Adsorptive bioremediation of soil highly contaminated with crude oil

Journal Pre-proof Adsorptive bioremediation of soil highly contaminated with crude oil

Galina Vasilyeva, Victoria Kondrashina, Elena Strijakova, JoseJulio Ortega-Calvo PII:

S0048-9697(19)35734-1

DOI:

https://doi.org/10.1016/j.scitotenv.2019.135739

Reference:

STOTEN 135739

To appear in:

Science of the Total Environment

Received date:

7 October 2019

Revised date:

20 November 2019

Accepted date:

23 November 2019

Please cite this article as: G. Vasilyeva, V. Kondrashina, E. Strijakova, et al., Adsorptive bioremediation of soil highly contaminated with crude oil, Science of the Total Environment (2019), https://doi.org/10.1016/j.scitotenv.2019.135739

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2019 Published by Elsevier.

Journal Pre-proof

1

Adsorptive bioremediation of soil highly contaminated with crude oil Dr. Galina Vasilyeva1* , Victoria Kondrashina1 , Dr. Elena Strijakova1 , Dr. Jose-Julio Ortega-Calvo2 1

Institute of Physicochemical and Biological Problems in Soil Science, RAS, Pushchino, Moscow region, Russia. 2

Instituto de Recursos Naturales y Agrobiologia de Sevilla (IRNAS), C.S.I.C., Avenida Reina Mercedes, 10, E-41012-Seville, Spain Running title: Adsorptive bioremediation of soil highly contaminated with crude oil. *Corresponding author. [email protected]

Jo u

rn

al

Pr

e-

pr

oo

f

Keywords: petroleum, metabolites, activated carbon, diatomite, phytotoxicity, leaching

1

Journal Pre-proof

2

Adsorptive bioremediation of soil highly contaminated with crude oil Dr. Galina Vasilyeva1* , Victoria Kondrashina1 , Dr. Elena Strijakova1 , Dr. Jose-Julio Ortega-Calvo2

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 3,429

oo

f

reported petroleum spills in the RF in 2017, mostly from commercial pipelines. As a result, 6,183 ha were contaminated by 10,278 m3 of crude oil and petroleum products (State Report,

pr

2017). This situation, which represents also the global problem of other oil-producing countries

e-

(Alvarez and Illman, 2006), requires the cost-effective, efficient and less labor-intensive methods

Pr

for remediation of petroleum-contaminated soils.

Bioremediation is a practical and cost-effective approach to solve a wide range of

al

problems of soil contamination. Bioremediation of petroleum-contaminated soils is widespread

rn

and environment friendly soil restoration method due to relatively low cost and low harmful

Jo u

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

hydrocarbons (TPH).

soils moderately contaminated

(<5% w/w) with total petroleum

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 water-soluble 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). 2

Journal Pre-proof

3

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

f

siliceous sediment composed of the skeletal remains of diatoms (microscopic plants) deposited

oo

in seas or lakes. Diatomite products may be applied as a soil conditioner to increase soil porosity

pr

and water holding capacity, as well as to reduce bulk density (Aksakal et al., 2012; Boyraz and

e-

Nalbant, 2015). It is characterized by a mesoporous structure: more than 90% of the pores are 250 nm, while activated carbons are mostly microporous adsorbents with pores <2 nm in size. The

Pr

presence of active hydroxyl groups on the surface of diatomite promotes adsorption of both

al

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

rn

in combination with aluminum sulfide is a highly effective flocculent, which enables a 100%

Jo u

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 biotransformation compared to suspended cells (Tessmer et al., 1997; Van der Loop et al., 1998; Carvalho et al., 2001; Shen 3

Journal Pre-proof

4

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 was 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

f

hydrocarbon-degrading bacteria Herbaspirillum chlorophenolicum strain FA1 immobilized on

oo

an alginate-diatomite matrix. This preparation is characterized by a high abundance of the

pr

microorganisms and a high crude oil degradation rate in soil compared to free-living cells or

e-

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

(a

commercial

product

based

on

diatomite

and

alumosilicates)

facilitated

al

adsorbent

Pr

petroleum-contaminated soils with a high concentration of hydrocarbons. Use of the “BioTer”

bioremediation of petroleum-contaminated soils (6.2 and 12.7% TPH), increased the number of

rn

indigenous petroleum degraders and water holding capacity from 0.6-0.8 to 36% compared to

Jo u

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; Friedman 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). 4

Journal Pre-proof

5

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 fuelcontaminated 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

f

(Kondrashina et al., 2018).

oo

The objective of the present study was to determine the potential for adsorptive

pr

bioremediation of a highly contaminated grey forest soil (5, 10 and 15% crude oil) by using the

e-

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

al

Pr

influence on leaching of hydrocarbons and polar metabolites from the soils has also been studied.

2.1 Materials

rn

2. MATERIALS AND METHODS

Jo u

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 phosphorus (46 mg P 2 O 5 kg-1 ) but moderate in exchangeable potassium (94 mg K 2 O kg-1 ). 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 200о C and 35% at 300о C, middle density (0.88 g cm-3 ), and moderate 5

Journal Pre-proof

6

hydrocarbon content (26.6% alkanes, 32.9% cycloalkanes, 26.5% aromatics, 10.0% resins, and 4.0% asphaltenes). Adsorbents. A mixed adsorbent (ACD) composed of granular activated carbon and diatomite (3:1 w/w) was used. The granular activated carbon of Agrosorb-AG3T M (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

f

shown in Table 1.

oo

Biopreparation. The association of two bacterial strains Pseudomonas putida B-2187 and

pr

Rhodococcus erytropolis Ac-859 (from the collection of the Institute of Microbiology, Russian

e-

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

Pr

Biotechinvest), respectively. These bacteria are highly efficient petroleum degraders and can

al

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

rn

suspension of the biopreparation was obtained as follows: the biomass of each microorganism

Jo u

was mixed in equal proportion and diluted with a saline buffer in a concentration about 5x10 8 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 6

Journal Pre-proof

7

temperature4.5°C (+6.7°to +2°C), and a daily average air temperature in the warm season of 720o 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 (33×33×60 cm3 ) 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

oo

f

compressed to natural state - approximately a 10-cm soil layer. The experiment design is given in Table 2, and the step-by-step scheme of all operations

pr

is shown in Table 3. The experimental soils were surficially contaminated with crude oil at 50,

e-

100 or 150 g kg-1 (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

Pr

fraction. The soils (excepting untreated controls, UnK) were thoroughly mixed and immediately

al

treated.

The pH of treated soils was adjusted from 5.8 to 6.5 by adding dolomite powder (DP) at

rn

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

Jo u

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 (N 17 P17 K 17 ) 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. 7

Journal Pre-proof

8

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 HNO 3 or H3 PO4 accumulation and result in the inhibition of bacterial growth (Chaıneau et al., 2005). To prevent the effect of soil acidification

f

during bioremediation, the soil should be fertilized fractionally with lower fertilizer doses and

oo

this should include liming.

pr

A single dose of mineral fertilizer N 17 P17 K17 ranged from 20 to 260 mg equivalent to N,

e-

P2 O5 and K 2 O 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

Pr

(depending on soil sample) were added at the beginning of spring at the second year. Table 2

al

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 second-year warm season, C:N:P:K ratios for contaminated 5P, 10P

rn

and 15P soils were approximately 46:0.5:1, 100:1:0.5:1, and 130:1:0.5:1, respectively. The

Jo u

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

8

Journal Pre-proof

9

hydrocarbons, TOPH), the numbers of hydrocarbon-degrading microorganisms, soil pH, and phytotoxicity were determined in the samples. 2.2.2 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 purpose, samples of about 1 kg (d.w.) of undisturbed soil were taken from each vessel (except UnK) by a plastic cylindrical sampler with

oo

f

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

pr

collected in glass flasks equipped with chemical funnels, filtered through paper filter and

e-

analyzed to determine concentrations of TPH and TOPH, as well as water phytotoxicity. The soil

Pr

samples were partially dried under ambient conditions and returned into the same vessels on the

rn

2.3 Analytical methods

al

next day.

