Biodegradation (2015) 26:259–269 DOI 10.1007/s10532-015-9732-7
ORIGINAL PAPER
Bioaugmentation of soil contaminated with high-level crude oil through inoculation with mixed cultures including Acremonium sp. Xiao-Kui Ma . Ning Ding . Eric Charles Peterson
Received: 21 February 2015 / Accepted: 27 April 2015 / Published online: 1 May 2015 Ó Springer Science+Business Media Dordrecht 2015
Abstract Heavy contamination of soil with crude oil has caused significant negative environmental impacts and presents substantial hazards to human health. To explore a highly efficient bioaugmentation strategy for these contaminations, experiments were conducted over 180 days in soil heavily contaminated with crude oil (50,000 mg kg-1), with four treatments comprised of Bacillus subtilis inoculation with no further inoculation (I), or reinoculation after 100 days with either B. subtilis (II), Acremonium sp.(III), or a mixture of both organisms (IV). The removal values of total petroleum hydrocarbons were 60.1 ± 2.0, 60.05 ± 3.0, 71.3 ± 5.2 and 74.2 ± 2.7 % for treatment (I–IV), respectively. Treatments (III–IV) significantly enhanced the soil bioremediation compared with treatments (I–II) (p \ 0.05). Furthermore, significantly (p \ 0.05) greater rates of degradation for petroleum hydrocarbon fractions were observed in
X.-K. Ma (&) N. Ding Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry, Ministry of Education, National Engineering Laboratory for Resource Developing of Endangered Chinese Crude Drugs in Northwest of China, College of Life Sciences, Shaanxi Normal University, Xi’an 710062, Shaanxi, China e-mail:
[email protected];
[email protected] E. C. Peterson Department of Chemical Engineering, Queen’s University, Kingston, Ontario, Canada
treatments (III–IV) compared to treatments (I–II), and this was especially the case with the degradative rates for polycyclic aromatic hydrocarbons and crude oil heavy fractions. Dehydrogenase activity in treatment (III–IV) containing Acremonium sp. showed a constant increase until the end of experiments. Therefore reinoculation with pure fungus or fungal-bacterial consortium should be considered as an effective strategy in bioaugmentation for soil heavily contaminated with crude oil. Keywords Bioaugmentation Acremonium sp. P0997 Reinoculation Fungal-bacterial consortia Crude oil Degradative rates
Introduction Crude oil is a complex mixture of aliphatic and aromatic compounds (Tang et al. 2010), and oil spills and contamination of soil with crude oil have happened frequently around the world during the exploitation and transportation of petroleum through unsuitable operation and leakage (Das and Mukherjee 2007). Pollution arising from oil-related accidents in soil has caused significant and negative environmental impacts and presented substantial hazards to human health (Borole et al. 1997; Das and Mukherjee 2007), and the need for remediation technologies for heavily contaminated sites is urgent.
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There are various physical–chemical approaches available for cleaning up contaminated soil such as incineration, solidification/stabilization, soil vapor extraction, and soil washing (Borole et al. 1997). However, these methods are relatively expensive, which may be ascribed to the fact that the extracted contaminants or incinerated soil need to be further treated or disposed of (Xu and Lu 2010). Compared with these methods, bioremediation demonstrates some advantages, as it is highly efficient and incurs reduced costs without generating further pollution (Das and Mukherjee 2007), with bioaugumentation in particular showing promise for soils heavily contaminated with petrochemicals (Hamdi et al. 2007; Megharaj et al. 2000). Bioaugmentation improves in situ biodegradation capacities under highly contaminated conditions in soils by the application of indigenous or exogenous microbial cultures exhibiting the ability to degrade target toxic molecules (Das and Mukherjee 2007). For instance, when bioaugmentation with Acinetobacter was applied in a bioremediation of soils polluted with 14,380 mg kg-1 total petroleum hydrocarbons (TPH), an increase of 30.0 % of TPH removal was reported compared with a bio-stimulation treatment without the addition of microbes (Ruberto et al. 2003). However, although some bioaugmentation studies showed a rapid degradation in the initial phase, strong decreases in degradative rates were observed over time, possibly due to low microbe number (Sabate´ et al. 2004). For instance, some measures, such as nutrients supplementation and inoculation with bacterial or fungal consortia or individual bacterium or fungus, had not shown improvements in degradation efficiency in the environments with recalcitrant toxic compounds or high-level crude oil contaminated (Ellegaard-Jensen et al. 2014; Rahman et al. 2003). Fungi can thrive under harsh environmental extremes including conditions such as temperature, water content, pH, or depletion of nutrients, permitting rapid proliferation in the soil matrix. This tolerance to a wide range of conditions may allow fungi to play a significant role in elimination of oil hydrocarbons and recalcitrant toxic compounds such as polycyclic aromatic hydrocarbons (PAHs) in bioremediation processes (Machin-Ramirez et al. 2010). Moreover, fungi also have the ability to synthesize relatively non-specific enzymes to degrade complex recalcitrant toxic compounds, including high molecular weight aromatic structures (Husaini et al. 2008).
