Bacterial Degradation of Petroleum Hydrocarbons in Saudi Arabia

Abstract

The Kingdom of Saudi Arabia (KSA) is the leading oil-exploring and -exploiting country in the world. As a result, contamination of the environment by petroleum products (mainly hydrocarbons) is common, necessitating strategies for their removal from the environment. Much work has been conducted on bacterial degradation of hydrocarbons in the KSA. This review comprehensively analyzed 43 research investigation articles on bacterial hydrocarbon degradation, mainly polyaromatic hydrocarbons (PAHs) within the KSA. More than 30 different bacterial genera were identified that were capable of degrading simple and complex PAHs, including benzo[a]pyrene and coronene. Different strategies for selecting and isolating these bacterial strains and their advantages and disadvantages were highlighted. The review also discussed the origins of sample inocula and the contributions of various research groups to this field. PAH metabolites produced by these bacteria were presented, and biochemical pathways of PAH degradation were proposed. More importantly, research gaps that could enrich our understanding of petroleum product biodegradation mechanisms were highlighted. Overall, the information presented in this paper will serve as a baseline for further research on optimizing bioremediation strategies in all petroleum-contaminated environments.

1. Introduction

Fossil fuels, encompassing crude oil, petroleum, coal, and natural gas, have historically been the world’s primary industrial energy production source. Furthermore, numerous crucial industrial products stem from these fossil fuels, including saturates, aromatics, bitumen, asphalt, and asphaltenes [1]. Derived from buried and fossilized remains of ancient plants and animals, fossil fuels primarily consist of hydrocarbons, compounds composed predominantly of hydrogen and carbon atoms. The high demand for energy and the aforementioned products has led to the development of major industries, consequently causing widespread environmental contamination. This contamination often occurs through oil or gas leakage during exploitation and transportation, oil spills resulting from accidents, and the release of by-products, such as plastic [2,3].

Crude oil is a complex mixture comprising various hydrocarbons, including alkanes or saturates, aromatics, resins, and asphaltenes [4]. Aromatics encompass mono-aromatic hydrocarbons (MAHs), such as phenol and its derivatives, as well as polycyclic aromatic hydrocarbons (PAHs) with two or more rings, such as naphthalene (two fused rings), phenanthrene (three fused rings), pyrene (four fused rings), and benzo[a]pyrene and benzo[k]fluoranthene (five fused rings), along with coronene (seven fused rings). Resins and asphaltenes are more complex compounds with higher molecular masses than saturates and aromatics. They comprise aromatic and non-aromatic rings, with small hydrocarbon side chains and occasionally one or more heteroatom(s) [4].

Alkanes or saturates are comparatively easier to degrade, followed by mono-aromatic hydrocarbons (MAHs), and then polycyclic aromatic hydrocarbons (PAHs), with the more complex PAHs posing greater challenges in degradation, thus contributing to their accumulation in the environment [5,6,7]. The toxicity of hydrocarbons in general, particularly PAHs, is well-documented. These compounds are carcinogenic, with some forming DNA adducts, leading to mutations, following transformation in animals [8,9]. Negative effects on development, reproduction, immune function, and endocrine systems have also been observed in experimental animals exposed to hydrocarbons [10]. Additionally, hydrocarbons have been found to bioaccumulate in plants and aquatic animals, resulting in contamination and disruption of entire ecosystems [11]. Thus, their removal from the environment remains a priority.

Several strategies for removing these pollutants have been proposed and evaluated; however, biodegradation (or bioremediation), which relies on the use of microorganisms, remains the best option since it is environmentally friendly and cost effective, compared to physical or chemical approaches [12,13]. In this regard, the literature is replete with work on the isolation and characterization of hydrocarbon-degrading bacteria (HDB), including those that degrade alkanes or saturates, MAHs, and PAHs, from various locations in the world where hydrocarbon contamination is common, and these HDB belong to diverse microbial genera [4,6,7,12,13,14]. Some HDBs have been developed as commercial products for the bioremediation of environments following accidental oil spills on land or sea [5].

The Kingdom of Saudi Arabia (KSA) possesses one of the world’s largest oil industries, with substantial oil reserves and a daily exploitation rate of one million barrels, positioning it as the foremost oil economy globally. Consequently, environmental contamination by oil or its derivatives is prevalent in the KSA [15,16]. For instance, the extraction of every one liter of oil generates three liters of “produced water” (PW), a byproduct of reservoir flushing, resulting in a staggering 30 million barrels of PW produced daily [3,17]. Although a majority of PW, which also contains petroleum products, is recycled, approximately 10–20% is discharged into the environment [3,17]. This, coupled with oil spills from leaks and accidents during extraction, refining, and transportation, as well as emissions from oil combustion, poses significant environmental challenges in the KSA.

As previously discussed, biodegradation or bioremediation represents the most effective and sustainable approach to removing these pollutants from the environment. In pursuit of this objective, extensive research has been conducted in the KSA, focusing on isolating and characterizing hydrocarbon-degrading bacteria (HDB). Various species of HDB have been identified, with some demonstrating the ability to degrade oil products in extremophilic conditions. To provide deeper insights into these HDB and their capacity to degrade oil products, we conducted a systematic literature review of research conducted on HDB in the KSA. This review encompasses (i) a summary of the nature of HDB and the extent of their distribution, (ii) an overview of the hydrocarbon compounds they degrade, (iii) a discussion of the biochemical mechanisms involved in hydrocarbon degradation, and (iv) an exploration of the unique characteristics of these HDB. Additionally, this review identifies and discusses research gaps that need to be addressed to enhance our understanding of PAH bioremediation, in light of the extensive research conducted globally on bioremediation

2. Approach to Data Collections

Published and peer-reviewed articles were systematically collected through an exhaustive search across five databases: PubMed (https://pubmed.ncbi.nlm.nih.gov, accessed on 20 July 2024), Scopus (https://www.scopus.com, accessed on 22 July 2024), ScienceDirect (https://www.sciencedirect.com, accessed on 22 August 2024), Web of Science (https://clarivate.libguides.com, accessed on 20 June 2024), and Google Scholar (https://scholar.google.com, accessed on 23 July 2024). The search utilized various combinations of keywords, including bacteria, degradation, hydrocarbons, and Saudi Arabia. Articles meeting the following four inclusion criteria were selected for further analysis: (i) original articles conducted in Saudi Arabia (the location where samples were collected) focusing on crude oil, petroleum, n-alkanes, BTEXs (benzene, toluene, ethyl-benzene, and xylene), monoaromatic hydrocarbons, PAHs, diesel, or kerosene (as degraded pollutants), (ii) featuring bacteria as the sole microorganism, (iii) published between 1983 and 2024, and (iv) written in English. Studies involving fungi, microalgae, or plants in hydrocarbon degradation were excluded, as were studies on bacterial degradation of hydrocarbons using samples not collected in Saudi Arabia. All data were meticulously recorded in a table using Microsoft^® Excel 4.3.4.28, and diagrams and graphs were generated using the same Microsoft^® Excel software.

The utilization of the aforementioned inclusion criteria resulted in the identification of 353 publications. After removing duplicated articles and applying eligibility criteria, a final set of 43 publications was obtained for thorough examination. These selected publications formed the basis of this review (Figure 1).

3. General Findings

3.1. Frequency of Publications, Research Institutions, and Site of Sample Collections

Research was conducted in nine institutions, including research centers and universities. Notably, King Fahd University of Petroleum and Minerals (KFUPM), King Abdulaziz University (KAU), and King Saud University (KSU) collectively contribute to over 65% of the reported studies. These universities are situated in the three major cities across three provinces in the KSA: KSU in Riyadh, Riyadh Province; KFUPM in Dhahran, Eastern Province; and KAU in Jeddah, Makkah Province (Figure 2).

The initial reported work on the biodegradation of hydrocarbons in the KSA dates back to the 1990s, with the first study meeting our inclusion criteria in 1999 [18]. Initially, an average of 1–2 publications were reported each year until 2014. However, from 2015 onwards, the annual number increased from 2 to 7, with an approximate average of 3 per year (Figure 3).