2.3.1 Determination of TPH and TOPH content in soil and lysimetric water

Jo u

TPH content in soil was measured using the Russian certified method (PND F 16.1:2.2.22-98). 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 CCl4 . The combined extract was separated from polar compounds in an Al2 O3 (3% H2 O) column. The total content of TPH in the purified extract was determined with an IR-Spectrometer KH-2 (Concentratomer KN-2M; SibEcopribor, Novosibirsk, Russia) and included the measurement of a total concentration of C-H containing groups of aliphatic and aromatic compounds absorbing in the infrared area of 2930±70 cm-1 . It

9

Journal Pre-proof

10

was calibrated with a petroleum standard solution in carbon tetrachloride (Russian State Reference Standard no. 7554_99). The content of polar C-H-containing petroleum fraction was estimated approximately as the difference between the total amounts of C-H-containing compounds determined before and after separation of polar organic molecules. According to Zavgorodnyaya et al. (2017), the nonpolar fraction is represented by aliphatic and aromatic petroleum hydrocarbons, while the polar fraction predominately by the initial non-hydrocarbon (hetero-atomic) oil compounds and the

oo

f

products of hydrocarbon biodegradation such as aliphatic and naphthenic acids, alcohols, and esters. Our results showed that this fraction can be used for approximate estimation of total content

e-

2.3.2 Count of degrading bacteria in the soil

pr

of oxidized products of petroleum hydrocarbons TOPH) in soil and lysimetric water.

The number of petroleum-degrading microorganisms (DM) was determined according to

Pr

Semenyuk et al. (2014) by inoculating soil suspensions at the appropriate dilutions on a solid

al

mineral medium containing 6.0 g Na2 HPO 4 ·Н2 O, 3.0 g КН2 PO4 , 0.5 g NaCl, 1.0 g NH4 Cl, 0.8 g

rn

MgSO 4 , and 20 g agar (per 1 L of distilled water), with diesel fuel vapors as a sole carbon and energy source. Diesel fuel (0.1 ml) was poured into the upper lead of Petri dish, which was

Jo u

covered upside down with a Petri dish with the inoculated agar medium. Petri dishes were incubated at 28о С for 5-7 days. The measurements were performed in triplicate for each sample. 2.3.3 pH of soil

Soil pH was measured in a 1:2.5 suspension of dry soil in distilled water with a pH meter. 2.3.4 Phytotoxicity of soil and lysimetric water Phytotoxicity of soil was determined by two methods. First, during the experiment, simultaneously with chemical analyses, we used an express test based on the germination rate of white clover (Trifolium repens) due to its high sensitivity to petroleum contaminants. A decrease in seeds germination compared to uncontaminated control was considered as an indicator of phytotoxicity. Phytotoxicity was measured in Petri dishes with 40 g soil samples taken from the 10

Journal Pre-proof

11

vessels as described previously (Kondrashina et al., 2018). Phytotoxicity in the experimental soil was calculated as: Phn = (Gn – Gps) × 100/Gps, where Gn and Gps are average values of germinated seeds in a given soil sample and in the PS sample, respectively. Phytotoxicity of lysimetric water was determined similarly. A Petri dish with a disk of filter paper was filled with 10 ml of the experimental water and seeded with 35 seeds of white clover. Germinated seeds were counted after 5-day incubation in closed Petri dishes at room temperature. Phytotoxicity of the lysimetric water was calculated as: Phnw = (Gnw – Gpw) ×

f

100/Gpw, where Gnw and Gpw are average values of germinated seeds in a sample of lysimetric

oo

water passed through contaminated and pure soil samples, respectively.

pr

In the second method, the soil phytotoxicity was evaluated by measuring the root length

e-

of wheat plants (Triticum vulgaris) harvested at the end of the warm season each year. For that purpose, 35 wheat seeds were sown into each vessel in the beginning of each September. After

Pr

two weeks, the seedlings were thinned to 20 per vessel. The plants were excavated one month after sowing and root lengths were measured. Phytotoxicity in the experimental soil (Phn ) was

al

calculated as: Phn = (RLn – RLps) × 100/RLps, where RLn and RLps are average root length of

rn

wheat plants grown in a soil sample and PS, respectively.

Jo u

2.3.5 Statistical analysis

One-way analysis of variance (ANOVA) was carried out to determine statistical differences in TPH content and soil phytotoxicity among treatments, where means were compared by post-hoc Fisher`s least significance test (LSD) using the STATISTICA software package version 8 (Statsoft Inc., USA). In addition, TPH content in soil and flashed waters (as well as their phytotoxicity) of the best treatments and their respective controls was compared using the Student’s t-test with STATISTICA 10 (TIBCO Software Inc., Palo Alto, California, USA). Differences were declared significant at p ≤ 0.05.

3. RESULTS 11

Journal Pre-proof

12

3.1. Influence of the ACD adsorbent and biopreparation on the dynamics of TPH and TOPH concentrations in the petroleum-contaminated soils The initial TPH content in the contaminated soil was 86% of the total petroleum contamination, while the remaining 14% was comprised of the resin-asphaltene fraction. Thus, the calculated contents of TPH in the soils initially contaminated with 5, 10 and 15% petroleum (5P, 10P and 15P) were 43.5, 87.6 and 130.5 g kg-1 , respectively. Initial TPH concentrations after weathering the surficially contaminated soils for five days were 33.5±1.5, 79.2±5.4 and

oo

3.1±0.5, 5.9±1.1 and 9.2±1.6 g kg-1 , respectively.

f

114.1±6.5 g kg-1 , while concentrations of CH-containing polar compounds were approximately

pr

Figure 1 shows the effect of the mixed adsorbent (ACD) and biopreparation (BP),

e-

separately or in combination (ACD+BP), on the dynamics of the TPH content and fraction of

Pr

CH-containing polar compounds. The natural attenuation of TPH in the UnK soils was slow, especially in the highly contaminated soils. The TPH content decreased by 64, 56 and 47% in 5P,

al

10P and 15P soils for two years, while the residual TPH content remained relatively high:

rn

approximately 12, 35 and 60 g kg-1 , respectively. Soil bioremediation sharply increased petroleum degradation. The accelerated TPH

Jo u

degradation during the first vegetative periods (first 6 months from May to October) was followed by a slow TPH degradation during the cold season (the next 5 months, from November to March) with the next accelerated degradation during the second vegetative period for 7 months (from April to October). The TPH concentrations were reduced mostly during the first year while that process slowed during the next year, probably because of the slower degradation rate of high- molecular-weight hydrocarbons. Reductions of residual TPH concentrations in the treated soils by the end of the first and second warm seasons are given in Table 4. During these periods, in control samples treated by activation of indigenous petroleum-degrading microorganisms, TPH concentrations decreased by

12

Journal Pre-proof

13

67-68 and 82-85%, respectively, but at the end of experiment, these were too high: 5.5, 14.0 and 17.1 g kg-1 in 5K, 10K and 15K, respectively. The best results were obtained with the combined addition of the adsorbent and biopreparation

(ACD+BP),

especially at the highest pollutant concentration.

The TPH

concentrations in those treatments were reduced by 77% and 90-91% in the first and second years, respectively. Statistical analyses revealed that TPH degradation was significantly higher in the combined treatment at all pollution levels compared to the respective controls that received

f

the fertilizer only (K): by 9.4-10.2% and 4.8-8.2%, respectively. Finally, the TPH concentration

oo

in the best variant of the less contaminated sample (5ACD+BP) was reduced to 4.5 g kg-1 , which

pr

was lower than the Russian Federation maximal permissible level (<5 g kg-1 ) in recultivated

e-

technogenic contaminated soils in taiga-forest region (Vasilyeva et al., 2013). Bioaugmentation alone (BP treatment) significantly affected the TPH degradation rate in

Pr

the medium contaminated soil only, where the TPH content in the 10BP sample was significantly

al

lower by 6.3 or 5.5%, as compared to the control sample 10K after 1 and 2 years, respectively. The addition of only the ACD adsorbent positively affected TPH degradation in the highly

rn

contaminated samples 10P and 15P during both years, but its influence was significantly lower

Jo u

compared to treatments ACD+BP (Table 4). Figure 1 shows the dynamics of the CH-containing polar fraction (TOPH) in the petroleum-contaminated samples during the experiment. A significant reduction of that fraction in the soil samples during the first two months was followed by its accumulation with a maximum at the end of the first year warm season (about 6 months), which corresponded to degradation of 70 to 75% of initial TPH. During the next warm season, some reduction of TOPH content in samples 10P and 15P was followed by their secondary accumulation, which coincided with degradation of the remained TPH fraction. There was not significant deviation of TOPH content in the sample 5P, and its accumulation in samples 10P and 15P amended with ACD adsorbent was substantially less compared to the treatments without adsorbent. To the end of the 13

Journal Pre-proof

14

experiment, the TOPH content in the soil samples 5P, 10P and 15P deviated in the intervals 4-6, 8-12 and 12-16 g kg-1 , respectively.

