The ef fects of electrical heating and additives on the microbial remediation of petroleum-contaminated soils

Hongbai Jia · Yu Zhang · Guocai Zhang · Li Zou ·Bowen Zhang

Abstract Although petroleum is an important source of energy and an economic driver of growth, it is also a major soil pollutant that has destroyed large swathes of vegetation and forest cover. Therefore, it is vital to develop af fordable and ef ficient methods for the bioremediation of petroleumcontaminated forest soils to restore vegetation and improve tree survival rates. In this study, bioremediation experiments were performed in an electrically heated thermostatic reactor to test the ef fects of organic matter additives, surfactants,and oxygen providers of nine hydrocarbon-degrading fungal strains on crude oil removal rates. In the three soil temperatures tested (20 °C, 25 °C, and 30 °C), the highest average crude oil removal rate was at 25 °C (74.8%) and the lowest at 30 °C (49.4%). At each temperature, variations in the addition of organic matter and oxygen providers had signif icant ef fects on crude oil removal rate. Variations in surfactant addition was signif icant at 20 °C and 25 °C but insignif icant at 30 °C. Given the same surfactant treatment, variations in temperature, organic additives, and oxygen providers was signif icant for crude oil removal rate. Treatments without surfactants and treatments with Tween80 exhibited their highest crude oil removal rates at 25 °C. However, treatments that included the SDS surfactant exhibited their highest crude oil removal rates at 30 °C. Amongst the treatments without surfactants, treatments with corn cob addition had the highest crude oil removal rates, and with surfactants,treatments that included the organic fertilizer exhibited the highest crude oil removal rates. Given the same organic fertilizer treatment, the highest crude oil removal rate was at 25 °C. At each level of oxygen availability, the maximum crude oil removal rate always occurred at 25 °C, and the treatments that included organic fertilizer exhibited the highest crude oil removal rates. Amongst the treatments without oxygen providers, treatments without surfactants had the highest crude oil removal rates, and with an oxygen provider, treatments with SDS addition exhibited the highest crude oil removal rates. Based on the crude oil removal rates of the treatments, we determined that S 1 W1 O1 (addition of Tween80, organic fertilizers, and H 2 O2 ) was optimum for remediating petroleum-contaminated forest soils in cold,high-altitude regions. This study is helpful to vegetation restoration and reforestation on petroleum contaminated forestlands.

Keywords Bioremediation · Petroleum-contaminated soil · Hydrocarbon-degrading fungi · Crude oil removal rate · Inf luencing factors

Introduction

The range of uses for petroleum products continues to expand, leading to increasing consumption rates. However,the pollution and destruction of vegetation such as forests and grasslands during oilf ield exploitation has become a problem to be addressed.

Limited by exploitation techniques, the development of the Daqing Oilf ield in 1959, Heilongjiang province, (especially its early development beginning in 1960), has resulted in serious pollution to soils of this oilf ield and surrounding areas, which has a signif icant impact on vegetation. Previous researchers have compared the LANDSAT-TM remote sensing image data during August and September in 1990, 2001,and 2007, and found that the average value of the vegetation coverage index NDVI in the Daqing area was decreasing due to direct and indirect soil contamination by petroleum due to oilf ield exploitation (Yang Mingjie 2008). Other studies have shown that the vegetation communities in the oilf ield and surrounding areas have continued to decline during the development of the oilf ield and the areas may eventually degrade into saline-alkali or bare alkali soil patches. During this degradation process, the height of vegetation can be reduced by 10–30 cm, and its biomass drastically to 0.755 t/hm2(Fu and Yang 2012). Other studies report that the area of land use related to the oilf ield such as oil and water wells,pipeline routes, and roads has increased annually to its current area exceeding 35,300 km2.

These areas are either already contaminated by petroleum or under the risk of contamination. Once there are oil residues, and corrosion perforation or fracture of pipelines due to various factors, serious soil contamination will be the result. The deposit of petroleum substances into soils can change the composition and structure of organic matter,changing of carbon to nitrogen ratios (C/N) and carbon to phosphorus (C/P). Since petroleum has relatively low density, strong adhesion, and low emulsifying capacity, it easily adheres to soil particles, blocks soil pores, and af fects soil permeability (Yuniati 2018). As a result, vegetation restoration of contaminated sites is dif ficult. The failure rate of af forestation is high, and the ef fects of af forestation worse than in non-contaminated areas. Therefore, attention should be given to the remediation of petroleum-contaminated soils by microorganisms.

