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Optimizing biohydrogen production from mushroomcultivation waste using anaerobic mixed cultures

Chyi-How Lay a,b, I-Yuan Sung a, Gopalakrishnan Kumar a, Chen-Yeon Chu b,c,d,Chin-Chao Chen e, Chiu-Yue Lin a,b,c,d,*aDepartment of Environmental Engineering and Science, Feng Chia University, TaiwanbGreen Energy Development Center, Feng Chia University, TaiwancDepartment of Chemical Engineering, Feng Chia University, TaiwandMaster’s Program of Green Energy Science and Technology, Feng Chia University, TaiwaneDepartment of Landscape Architecture, Chungchou University of Science and Technology, Taiwan

a r t i c l e i n f o

Article history:

Received 7 December 2011

Received in revised form

19 February 2012

Accepted 23 February 2012

Available online 1 April 2012

Keywords:

Biohydrogen

Mushroom cultivation waste

Anaerobic mixed cultures

* Corresponding author. Department of EnvTaichung, Taiwan 40724, Taiwan. Tel.: þ886

E-mail address: cylin@fcu.edu.tw (C.-Y. L0360-3199/$ e see front matter Copyright ªdoi:10.1016/j.ijhydene.2012.02.135

a b s t r a c t

The mushroom bag is a polypropylene bag stuffed with wood flour and bacterial nutrients.

After being used for growing mushroom for one to two weeks this bag becomes mushroom

cultivation waste (MCW). About 150 million bags (80,000 tons) of MCW are produced

annually in Taiwan and are usually burned or discarded. The cellulosic materials and

nutrients in MCW could be used as the feedstock and nutrients for anaerobic biohydrogen

fermentation. This study aims to select the inoculum from various waste sludges (sewage

sludge I, sewage sludge II, cow dung and pig slurry) with or without adding any extra

nutrients. A batch test was operated at a MCW concentration of 20 g COD/L, temperature

55 �C and an initial cultivation pH of 8. The results show that extra nutrient addition

inhibited hydrogen production rate (HPR) and hydrogen production yield (HY) when using

cow dung and pig slurry seeds. However, nutrient addition enhanced the HPR and HY in

case of using sewage sludge inoculum and without inoculum. This related to the inhibition

caused by high nutrient concentration (such as nitrogen) in cow dung and pig slurry. Peak

HY of 0.73 mmol H2/g TVS was obtained with no inoculum and nutrient addition. However,

peak HPR and specific hydrogen production rate (SHPR) of 10.11 mmol H2/L/d and

2.02 mmol H2/g VSS/d, respectively, were obtained by using cow dung inoculum without

any extra nutrient addition.

Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights

reserved.

1. Introduction as clean, efficient, renewable, and does not generate any toxic

Today’s energy system is mainly based on the fossil fuels

which are depleting and cannot be sustainable. In view of the

alternative fuels to overcome the future energy demand,

hydrogen is an attractive fuel because of various features such

ironmental Engineering a4 24517250x6200; fax: þ8in).2012, Hydrogen Energy P

by product on its combustion [1]. Biological hydrogen

production process is one of the main alternative methods.

Producing H2 via fermentative route is more environmental

friendly and less energy intensive, thereby being competitive

to conventional H2 production methods such as thermo-

nd Science, Feng Chia University, 100 Wenhwa Road, Seatwen,86 4 35072114.

ublications, LLC. Published by Elsevier Ltd. All rights reserved.

i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 7 ( 2 0 1 2 ) 1 6 4 7 3e1 6 4 7 816474

chemical means [2]. The substrates used in fermentative

hydrogen production are generally rich in carbohydrates like

glucose, sucrose and starch [3]. Currently, biohydrogen and

other biofuels are produced from various agricultural and

bioenergy-generation residues.

At present the biomass from crops like sweet sorghum,

potatoes, sugarcane, soybean and palm oil have been used as

rawmaterials for bioethanol or biohydrogen production [4e8].

The mushroom waste has attained considerable attention

because it is available abundantly and easy to collect and use

as feedstock for the biohydrogen process. Mushroom bag is

a polypropylene bag stuffed with wood flour and nutrients.

