Impact of organic loading rate on biohydrogen

Bioresource Technology 164 (2014) 371–379

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Impact of organic loading rate on biohydrogen production in an up-flow anaerobic packed bed reactor (UAnPBR) Antônio Djalma Nunes Ferraz Júnior a,b, Marcelo Zaiat a, Medhavi Gupta b, Elsayed Elbeshbishy c,⇑, Hisham Hafez d, George Nakhla b a Biological Processes Laboratory, Center for Research, Development and Innovation in Environmental Engineering, São Carlos School of Engineering (EESC), University of São Paulo (USP), Engenharia Ambiental – Bloco 4-F, Av. João Dagnone, 1100, Santa Angelina, 13563-120 São Carlos, SP, Brazil b Dept. of Chemical and Biochemical Engineering, University of Western Ontario, London, Ontario N6A 5B9, Canada c Dept. Civil and Environmental Engineering, University of Waterloo, London, Ontario N2L 3G1, Canada d Greenfield Ethanol, 540 Park Avenue East, Chatham, Ontario N7M 5J4, Canada

h i g h l i g h t s Biohydrogen from glucose in an up-flow anaerobic packed bed reactor was studied. Design modification were made in the settling zone to capture the detached biomass. Impact of organic loading rate on hydrogen yield were assessed. 1

Maximum hydrogen yield of 2.1 mol-H2 mol

-glucose was achieved.

The biomass yield in the modified system was lower than the conventional system.

a r t i c l e

i n f o

Article history: Received 25 February 2014 Received in revised form 2 May 2014 Accepted 4 May 2014 Available online 14 May 2014 Keywords: Biohydrogen Packed bed reactor Attached biomass Organic loading rate Settling zone

a b s t r a c t This study assesses the impact of organic loading rate on biohydrogen production from glucose in an up-flow anaerobic packed bed reactor (UAnPBR). Two mesophilic UAPBRs (UAnPBR1 and 2) were tested at organic loading rates (OLRs) ranging from 6.5 to 51.4 gCOD L1 d1. To overcome biomass washout, design modifications were made in the UAnPBR2 to include a settling zone to capture the detached biomass. The design modifications in UAnPBR2 increased the average hydrogen yield from 0.98 to 2.0 mol-H2 mol1-glucose at an OLR of 25.7 gCOD L1 d1. Although, a maximum hydrogen production rate of 23.4 ± 0.9 L H2 L1 d1 was achieved in the UAnPBR2 at an OLR of 51.4 gCOD L1 d1, the hydrogen yield dropped by 50% to around 1 mol-H2 mol1-glucose. The microbiological analysis (PCR/DGGE) showed that the biohydrogen production was due to the presence of the hydrogen and volatile acid producers such as Clostridium beijerinckii, Clostridium butyricum, Megasphaera elsdenii and Propionispira arboris. Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Abbreviations: AnFBR, anaerobic fluidized bed reactors; CSTR, continuous stirred tank reactors; DGGE, denaturing gradient gel electrophoresis; HRT, hydraulic retention time; IBRCS, integrated biohydrogen reactor clarifier system; LAPB, lactic acid-producing bacteria; LDPE, low density polyethylene; MEGA, Molecular Evolutionary Genetic Analysis; OLR, organic loading rate; SCOD, soluble chemical oxygen demand; SRT, solids retention time; STP, standard temperature and pressure; TCOD, total chemical oxygen demand; TSS, total suspended solids; UAnPBR, up-flow anaerobic packed bed reactor; VSS, volatile suspended solids; PCR, polymerase chain reaction. ⇑ Corresponding author. Tel.: +1 519 860 3556. E-mail address: [email protected] (E. Elbeshbishy). http://dx.doi.org/10.1016/j.biortech.2014.05.011 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.

Hydrogen as an energy carrier has been extensively studied. Its energy content is 2.7 times more than fossil fuels (i.e., gasoline, propane, methane and others), and is also considered a clean energy as its combustion produces only water (Chen et al., 2001). Among the various processes for hydrogen production, i.e., steam reforming, partial oxidation of hydrocarbons (Dashliborun et al., 2013) and alcohols (Carotenuto et al., 2013), and electrolysis (Tuomi et al., 2013), biological hydrogen production is receiving increasing attention. Biological hydrogen production can occur via two processes: photosynthesis and dark fermentation. However, the efficiency of hydrogen production via photosynthesis is

