Bacterial community response to modified-biochar and exogenous fungi inoculation during degradation of oil-contaminated clayey sediment in slurry phase

doi:10.21203/rs.3.rs-3087242/v1

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Bacterial community response to modified-biochar and exogenous fungi inoculation during degradation of oil-contaminated clayey sediment in slurry phase

https://doi.org/10.21203/rs.3.rs-3087242/v1

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published 21 Feb, 2024

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Bioremediation strategies for oil-contaminated clayey sediment are still a major environmental concern due to their reluctance to conventional treatment techniques. The main objective of this study was to evaluate the sequential application of modified biochar and exogenous fungus in slurry phase degradation of crude oil-contaminated clayey sediment. Biochar modification using acid and rhamnolipid increased its surface area from 2.544 to 25.378 m²g^-1, followed by a corresponding increase in the hydrogen-carbon and oxygen-carbon ratio, indicating improved stability, polarity, and oxygen contents. The maximum TPH removal efficiency of 82.4% was achieved in treatments with the acid-rhamnolipid-treated biochar, followed by a corresponding increase in FDA hydrolysis activity. Furthermore, high-throughput 16S RNA gene sequencing was employed to identify changes in the bacterial community structures of the original sediment and treatments before and after fungal inoculation. This highlighted Proteobacteria as the dominant phylum, followed by Campilobacterota, Bacteroidota, Actinobacteriota, and Desulfobacterota. In addition, it was observed that fungal inoculation promoted the proliferation of some specific TPH-degraders. Specifically, ASVs associated with Hyphomonas adhaerens and Marinobacter mobilis accounted for 77% and 5% of the total degrading populations in treatments where TPH removal was highest. Findings in this study provide valuable insights into the effect of biochar in the treatment of crude oil-contaminated marine sediments, and a better understanding of the fundamental role of exogenous fungus in the proliferation of efficient TPH-degraders towards effective degradation.

Modified biochar

Clayey sediment

Bacterial community

Exogenous microorganisms

Slurry soils

Bioremediation

Acid-treated biochar accelerated degradation rate within the first 21 days
Taxonomic groups originally present in low abundance improved predominantly after fungal addition.
Fungi promoted the abundance of pollutant-degrading bacterial species while impeding the abundance of non-degraders.
Overall degradation process impeded bacterial diversity.

Crude oil pollution has impacted the global ecosystem in the last three decades since its first major spill at Prince William Sound in 1989 to the most recent significant pipeline explosion on July 6, 2021, along the Gulf of Mexico. This has motivated intensive research on discovering and developing several mitigative and remediation approaches for petroleum hydrocarbon contamination. Bioremediation is one of the most promising technological advancements, relying on microorganisms to degrade hazardous wastes into less toxic or nontoxic substances (Khudur et al., 2018). This biological transformation offers a cost-effective, eco-friendly alternative to physical and chemical remediation techniques. Lately, clayey soils contaminated with organic pollutants are a critical environmental concern following their high reluctance to conventional bioremediation techniques (Wong et al., 2022). The large surface area and presence of nano and micro-pores in clayey soils result in poor availability of pollutants, nutrients, and oxygen, thus limiting the extent and rate of pollutant biodegradation (Akbari & Ghoshal, 2014). Difficulty in the remediation of clayey soils is attributed mainly to the high absorbance of organic compounds by significant portions of the soil fractions. Therefore, several researchers have focused on microbial remediation technologies that can safely and efficiently remediate clayey soil matrices (Partovinia et al., 2021).

Biochar, a porous carbonaceous material produced under pyrolysis from feedstock biomass, continues to receive increasing attention in its application to petroleum-contaminated sites. Its key physicochemical characteristics, such as high porosity, large surface area, and the presence of functional groups, makes it very suitable for the bioremediation of environmental pollutants. However, the poor performance of pristine biochar has led to the emergence of several biochar amendment strategies derived from physical, chemical, and biological modifications to improve its surface properties and adsorption capacity (Singh et al., 2021). Chemical modification usually involves treatment with various acids (e.g., Hydrochloric acid), bases, or surfactants ( e.g., rhamnolipid), which can enhance biochar's porosity, aid the addition of oxygen-containing functional groups, increase its polarity and surface area (Anae et al., 2021; Liu et al., 2015; Singh et al., 2021). Despite the multifunctional role of biochar in soil amendment, limited studies exist on its application to bioremediation of petroleum contamination in fine-textured clayey soils (Hua & Wang, 2014). So far, only two studies have reported the application of pristine biochar in the remediation of clayey soils (Beiyuan et al., 2020; Ma et al., 2021).