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For instance, fungal benzo[a]pyrene degradation has been shown to be more effective than that of bacterial cultures (Machin-Ramirez et al. 2010). Furthermore, bioaugmentation with fungi or fungal-bacterial consortia has been confirmed to be more efficient than pure bacterial culture approaches (Vogel 1996; Zhou et al. 2014). Thus, degradative techniques using fungi or mixed consortia have attracted attention in the bioremediation and bioaugmentation of some PAHs in solution (Heinaru et al. 2005; Llado et al. 2013; Machin-Ramirez et al. 2010). However, although some successful fungal applications have been reported (Rodriguez–Rodriguez et al. 2014), generally speaking, fungal bioaugmentation has been less studied than bacterial-mediated bioremediation processes. In addition, to date, no studies have investigated the bioaugmentation with reinoculation of either fungi or fungal– bacterial consortia for bioremediation of soils contaminated with high levels of crude oil (Rodriguez– Rodriguez et al. 2014). In a previous study in solid-phase sewage sludge systems under nonsterile conditions, the reinoculation of biopiles with blended mycelium (Trametes versicolor) exerted a favorable effect on the removal of pharmaceuticals (Rodriguez–Rodriguez et al. 2014). In this context, the present work aims to investigate the possibility of reinoculation of different cultures including pure bacterial or fungal culture and a consortium of these two microbes to enhance the global elimination in the system with bacterium firstly inoculated. In our previous study, the fungus Acremonium sp. was identified and confirmed to degrade PAHs and crude oil with relatively high efficiency in our laboratory (Ma et al. 2014). And, a strain of Bacillus subtilis possessing ability to degrade crude oil was isolated as well, and was used as a bacterial model organism in this study. After a first inoculation with the B. subtilis pre-grown in soil with high level crude oil contaminated, three different biomass reinoculation approaches, i.e., with pure B. subtilis or pure Acremonium sp. or a consortium of both these two strains, are discussed.
Materials and methods Soil Soils freshly contaminated with crude oil were collected from the top 10 cm layer in Northern China
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oil field, Yan’an, China, with a total TPH concentration of 60,000 mg kg-1. The physico-chemical characteristics of the soil were as follows: sand, 21.4 %; silt, 40.6 %; clay, 38.0 %; pH, 7.2; total nitrogen, 490 mg kg-1; total phosphorus, 370 mg kg-1. The average contamination in soil was adjusted to 50,000 mg of crude oil per kilogram of soil (50,000 mg kg-1 TPH) by the addition of lowcontaminated soil from same area, which was consisted of 65.0 % aliphatic hydrocarbons, 18.0 % PAHs and 20.0 % asphaltenes. To ensure the reproducibility of results, the soil was air-dried under a ventilation hood at 25 °C for 48 h, and carefully sieved with a 2 mm mesh sieve (Sabate´ et al. 2004). The soil was then homogenized by hand with shovels and stored at 4 °C for bioaugmentation experiments.
