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High efficient biohydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis under thermophilic condition
international journal of hydrogen energy xxx (xxxx) xxx
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High efficient biohydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis under thermophilic condition Peerawat Khongkliang a, Aminee Jehlee a, Prawit Kongjan b, Alissara Reungsang c,e, Sompong O-Thong a,d,* a Biotechnology Program, Department of Biology, Faculty of Science, Thaksin University, Phatthalung, 93210, Thailand b Chemistry Division, Department of Science, Faculty of Science and Technology, Prince of Songkla University (PSU), Muang, Pattani, 94000, Thailand c Department of Biotechnology, Faculty of Technology, Khon Kaen University, Khon Kaen, 40002, Thailand d Research Center in Energy and Environment, Faculty of Science, Thaksin University, Phatthalung, 93210, Thailand e Research Group for Development of Microbial Hydrogen Production Process from Biomass, Khon Kaen University, Khon Kaen, 40002, Thailand
highlights The two-stage fermentation and microbial electrolysis were investigated for biohydrogen production. H2 yield of 236 mL-H2$gCOD1 was achieved from two-stage biohydrogen processes. COD removal of 86% was achieved from two-stage biohydrogen processes. The overall energy yield of 4.48 kJ$gCOD1 was achieved in the two-stage biohydrogen processes. The hydrogen yield was 3 times increased when compared with dark fermentation alone.
article info
abstract
Article history:
Biohydrogen production from palm oil mill effluent by two-stage dark fermentation and
Received 29 June 2019
microbial electrolysis was investigated under thermophilic condition. The optimum
Received in revised form
chemical oxygen demand (COD) concentration and pH for dark fermentation were 66 g$L1
23 September 2019
and 6.5 with a hydrogen yield of 73 mL-H2$gCOD1. The dark fermentation effluent con-
Accepted 3 October 2019
sisted of mainly acetate and butyrate. The optimum voltage for microbial electrolysis was
Available online xxx
0.7 V with a hydrogen yield of 163 mL-H2$gCOD1. The hydrogen yield of continuous twostage dark fermentation and microbial electrolysis was 236 mL-H2$gCOD1 with a hydrogen
Keywords:
production rate of 7.81 L$L1$d1. The hydrogen yield was 3 times increased when
Palm oil mill effluent
compared with dark fermentation alone. Thermoanaerobacterium sp. was dominated in the
Biohydrogen
dark fermentation stage while Geobacter sp. and Desulfovibrio sp. dominated in the micro-
Dark fermentation
bial electrolysis cell stage. Two-stage dark fermentation and microbial electrolysis under
Microbial electrolysis cell
* Corresponding author. Biotechnology Program, Department of Biology, Faculty of Science, Thaksin University, Phatthalung, 93210, Thailand. E-mail address: [email protected] (S. O-Thong). https://doi.org/10.1016/j.ijhydene.2019.10.022 0360-3199/© 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved. Please cite this article as: Khongkliang P et al., High efficient biohydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis under thermophilic condition, International Journal of Hydrogen Energy, https://doi.org/ 10.1016/j.ijhydene.2019.10.022
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international journal of hydrogen energy xxx (xxxx) xxx
Thermophilic condition
thermophilic condition is a highly promising option to maximize the conversion of palm oil
Two-stage process
mill effluent into biohydrogen. © 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
Introduction Hydrogen production from organic waste has a high attraction in terms of environmentally friendly and carbon dioxideneutral fuel. Hydrogen production from renewable resources via dark fermentation is an intensive study in recent years [1]. Dark fermentation has the potential to convert low-cost residues or organic waste and wastewater to hydrogen [2]. Hydrogen production from wastewater and organic waste through fermentation is a promising commercially feasible method in term of low technical complexity and high hydrogen production rate [3]. Various real-world substrates such as starch processing wastewater, food waste, paper sludge hydrolysate, wheat straw hydrolysate, and palm oil mill wastewater showed a high potential for hydrogen gas production by the dark fermentation process [4]. Palm oil mill effluent is a proper feedstock for thermophilic hydrogen fermentation with a high hydrogen production rate of 16.9 mmol-H2$L1$h1 and huge volume availability [5]. Hydrogen yields could be enhanced through the acetate pathway and decreasing or preventing butyrate, ethanol, and propionate pathways by operating under thermophilic condition [4,6]. The hydrogen yields in a range of 2.5e3.7 mol-H2$mol1C6 was achieved by thermophiles (Clostridium thermocellum and Thermoanaerobacterium thermosaccharolyticum) [4]. Dark thermophilic fermentation for H2 production from palm oil mill effluent can yield 5-7 L-H2$L1 POME and 100-150 mL-H2$gVS1 by Thermoanaerobacterium-rich sludge [7]. However, only the hydrogen yield of 2.4e3 mol-H2 mol1C6 achieved from real substrates or wastewater via fermentation. Only 15% of energy-containing in the substrate is recovered as hydrogen gas by the dark fermentation process. The unrecovered energy remained in the soluble fermentation products such as acetic acid, ethanol, lactic acid, butyric acid, and propionic acid [8]. Thus, further conversion of volatile fatty acids (VFAs) in hydrogen fermentation effluent is needed to maximize the conversion efficiency of wastewater and organic waste into hydrogen [9]. The hydrogen yields also improve by a combined process of dark fermentation with microbial electrolysis cells (MEC) [10e13]. Lu et al. [10] reported that an overall hydrogen recovery of 96% of the maximum theoretical yield of 0.125 g H2$gCOD1 was obtained by the combined process. Lalaurette et al. [11] showed an overall hydrogen yield of 9.95 mol-H2$mol1glucose from cellobiose as feedstock by the combined process. Wang et al. [12] also reported a 41% increase in overall hydrogen yield (9.1 mmol-H2$gCOD1 to 33.2 mmol-H2$gCOD1) from cellulose by the combined process. It is apparent to improve hydrogen yield from synthetic wastewater using the combined dark fermentation and microbial electrolysis, but lower hydrogen yield for the complex organic waste and wastewater [13e15]. Low hydrogen yield of complex organic waste was occurred in
MECs stage due to low butyric acid degradation [14]. Liu et al. [14] tested the integrated biohydrogen process using pretreated waste activated sludge as feedstock achieving hydrogen yield of 10 mmol-H2$g1VSS with 90% of acetate removal, but only 21e55% of n-butyrate removal. Li et al. [13] reported a hydrogen yield of 15 mmol-H2$g1 from corn stalk with 81e91% acetate removal. However, butyrate and propionate removals were as small as 4e16%. The thermophilic fermentation is energetically more favorable for hydrogen production via the acetate pathway and enhanced the hydrolysis rate of the substrate than mesophilic fermentation [16]. Thus, operating two-stage dark fermentation process and microbial electrolysis under thermophilic condition could be enhanced hydrogen yields through the acetate pathway and decreasing or preventing butyrate. To date, only a few studies have dealt with hydrogen production in the dark fermentation process coupled with microbial electrolysis under thermophilic condition. The hydrogen production from POME by two-stage dark fermentation with microbial electrolysis under thermophilic condition has not been reported. The aim of this research was to evaluate the condition for hydrogen production from POME by coupling dark fermentation and microbial electrolysis under thermophilic condition. The optimum COD concentration, initial pH, and applied voltage, and feasibility of continuous two-stage dark fermentation and microbial electrolysis for hydrogen production from POME were also investigated.
Materials and methods Palm oil mill effluent and inoculums POME was collected from palm oil extraction process at Univanich Palm Oil Co., Ltd., Krabi, Thailand. The chemical
Table 1 e Chemical characteristic of palm oil mill effluent used in this study. Parameter pH Temperature ( C) Chemical oxygen demand (g$L1) Total carbohydrate (g$L1) Total solids (g$L1) Volatile solids (g$L1) Total nitrogen (g$L1) Total acids (mg$L1) Alkalinity (mg-CaCO3$L1) Oil (g$L1) Acetic acid (mg$L1) Butyric acid (mg$L1) Propionic acid (mg$L1)
POME 4.49 ± 0.04 79 ± 0.82 82.91 ± 0.95 19.44 ± 0.66 54.92 ± 0.08 45.48 ± 0.05 1.90 ± 0.02 450.68 1825 ± 106 9.5 ± 0.1 139.48 197.31 86.56
Please cite this article as: Khongkliang P et al., High efficient biohydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis under thermophilic condition, International Journal of Hydrogen Energy, https://doi.org/ 10.1016/j.ijhydene.2019.10.022
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and physical composition of POME was shown in Table 1. The anaerobic microbial sludge was taken from a POME based biogas plant. The anaerobic microbial sludge was enriched according to Mamimin et al. [17] for thermophilic dark fermentation inoculum. The bacteria inoculum for MEC was enriched from peatland soil, according to Khongkliang et al. [18]. The inoculum for dark fermentation and microbial electrolysis was enriched with the basal anaerobic medium (BA medium). The composition of the BA medium was contained of (g$L1) sucrose 2.0, yeast extract 1.0; NH4NO3 1.0; KH2PO4 1.0; K2HPO4 1.0; MgSO4$7H2O 0.2; FeCl$6H2O 0.05; CaCl2 0.02 and resazurin 0.5 mg$L1 in 1 L deionized water [18]. The pH of the medium was adjusted to 6.5 by adding 1 M HCl or NaHCO3. The medium was flushed with N2 gas for 3e5 min to obtain completely anaerobic conditions.
