hydrogen production characteristics of the organic fraction of municipal solid wastes by anaerobic...

$: Hydrogen production characteristics of the organic fraction of municipal solid wastes by anaerobic mixed culture fermentation$
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 3 4 ( 2 0 0 9 ) 8 1 2 – 8 2 0

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Hydrogen production characteristics of the organicfraction of municipal solid wastes by anaerobic mixedculture fermentation

Li Donga,b,*, Yuan Zhenhonga, Sun Yongminga, Kong Xiaoyinga, Zhang Yua,b

aGuangzhou Institute of Energy Conversion, Chinese Academy of Sciences, Guangzhou 510640, ChinabGraduate School of the Chinese Academy of Sciences, Beijing 100049, China

a r t i c l e i n f o

Article history:

Received 8 October 2008

Received in revised form

12 November 2008

Accepted 13 November 2008

Available online 13 December 2008

Keywords:

OFMSW

Hydrogen

Anaerobic fermentation

Mixed culture

* Corresponding author. Guangzhou InstituteNo. 1, Nengyuan Road, Wushan, Tianhe Dis

E-mail address: [email protected] (L.0360-3199/$ – see front matter ª 2008 Interndoi:10.1016/j.ijhydene.2008.11.031

a b s t r a c t

The hydrogen production from the organic fraction of municipal solid waste (OFMSW) by

anaerobic mixed culture fermentation was investigated using batch experiments at 37 �C.

Seven varieties of typical individual components of OFMSW including rice, potato, lettuce,

lean meat, oil, fat and banyan leaves were selected to estimate the hydrogen production

potential. Experimental results showed that the boiling treated anaerobic sludge was

effective mixed inoculum for fermentative hydrogen production from OFMSW. Mechanism

of fermentative hydrogen production indicates that, among the OFMSW, carbohydrates is

the most optimal substrate for fermentative hydrogen production compared with proteins,

lipids and lignocelluloses. This conclusion was also substantiated by experimental results

of this study. The hydrogen production potentials of rice, potato and lettuce were 134 mL/

g-VS, 106 mL/g-VS, and 50 mL/g-VS respectively. The hydrogen percentages of the total gas

produced from rice, potato and lettuce were 57–70%, 41–55% and 37–67%.

ª 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights

reserved.

1. Introduction produce electricity through fuel cells [1,2]. Generally, there are

The excessive use of fossil fuels is one of the primary causes of

global warming and acid rain, which have started to affect the

earth’s climate, weather condition, vegetation and aquatic

ecosystems. Considering the global environment, there is

a pressing need to develop non-polluting and renewable

energy source. As a sustainable energy source, hydrogen is

a promising alternative to fossil fuels. It is a clean and envi-

ronmentally friendly fuel, which produces water instead of

greenhouse gases when combusted. Furthermore, it has

a high energy yield (122 kJ/g), which is about 2.75 times greater

than that of hydrocarbon fuels, and could be directly used to

of Energy Conversion, Ctrict, Guangzhou 510640,Dong).ational Association for H

four available basic processes for the production of hydrogen

from non-fossil primary energy sources. These processes

include: (1) water electrolysis; (2) thermo-chemical processes;

(3) radiolytic processes; and (4) biological processes. For global

environmental considerations, biohydrogen production from

renewable organic waste represents an important area of

bioenergy production [3–5]. Biological hydrogen can be

generated by several ways. Hallenbeck and Benemann [6,7]

described the fundamentals of biological hydrogen produc-

tion: light-driven processes and dark fermentations. Nandi

and Sengupta reviewed the photosynthetic and fermentative

biological routes of hydrogen production [8]. Many studies

hinese Academy of Sciences, Research Center of Biomass Energy,Guangdong, China. Tel.: þ86 20 8705 1423; fax: þ86 20 8705 7737.

ydrogen Energy. Published by Elsevier Ltd. All rights reserved.

mailto:[email protected]

http://www.elsevier.com/locate/he

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 3 4 ( 2 0 0 9 ) 8 1 2 – 8 2 0 813

have reported on the groundwork for biohydrogen production

systems through photosynthesis [9–11]. However, hydrogen

production by fermentative bacteria is technically simpler

than that by photosynthetic bacteria. Because it does not rely

on the availability of solar energy with large surface area and

transparency of the mixed liquor of the wastewaters stream

[12].

