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 Bioresource Technology 135 (2013) 610–615

Contents lists available at SciVerse ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Co-fermentation of water hyacinth and beverage wastewater in powder

and pellet form for hydrogen production
Chyi-How Lay a,f, Biswarup Sen b,c,d,f,⇑, Chin-Chao Chen e,f, Jou-Hsien Wu e,f, Shih-Chi Lee g,f,
Chiu-Yue Lin b,c,d,f
a
Department of Chemistry and Bioengineering, Tampere University of Technology, Tampere, Finland
b
Department of Environmental Engineering and Science, Feng Chia University, Taichung, Taiwan, ROC
c
Green Energy Development Center, Feng Chia University, Taichung, Taiwan, ROC
d
Master’s Program of Green Energy Science and Technology, Feng Chia University, Taichung, Taiwan, ROC
e
Environmental Resources Laboratory, Department of Landscape Architecture, Chung Chou University of Science and Technology, Changhwa, Taiwan, ROC
f
Stone & Resource Industry R&D Center, Hualien, Taiwan, ROC
g
Green Energy and Eco-technology Center, Industrial Technology Research Institute South, Tainan, Taiwan, ROC

h i g h l i g h t s

" Co-fermentation efficiently converted solid waste and wastewater mixture into H2.
" Water hyacinth and beverage mixture (1.6:2.4) pellet was effective in H2 production.
" C/N was key factor in H2 production from water hyacinth and beverage mixture.
" Maximum H2 yield was 13.65 mL H2/g mixture feedstock from co-fermentation.

a r t i c l e i n f o a b s t r a c t

Article history: Hydrogen (H2) production potential of water hyacinth (WH) and beverage wastewater (BW) mixture in
Available online 4 July 2012 powder and pellet form at various combination ratios were evaluated. Batch co-fermentation results
showed peak biogas production of 105.5 mL and H2 production of 55.6 mL at the combination ratio of
Keywords: 1.6 g WH and 2.4 g BW in pellet form. With the same ratio in pellet form, the maximum H2 production
Anaerobic rate 542 mL H2/L-d, maximum specific H2 production rate 869 mL H2/g VSS-d and H2 yield 13.65 mL/g
Hydrogen yield feedstock were obtained, and were 88, 88 and 34% higher than its powder form. The predominant soluble
Soluble metabolites
metabolite was acetate in the concentration of 1059–2639 mg COD/L (40–79% of total metabolites) in
Carbon-to-nitrogen ratio
Pellet
most runs during co-fermentation of mixed feedstock. Carbon-to-nitrogen ratio and the physical form
of the combined feedstock are essential criteria for optimum H2 production. Co-fermentation also allevi-
ates the waste disposal problem of the industries.
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction anaerobic microorganisms. Thus, the exploration of solid wastes

Hydrogen is a promising alternative energy carrier for the fu-
ture because it is clean, renewable and generates harmless by-
products while combustion. Dark fermentative H2 production is a
very efficient method to effectively convert organic solid wastes
and wastewaters into H2 with net energy gain and high commer-
cial potential (Lee and Chung, 2010; Perera et al., 2010). Moreover,
organic solid wastes and wastewaters contain various amino acids
and trace nutrients, which could serve as growth factors for
⇑ Corresponding author at: Department of Environmental Engineering and
Science, Feng Chia University, Taichung, Taiwan, ROC. Fax: +886 4 35072114.
E-mail address: bsen@fcu.edu.tw (B. Sen).
and wastewaters as substrate for H2 production with concurrent
wastewater treatment is an attractive and effective way of tapping
clean energy from renewable sources in a sustainable manner.
Co-fermentation of multiple solid wastes and wastewaters for
H2 production by anaerobic bacteria has been studied previously
(Gómez et al., 2006; Tenca et al., 2011; Venkata Mohan et al.,
2007). In the present study, two wastes (water hyacinth (WH)
and beverage wastewater (BW)) of different origin and composition
were selected and combined to evaluate the collective H2 produc-
tion potential. Water hyacinth, scientifically known as Eichhornia
crassipes, has worldwide distribution and has become a persistent
and expensive aquatic problem damaging the environment, irriga-
tion systems and crops (Chuang et al., 2011). Although WH is

