Improved bioreduction of nitrobenzene by black carbon/biochar derived from crop residues

Abstract

Black carbon (BC) and biochar were produced from wheat straw and cotton stalk through open-field combustion and oxygen-limited pyrolysis, respectively. Different BC/biochar samples were characterized for their morphology, elemental composition, electrical conductivity, surface area and functional groups, etc., and assayed for their stimulating effects on microbial reduction of nitrobenzene by Shewanella oneidensis MR-1. Here, the preparation method was found to have significant effects on the physicochemical properties and stimulating capacities of BC/biochar samples, whereas the feedstock biomass did not demonstrate an obvious impact on these properties as anticipated. Better stimulation of bioreduction of nitrobenzene (10 to 400 mg L⁻¹) was normally observed with BC samples, which have more oxygenated groups than their biochar counterparts. The stimulating effects generally increased with the increase of BC/biochar dosage. And biochar obtained at higher pyrolysis temperatures generally showed better stimulating performance. Moreover, the addition of BC/biochar samples together with anthraquinone-2,6-disulfonate, which is a typical soluble quinone redox mediator, into bioreduction systems resulted in better stimulating effects than the use of quinone alone. BC/biochar prepared from crop residues therefore may be exploited as a low-cost redox mediator to stimulate nitroaromatic bioreduction.

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Introduction

Black carbon (BC), including biochar, soot, charcoal, activated carbon, coke and kerogen, etc., is generated through incomplete combustion or pyrolysis of fossil fuels and biomass¹ and is widespread in different environmental media including air, soil, sediment and rock. It is estimated that BC represents 2–18% of total organic carbon in soil and sediment. For fire-impacted soils, this value can even be as high as 30–45%.² As a specific subset of BC, the carbon-rich solid material produced by thermal decomposition of biomass under O₂-limited conditions is called biochar.³ In China and many other areas around the world, large amounts of crop residues are burned after harvest in open fields every year, resulting in not only negative effects on human health and climate, but also the production and accumulation of BC/biochar in soil and sediment.⁴ In many countries biochar has also been traditionally applied to fields to modify soil structure and increase crop yields.⁵

BC has been believed to be an important geosorbent, which controls the transport, fate, bioavailability and ecotoxicity of organic pollutants in soil and sediment.⁶ The application of biochar as a sorbent in water and wastewater treatment has attracted lots of interest recently. In fact, various kinds of organic and inorganic pollutants including dyes, phenols, antibiotics, pesticides, polynuclear aromatics and heavy metal ions (e.g. Pb²⁺, Cu²⁺, Cd²⁺, As³⁺, Hg²⁺ and Cr⁶⁺, etc.) could be efficiently removed by original or modified biochar sorbents.^7–9

For a long time, BC/biochar has been considered as inert material. And chemicals adsorbed onto BC/biochar were also assumed to be chemically inactive.⁶ However, during the past decade, several studies have indicated that when elemental iron, sulfide and thiol compounds were used as reductants, biochar and many other BC materials including activated carbon, graphite, carbon fiber, diesel soot, graphene oxide and carbon nanotubes, could catalyze the abiotic reduction of electrophilic contaminants such as azo dyes, nitroaromatic compounds, heterocyclic nitramines and halogenated compounds, through accelerating electron transfer via surface functional groups or graphene moieties.^1,3,6,10,11

Interactions between carbon materials and microorganisms have gradually raised concerns of researchers during past years. van der Zee et al.¹² firstly found that activated carbon could act as electron acceptor/redox mediator of granular sludge and Geobacter sp. enrichment culture to stimulate the anaerobic biotransformation of azo dyes. Addition of carbon nanotube in cell-immobilized alginate beads was observed to promote the reduction of nitrobenzene and Cr(VI) by Shewanella oneidensis MR-1.^13,14 Our previous study indicated that the presence of graphene could stimulate the reduction of nitrobenzene by anaerobic sludge.¹⁵ As for biochar, increasing evidence has suggested that it could play an important role in extracellular electron transfer of microorganisms. Microbial cells could attach themselves to biochar, which stimulated direct interspecies electron transfer under anaerobic conditions.¹⁶ Kappler et al.¹⁷ and Xu et al.¹⁸ reported that biochar could function as electron shuttle to stimulate both the rate and extent of microbial reduction of Fe(III) minerals by S. oneidensis MR-1. The coexistence of elemental iron and biochar enhanced the anaerobic denitrification by microbes.⁶ Very recently, Yu et al.¹⁹ have indicated that biochar prepared from rice straw could act as electron shuttle to stimulate reductive dechlorination of pentachlorophenol by Geobacter sulfurreducens. However, information about the difference between BC generated by combustion and biochar obtained through pyrolysis in impacting biotransformation is lacking. And the impacts of BC/biochar on microbial transformation of other contaminants remain unknown.