The onset of studies in the 1990s in the KSA may not be coincidental. In the year 1991, during the Gulf War, a substantial quantity of crude oil was released into the Arabian Gulf, impacting the entire eastern coastline of the KSA. Estimates suggest that between 4 to 10 million barrels of oil were released, forming a slick measuring 101 by 68 km and penetrating up to 40 cm into the sand and mud flats of Saudi Arabia’s shorelines, marking it the largest oil spill in history [15]. Following the war, the KSA initiated numerous studies to investigate the short and long-term impacts of this pollution on the environment [15].

This prompted the exploration and characterization of bacteria capable of degrading petroleum products, which could be utilized in bioremediation strategies. This could potentially account for the increased number of publications from the 1990s onwards.

Supporting this hypothesis, most samples or inocula were collected in the Eastern Province of the KSA, the region most affected by the 1991 oil spill in the Arabian Gulf (Figure 4). Additionally, the Arabian Gulf is recognized as the world’s largest oil reserve [16].

3.2. Bacterial Strain Identification

Figure 5 presents a summary of the frequency of identified bacterial genera. In total, 109 bacterial strains were identified. Among these, eighteen strains were attributed to the genera Bacillus and Pseudomonas. Additionally, seven strains were classified under the genus Ochrobactrum, while four strains belonged to Stenotrophomonas and Klebsiella genera. Furthermore, three strains were associated with each genera Acinetobacter, Halomonas, Marinobacter, Rhodococcus, and Staphylococcus. Genera such as Achromobacter, Arthrobacter, Burkholderia, Martelella, Micrococcus, Proteus, and Sphingomonas each comprised two strains. Notably, bacterial strains belonging to 20 different genera were identified only once (Figure 5).

Among the seventeen bacterial strains within the genus Pseudomonas, six were identified as Pseudomonas sp., indicating an inability to assign them to a specific species. Notably, the most prevalent species was Pseudomonas aeruginosa, with five strains identified, followed by Pseudomonas citronellolis and Pseudomonas stutzeri, each represented by two strains. Concerning the genus Bacillus, out of the eighteen strains, Bacillus subtilis and Bacillus cereus emerged as the dominant species, each with three strains. In comparison, three strains were categorized as Bacillus sp. The remaining nine strains were each associated with a distinct species (Table S1).

The strains attributed to the Pseudomonas and Bacillus genera stand out as the most predominant in the degradation of petroleum hydrocarbons in the KSA. Pseudomonas species are renowned for their remarkable biochemical versatility, particularly in hydrocarbon degradation, and have been extensively documented in the remediation of diverse contaminated environments [19]. The initial evidence showcasing the capability of Pseudomonas bacteria to degrade petroleum products dates back a century ago [20]. Since then, significant research efforts have elucidated their capacity to degrade a wide range of compounds, including alkanes, aromatics [19,21,22], chloroaromatics [23], synthetic plastics and pesticides [24,25]. Similarly, the genus Bacillus is recognized for its biochemical versatility, with numerous studies highlighting its proficiency in degrading various pollutants, including hydrocarbons (monoaromatics and polyaromatics), crude oil waste, and plastics, among others [26].

One notable attribute contributing to the biodegradative properties of Bacillus and Pseudomonas is their ability to express and release biosurfactants. These molecules enhance the solubility of pollutants, facilitating their absorption and subsequent degradation [27,28,29]. Intriguingly, a similar study involving a systematic review of bacterial hydrocarbon degradation in Colombia also underscored the predominance of the Pseudomonas and Bacillus genera [29]. This pattern may be a recurrent feature in various oil-contaminated environments.

3.3. In Vitro Culturing of the Bacterial Strains

The rationale of most studies discussed in this review was the search for active bacterial strains suitable for bioremediation. To this end, two methods of bacterial isolation are generally used. The “enrichment protocol” in a selective medium and the “prior growth” in a rich medium or in a non-selective medium. Both approaches require prior in vitro culturing of the bacterial strains, a process that is known to reduce bacterial diversity since only a tiny proportion of bacterial strains, less than 1%, are cultivable in vitro [30,31]. However, despite this common limitation, each approach has advantages and disadvantages.

In the “enrichment protocol”, strains in the inoculum (contaminated samples) are cultured in a medium containing the pollutant of interest as the sole carbon source. This progressively leads to the enrichment of bacteria strains capable of degrading the pollutant after several culture transfers or passages. Bacterial stains that cannot utilize the selected carbon source will be eliminated because their growth will be lower or null, while those that prefer the selected carbon source will be favored in the culture, leading to their isolation. At the end of the enrichment process, only active strains degrading the selected pollutant as a source of carbon will be isolated [32].

In the second approach, the “prior growth” in rich medium, strains from contaminated samples are first grown in a rich medium, a non-selective medium, which permits the growth of all microorganisms, so long as they can grow in vitro. Thereafter, each strain is isolated and its ability to individually degrade the pollutant of interest is evaluated. As a result, not all isolated bacterial strains (from the initial culture in a rich medium) will be active in the degradation of the pollutant of interest.

Additionally, in the initial culture (in rich medium), active strains can outcompete those that can degrade the pollutant of interest (since the culture is not selective), leading to low efficiency in selecting pollutant-degrading bacterial strains. Consequently, an “enrichment protocol” is commonly used for the selection and isolation of active bacterial strains degrading a specific pollutant [32]. In support of this, more than 75% of bacterial strains reported in this review were derived from an enrichment protocol (ST10–ST41) [18,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65], while “prior growth” in the rich medium was employed in only seven studies (ST1–ST3, ST6–ST9) [66,67,68,69,70,71,72] In three studies, the protocol of bacterial isolation was not mentioned (ST5, ST42–ST43) [62,64,73].

As mentioned earlier, “prior growth” in a rich medium leads to the selection of many strains, and some may not necessarily be active in pollutant degradation. This is illustrated in ST7 [70], in which a total of 43 strains were isolated, and out of these, only six, belonging to the genera Pseudomonas, Bacillus, Staphylococcus, Erwinia, and Nitratireductor, were active in degrading a crude oil product [70]. Thus, unlike “the enrichment protocol”, “prior growth” in the rich medium does not necessarily guarantee that the initially selected strains (after the culture in the rich medium) will be active to degrade a given pollutant.

In addition to the culturing methods, non-culturing strategies can be employed to study the degradation of petroleum products. They involved the identification of bacteria strains using various molecular techniques such as denaturing gradient gel electrophoresis (DGGE), restriction fragment length polymorphism (RFLP), 16S rRNA sequencing, or whole genome analysis [74]. This approach provides information on non-culturable and culturable bacterial strains in their natural environment, without any prior selection. As a result, it does not allow the isolation of bacterial strains and, thus, has limited interest in bioremediation strategy. This approach identifies bacterial communities in given samples but does not provide information on the ability of each of these strains to degrade pollutants.

Several reports have been dedicated to the use of this approach to study crude oil degradation [75]; however, so far, only one study has employed this approach in Saudi Arabia. In this study, the DGGE approach was used to identify the existing bacteria in contaminated samples, and the results showed very diverse genera, including Thiobacillus, Thiobaca, Halochromatium, Pseudomonas, Alcanivorax, Deinococcus, Desulfosarcina, Cytophaga, Holophaga, Acidobacteria, Geothrix, Verrumicrobia, Chloroflexus Spirochetes, and Planctomycetes, among others [76]. The identification of diverse genera is a common feature of this approach since there is no prior in vitro bacterial culturing [74,75,77]. However, as mentioned before, the contribution of each of these strains to the degradation cannot be established since none of the strains was cultured; thus, their potential for bioremediation cannot be evaluated.

3.4. Substrates Used in Studies: Crude Oil, Aliphatic, and Polyaromatic Compounds

As discussed earlier, in the “Prior Growth protocol” using a rich medium, bacteria are isolated first, and then their ability to degrade a given substrate is evaluated afterward. However, in the “Enrichment Protocol”, the selection of bacterial strains is carried out in the presence of a specific substrate, ensuring that the resulting bacterial strains will actively degrade this particular substrate. Subsequently, their ability to degrade other substrates can be evaluated.

Strains isolated using the “Prior Growth protocol” are not always associated with the degradation of the substrate of interest. However, in the KSA, some of these strains have been associated with the degradation of crude oil; the aliphatic compounds n-hexadecane, pristane, and n-octadecane; the monocyclic aromatic hydrocarbons (MAHs) phenol and benzene; and the polycyclic aromatic hydrocarbons (PAHs) phenanthrene, fluoranthene, pyrene, and dibenzothiophene (Table 1, ST1-ST9).

