Bioremediation of Soils Contaminated with Petroleum Solid Wastes and Drill Cuttings by Pleurotus Sp

Home , Drill cuttings

bioRxiv preprint doi: https://doi.org/10.1101/588673; this version posted March 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Bioremediation of soils contaminated with petroleum solid wastes and drill cuttings by Pleurotus sp. strains under diﬀerent treatment scales

Roberto Romero-Silvaa, Ayixon Sánchez-Reyesb,∗, Yuletsis Díaz-Rodrígueza, Ramón Alberto Batista-Garcíac, Danai Hernández-Hernándeza, Judith Tabullo de Roblesd

a Centro de Investigación del Petróleo, Churruca 481, El Cerro, La Habana, Cuba b Cátedras Conacyt, Instituto de Biotecnología, Av. Universidad 2001,Cuernavaca, Morelos, 62210, Morelos, México. Tel.: +527771031529 cCentro de Investigación en Dinámica Celular, Instituto de Investigaciones en Ciencias Básicas y Aplicadas, Universidad Autónoma del Estado de Morelos (UAEM), Ave. Universidad 1001, Col. Chamilpa, Cuernavaca, 62209, Morelos, México d Centro de Investigación en Biotecnología, Universidad Autónoma del Estado de Morelos, Av. Universidad 1001, Colonia Chamilpa, Cuernavaca, 62210, Morelos, México

Abstract Wastes from the oil industry represent one of the sources of soil pollution with the greatest environmental impact. Both drill cuttings and crude residues are delivered to the soil and produce severe toxic effects, mainly due to the presence of polycyclic aromatic hydrocarbons. Various bioremediation technologies have been implemented in order to restore the soil quality and the natural auto depuration capabilities, amongst them: composting, bioaugmentation and biostimulation. All of these bioremediation techniques promise to be eco-friendlier and cheaper alternatives than other approaches. In this work we have evaluated several strains of Pleurotus sp. for their effect on the bioremediation of oil- contaminated wastes and drill cuttings disposed in storage tanks or in open-air soil lots for many years. Our results suggest that combined natural attenuation mechanism and directed fungal biodegradation activities, could be promising strategies to remediate heavily petroleum polluted soils and drilling wastes both at the laboratory and in field conditions. Furthermore, we present new data that supporting Pleurotus genera as able to degrade asphaltenes, the most recalcitrant fraction of petroleum. This study proposes an approach that at the same time can treat soils contaminated with waste from drill cuttings and bottoms of crude storage tanks. Keywords: bioremediation, Pleurotus, drilling cuttings, petroleum wastes, natural attenuation

1. Introduction

∗ Corresponding author. The exploitation of petroleum resources is one Email addresses: [email protected] (Roberto Romero-Silva), of the anthropogenic activities with the great- [email protected] (Ayixon est environmental impact on aquatic and terres- Sánchez-Reyes), [email protected] (Yuletsis trial ecosystems. Cleaning of crude oil storage Díaz-Rodríguez), [email protected] (Ramón Alberto tanks and drilling operations, among other ac- Batista-García), [email protected] (Danai tivities, produce petroleum wastes that are ac- Hernández-Hernández), [email protected] (Judith Tabullo de Robles) cumulated in open-air lots, negatively impact- ing the soil quality. Petroleum-contaminated drill cuttings regularly contain a mix of wa-

