CHEMICALS

Phenanthrene (98+%), anthracene (99%), fluorene (99%), naphthalene (99+%), n­pentadecane (99+%), n­hexadecane (99%), n-octadecane (99%), and pristane (98%) were obtained from Aldrich Chemical Co., Inc. (Milwaukee, Wis.). Dibenzothiophene (Johnson Matthey Electronics, Ward Hill, Mass.) and iodonitrotetrazolium violet (Research Organics, Inc., Cleveland, Ohio) were research and development grade. High purity n­pentane was from Baxter Healthcare Corp. (Muskegon, Mich.).

MPN METHODS

PAH degraders, alkane degraders, and "total" hydrocarbon degraders were enumerated in separate 96­well microtiter plates. The most important difference among these MPN procedures is the selective growth substrates that were used. The alkane degrader MPN plates were provided with n­hexadecane as the selective growth substrate, whereas a mixture of four PAHs was used in the PAH degrader MPN plates. Number 2 fuel oil (F2) was the selective substrate for determination of total hydrocarbon degraders (Haines et al. l 996). Hexadecane and F2 were filter sterilized before use. The PAH substrate mixture was added to the microliter plates as a solution in pentane (10 ~L/well) before the plates were filled with growth medium. The mixture consisted of 10 g phenanthrene/L, I g anthracene/L, l g fluorene/L, and I g dibenzothiophene/L. Pentane evaporates rapidly, depositing the PAHs onto the surfaces inside each well. Hexadecane and F2 (5 pL/well) were added to the alkane and total hydrocarbon degrader plates after the wells were filled with growth medium, but before they were inoculated. Bushnell­Haas medium (Difco Products, Detroit, Mich.) supplemented with 2% NaCI (B­H) was used as the growth medium for all three hydrocarbon degrader MPN procedures (180 p~L B­H/well).

Samples were diluted in a saline buffer solution that contained 0.1 % sodium pyrophosphate (pH 7.5) and 2% NaCI. Tenfold serial dilutions were performed, and the plates were inoculated by adding 20 ~L of each dilution to I of the 12 rows of eight wells. The 10­~°dilution was inoculated into row 11, the 10­9 dilution was inoculated into row 10, and so on. The first row of each plate was inoculated with 20 ~L of undiluted sample, and row 12 remained un-inoculated to serve as a sterile control.

The alkane and total hydrocarbon degrader MPN plates were incubated for 2 weeks at room temperature and the PAH degrader plates were incubated for 3 weeks. In the PAH degrader procedure, positive wells turned yellow or brown owing to the accumulation of partial oxidation products of the aromatic substrates. Iodonitrototn~­zolium violet (INT) was used to identify positive wells in the alkane and total hydrocarbon degrader procedures (Haines et al. l996). After 2 weeks of incubation, 50 ~L of filter sterilized INT (3 g/L) was added to each well in these plates. In positive wells, INT is reduced to an insoluble formazan that deposits intracellularly as a red precipitate. Positive wells were scored after an overnight, room temperature incubation with INT. A computer program (Klee 1993) was used to calculate the MPN for each category of hydrocarbon degrader. The numbers that are reported have been corrected for the positive bias that is characteristic of published MPN tables (Salama et al., 1978).

INT REDUCTION KINETICS

A crude oil degrading enrichment culture, originally obtained from a beach in southwestern Texas, was transferred into 100 mL of B­H containing 500 mg/L of F2, Alaskan North Slope crude oil (NS), hexadecane, or phenanthrene, and the cultures were grown for 10 days at 20°C with shaking at 200 rpm. The cells were harvested by centrifugation, washed three times with 0.1 % sodium pyrophosphate (pH 7.5) plus 2% NaCI, and then resuspended in 20 mL of this saline buffer. Five milliliters of each resuspended culture was added to triplicate flasks containing 100 mL of B­H plus INT (0.3 g/L). Samples were collected and analyzed for INT reduction periodically for 24 h. The extent of INT reduction was determined by filtering 2 mL of each culture through 0.5­~m glass fiber filters (Micro Separations, Inc.) and then extracting the formazan with 95% ethanol. The concentration of INT­formazan in the ethanol extracts was determined spectrophotometrically by measuring the absorbence at 480 nm.

