Microbial community evolution during the aerobic

biodegradation of petroleum hydrocarbons in marine sediment

microcosms: Effect of biostimulation and seasonal variations

Hamdan Z. Hamdana, Darine A. Salama*

aDepartment of Civil and Environmental Engineering, Maroun Semaan Faculty of Engineering and Architecture,

American University of Beirut, Beirut, Lebanon.

*Corresponding Author: Darine A. Salam. American University of Beirut, Maroun Semaan Faculty of Engineering

and Architecture, Munib and Angela Masri Bldg, M418. P.O.Box: 11-0236, Riad El Solh 1107 2020. Beirut,

Lebanon. Email: ds40@aub.edu.lb, Phone: +961-1-350000-Ext: 3609, Fax: +961-1-744462

Pages: 9

Figures: 1

1. Dynamics of mostly active bacterial genera

Figure S1 represents the temporal microbial dynamics of the genera as they changed throughout the biodegradation experiments.

Gammaproteobacteria

In the wet season experiments, Glaciecola was prevailing up to day 11 in the nutrient enriched microcosms and under natural attenuation conditions. With and initial relative abundance of 2.9%, it increased rapidly to reach a maximum of 41% and 35.6% after 4 days, then gradually decreased to 14.9% and 3.3% after 42 days in BS and NA treatments, respectively. Glaciecola was reported to be involved in the biodegradation of crude oil hydrocarbons in marine environments, namely at colder temperatures, with not much more details being provided in the literature (Tremblay et al., 2017). The high occurrence of Glaciecola in the wet season microcosms indicates its major biodegradation role under the wet season experimental conditions, namely under the relatively lower incubation temperature. Glaciecola was also

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Figure S1. Microbial genera evolution

prominent in the dry season experiments with a higher relative abundance being observed in the BS treatment. Glaciecola decreased slowly from an original background of 14.8% to 1.8% and 2.5% in the BS and NA treatments, respectively.

Marinobacter was also observed at a relatively high abundance in the dry season experiments and was more significant under biostimulation conditions, increasing from 2.6% to a maximum of 15.4% and 13.3% after 4 days, then decreasing gradually to 10.4% and 10.3% after 42 days in BS and NA treatments, respectively. Marinobacter fluctuated in both treatments in the wet season experiments between 4.8 and 8.0% throughout the biodegradation experiments, and between 7% and 15.4% in the case of the dry season experiments. Marinobacter, a common and widely spread marine bacterial genus, is known for hosting efficient degraders of aliphatic and aromatic hydrocarbons (Duran, 2010; Handley and Lloyd, 2013). This explains such enrichment of this genus in both the dry and the wet season experiments.

Alteromonas was also a highly represented genus of Gammaproteobacteria. In the wet season experiments, Alteromonas increased initially from 5.0% to reach a maximum of 17.4% and 15.5% after 4 days in the BS and NA treatments, respectively. The relative high abundance of Alteromonas was maintained up to day 11 after which, it decreased to reach 5.7% and 5.5% after 21 days, in the BS and NA respective treatments. After 42 days, Alteromonas further decreased to 0.9% in the NA treatment and was maintained at 5.6% under biostimulation conditions. In the dry season experiments, Alteromonas, which composed 5.7% of the original microbial community, decreased rapidly during the biodegradation experiments to less than 2% under both BS and NA conditions, and reached less than 0.4% after 42 days. Alteromonas is a widely distributed aerobic bacterium in oil polluted marine sediments and was reported for its capability of degrading oil hydrocarbons, namely PAHs (Math et al., 2012). Jin et al. (2012) reported Alteromonas as a key agent for biodegradation of PAHs on contaminated shoreline sediments. The observed increase in the relative abundance of Alteromonas in the wet season experiment compared to the diminishing percentage in the dry season experiment as the biodegradation proceeded, could indicate a possible temperature related effect, facilitating the proliferation of this microbe at lower temperatures in oil contaminated sediment.

Furthermore, as the relative abundance of Glaciecola was decreasing during the wet season experiments, another member of Gammaproteobacteria, Alcanivorax, was taking over. This genus which had initially a negligible relative abundance during the first 8 days of the experiments, increased dramatically to reach a maximum of 30.6% and 20.8% after 21 days in BS and NA treatments, then decreased to 5.3% and 20.5% in both treatments, respectively, after 42 days. Alcanivorax is well known for its capability of utilizing a wide range of alkanes as a carbon source in marine environments and is an important factor in marine oil biodegradation of alkanes (Barbato et al., 2016; Naether et al., 2013). The late involvement of Alcanicorax in the bioremediation process indicates its role in the biodegradation of more persistent compounds; as the easily degradable hydrocarbons were consumed from the sediments, Alcanivorax emerged as a highly active bacterial genus during the later stages of biodegradation of more persistent hydrocarbons. In the dry season experiments, Alcanivorax was the major member representing the class Gammaproteobacteria, being more noticeable in the NA treatment. Alcanivorax

