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
Masters 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 efciently converted solid waste and wastewater mixture into H
2
.
" Water hyacinth and beverage mixture (1.6:2.4) pellet was effective in H
2
production.
" C/N was key fa ctor in H
2
produc tion from water hyacinth and beverage mixture.
" Maximum H
2
yield was 13.65 mL H
2
/g mixture feedstock from co-fermentation.
a r t i c l e i n f o
Article history:
Available online 4 July 2012
Keywords:
Anaerobic
Hydrogen yield
Soluble metabolites
Carbon-to-nitrogen ratio
Pellet
a b s t r a c t
Hydrogen (H
2
) production potential of water hyacinth (WH) and beverage wastewater (BW) mixture in
powder and pellet form at various combination ratios were evaluated. Batch co-fermentation results
showed peak biogas production of 105.5 mL and H
2
production of 55.6 mL at the combination ratio of
1.6 g WH and 2.4 g BW in pellet form. With the same ratio in pellet form, the maximum H
2
production
rate 542 mL H
2
/L -d, maximum specic H
2
production rate 869 mL H
2
/g VSS-d and H
2
yield 13.65 mL/g
feedstock were obtained, and were 88, 88 and 34% higher than its powder form. The predominant soluble
metaboli te was acetate in the concentration of 10592639 mg COD/L (4079% of total metabolites) in
most runs during co-fermentation of mixed feedstock. Carbon-to -nitrogen ratio and the physical form
of the combined feedstock are essent ial criteria for optimum H
2
production. Co-fermentat ion also allevi-
ates the waste disposal problem of the industries.
2012 Elsevier Ltd. All rights reserved.
1. Introduction
Hydrogen is a promising alternative energy carrier for the fu-
ture because it is clean, renewable and generates harmless by-
products while combusti on. Dark fermentative H
2
production is a
very efcient method to effectively convert organic solid wastes
and wastewa ters into H
2
with net energy gain and high commer-
cial potential (Lee and Chung, 2010; Perera et al., 2010 ). Moreover,
organic solid wastes and wastewater s contain various amino acids
and trace nutrients, which could serve as growth factors for
anaerobic microorganism s. Thus, the exploration of solid wastes
and wastewa ters as substrate for H
2
production with concurrent
wastewa ter treatment is an attractive and effective way of tapping
clean energy from renewable sources in a sustainable manner.
Co-ferme ntation of multiple solid wastes and wastewaters for
H
2
production by anaerobic bacteria has been studied previousl y
(Gmez 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 H
2
produc-
tion potential . Water hyacinth, scientically known as Eichhorni a
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 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biortech.2012.06.094

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).
Bioresource Technology 135 (2013) 610615
Contents lists available at SciVerse ScienceDi rect
Biore source Tec hnology
j our nal homepage: www. el sevi er . com/ l ocat e/ bi or t ech
generally considered as an aquatic weed, it is rich in nitrogenous
compounds and can replace synthetic nitrogen source needed for
H
2
fermentati on. 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 hemicellulos e 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 concentratio n
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 requiremen t of certain essential micro-nutr i-
ents such as nitrogen (N), phosphorus (P) and some trace elements
for bacterial metabolism, growth and activity (Kawano et al.,
2004), carbon-to-nitr ogen ratio (C/N) is highly crucial in microbial
co-fermentati on. An appropriate C/N of the collective feedstock for
co-fermentati on is central to the biological activity and the growth
of the fermentative microorgan isms. 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 H
2
production potential from co-fermentati on
of WH and BW in form of powder and pellet at various combina-
tion ratios. To our knowled ge, this is the rst report on production
of H
2
using mixture of powder and pellet form of WH and BW.
2. Methods
2.1. Seed inoculum
Pig slurry, used for H
2
production in this study, was obtained
from a pig farm wastewater treatment plant in Central Taiwan.
The characterist ics of the seed inoculum were: pH 6.6, alkalinity
0.71 g/L as CaCO
3
, volatile suspended solids (VSS) 25.7 g/L, total
chemical oxygen demand (TCOD) 19.8 g/L and total carbohydrat e
1.67 mg/L. The pig slurry was seeded into a 1 L continuo us stir-
red-tank reactor (CSTR), fed intermitten tly (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 efuent from the CSTR was used as seed inoculum
in this study.
2.2. Substrates
Water hyacinth was harvested from a waste sh 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 760860 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.
Briey, 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
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 pressure d 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. Experimen tal design and procedure
Batch H
2
production experime nts were performed in serum
bottles (volume of 125 mL) with anaerobic head space. The serum
vials were rst 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 experime ntal test was carried out in triplicate.
2.4. Analytica l methods
The analytical procedures of APHA Standard Methods (AHPA,
1995) were used to determine pH, oxidationreduction potential
(ORP), TCOD, ammonia nitrogen (NH
3
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 temperat ure
(20 C) and a pressure of 760 mm Hg. The biogas compositi on in
the batch enrichment assays was measured with a CHINA Chroma-
tography 8700T gas chromatograp h equipped with a packed (pack-
ing, Porapak Q), stainless steel column and a thermal conductivi ty
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) concentratio ns were
analyzed using a gas chromatography equipped with ame 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, respectivel y. Anthrone-su lfuric acid method was used
to measure total carbohydrat e concentr ation. 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 (R
m
, mL/h) and lag phase time (k, h) obtained from
the modied 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 .
Ht P exp exp
R
m
e
P
k t 1