Batch hydrogen production by thermophilic dark fermentation and MEC A batch H2 production by dark fermentation was carried out in 200 mL working volume. The various initial COD loadings of 83, 74, 66, 58, 50, and 41 g$L1 at an initial pH of 6.0 was investigated for H2 production at a temperature of 55 C. The optimum COD loading of 66 g$L1 was investigated for the effect of initial pH of 5.50, 5.75, 6.0, 6.25, 6.50, 6.75, and 7.0 on H2 production by dark fermentation. POME used as a substrate and inoculated with dark fermentation inoculum at food to microorganism ratio (F/M ratio) of 4:1 based on VS basis. All serum bottles were flushed with N2 gas for 3e5 min to remove the oxygen and sealed with rubber stoppers and aluminum cap. The serum bottles were placed in an incubator at a temperature of 55 C. During the experiments, the H2 gas content in biogas was measured at regular interval time. The volume of biogas production was measured through the use of the water replacement method. The dark fermentation effluent was analyzed for pH, chemical oxygen demand (COD), total solids (TS), volatile solids (VS), and VFAs concentrations [19]. Single-chambers membrane-less MEC was carried out in glass bottles with working volumes of 70 mL [18]. A MEC consisted of a graphite fiber felt as an anode (surface area 46 cm2) and carbon cloth as a cathode (surface area 30 cm2). The dark fermentation effluent at the COD loading of 66 g$L1 and initial pH of 6.50 was used as a substrate for H2 production by MEC. The initial pH was 6.0 adjusting by adding 1 M HCl or NaHCO3. The dark fermentation effluent and MEC inoculum ratio were 4:1 based on VS basis. All serum bottles were flushed with N2 gas for 3e5 min to remove the oxygen. The serum bottles were closed with rubber stoppers and aluminum cap to avoid gas leakage. The serum bottles were incubated at a temperature of 55 C. The MEC system was applied voltage by connecting to the power supply via copper wires. The effect of applied voltages (0.2e0.9 V) to the MEC system on hydrogen production was investigated. The biogas production was measured through the use of the water replacement method. After the H2 production ceased, the pH, COD, TS, VS, and VFAs concentrations were analyzed.
3
Continuous hydrogen production by integrated thermophilic dark fermentation and MEC Fig. 1 illustrates the two-stage dark fermentation and MEC for H2 production. The two-stage reactor consisted of a mixing tank, up-flow anaerobic sludge blanket (UASB) reactor for dark fermentation, fermentation effluent tank, and up-flow membrane-less MEC reactor for microbial electrolysis. The first reactor was double jacket glass UASB with a total capacity of 240 mL and working volume 200 mL. The second reactor was double jacket glass up-flow membrane-less MEC with a graphite fiber felt as an anode (40 mm diameter, 1.5 mm height, 138 cm2 surface area) and carbon cloth as a cathode (40 mm diameter, 0.5 mm height, 113 cm2 surface area 30 cm2). The MEC reactor was connected with the DC power supply via copper wires for voltage supply [18]. The MEC reactor volume was 1000 mL with a working volume of 600 mL. The anode biofilms were cultivated for 60 days before the experiment. The reactor was closed tightly by silicone stoppers to avoid gas leakage. The reactor was equipped with a water bath for temperature control at 55 C by circulating hot water within both reactors. Inoculum for dark fermentation process was taken from batch cultivation in serum bottles. POME was adjusted pH to 6.5 ± 3 before feeding into a dark fermentation reactor. The dark fermentation reactor was operated at a hydraulic retention time (HRT) of 2 days. The dark fermentation effluent was fed to the MEC reactor by a peristaltic pump. The MEC reactor was operated at HRT of 6 days. MEC reactor has applied a voltage of 0.7 V. The continuous operation was achieved stability stage when the H2 production variation less than 10%. The two-stage UASB and up-flow membrane-less MEC reactor were monitored for the biogas composition and biogas volume. The effluents from UASB and up-flow membrane-less MEC reactors were sampled every 2 days to determine pH, COD, TS, VS, and VFAs concentrations. Microbial community responsible for hydrogen production from both reactors was collected from the steady stage and analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) techniques.
Analytical methods and calculations The biogas compositions were monitored by gas chromatography connected with Packed Column (Shin-Carbon ST100/ 120, 2 m 1 mm, Restex) and thermal conductivity detector (GC-TCD, Shimadzu 8A), as previously described by Mamimin et al. [17]. Soluble metabolites were analyzed by gas chromatography connected with Crossbond Carbowax Polyethylene Glycol Column (Stabilwax-DA, 30 m 0.53 mm, Restex) and flame ionization detector (GC-FID, Shimadzu 17A) as previously described by Hniman et al. [20]. The COD, TS, VS, and pH were determined in accordance with the procedures described in the APHA [21]. The total carbohydrate in POME and effluent was analyzed by the Anthrone method [22]. The microbial community in two-stage dark fermentation and microbial electrolysis was analyzed by PCR-DGGE techniques, as previously described by Kongjan et al. [23]. The partial 16s rDNA gene sequences were identified by the ribosomal database project (RDP) and the national center for biotechnology
Please cite this article as: Khongkliang P et al., High efficient biohydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis under thermophilic condition, International Journal of Hydrogen Energy, https://doi.org/ 10.1016/j.ijhydene.2019.10.022
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Fig. 1 e Schematic description for H2 production by two-stage dark fermentation and MEC. information (NCBI). The energy yield and energy recovery of integrated two-stage thermophilic fermentation and MEC were calculated on the basis of electricity input as equation (1). The hydrogen energy recovery based on combustion energy is WH2 ¼ hH2 DHH2 , where hH2 are the moles of hydrogen production. TheDHH2 is the energy content of hydrogen (285.83 kJ mol1). The Win is the electricity input calculated from cp (the total coulombs) divided by Eap (the applied voltage) [24]. The cumulative hydrogen yield, maximum hydrogen potential, the lag-phase was fitted from the modified Gompertz equation (2) [25]. hw ¼ WH2 Win
(1)
Rm ðl tÞe þ 1 Ht ¼ Pmax exp exp p
(2)
Results and discussion Hydrogen production by the thermophilic fermentation The H2 yield of initial COD loading of 83, 74, 66, 58, 50, and 41 g$L1 was 47, 50, 56, 40, 27, and 31 mL-H2$gCOD1, respectively (Table 2). The maximum H2 production was 2.73 L$L1 obtaining at initial COD loading of 83 g$L1 with the H2 production rate of 5.8 mL$gCOD1$h1 (Fig. 2a). The lag-phase increased from 1.2 to 6.5 h when increased initial COD loading, suggesting that microorganisms required more time
for adapting to the higher initial COD loading of POME for H2 production [26]. The H2 production of 0.89e2.73 L$L1 with an H2 production rate of 1.39e2.33 mL$gCOD1$d1 was obtained from the dark fermentation stage. Further increase of initial COD loading, increasing hydrogen production, but not improved H2 yield and H2 production rate. The initial COD loading of 66 g$L1 was selected as the optimum for H2 production with efficient substrate conversion. The optimal initial COD loading of POME for dark fermentation stage was inlined with Mamimin et al. [27] reported that the maximum H2 production of 4.9 L$L1 was obtained from POME at COD loading 65 g$L1. The accumulation of VFAs products at high initial COD loading (74 and 83 g$L1) was over-acidification resulting in inhibition of the fermentation process. The measurement of VFAs showed that 3.51 g$L1 of total VFAs were detected at the initial COD loading of 83 g$L1, while only 1.40 g$L1 of VFAs were detected at the initial COD loading of 41 g$L1. The distributions of VFAs during dark fermentation were similar in all initial COD loading. Butyrate was the main soluble metabolites (37e63%) with followed by acetate (29e51%). The fermentation effluent was composed of propionate, iso-butyrate, iso-valerate, and valerate as a minor soluble metabolites. High VFAs concentration has negative affect on H2 production. The VFAs concentration above 2.5 g$L1 could inhibit hydrogen-producing bacteria [28]. Argun et al. [29] reported that the high initial COD loading above 20 g$L1 shifted the bacterial metabolism towards VFAs formation rather than hydrogen formation.
Please cite this article as: Khongkliang P et al., High efficient biohydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis under thermophilic condition, International Journal of Hydrogen Energy, https://doi.org/ 10.1016/j.ijhydene.2019.10.022
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Table 2 e Hydrogen production from POME by dark fermentation at various initial COD loading. Initial COD (g L1)
83 74 66 58 50 41
l (h)
Rmax (mL-H2$gCOD1$d1)
H2 yield (mL-H2$gCOD1)
3.5 2.1 2.3 1.9 1.2 1.4
5.8 6.4 6.9 5.3 2.6 2.5
47 50 56 40 27 31
Soluble metabolites (g$L1)
COD removal (%)
HAc
HPr
HBu
1.31 1.11 1.14 1.11 0.85 0.89
0.22 0.11 0.09 0.06 0.06 0.07
1.56 1.46 1.35 1.09 0.67 0.41
25 28 31 22 17 15
Cumulative H2 production (L·L-1)
HAc; Acetate, HPr; Propionate, HBu; Butyrate.
3 41
a
50
58
66
74
83
2
1
0
Cumulative H2 production (L·L-1)
0
1
2
4
3
4
5 6 Time (day)
5.50 6.50
b
5.75 6.75
7
8
6.00 7.00
9
10
6.25
3
2
1
0 0
1
2
3
4
5 6 Time (days)
7
8
9
10
Fig. 2 e Cumulative hydrogen production from palm oil mill effluent by dark fermentation at various initial COD loading(a) and initial pH (b).
