EFFECTS OF WASTE DRILLING FLUID ON BACTERIAL ISOLATES
FROM A MANGROVE SWAMP OILWELL IN
THE NIGER DELTA OF NIGERIA

BY
BENKA-COKER, M.O. & OLUMAGIN, A
DEPARTMENT OF MICROBIOLOGY
UNIVERSITY OF BENIN
BENIN CITY
NIGERIA

ABSTRACT

Four bacterial strains isolated from drilling mud cuttings collected
from a mangrove swamp oilwell in the Niger Delta of Nigeria were
cultured aerobically in the presence of 1.0% waste drilling fluid to
determine the effect of the waste on their growth. A 2-h lag phase
of growth was produced by the waste in cultures Micrococcus and
Pseudomonas species, while the waste increased the lag phases of
Alcaligenes and Staphylococcus species to 4 h. The exponential
phases of growth of Staphylococcus and Pseudomonas spp were
depressed by the waste drilling fluid but the waste stimulated the
exponential phases of Micrococcus and Alcaligenes spp. There was
enhancement of the growth rate of Alcaligenes and Mircococcus spp
while those of Staphylococcus and Pseudomonas spp were decreased.
The depression in growth of Staphyloccus and Pseudomonas spp in
the presence of the waste drilling fluid might lead to a degree in
their contribution to the removal of the waste from the environment
during spillage or disposal, and therefore, may lead to an
accumulation of the waste in the environment. 

WASTE DRILLING
ADVERSE EFFECTS OF WASTE DRILLING

INTRODUCTION

The increase in activities of petroleum exploration and production
has brought about an increase in the discharge of operational
materials such as drilling fluids and mud cuttings into our
environment. The impact of these waste in the Niger Delta
ecosystems of Nigeria is an obvious environmental concern 
particularly with regards to the persistence and ecotoxicity of these
wastes (Okpokwasili & Njoku, 1989; Benka-Coker & Ekundayo, 1995).
Drilling muds are primarily introduced with rotary drilling to
continuously remove drill cuttings during oil and gas extraction
(Ranney, 1979). Drilling muds contain suspensions of solids in
liquids or in liquid emulsion, which chemical additives to modify
their properties.

In off-shore drilling, waste are disposed into surrounding waters;
For wells in swampy locations, the wastes are disposed into swamp
cuts adjacent to the rig; while on land-based operations, even though
wastes are disposed into burrow or waste pits, quite often they
overflow into adjacent water bodies and arable farmlands (ifeadi  et
al 1985). The effect of waste drilling fluids on swamp ecology may
be more serious than on the marine environment because swamp cuts
are stagnant with only occasional seasonal flooding or tidal actions.
Seepages and infiltration to groundwater as well as seasonal
flooding and tidal action provide a direct communication with and
thus, pollution of, ground- and surface-waters and nearby
terrestrial environment (ifeadi et al. 1985). 

Discharged drilling mud represents a potential source of trace
metals, hydrocarbons and suspended solids to the environment.
Moreover, drilling muds contain surfactants, hydrocarbon solvents
and stablizers (Muhlemann, 1986) which may confer recalcitrance on
the drilling muds (Odokuma & Okpokwasili, 1992). 

Little research has been done on the fate of organic components or
drilling fluid following its discharge into tropical environment. Of
particular concern are the lignosulonates and their degradation
products, as well as lubricants, which have been identified as the
most toxic component of water-based drilling fluids (Boesch &
Rabalais, 1987) which are routinely used in Nigeria. The major
additives used in water-based drilling fluids have been found to be
slowly degraded by microorganisms  (Boesch & Rabalais, 1987) and
in most cases these compounds remain in the environment long after
being introduced. However, studies have shown that naturally
occuring microbial degradation mechanisms in the environment
result in the biodestruction of toxic substances such as
hydrocarbons (Colwell & Walker, 1977; Atlas, 1981, Okpokwasili et
al, 11986; Amanchukwu et al, 1989), surfactants (Higgins & Burns,
1975; Okpokwasili & Nwabuzor, 1988; Okpokwasili & Olisa 1990) and
dispersants (Odokuma & Okpokwasili, 1992). 

The current interest on the effect of waste drilling fluids
discharged into ecosystems is as a result of its impact on the biota
exposed to it. The study reported here was undertaking to determine
the effects of waste drilling fluid obtained from a mangrove swamp
oilwell, on bacteria isolated from drill mud cuttings from the same
oilwell. 