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A.D.N. Ferraz Júnior et al. / Bioresource Technology 164 (2014) 371–379

low and light-dependent, while the dark fermentation process is technically simpler and the hydrogen can be produced continuously from various types of substrates present in wastewater (Wang and Wan, 2009). Hydrogen production via dark fermentation is considered one of the most attractive processes because it is a low-cost technology, based on widely known fundamentals of hydrolysis, acidogenesis, and acetogenesis. In addition, it requires simple reactors and less energy input when compared to other technologies (Hallenbeck, 2009). Batch bioreactors have been frequently used in hydrogen production for process evaluation i.e., determining the biohydrogen potential from organic substrates (Fernandes et al., 2010), buffer requirements (Lin and Lay, 2004), and inhibitory effects (Lee et al., 2012). However, for practical reasons and economic considerations, continuous bioreactors are recommended (Guo et al., 2010). Continuous stirred tank reactors (CSTR) are simple to operate and thus have been used for model studies since they ensure maximum mixing and homogeneity (Hallenbeck, 2009). In CSTR, since biomass has the same retention time as the liquid, washout of biomass may occur at short hydraulic retention times (HRTs) (Wang and Wan, 2009). In contrast, in fixed-film reactors, bacterial growth rates and HRT are independent of each other which can facilitate maintaining long solids retention times (SRT) (Gavala et al., 2006). Furthermore, high biomass densities provide greater resistance to any inhibitory substances in the influent and shock loads (Metcalf and Eddy, 2003). While successful biohydrogen production has been achieved in CSTRs coupled with gravity settlers that decouple SRT from HRT (Hafez et al., 2010), other promising fixed-film processes such as packed bed reactors have not received much attention. Although packed bed reactors (PBR) have been used for the treatment of both dilute and high strength soluble wastewaters (Perna et al., 2013), their application to biohydrogen production merits further investigation. Wu et al. (2007) who evaluated both fluidized bed and packed bed bioreactors with polyethylene beads as carrier media, for biohydrogen and bioethanol production from sugars, observed biohydrogen production yields of 0.65 and 1.04 mol H2/mol hexose at OLR of 120 g COD/L d and an HRT of 4.0 h. Scoma et al. (2013) observed that in PBR with ceramic tubes, at HRTs of 1.7 days and OLRs of 5.5–38.8 g COD/L d, biohydrogen production increased with the reduction in HRT. Similarly, Leite et al. (2008) reported biohydrogen yields of 1.8 and 2.5 mol H2/mol glucose from the fermentation of glucose in a PBR with expanded 0.5–0.6 cm clay beads as support material at HRTs of 0.5–2.0 h. The focus of the limited aforementioned studies on biohydrogen using PBR was primarily on acidification efficiency and hydrogen yields with no particular discussion of biomass yields, attachment, and detachment which significantly impact bioreactor design. The main objectives of this study are to assess the impact of organic loading on biohydrogen production from glucose in an up-flow anaerobic packed bed reactor (UAnPBR) and evaluate biomass yields and detachment characteristics. Furthermore, the attached biomass developed in the reactors was characterized using PCR/ DGGE.

2. Methods Anaerobically digested sludge from the St. Marys wastewater treatment plant (St. Marys, Ontario, Canada) was used as seed. In order to select for hydrogen producing bacteria, the sludge was heat treated at 70 °C for 30 min. The characteristics of the seed were as follows: total suspended solids (TSS) – 14,700 mg L1; volatile suspended solids (VSS) – 10,900 mg L1; total chemical oxygen demand (TCOD) – 15,600 mg L1; soluble chemical oxygen demand (SCOD) – 900 mg L1; and pH – 7.1. Low density polyethylene

