Response of Pollution Bio-monitors : Pseudomonas and Bacillus species to Local “ Kpo-fire diesel ” and Industrial Refined Diesel in Freshwater Environment

This paper reports the response of pollution bio-monitors such as Pseudomonas and Bacillus species to local and industrial diesel in freshwater environments of the Niger Delta, Nigeria. The illegal local refined diesel is popularly referred to as 'Bunkery diesel' or 'Kpo-fire diesel' in Niger Delta, Nigeria. It uses simply three major step/process; cooking or 'boiling' of crude oil, distillation and extraction at different temperatures into Petrol, Diesel and Kerosene but majorly Diesel and Kerosene are the final products. Standard ecotoxicological bioassay using pollution bio-monitors as indicator tools with different concentrations of local and industrial refined diesel were employed. Local and Industrial refined diesel concentrations of 0, 1.625, 3.25, 6.5, 12.5 and 25 mg/L were tested on Pseudomonas and Bacillus species at exposure intervals of 0, 4, 8, 12 and 24 hours for twenty four (24) separate set-ups. Percentage (%) log mortality (derived from log survival of the test organism) expressed as Median Lethal Concentration (LC 50 mg/L) was used as indices to monitor toxicity. The research shows that local and industrial refined diesel cause cell mortality. The 24 h LC 50 of the diesel were Bacillus +Local refined Diesel (20.54 mg/L) >Pseudomonas + Local refined Diesel (21.36 mg/L) > Bacillus +Industrial refined Diesel (21.88 mg/L) > Pseudomonas + Industrial refined Diesel (40.90 mg/L) (noting; the lesser the LC 50 the more toxic the toxicant). Comparatively, Local refined diesel is more toxic than Industrial refined diesel to all the test organisms. Also Bacillus sp. is more susceptible than Pseudomonas sp. to both local and industrial refined diesel. Based on these findings, the Local “illegal” diesel popularly called 'Bunkery diesel' or 'Kpo-fire diesel' refining should be discouraged in the Niger Delta of Nigeria especially in freshwater environment. As an industrial environment with high population which is prone to diesel spill impact, toxicity to Pseudomonas and Bacillus species could become a simple and fast bioassay for monitoring ecosystem response to these pollutants. Keywords: Local refined diesel, 'Kpo-fire diesel”, “Bunkery diesel”, Ecotoxicity, Freshwater, Pseudomonas , Bacillus sp.


Introduction
350 C at atmospheric pressure, resulting in a Diesel like all other fossil fuels primarily mixture of carbon chains that typically contain consists of complex mixture of molecules called between 8 and 21 carbon atoms per molecule hydrocarbons, which is composed of 75% during petroleum separation (Collins, 2007).alkenes or saturated hydrocarbons (primary Diesel is a petroleum-based fuel for diesel paraffin's including n-, Iso-, and cycloparaffins), engines with chemical formula C H .It is a thick 12 23 and 25% of aromatic compounds (including light oily fuel that has its color varies from Nepthalenes and alkybenzenes).This is obtained colorless to brown (Sakar et al., 2005).
o from middle distillate fraction between 200 C and bacteria could be used as bio-indicator reflecting The ability to isolate high or low the level of ecosystem pollution by petroleum proportion for certain oil utilizing microorganism hydrocarbon (Leahy and Cowell, 1990; Nrior et from an environment depends on the nutrients al, 2017).available in the contaminated site, and the age of The aim of this study therefore was to the contamination (Atlas and Bartha, 1998;Nrior assess the response of pollution bio-monitors like Pseudomonas and Bacillus species to Local and Odokuma, 2017).The most abundant and "Kpo-fire diesel" and Industrial Refined Diesel in active petroleum hydrocarbon utilizing freshwater ecosystem in Niger Delta.As an microorganisms belong to the following genera; industrial environment with high population Pseudomonas, Staphylococcus, Streptococcus, which is prone to diesel spill impact, toxicity to Gordonia, Aeromicrobium, Dietzia, Sarcina, Pseudomonas and Bacillus species could become Vibrio, Flavobacteruim, Micrococcus, Bacillus a simple and fast bioassay for monitoring ((Choi, 2013;Nrior and Mene, 2017;Nrior and ecosystem response to these pollutants.Odokuma, 2017).

