In this study we checked the effect of soil texture in the rhizodegradation of crude oil by Sesbania cannabina plant, which has efficient TPH degradation ability. The soil used has clayey textured bioaugmented with microbial consortium originally isolated from the Yellow river delta (YRD) China. There are many studies which show the effects of nutrient addition and bioaugmentation but very few literatures shows the effect of physical texture of soil in rhizodegradation experiment. For the field scale treatment of polluted soil texture amendment is difficult to carry out so the soil texture used in its original form. The result shows no significant difference in the TPH concentration after 120days of plant growth. So it is concluded that the Sesbania cannabina has fibrous root structure which are not strong enough to support constricting and stretching forces of clayey textured soil so texture amendment is indispensible along with other amendments for optimizing rhizodegradation of crude oil in soil.
Combination of two approaches rhizoremediation and bioaugmentation, resulting in rhizodegradation. During rhizodegradation, exudates derived from the plant can help to stimulate the survival and action of bacteria. The root system of plants can help to spread bacteria through the soil and help to penetrate otherwise impermeable soil layers. (Kuiper, I. et al, 2004). Soil texture, compactness, and drainage are the main soil characteristics for plant growth. Compact soil layers limit root growth and affect the properties related to water and air movement. According to Gerhardt, K. E. et al 2009 found in spiked soils, chemicals tend to be bioavailable, whereas contaminants in naturally weathered soils are often not readily bioavailable. Various methods to enhance the biodegradation of the heavier compounds through the addition of nutrients, inoculums, and growth inducers have met with only limited success (Sherman, D.F. and Stroo, H.F. 1989). The hydrophobic characteristics of crude oil retard mass transfer of air, water, and contaminants from particles to microorganisms in soil, which limits the rate of uptake and metabolism of contaminants by hydrocarbon-degrading bacteria (Bosma et al., 1997; Semple et al., 2003). The moisture content and aeration were determined to be the key factors associated with PAH bioremediation. Neither biosurfactant addition, bioaugmentation, nor ferric octate addition led to differences in PAH or TPH biodegradation compared to biodegradation with nutrient treatment (Vin˜as et al, 2005). The specificity of the plant-bacteria interaction is dependent upon soil conditions, which can alter contaminant bioavailability, root exudates composition, and nutrient levels (Siciliano and Germida, 2009).
Crude oil contamination is very much serious problem in Yellow River Delta in Dongying, Shandong due to its exploitation, manufacturing and transportation. The size of contaminated site is very long so need of cost effective technique which can remediate such long area. In the present study soil was bioaugmented without any other amendment. Physicochemical properties of soil has been shown in table 1.
Physicochemical properties of the soils
WHC (water holding capacity in ml /gm) 0.4315
Moisture content 13.75%
*aSoil texture clayey
sand 11.516 % wt
silt 39.290 % wt
clay 42.452 % wt
Crude oil concentration (ppm) 3040
Total N (g kg−1) 0.816
Available N (mg kg−1) 1.68
Total P (g kg−1) 0.0288
Available P (mg kg−1) 13.44
Total C (gm kg-1) 12.70017
CEC (cation exchange capacity c mol kg-1) 6.0996
*aTotal oil degraders load in soil ( Num gm-1) 7.85 x 105
*b Total oil degraders (CFU/ gm of dried soil) 1.093 x 103
*a by DAPI method
*b by MPN method
Materials and methods
Soil sample was initially air dried for one week, then pulverized and passes through 2mm sieve. 1.5 Kg soil was used in each pot of size--------------. Initial pot weight was analyzed in order to keep the 60% of moisture content of soil . ---------pots were prepared 6 for each treatment i-e plants in sterilized soil, plants with free culture inoculation and two control was run, one was plants without inoculation and soil without plants. Each pot had 8 plants. Analysis was done after 80 and 120 days of plant growth, each pots used in triplicate. For required amount of inoculums initial soil load was checked by MPN, total bacterial load in mixed consortium was checked by direct microscopic count with Petroff-hausser counting chamber. The bacteria were introduced to ï¬nal concentration of 107CFU / kg of dry soil, for this calculated amount 37ml of culture suspension was used. First culture was fresh in flask containing 150ml of MSM, 100 uL of diesel oil and 2ml of old culture, after 2.5 days of incubation at 37°C at 150 rpm culture in log phase was used. First it was centrifuged at 4500 rpm for 10 minutes then pellet was washed 3 times with 0.9% saline and mixed thoroughly with hands in 1.5 kg of contaminated soil and water content was adjusted to 60% of the WHC. Sterilization of soil for control, soil samples was steam-sterilized (three successive sterilizations 24 h apart in order to killed spores, at 100°C for 1 h each) and the soil humidity was adjusted with sterile distilled water.
