Capabilities of pure culture of bacteria in the biodegradation of polycyclic aromatic hydrocarbons and total petroleum hydrocarbons in oilfield wastewater

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Int.J.Curr.Microbiol.App.Sci (2021) 10(03): 810-822 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 10 Number 02 (2021) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2021.1003.084 Capabilities of Pure Culture of Bacteria in the Biodegradation of Polycyclic Aromatic Hydrocarbons and Total Petroleum Hydrocarbons in Oilfield Wastewater O. Aleruchi* and O. Obire Department of Microbiology, Rivers State University, P.M.B 5080, Port Harcourt, Nigeria *Corresponding author ABSTRACT Keywords Polycyclic aromatic hydrocarbon, Total petroleum hydrocarbon, Oilfield wastewater, Biodegradation, Gas chromatography Article Info Accepted: 18 March 2021 Available Online: 10 April 2021 Polycyclic aromatic hydrocarbons (PAHs) and total petroleum hydrocarbons (TPHs) in oilfield wastewater are of environmental importance because of its negative impact in the environment where they are discharged. Therefore it is important to efficiently treat oilfield wastewater before its discharge into the environment. This study therefore investigated the capabilities of pure cultures of bacteria in the biodegradation of polycyclic aromatic hydrocarbons (PAHs) and total petroleum hydrocarbons (TPHs) in oilfield wastewater. Standard procedures where observed in the collection of oilfield wastewater samples and its investigations. The bacteria used for the study were isolated from soil enriched with oilfield wastewater. Four bacteria isolates were molecularly identified using 16S rRNA method as Morganella morganii (MN094330), Pseudomonas xiamenensis (MN094331), Chryseobacterium cucumeris (MN094332) and Staphylococcus sp (MN094333). Each biodegradation experimental 250 ml flask contained 125 ml of oilfield wastewater (ofww) and 6.25ml (5%) of the bacteria culture. The control contained only the ofww (125 ml). The set up were placed in a shaker incubator at 28oC with 200 rpm for aeration. The experimental samples were periodically analyzed at day 1, 7 and 21 intervals for PAHs and TPHs using Gas chromatography (GC). The initial total amount of PAHs and TPHs in the oilfield wastewater on day 1 was 101.72992 mg/l and 342.89053 mg/l, respectively. At the end of the experiment (day 21), the treatment with Pseudomonas xiamenensis recorded the least remaining of 22.23959 mg/l of PAHs with 78.1% removal while the control recorded the highest remaining of 75.40663 mg/l of PAHs remaining with 25.9% removal. There was complete absence of Chrysene in the treatments with Pseudomonas xiamenensis, Staphylococcus sp and Chryseobacterium cucumeris. There were reductions in the peaks of the various PAHs on day 21 in all the treatments. The least and highest amount of TPHs remaining on day 21 was observed in the Chryseobacterium cucumeris (58.18741 mg/l) and control (240.74905 mg/l), respectively with percentage removal of 84.8% and 36.9%, respectively. The treatment with Morganella morganii on day 21 showed total clearance of C12, Pr, C22 and C26. After 21 days of treatment, Pseudomonas xiamenensis showed removal of C12, C19, C22 and C26. Staphylococcus sp recorded removal of C12, Pr, C19, C20, C22 and C26. Chryseobacterium cucumeris completely removed C10, C12, Pr, C19, C20, C22, C23 and C26. At the end of the experiment, the ability of the individual bacterium to biodegrade PAHs and TPH were revealed by Gas chromatography (GC). However, some organisms’ biodegraded PAHs faster than TPH and vis versa. 