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Hydrogen Production by Fermentative Consortia - Valdez-Vazquez
Hydrogen Production by Fermentative Consortia - Valdez-Vazquez
Centro de Investigacion Cientca y de Educacion Superior de Ensenada (CICESE), Department of Marine Biotechnology, Ensenada, B.C. Mexico
CINVESTAV-IPN, Department of Biotechnology and Bioengineering, PO Box 14-740, Mexico D.F. 07000, Mexico
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 7 February 2008
Accepted 25 March 2008
In this work, H2 production by anaerobic mixed cultures was reviewed. First, the different anaerobic
microbial communities that have a direct relation with the generation or consumption of H2 are
discussed. Then, the different methods used to inhibit the H2-consuming bacteria are analyzed (mainly in
the methanogenesis phase) such as biokinetic control (low pH and short hydraulic retention time), heatshock treatment and chemical inhibitors along with their advantages/disadvantages for their application
on an industrial scale. After that, biochemical pathways of carbohydrate degradation to H2, organic acids
and solvents are showed. Fourth, structure, diversity and dynamics of H2-producers communities are
detailed. Later, the hydrogenase structure and activity is related with H2 production. Also, the causes for
H2 production inhibition are analyzed along with strategies to avoid it. Finally, immobilized-cells systems
are presented as a way to enhance H2 production.
2008 Elsevier Ltd. All rights reserved.
Keywords:
Anaerobic
Consortia
Hydrogen
Hydrogenases
Immobilized-cells
Inhibition
Methanogenesis
Solventogenesis
Contents
1.
2.
3.
4.
5.
6.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Hydrogen: a key intermediate in anaerobic environments . . . . . . . . . . . . . . . .
Induction of H2 accumulation in anaerobic consortia . . . . . . . . . . . . . . . . . . . .
3.1.
Biokinetic control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.1.
Acidogenic conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1.2.
Short hydraulic retention times . . . . . . . . . . . . . . . . . . . . . . . .
3.2.
Heat-shock treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.1.
Nutritional and environmental requirements for germination
3.3.
Chemical inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Structure, diversity and dynamics of H2-producers communities . . . . . . . . . . .
Biochemical pathways for H2 production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Hydrogenases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1.
Structure of Fe-Hase I from C. pasteurianum . . . . . . . . . . . . . . . . . . . . . .
6.2.
Hydrogenase activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.1.
pH. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.2.
Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.3.
Iron concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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* Corresponding author. Tel.: +52 646 17 505 69; fax: +52 646 17 505 69.
E-mail addresses: valdez_idania@yahoo.com (I. Valdez-Vazquez), hectorpoggi2001@gmail.com (H.M. Poggi-Varaldo).
Abbreviations: AC, activated carbon; ALSC, acrylic latex plus silicone; ATP, adenosine triphosphate; BES, 2-bromoethanesulfonate; CoA, coenzyme A; COD, chemical
oxygen demand; CpI, bidirectional hydrogenase from Clostridium pasteurianum; CpII, H2-oxidizing hydrogenase from Clostridium pasteurianum; CSTR, continuous stirred tank
reactors; D, dilution rate; DGGE, denaturing gradient gel electrophoresis; DNA, deoxyribonucleic acid; EA, activation energy; EC, expanded clay; HRT, hydraulic retention
time; HSP, heat-shock pretreatment; IV-SSAH, intermittently vented solid substrate for anaerobic H2 generation; LAB, lactic acid bacteria; NADH, reduced nicotinamide
adenine dinucleotide; NRB, nitrate-reducing bacteria; OFMSW, organic fraction of municipal solid waste; PCR, polymerase chain reaction; PU, polyurethane; rRNA, ribosomal
ribonucleic acid; SEM, scanning electron microscopy; S0, substrate concentration; SRB, sulfate-reducing bacteria; T-RFLP, terminal restriction fragment length polymorphism; TS, total solids; X0, initial sludge density.
1364-0321/$ see front matter 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.rser.2008.03.003
7.
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I. Valdez-Vazquez, H.M. Poggi-Varaldo / Renewable and Sustainable Energy Reviews 13 (2009) 10001013
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1. Introduction
For decades, the use and abuse of fossil fuels (either in liquid,
gaseous or solid form) has caused contamination of our soil, air and
water. Recently, diverse alternative fuels have been proposed to
substitute fossil fuels. Hydrogen is one of these alternative fuels
that is recognized as a promising future energy carrier. It is
considered a clean fuel since it does not have carbon, sulfur or
nitrogen that cause pollution during combustion [96]. Today, H2 is
principally produced from fossil fuels such as natural gas and
naphtha. However, this practice is an environmental contradiction
since a clean fuel is generated from a polluting and limited source.