Although waste oil can be treated in various ways such as by burying or burning, bioremediation is a promising treatment, as it is safe, cost-ef ficient, simple, and does not produce secondary pollution (Zhan and Ma 2017; Shaeyan et al. 2018). Numerous bioremediation techniques have been developed in recent years such as landfarming (an ex situ waste treatment process), composting (Ekperusi and Aigbodion 2015), bioreactors, biostimulation (stimulating the environment of oil-degrading bacteria), and oil removal by earthworms (Roubalova et al. 2018; Li et al. 2021). Various studies have also been performed on the use of microbial metabolites, e.g., on the ef fects of biosurfactants and dehydrogenase activity on waste oil biodegradation (Li et al.2020).

Zhan et al. ( 2018) noted that bioremediation is an intrinsically complex process that involves a number of factors,especially in the case of petroleum-polluted soils. Therefore,it may be possible to increase the ef ficiency of petroleum pollutant degradation by optimizing these factors. Most previous research on this subject, regardless of whether on in situ or ex situ bioremediation, generally focused on the development of af fordable and ef ficient methodologies. The focal points of these studies were: the selection and domestication of microbial strains (Yu et al. 2014; Barkusaraey et al.2020; Zhang et al. 2010; Wang et al. 2012), plant-mediated bioremediation and plant species selection (Zhang et al.2010); the use of fauna (especially earthworms) (Hanna and Weaver 2002; Li et al. 2008; Contreras-Ramos et al. 2009);optimization of factors that govern hydrocarbon metabolism by microbes, plants, and fauna, including the selection of oxygen donors and electron acceptors, biosurfactant selection, the proportioning of nutrient additions, and the selection and addition of organic matter. In the present work,bioremediation experiments were carried out on petroleumcontaminated understory soils using nine hydrocarbondegrading fungal strains to determine an optimal microbial remediation treatment. Our f indings will provide a practical foundation for the bioremediation of contaminated forest soils.

Materials and methods

Fungal strains and crude oil

Nine hydrocarbon-degrading fungal strains were selected from petroleum-contaminated soils (upper 20 cm) near the Daqing Agricultural Park under a poplar forest. The sibling species to strains 1#, 4#, 7#, and 8# isFusarium subglutinansand for strains 3# and W9Aspergillus terreus(strain US3 MTCC9921) andA. protuberus(strain SF 5044),respectively. The sibling species of strains 19#, W2, and W19 arePseudallescheria boydii,Eupenicillium crustaceum, andBeauveria bassiana, respectively.

Crude oil was obtained from production plant #2 of the Daqing Oilf ield Co., Ltd. The experimental soils were from the upper 20 cm soil layer from Daqing City. Soil samples were f iltered through a 1-mm mesh prior to analysis.

Culture media formulae

Electrical heating device and thermostat

The reactor was a 34 cm × 25 cm × 12 cm white plastic box purchased from the market (Yiwu Quanbei Plastic Industry Co., Ltd, Jinhua, Zhejiang province). The electrical heating wire (resistance 66 Ω/m) and thermostat (range 0–50 °C)were purchased from the Jiuzhou Electric Heating Instrument Co., Ltd., Mianyang, Sichuan). The 5-m-long electrical heating line was woven onto two identical metallic meshes(slightly smaller than the internal dimensions of the reactor),in a serpentine fashion, with a 4-cm spacing between parallel lines. Care was taken to ensure that the heating lines were parallel to the vertical direction of the meshes and parallel to each other on each of the meshes. The lines were connected in series to the thermostat by conductive wires. Each end of the heating line was connected to the thermostat by a wire and plug (Fig. 1). Sample IDs and the design of each treatment are shown in Table 1.

