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Continuous thermophilic hydrogen production andmicrobial community analysis from anaerobicdigestion of diluted sugar cane stillage

Samantha Christine Santos a, Paula Rubia Ferreira Rosa b,Isabel Kimiko Sakamoto a, Maria Bernadete Amancio Varesche a,Edson Luiz Silva b,*aDepartment of Hydraulics and Sanitation, School of Engineering of Sao Carlos, University of Sao Paulo,

Av. Trabalhador Sao-carlense, 400, 13566-590 Sao Carlos, Sao Paulo, BrazilbDepartment of Chemical Engineering, Federal University of Sao Carlos, Rod. Washington Luis, km 235,

13565-905, Sao Carlos, Sao Paulo, Brazil

a r t i c l e i n f o

Article history:

Received 21 October 2013

Received in revised form

27 March 2014

Accepted 30 March 2014

Available online xxx

Keywords:

Fermentation

Thermoanaerobacterium sp.

Clostridium sp.

Expanded clay

Co-substrate

* Corresponding author. Tel.: þ55 16 3351826E-mail address: [email protected] (E.L. Si

Please cite this article in press as: Santosanalysis from anaerobic digestion of didx.doi.org/10.1016/j.ijhydene.2014.03.241

http://dx.doi.org/10.1016/j.ijhydene.2014.03.20360-3199/Copyright ª 2014, Hydrogen Ener

a b s t r a c t

The aim of this study was to promote biohydrogen production in an thermophilic anaer-

obic fluidized bed reactor (AFBR) at 55 �C using a mixture of sugar cane stillage and glucose

at approximately 5000e5300 mg COD L�1. During a reduction in the hydraulic retention

time (HRT) from 8, 6, 4, 2 and 1 h, H2 yields of 5.73 mmol g CODadded�1 were achieved (at HRT

of 4 h, with organic loading rate of 52.7 kg COD m�3 d�1). The maximum volumetric H2

production of 0.78 L H2 h�1 L�1 was achieved using stillage as carbon source. In all oper-

ational phases, the H2 average content in the biogas was between 31.4 and 52.0%. Butyric

fermentation was the predominant metabolic pathway. The microbial community in

accordance with the DGGE bands profile was found similarity coefficient between 91 and

95% without significant changes in bacterial populations after co-substrate removal. Bac-

teria like Thermoanaerobacterium sp. and Clostridium sp. were identified.

Copyright ª 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights

reserved.

Introduction

Population growth associated with the development of eco-

nomic activity and rising income levels leads to a need for

increased energy production [1]. The use of fossil fuelsmust be

minimized due to emissions of greenhouse gases and their

impactonglobalwarming [2]. It is in this context that, according

to Souza et al. [3], the use of ethanol has attracted great atten-

tion in countries that are interested in reducing these effects.

4; fax: þ55 16 33518266.lva).

SC, et al., Continuous tluted sugar cane stillag

41gy Publications, LLC. Publ

Brazil is the second largest producer of ethanol and pro-

duced 25,780,404 m3 of ethanol in 2010/2011 [4]. Nevertheless,

with each liter of ethanol produced, approximately 5e10 L of

stillage is generated, and the disposal of this effluent, released

at temperatures between 85 and 90 �C, poses a high risk of

environmental impact due to high organic loads, a pH of

approximately 4e5, and the presence of recalcitrant com-

pounds [5]. As a solution to this issue, the anaerobic digestion

of stillage may result in the use of high organic loads con-

tained in this agro-industrial wastewater for biogas energy

hermophilic hydrogen production and microbial communitye, International Journal of Hydrogen Energy (2014), http://

ished by Elsevier Ltd. All rights reserved.

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http://dx.doi.org/10.1016/j.ijhydene.2014.03.241



Table 1 e Maximum and minimum values ofphysicalechemical parameters of raw sugar cane stillagecharacterization.

Parameters Minimum Maximum

pH 4.61 � 0.13 5.06 � 0.22

TOCa (ppm) 2342 � 136 5235 � 426

mg L�1

totalCODb 30.406 � 3217 33.797 � 3019

totalN (Kjedahl) 436 � 59 861 � 72

totalP as PO4�3 147 � 12 181 � 15

totalS as SO4�2 1400 � 156 2600 � 242

Malic acid 2639 � 395 6168 � 1040

Succinic acid 967 � 140 3624 � 518

Lactic acid 4558 � 397 12,697 � 1054

Formic acid 978 � 54 3556 � 210

Acetic acid 348 � 29 1617 � 112

Butyric acid 269 � 42 885 � 157

Iso-butyric acid 1547 � 433 4597 � 849

Propionic acid 314 � 21 911 � 67

a TOC: total organic carbon.b COD: chemical oxygen demand.

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 x x x ( 2 0 1 4 ) 1e1 22

generation, through conversion of organic substrates to

methane, performed by methanogenic archaea, and to H2,

performed by acidogenic bacteria, primarily with regard to

possible use of this biogas as an alternative energy source to

the use of CH4.

Accordingly, the feasibility of generating hydrogen from

organic wastewater, which is similar in composition to

sugar cane stillage, such as from alcohol distilleries [6],

and from molasses has been demonstrated over the last

decade [7e9]. The potential of these effluents has been

shown under mesophilic conditions in continuous reactors

[10,11].