Petroleum is still the principal source of energy for industries and industrial uses, even
Source of Sample for some domestic uses.Despite its importance Water sample was collected with sterile in the society, petroleum is a major source of sample bottles (container) from a fresh water pollution in the environment.Diesel exerts toxic stream at Ibaa in Emohua Local Government effects because some of the chemical Area in Rivers State, Nigeria.The container was compounds in diesel oil are slightly soluble in rinsed thrice with the water sample to be water.Its elevated concentration of the lower collected at site before collection was made.The molecular weight aromatic hydrocarbons i.e, bottle cap was replaced after collection and the monoaromatics hydrocarbon (MAHs) and mouth of the bottle was faced up, labeled and polynuclear aromatic hydrocarbons (PAHs) are taking to the laboratory for analysis within two absorbed by water organisms.The principal (2) hours of collection.acute toxic effect caused by these compounds is narcosis (Milinkovitoh and Godefery, 2011).The Identification of bacterial isolates harmful effect that chemicals have upon The isolates were obtained and individual organism depends on many different subjected to various characterization factors, not only on the fresh water organisms procedures.Pure isolates of bacteria were but also in the form in which population occurs identified on the basis of the colonial, (Richard et al., 2011).Microorganisms found in morphological, microscopic examination, fresh water environments such as bacteria, p h y s i c o -c h e m i c a l a n d b i o c h e m i c a l viruses and protozoa; influence the fresh water characteristics (Buchanan and Gibbons, 1974; ability to sustain life on earth (Futherman, 2004).Cowan, 1974).The following standard Microbial monitoring specifically for characterization tests were performed: Gram's petroleum hydrocarbon is the concurrent staining reaction, motility test, oxidase test, stimulation and inhibition effect of petroleum catalase test, coagulase test, starch hydrolysis, hydrocarbons on bacteria, which are used for methyl red and voges-proskauer tests, indole toxicity assessments (Macnaughton et al., 1999; test, Nitrate reduction test, sugar fermentation Nrior and Obire, 2015;Smalla et al., 2007).Some test (Glucose, Sucrose, Lactose, and Maltose).microorganism, like the bacteria are able to Morphological studies were carried out on utilize petroleum hydrocarbons in the different colonies on media plates.Pure colonies environment as their sole source of carbon and were isolated based on colony size, shape, energy (Bundy et al., 2002).Bacteria have been pigmentation, elevation and texture of the considered as one of the predominant individual organisms after 48hours of growth at o hydrocarbon utilizing agent found in the 30 C. The morphology was determined by environment (Venosa et al., 2001).The examination of the plates directly under the abundance of petroleum hydrocarbons utilizing microscope at low power (10x).
shown in Tables 1 and 2 respectively.A total of 24 Physico-chemical parameters of Freshwater set-ups were carried out; 12 for each organism.

