DSpace at VNU: Thermophilic fermentative hydrogen production from xylose by Thermotoga neapolitana DSM 4359

Renewable Energy 37 (2012) 174e179

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Renewable Energy
journal homepage: www.elsevier.com/locate/renene

Thermophilic fermentative hydrogen production from xylose by Thermotoga
neapolitana DSM 4359
Tien Anh Ngo a, b, *, Tra Huong Nguyen b, Ha Thi Viet Bui b
a
b

Institute of Advanced Energy, Kyoto University, Uji, Kyoto 611-0011, Japan
Department of Microbiology, Hanoi University of Science, Hanoi, Viet Nam

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 3 September 2010
Accepted 11 June 2011
Available online 2 July 2011

Biohydrogen production from xylose by Thermotoga neapolitana was investigated in batch culture using
serum bottles and a continuously stirred anaerobic bioreactor (CSABR). The effect of various xylose
concentrations on growth and H2 production were studied in small batch culture for highly efficient H2
production. The highest hydrogen production of 32.1 Æ 1.6 mmol-H2/L and maximum biomass
concentration of 959.63 Æ 47.9 mg/L were obtained at initial xylose concentration of 5.0 g/L. To develop
a large-scale biohydrogen production system as well as overcome the problems in small batch culture,

a continuously stirred anaerobic bioreactor was tested on T. neapolitana in both pH-uncontrolled batch
culture and pH-controlled batch culture. The results showed that the production level of H2 from
fermentation in a pH-controlled batch culture was much higher than those from a pH-uncontrolled batch
culture for H2 production from xylose. The H2 yield in a pH-controlled batch culture on xylose substrate
was 2.22 Æ 0.11 mol-H2 molÀ1 xyloseconsumed, which was nearly 1.2-fold higher than pH-uncontrolled
batch cultures. In order to study the precise effect of a stable pH on hydrogen production, and metabolite pathway involved, cultures was conducted with pH-controlled at different levels ranging from 6.5 to
7.5. The maximum H2 yield of 2.8 Æ 0.14 mol-H2 molÀ1 xyloseconsumed was measured while the pH was
maintained at 7.0. The acetic acid and lactic acid production were 2.98 Æ 0.15 g/L and 0.36 Æ 0.02 g/L,
respectively.
Ó 2011 Elsevier Ltd. All rights reserved.

Keywords:
Batch culture
Biohydrogen
Thermotoga neapolitana
Xylose
CSABR

1. Introduction
Biohydrogen is a green energy with the greatest potential to
replace fossil fuels in the future, given its high energy content, lack
of CO2 emissions, and readily available production sources from
various renewable feedstocks [1e3]. Carbohydrate rich wastes such
as lignocellulosic agricultural residues are promising feedstocks
with huge biofuel potential [4]. Pentose sugar (xylose) accounts for
up to 35e45% of the total sugars in the lignocellulosic hydrolysate
derived from wood, agricultural by products or crops [5]. To convert
cellulosic feedstock to high value hydrogen, full fermentation of
pentose (xylose) or hexoses (glucose, sucrose) in the hydrolysates
of cellulose and hemicelluloses is very important [6].

The hyperthermophilic bacteria Thermotoga neapolitana has
garnered increasing interest for potential biohydrogen production
with high yield from a wide range of carbohydrates, such as

* Corresponding author. Institute of Advanced Energy, Kyoto University, Uji,
Kyoto 611-0011, Japan.
E-mail address: (T.A. Ngo).
0960-1481/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.renene.2011.06.015

glucose, sucrose [7,8], and especially direct fermentation of starch,
hemicelluloses, cellulose [9,10] and glycerol wastes in our previous
research [11]. Moreover, the biohydrogen fermentation of
T. neapolitana at an ambient 75  C makes its H2 fermentation less
sensitive to contamination from methanogenic archaea, with
a higher rate of hydrolysis, and H2 yield [12e14,25]. Most studies on
biological hydrogen production in Thermotogales are based on
small batch fermentation in serum bottle and few of works have
used glucose as carbon source in pH-controlled bioreactors [26,27].
Investigations of xylose fermentation to biohydrogen were mainly
concentrated on fermentation using anaerobic mix culture at
mesophilic temperatures [15e17]. However, only limited study has
been carried out on the possibility of xylose utilisation for hydrogen
production by T. neapolitana [8]. Because of the main products
produced from the metabolic activities of T. neapolitana from
strictly anaerobic fermentation, including organic acids (such as
lactic and acetic acid), carbon dioxide (CO2) and H2 [10,18], growth
and H2 production by T. neapolitana in small batch cultures were
reported to be limited by a rapid decrease in pH and the effect from
high hydrogen partial pressure [8,12,19]. To overcome this problem,