The production of energy from stillage is highly variable

and depends on the raw materials and various operational

aspects of the ethanol production process. The presence of

recalcitrant substances, such as phenolic compounds, high

sulfate levels, heavy metals, melanoidins, glycerol and

other xenobiotics [5] can impair or inhibit microbial degra-

dation. Xia et al. [12] found that the treatability and

bioavailability of complex substrates may be improved by

adding a co-substrate, thus representing an optimal strat-

egy for auxiliary microbial degradation. A number of studies

of hydrogen production have been performed, particularly

regarding the use of glucose plus xylose [12] and galactose

[13].

The temperature represents another factor to be consid-

ered in improving the microbial conversion to H2. A variety of

real and synthetic substrates has been studied for possible H2

production by thermophilic fermentation processes, primarily

in batch reactors using palm oil [14], cheesewhey [15], cassava

[16], cellulose [12], rice straw [17] and wastewater from dis-

tilleries [6]. The effectiveness of thermophilic hydrogen pro-

duction was observed in continuous reactors digesting cheese

whey [18] and distillery wastewater [19].

The temperature has been a determining factor in the

biological production of hydrogen. The low solubility of this

biogas in the aqueous phase and the subsequent H2 facilitated

transfer from the liquid to the gas phase promotes high rate of

H2 production under thermophilic conditions [20,21] and in-

creases in hydrogen yields under low partial pressure condi-

tions [21]. At high temperatures, dark fermentation can

promote the hydrolysis of organic compounds and simplifi-

cation of microbial communities favorable for the production

of hydrogen [22].

Although it has been shown that stillage has potential in

fermentative production of hydrogen, the production of this

biogas from glucose and sugar cane stillage co-fermentation

under thermophilic conditions has not been extensively

explored. Glucose is an easily degradable sugar, and the

adaptation of microbial communities from this single sub-

strate may favor and facilitate the establishment of microbial

communities for degradation of complex substrates. In this

sense, the possibility of producing hydrogen under such

conditions becomes attractive. The aim of this study was to

promote the production of H2 in a thermophilic anaerobic

fluidized bed reactor (AFBR) at 55 �C using a concentration of

5000 mg COD L�1 obtained from the application of various

proportions of diluted stillage and glucose as carbon sources

until the sugar cane stillage became the only available sub-

strate for dark fermentation.

Please cite this article in press as: Santos SC, et al., Continuous tanalysis from anaerobic digestion of diluted sugar cane stillagdx.doi.org/10.1016/j.ijhydene.2014.03.241

Material and methods

Inoculum

The inoculumused to start the reactor operationwas obtained

from a granular sludge of a thermophilic upflow anaerobic

sludge blanket reactor (UASB), used for the treatment of stil-

lage from sugar cane, located at the Sao Martinho distillery

plant (Pradopolis, SP, Brazil). To obtain the hydrogen-

producing cultures, a thermic pretreatment of the biomass

was performed to promote the inhibition of methanogenesis

by eliminating methanogenic vegetative cells and enhance

the retention of acidogenic cells through the formation of

endospores.

Continuous substrate feeding

The stillage used as a substrate for H2 production was

collected at the Sao Martinho distillery plant, which is a

producer of sugar and ethanol that uses sugar cane as a

raw material. The physicalechemical characteristics of the

raw stillage, with minimum and maximum values, in

terms of pH, total organic carbon (TOC), COD, total nitro-

gen, total phosphorus (as PO4�3), total sulfur (as SO4

�2), and

organic acid composition and distribution, are presented in

Table 1.

The high organic content of the raw wastewater, of

30,000 mg COD L�1, was diluted to approximately

5000e5300 mg COD L�1, represented in the influent of the

AFBR reactor by decreasing the glucose percentages and

increasing the stillage percentage in the mixture of these

two organic substrates. Nutrients necessary for cell

growth were added [23], and there was no addition of

acidifying or alkalizing agents in the influent to the

reactor.




Effluent

65 ºC Water bath

Feeding pump

Feeding solution

Gas flowmeterWater trap (NaOH)

Expanded clay

Recycling pump

Fig. 1 e Schematic diagram of the thermophilic anaerobic fluidized bed reactor.

Table 2 e AFBR operating conditions for H2 productionfrom glucose and stillage, to 5000 mg COD LL1, andapplied organic loading rate (OLR).

Phases HRT (h) Substrate mixture (%) OLR(kg COD m�3 d�1)Glucose Stillage

1 8 67 33 26.6

2 8 33 67 30.5

3 6 33 67 35.6

4 4 33 67 52.7

5 2 33 67 107.2

6 1 33 67 225.3

Sugar cane stillage as sole organic source

7 2 0 100 120.8

8 1 0 100 216.8

i n t e r n a t i o n a l j o u r n a l o f h yd r o g e n e n e r g y x x x ( 2 0 1 4 ) 1e1 2 3

Experimental setup: thermophilic anaerobic fluidized bedreactor

The reactorwas constructed from transparent acrylic with the

following dimensions: a thickness of 5 mm, height of 120 cm,

internal diameter of 5.3 cm and a volumetric capacity of

2646 cm3 (Fig. 1).

The characteristics of the expanded clay used as the sup-

port material for biomass immobilization and adhesion were

as follows: pebble diameter between 2.8 and 3.5 mm, real

density of 1.5 g cm�3 and porosity of 23% [24,25]. Approxi-

mately 800 g of expanded claywas introduced into the reactor,

providing a static bed 40 cm tall in the AFBR reactor. The

placement of a U-shaped tube in the bed of the reactor and the

use of a thermostatic jacket, inside of which circulated water

from a thermostatic bath at 65 �C, operating alone and/or

simultaneously, maintained a uniform thermophilic temper-

ature of 55 � 1 �C.

