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Effect of bio-char on methane generation from glucose and aqueous phase of algae liquefaction using mixed anaerobic cultures
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Research paper
Effect of bio-char on methane generation from glucose and aqueous phase of algae liquefaction using mixed anaerobic cultures Saravanan R. Shanmugam, Sushil Adhikari∗, Hyungseok Nam, Sourov Kar Sajib Department of Biosystems Engineering, Auburn University, Auburn, AL 36849, USA
A R T I C L E I N F O
A B S T R A C T
Keywords: Biochar Activated carbon Methane production Microbial analysis Interspecies electron transfer
Activated carbon is known to enhance methane formation in anaerobic reactors via interspecies electron transfer between fermentative bacteria and methanogenic archaea. Biochar, a by-product of biomass pyrolysis process, could also perform similar functions due to its conductive properties and the presence of redox active moieties. Hence, this study was conducted to evaluate the effectiveness of different types of activated carbons and biochars on anaerobic digestion. Biochars obtained from canola meal, switchgrass and Ashe juniper were tested for methane production from both glucose and aqueous phase of bio-oil generated via hydrothermal liquefaction of algae. The results suggested that absorbents enhanced methane production. Furthermore, biochars synthesized at intermediate temperatures significantly increased methane yield and reduced the lag time required for methane formation. In addition, the results suggested that the redox active moieties such as quinones and phenazines in biochars are responsible for electron transport, which ultimately enhanced methane production.
1. Introduction Anaerobic digestion (AD) is an effective method to treat organic wastes and recover energy in the form of biogas (methane). Fossil fuels utilized for power generation can be substituted with biogas produced via AD process [1,2]. Methane generation via AD is carried out by several groups of microorganisms involved in hydrolysis, acidogenesis, acetogenesis and methanogenesis [3]. Typically, about 25–65% of waste organics are converted to methane in a mesophilic AD process [4]. Performance of the AD process is determined by the effectiveness of interspecies electron transfer (IET) between secondary fermenting bacteria producing diffusive electron carriers (such as formate and hydrogen) and methanogenic archaea [5]. Disruption of syntrophic association between these bacteria often results in instabilities in reactor performances due to a decrease in pH followed by the accumulation of volatile fatty acids (VFAs). Adsorbents such as granular activated carbon (GAC), powdered activated carbon (PAC), silica gel, gelatin, pectin, aluminium powder, and bentonite are added to methanogenic reactors to overcome the disruption problem and stabilize the AD process [5,6]. In addition, these adsorbents were found to increase the methane yields in the AD process. For example, Lee et al. [5] observed a 1.8-fold higher methane production rate in GAC added reactors in comparison to control reactors with no GAC addition. Desai and Madamwar [6] claimed that addition of silica gel (4 g/L) resulted in a two-fold enhancement in total gas production and
∗
17% increase in methane composition of biogas. Addition of adsorbents helps in sorption of toxic organic compounds, which are inhibitory to methanogenesis, and provides a high surface area for microbial growth, which in turn favors higher methane production rates. In addition, activated carbon (AC) adsorbent acts as a good conductive material (such as an electrode) to facilitate direct interspecies electron transfer (DIET) between secondary fermenting bacteria (acting as anode-reducing microorganisms) and methanogenic archaea (acting as cathode oxidizing microorganisms) [5,7,8]. For example, a study conducted by Liu et al. [7] showed that DIET occurred between Geobacter sp. (anode reducing bacteria) and Methanosarcina barkeri (cathode oxidizing bacteria). Thus, the addition of conductive materials leads to an increase in the energy efficiency and biogas production via DIET by removing several steps associated with hydrogen production and consumption [5,7]. Further, a study conducted by Kato et al. [8] concluded that electron transport via conductive materials is much faster than molecular transport of electron carriers. Similar to activated carbon adsorbents, biochar (BC) could also be used to enhance methane production via DIET during AD process due to its conductive properties similar to that of AC. BC is a carbon rich residue produced during the thermochemical decomposition of biomass via gasification or pyrolysis. BC is an inexpensive and eco-friendly solid material used for a number of purposes, such as soil remediation, waste management, greenhouse gas reduction and energy production [9]. The major element of BC is carbon (C), along with minor amounts of
Corresponding author. E-mail address: [email protected] (S. Adhikari).
http://dx.doi.org/10.1016/j.biombioe.2017.10.034 Received 2 March 2017; Received in revised form 16 October 2017; Accepted 24 October 2017 0961-9534/ © 2017 Elsevier Ltd. All rights reserved.
Please cite this article as: Shanmugam, S.R., Biomass and Bioenergy (2017), http://dx.doi.org/10.1016/j.biombioe.2017.10.034
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similar to what was described in the published document [20]. The initial study using glucose was conducted to compare the effectiveness of BCs (ABC-400 and SBC-500) with ACs (GAC and PAC). Addtional anaerobic digestion experiments were also performed using biochars (BCs) synthesized at different temperatures (400 °C, 600 °C, 700 °C, and 900 °C) to understand the effect of these BCs in treatment of complex organic wastes. BCs synthesized from two different materials such as Ashe Juniper biomass (ABC-400 and ABC-600) and canola meal (CBC700 and CBC-900) were evaluated. BOAP collected during HTL of algae was used as a complex organic substrate in this study. Information regarding algal composition, HTL operation, and BOAP characteristics can be found elsewhere [20,21]. AD experiments were conducted using a 160 mL serum bottle batch reactor with a 55 mL working volume comprising basal medium, inoculum and substrate. In order to study the effect of adsorbents on the performance of methane production process from glucose and BOAP, batch reactors (serum bottles) were supplemented with 1% of each adsorbent (0.55 g in 55 mL). Control reactors for each type of adsorbent were also ran in parallel with no adsorbent addition. AD experiments were conducted at mesophilic temperature (37 ± 1 °C) in an orbital shaker (with no shaking). The initial volatile suspended solids (VSS) used for AD experiments were 3000 ± 105 mg/L (4260 ± 149 mg COD/L) using a conversion factor of (1.42 g COD/g VSS). The initial pH and substrate concentration of all experimental sets were 7.8 ± 0.05, and 1 g COD/L, respectively. The substrate to inoculum ratio was maintained at 0.24 in all experimental sets (on COD basis). Separate culture control (no substrate addition) and adsorbent control (adsorbent with no substrate addition) experiments were also conducted to analyze any background CH4 production. Background CH4 yields of the corresponding experimental controls were subtracted from the experimental sets in the data presented. The batch reactors (both with different types of adsorbents and no adsorbents) were prepared and left for incubation in an orbital shaker (at 250 rpm) for a period of 7 days prior to the initial experiment to allow acclimation. At the end of 7 days, each batch reactor was fed with 1 g COD/L of glucose and BOAP and the reactors were not shaken. This procedure was followed to understand the role of IET in adsorbent added and non-adsorbent added cultures. However, the batch reactors were shaken only once at 250 rpm briefly (for 1 min) before performing the gas sampling. The CH4 yields from all experimental sets were reported in mL/g COD.
hydrogen (H), oxygen (O) and trace amounts of sulfur (S) and nitrogen (N). The elemental composition of BC depends upon the nature of raw biomass material and on the carbonization process [10]. Owing to its porous structure, specific surface area, surface functional groups, and high nutrient content (both micro- and macro-nutrients), BC, could be used to support bacterial growth and enhance biogas production. Addition of BC to enhance biogas production during AD process has been gaining some research attention [11–14]. BC, however, has low surface area in comparison to AC, but it contains certain redox active moieties (RAMs) such as quinones and phenazines, which act as electron transfer catalysts during redox reactions in many soil and biogeochemical environments. In addition, BCs produced at different temperature were found to have different characteristics [15]. Although there is a growing interest of utilizing BCs in AD, studies focused on the treatment of complex organic wastes with addition of BC are very limited in the open literature. To the best of our knowledge, no studies have been conducted to compare the effectiveness of BC (derived from both herbaceous and woody biomass) versus AC (both powder and granular) on methane production. Hence, this study was conducted to investigate the effect of biochars produced from different biomass and process conditions on methane production with microbial community analysis. AD experiments were carried out using simple substrate such as glucose and complex organic waste such as aqueous phase of bio-oil (BOAP) generated during hydrothermal liquefaction (HTL) of algae. 2. Materials and methods 2.1. Materials Seven types of adsorbents (two types of AC and five types of BC) were investigated to understand the effect of adsorbents during AD of simple organics (glucose) and complex organics (BOAP) in this study. Both PAC (Product code: C3014) and GAC (product code: 31616) were purchased from Sigma Aldrich (St. Louis, MO, USA) and used without further modifications. Biochars such as CBC, SBC and ABC were produced using pyrolysis of canola meal, switchgrass and Ashe juniper, respectively. Briefly, CBC was collected during bench-scale slow pyrolysis of canola meal operating at a temperatures of 700 °C (CBC-700) and 900 °C (CBC-900), respectively with a residence time of 2 h. SBC was collected during pyrolysis of switchgrass in an auger reactor operating at a temperature of 500 °C (SBC-500) with a residence time of 72 s, and the details are explained in the published document [16]. Similarly, ABC was collected during bench-scale slow pyrolysis of Ashe juniper biomass operated at temperatures of 400 °C (ABC-400) and 600 °C (ABC-600) with a residence time of 30 min. The detailed operation of bench-scale pyrolysis system is described elsewhere [17]. All these seven types of adsorbents were characterized for proximate analysis [17], BET (Brunauer-Emmett-Teller) surface area [18], average pore diameter [18], and pore volume [18], electrical conductivity (EC), particle density [18], particle size distribution [18] and pH (at point of zero charge, pHPZC) [19]. The EC of BC and AC was measured according to the standard procedure used to measure electrical conductivity of soil. In addition, all these adsorbents were analyzed for the presence of metal elements using the inductively coupled plasma-optical emission spectroscopy (ICP-OES; PerkinElmer Life Sciences 9300-DV system). Adsorbents were also characterized using FT-IR using the methods previously described elsewhere [20] to detect the presence of functional groups such as quinones and phenazines known to involve in IET.