an N2 sparging method and appropriate buffering [7,8,11,18] were


T.A. Ngo et al. / Renewable Energy 37 (2012) 174e179

175

successfully applied to enhance anaerobic H2 fermentation
T. neapolitana in a small. To develop a large-scale biohydrogen
production system and overcome the problems in small batch
culture, T. neapolitana was studied in pH-controlled batch culture
using a continuously stirred anaerobic bioreactor.
In this work, we describe the fermentative production of biohydrogen from xylose substrate by the hyperthermophile
T. neapolitana in serum bottle cultures and a 3 L-CSABR with pHuncontrolled/controlled batch cultures and a continuous pure N2
gas flow. The optimization of initial substrate concentration for
growth and H2 production were determined in serum bottles.
Investigation of pH values was carried out in a bioreactor with pH
control.
2. Materials and methods
2.1. Strain and cultivation medium
The T. neapolitana strain DSM 4359 was obtained from Deutsche
Sammlung von Mikroorganismen und Zelhulturen, Germany. The
cultures were grown in 120 mL serum bottles containing 40 mL
culture medium of modified Thermotoga maritima basal medium
(TMB) at 75  C and pH 7.5, with 10% (v/v) inoculation [12]. The
medium used for H2 fermentation consisted of (amounts are in
grams per litre of deionised water): 1.5 g KH2PO4; 4.2 g Na2HPO4$12H2O (22 mM PO3À
4 ); 0.5 g NH4Cl; 0.2 g MgCl2$6H2O (1 mM);
20.0 g NaCl; 2.0 g yeast extract; 5.0 g carbon source (glucose, sucrose,
xylose); 15.0 mL of the trace element solution (DSM-TES, see DSMZ

medium 141); and 1.0 mg resazurin, which was used as a redox
indicator. The anaerobic conditions for growth were created by
adding 1.1 g cysteine hydrochloride as a reducing agent and flushing
the headspace of the serum bottles with pure N2 within 5 min.
A batch culture using a 3 L bioreactor (Biotron, Korea), charged
with 900 mL of fresh medium and a 100 mL inoculum of T. neapolitana, was performed under the controlled of temperature, pH and
agitation at 75  C, 7.5, and 300 rpm, respectively, using a Biotron
controller system. The pH was kept constant by the addition of
2.0 N NaOH. Temperature was kept at 75  C using a heating coil
wrapped around the bioreactor. The gas headspace was sparged
with a continuous and pure N2 gas flow; the gas outlet from the
reactor was connected to a condenser. The flow and partial pressure
of the gas headspace in the outlet gas was monitored by a gas
meter. The complete setup is illustrated on Fig. 1.
2.2. Sampling and analyses
The biomass was monitored by dry cell weight (DCW) and
optical density (OD600), with sterile medium as the control. The H2
gas in the headspace was sampled by a gas-tight syringe (100 mL
injection volume, Hamilton, USA) and determined by gas chromatograph (GC, Hewlett Packard 5890 Series II, USA) employing
a thermal conductivity detector (TCD) and a 2-m stainless column
packed with Carboxen 1000, 50/80 mesh (Supelco). The operational
temperatures of the injection port, the oven, and the detector were
120, 70, and 120  C, respectively. Nitrogen was used as the gas
carrier at the flow rate of 55 mL per min.
The moles of produced H2 gas (nH2, mol) and the partial pressure of H2 (pH2, atm) in the batch culture using serum bottles were
calculated from the mole percentage of H2, which was determined
by GC analysis.
The hydrogen yield (YH2) is the molar amount of H2 produced
from the consumed substrate. It was calculated using Eq. (1):

YH2 ¼

nH2
nS

(1)

Fig. 1. Scheme of the anaerobic fermentation bioreactor.

