Influence of Wood Biochar on Phenanthrene Catabolism in Soils

Abstract

The impact of increasing amendments of two particle sizes of biochar (≤2 mm and 3–7 mm), applied at 0%, 0.01%, 0.1% and 1% concentrations, on the development of indigenous phenanthrene catabolism was investigated in two soils with different soil organic matter contents. Mineralisation of ¹⁴C-phenanthrene was measured after 1, 20, 60 and 100 d soil-phenanthrene-biochar aging period. The presence of biochar in the pasture soil (low OM) resulted in a decrease in the lag phase of ¹⁴C-phenanthrene mineralisation, with higher maximum rates of mineralisation following 20 d aging. Higher extents of ¹⁴C-phenanthrene mineralisation were observed in the Kettering loam soil (high OM), which was more prominent with 0.01% biochar amendments (p < 0.05) at 61.2% and 64.9% in ≤2 mm and 3–7 mm biochar amended soils, respectively. This study illustrates the potential role for biochar to enhance microbial catabolic activity to degrade common petroleum contaminants. It however depends on contaminant concentration, aging period, and soil properties.

1. Introduction

Soils contain a wide variety of indigenous microflora that possess catabolic potential to degrade contaminants, such as polycyclic aromatic hydrocarbons (PAHs) [1]. Indeed, microbial degradation is a major loss pathway for PAHs from soil [1,2]. The ability of microbes to adapt to PAHs and the time required for adaptation to occur, in part, determines the persistence of organic contaminants [3]. For example, Johnson and Karlson [4] showed that previous exposure to PAHs at levels greater than background levels influenced adaptation and the ability of the indigenous microbes to degrade PAHs. Furthermore, prolonged exposure to a contaminant can lead to greater catabolic activity of indigenous microbes [5,6]. However, the catabolic potential of microbes is influenced by contaminant concentration, bioavailability, chemical stability [7,8], and the presence of co-contaminants [9] and non-aqueous phase liquids (NAPLs) [10]. The adaptation period may further be affected by soil organic matter (SOM) which may provide a more readily available nutrient source than some aromatic contaminants, thus delaying the microbial need for contaminant breakdown [11].

Biochar (black carbon derived from pyrolysis of biomass) is a form of organic matter that is ubiquitous in nature [12]. Biochars retain some of the chemical properties of the original feedstock used in production, the extent of which depends on the conditions of pyrolysis [13]. Its presence in soil has been shown to influence mobility and bioavailability of organic contaminants as biochar can sequester such compounds reducing their mobility and degradability [14]. Biochar has been shown to adsorb organic compounds more strongly and nonlinearly to its carbonised phase than SOM [15,16]. The sorption mechanism of aromatic hydrocarbons, such as PAHs, onto wood char is assisted by π -electron interaction and pore-filling mechanism [17]. A fraction of PAHs may be sorbed to exterior surfaces of biochar and other portions are trapped within internal nanopores, thereby limiting mass transfer to microorganisms [18].

Recent studies have investigated the influence of the combined roles SOM and biochar amendments on the sorption of organic and inorganic contaminants in soils and sediments [15,19]. Biochar is a more important sorbent than SOM [20]. This suggests that SOM may compete for adsorption sites and block pore sites on black carbon. Furthermore, Rhodes et al. [1] showed that higher concentrations (>0.1%) of activated charcoal (AChar) reduced the extent and rate of ¹⁴C-phenanthrene mineralisation in soils by reducing indigenous microbial catabolic activity. More recently, Bushnaf et al. [21] showed rapid biodegradation of alkanes in biochar amended soils. However, there is very limited information on the effect of different particle sizes of biochar on catabolic activity of indigenous microorganisms in soils varying in SOM content. Therefore, the aim of this current study is to investigate the role of different particle sizes and doses of wood-derived biochar on the catabolic activity of microorganisms in ¹⁴C-phenanthrene spiked soils with differing SOM content. It further explores the effect of pre-exposure of ¹²C-phenanthrene on microbial catabolic activity in biochar amended soils during aging.