Parameters such as pH was determined
The toxicant (local and industrial diesel) using pH meter, Total Dissolved Solids using TDS concentrations were prepared by setting up six meter (Jenway 3015 method), Dissolved Oxygen test tubes per set, aseptically covered with (DO) and Biochemical Oxygen Demand (BOD) cotton wool.The test was carried out in six were determined by modified Winkler method separate test tubes containing appropriate (APHA, 1998), Chemical Oxygen Demand (COD) filtered water (freshwater from the habitat of the was determined by permanganate oxidation organism) separately.In six test tubes per set, method from the biodegradation set-up on were toxicant concentrations (%); Control 0, various days 1, 7, 14, 21 and 28 days.1.625, 3.25, 6.5, 12.5, 25   sum of dose difference using standard statistical The percentage log survival and mortality analysis using the formula (equation iii) below: of the bacterial isolates for Pseudomonas and Bacillus species in the local and industrial refined diesel used in the study was calculated using the formula adopted by Williamson and Johnson (1981) and Nrior et al (2017)  but also in the form in which population occurs.This result shows that with increasing conforms to the findings of Nrior et al. ( 2017) exposure time at a constant concentration of a that local and industrial kerosene toxicity on particular toxicant leads to increase in the Nitrobacter was a function of both the contact mortality rate as reflected in the deleterious time and concentrations.effect on the microbial biota.This result  The values of some physical and findings of Okpokwasili and Olisa (1991).With chemical properties of the freshwater used for respect to sensory evaluation: General the study are presented in Table 5.The general appearance of the habitat freshwater was clear; appearance of the water were clear, odour Odour was unobjectionable, Total Dissolved unobjectionable, colour 2.00 Hazan were within solids, Total iron and Lead were above limits.World Health Organisation (WHO) limit, but the This could probably be due to oxidation of pH 5.70 which is outside WHO limit -standard of petroleum and other organic matter discharged pH 6.5-8.5 for drinking water.However, the on daily basis into the water body, since the acidic pH range observed does not favour the aquatic area of study serve multiple purposes for test organisms in the microcosms.These oil and gas industry, manufacturing and physico-chemical factors were particularly fabrication industries etc.This could also be important for the survival of petroleum product-attributed to the presence and amount of organic utilizing microbial consortium in the aquatic matter present in the sample at the point of systems.These findings corroborated with the collection (Nrior and Odokuma, 2015).
50   (2000).Degradation and Mineralization of Highplants and animals health.Accumulation of molecular weight polycyclic aromatic petroleum hydrocarbons in animals and plants hydrocarbons by defined fungal-bacterial tissues may cause progeny's death or mutation cultures.J. Appl.Env.Microbiol.7: 56 -60.thus leading to extensive alteration or damage of ecosystems (Alvarez et al., 1991;Nrior and Bossert, I. D. and Compeau, G. C. (1995).al., 2004;Nrior et al., 2017).Microorganisms New York, pp 77 -125.found in freshwater such as bacteria, viruses and Proteus can influence the fresh water ability to Bundy, J. G. Paton, G. I. and Campbell, C. D. sustain life on earth (Futherman, 2004). This (2002).Microbial Communities in different soil suggests that the mode of action of industrial types do not converge after diesel contamination and local diesel is not limited to inhibition of the J Appl.Microbiol.92:288 -976.organism.
Choi, Charles Q. ( 2013 (2004).Microbiological resilience of soils There is an evidence to show that diesel can be contaminated with crude oil Geodemma Pp121toxic to microorganisms; it has been observed 130. that industrial diesel is more toxic to Pseudomonas sp.(40.90%),Bacillus sp.
50 toxicant) revealed increasing toxic levels as during toxicity for Local and Industrial refined follows; Industrial refined diesel, the results diesel in freshwater are shown in Tables 3-4.The harmful effect that chemicals have upon show that Pseudomonas sp.(40.90%) <Bacillus individual organism depends on many different sp.(21.88%) while for local refined diesel, factors, not only on the fresh water organisms Pseudomonas sp.(21.36%) < Bacillus sp.

Fig. 1 :
Fig. 1: The Median Lethal concentration (LC mg/L) of Industrial and Local refined diesel on 50

Fig. 2
Fig. 2 Percentage (%) mortality of Pseudomonas sp. to Industrial Refined Diesel in freshwater Odokuma, 2015).Many toxic compound such as C l e a n u p o f P e t r o l e u m H y d r o c a r b o n polycyclic aromatic hydrocarbon, benzene Contamination in soil, In: Young L. Y, and compounds, and cycloalkane rings causes great Cerniglia C. E. (Microbial transformation and deleterious effect on microbial biota (Franco et degradation of toxic organic chemical: Wiley, ) "Microbes Thrive in Conclusion Deepest Spot on Earth" Live science.Diesel fuel Diesel is obtained in the fractional characteristics and resource UFA.com.UFA o o th distillation of crude oil between 250 C and 350 C determination Bacteriology (1974) 8 edition.at atmospheric pressure, and the purpose of this study was to observe the toxicity of both Franco, I. M., Contin, G., Bragato and De Nobill, industrial and local diesel in the microorganisms.M.

Table 1 :
added separately.The test tubes were covered with cotton wool; the Toxicity test procedure for Pseudomonas and control consists of freshwater from the habitat of Pseudomonas sp. and Bacillus sp. in freshwater is Illustrative toxicity set-up procedure of Industrial and Local Refined Diesel with Pseudomonas sp. in freshwater.

Table 2 :
Illustrative toxicity set-up procedure of Industrial and Local Refined Diesel with Bacillus sp. in freshwater.

Table 3 :
Log Survival Count of Pseudomonas sp. with Industrial and Local refined Diesel at different concentrations.

Table 4 :
Log Survival Count of Bacillus sp. with Industrial and Local refined Diesel at different concentrations.Set-up ID Conc.(mg/L)Log survival count of Bacillus sp. with exposure

Table 5 :
Physico-chemical Analysis of Habitat water (Freshwater) COMMENT: This Water Sample under the same condition does not conform with the stated WHO Specification for Potable Water in all the parameters remarked OL (Out of the Limit of Specification).Appropriate treatment for these OL (Out of the Limit of Specification) parameters is therefore recommended.