Nitrogen, Phosphorus, Potassium Status of Bioaugmented

Nitrogen, Phosphorus, Potassium Status of
Bioaugmented Versus Biostimulated Crude Oil
Contaminated Coastal Plain Sand of Southern
Etukudoh, Ndarake Emmanuel(2) Ikpe, F. N (3)Akpan, Joyce
Fidelis (4)Ayolagha, Gaskin
(1,2)Department of Soil Science, Faculty of Agric., Rivers State
University of Science and Technology, PMB 5080, Port
Harcourt, Rivers State Nigeria.
(3)Department of Soil Science, Faculty of Agriculture. University
of Calabar .Cross River State
(4)Department of Soil Science, Faculty of Agriculture, Niger
Delta University. Wilberforce Island, Bayelsa State.
• Biochemical Effects of crude oil Spill on the environment notwithstanding our
natural ecosystem has been subjected to series of disturbance crude oil exploration
and exploitation by man. Overpopulation and industrialization have also contributed
in various ways to the deterioration of the environment upon which humanity is
completely dependent for life (Odu, 1980). Crude oil contamination on a soil
environment is known to affect soil quality via changes in soil pH, air temperature,
soluble plant nutrients and disruption of metabolic processes of soil microorganisms
(Moller, 1991, Isirimah, 2004) hence, the vigor of plant nutrients mineralization.
As early as 1930, Scientists in Moscow noted the decreases in growth rate of
local trees and attributed it to wide spread oil pollution and began five-year
remediation plan. In a follow up experiment, George Robinson (known as father of
bioremediation) experimented the action of soil microorganisms in a crude oil
polluted soil in Santa Maria, California and achieved a huge success (Meagher,
2000). Since then bioremediation has been judged the best alternative method to
remedy hydrocarbon contaminated soil. Methods of bioremediation include
bioaugmentation (seeding of hydrocarbonoclastic organisms into the contaminated
soil) and Biostimulation (provision of appropriate microbial food to stimulate
indigenous microbial growth).
Materials and Methods
• Pot experiment was conducted in Green-house of the Rivers State University of Science and
Technology Teaching and Research farm, Port Harcourt in the Rivers State of Nigeria. It is
located on latitude 4o501 N and longitudes 7o011E and on elevation of 18m above mean sea
level (FAO,1984). The area experiences two distinct seasons (raining and dry season). The
raining season starts from April and lasts till October with a brief period of dryness (August
Break). The rainfall is heavy with estimated annual range which may vary from 2000 to
2484mm (FAO, 1984, MANR, 2005). Rainfall pattern is bimodal with peaks in June and
September (Ukpong, 1992).
Top soil (0-15cm) was excavated from the uncultivated area of the Rivers State
University of Science and Technology Teaching and Research farm for the experiment.
Forty five (45) kilograms of soil were respectively polluted to 0,2,5 and 10% with Bonny
light crude oil of 0.835 relative density(specific gravity) and left undisturbed for seven days
to settled, then fifteen (15) kilograms of each of the pollution level were respectively
amended with 100kgha-1 Urea, 10tha-1 poultry manure and left unamended. Three (3)
kilograms of each of the treatment options were transferred into five (5) poly bags
measuring 30x25cm2 and seeded with 130 litres of 48 hours nutrient broth of
Acinetobacter clavatus, Bacillus subtilis, Pseudomonas aeruginosa and Corynebacterium
diphtheriae, left unseeded and replicated three times, then completely randomized and
arranged in a Greenhouse. The experiment was kept moistened at field capacity during the
study period (Page et al., 1982).
Soil Sampling
Before treatments, soil samples were analyzed for physical and
chemical properties. At week nine (9), soil was sampled for percent
total nitrogen, a vailable phosphorus and potassium.
