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Enhancement of hydrogen production by optimization of pH adjustment and separation conditions following dilute acid pretreatment of lignocellulosic biomass
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Enhancement of hydrogen production by optimization of pH adjustment and separation conditions following dilute acid pretreatment of lignocellulosic biomass Ralph Rolly Gonzales a,b,c, Gopalakrishnan Kumar a,b, Periyasamy Sivagurunathan d, Sang-Hyoun Kim a,b,* a Sustainable Environmental Process Research Institute, Daegu University, Jillyang, Gyeongsan, Gyeongbuk 38453, South Korea b Department of Environmental Engineering, Daegu University, Jillyang, Gyeongsan, Gyeongbuk 38453, South Korea c School of Civil and Environmental Engineering, University of Technology Sydney, 15 Broadway, Ultimo, New South Wales 2007, Australia d Center for Material Cycles and Waste Management Research, National Institute of Environmental Studies, Tsukuba 305-0053, Japan
article info
abstract
Article history:
Biorefinery is the integration of various conversion and separation unit processes of
Received 6 March 2017
biomass to energy, among other products. Downstream processes link these unit pro-
Received in revised form
cesses; however, these are often overlooked to affect energy yield. In this study, use of
2 May 2017
different alkaline agents and separation techniques, and order of operations, was assessed
Accepted 3 May 2017
after conversion of processed sugar into hydrogen through dark fermentation. pH was
Available online xxx
adjusted to pH 6 using various basic agents; and vacuum filtration and centrifugation were performed to facilitate separation. Sugar loss of 7e40% due to the downstream processes
Keywords:
was recorded; however, optimization of the processes ensured high volume and sugar
Biohydrogen
recovery and low degradation byproduct production. Satisfactory volume recovery with
Dilute acid pretreatment
high sugar and low byproduct concentrations were achieved after vacuum filtration and pH
Lignocellulosic biomass
adjustment with aqueous base. H2 yield and production rate significantly increased after
pH adjustment
performing the downstream processes. Peak H2 production rate and yield were
Separation
1824 mL H2 L1 d1 and 1.27 mol H2 mol1 sugar, respectively, for the optimum condition of vacuum filtration, followed by pH adjustment using 8 N Ca(OH)2. © 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
Introduction Renewable energy sources are currently of great interest due to increasing demand for energy, caused by development and
increase in population. Due to non-renewability, fossil fuels are considered to be limited energy sources, which also pose adverse environmental effects, such as greenhouse gas emissions [1]. Biorefinery is the integration of various
* Corresponding author. Sustainable Environmental Process Research Institute, Daegu University, Jillyang, Gyeongsan, Gyeongbuk 38453, South Korea. Fax: þ82 53 850 6699. E-mail address: [email protected] (S.-H. Kim). http://dx.doi.org/10.1016/j.ijhydene.2017.05.021 0360-3199/© 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved. Please cite this article in press as: Gonzales RR, et al., Enhancement of hydrogen production by optimization of pH adjustment and separation conditions following dilute acid pretreatment of lignocellulosic biomass, International Journal of Hydrogen Energy (2017), http://dx.doi.org/10.1016/j.ijhydene.2017.05.021
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conversion and separation unit processes of biomass to fuel, chemicals, and energy. These processes enable the harnessing of renewable energy from carbohydrate-rich agricultural residues and biological matter by thermochemical conversion of biomass [2]. Thus, it is considered as one of the solutions to reduce carbon footprint and dependency on fossil fuel for sustainable development [3]. Among the currently studied renewable energy sources for biofuel production is lignocellulosic biomass, whose recalcitrant characteristic is due to its complex matrix structure composed of cellulose, hemicellulose, lignin and other components [4e6]. The use of raw lignocellulosic biomass yielded low production of biofuels and exhibited low production rates due to the low accessibility of the feedstock by microbial populations [7]. Pretreatment processes are needed to break the polysaccharideelignin complex links, thereby increasing the accessibility of cellulose and hemicelluloses for biorefinery [8e10]. Dilute acid pretreatment is one of the most widely performed pretreatment methods for lignocellulosic biomass [4]. Dilute acid is normally preferred over concentrated acid since it is cost effective and environmentally-friendly [11]. During dilute acid pretreatment, the biomass is exposed to harsh physicochemical conditions to obtain a solution rich with monomeric sugars. The lignin-carbohydrate complexes are removed or disrupted, making the cellulose easily accessible to enzymes and microorganisms [12]. Downstream processes, such as pH adjustment and separation, accompany the chemical pretreatment prior to the biological conversion steps [13,14]. pH adjustment is essential because biological conversion of biomass to biofuels is known to be significantly affected by pH, of which typical optimum values are near neutral [8,15e17]. Moreover, pH of lignocellulosic hydrolyzate is sometimes adjusted to a mild basic pH range to facilitate precipitation of potential inhibitory compounds such as furfural and 5-hydroxymethylfurfural (5-HMF) [18]. Separation is also inevitable to fractionate the hydrolyzate from lignin and insoluble compounds. An ideal separation method of the aqueous and solid fractions of the hydrolyzate must recover as much sugar into aqueous phase as possible. The physicochemical unit processes between dilute acid hydrolysis and biological conversion may affect the sugar and byproduct contents of the hydrolyzate, although they have been regarded not to affect the recovery of the soluble compounds [19]. Dilute acid pretreatment of lignocellulosic biomass has been conducted in various studies; however, each study made use of different methods for neutralization and fractionation for the hydrolyzate. The study of the best neutralization and fractionation methods is important since the pH, state, and composition of the hydrolyzate are integral to the fermentation process taking place after pretreatment. After the downstream processes, the objective is to produce an energyrich biomass hydrolyzate that can be used for biogas and biofuel production [20]. According to Sievers et al., solideliquid separations are useful to provide a solids-free sugar stream for fermentation. Solids are known to interfere with fermentation of sugars [21]. Moreover, neutralization and fractionation must be optimized as sustainable and costeffective treatment methods for lignocellulosic hydrolyzate.
pH adjustment and separation downstream processes for hydrolyzates of lignocellulosic biomass are commonly performed in biorefinery processes. However, little attention is provided to these methods. Only a few separation and pH adjustment studies have been published for dilute acid hydrolyzate of lignocellulosic biomass. Moreover, no studies have shown the direct effect of these processes to H2 fermentation, according to the authors' knowledge. Hydrogen is an environmentally friendly biofuel with high energy efficiency [22,23], making it a promising alternative energy carrier. Due to the high energy consumption requirement of conventional physico-chemical H2 production methods, interest in biohydrogen has increased significantly in the recent years. Dark fermentation, or light-independent process, uses genera Clostridium, Enterobacter, or mixed culture dominated by these microorganisms to convert sugars into H2 [24]. Among these microbial species, Clostridium species were identified as the most dominant H2-producing bacteria for mesophilic dark fermentation at pH 5.5 [1]. These bacteria make use of monomeric sugars present in the dilute acid hydrolyzate of lignocellulosic biomass as substrate for dark fermentative process [24]. Pine tree wood, an agricultural feedstock of the wood pulp and paper industry, was used as the substrate for this study. This particular biomass was chosen since the paper industry is important in South Korean economy and this biomass is just disposed as waste or incinerated. Hydrolysis of lignocellulosic biomass, such as pine tree wood, leads production of a number of useful biofuels as compared to combustion, which primarily produces heat [25]. The objective of this study was to investigate resultant volume, sugar and byproduct contents, and biohydrogen production of dilute acid lignocellulosic hydrolyzate after pH adjustment and separation with various neutralizing agents (calcium hydroxide and sodium hydroxide), agent forms (powder and aqueous solution), separation methods (centrifugation and vacuum filtration) and the sequence of the unit processes (neutralizationeseparation and separationeneutralization). This study puts light to the significance and relevance of often overlooked yet important downstream process conditions, particularly, pH adjustment and separation, on biological conversion of biomass to bioenergy.
Materials and methods Dilute acid pretreatment of lignocellulosic biomass Untreated pine tree wood pellet was acquired from a local paper production company and was the lignocellulosic biomass used in this study. The glucan, xylan, arabinan, lignin, ash, and extractives composition of the biomass were measured by following the NREL laboratory analytical procedure [26]. The biomass composition is presented, on dry basis, in Table 1. The pine tree wood pellet was initially milled to reduce the particle size to 1e2 mm. Dilute acid pretreatment was performed with 10% (w/v) milled woody biomass and 5% (w/v) sulfuric acid (Duksan Pure Chemicals, South Korea), and carried out in an autoclave (Hanbaek Scientific, South Korea) at
Please cite this article in press as: Gonzales RR, et al., Enhancement of hydrogen production by optimization of pH adjustment and separation conditions following dilute acid pretreatment of lignocellulosic biomass, International Journal of Hydrogen Energy (2017), http://dx.doi.org/10.1016/j.ijhydene.2017.05.021
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Table 1 e Composition of pine tree wood pellet. Component Glucan Xylan Arabinan Lignin Ash Extractives
Content, % (w/w) 39.48 ± 0.87 20.50 ± 0.49 1.60 ± 0.18 37.11 ± 2.05 1.25 ± 0.03 0.06 ± 0.01
121 C for 30 min. The severity factor of this pretreatment process was 1.83.
pH adjustment and separation of the dilute acid lignocellulosic hydrolyzate The pH adjustment and separation scheme is summarized in Fig. 1. The solution freshly obtained from the dilute acid pretreatment step undergoes a series of steps to adjust the pH and to separate the solid and liquid components of the solutions. NaOH or Ca(OH)2 was added to adjust the pH of the dilute acid hydrolyzate to 5.5e6 from initial pH of 0.5 to 1.0. The alkaline agents are delivered into the hydrolyzate solution either in solid form or as an 8 N aqueous solution. Separation of the aqueous and solid fractions was performed using either centrifugation or vacuum filtration. Centrifugation was carried out at 67.2 g relative centrifugal force (RCF) for 5 min using a high-speed centrifuge (Combi-514R, Hanil Science, South Korea). Vacuum filtration was performed with 47 mm glass-fiber filters (Grade GF/C, Whatman, MO, USA). The pH adjustment and separation scheme was done in triplicate.
