Sequential fermentative and phototrophic system for hydrogen production: An approach for Brazilian alcohol distillery wastewater

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Sequential fermentative and phototrophic system for hydrogen production: An approach for Brazilian alcohol distillery wastewater Carolina Zampol Lazaro a, Maria Bernadete A. Varesche b, Edson Luiz Silva a,* ~o Carlos, Rod. Washington Luis, km 235, Department of Chemical Engineering, Federal University of Sa ~o Carlos, Sa ~ o Paulo, Brazil 13565-905 Sa b ~o Carlos, University of Sa ~o Paulo, Department of Hydraulics and Sanitation, School of Engineering of Sa ~o-carlense, 400, 13566-590 Sa ~ o Carlos, SP, Brazil Av. Trabalhador Sa a

article info

abstract

Article history:

This study evaluated the possibility of using sugarcane vinasse for biological hydrogen

Received 28 February 2015

production under non-sterile conditions via sequential two-stage fermentative and pho-

Received in revised form

totrophic processes, which simultaneously allows bioenergy production and chemical

18 May 2015

oxygen demand (COD) removal. The fermentative process was performed in an anaerobic

Accepted 1 June 2015

fluidized bed reactor (AFBR) fed with a mixture of sucrose and sugarcane vinasse

Available online xxx

(5:10 g COD L
1). The phototrophic process was performed using a microbial consortium (Rhodopseudomonas related microorganisms) as inoculum and batch reactors fed with

Keywords:

different concentrations of the AFBR effluent (10, 20, 40, 70 and 80%, v/v). The hydrogen

Lactobacillus

yield (HY) (0.34 mol H2 g
1 CODinfluent) observed in the fermentative step was justified by

Rhodopseudomonas

the establishment of microbial community composed predominantly by Lactobacillus

Sulfate reduction

related

Sulfide

(5.5 mmol H2 g
1 CODremoved) was observed for the lowest AFBR concentration. Increasing

Butyric acid

the AFBR effluent concentration as the substrate for the phototrophic reactors had a

microorganisms.

The

highest

HY

observed

in

the

phototrophic

stage

negative impact on cell growth, hydrogen production and COD removal mostly due to the occurrence of sulfate reduction. Hydrogen production was not observed in batch reactors fed with 70 and 80% of AFBR effluent; however, under those conditions, the sulfate removal rate was 86.5 and 86.9%, respectively. Copyright © 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

Introduction Industrial processes and domestic activities are based on the consumption of fossil fuels. The dependence on fossil fuels

has an economic impact, causes increased vulnerability due to increasing fuel prices, fuel shortages and potential embargos, and has issues related to global climate change and environmental degradation [1]. The search for an effective

* Corresponding author. Tel.: þ55 16 3351 8264; fax: þ55 16 3351 8266. E-mail address: [email protected] (E.L. Silva). http://dx.doi.org/10.1016/j.ijhydene.2015.06.003 0360-3199/Copyright © 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

Please cite this article in press as: Lazaro CZ, et al., Sequential fermentative and phototrophic system for hydrogen production: An approach for Brazilian alcohol distillery wastewater, International Journal of Hydrogen Energy (2015), http://dx.doi.org/ 10.1016/j.ijhydene.2015.06.003

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alternative energy source to fossil fuels has been the subject of several studies [2,3]. Hydrogen (H2) has been considered a promising clean fuel due to its high energy content (122 kJ g
1) compared to carbon fuels and the lack of COx, NOx and SOx emissions [4]. H2 can be produced by chemical and biological processes. Most of the H2 produced (90%) comes from the steam reforming of natural gas, which occurs at high temperatures. Other industrial processes involve the gasification of coal and the electrolysis of water. Those processes consume fossil fuels as an energy source and sometimes hydroelectricity as well. Biological processes offer advantages over chemical processes because most of them occur at ambient temperature and pressure and are energetically less expensive [4,5]. Biological hydrogen production processes include direct and indirect biophotolysis, dark fermentation and photofermentation [4,6,7]. The requirement for light energy is the main drawback of the first two processes, whereas the fermentative process occurs in the dark and is carried out by anaerobic and facultative bacteria. The main advantages of fermentation are that it occurs in simply designed bioreactors, wastewater can be used as the substrate, and there is no need to supply light energy [8,9]. Despite these advantages, the principle obstacle to the economic viability of the process is obtaining better hydrogen yields [3]. In the fermentative process, the maximum yield is limited to 4 mol of hydrogen per mole of glucose consumed when acetate is produced or 2 mol of hydrogen per mole of glucose consumed by the butyrate fermentative pathway [10]. However, in theory, up to 12 mol of hydrogen per mole of glucose could be produced if the organic matter was completely oxidized [11,12]. Hydrogen production via a sequential two-stage system involves a fermentative step (Eq. (1)) followed by a phototrophic stage, and the process may permit the complete oxidation of organic matter (Eq. (2)), ending with a maximum theoretical yield of up to 12 mol H2 produced per mole of glucose consumed (Eq. (3)). The carbohydrates are consumed in the fermentation stage, and organic acid is produced. In the second stage, the organic matter is removed once the organic acids are consumed, producing H2 and CO2 [13].

C6H12O6 þ 2H2O / 4H2 þ 2CO2 þ 2CH3COOH

(1)

2CH3COOH þ 4H2O þ “light energy” / 8H2 þ 4CO2

(2)

C6H12O6 þ 6H2O / 12H2 þ 6CO2

(3)