On the other hand, indigenous microorganisms have specific metabolic abilities for crude oil degradation in contaminated sites. However, their bioremediation efficiency is often inhibited by the biotoxicity and poor mobility pollutants (Yuan et al., 2018). Because oil-degrading bacteria are key players in the biodegradation of petroleum hydrocarbons, introducing exogenous diversity genetically adept at degrading the target pollutants. This is an effective approach for improving the bioremediation efficiency of indigenous microorganisms through combined metabolic synergy with exogenous strain(s). More so, previous studies have established that exogenous microorganisms can improve microbial community diversity and richness and may enhance soil microbial functional and metabolic capabilities (Yang et al., 2017).

Filamentous fungi are particularly well-equipped with specific metabolic capabilities. Their extended mycelial network enables them to actively transport hydrophobic pollutants, such as petroleum products, thus enhancing the degradation efficiency of indigenous microorganisms (Schamfuß et al., 2013; Schlosser et al., 2017; Atakpa et al., 2022). Scedosporium spp. are soil saprophytes ubiquitous in contaminated sites. Makadia et al. (2011) provided strong experimental evidence that the high level of total petroleum hydrocarbon (TPH) reduction and the dominance of Scedosporium boydii exist concurrently in naturally attenuated microcosms, highlighting the significance of Scedosporium boydii to the natural degradation of TPH. Studies show that Scedosporium spp. can degrade medium to long-chain alkanes found in crude oil and strongly grows in the presence of crude oil as the sole source of carbon and energy (Rougeron et al., 2018). These fungi have demonstrated unique abilities for degrading high molecular weight aromatic pollutants. This was first proven by Claußen and Schmidt (1998) after Scedosporium apiospermum proliferated in a mineral salt medium containing phenol or p-cresol as the sole carbon and energy source. Studies on other aromatic compounds, specifically those found in crude oil, such as the volatile tetrahydrofuran (THF) and highly toxic polychlorinated biphenyls (PCBs), have been fully documented (Gupta et al., 2017; Skinner et al., 2008; Tigini et al., 2009). These unique capabilities make Scedosporium sp. a suitable exogenous strain for synergistic collaboration with indigenous microorganisms in polluted soils.

Since recent techniques designed for clayey sediment remediation mostly utilize biostimulation or bioaugmentation techniques (Decesaro et al., 2021; Li et al., 2021; Partovinia et al., 2021), it was hypothesized that the sequential utilization of amended biochar and exogenous fungus could overcome the limitations of individual application and improve the overall remediation process. Therefore, microcosms were set up, aimed to investigate the effect of chemically modified biochar and Scedosporium sp. ZYY addition at specified sampling points on TPH degradation. The sediment enzyme activity and the dynamic changes in microbial community structure using 16S rRNA gene amplicon sequencing were also examined under the test conditions.

2.1 Experimental materials

2.1.1 Chemicals, Media and Sediment

The crude oil used in this study was obtained from Xinjiang Keli New Technology Development Co., Ltd, China. Artificial seawater used in all microcosms was prepared from natural sea salt at 35 g/L. All chemicals used were of analytical grade or higher, and were products of Sigma-Aldrich Co. Ltd, Shanghai, China. Clayey marine sediment was collected from Puxi Bridge, Zhoushan Island, in Zhejiang Province, China.

2.1.2 Biochar Pre-treatment and Characterization.

The commercially purchased biochar used in this study was produced from corn-straw biomass pyrolyzed at 500^oC under limited oxygen conditions. The fibres were sieved using mesh size 16 to obtain particles ≥ 1.19 mm and then rinsed with distilled water to clean up the fibres. Biochar amendments were conducted first with 0.5M HCl and then with a rhamnolipid solution. A 250mL Erlenmeyer flask containing 10 g biochar + 150mL solution of 0.5M HCl was kept in a rotary shaker at 150 rpm/30 ^oC for 24 h. Afterwards, the fibres were rinsed thoroughly with distilled water until a pH of ≈ 7.50 was achieved and then soaked in 1% rhamnolipid solution at 150 rpm/30 oC for 24 h. The pretreated biochar fibres were subsequently rinsed twice with distilled water, dried at 60°C for 12 h, and sterilized at 121°C for 20 min.

