Production Of Lactobionic Acid Research Articles

Tunable decoupled overproduction of lactobionic acid in Pseudomonas taetrolens through temperature-control strategies

Triggering the switch from growth to lactobionic acid formation in Pseudomonas taetrolens is crucial to attain a high-yield bio-production system. This study elucidates how the growth-coupled and uncoupled lactobionic acid formation ratio can be modulated through the application of temperature-control strategies. Whereas the combination of high initial cell densities with a temperature value of 30 °C stimulated the onset of the lactobionic acid production phase from early cultivation times, temperature-stat conditions below 28 °C and above 30 °C led to a growth-coupled lactobionic acid formation with reduced cell growth. Further implementation of a temperature-control bioprocessing strategy at 28 °C with a pH-shift cultivation at 6.5 resulted in higher lactobionic acid yields (98%), volumetric productivity (2.04 g/L h), and specific productivity rates (1.73 g/g h) within only 24 h. Controlling the culture temperature at 28 °C via either temperature-stat or -shift bioprocessing strategies led to an overproduction of lactobionic acid, overcoming the low-yield mixed-growth-associated production patterns found at temperatures different than 28 °C. This novel temperature-driven approach provides a step forward in the bio-based production of lactobionic acid at high specific production rates and yields by uncoupling completely the production phase from the cell growth.

Microbial amensalism in Lactobacillus casei and Pseudomonas taetrolens mixed culture

Pseudomonas taetrolens has recently been revealed as an effective microbial producer of lactobionic acid from carbohydrates contained in dairy byproducts. In terms of food industrial applications, the implementation of lactobionic acid biosynthesis coupled with the classic bacterial production of lactic acid appears an important goal. This research paper studies the simultaneous fermentation of residual cheese whey by P. taetrolens and Lactobacillus casei to co-produce lactic and lactobionic acids. Experimental data showed the importance of the interactions established between the two microorganisms. Changes in physiology, viability, growth, and productive capacity were tested experimentally. Lactobacillus was not seen to suffer any appreciable stress, but considerable variations were observed in the Pseudomonas behavior presumably owing to inhibitory lactic metabolites, interaction that can be classified as microbial amensalism. As to production, lactic acid remained without significant changes in mixed fermentations, whereas the production of lactobionic acid decreased sharply due to the competitive exclusion of Pseudomonas.

Biotechnological Production of Organic Acids from Renewable Resources.

Biotechnological processes are promising alternatives to petrochemical routes for overcoming the challenges of resource depletion in the future in a sustainable way. The strategies of white biotechnology allow the utilization of inexpensive and renewable resources for the production of a broad range of bio-based compounds. Renewable resources, such as agricultural residues or residues from food production, are produced in large amounts have been shown to be promising carbon and/or nitrogen sources. This chapter focuses on the biotechnological production of lactic acid, acrylic acid, succinic acid, muconic acid, and lactobionic acid from renewable residues, these products being used as monomers for bio-based material and/or as food supplements. These five acids have high economic values and the potential to overcome the "valley of death" between laboratory/pilot scale and commercial/industrial scale. This chapter also provides an overview of the production strategies, including microbial strain development, used to convert renewable resources into value-added products.

Production of lactobionic acid by BMED process using porous P84 co-polyimide anion exchange membranes

Abstract Porous P84 co-polyimide anion exchange membranes are prepared by phase inversion process using isopropanol (IPA) or water as the non-solvent. Membrane area resistances are in the range of 0.7–2.2 Ω cm2, lower than those of commercial anion exchange membrane AMX (2.0–3.5 Ω cm2) and dense P84 membrane (>80 Ω cm2). The ion exchange capacities (IECs) are in the range of 0.6–0.9 mmol/g and the water uptake (WR) values are 100–160%, both of which are higher than those of dense P84 membrane. The porous P84 co-polyimide membranes are utilized in bipolar membrane electrodialysis (BMED) process to produce lactobionic acid (LBA) and NaOH from sodium lactobionic. The pore morphology and IECs influence the output of LBA significantly. Membrane with big tear-like pores shows higher WR value and can produce 0.0555–0.0565 mol/L LBA. Sponge-like pores with IEC of 0.9 mmol/g can yield 0.0724 mol/L LBA after 180 min running under 20 V, while sponge-like pores with IEC of 0.7 mmol/g yield reduced LBA concentration of 0.0593 mol/L, illustrating that higher IEC is in favour of the passage of LB−. The porous P84 membranes have improved efficiency as compared with commercial membrane AMX (0.0451 mol/L) and the dense P84 membrane (merely 0.0162 mol/L) in the production of LBA. Meanwhile, the Na+ leakage through the porous membrane is similar to that of the commercial membrane. Hence, the porous membranes, due to their low resistances, can have high potential for producing organic acid with high molecular weight.

