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Roles of microorganisms other than Clostridium and Enterobacterin anaerobic

fermentative biohydrogen production systems A review

Chun-Hsiung Hung a,, Yi-Tang Chang b, Yu-Jie Chang c

a Department of Environmental Engineering, National Chung Hsing University, 250 Kuo-Kuang Road, Taichung 402, Taiwanb Department of Microbiology, Soochow University, 70, Linxi Road, Shilin District, Taipei 111, Taiwanc Graduate School of Environmental Education and Resources, Taipei Municipal University of Education, 1, Ai-Guo West Road, Taipei 100, Taiwan

a r t i c l e i n f o

Article history:

Received 30 November 2010

Received in revised form 18 February 2011

Accepted 20 February 2011

Available online 23 February 2011

Keywords:

Anaerobic

Dark fermentation

Co-existed

Bacterial community

Hydrogen

a b s t r a c t

Anaerobic fermentative biohydrogen production, the conversion of organic substances especially from

organic wastes to hydrogen gas, has become a viable and promising means of producing sustainable

energy. Successful biological hydrogen production depends on the overall performance (results of inter-

actions) of bacterial communities, i.e., mixed cultures in reactors. Mixed cultures might provide useful

combinations of metabolic pathways for the processing of complex waste material ingredients, thereby

supporting the more efficient decomposition and hydrogenation of biomass than pure bacteria species

would. Therefore, understanding the relationships between variations in microbial composition and

hydrogen production efficiency is the first step in constructing more efficient hydrogen-producing con-

sortia, especially when complex and non-sterilized organic wastes are used as feeding substrates. In this

review, we describe recent discoveries on bacterial community composition obtained from dark fermen-

tation biohydrogen production systems, with emphasis on the possible roles of microorganisms that co-

exist with common hydrogen producers.

2011 Elsevier Ltd. All rights reserved.

1. Introduction

The design and operation of engineered processes in environ-

mental biotechnology arepractical ways in which microbial ecology

is manipulated so that specific microbial communities achieve par-

ticular goals. In particular, the production of hydrogen as a renew-

able energy source presents a good example. Many approaches

have been established to generate hydrogen biologically, including

direct and indirect photolysis, photo-fermentation, and dark-fer-

mentation. Based on the rates of hydrogen production by various

biohydrogen systems, dark-fermentation systems stand out by

offering an excellent potential for practical application and integra-

tion with emerging hydrogen technologies (Levin et al., 2004). The

successful operation of any type of dark fermentation bioreactor

depends on the performance of the microorganisms present in the

system; hence,understandingthestructure of biohydrogen-produc-

ingcommunities is a critical step toward optimizing microbial com-

munities and improving hydrogen production (Hallenbeck and

Ghosh, 2009; Karadag and Puhakka, 2010; Koskinen et al., 2007;

Lo et al., 2008; Wu et al., 2006). Sometimes, bacterial communities

with the same composition profile result in different hydrogen

production yields, indicating that production is not changed as a

response to bacterial community changes, but rather due to

metabolic pathway shifts in response to environmental conditions

(Hawkes et al., 2002). Most often, the proliferation of non-hydro-

gen-producing microorganisms causes a complete shift in microbial

ecology and directly or indirectly affects hydrogen production (Ren

et al., 2007a,b). To design effective anaerobic fermentative biohy-

drogen production systems, scientists must first understand the

microbial populations responsible for hydrogen production and

the role of co-existing non-hydrogen-producing microorganisms.

The dark fermentation of hydrogen-producing bioprocesses has

attracted much attention in recent years because it may be inte-

grated in the treatment of organic wastes, and it has the potential

to be scaled up for commercial purposes (Davila-Vazquez et al.,

2008). Anaerobic sludge is frequently used as inocula in hydrogen

production that uses organic waste as feeding substrate (Yang

et al., 2007). Hallenbeck and Ghosh (2009) asserted that future

commercialized hydrogen fermentative productions will have to

be carried out under non-sterile conditions and using readily avail-

able complex feedstock. Applying the concept of mixed culture is

one of the techniques for improving hydrogen production because

complexmicrobial communities are likely to containa suite of nec-

essary hydrolytic activities and are potentially more robust to

changes in operational conditions. However, it remains unclear

whether pretreated natural microflora, mainly for the purpose of

eliminating hydrogenotrophic methanogens, is the most efficient

mixed culture for hydrogen production. The shift in complete

0960-8524/$ - see front matter 2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.biortech.2011.02.084

Corresponding author. Tel.: +886 4 22856643; fax: +886 4 22862587.

E-mail addresses: [email protected] (C.-H. Hung), [email protected]

(Y.-T. Chang), [email protected](Y.-J. Chang).

Bioresource Technology 102 (2011) 84378444

Contents lists available at ScienceDirect

Bioresource Technology

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / b i o r t e c h
http://dx.doi.org/10.1016/j.biortech.2011.02.084mailto:[email protected]:[email protected]:[email protected]://dx.doi.org/10.1016/j.biortech.2011.02.084http://www.sciencedirect.com/science/journal/09608524http://www.elsevier.com/locate/biortechhttp://www.elsevier.com/locate/biortechhttp://www.sciencedirect.com/science/journal/09608524http://dx.doi.org/10.1016/j.biortech.2011.02.084mailto:[email protected]:[email protected]:[email protected]://dx.doi.org/10.1016/j.biortech.2011.02.084


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bacterial compositions from the original inocula to an entirely new

microbial community has also been observed (Koskinen et al.,

2007; Lin et al., 2010). Hence, product formation by anaerobic

microflora is significantly affected by the specific characteristics

of feed substances, reactor types, and operational conditions

(Prakasham et al., 2010).

