Vol. 3, No. 3 (2015): 652-657 Research Article
Open Access
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ISSSSNN:: 22332200--22224466
Optimization of Hydrocarbons Biodegradation by
Bacterial Strains Isolated from Wastewaters in
Ouagadougou, Burkina Faso: Case Study of Diesel and
Used Lubricating Oils
Adama Sawadogo
1*, Harmonie C. Otoidobiga
1, Aminata Kaboré
1, Joseph B.
Sawadogo
1, Alfred S. Traoré
1and Dayéri Dianou
21 Research Center for Biological, Alimentary and Nutritional Sciences, Research and Training Unit, Life and Earth Sciences,
University of Ouagadougou, Ouagadougou, Burkina Faso
2 National Center for Scientific and Technological Research, Institute for Health Sciences Research, Ouagadougou, Burkina Faso.
* Corresponding author:Adama Sawadogo; e-mail: damouss75@yahoo.fr
ABSTRACT
Diesel oil and lubricants are hydrocarbons commonly used in the world. Contamination of environment by used lubricating and diesel oil is rapidly increasing due to global increase in the usage of petroleum products, particularly in developing countries. This has been shown to have harmful effects on the environment and human beings. Thus, the present work aims to optimize diesel oil and used oil biodegradation by bacterial strains isolated in wastewaters. Two strains namely S2 and S7 were incubated alone or together with diesel oil or Total Quartz 9000 used oil supplemented or not with yeast extract, peptone and trace elements for 14 days. The level of both petroleum hydrocarbons degradation was determined by gravimetric assay. The results obtained showed that the presence of strain S2 or S7 increased significantly hydrocarbons degradation. The association of strains S2 and S7 increased the biodegradation of hydrocarbons over the one of strain S7 alone (p< 0.0001). The supplementation of the culture medium with yeast extract, peptone or trace elements also increased the diesel and Total Quartz used oils biodegradation rates. Strain S2 showed the highest biodegradation capability on the two hydrocarbons tested compared to Strain S7 (p< 0.0001 and p= 0.015). Diesel oil was more biodegraded than Total Quartz 9000 used oil by S2, S7 or S2+S7.
Keywords:
biodegradation, diesel oil, Total Quartz used oil, nutrient factor, bacterial strain1. INTRODUCTION
Diesel oil and lubricants are hydrocarbons commonly used in the world. This utilization leads to the production of used oil in case of lubricants. Contamination of soil and surface water by used oil is rapidly increasing due to the global increase in the usage of petroleum products [1]. Soil and surface water contamination by used oil is a common occurrence in most developing countries. This has been shown to have harmful effects on the environment and human beings [2]. Similarly, diesel oil is one of the major products of crude oil that constitutes the main source of pollution in the environment. With the increasing
demand for diesel oil in cars, trucks, generators and industrial machines, combined with large quantities of the oil being transported over long distances, the contamination of soils through accidental spillage, cleaning of machines and tankers could reach an alarming state [3].
deplete the oxygen of 1,000,000 L of water; therefore, a city discharge of 200,000 inhabitants is the same as 47 L of residual oil, which affects 47 million L of water [5]. 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 [6]. Petroleum products pose also problems in agriculture by reducing the contaminated soil water holding capacity [7]. Petroleum products can also lead to health problems. So, used motor oil contains metals and heavy polycyclic aromatic hydrocarbons (PAHs) that could contribute to chronic hazards including mutagenicity and carcinogenicity [8-9]. In addition, prolonged exposure to high oil concentration may cause the development of liver or kidney disease, possible damage to the bone marrow, and an increased risk of cancer [10]. In these conditions, the treatment of oil contaminated environment is necessary to protect water supplies, human health and environmental quality [11].
Fortunately, the degradation of these oils in the environment is possible through several techniques: physical [12], chemical [13] or biological [6, 14-15]. Mechanical method to reduce hydrocarbon pollution is expensive and time consuming [16]. 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.
It has been established that, the biodegradation of hydrocarbons and heavy metals contaminated soils, by exploiting the ability of microbes to degrade and/or detoxify organic/chemical contaminants, is an efficient, economic, versatile and environmentally sound treatment [17-19]. So, bioremediation can be an alternative green technology for remediation of such hydrocarbon and other pollutants-contaminated environment. However, a number of limiting factors have been recognized to affect the biodegradation of petroleum hydrocarbons along with, temperature, salinity, oxygen content, oil concentration, presence of nutrients and hydrocarbon chemical composition [20-22]. Lack of essential nutrients such as nitrogen and phosphorus is one of the major factors affecting biodegradation of hydrocarbon by microorganisms in soil and water environment [2]. However, for the implementation of bioremediation technique, one important requirement is the presence of indigenous microorganisms with the appropriate metabolic capabilities to degrade hydrocarbons.
