selection of bacteria and plant seeds for potential use in the remediation of diesel contaminated...

J. Basic Microbiol. 45 (2005) 4, 251–256 DOI: 10.1002/jobm.200410503

© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 0233-111X/05/0408-0251

(Department of Civil Engineering, College of Engineering, 1Department of Applied Biological Sciences, College of Science and Arts, Jordan University of Science and Technology, Irbid-22110, Jordan)

Selection of bacteria and plant seeds for potential use in the remediation of diesel contaminated soils

ZIAD AL-GHAZAWI, ISMAIL SAADOUN1* and ABDALLA AL-SHAK’AH

(Received 22 October 2004/Accepted 24 February 2005)

Enumeration and recovery of the dominant bacteria from a chronically fuel contaminated soil has been investigated. Bacterial counts from these polluted soils ranged between 0.70 × 108 and 28.20 × 108 CFU/g soil. Three different types of bacterial colonies have been recovered on the agar plates. Biochemical examination of the recovered bacteria revealed that they mainly belonged to the genus Pseudomonas, Micrococcus and Bacillus. Turbidity, cell biomass (dry weight basis), and physical appearance determined the growth of these bacteria on diesel. A noticeable decline in alfalfa (Medicago sativa) seeds germination of 15–30% was shown at 500 mg/kg diesel or higher. Under these contaminated conditions, fescue grass (Cyndon dactylon) exhibited a higher viability than alfalfa indicating that C. dactylon seeds are relatively tolerant to diesel and can possibly be used in phyto-remediation of diesel contaminated soils. Results of diesel phyotoxicity to seed germination of these two plants were based on filter paper media and therefore; should be considered as first indication only. Extrapolation of such results to actual soil conditions should be catiously approached taking into account diesel sorption on soil and mechanisms of its bioavailability.

Petroleum fuel spills are considered to be the most frequent organic pollutant in terrestrial and aquatic ecosystems (BOSSERT et al. 1984, MARGESIN and SCHINNER 1997). As landfills have become more scarce and the cost, prohibitive, interest in biological methods (bioremediation) to treat organic wastes particularly petroleum contaminants has increased. Bioremediation is a potentially important option for restoring of oil-polluted environments by exploiting the degradation capabilities of the natural inhabiting microorganisms. Many plant species have been also used in a process referred to as phytoremediation to clean up contaminated soils. For example, both fescue grass and squash can enhance the bioremediation of hydrocarbon contaminated soils (NOVAK and AL-GHAZAWI 1997). The specific mechanisms involved in phytoremediation include: enhanced rhizosphere activity and sub-sequence biodegradation; phytodegradation; phytoextraction; phytovolatilization, and hy-draulic pumping (USACOE 1997). However, before starting a phytoremediation project, determining the toxicity of the contamination toward the plants is essential. It is also impor-tant to select microorganisms with potential to grow on petroleum contaminants. Therefore, a auccessful approcah requires the selection of plant seeds and bacterial species that have high resistance to the pollutant to be treated. For this reason, the effect of diesel on the ger-mination of fescue grass (Cyndon dactylon) and alfalfa (Medicago sativa) seeds was deter-mined. Microorganisms with a potential to grow on diesel as a sole carbon source were also selected.

* Corresponding author. Dr. I. SAADOUN; e-mail: [email protected]

