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Evaluation of hydrogen and methane productionfrom sugarcane vinasse in an anaerobic fluidizedbed reactor
Cristiane Marques dos Reis a, Mariana Fronja Carosia b,Isabel Kimiko Sakamoto b, Maria Bernadete Amancio Varesche b,Edson Luiz Silva a,*
a Department of Chemical Engineering, Federal University of S~ao Carlos, Rod. Washington Luis, Km 235, 13565-905
S~ao Carlos, SP, Brazilb Department of Hydraulics and Sanitation, School of Engineering of S~ao Carlos, University of S~ao Paulo, Av.
Trabalhador S~ao Carlense, 400, 13566-590 S~ao Carlos, SP, Brazil
a r t i c l e i n f o
Article history:
Received 18 February 2015
Received in revised form
17 April 2015
Accepted 25 April 2015
Available online 23 May 2015
Keywords:
Distillery wastewaters
Co-fermentation
Hydrogen
Hydraulic retention time
Methane
* Corresponding author. Tel.: þ55 16 3351826E-mail address: edsilva@ufscar.br (E.L. Si
http://dx.doi.org/10.1016/j.ijhydene.2015.04.10360-3199/Copyright © 2015, Hydrogen Energ
a b s t r a c t
This study evaluated the hydrogen and methane production from sugarcane vinasse in an
anaerobic fluidized bed reactor. Two reactors were operated with two different substrate
concentrations: R5 (5 g COD L�1) and R10 (10 g COD L�1). During the first stage, glucose was
used as the primary carbon source; vinasse was then added from 0% to 100% of the organic
source in hydraulic retention time (HRT) of 6 h. Later, HRT was changed to 4, 2 and 1 h.
The best hydrogen production rate was 0.57 L h�1 L�1 (R5, HRT ¼ 1 h, 100% vinasse).
Thebest hydrogen yieldwas 3.07mmolH2g�1 CODadded (R5,HRT¼ 6h, vinasse:glucose¼ 1:3).
Main metabolites were ethanol, butyric acid, propionic acid and methanol. Denaturing
gradient gel electrophoresis analysis identifiedPrevotella sp. andMegasphaera sp. belonging to
the Bacteria domain and Methanobacterium sp. and Methanosphaera sp. belonging to the
Archaea domain.
Copyright © 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights
reserved.
Introduction
Sugar and ethanol industry is one of themain segments of the
agribusiness sector in Brazil. In addition to the sugar and
alcohol, the main products, nearly all of the sub-products of
the industry, including bagasse, molasses and sugarcane
vinasse, are processed [1]. Vinasse, one of the major byprod-
ucts of the ethanol production process with nearly 14 L of
4; fax: þ55 312 16 335182lva).36y Publications, LLC. Publ
vinasse produced per liter of ethanol, can cause extensive
pollution due to its high organic load (up to 40 g COD L�1), and
it is a potential anaerobic digestion source [2].
In general, vinasse is a low pH brown-colored residue con-
taining particulate matter and high concentrations of organic
and inorganic compounds [3]. Phenolic compounds (such as
humicacidand tannicacid), themelanoidins (resulting fromthe
reaction of sugars and proteins by the Maillard reaction),
caramel and the furfural components contribute to its color [4].
66.
ished by Elsevier Ltd. All rights reserved.
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 4 0 ( 2 0 1 5 ) 8 4 9 8e8 5 0 9 8499
Thepresenceof thesecompoundsmakesvinasseacomplexand
difficult compound for degradation.
The anaerobic digestion of vinasse has the potential to
reduce the organic load of this residue while generating bio-
gases such as methane (CH4) or hydrogen (H2). Few studies,
however, focus on the energy recovery in the form of H2.
In general, the production of CH4 and H2 is a two-stage pro-
cess involving separation of the acidogenic and the methano-
genic stages. The combination of H2 and CH4 is more efficient
than CH4 alone when used as combustion engine fuel. In addi-
tion, the CH4 and H2 mixture reduces the emission of green-
house gases such as CO, CO2 and unburnt hydrocarbons [5].
Biological H2 production can be accomplished using simple
or complex substrates.Wastewater fromanumerous industries
including thedairy [6], brewery [7], andethanol-sugar industries
[8e10] can be used for biological hydrogen production.
The presence of inhibitory compounds in the distillery
wastewaters canbeabarrier to theanaerobicdigestionprocess
despite the rich amount of organic matter in the wastewater.
