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Two-stage thermophilic fermentation and mesophilic methanogenic process for biohythane production from palm oil mill effluent with methanogenic effluent recirculation for pH control
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Two-stage thermophilic fermentation and mesophilic methanogenic process for biohythane production from palm oil mill effluent with methanogenic effluent recirculation for pH control Sompong O-Thong a,c,*, Wantanasak Suksong a, Kanathip Promnuan a, Mathavee Thipmunee a, Chonticha Mamimin a, Poonsuk Prasertsan b a Biotechnology Program, Department of Biology, Faculty of Science, Thaksin University, Phatthalung, 93210, Thailand b Department of Industrial Biotechnology, Faculty of Agro Industry, Prince of Songkla University, Songkhla, 90112, Thailand c Research Center in Energy and Environment, Thaksin University, Phatthalung, 93210, Thailand
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abstract
Article history:
Two-stage thermophilic fermentation and mesophilic methanogenic process with meth-
Received 31 March 2016
anogenic effluent recirculation to hydrogen reactor for biohythane production from palm
Received in revised form
oil mill effluent (POME) was investigated. Maximum hydrogen (4.1 L H2/L-POME) and
12 July 2016
methane production (16.6 L CH4/L-POME) from POME in batch tests were achieved at 30%
Accepted 13 July 2016
recirculation rate of methanogenic effluent. Continuous hydrogen production of without
Available online xxx
and with recirculation phase was 2.2 and 3.8 L H2/L-POME, respectively. Continuous methane production of without and with recirculation phase was 12.2 and 14 L CH4/L-
Keywords:
POME, respectively. Both reactor effluents were effective to keep at optimal pH with 2 times
Hydrogen and methane
increasing in hydrogen production. The hydrogen and methane yield were 135 mL H2/gVS
Palm oil mill effluent
and 414 mL CH4/gVS, respectively. Biohythane gas composition was composed 13.3% of
Thermophilic fermentation
hydrogen, 54.4% of methane and 32.2% carbon dioxide. Two-stage process with meth-
Two-stage anaerobic digestion
anogenic effluent recirculation flavored Thermoanaerobacterium sp. in the hydrogen reactor
Methanogenic effluent
and efficiently for energy recovery from POME. © 2016 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
Introduction Hydrogen and methane can be produced through a two-stage microbial fermentation. A two-stage microbial fermentation process is based on the significant differences between
acidogens and methanogens in terms of physiology, nutritional needs, growth kinetics, and sensitivity to environmental conditions [1]. Hydrolysis and acidogenesis takes place in the first stage of the anaerobic digestion process. An acidogenic bacteria are grown under an optimal pH range of 5e6 and optimal hydraulic retention time (HRT) of 1e3 days.
* Corresponding author. Biotechnology Program, Department of Biology, Faculty of Science, Thaksin University, Phatthalung, 93210, Thailand. E-mail address: [email protected] (S. O-Thong). http://dx.doi.org/10.1016/j.ijhydene.2016.07.095 0360-3199/© 2016 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved. Please cite this article in press as: O-Thong S, et al., Two-stage thermophilic fermentation and mesophilic methanogenic process for biohythane production from palm oil mill effluent with methanogenic effluent recirculation for pH control, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.07.095
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Acidogenic bacteria could convert carbohydrate to significant amounts of hydrogen via acetate and butyrate pathways [2]. In the second stage, the remaining organic content in the hydrogenic effluent was anaerobically converted to methane by methanogens under optimal pH range of 7e8 and optimal HRT of 15e20 days [3]. This method is environmentally friendly and capable to utilize the organic waste to eliminate the pollution [4]. Various types of waste materials can be used as substrate for biohythane production such as starch wastewater, palm oil mill effluent, food waste and organic solid waste [3,5,6]. Hydrogen and methane yield from two-stage biohythane production of starch wastewater were 130 mL H2/gCOD and 230 mL CH4/gCOD, respectively [5]. Hydrogen and methane yield from two-stage biohythane production of food waste were 205 mL H2/gVS and 464 mL CH4/gVS, respectively [7]. Hydrogen and methane yield from two-stage biohythane production of palm oil mill effluent (POME) were 201 mL H2/gCOD and 315 mL CH4/gCOD, respectively [3]. Successful biohythane production from POME by two stage microbial fermentation was achieved under thermophilic and follow by mesophilic condition. Mamimin et al. [3] obtained the continuous biohythane production rate (HPR) of 4.4 L/L/d with containing of 51% CH4, 14% H2 and 35% CO2 by two-stage thermophilic and mesophilic methanogenic process. Palm oil mill effluent (POME) is a suitable substrate for biohythane production in term of energy recovery and high biogas production volume. Energy analysis suggested that the two-stage fermentation process had greater net energy recovery than the single hydrogen fermentation and methane fermentation process [3,8]. However, hydrogen production in first stage is coupled with the generation of volatile fatty acids (VFAs), such as acetic acid, propionic acid, and butyric acid. VFAs were accumulation during fermentation and lower the pH in the reactor below the optimal range resulting to inhibiting the metabolism of hydrogen-producing bacteria. While the addition of NaOH, KOH, NaHCO3 and Na2CO3 as alkali solution were applied for pH control in hydrogen fermentation stage. The cost of alkali chemicals is relatively high and associated with overloading of Naþ and Kþ ions severely inhibits hydrogen-producing bacteria in hydrogen fermentation reactor [9]. Previously reported found that a two-stage hydrogen and methane fermentation process with methanogenic sludge recirculation (two-stage recirculation process), with part of the methanogenic sludge circulated to a hydrogen reactor, could be successfully operated maintaining pH levels around 5.5 without any alkaline addition [7,10,11]. Kim et al. [12] also reported that direct recycling of a methanogenic effluent to a hydrogen production system increased hydrogen production from 1.19 to 1.76 m3 H2/m3/d, and decreased the requirement for exogenous alkali. Hydrogen yield from two-stage recirculation process was 2.5e2.8 mol/mol-hexose of hydrogen yield [10], which were relatively high comparing to 4 mol/molhexose from the maximum theoretical hydrogen yield. Cavinato et al. [11] demonstrated that the methane sludge circulation could be substituted for alkaline compounds with maintained appropriate pH around 5.4. However, the twostage recirculation process does not always improve hydrogen productivity. Hydrogen production might be suppressed due to the methanogenic activity by methanogenic
effluent recirculation. Under the conditions tested by Kraemer and Bagley [13], the methanogenic effluent recirculation to the hydrogen reactor decreased the hydrogen yield from 1.38 to 0.18 mol H2/mol glucose, although reduced the amount of alkali required for pH control by approximately 40%. Cheng et al. [14] reported that methanogen invasion in a hydrogen reactor had a negative effect on hydrogen production in a pilot study. Considering what has been described above, there is still a need to explain in principal why the two-stage recirculation process achieved relatively high hydrogen yields in earlier studies. A two-stage recirculation process provides superior performance in hydrogen fermentation. However, it is still unclear whether the recirculation process is affect on the operation of the reactor and microbial community besides the buffering ability. The microbial community composition is an important factor affecting hydrogen yield in hydrogen fermentation reactor. Hydrogen reactor normally was seeded by mixed microflora since the co-existence of hydrogen producing bacteria and hydrogen-consuming microorganisms that reduces the net hydrogen production in a hydrogen reactor. A two-stage recirculation of methanogenic effluent that includes many of hydrogenotrophic methanogens and homoacetogens resulted in the settling of hydrogen consumers in a hydrogen reactor regardless of their growth rates. It has potential to decrease the hydrogen yield in a two-stage recirculation process. This study examined the hydrogen production in a two-stage fermentation process using POME as feedstock, with the highly alkaline methanogenic effluent recirculation to a hydrogen production system to control pH. The effects of recirculation of methanogenic effluent on hydrogen production, VFAs yields and composition, alkali supplementation, microbial communities was investigated.