(LDPE) pellets were used as support. The pellets length, diameter, and specific surface area were 4.4 mm, 7.5 mm and 12 cm2 g1, respectively. Two UAnPBR were built using acrylic tubes (internal diameter – 80 mm; outer diameter – 88 mm). Thus, the ratio of particle diameter to column diameter (øsupport/øinternal reactor) was 1/8 to reduce wall effects. UAnPBR 1 (Fig. 1A) was divided into four zones: the feed zone (L1 = 100 mm – 0.5 L); bed zone (L2 = 500 mm – 2.5 L); effluent collection zone (L3 = 100 mm – 0.5 L); and biogas collection zone (L4 = 50 mm – 0.25 L), resulting in total and liquid volumes of approximately 3.5 and 2.3 L, respectively. During the UAnPBR1 start-up, biomass washout was observed. To overcome this problem, design modifications were made in the UAnPBR2 to include a settling zone to capture the detached biomass. Thus, UAnPBR2 was divided into five zones: the feed zone (L1 + L5 = 100 mm – 0.5 L); bed zone (L2 = 500 mm – 2.5 L); settling biomass zone and effluent collection zone (L3 = 450 mm – 2.25 L); and biogas collection zone (L4 = 50 mm – 0.25 L), resulting in total and liquid volumes of approximately 5.5 and 3.6 L, respectively. In both reactors, the bed zone was separated by stainless steel screens fixed by rods (4 mm) of the same material (Fig. 1B). The composition of the nutrients and trace mineral solution was identified to that reported by Hafez et al. (2010). The initial pH of the glucose-based synthetic wastewater was adjusted to 6.5 by a phosphate buffer solution. The volume and composition of the biogas (hydrogen, methane and nitrogen) as well as glucose concentrations were determined as described by Hafez et al. (2010). The VSS and COD were measured according to standard methods (APHA, 2005). 3. Set-up and operational conditions of UAnPBR for H2 production Two lab-scale reactors were operated, separately, at 37 °C, at different organic loading rates ranging from 6.5 g-COD L1 d1 to 51.4 g-COD L1 d1. More details about the operational conditions are highlighted in Table 1. Initially, UAnPBR1 was seeded with 1.0 L of sludge and started up in a continuous-flow mode with the glucose-based synthetic wastewater. Thus, to increase the mass transfer rates from the bulk liquid to the biocatalyst and the liquid-to-gas mass transfer, a liquid recycle (at 60% of the feed flow rate) was applied based on the findings of Lima and Zaiat (2012), who observed a positive impact on hydrogen production in a reactor with the same configuration. Reactors temperature was maintained at 37 ± 1 °C by a water bath (Heat circulating bath, VWRÒ). The same start-up technique and operational conditions were repeated for the UAnPBR2. The systems were monitored for influent and effluent TCOD, SCOD, volatile fatty acids (VFA), glucose, VSS, and biogas composition including hydrogen, methane and nitrogen. The gas volumes were corrected to standard temperature (0 °C) and pressure (760 mm Hg), (STP). 4. Molecular analysis Samples for molecular analysis were collected from the bed zone (attached biomass) of UAnPBR1 and UAnPBR2, at the end of the operation. Biomass detachment from 35 g of the low density polyethylene pellets was carried out by sonication at 40 W for 5 min, followed by centrifugation (5 min, at 6000g and 25 °C). The total DNA was extracted using UltraClean Soil DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, USA). 16S rRNA gene was amplified using specific primers 968FGC (50 -AAC CGC GAA GAA CCT TAC CGC GGC CCG GGG CGC CCG GGG CGC CCG GGC GGG GCG GGG GCA CGG GGGG-30 ) and 1392R (50 -AACG GGC GGT GTG TAC-30 ), according to Nielsen et al. (1999). Polymerase chain reaction (PCR) amplification


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Fig. 1. Schematic description of anaerobic fixed bed bioreactor (AFBR) for continuous hydrogen production; (A) UAnFBR1, (B) UAnFBR2.

Table 1 Operational conditions.

1 2

Parameter

Units

Condition I Condition II Condition III

Glucose concentration OLR HTR Flow rate1 Recycle ratio (Qr/Qin)2 Sodium bicarbonate

g L1 g-COD L1 d1 h L d1 – g L1

2.2 6.5 8 6.9/10.8 0.6 1

8.6 25.7 8 6.9/10.8 0.5 2.5

17.1 51.4 8 6.9/10.8 0.6 5

UAnPBR1/UAnPBR2. Qr– recycle flow rate; Qin– Influent flow rate.

conditions were: initial denaturing (94 °C for 7 min); 35 cycles (denaturing – 94 °C for 5 s; annealing – 55 °C for 45 s; extension – 72 °C for 1 min.); and final extension (72 °C for 10 min.). Denaturing gradient gel electrophoresis (DGGE) was performed with a DCode universal mutation system (Bio-Rad laboratories, Hercules, CA, USA). The PCR products were applied directly to 8% (w/v) polyacrylamide gel with 40–60% denaturant gradients. Electrophoresis was performed at a constant voltage of 75 V at 65 °C for 16 h. The

DNA templates of the bands of interest were re-amplified and the PCR products were purified using QIAquick PCR purification Kit (QIAGEN Sciences, Maryland, USA) in accordance with the manufacturer’s protocol. The sequences of re-amplified DNA fragments were determined by dideoxy chain termination and compared with available sequences in the GenBank database using the BLAST program (Altschul et al., 1990). Phylogenetic analyses of the sequences were performed using the Molecular Evolutionary Genetic Analysis 5.2 (MEGA 5.2) software (Kumar et al., 2004). Evolutionary distances were based on the Kimura model (Kimura, 1980) and tree reconstruction on the neighbor-joining method with bootstrap values calculated from 500 replicate runs, using the routines included in MEGA software (Maintinguer et al., 2008).