In practice, the theoretical yield is not attained because of substrate is used for the cell multiplication, for example. Furthermore, it is known that other metabolic processes besides those that favor H2 production occur in the reactors due to the metabolic versatility of the microorganisms and the environmental conditions. In addition to this, the consumption of hydrogen by microorganisms from different physiological groups (methanogens or sulfate reducers) can occur, especially if the inoculum is not a pure culture [14,15]. Although the use of waste as substrate for biological hydrogen

production is desirable, it has two negative implications: (1) waste is a complex substrate and has a variable composition and (2) it can also serve as the inoculum, which contains microorganisms that contribute or do not contribute to the desired process [16]. The ethanol industry is of paramount importance in Brazil, ~ o Paulo, which is responsible for especially in the state of Sa 62% of the ethanol production [17]. The process generates solid and liquid residues, e.g., sugarcane bagasse and vinasse, respectively. Vinasse is also known as distillery wastewater, stillage, distillery spent wash and thin stillage; its main characteristics include a high chemical oxygen demand (COD), acidity (pH 3.5e5.5) and sulfate richness (2.1e15.8 kg m
3) [18,19]. Each liter of ethanol produced generates 13 L of vinasse [20]. According to the Brazilian Ministry of Mines and Energy, the ethanol production in 2013 was 27.4 billion liters [21]. Thus, the estimated amount of vinasse generated from this harvest was 356 billion liters. Currently, most of it is used as a soil fertilizer; however, because of environmental pollution, alternative methods for vinasse disposal are being investigated [20]. Hydrogen production from fermentative processes using vinasse as the substrate has been studied [22e26]. However, it is known that some organic matter is still present after the fermentative stage, so a second stage should be implemented to remove the COD. Fernandes et al. [23] and Peixoto et al. [24] evaluated a process that combines the hydrogen production in the first stage followed by a methanogenic phase. Another possibility to obtain higher organic matter utilization is a process that combines a fermentative process followed by a photo-fermentation (Argun & Kargi, 2011). This sequential process was studied by Chen et al. [13] using pure species of Clostridium pasteurianum and Rhodobacter palustris. In the first step, sucrose was used as the substrate for Clostridium, and the effluent from the first stage (containing an initial butyric and acetic acid concentration of 2.9 and 0.9 g COD L
1, respectively) was used by Rhodobacter and achieved an overall COD removal of 72%. Zong et al. [27] evaluated hydrogen production from cassava and food waste using an anaerobic consortia and Rhodobacter sphaeroides and observed a COD removal of 84.3 and 80.2% (for an initial 18 and 20 g hexose L
1), respectively. Su et al. [28] employed an anaerobic consortium and Rhodopseudomonas palustris for H2 production via dark and photo-fermentation from raw, gelatinized and hydrolyzed starch. Avcioglu et al. [29] evaluated two-step H2 production from molasses using Caldicellusiruptor saccharolyticus, Rhodobacter capsulatus wild type and (DSM 1710) R. capsulatus hup- (Y03). Most of the studies mainly used pure cultures for the photo-fermentation stages; however, the use of a bacterial consortium could be biotechnologically more attractive, especially for bioenergy production on an industrial scale [16]. Thus, the present study aimed to evaluate the production of hydrogen from sugarcane vinasse through a sequential fermentative/phototrophic system in which the effluent from the first reactor was used as the substrate for the second process. The impact of adding different concentrations of fermentative reactor effluent to the phototrophic reactors was evaluated. The microbial diversity of the fermentative and phototrophic microbial consortium was studied using molecular (16S rRNA gene fragment) methods.

Please cite this article in press as: Lazaro CZ, et al., Sequential fermentative and phototrophic system for hydrogen production: An approach for Brazilian alcohol distillery wastewater, International Journal of Hydrogen Energy (2015), http://dx.doi.org/ 10.1016/j.ijhydene.2015.06.003

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Materials and methods Seed inoculum and culture medium The seed inoculum for the AFBR was a granular sludge obtained from a UASB reactor used in the treatment of wastewater from a poultry slaughterhouse. The granular sludge was macerated to disrupt the granules and then was subjected to a heat treatment (90  C for 10 min followed by ice bath until the material reached 25  C). The mineral medium used for the AFBR was prepared by adding solutions of micro- and macronutrients [30] supplemented with yeast extract (0.5 g L
1) and sodium bicarbonate (0.5 g L
1). The carbon source was a mixture of sucrose and sugarcane vinasse in a proportion of 5:10 COD L g
1, respectively. The total COD of the influent was 15 g L
1. The seed inoculum for the batch reactors used for the phototrophic tests was provided by Lazaro et al. [31]. In the present study, the phototrophic hydrogen-producing consortium was adapted to a synthetic medium in which the carbon source was a mixture of sodium acetate and butyrate (1.93 g L
1 CH3COONa$3H2O and 1.4 g L
1 CH3CH2CH2COONa) in order to simulate a fermentation reactor effluent. The adapted biomass was centrifuged (10,000 rpm for 5 min), and the cell pellet was used as the inoculum for tests of hydrogen production with different AFBR effluent concentrations. The synthetic culture medium was prepared using the following components: 0.6 g L
1 KH2PO4; 0.9 g L
1 K2HPO4; 0.2 g L
1 MgSO4; 0.4 g L
1 NaCl; 0.05 g L
1 CaCl2; 0.2 g L
1 yeast extract; 5 mL L
1 of iron citrate solution (1 g L
1); 1 mL L
1 of trace metals solution [32]; and 0.1 mg L
1 of vitamin B12.

Experimental setup The first-stage, the fermentative process, was performed in an AFBR made of acrylic with the following dimensions: 3.5 cm inner diameter and 150 cm height. Grounded tire was used in the AFBR as support material for microbial adhesion. The particles had the following properties: grain sizes between 2.8 and 3.5 mm, and density of 1.14 g cm
3. The reactor was maintained at constant temperature (30  C) with a water jacket, piping water from a recirculating ultrathermostatical bath. The experimental setup of the fermentation process is shown in Fig. 1. The phototrophic process was performed in glass bottles (0.5 L) with a working volume of 0.3 L and 0.2 L of head space flushed with argon (Ar 99%) for 10 min. The reactors were maintained at a temperature of 30 ± 2  C. The batch reactors were kept under constant light illumination from tungsten filament lamps (60 and 100 W) with a light intensity of approximately 6 klux. The experimental setup is shown in Fig. 2.

Chemical analysis Analyses of pH, COD and volatile suspended solids (VSS) were performed according to Standard Methods [33] from samples collected throughout the incubation period of the reactors. The total carbohydrate concentration was measured according to Dubois et al. [34] only for the AFBR.