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increased from negligible levels in the background community to reach a maximum of 27.8% in the NA at day 8, decreasing then to 3.4% after 42 days. Under BS conditions, Alcanivorax reached its maximum of 7.2% at day 4, decreasing thereafter to 1.5% after 42 days. Thus, in the dry season experiments, Alcanivorax could have acted as generalist hydrocarbonoclastic bacteria being more involved in the removal of easily biodegradable hydrocarbons during the early stages of the experiments. The enrichment of Alcanivorax at different stages of the biodegradation experiments conducted at 18 °C (wet season) and 28 °C (dry season), being more active at earlier stages of incubation at 18 °C and at later stages in the higher temperature (28 °C) experiments, could suggest possible temperature correlated effects, or could be due to the specific characteristics of the prevailing microbial community in each case, where other competing genera within the sediments could have impacted their abundance differently.

Pseudospirillum increased dramatically from negligible levels at day 21 to reach 16.3 and 9.2% at the end of the wet season experiments under BS and NA conditions, respectively. There is no clear description of Pseudospirillum in the literature; however, it is suspected to be involved in oil degradation as a part of the total community given its enrichment in oil contaminated coastal water as reported by (Doyle et al., 2018). Such a late spike in its relative abundance, especially towards the end of the experiments, indicates a possible involvement in the biodegradation of highly persistent petroleum hydrocarbons and their intermediate biodegradation products. Pseudospirillum was negligible at all times during the dry season experiments.

Psychrosphaera was also representative, varying between 0.5% and 4.5% during the wet season experiment, and 0.2% and 7% during the dry season experiments. Information about Psychrosphaera is very limited (Lee et al., 2014).

Pseudomonas was highly enriched in the NA compared to the BS in the dry season experiments, increasing form an original relative abundance of 4.7% to a maximum of 17% in the NA after 11 days, then decreasing to 4.3% after 42 days. In the BS, Pseudomonas reached its maximum of 9% after 8 days, decreasing then to 2.1% after 42 days. Pseudomonas fluctuated between 3.8% and 8% throughout the wet season experiments. Pseudomonas is reported for having members capable of utilizing PAHs as their carbon source, and thus are considered important natural degraders of PAHs (Dasgupta et al., 2013; Kumari et al., 2016; Mangwani et al., 2015). Pseudomonas enrichment during the early stages of the experiments indicates a more active involvement in removal of easily biodegradable hydrocarbons.

In the dry season experiments, Kangiella was noticed mainly under BS conditions and increased from 1.2% to a maximum of 13% at day 11 compared to 8.9% under NA at day 8, decreasing thereafter slightly to 9.3% and 1.9% in these respective treatments after 42 days. Kangiella fluctuated between 0.4% and 5.1 % during the wet season experiments. Kangiella has been isolated previously from various environmental settings such as marine sand and coastal regions, with no clear description of its involvement in hydrocarbon biodegradation. Several identified strains of Kangiella were observed to grow optimally at higher temperatures in the range of 30 °C to 37 °C (J. Wang et al., 2018). Additionally, although Kangiella is not yet reported to be involved in degradation of petroleum hydrocarbons, it belongs to the order Oceanospirillales that

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contains families known for being associated with oil biodegradation following oil spills (J. Wang et al., 2018).

Idiomarina was enriched in both treatments in the first stages of the experiments, increasing from 2.9% to 5.4% and 5.9% under BS and NA conditions, respectively, at day 4, then decreasing gradually to less than 1% after 42 days in the dry season experiments. Idiomarina was negligible at all times during the wet season experiments. Having this genus enriched in the dry season experiments thus suggests a strong possible involvement in biodegradation of oil hydrocarbons in warmer settings.

Methylophaga was negligible until day 21 and thrived towards the end of the dry season experiments at day 42 to reached 3.1% and 7.3% in the BS and NA treatments, respectively. Methylophaga was negligible throughout the wet season experiments. Methylophaga are strictly aerobic marine bacteria with exclusive requirements of various C1 carbon sources, with various studies reporting enrichment of Methylophga in oil contaminated environmental sources and laboratory experiments; however, a clear evidence for their involvement in hydrocarbon degradation remains lacking (Kostka et al., 2014). The rapid increase of Methylophaga at later stages of the experiments, indicates its involvement in the degradation of small carbon chains metabolites, namely C1, resulting from earlier degradation stages of larger hydrocarbons by other bacteria (Gutierrez and Aitken, 2014). This stresses the importance of successive enrichment of various microbial genera in hydrocarbon bioremediation studies for complete mineralization of the pollutants, with certain genera consuming the metabolites produced through the biodegradation activity of other groups until complete biodegradation of the original compounds is achieved.

