Citation: Rodrigues, C.J.C.; de Carvalho, C.C.C.R. Process Development for Benzyl Alcohol Production by Whole-Cell Biocatalysis in Stirred and Packed Bed Reactors. Microorganisms 2022, 10, 966. https://doi.org/10.3390/ microorganisms10050966 Academic Editors: Irina Ivshina and Elena A. Tyumina Received: 7 April 2022 Accepted: 29 April 2022 Published: 3 May 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). microorganisms Article Process Development for Benzyl Alcohol Production by Whole-Cell Biocatalysis in Stirred and Packed Bed Reactors Carlos J. C. Rodrigues 1,2 and Carla C. C. R. de Carvalho 1,2, * 1 Department of Bioengineering, iBB—Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisbon, Portugal; carlos.junio@tecnico.ulisboa.pt 2 Associate Laboratory i4HB—Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisbon, Portugal * Correspondence: ccarvalho@tecnico.ulisboa.pt; Tel.: +351-21-841-9594 Abstract: The ocean is an excellent source for new biocatalysts due to the tremendous genetic diversity of marine microorganisms, and it may contribute to the development of sustainable industrial processes. A marine bacterium was isolated and selected for the conversion of benzaldehyde to benzyl alcohol, which is an important chemical employed as a precursor for producing esters for cosmetics and other industries. Enzymatic production routes are of interest for sustainable processes. To overcome benzaldehyde low water solubility, DMSO was used as a biocompatible cosolvent up to a concentration of 10% (v/v). A two-phase system with n-hexane, n-heptane, or n-hexadecane as organic phase allowed at least a 44% higher relative conversion of benzaldehyde than the aqueous system, and allowed higher initial substrate concentrations. Cell performance decreased with increasing product concentration but immobilization of cells in alginate improved four-fold the robustness of the biocatalyst: free and immobilized cells were inhibited at concentrations of benzyl alcohol of 5 and 20 mM, respectively. Scaling up to a 100 mL stirred reactor, using a fed-batch approach, enabled a 1.5-fold increase in benzyl alcohol productivity when compared with batch mode. However, product accumulation in the reactor hindered the conversion. The use of a continuous flow reactor packed with immobilized cells enabled a 9.5-fold increase in productivity when compared with the fed-batch stirred reactor system. Keywords: biocatalysis; two-phase biocatalysis; stirred tank reactor; packed bed reactor; whole cells; immobilization; benzaldehyde; benzyl alcohol; marine biocatalyst 1. Introduction Benzyl alcohol is an important aromatic alcohol used as a solvent in inks, paints, glues, and resins [1], in household cleaners and detergents [2], and as a food additive [3]. It is a member of the fragrance structural group aryl alkyl alcohols, being frequently used as a fragrance ingredient in several consumer products such as shampoos, soaps, and cosmetic products [2]. Benzyl alcohol has bacteriostatic and antiseptic properties with modest toxicity, which increases their versatility [2,4]. Benzyl alcohol has also been used as a substrate for the synthesis of esters applied as important building blocks for bulk and commodity chemicals used in pharmaceutical, fragrance, and food industries [5,6]. Industrial production of benzyl alcohol is traditionally made by hydrolysis of benzyl chloride or hydrogenation of benzaldehyde [7]. These processes use non-renewable sub- strates, metal catalysts, high temperatures, and high pressures, and produce by-products with deleterious environmental effects. Microorganisms may also be used for the produc- tion of benzyl alcohol. The conversion of benzaldehyde to benzyl alcohol was undertaken in 1983 by the fungus Rhodotorula muciluginosa immobilized in an ultrafiltration cell [8]. The conversion involved the NADP-oxidoreductase enzyme present in the cells and was used as an example of the detoxication of industrial wastewaters since benzaldehyde commonly Microorganisms 2022, 10, 966. https://doi.org/10.3390/microorganisms10050966 https://www.mdpi.com/journal/microorganisms