Hydrogen production and microbial kinetics of Clostridium termitidis in mono-culture and co-culture with Clostridium beijerinckii on cellulose
Gomez‑Flores et al. AMB Expr
Hydrogen production and microbial kinetics of Clostridium termitidis in mono‑culture and co‑culture with Clostridium beijerinckii on cellulose
Maritza Gomez‑Flores 0 2
George Nakhla 0 1 2
Hisham Hafez 1
0 Department of Chemical and Biochemical Engineering, Faculty of Engineering, University of Western Ontario , London, ON N6A 5B9 , Canada
1 Department of Civil and Environmental Engineering, Faculty of Engineering, University of Western Ontario , London, ON N6A 5B9 , Canada
2 Department of Chemical and Biochemical Engineering, Faculty of Engi‐ neering, University of Western Ontario , London, ON N6A 5B9 , Canada
Cellulose utilization by hydrogen producers remains an issue due to the low hydrogen yields reported and the pretreatment of cellulose prior to fermentation requires complex and expensive steps. Clostridium termitidis is able to breakdown cellulose into glucose and produce hydrogen. On the other hand, Clostridium beijerinckii is not able to degrade cellulose but is adept at hydrogen production from glucose; therefore, it was chosen to potentially enhance hydrogen production when co‑ cultured with C. termitidis on cellulose. In this study, batch fermentation tests were conducted to investigate the direct hydrogen production enhancement of mesophilic cellulolytic bacteria C. termitidis co‑ cultured with mesophilic hydrogen producer C. beijerinckii on cellulose at 2 g l−1 compared to C. termitidis monoculture. Microbial kinetics parameters were determined by modeling in MATLAB. The achieved highest hydrogen yield was 1.92 mol hydrogen mol−1 hexose equivalentadded in the co‑ culture compared to 1.45 mol hydrogen mol−1 hexose equivalentadded in the mono‑ culture. The maximum hydrogen production rate of 26 ml d−1 was achieved in the co‑ culture. Co‑ culture exhibited an overall 32 % enhancement of hydrogen yield based on hexose equivalent added and 15 % more substrate utilization. The main metabolites were acetate, ethanol, lactate, and formate in the mono‑ culture, with also butyrate in the co‑ culture. Additionally, the hydrogen yield of C. beijerinckii only in glucose was 2.54 mol hydrogen mol−1 hexose equivalent. This study has proved the viability of co‑ culture of C. termitidis with C. beijerinckii for hydrogen production directly from a complex substrate like cellulose under mesophilic conditions.
Clostridium termitidis; Clostridium beijerinckii; Co‑ culture; Hydrogen production; Cellulose; Microbial kinetics
Hydrogen (H2) is considered a clean and renewable
energy resource that does not contribute to the
greenhouse effect (Lee et al. 2014). The main source of H2
production from fermentation is carbohydrates, among
which, cellulose is widely available in agricultural wastes
and industrial effluents such as pulp/paper and food
industries (Lee et al. 2014). In comparison to the use of
natural mixed consortia, pure cultures have achieved
higher H2 yields (Masset et al. 2012). Artificial microbial
co-cultures and consortia can perform complex
functions (Masset et al. 2012), such as, simultaneous hexose
and pentose consumption (Eiteman et al. 2008),
maintaining anaerobic conditions for obligate H2 producers,
improving the hydrolysis of complex sugars, allowing
fermentation over a wider pH range (Elsharnouby et al.
2013), and could be more robust to changes in
environmental conditions (Brenner et al. 2008). Although,
thermophiles have shown higher H2 production yields than
mesophiles in the literature (Kumar and Das 2000; Lu
et al. 2007; Munro et al. 2009; Ngo et al. 2012),
mesophilic H2 production is more economical and reliable
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than thermophilic and hyperthermophilic production.
Four co-culture experiments for biohydrogen
production from pure cellulose, two at mesophilic and two at
thermophilic conditions (Geng et al. 2010; Liu et al.
2008; Wang et al. 2008, 2009) have been reported. All of
these studies have shown enhancement of H2 production
compared to mono-cultures, with the highest H2 yield
of 1.8 mol hydrogen mol−1 hexose achieved by the
coculture of Clostridium thermocellum JN4 and
Thermoanaerobacterium thermosaccharolyticum GD17 at 60 °C
(Liu et al. 2008), potentially due to synergism between
the two cultures.
Clostridium termitidis ATCC 51846 is an
anaerobic, mesophilic, cellulolytic bacterium isolated from the
gut of a termite (Hethener et al. 1992), with reported
H2 yields of 1.99 mol hydrogen mol−1 hexose from
glucose, 1.11 mol hydrogen mol−1 hexose
equivalent from cellobiose (Gomez-Flores et al. 2015), and
0.62 mol hydrogen mol−1 hexose equivalent from
cellulose (Ramachandran et al. 2008). On the other hand, C.
beijerinckii is a mesophilic H2 producer which is not able
to degrade cellulose but is adept at H2 production from
glucose (Masset et al. 2012). Clostridium beijerinckii H2
yields from glucose have been reported to be 1.9 and
2.8 mol hydrogen mol−1 hexoseadded or consumed (Lin et al.