5. Results and discussions 5.1. Reactors performance The reactors were operated for 47 days at three different loading conditions, as described in Table 1: condition 1 from day 5 to

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day 12, condition 2 from day 13 to 34, and condition 3 from day 35 to 47. The systems showed stable hydrogen production during steady-state operation. The coefficients of variation (calculated as standard deviation divided by the average) for hydrogen production rates and yields in all runs were approximately less than 10%. SCOD removal efficiencies ranged between 9% and 31% for UAnPBR1 and 15% and 34% for UAnPBR2. The glucose conversion efficiency to volatile fatty acids (acetic, butyric, propionic, mostly) was around 100% at OLR of 6.5 and 25.7 g-COD L1 d1 and around 85% at OLR of 51.4 g-COD L1 d1 for both reactors.

5.2. Hydrogen production The temporal variations of volumetric biohydrogen production rate and hydrogen yields are depicted in Fig. 2a and b, respectively. The biogas produced showed a maximum hydrogen production content of 37.3% and 43.2% for UAnPBR1 and UAnPBR2, respectively, with no methane detected, confirming that heat treatment inhibited methanogens. Figs. 2 and 3 show the temporal variation of hydrogen production rate and hydrogen yield (based on the amount of glucose converted) Hydrogen production rates increased

(a)

(b)

Fig. 2. (a) Temporal variation of hydrogen yield, (b) temporal variation of volumetric hydrogen production rate.


375

(a)

(b)

Fig. 3. (a) Temporal variation of volatile suspended solids, (b) biomass yields at the three loading conditions.

with the increase in OLR, with averages of 1.7 ± 0.1 L-H2 d1, 7.4 ± 0.7 L-H2 d1, and 9.8 ± 0.8 L-H2 d1 for UAnPBR 1 at OLRs of 6.5 g-COD L1 d1, 25.7 g-COD L1 d1 and 51.4 g-COD L1 d1, respectively, and 6.3 ± 0.3 L-H2 d1, 22.6 ± 0.8 L-H2 d1, and 23.4 ± 0.9 L-H2 d1 for UAnPBR2 at the same OLRs. On the other hand, the hydrogen yield (Fig. 2a) remained constant with the increase in OLR between 6.5 g-COD L1 d1 and 25.7 g-COD L1 d1 at around 1.0 mol-H2 mol1 glucose for UAnPBR1 and 2.0 mol-H2 mol1 glucose for UAnPBR2, decreasing to 0.7 mol-H2 1 mol1 glucose in UAnPBR1 and 1.0 mol-H2 molglucose in UAnPBR2 at an OLR of 51.4 g-COD L1 d1. Considering the maximum theoretical hydrogen yield from glucose conversion to acetate of 4 mol-H2 mol1 glucose, these values represent 25% (UAnPBR1) of the theoretical and 50% (UAnPBR2) at OLRs of 6.5 g-COD L1 d1, and 25.7 g-COD L1 d1 and 17.5% (UAnPBR1) and 25% (UAnPBR2) of the theoretical at OLRs of 51.4 g-COD L1 d1.

Normalizing the hydrogen production based on the reactor liquid volume, as depicted in Fig. 2b, the volumetric hydrogen production rates averaged 0.8 ± 0.1 L-H2 d1 L1 reactor, 3.2 ± 0.3 L-H2 1 1 d1 L1 Lreactor for UAnPBR1 at OLRs of reactor, and 4.3 ± 0.3 L-H2 d 6.5 g-COD L1 d1, 25.7 g-COD L1 d1, and 51.4 g-COD L1 d1, 1 1 respectively, and 1.7 ± 0.1 L-H2 d1 L1 Lreactor, reactor, 6.3 ± 0.2 L-H2 d 1 1 and 6.5 ± 0.2 L-H2 d Lreactor for UAnPBR2 at the same OLRs. The temporal variations of effluent VSS concentrations are shown in Fig. 3a. Generally effluent VSS concentrations in UAnPBR1 were 200 mg L1 higher than UAnPBR2, ranging on average from 459 mg/L at 6.5 gCOD L1 d1 to 759 mg L1 at 51.4 gCOD L1 d1. The data also indicates that effluent VSS concentrations increased initially with the increase in OLR for a period of one week and stabilized thereafter. It should be noted that the suspended biomass in both liquid sections represented 13% and 9% of the total biomass (as VSS) in UAnPBR1 and UAnPBR2,

