2,3-Butanediol plus acetoin obtention by Enterobacter aerogenes ATCC 13048: inhibition by target products and cells reuse during fed-batch cultivation.

2,3-Butanediol (2,3-BD) is a highly valued building block, and optimizing its production by fermentation, particularly with crude glycerol, is crucial. Enterobacter aerogenes is a key microorganism for this process; however, there are limited studies addressing the inhibition effects of products and by-products on 2,3-BD production. This study investigates these inhibition effects to maximize 2,3-BD production. Final concentrations of 2,3-BD plus acetoin reached 89.3, 92.7, and 71.1 g.L-1 with productivities of 1.22, 1.69, and 0.99 g.L-1.h-1 in pure glycerol, glucose, and crude glycerol media, respectively. Acetic acid was the main by-product, with concentrations ranging from 10 to 15 g.L-1. The reinoculation of E. aerogenes cells highlighted the strong effect of 2,3-BD and acetic acid on microbial growth and metabolism, with the cultivation environment exerting selective pressure. Notably, cells reuse enhanced performance in crude glycerol media, achieving a specific productivity in relation to biomass (YP/X) of 9.18 g.g-1; about 25% higher than in fed-batch without cells reuse. By combining results from two fed-batch cycles, the total final concentration of 2,3-BD plus acetoin reached 99.4 g.L-1, alongside a 33% reduction in total acetic acid production with reused cells.

Medium composition and aeration to high (R,R)-2,3-butanediol and acetoin production by Paenibacillus polymyxa in fed-batch mode.

Concerning the potential application of the optically active isomer (R,R)-2,3-butanediol, and its production by a non-pathogenic bacterium Paenibacillus polymyxa ATCC 842, the present study evaluated the use of a commercial crude yeast extract Nucel®, as an organic nitrogen and vitamin source, at different medium composition and two airflows (0.2 or 0.5 vvm). The medium formulated (M4) with crude yeast extract carried out with the airflow of 0.2 vvm (experiment R6) allowed for a reduction in the cultivation time and kept the dissolved oxygen values at low levels until the total glucose consumption. Thus, the experiment R6 led to a fermentation yield of 41% superior when compared to the standard medium (experiment R1), which was conducted at airflow of 0.5 vvm. The maximum specific growth rate at R6 (0.42 h-1) was lower than R1 (0.60 h-1), however, the final cell concentration was not affected. Moreover, this condition (medium formulated-M4 and low airflow-0.2 vvm) was a great alternative to produce (R,R)-2,3-BD at fed-batch mode, resulting in 30 g.L-1 of the isomer at 24 h of cultivation, representing the main product in the broth (77%) and with a fermentation yield of 80%. These results showed that both medium composition and oxygen supply have an important role to produce 2,3-BD by P. polymyxa.

High-sodium maltobionate production by immobilized Zymomonas mobilis cells in polyurethane.

The purpose of this study was the production of maltobionic acid, in the form of sodium maltobionate, by Z. mobilis cells immobilized in polyurethane. The in situ immobilized system (0.125-0.35 mm) was composed of 7 g polyol, 3.5 g isocyanate, 0.02 g silicone, and 7 g Z. mobilis cell, at the concentration of 210 g/L. The bioconversion of maltose to sodium maltobionate was performed with different cell concentrations (7.0-9.0 gimobilized/Lreaction_medium), temperature (30.54-47.46 °C), pH (5.55-7.25), and substrate concentration (0.7-1.3 mol/L). The stability of the immobilized system was evaluated for 24 h bioconversion cycles and storage of 6 months. The maximum concentration of sodium maltobionate was 648.61 mmol/L in 34.34 h process (8.5 gdry_cell/Lreaction_medium) at 39 °C and pH 6.30. The immobilized system showed stability for 19 successive operational cycles of 24 h bioconversion and 6 months of storage, at 4 °C or 22 °C.

High lactobionic acid production by immobilized Zymomonas mobilis cells: a great step for large-scale process.

Lactobionic acid and sorbitol are produced from lactose and fructose in reactions catalyzed by glucose-fructose oxidoreductase and glucono-δ-lactonase, periplasmic enzymes present in Zymomonas mobilis cells. Considering the previously established laboratory-scale process parameters, the bioproduction of lactobionic acid was explored to enable the transfer of this technology to the productive sector. Aspects such as pH, temperature, reuse and storage conditions of Ca-alginate immobilized Z. mobilis cells, and large-scale bioconversion were assessed. Greatest catalyst performance was observed between pH range of 6.4 and 6.8 and from 39 to 43 °C. The immobilized biocatalyst was reused for twenty three 24-h batches preserving the enzymatic activity. The activity was maintained during biocatalyst storage for up to 120 days. Statistically similar results, approximately 510 mmol/L of lactobionic acid, were attained in bioconversion of 0.2 and 3.0 L, indicating the potential of this technique of lactobionic acid production to be scaled up to the industrial level.

Correction to: High lactobionic acid production by immobilized Zymomonas mobilis cells: a great step for large-scale process.