Nitrobenzene has been extensively used as intermediate in the manufacturing of dyes, explosives, pesticides and pharmaceuticals, etc. It has been listed as a priority pollutant due to its toxicity and recalcitrance. The sequential anaerobic–aerobic biological process has been believed to be one of the most effective methods for the treatment of nitrobenzene in wastewater. And the initial anaerobic reduction of nitrobenzene was suggested to be the rate-limiting step.¹⁵ Previously we have successfully improved the biotransformation of nitrobenzene by the synergies of Shewanella species and mediator-functionalized polyurethane foam.²⁰ However, the immobilization of quinone redox mediator is generally time-consuming and cost-ineffective. The continuous searching of easily accessible, low-cost and efficient redox mediators to improve microbial transformation of pollutants is anticipated.

Here, BC and biochar samples were initially prepared from crop residues through combustion and pyrolysis respectively. Then the effects of BC/biochar on microbial reduction of nitrobenzene by S. oneidensis MR-1 were studied in detail. Factors including feedstock biomass, pyrolysis temperature, BC/biochar dosage and soluble redox mediator etc. were investigated in detail. To the best of our knowledge, this is the first study on stimulating microbial reduction of nitroaromatic compounds by BC/biochar.

Materials and methods

Chemicals and strains

Nitrobenzene and aniline were of reagent grade and purchased from Sinopharm. All other chemicals were of analytical grade, commercially available and used without further purification.

S. oneidensis MR-1 obtained from ATCC (700550) was routinely cultured in Luria–Bertani (LB) broth medium aerobically at 30 °C. Nitrobenzene reduction studies were performed in modified M-R2A medium²¹ containing (mg L⁻¹) KH₂PO₄, 250; K₂HPO₄, 400; KCl, 505; NH₄Cl, 800; CaCl₂·2H₂O, 15; MgCl₂·6H₂O, 20; FeSO₄·7H₂O, 7; Na₂SO₄, 5; MnCl₂·4H₂O, 5; H₃BO₃, 0.5; ZnCl₂, 0.5; CoCl₂·6H₂O, 0.5; NiSO₄·6H₂O, 0.5; and CuCl₂·2H₂O, 0.3. Lactate (20 mM) was added as electron donor and the pH of the medium was adjusted to 7.0.

Preparation of BC and biochar

Feedstock biomass including wheat (Triticum aestivum L.) straw and cotton (Gossypium hirsutum L.) stalk were collected from Shandong Province of China in 2015. Elemental analysis of feedstock biomass was carried out with CHN element analyzer (Elementar Vario EL). A process simulating the field combustion was applied to produce BC from wheat straw (BC_w) and cotton stalk (BC_c).⁴ Specifically, biomass (∼100 g) was stacked and burned on a steel plate (1 m²) in open field under uncontrolled conditions in a windless afternoon. Continuous stirring and mixing of the biomass was conducted to ensure even combustion. Then ashes were carefully collected as black carbon after combustion. To prepare biochar from wheat straw (biochar_w) and cotton stalk (biochar_c), each biomass sample was grounded, dried and pyrolyzed in a laboratory tube furnace at a pyrolysis temperature of 600 °C and with a continuous N₂ flow (30 mL min⁻¹). To study the effects of pyrolysis temperature, different biochar samples (biochar₃₀₀, biochar₆₀₀ and biochar₈₀₀) were obtained by pyrolysis of wheat straw at 300 °C, 600 °C and 800 °C, respectively. All BC/biochar samples were grounded and passed through a 100-mesh sieve before characterization and further use in microbial nitrobenzene reduction.