1
H(t) is the cumulative hydrogen production (mL); P is the
hydrogen production potential (mL); R
m
is the maximum hydrogen
production rate (mL/h); e is 2.71828; k is the lag phase time (h) and
Table 1
Characteristics of the solid and wastewater powder feedstocks.
Feedstock TS
*
(g/L) VS
*
(g/L) TCOD
*
(g-COD/L) Total carbohydrate
*
Carbohydrate polymer (wt.%)
**
Elemental analysis
(wt.%)
**
C/N
**
(g/L) Cellulose Hemicellulose Lignin C H N S
Water hyacinth powder 0.79 0.63 0.73 0.52 17.3 24.7 1.1 26.1 5.4 2.2 0.4 11.9
Beverage wastewater powder 0.85 0.49 3.94 0.76 NA NA NA 48.2 5.3 0.1 0.3 602
*
Tested with water hyacinth solution of 1 g dried biomass in 1 L solution.
**
Tested with dried biomass.
C.-H. Lay et al. / Bioresource Technology 135 (2013) 610615 611
t is the cultivation time (h). The maximum hydrogen production
rate (HPR
max
, mL H
2
/L-d) was dened as hydrogen production
per working volume per cultivatio n time and calculated based on
the maximum hydrogen production rate (R
m
, mL/h) obtained from
Gompertz equation. Specic hydrogen production rate (SHPR
max
,
mL H
2
/g VSS-d) was dened as HPR divided by initial VSS of seed
inoculum (5 g/L). Hydrogen production yield (HY, mL H
2
/g sub-
strate) was dened as hydrogen production per g of dried WH-
BW powder and pellet.
3. Results and discussion
3.1. Biohydrogen productio n potential of WH-BW powder and pellet
Fig. 1 illustrate s the cumulative biogas and H
2
production from
WH-BW powder and pellet at various combination ratios. The peak
biogas and H
2
productions were obtained from powder and pellet
form at the combination ratio of 1.6:2.4 (WH:BW). The hydrogen
content, nal pH and nal ORP values were quite similar for pow-
der and pellet fermentation. The maximum cumulative biogas and
H
2
production in this study were 105.5 mL and 55.6 mL from pellet
feedstock containing 1.6 g WH and 2.4 g BW, which were 28 and
20% higher than values obtained from powder feedstock of similar
composition, respectivel y (Table 2).
The kinetic parameters estimated from the modied Gompert z
equation (Eq. (1)) and the HPR
max
, SHPR
max
and HY values calcu-
lated from these parameters are shown in Table 3. The HPR
max
and SHPR
max
values increased with the increasing BW fraction
from 0.8 to 2.4 g in both powder and pellet feedstock fermentation.
However, when the BW fraction was increased to 3.2 g, the H
2
pro-
duction efciency decreased, reecting in reduced HPR
max
to 2 mL
H
2
/L-d and SHPR
max
to 3 mL H
2
/g VSS-d, respectively . The H
2
pro-
duction efciency (HPR, SHPR and HY) from pellet feedstock was
higher than powder feedstock at WH and BW combination ratios
(g:g) of 3.2:0.8, 2.4:1.6 and 1.6:2.4. The HPR
max
and SHPR
max
from
pellet with a mixture ratio of WH 1.6 g and BW 2.4 g were 542 mL
H
2
/L-d and 869 mL H
2
/g VSS-d respectively (Table 3). These values
were 88% higher than powder feedstock of similar compositi on.
The maximum HY value of 13.65 mL H
2
/g feedstock (WH 1.6 g
and BW 2.4 g) was similar to composting waste (13.3 mL H
2
/g
feedstock ) using anaerobic microora (Mohanakrishna et al.,
2010) and poplar leaves (15.04 mL H
2
/g feedstock) using anaerobic
mixed microora (Cui et al., 2010 ). Nevertheless, the maximum
HPR 0.54 L H
2
/L-d in this study was higher than sugarcane bagasse
(0.01 L H
2
/L-d) using elephant dung (Fangkum and Reungsang,
2010), organic fraction of municipal solid (0.06 L H
2
/L-d) waste
using anaerobic sludge (Valdez-V azquez et al., 2006 ) and poplar
leaves (0.38 L H
2
/L-d) using anaerobic mixed microora (Cui
et al., 2010 ) (Table 4). The longer lag phase during H
2
fermentation
led to high H
2
production efciency (Table 3).
3.2. Production of soluble metabolites during WH-BW fermentation
Fermenta tive conversion of WH and BW into H
2
was accompa-
nied by formation of a variety of soluble metabolic products (SMP)
as shown in Table 5. In some reports (Cui et al., 2010; Sung et al.,
2011), acetate was the major product, followed by butyrate and
ethanol, during the cellulosic material fermentation. In this study
also, the predominant soluble product was acetate with the con-
centration of 10592639 mg COD/L (4079% of SMP) during WH-
BW powder fermentati on. Similarly, acetate was the main SMP
with concentr ation of 12562084 mg COD/L (4559% of SMP) with
the pellet feedstock comprising WH:BW in 3.2:0.8 and 2.4:1.6 ra-
tios. Based on the metabolic prole of the WH-BW fermentation,
the H
2
production followed acetate pathway. Moreover, when the
BW fraction was increased to 2.4 and 3.2 g, the metabolic pathway
Time (h)
0 50 100 150 200
C
u
m
u
l
a
t
i
b
e

