•A new type of porous foam was employed in an adsorption immobilization system.•Morphological changes in the A. niger immobilization system were analyzed by SEM.•The highest concentration of citric ...acid reached 162.7 g/L in immobilization.•Citric acid productivity kept highly in repeated fed-batch cultures over 600 h.
The efficiency of current methods for industrial production of citric acid is limited. To achieve continuous citric acid production with enhanced yield and reduced cost, immobilized fermentation was employed in an Aspergillus niger 831 repeated fed-batch fermentation system. We developed a new type of material (PAF201), which was used as a carrier for the novel adsorption immobilization system. Hydrophobicity, pore size and concentration of carriers were researched in A. niger immobilization. The efficiency of the A. niger immobilization process was analyzed by scanning electron microscopy. Then eight-cycle repeated fed-batch cultures for citric acid production were carried out over 600 h, which showed stable production with maximum citric acid concentrations and productivity levels of 162.7 g/L and 2.26 g L−1 h−1, respectively. Compared with some other literatures about citric acid yield, PAF201 immobilization system is 11.3% higher than previous results. These results indicated that use of the new adsorption immobilization system could greatly improve citric acid productivity in repeated fed-batch fermentation. Moreover, these results could provide a guideline for A.niger or other filamentous fungi immobilization in industry.
•Low viscous DES-water blends can be used in continuous processes for biocatalysis.•The pH control increases the conversion, using DES in a fed-batch reactor.•Fed-batch reactor reached the highest ...concentration of product 33 g/L.•The semicontinuous and continuous bioreactors reached similar productivities.•Biocatalyst stability was high in a packed-bed reactor, without loss for 10 days.
This work explores for the first time the use of Deep Eutectic Solvents (DES) with phosphate buffer 100 mM pH 7 as cosolvent (10% v/v) in biocatalytic reactions in fed-batch and packed-bed bioreactors. The lipase-catalyzed esterification of glycerol and benzoic acid is studied, as it involves two substrates with different polarities (for which DES are needed). In the fed-batch bioreactor, the highest conversion (90%) was obtained at a substrate flow rate of 0.01 mL/min. The fed-batch operation increased the conversion by 59% compared to the batch mode. Regarding productivity, semi-continuous and continuous bioreactors showed analogous results. Upon recirculation of the reaction media in the continuous bioreactor, a conversion of 67% was achieved in 7 cycles of operation. The stability of the biocatalyst in the packed-bed bioreactor decreased only 2% in 10 days, demonstrating the attractiveness that low viscous DES-water mixtures with continuous processes may have.
Methods of producing medium‐chain‐length poly‐3‐hydroxyalkanoate (mcl‐PHA) with high content of the dominant subunit, 3‐hydroxydecanoate (HD), were examined with an emphasis on a high yield of ...polymer from decanoic acid. High HD content was achieved by using a β‐oxidation knockout mutant of Pseudomonas putida KT2440 (designated as P. putida DBA‐F1) or by inhibiting β‐oxidation with addition of acrylic acid (Aa) to wild type P. putida KT2440 in carbon‐limited, fed‐batch fermentations. At a substrate feed ratio of decanoic acid and acetic acid to glucose (DAA:G) of 6:4 g/g, P. putida DBA‐F1 accumulated significantly higher HD (97 mol%), but much lower biomass (8.5 g/L) and PHA (42% of dry biomass) than the wild type. Both biomass and PHA concentrations were improved by decreasing the ratio of DAA:G to 4:6. Moreover, when the substrate feed ratio was further decreased to 2:8, 18 g/L biomass containing 59% mcl‐PHA consisting of 100 mol% HD was achieved. The yield of PHA from decanoic acid was 1.24 (g/g) indicating that de novo synthesis had contributed to production. Yeast extract and tryptone (YET) addition allowed the mutant strain to accumulate 74% mcl‐PHA by weight with 97 mol% HD at a production rate of 0.41 g/L/hr, at least twice that of published data for any β‐oxidation knock‐out mutant. Higher biomass concentration was achieved with Aa inhibition of β‐oxidation in the wild type but the HD content (84 mol%) was less than that of the mutant. A carbon balance showed a marked increase in supernantant organic carbon for the mutant indicating overflow metabolism. Increasing the dominant monomer content (HD) greatly increased melting point, crystallinity, and rate of crystallization.
Carbon‐limited, fed‐batch fermentation can be used to produce mcl‐PHA with high 3‐hydroxydecanoate content in a fadBA knockout mutant of Pseudomonas putida KT2440.
Clostridium butyricum is one of the best 1,3‐propanediol producers due to the nonpathogenic, less byproducts, and energy‐efficient fermentation process. In fermentation process, the relationship ...among substrate, product, and byproducts is intricate and hard to be analyzed. The present study is aimed at establishing a novel kinetic model not only based on biomass, substrate, and 1,3‐propanediol, but also considering the byproduct concentration to describe 1,3‐propanediol fermentation process by C. butyricum. The simulative result of the model fit well with that in the batch fermentation process. Furthermore, the model was also used to predict the result of fed‐batch fermentation process after some modifications. The predicted result of model fit well with the data in experiment when glycerol was controlled at around 10 g/L. Thus, this novel kinetic model could serve as a tool for further optimization of the fermentation process, and could be improved for some other similar processes.
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•Lactulose was synthesized in repeated fed-batch with immobilized β-galactosidase.•Yield and productivity in enzymatic transgalactosylation reactions were assessed.•Fed-batch ...operation allows to achieve high selectivity of synthesis.•Fructose to lactose molar ratio have influence on the accumulated mass of lactulose.•Repeated fed-batch operation allowed obtaining a high efficiency of biocatalyst use.
