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Energy balance of dark anaerobic fermentation as a toolfor sustainability analysis

Bernardo Ruggeri*, Tonia Tommasi, Guido Sassi

Dept. of Material Science and Chemical Engineering, Politecnico di Torino Corso Duca Degli Abruzzi 24, 10129 Turin, Italy

a r t i c l e i n f o

Article history:

Received 8 April 2010

Received in revised form

4 August 2010

Accepted 4 August 2010

Keywords:

BioH2

Dark fermentation

Sustainable energy balance

Methane

* Corresponding author. Tel.: þ39 011 090 46E-mail addresses: bernardo.ruggeri@polit

0360-3199/$ e see front matter ª 2010 Profedoi:10.1016/j.ijhydene.2010.08.014

a b s t r a c t

A process aimed at producing energy needs to produce more energy than the energy

necessary to run the process itself in order to be energetically sustainable. In this paper, an

energy balance of a batch anaerobic bioreactor has been defined and calculated, both for

different operative conditions and for different reactor scales, in order to analyze the

sustainability of hydrogen production through dark anaerobic fermentation. Energy

production in the form of hydrogen and methane, energy to warm up the fermentation

broth, energy loss during fermentation and energy for mixing and pumping have been

considered in the energy balance. Experimental data and literature data for mesophilic

microorganism consortia have been used to calculate the energy balance. The energy

production of a mesophilic microorganism consortium in a batch reactor has been studied

in the 16e50 �C temperature range. The hydrogen batch dark fermentation resulted to only

have a positive net production of energy over a minimal reactor dimension in summer

conditions with an energy recovery strategy. The best working temperature resulted to be

20 �C with 20% of available energy. Hydrogen batch dark fermentation may be coupled with

other processes to obtain a positive net energy by recovering energy from the end products

of hydrogen dark fermentation. As an example, methane fermentation has been consid-

ered to energetically valorize the end products of hydrogen fermentation. The combined

process resulted in a positive net energy over the whole range of tested reactor dimension

with 45e90% of available energy.

ª 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.

1. Introduction processing and water electrolysis [3] potentiates the biological

The search for renewable energy sources is at the present

a necessity [1] as a consequence of the shortage of hydro-

carbon and of global warming due to CO2 emission. In this

context, the use of hydrogen, produced from non-fossil

sources, can be considered as a clean and sustainable source

of energy. Hydrogen can be produced in several ways, e.g.,

through the electrolysis of water, the steam reforming of

no-renewable hydrocarbons, and the gasification of biomass

and biological processes [2]. The high cost of hydrocarbon

47; fax: þ39 011 090 4699.o.it (B. Ruggeri), tonia.tomssor T. Nejat Veziroglu. P

production of hydrogen, especially when organic wastes are

used, as a component of the sustainable energy market [4].

The biological processes are classified as photo fermentation

and dark fermentation processes. The photosynthesis of H2

by bacteria or algae offers an opportunity for direct trans-

formation of solar energy. In spite of the theoretical oppor-

tunity, the real application is currently difficult [5] mainly due

to the bioreactor scale up [6] and to the low efficiency in the

use of solar light [7,8]. The dark fermentation approach is

based on the well-known technology of the anaerobic

masi@gmail.com (T. Tommasi).ublished by Elsevier Ltd. All rights reserved.

Nomenclature

cp specific heat of the broth, [MJ kg�1 K�1]

D diameter of the bioreactor, [m]

DB diameter of the bench scale bioreactor, [m]

di diameter of the impeller, [m]

EH2 energy produced as H2 per unit volume of the

reactor, [MJ m�3]

ECH4 energy produced as CH4 per unit volume of the

reactor, [MJ m�3]

Ew energy for the initial warming per unit of the

reactor, [MJ m�3]

El energy loss per unit of the reactor, [MJ m�3]

Ee energy formixing and pumping per unit volume of

the reactor, [MJ m�3]

En net energy production per unit volume of the

reactor, [MJ m�3]

F filling fraction of the bioreactor volume, [e]

g acceleration of gravity, [m s�2]

he external convective heat transfer coefficient,

[kJ h�1 m�2 K�1]

hi internal convective heat transfer coefficient,

[kJ h�1 m�2 K�1]

HCH4 lower heating value of methane, 36.18 MJ/Nm3,

[MJ Nm�3]

HH2 lower heating value of hydrogen, 10.8 MJ/Nm3,

[MJ Nm�3]

L height of the bioreactor, [m]

ksteel thermal conductivity of steel, [MJ h�1 m�1 K�1]

kfoam thermal conductivity of the expanded polystyrene

foam, [MJ h�1 m�1 K�1]

N impeller rotation velocity, [rpm]

NB impeller rotation velocity of the bench scale

bioreactor, [rpm]

PH2 ðTwÞ H2 production vs. temperature per unit volume of

broth, [Nm3 m�3]

PCH4 ðTwÞ CH4 production vs. temperature per unit volume

of broth, [Nm3 m�3]

Pn Power number, [e]

Pw electrical power, [MJ h�1]

Re rotational Reynolds number Re a ND2, [e]

ROP RedOx Potential, [mV]

ssteel Thickness of the reactor wall, [m]

sfoam thickness of the reactor insulator, [m]

Ta outdoor ambient temperature, [�C]Tw working temperature in the bioreactor, [�C]U global heat transfer coefficient, [kJ h�1m�2 K�1]

VFA volatile fatty acids, [mg L�1]

VSS volatile suspended solid, [mg L�1]

VG produced gas volume per broth volume, [L L�1]

Greek

r biomass density, [mg L�1]

h global thermal efficiency of the heating system,

[e]

hcomb combustion efficiency, [e]

he electrical/mechanical conversion efficiency, [e]

hheat exc efficiency of the heat exchanger, [e]

Dt total duration of the batch fermentation, [h]

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 5 ( 2 0 1 0 ) 1 0 2 0 2e1 0 2 1 1 10203

digestion of organic wastes for biogas production. Anaerobic

digestion has been reported to operate in continuous and in

batchmodes, depending on the carbon source availability and

seasonality, the reacting time, the mixing approach and the

technological approach [9].