2007; Masset et al. 2012), 2.5 mol hydrogen mol−1
hexoseconsumed (Pan et al. 2008), and 2 mol hydrogen mol−1
hexoseadded (Taguchi et al. 1992). These experiments
differ from each other in the reactor size, medium and
initial glucose concentration.
Additionally, reasonably accurate mathematical
models able to predict biochemical phenomena as well as the
determination of its parameters are essential since they
provide the basis for design, control, optimization and
scale-up of process systems (Huang and Wang 2010).
Therefore, this study has two goals (1) evaluate the effect
of co-culture of C. termitidis and C. beijerinckii on
biohydrogen production and, (2) determine the microbial
kinetics of C. termitidis in mono-culture and co-cultured
with C. beijerinckii on cellulose.
Materials and methods
Microbial strain and media
The strains used were C. termitidis ATCC 51846
(American Type Culture Collection) and C. beijerinckii DSM
1820 (Deutsche Sammlung von Mikroorganismen und
Zellkulturen). All chemicals for media and substrates were
obtained from Sigma-Aldrich Canada Co. (Oakville, ON,
Canada). Fresh cells of C. termitidis were maintained by
successively transferring 10 % (v/v) of inoculum to ATCC
1191 medium containing 2 g l−1 of cellulose, whereas fresh
cells of C. beijerinckii were maintained by successively
transferring 10 % (v/v) of inoculum to ATCC 1191 medium
containing 2 g l−1 of cellobiose. The ATCC 1191 medium
was prepared according to Gomez-Flores et al. (2015).
Batch fermentations were performed in media bottles
(Wheaton, NJ, USA) with a working liquid volume of 500
and 210 ml of headspace. For the co-culture experiments,
bottles containing 450 ml of ATCC 1191 medium and 1 g
cellulose were tightly capped with screw caps with butyl
septum, degassed by applying vacuum, sparged with high
purity N2 gas, and autoclaved. Mono-culture bottles were
inoculated with 10 % (v/v) of C. termitidis cultures, while
co-culture bottles were inoculated with 10 % (v/v) of C.
termitidis and C. beijerinckii cultures in a volumetric
ratio of 1:1. All bottles were incubated at 37 °C in
shakers (Max Q4000, Thermo Scientific, CA, USA). Three (3)
ml liquid samples were taken at specific times for pH,
metabolites, cellular protein content and cellulose
analyses. Fermentations ran for 45 and 40 days for the
monoculture and co-culture, respectively. A total of 24 samples
were taken for the mono-culture experiments whereas 21
samples were taken for the co-culture experiments. pH
was initially set to 7.2 but was not controlled. Data shown
are the averages of duplicate experiments. Additionally,
fermentation on glucose 2 g l−1 by C. beijerinckii in the
ATCC 1191 medium was performed in serum bottles
(Wheaton, NJ, USA) with a working volume of 500 and
210 ml of headspace. Duplicate bottles were inoculated
with 10 % (v/v) of fresh cultures. Bottles were incubated
at 37 °C and 100 rpm for 48 h. Also, the initial pH was set
to 7.2 but was not controlled.
Cell growth was monitored by measuring cellular
protein content, samples (1 ml) were placed in
microcentrifuge tubes (VWR®, Polypropylene) and centrifuged
(Corning® LSE™, NY, USA) at 10,000×g for 15 min.
Supernatants were used for soluble product analysis by
transferring to new microcentrifuge tubes. The pellets
were re-suspended with 0.9 % (w/v) NaCl and
centrifuged at the same aforementioned conditions.
Supernatants were discarded, and 1 ml of 0.2 M NaOH was added
to microcentrifuge tubes and vortexed to re-suspend the
pellet. Microcentrifuge tubes were placed in a water bath
at 100 °C for 10 min. After cooling, tubes were
centrifuged and supernatants were collected for Bradford assay
using bovine serum albumin (BSA) as standard, measured
by a UV–visible spectrophotometer (Cary 50 Bio, Varian,
Australia) at 595 nm. The cellulose pellet was quantified
gravimetrically after being dried overnight at 100 °C (Liu
et al. 2008). pH was measured using a B10P SympHony
pH meter (VWR®). Ethanol, glucose, cellobiose, and
lactic, formic, acetic, and butyric acids, were measured
as follows: supernatants for metabolites analysis were
filtered through 0.2 µm and measured using an HPLC
(Dionex, Sunnyvale, CA, USA) consisting of a Dionex
GP50 Gradient pump and a Dionex LC25
Chromatography oven equipped with an Aminex HPX-87H column
(Bio-Rad) at 30 °C and 9 mM H2SO4 at 0.6 ml min−1 as
mobile phase, connected to a Perkin Elmer 200 series
refractive index detector (RID). Standard curves of
metabolites, glucose and cellobiose were performed on
ATCC 1191 medium. Cellular protein content was then
converted to dry weight using the correlation dry weight
(g l−1) = 0.0051 × protein (µg ml−1) (Gomez-Flores et al.
2015). For the estimation of the COD equivalents for the
biomass dry weight, the empirical formula of the organic
fraction of the biomass of C5H7O2N (Metcalf and Eddy
2003), and an organic fraction of 90 % of the cell dry
weight (Pavlostathis et al. 1988), were assumed.