The effects of the most significant operational variables on reactor performance of fed-batch and repeated fed-batch were evaluated in the lactulose production by enzymatic transgalactosylation. Feed flowrate in the fed stage (F) and fructose to lactose molar ratio (Fr/L) were the variables that mostly affected the values of lactulose yield (YLu), lactulose productivity (πLu) and selectivity of transgalactosylation (SLu/TOS). Maximum YLu of 0.21 g lactulose per g lactose was obtained at 50% w/w inlet carbohydrates concentration (IC) of, 50 °C, Fr/L 8, F 1 mL⋅min−1, 200 IU∙gLactose-1 reactor enzyme load and pH 4.5. At these conditions the selectivity was 7.4, productivity was 0.71 gLu∙g−1∙h-1and lactose conversion was 0.66. The operation by repeated fed batch increases the efficiency of use of the biocatalysts (EB) and the accumulated productivity compared to batch and fed batch operation with the same biocatalyst. EB obtained was 4.13 gLu∙mgbiocatalyst protein−1, 10.6 times higher than in fed-batch.
•A feeding strategy via RQ-control for HCD in S. cerevisiae was developed.•The best performing fermentation process was transferred into a pilot plant.•A repeated fed-batch process with continuous ...cysteine feeding is demonstrated.•Continuous cysteine feeding leads to highest GSH/cysteine yields and biomasses.•Highest performance in GSH production was 1651±43mg per liter broth after 36h.
In the following work a high cell density fed-batch process with Saccharomyces cerevisiae coupled with a high efficient incorporation of cysteine for glutathione (GSH) overproduction was developed. Therefore, a feeding strategy based on the respiratory quotient (RQ) was applied to ensure high biomass (96.1g/l). Furthermore, the optimal cysteine concentration and time of cysteine addition were investigated. Low concentrations of cysteine at late fermentation phases resulted in relatively high incorporation yields of about 0.40mol/mol and maintained the physiology of cultivated yeast. By changing the cysteine feeding from standard single shot to continuous addition, an often observed cell specific toxicity, triggered by high cysteine concentrations, could be prevented and the cysteine incorporation yield (0.54±0.01mol/mol) and GSH content (1650.7±42.8mg/l; 1.76±0.08%) were maximized, respectively. The developed process was transferred from laboratory into pilot plant scale. Further, the reduced cell specific toxicity enabled the development of a repeated fed-batch procedure with a suitable performance concerning cysteine incorporation yield (0.40±0.1mol/mol), biomass (84.2±1.2g/l) and GSH content (1304.7±61.4mg/l).
Advanced fed-batch microbioreactors mitigate scale up risks and more closely mimic industrial cultivation practices. Recently, high throughput microscale feeding strategies have been developed which ...improve the accessibility of microscale fed-batch cultivation irrespective of experimental budget. This review explores such technologies and their role in accelerating bioprocess development. Diffusion- and enzyme-controlled feeding achieve a continuous supply of substrate while being simple and affordable. More complex feed profiles and greater process control require additional hardware. Automated liquid handling robots may be programmed to predefined feed profiles and have the sensitivity to respond to deviations in process parameters. Microfluidic technologies have been shown to facilitate both continuous and precise feeding. Holistic approaches, which integrate automated high-throughput fed-batch cultivation with strategic design of experiments and model-based optimisation, dramatically enhance process understanding whilst minimising experimental burden. The incorporation of real-time data for online optimisation of feed conditions can further refine screening. Although the technologies discussed in this review hold promise for efficient, low-risk bioprocess development, the expense and complexity of automated cultivation platforms limit their widespread application. Future attention should be directed towards the development of open-source software and reducing the exclusivity of hardware.
•Fed-batch micro bioreactors can mimic industrial processes and reduce scale-up risks.•Diffusion- and enzyme-controlled feedings provide an easy-to-setup substrate supply.•Automated liquid handling systems can respond sensitively to process deviations.•Microfluidic systems can be used for precise and continuous feeding.•Customized and advanced systems can be built by additional hardware integration.
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•Sugars derived from energy crops were evaluated for 2,3-butanediol fermentation.•Fed-batch fermentation process was performed to produce high 2,3-butanediol titer.•Mixed sugar medium ...was less efficient due to carbon catabolite repression.•Detoxification of biomass hydrolyzate was needed for efficient fermentation.•High 2,3-butanediol titer (8%) was achieved using single sugar in fed-batch process.
For this study, 2,3-butanediol (BD) fermentation from pure and biomass-derived sugar were optimized in shake-flask and 5-L bioreactor levels using Klebsiella oxytoca ATCC 8724. The results showed that 70g/L of single sugar (glucose or xylose) and 90g/L of mixed-sugar (glucose:xylose=2:1) were optimum concentrations for efficient 2,3-BD fermentation. At optimum sugar concentrations, 2,3-BD productivities were 1.03, 0.64 and 0.50gL−1h−1, and yields were 0.43, 0.36 and 0.35g/g in glucose, xylose and mixed-sugar medium, respectively. The lack of simultaneous utilization of glucose and xylose led to the lowest productivity in the mixed-sugar medium. Detoxification of biomass hydrolyzates was necessary for efficient 2,3-BD fermentation when sugar concentrations in the medium was 90g/L or higher, but not with sugar concentrations of 30g/L or less. A fed-batch fermentation using glucose medium led to an increase 2,3-BD titer to 79.4g/L and yields 0.47g/g, while productivity decreased to 0.79gL−1h−1. However, the fed-batch process was inefficient using mixed-sugar and biomass hydrolyzates because of poor xylose utilization. These results indicated that appropriate biomass processing technologies must be developed to generate separate glucose and xylose streams to produce high 2,3-BD titer from biomass-derived sugar using a fed-batch process.