Dark fermentation is a promisingway of using inexpensive

feedstock, from several organic waste streams, as a substrate

source for hydrogen production [10]. Reviews on recent

studies on anaerobic digestion to produce hydrogen are

available in Refs. [11,12]. Nevertheless, several non-linear

effects, which influence the fermentative hydrogen produc-

tion, still remain to be clarified. The main factors under study

are: microorganism inoculum procedures, carbon sources,

reactor types, nitrogen phosphate and metal concentrations

(especially Fe2þ), fermentation temperature, agitation and

fermentation pH [13,14]. Studies on the macro effects of these

factors on fermentative hydrogen production have recently

been reviewed [15]. Fermentation temperature and pH have

been demonstrated to be the most relevant parameters that

affect the process performances [15e17], because of their

influence on the activity of hydrogen producing bacteria.

Essential enzymes for fermentative hydrogen production,

e.g., Hydrogenase and Ferrodoxin, require adequate pH and

temperature ranges [18,19]. Basification is necessary to

maintain the pH and the redox potential in the broth at the

most appropriate range to enhance the activity of the Ferro-

doxin enzyme pool [20].

Temperature affects the general cell viability and the

catabolic pathways. A low temperature depletes cell activity

and the hydrogen production rate, while high temperature

leads to more oxidized compounds to be available as electron

acceptors. This low temperature depletes hydrogen produc-

tion [21,22]. The temperature range depends on the type of

microorganism consortium that is used. The lower boundary

of the temperature range is generally similar, i.e., around

12e16 �C, while the optimal temperature may vary in the

mesophilic range, i.e., around 37 �C, and the thermophilic

range, i.e., around 55 �C, [23]; however 22 �C [24] and 60 �C[25,26] have also been reported. From an energetic point of

view, the fermentation temperature also determines the

energetic level to which all the materials must be warmed to

and the driving force for heat dispersion to the external

environment. The best working temperature can be different

from the optimal temperature for the microbial consortium

activity. The total energy balance is the key equation for

fullscale reactor design and the fermentation temperature is

the most relevant parameter [27,28]. Nevertheless very few

papers have dealt with the total energy balance in hydrogen

production [28]. The relevance of the energetic reasoning is

evident for the production of hydrogen as an energetic vector;

the value of the fermentation temperature determines the net

energy produced. Hydrogen production through dark

fermentation involves a partial oxidation of the carbon source

to fatty acids [13]. The outlet stream from the reactor still has

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 5 ( 2 0 1 0 ) 1 0 2 0 2e1 0 2 1 110204

a chemical energetic content that can be used for further

energy production, e.g., through anaerobic digestion with

a resulting methane production. The overall process, e.g.,

hydrogen production and subsequent methane production,

should have a higher specific net energy production due to the

thermal integration, which leads to an energy saving. In the

present paper, the performances of a mesophilic consortium

that produces hydrogen in a batch reactor are studied as

a function of the fermentation temperature. The total energy

balance is applied to investigate the energetic performances of

the process as a function of the basic technological parame-

ters of a fullscale bioreactor, i.e., reactor dimension and

reactor insulation. The net energy produced by the hydrogen

process alone and by the hydrogen process coupled with

a subsequent anaerobic digestion, i.e., methane production

from the outlet stream of the hydrogen reactor, has been

calculated. The net energy is reported as a function of the

reactor dimension and fermentation temperature in order to

identify the acceptable conditions for both summer and

winter operations. The importance of energy recovery strat-

egies has been verified.

2. Materials and methods

2.1. Seed microflora for H2 and CH4 production

The seed microorganism consortium for H2 production was

prepared according to Chen et al. [29] and Mu et al. [30]. The

sludge of an anaerobic digester working at 38 �C fed by

municipal wastewater (SMAT S.p.A., Turin) was pretreated by

leaving for 24 h at pH 3 in anaerobic conditions. The pH value

was reached by adding a 1 N HCl solution to the sludge. The

initial conditions of the sludgewere: pH 7.2; density 890 kg/m3,

volatile suspended solid (VSS) 11,560 mg/L and total solid

concentration (TSS) 16,500 mg/L. The seed microorganism

consortium for CH4 production was prepared from the

untreated sludge of the same anaerobic digester (SMAT S.p.A.,

Turin). The sludge was withdrawn once from the anaerobic

digester and partially treated as previously described. The

treated and untreated sludge portions were divided into 50 ml

amounts and frozen. Each amount was unfrozen at ambient

temperature under anaerobic conditions before being inocu-

lated into the reactor.

2.2. Reactor and medium composition

A 2 L working volume stirred-batch reactor (Minifors HT,

Switzerland) was filled whit a glucose synthetic medium [31]

(NaHCO3 1.25 g/l, NH4Cl 2.5 g/l; KH2PO4 0.25 g/l; K2HPO4

0.250 g/l; CaCl2 0.5 g/l; NiSO4 32 mg/L; MgSO4$7H2O 320 mg/l;

FeCl3 20 mg/l; Na2BO4$H2O 7.2 mg/l; Na2MoO4$2H2O 14.4 mg/l;

CoCl2$6H20 21 mg/l; MnCl2$4H2O 30 mg/l; Yeast extract

50 mg/l). 10% v/v seed was inoculated into the medium, N2

was sprinkled for 10 min to reach anaerobic conditions and

the broth was warmed to the chosen temperature. The C/N

ratio was set at 30 and the glucose concentration at 60 g/L in

order to maximize the hydrogen production rate, according to

previously kinetic studies [19].

