Influence of a Dispersant on the Types and Growth of Microbial Hydrocarbon Degraders in a Crude Oil-contaminated Medium

Background: Dispersants are first order response strategies for oil spill cleanup in an aquatic environment. However, their effects on the biodegradation capacity of indigenous hydrocarbon-degrading microorganisms are little known. Objectives: The influence of a dispersant (DS/TT/066) on the type(s) and growth of hydrocarbon-degrading bacteria (HDB) and hydrocarbon-degrading fungi (HDF) in a crude oil-contaminated medium (water) was investigated in the laboratory for 28 days. Methods: The experiment was set up in duplicates with the first set containing Forcados light crude oil (FLCO) alone in water while the other was a mixture of FLCO and DS/TT/066 (ratio 9:1 v/v). Identification and enumeration of HDB and HDF were conducted according to standard methods. Total petroleum hydrocarbons (TPH) in the test media was analyzed using a gas chromatography/flame ionization detector. Results: The results showed that HDB identified in the FLCO alone included Pseudomonas aeruginosa (day 0), Proteus vulgaris (day 14), P. aeruginosa and Kliebsiella pneumoniae (day 28). However, in the mixture, Escherichia coli was identified on day 14 in addition to the other species observed in FLCO alone. HDF identified in FLCO alone were Candida krusei and Candida albicans (days 0 and 14), Trichosporon cutaneum and C. albicans (day 28). In the mixture, HDF identified were C. albicans (day 0), C. albicans and Aspergillus spp. (days 14 and 28)″ Furthermore, the mixture enhanced the growth of HDBF (average counts: 32.5 × 107 and 225 × 106 cfu/mL) compared to FLCO alone (17.5 × 107 and 17.5 × 106 cfu/mL) by day 14 respectively. Total petroleum hydrocarbon reduction was highest (85%) in the mixture compared to 5% in FLCO alone by day 14. Conclusions: The study demonstrated the biodegradation efficiency of E. coli, P. vulgaris (bacteria), C. albicans and Aspergillus spp. (fungi) in a crude oil-contaminated aquatic environment in conjunction with dispersant use. Further studies in the field are recommended in order to explore their potential for rapid and large scale crude oil clean-up operations.


Introduction
In the aquatic environment, crude oil spills have widespread deleterious effects and require the deployment of various control and clean-up strategies including the use of dispersants. Dispersants are chemicals that are applied to oil to break it up into tiny droplets and enhance biodegradation of bulk crude oil by increasing the bioavailable fraction of the hydrocarbon through mobilization of absorbed hydrocarbons or by increasing its effectiveness in aqueous solubility. 1 Chemical dispersants can be very effective tools in the management of crude oil spilled in aquatic environments. 2 However, several important factors must be considered in deciding whether to use dispersants, including their physical effectiveness under conditions of expected use, the effect of dispersants on the fate of spilled petroleum hydrocarbon products, and the type of joint action toxicity exhibited by the mixtures. 3- 6 Dispersant effectiveness has been defined as the amount of oil that the dispersant puts into the water column compared to the amount of oil that remains on the surface. 7 Microbial biodegradation assays of pollutants have intensified in recent years as efforts have been made to find sustainable ways to cleanup contaminated environments. 8 Biodegradation of petroleum hydrocarbon (crude oil) is a complex process that depends on the nature Research and amount of the hydrocarbons present. 9 Microbial degradation is the major and ultimate natural mechanism by which crude oil is cleaned up in the environment. 10 The mechanism for degradation of crude oil hydrocarbons has been studied extensively, and a number of microorganisms, including bacteria and fungi, have been isolated and characterized for their ability to degrade crude oil hydrocarbons. [11][12][13] Bacteria are the most active agents in petroleum degradation and work as primary degraders of spilled oil in the environment. In addition, several bacteria are known to feed exclusively on hydrocarbons. 14 Some fungi are also capable of utilizing crude oil. 15 Typical bacteria groups previously identified for their degrading capacity include Pseudomonas spp., Gordonia spp. and Cellulomonas spp., while fungi genera include Aspergillus spp., Penicillium spp., Fusarium spp. and Candida spp. 16,17 According to Mulyono et al., dispersants tend to increase oil biodegradation by increasing the surface area for microbial attack and encouraging migration of the droplets through the water column making oxygen and nutrients more readily available. 18 Venosa and Holder also reported that degradation was much more rapid for dispersed oil than for non-dispersed oil, because in the non-dispersed control, the microbial culture first had to generate its own biosurfactant to emulsify the oil before substantial degradation could occur. 19 In addition, Sogbanmu and Otitoloju reported that some dispersants used in Nigeria were capable of enhancing the microbial biodegradation of crude oil. 20 However, few studies have identified the types of microbial hydrocarbon degraders and the extent of degradation of crude oil in concert with dispersant use in an aquatic environment.
This research consequently aimed to investigate the influence of a named dispersant on the types and growth of hydrocarbon-degrading bacteria and fungi in a crude oilcontaminated medium (water) with a concomitant evaluation of the extent of biodegradation through measurement of leftover total petroleum hydrocarbons over a period of 28 days.