Therefore, it is necessary to use other sources and methods to
obtain H2 in a renewable, sustainable and environmentally friendly
way.
In this regard, biotechnology can provide H2 from renewable,
cheap and abundant sources such as wastewater or organic solid
wastes. This way, the use of pure cultures of anaerobes, aerobes,
photosynthetic bacteria and cyanobacteria have been reported
with the objective of generating H2 [60]. Also, it is possible to use
undened microbial consortia to generate H2 from a fermentation
process. The use of fermentative consortia presents several
advantages such as high H2 generation rates (100 times more
than with photosynthetic cultures), continuous H2 generation at a
sustained rate since it does not depend on light energy as a
photosynthetic process does, generation of metabolites of commercial interest (such as organics acids and solvents), oxygen
limitation does not exist because it is an anaerobic process and the
most important fact is it can use complex organic waste as a
substrate in non-sterile conditions [13,46,45]. The use of organic
wastes instead of pure carbohydrates is the main advantage of the
fermentative process utilizing consortia since the costs for
implementation to full scale is smaller. Thus, it is possible to
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I. Valdez-Vazquez, H.M. Poggi-Varaldo / Renewable and Sustainable Energy Reviews 13 (2009) 10001013
Table 1
H2-producing and H2-consuming reactions presents in anaerobic processes
Equations
1
2
3
4
5
6
7
Type of reaction
Fermentation
Fermentation
Anaerobic oxidation (syntrophy)
Anaerobic oxidation (syntrophy)
Hydrogenotrophic methanogenesis
Acetogenesis from CO2 and H2
Sulfate reduction
Reaction
DG8 (a)
DG8 (b)
135
207
+48.2
+76.2
136
105
NA
284
319
17.6
5.5
3.2
7.1
165
Notes: (a) standard conditions; (b) conditions prevailing in anaerobic ecosystems (Valdez-Vazquez et al., 2005).
Fig. 2. Relationship between Gibbs free energy of electron acceptor on the threshold of H2. Adapted from [12].
I. Valdez-Vazquez, H.M. Poggi-Varaldo / Renewable and Sustainable Energy Reviews 13 (2009) 10001013
1003
origin and substrate type, among others. Investigators have used one
or more of these inhibition methods in studies of H2 production by
anaerobic consortia although it is not still clear which is the best. In
this way, possibly the simplest and most economic method is the
biokinetic control, mainly utilization of low pH.
3.1. Biokinetic control
In this review, we refer to biokinetic control as methanogen
inhibition by acidogenesis due mainly to organic acid accumulation during fermentation. This is achieved by overloading the batch
reactors with organic matter generating a fast and large organic
acid accumulation which reduce pH to critical levels for
methanogens. On the other hand, the growth rates could also be
used to eliminate methanogens in continuous reactors with liquid
substrates. This is possible because the growth rate of methanogens is lower than that of H2-producing microorganisms. Therefore, methanogens are washed out of the reactor utilizing high
dilution rates [16].
3.1.1. Acidogenic conditions
When pH is not controlled, a drop in pH due to organic acid
accumulation is linked to H2 evolution as is shown in Fig. 3. Thus,
under certain low pHs, methane evolution stops and H2/CO2 are
the main gases produced [70]. Most methanogens grow over a
relatively narrow pH range (68). Some acidophilic species such as
Methanobacterium espanolae, grow at pHs between 5.6 and 6.2 but
are unable to grow and produce methane at pH 4.7 [23,84] showed
that inhibition of methanogenesis activity is necessary for avoiding
methane formation from evolved H2 when an anaerobic inoculum
is used. So low pH (around 5.0) is effective for inhibiting
methanogenesis activity and obtaining an inoculum rich in H2
producers. The acclimatization times reported to obtain a
methanogen-free inoculum generating H2 and organic acids are
variable among different work and fall in a range between 3 and 30
days [84,8,50,92]. In order to enhance H2 production, inoculum
enrichment relevance at low pH was established by [8] where
inoculum acid-enrichment during 80 h produced 330 times more
H2 compared with the control (without enrichment).