Thus, an adjustment period is advisable to promote favorable
consumption of complex substrates by themicroorganisms in
the environment. When added in small quantities, the micro-
bial culture may adapt to the complex residue or to the inhib-
itory compounds [11]. Thus, the use of a simple co-substrate
can improve the microbial degradation of refractory sub-
stances in complex residues such as stillage. Some authors
[8,12,13] who employed distillery residues have also started
adapting or conditioning the reactor to produce hydrogenwith
simplesubstrates suchasglucoseandsucrose.So far, theeffect
of varying proportions of vinasse-glucose in thermophilic
conditions has been tested [14]. However, a study employing
different vinasse-glucose ratios forhydrogenproduction in the
mesophilic range has not been performed. The aim of this
study was to evaluate the production of H2 and CH4 in two
anaerobic fluidized reactors (AFBR) at room temperature
(22 ± 3 �C) with concentrations of 5 and 10 g COD L�1 obtained
from several ratios of diluted vinasse and glucose. Further-
more, the band profiles of the microbial communities were
investigated using polymerase chain reaction-denaturing
gradient gel electrophoresis (PCR-DGGE) in both the AFBRs.
Materials and methods
AFBR
Two acrylic fluidized bed reactors with the following di-
mensions were used: height 151 cm, diameter 3.1 cm, and
total volume of 1452 mL (Fig. 1). The support material used for
immobilizing the microorganisms was expanded clay: grain
sizes between 2.8 and 3.35 mm, a real density of 1.50 g cm�3,
porosity of 23% and minimum fluidization velocity of
1.24 cm s�1. Along the reactors, four samplerswere distributed
in order to collect support material for microbial
characterization.
Reactor start-up procedure
The inoculum was obtained from an upflow anaerobic sludge
blanket (UASB) reactor used for the treatment of swine
wastewater. Swine manure is known for possessing
hydrogen-producing bacteria. The sludge underwent heat
treatment to activate the acidogenic cells, as described in
methods proposed by Ref. [15] and adapted from Ref. [16].
The reactors R5 and R10 worked with distinct substrate
concentrations of 5 g COD L�1 and 10 g COD L�1, respectively.
Some nutrients were also added according to Leite et al. [17] at
the following concentrations (mg L�1): CO(NH2)2 (125);
NiSO4$6H2O (1); FeSO4 $7H2O (5); FeCl3$6H2O (0.5); CaCl2$6H2O
(47.0); CoCl2$2H2O (0.08); SeO (0.07); KH2PO4 (85.0); K2HPO4
(21.7); Na2HPO4$2H2O (33.4). To maintain the pH of the me-
dium between 4 and 5, hydrochloric acid (30%) and sodium
bicarbonate were used as buffer solutions. Both reactors were
operated in batch mode for 96 h in a closed circuit. After this
activation period, the reactors were operated in continuous
mode. Glucose was the only carbon source in the beginning.
However, vinasse was added to the feed gradually (0, 25, 75
and 100% of g COD L�1) until vinasse replaced glucose in the
feedmaking the total concentration of 5 g COD L�1 or 10 g COD
L�1 during the hydraulic retention time (HRT) of 6 h. When
vinasse was the only carbon source, the HRT was varied be-
tween 4, 2 and 1 h. Table 1 illustrates the experimental stages.
To facilitate the discussion of results, each operation phase
was named according to the vinasse content in the substrate
mixture and the HRTs for which reactors were operated, e.g.,
III (75%, 6 h) indicates a phase III where the reactors were fed
with a substrate mixture containing 75% vinasse and were
operated for an HRT of 6 h.
The vinasse used in this experiment was collected at the
Usina S~aoMartinho distillery plant (Pradopolis, SP, Brazil), and
it was stored frozen. Table 2 illustrates the main physical and
chemical properties of vinasse employed. The raw vinasse
had an average concentration of 40 g COD L�1 which was
diluted to achieve the desired concentration in each reactor:
R5 (5 g COD L�1) and R10 (10 g COD L�1). Both reactors were
operated for 364 days and each stage lasted for 40 days in
average.
Analytical methods
The pH, COD, nitrogen, phosphate, sulfate, magnesium, cal-
cium, potassium and the amount of volatile suspended solids
were analyzed according to the Standard Methods for the
Examination of Water and Wastewater [18]. Analyses of total
volatile acids were performed according to the methodology
proposed by Dilalo and Albertson [19], whereas bicarbonate
alkalinity was estimated as described by Ripley et al. [20]. The
total reducing sugars were measured according to Dubois
et al. [21]. The organic acids and alcohol concentrations were
measured by liquid chromatography (HPLC Shimadzu)
equipped with a pump (LC-10ADVP), an autosampler (SIL-20A
HT), a column oven (CTO-20A) at 43 �C, a refractive index
detector (RID-10A), a system Controller (SCL-10AVP) and col-
umn HPX-87H Aminex (300 mm, 7.8 mm, BioRad). The mobile
phase consisted of H2SO4 (0.01 N) at 0.5 mL min�1. The biogas
hydrogen content was determined by gas chromatography
(GC-2010, Shimadzu, Japan) using a thermal conductivity de-
tector (TCD) with argon as the carrier gas and a Supelco Car-
boxen 1010 Plot packed column (30 m � 0.53 mm i.d.).