Fig. 1 e The fermentative stage performed in the AFBR. 1feed container kept inside a refrigerator (4  C); 2- feed pump; 3- recirculation pump; 4- water bath temperature control; 5- effluent exit; 6- biogas exit.

The content of H2, CO2 and CH4 in the biogas was measured by gas chromatography (GC 2010, Shimadzu®) equipped with a thermal conductivity detector (TCD) and a Carboxen™ 1010 PLOT column (30 m  0.53 mm, Supelco) with argon (chromatographic grade) as the carrier gas [35]. The distribution and quantification of organic acids and alcohols was analyzed by liquid chromatography (Shimadzu®) equipped with an HPInnowax column (30 m, 0.25 mm, 0.25 m) and a flame ionization detector (FID) using hydrogen as the carrier gas and synthetic air and nitrogen as auxiliary gases [25].

Phylogenetic characterization of the microbial consortia The biomass from both the fermentative and phototrophic reactors was collected for phylogenetic characterization and kept frozen until use. The amplification of 16S rRNA gene fragments for cloning and sequencing analysis was performed using a set of primers, 27F and 1100R, that were targeted to the Bacterial Domain [36]. The cloning of the 16S rRNA gene PCR products was performed and the inserts from the clones were sequenced in an automated sequencer (ABI3730XL; Applied Biosystems). The sequences were manually checked using the DNASTAR package (Lasergene Sequence Analysis). Verified sequences were grouped into operational taxonomic units (OTUs) using the COMPLETE LINKAGE CLUSTERING tool. Then, the REPRESENTATIVE SEQUENCE tool was used to determine the representative sequences from each OTU, which were then used for the phylogenetic analysis. Representative sequences from each OTU were compared with sequences deposited in

Please cite this article in press as: Lazaro CZ, et al., Sequential fermentative and phototrophic system for hydrogen production: An approach for Brazilian alcohol distillery wastewater, International Journal of Hydrogen Energy (2015), http://dx.doi.org/ 10.1016/j.ijhydene.2015.06.003

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Fig. 2 e The phototrophic experiments were performed in batch reactors under constant light illumination. 1- tungsten filament lamps (60 and 100 W); 2- glass bottles; 3- beaker with water; 4- thermometer.

the databases using the SEQMATCH tool. These analyses were performed using the tools available from the Ribosomal Database Project (http://rdp.cme.msu.edu/). Sequences of representative OTUs and their closest relatives were used to construct a phylogenetic tree based on Jukes-Cantor distances [37] and the neighbor-joining method [38] with 1000 bootstrap resamplings [39]. Sulfurospirillum cavolei (AB246781) and Thermotoga elfii (X80790) was selected as the outgroup species. These analyses were performed using MEGA software, version 6 [40]. The representative sequence of each fermentative OTU (OTU_1-OTU_14) and phototrophic OTU (OTU_1-OTU_6) were deposited in the NCBI database under the following accession numbers: KP193028-KP193041 and KP193022e KP193027, respectively.

Results and discussion The present study evaluated hydrogen production via a twostage process using sugarcane vinasse as the substrate. The first stage was a fermentative process that was performed in an AFBR fed with diluted vinasse. The second stage was a phototrophic process that was performed in batch reactors using the AFBR effluent as the substrate after pH adjustment (NaOH solution). The results of each step will be presented and discussed separately.

Fermentative reactor performance

period (30 days), the percentages remained constant (Fig. 3B). We did not detect methane in the biogas during the experimental period. Thus, the conditions for the methanogenic microorganisms to colonize the reactor were not favorable.  n and Carvajal [22], methane was According to Buitro concomitantly produced with hydrogen at 24 h HTR at 25 and 35  C. Most likely, the HTR applied (8 h) was appropriate to inhibit the growth and the settlement of methanogenic archaea, which could be a contaminant microorganism in the vinasse. According to Pattra et al. [16], at 4 h HRT in a nonsterile CSTR fed with sugarcane juice, augmented Clostridium butyricum could compete with contaminating microorganisms. However, at 36 h HTR, species related to Lactobacillus harbinensis and Klebseilla pneumoniae were present as a major group in the reactor. The authors did not detect methane in the biogas or report the presence of methanogenic species in the reactor. The effluent produced by the fermentative process was composed mainly of butyric acid (36.4%), isobutyric acid (18%) and propionic acid (19.4%) (Table 1). The effluent composition explains the reduced hydrogen yield (0.47 mol H2 mol
1 carbohydrate) by the AFBR because reduced compounds were produced. It is known that the maximum hydrogen yield in fermentative systems occurs via the acetic acid pathway.

Table 1 e First-stage for hydrogen production in the AFBR from a mixture of sucrose and sugarcane vinasse. Parameters

Hydrogen generation and organic acid production In the first-stage, performed in the AFBR, hydrogen production occurred via the metabolism of fermentative microorganisms that consumed carbohydrates and produced biogas and organic acids (Table 1). The reactor had a high efficiency of carbohydrate consumption (5.1 g L
1 or 84.8%), while COD removal was approximately 16.5% (2.7 g L
1) (Fig. 3A). Lower COD removal is a characteristic of the fermentative stage; if the organic matter is not completely oxidized, the production of reduced compounds, such as organic acids and alcohols, occurs [3,12]. Therefore, the fermentative reactor effluent must undergo another process in which organic matter is efficiently removed [9,32]. The biogas produced in the AFBR was composed of H2 and CO2, 23.3 and 74.9%, respectively. During the experimental


1

CODI (g L ) CODE (g L
1) COD removal (%) Total carbohydratesI (g L
1) Total carbohydratesE (g L
1) Carbohydrates removal (%) Hydrogen yield (mol H2 mol
1 carbohydrate) Hydrogen yield (mmol H2 g
1 CODI) Biogas composition (%) Effluent composition (g L
1)

Values 16.3 ± 3.1 13.8 ± 2.5 16.5 ± 4.9 6.0 ± 0.9 0.9 ± 0.4 84.8 ± 8 0.47 ± 0.16 0.34 ± 0.12 H2: 23.3 ± 3.7; CO2: 74.9 ± 4.9 HAc: 0.4; HBu: 3.7; HPr: 2.0 HIsoBu: 1.8; HLa: 0.7; HMa: 0.8

HAc- acetic acid, HBu- butyric acid, HMa-malic acid, HLa- lactic acid, HPr-propionic acid, HIsoBu-isobutyric acid, CODI ¼ influent, CODE effluent.

