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Title: Influence of support materials on continuous hydrogen
production in anaerobic packed-bed reactor with immobilized
hydrogen producing bacteria at acidic conditions
Authors: Petra Muri, Romana Marinšek-Logar, Petar
Djinovića, Albin Pintar
EMT 9147
To appear in:
Enzyme and Microbial Technology
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Please cite this article as: Muri Petra, Marinšek-Logar Romana, Djinovića
Petar, Pintar Albin.Influence of support materials on continuous hydrogen
production in anaerobic packed-bed reactor with immobilized hydrogen
producing bacteria at acidic conditions.Enzyme and Microbial Technology
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Influence of support materials on continuous hydrogen production in anaerobic packedbed reactor with immobilized hydrogen producing bacteria at acidic conditions
Petra Muria, Romana Marinšek-Logarb, Petar Djinovića, Albin Pintara,*
Department for Environmental Sciences and Engineering, National Institute of Chemistry,
Hajdrihova 19, SI-1001 Ljubljana, Slovenia
Division of Microbiology and Microbial Biotechnology, Department of Animal Science,
Biotechnical Faculty, University of Ljubljana, Groblje 3, SI-1230 Domžale, Slovenia
Title: Influence of support materials on continuous hydrogen production in anaerobic packedbed reactor with immobilized hydrogen producing bacteria at acidic conditions
Petra Muri, Romana Marinšek-Logar, Petar Djinović, Albin Pintar
Corresponding author. Tel.: +386 1 47 60 237; fax: +386 1 47 60 460. E-mail address: (A.
H2 production was investigated in anaerobic packed-bed reactor at acidic conditions
Produced hydrogen yields (mol H2/mol glucose) were 1.80 (R1)>1.74 (R2)>1.46 (R3)
Main metabolic products were found to be acetic acid, butyric acid and ethanol
Higher hydrogen yield correlates with low acetate-to-butyrate (HAc/HBu) ratio
Quantity of attached biomass (gTVS/gsupport) were 0.06 (R1), 0.04 (R2) and 0.035 (R3)
Abstract - This study assesses the impact of different support materials (Mutag BioChipTM,
expanded clay and activated carbon) on microbial hydrogen production in an anaerobic packedbed reactor (APBR) treating synthetic wastewater containing glucose as the main carbon source
at low pH value. The APBRs were inoculated with acid pretreated anaerobic sludge and
operated at pH value of 4 ± 0.2 and hydraulic retention time (HRT) of 3 h. The maximum
hydrogen yield of 1.80 mol H2/mol glucose was achieved for the APBR packed with Mutag
BioChipTM (R1), followed by expanded clay (R2, 1.74 mol H2/mol glucose) and activated
carbon (R3, 1.46 mol H2/mol glucose). It was observed that the investigated support materials
influenced the immobilization of hydrogen producing bacteria and consequently hydrogen
production performance as well as composition of soluble metabolites. The main metabolic
products were acetic acid and butyric acid accompanied with a smaller content of ethanol. The
data indicated that in reactors with higher hydrogen yield (R1 and R2), acetate/butyrate
(HAc/HBu) ratios were 1.7 and 1.6, respectively, while in the reactor with the lowest hydrogen
yield (R3) the obtained HAc/HBu ratio was 4.8. Finally, stable hydrogen and organic acids
production throughout the steady-state operation period at low pH values was achieved in all
Keywords: biohydrogen; hydrogen producing bacteria; anaerobic packed-bed reactor;
immobilization; support materials; low pH value
1. Introduction
The use of alternative biofuels represents a possibility to decrease our dependence on fossil
fuels. A promising alternative fuel that can replace conventional fossil fuels and therefore
contribute towards a low carbon economy development is biologically generated hydrogen [1].
Hydrogen has been envisaged as a clean fuel, due to its combustion into water as the only
reaction product. Furthermore, hydrogen has high energy content per unit weight (122 kJ/g)
and can be therefore used either in combustion engines or for electricity production through
fuel cells [2]. On the other hand, when comparing volumetric densities with other gaseous fuels
at normal conditions, hydrogen contains only 3.00 kWh/m3, while this value for methane equals
to 9.97 kWh/m3 [3]; extremely low hydrogen volumetric density therefore poses a challenge
for its storage.
Hydrogen can be produced by several techniques such as thermochemical processes, water
electrolysis and various biological processes [4]. Currently, hydrogen is mainly produced by
steam reforming of natural gas and water electrolysis that are very energy intensive processes,
but can be simply performed. Biological processes of hydrogen production via bio-photolysis,
photo-fermentation and dark fermentation have an advantage over previously mentioned
methods, due to operation at ambient temperatures and utilization of renewable organic
compounds, but they are more sophisticated in design and performance [5]. Microbial
fermentative hydrogen production exhibits several advantages over other biological hydrogen
production processes, as it enables higher conversion yields and hydrogen evolution rates as
well as a wider range of substrate utilization. Further, it requires no light and exhibits a potential
for cost-effective hydrogen production [6]. However, hydrogen production at the industrial
scale still faces many drawbacks, such as too low hydrogen yields and often unstable hydrogen
production during longer operation periods, since evolutionary metabolic pathways of substrate
degradation mainly result in the generation of energy-rich compounds, rather than hydrogen
synthesis [7]. A maximum of 4 mol H2/mol glucose is obtained when acetate is the end product
of fermentation, while studies show that for most of the mesophiles, hydrogen yields are limited
to 2 mol H2/mol glucose. This is due to the fact that under standard conditions, complete
oxidation of glucose into hydrogen and carbon dioxide is thermodynamically unfavorable.