Materials
Crude Oil: Forcados light crude oil (FLCO) used for this experiment was obtained from the Shell production platform in Forcados, Burutu Local Government Area of Delta State, Nigeria. The physico-chemical properties of the crude oil include: sulphur content = 0.2%, American Petroleum Institute gravity = 60/60 F, rapid vapor pressure = 2.5 psi, and pour point = 25.
It was stored in a sealed plastic vessel in the laboratory at room temperature and used within a period of 30 days.

Dispersant (DS/TT/066):
Browncolored liquid containing surfactant mixed with a hydrocarbon solvent. Manufactured by Oil Pollution Environmental Control Limited, West Yorkshire, Great Britain.

Test Media Preparation
Mixtures containing FLCO alone and a binary mixture of FLCO:DS/TT/066 (ratio 9:1 v/v) were prepared and made up to 5 mL/L. For the FLCO:dispersant mixture, the proportion of each constituent compound dictated by the ratio was computed and measured out.
FLCO was applied to the substrate (water) before transferring the dispersant at the desired concentration into the test container. There were two replicates per mixture and these were exposed over a period of 28 days.

Isolation of Hydrocarbon Degrading Bacteria
Isolation of hydrocarbon degrading bacteria (HDB) was conducted as described by Sogbanmu and Otitoloju. 20 Briefly, diluent (distilled water) for serial dilution was prepared by pipetting 9 mL into test tubes. They were covered with aluminum foilwrapped cotton wool and autoclaved at 121°C for 15 minutes. A mineral salts medium (100 mL) to which was added an appropriate quantity of agaragar (solidifying agent) was brought to boil in a water bath and autoclaved at 121°C for 15 minutes. This was allowed to cool to about 45°C, then aseptically poured into sterile petri dishes and allowed to set. The mineral salts medium used for this study was adapted from Adebusoye  Research for 3 days (optimal growth time for hydrocarbon degrading bacteria).

Isolation of Hydrocarbon-Degrading Fungi
Briefly, 9 mL of distilled water was pipetted into a test tube and the tubes were covered with aluminum foil wrapped cotton wool and autoclaved at 121°C for 15 minutes and allowed to cool. Potato dextrose agar (Lab M) was prepared according to the manufacturer's instructions and then autoclaved at 121°C for 15 minutes. The agar was allowed to cool and then poured into sterile petri dishes. For isolation, 1 mL of sample mixtures was taken with the aid of a micropipette and transferred into a test tube containing 9 mL of sterile distilled water (after cooling). Ten-fold serial dilution was done up to 6 times (10 -6 ) and then 0.1 mL of sample (6 th dilution) was inoculated onto sterile plates containing cooled agar. The sample was spread on the agar surface with the aid of a sterile glass spreader for even distribution of colonies. Incubation was carried out at 28°C for 7 days (optimal growth time for hydrocarbon degrading fungi).