Novel molecular techniques have been used in recent studies to

reveal important contrasts in the bacterial community structures

of hydrogen-producing communities in reactors operated under

various conditions (Hung et al., 2008; Rittmann et al., 2008). The

awareness of the effects of operational parameters and community

structure on hydrogen production rates and yields during dark fer-

mentation can offer useful insights into the optimization of hydro-

gen generation. However, most related studies have focused on

predominant hydrogen producers, such as ClostridiumandEntero-

bacter. Studies on co-existing community structures and interac-

tions with predominant hydrogen producers are limited (Koskinen

et al., 2007). The overall population interactions may have positive

or negative effectson nethydrogenproduction.Thisis a concernthat

reflects fermentativemetabolic changes withinparticular microbial

populations (community function) caused by changes in the micro-

flora population itself (species and numbers, or a change in both).

Hydrogen utilization results in pure water; hence, the major

advantage of energy from hydrogen is the absence of polluting

emissions. With the use of the appropriate microbial mechanisms

and interactions of anaerobic biohydrogen-producing microorgan-

isms, hydrogen (biohydrogen) would be the desired source of fu-

ture energy. Overviews on predominant hydrogen-producing

microorganisms have already been published (Davila-Vazquez

et al., 2008; Li and Fang, 2007). To obtain better insights into the

optimization of reactor performance and the corresponding micro-

bial interactions in the use of mixed culture on non-sterile organic

waste, we describe recent discoveries on bacterial community

structures from dark fermentation biohydrogen production sys-

tems, with emphasis on the role of co-existing microorganisms

and their effects on system performance. An overall schematic of

possible roles of these microorganisms is given inFig. 1.

2. Improving hydrogen production by the granular formation/

retention of biomass

Organic loading rate (OLR) is an important parameter in study-

ing hydrogen bioreactors. To optimize a system for hydrogen

production, the reactor must be operated either in a range of the

organic loading rates that the system can handle effectively, or in

an optimal organic loading rate for a maximum hydrogen yield.

Hydraulic retention time (HRT) controls the microbial growth rate

(dilution rate of the reactor); hence, HRTs must be greater than the

maximum growth rate of the microorganisms because faster dilu-

tion rates cause washouts. OLR or HRT adjustment is a common

strategy for operators; shorter HRT and higher OLR may raise the

hydrogen production rate. A category of continuous flow reactors,

characterized by the retention of the microbial biomass or the

stimulation of granular sludge formation can overcome the wash-

out problem and result in high cell concentrations, fostering high

volumetric production rates (Abreu et al., 2010; Chang and Lin,

2004; Lee et al., 2006; Lin et al., 2010; Show et al., 2007).

Fang et al. (2002a) investigated the influence of HRT and su-

crose concentration on hydrogen production by acidogenic granu-

lar sludge at a constant loading rate of 25 g-sucrose/(l day), and

demonstrated that hydrogen-producing acidogenic sludge could

agglutinate into granules in a well-mixed reactor that treats syn-

thetic sucrose wastewater at 26 C, pH 5.5, and 6 h HRT. Phyloge-

netic analysis by molecular method on this sludge sample

showed that 69.1% of the clones are affiliated with Clostrisiumspe-

cies, 13.5% withSporolactobacillus racemicusin theBacillus/Staphy-

lococcusgroup, and a few unidentified ones. Whether there are any

microbial species in the system responsible for the formation of

hydrogen-producing granulars is unclear. However, a significant

amount of extracellular polymer substance (EPS) produced by the

biomass has been observed (Fang et al., 2002a). TheBacillus/Staph-

ylococcusgroup can produce EPS at different incubation conditions

(Mowad et al., 1995; Soares et al., 2005); hence, their role may be

hypothesized to be contributory to the formation of granulars.

Zoutberg et al. (1989)reported aggregation formation by a pure

culture ofClostridium byturicum at high glucose concentrations as

the sole carbon and energy source. As for the mixed-growth bacte-

rial community, phylogenetic analyses from several dark fermen-

tation sludge granular samples have identified the existence of

Streptococcus sp. with the bacterial community. Fang et al.(2002b) reported that hydrogen-producing sludge degrades 99%

of glucose at 36 C and pH 5.5, and produces methane-free biogas.

Based on the phylogenetic analysis of rDNA sequences, 64.6% of all

the clones are affiliated with three Clostridium species (Clostridia-

ceae), 18.8% with Enterobacteriaceae, and 3.1% with Streptococcus

bovis (Streptococcaceae). The remaining 13.5% belongs to eight

operational taxonomic units, the affiliations of which have not

been identified. In their system, it remains unclear whether the

Enterobacteriaceae and Streptococcaceae species produce hydro-

gen.Davila-Vazquez et al. (2009) reported an interesting finding

when they investigated a fermentative biohydrogen-producing

continuous stirred tank reactor (CSTR) with cheese whey as sub-

strate. In their system, Enterococcus faecium and Streptococcus sp.