In contrast, in the “Enrichment Protocol”, substrates that needs to be degraded are used during the enrichment process, leading to the isolation of bacterial strain that selectively degrade these substrates. As shown in Table 1, the initial substrates used included: crude oil (ST10–ST14); diesel (ST15); the aliphatics hexadecane, pristane, n-hexadecane, and n-tetradecane (ST16–ST18); the MAHs phenol and BTEX (ST19, ST20); and the PAHs anthracene (ST21, ST22), phenanthrene (ST23–ST30), a mixture of anthracene/phenanthrene or anthracene/phenanthrene/fluorene (ST31–ST34), pyrene (ST35–ST38), benzo(a)pyrene (ST39, ST40), and coronene (ST41). Thus, in order of importance, the substrates used to select active bacteria were PAHs (21 out of 43 studies), followed by crude oil and diesel (6 studies), and MAHs (2 studies). Figure 6 shows the chemical structure of PAHs discussed in this review.

Aliphatic degradation was carried out in 3 studies only (ST16–ST18). As discussed earlier, investigations on aliphatic degradation are of limited interest since these compounds are generally easy to degrade. Thus, they do not accumulate in the environment, which explains why there are fewer studies compared to those on aromatics. In addition, their mechanisms of degradation have already been extensively studied [78].

In the 23 studies where PAHs were used, phenanthrene alone was employed in 11 studies (ST23–ST34), and phenanthrene was used in combination with anthracene in 3 studies (ST32–ST34). In comparison, only two studies used anthracene as the sole carbon source (ST21, ST22). The four-ring PAH pyrene was employed in 4 studies (ST35–ST38) and the five rings benzo[a]pyrene in two studies (ST39, ST40), and finally, only one study was undertaken with the use of coronene (ST41). Thus, it is clear that phenanthrene was the most used PAH, followed by pyrene, and limited work has been carried out on benzo[a]pyrene and coronene. These observations are, overall, in line with the many studies carried out on the degradation of PAHs all over the world [5,6,7,79,80,81,82]. The PAH coronene is one the most recalcitrant PAHs, and so far, its biodegradation has been reported in only one other study [83].

Crude oil was used as an enrichment substrate in 5 studies (ST10–ST14), while diesel was employed in one study (ST15). As stated earlier, crude oil consists of various aliphatic and aromatic compounds, and diesel also, to a lesser extent [4,84]. When used as a carbon source, bacteria will degrade components that are more amenable to degradation, mainly the aliphatic ones. Thus, such a mixture of petroleum products in enrichment does not guarantee the selection of bacteria active in degrading complex petroleum products. Supporting this, in 4 studies (ST10–ST13), the isolated bacteria were tested on crude oil only, and only one study reported the degradation of PAHs by bacterial strains isolated under these conditions (using crude oil as an enrichment substrate) [ST14].

Table 1. Summary of work published on bacterial isolates collected from the Kingdom of Saudi Arabia. GC and HPLC represent gas chromatography and high performance liquid chromatography respectively.