The H2 yield from an initial pH of 5.50, 5.75, 6.0, 6.25, 6.50, 6.75, and 7.0 at the initial COD loading of 66 g$L1 was 3, 50, 54, 57, 71, 50, and 43 mL-H2$gCOD1, respectively (Table 3). The highest cumulative of H2 production and yield was 3.28 L$L1 and 71 mL-H2$gCOD1 at the initial pH of 6.50. While the initial pH value below 5.50 resulted in negligible H2 production (Fig. 2b). The thermophilic hydrogen-producing bacteria, Thermoanaerobacterium thermosaccharolyticum has an optimal
pH of 5.5e6.5 [30]. Xu et al. [31] also reported that at pH lower than 5.5 could be affected on the ability of the microbial cells to maintain of the internal pH, which resulted in the quantity of intracellular adenosine triphosphate (ATP) and inhibit the substrate uptake. The short lag-phase of 3.6 h was observed at an optimum pH of 6.5. Lag-phase for fermentative H2 production shorter than previously reported with sucrose and POME as substrate (10.1 h) [32]. The H2 production and soluble
Please cite this article as: Khongkliang P et al., High efficient biohydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis under thermophilic condition, International Journal of Hydrogen Energy, https://doi.org/ 10.1016/j.ijhydene.2019.10.022
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Table 3 e Hydrogen production from POME by dark fermentation of various initial pH. Initial pH
5.50 5.75 6.00 6.25 6.50 6.75 7.00
Final pH
l (h)
Rmax (mLH2$gCOD1$d1)
H2 yield (mL-H2 $gCOD1)
4.28 4.34 4.46 4.73 5.47 5.53 5.84
7.4 6.4 4.9 4.6 3.6 3.6 3.8
0.7 5.6 5.2 6.3 7.6 6.9 3.9
3 50 54 57 71 50 43
Soluble metabolites (g$L1) HAr
HPr
HBu
0.12 1.12 1.26 1.26 1.31 1.48 1.75
0.04 0.11 0.10 0.12 0.18 0.32 0.37
0.32 1.88 2.19 2.23 2.64 2.64 2.44
COD removal (%)
1 22 24 26 32 22 19
HAc; Acetate, HPr; Propionate, HBu; Butyrate.
metabolite of various pH were varied depending on pH. The pH of 6.0e6.5 was identified as a suitable pH for H2 production from POME. The metabolic pathway was shifted from butyrate to ethanol, when pH lower than 5.0, resulting in an unfavorable impact on H2 production [33]. Overall, the pH range of 5.5e6.5 was related to high H2 production under the thermophilic condition [34]. A mixture of acetate and butyrate was dominated in soluble metabolites at an optimum pH of 6.5. The butyrate concentration at an initial pH of 6.25e6.75 was while acetate concentration was 2.23e2.64 g$L1, 1.26e1.48 g$L1 (Table 3). The soluble metabolites show that the metabolic pathway of the hydrogen-producing bacteria was sensitive to the pH leading to the importance of pH controlling during reactor operation for H2 production [35].
Hydrogen production by a microbial electrolysis The dark fermentative effluent at the optimum of initial COD loading of 66 g$L1 and initial pH 6.50 was used for H2 production by MEC. H2 yield from applied voltages of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9 V was 25, 68, 102, 114, 116, 134, 76, and 69 mL-H2$gCOD1, respectively (Table 4). The H2 production increased at the applied voltage of 0.2e0.7 V, but hydrogen production was decreased when the applied voltage was over 0.8 V (Fig. 3). Table 4 summarizes the kinetic parameters of H2 production by MEC at various applied voltage. Lag-phase for H2 production by MEC was less than 24 h when applied voltage was higher than 0.5 V. While the lag-phase was higher than 24 h at an applied voltage lower than 0.5 V. The maximum H2
production and H2 yields were 4.43 L$L1 and 134 mL-H2$gCOD1, respectively at the applied voltage of 0.7 V. Khongkliang et al. [18] reported that the optimum of applied voltage for hydrogen production from cassava starch processing wastewater was 0.6 V with H2 yield of 245 mL-H2$gCOD1. The protons releasing from the decomposition of the substrate was reacted with electrons at an anode to form H2 gas. These data demonstrated that excessive applied voltage could restrain the activities of exoelectrogenic bacteria and negative effect on the electron transfer of exoelectrogenic bacteria. In addition, the electrode material could play an important role in the conductivity. The discharging and size of the porosity of the electrode could improve hydrogen production [36,37]. Wang et al. [38] reported that the energy loss was high at high voltage applied due to the high current resulting in more internal resistance voltage of MEC system. A voltage lower than 0.7 V, the electrons provided was not enough to neutralize a huge number of protons released from the microbial decomposition [39]. The consumption of butyrate and acetate was high for all voltages applied. Since acetic acid is the easiest utilized carbon source by electron transfer bacteria for produced hydrogen, resulting in high consumption of acetic acid among others VFAs [40]. The utilization efficiency of acetate, butyrate, propionate, valerate, iso-butyrate, and iso-valerate in MEC was 10e19%, 14e32%, 2e19%, 3e17%, 2e17%, and 4e11%, respectively. The maximum VFAs utilization was 29% at 0.7 V. The COD removal efficiency was enhanced when increased applied voltage from 0.2 V to 0.7 V. The COD removal efficiency was decreased at a high voltage applied of
Table 4 e Hydrogen production from dark fermentation effluent at various applied voltage by microbial electrolysis cell. Applied voltage
l (h)
0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
57.7 43.1 29.9 23.4 21.6 21.9 16.9 15.9
Rmax (mL-H2$gCOD1 d1)
H2 yield (mLH2$gCOD1)
COD removal (%)
3.0 9.8 17.6 16.7 17.6 22.7 10.0 10.5
25 68 102 114 116 134 76 69
9 25 37 42 43 49 28 26
Utilization (%) HAr
HPr
HBu
11 10 15 15 17 19 19 17
2 16 12 19 16 16 15 12
14 20 21 27 29 30 32 31
Energy recovery (%) 89 239 357 402 409 471 267 243
HAc; Acetate, HPr; Propionate, HBu; Butyrate.
Please cite this article as: Khongkliang P et al., High efficient biohydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis under thermophilic condition, International Journal of Hydrogen Energy, https://doi.org/ 10.1016/j.ijhydene.2019.10.022
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Cumulative H2 production (L·L-1)
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5 0.2 V 0.3 V 0.4 V 0.5 V 0.6 V 0.7 V 0.8 V 0.9 V
4 3 2 1 0 0
1
2
3 4 Time (days)
5
6
7
Fig. 3 e Cumulative hydrogen production from dark fermentation effluent by MEC at various applied voltage. 0.9 V. The maximum energy recovery was 471% at the applied voltage of 0.7 V. The energy recovery was higher than 100%, indicating that the energy obtained from the systems was higher than the energy input. Lu et al. [10] reported that the energy recovery of 287% was achieved from MEC feeding with ethanol effluent as a substrate at an applied voltage of 0.6 V. The applied voltage of 0.2e0.6 V has an energy input of 0.098 kWh$me3 to 1.39 kWh$me3, which are lower than that those types of water electrolysis for H2 production (4e5 kWh$me3) [41].
Continuous hydrogen production by two-stage dark fermentation and MEC The H2 production from POME by two-stage dark fermentation and MEC under thermophilic condition were shown in Fig. 4. The maximum H2 production rate from dark fermentation stage was 2.89 L$L1$d1 and corresponding to the COD removal efficiency of 23 ± 5%. The average H2 production rate was 2.43 ± 0.3 L$L1$d1 (Table 5) with an average hydrogen
concentration of 47 ± 2%. The H2 content obtained is similar to other studies conducted for H2 production using POME as a substrate under thermophilic temperature. Ottaviano et al. [42] observed the average hydrogen composition of 45% in biogas from the dark fermentation process. The thermophilic temperature most likely produces higher H2 yields and H2 production rate due to the elimination of bacteria consuming H2 and selection of more efficient H2 producing bacteria [43]. The distribution of VFAs after dark fermentation was shown in Fig. 5a. The fermentation effluent from dark fermentation reactor was mainly composed of acetate (1.86 ± 0.2 g$L1), and butyrate (2.66 ± 0.2 g$L1) with account for 95% of the total VFAs in soluble metabolites. The propionate, iso-butyrate, isovalerate and valerate has a little amount in soluble metabolites. The organic was converted to H2 via acetate and butyrate production with a theoretical H2 yield of 4 and 2 mol-H2$mol1C6, respectively [44]. Another soluble metabolites such as lactate, propionate, and ethanol was produced without hydrogen production [45]. VFAs production was found to increase along with operation time with an average
H2 production rate (L-H2·L-1·d-1)
7 6 5 4 3 2 1
Dark fermentation
Microbial electrolysis
0 0
10
20
30 Time (days)
40
50
60
Fig. 4 e Continuous hydrogen production from palm oil mill effluent by two-stage dark fermentation and MEC. Please cite this article as: Khongkliang P et al., High efficient biohydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis under thermophilic condition, International Journal of Hydrogen Energy, https://doi.org/ 10.1016/j.ijhydene.2019.10.022
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Table 5 e Process performance and effluent characteristics of two-stage dark fermentation and microbial electrolysis cell. Dark fermentation
Microbial electrolysis
Two-stage process
2 6.61 ± 0.1 32 ± 1 2.43 ± 0.3 73 ± 8 1.39 23 ± 5 e 5.75 ± 0.2 1.86 ± 0.2 2.66 ± 0.2 0.16 ± 0.03 0.02 ± 0.01
6 6.10 ± 0.1 10 ± 0.4 5.38 ± 0.5 163 ± 16 3.09 63 ± 7 37 ± 18 6.18 ± 0.1 0.93 ± 0.3 1.86 ± 0.3 0.09 ± 0.04 0.02 ± 0.01
8 6.61 ± 0.1, 6.10 ± 0.1 32 ± 1 7.81 236 4.48 86 37 ± 18 6.18 ± 0.1 0.93 ± 0.3 1.86 ± 0.3 0.09 ± 0.04 0.02 ± 0.01
Acetic Butyric Total VFA
a 5
Propionic Isovaleric
8
Isobutyric Valeric
7 6
4
5
3
4 3
2
2 1
1
0
0 0
10
VFAs concentration (g·L-1)
6
20
30
Acetic Butyric Total VFA
b 5 4
40
Propionic Isovaleric
50
60 8
Isobutyric Valeric
7 6 5
3
4 3
2
2 1
Total VFA (g·L-1)
VFAs concentration (g·L-1)
6
Total VFA (g·L-1)
Parameters HRT (days) pH (initial) OLR (g$L1$d1) Gas production (L$L1$d1) H2 yield (mL-H2$gCOD1) Energy yield (kJ$gCOD1) COD removal (%) VFAs utilization (%) pH Acetate (g$L1) Butyrate (g$L1) Propionate (g$L1) Valerate (g$L1)
1
0
0 0
10
20
30 Time (days)
40
50
60
Fig. 5 e Profiles of volatile fatty acids from the dark fermentation stage (a) and MEC stage (b) of hydrogen production from palm oil mill effluent by two-stage dark fermentation and MEC.