In a previous work performed in our country [18], two bacterial strains able to degrade hydrocarbons were isolated. The optimal temperature, salinity, oil concentration and pH witch permitted to obtain high growth rates of these bacterial strains on hydrocarbons were performed. However, the effects of nutrient factors (yeast extract, peptone, and traces elements) on hydrocarbons degradation by these strains are not
documented. Thus, the present work focused on the optimization of two hydrocarbons biodegradation using nutrient factors. This study also aims to investigate the synergic effect of two bacterial strains on hydrocarbon biodegradation.
2. MATERIALS AND METHODS
2.1 Bacterial strains
Two bacterial strains, namely S2 and S7 used in this study were isolated during our previous work from wastewaters in Ouagadougou, Burkina Faso. Based on their morphological, biochemical and physiological characteristics, the isolates appeared to be related to
Pseudomonas and Acinetobacter genera, respectively [18].
2.2 Hydrocarbons used in this study
The diesel oil used in this experiment was purchased from a local oil filling station and stored in the dark at ambient temperature throughout the study. The Total Quartz 9000 used oil was obtained from a local garage. Before use, the oils were sterilized using 0.2μm pore size membrane filter.
2.3 Culture media and incubation condition
Haas Broth was used for incubation.
Bushnell-Hass medium consisted of: K2HPO4 1.0 g/L, KH2PO4 1.0
g/L, NH4NO3 1.0 g/L, MgSO4 0.2 g/L, CaCl2 0.02 g/L and
FeCl3 0.005 g/L. Flasks (120 ml) containing 35.6 ml of
Bushnell-Haas Broth supplemented with 3% (v/v) 0.22 μm pore size filter-sterilized as hydrocarbon substrate were inoculated in triplicate with 4 ml inoculum of strains S2, S7 or their mixture (S2+S7), respectively and then incubated at 37˚C for 14 days. Controls without bacterial inoculation were prepared similarly. The pH was adjusted to 8.00, 7.50 and 7.75, respectively for cultures containing S2, S7 and S2+S7. In cultures supplemented with nutrient factors, 0.5% (w/v) yeast extract or peptone and 1% (v/v) trace elements of Widdel and Pfennig [23], were added to the Bushnell-Haas Broth media described above. The Widdel and Pfennig trace elements consists of: HCl (25%: v/v) 6.5 ml, FeCl2.4H2O 1.5 g, H3BO3 60 mg,
MnCl2.4H2O 100 mg, CoCl2.6H2O 120 mg, ZnCl2 70 mg,
NiCl2.H2O 25 mg, CuCl2.2H2O 15 mg, Na2MoO4.2H2O 25
mg, distilled water 1000 ml final volume.
2.4 Biodegradation study
Diesel oil and Total Quartz 9000 used oil degradations were studied by gravimetric analysis according to
Marquez-Rocha [24] and Panda [25]. After 14 days of
biosurfactant in soluble form. The lower two layers were spread out while the top layer containing petroleum ether mixed with diesel oil or Total Quartz used oil and acetone was taken in a preweighed clean beaker. The extracted oil was passed through anhydrous sodium sulphate to remove moisture. The petroleum ether and acetone was evaporated on a water bath. The gravimetric estimation of residual oil left after biodegradation was made by weighing the quantity of oil in a tarred beaker. The percentage of biodegraded oil was then evaluated in comparison to the initial hydrocarbon amount in the control funnel
according to Fusey and Oudot [26], as described by
Sawadogo [18].
2.5 Statistical analysis
The data collected were subjected to analysis of variance (ANOVA) with regards to hydrocarbons used, growth factor and bacterial strain using XLSTAT-Pro 7.5 software. Mean variables were compared using the Newman Keuls test at probability level p = 5% and differences in strains degradation rates were compared through the Least Significant Difference test at p= 5%.
3. RESULTS AND DISCUSSION
3.1 Biodegradation of diesel oil
The statistical analysis showed a significant difference between strain S2 and S7 for diesel oil biodegradation
(p< 0.0001, table 1). The combined effects of strain and growth factor showed also a significant effect on the oil degradation (p<0.0001, table 1). However, no significant difference was found among nutrient factors for diesel oil biodegradation (p= 0.151, table 1).
