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 6 ( 2 0 1 1 ) 1 5 1 8 7 e1 5 1 9 4

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Fermentative hydrogen production from different sugars

by Citrobacter sp. CMC-1 in batch culture

Rahul Mangayil*, Ville Santala, Matti Karp

Department of Chemistry and Bioengineering, Tampere University of Technology, P.O. Box 541, 33101 Tampere, Finland

article info abstract

Article history: In this study, production of hydrogen (H2) from glucose, xylose, galactose, mannose,
Received 1 July 2011 arabinose and rhamnose by a strain isolated from activated sludge was investigated. The
Received in revised form strain, named as Citrobacter sp. CMC-1, was enriched in cellobiose amended minimal
15 August 2011 media. Based on 16S rRNA sequence, the CMC-1 strain is a close relative of Citrobacter
Accepted 23 August 2011 amalonaticus strain SA01 (99%). Optimal cultivation parameters for H2 production and
Available online 13 September 2011 growth such as pH and temperature were investigated. H2 yields from glucose at optimal
conditions (pH 6.0 and 34
C) were 1.82  0.02 mol-H2/mol-glucose. Strain CMC-1 fermented
Keywords: galactose, mannose, xylose, arabinose and rhamnose. After 48 h incubation, the strain
Citrobacter sp. CMC-1 completely fermented all sugars tested, except arabinose. Increase in fermentation
Biohydrogen period lowered residual formate level in the media and improved H2 production for
Xylose galactose, mannose and xylose (1.68  0.24, 1.93  0.14 and 1.63  0.07 mol-H2/mol-
Cellobiose substrate respectively).
Carboxymethyl cellulose Copyright 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights
reserved.

1. Introduction efficient fermentation when xylose was used as substrate

Microbe-assisted conversion of lignocellulosic waste material
to energy is considered as a promising alternative for renew-
able energy [1]. Lignocellulosic biomass comprises of cellu-
lose, hemicellulose and lignin. Cellulose is a macromolecule
composed of glucose units, whereas hemicellulose contains
xylose, glucose, arabinose, mannose, galactose and rhamnose
[2]. Several strict and facultative anaerobic microbes are able
to naturally produce energy molecules such as ethanol and H2
[3]. Lately, investigations on effective monomeric hemi-
cellulosic sugar utilization and end product metabolite
production have been reported for microbes such as Klebsiella
pneumonia and Enterobacter sp [4,2,5]. Niu et al. (2010) investi-
gated substrate utilization and H2 production by K. pneumonia
ECU-15 strain in glucose and corn stalk hydrolysate [6].
Enterobacter aerogenes HGN-2 and HT 34 isolates have reported
yielding 1.98 mol-H2/mol-xylose [4]. Jayasinghearachchi et al.
(2010) isolated Clostridium amygdalinum strain C9 and reported
H2 production from xylose and arabinose at slightly alkaline
pH and mesophilic conditions [7]. Over the years, several H2
producing Citrobacter strains have been isolated. Oh et al.
(2003) isolated Citrobacter sp. Y19 from sludge digester and
reported a H2 production efficiency of 2.49 mol-H2/mol-
glucose [8]. Citrobacter intermedius isolated from sewage sludge
was tested for H2 production under anaerobic batch condi-
tions and observed a yield of 1.1 mol-H2/mol-glucose [9].
Biological H2 fermentation process efficiency is greatly
affected by environmental factors such as medium pH, cultiva-
tion temperature, substrate, etc. In batch cultivations, initial
medium pH is an important factor as medium pH affects cellular
enzyme activities, lag phase and end-metabolite production.
Cultivation temperature also greatly affects specific growth,