(where nH2 is the moles of hydrogen produced, and nS is the moles
of substrate consumed.)
The volumetric gas flow rate (F, mL minÀ1) from the bioreactor
and pH2 were used to calculate the molar flow rate of H2 (Q H2 ,
mmol.hÀ1) using Eq. (2):

Q H2 ¼

FpH2 min
60
RT
h

(2)

(where R is the gas constant for ideal gas (0.08206 L atm molÀ1 KÀ1), and T is 298 K.)
The amount of H2 (nH2 , mole) produced at the time (t) of inoculation (to ¼ 0) was calculated by numerical integration of the
molar H2 flow rate, Q H2 ;t , with respect to time (Eq. (3)):

NH2 ;tiþ1 ¼



X
Q H2 ;ti þ Q H2 ;tiþ1
ðtiþ1 À ti Þ
2

(3)

The organic acid concentration was quantified using an HPLC
system equipped with a reflective index detector (Agilent 1100,
USA): 50 mL of 0.2 mm filtered-culture supernatant was separated
on a Rezex ROA-Organic acid Hþ (8%) 300 Â 7.80 mm column
(Phenomenex, USA) and eluted with 0.5 mL minÀ1 of 0.005 M
H2SO4 at room temperature. The residual xylose concentration in
the culture supernatant was quantified using HPLC: 50 mL of 0.2 mm
filtered-culture supernatant was injected and analysed on a Rezex
RCM-Monosaccharide column (Phenomenex, USA) and eluted with
0.5 mL minÀ1 water at 60  C. Detection was performed with
a refractive index detector (Agilent 1100, USA).
3. Results and discustion
3.1. Investigation of H2 production of T. neapolitana from xylose
T. neapolitana were grown in serum bottles for application of
converting xylose into biohydrogen. When T. neapolitana was
grown anaerobically on xylose, a mixture of acetic and lactic acid
was produced (data not shown). H2 accumulated in the headspace
and accumulation of cells increased dry cell weight (DCW) from
0.08 to 0.73 Æ 0.03 g/L within 20 h and almost maintained from 25
to 55 h of cultivation (Fig. 2). H2 production was observed soon after
5 h upon initially entering into the log phase of growth, achieving

35

1.0

H2 content
Cell growth

A

0.8

30
25

0.6

20
0.4

15
10

0.2

Cell growth (g DCW/L)

H content (%, v/v headspace)
2

40

5
0

36

54

32

48

28

42

24

36

20

30

16

24

12

18

H2 content

8

12

H2 production

4

H2 production (mmol/L)

T.A. Ngo et al. / Renewable Energy 37 (2012) 174e179

H 2 content (% v/v, headspace)

176

6

0.0
10

20

30

40

50

0

0
2

Cultivation time (hour)

3

4

5

6

7

8

Xylose concentration (g/L)
Fig. 2. Investigation of H2 production by T. neapolitana from xylose in serum bottle
without pH control. All data points are averages of three replicate bottles.

B 1.4

3.2. Fermentative H2 production in batch anaerobic bioreactor
In T. neapolitana fermentations there was a rapid decrease in pH,
resulting in increasing pH stress. In some cultures, the process
stopped before all of substrate was consumed. This has previously
been observed also in cultures of T. neapolitana [7,10,12] and
T. neapolitana [9] from glucose substrate. In this study,
T. neapolitana was investigated in both pH-uncontrolled and pHcontrolled batch cultures from xylose substrate using a 3 L-CSABR
system at an initial pH of 7.5, and at temperature of 75  C. A mixture
of acetic and lactic acid was also produced when T. neapolitana was

100

4

0.8
2
0.6
0.4

1
Cell growth

0.2

80

60

40

Xylose utilization (%)

3

1.0

Residual xylose (g/L)

