- Email: [email protected]
Hydrogen production from crude glycerol in an alkaline microbial electrolysis cell
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 4 ( 2 0 1 9 ) 1 7 2 0 4 e1 7 2 1 3
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/he
Hydrogen production from crude glycerol in an alkaline microbial electrolysis cell Marina Badia-Fabregat a, Laura Rago a,b, Juan A. Baeza a, Albert Guisasola a,* Departament d’Enginyeria Quı´mica, Biologica i Ambiental, Escola d’Enginyeria, Universitat Autonoma de Barcelona, 08193, Bellaterra (Barcelona), Spain b Helmholtz Centre for Environmental Research GmbH e UFZ, Permoserstraße 15, 04318 Leipzig, Germany a
article info
abstract
Article history:
Crude glycerol is an undesired by-product of biodiesel production with a low commercial
Received 20 December 2018
value (i.e. a ton of biodiesel results in around 110 kg of crude glycerol) and, thus, glycerol
Received in revised form
needs valorization. In particular, there is a need of providing a benefit to alkaline waste-
18 March 2019
waters from biodiesel production with excess of glycerol. Bioelectrochemical systems (BES)
Accepted 20 March 2019
are an emerging technique to recover the energy contained in a substrate either as elec-
Available online 25 April 2019
tricity or as other added-value products such as hydrogen. Moreover, promising results have been reported with alkaline BES showing higher current intensities than neutral pH
Keywords:
conditions. This study is the first experimental evaluation of alkaline bioelectrochemical
Alkaline bioelectrochemical system
production of hydrogen from real crude glycerol as sole carbon source. The results show
Crude glycerol
that alkaline glycerol degradation is feasible under both microbial fuel cell mode (2 mA,
Hydrogen production
71.4 A/m3 and 55% of CE) and microbial electrolysis mode (maximum of 0.46 LH2/L/d and
Illumina 16S rDNA sequencing
85% of rCAT). The values obtained are promising since they are in the range of those ob-
Bioanode
tained with other simpler carbon sources such acetate. A complex consortium involving fermentative bacteria (such as Enterococcaceae), alkaline exoelectrogens (such as Geoalkalibacter) and homoacetogens (such as Acetobacterium) was naturally developed in the anode of the MEC. © 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
Introduction Global climate change challenges the current dependence on fossil fuels. Among the different strategies for the climate change mitigation, the use of biodiesel is an alternative fuel for diesel engines because of: i) its environmental advantages; ii) its similarity with conventional diesel in energy content and chemical structure; iii) the reduction of the carcinogenic compound emissions by approximately 85% when compared
with diesel fuel; iv) the absence of sulfur and polycyclic aromatic hydrocarbons [1e3] and v) better engine performance (better lubrication, high number and more complete combustion [4]). Biodiesel is a liquid fuel obtained from animal fats or vegetable oils through the process of transesterification of different triglycerides. These react with an alcohol to produce methyl ester, biodiesel, ethyl ester and glycerol under a catalytic process. The main drawback of biodiesel production is the generation of glycerol, an undesired by-product with a low
* Corresponding author. E-mail address: [email protected] (A. Guisasola). https://doi.org/10.1016/j.ijhydene.2019.03.193 0360-3199/© 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 4 ( 2 0 1 9 ) 1 7 2 0 4 e1 7 2 1 3
commercial value [5]. With the fast increase of glycerol supply in the world market, the price of refined glycerol dropped from $1.20 kg in 2003 to $0.60/kg in 2006 [4]. The price of crude glycerol in the United States reached a minimum value of $0.04e0.11/kg [6] while in 2018 the price has risen up to $0.3e0.4 [7]. The production of a ton of biodiesel results in around 110 kg of crude glycerol [8] and, hence, large surpluses of this material are expected in the future. When glycerol production is higher than its demand, glycerol becomes a highly loaded waste rather than a resource. For example, the production of biodiesel (B100) in Brazil in 2014 generated 3.45$105 m3 of crude glycerol [9]. Therefore, new technologies for glycerol valorization are needed in order to increase the value of this compound, which should become an energetic resource rather than an undesired waste. Nowadays, glycerol valorization technologies are based on the chemical production of high-added values in three different directions: (i) glycerol oxidation, (ii) glycerol hydrogenolysis and (iii) glycerol aqueous reforming [10]. Alkaline catalysts (i.e. alkaline earth metal hydroxides or alkoxides of sodium or potassium) are the most common catalysts in the transesterification process. However, homogeneous alkaline catalysis requires complex downstream processes including the removal of inorganic salts from the final product, the recovery of salt-containing glycerol, and the treatment of alkaline wastewater. Hence, there is a niche in the valorization of alkaline wastewaters from biodiesel production with excess of glycerol. Among the different alternatives for glycerol valorization, hydrogen production is a preferred option since hydrogen is a clean source of energy with no harmful effect during its combustion process. Crude glycerol is a suitable source of hydrogen via physicochemical processes such as catalytic conversion [11] or reforming [12]. Moreover, crude glycerol has also been reported as an adequate substrate for microbial conversion processes such as fermentation [13e18]. Bioelectrochemistry aims at recovering the energy contained in the substrate either as electricity or as reducing power to obtain added-value products. Bioelectrochemical systems (BES) combine electrochemistry with the metabolism of anode respiring bacteria (ARB), also known as exoelectrogenic bacteria, which can transfer the electrons obtained in their metabolism to an external solid anode. Then, electrons flow from the anode to the cathode where a reduction reaction takes place [19]. If the cathodic reaction has a higher reduction potential than the anodic reaction, the electromotive force of the system is positive, free Gibbs energy is negative and, thus, electrons flow spontaneously from the anode to the cathode. This is usually observed when oxygen reduction occurs at the cathode. This device is known as Microbial Fuel Cell (MFC) and the substrate is oxidized at the anode linked to electricity generation. On the other hand, if oxygen is replaced in the cathodic chamber for other reductive process with lower reduction potential, the system becomes thermodynamically non-spontaneous and, then, additional energy supply is required to drive the process. These devices are known as Microbial Electrolysis Cell (MEC) and the substrate is oxidized in the anode linked to, for example, hydrogen production in the cathode [20e22]. Most of the research conducted so far with BES is under neutral conditions. However, the few works on
17205
alkaline BES already showed that alkaline bioelectrochemistry envisages a promising scenario since higher current intensities can be obtained when compared to those obtained under neutral conditions [23e26]. Moreover, alkaline BES promote the growth of microorganisms that can also grow under highsaline conditions such as Geoalkalibacter or Oxalobacteraceae [25e30] and alkaliphilic environments may also be favorable to prevent a potential acidity buildup in the anode [31,32]. The use of glycerol in BES has been already reported since the fermentation products of glycerol can have an addedvalue. However, its use only in MFC rather than MEC has been reported [33e40]. Thus, the aim of this study is to be the first experimental evaluation of alkaline bioelectrochemical production of hydrogen from crude glycerol as sole carbon source as a potential technology for its valorization. Electrochemical and advanced microbiological tools are also used to gain insight into the process performance.
Materials and methods Medium and substrate The medium used was the same as in Rago et al. [23] except for the carbonate/bicarbonate buffer used, that was the same as in Badalamenti et al. [30]. Briefly, 100 mL of a stock solution of 30 g/L Na2CO3 and 100 g/L of NaHCO3 were added for each liter of medium. 10 mM of 2-bromoethanesulfonate were added to prevent methanogenesis. Glycerol dosage was initially low and it was increased during the start-up period. We considered stable period and, thus, end of start-up when two consecutive cycles had the same maximum current intensity. Glycerol was added so that an initial concentration of 3.1 g/L was obtained. Crude glycerol , Barcelona, was obtained from Stocks del Valles S.A., Montmelo a company that produces biodiesel from recycled animal fats and vegetables oils.
Reactors description, inoculation and operation An air-cathode MFC (A-MFC) of 400 mL as in [22,23] was used for enrichment of a consortium able to degrade glycerol. The materials of the A-MFC are described in the supplementary information. A-MFC was inoculated with sludge (1:1 v/v with fresh media) from a running alkaline air cathode MFC [23] fed with acetate and operated at pH ¼ 9.3. All the cells were operated at room temperature (around 20 C). An air-cathode cube MFC (B-MFC) of 28 mL built as in Rago et al. [23] was also used in the experimental period and operated at alkaline pH ¼ 9.3. The materials of the B-MFC are described in the supplementary information. The alkaline BMFC was inoculated by placing an anode into A-MFC around 2e4 weeks until a stable response was obtained. Then, the colonized anode was transferred to the B-MFC. After 49 days of operation, the B-MFC was converted to an alkaline MEC (B-MEC). B-MEC were analogous to B-MFC, but the cathode was not exposed to air and the cell had a glass cylinder at the top (16 mL), tightly sealed with a PTFE rubber cap and an aluminium crimp. The gas produced was further collected in a gas-tight bag (Ritter, Cali-5-bond) connected to
17206
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 4 ( 2 0 1 9 ) 1 7 2 0 4 e1 7 2 1 3
the PTFE rubber cap by means of a PVC hosepipe. Both electrodes were connected to a power source (HQ Power, PS-23023) applying a potential of 0.8 V except for days 15e20 when the potential applied was 1.2 V. Current production was measured quantifying the voltage drop across a 12 U external resistance serially connected to the circuit. All the cells were operated at room temperature (around 20 C). pH was monitored on-line with pH electrodes (HACH pH CRI-5233) connected to a pH meter (HACH CRI-MM44) as previously described [41] and controlled with base dosage (NaOH 3 M) using an automatic burette (Crison MultiBurette 2S) handled by the software Addcontrol developed using Labwidows CVI (National Instruments, USA). pH control experiments were performed on an orbital agitator at 100 rpm (DOS20L ELMY Sky Line digital orbital shaker).
Electrochemical calculations and cyclic voltammetry analysis The equations for the coulombic efficiency (CE), the cathodic recovery (rCAT) and the energy recovery of the cell calculated as the amount of energy produced as hydrogen with respect to the electrical input (rE) and the electrical input and the energy content of the substrate (rEþS) can be found in the supplementary information. The anode potential was measured using an AgjAgCl reference electrode (NaCl 3 M, 209 mV vs. standard hydrogen electrode). Low scan cyclic voltammetry analyses (CV) were conducted at a scan rate of 1 mV/s (Autolab Potentiostat Galvanostat). The medium in each CV was fresh to guarantee that the MFC was working under high substrate conditions and, prior to the CV, the circuit was opened for 1 h.
Chemical analyses Glycerol concentration in 0.22 mm filtered samples was determined by high performance liquid chromatography (HPLC, Dionex Ultimate 3000) provided with a refractive index detector and using an ionic exchange column (ICSep ICECOREGEL 87H3, Transgenomic). The mobile phase was 6 mM sulphuric acid. Volatile fatty acids (propionic, acetic and butyric acid), hydrogen and methane were analysed as in [23]. A description of the methodologies can be found in the supplementary information.