3.2 Influence of the ACD adsorbent and biopreparation on the dynamics of petroleumdegrading microorganisms, soil phytotoxicity, and pH Figure 2 shows the dynamic of petroleum-degrading microorganisms (DM) in soils. During the experiment, the number of these microorganisms (mostly degrading bacteria) in the

f

background soil varied from 104 to 106 CFU g-1 (data not presented). The DM number sharply

oo

increased and varied between 108 and 109 cells g-1 in the treated contaminated soils. There were

e-

followed 1 or 2 months after every fertilizing.

pr

three main maximal counts of those DM in 1-2, 4-5, and 12-13 months after the start, which

At 1-2 months after starting the experiment, the DM count was about 2 times higher in

Pr

the inoculated sample 5BP compared to control 5K, while similar exceed of maximal DM count

al

was detected in the highly contaminated samples 10BP and 15BP over appropriate control samples 10K and 15K after the second BP amendment (at 4-5 months) only. Soil amendment

rn

with the ACD adsorbent also resulted in some increase in degraders, especially in the 10P soil.

Jo u

However, the highest growth of DM number occurred in soils amended with ACD+BP, especially after the second BP treatment. The maximal count in 5P, 10P and 15P soils were 0.9×109 , 1.3×109 and 1.5×109 CFU g-1 , respectively. At the end of the experiment, counts in those soils were decreased to (0.2-0.4)×108 , (0.5-0.9)×108 and (1.5-2)×108 CFU g-1 , respectively. These results are consistent with the dynamics of the soil pH and phytotoxicity measured by the express method on white clover germination, presented in Figure 3. Soil phytotoxicity during the first months of bioremediation corresponded to changes in soil pH and TPH degradation, as well as was followed by accumulation of oxidized petroleum products. The significant soil acidification (up to pH 5.5) was mostly a result of the introduction of high doses of mineral fertilizer N 17 P17 K17 for maintaining an adequate level of macroelements for DM in the 14

Journal Pre-proof

15

contaminated soils. The increase of soil pH can be explained by liming effect. Soil liming and fractionated introduction of the fertilizer, especially during the first year, created conditions for generally maintaining soil pH between 5.8 and 6.7. The soil pH in the control (K) and BP-amended (BP) treatments of soil samples 5P, 10P and 15P were rather close and varied similarly during the course of the experiment. The pH of adsorbent-amended soils (ACD and ACD+BP) also deviated similarly, but their values were significantly higher compared to the other treatments, especially in highly contaminated soils

f

receiving larger doses of the adsorbent.

oo

Initial soil phytotoxicity has depended on petroleum concentration and increased from 60

pr

to 100% in contaminated soils, with 5P<10P<15P (Fig. 3). During the first months of

e-

bioremediation, soil phytotoxicity in almost all treatments without ACD (except some periods for 5K) remained high (30-100%) and was associated with soil pH and TPH degradation

Pr

followed by the accumulation of petroleum oxidized products. Soil phytotoxicity in the only BP-

al

amended samples was usually close to control except the first year, when these samples revealed even higher phytotoxicity compared to control. Conversely, the phytotoxicity of soils amended

rn

with the ACD adsorbent and especially in combination with biopreparation (ACD+BP) was

Jo u

significantly lower compared to soils without the adsorbent. Phytotoxicity was rather low (<2030%) in treatments ACD+BP of all soil samples during whole experiment except some short periods after soil fertilizing because of temporal soil acidification. During the experiment (in 1-2, 4-5 and 12-13 months) there were some deviations of phytotoxicity with temporal increase for one or two months. Usually these shifts coincided with soil acidification to pH <6, which often accompanied the soil amendment with mineral fertilizer. These results are also consistent with the phytotoxicity determined by the measuring of root lengths of 30-day wheat plants grown in these soils at the end of the warm seasons (Fig. 4). The soil phytotoxicity in all samples determined by the certified method mostly coincided with the values determined by our express method. The figure indicates that all control samples 5K, 15

Journal Pre-proof

16

10K and 15K demonstrated elevated phytotoxicity (30-88%) at the end of the treatment years. The samples 10BP and 15BP demonstrated the significantly lower (except 5BP) but still high phytotoxicity: 29-64%. From the other side, phytotoxicity of these samples amended with the adsorbent (ACD and ACD+BP) was significantly (p<0.05) lower compared to the respective controls. Soil phytotoxicity of ACD-amended treatments in the less contaminated soil 5P was reduced to practically nontoxic level (<20%) already by the end of the first year, while in the soil sample 10P by the end of the second year. Phytotoxicity of the ACD-amended samples 15P

oo

f

still remained increased (27-35%) at the end of treatment, but 2 or 3 times lower compared to

pr

control 15K.

e-

3.3. Influence of the ACD adsorbent on wash-out of hydrocarbons and polar products of petroleum biodegradation

Pr

Figure 5 shows the residual concentrations of TPH and TOPH as well as phytotoxicity of

al

soils 5P, 10P and 15P in treatments K and ACD+BP determined in 6 and 12 months after the

rn

beginning. These results were compared with the content of the same contaminants and phytotoxicity in lysimetric waters. Similar results have been received for treatments K and BP as

Jo u

well as for treatments ACD and ACD+BP. Therefore, they are not presented in the Figure 5. The content of TPH and total petroleum contaminants (TPH+TOPH) in soils increased in a row 5P<10P<15P in all treatments, with less content in ACD+BP treatments in comparison to their controls K. Hereby, the relative content of the polar fraction TOPH in these soils in 6 and 12 months varied in intervals 28-39% and 39-49% of total contaminants, respectively. Phytotoxicity of these soils in K treatments increased at the same row: from 20 to 80%, while soil phytotoxicity in ACD+BP treatments was about two times less or more than in the respective controls. On the other side, petroleum contaminants in lysimetric water were mostly comprised of polar fraction 60-100% of the total. Hereby, the concentration of total petroleum contaminants 16

Journal Pre-proof

17

in the lysimetric water of 5P soil and its phytotoxicity was low: <2 mg L-1 and <20%, respectively. However, the TPH content in lysimetric water of 10K and 15K soils varied between 17 mg L-1 and 20 mg L-1 , and 6 mg L-1 and 15 mg L-1 in the samples collected in 6 and 12 months, respectively. Besides, phytotoxicity of the water samples was extremely high (72-99%) during the first determination and elevated (25-51%) during the second one. Conversely, the content of the petroleum contaminants in the water samples from ACD+BP treatments was sufficiently lower than in the respective control water samples (about 3-4 and 2 times in 6 and 12

oo

f

months, respectively), and their phytotoxicity was insignificant (<10-20%).

pr

4. DISCUSSION

e-

Significant faster degradation of TPH in all the soils 5P, 10P and 15P was observed in the ACD-treatments, especially in combination with the biopreparation (ACD+BP), with a larger

Pr

effect at the highest petroleum concentration. Meantime, the separate use of BP significantly

al

affected the TPH degradation rate in the medium contaminated soil 10P only. Hereby, elevated TPH concentrations remained in the highly contaminated soils treated through bioaugmentation,

rn

both 10BP and 15BP (10 and 15 g kg-1 , respectively) to the end of the second year, whereas the

Jo u

best variant included the combined use of ACD and BP, that resulted in residual concentrations of 7 and 12 g kg-1 , respectively. There are several reports indicating faster petroleum biodegradation in soils treated by bioactivation or bioaugmentation. However, in situ bioremediation is recommended only for soils with petroleum contamination <5-10% because of high toxicity of heavily contaminated soils to petroleum-degrading microorganisms, as indigenous and inoculated with biopreparation (Banerjee1 et al., 2016; Macaulay and Rees, 2014; Alvarez and Illman, 2006; Alexander, 1999). A weak or sometimes negative impact of biopreparations on the bioremediation rate of petroleum-contaminated soils were observed in various publications. This was because the