Gas volume was measured by releasing the gas pressure
in the bottles using appropriately sized glass syringes in
the range of 5 to 100 ml to equilibrate with the ambient
pressure (Owen et al. 1979). H2 analysis was conducted
by employing a gas chromatograph (Model 310, SRI
Instruments, Torrance, CA, USA) equipped with a
thermal conductivity detector (TCD) and a molecular sieve
column (Mole sieve 5A, mesh 80/100, 1.83 m × 0.32 cm).
The temperatures of the column and the TCD detector
were 90 and 105 °C, respectively. Argon was used as the
carrier gas at a flow rate of 30 ml/min.
Modified Gompertz model
The following modified Gompertz model (Lay et al. 1999)
was used to describe the H2 production.
H = P exp
− t) + 1
where H is the cumulative H2 production (ml), P is the H2
production potential (ml), Rmax is the maximum H2
production rate (ml d−1) and λ is the lag time (d).
Kinetic equations and modeling
As shown in Fig. 1, there are mainly 2 steps: hydrolysis
of cellulose and fermentation of soluble sugars (glucose).
In both cases, C. termitidis’ putative cellulosome (Munir
et al. 2014) is responsible for the cellulose hydrolysis.
Fermentation of soluble sugars is performed by C. termitidis
in mono-culture, whereas in co-culture both, C.
termitidis and C. beijerinckii ferment the soluble sugars. The
soluble products in mono-culture are acetate, ethanol,
lactate and formate. In the co-culture, the lactate present
in the C. beijerinckii growth media acted as substrate,
and butyrate was an additional soluble product.
where S is cellulose concentration (g COD l−1) and So is
the non-biodegradable cellulose concentration
remaining at the end of the fermentation. Soluble sugars from
cellulose hydrolysis (cellobiose and glucose) were not
detected in any of the fermentations, implying that
cellulose hydrolysis was the rate-limiting step.
Nevertheless, cellulose is an insoluble substrate and Monod model
cannot be used. Therefore, a modified Monod approach,
incorporating particulate organic matter (POM) (Metcalf
and Eddy 2003) was used (Eq. 6).
where µmax (d−1) is the maximum specific growth
rate, Kx is the half-velocity degradation coefficient
(g COD PO g−1 COD biomass), PO is the particulate
organic (cellulose) concentration (g COD l−1) and X is
biomass concentration (g COD l−1) (Metcalf and Eddy
2003). The POM modeling approach considers the
particulate substrate conversion rate as the rate-limiting
process that is dependent on the particulate substrate
and biomass concentrations. The particulate
degradation concentration is expressed relative to the biomass
because the particulate hydrolysis is related to the
relative contact area between the non-soluble organic
material and the biomass (Metcalf and Eddy 2003). All
concentrations were expressed as g COD; for biomass
the factor of 1.42 g COD g−1 biomass based on the
empirical formula of C5H7O2N was used (Metcalf and
Among the various reactions involving glucose, only
acetate and butyrate pathways involve H2 production
according to Eqs. 2 and 3, respectively, while ethanol and
lactate are involved in a zero-H2 balance (Guo et al. 2010)
C6H12O6 + 2H2O → 2CH3COOH + 2CO2 + 4H2
C6H12O6 → CH3CH2CH2COOH + 2CO2 + 2H2. (3)
Lactate utilization is represented by Eq. 4 (Thauer et al.
CH3CHOHCOOH + H2O → CH3COOH + CO2 + 2H2.
Because cellulose was not completely biodegraded, the
use of a non-biodegradable factor So (g COD l−1) was
needed as presented in Eq. 5.
Fig. 1 Schematic representation of the steps involved in cellulose fermentation in a mono‑ culture and b co‑ culture
where P and YP/PO are acetate, ethanol, lactate and
formate concentrations (g COD l−1) and yields
(g COD g−1 COD PO), respectively.
b. Co-culture (C. termitidis and C. beijerinckii). No
distinction in biomass measurement was done for
each strain. Co-culture was modeled as a single
strain with the addition of lactate as substrate and
butyrate as product. Consequently, PO
consumption is described in Eq. 8, biomass growth from
cellulose and lactate is modeled by Eq. 10, and lactate
consumption was considered a first order reaction
where YX/L is the biomass yield from lactate (as
g COD g−1 COD) and KL is the lactate consumption
constant (l g−1 COD biomass d−1). Based on Eq. 4,
The two models are described as follows:
a. Mono-culture (C. termitidis only). Biomass growth
and PO consumption are described in Eqs. 7 and 8,
where YX/PO (g COD biomass g−1 COD PO) is the
biomass yield (Shuler and Kargı 2002). Acetate,
ethanol, lactate and formate production was modeled as
described by Eq. 9.
acetate is also produced from lactate. Thus acetate
kinetics are modeled by Eq. 12.
where YA/L is the acetate yield from lactate
(g COD g−1 COD).
Ethanol, formate and butyrate were described by
Eq. 9, where P and YP/PO are also butyrate concentration
(g COD l−1) and yield (g COD g−1 COD PO).
Microbial kinetics were estimated from the growth
phase only, ignoring the lag phase. Kinetic parameters
were estimated using MATLAB® R2014a. The solver
function used for numerical integration of the ordinary
differential equations i.e. Ode45, implemented fourth/
fifth order Runge–Kutta methods. Initial guesses were
manually adjusted to obtain a good fit to the data, and
average percentage errors (APE) and root mean square
errors (RMSE) were calculated. The complete
nomenclature is shown in Table 1.
