*Article* **A Case of Adaptive Laboratory Evolution (ALE):Biodegradation of Furfural by** *Pseudomonas pseudoalcaligenes* **CECT 5344**

#### **M. Isabel Igeño 1,2,**†**, Daniel Macias 1,**† **and Rafael Blasco 1,2,\***


Received: 30 April 2019; Accepted: 27 June 2019; Published: 29 June 2019

**Abstract:** *Pseudomonas pseudoalcaligenes* CECT 5344 is a bacterium able to assimilate cyanide as a nitrogen source at alkaline pH. Genome sequencing of this strain allowed the detection of genes related to the utilization of furfurals as a carbon and energy source. Furfural and 5-(hydroxymethyl) furfural (HMF) are byproducts of sugars production during the hydrolysis of lignocellulosic biomass. Since they inhibit the yeas<sup>t</sup> fermentation to obtain bioethanol from sugars, the biodegradation of these compounds has attracted certain scientific interest. *P. pseudoalcaligenes* was able to use furfuryl alcohol, furfural and furoic acid as carbon sources, but after a lag period of several days. Once adapted, the evolved strain (R1D) did not show any more prolonged lag phases. The transcriptomic analysis (RNA-seq) of R1D revealed a non-conservative punctual mutation (L261R) in BN5\_2307, a member of the AraC family of activators, modifying the charge of the HTH region of the protein. The inactivation of the mutated gene in the evolved strain by double recombination reverted to the original phenotype. Although the bacterium did not assimilate HMF, it transformed it into value-added building blocks for the chemical industry. These results could be used to improve the production of cost-effective second-generation biofuels from agricultural wastes.

**Keywords:** *Pseudomonas*; bioethanol; furfural; ALE; AraC
