Introduction Lubricants are manufactured in various formulations for different applications. Most formulas generally consist of two fractions: chemicals additives and basic oils.
Basic oils of lubricants are of mineral origin and derived from crude oil refining. They consist of three hydrocarbons families: paraffinic, naphthenic and aromatic . Lubricants can also contain vegetable-base oils triglycerides in non-esterified forms as complex esters. Release of hydrocarbons into the environment whether accidentally or due to human activities is a main cause of water and soil pollution  .
In many cases these lubricants are very often found in nature under used oil forms. Thus, in the European Union, 4. The situation is especially worrying for most developing countries in general and those of Sub-Saharan Africa in particular because of the absence of policy for regulation and control.
The presence of these products in the environment is a permanent threat, which can measure the extent of considering a liter of mineral oil polluting one million liters of water . Haut du formulaireBas du formulaire Soil contamination with hydrocarbons causes extensive damage of local system since accumulation of pollutants in animals and plant tissue may cause death or mutations  .
In the environment, non-volatile fractions of oils disperse in the aquatic environment or are absorbed into the ground creating a possible pollution of surface and ground waters . These pollutions can affect a catchment area of drinking water. In addition, lubricants can cause a malfunction of biological sewage treatment plants and sewage sludge contamination. Finally, lubricants pose problems in agriculture by reducing the contaminated soil capacity of water retention .
Similarly, the spreading of polycyclic aromatic hydrocarbons PAHs on agricultural crops can result in a decrease of germination, stunting and yield reduction .
From the environment, hydrocarbons can also enter in food chains, leading to health problems. Thus, the soluble fractions of some of these compounds, in particular those containing mainly aromatic hydrocarbons and polar compounds are toxic carcinogenic for many species . Similarly, oil particles inhaled in aerosol form can cause the occurrence of lipid pneumonia . In the body, PAHs are mutagenic and can cause a decrease in the immune response with an increased risk of infections .
Fortunately, the degradation of these oils in the environment is possible through several techniques: physical  , chemical  or biological   - .
The technology commonly used for the soil remediation includes mechanical, burying, evaporation, dispersion, and washing. However, these technologies are expensive and can lead to incomplete decomposition of contaminants. The process of bioremediation, defined as the use of microorganisms to detoxify or remove pollutants owing to their diverse metabolic capabilities is an evolving method for the removal and degradation of many environmental pollutants including the products of petroleum industry .
In addition, bioremediation technology is believed to be noninvasive and relatively cost-effective . Biodegradation by natural populations of microorganisms represents one of the primary mechanisms by which petroleum and other hydrocarbon pollutants can be removed from the environment  and is cheaper than other remediation technologies and the most efficient for environment safe depollution .
Among the microorganisms able to grow on hydrocarbons, bacteria remain qualitatively and quantitatively the most active agents   - . Based on the frequency of isolation, the predominant bacterial genera found on this issue are Pseudomonas, Acinetobacter, Alcaligenes, Vibrio, Flavobacterium, Achromobacter, Micrococcus, Nocardia and Corynebacteria   - . However, a number of limiting factors have been recognized to affect the biodegradation of petroleum hydro- carbons along with, temperature, salinity, oxygen content, oil concentration, presence of nutrients and hydro- carbon chemical composition   - .
Many of these factors have been discussed by Brusseau . The composition and inherent biodegradability of the petroleum hydrocarbon pollutant is the first and foremost important consideration when the suitability of a remediation approach is to be assessed.
Among physical factors, temperature plays an important role in biodegradation of hydrocarbons by directly affecting the chemistry of the pollutants as well as affecting the physiology and diversity of the microbial flora  - .
Atlas  found that at low temperatures, the viscosity of the oil increased, while the volatility of the toxic low molecular weight hydrocarbons were reduced, delaying the onset of biodegradation. Temperature also affects the solubility of hydrocarbons . Although hydrocarbon biodegradation can occur over a wide range of temperatures, the rate of biodegradation generally decreases with the decreasing temperature.
Venosa and Zhu  reported that ambient temperature of the environment affected both the properties of spilled oil and the activity of the microorganisms.