C. beijerinckii on glucose experiment
Clostridium beijerinckii degraded glucose in 46 h with an
initial lag phase of 22 h and had a yield of
2.54 mol hydrogen mol−1 glucose (Additional file 1: Figure S1a). pH
dropped from 7.1 to 6.2. With a 28 % higher H2 yield over
C. termitidis for the same substrate (Gomez-Flores et al.
2015) and under the same operating conditions, with the
exception of using 500 ml of working volume instead of
400 ml, C. beijerinckii was chosen to potentially enhance
Table 1 Abbreviations
Parameter Meaning and units
Lactate consumption constant (l g−1 COD biomass d−1)
Substrate utilization rate (g COD PO g−1 COD biomass d−1)
Half‑ velocity degradation coefficient (g COD PO g−1 COD
Maximum specific growth rate (d−1)
Non‑biodegradable factor (g COD l −1)
Acetate yield from lactate (g COD g−1 COD lactate)
Acetate yield from particulate organic (g COD g−1 COD PO)
Butyrate yield from particulate organic (g COD g−1 COD PO)
Ethanol yield from particulate organic (g COD g−1 COD PO)
Formate from particulate organic (g COD g−1 COD PO)
Lactate yield from particulate organic (g COD g−1 COD PO)
Biomass yield from lactate (g COD g−1 COD lactate)
Biomass yield from particulate organic (g COD bio‑
mass g−1 COD PO)
H2 production when co-cultured with C. termitidis on
cellulose by serving as a high H2 producer from glucose
formed from cellulose hydrolysis by C. termitidis.
At the same time, a correlation between dry weight
and cellular protein content was developed for C.
beijerinckii in a similar way to the correlation for C. termitidis
(Gomez-Flores et al. 2015). A 20 % cellular protein
content was obtained, in close agreement with the 19 %
obtained for C. termitidis in the aforementioned study
(Additional file 1: Figure S1b).
Hydrogen production from cellulose
The H2 production profiles in Fig. 2a clearly depict the
enhancement in H2 production from co-culture over
mono-culture. H2 production showed long lag phases
of up to 17 days. The results of the modified Gompertz
model are shown in Table 2. The overall H2 production
for the co-culture compared with the mono-culture
increased by 30 % to 326 ml. Moreover, the H2
production rate in the co-culture of 26 ml d−1 was double the
12 ml d−1 observed in the mono-culture.
0 5 10 15 20 25 30 35 40 45
Fig. 2 C. termitidis mono‑ cultured in 2 g l−1 cellulose and co‑ cultured
with C. beijerinckii 2 g l−1 cellulose. a Cumulative H2 production
profiles. b pH profiles. Data points are the averages of duplicates, lines
above and below represent the actual duplicates
mol H2 mol−1 hexose eq.added
mol H2 mol−1 hexose eqconsumed
Figure 2b shows the pH profiles. During the lag phases,
all cultures exhibited a marginal decrease in pH from 7.2
to around 7. Concurrent with the H2 production, the pH
dropped to around 6.1. As the optimum pH range for
C. termitidis growth has been reported to be >5 to <8.2
(Hethener et al. 1992), the pH changes observed in
mono-culture fermentations were assumed not to impact
the microbial kinetics. For C. beijerinckii DSM 1820
growth, the pH range reported is from 5.2 to 7.3, with
the former reported as inhibitory (Masset et al. 2012). As
the observed pH changes in the co-culture fermentation
were within the growth range reported for both strains,
pH changes were assumed not to affect the microbial
Cellulose was not completely consumed in neither case
but co-culture enhanced the extent of cellulose
utilization by 15 % to about 93 % (Table 2).
Table 2 also shows the H2 yields based on
hexose equivalent added and consumed. The H2 yield
of 1.92 mol hydrogen mol−1 hexose equivalentadded
obtained in the co-culture was 32 % greater than the H2
yield obtained by the mono-culture of 1.45 mol
hydrogen mol−1 hexose equivalentadded. Also, the H2 yield of
2.05 mol hydrogen mol−1 hexose equivalentconsumed in the
co-culture was 14 % greater than the H2 yield obtained
by the mono-culture of 1.8 mol hydrogen mol−1 hexose
Microbial products and kinetics
The experimental and modeled biomass and cellulose
profiles are illustrated in Fig. 3, which emphatically
demonstrates that the co-culture was able to utilize more
cellulose than mono-culture and the ultimate biomass
growth was similar in all cases.
Figure 4 shows the experimental and modeled
metabolites profiles. Neither glucose nor cellobiose from
cellulose hydrolysis were detected in any of the fermentations,
implying that cellulose hydrolysis was the rate limiting
factor. Clostridium termitidis metabolites on cellulose
were acetate, ethanol, lactate, and formate, in
agreement with Ramachandran et al. (2008). In mono-culture
experiments, acetate and ethanol were produced during
biomass growth, while, formate and lactate exhibited lag
phases and were not detected until day 38. H2 production
peaked around day 44 for the mono-culture experiment,
concurrent with all metabolites peak.
Clostridium beijerinckii DSM 1820 soluble
products from glucose have been reported by Masset et al.