0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2012.06.094
 C.-H. Lay et al. / Bioresource Technology 135 (2013) 610–615
generally considered as an aquatic weed, it is rich in nitrogenous
compounds and can replace synthetic nitrogen source needed for
H2 fermentation. Furthermore, it is a suitable feedstock for biogas
and bioethanol production because it is a fast-growing aquatic
weed and is rich in cellulose and hemicellulose without any lignin,
thus could be hydrolyzed easily compared to other agricultural
wastes by chemical or biological methods (Aswathy et al., 2010;
Forrest et al., 2010; Guragain et al., 2011; Ma et al., 2010) On the
other hand, BW is condensed beverage that is not used for con-
sumption due to long storage and expiry. Traditional concentration
technology is more often used to obtain a viscous form of con-
densed beverage, which also stabilizes the organic compounds
present in the beverage. BW is routinely used for commercial pur-
pose as it minimizes the transportation from the source.
In addition to the requirement of certain essential micro-nutri-
ents such as nitrogen (N), phosphorus (P) and some trace elements
for bacterial metabolism, growth and activity (Kawano et al.,
2004), carbon-to-nitrogen ratio (C/N) is highly crucial in microbial
co-fermentation. An appropriate C/N of the collective feedstock for
co-fermentation is central to the biological activity and the growth
of the fermentative microorganisms. It is therefore essential to ob-
tain an optimum value of C/N prior to fermentation of the collec-
tive feedstock, and for which a combination of two different
wastes with different C/N should be mixed in the right proportion.
We investigated the H2 production potential from co-fermentation
of WH and BW in form of powder and pellet at various combina-
tion ratios. To our knowledge, this is the first report on production
of H2 using mixture of powder and pellet form of WH and BW.
2. Methods
2.1. Seed inoculum
Pig slurry, used for H2 production in this study, was obtained
from a pig farm wastewater treatment plant in Central Taiwan.
The characteristics of the seed inoculum were: pH 6.6, alkalinity
0.71 g/L as CaCO3, volatile suspended solids (VSS) 25.7 g/L, total
chemical oxygen demand (TCOD) 19.8 g/L and total carbohydrate
1.67 mg/L. The pig slurry was seeded into a 1 L continuous stir-
red-tank reactor (CSTR), fed intermittently (5 min feeding at every
1 h) and operated at HRT of 4 h, temperature 45 ± 1 °C and pH 5.5
for 2 weeks. The effluent from the CSTR was used as seed inoculum
in this study.
2.2. Substrates
Water hyacinth was harvested from a waste fish pool in Chang-
hwa (Central Taiwan). The collected water hyacinth was sun-dried
and powdered to 0.8 mm-mesh size. The wastewater was con-
densed beverage with COD concentration of 760–860 g/L. In order
to minimize the volume for transportation to the biofuel plants, a
drying process was used to make BW powder from wastewater.
Briefly, the wastewater was dried in an air-bath oven, and then
powdered to 0.8 mm-mesh size similar to WH powder. The charac-
teristics of these powders are listed in Table 1. The WH and BW
Table 1
Characteristics of the solid and wastewater powder feedstocks.
Feedstock TS* (g/L) VS* (g/L) TCOD* (g-COD/L)
Water hyacinth powder 0.79 0.63 0.73
Beverage wastewater powder 0.85 0.49 3.94
*
Tested with water hyacinth solution of 1 g dried biomass in 1 L solution.
**
Tested with dried biomass.
611
powders were mixed in the ratios of 0.8:0.2, 0.6:0.4, 0.4:0.6 and
0.2:0.8 (g:g), then pressured into pellets each of 1 g weight. The
size of each cylindrical pellet feedstock had diameter of 1.0 cm
and height of 1.5 cm. The mixture of WH and BW is hereafter
shown as WH-BW.
2.3. Experimental design and procedure
Batch H2 production experiments were performed in serum
bottles (volume of 125 mL) with anaerobic head space. The serum
vials were first seeded with 20 mL of seed inoculum, 70 mL of de-
ionized water, 10 mL of pH adjustment solution (1 N HCl or 1 N
NaOH), WH and BW powders and pellets of 4 g per 100 mL work-
ing volume, followed by purging with argon gas. No extra nutrients
were added. The vials were sealed with aluminum cap and then
placed in a reciprocal air-bath shaker (150 rpm). No pH adjustment
was done. Gas samples were taken using a gas tight syringe to
analysis the biogas production and composition during fermenta-
tion. Each experimental test was carried out in triplicate.
2.4. Analytical methods
The analytical procedures of APHA Standard Methods (AHPA,
1995) were used to determine pH, oxidation–reduction potential
(ORP), TCOD, ammonia nitrogen (NH3–N), total solids (TS), sus-
pended solids (SS) and volatile suspended solids (VSS). Biogas vol-
ume was determined by a gas tight syringe at room temperature
(20 °C) and a pressure of 760 mm Hg. The biogas composition in
the batch enrichment assays was measured with a CHINA Chroma-
tography 8700T gas chromatograph equipped with a packed (pack-
ing, Porapak Q), stainless steel column and a thermal conductivity
detector. Oven, injector and detector temperatures were 40, 40
and 40 °C, respectively, and argon as the carrier gas (Lay et al.,
2011). Ethanol and volatile fatty acid (VFA) concentrations were
analyzed using a gas chromatography equipped with flame ioniza-
tion detector (Shimadzu GC-2014, Japan) and a glass column (pack-
ing, FON). Oven, injector and detector temperatures were 145, 175
and 185 °C, respectively. Anthrone-sulfuric acid method was used
to measure total carbohydrate concentration. Cellulose, hemicellu-
loses and lignin were determined by FIBERTECTM 1020 (M6). Ele-
mental analysis was performed on an Elemental Analyzer (Vario
EL Model).
Hydrogen production potential (P, mL), maximum hydrogen
production rate (Rm, mL/h) and lag phase time (k, h) obtained from
the modified Gompertz equation (Eq. (1)) (Lay et al., 1999) were
used as the response variable. STATISTIC software (version 6.0,
Statsoft Inc., USA) and Sigmaplot software (trial version 9.0, Systat
Software Inc., USA) were used for regression and graphical analyses
of the data, respectively.
  
