Identification of a Bacillus amyloliquefaciens (BA) strain able to bioremediate methyl tertiary-butyl ether (MTBE) in vitro and in situ

ABSTRACT

Methyl tertiary-butyl ether (MTBE) is an oxygenate that allows the complete combustion of gasoline. Although it is not carcinogenic, there is rising concern about its effects on human health because of its high water solubility and some proven systemic toxicities in animals. MTBE has become a target for many bioremediation studies with some microbes already proven to be effective in bioremediating it. Recently we isolated a Bacillus amyloliquefaciens (BA) strain from pooled, MTBE-contaminated agar that can be used as a potential bioremediant microbe for MTBE. We cultured the isolated strain of BA in agar containing MTBE as sole carbon source and compared it with the control groups that did not have MTBE. The Gas chromatography/ Mass Spectrometry was used to determine the amount of MTBE used up by the bacteria. We seeded four local surface soil samples with different pH with BA strain. We found that BA grew on agar containing MTBE as a sole carbon source and addition of MTBE (0.01- 11.3 μmol) to BA growth medium produced dose-dependent, statistically significant (p < 0.01) increases in BA numbers after 2 and 12 hours of incubation compared to control. The data from Gas Chromatography/ Mass Spectrometry demonstrated that BA reduced MTBE from 100 ppm to 9 ppm after 4 hours of incubation at 37°C. One hundred ppm MTBE was rapidly removed from the three local soil samples with neutral to alkaline pH (7.0-11.0), by between 2 and 4 days post introduction. These results suggest that BA has potential to be an effective terrestrial bioremediant for MTBE and given the diversity of ecosystems bacilli can tolerate may prove to have a wide range of applications.

INTRODUCTION

In 2004, the United States consumed 21.93 million barrels oil per day and this was used for fuel, to produce gasoline and manufacture plastics (Gibson 2006). Since 1970, the only oxygenate generally accepted as a gasoline additive, due its ability to blend with other gasoline components, namely hydrocarbons of the BTEX group, (Benzene, Toluene, Ethylbenzene and Xylenes) has been methyl tertiary-butyl ether (MTBE) with its current production of over 295,000 barrels per day in the United States alone (Gibson 2006).

International Agency for Research on Cancer based on the reported data concluded that there is inadequate evidence in humans and limited evidence in experimental animals for carcinogenicity of MTBE (IARC 1999). Some animal studies have shown increased incidence of tumors with MTBE but there is lack of consistency in outcome (McGregor 2006). An animal study has shown it to increase the severity of chronic nephropathy in mice (Clarry 1997) and another has shown it to be cytotoxic to isolated spermatogenic cells of rat (Li, Yin and Han 2007). The health and environmental concerns have arisen over this product due to its high water solubility and easy access to environmental niches (Mehlman 1998). Indeed, several senators have suggested a moratorium on its use in the near future with the related compound ethyl tertiary butyl ether (ETBE) being suggested as a more "environmentally friendly" motor fuel (Kerrey 2000).

Interestingly, although only around 6% of the gasoline entering the environment through leaking underground storage tanks or damaged railcars is MTBE (Uhler et al 2001) media coverage has ensured that a great deal of research has been focused on safely and rapidly removing MTBE from contaminated terrestrial and aquatic sources. Removal of MTBE via bioremediation using algal (Cundell and Brendley 2003), microbial (Ramsden 2000) and phytoremediation (Rubin and Ramaswami 2001) methods is proving invaluable in this regard as it provides a rapid and significantly cheaper method of removal of biohazards than traditional methods, which often employ applying caustic, solvent-based extractions or excavating and hauling of soil.