17

Journal Pre-proof

18

introduced exogenous microbes often failed to compete favorably with the indigenous microbes at the polluted sites (Macaulay and Rees, 2014). In our experiment, the number of indigenous DM increased (from 10 4 CFU g-1 in background soil) by several orders of magnitude in all the control samples 5K, 10K and 15K and varied from 108 to 109 CFU g-1 during two years of bioremediation. The initial population of inoculated DM in the BP-amended soils should be approximately 107 CFU g-1 . However, the detected initial DM number in the BP-amended soils and their maximum were very close to the

oo

f

concentrations in pure soilno more than twice higher compared to unamended controls. Hereby, their amount in highly contaminated soils 10BP and 15BP was usually several times

pr

lower than in 5BP soil. Considering the calculated number of the introduced petroleum-

e-

degrading strains was about two orders of magnitude higher than in background soil, we can conclude that the introduced strains were substantially inhibited in highly contaminated soils due

Pr

to their high toxicity. It can be explained by comparatively high activity of indigenous petroleum

rn

due to their elevated toxicity.

al

degraders in soil 5K and by inhibition of introduced strains in soils 10BP and especially in 15BP

Petroleum-degrading microorganisms can be inhibited or killed in highly and chronically

Jo u

contaminated soils, while degraders are usually preserved in freshly spiked soils with moderate doses of petroleum (Cho et al., 1997; Van Hamme et al., 2003; Liu et al., 2013). During bioremediation of moderately contaminated soils through activation of indigenous petroleumdegrading bacteria, their amount used to be increased up to 10 7 -109 CFU g-1 or higher (Agamuthu and Dadrasnia, 2013; Coulon and Delille, 2003) that is consistent with our results. The elevated toxicity of 10P and 15P soils was also confirmed by the dynamic of their phytotoxicity. In all the control soils (except some periods in 5K), the toxicity remained rather high during the experiment, hereby, this value in the BP-amended soils was close to the respective controls K or even higher. This fact can be explained by better availability of toxic petroleum metabolites in BP soils due to excretion of biosurfactants by the introduced microbial 18

Journal Pre-proof

19

strains. Bacteria of genera Rhodococcus and Pseudomonas, like many other petroleum-degrading strains used for biopreparations, are known to produce biosurfactants (Matvyeyeva et al., 2014). The main reason for reduced toxicity in ACD-amended soils is the adsorption of some hydrocarbons and especially highly toxic petroleum metabolites. The dynamic of TPH and TOPH content in the higher contaminated soils suggests that this C-H-containing polar fraction can be used as an approximate estimation of the total content of oxidized products of hydrocarbon biodegradation in soils or lysimetric water. We could suggest that hydrocarbon

f

degradation during the first year was accompanied by accumulating a significant amount of a

oo

polar fraction especially in highly contaminated soils. To the end of the experiment, the polar

pr

fraction ratio increased from 14-15 to 40-50% of total content of oil contaminants. In these soils,

e-

a polar petroleum fraction was washed out into lysimetric water and tremendously dominated among hydrocarbons. After 6 months, this fraction varied between 79 and 99% of all detected

Pr

petroleum contaminants, also, the lysimetric waters in 10K and 15K samples strongly inhibited

al

white clover germination (by 72-95%). The lower TOPH accumulation in soil samples 5P and in ACD-treated soils 10P and 15P may be explained by faster microbial degradation of those

rn

intermediates under less toxic conditions.

Jo u

It is known that bioremediation of diesel and petroleum contaminated soils is accompanied by accumulation of various intermediates, mostly oxygenated hydrocarbons such as alcohols, ketones and carboxylic acids. These polar compounds are highly toxic and more water-soluble and thus, more leachable to ground water than the original hydrocarbons (Mao et al., 2009; Watson et al., 2002). High amounts of toxic and mobile oxygenated aromatic hydrocarbons have been also identified in soils contaminated with various polycyclic aromatic hydrocarbons (Boll et al., 2015). The ACD-adsorbed petroleum hydrocarbons and their oxidized metabolites evidently remained mostly available to microbial degradation, what was confirmed by an increased count of petroleum-degrading microorganisms in the ACD-amended soils. Previously, we have proved 19

Journal Pre-proof

20

the approximately complete availability of 3,4-dichloroaniline and its toxic metabolite 4,5dichloropyrochatehine adsorbed in GAC to a bacterial strain able to use this compound as the sole source of carbon and energy (Bakhaeva et al., 2001). Several studies, such as Farhadian et al. (2008), have also demonstrated the availability of some hydrocarbons adsorbed to GAC for degrading microorganisms. However, the GAC adsorption of some contaminants, such as polycyclic petroleum hydrocarbons, may slow their microbial degradation. Nevertheless, these toxic adsorbed compounds did not reveal their toxic effect.

f

Cho et al. (1997) showed better bioremediation of soil contaminated with crude oil (6%

oo

TPH) collected in Kuwait in samples amended with 5% GAC from coconut chips or (in less

pr

degree) with baked diatomite (particle size 0.9-1,5 mm) introduced in a combination with

e-

petroleum degraders. Despite the lower adsorption capacity of diatomite compared to GAC and taking into account the low cost and polyfunctionality of diatomite, its potential for use in

Pr

permeable barriers and for groundwater treatment is high (Aivalioti et al., 2010). There are also

al

prepared hybrid carbon/diatomite adsorbents. Due to their open pore structure, these composites showed an extremely fast p-cresol adsorption rate compared to activated carbon with much

rn

larger porous development, and adsorptive capacities were comparable to those of activated

Jo u

carbon (Hadjar et al., 2011). A number of authors suggest that the action mechanism of GAC and diatomite includes increasing the oxygen content in the soil, accelerating mass transfer of nutrients, and increasing moisture capacity, which provides favorable conditions for maintaining active microbial colonies (Liang et al., 2009). Here, we extend those findings by showing that GAC and diatomite can accelerate bioremediation through a reduction in contaminant toxicity. Crooks and Prentice (2017) demonstrated that soil amendment with diatomite increased the resistance of plants and other living organisms to various stresses: biogenic or abiogenic, such as low or high temperatures, salt stress, and negative effect of heavy metals and other pollutants, insufficient moisture, etc. The main mechanism was related to exudation of monoand polysilicic acids accumulated in living cells. We suspect that adding diatomite to activated 20

Journal Pre-proof

21

carbon might reduce strong binding of some petroleum components (such as polycyclic aromatic compounds and others) in nanoporous space of GAC. Perhaps the weaker sorption of these hydrocarbons in pores of activated carbon mixed with diatomite can be also explained by release of mono- and polysilicic acids permeating into its nanoporous space. As a result, petroleum hydrocarbons adsorbed to ACD might be more readily degraded, as compared to those adsorbed to GAC (Kondrashina et al., 2018). Here, we conclude that most of the petroleum hydrocarbons adsorbed to the mixed ACD adsorbent were available to microbial degradation that was also

f

confirmed by the higher amounts of petroleum-degrading microorganisms at presence of the

oo

adsorbent.

pr

These results show that the positive influence of the mixed ACD adsorbent on the

e-

bioremediation rate of petroleum-contaminated soils is mostly related to high porosity and sorption capacity of the materials. The higher phytotoxicity of the highly contaminated soils in

Pr

control samples at the end of experiment indicates the presence of mobile toxic petroleum

al

components and/or their metabolites. The presence of the ACD adsorbent decreased their mobility in soil due to mostly reversible adsorption and, thus, minimized toxicity to the plants

Jo u

rn

and microorganisms remaining available to petroleum degraders.

5. ENVIRONMENTAL IMPLICATIONS Our results indicate that activity of degrading microorganisms (as indigenous, and inoculated with biopreparation) and pant growth during bioremediation of soils contaminated with crude oil, especially at high level (> 5%) can be strongly inhibited due to high toxicity of these soils. Besides, there is a risk of surface and ground water contamination both with petroleum hydrocarbons and especially with their polar microbial metabolites (mainly oxidized products of alkanes and aromatic hydrocarbons such as alcohols, aldehydes, ketones, carboxylic acids, phenols, etc.) with higher water solubility and toxicity.

