Biosorption performance evaluation of heavy metal onto aerobic granular sludge-derived biochar in the presence of effluent organic matter via batch and fluorescence approaches

In present study, the biosorption process of Cu(II) onto aerobic granular sludge-derived biochar was evaluated in the absence and presence of effluent organic matter (EfOM) by using batch and fluorescence approaches. It was found that EfOM gave rise to enhancement of Cu(II) removal efficiency onto biochar, and the sorption data were better fitted with pseudo-second order model and Freundlich equation, in despite of the absence and presence of EfOM. According to excitation-emission matrix (EEM), EfOM was mainly comprised by humic-like substances and fulvic-like substances and their intensities were reduced in the addition of biochar and Cu(II) from batch biosorption process. Synchronous fluorescence spectra coupled to two-dimensional correlation spectroscopy (2D-COS) further implied that a successive fluorescence quenching was observed in various EfOM fractions with the increasing Cu(II) concentration. Moreover, fulvic-like fraction was more susceptibility than other Corresponding author. Tel: +86 531 8276 7370; fax: +86 531 8276 7370. E-mail address: dubin61@gmail.com (B. Du) 2 fractions for fluorescence quenching of EfOM.


Introduction
With the rapid development of the economy, a great number of industries, such as metallurgy, machinery manufacturing, chemical, electronics, textile, instrument and printing etc., produce and discharge hazardous wastes based various heavy metals into the environment. The increasing heavy metal pollution has become one of the most worldwide environmental problems facing humanity, which brings about serious water pollution, threatens human health and ecosystem ). Due to their non-degradable, accumulation and persistent characteristics, the removal and elimination of toxic metal ions from contaminated water is significant necessary prior to discharge into receiving water.
Till now, numerous methods have been proposed for efficient heavy metal removal from aqueous solution, including chemical precipitation, ion exchange, adsorption, membrane filtration and electrochemical technologies etc. (Fang et al., 2017;Ming et al., 2012). Among all above these techniques, adsorption is regarded as one of the most important physico-chemical treatment processes due to its advantages of high efficiency, simplicity of design and ease of operation (Cui et al., 2015;Fomina and Gadd, 2014;He and Chen, 2014). As one kind of carbon-enriched biosorbents, biochar has been recently attracted considerable attention and successfully applied for treating various heavy metals because of its low-cost, resource abundant, eco-friendly, and mechanical and thermal stability (Tavakoli et al., 2017). At present, a wide range of biological raw materials (e.g. bacteria, sludge, biomass, algae and fungi etc.), either in original or modified forms, have been developed as precursors for preparing biochar and subsequent heavy metal sorption ( (Vigneswaran, 2006). It is generally accepted that HA and FA are chemically heterogeneous compounds that can interact with the surface of biosorbents, alter their physicochemical properties, and therefore influence their sorption behaviors (Yang and Xing, 2009). Moreover, a variety of functional groups are present in EfOM at different proportions and configurations, such as carbonyl, carboxyl, aromatic, acetal, and phenolic groups etc., which could also allow them to complex with heavy metal ions (Tian et al., 2012). Although the presence of HA or FA on the influence of metal sorption performance has already been acknowledged in recent literatures, there is still lack of information regarding to the effect of EfOM on metal sorption by using biochar. Especially, both FA and HA coexisted in EfOM have various binding sites that could compete with the target heavy metal during sorption process, which would be much more complicated than that of single system containing only FA or HA. Therefore, it is of great significance to investigate the adsorption behavior of heavy metal onto biochar in the presence of EfOM.
Hence, the objective of this study was to evaluate the presence of EfOM on the influence of metal sorption onto biochar via batch and spectral approaches. To achieve this purpose, biochar was prepared by using aerobic granular sludge (AGS) as raw material. Cu(II) was selected as the target metal ion for evaluating the sorption performance and difference in the presence and absence of EfOM. Moreover, the interaction between EfOM and Cu(II) in the sorption process was evaluated by using a combined use of 3D-EEM, fluorescence regional integration (FRI), synchronous fluorescence and two-dimensional correlation spectroscopy (2D-COS). The obtained results could provide an insightful understanding of metal bisorption in the presence of EfOM by application of fluorescence spectroscopy.

Effluent organic matter
The raw EfOM sample was collected before the disinfection step at the secondary settling tank in a municipal wastewater treatment plant (WWTP) located at Jinan, China. The WWTP used a combined anaerobic/anoxic/aerobic activated sludge process with a treatment capacity of 50,000 m 3 /day. Prior to experiment, the EfOM sample was filtered through a 0.45 nm filter to remove particles and then stored in the refrigerator at 4 o C before use. The total organic carbon (TOC) of the EfOM sample was typically around 9.0 mg/L.