Significant biodegradation of hydrocarbons have been reported in psychrophilic environments in temperate regions  . For the implementation of bioremediation technique, one important requirement is the presence of microorganisms with the appropriate metabolic capabilities.
If these microorganisms are present, then optimal rates of growth and hydrocarbon biodegradation can be sustained by ensuring that adequate concentrations of nutrients and oxygen are present and that the pH is between 6 and 9 .
Unfortunately, at Sub-Saharan African countries level in general and Burkina Faso in particular, few studies focused on bioremediation technology for the depollution of hydrocarbon contaminated environments. To our knowledge, only one study relied on the biodegradation of hydrocarbons by a mixed inoculum containing bacteria, yeasts and fungi .
Unfortunately, the identity of microbes in the inoculum, and the conditions to optimize the process as well were not investigated. As also mentioned Das and Chandran  , the scope of current understanding of oil bioremediation is also limited because the emphasis of most of these field studies and reviews has been given on the evaluation of bioremediation technology for dealing with large-scale oil spills on marine shorelines.
However, an understanding of the impacts of oil on indigenous microbial communities and identification of oil-degrading microbial groups are prerequisite for directing the management and cleanup of oil-contaminated ecosystems . Thus, in order to get insight of the bioremediation process of hydrocarbons in oil-contaminated environments of Ouagadougou city, the present study focused on the isolation, characterization and identification of indigenous hydrocarbon-degrading bacterial strains with regards to the conditions for optimizing their activities and the efficient cleanup of the hydrocarbon pollutants.
Materials and Methods 2. Source of isolation The source for bacterial isolation consisted of wastewaters contaminated with used motor oil, diesel and lubricating oils. Culture media and strains isolation For strains isolation, enriched cultures were prepared according to Malatova  , Mittal and Singh  and Kostka et al. Bushnell-Haas Broth was used in the enrichment technique. After one week incubation period, 1 ml of sample from primary enrichment was transferred to a fresh Bushnell-Haas Broth containing the same hydrocarbon and incubated as above.
The bacterial growth was monitored by culture turbidity and depletion of added oil at regular intervals compared to controls with 0. Location of the wastewater sampling sites in Ouagadougou, Burkina Faso. Figure 1. After four successive transfers to fresh medium, 0.
After 48 hours incubation period and later, single colonies appearing on the PCA agar were transferred on a fresh PCA agar and incubated. Pure colonies were obtained by using a single colony isolation procedure. The purity of cultures was confirmed by microscopic observations. Growth studies Experiments were conducted in the liquid MS medium described above with used motor oil SAE40 as sole carbon and energy source. Morphology The Gram stain reaction was determined with 4-day-old cultures by the modified method of Hucker described by Doetsch .
Cell morphology of 4-day-old colonies on PCA agar cultures and 4-day-old colonies cultures in the MS liquid medium as well were determined by oil immersion phase-contrast microscopy. Colony morphology and pigmentation were determined using 4-day-old PCA agar cultures. Physiological tests Growth at pH ranging from 4. Tolerance to NaCl was monitored at concentrations ranging from 0. Controls without bacterial inoculation were prepared similarly.
Experiments were performed in triplicate in ml flasks. The biodegradation capability was evaluated by monitoring bacterial growth through optical density measurement at nm and by a gravimetric analysis according to Fusey and Oudot  as follow: 40 ml of bacterial culture was added to 40 ml of chloroform in a separating ml funnel which was then shaken vigorously to obtain two emulsified layers a top layer consisting of a mixture of hydro- carbon and chloroform and a bottom layer containing water and biosurfactant in solution.
Oxidase, catalase and respiratory mode were investigated according to Kovacs  and Smibert and Krieg . Enzymes for sugars glucose, lactose fermentation and H2S production were studied using Kligler Hajna medium. Tubes were then inoculated with 1 ml of bacterial suspension before they were incubated at the optimal growth temperature as defined through the physiological tests described above. Bacterial growth was monitored per 24 h-incubation period up to 12 days by measuring the optical density at nm.