810 Int.J.Curr.Microbiol.App.Sci (2021) 10(03): 810-822 Introduction Hydrocarbons are a ubiquitous family of several chemically related environmental importunate organic compounds of various structures and with different levels of toxicity. Oilfield wastewater generated by petrochemical industries are characterized by the presence of large quantity of polycyclic and aromatic hydrocarbons, phenols, metal derivatives, surface active substances, sulphides, naphthylenic acids and other chemicals (Aleruchi and Obire, 2018; Suleimanov, 1995). Due to the ineffectiveness of purification systems, wastewater may become dangerous, leading to the accumulation of toxic products in the soil or receiving water bodies with potentially serious consequences on the ecosystem (Bay et al., 2003). Crude oil is a complex mixture of several polycyclic aromatic compounds and other hydrocarbons. The major hydrocarbon classes found in crude oil are the normal alkanes which are easily degraded, branched alkanes and cycloalkanes, (difficult to identify), the isoprenoids (very resistant to biodegradation), the aromatics (fairly identified and much more soluble than other hydrocarbons), and finally the polar ones containing mainly sulphur, oxygen and/or nitrogen compounds. Non hydrocarbon compounds may also be found in crude oil and they include porphyrins and their derivatives (Callot and Ocampo, 2000). Bioremediation can be applied as green technologies which are environmentally friendly and cost effective response to oil pollution. In recent years, there has been increasing interest in developing cost effective in-situ technique for bioremediation of oil contaminated sites. Three main approaches of this technique: natural attenuation (reliance on natural biodegradation activities and rates), which is sometimes called intrinsic bioremediation; biostimulation (stimulation of natural activities by environmental modification such as fertilizer addition to increase rates of biodegradation); and bioaugmentation (addition of exogenous microorganisms to supplant the natural degradative capacity of the hydrocarbonimpacted ecosystem) (Kaplan and Kitts 2004; Prince and Atlas 2005; Chikere et al., 2009a, b; Gertler et al., 2009a). Microorganisms are the major agents in the degradation of petroleum hydrocarbons. The organisms include bacteria, yeast, filamentous fungi and algae (Prince, 1993; Atlas, 1981). The principal bacteria and fungi genera responsible for oil degradation in both soils and aquatic environment have been identified as comprising mainly Pseudomonas, Achrobacter, Bacillus, Micrococcus, Nocardia, Trichoderma, Penicillium, Aspergillus and Morteilla (Atlas, 1981; Bossert and Bartha, 1984; Okpokwasili and Amanchukwu, 1988). This study therefore compares the potentials of individual isolates in the biodegradation of polycyclic aromatic hydrocarbons and total petroleum hydrocarbon in an oilfield wastewater. Materials and Methods Sample Collection and Isolation Oilfield wastewaters were collected from Ogbugu flow station; an onshore oil production platform located in Ogba/Egbema/Ndoni local government Area (ONELGA) of Rivers State, Nigeria. The Oilfield wastewater samples were collected using 4 Litre capacity sterile sample bottles and stored in an ice packed cooler. The soil samples were collected 80 meters away from the discharge pond at a depth of 0 - 15 cm with a clean hand auger into sterile polythene bags and stored in an ice packed cooler. The collected oilfield wastewater and soil samples were immediately transported to the laboratory for analysis within 24 hours. 811 Int.J.Curr.Microbiol.App.Sci (2021) 10(03): 810-822 Bacteria were isolated from the soil (100g each) enriched with various concentrations (10%, 25%, 50%, 75%) of oilfield wastewater. The enriched soil sample was incubated in a rotary shaker incubator at 28oC with 200 rpm for aeration and withdrawn seven (7) days interval for analyses. Preparation Inoculum of Enriched Soil Sample One gram (1g) of the enriched soil samples were serially diluted onto 9 ml of sterile normal saline in a test tube to give an initial dilution of 1:10 ml (10-1 dilution). Subsequent dilutions were done up to 10-3dilution (Prescott et al., 2005). Isolation of Bacteria Isolation of heterotrophic bacteria was done using nutrient agar by the spread plate technique as described by Prescott et al., (2005). Aliquots (0.1ml) of serially diluted samples of 10-2dilution were spread plated onto dried sterile nutrient agar plates in duplicates. The plates were incubated at 370C for 24 hours. Representative colonies were selected and sub-cultured to purify them into pure isolates for characterization. The purified colonies represented the bacteria isolated from the enriched soil samples. The individual isolate were labeled OA1, OA2, OA3 and OA4. Molecular Isolates Identification of Bacterial The 