Batch H2 fermentation Anaerobic granular sludge was obtained from an upflow sludge blanket reactor treating brewery wastewater in South Korea. The pH, volatile suspended solids (VSS), and total suspended solids (TSS) concentration of the sludge were 6.8,
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12.6 g L1, and 22.6 g L1, respectively. The sludge was heattreated at 90 C for 30 min to harvest only anaerobic sporeforming H2-producing bacteria [27] and was used as the inoculum of the following batch H2 fermentation. H2 fermentation was conducted in 100 mL serum bottles. The hydrolyzate solutions obtained after all separation and neutralization schemes were used as the substrate. The hydrolyzate solutions were diluted to obtain an initial sugar concentration of 10 g L1. 30 mL of the hydrolyzate solution with 10 g L1 initial sugar concentration was added to the serum bottle. This initial sugar concentration was previously found by Gonzales et al. [25] to provide the highest H2 production using pine tree wood as substrate. Mineral medium was supplied as follows: 6.72 g L1 NaHCO3, 3.00 g L1 NH4CO3, 0.125 g L1 KH2PO4, 0.100 g L1 MgCl2$6H2O, 0.015 g L1 MnSO4$6H2O, 0.025 g L1 FeSO4$7H2O, 0.005 g L1 CuSO4$5H2O, and 0.001 g L1 CoCl2$5H2O. 5 mL of seed inoculum and 5 mL mineral medium were added to obtain a solution with total working volume of 40 mL and initial pH of 7.0e7.5. The serum bottle was purged with N2 gas for 3 min and then agitated at 150 rpm and 35 C. The fermentation process was performed in duplicate.
Analytical methods Concentrations of monomeric sugars glucose, xylose, and arabinose in the dilute acid pine tree wood hydrolyzate were quantified using high performance liquid chromatography (Waters 717plus, MA, USA) with Aminex HPX-87P column (BioRad Laboratories, CA, USA) and a refractive index detector (Waters 410, MA, USA) with deionized water mobile phase. The organic acids and furan compounds contents were measured by HPLC system with Aminex HPX-87H (Bio-Rad Laboratories, USA) and an ultraviolet detector (Waters 2487, MA, USA) with 5 mM H2SO4 mobile phase, at 210 nm. SO2 4 ion concentration was analyzed by ion chromatography (Dionex Aquion IC System, Thermo Fisher Scientific, MA, USA) with Dionex IonPac AS14 anion exchange column and Dionex ICS-
Fig. 1 e Scheme of pH adjustment and separation experimental conditions. Please cite this article in press as: Gonzales RR, et al., Enhancement of hydrogen production by optimization of pH adjustment and separation conditions following dilute acid pretreatment of lignocellulosic biomass, International Journal of Hydrogen Energy (2017), http://dx.doi.org/10.1016/j.ijhydene.2017.05.021
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5000þ CD conductivity detector (Thermo Fisher Scientific, MA, USA), with a carbonate-bicarbonate buffer mobile phase. H2 content was analyzed by gas chromatography (SRI Instruments, USA) with a thermal conductivity detector (TCD) and a 1.8 m 3.2 mm stainless-steel column packed with mole sieve 13 and high purity N2 as the carrier gas. The temperatures of the injector, column, and detector were maintained at room temperature, 80 C, and 90 C, respectively. TSS and VSS were measured according to standard procedures [28].
Assay The H2 production with respect to time was fitted to the values estimated by a modified Gompertz equation (Eq. (1)) to estimate maximum H2 volume, maximum H2 production rate, lag time of H2 production, and H2 production yield [29,30]: RH ðl tÞ þ 1 H ¼ P exp exp P
(1)
where H, P, RH, l, t, and e are cumulative H2 production (mL), ultimate H2 production (mL), H2 production rate (mL d1), lag time (h), time (h), and exponential 1, respectively.
Results and discussion Neutralization of the dilute acid lignocellulosic hydrolyzate The concentrations of sugar and byproducts present in the hydrolyzate solutions after neutralization using powder and aqueous solutions of Ca(OH)2 and NaOH were compared in Table 2, Figs. 2 and 3. The data table and figures present the following terms, total sugar recovery, relative sugar recovery, and volume recovery, which were all calculated parameters in this study. Total sugar recovery of the hydrolyzate solution is the ratio of the total sugar content in
the hydrolyzate solution and the total sugar content in the pine tree wood pellet. Relative sugar recovery, on the other hand, is the ratio of the total sugar content in the hydrolyzate solution following the downstream processes and the total sugar content in the hydrolyzate prior to the downstream processes. This parameter shows that as we go down through the biological conversion process and prepare the substrate for fermentation, the loss of sugar is inevitable. Lastly, volume recovery is the ratio of the volume of the hydrolyzate solution following the downstream processes and the volume of the hydrolyzate prior to the said processes. This study aims to find the pH adjustment and separation scheme with high relative sugar recovery and volume recovery. Furthermore, this study hopes to bring light to certain issues of using lignocellulosic biomass as feedstock for bioenergy production, such as cost advantages and process handling operations. Hydrolyzate solutions treated with both powder and aqueous NaOH showed slightly higher concentrations of xylose and glucose compared to those treated with Ca(OH)2. NaOH showed more volume recovery compared to Ca(OH)2. The slurry form of Ca(OH)2 makes it a difficult basic substance to handle, as it rapidly separates from solution, not providing accurate concentration of the base at all times. Thus, if the subsequent fermentation can accept the sulfate and sodium concentration of the hydrolyzate [31], then NaOH would be better neutralizing agent. For both agents, the hydrolyzate treated with aqueous basic solutions showed higher sugar concentrations than the powder-treated hydrolyzate. As concentrated basic solutions were used, decrease of sugar concentration by dilution was only 5e9%. Nissila et al. [32] reported that the acid hydrolyzate neutralized with aqueous Ca(OH)2 solution provided the highest sugar recovery, compared to solid basic substances Ca(OH)2 and CaO. The same study explained that the use of solid alkaline agents can cause precipitation and degradation of sugars in solution [32].
Table 2 e The summary of sugar recovery (based on dry biomass weight) according to neutralization and separation. Conditions
Glucose, %
Xylose, %
Arabinose, %
Total sugar recovery, %a
Hydrolyzate without neutralization nor separation Vacuum filtration Vacuum filtration þ 8 N Ca(OH)2 Vacuum filtration þ 8 N NaOH Solid Ca(OH)2 þ vacuum filtration Solid NaOH þ vacuum filtration 8 N Ca(OH)2 þ vacuum filtration 8 N NaOH þ vacuum filtration 1000 rpm centrifugation 1000 rpm centrifugation þ 8 N Ca(OH)2 1000 rpm centrifugation þ 8 N NaOH Solid Ca(OH)2 þ 1000 rpm centrifugation Solid NaOH þ 1000 rpm centrifugation 8 N Ca(OH)2 þ 1000 rpm centrifugation 8 N NaOH þ 1000 rpm centrifugation
24.93 ± 0.63
66.81 ± 1.97
37.78 ± 5.17
38.22 ± 0.25
e
17.89 ± 0.74 15.12 ± 0.73 18.45 ± 1.33 12.94 ± 0.45 14.34 ± 1.83 14.27 ± 0.28 16.11 ± 1.18 17.41 ± 0.06 9.96 ± 0.85 12.78 ± 0.48 9.22 ± 0.45 9.94 ± 0.13 9.59 ± 0.23 10.33 ± 0.38
64.63 ± 57.76 ± 63.89 ± 50.65 ± 48.76 ± 51.72 ± 52.06 ± 65.33 ± 47.41 ± 53.13 ± 47.94 ± 49.51 ± 48.35 ± 48.36 ±
35.36 26.81 24.38 20.72 28.44 23.16 20.31 34.78 21.13 25.19 22.75 23.16 24.38 19.50
± 1.82 ± 1.15 ± 1.15 ± 1.72 ± 1.15 ± 2.87 ± 4.60 ± 1.36 ± 2.30 ± 4.60 ± 1.15 ± 2.87 ± 2.30 ± 0.00
35.23 ± 4.62 28.91 ± 0.55 33.09 ± 1.37 25.15 ± 0.11 25.42 ± 0.92 26.36 ± 0.91 27.65 ±1.29 33.79 ± 1.29 22.16 ± 1.33 25.87 ± 0.72 21.86 ± 0.79 22.85 ± 0.63 22.24 ± 1.47 22.72 ± 0.30
93.28 75.65 86.57 65.80 66.50 68.96 72.35 89.46 57.98 67.69 57.19 59.79 58.18 59.44
a b
3.62 3.06 1.55 1.20 0.83 2.19 1.62 1.67 2.35 3.08 1.52 1.66 3.99 0.19
Relative sugar recovery, %b
Computed from sugar content (dry basis) of the lignocellulosic biomass. Relative to the sugar recovery before separation and neutralization.