Lactobionic and cellobionic acid production profiles of the resting cells of acetic acid bacteria.

Lactobionic acid was produced by acetic acid bacteria to oxidize lactose. Gluconobacter spp. and Gluconacetobacter spp. showed higher lactose-oxidizing activities than Acetobacter spp. Gluconobacter frateurii NBRC3285 produced the highest amount of lactobionic acid per cell, among the strains tested. This bacterium assimilated neither lactose nor lactobionic acid. At high lactose concentration (30%), resting cells of the bacterium showed sufficient oxidizing activity for efficient production of lactobionic acid. These properties may contribute to industrial production of lactobionic acid by the bacterium. The bacterium showed higher oxidizing activity on cellobiose than that on lactose and produced cellobionic acid.

Simultaneous production of lactobionic and gluconic acid in cheese whey/glucose co-fermentation by Pseudomonas taetrolens

Substrate versatility of Pseudomonas taetrolens was evaluated for the first time in a co-fermentation system combining cheese whey and glucose, glycerol or lactose as co-substrates. Results showed that P. taetrolens displayed different production patterns depending on the co-substrate supplied. Whereas the presence of glucose led to a simultaneous co-production of lactobionic (78g/L) and gluconic acid (8.8g/L), lactose feeding stimulated the overproduction of lactobionic acid from whey with a high specific productivity (1.4g/gh) and yield (100%). Co-substrate supply of glycerol conversely led to reduced lactobionic acid yield (82%) but higher cell densities (1.8g/L), channelling the carbon source towards cell growth and maintenance. Higher carbon availability impaired the metabolic activity as well as membrane integrity, whereas lactose feeding improved the cellular functionality of P. taetrolens. Insights into these mixed carbon source strategies open up the possibility of co-producing lactobionic and gluconic acid into an integrated single-cell biorefinery.

Lactobionic acid production by glucose-fructose oxidoreductase from Zymomonas mobilis expressed in Escherichia coli.

ObjectivesTo use the glucose–fructose oxidoreductase (GFOR) from Zymomonas mobilis and expressed in Escherichia coli for lactobionic acid production by conversion of lactose from whey.ResultsThe highest concentrations of lactobionic acid (3.2 mg ml−1) during oxidation of whey-derived lactose by E. coli was at 24 h. Introduction of GFOR gene from Z. mobilis, into E. coli improved enzyme yields compared to what is obtainable by fermentation of the donor strain. The production of lactobionic acid by E. coli was 2.6-times higher than by Z. mobilis.ConclusionsRecombinants of E. coli overexpressing the GFOR gene from Z. mobilis produced higher amount of lactobionoic acid from whey-derived lactose.

A novel approach to monitor stress-induced physiological responses in immobilized microorganisms

Microbial cell immobilization has long been considered as a potential bioprocessing strategy to increase both microorganisms' tolerance and fitness in fermentation systems. To date, little emphasis has been put on how the entrapped cells respond to the bioprocessing stresses encountered during the cultivation. The present work presents for the first time a methodology to decipher the real health status of the entrapped microorganisms by combining multiparameter flow cytometry with confocal fluorescence microscopy as monitoring tools. Comparison between resting free and immobilized cell-based systems enabled to characterize the spatial-temporal physiological response of entrapped Pseudomonas taetrolens cells during lactobionic acid production in submerged cultivation. Whereas cellular leakage from beads led to planktonic cells that faced a progressive loss of membrane integrity, immobilized cells underwent a prompt stress-induced physiological response featured by the predominance of cellular damaging. Moreover, visualization without matrix de-entrapment through confocal fluorescence microscopy revealed the overtime formation of cellular micro-colonies inside the beads. These micro-colonies comprised a shell made of dead cells, whereas the inward cells remained metabolically active. The proposed approach herein raises the possibility of using flow cytometry and confocal fluorescence microscopy as indicators of microbial cell immobilization, providing further key information on the health status and robustness of entrapped microorganisms.