(2012) to be butyrate, acetate, formate, lactate, in
addition to butanol, acetone and isopropanol by Chen and
0 5 10 15 20 25 30 35 40 45
Fig. 3 Experimental and modeled growth kinetics. a Mono‑ culture.
b Co‑ culture
10 15 20 25 30 35 40 45
Hiu (1986), although, other strains of C. beijerinckii (i.e.
L9 and Fanp3) have been demonstrated to produce
ethanol from glucose (Lin et al. 2007; Pan et al. 2008). In the
co-culture experiment acetate and butyrate were
produced as lactate was consumed. It is noteworthy that only
butyrate production peaked on day 40, concurrent with
the H2 peak in the co-culture.
Mathematical models that accurately predict
biochemical phenomena provide the basis for design, control,
optimization and scale-up of process systems (Huang
and Wang 2010). Kinetic parameters of the
mathematical model are shown in Table 3. The co-culture exhibited
the highest µmax (0.2 d−1), thus rationalizing the end of
the fermentation test before the mono-culture. In this
regard, the impact of the synergy in microbial kinetics
was notorious, with µmax in co-culture of 0.2 d−1
double the 0.1 d−1 observed in mono-culture. It is
noteworthy that the maximum specific growth rates achieved
on glucose and cellobiose by C. termitidis of 0.22 and
0.24 h−1, respectively (Gomez-Flores et al. 2015), are
more than 50 times greater than those achieved by the
same strain on cellulose. The half-saturation constant, Kx,
varied between 0.42 and 1.1 g COD cellulose g−1 COD
biomass. PO/X values (Additional file 1: Figure S2) are
significantly greater than the Kx values, i.e. the growth
rate throughout the experiments equals µmax. The
recommended value for the hydrolysis rate of carbohydrates in
the anaerobic digestion model (ADM1) (Batstone et al.
2002) is 0.25 d−1 at mesophilic conditions which is
comparable to the growth rates obtained in the present study,
clearly emphasizing that the biodegradation of cellulose
Co-culture experiment reflected a slightly lower
biomass yield than monoculture (0.25 vs 0.3 g COD g−1
COD cellulose). YX/L (biomass yield from lactate) was
assumed to be the same as YX/PO (biomass yield from
cellulose) and YA/L (acetate yield from lactate) was
calculated as follows:
YA/L = fA/L(1 − YX/L)
where fA/L is the stoichiometric relationship based on
Eq. 4 of 1 mol acetate per mol lactate, calculated in g
COD as 0.66. YA/L was calculated to be 0.49 g COD
acetate g−1 COD lactate and the theoretical H2 production
from lactate was also calculated based on Eq. 4 and
subtracted from the measured H2 produced. The modified
Table 3 Kinetic parameters obtained in MATLAB of C.
termitidis mono-cultured and co-cultured with C. beijerinckii
on 2 g l−1 cellulose
Soa(g COD l−1)
H2 yields from cellulose in the co-culture experiment
were 1.72 mol hydrogen mol−1 hexose equivalentadded
and 1.84 mol hydrogen mol−1 hexose equivalentconsumed,
approximately 19 % higher than the mono-culture based
on hexose added. Nevertheless, the calculated H2 from
lactate may be overestimated since it is theoretical.
The average percentage errors (APE) and RMSE
calculated for the modeled biomass, substrate and
metabolites are the in Additional file 1: Table S1. Biomass and
cellulose exhibited the lowest average percentage errors,
within the range of 4–8 %, followed by PO/X with the
highest value of 11 % in co-culture. For both lactate and
formate in mono-culture, the model significantly under
estimated the lag phase, as evident from Fig. 4a.
Accordingly, the APE excluding the lag phase for lactate and
formate were 12 and 11 % and including lag phases was 81 %
in both cases.
COD balances calculated by summation of metabolites,
H2, cellulose and cells as g COD l−1 at the beginning and
end of fermentations are presented in Table 4. The COD
balances closed within 3–8 % of the initial, thus
confirming the reliability of the data. Theoretical H2
production from acetate and butyrate shown in Table 4 was
calculated based on 848 ml hydrogen g−1 acetate and
578 ml hydrogen g−1 butyrate (Eqs. 2, 3). The theoretical
values were consistent with the H2 measured during the
experiment with an average percent difference of 1 % of
the theoretical H2. C. beijerinckii DSM 1820 produced
a H2 yield of 2.54 mol hydrogen mol−1 glucose, added
or consumed, in line with the 1.9 and 2.8 mol
hydrogen mol−1 hexoseadded or consumed (Lin et al. 2007;
Masset et al. 2012), 2.5 mol hydrogen mol−1 hexoseconsumed
(Pan et al. 2008), and 2 mol hydrogen mol−1
hexoseadded (Taguchi et al. 1992). On the other hand, while the
highest reported mesophilic H2 yield by co-culture on
cellulose is 1.31 mol H2 mol−1 hexose with Clostridium
acetobutylicum X9 and Ethanoigenens harbinense B49
(Wang et al. 2008), and the highest thermophilic H2
yield is 1.8 mol H2 mol−1 hexose with C.
thermocellum JN4 and T. thermosaccharolyticum GD17 (Liu et al.
2008), the results from this study (Table 2) reveal a
significantly improved H2 yield in the co-culture of C.
termitidis and C. beijerinckii compared to the literature.