2.3. Analytical methods Liquid samples (1.5 mL) collected at the beginning and the end of AD experiments using pure glucose and complex organic waste (BOAP) were analyzed for TOC and COD according to the methods described previously [20]. In addition, volatile fatty acids (VFAs) composition was examined using a Shimadzu high performance liquid chromatograph (HPLC) equipped with UV and RI detectors. Both glucose and VFAs (lactic acid, acetic acid, propionic acid, formic acid and butyric acid) were analyzed using Aminex HPX-87H column with 5 mM sulfuric acid as eluent. The column temperature was set at 50 °C with an eluent flow rate of 0.6 mL/min. Headspace biogas composition was analyzed according to the method described previously [20]. Statistical difference between multiple means of the gas metabolite data were evaluated using a Tukey's paired comparison procedure at 95% confidence level [22]. 2.4. Microbial community analysis At the end of the batch experiment (using glucose), microbial samples were collected for community analysis using sterile 1.5 mL Eppendorf tubes from all experimental sets of glucose study. The liquid supernatant was discarded and 1.5 mL of settled microbial cultures were collected in these tubes. In batch reactors with adsorbents, both the adsorbents along with microbial cultures were taken in order to
2.2. Anaerobic digestion experimentation Mixed anaerobic culture obtained from Jackson Pike wastewater treatment facility (Columbus, Georgia, USA) was used for AD experiments of glucose and BOAP. Inoculum source, inoculum characterization, and basal media preparation for conducting AD experiments were 2
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compared to other types of adsorbents. The elements such as Ni, B, Co, Zn, and Cu were present in low concentrations. Results from FT-IR analysis showed absorbance around wavenumbers 1690 cm−1 (γC = O stretch), 1580 cm−1 (γC = C stretch), and 1300-1000 cm−1 (CeO stretch) in ABC-400, ABC-600 and SBC-500 adsorbents (Fig. 1a). In addition, strong absorbance between 2850 and 2920 cm−1 was observed in CBC-700, CBC-900 and ABC-400 samples due to CeH stretching (Fig. 1b). Study conducted by Burie et al. [25] indicated that absorbance at wavenumbers between 1500 and 1700 cm−1 correspond to the characteristic peak of quinone moieties (due to γC = O and γC = C stretching). Recent study by Yu et al. [26] concluded that quinone moieties in biochar play an important role in bacterial IET. In addition, absorbance peaks at wavenumbers 1100 cm−1 in SBC-500 and 1260 cm−1 in SBC-500 and ABC-400 might be due to presence of phenazines. Study conducted by Stammer and Taurins [27] showed characteristic peaks of phenazines at these wavenumbers (1100 and 1260 cm−1). However, the intensity of peak corresponding to both quinones and phenazines were found to be very low/absent in activated carbon adsorbents (PAC and GAC) and also from biochar adsorbents collected at high temperatures (CBC-700 and CBC-900) (Fig. 1a).
quantify the microorganisms attached to each adsorbent facilitating IET. DNA extraction was conducted using the MoBio PowerSoil DNA extraction kit (MoBio Laboratories, Solana Beach, CA, USA) according to the protocol provided by the supplier. The extracted DNA was then immediately stored at −20 °C until further used. DNA samples were shipped for community characterization to molecular research (MR DNA) laboratory (Shallowater, TX, USA). Briefly, amplification of prokaryotes (bacteria and archaea) was performed using paired-end 16S community sequencing on the Illumina platform (MiSeq). The 16S rRNA gene V4 variable region PCR primers 515/806 with barcode on the forward primer was used in a 28 cycle PCR using the HotStar Taq plus Master Mix Kit (Qiagen, USA) using the following conditions: 94 °C for 3 min, followed by 28 cycles of 94 °C for 30 s, 53 °C for 40 s, and 72 °C for 1 min, after which a final elongation step at 72 °C for 5 min was performed. Amplified DNA samples are purified using calibrated Ampure XP beads. Sequencing was conducted according to the manufacturer's guidelines. Sequence data were processed using MR DNA analysis pipeline. Operational taxonomic units (OTUs) were defined by clustering at 3% divergence (97% similarity). Final OTUs were taxonomically classified using basic local alignment search tool for nucleotides (BLASTn) against a curated database derived from RDPII and NCBI.
3.2. Biogas production using pure glucose 3. Results and discussion Biogas (CH4) production results under different experimental conditions are shown in Fig. 2. It is important to note that the methane production from glucose is well below theoretical limit (350 mL/g COD) mainly due to the lack of shaking. The goal here was not to obtain complete conversion of glucose to methane but rather to understand the effect of absorbents on methane conversion. The results indicate that ABC-400 and SBC-500 added cultures showed a shorter lag time required for CH4 production in comparison to both activated carbon added cultures (GAC and PAC) and no adsorbent added cultures (GLU). For example, the CH4 yields obtained in ABC-400 and SBC-500 at 24 h were found to be 39 ± 16 mL/g COD and 22 ± 4 mL/g COD, respectively. In comparison, the CH4 yields of GAC, PAC and GLU added cultures at 24 h were found to be 2 ± 0.6 mL/g COD, 10 ± 0.4 mL/g COD and 0 ± 0 mL/g COD, respectively. These results indicated that BC effectively reduced the lag time of CH4 generation compared to ACs. Study conducted by Sunyoto et al. [14] also concluded that BC addition reduced the lag phase of CH4 formation by 41% during AD of aqueous carbohydrates food waste. At the end of 48 h, the CH4 yields using PAC and GAC were similar in the range of 25–30 mL/g COD. However, both SBC-500 and ABC-400 showed higher CH4 production (71 ± 15 mL/g COD and 100 ± 27 mL/g COD, respectively) at the end of 48 h. After 48 h, GAC showed higher CH4 yields in comparison to PAC added cultures until 240 h. In comparison, PAC added cultures showed similar CH4 yields compared to GLU cultures until 96 h. The final CH4 yields at the end of 240 h showed the following trend: SBC-500 (332 ± 16 mL/ g COD) = ABC-400 (330 ± 2 mL/g COD) > GAC (271 ± 8 mL/g COD) > PAC (239 ± 10 mL/g COD) > GLU (193 ± 21 mL/g COD). From these results, it can be concluded that biochar derived from both herbaceous biomass (SBC-500) and woody biomass (ABC-400) showed similar levels of CH4 yields irrespective of the nature of pyrolysis. Study conducted by Yu et al. [26] mentioned that EC of adsorbent material is one of the important parameters affecting electron transfer between bacterial cells. It is interesting to note that the EC of GAC, SBC500 and ABC-400 was in the range of 322 and 350 μS cm−1. However, the CH4 yields of both SBC-500 and ABC-400 were found to be higher in comparison to GAC (Fig. 2). Between the ACs, the CH4 yield using GAC was found to be slightly higher as compared to PAC although the EC of GAC was 19 times higher than the EC PAC. In addition, EC of ABC-400 and SBC-500 was similar to that of GAC but CH4 yield with biochars was much higher than with GAC. These results indicate that IET between fermentative bacteria and methanogenic archaea in adsorbent added cultures could be influenced by some other parameters besides
3.1. Characterization of adsorbents used in this study Table 1 summarizes the characteristics of adsorbents investigated in this study. Biochars such as ABC-400 and SBC-500 showed higher volatile content (41.0 ± 0.3% and 40.0 ± 0.2%, respectively) due to low temperature pyrolysis (400 °C and 500 °C) in comparison to biochars produced at high temperatures such as ABC-600 (18.8 ± 0.3%), CBC-700 (17.9 ± 1.5%) and CBC-900 (17.8 ± 0.2%). In comparison, both GAC and PAC showed 7.0 ± 0.2% and 8.0 ± 0.2% volatile content, respectively indicating that they might have been prepared at higher temperatures. Both CBC-700 and CBC-900 showed high ash composition in comparison to all other adsorbents investigated in this study. The pHPZC for different type of adsorbents indicated that the pHs of ABC-400, SBC-500 and CBC-900 were in the neutral range whereas that of ABC-600, CBC-700, GAC and PAC were in the basic range. Electrical conductivity (EC) (in μS/cm) of adsorbents showed the following trend: ABC-400 (350 ± 14) = SBC (338 ± 12) > GAC (322 ± 1) > CBC-900 (273 ± 6) > CBC-700 (180 ± 11) > ABC600 (60 ± 10) > PAC (17 ± 0). No clear trend in EC data was observed in the case of BCs collected at different temperatures. EC of ABC-600 was found to be lower in comparison to ABC-400. On the other hand, EC of CBC-900 was found to be higher when compared to CBC-700. These results indicate that EC of BC might be affected by the ash (metal) composition of each material. The EC of BC obtained in this study (60–350 μS/cm) was similar to the values reported in literature (70–240 μS/cm) [19]. However, the EC of GAC was lower than the values reported in literature (> 1000 μS/cm) [23]. However, the surface area of BC was comparable with the surface area previously reported in literature (around 15 m2/g) [10]. In general, the surface area of ACs was found to be higher than BCs. Similarly, the pore volume and pore diameter of ACs were also found to be higher compared to BCs. In the case of CBC-700 and CBC-900, it was very difficult to measure the surface area and Azargohar et al. [24] previously reported a similar problem. The BET surface area and total pore volume for CBC synthesized at various temperatures were found to be < 2 m2/g and 0.001 cm3/g, respectively [24]. We could not also measure the surface area of ABC-600. The metal composition of both BCs and ACs is provided in Supplementary Information (Table S1). In general, higher concentrations of Ca, Mg, K and P were found in all the adsorbents. Fe concentration in PAC (915 ± 4 mg/kg) was found to be higher when 3
4
c
b
a
7.26 ± 0.05 & 0.669 ± 0.453
5.7 ± 2.0
N.P.c & 0.644± 0.339
763.0 ± 8.0
289.0 ± 15.0
7.3 ± 0.3 338.0 ± 12.0
7.27 ± 0.55 & 0.722 ± 0.221
8.9 ± 0.1 17.0 ± 0.0
12.0 ± 0.2 322.0 ± 1.0
6.8 ± 0.1 54.0 ± 1.0
1.04 & 0.002
1.0 ± 0.2 92.0 ± 1.8
7.1 ± 0.4 86.0 ± 3.0
40.0 ± 0.2
3.94 & 0.752
8.0 ± 0.2
7.0 ± 0.2b
SBC-500
2.6 & 0.188
PAC
GAC
Values
Dry weight-basis. a ± b indicate average ± standard deviation for triplicate samples. Could not measure.