Thermophilic AFBR startup and operational conditions forfermentative hydrogen production

Initially, the thermophilic anaerobic fluidized bed was oper-

ated in batch mode for a period of 48 h, promoting the acti-

vation of the hydrogen producer biomass. Analyses of the

substrate consumption by microorganisms were performed

during this period. Following this activation step, the reactor

was operated for 308 days in continuous mode over 8 experi-

mental phases. The strategy adopted in the thermophilic

reactor refers to the stabilization of H2 produced over ranges

of HRTs varying from 8 to 1 h during experimental phases 1

through 6 (Table 2), achieved by the application of various

percentages of glucose and stillage in the substrate feed.

Then, to evaluate the capacity of producing hydrogen from

pure stillage, glucose was completely removed from the

influent feed mixture, after which the complex substrate


(stillage) served as the sole carbon source for the thermophilic

production of hydrogen, during phases 7 and 8.

The recirculation was maintained at a rate of 133 L h�1

(expansion of 1/4 30%), and a superficial velocity of 1.30 times

the minimum fluidization velocity was maintained. The

reactor was operated without the addition of alkalizing or

acidifying agents, which could result in increased costs for the

biological production of hydrogen [25].

Analytical methods

The volumetric production of hydrogen wasmeasured using a

gas meter (TG-1, Ritter Inc., Germany), and the determination

of hydrogen gas was performed by gas chromatography (Shi-

madzu GC-2010) using a Supelco Carboxen 1010 Plot column

(30 m long, 0.53 mm internal diameter). The influent and

effluent samples were collected for physicalechemical and

metabolic analyses throughout the operational period of the

reactor. Organic acid concentrations were analyzed using

high-performance liquid chromatography (HPLC, Shimadzu),




Fig. 2 e Mean values of organic loading rate (OLR) and

removal efficiencies of COD and total carbohydrates during

hydrogen production in the thermophilic AFBR.


equipped with a pump (LC-10ADVP), autosampler (SIL-20A

HT), CTO-20A column at 43 �C, drag detector (SDP-M10 AVP)

and Aminex HPX-87H column (300 mm; 7.8 mm, BioRad). The

analyses for COD, pH, suspended solids, total nitrogen, total

phosphate, sulfate, zinc, manganese, magnesium, calcium

and potassium were performed in accordance with APHA:

Standard Methods for the Examination of Water and Waste-

water [26]. The TOC analyses were performed using a Shi-

madzu TOC 5000A total organic carbon analyzer [27]. The

analyses for total carbohydrates were performed using the

colorimetric method of Dubois et al. [28].

Molecular analysis of the microbial community

Molecular analyses were performed to evaluate the bacterial

communities present throughout the operational phases with

HRTs of 2 and 1 h, using the stillage and glucose mixture, and

the community present during these same HRTs when the

stillage served as the sole carbon source.

Nucleic acid extraction, PCR and DGGEGenomic DNA was extracted by cell lysis with glass beads

(Sigma), phenol, chloroform and phosphate buffer, using an

experimental procedure modified according to Griffths et al.

[29]. The amplification of the polymerase chain reaction (PCR)

was performed with a 968FGC e 1401R primer set to Domain

Bacteria [30] synthesized by Invitrogen. The amplified DNA

fragments were separated by the technique of denaturing

gradient gel electrophoresis (DGGE). The gels containing a

45e65% linear denaturing gradient (100% denaturant was 7 M

urea and 40% (v/v) deionized formamide). The gels were run at

75 V and 60 �C for 16 h in a 1� TAE buffer. The profile of the

DGGE bands was presented graphically on an Eagle Eye TM III

(Stratagene), at UV from 254 nm, connected to a computer

running the software Eagle Sight. The DGGE band patterns

were analyzed using Bionumerics software 2.5 (Applied

Maths, Kortrijk, Belgium). The similarities were calculated

based on the Pearson correlation coefficient.

Cloning and rRNA 16S sequence determinationThe PCR products were obtained with a 27F and 1100R primer

set [31] and purified using an Ilustra GFX PCR DNA kit and Gel

Band Purification product (GE Healthcare). The vector used

was the pGEM� Easy Vector System (Promega) and was

transformed into competent cells of Escherichia coli for creating

the clone’s library. The recovery of the fragment of interest

was performed by PCR using the primers M13F and M13R [32].

The PCR products were sent to Macrogen Inc� for nucleotide

sequence analysis. Sequence similarity searches were per-

formed using the Ribosomal Database Project (RDP e http://

rdp.cme.msu.edu/), and the Basic Local Alignment Search

Tool (BLAST) was used to search the National Center for

Biotechnology Information sequence database (http://www.

ncbi.nlm.nih.gov/BLAST/). The phylogenetic tree for each

sample was developed using the software MEGA Version 4.0.

Bootstrap analysis for 1000 replicates was performed to esti-

mate the confidence of the tree topologies. The sequences

representing each OTUs (operational taxonomic units) were

selected (dereplicate sequence). For the taxonomic classifica-

tion of sequences, a representative of each OTU was


developed using the RDP-Classifier. The confidence threshold

adopted was 99% for the specie taxonomic level and 88% for

the genus taxonomic level. Fifty-six sequenceswithin 13 OTUs

were found in this study. The sequences in this study were

deposited in the Genbank, with accession numbers ranging

from KF684949 to KF684961.