Extraction of crude oil samples and calculation of degradation ef ficiency (rate of removal)

For the extraction of crude oil from the liquid culture medium, the pH was adjusted to 2–2.5 using 1:1 H2 SO4.Then, 10 mL of a 1:1 (V/V) mix of petroleum ether and hexane was added. The mixture was shaken until the crude oil was dissolved. The culture medium and petroleum ether solution were transferred to a 50-mL centrifuge tube and vortexed for 3 min, followed by centrifugation at 8000 revolutions per min for 3 min. The supernatant was then extracted, and 10 mL of the petroleum ether-hexane solution used to wash the conical f lask that contained the culture.The wash was transferred to a centrifuge tube, vortexed and centrifuged and the supernatant extracted. This washing process was repeated. A 15-mL amount of trichloromethane was added to the used centrifuge tubes, vortexed for 5 min, and the extract was drawn of f. Another 10 mL of trichloromethane was added to the residual solution in the centrifuge tube, followed by vortexing, centrifugation, and supernatant extraction. The extracts were combined and f iltered through f ilter paper soaked in trichloromethane with 10 mM anhydrous Na2 SO4, with the f iltrate collected in a beaker of known weight. The used f ilter paper was soaked twice in 20 mL trichloromethane and the wash f iltered in the same way, with the f iltrate being collected in the same beaker. The beaker was then placed in a 60 °C oven to volatilize the organic solvent until it reached a constant weight.Finally, the beaker was removed from the oven, cooled for half an hour, and then weighed.

The crude oil extracted from the fungi-free culture medium at the beginning of the experiment was def ined as its initial weight. The composition of the crude oil was also analyzed.

For the extraction of crude oil from contaminated soils,the soil samples were air- dried, ground, and loaded into a 50-mL centrifuge tube. A 15 mL petroleum ether-hexane solution (1:1 V/V) was added to the centrifuge tube, followed by 5 min of vortexing, ultrasonication at 70 W for 25 min, and centrifugation at 8000 revolutions per min for 3 min. The supernatant was extracted, and this procedure was repeated twice. The samples were extracted with trichloromethane until colorless. Finally, the extracts were combined, and the remainder of the extraction process was the same as that described above for the extraction of crude oil from the liquid culture medium.

The gravimetric method for calculating the crude oil removal rate (CORR) was:

Fig. 1 Control-temperature system by heating

Table 1 Sample identities (IDs) and treatments

Statistical analysis

The results shown in the Fig. 2 represent the average of three independent replicates. Data are expressed as mean ± standard deviation. Statistical analysis was performed using IBM SPSS Statistics (IBM Corp., Armonk, NY, USA) software to evaluate the statistical dif ferences between treatments atp< 0.05 signif icance level.

Results and discussion

Crude oil removal rates at dif ferent temperatures

Figure 2 shows the crude oil removal rate of each treatment at 20 °C and indicates that the crude oil removal rate of the 14-days period accounted for most of the total crude oil removed in all experimental groups and controls (CK). crude oil removal rate was always higher in the treatment groups with hydrocarbon-degrading fungi than in the controls, indicating that the addition of exogenous fungi promoted the removal of crude oil from understory soils.

Fig. 2 Rate of crude oil removal from understory soils. a Treatment at 20 °C, b treatment at 25 °C, c treatment at 30 °C

The crude oil removal rate of each treatment at 25 °C shows that all treatments with the addition of exogenous fungi resulted in higher crude oil removal rates than the controls. In treatments 1–4 and 13–18, the 14-days rate of crude oil removal accounted for most of the total amount removed. The other treatments (including CK) had relatively high 14-days and 15–28 days removal rates, especially treatments that included the Tween80 surfactant.At 30 °C, the crude oil removal rates of CK and treatments 1, 2, and 13–16 had high 15–28 days rates of removal,whereas all other treatments had high removal rates in the 14-days period. When the soil temperature was 30 °C, the addition of SDS, organic fertilizer, and an oxygen provider promoted hydrocarbon degradation in the 15–28-days stage(treatments 13–16). This ef fect was especially pronounced when organic fertilizer and oxygen providers were both added to the soil (treatment 16), as this treatment led to the highest total crude oil removal rates at 30 °C (79.9%).

Factors af fecting the remediation of petroleum-contaminated understory soils

In this study, two dif ferent organic supplements were added:bio-organic fertilizer and crushed corn cobs. These added to the soil’s organic matter content as well as acted as leavening agents. Since the organic fertilizer particles were quite small, they only slightly af fect soil ventilation. Conversely,large-grain corn cob particles had a signif icant ef fect on ventilation. To explain the ef fects of oxygen availability on the remediation of petroleum-contaminated soils, in our analysis oxygen availability was def ined as 2 in the treatments with the addition of corn cobs and oxygen providers, as 1 in thetreatments with the addition of corn cobs or oxygen providers, and 0 in all other treatments. The oxygen availability of all treatments is shown in Table 2 .

