Comparison of biodegradative efficiency of wildtype versus mutagenised Scenedesmus vacuolatus of spent coolant waste: dehydrogenase activity and total petroleum degradation studies

ABSTRACT This study aims to compare the biodegradability efficiency of spent coolant waste by wild-type microalga versus mutagenised Scenedesmus vacuolatus. The biodegradation efficiency of both groups microalgae was evaluated using dehydrogenase activity (DHA) and total petroleum hydrocarbon (TPH) degradation using gas chromatography and mass spectrometry (GCMS). The DHA of both microalgae was conducted in relation to the triphenyl formazan production (TPF). The TPH of extracellular hydrocarbon degradation and intracellular hydrocarbons were analysed using GCMS. The data obtained from DHA and GCMS were analysed using analysis of variance at a 5% level of significance (0 < 0.05). The wild-type and mutagenised S. vacuolatus showed biodegradation of spent coolant waste after five weeks. The highest TPF produced by wild-type microalgae after five weeks was 1.139 mg/mL ± 0.009. The highest TPF produced by mutagenised S. vacuolatus after five weeks was 1.771 mg/mL ± 0.006 which was 1.55-fold higher than the TPF produced by the wild-type microalgae in the treatment weeks. The highest TPH degraded by wild-type after five weeks was 85.87% ± 0.20, while the highest TPH degradation of 100% was observed for the mutagenised S. vacuolatus after five weeks of spent coolant waste treatment. These results suggest that the mutagenised S. vacuolatus was better in utilising and degrading spent coolant waste and, therefore, can be used to mitigate spent oil pollution.