Rm  e
HðtÞ ¼ P  exp  exp ðk  tÞ þ 1 ð1Þ
P
H(t) is the cumulative hydrogen production (mL); P is the
hydrogen production potential (mL); Rm is the maximum hydrogen
production rate (mL/h); e is 2.71828; k is the lag phase time (h) and
Total carbohydrate* Carbohydrate polymer (wt.%)** Elemental analysis C/N**
(wt.%)**
(g/L) Cellulose Hemicellulose Lignin C H N S
0.52 17.3 24.7 1.1 26.1 5.4 2.2 0.4 11.9
0.76 NA NA NA 48.2 5.3 0.1 0.3 602
612 C.-H. Lay et al. / Bioresource Technology 135 (2013) 610–615

t is the cultivation time (h). The maximum hydrogen production higher than powder feedstock at WH and BW combination ratios
rate (HPRmax, mL H2/L-d) was defined as hydrogen production (g:g) of 3.2:0.8, 2.4:1.6 and 1.6:2.4. The HPRmax and SHPRmax from
per working volume per cultivation time and calculated based on pellet with a mixture ratio of WH 1.6 g and BW 2.4 g were 542 mL
the maximum hydrogen production rate (Rm, mL/h) obtained from H2/L-d and 869 mL H2/g VSS-d respectively (Table 3). These values
Gompertz equation. Specific hydrogen production rate (SHPRmax, were 88% higher than powder feedstock of similar composition.
mL H2/g VSS-d) was defined as HPR divided by initial VSS of seed The maximum HY value of 13.65 mL H2/g feedstock (WH 1.6 g
inoculum (5 g/L). Hydrogen production yield (HY, mL H2/g sub- and BW 2.4 g) was similar to composting waste (13.3 mL H2/g
strate) was defined as hydrogen production per g of dried WH- feedstock) using anaerobic microflora (Mohanakrishna et al.,
BW powder and pellet. 2010) and poplar leaves (15.04 mL H2/g feedstock) using anaerobic
mixed microflora (Cui et al., 2010). Nevertheless, the maximum
3. Results and discussion HPR 0.54 L H2/L-d in this study was higher than sugarcane bagasse
(0.01 L H2/L-d) using elephant dung (Fangkum and Reungsang,
3.1. Biohydrogen production potential of WH-BW powder and pellet 2010), organic fraction of municipal solid (0.06 L H2/L-d) waste
using anaerobic sludge (Valdez-Vazquez et al., 2006) and poplar
Fig. 1 illustrates the cumulative biogas and H2 production from leaves (0.38 L H2/L-d) using anaerobic mixed microflora (Cui
WH-BW powder and pellet at various combination ratios. The peak et al., 2010) (Table 4). The longer lag phase during H2 fermentation
biogas and H2 productions were obtained from powder and pellet led to high H2 production efficiency (Table 3).
form at the combination ratio of 1.6:2.4 (WH:BW). The hydrogen
content, final pH and final ORP values were quite similar for pow- 3.2. Production of soluble metabolites during WH-BW fermentation
der and pellet fermentation. The maximum cumulative biogas and
H2 production in this study were 105.5 mL and 55.6 mL from pellet Fermentative conversion of WH and BW into H2 was accompa-
feedstock containing 1.6 g WH and 2.4 g BW, which were 28 and nied by formation of a variety of soluble metabolic products (SMP)
20% higher than values obtained from powder feedstock of similar as shown in Table 5. In some reports (Cui et al., 2010; Sung et al.,
composition, respectively (Table 2). 2011), acetate was the major product, followed by butyrate and
The kinetic parameters estimated from the modified Gompertz ethanol, during the cellulosic material fermentation. In this study
equation (Eq. (1)) and the HPRmax, SHPRmax and HY values calcu- also, the predominant soluble product was acetate with the con-
lated from these parameters are shown in Table 3. The HPRmax centration of 1059–2639 mg COD/L (40–79% of SMP) during WH-
and SHPRmax values increased with the increasing BW fraction BW powder fermentation. Similarly, acetate was the main SMP
from 0.8 to 2.4 g in both powder and pellet feedstock fermentation. with concentration of 1256–2084 mg COD/L (45–59% of SMP) with
However, when the BW fraction was increased to 3.2 g, the H2 pro- the pellet feedstock comprising WH:BW in 3.2:0.8 and 2.4:1.6 ra-
duction efficiency decreased, reflecting in reduced HPRmax to 2 mL tios. Based on the metabolic profile of the WH-BW fermentation,
H2/L-d and SHPRmax to 3 mL H2/g VSS-d, respectively. The H2 pro- the H2 production followed acetate pathway. Moreover, when the
duction efficiency (HPR, SHPR and HY) from pellet feedstock was BW fraction was increased to 2.4 and 3.2 g, the metabolic pathway

120 120
WH : BW
(a) (b)
Cumulatibe H2 production (mL)

WH : BW
Cumulative H production (mL)

(g : g)
(g : g) 100
100 3.2 : 0.8
3.2 : 0.8 2.4 : 1.6
2.4 : 1.6 1.6 : 2.4
80 1.6 : 2.4 80 0.8 : 3.2
0.8 : 3.2

60
2

60

40 40

20 20

120 0
120
Cumulatibe biogas production (mL)

Cumulative biogas production (mL)

100 100

80 80

60 60

40 40

20 20

0 0
0 50 100 150 200 0 50 100 150 200

Time (h) Time (h)

Fig. 1. Cumulative biogas productions from combination of water hyacinth and beverage wastewater (a) powder and (b) pellet.
 C.-H. Lay et al. / Bioresource Technology 135 (2013) 610–615 613

Table 2
Biogas production and water quality after co-fermentation of water hyacinth and beverage wastewater.