Aerobically, a number of native soil bacterial mixtures have been demonstrated as being able to utilize MTBE as a carbon source (Steffan et al 1997; Jennings and Tanner 2000; Ramsden D, 2000) with optimization of biotic factors necessary to stimulate normal microbial soil activity. Several terrestrial and aquatic bacterial and fungal strains have also been shown to be able to individually bioremediate MTBE and its major metabolite tert-butyl alcohol, including Arthrobacter, Rhodococcus, Streptomyces and (Ramsden 2000; Jennings and Tanner 2000; Mo et al 1997). In contrast, only one major bacterial species namely PM-1 of Methylobium petroleophilum, is able not only to rapidly degrade MTBE but also use the compound as a sole carbon and energy source (Hanson et al 1999).

Recently, we were able to recover three bacterial isolates from agar contaminated with high levels of MTBE (1,000-5,000 ppm vol/vol; 11.4-56.8 μmoles), which were commercially confirmed by Environmental Microbiology Laboratory Inc, San Bruno, CA as separate Bacillus species. Whether these represent novel strains or a newly discovered property of an existing strain was not able to be established by the commercial laboratory, however one of the bacilli, identified as Bacillus amyloliquefaciensl, was identified as the dominant organism present on the contaminated agar and further research demonstrated that this strain of BA was able to grow on agar containing MTBE or its metabolite tert butyl alcohol (TBA) as sole carbon source, suggesting it might be an excellent bioremediant of the ether. The Environmental Protection Agency (EPA) has not set official safe limits for MTBE but maxima of 20 to 40 ppm have been recommended (Mehlman 1998). As our strain of BA was able to survive on agar containing up to 25 times recommended levels, we focused on evaluating its ability to act as a bioremediant of MTBE, using a combination of biological and chemical assays.

MATERIALS AND METHODS

Identification and culture of Bacillus amyloliquefaciens (BA)

Confirmation of the genus was made commercially (Environmental Microbiology Laboratory Inc., San Bruno, CA) with speciation performed using a commercial miniaturized API 50CHB test system (Biomerieux-Vitek, Hazelwood, MO). Culture media and sterile saline were obtained from Sigma Chemical Company (St. Louis, MO). BA was cultured in nutrient broth at 37°C in the presence or absence of MTBE (1-1,000 ppm vol/vol; equivalent to 0.01-11.3 μmoles). Prior to and after 1, 2, 3, 4, 8, and 12 hours of incubation 100 μl of the culture medium was removed using a pipette and serial ten-fold dilutions between 10-2 - 10-6 were prepared using sterile saline. Separate spread nutrient agar plates were prepared using 100 μl of each dilution, according to standard protocols (Tortora et al 1998). All plates were incubated at 37ºC and the number of colonies appearing after 48 hours determined by visual assessment. Growth curves were prepared on six occasions to determine consistent bacterial numbers.

The ability of BA to grow on MTBE and its major metabolite TBA as sole carbon source was also tested using agar plates prepared from low gelling agarose (Sigma Chemical Company, St. Louis, MO). Separate agarose plates were prepared to which 1,000 ppm MTBE or 500 ppm TBA (vol/vol) had been added and two individual colonies of BA were then inoculated onto the surface of each plate. For comparison, two colonies of a second bacillus species (Bacillus licheniformis) also previously isolated from agar heavily contaminated with MTBE were also inoculated to the surface of the agarose plates. The inoculated plates were incubated at 37ºC for up to 72 hours with appearance of growth being checked daily.

Gas Chromatography/ Mass Spectrometry

A PerkinElmer Autosystem XL Gas Chromatograph equipped with a Turbo Mass Gold Mass Spectrometer (GC/MS; Perkin Elmer, Boston, MA) was used to measure levels of MTBE (100 ppm vol/vol) and its metabolite TBA over a 4 hour incubation period in the presence of live and autoclaved BA. The GC/MS used PerkinElmer Elite-5 (5% phenyl) methylpolysiloxane series capillary column with length of 30 meters, inner diameter of 0.25 mm, and film thickness of 0.25 mm (Perkin Elmer, Boston, MA). One ml samples were injected into the instrument. The GC/MS operating parameters maintained the GC oven temperature at 400C for 5 minutes, ramped to 2200C at 400C/min, and then held at 2200C for one minute. The instrument separates and analyzes each component based on the mass to charge ratio and limits of detection were > 10 ppm for both MTBE and TBA (0.1 and 0.08 μmol, respectively)