1.2

Cell growth (g DCW/L)

high level of H2 from 20 h to the end of the cultivation, with
approximately 30% of H2 content in the headspace (Fig. 2). This
result indicated that there were no significant differences in the H2
production released in xylose fermentation and glucose fermentation which was studied in the previous research [12,7].
To determine the sole effect of the xylose concentration on H2
production and substrate utilisation of T. neapolitana, a complex
medium containing a fixed yeast extract concentration 2.0 g/L
supplemented with different initial xylose concentrations was
applied to the small batch cultures. Fig. 3 shows the effect of the
different initial xylose concentrations versus H2 production and
xylose utilisation. The cumulative H2 production increases with the
rising of xylose concentration in the range of 2.0e5.0 g/L, the
maximum H2 production of 32 Æ 1.6 mmol-H2/L culture and
maximum H2 content of 30% occurring at a xylose concentration of
5.0 g/L. Then, H2 production gradually decreased as the xylose
concentration increased (Fig. 3A). At an initial xylose concentration
of 5.0 g/L, the biomass of T. neapolitana reached to the highest value
of 0.96 Æ 0.05 g/L culture after 24 h of cultivation (Fig. 3B).

However, at the initial xylose concentration of 5.0 g/L, converted H2
yeild from xylose of 1.1 Æ 0.05 mol-H2 molÀ1 xylose, was lower than
the initial xylose concentration of 2.0 g/L, with the highest yield of
1.7 Æ 0.08 mol-H2 molÀ1 xylose. Accordingly, a high concentration
of xylose was not favorable for T. neapolitana growth and H2
production. The results herein indicate that the change in xylose
concentration remarkably affected H2 production and substrate
utilisation. Obviously, hydrogen production specific to the amount
of xylose added depends both on the degree of substrate conversion, as well as the metabolic conversion pathway, giving information about the potential of substrate to release a specific amount
of H2. Expression of H2 production per amount of substrate added is
often used to describe H2 production efficiency [20].

20

xylose utilization
Residual xylose

0.0

0
2

3

4

5

6

7

0

8

Xylose concentration (g/L)
Fig. 3. Growth of T. neapolitana from batch cultures in serum bottle at different initial
xylose concentrations: (A) H2 content in biogas and molar amount H2 production; (B)
biomass concentration as DCW and xylose initialization. All data points are averages of
three replicate bottles.

grown on xylose substrate. H2 production process started at around
3 h and the pH rapidly decreased from 7.5 to below 5.5 after 18 h of
cultivation (Fig. 4A and B). As shown in Fig. 4A, in the first 24 h of
cultivation, the cellular metabolism of T. neapolitana was robust.
With xylose as the sole carbon source, the biomass concentration
reached the maximum value of 1.4 Æ 0.07 g DCW/L and culture pH
almost unchanged after dropping to 5.17 at 30 h of cultivation. After
fermenting for 10 h, the CSABR produce biogas with a H2 content of
over 30%. Steady-state operation was achieved from 10 to 17 h and
the maximum of H2 production rate and H2 yield were respectively
3.1 Æ 0.1 mmol-H2 hÀ1 and 1.8 Æ 0.1 mol-H2 molÀ1 xylose at 15 h,
(Fig. 4B). The increase in H2 production was accompanied with
a proportional increase in xylose utilisation and acetic acid
production. Fig. 4B shows that approximately 86% of the xylose was
consumed within the first 24 h of cultivation. The acetic acid
production began at 3 h, while the lactic acid production started at
around 12 h from medium containing xylose. At the end of
fermentation, 2.1 Æ 0.1 g/L acetic acid, 0.34 Æ 0.02 g/L lactic acid was

obtained from xylose (Table 1). This result shows that the obtained
acetic acid production in medium was consistently shown to be
above ten fold higher than lactic acid production. It implies the H2acetic acid production pathway predominated over the main
compete lactic acid production pathway. In this study, the pH


T.A. Ngo et al. / Renewable Energy 37 (2012) 174e179

Cell growth
Culture pH

1.0

7.5

7.0

6.5

0.5

6.0

Culture pH

Cell growth (g DCW/L)

B

8.0

5.5

0.0

5.0
6

12

18

24

30

36

42

Xylose, H 2 , Lactic acid, acetic acid (mmol/L)

1.5

Cultivation time (hour)