In the original publication, table captions were incorrectly published. The correct captions are given here.

Assessment of different systems for the production of aldonic acids and sorbitol by calcium alginate-immobilized Zymomonas mobilis cells.

Equimolar amounts of lactobionic acid and sorbitol may be obtained in a reaction catalyzed by the enzymes glucose-fructose oxidoreductase and glucono-δ-lactonase, which are found in the periplasm of Zymomonas mobilis. These reactions are generally conducted using immobilized bacterial cells, and the cell treatment and immobilization steps are costly and time-consuming. This study evaluated alternatives to simplify the preparation of calcium alginate-immobilized biocatalyst and its application in different operation modes and types of reactors. It was possible to eliminate cell permeabilization with cetyltrimethylammonium bromide, and the reticulation of Z. mobilis cells with glutaraldehyde sufficed to inhibit the fermentative metabolism of carbohydrates by the bacterium, with accumulation of bioconversion products. When the process was carried out in a mechanically stirred reactor in batch mode, 530 mmol L- 1 of products were obtained in 24 h. The process was also tested in fed-batch mode so as to use of a larger amount of lactose, since it could not be used in the batch because of its low solubility in water. Under this condition, final products concentration reached 745 mmol L- 1 within 42 h. Similar results were obtained for reactions conducted in a pneumatically stirred reactor in batch and fed-batch modes, proving the potential use of this process in several industrial settings.

Purification, bioactivity and application of maltobionic acid in active films.

The objective of this study was to purify sodium maltobionate using Zymomonas mobilis cells immobilized in situ on flexible polyurethane (PU) and convert it into maltobionic acid for further evaluation of bioactivity (iron chelating ability, antibacterial potential and cytoprotection) and incorporation into films based on cassava starch, chitosan, and cellulose acetate. Sodium maltobionate exhibited a purity of 98.1% and demonstrated an iron chelating ability of approximately 50% at concentrations ranging from 15 to 20 mg mL-1. Maltobionic acid displayed minimal inhibitory concentrations (MIC) of 8.5, 10.5, 8.0, and 8.0 mg mL-1 for Salmonella enterica serovar Choleraesuis, Escherichia coli, Staphylococcus aureus, and Listeria monocytogenes, respectively. Maltobionic acid did not exhibit cytotoxicity in HEK-293 cells at concentrations up to 500 µg mL-1. Films incorporating 7.5% maltobionic acid into cassava starch and chitosan demonstrated inhibition of microbial growth, with halo sizes ranging from 15.67 to 22.33 mm. These films had a thickness of 0.17 and 0.13 mm, water solubility of 62.68% and 78.85%, and oil solubility of 6.23% and 11.91%, respectively. The cellulose acetate film exhibited a non-uniform visual appearance due to the low solubility of maltobionic acid in acetone. Mechanical and optical properties were enhanced with the addition of maltobionic acid to chitosan and cassava films. The chitosan film with 7.5% maltobionic acid demonstrated higher tensile strength (30.3 MPa) and elongation at break (9.0%). In contrast, the cassava starch film exhibited a high elastic modulus (1.7). Overall, maltobionic acid, with its antibacterial activity, holds promise for applications in active films suitable for food packaging. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03879-3.

Production of organic acids by periplasmic enzymes present in free and immobilized cells of Zymomonas mobilis.

In this work the periplasmic enzymatic complex glucose-fructose oxidoreductase (GFOR)/glucono-δ-lactonase (GL) of permeabilized free or immobilized cells of Zymomonas mobilis was evaluated for the bioconversion of mixtures of fructose and different aldoses into organic acids. For all tested pairs of substrates with permeabilized free-cells, the best enzymatic activities were obtained in reactions with pH around 6.4 and temperatures ranging from 39 to 45 °C. Decreasing enzyme/substrate affinities were observed when fructose was in the mixture with glucose, maltose, galactose, and lactose, in this order. In bioconversion runs with 0.7 mol l(-1) of fructose and with aldose, with permeabilized free-cells of Z. mobilis, maximal concentrations of the respective aldonic acids of 0.64, 0.57, 0.51, and 0.51 mol l(-1) were achieved, with conversion yields of 95, 88, 78, and 78 %, respectively. Due to the important applications of lactobionic acid, the formation of this substance by the enzymatic GFOR/GL complex in Ca-alginate-immobilized cells was assessed. The highest GFOR/GL activities were found at pH 7.0-8.0 and temperatures of 47-50 °C. However, when a 24 h bioconversion run was carried out, it was observed that a combination of pH 6.4 and temperature of 47 °C led to the best results. In this case, despite the fact that Ca-alginate acts as a barrier for the diffusion of substrates and products, maximal lactobionic acid concentration, conversion yields and specific productivity similar to those obtained with permeabilized free-cells were achieved.

Sodium, potassium, calcium lactobionates, and lactobionic acid from Zymomonas mobilis: A novel approach about stability and stress tests.