Field tests

Surface soil samples were obtained from four separate sites, one acidic (pH 4.0), one neutral (pH 7.0) and two alkaline (pH 9.0 and 11.0) on the Philadelphia University campus, with the following characteristics:-

Site 1 (pH 4.0) Loam-clay soil, conifer leaf litter, near major road

Site 2 (pH 7.0) Mold-covered, loam-clay soil located near a driveway

Site 3 (pH 9.0) Mold-covered, loam-sand soil with some grass cover, near a walkway

Site 4 (pH 11.0) Mold-covered clay soil partially colonized by ivy (Hedera helix), next to a parking lot.

Soil was seeded with BA (2 x 106 CFU/ml per gram of soil) in the presence or absence of 100 ppm (vol/vol) of MTBE per gram of soil, covered, maintained at room temperature (25ºC) and sampled daily to determine MTBE levels by GC/MS using the methodology described above. As a negative control MTBE (100 ppm vol/vol/g) was also added to previously autoclaved soils (20 minutes at 120°C at 15 p.s.i.), incubated as above and assessed for spontaneous loss of the ether.

Statistical analysis

Bivariate correlation statistics using the Pearson coefficient were performed using SPSS for Windows Version 6.13. Comparison statistics were made, where appropriate, using the nonparametric Wilcoxon Signed Ranks Test and parametric paired T-tests from the Minitab for Windows 2000 Version 5.0 program.

RESULTS

Ability of Bacillus amyloliquifaciens (BA) to grow on MTBE and TBA as sole carbon sources

BA was found to be able to grow on MTBE- and TBA-containing agarose, where the ether and its major metabolite were the sole carbon sources, after 48 and 72 hours of culture at 37°C, respectively (Figure 1). Both of the individual colonies tested demonstrated strong growth on both MTBE and TBA (Figure 1).

Figure 1 – Growth of individual Bacillus amyloliquifaciens (BA) colonies (isolates 1 and 2) and B. licheniformis (isolates 3 and 4) on agarose containing MTBE (1,000 ppm vol/vol) or tert butyl alcohol (500 ppm vol/vol) as sole carbon sources

Figure 1 – Growth of individual Bacillus amyloliquifaciens (BA) colonies (isolates 1 and 2) and B. licheniformis (isolates 3 and 4) on agarose containing MTBE (1,000 ppm vol/vol) or tert butyl alcohol (500 ppm vol/vol) as sole carbon sources

Effect of MTBE on the growth curve of Bacillus amyloliquefaciens (BA)

Six separate experiments were performed in which the growth of BA in nutrient broth over a 12-hour period at 37ºC was compared in the presence and absence (control) of MTBE at 1, 10, 100 or 1,000 ppm (vol/vol 0.01, 0.1, 1.1 and 11.3 μmol) (Figure 2). In all experiements, viable counts were performed at the onset of the experiment (0), hourly between 0 and 4 hours and at 8 and 12 hours of incubation. MTBE at between 1 and 1,000 ppm (vol/vol) produced dose-dependent and statistically significant (p < 0.01) increases in BA between 2 and 4 hours of incubation compared with control (Figure 2).

Figure 2 – Effect of MTBE on the growth of Bacillus amyloliquefaciens (BA) over a 12 hour incubation period

Figure 2 – Effect of MTBE on the growth of Bacillus amyloliquefaciens (BA) over a 12 hour incubation period

BA numbers showed no further increase between 8 and 12 hours of incubation either in control medium or with any concentration of MTBE tested, indicating that the bacterium had reached stationary phase (Figure 2). After 8 hours of incubation there was no significant difference in BA growth in medium to which 10, 100 or 1000 ppm MTBE was added (Figure 2) and at all times sampled, 1,000ppm (vol/vol 11.3 μmol ) MTBE did not produce statistically significant greater increases in bacterial growth than did 100 ppm (p > 0.5; vol/vol) (Table 1).