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related to the influent vinasse concentration. However, Santos et al. [26], who applied sugarcane vinasse to produce H2 at 55  C, observed mainly lactic (34.7%), isobutyric (23.1%), butyric (15.8%) and propionic acids (15.5%) (Table 2). Fernandes et al. [23] and Peixoto et al. [24] studied sequential hydrogen and methane production from corn and sugarcane vinasse, respectively. The production of acetic, butyric, propionic acids and ethanol was observed by both authors. However, lactic acid was also measured by Peixoto et al. [24]. Although the production of propionic acid is detrimental to hydrogen production, its generation is often reported. Furthermore, the accumulation of propionic acid in the fermentative effluent could result in a low efficiency of the methanogenic phase; thus, the production of propionic acids should be avoided for both hydrogen and methane production [42]. Nevertheless, even if propionic acids are undesirable in the production processes of hydrogen and methane, it is known that such compounds can be consumed by phototrophic bacteria for hydrogen production through the photofermentation process [43].

Phylogenetic characterization of the microbial consortium

Fig. 3 e A) Initial concentration and removal efficiency of total carbohydrate and COD during the fermentative process in the AFBR, - total carbohydrate removal, , initial carbohydrate (g L¡1); A COD removal, ◊ initial COD (g L¡1). B) Biogas composition during the fermentative process in the AFBR, - % CO2; A % H2.

Other metabolic routes that produce butyric and propionic acids are responsible for lower yields [41]. Vinasse from diverse sources has been used as a substrate  n and Carvajal [22] observed the for biological processes. Buitro production of acetic, butyric, isobutyric and propionic acids during hydrogen production using tequila vinasse. However,  n and Carvajal [22] were the concentrations observed by Buitro lower than those observed in the present study. This could be

A biomass sample from the AFBR fed with vinasse was used for phylogenetic microbial characterization. Sequences from 85 clones were grouped into 14 OTU (95% similarity) and assigned to the phylum Firmicutes (75.3%) and Bacteroidetes (24.7%) (confidence threshold of 95%). There was a predominance of microorganisms closely related to Lactobacillus sp. (Lactobacillaceae e OTU_2, 4, 5, 7, 8, 10, 11, 12 and 14) and uncultured Prevotella sp. (Prevotellaceae e OTU_1, 3 and 6) (Table 3, Fig. 4). Contrary to what was expected, the presence of Clostridium species that are commonly reported as hydrogen-producing microorganisms in bioreactors was scarce (OTU_9). The granular sludge used as seed inoculum was heat-treated to stimulate endospore-forming microorganisms, such as Clostridium species. Nevertheless, these species were not predominantly found by the molecular biology analysis. One possible explanation is that during the operational period, Clostridium species were washed out from the reactor, whereas Lactobacillus species became established because of their continuous inoculation via vinasse. Considering the use of complex substrates to produce  et al. [44] evaluated such production using hydrogen, Castello

Table 2 e Fermentative hydrogen production using vinasse as substrate. Inoculum source

Reactor and temperature

Substrate

Metabolites

Reference

Anaerobic granular sludge heat treated Hydrogen-producing biomass

Sequencing batch reactor, 35  C Batch, 25  C

Tequila vinasse (5 g COD L
1)

HAc, HBu

[22]

Corn vinasse (0.25 g COD L
1)

[23]

Acidogenic inoculum

Batch, 25  C

Sugarcane vinasse (0.3 g COD L
1)

Enriched microbial consortium Thermophilic sludge

Batch, 37  C AFBR, 55  C

Sugarcane vinasse (12 g COD L
1) Sugarcane vinasse (10 g COD L
1)

Pre-treated mesophilic granular sludge

AFBR, 30  C

Sugarcane vinasse (16 g COD L
1)

HAc, HBu, HIsoBu, HPr, EtOH HAc, HBu, HLa, HPr, EtOH HAc, HBu HLa, HBu, HIsoBu, HPr Mainly HBu, HPr, HIsoBu

[24] [25] [26] Present study

HAc- acetic acid, HBu- butyric acid, HIsoBu-isobutyric acid, HPr-propionic acid, EtOH- ethanol, HLa- lactic acid.

Please cite this article in press as: Lazaro CZ, et al., Sequential fermentative and phototrophic system for hydrogen production: An approach for Brazilian alcohol distillery wastewater, International Journal of Hydrogen Energy (2015), http://dx.doi.org/ 10.1016/j.ijhydene.2015.06.003

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Table 3 e Phylogenetic affiliation of the clone library of the mesophilic bacterial consortium. Phylogenetic affiliation

Prevotellaceae Lactobacillaceae Prevotellaceae Lactobacillaceae Lactobacillaceae Prevotellaceae Lactobacillaceae Lactobacillaceae Clostridiaceae Lactobacillaceae Lactobacillaceae Lactobacillaceae Bacilli Lactobacillaceae

OTU

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Phylogenetic relationship

Number of clones

Closest species in GenBank

Similarity

Accession number

uncultured Prevotella sp; J28 Lactobacillus paracasei; NRIC 0625 uncultured Prevotella sp; J28 Lactobacillus casei; FJAT-CF9 Lactobacillus casei; TJ6 uncultured bacterium; otu9-c17 Lactobacillus casei; FJAT-CF9 Lactobacillus plantarum; Chikuso-1; uncultured bacterium; FHPH5A28/1-7682 Lactobacillus casei; YIT 0180 Lactobacillus satsumensis (T); NRIC 0604 uncultured Lactobacillus sp.; BB16_T30-12 Lactobacillus casei; FJAT-CF9 Lactobacillus rhamnosus; AL3

1.000 1.000 1.000 1.000 1.000 0.987 0.999 1.000 0.963 0.998 0.990 1.000 0.976 0.996

