ResearchGate See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/337522935 Adsorptive bioremediation of soil highly contaminated with crude oil Article in Science of The Total Environment • November 2019 DOI: 10.1016/j.scitotenv.2019.135739 CITATIONS READS 41 82 4 authors, including: Russian Academy of Sciences Institute of physico-chemical and biological problems in soil sciences, RAS 55 PUBLICATIONS 909 CITATIONS 10 PUBLICATIONS 319 CITATIONS SEE PROFILE SEE PROFILE jose julio ortega calvo CSIC - Institute of Natural Resources and Agrobiology of Seville 121 PUBLICATIONS 4,459 CITATIONS SEE PROFILE Some of the authors of this publication are also working on these related projects: Call for Papers: Special Issue on "Nano-bioremediation Approaches for Sustainable Agriculture" in ESPR (IF 4.223) View project FUNCTIONAL SYNERGIES BETWEEN MICROORGANISMS AND PLANTS IN SUSTAINABLE REMEDIATION OF SOILS CONTAMINATED BY PAH5 View project All content following this page was uploaded by Galina K. Vasilyeva on 05 May 2022. The user has requested enhancement of the downloaded file. Science of the Total Environment 706 (2020) 135739 Contents lists available at ScienceDirect Science of the Total Environment ELSEVIER Jou rnal h omep age: w w w . e l s e v i e r . c o m / l o c a t e / s c i t o t e n v Adsorptive bioremediation of soil highly contaminated with crude oil Check for updates Galina Vasilyeva a Victoria Kondrashina a Elena Strijakova a Jose-Julio Ortega-Calvo b 'c, , , a Institute of Physicochemical and Biological Problems in Soil Science, RAS, Pushchino, Moscow region, Russia h Instituto de Recursos Naturales y Agrobiologia de Sevilla (IRNAS), C.S.I.C, Avenida Reina Mercedes, 10, E-41012 Seville, Spain HIGHLIGHTS GRAPHICAL ABSTRACT Bioremediation can be applied for soils with petroleum contamination <5% only. Adsorptive bioremediation in situ can extend possibilities of this approach. It is based on the use of a mixed adsorbent in combination with a biopreparation. The adsorbent (activated carbon + diatomite) reduces toxicity of those soils. The adsorbent may decrease leaching of toxic polar metabolites from the soils. TPH and polar metabolites TOPH, g kg-1 120 5 —6— Control 0 — Count of degrading bacteria x10 8 CFU 100 6 Adsorben tBsioo— rpbreenpt14 . 80 12 60 30 20 l ‘leactii: TOPH Q.._ 0 Phytotoxicity, % j, 16 40 20 10 _______ 0 8 6 6 12 18 0 6 12 18 Months 40 10 ARTICLE INFO 4 -0 e - -... 2 ABSTRACT Article history: Received 7 October 2019 Received in revised form 20 November 2019 Accepted 23 November 2019 Available online 25 November 2019 Editor: Damia Barcelo Keywords: Petroleum Metabolites Activated carbon Diatomite Phytotoxicity Leaching Due to the extended oil extraction and transportation in Russia and other oil-producing countries, many lands remain contaminated because of accidental spills. This situation requires the cost-effective and efficient remediation of petroleum-contaminated soils. Bioremediation of soils contaminated with high concentrations of crude oil is usually hampered by high toxicity thresholds for microbial degraders. We have performed a two-year microfield experiment on the influence of a mixed adsorbent (ACD) composed of granular activated carbon and diatomite on bioremediation of a grey forest soil contaminated with crude oil at concentrations (5-15 % w/w) that would theoretically not result in a successful pollutant removal due to toxicity. Remediation of these soils was evaluated after treating with the ACD adsorbent (from 4 to 12% w/w) and a biopreparation (BP) containing hydrocarbon-degrading bacteria, separately or in combination. Reduction of total petroleum hydrocarbons content was significantly greater in highly contaminated soils with the combined amendments than in the respective controls (through the activation of indigenous degrading microorganisms by fertilizing and mixing) by 9-10% and 5-8% at the end of the first and second years, respectively, depending on the contamination level. Significantly higher counts of petroleum-degrading microorganisms (as indigenous and introduced by the BP), as well as much less phytotoxicity was detected in the ACD-amended soils, as compared with the samples without adsorbent. In addition, the ACD mixture drastically reduced the wash-out of polar petroleum metabolites (evidently oxidized hydrocarbons) and the phytotoxicity of the lysimetric waters, especially in highly contaminated soils. The results indicate that the mixture of activated carbon and diatomite is a prospective adsorbent for the in situ bioremediation of soils highly contaminated with crude oil. © 2019 Elsevier B.V. All rights reserved. Corresponding author. E-mail address: gkvasilyeva@ramblersu (G. Vasilyeva). https://doi.org/10.1016/j.scitotenv.2019.135739 0048-9697/© 2019 Elsevier B.V. All rights reserved. 2 G. Vasilyeva et al. / Science of the Total Environment 706 (2020) 135739 1_ Introduction Environmental contamination by crude oil and petroleum products is one of the most serious problems in the Russian Federation (RF). According to the Ministry of Natural Resources, the volume of oil spills in Russia is 17-20 million tons annually, what accounts to approximately 7% of oil production in the country (Nikiforov et al., 2016). There were 3429 reported petroleum spills in the RF in 2017, mostly from commercial pipelines. As a result, 6183 ha were contaminated by 10,278 m3 of crude oil and petroleum products (State report, 2017). This situation, which represents also the global problem of other oil-producing countries (Alvarez and Illman, 2006), requires the cost-effective, efficient and less labor-intensive methods for remediation of petroleumcontaminated soils. Bioremediation is a practical and cost-effective approach to solve a wide range of problems of soil contamination. Bioremediation of petroleum-contaminated soils is widespread and environment friendly soil restoration method due to relatively low cost and low harmful impact to the nature in comparison with the physical and chemical soil restoration methods (Liu et al., 2011). Although bioremediation is prospective method for petroleum-contaminated soils, the success was noted only for 33% of sites contaminated with petroleum hydrocarbons. This is related with the high site specificity, treatment period (1-3 years or more), as well as its generally limited application to soils moderately contaminated (<5% w/w) with total petroleum hydrocarbons (TPH). Oil-degrading microorganisms may be strongly inhibited in highly contaminated soils (Alvarez and Illman, 2006). There is also a high probability of ground water contamination during the bioremediation process. Various watersoluble oxidized hydrocarbon metabolites, which are highly toxic and mobile in soil, were shown to accumulate during biodegradation of diesel fuel and various polycyclic aromatic hydrocarbons (Mao et al., 2009; Boll et al., 2015). In this context, we have developed a method based on adsorptive bioremediation for soils highly contaminated with various organic contaminants including chloroanilines and derivative herbicides, the explosive 2,4,6-trinitrotoluene, and polychlorinated biphenyls (PCB). This method is based on the use of adsorbents like activated carbon (charcoal) that creates better conditions for microbial degradation of the contaminants by reducing their toxicity (Vasilyeva et al., 1994, 2001, 2006, 2010). Diatomite is another widely used adsorbent for environmental protection. It is a natural siliceous sediment composed of the skeletal remains of diatoms (microscopic plants) deposited in seas or lakes. Diatomite products may be applied as a soil conditioner to increase soil porosity and water holding capacity, as well as to reduce bulk density (Aksakal et al., 2012; Boyraz and Nalbant, 2015). It is characterized by a mesoporous structure: >90% of the pores are 2-50 nm, while activated carbons are mostly microporous adsorbents with pores <2 nm in size. The presence of active hydroxyl groups on the surface of diatomite promotes adsorption of both hydrophobic and polar compounds such as heavy