After being used for growing mushroom for one to two weeks

this bag becomes mushroom cultivation waste (MCW) About

150 million bags (80,000 tons) of MCW are produced annually

in Taiwan and are usually burned or discarded. Therefore,

proper treatment or usage of this waste could avoid the

environmental problems. Moreover, recent studies show that

this waste could be a feedstock for biohydrogen production in

effective way [11]. The advantages of usingMCW feedstock for

hydrogen production include cost effective because of this

waste being needed to be treated for reducing environmental

pollution problems. However, it has disadvantages such as

further disposal of fermentation residue and solid fermenta-

tion is different from liquid fermentation in operation.

Fermentative hydrogen production process is a tedious

process and requires optimization of inoculums type,

pretreatment, substrate nature and composition, pH and

temperature to scale-up the process. Inoculum selection and

pretreatment are important. Several types of inoculum have

been reported such as sewage sludge [9] and cow dung [10].

Pretreatment of the mushroom cultivation waste by using

acid was also studied [11].

2. Materials and methods

2.1. Seed inocula and substrate

The seed inocula were collected from two municipal waste-

water treatment plants (SS1and SS2), cow dung compost (CD)

and pig slurry (PS) located in central Taiwan. The pH, total

COD (chemical oxygen demand), soluble COD, total carbohy-

drate, volatile suspended solids (VSS, to express the biomass

concentrations), total solids and NH3eN concentrations of the

seed inoculaums are listed in Table 1. The collected sludges

Table 1 e Characteristics of seed inoculms.

Seed pH Total COD Soluble COD

(mg COD/L)

S1 6.9 � 0.1 54,400 � 226 2020 � 28

S2 7.1 � 0.1 70,480 � 1471 2027 � 46

C 7.4 � 0.1 65,680 � 113 7200 � 339

P 7.0 � 0.1 77,200 � 1018 17,280 � 339

S1, Sewage sludge1 (C.H.); S2, Sewage sludge2 (L.M.); C, Cow dung; P, Pig

were heat pretreated at 95 �C for 1 h to inhibit hydrogen-

consuming bacteria.

MCW was collected from a mushroom farm in Changhwa

(central Taiwan). The waste was dried at 105 �C for zero

moisture and then powdered to pass 0.297 mm-mesh sieve.

The characteristics of the mushroom waste are shown in

Table 2.

2.2. Experimental design and procedure

Batch hydrogen production experiments were performed

using serum bottles (125 mL) with anaerobic head space. The

vials were first purged with argon gas followed by adding

10 mL of seed inocula, 40 mL of de-ionized water or nutrient

solution, 10 mL of pH adjustment solution (1 N HCl or 1 N

NaOH) and tested dried MCW agricultural powder ranging

1.2 g per 60 mL working volume (20 g/L). These vials were

placed in a reciprocal air-bath shaker (150 rpm) with a culti-

vation temperature of 55 � 1 �C. The tested initial pH value

was 8.0. The nutrient solution contained inorganic supple-

ments (mg/L): NH4HCO3 5240, K2HPO4 125, MgCl2$6H2O 100,

MnSO4$6H2O 15, FeSO4$7H2O 25, CuSO4$5H2O 5, CoCl2$5H2O

0.125 and NaHCO3 6720 [12]. Each experimental condition was

carried out with duplicate.

2.3. Analytical method

The analytical procedures of APHA Standard Methods [13]

were used to determine pH, oxidation-reduction potential

(ORP), total chemical oxygen demand (TCOD), ammonia

nitrogen (NH3eN), suspended solids (SS) and volatile SS (VSS).

Biogas volume 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 Chromatography 8700T gas chromatograph

equipped with a packed (packing, Porapak Q), stainless steel

column and a thermal conductivity detector. Oven, injector

and detector temperatures were 40, 40 and 40 �C, respectivelyand argon as the carrier gas. Same methods are indicated in

our previous studies [14]. Anthrone-sufuric acid method was

used to measure total carbohydrate concentration [14].

Cellulose, hemicelluloses and lignin were determined by

FIBERTECTM 1020 (M6). Elemental analysis was performed on

an Elemental Analyzer (Model: Vario EL).

Hydrogen production potential (P, mL), maximum

hydrogen production rate (Rm, mL/h) and lag phase time (l, h)

obtained from the modified Gompertz equation (Eq. (1)) [14]

Total carbohydrate(mg/L as glucose)

VSS(g/L)

NH3eN(mg/L)

6424 � 92 21 75

7054 � 215 32 124

10,641 � 226 22 276

2380 � 31 17 243

dung; COD, chemical oxygen demand.