2. Materials and Methods

2.1. Materials

Both non-labelled and [9-¹⁴C] phenanthrene (98% radioactive purity) were obtained from Sigma-Aldrich, UK. Goldstar multipurpose scintillation cocktail was obtained from Meridian, UK. Nutrient agar and purified agar were obtained from Oxoid, UK. Biochar was provided by Yorkshire Charcoal Co., UK and was produced by slow pyrolysis (16–18 h duration at 450–500°C) of mixed wood feedstock, containing approximately 90% Acer, and the remaining 10% a mixture of Quercus and Fraxinus species. The biochar was separated to obtain two different ranges of particle sizes (≤2 mm and 3–7 mm) designated as BioC1 and BioC2, respectively.

2.2. Soil Preparation for Aging and Spiking

Two soils were used, chosen for their differing SOM contents. The physical properties of both soils are listed in Table 1. The low organic matter soil was obtained from a Myerscough pasture field at Myerscough Agricultural College, UK, and had a sandy loam texture. Kettering loam was the higher OM soil and was provided by Boughton Loam Ltd (Kettering, UK) as shown in Table 1 [22]. Soils were sieved through a 2 mm mesh to homogenise the soils and remove large debris such as plant roots and stones. Each of the soils were rehydrated with deionised water to their required moisture content and individually amended with biochar. The ≤2 mm (BioC1) and 3–7 mm (BioC2) biochar fractions were amended into the soils in triplicate in the following concentrations: 0%, 0.01%, 0.1% and 1%. To ensure homogeneity, samples were blended using stainless spoon for 8 min [1,23]. The soils were then spiked with non-labelled ¹²C-phenanthrene standards prepared in toluene to achieve a concentration of 50 mg kg⁻¹ as per Doick et al. [23]. The treatments were stored in separate amber jars in the dark at 21 ± 1 °C for the duration of aging (1, 20, 60 and 100 days). NMR-Cryoporometry analysis was performed to know total pore volume and liquid per unit mass of individual biochars as per Webber et al. [24]. Ash content was measured by heating biochar samples at 760 °C for 6 h [25] using a Carbolite Furnace RHF 1400 and calculated using the equation: Ash content (%) = (Bb − Ba/Bb) × 100, whereby Bb and Ba were biochar weight before and after heating, respectively [26]. Biochar pH analysis was measured in triplicate at 1% (d/w). The mixture was shaken for 24 h at 100 rpm and then measured using a digital pH meter. Results of biochar analysis are shown in Table 2.

[ Table omitted. See PDF. ]

[ Table omitted. See PDF. ]

2.3. Measuring ¹⁴C-Phenanthrene Mineralisation by Indigenous Soil Microorganisms

After each aging period, soils were spiked with ¹²C- and ¹⁴C-phenanthrene (50 mg kg⁻¹ at 41.67 Bq g⁻¹ of soil) with toluene as the carrier solvent [27]. The mineralisation of ¹⁴C-phenanthrene was determined by measuring the evolution of ¹⁴CO₂ using modified 250 mL Schott bottles containing 1 M NaOH (1 mL) as a CO₂ trap, which was replaced daily [27]. After 1, 20, 60 and 100 d aging in the dark at 21 ± 1 °C, respirometers were prepared in triplicate, with 10 ± 0.2 g soil (w/w) and 30 mL sterilized minimal basal salts (MBS) solution. MBS solution was made as follows: 0.3 g L⁻¹ NaCl, 0.6 g L⁻¹ (NH₄)₂SO₄, 0.6 g L⁻¹ KNO₃, 0.25 g L⁻¹ KH₂PO₄, 0.75 g L⁻¹ K₂HPO₄, 0.15 g L⁻¹ MgSO₄·7H₂O, it also contained required micronutrients: 20 μg L⁻¹ LiCl(LiBO₂), 80 μg L⁻¹ CaSO₄·5H₂O, 100 μg L⁻¹ ZnSO₄·7H₂O, 100 μg L⁻¹ Al(SO₄)₃·16H₂O, 100 μg L⁻¹ NiCl·6H₂O(CoNO₃), 100 μg L⁻¹ CoSO₄·7H₂O(CoNO₃), 30 μg L⁻¹ KBr, 30 μg L⁻¹ KI, 600 μg L⁻¹ MnCl₂·2H₂O, 40 μg L⁻¹ SnCl₂·2H₂O and 300 μg L⁻¹ FeSO₄·7H₂O. Doick and Semple [28] recommend a 1:3 soil:liquid ratio for greater reproducibility and overall extents of mineralisation. The respirometers were then secured onto a SANYO Gallenkamp orbital shaker at 100 rpm, at 21 ± 1 °C in the dark for 14 days. At each sampling time (24 h) the trapped ¹⁴CO₂ was determined by adding 5 mL of Goldstar scintillation cocktail. These samples were counted using a Canberra Packard Tri-Carb 2250CA liquid scintillation analyser, utilising standard protocols and automatic quench correction.