Results and Discussion 3. Enrichment and isolation From the enrichment cultures, a total of six isolates able to use SAE40 used oil as carbon and energy source were obtained. Among the isolates, two strains namely S2 and S7 which showed the best growth highest OD at nm were selected for further characterization. Morphology Colony morphology on agar medium revealed a translucent round form with 3 - 5 mm in size for strain S2 while strain S7 appeared yellowish, round and 2 - 3 mm in size.
Both isolates stained Gram-negative and were non- motile and motile for S2 and S7, respectively. Cells of strain S2 were short rods, while slightly coccobacillus-like cells were also observed Figure 2 a , contrasting with the long rods bacillus-like cells of strain S7 Figure 2 b.
Physiology 3. Under and above this temperature range, strains growth is rather low. As underlined Atlas and Bartha  , Atlas  and Leahy and Colwell  , temperature influences hydrocarbon biodegradation by its effect on the physical nature and chemical composition of the oil and the rate of hydrocarbon metabolism by microorganisms.
According to Atlas and Bartha  , at low temperatures, the viscosity of the oil increases, the volatilization of toxic short-chain alkanes is reduced, and their water solubility is increased, delaying the onset of biodegradation. The findings of these authors supported partially our results.
The pH ranges for growth of isolates S2 and S7 are in agreement with the results on hydrocarbon-degrading strains of Dibble and Bartha  and Hambrick et al.
Indeed, many studies have revealed that there is a large number of hydrocarbon-degrading bacteria in oil-rich environments, such as oil spill areas and oil reservoirs Hazen et al. Many normal and extreme bacterial species have been isolated and utilized as biodegraders for dealing with petroleum hydrocarbons.
The degradation pathways of a variety of petroleum hydrocarbons e. For instance, some bacteria can metabolize specific alkanes, while others break down aromatic or resin fractions of hydrocarbons.
This phenomenon is related to the chemical structure of petroleum hydrocarbon components. Petroleum hydrocarbon-degrading bacteria and the type of petroleum components they degrade are listed in Table 1. Recent studies have identified bacteria from more than 79 genera that are capable of degrading petroleum hydrocarbons Tremblay et al. Similarly, some obligate hydrocarbonoclastic bacteria OHCB , including Alcanivorax, Marinobacter, Thallassolituus, Cycloclasticus, Oleispira and a few others the OHCB , showed a low abundance or undetectable status before pollution, but were found to be dominant after petroleum oil contamination Yakimov et al.
These phenomena suggest that these microorganisms are crucial to the degradation of petroleum hydrocarbons, and that they significantly influence the transformation and fate of petroleum hydrocarbons in the environment. Although some bacteria have been reported to have a broad spectrum of petroleum hydrocarbon degradation ability, Dietzia sp. DQb utilizes n-alkanes C6—C40 and other compounds as the sole carbon sources Wang et al. Indeed, most bacteria can only effectively degrade or utilize certain petroleum hydrocarbon components, while others are completely unavailable Chaerun et al.
This can be attributed to the fact that different indigenous bacteria have different catalytic enzymes; thus, their roles in oil-contaminated sites also vary widely. This also implies that the remediation of petroleum hydrocarbon contamination requires the joint action of multiple functional bacteria to achieve the best environmental purification effect Dombrowski et al.
Based on this view, Varjani et al. Tao et al. Wang C. A field study showed that bioaugmentation with an artificial consortium containing Aeromonas hydrophila, Alcaligenes xylosoxidans, Gordonia sp. Taken together, these studies indicate that improving the biodegradation potential via the application of bacterial consortia possessing multiple catabolic genes is a reasonable and feasible strategy for accelerating the removal efficiency of petroleum hydrocarbons from contaminated environments.
Petroleum hydrocarbon-degrading bacteria and their preferred degradation substrates. Toxic Impact of Petroleum Hydrocarbons The harm that oil pollution causes to the ecological environment is well known Sikkema et al. Labud et al. In diesel exposure experiments, researchers found that the primary effects of diesel fuel toxicity were reductions in species richness, evenness and phylogenetic diversity, with the resulting community being heavily dominated by a few species, principally Pseudomonas.