After 14 days incubation period at 37°C, the biodegradation rates obtained with strain S2 on diesel oil, and on diesel oil supplemented with yeast extract, peptone or trace elements were 30.57% ± 1.09, 35.21% ± 3.28, 31.14% ± 2.19, and 31.45% ± 0.51, respectively
(figure 1, table 3). Biodegradation rates of 18.25% ±
1.30, 28.70% ± 1.99, 23.31% ± 0.66, 19.74% ± 1.84
were obtained with strain S7 on the previous substrates after 14 days incubation period, respectively (figure 1, table 3). As underlined the statistical analysis, strain S2 showed the best biodegradation rate on diesel oil supplemented or not with nutrient factor (Figure 1, table 3). Our previous study showed that the maximum growth rate (µmax) of strain S2 is higher than the one of strain S7 on gasoline, diesel oil and Total Quartz 9000 used oil [18]. Thus, the highest growth of strain S2 could be explained the highest biodegradations of hydrocarbon with this strain compared to those of strain S7. This relation between strain growth rate and hydrocarbon biodegradation was demonstrated by Mandri and Lin [1].
Table 1: Variance of diesel oil biodegradation with regards to bacterial strain (S2 or S7) and nutrient factors (means of 3 replicates).
Source of variation df p f
strain 1 0.0001 s
Nutrient factors 1 0.151 ns
Strain x nutrient factors 3 0.0001 s
ns: not significant; s: significant; df: degree of freedom; p = probability at a risk of 5%; f: Fischer’ test.
Figure 1: Biodegradation rates of strains S2 and S7 on diesel oil supplemented or not with nutrient factors after 14 days of incubation. For both strains and for all the nutrient factors experienced, values sharing the same letter are not significantly
different according to the Newman-Keuls’ test at p = 5%.
The results showed also that the presence of yeast extract and peptone increases significantly the hydrocarbon biodegradation rates for both strains and for strain S7, respectively (figure 1). However, no significant difference was observed for trace elements and peptone on the diesel biodegradation rate with strain S2. Over all, the degradation rates were higher
known as the most important nutrients needed by hydrocarbon-utilizing bacteria to carry out effective and efficient biodegradative activities of xenobiotics in the environment. So, the presence of these nutrients in yeast extract and peptone could explain the increase of biodegradation in presence of these nutrient factors.
The results also showed low biodegradation rates on this hydrocarbon supplemented with trace elements compared to the ones observed on the hydrocarbon supplemented with yeast extract or peptone (figure 1, table 3). According to Springgate [32], trace elements can increase bacterial enzyme activity as cofactors and then, leading to bacterial growth. This increase of bacterial growth could explain the low increase in the biodegradation of hydrocarbons supplemented with trace elements.
3.2 Biodegradation of Total Quartz 9000used oil
The variance of biodegradation rate showed a significant difference between both strains for Total
Quartz used oil (p=0.015, table 2). The combined effects of bacterial strain and nutrient factor for this substrate were also significant (p< 0.009, table 2). Similarly, a significant effect was found for nutrient factor on the biodegradation of this substrate (p= 0.042, table 2). After 14 days incubation period, biodegradation rates of 15.46%± 1.24, 24.52% ± 2.34, 19.66% ± 2.15 and
16.43% ± 0.54 were obtained with strain S2 on Total
Quartz used oil, and on Total Quartz used oil supplemented with yeast extract, peptone or trace elements, respectively (figure 2, table 3), while
biodegradation rates of 13.73%± 1.51, 19.05%± 1.25,
15.22%± 1.09 and 13.67%± 0.82 were obtained with
strain S7 on the same substrates after 14 days incubation period, respectively (figure 2, table 3). As underlined in the case of diesel oil, strain S2 showed higher biodegradation rates than strain S7 on Total Quartz used oil. Nutrient factors also increased the biodegradation of this hydrocarbon by both strains.
Table 2: Variance of Total Quartz 9000 used oil biodegradation with regards to bacterial strain (S2 or S7) and nutrient factors (means of 3 replicates).
Source of variation df p f
strain 1 0.015 s
Nutrient factors 1 0.042 s
Strain x nutrient factors 3 0.009 s
ns: not significant; s: significant; df: degree of freedom; p = probability at a risk of 5%; f: Fischer’ test.
The results also showed that for both strains, the highest biodegradation rates were recorded on diesel and the lowest on used oil. These results could be explained by the composition of these hydrocarbons and their solubility. According to Marchal [33], diesel oil contains many components which the carbon number is between 12 and 25. In contrast, the carbon number of used oil component is between 20 and 35
[34]. So, diesel oil solubility in aqueous phase is higher than used oil. This high solubility can increase microbial access to hydrocarbons and thus, the biodegradation rate [4, 35].