• Analytical methods for soil
Soil pH was determined in water with glass electrodes in 1:2.50 soil
water ratio (Udo and Ogunwale, 1978); organic matter was
determined by methods of Nelson and Summers (1982): Total
nitrogen was determined by methods described by Udo et al.,
(2009); Calcium (Ca) and Magnesium(Mg) were extracted with
molar ammonium acetate; Potassium and Sodium concentrations
were determined by flame photometry as described by Udo et
al.,(2009); Aluminum plus hydrogen, was extracted with molar
Potassium chloride(KCL) and acidity determined by
titration(Mclean, 1965); effective cation exchange capacity was
determined according to Kamprath (1984). Av.P was determined by
methods described by Sparks (1996).
Statistical Analysis
• Analysis of variance according to the completely
randomized design of 0.05% probability (LSD)
level was used to compare the treatments effects.
• Table 1: Physicochemical Properties of the soil used for the study
Parameters Measured
pH (H2O): ratio 1:2.50
% organic carbon
Percent total N
Available phosphorus (mg/kg)
Ca (Cmol/kg)
Mg (Cmol/kg)
K (Cmol/kg)
% sand
% silt
% clay
Textural class
Sandy loan
• Table 2. Chemical Properties of Poultry
Manure used as soil amendment
Total N (%)
Phosphorus (mg/kg)
Calcium (cmol/kg)
Sodium (cmol/kg)
Potassium (cmol/kg)
• Table 3: Effect of treatments on soil available phosphorus (Av.P) (ppm)
Crude Oil Level
Week 0
week 9
0 1
267 314 388
1 2
208 430 490
220 227 245
189 300 310
218 225 255
200 260 330
225 249 260
199 284 390
230 250 273
210 300 360
353 399 434
400 480 510
332 363 388
360 430 440
309 323 354
320 350 400
333 327 384
340 370 480
315 336 390
332 420 468
309 315 387
500 580 640
206 233 243
435 485 458
210 225 251
403 429 463
255 239 268
397 440 470
219 206 247
434 460 461
220 242 254
240 280 340
208 221 230
300 319 396
183 207 221
299 356 378
85 110 227
282 393 360
181 199 205
309 337 383
LSD (0.05)
15.27 16.32 12.81
32.17 24.23 40.30
• Key: B0,B1,B2,B3,B4 = Soil without inculcation, Soil inoculated with A. clavatus, B.
Subtilis, P. aeruginsona and C. diphtheriae respectively.:0,1,2, = Soil without
amendment, Soil amended with urea and PM respectively
Table 4: Effects of treatments on total nitrogen
Treatment combination
Week 0
Week 9
Crude Oil Level
0 1
0.12 0.11 0.10
0.07 0.08 0.09
*0.10 0.12 0.12
0.04 0.06 0.08
*0.10 0.12 0.11
0.03 0.05 0.07
*0.10 0.10 0.12
0.04 0.05 0.06
*0.11 0.11 0.11
0.04 0.05 0.07
0.09 0.08 0.09
0.12 0.14 0.15
0.09 0.06 0.07
0.10 0.11 0.12
0.09 0.07 0.08
0.11 0.08 0.11
0.10 0.08 0.07
0.09 0.08 0.08
0.10 0.07 0.08
0.10 0.10 0.11
0.08 0.09 0.09
0.13 0.15 0.16
0.07 0.07 0.08
0.11 0.13 0.12
0.07 0.06* 0.08
0.11 0.12 0.12
0.07 0.07* 0.07
0.11 0.12 0.11
0.07 0.07 0.08
0.12 0.13 0.13
0.06 0.08 0.08
0.08 0.10 0.09
0.06 0.06 0.07
0.09 0.10 0.10
0.06 0.06 0.07
0.09 0.11 0.10
0.06 0.05* 0.06
0.10 0.12 0.10
0.06 0.06 0.06
LSD (0.05)
0.01 0.02 0.02
0.1 N.S N.S
Key: B0,B1,B2,B3,B4 = Soil without inoculcation, Soil inoculated with A. clavatus, B. Subtilis, P. aeruginsona and C. diphtheriae
respectively. :0,1,2, = Soil without amendment, Soil amended with urea and PM respectively.
:*= significant, NS= Not significant..