However, production of molecular hydrogen is important in achieving the redox balance in
several fermentations. On the other hand, conversion of glucose into reduced metabolic
products is thermodynamically more favorable as generation of ATP during the production of
short chain fatty acids (SCFA) and/or alcohols supports microbial growth.
For most studies on dark fermentative hydrogen production, batch reactors have been used due
to their simple operation and efficient control. However, to realize an industrial application of
hydrogen production, continuous-flow bioreactors are recommended. Among them, the
continuous stirred tank reactor (CSTR) is still the most widely used type of reactor for model
studies, since it enables efficient mixing and homogeneity [8]. However, the CSTR is unable to
maintain high levels of fermentative microbial biomass at short HRTs as rapidly mixing pattern
causes washout of biomass. Therefore, granular type or fixed-bed reactors such as upflow
anaerobic sludge blanket (UASB), anaerobic packed-bed reactor (APBR) and anaerobic
fluidized-bed reactors (AFBR) appear to be more convenient. These reactors allow to achieve
high cell densities by immobilization and permit the unit to be run at HRTs that are independent
of the bacterial growth rate [9,10]. Thus, high hydrogen volumetric flow rates can be achieved
with these types of reactors. Furthermore, high biomass densities provide greater resistance to
inhibitory substances in the influent and shock loads.
Among reactor types with immobilized microbial cells, APBR is characterized by simple
configuration and high efficiency of hydrogen production. Moreover, the APBR configuration
with microbial biofilm attached to inert carriers appears to result in lower construction and
operation costs, since the reactor operates without mechanical agitation and does not require
external sedimentation tank to separate the biomass [11]. Although APBR reactors were studied
using different support materials for biofilm growth and model as well as real substrates, their
application for fermentative hydrogen production requires further investigation [12,13]. Several
previous studies on biohydrogen production in APBRs have indicated an influence of different
operating conditions on hydrogen yield, however, without a detailed assessment on microbial
cell attachment and detachment processes in formed biofilms, although stability of the formed
biofilm is crucial for long-term operation of reactors. Moreover, a thorough review of the
literature has revealed that dark fermentation studies mostly provide data obtained for the pH
range of 5.0-7.0 [14], with a few studies evaluating the aforementioned process at low pH
conditions, although hydrogen production at low pH values could bring added advantage by
cost savings for pH adjustments, eliminated pretreatments for restriction or termination of
methanogens and provided capability to treat acidic waste. In this context, the main objective
of this study was to investigate hydrogen production and microbial biofilm stability at low pH
values (4 ± 0.2) for different support materials (Mutag BioChipTM, expanded clay and activated
carbon) in the APBR reactor system. While assessing biohydrogen production over various
support materials, an influence of support materials on the immobilization of hydrogen
producing bacteria was investigated as well.
2. Experimental
2.1. Seeding sludge
The microbial inoculum used in this study was obtained from the anaerobic biogas digester
treating waste sludge from a municipal wastewater treatment plant operating under mesophilic
temperature conditions and CSTR mode. The collected sludge was sieved using 90 μm mesh
(Retsch, S/N 05036692) in order to remove large solid particles, and then stored at 4 °C. The
characteristics of inoculum used in the present study are summarized in Table 1. Prior to
immobilization process, hydrogen productivity of the seeding sludge was enhanced by acid
pretreatment with the following conditions: pH = 5.5 for 24 h at 37 °C.
2.2. Medium composition
The medium used for H2 production consisted of glucose (5000 mg/L) as a carbon source and
of inorganic supplements including (mg/L) 125 CH4N2O, 2 NiSO4·6H2O, 5 FeSO4·7H2O, 2
FeCl3·6H2O, 1.5 CoCl2·6H2O, 5 CaCl2·2H2O, 85 KH2PO4, 22 K2HPO4 and 50 Na2HPO4·7H2O.
The pH value in the range of 4 ± 0.2 was maintained by means of NaHCO3.
2.3. Support materials
The APBRs were packed with three different types of support materials for hydrogen producing
bacteria: Mutag BioChipTM (Umwelttechnologie AG, Germany; R1), expanded clay (R2) and
activated carbon (Comelt S.p.A., Italy; R3) with the characteristics presented in Table 2.