The achievement of a yield of 1.92 mol hydrogen mol−1
hexose using two mesophilic cultures represents about
50 % improvement of the literature at similar
conditions. Although the aforementioned yield is only 7 %
higher than the maximum thermophilic yield, the
balance of thermal energy input and output based on
hydrogen in this study is still more favorable than
reported elsewhere in the literature.
Based on the modeled acetate and butyrate profiles,
modeled H2 profiles shown in Fig. 5 were calculated in
a similar manner as the theoretical H2 shown in Table 4,
with 848 ml H2 g−1 acetate and 578 ml hydrogen g−1
butyrate (from stoichiometry of Eqs. 2 and 3), and
1.067 g COD g−1 acetate and 1.82 g COD g−1 butyrate.
The modeled H2 profiles closely match the experimental
H2, as verified with the low APE values ranging from 10
to 15 % and RMSE values (9–13 ml).
Microbial products and kinetics
Anaerobic lactate consumption has been reported by
different inoculums, such as soil, kitchen waste compost,
The evident lactate consumption in co-culture
fermentations shown in Fig. 4b, could be assumed to follow Eq. 4
since acetate was produced simultaneously.
Apparently, co-culture fermentation exhibited
acetate consumption after day 27 (Fig. 4b), which could
be explained by Eqs. 15 and 16, although lactate was
below the detection limit during this period of time. In
contrast, mono-culture fermentation did not exhibit
this phenomenon because C. termitidis does not
produce butyrate; thus acetate consumption in co-culture
fermentations could be attributed to the presence of C.
beijerinckii. Interestingly, the co-culture experiment
of C. thermocellum JN4 and T. thermosaccharolyticum
GD17 on cellulose reported by Liu et al. (2008) also
consumed lactate with acetate production whereas C.
thermocellum JN4 in mono-culture did not; no
explanation of this phenomenon was attempted by the
Desvaux et al. (2000) found a µmax of 0.056 h−1 with
C. cellulolyticum grown on 2.4 g cellulose l−1 with a
biomass yield of 36.5 g of cells mol−1 hexose equivalent (or
0.2 g cells g−1 hexose). Kinetics on cellulose have been
also explained by alternative models to Monod. For
example, Holwerda and Lynd (2013) found that the best
fit to their results on C. thermocellum was with a
substrate utilization rate that is both first order with respect
to substrate and first order in cells. Recently, Gupta
et al. (2015) found a µmax of 0.05 d−1 on cellulose using
mesophilic anaerobic digested sludge (ADS) and a Ks of
2.1 g l−1, which is four times lower than that achieved by
C. termitidis in the present study.
This study is the first to model C. termitidis microbial
kinetics on cellulose and in co-culture with C.
beijerinckii. High H2 yields at mesophilic temperature directly
from cellulose of 1.8 and 2.05 mol hydrogen mol−1
hexose equivalentconsumed in mono-culture and co-culture,
respectively, were achieved as compared to the literature.
Cellulose degradation by the co-culture was 15 % higher
than the mono-culture of C. termitidis. The viability of C.
termitidis and C. beijerinckii producing H2 together was
Clostridium diolis JPCC H-3, Clostridium butyricum
JPCC H-1, C. acetobutylicum P262, and also C.
beijerinckii JPCC H-4 (Diez-Gonzalez et al. 1995; Grause et al.
2012; Lee et al. 2010; Matsumoto and Nishimura 2007).
Nevertheless, in some cases, acetate has been
simultaneously consumed. The metabolic pathways reported in the
literature are shown in Eqs. 4, 15 and 16 (Costello et al.
1991; Diez-Gonzalez et al. 1995; Grause et al. 2012;
Matsumoto and Nishimura 2007; Thauer et al. 1977):
CH3CH (OH )COOH + 0.5CH3COOH
→ 0.75CH3CH2CH2COOH + 0.5H2 + CO2 + 0.5H2O
CH3CH (OH )COOH + 0.43CH3COOH
→ 0.7CH3CH2CH2COOH + 0.57H2 + CO2 + 0.7H2O
Additional file 1. Additional figures and tables.
MGF did the experimental design, laboratory work, data analysis, develop‑
ment of the code in Matlab, modeling, and paper writing. GN contribution
was supervision, critical and data interpretation, paper review, and corrections.
HH did paper review. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Ethics approval and consent to participate
Not applicable since this article does not contain any studies with human
participants or animals performed by any of the authors.
This work was supported by the Eastern platform of the Biofuel Network.