MATERIALS AND METHODS

Isolation of test microorganisms 

The source of microorganisms tested was drilling mud cuttings
obtained from a mangrove swamp oilfield in the Niger Delta of
Nigeria. The isolation medium was mineral salts medium previously
described by Mills et al. (1978), and modified by Okpokwasili &
Okorie (1988). The medium contained : 2.0 g NaCl; 0.42 g MgSO4,
7H2O; 0.29 g KCI; 0.89 g KH2PO4; 1.25 g. Na2HPO4; o.42 g NaNO3; 1 L 
deionised water. The pH of the medium was adjusted to 7.4.
Bacteriological ager (Oxoid) was added to obtain solid medium at a
concentration of 1.5% (w/v) when necessary. The vapour phase
transfer method described by Amund and Igiri (1990) was used in the
isolation of the bacteria present in the drill mud cuttings. Twenty
grammes (20 g) of composite samples of the drill mud cuttings were
added to 180 ml sterile mineral salts solution and incubated at 30û
C ffor 48 h. Decimal serial dilutions of the broth were then prepared
with aliquots of sterile mineral salts solution in test tubes as
diluent. Dilutions of the drill mud cuttings in 0.1 ml  volumes were
inoculated onto plates of mineral salts agar by the spread plate
technique in duplicates. The inoculated ager plates were inverted
onto the lid of the same Petri dish in which 9.0 cm sterile Whatman
No.1  filter paper saturated with sterile waste drilling fluid was
placed. The filter paper supplied the drilling fluid by vapour transfer
to the inoculum. The inoculated plates were incubated at 30û C for
14 days (Okpokwasili & Okorie, 1988). Control were set up with
duplicate mineral salts ager plates containing : 

no drill mud cuttings and no waste drilling fluid.

No waste drilling fluid but containing drill mud cuttings; and no drill
mud cuttings but containing waste drilling fluid. 

The bacteria growing on the agar plates were purified and isolated.
The pure isolates were stored on nutrient agar slants for further
tests. They were examined for colonial morphology,
micromorphology and biochemical characteristics by means of
determinative schemes of Buchanan & Gibbons (1974) and Cowan
(1974).

Effect of waste drilling fluid on growth of bacterial
isolates 

Four isolates, identified as species of Alcaligenes Micrococcus,
Pseudomonas  and Staphylococcus, obtained from the drill mud
cuttings, were used for the tests. The culture medium used was that
described by Nnubia and Okpokwasili (1991) and it contained;  0.05 g
sodium glycerophosphate; 0.05 ml glycerol; 0.1 g bacteriological
peptone; 0.1 g yeast extract; 1 L mineral salts solution. The
physico-chemical characteristics of the waste drilling fluid used
are shown in Table 1. 

The isolates were streaked-inoculated onto nutrient agar plates
from stock culture slants and incubated at 30ûC for 48 h to check
viability and purity.  A loopfull of each of the test isolates from the
purity plates was inoculated into test tubes containing 9.0 ml of
sterile nutrient broth. These were incubated at 30C for 48 h. The
nutrient broth culture were used to prepare a standard calibration
curve using the method described by Pawsey (1974). 

Two sets of duplicate 250 ml sterile cotton-stoppered Erlenmeyer
flasks, each containing 90.0 ml of the culture medium, were
prepared for each bacterial isolate. The bacterial cells were diluted
appropriately (1.4 x 10 (5), 1.7 x 10 (7), 1.2 x 10(7) and 1.9 x 10 (5)
cfu ml-1 for Alcaligenes, Micrococcus, Pseudomonas and
Staphylococcus species respectively ) and 10.0 ml of each of the
dilutions added to both sets of duplicate flasks, each containing 90.0
ml culture medium, as inoculum. Waste drilling fluid was added to a
set of the inoculated flasks at a 1.0 % concentration. This set of
flasks served as the test flasks while the second set with no waste
drilling fluid served as the control flasks. Both  sets of flasks (test
and control) were incubated with shaking at 120 rpm at 30C for 14
h. The absorbance was read at a 2 h interval to follow the growth
curve. Also determined was the viable bacterial counts on nutrient
agar plates every two hours. 