The bioproduction of lactobionic acid and its salts can be performed by enzymatic complex glucose-fructose oxidoreductase (GFOR) and glucono-δ-lactonase (GL) of Zymomonas mobilis. Considering the applicability of these compounds in pharmaceutical area, the aim of this study was to assess the accelerated and long-term stability studies of sodium, potassium, calcium lactobionate, and lactobionic acid. Furthermore, stress tests were performed to evaluate the stability against pH, temperature and oxidation. The samples submitted to degradation tests were analyzed by high-performance liquid chromatography (HPLC) and high-resolution mass spectrometry analysis (HRMS-ESI-QTOF). Sodium, potassium, and calcium lactobionate were stable for six months of analyses considering the accelerated (40 °C and 75% RH) and long-term (30 °C and 75% RH) stability studies. The presence of lactobiono-δ-lactone and a significant increase in moisture were observed for both biosynthesized and commercially available lactobionic acid samples. Against the forced degradation tests, all the lactobionate salts and lactobionic acid showed to be stable upon alkaline and acid pH conditions, at 60 and 80 °C, and also against UV light exposition. Furthermore, the presence of lactobiono-δ-lactone form was observed in lactobionic acid samples. However, the degradation of both lactobionic acid and lactobionate salts was evident in the presence of hydrogen peroxide. This degradation kinetic profile suggests, that lactobionate salts follows a zero-order reaction model and lactobionic acid follows a second-order kinetic. The MS analysis of the main degradation product suggests a molecular formula C11H20O10 resulting from the oxidative decarboxylation. This report brings an amount of results as contribution to the scarce information regarding the chemical and physical-chemical stability of sodium, potassium, calcium lactobionate, and lactobionic acid. These data may be useful and serve as reference, in view of the multipurpose applications of the cited compounds.

Quantification of lactobionic acid and sorbitol from enzymatic reaction of fructose and lactose by high-performance liquid chromatography.

Experimental conditions for complete separation and quantification of mixtures containing lactobionic acid, sorbitol, lactose and fructose are discussed for the first time. These mixtures appear in the enzymatic bioconversion of fructose and lactose catalyzed by glucose-fructose oxidoreductase (GFOR) and glucono-delta-lactonase (GL) enzymes of Zymomonas mobilis cells. The high-performance liquid chromatography (HPLC) separation was carried out in a strong cation ion exchange resin (hydrogen form) based on a copolymer of styrene divinylbenzene (PS-DVB). A stationary phase of beta-cyclodextrin was also evaluated. An efficient separation was obtained with PS-DVB column eluted with sulfuric acid 0.450 mM solutions (pH 3.0-3.2) at 75 degrees C. The formation of lactones was observed, which is associated with the dissolution of lactobionic acid crystals; however, by dissolving the lactobionic acid crystals on alkaline calcium hydroxyde solution in equimolar ratio a single lactobionic acid chromatographic peak without lactobionolactone is obtained.

Production and characterization of endo-polygalacturonase from Aspergillus niger in solid-state fermentation in double-surface bioreactor

Endo-polygalacturonase (endo-PG) production by Aspergillus niger T0005/007-2 in solid medium with 170 mm of height was evaluated in a cylindrical double surface bioreactor in 96-h experiments. Cell concentration close to 92 mg.g -¹ dm (mg per g of dry medium) in the standard condition (static) was achieved, whereas in tests under forced aeration of 1.4 and 2.8 L.min-1. Kg-1 mm (L of air per minute per Kg of moist medium) and with the central shaft fungal biomass attained approximately 100 mg.g-1 dm. Superior endo-PG activity was obtained with the central-shaft system, 78 U.g-1 dm (units per g of dry medium). Forced aeration and pressure pulse showed no positive effect on the production of endo-PG, 45 U.g-1 dm and 28 U.g-1 dm, respectively. None of the conditions evaluated was efficient for medium temperature control. Endo-PG was stable up to 40ºC. The activity decreased in 50 percent after 120 minutes at 50ºC, which is a temperature normally found during this process.

Influence of medium composition and pH on the production of polygalacturonases by Aspergillus oryzae

Um meio líquido contendo farelo de trigo, sais e fonte de indutor (pectina) foi definido para a produção de exo e endo-poligalacturonases por Aspergillus oryzae CCT3940. A indução por pectina purificada foi significativamente maior que a observada com cascas de cítricos. O crescimento de A. oryzae é favorecido por valores de pH próximos a 4, embora uma queda até valor em torno de 3 seja necessária para a produção das enzimas. Posteriormente, atividades decrescentes foram observadas com a subida normal do pH até próximo à neutralidade. As maiores atividades foram alcançadas quando o processo foi iniciado em pH 4 e controlado quando decresceu até níveis ligeiramente abaixo de 3 (159 unidades.mL-1 para endo-PG, em 83 h, e 45 unidades.mL-1, em 64 h, para exo-PG), com a perda de atividades sendo drasticamente reduzida. Os melhores valores de pH e temperatura para a ação de exo-PG (4,5/57°C) e endo-PG (4,3/40ºC) foram estimados.