Study Number (ST #)	Pollutant Used to Isolate Bacterial Strains	Type of Inoculum	Location of Inoculum Collection	Research Center or University	Method of Strain Selection	List of Isolated Bacteria	Other Pollutants Degraded by Bacterial Strains	Type of Bacteria	Main Results	Reference
ST 1	None	Contaminated Soil	Dammam	King Saud University (KSU)	Prior growth but medium not mentioned	Staphylococcus Corynebacterium	Crude Oil	Mesophile	30–70% of degradation rate after 28 days, using weighing method	[66]
ST 2	None	Crude Oil Samples	No Information	Taif University (TU)	Prior growth in rich medium	Klebsiella Acinetobacter	Phenanthrene, Fluoranthene, Pyrene, Benzene	Mesophile	58–83% degradation, after 48 h, by weighing method	[67]
ST 3	None	Petroleum Sludge	Jeddah	Taif University (TU)	Prior growth in wastewater	Pantoea Acinetobacter Bacillus	n-Hexadecane, Phenol, Phenanthrene	Mesophile	Almost 100% degradation within 4 days, GC	[68]
ST 4	None	Crude Oil contaminated Microbial Mats	Dawhat, Al-Daffi, Jubail	King Fahd University of Petroleum and Minerals (KFUPM)	Metagenomic Analysis	Cyanobacterial mat.	Pristane, n-Octadecane, Phenanthrene, Dibenzothiophene	Mesophile	14–25% of degradation, by GC	[76]
ST 5	None	Soil	Not mentioned	Imam Abdul Rahman bin Faisal University (IAU)	Not Specified	Alcanivorax	Crude Oil	Mesophile	Up to 73% reduction, by GC	[73]
ST 6	None	Soil	Dammam	Imam Abdul Rahman bin Faisal University (IAU)	Prior growth in rich medium	Bacillus Pseudomonas	Crude Oil	Mesophile	40–56% of oil degradation after 7 days, by spectrophotometry	[69]
ST 7	None	Contaminated Sediments	ARAMCO, Jazan	King Abdulaziz University (KAU)	Prior growth in Bushnell-Haas medium	Pseudomonas Bacillus Staphylococcus Erwinia Nitratireductor	Crude Oil	Mesophile	67–77% of oil degradation by after 7 days, by spectrophotometry	[70]
ST8	None	Marine Soil Sediment	Eastern Province	King Saud University (KSU)	Prior growth in rich medium	Bacillus	Crude Oil	Mesophile	>90% of degradation after 14 days, by HPLC	[71]
ST 9	None	Bilge Wastewater	Aljubail Port	King Saud University (KSU)	Prior growth in Bushnell-Haas medium	Acinetobacter Klebsiella Pseudomonas Bacillus Brevibacterium	Crude Oil	Mesophile	90% of reduction of crude within 3 days, by weighing	[72]
ST 10	Crude Oil	Contaminated Soil	Arabian Gulf	King Saud University (KSU)	Enrichment Culture	Pseudomonas	Crude Oil	Mesophile	>80% degradation within 10 days, GC	[18]
ST 11	Crude Oil	Sediments	Yanbu	King Abdulaziz University (KAU)	Enrichment Culture	Pseudomonas Nitratireductor	Crude Oil	Mesophile	65–95% degradation 14 days, by GC	[33]
ST 12	Crude Oil	Contaminated Sediment	Jeddah	Taif University (TU)	Enrichment Culture	Enterobacter	Crude Oil	Mesophile	87–97% of degradation after 14 days, by gravimetric methods	[34]
ST 13	Crude Oil	Contaminated Soil	Dhahran	Shaqra University (SU)	Enrichment Culture	Bacillus Pseudomonas	Crude Oil	Mesophile	Not mentioned	[35]
ST 14	Crude oil	Soil	Al-Ahsa	King Faisal University (KFU)	Enrichment Culture	Georgina Arthrobacter	Crude Oil, n-alkanes, PAHs (biphenyl, naphthalene, and anthracene)	Mesophile	67% of PAH degradation within 14 days, by GC	[36]
ST 15	Diesel	Contaminated Soil	Al-Majmaah	Majmaah University	Enrichment Culture	Microbacterium	Diesel	Mesophile	21–78% of degradation after 5 days, by GC-MS	[37]
ST 16	Hexadecane	Contaminated Soil	Riyadh	King Saud University (KSU)	Enrichment Culture	Pseudomonas Rhodococcus Bacillus	Hexadecane	Mesophile	100% degradation in 5–9 days, using GC	[38]
ST 17	Pristane and hexadecane	Contaminated Sand Samples	No Information	Vietnam Academy of Science and Technology (VAST)	Enrichment Culture	Nocardia	Pristane, various C6-C16 alkanes	Mesophile	Up to 90% within 3 weeks, using GC	[39]
ST 18	n-tetradecane	Contaminated Soil	No Information	Taif University (TU)	Enrichment Culture	Pseudomonas Bacillus		Mesophile	50–90% within 5 days, by GC	[40]
ST 19	Phenol	Wastewater from Industrial Treatment Plant	Yanbu	King Abdulaziz University (KAU)	Enrichment Culture	Ochrobactrum Pseudomonas	Phenol	Mesophile	100% degradation within 4 days by Ultra-Fast Liquid Chromatography	[41]
ST 20	Benzene, Toluene, Ethylbenzene, Xylene s	Contaminated Soil, Water and Oily Sludge	Eastern Region	King Abdulaziz City for Science and Technology (KACST)	Enrichment Culture	Bacillus Paenibacillus Burkholderia Proteus	Benzene, Toluene, Ethylbenzene, Xylene	Mesophile	100% degradation except for Benzene, within 21 days, by HPLC	[42]
ST 21	Naphthalene	Contaminated Soil Sample	Dhahran	King Fahd University of Petroleum and Minerals (KFUPM)	Enrichment Culture	Methylobacterium Pseudomonas	Naphthalene	Mesophile	Not directly determined. Degradation was assessed by bacteria growth	[43]
ST 22	Anthracene	Marine Water	Abhor, Red Sea, Jeddah	King Saud University (KSU)	Enrichment Culture	Sphingomonas	Anthracene	Psychrophile		[44]
ST23	Phenanthrene	Contaminated Soil	Abha	King Khalid University (KKU)	Enrichment Culture	Sphingomonas Pseudomonas Micrococcus Arthrobacter Stenotrophomonas Kocuria Shinella	Naphthalene, Phenanthrene	Mesophile	Not direct assessment (assessment by bacterial growth)	[45]
ST 24	Phenanthrene	Marine Water	Abhor, Red Sea, Jeddah	King Abdulaziz University (KAU)	Enrichment Culture	Ochrobactrum Propionispira Martelella Bacillus Marinobacter Azospira	Phenanthrene, Pyrene, Fluorene, Hexadecane	Halophile	88–100% degradation within 12 days, by HPLC and GC	[46]
ST 25	Phenanthrene	Sediments from Mineral Mining Site	No Information	King Abdulaziz University (KAU)	Enrichment Culture	Stenotrophomonas	Anthracene, Phenanthrene, Naphthalene, Fluorene, Pyrene, Benzo(e)pyrene, Benzo(k)fluoranthene	Acidophile	80–95% degradation of PAHs by GC	[47]
ST 26	Phenanthrene	Contaminated Sediment	Jubail	King Fahd University of Petroleum and Minerals (KFUPM)	Enrichment Culture	Pseudomonas Ochrobactrum	Phenanthrene, Pyrene	Mesophile	62–94% degradation, by GC	[48]
ST 27	Phenanthrene	Brine Water Sample	Jeddah	King Abdulaziz University (KAU)	Enrichment Culture	Ochrobactrum Pseudomonas	Phenanthrene Naphthalene, Anthracene, Fluorene, Pyrene, Benzo(e)pyrene, Benzo(k)fluoranthene	Halothermophile	81–90% degradation, within 14 days, by GC	[49]
ST 28	Phenanthrene	Drilling Site Sediment	Al-Khobar	King Abdulaziz University (KAU)	Enrichment Culture	Pseudomonas Bacillus	Naphthalene, Phenanthrene, Fluorene, Anthracene, Pyrene, Benzo(e)pyrene, Benzo(k)fluoranthene	Thermophile (60 °C)	81–95% degradation, by HPLC	[65]
ST 29	Phenanthrene	Saline Seawater and Sediment	Abhor, Red Sea, Jeddah	King Abdulaziz University (KAU)	Enrichment Culture	Ochrobactrum Stenotrophomonas Achromobacter Mesorhizobium	Phenanthrene, Fluorene, Pyrene	Halophile	50–90% degradation within 12 days, by GC	[50]
ST 30	Phenanthrene	Briny Water and Sediment	Red Sea	King Abdulaziz University (KAU)	Enrichment Culture	Marinobacter (3 strains)	Phenanthrene, Pyrene	Halophile	71–90% degradation within 12 days, by GC	[51]
ST 31	Phenanthrene or Anthracene	Contaminated Soil Sample	Dhahran	King Fahd University of Petroleum and Minerals (KFUPM)	Enrichment Culture	Pseudomonas Stenotrophomonas Ralstonia Thermomonas	Anthracene, Phenanthrene	Mesophile	Up to 75% degradation within 15 days, by GC	[58]
ST 32	Phenanthrene or Anthracene	Contaminated Soil	Aseer	King Khalid University (KKU)	Enrichment Culture	Bacillus Ochrobactrum	Anthracene, Phenanthrene	Mesophile	Assessment by bacterial growth	[53]
ST 33	Phenanthrene and Anthracene	Contaminated Soil	No Information	King Saud University (KSU)	Enrichment Culture	Halomonas	Anthracene, Phenanthrene	Halophile	95–100% degradation within 1–2 days, by GC-MS	[55]
ST 34	Fluorene	Drilling Site Sample	No Information	King Abdulaziz University (KAU)	Enrichment Culture	Ochrobactrum Bacillus Marinobacter Pseudomonas Martelella Stenotrophomonas Rhodococcus	Anthracene, Phenanthrene, Fluorene, Naphthalene, Pyrene, Benzo(a)pyrene, Benzo(e)pyrene, Benzo(k)fluoranthene.	Halo-alkalo-thermophile (60 °C)	55–100% degradation within 16 days	[56]
ST 35	Pyrene	Contaminated Soil	Jubail	King Fahd University of Petroleum and Minerals (KFUPM)	Enrichment Culture	Burkholderia Caulobacter	Pyrene	Mesophile	21–59% of degradation within 18 days, by GC	[57]
ST 36	Pyrene	Wastewater Sludge	Jubail	King Fahd University of Petroleum and Minerals (KFUPM)	Enrichment Culture	Halomonas	Pyrene, Naphthalene, Anthracene, Phenanthrene,	Halophile	50% degradation within 18 days, by GC	[52]
ST 37	Pyrene	Contaminated Soil	Jubail	King Fahd University of Petroleum and Minerals (KFUPM)	Enrichment Culture	Achromobacter	Pyrene, Naphthalene, Anthracene, Phenanthrene	Mesophile	50% degradation within 15 days, by GC	[54]
ST 38	Pyrene	Contaminated Soil	Abha	King Khalid University (KKU)	Enrichment Culture	Klebsiella	Pyrene, Naphthalene, Anthracene, Phenanthrene, Phenanthrene	Mesophile	70% degradation after 12 days, by HPLC	[59]
ST 39	Benzo(a)Pyrene.	Contaminated Sediment	Dhahran	King Fahd University of Petroleum and Minerals (KFUPM)	Enrichment Culture	Staphylococcus	Benzo(a)pyrene, Pyrene, Naphthalene, Anthracene, Phenanthrene	Halophile	44–80% degradation within 30 days, by GC	[60]
ST 40	Benzo(a)Pyrene.	Contaminated Sediment	Dhahran	King Fahd University of Petroleum and Minerals (KFUPM)	Enrichment Culture	Bradyrhizobium Micrococcus Bacillus	Benzo(a)pyrene, Pyrene, Naphthalene, Anthracene, Phenanthrene	Mesophile	44–75% within 30 days, by GC	[61]
ST 41	Coronene	Contaminated Sediment	Dhahran	King Fahd University of Petroleum and Minerals (KFUPM)	Enrichment Culture	Halomonas,	Coronene, Benzo(a)pyrene, Pyrene, Naphthalene, Anthracene, Phenanthrene	halophilic	48% degradation within 20 days, and 76% within 80 days, by GC	[63]
ST 42	Not mentioned	Sediments from Industrial Area	Taif	Taif University (TU)	Not mentioned	Bacillus Actinomyces Pseudomonas	Crude Oil	Mesophile	52–63% degradation, by gravimetric methods	[64]
ST 43	Not mentioned	Contaminated Sediment	Jubail	King Fahd University of Petroleum and Minerals (KFUPM)	Not mentioned	Brevibacillus Proteus Rhodococcus	Naphthalene, Pyrene.	Mesophile	35–62% within 18 days, by GC	[62]

Table 1. Summary of work published on bacterial isolates collected from the Kingdom of Saudi Arabia. GC and HPLC represent gas chromatography and high performance liquid chromatography respectively.