Nitrobenzene bioreduction

The S. oneidensis MR-1 cells cultured aerobically overnight were harvested by centrifugation (10 000g, 5 min), washed thrice with and resuspended in sterile phosphate buffer solution (20 mM, pH 7.0) before use.

Nitrobenzene reduction experiments were carried out in 100 mL glass serum bottles in an anaerobic glove box under nitrogen atmosphere. Normally, each bottle contained 50 mL M-R2A medium added with 0.5 g L⁻¹ BC/biochar and 100 mg L⁻¹ nitrobenzene. After inoculation of harvested MR-1 cells to achieve an initial biomass concentration of 0.03 g L⁻¹, aluminum and rubber-capped bottles were placed in a shaker (30 °C, 150 rpm).

Samples (2 mL) were periodically taken with sterile needle and syringe. The sample solutions were centrifuged to separate BC/biochar particles. Then the adsorbed nitrobenzene on BC/biochar particles was extracted three times with methanol (2 mL).⁴ The extracted solution was mixed with the supernatant solution, and filtered for high performance liquid chromatography (HPLC) analysis as described in following section.

In order to assess the stimulating effects of BC_w/biochar_w on reduction of different concentrations of nitrobenzene, 10 to 400 mg L⁻¹ nitrobenzene was applied in the experiment. The BC_w/biochar_w concentration was varied from 0.17 to 0.67 g L⁻¹ to study the effects of BC_w/biochar_w dosage on nitrobenzene reduction. Stimulating effects of biochar samples that obtained at different pyrolysis temperatures (biohcar₃₀₀, biochar₆₀₀ and biochar₈₀₀) were also compared. Reduction systems containing different concentrations of BC_w/biochar_w (0.17, 0.33, 0.50 and 0.67 g L⁻¹) and 50 μM anthraquinone-2,6-disulfonate (AQDS) were used to investigate the effects of coexisting insoluble and soluble redox mediator on microbial nitrobenzene reduction. Abiotic controls without inoculation of cells, and biotic controls without BC/biochar were also performed. All treatments and controls were run in triplicate.

BC/biochar characterization and chemical analysis

The structure and morphology of BC/biochar samples were examined by scanning electron microscopy (SEM, Jeol JSM-7800F at an accelerating voltage of 20.0 kV for BC and biochar, and Hitachi SU8000 at an accelerating voltage of 5.0 kV for BC and associated cells) and transmission electron microscopy (TEM, Tecnai G2 operated at 120 kV). The BET specific surface areas of all samples were measured based on N² adsorption at −196 °C by surface area analyzer (Quantachrome NOVA4200e). The element contents of the samples were determined using CHN element analyzer (Elementar Vario EL) and X-ray fluorescence (XRF) spectrophotometer (Shimadzu Lab Center XRF-1800). The ash content was determined by a proximate analyser (Changsha Sande Industrial Co., Ltd, SDTGA5000) by holding the temperature at 550 °C for 30 min, then rising the temperature with 5 °C min⁻¹ to 815 °C and holding until constant weight (mass difference ± 0.05%) was reached. The functional groups of samples were analyzed with Fourier transform infrared spectroscopy (FTIR, Bruker Equinox 55 FTIR spectrometer) over the wavenumber range of 4000–400 cm⁻¹ and ¹³C nuclear magnetic resonance spectroscopy (NMR) performed on Bruker AVANCE III 600 spectrometer at a resonance frequency of 150.9 MHz. ¹³C cross-polarization magic angle spinning (CP/MAS) NMR spectra were recorded using a 4 mm MAS probe and a spinning rate of 14 kHz. A contact time of 2 ms and a recycle delay of 5 s were used for the ¹H–¹³C CP/MAS measurement. The chemical shifts of ¹³C were externally referenced to tetramethylsilane. To determine the electrical conductivity (EC) of prepared BC/biochar, the samples were firstly compacted into circular tablets with a diameter of 10 mm at 20 MPa. Then the measurement of EC was performed using a four-point probe apparatus (RTS-4, 4-probes Tech) at room temperature.