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culture in the bioaugmentation experiments. Similarly, to generate fungal cultures for bioaugmentation, Acremonium sp. was grown for 72 h in Potato Dextrose Agar (PDA) liquid medium at 30 °C/ 100 rpm. The culture was centrifuged and the obtained fungal mycelia were washed twice with 0.1 M phosphate buffer, and were utilized as fungal inocula. Experimental design and treatments
The hydrocarbon-degrading bacterial and fungal strains used in this study were isolated from petroleum contaminated soil near a waste drilling pool in a Northern China oil field, Yan’an, China and selected based on their ability to degrade crude oil and PAHs in our laboratory. The bacterial and fungal isolates were identified as B. subtilis (CCTCC AB 2014248) and Acremonium sp P0997 (CCTCC M 2013569), based on evaluation by standard biochemical tests, observation of morphological characteristics, and molecular methods. B. subtilis has been confirmed to degrade separate PAHs (e.g. naphthalene, fluorene, phenanthrene, anthracene, fluoranthene) and crude oil with 95.5 ± 4.3, 85.7 ± 2.2, 72.5 ± 3.0, 57.4 ± 2.7, 27.4 ± 1.8 and 52.0 ± 3.8 %, respectively. Furthermore, Acremonium sp P0997 can degrade PAHs simultaneously existing in media with high efficiency (Ma et al. 2014), also achieving hydrocarbon degradation of 54.0 ± 4.2 % in our laboratory.
All biodegradation experiments to determine the degradation efficiency of different treatments were carried out in a series of identical plastic trays with a total volume of about 10 L (17 cm length, 22 cm width, and 32 cm height). The soil was stirred in an agitator to obtain a homogeneous soil-contaminant mixture. It was assumed that abiotic and volatilization losses were equal in all the treatments. Approximate 2.5 kg of contaminated soil was individually stacked in each tray (8–10 cm height). Deionized water was added twice per week to maintain water content in the soil to approximately 60.0 % of the water-holding capacity (Chagas-Spinelli et al. 2012). To investigate the role of reinoculation of fungal species in bioaugumentation, at day 100, the biopiles inoculated with B. subtilis (108 cells g-1 soil) were separated into four series that were subject to different reinoculation strategies to compare degradative rates in contaminated soil: with no further inoculation (I), and further treatments involving a second inoculation with B. subtilis (108 cells g-1 soil) (II), Acremonium sp. mycelia at a concentration of 10 mg g-1 soil (wet weight) (III) or a consortium comprised of both B. subtilis (0.5 9 108 cells g-1 soil) and Acremonium sp. mycelia (5 mg g-1 soil) (IV). Remediation experiment at the same conditions without any inoculation was used as negative control for physicochemical degradation of crude oil during experiments.
Culture preparation
Sampling
To generate bacterial inocula for bioaugmentation, B. subtilis was grown in nutrient broth at 28 °C and 160 rpm for 24 h. Then, the culture was centrifuged at 12,000 rpm for 10 min. The cell pellet was washed twice with 0.1 M phosphate buffer pH 7.0 (Mahmoud et al. 2014), and re-suspended in the fresh phosphate buffer to obtain a same volume as the broth volume used, and the resulted suspension was used as bacterial
Six subsamples from each treatment were combined as one sample for a given treatment at different time points (on days 0, 20, 40, 60, 80, 100, 120, 140, 160) (Chagas-Spinelli et al. 2012). All samples were stored at -20 °C and thawed 3 days before analyses as described previously (Stemmer et al. 1998). The residual oil in 10 g samples was extracted by the conventional Soxhlet extraction (Lu et al. 2009).
Microorganisms
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Aliphatic, aromatic and polar fractions were separated according to the methods given previously (Bastow et al. 2007). GC/MS analyses The saturated hydrocarbons and aromatic hydrocarbons in crude oil were analyzed by GC/MS. The GC/ MS was performed with a Shimadzu QP2010 GC–MS coupled to a DSQ II mass spectrometer. GC/MS spectra were obtained in the electron impact mode (70 eV), scanning from m/z 40 to 1000 every second. A RTX-5MS elastic silica capillary column was used with an injection volume of 1 lL and n-helium as a carrier gas (37 kPa, 1 mL min-1). The light and heavy aliphatic contents were calculated from areas with \C22 and CC22, respectively. The identification of the most relevant peaks such as PAHs, light and heavy aliphatic compounds was grounded on total mass spectra, working with the mass spectra databases (NIST/EPA/NIH Mass spectral library NIST2000, Wiley/NBS Registry of Mass spectral Data 7th Ed., electronic versions).