21

Journal Pre-proof

22

We suggest a new approach of adsorptive bioremediation for petroleum-contaminated soils based on the use of a mixed adsorbent composed of activated carbon and diatomite. It has several advantages compared to other related methods such as bioremediation through bioactivation or bioaugmentation. First of all, this approach can be applied to highly contaminated soils (up to 15% crude oil at least) due to reduction of soil toxicity for microorganisms and plants because of mostly reversible adsorption of petroleum components and their toxic microbial metabolites, due to reduction of the soil hydrophobicity, as well as due

f

to increased persistence of the plants to various stress at presence of diatomite. This allows

oo

phytoremediation to be carried out at an earlier period, which in turn can reduce time of

pr

remediation.

e-

Besides, the adsorptive bioremediation approach minimizes the risk of the contaminant leaching to underground waters, that allows to apply an in situ soil biorecultivation. Soil cleaning

Pr

by in situ bioremediation can last from 1 to 3 plant growing seasons, but it does not require

al

excavation, transportation, and disposal of hazardous waste and does not destroy the soil cover. Our analysis presented in (Slyusarevsky et al., 2018) has demonstrated that the cost of adsorptive

rn

bioremediation based on the use of activated carbon varies in the interval 2.53.9 million rubles

Jo u

ha-1 , which might be higher compared to that of regular bioremediation 1.0–4.3 million rubles ha-1 . Nevertheless, the implementation of adsorbents for bioremediation of oil-contaminated soils is often justified, since they significantly extend the capabilities of the in situ biotreatment. It is also calculated that the mechanical remediation of oil-contaminated soils (often used in Russia) is carried out quickly and radically. However, the cost of this approach varies from 3.8 to 62.4 million rubles ha-1 depending on a distance and price list of a company certified for toxic grounds utilization. Thus, we suppose that the adsorptive bioremediation could be one of the most ecological, cost-effective,

efficient

and

less

labor

intensive

22

methods

for

remediating

petroleum-

Journal Pre-proof

23

contaminated soils, which can help to solve the problems with petroleum-contaminated lands in Russia and other oil-producing countries.

6. CONCLUSIONS We propose a new approach of adsorptive bioremediation for soils highly contaminated with crude oil based on the use of a mixed adsorbent (ACD) composed of activated carbon and diatomite. A microfield, two-year experiment was carried out with grey forest soil contaminated

f

with 5, 10 and 15% crude oil. The most notable impact on TPH degradation was detected in soils

oo

amended with the ACD adsorbent, and especially in combination with BP, moreover in the

pr

highly contaminated soils. The total petroleum hydrocarbons in the best treatments were reduced

e-

by 90-91% compared to 82-88% in the controls. A positive influence of BP alone was significant only in the intermediate contaminated sample. In addition to the faster reduction of TPH in

Pr

ACD-amended soils, less oxygenated petroleum products were formed in highly contaminated

al

soils, as compared to soils without adsorbent. Introduction of the adsorbent in combination with BP into these soils significantly (2-7-times) increased the degrader amount compared to

rn

respective controls without any amendments. This corresponds to a sharp reduction in soil

Jo u

phytotoxicity in the presence of ACD, especially in highly contaminated soils.

Acknowledgements

This work was financed by the Russian Foundation of Basic Research (Project No. 16-0500617). We are grateful to Prof. P.J. Shea (UNL, USA) for valuable remarks and proofreading of the manuscript, and to Dr. M.A. Pukalchik (Skolkovo IST, Russia) for the help in statistical analyses. We thank the Spanish Ministry of Science, Innovation and Universities (CGL201677497-R), for supporting the work of J.J Ortega-Calvo.

Conflict of interest 23

Journal Pre-proof

24

No conflict of interest declared. REFERENCES Agamuthu, P., Dadrasnia, A., 2013. Potential of biowastes to remediate diesel fuel contaminated soil. Global NEST Journal 15(4), 474-484. https//doi.org/10.30955/gnj.001031. Agarry, S.E., 2018. Evaluation of the effects of Inorganic and organic fertilizers and activated carbon on bioremediation of soil contaminated with weathered crude oil. J. Appl. Sci. Environ. Managm. 22(4), 587-595. https//doi.org/10.4314/jasem.v22i4.27.

f

Agarry, S.E., Oghenejoboh, K.M., Solomon, B.O., 2015. Kinetic modeling and half-life study of

oo

adsorptive bioremediation of soil artificially contaminated with Bonny light crude oil. J.

pr

Ecol. Engin. 16(3), 1–13. https//doi.org/10.12911/22998993/2799.

thermally

e-

Aivalioti, M., Vamvasakis, I., Gidarakos, E., 2010. BTEX and MTBE adsorption onto raw and modified

diatomite.

J.

Hazard.

Mater.

178,

136–143.

Pr

https//doi.org/10.1016/j.jhazmat.2010.01.053.

al

Aksakal, E.L., Angin, I., Oztas, T., 2012. Effects of diatomite on soil physical properties. Catena 88(1), 1-5. https//doi.org/10.1016/j.catena2012.09.001.

Alvarez,

Jo u

Press. -453 p.

rn

Alexander, M. 1999. Biodegradation and bioremediation. 2nd edition. CA, USA: Academic

P.J.J.,

Illman,

W.A.,

2006.

Bioremediation

and

natural attenuation: Process

fundamentals and mathematical models. (Eds. J.L. Schnoor and A. Zehnder). New Jersy: Willey-InterScience, - 612 p. Alvaro, C.E., Olave, A.J., Schmid, P.M., Nudelman, N.S., 2014. Microbial remediation performance of environmental liabilities from oil spills in Patagonian soils: A laboratory – scale. International Journal of Research in Chemistry and Environment 4(3), 119-126. Ameh, A., Abdulkareem, J.A., Otaru, A.S., 2013. Remediation of petroleum polluted site in Nigeria using granulated activated carbon (GAC). Journal of Environmental Science,

24

Journal Pre-proof Toxicology

and

Food

Technology

3(5),

25 100-109.

https//doi.org/10.9790/2402-

035100109. Bakhaeva, L.P., Vаsilyeva, G.K., Surovtseva, E.G., Mukhin, V.M., 2001. Microbial degradation of 3,4-dichrloroaniline sorbed by activated carbon. Microbiology 70(3), 277-284. Banerjee1, A., Roy, A., Dutta, S., Mondal, S., 2016. Bioremediation of hydrocarbon – a review. International

Journal

of

Advanced

Research

4(6).

1303-1313.

https//doi.org/10.21474/IJAR01/734.

f

Brennan, A., Jimenez, E., Alburquerque, J.A., Knapp, C.W., Switzer, C., 2014. Effects of

oo

biochar and activated carbon amendment on maize growth and the uptake and measured

pr

availability of polycyclic aromatic hydrocarbons (PAHs) and potentially toxic elements

e-

(PTEs). Environ. Pollut. 193, 79-87. https//doi.org/10.1016/j.envpol.2014.06.016. Boll, E.S., Johnsen, A.R., Christensen, J.H., 2015. Polar metabolites of polycyclic aromatic

Pr

compounds from fungi are potential soil and groundwater contaminants. Chemosphere

al

119, 250-257. https//doi.org/10.1016/j.chemosphere.2014.06.033. Boyraz, D., Nalbant, H., 2015. Comparison of zeolite (clinoptilolite) with diatomite and pumice

rn

as soil conditioners in agricultural soils. Pak. J. Agr. Sci. 52(4), 923-929.

Jo u

Chaıneau, C.H., Rougeux, G., Yepremian, C., Oudot, J., 2005. Effects of nutrient concentration on the biodegradation of crude oil and associated microbial populations in the soil. Soil Biol. Biochem. 37, 1490-1497. https//doi.org/10.1016/j.soilbio.2005.01.012. Carvalho, M.F., Vasconcelos, I., Bull, A.T., Castro, P.M.L., 2001. A GAC biofilm reactor for the continuous degradation of 4-chlorophenol: treatment efficiency and microbial analyses. Appl. Microbiol. Biotechnol. 57, 419-426. https//doi.org/10.1007/s002530100794. Chen, Y., Yu, B., Lin J., Naidu, R., Chen, Z., 2016. Simultaneous adsorption and biodegradation of diesel oil using immobilized Acinetobacter venetianus on porous material. Chem. Eng. J. 289, 463-470. https//doi.org/10.1016/j.cej.2016.01.010.

