Uptake of crude petroleum hydrocarbons by mudflat bacteria exposed to nitrogenous fertilizer plant effluents

In the Niger delta, due to the large-scale exploration and exploitation of crude petroleum, hydrocarbon spills frequently occur. The adverse impacts of these occurrences have frequently been causes for concerns in the area. However, in open estuarine environments, minor spills have short-term effects due to washout by stream water and increased microbial action. The microbial degradation of low level spills is enhanced by the ‘continuous’ input of nitrogenous fertilizer (NPK) components. The ‘continuous’ input assists in mitigating dilution effects of the stream water thus increasing the availability of nutrients to the petroleum degrading microbes. The net result is the increased recovery potential of this estuarine environment in the event of pollution by crude oil hydrocarbons. Enumeration of viable aerobic heterotrophic bacteria showed that counts were higher in hydrocarbon amended nitrogenous fertilizer plant effluent than in the raw effluent. The higher counts were accompanied by a rapid decline in the level of crude petroleum in the amended effluent. The ability of aerobic heterotrophic mudflat bacteria to grow on three different refined petroleum products namely kerosine, diesel and engine oil was also studied. At the levels tested, there were no appreciable differences in growth patterns of petroleum utilizing bacteria in three refined products after 5 weeks incubation at ambient temperature.


INTRODUCTION
Aquatic bacteria exhibit a variety of responses to pollutants.Whereas organic wastes frequently increase bacterial abundance, e.g. in river waters (Rheinheimer, 1965;Deufel, 1972;Coleman et al., 1974;Kachan, 1976;Starzecka, 1979) and sediments (Ossowska-Cypryk, 1981), inorganic wastes may provoke a different response.Inorganic wastes such as phosphates and nitrogenous materials that may be derived from fertilizer factory effluent discharges may result in algal blooms in streams and rivers.Pollution may also cause change in the taxonomic composition of aquatic bacterial communities.
For bacteria to grow efficiently they require about ten parts carbon to one part nitrogen (Olivieri et al., 1976).If the ratio is greater such as can occur during oil spills, e.g. 100 : 1 or 1000 : 1, growth of the bacteria and utilization of the carbon(s) will be retarded.Biodegradation by aquatic micro organisms plays an important role in the fate of organic pollutants.Biodegradation of petroleum hydrocarbons in the aquatic and terrestrial environments has been studied extensively (Blumer and Sass, 1972;Farrington, 1980;Lee, 1980;Fusey and Oudot, 1984;Oudot, 1984).
Several approaches at solving the problem of oil pollution have been described (Garret, 1969;Meijs et al., 1969;Wang et al., 1975;Olivieri et al., 1976), but each has its limitations.Numerous investigators have studied the possibility of using micro-organisms to increase the natural biodegradation of petroleum (Le-Petit and Barthelemy, 1968;Byrom and Beastall, 1971;Atlas and Bartha, 1972;Mulkins-Philips and Stewart, 1972;Soli and Bens, 1972;Schwarz et al., 1974), but it is too slow to have any practical importance for oil clean up.Inoculation of oilpolluted areas with oil-decomposing micro-organisms seems to be ineffective because of growth-limiting nitrogen and phosphorus concentrations in seawater (Le-Petit and Barthelemy, 1968;Zobell, 1969).Biodegradation of crude petroleum by heterotrophic bacteria in systems receiving NPK fertilizer factory effluents has been studied (Adoki et al., 1999).Reports show that urea and phosphorus sources were able to enhance biodegradation of crude petroleum.The present study further examines biodegradation of crude and refined petroleum by sediment bacteria in NPK fertilizer factory effluent.The results should provide an insight to the fate of crude petroleum and refined petroleum in an estuarine system receiving these fertilizer effluents.

Samples
Effluent and sediment samples from the outfall point of the National Fertilizer Factory at Onne in Rivers State, Niger delta were collected using procedures outlined by APHA (1975).

Biodegradation tests
Biodegradation tests were carried out in 1000 ml bottles.Only batch cultivations were monitored.To each of twelve 1000 ml presterilized bottles was added 495 ml of fertilizer factory effluent as nutrient medium, 5 ml of Bonny medium crude oil using a sterile glass pipette and 2 g sediment as inoculant.The bottles were subsequently stoppered and incubated at room temperature (28 ± 2ºC) with aeration/agitation through Teflon tubes by means of Teacax AP-1500 air pump.Seawater with no sediment addition was also included in the tests.

