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High cell density cultivation of Escherichia coli K4 in a microfiltration bioreactor: a step towards improvement of chondroitin precursor production.

Restaino OF, Cimini D, De Rosa M, Catapano A, De Rosa M, Schiraldi C - Microb. Cell Fact. (2011)

Bottom Line: It is usually obtained by extraction from animal tissues, but the risk of virus contaminations, as well as the scarceness of raw material, makes this productive process unsafe and unable to satisfy the growing market demand.In this work, on the trail of these results, we exploited new fermentation strategies to further improve the capsular polysaccharide production.High polysaccharide concentrations (4.73 ± 0.2 g·L(-1)), with corresponding average yields (0.13 ± 0.006 gK4 CPS·gcdw(-1)), were obtained; the increase of K4 CPS titre, compared to batch and fed-batch results, was of 16-fold and 3.3-fold respectively, while average yield was almost 3.5 and 1.4 fold higher.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, Second University of Naples, Via de Crecchio 7, 80138, Naples, Italy.

ABSTRACT

Background: The bacteria Escherichia coli K4 produces a capsular polysaccharide (K4 CPS) whose backbone is similar to the non sulphated chondroitin chain. The chondroitin sulphate is one of the major components of the extra-cellular matrix of the vertebrate connective tissues and a high value molecule, widely employed as active principle in the treatment of osteoarthritis. It is usually obtained by extraction from animal tissues, but the risk of virus contaminations, as well as the scarceness of raw material, makes this productive process unsafe and unable to satisfy the growing market demand. In previous studies a new biotechnological process to produce chondroitin from Escherichia coli K4 capsular polysaccharide was investigated and a 1.4 g·L(-1) K4 CPS concentration was reached using fed-batch fermentation techniques. In this work, on the trail of these results, we exploited new fermentation strategies to further improve the capsular polysaccharide production.

Results: The inhibitory effect of acetate on the bacterial cells growth and K4 CPS production was studied in shake flask conditions, while a new approach, that combined the optimization of the feeding profiles, the improvement of aeration conditions and the use of a microfiltration bioreactor, was investigated in three different types of fermentation processes. High polysaccharide concentrations (4.73 ± 0.2 g·L(-1)), with corresponding average yields (0.13 ± 0.006 gK4 CPS·gcdw(-1)), were obtained; the increase of K4 CPS titre, compared to batch and fed-batch results, was of 16-fold and 3.3-fold respectively, while average yield was almost 3.5 and 1.4 fold higher.

Conclusion: The increase of capsular polysaccharide titre confirmed the validity of the proposed fermentation strategy and opened the way to the use of the microfiltration bioreactor for the biotechnological production of chondroitin.

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Acetate inhibition in shake flasks. Effect of different acetate concentration addition to the conventional medium on the specific growth rates (μmax) of E. coli K4 during exponential phase, in shake flask conditions.
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Figure 1: Acetate inhibition in shake flasks. Effect of different acetate concentration addition to the conventional medium on the specific growth rates (μmax) of E. coli K4 during exponential phase, in shake flask conditions.