The pH was determined using a pH meter (Mettler-Toledo FiveEasy Plus, USA). The C, H, N, and S elements of the biochar were measured using an elemental analyzer (Vario MACRO cube). Fourier transform infrared (FT-IR) analysis at a wavenumber ranging from 4000 to 400 cm^− 1 was used to determine the surface functional groups of biochars. Brunauer-Emmett-Teller (BET) was employed for surface area analysis, while the micrographs of the surface morphology were obtained using Sigma 500 Scanning Electron Microscopy (SEM). All characterization analyses were conducted using standard methods.

2.2 Experimental design

Five treatment conditions were set up to study the initial effect of the biochar amendment on crude oil degradation in slurry-phase contaminated sediment. The treatments are as follows: (1). Acid-Rhamnolipid treated biochar + Soil + oil (ARB); (2). Pristine biochar + rhamnolipids + soil + oil (PB + R); (3). Rhamnolipid + soil + oil (Rha); (4). Pristine biochar + soil + oil (PB); (5). Soil + oil (control). The concentration of rhamnolipid used in treatments PB + R and Rha was based on the weight difference between the pre-treated and pristine biochar. Each microcosm was prepared in 250 Erlenmeyer flasks containing 100g of clayey sediment, 100 mL of artificial seawater, a TPH concentration of 5,000mg/kg, and 1 g of biochar. All microcosms were agitated at 25^oC/150 rpm for 6 weeks. 1% microbial cell suspension of Scedosporium sp. ZYY was introduced into treatments ARB, PB + R, Rha, and PB, at the beginning of the 5th week (day 29) of the incubation period, to study the effect of the fungus on the microbial community structure. At each sampling point (days: 14, 21, 28, 35, and 42), slurry microcosms from all treatments were sacrificed for TPH removal, sediment enzymes, and microbial community structure analysis. All experiments were conducted in triplicate microcosms for each treatment on each sampling day. The slurry phase system was chosen for experiments as it has been previously established that enhanced mixing in slurries leads to a higher biodegradation rate (Akbari et al., 2021).

2.3 Determination of residual TPH concentration

Residual TPH was extracted using a dichloromethane/acetone (1:1v/v) mixture and determined gravimetrically after solvent evaporation. Briefly, 1 g of samples previously dried at 30 ^oC/24 h were mixed with 10 mL of 1:1 dichloromethane/acetone in a 20 mL centrifugal tube (Schwab et al., 1999). The mixture was agitated in a rotary shaker for 30 min and centrifuged for 10 min at 5000 rpm to obtain the supernatant. Each treatment sample was extracted four times, and no visible color was observed in the fourth extracted solvent. The extraction efficiency was determined to be 82%. All extracted filtrates were combined and transferred into a pre-weighted volumetric flask and kept under the chemical fume hood for complete dissipation of the solvent. Eventually, the difference between the initial and final weights of the flask was used to determine the residual TPH using a balance with 0.01 mg readability (SQP, Sartorius, Germany) (Zhou et al., 2019).

2.4 Enzymatic activity determination

Fluorescein diacetate (FDA) hydrolysis assay was carried out to estimate the total microbial activity in each treatment. Briefly, 1 mL slurry mixture was placed in 50 mL Erlenmeyer flasks, then 10 mL of phosphate buffer solution (PBS) at pH 7.6, and 0.25 mL FDA solution (2 g/L FDA in acetone) was added. Thereafter, the mixture was cultivated in a rotary shaker for 1h at 150 rpm/25°C. After incubation, the mixture was centrifuged at 4000 rpm for 5 min to obtain clear supernatant. Absorbance was determined at 490 nm and expressed as µg FDA g^− 1 of soil.

2.5 16s rRNA gene sequencing and Bioinformatic analysis

Microbial community analysis of the original sediment (OS) and 2 mL slurry at sampling days 21 and 42 was conducted by Novo Gene Technology (Shanghai, China), based on 16S rRNA sequencing technologies. The raw data were quality-filtered using Trimmomatic (version 0.33) (Bolger et al., 2014), and primer sequences were identified and removed using Cutadapt (version 1.9.1) (Martin, 2011). Qiime2 with DADA2 was used for denoising and chimera analysis, and valid sequences were clustered into amplicon sequence variants (ASVs). The Silva 138.1 database was used as a reference database for the identification of bacterial ASVs, while the annotation of each ASVs was conducted according to classification-Sklearn algorithm. Furthermore, community diversity indexes (Chao, Ace, Shannon) were generated using the MOTHUR program (version 1.31.2, http://www.mothur.org/).