concentration of 4.77 ± 0.4 g$L1. The pH in the reactor decreased from 6.61 ± 0.1 to 5.75 ± 0.2 during the operation due to the accumulation of VFAs, while H2 production and H2 yield are fluctuations not more than 10%. The pH in the reactor has an impact on the growth of microbial and the reactor pH should maintain above the inhibitory levels of pH 4.5 [46]. The MEC reactor was operated at HRT 6 day for H2 production from the first stage effluent. The fermentation effluent was rich in VFAs (range of 3.96e5.44 g$L1) served as a good substrate for H2 production by MEC under an applied voltage 0.7 V. The H2 production from the MEC reactor was
shown in Fig. 4. During the initial operation period, bacteria were not able to utilize VFAs from dark fermentation effluent leading to low H2 production rate. Thereafter, the bacteria were gradually adapted to the VFAs in dark fermentation effluent and started to utilizing the VFAs for hydrogen production. The maximum H2 production rate from MEC reactor was 6.49 L$L1$d1. The average H2 production rate was 5.38 L$L1$d1, with an average H2 concentration of 44 ± 3%. Fig. 5b shows the VFAs consumption during 60 days of operation for continuous hydrogen production by MEC reactor. Acetate and butyrate concentration sharply decreased from 1.86 ± 0.2 and 2.66 ± 0.2 g$L1 to 0.93 ± 0.3, and 1.86 ± 0.3 g$L1
Please cite this article as: Khongkliang P et al., High efficient biohydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis under thermophilic condition, International Journal of Hydrogen Energy, https://doi.org/ 10.1016/j.ijhydene.2019.10.022
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Table 6 e COD balance of hydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis cell. COD distribution
COD (mgCOD$L1)
First stage COD input H2 production Acetate Butyrate Propionate Valerate Isobutyrate Isovalerate Cell mass Complex COD in dark fermentation effluent Balance Second stage COD input H2 production Acetate Butyrate Propionate Valerate Isobutyrate Isovalerate Cell mass Complex COD of MEC effluent Balance
82911 19412 2407 5566 236 243 144 39 8291 36337
COD distribution (%) 100 23.41 2.90 6.71 0.28 0.29 0.17 0.05 10.00 43.83 12.35
61183 43060 987 3391 147 32 77 71 6118 10823
100 70.38 1.61 5.54 0.24 0.05 0.13 0.12 10.00 17.69 5.76
but propionate, iso-butyrate, iso-valerate, and valerate concentration did not change significantly. Propionate utilization rate was relatively slower than acetate and butyrate. Acetate was highly utilized by the microbial consortium in MEC due to its simpler form than propionate. The hydrogen production in MEC obtained from the reduction of Hþ with e from applied voltage [47]. The VFAs utilization of 37 ± 18% was achieved from MEC reactor. The result indicated that the acetate was the main substrate at an anode for H2 production in the MEC reactor. An acetate containing two carbon atoms could more easily electron transfers than butyrate, which containing four carbon atoms [13]. The hydrogen production from POME by two-stage dark fermentation and MEC was 3 times higher than dark fermentation alone and comparable with other
organic waste via two-stage dark fermentation and MEC (Table 7). The H2 yield and H2 production rate from POME by two-stage dark fermentation and MEC were 236 mL-H2$gCOD1 and 7.81 L$L1$d1, respectively. The H2 yield from corn stalk, cassava starch wastewater, crude glycerol and sugar wastewater by two-stage dark fermentation and MEC was 387, 465, 149, and 396 mL-H2$gCOD1, respectively [13,18,48,49]. Li et al. [13] reported that the H2 production rate of 5.16 L$L1$d1 was obtained from corn stalk at 20 g$L1 as substrate by two-stage dark fermentation and MEC. The COD removal efficiency of two-stage dark fermentation and MEC was 86%. Two-stage dark fermentation and MEC reactor have the potential to increase the H2 yield gone beyond the current maximum of 4 mol-H2$mol1C6 and address technical barriers that currently limit the techno-economic feasibility of fermentative H2 production. The integrated thermophilic fermentative with MEC reactor could be a very high potential method as it has a practical highest H2 yield of 8e10 mol-H2$mol1C6. It is likely that the hot wastewater like palm oil mill effluent will initially prove most attractive as substrates for H2 production at thermophilic conditions. The remaining major challenge is to determine whether the economics and reliability of integrated thermophilic fermentative with MEC reactor for hydrogen production are sufficiently attractive for commercial production. The high efficiency for hydrogen production of the two-stage dark fermentation and MEC was evaluated in terms of energy output (Table 5). The total energy output from the dark fermentation of POME (first stage) was 1.39 kJ$gCOD1, while from the second stage by MEC was 3.09 kJ$gCOD1. The COD balance of the two-stage dark fermentation and MEC process was shown in Table 6. 23.41% of COD in POME was removed in the first stage. Fermentation effluent with a contained high amount of VFAs could be utilized as a substrate for H2 production in MEC. 70.38% of overall COD was converting to hydrogen gas. The unidentified COD in the two-stage dark fermentation and MEC was 12.35% and 5.75%, respectively.
Microbial community of two-stage dark fermentation and MEC The bacterial community structure of the two-stage dark fermentation and MEC process was shown in Fig. 6. The dark fermentation stage (Fig. 6a) was dominated by
Table 7 e Comparison of hydrogen yields from the various substrate by two-stage dark fermentation and MEC. Substrate
Dark fermentation Type Yield (mLreactor H2$gCOD1)
Corn stalk Cassava starch wastewater Crude glycerol Sugar wastewater Palm oil mill effluent Palm oil mill effluent
Microbial electrogenesis Type reactor
Applied Yield (mLvoltage (V) H2$gCOD1)
COD removal (%)
Overall Yield References (mL-H2$gCOD1)
CSTR UASB
130 260
Single chamber Up-flow membrane less
0.8 0.6
257 205
44 70
387 465
[13] [18]
Batch Batch
43 52
Two-chamber Two-chamber
1.0 0.2
106 344
50 67
149 396
[48] [49]
Batch
71
Single chamber
0.7
134
81
205
This study
UASB
73
Up-flow membrane less
0.7
163
86
236
This study
Please cite this article as: Khongkliang P et al., High efficient biohydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis under thermophilic condition, International Journal of Hydrogen Energy, https://doi.org/ 10.1016/j.ijhydene.2019.10.022
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Fig. 6 e DGGE profile of bacteria community in the sludge from dark fermentation stage (a) and MEC stage (b) of hydrogen production from palm oil mill effluent by two-stage dark fermentation and MEC. Thermoanaerobacterium sp., Thermoanaerobacterium thermosaccharolyticum, and Thermoanaerobacterium aciditolerans. Thermoanaerobacter brockii was dominant during the 25 days and gradually decreased when operating in a long time, while Clostridium sp. was gradually increased of overtime. Clostridium sp. and Thermoanaerobacterium sp. was commonly found in the hydrogen production reactor feeding with POME operating under thermophilic condition [5,50] Prasertsan et al. [5] reported that Thermoanaerobacterium sp. was dominated in the hydrogen production reactor feeding with POME at 60 C. The optimum temperature and pH for growth of Thermoanaerobacterium sp. was 55e60 C and 5.2e6.5, respectively [51]. The dominant bacteria in the second-stage microbial electrolysis are mainly dominated by Geobacter sp., Desulfovibrio sp. and Thermoanaerobacterium thermosaccharolyticum. Besides, the bacterial community in second-stage were changed when operating for a long time. Shewanalla sp. was the dominant population during the first 25 days, while Geobacillus sp. was more prominent in a long time of operation. The Geobacillus sp. and Desulfovibrio sp. were dominated at an anode biofilm with the ability to produce hydrogen as exoelectrogens [52]. Geobacter sp. and Shewanalla sp. has been well reported as electrochemically active bacteria able to utilize acetate as an electron donor. The dominant of Geobacter sp. on the anode electrode, indicating that this bacterial played a significant role in the electron generation [53]. Desulfovibrio sp. has electrons transfer cytochromes and can form biofilms on
electrodes for harvesting electrons for hydrogen formation. Furthermore, Desulfovibrio sp. has been shown to interact with a cathode to facilitate hydrogen production [54].
Conclusions The two-stage dark fermentation and microbial electrolysis operating under thermophilic condition is a highly promising option to maximize the conversion of POME into biohydrogen. The optimum COD concentration and pH by dark fermentation were 66 g$L1 and 6.5, respectively. The optimum applied voltage for hydrogen production from dark fermentation effluent by MEC was of 0.7 V. The hydrogen yield of continuous two-stage dark fermentation and MEC was 236 mL-H2$gCOD1 with a hydrogen production rate of 7.81 L$L1$d1. The hydrogen yield was 3 times increased when compared with dark fermentation alone.
Acknowledgments The authors would like to thank the Research and Development Institute Thaksin University, Thailand through the graduate research fund (Grant No. 04-4/2559), Thailand Science Reserach and Inovation, Thailand; Thailand Research Fund, Thailand through the Research and Researcher for
Please cite this article as: Khongkliang P et al., High efficient biohydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis under thermophilic condition, International Journal of Hydrogen Energy, https://doi.org/ 10.1016/j.ijhydene.2019.10.022
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Industry (PHD60I0036), TRF Senior Research Scholar (Grant No. RTA6280001), TRF Mid-Career Research Grant (Grant No. RSA6180048) and Waste and Energy Management Co., Ltd. for financial support.