2.4. Anaerobic packed-bed reactor for H2 production
Fig. 1 shows a schematic drawing of the reactor set-up. Initially, reactors were seeded with the
acid treated sludge diluted with MQ water in ratio of 1:1 and circulated through the reactor unit
for 24 h. The reactor and inoculum were initially purged with N2 to assure anaerobic conditions.
The procedure of inoculum circulation through reactors has been repeated till the reactor
reached steady state. After the steady state was reached, the APBRs with HRT of 3 h were
continuously fed for 25 days with synthetic wastewater containing glucose (5000 mg/L),
nutrients and sodium bicarbonate using a peristaltic pump. The reactors had a total volume of
230 mL and a working volume of 110, 100 and 70 mL after packing with Mutag BioChipTM,
expanded clay and activated carbon, respectively. The glass reactors had the internal diameter
of 2.9 cm and length of 35 cm. The temperature in the APBRs was maintained at 37 °C by
recirculating heated water from a thermostatic bath over the reactor body.
A gas-liquid separator was used at the outlet of the reactor to collect gaseous and soluble
products separately. Cumulative production of biogas was measured by flow-through liquid
displacement cells. Reactors were connected via a multi-channel automatic valve to a gas
chromatograph, which enabled real-time analysis of the discharged gas stream. In order to
assess performance of the reactors, glucose conversion, SCFAs, biomass yield, cell mass
concentration in the effluent, cumulative volume of produced biogas and its composition were
2.5. Analytical methods
Composition of gas phase was determined by means of a micro GC analyzer (Agilent
Technologies, model 490) equipped with a TCD detector calibrated for hydrogen, nitrogen,
methane, carbon dioxide and hydrogen sulfide. CP-MolSieve 5A column (Agilent) with Ar as
a carrier gas was used to analyze hydrogen, nitrogen and methane, while carbon dioxide and
hydrogen sulfide were analyzed using a CP-PoraPLOT U column (Agilent) with He as a carrier
gas. The temperature of the injector and column were kept at 80 and 100 °C, respectively.
Sugars, SCFAs (i.e. malonate, succinate, lactate, formate, acetate, propionate, butyrate, oxalate
and valerate) and alcohol (i.e. ethanol) in liquid-phase samples were determined by HPLC
technique (Agilent Technologies, model 1260 Infinity) equipped with a cation-exchange HiPlex H column (7.7 × 300 mm, 8 μm). The column temperature was set to 50 °C. The analyses
of SCFAs, ethanol and sugars in the fermentation broth were performed using 0.01 M H2SO4
as a mobile phase with a flow rate of 0.6 mL/min. SCFAs were analyzed by UV detection at
210 nm, while concentration of ethanol and glucose were determined by using a RI detector
kept at 35 °C.
TS and VS (to represent microbial biomass concentration) were measured in accordance with
ISO 15705:2002-11 and SIST EN 14346:2007 standardized procedures, respectively. Support
materials and microbial morphology at the end of immobilization and long-term operation
period were examined using Carl Zeiss (SUPRA 35 VP) scanning electron microscope (SEM).
For SEM analysis, support materials with attached biofilm were firstly freeze dried, attached to
the aluminium sample carrier using conductive carbon tape and pretreated by carbon sputtering.
In order to characterize materials for hydrophilicity/hydrophobicity, contact angles (Θ) of
support materials were measured using a tensiometer Krüss DSA 20 (Krüss GmbH, Germany)
according to the method described elsewhere [15]. Hydrophobicity of bacterial cells was
evaluated by measuring their adherence to chloroform drops [16]. Furthermore, surface charge
characteristics of the examined support materials were analyzed by point of zero charge
measurements [17].
2.6. Microbial community analysis
Temporal variations of bacterial and archaeal microbial communities were assessed with
Terminal Restriction Fragment Length Polymorphism (T-RFLP) method. Before the analysis,
all samples were dried in N2 atmosphere and stored at -20 °C.
Microbial biomass on the examined supports has been mechanically pretreated in order to
release microbial biofilm in the suspension. Samples were added to 2 mL tubes with sterile
zirconium beads of various sizes (0.3 g with diameter of 0.1 mm and 0.1 g with diameter of 0.5
mm, Roth). Samples were then immersed into TE buffer and homogenized (3-4 min) on a
shaker (Vortex Genie2) equipped with the microcentrifuge tube adapter (Mobio Laboratories,
Carlsbad, USA). After shaking, 200 µL of suspension was used for DNA isolation. Microbial
DNA was extracted and purified using Power Soil DNA Isolation Kit (Mobio Laboratories,
Carlsbad, USA). The quantity of the extracted DNA was checked by measuring its absorbance
on NanoVue-Plus spectrophotometer (GE Healthcare, UK).