Table 2 e Characteristics of mushroom waste.

TS*(g/L)

VSa

(g/L)TCODa

(G-COD/L)Total

Carbohydratea

(g/L)

Celluloseb

(%)Hemicelluloseb

(%)Ligninb

(%)Ultimate analysis wt % b C/N

ratiobC H N S

0.84 0.78 1.01 0.46 31.2 6.9 14 40.2 5.0 1.4 0.2 28.7

a Tested by the agricultural waste solution with 1 g dried biomass in 1 L solution.

b Tested by dried biomass.

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 en e r g y 3 7 ( 2 0 1 2 ) 1 6 4 7 3e1 6 4 7 8 16475

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 regres-

sion and graphical analyses of the data, respectively.

HðtÞ ¼ P$exp

�� exp

�Rm$eP

ðl� tÞ þ 1

��(1)

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; l is the lag

phase time (h) and t is the cultivation time (h). The maximum

hydrogen production rate (HPRmax, mmol H2/L-d) was defined

as hydrogen production per working volume per cultivation

time and calculated based on the maximum hydrogen

production rate (Rm, mL/h) obtained from Gompertz equation.

Specific hydrogen production rate (SHPRmax, mmol H2/g VSS-

d) was defined as HPR divided by initial VSS of seed inocu-

lums (5 g/L). Hydrogen production yield (HY, mmol H2/g TVS)

was defined as hydrogen production per gram of dried MCW.

3. Results and discussion

3.1. Hydrogen generation

Table 3 shows that the nutrient addition could enhance the

total biogas production comparingwithwithout extra nutrient

for the same seed inoculum. On the contrary, the hydrogen

contents in biogas of no nutrient addition were higher than

that of with extra nutrients for the same seed inoculum. The

Table 3 e Biogas production using various seed inocula witho

Seed Nutrientformulation

Totalbiogas(mL)

H2

conc.(%)

H2

(mL)Modified Gompertequation paramete

P(mL)

R(mL/h)

l

(h)

SS1 Without 21.0 38.1 8.0 7.8 0.20 3.8 0

With 67.3 25.6 17.2 17.7 0.21 3.8 0

SS2 Without 19.7 38.1 7.5 7.5 0.15 4.6 0

With 73.0 27.1 19.8 20.4 0.20 4.3 0

CD Without 51.5 47.0 24.2 24.3 0.68 26.2 0

With 72.3 16.7 12.1 12.1 0.21 18.1 0

PS Without 20.0 32.0 6.4 6.8 0.05 3.2 0

With 34.3 7.0 2.4 2.3 0.03 9.9 0

EB Without 13.7 30.7 4.2 4.4 0.07 5.9 0

With 150.0 26.3 39.5 40.5 0.27 21.5 0

S1, Sewage sludge1 (C.H.); S2, Sewage sludge2 (L.M.); C, Cow dung; P, Pig

peak hydrogen production of 24.2 mL was obtained using

cow dung seed inoculum without any Endo nutrient

(Fig. 1a and Table 3). However, the hydrogen production was

less than 10 mL for other seed inoculum. Moreover, the

maximum biogas and hydrogen production were 150 mL and

39.5 mL, respectively, with nutrient addition and no seed

inoculum (EB, Endogenous bacteria) (Fig. 2b and Table 3).

A peak HY of 0.73 mmol H2/g TVS was obtained with no

inoculum at nutrient addition (Table 3). This HY value was

about 3 times of HY 0.28 mmol H2/g TVS which was obtained

in beer lees fermentation at 36 �C and pH 7.0 by cow dung

compost [15]. However, peak HPR and SHPR of 10.11 mmol H2/

L/d and 2.02 mmol H2/g VSS/d, respectively, were obtained by

using cow dung inoculum without extra nutrients. This value

is higher than that of converting other agricultural wastes into

hydrogen (Table 4). Fig. 3 depicts that nutrient addition

inhibited HPR and HY when using cow dung and pig slurry

seeds. Biohydrogen production requires certain essential

micro-nutrients such as nitrogen (N), phosphate (P), and some

trace elements for bacterial metabolism, growth and activity

[16]. A proper C/N-ratio value for mixed microflora is neces-

sary to optimize anaerobic hydrogen production from organic

wastewater. The reason of low HY for cow dung and pig slurry

might be that too high nutrient concentration (such as

nitrogen) which inhibited the fermentation (Fig. 3). However,

nutrient addition elevated HPR and HY in the case of using

sewage sludge inoculum and without inoculum. Using cow

dung seed has themost significant increase (240%) in HPR and

SHPR. On the other hand, the HY increased from 0.04 mmol

H2/g TVS with extra nutrients to 0.12 mmol H2/g TVS without

ut and with nutrients.