Introduction
The extensive increase in the demands of lubricant oil emulsions as a source of energy has increased worldwide.About four trillion tons of lubricant oil emulsions are produced each year globally [1].Lubricant oil emulsions are commonly made from petroleum extract, one of the crude oil-derived products.Spent oil emulsions as lubricant emulsions are utilised as a cooling agent for mechanical operations such as lubrication and maintenance of various machine components [1,2].Every time spent oil emulsion is made use of during industrial routine processes, it produces spent oil waste that is hazardous as a result of the accumulation of impurities such as carbon, water, metals, ash, dust, hydrocarbons (HCs), and other compounds [2,3].These impurities reduce the oil emulsion efficiency and quality, which become unsuitable for use [4].More than 28,220 tons of spent oil waste are produced yearly by oil and manufacturing industries worldwide [2].For instance, it was reported that each year between 1.7 and 8.8 million tons of spent oil waste are deposited in water and on land, of which 90% are linked to industrial activities, including the intentional release of oil waste [5].It was also reported that about 30% of the spent oil waste flow into groundwater [6], and nearly six million deaths are registered every year due to the pollution of groundwater [7].
Poor management and illegal disposal of spent oil wastes have been reported to contribute to environmental pollution [8,9].Spent oil waste contains harmful pollutants such as aliphatic compounds, monoaromatics, polycyclic aromatic hydrocarbons (PAH), and chlorinated compounds that end up in water bodies and lands and result in severe pollution [10,11].These harmful pollutants persist in the contaminated sites for a long time without being biodegraded due to their recalcitrant properties [12].Studies have also shown that their persistence in the contaminated sites causes these pollutants' transportation into water systems via runoff and across the atmosphere via volatilisation and condensations [1,12,13].Oil leaks resulting from the disposal of spent oil wastes are becoming a more visible issue and are regarded as one of the most prevalent environmental contaminants, much more than other petroleum-based products [13].Scientists reported that the accumulation of spent oil waste contaminants in the environment causes pollution in several ways.For example, the incineration of the spent oil waste emits toxic greenhouse gases and other harmful compounds, including furans, dioxins, carbon monoxide, and heavy metals, contributing significantly to the ozone layer depletion [14].Moreover, some of the compounds have negative health implications.For instance, dioxins cause damage to the endocrine hormone in humans.Additives and hydrocarbons pollutants present in the spent oil wastes cause different kinds of cancers, respiratory and nervous system disorders in humans, and endocrine, developmental, and epigenetic disorders in marine animals [1,11].These compounds also cause severe damages and problems to residents living around the area where the oil waste has been released [5,15].Many traditional and chemical treatment methods have been used to treat and remove organic pollutants in spent oil waste [13].These methods are considered environmentally not safe, inefficient, incomplete decomposition of pollutants, and the operational costs make applying these treatment methods costly [8].According to past research, spent oil waste pollution is becoming a highly sought-after field of study due to its impact on the natural ecosystem [5,16].Due to the rise in environmental pollution of spent oil wastes, the need for a cost-effective method that can be used to treat and remove these pollutants across a wide range of industrial applications is of great concern.
The use of microalgae either as wild-type or modified, have sparked a lot of scientific interest in the past years as an innovative treatment as a result of their crucial role in the ecosystem used as biofuel, food, feeds, and pharmaceuticals [12].Microalgal treatment has proven to be a simple, safe, viable, efficient, cost-effective, and environmentally friendly method compared to the traditional and chemical treatment [10,14].Microalgae are photosynthetic and highly adaptive to any environment with extreme conditions.They get accustomed to the environment by resisting toxic pollutants and using them as the carbon source to release oxygen for the breakdown of the pollutants [14,17,18].Their by-products and biomass are also used as feedstocks for animals and biofuel production [19].These bioremediation potentials of microalgae are beneficial to the ecosystem, especially with regard to the removal of contaminants and other hazardous chemicals.In the past years, the use of microalgal treatment of lubricant oil waste has been successfully demonstrated, with the primary focus on hydrocarbon pollutant removal [12,19,20].It has been reported that microalgae completely remove HCs present in oil waste [12,19,20].For instance [21] reported the complete removal of HCs detected in spent lubricant oil emulsion waste by wild-type microalgae Scenedesmus vacuolatus [21,22].In another study [23] reported the total oxidation of HCs found in the oil waste by the wild-type microalgae S. obliquus and Chlorella vulgaris.The catabolic abilities and the utilisation of oil wastes by these microalgal species, further demonstrates that microalgae possess extraordinary capabilities that could be used for bioremediation of oil contaminated sites.Although studies on biodegradation have been limited to wild-type microalgae, no studies yet have been reported on the use of mutagenised microalgae Scenedesmus vacuolatus for biodegradation of spent coolant oil.Thus, making this study the first to report for the biodegradation of oil waste using mutagenised microalgae S. vacuolatus.
The microalgal biodegradation treatment uses microalgae to transform toxic pollutants into compounds that are non-hazardous than the initial compounds.Microalgal biodegradation takes place through two mechanisms; the first is by metabolic degradation, in which the pollutants are used as substrates for the microalgae, and the second is by co-metabolism, in which the pollutants are broken down by enzymes that are catalysing other existing hydrocarbons [24].Microalgal degradation can also occur intracellularly or extracellularly via various catabolic processes; these include methylation, isomerisation, subterminal oxidation, decarboxylation, hydroxylation, carboxylation, and dehydrogenation.However, these catabolic processes are yet to be fully understood [25,26].The first phase of degradation starts in the extracellular, and the transformed products are then mineralised within the cells (intracellular) [24].The degradation pathway for aliphatic compounds is initiated by a methyl group's terminal oxidation to form an alcohol, further transform into fatty acid.The fatty acid is then broken down via decarboxylation to release carbon dioxide and produce a secondary fatty acid through β-oxidation [27].In some instances, the aliphatic compound is oxidised via a subterminal oxidation pathway to form an alcohol, transformed into a ketone, and further converted into fatty acid.This metabolic pathway is carried out by the class of enzymes called oxygenases [21].The metabolic pathway for aromatic compounds involves forming a diol via cis hydroxylation of the aromatic ring.The aromatic ring is further cleaved to form a dicarboxylic acid by the enzyme oxygenases [27].
Over the years, several methods have been reported in evaluating microorganisms' capabilities and efficiencies during the biodegradation process.These include the enumeration method, dehydrogenase activity (DHA), gas chromatography, and mass spectrometry (GCMS).These methods have also been used as indicators to monitor microbial oxidative activity during degradation [28].Among the methods mentioned above, DHA and GCMS are of much interest for this study.DHA has been successfully used to determine the oxidative activity of microorganisms with the majority of studies focusing on bacteria and fungi [29][30][31][32].Thus far, there are limited studies on the use of DHA to quantify the biodegradation efficiency of microalgae in oil waste contaminated environment.Two approaches using triphenyl tetrazolium chloride (TTC) and iodonitrotetrazolium chloride (INT) are used to assess dehydrogenase activity, particularly TTC [30].In this study, DHA using the TTC method is of significant interest because it is considered a simple and efficient technique for assessing oxidative activity and degradation ability of microorganisms .The INT method is costly and insoluble in water compare to TTC, which has led to this method not being commonly used [30].Dehydrogenases are oxidoreductase class of enzymes, which forms an essential part of the enzyme system found in all living microorganisms [30,32].In aerobic degradation, dehydrogenase enzymes facilitate the oxidation of HCs via the dehydrogenation mechanism by transferring electrons through a co-enzyme, which acts as an electron acceptor.After the dehydrogenation of HCs by these enzymes, the HCs become more hydrophilic, hydrophobic, and available to microorganisms by substituting the HCs with methyl groups via methylation and subterminal oxidation [26,30].The use of GCMS to monitor the total petroleum hydrocarbon (TPH) degradation of various oil wastes, ranging from crude oil to lubricant emulsion has been reported in several biodegradation studies [17,23,[34][35][36].The TPH is an important parameter in oil biodegradation, it represents the total amount of oil degradation [23,34].In this study, the TPH further elucidate the microalgae ability and potential degradation metabolic pathway to degrade SCW.This study aimed to evaluate the biodegradation capabilities of wild-type versus mutagenised Scenedesmus vacuolatus for the biodegradation of spent coolant wastes (SCW).The degradation efficiency of both groups of microalgae was evaluated based on the DHA and TPH biodegradation using GCMS.