Preparing of AGS-BC
Aerobic granular sludge (AGS) was used as raw material for biochar preparation, which was collected from our lab-scale SBR at the end of aeration process. The detailed synthetic wastewater of SBR could be found elsewhere (Wei et al., 2014).
Previous literature has been proved that AGS based biochar has a better sorption capacity than that of activated sludge . The raw AGS for carbonization had a regular and round-shaped outer structure with an average size of about 2 mm. All the sludge samples were washed three times by using deionized water to remove the surface soluble ions.
AGS-BC was prepared by using a chemical activation method according to the literature reported by Tay et al. (2001). The raw sludge sample was firstly dried at 105 o C for 24 h to achieve constant weight. Next, 10 g of samples were added into 25 mL of 5 mol/L ZnCl 2 solution for 24 h at temperature. After that, the sample was carbonized at 650 o C for 2h in a quartz tube under N 2 atmosphere. After cooling down to room temperature, the obtained products were wished with 3M HCl, followed by filtration and dried at 105 o C. 6

Experimental design
For comparing the effect of EfOM on the metal bisorption performance, batch sorption tests were carried out in 50-mL conical flasks at 25 o C, in which Cu(II) and EfOM were added and pre-equilibrated for 24 h before AGS-BC was added. More

Fluorescence analysis
3D-EEM spectra were scanned with a luminescence spectrometer (LS-55, Perkin-Elmer Co., USA). Detailed, EEM scans were made at the emission wavelengths (Em) from 280 to 550 nm with a 0.5 nm-increment and at the excitation wavelengths (Ex) from 220 nm to 400 nm with a 10 nm-step. The excitation and emission slits were both 10 nm. The scanning speed was set at 1200 nm/min for all the fluorescence measurements.

Analytical methods
The specific surface area and pore structure parameters of AGS-BC were determined by using Micromeritics ASAP 2020 surface area and porosity analyzer (Quantachrome, United States). Fourier Transform infrared spectroscopy (FTIR) in the spectral range of 4000-400 cm -1 was measured by using a Perkin-Elmer Spectrum One spectrometer (United States). The surface physical morphology of biochar was observed by using a scanning electron microscope (SEM, Quanta 250 FEG). The surface charge of the samples was determined by measuring the zeta potential by using a Malvern Zetameter (Zetasizer 2000). The adsorption experimental results of Cu(II) were analyzed in triplicate, and the averaged data were presented here. The synchronous and the asynchronous maps in 2D-COS for extending the spectra along the second dimension could be obtained from the method reported by Noda and Ozaki (2005).

Characterization of AGS-BC
The physical and chemical characteristics of prepared AGS-BC were observed by using BET, FTIR spectra, SEM and Zeta potential. It was found that BJH desorption cumulative volume of pores and BET surface area of biochar were 0.6985 cm 3 /g and 1175.1 m 2 /g, respectively. The surface area of AGS-BC was much higher than the previous literature reported by using activated sludge as raw material (Al-Malack and Dauda, 2017), which is beneficial for surface sorption. The main functional groups in AGS-BC included -OH at 3436 cm -1 , C=O at 1631 cm -1 and C-O-C at 1212 cm -1 . It was found from SEM that different pores and cavities were observed in the surface of biochar, which might be resulted from the decomposition of ZnCl 2 during the thermal treatment process. Zeta potential of biochar decreased from 11.6 to -4.7 mV in the pH range of 2-10, respectively. The negative Zeta potential (pH>5.0) of the sludge-based biochar implied that the positively charged metal pollutants may be more easily adsorbed.

Effect of contact time on Cu(II) sorption
The effect of contact time on Cu(II) sorption onto AGS-BC was evaluated in the absence and presence of EfOM. It is clearly observed that Cu(II) sorption onto AGS-BC occurred in two steps as a function of contact time: a very rapid initial sorption, followed by a long period of much slower uptake, as similarly reported by Wei et al. (2016c). Cu(II) sorption rate was much higher in the beginning due to larger surface area available of adsorbent, in spite of the presence and absence of EfOM.
However, Cu(II) sorption onto AGS-BC achieving equilibrium were at 60 and 300 min in the presence and absence of EfOM, respectively. The presence of EfOM gave rise to enhancement of Cu(II) removal, whose removal increased almost from 57.4% to 76.8%. The result suggested that the addition of EfOM increased the sorption property of Cu(II) onto AGS-BC.
In order to elucidate the potential rate-limiting steps of biosorption, the batch results of Cu(II) onto AGS-BC in the presence and absence of EfOM were analyzed according to pseudo-first-order and pseudo-second-order models. The above two kinetic models can be expressed as follows (Eqs (1-2)): Where q e (mg/g) is the adsorbed amount at time t, q t (mg/g) is the adsorbed amount at equilibrium, k 1 (1/min) is the rate constant of pseudo-first-order kinetic model, and k 2 (mg/ (g min)) are the equilibrium rate constant of the pseudo-second-order kinetic model. Fig.1 shows the pseudo-first-order and pseudo-second-order kinetics fit of Cu(II) sorption onto AGS-BC in the absence and presence of EfOM. Table 1  suggesting that sorption kinetic data were better fitted to pseudo-second-order model than the pseudo-second-order and the intra-particle diffusion models.