2.4. Statistical Analyses

Statistical significances for overall extents of mineralisation, fastest rates of mineralisation and lag phases were evaluated using an analysis of variance (ANOVA). ANOVA (p < 0.05) was used to investigate significant differences amongst biochar amendments and particle sizes within each time points. Statistical analysis was done using SigmaStat software (Version 2.0).

3. Results

3.1. Effects of Biochar Amendment on Mineralisation of ¹⁴C-Phenanthrene

The mineralisation of ¹⁴C-phenanthrene to ¹⁴CO₂ over a 14 d experimental period was measured in both the Kettering loam and Myerscough soils amended with 0%, 0.01%, 0.1% and 1% biochar (BioC1 & BioC2), after 1, 20, 60 and 100 d soil-phenanthrene aging period. The effect of each biochar focused on changes in the lag phase, rate and extent of ¹⁴C-phenanthrene mineralisation in the soils following pre-exposure to ¹²C-phenanthrene.

3.1.1. Impact of Biochar on the Lag Phase Prior to Mineralisation of ¹⁴C-Phenanthrene in Soil

Lag phases were defined and measured as the time taken for the extent of mineralisation of ¹⁴C-phenanthrene to exceed 5% of the amount applied in each respirometry bottle. Differences in soils showed that lag phase was greater in the Myerscough soil compared to the Kettering loam (Table 3 and Table 4) and was further evident in biochar amended soils. For instance at 1 d incubation, 0.01%, 0.1% and 1% BioC2 amendments in the Myerscough soil showed statistically greater (p < 0.01) lag phases than 0.01%, 0.1% and 1% BioC2 in the Kettering loam, the lag phases lasted between 4 and 9 d for the Myerscough soil and remained at approximately 3 d for Kettering loam soil (Table 3 and Table 4). When particles sizes were compared over time, there was statistically insignificant difference (p > 0.05) in the lag phases. Following 20 d aging period in both soils (Myerscough and Kettering loam), there was very little real difference in lag phases but these were shown to decline significantly (p < 0.05) to a maximum of 2.9 and 2.7 d, respectively. Noticeably, the 20 d soil-phenanthrene aging period exhibited the shortest lag phases, irrespective of biochar concentration (Table 3 and Table 4; Figure 1 and Figure 2). The reduction in lag phase was not consistent with an increase in aging period, rather, a minimum of 30% increase in lag phase was observed following 60 and 100 d time point compared to 20 d time point. The addition of biochar to the soils had an effect after 20 d, where increasing concentrations of biochar led to a decreasing lag phase in the Myerscough soil and an increased lag phase in the Kettering loam after 100 d aging period (Table 3 and Table 4), when compared to controls.

[ Table omitted. See PDF. ]

^a: No statistical significant difference (p > 0.05) in soils amended with biochar compared to control; ^A: No statistical significant difference (p > 0.05) in same biochar dose but different soil types; ^b: Statistical significant difference (p < 0.05) in soils amended with biochar compared to control; ^B: Statistical significant difference (p < 0.05) in same biochar dose but different soil types.

[ Table omitted. See PDF. ]

^a: No statistical significant difference (p > 0.05) in soils amended with biochar compared to control; ^A: No statistical significant difference (p > 0.05) in same biochar dose but different soil types; ^b: Statistical significant difference (p < 0.05) in soils amended with biochar compared to control; ^B: Statistical significant difference (p < 0.05) in same biochar dose but different soil types.