metals, dyes and phenols (Ma et al., 2015). Due to a specific porous structure, it can also adsorb petroleum compounds. Powdery diatomite in combination with aluminum sulfide is a highly effective flocculent, which enables a 100% reduction of crude oil and diesel oil in water emulsion (Puszkarewicz, 2008). Activated carbon and diatomite amendments (separately or in combination) have been employed previously for the remediation of petroleum-contaminated soils and other media, mostly as biocarriers for microbial cells in biopreparations. For example, Hodge et al. (1991) used those adsorbents as the carriers for microbial strains degrading diesel fuel vapors and showed that the efficiency of granular activated carbon (GAC) was significantly greater than diatomite. It has also been shown that microbial cells immobilized on GAC were highly stable to gradients of temperature, pH, salinity, and high content of toxicants ( phenol, 4-chlorophenol and other organic compounds), as well as demonstrated better ability to biotrans formation compared to suspended cells (Tessmer et al., 1997; Van der Loop et al., 1998; Carvalho et al., 2001; Shen et al., 2015; Chen et al., 2016). Muangchinda et al. (2018) also used activated carbon in combination with chitosan for that purpose. High densities of mixed culture biomass of petroleum-degraders isolated from contaminated soils were achieved by culturing microorganisms with GAC in a bioreactor. Introduction of the resultant biomass into soils containing 5 to 20% aged oil increased TPH degradation (Liang et al., 2009). Diatomite has been used as the carrier for microorganisms in purification of household wastes (Zhang et al., 2009; Chu et al., 2010). There was a biopreparation obtained from hydrocarbon-degrading bacteria Herbaspirillum chlorophenolicum strain FA1 immobilized on an alginate-diatomite matrix. This preparation is characterized by a high abundance of the microorganisms and a high crude oil degradation rate in soil compared to free-living cells or immobilized on alginate (Hu et al., 2011; Wang et al., 2012, 2015). Microbial inocula with nutrients immobilized on diatomaceous earth constitute an effective formulation to remediate petroleum-contaminated soils with a high concentration of hydrocarbons. Use of the "BioTer" adsorbent (a commercial product based on diatomite and alumosilicates) facilitated bioremediation of petroleum-contaminated soils (6.2 and 12.7% TPH), increased the number of indigenous petroleum degraders and water holding capacity from 0.6-0.8 to 36% compared to 20% in control (Alvaro et al., 2014; Silvana et al., 2014). The GAC was also used in permeable reactive barriers, in particular to capture and degrade hydrocarbon contaminants at fuel spill sites in Antarctica. The bacterial cells were observed within a uniform microbial biofilm layer, embedded within extracellular polymer substances on the surface of the GAC particles (Mumford et al., 2015; Freidman et al., 2017). Positive influence of GAC and biochar was demonstrated in recent short investigations (2-4 weeks) where those adsorbents amendment accelerated TPH biodegradation in Nigerian soils spiked with 5 or 10% weathered crude oil treated through bioactivation (Ameh et al., 2013; Agarry et al., 2015; Agarry, 2018). Formation of microbial biofilm communities on GAC in treatment of oil sands was shown positively affected petroleum degradation (Islam et al., 2015). It was also shown that activated carbon and biochar amendments reduced toxic contaminants bioavailability and improved plant growth characteristics (chlorophyll content and shoot or root biomass) of maize (Zea mays) grown in PAHs contaminated soils (Brennan et al., 2014). In our study, the amendment with GAC has accelerated bioremediation of a diesel fuel-contaminated soil (Semenyuk et al., 2014). In a two-year experiment, we have demonstrated an even more significant influence of a mixed adsorbent ACD (composed of GAC and diatomite) on the bioremediation rate of three types of mineral soils spiked with 4.5% crude oil (Kondrashina et al., 2018). The objective of the present study was to determine the potential for adsorptive bioremediation of a highly contaminated grey forest soil (5, 10 and 15% crude oil) by using the mixed adsorbent ACD in combination with a biopreparation. In addition to studying the effect of the adsorbent on the rate of bioremediation, phytotoxicity and microbial count, the ACD influence on leaching of hydrocarbons and polar metabolites from the soils has also been studied. 