March 24, 2019 bioRxiv preprint doi: https://doi.org/10.1101/588673; this version posted March 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

ter, oil or synthetic-based fluids with significant strains, as part of the environmental management amounts of petroleum hydrocarbons. The hy- and final disposal of pollutants from the Cuban drocarbon content includes a wide range of sat- oil industry (Romero et al. 2011). The effect urated and high-molecular-weight aromatic com- of four strains of Pleurotus sp. has been eval- pounds (Broni-Bediako and Amorin 2010). Crude uated on the bioremediation of oil-contaminated oil and drill cuttings are toxic, mainly due to soils and drill cuttings wastes disposed in storage polycyclic aromatic hydrocarbons (PAH) present tanks or in open-air soil lots for years, and three in the muds (5-10%) (Petri et al. 2015). There scales were implemented for their study at lab- are several physicochemical methods for drill cut- oratory, microcosm and field levels. Our results tings treatment: cuttings re-injection, microwave showed that several fungal strains are capable to drying, and thermal desorption. Unfortunately, reduce asphaltenes and resins content in the sam- all of these methods have serious technical and ples tested, a task that only a few microorganisms environmental challenges, such as expensive im- can do. plementation, high energy demand, risk of acci- dental releases to the environment and high level 2. Materials and Methods of pollutant residues. Many biological processes have been investigated in order to overcome these Fungi isolation, culture and identification drawbacks, e.g. composting (Ahmadi et al. 2016), Four Pleurotus sp. fungal strains with abun- bioaugmentation (Rezaei Somee et al. 2018), or dant fruiting body, oyster-shaped and white or biostimulation (Kogbara et al. 2016). All of these pale yellow in color were isolated from different bioremediation techniques, promise to be better decaying woods pieces collected in May 2010, from alternatives, eco-friendly and moneywise, than a Zoological park in Habana, Cuba. The location ◦ ◦ physicochemical approaches. Although bioaug- coordinates were 23 06’40.6"N 82 23’48.8"W. In mentation (the practice of adding cultured mi- order to obtain axenic cultures, pieces of the fruit- croorganisms for biodegrading soil or water con- ing body were inoculated in Petri dishes with taminants), has been applied successfully in sev- Sabouraud Dextrose Agar (BioCen Cat. 4018) eral studies of petroleum hydrocarbons biodegra- and 25 µg/µL of streptomycin, at room temper- ◦ dation by bacteria, there are few reports of the ature (26 ± 2 C) for seven days. Agar plugs use of processes that combine degradative activ- with pure mycelium were passed to new dishes ity of indigenous bacteria and the bioaugmen- and grown for another seven days. Besides the tation with fungal specimens in soils contami- evidence offered by the fructification bodies, the nated with oil-based drill cuttings. Furthermore, isolated strains showed morphological features fungi gather valuable metabolic characteristics as (mycelia growth pattern and conidial morphol- they are natural xenobiotic degraders and in fact, ogy) consistent with the genus Pleurotus. The many species have been isolated from oil-polluted strains were coded as B-1, B-7, B-10, B-15 and sources (Dantán et al. 2008; Marchand et al. pure cultures were conserved in the Chemistry 2017). On the other hand, they contribute with and Biotechnology Laboratory of the Petroleum overall geomicrobial activities that play key roles Research Center, Cuba. in the cycling of organic matter and diagenesis Solid State Fermentation (SEF). Laboratory (Ehrlich 2006). Their ability to be massively scale assay cultured on industrial wastes with conventional Three grams of sugarcane bagasse (particle microbiological techniques, makes fungi attrac- size <1.6 mm) and 17 grams of soil contaminated tive candidates for use in soil bioremediation pro- with petroleum solid wastes were added to conical cesses. flasks of 100 mL capacity. They were sterilized for ◦ This paper describes a comprehensive analy- 15 min at 121 C. A solution of ammonium sul- sis in order to assess the applicability for large- fate (0.25%) sterilized independently was used to scale bioremediation processes of several fungal moisten each flask (32 mL). As inoculant we used March 24, 2019 bioRxiv preprint doi: https://doi.org/10.1101/588673; this version posted March 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