Please cite this article in press as: Gonzales RR, et al., Enhancement of hydrogen production by optimization of pH adjustment and separation conditions following dilute acid pretreatment of lignocellulosic biomass, International Journal of Hydrogen Energy (2017), http://dx.doi.org/10.1016/j.ijhydene.2017.05.021
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Fig. 2 e The total sugar concentrations and final hydrolyzate volumes for (a) filtration and pH adjustment conditions and (b) centrifugation and pH adjustment conditions.
Separation of the solid and aqueous hydrolyzate fractions An ideal separation of the aqueous and solid in the hydrolyzate would efficiently separate the two phases and recover sugar into aqueous phase as much as possible. Separation of lignocellulosic biomass hydrolyzate is difficult due to formation of impermeable filter cakes and fibrous properties of the residues [21]. Particle size distribution in the hydrolyzate is a very important factor in solideliquid fractionation. In filtration, only particles larger than the filter pore size are retained; and during centrifugation, particles larger than 1 mm settle, while particles between 1 nm and 1 mm form colloids, which then suspend in mixture due to Brownian motion [20]. Centrifugation and vacuum filtration are the two most widely performed methods for separation of the hydrolyzate fractions [33]. Centrifugation causes separation by means of
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centrifugal force. The solids and liquids are transported to either end of a centrifuge tube by rotating the centrifuge at a high speed [20]. Pressurized filtration, under which category vacuum filtration falls in, separates solids and liquids through a filter, with the aid of applied pressure. The filter cake is compressed during pressure filtration, ensuring a solid fraction with high dry matter content and high recovery of liquor [20,21]. The use of centrifugation requires costly centrifugation equipment and electrical energy, and solideliquid separation is highly dependent on centrifugal force and efficiency of decantation. The use of centrifugation, therefore, is not cost-effective in large scale operations typical of bioenergy production, especially because complete separation is not always ensured when centrifugation is performed. Vacuum filtration, on the other hand, requires intricate equipment, vacuum conditions and the use of specialized filters with various pore sizes, but this method usually shows better separation performance [33]. Also, industrial scale vacuum filters can be reused by washing between cycles and can be changed only during maintenance and occurrence of fouling. However, little information has been available if these two methods can be used interchangeably without affecting sugar recovery significantly. Sugar analysis of the hydrolyzate fractions obtained for both methods are shown in Table 2 and Fig. 2. In any case, the downstream processes could not completely recover all the sugars in hydrolyzate. Vacuum filtration exhibited better dewatering performance than centrifugation, ensuring better separation of the solid and aqueous fractions, thus higher volumes were obtained for filtration as compared to centrifugation. The sugar concentrations obtained from filtration also showed slightly higher glucose and xylose recovery than centrifuged hydrolyzate fraction comparatively. On the other hand, there was no significant difference on the concentrations of organic acids, furfural, and 5-HMF. These compounds are known to be potential inhibitors to fermentative organisms [34]. Therefore, vacuum filtration would be the preferred method if a high-volume dilute acid hydrolyzate with high sugar yield is required [35]. This is validated by Hjorth et al. [20], who reported that the advantage of pressurized filtration over centrifugation is the production of a solid fraction with a high solids concentration. It would then be advantageous to choose vacuum filtration since this process concentrated the solids the most, liberating the most of the sugar-laden hydrolyzate liquor. Fig. 1 and Table 2 show that hydrolyzate obtained from separation followed by neutralization showed slightly higher sugar recovery as compared to the hydrolyzate which were neutralized first. Furthermore, no significant variations were observed for the presence of the degradation byproducts for all samples. Performing vacuum filtration before neutralization ensures higher sugar concentration and volume recovery, due to complete separation of the solid and liquid fractions. More so, performing separation first avoids the alkalinity of the solid fraction, which is essential for further enzymatic saccharification experiments using the biomass.
Batch H2 fermentation Modified Gompertz equation (Eq. (1)) was used to fit the cumulative H2 production curves during the batch fermentation.
Please cite this article in press as: Gonzales RR, et al., Enhancement of hydrogen production by optimization of pH adjustment and separation conditions following dilute acid pretreatment of lignocellulosic biomass, International Journal of Hydrogen Energy (2017), http://dx.doi.org/10.1016/j.ijhydene.2017.05.021
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Fig. 3 e The concentrations of the degradation products formic acid, acetic acid, levulinic acid, furfural, and 5-HMF for (a) filtration and pH adjustment conditions, and (b) centrifugation and pH adjustment conditions.
The correlation coefficients, R2, are larger than 0.99, indicating accuracy of the fit. The H2 fermentation parameters, volumetric HPR (mL L1 d1) and H2 yield (L H2 mol1 sugar), were estimated and shown in Table 3. H2 yield was computed based on sugar equivalents consumed and H2 production. Generally favorable H2 production was observed in this study, due to the use of inoculum sourced from a municipal wastewater treatment plant. Sludge sourced from wastewater treatment plants contain more potential hydrolytic and fermentative microbial population, compared to other inoculum sources, such as cow dung and compost [36]. While the highest sugar recovery was found for the dilute acid hydrolyzate obtained after vacuum filtration and dilution with aqueous NaOH, the highest H2 yield and H2 production rate were obtained when the hydrolyzate obtained after vacuum filtration and dilution with Ca(OH)2 was used as substrate. Based on the H2 yield and H2 production rate values tabulated in Table 3, hydrolyzates obtained after filtration did not show significant difference when used for H2 fermentation, when compared to hydrolyzates obtained after
centrifugation. When sugar concentration in the hydrolyzate was fixed to 10 g L1, the concentrations of inhibitors, such as furfural and 5-HMF, are likewise diluted. The concentrations of these inhibitors did not vary significantly with either use of filtration and centrifugation as means of separation. However, it was noticeable that the H2 yield and H2 production rate are significantly higher for hydrolyzates neutralized with Ca(OH)2. The use of Ca(OH)2 as a neutralizing agent forms CaSO4, an insoluble salt; using NaOH would then form Na2SO4, a soluble compound, leaving SO2 4 present in the solution [32]. Higher SO2 4 concentrations at pH 5.5e6 generally inhibit H2 production due to increased activity of sulfate-reducing bacteria (SRB) [31]. Although the heat treatment was used in this study, all SRB in activated sludge might not be inactivated completely. It was reported that some SRB species were able to survive at extremely high temperature, even up to 140 C [37]. The reduced H2 production is attributed to acclimation of the SRB to SO2 4 -reducing conditions as a response to increased SO2 4 level. Since H2S content of the biogas was not measured in this study, it could not be confirmed if SO2 in the 4
Please cite this article in press as: Gonzales RR, et al., Enhancement of hydrogen production by optimization of pH adjustment and separation conditions following dilute acid pretreatment of lignocellulosic biomass, International Journal of Hydrogen Energy (2017), http://dx.doi.org/10.1016/j.ijhydene.2017.05.021
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Table 3 e H2 production rate (HPR) and H2 yield (HY) of hydrolyzates from dilute acid pretreatment of pine tree wood. Conditions
Without separation nor neutralization Vacuum filtration þ 8 N Ca(OH)2 Vacuum filtration þ 8 N NaOH Solid Ca(OH)2 þ vacuum filtration Solid NaOH þ vacuum filtration 8 N Ca(OH)2 þ vacuum filtration 8 N NaOH þ vacuum filtration 1000 rpm centrifugation þ 8 N Ca(OH)2 1000 rpm centrifugation þ 8 N NaOH Solid Ca(OH)2 þ 1000 rpm centrifugation Solid NaOH þ 1000 rpm centrifugation 8 N Ca(OH)2 þ 1000 rpm centrifugation 8 N NaOH þ 1000 rpm centrifugation
5-HMF concentration, g L1 0.80 ± 0.34 ± 0.35 ± 0.65 ± 0.54 ± 0.67 ± 0.62 ± 0.56 ± 0.45 ± 0.78 ± 0.69 ± 0.73 ± 0.64 ±