Study Number (ST #)	Pollutant Used to Isolate Bacterial Strains	Type of Inoculum	Location of Inoculum Collection	Research Center or University	Method of Strain Selection	List of Isolated Bacteria	Other Pollutants Degraded by Bacterial Strains	Type of Bacteria	Main Results	Reference
ST 1	None	Contaminated Soil	Dammam	King Saud University (KSU)	Prior growth but medium not mentioned	Staphylococcus Corynebacterium	Crude Oil	Mesophile	30–70% of degradation rate after 28 days, using weighing method	[66]
ST 2	None	Crude Oil Samples	No Information	Taif University (TU)	Prior growth in rich medium	Klebsiella Acinetobacter	Phenanthrene, Fluoranthene, Pyrene, Benzene	Mesophile	58–83% degradation, after 48 h, by weighing method	[67]
ST 3	None	Petroleum Sludge	Jeddah	Taif University (TU)	Prior growth in wastewater	Pantoea Acinetobacter Bacillus	n-Hexadecane, Phenol, Phenanthrene	Mesophile	Almost 100% degradation within 4 days, GC	[68]
ST 4	None	Crude Oil contaminated Microbial Mats	Dawhat, Al-Daffi, Jubail	King Fahd University of Petroleum and Minerals (KFUPM)	Metagenomic Analysis	Cyanobacterial mat.	Pristane, n-Octadecane, Phenanthrene, Dibenzothiophene	Mesophile	14–25% of degradation, by GC	[76]
ST 5	None	Soil	Not mentioned	Imam Abdul Rahman bin Faisal University (IAU)	Not Specified	Alcanivorax	Crude Oil	Mesophile	Up to 73% reduction, by GC	[73]
ST 6	None	Soil	Dammam	Imam Abdul Rahman bin Faisal University (IAU)	Prior growth in rich medium	Bacillus Pseudomonas	Crude Oil	Mesophile	40–56% of oil degradation after 7 days, by spectrophotometry	[69]
ST 7	None	Contaminated Sediments	ARAMCO, Jazan	King Abdulaziz University (KAU)	Prior growth in Bushnell-Haas medium	Pseudomonas Bacillus Staphylococcus Erwinia Nitratireductor	Crude Oil	Mesophile	67–77% of oil degradation by after 7 days, by spectrophotometry	[70]
ST8	None	Marine Soil Sediment	Eastern Province	King Saud University (KSU)	Prior growth in rich medium	Bacillus	Crude Oil	Mesophile	>90% of degradation after 14 days, by HPLC	[71]
ST 9	None	Bilge Wastewater	Aljubail Port	King Saud University (KSU)	Prior growth in Bushnell-Haas medium	Acinetobacter Klebsiella Pseudomonas Bacillus Brevibacterium	Crude Oil	Mesophile	90% of reduction of crude within 3 days, by weighing	[72]
ST 10	Crude Oil	Contaminated Soil	Arabian Gulf	King Saud University (KSU)	Enrichment Culture	Pseudomonas	Crude Oil	Mesophile	>80% degradation within 10 days, GC	[18]
ST 11	Crude Oil	Sediments	Yanbu	King Abdulaziz University (KAU)	Enrichment Culture	Pseudomonas Nitratireductor	Crude Oil	Mesophile	65–95% degradation 14 days, by GC	[33]
ST 12	Crude Oil	Contaminated Sediment	Jeddah	Taif University (TU)	Enrichment Culture	Enterobacter	Crude Oil	Mesophile	87–97% of degradation after 14 days, by gravimetric methods	[34]
ST 13	Crude Oil	Contaminated Soil	Dhahran	Shaqra University (SU)	Enrichment Culture	Bacillus Pseudomonas	Crude Oil	Mesophile	Not mentioned	[35]
ST 14	Crude oil	Soil	Al-Ahsa	King Faisal University (KFU)	Enrichment Culture	Georgina Arthrobacter	Crude Oil, n-alkanes, PAHs (biphenyl, naphthalene, and anthracene)	Mesophile	67% of PAH degradation within 14 days, by GC	[36]
ST 15	Diesel	Contaminated Soil	Al-Majmaah	Majmaah University	Enrichment Culture	Microbacterium	Diesel	Mesophile	21–78% of degradation after 5 days, by GC-MS	[37]
ST 16	Hexadecane	Contaminated Soil	Riyadh	King Saud University (KSU)	Enrichment Culture	Pseudomonas Rhodococcus Bacillus	Hexadecane	Mesophile	100% degradation in 5–9 days, using GC	[38]
ST 17	Pristane and hexadecane	Contaminated Sand Samples	No Information	Vietnam Academy of Science and Technology (VAST)	Enrichment Culture	Nocardia	Pristane, various C6-C16 alkanes	Mesophile	Up to 90% within 3 weeks, using GC	[39]
ST 18	n-tetradecane	Contaminated Soil	No Information	Taif University (TU)	Enrichment Culture	Pseudomonas Bacillus		Mesophile	50–90% within 5 days, by GC	[40]
ST 19	Phenol	Wastewater from Industrial Treatment Plant	Yanbu	King Abdulaziz University (KAU)	Enrichment Culture	Ochrobactrum Pseudomonas	Phenol	Mesophile	100% degradation within 4 days by Ultra-Fast Liquid Chromatography	[41]
ST 20	Benzene, Toluene, Ethylbenzene, Xylene s	Contaminated Soil, Water and Oily Sludge	Eastern Region	King Abdulaziz City for Science and Technology (KACST)	Enrichment Culture	Bacillus Paenibacillus Burkholderia Proteus	Benzene, Toluene, Ethylbenzene, Xylene	Mesophile	100% degradation except for Benzene, within 21 days, by HPLC	[42]
ST 21	Naphthalene	Contaminated Soil Sample	Dhahran	King Fahd University of Petroleum and Minerals (KFUPM)	Enrichment Culture	Methylobacterium Pseudomonas	Naphthalene	Mesophile	Not directly determined. Degradation was assessed by bacteria growth	[43]
ST 22	Anthracene	Marine Water	Abhor, Red Sea, Jeddah	King Saud University (KSU)	Enrichment Culture	Sphingomonas	Anthracene	Psychrophile		[44]
ST23	Phenanthrene	Contaminated Soil	Abha	King Khalid University (KKU)	Enrichment Culture	Sphingomonas Pseudomonas Micrococcus Arthrobacter Stenotrophomonas Kocuria Shinella	Naphthalene, Phenanthrene	Mesophile	Not direct assessment (assessment by bacterial growth)	[45]
ST 24	Phenanthrene	Marine Water	Abhor, Red Sea, Jeddah	King Abdulaziz University (KAU)	Enrichment Culture	Ochrobactrum Propionispira Martelella Bacillus Marinobacter Azospira	Phenanthrene, Pyrene, Fluorene, Hexadecane	Halophile	88–100% degradation within 12 days, by HPLC and GC	[46]
ST 25	Phenanthrene	Sediments from Mineral Mining Site	No Information	King Abdulaziz University (KAU)	Enrichment Culture	Stenotrophomonas	Anthracene, Phenanthrene, Naphthalene, Fluorene, Pyrene, Benzo(e)pyrene, Benzo(k)fluoranthene	Acidophile	80–95% degradation of PAHs by GC	[47]
ST 26	Phenanthrene	Contaminated Sediment	Jubail	King Fahd University of Petroleum and Minerals (KFUPM)	Enrichment Culture	Pseudomonas Ochrobactrum	Phenanthrene, Pyrene	Mesophile	62–94% degradation, by GC	[48]
ST 27	Phenanthrene	Brine Water Sample	Jeddah	King Abdulaziz University (KAU)	Enrichment Culture	Ochrobactrum Pseudomonas	Phenanthrene Naphthalene, Anthracene, Fluorene, Pyrene, Benzo(e)pyrene, Benzo(k)fluoranthene	Halothermophile	81–90% degradation, within 14 days, by GC	[49]
ST 28	Phenanthrene	Drilling Site Sediment	Al-Khobar	King Abdulaziz University (KAU)	Enrichment Culture	Pseudomonas Bacillus	Naphthalene, Phenanthrene, Fluorene, Anthracene, Pyrene, Benzo(e)pyrene, Benzo(k)fluoranthene	Thermophile (60 °C)	81–95% degradation, by HPLC	[65]
ST 29	Phenanthrene	Saline Seawater and Sediment	Abhor, Red Sea, Jeddah	King Abdulaziz University (KAU)	Enrichment Culture	Ochrobactrum Stenotrophomonas Achromobacter Mesorhizobium	Phenanthrene, Fluorene, Pyrene	Halophile	50–90% degradation within 12 days, by GC	[50]
ST 30	Phenanthrene	Briny Water and Sediment	Red Sea	King Abdulaziz University (KAU)	Enrichment Culture	Marinobacter (3 strains)	Phenanthrene, Pyrene	Halophile	71–90% degradation within 12 days, by GC	[51]
ST 31	Phenanthrene or Anthracene	Contaminated Soil Sample	Dhahran	King Fahd University of Petroleum and Minerals (KFUPM)	Enrichment Culture	Pseudomonas Stenotrophomonas Ralstonia Thermomonas	Anthracene, Phenanthrene	Mesophile	Up to 75% degradation within 15 days, by GC	[58]
ST 32	Phenanthrene or Anthracene	Contaminated Soil	Aseer	King Khalid University (KKU)	Enrichment Culture	Bacillus Ochrobactrum	Anthracene, Phenanthrene	Mesophile	Assessment by bacterial growth	[53]
ST 33	Phenanthrene and Anthracene	Contaminated Soil	No Information	King Saud University (KSU)	Enrichment Culture	Halomonas	Anthracene, Phenanthrene	Halophile	95–100% degradation within 1–2 days, by GC-MS	[55]
ST 34	Fluorene	Drilling Site Sample	No Information	King Abdulaziz University (KAU)	Enrichment Culture	Ochrobactrum Bacillus Marinobacter Pseudomonas Martelella Stenotrophomonas Rhodococcus	Anthracene, Phenanthrene, Fluorene, Naphthalene, Pyrene, Benzo(a)pyrene, Benzo(e)pyrene, Benzo(k)fluoranthene.	Halo-alkalo-thermophile (60 °C)	55–100% degradation within 16 days	[56]
ST 35	Pyrene	Contaminated Soil	Jubail	King Fahd University of Petroleum and Minerals (KFUPM)	Enrichment Culture	Burkholderia Caulobacter	Pyrene	Mesophile	21–59% of degradation within 18 days, by GC	[57]
ST 36	Pyrene	Wastewater Sludge	Jubail	King Fahd University of Petroleum and Minerals (KFUPM)	Enrichment Culture	Halomonas	Pyrene, Naphthalene, Anthracene, Phenanthrene,	Halophile	50% degradation within 18 days, by GC	[52]
ST 37	Pyrene	Contaminated Soil	Jubail	King Fahd University of Petroleum and Minerals (KFUPM)	Enrichment Culture	Achromobacter	Pyrene, Naphthalene, Anthracene, Phenanthrene	Mesophile	50% degradation within 15 days, by GC	[54]
ST 38	Pyrene	Contaminated Soil	Abha	King Khalid University (KKU)	Enrichment Culture	Klebsiella	Pyrene, Naphthalene, Anthracene, Phenanthrene, Phenanthrene	Mesophile	70% degradation after 12 days, by HPLC	[59]
ST 39	Benzo(a)Pyrene.	Contaminated Sediment	Dhahran	King Fahd University of Petroleum and Minerals (KFUPM)	Enrichment Culture	Staphylococcus	Benzo(a)pyrene, Pyrene, Naphthalene, Anthracene, Phenanthrene	Halophile	44–80% degradation within 30 days, by GC	[60]
ST 40	Benzo(a)Pyrene.	Contaminated Sediment	Dhahran	King Fahd University of Petroleum and Minerals (KFUPM)	Enrichment Culture	Bradyrhizobium Micrococcus Bacillus	Benzo(a)pyrene, Pyrene, Naphthalene, Anthracene, Phenanthrene	Mesophile	44–75% within 30 days, by GC	[61]
ST 41	Coronene	Contaminated Sediment	Dhahran	King Fahd University of Petroleum and Minerals (KFUPM)	Enrichment Culture	Halomonas,	Coronene, Benzo(a)pyrene, Pyrene, Naphthalene, Anthracene, Phenanthrene	halophilic	48% degradation within 20 days, and 76% within 80 days, by GC	[63]
ST 42	Not mentioned	Sediments from Industrial Area	Taif	Taif University (TU)	Not mentioned	Bacillus Actinomyces Pseudomonas	Crude Oil	Mesophile	52–63% degradation, by gravimetric methods	[64]
ST 43	Not mentioned	Contaminated Sediment	Jubail	King Fahd University of Petroleum and Minerals (KFUPM)	Not mentioned	Brevibacillus Proteus Rhodococcus	Naphthalene, Pyrene.	Mesophile	35–62% within 18 days, by GC	[62]

