Production of B-group vitamins by two Azotobacter strains with phenolic compounds as sole carbon source under diazotrophic and adiazotrophic conditions J.J. Revillas 1 , B. Rodelas 1 , C. Pozo 2 , M.V. Martõ Ânez-Toledo 2 and J. Gonza  lez-Lo  pez 1 1 Departamento de Microbiologõ Âa (Facultad de Farmacia), and 2 Instituto del Agua, Universidad de Granada, Spain 228/3/2000: received 14 March 2000, revised 15 May 2000 and accepted 17 May 2000 J . J . REVILLAS , B . RODELAS , C . POZO , M . V . MARTI  NEZ - TOLEDO AND J . GONZA  LEZ - LO  PEZ . 2000. Azotobacter vinelandii strain ATCC 12837 and A. chroococcum strain H23 (CECT 4435) were able to grow on N-free or NH 4 Cl-amended chemically-de®ned (Burk's) media, with protocatechuic acid (1±2 mmol l 1 ) or sodium p-hydroxybenzoate (1±10 mmol l 1 ) as sole carbon (C) sources. At a concentration of 2 mmol l 1 , both substrates supported nitrogen ®xation (acetylene reduction assay) at similar or higher rates than bacteria grown in control media amended with 2 mmol l 1 sodium succinate as C source. The two strains produced the B-group vitamins niacin, pantothenic acid, thiamine, ribo¯avin and biotin after 72 h of growth in chemically-de®ned media with 2 mmol l 1 protocatechuic acid, sodium p- hydroxybenzoate or sodium succinate as sole C source, either in N-free media or in media amended with 01% NH 4 Cl. Quantitative production of all vitamins was affected by the use of the different C and N substrates. INTRODUCTION Obligate aerobes of the genus Azotobacter are common inhabitants of soils of neutral pH in cold and temperate regions, where they constitute a major fraction of the free- living N 2 -®xing population that keeps nitrogen cycling in the biosphere (Tchan and New 1984). Azotobacter spp. have been widely used as indicators of the effects of xeno- biotics on biological N 2 -®xation in soil (Gonza Âlez-Lo Âpez 1992). In addition, strains of this genus are often able to colonize the rhizosphere and root surfaces of many agrono- mically-important plants and establish, under natural con- ditions, non-endosymbiotic root associations that lead to plant growth promotion (Becking 1992). One of the factors that most in¯uences the growth and biological activities of Azotobacter spp. in their natural habitat is the availability of carbon substrates. Sugars and organic acids supplied in plant root exudates are readily incorporated by these organisms (Tchan and New 1984). However, survival and persistence of Azotobacter in the soil and rhizosphere is believed to be highly dependent on their ability to metabolize simple phenolic compounds, which are commonly present in soils at concentrations of 1±2 mmol l 1 (Wu et al. 1987). Phenolic compounds in the soil come mainly from plant litter decomposition or soil con- tamination by agrochemicals and are toxic for most micro- organisms, but a few genera of bacteria and fungi are able to degrade and use them as carbon sources (Hardisson et al. 1969). Growth and N 2 -®xation supported by some phenolic monomers, at concentrations well over the range commonly found in natural soils, have been reported for A. vinelandii strains (Peterson and Peterson 1988; Moreno et al. 1999). Agronomical exploitation of Azotobacter±plant interac- tions has been successfully tested under laboratory and ®eld conditions (Becking 1992), and the positive effect of inoculation of plants with these bacteria has been attributed to the cession of ®xed N to the plant, enhancement of solu- ble P availability and antibiosis to pathogenic bacteria and fungi (Brown 1974; Jagnow 1987; Abbas and Okon 1993). Production of phytohormones and water-soluble vitamins has also been related to the ability of Azotobacter spp. to enhance crop yielding, as exogenous supply of these biolo- gically-active substances affects plant growth at different levels (Oertli 1987; Okon and Itzigsohn 1995). Phytohormones and vitamins are also linked to the positive in¯uence of these rhizobacteria on the function of rhizo- bial±legume symbiosis and mycorrhiza formation (Brown and Carr 1984; Martõ Ânez-Toledo et al. 1991; Rodelas et al. 1999). Qualitative and quantitative production of B-group vita- mins by A. vinelandii in laboratory media supplied with Correspondence to: Dr J. Gonza Âlez Lopez, Environmental Microbiology Group, Departamento de Microbiologõ Âa (Facultad de Farmacia), Universidad de Granada, 18071 Granada, Spain (e-mail: mrodelas@ugr.es). Journal of Applied Microbiology 2000, 89, 486493 = 2000 The Society for Applied Microbiology