[14]
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Please cite this article as: Khongkliang P et al., High efficient biohydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis under thermophilic condition, International Journal of Hydrogen Energy, https://doi.org/ 10.1016/j.ijhydene.2019.10.022
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High efficient biohydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis under thermophilic condition Peerawat Khongkliang a, Aminee Jehlee a, Prawit Kongjan b, Alissara Reungsang c,e, Sompong O-Thong a,d,* a Biotechnology Program, Department of Biology, Faculty of Science, Thaksin University, Phatthalung, 93210, Thailand b Chemistry Division, Department of Science, Faculty of Science and Technology, Prince of Songkla University (PSU), Muang, Pattani, 94000, Thailand c Department of Biotechnology, Faculty of Technology, Khon Kaen University, Khon Kaen, 40002, Thailand d Research Center in Energy and Environment, Faculty of Science, Thaksin University, Phatthalung, 93210, Thailand e Research Group for Development of Microbial Hydrogen Production Process from Biomass, Khon Kaen University, Khon Kaen, 40002, Thailand
highlights The two-stage fermentation and microbial electrolysis were investigated for biohydrogen production. H2 yield of 236 mL-H2$gCOD1 was achieved from two-stage biohydrogen processes. COD removal of 86% was achieved from two-stage biohydrogen processes. The overall energy yield of 4.48 kJ$gCOD1 was achieved in the two-stage biohydrogen processes. The hydrogen yield was 3 times increased when compared with dark fermentation alone.
article info
abstract
Article history:
Biohydrogen production from palm oil mill effluent by two-stage dark fermentation and
Received 29 June 2019
microbial electrolysis was investigated under thermophilic condition. The optimum
Received in revised form
chemical oxygen demand (COD) concentration and pH for dark fermentation were 66 g$L1
23 September 2019
and 6.5 with a hydrogen yield of 73 mL-H2$gCOD1. The dark fermentation effluent con-
Accepted 3 October 2019
sisted of mainly acetate and butyrate. The optimum voltage for microbial electrolysis was
Available online xxx
0.7 V with a hydrogen yield of 163 mL-H2$gCOD1. The hydrogen yield of continuous twostage dark fermentation and microbial electrolysis was 236 mL-H2$gCOD1 with a hydrogen
Keywords:
production rate of 7.81 L$L1$d1. The hydrogen yield was 3 times increased when
Palm oil mill effluent
compared with dark fermentation alone. Thermoanaerobacterium sp. was dominated in the
Biohydrogen
dark fermentation stage while Geobacter sp. and Desulfovibrio sp. dominated in the micro-
Dark fermentation
bial electrolysis cell stage. Two-stage dark fermentation and microbial electrolysis under
Microbial electrolysis cell
* Corresponding author. Biotechnology Program, Department of Biology, Faculty of Science, Thaksin University, Phatthalung, 93210, Thailand. E-mail address: [email protected] (S. O-Thong). https://doi.org/10.1016/j.ijhydene.2019.10.022 0360-3199/© 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved. Please cite this article as: Khongkliang P et al., High efficient biohydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis under thermophilic condition, International Journal of Hydrogen Energy, https://doi.org/ 10.1016/j.ijhydene.2019.10.022
2
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Thermophilic condition
thermophilic condition is a highly promising option to maximize the conversion of palm oil
Two-stage process
mill effluent into biohydrogen. © 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
Introduction Hydrogen production from organic waste has a high attraction in terms of environmentally friendly and carbon dioxideneutral fuel. Hydrogen production from renewable resources via dark fermentation is an intensive study in recent years [1]. Dark fermentation has the potential to convert low-cost residues or organic waste and wastewater to hydrogen [2]. Hydrogen production from wastewater and organic waste through fermentation is a promising commercially feasible method in term of low technical complexity and high hydrogen production rate [3]. Various real-world substrates such as starch processing wastewater, food waste, paper sludge hydrolysate, wheat straw hydrolysate, and palm oil mill wastewater showed a high potential for hydrogen gas production by the dark fermentation process [4]. Palm oil mill effluent is a proper feedstock for thermophilic hydrogen fermentation with a high hydrogen production rate of 16.9 mmol-H2$L1$h1 and huge volume availability [5]. Hydrogen yields could be enhanced through the acetate pathway and decreasing or preventing butyrate, ethanol, and propionate pathways by operating under thermophilic condition [4,6]. The hydrogen yields in a range of 2.5e3.7 mol-H2$mol1C6 was achieved by thermophiles (Clostridium thermocellum and Thermoanaerobacterium thermosaccharolyticum) [4]. Dark thermophilic fermentation for H2 production from palm oil mill effluent can yield 5-7 L-H2$L1 POME and 100-150 mL-H2$gVS1 by Thermoanaerobacterium-rich sludge [7]. However, only the hydrogen yield of 2.4e3 mol-H2 mol1C6 achieved from real substrates or wastewater via fermentation. Only 15% of energy-containing in the substrate is recovered as hydrogen gas by the dark fermentation process. The unrecovered energy remained in the soluble fermentation products such as acetic acid, ethanol, lactic acid, butyric acid, and propionic acid [8]. Thus, further conversion of volatile fatty acids (VFAs) in hydrogen fermentation effluent is needed to maximize the conversion efficiency of wastewater and organic waste into hydrogen [9]. The hydrogen yields also improve by a combined process of dark fermentation with microbial electrolysis cells (MEC) [10e13]. Lu et al. [10] reported that an overall hydrogen recovery of 96% of the maximum theoretical yield of 0.125 g H2$gCOD1 was obtained by the combined process. Lalaurette et al. [11] showed an overall hydrogen yield of 9.95 mol-H2$mol1glucose from cellobiose as feedstock by the combined process. Wang et al. [12] also reported a 41% increase in overall hydrogen yield (9.1 mmol-H2$gCOD1 to 33.2 mmol-H2$gCOD1) from cellulose by the combined process. It is apparent to improve hydrogen yield from synthetic wastewater using the combined dark fermentation and microbial electrolysis, but lower hydrogen yield for the complex organic waste and wastewater [13e15]. Low hydrogen yield of complex organic waste was occurred in
MECs stage due to low butyric acid degradation [14]. Liu et al. [14] tested the integrated biohydrogen process using pretreated waste activated sludge as feedstock achieving hydrogen yield of 10 mmol-H2$g1VSS with 90% of acetate removal, but only 21e55% of n-butyrate removal. Li et al. [13] reported a hydrogen yield of 15 mmol-H2$g1 from corn stalk with 81e91% acetate removal. However, butyrate and propionate removals were as small as 4e16%. The thermophilic fermentation is energetically more favorable for hydrogen production via the acetate pathway and enhanced the hydrolysis rate of the substrate than mesophilic fermentation [16]. Thus, operating two-stage dark fermentation process and microbial electrolysis under thermophilic condition could be enhanced hydrogen yields through the acetate pathway and decreasing or preventing butyrate. To date, only a few studies have dealt with hydrogen production in the dark fermentation process coupled with microbial electrolysis under thermophilic condition. The hydrogen production from POME by two-stage dark fermentation with microbial electrolysis under thermophilic condition has not been reported. The aim of this research was to evaluate the condition for hydrogen production from POME by coupling dark fermentation and microbial electrolysis under thermophilic condition. The optimum COD concentration, initial pH, and applied voltage, and feasibility of continuous two-stage dark fermentation and microbial electrolysis for hydrogen production from POME were also investigated.
Materials and methods Palm oil mill effluent and inoculums POME was collected from palm oil extraction process at Univanich Palm Oil Co., Ltd., Krabi, Thailand. The chemical
Table 1 e Chemical characteristic of palm oil mill effluent used in this study. Parameter pH Temperature ( C) Chemical oxygen demand (g$L1) Total carbohydrate (g$L1) Total solids (g$L1) Volatile solids (g$L1) Total nitrogen (g$L1) Total acids (mg$L1) Alkalinity (mg-CaCO3$L1) Oil (g$L1) Acetic acid (mg$L1) Butyric acid (mg$L1) Propionic acid (mg$L1)
POME 4.49 ± 0.04 79 ± 0.82 82.91 ± 0.95 19.44 ± 0.66 54.92 ± 0.08 45.48 ± 0.05 1.90 ± 0.02 450.68 1825 ± 106 9.5 ± 0.1 139.48 197.31 86.56
Please cite this article as: Khongkliang P et al., High efficient biohydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis under thermophilic condition, International Journal of Hydrogen Energy, https://doi.org/ 10.1016/j.ijhydene.2019.10.022
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and physical composition of POME was shown in Table 1. The anaerobic microbial sludge was taken from a POME based biogas plant. The anaerobic microbial sludge was enriched according to Mamimin et al. [17] for thermophilic dark fermentation inoculum. The bacteria inoculum for MEC was enriched from peatland soil, according to Khongkliang et al. [18]. The inoculum for dark fermentation and microbial electrolysis was enriched with the basal anaerobic medium (BA medium). The composition of the BA medium was contained of (g$L1) sucrose 2.0, yeast extract 1.0; NH4NO3 1.0; KH2PO4 1.0; K2HPO4 1.0; MgSO4$7H2O 0.2; FeCl$6H2O 0.05; CaCl2 0.02 and resazurin 0.5 mg$L1 in 1 L deionized water [18]. The pH of the medium was adjusted to 6.5 by adding 1 M HCl or NaHCO3. The medium was flushed with N2 gas for 3e5 min to obtain completely anaerobic conditions.