3.5. Mesophilic, Halophilic, and Thermophilic Bacteria

In both the “enrichment protocol” and “prior growth”, bacterial cultures are generally grown at a temperature range of 30–40 °C, low salinity of <4% NaCl, and neutral pH. Bacterial strains selected under these conditions are known as mesophiles [61]. These conditions are considered the most prevalent in most contaminated sites. As a result, most of the reported bacteria-degrading pollutants in general, and petroleum products in particular, have been selected under these mesophilic conditions. The same observation can be made in studies carried out in Saudi Arabia, where more than 71% of reported bacteria are mesophilic (Table 1).

However, contamination also occurs in extreme environments, primarily characterized by high salinity (for halophilic bacteria) and high temperatures (for thermophilic bacteria). Thermophiles are categorized into three groups based on the optimal temperature ranges for bacterial growth: moderate thermophiles (40–59 °C), extreme thermophiles (60–85 °C), and hyper-thermophiles (>85 °C) [85]. In relation to salinity, moderately halophilic bacteria grow in the range of 3–5% (w/v) NaCl, while extremely halophilic bacteria grow at >15% (w/v) NaCl [86].

Nine studies investigated the degradation of PAHs in halophilic conditions, ranging from 4% to 30% NaCl (ST24, ST27, ST29, ST30, ST33, ST34, ST36, ST39, ST41). Interestingly, in most of these studies, bacterial consortia were used rather than single bacterial strains. For instance, the consortium of Ochrobactrum halosaudia AJH1, O. halosaudia AJH2, and P. aeruginosa AJH3 was shown to degrade phenanthrene, the 3-ring polyaromatic hydrocarbon, from 20% up to 30% NaCl, although the efficiency of degradation decreased significantly at 30% (ST27). Furthermore, this consortium could also degrade phenanthrene at temperatures between 30 and 60 °C, making it a halo-thermophilic bacterial consortium (ST27) [65]. Other studies used two or more bacterial strains (ST24, ST26, ST29, ST30, ST33, ST34).

The use of consortia shows that salinity conditions reduce or limit bacterial growth. As a result, different strains are used as consortia, each providing a small contribution to the degradation process. This is a common feature of pollutant degradation in extreme conditions, particularly in the context of PAH degradation in high salinity [86]. However, our group reported three studies in which single halophilic bacterial strains were used to degrade PAHs (ST36, ST39, ST41). More specifically, Budiyanto et al. isolated two single bacterial strains, Halomonas shengliensis 10PY2B and Halomonas smyrnensis 20PY1A, that could each degrade pyrene at 10–20% NaCl (ST36). A strain of Staphylococcus haemolyticus 10SBZ1A could degrade the highly complex benzo[a]pyrene at 10% NaCl (ST39), and finally, the degradation of the 7-ring coronene was reported using Halomonas caseinilytica (ST41). Thus far, the degradation of benzo[a]pyrene in halophilic conditions has been reported only in another study carried out in India, using a single strain, Ochrobactrum sp. VA1, but at a relatively low salinity of 3% NaCl [87]. Thus, these Saudi strains are among the most active reported so far for the degradation of benzo[a]pyrene in a hypersaline condition of 20% NaCl.

In relation to thermophilicity, three studies investigated the degradation of PAHs in thermophilic conditions (ST27, ST28, ST34). As with halophilic conditions, degradation was observed using consortia, and it occurred at temperatures as high as 60 °C (ST27-28, ST34). The degradation of petroleum products, including aliphatics, MAHs, and PAHs, has been reported in different parts of the world and can occur at temperatures as high as 80 °C [7]. Interestingly, almost 60% of these bacteria belong to the Geobacillus genus, and 80% belong to the Bacillaceae family [7]. However, in Saudi samples, as shown in Table 1, out of 11 genera identified for thermophilic bacterial strains (ST27, ST28, ST34), no Geobacillus was present, and only 2 belonged to the Bacillaceae family (Bacillus strains in ST27 and ST34).