3.1.2. Impact of Biochar on the Maximum Rates of ¹⁴C-Phenanthrene Mineralisation in Soil

The mean maximum rates of mineralisation over the 14 d experimental period for each biochar amendment in both soils at all soil-phenanthrene aging periods was calculated (Table 3 and Table 4). The maximum rates of mineralisation were attained within 3–10 days and ≤3 days of mineralisation for the Myerscough and Kettering loam soils, respectively. After 1 d soil-phenanthrene aging period, there was generally insignificant difference (p > 0.05) in the maximum rate of ¹⁴CO₂ evolution per day in both soils and particle size amendments. However, the presence of biochar during soil-phenanthrene aging maintained maximum rates of mineralisation in both soils until there was increase in aging where biochar increased maximum rates in both soils following 20 and 60 d soil-phenanthrene aging period (Table 3 and Table 4). For instance, the highest rate of mineralisation (30%–33% d⁻¹) was observed in 0.01% of both particle sizes of biochar-amended to Kettering loam. Afterwards (100 d), rates of mineralisation decreased (p < 0.05) to be consistent with those in the control soil (Table 3 and Table 4).

3.1.3. Impact of Biochar on the Total Extents of ¹⁴C-Phenanthrene Mineralisation

The extent of ¹⁴CO₂ production over 14 d varied in both soils, with increasing soil-phenanthrene aging period (Figure 1 and Figure 2; Table 3 and Table 4). When the extent of mineralisation was compared amongst the different soil-phenanthrene aging periods (1, 20, 60 and 100 d), there were statistical differences. The extent of mineralisation did not exceed 50% of the dose applied to the controls; however, the addition of both biochars (BioC1 and BioC2) resulted in statistical increases (p < 0.05) in the extent of ¹⁴C-phenanthrene mineralisation in Kettering loam soil after 1 d soil-phenanthrene aging period. However, this was not consistent with increasing soil aging. Although 1% biochar amendment of both particle sizes (≤2 mm and 3–7 mm) resulted in a lower extent of ¹⁴C-phenanthrene mineralisation when compared to the control soil; this was not statistically significant (p > 0.05) (Table 3). Following an increase in soil-phenanthrene aging period, biochar maintained the extent of ¹⁴C-phenanthrene mineralisation in both soils in comparison to the control soil, with no further statistical increase (p > 0.05). For example, the extent of mineralisation in the biochar-amended Kettering loam soil ranged between 55%–59%, 40%–44% and 41%–48% after 20, 60 and 100 d aging, respectively, whilst in the Myerscough soil, it ranged between; 50%–55%, 49%–56% and 36%–44% after 20, 60 and 100 d aging, respectively. There were, however insignificant differences (p > 0.05) between the two particle sizes on the extent of ¹⁴C-phenanthrene mineralisation in both soils. In the Myerscough soil, there was a minimum of 7% increase in the extent of mineralisation after 20 d aging compared to 1 d aging. Following further aging (60 and 100 d), the extent of mineralisation after 20 d aging were statistically greater (p < 0.05) than 100 d aging irrespective of biochar concentration and particle size (Table 4; Figure 1). In contrast, biochar amendments in Kettering loam showed that extents of mineralisation after 20 d aging was statistically greater (p < 0.05) than mineralisation after 60 and 100 d aging, but similar to 1 d aging (Table 3; Figure 2). Despite an increase in the extent of mineralisation after 20 d soil aging in the Kettering loam, it showed a statistically greater (p < 0.05) extent of mineralisation when amended with biochar than the Myerscough soil at 1 and 100 d soil aging.

