A Mittal, P Singh
biodegradation, polycylic aromatic hydrocarbon pah, pseudomonas
A Mittal, P Singh. Polycyclic aromatic hydrocarbon degradation by developed consortium in microcosms study.. The Internet Journal of Microbiology. 2008 Volume 7 Number 1.
A feasibility study was conducted prior to onsite full-scale bioremediation study. This feasibility study has investigated the possibility of bioaugmentation of polycyclic aromatic hydrocarbon (PAH)-polluted soil with PAH-degrading strains or consortia. Five treatment options- Oil alone, Oil + Indigenous microflora, Oil + nutrients, Oil + nutrient + tilling, Oil + nutrient + tilling + developed consortium were evaluated. Out of five treatments the application of bacterial consortium, nutrients and environmental factors resulted in 79.16% removal of TPH in 60days, compared to 30.24% removal of TPH carried out by indigenous microflora. The results of High-pressure liquid chromatographic analysis showed that the ratio of di / tri aromatics decreased from initial 0.63 to 0.25 with progressive treatment of nutrient addition, nutrient+tilling, nutrient+tilling+microbial seeding. Similar effect was observed in di / di+tri aromatics ratios which also decreased 0.31 to 0.20 by bioaugmentation only.
Polycyclic aromatic hydrocarbons (PAHs) constitute a group of priority environmental pollutants, which are ubiquitous contaminants in soils and sediments and are of environmental concern because of their toxic, mutagenic and/or carcinogenic effects (Mastrangelo
Bioremediation is a technique that enhances the natural rate of biodegradation of pollutants through reactions carried out by selected microorganisms (Providenti
There are many definitions of 'microcosm' a typical one is that of an intact, minimally disturbed piece of an ecosystem brought into the laboratory for study in its natural state (Prichard and Bourquin, 1984). Microcosms can vary in complexity from simple static soil jars of contaminated soil to highly sophisticated systems designed to enable variations in various environmental parameters encountered on site to be more accurately simulated in the laboratory.
A feasibility study was conducted to assess the short term bioremediation potential of a sandy loam soil of Haridwar region, freshly contaminated with un-weathered Sobhasan crude oil well # Y. Five treatment options- Oil alone, Oil + Indigenous Microflora, Oil + Nutrients,
Material And Methods
Crude oil samples from Sobhasan oil well # Y was collected with the help of O.N.G.C. authorities.
Analysis of soil at the bioremediation site
Physical and chemical properties of soil samples were analyzed. The samples were drawn with a hollow pipe, 3.5 cm in diameter. Air-dried and pulverized soil samples were analyzed for pH, temperature, moisture level, organic carbon, nitrogen, and available phosphorus with standard methods.
Scaling up of Bioinoculant consortium
To prepare the inoculums, bacterial isolates were grown separately in 2-liter Erlenmeyer flasks containing 500 ml of mineral medium and molasses (2%) as the sole carbon and energy source. All the isolates were grown to mid-log phase (108CFU/ml) and they mixed in equal proportions. The mixed culture was used as the inoculum (5%) for large-scale culture in a bioreactor (Bioflow 3000, New Brunswick) with a working volume of 10 liters. The same minimal salt medium with molasses (2% wt/vol) as sole carbon source was used in the bioreactor for large-scale culture. The growth conditions in the bioreactor were as follows: temperature 32°C; aeration, 0.75 volume of air/volume of medium / min; agitation, 250 rpm; pH 7.0 (adjusted with 1N HCl-NaOH); and duration of growth, 15 h. Silicone oil was added to control excessive foaming in the bioreactor. After growth, the culture was immobilized onto the selected carrier material, corncob powder (a biodegradable agriculture residue) by simple mixing of carrier material and culture in ratio of 1:3. The carrier-based culture was dispensed into sterile reusable polythene bags (4 kg of culture immobilized onto carrier material in each 10 kg polythene bag) and stored at 4°C after the bag was aseptically sealed. In previous study, various carrier materials were screened to determine the survival of carrier-based culture by monitoring the CFU counts (on LBA plates) at 15-days intervals,upto 90 days (Mittal
Experimental design and treatments
The total area (225 m2) of the feasibility study was divided into 20Blocks (four replicate Block for each treatment), 1 by 1 m each and separated by a gap of 2 m. The experimental design chosen was a completely randomized block design. The treatments were as follows: (i) Oil alone; control where no treatment was done. (ii)
Block 2-Oil + Indigenous Microflora: It is essentially a no action control, wherein the initial state of the microcosm is left unmanipulated throughout the course of the investigation. In this Block no treatment was given except ½ to 1-liter water was added regularly.