Volatile content (% db)a Ash (% db)a Fixed carbon (% db)a pHpzc Electrical conductivity (μS cm−1) BET Surface area (m2 g−1) Average pore diameter (nm) & pore volume (cm3 g−1) Particle density (g/ cc) & Avg. size distribution (mm)
Characteristics (units)
Table 1 Characteristics of adsorbents used in this study.
1.45 ± 0.01 & 1.068 ± 0.838
0.90 & 0.001
8.0 ± 3.0
7.0 ± 0.2 350.0 ± 14.0
4.7 ± 0.2 54.0 ± 0.8
41.0 ± 0.3
ABC-400
1.45 ± 0.13 & 0.903 ± 0.987
1.68 ± 0.00 & 0.541 ± 0.548
N.P.c
N.P.c
N.P.c
N.P.c
8.9 ± 0.1 180.0 ± 11.0
24.4 ± 0.4 55.1 ± 1.1
17.9 ± 1.5
CBC-700
8.0 ± 0.1 60.0 ± 10.0
4.2 ± 0.9 75.7 ± 0.6
18.8 ± 0.3
ABC-600
1.69 ± 0.00 & 2.548± 1.359
N.P.c
N.P.c
7.3 ± 0.3 273.0 ± 6.0
24.9 ± 0.4 56.0 ± 0.4
17.8 ± 0.2
CBC-900
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Fig. 1. FT-IR spectra of adsorbents investigated in this study.
associated with condensed aromatic sub-structures of the biochar were found to be key players involved in electron transfer mechanism [15]. In addition, the phenolic moieties in biochar were found to function as electron donors in redox reactions. Redox properties of biochar may originate from both organic and inorganic components. Metals in particular Fe and Mn are known for their ability to participate in redox reactions [15]. The sum of Fe and Mn was almost three times higher in PAC as compared to other adsorbents in this study. Although the CH4 yield with adsorbents was higher than no adsorbent added cultures, PAC gave low CH4 yield among adsorbents. The results indicate that organic electron accepting and donating moieties might be dominating the biochar redox properties in addition to the inorganic moieties. FTIR analysis shows the presence of quinone moieties in both biochars (ABC-400 and SBC-500) used in this study (Fig. 1). In addition, the presence of phenazine moieties (at 1100 cm−1) was also identified in these biochars. This indicates that these functional groups in biochar participated in electron transfer during substrate/fermentation metabolite oxidation. Similar to an increase in CH4 yields, the addition of adsorbents also favored a higher COD reduction compared to non-adsorbent cultures. COD reduction data in cultures fed glucose showed the following trend: GAC (94 ± 1%) = PAC (93 ± 1%) = SBC-500 (94 ± 3%) = ABC400 (93 ± 1%) > GLU (81 ± 0%) (data not shown). Table 2 shows the comparison of CH4 yields and COD removal results obtained with various types of adsorbents reported in the literature along with the results from this study. In general, higher CH4 yields were observed in biochar added cultures in this study compared to the previous studies in literature.
Fig. 2. Methane production profiles from glucose [Note: Negligible CH4 production was observed in CON cultures].
EC. One possible reason for this could be due to the adsorption of VFAs produced during glucose degradation by ACs that have high surface area. Study conducted in literature showed that VFAs such as acetic, propionic and butyric acids showed good adsorption characteristics on AC [28]. Previous studies in literature found that biochars play an important role in reductive transformation of organic contaminants by facilitating the electron transfer between the electron donors and receiving organic compounds [29,30]. Two types of redox active structures namely quinone-hydroquinone moieties and conjugated pi-electron systems 5
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Table 2 Comparison of methane yields obtained in this study with previous studies reported in literature. S.No.
Adsorbent type
Inoculum source
Relative increase in CH4 yieldsa
Relative increase in COD removal
Reference
1 2 3 4 5 6 7 8 9 10 11 12
Granular activated carbon Biochar (pine sawdust) Powdered activated carbon Biochar (holm oak residues) Biochar (straw digestate) Biochar (rice husk) Charcoal Powdered activated carbon GAC PAC SBC-500 (Switch grass) ABC-400 (Ashe Juniper)
Anaerobic digester wastewater plant Anaerobic digester wastewater plant Activated sludge from sewage Anaerobic digester treating biowastes Anaerobic digester cattle manure wastewater Digested cow dung Cattle dung, poultry waste, cheese whey Anaerobic digester wastewater plant Anaerobic digester wastewater plant Anaerobic digester wastewater plant Anaerobic digester wastewater plant
78% 10% N.P.b 5% 32% 31% 17% 38% 40%c 24%c 72%c 71%c
47% N.P. 155% N.P.b N.P.b N.P.b 25% 29% 16%c 15%c 16%c 15%c
[5] [14] [31] [32] [11] [33] [34] [6] This study This study This study This study
a b c
Relative yields are calculated based on the corresponding yields obtained in control cultures (no adsorbent addition). N.P. = Not provided. Indicates results obtained from AD of glucose.
under all experimental conditions in comparison to aceticlastic methanogens such as Methanosaeta sp. and Methanosarcina sp. (utilizing acetate as substrate for their growth). Absence of Methanosaeta sp. indicates that aceticlastic methanogenesis are not the dominant CH4 production pathway in this experiment. Studies conducted in literature reported evidence for IET involving Methanosarcina sp. [7,10]. Unlike Methanosaeta sp. which is a strict acetate utilizing methanogen, Methanosarcina sp. are mixotrophic in nature capable of utilizing a variety of substrates such as H2, CO2, methanol, and methylamines in addition to acetate [36]. In addition, Methanosarcina sp. are capable of performing acetate oxidation themselves and could therefore mediate the entire process of acetate oxidation and subsequent methanogenesis rather than functioning only as an acetate sink [37]. Absence of acetate in all adsorbent added cultures (Supplementary Table S4) also supports the evidence for acetate oxidation by Methanosarcina sp. in adsorbent added cultures. On the other hand, acetate was observed in GLU cultures, which also showed low abundance of Methanosarcina sp. Methanosarcina sp. was reported to be abundant in tightly-bound fractions on the biochar surface [13]. Similar results of higher abundance of Methanosarcina sp. was observed in all adsorbent added glucose-fed cultures in this study. For example, higher relative abundance of Methanosarcina sp. was obtained from the adsorbents added cultures of ABC-400 by 215%, SBC-500 by 141%, PAC by 316%, and GAC by 74% in comparison to the species of the corresponding controls (ABCCON, SBC-CON, PAC-CON and GAC-CON) (Table S3). In comparison, GLU cultures showed a 64% decrease in Methanosarcina sp. in comparison to GLU-CON cultures. A study conducted by Wang et al. [38] on
3.3. Microbial community profiles Microbial community characterization of bacteria and archaea of both adsorbent and non-adsorbent added glucose-fed cultures is presented in Supplementary Tables S2 and S3, respectively. Bacteria such as Clostridium sp. Bacillus sp. Bacteroides sp. and Anaerobaculum sp. that are known for their ability to utilize carbohydrates (such as glucose) to make acetate, H2 and CO2 were present in all experimental conditions. Shanmugam et al. [35] observed the presence of Clostridium sp., Bacillus sp. and Bacteroides sp. in the mesophilic mixed anaerobic cultures producing H2 and acetate by utilizing glucose as substrate. However, the relative abundance of these bacteria varied under different experimental conditions. Geobacter sp. was detected under all experimental conditions (Table S2). High abundance of Wolinella sp. and Geobacter sp. in both GLU and GAC added cultures indicate that substrate (acetate) derived electrons by Geobacter sp. were transferred to anaerobic partner Wolinella sp. On the other hand, the redox active moieties in biochar might have played an important role in transfer of electrons during acetate oxidation. This was clearly evident from the low abundance of Wolinella sp. in biochar added cultures compared to their corresponding controls. Results by Chen et al. [10] concluded that the association of bacterial cells to the biochar conducted electrons through the biochar rather than biological electrical connections. Similar to bacterial profiles, the archaeal profiles showed diversity under each experimental condition (Fig. 3 and Table S3). In general, hydrogenotrophic methanogens (utilize H2/CO2 for their growth) such as Methanothermobacter sp. and Methanobrevibacter sp. were abundant
Fig. 3. Composition of aceticlastic and hydrogenotrophic methanogens under different experimental conditions.