Genomic DNA was used as a template for PCR reactions with primers specific for bacteria (fD1
[18] and 926r [19]) and archaea (A109f [20] and Ar915r [21]). PCR mixtures (25 µL) contained
1X PCR buffer with KCl, 1.5 mM MgCl2, 0.2 µM dNTP, 0.3 µM of each primer and 0.5 U of
Taq DNA polymerase. The PCR protocol used for bacteria was: 95 °C for 5 min, 25 cycles of
95 °C for 30 s, 55 °C for 30 s and 72 °C for 1 min, while PCR protocol used for archaea was:
95 °C for 5 min, 28 cycles of 95 °C for 30 s, 52 °C for 45 s and 72 °C for 1.3 min. The reactions
were subsequently exposed to 72 °C for 7 min and cooled to 4 °C. PCR products were purified
using High Pure PCR Product Purification Kit (Roche, Switzerland) and quantified using
NanoVue-Plus spectrophotometer (GE Healthcare, UK).
The purified product (100-150 ng) with 10 U of two restriction enzymes (BsuRI and Mspl) was
digested at 37 °C for 16 h. The restriction enzymes were inactivated by incubation at 80 °C for
20 min. Next, the restriction products were purified with the kit mentioned above and 6 µL was
mixed with 8.5 µL HiDi formamide and 0.5 µL GeneScan ROX-500 internal size standard
(Applied Biosystems, USA) and denaturated at 95 °C for 3 min followed by rapid cooling on
ice. T-RFs were separated by capillary gel electrophoresis on an ABI PRISM 3130xl Genetic
Analyser (Applied Biosystems, USA) and electropherograms were analyzed by BioNumerics
software 5.1 (Applied Maths, Belgium). Pearson’s correlation coefficient and UPGMA
clustering method were used to construct the dendrograms.
3. Results and discussion
3.1. Immobilization of hydrogen producing bacteria on selected supports
Immobilized cell culture systems for fermentative hydrogen production can be operated in
batch, fed batch and continuous-flow modes. Although most of the studies for hydrogen
production by immobilized systems are evaluated in continuous-flow operation, assessments of
microorganism immobilization for use in aforementioned processes are mainly done in
(repeated) batch operating mode [22-25]. By considering these facts, one of the aims of this
study was to investigate an influence of support materials on the immobilization process of
hydrogen producing bacteria. In the given range of operating conditions, the bioreactors
exhibited the steady-state operation after 4-5 days of immobilization. Fig. 2 shows production
of soluble metabolic products and hydrogen production rate during the steady-state period of
immobilization process. In R1 reactor system, we observed the highest concentration of soluble
metabolic products formed (618 mg/L) as well as highest H2 production rate (32.5-35.3 NmL/(L
h)). On the other hand, concentrations of soluble metabolic products formed in reactors filled
with expanded clay (R2) and activated carbon (R3) were 390 and 438 mg/L, while H2
production rates were found to be 29.6-33.5 and 27.4-30.1 NmL/(L h), respectively. Among
soluble metabolites formed during the immobilization process, acetic and butyric acid were
dominant products in all three cases. However, their molar ratio varied significantly;
acetic/butyric acid ratio was the highest for R3 (7.44), followed by R2 (3.35) and R1 (2.14).
The obtained results indicate that immobilization support materials affect production of
hydrogen and soluble metabolites, because different composition and/or dynamics of microbial
communities could develop on examined support materials.
The performance of APBRs during immobilization period could be strongly influenced by
characteristics of support materials. Surface hydrophobicity has been described as one of the
most important factors involved in bacterial adhesion process [26]. The results of contact angle
measurements revealed that Θ value for Mutag BioChipTM was 114°, while high porosity of
expanded clay and activated carbon (i.e. materials predominantly containing micro- and
mesopores) prevented formation of water droplets and subsequent Θ measurements. Therefore,
the results of this analysis have shown that Mutag BioChipTM carriers were hydrophobic, while
expanded clay and activated carbon were found to be hydrophilic. In regard to cell affinity for
different support materials, hydrophobicity index of inoculum was measured. Its value of 69.4
showed hydrophobic nature of present microbial cells in the inoculum, suggesting higher
adhesion of bacteria on Mutag BioChipTM, as this material was shown to be hydrophobic.
Moreover, we assume that better adsorption in R1 was achieved due to numerous macropores
on the surface of Mutag BioChipTM (see Fig. 6a below), as these pores can be one of the crucial
parameters affecting binding of microorganisms on the surface. Accordingly to Basile et al.
[27], cell affinity could also be affected by charged functional chemical groups on the surface.
As bacterial cells tend to have a net negative charge on the cell wall [28], their adsorption would
be enhanced in the case of positively charged surface. As listed in Table 2, point of zero charge
measurements revealed that all the support materials examined were positively charged at the
employed experimental conditions, and exhibited therefore similar influence on microbial cells
An analysis of anaerobic mixed culture on different support materials acquired from
experiments at the end of immobilization was conducted using SEM imaging technique. SEM
observations showed that mostly micro-shaped bacilli were found on investigated support
materials (Fig. 3). The characteristic layer of extracellular polymeric substances (EPS) in the
form of net-like structure that is a typical component of biofilms, was at the end of
immobilization evidently detected only on the surface of Mutag BioChipTM particles.