The microbial population densities in the washed, resuspended cultures were determined by MPN (total hydrocarbon degraders, alkane degraders, and PAH degraders) and heterotrophic plate counts (HPC). Marine agar (Difco Products, Detroit, Mich.) was used for the HPCs.

TABLE 1. Extent of INT reduction by hydrocarbon-degrading bacteria grown on different substrates.
Growth Substrate	HPC (CFU/mL)	Amount of INT reduced* (fmol INT-formazan/CFU)
Alaskan North Slope crude (NS)	3.1x108	0.69
No. 2 Fuel Oil (F2)	3.4x108	0.46
Phenanthrene	1.6x108	0.77
Hexadecane	3.5x108	1.8
*Amount of INT - formazan produced following a 24-h incubation.

OPTIMIZATION OF THE PAH SUBSTRATE MIXTURE

The composition of the PAH substrate mixture was optimized by determining the effects of substrate concentration on the growth of an oil degrading enrichment culture on phenanthrene. The accumulation of colored products was also monitored. A washed suspension of bacteria that had been grown for 2 weeks on NS was inoculated into I 00 mL of B­H containing 50 mg of phenanthrene and several different concentrations of fluorene, anthracene, and dibenzothiophene. The absorbency of culture filtrate at 400 nm was used as a measure of colored product formation. The pH of the culture filtrates was always between 7 and 7.5. Growth was monitored by measuring the increase in biomass protein by pelleting cells from 1.0 mL of culture medium in a microcentrifuge. Protein was extracted from the pelleted biomass by heating in 0.1 M NaOH at 90°C for 10 min. The extracted protein was quantified using the bicinchoninic acid (BCA) method (Pierce Biochemicals, Rockford, Ill.).

ACCURACY AND SELECTIVITY OF THE HYDROCARBON DEGRADER MPN PROCEDURES

Several pure cultures of hydrocarbon and nonhydrocarbon degrading bacteria were used to determine the accuracy of the hydrocarbon degrader MPN procedures by comparing population estimates obtained for these cultures with the alkane, PAM, and total hydrocarbon degrader MPN with those obtained by HPC. The selectivity of the hydrocarbon degrader MPN procedures was evaluated by comparing alkane, PAM, and total hydrocarbon degrader MPN results for these hydrocarbon degrading pure cultures, pure cultures of two nonhydrocarbon degrading bacteria, and several hydrocarbon degrading enrichment cultures. The enrichment cultures were grown on crude oil, normal and branched alkanes (n­pentadecane and pristane), and PAHs (either phenanthrene or a mixture of naphthalene, phenanthrene, and fluorene). The pure cultures were isolated from enrichment cultures that had been grown on crude oil, pentadecane, or a mixture of naphthalene, phenanthrene, and fluorene. Isolates were obtained by plating on B­H solidified with noble agar. Inoculated plates were sprayed with dilute solutions of phenanthrene or n­octadecane in pentane. Colonies that were surrounded by clear zones were picked and restreaked on B­H­noble agar plates sprayed with the appropriate substrate. Isolated colonies were streaked on marine agar plates to cheek for purity.

RESULTS

Reduction of INT was used to detect positive dilutions in a hydrocarbon degrader MPN procedure (Haines et al. 1996). Tetrazolium dyes, like INT, are reduced by respiratory electron transport systems to insoluble formazans that are highly colored (Packard 1971; Rodriguez et al. l 992). Thus, INT reduction can be a sensitive indicator of growth and should be generally useful for detection of positive dilutions in MPN assays. When hexadecane and F2 were provided as the selective substrates, INT was very effectively reduced in positive wells. When phenanthrene was used, however, it was reduced very poorly in positive wells, and they were difficult to distinguish from negative wells.