Moreover, they found that the decline in richness and phylogenetic diversity was linked to the disruption of the nitrogen cycle, with species and functional genes involved in nitrification being significantly reduced van Dorst et al. Cerniglia et al. However, the phenolic and quinonic naphthalene derivatives inhibited bacterial growth. This could be explained by phenols and quinones with higher solubility, enhancing the mass transfer of molecules to bacterial cells, resulting in higher toxic effects than the former compounds.
Several studies have also reported that certain metabolic intermediates with relatively high solubility produced from the degradation of petroleum hydrocarbons by bacteria may have higher cytotoxicity than the parent molecules and therefore damage the bacteria Hou et al.
However, indigenous bacteria form very large aggregates, and each species has its own function. Accordingly, while some bacteria that are sensitive to petroleum hydrocarbons are greatly inhibited upon exposure to petroleum hydrocarbons, others that can efficiently degrade petroleum hydrocarbons, as well as bacteria that can take advantage of cytotoxic intermediate metabolites, will flourish. However, clean-up of petroleum oil pollutants by relying on the strength of these indigenous microorganisms alone will take a long time; therefore, it is necessary to develop intervention measures to speed the process up.
Restriction of Physical Contact Between Bacteria and Petroleum Hydrocarbons Due to the hydrophobicities and low water solubilities of most petroleum hydrocarbons, the biodegradation rate is generally limited in the environment. This is because the first step in the degradation process of petroleum oil often requires the participation of bacterial membrane-bound oxygenases, which require direct and effective contact between bacterial cells and petroleum hydrocarbon substrates.
The primary factors restricting the biodegradation efficiency of petroleum hydrocarbons are as follows: 1 limited bioavailability of petroleum hydrocarbons to bacteria, and 2 the fact that bacterial cell contact with hydrocarbon substrates is a requirement before introduction of molecular oxygen into molecules by the functional oxygenases Vasileva-Tonkova et al.
However, bacteria have evolved countermeasures against petroleum contaminants, such as improving the adhesion ability of cells by altering their surface components and secreting bioemulsifier to enhance their access to target hydrocarbon substrates. Bacteria with such functions are often screened for use as environmental remediation agents, accelerating the removal of petroleum hydrocarbon pollutants from the environment Kaczorek et al.
Bacterial surface properties are essential to the effective biodegradation of hydrophobic hydrocarbon substrates Figure 2 and their adhesion mechanisms are of great importance Zhang et al. Ron and Rosenberg found that adherence of hydrophobic pollutants to bacterial cells is mainly related to hydrophobic fimbriae, fibrils, outer-membrane proteins and lipids, as well as certain small molecules present in cell surfaces such as gramicidin S and prodigiosin.
Fimbriae present on bacterial surfaces were confirmed to be necessary for the growth of Acinetobacter sp. RAG-1, with C16 alkane as the carbon source and beneficial to bacterial adherence, assimilation hydrophobic substrates and their metabolic activity Rosenberg and Rosenberg, Nevertheless, bacterial capsules and several anionic exopolysaccharides produce inhibitory effects on hydrocarbon substrate adhesion.
For example, Bacillus licheniformis decreases cell surface hydrophobicity in response to exposure to organic solvents and has little affinity for toxic organic compounds Torres et al.
Although bacterial adherence can enhance the biodegradation of hydrophobic hydrocarbons, it is not necessary to attach bacterial cells to targeted substrates Abbasnezhad et al.
Although chemical-physical phenomena play an important role in the process of oil detoxification, the ultimate and complete degradation is mainly accomplished by marine microflora, dominant bacteria in this role Della Torre et al.
In the natural environment, biodegradation of crude oil involves a succession of species within the consortia of the present microbes Alkatib et al.
Indeed, since a single species can metabolize only a limited range of hydrocarbon substrates, a consortium of many different bacterial species, with broad enzymatic capacities, is usually involved in oil degradation Rooling et al.
Although some bacteria, belonging to Pseudomonas Das and Chandar, and Rhodococcus genera Hassanshahian et al. On the above mentioned basis, bioremediation techniques have been developed and improved for cleaning up oil-polluted marine environments as an alternative to chemical and physical techniques Alkatib et al. Bioremediation can be described as the conversion of pollutants hydrocarbons by micro-organisms bacteria into energy, cell mass and biological waste products Nikolopoulou and Kalogeraki, Nevertheless, the rates of uptake and mineralization of many organic compounds hydrocarbons by bacteria in polluted seawater is limited due to the poor availability of nitrogen and phosphorus Yakimov et al.