* Corresponding author. Tel.: 358 3 3115 2052; fax: 358 3 3115 2869.
E-mail address: rahul.mangayil@tut.fi (R. Mangayil).
0360-3199/$ e see front matter Copyright 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijhydene.2011.08.076
15188 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 6 ( 2 0 1 1 ) 1 5 1 8 7 e1 5 1 9 4
microbial assisted hydrogen production, metabolite distribu-
tion. Studies on fermentative H2 production have been con-
ducted at ambient [10], mesophilic [2,4,5,8] and thermophilic
range [11,12]. Thus it is important to elucidate the initial pH and
cultivation temperature for optimal H2 production.
The aim of this study was to isolate cellobiose utilizing effi-
cient H2 producer from activated sludge. Although several
physio-chemical parameters influence H2 production, it has
been reported that pH and temperature are the most important
factors that affect H2 production efficiency [13]. In this study,
optimal initial pH and temperature was investigated for
maximal H2 and end-metabolite production. Further, cumula-
tive H2 volume and H2 production rate at optimal culture
conditions and glucose as the sole carbon source were studied.
The bacterium was tested for substrate utilization and end-point
H2, volatile fatty acids and alcohol production from galactose,
mannose, xylose, arabinose and rhamnose. Finally, effect of
increase in fermentation period on end-metabolite production
was investigated for the above mentioned monomeric sugars.
2. Materials and methods
2.1. Isolation of the strain CMC-1
The enrichment system was designed as described by
Ref. [14]. Eight 25 ml serum bottles were filled with 8 ml of M9
medium [15] containing 0.5 g/L cysteine-hydrochloride and
a water-lock connected to each other with gas tight tubing.
Cellobiose was used as the sole carbon source. Cellobiose
concentrations were increased stepwise in seven bottles with
an initial and final concentration of 1 and 60 g/L respectively.
The bottles were purged with oxygen free nitrogen for 15 min,
capped with septum rubber stoppers (20 mm), sealed with
aluminum crimps and autoclaved at 121
C for 15 min. After
autoclaving, the serum bottles were placed in the flask clamps
fixed on steel stage. The bottles were connected using tubing
(13 cm, Masterflex Norprene tubing (A60 G)) with needles
fixed at either ends. One end contained 1.2 mm  50 mm
needle and the other end 0.6 mm  25 mm needle. A water
lock was placed at the end of the system to allow the gas to get
through and maintain anoxic environment in the system.
Activated sludge collected from waste water treatment
plant (Viinikanlahti, Finland) was used as inoculum. The
sludge was cultivated in 120 ml serum bottles with working
volume of 50 ml sterile anoxic glucose amended LB medium at
37
C for 4 days. By the onset of H2 production, 5 ml of culture
(10%) were inoculated to the first bottle of the enrichment
system and incubated at 37
C until the inoculum was trans-
ferred to the last bottle. After the run, culture samples from
the last bottle were spread on M9 minimal agar plates con-
taining 3 g/L carboxymethyl cellulose (CMC). CMC was used as
the sole substrate in order to ensure growth of microbes
capable of utilizing complex substrates. The plates were then
incubated in anaerobic jars at 37
C.
2.2. Experimental procedure
Batch experiments were performed in 120 ml serum bottles
with a working volume of 50 ml. The substrate concentration
was kept at 5 mM and 500 ml of pre-culture was used as an
inoculum. Glucose was used as the sole carbon source for all
experiments unless otherwise stated. Batch experiments to
investigate the effect of initial pH (4e7) on H2 production and
cell growth were performed at 30
C and 150 rpm. Growth and
H2 production at different temperatures (25
Ce40
C) were
performed using a temperature gradient incubator (Test Tube
Oscillator, Terra-tec, Australia) at optimal pH and 100 oscilla-
tion/min. Twenty-five milliliter tubes with 10 ml culture
medium, supplemented with glucose (5 mM) was inoculated
with 100 ml pre-culture and incubated in temperature gradient
at the optimal initial pH (observed from pH experiment). The
incubation time for optimal pH and temperature was 24 h. H2
production at optimal pH and temperature was conducted in
duplicate cultivations.
Carbon material balance was calculated by multiplying the
yield obtained for each carbon containing metabolite by the
respective number of carbons present in the molecular
formula. The sum of total carbon mass obtained for individual
metabolite was then divided by the number of carbon atoms
in 1 mol of substrate used. The cellular composition of CMC-1
isolate was assumed to be CH1.67 O0.20 N0.27 [16]. The CO2 in the
liquid phase was ignored during the carbon balance
calculations.
Cell growth and cumulative H2 production kinetic studies
on glucose were performed at optimal pH and temperature.
Specific growth rate (h1) was calculated by substituting the
data points at exponential curve in equation m ln OD600/t.
Cumulative H2 production was calculated as described by Lee
and Rittmann (2009) [17]. The H2 yield values were calculated
by converting the amount of end-point H2 gas produced (ml) to
moles (mol) using the ideal gas equation for respective culti-
vation temperatures, dividing by the product of initial milli
molar glucose concentration (mM) and the culture media
volume (ml). The fermentation time for kinetic study was 24 h
and was conducted in duplicate cultivations.
The isolate was tested for substrate utilization efficiency
and end product metabolite production when grown in
galactose, mannose, xylose and arabinose for 24 and 48 h.
Batch fermentations with monomeric hemicellulose sugars
were performed in triplicate cultivations.
2.3. 16S rRNA gene sequencing
Genomic DNA was isolated using E.Z.N.A Bacterial DNA Kit
(Omega bio-tek, USA). Amplification of 16S rRNA gene was
done using 50 -CCT ACG GGA GGC AGC AG-30 and 50 -CCG TCA
ATT CMT TTG AGT TT-30 primers. The amplified product was
sequenced (Macrogen, Korea). The sequence was compared
with existing related sequences available in GenBank using
BLAST program.
2.4. Analytical techniques
Biomass for each batch fermentation experiments was
determined by measuring the optical absorbance at 600 nm
using a spectrophotometer. The value of the optical density
was converted to dry cell weight (DCW) using a calibration
equation (DCW 0.294  OD600 0.0717).
 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 6 ( 2 0 1 1 ) 1 5 1 8 7 e1 5 1 9 4 15189