2. Materials and methods 2.1. Materials 2.1.1. Soil A grey forest soil was obtained from a grassy site near Pushchino town (Moscow region, Russia). The sample was collected on an uncontaminated site at 20-cm depth and partially dried. Stones and roots were removed; the soil was passed through a 1-cm sieve and thoroughly mixed. That is a loamy soil with 1.7% organic matter and pH 5.8. The soil was low in nitrogen (13 mg N kg 1) and available G. Vasilyeva et al. / Science of the Total Environment 706 (2020) 135739 phosphorus (46 mg P 205 kg-1) but moderate in exchangeable potassium (94 mg K2O kg 1). 2.1.2. Contaminant The crude oil used to contaminate the soil samples was obtained from the Moscow refinery in Kopotnya (Russia). The oil was classified as sulfurous (1.0% sulfur mass fraction) with a low content of hydrogen sulphide, chlorides, methyl- and ethylmercaptans, a fraction yield of 21% at 200°C and 35% at 300°C, middle density (0.88 g cm 3), and moderate hydrocarbon content (26.6% alkanes, 32.9% cycloalkanes, 26.5% aromatics, 10.0% resins, and 4.0% asphaltenes). 2.1.3. Adsorbents A mixed adsorbent (ACD) composed of granular activated carbon and diatomite (3:1 w/w) was used. The granular activated carbon of Agrosorb-AG3Tm (GAC) produced from a backed powder coal with granule size 1-2 mm was purchased from NPO Neorganika (Elektrostal, Russia). Diatomite from the field Inzenskoe (Ulyanovsk region, Russia) was powdered in a mortar to <0.5 mm grain size. Characteristics of the GAC and diatomite are shown in Table 1. 2.1.4. Biopreparation The association of two bacterial strains Pseudomonas putida B-2187 and Rhodococcus erytropolis Ac-859 (from the collection of the Institute of Microbiology, Russian Academy of Sciences) was used as a biopreparation. These microorganisms are included to a mixture of commercial biopreparations Putidoil (ZSNIIGG, Tyumen, Russia) and Devoroil (NPO Biotechinvest), respectively. These bacteria are highly efficient petroleum degraders and can utilize aliphatic and aromatic hydrocarbons as growth substrates. Microbial biomass was obtained in a complete medium as described by Filonov et al. (2008). The concentrated suspension of the biopreparation was obtained as follows: the biomass of each microorganism was mixed in equal proportion and diluted with a saline buffer in a concentration about 5 x 108 CFU mL 1 for each strain (CFU - colony forming units). The total initial cell density in the soil reached 107 CFU g 1. Soil amendment with the BP (treatments BP and ACD + BP) was repeated in 3.5 months. 2.2. Experiment design 2.2.1. Influence of ACD adsorbent on the rate of soil bioremediation The microfield experiment was performed at the experimental station of the Institute of Physicochemical and Biological Problems in Soil Science RAS (Pushchino, Russia). The climatic conditions included the average annual precipitation 560 mm (380-770 mm), average annual air temperature 4.5 °C (+6.7° to +2 °C), and a daily average air temperature in the warm season of 7-20 °C (Vasilyeva et al., 1996). The experiment continued for 18 months: from the beginning of May 2017 until the end of October 2018. Bottomless polyvinyl chloride vessels (33 x 33 x 60 cm 3) were dug in the soil so that the top edge of the vessels would be 5 cm above the soil level. An initial soil was removed from the vessels at a depth of 15 cm below the top edge, and the bottom was covered with a plastic polyvinyl mesh. The vessels were filled with the experimental soils (12 kg d.w.), which were compressed to natural state - approximately a 