6
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CH4 yield (mL/g COD)
400
Fig. 4. Methane production profiles from BOAP.
24 h
67 h
117 h
240 h
432 h
768 h
350
300 250 200 150
100 50 0
ABC-400
ABC-600
CBC-700
CBC-900
Control (BOAP)
Experimental Set
achieved using ABC-400 addition. From these results, it can be concluded that presence of RAMs in addition to inorganic moieties in biochars might play an important role by acting as a catalyst mediating the electron transfer during fermentation of glucose and BOAP. Although bioenergy generation via AD for waste treatment is promising, the long retention times required for optimal operation (due to slower methanogenesis reaction) is the major bottleneck in designing compact digesters. Addition of adsorbents, especially intermediate temperature BCs (i.e. BCs collected at 400 °C and 500 °C), could help overcome this limitation by significantly reducing the lag times and increasing the methane production rates, which could ultimately reduce the volume of anaerobic digesters. Though the results obtained from this study looks promising, future studies must focus on investigating the effect of biochar addition in different anoxic habitats together with microbial community analysis.
Methanosarcina sp. suggested that diverse membrane-bound electron transport pathways have evolved involving phenazine moieties donating electrons to the heterodisulfide reductase for the reduction of coenzyme M and coenzyme B (CoM-S-S-CoB) involved in the CH4 formation pathway. Presence of quinone and phenazine moieties in biochar (Fig. 1) indicate that they are able to accept electrons during substrate oxidation. These groups could then donate electrons to the coenzyme complex CoM-S-S-CoB in methanogens necessary for CH4 formation. This could be the other possible reason for higher CH4 yields in biochar added cultures in comparison to other cultures. 3.4. Biogas production using BOAP Biogas production results from glucose indicate BCs derived from woody and herbaceous biomass gave similar CH4 yields (Fig. 1). Hence, ABC-400 was chosen to evaluate the biogas production from complex organic waste such as BOAP. Similar to the results from glucose, CH4 production from BOAP showed increase in CH4 yields with the addition of biochars (Fig. 4). Methane yields (in mL/g COD) from BCs produced at different temperatures shows the following trend: ABC-400 (296 ± 23) > ABC-600 (88 ± 8) > CBC-700 (43 ± 11) = CBC900 (37 ± 7) > control (BOAP) (24 ± 1). These results indicate that the cultures using BC produced at intermediate temperature (ABC-400) showed better biogas production as compared to the cultures with BCs produced at higher temperatures (ABC-600, CBC-700 and CBC-900). A study conducted by Klupfel et al. [15] showed that concentrations of these redox active moieties (RAMs) was found to be higher in intermediate to high temperature biochars (400–500 °C), which corroborated with this study. High levels of RAMs (identified by FT-IR analysis) were observed in ABC-400 correlating very well with the CH4 results (Fig. 4). Absence of peaks corresponding to RAMs in CBC-700 and CBC-900 resulted in low CH4 yields. Due to these abilities, application of biochar in environmental engineering applications is gaining significant research attention. Although increase in aromaticity and extent of ring condensation increases with increasing temperature of biochar (collected during thermochemical conversion process), it decreases the char oxygen content and the yield [39]. This resulted in a decrease in quinone and hydroquinone moieties with increasing temperature of biochars [15]. Similar to the biogas production results, COD reduction results from BOAP showed the following trend: ABC-400 (85 ± 2%) > ABC-600 (15 ± 0%) > CBC-700 (8 ± 0%) > CBC-900 (7 ± 0%) > Control (BOAP) (4 ± 0%) (data not shown). Earlier study conducted by Shanmugam et al. [20] indicated that BOAP consists of complex organics and showed low levels of COD reduction. The results obtained from this study indicate that high levels of COD reduction can be
4. Conclusions Addition of adsorbents reduced the lag times and increased the methane yields in comparison to no adsorbent added cultures during anaerobic digestion of glucose and bio-oil aqueous phase. Biochars showed higher methane yields in comparison to activated carbon adsorbents. Biochars produced at intermediate temperatures such as 400 °C and 500 °C were found to be more effective compared to those produced at higher temperatures (≥600 °C). The study showed that the presence of redox active moieties such as quinones and phenazines in biochar played an important role in facilitating electron transfer between fermentative bacteria and methanogens in comparison to other properties such as composition of inorganic moieties (Fe and Mn), surface area and electrical conductivity. Addition of adsorbents selectively enriched aceticlastic methanogen (Methanosarcina sp.) whereas hydrogenotrophic methanogens (Methanothermobacter sp. and Methanobrevibacter sp.) were enriched in no adsorbent added cultures during anaerobic digestion of glucose. Abbreviation S.No. Abbreviations Full Form 1 2 3 4 5
7
ABC AC AD BC BET
Ashe juniper biochar Activated carbon Anaerobic digestion Biochar Brunauer-Emmett-Teller surface area analyzer
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BOAP
7 8 9 10
CBC CH4 COD CON
11 12 13 14 15 16 17 18 19
DIET EC FT-IR GAC GC GLU HPLC HTL ICP-OES
20 21 22 23 24 25 26
IET PAC pHPZC SBC TN TOC VSS
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Aqueous phase of bio-oil generated via Hydrothermal Liquefaction of Algae Canola meal biochar Methane Chemical oxygen demand Control cultures (No adsorbent and Substrate) Direct interspecies electron transfer Electrical conductivity Fourier Transform - Infrared Spectroscopy Granular activated carbon Gas chromatography Glucose High performance liquid chromatography Hydrothermal liquefaction Inductively coupled plasma-optical emission spectroscopy Interspecies electron transfer Powdered activated carbon pH at point of zero charge Switchgrass biochar Total nitrogen Total organic carbon Volatile suspended solids
Acknowledgments The authors would like to acknowledge Alabama Agricultural Experiment Station (ALA014-1-13006) and Auburn UniversityIntramural Grant Program (AU-IGP-150200) for funding this study. Also, the authors would like to thank Dr. Niki Labbe of University of Tennessee-Knoxville for providing switchgrass biochar samples for this study. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx. doi.org/10.1016/j.biombioe.2017.10.034. References [1] S. Soda, Y. Iwai, K. Sei, Y. Shimoda, M. Ike, Model analysis of energy consumption and greenhouse gas emissions of sewage sludge treatment systems with different processes and scales, Water Sci. Technol. 61 (2010) 365–373. [2] O. Nowak, S. Keil, C. Fimml, Examples of energy self-sufficient municipal nutrient removal plants, Water Sci. Technol. 64 (2011) 1–6. [3] S.H. Lee, H.J. Kang, Y.H. Lee, T.J. Lee, K. Han, Y. Choi, H.D. Park, Monitoring bacterial community structure and variability in time scale in full-scale anaerobic digesters, J. Environ. Monit. 14 (2012) 1893–1905. [4] G.F. Parkin, W.F. Owen, Fundamentals of anaerobic digestion of wastewater sludge, J. Environ. Eng. 112 (1986) 867–920. [5] J.Y. Lee, S.H. Lee, H.D. Park, Enrichment of specific electro-active microorganisms and enhancement of methane production by adding granular activated carbon in anaerobic reactors, Bioresour. Technol. 205 (2016) 205–212. [6] M. Desai, D. Madamwar, Anaerobic digestion of a mixture of cheese whey, poultry waste and cattle dung: a case study of the use of adsorbents to improve digester performance, Environ, Pollution 86 (1994) 337–340. [7] F. Liu, A.E. Rotaru, P.M. Shrestha, N.S. Malvankar, K.P. Nevin, D.R. Lovely, Promoting direct interspecies electron transfer with activated carbon, Energy Environ. Sci. 5 (2012) 8982–8989. [8] S. Kato, K. Hashimoto, K. Watanabe, Methanogenesis facilitated by electric syntrophy via (semi) conductive iron-oxide minerals, Environ. Microbiol. 14 (2012) 1646–1654. [9] J.S. Cha, S.H. Park, S.C. Jung, C. Ryu, J.K. Jeon, M.C. Shin, Y.K. Park, Production and utilization of biochar: a review, J. Ind. Eng. Chem. 40 (2016) 1–15. [10] S. Chen, A.E. Rotaru, P.M. Shrestha, N.S. Malvankar, F. Liu, W. Fan, K.P. Nevin, D.R. Lovley, Promoting interspecies electron transfer with biochar, Sci. Rep. 4 (2014) 1–7.