have been occasionally detected during operations. These microor-ganisms have not been detected in the period immediately before

system washout, possibly explaining the slight improvement in

hydrogen production. Several studies (Cheng et al., 2008; Lo

et al., 2008; Wu et al., 2006, 2008) on the use of granular sludge

to enhance dark fermentation biohydrogen production have all

identified the existence ofStreptococcus sp. in the bacterial com-

munity, especially when the systems are operated under low HRTs,

using starch, xylose, glucose, and sucrose as substrates. The role of

the co-existence ofStreptococcussp. withClostridiumwas not clear

when the co-existence was first identified. A recent study on EPS

production capability and bacterial composition using fluorescence

in situ hybridization and denaturing gradient gel electrophoresis

targeted granular sludge from the above-mentioned reactors, and

demonstrated that among self-forming granular sludge species,Clostridium pasteurianum is the predominant hydrogen-producing

Fig. 1. Possible roles of microorganisms co-existing with Clostridium and Entero-

bacter, the well-accepted hydrogen-producers, in the dark fermentation biohydro-gen systems.

8438 C.-H. Hung et al. / Bioresource Technology 102 (2011) 84378444


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bacterium. The FISH results on sludge granules showed thatStrep-

tococcusspecies aggregate inside the granules and are surrounded

by rod-like cells; Clostridium. Clostridium sp. and Streptococcus sp.

aggregate and form a meshed structure during the formation of

the granular. The ratio ofClostridium sp. and Streptococcus sp. to

the overall cell count is 85% and 13%, respectively. The presence

ofStreptococcussp. is possibly the most important factor in granu-

lar sludge formation in these efficient dark fermentation granular

sludge systems (Hung et al., 2010).

3. Maintaining an anaerobic environment by oxygen depletion

The predominant biohydrogen producer Clostridium is among

the distinctly strict anaerobic microorganisms. Facultative anaer-

obes, such asEnterobactersp., are also capable of producing hydro-

gen through the fermentation of organic substrates (Das and

Veziroglu, 2001). The ideal reactor design should consider remov-

ing oxygen as much as possible from feedstock before it enters the

bioreactor. However, considering the size of industrially feasible

operations, this task is extremely difficult. IfClostridiumis the only

microorganism seeded in the start-up of the biohydrogen produc-

tion system, it may not be able to consume oxygen by aerobic res-

piration, thereby lowering the redox potential. Therefore, the

amount of dissolved oxygen entering the reactor must be removed

by the previously mentioned hydrogen-producing facultative

anaerobic microorganisms or by non-hydrogen-producing ones.

Yokoi et al. (1998)demonstrated that a mixed culture of two pure

strains of C. butyricum andE. aerogenes could produce hydrogen

from starch, and provide high hydrogen yields without the need

to add any reducing agents to the medium because E. aerogenes,

a facultative anaerobe, removes oxygen and generates anaerobic

conditions in the reactor.

Similar observations are obtained from much more complex

biohydrogen-producing microbial communities. Zhu and Bland

(2006) investigated the effect of microorganism seeding in dark-

fermentation systems. They concluded that heat-shock pretreat-ment may result in the destruction of other non-spore-forming

bacteria, resulting in a reduced system capacity to consume oxy-

gen. This inability to consume oxygen from the cultivation mixture

results in the decreased conversion of the substrate into hydrogen.

Huang et al. (2010) conducted a sucrose-feeding hydrogen produc-

tion experiment using a 10 L CSTR with no specific procedure to re-

move oxygen from the culture and headspace. Cattle dung compost

was pretreated by boiling for 5 min and then used as inoculum. The

predominant microorganisms in this study were BacillusandClos-

tridium. The bioreactor was not designed to remove oxygen in the

headspace and in the culture; hence, oxidation reduction potential

(ORP) was above 100 mV in the beginning.Bacillussp. and faculta-

tive anaerobes dominated in the lag phase, and with the growth of

Bacillussp., oxygen was exhausted and the ORP began to decreaserapidly. Subsequently, an anaerobic environment suitable to the

growth of the anaerobic and hydrogen-producing bacteria Clostrid-

ium was established in the system. Hung et al. (2010) explored bac-

terial community structures in several granular sludge-forming

bioreactors to confirm how bacteria interact. The most important

species in granular bioreactors are C. pasteurianum, Streptococcus

sp., and Klebsiella sp. While C. pasteurianum is the predominant

hydrogen-producing species within the group, Streptococcus sp.

andKlebsiella sp. may function as facultative anaerobes. After the

successful pure-culture isolations ofStreptococcussp. and Klebsiella

sp. from the same sludge sample, significant oxygen consumption

was found inKlebsiellasp. incubation, as the ORP decreased signif-

icantly under aerobic and anaerobic conditions. These results con-

firm that facultative anaerobes, especially Klebsiella sp., assistobligate anaerobes by maintaining the conditions necessary to

promote fermentation and by enhancing hydrogen production. De-

spite being a facultative anaerobe, Streptococcus sp. produces no

obvious ORP changes under either aerobic or anaerobic incubation

conditions.

