The Caspian Sea is the world’s largest brackish lake with a salinity of approximately one-third of normal seawater (Leroy et al., 2007; Owen et al., 2023). This lake lies between Europe and Asia and borders five (5) countries along its coast – Russia, Azerbaijan, Iran, Kazakhstan, and Turkmenistan ( Figure 1 ). The Caspian Sea is an endorheic lake with no outlets or direct connection to an ocean; however, more than 130 rivers flow into the Caspian Sea (Owen et al., 2023). The Caspian Sea is divided into three main geological basins – the northern, middle, and southern basins. Water depths increase towards the southern end of this basin, reaching a maximum depth of 1025 meters (Dumont et al., 2004; EIA, 2013; Owen et al., 2023).

The Caspian basin is one of the oldest oil-producing regions in the world (Effimoff, 2000; Katz et al., 2000). This region is well known for its considerable oil reserves with an estimated 48 billion barrels of oil and 292 trillion cubic feet of natural gas in proven and probable reserves (EIA, 2013; Holstein et al., 2018). The Caspian basin also contributes to the global energy supply with an average production of 2.6 million barrels/day according to the US Energy Information Administration EIA (2013). Azeri light crude oil is extracted offshore near Azerbaijan’s shoreline of the Caspian Sea ( Figure 1 ). It is a well-balanced, sweet (low sulfur content) crude that is valued since a greater percentage of it can be easily refined into desirable petroleum products, for example naphtha (for solvents), gasoline and other fuel oils. The large scale of this industry has contributed to the waterbody’s environmental decline. This region faces challenges with the transportation of the oil extracted and produced since its hydrocarbon reserves are relatively far from export markets. These long distances require shipping and pipeline infrastructure to transport oil which a significant source of oil pollution (Holstein et al., 2018). Crude oil is both a natural and anthropogenic contaminant within the Caspian Sea. Oil also enters the Caspian through natural seepages from the seabed and mud volcanoes (Mityagina et al., 2019). Approximately one million tons of this oil leaked into the Caspian annually from these various sources (Mityagina et al., 2019; Modabberi et al., 2020). The Caspian Sea is surrounded by urban, industrial, and agricultural areas. In addition to oil pollution from the petroleum industry, drainage from various river basins brings an influx of untreated sewage, industrial and agricultural effluents that induce eutrophication which causes anoxic (low oxygen) conditions in deeper, colder waters (Miller et al., 2020; Modabberi et al., 2020). Oxygen saturation has dropped to around ~3% in deeper waters ( Supplementary Table 1 ). Such anoxic conditions affect the mortality of benthic marine organisms and alter biogeochemical cycling (Aladin and Plotnikov, 2004). Since the Caspian Sea is an enclosed waterbody, it acts as a reservoir for pollutants where they begin to accumulate, making it particularly vulnerable to human influence. The lack of outlets, weak tidal forces, and increasing evaporation rates encourage the accumulation of pollutants in the waters and benthic sediments of the Caspian Sea (Nasrollahzadeh, 2010; Medvedev et al., 2017; Mishra et al., 2017).

Crude oil is a complex mixture of thousands of organic compounds containing mainly hydrogen and carbon, which is highlighted in Figure 2 (Hassanshahian and Cappello, 2013; Mahjoubi et al., 2018; Wang et al., 2021). Depending on their chemical structure, these hydrocarbons can be classified into alkanes, alkenes, cycloalkanes, and aromatics (Stauffer et al., 2008). Crude oil also contains derivatives of nitrogen-, sulfur- and oxygen-containing compounds (Morgan and Watkinson, 1994; Stauffer et al., 2008). Metals and other inorganic compounds may also be present at very low concentrations. The complex chemistry of crude oil needs a complex community of microorganisms to degrade it since each component has a distinct chemical property that influences its biodegradability. No single organism is able to metabolize all hydrocarbon fractions in crude oil. Microbial degradation of the crude oil fractions follows an ease of degradation from bioavailable to recalcitrant compounds, typically in the order of n-alkanes > branched alkanes > aromatics > cyclic alkanes > polycyclic aromatic hydrocarbons > resins and asphaltenes (Das and Chandran, 2011; Varjani, 2017; Varjani and Upasani, 2017). This results in a succession of microbial bloom and death as compounds are consumed (Hazen et al., 2010; Olajire and Essien, 2014; Mahjoubi et al., 2018). Hence, these microbes are working in conjunction with each other with their varying enzymatic capabilities to provide a dynamic community response to this contaminant.