Materials and methods Palm oil mill effluent and inoculums The palm oil mill effluent (POME) used in this study was collected from Suksomboon Palm Oil Co., Ltd., Chonburi, Thailand. POME was stored at the temperature of 4 C for later use. POME is brownish, slurry, viscous, acidic and high oil and grease containing. The characteristics of POME and inoculums were shown in Table 1. Seed sludge for hydrogen production was collected from a palm oil mill biogas plant. The sludge was enriched for thermophilic hydrogen producing bacteria according to Mamimin et al. [15]. The enriched sludge has a volatile suspended solids (VSS) concentration of 6.0 g/L was gradually acclimatized with POME by successive transfer to increasing concentration of POME from 50% to 100%. The acclimatized sludge was operated by discharged of 50% cultured broth and addition of 50% fresh POME every 24 h for 10 times. Then, the sludge was used in batch test and seeded into a CSTR reactor for continuous operation. An anaerobic digester sludge obtained from a palm oil mill biogas plant was used as a seed sludge for biomethane production (BMP) and methanogenic reactor without treatment. Anaerobic digester sludge was placed in an incubator for 5 days until the biogas production was stopped in order to minimize the contribution of
Please cite this article in press as: O-Thong S, et al., Two-stage thermophilic fermentation and mesophilic methanogenic process for biohythane production from palm oil mill effluent with methanogenic effluent recirculation for pH control, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.07.095
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Table 1 e Chemical characteristics of palm oil mill effluent. Parameter Biochemical oxygen demand (BOD) Chemical oxygen demand (COD) Total carbohydrate Total nitrogen Ammonium-nitrogen Total phosphorus Phosphate Oil Total solids (TS) Volatile solids (VS) Suspended solids (SS) Volatile fatty acids Alkalinity Ethanol Acetic acid Propionic acid Butyric acid Ash pH
Concentration (g/L) 48.3 ± 0.6 85.85 ± 2.0 16.54 ± 0.2 0.83 ± 0.1 0.03 ± 0.001 0.13 ± 0.001 0.021 ± 0.001 10.6 ± 0.02 63.2 ± 0.3 52.8 ± 0.4 30.53 ± 0.2 1.54 ± 0.35 0.53 ± 0.15 0.2 ± 0.06 1.18 ± 0.03 0.34 ± 0.05 0.35 ± 0.04 10.4 ± 0.2 4.53 ± 0.13
organic materials containing in the inoculum. The methane inoculum was contained of 15.4 g/L total solids (TS), 13.6 g/L volatile solids (VS), and 11.2 g/L volatile suspended solids (VSS).
Batch tests Two-stage biochemical hydrogen production (BHP) and BMP of recirculation methanogenic effluent with POME were identified in batch assays under thermophilic and mesophilic conditions as described previously by Giordano et al. [16]. The experiments were carried out in batch condition using 500 mL glass serum bottles. BHP from POME mixed with methanogenic effluent at recirculation rate of 50%, 40%, 30%, 25%, 20% and 15% was investigated. Additionally, for testing the inoculum quality, positive controls with 20 g/L of cellulose and 20 g/L of glucose instead of samples were also included for observation. Serum bottles were manually mixed every day and maintained at constant temperature (55 C) in a thermostatic cabinet for 5 days. When the biological hydrogen production ceased, the serum bottles were opened and another 120 g of anaerobic digestion sludge were introduced into the reactors and incubated for 45 days at mesophilic condition (35 C) in order to evaluate methane production. The reactors were manually mixed every day during the first 7 days and every 2 days for the rest of the experimental with maintained at static conditions. Biogas production was determined through the use of the water replacement method [17]. Biogas composition in the headspace of the bottles was monitored by GC-TCD according to Hniman et al. [18]. The background of hydrogen and methane production from the inoculum was determined in blank assays with water instead of samples. Hydrogen and methane from blank was subtracted from substrate assays [19].
Experimental set up and operations Continuous hydrogen and methane production were carried out in the completely stirred tank reactor (CSTR) and upflow
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anaerobic sludge blanket reactor (UASB) reactors with a working volume of 5 L and 35 L, respectively. CSTR were maintained temperature at 55 C by circulating hot water inside a coil surrounding each reactor, while UASB reactor were maintained temperature at 35 C. Fig. 1 illustrates the schematic diagram of the experimental apparatus used in this study. The apparatus was composed of four parts including mixing tank, CSTR reactor for hydrogen production, UASB reactor for methane production and setting tank for recycle methanogenic effluent. POME and methanogenic effluent was mixed in mixing tank before feed into CSTR reactor. CSTR was operated at pH of 5.5 and HRT of 2 days. The operation reached steady state when hydrogen gas content, biogas volume and pH were stable (less than 10% variation). The third column was a thermophilic methanogenic reactor (UASB). A gas meter was installed in CSTR and UASB reactor to record gas volume automatically. Second stage was operated at mesophilic condition, pH of 7.5 and HRT of 15 days. Organic loading rate (OLR) of CSTR and UASB reactor was 14.3 g-VS/L/d and 1.58 g-VS/L/d. Last tank was setting tank for separating of bacterial sludge from UASB reactor effluent. The operation of process was divided into two periods with no recirculation and 30% recirculation rate of methanogenic effluent. The two-stage CSTR and UASB were routinely monitored for the pH, gas production and composition, volatile fatty acids (VFAs) distributions, chemical oxygen demand (COD) and suspended solids (SS). The steady-state condition (pH over 7.3 and VFA less than 1000 mg/L) was confined from the methanogenic reactor. The sludge samples from each stage of operation were analyzed for microbial community structure using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE).
Analytical methods The biogas volume and composition were measured by the displacement of water and gas chromatography (GC-TCD, Shimadzu 8A) equipped with thermal conductivity detector and filled with 2.0 m packed column (Shin-Carbon ST 100/120 Restex) as previously described by Mamimin et al. [15]. The gas samples of 100 ml for methane and 500 ml for hydrogen were injected in duplicate. Volatile fatty acids (VFAs) were analyzed by gas chromatography (GC-FID, Shimadzu 17A) as previously described by Hniman et al. [18]. Chemical oxygen demand (COD), pH, suspended solid (SS), oil concentration, total phosphorus and total Kjeldahl nitrogen (TKN) were determined in accordance with the procedures described in the standard methods for the examination of water and wastewater [20]. Ammonium-nitrogen and phosphate concentrations were analyzed using commercial test kits from Spectroquant (Merck Ltd., Germany). The total carbohydrate content was analyzed by the Anthrone method [21]. The hydrogen and methane yield obtained from each gram of COD in POME was calculated according to Zhu et al. [22]. The densities of hydrogen and methane gas used for the calculation were 0.09 mg/mL and 0.72 mg/mL. The heating values of the hydrogen and methane were 142 kJ/g and 55.6 kJ/g [23]. Microbial community structure in the hydrogen production stage and methane production stage was analysis by PCRDGGE as pervious described by Kongjan et al. [1]. Most of the bands were excised from the gel and re-amplified. After re-
Please cite this article in press as: O-Thong S, et al., Two-stage thermophilic fermentation and mesophilic methanogenic process for biohythane production from palm oil mill effluent with methanogenic effluent recirculation for pH control, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.07.095
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Fig. 1 e Schematic diagram of two-stage recirculation systems for hydrogen and methane experimental apparatus.
amplification, PCR products were purified and sequenced by Macrogen Inc. (Seoul, Korea). Closest matches for partial 16S rRNA gene sequences were identified by database searches in Gene Bank using BLAST [24].