16S rRNA regions of the rRNA gene of the isolates (OA1- OA4) were amplified after extraction and quantification of the DNA using the 27F: 5'-AGAGTTTGAT CMTGGCTCAG-3' and 1492R: 5'-CGGTT ACCTTGTTACGACTT-3' primers on an ABI 9700 Applied Bio systems thermal cycler at a final volume of 40 microlitres for 35 cycles. The PCR mix included: the X2 Dream taq Master mix supplied by Inqaba, South Africa (taq polymerase, DNTPs, MgCl), the primers at a concentration of 0.5µM and the extracted DNA as template. The PCR conditions were as follows: Initial denaturation, 95ºC for 5 minutes; denaturation at 95 ºC for 30 seconds; annealing at 52ºC for 30 seconds; extension at 72 ºC for 30 seconds for 35 cycles and final extension at 72ºC for 5 minutes. The product was resolved on a 1% agarose gel at 130V for 30 minutes and visualized on a blue light transilluminator. The sequences of the isolates were edited using the bioinformatics algorithm Trace edit; similar sequences were downloaded from the National Center for Biotechnology Information (NCBI) data base using BLASTN and was further aligned using ClustalX. The evolutionary history was inferred using the Neighbour-Joining method in MEGA 6.0 (Saitou and Nei, 1987). The bootstrap consensus tree inferred from 500 replicates (Felsenstein, 1985) was taken to represent the evolutionary history of the taxa analysed. The evolutionary distances were computed using the Jukes-Cantor method (Jukes and Cantor, 1969). The identified isolates were submitted to the Gene bank and were assigned accession numbers. Biodegradation Experiment The experiment was designed to analyze the potential of the selected organisms to biodegrade polycyclic aromatic hydrocarbons (PAHs) and total petroleum hydrocarbon in oilfield wastewater using Gas Chromatograph. Preparation of Inoculum The microbial inocula consisted of indigenous organisms (HUB) obtained earlier from the study of enrichment of various concentration of oilfield wastewater on soil microorganisms. 812 Int.J.Curr.Microbiol.App.Sci (2021) 10(03): 810-822 The method described by El-Borai et al., (2016) was adopted. Bacteria were sub cultured on a sterile nutrient agar and incubated for 24 hours at 37 oC. A loopful of each bacterium isolates (OA1- OA4) were inoculated into 4 ml nutrient broth medium at 35 oC for activation of the organisms for biodegradation. The different strains from overnight cultures at the log phase of growth were transferred to 250 ml conical flasks each containing 50 ml of sterile defined mineral salts medium (MSM) for 24 hours at 35 oC in a shakers incubator. The bacterial suspension turbidity was adjusted to 0.5 McFarland standards (1.5×108). Composition of Biodegradation Set up The biodegradation experimental set up was made up of five conical flasks (250 ml) each, labeled A1 to A5 (Table 1). Each flask contained 125ml of oilfield wastewater (ofww) and 6.25 ml (5%) of the bacteria culture. The set up were placed in a shaker incubator at 28oC with 200 rpm for aeration and were as presented on Table 1. Results and Discussion Figure 1 shows the phylogenetic tree and the evolutionary relationship of the individual isolates. The isolates labelled OA1 to OA4 showed 100% relatedness to their relatives in the gene bank and were assigned accession numbers. The isolates labelled OA1, OA2, OA3 and OA4 were identified as Morganella morganii (MN094330), Pseudomonas xiamenensis (MN094331), Chryseobacterium cucumeris (MN094332) and Staphylococcus sp (MN094333), respectively. Figure 2 shows the initial concentration of the PAHs and the biodegradation by the single bacterium. On day 7 Chryseobacterium cucumeris recorded the least remaining while the control recorded the highest remaining. On day 21 Pseudomonas xiamenensis showed the least remaining. The concentration of the PAHs on the initial was 101.72992 mg/l. The treatment option containing Pseudomonas xiamenensis had the least amount of 22.23959 mg/l remaining on day 21 with removal of 78.1%, which was followed by the treatment option with Staphylococcus sp which recorded remaining of 27.31228 mg/l with 73.2% removal, Chryseobacterium cucumeris had 34.98499 mg/l remaining with 65.6% removal and Morganella morganii recorded 35.50295 mg/l remaining with 