4. Potential increase in hydrogen production from the

breakdown of complex organic substrates

The anaerobic digestion of organic wastes is generally consid-

ered as a simple and effective biotechnological means of reducing

and stabilizing organic wastes. The anaerobic degradation of or-

ganic matter first to hydrogen and simpler organics then to meth-

ane and carbon dioxide by microorganisms occurs in various

habitats, such as sludge anaerobic bioreactors. The digestive

metabolism of anaerobic microorganisms is complex, involving

several intermediate steps as organic polymers are decomposed

into methane as the end product. The same principle also applies

to biohydrogen production through dark fermentation processes,

wherein methane production is avoided. Complex organics, such

as agricultural wastes, have received increasing attention in at-

tempts to utilize them in biohydrogen production. Dark fermenta-

tion is relatively inexpensive and efficient, and it has low energy

demands. Moreover, it has the advantage of simultaneous waste

reduction and hydrogen generation. Therefore, improvements in

hydrogen production by mixed cultures that can hydrolyze com-

plex organics can be sought by increasing the flux of easily biode-

gradable substrates involved in hydrogen-generating pathways.

Ueno et al. (2006) investigated changes in product formation by

thermophilic anaerobes in enriched-sludge compost and artificial

garbage slurry as the model solid wastes. Hydrogen fermentation

with acetate/butyrate formation was optimized at certain HRT

and pH values. A complexbacterial community was observed. They

concluded that Thermoanaerobacterium thermosaccharolyticum is

the dominant hydrogen-producing microorganism, and unidenti-

fied organisms that are phylogenetically related to several cellulo-

lytic microorganisms, such asBacillus, become dominant after thesystem changes operation to longer HRT and high pH where the

relatively high-solubilization efficiency of solid materials is ob-

served. Using starch, a relatively complex organic compound, as

the feeding substrate for dark fermentation,Cheng et al. (2008)re-

ported that whether the diversity of theClostridium community

could directly affect reactor performance is inconclusive. Bacterial

cell counts showed that the viable number of dominant Clostridium

sp. changes with hydrogen production rate (HPR); hence, cell count

could directly affect hydrogen production efficiency. The highest

HPR and hydrogen yield (HY) were obtained when the reactor

was operated at 0.5 h HRT, and cell counts ofClostridium sp. and

Bifidobacteriumsp. accounted for 40% and 4060% of the total com-

munity in the system, respectively. They suggested that bacterial

species that could degrade starch, such as Bifidobacterium sp.,breaks down starch into small molecules first and then these sim-

pler compounds are utilized by Clostridium species for hydrogen

production.

The selection of carbon substrates and the operational condi-

tions of bioreactors are critical selection parameters determining

the evolution of characteristic hydrogen-producing communities.

Lo et al. (2008) investigated bioproduction using xylose as sub-

strate, and they observed that bacterial composition seems to

change with HRT. Aside from hydrogen-producing Clostridium,

other microorganisms, such as Bifidobacterium sp. and Olsenella

sp., were found when the reactor was operated at a high dilution

rate (short HRT). Meanwhile, K. oxytoca and Pseudomonas sp.

thrived when the culture was operated at a long HRT. Most of these

non-clostridia bacteria are not hydrogen producers, but they maycontribute to the degradation of carbon substrates. In a sucrose-

C.-H. Hung et al. / Bioresource Technology 102 (2011) 84378444 8439


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feeding reactor studied byMaintinguer et al. (2008), one microor-

ganism that is phylogenetically related toBurkholderia cepaciawas

found. There are no reports associating this bacterium with hydro-

gen production. However, this bacterium is commonly found in the

environment and is utilized in organic degradation processes. Doi

et al. (2009)reported a bacterial community analysis from a reac-

tor fed with waste bread. Two microorganisms, Megasphaera sp.

andBifidobacterium

sp., which might contribute to substrate break-

down, are present in the microflora from the preparation stage un-

til the pilot-plant operation. Lay et al. (2010) reported a dark

fermentation biohydrogen production process using soluble

condensed molasses fermentation (CMS) as the substrate. Phyloge-

netic analysis on the microbial community revealed a complex

diversity of Clostridium, including Clostridium acetobutylicum,

C. pasteurianum, C. saccharobutylicum, and C. sporosphaeroides, as

well as one non-clostridia microorganism, Acidaminococcus sp.

They concluded that co-cultivating Clostridium and Acidaminococ-

cus strains might efficiently consume the carbohydrates and mono-

sodium glutamate contained in CMS, thereby enhancing hydrogen

productivity. Lu et al. (2009) used cornstalks as substrate to

produce hydrogen and methane, and found similar results. They

identified diverse bacterial communities, and concluded thatCyto-

phagales str., Acetivibrio cellulolyticus, and Clostridium sp. may be

useful in degrading cellulose, whereas Clostridium sp. may be

beneficial to hydrogen production. Doi et al. (2009) reported an

interesting conclusion regarding the interactions of hydrogen-

producingClostridiumwith acetate producers. They suggested that

hydrogen gas is primarily produced by Clostridium sp. and that

Clostridium sp. might use acetate converted from lactic acid be-

cause of its coexistence withBifidobacterium pseudocatenulatum.

Also, not every clostrida species existed in the dark fermentation

system is responsible for hydrogen production. For example, a re-

cent study by Nissil et al. (2011) concluded that bacterium closely

related to Thermoanaerobium thermosaccharolyticum is the pre-

dominant hydrogen producer and bacteria closely related to Clos-

tridium cellulosi and Clostridium stercorarium were responsible for

cellulose degradation and therefore promoting the hydrogenproduction.