Results and discussion Effect of recirculation rate on hydrogen and methane production Hydrogen and methane production tests were started immediately after incubation indicating that a short lag phase. It is noteworthy that more than 90% of the hydrogen was produced in approximately 2 days. The hydrogen concentration in the biogas of the BHP tests ranged between 45% and 60% with CO2 as the remaining fraction and no CH4 was detected. Hydrogen production from POME with recirculation of methanogenic effluent at recirculation rate of 0%, 10%, 20%, 25%, 30%, 40% and 50% was 1.1, 2.8, 3.3, 3.6, 4.1, 3.8 and 2.8 L H2/L-POME, respectively. Hydrogen production from POME mixed with methanogenic effluent at 30% recirculation rate has highest hydrogen yield of 188 mL H2/g COD and highest hydrogen production of 4.1 L H2/L-POME (Table 2), which is within the range obtained in other studies under thermophilic conditions [25]. When the recirculation rate was increased from 40% to 50%, the hydrogen yield was decreased from 188 to 140 mL H2/g COD. The VFAs production caused a significant
pH decrease from 4.5 to 4.0 after 4 days of incubation of without recirculation and recirculation rate of 15e25%. The recirculation rate of 30e50% could maintain the pH in the ranged of 5.0e5.6. This pH was in the optimal range for hydrogenase enzyme of 5.5e6.5 [26], resulting high hydrogen production. This result indicating that methanogenic effluent recirculation shown a considerable positive effect on alleviating VFA inhibition and pH decrease (alkalinity 3.0e3.6 gCaCO3/L) in two-stage recirculation process. Methane yield in second stage yield was 279e345 mL CH4/gCOD. Methane production in second stage from POME with recirculation of methanogenic effluent at recirculation rate of 0%, 10%, 20%, 25%, 30%, 40% and 50% was 9.8, 13.9, 15.1, 15.1, 16.6, 15.6 and 14.8 L CH4/L-POME, respectively. The methane concentration in the biogas ranged between 64 and 70%. Maximum hydrogen (4.1 L H2/L-POME) and methane production (16.6 L CH4/LPOME) from POME in batch tests were achieved at 30% recirculation rat of methanogenic effluent. VFA from hydrogen production process was completely degraded in second stage methane production with VFAs concentration in effluent below 30 mg/L. Although methane production lasted approximately 30 days, about 90% of the total methane was produced during the first 15 days. Hydrogen and methane recovery from two-stage recirculation process of POME was 95% at 30% recirculation rate (Table 3). This indicating that almost all COD in the POME was consumed by conversion into both fuel gas and microbial biomass. Energy recovery demonstrated that much more energy could be gained by two-stage recirculation
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Table 2 e Summary of hydrogen and methane production from two-stage recirculation methanogenic effluent process of POME at different recirculation rates. Recirculation rate (%) 50 40 30 25 20 15 0
Alkalinity (gCaCO3/L)
Initial pH
Final pH
H2 yield (mL H2/g-COD)
CH4 yield (mL CH4/g-COD)
H2 production (L H2/L-POME)
CH4 production (L CH4/L-POME)
3.3 2.6 2.0 1.4 1.2 0.8 0.5
7.3 6.8 6.5 5.8 5.5 5.0 4.5
5.6 5.2 5.0 4.5 4.2 4.1 4.0
140 187 188 181 166 140 60
280 321 345 308 307 279 200
2.8 3.8 4.1 3.6 3.3 2.8 1.2
12 15.6 16.6 15.1 15.1 13.9 9.8
process. This suggests that two-stage recirculation process was much more efficient in biodegradation than the without recirculation [3,27].
Continuous biohythane production by two-stage recirculation process Two-stage recirculation process with 30% recirculation rate was operated with OLR of 15 kg-VS/m3/d and HRT days for biohythane production from POME. Hydrogen production from no recirculation and 30% recirculation was 2.2 and 3.8 L H2/L-POME, respectively. The hydrogen gas production observed from the CSTR reactor was show in Fig. 2. The maximum hydrogen production record from this reactor was 3.9 L H2/L-POME, which was observed in 5 days after recirculation methanogenic effluent. The hydrogen concentration in the biogas was 48e60% with an average concentration of 55%. The methane concentration was not observed in the biogas, indicating that a no methanogenic activity existed in the hydrogen reactor. The average and maximum hydrogen yields were calculated to be 180 mL H2/gCOD respectively, with acetic acid and butyric acid as main products in fermentation soluble metabolites. The characteristics of the hydrogenic effluent such as pH, alkalinity and COD were presented in Fig. 2. COD remaining was 39.5 g/L on average, resulting in a COD removal efficiency of 35%. The average hydrogen production over the entire recirculation experiment was 3.8 L H2/ L-POME. The hydrogen production was similar to than hydrogen production of 4.7 L H2/L-POME from POME in batch reactor under thermophilic condition [28]. The hydrogen production was also higher than hydrogen production (2.64 L H2/L-POME) from POME in CSTR operating at HRT 4 days under thermophilic condition [29]. Thermophilic hydrogen
fermentation is enhancing biogas production yields by reducing biogas solubility and lowering the H2 partial pressure in the headspace. Thermophilic hydrogen fermentation can improve the degradation efficiency at high temperatures [25]. Two-stage recirculation process was operated at HRT 2 days for hydrogen production and HRT of 15 days for methane production. The operation conditions agreed with Liu et al. [23] that reported HRT for first stage for hydrogen from organic fraction of municipal solid wastes are 1e2 days and HRT second stage for methane production are 10e15 days, respectively. The volume of reactor for methane production is about 7 times as large than of hydrogen reactor. Methane production from no recirculation and 30% recirculation was 12.2 and 14 L H2/L-POME, respectively. Methane gas production from effluent of hydrogen stage was examined using UASB. The gas production observed from the methane reactor is shown in Fig. 3. There was no delay of methane production observed in the reactor during start-up stage, demonstrating that the effluent of hydrogen reactor was readily usable for methanogens. During steady state, biogas productions were stable and COD removal greater than 90%. The methane concentration in the biogas was 70e76% (v/v) with an average of 73%. The methane yield based on COD basis in the reactor was 271 mL CH4/gCOD. Two-stage hydrogen and methane enhanced degradation of organic waste and favors a high product yields and biogas quality. The hydrogen and methane yield were 135 mL H2/gVS and 414 mL CH4/gVS, respectively. Biohythane gas composition was composed 13.3% of hydrogen, 54.4% of methane and 32.2% carbon dioxide. The characteristics of the effluent of the hydrogen and methane reactor are presented in Table 4. The overall removal efficiencies of COD and SS of two-stage were 93% and 69%,
Table 3 e Summary of energy recovery from hydrogen and methane production of two-stage recirculation methanogenic effluent process of POME at different recirculation rates. Recirculation rate (%) 50 40 30 25 20 15 0
Biohythane production (L/L-POME)
Energy recovery of H2 (%)
Energy recovery of CH4 (%)
Total energy recovery (%)
14.8 19.4 20.7 18.7 18.4 16.6 11.0
9.07 12.37 13.36 11.88 10.89 9.074 3.8
72.05 80.62 85.79 78.04 78.04 71.83 50.6
81.5 93 95.2 89.9 88.9 80.9 54.4
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Fig. 2 e Hydrogen production, pH, alkalinity, COD and OLR during thermophilic hydrogen production from POME by twostage recirculation process. Please cite this article in press as: O-Thong S, et al., Two-stage thermophilic fermentation and mesophilic methanogenic process for biohythane production from palm oil mill effluent with methanogenic effluent recirculation for pH control, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.07.095
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Fig. 3 e Methane production, pH, alkalinity, COD and OLR during mesophilic methane production from POME by two-stage recirculation process.