Alphaproteobacteria:

Alphaproteobacteria was mainly represented in the dry season experiments by Litorimonas, Erythrobacter, Altererythrobacter, Algimonas, Maritimibacter, Primorskyibacter and Tropicibacter.

Litorimonas was reported to be enriched as a part of the microbial community involved in the biodegradation of oil in seawater, with no specific correlation of its role in the biodegradation process (Brakstad et al., 2018). A member of Erythrobacter was reported in a previous study to be involved in PAHs biodegradation and was isolated from deep sea sediments (Zhuang et al., 2015). Altererythrobacter are known for degrading recalcitrant hydrocarbons (Maeda et al., 2018). Algimonas is an aerobic bacterium that is poorly described in the literature (Zhang et al., 2015). Maritimibacter is reported for its capability of growing using hexadecane as a sole carbon source (Prince et al., 2018). Members of Primorskyibacter are reported to be novel aerobic bacteria that are isolated from coastal shallow sediments (Romanenko et al., 2011; N.-N. Wang et al., 2018). Species belonging to Tropicibacter, isolated from seawater, were previously reported to be capable of degrading PAHs as well as alkylated PAHs (Harwati et al., 2009).

In the dry season experiments, Litorimonas increased as the biodegradation proceeded, from less than 0.1% at the start, to significant enrichment observed after day 21, to reach a maximum of 16.1% and 10.2% in BS and NA treatments, respectively, after 42 days. Erythrobacter also

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increased from 0.3% to 3.3 and 2% in BS and NA treatments, respectively, after 42 days. The other genera fluctuated between less than 1% and 7% throughout the experiments, with no clear trend in their relative abundances.

In the wet season experiments, Alphaproteobacteria was represented mainly by the members Sulfitobacter and Altererythrobacter. Sulfitobacter increased slightly from 0.9% to reach 3.3% and 2.5% after 42 days in the BS and NA treatments, respectively. Altererythrobacter increased from 0.05% to 2.7 and 1.7% in these respective treatments towards the end of the experiments. Members belonging to Sulfitobacter are reported to be involved in oil biodegradation (Marietou et al., 2018). Various species under the Altererythrobacter genus were isolated from marine environments, including sediments, and are reported for their capability of degrading recalcitrant petroleum hydrocarbons such as alkanes and PAHs (Maeda et al., 2018). The increase in this genus towards the end of experiments is mostly correlated with the degradation of the persistent hydrocarbons at later stages of the biodegradation process.

The results observed for the class Alphaproteobacteria indicates the involvement of this class in the degradation of more persistent compounds, with members belonging to this class being more specialized rather than being generalist hydrocarbon degraders.

Actinobacteria:

Actinobacteria was mainly represented at the genus level by Arthrobacter in the wet season experiments, and Arthrobacter and Gordonia in the dry season experiments. In the case of the wet season experiments, the relative abundance of Arthrobacter increased from 1% to reach a maximum of 4.8% and 8.3% at day 8 under BS and NA conditions, respectively, and decreased to less than 0.8% in both treatments after 42 days. Arthrobacter is well known for its capability of degrading a wide range of environmental organic pollutants such as alkanes and PAHs (Ren et al., 2018), and its enrichment during the earlier stages of the experiments indicates its primarily involvement in the removal of the easily biodegradable components of the crude oil.

In the dry season experiments, Arthrobacter composed a major portion of the background microbial community (25.7%), and decreased slightly after 4 days. Its relative abundance fluctuated later between 3.1% and 10% in both BS and NA treatments, with no clear observed trend. On the other hand, Gordonia, which was more prominent in the NA treatment, was mostly noticeable at the early (day 4) and late (day 42) stages of the experiments, with measured relative abundances of 1.5% and 4.5% at day 4, and 2.4% and 3.6% at day 42 the in BS and NA treatments, respectively. Gordonia is known for the ability of degrading n-alkanes (Kim et al., 2018; Quatrini et al., 2007).

Flavobacteria:

Genera belonging to Flavobacteria were negligible at all times during the dry season experiments, while Dokdonia and Winogradskyella being noticeable in the wet seasons experiment. Dokdonia was mainly observed at day 21, reaching 2.25% and 3.9% in the BS and NA treatments, respectively. Winogradskyella, on the other hand, fluctuated between 0.2% and 3.3% in both treatments throughout the experiments. No information about the involvement of

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Dokdonia in oil biodegradation was found. Winogradskyella was reported to dominate during oil degradation in polluted seawater (Wang et al., 2014).

Deltaproteobacteria

Deltaproteobacteria represented 5.6% and 2.9% of the background microbial communities in the wet and dry season experiments, respectively, decreasing to negligible levels after day 0. The only detected genus under this class, Anaeromyxobacter, represented less than 0.3% of the total microbial community after initialization of the experiments, indicating insignificant involvement of this class in oil biodegradation.

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