Fig. 1 e (A) Effect of initial pH on cell growth, H2 and ethanol yields (B) End product metabolite produced over the pH range
tested. Error bars indicate standard deviation from duplicate cultivations. In some cases, the error bars are smaller than the
symbol.

Carbon substrates, organic acids and alcohols were
analyzed using High-Performance Liquid Chromatography
(HPLC) (LC-20AD, Shimadzu, Japan) equipped with Shodex
SUGAR (SH1011) column (300  8 mm) and refractive index
detector (RID, RID-10A). Prior to analyses, the culture samples
were centrifuged at 7600g for 10 min and the supernatant was
filtered through 0.45 mm disposable filters. A solution of 0.01N
H2SO4 was used as the mobile phase at 0.9 ml/min and the
column temperature was kept at room temperature. Sample
injection volume was 100 ml.
Gas chromatograph (GC-2014, Shimadzu GC), equipped
with thermal conductivity detector (TCD) and PORAPAK
column (2 m length and an inner diameter of 2 mm) were used
to determine the gaseous content. N2 (Instrument N2 5.0) was
used as carrier gas at the flow rate of 20 ml/min. The operating
temperatures of the column, detector and oven were 80
C,
110
C and 80
C respectively.
3. Results and discussions
small rod shaped gram-negative bacteria. The strain is
referred here as Citrobacter sp. CMC-1. Specific growth rates of
CMC-1 isolate grown in M9 media supplemented with
different carbon sources were: glucose 0.32 OD600 h1, cello-
biose 0.13 OD600 h1 and CMC 0.08 OD600 h1. Mean generation
time of CMC-1 strain on glucose, cellobiose and CMC were
calculated to be 2.1, 5.5 and 8.3 h respectively. Preliminary
investigations on H2 production from cellobiose and CMC
revealed that CMC-1 strain produced 1.4  0.2 mol-H2/mol-
cellobiose and 0.02 mmol-H2/g-CMC with mixed acid
fermentation profile.
Nucleotide BLAST searches against GeneBank database
revealed that CMC-1 isolate belongs to the family Enter-
obacteriaceae. Within the Enterobacteriaceae family, the query
sequence showed significant alignments with Citrobacter,
Enterobacter, Klebsiella and Salmonella spp. The strain closely
resembled to Citrobacter amalonaticus strain SA01 with 99%
query coverage. The 16S rRNA gene sequence of CMC-1
isolate was deposited in GenBank with accession number
GU324417.