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Table 4 e Process performance and effluent characteristics of two-stage methanogenic effluent recirculation process of POME at 30% recirculation rate. Two stage process
Conditions HRT (d) Temperature ( C) Flow rate (L/d) Working Volume (L) Organic loading rate (kg-VS/m3/d) pH (initial) Gas production (Lbiogas/L/d) Gas Yield (ml H2 or CH4/g-VS) Removal efficiency Chemical oxygen demand (%) Total volatile solid (%) Total suspended solid (%) Carbohydrates consumption (%) Effluent pH Chemical oxygen demand (g/L) Alkalinity (g CaCO3/L) Volatile fatty acids (g/L) Ethanol (g/L) Acetic acid (g/L) Propionic acid (g/L) Butyric acid (g/L)
Hydrogen reactor (CSTR)
Methane reactor (UASB)
2 55 2.5 5 14.3 6.5 1.31 135
15 35 2.3 35 1.58 7.50 0.16 414
23.61 42.08 34.45 71.9
93.38 68.11 69.6 90.49
5.0 37.47 2.6 3.34 0.13 1.71 0.57 0.93
7.62 1.63 7.6 0.15 0.02 0.07 0.08 0.02
Fig. 4 e DGGE profile of bacterial community in sludge of hydrogen reactor from no recirculation phase (5, 15 and 25 day) and recirculation phase (35, 45 and 60 day) under thermophilic condition.
respectively. The maximum hydrogen production rate of 1.31 L H2/L/d and maximum methane production rate were 1.18 L CH4/L/d were achieved by two-stage recirculation process. The hydrogen and methane yield from POME by twostage recirculation process, which is within the range obtained in other studies from previous report (Table 5). Both reactor effluents were effective to keep at optimal pH with 2 times increasing in hydrogen production. It was previously reported that recirculation methanogenic effluent into hydrogen reactor of food waste could maintained the pH of the systems around 5.5 without addition of exogenous alkali [10,11].
Microbial population dynamics in two-stage recirculation process Two-stage recirculation process has higher hydrogen production than without recirculation, indicating different microbial community in hydrogen reactor. The DGGE results indicated a shift in the predominant bacteria phylum responsible for hydrogen production, from Clostridium sp., Enterococcus sp. and Marinomonas sp. to Thermoanaerobacterium sp., after methanogenic effluent recirculation began (Fig. 4). Two-stage process with methanogenic effluent recirculation flavored Thermoanaerobacterium sp. in the hydrogen reactor. Two-stage
Table 5 e Hydrogen and methane yield from two-stage anaerobic digestion process of various organic waste. Feedstocks
Skim latex serum Food waste Grass Agave tequilana OFMSW Dairy processing waste POME POME
Hydrogen reactor
Methane reactor
Yield
Reactor types
Yield
41.3 mL/gVS 50.0 mL/gVS 6.7 mL/gVS 60.0 mL/gCOD 29.0 mL/gVS 160.7 mL/gCOD 210.0 mL/gCOD 180 mL/gCOD
Batch CSTR CSTR Batch CSTR IBR ASBR CSTR
321.1 440.0 349.4 240.0 287.0 178.1 315.0 271.2
Reactor types
mL/gVS mL/gVS mL/gVS mL/gCOD mL/gVS mL/gCOD mL/gCOD mL/gCOD
Batch CSTR CSTR Batch CSTR IBR UASB UASB
Total energy yield
Temperature ( C)
References
11.9 kJ/gVS 16.3 kJ/gVS 12.6 kJ/gVS 9.2 kJ/gCOD 10.6 kJ/gVS 8.1 kJ/gCOD 13.4 kJ/gCOD 11.6 kJ/gCOD
55/55 35/35 35/35 35/35 55/55 55/55 55/35 55/35
[27] [30] [31] [32] [33] [34] [3] This study
OFMSW ¼ organic fraction of municipal solid waste; IBR ¼ induced bed reactors.
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recirculation process perform well in terms of hydrogen production or carbohydrates degradation compared to those without recirculation, perhaps due to an available alkalinity and nitrogen source. Methanogenic effluent recirculation was supplemented the NHþ 4 into the hydrogen reactor [6]. Nitrogen source is nutrient species specifically for Thermoanaerobacterium sp. [25]. The enhance of hydrogen production obtained with recirculation under thermophilic condition, which may not flavored mesophilic hydrogen consumption bacteria and archaea containing in methanogenic effluent [5]. Thermoanaerobacterium is one of the most important hydrogenproducing species [3,18,25]. For the growth of Thermoanaerobacterium sp., the optimal temperature and pH ranges are 55e60 C and 5.2e6.5, which together allowed efficient hydrogen production when POME as substrates [5]. Analysis of the microbial communities showed that methanogenic effluent recirculation also enriched bacterial communities in the methane reactor. DGGE profiling of sludge from methane reactor illustrated that Clostridium sp., Fervidobacterium sp., and Ruminococcus sp. were dominant bacteria in methane production stage. Methanosarcina sp. and Methanoculleus sp. were dominant and played an important role in methane production (Fig. 5). Methanosarcina species were reported to be dominant at high acetate concentration (>1.2 mM), and the results were consistent with the high acetate concentrations in hydrogen effluent that feed to methane reactors. Methanoculleus species were responsible for hydrogenotrophic methanogenesis [1].
Conclusions Recirculation of methanogenic effluent into hydrogen reactor promoted the generation of significant amount of hydrogen gas. Both hydrogen production rate and yield increased in methanogenic effluent recirculation systems. Maximum hydrogen (4.1 L H2/L-POME) and methane production (16.6 LCH4/L-POME) from POME in batch tests were achieved at 30% recirculation rat of methanogenic effluent. The recirculation of methanogenic effluent at 30% recirculation rate could compensate for alkalinity required by hydrogen reactor. Both reactor effluents were effective to keep at optimal pH with 2 times increasing in hydrogen production. Continuous hydrogen and methane production of two stage recirculation process was 3.8 L H2/L-POME and 14 L CH4/L-POME, respectively. The hydrogen and methane yield were 135 mL H2/gVS and 414 mL CH4/gVS, respectively. Biohythane gas composition was composed 13.3% of hydrogen, 54.4% of methane and 32.2% carbon dioxide. Thermoanaerobacterium sp. was dominated during hydrogen production, whereas archaea belonging to Methanosarcina sp. and Methanoculleus sp. were dominated in the methane reactor. Two-stage process with methanogenic effluent recirculation for biohythane production flavored Thermoanaerobacterium sp. in the hydrogen reactor and could efficiently for energy recovery from POME. Fig. 5 e DGGE profile of bacterial (A) and archaea (B) community in sludge of methane reactor from no recirculation phase (5, 15 and 25 day) and recirculation phase (35, 45 and 60 day) under mesophilic condition.
