Bioremediation & Biodegradation Microalgal-Bacterial Consortium in Polyaromatic Hydrocarbon Degradation of Petroleum – Based Effluent

The recurrent and indiscriminate discharge of flow stations' crude oil effluent has led to an increased polyaromatic hydrocarbons (PAHs) level in the environment. PAHs are high-priority environmental pollutants of significant human health concerns, thus considerable effort has been made in this study to biodegrade these PAHs using microalgal- bacterial consortia. Crude oil effluent samples collected from the saver pit at Egbaoma flow station in the Niger Delta region of Nigeria were analysed biochemically and microbiologically. Three species of bacteria and two species of microalgae isolated, characterized and screened emerged as best hydrocarbon utilizers. The consortia comprised of Chlorella minutissimma and Aphanocapsa sp. as microalgae inoculants, while Citrobacter sp. SB9, Pseudomonas aeruginosa SA3 and Bacillus subtilis SA7 as bacterial inoculants. Growth dynamics, pH and degradation of the effluents' PAH content were analyzed for a period of ten days. Consortium BCC (bacteria inoculants and Chlorella minutissimma ), BCCA (all inoculants) and BCA (bacteria inoculants and Aphanocapsa sp.) had PAH degradation percentage of 92.09%, 67.76% and 47.19% and optical density (OD) of 0.1255°A, 0.0669°A and 0.0703°A from an initial OD of 0.0126°A respectively. Only BCA had an acidic pH after ten days. Effective synergism and excellent PAH degradation is achievable and highly recommendable with microalgal-bacterial consortium yet its success is highly dependent on the consortium assembling, therefore, proper selection of the microbes for biodegradation is paramount to the success and efficiency of the degradation process.


Introduction
Petroleum-based effluent is the largest waste stream generated in the petroleum industry. On shore, crude oil effluent generated in flow stations is collected in a series of pit where it is mechanically treated with API (American Petroleum Institute) oil-water separator before being discharged into surrounding water bodies [1]. The effluent contains significant amount of oil and other impurities [2]. It has been reported that toxic compounds such as aliphatic hydrocarbons, aromatic hydrocarbons, phenols, heavy metals/metal derivatives and other chemicals used during processes such as de-emulsification, are always present in crude oil effluent [3,4]. The accumulation of the toxic compounds from the discharged effluent in the receiving water bodies and its surrounding environment results in serious environmental and health consequences [5]. Polycyclic aromatic hydrocarbons (PAHs), a major constituent of petroleum-based effluent, are a group of hydrocarbons containing two or more fused aromatic rings in linear, angular or clustered arrangements. They are naturally present in crude-oil and generated during fuel combustion, waste incineration or as by-product of industrial processes such as petroleum exploration [6]. PAHs have been reported as high priority pollutants of the environment and are of significant health concerns because of their ability to solubilized in water, toxicity, recalcitrant nature and bioaccumulation potential [7][8][9] amongst other researcher, have reported that the toxicity and recalcitrant nature of PAHs are the major reasons for the relative inefficiency in PAHs degradation by bacteria and fungi both in pure form and in consortium, therefore researchers have reportedly explored the bioaccumulation and biodegradation potentials of microalgae [10][11][12][13]. Microalgae play an important role in the environmental cleaning of pollutants. These are a diverse group of prokaryotes with varying morphological and physiological properties, and have attracted increasing interests because of their immense potentials [7,[14][15][16][17][18]. Their most common feature is their ability to carry out oxygenic photosynthesis which is similar to that in higher plants, and this makes a large contribution to the equilibrium of the earth's atmosphere by producing oxygen and removing carbon (IV) oxide [19]. Vast interest in microalgae obtained from the environment has grown considerably because they represent an innovative approach to obtain new underexplored resources [20]. In nature and long term laboratory algal cultures, most microalgae are found in association with other aerobic or anaerobic microorganisms. The bacterial assemblages are known to influence the development or decline of algal blooms [21]. The molecular oxygen from algal photosynthesis is used as an electron acceptor by bacteria to degrade organic matter while the carbon dioxide (CO 2 ) from the bacterial mineralization completes the photosynthetic cycle. The symbiotic interactions of microalgae and bacteria form the basis of the biological oxygen demand (BOD) removal in the wastewater treatment ponds, first reported by Ref. [22]. Furthermore, microalgae can help in co-metabolic degradation or produce biosurfactants and extracellular matters to enhance bacterial activity for increasing pollutant bioavailability [23]. Additionally, microalgae can accumulate hydrocarbons, and subsequently make those compounds available to the associated hydrocarbon-utilizing bacteria [24]. The cooperation of phototrophic microalgae and heterotrophic bacteria could potentially improve the degradation of environmental contaminants including oil pollutants [25,26]. Since microalgae and bacteria have been reported to biodegrade environmental pollutants with varying degrees of success, this study has been conducted to ascertain