Microbiological analyses Next generation sequencing (NGS) was performed to analyse the microbial community developed in the anode of A-MFC, BMFC and B-MEC, and in the cathode of B-MFC. The anode graphite fibers and the carbon cloth cathodes were rinsed with 1 mL of sterile MilliQ water to remove any residue and then were cut and combined for DNA extraction. Total DNA was extracted from approximately 0.2 g of samples using a PowerBiofilm DNA Isolation Kit (MoBio Laboratories, Inc., Carlsbad, CA) according to the manufacturer's instructions. Quality and quantity of the DNA were measured using a NanoDrop® spectrophotometer (ThermoScientific). NGS of 16S rRNA gene sequencing was performed in Illumina MiSeq sequencer by
the Research and Testing Laboratory (Lubbock, TX). Each DNA sample (20 ng/mL, quality ratio of 1.8) was analyzed with an average of 10000 reads/assay with 2 300 bp technology and using the primers couple 28F and 388R for bacterial 16S rRNA gene. Sequences were assembled, sorted and trimmed using the Pipeline Initial Process at the Ribosomal Database Project (RDP) Pyrosequencing Pipeline (http://rdp.cme.msu.edu/ index.jsp [42]) with the default settings. Chimeras were checked using Uchime in the same website platform. The RDP Classifier was used to assign 16S rRNA gene sequences to a taxonomical hierarchy with a confidence threshold of 95%. The relative abundance of a given phylogenetic group was calculated as the number of sequences associated with that group divided by the total number of sequences per sample. Representative sequence was obtained for each Operational Taxonomic Unit (OTU), rarefaction tool was used to assess the sequencing depth and, finally, ecological metrics such as Shannon and Chao1 alpha diversity were estimated using the pertinent RDPipeline tools [43].
Results and discussion Start-up and operation of an alkaline glycerol microbial fuel cell The first objective of this work was to obtain an anodic consortium able to degrade glycerol under alkaline conditions. It is commonly accepted that exoelectrogens can use a limited range of carbon sources. Thus, a consortium between fermenters and exoelectrogens was required so that exoelectrogens would degrade the complex substrates and exoelectrogens would live off these products [44]. Different strategies to obtain this consortium have been reported at neutral pH [22], however, to the best of our knowledge, this is the first report of this consortium able to use glycerol as sole carbon source under alkaline conditions. Fig. 1 shows the start-up and operation of an alkaline aircathode MFC (A-MFC) with crude glycerol as sole carbon source. An anode from an acetate-fed alkaline air cathode MFC [23] was used as inoculum. Preliminary batch experiments with glycerol showed that this anodic consortium could use glycerol despite it had been fed with only acetate for more than two months. The fact that the A-MFC was previously inoculated with biomass from an anaerobic digester could explain this instantaneous acclimation to glycerol. The intensity obtained under these conditions (i.e. raw glycerol as sole carbon source and alkaline conditions, pH ¼ 9.3) was between 1.5 and 2 mA (3.75 and 5 A/m3 or 1.875 and 2.5 mA/ m2) during all the experimental period, which was more than two-fold higher than that obtained with the same cell under neutral conditions [45]. Rago et al. [23] already observed that alkaline conditions in the same experimental BES set-up could lead to higher current intensity values. Two cycles were monitored to understand the fate of glycerol and the potential fermentation products (Fig. 1, down). A first cycle under conventional A-MFC operation showed that glycerol was rapidly consumed in less than two days and that part of this glycerol was transformed into
17207
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 4 ( 2 0 1 9 ) 1 7 2 0 4 e1 7 2 1 3
2.5
A Intensity (mA)
2.0 1.5 1.0 0.5 0.0 20
40
Time (d) 60
1.4 Closed circuit
0.8
8
0.6
7
0.4 6
0.2 0.0
5 0
2
4 Time (d)
6
8
Concentration (g/L)
1.0
120 10
Open circuit
C
1.2
9
pH
Concentration (g/L)
1.2
100
1.4
10
B
80
9
1.0 0.8
8
0.6
7
pH
0
0.4 6
0.2 0.0
5 0
2
4
6
8
Time (d)
Fig. 1 e A): Evolution of current intensity for the whole experimental period of A-MFC with crude glycerol as sole carbon source. Vertical lines show the points of glycerol addition. DOWN: Evolution of glycerol and potential fermentation products under closed (B) and open (C) circuit conditions. Glycerol (C), acetic acid (B), propionic acid (;), butyric acid (△) and pH (–).
volatile fatty acids (mainly acetic and propionic acids). The same experiment conducted under open-circuit conditions showed very similar results in terms of glycerol degradation and VFA accumulation indicating that glycerol fermentation was not the limiting step. Thus, VFA consumption by exoelectrogens was the limiting step and should be enhanced for a higher cell performance. Moreover, direct glycerol utilization by exoelectrogens could be neglected since glycerol degradation did not increase when closing the circuit. VFA degradation was also observed under open circuit conditions, which could be attributed to anaerobic activity or to aerobic degradation with oxygen leakages from the cathode. Thus, there existed a competition between exoelectrogens and VFAdegraders under closed circuit conditions. The occurrence of this competition agrees with the observed low CE values (18 ± 8%): around of the 80% of the electrons contained in the initial substrate were not transferred from the anode to the cathode. Hence, a possibility to enhance bioelectrochemical glycerol degradation was to use a different set-up where exoelectrogens were favored against anaerobic VFAdegraders. The widely used cube-shaped MFC, B-MFC [19,46], seemed a reasonable alternative. These cells are smaller, the anode is placed closer to the cathode and they are much more efficient in terms of CE. Thus, a small brush was placed in the A-MFC for two weeks in view of its inoculation. Then, it was moved to a B-MFC and fed with raw glycerol. Fig. 2 shows the experimental evolution of this cell during almost two months. As can be observed, the cell performance was higher than that
observed in A-MFC. Similar absolute current intensity levels were attained (i.e. around 2 mA) despite the volume was one order of magnitude lower, and hence much higher volumetric intensity was obtained, i.e. 71.2 A/m3 (2.5 mA/m2) in B-MFC vs 3.75e5 A/m3 in A-MFC. In addition, much higher CE was obtained in B-MFC (55 ± 17%) vs A-MFC (18 ± 8%). Table 1 compares the results obtained in this work with similar works using the same experimental set-up with glycerol and acetate under neutral and alkaline conditions. As can be observed, alkaline conditions usually lead to higher CE and higher intensities with respect to those working at neutral pH [23]. On the other side, the use of a more complex substrate, in this case glycerol, also results in slightly lower CEs when compared to acetate since part of the initial electrons contained in the complex substrate are lost in the first fermentation steps. Fig. 3 shows the CVs conducted during the B-MFC operation to gain more insight on the process. The intensity obtained with glycerol is only 20% higher than that with acetate suggesting that glycerol fermentation may lead to VFA other than acetate that could be consumed simultaneously by the anodic consortia. Fermentation does not seem the limiting step and, thus, actions aiming at enhancing the bioelectrochemical usage of acetate are necessary to upgrade the cell performance. Propionate shows very low intensity since it is hardly produced as intermediary in the process. Kiely et al. [47] already showed that acetate is a preferred electron donor for exoelectrogens when compared to propionate.
17208
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 4 ( 2 0 1 9 ) 1 7 2 0 4 e1 7 2 1 3
Fig. 2 e Current intensity for the whole experimental period of B-MFC with crude glycerol as sole carbon source under alkaline conditions.
Start-up and operation of an alkaline glycerol-MEC The B-MFC was then converted to B-MEC to assess the potential bioelectrochemical alkaline hydrogen production with glycerol as sole carbon source. Fig. 4 shows the evolution of the current intensity in the B-MEC during the whole experimental period. The current intensity varied between 1.5 and 2 mA under different applied voltages. CE increased with respect to that obtained under MFC conditions up to 66 ± 22%. Obtaining lower CE values under MFC conditions is probably linked to some oxygen diffusion in the anodic chamber. Another possibility for the increased CE is that some hydrogen was recycled from the cathode to the anode since the MEC was operating as a single chamber. This hydrogen recycling would explain the high presence of acetogenic bacteria at the anode that, besides glycerol degradation, can use hydrogen as electron donor (see next section). The “fake” increase of CE due to hydrogen recycling in single-chamber MEC has already been discussed and reported in similar cells [48e52]. A twochamber system would solve this hydrogen recycling at expenses of a lower MEC performance due to higher internal resistances [48]. The highest cathodic recovery, rCAT, obtained was 84 ± 7% in the last cycles. These high values indicate that most of the electrons flowing from the anode to the cathode could be recovered as hydrogen. These percentages are close to the theoretically 100% of rCAT provided that there is not hydrogen scavenging in the cathode. In any case, small hydrogen losses could explain the rCAT obtained. With respect to the energy
recovery indexes, rE was 110 ± 14% indicating the amount of hydrogen obtained contains more energy than that used to produce it. However, if we consider the combustion heat of the substrate (rEþS), this value decreases to 40 ± 12%, indicating that the amount of hydrogen production needs to be increased to have a net substrate valorization. Fig. 4 also shows the amount of hydrogen produced in each cycle, which
Table 1 e Comparison of intensity and CE for the same Cube-MFC (REXT ¼ 220U) fed with acetate and glycerol under alkaline and neutral conditions. pH
Carbon source
Intensity (mA/m2 cathode)
CE (%)
Ref
Neutral Neutral Alkaline Alkaline
Acetate Glycerol Acetate Glycerol
2.14 1.42e2.14a 2.14 2.14e2.84
47 35 60 55
[23,45] [22] [23] This work
a
REXT ¼ 100U.
Fig. 3 e Turnover CVs as function of cell (up) and anode (down) potential with different substrates: glycerol (solid), acetic acid (dash-dotted) and propionic (dashed).
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 4 ( 2 0 1 9 ) 1 7 2 0 4 e1 7 2 1 3
17209
Fig. 4 e Current intensity (solid) and hydrogen production (C) for the whole experimental period of B-MEC. Dotted bars indicate the end of each cycle.
showed some variability. In this sense, the highest hydrogen production was 0.46 L H2/L/d which is in the range of similar works using the same cube-shaped MECs [31,42]. However, the value is lower than that reported under alkaline conditions in the same set-up with acetate as sole carbon source [23]. Both this observation and the electrochemical measurements conducted under MFC mode suggest that exoelectrogenic activity is the limiting step of the cell configuration used in the present work and, thus, enhancing anodic exoelectrogenesis would lead to higher hydrogen production values.
High-throughput sequencing and microbial community analysis In order to go in depth in the knowledge of the bacterial community developed in the reactors to help explain their behaviour, DNA was extracted from the biofilms sampled and 16S sequencing analysis were performed from both anodes during MFC and MEC operation. Rarefaction curves of sequencing data showed gentle slopes under the obtained sequencing depth. This fact indicates that the sequencing libraries could correctly reflect the microbial community diversity. Alpha-diversity analysis showed that both richness and diversity of Bacteria had similar values at B-MEC and BMFC anode samples (Table 2). The results of the Illumina 16S rRNA gene amplicon sequencing showed also many similarities between both samples, with only a slight shift in anodic communities’ composition in B-MFC and B-MEC samples (Fig. 5). Most bacteria detected in both anodes belonged to the Enterococcaceae family, accounting for 45.4 and 40.9% at BMFC and B-MEC, respectively. Among them, 36.6 and 32.4% (at B-MFC and B-MEC) were affiliated to the genus Enterococcus.