2.3. Test runs for H2 and CH4 production

The batch test runs were conducted at 16, 20, 35, 40 and 50 �C.The initial pH was set at 7.2 by 1 N HCl solution. After the

initial free decrease till 5.2, the pH value was maintained by

pumping in a 2 N NaOH solution [20]. The test runs were shut

down when the biogas production stopped.

The broth of one of the test runs, conducted at 35 �C, was

inoculated with 10% untreated sludge after the end of the run

when the pH reached 7 by free evolution of the broth. The

test run was shut down when biogas production stopped

again.

2.4. Analytical tests

The pH (HA405-DXK-S8, Mettler Toledo), RedOx Potential

(ROP, Pt4805-DXK-S8, Mettler Toledo), temperature, NaOH

flow rate (Iris, Infors HT) and total gas produced volume

(Milligas Counter, Ritter) were monitored and registered every

5 min. Liquid samples were withdrawn twice a day during the

test runs, the glucose concentration was measured by an

enzymatic method (Biopharm-Roche) and the volatile fatty

acids and ethanol concentrations were measured by a gas-

chromatographic method (Model 6580, Agilent). The gas was

collected in bags whichwere changed on the basis of time and

the gas quantity; average concentrations of H2, O2, CH4, CO2,

CO, N2 were measured in each bag by means of off-line gas-

chromatographic analysis (Varian, CP 4900).

2.5. Uncertainty evaluation

The uncertainty of net energy estimated values was evalu-

ated in accord to the rules reported in Ref. [40]. At the end

to evaluate the uncertainty, considering that Guide to

Uncertainty Measurement (GUM) defines uncertainty as

a quantifiable parameter associated with the results of

a measurement procedure, the suggested approach has been

utilized either to evaluate the uncertainty or to estimate the

most affecting parameters. This latest was obtained by using

the expression reported in Ref. [41] known as the law of the

propagation of uncertainty based on the evaluation of the

partial derivatives of the parameters on the estimation of

the net energy, called sensitivity coefficients which describe

how the output estimate, varies with changes in the value of

the input estimates.

3. Energy balance methodology

The net energy produced (En) in a bioreactor by dark fermen-

tation is the difference between the energy contained in the

generated gas, i.e., contained in the hydrogen (EH2 ) and/or the

methane (ECH4 ) produced, and the energy spent to obtain and

maintain the reaction conditions. The last term involves the

warm up of the inlet materials to start fermentation (Ew), the

heat loss to the environment (El) during fermentation and the

electrical energy for mixing and pumping (Ee).

The net energy production may be expressed as:

En ¼ EH2þ ECH4

� ðEw þ El þ EeÞ (1)

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The energy balance considers a reference volume and

a reference time period onwhich each termmust be evaluated

and calculated. The system under study is a batch reactor into

which raw materials are introduced, agitated, warmed to the

working temperature, inoculated by seed and left to evolve,

controlling the pH by adding an alkaline solution till the gas

production stopped. In this case, the reference volume is the

reactor volume and the reference time period is the duration

of a single batch run. Intensive terms may be calculated,

referring to the unit working volume of the reactor. A batch

process was considered because hydrogen produced as

a function of the working temperature has mainly been

studied in detail and recognized in Ref. [15] for batch

processes. Table 1 summarizes the data from several litera-

ture cases of batch hydrogen production at the optimal

working fermentation temperature. In order to investigate the

temperature and scale-up effects, the net energy per unit

volume of the reactor was calculated at different working

fermentation temperatures and different dimensions of the

reactor. The reactor diameter D was chosen as the reactor

dimension parameter, and the reactor was considered to be

cylindrical with a height-to-diameter ratio equal to 4. Two

different outdoor ambient conditions were considered in

order to evaluate the effect of seasonal variations of the

ambient temperature. Each term of Equation (1) is discussed

hereafter.

3.1. Energy production

The energy produced per unit volume of the reactor is the total

energy embedded in the gas produced by a single batch run

referring to the reactor volume, i.e., the energy contained in

the amount of hydrogen and methane retrieved from a single

batch run referring to the reactor volume.

For hydrogen and methane, this can be calculated as:

EH2¼ F$PH2

ðTwÞ$HH2; ECH4 ¼ F$PCH4 ðTwÞ$HCH4 (2)

where PH2ðTwÞ and PCH4 ðTwÞ are the specific production of H2

and CH4 respectively, i.e., the amount of H2 or CH4 produced in

a single batch run per unit volume of broth at the working

temperature. These are expressed as Nm3 of H2 or CH4 per m3

of fermenting broth, and their value, which was determined

experimentally, depends on the working temperature. In

Table 1 e Highest specific hydrogen production and duration fmicroorganism consortium producing hydrogen.

References Substrate Microorganisms In

[15] Glucose Mixed culture

[38] Sucrose Mixed culture

[19] Glucose Mixed culture

[17] Glucose Mixed culture

[Present study] Glucose Mixed culture

[26] Sucrose Mixed culture

[26] Sucrose Mixed culture

[18] Cattle wastewater Mixed culture

[39] Glucose Geothermal hot

spring sediments

addition, HH2is the lower heating value of hydrogen

(10.8 MJ/Nm3) and HCH4 that of methane (36.18 MJ/Nm3). F is

the liquid hold up in the reactor, i.e., the fraction of reactor

volume filled by liquid.