Methodology
All chemicals were of analytical grade; crude oil was obtained from Egbaoma flow station, Nigeria. Unless otherwise specified, all experiments were conducted in triplicate at 28 ± 2°C under sterile conditions. The microbial strains were isolated from the saver pit sample.

Sample site and collection
Sampling was conducted in June and September at the Egbaoma flow station's saver pit in Umutu, Niger Delta region of Nigeria. Subsurface effluent samples were collected into 250 mL amber bottles for hydrocarbon analysis and 1 L plastic bottle for physicochemical and microbiological analysis, although, pH and temperature were recorded in situ. Samples were transported in ice packs containing dry ice at 4°C to the laboratory for analysis.

Sample analysis
Physicochemical assessment: The physicochemical analysis of the waste water samples were conducted using standard methods [27,28]. The pH and electrical conductivity were measured using pH meter 3015, Jenway, and conductivity meter as described by Ref. [29] while dissolved oxygen (DO) and total dissolved solids (TDS) were measured using [30] DO and TDS meter respectively. Turbidity was measured using ref [30]  ) and total petroleum hydrocarbon (TPH) were determined using [17] spectrophotometer while total hardness, alkalinity, and biological oxygen demand (BOD) were determined volumetrically [27,28]. Salinity was determined using Ref. [31] equipment while temperature was determined with a mercury thermometer.
PAH assessment: PAHs were analyzed using a gas chromatography fitted with flame ionization detector (GC-FID). The extractions were carried out using n-hexane and methanol followed by procedures [27,28].
Microbiological assessment: Total cultural heterotrophic bacteria and microalgae counts were done using the pour plate techniques on nutrient agar (Oxoid, UK) as described by Ref. [32,33] on Allen medium respectively. The total hydrocarbon utilizing bacteria and microalgae counts were determined using the vapour phase transfer method [8,34] Hydrocarbon utilizing microalgae and bacteria were screened for pollutant tolerance/utilization using Allen medium and minimal salt medium of Ref. [35] respectively supplemented with 1% (v/v), 1.5% (v/v) and 3% (v/v) crude oil. This was done by subculturing the isolates from 1% (v/v) to 1.5% (v/v) and finally to 3% (v/v) after an incubation period of 5 days each for bacteria and 7 days each for microalgae. Isolates with the best hydrocarbon utilizing ability were characterized and identified based on their morphological, physiological and biochemical characteristics presented in Bergey's Manual of Determinative Bacteriology [36] and the API kit profiles [37] while that of microalgae was done by comparison with those documented in the Identification Guide to freshwater and terrestrial algae [38]. These isolates were used to make up three consortia in the following order, The ability of isolates to degrade PAHs was determined using BG11 medium [39] as described by Ref. [12] with a slight modification. Thirty milliliters of BG11 medium and ten milliliters of filtered effluent sample was dispensed aseptically into one hundred milliliters Erlenmeyer flasks after which three milliliters suspension of isolates in a ratio of 2:1 for microalgae and bacteria was aseptically dispensed into the flask. Polyaromatic hydrocarbon degradation assessment was carried out for 240 hrs in 120 hrs intervals on a rotary shaker (G24 environmental incubator shaker, New Brunswick Scientific Co. Inc. Edison, New Jersey, USA.) at 160 rpm. The initial absorbance of the medium was measured at 600 nm on a UV-Visible Spectrophotometer (Camspec M105) and standardized at 0.8°A while pH was adjusted to 6.5.