b
i
o
g
a
s

p
r
o
d
u
c
t
i
o
n

(
m
L
)
0
20
40
60
80
100
120
C
u
m
u
l
a
t
i
b
e

H
2

p
r
o
d
u
c
t
i
o
n

(
m
L
)
20
40
60
80
100
120
3.2 : 0.8
2.4 : 1.6
1.6 : 2.4
0.8 : 3.2
WH : BW
(g : g)
(a)
C
u
m
u
l
a
t
i
v
e

H
2

p
r
o
d
u
c
t
i
o
n

(
m
L
)
0
20
40
60
80
100
120
3.2 : 0.8
2.4 : 1.6
1.6 : 2.4
0.8 : 3.2
Time (h)
0 50 100 150 200
C
u
m
u
l
a
t
i
v
e

b
i
o
g
a
s

p
r
o
d
u
c
t
i
o
n

(
m
L
)
0
20
40
60
80
100
120
WH : BW
(g : g)
(b)
Fig. 1. Cumulative biogas productions from combination of water hyacinth and beverage wastewater (a) powder and (b) pellet.
612 C.-H. Lay et al. / Bioresource Technology 135 (2013) 610615
changed to ethanol production (Eq. (2)) contributi ng to 61100% of
SMP (Fig. 2).
C
6
H
12
O
6
2H
2
O !2C
2
H
5
OH2HCO

