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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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 L
1 d
1 and 1.27 mol H2 mol
1 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.

increase in population. Due to non-renewability, fossil fuels

Introduction are considered to be limited energy sources, which also pose
adverse environmental effects, such as greenhouse gas
Renewable energy sources are currently of great interest due emissions [1]. Biorefinery is the integration of various
to increasing demand for energy, caused by development and

* Corresponding author. Sustainable Environmental Process Research Institute, Daegu University, Jillyang, Gyeongsan, Gyeongbuk 38453,
South Korea. Fax: þ82 53 850 6699.
E-mail address: sanghkim1@daegu.ac.kr (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
2 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

conversion and separation unit processes of biomass to fuel, pH adjustment and separation downstream processes for
chemicals, and energy. These processes enable the harness- hydrolyzates of lignocellulosic biomass are commonly per-
ing of renewable energy from carbohydrate-rich agricultural formed in biorefinery processes. However, little attention is
residues and biological matter by thermochemical conversion provided to these methods. Only a few separation and pH
of biomass [2]. Thus, it is considered as one of the solutions to adjustment studies have been published for dilute acid hy-
reduce carbon footprint and dependency on fossil fuel for drolyzate of lignocellulosic biomass. Moreover, no studies
sustainable development [3]. have shown the direct effect of these processes to H2
Among the currently studied renewable energy sources for fermentation, according to the authors' knowledge.
biofuel production is lignocellulosic biomass, whose recalci- Hydrogen is an environmentally friendly biofuel with high
trant characteristic is due to its complex matrix structure energy efficiency [22,23], making it a promising alternative
composed of cellulose, hemicellulose, lignin and other com- energy carrier. Due to the high energy consumption require-
ponents [4e6]. The use of raw lignocellulosic biomass yielded ment of conventional physico-chemical H2 production
low production of biofuels and exhibited low production rates methods, interest in biohydrogen has increased significantly
due to the low accessibility of the feedstock by microbial in the recent years. Dark fermentation, or light-independent
populations [7]. Pretreatment processes are needed to break process, uses genera Clostridium, Enterobacter, or mixed cul-
the polysaccharideelignin complex links, thereby increasing ture dominated by these microorganisms to convert sugars
the accessibility of cellulose and hemicelluloses for bio- into H2 [24]. Among these microbial species, Clostridium spe-
refinery [8e10]. cies were identified as the most dominant H2-producing bac-
Dilute acid pretreatment is one of the most widely per- teria for mesophilic dark fermentation at pH 5.5 [1]. These
formed pretreatment methods for lignocellulosic biomass [4]. bacteria make use of monomeric sugars present in the dilute
Dilute acid is normally preferred over concentrated acid since acid hydrolyzate of lignocellulosic biomass as substrate for
it is cost effective and environmentally-friendly [11]. During dark fermentative process [24].
dilute acid pretreatment, the biomass is exposed to harsh Pine tree wood, an agricultural feedstock of the wood pulp
physicochemical conditions to obtain a solution rich with and paper industry, was used as the substrate for this study.
monomeric sugars. The lignin-carbohydrate complexes are This particular biomass was chosen since the paper industry
removed or disrupted, making the cellulose easily accessible is important in South Korean economy and this biomass is
to enzymes and microorganisms [12]. just disposed as waste or incinerated. Hydrolysis of lignocel-
Downstream processes, such as pH adjustment and sepa- lulosic biomass, such as pine tree wood, leads production of a
ration, accompany the chemical pretreatment prior to the number of useful biofuels as compared to combustion, which
biological conversion steps [13,14]. pH adjustment is essential primarily produces heat [25].
because biological conversion of biomass to biofuels is known The objective of this study was to investigate resultant
to be significantly affected by pH, of which typical optimum volume, sugar and byproduct contents, and biohydrogen
values are near neutral [8,15e17]. Moreover, pH of lignocellu- production of dilute acid lignocellulosic hydrolyzate after pH
losic hydrolyzate is sometimes adjusted to a mild basic pH adjustment and separation with various neutralizing agents
range to facilitate precipitation of potential inhibitory com- (calcium hydroxide and sodium hydroxide), agent forms
pounds such as furfural and 5-hydroxymethylfurfural (5-HMF) (powder and aqueous solution), separation methods (centri-
[18]. Separation is also inevitable to fractionate the hydroly- fugation and vacuum filtration) and the sequence of the unit
zate from lignin and insoluble compounds. An ideal separa- processes (neutralizationeseparation and separationeneu-
tion method of the aqueous and solid fractions of the tralization). This study puts light to the significance and
hydrolyzate must recover as much sugar into aqueous phase relevance of often overlooked yet important downstream
as possible. The physicochemical unit processes between process conditions, particularly, pH adjustment and separa-
dilute acid hydrolysis and biological conversion may affect the tion, on biological conversion of biomass to bioenergy.
sugar and byproduct contents of the hydrolyzate, although
they have been regarded not to affect the recovery of the
soluble compounds [19]. Materials and methods
Dilute acid pretreatment of lignocellulosic biomass has
been conducted in various studies; however, each study made Dilute acid pretreatment of lignocellulosic biomass
use of different methods for neutralization and fractionation
for the hydrolyzate. The study of the best neutralization and Untreated pine tree wood pellet was acquired from a local
fractionation methods is important since the pH, state, and paper production company and was the lignocellulosic
composition of the hydrolyzate are integral to the fermenta- biomass used in this study. The glucan, xylan, arabinan,
tion process taking place after pretreatment. After the lignin, ash, and extractives composition of the biomass were
downstream processes, the objective is to produce an energy- measured by following the NREL laboratory analytical pro-
rich biomass hydrolyzate that can be used for biogas and cedure [26]. The biomass composition is presented, on dry
biofuel production [20]. According to Sievers et al., solid- basis, in Table 1.
eliquid separations are useful to provide a solids-free sugar The pine tree wood pellet was initially milled to reduce the
stream for fermentation. Solids are known to interfere with particle size to 1e2 mm. Dilute acid pretreatment was per-
fermentation of sugars [21]. Moreover, neutralization and formed with 10% (w/v) milled woody biomass and 5% (w/v)
fractionation must be optimized as sustainable and cost- sulfuric acid (Duksan Pure Chemicals, South Korea), and car-
effective treatment methods for lignocellulosic hydrolyzate. ried 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
 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 3