Batch hydrogen production by thermophilic dark fermentation and MEC A batch H2 production by dark fermentation was carried out in 200 mL working volume. The various initial COD loadings of 83, 74, 66, 58, 50, and 41 g$L1 at an initial pH of 6.0 was investigated for H2 production at a temperature of 55 C. The optimum COD loading of 66 g$L1 was investigated for the effect of initial pH of 5.50, 5.75, 6.0, 6.25, 6.50, 6.75, and 7.0 on H2 production by dark fermentation. POME used as a substrate and inoculated with dark fermentation inoculum at food to microorganism ratio (F/M ratio) of 4:1 based on VS basis. All serum bottles were flushed with N2 gas for 3e5 min to remove the oxygen and sealed with rubber stoppers and aluminum cap. The serum bottles were placed in an incubator at a temperature of 55 C. During the experiments, the H2 gas content in biogas was measured at regular interval time. The volume of biogas production was measured through the use of the water replacement method. The dark fermentation effluent was analyzed for pH, chemical oxygen demand (COD), total solids (TS), volatile solids (VS), and VFAs concentrations [19]. Single-chambers membrane-less MEC was carried out in glass bottles with working volumes of 70 mL [18]. A MEC consisted of a graphite fiber felt as an anode (surface area 46 cm2) and carbon cloth as a cathode (surface area 30 cm2). The dark fermentation effluent at the COD loading of 66 g$L1 and initial pH of 6.50 was used as a substrate for H2 production by MEC. The initial pH was 6.0 adjusting by adding 1 M HCl or NaHCO3. The dark fermentation effluent and MEC inoculum ratio were 4:1 based on VS basis. All serum bottles were flushed with N2 gas for 3e5 min to remove the oxygen. The serum bottles were closed with rubber stoppers and aluminum cap to avoid gas leakage. The serum bottles were incubated at a temperature of 55 C. The MEC system was applied voltage by connecting to the power supply via copper wires. The effect of applied voltages (0.2e0.9 V) to the MEC system on hydrogen production was investigated. The biogas production was measured through the use of the water replacement method. After the H2 production ceased, the pH, COD, TS, VS, and VFAs concentrations were analyzed.
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Continuous hydrogen production by integrated thermophilic dark fermentation and MEC Fig. 1 illustrates the two-stage dark fermentation and MEC for H2 production. The two-stage reactor consisted of a mixing tank, up-flow anaerobic sludge blanket (UASB) reactor for dark fermentation, fermentation effluent tank, and up-flow membrane-less MEC reactor for microbial electrolysis. The first reactor was double jacket glass UASB with a total capacity of 240 mL and working volume 200 mL. The second reactor was double jacket glass up-flow membrane-less MEC with a graphite fiber felt as an anode (40 mm diameter, 1.5 mm height, 138 cm2 surface area) and carbon cloth as a cathode (40 mm diameter, 0.5 mm height, 113 cm2 surface area 30 cm2). The MEC reactor was connected with the DC power supply via copper wires for voltage supply [18]. The MEC reactor volume was 1000 mL with a working volume of 600 mL. The anode biofilms were cultivated for 60 days before the experiment. The reactor was closed tightly by silicone stoppers to avoid gas leakage. The reactor was equipped with a water bath for temperature control at 55 C by circulating hot water within both reactors. Inoculum for dark fermentation process was taken from batch cultivation in serum bottles. POME was adjusted pH to 6.5 ± 3 before feeding into a dark fermentation reactor. The dark fermentation reactor was operated at a hydraulic retention time (HRT) of 2 days. The dark fermentation effluent was fed to the MEC reactor by a peristaltic pump. The MEC reactor was operated at HRT of 6 days. MEC reactor has applied a voltage of 0.7 V. The continuous operation was achieved stability stage when the H2 production variation less than 10%. The two-stage UASB and up-flow membrane-less MEC reactor were monitored for the biogas composition and biogas volume. The effluents from UASB and up-flow membrane-less MEC reactors were sampled every 2 days to determine pH, COD, TS, VS, and VFAs concentrations. Microbial community responsible for hydrogen production from both reactors was collected from the steady stage and analyzed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) techniques.
Analytical methods and calculations The biogas compositions were monitored by gas chromatography connected with Packed Column (Shin-Carbon ST100/ 120, 2 m 1 mm, Restex) and thermal conductivity detector (GC-TCD, Shimadzu 8A), as previously described by Mamimin et al. [17]. Soluble metabolites were analyzed by gas chromatography connected with Crossbond Carbowax Polyethylene Glycol Column (Stabilwax-DA, 30 m 0.53 mm, Restex) and flame ionization detector (GC-FID, Shimadzu 17A) as previously described by Hniman et al. [20]. The COD, TS, VS, and pH were determined in accordance with the procedures described in the APHA [21]. The total carbohydrate in POME and effluent was analyzed by the Anthrone method [22]. The microbial community in two-stage dark fermentation and microbial electrolysis was analyzed by PCR-DGGE techniques, as previously described by Kongjan et al. [23]. The partial 16s rDNA gene sequences were identified by the ribosomal database project (RDP) and the national center for biotechnology
Please cite this article as: Khongkliang P et al., High efficient biohydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis under thermophilic condition, International Journal of Hydrogen Energy, https://doi.org/ 10.1016/j.ijhydene.2019.10.022
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Fig. 1 e Schematic description for H2 production by two-stage dark fermentation and MEC. information (NCBI). The energy yield and energy recovery of integrated two-stage thermophilic fermentation and MEC were calculated on the basis of electricity input as equation (1). The hydrogen energy recovery based on combustion energy is WH2 ¼ hH2 DHH2 , where hH2 are the moles of hydrogen production. TheDHH2 is the energy content of hydrogen (285.83 kJ mol1). The Win is the electricity input calculated from cp (the total coulombs) divided by Eap (the applied voltage) [24]. The cumulative hydrogen yield, maximum hydrogen potential, the lag-phase was fitted from the modified Gompertz equation (2) [25]. hw ¼ WH2 Win
(1)
Rm ðl tÞe þ 1 Ht ¼ Pmax exp exp p
(2)
Results and discussion Hydrogen production by the thermophilic fermentation The H2 yield of initial COD loading of 83, 74, 66, 58, 50, and 41 g$L1 was 47, 50, 56, 40, 27, and 31 mL-H2$gCOD1, respectively (Table 2). The maximum H2 production was 2.73 L$L1 obtaining at initial COD loading of 83 g$L1 with the H2 production rate of 5.8 mL$gCOD1$h1 (Fig. 2a). The lag-phase increased from 1.2 to 6.5 h when increased initial COD loading, suggesting that microorganisms required more time
for adapting to the higher initial COD loading of POME for H2 production [26]. The H2 production of 0.89e2.73 L$L1 with an H2 production rate of 1.39e2.33 mL$gCOD1$d1 was obtained from the dark fermentation stage. Further increase of initial COD loading, increasing hydrogen production, but not improved H2 yield and H2 production rate. The initial COD loading of 66 g$L1 was selected as the optimum for H2 production with efficient substrate conversion. The optimal initial COD loading of POME for dark fermentation stage was inlined with Mamimin et al. [27] reported that the maximum H2 production of 4.9 L$L1 was obtained from POME at COD loading 65 g$L1. The accumulation of VFAs products at high initial COD loading (74 and 83 g$L1) was over-acidification resulting in inhibition of the fermentation process. The measurement of VFAs showed that 3.51 g$L1 of total VFAs were detected at the initial COD loading of 83 g$L1, while only 1.40 g$L1 of VFAs were detected at the initial COD loading of 41 g$L1. The distributions of VFAs during dark fermentation were similar in all initial COD loading. Butyrate was the main soluble metabolites (37e63%) with followed by acetate (29e51%). The fermentation effluent was composed of propionate, iso-butyrate, iso-valerate, and valerate as a minor soluble metabolites. High VFAs concentration has negative affect on H2 production. The VFAs concentration above 2.5 g$L1 could inhibit hydrogen-producing bacteria [28]. Argun et al. [29] reported that the high initial COD loading above 20 g$L1 shifted the bacterial metabolism towards VFAs formation rather than hydrogen formation.
Please cite this article as: Khongkliang P et al., High efficient biohydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis under thermophilic condition, International Journal of Hydrogen Energy, https://doi.org/ 10.1016/j.ijhydene.2019.10.022
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Table 2 e Hydrogen production from POME by dark fermentation at various initial COD loading. Initial COD (g L1)
83 74 66 58 50 41
l (h)
Rmax (mL-H2$gCOD1$d1)
H2 yield (mL-H2$gCOD1)
3.5 2.1 2.3 1.9 1.2 1.4
5.8 6.4 6.9 5.3 2.6 2.5
47 50 56 40 27 31
Soluble metabolites (g$L1)
COD removal (%)
HAc
HPr
HBu
1.31 1.11 1.14 1.11 0.85 0.89
0.22 0.11 0.09 0.06 0.06 0.07
1.56 1.46 1.35 1.09 0.67 0.41
25 28 31 22 17 15
Cumulative H2 production (L·L-1)
HAc; Acetate, HPr; Propionate, HBu; Butyrate.
3 41
a
50
58
66
74
83
2
1
0
Cumulative H2 production (L·L-1)
0
1
2
4
3
4
5 6 Time (day)
5.50 6.50
b
5.75 6.75
7
8
6.00 7.00
9
10
6.25
3
2
1
0 0
1
2
3
4
5 6 Time (days)
7
8
9
10
Fig. 2 e Cumulative hydrogen production from palm oil mill effluent by dark fermentation at various initial COD loading(a) and initial pH (b).