0.26 0.23 0.27 0.39 0.34 0.23 0.42 0.27 0.18 0.29 0.35 0.22 0.28
Furfural concentration, g L1 0.26 0.13 0.03 0.26 0.03 0.35 0.13 0.24 0.20 0.24 0.42 0.42 0.19
hydrolyzate solutions was indeed reduced by SRB. The SO2 4 concentrations of the hydrolyzates are shown in Table 3. 1 optimal value While all values are below the 3000 mg SO2 4 L for H2 production at pH 5.5 [27], the hydrolyzate solutions treated with Ca(OH)2 have significantly less SO2 4 levels than those treated with NaOH. Presence of lower amounts SO2 4 in the solutions decreases the probability of inhibition of H2 production [38]. Comparison of the two alkaline agents shows that while NaOH was found to be better for sugar recovery, Ca(OH)2 was found to be better for H2 production. If the objective of a study is to generate a hydrolyzate solution with high sugar content via dilute acid pretreatment, NaOH proved to be the better alkali for pH adjustment. In studies like this one, wherein the dilute acid pretreatment is a preliminary step to obtain a monomeric sugar-rich solution from biomass prior to biological conversion to energy, Ca(OH)2 must be used as the alkali for pH adjustment downstream process. The use of Ca(OH)2 also eliminates the presence of SO2 4 ; the presence of which increases the activity of SRB, hereby competing with H2 fermentative bacteria, and ultimately inhibiting H2 fermentation. Butyric and acetic acids were the main volatile fatty acids (VFA) produced during the fermentation as shown in Fig. 4. The figure also shows a comparison of the total concentration of soluble metabolic products (SMP), which include the VFA and the furan compounds. Chaganti et al. [39] mentioned that these metabolites are typically observed with H2 fermentation processes which make use xylose and glucose as substrates at pH 5.5. Acetic and butyric acids are known to be byproducts of H2 fermentation. This suggests that acetic and butyric acid pathways were the dominant routes for H2 production [39] and the fermentable sugars in the solutions were utilized only in H2 production. Peak H2 yields from dark H2 fermentation using various lignocellulosic biomass in previous studies are shown in Table 4. All of these studies explicitly performed acid pretreatment of the biomass and downstream separation and pH adjustment processes prior to H2 fermentation. The units for hydrogen yield for all studies were converted to mol H2 g1 sugar, for uniformity. Cui et al. [40] reported H2 yield of 53 mol H2 g1 total sugar (TS) with beer lees, whose acid
± 0.01 ± 0.00 ± 0.00 ± 0.08 ± 0.01 ± 0.17 ± 0.00 ± 0.14 ± 0.02 ± 0.09 ± 0.17 ± 0.05 ± 0.02
SO2 4 concentration, g L1
HPR, mL H2 L1 d1
1.79 1.52 2.49 1.64 2.94 1.68 2.54 1.74 2.55 1.86 2.58 1.91 2.72
1001 ± 0.24 1824 ± 1.44 1774 ± 2.64 1138 ± 3.12 1208 ± 1.68 1815 ± 6.00 1198 ± 2.64 1590 ± 1.20 1268 ± 4.08 1192 ± 0.48 963 ± 1.20 1552 ± 1.92 961 ± 2.40
HY, mol H2 mol1 sugar 0.31 ± 1.27 ± 1.27 ± 0.70 ± 0.88 ± 0.99 ± 0.89 ± 0.62 ± 0.68 ± 0.76 ± 0.60 ± 0.97 ± 0.75 ±
0.11 0.15 0.18 0.05 0.35 0.02 0.03 0.08 0.14 0.01 0.06 0.05 0.06
hydrolyzate was treated with aqueous NaOH prior to H2 fermentation with mixed culture. The dilute acid hydrolyzate of corn stover was first centrifuged, treated with 2 N Ca(OH)2, and then centrifuged once more before H2 fermentation with T. thermosaccharolyticum W16 as inoculum in a study by Cao et al. [41]. This study was able to produce 291.3 mL H2 g1 TS. Chong et al. [38] used acid-pretreated oil palm empty fruit bunch which was filtered using a 0.45 mm pore size filter and treated with 5 N NaOH to produce 278.0 mL H2 g1 xylose after fermentation with anaerobic sludge. Acid hydrolyzate of poplar leaves was simply treated with aqueous NaOH to make its pH suitable for H2 fermentation, and produced 44.9 mL H2 g1 TS in a study conducted by Cui et al. [42]. Centrifugation followed by pH adjustment with 0.1 M TriseHCl buffer was the downstream separation and pH adjustment processes for rice straw hydrolyzate in another study by Loo et al. [43], which produced 106.7 mL H2 g1 reducing sugars (RS). Monlau et al. [44] performed filtration with a 0.25 m pore size filter, followed by pH adjustment with 1 N NaOH on acid hydrolyzate of sunflower stalks. This study produced 286.5 mL H2/g hexose using anaerobic sludge as inoculum. Pattra et al. [45] employed two separation processes for its acid hydrolyzate of sugarcane bagasse. The hydrolyzate was filtered, after which Ca(OH)2 was added, and it was centrifuged before H2 fermentation producing 242.9 mL H2 g1 TS. The use of pure culture, such as Thermoanaerobacterium thermosaccharolyticum W16 and Clostridium butyricum, gave generally high H2 yields. Using initial 5 g L1 TS concentration in this study, dilute acid pretreated pine tree wood gave H2 yield of 178.3 and 179 mL H2 g1 TS, for hydrolyzate filtered and treated with 8 N NaOH and Ca(OH)2, respectively. concentrations ranged from 1.52 to The initial SO2 4 1 as shown in Table 3. The comparatively high 2.94 g SO2 4 L hydrogen yield implies that there was no severe inhibition by sulfate reducing bacteria and/or sulfide in this study. The heat-treatment of inoculum would have prevented the existence of sulfate reducing bacteria, as well as methanogens. The difference in concentrations of the SMP for the hydrolyzate treated with either NaOH or Ca(OH)2 is statistically insignificant. Based on this study, we can adjust the pH of the dilute acid hydrolyzate with aqueous solutions of either NaOH
Please cite this article in press as: Gonzales RR, et al., Enhancement of hydrogen production by optimization of pH adjustment and separation conditions following dilute acid pretreatment of lignocellulosic biomass, International Journal of Hydrogen Energy (2017), http://dx.doi.org/10.1016/j.ijhydene.2017.05.021
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Fig. 4 e The VFA production during the hydrogen fermentation using the dilute acid pine tree wood hydrolyzate.
Table 4 e Dark fermentative H2 production from various dilute acid pretreated lignocellulosic biomass in previous studies. Substrate
Downstream processes
H2 fermentation conditions
Inoculum
Beer lees Corn stover
Aqueous NaOH Centrifugation, 2 N Ca(OH)2, centrifugation Filtration (0.45 mm pore size), 5 N NaOH Aqueous NaOH Centrifugation, 0.1 M Tris eHCl buffer Filtration (0.25 mm pore size), 1 N NaOH Filtration, Ca(OH)2, centrifugation Filtration, 8 N NaOH
Batch, 35 C, pH 7.0 Batch, 60 C
Mixed culture T. thermosaccharolyticum W16
53.0 mL H2/g TSa 291.3 mL H2/g TS
[40] [41]
Batch, 35 C, 5 g TS/L Batch, 35 C, Batch, 37 C, 14 g RSb/L Batch, 35 C,
pH 7.0,
Anaerobic sludge
278.0 mL H2/g xylose
[38]
pH 7.0 pH 7.0,
Mixed culture Clostridium butyricum CGS5
44.9 mL H2/g TS 106.7 mL H2/g RS
[42] [43]
pH 5.5
Anaerobic sludge
286.5 mL H2/g hexose
[44]
Batch, 37 C, pH 7.0
Clostridium butyricum
242.9 mL H2/g TS
[45]
Batch, 35 C, pH 7.0, 10 g TS/L Batch, 35 C, pH 7.0, 10 g TS/L
Anaerobic granular sludge
178.3 mL H2/g TS
This study
Anaerobic granular sludge
179.0 mL H2/g TS
This study
Oil palm empty fruit bunch Poplar leaves Rice straw Sunflower stalks Sugarcane bagasse Pine tree wood Pine tree wood a b
Filtration, 8 N Ca(OH)2, filtration
H2 yield
Reference
Total sugar. Reducing sugar.
Please cite this article in press as: Gonzales RR, et al., Enhancement of hydrogen production by optimization of pH adjustment and separation conditions following dilute acid pretreatment of lignocellulosic biomass, International Journal of Hydrogen Energy (2017), http://dx.doi.org/10.1016/j.ijhydene.2017.05.021
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or Ca(OH)2 interchangeably, if and only if SO2 4 concentration is well below the threshold limit of the anaerobic bacteria. [8]
Conclusion [9]
A series of pH and separation downstream processes were performed to determine the best option to obtain liquid feedstock for fermentation of dilute acid pretreated lignocellulosic biomass. The downstream unit processes and their sequence significantly affected sugar recovery. Filtration followed by pH adjustment using aqueous NaOH recovered 95.29% of sugars liberated from dilute acid pretreatment. The dilute acid hydrolyzate solutions of pine tree wood were then used for dark hydrogen fermentation processes. The substrate concentration of the hydrolyzate solutions was adjusted to 10 g L1. The maximum H2 yield and H2 production rate were 1824 mL H2 L1 d1 and 1.27 mol H2 mol1 sugar, respectively, for the hydrolyzate solution obtained after vacuum filtration and pH adjustment using 8 N Ca(OH)2. The downstream processes would be applied to dilute acid hydrolyzate of other biomass as well for high sugar recovery and high H2 yield and production rate during H2 fermentative processes.
[10]
[11]
[12]
[13]
[14]
[15]
Acknowledgement This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2017R1A2A2A07000900).
