Formation of hydrogen-producing granules and microbial community analysis in a UASB reactor

Renewable Energy 53 (2013) 12e17

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Formation of hydrogen-producing granules and microbial community analysis in a UASB reactor Yan-Yan Ning a, Shao-Feng Wang a, Da-Wei Jin a, Hideki Harada b, Xian-Yang Shi a, * a b

College of Resources and Environmental Engineering, Anhui University, Hefei 230039, China Department of Civil Engineering, Tohoku University, Sendai 980-8579, Japan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 May 2012 Accepted 28 October 2012 Available online 3 December 2012

The effect of substrate concentration on continuous H2 production from a sucrose-rich synthetic wastewater was investigated in this work. The maximum H2 production rate of 2.89 m3 H2/m3/d achieved at 7000 mg COD/L while the peak ethanol concentration of 2840 mg/L was observed at 5000 mg COD/L. The H2-producing granules with ethanol-type fermentation were successfully cultivated in an upflow anaerobic sludge blanket (UASB) reactor. Denaturing gradient gel electrophoresis analysis demonstrates that the substrate concentration had a considerable effect on the microbial community. After the mature H2-producing granules were formed, the microbial community structure in the UASB reactor tends to stabilize. The dominant bacterium in the mature H2-producing granules were composed of Janthinobacterium sp. WPCB148, Clostridium sp. HPB-4, Variovorax paradoxus strain SJ100, Variovorax sp. CN3b and Uncultured bacterium clone C2. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: Hydrogen production Upflow anaerobic sludge blanket (UASB) reactor Granule PCR-DGGE

1. Introduction Hydrogen is regarded as a clean energy source since the only product is water after its combustion [1]. Physical, chemical and biological processes have been used to generate H2 [2]. Among them, biological H2 production by dark fermentative bacteria has attracted increasing interests because of its low energy input and costs as well as applicability to different types of organic wastes [3]. An anaerobic continuous stirred tank reactor (CSTR) is always used for H2 production in most dark H2 fermentation [4e6]. However, the CSTR is unable to maintain a high level fermentative biomass at a high dilution rate. The bio-H2 generation with CSTR is sensitive to the fluctuation of operating parameters such as temperature, hydraulic retention time (HRT) and pH [7]. Several strategies have been applied to overcome this problem. An upflow anaerobic sludge blanket (UASB) reactor with H2-producing granules is desirable for high rate bio-H2 generation. The H2-producing granules with butyrate-type fermentation in the UASB reactor showed high and stable performance [3]. However, the ethanoltype fermentation has been proved the optimal fermentation type of bio-H2 production in several researches [4]. Up to now, the information about H2-producing granules with ethanol-type fermentation in a UASB reactor is not available.

* Corresponding author. Fax: þ86 551 3861970. E-mail address: [email protected] (X.-Y. Shi). 0960-1481/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.renene.2012.10.051

Dark H2 production is a complex process and is influenced by many factors such as HRT [8], influent substrate concentration [9], pH [10], temperature [11], and nutritional requirements, etc [12]. Among these operating parameters, the substrate concentration is an especially important factor because the distribution of metabolic products during fermentation depends on variation of the influent organic concentration [3]. It is essential to study its influence on H2 production for getting continuous and stable bio-H2 generation. Thus, this study was conducted to investigate the effect of substrate concentration (recorded as COD) on the H2 production by H2-producing granules from organic synthetic wastewater in a UASB reactor. The microbial community structure was also examined using denaturing gradient gel electrophoresis (DGGE). 2. Materials and methods 2.1. Synthetic wastewater and seed sludge A synthetic wastewater containing sucrose and nutrients was used as the substrate. The composition of nutrients was as follows (mg/L): NH4HCO3 2024; K2HPO4$3H2O 800; CaCl2 50; MgCl2$6H2O 100; FeCl2 25; NaCl 10; CoCl2$6H2O 5; MnCl2$4H2O 5; AlCl3 2.5; (NH4)6MO7O24 15; H3BO4 5; NiCl2$6H2O 5; CuCl2$5H2O 5; ZnCl2 5. The pH of the synthetic wastewater was adjusted to 7.0 by adding 3.0 mol/L HCl or 3.0 mol/L NaOH. The seed sludge was collected from an internal circulation reactor treating citrate-producing

Y.-Y. Ning et al. / Renewable Energy 53 (2013) 12e17 Table 1 Operating parameters of the reactor for biological H2 production. Day

COD (mg/L)

0e6 7e15 16e31 32e51 52e66 67e96 97e161

1000 3000 5000 6000 7000 8000 9000

wastewater with volatile suspended solid (VSS) of 13.80 g/L. Before being seeded into UASB, the sludge was sieved through 2.0 mm screen to remove big grain-size matters.