3.2. Hydrogen production
There was a slight difference in biohydrogen production among tested bioreactors filled with
different support particles during 25 days long operation period (Table 3). The highest hydrogen
production rate and yield reached in R1 unit were 16.65 mmol H2/(L h) and 1.80 mol H2/mol
glucose, respectively. In R2 reactor hydrogen production rate and yield were found to be 15.55
mmol H2/(L h) and 1.74 mol H2/mol glucose, while in R3 system hydrogen production rate and
yield decreased to 13.65 mmol H2/(L h) and 1.46 mol H2/mol glucose.
As one can see in Table 3, support materials exhibited no influence on biogas composition, as
in all cases H2 content in the biogas was 52.3-53.2 vol. %. Moreover, high hydrogen yields
correlated with glucose conversion over 98.0 % in all reactors (Table 3, Fig. 4). The findings
of this study are comparable to the results of Sivagurunathan et al. [29], who measured 99.6 %
galactose removal at HRT of 2 h with hydrogen yield of 2.25 mol H2/mol galactose.
Furthermore, Ferraz Júnior et al. [30] obtained similar results with 100 % glucose removal at
HRT of 8 h and hydrogen yield of 2.1 mol H2/mol glucose.
3.3. Production of soluble metabolites
Production of soluble metabolites formed during the dark fermentation process associated with
different support materials in the APBRs is depicted in Fig. 4. The steady-state operation in all
reactors was observed after approximately 5 days of operation, which is attributed to the
required time for biofilm development. It appears that acetate and butyrate were the main
metabolites during the operation under consideration, suggesting that the primary hydrogen
producers in the culture were acetate and butyrate producing bacteria. In R1 reactor,
concentrations of acetate and butyrate were in the range of 1033-1246 and 890-1041 mg/L,
respectively. Similarly, in R2 unit measured values of acetate and butyrate were 947-1091 and
895-1011 mg/L, respectively. On the other hand, in R3 reactor which exhibited lower H2
production performance, concentrations of acetate and butyrate were found to be 804-995 and
225-333 mg/L, respectively. As SCFAs dominated among soluble metabolites and the
production of solvents (e.g., ethanol) was negligible at low pH value, the production of H 2 in
the culture was metabolically favored. Finally, production of lactate, formate and propionate
which are typical soluble metabolites that lower H2 production, was minor in this study (below
50 mg/L).
The comparison of hydrogen yield and distribution of general metabolites between studies taken
from the literature and this work is presented in Table 4. As one can see, acetic and butyric acid
fermentation usually occurs in the pH range of 5.5-6.5, while the pH range of 4.0-5.5 is typical
for ethanol fermentation. In the study of Reis and Silva [31], dominant metabolic products in
the pH range of 4.0-5.0 were acetate (41.8 %) and ethanol (38.8 %), while Chu et al. [32]
reported formation of ethanol (31.0 %), acetate (16.9 %) and butyrate (46.8 %) at pH = 5.5. On
the other hand, Barros et al. [33] obtained low ethanol production (15.3 %), as mainly acetate
(45.1 %) and butyrate (37.3 %) were produced at pH value of 5.5. However, in this study,
ethanol production was at selected operational conditions found to be low in all the reactors
(5.1-7.9 %).
Predominance of acid end products during hydrogen production is however known to occur in
the acidogenic phase, which decreases the pH value of culture medium. As low pH value
disfavors growth of microorganisms, it has been suggested that the uptake of acids (normally
accompanied by an increase of pH value) which occurs during solvent producing phase,
functions as a detoxification process initiated in response to the accumulation of acid end
products [37]. Solvent production is not guaranteed under low pH values and becomes brief
and unproductive if pH value decreases below the value of 4.5 before enough acids are formed
[38]. This is supported by the study of Shida et al. [36], where similar results to those obtained
in this work are reported. The main soluble metabolites reported were acetate (52.3 %) and
butyrate (40.1 %), with 7.5 % of ethanol leading to hydrogen yield of 2.29 mol H2/mol glucose
at pH value of about 3.8. The findings obtained in this study suggest that high hydrogen yields
can be obtained at low pH values. Knowing that low pH values suppress hydrogenase activity,
one can speculate that pH value within the biofilm where microbial community is present, is
higher than in outer regions where the conditions are acidic [39].