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Available online at www.sciencedirect.com
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Enhancement of hydrogen production by optimization of pH adjustment and separation conditions following dilute acid pretreatment of lignocellulosic biomass Ralph Rolly Gonzales a,b,c, Gopalakrishnan Kumar a,b, Periyasamy Sivagurunathan d, Sang-Hyoun Kim a,b,* a Sustainable Environmental Process Research Institute, Daegu University, Jillyang, Gyeongsan, Gyeongbuk 38453, South Korea b Department of Environmental Engineering, Daegu University, Jillyang, Gyeongsan, Gyeongbuk 38453, South Korea c School of Civil and Environmental Engineering, University of Technology Sydney, 15 Broadway, Ultimo, New South Wales 2007, Australia d Center for Material Cycles and Waste Management Research, National Institute of Environmental Studies, Tsukuba 305-0053, Japan
article info
abstract
Article history:
Biorefinery is the integration of various conversion and separation unit processes of
Received 6 March 2017
biomass to energy, among other products. Downstream processes link these unit pro-
Received in revised form
cesses; however, these are often overlooked to affect energy yield. In this study, use of
2 May 2017
different alkaline agents and separation techniques, and order of operations, was assessed
Accepted 3 May 2017
after conversion of processed sugar into hydrogen through dark fermentation. pH was
Available online xxx
adjusted to pH 6 using various basic agents; and vacuum filtration and centrifugation were performed to facilitate separation. Sugar loss of 7e40% due to the downstream processes
Keywords:
was recorded; however, optimization of the processes ensured high volume and sugar
Biohydrogen
recovery and low degradation byproduct production. Satisfactory volume recovery with
Dilute acid pretreatment
high sugar and low byproduct concentrations were achieved after vacuum filtration and pH
Lignocellulosic biomass
adjustment with aqueous base. H2 yield and production rate significantly increased after
pH adjustment
performing the downstream processes. Peak H2 production rate and yield were
Separation
1824 mL H2 L1 d1 and 1.27 mol H2 mol1 sugar, respectively, for the optimum condition of vacuum filtration, followed by pH adjustment using 8 N Ca(OH)2. © 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.
Introduction Renewable energy sources are currently of great interest due to increasing demand for energy, caused by development and
increase in population. Due to non-renewability, fossil fuels are considered to be limited energy sources, which also pose adverse environmental effects, such as greenhouse gas emissions [1]. Biorefinery is the integration of various
* Corresponding author. Sustainable Environmental Process Research Institute, Daegu University, Jillyang, Gyeongsan, Gyeongbuk 38453, South Korea. Fax: þ82 53 850 6699. E-mail address: [email protected] (S.-H. Kim). http://dx.doi.org/10.1016/j.ijhydene.2017.05.021 0360-3199/© 2017 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved. Please cite this article in press as: Gonzales RR, et al., Enhancement of hydrogen production by optimization of pH adjustment and separation conditions following dilute acid pretreatment of lignocellulosic biomass, International Journal of Hydrogen Energy (2017), http://dx.doi.org/10.1016/j.ijhydene.2017.05.021
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conversion and separation unit processes of biomass to fuel, chemicals, and energy. These processes enable the harnessing of renewable energy from carbohydrate-rich agricultural residues and biological matter by thermochemical conversion of biomass [2]. Thus, it is considered as one of the solutions to reduce carbon footprint and dependency on fossil fuel for sustainable development [3]. Among the currently studied renewable energy sources for biofuel production is lignocellulosic biomass, whose recalcitrant characteristic is due to its complex matrix structure composed of cellulose, hemicellulose, lignin and other components [4e6]. The use of raw lignocellulosic biomass yielded low production of biofuels and exhibited low production rates due to the low accessibility of the feedstock by microbial populations [7]. Pretreatment processes are needed to break the polysaccharideelignin complex links, thereby increasing the accessibility of cellulose and hemicelluloses for biorefinery [8e10]. Dilute acid pretreatment is one of the most widely performed pretreatment methods for lignocellulosic biomass [4]. Dilute acid is normally preferred over concentrated acid since it is cost effective and environmentally-friendly [11]. During dilute acid pretreatment, the biomass is exposed to harsh physicochemical conditions to obtain a solution rich with monomeric sugars. The lignin-carbohydrate complexes are removed or disrupted, making the cellulose easily accessible to enzymes and microorganisms [12]. Downstream processes, such as pH adjustment and separation, accompany the chemical pretreatment prior to the biological conversion steps [13,14]. pH adjustment is essential because biological conversion of biomass to biofuels is known to be significantly affected by pH, of which typical optimum values are near neutral [8,15e17]. Moreover, pH of lignocellulosic hydrolyzate is sometimes adjusted to a mild basic pH range to facilitate precipitation of potential inhibitory compounds such as furfural and 5-hydroxymethylfurfural (5-HMF) [18]. Separation is also inevitable to fractionate the hydrolyzate from lignin and insoluble compounds. An ideal separation method of the aqueous and solid fractions of the hydrolyzate must recover as much sugar into aqueous phase as possible. The physicochemical unit processes between dilute acid hydrolysis and biological conversion may affect the sugar and byproduct contents of the hydrolyzate, although they have been regarded not to affect the recovery of the soluble compounds [19]. Dilute acid pretreatment of lignocellulosic biomass has been conducted in various studies; however, each study made use of different methods for neutralization and fractionation for the hydrolyzate. The study of the best neutralization and fractionation methods is important since the pH, state, and composition of the hydrolyzate are integral to the fermentation process taking place after pretreatment. After the downstream processes, the objective is to produce an energyrich biomass hydrolyzate that can be used for biogas and biofuel production [20]. According to Sievers et al., solideliquid separations are useful to provide a solids-free sugar stream for fermentation. Solids are known to interfere with fermentation of sugars [21]. Moreover, neutralization and fractionation must be optimized as sustainable and costeffective treatment methods for lignocellulosic hydrolyzate.
pH adjustment and separation downstream processes for hydrolyzates of lignocellulosic biomass are commonly performed in biorefinery processes. However, little attention is provided to these methods. Only a few separation and pH adjustment studies have been published for dilute acid hydrolyzate of lignocellulosic biomass. Moreover, no studies have shown the direct effect of these processes to H2 fermentation, according to the authors' knowledge. Hydrogen is an environmentally friendly biofuel with high energy efficiency [22,23], making it a promising alternative energy carrier. Due to the high energy consumption requirement of conventional physico-chemical H2 production methods, interest in biohydrogen has increased significantly in the recent years. Dark fermentation, or light-independent process, uses genera Clostridium, Enterobacter, or mixed culture dominated by these microorganisms to convert sugars into H2 [24]. Among these microbial species, Clostridium species were identified as the most dominant H2-producing bacteria for mesophilic dark fermentation at pH 5.5 [1]. These bacteria make use of monomeric sugars present in the dilute acid hydrolyzate of lignocellulosic biomass as substrate for dark fermentative process [24]. Pine tree wood, an agricultural feedstock of the wood pulp and paper industry, was used as the substrate for this study. This particular biomass was chosen since the paper industry is important in South Korean economy and this biomass is just disposed as waste or incinerated. Hydrolysis of lignocellulosic biomass, such as pine tree wood, leads production of a number of useful biofuels as compared to combustion, which primarily produces heat [25]. The objective of this study was to investigate resultant volume, sugar and byproduct contents, and biohydrogen production of dilute acid lignocellulosic hydrolyzate after pH adjustment and separation with various neutralizing agents (calcium hydroxide and sodium hydroxide), agent forms (powder and aqueous solution), separation methods (centrifugation and vacuum filtration) and the sequence of the unit processes (neutralizationeseparation and separationeneutralization). This study puts light to the significance and relevance of often overlooked yet important downstream process conditions, particularly, pH adjustment and separation, on biological conversion of biomass to bioenergy.
Materials and methods Dilute acid pretreatment of lignocellulosic biomass Untreated pine tree wood pellet was acquired from a local paper production company and was the lignocellulosic biomass used in this study. The glucan, xylan, arabinan, lignin, ash, and extractives composition of the biomass were measured by following the NREL laboratory analytical procedure [26]. The biomass composition is presented, on dry basis, in Table 1. The pine tree wood pellet was initially milled to reduce the particle size to 1e2 mm. Dilute acid pretreatment was performed with 10% (w/v) milled woody biomass and 5% (w/v) sulfuric acid (Duksan Pure Chemicals, South Korea), and carried out in an autoclave (Hanbaek Scientific, South Korea) at
Please cite this article in press as: Gonzales RR, et al., Enhancement of hydrogen production by optimization of pH adjustment and separation conditions following dilute acid pretreatment of lignocellulosic biomass, International Journal of Hydrogen Energy (2017), http://dx.doi.org/10.1016/j.ijhydene.2017.05.021
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Table 1 e Composition of pine tree wood pellet. Component Glucan Xylan Arabinan Lignin Ash Extractives
Content, % (w/w) 39.48 ± 0.87 20.50 ± 0.49 1.60 ± 0.18 37.11 ± 2.05 1.25 ± 0.03 0.06 ± 0.01
121 C for 30 min. The severity factor of this pretreatment process was 1.83.
pH adjustment and separation of the dilute acid lignocellulosic hydrolyzate The pH adjustment and separation scheme is summarized in Fig. 1. The solution freshly obtained from the dilute acid pretreatment step undergoes a series of steps to adjust the pH and to separate the solid and liquid components of the solutions. NaOH or Ca(OH)2 was added to adjust the pH of the dilute acid hydrolyzate to 5.5e6 from initial pH of 0.5 to 1.0. The alkaline agents are delivered into the hydrolyzate solution either in solid form or as an 8 N aqueous solution. Separation of the aqueous and solid fractions was performed using either centrifugation or vacuum filtration. Centrifugation was carried out at 67.2 g relative centrifugal force (RCF) for 5 min using a high-speed centrifuge (Combi-514R, Hanil Science, South Korea). Vacuum filtration was performed with 47 mm glass-fiber filters (Grade GF/C, Whatman, MO, USA). The pH adjustment and separation scheme was done in triplicate.