5. Hindering hydrogen production or hydrogen consumption

Hydrogen-producing microflora in dark-fermentation systems

must be able to thrive on non-sterile substrates. For sustainable

hydrogen production, hydrogen consumers should be eliminated

from the microflora, and this is easily achieved through the pre-

treatment of seeding sludge. However, the sensitivity of these bac-

teria to unstable feeding conditions can be disadvantageous in

continuous cultures because substrate metabolism may be chan-

ged from hydrogen production to the production of methane and

other organic compounds. Based on observations on the composi-

tion of fermentative products, long-term cultures under non-sterileconditions might cause a population shift from hydrogen-produc-

ing bacteria to hydrogen-consuming or non-hydrogen-producing

bacteria, such as homoacetogens or propionate producers (Hussy

et al., 2003). However, with the application of advanced molecular

techniques, researchers can now identify some potential hydrogen

consumers that co-exist with Clostridium in dark fermentation. The

careful control of the design parameters, including substrate con-

centration, is necessary to suppress the activity of such bacteria.

In their fermentation system,Kawagoshi et al. (2005)observed a

relatively simple bacterial diversity composed of only three micro-

organisms. They suggested thatClostridium, followed by Coprother-

mobacterspecies, plays an important role in hydrogen production.

The third microorganism,Lactobacillus species, could have an ad-

verse effect on hydrogen production. Saraphirom and Reungsang(2010) reported that in their sweet sorghum syrup feeding fermen-

tation system, the existence ofSporolactobacillus sp., which could

excrete bacteriocins, might cause an adverse effect on hydrogen-

producing bacteria, and might be responsible for the observed rel-

atively low hydrogen production.

Koskinen et al. (2007)used a biohydrogen-producing fluidized-

bed bioreactor (FBR) to explore microbial community dynamics. A

rumen bacterium, Schwartzia succinivorans, which is a potential

hydrogen consumer, was detected in both the attached- and sus-

pended-growth phases of the FBR. The growth trend of this organ-

ism in the attached- and suspended-growth phases coincide with

the low production of hydrogen and elevated concentrations of

propionate in the FBR. They also suggested the presence of possible

hydrogen consumers, including Megasphaera sueciensis-affiliated

strains that are distantly related to classBacteroidetesand another

strain affiliated with Lachnospiraceae. The proportion trends of

these strains do not correlate with effluent propionate concentra-

tions, suggesting that the strains are more likely homoacetogens

than propionate producers. A converse observation was proposed

byCastell et al. (2009). Accordingly, in their dark fermentation

system, which uses unsterilized whey as feeding substrate,

Megasphaeracould be a hydrogen producer.

The presence of the well-known hydrogen-consuming metha-

nogens in dark-fermentation systems is excluded from this review

because it is possible to eliminate them through different opera-

tion techniques including heat-shock pretreatment (temperatures

ranging from 80 to 121 C), acid pretreatment (pH 24), and alka-

line pretreatment (pH 11) (Ren et al., 2008).

6. Hydrogen producers

Hydrogen production reactor performance is closely correlated

with the predominant hydrogen-producer community structure.

Among dark fermentation hydrogen-producing bacteria,Clostrid-

ium sp. and Enterobacter, as well as some microorganisms that

are phylogenetically related to these two, are the most widely

studied and identified (Prasertsan et al., 2009). Studies on hydro-

gen production by these microorganisms have been mostly con-ducted at different incubation conditions with pure or mixed

cultures of these two. However, when complex unsterilized organ-

ic wastes are used as substrate, the hydrogen-production commu-

nity evolves. Microorganisms other than the common hydrogen

producers have been identified from various dark-fermentation

systems. If further studies show that higher hydrogen yields can

be achieved in the presence of these co-existing populations, at-

tempts to isolate and characterize them would be warranted.

Luo et al. (2008)reportedSelenomonassp. in a glucose-feeding

steady-state fermentative hydrogen production system and con-

cluded it could contribute to greater hydrogen production at low

OLRs.Kawagoshi et al. (2005)investigated the effects of condition-

ing for a variety of inocula on fermentative hydrogen production.

Clostridium and co-existing Coprothermobacter appear to play animportant role in hydrogen fermentation. Coprothermobacter is

phylogenetically classified as one of the Thermoanaerobacterium

species. Microorganisms from the Thermoanaerobacteriales family,

such as T. thermosaccharolyticum, Thermoanaerobacterium bryantii

andThermoanaerobacter wiegelii have been identified as potential

hydrogen producers (Basile et al., 2010; Karadag and Puhakka,

2010; Kongjan et al., 2011; Ueno and Tatara, 2008; Zhang et al.,

2003) in dark-fermentation systems operated under thermophilic

mode. In a dark fermentation UASB reactor using unsterilized

cheese whey as substrate,Castell et al. (2009)reported an extre-

mely complex bacterial community. Microbiological studies using

both culture and non-culture methods (Terminal restriction frag-

ment length polymorphism, 16S rRNA cloning library, and isola-

tion) have shown the prevalence of fermentative organisms fromthe generaMegasphaera, Anaerotruncus, Pectinatus, andLactobacil-



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lus, which they concluded that may all be responsible for hydrogen

production. However, this conclusion about Lactobacillus hydrogen

producing ability is wrong since it has not yet been reported carry-

ing the ability of hydrogen production (Stiles and Holzapfel, 1997).

Ren et al. (2007a,b)studied bacterial communities in several etha-

nol-type biohydrogen production reactors. They confirmed the

existence of several putative hydrogen-producing bacteria besides

severalClostridium

strains, includingAcetanaerobacterium elonga-tum, Ethanoligenens harbinense, andMegasphaera cerevisiae.