12.6 g L
1, and 22.6 g L
1, respectively. The sludge was heat-
Table 1 e Composition of pine tree wood pellet.
treated at 90  C for 30 min to harvest only anaerobic spore-
Component Content, % (w/w) forming H2-producing bacteria [27] and was used as the
Glucan 39.48 ± 0.87 inoculum of the following batch H2 fermentation.
Xylan 20.50 ± 0.49 H2 fermentation was conducted in 100 mL serum bottles.
Arabinan 1.60 ± 0.18 The hydrolyzate solutions obtained after all separation and
Lignin 37.11 ± 2.05
neutralization schemes were used as the substrate. The hy-
Ash 1.25 ± 0.03
drolyzate solutions were diluted to obtain an initial sugar
Extractives 0.06 ± 0.01
concentration of 10 g L
1. 30 mL of the hydrolyzate solution
with 10 g L
1 initial sugar concentration was added to the
121  C for 30 min. The severity factor of this pretreatment serum bottle. This initial sugar concentration was previously
process was 1.83. found by Gonzales et al. [25] to provide the highest H2 pro-
duction using pine tree wood as substrate. Mineral medium
pH adjustment and separation of the dilute acid was supplied as follows: 6.72 g L
1 NaHCO3, 3.00 g L
1 NH4CO3,
lignocellulosic hydrolyzate 0.125 g L
1 KH2PO4, 0.100 g L
1 MgCl2$6H2O, 0.015 g L
1
MnSO4$6H2O, 0.025 g L
1 FeSO4$7H2O, 0.005 g L
1 CuSO4$5H2O,
The pH adjustment and separation scheme is summarized in and 0.001 g L
1 CoCl2$5H2O. 5 mL of seed inoculum and 5 mL
Fig. 1. The solution freshly obtained from the dilute acid pre- mineral medium were added to obtain a solution with total
treatment step undergoes a series of steps to adjust the pH working volume of 40 mL and initial pH of 7.0e7.5. The serum
and to separate the solid and liquid components of the solu- bottle was purged with N2 gas for 3 min and then agitated at
tions. NaOH or Ca(OH)2 was added to adjust the pH of the 150 rpm and 35  C. The fermentation process was performed
dilute acid hydrolyzate to 5.5e6 from initial pH of 0.5 to 1.0. in duplicate.
The alkaline agents are delivered into the hydrolyzate solu-
tion either in solid form or as an 8 N aqueous solution. Sepa- Analytical methods
ration of the aqueous and solid fractions was performed using
either centrifugation or vacuum filtration. Centrifugation was Concentrations of monomeric sugars glucose, xylose, and
carried out at 67.2 g relative centrifugal force (RCF) for 5 min arabinose in the dilute acid pine tree wood hydrolyzate were
using a high-speed centrifuge (Combi-514R, Hanil Science, quantified using high performance liquid chromatography
South Korea). Vacuum filtration was performed with 47 mm (Waters 717plus, MA, USA) with Aminex HPX-87P column (Bio-
glass-fiber filters (Grade GF/C, Whatman, MO, USA). The pH Rad Laboratories, CA, USA) and a refractive index detector
adjustment and separation scheme was done in triplicate. (Waters 410, MA, USA) with deionized water mobile phase.
The organic acids and furan compounds contents were
Batch H2 fermentation measured by HPLC system with Aminex HPX-87H (Bio-Rad
Laboratories, USA) and an ultraviolet detector (Waters 2487,
Anaerobic granular sludge was obtained from an upflow MA, USA) with 5 mM H2SO4 mobile phase, at 210 nm. SO2
4 ion