Production of Lactobionic Acid from its Sodium Salt Solution by Ion-Exchange on a Commercial Strong Acid Resin: Kinetic Data and Modeling

Lactobionic acid (LBA) is a high value added product obtained from lactose oxidation. Since this reaction should be conducted in alkaline media by adequate addition of a Na-, Ca- or K-base, the final product is a lactobionate salt solution instead of the acid form. In this study, the behavior of a strong cation exchange resin (AmberliteTM FPC23 H) for the production of a LBA solution from its sodium lactobionate salt (LBNa), was investigated. The sodium exchange efficiency was evaluated at three different temperatures (5, 25, and 35ºC). The resin exhibited a good performance in the removal of sodium from the LBNa solution. In all cases, sodium concentration in LBA solution was reduced below 5 mg/L. A complete sodium removal was achieved after about 30 min at 5ºC, and after circa 10 min at 25 and 35ºC. Various kinetic models were used for the evaluation of experimental ion-exchange kinetic data. The rate constants, transient capacities, and related correlation coefficients for each kinetic model were calculated and discussed. Results showed that the pseudo-second order and the reversible reaction models adequately describe the experimental kinetic data. The activation energies computed using the rate constants obtained with these models were 50.87 and 36.12 kJ/mol, respectively.

Removal of lactobionic acid by electrodialysis

Lactobionic acid has a number of applications, such as in cosmetic formulations and detergents, as well as in the medical field, where it is used for the preservation of organs destined for transplantation. Previous studies have reported that a promising alternative procedure for the production of lactobionic acid is the biotechnological route, using permeabilized cells of Zymomonas mobilis to produce sorbitol and lactobionic acid from fructose and lactose. However, the acid produced during the process accumulates in the reaction medium, causing enzyme deactivation. It was found that this problem can be avoided by coupling an electrodialysis unit to the reaction vessel, resulting in efficient removal of the acid from the reaction medium and improved the stability of the enzyme. These tests employed a synthetic mixture containing lactobionic acid, sorbitol, lactose, and fructose, and a factorial design was performed to identify the most influential variables. The NaCl concentration in the concentrate stream, together with the potential difference, exerted the greatest effects on the rate of removal of lactobionic acid. In all experiments, the removal efficiency exceeded 95%. The best conditions for the system investigated were a potential of 60 V, and NaCl concentrations of 3 and 25 g L-1 in the concentrate stream and the electrode compartment, respectively.

Optimization of production, purification and lyophilisation of cellobiose dehydrogenase by Sclerotium rolfsii.

BackgroundThe enzyme cellobiose dehydrogenase (CDH) can be used to oxidize lactose to lactobionic acid. As Sclerotium rolfsii is known to be a good producer of CDH, the aim of this paper was to simplify its production and secondly to systematically study its purification aiming for a high yield. Two preservation methods (freezing and freeze-drying) and the influence of several protectants were investigated.ResultsProduction of cellobiose dehydrogenase was optimized leading to a more simplified medium composition. Purification of the enzyme was evaluated by determining breakthrough profiles on different ion exchange (IEX) and hydrophobic interaction (HIC) materials with regard to buffer composition. Highest purification with an acceptable loss during the capture step using IEX was obtained with a Q Sepharose XL medium and a 100 mM sodium acetate buffer at pH 4.5. Subsequent purification using hydrophobic interaction chromatography was done at 1.1 M ammonium sulfate concentration. Purification was moderate, yielding a specific activity of 11.9 U/mg (56% yield). However, as could be shown in a preliminary experiment, purity of the obtained enzyme solution was sufficient for its intended use to oxidize lactose to lactobionic acid. Various sugars and sugar alcohols were investigated to study their protective effect during lyophilisation and freezing at -20°C. Glucose and lactulose could be identified to have a high lyoprotective effect while loss of enzyme activity was high (77%) when using no additives.ConclusionBy simplifying the cultivation medium of Sclerotium rolfsii, the costs of cellobiose dehydrogenase production could be reduced. Simultaneously, CDH production was increased by 21%. The production of lactobionic acid from lactose is possible using partially purified and unpurified enzyme. Storage at -20°C using 50% (w/v) glycerol was considered to be most suited for preservation of the enzyme.