Results and discussion

Thermophilic AFBR reactor performance

Regarding the wastewater biodegradability used for hydrogen

production, the COD/SO4�2 ratio was between 12 and 21, and

thus the biological conversion to hydrogen was favorable

(Table 1). It has been reported that 3000 mg L�1 of this salt

represents the maximum concentration that does not impair

the efficiency of hydrogen production [33] and that a COD/

SO4�2 ratio between 3 and 50 obtained from glucose allows

stable hydrogen production (from 0.39 L H2 h�1 L�1) [34],

similar to the values obtained in this study (0.78 L H2 h�1 L�1).

The removal efficiencies of COD and total carbohydrates

associated with the various OLRs are shown in Fig. 2. The pH

values of the influent and effluent remained similar and

constant in all of the phases (4.3 and 4.1 in the influent and

effluent, respectively). Therefore, maintaining a pH of

approximately 4.0 in the thermophilic AFBR demonstrated

good intrinsic buffering capacity in the anaerobic reactor, as

observed by Amorim et al. [24] in a mesophilic AFBR using

glucose as the organic source.

During the operational phases, there were no significant

changes in pH values, thus avoiding modifications in the mi-

crobial metabolism interrelations and competition for new

substrate material when added to the overall percentage. This

pH stability was also observed by other authors in thermo-

philic conditions when no alkalizing or acidifying agents were

used for pH control [18,20,22].

Although hydrogen production was observed during all of

the operating phases, the carbohydrate removal efficiencies

during AFBR operation indicated that there was incomplete

consumption of the substrate (Fig. 2). The strategy selected for

the thermophilic reactor, i.e., applying 5000 mg COD L�1 by

means of the addition of a mixture of glucose and stillage at

various relative concentrations during the stabilization of H2

production, caused the system conversion efficiencies to be


http://rdp.cme.msu.edu/

http://rdp.cme.msu.edu/

http://www.ncbi.nlm.nih.gov/BLAST/

http://www.ncbi.nlm.nih.gov/BLAST/



Fig. 3 e Organic loading rate, H2 yield (a), H2 volumetric

productivity and H2 content (b) during HRT reduction.


between 27.1 and 52.6%. The lowest values of total carbohy-

drate removal were obtained during the HRT of 8 h (Phase 2),

using a mixture of 33% glucose and 67% stillage. While this

proportion was kept constant and the HRT was reduced, a

total carbohydrate conversion efficiency of approximately

50% was observed.

The verification and proven feasibility of hydrogen pro-

duction, even in situations of incomplete total carbohydrate

conversion efficiency, was also observed by Peintner et al. [35]

in a trickling bed reactor and AFBR reactor, both under ther-

mophilic conditions. Lowering the HRT from 10 to 7.5 h, the

authors found residual glucose (1.9 g L�1). In this study, a

similar level of residual carbohydrate was observed in the

AFBR effluent (2.5 g L�1) during an HRT of 8 h.

The organic matter removal remained constant during the

operation phases, reaching efficiency values between 7.8 and

13.9% (Fig. 2). These low observed efficiencies may be attrib-

uted to the presence of organic acids derived from fermenta-

tion. Studying the potential for anaerobic treatment of

wastewater with high amounts of organic matter, Intanoo

et al. [6] observed that the use of wastewater from alcohol

distilleries for hydrogen production under thermophilic tem-

peratures in a batch reactor yielded a removal efficiency of

32% (OLR of 68 kg COD m�3 d�1), suggesting that higher rates

may cause an increase in the total amount of volatile acids

and a consequent decrease in the organic matter removal ef-

ficiency. This trend was also observed in this study, with an

OLR of up to 225.3 kg COD m�3 d�1 and an average COD

removal of 13.4%.

Hydrogen production: yield, volumetric productivity andcontent

The H2 yield, volumetric productivity and constituents of the

biogas obtained during the operational phases are presented

in Fig. 3. The H2 yield (HY) that was achieved ranged between

1.97 and 5.73 mmol g CODadded�1 . Similar values were observed

by Yang et al. [36] using cheese processing wastewater. Those

authors obtained 2.3 mmol H2 g CODadded�1 with an HRT of 24 h

under mesophilic conditions in a CSTR reactor; however,

methane was detected in the biogas produced, which did not

occur in this study. Lee et al. [37] verified HY values of

1.7 mmol H2 g CODadded�1 at an OLR of 28 g COD L�1 d�1, i.e.,

close to those obtained in this study, in an intermittent CSTR

reactor fed with vegetable kitchen waste at 60 �C. Hsiao et al.

[38] obtained an anaerobic continuous flow of hydrogen at a

rate of 2.0 mmol H2 g CODadded�1 from the fermentation of

condensed molasses solubles at a concentration of

40 g COD L�1.

The highest mean value of H2 yield (5.73 mmol g CODadded�1 )

was obtained by applying an HRT of 4 h, during which it was

possible to achieve up to 50.8% of hydrogen in the biogas. In

this experimental phase, 45.5 COD and 7.8% total carbohy-

drate removal efficiencies were observed. A reduction in the

H2 yield with the application of lower HRTs of 2 and 1 h using

only stillage as the influent was observed (OLR of 120.8 and

216.8 kg COD m�3 d�1, respectively). The highest H2 produc-

tivity was associated with the lowest HRT (0.78 L H2 h�1 L�1).