The concentrations of nitrobenzene and aniline were analyzed using HPLC (Shimadzu LC-20AT, Japan) equipped with an Elite hypersil BDS C18 column (250 × 4.6 mm, 25 μm) for separation at 40 °C and a diode array detector (SPD-M20A, Japan) for measurement at 254 nm. A methanol water mixture (55/45, v/v) was prepared as the mobile phase at a flow rate of 1.0 mL min⁻¹.

Results and discussions

Characterization of BC/biochar samples

Different morphologies were found with BC and biochar samples prepared from crop residues (Fig. 1). Biochar samples generally maintained more integral tissue structures of original biomass and were rich in pores and channels. On the other hand, most BC samples were found as fragments with much smoother surfaces. When the same feedstock biomass was used, biochar samples usually possessed higher BET surface areas (269.5 m² g⁻¹ for biochar_w and 169.9 m² g⁻¹ for biochar_c) than their BC counterparts (209.6 m² g⁻¹ for BC_w and 148.9 m² g⁻¹ for BC_c). This was in line with the more porous structures of biochar samples.

Fig. 1 SEM and TEM (insets) images of biochar_w (a), BC_w (b), biochar_c (c) and BC_c (d) samples prepared and used in this study.

Elemental analysis revealed that the C, H, and N contents of wheat straw were 38.25%, 5.62% and 0.69%, respectively, which were similar to those of cotton stalk (42.49%, 5.81% and 1.07%, respectively). Basically, after pyrolysis or combustion, the carbon content would be enhanced due to decomposition of biomass components such as lignin, cellulose, and hemicelluloses. In consideration of different preparation methods, the carbon contents of biochar samples were generally higher than those of BC samples (Table 1). The H/C ratios of all the BC/biochar samples were less than 1.0, suggesting that they were highly carbonized and exhibited highly aromatic structures.²² The H/C ratios of BC samples were almost twice as high as those of biochar samples, suggesting the lower aromaticity of BC samples.²³ The O/C values of BC samples were several times larger than those of biochar samples, revealing that BC samples had more oxygenated functional groups and were more hydrophilic. The (N + O)/C values of BC samples were also higher than those of biochar samples, which again indicated the higher polarity and contents of functional groups of BC samples.^22,24 In addition, the XRF analysis showed that ashes in BC and biochar samples predominantly contained silica, chlorine salt and alkali metals. Moreover, after obtaining ash contents of different samples, the oxygen contents of BC/biochar samples were also determined by difference (Table S1†). The slightly higher oxygen contents obtained with XRF in comparison to those calculated by difference could be attributed to oxygen contained in ash contents.

Table 1 Elemental analysis of BC and biochar samples

Elemental analysis	BCw	Biocharw^c	BCc	Biocharc^c	Biochar300	Biochar800
a Element content determined by CHN element analyzer.b Element content determined by XRF analysis and corrected by calcium carbonate as standard sample.c Biocharw and biocharc were produced at 600 °C.
C^a (wt%)	20.99	66.92	53.09	77.18	58.66	66.79
N^a (wt%)	0.87	0.93	1.12	1.15	1.31	1.08
H^a (wt%)	1.12	1.74	2.67	2.29	4.63	1.19
O^b (wt%)	64.27	28.39	43.94	13.27	34.53	26.03
Si^b (wt%)	5.4	2.06	0.34	2.99	2.76	2.24
K^b (wt%)	3.92	0.59	0.72	2.56	1.65	0.79
Cl^b (wt%)	1.82	0.11	0.14	1.43	1.03	0.77
Mg^b (wt%)	0.61	0.21	0.31	0.40	0.34	0.32
Ca^b (wt%)	0.57	0.11	0.21	0.27	0.23	0.21
Fe^b (wt%)	0.55	0.33	0.28	0.36	0.29	0.60
S^b (wt%)	0.39	0.07	0.09	0.30	0.20	0.31
Al^b (wt%)	0.25	0.10	0.18	0.27	0.16	0.12
P^b (wt%)	0.19	0.11	0.13	0.19	0.13	0.10
Pb^b (wt%)	0.07	0.15	0.04	0.17	0.04	0.30
H/C	0.64	0.31	0.60	0.36	0.95	0.21
O/C	2.30	0.32	0.62	0.13	0.44	0.29
(O + N)/C	2.33	0.33	0.64	0.14	0.46	0.31