DQ168844 AB362692 DQ168844 HQ318715 FJ476122 JF281143 HQ318715 AB104855 FJ430233 AB008204 AB154519 JX950981 HQ318715 AF261765

cheese whey in a UASB and reported that the reduced hydrogen yield obtained was due to the establishment of a mixed microbial consortium comprising both hydrogenproducing organisms (Clostridium, Ruminococcus and Enterobacter) and also non-hydrogen-producing species (Lactobacillus, Dialister and Prevotella). Fernandez et al. [45] also evaluated hydrogen production from cheese whey in a fixed bed reactor and explained the reduced hydrogen yield by the predominance of non-hydrogen-producing microorganisms such as Sporolactobacillus sp. and Prevotella. Lactobacilli were also a predominant population in a thermophilic AFBR fed with sugarcane vinasse (20 g COD L
1) under a HRT of 4 h [26]. In another study, Mariakakis, Meyer and Steinmetz [46] evaluated the effect of HRT, organic loading and microbial dynamics during hydrogen production from molasses. According to these authors, many studies have identified the co-

10 13 9 19 6 2 6 3 1 9 2 1 2 2

existence of hydrogen-producing microorganisms and other microbial groups that often compete with the former for substrate availability. Some of these competing groups are homoacetogens that produce acetic acid, bacteria that produce propionic acid, the sulfate-reducing bacteria (SRB), and especially, the lactic acid bacteria (LAB). In most of the studies, the presence of LAB had a negative influence on hydrogen production which may be explained by (1) competition for substrate or (2) excretion of bacteriocins that inhibit the growth of other microorganisms, such as Clostridium [47]. Thus, analyzing the data available in literature, it can be inferred that the microbial groups identified in the present study are in accordance to what was found by other researchers working on the same subject. Regarding the predominant species observed in the pre is et al. [48] affirmed that Lactobacillus species sent work, Go

Fig. 4 e Phylogenetic tree of key OTUs and their closest relatives. The tree is based on Jukes-Cantor distances and was constructed using the neighbor-joining method with 1000 bootstrap resamplings. Sulfurospirillum cavolei was selected as the outgroup. Numbers close to the nodes represent the bootstrap values (>50%). Please cite this article in press as: Lazaro CZ, et al., Sequential fermentative and phototrophic system for hydrogen production: An approach for Brazilian alcohol distillery wastewater, International Journal of Hydrogen Energy (2015), http://dx.doi.org/ 10.1016/j.ijhydene.2015.06.003

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effectively compete with other microorganisms due to their fast growth and considerable tolerance to alcohol and low pH. They are the most common contaminant bacteria in fermentation cultures performed under non-sterile conditions in ethanol factories. Acid-resistant Lactobacillus species are also known to be indigenous flora not just in fermentation broth but also in vinasse [49]. The presence of Lactobacillusrelated species explains the consumption of the carbohydrates and the production of organic acids, such as lactic acid and acetic acid in the present study. According to Shah et al. [50], microorganisms from the genus Prevotella can ferment glucose, sucrose, and starch while producing acetic, isobutyric, isovaleric and succinic acids as byproducts. In the present study, the operational conditions (substrate and temperature) favored the establishment of these organisms, and it is believed that they consumed the carbohydrate present in vinasse and were responsible for the production of the above mentioned acids.

Phototrophic reactor performance Phylogenetic characterization of the microbial consortium After reactivation step, adaptation and determination of the ideal growing conditions of the microbial consortium [51,52], a composite sample was collected, and the biomass was used for phylogenetic characterization to identify the microorganisms present in the consortium. The sequences of 89 clones from the biomass were analyzed. These sequences were grouped into 6 OTUs with 95% similarity. The microorganisms present in the reactor belonged to the Bacteria Domain, Proteobacteria phylum and Bradyrhizobiaceae family. Sequences of the clones were closely related to Rhodopseudomonas sp. and Bradyrhizobiaceae bacterium (Table 4, Fig. 5). The phylogenetic characterization analysis showed that the composition of the phototrophic microbial consortium changed significantly after the reactivation phase, adaptation and determination of the optimal growing conditions. According to data presented by Lazaro et al. [31] for the microbial consortium for their study, only a third of the sequenced clones were related species of phototrophic bacteria: Rhodobacter, Rhodospirillum and Rhodopseudomonas and Sulfurospirillum. In the present study, after the reactivation phase, adaptation and determination of optimal culture conditions, the microbial consortium showed 95.5% of sequences of clones related to Rhodopseudomonas. This result could be explained by the adaptation of the microorganisms to a new light source, which may have resulted in enrichment for a

microbial consortium consisting primarily of microorganisms of interest to the phototrophic hydrogen production process. Rhodopseudomonas are gram-negative microorganisms that are often used in studies of photo-fermentative production of hydrogen from organic acids [13,53e55]. Due to their metabolic versatility, these microorganisms can be found in many habitats. An instructive example of this metabolic diversity is the ability of the bacterium Rhodopseudomonas palustris to grow under different metabolic modes: photoautotrophic or photosynthetic metabolism, in which light is the energy source and CO2 is the carbon source; photoheterotrophic metabolism, in which the carbon source is organic compounds; chemoheterotrophic metabolism, in which both the carbon and energy sources are organic compounds; and chemoautotrophic metabolism, in which the energy source is an inorganic compound and the carbon source is CO2 [56]. The role of the purple non-sulfur (PNS) bacteria in sulfur metabolism should also be highlighted. Initially, it was believed that these microorganisms were unable to tolerate toxic sulfur compounds such as sulfides like the purple sulfur bacteria; however, recent studies have clearly shown that oxidation of these compounds is common in this group. In some species, sulfur formed outside the cell is the end product of sulfide oxidation; for others (Rhodopesudomonas palustris), sulfate is the end product [57]. Most of the photobiological hydrogen production experiments were performed with pure cultures under sterile conditions, and there was no need to perform a phylogenetic characterization of the microorganisms; however, when using a microbial consortium, it is quite interesting to find the closest relatives of the microorganisms present in the bioreactor. Zang, Liu and Fang [58] evaluated the production of hydrogen from acidic wastewater and found that 81% of the microorganisms were similar to R. capsulatus (99.2% similarity), and a lesser percentage of microorganisms were related to Bacillus/Clostridium species. In addition to Rhodopseudomonas, we found some clone sequences related to Bradyrhizobium, which is a bacterium that is frequently associated with plant roots and establishes a symbiotic relationship with them, mainly due to their ability to fix atmospheric nitrogen. It is known that in addition to photo-fermentation, biophotolysis is another light-dependent biological process used for the production of hydrogen. Biophotolysis performed by green algae such as Chlamydomonas reinhardtii produces molecular oxygen. It is known that the Fe-hydrogenase enzyme responsible for hydrogen production in these organisms is oxygen