Volumetric hydrogen production was measured by the Ritter
Fig. 1 e Schematic description of the mesophilic AFBR.
Table 1 e AFBR operating conditions for H2 and CH4
production from glucose and vinasse in R5 and R10.
Phases Substrate mixture HRT(h)
Glucose/Vinasse
variation
I 100% glucose 6
II 75% glucose; 25% vinasse 6
III 25% glucose; 75% vinasse 6
IV 100% vinasse 6
HRT variation V 100% vinasse 4
VI 100% vinasse 2
VII 100% vinasse 1
i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 4 0 ( 2 0 1 5 ) 8 4 9 8e8 5 0 98500
MilliGas-counter (Type: MGC-1). Hydrogen yield and the
hydrogen production rate were measured as it follows:
� Hydrogen production rate (HPR) ¼ {[Biogas production
measured in the MilliGas-counter (L.h�1)]*(% H2)/[Real vol-
ume of reactor based on bed height of particules (L)]}.
� Hydrogen Yield (HYadded) ¼ {[HPR (L h�1 L�1)]/[Organic
Loading Rate (g L�1 h�1)*22.4 L mol�1]}.
Table 2 e Main physical and chemical properties of rawsugarcane vinasse.
Parameters Vinasse
pH 5.0 ± 0.18
COD (mg L�1) 42,818 ± 4391
Total volatile acids (mg L�1) 3012 ± 512
Total alkalinity (mg CaCO3 L�1) 292 ± 38
Volatile suspended solids (mg L�1) 287 ± 72
Total Kjeldahl nitrogen (mg L�1) 244 ± 36
Phosphate (mg L�1) 3796 ± 387
Sulfate (mg L�1) 1400 ± 130
Magnesium (mg L�1) 580 ± 52
Calcium (mg L�1) 757 ± 151
Potassium (mg L�1) 4500 ± 670
Molecular analysis of the microbial community
At the end of each operation stage of the reactors R5 (5 g COD
L�1) and R10 (10 g COD L�1), particle samples were collected
through the samplers distributed laterally along the reactors.
To disengage the biomass attached to the support material,
the particles were washed three times with PBS solution
(NaCl, KCl, Na2POH4, KH2PO4) and centrifuged for 3 min at
3000 rpm.
The extraction of genomic DNA from the samples was
performed by cell lysis [22]. For this procedure we used glass
beads, phenol, chloroform and buffer solution. The 16S rRNA
fragments were amplified by PCR. The primers used were the
following: 968FGC-1401R for the Domain Bacteria [23], and
Parch519fGC-Arch915r for the Domain Archaea [24]. The PCR
amplification product was subjected to electrophoresis in
DGGE for both domains, Bacteria and Archaea, in order to
separate the amplified DNA fragments. The gels contained
45e60% linear denaturing gradient (100% denaturant of 7 M
urea and 40% (v/v) of deionized formamide). Electrophoresis
was run under 75 V and 60 �C for 16 h. The band profiles were
visualized in a chamber with a transilluminator Eagle Eye III
TM (Stratagene) at 254 nm and the images were captured by
Eagle Vista. The BioNumerics 2.5 software was used to
calculate the similarity coefficient (Pearson correlation) from
the band profiles. Clustering was performed using the UPGMA
(unweighted Pair Group Method using Average).
The best condition for biogas production was obtained
from cloning and sequencing the 16s rRNA for which we used
primer 27F-1100R [25] for the Bacteria Domain and primer 21F
Arch958R-for the Archaea Domain [24]. The vector pGEM®
Easy Vector System (Promega) was used and transformed into
competent cells of Escherichia coli. The cloned DNA was then
subjected to PCR using the M13F and M13R primers.
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 4 0 ( 2 0 1 5 ) 8 4 9 8e8 5 0 9 8501
The PCR products were sent to Macrogen® (www.
macrogen.com) for the analysis of the nucleotide sequences.
The sequences were compared with the data from the Na-
tional Center for Biotechnology Information (NCBI) database
Ribosomal Database Project and http://blast.ncbi.nlm.nih.gov/
(http://rdp.cme.msu.edu/seqmatch). The phylogenetic trees
were developed using the MEGA 6.0 software using the
“neighbor-joining” method in a Bootstrap analysis method
with 1000 replications. The sequences from this study were
deposited in GenBank with accession numbers ranging from
KM820898 to KM820906.
Fig. 3 e Biogas content throughout the operation phases in
R10 (10 g COD L¡1).
Results and discussion
Biogas production
Figs. 2 and 3 show the biogas content for the reactors R5 (5 g
COD L�1) and R10 (10 g COD L�1), respectively. Both reactors
produced only H2 and CO2 till phase III (75%, 6 h). However,
CH4 was generated after vinasse became the only carbon
source.