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respectively, confirming indeed that the reactors behaved as fixedfilm systems. The attached biomass in UAnPBR2 of 33 g was 2.5 times higher than the 13 g in UAnPBR1, noting that the ratio of influent glucose mass rate to UAnPBR2 to UAnPBR1 was only 1.56 times. This clearly emphasizes that either the biomass yield in UAnPBR2 was higher than UAnPBR1 or the biomass detachment rate in UAnPBR2 was lower than UAnPBR1, resulting in better biomass attachment. The biomass yields, calculated as the sum of attached biomass and effluent suspended biomass per unit influent COD converted, are depicted in Fig. 3b. The biomass yields in UAnPBR1 remained relatively constant, varying narrowly, from 0.38 to 0.41 gVSS g1 CODconverted while the biomass yields for UAnPBR2 were significantly lower at 0.19–0.28 gVSS g1CODconverted. It is noteworthy that generally the hydrogen yield decreased with increasing biomass yields, consistent with the observations of Hafez et al. (2010). The first-order biomass detachment rate coefficient at 51.4 gCOD L1 d1for UAnPBR1 and UAnPBR2 were computed as 0.31 and 0.16 d1, corresponding to SRTs of 3.3 and 6.3 days, respectively. It is thus evident that the improved performance of UAnPBR2 relative to UAnPBR1 is not due to a higher biomass yield, but rather a lower biomass detachment rate, resulting in higher biomass retention. 5.3. Soluble metabolic products Table 3 shows the COD mass balances that considered the measured influent and effluent CODs and the equivalent CODs for both hydrogen gas (8 g-COD g1-H2, at 37 °C) and biomass synthesis (1.42 gCOD g1-VSS). COD mass balance closures ranged between 87% and 101% validating the reliability of the data. At OLRs of 6.5

and 25.7 g COD/L d, acetate, butyrate and propionic acids were the main soluble products in both reactors, while at an OLR of 51.4 g COD/L d, isobutyric acid increased significantly (Table 3). Stoichiometric reactions illustrate the hydrogen, acetic (I) and butyric (II) acids production from glucose, with reaction (III) and (IV) illustrating the hydrogen consumption from propionic acid and carbon dioxide (Antonopoulou et al., 2008).

Acetic acid production : C6 H12 O6 þ 2H2 O ! 2CH3 COOH þ 2CO2 þ 4H2

ðIÞ

Butyric acid production : C6 H12 O6 ! CH3 CH2 CH2 COOH þ 2CO2 þ 2H2

ðIIÞ

Propionic acid production : C6 H12 O6 þ 2H2 ! 2CH3 CH2 COOH þ 2H2 O

ðIIIÞ

Acetic acid production by=C:ljungdahlii : 4H2 þ 2CO2 ! CH3 COOH þ 2H2 O

ðIVÞ

The measured average concentrations of acetate and butyrate were used to estimate the theoretical hydrogen production (Table 4) based on reactions I and II. It is apparent from Table 4 that the theoretical hydrogen production through the acetate and butyrate pathways closely matches the experimental data, thus confirming that hydrogen scavenging pathways such as the propionate pathway (Eq. 3) and the homoacetogens pathway (reaction IV) do not play any role. Furthermore, the contribution of the acetate pathway to hydrogen production in UAnPBR1 was only 50% and 68%, respectively at OLRs of 6.5 and 25.7 g COD L1 d1, as compared with 87%, and 77%, for UAnPBR2. At all three loadings, much higher propionate concentrations were observed in UAnPBR1 than UAnPBR2.