3
2H

2
Brock et al. (Brock et al., 1994 ) reported that Clostridium cellobi-
oparum and C. thermocellum could convert cellulose to acetate,
lactate, succinate, ethanol, CO
2
and H
2
. Moreove r, sugars, starch
and pectin could be converted by C. butyricum , C. acetobutylicum,
C. pasteurian um, C. perfringens and C. thermosulfurogen s to acetone,
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) H
2
(%) H
2
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 4
Comparison of the biohy drogen production from solid wastes.
Substrate Seed Temp (C) pH H
2
Yield (mL/g VS) HPR (L H
2
/L-d) Reference
Beer lees Anaerobic mixed microora 35 7.0 3.16 1.14 (Cui et al., 2009 )
Composite vegetable waste Anaerobic microora 32 6.0
i
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 5.06.0 60.1 - (Kim et al., 2004 )
Household solid waste Household solid waste 37 5.05.5 43 - (Liu et al., 2006 )
OFMSW Anaerobic sludge 35 - 10.07 0.06 (Valdez-Vazquez et al., 2006 )
Pineapple waste Anaerobic microora 37 7.5 5.92 mL/g COD 9.20 (Wang et al., 2006 )
Popular leaves Anaerobic mixed microora 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 microora 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 bevera ge 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.
Table 3
Hydrogen production performance and the modied Gompertz equation parameter values.
Feedstock type WH:BW ratio (g:g) C/N Modied Gompertz equation parameter
values
HPR
max
(mL H
2
/L-d) SHPR
max
(mL H
2
/g VSS-d) HY (mL/g feedstock)
P (mL) R
m
(mL/h) k (h) R
2
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
C.-H. Lay et al. / Bioresource Technology 135 (2013) 610615 613
butanol, ethenaol, isopropanol , butyrate, acetate, propionate, suc-
cinate, CO
2
and H
2
. Our results on metabolic products indicate that
the pig slurry inoculum used in the WH-BW co-ferme ntation sys-
tem might contain such species of Clostridium.
(Cheng et al., 2010 ) calculated the theoretical maximum poten-
tial of H
2
production from water hyacinth. The carbon sources for
anaerobic fermentation to produce biogas in water hyacinth were
glucose and xylose that were hydrolyz ed from cellulose and hemi-
cellulose. The theoretical H
2
production from glucose and xylose
were based on the following mechanism s:
Glucose : C
6
H
12
O
6
2H
2
O !2CH
3
COOH2CO
2
4H
2
3
Xylose : C
5
H
10
O5 H
2
O !2CH
3
COOHCO
2
H
2
4
According to Eq. (3), 1 mol of glucose can be converte d into
4 mol of H
2
. Thus, 1 g glucose can be converted into 498 mL H
2
,
theoreticall y. When cellulose is completely hydrolyz ed into glu-
cose with a H
2
O 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 H
2
produc-
tion yield from 0.08 g (0.192 g/g WH 40% of pellet feedstock)
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
H
2
(Eq. (4)). Thus, 1 g xylose can be converte d into 298 mL H
2
.
When hemicellulos e in WH is complete ly hydrolyzed into xylose
with a H
2
O 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
maximum HY from 0.2806 g xylose hydrolysate in the 1 g pellet
feedstock was 32.78 mL (298 mL 0.11), respectivel y. To sum up
the biogas production from glucose and xylose hydrolysate s, the
theoretical maximum HY from 1 g pellet feedstock was 301.7 mL
H
2
/g feedstock . However, the real maximum HY of 13.65 mL/g
feedstock was only 5% of theoretical maximum HY (301.7 mL H
2
/
g feedstock ). The practical low yield might be due to lack of ef-
cient fermentative microorganism s 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. Signicance of the experimen tal results
The results of this study indicate that pellet form of the mixture
gives higher bioH
2
production than the powder form. The reason
S
M
P

c
o
m
p
o
s
i
t
i
o
n

(
%
)
20
40
60
80
100
EtOH
BuOH
HAc
HPr
HBu
HVa
TVFA
(a)
Water hyacinth : Beverage wastewater (w:w)
3.2:0.8 2.4:1.6 1.6:2.4 0.8:3.2
S
M
P

c
o
m
p
o
s
i
t
i
o
n

(
%
)
0
20
40
60
80
100
C/ N ratio
17 26 42 87
(b)
Fig. 2. Soluble metabolites production prole 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.
614 C.-H. Lay et al. / Bioresource Technology 135 (2013) 610615
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 characteri stic 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 consump tion. 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 efciency. This result was similar to the
optimal C/N ratio for hydrogen production from sucrose using
anaerobic mixed microora in our previous study (Lin and Lay,
2004). The present study demonstrat ed that a mixture of solid
waste and wastewater would be suitable feedstock for biohydrogen
production. The compositi ons 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 commerciall y potential hydrogen /
methane production system, further process optimization is also
crucial.
4. Conclusions
The evaluation of the H
2
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 H
2
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 decrease d H
2
production. Therefore,
C/N is an important index for selecting or equalizing a mixture
feedstock while developing a low cost H
2
production process.
Acknowled gements
The authors gratefully acknowledge the nancial support by
Taiwans Bureau of Energy (Grant no. 101-D0204-3), Taiwans
National Science Council (NSC-99-2221-E-035-02 4-MY3, NSC-99-
2221-E-035- 025-MY3, NSC-99-2632- E-035-001-MY 3, NSC-101-
2218-E-035- 03-MY3). Feng Chia University (FCU-11G27150) and
APEC Research Center for Advanced Biohydrogen Technology .
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