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The FTIR spectra of samples were shown in Fig. 2. The series of peaks in the range of 400 to 900 cm⁻¹ indicated the presence of carbonates (at 875 and 712 cm⁻¹)²⁵ or/and silicates (at 490 and 800 cm⁻¹).^24,26 The stronger absorbance at 1110 cm⁻¹ of two BC samples indicated that they have more aliphatic hydroxyl groups or stretching vibration of C–O–C than biochar samples. This could be attributed to oxygenated groups on carbon skeleton and organic compounds not destroyed during pyrolysis and combustion process.²⁴ The stronger absorbance band at 1430 cm⁻¹ indicated the presence of more aliphatic hydrocarbons in BC samples. The stronger absorbance peak of BC samples at 1640 cm⁻¹ was attributed to C=O stretching vibration and suggested that BC samples might possess more conjugated ketone and quinone groups than biochar samples. The FTIR spectra of biochar samples prepared at different pyrolysis temperatures were also compared. The overall intensity of the spectra became weaker in samples prepared at increased pyrolysis temperatures, indicating the condensed aromatic structures (C=C stretching vibration at 1510 and 1600 cm⁻¹) and lower contents of oxygenated functional groups of biochar₆₀₀ and biochar₈₀₀.^4,24,27

Fig. 2 FTIR spectra of BC and biochar samples produced at different temperatures (biochar_w and biochar_c were produced at 600 °C).

¹³C CP/MAS NMR were compiled to accurately determine the composition of functional moieties in biochar samples (biochar₃₀₀ and biochar₆₀₀) (Fig. S1 and Table S2†). The chemical shift regions assigned to the major types of C were 0–45 ppm for alkyl C, 45–90 ppm for O-alkyl C, 90–145 ppm for aryl C, 145–165 ppm for O-aryl C, 165–190 ppm for carboxyl/ester/quinone C, and 190–220 ppm for ketone/quinone/aldehyde C.^28,29 The well-defined aromatic C resonance centred at δ = 125 ppm was observed in the ¹³C NMR spectra of biochar₆₀₀. Accordingly, attenuation of signals corresponding to oxygenated aromatic C (δ = 145–190 ppm), alkyl C (δ = 0–45 ppm) and O-alkyl C (δ = 0–45 ppm) was observed in NMR spectra of biochar₆₀₀ in comparison to those of biochar₃₀₀. When the charring temperature was increased from 300 to 600 °C, the trends of total alkyl C (0–90 ppm) contents decreased from 36.1% to 9.1%, while the total aryl C (90–165 ppm) contents increased from 51.5% to 86.4%. The initial loss of biomass over 300 °C was attributable to aliphatic components, which were either lost or converted to aromatic carbon in the pyrolysis process.²⁹ In addition, the peak intensity of other functional groups (165–220 ppm for carboxyl/ester/quinone/ketone C) of biochar₆₀₀ was also lower than that of biochar₃₀₀. This was similar to the result of FTIR.

Based on the above characterization, it seems that the preparation method (oxygen-limited pyrolysis or open-field combustion) played a more important role than feedstock biomass (wheat or cotton) in determining properties like structure, morphology, aromaticity, polarity, and elemental and functional group composition of BC/biochar samples.

Effects of different BC and biochar samples on microbial nitrobenzene reduction

Abiotic controls were conducted to determine the contribution of adsorptive nitrobenzene removal by BC and biochar samples. As shown in Fig. 3a, around 5.4%, 2.4%, 7.8% and 4.0% nitrobenzene was removed from the aqueous solution in 120 h by biochar_w, BC_w, biochar_c and BC_c, respectively. Biochar samples demonstrated a little higher adsorption properties than their BC counterparts, which may due to their larger surface areas. However, the adsorptive removal of nitrobenzene by BC/biochar was generally very limited, which was similar with the results reported before.^4,22 Moreover, no aniline was detected in abiotic systems (Fig. 3b), suggesting that the BC and biochar samples had no reducing capacity towards nitrobenzene.

Fig. 3 Effects of different BC and biochar samples on bioreduction of nitrobenzene (a) to aniline (b). And plot of ln(C_t/C₀) versus time for bioreduction of nitrobenzene (c). Open and solid symbols represent abiotic control and bioreduction systems containing S. oneidensis MR-1, respectively.