Block 3-Oil + Nutrients: This microcosm was amended with inorganic nutrients in the form of Diammonium phosphate (DAP) and urea fertilizer, in order to have a C: N: P ratio of 120:10:1 as per Gibb’s formula (assuming crude oil is 78% carbon). Nutrients (fertilizer) were applied two times, after one-month time interval.
Block 4-Oil + Nutrient (Fertilizer) + Tilling (Aeration): It entails the two or three times per weekly tilling of the microcosm contents to promote soil aeration, in addition to nutrient amendment.
Block 5-Oil + Nutrient (Fertilizer) + Tilling (Aeration) + Developed consortium: The developed mixed culture consortium of hydrocarbon degraders (
Throughout the course of study in field treatments were open to air and sufficient water was added to hydrate the soil to 50% of its water holding capacity. In biostimulation treatment, a dry mix fertilizer (N: P) was applied to select blocks (Block3, Block4 and Block5).
Extraction of Total Petroleum Hydrocarbons (TPH)
To assess the rate at which the TPH was being degraded, samples were collected at time Zero (just before initiating the bioremediation), 30 days later, and at the end of the study (60 days after initiating the process). The sample was weighed and mixed with anhydrous sodium sulfate in a 1:1 ratio and was then used for soxhlet extraction. Because the results were desired using dry-weight basis, the sample weight was adjusted to account for the moisture and this weight was used for analysis.
Total petroleum hydrocarbon from 10 gm soil was then consecutively extracted with hexane, methylene chloride and chloroform (100 ml each) for 16 hours in soxhlet apparatus. The extraction cycle around the thimble were approximately 6~8 cycles per hour. The solvent extract containing soluble oil from the soil was evaporated in Rota evaporator. The residual oil content was estimated.
Fractionation of TPH and analysis of fractions
The group type (saturate, aromatic and NSOs) analysis of whole oils has been carried out in chemito-1000 gas chromatography. For isolation of various compound class fractions (saturate, aromatic and NSOs), column chromatographic technique was used. The separation was carried out on dual packed activated silica-gel/alumina columns by successive elution with petroleum ether for saturates, benzene for aromatics and methanol for NSO compounds.
Aromatic fraction was further fractionated as follows:
Shimadzu VP-3 HPLC system using normal isocratic mode was used for the fractionation of aromatic hydrocarbons into mono, di and tri-aromatics on a silica amino (NH2) column (30cmX4.5mm). Elution was carried out using n-hexane (HPLC grade) and the separation was monitored with UV-visible absorbance detector operated at 245 m wavelength. The flow rate was maintained at 6.5ml/min.
Survival of Introduced consortium
Survival of introduced bacterial consortium during the course of study was indicated indirectly by observing the increase in population of consortium in soil over the period of study. Soil samples (1 gm) were collected before and after the application of bacterial consortium i.e. after 15 Days, 30 Days, 45 Days and 60 Days, suspended in saline water (0.85% NaCl). The suspension after appropriate dilution was plated on Luria Bertani agar (LBA) media plates.
Results and Discussion
The first and foremost criterion for designing a bioremediation program is to study the native microflora of the system and to analyses the physico-chemical composition of soil. The soil sample taken for feasibility study (garden soil of Kanya Gurukula Mahavidyalaya, Haridwar) was analyzed for detection of different groups of microbes. The soil contains 8 X 108 /g of total heterotrophs, 4.5 X 105 /g of actinomycetes and 1.4 X 104 /g of fungi. The hydrocarbon degrader’s count is 2.2 X 104 /g of soil.