Physicochemical analysis of soil
Soil texture was analyzed by Ted Sammis method. Briefly a jar was half filled with soil and wet to a muddy consistency, soil level was marked and then water filled in the jar about ¾, soil completely mixed up in the water. After 40 seconds level marked showed sand then after 6hrs level showed silt portion of soil. The percentage sand, silt and clay was measured by measuring the distance from bottom to the first mark was sand fraction, the distance from the first mark to the second mark was silt fraction and the distance from the second to the third mark was clay fraction. From the distances percentage volume was calculated by dividing the distance of each fraction with total height. Percent volume converted to percent weight by multiplying the percentage of sand by 1.19, the percentage of silt by 0.87 and the percentage of clay by 0.94. These numbers are the weight ratio's of bulk density compared to average bulk density of the material. Soil texture was estimated by using the table below
Soil pH was measured in 0.01 mol L−1 CaCl2 with soil-to-solution ratio of 1:2.5. Water holding capacity was calculated by the experiment in which the difference of the weight of dry and wet soil after 4 to 8 hours (until the absorbent paper on the soil surface was completely wet) was taken. The percentage of WHC was calculated by the formula. Percentage of moisture content was calculated by dividing the loss in weight on drying for 2 hrs at 105°C with initial sample weight and multiplying with 100. Soil samples was steam-sterilized (three successive sterilizations 24 h apart, at 100°C for 1 h each) and 60% soil moisture was adjusted with sterile tap water.
TPH was detected ultrasonically using a solvent extraction method. Briefly, air-dried soil samples (1 g) were mixed with 1 g anhydrous sodium sulphate, and extracted 3 times in 10 mL hexane by ultrasonication for 1 h each time. Then analysis was conducted with the method of Wang et al (2011).
Cation exchange capacity (CEC) was determine by using ammonium chloride standard method with some changes, briefly soil samples were treated with ammonium chloride solution and boiled till NH3 evolved, which dissolved all calcium carbonate and form calcium chloride, then repeated treatment of soil samples untill the soil saturated with ammonium, and then wash away the excess ammonium with ethanol. In order to confirm that all Ca ion conversion 5ml clear supernatant was mixed with 1 ml of buffer solution and small amount of K-B powder, blue color means no Ca if pink color means Ca is present then repeat the step. Then ammonium ion removed by washing with 95% ethanol and centrifugation for 3min ~ 5min at speed 3000r/min ~ 4000r/min, discard the ethanol, washed 3 times 4 times, until the final ethanol solution has no ammonium ion-free (checked by Nessler reagent no yellow color means no ammonium ion otherwise repeat step).
Sample was transferred into the Kjeldahl's flask by dissolving with distill water, after sufficient distillate had been collected then titrated with hydrochloric acid standard solution, and calculated the amount of ammonium in the soil shows the cation exchange capacity.
Total and available N and total P was detected by Kjeldahl's method. (Bremner and Mulvaney, 1982). Available P was detected by Olsen P method (Pansu and Gautheyrou, 2006), briefly 20 ml extracting solution of bicarbonate (0.5M NaHCO3, pH 8.5) for 1 gm of soil was used, flask was shaken for 30min at 200rpm at room temperature and then filtered. To 10ml of filtered sample 20-30ml of distill water was added along with 2 drops of dinitrophenol indicator, then 50% NaOH was used to gain yellowish color, and 5ml of Molybdenum-antimony anti reagent was added and 50ml volume made by distill water then left for 30 minutes. UV-probe spectroscope at 700nm was used to check the absorbance. With the help of standard curve concentration of P (mg/L) of sample was calculated.
Petroleum degraders were enumerated by the most-probable-number (MPN) technique Krik et al 2005. A 96-well microtiter plate containing sample, mineral salt medium (MSM) and crude oil was used to calculate the MPN of hydrocarbon degraders in colony forming units (CFU) g-1 dry soil. Total microbial activity in soil was measured using fluorescein diacetate [3', 6'-diacetylfluorescein (FDA)] hydrolysis rate (Green and Diack 2006). The amount of FDA hydrolyzed was measured by absorbance at 490 nm (A490). The value of A490 per gram soil was referred to the microbial activity in the soil.
Plant cultivation and analysis
Local dominant specie of plant Sesbania cannabina was selected for biodegradation tests in lab experiment. First seed were selected then sterilized with 10% H2O2 for 30 minutes and kept in saturated CaSO4 solution for overnight. After 80 days and then 120 days of growth, plants were harvested and then analyzed. Plant samples were separated to shoots and roots, while the soils were divided to rhizosphere and non-rhizosphere soil by hand shaking method (Phillips et al., 2009). The roots of the plants were scanned and analyzed by Epson Scanning and WinRHIZO Pro. 2005 to obtain the root parameters such as root length, root surface area, root volume and number of root tips (Bauhus and Messier, 1999). The plant samples were oven dried at 105oC for 15 min then 60oC for 48 h, and then weighed to get the biomass.