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Microbial dehydrogenase activity (DHA) in soil samples was determined by monitoring the rates of reduction of 2, 3, 5-triphenyltetrazolium chloride to triphenylformazan according to a modified method described previously (Lu et al. 2009). Specifically, 5 mL acetone was used to terminate the reaction after 10 min, and then the samples were centrifuged and the supernatant was measured by absorbance at 485 nm. Results were expressed as lg triphenyl tetrazolium formazan g-1 soil 3 h-1. Statistical analysis Results were evaluated for statistical significance by a one way analysis of variance ANOVA using the Statistical Package of the Social Science (SPSS) program. The variance and significant differences of TPH concentration and enzymatic activity among various treatments were analyzed by least significant difference (LSD). The level of statistical significance was defined at p \ 0.05. All experiments were run in triplicate.
Microbial enumeration
Results
Quantification of the total number of separate bacteria and fungi in the soil samples was performed as a standard method described previously (Salminen et al. 2004). For each replicate treatment, 2 g of fresh soil sample was mixed with 20 mL of autoclaved NaCl solution (0.9 %), including 1 mL Na4P2O7 (0.2 g L-1) and 0.1 mL Tween 80 (2.0 %), with subsequent serial dilution. Bacteria and fungi were grown on nutrient agar and PDA, respectively, with the addition of 50 mg mL-1 of the fungicide cycloheximide or the bactericide streptomycin. The plates were incubated at 30 °C, and colonies were enumerated after 24 and 48 h separately for bacteria and fungi (Trindade et al. 2005). Results were expressed as the number of colony-forming units per g of dry soil (CFU g-1 soil).
Removal of TPHs and microbial growth
Biochemical analyses The total hydrolytic activity in soil samples was measured by fluorescein diacetate (FDA) hydrolysis using a previous method (Adam and Duncan 2001), with results expressed as lg fluorescein g-1 soil 3 h-1.
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As shown in Fig. 1a, the most rapid degradation of crude oil across all treatments was obtained during the initial 20 days of bioremediation, followed by a relatively stable increase over time before reinoculation. A maximum degradation of 10.0 % TPH was achieved in the negative control after 180 days. In comparison, the total TPH removal efficiency for treatments (I–IV) was observed to be 60.1 ± 2.0, 60.1 ± 3.0, 71.3 ± 5.2 and 74.2 ± 2.7 % after 180 days, respectively. It is interesting to note that bioaugmentation in treatments (III–IV), which utilize fungal species for reinoculation, were both shown to have a significantly higher TPH removal efficiency compared to that in treatment (I–II), which involved bacterial augmentation only. Furthermore, the reinoculation of treatment (II) with B. subtilis displayed no positive influence on TPH removal compared with that in treatment (I), which was only inoculated initially, suggesting the introduction of fungi is responsible for the improved TPH removal observed in treatments (III–IV).
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263 b Fig. 1 Removal of total petroleum hydrocarbons (TPH) and
microbial growth. a TPH degradation levels in soil as function of sampling time. b Growth patterns in colony-forming units of bacteria in soil contaminated with crude oil. c Growth patterns in colony-forming units of fungi in soil contaminated with crude oil. Treatments: inoculation with B. subtilis (108 cells g-1 soil) with no further inoculation (I), and further treatments involving a second inoculation with B. subtilis (108 cells g-1 soil) (II), Acremonium sp. mycelia at a concentration of 10 mg g-1 soil (wet weight) (III) or a consortium comprised of both B. subtilis (0.5 9 108 cells g-1 soil) and Acremonium sp. mycelia (5 mg g-1 soil) (IV). Remediation experiment without any inoculation was used as negative control for physicochemical degradation of crude oil during experiments
reinoculation. However, the total number of separate bacteria and fungi for individual reinoculation treatments (II–IV) was observed to increase after reinoculation on day 100 (Fig. 1b, c), followed by a sharp decrease on day 120 and stabilization on days 140–160. Interestingly, at the end of the experiment on day 160, the highest populations for both organisms were observed in treatment (IV), which involved reinoculation with both bacteria and fungi, and the highest TPH removal were also demonstrated (Figs. 1a, c, 2a). These results suggest that both bacteria and fungi play an important role in TPH removal, and that the presence of both types of organisms may result in synergistic degradation activity during bioaugumentation. Degradation of petroleum hydrocarbon fractions and PAHs
