Effects of bacterial lignin peroxidase on organic carbon mineralization in soil, using recombinant Streptomyces strains

Abstract

To study the effects of bacterial lignin peroxidase ALip-P3 of Streptomyces viridosporus T7A on the rate of organic carbon turnover in soil, purified lignin peroxidase, with and without addition of H2O2, was added to sterile and nonsterile silt loam soil. Recombinant Streptomyces lividans strains expressing plasmid-encoded ALip-P3 were also inoculated into the soil. Carbon mineralization was monitored by measuring the rate of CO2 evolution from the soil. In sterile soil, lignin peroxidase addition altered carbon turnover, by increasing the CO2 evolution rate above the near-zero rate of sterile, uninoculated soil. H2O2, when added alone, had no effect, and its addition in combination with peroxidase gave results similar to peroxidase alone. This effect was also observed upon addition of lignin peroxidase to sterile soil already inoculated with S. viridosporus T7A. The increases in soil CO2 evolution rates were also observed in experiments using nonsterile soil. However, results showed more variation, and the effect was shorter lived as a result of lessened peroxidase stability. Three recombinant S. lividans strains expressing the ALip-P3 gene in plasmid pIJ702.LP were also inoculated into soil. There were no significant differences in CO2 evolution rates for sterile soil inoculated with recombinants as compared with sterile soil inoculated with wild-type S. lividans strains. However, in nonsterile soil, addition of the recombinants caused a significantly greater increase in the CO2 evolution rate as compared with the corresponding wild types or S. viridosporus T7A. The effect was short lived, lasting about 5 days. Both the recombinant and wild-type Streptomyces survived in the soil for at least 30 days, and pIJ702.LP was stable in the recombinants in soil. Plasmid pIJ702.LP was transformed into three mutants of S. viridosporus T7A that lacked lignin peroxidase. Plasmid-expressing transformants regained the ability to produce lignin peroxidase. The results show that addition of lignin peroxidase ALip-P3 to soil transiently enhanced the short-term rate of carbon mineralization in the soil. The enhancement was lignin peroxidase specific, since substitution of horseradish peroxidase for lignin peroxidase in the soil addition studies resulted in no enhancement of CO2 evolution. In addition, pIJ702.LP-expressing S. lividans strains also caused the effect, which was significant only in nonsterile soil. Thus, lignin peroxidase ALip-P3 appears to affect the short-term turnover rate of lignin-derived organic carbon in soil, and normal, low lignin peroxidase concentrations in soil may limit the initial turnover rate of lignified plant residues in soil. This is the first report of a genetically engineered microorganism having a measurable effect on a biogeochemical cycle in soil. Key words: Streptomyces, recombinant, lignin, peroxidase, soil.