Batch H2 fermentation Anaerobic granular sludge was obtained from an upflow sludge blanket reactor treating brewery wastewater in South Korea. The pH, volatile suspended solids (VSS), and total suspended solids (TSS) concentration of the sludge were 6.8,
3
12.6 g L1, and 22.6 g L1, respectively. The sludge was heattreated at 90 C for 30 min to harvest only anaerobic sporeforming H2-producing bacteria [27] and was used as the inoculum of the following batch H2 fermentation. H2 fermentation was conducted in 100 mL serum bottles. The hydrolyzate solutions obtained after all separation and neutralization schemes were used as the substrate. The hydrolyzate solutions were diluted to obtain an initial sugar concentration of 10 g L1. 30 mL of the hydrolyzate solution with 10 g L1 initial sugar concentration was added to the serum bottle. This initial sugar concentration was previously found by Gonzales et al. [25] to provide the highest H2 production using pine tree wood as substrate. Mineral medium was supplied as follows: 6.72 g L1 NaHCO3, 3.00 g L1 NH4CO3, 0.125 g L1 KH2PO4, 0.100 g L1 MgCl2$6H2O, 0.015 g L1 MnSO4$6H2O, 0.025 g L1 FeSO4$7H2O, 0.005 g L1 CuSO4$5H2O, and 0.001 g L1 CoCl2$5H2O. 5 mL of seed inoculum and 5 mL mineral medium were added to obtain a solution with total working volume of 40 mL and initial pH of 7.0e7.5. The serum bottle was purged with N2 gas for 3 min and then agitated at 150 rpm and 35 C. The fermentation process was performed in duplicate.
Analytical methods Concentrations of monomeric sugars glucose, xylose, and arabinose in the dilute acid pine tree wood hydrolyzate were quantified using high performance liquid chromatography (Waters 717plus, MA, USA) with Aminex HPX-87P column (BioRad Laboratories, CA, USA) and a refractive index detector (Waters 410, MA, USA) with deionized water mobile phase. The organic acids and furan compounds contents were measured by HPLC system with Aminex HPX-87H (Bio-Rad Laboratories, USA) and an ultraviolet detector (Waters 2487, MA, USA) with 5 mM H2SO4 mobile phase, at 210 nm. SO2 4 ion concentration was analyzed by ion chromatography (Dionex Aquion IC System, Thermo Fisher Scientific, MA, USA) with Dionex IonPac AS14 anion exchange column and Dionex ICS-
Fig. 1 e Scheme of pH adjustment and separation experimental conditions. Please cite this article in press as: Gonzales RR, et al., Enhancement of hydrogen production by optimization of pH adjustment and separation conditions following dilute acid pretreatment of lignocellulosic biomass, International Journal of Hydrogen Energy (2017), http://dx.doi.org/10.1016/j.ijhydene.2017.05.021
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5000þ CD conductivity detector (Thermo Fisher Scientific, MA, USA), with a carbonate-bicarbonate buffer mobile phase. H2 content was analyzed by gas chromatography (SRI Instruments, USA) with a thermal conductivity detector (TCD) and a 1.8 m 3.2 mm stainless-steel column packed with mole sieve 13 and high purity N2 as the carrier gas. The temperatures of the injector, column, and detector were maintained at room temperature, 80 C, and 90 C, respectively. TSS and VSS were measured according to standard procedures [28].
Assay The H2 production with respect to time was fitted to the values estimated by a modified Gompertz equation (Eq. (1)) to estimate maximum H2 volume, maximum H2 production rate, lag time of H2 production, and H2 production yield [29,30]: RH ðl tÞ þ 1 H ¼ P exp exp P
(1)
where H, P, RH, l, t, and e are cumulative H2 production (mL), ultimate H2 production (mL), H2 production rate (mL d1), lag time (h), time (h), and exponential 1, respectively.
Results and discussion Neutralization of the dilute acid lignocellulosic hydrolyzate The concentrations of sugar and byproducts present in the hydrolyzate solutions after neutralization using powder and aqueous solutions of Ca(OH)2 and NaOH were compared in Table 2, Figs. 2 and 3. The data table and figures present the following terms, total sugar recovery, relative sugar recovery, and volume recovery, which were all calculated parameters in this study. Total sugar recovery of the hydrolyzate solution is the ratio of the total sugar content in
the hydrolyzate solution and the total sugar content in the pine tree wood pellet. Relative sugar recovery, on the other hand, is the ratio of the total sugar content in the hydrolyzate solution following the downstream processes and the total sugar content in the hydrolyzate prior to the downstream processes. This parameter shows that as we go down through the biological conversion process and prepare the substrate for fermentation, the loss of sugar is inevitable. Lastly, volume recovery is the ratio of the volume of the hydrolyzate solution following the downstream processes and the volume of the hydrolyzate prior to the said processes. This study aims to find the pH adjustment and separation scheme with high relative sugar recovery and volume recovery. Furthermore, this study hopes to bring light to certain issues of using lignocellulosic biomass as feedstock for bioenergy production, such as cost advantages and process handling operations. Hydrolyzate solutions treated with both powder and aqueous NaOH showed slightly higher concentrations of xylose and glucose compared to those treated with Ca(OH)2. NaOH showed more volume recovery compared to Ca(OH)2. The slurry form of Ca(OH)2 makes it a difficult basic substance to handle, as it rapidly separates from solution, not providing accurate concentration of the base at all times. Thus, if the subsequent fermentation can accept the sulfate and sodium concentration of the hydrolyzate [31], then NaOH would be better neutralizing agent. For both agents, the hydrolyzate treated with aqueous basic solutions showed higher sugar concentrations than the powder-treated hydrolyzate. As concentrated basic solutions were used, decrease of sugar concentration by dilution was only 5e9%. Nissila et al. [32] reported that the acid hydrolyzate neutralized with aqueous Ca(OH)2 solution provided the highest sugar recovery, compared to solid basic substances Ca(OH)2 and CaO. The same study explained that the use of solid alkaline agents can cause precipitation and degradation of sugars in solution [32].
Table 2 e The summary of sugar recovery (based on dry biomass weight) according to neutralization and separation. Conditions
Glucose, %
Xylose, %
Arabinose, %
Total sugar recovery, %a
Hydrolyzate without neutralization nor separation Vacuum filtration Vacuum filtration þ 8 N Ca(OH)2 Vacuum filtration þ 8 N NaOH Solid Ca(OH)2 þ vacuum filtration Solid NaOH þ vacuum filtration 8 N Ca(OH)2 þ vacuum filtration 8 N NaOH þ vacuum filtration 1000 rpm centrifugation 1000 rpm centrifugation þ 8 N Ca(OH)2 1000 rpm centrifugation þ 8 N NaOH Solid Ca(OH)2 þ 1000 rpm centrifugation Solid NaOH þ 1000 rpm centrifugation 8 N Ca(OH)2 þ 1000 rpm centrifugation 8 N NaOH þ 1000 rpm centrifugation
24.93 ± 0.63
66.81 ± 1.97
37.78 ± 5.17
38.22 ± 0.25
e
17.89 ± 0.74 15.12 ± 0.73 18.45 ± 1.33 12.94 ± 0.45 14.34 ± 1.83 14.27 ± 0.28 16.11 ± 1.18 17.41 ± 0.06 9.96 ± 0.85 12.78 ± 0.48 9.22 ± 0.45 9.94 ± 0.13 9.59 ± 0.23 10.33 ± 0.38
64.63 ± 57.76 ± 63.89 ± 50.65 ± 48.76 ± 51.72 ± 52.06 ± 65.33 ± 47.41 ± 53.13 ± 47.94 ± 49.51 ± 48.35 ± 48.36 ±
35.36 26.81 24.38 20.72 28.44 23.16 20.31 34.78 21.13 25.19 22.75 23.16 24.38 19.50
± 1.82 ± 1.15 ± 1.15 ± 1.72 ± 1.15 ± 2.87 ± 4.60 ± 1.36 ± 2.30 ± 4.60 ± 1.15 ± 2.87 ± 2.30 ± 0.00
35.23 ± 4.62 28.91 ± 0.55 33.09 ± 1.37 25.15 ± 0.11 25.42 ± 0.92 26.36 ± 0.91 27.65 ±1.29 33.79 ± 1.29 22.16 ± 1.33 25.87 ± 0.72 21.86 ± 0.79 22.85 ± 0.63 22.24 ± 1.47 22.72 ± 0.30
93.28 75.65 86.57 65.80 66.50 68.96 72.35 89.46 57.98 67.69 57.19 59.79 58.18 59.44
a b
3.62 3.06 1.55 1.20 0.83 2.19 1.62 1.67 2.35 3.08 1.52 1.66 3.99 0.19
Relative sugar recovery, %b
Computed from sugar content (dry basis) of the lignocellulosic biomass. Relative to the sugar recovery before separation and neutralization.