zr

HPR(mmol H2/L/d)

SHPR(mmol H2/g VSS/d)

HY(mmol H2/g TVS)

R2

.9707 2.97 0.59 0.15

.9795 3.12 0.62 0.32

.9785 2.23 0.45 0.14

.9831 2.97 0.59 0.37

.992 10.11 2.02 0.45

.9981 3.12 0.62 0.22

.9834 0.74 0.15 0.12

.9789 0.45 0.09 0.04

.9674 1.04 N.A 0.08

.9966 4.02 N.A 0.73

dung, EB, Endogenous bacteria.

HY

(m

mol

H2/

g T

VS)

0.0

0.2

0.4

0.6

0.8

1.0

WithoutWith

HP

R (

mm

ol H

2/L

-d)

0

2

4

6

8

10

12

seed

SS1 SS2 CD PS EB

SHP

R (

mm

ol H

2/g

VSS

-d)

0.0

0.5

1.0

1.5

2.0

2.5

Fig. 2 e HY, HPR, and SHPR using various seed inocula (SS1,

Sewage sludge 1 (C.H.); SS2, Sewage sludge 2 (L.M.); C, Cow

dung; PS, Pig dung; EB, endogenous bacteria) without and

with nutrient.

Time (h)

0 50 100 150 200 250 300 350

Acc

umul

ativ

e H

2 pr

oduc

tion

(m

L)

0

10

20

30

40

50

Acc

umul

ativ

e H

2 pr

oduc

tion

(m

L)

0

10

20

30

40

50

SS1 SS2CD PD EB

a

b

Fig. 1 e Hydrogen production using various seed

inoculaums (SS1, Sewage sludge 1 (C.H.); SS2, Sewage

sludge 2 (L.M.); CD, Cow dung; PS, Pig slurry; EB, endogens

bacteria) (a) without and (b) with nutrient formulation.

i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 7 ( 2 0 1 2 ) 1 6 4 7 3e1 6 4 7 816476

extra nutrient (Table 3 and Fig. 3). Table 3 also indicates that

long lag phase time (l) values were obtained for the fermen-

tation using pig dung seed with and without extra nutrients

and endogenous bacteria with extra nutrients. Therewere low

C/N ratios because of low carbohydrate concentration and

high NH3eN concentration in these conditions.

Table 4 e Biohydrogen production from various rawagriculture wastes.

Feedstock HPR(mmol H2/L/d)

HY(mmol H2/g TVS)

Reference

Mushroom

waste

10.11 0.45 This study

Mushroom

waste

4.02 0.73 This study

Polar leaves 24.01 0.61 [17]

Beer Lees 46.53 0.13 [20]

Beer lees NA 0.28 [15]

Cornstalk NA 0.13 [21]

3.2. Productions of ethanol and volatile fatty acids

Fig. 3 indicates that the main soluble metabolic product (SMP)

was acetate (1440e4570 mg COD/L, 60.4e86.8% of SMP) from

mushroom MCW wastes substrate after 308 h cultivation.

Similar results were reported during cellulosic materials

fermentation [17,18]. Propionate concentration was high

(1142 mg COD/L, 20.0% of SMP) using pig slurry inoculumwith

low hydrogen production performance (0.04 mmol H2/g TVS)

(Table 5). The reason was that hydrogen production was

inhibited by the cumulated propionate. Similar results were

reported by Vavilin et al. [19]. The peak SMP concentration of

5708mg COD/Lwithout any extra nutrient was obtained using

pig slurry inoculum. However, cow dung and pig slurry seed

inoculum and endogenous bacteria seed could convert the

MCW into high SMP concentrations ranging from 5437 to

5807 mg COD/L.