252 Z. AL-GHAZAWI et al.

© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Materials and methods

Collection of samples: Soil samples were collected from fuel oil-polluted soils at 4 gas stations in Jordan. The four stations had a historic exposure to fuel spills for more than 10 years. Samples of 250 g (fresh weight) were obtained from the subsurface after removing the upper 3 cm of the surface soil. Sample processing: Each soil sample was crushed, thoroughly mixed, and then sieved through a 2 mm pore size sieve (RETSCH, Germany). The soil samples were placed in polyethylene bags, closed tightly and then stored in a refrigerator at 4 °C ± 1 prior to analysis. Bacteria were then isolated from the sieved soil by suspending 1 g of the soil in 100 ml of sterile distilled water, agitating in a water-bath shaker at 100 rev/min for 30 min, then serially diluting up to 10–6. Aliquots of 0.1 ml from each dilution were spread over the surface of nutrient agar (HIMEDIA, India) plates. The plates were incubated at 28 °C for 2 days and thereafter the total number of colonies counted. Characterization and identification of bacteria: The morphological features such as colour, size, form, margin, and elevation of each colony were determined. Gram stain test was performed for each isolate. The bacterial strains were identified on the basis of BERGEY’s Manual of Systematic Bacteri-ology (HOLT et al. 1994). A culture of Rhodococcus erythropolis, that is capable of degrading different hydrocarbons (SAADOUN et al. 1999) was used as a control reference. Biochemical tests: The following biochemical tests were used in the identification of the bacterial isolates: gelatin liquefaction; citrate utilization; oxidase; catalase; growth at 6.5% sodium chloride; fluorescent pigment production; indole formation and nitrate reduction (CAPPUCCINO and SHERMAN 1996). To determine glucose fermentation as well as gas production, the isolates were placed in phenol red glucose broth. Growth on diesel: Colonies of different bacteria were inoculated into 50 ml sterile mineral salts medium (MSM) of LEADBETTER and FOSTER (1958) supplemented with 0.05% (v/v) filter-sterilized diesel that has been sterilized through 0.45 µm pore size millipore filters (MILLIPORE corp., Bedford, MA, USA) and incubated at 28 °C and 200 rpm for 14 days. The growth response of each of the bacterial isolate on diesel was initially determined at 7 days intervals by physical appearance (turbidity) and measuring the optical density (O.D.) at 540 nm using a colorimeter (BAUSCH and LOMB Inc., Rochester, NY). To determine the dry weight of cells/ml of the cell suspension, 2 ml aliquots of the final cell suspension was placed in pre-weighed aluminum tares and dried over night at 65 °C before weighting. Growth on diesel was also determined by the ‘hole-plate diffusion method’. Twenty milliliters of mineral salts agar medium (MSM) was plated and the plates inoculated with the above test organisms using a sterile swab. Cores of 6 mm diameter were removed aseptically from the agar using an alcohol flamed test tube and the holes filled up with 50 µl of filter sterilized diesel. In the treatment, the control hole was filled with sterile distilled water instead of filter-sterilized diesel. The inoculated agar plates were incubated at 28 °C and checked after 48 hrs for physical appearance of growth sur-rounding the holes. Growth response by this method was rated as no growth (–), weak growth (+) and strong growth (++). Plant seeds: Seeds of two plants, fescue grass (Cyndon dactylon) and alfalfa (Medicago sativa) were used in this investigation. The seeds were purchased from the local market. Two replicates from each type of seed were grown at different diesel concentrations (0.0, 100, 500, 1000, 2000, 4000 and 8000 mg/kg). The germinated seeds under these conditions are expected to be tolerant to diesel and thus may be recommended for phytoremediation of diesel contaminated soils. However; growth on cotton-based filter papers is not the same as growth in soil and results of this study are only first indication of diesel phytotoxicity of seeds under real soil conditions. Phytotoxicity and indexing the samples: An index was formulated to assign the sample number and diesel concentration. The F and A letters were assigned to Cyndon dactylon and alfalfa, respectively. Digits 00, 01, 05, 10, 20, 40 and 80 were assigned to diesel concentration of 0.00, 100, 500, 1000, 2000, 4000 and 8000 mg/kg, respectively. For example, the A205 index refers to Alfalfa, replicate number 2 with diesel concentration of 500 mg/kg.

Growth of bacteria and plant seeds on diesel 253


Two filter papers were placed in a PETRI dish, and the required volume of diesel was added. Diesel was dissolved in hexane to provide larger volume and thus make sure that the diesel is fully and homogeneously distributed all over the entire filter papers. After adding the diesel contamination, the dishes were opened inside a fume hood to evaporate the hexane. The zero diesel concentration was used as a control to determine the viability of the seeds. For each species fifty seeds were introduced to each dish, and then 12 ml of sterile distilled water added. The dishes were sealed and incubated in the dark at room temperature to simulate below ground soil conditions. After 14 days, the dishes were opened and the germinated seeds quantified.

Results and discussion

The population of bacteria in the soil samples polluted with gasoline, diesel fuel and engine oils ranged between 0.70 × 108 and 28.20 × 108 CFU/g soil (Table 1). These densities are similar to those found by SAADOUN (2004). The maximum recovered colonies on agar plates from these soils were of 3 different types of bacterial strains (Table 1). Biochemical examination of the recovered bacteria revealed that they belonged mainly to the genus Pseudomonas. Based on biochemical tests, the isolates belong to the Pseudomonas, Micrococcus and Bacillus (Table 2). The genus Pseudomonas was the most prevalent in fuel-oil contaminated soils than the other genera. The prevalence of this genus in these hydrocarbon-polluted soils reflects their potential in utilizing these contaminants for growth, thus cleaning up these sites (CORK and KRUEGER 1991). Therefore, the different bacterial isolates of this study were visually evaluated for such potential using turbidity and the hole-plate diffusion method. Using these methods in comparison with Rhodococcus erythropolis, only 3 isolates out of the 8 were able to grow on diesel as a sole carbon source (Table 3). Physical appear-ance (Fig. 1) and both, dry weight as well as optical density (O.D.) as summarized in Table 3 represent the growth response of the bacterial cultures on diesel fuel. Based on these data, growth was more prominent in Bacillus subtilis than the other isolates. The maximum % increase in the OD of the tested bacteria after 14 days was about 15 whereas the increase in biomass over that same period raneged from 14% to 30%. By comparison R. erythropolis increased in biomass by 65%. A noticeable retardation in the germination of alfalfa (Medicago sativa) seeds occurred at 500 mg/kg diesel and above, which accounts for 15 to 30% decrease in seed germination (Table 4). Alfalfa exhibited a comparatively low viability with only 72% in the presence of water only (“no diesel” Control). When the lowest diesel concentration tested (i.e. 100 mg/kg) germinating was present, but germination percentage was decreased even fur-ther by 2.8%. Alfalfa also suffered from the rot conditions. In comparison, germination percentage for Cyndon dactylon seeds was 97% when no diesel contamination was present and decreased by less than 8% even at diesel concentration as high as 8000 mg/kg. Further-more, no rot signs were found on Cyndon dactylon. Table 1 Total bacterial count and diversity of bacteria in soils contaminated with fuel oil