The experimental observation that H2 production is improved
under acidogenic conditions were also conrmed by [99] in
anaerobic reactors during shock loads. They proposed that this
behavior could be corroborated by theoretical analysis of an
equilibrium between formate and H2 through a formate:hydrogen
lyase:
H2g HCO3 ! HCOO H2 O
(8)
(9)
10pH
H2 aq
K H2
(10)
Fig. 4. Effect of high hydraulic retention times on hydrogen and methane yields.
(&), (~) hydrogen yields from two different works; (*) methane yield (adapted
from data published by [85,7]).
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Fig. 5. Wide range of hydraulic retention time affecting hydrogen production rates.
Arrow indicates probable wash-out of the reactor by extreme high hydraulic
retention time (based on data reported by [85,47,7,30]).
Fig. 6. The transition of dormant spores to active vegetative forms. Adapted from
Ref. [75].
most frequent nutrient germinant being L-alanine. Finally, outgrowth leads to the formation of a new vegetative cell [22]. In the
literature, there are a wide number of reports studying these three
phases with species of Bacillus spores but in smaller proportion
with H2-producing bacteria such as Clostridium [71]. Reports that
have studied the most favorable conditions for spore germination
along with the H2 production are scarce in spite of its signicance
for fermentative H2 production processes.
The HSP effectiveness for H2 production was demonstrated by
[42] using a heat-treated anaerobic digested sludge during 15 min
degrading the organic fraction of municipal solid waste (OFMSW)
incubated at 37 8C. The H2 yield obtained with this heat-treated
anaerobic sludge was similar to those found with a H2-producing
bacteria of the genus Clostridium (140 and 180 mL H2/g OFMSW,
respectively). In both cases, 60% of hydrogen and trace elements of
methane were found. Later on, [40] determined that clostridia was
the predominant H2-producing bacteria in the heat-treated sludge
by means of metabolite proling. They found that the activity of
heat-shocked sludge was signicantly inhibited when the initial
cellulose concentration exceeded 25 g/L. Also, the sludge activity
was inuenced mainly by the ratio of the initial substrate
concentration (S0) to initial sludge density (X0). Thus, high H2
generation was obtained from cellulose at low S0/X0 ratio. [64]
presented an interesting work where effectiveness of low pH and
HSP for methanogen elimination were compared. They found that
greater H2 yields were found with heat-treated inocula (pH 6.2 or
7.5) than that at low pH (6.2) without HSP. These results were
important to establish HSPs superiority over low pH. However, the
pHs used were not those reported as the best for H2 production
(pH 5.5). Therefore, the HSP superiority over low pH is only
relevant at pH 6.2 and 7.5 since H2 performance could change at pH
5.5.
However, HSP has been used by several authors during short
operation periods. Increasing evidence shows that a stable H2
production and methanogen repression is not possible for longterm continuous operation [36]. In this respect, [82] utilized an
anaerobic sludge treated thermally at 100 8C for 15 min for H2
production using sucrose as the substrate in two types of
continuous reactors. The rst reactor was equipped with an
activation chamber which exposed a third of the biomass at 90 8C
for 20 min, while the second reactor was not equipped with an
activation chamber but the biomass was exposed to an initial
heat-treatment. Their results indicated that the performance of
the rst reactor was better than that of the second reactor. Both
initial and repeated heat treatments of the biomass during
operation enhanced H2 percentage. However, the highest H2%
I. Valdez-Vazquez, H.M. Poggi-Varaldo / Renewable and Sustainable Energy Reviews 13 (2009) 10001013
was obtained from the rst reactor (51% vs. 43%). From these
results, it seems to be that the initial HSP is not enough to
maintain free methanogen reactors during the operation even
when the pH was 5.5. Although repeated heat treatments seemed
to be effective for eliminating methanogens during operation, it
was not reasonable.
HSP has also been used for eliminating lactic acid bacteria (LAB)
that is present in some waste such as that produced in bean curd
manufacturing. LAB may cause the inhibition of H2-producing
bacteria by excretion of bacteriocins [63]. Bacteriocins are proteins
with bactericidal activity directed against many Gram-positive
bacteria, including Clostridium. These compounds are frequently
found as secondary metabolites produced by diverse microorganisms such as LAB and genus Bacillus [28]. Several authors have
reported that in decreasing the pH there was an increase in
bacteriocin production which are more active at low pH [68]. From
these observations, it can be pointed out that the heat treatment of
organic waste could be effective for preventing H2 production
inhibition by LAB.