Please cite this article in press as: Gonzales RR, et al., Enhancement of hydrogen production by optimization of pH adjustment and separation conditions following dilute acid pretreatment of lignocellulosic biomass, International Journal of Hydrogen Energy (2017), http://dx.doi.org/10.1016/j.ijhydene.2017.05.021
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Fig. 2 e The total sugar concentrations and final hydrolyzate volumes for (a) filtration and pH adjustment conditions and (b) centrifugation and pH adjustment conditions.
Separation of the solid and aqueous hydrolyzate fractions An ideal separation of the aqueous and solid in the hydrolyzate would efficiently separate the two phases and recover sugar into aqueous phase as much as possible. Separation of lignocellulosic biomass hydrolyzate is difficult due to formation of impermeable filter cakes and fibrous properties of the residues [21]. Particle size distribution in the hydrolyzate is a very important factor in solideliquid fractionation. In filtration, only particles larger than the filter pore size are retained; and during centrifugation, particles larger than 1 mm settle, while particles between 1 nm and 1 mm form colloids, which then suspend in mixture due to Brownian motion [20]. Centrifugation and vacuum filtration are the two most widely performed methods for separation of the hydrolyzate fractions [33]. Centrifugation causes separation by means of
5
centrifugal force. The solids and liquids are transported to either end of a centrifuge tube by rotating the centrifuge at a high speed [20]. Pressurized filtration, under which category vacuum filtration falls in, separates solids and liquids through a filter, with the aid of applied pressure. The filter cake is compressed during pressure filtration, ensuring a solid fraction with high dry matter content and high recovery of liquor [20,21]. The use of centrifugation requires costly centrifugation equipment and electrical energy, and solideliquid separation is highly dependent on centrifugal force and efficiency of decantation. The use of centrifugation, therefore, is not cost-effective in large scale operations typical of bioenergy production, especially because complete separation is not always ensured when centrifugation is performed. Vacuum filtration, on the other hand, requires intricate equipment, vacuum conditions and the use of specialized filters with various pore sizes, but this method usually shows better separation performance [33]. Also, industrial scale vacuum filters can be reused by washing between cycles and can be changed only during maintenance and occurrence of fouling. However, little information has been available if these two methods can be used interchangeably without affecting sugar recovery significantly. Sugar analysis of the hydrolyzate fractions obtained for both methods are shown in Table 2 and Fig. 2. In any case, the downstream processes could not completely recover all the sugars in hydrolyzate. Vacuum filtration exhibited better dewatering performance than centrifugation, ensuring better separation of the solid and aqueous fractions, thus higher volumes were obtained for filtration as compared to centrifugation. The sugar concentrations obtained from filtration also showed slightly higher glucose and xylose recovery than centrifuged hydrolyzate fraction comparatively. On the other hand, there was no significant difference on the concentrations of organic acids, furfural, and 5-HMF. These compounds are known to be potential inhibitors to fermentative organisms [34]. Therefore, vacuum filtration would be the preferred method if a high-volume dilute acid hydrolyzate with high sugar yield is required [35]. This is validated by Hjorth et al. [20], who reported that the advantage of pressurized filtration over centrifugation is the production of a solid fraction with a high solids concentration. It would then be advantageous to choose vacuum filtration since this process concentrated the solids the most, liberating the most of the sugar-laden hydrolyzate liquor. Fig. 1 and Table 2 show that hydrolyzate obtained from separation followed by neutralization showed slightly higher sugar recovery as compared to the hydrolyzate which were neutralized first. Furthermore, no significant variations were observed for the presence of the degradation byproducts for all samples. Performing vacuum filtration before neutralization ensures higher sugar concentration and volume recovery, due to complete separation of the solid and liquid fractions. More so, performing separation first avoids the alkalinity of the solid fraction, which is essential for further enzymatic saccharification experiments using the biomass.
Batch H2 fermentation Modified Gompertz equation (Eq. (1)) was used to fit the cumulative H2 production curves during the batch fermentation.
Please cite this article in press as: Gonzales RR, et al., Enhancement of hydrogen production by optimization of pH adjustment and separation conditions following dilute acid pretreatment of lignocellulosic biomass, International Journal of Hydrogen Energy (2017), http://dx.doi.org/10.1016/j.ijhydene.2017.05.021
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Fig. 3 e The concentrations of the degradation products formic acid, acetic acid, levulinic acid, furfural, and 5-HMF for (a) filtration and pH adjustment conditions, and (b) centrifugation and pH adjustment conditions.
The correlation coefficients, R2, are larger than 0.99, indicating accuracy of the fit. The H2 fermentation parameters, volumetric HPR (mL L1 d1) and H2 yield (L H2 mol1 sugar), were estimated and shown in Table 3. H2 yield was computed based on sugar equivalents consumed and H2 production. Generally favorable H2 production was observed in this study, due to the use of inoculum sourced from a municipal wastewater treatment plant. Sludge sourced from wastewater treatment plants contain more potential hydrolytic and fermentative microbial population, compared to other inoculum sources, such as cow dung and compost [36]. While the highest sugar recovery was found for the dilute acid hydrolyzate obtained after vacuum filtration and dilution with aqueous NaOH, the highest H2 yield and H2 production rate were obtained when the hydrolyzate obtained after vacuum filtration and dilution with Ca(OH)2 was used as substrate. Based on the H2 yield and H2 production rate values tabulated in Table 3, hydrolyzates obtained after filtration did not show significant difference when used for H2 fermentation, when compared to hydrolyzates obtained after
centrifugation. When sugar concentration in the hydrolyzate was fixed to 10 g L1, the concentrations of inhibitors, such as furfural and 5-HMF, are likewise diluted. The concentrations of these inhibitors did not vary significantly with either use of filtration and centrifugation as means of separation. However, it was noticeable that the H2 yield and H2 production rate are significantly higher for hydrolyzates neutralized with Ca(OH)2. The use of Ca(OH)2 as a neutralizing agent forms CaSO4, an insoluble salt; using NaOH would then form Na2SO4, a soluble compound, leaving SO2 4 present in the solution [32]. Higher SO2 4 concentrations at pH 5.5e6 generally inhibit H2 production due to increased activity of sulfate-reducing bacteria (SRB) [31]. Although the heat treatment was used in this study, all SRB in activated sludge might not be inactivated completely. It was reported that some SRB species were able to survive at extremely high temperature, even up to 140 C [37]. The reduced H2 production is attributed to acclimation of the SRB to SO2 4 -reducing conditions as a response to increased SO2 4 level. Since H2S content of the biogas was not measured in this study, it could not be confirmed if SO2 in the 4
Please cite this article in press as: Gonzales RR, et al., Enhancement of hydrogen production by optimization of pH adjustment and separation conditions following dilute acid pretreatment of lignocellulosic biomass, International Journal of Hydrogen Energy (2017), http://dx.doi.org/10.1016/j.ijhydene.2017.05.021
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Table 3 e H2 production rate (HPR) and H2 yield (HY) of hydrolyzates from dilute acid pretreatment of pine tree wood. Conditions
Without separation nor neutralization Vacuum filtration þ 8 N Ca(OH)2 Vacuum filtration þ 8 N NaOH Solid Ca(OH)2 þ vacuum filtration Solid NaOH þ vacuum filtration 8 N Ca(OH)2 þ vacuum filtration 8 N NaOH þ vacuum filtration 1000 rpm centrifugation þ 8 N Ca(OH)2 1000 rpm centrifugation þ 8 N NaOH Solid Ca(OH)2 þ 1000 rpm centrifugation Solid NaOH þ 1000 rpm centrifugation 8 N Ca(OH)2 þ 1000 rpm centrifugation 8 N NaOH þ 1000 rpm centrifugation
5-HMF concentration, g L1 0.80 ± 0.34 ± 0.35 ± 0.65 ± 0.54 ± 0.67 ± 0.62 ± 0.56 ± 0.45 ± 0.78 ± 0.69 ± 0.73 ± 0.64 ±
0.26 0.23 0.27 0.39 0.34 0.23 0.42 0.27 0.18 0.29 0.35 0.22 0.28
Furfural concentration, g L1 0.26 0.13 0.03 0.26 0.03 0.35 0.13 0.24 0.20 0.24 0.42 0.42 0.19