two agar slants (5 mm in diameter) extracted from of the inoculum were authorized by the Biosecu- the active edge of colony growth of the four Pleu- rity Licenses CH 17-P (78) 10 and CH17-P (81) rotus strains. The fermentation lasted 15 days in 10 granted by the National Center for Biological static condition at room temperature (26 ± 2◦C). Safety of Cuba (CSB). The analytical monitor- Five replicates were used for each treatment, in- ing of the bioremediation process was assessed by cluding an abiotic control (without fungal treat- extracting a subset of field topsoil samples (~10 ment). grams of soil) for chemical and microbiological Hydrocarbon Degradation in Microcosms analysis. The following variables were recorded The microcosm assay was carried out in com- at 0, 30 and 70 days: total petroleum hydrocar- posters (diameter 35 cm, depth 18 cm) with ster- bons (TPH) (APHA 1998), oils and grease (EPA ilized petroleum solid wastes (1 kg), sugarcane 1999), saturated, aromatic, resins and asphaltenic bagasse (16 g) and then brought up to 60% of components (SARA) (García-Rivero et al. 2007; moisture with ammonium sulfate solution (0.25%) Drews A 2008), total nitrogen (ISO 1995), to- sterilized independently. The composters were in- tal phosphorus (APHA 1998), total microorgan- oculated with circular sections of mycelia (~ 20 ism count, hydrocarbon-degrading microorgan- cm2) from the four Pleurotus strains already de- isms (Margesin et al. 2003). scribed, with 4 fragments for each composter. The Biodegradation analysis incubation period lasted 30 days at room temper- The rates of biodegradation expressed as a ature (26 ± 2◦C) on static regime, with periods (6 percentage were determined by monitoring the hours) of sunlight in order to simulate field con- reduction of petroleum components for all sam- ditions. The microcosms were covered with alu- ples at the initial and final stages of each exper- minum foil and the soil was moistened by the ad- iment. Uninoculated controls supplemented with dition of 250 mL sterile distilled water every week contaminated soils samples were always consid- until the end of the experiment. ered. The following equation was used for calcu- Bioaugmentation of parcels contaminated with lations: (Ci−Cf) petroleum solid wastes and drill cuttings using a Biodegradation rate (%) = Ci × 100 Pleurotus sp. strain Where Ci indicates initial concentration; Cf Two parcels (15x15x0.30 m) from a long-time indicates final concentration contaminated (~10 years) region with petroleum Statistical analysis solid wastes and drill cuttings, in La Cantera Bi- For each experiment a completely randomized rama, located on Matanzas province, Cuba; were design was carried out. The Kolmogorov-Smirnov selected for a field-based bioremediation experi- test was used to check the normality premise. ment. First, the soil was manually conditioned The homogeneity of variance was verified by the by removing the surface layer. The field contained Chochran- Hartley and Bartlett test. The Dun- aged hydrocarbon residues and a significant rock can test was used for “a posteriori” means com- content. Then, the parcels were biostimulated by parison. The ANOVA, and two-way joining clus- adding sawdust according to Díaz et al. (2013) tering analysis were assessed using the statistical and moistened with a natural surfactant solu- package Statistica 7.0 StatSoft, Inc. (2004). Ad- tion obtained as a byproduct of the processing ditional data are given in Supplementary material of henequen fibers (Agave fourcroydes). Subse- 1. quently, one parcel was bioaugmented with a mix- ture of spores and mycelium -in Sabouraud liquid 3. Conclusions medium- from Pleurotus sp. B-7 strain (10E+06 spores. mL−1) and the other parcel was left in- One of the drawbacks in bioremediation lies in tact, as a natural attenuation control. No phys- achieving comparable results in laboratory as in ical barrier was established between the parcels. the field. Although selected and adapted micro- The specifications for transportation and release bial strains are useful in hydrocarbon biodegra- March 24, 2019 bioRxiv preprint doi: https://doi.org/10.1101/588673; this version posted March 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