Significant decline of nitrobenzene concentration was obtained in biotic systems. In 120 h, around 55.5%, 77.5%, 59.8% and 73.7% nitrobenzene were removed in biotic systems containing biochar_w, BC_w, biochar_c and BC_c, respectively, which were all higher than the reduction efficiency (40.5%) observed in system containing MR-1 alone. Obviously, much higher stimulating effects were achieved with the addition of BC samples. The concentration of aniline, which was detected as the sole product in bioreduction system, increased gradually with the reduction of nitrobenzene (Fig. 3b). In 120 h, 30.8 mg L⁻¹ aniline was generated by MR-1 alone, whereas around 41.3, 58.2, 46.2 and 53.8 mg L⁻¹ aniline was formed in biotic systems added with biochar_w, BC_w, biochar_c and BC_c, respectively. Thus the presence of BC and biochar samples derived from crop residue effectively stimulated nitrobenzene bioreduction to aniline. Based on molar concentration, the amount of nitrobenzene disappeared matched well with that of aniline formed (data not shown). Similar mass balance has also been found in a previous study on BC-catalyzed chemical reduction of nitrobenzene to aniline by sulfide.⁴

The kinetics of nitrobenzene reduction could be plotted as ln(C_t/C₀) versus time (Fig. 3c), where C_t and C₀ were the nitrobenzene concentrations at time t and 0, respectively. According to the pseudo-first-order reaction equation ln(C_t/C₀) = −k_obst, the values of reduction rate constant k_obs were calculated to be 6.53 × 10⁻³ h⁻¹, 14.80 × 10⁻³ h⁻¹, 7.57 × 10⁻³ h⁻¹ and 14.11 × 10⁻³ h⁻¹ for biochar_w, BC_w, biochar_c and BC_c, respectively, which were all higher than that of nitrobenzene reduction mediated by MR-1 cells alone (3.77 × 10⁻³ h⁻¹). It should be noted that the k_obs values determined here for microbial nitrobenzene reduction in the presence of BC/biochar was similar with those reported previously for BC/biochar-mediated abiotic nitrobenzene reduction by sulfide.^4,22 Therefore, it seemed that BC/biochar could improve the transfer of both chemically and biologically generated electrons to nitrobenzene at a similar pace.

It has been suggested that BC/biochar produced from different feedstock biomass species generally processed different physicochemical properties.^5,30,31 However, initial biomass species (wheat straw or cotton stalk) seemed to have little impact on the stimulation capacity of BC/biochar samples here (Fig. 3). On the other hand, BC samples generally demonstrated higher stimulating effects than biochar samples prepared from the same crop residues. Thus, preparation procedure (pyrolysis vs. combustion) seemed to play a vital role in determining the stimulating performance of carbon materials.

Additionally, the stimulating effects of wheat biochar samples prepared at different pyrolysis temperatures on nitrobenzene bioreduction were compared. As shown in Fig. 4, the addition of biochar₃₀₀, biochar₆₀₀, and biochar₈₀₀ could all improve microbial reduction of nitrobenzene. Only 65.8% reduction was achieved in systems added with biochar₃₀₀ in 240 h, which is much poorer than that (94.1%) of system containing equal concentration of biochar₆₀₀. However, stimulating capacity of biochar₈₀₀ was only a little better than that of biochar₆₀₀. Therefore, a pyrolysis temperature at 600 °C might be high enough for the preparation of redox active biochar samples. A recent study on microbial dechlorination of pentachlorophenol by G. sulfurreducens in the presence of rice straw-derived biochar also found that biochar samples produced at higher pyrolysis temperature possessed better stimulating capacity.¹⁹

Fig. 4 Effects of 0.50 g L⁻¹ biochar_w prepared at different temperatures (300 °C, 600 °C, 800 °C) on microbial nitrobenzene reduction by S. oneidensis MR-1.