Identification of Isolates
The distinct colonies obtained after incubation were sub-cultured onto new (freshly-prepared) nutrient agar plates ( For isolation, 1 mL of sample mixture was aseptically transferred into a test tube containing 9 mL of distilled water (after cooling) using a micropipette. Ten-fold serial dilution was done up to 7 times (10 7 ) and then 0.1 mL of sample (7 th dilution) was inoculated onto sterile plates containing cooled agar. The sample was spread on the surface with the aid of a sterile glass spreader for even distribution of colonies or cells. A sterile filter paper to which crude oil had been added and spread evenly was placed on the inside top cover of the petri dish before covering. These plates were stored in an incubator at room temperature

DAY 14
FLCOa software and utilized automated integration to obtain total area counts of chromatogram peaks. The data for total petroleum hydrocarbon concentration measured in the samples by gas chromatography/flame ionization detector were expressed in mg/L.

Data Analysis
The results of the analysis of total hydrocarbon degrading bacteria and hydrocarbon degrading fungi were expressed in colony forming units per milliliter (cfu/mL) and represent average counts of colonies. Microsoft Excel version 2010 was used to obtain the mean and standard deviation of the data as well as plot the graphs.
Identification was done with the use of a flow chart based on the manual for the identification of bacteria. 23 Biochemical tests for identification of fungi isolates are presented in Table 2.

Enumeration of Hydrocarbon-Degrading Bacteria and Fungi
The cultured samples of bacteria and fungi which were incubated at room temperature for 3 days and 28°C for 7 days, respectively, were brought out and colonies in the different petri dishes were observed, counted and recorded. 20

Collection, Extraction and Analysis of Total Petroleum Hydrocarbon in Water Samples
With the aid of a micropipette, 100 mL of water sample was obtained and

Identification of Hydrocarbon-Degrading Bacteria and Fungi in the Test Media
The results of the HDB showed that FLCO alone supported the growth of Pseudomonas aeruginosa at day 0, Proteus vulgaris at day 14, and P. aeruginosa and Kliebsiella pnuemoniae by day 28. The FLCO:DS/TT/066 mixture also supported the growth of the same bacteria species as FLCO on days 0 and 28. However, for day 14, in addition to P. vulgaris, Escherichia coli was identified (Tables 1 and 3).
The HDF identified in the medium containing FLCO alone was Candida krusei and Candida albicans at day 0, Candida albicans at day 14, Trichosporon cutaneum and Candida albicans at day 28. The FLCO:DS/ TT/066 mixture favored the growth of C. albicans alone at day 0, C. albicans and Aspergillus spp. at days 14 and 28 (Tables 2 and 3).   (Figure 1 and 2).

Enumeration of Hydrocarbon-Degrading Bacteria and Fungi in the Test Media
Comparison of the percentage increase in HDB from day 0 to day 14 in the test media showed a ~29% and ~54% increase for FLCO alone and the FLCO:DS/TTT/066 mixture, respectively. A decrease of 9% and ~60%, respectively, was observed in the test media from day 14 to day 28 ( Figure 1). For HDF, the percentage increase from day 0 to day 14 in the test media was ~89% and ~94% for FLCO alone and FLCO:DS/TTT/066 mixture, respectively. A decrease of ~86% and ~97%, respectively, was observed in the test media from day 14 to day 28. Hence, in both media, the highest HDB and HDF was observed on day 14 (Figure 2).

Total Petroleum Hydrocarbons Analysis
The results of the analyses of TPH in the test media revealed that the FLCO:DS/TT/066 mixture had a higher reduction in the TPH at days 14 and 28 (86% and 93%, respectively) compared to the mixture containing FLCO alone (5% and 38% respectively). Consequently, the highest reduction in TPH levels was achieved in the FLCO:DS/TT/066 mixture at day 28 compared to FLCO alone (Figure 3).