The authors acknowledge the support by Consejo Nacional de Ciencia y
Tecnologia de Mexico (CONACYT) and Alianza para la Formacion e Investi‑
gacion en Infraestructura para el Desarrollo de Mexico, awarded to Maritza
Batstone DJ , Keller J , Angelidaki I , Kalyuzhnyi S , Pavlostathis SG , Rozzi A , Sanders W , Siegrist H , Vavilin V ( 2002 ) Anaerobic digestion model no . 1 (ADM1). IWA Publishing , London
Brenner K , You L , Arnold FH ( 2008 ) Engineering microbial consortia: a new frontier in synthetic biology . Trends Biotechnol 26 : 483 - 489 . doi:10.1016/j. tibtech. 2008 .05.004
Chen JS , Hiu SF ( 1986 ) Acetone‑butanol‑isopropanol production by Clostridium beijerinckii (synonym, Clostridium butylicum) . Biotechnol Lett 8 : 371 - 376 . doi:10.1007/BF01040869
Costello DJ , Greenfield PF , Lee PL ( 1991 ) Dynamic modelling of a single‑stage high‑rate anaerobic reactor-I. Model derivation . Water Res 25 : 847 - 858 . doi:10.1016/ 0043 ‑1354(91) 90166 ‑ N
Desvaux M , Guedon E , Petitdemange H ( 2000 ) Cellulose catabolism by Clostridium cellulolyticum growing in batch culture on defined medium . Appl Environ Microbiol 66 : 2461 - 2470 . doi:10.1128/AEM.66.6. 24612 ‑ 470 . 2000
Diez‑ Gonzalez F , Russell JB , Hunter JB ( 1995 ) The role of an NAD‑independent lactate dehydrogenase and acetate in the utilization of lactate by Clostridium acetobutylicum strain P262 . Arch Microbiol 164 : 36 - 42 . doi:10.1007/ BF02568732
Eiteman MA , Lee SA , Altman E ( 2008 ) A co‑fermentation strategy to consume sugar mixtures effectively . J Biol Eng 2 : 3 . doi:10.1186/ 1754 ‑ 1611 ‑2‑3
Elsharnouby O , Hafez H , Nakhla G , El Naggar MH ( 2013 ) A critical literature review on biohydrogen production by pure cultures . Int J Hydrogen Energy 38 : 4945 - 4966 . doi:10.1016/j.ijhydene. 2013 .02.032
Geng A , He Y , Qian C , Yan X , Zhou Z ( 2010 ) Effect of key factors on hydrogen production from cellulose in a co‑ culture of Clostridium thermocellum and Clostridium thermopalmarium . Bioresour Technol 101 : 4029 - 4033 . doi:10.1016/j.biortech. 2010 .01.042
Gomez‑Flores M , Nakhla G , Hafez H ( 2015 ) Microbial kinetics of Clostridium termitidis on cellobiose and glucose for biohydrogen production . Biotechnol Lett 37 : 1965 - 1971 . doi:10.1007/s10529‑ 015 ‑ 1891 ‑4
Grause G , Igarashi M , Kameda T , Yoshioka T ( 2012 ) Lactic acid as a substrate for fermentative hydrogen production . Int J Hydrogen Energy 37 : 16967 - 16973 . doi:10.1016/j.ijhydene. 2012 .08.096
Guo XM , Trably E , Latrille E , Carrère H , Steyer JP ( 2010 ) Hydrogen production from agricultural waste by dark fermentation: a review . Int J Hydrogen Energy 35 : 10660 - 10673 . doi:10.1016/j.ijhydene. 2010 .03.008
Gupta M , Gomez‑Flores M , Nasr N , Elbeshbishy E , Hafez H , Hesham El Naggar M , Nakhla G ( 2015 ) Performance of mesophilic biohydrogen‑producing cultures at thermophilic conditions . Bioresour Technol 192 : 741 - 747 . doi:10.1016/j.biortech. 2015 .06.047
Hethener P , Brauman A , Garcia JL ( 1992 ) Clostridium termitidis sp. nov., a cellulolytic bacterium from the gut of the wood‑feeding termite, Nasutitermes lujae . Syst Appl Microbiol 15 : 52 - 58 . doi:10.1016/ S0723‑2020(11)80138‑4
Holwerda EK , Lynd LR ( 2013 ) Testing alternative kinetic models for utilization of crystalline cellulose (Avicel) by batch cultures of Clostridium thermocellum . Biotechnol Bioeng 110 : 2389 - 2394 . doi:10.1002/bit.24914
Huang WH , Wang FS ( 2010 ) Kinetic modeling of batch fermentation for mixed‑sugar to ethanol production . J Taiwan Inst Chem Eng 41 : 434 - 439 . doi:10.1016/j.jtice. 2010 .03.002
Kumar N , Das D ( 2000 ) Enhancement of hydrogen production by Enterobacter cloacae IIT‑BT 08 . Process Biochem 35 : 589 - 593 . doi:10.1016/ S0032‑9592(99)00109‑ 0
Lay JJ , Lee YJ , Noike T ( 1999 ) Feasibility of biological hydrogen production from organic fraction of municipal solid waste . Water Res 33 : 2579 - 2586 . doi:10.1016/S0043‑1354(98)00483‑ 7
Lee ZK , Li SL , Kuo PC , Chen IC , Tien YM , Huang YJ , Chuang C , Wong SC , Cheng SS ( 2010 ) Thermophilic bio‑ energy process study on hydrogen fermentation with vegetable kitchen waste . Int J Hydrogen Energy 35 : 13458 - 13466 . doi:10.1016/j.ijhydene. 2009 .11.126