25

Journal Pre-proof

26

Cho, B.H., Chino, H., Tsuji, H., Kunito, T., Nagaoka, K., Otsuka, S., Yamashita, K., Matsumoto, S., Oyaizu, H., 1997. Laboratory-scale bioremediation of oil-contaminated soil of Kuwait with

soil

amendment

materials.

Chemosphere

35(7).

1599-1611.

https//doi.org/10.1016/S0045-6535(97)00220-8. Chu, H., Cao, D., Dong, B., Qiang, Z., 2010. Bio-diatomite dynamic membrane reactor for micro-polluted

surface

water

treatment.

Water

Res.

44,

1573-1579.

https//doi.org/10.1016/j.watres.2009.11.006.

f

Coulon, F., Delille, D., 2003. Effects of biostimulation on growth of indigenous bacteria in Sub-

oo

Antarctic soil contaminated with oil hydrocarbons. Oil Gas Sci. Technol. 58(4), 469-479.

pr

https//doi.org/10.2516/ogst:2003030.

e-

Crooks, R., Prentice, P., 2017. Extensive investigation into field based responses to a silica fertilizer. Silicon 9(2), 301-304. https//doi.org/10.1007/s12633-015-9379-3.

Pr

Dados, A., Omirou, M., Demetriou. K., Papastephanow, C., Ioannides, I.M., 2015. Rapid

al

remediation of soil heavily contaminated with hydrocarbons: a comparison of different approaches. Ann. Rev. Microbiol. 65, 241-251. https//doi.org/10.1007/s13213-014-0856-

rn

5.

through

Jo u

Farhadian, M., Duchez, D., Vachelard, C., Larroche, C., 2008. BTX removal from polluted water bioleaching

processes.

Appl.

Biochem.

Biotechnol.

151,

295–306.

https//doi.org/10.1007/s12010-008-8189-0. Filonov, A.E., Petrikov, J.V., Yakshina, T.V., Puntus, I.E., Vlasova, E.P., Nechaeva, I.A., Samoilenko V.A 2008. Regimes of separated and combined cultivation of oil-degrading microorganisms of Pseudomonas and Rhodococcus genera. Biotechnology in Russia 6, 110-117. Freidman, B.L., Speirs, L.B.M., Churchill, J., Gras, S.L., Tucci, J., Snape, I., Stevens, G.W., Mumford, K.A., 2017. Biofilm communities and biodegradation within permeable

26

Journal Pre-proof

27

reactive barriers at fuel spill sites in Antarctica. Int. Biodeterrior. Biodegrad. 125, 45-53. https//doi.org/10.1016/j.ibiod.2017.08.008. Hadjar, H., Hamdi, B., Ania, C.O., 2011. Adsorption of p-cresol on novel diatomite/carbon composites.

J.

Hazard.

Mater.

188(1),

304-310.

https//doi.org/10.1016/j.jhazmat.2011.01.108 Hodge, D.S., Medina, V.F., Islander, R.L., Devinny, J.S., 1991. Treatment of hydrocarbon fuel vapors

in

biofilters.

Environ.

Technol.

12,

655-662.

f

https//doi.org/10.1080/09593339109385053.

oo

Hu, W.W., Wang, Z.Y., Liu, J.D., Xu, Y., Li, F.M., Xing, B.S., Gao, D.M., 2011.

river

delta.

Huanjing

Kexue

yu

Jishu

34(3),

116-120.

e-

Yellow

pr

Bioremediation of crude oil contaminated soil with immobilized microorganism in

https//doi.org/10.1016/S1002-0160(12)60057-5.

Pr

Islam, Md.S., Zhang, Y., Mc. Phedran, K.N., Liu, Y., El-Din, M.G., 2015. Next-generation

al

pyrosequencing analysis of microbial biofilm communities on granular activated carbon in treatment of oil sands process-affected water. Appl. Environ. Microbiol. 81, 4037-

rn

4048. https//doi.org/10.1128/AEM.04258-14.

Jo u

Kondrashina, V., Strijakova, E., Zinnatshina, L., Bocharnikova, E., Vasilyeva, G. 2018. Influence of activated

carbon and

other additives on bioremediation rate and

characteristics ofpetroleum-contaminated soils. Soil Sci. (Special issue) 138(4), 150-159. https//doi.org/10.1097/SS.0000000000000234. Liang, Y., Zhang X., Dai, D., Guanghe, L., 2009. Porous biocarrier-enhanced biodegradation of crude

oil

contaminated

soil.

Int.

Biodeterior.

Biodegrad.

63(1),

80-87.

https//doi.org/10.1016/j.ibiod.2008.07.005. Liu, P.W.G., Chang, T.C., Whang, L.M., Kao, C.H., Pan, P.T., Cheng, S.S., 2011. Bioremediation of petroleum hydrocarbon contaminated soil: effects of strategies and

27

Journal Pre-proof microbial

community

shift.

Int.

28

Biodeterior.

Biodegrad.

65,

1119-1127.

https//doi.org/10.1016/j.ibiod.2011.09.002. Liu, P.W.G., Chang, T.C., Chen, C.H., Wang, M.Z., Hsu, H.W., 2013. Effects of soil organic matter and bacterial community shift on bioremediation of diesel-contaminated soil. Int. Biodeterior. Biodegrad. 85, 661-670. https//doi.org/10.1016/j.ibiod.2013.01.010. Ma, S.C., Wang, Z.G., Zhang, J.L., Sun, D.H., Liu, G.X., 2015. Detection analysis of surface hydroxyl active sites and simulation calculation of the surface dissociation constants of suspensions.

Appl.

https//doi.org/10.1016/j.apsusc.2014.12.006.

Surf.

Sci.

327,

453-461.

f

diatomite

oo

aqueous

pr

Macaulay, B.M., Rees, D., 2014. Bioremediation of oil spills: A review of challenges for

e-

research advancement. Annals of Environmental Science 8, 9-37. Mao, D., Lookman, R., Van de Weghe, H., Weltens, R., Vanermen, G. , De Brucker, N. Diels,

Pr

L., 2009. Combining HPLC-GCXGC, GCXGC/TOF-MS, and selected ecotoxicity assays

al

for detailed monitoring of petroleum hydrocarbon degradation in soil and leaching water. Environ. Sci. Technol. 43, 7651–7657. https//doi.org/10.1021/es9015603.

biodegradation.

Jo u

products

rn

Matvyeyeva, O.L, Vasylchenko, О.А., Aliievа, O.R., 2014. Microbial biosurfactants role in oil International Journal of Environmental Bioremediation &

Biodegradation 2(2), 69-74. https//doi.org/10.12691/ijebb-2-2-4. Muangchinda, C., Chamcheun, C., Sawatsing, R., Pinyakong, O., 2018. Diesel oil removal by Serratia sp. W4-01 immobilized in chitosan-activated carbon beads. Environ. Sci. Pollut. R. 25(27), 26927-26938. Mumford, K.A., Powell, S.M., Rayner, J.L., Hince, G., Snape, I., Stevens, G.W., 2015. Evaluation of a permeable reactive barrier to capture and degrade hydrocarbon contaminants. Environ. Sci. Pollut. R. 22, 12298–12308. https//doi.org/10.1007/s11356015-4438-2.

28

Journal Pre-proof

29

Nikiforov, A., Ponomarev, A., Parfenov, V., 2016. Modeling of processes of oil migration in the soil with the use of X-ray microtomography to optimize the process of liquidation of oil spills. Ecology, economics, education and legislation conference proceedings, SGEM 2016, V. II. International Multidisciplinary Scientific GeoConference-SGEM, p. 589-596. PND F 16.1:2.2.22-98. (Environmental Save Regulation Documents) Method for determination of mass share of petroleum products in soil and bottom sediments by IR spectrometry. Quantitative analysis of soils. Moscow. 1998. (in Russian)

f

Puszkarewicz, A. 2008. Removal of petroleum compounds from water in coagulation process.

E.

Remediation of petroleum-contaminated

Yatsenko,

Zavgorodnyaya,

of

Strijakova,

Vasilyeva,

diesel

fuel

E.R.,

Filonov,

A.E.,

Petrikov,

K.V.,

G.K., 2014. Effect of activated charcoal on

contaminated

soil.