Gustavo et al. [13] reviewed fermentative hydrogen

production from various substrates by different inoculum.

These substrates not only included pure carbohydrates, such

as glucose [14–16], xylose [17], sucrose [18–20] and lactose [21],

but also molasses [22] and starch [23]. Besides, fruit waste [24],

palm oil mill effluent [25], food waste [26–28] and other organic

solid waste [29,30] was studied focusing on feasibility and

process parameters of fermentative hydrogen production.

Most of the inoculums for fermentative hydrogen produc-

tion were pure culture (such as Clostridium sp.). Clostridium sp.

is obligate anaerobes which are very sensitive to minute

amounts of dissolved oxygen. In order to grow Clostridium sp.

for hydrogen production, addition of expensive reducing

agents (such as L-cysteine) may be required, reducing the

feasibility in practical applications [31]. Therefore, some

researchers use a mixed culture as inoculum. This mixed

culture includes other fermentative hydrogen production

facultative anaerobes (such as Enterobacter sp.). Yokoi et al. [32]

have utilized a mixture of Enterobacter sp. and Clostridium sp. to

produce hydrogen under a reducing agent-free culture

medium and thereby lowering the cost of hydrogen produc-

tion. Treatments of sludge used as mixed inoculum (including

heating, chemical acidification, adding methanogens inhib-

itor, freezing and thawing, sterilizing and sonication) have

been investigated as a method for increasing hydrogen

production by altering the nature of the anaerobic microbial

communities [33,34]. Hydrogen-producing anaerobic bacteria

such as Clostridium species form endospores when unfavor-

able environmental conditions are encountered by bacterial

stresses (e.g., elevated temperature, chemicals, and radiation)

[35,36]. If the activities of non-spore-forming hydro-

genotrophic methanogens are inhibited by the dominance of

spore-forming hydrogen- producing bacteria, the fermenta-

tion may possess a significant capacity for the conversion of

organic wastes into hydrogen [14,20].

Selection of a proper pH is also crucial to enhance

hydrogen production, due to the effects of pH on hydrogenase

activity or metabolic pathways [37]. Some investigators [38,39]

reported that maximum hydrogen yield was determined at

a pH value of 5.5. Whereas Lee et al. [40] reported that the

maximum hydrogen yield was achieved at initial pH 9.0.

Cheong et al. [41] found maximum hydrogen yield under

culture conditions of initial pH 7. These conflicting results

seem to be due to the initial pH of the culture without main-

tained buffering capacity for preventing a decrease of pH.

Little information is available on the biohydrogen produc-

tion characteristics and mechanism from the organic fraction

of municipal solid waste (OFMSW) by mixed culture. As

a major burden to the environment, the generation of

municipal solid waste (MSW) amounts to 170 million tons in

2006 in China, and this number is growing by 6% per year [42].

Nearly 60% of MSW is organic fraction such as kitchen waste,

waste paper and urban greening waste. To treat OFMSW by

fermentative hydrogen production, not only alleviates conflict

between energy supply and demand in a certain extent, but

also improves economic feasibility for MSW treatment.

The purpose of this study is to investigate the character-

istics and mechanism of biohydrogen production from the

individual OFMSW by batch experiments using mixed culture

as inoculum. For this purpose, seven varieties of typical

organic materials were selected and their hydrogen produc-

tion characteristics were studied. These materials included

rice, potato, lettuce, lean meat, oil, fat and Banyan leaves

which represent natural starches, cellulose, protein, vegetable

lipid, animal lipid and lignocellulose materials widely existed

in OFMSW.

2. Materials and methods

2.1. Seed microorganisms

The seed used in this study was heat treated anaerobic sludge.

The sludge was originally obtained from an swine manure

anaerobic digester and acclimatized with kitchen waste for

2 month at 37 �C. Firstly, the sludge was introduced into the

anaerobic reactor, then the kitchen waste was added once

a week and the adding amount increased step by step. Prior

to use, the sludge was sieved to remove bone, sand and

other coarse matters. Thereafter, the sludge was boiled for

15 min to inactivate the hydrogentrophic methanogens and to

enrich the hydrogen-producing bacteria. The pH, ammonia

nitrogen, alkalinity, volatile fatty acids (VFAs) and volatile

suspended solid (VSS) were 9.2, 230 mg/L, 860 mg/L, 119 mg/L

and 3750 mg/L.