2.6 Statistical analysis

Data are expressed as means ± standard deviation (SD). One-way ANOVA was performed to investigate the significant differences (P < 0.05) between treatments using SigmaStat 3.5 program. Data processing was conducted using Origin Pro, 2022b software, and Microsoft Excel 2016. The differences in community structure between different samples or groups were explored through dimensionality reduction analysis and sample cluster tree display such as PCoA and NMDS. To further explore the differences in community structure between the grouped samples, T-test, MetaStat, LEfSe, and other statistical analysis methods were used to test the differences in species composition and community structure of the grouped samples.

3.1 Physicochemical characteristics of pretreated biochars

The main mechanism of organic pollutants removal for biochar is adsorption (Wang & Wang, 2019), which is highly influenced by its physiochemical properties such as surface area, functional groups, and pore size distribution. These physiochemical properties can be improved through various amendment strategies, such as chemical modifications (Wang & Wang, 2019). Chemically pre-treated biochar fibres have been found to possess increased surface areas, hydrophilicity, and oxygen-containing functional groups (Shen et al., 2008; Wang & Wang, 2019; Zhen et al., 2021). Results of the FTIR analysis shown in Fig. 1 (a) revealed that both pristine and modified fibres showed absorption in the region covering 400 to 4000 cm^− 1 wavenumbers. Prominent peaks observed at 3421 cm^− 1 can be assigned to –OH stretching of aromatic and aliphatic groups, while those at 2919 cm^− 1 can be attributed to –CH asymmetric stretching vibration of methyl/methylene groups (Antonino et al., 2021). Shoulders related to C ≡ C and C-O-C stretching of aromatic groups can be seen at 1618 and 1085 cm^− 1, respectively (Guo et al., 2021; Jiménez et al., 2020), whereas small broad peaks at 475 cm^− 1 correspond to the stretching of C-H bonds of some aromatic and aliphatic functional groups (Guo et al., 2021). All fibres generally exhibited well-defined structures at similar positions. However, the acid-pretreated biochar exhibited slightly higher band intensities, indicating the relative abundance and concentration of existing functional groups.

BET results revealed a remarkable increase in surface area from 2.544 to 25.378 m²g^− 1 for ARB (Table 1). In addition, the H/C ratio increased from 0.047 to 0.061. In contrast, the O/C ratio increased from 0.021 to 0.032 for the acid-rhamnolipid treated biochar (ARB), indicating that modifications with HCl improved the stability, polarity and the oxygen contents of the pretreated biochar. Finally, the SEM result of ARB at the end of the incubation period (Fig. 1b) indicated the presence of enlarged pore sizes of the biochar, likely caused by the acid modification.

Table 1

Physicochemical properties of pristine and modified biochar used in this study.
Biochar	Surface area (m²/g)	Pore size (nm)	Total pore volume (cm³/g)	Total elemental content (g/kg)					H/C ratio	O/C ratio
Biochar	Surface area (m²/g)	Pore size (nm)	Total pore volume (cm³/g)	C	H	N	S	O	H/C ratio	O/C ratio
PB	2.544	10.5	0.01	548.7	26.0	21.5	2.2	11.4	0.047	0.021
ARB	25.378	17.8	0.24	452.1	27.7	21.3	1.9	14.6	0.061	0.032

3.2 TPH Degradation

Petroleum hydrocarbon removal in soils is generally characterized by rapid removal during the initial stage, followed by a slower and even plateau phase (Alexander, 2002; Qin et al., 2013). As shown in Fig. 2a, more than 51.8, 43.7, 39.3, and 25.8% of initial TPH was removed in ARB, PB + R, Rha, and PB treatment, respectively, within the first 21 days of incubation. This was mainly attributed to the metabolic consumption of the more readily available substrate by the bacterial community, facilitated by the presence of biochar or rhamnolipid in these treatments. A similar degradation pattern involving biochar and rhamnolipid has been reported by Wei et al. (2020). However, at week 4, the degradation rate slowed down (Fig. 2a) with a slight decline in removal amount from 123.3 to 96.7 mg TPH kg^− 1 d^− 1 in ARB, 104 to 85.3 mg TPH kg^− 1 d^− 1 in PB + R, 93.8 to 74.8 mg TPH kg^− 1 d^− 1 in Rha, and from 61.4 to 49.2 mg TPH kg^− 1 d^− 1 in PB treatments. After the introduction of the exogenous Scedosporium sp. ZYY at the beginning of the 5th week (day 29), degradation rate began to improve steadily in these treatments. It continued until the remaining days of the study, achieving a final removal rate of 82.4%, 73.6%, 65.7%, and 47.6% in ARB, PB + R, Rha, and PB, respectively, at 42 d. The physical dissipation of crude oil in the treatments from 1 d and at 42 d (week 6) of the experiment period is shown in Fig. S1.