3.1. General characteristics and identification of isolated

strain CMC-1
Table 1 e H2 content in biogas (%), biomass (g/L) and final
Sample from activated sludge was enriched in cellobiose pH (mean standard deviation) of Citrobacter sp. CMC-1
amended minimal medium and plated on agar plates con- as a function of the initial pH.
taining CMC. Single colonies that appeared on CMC agar Initial pH Final pH Biomass (g/L) H2 content (%)
plates were eventually sub cultured for five rounds and grown
4.0 3.64  0.01 0.09  0.03 1.84  0.03
in anaerobic jars at 37
C. The isolate appeared to be yellow
5.0 4.13  0.02 0.15  0.02 14.63  0.00
colored and round shaped colonies with irregular edges in M9
6.0 5.61  0.01 0.16  0.04 15.84  0.01
agar supplemented with CMC. From microscopic analysis and 7.0 6.60  0.01 0.13  0.02 8.70  0.04
gram staining the isolated bacterial strain was found to be
15190 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 6 ( 2 0 1 1 ) 1 5 1 8 7 e1 5 1 9 4

Fig. 2 e (A) Effect of temperature on cell growth, H2 and ethanol yields (B) End product metabolite produced over the pH range
tested. Error bars indicate standard deviation from duplicate cultivations. In some cases, the error bars are smaller than the
symbol. The error bars at 38
C, 39
C and 40
C are for H2 yield.

3.2. Effect of initial pH on H2 production
The optimal initial pH of the culture medium that yielded the
maximum H2 production on glucose fermentation by CMC-1
strain was investigated. CMC-1 isolate was observed to
produce H2 at wide pH ranges as reported for several Cit-
robacter sp. [18,8]. Though H2 was produced, a drastic decrease
in production yield was observed at pH values below 5.0. H2
production is favorable at slightly acidic pH but fermentation
at pH values below 5.0, decreased H2 production efficiency
also by others [19]. The optimal pH value of pH 6.0 identified
from this study is in line with the work of Oh et al. (2003) for
Citrobacter sp. Y19 and Hamilton et al. (2010) for Citrobacter
freundii CWBI952 [8,20]. At optimal pH, a biomass amount of
0.16  0.04 g/L was produced with H2 yield of 1.75  0.08 mol-
H2/mol-glucose. An increase in initial pH value (pH 7.0)

Table 2 e Carbon mass balance and carbon recovery for anaerobic metabolism of 1 mol of glucose by CMC-1 isolate at pH
6.0 and 34
C.a
Number of Carbon mass Carbon mass Metabolite
carbon atoms (per mol-carbon) recovery (%) recovery (%)

Reactant
Glucose 6 99.7
Products
Acetate 2 1.26  0.01 21.06
Ethanol 2 2.66  0.3 44.53
Lactate 3 0.36  0.45 6
Formate 1 1.45  0.9 24.28
CO2b 1 0.25  0.11 4.12
H2 0 1.8  0.34 e
Biomassc 1 0.38  0.00 0.01
Total products 5.98 100
Total error % 2.0