2.2. Experimental substrate

The materials used in this study are given in Table 1. All feed

amount of substrate was 8.0 g (calculated by VS) except meat

of 5.0 g in order to avoid ammonia inhibition [43].

2.3. Experimental setup and procedure

A 500 ml serum bottle used as a reactor was placed in the

water bath at 37 � 1 �C. The substrate and 200 mL inoculum

were added into the bottle. For the nitrogen-poor substrates

(including rice, potato, oil, fat and banyan leaves), 5 ml

NH4HCO3 solution of 200 g/L was added to supplement

nitrogen source. Total substrate concentrations in reactor

were adjusted to 20 g-VS/L by adding distilled water except

meat of 12.5 g-VS/L. The initial pH was adjusted to 5.5 by

adding 2 mol/L HCl or 2 mol/L KOH in this study. The head-

space of the reactor was filled with pure N2 to assure the

anaerobic condition. Mixing was conducted twice a day

manually. The anaerobic digestion was finished until no gas

was produced. Each experimental condition was carried out in

duplicate.

2.4. Analytical methods

Total solid (TS), volatile solid (VS), ammonia nitrogen and

alkalinity were determined using standard techniques [44].

Table 1 – Characteristics of substrates

Substrate TS (g) Heat value (J/g TS) VS (%TS) VS (g) [C] (%TS) [H] (%TS) [O] (%TS) [N] (%TS) [C]/[N]

Rice 8.0 17053 99.5 8.0 42.63 5.74 50.06 0.89 47.7

Potato 8.0 16324 99.5 8.0 41.36 5.59 51.16 1.17 35.4

Lettuce 9.5 16669 84.6 8.0 42.12 4.84 33.66 3.26 12.9

Lean meat 5.3 24077 94.9 5.0 50.97 6.10 23.04 13.11 3.9

Oil 8.0 38196 100 8.0 76.40 7.90 12.90 0.01 6967

Fat 8.0 38917 100 8.0 78.20 10.0 9.00 0.01 8279

Banyan leaves 9.3 17563 85.9 8.0 45.34 4.98 34.48 0.36 125.7


Heat values were determined by WGR-1 heat value

analyzer. Elementary analysis was determined by Vario EL

element analyzer. The pH was determined by pHS-3C pH

meter.

Biogas production was measured by the displacement of

saturated brine solutions. The gas volumes were corrected to

a standard temperature (0 �C) and pressure (1 atm) (STP). The

compositions of biogas (H2, CH4 and CO2) were analyzed by

a gas chromatograph (Agilent 6890) equipped with a thermal

conductivity detector (TCD) and a 2 m stainless column

packed with Porapak Q (50/80 mesh). The operational

temperature at the injection port, the column oven and the

detector were 100 �C, 70 �C and 150 �C respectively. Argon was

used as a carrier gas at a flow rate of 30 mL/min.

Liquid samples were centrifuged with 8000 rpm at 0–4 �C

and filtrated with 0.45 mm filter. The concentrations of VFAs

and alcohols were determined using a gas chromatograph

(Agilent 6820) equipped with a flame ionization detector (FID).

The packed capillary column (DB-FFAP) was 30 m � 0.25 mm

with inner diameter 0.25 mm. Nitrogen was used as a carrier

gas at a flow rate of 30 mL/min and split ratio was 1:50. The

operational temperature of injection port and detector were

250 �C and 300 �C. The initial temperature of oven was 40 �C

for 5 min, then increased to 140 �C at rate of 10 �C/min and

maintained for 1 min, subsequently increased to 250 �C at rate

of 5 �C/min and maintained for 3 min. The VFAs and alcohols

analyzed included acetate, propionate, butyrate, isobutyrate,

valerate, isovalerate, methanol, ethanol, propanol and

butanol.