3.6. Identification of Metabolites, Enzymes, and Biochemical Degradation Pathways of PAHs

Biochemical pathways associated with PAH degradation have been investigated by identifying PHA metabolites and enzymes involved in this biodegradation. This knowledge can be utilized to develop microbial biocatalysts with enhanced degradation capabilities by using genetic engineering and synthetic biology strategies [88,89]. These engineered strains could then be used to enhance pollutant biodegradation as part of the bioremediation strategies.

Numerous studies have focused on the identification of PAH metabolites using gas chromatography and liquid chromatography, coupled with mass spectrometry (GC- and LC-MS), and enzyme detection using various techniques, including whole genome analysis, proteomics, and transcriptomes [81,88].

The biodegradation of PAHs is generally initiated by the action of mono- or di-oxygenase enzymes that hydroxylate a ring in PAHs, leading to ring cleavage and the subsequent formation of hydroxylic or carboxylic derivatives with fewer rings. The aliphatic moieties of these derivatives are then removed, allowing them to enter Krebs cycles for energy generation in the form of ATP (adenosine triphosphate). This process continues until all rings are cleaved [82,90].

In the KSA, 11 investigations have reported the identification of PAH metabolites. Six were carried out at the KFUPM (ST21, ST31, ST36, ST37, ST39, ST40), three at KAU (ST25, ST28, ST29), and two at KSU (ST22, ST33). These studies investigated the metabolites of various PAHs, such as naphthalene, phenanthrene, anthracene, fluorene, pyrene, and benzo[a]pyrene.

For instance, the degradation of naphthalene, considered the simplest among PAHs, involves hydroxylation of one of the rings followed by ring cleavage to produce a benzoic acid or benzaldehyde derivative. When Methylobacterium radiotolerans N7A and Pseudomonas aeruginosa N7B1 (ST21) were used for naphthalene degradation, o-phthalic acid and 4-hydroxy-2-oxovaleeric acid were obtained as metabolites. Furthermore, a consortium of P. aeruginosa, Bacillus thermosaudia, and Stenotrophomonas maltophilia (ST28) produced 1,2-dihydroxynaphthalene and 3,4-dihydroxybenzoate as metabolites in naphthalene degradation. Additionally, 3,4-dihydroxybenzoate was obtained during naphthalene degradation by St. maltophilia strain AJH1 (ST25). A tentative metabolic pathway for the biodegradation of naphthalene using bacterial strains isolated from the KSA is shown in Figure 7 (ST21, ST25, ST28).

The degradation of anthracene and phenanthrene commences with hydroxylation at either the terminal or the inner ring. For instance, anthracene-1,2-diol was observed in the degradation of anthracene using S. maltophilia AJH1 (ST25) or a consortium of P. aeruginosa CEES1 and B. thermosaudia CEES2 (ST28), while anthracene-9,10-dione was obtained in the degradation of anthracene using Halomonas sp. BR04 (ST33b). Subsequent ring cleavage of the middle ring of anthracene-9,10-dione led to the generation of o-phthalate derivative, which is further degraded to benzoic acid before entering the TCA cycle. Degradation pathways based on the identified metabolites for anthracene are illustrated in Figure 7 (ST22, ST25, ST28, ST33). Similarly, phenanthrene degradation using various strains isolated in the KSA revealed both oxidations of the middle ring and side rings (ST25, ST28, ST30, ST31, ST33b), as depicted in Figure 7.

In the case of pyrene degradation by Halomonas shengliensis and Halomonas smyrnensis (ST36), several metabolites were identified, including 4-phenanthrenecarboxylic acid, 4-(1-hydroxynaphthalen-2-yl)-2-oxo-but-3-enoic acid, and phthalic acid. Likewise, the degradation of pyrene by Achromobacter xylosoxidans PY4 resulted in the identification of monohydroxypyrene, 4-(1-methoxynaphthalen-2-yl)-2-oxo-but-3-enoic acid, 9,10-phenanthrenequinone, 2-methoxybenzalpyruvic acid, and dibutyl phthalate (ST37). These identified metabolites in the metabolic pathway of pyrene in ST36 and ST37 suggest that pyrene oxidation takes place at C4 and C5 positions, followed by ring cleavage at these positions (Figure 8). 4-Phenanthrenecarboxylic acid was also observed in pyrene degradation using S. maltophilia strain AJH1 (ST25), supporting the C4-C5 ring cleavage of pyrene. This metabolite was also observed using a consortium of P. aeruginosa CEES1 and B. thermosaudia CEES2 (ST28). However, the identification of pyrene-1,2-oxide and 1-hydroxypyrene in the degradation of pyrene in studies ST25 and TS28 suggests a ring cleavage at C1 and C2, but no further phenalene-based metabolites were identified. It is not uncommon for a single strain to follow more than one pathway in the degradation of complex PAHs. A tentative metabolic pathway for pyrene by bacterial strains isolated from the KSA is shown in Figure 8 (ST25, ST28, ST36, ST37)

In relation to the degradation of benzo[a]pyrene using Staphylococcus haemoliticus 10SBZ1A (ST39), the metabolites dihydroxy-benzo[a]pyrene and benzo[a]pyrene-quinone were observed. However, the specific isomer was not identified. Additionally, either 4,5-chrysene-dicarboxylic acid, 4-(8-hydroxypyren-7-yl)-2-oxobut-3-enoic acid, or 4-(7-hydroxypyren-8-yl)-2-oxobut-3-enoic acid was observed, and either 10-oxabenzo[def]chrysene-9-one or 7-oxabenzo[def]chrysene-8-one was also observed. The structural similarity of these metabolites, their exact molar masses, and the lack of standard samples for comparison restricted the proper identification of these metabolites. Additionally, 4-formylchrysene-5-carboxylic was also observed in the degradation of benzo[a]pyrene in study ST39, indicating a ring cleavage at C4-C5 of benzo[a]pyrene. Dihydroxy-benzo[a]pyrene, methylated-dihydrodiol-benzo[a]pyrene, and 4-formylchrysene-5-carboxylic acid were also detected when using individual and consortium of Bradyrhizobium japonicun JBZ1A, Micrococcus luteus JBZ2B, and Bacillus cereus JBZ5E (ST40). A tentative degradation pathway is shown for benzo[a]pyrene in Figure 8 for ST39 and ST40, although no early metabolites (hydroxylated form of benzo[a]pyrene) were identified.

These findings highlight the complexity of biodegradation of PAHs and the diverse metabolites produced during their degradation by various microbial strains. A detailed understanding of the biodegradation pathways of these PAHs will provide crucial insights for advancing future bioremediation strategies and discovering novel enzymes with unique functions. However, the limited number of studies on PAH biodegradation constrains our understanding of the complete metabolic pathways; therefore, there is a need for further research on this topic to elucidate the biodegradation pathways for PAHs.

4. Knowledge Gaps

As discussed earlier, many studies have been dedicated to the biodegradation of crude oil products, mainly PAHs. Various bacterial strains have been isolated and characterized, and metabolites have been identified, and this has contributed to improving our knowledge of this research topic [81]. However, several fields in biodegradation have not received much attention, and they are discussed below.

4.1. Studies on Higher PAHs and Complex Oil Products

The aforementioned studies in the review in particular, and those published globally in general, have given limited attention to PAHs with more than five fused rings. For instance, coronene, a PAH with seven fused rings, has been reported in only two studies, one of which was conducted in the KSA [63,83]. However, no studies have focused on metabolite identification or biotechnical degradation pathways for this compound. Furthermore, no investigations have been carried out on the biodegradation of complex PAHs with more than seven rings.