Table 2 e Characteristics of sequencing libraries. Sample B-MFC B-MEC
Nr of aligned sequences
Number of OTUs
Chao 1 value
Shannon Index
44720 17031
493 413
687 594
2.83 3.09
Enterococcus spp. are gram positive bacteria and facultative aerobes, belonging to the so-called lactic acid bacteria group. Their usual habitat is the digestive system of mammals, and some species are even opportunistic human pathogens. However, their presence and persistence in secondary habitats, such as surface waters, not only as a signal of faecal contamination but as a regular habitat, has been reviewed recently by some authors [53]. Enterococcus faecalis has even been reported in some bioelectrochemical systems [54]. In fact, some studies revealed its capacity to transfer electrons derived from the fermentation process directly and indirectly to an electrode [55]. On the other hand, Enterococcus spp., being an acid lactic bacteria, can be commonly found in acidic environments. It might be surprising, thus, to find them in alkaline systems of the present study. However, some previous works already pointed out their ability to survive in alkaline environments up to pH 10 [56]. Moreover, local acidic environments in the anode could favour their growth. Therefore, the role of Enterococcus sp. might be both the fermentation of glycerol and the transfer of electrons to the anode through its exoelectrogenic activity. The next main bacterial families found in B-MFC and BMEC anodes in terms of relative abundance were Eubacteriaceae and Geobacteraceae families: 9.8% each one at B-MFC and 16.6% and 9.9%, respectively at B-MEC. All members of both families are strict anaerobes [57,58], which confirms that no oxygen reached the anode biofilm (or at least some sections of it) under the studied conditions. Thus, any possible aerobic consumption of glycerol due to cathode leaks, occurred either in the bulk liquid phase or in the external layer of the biofilm (for example by the abovementioned Enterococcus sp). Geoalkalibacter sp. accounts for 9.2 and 9.8% at B-MFC and B-MEC, respectively. It belongs to the Geobacteraceae family (Desulfuromonadales class), which was already studied for its exoelectrogenic ability in pure culture [29,30,59] and it was also isolated in mixed culture alkaline bioelectrochemical reactors [23,27]. Thus, its main role might be extracellular electron transfer from fermentation products (i.e. acetate) to the anode. Its relative abundance remained constant when the bioelectrochemical system was switched from MFC to MEC. Acetobacterium sp. was the genus that exhibited higher changes in their relative abundance from B-MFC to B-MEC.
17210
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 4 ( 2 0 1 9 ) 1 7 2 0 4 e1 7 2 1 3
Fig. 5 e Family representation of Illumina 16S rRNA gene amplicon sequencing results of anodic biofilms samples of B-MFC and B-MEC. Sequences accounting for less than 1% of the total reads have been included in the “Other unclassified” category.
Specifically, it doubled its percentage, from 8.2% in B-MFC to 16.2% in B-MEC. Affiliated to the Eubacteriaceae family (Clostridiales class), it is a well-known homoacetogenic bacteria [60], which means that can produce acetate from hydrogen and carbon dioxide. It was already demonstrated to be capable to grow with glycerol as sole carbon and energy source and to produce then acetic acid [61]. Moreover, Acetobacterium genus was previously found in the anodic samples producing acetic acid, then used by exoelectrogenic bacteria in bioelectrochemical systems fed with different substrates (e.g. methanol [48]). Thus, Acetobacterium might be responsible of acetic acid production that was then used, for example, by Geoalkalibacter sp., to generate electrons. The increase of Acetobacterium sp. at B-MEC could be due to the higher production of hydrogen (in the cathode) in the MEC, which was singlechamber configuration. Alkalibacter genus (3% in B-MFC and 1% in B-MEC), also in the Eubacteriaceae family, is strictly anaerobic and an alkaliphilic genus of Firmicutes phylum [62]. In a previous study [23], Alkalibacter genus was enriched in similar reactors and its exoelectrogenic activity was supposed but not fully demonstrated. Also other bacteria from Desulfuromonadaceae family (approximately 5% of unclassified in both B-MFC and B-MEC) were often found in BES with an important role in exoelectrogenesis [63e65]. Some bacteria from the Alcaligenaceae family, which accounted for 0.2% and 2.7% of total bacteria at B-MFC and BMEC anode biofilm, respectively, have been reported to have extracellular electron transfer capacity as well. In particular, Alcaligenes faecalis was reported to generate electricity at a posed potential of 0.23 V vs. SHE [66]. Unclassified Clostridiales (2.2% in B-MFC and 4.6% in B-MEC) and unclassified Firmicutes (1.7% in B-MFC and 2.7% in B-MEC), represented also an important amount of the sequences. Many genera and species from Clostridiales order and from Firmicutes phylum, have showed exoelectrogenic activity [63,67,68]. In summary, almost all the bacterial families identified in the anode biofilm of B-MFC and B-MEC have previously shown exoelectrogenic activity. Therefore, we can conclude that we achieved a highly adapted biofilm to the dual activity of
glycerol fermentation and external electron transfer to the anode.
Conclusions This work evaluates, for the first time, the use of crude glycerol as sole carbon source in alkaline BES. This would provide a valorization opportunity for glycerol, an undesired byproduct of alkaline biodiesel production with low commercial value. Promising results have been reported with alkaline BES: current intensities higher than those found under neutral pH conditions. This work shows a successful glycerol degradation in an alkaline MFC with intensities similar to those obtained in the same systems with acetate (2 mA, 71.4 mA/m3 and 2.84 mA/m2 and a CE of 55%). Hydrogen production in a MEC was also feasible and with performance values close to those obtained with acetate (0.46 LH2/L/d and 85% of rCAT) indicating, for the first time, the suitability of this waste in being transformed to hydrogen. Finally, a deep analysis on the microbiota found in the anode indicated that the MEC was performing successfully due to a consortium of glycerol fermenters and exoelectrogens mainly composed of Enterococcaceae and Geoalkalibacter, respectively.
Acknowledgements This study was financed by the project VALTEC13-1-0140 granted by ACCIO (Generalitat de Catalunya). The authors are members of the GENOCOV group (Grup de Recerca Consolidat de la Generalitat de Catalunya, 2017 SGR 1175). Laura Rago is grateful for the FPI grant BES-2011-051308 from the Spanish Ministerio de Economı´a y Competitividad.
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijhydene.2019.03.193.
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 4 ( 2 0 1 9 ) 1 7 2 0 4 e1 7 2 1 3
references
[1] Atabani AE, Silitonga AS, Badruddin IA, Mahlia TMI, Masjuki HH, Mekhilef S. A comprehensive review on biodiesel as an alternative energy resource and its characteristics. Renew Sustain Energy Rev 2012;16:2070e93. https://doi.org/10.1016/j.rser.2012.01.003. [2] Zhang Y. Biodiesel production from waste cooking oil: 1. Process design and technological assessment. Bioresour Technol 2003;89:1e16. https://doi.org/10.1016/S0960-8524(03) 00040-3. [3] Christopher LP, Kumar Hemanathan, Zambare VP. Enzymatic biodiesel: challenges and opportunities. Appl Energy 2014;119:497e520. https://doi.org/10.1016/ j.apenergy.2014.01.017. [4] Quispe CAG, Coronado CJR, Carvalho Jr JA. Glycerol: production, consumption, prices, characterization and new trends in combustion. Renew Sustain Energy Rev 2013;27:475e93. https://doi.org/10.1016/j.rser.2013.06.017. [5] Thompson J, He B. Characterization of crude glycerol from biodiesel production from multiple feedstocks. Appl Eng Agric 2006;22:261e5. [6] Werpy T, Petersen G. Top value added chemicals from biomass volume Idresults of screening for potential candidates from sugars and synthesis gas. 2004. [7] Qing Y, Lu H, Liu Y, Liu C, Liang B, Jiang W. Production of glycerol carbonate using crude glycerol from biodiesel production with DBU as a catalyst. Chin J Chem Eng 2018;26:1912e9. https://doi.org/10.1016/j.cjche.2018.01.010. [8] Adhikari S, Fernando SD, Haryanto A. Hydrogen production from glycerin by steam reforming over nickel catalysts. Renew Energy 2008;33:1097e100. https://doi.org/10.1016/ j.renene.2007.09.005. [9] Schultz E, Tatiane de Souza D, Caramez Triches Damaso M. The glycerol biorefinery: a purpose for Brazilian biodiesel production. Chem Biol Technol Agric 2014;1. https://doi.org/ 10.1186/s40538-014-0007-z. [10] Dimitratos Nikolaos, Villa Alberto, Carine E, Chan-Thaw CE, Prati L. Valorisation of glycerol to fine chemicals and fuels. In: Petrochemical catal. Mater. Process. Emerg. Technol. Igiglobal; 2016. p. 352e84. [11] Lin Y-C. Catalytic valorization of glycerol to hydrogen and syngas. Int J Hydrogen Energy 2013;38:2678e700. https://doi. org/10.1016/j.ijhydene.2012.12.079. [12] Patcharavorachot Y, Chatrattanawet N, Arpornwichanop A, Assabumrungrat S. Optimization of hydrogen production from three reforming approaches of glycerol via using supercritical water with in situ CO2 separation. Int J Hydrogen Energy 2019;44:2128e40. https://doi.org/10.1016/j. ijhydene.2018.07.053. [13] Rodrigues CV, Santana KO, Nespeca MG, Eduardo de Oliveira J, Maintinguer SI. Crude glycerol by transesterification process from used cooking oils: characterization and potentialities on hydrogen bioproduction. Int J Hydrogen Energy 2016;41:14641e51. https://doi.org/10.1016/j.ijhydene.2016.06.209. [14] Mangayil R, Karp M, Santala V. Bioconversion of crude glycerol from biodiesel production to hydrogen. Int J Hydrogen Energy 2012;37:12198e204. https://doi.org/10.1016/ j.ijhydene.2012.06.010. [15] Rossi DM, Berne da Costa J, Aquino de Souza E, Peralba M, do chia Ayub MA. Comparison of different CR, Samios D, Za pretreatment methods for hydrogen production using environmental microbial consortia on residual glycerol from biodiesel. Int J Hydrogen Energy 2011;36:4814e9. https://doi. org/10.1016/j.ijhydene.2011.01.005.