Feedstock type WH:BW ratio (g:g) C/N Final pH Final ORP (-mV) Total biogas production (mL) H2 (%) H2 production (mL)
Powder 3.2:0.8 17 5.92 302 40.5 20.7 8.4
2.4:1.6 26 5.21 246 70.0 42.9 30.0
1.6:2.4 42 5.44 209 82.5 50.8 41.9
0.8:3.2 87 4.47 43 7.5 2.7 0.2
Pellet 3.2:0.8 17 5.45 266 39.5 24.1 9.5
2.4:1.6 26 5.34 247 71.0 45.8 32.5
1.6:2.4 42 5.35 218 105.5 52.7 55.6
0.8:3.2 87 4.50 60 10.0 3.0 0.3

Table 3
Hydrogen production performance and the modified Gompertz equation parameter values.

Feedstock type WH:BW ratio (g:g) C/N Modified Gompertz equation parameter HPRmax (mL H2/L-d) SHPRmax (mL H2/g VSS-d) HY (mL/g feedstock)
values
P (mL) Rm (mL/h) k (h) R2
Powder 3.2:0.8 17 8.15 0.78 1.0 0.9919 187 300 2.04
2.4:1.6 26 29.6 0.78 8.7 0.9762 187 300 7.40
1.6:2.4 42 40.7 1.20 23.8 0.9952 288 462 10.18
0.8:3.2 87 0.2 0.01 3.1 0.9936 2 3 0.05
Pellet 3.2:0.8 17 9.4 0.70 4.2 0.9971 168 269 2.35
2.4:1.6 26 32.0 1.42 10.7 0.9895 341 546 8.00
1.6:2.4 42 54.6 2.26 16.0 0.9987 542 869 13.65
0.8:3.2 87 0.3 0.01 44.0 0.9200 2 3 0.08

Table 4
Comparison of the biohydrogen production from solid wastes.

Substrate Seed Temp (°C) pH H2 Yield (mL/g VS) HPR (L H2/L-d) Reference
Beer lees Anaerobic mixed microflora 35 7.0 3.16 1.14 (Cui et al., 2009)
Composite vegetable waste Anaerobic microflora 32 6.0i 13.3 3.59 (Mohanakrishna et al., 2010)
Cornstalk Cow dung compost 36 7.0 3.2 - (Zhang et al., 2007)
Food waste and sewage sludge Anaerobic sludge 35 5.0–6.0 60.1 - (Kim et al., 2004)
Household solid waste Household solid waste 37 5.0–5.5 43 - (Liu et al., 2006)
OFMSW Anaerobic sludge 35 - 10.07 0.06 (Valdez-Vazquez et al., 2006)
Pineapple waste Anaerobic microflora 37 7.5 5.92 mL/g COD 9.20 (Wang et al., 2006)
Popular leaves Anaerobic mixed microflora 35 7.0 15.04 0.38 (Cui et al., 2010)
Spent brewery grains Compost 40 6.0 20.4 6.42 (Chou et al., 2008)
Sugarcane bagasse Elephant dung 55 7.1 97.88 0.01 (Fangkum and Reungsang, 2010)
Wheat straw Cow dung compost 36 7.0 0.5 - (Fan et al., 2006)
Wheat straw Anaerobic microflora 35 - 5.69 - (Nasirian et al., 2011)
Water hyacinth and CMS Pig slurry 35 7.0 13.65 0.54 This study

OFMSW, organic fraction of municipal solid waste; CMS, condensed molasses fermentation solubles.

Table 5
Variations in soluble metabolic products during co-fermentation of water hyacinth and beverage wastewater.