sludge blanket reactor treating brewery wastewater in South concentration was analyzed by ion chromatography (Dionex
Korea. The pH, volatile suspended solids (VSS), and total Aquion IC System, Thermo Fisher Scientific, MA, USA) with
suspended solids (TSS) concentration of the sludge were 6.8, 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
4 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

5000þ CD conductivity detector (Thermo Fisher Scientific, MA, the hydrolyzate solution and the total sugar content in the
USA), with a carbonate-bicarbonate buffer mobile phase. H2 pine tree wood pellet. Relative sugar recovery, on the other
content was analyzed by gas chromatography (SRI In- hand, is the ratio of the total sugar content in the hydroly-
struments, USA) with a thermal conductivity detector (TCD) zate solution following the downstream processes and the
and a 1.8 m  3.2 mm stainless-steel column packed with total sugar content in the hydrolyzate prior to the down-
mole sieve 13 and high purity N2 as the carrier gas. The stream processes. This parameter shows that as we go down
temperatures of the injector, column, and detector were through the biological conversion process and prepare the
maintained at room temperature, 80  C, and 90  C, respec- substrate for fermentation, the loss of sugar is inevitable.
tively. TSS and VSS were measured according to standard Lastly, volume recovery is the ratio of the volume of the
procedures [28]. hydrolyzate solution following the downstream processes
and the volume of the hydrolyzate prior to the said pro-
Assay cesses. This study aims to find the pH adjustment and sep-
aration scheme with high relative sugar recovery and
The H2 production with respect to time was fitted to the values volume recovery. Furthermore, this study hopes to bring
estimated by a modified Gompertz equation (Eq. (1)) to esti- light to certain issues of using lignocellulosic biomass as
mate maximum H2 volume, maximum H2 production rate, lag feedstock for bioenergy production, such as cost advantages
time of H2 production, and H2 production yield [29,30]: and process handling operations.
   Hydrolyzate solutions treated with both powder and
RH
H ¼ P  exp
exp  ðl
tÞ þ 1 (1) aqueous NaOH showed slightly higher concentrations of
P
xylose and glucose compared to those treated with Ca(OH)2.
where H, P, RH, l, t, and e are cumulative H2 production (mL), NaOH showed more volume recovery compared to Ca(OH)2.
ultimate H2 production (mL), H2 production rate (mL d
1), lag The slurry form of Ca(OH)2 makes it a difficult basic substance
time (h), time (h), and exponential 1, respectively. 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
Results and discussion concentration of the hydrolyzate [31], then NaOH would be
better neutralizing agent.
Neutralization of the dilute acid lignocellulosic hydrolyzate For both agents, the hydrolyzate treated with aqueous
basic solutions showed higher sugar concentrations than the
The concentrations of sugar and byproducts present in the powder-treated hydrolyzate. As concentrated basic solutions
hydrolyzate solutions after neutralization using powder and were used, decrease of sugar concentration by dilution was
aqueous solutions of Ca(OH)2 and NaOH were compared in only 5e9%. Nissila et al. [32] reported that the acid hydrolyzate
Table 2, Figs. 2 and 3. The data table and figures present the neutralized with aqueous Ca(OH)2 solution provided the
following terms, total sugar recovery, relative sugar recov- highest sugar recovery, compared to solid basic substances
ery, and volume recovery, which were all calculated pa- Ca(OH)2 and CaO. The same study explained that the use of
rameters in this study. Total sugar recovery of the solid alkaline agents can cause precipitation and degradation
hydrolyzate solution is the ratio of the total sugar content in 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 Relative sugar
recovery, %a recovery, %b
Hydrolyzate without neutralization 24.93 ± 0.63 66.81 ± 1.97 37.78 ± 5.17 38.22 ± 0.25 e
nor separation