3.2. Warming energy

The energy required to warm the fermenting broth mainly

depends on its specific heat (cp), the difference between the

working temperature (Tw) and the outdoor ambient tempera-

ture (Ta) and the efficiency of the heating system (h). The

heating energy per unit volume of the bioreactor may be

calculated as:

Ew ¼�r$cp$ðTw � TaÞ$F

�h

(3)

Where the r and cp of water were used for the fermenting

broth. The warming device was considered to be composed

of a combustion boiler (hcomb z 0.8) and a heat exchanger

(hheat exc z 0.6). The global efficiency of the warming system

was calculated as the product (hz 0.48). The outdoor ambient

temperaturewas considered for different seasonal conditions,

i.e., summer and winter conditions. Ta was calculated on the

basis of historical data from northern Italy; mean night and

day values over the season were considered in order to avoid

an increase in the computational complexity: Ta ¼ 5 �C for the

winter and Ta ¼ 15 �C for the summer. A heat recovery of 50%

was considered, i.e., at the end of the batch run 50% of the heat

of the broth is considered to have been recovered. The

warming of the reactor wall and insulator and that of the

NaOH solution was neglected in order to consider the most

optimistic situation in which specific strategies are performed

to enhance energy saving.

3.3. Heat loss

The difference between the working temperature of broth Tw

and the ambient temperature Ta outside the reactor is

responsible for the heat loss from the fermenting broth. The

energy lost must be supplied from the heating system of the

temperature control system and it depends on the insulation

of the fermenting broth from the external environment, the

surface area exposed to the environment and the duration of

or batch operative conditions with mesophilic

itialpH

pHControl

Tw (�C) Dt (h) Produced H2

(mmol/L)

7.0 no 40 25 123

8.0 no 35 25 207

at 5.5 41 24 91

7.5 no 25e26 50 60

7.2 at 5.2 35 340 442

8.5 no 22 466 120

8.5 no 37 90 50

at 5.5 45 30 8

6.5 no 51.7 16 625

Table 2 e Experimental H2 production results: H2 produced, run duration and relative mass abundance of the measuredVOC.

Tw

(�C)H2 produced(mmol/L)

Dt(days)

Acetate(%)w

Butyrate(%)w

Propionate(%)w

Formate(%)w

Lactate(%)w

Ethanol(%)w

16 15.4 6.6 1.2 60.2 0.4 1.5 18.0 18.4

20 215.3 23.9 2.9 81.1 0 2.8 0.04 13.3

35 442.0 13.8 7.9 90.8 0.1 0.6 0 0.6

40 96.0 8.4 4.9 15.3 0.1 10.0 68.2 1.5

50 1.1 12.5 1.7 4.0 0.01 3.1 90.4 0.9

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 5 ( 2 0 1 0 ) 1 0 2 0 2e1 0 2 1 110206

the batch run (Dt). The energy loss per unit volume of reactor

may be calculated as:

El ¼

�4:5D

$kfoam

sfoam$DtðTwÞ$ðTw � TaÞ

�

h(4)

in which the bottom and the top of the bioreactor walls were

considered insulated with the same thickness as the vertical

wall. The bioreactor walls were considered to be constituted

by a steel 2.5 mm thick wall, as the structural material and

300 mm thick expanded polystyrene foam as the insulating

material. The total duration of fermentation Dt depends on

the working temperature, which was experimentally deter-

mined as reported in Tables 1e3.

The total resistance, i.e., the reciprocal of the total heat

transfer coefficient U, accounts for the total insulation of the

broth from the outside environment, and it can be calculated

as the sum of the single resistances, i.e., internal broth, steel,

foam and external air resistances:

U�1 ¼ h�1i þ ssteel

ksteelþ sfoamkfoam

þ h�1e (5)

where h is the convective heat transfer coefficient for the

internal and external fluid, s the thickness of the steel and

foamand k the thermal conductivity of the steel and foam [32].

Very thick polystyrene foam makes the foam resistance the

only relevant contribution to the total resistance, as graphi-

cally reported in Fig. 1, therefore both the convective internal

and external fluid resistance, as well as the steel resistance,

may be neglected to simplify the calculation. The resistance to

heat transport is here only considered in the insulating

material (sfoam/kfoam). This assumption leads to over-

estimating the insulator thickness at the same energy loss.

The term 4.5/D accounts for the exchange surface per unit

volume of the reactor, and it depends on geometrical

assumptions, i.e., the height-to-diameter ratio.

Table 3 e Numerical values to calculate the net energyproduction for integrated H2 and CH4 production.

Tw

(�C)H2 produced(mmol H2/L)

CH4 produced(mmolCH4/L)

Total Energy(kJ/L)

Test RunDurationDt (days)

16 15.4 391 317.5 22.6

20 220 452 409.0 39.9

35 442 530 519.2 29.8

3.4. Electrical energy for mixing and pumping

The electrical energy consumed to run the bioreactor is for

mixing, filling up and empting the bioreactor using a pump. In

batch fermentation, the raw material and the inoculum are

pumped in at the beginning of a run and the broth is pumped

out at the end of the run. The reactor ismixed all along the run.

The energy for pumping may roughly be calculated as the

energy necessary to lift the broth to the top of the reactor, i.e.,

E ¼ rgL; it accounts for the efficiency due to the pressure drop

and the partial recovery from empting the reactor, where L is

the height of the reactor.

The power number and the rotational Reynolds number

were considered to evaluate the mixing performances of the

bioreactor [33]. As the turbulence scale-up criteria, the rota-

tional Reynolds number was considered independent of the

reactor diameter in order to evaluate the energy necessary to

mix the fermenting broth at different diameters, i.e., the

power number is independent of the reactor diameter [33,34].

Geometrical similitude was assumed for the vessel and

impeller scale up, i.e., an equal impeller-to-reactor diameter

ratio. The impeller-to-reactor diameter ratio in the bench

scale bioreactor was set at 0.5. The following equation allows

us to estimate the electrical power necessary tomix the broth:

Pw ¼ Pn$r

8gp$N3

B$D6B

D4(6)

The procedure reported by Bailey and Ollis [34] was used to

calculate the power number (Pn) for the bench scale reactor. In

Fig. 1 e (a) Geometrical similarity used in the scale-up

procedure D/di [ 2 (b) Assumption used for the evaluation

of the heat loss through the bioreactor wall.