dation, not all fractions are degraded in similar 46.34% by B15 strain) fractions were degraded by extension, the combination of different strategies all strains. However, the aromatic fraction was becomes necessary in order to significantly re- highly elusive to biodegradation, with just 9.2% duce contaminant levels under realistic field con- achieved by B-7 strain. Remarkably, we observed ditions. Our results point toward a combined use asphaltenes biodegradation in the experiment, be- of bioaugmentation and natural attenuation with ing B-7 the strain with the higher biodegradation the purpose of obtaining improved outcomes in rate (46.45%) followed by B-10 strain with 21. bioremediation processes. Currently, we are car- 6%. rying out studies on this site to understand how There is very little evidence of fungi that autochthonous microbial communities interact in can degrade asphalts and resins; to date, some the hydrocarbon-degradation processes, through examples have been described in detail e.g., metagenomic approaches. It is worth to highlight the ascomycetes Neosartorya fischeri (Hernández- the isolation of a very efficient strain of Pleurotus López et al. 2016), Pestalotiopsis sp. (Yanto and capable of asphaltene degradation (B-7 strain), Tachibana 2013) and recently the basidiomycete since very few organisms can achieve this capac- Daedaleopsis sp. (Pourfakhraei et al. 2018). ity. Asphalthenes biodegradation rates achieved by Pleurotus sp. B-7 and B-10 are comparable to 4. Results and Discussion these former examples. Pleurotus sp. B-7 was the only strain capable to modify all fractions in Bioremediation of soils contaminated with the polluted soil, a remarkable fact considering petroleum solid wastes (Lab scale) the petroleum hydrocarbons chemical complex- In order to characterize the petroleum-based ity. For example, the resins influence the struc- hydrocarbon content in the soil, the samples were tural stability of petroleum, asphaltenes contain subjected to an initial analysis by standard labo- the highest molecular weight constituents in the ratory methods for the fractionation of petroleum crude (Andersen and Speight 2001), and aromatic (Table 1). The soil samples were rich in com- compounds are very stable and resistant to bio- pounds that primarily come from crude oil, be- logical degradation; all of these factors play a ma- ing the resin, and the oils and grease, the major jor role preventing biodegradation processes by 4 −1 fractions with a content of 10 mg.kg in av- microorganisms therefore, finding microbes capa- erage. The total petroleum hydrocarbons, satu- ble to degradation of these fractions is valuable rated, aromatics and asphaltenic components os- for the development of effective bioremediation 3 −1 cillated in the 10 mg.kg order. This confirmed strategies. These findings are not uncommon, that the soil samples were composed almost exclu- for several studies support the role of Pleurotus sively by complex mixtures of hydrocarbons de- for bioremediation of petroleum hydrocarbon con- rived from crude oil. taminants in soil (Harms et al. 2011; Sukor et al. After 15 days of treatment with four strains 2012; Mohammadi et al. 2017). The degrada- of the basidiomycete Pleurotus sp. (as described tive activity is often associated with the ability in MM) we observed that all of the strains tested of fungi to produce extracellular hydrolases and decreased the content of several of the oil frac- oxidoreductases (Varjani and Upasani 2017). We tions contained in the polluted soil, in contrast to conclude that Pleurotus sp. B7 is a good candi- the abiotic control for which no significant change date for deeper studies in bioremediation process. was detected. The highest degradation rates were Microcosm bioremediation of soils contami- reached for saturated hydrocarbons (36%, 41.6%, nated with petroleum solid wastes 54.4% and 53.56% for the B1; B7; B10 and B15 In order to scale up the laboratory soil strains, respectively) (Figure 1). The resins (max- biodegradation experiment, we carried out a imum biodegradation of 40.5% by B10 strain), microcosm soil biodegradation study for 30 days. and oils and grease (maximum biodegradation of The treatments assessed were: a non-sterile nat- March 24, 2019 bioRxiv preprint doi: https://doi.org/10.1101/588673; this version posted March 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Table 1: Initial characterization of soils contaminated with petroleum solid wastes Oils and TPH Saturated Aromatics Resins Asphaltenic Treatments grease (mg.kg −1) (mg.kg −1) (mg.kg −1) (mg.kg −1) components (mg.kg (mg.kg −1) −1)