Enterobacter, along with Clostridium in dark fermentation biohy-

drogen production reactors, is also believed to function as a hydro-

gen producer (Koskinen et al., 2007; Maintinguer et al., 2008).

However, several reports have concluded that the role ofEntero-

bacter as a hydrogen producer in mixed-culture fermentation is

not clear (Fang et al., 2002a; Hung et al., 2010).

7. Competition for substrate

Not all of feeding substrates in dark fermentation reactors are

utilized in hydrogen production. Organics could be incorporated

into the biomass and transformed into various fermentation prod-

ucts, including acetic, propionic, and butyric acids, as well as etha-

nol. Therefore, biohydrogen production is strongly correlated with

the composition of the bacterial community as the amount of pre-

dominant hydrogen producers evolves. In other words, the exis-

tence of non-hydrogen producing carbohydrate consumers has a

negative effect on the overall performance of reactors. These

microorganisms could outcompete the hydrogen-producing ones,

thereby decreasing hydrogen yields (Jo et al., 2007). This is an ex-

tremely important concern in the feasibility of producing hydrogen

based on unsterilized substrates, inasmuch as the bacterial com-

munity is influenced by different inocula, reactor operational con-

ditions, and feeding substrate strength. Thus, to improve hydrogen

production, it is necessary to study the physiology of these micro-

organisms and find the optimal reactor operation conditions that

inhibit their growth. Kim et al. (2006) investigated the effect ofsubstrate concentration, sucrose in this case, on hydrogen produc-

tion. From a rich and diverse bacterial community with Clostridium,

they identified a strain of spore-forming lactic acid bacterium,

Bacillus racemilacticus, and concluded that this bacterium might

slightly increase lactate concentration and decrease hydrogen

andn-butyrate at sucrose concentrations greater than 30 g COD/l.

In a study on bacterial community changes during fermentative

hydrogen and acid production from organic waste under thermo-

philic conditions,Ueno et al. (2006)reported a significant shift in

microorganism community and various fermentation patterns.

They concluded that hydrogen fermentation with acetate/butyrate

formation and with T. thermosaccharolyticum as the dominant

hydrogen-producing microorganism is optimized at an HRT lower

than 1 d and at pH 5.0 and 6.0. However, unidentified organismsbecome dominant after 4.0 d HRT at pH 7.0 and 8.0, where the rel-

atively high-solubilization efficiency of solid organic materials and

no hydrogen production are observed.Castell et al. (2009)inves-

tigated a dark fermentation systemusing cheese whey as substrate

and concluded that during the operation, low hydrogen yields

could be caused by the presence of fermentative organisms with

low yields, such as propionate producers Megasphaera andPectin-

atus, and by fermenters that cannot produce hydrogen. Competi-

tion for the substrate by species, such as Prevotella, Olsenella,

Bulleidia, Mitsoukella and Selenomonas, may affect the desired

hydrogen production output.Lin et al. (2010)operated a 400 L pi-

lot-scale dark fermentation system using sucrose as feeding sub-

strate, and concluded that C. pasteurianum is the only dominant

species showing high hydrogen production efficiency in their sys-tem. However, otherClostridium species, such as C. butyricum and

C. tyrobutyricum, present in the reactor occasionally are considered

as substrate competitors that reduce substrate amount.

Renet al. (2007a,b) reported that a small amountofLactococcus is

believed to have an inhibiting effect on producing hydrogen by eth-

anol productionin their ethanol + butyrictypefermentationreactor.

After investigating the effect of changing temperature on anaerobic

hydrogen production in an open mixed-culture bioreactor, Karadag

and Puhakka (2010) reported that the operational temperature sig-

nificantly affects the dynamics of microbial communities, which in

turn affects hydrogen production and competition for substrate

through different metabolic pathways. High lactate production at

50and55 C, which occurs with lowhydrogen production, is associ-

ated with thepresence ofBacillus coagulans. Thermoanaerobacterium

dominated at 60 and 65 C and lower H2production was obtained

with ethanol type fermentation.Ahn et al. (2005)reported that the

productionof reducedendproducts,lactatein this case, is associated

with low hydrogen yields from a thermophilic trickling biofilter

used for continuous biohydrogen production. Ribosomal gene

analysis revealed that microorganisms related to Bacillalesbut not

Lactobacillalesplay a role in lactate production in the given system.

8. Phylogenetic characterization of co-existing microorganisms

A phylogenetic classification comparison of the above-men-

tioned microorganisms as well as how they influence the hydrogen

productionprocess is given in Table1. Without comparingtheir via-

ble cell number in the dark fermentation biohydrogen reactor, we

can conclude that most of these co-existing microorganisms, other

thanthe well-accepted hydrogen-producing Clostridium andEntero-

bacter, are Gram-positive microorganisms. For example, microor-

ganisms that are suggested to participate in granular formation

such as S. racemicus(Sporolactobacillaceae),C. byturicum(Clostridia-

ceae), and Streptococcus sp. (Streptococcaceae) all belong to the

Gram-positive group. This may due to their capability to produce

EPS under anaerobic conditions (Mowad et al., 1995; Soares et al.,

2005). Non-hydrogen-producing clostrida species such asA. cellulo-lyticus (Ruminococcaceae),C. cellulosi, andC. stercorarium also benefit

hydrogengas productionby breaking downcomplexorganics. Other

microorganisms that phylogenetically relate to Clostridiumsuch as

Coprothermobacter sp. (Thermodesulfobiaceae), Anaerotruncus sp.,

and A. elongatum (both are Ruminococcaceae) can also produce

hydrogen.