13

Volatile fatty acids (VFAs) were analyzed by a gas chromatography (Agilent 7890) equipped with a flame ionization detector (FID) and a 30 m  0.25 mm  0.25 mm fused-silica capillary column (DB-FFAP). The temperature of oven was performed using the following program: 100  C for 3 min, 100  Ce180  C with a ramp of 20  C/min, 180  C for 3 min. The temperatures of injector and detector were 220  C and 230  C, respectively. Nitrogen was used as carrier gas at a flow rate of 50 mL/min [12]. The total carbohydrate content was analyzed by anthrone method [13]. The VSS and COD were measured according to Standard Methods [14]. The digital imaging of H2-producing granules was analyzed by using a photon microscope equipped with a digital camera (Leica DM 2500). A scanning electron microscopy (Hitachi S-4800) was used for observing the micro-structure and predominant bacterial morphologies of the granules.

2.2. Reactor operation 2.4. Microbial community analysis A laboratory-scale UASB reactor with a reaction portion of 4.0 L and a three-phase separator portion of 2.0 L was used for bio-H2 production. The reactor was operated at 36  1  C and with an HRT of 10 h. During operation, pH of the mixed liquor in the reactor was kept around 4.0 by adjusting the influent alkalinity through dosing NaHCO3. The substrate concentrations in every stage of reactor operation were increased gradually and were given in Table 1. 2.3. Analytical methods The volume of evolved gas was measured daily using waterreplace equipment and the biogas contents were analyzed by gas chromatography (Agilent 6890N) equipped with a thermal conductivity detector and a 1.5 m stainless steel column packed with GDX-102 (60/80 mesh). The temperatures of injector, column and detector were 100, 60 and 105  C, respectively. Argon was used as carrier gas at a flow rate of 30 mL/min.

Total genomic DNA was extracted and purified from samples of each phase. The primer set of 357F (50 -CCTACGGGAGGCAGCAG-30 ) with a GC clamp and 907R (50 -CCGTCAATTCCTTTRAGTTT-30 ) at the annealing temperature of 55  C were used for the PCR amplification of the variable V3eV5 region of 16S rDNA from the purified genomic DNA. DGGE of PCR products was performed with a D-code system (Bio-Rad, USA). The 6% polyacrylamide gel was used to cast a gel with denaturant gradients ranging from 45 to 65% (100% denaturant was defined as 7 M urea and 40% deionized formamide). Electrophoresis was conducted in a 1  TAE buffer solution at a constant temperature of 60  C. The DNA template of the bands of interest was re-amplified and then the PCR products were purified and cloned. The homology for the sequences of re-amplified DNA fragments was determined with available sequences in GenBank database using BLAST and phylogenetic trees were then constructed by MEGA 4.0 [15].

Fig. 1. Images of sludge in the H2-producin granulation process: (A) seed sludge; (B) sludge on day 15th; (C) sludge on day 60th; (D) sludge on day 150th.

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Y.-Y. Ning et al. / Renewable Energy 53 (2013) 12e17