Abiotic con- 3.62E+04 8.74E+03 4.81E+03 4.19E+03 2.69E+04 5.75E+03 trol Pleurotus sp. 3.92E+05 6.55E+04 1.00E+04 5.40E+05 1.09E+05 8.87E+04 B-1 Pleurotus sp. 2.34E+05 3.70E+04 9.53E+03 4.72E+04 7.91E+04 1.81E+04 B-7 Pleurotus sp. 2.15E+05 5.19E+04 5.17E+03 5.07E+05 4.63E+04 4.20E+04 B-10 Pleurotus sp. 9.16E+04 1.11E+05 3.99E+03 6.91E+05 2.11E+05 1.14E+05 B-15

Figure 1: Biodegradation rates (%) for soil samples contaminated with petroleum solid wastes during 15 days of treatment with four strains of Pleurotus sp. (n=5)

ural attenuation control (to supervise natural compared to natural attenuation in the polluted ability for intrinsic soil remediation in bios- soil. Furthermore, B-7 strain excels once again as timulation conditions); Pleurotus sp. strains a compelling candidate to perform escalated soil in contaminated soil as described in MM; and bioremediation studies in order to overcome nat- appropriate sterilized controls to assess abiotic ural attenuation shortcomings. degradation. The natural attenuation treatment Bioaugmentation of a field soil contaminated achieved significant biodegradation for all frac- with petroleum solid wastes and drill cuttings with tions in the polluted soil (maximum biodegrada- Pleurotus sp. B-7 strain tion for saturated hydrocarbons 30.6%), with the Pleurotus sp. B-7 was selected as study model exception of asphaltenes for which no biodegra- for a real field scale bioaugmentation experiment, dation was detected, reinforcing the fact that since it was the strain with better biodegrada- native microorganisms are rarely capable of as- tion performance in both the laboratory and in phaltene degradation (Figure 2). Biodegradation the microcosm experiments. Before the bioaug- by natural attenuation is not unusual, due to the mentation treatment, the soil was conditioned as presence of indigenous degrader microorganisms described in MM; a total of 10 L of the fungal in soil samples with potential effect in intrinsic inoculum was spread on the polluted soil surface. bioremediation (Lee et al. 2018). However, an The selected soils had relatively high concentra- effect of biostimulation should not be discarded tions of petroleum solid wastes. At the beginning since in real field conditions the nutritional status of experiment, the contaminant levels and hydro- can be that of oligotrophy. carbon degrading microorganism count were quite Pleurotus sp. B-1 could not degrade asphalts similar for control and treated parcel according to nor resins in the microcosm in contrast to our F test (p-value > 0.05), suggesting a similar po- laboratory experiment, which showed a modest tential for natural attenuation in both sites (Fig- behavior at this respect (11-23%). Pleurotus sp. ure 3A). B-7 and B-15 degrade resins fraction consider- Regardless of the treatment, the main effects ably more than any other treatment, and the were observed after 70 days in terms of reduc- aromatics compounds were degraded by all fun- ing pollutants. This was confirmed by the pres- gal strains, contrasting with the laboratory ex- ence of only two statistically-different homoge- periment. Pleurotus sp. B-10 and B-15 de- neous groups (days 0 and 70) (Figure 4), clus- grade aromatic fractions in similar extension ~ tered respect to the temporal dynamics of degra- 53.4-59.9% and significantly more than any other dation [Oils and grease Duncan test P-value: 0.00; treatment. Maybe the aromatics fractions be- TPH Duncan test P-value: 0.01; Saturated hy- come more biodegradable when longer degrada- drocarbons Duncan test P-value: 0.03); Resins tion times are applied to the process. Pleurotus Duncan test P-value: 0.01; Asphaltenic compo- sp. B-7 was the only one that degraded all frac- nents Duncan test P-value: 0.01 (α: 0.05)] Sup- tions, even asphalts (40.6%), which are elusive to plementary material 1. Interestingly, at 70 days of degradation by other treatments. The concentra- treatment the Resins, Saturated and Aromatics- tion of contaminants for the abiotic control by the hydrocarbons reached accepted levels according end of the assessment showed no significant differ- to National Standardization Body of the Repub- ences with the initial concentrations (in average lic of Cuba (NC 819: 2017; NC 1263: 2018) (≤10 4.2E+04 mg.kg−1 vs 3.9E+04 mg.kg−1; standard 000 mg. kg −1). error ~ 0.06%), suggesting that abiotic mecha- However, this period of time was not enough nisms like volatilization or dilution may not signif- to reduce Oils and grease, or Asphaltenes up to icantly contribute to degradation in these assay. standards accepted levels. Probably the high ini- These observations indicated that fungal treat- tial content of Oils and grease in treated parcels, ment improved the biodegradation of recalcitrant interfered drastically with the microbial metabolic fractions like aromatics, resins and asphaltenes processes due to its diverse and complex chemical March 24, 2019 bioRxiv preprint doi: https://doi.org/10.1101/588673; this version posted March 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