Microbiology

83(5),

589-598.

al

bioremediation

Yu.A.,

V.S.,

Pr

N.N.,

e-

Washington DC, - 526 p. Semenyuk,

soils 1998. Lewis Publishers,

pr

Riser-Roberts,

oo

Environ. Prot. Eng. 34(1), 5-14.

https//doi.org/10.1134/S0026261714050221.

rn

Shen, T., Youngrui, P., Mutai, B., Xu, N., Inren, L., 2015. Biodegradation of different petroleum

Jo u

hydrocarbons by free and immobilized microbial consortia. Environmental Science: Processes Impacts 17(12), 2022-2033. https//doi.org/10.1039/c5em00318k. Silvana, A.C.E., Olave, A.J., Schmid, P.M., Nudelman, N.S., 2014. Microbial remediation performance of environmental liabilities from oil spills in Patagonian soils: a laboratoryscale. International Journal of Research in Chemistry and Environment 4(3), 119-126. Slyusarevsky, A.V., Zinnatshina, L.V., Vasilyeva, G.K., 2017. Comparative environmental and economic analysis of methods for the remediation of oil-contaminated soils by in situ bioremediation and mechanical soil replacement. Ecology and Industry of Russia 22 (11), 40-45. https//doi.org/10.18412/1816-0395-2018-11-40-45.

29

Journal Pre-proof

30

State report. 2017. “Status and protection of the environment in Russian Federation in 2016 y. Moscow: Ministry of the Environment Protection, Scientific Information Agency – Nature 2017, -760 p. Tessmer, C.H., Vidic, R.D., Uranovski, L.J., 1997. Impact of oxygen-containing surface functional groups on activated carbon adsorption of phenols. Environ. Sci. Technol. 31, 1872-1878. https//doi.org/10.1021/es960474r. Van der Loop, S.L., Suidan, M.T., Moteleb, M.A., Maloney, S.W., 1998. Two-stage 2,4,6-trinitrotoluene

conditions.

Water

Environ.

nitrogen-rich Res.

and

nitrogen-limiting

70(2),

189-196.

pr

https//doi.org/10.2175/106143098X127035.

under

f

of

oo

biotransformation

Microbiol.

Mol.

Biol.

e-

Van Hamme, J.D., Singh, A., Ward, O.P., 2003. Recent advances in petroleum microbiology. R. 67(4), 503-549. https//doi.org/10.1128/MMBR.67.4.503-

Pr

549.2003.

Toxicol.

2,4,6-trinitritoluene Chem.

rn

decrease

al

Vasilyeva, G.K., Kreslavski, V.D., Shea, P.J., Oh, B-T. 2001. Potential of activated carbon to toxicity and

20(5),

accelerate soil decontamination. 965-971.

Environ

https//doi.org/10.1897/1551-

Jo u

5028(2001)020<0965:poactd>2.0.co;2. Vasilyeva, G.K., Strijakova, E.R., Bocharnikova, E.A., Semenyuk, N.N., Yatsenko, V.S., Slyusarevski, A.V., Barishnokova, E.A. 2013. Petroleum and petroleum products as soil contaminants. Technology of combined physical-biological remediation of contaminated soils. Russ J. Gen. Chem. 57(1), 79-104. (in Russian) Vasilyeva, G.K., Strijakova, E.R., Nikolaeva, S.N., Lebedev, A.T., Shea, P.J., 2010. Dynamics of PCB removal and detoxification in historically contaminated soils amended with activated

carbon.

Environ.

https//doi.org/10.1016/j.envpol.2009.10.010.

30

Pollut.

158(3),

770-777.

Journal Pre-proof

31

Vasilyeva, G.K., Strijakova, E.R., Shea, P.J., 2006. Use of activated carbon for soil bioremediation. In: Viable methods of soil and water pollution monitoring, protection and remediation. Serial NATO Collection, Springer, Netherlands, pp. 309-322. Vasilyeva, G.K., Surovtseva, E.G., Belousov, V.S., 1994. Microbial method for the clean-up of soil contaminated with propanil and 3,4-dichloroaniline. Microbiology 63(1), 75-80. Vasilyeva, G.K., Surovtseva, E.G., Ivannikova, L.A., Bakhaeva, L.P., 1996. Dynamic of chloroaniline degrading bacteria monitored for six years after inoculation into gray forest

f

soil. Microbiology 65(4), 487-492.

oo

Wang, X., Zhang, S., Wang, C., 2015. Biodegradation of spilled-oil by immobilization

pr

microorganisms on diatomite-based carrier material. Proceedings of the 5th international

Advances

in

Engineering

Research

21,

1541-1546.

https//doi.org/10.2991/icimm-

Pr

15.2015.282.

e-

conference on information engineering for mechanics and materials. Book seria: AER-

al

Wang, Z.Y., Xu, Y., Wang, H.Y., Zhao J., Gao, D.M., Li, F.M., Xing, B., 2012. Biodegradation of crude oil in contaminated soils by free and immobilized microorganisms. Pedosphere

rn

22(5), 717–725. https//doi.org/10.1016/S1002-0160(12)60057-5.

Jo u

Watson, J. S., Jones, D. M., Swannell, R. P. J., van Duin, A.C.T., 2002. Formation of carboxylic acids during aerobic biodegradation of crude oil and evidence of microbial oxidation of hopanes.

Org.

Geochem.

33(10),

1153–1169.

https//doi.org/10.1016/S0146-

6380(02)00086-4. Zavgorodnyaya Yu. A., Stepanov A. A., Trofimov S. Ya., Farkhodov Yu. R., Pervakova V. N., Sokolova T. A., and Aptikaev R. S. 2017. Effect of introduction of clays, mineral fertilizers, and soil ameliorants on decomposition of organic pollutants in oil-polluted sand under conditions of model experiment. Moscow University Soil Science Bulletin 72 (1), 35–41. https//doi.org/10.3103/S0147687417010070.

31

Journal Pre-proof

32

Zhang, W., Rao, P., Zhang, H., Jingli, X.U., 2009. The role of diatomite particles in the activated sludge system for treating coal gasification wastewater. Chin. J. Chem. Eng. 17(1), 167-

Jo u

rn

al

Pr

e-

pr

oo

f

170. https//doi.org/10.1016/S1004-9541(09)60050-1.

32

Journal Pre-proof

33

Concentration of TPH 120 80

5P

45

K ACD BP ACD+BP UnK

40 35 30 25 20 15

f

10

oo

5

Concentration of TOPH

25 20

pr

Concentration of TPH or TOPH, g kg-1

15P

10P

без МД

15

e-

10

0 3

6

9

12

15

18

0

3

6

9

12

15

18

0

3

6

9

12

15

18

Months

al

0

Pr

5

rn

Figure 1. Influence of soil treatments on the dynamics of total petroleum hydrocarbons (TPH) and total polar petroleum product (TOPH) concentrations in soils contaminated with 5, 10 or

Jo u

15% crude oil (5P, 10P and 15P, respectively) during bioremediation in according to Table 2: control (K), treated with adsorbent (ACD) or biopreparation (BP) separately and in combination (ACD+BP), in comparison to untreated control (UnK). Vertical lines indicate the beginning time of the second warm season. Green arrows indicate time of fertilizing, and blue arrows – time of liming.

33

Journal Pre-proof

34

Count of hydrocarbon degraders Samples without biopreparation 12

15P

K ACD

8 6 4 2 0

Samples with biopreparation

16 14

BP ACD+BP

f

12 10

oo

-1 Count of DM, х108 CFU g

10P

5P

10

8

pr

6 4

0 0

3

6

9

12 15 18

0

e-

2 3

6

9

12 15 18

0

3

6

9

12 15 18

Pr

Months

al

Figure 2. Influence of soil treatments on the dynamic of petroleum degrading microorganisms

rn

(DM) count in soils contaminated with 5, 10 or 15% crude oil (5P, 10P and 15P, respectively)

Jo u

during two years of bioremediation treated through bioactivation (without biopreparation, in control K and ACD-amended soils) and bioaugmentation (with biopreparation alone (BP) or in combination with the adsorbent – ACD+BP). Arrows indicate time of bioaugmentation.