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 5 ( 2 0 1 0 ) 1 0 2 0 2e1 0 2 1 1 10207

the Equation (6) where subscript B refers to the bench scale

bioreactor, Pw is the power per volumeunit volume required to

reach the target mixing performances, i.e., the value of the

Reynolds number equal to that of the bench reactor. The total

electrical energy is the sum of the pumping and the mixing

terms, and the mixing power must be multiplied by the

duration of the run:

Ee ¼ Pw$DtðTwÞhel

þ 2rgL ¼ Pn$r$N3B$D

6B

8gp$hel

$DtðTwÞD4

þ 8rgD (7)

An efficiency factor of electrical energy conversion into

mixing energy of 0.75 was considered. The pumping term

resulted to be around 100 J/L per meter of reactor diameter. In

the considered diameter range this term resulted to be negli-

gible with respect to the mixing term, which means that it

is not interesting to calculate the pumping term more

accurately.

4. Results

4.1. Experimental results

4.1.1. Hydrogen productionThe hydrogen evolution was measured in the bench scale

bioreactor at each investigated temperature and is reported in

Fig. 2, while Table 2 reports, for each temperature, the main

experimental results, i.e., the specific hydrogen productivity,

the batch test run duration and the relative abundance of the

main metabolites at the end of the fermentation. The meso-

philic microorganism consortium, obtained from a waste-

water treatment sludge operating at 38 �C and treated at

acid conditions, has resulted to have a maximal hydrogen

production at 35 �C; this result agrees with the literature data

reported in Table 1. The mesophilic microorganism consor-

tium stops hydrogen production at 50 �C and reduces the

production of hydrogen to a very low level under 16 �C. Themetabolites reported in Table 2 are those that were recognized

[19] and measured in the test runs at the end of fermentation.

The data are reported as the mass percentage of the total

measured metabolites at the end of the test run. Acetate,

propionate and butyrate are known to be the compounds that

are stoichiometrically related to hydrogen production [13,19]

Fig. 2 e Hydrogen production vs. time in the bench scale

bioreactor (2 L, pH 5.2; 60 g/l glucose, N [ 100 rpm).

their relative amount depends on the temperature, which is

in agreement with literature findings [22,35]. At a lower

temperature than 35 �C, the main product is butyrate; its

relative abundance diminishes as the temperature decreases

while the ethanol and lactate abundance increases. Over

35 �C, lactate becomes the main product and little ethanol is

observed. It is therefore evident that temperature variations

shift the metabolic pathways. Acetate and butyrate abun-

dances have been confirmed to be closely related to hydrogen

production [39].

4.1.2. Methane productionAcetate and butyrate constitute 84% and 99% of the total

volatile organic compounds at 20 and 35 �C respectively. Thus

the broth at the end of fermentation contains the main

residual chemical energy in the form of acetate and butyrate;

they constitute the most ideal substrate for the methanogen

consortium [9]. The methanogen consortium can be inocu-

lated at the end of hydrogen production to produce methane

from fatty acids in order to utilize the energy contained in the

initial carbon source asmuch as possible. The gas evolution of

a 35 �C test run with hydrogen and the subsequent methane

production are reported in Fig. 3a. The first part of the test run

involved the dark fermentation to produce hydrogen and

volatile fatty acids after an inoculum with sludge treated at

acid conditions. The second part was the secondary fermen-

tation of the fatty acids to producemethane after an inoculum

with untreated sludge. As an average, 80% v/v ofmethanewas

measured in the biogas produced during the second part of

Fig. 3 e Time curve evolution of glucose fermentation

(initial concentration 60 g/l) (a) cumulative gas production

H2 and CH4 (b) pH and ROP evolution.

Fig. 5 e Net energy production vs. diameter of a bioreactor

for H2 production with 50% warming up energy recovery:

(a) winter and (b) summer conditions.

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 5 ( 2 0 1 0 ) 1 0 2 0 2e1 0 2 1 110208

fermentation. Fig. 3b reports the pH and ROP evolution during

the 35 �C test run; the pH of the broth evolved naturally till

reaching neutrality after the end of the hydrogen production.

At neutral pH, the reactor was inoculated by the untreated

sludge. The ROP remains at a reductive range at around

�500mV over the duration of the whole run. The correction of

pH at the end of hydrogen production did not lead to

a subsequent methane production. The natural evolution of

pH seems to allow microorganism to modify their environ-

ment and activity to reach the right condition for secondary

inoculum. The methanogenic consortium lag phase was

around 10 days at pH 7.2. As the gas production restarted, the

pH increased till 9, and the methane concentration in the gas

reached 80e85%. In order to evaluate the methane production

in the range less than 35 �C, a methanogenic smoothing

function, retrieved from Ref. [27], was used to extrapolate data

from themethane production data experimentally obtained at

35 �C. The experimental data at 35 �C and the data extrapo-

lated at 16 and 20 �C using a smoothing function [36] are

reported in Table 3. The total energy production, as well as the

hydrogen and methane contribution, is reported in Fig. 4 as

a function of the working temperature; the methane contri-

bution to energy production was calculated to be greater than

80% over the whole temperature range.

4.2. Net energy evaluation

The net energy was calculated for a single batch run using

Equations (1)e(7) for each working temperature for the

summer and winter reference ambient temperatures for

a bioreactor diameter ranging from 0.5 to 10 m. An expanded

polystyrene foam thickness of 30 cm was considered to

insulate the bioreactor.