Figure 2: Biodegradation rates (%) achieved by four Pleurotus sp. strains and a natural attenuation control in soil microcosm bioremediation assay for 30 days. (n=2)

Figure 3: Microbial count, nitrogen and phosphorus levels in natural attenuation control and B-7 treated parcels. Total hydrocarbon-degrading bacteria (A), Total nitrogen (B), Total Phosphorus (C).

Figure 4: Two-Way joining graph of the oil-fractions remotion (mg.kg −1) in the field assay for the natural attenuation control and B-7 strain during 0, 30 and 70 days of treatment (left side on the heatmap). Significant temporal differences between treatments are clustered in the right side dendrogram homogeneous groups (See text for P-values details and Supplementary material 1) composition (coming from aged fatty matter, sul- in the parcel with the fungal inoculum, although fur compounds, and organic pigments) (Pawlak the contribution of indigenous microbiota is not and Rauckyte 2008). The percentage of resins re- ruled out, for example, there was a significant moval achieved by the inoculum and the control, increase in the biodegraders count with the B- amount to 56% and 27% respectively, while the 7 treatment (8.50E+04 CFU.g soil −1 at 0 day asphaltenes amounted to degrade a maximum of and 3.55E+08 CFU.g soil −1 at 70 day) (Figure 14% in B-7 treatment. These findings suggest a 3A). This increase -in four magnitude orders- may greater resins and asphaltenes biodegradation in be related with nutrient biostimulation applied to the parcel with the fungal inoculum, and confirm the treated parcel. the difficulties in degrading the asphaltenics com- In the parcel’s topsoil, the total phospho- ponents under our field conditions. Thus, longer rus concentrations were extremely low (attenua- treatments may be necessary for accomplish the tion control 0.152 mg.kg−1, B-7 treatment 0.224 oil-pollutants levels in impacted soils, required by mg.kg−1) at the beginning of the experiment (Fig- the normativity. ure 3C), maybe due to the historical character- Except for TPH, resins and asphaltenes, at 70 istics of the field, intrinsically dry, geologically days there were no differences between B-7 and young, little fertile with sparse vegetation mostly attenuation control. The percentage of resins re- composed by Gramineae, spiny crawling plants moval achieved by the inoculum and the control, like Ricinus communis. After 70 days we no- amount to 56% and 27% respectively, while the ticed a decrease of phosphorous, to almost un- asphaltenes amounted to degrade a maximum of detectable levels (~0.09 mg.kg−1). The total ni- 14% in B-7 treatment. These findings suggest trogen content in both plots also suffered a signif- a greater resins and asphaltenes biodegradation icant decrease over 70 days (Figure 3B), maybe as

