of mixed substrates by Clostridium pasteurianum and its physiological, metabolic and proteomic characterizations

BackgroundClostridium pasteurianum is becoming increasingly attractive for the production of chemicals and fuels such as n-butanol and 1,3-propanediol. Previously we have shown that dual substrate fermentation using glucose and glycerol enhanced the cell growth and butanol production significantly. Although C. pasteurianum can grow efficiently with either glucose or glycerol alone, under certain conditions, glucose limitation in the mixed substrate fermentation leads to growth cessation. To understand this phenomenon and for process optimization, fermentation experiments were performed in the presence of excess glycerol but with varied initial concentrations of glucose which were followed by physiological, metabolic and proteomic analyses.ResultsPhysiological characterization showed that the observed cease of growth is not due to the toxicity of n-butanol. Furthermore, the growth can be resumed by addition of glucose or the intermediate oxaloacetate. Proteomic analysis shed more light on the system-level regulation of many proteins directly or indirectly associated with this phenomenon. Surprisingly, it is found that the specific growth rate of C. pasteurianum in the different growth phases (e.g. before and after glucose limitation) correlated well with the expression level of the ATP dependent pyruvate carboxylase and with the expression level of biotin synthase which provides the cofactor biotin for the formation of oxaloacetate from pyruvate. Bioenergetic analysis based on the formation rates of metabolites further show that ATP supply is not a limiting factor for the pyruvate carboxylation to oxaloacetate.ConclusionsThe results of physiological and proteomic analyses clearly show that the anaplerotic synthesis of oxaloacetate plays a key role in determining the growth behaviour of C. pasteurianum in fermentations with mixed substrates of glucose and glycerol. This study provides interesting targets for metabolic engineering of this emerging industrial microorganism.

Comparative physiological study of the wild type and the small colony variant of Pseudomonas aeruginosa 20265 under controlled growth conditions

Small-colony variants (SCVs) of Pseudomonas aeruginosa are often found in chronically infected airways of patients suffering from cystic fibrosis. These slow-growing morphological variants have been associated with persistent and antibiotic-resistant infections. Nevertheless, the behavior of SCVs under varied availability of O2 and iron, two key variables relevant to the lung environment of CF patients and pathogenicity of P. aeruginosa, has not been systematically studied so far. In this work, the effects of O2 and iron were comparatively studied for a CF P. aeruginosa wild type (WT) strain and its SCV phenotype in a real-time controlled cultivation system. Significant differences in the behavior of these strains were observed and quantified. In general, SCV exhibited a higher fitness than the WT toward aerobic conditions. Under iron rich condition, and despite less release of total extracellular proteins, absence of flagellin and lower siderophore production, the SCV cells grown at fully aerobic conditions showed a higher specific growth rate and a significantly higher cytotoxicity in comparison with the WT cells. The strains behaved also differently towards iron limitation. The phenomena of limited O2 transfer from the gas to the liquid phase and enhancement of formation of virulence factors under conditions of iron limitation were much more profound in the SCV culture than in the WT culture. These results have important implications for better understanding the pathogenicity of P. aeruginosa and its small-colony variants.