a Calculated for the serum-bottle culture with the working volume of 50 ml, corresponding to 49 ml M9 media, 500 ml (5 mM) glucose and 500 ml
(1%) inoculum. The glucose supplied was completely consumed. Each value was measured after 24 h cultivation and was an average of
duplicate cultivations.
b CO2in the liquid phase was ignored.
c Biomass yield calculated as mmol/mmol-substrate.
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resulted in 0.93  0.03 mol-H2/mol-glucose (Fig. 1A). At
optimal pH, the biogas H2 content reached a value of
15.84%  0.01 (Table 1). Seol et al. (2008) observed a similar
decrease in H2 production efficiency at pH 7.0. At alkaline pH,
the H2 production of Enterobacteriaceae is affected by the
cellular formate regulation mechanism and subsequent
breakdown of formate to H2 and CO2 [21]. Cell growth and
glucose utilization were limited at pH 4.0. Glucose was
completely utilized at pH 5.0. The cell density increased when
raising the pH up to 6.0 and decreased at higher pH (Fig. 1A).
Low biomass yields (0.09e0.16 g/L) at all pH values could be
related to the absence of high substrate concentration, yeast
extract and vitamins from the cultivation medium [16].
At optimal pH, acetate (10.92  0.30 mM), ethanol
(7.61  0.10 mM), lactate (2.98  0.35 mM) and formate
(1.85  0.07 mM) were the main metabolites produced (Fig. 1B).
Lactate and ethanol were the sole metabolites produced at pH
4.0. An increase in pH from 4.0 resulted in a rise in acetate
concentration (0e10.92  0.30 mM). Formate accumulation
was not observed at pH 4.0 and 5.0 but was evident in culture
media from pH 6.0. This can be due to the formate transport
mechanism extracellular to the cultivation medium at alka-
line pH as explained by Seol et al. (2008) [21].
3.3. Effect of temperature on H2 production
The effect of temperature on H2 production was investigated
within the range of 25e40
C, with a stepwise increase of 0.5
C
between the cultivations. The initial pH was adjusted to be 6.0.
The H2 yield increased from 1.42  0.25 to 1.82  0.02 mol-H2/
mol-glucose as temperature increased from 25
C to 34
C.
Further increase in temperature lowered the H2 yields

Fig. 3 e Kinetic study on (A) cell growth and cumulative H2 production (B) and H2 production rate under glucose (5 mM)
fermentation conducted at 34
C and pH 6.0.
15192 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 6 ( 2 0 1 1 ) 1 5 1 8 7 e1 5 1 9 4