70

4.0
H2 production rate

Residual xylose
Accumulated H 2 production

60

3.5

Lactic acid
Acetic acid

50

3.0
2.5

40
2.0
30
1.5
20

1.0

10

H 2 production rate (mmol/h)

A

177

0.5

0

0.0
10

20

30

40

Cultivation time (hour)

1.0

7.0

6.5

0.5

6.0
Cell growth
Culture pH

0.0

Culture pH

Cell growth (g DCW/L)

7.5

5.5

5.0
6

12

18

24

30

36

42

80

4.0

H2 production rate
Residual xylose
Accumulated H 2 production

70

3.5

Lactic acid
Acetic acid

60

3.0

50

2.5

40

2.0

30

1.5

20

1.0

10

0.5

H 2 production rate (mmol/h)

D

8.0

Xylose, H 2 , Lactic acid, acetic acid (mmol/L)

C 1.5

0.0

0
10

Cultivation time (hour)

20

30

40

50

Cultivation time (hour)
Fig. 4. Growth of T. neapolitana from xylose as the main substrate in the batch culture using a 3 L-CSABR: (A) Cell growth (DCW) and pH in batch culture without pH control; (B)
Metabolites (xylose, H2, lactic acid, acetic acid) in batch culture without pH control; (C) Cell growth (DCW) and pH in batch culture with pH control; Metabolites (xylose, H2, lactic

acid, acetic acid) in batch culture with pH control.

decreased in the culture supernatants, resulting in cessation of the
H2 fermentation process prior to complete consumption of the
substrate. At the end of fermentation, approximately 89.4% of
xylose was consumed.

pH was initially 7.5 (Æ0.01) in all experiments. In the pHcontrolled batch culture, pH was kept constant by the addition of
2.0 N NaOH during cultivation. A very strong metabolism occurred in
bacterial cells within the initial 15 h-cultivation with xylose as the

Table 1
Performance and metabolite analysis of T. neapolitana fermentation.
Parameters

Working volume of 1000 mL of culture in a 3L CSABRa system
Without pH control

With pH controlb

31.9 Æ 1.6
3.1 Æ 0.1
1.84 Æ 0.09
2.1 Æ 0.1
0.34 Æ 0.02
89.4 Æ 4.5
5.17

38.3
3.76

2.71
2.98
0.21
97.8
6.5

6.5
Maximum H2 content (%)
Maximum H2 production rate (mmol-H2hÀ1)
H2 yieldc
Final [acetic acid] (g/L)
Final [lactic acid] (g/L)
Xylose consumption (%)
Final pH

Æ
Æ
Æ
Æ
Æ
Æ

1.9
0.18
0.14
0.15
0.01
0.8

7.0

7.5

39.6 Æ 1.98
3.89 Æ 0.19
2.8 Æ 0.1
3.02 Æ 0.15
0.16 Æ 0.01
98.8 Æ 1.1
7.0

33.5 Æ 1.7
3.3 Æ 0.16
2.2 Æ 0.11
2.5 Æ 0.12
0.15 Æ 0.01
95.4 Æ 4.4
7.5

Each measurement was repeated three times and averaged. Error indicates one standard deviation of uncertainty.
a
CSABR ¼ continuously stirred anaerobic bioreactor.
b
The culture was conducted with the pH-controlled at three different levels ranging from 6.5 to 7.5 with standard error in measurement of (Æ0.01).
c
H2 yield ¼ (amount (mol) of H2 formed)/(amount (mol) of substrate consumed).


