One of the great challenges of microbiology is to find efficient ways to monitor and thereby understand environmental microbial diversity. If environmental microbial diversity can be adequately monitored, then the direction and rate of processes catalyzed by environmental microbial communities may become better understood. This applies both to in situ processes and to processes in controlled environments (e.g., reactors).
With respect to in situ processes, Shi et al. (14
) defined the differences between microbial community structures in pristine and fuel-contaminated aquifers by using 16S rRNA probes. The fuel-contaminated areas were enriched in members of the β subclass of the class Proteobacteria
(β-proteobacteria) and γ-proteobacteria compared to α-proteobacteria (14
). Addition of toluene in microcosm studies enriched microorganisms that were only minor constituents of the fuel-contaminated community in situ. These results suggested that characterization of the microbial community change when toluene is added might help identify key toluene degraders in situ. In another recent study the in situ microbial population changes during oil spills were characterized by phospholipid fatty acid analysis and PCR-amplified 16S ribosomal DNA denaturing gradient gel electrophoresis (DGGE) (6
). DGGE indicated that oil enhanced the presence of gram-negative α-proteobacteria. Although phospholipid fatty acid analysis indicated that the microbial communities of oil-contaminated and noncontaminated plots were similar after 14 weeks, DGGE still revealed major population differences.
With respect to microbial communities in controlled environments, Stoffels et al. (17
) extensively characterized degradation of Solvesso 100, a complex mixture of aromatic hydrocarbons used in industrial painting. Inoculation of a fermentor with a sample from a Solvesso 100-laden waste stream resulted in a community in which γ-proteobacteria, especially Pseudomonas
spp., were dominant, even though the original inoculum was dominated by α- and β-proteobacteria. Interestingly, the original diversity did not appear to be lost during fermentor enrichment; use of the fermentor culture to inoculate a trickle-bed bioreactor resulted in a community in which α- and β-proteobacteria were dominant. These transitions were established by direct fluorescent in situ hybridization of samples with 16S and 23S rRNA-targeted probes and by performing probe analysis of colonies obtained following plating on different media. Both methods indicated that distinctly different populations derived from the same environmental sample were able to degrade Solvesso 100. Stoffels et al. (17
) concluded that establishment of an enrichment culture prior to inoculation of a reactor may result in enrichment of bacteria that do not effectively colonize the reactor. However, this would be a problem only if some of the original diversity in the sample was permanently lost during enrichment. Communities may be merely dynamic (i.e., capable of reestablishing dominance depending on the growth conditions).
Our results suggest that contaminated and uncontaminated soils at depths ranging from 0 to 14 m at the ethylene plant site investigated have populations in which specific C5+-degrading bacterial strains are typically community members. The RSGP results for community DNA without enrichment on hydrocarbons revealed community profiles (data not shown) that were more evenly distributed than those obtained following enrichment on hydrocarbons (Fig. ). Enrichment of similar organisms from diverse backgrounds occurs after exposure to C5+, suggesting that evolution of a single dynamic microbial community occurs during growth on hydrocarbons. Different conditions result in differential expression of community members. Enrichment under identical conditions leads to development of similar community compositions, irrespective of contamination history (Fig. and ). These results are similar to those of Shi et al. (14
), who demonstrated that similar microbial communities developed in soils exposed to toluene regardless of whether the soils were initially contaminated. The soil environment targeted in our study supports a variety of different culturable bacteria. The ability to degrade aromatic hydrocarbons is widely distributed in this community, and the community includes γ-proteobacteria. (Pseudomonas
spp.), β-proteobacteria (Alcaligenes
spp.), α-proteobacteria (Sphingomonas
spp.), high-G+C-content gram-positive bacteria, such as actinobacteria (Rhodococcus
spp.) and Microbacterium
, and low-G+C-content gram-positive bacteria (Bacillus
spp.). We used RSGP rather than rRNA-targeted probes to monitor the dynamics of this community. The main advantage of this technique is that multiple cultured bacteria can be tracked in a single hybridization step. This has not been accomplished yet with rRNA targeted probes. Some disadvantages of the technique are the fact that the microbial community is described solely in terms of its culturable component, although this is likely to be a significant fraction of the total bacteria in enrichment cultures, the fact that genomically similar microorganisms cannot be distinguished, and the fact that calculated fx
values contain cross-hybridization contributions for which we cannot correct (19
). Despite these shortcomings, the RSGP method demonstrated that enrichments from contaminated and uncontaminated soils from the same site developed along similar paths when they were exposed to similar conditions. The samples were initially dominated by γ-proteobacteria (Pseudomonas
spp.) but converged to a community dominated by β-proteobacteria (Alcaligenes
spp.), as shown in Fig. and . The reasons for the observed succession are not clear. In our experimental system the supply of C5+ was continuous at a constant concentration, whereas other required nutrients were replenished every 2 weeks when the culture was transferred. The increases and decreases in some of the early major community components (for instance, standard 11 [Pseudomonas syringae
LQ 20]) may be related to the gradual removal of soil from the enrichment.