Under thermophilic conditions using starch as the organic

substrate in a UASB with pH values similar to those of this


study (approximately 5.0), Akutsu et al. [20] found increasing

H2 volumetric productivity, i.e., from 0.04 to 0.16 L H2 h�1 L�1,

accompanied by an OLR increase from 8 to

127 kg COD m�3 d�1. Those authors observed, similarly to the

findings of the present study, a decrease in the H2 yield (from

1.68 to 0.20 mol mol glucose�1) with increasing OLR. During

the operational phases, this behavior was also observed

(Fig. 3), and the yield associated with an HRT of 4 h

(5.73 mmol H2 g CODadded�1 ) decreased with an HRT of 1 h

(1.97 mmol H2 g CODadded�1 ).

Variations in the volumetric H2 productivity with re-

ductions in the HRT from 8 h to 1 h (0.19 and 0.78 L H2 h�1 L�1,

respectively) were observed (Fig. 3). Similar behavior was

observed by Wang et al. [11]: 4 mmol H2 h�1 L�1 and

12 mmol H2 h�1 L�1 were observed with HRTs of 10 and 5 h,

respectively, in the digestion of sugar refinery molasses in a

mesophilic CSTR reactor. Thus, the values obtained in the

thermophilic AFBR reactor were found to favor the biological

hydrogen production, particularly with an HRT of 1 h. Oper-

ational stability was observed at 55 �C, with biohydrogen

production of 7.1e30.7 mmol H2 h�1 L�1.

Table 3 presents the data obtained in studies of thermo-

philic hydrogen production and the potential use of stillage in

the fermentative production of H2, including the results of this

study. The experiments performed under mesophilic condi-

tions, using molasses [8,10,11] and high temperatures and

using various types of industrial wastewater [6,15,13] ach-

ieved lower rates of hydrogen production compared to those

obtained in this study (0.78 L h�1 L�1). The comparison dem-

onstrates the feasibility of producing biogas from diluted

sugar cane stillage.

The hydrogen percentages obtained from the biogas

remained constant and high, with mean values ranging from

31.4 to 52.0% during the operational phases. Similarly, Kong-

jan et al. [9], in experiments using a batch mode and a UASB

reactor, both thermophilic, did not detect methane in the




Table 3 e Mesophilic and thermophilic fermentation production of H2 from various substrates, including the resultsobtained in this study.

Substrate Temperature (�C) Inoculum H2 volumetric productivity Reference

Alcohol distilleries wastewater 55 Sewage sludge 0.08 L h�1 L�1 [6]

Molasses wastewater 35 Sewage sludge 0.4 L h�1 L�1 [8]

Molasses wastewater 35 Sewage sludge 12.5 mmol h�1 L�1 [10]

Molasses wastewater 35 Sewage sludge 12.27 mmol h�1 L�1 [11]

Glucose and galactose mixture 37 E. coli WDHL 0.024 L h�1 L�1 [13]

Cheese whey 55 Sewage sludge 0.003 L h�1 L�1 [15]

Sugar cane stillage 55 Thermophilic UASB

from stillage treatment

0.78 L h�1 L�1 The present

study30.7 mmol h�1 L�1.

Fig. 4 e Soluble metabolites as relative percentages (a), H2

yield and the concentrations (mol LL1) of lactic and acetic

acids (b) during HRT reduction.


biogas formed from the wastewater treatment of stillage from

sugar beets and measured 61.2% H2 in the biogas. As in this

study, methanogenic archaea were completely suppressed by

the operating conditions and low pH established in the

reactor, i.e., approximately 5.0.

Under thermophilic conditions, Intanoo et al. [6] obtained

43% H2 in the biogas from a batch reactor. Azbar et al. [18]

obtained an average value of 45% in a CSTR reactor treating

cheese whey. These results are similar to those obtained in

this study, in which up to 52% H2 was achieved from dark

fermentation of sugar cane stillage.

In an AFBR mesophilic reactor, Barros and Silva [39] used

glucose as the carbon source and obtained H2 levels between

46 and 60%. These authors reported that higher percentages

were achieved with the application of lower HRTs, from 8 to

2 h. In this study, an HRT of 4 h produced values that were

similar or superior in terms of the H2 content (50.8%) using a

mixture of glucose and stillage in proportions of 33 and 67%,

respectively.

Energy assessment from diluted stillage wastewater

The performance of the thermophilic biological process in

terms of power generationwas calculated, taking into account

the standard conditions of temperature and pressure and the

energy in the hydrogen produced (141.87 kJ g�1) [40].

The maximum energy production rate in terms of

hydrogen gas was 4.93 kJ h�1 L�1 from diluted sugar cane

stillage with an OLR of 216.81 kg COD m�3 d�1, whereas the

lowest values, 1.15 kJ h�1 L�1, were obtained during the bio-

logical degradation of the mixture of substrates mixture

(glucose and stillage), with an OLR of 26.57 kg COD m�3 d�1

during an HRT of 8 h. These decreasing values can be attrib-

uted to the difference in the applied organic load and the type

of mixture of organic substrates available for microbial

conversion.

Lay et al. [41] reported a rate of energy production of

0.82 kJ h�1 L�1 using tofu-processing wastewater in a CSTR

reactor during an HRT of 8 h. Using concentrations similar to

those of this study (6000 mg COD L�1), Han et al. [42] reported

energy production of 1.41 kJ h�1 L�1 from sugar beet refinery

molasses.