Methodology
The SCW used in this study was supplied by a local manufacturing company in Pietermaritzburg, South Africa.The biodegradation analysis of SCW was conducted in large volumes (500 mL) studies under optima abiotic conditions established previously [21].The indigenous S. vacuolatus was mutagenised using the ultraviolet light radiation in a previous study.

Microorganism
The indigenous microalgae wild-type and mutagenised S. vacuolatus was used in this study are of the same species previously isolated from lubricant oil waste [21].

SCW biodegradation by microalgae experimental setup
To evaluate the degradation efficiency by wild-type and mutagenised indigenous S. vacuolatus, biodegradation analysis was carried out.The biodegradation experiments were conducted in large volumes (500 mL).The experimental analysis was carried out in BG 11 medium.The wild-type and mutagenised S. vacuolatus (50 mL) each were inoculated into a 500 mL BG11 media separately, supplemented with 50 mL of SCW as substrate.The flasks were agitated and incubated at 25°C for five weeks.The control experiment was made with BG11 medium having SCW without inoculum.All experimental analysis was performed in replicates (n = 6) and monitored for five weeks.Each week SCW samples were taken from the different flask and analysed for DHA and further evaluated via GCMS to monitor the SCW biodegradation by both group of microalgae.

Dehydrogenase activity (DHA)
The method by [33], was used to assess the DHA of the wild-type and mutagenised S. vacuolatus.This method thus serves as an indicator of the oxidative metabolic activity and degradation capability of microalgae.This experiment was replicated six times and conducted over a period of five weeks.After each week of incubation, the DHA were analysed.The incubated samples of SC waste (20 mL) were placed in a sterile centrifuge tubes with the addition of 2.5 mL distilled water, 0.2 g CaCO 3 , and 1 mL triphenyl tetrazolium chloride (TTC).The oil wastes samples were incubated at 37°C for 24 hours.To extract the triphenyl formazan (TPF) a red/pink colour, 10 mL of methanol was added to each of the centrifuge tubes and shake vigorously for 10 mins.The oil wastes samples were filtered using a cotton wool through Whatman paper in a funnel and the supernatants were collected.The obtained solution was decanted into clean centrifuge tubes, and the absorbance of the reddish/pinkish coloured solution was measured using a spectrophotometer at a wavelength of 485 nm.The concentration of the TPF produced was calculated using a standard calibration curve (see supplementary document 1, Fig. S1) for standard calibration curve.
The standard calibration curve was constructed by preparing a standard solution of TPF.0.1 g of TPF was dissolved in 80 ml of methanol and made up to 100 mL with methanol.A set of five TPF solutions with concentrations of 0.005, 0.010, 0.015, 0.020 and 0.025 mg/mL was prepared using 100 mL volumetric flasks.The absorbance of the prepared TPF solutions were measured (in triplicates) using spectrophotometer at a wavelength of 485 nm and against the known TPF concentration for the determination of the unknown concentration of TPF produced.