RESULTS AND DISCUSSION 

The effects of waste drilling fluid on species of Alcaligenes,
Micrococcus, Pseudomonas and Staphylococcus in batch culture are
shown in Figures 1 to 4. The waste drilling fluid increased the lag
phase of Alcaligenes sp to 4 h (Fig. 1) while the waste chemical
stimulated exponential phase of growth of the organism  (Fig. 1). The
waste drilling fluid produced a lag phase of 2 h for Micrococcus sp.
but stimulated the exponential phase of growth of the same
organism (Fig. 2). In the presence of the waste, the lag phase of
growth of Staphylococcus sp was 4 h (Fig. 3). The exponential growth
phase was also depressed (Fig. 3). The waste drilling fluid also
produced a 2 h lag phase for Pseudomonas sp and depressed the
exponential growth phase (Fig. 4). 

The growth rates at the exponential phase of growth of the
organisms between the6th and 110th hours of incubation, using the
method described by Stanier et al (1977) for calculation, are
presented in Table 2. The growth rates for controls were 0.16, 0.15
and 0.14 h-1 for species of Alcaligenes, Micrococcus, Pseudomonas
and Staphylococcus respectively. Comparative analysis of growth
rates relative to the control values showed that the waste drilling
fluid depressed the growth rates of Staphylococcus and
Pseudomonas spp while the growth rates of species of Alcaligenes
and Micrococcus were stimulated by the waste drilling fluid. 

The results obtained have shown that the effect of the waste
drilling fluid on the bacterial isolates showed differential
responses of the isolates to the waste. The pattern of increase and
decrease of lag and exponential growth phases of the isolates
corroborates the observation of Cadder and Lader (1976). Nnubia and
Okpokwasili (1991) working on the effect of dispersants on
bacterial isolates observed a similar trend in the bacterial response
to the different chemical dispersants used. It seems that the waste
drilling fluid elicited differential toxic effects on the four bacterial
isolates. While the growth rates of Alcaligenes and Micrococcus spp
may have been unaffected by the waste, those of Staphylococcus and
Pseudomonas spp were affected by the waste. The response showed
by the species of Alcaligenes and Micrococcus may be due to the
genetic ability of the organisms to resist pollutional stress and
detoxify  the waste drilling fluid and its chemical components
through biodegradative means (Westlake, 1982; Okpokwasili et al,
1986).

These bacterial isolates are among those forming the bacterial
community of the oilfield location studied. The depression in growth
of Staphylococcus and Pseudomonas spp in the presence of the waste
might lead to a decrease in their contribution to the removal of the
waste from the environment during spillage or disposal and
therefore may result in an accumulation of the waste in the
environment. This may be detrimental to the ecosystem as a whole
as a result of the toxic components of the waste chemical (Boesch &
Rabalais 1987). However, the depression in the role of
Staphylococcus and Pseudomonas in the removal of the waste from
the environment may not be apparent because Alcaligenes and
Micrococcus spp may assume an enhanced role in the biodegradation
of the waste, while Staphylococcus and Pseudomonas spp being
members of the same consortium, may then act on the breakdown
products of the waste drilling fluid after the initial attack had
occurred, as secondary metabolisers (Nnubia & Okpokwasili, 1993). 

The results obtained in this study suggest that the effects of the
waste drilling fluid are likely to result in selective enrichment for,
growth limitation of, or toxicity to, specific generic bacteria which
may be important in the ultimate removal of the waste from the
environment. The presence of Alcaligenes and Micrococcus is
therefore, of ecological significance. They can be multiplied and
used in the treatment of the drilling wastes before disposal or
employed as inoculants in clean up exercises when the environment
gets contaminated with drilling discharges thereby sustaining the
environment to its original stance. 

The authors are grateful to the Shell Petroleum Development
Company (Nigeria) Limited for providing the drill mud cutting and
waste drilling fluid used in this study. 

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Table 1. Chemical and physical parameters of waste drilling fluids
used in the experiments.
---------------------------------------------------------------
pH                                                   9.7
Electrical conductivity(gm/cm)                     300.0
Special gravity (g/cm)                               1.33
Bicarbonate hardness(ppm)                          375.0
Potassium(meq/100g)                                   3.1
Sodium(meq/100g)                                     2.7
Calcium (meq/100g)                                   0.25
Magnesium(meq/100g)                                  0.80
Nitrate-nitrogen                                     1.53
Ammonium-nitrogen(ppm)                               8.0         

Phosphate(ppm)                                     170.0
Sulphate(ppm)                                      260.0
Chloride(ppm)                                        7.0
Silicate(ppm)                                       28.0
Iron (ppm)                                         120.0
Zinc(ppm)                                           41.0
Manganese(ppm)                                      75.0
Copper(ppm)                                          0.65
Lead(ppm)                                           20.0
Chromium(ppm)                                        5.4
Barium(ppm)                                        178.9