Table 2 Oxygen availabilities by treatments

ANOVA was performed on the crude oil removal rate of each treatment after 28 days in SPSS 17.0 (Table S1).Based on the ANOVA, the addition of surfactant and interactions between surfactants, organic matter additives, and oxygen availability were insignif icant. However, all other factors and interactions were highly signif icant. Therefore,the crude oil removal rate was signif icantly af fected by variations in organic matter, oxygen availability, temperature,and inter-factor interactions. The selection of a suitable set of conditions will signif icantly increase the CORR and the speed of bioremediation in petroleum-contaminated soils.

Ef fects of inter-factor interactions

Ef fects of temperature on the remediation of petroleum-contaminated soils

In Fig. 2, the highest crude oil removal rates on average of the treatments were at 25 °C (74.8%), and the lowest 30 °C(49.4%). By performing an ANOVA between the crude oil removal rates of the treatments at each temperature(Table S2), it was found that surfactant addition was signif icant at 20 °C and 25 °C but insignif icant at 30 °C. The addition of organic matter and oxygen availability were highly signif icant at all three temperatures. For all temperatures,treatments that included the addition of organic fertilizer(treatments 3, 4, 9, 10, 15, and 16) always had the highest crude oil removal rates.

Ef fects of surfactant on bioremediation of petroleum-contaminated soils

The ef fects of the SDS and Tween80 surfactants on crude oil removal rates were relatively small and insignif icant(Table S3). Based on the ANOVA of the CORRs corresponding to each treatment, variations in temperature,organic additives and oxygen availability have extremely signif icant ef fects on crude oil removal rates.

Ef fects of organic matter addition on the remediation of petroleum-contaminated soil

Fig. 3 Removal rate crude oil from petroleum-polluted soils under dif ferent treatments

Treatments with the same type of organic matter addition always had their highest crude oil removal rate at 25 °C(Fig. 3). Therefore, this temperature is the optimum for the bioremediation of petroleum-contaminated soils. This is likely to be caused by lower temperatures (20 °C) inhibiting microbial growth and higher temperatures (30 °C) increasing the toxicity of petroleum hydrocarbons which negatively af fects microbial degradation. The ANOVA for each type of organic matter treatment showed that surfactant and temperature were highly signif icant factors for the rate of crude oil removal (Table S4).Between treatments without organic matter addition and treatments with organic matter, the addition of the SDS surfactant (treatments 13 and 14) led to the highest crude oil removal rates, whereas treatments without surfactant (treatments 1 and 2) had the lowest crude oil removal rates. This is because surfactants solubilized the crude oil in the soil and increased the utilization of hydrocarbons by the microbes,thus increasing the rate of removal. In treatments with corn cob addition, the highest CORR was in treatments without surfactant, and the lowest in treatments with SDS addition.This might be because the corn cobs increased local surfactant concentrations in the soil, which increased surfactant toxicity.

Ef fects of the addition of oxygen provider on the remediation of petroleum-contaminated soils

The addition of the H2O2oxygen provider led to higher crude oil removal rates. All fungi selected for this study were aerobic. During the oxidation of organic matter by aerobic microbes, atomic oxygen is the terminal electron acceptor in the respiratory electron transport chain (ETC),and oxygen ultimately binds with hydrogen to form water.Large amounts of energy are released during electron transfer processes and this energy is used for cellular physiological functions, growth, and metabolism. During the degradation of petroleum hydrocarbons, the microbes will extract hydrogen atoms from the carbon atoms they were bound to and will subsequently react with atomic oxygen.The energy produced by this reaction will then be used by the microbes to maintain their growth and biosynthetic reactions. Since petroleum hydrocarbons are virtually free from oxygen atoms, they are reduced to a greater degree than most other organic compounds. Therefore, petroleum hydrocarbons require a large amount of oxygen to be oxidized. It has been estimated that the decomposition of 1 g of petroleum requires 3–4 g of oxygen (Liu et al. 1993).The ANOVA on the CORRs associated with each oxygen provider treatment showed that temperature, organic matter addition, and surfactant addition had extremely high ef fects on the CORR (Table S5).