The hydrogen production efficiency was evaluated using

the hydrogen content (HC) in the biogas, hydrogen yield (the

ultimate hydrogen production per gram VS, HY, mL/g-VS),

volumetric hydrogen production rate (the hydrogen produc-

tion from 1 L culture per day, VHPR, mL/(Lculture � d)) and

specific hydrogen production rate (the hydrogen production

from 1 g of dry biomass per day, SHPR, mL/(g-VSS � d)). The

modified Gompertz equation was used to describe the prog-

ress of cumulative hydrogen production obtained from the

batch experiments [45]. Using the cumulative hydrogen

production data to fit the modified Gompertz equation, the

maximum hydrogen production rates were estimated.

HðtÞ ¼ P� exp

�� exp

�Rm � e

Pðl� tÞ þ 1

��; (1)

Where H (t) is cumulative hydrogen production (mL), P

hydrogen production potential (mL), Rm maximum hydrogen

production rate (mL/d), e ¼ 2.71828, l lag-phase time (d) and t

time (d).

3. Results and discussion

3.1. Hydrogen production potential for differentsubstrates

Fig. 1 shows the cumulative hydrogen production and

hydrogen content. For the carbohydrates substrate (including

rice, potato and lettuce), gas production began immediately

after the inoculation. The produced gas was found to consist of

hydrogen and carbon dioxide. Methane was never detected

during the incubation of each substrate. During the first

2 days, cumulative hydrogen production rapidly increased and

then reached a plateau for each substrate. Hydrogen concen-

tration increased sharply and reached a maximum value of

70% at 3 days, 55% at 1 day, and 67% at 1.5 days for rice, potato

and lettuce respectively. But they declined to 26%, 22% and

17% finally. The decrease in hydrogen concentration was likely

to be associated with the consumption of hydrogen. However,

no methanogenesis was observed throughout the experiment

due to the inhibition of methanogenic activity by thermal

treatment. This implies that hydrogen might be utilized by

some other microorganisms, such as homoacetogens [46,47].

The hydrogen yields for rice, potato and lettuce were 134 mL/

g-VS, 106 mL/g-VS and 50 mL/g-VS respectively. It is notice-

able that there was a lag time of about 15 h for rice. For the

protein substrate, only 18 mL gas was produced during the

hydrogen production fermentation of lean meat. No hydrogen

was detected during the incubation with carbon dioxide as

main gas component. For the lipid substrate (including oil and

fat), the gas production was less than that using carbohydrates

as the substrate. A hydrogen yield of 6.25 mL/g-VS indicates

that about 0.1% of oil was converted to hydrogen based on

COD. Unpredictably, no hydrogen was produced with fat as

a substrate. For the lignocellulose substrate, a hydrogen yield

of 1.75 mL/g-VS indicates that less than 0.1% of banyan leaves

was converted to hydrogen based on COD.

Fig. 2 shows the modified Gompertz equation curves fitted

using the cumulative hydrogen production data for rice,

potato and lettuce. The hydrogen production potentials, the

maximum hydrogen production rates and lag-phase time

were estimated using modified Gompertz equation. The

results are given in Table 2. The hydrogen production poten-

tial of lettuce was less than that of rice and potato, since

cellulosic carbohydrate was more recalcitrant to degradation

compared with amylaceous carbohydrate.

Several studies have also reported hydrogen production

from various carbohydrates by pure culture and mixed

0

500

1000

1500

2000Rice

cu

mm

ulative g

as p

ro

du

ctio

n (m

L)

cu

mm

ulative g

as p

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ulative g

as p

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as p

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ulative g

as p

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ulative g

as p

ro

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L)

cu

mm

ulative g

as p

ro

du

ctio

n (m

L)

total gas hydrogen hydrogen content

0

20

40

60

80

hyd

ro

gen

co

nten

t (%

v/v)

hy

dro

gen

co

nten

t (%

v/v

)

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20

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60

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hyd

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nten

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v/v)

hyd

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yd

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v/v

)

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nten

t (%

v/v)

0

400

800

1200

1600

2000Potato


0

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40

60

80

0 1 2 3 4 5 6 70

200

400

600

800

1000


Lettuce

time

0 1 2 3 4 5 6 7time

0 1 2 3 4 5 6 7time

0 1 2 3 4 5 6 7time

0 1 2 3 4 5 6 7time

0

50

100

150

200

250

300

350

400Lean meat


0

20

40

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0 1 2 3 4 5 6 70

100

200

300

400Oil

0

20

40

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80

time


0

100

200

300

400

0

20

40

60

80


Fat

0 1 2 3 4 5 6 70

50

100

150

200Banyan leaf

time

0

20

40

60

80


Fig. 1 – Cumulative hydrogen productions and hydrogen contents.