4.2.1. Hydrogen productionThe net energy production for a batch run producing

hydrogen is reported in Fig. 5 as a function of the reactor

diameter. Fig. 5a and b refers to winter and summer condi-

tions, respectively. As a first result, the net energy for a 20 �Cworking temperature is always greater than that for a 35 �C in

spite of the lower hydrogen production at 20 �C, in which the

energy production is about 50% of the maximum at 35 �C(See Table 2 and Fig. 4). The net energy production increases

with diameter, due to the diminution of the external surface

area per unit volume which enhances the insulation and the

Fig. 4 e Specific energy production vs. temperature: H2 and

CH4 productions.

reduction of energy loss per unit volume. The energy loss

becomes more relevant under a critical reactor dimension,

around 3m in diameter, for the geometrical assumptions here

considered, i.e., height-to-diameter ratio and foam thickness.

Over the critical reactor dimension, the relevance of the

energy loss is almost independent of the reactor dimension.

At the winter condition, the net energy production is negative

over the whole diameter range, while, at the summer condi-

tion with 50% heat recovery, the net energy becomes positive

over a 1 m diameter at a 20 �C working temperature and over

a 3 m diameter at 35 �C. At 20 �C, the net energy is around 20%

of the total energy produced, while at 35 �C, the net energy is

around 5% of the total energy produced. The main energy

consumption is due to the initial warming of the broth, even

though an energy recovery was considered and the warming

of the reactor structure was neglected. If the energy recovery

is not considered, the net energy production is also negative

for summer conditions. Similar results were obtained using

the specific production data of other researchers, reported in

Table 1. The production of hydrogen for energy purposes by

batch runs has proved to be critical, and particular care has to

be taken in the raw material treatment, the energy recovery

and the warming strategies in order to design a low energy

requirement pretreatment and warming process, e.g., the use

of solar warming or of thermal wastes to warm the broth

and more favorable climatic conditions. In general, from

a sustainable energy point of view, a secondary process

should be coupled to hydrogen production. An energetic

analysis of the whole process should be performed. On the

other hand the Volatile Fatty Acids (VFA) and the other ener-

getic metabolites available in the broth at the end of hydrogen

fermentation may be energetically valorized, and several

approaches are available [9,37,38].

Fig. 7 e Net energy production vs. diameter for integrated

H2 and CH4 production with 50% warming energy recovery:

(a) winter and (b) summer conditions.

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 5 ( 2 0 1 0 ) 1 0 2 0 2e1 0 2 1 1 10209

4.2.2. Coupled hydrogen and methane productionAnaerobic digestion to produce biogas at a high methane

concentration is a possible way of energetically valorizing

the VFAs and the other compounds available in the broth at

the end of acidogenic fermentation. The methanation of the

residue seems to be one of the easiest processes to perform,

because the same reactor can be used without any further

addition or modification. In a first case, the net energy was

calculatedwithout any heat recovery; the results are shown in

Fig. 6 for the winter (6a) and summer (6b) conditions at the

lowest working temperature, i.e., 16, 20 and 35 �C. A higher

working temperature leads to a lower net energy production,

due to the lower energy production and the higher energy

requirement. The net energy production is positive over the

reactor dimension whole range both for the winter and the

summer average conditions. A 3m diameter was confirmed to

be the critical reactor dimension under which energy loss

plays amore important role. The 20 �Cworking temperature is

the most convenient from an energetic point of view over the

whole diameter range. Nevertheless, over a 2 m reactor

diameter, the net energy production resulted to be at about

the same level at 20 and 35 �C, i.e., the greater energy

production at 35 �C is balanced by the greater energy neces-

sary to warm and maintain the working temperature. This

means that, over a 2 m reactor diameter, the energetic criteria

are not relevant in order to choose the working temperature.

The net energy resulted to be 45e88% of the produced energy

as H2 and CH4 forms, depending on the working temperature

and the average seasonal conditions, the maximal percentage

is at 20 �C for the summer.

In the second case, net energy was calculated considering

the recovery of 50% of the warming energy, the results are

shown in Fig. 7 for winter (7a) and summer (7b) conditions.

The 35 �C working temperature is the most convenient, from

Fig. 6 e Net energy production vs. diameter for integrated

H2 and CH4 production without energy recovery: (a) winter

and (b) summer conditions.

an energetic point of view, over almost the whole reactor

dimension range, because of the greater relevance of the

warming energy in the balance. Nevertheless for a very small

reactor dimension the energy loss becomes very relevant and

20 �C could be considered as an energetically convenient

choice. The net energy resulted to be 70e80% of the produced

energy, depending on the working temperature and the

average seasonal conditions, while themaximal percentage is

at 35 �C for the summer; in Fig. 8 is reported a Sankey diagram

for the best situation. Once again the importance of the

running strategy is stressed; the recovery of energy makes it

energetically convenient to work at higher temperatures.

Working at higher temperatures reduces the run time and

enhances the hydrogen production, and it also leads to

Fig. 8 e Sankey diagram for the best situation: summer

time, working temperature 35 �C, 50% heat recovery,

reactor diameter > 4 m.

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 5 ( 2 0 1 0 ) 1 0 2 0 2e1 0 2 1 110210

economic advantages. The optimal reactor dimension

depends on many factors such as economic considerations,

robustness, reliability and flexibility. The present paper has

offered a rough evaluation for energetic sustainability. The

same procedure may be considered for a design criterion to

calculate the optimal insulator thickness.