(Fig. 2A). This implies that the optimal temperature for H2
production is 34
C, in which H2 content of headspace was
16.83%  0.04. The lowest H2 yield of 0.61  0.03 mol-H2/mol-
glucose was observed at 40
C. The decrease in H2 production
can be explained by cell growth, because cell growth followed
a similar trend as observed for H2 yield with the investigated
temperature range (Fig. 2A).
The H2 evolution was accompanied by the production of
volatile fatty acids (VFAs) and ethanol as shown in Fig. 2B.
Acetate, ethanol, lactate and formate were observed to be the
major fermentation products. Ethanol concentrations
remained unchanged at the tested temperature range and H2
production occurred along with acetate fermentation. Accu-
mulated formate concentration was around 0.73e1.42 mM
and increased formate level was observed at cultivation
temperatures above 37
C (3.0e4.24 mM). Fermentation at
temperatures above 37
C resulted in increased lactate
production (4.93e6.09 mM), while acetate and ethanol levels
decreased. The sharp reduction in H2 yield observed after
37
C suggests that acetate production is accompanied with H2
production.
3.4. Carbon material balance in batch fermentation at
optimal culture conditions
Table 2 summarizes the results of carbon mass balance
calculations. Fermentation was performed in batch cultiva-
tions with a working volume of 50 ml corresponding to 49 ml
M9 media, 500 ml (5 mM) glucose and 500 ml (1%) inoculum. As
shown in Table 1, the carbon mass recovery was 99.7  2.0%.
The carbons from glucose were directed toward acetate,
ethanol, formate, lactate, CO2 and biomass.
3.5. Growth and H2 production kinetics in glucose
The kinetics of H2 production from glucose was studied for
24 h at 34
C and pH 6.0. Cumulative H2 production initiated
with a small lag phase. CMC-1 yielded the highest cumulative
H2 volume by the 12th hour (20.1 ml), after which the H2
content was observed to decrease (Fig. 3A). The strain CMC-1
yielded the highest H2 production rate (mmol-H2/h) by the
4th hour (0.2 mmol-H2/h, Fig. 3B). Kim et al. (2008) have re-
ported the presence of H2 evolving FHL complex at
exponential growth phase and membrane bound uptake
hydrogenase at stationary phase in C. amalonaticus Y19. The
decrease in cumulative H2 production can be due to the acti-
vation of uptake hydrogenases in CMC-1 by the onset of the
stationary phase [22]. The cell growth also followed the same
progress of cumulative H2 production. The exponential
growth phase for CMC-1 strain was from 2nd to 8th hour,
producing maximal biomass of 0.16 g/L. The specific growth
rate for CMC-1 strain on glucose was calculated to be 0.30 h1
with a doubling time of 2.9 h.
Acetate, lactate and ethanol were produced by the onset of
biomass production. Formate accumulation was observed in
the media at 6th hour but was absent in later time-point
samples, indicating formate breakdown during subsequent
fermentation. At the end of fermentation time, the concen-
tration of lactate and ethanol was 1.68  0.02 and
7.11  0.02 mM respectively.
3.6. H2 production from xylose
Xylose is the major pentose sugar residue of hemicelluloses.
Bioenergy production from xylose fermentation by facultative
anaerobic microbes has not been thoroughly studied [2].
Anand and Sripathi (2004) have isolated xylose utilizing
microbes from the gut of Pteropus giganteus, comprising
predominantly of C. freundii, Proteus vulgaris, Proteus mirabilis,
Serratia liquefaciens and Klebsiella oxytoca. Their finding indi-
cates the importance of facultative anaerobes in degrading
complex substrates to produce valuable metabolites [23].
Batch experiment was conducted at 34
C and pH 6.0 for 24
and 48 h. Xylose fermentation from 24 h cultivation yielded
1.22  0.1 mol-H2/mol-xylose (Table 3). CMC-1 strain yielded
0.24  0.01 mmol-biomass/mmol-xylose. When the fermen-
tation time was increased to 48 h, the H2 and biomass yield
increased to 1.63  0.07 mol-H2/mol-xylose and
0.27  0.02 mmol-biomass/mmol-xylose. H2 production
through formate has been reported for C. amalonaticus Y19,
mediated by FHL complex and hydrogenase. The increase in
H2 yield within the longer fermentation time is due to the
utilization of produced formate [22].
Analyzing the VFAs and ethanol produced, xylose
fermentation predominantly resulted in acetate, ethanol,
formate and lactate (Table 4). Formate accumulation of

Table 3 e H2 yield (mol/mol-substrate) and biomass yield (mmol-biomass/mmol-substrate) data from 24 to 48 h

fermentation (mean standard deviation) from galactose, mannose, xylose, arabinose and rhamnose by strain CMC-1 at
34
C and pH 6.0.a
Substrate 24 h fermentation 48 h fermentation

H2 yield (mol/ Biomass yield (mmol- H2 content H2 yield (mol/ Biomass yield (mmol- H2 content
mol-substrate) biomass/mmol-substrate) (%) mol-substrate) biomass/mmol-substrate) (%)

Galactose 1.18  0.04 0.25  0.01 10.72  0.00 1.68  0.24 0.32  0.02 14.31  0.01
Mannose 1.23  0.23 0.24  0.03 11.05  0.02 1.93  0.14 0.36  0.02 15.59  0.01
Xylose 1.22  0.10 0.24  0.01 10.53  0.01 1.63  0.07 0.27  0.02 13.76  0.01
Arabinose 0.94  0.07 0.20  0.01 9.59  0.01 0.79  0.03 0.22  0.03 7.89  0.03
Rhamnose 1.01  0.08 0.22  0.02 9.89  0.01 NAb NAb NAb

a H2 yield and H2 content data are from triplicate cultivations. Mean average value is reported with standard deviation from triplicate
cultivations.
b Not analyzed.
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Table 4 e Soluble metabolite profile from major sugar (mean standard deviation) residues and carbon material balance (%)
from 24 to 48 h fermentation by CMC-1 at 34
C and pH 6.0.a
Substrate Lactate Acetate Formate Ethanol Propionate 1-Propanol Carbon mass recoveryb,c (%)

