Cell Dry Matter Research Articles

Acetic Acid as an Indirect Sink of CO2 for the Synthesis of Polyhydroxyalkanoates (PHA): Comparison with PHA Production Processes Directly Using CO2 as Feedstock

White biotechnology is promising to transform CO2 emissions into a valuable commodity chemical such as the biopolymer polyhydroxyalkanaotes (PHA). Our calculations indicated that the indirect conversion of acetic acid from CO2 into PHA is an interesting alternative for the direct production of PHA from CO2 in terms of CO2 fixation, H2 consumption, substrate cost, safety and process performance. An alternative cultivation method using acetic acid as an indirect sink of CO2 was therefore developed and a proof-of-concept provided for the synthesis of both the homopolymer poly(3-hydroxybutyrate) (PHB) and the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). The aim was to compare key performance parameters with those of existing cultivation methods for direct conversion of CO2 to PHA. Fed-batch cultivations for PHA production were performed using a pH-stat fed-batch feeding strategy in combination with an additional Dissolved Oxygen (DO)-dependent feed. After 118 h of fermentation, 60 g/L cell dry matter (CDM) containing 72% of PHB was obtained, which are the highest result values reported so far. Fed-batch cultivations for PHBV production resulted in 65 g/L CDM and 48 g/L PHBV concentration with a 3HV fraction of 27 mol %. Further research should be oriented towards process optimisation, whole process integration and design, and techno-economic assessment.

Production of nitrogen-based platform chemical: cyanophycin biosynthesis using recombinantEscherichia coliand renewable media substitutes

Background Cyanophycin (CGP) is a sustainable polymer that can be converted to a derivative with reduced arginine content, or to completely biodegradable poly-aspartic acid, which can substitute for non-biodegradable polyacrylates. In nature, it is produced by most cyanobacteria; however, these microbes are not suitable for large-scale production due to slow growth and low polymer content. Results Cyanophycin synthetase gene (cphA) from Anabaena variabilis ATCC 29413 was PCR amplified and cloned into Escherichia coli. Different renewable media components, such as soybean meal, potato wastes and corn-derived zein hydrolysate, were evaluated for their feasibility for CGP production at shake flask level. The optimized conditions were then tested in a 7 L bioreactor; a maximum cell weight of 10.2 g L-1 was obtained. CGP comprised 23 g g-1 of cell dry matter (CDM) with molecular weight between 21.5 and 31 kDa and was composed of aspartic acid, arginine and lysine in the ratio of 1.05:1:0.2 (mass basis). Conclusion The efficient use of renewable biomass for cyanophycin production could achieve competitive price, and in return, promote the use of this platform chemical to produce innovative polymers and materials.© 2012 Society of Chemical Industry

Physiological conditions conducive to high cell density and high cyanophycin content in Ralstonia eutropha strain H16 possessing a KDPG aldolase gene-dependent addiction system

The recombinant strain of Ralstonia eutropha H16-PHB(-)4-∆eda (pBBR1MCS-2::cphA (6308)/eda (H16)) presenting a 2-keto-3-desoxy-phosphogluconate (KDPG) aldolase (eda) gene-dependent catabolic addiction system for plasmid maintenance when using gluconate or fructose as sole carbon source was used in this study. The effects of the initial pH, the nitrogen-to-carbon ratio, the inorganic components of medium, the oxygen supply, and the different carbon and nitrogen sources on the cell dry matter (CDM) and the cyanophycin granule polypeptide (CGP) content of the cells were studied in a mineral salts medium (MSM) without any additional amino acids or CGP precursor substrates. The experiments were designed to systematically find out the optimal conditions for growth of cells to high densities and for high CGP contents of the cells. Maximum contents of water-insoluble CGP and water-soluble CGP, contributing to 47.5% and 5.8% (w/w) of CDM, respectively, were obtained at the 30-L scale cultivation when cells were cultivated in MSM medium containing sufficient supplements of fructose, NH(3), K(2)SO(4), MgSO(4)[Symbol: see text]7H(2)O, Fe(Ш)NH(4)-citrate, CaCl(2)[Symbol: see text]2H(2)O, and trace elements (SL6). The molecular masses of water-insoluble and water-soluble CGP ranged from 25 to 31kDa and from 15 to 21kDa, respectively. High cell densities of up to 82.8g CDM/L containing up to 37.8% (w/w) water-insoluble CGP at the 30-L scale cultivation were also obtained. This is by far the best combination of high cell density and high cellular CGP contents ever reported, and it showed that efficient production of CGP at the industrial scale in white biotechnology could be achieved.

β-Carotene production by Saccharomyces cerevisiae with regard to plasmid stability and culture media

A recombinant Saccharomyces cerevisiae strain was used for the production of β-carotene. The episomal plasmid YEplac195YB/I/E was extended by a gene coding for the mevalonate kinase (mvaK1) from Staphylococcus aureus. The adh1 promoter was chosen for constitutive expression of mvaK1. The recombinant strain S. cerevisiae G175 (YEplac-CaroSA) synthesised β-carotene by expressing the carotenogenic genes of Xanthophyllomyces dendrorhous together with the mvaK1 gene. Cells of this strain were investigated for their carotenoid contents in YNB and YPD media. A corresponding mvaK1 transcript in the recombinant yeast host was verified. Growth experiments of a specific erg12 deletion mutant showed that the mevalonate kinase (MvaK1) was able to complement the function of the deleted native mevalonate kinase (Erg12) from S. cerevisiae in the MVA pathway under control of the constitutive adh1 promoter. Cells of S. cerevisiae G175 (YEplac-CaroSA) exhibited high plasmid stability under either selective or non-selective cultivation conditions. Time course experiments demonstrated high plasmid stability even over extended cultivation periods. Carotenoid production was therefore also stable in larger culture volumes. Due to the stability of the plasmid, cultivation of the cells in complex YPD medium was possible, and 14.3 mg β-carotene per litre and a cell density of 9 g cell dry matter (CDM) per litre were achieved. The highest amount of 3,897 μg β-carotene per gramme CDM at a cell density of 1 g CDM per litre was measured after cultivation of the cells in YNB medium with glucose as sole carbon source.

