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Studies on the biodegradation potential of some micro-organisms isolated from water systems of two petroleum producing areas in Nigeria. Nigerian Journal of Botany. 1: 81-90.

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Nigerian Journal of Botany, Vol. 1. pp. 81—90, 1988. 81
STUDIES ON THE BIODEGRADATION POTENTIAL OF SOME MICRO
ORGANISMS ISOLATED FROM WATER SYSTEMS OF TWO PETROLEUM
PRODUCING AREAS IN NIGERIA
by
0. OBIRE
Department of Microbiology*
University o f Benin
Benin, Bendel State
Nigeria
(Received April 6,1988)
ABSTRACT
Some microorganisms isolated from water systems of two petroleum producing areas of
Nigeria were tested for their ability to degrade crude oils. Population levels of tota l hetero-
trophic bacteria and hydrocarbon degrading bacteria were much higher than those of yeasts
and moulds. The oil degrading bacterial isolates were Pseudomonas fluorescens, Acinetobacter
Iw offi and Micrococcus varians while the yeasts were Candida Upolytica, Rhodotorula Sp.,
Saccharomyces Sp., and Sporobolomyces Sp. The moulds were Aspergillus niger, Aspergillus
terreus, Blastomyces Sp., Botryodiplodia theobromae, Fusarium Sp., Nigrospora Sp., Penici-
llium chrysogenum, Penicillium glabrum, Pleurophragmium Sp., and THchoderma harzianum.
The best oil degrading bacterium, yeast and mould were Pseudomonas fluorescens, Candida
Upolytica and THchoderma harzianum respectively. Forcados blend was more rapidly and
extensively degraded than the heavier Bonny medium crude oil with a low saturate content.
The studies also showed th at NO"^ and PO 4 supplements enhanced biodegradation of oil.
INTRODUCTION
The increase in demand for crude oil as a source of energy and as a primary raw material
for industries has resulted in an increase in its production, transportation and refining which in
turn has resulted in gross pollution of the environment (Gutnick and Rosenberg, 1977). The
yearly influx of petroleum pollutants into the world’s oceans has been estimated to be as high
as six million metric tonnes (National Academy of Sciences, 1975). Apparently, most of this
petroleum pollutants eventually undergoes microbial degradation (Lee, 1980) but at a rate that is
very slow under natural conditions. The current mechanical, physical and chemical methods
of ridding the environment of spilled oil have proved to have harmful effect on the ecosystems
(Nelson-Smith, 1973). Therefore, to conserve species diversity and ecosystem stability, micro
organisms may be the ultimate agents of getting rict of spilled oil from the environment. Degrada
tion can be stimulated by inoculating the oil slicks with efficient microbial species and by the
addition of nutrients.
Various commercial inocula, such as “Petrodeg”,Petrobac”,Ekolo-Gest”, and “DBC —
Bacteria” had appeared on the market in developed countries as being effective for oil cleanup
(Atlas and Bartha, 1973; Barthaand Atlas, 1977). Since microorganisms are ubiquitous in nature
and are habitat specific, there is therefore the need to isolate and study indigenous microbial
flora capable of utilizing petroleum hydrocarbons in the tropics.
Present Address
^Department of Biology, Federal University of Technology Akure, Ondo State, Nigeria.
82 OBIRE, O.
This study reports the presence of oil-degrading microorganisms in the water systems of
Forcados river and Chanomi creek of Bendel State in Nigeria; their oil biodegradation potential
and also the effect of nutrient supplement on their oil biodegradation ability.
MATERIALS AND METHODS
The experimental stations are the Forcados river and Chanomi creek, two oil producing areas
of Bendel State of Nigeria. The water in Chanomi creek was oil polluted eleven months before the experi
mental period. Water samples were taken from two sites on forcados river and from five sites on Chanomic
creek. The map of the study area is shown in Fig 1.
