Three distinct phases of isoprene formation during growth and sporulation of Bacillus subtilis.

Isoprene (2-methyl-1,3-butadiene) is a volatile hydrocarbon produced by a diverse group of organisms including bacteria, marine algae, animals, and a wide variety of plants (12, 13, 16, 23), sometimes in relatively large amounts. For example, global isoprene emission from green plants is estimated to be about 500 million tons per year (10), an amount which is comparable to global methane emissions of about 510 million tons per year (7). Surprisingly little is known about the biochemical rationale for isoprene production in any biological system (4, 14, 21). Our discovery of isoprene formation in bacteria (12) has created the possibility of studying isoprene biosynthesis at the physiological, biochemical, and genetic levels in prokaryotic systems. A wide variety of bacterial species have been shown to produce isoprene, including both gram-positive and gram-negative strains (12, 23). Bacillus species were shown to be the most active isoprene producers on a variety of growth media (12). Here, we describe a more detailed exploration of Bacillus subtilis 6051 as an experimental system for studying isoprene biosynthesis and show that this wild-type strain exhibits a unique pattern of isoprene production during cellular growth and sporulation. B. subtilis isoprene formation during aerobic growth. With a stirred fermentor in which a variety of growth conditions (pH, aeration, and temperature) could be closely monitored and controlled, the production of isoprene was measured over the complete life cycle of B. subtilis. Wild-type B. subtilis 6051 (Marburg strain) was obtained from the American Type Culture Collection (Manassas, Va.) and maintained on AB3 plates (17.5 g of antibiotic medium 3 and 15 g of agar per liter; Difco). Each fermentor experiment consisted of growing a bacterial culture in 1 liter of F medium, a glucose-tryptone-salts medium (see legend to Fig. 1), with a BioFlo 2000 fermentation system (New Brunswick Scientific). Besides monitoring the pH and dissolved oxygen of the culture, we also measured the cell growth (optical density at 600 nm [OD600]), spore formation, isoprene production, and various extracellular metabolites in an attempt to determine how growth and differentiation affect isoprene formation. Heat-resistant spores were measured in aliquots removed from the fermentor and heated at 80°C for 20 min. Dilutions of these samples in sterile water were then plated on NB plates (8 g of nutrient broth and 15 g of agar per liter; Difco), and single colonies were counted following overnight incubation at 37°C. Isoprene in the exit gas from the fermentor was analyzed with a gas chromatography (GC) system which is highly sensitive to isoprene (8, 12). Isoprene production rates (nanomoles per liter of oxygen) were calculated by converting GC area units to nanomoles of isoprene via a standard isoprene concentration calibration curve. Glucose was measured in cell culture extracts with an assay kit (glucose oxidase-peroxidase; Sigma Chemical). Acetoin production was analyzed by high-performance liquid chromatography (HPLC) analysis of cell culture extracts which had been derivatized with 2,4-dinitrophenylhydrazine, as described in detail elsewhere (22). For these experiments, the inoculum was a 100-ml precul