Vacuum filtration 17.89 ± 0.74 64.63 ± 3.62 35.36 ± 1.82 35.23 ± 4.62 93.28
Vacuum filtration þ 8 N Ca(OH)2 15.12 ± 0.73 57.76 ± 3.06 26.81 ± 1.15 28.91 ± 0.55 75.65
Vacuum filtration þ 8 N NaOH 18.45 ± 1.33 63.89 ± 1.55 24.38 ± 1.15 33.09 ± 1.37 86.57
Solid Ca(OH)2 þ vacuum filtration 12.94 ± 0.45 50.65 ± 1.20 20.72 ± 1.72 25.15 ± 0.11 65.80
Solid NaOH þ vacuum filtration 14.34 ± 1.83 48.76 ± 0.83 28.44 ± 1.15 25.42 ± 0.92 66.50
8 N Ca(OH)2 þ vacuum filtration 14.27 ± 0.28 51.72 ± 2.19 23.16 ± 2.87 26.36 ± 0.91 68.96
8 N NaOH þ vacuum filtration 16.11 ± 1.18 52.06 ± 1.62 20.31 ± 4.60 27.65 ±1.29 72.35
1000 rpm centrifugation 17.41 ± 0.06 65.33 ± 1.67 34.78 ± 1.36 33.79 ± 1.29 89.46
1000 rpm centrifugation þ 8 N Ca(OH)2 9.96 ± 0.85 47.41 ± 2.35 21.13 ± 2.30 22.16 ± 1.33 57.98
1000 rpm centrifugation þ 8 N NaOH 12.78 ± 0.48 53.13 ± 3.08 25.19 ± 4.60 25.87 ± 0.72 67.69
Solid Ca(OH)2 þ 1000 rpm centrifugation 9.22 ± 0.45 47.94 ± 1.52 22.75 ± 1.15 21.86 ± 0.79 57.19
Solid NaOH þ 1000 rpm centrifugation 9.94 ± 0.13 49.51 ± 1.66 23.16 ± 2.87 22.85 ± 0.63 59.79
8 N Ca(OH)2 þ 1000 rpm centrifugation 9.59 ± 0.23 48.35 ± 3.99 24.38 ± 2.30 22.24 ± 1.47 58.18
8 N NaOH þ 1000 rpm centrifugation 10.33 ± 0.38 48.36 ± 0.19 19.50 ± 0.00 22.72 ± 0.30 59.44
a
Computed from sugar content (dry basis) of the lignocellulosic biomass.
b
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
 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 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 frac-
tion with high dry matter content and high recovery of liquor
[20,21]. The use of centrifugation requires costly centrifuga-
tion equipment and electrical energy, and solideliquid sepa-
ration 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 al-
ways 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 vol-
umes were obtained for filtration as compared to centrifuga-
tion. 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
Fig. 2 e The total sugar concentrations and final sugar yield is required [35]. This is validated by Hjorth et al. [20],
hydrolyzate volumes for (a) filtration and pH adjustment who reported that the advantage of pressurized filtration over
conditions and (b) centrifugation and pH adjustment centrifugation is the production of a solid fraction with a high
conditions. 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.
Separation of the solid and aqueous hydrolyzate fractions Fig. 1 and Table 2 show that hydrolyzate obtained from
separation followed by neutralization showed slightly higher
An ideal separation of the aqueous and solid in the hydroly- sugar recovery as compared to the hydrolyzate which were
zate would efficiently separate the two phases and recover neutralized first. Furthermore, no significant variations were
sugar into aqueous phase as much as possible. Separation of observed for the presence of the degradation byproducts for
lignocellulosic biomass hydrolyzate is difficult due to forma- all samples. Performing vacuum filtration before neutraliza-
tion of impermeable filter cakes and fibrous properties of the tion ensures higher sugar concentration and volume recovery,
residues [21]. Particle size distribution in the hydrolyzate is a due to complete separation of the solid and liquid fractions.
very important factor in solideliquid fractionation. In filtra- More so, performing separation first avoids the alkalinity of
tion, only particles larger than the filter pore size are retained; the solid fraction, which is essential for further enzymatic
and during centrifugation, particles larger than 1 mm settle, saccharification experiments using the biomass.
while particles between 1 nm and 1 mm form colloids, which
then suspend in mixture due to Brownian motion [20]. Batch H2 fermentation
Centrifugation and vacuum filtration are the two most
widely performed methods for separation of the hydrolyzate Modified Gompertz equation (Eq. (1)) was used to fit the cu-
fractions [33]. Centrifugation causes separation by means of mulative 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
6 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