Gas chromatography and mass spectroscopy (GCMS) analysis
To ascertain the efficacy of the two groups of microalgae (wild-type and mutagenised S. vacuolatus) for SCW degradation, GCMS analysis was carried out.The SCW degradation was evaluated in terms of the total petroleum hydrocarbon (TPH) accompanied with the HCs degradation which was carried out in replicates (N = 6).Prior to the GCMS analysis, the following extraction procedure including extracellular and intracellular HCs extraction analysis was performed for each sample.For extracellular HCs extraction, 50 mL of each samples (treated SCW samples with mutagenised S. vacuolatus and wild-type and control) was extracted with dichloromethane.The extracellular oil extracts were filtered using sodium sulphate.Then, the oil extracts were placed under the fume mood for the dichloromethane to evaporated.
The intracellular HCs in the microalgae cells (wild-type and mutagenised S. vacuolatus) were extracted using the beads beating methodology by [37].Both groups of microalgae cultures (50 mL) were transferred into sterile centrifuge tubes each and centrifuge at 11,000 rpm for 5 mins to obtain the microalgae pallets.The pallets were transferred into 2 mL tubes with glass beads and nuclease free water.The microalgae solutions were agitated for 5 mins.The supernatants were obtained and decanted into sterile test tubes, incubated at 70°C for 10 mins and vortexed two times throughout the incubation.Lysis buffer and ethanol (one volume) was added to the supernatants, mixed thoroughly and centrifuge at 11,000 rpm for 5 mins.
To determine the intracellular metabolites and the TPH of the extracellular oil extracts, 1 µL of the intracellular extracts and extracellular oil extracts were dissolved in 20 ml n-hexane separately and analysed using GCMS.The method by [38] was used for the GCMS analysis.The GCMS (QP2010 GC) with HP-5 MS capillary column (30 mm, and 0.25 mm), helium gas (0.8 ml mn −1 ), and 70 eV mass detector was used.The injection temperature was set-up at 250°C, and oven temperature at 40°C to 260°C.All samples were injected using split mode with an injection ratio of 1:25.The extracellular and intracellular extracts constituents were identified based on their mass spectra and compared to the NIST library.To evaluate the TPH degradation, the total area percentage (%) of the untreated SCW (control) were subtracted from the total area % of the treated SCW.

Statistical analysis
The data were subjected to analysis of variance in GenStat software eighteenth edition.The results are means ± standard Error of six replicates (n = 6).A general analysis of variance (ANOVA) and fishers protected least significant difference (LSD) test was conducted to compare the means of DHA and TPH degradation of SCW by wild-type and mutagenised S. vacuolatus.The results are regarded significant if p < 0.05.

Wild-type microalgae DHA in spent coolant waste (SCW)
The DHA was performed to assess the oxidative activities and degradation efficiency of microalgae.The DHA was measured in terms of TPF produced, which was determined by means of a standard curve (see supplementary document).The colour of the DHA was also measured (see supplementary document 2, Fig. S2).The TPF produced is shown by the pink colour.Week one revealed a light pink colour, indicating low TPF production.TPF production was high, as evidenced by the pink colour noticed at week two.A dark pink colour was seen in week three, indicating that TPF production was very high.At the end of weeks four and five a pale pink colour was noticed, indicating a decrease in TPF production.In comparison to the control no DHA was observed.Dehydrogenase activity has been described as an effective method for evaluating the oxidative metabolism and degradation abilities of microorganisms [30,31].
The DHA of wild type microalgae by the TPF production at different weeks intervals (week one -week five) are shown in the supplementary document 3, Fig. S3.The DHA of wild-type algae augmented with SCW was relatively low at the beginning of the study in comparison to mutagenised algae.The DHA of wildtype showed a significant increase (p < 0.001) in TPF production (0.345 mg/mL ± 0.006 and 0.519 mg/mL ± 0.002) from the first two weeks to its maximum TPF production (1.139 mg/mL ± 0.009) by the third week, which also indicated the highest DHA.The observed gradual rise in the DHA from the first week to third week was ascribed to the metabolism of SCW as a carbon source.Similar increase in DHA of Streptomyces venezuelae was reported by [29].The author also reported that DHA varies depending on the duration of incubation.After the third week, the DHA declines in both fourth and fifth week, leading to a rapid decrease in the TPF production with 0.223 mg/mL ± 0.004 and 0.120 mg/mL ± 0.003, respectively.The possible reasons for the reduction in DHA at the higher weeks of treatment includes (1) the decrease in the uptake and metabolism of HCs of SCW, (2) decrease in the oxidative activities, that may have been caused by the long exposure to the SCW along with its toxic HCs including monoaromatics, PAHs, and other toxic HCs (3) cell decomposition or death of microalgae, possibly resulting from the accumulation of organic waste (organic acids/bases), thus making the medium highly toxic, concentrated and inhibiting dehydrogenase enzyme activities [30,39].A reduction in TPF production with increasing incubation treatment time was also noticed by [29].

