Phenanthrene-Degrader Community Dynamics in Rhizosphere Soil from a Common Annual Grass

Enhanced rates of phenanthrene biodegradation were observed in rhizosphere soils (17.2 and 15.5 mg/kg/d for initial and re-spiked additions, respectively) planted with slender oat (Arena barbara Pott ex Link) compared with unplanted bulk soil controls (12.4 and 10.7 mg/ kg/d). Soil microbial populations were characterized using a modified most probable number (MPN) method to determine quantitative shifts in heterotrophic and phenanthrene degrader communities while principal component analysis (PCA) of fatty acid methyl ester (FAME) data from isolated phenanthrene degraders was used to identify qualitative differences and degrader community diversity. The average heterotrophic bacterial population over time was about three times larger in rhizosphere soil than in bulk soil while phenanthrene degrading populations increased by as much as an order of magnitude between 24 and 28 days after planting (DAP). Thus, phenanthrene degraders were selectively enriched in rhizosphere soil compared with bulk soil. The greatest selection for degraders occurred during the later stages of plant development from 24 to 32 DAP. A PCA plot of the FAME data from phenanthrene degrader isolates indicated that the rhizosphere degraders were less diverse than bulk soil degraders. These results give us some insight into the mechanisms responsible for enhanced biodegradation and selective degrader enrichment in rhizosphere soils. T I~E rhizosphere, defined as the volume of soil under the influence of the plant root, is a dynamic environment characterized by increased microbial community size and activity compared with the bulk soil (Curl and Truelove, 1986). Recently, researchers have observed that the rhizosphere and its associated properties enhance degradation of pollutants. Investigators have been able to determine that (i) degradation and mineralization of a variety of environmental contaminants is enhanced in the rhizosphere (Hsu and Bartha, 1979; Crowley et al., 1996; G0nther et al., 1996; Narayanan et al., 1995; Federle and Schwab, 1989; Sahu et al., 1990; Sandmann and Loos, 1984; Anderson et al., 1995), (ii) various plant species have different impacts on pollutant degradation rates in rhizosphere soils (Aprill and Sims, 1990; Knaebel and Vestal, 1992; Anderson et al., 1993), and (iii) establishment of rhizosphere microbial communities need not precede contamination in order for enhanced biodegradation to occur (Anderson and Walton, 1995). While a substantial amount of work has demonstrated that the rhizosphere enhances bioremediation of many environmental contaminants, there is little information available on the mechanisms of enhancement. Polycyclic aromatic hydrocarbons (PAHs) are multiRyan K. Miya and Mary K. Firestone, Dep. of Environmental Science, Policy, and Management, Ecosystem Sciences Division, 151 Hilgard Hall #3110, Univ. of California at Berkeley, Berkeley, CA 947203110. Received 24 Feb. 1999. *Corresponding author (rmiya@ nature.berkeley.edu) Published in J. Environ. Qual. 29:584-592 (2000). ringed organic compounds that are widely distributed in the environment. Fossil fuel combustion and industrial processing often result in PAH contamination of soils and the environment. Plant stimulation of soil microbes resulting in enhanced PAH biodegradation has been found by using a variety of grass species (Aprill and Sims, 1990). Grasses generally provide a dense rooting area conducive to microbial activity enhancement and moderate rooting depths that influence treatment up to 3 m below the soil surface (Aprill and Sims, 1990). Using selected prairie grass species, benz(a)anthracene, chrysene, benzo(a)pyrene, and dibenz(a,h)anthracene were found at consistently lower concentrations in vegetated soils compared with unvegetated controls after 59 d and significantly lower concentrations were found after 151 d (Aprill and Sims, 1990). In a greenhouse study using alfalfa (Medicago sativa subsp, sativa), fescue ( Festuca ), sudangrass [Sorghum drummondii (Nees ex Steud.) Millsp. & Chase], and switchgrass (Panicum virgatum L.), the PAHs anthracene and pyrene were significantly degraded after 4 wk in vegetated soils (3044% more) compared with unvegetated controls (Reilley et al., 1996). Perennial ryegrass (Lolium perenne L.) also was found to enhance biodegradation of a hydrocarbon mixture containing phenanthrene, anthracene, fluoranthene, and pyrene (G0nther et al., 1996). These studies indicate that a large potential exists for the effective use of plant-enhanced bioremediation in PAH-contaminated soils. Few studies to date have investigated PAH-degrader community characteristics in rhizosphere soil. One study using alfalfa and alpine bluegrass (Poa alpina L.) grown for 14 wk in soil containing a mixture of organic chemicals including phenanthrene and pyrene reported selective enrichment of organic chemical degrader populations in contaminated rhizosphere soil (Nichols et al., 1997). While the authors implied that higher numbers of degraders in contaminated rhizosphere soils should result in potential stimulation of bioremediation around plant roots, they did not measure contaminant degradation. Others also report that enhanced disappearance of pollutants in rhizosphere soil is accompanied by increased microbial population sizes and soil respiration rates (1.6-1.8 times higher) compared with nonrhizosphere soils (Gt~nther et al., 1996). However, these are general characteristics of rhizosphere soils even in the absence of pollutant compounds. Finally, Radwan et al. (1995) observed zones of oil biodegradation around roots of Kuwaiti desert plants (primarily family Compositae) 4 yr after the Gulf War. Strains of Arthrobacter, Abbreviations: MPN, most probable number; PCA, principal component analysis; FAME, fatty acid methyl ester; DAP, days after planting; PAH, polycyclic aromatic hydrocarbon.