17211
[16] Sarma SJ, Brar SK, Sydney EB, Le Bihan Y, Buelna G, Soccol CR. Microbial hydrogen production by bioconversion of crude glycerol: a review. Int J Hydrogen Energy 2012;37:6473e90. https://doi.org/10.1016/j.ijhydene.2012.01.050. [17] Choi W. Glycerol-based biorefinery for fuels and chemicals. Recent Pat Biotechnol 2008;2:173e80. [18] Fukuda H, Kondo A, Noda H. Biodiesel fuel production by transesterification of oils. J Biosci Bioeng 2001;92:405e16. https://doi.org/10.1016/S1389-1723(01)80288-7. [19] Logan BE. Microbial fuel cells. 2008. [20] Cheng S, Logan BE. High hydrogen production rate of microbial electrolysis cell (MEC) with reduced electrode spacing. Bioresour Technol 2011;102:3571e4. https://doi.org/ 10.1016/j.biortech.2010.10.025. [21] Logan BE, Rabaey K. Conversion of wastes into bioelectricity and chemicals by using microbial electrochemical technologies. Science 2012;337:686e90. https://doi.org/ 10.1126/science.1217412. [22] Montpart N, Rago L, Baeza JA, Guisasola A. Hydrogen production in single chamber microbial electrolysis cells with different complex substrates. Water Res 2015;68:601e15. https://doi.org/10.1016/j.watres.2014.10.026. [23] Rago L, Baeza JA, Guisasola A. Increased performance of hydrogen production in microbial electrolysis cells under alkaline conditions. Bioelectrochemistry 2016;109:57e62. https://doi.org/10.1016/j.bioelechem.2016.01.003. [24] Zhang E, Zhai W, Luo Y, Scott K, Wang X, Diao G. Acclimatization of microbial consortia to alkaline conditions and enhanced electricity generation. Bioresour Technol 2016;211:736e42. https://doi.org/10.1016/j.biortech.2016.03.115. [25] Weerasinghe Mohottige TN, Ginige MP, Kaksonen AH, Sarukkalige R, Cheng KY. Rapid start-up of a bioelectrochemical system under alkaline and saline conditions for efficient oxalate removal. Bioresour Technol 2018;250:317e27. https://doi.org/10.1016/j.biortech.2017.11. 009. [26] Mohottige TNW, Ginige MP, Kaksonen AH, Sarukkalige R, Cheng KY. Bioelectrochemical oxidation of organics by alkalihalotolerant anodophilic biofilm under nitrogen-deficient, alkaline and saline conditions. Bioresour Technol 2017;245:890e8. https://doi.org/10.1016/j.biortech.2017.08.157. [27] Miceli JF, Parameswaran P, Kang DW, Krajmalnik-Brown R, Torres CI. Enrichment and analysis of anode-respiring bacteria from diverse anaerobic inocula. Environ Sci Technol 2012;46:10349e55. https://doi.org/10.1021/es301902h. [28] Carmona-Martı´nez A a, Pierra M, Trably E, Bernet N. High current density via direct electron transfer by the halophilic anode respiring bacterium Geoalkalibacter subterraneus. Phys Chem Chem Phys 2013;15:19699e707. https://doi.org/ 10.1039/c3cp54045f. [29] Pierra M, Carmona-Martı´nez AA, Trably E, Godon JJ, Bernet N. Specific and efficient electrochemical selection of Geoalkalibacter subterraneus and Desulfuromonas acetoxidans in high current-producing biofilms. Bioelectrochemistry 2015;106:221e5. https://doi.org/10.1016/ j.bioelechem.2015.02.003. [30] Badalamenti JP, Krajmalnik-brown R, Torres CI. Generation of high current densities by pure cultures of anode- respiring geoalkalibacter spp . under alkaline and saline conditions. mBio 2013;4:1e8. https://doi.org/10.1128/mBio.00144-13. [31] Torres CI, Kato Marcus A, Rittmann BE. Proton transport inside the biofilm limits electrical current generation by anode-respiring bacteria. Biotechnol Bioeng 2008;100:872e81. https://doi.org/10.1002/bit.21821. [32] Torres CI, Lee HS, Rittmann BE. Carbonate species as OHcarriers for decreasing the pH gradient between cathode and anode in biological fuel cells. Environ Sci Technol 2008;42:8773e7. https://doi.org/10.1021/es8019353.
17212
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 4 ( 2 0 1 9 ) 1 7 2 0 4 e1 7 2 1 3
[33] Guimaraes AQ, Linares JJ. Glycerol utilization in microbial fuel cells: conditioning stage and influence of the glycerol concentration. J Electrochem Soc 2013;161:F125e32. https:// doi.org/10.1149/2.068401jes. [34] Nimje VR, Chen C-Y, Chen C-C, Chen H-R, Tseng M-J, Jean JS, et al. Glycerol degradation in single-chamber microbial fuel cells. Bioresour Technol 2011;102:2629e34. https:// doi.org/10.1016/j.biortech.2010.10.062. [35] Reiche A, Kirkwood KM. Comparison of Escherichia coli and anaerobic consortia derived from compost as anodic biocatalysts in a glycerol-oxidizing microbial fuel cell. Bioresour Technol 2012;123:318e23. https://doi.org/10.1016/j. biortech.2012.07.005. n a, Go mez X, Guiot SR, [36] Escapa a, Manuel M-F, Mora Tartakovsky B. Hydrogen production from glycerol in a membraneless microbial electrolysis cell. Energy & Fuels 2009;23:4612e8. https://doi.org/10.1021/ef900357y. [37] Montpart N, Baeza JA, Guisasola A. From methanol to electricity and hydrogen through bioelectrochemical systems. 2017. https://doi.org/10.1016/B978-0-444-63903-5.00012-1. [38] Feng Y, Yang Q, Wang X, Liu Y, Lee H, Ren N. Treatment of biodiesel production wastes with simultaneous electricity generation using a single-chamber microbial fuel cell. Bioresour Technol 2011;102:411e5. https://doi.org/10.1016/j. biortech.2010.05.059. [39] Santos JBC, Barros VVS de, Linares JJ. Influence of sludge age on the performance of a cyclically fed microbial fuel cell using glycerol from biodiesel. Fuel Cells 2018;18:771e81. https://doi.org/10.1002/fuce.201800089. [40] Sharma Y, Parnas R, Li B. Bioenergy production from glycerol in hydrogen producing bioreactors (HPBs) and microbial fuel cells (MFCs). Int J Hydrogen Energy 2011;36:3853e61. https:// doi.org/10.1016/j.ijhydene.2010.12.040. [41] Ruiz Y, Baeza JA, Guisasola A. Enhanced performance of bioelectrochemical hydrogen production using a pH control strategy. ChemSusChem 2015;8:389e97. https://doi.org/ 10.1002/cssc.201403083. [42] Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ, et al. The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res 2009;37:D141e5. https://doi.org/10.1093/nar/gkn879. [43] Cole JR, Wang Q, Fish JA, Chai B, McGarrell DM, Sun Y, et al. Ribosomal Database Project: data and tools for high throughput rRNA analysis. Nucleic Acids Res 2014;42:D633e42. https://doi.org/10.1093/nar/gkt1244. [44] Montpart N, Rago L, Baeza JA, Guisasola A. Hydrogen production in single chamber microbial electrolysis cells with different complex substrates. Water Res 2015;68:601e15. [45] Ruiz Y, Baeza JA, Guisasola A. Microbial electrolysis cell performance using non-buffered and low conductivity wastewaters. Chem Eng J 2016;289. https://doi.org/10.1016/ j.cej.2015.12.098. [46] Logan BE. Scaling up microbial fuel cells and other bioelectrochemical systems. Appl Microbiol Biotechnol 2010;85:1665e71. https://doi.org/10.1007/s00253-009-2378-9. [47] Kiely PD, Regan JM, Logan BE. The electric picnic: synergistic requirements for exoelectrogenic microbial communities. Curr Opin Biotechnol 2011;22:378e85. https://doi.org/10.1016/ j.copbio.2011.03.003. [48] Montpart N, Ribot-Llobet E, Garlapati VK, Rago L, Baeza JA, Guisasola A. Methanol opportunities for electricity and hydrogen production in bioelectrochemical systems. Int J Hydrogen Energy 2014;39:770e7. https://doi.org/10.1016/ j.ijhydene.2013.10.151. [49] Xafenias N, Mapelli V. Performance and bacterial enrichment of bioelectrochemical systems during methane and acetate production. Int J Hydrogen Energy 2014;39:21864e75. https:// doi.org/10.1016/j.ijhydene.2014.05.038.
[50] Parameswaran P, Torres CI, Kang D-W, Rittmann BE, Krajmalnik-Brown R. The role of homoacetogenic bacteria as efficient hydrogen scavengers in microbial electrochemical cells (MXCs). Water Sci Technol 2012;65:1e6. https://doi.org/ 10.2166/wst.2011.519. s P. Microbial [51] Rago L, Ruiz Y, Baeza JA, Guisasola A, Corte community analysis in a long-term membrane-less microbial electrolysis cell with hydrogen and methane production. Bioelectrochemistry 2015;106:359e68. https:// doi.org/10.1016/j.bioelechem.2015.06.003. [52] Ruiz Y, Baeza JA, Guisasola A. Revealing the proliferation of hydrogen scavengers in a single-chamber microbial electrolysis cell using electron balances. Int J Hydrogen Energy 2013;38:15917e27. https://doi.org/10.1016/ j.ijhydene.2013.10.034. [53] Staley C, Dunny GM, Sadowsky MJ. Chapter four environmental and animal-associated enterococci. 2014. p. 147e86. https://doi.org/10.1016/b978-0-12-8002612.00004-9. [54] Read ST, Dutta P, Bond PL, Keller J, Rabaey K. Initial development and structure of biofilms on microbial fuel cell anodes. BMC Microbiol 2010;10:98. https://doi.org/10.1186/ 1471-2180-10-98. [55] Pankratova G, Leech D, Gorton L, Hederstedt L. Extracellular electron transfer by the gram-positive bacterium Enterococcus faecalis. Biochemistry 2018;57:4597e603. https://doi.org/10.1021/acs.biochem.8b00600. [56] Ran S, Liu B, Jiang W, Sun Z, Liang J. Transcriptome analysis of Enterococcus faecalis in response to alkaline stress. Front Microbiol 2015;6:795. https://doi.org/10.3389/ fmicb.2015.00795. [57] Ludwig W, Schleifer K, Whitman W. Eubacteriaceae fam. nov.. 2015. https://doi.org/10.1002/9781118960608.fbm00130. [58] Garrity G,A, Bell J, Lilburn T. Geobacteraceae fam. nov.. 2015. https://doi.org/10.1002/9781118960608.fbm00202. [59] Yoho RA, Popat SC, Rago L, Guisasola A, Torres CI. Anode biofilms of geoalkalibacter ferrihydriticus exhibit electrochemical signatures of multiple electron transport pathways. Langmuir 2015;31:12552e9. https://doi.org/ 10.1021/acs.langmuir.5b02953. [60] Drake H. Acetogenesis. New York - London: Chapman and Hall; 1994. [61] Emde R, Schink B. Fermentation of triacetin and glycerol by Acetobacterium sp. No energy is conserved by acetate excretion. Arch Microbiol 1987;149:142e8. https://doi.org/ 10.1007/BF00425080. [62] Garnova ES, Zhilina TN, Tourova TP, Kostrikina NA, Zavarzin GA. Anaerobic, alkaliphilic, saccharolytic bacterium Alkalibacter saccharofermentans gen. nov., sp. nov. from a soda lake in the Transbaikal region of Russia. Extremophiles 2004;8:309e16. https://doi.org/10.1007/ s00792-004-0390-7. [63] Koch C, Harnisch F. Is there a specific ecological niche for electroactive microorganisms? ChemElectroChem 2016;3:1282e95. https://doi.org/10.1002/celc.201600079. [64] Rago L, Cristiani P, Villa F, Zecchin S, Colombo A, Cavalca L, et al. Influences of dissolved oxygen concentration on biocathodic microbial communities in microbial fuel cells. Bioelectrochemistry 2017;116:39e51. https://doi.org/10.1016/ j.bioelechem.2017.04.001. [65] Heidrich ES, Edwards SR, Dolfing J, Cotterill SE, Curtis TP. Performance of a pilot scale microbial electrolysis cell fed on domestic wastewater at ambient temperatures for a 12month period. Bioresour Technol 2014;173:87e95. https:// doi.org/10.1016/j.biortech.2014.09.083. [66] Yu L, Yuan Y, Rensing C, Zhou S. Combined spectroelectrochemical and proteomic characterizations of bidirectional Alcaligenes faecalis-electrode electron transfer.