4.2.3. UncertaintyAs concerns the main affecting parameters on the net energy

estimation, the warming energy compared to other energy

terms has the highest predominance. It reaches almost the

same numerical value both in winter time and in summer

time: 85% and 70% respectively. In regard to themain affecting

parameter, by applying the procedure recalled in Section 2.5

the variability either of ambient temperature Ta or that of

the global thermal efficiency h are the main sources of the

uncertainty. With a Ta variability around 5 �C and a relative

variability of the efficiency around 10% on the used values for

the calculation, the uncertainty of the net energy is around

50 kJ/L; the evaluation of the uncertainty without any vari-

ability of Ta and h, i.e., considering only the uncertainty due to

the experimental data results on the range (5e15) kJ/L, this

confirms that the Ta is the main affecting parameter in

anaerobic digestion. According to the value of the net energy

estimation reported in Figs. 6 and 7, we can consider the

suggested energy balance sufficiently acceptable in the eval-

uation of the sustainability of biohydrogen and biogas

productions. In any case for a specific design of a detailed

plant the uncertainty could be reduced.

5. Conclusions and comments

This paper has considered the energy balance to evaluate the

net energy produced, by batch runs of a bioreactor producing

hydrogen, as a function of the working temperature and

reactor scale. The procedure proposed in this paper allows an

evaluation of the net energy in order to define themain criteria

to choose the scale up and design strategies. For detailed

design purposes, it is necessary to define a more refined

calculation procedure. The evaluation of the net energy allows

maximizing the energy produced by the plant differently

to maximize the yield of hydrogen, as reported in Fig. 6.

The most relevant conclusion is that particular attention

must be devoted to the process energy saving strategy to reach

a positive net energy production. The main part of the energy

is required for the warming of the broth; an adequate strategy

needs to be designed in order to reduce this energy cost, e.g.,

the use of solar warming or thermal wastes and heat recovery

and thermal integration if it is possible. However, the effects

of seasonal differences on the net energy are relevant; by

uncertainty analysis the ambient temperature and its varia-

tion play the utmost important role. In the case of the

production of H2 alone, the best calculated available energy

was around 20%.

The broth at the end of dark fermentation contains

substances which embed a part of the energy originally in the

substrate. These substances can be valorized by integrating

a process with the hydrogen production. The anaerobic

fermentation of the residual metabolites to methane can

easily be thermally integrated with hydrogen production

through a two-step hydrogenemethane production. Energetic

sustainability is reached, for the integrated process, over the

whole range of operative conditions considered in the anal-

ysis. The net energy was calculated to be higher than 50% and

to even reach 80% in the best case. However, it is possible to

work at higher temperatures by enhancing the process strat-

egies to save energy, this have a consequent positive

economic impact. The reactor scale plays an important role

under a critical dimension due to the higher weight of the

energy loss. All the calculations have been performed at the

typical climatic conditions of northern Italy; warmer climatic

conditions would lead to an increase in the net energy

production.

Finally, it has been observed that the shift of the pH

towards neutral values and the addition of methanogens

inoculum are able to start a methanogenic fermentation to

produce methane using the VFAs and the other products at

the end acidogenic fermentation as the substrate. However,

the way the shift of the pH towards neutral is performed, may

influence the lag phase of themethanogenic consortium. This

aspect needs to be evaluated in detail both from a biological

and from an energetic point of view.

Lastly, the present evaluation of the net energy does not

take into account the quantities of energy spent to construct

the plant as well the energy spent to produce the chemicals,

(mainly NaOH) to control the pH during the fermentation. This

would be accomplished by a more comprehensive analysis

using Life Cycle Approach (LCA) in order to evaluate the

amount of energy delivered to society. By LCA approach the

net energy produced needs to be compared with the total

energy required to produce thematerial, to assemble the plant

and process, deliver and upgrade otherwise the sources, the

organic refuse in the present contest, in a useful form to give

an energy service. In the present paper only the net energy

was evaluated. The evaluation of “useful” energy delivered

into the society reserves several advantages: it assesses the

change in the physical scarcity of energy resources, it is

a measure of the potential of such technology to do useful

work in sustainable way and finally it is possible to rank

alternative energy supply technologies according to their

capacity to do useful work. Researchers in this area are in due

course; reliable results are welcomed in order to acquire an

evaluation of energetic of biohydrogen and biogas

productions.

r e f e r e n c e s

[1] Balat M. Potential importance of hydrogen as a futuresolution environmental and transportation problem. Int JHydrogen Energy 2008;33:4013e29.

[2] Das D, Veziroglu TN. Advances in biological hydrogenproduction processes. Int J Hydrogen Energy 2008;33:6046e57.

[3] Turner JA. Sustainable hydrogen production. Science 2004;305:972e4.

[4] Westermann P, Jorgensen B, Lange L, Ahring BK,Christensen CH. Review: maximizing renewable hydrogenproduction from biomass in a bio/catalytic refinery. Int JHydrogen Energy 2007;32:4135e41.

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 5 ( 2 0 1 0 ) 1 0 2 0 2e1 0 2 1 1 10211

[5] Roger CP, Kheshgi HS. The photobiological production ofhydrogen: potential efficiency and effectiveness asa renewable fuel. Critic Rev Microbiol 2005;31(1):19e31.

[6] Akkerman I, Janssen M, Rocha J, Wijffeld RH. Photobiologicalhydrogen production: photochemical efficiency andbioreactor design. Int J Hydrogen Energy 2002;27:1195e208.

[7] Anastasios M. Green alga hydrogen production: progress,challenges and prospects. Int J Hydrogen Energy 2002;27:1217e28.

[8] Miyake J, Kawamura S. Efficiency of light energy conversionto hydrogen by the photosynthetic bacterium Rhodospirillumsphaeraides. Int J Hydrogen Energy 1987;12(3):147e9.

[9] Reith JH, Wijffeels RH, Barten H. Status and perspectives ofbiological methane and hydrogen production. Nederland:Dutch Biological Foundation; 2003.

[10] Kapdan IK, Kargi F. Review e biohydrogen production fromwaste material. Enzym Microb Technol 2006;38:569e82.