Forcados blend crude oil produced in Bendel State and Bonny medium crude oil produced in Rivers
State were used in the study for comparison. The Bonny medium crude oil is heavier than the Forcados blend
crude oil, which contains more saturate fraction (Obire, 1985)
Isolation, identification and enumeration o f oil-degrading microorganisms
Oil-degrading microorganisms were isolated after enrichment of w ater samples on Forcados blend
crude oil in the minimal salts medium of Mulkins—Phillips and Stewart (1974) and the trace elements stock
of Bauchop and Elsden (1969). Enrichment was carried out by inoculating two millilitres of each water
sample into 250ml aliquots of the medium containing 0.1% (v/v) millipore filter-sterilized crude oil as sole
carbon source. The fcnrichment cultures were incubated on a rotary shaker at 180rpm at 28 + 2°C. After
48hr, two millilitres of the enrichment culture were transferred to a new batch of the enrichment medium and
incubated for a further 48hrs. Pure cultures were isolated by plating out serial dilutions of 0.1ml aliquots of
the enriched cultures onto nutrient agar plates to obtain discrete bacterial colonies and onto potato dextrose
agar plates to obtain discrete fungi! isolates.
“Oil agar” was prepared by the addition of 1% (v/v) sterlized crude oil to sterilized minimal salts
agar which was cooled to 45 °C. The agar and the crude oil were mixed thoroughly and dispensed into Petri
dishes to solidify. The ability of isolated strains to degrade crude oil was tested by streaking single colonies
of bacteria or yeasts onto oil agar plates. Discs of moulds were, • however, used to inoculate oil agar plates.
Pure cultures th at developed and showed oil degradation potential by a zone of clearance of oil, were identi
fied on the basis of their cultural, morphological and physiological characteristics and by reference to Bucha
nan and Gibbon (1974), Cowan and Steel (1974), B arnett and Hunter (1972), and Pitt (1979).
Oil agar plates were inoculated with 0.1 ml aliquots of serial dilution of the water samples. Plates were
incubated at 28 + 2°C for 5—7 days. Colonies which developed and showed zone of clearance of oil were
counted as oil-degrading microorganisms. Oil agar plates without inoculum served as control.
Nutrient agar plates to which 5% actidione (Cycloheximide) was incorporated for inhibition o f fungi
were used fcr total viable counts for heterotrophic bacteria. Potato dextrose agar places to which 0.5%
streptomycin and 0.5% ampicillin were incorporated were used for total viable counts of yeasts and moulds.
Determination o f the Biodegradation of crude oil by the oil degrading isolates
The bacterial and yeast isolates were separately suspended in 0.01 M sodium phosphate buffer
(p ^ 7.0). An aliquot (0.1ml) of each suspension was inoculated into a univerasal bottle containing 10 ml
sterilized minimal salts liquid medium and 1.0 ml sterilized crude oil. Discs of moulds were used as inocula
in the set up for moulds. Uninoculated set ups served as controls. The degradation of the oil cultures were
monitored at 7 days intervals for 28 days using gravimetric method (Obrire, 1985) by extracting a definite
volume of the culture medium containing oil as sole carbon source with carbon tetrachloride. The solvent was
then allowed to evaporate and the residue was weighed by means of a sensitive chemical balance. The values
obtained were subtracted from the original weight of the oil a t the onset of the experiments and that o f the
control.
Determination o f the effect of nitrate and phosphate on oil biodegradation
The following varying concentrations of KNO3 jmd K2HP04 (Oppenheimer, et at, 1980) were pre
pared in one litre of charcoal filtered Forcados river water;
0m g/l NO3 + 10m g/l PQ3~4
5 mg/1 NO3 + 10 mg/1 P03"4
25 mg/1 NO3 + 10 mg/l P0 3_4
100 mg/1 NO3 + 0.1m g/l P 0 3"4
Microorganisms from Petroleum Producing Areas. 83
100 mg/l NO3 + 1.0mg /l P03"4
100 mg/l NO^ + 10 mg/l P 03"4
0 mg/l NO^ + 0 mg/l P 03"4
Ten ml samples ot each mixture were used as experimental culture solutions. Each culture series was
inoculated with 1.0 ml of sterilized Forcados blend crude oil. Unionoculated set ups served as controls. The
biodegradation of oil was monitored by the gravimetric method at 3 days intervals for 12 days.
RESULTS
Isolation, identification and enumeration of oil-degrading microorganisms
Three groups of oil-degrading bacteria were isolated. Pseudomonas fluorescens and Acin<>-
tobacter Iwoffi were isolated from both Forcados river and Chanomi creek. Micrococus varians
was isolated only from the Chanomi creek.