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 4 ( 2 0 1 9 ) 1 7 2 0 4 e1 7 2 1 3
Biosens Bioelectron 2018;106:21e8. https://doi.org/10.1016/j. bios.2018.01.032. [67] Park HS, Kim BH, Kim HS, Kim HJ, Kim G, Kim M, et al. A novel electrochemically active and Fe ( III ) -reducing bacterium phylogenetically related to Clostridium butyricum
17213
isolated from a microbial fuel cell. Scanning 2001;7:297e306. https://doi.org/10.1006/anae.2001.0399. [68] Logan BE. Exoelectrogenic bacteria that power microbial fuel cells. Nat Rev Microbiol 2009;7:375e81. https://doi.org/ 10.1038/nrmicro2113.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/he
Hydrogen production from crude glycerol in an alkaline microbial electrolysis cell Marina Badia-Fabregat a, Laura Rago a,b, Juan A. Baeza a, Albert Guisasola a,* Departament d’Enginyeria Quı´mica, Biologica i Ambiental, Escola d’Enginyeria, Universitat Autonoma de Barcelona, 08193, Bellaterra (Barcelona), Spain b Helmholtz Centre for Environmental Research GmbH e UFZ, Permoserstraße 15, 04318 Leipzig, Germany a
article info
abstract
Article history:
Crude glycerol is an undesired by-product of biodiesel production with a low commercial
Received 20 December 2018
value (i.e. a ton of biodiesel results in around 110 kg of crude glycerol) and, thus, glycerol
Received in revised form
needs valorization. In particular, there is a need of providing a benefit to alkaline waste-
18 March 2019
waters from biodiesel production with excess of glycerol. Bioelectrochemical systems (BES)
Accepted 20 March 2019
are an emerging technique to recover the energy contained in a substrate either as elec-
Available online 25 April 2019
tricity or as other added-value products such as hydrogen. Moreover, promising results have been reported with alkaline BES showing higher current intensities than neutral pH
Keywords:
conditions. This study is the first experimental evaluation of alkaline bioelectrochemical
Alkaline bioelectrochemical system
production of hydrogen from real crude glycerol as sole carbon source. The results show
Crude glycerol
that alkaline glycerol degradation is feasible under both microbial fuel cell mode (2 mA,
Hydrogen production
71.4 A/m3 and 55% of CE) and microbial electrolysis mode (maximum of 0.46 LH2/L/d and
Illumina 16S rDNA sequencing
85% of rCAT). The values obtained are promising since they are in the range of those ob-
Bioanode
tained with other simpler carbon sources such acetate. A complex consortium involving fermentative bacteria (such as Enterococcaceae), alkaline exoelectrogens (such as Geoalkalibacter) and homoacetogens (such as Acetobacterium) was naturally developed in the anode of the MEC. © 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
Introduction Global climate change challenges the current dependence on fossil fuels. Among the different strategies for the climate change mitigation, the use of biodiesel is an alternative fuel for diesel engines because of: i) its environmental advantages; ii) its similarity with conventional diesel in energy content and chemical structure; iii) the reduction of the carcinogenic compound emissions by approximately 85% when compared
with diesel fuel; iv) the absence of sulfur and polycyclic aromatic hydrocarbons [1e3] and v) better engine performance (better lubrication, high number and more complete combustion [4]). Biodiesel is a liquid fuel obtained from animal fats or vegetable oils through the process of transesterification of different triglycerides. These react with an alcohol to produce methyl ester, biodiesel, ethyl ester and glycerol under a catalytic process. The main drawback of biodiesel production is the generation of glycerol, an undesired by-product with a low
* Corresponding author. E-mail address: [email protected] (A. Guisasola). https://doi.org/10.1016/j.ijhydene.2019.03.193 0360-3199/© 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 4 ( 2 0 1 9 ) 1 7 2 0 4 e1 7 2 1 3
commercial value [5]. With the fast increase of glycerol supply in the world market, the price of refined glycerol dropped from $1.20 kg in 2003 to $0.60/kg in 2006 [4]. The price of crude glycerol in the United States reached a minimum value of $0.04e0.11/kg [6] while in 2018 the price has risen up to $0.3e0.4 [7]. The production of a ton of biodiesel results in around 110 kg of crude glycerol [8] and, hence, large surpluses of this material are expected in the future. When glycerol production is higher than its demand, glycerol becomes a highly loaded waste rather than a resource. For example, the production of biodiesel (B100) in Brazil in 2014 generated 3.45$105 m3 of crude glycerol [9]. Therefore, new technologies for glycerol valorization are needed in order to increase the value of this compound, which should become an energetic resource rather than an undesired waste. Nowadays, glycerol valorization technologies are based on the chemical production of high-added values in three different directions: (i) glycerol oxidation, (ii) glycerol hydrogenolysis and (iii) glycerol aqueous reforming [10]. Alkaline catalysts (i.e. alkaline earth metal hydroxides or alkoxides of sodium or potassium) are the most common catalysts in the transesterification process. However, homogeneous alkaline catalysis requires complex downstream processes including the removal of inorganic salts from the final product, the recovery of salt-containing glycerol, and the treatment of alkaline wastewater. Hence, there is a niche in the valorization of alkaline wastewaters from biodiesel production with excess of glycerol. Among the different alternatives for glycerol valorization, hydrogen production is a preferred option since hydrogen is a clean source of energy with no harmful effect during its combustion process. Crude glycerol is a suitable source of hydrogen via physicochemical processes such as catalytic conversion [11] or reforming [12]. Moreover, crude glycerol has also been reported as an adequate substrate for microbial conversion processes such as fermentation [13e18]. Bioelectrochemistry aims at recovering the energy contained in the substrate either as electricity or as reducing power to obtain added-value products. Bioelectrochemical systems (BES) combine electrochemistry with the metabolism of anode respiring bacteria (ARB), also known as exoelectrogenic bacteria, which can transfer the electrons obtained in their metabolism to an external solid anode. Then, electrons flow from the anode to the cathode where a reduction reaction takes place [19]. If the cathodic reaction has a higher reduction potential than the anodic reaction, the electromotive force of the system is positive, free Gibbs energy is negative and, thus, electrons flow spontaneously from the anode to the cathode. This is usually observed when oxygen reduction occurs at the cathode. This device is known as Microbial Fuel Cell (MFC) and the substrate is oxidized at the anode linked to electricity generation. On the other hand, if oxygen is replaced in the cathodic chamber for other reductive process with lower reduction potential, the system becomes thermodynamically non-spontaneous and, then, additional energy supply is required to drive the process. These devices are known as Microbial Electrolysis Cell (MEC) and the substrate is oxidized in the anode linked to, for example, hydrogen production in the cathode [20e22]. Most of the research conducted so far with BES is under neutral conditions. However, the few works on
17205
alkaline BES already showed that alkaline bioelectrochemistry envisages a promising scenario since higher current intensities can be obtained when compared to those obtained under neutral conditions [23e26]. Moreover, alkaline BES promote the growth of microorganisms that can also grow under highsaline conditions such as Geoalkalibacter or Oxalobacteraceae [25e30] and alkaliphilic environments may also be favorable to prevent a potential acidity buildup in the anode [31,32]. The use of glycerol in BES has been already reported since the fermentation products of glycerol can have an addedvalue. However, its use only in MFC rather than MEC has been reported [33e40]. Thus, the aim of this study is to be the first experimental evaluation of alkaline bioelectrochemical production of hydrogen from crude glycerol as sole carbon source as a potential technology for its valorization. Electrochemical and advanced microbiological tools are also used to gain insight into the process performance.
Materials and methods Medium and substrate The medium used was the same as in Rago et al. [23] except for the carbonate/bicarbonate buffer used, that was the same as in Badalamenti et al. [30]. Briefly, 100 mL of a stock solution of 30 g/L Na2CO3 and 100 g/L of NaHCO3 were added for each liter of medium. 10 mM of 2-bromoethanesulfonate were added to prevent methanogenesis. Glycerol dosage was initially low and it was increased during the start-up period. We considered stable period and, thus, end of start-up when two consecutive cycles had the same maximum current intensity. Glycerol was added so that an initial concentration of 3.1 g/L was obtained. Crude glycerol , Barcelona, was obtained from Stocks del Valles S.A., Montmelo a company that produces biodiesel from recycled animal fats and vegetables oils.
Reactors description, inoculation and operation An air-cathode MFC (A-MFC) of 400 mL as in [22,23] was used for enrichment of a consortium able to degrade glycerol. The materials of the A-MFC are described in the supplementary information. A-MFC was inoculated with sludge (1:1 v/v with fresh media) from a running alkaline air cathode MFC [23] fed with acetate and operated at pH ¼ 9.3. All the cells were operated at room temperature (around 20 C). An air-cathode cube MFC (B-MFC) of 28 mL built as in Rago et al. [23] was also used in the experimental period and operated at alkaline pH ¼ 9.3. The materials of the B-MFC are described in the supplementary information. The alkaline BMFC was inoculated by placing an anode into A-MFC around 2e4 weeks until a stable response was obtained. Then, the colonized anode was transferred to the B-MFC. After 49 days of operation, the B-MFC was converted to an alkaline MEC (B-MEC). B-MEC were analogous to B-MFC, but the cathode was not exposed to air and the cell had a glass cylinder at the top (16 mL), tightly sealed with a PTFE rubber cap and an aluminium crimp. The gas produced was further collected in a gas-tight bag (Ritter, Cali-5-bond) connected to
17206
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 4 ( 2 0 1 9 ) 1 7 2 0 4 e1 7 2 1 3
the PTFE rubber cap by means of a PVC hosepipe. Both electrodes were connected to a power source (HQ Power, PS-23023) applying a potential of 0.8 V except for days 15e20 when the potential applied was 1.2 V. Current production was measured quantifying the voltage drop across a 12 U external resistance serially connected to the circuit. All the cells were operated at room temperature (around 20 C). pH was monitored on-line with pH electrodes (HACH pH CRI-5233) connected to a pH meter (HACH CRI-MM44) as previously described [41] and controlled with base dosage (NaOH 3 M) using an automatic burette (Crison MultiBurette 2S) handled by the software Addcontrol developed using Labwidows CVI (National Instruments, USA). pH control experiments were performed on an orbital agitator at 100 rpm (DOS20L ELMY Sky Line digital orbital shaker).
Electrochemical calculations and cyclic voltammetry analysis The equations for the coulombic efficiency (CE), the cathodic recovery (rCAT) and the energy recovery of the cell calculated as the amount of energy produced as hydrogen with respect to the electrical input (rE) and the electrical input and the energy content of the substrate (rEþS) can be found in the supplementary information. The anode potential was measured using an AgjAgCl reference electrode (NaCl 3 M, 209 mV vs. standard hydrogen electrode). Low scan cyclic voltammetry analyses (CV) were conducted at a scan rate of 1 mV/s (Autolab Potentiostat Galvanostat). The medium in each CV was fresh to guarantee that the MFC was working under high substrate conditions and, prior to the CV, the circuit was opened for 1 h.
Chemical analyses Glycerol concentration in 0.22 mm filtered samples was determined by high performance liquid chromatography (HPLC, Dionex Ultimate 3000) provided with a refractive index detector and using an ionic exchange column (ICSep ICECOREGEL 87H3, Transgenomic). The mobile phase was 6 mM sulphuric acid. Volatile fatty acids (propionic, acetic and butyric acid), hydrogen and methane were analysed as in [23]. A description of the methodologies can be found in the supplementary information.