Mentions: In order to investigate the effect of acetate concentration on the growth of E. coli K4 and its capsular polysaccharide production ability, several shaking flask experiments were performed by using eight different acetate concentrations in the medium, in the range from 0 to 40 g·L-1. No sensible changes in the growth medium initial pH values were observed when acetate was added (from a pH value of 7.42 ± 0.01 for 0 g·L-1 to 7.35 ± 0.01 for 40 g·L-1 of added acetate). Growth rates (μmax) calculated during the exponential phase, showed a dependency on the acetate concentrations: compared with the control (without acetate the μmax μ= 0.746 ± 0.055·h-1), the addition of 1.5 g·L-1 of acetate to the medium seemed to slightly help bacterial replication increasing the μmax to 0.845 ± 0.017 h-1 (Figure 1). A growth rate value similar to the control experiment was observed in case of addition of 5 g·L-1 acetate (μmax μ= 0.795 ± 0.013 h-1), while a significant decrease in μmax (0.680 ± 0.022 h-1) was noticed when 10 g·L-1 of acetate were added (Figure 1). The capsular polysaccharide biosynthesis seemed to be influenced by the acetate addition too (Figure 2). Compared to the K4 CPS concentration in the conventional medium (0.117 ± 0.01 g·L-1) after 24 hours of growth, the addition of 1.5 g·L-1 of acetate caused an increase to 0.195 ± 0.02 g·L-1 (Figure 2). Similarly to the control, in this case, the whole glycerol amount was consumed during the growth (data not shown). Cultivation on 5 g·L-1 acetate added medium caused a similar K4 CPS production (0.204 ± 0.01 g·L-1) while on 10 g·L-1 even a slightly higher titre was observed (0.228 ± 0.02 g·L-1). Further increasing the acetate concentration in the medium reduced μmax, the final biomass yield and, consequently, the K4 CPS production that, at the highest acetate concentration tested (40 g·L-1), decreased to 0.020 ± 0.009 g·L-1 (Figure 2). In these cases the bacteria did not use the whole initial amount of glycerol (residual concentration between 3.2 and 3.9 g·L-1) and did not reach the same biomass maximum value obtained in the conventional medium. As already reported before [8], in all shake flask experiments, K4 CPS concentration reached the maximum after 24 hours of culture. Different experiments tested the ability of E. coli K4 to grow on acetate (from 1.5 to 20 g·L-1) in glycerol free medium. Also in this case the acetate addition did not cause any pH modification in the media. The bacteria metabolised acetate, as visible in Table 1, and a residue of acetate was observed at the end of the experiment only in case of the highest initial concentration. The higher the acetate titre was the greater the cell concentration achieved (passing from 0.92 to 1.99 gcdw·L-1), while growth rates quickly diminished from 0.84 to 0.51 h-1.


High cell density cultivation of Escherichia coli K4 in a microfiltration bioreactor: a step towards improvement of chondroitin precursor production.

Restaino OF, Cimini D, De Rosa M, Catapano A, De Rosa M, Schiraldi C - Microb. Cell Fact. (2011)

Acetate inhibition in shake flasks. Effect of different acetate concentration addition to the conventional medium on the specific growth rates (μmax) of E. coli K4 during exponential phase, in shake flask conditions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3050683&req=5

Figure 1: Acetate inhibition in shake flasks. Effect of different acetate concentration addition to the conventional medium on the specific growth rates (μmax) of E. coli K4 during exponential phase, in shake flask conditions.
Mentions: In order to investigate the effect of acetate concentration on the growth of E. coli K4 and its capsular polysaccharide production ability, several shaking flask experiments were performed by using eight different acetate concentrations in the medium, in the range from 0 to 40 g·L-1. No sensible changes in the growth medium initial pH values were observed when acetate was added (from a pH value of 7.42 ± 0.01 for 0 g·L-1 to 7.35 ± 0.01 for 40 g·L-1 of added acetate). Growth rates (μmax) calculated during the exponential phase, showed a dependency on the acetate concentrations: compared with the control (without acetate the μmax μ= 0.746 ± 0.055·h-1), the addition of 1.5 g·L-1 of acetate to the medium seemed to slightly help bacterial replication increasing the μmax to 0.845 ± 0.017 h-1 (Figure 1). A growth rate value similar to the control experiment was observed in case of addition of 5 g·L-1 acetate (μmax μ= 0.795 ± 0.013 h-1), while a significant decrease in μmax (0.680 ± 0.022 h-1) was noticed when 10 g·L-1 of acetate were added (Figure 1). The capsular polysaccharide biosynthesis seemed to be influenced by the acetate addition too (Figure 2). Compared to the K4 CPS concentration in the conventional medium (0.117 ± 0.01 g·L-1) after 24 hours of growth, the addition of 1.5 g·L-1 of acetate caused an increase to 0.195 ± 0.02 g·L-1 (Figure 2). Similarly to the control, in this case, the whole glycerol amount was consumed during the growth (data not shown). Cultivation on 5 g·L-1 acetate added medium caused a similar K4 CPS production (0.204 ± 0.01 g·L-1) while on 10 g·L-1 even a slightly higher titre was observed (0.228 ± 0.02 g·L-1). Further increasing the acetate concentration in the medium reduced μmax, the final biomass yield and, consequently, the K4 CPS production that, at the highest acetate concentration tested (40 g·L-1), decreased to 0.020 ± 0.009 g·L-1 (Figure 2). In these cases the bacteria did not use the whole initial amount of glycerol (residual concentration between 3.2 and 3.9 g·L-1) and did not reach the same biomass maximum value obtained in the conventional medium. As already reported before [8], in all shake flask experiments, K4 CPS concentration reached the maximum after 24 hours of culture. Different experiments tested the ability of E. coli K4 to grow on acetate (from 1.5 to 20 g·L-1) in glycerol free medium. Also in this case the acetate addition did not cause any pH modification in the media. The bacteria metabolised acetate, as visible in Table 1, and a residue of acetate was observed at the end of the experiment only in case of the highest initial concentration. The higher the acetate titre was the greater the cell concentration achieved (passing from 0.92 to 1.99 gcdw·L-1), while growth rates quickly diminished from 0.84 to 0.51 h-1.