0 1 2 3 4 5 6 70

200

400

600

800

1000

1200

time (d)

cu

mu

lative h

yd

ro

gen

p

ro

du

ctio

n (m

L)

ricepotatolettuce

Fig. 2 – Modified Gompertz equation fitting curves for rice,

potato and lettuce.

Table 2 – Modified Gompertz equation parameters forrice, potato and lettuce

Substrate P(mL)

Psa

(mL/g-VS)

Rm

(mL/d)Rmsb

[mL/(g-VS-d]

l (d) R2 T90c

(d)

Rice 1056 132 896 112 0.6 0.9990 1.9

Potato 816 102 744 93 0.2 0.9979 1.5

Lettuce 384 48 392 49 0.1 0.9960 1.3

a The hydrogen production potential per gram substrate.

b The maximum hydrogen production rate per gram substrate.

c The time for cumulative hydrogen production achieving 90% of P.


cultures in batch, continuous, and cell immobilization reac-

tors. Table 3 lists the results of these studies including HY,

VHPR, SHPR and HC for comparison. Generally, the unit of

hydrogen yield was mol H2 / mol substrate or mL H2 / mol

substrate which were reported in previous studies when

substrate was pure carbohydrate (such as glucose and

sucrose). But it is difficult to determine the molar amount of

complex mixture, so in this paper, the units of hydrogen yields

are unified to mL/g-VS at standard temperature (0 �C) and

pressure (1 atm) (STP).

As shown in Table 3, hydrogen yield is dependent on

substrate, seed, reactor type and culture conditions. More-

over, these factors are interactional, such as temperature, pH

and substrate concentration [19]. In general, the hydrogen

yield of cellulosic carbohydrate (such as microcrystalline

cellulose and lettuce) was less than pure and amylaceous

carbohydrate, because hydrogen production from them

requires hydrolysis reaction which requires long time.

3.2. Production of soluble intermediate metabolites

The production of hydrogen accompanied VFAs and alcohols

production during the anaerobic fermentation of organic

substrate. The concentration and distributions of VFAs have

been used as a useful indicator for monitoring hydrogen

production [48]. The VFAs and alcohols concentrations at the

end of fermentation were given in Table 4. The main inter-

mediate metabolites were VFAs with negligible alcohols. The

VFAs concentrations were in line with hydrogen yields since

VFAs were byproducts of hydrogen production fermentation.

From the results of intermediate metabolites, it is concluded

that the degradation of meat, oil, fat and banyan leaves were

limited. The production of VFAs lowered the pH for the rice,

potato, lettuce, oil and fat. Whereas the fermentation of meat

increased pH since the degradation of meat produced

ammonia. The same phenomenon was observed during the

fermentation of banyan leaves, it was likely some alkalescent

compounds were produced. However it is required to be

warranted.

The concentrations of dominating VFAs and their fraction

in SMP are given in Table 5. Among them, butyrate and

acetate were the main intermediate metabolites for rice,

potato and lettuce, accounting for more than 75% (w/w) of the

total VFA and alcohols, suggesting a butyrate-type fermenta-

tion in this experiment. The results also suggest that the

mixed culture used in this study possessed clostridial char-

acteristics as evidenced by the typical hydrogen/acid-

producing phases.

The VFAs can be stimulatory, inhibitory or even toxic to

the fermentative bacteria depending on their concentrations.

A low level of VFAs may have no effect or a stimulatory effect

on hydrogen production. However, at a high level, VFAs can

lead to severe inhibition on hydrogen fermentation. It is

presumed that the VFAs in their undissociated forms can

freely permeate the bacteria cell membrane [52]. If the

undissociated VFAs are pumped into the culture, they will

penetrate the plasma membrane and dissociate depending on

the pH inside cell. Thus, the pH inside the cell will be lowered.