Meanwhile, the control (natural attenuation) with a final TPH concentration of 4168 mg/kg did not achieve significant TPH removal due to the poor degradation capacity of the indigenous microorganisms in this treatment. This could be attributed to the slow acclimatization of the microorganisms to the presence of hydrocarbons and the overall substrate bioavailability limitation. The results suggest that the presence of biochar, especially in ARB, had a significant impact on TPH degradation within the first few weeks of the study. Biochars amended with acids result in increased surface area, improved hydrophilicity, and the introduction of oxygen-containing functional groups, which are crucial in activating the two main mechanisms (sorption and interaction between the functional groups) it employs for pollutant removal in contaminated soils. It is possible that the presence of ARB had an additional accelerative effect on hydrocarbon degradation, including providing a favorable surface for microbial colonization, which effectively shortened the contact distance between pollutants and microorganisms.

On the other hand, increased degradation rate and sediment enzyme activity (Fig. 2b) due to the addition of Scedosporium sp. ZYY highlights the effectiveness of bioaugmentation using specialized strains in bioremediation processes. Previous studies have shown fungi's advantages, such as the secretion of several low substrate specificity enzymes and the capacity of filamentous fungi to form mycelial networks to enhance access to hydrocarbon contaminants, among others, during hydrocarbons degradation in contaminated soils (Harms et al., 2011; Akbari et al., 2021). FDA activity assay conducted to monitor the degradation activities of microorganisms in different treatments revealed that microbial activities were higher in ARB, PB + R, and Rha treatments after fungi inoculation compared to the control treatment. This result corroborated with the TPH degradation results in these treatments.

Interesting findings were made in the present study, especially since no previous literature has reported on the sequential application of modified biochar and exogenous fungus in the slurry phase treatment of contaminated marine sediments. Notably, the degradation efficiencies using biochar, rhamnolipid, or bioaugmentation techniques reported in previous experiments on petroleum hydrocarbon-contaminated sediments in slurry systems are relatively lower than those obtained in the present work. For instance, Akbari et al. (2021) achieved the highest TPH removal rate of 21–40% at the end of 80 days from an initial TPH concentration of 2922 ppm, which was lower than the initial TPH amount used in this study. Few comparable studies where a higher TPH removal rate of 86% (Avona et al., 2022) and 100% (Smith et al., 2015) have been reported, involve the utilization of sophisticated laboratory bioreactor setups and longer experiment durations.

3.3 Changes in Bacterial Community Composition

High throughput analysis of the bacterial 16S rRNA gene was performed to describe the microbial dynamics in the OS and treated sediment at days 21 and 42 (Fig. 3). High-quality sequences detected from the OS and the five treatment groups revealed 95–99% bacterial composition, and 1% Archaea, while the remaining 4% were unassigned. Proteobacteria, Campilobacterota, Bacteroidota, Actinobacteriota, and Desulfobacterota represented the major phyla covering up to 96% of the total ASVs retrieved in the sample (Fig. 3a). Notably, ASVs affiliated with these phyla were more predominant in the treated samples than in the OS. Moreover, all treatments exhibited a slight increase in ASVs at 42 d for Bacteroidota, Actinobacteriota, and Desulfobacterota. However, an increased abundance of Proteobacteria at day 42 was only observed in the ARB treatment, while the abundance of Campilobacterota declined in ARB, Rha, and PB treatments. Proteobacteria are well-known efficient petroleum hydrocarbon degraders. Therefore, their high abundance in contaminated sites is an indication of their significant contribution to TPH removal (Zhen et al., 2021).

