Quantitative Assessment of Factors Affecting the Recovery of Indigenous and Released Thermophilic Bacteria from Compost

Abstract

Thermophilic actinomycetes and bacilli were recovered from mushroom compost by conventional dilution plating and sedimentation chamber-Andersen sampler methods. Excessive growth of thermophilic bacilli on dilution plates accounted for the poor recovery and limited diversity of actinomycete colonies, and this result was largely unaffected by the use of modified extraction procedures and diluents. Assessment of the actinomycete population was more successfully achieved by applying the sedimentation chamber method, by using selective media, or both. Background resistance of the compost microflora to selective agents (kanamycin, novobiocin, tetracycline, thiostrepton, and NaCl) was extremely varied, but both actinomycetes and bacilli were particularly sensitive to tetracycline. The selective isolation of Thermoactinomyces spp. and Thermomonospora chromogena by novobiocin and kanamycin, respectively, was shown to be reproducible, and the use of high concentrations of kanamycin resulted in the isolation of a novel group of unidentified thermophilic actinomycetes. Comparison of nonselective nutrient media demonstrated that the nutrient-rich protoplast regeneration medium R5 was surprisingly efficient for actinomycete recovery. This medium was found to be particularly appropriate for the recovery of Saccharomonospora viridis BD125, introduced as spores into both sterile and fresh samples of mushroom compost. This stable pigmented variant of the S. viridis strains indigenous to compost was released at concentrations of up to 10 7 spores g of compost −1 in order to provide information for future experiments on the release and recovery of genetically manipulated strains. The detection limits for this strain were in the region of 10 2 g −1 from sterilized compost but only 10 5 g −1 from nonsterile compost. These figures correspond to mean recovery efficiencies of approximately 70% (sterilized compost) and 53% (fresh compost) of viable spores released. Further improvements in the detection and recovery of S. viridis strains released into compost should be achieved by the introduction of selectable markers developed from this information on the antibiotic resistance profile of the indigenous compost microflora.