Composition and distribution of soluble metabolitesproduced

The primary metabolites found in the effluent (Fig. 4) with the

operation of the thermophilic AFBR reactor were succinic,


lactic and butyric acids at percentages between 3.4 and 44.3%,

7.0e30.9% and 10.3e28.9%, respectively. Acetic acid, iso-

butyric and propionic acids were observed at levels of

2.1e28.9%, 4.3e24.8%, and 2.4e19.8%, respectively. Ethanol

was observed only during phases 1 and 6, at percentages of

10.2 and 6.7%, respectively.

Succinic acid was present in all of the operational phases,

except the last phase, phase 8, which involved the use of 100%

stillage and anHRT of 1 h. The percentages were between 4.6%

and 44.3% in phase 7 (100% stillage) and phase 2 (67% stillage

and 33% glucose), respectively. Under such conditions, H2

yieldsof 3.53mmolH2gCODadded�1 and4.45mmolH2 gCODadded

�1 ,

respectively, were obtained. The performance of the thermo-

philic reactorwasnot affectedbyhighor lowconcentrations of

this secondary soluble product.

Lactic acid was observed at levels between 217.1 and

1357.7 mg L�1 during all of the operational phases. Kuo et al.

[43], using a thermophilic AFBR reactor treating food waste,

also demonstrated that the presence of lactic acid (at con-

centrations of 6000 mg L�1) played a special role during the

dark fermentative production of hydrogen. As observed in this

study, the thermophilic reactor used by those authors




Table

4eMeanvaluesofH

2yield,v

olum

etric

pro

ductivityandH

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exhibited an effective H2 yield of 2.50 mmol H2 g CODadded�1 and

similar percentages of hydrogen in the biogas (47%).

The mixture of glucose and stillage used in phases 1 and 4

(HRT of 8 and 4 h) produced the highest levels of lactic acid

(24.7% and 30.9%, respectively), the highest H2 yield (4.98 and

5.73mmol g CODadded�1 , respectively), and elevated percentages

of H2 in the biogas (49.0 and 50.8%, respectively). The impor-

tance of lactic acid was also confirmed in phase 6 (HRT of 1 h),

in which the lowest percentage of this metabolite (7%) was

obtained, associated with a consequent decrease in the yield

of H2 (1.97 mmol H2 g CODadded�1 ).

The use of stillage as the sole source of organic substrate

and subsequent removal of the glucose during phases 7 and 8

favored the production of butyric acid, acetic acid, and iso-

butyric acid and a reduction of succinic acid, suggesting a

metabolic pathway shift. Kim and Kim (2012) [44] reported

that carbon sources may control the metabolic pathways,

resulting in varying amounts of the organic acids produced

and therefore variations in the H2 yields. The apparent change

in the composition of secondary metabolites produced during

the operational phases resulted in various H2 productivity and

yields, reaching values between 0.19 and 0.78H2 h�1 L�1 and

between 1.97 and 5.73 mmol H2 g CODadded�1 , respectively.

The highest percentages of propionic acid (19.5 and 14.1%)

were reflected by the levels of H2 in the biogas; i.e., the lowest

values were obtained between operational phases (41.6 and

31.4%), associated with HRTs of 2 and 1 h, respectively.

Table 4 shows the mean values of H2 yield, volumetric

productivity and level of H2 in the biogas, and the concen-

trations of the secondary metabolites produced during dark

fermentation associated with the various substrate composi-

tions (mixtures), organic loading rate and HRTs.

Acetic acid at levels of 127.7e1033.8 mg L�1 was observed

during an HRT of 2 h (phase 5), reflecting decreases in the H2

levels and yield, from 50.8 to 42.8% and 5.73 to

3.44mmol H2 g CODadded�1 , respectively. The formation of acetic

acid via themetabolic pathway of homoacetogens (the H2/CO2

conversion to acetate) occurs due to consumption of hydrogen

and carbon dioxide [45], whichmay have occurred and caused

reduced H2 yields and H2 levels in the biogas during the OLR of

107.2 kg m�3 d�1.

Luo et al. [16] also observed an elevated acetic acid con-

centration of 689 mg L�1 (lower than that obtained in this

study of the mixture of glucose and stillage) in a thermophilic

CSTR reactor for H2 production from alcohol distillery waste-

water. However, in a mesophilic CSTR reactor, the authors

detected a high level of this metabolite, i.e., 1600 mg L�1.

Although homoacetogens were present, the authors observed

H2 production of 69.6mL gVS�1, whereas the production in the

mesophilic reactor was lower (14.0 mL gVS�1). The findings of

this study, related to the minor consequence of operating the

reactors at elevated temperatures, were also confirmed by

Akutsu et al. [20], who obtained 1.7 mol H2 mol glucose�1 from

the use of starch as the organic substrate in a thermophilic

UASB reactor, despite the presence of homoacetogens.

The highest butyric acid concentration, 2705.8 mg L�1,

occurredwhen the highest rate of organic loading was applied

(225.3 kg COD m�3 d�1). Under thermophilic conditions, Inta-

noo et al. [6] also obtained an increase in the concentration of

butyric acid, from 2000 to 10,000 mg L�1, when raising the OLR




Fig. 5 e Similarity coefficients (Pearson correlation) and the UPGMA clustering method, from the DGGE band profiles,

referring to the bacterial communities of the inoculum and from the operational phases in the thermophilic AFBR reactor.