Table 4 e Phylogenetic affiliation of the clone library from phototrophic bacterial consortium. Phylogenetic affiliation

Bradyrhizobiaceae

UTO

1 2 3 4 5 6

Phylogenetic relationship

Number of clones

Closest species in GenBank

Similarity

Accession number

Rhodopseudomonas palustris; DX-1 Rhodopseudomonas palustris; PB-a Rhodopseudomonas sp. AM6 Rhodopseudomonas sp. JA495 Bradyrhizobiaceae bacterium D235 Rhodopseudomonas palustris; NTUIOB-PS3

0.996 1.000 0.992 0.994 0.994 0.987

EU221586 EU282380 EU252494 FN543497 AB480397 AB689796

40 35 3 5 4 2

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Fig. 5 e Phylogenetic tree of key OTUs and their closest relatives. The tree is based on Jukes-Cantor distances and was constructed using the neighbor-joining method with 1000 bootstrap resamplings. Thermotoga elfii was selected as the outgroup. Numbers close to the nodes represent the bootstrap values.

sensitive, so the cultivation of these microorganisms with other oxygen-consuming species may contribute significantly to the production of hydrogen via that process. Wu et al. [59] evaluated this alternative and observed increased hydrogen production by a co-culture of C. reinhardtii and Bradyrhizobium japonicum. In the present study, other Bradyrhizobiaceae-related bacterium, as Bradyrhizobium, may have carried out a similar function as discussed by Wu et al. [59]. Most likely, the surface of the reactor was not completely anaerobic, although efforts were made to ensure a lack of oxygen. Thus, even though these microorganisms were not the objective of this study, the coculture is very interesting, especially in light of the production of hydrogen by photo-fermentation reactors on a larger scale for which ensuring anaerobic conditions could be difficult.

Hydrogen production, COD and sulfate removal, and sulfide production The organic acids produced in the first stage (AFBR) could be utilized for further hydrogen production and COD removal in the second stage. The second stage was carried out in anaerobic batch reactors that were fed with different concentrations of the AFBR

effluent. In general, we observed that an increase in the AFBR effluent concentration (from 10 to 80 %) (1.6e11.7 g COD L
1, respectively) negatively impacted cell growth, hydrogen production and COD removal. The negative impact could be due the high concentration of organic acids or the presence of inhibitors in the AFBR effluent because vinasse contains compounds such as phenols that are toxic to bacteria [60]. In addition to the experiment for hydrogen production with the AFBR effluent, we also carried out an experimental control (0% effluent) that was fed a mixture of acetate and butyrate. As expected, in the control, we observed the highest cumulative hydrogen production, the highest percentage of H2 in the biogas, and the lowest sulfate removal and sulfide production (Table 5). As higher concentrations of effluent were added to the culture medium, hydrogen production decreased. Cumulative hydrogen production and percentage of hydrogen in the biogas decreased from 6.2 to zero mmol H2 L
1 and 89.5% to the zero in the tests with 10 and 80% effluent, respectively. Conversely, the percentage of CO2 increased from 10.5 to 100% in experiments with 10 and 80% effluent, respectively. These results demonstrate that although there was no hydrogen production in reactors fed with higher

Table 5 e Phototrophic hydrogen production experiments with different concentrations of AFBR effluent. Parameters

Effluent concentration
1

H2 concentration (mmol H2 L ) CO2 concentration (mmol H2 L
1) % H2a % CO2a Initial COD (g L
1) COD removal (%)
1 Initial SO2
4 (mg L ) SO2
4 removal (%) b P (mmol H2 L
1) b Rm (mmol H2 L
1 h
1) b L (h) R2 a b

0% (control)

10%

20%

40%

70%

80%

11.8 ± 3.3 1.0 ± 0.2 92.2 ± 1.6 7.8 ± 1.6 1.8 61.2 ± 6.4 60 ± 15 16.7 ± 6 11.8 ± 0.4 0.16 ± 0.2 11.9 ± 4.1 0.992

6.2 ± 1.7 0.7 89.5 ± 2.6 10.5 ± 2.6 1.6 ± 0.1 66.1 ± 1.1 169 ± 25.5 68.8 ± 7 6.3 ± 0.2 0.07 30.2 ± 3.3 0.997

5.9 ± 1.5 0.9 86.3 ± 2.8 13.7 ± 2.8 3.0 ± 0.1 70.3 ± 1.6 278 ± 35.5 77.0 ± 9 6.0 ± 0.1 0.09 24.7 ± 2.8 0.997

0.8 ± 0.1 1.3 ± 0.1 35.5 ± 5.1 64.5 ± 5.1 6.0 ± 0.2 52.2 ± 1.9 496 ± 40.3 83.9 ± 3 0.7 0.01 46.8 ± 5.9 0.993

e 1.9 ± 0.1 e 100 10.2 ± 0.3 7.0 ± 3.5 823 ± 15.5 86.5 ± 1 e e e e

e 2.1 ± 0.1 e 100 11.7 ± 0.2 5.9 ± 2.1 932 ± 90 86.9 ± 3 e e e e

Percentage at the end of the experiments. Adjustment of the data by the modified Gompertz model [51,52].