The Utilization of Pseudomonas taetrolens to Produce Lactobionic Acid

Lactobionic acid is a relatively new product derived from lactose oxidation, with high potential applications as a bioactive compound. Conducted experiments confirmed that both the time and temperature influenced the production of lactobionic acid during bioconversion of lactose using the Pseudomonas taetrolens bacteria. The study also investigated the effect of inoculum concentration on the production of lactobionic acid as a result of oxidation of whey-derived lactose. The highest concentration of lactobionic acid during oxidation of whey-derived lactose at a temperature of 30 °C by microorganisms. P. taetrolens was obtained during 50-h oxidation of the medium, which contained 25 % addition of the inoculum, in which the count of live cells was 2.85 × 109 CFU/ml.

English

The focus of this work is on process systems engineering (PSE) methods and tools, and especially on how such PSE methods and tools can be used to accelerate and support systematic bioprocess development at a miniature scale. After a short presentation of the PSE methods and the bioprocess development drivers, three case studies are presented. In the first example it is demonstrated how experimental investigations of the bi-enzymatic production of lactobionic acid can be modeled with help of a new mechanistic mathematical model. The reaction was performed at lab scale and the prediction quality analyzed. In the second example a computational fluid dynamic (CFD) model is used to study mass transfer phenomena in a microreactor. In this example the model is not only used to predict the transient dynamics of the reactor system but also to extract material properties like the diffusion velocities of substrate and product, which is otherwise difficult to access. In the last example, a new approach to the design of microbioreactor layouts using topology optimization is presented and discussed. Finally, the PSE methods are carefully discussed with respect to the complexity of the presented approaches, the applicability with respect to practical considerations and the opportunity to analyze experimental results and transfer the knowledge between different scales.

Production of lactobionic acid by means of a process comprising the catalytic oxidation of lactose and bipolar membrane electrodialysis

Lactobionic acid (LBA) is a high value-added oxidation product of lactose with numerous potential applications in the food, pharmaceutical and chemical industries. This study aimed to investigate the viability of producing LBA from a sodium lactobionate (LBNa) solution obtained by the aerobic oxidation of lactose over gold nanoparticles supported on mesoporous silica, using a three-compartment bipolar membrane electrodialysis (BMED) stack with an effective area of 0.01m2. The results indicated that LBNa was continuously split in LB− (lactobionate anion) and Na+ ions during BMED, which formed LBA and NaOH in the acid and base compartments, respectively. A demineralization rate of about 50% was achieved after applying a voltage difference of 5.0V during 100min followed by 5.5V during 80min, while the LBA concentration increased about 2.5 times in the acid compartment. The specific energy consumption was 0.57kWhkg−1 over the whole stack. Although sodium was found in small amounts (17.5ppm) in the acid compartment, these results show for the first time the viability of producing LBA by means of an integral process comprising the catalytic oxidation of lactose and BMED, with the advantage that the NaOH could be efficiently recovered for its reutilization in further oxidation reactions.

Feeding strategies for enhanced lactobionic acid production from whey by Pseudomonas taetrolens

High-level production of lactobionic acid from whey by Pseudomonas taetrolens under fed-batch fermentation was achieved in this study. Different feeding strategies were evaluated according to the physiological status and fermentation performance of P. taetrolens. A lactobionic acid titer of 164g/L was obtained under co-feeding conditions affording specific and volumetric productivities of 1.4g/gh and 2.05g/Lh, respectively. Flow cytometry assessment revealed that P. taetrolens cells exhibited a robust physiological status, which makes them particularly well-suited for employing concentrated nutrient solutions to further prolong the growth and production phases. Such detailed knowledge of the physiological status has been revealed to be a key issue to further support the development of high-yield lactobionic acid production processes under feeding strategies. The present study has demonstrated the feasibility of P. taetrolens to achieve high-level bio-production of lactobionic acid from whey through fed-batch cultivation, suggesting its major potential for industrial-scale implementation.

Selection method of pH conditions to establish Pseudomonas taetrolens physiological states and lactobionic acid production