The oil-degrading yeasts isolated were Candida Upolytica, Saccharomyces sp, Sporobolo-
myces sp. and Rhodotorulasp. from both Forcados river and Chanomi creek.
The following moulds were isolated from both Forcados river and Chanomi creek: Asper
gillus terreus, Aspergillus niger, Blastomyces sp, Penicillium chrysogenum and Penicillium
glabrum while Botryodiplodia theohromae, Fusarium sp, Nigrospora sp, Pleurophragmium sp
aid Trichoderma harzianum were isolated from Chanomi creek.
Tlie mean densities of heterotrophic microorganisms in water samples from the different
sites at the two experimental stations are shown in Table 1. The bacteria were the most abun
dant isolates while the moulds were the least abundant.
The mean densities of hydrocarbon-degrading microorganisms in the water samples are
shown in Table 2. The highest hydrocarbon-degrading isolates were the bacteria and the least
were the moulds. Samples from Chanomi creek had more bacteria, yeast and mould degraders
than samples from Forcados river.
The percentage of hydrocarbon-degrading microorganisms in relation to the total popula
tion of heterotrophic microorganisms in the different stations is shown in Table 3, The percen
tage of hydrocarbon-degrading bacteria, yeast and moulds from Chanomi creek site 3 was higher
than corresponding values from other sites.
TABLE 1
Mean density of heterotrophic microorganisms in colony—forming—units per ml
(cfu/ml) o f water samples from Fore ados river and Chanomi creek
84 OBIRE, O.
Sampling Bacteria D ensity (cfu/m l)
Yeasts Moulds
Stations Sites
13.2 x 104 2.4 x 102 3.2 x 102
Forcados river 22.6 x 104 1.7 x 102 0.8 x 102
11.7 x 105 4.6 x 102 1.1 x 102
23.9 x 105 1.6 x 102 1.9 x 102
34.5 x 103 4„9 x 102 2.3 x 102
Chanomi creek 44.2 x 104 6.9 x 102 3.8 x 102
56.3 x 104 6.0 xlO 2 4.0 x 102
TABLE 2
Mean density of hydrocarbon—degrading microcoorganisms in colony—form ing units per ml
(cfu/ml) o f water samples fro m Forcados River and Chanomi creek
Density (cfufml)
Stations Sampling
sites Bacteria Yeasts Moulds
13.4'x 102 0.6 x 102 0.6 x 102
Forcados river 22.3 x 102 0.8 x 102 0„3 x 102
15.5 x 102 1.0 x 102 0.2 x 102
24.7 x 102 0.5 x 102 0.7 x 102
Chanomi creek 3 5.0 x 102 1.2 x 102 0.2 x 102
4 3.1 x 102 2.3 x 102 1.3 x 102
5 4.3 x 102 1.9 x 102 1.1 x 102
Determination of the biodegradation of crude oil by the oil-degrading isolates
The biodegradation of Forcados blend crude oil (mg oil/mg dry weight) by cultures of
bacteria, yeasts and moulds is shown in Table 4. The oil degrading ability of the organisms was
highest on the 7th day of incubation in liquid minimal salts crude oil medium. Of the bacterial
isolates, P. fluorescens from Chanomi creek had the highest oil biodegradation capability while
M. varians had the least. The bacterial isolates from Chanomi creek were better degraders than
corresponding isolates from Forcados river. The yeast isolate C. lipolytica had 530 mg oil per
mg dry weight being the highest among the yeast isolates.
The oil degrading ability of the moulds was highest on the 14th day of'incubation. T.
harziamm had the highest biodegradation of 692.5 mg oil/mg dry weight On the 14th day of
incubation. Generally the amount of biodegradation per unit time was less* than that in either
bactem oi yeast isolates.
The biodegradation of Bonny medium crude oil by pure cultures of bacteria and moulds
are shown in Table 5. The oil degrading ability of the organisms was highest on the 28th day of
Microorganism s from Petroleum Producing Areas. 85
incubation for both bacteria and moulds. Bonny medium crude oil was better degraded than
Forcados crude oil within the same period.