The ineffectiveness of INT for identifying positive wells in the MPN procedure for PAH degraders was probably due primarily to the growth yield on phenanthrene, which was lower than that achieved when crude oil or F2 were provided as the growth substrates. Table I shows that the amount of INT­formazan deposited in phenanthrene­grown cells was approximately the same as that deposited in NS­ and F2­grown cells, but the cell density was only about half that attained by the other cultures. Hexadecane­grown cells, however, reduced substantially more INT than did cells grown on the other substrates. This also was apparent in the MPN assays: positive wells in plates provided with hexadecane were very dark red and easy to score, whereas those provided with F2 were much less intense and ambiguous positives were occasionally observed.

Although INT reduction is not suitable for detecting positive wells in the PAH degrader MPN assays, the accumulation of products from the partial oxidation of PAHs is useful. Some of the partial oxidation products of PAHs have strong absorption maxima in the visible region. Fluorene, in particular, is converted to a bright yellow product by many PAH degrading cultures (Grifoll et al. 1992; Boldrin et al. 1993). These colored products have not been identified, but the mesa­cleavage path way for aromatic rings can produce yellow intermediates from some substrates (Ribbons and Eaton 1982; Smith 1990).

Since many PAHs are toxic, it was important to optimize the substrate concentrations to maximize growth and color production. Phenanthrene showed no inhibitory effects for any of the hydrocarbon degrading enrichment cultures that were tested, but naphthalene, anthracene, fluorene, dibenzothiophene. Acenaphthene, and fluoranthene all were inhibitory to some degree. Chrysene was not inhibitory, but it also was not biotransformed by any of the cultures that we tested. Although naphthalene, acenaphthene, and fluoranthene were biologically transformed, colored products did not accumulate to a significant extent.

The effects of fluorene concentration on growth and colored product formation are shown in Fig. 1. Fluorene inhibited growth of this oil degrading enrichment culture at all concentrations that were tested and the extent of inhibition increased with increasing fluorene concentration (Fig. IA), even though the solution was saturated with fluorene at the lowest concentration. Similar phenomena have been observed for other PAHs (Bauer and Capone 1985). The formation of colored products was greatest in the presence of 50 mg fluorene/L, but it decreased at higher fluorene concentrations (Fig. IB). Similar but less dramatic results were obtained when dibenzothiophene and anthracene were substituted for fluorene (data not shown). The composition of the substrate mixture that we used in the PAH degrader MPN procedure (500 mg phenanthrene/L, 50 mg/L each for fluorene, anthracene, and dibenzothiophene) maximized color production and minimized the inhibitory effects of the various PAH substrates.

Fig. 1. Growth (A) and colored product formation (B) of an oil-degrading enrichment culture on mixtures of phenanthrene (500 mg/L) and various concentrations of fluorene. The fluorene concentrations were 0(()),50(l),100((u)),250((n)), and 500 (+) mg/L. A 0.1% inoculum of the oil degrading culture was used.

ACCURACY AND SELECTIVITY OF THE ALKANE AND PAH DEGRADER MPN ASSAYS

Effective methods for enumerating alkane and PAH degraders in environmental samples must be highly selective for the target group and capable of accurately determining the number of bacteria that are present. Selectivity requires the method to respond to the target group but not to bacteria with other metabolic capabilities, even when those organisms constitute a large fraction of the microbial community. Many questions surround the accuracy of microbial enumeration techniques that rely on growth of the target organisms (Herbert 1990), but at a minimum, a method should be able to accurately quantify the number of organisms present when culturable strains are tested. For the MPN method, that means that inoculation of a single cell must produce a positive well (Colwell 1979; Alexander 1982).