Lee KS , Whang LM , Saratale GD , Chen SD , Chang JS , Hafez H , Nakhla G , El Naggar MH ( 2014 ) Biological hydrogen production: dark fermentation . In: Sherif SA, Goswami DY , Stefanakos EK , Steinfeld A (eds) Handbook of hydrogen energy. The CRC Press series in mechanical and aerospace engineering . CRC Press, Boca Raton
Lin PY , Whang LM , Wu YR , Ren WJ , Hsiao CJ , Chang SLLS ( 2007 ) Biological hydrogen production of the genus Clostridium: metabolic study and mathematical model simulation . Int J Hydrogen Energy 32 : 1728 - 1735 . doi:10.1016/j.ijhydene. 2006 .12.009
Liu Y , Yu P , Song X , Qu Y ( 2008 ) Hydrogen production from cellulose by coculture of Clostridium thermocellum JN4 and Thermoanaerobacterium thermosaccharolyticum GD17 . Int J Hydrogen Energy 33 : 2927 - 2933 . doi:10.1016/j.ijhydene. 2008 .04.004
Lu W , Wen J , Chen Y , Sun B , Jia X , Liu M , Caiyin Q ( 2007 ) Synergistic effect of Candida maltosa HY‑35 and Enterobacter aerogenes W ‑23 on hydro ‑ gen production . Int J Hydrogen Energy 32 : 1059 - 1066 . doi:10.1016/j. ijhydene. 2006 .07.010
Masset J , Calusinska M , Hamilton C , Hiligsmann S , Joris B , Wilmotte A , Thonart P ( 2012 ) Fermentative hydrogen production from glucose and starch using pure strains and artificial co‑ cultures of Clostridium spp . Biotechnol Biofuels 5 : 1 - 15 . doi:10.1186/ 1754 ‑ 6834 ‑ 5 ‑ 35
Matsumoto M , Nishimura Y ( 2007 ) Hydrogen production by fermentation using acetic acid and lactic acid . J Biosci Bioeng 103 : 236 - 241 . doi:10.1263/jbb.103.236
Metcalf L , Eddy HP ( 2003 ) Wastewater engineering: treatment and reuse, 4th edn. McGraw‑Hill , New York
Munir RI , Schellenberg J , Henrissat B , Verbeke TJ , Sparling R , Levin DB ( 2014 ) Comparative analysis of carbohydrate active enzymes in Clostridium termitidis CT1112 reveals complex carbohydrate degradation ability . PLoS ONE 9:e104260. doi:10.1371/journal.pone.0104260
Munro SA , Zinder SH , Walker LP ( 2009 ) The fermentation stoichiometry of Thermotoga neapolitana and influence of temperature, oxygen, and pH on hydrogen production . Biotechnol Prog 25 : 1035 - 1042 . doi:10.1002/btpr.201
Ngo TA , Nguyen TH , Bui HTV ( 2012 ) Thermophilic fermentative hydrogen production from xylose by Thermotoga neapolitana DSM 4359 . Renewable Energy 37 : 174 - 179 . doi:10.1016/j.renene. 2011 .06.015
Owen WF , Stuckey DC , Healy JB , Young LY , McCarty PL ( 1979 ) Bioassay for monitoring biochemical methane potential and anaerobic toxicity . Water Res 13 : 485 - 492 . doi:10.1016/ 0043 ‑13 5
Pan CM , Fan YT , Zhao P , Hou HW ( 2008 ) Fermentative hydrogen production by the newly isolated Clostridium beijerinckii Fanp3 . Int J Hydrogen Energy 33 : 5383 - 5391 . doi:10.1016/j.ijhydene. 2008 .05.037
Pavlostathis SG , Miller TL , Wolin MJ ( 1988 ) Kinetics of insoluble cellulose fermentation by continuous cultures of Ruminococcus albus . Appl Environ Microbiol 54 : 2660 - 2663
Ramachandran U , Wrana N , Cicek N , Sparling R , Levin DB ( 2008 ) Hydrogen production and end‑product synthesis patterns by Clostridium termitidis strain CT1112 in batch fermentation cultures with cellobiose or α‑ cellulose . Int J Hydrogen Energy 33 : 7006 - 7012 . doi:10.1016/j. ijhydene. 2008 .09.022
Shuler ML , Kargı F ( 2002 ) Bioprocess engineering: Basic concepts . Prentice Hall PTR, Upper Saddle River
Taguchi F , Hang JD , Takiguchi S , Morimoto M ( 1992 ) Efficient hydrogen production from starch by a bacterium isolated from termites . J Ferment Bioeng 73 : 244 - 245 . doi:10.1016/ 0922 ‑338X(92) 90172 ‑ Q
Thauer RK , Jungermann K , Decker K ( 1977 ) Energy conservation in chemotrophic anaerobic bacteria . Bacteriol Rev 41 : 100 - 180
Wang AJ , Ren NQ , Shi YG , Lee DJ ( 2008 ) Bioaugmented hydrogen production from microcrystalline cellulose using co‑ culture ‑ Clostridium acetobutylicum X‑9 and Etilanoigenens harbinense B‑49. Int J Hydrogen Energy 33 : 912 - 917 . doi:10.1016/j.ijhydene. 2007 .10.017
Wang A , Gao L , Ren N , Xu J , Liu C ( 2009 ) Bio‑hydrogen production from cellulose by sequential co‑ culture of cellulosic hydrogen bacteria of Enterococcus gallinarum G1 and Ethanoigenens harbinense B49 . Biotechnol Lett 31 : 1321 - 1326 . doi:10.1007/s10529‑ 009 ‑ 0028 ‑z