As stated earlier, in addition to saturates and mono- and polycyclic aromatic hydrocarbons, oil products contain resins and asphaltenes [4]. Relatively little research has been conducted on the biodegradation of these components, in contrast to the attention given to saturates and aromatics due to the high complexity of the structures of asphaltenes and resins [4]. Evidence of bacterial degradation of asphaltenes and resins has been reported in various parts of the world, although this degradation is only partial since it is primarily associated with aliphatic moieties only of these compounds [4]. However, so far, there is no report on the KSA’s biodegradation of resins and asphaltenes. Thus, the bacterial degradation of these oil components awaits elucidation in the KSA.

4.2. Omics Studies: Whole-Genome Analysis, Transcriptomics or Proteomics

Genetic studies involving gene expression, transcriptomics, proteomics, or whole genome analysis have been carried out on bacterial degradation of MAHs and PAHs [5]. From these studies, various genes encoding enzymes that degrade aromatic compounds have been identified, and their roles clarified; in addition, some of these genes have been used in genetic engineering to generate bacterial strains with high capabilities to degrade PAHs. For instance, the genes coding for dioxygenase (the first enzyme in MAH and PAH degradation) from Mycobacterium sp. PYR-1 was expressed in an Escherichia coli strain, enabling this strain to degrade PAHs [91]. A recombinant strain Pseudomonas fluorescens HK44 and Pseudomonas putida KT2442 were constructed with naphthalene-catabolic plasmids [92,93,94]. These genes have been used as probes to detect bacteria that degrade PAHs in various environments [95,96,97].

However, in the KSA, this area of research has received little attention. To date, only two studies have focused on “omics” analysis in the context of petroleum product biodegradation. In the first study, a whole-genome analysis of a bacterial strain, Idiomarina piscisalsi 10PY1A, that degrades pyrene was carried out. The results revealed the existence of several open-reading frames of putative enzymes that degrade aromatic compounds. These included Rieske non-heme iron aromatic ring-hydroxylating oxygenase, homogentisate 1,2-dioxygenase, 1-hydroxy-2-naphthoate hydroxylase, and phthalate 3,4-dioxygenase, among others [98]. In the second investigation (ST37), proteomic analysis of Achromobacter xylosoxidans PY4 was carried out in the context of pyrene degradation, and gene products of the lower pyrene degradation pathway were identified, including 4-hydroxyphenylpyruvate dioxygenase and homogentisate 1,2-dioxygenase, the latter being an enzyme involved in ring opening.

These two “omics” studies have permitted the identification of enzymes of lower PAH degradation pathways only. Thus, information on the upper PAH degradation pathways remains to be elucidated. In addition, no reports exist on omics investigations of PAHs with more than four rings, such as benzo[a]pyrene or coronene, in bacteria isolated in the KSA.

4.3. The Potential of Fungi and Algae in the Degradation of Petroleum Products

As discussed earlier, this review has focused only on the bacterial strains degrading petroleum products (as per the inclusion criteria). However, the degradation of petroleum products by fungal strains has also been reported in other parts of the world [99,100,101]. In the KSA, a fungal strain, Hortaea sp. B15, was shown to degrade phenanthrene, chrysene, and pyrene [102,103]. In another study (ST33), a fungal strain Cryptococcus s.p MR 22 was isolated from contaminated samples (along with the bacterial stain Holomonas sp. BR04), and found to degrade phenanthrene and anthracene [55]. This is in line with studies carried out in other parts of the world indicating the potential of fungi in the removal of petroleum pollutants [104].

Likewise, several reports have shown that microalgal organisms can degrade various petroleum products, including PAH [105,106,107]. These organisms are important since they are photosynthetic, using CO₂ to synthesize organic molecules, thus contributing to the removal of CO₂ from the environment. In addition, these organisms are associated with growth and biomass accumulation capability, great diversity, and robust adaptability in various environments [108]. In the KSA, so far, only one study has been dedicated to the use of microalgae in petroleum product degradation [109]. This study, carried out by our research team, involved using a Gonium pectorale strain to degrade phenanthrene and anthracene [109]. In other parts of the world, reports indicate that complex PAH, such as pyrene and benzo[a]pyrene, can efficiently be degraded by microalgal strains [105,106,107].

The use of the microalgae–bacteria consortium is emerging as a new strategy for pollutant removal [105,108,110]. On the one hand, algae produce O₂ through photosynthesis, which supports aerobic bacterial activity, crucial for breaking down organic pollutants. On the other hand, bacteria produce CO₂ that algae will utilize in the photosynthetic process. In addition, algae can absorb higher concentrations of pollutants with less negative effect on their growth compared to bacteria, thus allowing these bacteria to grow actively in environments contaminated with high concentrations of pollutants. This mutualistic relationship improves the overall effectiveness and rate of pollutant removal [105]. This concept awaits investigation in the context of cleaning the environment in the KSA.

4.4. Biodegradation Stimulation Methods

Several reports have been dedicated to increasing biodegradation by either adding nutrients, a process known as biostimulation, or external biosurfactants.

This increase in bacterial growth can lead to a higher degradation rate of organic pollutants, including hydrocarbons. The beneficial effects of biostimulation have been demonstrated in the removal of hydrocarbons in various studies conducted in both soil and marine environments [12,111,112]. However, the application of biostimulation for pollutant removal has not yet been explored in Saudi Arabia.

Another strategy to increase the efficiency of biodegradation is the use of the surfactant. They are surface-active agents that reduce the surface tension between water and hydrophobic pollutants (such as PHAs), thus enhancing pollutants’ solubility and availability, making them more accessible to microorganisms. As stated earlier, the active bacteria degrading petroleum products express surfactants (known as biosurfactants) to increase the solution of the pollutants [28]. Therefore, biodegradation or bioremediation can be facilitated by adding surfactants to the environment, thereby enhancing both pollutant solubility and degradation, a strategy that has been evaluated and tested already [113,114] but awaits evaluation in the context oil contaminated environments, including those of Saudi Arabia.

4.5. Degradation of Petroleum Products in Anaerobic Conditions

The biodegradation of pollutants is primarily carried out under aerobic conditions because oxygen serves as an electron acceptor, enabling faster and more efficient breakdown of complex organic pollutants by microorganisms. As discussed earlier, aerobic microbes have well-established pathways, primarily involving oxygenases, that directly facilitate the degradation of hydrocarbons. In contrast, anaerobic conditions present challenges due to the lack of oxygen, which is a key element in many metabolic pathways for degradation. Under anaerobic conditions, microbes must rely on alternative electron acceptors, such as sulfate, carbon dioxide, and nitrate, which leads to slower degradation rates [6]. The anaerobic degradation of petroleum products has been reported, however, to a much lesser extent than in aerobic conditions [6,79,115]. For example, in the case of Saudi Arabia, there have been no reports on the degradation of petroleum products under anaerobic conditions. All studies summarized in this review (see Table 1) were conducted under aerobic conditions. Overall, the metabolic pathways associated with anaerobic degradation are more complex and less well-understood; therefore, exploring these mechanisms could lead to the discovery of new biochemical pathways that may be leveraged in biocatalysis processes. Consequently, further research on anaerobic degradation is essential.

5. Conclusions and Future Perspective

This review has highlighted the research carried out so far in the KSA on the microbial degradation of petroleum products. Bacteria belonging to several genera have been identified, isolated, and characterized. Overall, the distribution (in terms of species or genera) and frequency of identification of these bacteria are similar to those reported in other parts of the world. The degradation of PAHs has been documented. However, more efforts need to be dedicated to the degradation of more complex PAHs. Likewise, the use of microalgae, either alone or in combination with bacteria, offers an alternative approach to improve the degradation of recalcitrant PAHs.

Extreme environmental conditions, including dryness, high temperature, and high salinity characterize the KSA. Thus, it is conceivable that bacteria have adapted to this environment by expressing enzymes with unique catabolic activities. Identifying and characterizing such enzymes will offer, for instance, new biocatalytic strategies or contribute to creating genetically engineered microorganisms (GEMs) with unique catalytic properties. Therefore, there is a need for more comprehensive studies on “Omics” on bacterial strains in the KSA.

Finally, the isolated and characterized active bacteria described in this review underscore the necessity of field studies on bioremediation. The concept of bioremediation for the removal of pollutants in natural conditions of contamination has been evaluated in various parts of the world, and some of these products are commercially available [5]. The active bacteria identified in the KSA have the potential to be used in real-world cleaning of oil-contaminated environments, which highlights the promising prospect of applying bioremediation to address environmental challenges.