(In) inoculum after heat pre-treatment; (Gli D Stillage; 2 h): in reference to phase 5, with 33% glucose and 67% stillage;

(Glu D Stillage; 1 h): in reference to phase 6, with 33% glucose and 67% stillage; (Stillage; 2 h): in reference to phase 7, with

100% stillage; (Stillage; 1 h): in reference to phase 8, with 100% stillage.


from 68 kg COD m�3 d�1 to 79 kg COD m�3 d�1. O-Thong et al.

[14], studying the digestion of palm oil processing wastewater

in a thermophilic sequencing batch reactor, obtained high

concentrations of butyric acid, i.e., from 2000 to 6200 mg L�1,

and high levels of H2, between 58 and 60%, similar to the

values obtained in this study.

Molecular characterization of microbial diversity

Differences were observed in the band profiles present in the

inoculum from the thermophilic UASB used for stillage

treatment compared to those obtained during the operational

phase of the thermophilic AFBR (Fig. 5), based on the bacterial

structures analyzed by PCR-DGGE. The analyses were per-

formed specifically for the operating phases of HRTs of 2 and

1 h, using the mixture of glucose and stillage (phases 5 and 6),

and the operating phases of HRTs of 2 and 1 h, using the

stillage as the only organic substrate source (phases 7 and 8).

The latter displayed the highest values of volumetric H2 pro-

ductivity, i.e., between 0.58 and 0.78 L h�1 L�1.

A 44% similarity (Pearson correlation) was observed be-

tween the band profiles of the inoculum and the operational

phases, indicating that significant changes occurred in the

bacterial community during the AFBR reactor operation dur-

ing the use of glucose and stillage as the substrate for the

production of hydrogen.

Table 5 e Genetic sequencing results of 16S rRNA fragments o

OTU Number ofsequences

Organism affiliation Sle

1 4 Clostridium cellulosi

2 1 Thermoanaerobacterium sp. MYST/2012-07

3 1 Thermoanaerobacterium thermosaccharolyticum




7 2 Uncultured bacterium clone VKW-TB-3.3 16S

8 11 Uncultured bacterium clone D8-50C-C4-3

9 1 Lactobacillus sp.

10 1 Lactobacillus sp.

11 1 Uncultured bacterium isolate d21l12b41

12 1 Moorella sp.

13 1 Caldanaerobius sp.


In contrast, the availability of stillage as the sole carbon

source did not produce modifications in the thermophilic

microorganism structure. The similarity coefficients between

the operational phases obtained with the substrate mixture

and operational phases using 100% stillage were high, i.e., 91,

92 and 95%. A level of H2 production of 0.78 L h�1 L�1 was

obtained during an HRT of 1 h, using stillage as the organic

source (phase 8). This level was associated with a 91% simi-

larity coefficient between this phase and the phases using the

mixture of glucose and stillage and to the phase using 100%

stillage (phase 2) during an HRT of 2 h. Thus, a reduced pop-

ulation change as a function of the imposed conditions was

found.

The phylogenetic sequence of the biomass formed in the

expanded clay support material of the thermophilic AFBR

after an operational phase of an HRT of 2 h (phase 7, 100%

stillage) was grouped into 13 operational taxonomic units

(OTUs), corresponding to only one phylum (Firmicutes) and

two classes, Clostridia (85%) and Bacilli (15%), totaling 56 se-

quences. Among these, a 99% similarity to Thermoanaer-

obacterium thermosaccharolyticum, Clostridium cellulosi and

uncultured bacteria was obtained. A similarity of 88e92% was

related to Lactobacillus sp.; Moorella sp. and Caldanaerobius sp.

OTUs 3 to 6 were related to T. thermosaccharolyticum,

belonging to the Thermoanaerobacterales family (Table 5).

Thermophilic bacteria, such as T. thermosaccharolyticum,

f the bacteria domain.

equencength (pb)

Identify(%)

Phylum GenBankaccession no

Relativeabundance

980 99 Firmicutes NR044624.1 7%

1032 99 Firmicutes JX442957.1 2%

1020 99 Firmicutes JX984979.1 2%

834 99 Firmicutes JX984974.1 43%

1037 99 Firmicutes HM585225.1 2%

1027 99 Firmicutes AF247003.1 13%

712 99 Firmicutes GQ849504.1 4%

954 99 Firmicutes HQ266872.1 20%

606 88 Firmicutes AB016864.1 2%

660 89 Firmicutes DQ523489.2 2%

1043 92 Firmicutes FR687166.1 2%

975 89 Firmicutes AB086398.1 2%

1019 91 Firmicutes NR044258.1 2%




Fig. 6 e Phylogenetic tree of the OTUs achieved from the thermophilic fermentative hydrogen-producing microorganisms

found in the AFBR reactor (support material), constructed using the neighbor-joining method based on a comparison of the

16S rRNA gene. Numbers at nodes represent bootstrap values (percentages of 1000 replicates). Bar, 95% substitutions in

nucleotide sequence (uncultured Chloroflexus sp.: outgroup).


display optimal growth at 60 �C and have ample capacity to

ferment various substrates (such as xylose, sucrose and

starch) to H2 and soluble byproducts [14]. These microorgan-

isms are obligate anaerobes that do not grow in the absence of

a fermentable carbohydrates and produce common fermen-

tation end-products, such as acetic, butyric, lactic and suc-

cinic acids [46], as observed in this study, with percentages of

up to 28.9, 29.9, 31.0 and 44.3% of acetic, butyric, lactic and

succinic acids, respectively.