Microbiological analyses Next generation sequencing (NGS) was performed to analyse the microbial community developed in the anode of A-MFC, BMFC and B-MEC, and in the cathode of B-MFC. The anode graphite fibers and the carbon cloth cathodes were rinsed with 1 mL of sterile MilliQ water to remove any residue and then were cut and combined for DNA extraction. Total DNA was extracted from approximately 0.2 g of samples using a PowerBiofilm DNA Isolation Kit (MoBio Laboratories, Inc., Carlsbad, CA) according to the manufacturer's instructions. Quality and quantity of the DNA were measured using a NanoDrop® spectrophotometer (ThermoScientific). NGS of 16S rRNA gene sequencing was performed in Illumina MiSeq sequencer by
the Research and Testing Laboratory (Lubbock, TX). Each DNA sample (20 ng/mL, quality ratio of 1.8) was analyzed with an average of 10000 reads/assay with 2 300 bp technology and using the primers couple 28F and 388R for bacterial 16S rRNA gene. Sequences were assembled, sorted and trimmed using the Pipeline Initial Process at the Ribosomal Database Project (RDP) Pyrosequencing Pipeline (http://rdp.cme.msu.edu/ index.jsp [42]) with the default settings. Chimeras were checked using Uchime in the same website platform. The RDP Classifier was used to assign 16S rRNA gene sequences to a taxonomical hierarchy with a confidence threshold of 95%. The relative abundance of a given phylogenetic group was calculated as the number of sequences associated with that group divided by the total number of sequences per sample. Representative sequence was obtained for each Operational Taxonomic Unit (OTU), rarefaction tool was used to assess the sequencing depth and, finally, ecological metrics such as Shannon and Chao1 alpha diversity were estimated using the pertinent RDPipeline tools [43].
Results and discussion Start-up and operation of an alkaline glycerol microbial fuel cell The first objective of this work was to obtain an anodic consortium able to degrade glycerol under alkaline conditions. It is commonly accepted that exoelectrogens can use a limited range of carbon sources. Thus, a consortium between fermenters and exoelectrogens was required so that exoelectrogens would degrade the complex substrates and exoelectrogens would live off these products [44]. Different strategies to obtain this consortium have been reported at neutral pH [22], however, to the best of our knowledge, this is the first report of this consortium able to use glycerol as sole carbon source under alkaline conditions. Fig. 1 shows the start-up and operation of an alkaline aircathode MFC (A-MFC) with crude glycerol as sole carbon source. An anode from an acetate-fed alkaline air cathode MFC [23] was used as inoculum. Preliminary batch experiments with glycerol showed that this anodic consortium could use glycerol despite it had been fed with only acetate for more than two months. The fact that the A-MFC was previously inoculated with biomass from an anaerobic digester could explain this instantaneous acclimation to glycerol. The intensity obtained under these conditions (i.e. raw glycerol as sole carbon source and alkaline conditions, pH ¼ 9.3) was between 1.5 and 2 mA (3.75 and 5 A/m3 or 1.875 and 2.5 mA/ m2) during all the experimental period, which was more than two-fold higher than that obtained with the same cell under neutral conditions [45]. Rago et al. [23] already observed that alkaline conditions in the same experimental BES set-up could lead to higher current intensity values. Two cycles were monitored to understand the fate of glycerol and the potential fermentation products (Fig. 1, down). A first cycle under conventional A-MFC operation showed that glycerol was rapidly consumed in less than two days and that part of this glycerol was transformed into
17207
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 4 ( 2 0 1 9 ) 1 7 2 0 4 e1 7 2 1 3
2.5
A Intensity (mA)
2.0 1.5 1.0 0.5 0.0 20
40
Time (d) 60
1.4 Closed circuit
0.8
8
0.6
7
0.4 6
0.2 0.0
5 0
2
4 Time (d)
6
8
Concentration (g/L)
1.0
120 10
Open circuit
C
1.2
9
pH
Concentration (g/L)
1.2
100
1.4
10
B
80
9
1.0 0.8
8
0.6
7
pH
0
0.4 6
0.2 0.0
5 0
2
4
6
8
Time (d)
Fig. 1 e A): Evolution of current intensity for the whole experimental period of A-MFC with crude glycerol as sole carbon source. Vertical lines show the points of glycerol addition. DOWN: Evolution of glycerol and potential fermentation products under closed (B) and open (C) circuit conditions. Glycerol (C), acetic acid (B), propionic acid (;), butyric acid (△) and pH (–).
volatile fatty acids (mainly acetic and propionic acids). The same experiment conducted under open-circuit conditions showed very similar results in terms of glycerol degradation and VFA accumulation indicating that glycerol fermentation was not the limiting step. Thus, VFA consumption by exoelectrogens was the limiting step and should be enhanced for a higher cell performance. Moreover, direct glycerol utilization by exoelectrogens could be neglected since glycerol degradation did not increase when closing the circuit. VFA degradation was also observed under open circuit conditions, which could be attributed to anaerobic activity or to aerobic degradation with oxygen leakages from the cathode. Thus, there existed a competition between exoelectrogens and VFAdegraders under closed circuit conditions. The occurrence of this competition agrees with the observed low CE values (18 ± 8%): around of the 80% of the electrons contained in the initial substrate were not transferred from the anode to the cathode. Hence, a possibility to enhance bioelectrochemical glycerol degradation was to use a different set-up where exoelectrogens were favored against anaerobic VFAdegraders. The widely used cube-shaped MFC, B-MFC [19,46], seemed a reasonable alternative. These cells are smaller, the anode is placed closer to the cathode and they are much more efficient in terms of CE. Thus, a small brush was placed in the A-MFC for two weeks in view of its inoculation. Then, it was moved to a B-MFC and fed with raw glycerol. Fig. 2 shows the experimental evolution of this cell during almost two months. As can be observed, the cell performance was higher than that
observed in A-MFC. Similar absolute current intensity levels were attained (i.e. around 2 mA) despite the volume was one order of magnitude lower, and hence much higher volumetric intensity was obtained, i.e. 71.2 A/m3 (2.5 mA/m2) in B-MFC vs 3.75e5 A/m3 in A-MFC. In addition, much higher CE was obtained in B-MFC (55 ± 17%) vs A-MFC (18 ± 8%). Table 1 compares the results obtained in this work with similar works using the same experimental set-up with glycerol and acetate under neutral and alkaline conditions. As can be observed, alkaline conditions usually lead to higher CE and higher intensities with respect to those working at neutral pH [23]. On the other side, the use of a more complex substrate, in this case glycerol, also results in slightly lower CEs when compared to acetate since part of the initial electrons contained in the complex substrate are lost in the first fermentation steps. Fig. 3 shows the CVs conducted during the B-MFC operation to gain more insight on the process. The intensity obtained with glycerol is only 20% higher than that with acetate suggesting that glycerol fermentation may lead to VFA other than acetate that could be consumed simultaneously by the anodic consortia. Fermentation does not seem the limiting step and, thus, actions aiming at enhancing the bioelectrochemical usage of acetate are necessary to upgrade the cell performance. Propionate shows very low intensity since it is hardly produced as intermediary in the process. Kiely et al. [47] already showed that acetate is a preferred electron donor for exoelectrogens when compared to propionate.
17208
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 4 ( 2 0 1 9 ) 1 7 2 0 4 e1 7 2 1 3
Fig. 2 e Current intensity for the whole experimental period of B-MFC with crude glycerol as sole carbon source under alkaline conditions.
Start-up and operation of an alkaline glycerol-MEC The B-MFC was then converted to B-MEC to assess the potential bioelectrochemical alkaline hydrogen production with glycerol as sole carbon source. Fig. 4 shows the evolution of the current intensity in the B-MEC during the whole experimental period. The current intensity varied between 1.5 and 2 mA under different applied voltages. CE increased with respect to that obtained under MFC conditions up to 66 ± 22%. Obtaining lower CE values under MFC conditions is probably linked to some oxygen diffusion in the anodic chamber. Another possibility for the increased CE is that some hydrogen was recycled from the cathode to the anode since the MEC was operating as a single chamber. This hydrogen recycling would explain the high presence of acetogenic bacteria at the anode that, besides glycerol degradation, can use hydrogen as electron donor (see next section). The “fake” increase of CE due to hydrogen recycling in single-chamber MEC has already been discussed and reported in similar cells [48e52]. A twochamber system would solve this hydrogen recycling at expenses of a lower MEC performance due to higher internal resistances [48]. The highest cathodic recovery, rCAT, obtained was 84 ± 7% in the last cycles. These high values indicate that most of the electrons flowing from the anode to the cathode could be recovered as hydrogen. These percentages are close to the theoretically 100% of rCAT provided that there is not hydrogen scavenging in the cathode. In any case, small hydrogen losses could explain the rCAT obtained. With respect to the energy
recovery indexes, rE was 110 ± 14% indicating the amount of hydrogen obtained contains more energy than that used to produce it. However, if we consider the combustion heat of the substrate (rEþS), this value decreases to 40 ± 12%, indicating that the amount of hydrogen production needs to be increased to have a net substrate valorization. Fig. 4 also shows the amount of hydrogen produced in each cycle, which
Table 1 e Comparison of intensity and CE for the same Cube-MFC (REXT ¼ 220U) fed with acetate and glycerol under alkaline and neutral conditions. pH
Carbon source
Intensity (mA/m2 cathode)
CE (%)
Ref
Neutral Neutral Alkaline Alkaline
Acetate Glycerol Acetate Glycerol
2.14 1.42e2.14a 2.14 2.14e2.84
47 35 60 55
[23,45] [22] [23] This work
a
REXT ¼ 100U.
Fig. 3 e Turnover CVs as function of cell (up) and anode (down) potential with different substrates: glycerol (solid), acetic acid (dash-dotted) and propionic (dashed).
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 4 ( 2 0 1 9 ) 1 7 2 0 4 e1 7 2 1 3
17209
Fig. 4 e Current intensity (solid) and hydrogen production (C) for the whole experimental period of B-MEC. Dotted bars indicate the end of each cycle.
showed some variability. In this sense, the highest hydrogen production was 0.46 L H2/L/d which is in the range of similar works using the same cube-shaped MECs [31,42]. However, the value is lower than that reported under alkaline conditions in the same set-up with acetate as sole carbon source [23]. Both this observation and the electrochemical measurements conducted under MFC mode suggest that exoelectrogenic activity is the limiting step of the cell configuration used in the present work and, thus, enhancing anodic exoelectrogenesis would lead to higher hydrogen production values.
High-throughput sequencing and microbial community analysis In order to go in depth in the knowledge of the bacterial community developed in the reactors to help explain their behaviour, DNA was extracted from the biofilms sampled and 16S sequencing analysis were performed from both anodes during MFC and MEC operation. Rarefaction curves of sequencing data showed gentle slopes under the obtained sequencing depth. This fact indicates that the sequencing libraries could correctly reflect the microbial community diversity. Alpha-diversity analysis showed that both richness and diversity of Bacteria had similar values at B-MEC and BMFC anode samples (Table 2). The results of the Illumina 16S rRNA gene amplicon sequencing showed also many similarities between both samples, with only a slight shift in anodic communities’ composition in B-MFC and B-MEC samples (Fig. 5). Most bacteria detected in both anodes belonged to the Enterococcaceae family, accounting for 45.4 and 40.9% at BMFC and B-MEC, respectively. Among them, 36.6 and 32.4% (at B-MFC and B-MEC) were affiliated to the genus Enterococcus.