As shown in Fig. 5, MR-1 cells were found to be in close contact with BC/biochar particles, which could facilitate the use of oxygen-containing groups of the carbon materials to improve and extend the transfer of biogenerated electrons. The higher contents of oxygen-containing groups in BC might be the determinant factors for its better accelerating effects.⁴ Specifically, phenolic and quinone groups were considered to be vital redox-active moieties determining the electron accepting and donating capacity of biochar.³² On the other hand, EC property of BC/biochar was also suggested to be crucial factor affecting their capacity of stimulating chemical and biological pollutant reduction. Xu et al.³³ found that the EC of BC correlated with its reactivity to catalyze the transformation of series of nitrated explosives by sulfide, whereas the oxygen-containing groups of BC seemed to have no contribution for its stimulation activity. It was recently suggested that both redox-active moieties and EC of biochar produced at higher charring temperatures (>600 °C) contributed to reductive dechlorination of pentachlorophenol by Geobacter sulfurreducens. However, very limited EC was found with biochar samples obtained at lower pyrolysis temperatures (≤600 °C), whose redox activity mainly depended on their redox moieties.¹⁹ The measured EC values of all the BC/biochar samples prepared in this study were less than 10⁻³ S cm⁻¹ (data not shown), which was extremely low and negligible. Thus the stimulating capacities of BC/biochar samples in our study might be determined only by their oxygenated groups.

Fig. 5 SEM and TEM images (insets) of S. oneidensis MR-1 cells attached to biochar_w (a) or BC_w (b) particles during nitrobenzene bioreduction.

Enhanced reduction of different concentrations of nitrobenzene

As shown in Fig. 6a, the presence of 0.5 g L⁻¹ BC_w/biochar_w could effectively promote the reduction of different concentrations of nitrobenzene ranging from 10 to 400 mg L⁻¹. And better stimulating effects were generally found with BC_w, especially when the nitrobenzene concentration was equal to or higher than 50 mg L⁻¹. Actually, no significant difference in reduction performance could be found between system containing only MR-1 cells and those added with BC_w/biochar_w for the reduction of 10 and 20 mg L⁻¹ nitrobenzene. It seemed that at lower nitrobenzene concentration, microbial cells alone could efficiently deal with nitrobenzene reduction and the electron shuttling activity of BC_w/biochar_w was not effectively utilized.

Fig. 6 Effects of nitrobenzene concentration (10–400 mg L⁻¹) on reduction efficiency at 48 h (a) and initial reduction rate (b) in the presence of BC_w/biochar_w.

Moreover, the relationship between reduction rate and nitrobenzene concentration could be described with Michaelis–Menten kinetics, ν = V_max[S]/(K_m + [S]), where V_max and K_m denoted maximum reduction rate and Michaelis constant, respectively, and [S] represented the concentration of nitrobenzene (Fig. 6b). The calculated V_max/K_m were 0.77, 1.13 and 1.58 L h⁻¹ g⁻¹ biomass for biotic systems containing MR-1 alone, and supplemented with biochar_w and BC_w, respectively, again indicating the better stimulating effects of BC_w over biochar_w.

Effects of BC/biochar dosage on nitrobenzene reduction

The stimulating effects of BC_w on biotic reduction of nitrobenzene increased obviously with the increase of its dosage (Fig. 7). MR-1 cell alone reduced about 42.0% nitrobenzene in 144 h. The presence of 0.17 g L⁻¹ BC_w slightly enhanced the reduction efficiency to 44.2%. Around 63.5% and 82.2% nitrobenzene were reduced by MR-1 in 144 h in the presence of 0.33 and 0.50 g L⁻¹ BC_w. When the BC_w dosage was increased to 0.67 g L⁻¹, complete reduction of nitrobenzene could be achieved in 120 h. On the other hand, the stimulating effects of biochar_w varied very little (55.5–62.9%) at different dosages. Surprisingly, when the biochar_w dosage was increased from 0.50 to 0.67 g L⁻¹, the stimulating effect even decreased. This might be caused by the aggregation of biochar particles at higher concentrations, which could decrease the available sites and moieties participating in reduction stimulation.

Fig. 7 Effects of BC_w (solid line)/biochar_w (dashed line) dosage (0.17–0.67 g L⁻¹) on nitrobenzene reduction by S. oneidensis MR-1.