After phase V (100%, 4 h), we observed a reduction in the H2
content along with HRT reduction and reached a minimum
value of 5% for the reactor R5 (5 g COD L�1). At the HRT 1 h, the
reactor stopped producing CH4 in R5 so the content of H2
reestablished and reached 34%. In reactor R10 (10 g COD L�1),
the CH4 production persisted despite the reduction of HRT,
even under turbulent conditions as the HRT 1 h.
Maintaining the pH in acidic conditions and performing
heat treatment of the inoculum are methods of inactivating
the methanogens [26]. In general, methanogenic archaea are
inhibited below pH 6. However, it was verified that H2-con-
sumers are more tolerant to acidic conditions than other CH4-
producers [16].
In this study, there was a variation in biogas composition
when the carbon source was changed from a mixture of
glucose/vinasse (phases I (0%, 6 h), II (25%, 6 h), III (75% 6 h)) to
a composition of vinasse (phase IV (100%, 6 h)).
Xia et al. [27] showed that the mixture cellulose/xylose led
to CH4 production, whereas a mixture cellulose/glucose
Fig. 2 e Biogas content throughout the operation phases in
R5 (5 g COD L¡1).
generated only H2 and CO2 as the biogases. Moreover, Cha-
ganti et al. [28] found that the use of the mixture glucose/
xylose resulted in a better result for H2 production than the
use of separatemediums of glucose and xylose. Oh andMartin
[29] found that the availability of glucose may facilitate or
impede the presence of certain populations in anaerobic
digestion of ethanol stillage. Thus, high values of glucose
decrease the presence of hydrogenotrophic microorganisms,
while facilitating the presence of acetoclastic microorgan-
isms. In contrast, low glucose availability leads to the devel-
opment of hydrogenotrophic archaea. In the present work,
CH4 was produced when glucose was removed from feed.
Some studies using distillery wastewater also verified the
production of CH4 and H2. Espinoza-Escalante et al. [30] re-
ported the production of CH4 with H2 from stillage processing
during the production of tequila with pH below 4.5. Buitron
et al. [8] reported the production of CH4 (35e44%) alongwithH2
at HRT 24 h. CH4 production stopped at HRT 12 h. Possibly, the
methanogenswerewashed reactor hydrodynamics under this
condition. Searmisirimongkol et al. [9] also produced CH4
under an acidogenic condition. It was found that the higher
the applied organic load, the lower the percentage of CH4
generated. In this study, the production of CH4 in reactor R5
ceased when HRT was changed from 2 to 1 h.
Despite being a limiting factor for the production of H2, this
production of CH4 can be valuable. For example, the mixture
of 20% by volume of H2 and CH4, referred to as hythane, is
patented by Hydrogen Consultants Inc [5]. Furthermore, ac-
cording to Bauer and Forest [5], the presence of H2 in the
natural gas used as vehicle fuel helps the engine performance
and reduces the greenhouse gas emissions. Thus, the conjoint
generation of the biological CH4 and H2 can serve as a new
source for this fuel mixture.
Figs. 4 and 5 illustrate the hydrogen production rate (HPR)
and hydrogen yield (HY) across the different phases. In both
reactors, the HPR decreasedwith the addition of vinasse in the
feed. This indicates that the reactors were affected by the
presence of a new substrate.
The HY showed an irregular behavior from phases I to IV.
In R5 (5 g COD L�1), HY was maximum when vinasse was
75% of the organic load, reaching 3.07 mmol H2 g COD�1added
Fig. 4 e HPR and HY throughout the operation phases in R5 (5 g COD L¡1).
i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 4 0 ( 2 0 1 5 ) 8 4 9 8e8 5 0 98502
(14.74 mmol H2 g COD�1removed). It can be seen that glucose
acted in a positive way for the consumption of organic
matter. However, because of the CH4 production, H2 yield
dropped significantly in phase IV (100%, 6H), reaching
0.3 mmol H2 g COD�1added (4.87 mmol H2 g COD�1
removed). In
R10 (10 g COD L�1) there was a peak in stage II (25%, 6H),
1.32 mmol H2 g COD�1added (6.60 mmol H2 g COD�1
removed),
and due to the appearance of CH4 there was a reduction of
approximately 50% in stage IV (100%, 6 h). In stage VII (100%,
1 h), CH4 production ceased and HY increased to approxi-
mately 1.96 mmol H2 g COD�1added. This indicates a good
performance of AFBR for the H2 production from sugarcane
vinasse when CH4 production is inhibited. The best yield
when the reactor is operated solely with vinasse was
1.96 mmol H2 g COD�1added.
HPR in reactor R10 (10 g COD L�1) was gradually decreased
as the HRT was decreased. CH4 production continued even in
low HRT. Similar to R5, HY in R10 showed a downward trend
with decreasing HRT. The minimum value was obtained in
phase VII (100%, 1 h), reaching 1.96 mmol H2 g COD�1added.