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Research paper
Effect of bio-char on methane generation from glucose and aqueous phase of algae liquefaction using mixed anaerobic cultures Saravanan R. Shanmugam, Sushil Adhikari∗, Hyungseok Nam, Sourov Kar Sajib Department of Biosystems Engineering, Auburn University, Auburn, AL 36849, USA
A R T I C L E I N F O
A B S T R A C T
Keywords: Biochar Activated carbon Methane production Microbial analysis Interspecies electron transfer
Activated carbon is known to enhance methane formation in anaerobic reactors via interspecies electron transfer between fermentative bacteria and methanogenic archaea. Biochar, a by-product of biomass pyrolysis process, could also perform similar functions due to its conductive properties and the presence of redox active moieties. Hence, this study was conducted to evaluate the effectiveness of different types of activated carbons and biochars on anaerobic digestion. Biochars obtained from canola meal, switchgrass and Ashe juniper were tested for methane production from both glucose and aqueous phase of bio-oil generated via hydrothermal liquefaction of algae. The results suggested that absorbents enhanced methane production. Furthermore, biochars synthesized at intermediate temperatures significantly increased methane yield and reduced the lag time required for methane formation. In addition, the results suggested that the redox active moieties such as quinones and phenazines in biochars are responsible for electron transport, which ultimately enhanced methane production.
1. Introduction Anaerobic digestion (AD) is an effective method to treat organic wastes and recover energy in the form of biogas (methane). Fossil fuels utilized for power generation can be substituted with biogas produced via AD process [1,2]. Methane generation via AD is carried out by several groups of microorganisms involved in hydrolysis, acidogenesis, acetogenesis and methanogenesis [3]. Typically, about 25–65% of waste organics are converted to methane in a mesophilic AD process [4]. Performance of the AD process is determined by the effectiveness of interspecies electron transfer (IET) between secondary fermenting bacteria producing diffusive electron carriers (such as formate and hydrogen) and methanogenic archaea [5]. Disruption of syntrophic association between these bacteria often results in instabilities in reactor performances due to a decrease in pH followed by the accumulation of volatile fatty acids (VFAs). Adsorbents such as granular activated carbon (GAC), powdered activated carbon (PAC), silica gel, gelatin, pectin, aluminium powder, and bentonite are added to methanogenic reactors to overcome the disruption problem and stabilize the AD process [5,6]. In addition, these adsorbents were found to increase the methane yields in the AD process. For example, Lee et al. [5] observed a 1.8-fold higher methane production rate in GAC added reactors in comparison to control reactors with no GAC addition. Desai and Madamwar [6] claimed that addition of silica gel (4 g/L) resulted in a two-fold enhancement in total gas production and
∗
17% increase in methane composition of biogas. Addition of adsorbents helps in sorption of toxic organic compounds, which are inhibitory to methanogenesis, and provides a high surface area for microbial growth, which in turn favors higher methane production rates. In addition, activated carbon (AC) adsorbent acts as a good conductive material (such as an electrode) to facilitate direct interspecies electron transfer (DIET) between secondary fermenting bacteria (acting as anode-reducing microorganisms) and methanogenic archaea (acting as cathode oxidizing microorganisms) [5,7,8]. For example, a study conducted by Liu et al. [7] showed that DIET occurred between Geobacter sp. (anode reducing bacteria) and Methanosarcina barkeri (cathode oxidizing bacteria). Thus, the addition of conductive materials leads to an increase in the energy efficiency and biogas production via DIET by removing several steps associated with hydrogen production and consumption [5,7]. Further, a study conducted by Kato et al. [8] concluded that electron transport via conductive materials is much faster than molecular transport of electron carriers. Similar to activated carbon adsorbents, biochar (BC) could also be used to enhance methane production via DIET during AD process due to its conductive properties similar to that of AC. BC is a carbon rich residue produced during the thermochemical decomposition of biomass via gasification or pyrolysis. BC is an inexpensive and eco-friendly solid material used for a number of purposes, such as soil remediation, waste management, greenhouse gas reduction and energy production [9]. The major element of BC is carbon (C), along with minor amounts of
Corresponding author. E-mail address: [email protected] (S. Adhikari).
http://dx.doi.org/10.1016/j.biombioe.2017.10.034 Received 2 March 2017; Received in revised form 16 October 2017; Accepted 24 October 2017 0961-9534/ © 2017 Elsevier Ltd. All rights reserved.
Please cite this article as: Shanmugam, S.R., Biomass and Bioenergy (2017), http://dx.doi.org/10.1016/j.biombioe.2017.10.034
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similar to what was described in the published document [20]. The initial study using glucose was conducted to compare the effectiveness of BCs (ABC-400 and SBC-500) with ACs (GAC and PAC). Addtional anaerobic digestion experiments were also performed using biochars (BCs) synthesized at different temperatures (400 °C, 600 °C, 700 °C, and 900 °C) to understand the effect of these BCs in treatment of complex organic wastes. BCs synthesized from two different materials such as Ashe Juniper biomass (ABC-400 and ABC-600) and canola meal (CBC700 and CBC-900) were evaluated. BOAP collected during HTL of algae was used as a complex organic substrate in this study. Information regarding algal composition, HTL operation, and BOAP characteristics can be found elsewhere [20,21]. AD experiments were conducted using a 160 mL serum bottle batch reactor with a 55 mL working volume comprising basal medium, inoculum and substrate. In order to study the effect of adsorbents on the performance of methane production process from glucose and BOAP, batch reactors (serum bottles) were supplemented with 1% of each adsorbent (0.55 g in 55 mL). Control reactors for each type of adsorbent were also ran in parallel with no adsorbent addition. AD experiments were conducted at mesophilic temperature (37 ± 1 °C) in an orbital shaker (with no shaking). The initial volatile suspended solids (VSS) used for AD experiments were 3000 ± 105 mg/L (4260 ± 149 mg COD/L) using a conversion factor of (1.42 g COD/g VSS). The initial pH and substrate concentration of all experimental sets were 7.8 ± 0.05, and 1 g COD/L, respectively. The substrate to inoculum ratio was maintained at 0.24 in all experimental sets (on COD basis). Separate culture control (no substrate addition) and adsorbent control (adsorbent with no substrate addition) experiments were also conducted to analyze any background CH4 production. Background CH4 yields of the corresponding experimental controls were subtracted from the experimental sets in the data presented. The batch reactors (both with different types of adsorbents and no adsorbents) were prepared and left for incubation in an orbital shaker (at 250 rpm) for a period of 7 days prior to the initial experiment to allow acclimation. At the end of 7 days, each batch reactor was fed with 1 g COD/L of glucose and BOAP and the reactors were not shaken. This procedure was followed to understand the role of IET in adsorbent added and non-adsorbent added cultures. However, the batch reactors were shaken only once at 250 rpm briefly (for 1 min) before performing the gas sampling. The CH4 yields from all experimental sets were reported in mL/g COD.
hydrogen (H), oxygen (O) and trace amounts of sulfur (S) and nitrogen (N). The elemental composition of BC depends upon the nature of raw biomass material and on the carbonization process [10]. Owing to its porous structure, specific surface area, surface functional groups, and high nutrient content (both micro- and macro-nutrients), BC, could be used to support bacterial growth and enhance biogas production. Addition of BC to enhance biogas production during AD process has been gaining some research attention [11–14]. BC, however, has low surface area in comparison to AC, but it contains certain redox active moieties (RAMs) such as quinones and phenazines, which act as electron transfer catalysts during redox reactions in many soil and biogeochemical environments. In addition, BCs produced at different temperature were found to have different characteristics [15]. Although there is a growing interest of utilizing BCs in AD, studies focused on the treatment of complex organic wastes with addition of BC are very limited in the open literature. To the best of our knowledge, no studies have been conducted to compare the effectiveness of BC (derived from both herbaceous and woody biomass) versus AC (both powder and granular) on methane production. Hence, this study was conducted to investigate the effect of biochars produced from different biomass and process conditions on methane production with microbial community analysis. AD experiments were carried out using simple substrate such as glucose and complex organic waste such as aqueous phase of bio-oil (BOAP) generated during hydrothermal liquefaction (HTL) of algae. 2. Materials and methods 2.1. Materials Seven types of adsorbents (two types of AC and five types of BC) were investigated to understand the effect of adsorbents during AD of simple organics (glucose) and complex organics (BOAP) in this study. Both PAC (Product code: C3014) and GAC (product code: 31616) were purchased from Sigma Aldrich (St. Louis, MO, USA) and used without further modifications. Biochars such as CBC, SBC and ABC were produced using pyrolysis of canola meal, switchgrass and Ashe juniper, respectively. Briefly, CBC was collected during bench-scale slow pyrolysis of canola meal operating at a temperatures of 700 °C (CBC-700) and 900 °C (CBC-900), respectively with a residence time of 2 h. SBC was collected during pyrolysis of switchgrass in an auger reactor operating at a temperature of 500 °C (SBC-500) with a residence time of 72 s, and the details are explained in the published document [16]. Similarly, ABC was collected during bench-scale slow pyrolysis of Ashe juniper biomass operated at temperatures of 400 °C (ABC-400) and 600 °C (ABC-600) with a residence time of 30 min. The detailed operation of bench-scale pyrolysis system is described elsewhere [17]. All these seven types of adsorbents were characterized for proximate analysis [17], BET (Brunauer-Emmett-Teller) surface area [18], average pore diameter [18], and pore volume [18], electrical conductivity (EC), particle density [18], particle size distribution [18] and pH (at point of zero charge, pHPZC) [19]. The EC of BC and AC was measured according to the standard procedure used to measure electrical conductivity of soil. In addition, all these adsorbents were analyzed for the presence of metal elements using the inductively coupled plasma-optical emission spectroscopy (ICP-OES; PerkinElmer Life Sciences 9300-DV system). Adsorbents were also characterized using FT-IR using the methods previously described elsewhere [20] to detect the presence of functional groups such as quinones and phenazines known to involve in IET.