Feedstock type WH:BW ratio (g/g) C/N ratio Ethanol Butanol Acetate Propionate Butyrate Valerate TVFA SMP
(mg COD/L) (% of SMP)
Powder 3.2:0.8 17 21 (0.6) 3 (0.1) 2639 (78.9) N.D. (0) 662 (19.8) 20 (0.6) 3321 (99.3) 3345 (100)
2.4:1.6 26 320 (8.6) 20 (0.5) 2595 (69.7) 115 (3.1) 653 (17.5) 21 (0.6) 3384 (90.9) 3724 (100)
1.6:2.4 42 274 (6.2) 938 (21.1) 1783 (40.2) 82 (1.9) 1329 (30.0) 31 (0.7) 3225 (72.7) 4437 (100)
0.8:3.2 87 61 (3.9) N.D. (0) 1059 (67.4) 66 (4.2) 367 (23.3) 18 (1.2) 1511 (96.1) 1571 (100)
Pellet 3.2:0.8 17 57 (2.0) 15 (0.5) 1256 (44.8) 290 (10.3) 923 (32.9) 264 (9.4) 2733 (97.4) 2805 (100)
2.4:1.6 26 272 (7.8) 24 (0.7) 2084 (58.9) 110 (3.1) 1023 (28.9) 26 (0.7) 3243 (91.6) 3539 (100)
1.6:2.4 42 210 (61.0) 43 (12.5) N.D. (0) N.D. (0) 83 (0) 8 (2.3) 92 (26.5) 344 (100)
0.8:3.2 87 74 (100) N.D. (0) N.D. (0) N.D. (0) N.D. (0) N.D. (0) N.D. (0) 74 (100)

TVFA, total volatile fatty acid; SMP, soluble metabolic products.