Acknowledgments This work was supported by the Core-to-Core Program, which was financially supported by aJapan Society for the Promotion
Please cite this article in press as: O-Thong S, et al., Two-stage thermophilic fermentation and mesophilic methanogenic process for biohythane production from palm oil mill effluent with methanogenic effluent recirculation for pH control, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.07.095
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of Science, National Research Council of Thailand, Vietnam Ministry of Science and Technology (MOST), the National University of Laos, Beuth University of Applied Sciences and Brawijaya University, Research and Development Institute Thaksin University (RDITSU), Agricultural Research Development Agency, Research Group for Development of Microbial Hydrogen Production Process from Biomass, Khon Kaen University and Thailand Research Fund through grant number MRG5580074 and RTA5780002 for financial support.
references
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Two-stage thermophilic fermentation and mesophilic methanogenic process for biohythane production from palm oil mill effluent with methanogenic effluent recirculation for pH control Sompong O-Thong a,c,*, Wantanasak Suksong a, Kanathip Promnuan a, Mathavee Thipmunee a, Chonticha Mamimin a, Poonsuk Prasertsan b a Biotechnology Program, Department of Biology, Faculty of Science, Thaksin University, Phatthalung, 93210, Thailand b Department of Industrial Biotechnology, Faculty of Agro Industry, Prince of Songkla University, Songkhla, 90112, Thailand c Research Center in Energy and Environment, Thaksin University, Phatthalung, 93210, Thailand
article info
abstract
Article history:
Two-stage thermophilic fermentation and mesophilic methanogenic process with meth-
Received 31 March 2016
anogenic effluent recirculation to hydrogen reactor for biohythane production from palm
Received in revised form
oil mill effluent (POME) was investigated. Maximum hydrogen (4.1 L H2/L-POME) and
12 July 2016
methane production (16.6 L CH4/L-POME) from POME in batch tests were achieved at 30%
Accepted 13 July 2016
recirculation rate of methanogenic effluent. Continuous hydrogen production of without
Available online xxx
and with recirculation phase was 2.2 and 3.8 L H2/L-POME, respectively. Continuous methane production of without and with recirculation phase was 12.2 and 14 L CH4/L-
Keywords:
POME, respectively. Both reactor effluents were effective to keep at optimal pH with 2 times
Hydrogen and methane
increasing in hydrogen production. The hydrogen and methane yield were 135 mL H2/gVS
Palm oil mill effluent
and 414 mL CH4/gVS, respectively. Biohythane gas composition was composed 13.3% of
Thermophilic fermentation
hydrogen, 54.4% of methane and 32.2% carbon dioxide. Two-stage process with meth-
Two-stage anaerobic digestion
anogenic effluent recirculation flavored Thermoanaerobacterium sp. in the hydrogen reactor
Methanogenic effluent
and efficiently for energy recovery from POME. © 2016 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
Introduction Hydrogen and methane can be produced through a two-stage microbial fermentation. A two-stage microbial fermentation process is based on the significant differences between
acidogens and methanogens in terms of physiology, nutritional needs, growth kinetics, and sensitivity to environmental conditions [1]. Hydrolysis and acidogenesis takes place in the first stage of the anaerobic digestion process. An acidogenic bacteria are grown under an optimal pH range of 5e6 and optimal hydraulic retention time (HRT) of 1e3 days.
* Corresponding author. Biotechnology Program, Department of Biology, Faculty of Science, Thaksin University, Phatthalung, 93210, Thailand. E-mail address: [email protected] (S. O-Thong). http://dx.doi.org/10.1016/j.ijhydene.2016.07.095 0360-3199/© 2016 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved. Please cite this article in press as: O-Thong S, et al., Two-stage thermophilic fermentation and mesophilic methanogenic process for biohythane production from palm oil mill effluent with methanogenic effluent recirculation for pH control, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.07.095
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Acidogenic bacteria could convert carbohydrate to significant amounts of hydrogen via acetate and butyrate pathways [2]. In the second stage, the remaining organic content in the hydrogenic effluent was anaerobically converted to methane by methanogens under optimal pH range of 7e8 and optimal HRT of 15e20 days [3]. This method is environmentally friendly and capable to utilize the organic waste to eliminate the pollution [4]. Various types of waste materials can be used as substrate for biohythane production such as starch wastewater, palm oil mill effluent, food waste and organic solid waste [3,5,6]. Hydrogen and methane yield from two-stage biohythane production of starch wastewater were 130 mL H2/gCOD and 230 mL CH4/gCOD, respectively [5]. Hydrogen and methane yield from two-stage biohythane production of food waste were 205 mL H2/gVS and 464 mL CH4/gVS, respectively [7]. Hydrogen and methane yield from two-stage biohythane production of palm oil mill effluent (POME) were 201 mL H2/gCOD and 315 mL CH4/gCOD, respectively [3]. Successful biohythane production from POME by two stage microbial fermentation was achieved under thermophilic and follow by mesophilic condition. Mamimin et al. [3] obtained the continuous biohythane production rate (HPR) of 4.4 L/L/d with containing of 51% CH4, 14% H2 and 35% CO2 by two-stage thermophilic and mesophilic methanogenic process. Palm oil mill effluent (POME) is a suitable substrate for biohythane production in term of energy recovery and high biogas production volume. Energy analysis suggested that the two-stage fermentation process had greater net energy recovery than the single hydrogen fermentation and methane fermentation process [3,8]. However, hydrogen production in first stage is coupled with the generation of volatile fatty acids (VFAs), such as acetic acid, propionic acid, and butyric acid. VFAs were accumulation during fermentation and lower the pH in the reactor below the optimal range resulting to inhibiting the metabolism of hydrogen-producing bacteria. While the addition of NaOH, KOH, NaHCO3 and Na2CO3 as alkali solution were applied for pH control in hydrogen fermentation stage. The cost of alkali chemicals is relatively high and associated with overloading of Naþ and Kþ ions severely inhibits hydrogen-producing bacteria in hydrogen fermentation reactor [9]. Previously reported found that a two-stage hydrogen and methane fermentation process with methanogenic sludge recirculation (two-stage recirculation process), with part of the methanogenic sludge circulated to a hydrogen reactor, could be successfully operated maintaining pH levels around 5.5 without any alkaline addition [7,10,11]. Kim et al. [12] also reported that direct recycling of a methanogenic effluent to a hydrogen production system increased hydrogen production from 1.19 to 1.76 m3 H2/m3/d, and decreased the requirement for exogenous alkali. Hydrogen yield from two-stage recirculation process was 2.5e2.8 mol/mol-hexose of hydrogen yield [10], which were relatively high comparing to 4 mol/molhexose from the maximum theoretical hydrogen yield. Cavinato et al. [11] demonstrated that the methane sludge circulation could be substituted for alkaline compounds with maintained appropriate pH around 5.4. However, the twostage recirculation process does not always improve hydrogen productivity. Hydrogen production might be suppressed due to the methanogenic activity by methanogenic
effluent recirculation. Under the conditions tested by Kraemer and Bagley [13], the methanogenic effluent recirculation to the hydrogen reactor decreased the hydrogen yield from 1.38 to 0.18 mol H2/mol glucose, although reduced the amount of alkali required for pH control by approximately 40%. Cheng et al. [14] reported that methanogen invasion in a hydrogen reactor had a negative effect on hydrogen production in a pilot study. Considering what has been described above, there is still a need to explain in principal why the two-stage recirculation process achieved relatively high hydrogen yields in earlier studies. A two-stage recirculation process provides superior performance in hydrogen fermentation. However, it is still unclear whether the recirculation process is affect on the operation of the reactor and microbial community besides the buffering ability. The microbial community composition is an important factor affecting hydrogen yield in hydrogen fermentation reactor. Hydrogen reactor normally was seeded by mixed microflora since the co-existence of hydrogen producing bacteria and hydrogen-consuming microorganisms that reduces the net hydrogen production in a hydrogen reactor. A two-stage recirculation of methanogenic effluent that includes many of hydrogenotrophic methanogens and homoacetogens resulted in the settling of hydrogen consumers in a hydrogen reactor regardless of their growth rates. It has potential to decrease the hydrogen yield in a two-stage recirculation process. This study examined the hydrogen production in a two-stage fermentation process using POME as feedstock, with the highly alkaline methanogenic effluent recirculation to a hydrogen production system to control pH. The effects of recirculation of methanogenic effluent on hydrogen production, VFAs yields and composition, alkali supplementation, microbial communities was investigated.