2.3. Analytical methods Liquid samples (1.5 mL) collected at the beginning and the end of AD experiments using pure glucose and complex organic waste (BOAP) were analyzed for TOC and COD according to the methods described previously [20]. In addition, volatile fatty acids (VFAs) composition was examined using a Shimadzu high performance liquid chromatograph (HPLC) equipped with UV and RI detectors. Both glucose and VFAs (lactic acid, acetic acid, propionic acid, formic acid and butyric acid) were analyzed using Aminex HPX-87H column with 5 mM sulfuric acid as eluent. The column temperature was set at 50 °C with an eluent flow rate of 0.6 mL/min. Headspace biogas composition was analyzed according to the method described previously [20]. Statistical difference between multiple means of the gas metabolite data were evaluated using a Tukey's paired comparison procedure at 95% confidence level [22]. 2.4. Microbial community analysis At the end of the batch experiment (using glucose), microbial samples were collected for community analysis using sterile 1.5 mL Eppendorf tubes from all experimental sets of glucose study. The liquid supernatant was discarded and 1.5 mL of settled microbial cultures were collected in these tubes. In batch reactors with adsorbents, both the adsorbents along with microbial cultures were taken in order to
2.2. Anaerobic digestion experimentation Mixed anaerobic culture obtained from Jackson Pike wastewater treatment facility (Columbus, Georgia, USA) was used for AD experiments of glucose and BOAP. Inoculum source, inoculum characterization, and basal media preparation for conducting AD experiments were 2
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compared to other types of adsorbents. The elements such as Ni, B, Co, Zn, and Cu were present in low concentrations. Results from FT-IR analysis showed absorbance around wavenumbers 1690 cm−1 (γC = O stretch), 1580 cm−1 (γC = C stretch), and 1300-1000 cm−1 (CeO stretch) in ABC-400, ABC-600 and SBC-500 adsorbents (Fig. 1a). In addition, strong absorbance between 2850 and 2920 cm−1 was observed in CBC-700, CBC-900 and ABC-400 samples due to CeH stretching (Fig. 1b). Study conducted by Burie et al. [25] indicated that absorbance at wavenumbers between 1500 and 1700 cm−1 correspond to the characteristic peak of quinone moieties (due to γC = O and γC = C stretching). Recent study by Yu et al. [26] concluded that quinone moieties in biochar play an important role in bacterial IET. In addition, absorbance peaks at wavenumbers 1100 cm−1 in SBC-500 and 1260 cm−1 in SBC-500 and ABC-400 might be due to presence of phenazines. Study conducted by Stammer and Taurins [27] showed characteristic peaks of phenazines at these wavenumbers (1100 and 1260 cm−1). However, the intensity of peak corresponding to both quinones and phenazines were found to be very low/absent in activated carbon adsorbents (PAC and GAC) and also from biochar adsorbents collected at high temperatures (CBC-700 and CBC-900) (Fig. 1a).
quantify the microorganisms attached to each adsorbent facilitating IET. DNA extraction was conducted using the MoBio PowerSoil DNA extraction kit (MoBio Laboratories, Solana Beach, CA, USA) according to the protocol provided by the supplier. The extracted DNA was then immediately stored at −20 °C until further used. DNA samples were shipped for community characterization to molecular research (MR DNA) laboratory (Shallowater, TX, USA). Briefly, amplification of prokaryotes (bacteria and archaea) was performed using paired-end 16S community sequencing on the Illumina platform (MiSeq). The 16S rRNA gene V4 variable region PCR primers 515/806 with barcode on the forward primer was used in a 28 cycle PCR using the HotStar Taq plus Master Mix Kit (Qiagen, USA) using the following conditions: 94 °C for 3 min, followed by 28 cycles of 94 °C for 30 s, 53 °C for 40 s, and 72 °C for 1 min, after which a final elongation step at 72 °C for 5 min was performed. Amplified DNA samples are purified using calibrated Ampure XP beads. Sequencing was conducted according to the manufacturer's guidelines. Sequence data were processed using MR DNA analysis pipeline. Operational taxonomic units (OTUs) were defined by clustering at 3% divergence (97% similarity). Final OTUs were taxonomically classified using basic local alignment search tool for nucleotides (BLASTn) against a curated database derived from RDPII and NCBI.
3.2. Biogas production using pure glucose 3. Results and discussion Biogas (CH4) production results under different experimental conditions are shown in Fig. 2. It is important to note that the methane production from glucose is well below theoretical limit (350 mL/g COD) mainly due to the lack of shaking. The goal here was not to obtain complete conversion of glucose to methane but rather to understand the effect of absorbents on methane conversion. The results indicate that ABC-400 and SBC-500 added cultures showed a shorter lag time required for CH4 production in comparison to both activated carbon added cultures (GAC and PAC) and no adsorbent added cultures (GLU). For example, the CH4 yields obtained in ABC-400 and SBC-500 at 24 h were found to be 39 ± 16 mL/g COD and 22 ± 4 mL/g COD, respectively. In comparison, the CH4 yields of GAC, PAC and GLU added cultures at 24 h were found to be 2 ± 0.6 mL/g COD, 10 ± 0.4 mL/g COD and 0 ± 0 mL/g COD, respectively. These results indicated that BC effectively reduced the lag time of CH4 generation compared to ACs. Study conducted by Sunyoto et al. [14] also concluded that BC addition reduced the lag phase of CH4 formation by 41% during AD of aqueous carbohydrates food waste. At the end of 48 h, the CH4 yields using PAC and GAC were similar in the range of 25–30 mL/g COD. However, both SBC-500 and ABC-400 showed higher CH4 production (71 ± 15 mL/g COD and 100 ± 27 mL/g COD, respectively) at the end of 48 h. After 48 h, GAC showed higher CH4 yields in comparison to PAC added cultures until 240 h. In comparison, PAC added cultures showed similar CH4 yields compared to GLU cultures until 96 h. The final CH4 yields at the end of 240 h showed the following trend: SBC-500 (332 ± 16 mL/ g COD) = ABC-400 (330 ± 2 mL/g COD) > GAC (271 ± 8 mL/g COD) > PAC (239 ± 10 mL/g COD) > GLU (193 ± 21 mL/g COD). From these results, it can be concluded that biochar derived from both herbaceous biomass (SBC-500) and woody biomass (ABC-400) showed similar levels of CH4 yields irrespective of the nature of pyrolysis. Study conducted by Yu et al. [26] mentioned that EC of adsorbent material is one of the important parameters affecting electron transfer between bacterial cells. It is interesting to note that the EC of GAC, SBC500 and ABC-400 was in the range of 322 and 350 μS cm−1. However, the CH4 yields of both SBC-500 and ABC-400 were found to be higher in comparison to GAC (Fig. 2). Between the ACs, the CH4 yield using GAC was found to be slightly higher as compared to PAC although the EC of GAC was 19 times higher than the EC PAC. In addition, EC of ABC-400 and SBC-500 was similar to that of GAC but CH4 yield with biochars was much higher than with GAC. These results indicate that IET between fermentative bacteria and methanogenic archaea in adsorbent added cultures could be influenced by some other parameters besides
3.1. Characterization of adsorbents used in this study Table 1 summarizes the characteristics of adsorbents investigated in this study. Biochars such as ABC-400 and SBC-500 showed higher volatile content (41.0 ± 0.3% and 40.0 ± 0.2%, respectively) due to low temperature pyrolysis (400 °C and 500 °C) in comparison to biochars produced at high temperatures such as ABC-600 (18.8 ± 0.3%), CBC-700 (17.9 ± 1.5%) and CBC-900 (17.8 ± 0.2%). In comparison, both GAC and PAC showed 7.0 ± 0.2% and 8.0 ± 0.2% volatile content, respectively indicating that they might have been prepared at higher temperatures. Both CBC-700 and CBC-900 showed high ash composition in comparison to all other adsorbents investigated in this study. The pHPZC for different type of adsorbents indicated that the pHs of ABC-400, SBC-500 and CBC-900 were in the neutral range whereas that of ABC-600, CBC-700, GAC and PAC were in the basic range. Electrical conductivity (EC) (in μS/cm) of adsorbents showed the following trend: ABC-400 (350 ± 14) = SBC (338 ± 12) > GAC (322 ± 1) > CBC-900 (273 ± 6) > CBC-700 (180 ± 11) > ABC600 (60 ± 10) > PAC (17 ± 0). No clear trend in EC data was observed in the case of BCs collected at different temperatures. EC of ABC-600 was found to be lower in comparison to ABC-400. On the other hand, EC of CBC-900 was found to be higher when compared to CBC-700. These results indicate that EC of BC might be affected by the ash (metal) composition of each material. The EC of BC obtained in this study (60–350 μS/cm) was similar to the values reported in literature (70–240 μS/cm) [19]. However, the EC of GAC was lower than the values reported in literature (> 1000 μS/cm) [23]. However, the surface area of BC was comparable with the surface area previously reported in literature (around 15 m2/g) [10]. In general, the surface area of ACs was found to be higher than BCs. Similarly, the pore volume and pore diameter of ACs were also found to be higher compared to BCs. In the case of CBC-700 and CBC-900, it was very difficult to measure the surface area and Azargohar et al. [24] previously reported a similar problem. The BET surface area and total pore volume for CBC synthesized at various temperatures were found to be < 2 m2/g and 0.001 cm3/g, respectively [24]. We could not also measure the surface area of ABC-600. The metal composition of both BCs and ACs is provided in Supplementary Information (Table S1). In general, higher concentrations of Ca, Mg, K and P were found in all the adsorbents. Fe concentration in PAC (915 ± 4 mg/kg) was found to be higher when 3
4
c
b
a
7.26 ± 0.05 & 0.669 ± 0.453
5.7 ± 2.0
N.P.c & 0.644± 0.339
763.0 ± 8.0
289.0 ± 15.0
7.3 ± 0.3 338.0 ± 12.0
7.27 ± 0.55 & 0.722 ± 0.221
8.9 ± 0.1 17.0 ± 0.0
12.0 ± 0.2 322.0 ± 1.0
6.8 ± 0.1 54.0 ± 1.0
1.04 & 0.002
1.0 ± 0.2 92.0 ± 1.8
7.1 ± 0.4 86.0 ± 3.0
40.0 ± 0.2
3.94 & 0.752
8.0 ± 0.2
7.0 ± 0.2b
SBC-500
2.6 & 0.188
PAC
GAC
Values
Dry weight-basis. a ± b indicate average ± standard deviation for triplicate samples. Could not measure.