As shown in Fig. 1b, c, the total number of separate bacteria and fungi in treatment (I–IV) displays significant variation compared with that in the negative control after reinoculation. The total number of separate bacteria and fungi across all treatments significantly increased after B. subtilis inoculation, reaching a peak on day 40 (Fig. 1b, c) before
To further understand the biodegradative differences when utilizing fungi and mixed cultures in bioaugumentation, light (C14–C21), heavy (C22–C31) and polyaromatic hydrocarbon (PAH) fractions in crude oil across all treatments at the end of experiments were analyzed by GC–MS. As shown in Fig. 2a, in the case of treatment (I), which involved a single inoculation of B. subtilis, the total degradation of the light fraction was observed to be up to 93.7 ± 2.7 %, while the heavy fraction and PAH fraction were degraded by 75.0 ± 1.1 and 62.1 ± 1.2 %, respectively. Moreover, highly similar fraction degradation was observed in treatment (II), which was reinoculated with B. subtilis. Interestingly, reinoculation with Acremonium sp. (treatment III) and the mixed Acremonium sp. and B. subtilis culture (treatment IV) significantly
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Fig. 2 Degradation of polycyclic aromatic hydrocarbons (PAHs) and petroleum hydrocarbon fractions. a The total degradation of petroleum fraction in soil after 180 days. b PAHs removed with time after reinoculation. The asterisk (*) means statistically significant
promoted degradation of all fractions, especially with PAH removals of 87.6 and 95.7 %, respectively (Fig. 2a), while in the case of treatment (II), 62.1 % removal was just obtained. Moreover, treatment (IV) not only showed the highest degradation rate of individual light, heavy and PAH fractions, but also demonstrated a significant enhancement in degradation (p \ 0.01) for heavy and for PAH fractions (Fig. 2a), with a 33.0–34.0 % increase in PAHs removal in treatment (IV) compared to treatments (I–II). Additionally, heavy fraction degradation in treatment (IV) increased by 10.0 and 9.0 % compared to that in treatment (I) and (II), while the light fraction removal was also enhanced by 4.0 and 5.0 %, respectively. Treatment (III) also showed a significant difference (p \ 0.01) in degradation compared to
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treatments (I–II), as the removal was increased by 25.0 and 24.0 % for PAH removal, and 4.0 and 3.0 % for heavy fraction removal, respectively (Fig. 2a). A significant difference (p \ 0.05) was also found in biodegradation of PAHs and heavy fractions between treatment (III) and treatment (IV) (Fig. 2a), wherein the PAHs and heavy fraction degradation in treatment (III) were 9.0 and 6.0 % less than that in treatment (IV). These results emphasized the apparent benefits of using mixed inocula for reinoculation during bioaugumentation compared to use of pure fungal cultures for the purposes of biodegradation of heavy hydrocarbons and PAHs. Degradation of PAHs in soil among different treatments from the 100th to 180th day (i.e. after reinoculation) of bioremediation experiments can be seen in Fig. 2b, in which a sustained increase in PAHs degradation is observed over time in treatment (IV). However, lower PAHs degradation in treatment (III) was observed compared to that in treatment (I) and (II) on the 140th day (Fig. 2b). These results suggest that the degradation process in soil may be very complex with factors such as interspecies interaction affecting the degradative rates. Furthermore, the sustained increase of PAHs degradation with time in treatment (IV) further confirmed the apparent benefits of using mixed inocula to re-inoculate compare to use of pure fungal or bacterial cultures. To better understand how the interaction of fungi and bacteria during reinoculation, GC–MS chromatographs were examined to investigate the dynamic changes in different hydrocarbon species fractions over time. On day 120, that is 20 days after the introduction of fungal cultures, a substantial increase in light fraction hydrocarbons (i.e. C16) in treatment (III) is apparent (Fig. 3b), compared to treatment (I) without the fungus reinoculation (Fig. 3a). This observed increase in hydrocarbon fractions may be explained by a previous study in which fungi cometabolically oxidize or mineralize some PAHs to less toxic products (Saraswathy and Hallberg 2002). That is, it has been well documented that fungal enzymes can effectively attack and open aromatic ring structure, facilitating further degradation and such activity may explain such a large increase in light fraction C16 hydrocarbons in treatment (III). This possibility is further supported by the fact that higher heavy fractions such as C22 are observed in the absence of fungal reinoculation (Fig. 3a). As can be seen in