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concentrations of effluent (70 and 80%), there was microbial metabolism responsible for CO2 generation, sulfate removal and sulfide production (Table 5, Fig. 6A). The main reason to use a two-stage process is COD removal. Organic matter removal ranged from 66.1 to 5.9 % in experiments with 10 and 80% of AFBR effluent concentration, respectively (Table 5). Higher COD removal (70.3%) occurred in the experiment with 20% of AFBR effluent concentration. The values measured in trials with 10 and 20 %AFBR effluent concentration were comparable to those reported by Tao el al [61]. that used a pure culture (Rhodobacter ZX-5) for hydrogen production from acid-rich residues (i.e., acetic, butyric and succinic acids). COD removal observed in the present study was also comparable to that measured in a study that evaluated methane production from vinasse (69%) [62]. It is important to emphasize that organic matter present in the fermentative reactor effluent could be removed by both phototrophic and methanogenic systems. The main disadvantage of the phototropic process is the requirement for light energy, whereas its main advantage is the capability of

Fig. 6 e A) Initial and final biomass concentration (expressed as VSS) for the phototrophic hydrogen production experiments in batch reactors with different AFBR effluent concentrations, - initial VSS, - final VSS. B) Cumulative hydrogen production during the experimental period, A control (without effluent); - 10% effluent (v/v); : 20% effluent (v/v); C 40% effluent (v/v).

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phototrophic bacteria to directly consume a wide range of organic acids as substrates for bioenergy production [32]. The methanogens are able to directly consume only some fermentation products, especially acetate, H2, CO2 and other one-carbon compounds. For the degradation of fatty acids longer than two carbon atoms chain, an additional group of microorganisms is needed. Thus, the main disadvantage of methanogenic stage reactors is the nutritional restriction of the methanogenic archaea, which are acetoclastics (acetic acid) and hydrogenotrophics (H2 and CO2, formate) [63]. In the present study, as seen in Fig. 6A, the biomass concentration was slightly higher for the control (0%) and 10 and 20% of AFBR effluent as substrate. For higher effluent concentrations (70 and 80%), we observed a decrease in biomass that reached the lowest (245 mg SSV L
1) value at the highest AFBR effluent concentration (80%). The limited growth could be related to the inhibition of the PNS bacterial growth due to the presence of sulfide or because the vinasse color could have impeded light penetration. The hydrogen yield, expressed as moles H2 per grams of COD removal, decreased with an increase in the AFBR effluent concentration (Fig. 7B). The maximum value was 10.1 mmol H2 g
1 CODremoved in the control. This value decreased to 5.5 mmol H2 g
1 CODremoved for the experiment with 10% AFBR effluent concentration. The COD influent for the control and the 10% AFBR effluent was similar (Table 5). This result makes it clear that the negative impact of AFBR effluent on phototrophic hydrogen production is not because of the COD concentration but is due to the AFBR effluent composition. In the control, the carbon source was acetate and butyrate; however, for the 10% AFBR effluent, the organic matter was composed mainly of butyric, isobutyric and propionic acids. This difference could explain the hydrogen yield results. We even observed a reduced hydrogen yield result for the experiment with 20% of AFBR (2.8 mmol H2 g
1 CODremoved). For the treatments with 70 and 80% AFBR effluent, there was no hydrogen production. Thus, we observed a reduction in the hydrogen yield with an increase in AFBR effluent. However, an opposite trend was observed for sulfate removal efficiency (Fig. 7B). The results imply that increasing the AFBR effluent concentration caused the preponderance of the sulfate reduction process over hydrogen production, as will be discussed further. In addition to hydrogen production and COD removal, we also analyzed sulfate removal and sulfide production in the phototrophic batch reactor. It is known that vinasse is a sulfate-rich wastewater and that sulfate reduction could detrimentally affect bioenergy production. Sulfate reduction and sulfide generation is a process that commonly occurs in anaerobic reactors (Eq. (4)) [64]. In the present study, we did not observe sulfate removal in the fermentative stage (i.e., in the AFBR). However, in the phototrophic stage, in which the AFBR effluent was used as the substrate for hydrogen production, sulfate removal reached 86.9% (for the 80% effluent treatment) (Table 5). Sulfide production was lower in the control (10.4 mg L
1) and increased to 30.9, 42.3, 55.7, 76.1 and 74.6 mg L
1 for the treatments with 10e80 % of the AFBR effluent, respectively (Fig. 7A). The ratio of COD and SO
2 4 for the control was 30, while for all the other treatments, it was approximately 10. Thus, in the AFBR

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effluent treatments, the concentration of sulfate was three times higher than the control. SO4
2 þ 4H2 þ Hþ /HS
4H2 O

Fig. 7 e A) Sulfate and sulfide concentration in the phototrophic hydrogen production experiments fed with AFBR effluent, - initial sulfate, - final sulfate, B produced sulfide. B) Hydrogen yield and percentage of sulfate removal in the phototrophic hydrogen production experiments fed with AFBR effluent, - % sulfate removal, A hydrogen yield.

(4)

Sulfate reduction in hydrogen production systems is undesirable because (1) the sulfate reduction process consumes hydrogen and (2) the sulfide concentration could be toxic to microorganisms [14,32,65]. SRB is a diverse group of anaerobic microorganisms which plays an important role in the cycling of carbon and sulfur. In the environment rich in sulfate, SRB are viewed as hydrogen-consuming microorganism. However, due to its metabolic diversity, in sulfate-limiting habitats, they are able to grow fermentatively by producing H2 and CO2 [66], e.g., Desulfovibrio vulgaris growing on formate [67]. Sulfate reduction with sulfide production has not often been reported in studies that evaluate the hydrogen production by two-stage processes. Possibly, this process has not attracted much attention because most of the studies were performed with pure cultures of PNS bacteria, instead of a mixed consortium under aseptic conditions. Furthermore, it may not have been noticed because not all wastewaters have high concentrations of sulfate. However, Lee et al. [32] affirmed that a medium with high sulfate concentrations is not suitable for hydrogen gas production. The reason is the rapid proliferation of sulfur-reducing bacteria that produce hydrogen sulfide, which should be toxic and inhibit the growth of PNS bacteria. On the other hand, the interference of the nitrogen source in phototrophic hydrogen production has been studied often due to the repression of nitrogenase, a key enzyme in purple bacteria [68]. Samples from the beginning and the end of the phototrophic stage were analyzed for their organic acid content. A higher concentration of organic acids was observed in treatments fed with higher AFBR effluent concentrations. At the beginning of the experiments, the organic acids detected were acetic, butyric, malic, lactic, propionic, isobutyric and caproic acids (Table 6). For all the treatments, the concentration of acetic and caproic acids increased, while the concentrations of butyric, isobutyric, lactic and propionic acids decreased. In general, the concentration of malic acid did not change