Moreover, the HAc/HBu ratio has been reported as an indicator for efficiency evaluation of
hydrogen producing cultures [33,40]. In general, a higher HAc/HBu ratio gives a higher
theoretical H2 yield, as hexose fermentation to acetic acid gives 4 mol of H2/mol hexose, while
fermentation to butyric acid gives 2 mol H2/mol hexose (reactions (1) and (2)):
6 12 6 + 22  → 23  + 22 + 42
6 12 6 → 3 2 2  + 22 + 22
The obtained HAc/HBu ratios from the APBR reactors are plotted in Fig. 4. HAc/HBu ratios
for the reactors packed with Mutag BioChipTM, expanded clay and activated carbon were 1.7,
1.6 and 4.8, respectively. These results indicated that butyrate was slightly dominated by acetate
production in the case of Mutag BioChipTM (R1) and expanded clay (R2) supports, while in the
reactor packed with activated carbon (R3) low production of butyrate compared to acetate was
observed. Although the HAc/HBu ratio in the R3 system was found to be the highest among
APBRs examined, its hydrogen yield was lower compared to R1 and R2. This could be due to
the presence of (i) autotrophic homoacetogenic bacteria that are consuming H2/CO2 (reaction
(3)) or CO and/or (ii) heterotrophic homoacetogenic bacteria that grow on a wide range of
sugars, alcohols, methoxylated aromatic compounds and one carbon compounds to form acetate
42 + 22 → 3  + 22 
There are, however, several studies reporting high levels of homoacetogenic bacteria in dark
fermentation process: for example, Dinamarca et al. [42] observed that homoacetogenic
hydrogen consumption was 4 to 62 mmol H2/(Lsludge day) in heat treated anaerobic mixed
culture at 35 °C. They also observed that simultaneous hydrogen production and consumption
occurred in non-methanogenic fermentation. Moreover, Nie et al. [43] found that the maximum
hydrogen consumption in pretreated sludge at 105 °C was equal to 1.3 mmol/h. Yet Luo et al.
[44] reported that 43 % of total acetate was produced by homoacetogens with acid-pretreated
mesophilic sludge cultivated under 37 °C and initial pH = 5.5.
3.4. Effect of different support materials on biofilm formation
The accumulation of biofilm that is primarily composed of microbial cells and extra-cellular
polymeric substances is the net result of attachment, growth and detachment. Fig. 5a shows the
attached biomass in investigated APBRs. After twelve days of operation, the reactor packed
with Mutag BioChipTM (R1) contained the highest amount of adhered biomass (i.e. 0.07 g
VSS/gsupport). This increased amount of biomass attached is suggested to contribute to higher
hydrogen production performance of R1, as there may be a greater quantity of hydrogenogenic
bacteria adhered to this support material compared to R2 and R3 reactor systems (with obtained
microbial biomass concentrations of 0.04 and 0.03 g VSS/gsupport, respectively). The temporal
variations of effluent VSS concentrations are illustrated in Fig. 5b. As shown, these values were
the highest in R3, followed by R2 and R1. This observed order might be attributed to different
generation times of microbial biomass that developed on the support materials. It was also
found out that the effluent VSS concentrations in the beginning of experiments were higher in
comparison to the later operating stage and that after 12th day the effluent VSS concentrations
stabilized; average steady-state values of effluent VSS concentrations in R1, R2 and R3 systems
were equal to 0.07, 0.15 and 0.22 g VSS/Leffluent, respectively.
Table 5 shows that attachment/detachment rates in R1 and R2 units were slightly lower
compared to R3 system, being 0.10, 0.12 and 0.16 g VSS/d, respectively. Biomass yields
calculated as the sum of attached biomass and effluent suspended biomass per unit of glucose
consumed, were 0.11, 0.08 and 0.18 g VSS/(g glucose consumed) for R1, R2 and R3 reactors,
respectively. The inverse relationship between the biomass and hydrogen yields shows that
higher biomass yield in R3 unit is associated with growth of non-hydrogen producing bacteria,
since hydrogen yield in this reactor system was lower compared to the ones obtained in R1 and
R2 units. It is noteworthy to emphasize that the reported biomass yield for hydrogen producing
bacteria of 0.1 g VSS/(g glucose consumed) [45] is consistent with our results. This clearly
reflects higher proportion of non-hydrogen producing bacteria in R3 unit compared to R1 and
R2 reactors.
Biofilm analysis of samples collected at the end of experiments shows that all support materials
examined in this study were appropriate for biomass immobilization and further formation of
biofilm. SEM micrographs depicted in Fig. 6 show that the biofilm fully covered the surface
and cavities of all support materials, which indicates high biomass retention in APBR reactors.
Based on observed biofilm morphologies (Fig. 6) and above-discussed production of acetate
and butyrate during the operation phase, the present microorganisms might belong to the genera
Clostridium, Enterobacter and Bacillus associated with carbohydrates conversion to hydrogen
and organic acids.
3.5. Microbial analysis
Terminal restriction fragments of 16 S rRNA genes were analyzed to evaluate changes in
microbial communities developed after 25 days of operation compared to initial microbial
communities (i.e. inoculum from biogas digester and acid pretreated inoculum used for cell
immobilization). Analysis of bacterial community with the T-RFLP method revealed small
changes in the structure between initial inoculum and acid pretreated inoculum, as their
similarity was 93.0 % (Fig. 7). Furthermore, their weak band intensity in bacterial as well as in
archaeal community showed that microbial activity in these two samples was low. Moreover,
bacterial diversity in initial inoculum and acid pretreated inoculum was considerably higher
than that after 25 days of operation, confirming that the employed operating conditions (pH =
4.0 ± 0.2 and HRT = 3 h) enabled formation of selectively enriched community capable of
hydrogen production. Bacterial microbial community developed on the carrier materials after
25 days of operation at low pH value and hydraulic retention time of 3 h was completely
different from initial inoculum and inoculum used for immobilization, since their similarity
ranged from 3.1 to 8.2 %. Bacterial communities developed on different support materials
exhibit high similarity between R1 and R2 reactors (similarity over 92.5 %), while similarity of
bacterial community in the R3 unit compared to R1 and R2 was found to be lower (84.4 %).