Among the nutrient factors, yeast extract showed the best positive effect on hydrocarbons biodegradation (figures 1, 2). So, it was used in further optimization.
Figure 2: Biodegradation rates of strains S2 and S7 on Total Quartz 9000 used oil supplemented or not with nutrient factors after 14 days incubation period. For both strains and for all the nutrient factors experienced, values sharing the same letter are
Table 3: Biodegradation rates of diesel oil and Total Quartz 9000 used oil supplemented or not with nutrient factors after 14 days incubation period (means of 3 replicates). The values sharing the same letter are not significantly different according to the
Newman-Keuls test at p = 5%.
Hydrocarbon Strain Biodegradation rate (%)
Diesel oil S2 30.57b ± 1.09
S7 18.25de ± 1.30
Diesel oil +Yeast extract S2 35.21a ± 3.28
S7 28.70b ± 1.99
Diesel oil+ Peptone S2 31.14b ± 2.19
S7 23.31c ± 0.66
Diesel oil +Traces elements S2 31.45b ± 0.51
S7 19.74d ± 1.84
Used oil S2 15.46e ± 1.24
S7 13.73f ± 1.51
Used oil+ Yeast extract S2 24.52c ± 2.34
S7 19.05de ± 1.25
Used oil+ Peptone S2 19.66d ± 2.15
S7 15.22ef ± 1.09
Used oil +Traces elements S2 16.43def ± 0.54
S7 13.67f ± 0.82
Table 4: Variance of the difference in hydrocarbons biodegradation with regards to bacterial strain (S2, S7 and S2+S7) (means of 3 replicates).
Source of variation df p f
S2+S7 x S2 x S7 2 0.003 s
S2+S7 x S2 1 0.165 ns
S2+S7 x S7 1 0.0001 s
S2 x S7 1 0.05 s
ns: not significant; s: significant; df: degree of freedom; p = probability at a risk of 5%; f: Fischer’ test
3.3 Biodegradation of hydrocarbons with strains S2, S7 or S2+S7
Statistical analysis showed a significant difference between S2 and S7, S2+S7and S7 for the hydrocarbons biodegradation (p= 0.05 and p<0.0001, respectively; table 4). However, no significant difference was found between S2 and S2+S7 for the hydrocarbons biodegradation (p= 0.165, table 4). After 14 days incubation period with strain S2, S7 or S2+S7 on diesel oil and Total Quartz used oil supplemented or not with yeast extract, biodegradation rates were evaluated by gravimetric assay. For strain S2, biodegradation rates of 30.57% ± 1.09, 35.21% ± 3.28, 15.47% ± 1.25, 25.35% ± 2.34 were found on diesel oil, diesel oil supplemented with yeast extract, Total Quartz used oil and Total Quartz used oil supplemented with yeast
extract, respectively, while for strain S7,
biodegradation rates of 18.26% ± 1.30, 30.00% ± 1.99, 14.40% ± 2.53, 19.88% ± 1.25 were obtained on the same substrates, respectively. For the association of both strains (S2+S7), biodegradation rates of 33.76% ± 1.52, 37.53% ± 1.75, 23.09% ± 1.83, 28.57% ± 2.23 were obtained on the previous substrates, respectively (figure 3). The biodegradation rates obtained with S2+S7 are higher than the biodegradation rate recorded with S2 or S7. Thus, the association of the two strains has positive effects on hydrocarbons biodegradation as revealed the statistical analysis. According to Solano-serena [36], synergies can be found between bacteria in mix inoculums, and these synergies can lead to an increase of hydrocarbons biodegradation. These synergies can result to an additional effect of enzymes present in each bacterial strain [21].
Figure 3: Biodegradation rate of strains S2, S7 and S2+S7 on hydrocarbons supplemented or not with yeast extract after 14 days of incubation. For all strains and for all the hydrocarbons experienced, values sharing the same letter are not significantly different according to the Least
Significant Difference test at p = 5%.
4. CONCLUSION
This study showed that strains S2 and S7 are able to degrade diesel oil and Total Quartz 9000 used oil after 14 days of incubation. It also showed that nutrient
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both strains. Therefore, nutrient factors (yeast extract and peptone) can be utilized effectively to reclaim soil or water contaminated with diesel and used oils. In further, biodegradation of these hydrocarbons supplemented or not with nutrient factors during a long incubation period are required in order to increase the biodegradation rates.
5.ACKNOWLEDGMENTS
The authors would like to express profound gratitude to ISP-SUEDE, PACER-UEMOA, CRSBAN-RABIOTECH, UFR-SVT/UO and CNRST/IRSS, for financial and technical supports.
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