Metabolites produced after 24 h fermentation (mM)

Galactose 0.69  0.12 3.72  0.05 7.36  0.18 4.07  0.09 NDc NDc 102  0.17
Mannose 0.35  0.06 3.89  0.12 8.09  0.22 3.9  0.11 NDc NDc 103  0.42
Xylose 1.30  0.05 2.88  0.17 5.79  0.28 2.15  0.13 NDc NDc 103  0.30
Arabinose 1.22  0.09 2.98  0.01 6.78  0.16 2.23  0.44 NDc NDc 97  0.28
Rhamnose 0.20  0.03 3.58  0.08 5.09  0.90 0.29  0.12 2.26  0.03 2.95  0.15 102  0.40
Metabolites produced after 48 h fermentation (mM)
Galactose 1.12  0.18 3.87  0.24 4.56  0.02 4.21  0.16 NDc NDc 101  0.67
Mannose 0.63  0.45 4.30  0.13 5.45  0.15 3.00  0.22 NDc NDc 97  0.39
Xylose 1.1  0.04 3.28  0.13 2.86  0.13 2.9  0.04 NDc NDc 102  0.34
Arabinose 1.95  0.10 2.98  0.10 7.91  0.28 2.43  0.09 NDc NDc 111  0.01

a Carbon balance calculated as total products e carbon from triplicate cultivations. Mean average value is reported with standard deviation
from triplicate cultivations.
b CO2 in the liquid phase was ignored.
c Not detected.