Discussion

The Crude Oil Removal Rate of each treatment at 20 °C indicates that the rate of crude oil removal over the f irst 14 days accounted for most of the total amount removed in all experimental groups and controls. The rate of removal was always higher in the treatment groups with hydrocarbon- degrading fungi than in the controls, indicating that the addition of exogenous fungi promoted the removal of crude oil from soils. The 15–28 days crude oil removal rates of treatments 1 and 2 were relatively high, which might be because the lack of organic matter slowed the rate of microbial growth, while the lack of surfactant limited the availability of the petroleum hydrocarbons which delayed the removal of crude oil. At 20 °C, treatment 14 had the highest rate of crude oil removal (81.8%) after 28 days of treatment.

Compared with the results obtained at 20 °C, the f irst 14 days and the 15–28 days crude oil removal rates of the controls at 25 °C were both high. It is possible that the higher temperature led to the proliferation of hydrocarbondegrading bacteria during the 15–28 days period, thus increasing its crude oil removal rate (Vasilyeva et al. 2020).Treatments 7–12 included the addition of the Tween80 surfactant, whereas treatments 13–18 included the addition of the SDS surfactant.

The results indicate that the non-ionic Tween80 surfactant inhibited hydrocarbon degradation in the f irst 14 days. This could be attributed to the surfactant enhancing oil adsorption by the soil, which obstructed and delayed the release of hydrocarbons and thus inhibited and delayed crude oil degradation. Non-ionic surfactants are readily biodegraded and they ef fectively serve as another source of carbon for petroleum hydrocarbons (Gaylarde et al. 1999; Allsopp et al.2004; Zhi 2006). Since the critical micelle concentration(CMC) of Tween80 is relatively low, the relative micelle concentration was quite high. As the microbial population in the Tween80 treatment increased, the rate of hydrocarbon degradation by the microbes also increased and that is why the overall crude oil removal rates of the Tween80 treatments were not signif icantly lower than those of other treatments. In treatments 5 and 6, corn cobs and hydrocarbons both served as carbon sources for microbial proliferation,especially in the early stages of growth, but carbon availability was lower with fresh corn cobs than with organic fertilizer. Consequently, the initial rate of microbial growth in the treatments with corn cobs added were slower than those with organic fertilizer, which resulted in lower 14-days crude oil removal rates in the former. Nevertheless, the rate of crude oil removal in the corn cob treatments increased after their microbial populations increased. At 25 °C, treatment 10 had the highest crude oil removal rate after 28 days(81.5%).

This might be because the degradation intermediates of Tween80 are more toxic than Tween80 itself (Ding et al.2008), and the increased concentration of these intermediates at 30 °C inhibited hydrocarbon degradation by the microbes. Furthermore, the increased toxicity of the hydrocarbons at high temperatures decreases the proportion of their biodegradable component, which then decreases rate of crude oil removal and microbial activity (Che and Yi 2003;Zhang et al. 2008a). In some treatments, the organic matter additives became the primary source of carbon instead of the more toxic hydrocarbons, which reduced hydrocarbon degradation especially in the early 14-days period, and thus decreased crude oil removal rate. At 30 °C, most of the experimental treatments had lower total crude oil removal rates than the controls. This may have been caused by the high temperature increasing the rate of soil microbe growth in the controls, which then increased the rate of hydrocarbon degradation. This could also be explained by the lack of additives in the controls which accelerated moisture evaporation at 30 °C compared with the other treatment groups.The resulting improvement in soil ventilation then increased hydrocarbon volatilization.

In treatment 2, which only included the addition of exogenous fungi, a thick mycelial membrane formed on the surface of the soil due to rapid microbial growth, (a result of ample nutrient availability in the early stages), which inhibited the volatilization of petroleum hydrocarbons. This may also explain the low rate of oil removal of other treatments compared with the controls.

The highest rate of crude oil removal were at 25 °C(74.8% average), and the lowest were at 30 °C (49.4% average). This might be because low temperatures are detrimental to microbial proliferation, whereas high temperatures can signif icantly increase the toxicity of crude oil. Both of these ef fects reduce or inhibit hydrocarbon degradation by microbes and negatively af fect microbial activity (Che and Yi 2003; Zhang et al. 2008b). Therefore, the selection of a suitable treatment temperature will signif icantly improve the rate of crude oil removal. An ANOVA between the CORRs of the treatments at each temperature, it was found that the addition of surfactant was signif icant at 20 °C and 25 °C but insignif icant at 30 °C (Table S2). The addition of organic matter and oxygen availability were highly signif icant at all three temperatures. The addition of organic fertilizer (treatments 3, 4, 9, 10, 15, and 16) always had the highest rates of crude oil removal, indicating that microbes require organic matter as a source of carbon and organic/inorganic products as electron acceptors for the biosynthetic reactions that occur during hydrocarbon degradation. The addition of organic fertilizer also provides the microbes with a highly available source of nutrients, which promotes their proliferation and thus increases the crude oil removal rate (Lopes et al. 2006).