10-cm soil layer. Table 1 Characteristics of the granular activated carbon and diatomite used in the experiments. Parameter AG-3A Diatomite Total pore volume by water content, cm3 Effective micropore volume, cm3 g-1 Surface area, m2 g Adsorption capacity by iodine, mg g Adsorption capacity by methylene blue, mg g 0.87 0.27 850 760 195 0.5 0.25 5.7 1265 285 1 3 The experiment design is given in Table 2, and the step-by-step scheme of all operations is shown in Table 3. The experimental soils were surficially contaminated with crude oil at 50, 100 or 150 g kg (5, 10 and 15% w/w; soil samples denoted as 5P, 10P and 15P, respectively) and left for 5 days (air temperature 10-23 °C; without precipitation) for volatilization of a light fraction. The soils (excepting untreated controls, UnK) were thoroughly mixed and immediately treated. The pH of treated soils was adjusted from 5.8 to 6.5 by adding dolomite powder (DP) at 0.5 g kg 1. Liming was repeated after 3.6 and 13.5 months by adding DP at 1 g kg 1 to prevent strong acidification of the soil after the application of mineral fertilizers in high doses. All variants of the soil samples (K, BP, ACD and ACD + BP) were treated similarly excepting untreated contaminated control soils (UnK), which were not mixed, limed or fertilized. The mixed adsorbent ACD (4, 8 or 12% w/w depending on soil contamination) and the biopreparation were added to some treated samples, separately or in combination. The mineral fertilizer Azophoska (N17P17K17) was added to every vessel (except UnK) in similar doses for each variant, but in increased doses for strongly contaminated soils. The fertilizer was added fractionally to avoid strong soil acidification. An uncontaminated (pure) soil (PS) was used to determine the phytotoxicity of the experimental soil. The PS soil was treated similarly to the others, but with reduced doses of the mineral fertilizer and dolomite powder. An optimal ratio of petroleum organic carbon to macroelements (C: N: P:K) is the most important factor for successful microbial remediation of highly contaminated soils. Ratios such as 100:1:1:1 or 50:1:1:1 (with some variations) have been suggested in (Riser-Roberts, 1998; Dados et al., 2015). Because of high concentrations of hydrocarbons (initially 34-114 g kg 1), the high doses of mineral fertilizers were needed (about 340-2300 mg NPK kg 1). Such high doses may cause strong acidification of soil due to HNO3 or H3PO4 accumulation and result in the inhibition of bacterial growth (Chalneau et al., 2005). To prevent the effect of soil acidification during bioremediation, the soil should be fertilized fractionally with lower fertilizer doses and this should indude liming. A single dose of mineral fertilizer N i71317K17 ranged from 20 to 260 mg equivalent to N, P205 and 1(30 per kg 1. During the first warm season, the fertilizer was added on 0.2, 1 and 3.9 months (260, 260, and 20 mg NPK per kg 1, respectively), then 20, 50 or 100 mg NPK per kg 1 (depending on soil sample) were added at the beginning of spring at the second year. Table 2 shows the total amount of fertilizer and calculated C:N:P:K ratios to the end of each warm season. By the end of the secondyear warm season, C:N:P:K ratios for contaminated 5P, 10P and 15P soils were approximately 46:0.5:1, 100:1:0.5:1, and 130:1:0.5:1, respectively. The uncontaminated soil sample (PS) was supplied with two-times less doses (compared to sample 5P) of dolomite lime and fertilizer every spring. All samples and treatments were in triplicates. Soils in all vessels (except UnK) were periodically moistened (with settled, desalted tap water) to avoid excessive soil drying. Soils were mixed periodically during fertilizing and sampling. All procedures (mixing and addition of BP, adsorbents and fertilizers) were performed before wetting the soils to avoid disaggregation. The maximum precautionary measures were taken: first noninoculated and then inoculated samples were mixed and sampled. During the experiment, the soils were sampled periodically for chemical and biological analyses. The content of TPH and their polar metabolites (mostly total oxidized petroleum hydrocarbons, TOPH), the numbers of hydrocarbon-degrading microorganisms, soil pH, and phytotoxicity were determined in the samples. 