The H2 yield from an initial pH of 5.50, 5.75, 6.0, 6.25, 6.50, 6.75, and 7.0 at the initial COD loading of 66 g$L1 was 3, 50, 54, 57, 71, 50, and 43 mL-H2$gCOD1, respectively (Table 3). The highest cumulative of H2 production and yield was 3.28 L$L1 and 71 mL-H2$gCOD1 at the initial pH of 6.50. While the initial pH value below 5.50 resulted in negligible H2 production (Fig. 2b). The thermophilic hydrogen-producing bacteria, Thermoanaerobacterium thermosaccharolyticum has an optimal
pH of 5.5e6.5 [30]. Xu et al. [31] also reported that at pH lower than 5.5 could be affected on the ability of the microbial cells to maintain of the internal pH, which resulted in the quantity of intracellular adenosine triphosphate (ATP) and inhibit the substrate uptake. The short lag-phase of 3.6 h was observed at an optimum pH of 6.5. Lag-phase for fermentative H2 production shorter than previously reported with sucrose and POME as substrate (10.1 h) [32]. The H2 production and soluble
Please cite this article as: Khongkliang P et al., High efficient biohydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis under thermophilic condition, International Journal of Hydrogen Energy, https://doi.org/ 10.1016/j.ijhydene.2019.10.022
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Table 3 e Hydrogen production from POME by dark fermentation of various initial pH. Initial pH
5.50 5.75 6.00 6.25 6.50 6.75 7.00
Final pH
l (h)
Rmax (mLH2$gCOD1$d1)
H2 yield (mL-H2 $gCOD1)
4.28 4.34 4.46 4.73 5.47 5.53 5.84
7.4 6.4 4.9 4.6 3.6 3.6 3.8
0.7 5.6 5.2 6.3 7.6 6.9 3.9
3 50 54 57 71 50 43
Soluble metabolites (g$L1) HAr
HPr
HBu
0.12 1.12 1.26 1.26 1.31 1.48 1.75
0.04 0.11 0.10 0.12 0.18 0.32 0.37
0.32 1.88 2.19 2.23 2.64 2.64 2.44
COD removal (%)
1 22 24 26 32 22 19
HAc; Acetate, HPr; Propionate, HBu; Butyrate.
metabolite of various pH were varied depending on pH. The pH of 6.0e6.5 was identified as a suitable pH for H2 production from POME. The metabolic pathway was shifted from butyrate to ethanol, when pH lower than 5.0, resulting in an unfavorable impact on H2 production [33]. Overall, the pH range of 5.5e6.5 was related to high H2 production under the thermophilic condition [34]. A mixture of acetate and butyrate was dominated in soluble metabolites at an optimum pH of 6.5. The butyrate concentration at an initial pH of 6.25e6.75 was while acetate concentration was 2.23e2.64 g$L1, 1.26e1.48 g$L1 (Table 3). The soluble metabolites show that the metabolic pathway of the hydrogen-producing bacteria was sensitive to the pH leading to the importance of pH controlling during reactor operation for H2 production [35].
Hydrogen production by a microbial electrolysis The dark fermentative effluent at the optimum of initial COD loading of 66 g$L1 and initial pH 6.50 was used for H2 production by MEC. H2 yield from applied voltages of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9 V was 25, 68, 102, 114, 116, 134, 76, and 69 mL-H2$gCOD1, respectively (Table 4). The H2 production increased at the applied voltage of 0.2e0.7 V, but hydrogen production was decreased when the applied voltage was over 0.8 V (Fig. 3). Table 4 summarizes the kinetic parameters of H2 production by MEC at various applied voltage. Lag-phase for H2 production by MEC was less than 24 h when applied voltage was higher than 0.5 V. While the lag-phase was higher than 24 h at an applied voltage lower than 0.5 V. The maximum H2
production and H2 yields were 4.43 L$L1 and 134 mL-H2$gCOD1, respectively at the applied voltage of 0.7 V. Khongkliang et al. [18] reported that the optimum of applied voltage for hydrogen production from cassava starch processing wastewater was 0.6 V with H2 yield of 245 mL-H2$gCOD1. The protons releasing from the decomposition of the substrate was reacted with electrons at an anode to form H2 gas. These data demonstrated that excessive applied voltage could restrain the activities of exoelectrogenic bacteria and negative effect on the electron transfer of exoelectrogenic bacteria. In addition, the electrode material could play an important role in the conductivity. The discharging and size of the porosity of the electrode could improve hydrogen production [36,37]. Wang et al. [38] reported that the energy loss was high at high voltage applied due to the high current resulting in more internal resistance voltage of MEC system. A voltage lower than 0.7 V, the electrons provided was not enough to neutralize a huge number of protons released from the microbial decomposition [39]. The consumption of butyrate and acetate was high for all voltages applied. Since acetic acid is the easiest utilized carbon source by electron transfer bacteria for produced hydrogen, resulting in high consumption of acetic acid among others VFAs [40]. The utilization efficiency of acetate, butyrate, propionate, valerate, iso-butyrate, and iso-valerate in MEC was 10e19%, 14e32%, 2e19%, 3e17%, 2e17%, and 4e11%, respectively. The maximum VFAs utilization was 29% at 0.7 V. The COD removal efficiency was enhanced when increased applied voltage from 0.2 V to 0.7 V. The COD removal efficiency was decreased at a high voltage applied of
Table 4 e Hydrogen production from dark fermentation effluent at various applied voltage by microbial electrolysis cell. Applied voltage
l (h)
0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
57.7 43.1 29.9 23.4 21.6 21.9 16.9 15.9
Rmax (mL-H2$gCOD1 d1)
H2 yield (mLH2$gCOD1)
COD removal (%)
3.0 9.8 17.6 16.7 17.6 22.7 10.0 10.5
25 68 102 114 116 134 76 69
9 25 37 42 43 49 28 26
Utilization (%) HAr
HPr
HBu
11 10 15 15 17 19 19 17
2 16 12 19 16 16 15 12
14 20 21 27 29 30 32 31
Energy recovery (%) 89 239 357 402 409 471 267 243
HAc; Acetate, HPr; Propionate, HBu; Butyrate.
Please cite this article as: Khongkliang P et al., High efficient biohydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis under thermophilic condition, International Journal of Hydrogen Energy, https://doi.org/ 10.1016/j.ijhydene.2019.10.022
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Cumulative H2 production (L·L-1)
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5 0.2 V 0.3 V 0.4 V 0.5 V 0.6 V 0.7 V 0.8 V 0.9 V
4 3 2 1 0 0
1
2
3 4 Time (days)
5
6
7
Fig. 3 e Cumulative hydrogen production from dark fermentation effluent by MEC at various applied voltage. 0.9 V. The maximum energy recovery was 471% at the applied voltage of 0.7 V. The energy recovery was higher than 100%, indicating that the energy obtained from the systems was higher than the energy input. Lu et al. [10] reported that the energy recovery of 287% was achieved from MEC feeding with ethanol effluent as a substrate at an applied voltage of 0.6 V. The applied voltage of 0.2e0.6 V has an energy input of 0.098 kWh$me3 to 1.39 kWh$me3, which are lower than that those types of water electrolysis for H2 production (4e5 kWh$me3) [41].
Continuous hydrogen production by two-stage dark fermentation and MEC The H2 production from POME by two-stage dark fermentation and MEC under thermophilic condition were shown in Fig. 4. The maximum H2 production rate from dark fermentation stage was 2.89 L$L1$d1 and corresponding to the COD removal efficiency of 23 ± 5%. The average H2 production rate was 2.43 ± 0.3 L$L1$d1 (Table 5) with an average hydrogen
concentration of 47 ± 2%. The H2 content obtained is similar to other studies conducted for H2 production using POME as a substrate under thermophilic temperature. Ottaviano et al. [42] observed the average hydrogen composition of 45% in biogas from the dark fermentation process. The thermophilic temperature most likely produces higher H2 yields and H2 production rate due to the elimination of bacteria consuming H2 and selection of more efficient H2 producing bacteria [43]. The distribution of VFAs after dark fermentation was shown in Fig. 5a. The fermentation effluent from dark fermentation reactor was mainly composed of acetate (1.86 ± 0.2 g$L1), and butyrate (2.66 ± 0.2 g$L1) with account for 95% of the total VFAs in soluble metabolites. The propionate, iso-butyrate, isovalerate and valerate has a little amount in soluble metabolites. The organic was converted to H2 via acetate and butyrate production with a theoretical H2 yield of 4 and 2 mol-H2$mol1C6, respectively [44]. Another soluble metabolites such as lactate, propionate, and ethanol was produced without hydrogen production [45]. VFAs production was found to increase along with operation time with an average
H2 production rate (L-H2·L-1·d-1)
7 6 5 4 3 2 1
Dark fermentation
Microbial electrolysis
0 0
10
20
30 Time (days)
40
50
60
Fig. 4 e Continuous hydrogen production from palm oil mill effluent by two-stage dark fermentation and MEC. Please cite this article as: Khongkliang P et al., High efficient biohydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis under thermophilic condition, International Journal of Hydrogen Energy, https://doi.org/ 10.1016/j.ijhydene.2019.10.022
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Table 5 e Process performance and effluent characteristics of two-stage dark fermentation and microbial electrolysis cell. Dark fermentation
Microbial electrolysis
Two-stage process
2 6.61 ± 0.1 32 ± 1 2.43 ± 0.3 73 ± 8 1.39 23 ± 5 e 5.75 ± 0.2 1.86 ± 0.2 2.66 ± 0.2 0.16 ± 0.03 0.02 ± 0.01
6 6.10 ± 0.1 10 ± 0.4 5.38 ± 0.5 163 ± 16 3.09 63 ± 7 37 ± 18 6.18 ± 0.1 0.93 ± 0.3 1.86 ± 0.3 0.09 ± 0.04 0.02 ± 0.01
8 6.61 ± 0.1, 6.10 ± 0.1 32 ± 1 7.81 236 4.48 86 37 ± 18 6.18 ± 0.1 0.93 ± 0.3 1.86 ± 0.3 0.09 ± 0.04 0.02 ± 0.01
Acetic Butyric Total VFA
a 5
Propionic Isovaleric
8
Isobutyric Valeric
7 6
4
5
3
4 3
2
2 1
1
0
0 0
10
VFAs concentration (g·L-1)
6
20
30
Acetic Butyric Total VFA
b 5 4
40
Propionic Isovaleric
50
60 8
Isobutyric Valeric
7 6 5
3
4 3
2
2 1
Total VFA (g·L-1)
VFAs concentration (g·L-1)
6
Total VFA (g·L-1)
Parameters HRT (days) pH (initial) OLR (g$L1$d1) Gas production (L$L1$d1) H2 yield (mL-H2$gCOD1) Energy yield (kJ$gCOD1) COD removal (%) VFAs utilization (%) pH Acetate (g$L1) Butyrate (g$L1) Propionate (g$L1) Valerate (g$L1)
1
0
0 0
10
20
30 Time (days)
40
50
60
Fig. 5 e Profiles of volatile fatty acids from the dark fermentation stage (a) and MEC stage (b) of hydrogen production from palm oil mill effluent by two-stage dark fermentation and MEC.