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 centrifugation. When sugar concentration in the hydrolyzate
accuracy of the fit. The H2 fermentation parameters, volu- was fixed to 10 g L
1, the concentrations of inhibitors, such as
metric HPR (mL L
1 d
1) and H2 yield (L H2 mol
1 sugar), were furfural and 5-HMF, are likewise diluted. The concentrations
estimated and shown in Table 3. of these inhibitors did not vary significantly with either use of
H2 yield was computed based on sugar equivalents filtration and centrifugation as means of separation. However,
consumed and H2 production. Generally favorable H2 pro- it was noticeable that the H2 yield and H2 production rate are
duction was observed in this study, due to the use of inoculum significantly higher for hydrolyzates neutralized with Ca(OH)2.
sourced from a municipal wastewater treatment plant. Sludge The use of Ca(OH)2 as a neutralizing agent forms CaSO4, an
sourced from wastewater treatment plants contain more po- insoluble salt; using NaOH would then form Na2SO4, a soluble
tential hydrolytic and fermentative microbial population, compound, leaving SO2
4 present in the solution [32]. Higher
compared to other inoculum sources, such as cow dung and SO2
4 concentrations at pH 5.5e6 generally inhibit H2 produc-
compost [36]. While the highest sugar recovery was found for tion due to increased activity of sulfate-reducing bacteria
the dilute acid hydrolyzate obtained after vacuum filtration (SRB) [31]. Although the heat treatment was used in this study,
and dilution with aqueous NaOH, the highest H2 yield and H2 all SRB in activated sludge might not be inactivated
production rate were obtained when the hydrolyzate obtained completely. It was reported that some SRB species were able to
after vacuum filtration and dilution with Ca(OH)2 was used as survive at extremely high temperature, even up to 140  C [37].
substrate. Based on the H2 yield and H2 production rate values The reduced H2 production is attributed to acclimation of the
tabulated in Table 3, hydrolyzates obtained after filtration did SRB to SO2
4 -reducing conditions as a response to increased
not show significant difference when used for H2 fermenta- SO2
4 level. Since H2S content of the biogas was not measured
tion, when compared to hydrolyzates obtained after in this study, it could not be confirmed if SO2
4 in the

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
 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 7

Table 3 e H2 production rate (HPR) and H2 yield (HY) of hydrolyzates from dilute acid pretreatment of pine tree wood.
Conditions 5-HMF Furfural SO2