Table 6 e Distribution of organic acids at the beginning and at the end of the experiments. Acids (mg L
1)

HAc HBu HMa HLa HPr HIsoBu HCa

I (initial)

Effluent concentration (v/v)

F (final)

0% (control)

10%

20%

40%

70%

80%

I F I F I F I F I F I F I F

407.1 167.1 590.7 313.7 e e e e e e e e e e

35 219 319 68 57 e 57 e 211 12 305 e 27 281

78 319 754 267 125 111 137 e 421 51 516 3 90 486

117 533 1682 1167 274 240 289 e 710 169 683 16 245 1546

276 1154 2708 1815 582 460 553 e 1336 906 1470 e 358 3197

247 1246 3287 2020 548 520 650 e 1536 1205 1330 e 658 3719

HAc- acetic acid, HBu- butyric acid, HMa-malic acid, HLa- lactic acid, HPr-propionic acid, HIsoBu-isobutyric acid, HCa-caproic acid.

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[28] [72] Present study

[13] [27]

90.3 84.3 80.2 e e 66.1 Centrifugation (4800 rpm) and filtration Centrifugation and vacuum filtration e

2.8 L 0.8 ml g
1 cassava 0.6 ml g
1 food waste 175.9 ml 755 ml L
1 6.2 mmol L
1 (152.5 ml L
1) Centrifugation (13,000 rpm) Centrifugation (8000 rpm)

Anaerobic consortium, Rhodopseudomonas palustris Enterobacter aerogenes, Rhodopseudomonas BHU01 Anaerobic consortium, phototrophic consortium

Batch Batch Batch

Sucrose Cassava Food waste Cassava starch Sugarcane bagasse Sugarcane vinasse Batch Batch Clostridium pasteurianum, Rhodopseudomonas palustris Anaerobic consortium, Rhodobacter sphaeroides

Substrate Reactor Microorganism

Table 7 e Phototrophic hydrogen production by two-stage process systems.

Dark effluent pre-treatment

H2 production at photo-fermentation

COD removal (%)

Reference

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significantly; except for the 10% AFBR effluent treatment, in which it was totally consumed (Table 6). The production of acetic acid could be associated with sulfate reduction and sulfide production. According to Muyzer and Stams [69], microorganisms such as Desulfotomaculum and Desulfovibrio can utilize hydrogen and diverse organic compounds (ethanol, formate, lactate, pyruvate, malate and succinate) as carbon sources, and they are incompletely oxidized with the concomitant formation of acetate. For all the treatments, we observed significant caproic acid production, which was highest in the treatments with the highest AFBR effluent concentrations (70 and 80%). This result could be related to cell lysis at high AFBR effluent concentrations because caproic acid is a known membrane compound in Gram-negative cells [70]. The two-stage process for biological hydrogen production has been evaluated using diverse substrates: food waste [27], cassava starch [28], molasses [29], starch [71], sugarcane bagasse [72], and sucrose [13]. As seen in the comparative table (Table 7), most of the studies used pure cultures for the photo-fermentation stages, and dark fermentation reactor effluent was pre-treated by centrifugation and/or filtration, which could increase hydrogen production in the second stage by removing contaminating microorganisms and particles. Thus, a pre-treatment that removes color and indigenous organisms from the effluent could significantly enhance photo-fermentation. Wastewaters with different degrees of transparency require different illumination intensities for optimum gas production [73]. In the present study, our results are lower than those reported in the literature; however, we did not use pure cultures for both the fermentative and phototrophic stages, nor was the dark fermentative effluent pretreated. Our results indicate a pre-treatment for the dark fermentative AFBR effluent to counter the negative influence of sulfate on photo-fermentation should be sulfate removal, e.g., treatment with CaO followed by filtration [74]. The use of CaO is often applied in studies on fermentative hydrogen production by acid hydrolyzed substrates, e.g., cellulosic materials. Nevertheless, any pre-treatment should be evaluated in terms of improvement in the hydrogen yield versus the cost and time consumed, i.e., the cost-benefit ratio.

Conclusions Non-sterile sugarcane vinasse with a high organic matter concentration (10 g COD L
1) was used as substrate for hydrogen production in a sequential system composed by fermentative and phototrophic stages. First, the vinasse was treated in a dark fermentation reactor and the hydrogen yield was 0.34 mmol H2 g
1 CODadded. Then, the effluent of such reactor was diluted to be treated by a phototrophic microbial consortium and the maximum hydrogen yield was 5.5 mmol H2 g
1 CODremoved (10% dilution). However, increasing the effluent concentration as the substrate for the phototrophic reactors had a negative impact on cell growth, hydrogen production and COD removal, probably because the sugarcane vinasse composition includes toxic compounds, such as phenols and melanoidins. Furthermore, a sulfate

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reduction (86.9%) coupled to sulfide production (74.6 mg L
1) observed during the phototrophic stage could have been responsible for reduced hydrogen yields and moderated organic matter removal due to sulfide toxicity or via the consumption of hydrogen in the reduction process. Thus, it can be concluded that although sugarcane vinasse is a suitable substrate for biological hydrogen production, sulfate reduction should be inhibited to assure more favorable results. Regarding the microbial phylogenetic characterization, despite the efforts made to enrich endospore-forming bacteria, microorganisms closely related to Lactobacillus species were predominant in the fermentative stage, which can explain the reduced hydrogen yield obtained. In the phototrophic stage, SRB were a possible contaminating microorganism present in the fermentative effluent and predominated in the second step, inhibiting the growth of the Rhodopseudomonas species from the seed inoculum.

Acknowledgements The authors gratefully acknowledge the financial support ~ o Paulo Research Foundation (Processes 2009/ of FAPESP - Sa 15984-0, 2012/03979-5 and 2012/21715-5)

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