**2. Phylogeny and taxonomy of** *Xanthobacter* **spp**

The phylogenetic position of *Xanthobacte*r based on 16S rRNA sequence analysis published in Bergey's Manual of Systematic Bacteriology, showed that genus Xanthobacter is part of phylum Proteobacteria, class Alpha proteobacteria order Rhizobiales family Xanthobacteraceae. However, using phylogenetic trees constructed on the basis of 16S rRNA sequence comparisons, the type strains of *Aquabacter spiritensis* and *Azorhizobium caulinodans* are intermingled with the otherwise well-defined genus cluster *Xanthobacter, Aquabacter* and *Azorhizobium* (both single species genera are recognized as separate genera V and VI within the same family Hyphomicrobiaceae, some of the key properties described for the type species *X. autotrophicus* it is suggested to keep the separate genera names despite the 16S rRNA sequence similarity [22]. The 16S rRNA sequence is more than 98%

#### **Figure 1.**

*Photographs of* X. autotrophicus *(a) macroscopic morphology in a mineral medium without sucrose either ammonium nitrate (MMWSA) under autotrophic conditions after 30 h incubation at 35°C, (b) and (c) microscopic morphology of* X. autotrophicus *according at Gram negative in MMWSA under the same incubation condition (photos from Environmental Laboratory-UMSNH, Sánchez-Yañez et al., 2020).*

similar to those of *X. flavus* and *X. autotrophicus* strains as is showed in **Figure 1** [2, 6, 8, 23, 27]. The morphology and some of the physiological proprieties are different to separate species, supported by the low below 50% DNA-DNA hybridization data as well as tricarboxylic acid or TCA cycle intermediate; (1) synthesis of the water insoluble zeaxanthin dirhamnoside, showed by the yellow colonies; (2) normally to grow chemolithoautotrophically; and (3) able to fix dinitrogen under microaerophilic chemolithoautotrophic or heterotrophic conditions [1, 28]. Other characteristics are given in **Table 1**, *Xanthobacter* is free-living in soil and water as well as root-associated but never noduling, exhibits acetylene reduction as an indirect technique for nitrogen fixing capacity. Other features of *Xanthobacter* a are the h i g h G+C related with some flavobacteria and Cytophaga spp: (1) antibiotic pattern sensitivity [17] (2) positive reaction for catalase, oxidase and phosphatase acid and alkaline types; (3) negative reaction for methyl red, gas from carbohydrates, and the Voges-Proskauer test; and (4) containing ubiquinones Q10 and Q8 like is in *Beijerinckia*, and *Azotobacter*, are important for truly identification of these species, demonstration of the pigment zeaxanthin dirhamnoside and acquisition of the 16S rDNA sequence are important [2, 6, 21].