Some of these co-existing microorganisms play multiple roles.

For example, microorganisms belonging to the Gram-positive

Bacillusgroup have both positive and negative effects when they

are identified from different dark fermentation biohydrogen sys-

tems. Their negative effect on competition in organic substrates

with hydrogen producers should be noted. Another interesting

finding that has never been discussed in biohydrogen produc-

tion-related literature involves the microorganisms in the Veillo-nellaceae family. In the current review, several microorganisms

belonging to this strict anaerobic bacteria family were identified

from the reactors operated under different conditions all over the

world. Their existence in the biohydrgen reactor was as complex

as the roles they play as hydrogen producers, hydrogen consumers,

or substrate consumers, which is worthy of further research.

The roles of Gram-negative microorganisms such as Enterobac-

ter aerogenes (Enterobacteriaceae), Klebsiella sp. (Enterobacteria-

ceae), Klebsiella oxytoca (Enterobacteriaceae), Pseudomonas sp.

(Pseudomonadaceae), and B. cepacia (Burkholderiaceae) focus on

the positive side of promoting hydrogen production by maintain-

ing a strict anaerobic environment and by breaking down complex

substrates. Therefore, if hydrogen production involves designing or

creating a bacterial community of functional members, theseGram-negative bacteria can be suitable candidates. Several



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Table 1

Phylogenetic characteristics and roles of microorganisms co-existed with the well-known dark fermentation biohydrogen producers, Clostridiumand Enterobacter.

Role/microorganisms Taxonomy Influence on the process Reference

Improving hydrogen production by the granular formation/retention of biomass

Sporolactobacillus

racemicus

Sporolactobacillaceae Significant amount of extracellular polymer substance produced by this bacteria could be

helpful on granular formation

Fang et al. (2002a)

Clostridium byturicum Clostridiaceae Self-aggregation formed when incubated under high concentration of glucose Zoutberg et al.

(1989)

Streptococcussp. Streptococcaceae It existed in dark-fermentation systems when improvement of hydrogen production wasobserved but apparently not a hydrogen-producer

Davila-Vazquezet al. (2009) and

Fang et al.

(2002b)

Streptococcussp. Streptococcaceae Clostridiumsp. and Streptococcus sp. aggregated and formed a meshed structure during the

formation of the granular

Hung et al. (2010)

Maintaining an anaerobic environment by oxygen depletion

Enterobacter aerogenes Enterobacteriaceae Enterobacter aerogenes, a facultative anaerobe, removes oxygen and generates anaerobic

conditions for hydrogen-producingClostridium

Yokoi et al. (1998)

Klebsiellasp. Enterobacteriaceae Incubation o f a s train o f Klebsiellasp. isolated from dark fermentation biohydrogen reactor

could significantly decreased ORP value and achieved an obligate anaerobic condition

Hung et al. (2010)

Bacillussp. Bacillaceae Growth of Bacillus sp. significantly reduced oxygen concentration and created an anaerobic

environment suitable toClostridium

Huang et al.

(2010)

Increasing hydrogen production from the breakdown of complex organic substrates

Bacillussp. Bacillaceae When organisms that are phylogenetically related to cellulolytic microorganisms, such as

Bacillus, become dominant, high-solubilization efficiency of solid materials was observed

in the biohydrogen reactor

Ueno et al. (2006)

Bifidobacteriumsp. Bifidobacteriaceae Bifidobacterium sp. breaks down starch into small molecules first and then simplifiedorganic compounds are utilized by Clostridiumspecies for hydrogen production Cheng et al.(2008)

Bifidobacteriumsp. Bifidobacteriaceae These non-clostridia bacteria which co-existed with hydrogen producers may contribute

to the degradation of carbon substrates

Lo et al. (2008)

Olsenellasp. Coriobacteriaceae

Klebsiella oxytoca Enterobacteriaceae

Pseudomonassp. Pseudomonadaceae

Burkholderia cepacia Burkholderiaceae Identified in a sucrose-feeding dark fermentation system and apparently not a hydrogen

producer but is commonlyfound in theenvironment andis utilized in organic degradation

processes

Maintinguer et al.

(2008)

Megasphaerasp. Veillonellaceae Presented in the microflora from the dark fermentation biohydrogen preparation stage

which might contribute to substrate breakdown

Doi et al. (2009)

Bifidobacteriumsp. Bifidobacteriaceae

Acidaminococcussp. Acidaminococcaceae Co-cultivating ofClostridiumand Acidaminococcus might efficiently consume the

carbohydrates and monosodium glutamate contained in CMS, thereby enhancing

hydrogen productivity

Lay et al. (2010)

Cytophagales str. Cytophagaceae Cytophagales str., Acetivibrio cellulolyticus, andClostridiumsp. degrade cellulose together,

whereas hydrogen-producingClostridiumsp. may be beneficiated

Lu et al. (2009)

Acetivibrio cellulolyticus Ruminococcaceae

Bifidobacterium

pseudocatenulatum

Bifidobacteriaceae Hydrogen gas is primarily produced byClostridiumsp. and that Clostridiumsp. might use

acetate converted from lactic acid because of its coexistence withBifidobacterium

pseudocatenulatum

Doi et al. (2009)

Clostridium cellulosiand

Clostridium

stercorarium

Clostridiaceae Clostridium cellulosiandClostridium stercorariumwere responsible for cellulose

degradation and therefore promoting the hydrogen production

Nissil et al.,

(2011)

Hindering hydrogen production or hydrogen consumption

Lactobacillussp. Lactobacillaceae Lactobacillus sp. co-existed with hydrogen-producing Clostridiumand Coprothermobacter

and might have an adverse effect on hydrogen production

Kawagoshi et al.