Figure 3 illustrates the changing biochemical demands of oil biodegradation with decreasing oxygen concentrations. Alternate electron acceptors (NO₃ ⁻, Fe³⁺, SO₄ ²⁻, CO₂) are needed to complete this process in the absence of oxygen (Hazen et al., 2016). Other environmental variables, such as temperature, can also influence oil biodegradation. Temperature normally affects the metabolic rate of these hydrocarbon-degrading organisms with metabolism decreasing with decreasing temperatures (Das and Chandran, 2011). However, over long periods of time, changes in enzymatic ability to degrade at low temperatures will increase and the organisms will become psychrophiles and will degrade oil faster at colder temperatures (Hazen et al., 2010; Hazen, 2018; Brown et al., 2020). The physicochemical characteristics of the oil are also affected by temperature where oil viscosity increases with decreasing temperature (Ribicic et al., 2018b). Hence, the biodegradation will proceed differently in surface waters (warmer, well-oxygenated) versus deep waters (cold, anoxic conditions) of the Caspian Sea.

This study aims to characterize the in-situ community structure of the Caspian Sea and to investigate the ability of the indigenous microbial community to degrade crude oil under oxic or hypoxic conditions. We observed changes in the community composition in response to crude oil amendment as well as a decrease in hydrocarbon fractions in both sets of microcosms. The environmental conditions (brackish water, oxygen/temperature gradient, eutrophication) in the Caspian Sea could potentially limit the extent of crude oil biodegradation (Varjani and Upasani, 2017). Hence, studying community structure and the potential “memory response” of these indigenous microbes will aid in elucidating their metabolic potential and adaptation to this pollutant.

Our seawater microcosms were kept at their ambient environmental conditions to potentially mimic the in situ temperature and oxygen conditions under which biodegradation would proceed at these varying depths. Figures 5 , 6 highlight the variation in the response of the microbial community to the crude oil amendment based on oxygen concentration and temperature. These differing community structures are indicative of the different metabolic strategies needed to degrade the hydrocarbons found in crude oil. Hydrocarbon degraders are typically present at low abundances in the environment; however, their presence will increase with increasing concentrations of hydrocarbons (Head et al., 2006). This trend was also observed in our experimental microcosms. The impact of crude oil amendment increased the relative abundance of different hydrocarbon-degrading taxa in our experiment microcosms when compared to the control microcosms with no oil.

Figures 5 , 6 show evidence of microbial bloom and succession in the experimental microcosms where the relative abundance of different taxa changed during days 5 to 15 in both oxic and hypoxic microcosms. Community dynamics shifted as the microbial community jointly acted on the different hydrocarbons found in crude oil (Hazen et al., 2016; Varjani and Upasani, 2017; Xu et al., 2018). As the preferred substrates are consumed the microbes specialized to metabolize those compounds exhaust their carbon source and are replaced by microbes able to utilize the remaining compounds resulting in a cycle of bloom and succession (Dubinsky et al., 2013; Hazen et al., 2016; Ribicic et al., 2018b). Secondary consumers are also necessary to metabolize the daughter products from the crude oil which contributes to this microbial network (Head et al., 2006).