Spills of C5+ have occurred occasionally at the ethylene plant site investigated, leading to exposure of microbial communities to nearly pure C5+ at the center of the spill and to lower C5+ concentrations away from the center of the spill. We demonstrated that communities derived from an uncontaminated location where a new ethylene plant is under construction can develop the ability to degrade most C5+ hydrocarbons, which is important for the plant operators in view of the potential for accidental contamination of this location. Benzene, toluene, and xylene are major constituents of C5+. Degradation of mixtures of BTEX compounds has been investigated by numerous authors. Oh et al. (10
) demonstrated that p
-xylene was cometabolically removed by benzene and toluene degraders. The presence of p
-xylene decreased the rates of degradation of these primary substrates. Similarly, Deeb and Alvarez-Cohen (1
) found that the rates of degradation of BTEX compounds in mixtures were lower than rates of degradation of the pure compounds. Also, the order of degradation of compounds in mixtures was different than the order of degradation of the pure compounds. The presence of o
-xylene enhanced benzene and toluene removal, but the latter two compounds inhibited removal of xylene. We determined the degradation kinetics only for the C5+ components present in the mixture, as exposure to pure components is unlikely to occur at sites where this mixture is generated. Benzene is the main component of C5+ (45%, wt/wt), followed by poorly degradable DCPD and cyclopentadiene (20%, wt/wt). Toluene, styrene, xylenes, and naphthalene, the other components that we monitored, are present at much smaller concentrations (6, 3, 2, and 2% [wt/wt], respectively). A large number of the standards isolated (31 of the 44 standards tested) were capable of benzene degradation, whereas degradation of the minor C5+ components was less widespread (Table ). The main community members identified in enrichment cultures (Fig. ) are all capable of benzene degradation (Table ). The degradation curves for all C5+ components suggested that there was an acclimation period (lag time), followed by degradation (9
). The lag time may involve adaptation to the C5+ concentration used in the degradation experiments; hydrocarbon degradation genes should have been induced in the inocula, which were all grown in desiccators with 1% C5+ in vpo as the sole carbon and energy source. Despite its much higher concentration, benzene was generally removed first, due to a shorter lag time and/or a higher first-order rate constant than those observed for the other degradable C5+ components (Table ). This was true for enrichment cultures derived from contaminated and uncontaminated soils and for synthetic consortia. Inhibition of xylene removal by benzene, as described by Deeb and Alvarez-Cohen (1
), may have contributed to the slower removal of xylene by some of our cultures. The fact that the benzene concentration was highest and the benzene supply was unlimited during growth of enrichments or synthetic consortia on 1% C5+ in vpo in the desiccators prior to the rate studies may have forced the communities to make benzene removal a priority, because benzene metabolism can reduce its toxicity (15
). The communities at field sites contaminated with C5+ may be similarly adapted to benzene removal. Our enrichment studies suggest that a variety of communities with different compositions are able to metabolize the benzene-dominated C5+ mixture (Fig. ). Hence, just as communities with different compositions have been shown to effectively degrade Solvesso 100 (17
), our results indicate that a variety of communities with different compositions are active in C5+ degradation. A single community structure that is effective in C5+ degradation therefore cannot be defined.