Materials and methods Palm oil mill effluent and inoculums The palm oil mill effluent (POME) used in this study was collected from Suksomboon Palm Oil Co., Ltd., Chonburi, Thailand. POME was stored at the temperature of 4 C for later use. POME is brownish, slurry, viscous, acidic and high oil and grease containing. The characteristics of POME and inoculums were shown in Table 1. Seed sludge for hydrogen production was collected from a palm oil mill biogas plant. The sludge was enriched for thermophilic hydrogen producing bacteria according to Mamimin et al. [15]. The enriched sludge has a volatile suspended solids (VSS) concentration of 6.0 g/L was gradually acclimatized with POME by successive transfer to increasing concentration of POME from 50% to 100%. The acclimatized sludge was operated by discharged of 50% cultured broth and addition of 50% fresh POME every 24 h for 10 times. Then, the sludge was used in batch test and seeded into a CSTR reactor for continuous operation. An anaerobic digester sludge obtained from a palm oil mill biogas plant was used as a seed sludge for biomethane production (BMP) and methanogenic reactor without treatment. Anaerobic digester sludge was placed in an incubator for 5 days until the biogas production was stopped in order to minimize the contribution of
Please cite this article in press as: O-Thong S, et al., Two-stage thermophilic fermentation and mesophilic methanogenic process for biohythane production from palm oil mill effluent with methanogenic effluent recirculation for pH control, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.07.095
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Table 1 e Chemical characteristics of palm oil mill effluent. Parameter Biochemical oxygen demand (BOD) Chemical oxygen demand (COD) Total carbohydrate Total nitrogen Ammonium-nitrogen Total phosphorus Phosphate Oil Total solids (TS) Volatile solids (VS) Suspended solids (SS) Volatile fatty acids Alkalinity Ethanol Acetic acid Propionic acid Butyric acid Ash pH
Concentration (g/L) 48.3 ± 0.6 85.85 ± 2.0 16.54 ± 0.2 0.83 ± 0.1 0.03 ± 0.001 0.13 ± 0.001 0.021 ± 0.001 10.6 ± 0.02 63.2 ± 0.3 52.8 ± 0.4 30.53 ± 0.2 1.54 ± 0.35 0.53 ± 0.15 0.2 ± 0.06 1.18 ± 0.03 0.34 ± 0.05 0.35 ± 0.04 10.4 ± 0.2 4.53 ± 0.13
organic materials containing in the inoculum. The methane inoculum was contained of 15.4 g/L total solids (TS), 13.6 g/L volatile solids (VS), and 11.2 g/L volatile suspended solids (VSS).
Batch tests Two-stage biochemical hydrogen production (BHP) and BMP of recirculation methanogenic effluent with POME were identified in batch assays under thermophilic and mesophilic conditions as described previously by Giordano et al. [16]. The experiments were carried out in batch condition using 500 mL glass serum bottles. BHP from POME mixed with methanogenic effluent at recirculation rate of 50%, 40%, 30%, 25%, 20% and 15% was investigated. Additionally, for testing the inoculum quality, positive controls with 20 g/L of cellulose and 20 g/L of glucose instead of samples were also included for observation. Serum bottles were manually mixed every day and maintained at constant temperature (55 C) in a thermostatic cabinet for 5 days. When the biological hydrogen production ceased, the serum bottles were opened and another 120 g of anaerobic digestion sludge were introduced into the reactors and incubated for 45 days at mesophilic condition (35 C) in order to evaluate methane production. The reactors were manually mixed every day during the first 7 days and every 2 days for the rest of the experimental with maintained at static conditions. Biogas production was determined through the use of the water replacement method [17]. Biogas composition in the headspace of the bottles was monitored by GC-TCD according to Hniman et al. [18]. The background of hydrogen and methane production from the inoculum was determined in blank assays with water instead of samples. Hydrogen and methane from blank was subtracted from substrate assays [19].
Experimental set up and operations Continuous hydrogen and methane production were carried out in the completely stirred tank reactor (CSTR) and upflow
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anaerobic sludge blanket reactor (UASB) reactors with a working volume of 5 L and 35 L, respectively. CSTR were maintained temperature at 55 C by circulating hot water inside a coil surrounding each reactor, while UASB reactor were maintained temperature at 35 C. Fig. 1 illustrates the schematic diagram of the experimental apparatus used in this study. The apparatus was composed of four parts including mixing tank, CSTR reactor for hydrogen production, UASB reactor for methane production and setting tank for recycle methanogenic effluent. POME and methanogenic effluent was mixed in mixing tank before feed into CSTR reactor. CSTR was operated at pH of 5.5 and HRT of 2 days. The operation reached steady state when hydrogen gas content, biogas volume and pH were stable (less than 10% variation). The third column was a thermophilic methanogenic reactor (UASB). A gas meter was installed in CSTR and UASB reactor to record gas volume automatically. Second stage was operated at mesophilic condition, pH of 7.5 and HRT of 15 days. Organic loading rate (OLR) of CSTR and UASB reactor was 14.3 g-VS/L/d and 1.58 g-VS/L/d. Last tank was setting tank for separating of bacterial sludge from UASB reactor effluent. The operation of process was divided into two periods with no recirculation and 30% recirculation rate of methanogenic effluent. The two-stage CSTR and UASB were routinely monitored for the pH, gas production and composition, volatile fatty acids (VFAs) distributions, chemical oxygen demand (COD) and suspended solids (SS). The steady-state condition (pH over 7.3 and VFA less than 1000 mg/L) was confined from the methanogenic reactor. The sludge samples from each stage of operation were analyzed for microbial community structure using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE).
Analytical methods The biogas volume and composition were measured by the displacement of water and gas chromatography (GC-TCD, Shimadzu 8A) equipped with thermal conductivity detector and filled with 2.0 m packed column (Shin-Carbon ST 100/120 Restex) as previously described by Mamimin et al. [15]. The gas samples of 100 ml for methane and 500 ml for hydrogen were injected in duplicate. Volatile fatty acids (VFAs) were analyzed by gas chromatography (GC-FID, Shimadzu 17A) as previously described by Hniman et al. [18]. Chemical oxygen demand (COD), pH, suspended solid (SS), oil concentration, total phosphorus and total Kjeldahl nitrogen (TKN) were determined in accordance with the procedures described in the standard methods for the examination of water and wastewater [20]. Ammonium-nitrogen and phosphate concentrations were analyzed using commercial test kits from Spectroquant (Merck Ltd., Germany). The total carbohydrate content was analyzed by the Anthrone method [21]. The hydrogen and methane yield obtained from each gram of COD in POME was calculated according to Zhu et al. [22]. The densities of hydrogen and methane gas used for the calculation were 0.09 mg/mL and 0.72 mg/mL. The heating values of the hydrogen and methane were 142 kJ/g and 55.6 kJ/g [23]. Microbial community structure in the hydrogen production stage and methane production stage was analysis by PCRDGGE as pervious described by Kongjan et al. [1]. Most of the bands were excised from the gel and re-amplified. After re-
Please cite this article in press as: O-Thong S, et al., Two-stage thermophilic fermentation and mesophilic methanogenic process for biohythane production from palm oil mill effluent with methanogenic effluent recirculation for pH control, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.07.095
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Fig. 1 e Schematic diagram of two-stage recirculation systems for hydrogen and methane experimental apparatus.
amplification, PCR products were purified and sequenced by Macrogen Inc. (Seoul, Korea). Closest matches for partial 16S rRNA gene sequences were identified by database searches in Gene Bank using BLAST [24].