All microcosms showed the presence of some hydrocarbon-degrading phyla, which have been previously identified in other oil-contaminated environments, most noticeably after the DeepWater Horizon oil spill (Mason et al., 2012; Dubinsky et al., 2013; King et al., 2015; Hazen, 2018). The greatest biodiversity was observed in microcosms without any crude oil amendment in both oxic and hypoxic conditions. This is unsurprising as some hydrocarbons and their intermediates are toxic to some microorganisms and the action of hydrocarbon-degraders is not induced until carbon enrichment occurs (Xu et al., 2018). The shift in community structure by day 5 shows that an influx of hydrocarbons will affect biodiversity regardless of oxygen condition. The observed reduction in biodiversity is due to the selection pressure for hydrocarbon-degraders after crude oil enrichment in these microcosms. Additionally, significant differences were noted in community structures in oxic versus hypoxic conditions. This was further indicated by our NMDS plot ( Figure 7 ), where there is a clear distinction in the clustering of samples based on oxygen condition and crude oil amendment. Figure 7 shows a difference in communities in our experimental microcosms at various time points. Initial sampling points are clustered closer together, indicating the early microbial communities share some similarities; however, after day 5, this community changes significantly. Later time points are clustered further away from each other, and distinct groupings between the oxic and hypoxic samples can be observed ( Figure 7 ).

Greater biodiversity was observed in oxic microcosms. This may be explained by the typical aerobic degradation pathway of hydrocarbons which involves an oxidative reaction via oxygenase enzymes. This initial oxidation causes the hydrocarbon to become more water soluble and introduces a new reaction point for other species (Hassanshahian and Cappello, 2013). In oil-spiked oxic microcosms, there was an increased presence of hydrocarbonoclastic bacteria, which are often described as obligate hydrocarbon degraders ( Figures 5A, B ). Alkane-degrading families, such as Alcanivorax and Flavobacterium, and PAH-degrading strains, such as Micrococcales and Oceanicola, could indicate the biodegradation of the different hydrocarbon fractions found in crude oil. In addition to the increased biodiversity, aerobic biodegradation has a lower energy input for a higher energy output when compared to anaerobic biodegradation of hydrocarbons (Olajire and Essien, 2014). These factors could play a role in the hydrocarbon degradation patterns measured by the GC-MS where the relative abundance of aliphatic and aromatic hydrocarbons were significantly reduced by day 115 ( Figures 8 , 9 ).

Anaerobic biodegradation of crude oil in the Caspian Sea is impacted by its environmental conditions: salinity, hypoxic to anoxic conditions and low temperatures. These harsher conditions may hinder the remediation potential of microbes by slowing their rate of metabolism. Colder temperatures also increase the viscosity of crude oil, making it more difficult to be solubilized and degraded (Das and Chandran, 2011; Ribicic et al., 2018a). Under these conditions, the anaerobic biodegradation of hydrocarbons may be incomplete, resulting in the accumulation of daughter products or intermediates (Parisi et al., 2009; Meckenstock et al., 2015). The complexity of anaerobic biodegradation is especially highlighted with PAHs. Their low solubility and stable ring structures make these compounds less bioavailable (Boll and Estelmann, 2018). Unlike aerobic biodegradation, which relies on the presence of oxygen as a terminal electron acceptor, anaerobic biodegradation uses alternative electron acceptors such as nitrate or sulfate ( Figure 3 ). Taxa related to sulfate reduction (Desulfocapsaceae, Desulfovibri) and nitrogen cycling (Nitrosococcales) were found in hypoxic microcosms spiked with crude oil ( Figure 6B ). These bacteria are possibly coupling these metabolic processes while using carbon as an energy source, indicating that anaerobic biodegradation may be occurring. Sulfate-reducing bacteria can degrade a wide range of substrates including alkanes, benzene, and PAHs (Mahmoudi et al., 2015). Our findings are consistent with other studies at the lower depths of the Caspian who also reported the dominance of Gammaproteobacteria in addition to the presence of sulfate-reducing taxa and ammonia-oxidizers (Mahmoudi et al., 2015; Hazen and Techtmann, 2018). Psychrophilic and oil-degrading taxa, such as Colwellia and Cycloclasticus, were primarily found in hypoxic microcosms which were kept at low temperatures ( Figure 6B ). The presence of hydrocarbon-degrading taxa after oil amendment should signal that degradation is occurring. Figure 8 shows evidence of the decrease in relative abundance of aliphatic hydrocarbons. The dampening of peaks within the GC-MS spectra and an increase in the unresolved complex mixture (UCM) in Supplementary Figure 2 might also indicate biodegradation of the oil.