Fig. 2. Comparison of HPC, total-hydrocarbon-degrader MPN (F2-MPN), alkane-degrader MPN (C16-MPN), and PAH-degrader MPN (PAH-MPN) for several hydrocarbon-degrading enrichment cultures. The growth substrates for the enrichment cultures are the following: NS-1 and NS-2, Alaskan North Slope crude oil; Prs, pristane (2,6,10,14-tetramethylpentadecane); C15, n-pentadecane; Phe, phenanthrene; NPF-1 and NPF-2, a mixture of naphthalene, phenanthrene, and fluorene (1:1:1). Error bars represent 1 SD for five replicate determinations for the MPNs and three replicate determinations for the HPC.

The selectivity of these MPN procedures was evaluated with enrichment and pure cultures of hydrocarbon degraders by comparing the numbers of heterotrophs, hydrocarbon degraders, alkane degraders, and PAH degraders that were present in each culture. For the enrichment cultures, the relative abundances of alkane and PAH degraders were correlated with the composition of the enrichment substrate (Fig. 2). PAH degraders were completely absent in the alkane degrading enrichment cultures (Prs and C15) and alkane degraders were much less abundant than aromatic degraders in the PAH degrading enrichment cultures (Pine, NPF­ I, and NPF­2). Alkane and aromatic degraders were both present in the crude oil enrichments (NS­1 and NS­2), but alkane degraders were much more abundant. In the crude oil enrichments, the alkane (C16­MPN) and total hydrocarbon degrader (F2 ­ MPN) populations were essentially identical. In fact, the total hydrocarbon degrader MPN was approximately equal to the MPN for the most abundant group (enumerated with either hexadecane or the mixture of PAHs) for most of the enrichment cultures. Thus, when a method for total hydrocarbon degraders is used, the population density of the minority group is masked by the dominant group. Since crude oil and roost relined petroleum products are composed primarily of alkanes and alkane degraders are more common than aromatic degraders in many environments, total hydrocarbon degrader estimates will usually reflect the population density of alkane degraders.

The selectivity of these procedures is also demonstrated by the pure culture data. Pure cultures of alkane degraders were not detected by the PAH degrader MPN procedure and pure cultures of PAH degraders gave no response in the alkane degrader procedure (Fig. 3). Furthermore, pure cultures of bacteria that cannot grow on hydrocarbons but were stable members of the hydrocarbon degrading enrichment cultures were not detected by any of the hydrocarbon degrader MPN methods.

Fig. 3. Comparison of HPC, total-hydrocarbon-degrader MPN (F2-MPN), alkane-degrader MPN (C16-MPN), and PAH-degrader MPN (PAH-MPN) for several pure cultures isolated from hydrocarbon-degrading enrichment cultures. The source of each isolate is indicated in its designation: Oil, crude oil degrading enrichment culture; PAH, culture grown on a mixture of naphthalene, phenanthrene, and fluorene (1:1:1).

The pure culture data also demonstrate the accuracy of these procedures. HPC, the total hydrocarbon degrader MPN, and one of the selective MPN procedures all gave similar estimates of the bacterial population density for five of the six hydrocarbon degrading cultures that were tested (Fig. 3). The population density of the sixth culture (PAH­3), a phenanthrene degrading isolate, was underestimated by the PAH degrader MPN procedure. The reason for this is not known, but similar results have been reported for several pure and mixed cultures that were analyzed with the total hydrocarbon degrader procedure (Haines et al. 1996).

DISCUSSION

The MPN procedures described in this paper permit the selective enumeration of aliphatic and aromatic hydrocarbon degrading bacteria. Although this capability can be useful for characterizing hydrocarbon degrading microbial populations and for monitoring the response of environments to oil contamination, currently available methods either cannot distinguish between these populations or are inconvenient for field applications.