34

Journal Pre-proof

35

Phytotoxicity

Phytotoxicity, %

10P

5P

100

15P

80 60 40 20

Pract. nontoxic 0

pH of soil

6,5

f

6,0 5,5 0

3

6

9

12

15

18

0

3

6

9

oo

рН of soil

7,0

12

15

18 0

3

6

9

12

15

18

e-

pr

Months

Pr

Figure 3. Influence of soil treatments on synchronic dynamics of pH and phytotoxicity of the soils contaminated with 5, 10 or 15% crude oil (5P, 10P and 15P, respectively) determined by

al

white clover (Trifolium repens) germination. Legend is similar to the Fig. 1. The horizontal line

Jo u

rn

in top panels indicates the level of practically nontoxic effects.

35

Journal Pre-proof

36

Phytotoxicity count on root lengh 100

5P K BP ACD ACD+BP

60

a

15P b

a b

a b b

b

b b

b

a 40

aa

a b

b

a

c c c

b

20

c

b Practic. nontoxic

Phytotoxicity, %

80

10P

b

0 1st

2nd

1st

1st

2nd

2nd

oo

f

Vegetative seasons

Figure 4. Influence of soil treatments on phytotoxicity of soils contaminated with 5, 10 or 15%

pr

crude oil (5P, 10P and 15P, respectively) determined by measuring root lengths of 1-month

e-

wheat plants grown in those soils at the end of the 1st and 2nd vegetative seasons: in control (K), treated with the adsorbent (ACD) or biopreparation (BP) separately, and in combination

Pr

(ACD+BP). The horizontal line indicates the level of practically nontoxic effects. Different

al

letters on the plot denote significant differences (a = 0.05) between the treatments of the same

Jo u

rn

soil sample in one year according to the Fisher’s test;

36

Journal Pre-proof

37

Phytotoxicity of soil

Concentration of TPH and TOPH in soil

12 months

6 months

80

50

* * *

10 0

*

in flashed waters TOPH-K TOPH-ACD+BP TPH-K TPH-ACD+BP

20 15

*

40 20 0 100

*

*

*

* of flashed waters

f

20

*

K ACD+BP

oo

* *

30

60

Phytotoxicity, %

40

80

pr

60

10

40

* *

5

10P

15P

5P

10P

*

20

15P

*

*

10P

15P

0

5P

* 5P

* 10P

15P

al

5P

*

Pr

0

e-

*

rn

Figure 5. Influence of soil treatments with the ACD adsorbent in combination with a biopreparation (ACD+BP) in comparison with control (K) on the content of total petroleum

Jo u

Conc. of TPH/TOPH, mg L

100

12 months

-1

Conc. of TPH/TOPH, g kg

-1

6 months 60

hydrocarbons (TPH) and polar petroleum products (TOPH) in the soil and the leaching water, as well as their phytotoxicity determined through the germination of white clover in soils initially contaminated with 5, 10 and 15% crude oil (5P, 10P and 15P, respectively) after 6 and 12 months of bioremediation. Black asterisks indicate values of TPH content in soil or flashed water, and red asterisks – values of TOPH content or phytotoxicity of the same samples significantly different (a=0.05) in comparison to the similar control samples without additives.

37

Journal Pre-proof

38

Table 1. Characteristics of the granular activated carbon and diatomite used in the experiments AG-3A

Diatomite

Total pore volume by water content, cm3 g-1

0.87

0.5

Effective micropore volume, cm3 g-1

0.27

0.25

Surface area, m2 g-1

850

5.7

Adsorption capacity by iodine, mg g-1

760

1265

Adsorption capacity by methylene blue, mg g-1

195

285

Jo u

rn

al

Pr

e-

pr

oo

f

Parameter

38

Journal Pre-proof

39

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 Soil sample

Total N 17 P17 K17

Total

Mixing,

The ACD

oil Treatment dose

adsorbent dose

amount of

liming,

inoculated

and

bacteria

(% w/w) (% w/w)

fertilizing

(CFU g-1 )*

(mg N, P2 O5 , or K2 O kg-1 ) over C:N:P:K ratio at the end of each year ** 1st year

2nd year

+

260

280

-

-

-

-

-

5UnK

-

-

5K

-

oo

-

+

107 + 107

+

__540__

__590__

-

+

50:1:0.5:1

46:1:0.5:1

4

107 + 107

+

-

-

-

-

-

-

-

+

-

107 + 107

+

__540__

__640__

8

-

+

120:1:0.5:1

100:1:0.5:1

10ACD+BP

8

107 + 107

+

15UnK

-

-

-

-

-

15K

-

-

+

-

107 + 107

+

__540__

__640__

15ACD

12

-

+

170:1:0.5:1

130:1:0.5:1

15ACD+BP

12

107 + 107

+

5BP

5

Pr

4

5ACD+BP

10K 10BP

rn

al

10UnK

10

Jo u

10ACD

15P

-

e-

-

5ACD

10P

pr

(pure) soil

5P

f

Background PS

15BP

15

* Total amount of inoculated bacteria in soils during both treatments with biopreparation. **Accounting for approximate initial carbon content in the TPH and total NPK content in the fertilized soils. 39

Journal Pre-proof

40

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)

Soil samples Months -0.17

PS

5P

10P

15P

0

5

10

15

0.25

0.5

0.5

0.5

-

4

8

12

-

107

107

107

260

260

260

260

-

260

260

260

-

107

107

107

0.5

1.0

1.0

1.0

-

20

20

20

Contamination with crude oil, % (w/w) Liming, g DP kg-1 Amendment with ACD adsorbent, % (w/w)

0.2

Amendment with biopreparation, CFU g-1

0.2

Fertilizing, mg NPK kg-1

1.0

Fertilizing, mg NPK kg-1

3.5

Amendment with biopreparation, CFU g-1

3.6

Liming, g DP kg-1

3.9

Fertilizing, mg NPK kg-1

4.0

Sowing of white clover, seeds per vessel

35

35

35

35

4.5

Plants thinning, to seedlings per vessel

20

20

20

20

5.0

Plants excavation and root measuring

+

+

+

+

6.0

Study of TPH and TOPH leaching

+

+

+

+

Fertilizing, mg NPK kg-1

20

50

100

100

Study of TPH and TOPH leaching

+

+

+

+

12.0

oo

pr

e-

Pr

rn

Jo u

11.0

f

0.1

al

0

Operation

13.5

Liming, g DP kg-1

0.5

1.0

1.0

1.0

16.0

Sowing of white clover, seeds per vessel

35

35

35

35

16.5

Plants thinning, to seedlings per vessel

20

20

20

20

17.0

Plants excavation and root measuring

+

+

+

+

40

Journal Pre-proof

41

Table 4. Percent reduction in TPH content in the grey forest soil samples initially contaminated with 5, 10 and 15% crude oil (5P, 10P and 15P, respectively) at end of the first and second years. Means (SD) (n=3) TPH Reduction Treatments

(% of initial content in the weathered soils) 1st year

2nd year

5BP

70.1 (1.5)a

5ACD

74.6 (1.4)b

5ACD+BP

77.3 (2.9)b

oo

67.9 (1.5)a

Pr

e-

pr

5K

f

5P

83.6 (1.8)a 86.0 (0.9)a

87.5 (0.6)ab 89.6 (0.9)b

10P

67.1 (1.7)a

rn

10BP

al

10K

73.4 (1.7)b

87.8 (1.3)b

72.2 (1.6)b

89.3 (0.6)bc 90.5 (0.8)c

Jo u

10ACD

82.3 (1.8)a

77.3 (1,3)c

15K

66.7 (2.1)a

85.1 (0.9)a

15BP

68.0 (1.8)a

87.3 (0.9)b

15ACD

72.4 (1.3)b

89.5 (0.4)c

15ACD+BP

76,6 (1.3)c

89.9 (0.4)c

10ACD+BP

15P

Note: different letters in the column (for the same year) denote significant differences (a = 0.05) between the treatments of one soil sample according to the Fisher’s LSD post hoc test (ANOVA).

41

Journal Pre-proof

42

Graphical abstract

HIGHLIGHTS 1 Bioremediation can be applied for soils with petroleum contamination <5% only 2 Adsorptive bioremediation in situ can extend possibilities of this approach 3 It is based on the use of a mixed adsorbent in combination with a biopreparation 4 The adsorbent (activated carbon + diatomite) reduces toxicity of those soils 5

Jo u

rn

al

Pr

e-

pr

oo

f

The adsorbent may decrease leaching of toxic polar metabolites from the soils 6

42

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5