Fig. 3 e Soluble metabolic products using various seed inoculums (SS1, Sewage sludge1 (C.H.); SS2, Sewage sludge2 (L.M.); C,

Cow dung; PS, Pig dung; EB, endogenous bacteria) (a) without and (b) with nutrient.

Table 5 e Soluble metabolic products using various seed inoculums without and with nutrient.

Seed Nutrientformulation

HY(mmol H2/g TVS)

EtOH(mmol/g TVS)

HAc(mmol/g TVS)

HPr(mmol/g TVS)

HBu(mmol/g TVS)

HVa(mmol/g TVS)

SS1 Without 0.15 0.02 1.54 0.09 0.10 N.D

With 0.32 0.01 3.22 0.16 0.12 0.00

SS2 Without 0.14 0.02 1.47 0.10 0.06 N.D

With 0.37 0.01 2.90 0.13 0.09 0.00

CD Without 0.45 0.03 2.68 0.11 0.08 N.D

With 0.22 0.01 3.57 0.19 0.13 0.00

PS Without 0.12 0.02 2.55 0.49 0.26 0.04

With 0.04 0.03 3.03 0.51 0.16 0.03

EB Without 0.08 0.03 1.13 0.09 0.02 N.D

With 0.73 0.06 3.29 0.26 0.29 N.D

S1, Sewage sludge1 (C.H.); S2, Sewage sludge2 (L.M.); C, Cow dung; P, Pig dung; EB, Endogenous bacteria EtOH, ethanol; HAc, acetic acid; HPr,

propionic acid; HBu, butyric acid; HVa, valeric acid; TVFA ¼ HAc þ HPr þ HBu þ HVa; SMP ¼ TVFA þ EtOH.

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 en e r g y 3 7 ( 2 0 1 2 ) 1 6 4 7 3e1 6 4 7 8 16477

i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 3 7 ( 2 0 1 2 ) 1 6 4 7 3e1 6 4 7 816478

The liquid product analysis shows that the major metab-

olites determined after fermentation were ethanol, acetic,

propionic and butyric acids. Peak hydrogen yieldwas obtained

at nutrient addition using endogenous bacteria seed because

the substrate was efficiently utilized for hydrogen production

by the endogenous bacteria. The possible metabolic pathway

of soluble sugar (mainly glucose) to produce hydrogen at the

maximum yield is shown as Eq. (2):

C6H12O6/0:285H2 þ 1:310CO2 þ 0:023C2H5OH

þ 1:287CH3COOHþ 0:101C2H5COOHþ 0:113C3H7COOH (2)

As shown in Eq. (2), the metabolic pathway was mainly

acetic acid fermentation, and the conversion efficiency of H

element in glucose into H2 was 4.75%.

Theoretically the metabolic pathways of producing acetic

acid from glucose and xylose are as follows:

C5H10O5 þ 1:67H2O/3:33H2 þ 1:67CH3COOHþ 1:67CO2 (3)

C6H12O6þ2H2O/4H2þ2CH3COOHþ 2CO2 (4)

The theoretical metabolic pathway followed mainly acetic

acid fermentation which favors high hydrogen production.

Comparing Eqs. (2)e(4), the acetic acid production from

mushroom wastes was 77.1% and 64.4% from glucose and

xylose. Shifting the metabolic pathway to acetate-butyrate

fermentation could lead to higher hydrogen production.

4. Conclusions

Mushroom cultivation waste could be used as the feedstock

for hydrogen production using waste sludges (sewage sludge,

cow dung and pig slurry) with or without adding any extra

nutrients. Peak HY of 0.73 mmol H2/g TVS was obtained with

nutrient addition by the endogenous bacteria of mushroom

waste. Cow dung inoculum could directly degrade mushroom

waste without any extra nutrients and has peak HPR of

10.11 mmol H2/L/d and SHPR of 2.02 mmol H2/g VSS/d. The

main soluble metabolic product was acetate (1440e4570 mg

COD/L, 60.4e86.8% of SMP) after 308 h cultivation.

Acknowledgments

The authors gratefully acknowledge the financial support by

Taiwan’s Bureau of Energy (grant no. 99-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), Feng

Chia University (FCU-10G27101) and APEC Research Center for

Advanced Biohydrogen Technology.

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