Mutagenised S. vacuolatus DHA in spent coolant waste (SCW)
The DHA of mutagenised S. vacuolatus with SCW are shown in the supplementary document 4 and 5, Fig. S4 and S5.The mutagenised S. vacuolatus treatment showed a significantly higher DHA.The TPF produced by the mutagenised S. vacuolatus were 0.635 mg/mL ± 0.001 (pink colour), 1.350 mg/mL ± 0.004 (dark pink colour), 1.771 mg/ mL ± 0.006 (very dark pink colour), 0.433 mg/mL ± 0.001 (pink colour), and 0.249 mg/ mL ± 0.002 (light pink colour) respectively for week one, two, three, four and five compared to the control.The TPF produced by the mutagenised S. vacuolatus were 1.84-fold, 2.60-fold, 1.55-fold, 1.94-fold, and 2.08-fold higher than the TPF produced by the wild-type microalgae in the treatment weeks respectively.The relatively higher DHA with a very dark pink colour was obtained in the mutagenised S. vacuolatus treatments.This could be attributed to the increased utilisation and metabolism of HCs of SCW and the high increase in the dehydrogenase enzymes involved in the HCs oxidative metabolic process [31,33,40,41].Evidently from our findings, the mutagenised S. vacuolatus exhibited higher DHA with SCW as substrate than the wildtype microalgal strain.

SCW extracellular biodegradation by wildtype microalgae
Biodegradation of SCW by wild-type microalgae after five weeks of treatment was evaluated by GCMS analysis.GCMS has been a commonly used methodology for analysing the chemical constituents of lubricant-based oil products [17,23,34,35].Table 1 shows the HCs present in the untreated SCW (control compounds).The HC constituents were aliphatic HCs (alkanes) alkenes, aromatic compounds (monoaromatic HCs, polycyclic aromatic hydrocarbons (PAHs), and chlorinated compounds), alcoholic compounds, and other organic acids.The major HC of the SCW were the aliphatic HCs.The monoaromatics, PAHs and chlorinated HCs were the major hazardous HCs found in the SCW.In a related study [5], reported lubricant oil waste containing a complex mixture of HCs.In this study, the untreated SCW (control compounds) were used to compare the experimental weeks to evaluate the microalgae (wild-type and mutagenised S. vacuolatus) biotransformation and biodegradation.
Table 1.Hydrocarbon compounds detected in the untreated spent coolant waste (SCW) control compounds.