Kongjan et al. [47], studying desugared molasses in a two-

stage thermophilic UASB for the biological production of

hydrogen, also obtained sequence similarity with Thermoa-

naerobacterium sp. The authors reported that this bacterium is

moderately thermoacidophilic, gram-positive and endospore

positive. In an earlier study, also under thermophilic condi-

tions, Kongjan et al. [9] detected the dominance of T. thermo-

saccharolyticum in the UASB granules, which was used for the

fermentative production of H2 from desugared molasses. In

the present study, H2 and organic acids production were

produced from sugar cane stillage.

A lower percentage, i.e., 7% (OTU 1), was related to the

species C. cellulosi, belonging to the Ruminococcacea family. C.

cellulosi is considered a thermophilic bacterium forming en-

dospores and able to degrade cellulose with production of H2,

CO2, butyrate, acetate and ethanol, with growth at tempera-

tures between 55 and 60 �C [48]. This thermophile bacterium

survives at a temperature of 100 �C for 20 min, heat shock

favors its germination, and it consists of rods with


lophotrichous flagella, spherical terminal endospores that

swell the cells [49]. Other biological reactors producing H2 also

identified this specie growing from various carbon sources,

such as sugar mill [50] and cassava plant ethanol [48]. In a

CSTR reactor involving thermophilic anaerobic digestion of

mixed swine slurry and market biowaste, Merlino et al. [51]

related the biological production of hydrogen at a rate of

0.06 L H2 h�1 L�1 to the presence of microorganisms similar to

C. cellulosi.

According to Kim and Kim [22], thermophilic reactors

simplify the microbial communities favorable for H2 produc-

tion, selecting for efficient hydrogen producers. These be-

haviors were observed in the present study, in which only two

families representing the effective hydrogen-producing mi-

crobial community were present, based on their species and

accession numbers (GenBank) shown in Table 5, with a 99%

degree of similarity.

The sequence ofmicroorganisms representedbyOTU7was

related to the uncultured bacteria, similar to those found by

Lee et al. [37] (GenBank accession number GQ849504.1) in an

intermittent thermophilic CSTR reactor used for hydrogen

production from vegetable kitchen waste. In that study, an H2

yield of 1.7 mmol g CODadded�1 was found, similar to that ob-

tained in this study,with values of 2.06e3.56mmol g CODadded�1 .

OTU 8 (GenBank accession number HQ266872.1) was also

reported by Liu and Conrad [52]. In a study of chemo-

lithotrophic acetogenic H2/CO2 utilization in Italian rice field

soil, those authors demonstrated that such microorganisms





as the Thermoanaerobacteriaceae family were dominant at a

temperature of 50 �C. Acetivibrio (OTU 11) was related to cel-

lulose degradation and the biological production of hydrogen

by Lu et al. [53] via anaerobic fermentation in three stages

using cornstalks as the organic substrate (GenBank accession

number FR687166.1).

The phylogenetic tree was developed with the 56 se-

quences obtained from the 16S rRNA gene fragment analysis

(Fig. 6), shown in Table 5.

Caldanaerobius (OTU 13) consists of anaerobic and thermo-

philic chemo-organotroph bacteria of the family Thermoa-

naerobacteraceae and the class Clostridia [54] of the phylum

Firmicutes. Theirmetabolic end products via fermentation are

ethanol, acetate, formate, lactate, carbon dioxide and

hydrogen [55]. However, to our knowledge, there are no pre-

vious studies that report the presence of this genus in bio-

logical hydrogen production. This family has also exhibited

results similar to those of Moorella (OTU 12) regarding the

production of H2, ethanol and acetate under thermophilic

conditions using fructose as the organic source [56].

The genus Lactobacillus (OTUs 9 and 10) consists of bacteria

that produce lactic acid as a major metabolic product from

carbohydrates through fermentation, and lactic acid has been

reported to have an inhibitory effect on hydrogen production

by Clostridium, in which reduced hydrogen output is associ-

ated with a simultaneous increase in lactate production [57].

However, Baghchehsaraee et al. [58] reported that lactate

degradation could lead to a greater residual amount of NADH

for hydrogen production via butyrate metabolism, thus

explaining the effective volumetric production of H2 (up to

0.78 L h�1 L�1) in the presence of high amounts of lactic acid

among the metabolites produced in the AFBR. Lactic acid

concentrations between 7.0 and 30.9% were observed, favor-

ing the presence of microorganisms related to Lactobacillus.

Conclusions

Stable H2 production from the co-fermentation of glucose and

stillage in a thermophilic AFBR was verified. High concentra-

tions of lactic acid, among variousmetabolites, were produced

during the fermentation, although no volumetric decrease in

hydrogen production or H2 percentage in the biogas was

detected.

The stillage supply as the sole substrate caused an increase

in H2 volumetric productivity, and the highest average value

under this condition was 0.78 L H2 h�1 L�1.

Significant changes in microbial diversity were not

observed after removal of the co-substrate. Similarities in

biomass digestion were verified to Thermoanaerobacterium sp.

and Clostridium sp., which were both efficient hydrogen pro-

ducers at elevated temperatures.

Acknowledgments

The authors gratefully acknowledge the financial support of

FAPESP e Sao Paulo Research Foundation and CNPq e Con-

selho Nacional de Desenvolvimento Cientıfico e Tecnologico,

Brazil.


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