The results of the present study are in accordance with the
literature on H2 production from complex wastes. Gahde et al.
[31] reported a yield of 10.95 mmol H2 g COD�1removed from
dairy wastewater. Compared with the present study, during
Fig. 5 e HPR and HY throughout the ope
the best conditions for biogas generation, a yield of
18.37 mmol H2 g COD�1removed in phase VII (100% vinasse, 1 h)
in reactor R5 was obtained.
A comparison between some studies using distillery
wastewaters for H2 production is shown in Table 3.
Intermediate metabolites production
Reactor R5 and R10 presented a similar trend in relation to
metabolites. The distribution values can be found in the
Supplementary material Table S1 and Table S2, respectively.
In phase I (0%, 6 h), when glucose was the only carbon source,
there was a preponderance of ethanol and butyric acid,
together amounting to more than 85% of the metabolites
produced. In Phase II (25%, 6 h), with the addition of vinasse
there was a shift in metabolic pathways. Ethanol production
ceased and a predominance of propionic acid production was
observed. When vinasse became the main substrate in phase
III (75%, 6 h), ethanol was again produced by the reactor and
methanol appeared as the predominant metabolite. From
phase IV to VII, both reactors presented mixed fermentation.
Recent studies [34e36] observed that ethanol can be pro-
duced concurrently with H2 without any loss in the produc-
tivity of the reactor. An alternative route (Equation (1)) was
ration phases in R10 (10 g COD L¡1).
Table 3 e Comparison of H2 production from distillery wastewaters in various studies.
Reactor HY Temperature (�C) Metabolites Reference
ASBRa 7.98 mmol H2 g COD�1d 35 HAc, HPr, HBu, HiBu, EtOH, Acetone [8]
ASBRa 6.77 mmol H2 g COD�1removed
d 37 HAc, HPr, HBu, HVal [9]
ASBRa 4.83 mmol H2 g COD�1removed
d 55 HBu, HVal, HAc, HPr, EtOH [10]
Batch 2.31 mmol H2 g COD�1added 55 HBu, HAc, HPr,EtOH [32]
APBRb 0.7 mmol H2 g COD�1added 55 HAc, HBu, EtOH, HPr [33]
AFBRc 2.23 mmol H2 g COD�1added 55 HLa, HBu, HiBu, HPr [14]
AFBRc 1.96 mmol H2 g COD�1added
18.37 mmol H2 g COD�1removed
22 MeOH, EtOH, HBu, HMal, HVal, HPr This study
a ASBR-anaerobic sequencing batch reactor.b APBR-anaerobic packed bed reactor.c AFBR-anaerobic fluidized bed reactor.d Based on data from article.
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 4 0 ( 2 0 1 5 ) 8 4 9 8e8 5 0 9 8503
launched [37] as a possible deviation followed during the
acidogenic process for producing H2 and ethanol:
C6H12O6 þH2O/C2H5OHþ CH3COOHþ 2H2 þ 2CO2 (1)
Propionic acid is harmful for anaerobic digestion because it
consumes the H2 generated by the system. In fact, during
Phase II (25%, 6 h) there was a decrease in HY. This can also be
attributed to the introduction of vinasse. According to equa-
tion (2), for each mole of propionic acid generated, there are
2 mol of H2 consumed:
C6H12O6 þ 2H2/2CH3CH2COOHþ 2H2O (2)
Yu et al. [38] also found considerable amounts of propionic
acid in an acidogenic system producing H2 from rice wine
vinasse. The authors showed 40% propionic acid among the
metabolites generated under pH 4, which decreased as the pH
was increased to 6 (20%).
Methanol was produced in large proportions when vinasse
was introduced to the system. Methanol can be produced
when CH4 is generated and oxidized by ruminant animals,
stabilization ponds, lakes and landfills. Furthermore, meth-
anol, formate and formaldehyde are intermediates in the
process of CH4 oxidation according to the equation (3) [39].
Thus, methanol is then used for the growth of methylotrophic
microorganisms and is readily converted into formaldehyde
dehydrogenase and the formate. There is little information
regarding methanol production such that both the process of
production and consumption are favorable, and preferably in
liquid media [39].
CH4/CH3OH/HCHO/HCOOH/CO2 (3)
In relation to the metabolites generated, the use of single
substrates such as glucose favored the production of butyrate.
While the use of a complex wastewater such as vinasse
favored the production of organic acids and other solvents
(mixed fermentation).
Composition of the microbial communities
In Fig. 6 a lower similarity coefficient of 55% was found for
biomass of phase I (0%, 6 h) compared with other phases of
operation for the populations of bacteria. At this stage, only
glucose was used as organic source at HRT 6 h. It was found
that between phase II (25%, 6 h) and phase III (75%, 6 h), the
similarity coefficient increased to 83%, probably due to the
presence of a new substrate, vinasse (25%), favoring the se-
lection of better adaptedmicroorganisms. Relative to phase IV
(100%, 6 h), there was a decrease in the similarity between the
populations in comparison with phase III, where a similarity
of 76% was observed. Possibly because of the use of vinasse as
the only carbon source, new microorganisms were favored.