Analyzing the bacterial population at the genus level into Proteobacteria phylum also highlighted an increase in the total sequences, especially for the genera Erythrobacter sp., Sulfurovum sp., Alcanivorax sp., and Pseudomonas sp., accounting for 24.9%, 7.8%, 8.3% and 6.8% of the total ASVs, respectively (Fig. 3b). The genus Erythrobacter has been identified as a key degrader of petroleum hydrocarbons in marine sediments (Gao et al., 2015). Sulfurovum has been found to dominate microbial communities in the sediment–seawater interface, surface sediments, and deep-sea hydrothermal vents (Sun et al., 2020); while Alcanivorax and Pseudomonas genera represent two of the most studied groups of n-alkane degraders with high efficiency for crude oil bioremediation.

At the species level, a marked increase for sequences associated with Hyphomonas adhaerens (77%), Marinobacter mobilis (5%), and Erythrobacter seohaensis (4%), all belonging to Proteobacteria phylum was observed, particularly in ARB treatment at 42 d (Fig. 3c). Hyphomonas spp occupy various habitats in the marine environment and have been reported to belong to an opportunistic community of "fast" degraders in the surface environment (Tamburini et al., 2021). Members of Marinobacter spp. isolated from marine sediments produce highly stable surface-active agents for combatting marine oil spills (Raddadi et al., 2017) and have been regarded as specialists in degrading hydrocarbons, especially n-alkanes. Whereas, Erythrobacter spp. have been identified as potential degraders of PAHs (Kahla et al., 2021). Additionally, a significant (P < 0.05) increase in sequences affiliated with Methylophaga pinxianii was observed in the OS compared to the treated samples. Although previous studies have proven that Methylophaga spp. (Proteobacteria phylum) possess genes involved in the assimilation of aliphatic and aromatic compounds (Sauret et al., 2015; Song et al., 2022; Vila et al., 2010), their sparseness in the contaminated treatments is an indication that their proliferation is affected by higher degrees of oil pollution, such as the amount utilized in the present study. Overall, the abundance of hydrocarbon degraders and TPH removal in the ARB treatment suggested the presence of modified biochar and the exogenous fungus, Scedosporium sp. ZYY enhanced microbial-mediated degradation of TPH in the contaminated slurry sediment.

3.4 Diversities of Bacterial Communities during Degradation

The common and unique ASVs between the different sample groups are presented in Fig. 4. Each circle represents the various treatments at the specific sampling time. The number of circles and overlapping parts of the circle represents the number of common ASVs between the sample groups, while the numbers without overlapping parts represent the number of unique ASVs of the sample groups. Sample coverages were all > 99%, indicating the reliability of the data for diversity analysis (Fig. 5a). OS showed the highest α diversity with the largest Chao1, Shannon, and Pielou_e indexes (Fig. 5b-d). Previous studies in oil-contaminated samples have also observed decreased diversity due to crude oil (Looper et al., 2013; Wei et al., 2020). This can be attributed to the out-competition of other bacterial populations by specific oil degraders. In addition, an increase in the Chao1 index was only observed in ARB, PB, and Control treatments at 42 d compared to 21 d, while the Shannon index decreased at 42 d in all treatments except the Control and PB treatment. A high Chao1 index represents high species richness or diversity, while a high Shannon index represents high microbial community diversity (Liu et al., 2022). However, a slightly different trend in evenness was highlighted by the Pielou_e index (Fig. 5d), which increased in the Control, PB, and PB + R treatments but decreased in Rha and ARB. Overall, these results imply that Scedosporium sp. ZYY had little influence on the bacterial community evenness. Nevertheless, the presence of the fungus increased the species' diversity.

Principal coordinate analysis (PCoA) of bacterial community compositions based on the weighted UniFrac distance matrix was employed to study the β-diversity between samples. Principal coordinates accounted for 14·55% and 63·61% of the variance, respectively. As shown in Fig. 6a, treatments with high community structure similarity tend to cluster together, while treatments with significant community differences were farther apart. Samples from the OS were relatively concentrated in the area below the vertical axis' middle line, while samples from the treatment groups were concentrated in the area above, indicating the shifting bacterial community patterns between the OS and the treatment groups caused by the presence of oil. Similarly, Non-Metric Multi-Dimensional Scaling (NMDS) analysis was employed to highlight species abundance and distribution in the samples (Fig. 6b). According to NMDS analysis, the variations within the sample groups were small enough to aggregate into a cluster except in OS and some species in ARB.21 and PB.42. These findings indicated that the bacterial community composition in the original sediment was different from those in the treatments, and this corroborated with the result in Fig. 3c.