• After treatments, lower Av.P values obtained in bacterial seeded soil as
compared to that of unseeded soil may be due to immobilization of soil Av.P
by the Soil microorganisms utilizing Av.P as bacterial seeding is likely to
increase microbial cell in the soil. Also higher Av.P values obtained in the
amended soil as compared to the unamended soil suggests lack of nutrient
resources to support seeded microbial populations in the unamended soil.
Subsequent increase in Av.P at 2% crude oil pollution level in week 9 in
comparison to that of 5-10 crude oil pollutant level; therefore mean that at
2% crude oil pollution level effect on soil biochemical processes was
minimal at 2%, while at 5-10% some microbial vital functions may have
been inhibited in the presence of/and toxicity of high crude oil pollution
level as earlier reported by Xu and schurri (1990).
• In week 9, soil Av.P status improved at all levels of crude oil pollution level.
This may be due to subsequent changes in the Soil biochemical processes
with time to as degradation proceed. It also means that toxicity of crude oil
get reduced with time and that the degradation and mineralization of
phosphorus out of hazardous substances also improved with time as earlier
reported by Xu and Schurri, (1990). Isrimah et al., (1989) also observed
that availability of phosphorus is pH related.
Discussion conti.
• Etukudoh, (2011) observed that the presence of soil amendment (feed stuff)
and crude oil significantly stimulated bacterial growth, thus immobilization.
Therefore, lower Av.P values in the Urea amended soil as compared to that
of PM amended soil in week 9 indicated that the former was more effective
in regulating soil reaction (pH) to suite microbial activities. Considering the
Study period, this Suggests that PM (organic fertilizer) is better than the
Urea (inorganic fertilizer) in making phosphorus more available.
• Decrease in total N in the soil after bacterial seeding as compared to
unseeded soil may be due to increase in bacterial activities. This therefore
indicated that bacterial seeding and application of soil amendments were
effective for the process of phosphorus mineralization to take place.
Subsequent increase in total N at week 9 during the study period may be
due to increased in the rate of mineralization and vigor of nitrification as
earlier reported by Odu (1980) and Isirimah et al; (1989). Lower total N
values recorded at 2% crude oil pollution level as compared to other
pollution levels may be due to the attraction and spontaneous increase in
microbial population due to the presence of crude oil and soil amendments.
This was earlier observed by Ita and Essien (2002), who reported that at a
lower level of crude oil pollution level, hydrocarbon utilizers are attracted
and their increase stimulated more than in the highly contaminated or in
the uncontaminated soil.
Discussion conti.
• Decreased in soil K in the soil with increase in crude oil
pollution level was observed with significant differences
among treatments option in both week 1 and in week 9.
This indicated that crude oil pollution level as low as 2%
could affect K dynamic in the Soil. According to
Alexander (1979), the quantity of water soluble nonexchangeable K fraction is microbial in origin. Therefore
depression of soil organism due to crude oil and
presence of soil amendments and bacterial seeding are
synonymous with K depletion in the Soil.
In week 9, potassium (K) values were higher in the
Urea amended soil. It therefore means that PM could
better raise K status in the soil than that of Urea and that
bacterial seeding will result in the depletion of soil K.
• Effects of bioaugmentation and biostimulation on the availability of
phosphorus, nitrogen, and potassium in a crude oil contaminated
Coastal Plain Sands of southern Nigerian were evaluated.
Bioaugmentation resulted in the decrease of all tested parameters in
all treatment options. Soil seeded with C. diptheriae and A.
clavatus, P.aeruginosa made more phosphorus available at 0 and 2,
5 and 10% crude oil pollution levels respectively in all treatment
options while values of total N and Potassium were higher in the
unseeded soil. In general, Av.P and K values were higher in the PM
amended soil, total N values were also higher in the 0- 5% crude oil
pollution level amended with PM but at 10% crude oil pollution
levels values were higher in the urea amended
• This study suggests that 2-5% crude oil concentrated Coastal Plain
Sands is better amended with PM and Seeded with C. diphtheriae
while above 5% crude oil contamination should be amended with
Urea and Seeded with P. aeruginosa to bring about much desirable
NPK status in the soil..
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