(2005)

Sporolactobacillussp. Sporolactobacillaceae It could excrete bacteriocins and cause an adverse effect on hydrogen-producing bacteria Saraphirom and

Reungsang (2010)

Schwartzia succinivorans Veillonellaceae Potential hydrogen consumers Koskinen et al.

(2007)Megasphaera sueciensis

affiliated strains

Veillonellaceae

Hydrogen producers

Selenomonassp. Veillonellaceae It could contribute to greater hydrogen production at low OLRs using glucose as substrate Luo et al. (2008)

Coprothermobactersp. Thermodesulfobiaceae Microorganisms from the Thermoanaerobacteriales family have been identified as

potential hydrogen producers in dark-fermentation systems operated under thermophilic

mode

Kawagoshi et al.

(2005)

Megasphaerasp. Veillonellaceae All might be responsible for hydrogen production Castell et al.(2009)Anaerotruncussp. Ruminococcaceae

Pectinatussp. Veillonellaceae

Acetanaerobacterium

elongatum

Ruminococcaceae Possible hydrogen producers co-existed withClostridium Ren et al.

(2007a,b)

Ethanoligenens

harbinense

Ruminococcaceae

Megasphaera cerevisiae Veillonellaceae

Competition for substrate

Bacillus racemilacticus Bacillaceae This bacterium might increase lactate concentration and decrease hydrogen production Kim et al. (2006)

Prevotellasp. Prevotellaceae These microorganisms may compete for substrate and affect the desired hydrogen

production

Castell et al.

(2009)Olsenellasp. Coriobacteriaceae

Bulleidiasp. Erysipelotrichaceae

Mitsoukellasp. Veillonellaceae

Selenomonassp. Veillonellaceae



7/8

microorganisms such as Cytophagaceae andPrevotellaceae which

belong to phylum CytophagaFlexibacterBacteroides are occa-

sionally identified within dark fermentation biohydrogen reactors.

Their roles are all substrate related in terms of either breaking

down complex organics or competing for these.

9. Microorganisms with unidentified functions

Thecomposition of microbial ecologyin a dark fermentation sys-tem receiving unsterilized organic wastes is strongly influenced by

incoming microorganisms originating from the feedstock. Unless

proper operational parameters, such as pH, temperature, HRT, and

OLR, are employed to avoid the proliferation of undesired microor-

ganisms, competition among predominant hydrogen producers

and co-existing ones are foreseeable. For example, microorganisms,

such as Bacillus sp. and Escherichia coli, are identified either as help-

ers or spoilers across different fermentation systems. To overcome

this instability and ensure high hydrogen production efficiency un-

der continuousoperation, hydrogen fermentationmicrobiology and

the factors involved in the stabilization/destabilization of the pro-

cess should be further investigated. With the application of cul-

ture-independent molecular methods, microorganisms that exist

in dark fermentation system at previous undetectable quantitiescan now be identified. However, their role in the biohydrogen pro-

duction ecology is still unclear. For example, in a biohydrogen pro-

duction system using starch as substrate under thermophilic

conditions,Zhang et al. (2003) reported the existence of an uncul-

tured Saccharococcus sp. Saccharococcus, a Gram-positivefacultative

anaerobic thermophile, cannot degrade feedingstarch, andwhether

it has the ability to produce hydrogen is also unclear. Koskinen et al.

(2007) reported a detailed bacterial communitystudy on a dark fer-

mentation fluidized-bed reactor. They concluded that within that

complex ecology, some of the organisms detected likely carried

out metabolism without the production or consumption of H2, such

asDesulfovibrio desulfuricans,Anaerofilum agileandBifidobacterium

dentiumaffiliated strains. Furthermore, many of the microorgan-

isms, including affiliated strains from M. sueciensis, Bacteroidetes,

andLachnospiraceae, present in that reactor have not been isolated

or characterized and their potential functions remain unclear. Most

of the studieshave only obtained qualification results on howmany

types of microorganisms exist. To facilitate the creation of better

hydrogen production systems, research on obtaining structural

quantification results, i.e., species, number, and the special location

of microorganisms, including predominant hydrogen producers and

co-existing ones, is in order.

10. Conclusion

Hydrogen production efficiency is dependent on both hydro-

gen-producing bacteria and other populations. This indicates that

co-metabolism within the microbial community play a vital rolein biohydrogen production in reactors because population interac-

tions could have positive or negative effects on net hydrogen

production. In this review, we conclude that besides the predomi-

nant hydrogen producers, such as Clostridium and Enterobacter,

other co-existing microorganisms participate in overall reactor

performance as helpers in granular formation, oxygen depletion,

additional hydrogen production, and hydrogen production

enhancement by breaking down complex compounds. They might

also hinder hydrogen production by acting as hydrogen consumers

or substrate competitors.

Acknowledgements

We are grateful for the helpful comments from two anonymous

reviewers especially for pointing out the obvious mistake that we

made.

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