Volatile content (% db)a Ash (% db)a Fixed carbon (% db)a pHpzc Electrical conductivity (μS cm−1) BET Surface area (m2 g−1) Average pore diameter (nm) & pore volume (cm3 g−1) Particle density (g/ cc) & Avg. size distribution (mm)
Characteristics (units)
Table 1 Characteristics of adsorbents used in this study.
1.45 ± 0.01 & 1.068 ± 0.838
0.90 & 0.001
8.0 ± 3.0
7.0 ± 0.2 350.0 ± 14.0
4.7 ± 0.2 54.0 ± 0.8
41.0 ± 0.3
ABC-400
1.45 ± 0.13 & 0.903 ± 0.987
1.68 ± 0.00 & 0.541 ± 0.548
N.P.c
N.P.c
N.P.c
N.P.c
8.9 ± 0.1 180.0 ± 11.0
24.4 ± 0.4 55.1 ± 1.1
17.9 ± 1.5
CBC-700
8.0 ± 0.1 60.0 ± 10.0
4.2 ± 0.9 75.7 ± 0.6
18.8 ± 0.3
ABC-600
1.69 ± 0.00 & 2.548± 1.359
N.P.c
N.P.c
7.3 ± 0.3 273.0 ± 6.0
24.9 ± 0.4 56.0 ± 0.4
17.8 ± 0.2
CBC-900
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Fig. 1. FT-IR spectra of adsorbents investigated in this study.
associated with condensed aromatic sub-structures of the biochar were found to be key players involved in electron transfer mechanism [15]. In addition, the phenolic moieties in biochar were found to function as electron donors in redox reactions. Redox properties of biochar may originate from both organic and inorganic components. Metals in particular Fe and Mn are known for their ability to participate in redox reactions [15]. The sum of Fe and Mn was almost three times higher in PAC as compared to other adsorbents in this study. Although the CH4 yield with adsorbents was higher than no adsorbent added cultures, PAC gave low CH4 yield among adsorbents. The results indicate that organic electron accepting and donating moieties might be dominating the biochar redox properties in addition to the inorganic moieties. FTIR analysis shows the presence of quinone moieties in both biochars (ABC-400 and SBC-500) used in this study (Fig. 1). In addition, the presence of phenazine moieties (at 1100 cm−1) was also identified in these biochars. This indicates that these functional groups in biochar participated in electron transfer during substrate/fermentation metabolite oxidation. Similar to an increase in CH4 yields, the addition of adsorbents also favored a higher COD reduction compared to non-adsorbent cultures. COD reduction data in cultures fed glucose showed the following trend: GAC (94 ± 1%) = PAC (93 ± 1%) = SBC-500 (94 ± 3%) = ABC400 (93 ± 1%) > GLU (81 ± 0%) (data not shown). Table 2 shows the comparison of CH4 yields and COD removal results obtained with various types of adsorbents reported in the literature along with the results from this study. In general, higher CH4 yields were observed in biochar added cultures in this study compared to the previous studies in literature.
Fig. 2. Methane production profiles from glucose [Note: Negligible CH4 production was observed in CON cultures].
EC. One possible reason for this could be due to the adsorption of VFAs produced during glucose degradation by ACs that have high surface area. Study conducted in literature showed that VFAs such as acetic, propionic and butyric acids showed good adsorption characteristics on AC [28]. Previous studies in literature found that biochars play an important role in reductive transformation of organic contaminants by facilitating the electron transfer between the electron donors and receiving organic compounds [29,30]. Two types of redox active structures namely quinone-hydroquinone moieties and conjugated pi-electron systems 5
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Table 2 Comparison of methane yields obtained in this study with previous studies reported in literature. S.No.
Adsorbent type
Inoculum source
Relative increase in CH4 yieldsa
Relative increase in COD removal
Reference
1 2 3 4 5 6 7 8 9 10 11 12
Granular activated carbon Biochar (pine sawdust) Powdered activated carbon Biochar (holm oak residues) Biochar (straw digestate) Biochar (rice husk) Charcoal Powdered activated carbon GAC PAC SBC-500 (Switch grass) ABC-400 (Ashe Juniper)
Anaerobic digester wastewater plant Anaerobic digester wastewater plant Activated sludge from sewage Anaerobic digester treating biowastes Anaerobic digester cattle manure wastewater Digested cow dung Cattle dung, poultry waste, cheese whey Anaerobic digester wastewater plant Anaerobic digester wastewater plant Anaerobic digester wastewater plant Anaerobic digester wastewater plant
78% 10% N.P.b 5% 32% 31% 17% 38% 40%c 24%c 72%c 71%c
47% N.P. 155% N.P.b N.P.b N.P.b 25% 29% 16%c 15%c 16%c 15%c
[5] [14] [31] [32] [11] [33] [34] [6] This study This study This study This study
a b c
Relative yields are calculated based on the corresponding yields obtained in control cultures (no adsorbent addition). N.P. = Not provided. Indicates results obtained from AD of glucose.
under all experimental conditions in comparison to aceticlastic methanogens such as Methanosaeta sp. and Methanosarcina sp. (utilizing acetate as substrate for their growth). Absence of Methanosaeta sp. indicates that aceticlastic methanogenesis are not the dominant CH4 production pathway in this experiment. Studies conducted in literature reported evidence for IET involving Methanosarcina sp. [7,10]. Unlike Methanosaeta sp. which is a strict acetate utilizing methanogen, Methanosarcina sp. are mixotrophic in nature capable of utilizing a variety of substrates such as H2, CO2, methanol, and methylamines in addition to acetate [36]. In addition, Methanosarcina sp. are capable of performing acetate oxidation themselves and could therefore mediate the entire process of acetate oxidation and subsequent methanogenesis rather than functioning only as an acetate sink [37]. Absence of acetate in all adsorbent added cultures (Supplementary Table S4) also supports the evidence for acetate oxidation by Methanosarcina sp. in adsorbent added cultures. On the other hand, acetate was observed in GLU cultures, which also showed low abundance of Methanosarcina sp. Methanosarcina sp. was reported to be abundant in tightly-bound fractions on the biochar surface [13]. Similar results of higher abundance of Methanosarcina sp. was observed in all adsorbent added glucose-fed cultures in this study. For example, higher relative abundance of Methanosarcina sp. was obtained from the adsorbents added cultures of ABC-400 by 215%, SBC-500 by 141%, PAC by 316%, and GAC by 74% in comparison to the species of the corresponding controls (ABCCON, SBC-CON, PAC-CON and GAC-CON) (Table S3). In comparison, GLU cultures showed a 64% decrease in Methanosarcina sp. in comparison to GLU-CON cultures. A study conducted by Wang et al. [38] on
3.3. Microbial community profiles Microbial community characterization of bacteria and archaea of both adsorbent and non-adsorbent added glucose-fed cultures is presented in Supplementary Tables S2 and S3, respectively. Bacteria such as Clostridium sp. Bacillus sp. Bacteroides sp. and Anaerobaculum sp. that are known for their ability to utilize carbohydrates (such as glucose) to make acetate, H2 and CO2 were present in all experimental conditions. Shanmugam et al. [35] observed the presence of Clostridium sp., Bacillus sp. and Bacteroides sp. in the mesophilic mixed anaerobic cultures producing H2 and acetate by utilizing glucose as substrate. However, the relative abundance of these bacteria varied under different experimental conditions. Geobacter sp. was detected under all experimental conditions (Table S2). High abundance of Wolinella sp. and Geobacter sp. in both GLU and GAC added cultures indicate that substrate (acetate) derived electrons by Geobacter sp. were transferred to anaerobic partner Wolinella sp. On the other hand, the redox active moieties in biochar might have played an important role in transfer of electrons during acetate oxidation. This was clearly evident from the low abundance of Wolinella sp. in biochar added cultures compared to their corresponding controls. Results by Chen et al. [10] concluded that the association of bacterial cells to the biochar conducted electrons through the biochar rather than biological electrical connections. Similar to bacterial profiles, the archaeal profiles showed diversity under each experimental condition (Fig. 3 and Table S3). In general, hydrogenotrophic methanogens (utilize H2/CO2 for their growth) such as Methanothermobacter sp. and Methanobrevibacter sp. were abundant
Fig. 3. Composition of aceticlastic and hydrogenotrophic methanogens under different experimental conditions.