TABLES
Percentage o f hydrocarbon—degrading microorganisms in relation to the total population of
heterotropic microorganisms in water samples from Forcados River and Chanomic creek
Stations Sampling Bacteria Ye&sts Moulds
sites (%) (%) (%)
11.04 0.18 0.18
Forcados river 2 0.88 0.30 0.11
10.32 0.06 0.01
20.12 0.01 0.02
Chanomi creek 39.58 2.30 0.38
40.72 0.53 0.30
50.67 0.30 0.17
Effect o f nitrate and phosphate on oil biodegradation
The effect of varying concentrations of nitrate and phosphate on the biodegradation of
oil by P. fluorescens, C. Upolytica and T. harzianum is as shown in Table 6. For maximal oil
biodegradation, both bacteria and yeasts required a higher concentration of nitrate and pho
sphate than the mould.
DISCUSSION
The hydrocarbon-degrading bacterial species isolated in this study are similar to those
reported by Atlas (1981). The yeast isolates are also comparable to those reported by Ahearn
et al (1971). Some of the moulds,Penicillium sp, Aspergillus sp, Fusarium sp and Trichoderma
sp were reported as hydrocarbon-degraders by previous workers (Llanos and Kjoller, 1976),
However, the other moulds such as Botryodiplodia theobromae, Blastomyces sp, Nigrospora
sp and Pleurophragmium sp have not been reported as hydrocarbon degraders.
The percentage of hydrocarbon-degrading microorganisms was low relative to total
heterotrophic microorganisms (Table 3). The values are similar to those obtained by Oppen-
heimer et al (1980) for the Georgia Shelf area of United States of America where oil explora
tion activity was being carried out and those reported by Atlas (1981). The presence of micro
organisms in an environment is directly related to the availability of nutrients.
Oppenheimer et al (1980) affirmed that due to the continuity and dynamic nature of
water systems, statistical analysis of the distribution of microorganisms in water systems may
be misleading or entirely wrong. Statistical analysis was therefore not used in this study.
Oil is a complex substrate and its rate of degradation by microorganisms depends on the
type of microorganisms. The biodegradation of crude oil in this study, by the bacterial and
yeast isolates was highest in the first week of incubation while that of the moulds was highest
after the second week of incubation. It is likely that the bacterial and yeast isolates secrete
digestive enzymes or emulsifiers capable of degrading oil, faster than the secretion of such sub
stances by moulds. They were therefore able to degrade crude oil faster than the moulds.
u
TABLE 5
Cumulative biodegradation o f Bonny medium crude oil (mg oil/mg dry weight) by pure cultures o f
bacteria and mould at 28 + 2°C for 28 days
Bacteria Moulds
Incubation
Period
(days)
P. fluorescens*
A. Iwoffi*
M, Parians *
.. .
*
&
.+. * S t
§4 % * .g | *
« . 5 .so &
^ ^ 3 « £ * 4 *• o; «
710 6 3 6' 3 2 6 - 1 2 1 2 1 8
U30 26 15 11 8 8 14 4 5 3 5 3 18
21 6? 57 35 33 28 38 48 24 13 12 26 22 55
28 104 92 67 73 74 77 84 50 28 26 64 59 13
Isolates from Chanomi creek
+ Isolates from Forcados river oo
-4
Micro-organisms from Petroleum Producing Areas
88 OBIRE, O.
The bacteria were the most abundant and the most effective hydrocarbon-degraders while
the moulds were the least effective degraders. The fast generation rate of bacteria and yeast
might be responsible for this. The surface area/volume ratios of bacteria and yeast are higher
than that of moulds. Bacteria and yeast will therefore have more of their surfaces in direct con
tact with the substrate (crude oil), thus resulting in faster rate of degradation.
The presence of oil as substrate in the water systems seemed to determine the rate and
activities of degraders. Chanomi creek was oil polluted a few months before this study. There
were more oil degraders in ihe water system than in the water from Forcados river. There were
also more hydrocarbon-degraders at the site of the burst pipeline on Chanomi creek than from
the other sites (Table 3). The available oil must have served as a carbon source for the initial
nultiplication and growth of the degraders.