22.2. Influence of the ACD adsorbent on wash-out of petroleum hydrocarbons and their polar metabolites from the soils In 6 and 12 months after initial treatment, the risk of leaching of petroleum contaminants was studied using laboratory lysimeters. For this G. Vasilyeva et at / Science of the Total Environment 706 (2020) 135739 4 Table 2 Scheme of the micro field experiment with grey forest soil initially contaminated with 5, 10 and 15% crude oil (5P, 10P and 15P, respectively) and background soil. Crude The ACD Soil oil adsorbent dose dose Treatment sample (% w/w) (% wily) Total amount of inoculated Mixing, Total Nt7P171(17 liming, (mg N, P205, or K20 kg-1) and bacteria over C:N:P:K ratio at the end of each year ** (CFU g-1)* fertilizing 1St year 2nd year 260 280 540 590 46:1:0.5:1 Background PS (pure) soil 5UnK 5K 5P 107+ 5 5BP 5ACD 4 5ACD+BP 4 50:1:0.5:1 107+ 107 10UnK 10K 10P 10BP 540 107+ 10' 10 10ACD 8 10ACD+BP 8 + 640 100:1:0.5:1 120:1:0.5:1 107+ 107 15UnK 15K 15P 15 15BP 540 107+ 107 15ACD 12 15ACD+BP + 7 12 10 + 10 640 130:1:0.5:1 170:1:0.5:1 7 'Total amount of inoculated bacteria in soils during both treatments with biopreparation. b Accounting for approximate initial carbon content in the TPH and total NPK content in the fertilized soils. Table 3 Step-by-step scheme of the micro field experiment with grey forest soil initially contaminated with 5, 10 and 15% crude oil (5P, 10P and 15P, respectively) and background soil (PS). Months Operation Soil samples PS 5P 10P 15P 0 0.25 5 10 15 0.5 4 0.5 0.5 107 8 107 12 107 260 260 107 1.0 20 260 260 107 1.0 20 260 260 107 1.0 20 35 35 35 35 20 20 20 20 Study of TPH and TOPH leaching + + + + Fertilizing, mg NPK 20 50 100 100 Liming, g DP kg -1 0.5 1.0 1.0 1.0 Sowing of white clover, seeds per vessel Plants thinning, to seedlings per vessel Plants excavation and root measuring 35 35 35 35 20 20 20 20 -0.17 Contamination with crude oil, % (w/w) 0 Liming, g DP kg -1 0.1 0.2 Amendment with ACD adsorbent, % (w/w) Amendment with biopreparation, CFU 0.2 1.0 Fertilizing, mg NPK Fertilizing, mg NPK kg -1 3.5 Amendment with biopreparation, CPU g 3.6 3.9 Liming, g DP kg-, Fertilizing, mg NPK kg -1 0.5 4.0 4.5 5.0 6.0 Sowing of white clover, seeds per vessel Plants thinning, to seedlings per vessel Plants excavation and root measuring 11.0 12.0 Study of TPH and TOPH leaching 13.5 16.0 16.5 17.0 260 purpose, samples of about 1 kg (d.w.) of undisturbed soil were taken from each vessel (except UnK) by a plastic cylindrical sampler with an internal diameter and a high of 10 cm. These cylinders were mounted on tripods with plastic mesh. Then 200 mL of distilled water was run through the soil sample. The lysimetric water was collected in glass flasks equipped with chemical funnels, filtered through paper filter and analyzed to determine concentrations of TPH and TOPH, as well as water phytotoxicity. The soil samples were partially dried under ambient conditions and returned into the same vessels on the next day. 2.3. Analytical methods 23.1. Determination of TPH and TOPH content in soil and lysimetric water TPH content in soil was measured using the Russian certified method (PND F 16.1:2.2.22-98, 1998). For that purpose, a sample of 5 g air-dried soil was stirred and passed through 1 mm sieve. Further, 1 g of the sieved sample was extracted with 30 mL of carbon tetrachloride ( for chromatography) by shaking for 1 h. After separation of the extract by filtering through paper filter, the sample was extracted again with 10 mL CCI4. The combined extract was separated from polar compounds in an Al203 (3% H2O) column. The total content of TPH in the purified extract was determined with an IR-Spectrometer KH-2 (Concentratomer KN-2 M; SibEcopribor, Novosibirsk, Russia) and included the measurement of a total concentration of C-H containing