6
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CH4 yield (mL/g COD)
400
Fig. 4. Methane production profiles from BOAP.
24 h
67 h
117 h
240 h
432 h
768 h
350
300 250 200 150
100 50 0
ABC-400
ABC-600
CBC-700
CBC-900
Control (BOAP)
Experimental Set
achieved using ABC-400 addition. From these results, it can be concluded that presence of RAMs in addition to inorganic moieties in biochars might play an important role by acting as a catalyst mediating the electron transfer during fermentation of glucose and BOAP. Although bioenergy generation via AD for waste treatment is promising, the long retention times required for optimal operation (due to slower methanogenesis reaction) is the major bottleneck in designing compact digesters. Addition of adsorbents, especially intermediate temperature BCs (i.e. BCs collected at 400 °C and 500 °C), could help overcome this limitation by significantly reducing the lag times and increasing the methane production rates, which could ultimately reduce the volume of anaerobic digesters. Though the results obtained from this study looks promising, future studies must focus on investigating the effect of biochar addition in different anoxic habitats together with microbial community analysis.
Methanosarcina sp. suggested that diverse membrane-bound electron transport pathways have evolved involving phenazine moieties donating electrons to the heterodisulfide reductase for the reduction of coenzyme M and coenzyme B (CoM-S-S-CoB) involved in the CH4 formation pathway. Presence of quinone and phenazine moieties in biochar (Fig. 1) indicate that they are able to accept electrons during substrate oxidation. These groups could then donate electrons to the coenzyme complex CoM-S-S-CoB in methanogens necessary for CH4 formation. This could be the other possible reason for higher CH4 yields in biochar added cultures in comparison to other cultures. 3.4. Biogas production using BOAP Biogas production results from glucose indicate BCs derived from woody and herbaceous biomass gave similar CH4 yields (Fig. 1). Hence, ABC-400 was chosen to evaluate the biogas production from complex organic waste such as BOAP. Similar to the results from glucose, CH4 production from BOAP showed increase in CH4 yields with the addition of biochars (Fig. 4). Methane yields (in mL/g COD) from BCs produced at different temperatures shows the following trend: ABC-400 (296 ± 23) > ABC-600 (88 ± 8) > CBC-700 (43 ± 11) = CBC900 (37 ± 7) > control (BOAP) (24 ± 1). These results indicate that the cultures using BC produced at intermediate temperature (ABC-400) showed better biogas production as compared to the cultures with BCs produced at higher temperatures (ABC-600, CBC-700 and CBC-900). A study conducted by Klupfel et al. [15] showed that concentrations of these redox active moieties (RAMs) was found to be higher in intermediate to high temperature biochars (400–500 °C), which corroborated with this study. High levels of RAMs (identified by FT-IR analysis) were observed in ABC-400 correlating very well with the CH4 results (Fig. 4). Absence of peaks corresponding to RAMs in CBC-700 and CBC-900 resulted in low CH4 yields. Due to these abilities, application of biochar in environmental engineering applications is gaining significant research attention. Although increase in aromaticity and extent of ring condensation increases with increasing temperature of biochar (collected during thermochemical conversion process), it decreases the char oxygen content and the yield [39]. This resulted in a decrease in quinone and hydroquinone moieties with increasing temperature of biochars [15]. Similar to the biogas production results, COD reduction results from BOAP showed the following trend: ABC-400 (85 ± 2%) > ABC-600 (15 ± 0%) > CBC-700 (8 ± 0%) > CBC-900 (7 ± 0%) > Control (BOAP) (4 ± 0%) (data not shown). Earlier study conducted by Shanmugam et al. [20] indicated that BOAP consists of complex organics and showed low levels of COD reduction. The results obtained from this study indicate that high levels of COD reduction can be
4. Conclusions Addition of adsorbents reduced the lag times and increased the methane yields in comparison to no adsorbent added cultures during anaerobic digestion of glucose and bio-oil aqueous phase. Biochars showed higher methane yields in comparison to activated carbon adsorbents. Biochars produced at intermediate temperatures such as 400 °C and 500 °C were found to be more effective compared to those produced at higher temperatures (≥600 °C). The study showed that the presence of redox active moieties such as quinones and phenazines in biochar played an important role in facilitating electron transfer between fermentative bacteria and methanogens in comparison to other properties such as composition of inorganic moieties (Fe and Mn), surface area and electrical conductivity. Addition of adsorbents selectively enriched aceticlastic methanogen (Methanosarcina sp.) whereas hydrogenotrophic methanogens (Methanothermobacter sp. and Methanobrevibacter sp.) were enriched in no adsorbent added cultures during anaerobic digestion of glucose. Abbreviation S.No. Abbreviations Full Form 1 2 3 4 5
7
ABC AC AD BC BET
Ashe juniper biochar Activated carbon Anaerobic digestion Biochar Brunauer-Emmett-Teller surface area analyzer
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6
BOAP
7 8 9 10
CBC CH4 COD CON
11 12 13 14 15 16 17 18 19
DIET EC FT-IR GAC GC GLU HPLC HTL ICP-OES
20 21 22 23 24 25 26
IET PAC pHPZC SBC TN TOC VSS
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Aqueous phase of bio-oil generated via Hydrothermal Liquefaction of Algae Canola meal biochar Methane Chemical oxygen demand Control cultures (No adsorbent and Substrate) Direct interspecies electron transfer Electrical conductivity Fourier Transform - Infrared Spectroscopy Granular activated carbon Gas chromatography Glucose High performance liquid chromatography Hydrothermal liquefaction Inductively coupled plasma-optical emission spectroscopy Interspecies electron transfer Powdered activated carbon pH at point of zero charge Switchgrass biochar Total nitrogen Total organic carbon Volatile suspended solids
Acknowledgments The authors would like to acknowledge Alabama Agricultural Experiment Station (ALA014-1-13006) and Auburn UniversityIntramural Grant Program (AU-IGP-150200) for funding this study. Also, the authors would like to thank Dr. Niki Labbe of University of Tennessee-Knoxville for providing switchgrass biochar samples for this study. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx. doi.org/10.1016/j.biombioe.2017.10.034. References [1] S. Soda, Y. Iwai, K. Sei, Y. Shimoda, M. Ike, Model analysis of energy consumption and greenhouse gas emissions of sewage sludge treatment systems with different processes and scales, Water Sci. Technol. 61 (2010) 365–373. [2] O. Nowak, S. Keil, C. Fimml, Examples of energy self-sufficient municipal nutrient removal plants, Water Sci. Technol. 64 (2011) 1–6. [3] S.H. Lee, H.J. Kang, Y.H. Lee, T.J. Lee, K. Han, Y. Choi, H.D. Park, Monitoring bacterial community structure and variability in time scale in full-scale anaerobic digesters, J. Environ. Monit. 14 (2012) 1893–1905. [4] G.F. Parkin, W.F. Owen, Fundamentals of anaerobic digestion of wastewater sludge, J. Environ. Eng. 112 (1986) 867–920. [5] J.Y. Lee, S.H. Lee, H.D. Park, Enrichment of specific electro-active microorganisms and enhancement of methane production by adding granular activated carbon in anaerobic reactors, Bioresour. Technol. 205 (2016) 205–212. [6] M. Desai, D. Madamwar, Anaerobic digestion of a mixture of cheese whey, poultry waste and cattle dung: a case study of the use of adsorbents to improve digester performance, Environ, Pollution 86 (1994) 337–340. [7] F. Liu, A.E. Rotaru, P.M. Shrestha, N.S. Malvankar, K.P. Nevin, D.R. Lovely, Promoting direct interspecies electron transfer with activated carbon, Energy Environ. Sci. 5 (2012) 8982–8989. [8] S. Kato, K. Hashimoto, K. Watanabe, Methanogenesis facilitated by electric syntrophy via (semi) conductive iron-oxide minerals, Environ. Microbiol. 14 (2012) 1646–1654. [9] J.S. Cha, S.H. Park, S.C. Jung, C. Ryu, J.K. Jeon, M.C. Shin, Y.K. Park, Production and utilization of biochar: a review, J. Ind. Eng. Chem. 40 (2016) 1–15. [10] S. Chen, A.E. Rotaru, P.M. Shrestha, N.S. Malvankar, F. Liu, W. Fan, K.P. Nevin, D.R. Lovley, Promoting interspecies electron transfer with biochar, Sci. Rep. 4 (2014) 1–7.
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