3.5 Implication of the Study

In addition to providing sufficient nutrients and conducive reproductive space for microorganisms (Zhang et al., 2021), previous studies have reported on the prospects of biochar in the remediation of soil organic pollutants (Kalsi et al., 2020). This study improved the typical adsorption properties of pristine biochar by introducing preparation conditions that influenced the adsorption and degradation of petroleum hydrocarbons. It has been reported that the diameter of most soil microorganisms is smaller than the average pore diameter of biochar (Zheng et al., 2022). Therefore, the increased rough surfaces and enlarged porous structures of biochar resulting from the modifications (Fig. 1b) enhanced soil aeration, nutrients, and water retention capacity. More so, it provided sufficient space for microbial colonization and a larger surface area for oil absorption, which eventually enhanced TPH removal (Fig. 2a).

Furthermore, previous reports have linked the significant increase in hydrocarbonoclastic bacteria and pollutant degradation to bioaugmentation with allochthonous fungi (Medaura et al., 2021). In this study, the subsequent addition of Scedosporium sp. ZYY into the treatments after 21 d of incubation improved the degradation rate by promoting the growth of TPH-degrading populations (Fig. 3c). These results suggest that biochar and exogenous fungus generally imposed synergistic functions in that biochar provided the microorganisms a conducive surface area for interfacial uptake of hydrocarbons. At the same time, the fungus promoted the proliferation of relevant species and increased their bioaccessibility to the target pollutants. This is the first study that reports the sequential application of biochar and exogenous fungus in the treatment of oil-contaminated clayey sediment. The findings of this study demonstrate the prospects of this application in the remediation of oil-contaminated clayey sediments. However, there remains a scarcity of research focused on the electron transfer mechanism of biochar in promoting microbial soil remediation and its application in removing various pollutants, which deserves further in-depth discussion.

Treatment of crude oil-contaminated clayey sediment represents a challenge caused by low biodegradability in natural conditions due to weathered oil residues usually present. This study demonstrated the high efficiency of the sequential application of modified biochar and exogenous fungus remediation of oil-polluted clayey sediment. All treatments with biochar caused significant (P < 0.05) reduction in TPH content during the initial stage of the degradation experiment. However, during the latter portion of the degradation period, when degradation rates began to decline, the subsequent inoculation of Scedosporium sp. ZYY into the microcosms enhanced the mobilization of pollutants to the indigenous bacterial community, which stimulated their activities. Furthermore, the presence of the fungi eventually caused a shift in the bacterial community structure by promoting the proliferation of important TPH-degraders, specifically, Hyphomonas adhaerens and Marinobacter mobilis, which enhanced TPH removal in the final stages of the experiment period. The results obtained in this work affirm the prospects of modified biochar and exogenous species application for the degradation of petroleum hydrocarbons in polluted matrices such as oil-contaminated marine sediments.

Ethical Approval

We declare that current research fully abides by both local and international guidelines of ethical research regulations.

Consent to participate

Not applicable.

Consent to Publish

Not applicable.

Authors contribution:

Edidiong Okokon Atakpa: Conceptualization, Methodology, Investigation, Formal analysis, Data curation, Writing - original draft. Bozhi Yan: Investigation, Formal analysis, Data curation. Samuel Ukpong Okon: Investigation, editing. Qing Liu: Investigation. Dongdong Zhang: Investigation. Chunfang Zhang: Validation, Writing - review & editing, Project administration.

Funding

This work was supported by the Research Funds of the Guangxi Key Laboratory of Theory and Technology for Environmental Pollution Control (No.2001K004), National Natural Science Foundation of China (No. 51978189).

Competing interests

The authors declare no conflict of interest.

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Bacterial community response to modified-biochar and exogenous fungi inoculation during degradation of oil-contaminated clayey sediment in slurry phase

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Abstract

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Highlights

1. Introduction

2. Materials and Methods

2.1 Experimental materials

2.1.1 Chemicals, Media and Sediment

2.1.2 Biochar Pre-treatment and Characterization.

2.2 Experimental design

2.3 Determination of residual TPH concentration

2.4 Enzymatic activity determination

2.5 16s rRNA gene sequencing and Bioinformatic analysis

2.6 Statistical analysis

3. Results and Discussion

3.1 Physicochemical characteristics of pretreated biochars

3.2 TPH Degradation

3.3 Changes in Bacterial Community Composition

3.4 Diversities of Bacterial Communities during Degradation

3.5 Implication of the Study

4. Conclusion

Declarations

References

Supplementary Files

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