5.79  0.28 mM was observed after 24 h cultivation. An
increase in incubation time resulted in the decrease of
formate concentration (5.79  0.28e2.86  0.13 mM) in the
culture media. Increase in acetate and ethanol concentrations
indicate more complete utilization of the sugar tested with
prolonged fermentation time and mixed acid fermentation
route of CMC-1 isolate. The decrease in formate concentration
can be correlated with the increase in H2 production.
3.7. H2 production by strain CMC-1 from galactose,
mannose, arabinose and rhamnose
Galactose, mannose, arabinose and rhamnose fermentation
studies were conducted to explore if CMC-1 isolate could produce
H2 directly from monomeric hemicellulose sugars. Batch exper-
iment was conducted at 34
C and pH 6.0 for 24 and 48 h.
CMC-1 strain produced H2 from all the above listed sugars
and yielded 1.18  0.04 mol-H2/mol-galactose, 1.23  0.23 mol-
H2/mol-mannose, 0.94  0.07 mol-H2/mol-arabinose and
1.01  0.08 mol-H2/mol-rhamnose (Table 3). Rhamnose
fermentation was not investigated for 48 h. Prolonged culti-
vation time improved H2 yields from galactose
(1.68  0.24 mol) and mannose (1.93  0.14 mol), when CMC-1
was able to completely utilize carbon sources. As explained
above, this increase in H2 yield was due to residual formate
breakdown. For arabinose fermentation, the H2 production
trend was in the opposite fashion as that of other sugars. An
increase in fermentation time decreased the H2 yield from
0.94  0.07 to 0.79  0.03 mol-H2/mol-arabinose. This can be
due to the energy intensive arabinose utilization route,
involving complex enzymatic reactions before entering the
pentose phosphate pathway [24,25].
At the end of the 24 h mannose fermentation, formate
(8.09  0.22 mM), ethanol (3.9  0.11 mM), acetate
(3.89  0.12 mM) and lactate (0.35  0.06 mM) were the main
metabolites produced with a biomass yield of
0.24  0.03 mmol-biomass/mmol-mannose. Approximately
103  0.42% of carbon was recovered from measured metab-
olites together with the biomass. After 48 h of fermentation,
a rise in acetate concentration (4.30  0.13 mM) was followed
by a decrease in ethanol (4.4  0.0 mM) and formate
(5.45  0.15 mM) accumulation compared to the values ob-
tained after 24 h cultivation. Lactate concentration remained
low even after prolonged fermentation (0.63  0.45 mM). Low
lactate production was due to low carbon source and thereby
low intracellular pyruvate concentrations [12]. After 48 h
cultivation, CMC-1 strain yielded 0.36  0.02 mmol-biomass/
mmol-mannose with a carbon recovery of 97  0.39%.
Galactose fermentation accumulated formate
(7.36  0.18 mM), ethanol (4.07  0.09 mM), acetate
(3.72  0.05 mM) and lactate (0.69  0.12 mM) as the main
metabolites and yielded 0.25  0.01 mmol-biomass/mmol-
galactose. As in the case with mannose fermentation, pro-
longed incubation time with galactose resulted in improved
acetate production (3.87  0.24 mM) and biomass yield
(0.32  0.02 mmol-biomass/mmol-galactose), with a decrease
in formate (4.56  0.02 mM) concentration. Galactose was
completely utilized at the end of fermentation and carbon
mass recovery was calculated to be 101  0.67%.
The major VFAs produced from arabinose breakdown were
acetate, ethanol, lactate and formate. An approximate
amount of 99  0.26% carbon was recovered from the 24 h
arabinose fermentation. Acetate was the main metabolite and
the concentration increased from 2.98  0.01 to 3.28  0.13 mM
when the incubation time was increased (Table 4). Formate
accumulation was observed to increase in the culture medium
(from 6.78  0.16 to 7.91  0.28) and H2 production from
extracellular formate was not observed. H2 and metabolite
production from arabinose followed an opposite trend when
compared with the other hexose and/or pentose sugars tested.
Though ethanol was the only alcohol produced during the
xylose, arabinose, galactose and mannose fermentation,
a slightly different VFA profile was detected for rhamnose
fermentation (Table 4). From rhamnose fermentation, CMC-1
strain produced small amounts of lactate (0.2  0.03 mM).
However, acetate and formate concentrations were 3.58  0.08
and 5.09  0.90 mM respectively. In addition to ethanol
(0.29  0.12 mM), propionate (2.26  0.03 mM) and 1-propanol
(2.95  0.15) were observed in the culture media (Table 4). At
the end of fermentation, CMC-1 yielded 0.51  0.02 mol-1-
propanol/mol-rhamnose and 0.22  0.02 mmol-biomass/
mmol-rhamnose.
15194 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 6 ( 2 0 1 1 ) 1 5 1 8 7 e1 5 1 9 4
4. Conclusions
In this study, the isolation of a mesophilic H2 producing strain
from activated sludge by using minimal media supplemented
with cellobiose is described. The strain was identified as Cit-
robacter sp. based on 16S rRNA gene sequence analysis. The
doubling time of CMC-1 strain on cellobiose and CMC were
calculated to be 5.5 and 8.3 h respectively, registering a H2
production with yield of 1.4  0.2 mol-H2/mol-cellobiose. The
bacterium produced H2 at wide pH and temperature ranges.
The maximal H2 yield from glucose at pH 6.0 and 34
C was
1.82  0.02 mol-H2/mol-glucose, with a specific growth rate of
0.30 h1 and the highest H2 production rate of 0.2 mmol-H2/h.
The strain can utilize monomeric hemicellulose sugars effi-
ciently to produce H2 and value-added alcohols.
Acknowledgments
The research was funded by the Maj and Tor Nessling Foun-
dation (project no: 620110), The Academy of Finland (A ar-
imikro, project no. 126974).
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