4. Discussion

The microbial communities within both soils were able to mineralise the ¹⁴C-phenanthrene over 14 days at each time point (1, 20, 60 and 100 d). However, this was not achieved without the adaptation of the indigenous microorganisms within each soil. All the curves of mineralisation in this study were sigmoidal (Figure 1 and Figure 2). Suggesting that the phenanthrene-degrading populations were continually growing [5,9]. The adaptation was probably through one or more mechanisms, such as an increase in microbial population, induction of catabolic enzymes, transgenic manipulations via horizontal gene transfer within the degrading populations [29]. After 1 d soil-phenanthrene aging, biochar amendments had no impact on the lag phase in both Myerscough and Kettering loam soils. It has been suggested that longer initial adaptation periods results in shorter lag phases and greater rates of mineralisation [5]. However, the shorter lag phases experienced in the Kettering loam soil compared to the Myerscough soil was apparently due to the difference in soil properties and indigenous microflora [1,30]. After 20 d aging of ¹²C-phenanthrene, both the Myerscough and Kettering loam soils experienced significant decreases in lag phases which support Rhodes et al. [1,30], where lag phases decreased after 42 and 25 d aging periods, respectively. Such increase in soil-phenanthrene contact time allowed continued proliferation of microbial degrading organisms that eventually enhanced degradation of the phenanthrene bioavailable fraction [1,4]. Despite decreases in the lag phase in this current study, indigenous soil populations never fully acclimatised to the addition of phenanthrene since the sigmoidal shape was always apparent (Figure 1 and Figure 2). To explain this, the time taken to mineralise a particular PAH depends on level of contamination in soil and soil type [1,4]. With respect to concentration effects, higher concentrations of PAHs can be toxic to degraders and inhibit their growth [9], also, 10 mg kg⁻¹ of phenanthrene was spiked into soils by Rhodes et al. [1,30], whilst 50 mg kg⁻¹ of phenanthrene was spiked in soils in this current study. After 20 d aging, biochar amendments (0.1% and 1%) decreased the lag phase in the Myerscough soil to a greater extent compared to the control soil. Biochar has the potential to interact with the soil matrix and adsorb growth-inhibiting substances [31]. Furthermore, the presence of biochar alters nutrient availability for microbial utilisation within soils following aging [32,33]. In the Kettering loam soil, it took 20 days of soil-phenanthrene aging to reduce the lag phase from 3.3 d to 2 d. This means that after pre-exposure of phenanthrene to indigenous microorganisms in biochar amended soils, the lag phase will be decreased depending on soil-phenanthrene aging period, and soil characteristics.

Figure 1. Mineralisation of 14C-phenanthrene in Myerscough soil amended with biochar at 0% (○), 0.01% (▽), 0.1% (□) and 1% (◊) after 1, 20, 60 and 100 d soil-phenanthrene aging periods. Error bars represent standard error of mineralisation (SEM) (n = 3).

Figure 2. Mineralisation of 14C-phenanthrene in Kettering loam soil amended with biochar at 0% (○), 0.01% (▽), 0.1% (□) and 1% (◊) after 1, 20, 60 and 100 d soil-phenanthrene aging periods. Error bars represent standard error of mineralisation (SEM) (n = 3).

Maximum rates of mineralisation showed neither significant differences between soils nor between the control and biochar amended samples at 1 d soil-phenanthrene aging, which concurs with the lag phase results. The fastest rates of ¹⁴CO₂ evolution commonly occur during the exponential phase of mineralisation (Table 3 and Table 4; Figure 1 and Figure 2). In this study, the addition of biochar of both particle sizes (≤2 mm and 3–7 mm) produced higher maximum rates of mineralisation in both the Myerscough and Kettering loam soils than in the control following increased soil-phenanthrene aging. Similarly, Bushnaf et al. [21] showed that biodegradation of water-dissolved and available gasoline constituents were faster in 2% biochar-amended soils than in unamended soils. Kolb et al. [34] equally demonstrated that the addition of manure/wood biochar (500 °C) to 4 different soil types expressed an increasing microbial activity at 1, 2.5, 5 and 10% charcoal amendment, but depended on soil type. In addition, increasing concentrations of biochar in Walla Walla loam soils resulted in increased microbial activity and respiration in first few days of incubation, and then decreased after 6 d [35]. Although this is contrary to Rhodes et al. [1], where 1% AChar resulted in reduction in maximum rates of mineralisation and created long lag phases. This difference is apparently due to the difference in black carbon material used, phenanthrene concentration and soil properties. The biochar used in this study was not subject to any form of chemical activation to increase microporosity. Also, biochar is a preferred habitat for microbial growth than some activated carbon [36] and enhances microbial catabolic ability in soils [37,38]. Similarly, the availability of C, N and other nutrients from biochar amended soils increases microbial colonisation, ATP production and microbial production [39,40]. The biochar used in this study shows that amongst its pores, it contains more macropores (>50 nm) that encourage microbial colonisation and reproduction [41] (Table 2). Additionally, the biochars also contain liquid hydrocarbons (Table 2) within the pores that can serve as substrates for growth and metabolism [42]. Despite presence of OM in soils, the degree of competition for sorption site between contaminant and OM felt by the adsorbate will decrease from low to high concentrations [43]. It thus means that biochar does not discourage catabolic activity in soils.