changed to ethanol production (Eq. (2)) contributing to 61–100% of
SMP (Fig. 2).
C6 H12 O6 þ 2H2 O ! 2C2 H5 OH þ 2HCO3 þ 2Hþ
Brock et al. (Brock et al., 1994) reported that Clostridium cellobi-
oparum and C. thermocellum could convert cellulose to acetate,
lactate, succinate, ethanol, CO2 and H2. Moreover, sugars, starch
ð2Þ and pectin could be converted by C. butyricum, C. acetobutylicum,
C. pasteurianum, C. perfringens and C. thermosulfurogens to acetone,
614
100
(a)
80
SMP composition (%)
60
40
20
100
80 (b)
SMP composition (%)
60
40
20
0
3.2:0.8
Water hyacinth : Beverage wastewater (w:w)
Fig. 2. Soluble metabolites production profile at various combination ratios and C/N values during from co-fermentation of water hyacinth (WH) and beverage wastewater
(BW) (a) powder and (b) pellet.
butanol, ethenaol, isopropanol, butyrate, acetate, propionate, suc-
cinate, CO2 and H2. Our results on metabolic products indicate that
the pig slurry inoculum used in the WH-BW co-fermentation sys-
tem might contain such species of Clostridium.
(Cheng et al., 2010) calculated the theoretical maximum poten-
tial of H2 production from water hyacinth. The carbon sources for
anaerobic fermentation to produce biogas in water hyacinth were
glucose and xylose that were hydrolyzed from cellulose and hemi-
cellulose. The theoretical H2 production from glucose and xylose
were based on the following mechanisms:
Glucose : C6 H12 O6 þ 2H2 O ! 2CH3 COOH þ 2CO2 þ 4H2
Xylose : C5 H10 O5 þ H2 O ! 2CH3 COOH þ CO2 þ H2
According to Eq. (3), 1 mol of glucose can be converted into
4 mol of H2. Thus, 1 g glucose can be converted into 498 mL H2,
theoretically. When cellulose is completely hydrolyzed into glu-
cose with a H2O molecule, the weight ratio of glucose to cellulose
comes to 1.1 (180/162) (Cheng et al., 2010). Hence, the theoretical
maximum glucose hydrolysate obtained from 17.3% cellulose in
water hyacinth (Table 1) is determined as 17.3  1.1 = 19.2%. The
other glucose source should be from solid BW with a glucose con-
tent of 0.76 g/g BW (Table 1). The optimal combination ratio con-
tained 40% WH and 60% BW (1.6 g WH and 2.4 g BW in 4 g pellet
feedstock). Based on these, the theoretical maximum H2 produc-
tion yield from 0.08 g (0.192 g/g WH  40% of pellet feedstock)
C.-H. Lay et al. / Bioresource Technology 135 (2013) 610–615
EtOH
BuOH
HAc
HPr
HBu
HVa
TVFA
2.4:1.6 1.6:2.4 0.8:3.2
17 26 42 87
C/ N ratio
and from 0.46 g (0.76 g/g solid BW  60% of pellet feedstock) of
glucose hydrolysate in the 1 g pellet feedstock was 268.92 mL
(498 mL  0.54).
On the other hand, 1 mol xylose can be converted into 2 mol of
H2 (Eq. (4)). Thus, 1 g xylose can be converted into 298 mL H2.
When hemicellulose in WH is completely hydrolyzed into xylose
with a H2O molecule, the weight ratio of xylose to hemicellulose
comes to 1.136 (150/132) (Cheng et al., 2010). Hence, the theoret-
ical maximum xylose hydrolysate obtained from 24.7% hemicellu-
lose in water hyacinth (Table 1) is determined as 24.7 
1.136 = 28.06%. Moreover, the WH fraction is only 40% in the opti-
mal mixture ratio in the pellet feedstock. Therefore, the theoretical
ð3Þ
maximum HY from 0.2806 g xylose hydrolysate in the 1 g pellet
feedstock was 32.78 mL (298 mL  0.11), respectively. To sum up