[11] Nishio N, Nakashimada Y. High rate production of hydrogen/methane from various substrate and wastes. Adv BiochemEng Biotechnol 2004;90:63e87.

[12] Hawkes FR, Hussy I, Kyazze G, Dinsdale R, Hawkes DL.Continuous dark fermentative hydrogen production bymesophilic microflora: principles and progress. Int JHydrogen Energy 2007;32:172e84.

[13] Das D, Veziroglu TN. Hydrogen production by biologicalprocesses: a survey of literature. Int J Hydrogen Energy 2001;26:13e28.

[14] Sen U, Shakdwipee M, Banerjee R. Status of biologicalhydrogen production. J Sci Ind Res 2008;76(11):980e93.

[15] Wang JL, Wan W. Factor influencing fermentative hydrogenproduction: a review. Int J Hydrogen Energy 2009;34:799e811.

[16] Kaual SK, Chen WH, Li L, Sung S. Biological hydrogenproduction: effect of pH and intermediate product. Int JHydrogen Energy 2004;29:1123e31.

[17] Wang JL, Wan W. Effect of temperature on fermentativehydrogen production by mixed cultures. Int J HydrogenEnergy 2008;33:5392e7.

[18] Batie CJ, Kamin H. The relation of pH andoxidationereduction potential to the association state of theferrodoxin.ferrodoxin:NADPþ reductase complex. J BiolChem 1981;256(15):7756e63.

[19] Ruggeri B, Tommasi T, Sassi G. Experimental kinetics anddynamics of hydrogen production on glucose by hydrogenforming bacteria (HFB) culture. Int J Hydrogen Energy 2009;34:753e63.

[20] Adams MWW, Mortenson LE, Chen TS. Hydrogenase.Biochem Biophys Acta 1981;594:105e16.

[21] Tang GL, Huang J, Sun ZJ, Tang QQ, Yan CH, Liu GQ.Biohydrogen production from cattle wastewater by enrichedanaerobic mixed consortia: influence of fermentationtemperature and pH. J Biosci Bioeng 2008;106(1):80e7.

[22] Mu Y, Zhung XJ, Yu HQ, Zhu RF. Biological hydrogenproduction by anaerobic sludge at various temperatures.Int J Hydrogen Energy 2006;31:780e5.

[23] Li CL, Fang HHP. Fermentative hydrogen production fromwastewater and solid waste by mixed culture. Crit RevEnviron Sci Technol 2007;31:1e39.

[24] Gadhamshetty V, Johnson Dc DC, Nirmalakhandan N,Smith GB, Deng S. Feasibility of biohydrogen production atlow temperatures in unbuffered reactor. Int J HydrogenEnergy 2009;34:1233e43.

[25] Yokoyama H, Waki M, Moriya N, Yasuda T, Tanaka Y,Haga K. Effect of fermentation temperature on hydrogenproduction from cow waste slurry by using anaerobicmicroflora within the slurry. Appl Microbiol Biotechnol 2007;74(2):474e83.

[26] O-Thong S, Presertsan P, Karakashev D, Angelidaki I.Thermophilic fermentative hydrogen production by thenewly isolated Thermoanaerobacterium thermosaccharolyticumPSU-2. Int J Hydrogen Energy 2008;33(4):1204e14.

[27] Ruggeri B. Thermal analysis of anaerobic digesters. Chem Ind1984;66(7e8):477e83.

[28] Bonallagui H, Haonari O, Tauhami Y, Ben Cheikh R,Maronani L, Hamdi M. Effect of temperature on theperformance of an anaerobic tubular reactor treatingfruit and vegetable waste. Process Biochem 2004;39:2143e8.

[29] Chen CC, Lin CY, Liu MC. Acidebase enrichment enhancesanaerobic hydrogen production process. Appl MicrobiolBiotechnol 2002;58:224e8.

[30] Mu Y, Yu HQ, Wang G. Evaluation of three methods forenriching H2-producing cultures from anaerobic sludge.Enzyme Microb Technol 2006;40(4):947e53.

[31] Fang HP, Li C, Zhang M. Acidophilic biohydrogen productionfrom rice slurry. Int J Hydrogen Energy 2006;31:683e92.

[32] Rohsenaw WM, Hartnett JP. Handbook of heat transfer.New York: McGraw Hill; 1973.

[33] Nagata S. Mixing: principles and applications. New York:John Wiley & Sons; 1975.

[34] Bailey JE, Ollis DF. Biochemical engineering fundamentals.2nd ed. Singapore: McGraw Hill Education InternationalEditions; 1986.

[35] Lee KS, Lin PJ, Chang JS. Temperature effects onbiohydrogen production in a granular sludge bed inducedby activated carbon carriers. Int J Hydrogen Energy 2006;31:465e72.

[36] Ruggeri B. Thermal and kinetic aspects of biogas production.Agric Wastes 1986;16:183e91.

[37] Pham TH, Rabaey K, Aelterman P, Clauwaert P, DeShampelaire L, Boon N, Verstraete W. Microbial fuel cells inrelation to conventional anaerobic digestion technology. EngLife Sci 2006;6(3):285e92.

[38] Kapdan IK, Kargi F, Oztekin R, Argun H. Bio-hydrogenproduction from acid hydrolyzed wheat starch by photo-fermentation using different Rhodobacter sp. Int J HydrogenEnergy 2009;34:2201e7.

[39] Lee HS, Rittmann BE. Evaluation of metabolism usingstoichiometry in fermentative biohydrogen. BiotechnolBioeng 2009;102:749e58.

[40] ISOTAG4. Guide to the expression of the uncertainty inmeasurements (GUM). Geneva: ISO; 1994.

[41] Ruggeri B. Chemicals exposure: scoring procedure anduncertainty propagation in scenario selection for riskanalysis. Chemosphere 2009;77:330e8.