Although we identified the presence of PAH-degrading taxa, reduced biodiversity coupled with the complexities of anaerobic biodegradation of PAHs may contribute to the inconclusive trend noticed in Figure 9 . The increase in the relative concentration of naphthalene, its alkylated homologs, and cycloalkane intermediates suggests the possible occurrence of biotransformation of crude oil rather than complete degradation within these microcosms (Meckenstock et al., 2004). Incomplete biodegradation of aromatic hydrocarbons could occur under these hypoxic conditions leading to the production of daughter products or dead-end metabolites which can inhibit biodegradation (Nzila, 2018). These products typically have similar structures to their parent compound causing difficulties during separation and identification using the GC-MS (Annweiler et al., 2002; Marshall and Rodgers, 2008; Nzila, 2018). These results are contrary to a previous study carried out by previous members of this group. Miller et al. (2019) found that rates of anaerobic biodegradation exceeded those of aerobic biodegradation in Caspian seawater microcosms amended with oil and a dispersant over a time period of seventeen (17) days. Dispersants can enhance the biodegradation of crude oil by reducing the surface tension to cause the formation of smaller oil droplets. These smaller droplets provide a larger surface area for microbial interaction (National Academies of Sciences and Medicine, 2020). The “bottle effect” due to long travel and holding times during the global pandemic may have also played a role in these differing results. However, this warrants further study into the mechanisms behind anaerobic degradation within this environment.

Indicator species can be used as ecological indicators of habitat types or environmental conditions due to their niche preferences. They are usually determined by analyzing the relationship between the observed species (based on abundance data) in a set of sampled sites and a user-defined classification of those sites. (De Cáceres et al., 2010). We wanted to further elucidate the microbial response to our oxygen conditions in the presence of crude oil. The identification of halotolerant species in both oxic and hypoxic indicates the adaptation of the indigenous microbes to the brackish conditions within the Caspian. Oxic microcosms ( Table 1 ) show the presence of aerobic bacteria previously correlated to hydrocarbon degradation. Hypoxic microcosms ( Table 2 ) were more correlated with the presence of mixotrophs and psychrophilic taxa which could indicate possible microaerophilic conditions in these microcosms. Taxa involved in sulfate cycling were also more correlated with hypoxic microcosms, potentially highlighting a metabolic strategy for hydrocarbon degradation via an alternate terminal electron ( Figure 3 ). Notably, our indicator species analysis also identified an ASV associated with the unclassified Gammaproteobacteria (stat = 0.885) that dominated hypoxic microcosms.

Microcosms showed greater similarity to each other based on oxygen concentration as opposed to hydrocarbon amendment. The variation in metabolic strategies needed to degrade oil in oxic and hypoxic conditions could account for these differences. The microbes within assembled microcosms showed some adaptation to their simulated environmental conditions and displayed degradation of hydrocarbon fractions found within crude oil. Distinct communities were present in surface waters and deeper waters of the Caspian Sea. These communities have been influenced by the continuous presence of oil in the water body and contain a mix of different microorganisms that have been shown to degrade hydrocarbons themselves or have been found in other hydrocarbon-enriched environments. The structure of these communities changed over time after the crude oil amendment, which was indicative of a pattern of bloom and succession typically noticed in places that have experienced an oil spill. Metagenomic analysis of these microcosms is pending to further elucidate the possible functional role of the identified species to crude oil amendment in differing oxygen conditions.

The original contributions presented in the study are included in the article/ Supplementary Materials , further inquiries can be directed to the corresponding author/s.

ZG: Formal analysis, Investigation, Methodology, Visualization, Writing – original draft, Writing – review & editing. AP: Data curation, Software, Writing – review & editing. JM: Methodology, Resources, Conceptualization, Software, Writing – review & editing. MFC: Formal analysis, Investigation, Resources, Conceptualization, Writing – review & editing. DJ: Funding acquisition, Project administration, Resources, Writing – review & editing. OP: Funding acquisition, Project administration, Resources, Writing – review & editing. NG: Investigation, Project administration, Resources, Supervision, Writing – review & editing. MC: Formal analysis, Investigation, Methodology, Writing – review & editing. PG: Formal analysis, Investigation, Resources, Writing – review & editing. TCH: Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Visualization, Writing – original draft, Writing – review & editing.