Table 2 Comparative study of different design of reactor, operating conditions and efficiency of dark fermentative hydrogen production.

a

Reactor

Support material

Substrate

pH, temperature (°C)

HRT (h)

OLR (g-COD m3 d1)

Hydrogen yield (mol-H2 mol1 glucose)

References

IBRCS AFBR HAIB Chemostat Chemostat UAnPBR1 UAnPBR2

– Clay expanded Clay expanded – – Low density polyethylene Low density polyethylene

Glucose Glucose Glucose Glucose Glucose Glucose Glucose

5.5, 37 5.5, 30 3.3–7.3, 30 5.7, 35 –, 15–34 6.5, 37 6.5, 37

8 1–8 0.5 60–6 60–6 8 8

6.5–154 15.7–116.6 96 8–80 8–80 6.5–51.4 6.5–51.4

2.8 2.49 2.48 1.7 1.42 1.11/0.98a 2.26/2.0a

Hafez et al. (2010) Noike et al. (2002) Fang et al. (2006) Ohnishi et al. (2010) Ohnishi et al. (2012) This study This study

Hydrogen yield at STP.

Table 3 Summary of products and COD mass balance. Variables

Condition I

1

VSSout (g L ) CODin (g L1) TCODout (g L1) SCODout (g L1) Acetic acid (gCOD L1) Propionic acid (gCOD L1) Iso butyric acid (gCOD L1) Butyric acid (gCOD L1) Iso valeric acid (gCOD L1) Valeric acid (gCOD L1) VFA (gCOD L1) Glucoseout (g L1) Hydrogen gas (L d1)a Hydrogen gas (gCOD d1)b COD balance (%) a b

Condition II

Condition III

UAnPBR1

UAnPBR2

UAnPBR1

UAnPBR2

UAnPBR1

UAnPBR2

0.3 ± 0.1 2.3 ± 0.0 1.63 ± 0.2 1.5 ± 0.1 0.3 ± 0.1 0.7 ± 0.2 0.0 ± 0.0 0.2 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 1.2 0.0 ± 0.0 1.7 ± 0.1 1.2 ± 0.1 88

0.2 ± 0.1 2.3 ± 0.0 1.56 ± 0.1 1.4 ± 0.1 0.7 ± 0.1 0.2 ± 0.0 0.0 ± 0.0 0.5 ± 0.1 0.0 ± 0.0 0.0 ± 0.0 1.3 0.0 ± 0.0 6.3 ± 0.3 4.5 ± 0.2 87

0.6 ± 0.2 9.1 ± 0.0 7.7 ± 0.4 6.9 ± 0.4 0.7 ± 0.3 1.8 ± 0.5 0.5 ± 0.2 3.5 ± 0.7 0.0 ± 0.0 0.0 ± 0.0 6.6 0.0 ± 0.0 7.4 ± 0.7 5.3 ± 0.5 93

0.4 ± 0.1 9.1 ± 0.0 7.6 ± 0.0 7.1 ± 0.1 2.4 ± 0.2 1.2 ± 0.2 1.3 ± 0.1 1.8 ± 0.5 0.0 ± 0.0 0.0 ± 0.0 6.7 0.0 ± 0.0 22.6 ± 0.8 16.1 ± 0.8 101

0.6 ± 0.1 18.3 ± 0.0 16.6 ± 0.3 14.6 ± 0.4 1.6 ± 0.2 4.4 ± 0.5 2.5 ± 0.4 3.6 ± 0.5 0.0 ± 0.0 0.0 ± 0.0 12.1 1.5 ± 0.1 11.1 ± 0.7 7.9 ± 0.5 91

0.5 ± 0.1 18.3 ± 0.0 15.5 ± 0.6 14.4 ± 0.5 2.2 ± 0.9 3.2 ± 0.6 3.6 ± 0.5 3.1 ± 0.5 0.0 ± 0.0 0.0 ± 0.0 12.2 1.3 ± 0.1 23.4 ± 0.9 16.7 ± 0.6 91

Hydrogen production at 37 °C. COD balance (%) = ((SCODout (gCOD d1) + H2 (gCOD d1) + VSSout (gCOD d1)/(TCODin (gCOD d1)).

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A.D.N. Ferraz Júnior et al. / Bioresource Technology 164 (2014) 371–379 Table 4 Theoretical hydrogen production from acetic and butyrate pathways. Condition

Hydrogen production rate (L-H2 d1) UAnPBR1

I II III

2 8.2 11.1

Hydrogen production from acetate (L-H2 d1)

Hydrogen production from butyrate (L-H2 d1)

Total theoretical hydrogen production rate (L-H2 d1)

THPR/HPr (%)

UAnPBR2

UAnPBR1

UAnPBR2

UAnPBR1

UAnPBR2

UAnPBR1

UAnPBR2

UAnPBR1

UAnPBR2

7.1 25.6 26.5

1.7 4.1 8.9

5.9 20.1 19.1

0.2 3.9 4.1

0.8 3.2 5.4

1.9 7.9 12.9

6.7 23.2 24.5

97.5 96.7 116.3

94.6 90.7 92.6

The value in this table was calculated at 37 °C.