Most hydrocarbon degrader MPN methods use complex substrates, such as crude oil or refined petroleum products, as the selective substrate (Mulkins­Phillips and Stewart 1974; Walker and Colwell 1976; Brown and Braddock 1990; Song and Bartha 1990; Haines et al. 1996). These methods probably enumerate alkane degraders in most situations, because the substrates are composed primarily of aliphatic hydrocarbons and alkane degraders are often more common than PAH degraders (Heitkamp and Cerniglia 1987; Fopht et al. 199()). Furthermore, some methods detect positive wells by applying criteria that are biased towards alkane degraders. For example, the Sheen screen uses dispersion or emulsification of the crude oil substrate to identify positive wells (Brown and Braddock 1990), but these effects are associated primarily with growth on aliphatic hydrocarbons (Hommel 1990). Although aromatic hydrocarbons are of most concern, in many cases esthnatcs of the total hydrocarbon degrading population provide no useful information regarding the potential for PAH degradation.

Alkane and PAH degrading bacteria can be separately enumerated by using two MPN assays with either hexadecane or a mixture of PAHs as the selective substrates. Positive wells are detected by different methods in these two procedures, because INT was reduced poorly in positive wells of the PAH degrader MPN plates. Similarly poor discrimination of positive and negative wells was observed when pure cultures of PAH degraders were enumerated with F2 as the selective substrate. INT reduction very effectively distinguished positive and negative wells when hexadecane was provided as the growth substrate, however. Although the yield on hexadecane was similar to that observed for growth on F2 and crude oil, hexadecane grown cultures deposited nearly three times as much INT formazan as the cultures grown on the complex hydrocarbons. The difference might be due to accumulation of larger amounts of intracellular storage products by bacteria that are grown on normal alkanes.

INT reduction is superior to some of the traditional methods, such as turbidity and measurement of biomass protein (Atlas 1979; Colwell 1979), that have been used to identify positive dilutions in hydrocarbon degrader MPN procedures. Reliance on turbidity and biomass protein can lead to false negatives if growth is closely associated with the water hydrocarbon interface (Roubal and Atlas 1978; Atlas 1979), but INT reduction will occur whenever cells are metabolically active. If that activity is restricted to the interracial region, INT­formazan deposition will occur and be visible at the interface. Also, deposition of the INT­formazan increases the contrast between positive and negative wells over that achieved as a result of turbidity alone. This is particularly important for the 96­well microtiter plates, which have a relatively short optical path length.

The accumulation of colored products during metabolism of PAHs is a simple, sensitive, and highly selective method for identifying positive wells in the PAH degrader MPN procedure. Colored products are frequently produced during growth on PAHs (Mueller et al. 1990; Grifoll et al. l992; Boldrin et al. 1993), especially when substrates such as fluorene and dibenzothiophene are cometabolized. The color that we observe is probably due to a variety of colored products that are formed from different substrates by different bacteria. In practice, the color of positive wells can range from bright yellow to brown. Color production in the PAH degrader MPN plates is highly selective. We have never observed formation of colored products by non-PAH degrading bacteria, even when alkane or nonhydrocarbon degraders were inoculated at high cell densities (e.g., cell densities were greater than 108 mL^-1 in the first row of some plates).

The selectivity of the PAH degrader MPN procedure is also shown by its responses to enrichment cultures grown on alkanes and crude oil. Alkane degrading enrichment cultures (Prs and C15) contained no PAH degraders as measured by this assay, and it was not possible to isolate PAH degraders or to obtain PAH degrading enrichment cultures using alkane degrading cultures as inocula. The crude oil cultures (NS­ I and NS­2) both contained PAH degraders, but their relative abundance in the two microbial communities was quite different (Fig. 3). The relative abundance of PAH degraders in each community was in good agreement with the extent of PAH degradation that was observed. Approximately 35% of total PAHs were degraded in 3 weeks in the NS­ I culture, in which 10% of the hydrocarbon degraders could grow on PAHs, but no PAH degradation could be seen in the NS­2 culture after a similar incubation period (data not shown). PAH degraders represented less than 0.01% of the hydrocarbon degrading population in NS­2. Nevertheless, it was possible to isolate phenanthrene and naphthalene degraders from both of these oil degrading enrichment cultures. Therefore, results from the PAH degrader MPN assay were consistent with other indicators of the presence of PAH degraders. Since some of the enrichment cultures that were tested also contained large populations of nonhydrocarbon degraders, it is clear that false positives do not result from interactions between alkane degraders and other oil­associated heterotrophs.