178

T.A. Ngo et al. / Renewable Energy 37 (2012) 174e179

main substrate (Fig. 4C). The cell growth significantly increased and
reached the highest concentration of 1.42 Æ 0.07 g DCW/L at 15 h and
was nearly maintained from 15 to 33 h of cultivation. The residual
xylose concentration in medium was 0.6 Æ 0.03 g/L, that means about
90% of xylose was consumed only at the first 24 h (Fig. 4D). These
results indicated that growth and xylose utilisation of T. neapolitana
in the pH-controlled batch cultures greater than that in pHuncontrolled batch culture. As shown in Fig. 4D, the H2 production
rate achieved the best value of 3.3 Æ 0.16 mmol-H2 hÀ1 responding to
H2 content in the mixture gas was 33.5 Æ 1.7% at 13 h. After that, the
H2 production rate was gradually decreased over time and negligible
after 33 h. Therefore, a decrease in H2 production of T. neapolitana in
the pH-controlled cultures was due to exhaustion of the growthlimiting substrate which was xylose in this case. The acetic acid
production also observed after 3 h, while the lactic acid production
started at 42 h from xylose (Fig. 4D). Similar to the H2 production and
xylose utilisation results, the levels of acetic acid from fermentation
with pH control were much higher than those of the fermentation
without pH control. In contrast to acetic acid production, lactic acid
production was the lowest under conditions of pH control. In other
words, the acetic acid concentration was much higher than the lactic
acid concentration. This result implies that the batch culture with pH
control was found for highly efficient hydrogen production. The
observed correlation between the acetic acid/lactic acid ratio and the
H2 yield implies a metabolic association between acetic acid and H2,
as is well-described in prokaryotic fermentation such as the Clostridia and Enteric bacteria. Results from Table 1 show that maximum
H2 yield, acetic acid production, and xylose utilisation in pHcontrolled culture was higher than those in pH-uncontrolled
culture. The best of H2 yield of T. neapolitana obtained in the pHcontrolled culture was 2.2 Æ 0.11 mol-H2 molÀ1 xylose.
3.3. Determination of optimal pH for H2 production from xylose
using a 3 L-CSABR system

The effect of pH on fermentative H2 production by T. neapolitana
strain was investigated in a 3 L-CSABR system with pH-controlled at
three different levels ranging from 6.5 to 7.5 while keeping other
operating conditions constant (stirring, temperature, pressure, and
initial culture medium). In Table 1, the maximum H2 production yields
are indicated for different pH conditions, with the related final acetic
and lactic acid concentrations, and xylose utilisation. The maximum
H2 production yield and H2 production rate, 2.8 Æ 0.1 mol-H2 molÀ1
xylose and 3.89 Æ 0.19 mmol-H2 hÀ1 respectively, were obtained at
a pH of 7.0. These optimum pH and yields were in accordance with
previous studies with the T. neapolitana culture [12]. Since at this pH
level utilisation of xylose and organic acid concentration also peaked,
overall performance for the process was at a maximum (Table 1).
Subsequently, the H2 production rate increased exponentially reaching the maximum level in comparing with those of T. neapolitana
cultured at other pH levels until substrate depletion (data not shown).
The H2 yield of 2.8 Æ 0.1 mol-H2molÀ1 xylose in this study was
substantially higher than in other studies using xylose as carbon
source from pure cultures of bacteria (0.14 mol-H2molÀ1 xylose by
Thermoanaerobacter finnii [21], 0.77 mol-H2molÀ1 xylose by Clostridium tyrobutyricum ATCC 25755 [22], and 0.73 mol-H2molÀ1 xylose
by Clostridium butyricum CGS5 [16]) and mixed culture fermentation
(1.36 mol-H2molÀ1 xylose [23], 1.84 mol-H2molÀ1 xylose [24]). The
successful investigation in this study may be a potential culture
technique for hydrogen production systems from T. neapolitana.
4. Conclusions
H2 production obtained from xylose fermentation of
T. neapolitana in the batch culture. During the cultivations at different

initial xylose concentrations, the optimal xylose concentration for
T. neapolitana growth and H2 production were defined in the medium
at an initial xylose concentration of 5.0 g/L. We successfully conducted biohydrogen fermentation by T. neapolitana from xylose in

a continuously stirred anaerobic bioreactor system. The results
indicated that the H2 yield and H2 production rate in pH-controlled
batch culture was much higher than those from pH-uncontrolled
batch culture. The optimal condition of pH 7.0 obtained from the
3 L reactor by considering H2 yield. The maximum H2 yield of
2.8 Æ 0.1 mol-H2molÀ1 xylose was achieved in pH-controlled batch
culture at a constant pH of 7.0. The successful investigation in this
study may be a potential culture technique for hydrogen production
systems using T. neapolitana in converting H2 from pentose (xylose).

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