At 1 d soil-phenanthrene aging, the two soil types exhibited contrary responses to the effect of biochar amendment on total extents of ¹⁴C-phenanthrene mineralisation. Higher extents of mineralisation in 0.01% and 0.1% biochar-amended Kettering loam soil showed that low concentrations of wood biochar amendment can influence extent of phenanthrene biodegradation due to potential role of humic substances on biochar sorption sites and surface properties [19,43,44,45]. The biochar dose that led to response was low and a proper homogenous mixture with the soil would not be certain. Therefore there would have been some biochar hotspots within the soil-biochar mixture. However it is worthy to note that ¹⁴C-phenanthrene was spiked directly into aqueous solution of the respirometer assay and not into soil. Furthermore, the slurry method used enables agitation which tends to aid distribution of microorganisms evenly [4,27], and facilitates degradation of phenanthrene, while soil particles separate into suspension [27]. In so doing, microorganisms adhere to BC particles to reduce distance to substrate and mineralise phenanthrene [9,46,47], whilst presence of microbes on pore sites and native SOM may attenuate sorption capacity of biochar [20]. Although not investigated, attachment of microorganisms to biochar is often through one of the following processes: (1) flocculation; (2) adsorption; (3) covalent bonding; (4) cross-linking of cells; (5) encapsulation in a polymer-gel; and (6) entrapment within matrix [31]. Zhou et al. [48] illustrated that modification with humic acids changes charcoal properties, which will eventually affect PAH desorption dynamics. PAH concentration is a determining factor governing the extent of mineralisation in soils when investigating microbial catabolic activities in slurry method. This study supports Couling et al. [9], where greatest extents of ¹⁴C-phenanthrene mineralisation did not exceed 60% at higher concentrations, despite aging in unamended biochar soils. A decline in microbial activity caused the lower extent of mineralisation due to toxicity of the higher concentration of phenanthrene.

5. Conclusions

This study shows soils contain an intrinsic microbial catabolic ability to degrade PAHs over time, but that this differs with soil characteristics and soil-PAH exposure time. The ability to degrade phenanthrene (a known PAH) was significantly enhanced (albeit to a minor extent) due to amendment of wood-derived biochar in soils [37,39]. This was expressed by shorter lag phases and higher maximum rates of ¹⁴C-phenanthrene mineralisation in both soils, following increased soil-phenanthrene aging period. The findings also showed that the presence of low concentrations of such biochar does not inhibit the catabolic activity of indigenous microorganisms. It, however, depends on contaminant concentration, soil-contaminant pre-exposure time, and soil properties. Biochar retains much of the original chemical properties and features of the feedstock [13], which sustains PAH-degrading microorganisms. However, at higher concentrations of biochar, it is assumed there will be more available pore sites that will cause further sequestration of contaminants. This shows that biochar could be applied as a tool in the bioremediation process of contaminated land. Further studies will investigate the effect of biochar on the biodegradation of aged ¹⁴C-PAHs in soils.

Acknowledgments

The authors are thankful to Petroleum Development and Technology Fund for partial funding. The biochar analysis (pore size distribution) was done by Beau Webber of Lab-Tools Ltd., Kent, UK. Additionally, we thank Aller Fernandez for her comments and advice in this research.

Author Contributions

Uchenna Ogbonnaya and Ayodeji Oyelami developed the initial idea of the research and designed the whole experiment. Justin Matthews was involved in the experimental sampling stage, data collection and initial presentation of data. Uchenna Ogbonnaya was responsible for following up on study participant and data cleaning. Kirk Semple and Olusoji Adebisi provided technical advice on the research. Uchenna Ogbonnaya drafted the manuscript and co-authors read and approved the final manuscript.

Conflict of Interest

The authors declare no conflict of interest.

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