The alkane degrader MPN procedure also was highly selective. The pure cultures of PAH degraders that we tested were unable to grow on alkanes and they did not produce any positive wells on alkane degrader MPN plates. Bacteria that were unable to grow on hydrocarbons also produced no positive wells on these plates (Fig. 3). Furthermore, we observed no growth when the PAH degrading enrichment culture that was devoid of alkane degraders was inoculated into B­H containing hexadecane as the only carbon source, and no colonies produced clearing zones when this culture was plated onto octadecane spray plates. Thus, even when the cell density of the inoculum was very high, INT reduction did not occur unless the bacteria actually grew during the 2­week incubation period.

In addition to their selectivity, the alkane and PAH degrader MPN methods provided accurate estimates of the number of organisms present in these cultures. In most cases, the cell densities for pure cultures of alkane and PAH degrading bacteria that were measured with these MPN procedures were essentially identical to the cell densities estimated using plate counts on marine agar. This does not imply that all hydrocarbon degraders that are present in natural environments will be detected by these procedures. These MPN methods suffer from all of the familiar problems associated with growth­based enumeration methods (Herbert 1990). Nevertheless, these methods are accurate for those organisms that can be grown in the laboratory. It is particularly important to demonstrate that this is true for methods that enumerate organisms that grow on insoluble substrates, because substrate availability might be dependent on cell density in some cases. For example, if cells must attach to or solubilize the substrate to metabolize it, single cells might do this less effectively than larger populations. Under these conditions, single cells might not initiate growth, and the MPN procedure will underestimate the target population density. This does not appear to be a serious problem with the methods developed in this study.

The ability to distinguish between alkane and PAH degrading bacteria is an important feature of these MPN procedures, because aliphatic and aromatic hydrocarbons appear to be degraded by two distinct subsets of hydrocarbon degrading microbial populations. Foght et al. (1990) tested 61 hexadecane­ and 21 phenanthrene­mineralizing bacteria and none of these isolates mineralized both compounds. This is consistent with our observations. None of the alkane degraders that we isolated from crude oil enrichment cultures could grow on phenanthrene, and none of the phenanthrene or naphthalene degraders that we isolated could grow on hexadecane. When bacteria from oil­contaminated sediment from Prince William Sound, Alaska, were plated on oil agar, about one third of the colonies contained genes for both alkane (alkB) and aromatic (xyIE) hydrocarbon metabolism (Sotsky et al. 1994). This implies, of course, that two thirds of the hydrocarbon degraders present in these sediment samples could grow on alkanes or aromatics but not both. Thus, even when bacteria with the capability to degrade both aliphatic and aromatic hydrocarbons are present, important information regarding the structure of the microbial community can be obtained by separately enumerating the alkane and PAH degrading populations.

The MPN procedures that have been described here provide a simple means for simultaneously determining the population densities for aliphatic and aromatic hydrocarbon degrading bacteria. The alkane and PAH degrader MPN plates can be prepared at the same time from the same series of dilutions and the same growth conditions are used for both analyses. Therefore, there is no possibility that differences can result from sample heterogeneity or differences in selective pressure imposed by the cultivation technique (e.g., cultivation in liquid medium versus solid medium). Both procedures are highly selective for their target populations and they accurately determine the number of culturable hydrocarbon degraders that are present. Simple enough for the field, these methods should provide useful tools for characterizing petroleum-contaminated environments or for monitoring the progress of bioremediation.

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