Sucrose Hydrolysis in a Continuous Packed-Bed Reactor with Auto-immobilise Aspergillus niger Biocatalyst Obtained by Solid-State Fermentation.

Invertase from Aspergillus niger C28B25 was produced by solid-state fermentation (SSF). Fermented solids were used directly as a biocatalyst for batch and continuous hydrolysis of sucrose in a packed-bed reactor under different operational conditions with various temperatures, sucrose concentrations, and feed flow rates. The SSF allowed obtaining a biocatalyst with an invertase activity of 82.2 U/g db. The biocatalyst maintained its activity in the range of 40 to 70 °C for at least 70 h of continuous operation. In a 20-mL packed bed reactor, the highest hydrolysis rate (12.3 g/g db h) was obtained at 40 °C with 2 M sucrose. Continuous hydrolysis in 20-mL and 200-mL reactors at 60 °C led to sucrose hydrolysis above 60% (8.5 residence times) and above 55% (4.5 residence times), respectively. The auto-immobilised biocatalyst produced by SSF without recovery, purification, and immobilisation stages offers an economical alternative for developing accessible biocatalysts that can be applied in batch or continuous sucrose hydrolysis processes. This study shows the potential of biocatalyst production by SSF for other enzymatic systems.

Continuous ethyl oleate synthesis by lipases produced by solid-state fermentation by Rhizopus microsporus.

Lipases produced by solid-state fermentation were used directly as biocatalysts for continuous synthesis of ethyl oleate in a continuously stirred tank reactor. The effect of biocatalyst reutilisation, molar ratio of substrates, agitation rate and feed rate on the esterification of oleic acid with ethanol were investigated. The catalyst maintained 90% conversion for four batch cycles with a 1:2 molar ratio (oleic acid:ethanol). Mechanical agitation at 200 and 300 rpm during 12 h of continuous reaction did not affect the biocatalytic conversion, allowing substrate conversions greater than 90% that were obtained with 50 mM oleic acid at a molar ratio of 1:2 during 14 h reaction. In contrast, substrate conversion was 70% with 100 mM oleic acid at a flow rate of 2 mL/min during 25 h of reaction. These results are promising and offer a technical alternative for the development of accessible biocatalysts that can be used in continuous operations.

Improvement of covalent immobilization procedure of ß-galactosidase from Kluyveromyces lactis for galactooligosaccharides production: Modeling and kinetic study.

Galactooligosaccharides (GOS) are prebiotics produced from lactose through an enzymatic reaction. Employing an immobilized enzyme may result in cost reductions; however, the changes in its kinetics due to immobilization has not been studied. This study experimentally determined the optimal reaction conditions for the production of GOS from lactose by ß-galactosidase (EC 3.2.1.23) from Kluyveromyces lactis covalently immobilized to a polysiloxane-polyvinyl alcohol (POS-PVA) polymer activated with glutaraldehyde (GA), and to study the transgalactosylation kinetics. Yield immobilization was 99 ± 1.1% with 78.5 ± 2.4% enzyme activity recovery. An experimental design 24 with 1 center point and 2 replicates was used. Factors were lactose [L], enzyme concentration [E], pH and temperature (T). Response variables were glucose and galactose as monosaccharides [G1], residual lactose [Lac]r and GOS as disaccharides [G2] and trisaccharides [G3]. Best conditions were pH 7.1, 40 °C, 270 gL-1 initial lactose concentration and 6 U mL-1 enzyme concentration, obtaining 25.46 ± 0.01 gL-1 yield of trisaccharides. Although below the HPLC-IR detection limit, tetrasaccharides were also identified after 115 min of reaction. The immobilization protocol was then optimized by diminishing total reactant volumes : support ratio, resulting in improved enzyme activity synthesizing 43.53 ± 0.02 gL-1 of trisaccharides and 13.79 ± 0.21 gL-1 of tetrasaccharides, and after four cycles remaining relative activity was 94%. A reaction mechanism was proposed through which a mathematical model was developed and rate constants were estimated, considering a pseudo steady-state hypothesis for two concomitant reactions, and from this simplified analysis, the reaction yield could eventually be improved. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1568-1578, 2017.

Lactase production by solid-state cultivation of Kluyveromyces marxianus CDBBL 278 on an inert support: effect of inoculum, buffer, and nitrogen source.

A study was carried out to select the conditions for cultivation of Kluyveromyces marxianus CDBBL 278 in solid-state culture (SSC) using polyurethane foam (PUF) as an inert support. PUF was impregnated with culture media containing lactose (50 g/L) as the carbon and energy source. Evaluation of culture parameters during different growth phases was carried out by respirometry. The effect of inoculum level, buffer capacity of the medium, and nitrogen source upon the yield of biomass on lactose (Yx/s) and production of lactase and inulinase was investigated. The highest lactase titre was achieved with an inoculum level of 1 x 10(7) cells per gram of wet matter (gwm) and 20% of the total nitrogen source provided as urea. The best biomass yield (0.37) was obtained when less than 40% of the total nitrogen was provided as urea. Using potassium phosphate allowed 90% substrate consumption in 30 h. In the best conditions, intracellular lactase and extracellular inulinase activities of 1147.7 IU/gX and 241.6 IU/gX were obtained, respectively, with a lag phase of 13.8 h and a rate of respiratory activity (microCO2) of 0.23 +/- 0.01 h(-1). To our knowledge, this is the first report on lactase production by K. marxianus CDBBL 278 in SSC. This study gives basic information about biomass yield and enzyme production using lactose as the sole carbon source in SSC on an inert support.