To prevent unfavorable physiological conditions within the

bacteria cell, excess energy must be used to pump ions, e.g.

potassium ions, from the culture. Therefore, the energy used

for bacteria growth is reduced and the bacteria growth rate

will be lowered [53]. On the other hand, if a high level of

dissociated VFAs is present in the culture, the ionic strength

in solution will increase. Such an increase can result in cell

lysis. The effects of VFAs on the fermentative bacteria are

associated with the buffer capacity (such as pH and alkalinity

values). One of the criteria for judging fermentation stability

is the ratio of VFAs/alkalinity. There are three critical values

for this [54]:

<0.4 fermentation should be stable;

0.4–0.8 some instability will occur;

>0.8 significant instability.

In this study, the ratio of VFA/alkalinity at the end of

fermentation for rice, potato and lettuce substrate were far

more than 0.8 which resulted in severe inhibition. It can be

concluded that the removal of VFAs is required in order to

produce hydrogen continuously. On the other hand, the

energy contained in VFAs and alcohols should be further

recovered. The hydrogen production fermentation followed

by methane fermentation or photo-fermentation can realize

this objective. During the following step, organic acids are

converted to methane by methanogen or to hydrogen by

photosynthetic bacteria.

Table 3 – Comparison of hydrogen production efficiency from various substrate by different seed

Substrate(concentration)

Seed Reactor type andculture conditions

HY(mL/g-VS)

VHPR[mL/

(Lculture � d)]

SHPR[mL/

(g-VSS � d)]

HC(%)

Reference

Glucose (10 g-VS/L) Heat treated anaerobic

sludge from sewage

treatment plant

Serum bottles, 37 �C,

initial pH 6.0

217 – 1483 42 [49]

Sucrose (30 g-VS/L) Heat treated sludge from

UASB digester

5 L agitated fermenter,

35�C, controlled pH 5.5

143 4824 600 – [50]

Microcrystalline

cellulose (25 g-VS/L)

Heat treated sludge from

anaerobic high-solids

digester


initial pH 7.0

36 350 303 50 [51]

Jackfruit peel (33 g-VS/L) Heat treated microflora

from cow dung

Anaerobic contact

filter, room temperature,

controlled pH 5.3

198 – – 55 [24]

Food waste (10 g-VS/L) Thermophilic acidogenic

culture form CSTR


controlled pH 5.5

70 – 456 69% [26]

Cabbage (30 g TS/L) Heat treated sludge from

anaerobic digester


initial pH 7.0

62 – – 55% [30]

Carrot (20 g TS/L) Heat treated sludge from

anaerobic digester


initial pH 7.0

71 – – 47% [30]

Rice (40 g TS/L) Heat treated sludge from

anaerobic digester


initial pH 7.0

96 – – 46% [30]

Rice (20 g-VS/L) Boling treated sludge from

anaerobic digester


initial pH 5.5

134 2240 1990 57–70 This study

Potato (20 g-VS/L) Boling treated sludge from

anaerobic digester


initial pH 5.5

106 1860 1650 41–55 This study

Lettuce (20 g-VS/L) Boling treated sludge from

anaerobic digester


initial pH 5.5

50 980 870 37–67 This study


3.3. Biohydrogen production characteristic ofcomponents of OFMSW

3.3.1. CarbohydratesFermentative hydrogen production usually proceeds from the

anaerobic glycolytic breakdown of sugars. The majority of

microbial hydrogen production is driven by the anaerobic

metabolism of pyruvate, formed during the catabolism of

various substrates. Acetate and butyrate are formed during

hydrogen -producing fermentations.

Glucoseþ2H2O/2Acetateþ2CO2þ4H2 DG0¼�206:3kj=mol

(2)

Glucose/Butyrateþ2CO2þ2H2 DG00 ¼�254:8kj=mol (3)

Table 4 – Final pH, VFAs, Alcohols, NH3-N, and Alkalinity at th

Substrate Final pH VFAsa (mg/L) Alcoholsb (mg/L) SMPc

Rice 4.56 14405 � 79 198 � 18 1460

Potato 4.81 5288 � 49 473 � 20 576

Lettuce 5.14 5013 � 36 182 � 8 519

Meat 5.74 2324 � 11 42 � 10 236

Oil 5.48 2212 � 10 118 � 16 233

Fat 5.28 1019 � 8 106 � 10 112

Banyan leaves 5.72 2181 � 16 141 � 4 232

a Including acetate, propionate, butyrate, i-butyrate, valerate, i-butyrate

b Including methanol, ethanol, propanol and butanol.

c SMP ¼ VFAs þ Alcohols.

d Total alkalinity calculated by CaCO3.