4 HPR, HY,
concentration, concentration, concentration, mL H2 L
1 d
1 mol H2 mol
1 sugar
g L
1 g L
1 g L
1
Without separation nor neutralization 0.80 ± 0.26 0.26 ± 0.01 1.79 1001 ± 0.24 0.31 ± 0.11
Vacuum filtration þ 8 N Ca(OH)2 0.34 ± 0.23 0.13 ± 0.00 1.52 1824 ± 1.44 1.27 ± 0.15
Vacuum filtration þ 8 N NaOH 0.35 ± 0.27 0.03 ± 0.00 2.49 1774 ± 2.64 1.27 ± 0.18
Solid Ca(OH)2 þ vacuum filtration 0.65 ± 0.39 0.26 ± 0.08 1.64 1138 ± 3.12 0.70 ± 0.05
Solid NaOH þ vacuum filtration 0.54 ± 0.34 0.03 ± 0.01 2.94 1208 ± 1.68 0.88 ± 0.35
8 N Ca(OH)2 þ vacuum filtration 0.67 ± 0.23 0.35 ± 0.17 1.68 1815 ± 6.00 0.99 ± 0.02
8 N NaOH þ vacuum filtration 0.62 ± 0.42 0.13 ± 0.00 2.54 1198 ± 2.64 0.89 ± 0.03
1000 rpm centrifugation þ 8 N Ca(OH)2 0.56 ± 0.27 0.24 ± 0.14 1.74 1590 ± 1.20 0.62 ± 0.08
1000 rpm centrifugation þ 8 N NaOH 0.45 ± 0.18 0.20 ± 0.02 2.55 1268 ± 4.08 0.68 ± 0.14
Solid Ca(OH)2 þ 1000 rpm centrifugation 0.78 ± 0.29 0.24 ± 0.09 1.86 1192 ± 0.48 0.76 ± 0.01
Solid NaOH þ 1000 rpm centrifugation 0.69 ± 0.35 0.42 ± 0.17 2.58 963 ± 1.20 0.60 ± 0.06
8 N Ca(OH)2 þ 1000 rpm centrifugation 0.73 ± 0.22 0.42 ± 0.05 1.91 1552 ± 1.92 0.97 ± 0.05
8 N NaOH þ 1000 rpm centrifugation 0.64 ± 0.28 0.19 ± 0.02 2.72 961 ± 2.40 0.75 ± 0.06

hydrolyzate solutions was indeed reduced by SRB. The SO2
4 hydrolyzate was treated with aqueous NaOH prior to H2
concentrations of the hydrolyzates are shown in Table 3. fermentation with mixed culture. The dilute acid hydrolyzate

1
While all values are below the 3000 mg SO2
4 L optimal value of corn stover was first centrifuged, treated with 2 N Ca(OH)2,
for H2 production at pH 5.5 [27], the hydrolyzate solutions and then centrifuged once more before H2 fermentation with
treated with Ca(OH)2 have significantly less SO2
4 levels than T. thermosaccharolyticum W16 as inoculum in a study by Cao
those treated with NaOH. Presence of lower amounts SO2
4 in et al. [41]. This study was able to produce 291.3 mL H2 g
1 TS.
the solutions decreases the probability of inhibition of H2 Chong et al. [38] used acid-pretreated oil palm empty fruit
production [38]. bunch which was filtered using a 0.45 mm pore size filter and
Comparison of the two alkaline agents shows that while treated with 5 N NaOH to produce 278.0 mL H2 g
1 xylose after
NaOH was found to be better for sugar recovery, Ca(OH)2 was fermentation with anaerobic sludge. Acid hydrolyzate of
found to be better for H2 production. If the objective of a study poplar leaves was simply treated with aqueous NaOH to make
is to generate a hydrolyzate solution with high sugar content its pH suitable for H2 fermentation, and produced
via dilute acid pretreatment, NaOH proved to be the better 44.9 mL H2 g
1 TS in a study conducted by Cui et al. [42].
alkali for pH adjustment. In studies like this one, wherein the Centrifugation followed by pH adjustment with 0.1 M
dilute acid pretreatment is a preliminary step to obtain a TriseHCl buffer was the downstream separation and pH
monomeric sugar-rich solution from biomass prior to biolog- adjustment processes for rice straw hydrolyzate in another
ical conversion to energy, Ca(OH)2 must be used as the alkali study by Loo et al. [43], which produced 106.7 mL H2 g
1
for pH adjustment downstream process. The use of Ca(OH)2 reducing sugars (RS). Monlau et al. [44] performed filtration
also eliminates the presence of SO2
4 ; the presence of which with a 0.25 m pore size filter, followed by pH adjustment with
increases the activity of SRB, hereby competing with H2 1 N NaOH on acid hydrolyzate of sunflower stalks. This study
fermentative bacteria, and ultimately inhibiting H2 produced 286.5 mL H2/g hexose using anaerobic sludge as
fermentation. inoculum. Pattra et al. [45] employed two separation processes
Butyric and acetic acids were the main volatile fatty acids for its acid hydrolyzate of sugarcane bagasse. The hydrolyzate
(VFA) produced during the fermentation as shown in Fig. 4. was filtered, after which Ca(OH)2 was added, and it was
The figure also shows a comparison of the total concentration centrifuged before H2 fermentation producing 242.9 mL H2 g
1
of soluble metabolic products (SMP), which include the VFA TS.
and the furan compounds. Chaganti et al. [39] mentioned that The use of pure culture, such as Thermoanaerobacterium
these metabolites are typically observed with H2 fermentation thermosaccharolyticum W16 and Clostridium butyricum, gave
processes which make use xylose and glucose as substrates at generally high H2 yields. Using initial 5 g L
1 TS concentration
pH 5.5. Acetic and butyric acids are known to be byproducts of in this study, dilute acid pretreated pine tree wood gave H2
H2 fermentation. This suggests that acetic and butyric acid yield of 178.3 and 179 mL H2 g
1 TS, for hydrolyzate filtered
pathways were the dominant routes for H2 production [39] and treated with 8 N NaOH and Ca(OH)2, respectively.
and the fermentable sugars in the solutions were utilized The initial SO2
4 concentrations ranged from 1.52 to