TABLE 6
Effects of varying concentrations of nitrate and phosphate on biodegradation o f oU (mg oilmg dry wt o f
isolate) by P. fluorescens, G. lipoltica and T. harzianum at 28 + 2 °C fpr 12 days
Amount of biodegradetion (mg oil/mg dry wt of isolate) Concentration of nutrients/litre in die medium
0 mg NO j 5 mg NO j 25 mg N OJ lOOmg NOf WOmgN OJ' lOOmg NOj Omg N O J
+++++ + +
Isolates Incubation
Period (days)
3-
lOmg PO 4 3.
lOmgPO 4 lOmg P 0^4 0 jm g p0^4 1.0mgPQ ?~4 lOmg PO^~4 omg PO^~4
3 10 20 10 5 10 55
*seudomona$ 6 30 30 30 20 60 20 20
fluoresces \ 9 590 610 620 520 630 540 580
12 40 20 30 50 50 50 20
3 10 20 10 520 510
Candida 630 30 40 30 30 20 30
lipotytica 9530 610 620 610 670 590 600
12 10 20 20 20 20 20 10
310 20 20 20 20 20 10
Trkfooderma 620 40 40 10 30 10 25
harzianum 9 580 630 620 610 600 550 610
12 50 50 30 50 50 50 30
The study also showed that degraders are capable of adaptation. The isolates rapidly degra
ded Forcados blend within 14 days of incubation while degradation of Bonny crude oil was
slow within 14 days of incubation but increased afterwards (Table 5). In addition, the rate of
degradation might also have been affected by the differences in the chemical composition of
the crude oils. The Forcados blend, a lighter crude oil with high saturate content, was more
rapidly and extensively degraded than the heavier Bonny medium crude oil with a low saturate
content. Westlake et al., 1974 aftd Higgins et al., 1981 also reported similar findings.
Micro-orginisms from Petroleum Producing Areas. 89
Microbial oil degradation responded positively to nitrogen and phosphorus addition.
However, the non-enriched cultures showed appreciable oil degradation which showed that the
available nutrient concentrations in the water samples were adequate to support hydrocarbon
degradation. Nutrient concentration usually becomes limiting in times of oil spills (Atlas, 1981;
Oppenheimer et aL, 1980), during which period additional nitrogen and phosphorus would be
needed to enhance metabolic degradation of hydrocarbons (Oppenheimer et al., 1980).
The understanding of microbial hydrocarbon degradation process in the tropics, should
make the development of models of highly effective hydrocarbon-degrading inocula possible.
Such will bring about the least undesirable ecological side effects in the environment.
I am grateful to Prof. J. A. Ekundayo for his advice, guidance and encouragement while
on this project.
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90 OBIRE, 0.
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In the previous decades, the pollutionPollution of the environmentEnvironment has increased due to rapid growth in human population, urbanization, and industrialization. The indiscriminate use of the resources by the ever-increasing human population has increased the pollutionPollution of environmental sites mainly by industrial effluents, sewage, surface runoff from agricultural fields, fertilizers, and pesticidesPesticides. The widespread degradationDegradation of natural ecosystems by pollutants cause serious threatsThreats as they get into various environmentsEnvironment (air, soil, and water) and accumulate in food chains. Conventional methods used in the remediation/treatment processes of contaminated environmentsEnvironment are expensive, inefficient, leave toxic residues in the environmentEnvironment, and are not reliable. Therefore, bioremediationBioremediation is viewed as an evolving and promising technique in the treatment of various types of contaminants. BioremediationBioremediation of polluted environmentsEnvironment has proven successful, efficient, and reliable because of its environmentEnvironment-friendly features. The basic principle of bioremediationBioremediation is natural attenuation also known as intrinsic bioremediationBioremediation, which involves the remediation of polluted sites naturally in the environmentsEnvironment without any anthropogenicAnthropogenic interference to reduce the toxicity, mass, and concentration of pollutants in those media. BioremediationBioremediation may involve the use of plants, fungi, bacteria, and other algae for remediation but the process mainly focuses on the use of microorganisms. This chapter, therefore, tries to provide a comprehensive knowledge of the potential role of bioremediationBioremediation technologies and processes for the potential remediation of contaminated sites.
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