It is favorable thermodynamically using carbohydrate

substrate to produce hydrogen by anaerobic fermentation.

In theory, acetate fermentation is capable of generating

4 mol H2 /mol glucose. This is the so-called ‘‘Thauer limit’’ [55].

Yield of hydrogen by butyrate fermentation carried out by some

clostridia are lower than that of acetate fermentation. Several

study results indicated that actual hydrogen yields were lower

than 4 mol that were theoretically possible, typically ranging

from 0.5 to 2.5 mol H2/mol hexose. In fact, the intermediate

metabolites often are a mixture of acetate and butyrate:

4Glucose/2Acetateþ3Butyrateþ8CO2þ8H2 (4)

For this fermentation, the yield is 2 mol H2/mol glucose.

Approximately 50% of clostridia isolated to date carry out this

mixed acids fermentation. Other fermentation pathways were

e end of fermentation

(mg/L) NH3-N (mg/L) Alkalinityd (mg/L) VFAs/alkalinity

3 � 98 810 � 16 1828 � 30 7.88

1 � 69 780 � 7 2944 � 14 1.80

5 � 45 850 � 13 2287 � 20 2.19

6 � 21 1600 � 43 4385 � 25 0.53

0 � 25 420 � 17 4659 � 54 0.47

5 � 18 510 � 13 4291 � 74 0.24

2 � 20 730 � 21 5108 � 47 0.43

.

Table 5 – Distribution of intermediate metabolites

Substrate HAc (mg/L) HPr (mg/L) HBu (mg/L) EtOH (mg/L) HBu/HAc

Rice 7098 � 45 48.6%a 2795 � 36 19.1% 3899 � 39 26.7% 145 � 11 1.0% 0.55

Potato 2906 � 32 50.4% 193 � 15 3.4% 2179 � 40 37.8% 426 � 55 7.4% 0.75

Lettuce 3416 � 39 65.8% 280 � 16 5.4% 1312 � 36 25.3% 86 � 9 1.7% 0.38

Meat 744 � 24 31.4% 462 � 24 19.5% 744 � 27 31.4% 42 � 12 1.8% 1.00

Oil 196 � 15 8.4% 163 � 20 7.0% 1789 � 18 76.8% 45 � 7 1.9% 9.13

Fat 860 � 21 76.4% 152 � 19 13.5% 7 � 6 0.6% 54 � 15 4.8% 0.01

Banyan leaves 1643 � 19 70.8% 223 � 11 9.6% 301 � 22 13.0% 71 � 6 3.1% 0.18

a % are percentage of individual HAc, HPr, HBu and EtOH in SMP.


found in sacchrolytic clostridia, such as the production of

propionate by Clostridium arcticum, succinate by Clostridium

coccoides, and lactate by Clostridium barkeri.

3.3.2. LipidsGlycerol and long chain fatty acids (LCFA) are mainly

produced from anaerobic hydrolysis of lipids. Glycerol could

be a substrate for hydrogen production and for solvent

production with saccharolytic clostridia [56]. It is apparent

that glycerol may be suitable substrate for solvent production

rather than hydrogen production. The degradations of LCFA

(b-oxidation) are thermodynamically unfavorable reactions

unless the hydrogen partial pressure is maintained to an

extremely low level:

n� LCFA/ðn� 2Þ � LCFAþ 2Acetateþ 2H2 DG0

¼ þ48 mj=mol (5)

PyruvateþAcetateþ2H2/þCO2þH2O DG0¼�95:4kj=mol (6)