Discussion
The identified HDB in the test media showed the types of bacteria that

Figure 3 -Total petroleum hydrocarbons levels in test media over a period of 28 days
Research were capable of utilizing the crude oil as a carbon source and thus degrading it. These microorganisms have also been reported in other studies to be capable of hydrocarbon degradation. 26 However, in addition to the identified bacteria in both test media, E. coli was also identified in the dispersed crude oil mixture on day 14 compared to the undispersed oil. This suggests that E. coli was able to degrade the oil in the presence of a dispersant. The mechanism of hydrocarbon degradation in bacteria has been studied and shown to involve the production of biosurfactant which promotes the cracking of hydrocarbon molecules by micelle formation, increasing their mobility, bioavailability and exposure to bacteria, thus favoring hydrocarbon biodegradation. 27 The presence of the dispersant seems to have hastened the process of biosurfactant production so that the bacteria were able to act faster than in the undispersed medium.
In addition, the presence of E. coli in the dispersed mixture and not in the undispersed mixture could also mean that some components of the crude oil which E. coli specializes in degrading were available in the dispersed medium, but not the undispersed medium. Other bacteria (Pseudomonas aeruginosa, Proteus vulgaris and Klebsiella pneumoniae) identified in both media shows that these species are broad spectrum and hence can act both in the presence and absence of a dispersant. P. aeruginosa has been used for bioremediation of places contaminated by petroleum. 28 In addition, the different bacteria identified showed some level of specificity in the timing of occurrence. P. aeruginosa were only identified on day 0 and 28, which suggests that they were only available to act on components of the oil that were present in the beginning and towards the end of the degradation process. It In order to certify that actual biodegradation had occurred, total petroleum hydrocarbons in the test media was measured. The results showed a reduction of TPH in both media from days 0 to 28. However, the dispersed oil medium showed a very sharp decrease in TPH by day 28 in particular, compared to the undispersed oil. These results suggest that the HDB and HDF in the media, especially those identified on day 14, did the greater work of degradation as the percentage reduction in TPH between day 0 and 14 was several folds higher than that observed between days 14 and 28 in the dispersed oil medium. Also, the level of TPH left over in the dispersed oil medium after 28 days revealed that biodegradation was several times faster in the presence of the dispersant. The results of the TPH levels correlate with the results of the HDB and HDF counts in the dispersed oil medium. The level of TPH in the dispersed oil medium was higher on day 0 which suggests that the crude oil was more available in the medium, thus stimulating the HDB and HDF growth as seen in the counts on day 14. This is in accordance with the report by Souza et al., which argued that the larger the degrading microorganism population, the quicker and more efficient the Research bioremediation process. 27 The increase in HDB and HDF counts means that the microorganisms were actively degrading the TPH which resulted in a very steep reduction in TPH by day 14. Suwansukho et al. noted that biosurfactants are usually produced in the exponential or stationary phase of microbial growth when there is a high cellular density. 30 These biosurfactants synthesized as metallic products of different microorganisms degrade hydrocarbons and use them as a carbon source. 31,32 However, the gradual depreciation in TPH in the dispersed oil medium resulted in the reduction in HDB and HDF counts between days 14 and 28. This was shown in the slight decrease in TPH between days 14 and 28 compared to days 0 and 14.

Conclusions
The diversity of microorganisms capable of biodegrading pollutants such as oil is vast and yet to be fully exploited. In addition, with advances in research, the number of identified species grows every year. 33 The present study identified the types of HDB and HDF in a dispersed oil medium, with a focus on the identification of specific bacteria and fungi in the dispersed medium as opposed to the undispersed medium. The efficacy of degradation seen in the dispersed oil medium suggests that these specific bacteria and fungi might possess greater biodegradation potentials which have yet to be explored in the presence of a dispersant. Therefore, we recommend that further studies, both laboratory and field based, be carried out to take advantage of the biodegradation potentials of these naturally occurring microorganisms in tandem with responsible dispersant use to combat crude oil pollution in aquatic ecosystems. In particular, we recommend further research to better understand the specific roles of the identified HDB and HDF colonies in the degradation of crude oil in concert with dispersant use under natural conditions.