3.2.1. Nutritional and environmental requirements for germination
When HSP is used, spores of H2-producing microorganisms are
selected. The critical step to obtaining vegetative cell producers of H2
from dormant spores is the germination step (Fig. 6). In consequence, it is necessary to provide conditions and nutriments
required for a fast and optimal germination. Only a few authors have
studied the effect of initial substrate and pH levels for germination of
H2-producing bacteria spores. In spite of using different heat-treated
inocula, three different studies conclude in that the optimal pH for
H2 production is 5.5, where optimal substrate concentration was
between 7.5 and 15 g COD/L in liquid cultures [94,95,17].
When all vegetative cells are killed, it is normal to nd a lag time
of several days for H2 production to begin. This lag time will
depend on several factors such as initial pH, nutrient concentrations, temperature, germinant availability, among others. Typical
lag times for H2 production under mesophilic conditions are 24
days. However, [90] observed a much bigger lag time under
thermophilic conditions (>4 days), utilizing heat-treated micloora fermenting solid organic sustrate. To this respect, [89] found
that the addition of specic nutrients (termed germinants such as
aminoacids) was necessary for optimal and complete germination
at thermophilic incubation obtaining lag times <1 day for H2
production. Thus, in absence of germinants, the best temperature
for spore germination seems to be at 37 8C.
It is possible to say that HSP is effective and fast to obtain
inocula for H2 production although it is necessary that further
studies on nutritional and environmental requirements for optimal
germination of selected spores achieving a large H2 production be
conducted. On the other hand, this method does not avoid the
proliferation of H2-consuming microorganisms coming from nonsterile feedstock which could cause H2 production depletion [82].
It is then necessary to apply HSP whenever the H2 production
decreases, causing an increase in production costs.
3.3. Chemical inhibitors
In literature, there have been reports of many chemical
substances that inhibit methane formation for methanogenic
archaea that have different specicities and act at different
concentrations. These compounds have been applied to study the
importance of methanogenesis in the environment and can be
classied into two major groups: nonspecic and specic inhibitors.
Chloroform, uoroacetate and acetylene are some examples of
nonspecic inhibitors for methanogens. Chloroform (CHCl3) is
known to block the function of corrinoid enzymes and to inhibit
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Fig. 7. Biochemical pathways utilized by Clostridia genera for the conversion of carbohydrates to hydrogen, carbon dioxide, organic acids and solvents. Adapted from Ref. [34].
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(11)
Fig. 8. Structure of iron-sulphur clusters from CpI of Clostridium pasteurianum. (a) irons tetrahedrally coordinated from [Fe2S2] cluster in N-terminal FS2 domain; 9b) two
[Fe4S4] clusters from FS4A-FS4B domain which is adjacent to the active site domain; (c) single [Fe4S4] cluster from FS4C domain which is positioned between the FS2 and
FS4A-FS4B domains and (d) two subclusters [Fe4S4] and [Fe2] (named H-cluster) from the active site domain. Adapted from Ref. [33].
I. Valdez-Vazquez, H.M. Poggi-Varaldo / Renewable and Sustainable Energy Reviews 13 (2009) 10001013
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6.2.1. pH
Several reports observed that the H2 production initiation was
carried out only after pH decreased to 5.5. Studies found that the
hydrogenase activity measured in whole cells from acid-producing
cultures maintained at pH 5.8 was about 2.2 times higher than that
measured in cultures maintained at pH 4.5 [4]. In general,
hydrogenase activity (uptake and evolution) is low in cells
maintained at a pH < 5.2 [26]. Also, [1] found that the activity
of hydrogenase I increased steadily with decreasing pH with an
optimum pH of 6.3. Thus, these studies on hydrogenase activity are
directly correlated with those of H2 fermentation showing that pH
plays multiple roles in H2 production.
6.2.2. Temperature
Adams and Mortenson [1] determined the effect of temperature
on the rate of H2 catalysis by hydrogenases I and II from mesophilic
C. pasteurianum. The authors obtained the Arrhenius plot for
calculating the activation energy (EA) values and optimum
temperatures for the reaction. With both enzymes in both assay
systems, the plots were linear in the range of 1550 8C. The rate of
reaction decreased between 50 and 70 8C.
These in vitro results, together with the in vivo observations,
suggest that the optimum temperature is approximately 50 8C. In
different systems (reactors using soluble and solid substrates), it
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