Microbial physiological responses resulting from inappropriate bioprocessing conditions may have a marked impact on process performance within any fermentation system. The influence of different pH-control strategies on physiological status, microbial growth and lactobionic acid production from whey by Pseudomonas taetrolens during bioreactor cultivations has been investigated for the first time in this work. Both cellular behaviour and bioconversion efficiency from P. taetrolens were found to be negatively influenced by pH-control modes carried out at values lower than 6.0 and higher than 7.0. Production schemes were also influenced by the operational pH employed, with asynchronous production from damaged and metabolically active subpopulations at pH values lower than 6.0. Moreover, P. taetrolens showed reduced cellular proliferation and a subsequent delay in the onset of the production phase under acidic conditions (pH < 6.0). Unlike cultivations performed at 6.5, both pH-shift and pH-stat cultivation strategies performed at pH values lower than 6.0 resulted in decreased lactobionic acid production. Whereas the cellular response showed a stress-induced physiological response under acidic conditions, healthy functional cells were predominant at medium operational pH values (6.5-7.0). P. taetrolens thus displayed a robust physiological status at initial pH value of 6.5, resulting in an enhanced bioconversion yield and lactobionic acid productivity (7- and 4-fold higher compared to those attained at initial pH values of 4.5 and 5.0, respectively). These results have shown that pH-control modes strongly affected both the physiological response of cells and the biological performance of P. taetrolens, providing key information for bio-production of lactobionic acid on an industrial scale.

Physiological heterogeneity of Pseudomonas taetrolens during lactobionic acid production

Physiological heterogeneity constitutes a critical parameter in biotechnological systems since both metabolite yield and productivity are often hampered by the presence of undesired physiological cell subpopulations. In the present study, the physiological status and functionality of Pseudomonas taetrolens cells were monitored by multiparameter flow cytometry during fermentative lactobionic acid production at the shake-flask and bioreactor scale. In shake-flask fermentation, the onset of the lactobionic acid production phase was accompanied by a progressive loss of cellular metabolic activity, membrane polarization, and membrane integrity concomitantly to acidification. In fact, population dynamics has shown the prevalence of damaged and dead subpopulations when submitted to a pH < 4 from 16 h onwards. Furthermore, fluorescence-activated cell sorting revealed that these sublethally injured cells were nonculturable. In contrast, P. taetrolens cells exhibited a robust physiological status during bioreactor cultivations performed with a pH-shifted strategy at 6.5, remaining predominantly healthy and metabolically active (>96 %) as well as maintaining bioconversion efficiency throughout the course of the fermentation. Additionally, an assessment of the seed culture's physiological robustness was carried out in order to determine the best seed culture age. Results showed that bioreactor culture performance, growth, and lactobionic acid production efficiency were strongly dependent on the physiological heterogeneity displayed by the seed culture. This study provides the most suitable criteria for optimizing lactobionic acid production efficiency through a novel flow cytometric-based approach based on the physiological status of P. taetrolens. It also constitutes a valuable, broad-ranging methodology for the enhancement of microbial bioprocesses involved in the production of secondary metabolites.

Sugar oxidation profiles of membrane-bound dehydrogenase from Acetobacter orientalis

Abstract Previously we reported the production of lactobionic acid by resting cells of the Acetobacter orientalis strain KYG22. In the present study we have prepared membrane fraction of this bacterium and demonstrated that the enzyme responsible for D -glucose and lactose oxidation was present in this fraction, and it effectively oxidized D-glucose and D-allose. The activity against other monosaccharides was relatively low. The enzyme also displayed low activity against disaccharides, with the exception of melibiose.

Optimization of Lactobionic Acid Production byAcetobacter orientalisIsolated from Caucasian Fermented Milk, “Caspian Sea Yogurt”

We have reported that lactobionic acid is produced from lactose by Acetobacter orientalis in traditional Caucasian fermented milk. To maximize the application of lactobionic acid, we investigated favorable conditions for the preparation of resting A. orientalis cells and lactose oxidation. The resting cells, prepared under the most favorable conditions, effectively oxidized 2-10% lactose at 97.2 to 99.7 mol % yield.

Role of dissolved oxygen availability on lactobionic acid production from whey by Pseudomonas taetrolens

The influence of dissolved oxygen availability on cell growth and lactobionic acid production from whey by Pseudomonas taetrolens has been investigated for the first time. Results from pH-shift bioreactor cultivations have shown that high agitation rate schemes stimulated cell growth, increased pH-shift values and the oxygen uptake rate by cells, whereas lactobionic acid production was negatively affected. Conversely, higher aeration rates than 1.5Lpm neither stimulated cell growth nor lactobionic acid production (22% lower for an aeration rate of 2Lpm). Overall insights into bioprocess performance enabled the implementation of 350rpm as the optimal agitation strategy during cultivation, which increased lactobionic productivity 1.2-fold (0.58–0.7g/Lh) compared to that achieved at 1000rpm. Oxygen supply has been shown to be a key bioprocess parameter for enhanced overall efficiency of the system, representing essential information for the implementation of lactobionic acid production at a large scale.