Characterization of soil at the bioremediation site
Physical and chemical properties of soil samples taken were analyzed for pH, temperature, moisture level, organic carbon, nitrogen and available phosphorus in each block at zero time intervals i.e. initially and after 60 days of bioremediation after application of microbial consortium.
The soil used is sandy loam in texture and its pH was 7.88. Moisture content of soil was 7.30 %, while water holding capacity was 40.55. The temperature recorded during the study varied from 25°C- 33°C. Organic carbon content (TOC) of soil was 1.23%, nitrogen content - 0.126%, initially. After contamination with crude oil Organic carbon content (TOC) of soil reached 2.79% and amendment by fertilizer nitrogen content reached to 0.235%.
Crude oil used
The crude oil deployed for microcosm study (Sobhasan crude oil # y) was analyzed for its gross composition. Saturates, aromatics and NSO percentage was found to be 71.7%, 20.0% and 7.4% respectively.
The TPH decreased from 0.1885 gm to 0.1315 gm in 60 days, indicating 30.24% degradation of crude oil (Table-2). During the course of biodegradation, the pH dropped from 7.88 - 7.71. . Many other workers have also been reported the pH alteration during crude oil degradation (Zaidi
The soil tilling stimulates the microbial activity due to breakdown of soil aggregates and better exposure and aeration of degradable material (Paul and Clark, 1989).
The temperature recorded during the study varied from 25°C - 33°C. Since maintenance of temperature in open soil system is not feasible, the bioremediation efforts should be concentrated during such a period of year when the temperature is suitable for treatment. Brady (1990) suggested that biodegradation rates increase with increasing soil temperature upto a certain maximum temperature, which would be optimum for indigenous hydrocarbon degraders (Barker and Neumann, 1993). Moisture content of soil is critical for microbial activities, since nutrients, organic constituents, Oxygen and metabolic wastes are transported to and from microbial cells in water. The optimum level of soil water for microbial activity is 10-13% i.e. 65–80% of water holding capacity. (Bossert and Bartha, 1984; Sims
Survival of Introduced consortium
Survival of the microorganisms in the soil after their application is a deciding factor in rate of degradation of hydrocarbons (Ramos
Aromatic hydrocarbon degradation study
HPLC analysis of aromatic fractions of Sobhasan crude oil (deployed in microcosm study) was done after 60 days period only (fig.1). The effect of indigenous microflora as well as seeded consortium was observed. The widely accepted ratios of biodegradability di/tri aromatic and di / di + tri aromatic were calculated & are given in table 3. The results showed that the ratio of di / tri aromatics decreased from initial 0.63 to 0.25 with progressive treatment of nutrient addition, nutrient+tilling, nutrient+tilling+microbial seeding. Similar effect was observed in di / di+tri aromatics ratios which also decreased from 0.38 to 0.20.The results indicate that the developed consortium could help in degradation of higher aromatics, as ratio was decreased from 0.31 to 0.20 by bioaugmentation only. Thus developed consortium could help in detoxification of the harmful and carcinogenic aromatics specially polyaromatic hydrocarbons.
Crude oil used: Sobhasan crude oil well # Y.
A carrier-based formulation made it easy to transfer the microorganisms from the laboratory to the field. Corncob powder (agricultural byproduct) was found to be the best carrier for this purpose as survival of 7.2 X 108 cfu/g was recorded during storage over 3 months. Corncob powder, being a good porous material facilitates growth and exposes a grater surface area to microbial action.
The present study clearly demonstrates that application of a carrier-based bacterial consortium, improvised after isolation of potent hydrocarbon degraders from the oil contaminated site, can be used to remediate soil contaminated with crude oil. Maintenance of proper environmental factors is an essential aspect for treating an oil spill by biotechnological methods. In view of synergistic and antagonistic relationship among the various strains of bacteria, bioremediation is considered as site-specific approach.