Reactor R10 presented a similar behavior as compared to
reactor R5. The lowest similarity coefficient was obtained in
phase R10 I, where it had a value of 67% over the following
phases. The introduction of vinasse in the system led to an
increase in the coefficient of similarity, reaching 87% between
phases II and III, which were the most similar among the
samples. Between Phase IV and V, the coefficient of similarity
decreased to 84%, but stayed greater than 80%. Between
phases VI and VII the similarity coefficient was 77%. It was
found that increasing vinasse concentration in phases II, III
and IV, as well reducing HRT from 6 to 1 h in phases V, VI and
VII resulted in a decrease in the similarity. This is an indica-
tion that there may have been changes in bacterial pop-
ulations caused bywashing in lowHRT or by adaptation to the
presence of a more complex substrate such as vinasse.
Fig. 7a shows the bands profile of the reactor R5 (5 g COD
L�1) for the Archaea domain. At the beginning of the reactor
operation, the similarity between this phase and the other
was 70% and was the most distinctive of the operation. As the
operating phases were changed, there was an increase in
similarity between phases, indicating that there was a selec-
tion of Archaea as a function of the operating conditions
imposed. The greatest similarity in the latter stages was 95%,
which coincided with the highest CH4 production in the
reactor. The band profile for the reactor R10 (Fig. 7b) shows
that Phase II presents a similarity (55%) lower than the
following operational phases. During this step vinasse was
introduced in the system. Possibly, owing to a shock in this
population, only the most adapted were maintained under
this condition. From Phase IV, there are similarities ranging
from 77 to 97% between phases I, VI, VII and V, which may
indicate the adaptation of the microbial population to a
complex substrate such as vinasse.
Samples from phase VII (100%, 1 h) of reactor R5 were
chosen to be subjected to cloning and sequencing of frag-
ments of 16S rRNA gene, once it reached the best stage for H2
Fig. 6 e Coefficient of similarity by Pearson correlation of DGGE profile bands in relation to the operational steps applied in
reactors R5 (a) and R10 (b) for the domain Bacteria.
i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 4 0 ( 2 0 1 5 ) 8 4 9 8e8 5 0 98504
production with no CH4 production. For Bacteria Domain, 164
clones belonging to the phylum Firmicutes and Bacteroidetes
were obtained. Clones were grouped into five operational
taxonomic units (OTUs), as shown in Fig. 8.
Among all of the sequences obtained and shown in Table 4,
55% belonged to the phylum Bacteroidetes and uncultured
Prevotella, and 28% belonged to the phylum Firmicutes genus
Megasphaera. The presence of 3% of uncultured Clostridia also
belonging to the phylum Firmicutes was observed.
OTUs 1 and 2 were identified as belonging to the phylum
Bacteroidetes. OTU 1were related to uncultured Prevotella. The
Fig. 7 e Coefficient of similarity by Pearson correlation of DGGE
reactors R5 (a) and R10 (b) for the domain Archaea. NOTE: R10 II
populations were below the detection limit of the technique.
microorganisms affiliated to Prevotella spp. are non-
endospore-forming bacteria and strictly anaerobic. In gen-
eral, the Bacteroidia class, towhich Prevotella sp. is affiliated, is
characterized by the ability to grow on a variety of carbohy-
drates, and it can convert cellulose into acetate, propionate
and succinic acid [40]. During sucrose fermentation, succinic
acid is generated. Lactic acid and acetic acid are produced in
minor quantities. Some species can produce H2 at low con-
centrations when the process is directed to the acetate-
pathway [41]. In biogas production from food waste in batch
reactor, Shin et al. [42] also found Prevotella sp. According to
profile bands in relation to the operational steps applied in
I - this sample could not be amplified. Probably the archeal
Fig. 8 e Phylogenetic tree based on the consensus sequences of the microbial consortium clones with primers for Domain
Bacteria. Methanosarcina acetivorans was used as outgroup.
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 4 0 ( 2 0 1 5 ) 8 4 9 8e8 5 0 9 8505
the study, this species can produce acetic acid, succinic acid,
isobutyric acid and isovaleric acid from the degradation of
starch, sucrose and glucose. Li et al. [43] found the presence of
Prevotella spp. in fixed bed reactors used for H2 production
from sucrose at HRT 2 h. Won et al. [44] verified that Prevotella
spp. was present in greatest proportions at a pH of approxi-
mately 4.5, similar to the present study. The authors verified a
satisfactory H2 production (2.18 L H2 L�1) and hydrogen yield
(1.29 mol�1 H2 mol sucrose). Mariakakis et al. [41] observed
that Prevotella sp. wasmore frequent by increasing the organic
load under low HRT when producing H2 from hexose. Similar
to the present study, the best conditions for H2 production
occurred at the phase of the highest organic load, indicating
that these species is able to produce H2 in conditions of high
organic load. The cited studies demonstrated the feasibility of
H2 production by Prevotella sp. concomitant with organic acids
in a suitable environment, i.e., with a pH approximately 4.5
and availability of organic matter, such as observed in this
study with sugarcane vinasse.