Formula/Isomers
Alkenes compounds Formula/Isomers Monoaromatic compounds Formula/Isomer Hexane,2,3-dimethyl The table shows the hydrocarbon compounds with their molecular formular and isomer present in the spent coolant waste.
To determine the biodegradation efficiencies of SCW by wild-type microalgae, the TPH degradation was evaluated.The TPH was targeted in this study instead of the different groups of HCs because SCW contains a variety of complex HCs.Several studies on oil waste degradation have shown that TPH is a significant indicator of total oil degradation [34,36,42].The TPH degradation of SCW after five weeks of treatment by wild-type microalgae are shown in supplementary document 6, Fig. S6.The ANOVA analysis showed there was a significant difference (p < 0.001) among the TPH degradation.The TPH degradation obtained were 45.89% ± 0.61, 62.30 ± 0.45, 71.59% ± 0.09, 78.41% ± 0.18, and 85.87% ± 0.20 for week one, two, three, four and five respectively.The significant increase in TPH degradation observed after the fifth week of SCW treatment was due to increased metabolic activity of the wild-type microalgae because of the prolonged treatment period [23,43].The decrease in the TPH degradation rate at the early stage of treatment, was probably caused by the slow adaptation rate by the wild-type microalgae to the carbon source [44].
Tables 2a and 2b shows the degradation of extracellular HCs of SCW by wild-type microalgae after five weeks of treatments.The result obtained revealed that the HCs present were significantly different from the control (Table 1).The alkanes belonging to C8, C11, C14, C20, C24 and C43 groups with their isomers present in the control were completely degraded by the wild-type microalgae in all the treatments.While alkanes ranging from C9 -C18 were transformed into their derivatives via isomerisation mechanism by the wild-type microalgae in all the treatment weeks.This shows isomerisation mechanism as one of the many mechanisms used by the wildtype microalgae to breakdown alkane HCs present in the SCW [45].The oxidative biodegradation of alkanes into their corresponding derivatives by microorganisms have been reported [28,46].All the alkenes detected in the control were removed in all the treatment weeks, indicating that the alkenes HCs were completely degraded by the wild-type microalgae.In addition, several aliphatic products such as alcoholic compounds, fatty acids, and carboxylic acids were detected in all the treatment weeks.The production of these compounds suggests that the aliphatic portions of the SCW are being used as substrate.These intermediate compounds are also known to have no harmful effects on the environment [22,47,48].These aliphatic compounds were identified as intermediate/oxidised products from the degradation of the alkanes and its derivatives.The formation of these compounds suggests biodegradation of the alkanes HCs occurring through oxidation/subterminal oxidation pathway by wildtype microalgae [36,49].For example, this was evidenced by the formation of the alkane fatty alcohols such as n-tridecan-1-ol, 1-nonanol,4,8-dimethyl, 11-methyl dodecanol, n-heptadecanol and fatty acids tridecanoic acid methyl ester, nonanoic acid, dodecanoic acid methyl ester etc., observed as the degradation products of the most degraded alkanes such as tridecane, nonane, and dodecane, respectively.The addition of the methyl groups in the alkane's oxidation products, suggests that the alkane degradation by wild-type microalgae is initiated by methylation oxidation pathway [36].The production of alcohols and fatty acids recorded in this study has been described by different authors to be the intermediate products of alkanes occurring via different catabolic pathways such as terminal oxidation or subterminal oxidation pathway [36,42,49].The oxidation pathway of alkanes HCs is the transformation of the alkane HCs into alcohol, which is converted into aldehyde and ketone and then is further broken down into fatty acids [36,49].The table shows the list of extracellular hydrocarbons present with their molecular formula after wild-type microalgae treatment of SCW.
Aromatic compounds, including chlorinated compounds, monoaromatics, and PAHs, were also found in the control.The chlorinated compounds (behenyl chloride and 1-octadecanesulphonyl chloride) detected were efficiently removed by wild-type microalgae in all the treatment weeks.Similarly, the microalgae Fucus vesiculosus, Caepidium antarcticum and Desmarestia species have also been reported to degrade chlorinated HCs in past studies [27,50,51].All the monoaromatics were completely degraded into new compounds in the first two weeks of treatments by the wild-type microalgae.The new monoaromatic compounds formed were the oxidised products of the benzene HCs compared to the control.This includes benzene derivatives benzene,1,3-dimethyl, benzene,1-ethyl-3-methyl, and benzene,2-ethyl-1,4-dimethyl, formed via isomerisation mechanism; benzene alcohols and carboxylic acids such as 1,4-benzenediol,2,6-bis (1,1-dimethylethyl), benzene acetic acid, 4-tridecyl ester, 1,2-benzenedicarboxylic acid, butyl 2-methylpropyl ester, 3-trifluoromethylbenzoic acid, dodecyl ester, and 1,2-benzene dicarboxylic acid, possibly formed by oxidation pathway [12,25,49].The inclusion of the methyl groups to the benzene degradation products again suggests methylation oxidation pathway as one of the mechanisms used by the wild-type microalgae to degrade The table shows the list of extracellular hydrocarbons present with their molecular formula after wild-type microalgae treatment of SCW.

Table 3a
. Extracellular compounds detected after week one, two and three of treatment of spent coolant waste by mutagenised S. vacuolatus. .

C18H36O2
The table shows the list of extracellular hydrocarbons present with their molecular formula after mutagenised microalgal treatment of SCW.monoaromatic HCs.Furthermore, the production of these benzene alcohol, monocarboxylic and dicarboxylic acids may serve as a mechanism or a strategy by which the microalgae protect its cells from the toxicity of the parent benzene HCs [11].Similar to the finding of [52], the authors reported the degradation of BTEX compounds (benzene, toluene, ethylbenzene, and p-xylene) into different metabolic products by the microalga Paurachlorella kessleri after 72 hours of treatment.
The PAHs detected were also efficiently degraded into their naphthalene derivatives after two weeks of SCW treatment compared to the control.The naphthalene derivatives including, naphthalene,1-methyl, naphthalene,1,3-dimethyl, 1,2-naphthalenediol, 1-naphthol,1,2,3,4-tetrahydro-2-methyl, 1,2,3,4-tetrahydro-1,2-naphthalenediol, and 2-naphthalenecarboxylic acid, 4,4ʹ-methylenebis [3-methyl] were formed.The presence of these compounds shows the degradation of the PAHs which were probably produced by hydroxylation and methylation pathway [12].After the third, fourth, and fifth weeks of SCW treatment (Tables 2a and 2b), no monoaromatic HCs and PAHs or their oxidised products were detected, only the non-toxic degradation products of alkanes were observed.This therefore clearly confirms the complete degradation of the aromatic HCs and the utilisation of the oxidised products.Naphthalene and its derivatives have been reported to be degraded by the Ankistrodesmus species [53].The degradation of both monoaromatics and PAHs by microorganisms into different extracellular metabolites such as their corresponding diol and carboxylic acids have been reported to occur by hydroxylation and carboxylation mechanism [12,25,53,54,55].It should be noted that monoaromatic (benzene) and PAHs compounds (naphthalene and chlorinated HCs) detected in the control as stated above, are highly toxic and carcinogenic.They cause different kinds The table shows the list of extracellular hydrocarbons present with their molecular formula after mutagenised S. vacuolatus treatment of SCW.