A novel plasmid addiction system for large-scale production of cyanophycin in Escherichia coli using mineral salts medium

Hitherto the production of the biopolymer cyanophycin (CGP) using recombinant Escherichia coli strains and cheap mineral salts medium yielded only trace amounts of CGP (<0.5%, w/w) of the cell dry matter (CDM). This was probably due to the instability of the plasmids encoding the cyanophycin synthetase. In this study, we developed an anabolism-based media-dependent plasmid addiction system (PAS) to enhance plasmid stability, and we established a process based on a modified mineral salts medium yielding a CGP content of 42% (w/w) at the maximum without the addition of amino acids to the medium for the first time. This PAS is based on different lysine biosynthesis pathways and consists of two components: (1) a knockout of the chromosomal dapE disrupts the native succinylase pathway in E. coli and (2) the complementation by the plasmid-encoded artificial aminotransferase pathway mediated by the dapL gene from Synechocystis sp. PCC 6308, which allows the synthesis of the essential lysine precursor L,L-2,6-diaminopimelate. In addition, this plasmid also harbors cphAC595S, an engineered cyanophycin synthetase gene responsible for CGP production. Cultivation experiments in Erlenmeyer flask and also in bioreactors in mineral salts medium without antibiotics revealed an at least 4.5-fold enhanced production of CGP in comparison to control cultivations without PAS. Fermentation experiments with culture volume of up to 400 l yielded a maximum of 18% CGP (w/w) and a final cell density of 15.2 g CDM/l. Lactose was used constantly as an effective inducer and carbon source. Thus, we present a convenient option to produce CGP with E. coli at a technical scale without the need to add antibiotics or amino acids using the mineral salts medium designed in this study.

Physiological Conditions Conducive to High Cyanophycin Content in Biomass ofAcinetobacter calcoaceticusStrain ADP1

The effects of the inorganic medium components, the initial pH, the incubation temperature, the oxygen supply, the carbon-to-nitrogen ratio, and chloramphenicol on the synthesis of cyanophycin (CGP) by Acinetobacter calcoaceticus strain ADP1 were studied in a mineral salts medium containing sodium glutamate and ammonium sulfate as carbon and nitrogen sources, respectively. Variation of all these factors resulted in maximum CGP contents of only about 3.5% (wt/wt) of the cell dry matter (CDM), and phosphate depletion triggered CGP accumulation most substantially. However, addition of arginine to the medium as the sole carbon source for growth promoted CGP accumulation most strikingly. This effect was systematically studied, and an optimized phosphate-limited medium containing 75 mM arginine and 10 mM ammonium sulfate yielded a CGP content of 41.4% (wt/wt) of the CDM at 30 degrees C. The CGP content of the cells was further increased to 46.0% (wt/wt) of the CDM by adding 2.5 microg of chloramphenicol per ml of medium in the accumulation phase. These contents are by far the highest CGP contents of bacterial cells ever reported. CGP was easily isolated from the cells by using an acid extraction method, and this CGP contained about equimolar amounts of aspartic acid and arginine and no detectable lysine; the molecular masses ranged from 21 to 29 kDa, and the average molecular mass was about 25 kDa. Transmission electron micrographs of thin sections of cells revealed large CGP granules that frequently had an irregular shape with protuberances at the surface and often severely deformed the cells. A cphI::OmegaKm mutant of strain ADP1 with a disrupted putative cyanophycinase gene accumulated significantly less CGP than the wild type accumulated, although the cells expressed cyanophycin synthetase at about the same high level. It is possible that the intact CphI protein is involved in the release of CGP primer molecules from initially synthesized CGP. The resulting lower concentration of primer molecules could explain the observed low rate of accumulation at similar specific activities.

High cell density cultivation of Rhodococcus opacus for lipid production at a pilot-plant scale.

The triacylglycerol (TAG)-accumulating bacterium Rhodococcus opacus strain PD630 was investigated with respect to the fermentative production of TAGs consisting of an unusually high fraction of fatty acids with an odd-number of carbon atoms and unsaturated monoenic fatty acids from sugar beet molasses and sucrose. Fed-batch fermentations were optimized at the 30-1 scale in a stirred tank bioreactor at 30 degrees C using a mineral salts medium, which contained sugar beet molasses and sucrose as sole carbon sources. Approximately 37.5 g cell dry matter (CDM) per liter was the highest cell density that was obtained at that scale with a TAG content in the cells of 52%. This fermentative process was also applied to a 500-1 pilot-plant scale. Cell densities as high as 18.4 g CDM per liter were obtained, and 42% of the sucrose present in the medium was converted into cell mass which consisted of 38.4% TAGs.