Treatments without the addition of surfactant and treatments with Tween80 had their highest CORRs at 25 °C,whereas the treatments with SDS addition had their highest rates of crude oil removal at 30 °C (Fig. 2). This indicates that the optimal temperature for the use of Tween80 is 25 °C,whereas the optimal temperature for SDS is 30 °C, which is consistent with the f indings of Abayneh and Chen ( 2018).

Among the treatments without the addition of surfactant,adding corn cobs led to the highest crude oil removal rates,but with the addition of surfactant, the addition of the organic fertilizer was associated with the highest crude oil removal rates. This might be because the soil ventilation and nutrients provided by corn cobs in surfactant-less soils are important for microbial growth and the electron transfer processes that occur during hydrocarbon degradation. In treatments with the addition of surfactant, high surfactant concentrations may have inhibited the microbial degradation of hydrocarbons in the micelles. Under these circumstances,the addition of organic fertilizer, the corn cobs, provided the nutrients necessary for microbial growth which increased the rate of crude oil removal. Between the treatments without the addition of surfactant and treatments with Tween80, an oxygen availability of 2 (treatments 4 and 6) was associated with the highest crude oil removal rates. As for the treatments with the addition of SDS, an oxygen availability of 1(treatments 2 and 5) led to the highest rates of removal. This is possibly because the addition of the H2O2oxygen provider with SDS and corn cobs present was toxic for the microbes which decreased the crude oil removal rate.

All treatments with the same addition of organic matter always had their highest rate of removal at 25 °C (Fig. 3).Treatments with the addition of H2O2had higher crude oil removal rates than those without H2O2. This may be because the oxygen provider increased oxygen availability for microbial growth and hydrocarbon metabolism, which accelerated the aerobic metabolism of hydrocarbons and increased crude oil removal. In a study of microbe-mediated hydrocarbon degradation in oil-polluted groundwater, Li et al. ( 2018)found that nutrient addition without additional ventilation led to low hydrocarbon degradation rates. Conversely,hydrocarbon degradation rapidly accelerated when both nutrients and oxygen were provided.

Xu et al. ( 2019) found that the rate of hydrocarbon degradation in estuary sediments is largely dependent upon oxygen availability as it decreases in proportion with oxygen availability. It has been shown that H2O2addition increases the rate of degradation of petroleum hydrocarbons because hydrogen peroxide provides oxygen to the microbes and directly oxidizes some of the petroleum hydrocarbons (Xiao 2005). H2O2spontaneously decomposes to release O2which may then be directly consumed by the microbes or after it has been solubilized in water.

The ANOVA on crude oil removal rates associated with each oxygen provider showed that temperature, the addition of organic matter, and use of surfactant were highly ef fective (Table S5). Crude oil removal rates were always the highest at 25 °C at both oxygen availability levels (Fig. 3).Treatments that included the addition of organic fertilizer also had the highest rates of oil removal. Therefore, at each level of oxygen availability, 25 °C was the optimal temperature for microbe-mediated hydrocarbon degradation, and the addition of organic fertilizer had the largest positive ef fect on this process. In treatments without an oxygen provider,those without a surfactant had the highest crude oil removal rates. Treatments with an oxygen provider and with SDS surfactant addition had the highest crude oil removal rates.In the absence of an oxygen provider, microbial growth was slow due to low oxygen levels in the soil. A high concentration of surfactant will, in this scenario, suppress the degradation of petroleum hydrocarbons in the micelles and act as an alternate source of carbon to further slow microbial degradation. With the addition of an oxygen provider,microbial growth will be more rapid due to the ample supply of oxygen, which results in rapid metabolization of the surfactants, and also increases the rate of microbe-mediated hydrocarbon degradation.