Table 4a
. Intracellular metabolites extracted from the cells of the wild-type microalgae after three weeks of treatment of spent coolant waste.

C6H10O2
The table shows the list of intracellular hydrocarbons with their molecular formula found in the cells wild-type microalgae.
of cancers, respiratory and nervous system disorders, and genetic defects in humans [1,11,56,57].Hence, the ability of the wild-type microalgae to completely break down these hazardous HCs and transform them into non-toxic compounds is seen as a unique ability, that may be beneficial to the health of the natural ecosystem.The table shows the list of intracellular hydrocarbons with their molecular formula found in the cells of the wild-type microalgae.

Table 5a
. Intracellular metabolites extracted from the mutagenised indigenous S. vacuolatus cells after three weeks of treatments of SCW.

C16H30O2
The table shows the list of intracellular hydrocarbons with their molecular formula found in the cells of mutagenised S. vacuolatus

SCW extracellular biodegradation by mutagenised S. vacuolatus
In this study, the mutagenised S. vacuolatus treatments of SCW at various weeks caused increase in the TPH degradation after five weeks of treatment.This degrading trend was also seen in the SCW treatment with the wild-type microalgae.The table shows the list of intracellular hydrocarbons with their molecular formula found in the cells of the mutagenised S. vacuolatus respectively.TPH degradation was highest in weeks five and four, and lowest in week one.The increased TPH degradation was probably due to improved metabolic activities of the mutagenised S. vacuolatus [58].Tables 3a and 3b shows the HCs degradation by mutagenised S. vacuolatus after five weeks of treatments.The result obtained revealed higher oxidation of extracellular HCs of SCW.An increase in the oxidation activity with increasing degradation products were observed from week one to week four in comparison to the control.The alkanes ranging from C8 -C43 and their isomers were completely degraded resulting to new alkanes derivative ranging from C12 -C54 were observed in week one -week four.The synthesis of the new alkanes suggests the metabolic activity of the mutagenised S. vacuolatus in the course of utilising SC waste as carbon source.Alkanes compounds C8 -C15 and C16 -C36 have been reported to be easily oxidised by microorganisms as a result of the non-complex nature of these compounds [46].Similarly, Microcoleus chthonoplastes and Phormidium corium has been reported to oxidise n-alkanes [59].Another interesting result observed were the complete removal of all alkenes (C7 -C20) and cycloalkanes (C8 -C13) in all the weeks of treatment by mutagenised S. vacuolatus.The degradation of these compounds clearly proves the excellent extraordinary ability of this mutagenised S. vacuolatus.Our finding also shows that the mutagenised S. vacuolatus successfully utilised and metabolised the alkanes, alkenes, and cycloalkanes fractions of SCW.
Apart from degrading the alkanes and cyclic alkanes, mutagenised S. vacuolatus, completely degraded all monoaromatics, PAHs and chlorinated compounds after week one with only the degradation products present in the treated oil sample.The early breakdown of the aromatics could be explained by their solubility [59] and utilisation these HCs as substrates, which in turn stimulated the degradation of SCW.Among the monoaromatics degradation products formed were the benzene alcohols and carboxylic acids including, benzyl alcohol,4-methoxy-6-fluoro, 2,5-di-tert-Butyl-1,4-benzoquinone, dihydroartemisininoxymethyl benzoic acid, 1,2-benzenedicarboxylic acid, butyl,2-methyl propyl ester, benzyloxy tridecanoic acid, 4-trifluoromethylbenzoic acid, tridecyl ester, etc., (Tables 3a and 3b).The PAHs degradation products detected were also the naphthalene alcohols ketone and carboxylic acids such as 1-naphthol,1,2,3,4-tetrahydro-2-methyl, 3,4-dihydro-3-methyl, naphthalen-1-one, 2-naphthalenecarboxylic acid, 4,4ʹmethylenebis, 2-naphthoic acid, 3-methoxy-4-methyl etc.Some of the monoaromatic and PAHs products produced by the mutagenised S. vacuolatus were similar to those produced by the wild-type microalgae.Again, these aromatic alcohols, ketones and carboxylic compounds were identified as the degradation products of the monoaromatics, and PAHs as stated above.Furthermore, the presence of these compounds demonstrates that the aromatic HCs degradation of SCW is influenced by the hydroxylation and methylation mechanism [25].More so, the aromatics products detected in this present study were similar to the aromatic metabolic products reported by [53] and [12].After week two, and three, no aromatic HCs were detected, the compounds formed were the alkane derivatives alcohols, fatty acids and other oxidised products which are considered to be non-hazardous to the ecosystem.By week four, only fatty acids and carboxylic acids were present.This proved the total degradation of HCs present in the SCW.After week five, no HCs were detected, indicating that the HCs in the SCW were efficient removed by the mutagenised S. vacuolatus (see supplementary document 9, Fig. S9) for the gas chromatogram.This therefore provides an opportunity for the exploitation of this mutagenised S. vacuolatus for bioremediation.