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Fig. 3 Gas chromatography fingerprinting of saturate fraction. a Bioaugmentation in treatment (I) on 120 day. b Biodegradation in treatment (III) on day 120. c Bioaugmentation in treatment (III) on day 140. d Bioaugmentation in treatment (IV) on day 120. e Bioaugmentation in treatment (IV) on day 140
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Fig. 3c, by day 140, this large spike of C16 hydrocarbons has been substantially metabolized, while, a dramatic increase in C22 was detected in treatment (III), which may depict a change of aliphatic hydrocarbon chromatograms corresponding to treatments (I–II) after 100 days. Interestingly, after the re-introduction of both bacterial and fungal species in treatment (IV), no dramatic C16 spike was observed on day 120 (Fig. 3d), and by day 140 all fractions showed the lowest concentrations across all treatments, indicating excellent hydrocarbon degradation (Fig. 3e). The difference between treatments (III) and (IV) may be explained by the fact that while fungi are thought to be capable of metabolizing some aromatic compounds, they may lack the essential enzymes to transform the co-oxidation products (Atlas and Cerniglia 1995). Thus, these results suggest the introduction of bacteria alongside fungi in bioaugmentation can promote further hydrocarbon degradation by utilization of fungal co-oxidation products. FDA activity and dehydrogenase activity FDA hydrolysis has been widely used as an accurate and simple method to determine amounts of enzymatically active fungi (So¨derstro¨m 1977) and bacteria (Schnu¨rer and Rosswall 1982) in soil, and has been employed here to investigate the enzymatic degradation activity of TPH across treatments. As shown in Fig. 4a, after a rapid decline of FDA activity on day 40, a gradual increase in activity was observed before reinoculation, likely due to microbial adaption to hydrocarbon utilization. Furthermore, on the 120th day, in spite of a significant increase in the microbial population after addition of Acremonium sp. (Fig. 1b, c), FDA activity showed a downward trend (Fig. 4a) in treatment (III). However, after a sharp decrease to the background level between 20 and 40 days, FDA activity in the control remained unchanged until the end of the experiment. These results may suggest that reinoculation treatments may bolster significantly microbial counts, but the increase of microbial population may not always result in the prompt increase of FDA activity and the associated high degradation activity. DHA in soil is an index for overall microbial activity such as total oxidative, presenting a more accurate measure of microbial capability for petroleum hydrocarbon degradation (Lu et al. 2009). As
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Fig. 4 a Fluorescein diacetate (FDA) activity in soil samples over 180 days of bioremediation. b Microbial dehydrogenase activity (DHA) in soil samples over 180 days of bioremediation. DHA results are expressed as lg triphenyl tetrazolium formazan (TPF) g-1 soil 3 h-1
shown in Fig. 4b, before reinoculation, the DHA reached a high point on day 40 and then dropped quickly on day 60, then maintained a relatively stable period before reinoculation. After reinoculation, a relatively constant increase in DHA in treatments III and IV was observed and which was shown to correlate with the high degradation efficiency (Figs. 1b, c, 4b). Combined with the above results of TPH degradation experiments in Fig. 1a, these results demonstrate that the high degradation efficiency in treatment (III–IV) may not correlate with the FDA activity in soil during bioaugmentation, but rather to DHA and the increase of microbial population, especially the number of the functional microbes. The high degradation efficiency in treatment (III–IV) may be
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primarily from the introduction of the fungus Acremonium sp. with the enzymatic degradation activity.