(a) UAnPBR 1 UAnPBR 2

E

A

F

B C D

G

(b) 80 Propionispira arboris (NR 02935.7)

Band A

40

Propionispora hippei (NR 036875.1) 99

99

Propionispora vibrioides (NR 025418.1) Megasphaera micronuciformis (NR 025230.1) Megasphaera elsdenii (NR 102980.1)

97 65

Band D

99

96 Band G

Clostridium butyricum (NR 04214.4) Band E 100

Clostridium beijerinckii (NR 07443.4) 82

Band B 86

Band F Lactobacillus harbinensis (NR 04126.3) Lactobacillus casei (NR 07503.2)

99 94

Band C

Gluconobacter roseus (NR 04104.9)

0.05 Fig. 4. (a) DGGE-profiles of 16S rRNA gene fragment of the bacterial populations at the end of the operation of the reactors, (b) Consensus phylogenetic tree based on the DGGE excised sequences of bands with primers for bacteria domain obtained from the operation of the reactor UAnPBR1 and UAnPBR2. The bootstrap values indicate the repetition percentages (500 replicate runs). GenBank accession numbers are listed after species names.

378


5.4. Microbial community analysis Fig. 4a presents DGGE-profiles of the 16S rRNA gene fragments of the bacterial populations at the end of the operation of the reactors (UAnPBR1 and UAnPBR2). As expected, there was a predominance of bands affiliated with notable hydrogen producers (bands A, B, E, and F) such as Clostridium and Propionispira. The aforementioned bands were excised and identified as described in Table 5. Band A showed 99% phylogenetic similarity with Propionispira arboris. There are no reports in the literature that associate this species to hydrogen production, however, representatives of Propionispora genus, such as P. vibrioides and P. hippei are reported as Gram-negative, spore-forming, responsible for the conversion of sugars into propionic acid, acetic acid, carbon dioxide, and small amounts of hydrogen (Abou-Zeid et al., 2004). Bands B and F were considered as the same population. The population represented by these bands (B and F) showed 100% phylogenetic similarity to Clostridium beijerinckii. Band E showed 98% phylogenetic similarity to Clostridium butyricum. This bacterium may be associated with the high concentrations of isobutyric and butyric acid of 1.98 ± 0.27 g L1 and 1.7 ± 0.33 g L1 observed at the end of the operation of UAnPBR2. However, it is noteworthy that both C. beijerinckii and C. butyricum have some of the greatest hydrogen production potential reported in the literature (Ye et al., 2012). Band C showed 99% phylogenetic similarity to Lactobacillus casei. Although, the Lactobacillus genus is a lactic acid-producing bacteria (LAPB), there is a contradiction in the literature about the role of these microorganisms in hydrogen production. Some authors have reported inhibitory effects of LAPB on hydrogen production (Baghchehsaraee et al., 2010), while, others believe that LAPB may be associated with hydrogen production in small amounts via medium acidification (Baghchehsaraee et al., 2010). A third group believes that, in fact, there is a competition between LAPB and hydrogen producing bacteria for the substrate, which would lead to an unstable system (Baghchehsaraee et al., 2010). Bands D and G were considered as the same population as well. The populations represented by bands D and G showed 98% phylogenetic similarity to Megasphaera elsdenii. This strain produces acetate, propionate, butyrate, valerate, carbon dioxide, and small amounts of hydrogen from glucose, fructose and lactate (Ohnishi et al., 2010). Fig. 4b presents the consensus phylogenetic tree obtained from the information of sequences at the end of the operation of the reactor UAnPBR1 and UAnPBR2 (Maintinguer et al., 2008). The sequences related to bands A and C were related phylogenetically to P. arboris and Lactobacillus casei, respectively. The sequences of the bands B, E and F were associated with Clostridium species

Table 5 Affiliation of DGGE fragments determined by 16S rRNA sequence. Band

Affiliation

Access number (Genbank)

A

Propionispira arboris

B C

Clostridium beijerinckii Lactobacillus casei

D

Megasphaera elsdenii

E

Clostridium butyricum Clostridium beijerinckii Megasphaera elsdenii

BankIt1640036 KF274052 BankIt1640036 KF274053 BankIt1640036 KF274054 BankIt1640036 KF274055 BankIt1640036 KF274056 BankIt1640036 KF274057 BankIt1640036 KF274058