At each temperature, the rate of crude oil removal associated with treatments that included an oxygen provider, (high oxygen availability), were higher than treatments without an oxygen provider. The microbe-mediated degradation of petroleum hydrocarbons mainly occurs through oxidation.In bioremediation, the oxygen transfer rate is often the limiting factor for biodegradation (Xiao 2005). Therefore, the addition of an oxygen provider will enhance the microbial degradation of petroleum hydrocarbons. At low temperatures(20 °C), treatments with SDS had the highest rate of crude oil removal, while treatments with Tween80 had the lowest. The opposite was observed at 25 °C. Since the critical micelle concentration of Tween80, (a non-ionic surfactant),is lower than that of SDS (an anionic surfactant), the actual concentration of the former is higher.

At low temperatures, both the surfactant and microbes will be less active. High surfactant concentrations will then inhibit microbial utilization of petroleum hydrocarbons in the micelles (Tiehm 1994; Sobisch et al. 2000). Non-ionic surfactants will greatly enhance the solubility of hydrophobic compounds but the self-adsorption of these surfactants could also reduce their adsorption by petroleum pollutants.SDS, which has a higher critical micelle concentration,helps to solubilize petroleum hydrocarbons and promotes their degradation by microbes. At a temperature such as 25 °C, the ability of the surfactants to increase solubility and reduce surface tension will cause the soil-adsorbed petroleum hydrocarbons to migrate into the surfactant-containing aqueous phase, thus increasing their bioavailability (Zhao et al. 2005).

Biological membranes consist of numerous phospholipid molecules, with a strong tendency to adsorb surfactants since they are structurally similar to phospholipids. The permeability of biomembranes changes when they adsorb surfactants, and this increases the rate at which hydrophobic compounds pass through the membrane, thus increasing the rate of hydrocarbon degradation by the microbes (Tan and Zhou 2007). Che and Yi ( 2003) demonstrated that the rate of organic compound degradation increases with surfactant concentration in soils at suitable temperatures. Furthermore,PAHs (Polycyclic aromatic hydrocarbons) degradation rate was higher with non-ionic surfactants than with anionic surfactants, and these f indings are consistent with the results of this study. At high temperatures, high surfactant concentrations become toxic to microbes. Although the microbes can utilize Tween80 and petroleum hydrocarbons as sources of carbon, the degradation intermediates of Tween80 may be more toxic than Tween80 itself (Ding et al. 2008). Therefore,the generation of these intermediates at high temperatures will reduce the ef ficiency of petroleum hydrocarbon degradation by microbes.

Conclusions

The microbial remediation of petroleum-contaminated soils was evaluated with electrical heating and a variety of additive combinations. By analyzing the rate of crude oil removal of each treatment, it was found that:

At 20 °C, the 14 days rates of removal accounted for most of the total crude oil removal in all treatments; only treatments 1 and 2 showed relatively high removal rates in the 15–28 days period. At 25 °C, the 14 days rate of removal was dominant for treatments 1–4, 13, and 18. At 30 °C, the 15–28 days removal rate was dominant for the controls and for treatments 1, 2, 13, 14, 15, and 16. The f irst 14-days crude oil removal rate accounted for most of the total crude removal in other treatments. Other than surfactant and the interactions between organic matter, surfactant and oxygen availability, all other factors and interactions were highly significant for crude oil removal rate. Therefore, variation in organic matter, oxygen availability, and temperature and inter-factor interactions signif icantly af fected the rate of crude oil removal. Of the three temperatures, 25 °C and 30 °C had the highest and the lowest average rates of crude oil removal, respectively. For all three temperatures,treatments that included the addition of organic fertilizer always had the highest rate of removal, and those that included an oxygen provider always had higher crude oil removal rates than treatments without an oxygen provider.In terms of the rate of removal corresponding to surfactant treatment, variations in temperature, the addition of organic matter, and oxygen availability had highly significant ef fects. Between treatments with the same organic matter,the highest removal rate was at 25 °C. Treatments with the addition of H2O2had higher crude oil removal rates than treatments without H2O2. Based on the rates of crude oil removal, the optimum treatments for microbial remediation of petroleum-contaminated forest soils in high and cold regions are: S2W1O1(SDS, organic fertilizer, and H2O2) if soil temperatures are 20 °C (28 days removal rate of 81.8%),S1W1O1(Tween80, organic fertilizer and H2O2) if the soil temperatures are 25 °C (28 days removal rate of 81.5%), and S2W2O1(SDS, corn cob, and H2O2) if soil temperatures are 30 °C (28 days removal rate of 79.9%).