Discussion The most extensive studies in bioaugmentation have focused on treatment of sites contaminated with recalcitrant toxic substances such as aromatic compounds by bacteria as well as fungi and microbial consortia (Zhou et al. 2014), but there are few reports on the bioaugmentation with a reinoculation of fungal–bacterial consortia or fungi and their use in cleaning up soils with high levels of crude oil contamination (Rodriguez–Rodriguez et al. 2014). This work presented hereby has evaluated the potential use of reinoculation of filamentous fungal culture or mixed fungal–bacterial culture for biodegradation assays, demonstrating reinoculation with pure Acremonium sp. and the fungal–bacterial consortium have showed high degradation efficiency in soil heavily contaminated with crude oil. Therefore, reinoculation with the pure fungus or the fungal-bacterial consortium should be considered as an effective strategy in bioaugmentation process for the treatment of soil heavily contaminated with crude oil. There were variations in the levels of crude oil depletion and PAHs degradation for the two reinoculation treatments involving Acremonium sp.. However, when compared among treatments (I–IV), much higher removal values were found to be consistently achieved in the specific treatment (IV). These results suggest that the reinoculation of specific fungalbacterial co-cultures enhance biodegradation compared with that of respective pure cultures. Filamentous fungi have demonstrated the ability to degrade complex recalcitrant toxic compounds such as PAHs (Machin-Ramirez et al. 2010) and while pyrene (4-ring PAH) served as a sole carbon source, a maximum of 75.0 % removal for pyrene at 50 mg L-1 was achieved for axenic cultures of Trichoderma sp. (Saraswathy and Hallberg 2002). In our study, the reinoculation in treatments (III) and (IV) had a distinct positive impact on degradation efficiency, while no notable change in degradation was observed in treatment (II) with pure bacterial strain reinoculation. These results suggest that reinoculation with pure specific pure fungus or fungal-bacterial consortium represents an attractive alternative for the elimination
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of high-level crude oil in soil, which provided more information than a previous report about fungal reinoculation (Rodriguez–Rodriguez et al. 2014). The possibility that a new addition of pure fungal or fungal-bacterial biomass presented hereby could enhance the global elimination in the system becomes important in the design of a bioaugmentation process. Further removal of crude oil was accomplished after the reinoculation step with treatment (III–IV); however, no significant additional effect was obtained with reinoculation of pure bacterium. The total elimination of 71.3 ± 5.2 and 74.2 ± 2.7 % for crude oil in the reinoculation of separate pure fungus and the consortium of both B. subtilis and Acremonium sp. is much remarkable, considering the strikingly high concentration of crude oil in the soil. Compared with other reported studies, the degree of TPH reduction in treatment (III–IV) was relatively high (Sabate´ et al. 2004). This result resembles the 63.0 % elimination reported previously from a biopile with a microcosm with B. subtilis DM-04 and Pseudomonas aeruginosa (Das and Mukherjee 2007). According to one study, the high degree of biodegradation of TPH (more than 90.0 %) was observed after ex situ bioremediation, but the total TPH content in the contaminated soil was just 5.2 g kg-1 (about 0.05 %), much less than that (5.0 %) in this study (Das and Mukherjee 2007). Overall, the reinoculation step presented hereby enhanced the removal of TPH in soil bioaugmentation using the pure fungus or the consortium of fungus and bacterium. Moreover, for PAH removals, reinoculation with the pure fungus or the consortium of fungusbacterium was more efficient than with pure bacterial reinoculation. The elimination of the heavy fraction and PAH fraction in crude oil during the postreinoculation period was especially efficient for reinoculation with Acremonium sp. (treatment III) and the mixed Acremonium sp. and B. subtilis culture (treatment IV). Interestingly, the apparent benefits of using the mixed inocula or the pure fungal culture to re-inoculate compare to use of the pure bacterial culture presented hereby provided more information about reinoculation bioaugmentation than a previous report concerning the reinoculation of solely blended mycelium (Rodriguez–Rodriguez et al. 2014). The enhanced performance in removal of crude oil was achieved with the application of the pure fungal culture and the mixed culture for reinoculation, thereby improving the cost-effectiveness of the
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process. Therefore, reinoculation of using mixed inocula with fungus and bacterium or pure fungus culture may be an alternative considered as a step in bioaugmentation process design for the treatment of high-level crude oil in soil. This result could be ascribed to enhanced DHA and the increase of microbial population after these reinoculations. In some cases (especially for removal efficacy), the 180-d treatment time was a little long to completely remove most of the compounds. In this respect, a proper optimization in terms of the treatment period especially the time after reinoculation among other aspects should be performed, considering the degree of removal or time required for the potential application of the process. Conclusions In summary, an efficient and enhanced removal was achieved with reinoculation of Acremonium sp. and a mixed culture of Acremonium sp. and B. subtilis for the bioremediation of soil heavily contaminated with crude oil. Thus, this work showcases the value of pure fungi and consortium of fungus and bacterium in bioaugmentation techniques to achieve improved bioremediation of heavily contaminated soil. Ongoing efforts are dedicated to understanding the mechanisms of the enhanced bioremediation of the mixed fungalbacterial culture, so as to guide future design for optimal bioaugmentation strategies. Acknowledgments The project was supported by the Fundamental Research Funds for the Central Universities of China (No. GK201402022). Conflict of interest The authors declare that they have no conflict of interest.
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