Table 2 e Characteristics of sequencing libraries. Sample B-MFC B-MEC
Nr of aligned sequences
Number of OTUs
Chao 1 value
Shannon Index
44720 17031
493 413
687 594
2.83 3.09
Enterococcus spp. are gram positive bacteria and facultative aerobes, belonging to the so-called lactic acid bacteria group. Their usual habitat is the digestive system of mammals, and some species are even opportunistic human pathogens. However, their presence and persistence in secondary habitats, such as surface waters, not only as a signal of faecal contamination but as a regular habitat, has been reviewed recently by some authors [53]. Enterococcus faecalis has even been reported in some bioelectrochemical systems [54]. In fact, some studies revealed its capacity to transfer electrons derived from the fermentation process directly and indirectly to an electrode [55]. On the other hand, Enterococcus spp., being an acid lactic bacteria, can be commonly found in acidic environments. It might be surprising, thus, to find them in alkaline systems of the present study. However, some previous works already pointed out their ability to survive in alkaline environments up to pH 10 [56]. Moreover, local acidic environments in the anode could favour their growth. Therefore, the role of Enterococcus sp. might be both the fermentation of glycerol and the transfer of electrons to the anode through its exoelectrogenic activity. The next main bacterial families found in B-MFC and BMEC anodes in terms of relative abundance were Eubacteriaceae and Geobacteraceae families: 9.8% each one at B-MFC and 16.6% and 9.9%, respectively at B-MEC. All members of both families are strict anaerobes [57,58], which confirms that no oxygen reached the anode biofilm (or at least some sections of it) under the studied conditions. Thus, any possible aerobic consumption of glycerol due to cathode leaks, occurred either in the bulk liquid phase or in the external layer of the biofilm (for example by the abovementioned Enterococcus sp). Geoalkalibacter sp. accounts for 9.2 and 9.8% at B-MFC and B-MEC, respectively. It belongs to the Geobacteraceae family (Desulfuromonadales class), which was already studied for its exoelectrogenic ability in pure culture [29,30,59] and it was also isolated in mixed culture alkaline bioelectrochemical reactors [23,27]. Thus, its main role might be extracellular electron transfer from fermentation products (i.e. acetate) to the anode. Its relative abundance remained constant when the bioelectrochemical system was switched from MFC to MEC. Acetobacterium sp. was the genus that exhibited higher changes in their relative abundance from B-MFC to B-MEC.
17210
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 4 ( 2 0 1 9 ) 1 7 2 0 4 e1 7 2 1 3
Fig. 5 e Family representation of Illumina 16S rRNA gene amplicon sequencing results of anodic biofilms samples of B-MFC and B-MEC. Sequences accounting for less than 1% of the total reads have been included in the “Other unclassified” category.
Specifically, it doubled its percentage, from 8.2% in B-MFC to 16.2% in B-MEC. Affiliated to the Eubacteriaceae family (Clostridiales class), it is a well-known homoacetogenic bacteria [60], which means that can produce acetate from hydrogen and carbon dioxide. It was already demonstrated to be capable to grow with glycerol as sole carbon and energy source and to produce then acetic acid [61]. Moreover, Acetobacterium genus was previously found in the anodic samples producing acetic acid, then used by exoelectrogenic bacteria in bioelectrochemical systems fed with different substrates (e.g. methanol [48]). Thus, Acetobacterium might be responsible of acetic acid production that was then used, for example, by Geoalkalibacter sp., to generate electrons. The increase of Acetobacterium sp. at B-MEC could be due to the higher production of hydrogen (in the cathode) in the MEC, which was singlechamber configuration. Alkalibacter genus (3% in B-MFC and 1% in B-MEC), also in the Eubacteriaceae family, is strictly anaerobic and an alkaliphilic genus of Firmicutes phylum [62]. In a previous study [23], Alkalibacter genus was enriched in similar reactors and its exoelectrogenic activity was supposed but not fully demonstrated. Also other bacteria from Desulfuromonadaceae family (approximately 5% of unclassified in both B-MFC and B-MEC) were often found in BES with an important role in exoelectrogenesis [63e65]. Some bacteria from the Alcaligenaceae family, which accounted for 0.2% and 2.7% of total bacteria at B-MFC and BMEC anode biofilm, respectively, have been reported to have extracellular electron transfer capacity as well. In particular, Alcaligenes faecalis was reported to generate electricity at a posed potential of 0.23 V vs. SHE [66]. Unclassified Clostridiales (2.2% in B-MFC and 4.6% in B-MEC) and unclassified Firmicutes (1.7% in B-MFC and 2.7% in B-MEC), represented also an important amount of the sequences. Many genera and species from Clostridiales order and from Firmicutes phylum, have showed exoelectrogenic activity [63,67,68]. In summary, almost all the bacterial families identified in the anode biofilm of B-MFC and B-MEC have previously shown exoelectrogenic activity. Therefore, we can conclude that we achieved a highly adapted biofilm to the dual activity of
glycerol fermentation and external electron transfer to the anode.
Conclusions This work evaluates, for the first time, the use of crude glycerol as sole carbon source in alkaline BES. This would provide a valorization opportunity for glycerol, an undesired byproduct of alkaline biodiesel production with low commercial value. Promising results have been reported with alkaline BES: current intensities higher than those found under neutral pH conditions. This work shows a successful glycerol degradation in an alkaline MFC with intensities similar to those obtained in the same systems with acetate (2 mA, 71.4 mA/m3 and 2.84 mA/m2 and a CE of 55%). Hydrogen production in a MEC was also feasible and with performance values close to those obtained with acetate (0.46 LH2/L/d and 85% of rCAT) indicating, for the first time, the suitability of this waste in being transformed to hydrogen. Finally, a deep analysis on the microbiota found in the anode indicated that the MEC was performing successfully due to a consortium of glycerol fermenters and exoelectrogens mainly composed of Enterococcaceae and Geoalkalibacter, respectively.
Acknowledgements This study was financed by the project VALTEC13-1-0140 granted by ACCIO (Generalitat de Catalunya). The authors are members of the GENOCOV group (Grup de Recerca Consolidat de la Generalitat de Catalunya, 2017 SGR 1175). Laura Rago is grateful for the FPI grant BES-2011-051308 from the Spanish Ministerio de Economı´a y Competitividad.
Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.ijhydene.2019.03.193.
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 4 ( 2 0 1 9 ) 1 7 2 0 4 e1 7 2 1 3
references
[1] Atabani AE, Silitonga AS, Badruddin IA, Mahlia TMI, Masjuki HH, Mekhilef S. A comprehensive review on biodiesel as an alternative energy resource and its characteristics. Renew Sustain Energy Rev 2012;16:2070e93. https://doi.org/10.1016/j.rser.2012.01.003. [2] Zhang Y. Biodiesel production from waste cooking oil: 1. Process design and technological assessment. Bioresour Technol 2003;89:1e16. https://doi.org/10.1016/S0960-8524(03) 00040-3. [3] Christopher LP, Kumar Hemanathan, Zambare VP. Enzymatic biodiesel: challenges and opportunities. Appl Energy 2014;119:497e520. https://doi.org/10.1016/ j.apenergy.2014.01.017. [4] Quispe CAG, Coronado CJR, Carvalho Jr JA. Glycerol: production, consumption, prices, characterization and new trends in combustion. Renew Sustain Energy Rev 2013;27:475e93. https://doi.org/10.1016/j.rser.2013.06.017. [5] Thompson J, He B. Characterization of crude glycerol from biodiesel production from multiple feedstocks. Appl Eng Agric 2006;22:261e5. [6] Werpy T, Petersen G. Top value added chemicals from biomass volume Idresults of screening for potential candidates from sugars and synthesis gas. 2004. [7] Qing Y, Lu H, Liu Y, Liu C, Liang B, Jiang W. Production of glycerol carbonate using crude glycerol from biodiesel production with DBU as a catalyst. Chin J Chem Eng 2018;26:1912e9. https://doi.org/10.1016/j.cjche.2018.01.010. [8] Adhikari S, Fernando SD, Haryanto A. Hydrogen production from glycerin by steam reforming over nickel catalysts. Renew Energy 2008;33:1097e100. https://doi.org/10.1016/ j.renene.2007.09.005. [9] Schultz E, Tatiane de Souza D, Caramez Triches Damaso M. The glycerol biorefinery: a purpose for Brazilian biodiesel production. Chem Biol Technol Agric 2014;1. https://doi.org/ 10.1186/s40538-014-0007-z. [10] Dimitratos Nikolaos, Villa Alberto, Carine E, Chan-Thaw CE, Prati L. Valorisation of glycerol to fine chemicals and fuels. In: Petrochemical catal. Mater. Process. Emerg. Technol. Igiglobal; 2016. p. 352e84. [11] Lin Y-C. Catalytic valorization of glycerol to hydrogen and syngas. Int J Hydrogen Energy 2013;38:2678e700. https://doi. org/10.1016/j.ijhydene.2012.12.079. [12] Patcharavorachot Y, Chatrattanawet N, Arpornwichanop A, Assabumrungrat S. Optimization of hydrogen production from three reforming approaches of glycerol via using supercritical water with in situ CO2 separation. Int J Hydrogen Energy 2019;44:2128e40. https://doi.org/10.1016/j. ijhydene.2018.07.053. [13] Rodrigues CV, Santana KO, Nespeca MG, Eduardo de Oliveira J, Maintinguer SI. Crude glycerol by transesterification process from used cooking oils: characterization and potentialities on hydrogen bioproduction. Int J Hydrogen Energy 2016;41:14641e51. https://doi.org/10.1016/j.ijhydene.2016.06.209. [14] Mangayil R, Karp M, Santala V. Bioconversion of crude glycerol from biodiesel production to hydrogen. Int J Hydrogen Energy 2012;37:12198e204. https://doi.org/10.1016/ j.ijhydene.2012.06.010. [15] Rossi DM, Berne da Costa J, Aquino de Souza E, Peralba M, do chia Ayub MA. Comparison of different CR, Samios D, Za pretreatment methods for hydrogen production using environmental microbial consortia on residual glycerol from biodiesel. Int J Hydrogen Energy 2011;36:4814e9. https://doi. org/10.1016/j.ijhydene.2011.01.005.