65.1% removal. The control had the highest amount of 75.40663 mg/l remaining with 25.9% removal at the end of the experiment (Table 2). The GC profile in Figure 3 (day 1) showed the presence of Naphthalene, Acenephthylene, Acenaphthene, Anthracene and Chrysene. The control and treatment with Morganella morganii did not show any clearance of the individual polycyclic aromatic hydrocarbons on day 21 (Figures 4 and 5). There was complete absence of Chrysene on day 21 in the treatments with Pseudomonas xiamenensis, Staphylococcus sp and Chryseobacterium cucumeris as shown in the GC (Figures 6, 7 and 8). There were reductions in the peaks of the various polycyclic aromatic hydrocarbons on day 21 in all the treatments. The results in Figure 9 showed the concentration of the total petroleum hydrocarbon on day 1, 7 and 21. On day 7, the Staphylococcus sp, treated sample recorded the highest remaining while the least remaining was observed in the Chryseobacterium cucumeris. The highest and least remaining on day 21 was observed in control and Chryseobacterium cucumeris, respectively. Table 3 showed the initial, final concentration and the percentage removal of 813 Int.J.Curr.Microbiol.App.Sci (2021) 10(03): 810-822 the treatment options. The initial concentration on day 1 was 342.89053 mg/l. The final concentration recorded on day 21 for the control was 240.74905 mg/l with 36.9% percentage removal. Morganella morganii recorded remaining of 129.47221 mg/l with 66.1% removal. Pseudomonas xiamenensis on final day recorded 119.29648 mg/l and obtained percentage removal of 68.8%. Staphylococcus sp recorded 85.04915 mg/l on day 21 with 77.7% percentage removal. Chrysebacterium cucumeris recorded final concentration of 58.18741 mg/l with percentage removal of 84.8%. The GC profiles of the various treatments are shown in Figure 9, 10, 11, 12, 13 and 14. Figure 9 show the individual n-alkanes and their peaks on day 1. The n-alkanes recorded were C8, C9, C10, C12, C14, C15, Pr, C18, C19, C20, C22, C23 and C26. Figure 10 showed the GC of the control on day 21, there was no clearance of n-alkanes as observed. Figure 11 showed the GC profile of the treatment with Morganella morganii on day 21, there was total clearance of C12, Pr, C22 and C26. Treatment with Pseudomonas xiamenensis on day 21 showed removal of C12, C19, C22 and C26 as shown in Figure 12. Staphylococcus sp treatment option on day 21 recorded removal of C12, Pr, C19, C20, C22 and C26 as shown in Figure 13. Treatment option with Chryseobacterium cucumeris as shown in Figure 14 completely removed C10, C12, Pr, C19, C20, C22, C23 and C26. Generally there was reduction in the level of peaks. Microorganisms obtained from hydrocarbon polluted environment have been known to be efficient in using hydrocarbons as carbon and energy sources (Obire et al., 2020; Aleruchi and Abu, 2015; Cui et al., 2008). The results clearly showed that the bacteria isolates were capable of growing in hydrocarbon polluted environment as they were isolated from soil enriched with oilfield wastewater. Bacteria isolate showed 100% relatedness to their relatives in the gene bank. Microorganisms capable of utilizing hydrocarbon are widely distributed in nature and have been found in areas not directly contaminated with hydrocarbon (Yousseff et al., 2010). The biodegradative capabilities of the single isolates to biodegrade polycyclic aromatic and total petroleum hydrocarbons were compared. For polycyclic aromatic hydrocarbon, Chryseobacterium cucumeris recorded the least remaining on day 7, this was followed by Staphylococcus sp, Morganella morganii, Pseudomonas xiamenensis and control. Pseudomonas xiamenensis on day 21 had the least remaining and the highest percentage removal, followed by Staphylococcus sp, Chryseobacterium cucumeris, Morganella morganii and the control. The results indicate that some organisms have different strategy they use to attack polycyclic aromatic hydrocarbon, while some may attack faster, some slowly. This was seen in the case of treatment with Pseudomonas xiamenensis which had highest remaining concentration of polycyclic aromatic hydrocarbon among the treatment options on day 7 but on day 21 it recorded the least remaining, Chryseobacterium cucumeris and Morganella morganii reduced in its degradation rate after day 7. Staphylococcus sp maintained its degradation rate. The control recorded the highest remaining concentration of polycyclic aromatic hydrocarbons on day 21. The Gas Chromatography on day 21 showed total clearance of chrysene by Pseudomonas xiamenensis, Staphylococcus sp and Chyrseobacterium cucumeris. 