A previously developed UV-vis assay method for lignin degradation activity has been developed for use as a spray assay on agar plates. Nine mesophilic strains were isolated using this method from woodland soil incubated in enrichment cultures containing wheat straw lignocellulose: four Microbacterium isolates, two Micrococcus isolates, Rhodococcus erythropolis (all Actinobacteria) and two Ochrobactrum isolates (Alphaproteobacteria). Three thermotolerant isolates were isolated from the same screening method applied at 45°C to samples of composted wheat straw from solid-state fermentation: Thermobifida fusca and two isolates related to uncharacterized species of Rhizobiales and Sphingobacterium (Bacteroidetes), the latter strain showing tenfold higher lignin degradation activity than other isolates. The isolated strains were able to depolymerize samples of size-fractionated high molecular weight and low molecular weight Kraft lignin, and produced low molecular weight metabolites oxalic acid and protocatechuic acid from incubations containing wheat straw lignocellulose.