17211
[16] Sarma SJ, Brar SK, Sydney EB, Le Bihan Y, Buelna G, Soccol CR. Microbial hydrogen production by bioconversion of crude glycerol: a review. Int J Hydrogen Energy 2012;37:6473e90. https://doi.org/10.1016/j.ijhydene.2012.01.050. [17] Choi W. Glycerol-based biorefinery for fuels and chemicals. Recent Pat Biotechnol 2008;2:173e80. [18] Fukuda H, Kondo A, Noda H. Biodiesel fuel production by transesterification of oils. J Biosci Bioeng 2001;92:405e16. https://doi.org/10.1016/S1389-1723(01)80288-7. [19] Logan BE. Microbial fuel cells. 2008. [20] Cheng S, Logan BE. High hydrogen production rate of microbial electrolysis cell (MEC) with reduced electrode spacing. Bioresour Technol 2011;102:3571e4. https://doi.org/ 10.1016/j.biortech.2010.10.025. [21] Logan BE, Rabaey K. Conversion of wastes into bioelectricity and chemicals by using microbial electrochemical technologies. Science 2012;337:686e90. https://doi.org/ 10.1126/science.1217412. [22] Montpart N, Rago L, Baeza JA, Guisasola A. Hydrogen production in single chamber microbial electrolysis cells with different complex substrates. Water Res 2015;68:601e15. https://doi.org/10.1016/j.watres.2014.10.026. [23] Rago L, Baeza JA, Guisasola A. Increased performance of hydrogen production in microbial electrolysis cells under alkaline conditions. Bioelectrochemistry 2016;109:57e62. https://doi.org/10.1016/j.bioelechem.2016.01.003. [24] Zhang E, Zhai W, Luo Y, Scott K, Wang X, Diao G. Acclimatization of microbial consortia to alkaline conditions and enhanced electricity generation. Bioresour Technol 2016;211:736e42. https://doi.org/10.1016/j.biortech.2016.03.115. [25] Weerasinghe Mohottige TN, Ginige MP, Kaksonen AH, Sarukkalige R, Cheng KY. Rapid start-up of a bioelectrochemical system under alkaline and saline conditions for efficient oxalate removal. Bioresour Technol 2018;250:317e27. https://doi.org/10.1016/j.biortech.2017.11. 009. [26] Mohottige TNW, Ginige MP, Kaksonen AH, Sarukkalige R, Cheng KY. Bioelectrochemical oxidation of organics by alkalihalotolerant anodophilic biofilm under nitrogen-deficient, alkaline and saline conditions. Bioresour Technol 2017;245:890e8. https://doi.org/10.1016/j.biortech.2017.08.157. [27] Miceli JF, Parameswaran P, Kang DW, Krajmalnik-Brown R, Torres CI. Enrichment and analysis of anode-respiring bacteria from diverse anaerobic inocula. Environ Sci Technol 2012;46:10349e55. https://doi.org/10.1021/es301902h. [28] Carmona-Martı´nez A a, Pierra M, Trably E, Bernet N. High current density via direct electron transfer by the halophilic anode respiring bacterium Geoalkalibacter subterraneus. Phys Chem Chem Phys 2013;15:19699e707. https://doi.org/ 10.1039/c3cp54045f. [29] Pierra M, Carmona-Martı´nez AA, Trably E, Godon JJ, Bernet N. Specific and efficient electrochemical selection of Geoalkalibacter subterraneus and Desulfuromonas acetoxidans in high current-producing biofilms. Bioelectrochemistry 2015;106:221e5. https://doi.org/10.1016/ j.bioelechem.2015.02.003. [30] Badalamenti JP, Krajmalnik-brown R, Torres CI. Generation of high current densities by pure cultures of anode- respiring geoalkalibacter spp . under alkaline and saline conditions. mBio 2013;4:1e8. https://doi.org/10.1128/mBio.00144-13. [31] Torres CI, Kato Marcus A, Rittmann BE. Proton transport inside the biofilm limits electrical current generation by anode-respiring bacteria. Biotechnol Bioeng 2008;100:872e81. https://doi.org/10.1002/bit.21821. [32] Torres CI, Lee HS, Rittmann BE. Carbonate species as OHcarriers for decreasing the pH gradient between cathode and anode in biological fuel cells. Environ Sci Technol 2008;42:8773e7. https://doi.org/10.1021/es8019353.
17212
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 4 ( 2 0 1 9 ) 1 7 2 0 4 e1 7 2 1 3
[33] Guimaraes AQ, Linares JJ. Glycerol utilization in microbial fuel cells: conditioning stage and influence of the glycerol concentration. J Electrochem Soc 2013;161:F125e32. https:// doi.org/10.1149/2.068401jes. [34] Nimje VR, Chen C-Y, Chen C-C, Chen H-R, Tseng M-J, Jean JS, et al. Glycerol degradation in single-chamber microbial fuel cells. Bioresour Technol 2011;102:2629e34. https:// doi.org/10.1016/j.biortech.2010.10.062. [35] Reiche A, Kirkwood KM. Comparison of Escherichia coli and anaerobic consortia derived from compost as anodic biocatalysts in a glycerol-oxidizing microbial fuel cell. Bioresour Technol 2012;123:318e23. https://doi.org/10.1016/j. biortech.2012.07.005. n a, Go mez X, Guiot SR, [36] Escapa a, Manuel M-F, Mora Tartakovsky B. Hydrogen production from glycerol in a membraneless microbial electrolysis cell. Energy & Fuels 2009;23:4612e8. https://doi.org/10.1021/ef900357y. [37] Montpart N, Baeza JA, Guisasola A. From methanol to electricity and hydrogen through bioelectrochemical systems. 2017. https://doi.org/10.1016/B978-0-444-63903-5.00012-1. [38] Feng Y, Yang Q, Wang X, Liu Y, Lee H, Ren N. Treatment of biodiesel production wastes with simultaneous electricity generation using a single-chamber microbial fuel cell. Bioresour Technol 2011;102:411e5. https://doi.org/10.1016/j. biortech.2010.05.059. [39] Santos JBC, Barros VVS de, Linares JJ. Influence of sludge age on the performance of a cyclically fed microbial fuel cell using glycerol from biodiesel. Fuel Cells 2018;18:771e81. https://doi.org/10.1002/fuce.201800089. [40] Sharma Y, Parnas R, Li B. Bioenergy production from glycerol in hydrogen producing bioreactors (HPBs) and microbial fuel cells (MFCs). Int J Hydrogen Energy 2011;36:3853e61. https:// doi.org/10.1016/j.ijhydene.2010.12.040. [41] Ruiz Y, Baeza JA, Guisasola A. Enhanced performance of bioelectrochemical hydrogen production using a pH control strategy. ChemSusChem 2015;8:389e97. https://doi.org/ 10.1002/cssc.201403083. [42] Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ, et al. The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res 2009;37:D141e5. https://doi.org/10.1093/nar/gkn879. [43] Cole JR, Wang Q, Fish JA, Chai B, McGarrell DM, Sun Y, et al. Ribosomal Database Project: data and tools for high throughput rRNA analysis. Nucleic Acids Res 2014;42:D633e42. https://doi.org/10.1093/nar/gkt1244. [44] Montpart N, Rago L, Baeza JA, Guisasola A. Hydrogen production in single chamber microbial electrolysis cells with different complex substrates. Water Res 2015;68:601e15. [45] Ruiz Y, Baeza JA, Guisasola A. Microbial electrolysis cell performance using non-buffered and low conductivity wastewaters. Chem Eng J 2016;289. https://doi.org/10.1016/ j.cej.2015.12.098. [46] Logan BE. Scaling up microbial fuel cells and other bioelectrochemical systems. Appl Microbiol Biotechnol 2010;85:1665e71. https://doi.org/10.1007/s00253-009-2378-9. [47] Kiely PD, Regan JM, Logan BE. The electric picnic: synergistic requirements for exoelectrogenic microbial communities. Curr Opin Biotechnol 2011;22:378e85. https://doi.org/10.1016/ j.copbio.2011.03.003. [48] Montpart N, Ribot-Llobet E, Garlapati VK, Rago L, Baeza JA, Guisasola A. Methanol opportunities for electricity and hydrogen production in bioelectrochemical systems. Int J Hydrogen Energy 2014;39:770e7. https://doi.org/10.1016/ j.ijhydene.2013.10.151. [49] Xafenias N, Mapelli V. Performance and bacterial enrichment of bioelectrochemical systems during methane and acetate production. Int J Hydrogen Energy 2014;39:21864e75. https:// doi.org/10.1016/j.ijhydene.2014.05.038.
[50] Parameswaran P, Torres CI, Kang D-W, Rittmann BE, Krajmalnik-Brown R. The role of homoacetogenic bacteria as efficient hydrogen scavengers in microbial electrochemical cells (MXCs). Water Sci Technol 2012;65:1e6. https://doi.org/ 10.2166/wst.2011.519. s P. Microbial [51] Rago L, Ruiz Y, Baeza JA, Guisasola A, Corte community analysis in a long-term membrane-less microbial electrolysis cell with hydrogen and methane production. Bioelectrochemistry 2015;106:359e68. https:// doi.org/10.1016/j.bioelechem.2015.06.003. [52] Ruiz Y, Baeza JA, Guisasola A. Revealing the proliferation of hydrogen scavengers in a single-chamber microbial electrolysis cell using electron balances. Int J Hydrogen Energy 2013;38:15917e27. https://doi.org/10.1016/ j.ijhydene.2013.10.034. [53] Staley C, Dunny GM, Sadowsky MJ. Chapter four environmental and animal-associated enterococci. 2014. p. 147e86. https://doi.org/10.1016/b978-0-12-8002612.00004-9. [54] Read ST, Dutta P, Bond PL, Keller J, Rabaey K. Initial development and structure of biofilms on microbial fuel cell anodes. BMC Microbiol 2010;10:98. https://doi.org/10.1186/ 1471-2180-10-98. [55] Pankratova G, Leech D, Gorton L, Hederstedt L. Extracellular electron transfer by the gram-positive bacterium Enterococcus faecalis. Biochemistry 2018;57:4597e603. https://doi.org/10.1021/acs.biochem.8b00600. [56] Ran S, Liu B, Jiang W, Sun Z, Liang J. Transcriptome analysis of Enterococcus faecalis in response to alkaline stress. Front Microbiol 2015;6:795. https://doi.org/10.3389/ fmicb.2015.00795. [57] Ludwig W, Schleifer K, Whitman W. Eubacteriaceae fam. nov.. 2015. https://doi.org/10.1002/9781118960608.fbm00130. [58] Garrity G,A, Bell J, Lilburn T. Geobacteraceae fam. nov.. 2015. https://doi.org/10.1002/9781118960608.fbm00202. [59] Yoho RA, Popat SC, Rago L, Guisasola A, Torres CI. Anode biofilms of geoalkalibacter ferrihydriticus exhibit electrochemical signatures of multiple electron transport pathways. Langmuir 2015;31:12552e9. https://doi.org/ 10.1021/acs.langmuir.5b02953. [60] Drake H. Acetogenesis. New York - London: Chapman and Hall; 1994. [61] Emde R, Schink B. Fermentation of triacetin and glycerol by Acetobacterium sp. No energy is conserved by acetate excretion. Arch Microbiol 1987;149:142e8. https://doi.org/ 10.1007/BF00425080. [62] Garnova ES, Zhilina TN, Tourova TP, Kostrikina NA, Zavarzin GA. Anaerobic, alkaliphilic, saccharolytic bacterium Alkalibacter saccharofermentans gen. nov., sp. nov. from a soda lake in the Transbaikal region of Russia. Extremophiles 2004;8:309e16. https://doi.org/10.1007/ s00792-004-0390-7. [63] Koch C, Harnisch F. Is there a specific ecological niche for electroactive microorganisms? ChemElectroChem 2016;3:1282e95. https://doi.org/10.1002/celc.201600079. [64] Rago L, Cristiani P, Villa F, Zecchin S, Colombo A, Cavalca L, et al. Influences of dissolved oxygen concentration on biocathodic microbial communities in microbial fuel cells. Bioelectrochemistry 2017;116:39e51. https://doi.org/10.1016/ j.bioelechem.2017.04.001. [65] Heidrich ES, Edwards SR, Dolfing J, Cotterill SE, Curtis TP. Performance of a pilot scale microbial electrolysis cell fed on domestic wastewater at ambient temperatures for a 12month period. Bioresour Technol 2014;173:87e95. https:// doi.org/10.1016/j.biortech.2014.09.083. [66] Yu L, Yuan Y, Rensing C, Zhou S. Combined spectroelectrochemical and proteomic characterizations of bidirectional Alcaligenes faecalis-electrode electron transfer.
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 4 ( 2 0 1 9 ) 1 7 2 0 4 e1 7 2 1 3
Biosens Bioelectron 2018;106:21e8. https://doi.org/10.1016/j. bios.2018.01.032. [67] Park HS, Kim BH, Kim HS, Kim HJ, Kim G, Kim M, et al. A novel electrochemically active and Fe ( III ) -reducing bacterium phylogenetically related to Clostridium butyricum
17213
isolated from a microbial fuel cell. Scanning 2001;7:297e306. https://doi.org/10.1006/anae.2001.0399. [68] Logan BE. Exoelectrogenic bacteria that power microbial fuel cells. Nat Rev Microbiol 2009;7:375e81. https://doi.org/ 10.1038/nrmicro2113.