Results
The physicochemical analysis of the crude oil effluent sample showed that total nitrogen (September samples only), sulphur, conductivity, salinity and total petroleum hydrocarbon, for both samples, were well above the [40] limit while other parameters were relatively within the allowable limits ( Table 1). The mean heterotrophic bacteria and microalgae counts were 30.0 × 10 6 cfu/ml and 530 cells/ml respectively while the of hydrocarbon utilizing bacteria and microalgae counts were 6.01 × 10 3 cfu/ ml and 189 cells/ml respectively. The ability of isolates to tolerate and utilize hydrocarbon showed three bacteria isolates, Pseudomonas aeruginosa SA3, Bacillus subtilis SA7 and Citrobacter sp. SB9, grew very well on all concentrations of crude oil while seven microalgae isolates survived at 3%v/v crude oil with two isolates, Chlorella minutissima and Aphanocapsa sp, emerging as the most dominant having counts of 66 cells/ml and 52 cells/ml respectively. BCCA -Pseudomonas aeruginosa SA3, Bacillus subtilis. SA7, Citrobacter sp. SB7, Chlorella minutissima SA1 and Aphanocapsa sp. SB5+MSM+1% (v/v) crude oil effluent BCA, BCC and BCCA respectively (Figure 2) with consortium BCA falling below the initial pH level. The optical density (OD) of the degradation medium increased from 0.0126°A to 0.1255°A, 0.0703°A and 0.0669°A for consortium BCC, BCA and BCCA respectively (Figure 1) with consortium BCC having the highest optical density after 240 hrs while pH change observed ranged from 6.5 to 6.3, 6.8 and 6.6 for consortium. Figure 3 shows the percentage degradation of total PAH by the consortia. Consortium BCC had the highest percentage of 92.07%, preceded by BCCA with a percentage of 67.76% while the least was observed in BCA with percentage of 47.19%. Consortium BCC was able to remove over 90% of sixteen PAHs detected in the crude oil effluent after 240 hrs with an exception of acenaphthylene, anthracene and benzo (a) anthracene ( Figure 4).

Discussion
According to Ref. [41,42], the percentage of hydrocarbon utilizing microbial counts as compared to the heterotrophic microbial counts obtained from the saver pit sample could be attributed to the percentage of total petroleum hydrocarbon in the sample. These researchers proved that there's always a correlation between the level of hydrocarbon pollution and the ratio of heterotrophic microorganisms to hydrocarbon utilizing microorganisms present in the effluent. The emergence of Pseudomonas aeruginosa SA3, Bacillus subtilis. SA7, Citrobacter sp. SB7, Chlorella minutissima SA1 and Aphanocapsa sp. SB5 as best hydrocarbon utilizers indicates that these species are capable of utilizing and metabolizing the various hydrocarbon components contained in the crude oil [10,23,43]. The optical density, pH and PAH degradation percentage observed for the consortia in this study indicates successful synergistism of inoculant, utilization and degradation of the PAH fraction of the effluent. This is affirmative to the findings of Ref. [12,44,45] who achieved similar results with different species of bacteria and microalgae in consortium. The increased concentration of some PAHs such as anthracene and benzo(a)anthracene by the consortia (Figure 4) affirms the findings of Ref. [13,30] who proved that these PAHs are refractory organic compounds. The accumulation of some PAHs such as acenaphthene, dibenzo(a,h)anthracene, indicated by increased concentrations in Figure 4 is in accordance with the findings of Ref. [46,47] who proved that some hydrocarbons especially PAHs tend to increase during biodegradation in relative or absolute amounts suggesting that these compounds do not only tend to resist biodegradation but are formed de novo by condensation and/or displacement reactions of the biodegradation derivatives. The fluctuations observed for the control samples does not indicate degradation or accumulation, it simply indicates the condensation and/or displacement reactions of the biodegradation components that occurs in the natural environment.

Conclusion
This study has proven that microalgae-bacteria consortium isolated from a crude oil polluted site possess the ability to biodegrade crude oil effluent based-PAHs and preferentially attack high molecular PAHs that has been branded by the US EPA as high priority pollutants.