F G

Similarity (%)

Seq1

99

Seq2

100

Seq3

99

Seq4

99

Seq5

98

Seq6

100

Seq7

99

and the sequences related to bands D and G were related phylogenetically to M. elsdenii. Gluconobacter roseus is presented as an outgroup. It is known that PCR/DGGE analysis is a qualitative technique. However, as previously discussed, the improved performance of UAnPBR2 relative to UAnPBR1 was due to the low biomass detachment rate, resulting in higher biomass retention. Based on this, the modification in the feed zone and the extra column for settling biomass added to the reactor changed the population dynamics of microorganisms, probably, by increasing the microbial group that has the greatest hydrogen production potential i.e., Clostridium species. 5.5. Comparative system performance In this study, the seed, the synthetic wastewater, and the operational conditions were the same as those adopted by Hafez et al. (2010) except for the OLR which ranged from 6.5 g-COD L1 d1 to 154 g-COD L1 d1 in the integrated biohydrogen reactor clarifier system (IBRCS). The aforementioned authors observed hydrogen production rates of 12 L d1, 48 L d1 and 97 L d1 at OLR of 6.5 g-COD L1 d1, 25.7 g-COD L1 d1 and 51.4 g-COD L1 d1, respectively, corresponding to volumetric hydrogen production rates of 2.4, 9.6, and 19.5 L-H2 d1 L1 reactor, about 50% higher than UAnPBR2 at the two lowest OLRs and three times higher at the highest OLR. Also, the much higher hydrogen yield of 2.8 mol-H2 mol1 glucose observed in the IBRCS was independent of OLRs. Amorim et al. (2009) evaluated the effect of HRT and OLR on hydrogen production in anaerobic fluidized bed reactors (AnFBR) with expanded clay as a support media and fed with synthetic wastewater containing 4 g L1 of glucose. The AnFBR was inoculated with heated anaerobic sludge (90 °C for 10 min.) and operated at a pH around 5.5, at 30 °C. The HRT and OLR ranged from 1 to 8 h, and from 15.7 to 116.6 g-COD L1 d1, respectively. The aforementioned authors reported that the hydrogen yield increased from 1.41 to 2.23 mol H2 mol1 glucose when HRT decreased from 8 to 4 h (OLR from 15.7 to 33.6 g-COD L1 d1), and stabilized between 2.49 and 2.41 mol-H2 mol1 glucose for HRTs from 2 to 1 h (OLR from 66.5 to 116.6 g-COD L1 d1). The aforementioned values are in agreement with this study despite the much lower OLR for UAnPBR2 of 27.5 g-COD L1 d1. Leite et al. (2008) operated a horizontal packed-bed bioreactor with expanded clay as support media, and fed with synthetic wastewater containing 4 g L1 of glucose. The reactor was operated at 30 ± 1 °C. At HRT and OLR of 0.5 h and 96 g-COD L1 d1, the authors reported a maximum hydrogen yield of 2.48 mol-H2 mol1 glucose without addition of buffer solution which is higher than the reported yields in this study. Lin and Chang (1999) operated a chemostat-type anaerobic reactor for hydrogen production, with synthetic wastewater (20 g L1 of glucose) at a pH of 5.7, and 35 °C. The chemostat reactor was inoculated with sewage sludge and the SRT ranged from 0.25 to 2.5 days. At SRTs of 1 and 0.50 day, the organic loading rates were 20 g-COD L1 d1 and 40 g-COD L1 d1, respectively. The observed hydrogen yield increased from 1.21 mol-H2 mol1-glucose at an OLR of 20 g-COD L1 d1 to 1.70 mol-H2 mol1-glucose at an OLR of 40 g-COD L1 d1. The same experiment was repeated at ambient temperature and without pH control of the feed, resulting in an average hydrogen yield of 1.30 mol-H2 mol1-glucose at an OLR of 20 g-COD L1 d1 and temperatures varying from 25 to 29 °C, and 1.42 mol-H2 mol1-glucose at an OLR of 40 g-COD L1 d1 and temperatures varying from 28 to 32 °C (Lin and Chang, 2004). These values are slightly lower compared to this study. Table 2 compares the results using different reactor designs fed with glucose. It appears that generally the performance of UAnPBR2 was better than chemostats but inferior to both the AnFBR and IBRCS processes. It is noteworthy that the frequently reported problems for this reactor


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