814 Int.J.Curr.Microbiol.App.Sci (2021) 10(03): 810-822 Table.1 Biodegradation Set up Set up A1 A2 A3 A4 A5 Content Control (ofww only) Morganella morganii + ofww Pseudomonas xiamenensis + ofww Staphylococcus sp + ofww Chryseobacterium cucumeris + ofww Table.2 Biodegradation of PAHs by Single Isolates (Bacterium) Treatments Initial (Day 1)(mg/l) Final (Day 21)(mg/l) % Removal Control (ofww) Morganella morganii + ofww Pseudomonas xiamenensis + ofww Staphylococcus sp + ofww Chryseobacterium cucumeris + ofww 101.72992 101.72992 101.72992 101.72992 101.72992 75.40663 35.50295 22.23959 27.31228 34.98499 25.9 65.1 78.1 73.2 65.6 KEY: offw= oilfield wastewater Fig.1 Phylogenetic tree showing the evolutionary distance between the bacterial Isolates 815 Int.J.Curr.Microbiol.App.Sci (2021) 10(03): 810-822 Table.3 Biodegradation of TPHs by Single Isolates (Bacterium) Treatments Control (ofww) Morganella morganii + ofww Pseudomonas xiamenensis + ofww Staphylococcus sp + ofww Chryseobacterium cucumeris + ofww Initial (Day 1)(mg/l) 342.89053 342.89053 342.89053 342.89053 342.89053 Final (Day 21)(mg/l) 240.74905 129.47221 119.29648 85.04915 58.18741 % Removal 36.9 66.1 68.8 77.7 84.8 KEY: offw= oilfield wastewater Fig.2 Biodegradation of PAH by single bacterium (Morganella morganii, Pseudomonas xiamenensis, Staphylococcus sp and Chryseobacterium cucumeris) PAHs (mg/l) 150 Control Mm 100 Px Ss Cc 50 0 1 7 21 Duration of incubation (Days) Fig.3 GC profile showing the polycyclic aromatic hydrocarbon (PAH) on Day 1 Fig.4 GC profile showing the biodegradation of polycyclic aromatic hydrocarbon (PAH) by the control on day 21 816 Int.J.Curr.Microbiol.App.Sci (2021) 10(03): 810-822 Fig.5 GC profile showing the biodegradation of PAHs by Morganella morganii on day 21 Fig.6 GC profile showing the biodegradation of PAH by Pseudomonas xiamenensis on day 21 Fig.7 GC profile showing the biodegradation of Staphylococcus sp PAHs by on day 21 Fig.8 GC profile showing the biodegradation of PAHs by Chryseobacterium cucumeris on day 21 817 Int.J.Curr.Microbiol.App.Sci (2021) 10(03): 810-822 Fig.9 Biodegradation of TPH by single bacterium (Morganella morganii, Pseudomonas xiamenensis, Staphylococcus sp and Chryseobacterium cucumeris) 500 Control Mm Px Ss Cc TPH (mg/l) 400 300 200 100 0 1 7 21 Durati on of Incubati on (Days) Fig.10 GC profile showing the total petroleum hydrocarbon (TPH) of the control on day 1 Fig.11 GC profile showing the biodegradation of total petroleum hydrocarbon (TPH) by the control on day 21 Fig.12 GC profile showing the biodegradation of TPH by Morganella morganii on day 21 818 Int.J.Curr.Microbiol.App.Sci (2021) 10(03): 810-822 Fig.13 GC profile showing the biodegradation of TPH by Pseudomonas xiamenensis on day 21 Fig.14 GC profile showing the biodegradation of TPH by Staphylococcus sp on day 21 Fig.15 GC profile showing the biodegradation of TPH by Chryseobacterium cucumeris on day 21 On day 7, the biodegradation of total petroleum hydrocarbon recorded least remaining in the treatment with Chyrseobacterium cucumeris which was followed by Pseudomonas xiamenensis, Morganella morganii, control and Staphylococcus sp. Staphylococcus sp did not reduce the total petroleum hydrocarbon on day 7 however it recorded the second best in the reduction of total petroleum hydrocarbon on day 21, while the best or least reduction was observed in the treatment with Chyrseobacterium cucumeris. The control on day 21 did not show any clearance of the individual n alkane but removal most n alkanes were observed in other treatment options. Chrseobacterium cucumeris showed more clearance of the n alkane which was followed by Staphylococcus sp. Comparing the degradative capabilities of the individual isolates to biodegrade polycyclic aromatic hydrocarbon and total petroleum hydrocarbon. 819
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