In the present study, Megasphaera sp. accounted for 28% of
microorganisms found (OTU 2). According to Marchadin et al.
[45], these microorganisms belong to the phylum Firmicutes
and are able to metabolize carbohydrates producing lactate,
succinic acid, butyric acid, valeric acid and caproic acid. Acetic
acid and propionic acid can also be produced in low amounts.
Furthermore, this specie is sensitive to low pH and high con-
centrations of ethanol.
Castell�o et al. [46] used non-sterilized cheese whey in a
UASB reactor and pointedMegasphaera sp. as a possible source
of microorganisms for H2 production. However, it can also
convert lactic acid into propionic acid. The authors also found
Table 4 e 16S rRNA clone analysis of Bacteria.
OTU Organism affiliation Identity(%)
Phylum GeAcce
1 Uncultured bacterium 97 Bacteroidetes GQ4
Uncultured Prevotella sp. 96 JX57
2 Uncultured Prevotellaceae
bacterium
100 Bacteroidetes JF80
3 Megasphaera sp. 98 Firmicutes HM9
4 Uncultured bacterium 98 Firmicutes JQ07
5 Uncultured Clostridia bacterium 99 Firmicutes EU8
Prevotella sp. Similarly, in the present study, the authors found
out that H2 productionwas reduced due to the CH4 generation.
On the other hand, the H2 remained constant, showing that
thesemicroorganisms are also capable of generating H2. In the
study by Ohnishi et al. [47], Megasphaera sp. could produce H2
in low amounts, besides the organic acids cited. Santos et al.
[14] used vinasse from sugarcane produced in the same dis-
tillery plant of the present study (S~aoMartinho, Prad�opolis, SP,
Brazil) and verified the presence of the following microor-
ganisms: Lactobacillus sp. and Megasphaera sp. in thermophilic
AFBR for H2 production.
OTUs 4 and 5 were also related to the Firmicutes phylum.
OTU 5 is represented as an Uncultured Clostridia Bacterium
and these microorganisms corresponded to 3% of clones
present in this sample. Microorganisms belonging to the
Clostridia class are gram-positive and endospore-forming.
Microorganisms belonging to this class are able to degrade a
variety of organic substrates producing acetate, propionate
and butyrate. In minor proportions, these microorganisms
can also generate formate, lactate, succinate and caproate
[48,49].
The presence of these bacteria related to Clostridium spp. is
common in the process of H2 production from complex waste,
such as the vinasse that was used in the present study, and
are observed in other studies in the literature [13,32,50].
Table 5 illustrates the fermentation products generally
generated by the predominant microorganisms found in the
present study according to literature. Propionic acid and lactic
acid are among these products. In the present work, the
concentration (721 ± 360 mg L�1) of propionic acid from phase
II (25%, 6 h) was the highest among the metabolites.
nBankssion n�
Number of sequence Sequencelength (pb)
Relativeabundance
(%)
77873.1 11 399 7
5984.1
6757.1 91 321 55
90965.2 46 525 28
2158.1 11 576 7
87973.1 5 471 3
Table 5 e Comparison among the fermentation products from microorganisms predominantly found in R5 reactor(5 g COD L¡1).
Organism affiliation Relativeabundance (%)
Fermentationproducts
Substrate Reference
Uncultured Prevotellaceae
bacterium
55 HSu,HAc,HLa,H2a Sucrose [41]
Megasphaera sp. 28 HBu, HVal, HAc, HPr, H2a Food waste [47]
a Low amounts.
i n t e rn a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 4 0 ( 2 0 1 5 ) 8 4 9 8e8 5 0 98506
Megasphaera sp. is able to synthesize propionate from lactic
acid, increasing the quantity of this acid in the system [46]. A
decrease in lactic acid concentration was verified in both re-
actors while the concentration of propionic acid increased in
the system.
In Phase IV (100%, 1 h), the reactor R5 resulted in a wide
distribution of metabolites in terms of soluble microbial
products (SMP) such as propionic acid (14.37% SMP), isobutyric
acid (13.09% SMP) butyric acid (11.95% SMP), propionic acid
(10.71% SMP), malic acid (9.99% SMP), valeric acid (6.37% SMP),
acetic acid (6.34% SMP) and lactic acid (1% SMP). These me-
tabolites are consistent with the major metabolites generated
by the microorganisms in the reactor R5 (Table 5).