Soil sample

Site of collection

Time of exposure (Year)

Colour of soil sample

C.F.U. × 108/gm

Colony types

5B Al-Husn 10 Light Black 0.70 2 6B Industrial

City/Irbid 15 Black 28.20 1

8B Amman 20 Light Brown 9.60 2 12B Aramtha 30 Black 0.89 3 Total 8



Table 2 Morphological and physiological properties of the different bacterial isolates and their potential to grow on diesel

Species identified Biochemical and cultural criteria* Growth on diesel**

Motility Gelatin Citrate Nitrate Fluorescent pigment

Glucose

5B1: P. vesicularis – – – + – + – 5B2: P. putrefaciens + – – – – – – 6B1: P. putrefaciens + – – – – – + 8B1: Bacillus subtilis + ND – + – + ++ 8B2: P. fluorescense + + + + + – – 12B1: P. fluorescense + + + + + – – 12B2: P. cepacia + + – + – + – 12B3: M. luteus + ND ND ND ND + ++

* All of the Pseudomonas and Micrococcus isolates were able to ferment glucose and were oxidase positive.

** Growth of different bacteria arround the wells containing diesel on MS agar plates, –: No growth; +: Weak growth; ++: Strong growth; R. erythropolis exhibited a strong growth on diesel

ND: Not determined

Fig. 1 Growth response of the isolates (a): 6B1: P. putrefaciens (weak growth, +); (b): B81: Bacillus subtilis (strong growth, ++); (c): Rhodococcus erythropolis (top view, 5 days), and (d): R. erythropolis (top view, 10 days) on diesel as tested by hole plate diffusion method

Growth of bacteria and plant seeds on diesel 255


Table 3 Growth response of different bacterial isolates on diesel as measured by turbidity and dry weight at different time intervals

Bacteria Growth measurements over time

O.D. (540 nm) Dry weight (mg/ml)

7 days 14 days 7 days 14 days

6B1: P. putrefaciens 0.148 0.172 (14%) 29 33 (14%) 12B3: Micorococcus luteus 0.128 0.147 (15%) 40 50 (25%) B81: Bacillus subtilis 0.206 0.237 (15%) 20 26 (30%) R. erythropolis ND ND 85 140 (65%)

Numbers in paretheses represent the % increase in O.D. or biomass from day 7 Table 4 Germination of alfalfa (Medicago sativa) and fescue grass (Cyndon dactylon) seeds at different diesel concentration1

Plant species Diesel conc. mg/kg

Average no. of germinated seeds

Average % of germinated seeds

% Decrease in seed germination as compared with control (0.0 diesel)

Alfalfa 0.00 36 72 0.0 (Medicago sativa) 100 35 70 2.8 500 30.5 61 15.3 1000 28.5 57 20.8 2000 28 56 22.2 4000 25 50 30.5 8000 25 50 30.5 Fescue grass 0.00 48.5 97 0.0 (Cyndon dactylon) 100 46.5 93 4.1 500 45 90 7.2 1000 47 94 3.1 2000 47 94 3.1 4000 49 98 –1.0 8000 45 90 7.2

1 A total of 50 seeds were tested on each plate per plant. Reported results are averages of two replicates

Results of diesel phyotoxicity to seed germination of these two plants were based on filter paper media and therefore; should be considered as first indication only. Interpretation of the results of seed germination retardation can also be challenged by the density difference of the filter paper (ρ < 0.5 gm/cm3) compared to the soil (ρs = 2.5–2.7 gm/cm3). The varia-tion in the density will affect the distribution of the contaminants and its subsequent uptake by the seeds. Although the presented results can shed some light on how the seeds of these two plants respond to diesel phytotoxicity; retardation of seed germination should be studied using actual soil media instead of the cotton-based filter papers. Extrapolation of the seed germination results of this study to actual soil conditions should be catiously approached taking into account diesel sorption on soil and mechanisms of its bioavailability. The pre-liminary phytotoxicity results presented here, however; indicate that Cyndon dactylon is tolerant to diesel and can be used in phytoremediating this contaminant. Nevertheless, this plant has short roots and thus cannot be used to extract the pollutant from deeper surfaces.



Acknowledgements

Appreciation is extended to Jordan University of Science and Technology for administrative support.

References

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Mailing address: Dr. ISMAIL SAADOUN, Department of Applied Biology, Jordan University of Science and Technology, P.O. Box 3030, Irbid – 22110, Jordan Phone: 962-2-7201000-Ext. 23494, Fax: 962-2-7095014 E-mail address: [email protected]

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