hydrolyzate solutions was indeed reduced by SRB. The SO2 4 concentrations of the hydrolyzates are shown in Table 3. 1 optimal value While all values are below the 3000 mg SO2 4 L for H2 production at pH 5.5 [27], the hydrolyzate solutions treated with Ca(OH)2 have significantly less SO2 4 levels than those treated with NaOH. Presence of lower amounts SO2 4 in the solutions decreases the probability of inhibition of H2 production [38]. Comparison of the two alkaline agents shows that while NaOH was found to be better for sugar recovery, Ca(OH)2 was found to be better for H2 production. If the objective of a study is to generate a hydrolyzate solution with high sugar content via dilute acid pretreatment, NaOH proved to be the better alkali for pH adjustment. In studies like this one, wherein the dilute acid pretreatment is a preliminary step to obtain a monomeric sugar-rich solution from biomass prior to biological conversion to energy, Ca(OH)2 must be used as the alkali for pH adjustment downstream process. The use of Ca(OH)2 also eliminates the presence of SO2 4 ; the presence of which increases the activity of SRB, hereby competing with H2 fermentative bacteria, and ultimately inhibiting H2 fermentation. Butyric and acetic acids were the main volatile fatty acids (VFA) produced during the fermentation as shown in Fig. 4. The figure also shows a comparison of the total concentration of soluble metabolic products (SMP), which include the VFA and the furan compounds. Chaganti et al. [39] mentioned that these metabolites are typically observed with H2 fermentation processes which make use xylose and glucose as substrates at pH 5.5. Acetic and butyric acids are known to be byproducts of H2 fermentation. This suggests that acetic and butyric acid pathways were the dominant routes for H2 production [39] and the fermentable sugars in the solutions were utilized only in H2 production. Peak H2 yields from dark H2 fermentation using various lignocellulosic biomass in previous studies are shown in Table 4. All of these studies explicitly performed acid pretreatment of the biomass and downstream separation and pH adjustment processes prior to H2 fermentation. The units for hydrogen yield for all studies were converted to mol H2 g1 sugar, for uniformity. Cui et al. [40] reported H2 yield of 53 mol H2 g1 total sugar (TS) with beer lees, whose acid
± 0.01 ± 0.00 ± 0.00 ± 0.08 ± 0.01 ± 0.17 ± 0.00 ± 0.14 ± 0.02 ± 0.09 ± 0.17 ± 0.05 ± 0.02
SO2 4 concentration, g L1
HPR, mL H2 L1 d1
1.79 1.52 2.49 1.64 2.94 1.68 2.54 1.74 2.55 1.86 2.58 1.91 2.72
1001 ± 0.24 1824 ± 1.44 1774 ± 2.64 1138 ± 3.12 1208 ± 1.68 1815 ± 6.00 1198 ± 2.64 1590 ± 1.20 1268 ± 4.08 1192 ± 0.48 963 ± 1.20 1552 ± 1.92 961 ± 2.40
HY, mol H2 mol1 sugar 0.31 ± 1.27 ± 1.27 ± 0.70 ± 0.88 ± 0.99 ± 0.89 ± 0.62 ± 0.68 ± 0.76 ± 0.60 ± 0.97 ± 0.75 ±
0.11 0.15 0.18 0.05 0.35 0.02 0.03 0.08 0.14 0.01 0.06 0.05 0.06
hydrolyzate was treated with aqueous NaOH prior to H2 fermentation with mixed culture. The dilute acid hydrolyzate of corn stover was first centrifuged, treated with 2 N Ca(OH)2, and then centrifuged once more before H2 fermentation with T. thermosaccharolyticum W16 as inoculum in a study by Cao et al. [41]. This study was able to produce 291.3 mL H2 g1 TS. Chong et al. [38] used acid-pretreated oil palm empty fruit bunch which was filtered using a 0.45 mm pore size filter and treated with 5 N NaOH to produce 278.0 mL H2 g1 xylose after fermentation with anaerobic sludge. Acid hydrolyzate of poplar leaves was simply treated with aqueous NaOH to make its pH suitable for H2 fermentation, and produced 44.9 mL H2 g1 TS in a study conducted by Cui et al. [42]. Centrifugation followed by pH adjustment with 0.1 M TriseHCl buffer was the downstream separation and pH adjustment processes for rice straw hydrolyzate in another study by Loo et al. [43], which produced 106.7 mL H2 g1 reducing sugars (RS). Monlau et al. [44] performed filtration with a 0.25 m pore size filter, followed by pH adjustment with 1 N NaOH on acid hydrolyzate of sunflower stalks. This study produced 286.5 mL H2/g hexose using anaerobic sludge as inoculum. Pattra et al. [45] employed two separation processes for its acid hydrolyzate of sugarcane bagasse. The hydrolyzate was filtered, after which Ca(OH)2 was added, and it was centrifuged before H2 fermentation producing 242.9 mL H2 g1 TS. The use of pure culture, such as Thermoanaerobacterium thermosaccharolyticum W16 and Clostridium butyricum, gave generally high H2 yields. Using initial 5 g L1 TS concentration in this study, dilute acid pretreated pine tree wood gave H2 yield of 178.3 and 179 mL H2 g1 TS, for hydrolyzate filtered and treated with 8 N NaOH and Ca(OH)2, respectively. concentrations ranged from 1.52 to The initial SO2 4 1 as shown in Table 3. The comparatively high 2.94 g SO2 4 L hydrogen yield implies that there was no severe inhibition by sulfate reducing bacteria and/or sulfide in this study. The heat-treatment of inoculum would have prevented the existence of sulfate reducing bacteria, as well as methanogens. The difference in concentrations of the SMP for the hydrolyzate treated with either NaOH or Ca(OH)2 is statistically insignificant. Based on this study, we can adjust the pH of the dilute acid hydrolyzate with aqueous solutions of either NaOH
Please cite this article in press as: Gonzales RR, et al., Enhancement of hydrogen production by optimization of pH adjustment and separation conditions following dilute acid pretreatment of lignocellulosic biomass, International Journal of Hydrogen Energy (2017), http://dx.doi.org/10.1016/j.ijhydene.2017.05.021
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Fig. 4 e The VFA production during the hydrogen fermentation using the dilute acid pine tree wood hydrolyzate.
Table 4 e Dark fermentative H2 production from various dilute acid pretreated lignocellulosic biomass in previous studies. Substrate
Downstream processes
H2 fermentation conditions
Inoculum
Beer lees Corn stover
Aqueous NaOH Centrifugation, 2 N Ca(OH)2, centrifugation Filtration (0.45 mm pore size), 5 N NaOH Aqueous NaOH Centrifugation, 0.1 M Tris eHCl buffer Filtration (0.25 mm pore size), 1 N NaOH Filtration, Ca(OH)2, centrifugation Filtration, 8 N NaOH
Batch, 35 C, pH 7.0 Batch, 60 C
Mixed culture T. thermosaccharolyticum W16
53.0 mL H2/g TSa 291.3 mL H2/g TS
[40] [41]
Batch, 35 C, 5 g TS/L Batch, 35 C, Batch, 37 C, 14 g RSb/L Batch, 35 C,
pH 7.0,
Anaerobic sludge
278.0 mL H2/g xylose
[38]
pH 7.0 pH 7.0,
Mixed culture Clostridium butyricum CGS5
44.9 mL H2/g TS 106.7 mL H2/g RS
[42] [43]
pH 5.5
Anaerobic sludge
286.5 mL H2/g hexose
[44]
Batch, 37 C, pH 7.0
Clostridium butyricum
242.9 mL H2/g TS
[45]
Batch, 35 C, pH 7.0, 10 g TS/L Batch, 35 C, pH 7.0, 10 g TS/L
Anaerobic granular sludge
178.3 mL H2/g TS
This study
Anaerobic granular sludge
179.0 mL H2/g TS
This study
Oil palm empty fruit bunch Poplar leaves Rice straw Sunflower stalks Sugarcane bagasse Pine tree wood Pine tree wood a b
Filtration, 8 N Ca(OH)2, filtration
H2 yield
Reference
Total sugar. Reducing sugar.
Please cite this article in press as: Gonzales RR, et al., Enhancement of hydrogen production by optimization of pH adjustment and separation conditions following dilute acid pretreatment of lignocellulosic biomass, International Journal of Hydrogen Energy (2017), http://dx.doi.org/10.1016/j.ijhydene.2017.05.021
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or Ca(OH)2 interchangeably, if and only if SO2 4 concentration is well below the threshold limit of the anaerobic bacteria. [8]
Conclusion [9]
A series of pH and separation downstream processes were performed to determine the best option to obtain liquid feedstock for fermentation of dilute acid pretreated lignocellulosic biomass. The downstream unit processes and their sequence significantly affected sugar recovery. Filtration followed by pH adjustment using aqueous NaOH recovered 95.29% of sugars liberated from dilute acid pretreatment. The dilute acid hydrolyzate solutions of pine tree wood were then used for dark hydrogen fermentation processes. The substrate concentration of the hydrolyzate solutions was adjusted to 10 g L1. The maximum H2 yield and H2 production rate were 1824 mL H2 L1 d1 and 1.27 mol H2 mol1 sugar, respectively, for the hydrolyzate solution obtained after vacuum filtration and pH adjustment using 8 N Ca(OH)2. The downstream processes would be applied to dilute acid hydrolyzate of other biomass as well for high sugar recovery and high H2 yield and production rate during H2 fermentative processes.
[10]
[11]
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[15]
Acknowledgement This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No. 2017R1A2A2A07000900).
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i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 7 ) 1 e1 0
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Please cite this article in press as: Gonzales RR, et al., Enhancement of hydrogen production by optimization of pH adjustment and separation conditions following dilute acid pretreatment of lignocellulosic biomass, International Journal of Hydrogen Energy (2017), http://dx.doi.org/10.1016/j.ijhydene.2017.05.021