For that reason, in the application of biostimulation techniques the growth of oil-degrading bacteria can be strongly enhanced by fertilization with inorganic nutrients Nikolopoulou and Kalogeraki, By using these consortia, we have been able to investigate the capability of efficient biodegradation of crude oil could be accomplished by the mixed populations.
At an early stage light fractions of oil are naturally removed; mostly by evaporation, thence by photo-oxidation and by geo-chemicals reactions.
Enrichment and isolation From the enrichment cultures, a total of six isolates able to use SAE40 used oil as carbon and energy source were obtained. However, the phenolic and quinonic naphthalene derivatives inhibited bacterial growth. Leahy, J.
MacNaughton, S. For aerobic degradation processes, using oxygen as an electron acceptor is quite important, but it is usually not adequate in petroleum oil-contaminated environments because of the limited air permeability.
Results and Discussion 3.
Strain isoSS belong to a collection of hydrocarbon-degrading bacteria hold at IAMC-Messina, strains isoSS-2 and iso-SS03 were isolated from natural seawater from crude oil enrichments in previously research. Received 2 April ; revised 1 May ; accepted 26 May ABSTRACT Lubricants are very often found in nature under waste-oil forms and represent for the environment a real danger of pollution due to the difficulty of their biodegradation. The situation is especially worrying in most developing countries in particular those of Sub-Saharan Africa due to the absence of regulation or control. The pH ranges for growth of isolates S2 and S7 are in agreement with the results on hydrocarbon-degrading strains of Dibble and Bartha  and Hambrick et al.
Unfortunately, the identity of microbes in the inoculum, and the conditions to optimize the process as well were not investigated. Canadian Journal of Microbiology, 46, However, bacteria have evolved countermeasures against petroleum contaminants, such as improving the adhesion ability of cells by altering their surface components and secreting bioemulsifier to enhance their access to target hydrocarbon substrates.
San Raineri 86, Messina, Italy. After 48 hours incubation period and later, single colonies appearing on the PCA agar were transferred on a fresh PCA agar and incubated. Dibble, J. The advantages of microbial communities are presented because there are a variety of catabolic genes in a bacterial consortium, and the synergistic effects of these genes are beneficial to achieving the purification of pollutants Gurav et al. Bacterial growth was monitored per 24 h-incubation period up to 12 days by measuring the optical density at nm. The enrichment technique was used for selecting hydrocarbon degraders.
All these findings suggest that the concept of a maximum or threshold concentration for microbial degradation of hydrocarbons may be related to the pollutant type and environmental factors. Send correspondence to S.
Ron and Rosenberg found that adherence of hydrophobic pollutants to bacterial cells is mainly related to hydrophobic fimbriae, fibrils, outer-membrane proteins and lipids, as well as certain small molecules present in cell surfaces such as gramicidin S and prodigiosin. The "fate" of petroleum in the sea water largely depends on mechanical wave, wind , physical temperature, UV and chemical pH, dissolved oxygen and nutrient concentration factors which may differently influence its natural transformation oil weathering and bio-degradation Nikolopoulou and Kalogeraki,
Wodzinski, R. In: Bianchi, M. In addition, lubricants can cause a malfunction of biological sewage treatment plants and sewage sludge contamination. Soil Decontamination Biotechnology, Vol. The rates of uptake and mineralization of many organic compounds by microbial populations in the aquatic environment are proportional to the concentration of the compound, generally conforming to Michaelis-Menten kinetics   . Ayed et al.
One reason of the difference with their data could be explained by the fact that these authors focused on marine bacteria, known to support high salt concentration and hypersaline environments as well. In a study of hypersaline salt evaporation ponds, Ward and Brock  showed that rates of hydrocarbon metabolism decreased with increasing salinity in the range 3. Send correspondence to S. Bioresource Technology, 90, Biotechnology and Bioengineering, 13,