It only proceeds at very low partial pressures of hydrogen, below

10�3 atm, when the free energy change is negative. This low

partial pressure is realized in some natural anaerobic digestions

where the produced hydrogen is consumed timely by hydro-

genotrophic methanogens permits the reaction thermody-

namically favorable. But hydrogenotrophic methanogens are

absence in hydrogen production reactor where extremely low

hydrogen partial pressure can not be maintained. Moreover,

LCFA are one of the inhibitors for anaerobic bacteria [57]. They

adhere to cell wall of bacteria which restricts the transportation

of nutrition. Therefore, it may be difficult to produce hydrogen

from long chain fatty acids. Because a large portion of chemical

oxygen demand (COD) of lipid is converted to long chain fatty

acids during the hydrolysis reaction, even if glycerol could have

high hydrogen production potential, hydrogen production from

lipid may not be high. In fact, hydrogen production yields of oil

and fat were very low in this study.

3.3.3. ProteinsProtein is hydrolyzed to various amino acids by extracellular

enzymes. There are three types of amino acid degradation

reactions under anaerobic condition [55]:

1) Stickland reaction

Alanineþ 2glycine/3acetateþ 3NH3 þ CO2 (7)

2) Oxidative deamination from sole amino acid

Leucineþ 3H2O/isovalerateþHCO�3 NHþ4 þ 2H2 DG0

¼ þ4:2 kj=mol (8)

3) Reductive deamination from sole amino acid

GlycineþH2/acetateþNH3 DG0 ¼ �77:8 kj=mol (9)

All degradation of amino acids involves production of volatile

fatty acids and ammonia. The concentration of ammonia

produced correlated with the amount of amino acids

(proteins) degraded. Therefore, the degree of protein degra-

dation can be known by observing ammonia concentration. In

this study, when lean meat was used as a substrate of

hydrogen production fermentation without NH4HCO3 added,

ammonia was produced and the concentration was higher

than that of other substrate fermentation with NH4HCO3

added. It can be concluded that the lean meat was degraded

partially. An 18 mL gas also testified this conclusion although

the gas was carbon dioxide without hydrogen produced. There

are some reasons why hydrogen was not produced. Firstly,

hydrogen is not produced by Stickland reaction. Moreover,

approximately 90% of amino acids degradation is carried out

by Stickland reaction [58]. Secondly, oxidative deamination

from sole amino acid is thermodynamically unfavorable

reactions unless the hydrogen partial pressure is maintained

to an extremely low level. Thirdly, amino acids such as

glycine, which was degraded by reductive deamination can

consume hydrogen as an electron donor [58]. Therefore, even

if hydrogen is produced from oxidative deamination, the

hydrogen can be utilized by reductive deamination. From the

above, it may be difficult to produce hydrogen from substrate

which includes a large quantity of protein.

3.3.4. LignocellulosesCellulose, hemicellulose and lignin compose lignocellulose

which has a complicated structure. For untreated natural

lignocellulose materials, it is difficult to degrade. In most

cases, the cellulose fibers are embedded in a matrix of other

structural biopolymers, primarily hemicelluloses and lignin.

Complicated structure feature limits the rate and extent of

hydrolysis [59]. Therefore, it is very difficult to produce

hydrogen from lignocellulose substrate unless suitable

pretreatment is adopted.

4. Conclusion

1) Boiling treated anaerobic sludge is an effective mixed inoc-

ulum for fermentative hydrogen production from OFMSW.

2) Among the OFMSW, carbohydrates is the most optimal

substrate for fermentative hydrogen production compared

with proteins, lipids and lignocelluloses.


3) The hydrogen production potentials of three individual

carbohydrates were measured: rice 134 mL/g-VS, potato

106 mL/g-VS, and lettuce 50 mL/g-VS. The hydrogen percent-

ages of the total gas produced from rice, potato and lettuce

were 57–70%, 41–55% and 37–67%, respectively.

4) In order to produce biological hydrogen continuously,

the intermediate metabolites (including VFAs and alcohols)

should be removed timely to avoid inhibition. On the other

hand, intermediate metabolites should be utilized by a suit-

able secondary process step to increase the energy yields of

the waste. The methane fermentation or photo-fermentation

is suggested as secondary step to produce methane or

hydrogen.

Acknowledgment

This work was financially supported by the Guangdong

Science and Technology Program (No. 0711031100011).

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