Quite high similarity values among the bacterial communities allude that different support
materials don’t exhibit a major impact on the microbial community structure. Bacterial
diversity, assessed by the number of clear T-RFLP bands, was similar in R1 and R2. Markedly
lower diversity was observed for bacterial community in R3. Considering the fact that higher
hydrogen yields were achieved in R1 (1.80 mol H2/mol glucose) and R2 (1.74 mol H2/mol
glucose) compared to R3 (1.46 mol H2/mol glucose), it is suggested that higher bacterial
diversity enables higher hydrogen yields. Similar results about the microbial diversity were
obtained by Ren et al. [46], where hydrogen production was evaluated for pH ranges of 6.0-6.5,
5.5-6.0 and 4.0-4.5, corresponding to propionate type, ethanol-butyrate type and ethanol type
fermentation, respectively. DGGE analysis further showed higher microbial diversity during
ethanol type and ethanol-butyrate type fermentations that were accompanied with higher
hydrogen production rates. Although in this work taxonomical confirmation of presumptive
bacteria such as Clostridium, Bacillus, Enterobacter and Klebsiella was not identified, it is well
known that these populations are involved in stable and efficient hydrogen production [29,30].
Analysis of archaeal community revealed similar quantity of archaea in initial inoculum and
acid pretreated inoculum as well as lower quantity after 25 days of operation in all APBRs,
which was accompanied with the fact that archaeal community had much lower abundance than
bacterial community. Slightly lower diversity of archaeal community in R1 unit compared to
R2 and R3 reactors could be associated with higher hydrogen yield in R1, as archaea could be
associated with substrate depletion. As in the case of bacterial community, significant changes
at the end of long-term operation were observed also in archaeal community. The similarity of
archaeal community in initial inoculum and inoculum used for immobilization compared to
archaeal community after 25 days of operation was namely 26.6 % (Fig. 8). There was no
methane detected in the biogas formed, which is in agreement with the findings of Han et al.
[47], who reported relatively narrow pH range of 5.5-8.2 for methane production and its
inhibition at pH values lower than 6. Thus, we could reasonably assume that the employed
operating conditions allowed only the development of non-methanogenic archaeal community,
or T-RFLP bands were due to inactive archaeal cells or DNA of dead cells that is amplified
during T-RFLP analysis.
4. Conclusions
This study has demonstrated that the examined support materials (Mutag BioChipTM, expanded
clay and activated carbon) were adequate for cell immobilization and further formation of
biofilm containing hydrogenogenic bacteria at low pH values. The results showed that APBRs
packed with either Mutag BioChipTM (R1) or expanded clay (R2) achieved higher hydrogen
production performance compared to the reactor filled with activated carbon (R3). The
hydrogen yield (1.80 mol H2/mol glucose), production of HAc (1033-1246 mg/L) and HBu
(890-1041 mg/L) as well as the amount of biomass adhered (0.07 g VSS/gsupport) were found to
be the highest in R1. Moreover, the biomass detachment rates in R1 were lower than in R2 and
R3. The better performance of R1 reactor system might be attributed to the properties of Mutag
BioChipTM carrier and used inoculum, as both Mutag BioChipTM and inoculum exhibited
hydrophobic characteristics, while the other two supports (expanded clay and activated carbon)
were hydrophilic. Moreover, better adsorption in R1 could be achieved due to numerous
macropores on the surface of Mutag BioChipTM. Finally, in the given range of operating
conditions stable performance of APBRs for biohydrogen production (20 days) was obtained.
Acknowledgements - This research was financially supported by the Ministry of Education,
Science and Sport of the Republic of Slovenia through Research program P2-0150. The authors
gratefully acknowledge Dr. Muhammad Shahid Arshad for his work on the SEM microscope
and Dr. Gregor Žerjav for performing the hydrophilicity/hydrophobicity measurements.
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Table 1. Characteristics of anaerobic sludge used as inoculum.
Density (g/mL)
1.01 ± 0.07
pH value (/)
8.15 ± 0.14
TS (g/kg)
25.0 ± 2.8
TVS (g/kg)
13.8 ± 1.3
TVS (%)
67.7 ± 5.3
C/H/N/S (wt. %)
34.7 ± 1.9/4.9 ± 0.6/5.5 ± 1.8/1.6 ± 1.2
Hydrophobicity index
69.4 ± 7.2
Table 2. Characteristics of support materials.