concentration of 4.77 ± 0.4 g$L1. The pH in the reactor decreased from 6.61 ± 0.1 to 5.75 ± 0.2 during the operation due to the accumulation of VFAs, while H2 production and H2 yield are fluctuations not more than 10%. The pH in the reactor has an impact on the growth of microbial and the reactor pH should maintain above the inhibitory levels of pH 4.5 [46]. The MEC reactor was operated at HRT 6 day for H2 production from the first stage effluent. The fermentation effluent was rich in VFAs (range of 3.96e5.44 g$L1) served as a good substrate for H2 production by MEC under an applied voltage 0.7 V. The H2 production from the MEC reactor was
shown in Fig. 4. During the initial operation period, bacteria were not able to utilize VFAs from dark fermentation effluent leading to low H2 production rate. Thereafter, the bacteria were gradually adapted to the VFAs in dark fermentation effluent and started to utilizing the VFAs for hydrogen production. The maximum H2 production rate from MEC reactor was 6.49 L$L1$d1. The average H2 production rate was 5.38 L$L1$d1, with an average H2 concentration of 44 ± 3%. Fig. 5b shows the VFAs consumption during 60 days of operation for continuous hydrogen production by MEC reactor. Acetate and butyrate concentration sharply decreased from 1.86 ± 0.2 and 2.66 ± 0.2 g$L1 to 0.93 ± 0.3, and 1.86 ± 0.3 g$L1
Please cite this article as: Khongkliang P et al., High efficient biohydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis under thermophilic condition, International Journal of Hydrogen Energy, https://doi.org/ 10.1016/j.ijhydene.2019.10.022
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Table 6 e COD balance of hydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis cell. COD distribution
COD (mgCOD$L1)
First stage COD input H2 production Acetate Butyrate Propionate Valerate Isobutyrate Isovalerate Cell mass Complex COD in dark fermentation effluent Balance Second stage COD input H2 production Acetate Butyrate Propionate Valerate Isobutyrate Isovalerate Cell mass Complex COD of MEC effluent Balance
82911 19412 2407 5566 236 243 144 39 8291 36337
COD distribution (%) 100 23.41 2.90 6.71 0.28 0.29 0.17 0.05 10.00 43.83 12.35
61183 43060 987 3391 147 32 77 71 6118 10823
100 70.38 1.61 5.54 0.24 0.05 0.13 0.12 10.00 17.69 5.76
but propionate, iso-butyrate, iso-valerate, and valerate concentration did not change significantly. Propionate utilization rate was relatively slower than acetate and butyrate. Acetate was highly utilized by the microbial consortium in MEC due to its simpler form than propionate. The hydrogen production in MEC obtained from the reduction of Hþ with e from applied voltage [47]. The VFAs utilization of 37 ± 18% was achieved from MEC reactor. The result indicated that the acetate was the main substrate at an anode for H2 production in the MEC reactor. An acetate containing two carbon atoms could more easily electron transfers than butyrate, which containing four carbon atoms [13]. The hydrogen production from POME by two-stage dark fermentation and MEC was 3 times higher than dark fermentation alone and comparable with other
organic waste via two-stage dark fermentation and MEC (Table 7). The H2 yield and H2 production rate from POME by two-stage dark fermentation and MEC were 236 mL-H2$gCOD1 and 7.81 L$L1$d1, respectively. The H2 yield from corn stalk, cassava starch wastewater, crude glycerol and sugar wastewater by two-stage dark fermentation and MEC was 387, 465, 149, and 396 mL-H2$gCOD1, respectively [13,18,48,49]. Li et al. [13] reported that the H2 production rate of 5.16 L$L1$d1 was obtained from corn stalk at 20 g$L1 as substrate by two-stage dark fermentation and MEC. The COD removal efficiency of two-stage dark fermentation and MEC was 86%. Two-stage dark fermentation and MEC reactor have the potential to increase the H2 yield gone beyond the current maximum of 4 mol-H2$mol1C6 and address technical barriers that currently limit the techno-economic feasibility of fermentative H2 production. The integrated thermophilic fermentative with MEC reactor could be a very high potential method as it has a practical highest H2 yield of 8e10 mol-H2$mol1C6. It is likely that the hot wastewater like palm oil mill effluent will initially prove most attractive as substrates for H2 production at thermophilic conditions. The remaining major challenge is to determine whether the economics and reliability of integrated thermophilic fermentative with MEC reactor for hydrogen production are sufficiently attractive for commercial production. The high efficiency for hydrogen production of the two-stage dark fermentation and MEC was evaluated in terms of energy output (Table 5). The total energy output from the dark fermentation of POME (first stage) was 1.39 kJ$gCOD1, while from the second stage by MEC was 3.09 kJ$gCOD1. The COD balance of the two-stage dark fermentation and MEC process was shown in Table 6. 23.41% of COD in POME was removed in the first stage. Fermentation effluent with a contained high amount of VFAs could be utilized as a substrate for H2 production in MEC. 70.38% of overall COD was converting to hydrogen gas. The unidentified COD in the two-stage dark fermentation and MEC was 12.35% and 5.75%, respectively.
Microbial community of two-stage dark fermentation and MEC The bacterial community structure of the two-stage dark fermentation and MEC process was shown in Fig. 6. The dark fermentation stage (Fig. 6a) was dominated by
Table 7 e Comparison of hydrogen yields from the various substrate by two-stage dark fermentation and MEC. Substrate
Dark fermentation Type Yield (mLreactor H2$gCOD1)
Corn stalk Cassava starch wastewater Crude glycerol Sugar wastewater Palm oil mill effluent Palm oil mill effluent
Microbial electrogenesis Type reactor
Applied Yield (mLvoltage (V) H2$gCOD1)
COD removal (%)
Overall Yield References (mL-H2$gCOD1)
CSTR UASB
130 260
Single chamber Up-flow membrane less
0.8 0.6
257 205
44 70
387 465
[13] [18]
Batch Batch
43 52
Two-chamber Two-chamber
1.0 0.2
106 344
50 67
149 396
[48] [49]
Batch
71
Single chamber
0.7
134
81
205
This study
UASB
73
Up-flow membrane less
0.7
163
86
236
This study
Please cite this article as: Khongkliang P et al., High efficient biohydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis under thermophilic condition, International Journal of Hydrogen Energy, https://doi.org/ 10.1016/j.ijhydene.2019.10.022
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Fig. 6 e DGGE profile of bacteria community in the sludge from dark fermentation stage (a) and MEC stage (b) of hydrogen production from palm oil mill effluent by two-stage dark fermentation and MEC. Thermoanaerobacterium sp., Thermoanaerobacterium thermosaccharolyticum, and Thermoanaerobacterium aciditolerans. Thermoanaerobacter brockii was dominant during the 25 days and gradually decreased when operating in a long time, while Clostridium sp. was gradually increased of overtime. Clostridium sp. and Thermoanaerobacterium sp. was commonly found in the hydrogen production reactor feeding with POME operating under thermophilic condition [5,50] Prasertsan et al. [5] reported that Thermoanaerobacterium sp. was dominated in the hydrogen production reactor feeding with POME at 60 C. The optimum temperature and pH for growth of Thermoanaerobacterium sp. was 55e60 C and 5.2e6.5, respectively [51]. The dominant bacteria in the second-stage microbial electrolysis are mainly dominated by Geobacter sp., Desulfovibrio sp. and Thermoanaerobacterium thermosaccharolyticum. Besides, the bacterial community in second-stage were changed when operating for a long time. Shewanalla sp. was the dominant population during the first 25 days, while Geobacillus sp. was more prominent in a long time of operation. The Geobacillus sp. and Desulfovibrio sp. were dominated at an anode biofilm with the ability to produce hydrogen as exoelectrogens [52]. Geobacter sp. and Shewanalla sp. has been well reported as electrochemically active bacteria able to utilize acetate as an electron donor. The dominant of Geobacter sp. on the anode electrode, indicating that this bacterial played a significant role in the electron generation [53]. Desulfovibrio sp. has electrons transfer cytochromes and can form biofilms on
electrodes for harvesting electrons for hydrogen formation. Furthermore, Desulfovibrio sp. has been shown to interact with a cathode to facilitate hydrogen production [54].
Conclusions The two-stage dark fermentation and microbial electrolysis operating under thermophilic condition is a highly promising option to maximize the conversion of POME into biohydrogen. The optimum COD concentration and pH by dark fermentation were 66 g$L1 and 6.5, respectively. The optimum applied voltage for hydrogen production from dark fermentation effluent by MEC was of 0.7 V. The hydrogen yield of continuous two-stage dark fermentation and MEC was 236 mL-H2$gCOD1 with a hydrogen production rate of 7.81 L$L1$d1. The hydrogen yield was 3 times increased when compared with dark fermentation alone.
Acknowledgments The authors would like to thank the Research and Development Institute Thaksin University, Thailand through the graduate research fund (Grant No. 04-4/2559), Thailand Science Reserach and Inovation, Thailand; Thailand Research Fund, Thailand through the Research and Researcher for
Please cite this article as: Khongkliang P et al., High efficient biohydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis under thermophilic condition, International Journal of Hydrogen Energy, https://doi.org/ 10.1016/j.ijhydene.2019.10.022
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Industry (PHD60I0036), TRF Senior Research Scholar (Grant No. RTA6280001), TRF Mid-Career Research Grant (Grant No. RSA6180048) and Waste and Energy Management Co., Ltd. for financial support.
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Please cite this article as: Khongkliang P et al., High efficient biohydrogen production from palm oil mill effluent by two-stage dark fermentation and microbial electrolysis under thermophilic condition, International Journal of Hydrogen Energy, https://doi.org/ 10.1016/j.ijhydene.2019.10.022