Table 1 – Effect of 100 and 1,000 ppm on the growth of Bacillus amyloliquefaciens over a 12 hour incubation period

Table 1 – Effect of 100 and 1,000 ppm on the growth of Bacillus amyloliquefaciens over a 12 hour incubation period

Gas chromatrography/ mass spectrometry assessment of levels of MTBE and TBA in growth medium of Bacillus amyloliquefaciens (BA)

Measurement of nutrient broth spiked with 100 ppm MTBE (vol/vol; 1.1 μmol) and monitored over a 4-hour period demonstrated that in the absence of BA, loss and spontaneous breakdown of the ether were minimal at 37°C ( < 5%). Adding live BA to the medium resulted in a decrease in measurable MTBE levels from 100 ppm (vol/vol) to 40 ppm (vol/vol; 0.4 μmol) after 2 hours of incubation and 9 ppm (vol.vol; 0.1 μmol) after 4 hours of incubation at 37°C (Figure 3). In contrast, dead BA (autoclaved for 15 minutes at 121ºC, 20 psi) produced no measurable decrease in MTBE levels (94.7 + 4.3 ppm vol/vol after 4 hours of incubation at 37°C). No TBA was detectable following either experiment. Regression analysis demonstrated that the growth of Bacillus amyloliquefaciens directly correlated to the disappearance of MTBE (r = 0.91; p < 0.01).

Figure 3 – Correlation between Bacillus amyloliquefaciens growth and reduction in MTBE levels

Figure 3 – Correlation between Bacillus amyloliquefaciens growth and reduction in MTBE levels

Gas chromatrography/ mass spectrometry assessment of levels of MTBE and TBA in soil samples seeded with Bacillus amyloliquefaciens (BA)

Measurement of MTBE levels in soil samples spiked with 100 ppm MTBE (vol/vol/ g; 1.1 μmol) demonstrated that over a six day period in the absence of BA, removal of MTBE was maximal at pH values of 7.0 and 9.0 (complete elimination by 4 and 5 days respectively; Figure 4). When BA was added to the extant soil flora MTBE was removed from the soil between 17 and 40%, more rapidly (Figure 4). Again, removal of MTBE was far more rapid in soils that were neutral to alkaline pH (removal by 2 and 3 days respectively in the presence of BA versus 4 and 5 days in its absence for pH 7.0 and 9.0 soils, respectively). Spontaneous loss of MTBE from the soils due to volatilization was minimal during the six day period of assessment (98.7 + 3.6 ppm vol/vol).

Figure 4 – Gas chromatography/ mass spectrometry (GC/MS) assessment of rate of MTBE degradation of soils from four sites seeded with Bacillus amyloliquefaciens

Figure 4 – Gas chromatography/ mass spectrometry (GC/MS) assessment of rate of MTBE degradation of soils from four sites seeded with Bacillus amyloliquefaciens

DISCUSSION

As Bacillus amyloliquifaciens was the predominant bacterium among three isolates from the MTBE contaminated agar, we hypothesized that the strain of the bacteria metabolizes MTBE and hence can be used as a bioremediant. Although it was not clear from our study whether this isolate is a novel BA or represents an adaptation from an existing strain, we were able to establish that this isolate metabolized rather than sequestered MTBE based on three separate findings. Firstly, the strain of BA was able to grow on MTBE and its major metabolite – tert butyl alcohol (TBA) as sole carbon sources (Figure 1). Second, this strain of BA demonstrated a significant increase in growth, which was associated with a simultaneous disappearance in MTBE from the growth medium (Figure 2). No tert butyl alcohol was found in the external growth medium and non-specific binding of MTBE to dead bacteria was minimal; results indicating BA is internally metabolizing MTBE. Thirdly, the removal of MTBE from the growth medium was extremely rapid; medium spiked with 100 ppm MTBE (vol/vol; 1.1 μmol) contained only 9 ppm (vol/vol; 0.1 μmol), as assessed by GC/MC after only 4 hours of incubation at 37°C (Figure 3).