Mutag BioChipTM
Expanded clay
Activated carbon
Diameter (mm)
Length (mm)
Macropore size (μm)
50 - 300
Point of zero charge
Material: polyethylene. bChemical composition: 67.7 % O, 13.4 % Si, 10.2 % Fe, 8.7 % Al.
Table 3. Performance of hydrogen production in APBRs during 25 days operation.
Support material
H2 content
H2 production
H2 yield
(vol. %)
(mmol/(L h))
Mutag BioChipTM (R1)
99.1 ± 6.2
52.9 ± 2.5
16.65 ± 2.65
1.80 ± 0.22
Expanded clay (R2)
98.9 ± 4.6
53.2 ± 2.8
15.55 ± 2.06
1.74 ± 0.18
Activated carbon (R3)
98.0 ± 7.3
52.3 ± 3.4
13.65 ± 1.78
1.46 ± 0.12
Table 4. Soluble metabolites distribution in different APBR processes for dark fermentative
hydrogen production.
pH value
Y H2
4.0 - 5.0
This study
This study
This study
HAc: acetic acid; HBu: butyric acid; HPr: propionic acid; Eth: ethanol; HAc/HBu: molar
acetate to butyrate ratio; HAc/Eth: molar acetate to ethanol ratio.
Table 5. Summary of microbial biomass dynamics in APBRs during 25 days operation.
Support material
VSS/ (g VSS/L)
(g VSS/d)
Mutag BioChipTM (R1)
0.07 ± 0.01
0.07 ± 0.02
0.10 ± 0.05
0.11 ± 0.01
Expanded clay (R2)
0.04 ± 0.01
0.15 ± 0.03
0.12 ± 0.06
0.08 ± 0.02
Activated carbon (R3)
0.03 ± 0.01
0.22 ± 0.03
0.16 ± 0.05
0.18 ± 0.04
Figure 1. Schematic drawing of anaerobic packed-bed reactor (APBR) for continuous
hydrogen production. Legend: (1) synthetic wastewater reservoir, (2) peristaltic pump, (3)
APBR reactor filled with different support materials, (4) gas-liquid separator, (5) gas measuring
system connected to -GC analyzer.
lactic acid
formic acid
acetic acid
propanoic acid
butyric acid
H2 production rate (NmL/(L h))
H2 production rate (NmL/(L h))
Soluble metabolites concentration (mg/L)
malonic acid
pentanoic acid
succinic acid
Mutag BioChip
Expanded clay
Activated carbon
Figure 2. Concentration of soluble metabolites during the steady state of immobilization (after
4 days for Mutag BioChipTM and activated carbon, and after 5 days for expanded clay) (a) and
H2 production rate (b) in APBRs containing different support materials.
Figure 3. SEM images of the surfaces on Mutag BioChipTM (a), expanded clay (b) and activated
carbon (c) at the end of immobilization period.
H2 yield
HAc/HBu ratio
HAc/HBu ratio
lactic acid
butyric acid
H2 yield (mol H2/mol glucose)
Glucose concentration (g/L)
malonic acid
succinic acid
formic acid
acetic acid
propionic acid
oxalic acid
valerianic acid
Soluble metabolites concentration (mg/L)
Time (day)
Time (day)
H2 yield
HAc/HBu ratio
Time (day)
Time (day)
lactic acid
butyric acid
H2 yield
HAc/HBu ratio
H2 yield (mol H2/mol glucose)
malonic acid
succinic acid
formic acid
acetic acid
propionic acid
oxalic acid
valerianic acid
Soluble metabolites concentration (mg/L)
Time (day)
HAc/HBu ratio
Glucose concentration (g/L)
HAc/HBu ratio
lactic acid
butyric acid
H2 yield (mol H2/mol glucose)
Glucose concentration (g/L)
malonic acid
succinic acid
formic acid
acetic acid
propionic acid
oxalic acid
valerianic acid
Soluble metabolites concentration (mg/L)
Time (day)
Figure 4. Variations in glucose conversions and soluble metabolites production (Mutag
BioChipTM (a), expanded clay (c), activated carbon (e)) as well as H2 yield and HAc/HBu ratio
(Mutag BioChipTM (b), expanded clay (d), activated carbon (f)). HAc/HBu: molar acetate to
butyrate ratio.
Biomass attached (g VSS/gsupport)
Time (day)
Biomass washout (g VSS/Leffluent)
Time (day)
Figure 5. Temporal variations in attached biomass (a) and effluent VSS concentrations (b).
Figure 6. SEM images of support materials (Mutag BioChipTM (a), expanded clay (c) and
activated carbon (e)) and biofilms in APBRs at HRT of 3 h (R1 (b), R2 (d) and R3 (f)).
Figure 7. Pearson correlation dendrogram of bacterial 16S rRNA gene T-RFLP profiles (AS:
anaerobic sludge; AS 5.5: anaerobic sludge with pH value adjusted to 5.5).
Figure 8. Pearson correlation dendrogram of archaeal 16S rRNA gene T-RFLP profiles (AS:
anaerobic sludge; AS 5.5: anaerobic sludge with pH value adjusted to 5.5).
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