a response to the increased metabolic demands of ment an asphalt bioremediation scheme with this autochthonous and aloctone populations. Phos- strain. However, these kinds of realistic field ap- phorus and nitrogen are essential nutrients re- proaches require extensive environmental moni- quired by microorganism in the synthesis of cel- toring to weigh the real effectiveness of the soil lular structures and for maintenance of adequate bioremediation process in order to meet interna- metabolic equilibria. The low levels detected in tional standards. This study is the first large- this study may be caused by a weak ability to scale approach to clean up soils contaminated release parent material rock-derived, like phos- with petroleum hydrocarbons through combined phorus, or by sequestration effects mediated by natural attenuation and bioaugmentation in the hydrophobic soil matrix contaminants (Eschen- Cuban oil industry, and confirms that long term bach et al. 2013). This suggest that biostimu- monitoring is a necessary condition in the current lation plays an important role in bioremediation efforts for bioremediation of petroleum hydrocar- so maybe it should be considered for treatments bons polluted soils. at field level by adding nutrients every 70 days (in the case of this study). Acknowledgments There are several reports suggesting Pleuro- tus genus can effectively degrade petroleum hy- We thank to Unión Cuba-Petróleo (CUPET) drocarbons in the presence of diverse indigenous for the financial support; and to Jorge Luis Folch- microflora (Hestbjerg et al. 2003; Mohammadi, Mallol (PhD) for his helpful comments. et al. 2017). In this study, we evaluated the Funding: This work was supported by the behavior of four strains of Pleurotus sp. on the Unión Cuba-Petróleo (CUPET) through project degradation of polluted soils with petroleum hy- 2416 granted to Centro de Investigación del drocarbons and drill cuttings. Our experimental Petróleo, Cuba. approximation contemplated several scales to se- Conflict of Interest: The authors declare lect the strain with the best performance in the that they have no conflict of interest. bioremediation of contaminated soil. Finally, the References selected strain (B-7) was tested in the field against Adams GO, Fufeyin PT, Okoro SE, et al a natural attenuation control in order to evaluate (2015) Bioremediation , Biostimulation and its possible use on an industrial scale to the biore- Bioaugmention : A Review. International mediation of soils impacted with wastes from the J Environ Bioremediation Biodegrad. doi: petroleum industry. Our strictly controlled lab- 10.12691/ijebb-3-1-5. oratory and microcosm studies yielded promising Ahmadi M, Dashtestani M, Takdastan A, results for biodegradation of the most of the hy- et al (2016) Data on biodegradation of total drocarbon fractions in crude oil and waste. Fur- petroleum hydrocarbons using co-composting of thermore, the field study achieved significant de- cow manure/oily drill wastes. Data Br. doi: crease of certain contaminants to acceptable lev- 10.1016/j.dib.2016.10.008 els at 70 days, according to the Cuban standards American Public Health Association (APHA) for crude oil residues and water-based drilling cut- (2005) Standard Methods for the Examination of tings treatment (<10, 000 mg.kg −1: NC 819:2017 Water and Wastewater, 21st ed.; APHA: Wash- and NC 1263: 2018). As other studies have ington, DC, USA, 2005 pointed out, the combination of several bioreme- Andersen SI, Speight JG (2001) Petroleum diation strategies could improve the hydrocarbon resins: Separation, character, and role in removal (Adams et al. 2015; Rodriguez-Campos petroleum. Pet Sci Technol. doi: 10.1081/LFT- et al. 2018). Remarkably, asphaltenic compo- 100001223 nents in the polluted soils, were consistently mod- APHA A-W (1998) Standard methods for the ified by Pleurotus B-7 in the different experiments examination of water performed, supporting the possibility to imple- March 24, 2019 bioRxiv preprint doi: https://doi.org/10.1101/588673; this version posted March 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

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