Methanol (22% SMP) and ethanol (12.26% SMP) were also
produced in large quantities, but they are not reported as
fermentation products in Table 5. Some species relating to this
class are able to generate methanol under the conditions
shown in present study, for example, Clostridium butyricum in
pectin fermentation [49]. As already mentioned, microorgan-
isms affiliated to the Clostridia class were also observed in the
samples of present study.
Zeikus and Schink [51] conducted an experiment to
demonstrate the degradation of pectin by various microbial
cultures such as C. butyricum. It was found that the major
fermentation product from this strain was methanol. The
control sample with glucose in the absence of methanol led to
generation of other metabolites. In contrast with the present
study, vinasse sample can contain pectin residue in its
composition as a result of bagasse processing in the ethanol
production stage. Then, pectin can be used by certain Clos-
tridium species for methanol formation.
Table 6 shows the 16S rRNA clone analysis of Archaea
found in this sample. For the Domain Archaea, four OTUS
were identified (Fig. 9). OTUs 1, 2 and 3 were related to un-
cultured archaeon affiliated to genus Methanobacterium. These
microorganisms are hydrogenotrophic methanogens, i.e.,
they use CO2 and H2 to form CH4 [52]. As expected, the
presence of this microorganism is responsible for CH4 pro-
duction, and consequently the decrease in H2 production in
Table 6 e 16S rRNA clone analysis of Archaea.
OTU Organism affiliation Identity(%)
Genus
1 Uncultured Methanobacterium sp. 99 Methanobacterium
2 Uncultured archaeon 97 Methanobacterium
3 Uncultured archaeon 98 Methanobacterium
4 Uncultured Methanosphaera sp.
Uncultured archaeon
94 Methanosphaera
the present study. The production of CH4 arises from the
consumption of 4 mol of H2 and 1 mol of CO2 releasing CH4
and H2O.
In a study employing synthetic stillage, Rodriguez et al. [53]
also noted the presence of microorganisms belonging to the
genus Methanobacterium in low sulfate concentrations (higher
ratio of substrate/sulfate). In contrast, when there was a
smaller substrate/sulfate ratio, these organisms disappeared
from the reactor. Rosa et al. [54] found this microbial culture
(Methanobacterium) in a study using cheese whey as organic
substrate for H2 production in a fluidized bed reactor. Simi-
larly, in the present study, the presence of CH4 occurred at low
pH of approximately 4.5.
OTU 4 was related to the uncultured Methanosphaera,
accounting for 1% of the representative sequences. This
archaea is capable of reducing CH4 to methanol using H2. It is
present in animal gut. The presence of such microorganism
explains the appearance of CH4 in the reactor due to the large
presence of methanol in the system. Kim et al. [55] also noted
the presence of Methanosphaera stadtmanae in sludge from the
treatment of swine manure.
Low pH (approximately 4.5) and low HRT (1 h) are harsh
conditions for the CH4 formation by methanogenic archaea.
However, this adverse condition can be offset by other con-
ditions such as the provision of substrate, favoring the
development of this species. The presence of important me-
tabolites can lead to the growth of these microorganisms in
the reactor, beyond the duration of operation of the reactors,
up to 200 days, favoring the adaptation of the methanogenic
archaea in the reactor.
Conclusions
From this study, it was possible to verify the feasibility of
producing biogas from sugarcane vinasse using anaerobic
fluidized-bed reactors. However, the maintenance of a low pH
(4e5) was not sufficient to inactivate the methanogenic
organisms.
GenBankaccession n�
Number ofsequence
Sequencelength (pb)
Relativeabundance (%)
KC533586.1 81 385 94
EU447673 1 540 1
KF848976.1 3 480 3
AB935212.1
KJ676678.1
1 360 1
Fig. 9 e Phylogenetic tree based on the consensus sequences of the microbial consortium clones with primers for Domain
Archaea. Uncultured Prevotella was used as outgroup.
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n en e r g y 4 0 ( 2 0 1 5 ) 8 4 9 8e8 5 0 9 8507
The use of glucose as an auxiliary substrate was effective
as a co-substrate for H2 production. In general, the maximum
H2 production in the concentration of 5 g COD L�1 and under
the hydraulic retention time of 1 h: 0.57 L h�1 L�1 and
1.96 mmol H2 g COD�1removed was obtained when vinasse was
the only organic source.
Mainmetabolites were ethanol, butyric acid andmethanol.
Microbial characterization identified Prevotella sp. and Mega-
sphaera sp. belonging to the domain Bacteria and Meth-
anobacterium sp. and Methanosphaera sp. belonging to the
domain Archaea.
Acknowledgements
The authors gratefully acknowledge the financial support of
CNPq e National Council for Scientific and Technological
Development, CAPES e Coordination for the Improvement of
Higher Education Personnel, and FAPESPe S~ao Paulo Research
Foundation.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.ijhydene.2015.04.136.
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