1
only in H2 production. 2.94 g SO2
4 L as shown in Table 3. The comparatively high
Peak H2 yields from dark H2 fermentation using various hydrogen yield implies that there was no severe inhibition by
lignocellulosic biomass in previous studies are shown in Table sulfate reducing bacteria and/or sulfide in this study. The
4. All of these studies explicitly performed acid pretreatment heat-treatment of inoculum would have prevented the exis-
of the biomass and downstream separation and pH adjust- tence of sulfate reducing bacteria, as well as methanogens.
ment processes prior to H2 fermentation. The units for The difference in concentrations of the SMP for the hydroly-
hydrogen yield for all studies were converted to mol H2 g
1 zate treated with either NaOH or Ca(OH)2 is statistically
sugar, for uniformity. Cui et al. [40] reported H2 yield of insignificant. Based on this study, we can adjust the pH of the
53 mol H2 g
1 total sugar (TS) with beer lees, whose acid 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),
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8 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

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 Inoculum H2 yield Reference
conditions
Beer lees Aqueous NaOH Batch, 35  C, pH 7.0 Mixed culture 53.0 mL H2/g TSa [40]
Corn stover Centrifugation, 2 N Ca(OH)2, Batch, 60  C T. thermosaccharolyticum W16 291.3 mL H2/g TS [41]
centrifugation
Oil palm empty Filtration (0.45 mm pore Batch, 35  C, pH 7.0, Anaerobic sludge 278.0 mL H2/g xylose [38]
fruit bunch size), 5 N NaOH 5 g TS/L
Poplar leaves Aqueous NaOH Batch, 35  C, pH 7.0 Mixed culture 44.9 mL H2/g TS [42]
Rice straw Centrifugation, 0.1 M Tris Batch, 37  C, pH 7.0, Clostridium butyricum CGS5 106.7 mL H2/g RS [43]
eHCl buffer 14 g RSb/L
Sunflower stalks Filtration (0.25 mm pore Batch, 35  C, pH 5.5 Anaerobic sludge 286.5 mL H2/g hexose [44]
size), 1 N NaOH
Sugarcane bagasse Filtration, Ca(OH)2, Batch, 37  C, pH 7.0 Clostridium butyricum 242.9 mL H2/g TS [45]
centrifugation
Pine tree wood Filtration, 8 N NaOH Batch, 35  C, pH 7.0, Anaerobic granular sludge 178.3 mL H2/g TS This study
10 g TS/L
Pine tree wood Filtration, 8 N Ca(OH)2, Batch, 35  C, pH 7.0, Anaerobic granular sludge 179.0 mL H2/g TS This study
filtration 10 g TS/L
a
Total sugar.
b
Reducing sugar.

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separation conditions following dilute acid pretreatment of lignocellulosic biomass, International Journal of Hydrogen Energy (2017),
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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 9

or Ca(OH)2 interchangeably, if and only if SO2

4 concentration perspectives of lignocellulose-based biohydrogen
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separation conditions following dilute acid pretreatment of lignocellulosic biomass, International Journal of Hydrogen Energy (2017),
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