Bottom Line: It is usually obtained by extraction from animal tissues, but the risk of virus contaminations, as well as the scarceness of raw material, makes this productive process unsafe and unable to satisfy the growing market demand.In this work, on the trail of these results, we exploited new fermentation strategies to further improve the capsular polysaccharide production.High polysaccharide concentrations (4.73 ± 0.2 g·L(-1)), with corresponding average yields (0.13 ± 0.006 gK4 CPS·gcdw(-1)), were obtained; the increase of K4 CPS titre, compared to batch and fed-batch results, was of 16-fold and 3.3-fold respectively, while average yield was almost 3.5 and 1.4 fold higher.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Experimental Medicine, Section of Biotechnology and Molecular Biology, Second University of Naples, Via de Crecchio 7, 80138, Naples, Italy.

ABSTRACT

Background: The bacteria Escherichia coli K4 produces a capsular polysaccharide (K4 CPS) whose backbone is similar to the non sulphated chondroitin chain. The chondroitin sulphate is one of the major components of the extra-cellular matrix of the vertebrate connective tissues and a high value molecule, widely employed as active principle in the treatment of osteoarthritis. It is usually obtained by extraction from animal tissues, but the risk of virus contaminations, as well as the scarceness of raw material, makes this productive process unsafe and unable to satisfy the growing market demand. In previous studies a new biotechnological process to produce chondroitin from Escherichia coli K4 capsular polysaccharide was investigated and a 1.4 g·L(-1) K4 CPS concentration was reached using fed-batch fermentation techniques. In this work, on the trail of these results, we exploited new fermentation strategies to further improve the capsular polysaccharide production.

Results: The inhibitory effect of acetate on the bacterial cells growth and K4 CPS production was studied in shake flask conditions, while a new approach, that combined the optimization of the feeding profiles, the improvement of aeration conditions and the use of a microfiltration bioreactor, was investigated in three different types of fermentation processes. High polysaccharide concentrations (4.73 ± 0.2 g·L(-1)), with corresponding average yields (0.13 ± 0.006 gK4 CPS·gcdw(-1)), were obtained; the increase of K4 CPS titre, compared to batch and fed-batch results, was of 16-fold and 3.3-fold respectively, while average yield was almost 3.5 and 1.4 fold higher.

Conclusion: The increase of capsular polysaccharide titre confirmed the validity of the proposed fermentation strategy and opened the way to the use of the microfiltration bioreactor for the biotechnological production of chondroitin.

Show MeSH
Related in: MedlinePlus