Intracellular compounds in the cells of wild-type and mutagenised microalgae in spent coolant waste
Tables 4a and 4b and Tables 5a and 5b shows the list of intracellular compounds detected in the cells of wild-type and mutagenised S. vacuolatus.The results showed the intracellular bioaccumulation and biosynthesis of HCs by both microalgae groups in comparison to the control and extracellular degradation compounds.The accumulated and synthesised HCs were variation of alkanes, alkenes, alcohols, fatty acids, carboxylic acids, and other carbon-based organic acids.In the wild-type microalgal cell extract after week oneweek five, alkanes HCs that range from C7 -C54; alkenes C7 -C35; alcohols C11 -C33; fatty acids C13 -C27 and other carboxylic compounds C6 -C24 were detected.Whereas alkanes ranging from C5 -C60; alkenes C5 -C35; alcohols C5 -C36; fatty acid C16 -C69 and carboxylic acids C6 -C38 were detected in the mutagenised S. vacuolatus cells extracts.The presence of these compounds in the cell extracts of both microalgae groups indicates intracellular utilisation and biodegradation of SCW [24].
The alkanes such as dodecane, pentadecane, tetradecane, hexadecane, etc., present in the control were transformed by both microalgae groups.For instance, dodecane, pentadecane, tetradecane and hexadecane were transformed into their respective fatty acid derivatives such as dodecanoic acid methyl ester, pentadecanoic acid, tetradecanoic acid and hexadecanoic acid.The cycloalkanes cyclohexane and cyclopentane were also broken down intracellularly into their respective carboxylic acid derivatives.These alkanes and cycloalkanes compounds' transformation products further reveals intracellular degradation occurring via intracellular methylation and oxidation [13,21,24].
Alkenes compounds such as 7-heptadecene, 3-hexadecene, 3-hexene,3-ethyl-2-5-dimethyl, squalene, 17-pentatriacontene, etc., were common to both microalgae groups.These alkenes could have been produced from the intracellular microalgae metabolism of the fatty acids detected in this study [60].Also, the alkenes produced in this study, were identified as those of the long-chain alkenes which have been detected in different microalga species and believed to have great industrial and biotechnological importance [60,61].In addition, these alkenes have been reported to contribute majorly to the hydrocarbon cycle of aquatic environment [60].

Conclusion
In this study, the wild-type microalgae, and mutagenised indigenous microalgae S. vacuolatus exhibited efficient multi-degradation abilities of various HCs fractions of SCW efficiently.The mutagenised S. vacuolatus exhibited higher DHA and TPH with SCW as carbon source when compared to the wild-type microalgae.The identified intermediate degradation products showed the methylation, isomerisation, oxidation/subterminal oxidation, hydroxylation and carboxylation for alkanes, cyclic alkanes, monoaromatic and PAHs biodegradation.With the excellent degradative abilities for HCs possessed by the mutagenised S. vacuolatus, this microalgal strain could be potential candidate that could be employed for bioremediation of oil contaminated sites.

Table 2a .
Extracellular compounds detected after week one, two, and three of treatment of SCW by wild-type microalgae.

Table 2b .
Extracellular compounds detected after week four and five of treatment of SCW by wild-type microalgae.

Table 3b .
Extracellular compounds detected after week four and five treatment of SCW by mutagenised indigenous S. vacuolatus.

Table 4b .
Intracellular metabolites extracted from the cells of the wild-type microalgae after four and five weeks of treatments of spent coolant waste.

Table 5b .
Intracellular hydrocarbon extracted from the mutagenised S. vacuolatus cells after four and five weeks of treatments of spent coolant waste.