Removal of MTBE by our isolate of BA is between 6 and 20 times faster than the majority of other individual bacterial species, where complete removal of 60 ppm MTBE (0.7 μmol) from in vitro cultures required between 7 and 28 days (Mo et al 1997; Okeke and Frankenberger 2003). Indeed, to date only the PM-1 bacterium Methylobium petroleophilum has been shown to degrade MTBE within a few hours and at a similar rapid rate of 0.23 μmol MTBE/ hour (Hanson et al 1999). Indeed MTBE removal by PM-1 is also at a similar rate to our own strain; 20 μg/ml/hour i.e. for PM-1 versus 0.3 μmol MTBE/ hour for BA (Hanson et al 1999). It is also of interest to note that prior to the current study PM-1 was also the sole bacterial strain shown to be able to grow directly on MTBE as a carbon source and studies have suggested that the reason it is able to utilize the ether is that it can metabolize the ether via the TBA pathway (Hanson et al 1999). Although the enzyme systems possessed by PM-1 and BA may therefore be similar and both appear to be terrestrial bacteria their shared properties seem to end at this point. Recent studies have shown that PM-1 is a gram negative organism belonging to the methylotroph subgroup of the beta Proteobacteria (Nakatsu et al, 2004), whereas BA is a gram positive saprobic spore forming bacillus.

In terms of its abilities to bioremediate in situ our studies are currently ongoing but preliminary data have been obtained at four locations around Philadelphia University with one acidic (pH 4.0) and three alkaline (pH 7.0-11.0). These suggested that BA seeded at an initial 2 x 106 CFU/g was able to remove 100 ppm (vol/vol; 1.1 μmol) from alkaline soils within 2-4 days, compared with 4-6 days in soils to which no BA was added (Figure 4). In contrast BA appeared to be less active in the single acidic soil site examined (pH 4.0), from which the 100 ppm MTBE was removed only one day quicker in the presence of this bacterium versus its absence (Figure 4).

The pH activity spectrum of BA is in keeping with previous data by Kroll (1990) who found that although the endospores of Bacili were well distributed across a variety of soils, active cells were usually only recovered from soils with alkaline or extremely alkaline culture conditions (7.0-9.0). Thus future usage of BA may need to be limited to neutral to alkaline terrestrial environments.

Bacillus amyloliquefaciens is an aerobic, gram positive spore forming rod that is able to occupy a variety of terrestrial sites due to the versatility of the enzymes it secretes (Wikipedia 2006). Enzymes from BA have been previously utilized for a variety of industrial purposes, including paper and linen manufacture (xylanolytic enzymes; Breccia et al, 1998), enzymic digestion of DNA (BAMH-1; Wikipedia, 2006), biological laundry detergents (Wells et al, 1983 subtilisin) and in wastewater cleanup as a consortium member with other bacteria to clean up varied hydrocarbons and chemicals (Roetech 106™. This is, however, the first study to date that has directly associated this species with bioremediant activity against MTBE. The bioremediant property of BA strain for MTBE is of practical significance because our results show that BA can rapidly remove and metabolize methyl tertiary butyl ether (MTBE) at levels expected to be found in severely contaminated groundwater (upto100 ppm).

Further studies are currently underway to delineate the absolute pH and temperature limitations of BA and also whether this bacillus is able to bioremediate other components of gasoline. We are also evaluating the capacities of the Bacillus licheniformis strain isolated as a bioremediant given its capacity, albeit weaker, to also survive on MTBE and TBA as sole carbon sources.

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