Results and discussion Effect of recirculation rate on hydrogen and methane production Hydrogen and methane production tests were started immediately after incubation indicating that a short lag phase. It is noteworthy that more than 90% of the hydrogen was produced in approximately 2 days. The hydrogen concentration in the biogas of the BHP tests ranged between 45% and 60% with CO2 as the remaining fraction and no CH4 was detected. Hydrogen production from POME with recirculation of methanogenic effluent at recirculation rate of 0%, 10%, 20%, 25%, 30%, 40% and 50% was 1.1, 2.8, 3.3, 3.6, 4.1, 3.8 and 2.8 L H2/L-POME, respectively. Hydrogen production from POME mixed with methanogenic effluent at 30% recirculation rate has highest hydrogen yield of 188 mL H2/g COD and highest hydrogen production of 4.1 L H2/L-POME (Table 2), which is within the range obtained in other studies under thermophilic conditions [25]. When the recirculation rate was increased from 40% to 50%, the hydrogen yield was decreased from 188 to 140 mL H2/g COD. The VFAs production caused a significant
pH decrease from 4.5 to 4.0 after 4 days of incubation of without recirculation and recirculation rate of 15e25%. The recirculation rate of 30e50% could maintain the pH in the ranged of 5.0e5.6. This pH was in the optimal range for hydrogenase enzyme of 5.5e6.5 [26], resulting high hydrogen production. This result indicating that methanogenic effluent recirculation shown a considerable positive effect on alleviating VFA inhibition and pH decrease (alkalinity 3.0e3.6 gCaCO3/L) in two-stage recirculation process. Methane yield in second stage yield was 279e345 mL CH4/gCOD. Methane production in second stage from POME with recirculation of methanogenic effluent at recirculation rate of 0%, 10%, 20%, 25%, 30%, 40% and 50% was 9.8, 13.9, 15.1, 15.1, 16.6, 15.6 and 14.8 L CH4/L-POME, respectively. The methane concentration in the biogas ranged between 64 and 70%. Maximum hydrogen (4.1 L H2/L-POME) and methane production (16.6 L CH4/LPOME) from POME in batch tests were achieved at 30% recirculation rat of methanogenic effluent. VFA from hydrogen production process was completely degraded in second stage methane production with VFAs concentration in effluent below 30 mg/L. Although methane production lasted approximately 30 days, about 90% of the total methane was produced during the first 15 days. Hydrogen and methane recovery from two-stage recirculation process of POME was 95% at 30% recirculation rate (Table 3). This indicating that almost all COD in the POME was consumed by conversion into both fuel gas and microbial biomass. Energy recovery demonstrated that much more energy could be gained by two-stage recirculation
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Table 2 e Summary of hydrogen and methane production from two-stage recirculation methanogenic effluent process of POME at different recirculation rates. Recirculation rate (%) 50 40 30 25 20 15 0
Alkalinity (gCaCO3/L)
Initial pH
Final pH
H2 yield (mL H2/g-COD)
CH4 yield (mL CH4/g-COD)
H2 production (L H2/L-POME)
CH4 production (L CH4/L-POME)
3.3 2.6 2.0 1.4 1.2 0.8 0.5
7.3 6.8 6.5 5.8 5.5 5.0 4.5
5.6 5.2 5.0 4.5 4.2 4.1 4.0
140 187 188 181 166 140 60
280 321 345 308 307 279 200
2.8 3.8 4.1 3.6 3.3 2.8 1.2
12 15.6 16.6 15.1 15.1 13.9 9.8
process. This suggests that two-stage recirculation process was much more efficient in biodegradation than the without recirculation [3,27].
Continuous biohythane production by two-stage recirculation process Two-stage recirculation process with 30% recirculation rate was operated with OLR of 15 kg-VS/m3/d and HRT days for biohythane production from POME. Hydrogen production from no recirculation and 30% recirculation was 2.2 and 3.8 L H2/L-POME, respectively. The hydrogen gas production observed from the CSTR reactor was show in Fig. 2. The maximum hydrogen production record from this reactor was 3.9 L H2/L-POME, which was observed in 5 days after recirculation methanogenic effluent. The hydrogen concentration in the biogas was 48e60% with an average concentration of 55%. The methane concentration was not observed in the biogas, indicating that a no methanogenic activity existed in the hydrogen reactor. The average and maximum hydrogen yields were calculated to be 180 mL H2/gCOD respectively, with acetic acid and butyric acid as main products in fermentation soluble metabolites. The characteristics of the hydrogenic effluent such as pH, alkalinity and COD were presented in Fig. 2. COD remaining was 39.5 g/L on average, resulting in a COD removal efficiency of 35%. The average hydrogen production over the entire recirculation experiment was 3.8 L H2/ L-POME. The hydrogen production was similar to than hydrogen production of 4.7 L H2/L-POME from POME in batch reactor under thermophilic condition [28]. The hydrogen production was also higher than hydrogen production (2.64 L H2/L-POME) from POME in CSTR operating at HRT 4 days under thermophilic condition [29]. Thermophilic hydrogen
fermentation is enhancing biogas production yields by reducing biogas solubility and lowering the H2 partial pressure in the headspace. Thermophilic hydrogen fermentation can improve the degradation efficiency at high temperatures [25]. Two-stage recirculation process was operated at HRT 2 days for hydrogen production and HRT of 15 days for methane production. The operation conditions agreed with Liu et al. [23] that reported HRT for first stage for hydrogen from organic fraction of municipal solid wastes are 1e2 days and HRT second stage for methane production are 10e15 days, respectively. The volume of reactor for methane production is about 7 times as large than of hydrogen reactor. Methane production from no recirculation and 30% recirculation was 12.2 and 14 L H2/L-POME, respectively. Methane gas production from effluent of hydrogen stage was examined using UASB. The gas production observed from the methane reactor is shown in Fig. 3. There was no delay of methane production observed in the reactor during start-up stage, demonstrating that the effluent of hydrogen reactor was readily usable for methanogens. During steady state, biogas productions were stable and COD removal greater than 90%. The methane concentration in the biogas was 70e76% (v/v) with an average of 73%. The methane yield based on COD basis in the reactor was 271 mL CH4/gCOD. Two-stage hydrogen and methane enhanced degradation of organic waste and favors a high product yields and biogas quality. The hydrogen and methane yield were 135 mL H2/gVS and 414 mL CH4/gVS, respectively. Biohythane gas composition was composed 13.3% of hydrogen, 54.4% of methane and 32.2% carbon dioxide. The characteristics of the effluent of the hydrogen and methane reactor are presented in Table 4. The overall removal efficiencies of COD and SS of two-stage were 93% and 69%,
Table 3 e Summary of energy recovery from hydrogen and methane production of two-stage recirculation methanogenic effluent process of POME at different recirculation rates. Recirculation rate (%) 50 40 30 25 20 15 0
Biohythane production (L/L-POME)
Energy recovery of H2 (%)
Energy recovery of CH4 (%)
Total energy recovery (%)
14.8 19.4 20.7 18.7 18.4 16.6 11.0
9.07 12.37 13.36 11.88 10.89 9.074 3.8
72.05 80.62 85.79 78.04 78.04 71.83 50.6
81.5 93 95.2 89.9 88.9 80.9 54.4
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Fig. 2 e Hydrogen production, pH, alkalinity, COD and OLR during thermophilic hydrogen production from POME by twostage recirculation process. Please cite this article in press as: O-Thong S, et al., Two-stage thermophilic fermentation and mesophilic methanogenic process for biohythane production from palm oil mill effluent with methanogenic effluent recirculation for pH control, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.07.095
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Fig. 3 e Methane production, pH, alkalinity, COD and OLR during mesophilic methane production from POME by two-stage recirculation process.