the biogas production from glucose and xylose hydrolysates, the
ð4Þ
theoretical maximum HY from 1 g pellet feedstock was 301.7 mL
H2/g feedstock. However, the real maximum HY of 13.65 mL/g
feedstock was only 5% of theoretical maximum HY (301.7 mL H2/
g feedstock). The practical low yield might be due to lack of effi-
cient fermentative microorganisms in terms of both types and
numbers in the seed inoculum. Another cause would be the size
of the pellet; probably a smaller size of pellet could help in achiev-
ing yield close to the theoretical maximum.
3.3. Significance of the experimental results
The results of this study indicate that pellet form of the mixture
gives higher bioH2 production than the powder form. The reason
 C.-H. Lay et al. / Bioresource Technology 135 (2013) 610–615
might be that the pellet feedstock could release the carbon and
nitrogen sources gradually similar to the drugs release mechanism
of tablets after ingestion in our body to extend the therapy period.
Using the characteristic of the pellet feedstock in the bioenergy pro-
duction process we can replace the continuous feeding unit with
sequencing fed-batch unit to save the energy consumption. Further-
more, a proper C/N value of feedstock is necessary to obtain the
maximum yield of hydrogen and for effective microbial activity. In
our previous study, C/N of 47 was optimum for hydrogen produc-
tion from sucrose (Lin and Lay, 2004). In this study, the C/N ratios
of the WH-BW mixtures ranged from 17 to 87. A combination ratio
of 1.6 g WH and 2.4 g BW having C/N ratio of 42, showed the max-
imum hydrogen production efficiency. This result was similar to the
optimal C/N ratio for hydrogen production from sucrose using
anaerobic mixed microflora in our previous study (Lin and Lay,
2004). The present study demonstrated that a mixture of solid
waste and wastewater would be suitable feedstock for biohydrogen
production. The compositions in the waste materials are very com-
plex; therefore, the C/N ratio could play an important index for
selecting or equalizing a mixture feedstock for co-fermentation.
However, in order to develop a commercially potential hydrogen/
methane production system, further process optimization is also
crucial.
4. Conclusions
The evaluation of the H2 production potential of a mixture of
water hyacinth and beverage wastewater in powder and pellet
form showed that pellet form of the mixture gives higher H2
production than the powder form. While using pellet form of the
mixture the HPR, SHPR and HY were enhanced. The optimal com-
bination ratio was 1.6 g WH and 2.4 g BW with a C/N of 42. C/N
higher or lower than 42 led to decreased H2 production. Therefore,
C/N is an important index for selecting or equalizing a mixture
feedstock while developing a low cost H2 production process.
Acknowledgements
The authors gratefully acknowledge the financial support by
Taiwan’s Bureau of Energy (Grant no. 101-D0204-3), Taiwan’s
National Science Council (NSC-99-2221-E-035-024-MY3, NSC-99-
2221-E-035-025-MY3, NSC-99-2632-E-035-001-MY3, NSC-101-
2218-E-035-03-MY3). Feng Chia University (FCU-11G27150) and
APEC Research Center for Advanced Biohydrogen Technology.
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