Scale-down and parallel operation of the riboflavin production process with Bacillus subtilis

Knorr, Bettina; Schlieker, H.; Hohmann, Hans‐Peter; Weuster‐Botz, Dirk

doi:10.1016/j.bej.2006.10.023

Cited by 63 publications

(36 citation statements)

References 74 publications

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“…Another reason for the low riboXavin production in C. acetobutylicum might be due to the low cell density obtained in the static culture, with biomass in terms of A 600 generally being less than 10. In comparison, in B. subtilis, fed-batch cultures in bioreactors can accumulate biomass up to 50 g/l [25]. Improvement of the culture conditions with the aim of increasing biomass could deWnitely be an approach to eVect an increase in riboXavin concentration in C. acetobutylicum cultures.…”

Section: Discussionmentioning

confidence: 99%

Improving the Clostridium acetobutylicum butanol fermentation by engineering the strain for co-production of riboflavin

Cai

Bennett

2010

J Ind Microbiol Biotechnol

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Solvent-producing clostridia are well known for their capacity to use a wide variety of renewable biomass and agricultural waste materials for biobutanol production. To investigate the possibility of co-production of a high value chemical during biobutanol production, the Clostridium acetobutylicum riboflavin operon ribGBAH was over-expressed in C. acetobutylicum on Escherichia coli-Clostridium shuttle vector pJIR750. Constructs that either maintained the original C. acetobutylicum translational start codon or modified the start codons of ribG and ribB from TTG to ATG were designed. Riboflavin was successfully produced in both E. coli and C. acetobutylicum using these plasmids, and riboflavin could accumulate up to 27 mg/l in Clostridium culture. Furthermore, the C. acetobutylicum purine pathway was modified by over-expression of the Clostridium purF gene, which encodes the enzyme PRPP amidotransferase. The function of the plasmid pJaF bearing C. acetobutylicum purF was verified by its ability to complement an E. coli purF mutation. However, co-production of riboflavin with biobutanol by use of the purF over-expression plasmid was not improved under the experimental conditions examined. Further rational mutation of the purF gene was conducted by replacement of amino acid codons D302 V and K325Q to make it similar to the feedback-resistant enzymes of other species. However, the co-expression of ribGBAH and purFC in C. acetobutylicum also did not improve riboflavin production. By buffering the culture pH, C. acetobutylicum ATCC 824(pJpGN) could accumulate more than 70 mg/l riboflavin while producing 190 mM butanol in static cultures. Riboflavin production was shown to exert no effect on solvent production at these levels.

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Section: Discussionmentioning

confidence: 99%

Improving the Clostridium acetobutylicum butanol fermentation by engineering the strain for co-production of riboflavin

Cai

Bennett

2010

J Ind Microbiol Biotechnol

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“…The riboflavin-producing plasmids pRF69 and pRF93 contain the rib operon and either a cloramphenicol or tetracycline resistance marker, respectively, were integrated chromosomally into the bacterial genome. Additionally, the native promoters ribP 1 and ribP 2 of the rib operon were replaced with a strong constitutive promoter P 15 derived from the Bacillus Bacteriophage SPO1 (Bretzel et al, 1999;Knorr, 2005;Perkins et al, 1990Perkins et al, , 1999. B. subtilis RB50 is deposited at NRRL (B-18502) and the plasmid pRF69 at ATCC (68338).…”

Section: Bacterial Strainmentioning

confidence: 99%

“…The feed solution containing 795 g/L glucose was supplied at an initial rate of 13.3 mL/L/h for 2 h and at 14.7 mL/L/h for the remaining time of the cultivation. Stirring speed at 1,200 rpm and air-flow between 3 and 5 L/min ensured dissolved oxygen levels above 15% troughout the cultivations (Knorr, 2005;Zamboni et al, 2003). Samples were taken every 6 h and analyzed for cell dry mass (CDM), riboflavin, DMRL, acetate, acetoin, and 2,3 butanediol.…”

Section: Growth Conditions and Mediamentioning

confidence: 99%

Biomass recycling from a riboflavin cultivation withB. subtilis: Lysis, extract production and testing as substrate in riboflavin cultivation

Bretz

Ilijevic

Grüneberg

et al. 2006

Biotechnol. Bioeng.

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Autolysis of riboflavin-producing B. subtilis can be induced by pH, lack of carbon source, and the buffer system. Stress factors like temperature shift or oxygen dearth enhance the autolysis process. After cultivation of a riboflavin-producing strain, the pH of the whole culture broth was adjusted to 6.5-7.5. At a temperature of 40 degrees C, autolysis started after 1 h. Adding a defined amount of commercially available endo- and exo-proteases enhanced both auto- and proteo-lysis. Optimization of endo- and exo-protease concentrations and of the time increased the degree of proteolysis. Additionally, the amount of DNA and Protein trapped in the riboflavin crystals could be significantly reduced by autolysis. After autolysis, the cultivation broth was centrifuged and the supernatant was cross-flow filtrated with a cut off of 10 kDa. Using this autolysate instead of yeast extract as a medium component for riboflavin production with B. subtilis, a riboflavin yield of 77% was obtained in comparison with the standard cultivation on yeast extract.

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“…Parallel batch and fed-batch fermentations of Escherichia coli were performed with high parallel reproducibility making use of a standard liquid handler for intermittend substrate and base additions [13]. The successful scale-down into the 10 mL-scale of an industrial fedbatch process for the microbial production of riboflavin with Bacillus subtilis was demonstrated recently [6].…”

Section: Introductionmentioning

confidence: 99%

Fully automated single-use stirred-tank bioreactors for parallel microbial cultivations

Kusterer

Krause

Kaufmann³

et al. 2008

Bioprocess Biosyst Eng

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Single-use stirred tank bioreactors on a 10-mL scale operated in a magnetic-inductive bioreaction block for 48 bioreactors were equipped with individual stirrer-speed tracing, as well as individual DO- and pH-monitoring and control. A Hall-effect sensor system was integrated into the bioreaction block to measure individually the changes in magnetic field density caused by the rotating permanent magnets. A restart of the magnetic inductive drive was initiated automatically each time a Hall-effect sensor indicates one non-rotating gas-inducing stirrer. Individual DO and pH were monitored online by measuring the fluorescence decay time of two chemical sensors immobilized at the bottom of each single-use bioreactor. Parallel DO measurements were shown to be very reliable and independently from the fermentation media applied in this study for the cultivation of Escherichia coli and Saccharomyces cerevisiae. The standard deviation of parallel pH measurements was pH 0.1 at pH 7.0 at the minimum and increased to a standard deviation of pH 0.2 at pH 6.0 or at pH 8.5 with the complex medium applied for fermentations with S. cerevisiae. Parallel pH-control was thus shown to be meaningful with a tolerance band around the pH set-point of +/- pH 0.2 if the set-point is pH 6.0 or lower.

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Scale-down and parallel operation of the riboflavin production process with Bacillus subtilis

Abstract: DanksagungIch möchte mich an dieser Stelle bei all denjenigen bedanken, die auf verschiede-

Cited by 63 publications

References 74 publications

Improving the Clostridium acetobutylicum butanol fermentation by engineering the strain for co-production of riboflavin

Improving the Clostridium acetobutylicum butanol fermentation by engineering the strain for co-production of riboflavin

Biomass recycling from a riboflavin cultivation withB. subtilis: Lysis, extract production and testing as substrate in riboflavin cultivation

Fully automated single-use stirred-tank bioreactors for parallel microbial cultivations

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