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Table 4 e Process performance and effluent characteristics of two-stage methanogenic effluent recirculation process of POME at 30% recirculation rate. Two stage process
Conditions HRT (d) Temperature ( C) Flow rate (L/d) Working Volume (L) Organic loading rate (kg-VS/m3/d) pH (initial) Gas production (Lbiogas/L/d) Gas Yield (ml H2 or CH4/g-VS) Removal efficiency Chemical oxygen demand (%) Total volatile solid (%) Total suspended solid (%) Carbohydrates consumption (%) Effluent pH Chemical oxygen demand (g/L) Alkalinity (g CaCO3/L) Volatile fatty acids (g/L) Ethanol (g/L) Acetic acid (g/L) Propionic acid (g/L) Butyric acid (g/L)
Hydrogen reactor (CSTR)
Methane reactor (UASB)
2 55 2.5 5 14.3 6.5 1.31 135
15 35 2.3 35 1.58 7.50 0.16 414
23.61 42.08 34.45 71.9
93.38 68.11 69.6 90.49
5.0 37.47 2.6 3.34 0.13 1.71 0.57 0.93
7.62 1.63 7.6 0.15 0.02 0.07 0.08 0.02
Fig. 4 e DGGE profile of bacterial community in sludge of hydrogen reactor from no recirculation phase (5, 15 and 25 day) and recirculation phase (35, 45 and 60 day) under thermophilic condition.
respectively. The maximum hydrogen production rate of 1.31 L H2/L/d and maximum methane production rate were 1.18 L CH4/L/d were achieved by two-stage recirculation process. The hydrogen and methane yield from POME by twostage recirculation process, which is within the range obtained in other studies from previous report (Table 5). Both reactor effluents were effective to keep at optimal pH with 2 times increasing in hydrogen production. It was previously reported that recirculation methanogenic effluent into hydrogen reactor of food waste could maintained the pH of the systems around 5.5 without addition of exogenous alkali [10,11].
Microbial population dynamics in two-stage recirculation process Two-stage recirculation process has higher hydrogen production than without recirculation, indicating different microbial community in hydrogen reactor. The DGGE results indicated a shift in the predominant bacteria phylum responsible for hydrogen production, from Clostridium sp., Enterococcus sp. and Marinomonas sp. to Thermoanaerobacterium sp., after methanogenic effluent recirculation began (Fig. 4). Two-stage process with methanogenic effluent recirculation flavored Thermoanaerobacterium sp. in the hydrogen reactor. Two-stage
Table 5 e Hydrogen and methane yield from two-stage anaerobic digestion process of various organic waste. Feedstocks
Skim latex serum Food waste Grass Agave tequilana OFMSW Dairy processing waste POME POME
Hydrogen reactor
Methane reactor
Yield
Reactor types
Yield
41.3 mL/gVS 50.0 mL/gVS 6.7 mL/gVS 60.0 mL/gCOD 29.0 mL/gVS 160.7 mL/gCOD 210.0 mL/gCOD 180 mL/gCOD
Batch CSTR CSTR Batch CSTR IBR ASBR CSTR
321.1 440.0 349.4 240.0 287.0 178.1 315.0 271.2
Reactor types
mL/gVS mL/gVS mL/gVS mL/gCOD mL/gVS mL/gCOD mL/gCOD mL/gCOD
Batch CSTR CSTR Batch CSTR IBR UASB UASB
Total energy yield
Temperature ( C)
References
11.9 kJ/gVS 16.3 kJ/gVS 12.6 kJ/gVS 9.2 kJ/gCOD 10.6 kJ/gVS 8.1 kJ/gCOD 13.4 kJ/gCOD 11.6 kJ/gCOD
55/55 35/35 35/35 35/35 55/55 55/55 55/35 55/35
[27] [30] [31] [32] [33] [34] [3] This study
OFMSW ¼ organic fraction of municipal solid waste; IBR ¼ induced bed reactors.
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recirculation process perform well in terms of hydrogen production or carbohydrates degradation compared to those without recirculation, perhaps due to an available alkalinity and nitrogen source. Methanogenic effluent recirculation was supplemented the NHþ 4 into the hydrogen reactor [6]. Nitrogen source is nutrient species specifically for Thermoanaerobacterium sp. [25]. The enhance of hydrogen production obtained with recirculation under thermophilic condition, which may not flavored mesophilic hydrogen consumption bacteria and archaea containing in methanogenic effluent [5]. Thermoanaerobacterium is one of the most important hydrogenproducing species [3,18,25]. For the growth of Thermoanaerobacterium sp., the optimal temperature and pH ranges are 55e60 C and 5.2e6.5, which together allowed efficient hydrogen production when POME as substrates [5]. Analysis of the microbial communities showed that methanogenic effluent recirculation also enriched bacterial communities in the methane reactor. DGGE profiling of sludge from methane reactor illustrated that Clostridium sp., Fervidobacterium sp., and Ruminococcus sp. were dominant bacteria in methane production stage. Methanosarcina sp. and Methanoculleus sp. were dominant and played an important role in methane production (Fig. 5). Methanosarcina species were reported to be dominant at high acetate concentration (>1.2 mM), and the results were consistent with the high acetate concentrations in hydrogen effluent that feed to methane reactors. Methanoculleus species were responsible for hydrogenotrophic methanogenesis [1].
Conclusions Recirculation of methanogenic effluent into hydrogen reactor promoted the generation of significant amount of hydrogen gas. Both hydrogen production rate and yield increased in methanogenic effluent recirculation systems. Maximum hydrogen (4.1 L H2/L-POME) and methane production (16.6 LCH4/L-POME) from POME in batch tests were achieved at 30% recirculation rat of methanogenic effluent. The recirculation of methanogenic effluent at 30% recirculation rate could compensate for alkalinity required by hydrogen reactor. Both reactor effluents were effective to keep at optimal pH with 2 times increasing in hydrogen production. Continuous hydrogen and methane production of two stage recirculation process was 3.8 L H2/L-POME and 14 L CH4/L-POME, respectively. The hydrogen and methane yield were 135 mL H2/gVS and 414 mL CH4/gVS, respectively. Biohythane gas composition was composed 13.3% of hydrogen, 54.4% of methane and 32.2% carbon dioxide. Thermoanaerobacterium sp. was dominated during hydrogen production, whereas archaea belonging to Methanosarcina sp. and Methanoculleus sp. were dominated in the methane reactor. Two-stage process with methanogenic effluent recirculation for biohythane production flavored Thermoanaerobacterium sp. in the hydrogen reactor and could efficiently for energy recovery from POME. Fig. 5 e DGGE profile of bacterial (A) and archaea (B) community in sludge of methane reactor from no recirculation phase (5, 15 and 25 day) and recirculation phase (35, 45 and 60 day) under mesophilic condition.
Acknowledgments This work was supported by the Core-to-Core Program, which was financially supported by aJapan Society for the Promotion
Please cite this article in press as: O-Thong S, et al., Two-stage thermophilic fermentation and mesophilic methanogenic process for biohythane production from palm oil mill effluent with methanogenic effluent recirculation for pH control, International Journal of Hydrogen Energy (2016), http://dx.doi.org/10.1016/j.ijhydene.2016.07.095
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of Science, National Research Council of Thailand, Vietnam Ministry of Science and Technology (MOST), the National University of Laos, Beuth University of Applied Sciences and Brawijaya University, Research and Development Institute Thaksin University (RDITSU), Agricultural Research Development Agency, Research Group for Development of Microbial Hydrogen Production Process from Biomass, Khon Kaen University and Thailand Research Fund through grant number MRG5580074 and RTA5780002 for financial support.
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