Water chemistry
The following physico-chemical parameters, which include pH, conductivity, turbidity, salinity, dissolved oxygen (fresh sample only) ammonium nitrogen, phosphate, nitrate and sulphate were determined by methods and procedures outlined by APHA (1975).

RESULTS AND DISCUSSION
Figure 1 shows changes in pH of fertilizer factory effluent by stream sediment bacteria induced by the addition of crude petroleum hydrocarbons.The figure shows that for about 4 weeks no significant trend in pH was observed in both the unpolluted and polluted conditions.However, beyond 4 weeks reversed trends were recorded.By contrast, no such reverse trends were recorded for phosphate and nitrate levels (Figure 2).The levels of these nutrients did not show very significant differences in the polluted and unpolluted conditions.However, a very sharp drop in phosphate level was recorded in the unpolluted condition after 5 weeks incubation at room temperature.Beyond 5 weeks, a very sharp decline was recorded in phosphate level in the unpolluted culture.Uptake of nitrate was generally higher in the unpolluted culture as reflected in the lower levels recorded between 2 and 6 weeks of incubation.Nutrient uptake was adversely affected by amendment with petroleum hydrocarbons.
Figure 3 shows that sulphate levels were generally low in hydrocarbon polluted samples.Uptake patterns were   however similar in both cultures.These trends were also exhibited in counts of viable aerobic heterotrophic bacteria.Counts were slightly higher in cultures exposed to petroleum hydrocarbons.This could be ascribed to the additional carbon source provided by the hydrocarbons (Figure 4).Similar trends were recorded in counts of petroleum-utilizing bacteria (Figure 4).Enumeration of viable aerobic heterotrophic bacteria showed that counts were higher in hydrocarbon amended effluent.The higher count was accompanied by a rapid decline in the level of crude petroleum in the amended effluent as shown in Figure 5.The ability of aerobic heterotrophic mudflat bacteria to grow on refined petroleum products was also studied.The results (Figure 6) show that there was no significant difference in growth patterns of petroleum utilizing bacteria in the three refined products tested after 5 weeks incubation at room temperature.
Several approaches at solving the problem of oil pollution have been described (Garret, 1969;Meijs et al., 1969;Wang et al., 1975;Olivieri et al., 1976), but each has its limitations.The possibility of using microorganisms to increase the natural biodegradation of petroleum (Le-Petit and Barthelemy, 1968, Byrom andBeastall, 1971;Atlas and Bartha, 1972;Mulkins-Philips and Stewart, 1972;Soli and Bens, 1972;Schwarz et al., 1974) has been investigated, but this process is too slow to have any practical importance for oil clean-up.Inoculation of oil-polluted areas with oil-decomposing micro-organisms seems to be ineffective because of growth-limiting nitrogen and phosphorus concentrations in seawater (Le-Petit and Barthelemy, 1968;Zobell, 1969).
In the Niger delta, due to the large-scale exploration and exploitation of crude petroleum hydrocarbon spills frequently occur.The adverse impacts of these occurrences have frequently been causes for concerns in the area.However, in open estuarine environments such as the NAFCON creek, minor spills have short-term effects due to washout by stream water and increased microbial action.The microbial degradation of low level spills is enhanced by the 'continuous' input of nitrogenous fertilizer (NPK) components.The 'continuous' input assists in mitigating dilution effects of the stream water thus increasing the availability of nutrients to the petroleum degrading microbes.The net result is the increased recovery potential of this estuarine environment in the event of pollution by crude oil hydrocarbons.

Figure 1 .
Figure 1.Bacteria induced changes in pH of fertilizer factory effluent amended with crude petroleum hydrocarbons.

Figure 2 .
Figure 2. Nitrate and phosphate profiles of fertilizers factory effluent amended with crude petroleum hydrocarbons.

Figure 3 .
Figure 3. Sulphate profile of fertilizer factory effluent amended with crude petroleum hydrocarbons.

Figure 4 .
Figure 4. Viable counts of bacteria in crude petroleum hydrocarbon amended effluent.

Figure 5 .
Figure 5. Uptake of crude petroleum hydrocarbons by aerobic heterotrophic bacteria in fertilizer effluent.

Figure 6 .
Figure 6.Growth profile of aerobic heterotrophic bacteria in cultures amended with refined petroleum products.