Adsorption isotherm
The adsorption isotherm could provide the detailed information about the surface properties of adsorbent, the adsorption behavior and the design of adsorption systems (Fu et al., 2015). For better predicting the isotherm results, two common models including Langmuir and Freundlich are described based on the adsorption equilibrium data. Langmuir and Freundlich models can be described in linear form as follows: m e e e 1 bq c q bc 1/ e f e n q K c Where c e (mg/L) is the equilibrium concentration of Cu(II), q e (mg/g) is the adsorption capacity, q m (mg/g) is the theoretical maximum sorption capacity, b (L/mg) is the Langmuir constant related to adsorption energy, K f is the binding energy constant reflecting affinity of adsorbents to Cu(II), and n is the Freundlich constant.  Table 2 summarizes the constants and correlation coefficients of adsorption isotherms for the sorption of Cu(II) onto AGS-BC with and without EfOM. It was found that Cu(II) sorption onto biochar in the presence of EfOM was significantly higher than in the absence system. The found that the presence of EfOM significantly reduced the adsorption capacities and sorption rates of perfluorinated compounds onto activated carbon.

3D-EEM
It is well reported that the interaction between HA or FA and sorbent not only alter the surface properties and environmental behavior of these sorbent materials, but also affect the adsorption of coexisting contaminants (Tian et al., 2012). Therefore, The presence of humic-like substances may be related to a biological production and activity of microorganisms, which could be referred to the non-biodegradable component in EfOM samples (Wei et al., 2016c). Compared to previous literature (Phong and Hur, 2016), Peak C at Ex/Em of about 250/350 nm was assigned to protein-like substances, whose fluorescence intensity was much lower than those of humic-like substances and fulvic -like substances. The reason may be attributed that protein-like substances were representative of the biodegradable EfOM and therefore could be utilized by activated microorganism (Yang et al., 2017).  Fig. 4 shows the FRI distribution of EfOM samples during the biosorption process. In the raw EfOM (sample A in Fig. 4), Region III (fulvic acid-like substances)

FRI analysis
and Region V (humic acid-like organics) expressed significant higher percentages (27.8% and 34.7%) than those of other Regions, suggesting that they had the relatively high amounts in EfOM. The FRI observation was consistent with the Peak A and Peak B fluorescence intensities in 3D-EEM (Fig. 3A). It was found that the percentages of Region III and Region V were reduced relatively higher degrees after interaction with single Cu(II) and AGS-BC, whose values were changed to 24.8% and 24.4%, respectively. Therefore, the coexisted fulvic-like and humic-like substances in EfOM may be referred to react with the adsorbate and adsorbent. Additionally, there was a highest reduced percentage of Region V in the presence of both Cu(II) and AGS-BC, whose value was accounted for 18.2 % of the total fluorescence percentages, suggesting that humic-like substances played significant role in metal sorption process.

Synchronous fluorescence spectra
Since both FA and HA in EfOM containing different types of functional groups could react with Cu(II), therefore, the interaction between FA/HA and Cu(II) was important and complex than the single system. In present study, synchronous fluorescence spectra were further applied for better understand the interaction between EfOM and Cu(II), as displayed in Fig. 5. which was similar to our observation.

2D-COS
To provide more information on the heterogeneous distribution of metal binding sites within EfOM, 2D-COS was applied to synchronous fluorescence spectra of EfOM with Cu(II) addition as the external perturbation. Two types of the maps, synchronous 2D and asynchronous 2D spectra, were generated from 2D-COS (Wei et al., 2016b). As shown in Fig. 6A, one positive autopeak (345 nm) was indentified along the diagonal line of the synchronous map, indicating that the spectral changes proceed in the same direction for the corresponding areas. Moreover, fulvic-like fraction was more susceptibility than thoses of other fractions in EfOM (e.g. protein-like and humic-like fractions).
Asynchronous map reveals a sequential or successive change in the spectral intensities in response to external perturbation (Yu et al., 2012a). As shown in Fig. 6b, one obvious negative area (250-300, 300-400 nm) was observed. According to Noda' rule, EfOM fluorescence quenching took place in the order of 300-400 nm >250-300 nm. Therefore, it could be concluded from asynchronous map that, for EfOM quenching, fulvic-like fraction and the short wavelength of humic-like fraction took place earlier than other fluorescence components.

Conclusions
In summary, the influence of EfOM sorption onto sludge-based biocher was evaluated by using batch and fluorescence approaches. For sorption process, Cu(II) sorption onto biochar followed pseudo-second order kinetic model and Freundlich model in both absence and presence of EfOM. Spectroscopic observation implied that FA and HA were two main components in EfOM and expressed decreased trends in the addition of Cu(II), which may be the reason for enhanced biosorption performance.
The result of present study is helpful to provide insightful information on the meal treatment process by using biochar.

Acknowledgements
This study was supported by Natural Science Foundation of Shan dong Province