Effects of coexisting AQDS and BC/biochar on bioreduction of nitrobenzene

AQDS has been well recognized as a model soluble redox mediator and could greatly improve microbial reduction of nitroaromatics and many other oxidative pollutants.^34–37 It was found that the presence of 50 μM AQDS could effectively stimulate nitrobenzene bioreduction. Over 98% nitrobenzene was reduced in 72 h, which was much higher than that (30.1%) observed in system containing MR-1 alone (Fig. 8 and 3a). Almost no additional improvement in nitrobenzene reduction efficiency was observed when BC_w at a concentration of 0.17 g L⁻¹ was added together with AQDS into the bioreduction system. However, the coexistence of 0.33–0.67 g L⁻¹ BC_w with AQDS further stimulated the bioreduction performance. Most notably, almost 90% nitrobenzene was reduced in 24 h when 50 μM AQDS and 0.67 g L⁻¹ BC_w coexisted in the bioreduction system. During the same period, 48.3% and 60.0% reduction were reached in systems containing 50 μM AQDS and 0.33 or 0.50 g L⁻¹ BC_w, respectively, both of which were still higher than that (39.0%) obtained in systems having AQDS as sole redox mediator. In 48 h, less than 80% reduction occurred in bioreduction systems only added with AQDS, whereas over 95% reduction was observed in systems containing both AQDS and over 0.33 g L⁻¹ BC_w.

Fig. 8 Effects of coexisting BC_w (a) and biochar_w (b) samples (0.17–0.67 g L⁻¹) and AQDS (50 μM) on nitrobenzene reduction by S. oneidensis MR-1.

As shown in Fig. 8b, biochar_w could also further accelerate the AQDS-mediated nitrobenzene bioreduction. In 36 h, 57.2% nitrobenzene was reduced in bioreduction system containing 50 μM AQDS. The simultaneous addition of 0.17, 0.33 and 0.50 g L⁻¹ biochar_w with 50 μM AQDS resulted in 64.9%, 75.7% and 83.7% reduction of nitrobenzene in 36 h, respectively. However, a further increase of biochar_w dosage to 0.67 g L⁻¹ did not cause continuous enhancement of the reduction efficiency. In 48 h, over 95% reduction was achieved in systems containing AQDS and more than 0.33 g L⁻¹ biochar_w. And the reduction efficiency of system added with 0.17 g L⁻¹ biochar_w was around 87%, which was still higher than that of bioreduction system added with only AQDS (78.5%).

Kappler et al.¹⁷ found that compared to AQDS-supplemented bioreduction system, systems added with high concentrations of biochar (5–10 g L⁻¹) together with 100 μM AQDS demonstrated high ferrihydrite reduction rates and extents. It was suggested that the combination of soluble AQDS and solid-state redox-active biochar could further facilitate electron transfer between MR-1 cell and ferrihydrite. More recently, rice straw biochar adsorbed by AQDS was demonstrated to have better stimulating effects than unmodified biochar on pentachlorophenol dechlorination by G. sulfurreducens.¹⁹ Considering the wide distribution of redox-active quinone compounds and humic substances in soils and sediments, the cooperation of BC/biochar and naturally-occurring soluble redox mediator might contribute to in situ reduction and bioremediation of pollutant. Also, this coupling stimulation effect could be utilized to speed up the treatment of oxidative pollutants in engineered systems.

Conclusions

For the first time, BC/biochar prepared from crop residues were found capable of effectively stimulating nitrobenzene bioreduction. The preparation method was crucial in determining the physicochemical properties and stimulating capacity of BC/biochar. BC samples possessing more oxygenated groups generally demonstrated better stimulating capacity. And the stimulating performance of biochar usually increased with the increase of its charring temperature. Effective stimulation by BC/biochar was observed for nitrobenzene concentration ranging from 10 to 400 mg L⁻¹. Higher dosage of BC/biochar generally resulted in better stimulating effects. The presence of BC/biochar also helped further enhance AQDS-mediated nitrobenzene bioreduction. According to these findings, BC/biochar prepared from crop residues could be used as cost-effective and efficient redox mediators to stimulate pollutant bioreduction.

Acknowledgements

This work was supported by Open Project of State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology (No. QAK201530), National Natural Science Foundation of China (No. 51478076) and “Twelfth Five-year Plan” for Science & Technology Research of China (2012BAD15B05).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra11671j

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