3. Results and discussions 3.1. Formation of H2-producing granules As showed in Fig. 1, the seed sludge was in suspension and H2-producing granules formed gradually by adjusting substrate concentration. The granulation process was approximately divided into three stages. The first stage (1e15 d) was the process of microbial proliferation under anaerobic environment with a low H2-producing performance. In the second stage (16e59 d), small granules began to form and the performance of H2 production increased gradually. In the third stage (60e161 d), the diameter of granules increased and their growth rate became slower, indicating that mature and stable granules with an average diameter of 1.2 mm were finally formed. A good H2-production performance was observed at late-operation phase with the formation of anaerobic granules. The SEM analysis showed that a large number of filamentous microorganisms were displayed in seed sludge whereas there were three bacterial morphologies in the H2-producing granules: rod-coccus-filament (Fig. 2). 3.2. Effect of substrate concentration (COD) on H2 production The H2 content and H2 production rate depend on considerably substrate concentration for its significant effects on bacterial growth and enzymatic activity [16]. As showed in Fig. 3, the UASB reactor was operated for 160 days and no methane was detected. After start-up of UASB, the H2 content showed an apparent fluctuation and then returned to previous level varying between 35% and 55% with the change of COD concentration. The H2 production rate was low at initial period of the experiment (Fig. 3) because the consumption of substrate was mainly used for microbial proliferation. The H2 production rate increased with an increase in substrate concentration after the reactor was started up and then tended to be stable after 61 days of UASB operation. For comparison, Table 2 lists the maximum H2 production rate in this work with those found in literature [12,17,18].

Fig. 3. The H2 content and H2 production rate during operation of UASB reactor.

As showed in Table 2, the maximum H2 production rate of 2.89 m3 H2/m3/d in our work was comparable with those of other wellknown studies. These results suggested that H2 production system by H2-producing granules displayed a good stability and self-regulation. 3.3. Production of VFAs Dark H2 production was accompanied by the formation of large quantities of VFAs. During the first period of the UASB reactor operation, the major soluble metabolites were ethanol, acetate and butyrate and propionate concentration was very low. As showed in Fig. 4, the acetate concentration varied rapidly with increasing COD. After 17-day operation, ethanol was gradually examined as the main metabolites while acetate and butyrate concentrations began to decrease. The maximum ethanol concentration of 2840 mg/L accounted for 84% of total VFAs was observed at a COD concentration of 5000 mg/L (16e31 d) and the average level was

Fig. 2. SEM image for interior porous structure and predominant bacterial morphologies of the inoculum (a) and the H2-producing granules (b, c, d).

Y.-Y. Ning et al. / Renewable Energy 53 (2013) 12e17

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Table 2 Comparison for H2 production rate with other studies. Reference

Reactor type

Maximum H2 production rate (m3 H2/m3/d)

Substrate

Fermentation

[12] [17] [18] This study

CSTR UASB UASB UASB

2.39 1.17 3.48 2.89

Sucrose Glucose Sucrose Sucrose

Butyrate-type Butyrate-type Butyrate-type Ethanol-type

1000 mg/L during the UASB operation. The amount and component of VFAs reflect a shift of metabolic pathways that occurred with the change of substrate concentration. The ethanol-type fermentation with many advantages for H2 generation was formed according to the ethanol and acetate concentration [4]. The results in Table 2 also demonstrated that ethanol-type fermentation had good ability of H2 production compared with other fermentative types. 3.4. Structure of microbial community in UASB The DGGE profiles (Fig. 5) revealed that the microbial community structure of H2 production system varied gradually in response to the increase in substrate concentration. Two Bands (4 and 6) in the seed sludge disappeared or their intensity reduced, while four bands (1, 9, 10 and 15) were enriched. Two bands (14 and 17) always existed in DGGE profiles no matter how the changes in COD concentration, suggesting that the microbial population could be selected by controlling substrate concentration in a reasonable level. To identify the microbial species, 16S rDNA sequences of each band were determined by comparing with the GenBank database (Table 3) and a phylogenetic tree was presented in Fig. 6. In these sequences, 21 bacteria clones respectively belonged to a-proteobacteria, b-proteobacteria and Clostridia. Variovorax paradoxus strain SJ100 and Variovorax sp. CN3b (band 14 and 17) in DGGE profiles were found throughout the operation of UASB under the changes in COD concentration, suggesting that they could grow at high concentration organic wastewater. On the other hand, the physiology activity of Variovorax in H2 production has not been reported. The relative quantities of Janthinobacterium sp. WPCB148 (band 9) and Clostridium sp. HPB-4 (band 10) increased with increasing COD concentration. Both Janthinobacterium and Clostridium sp. HPB-4 was tightly related to H2 production [19e21]. Band 13 (Undibacterium sp. EM 1) and 16 (Uncultured bacterium) clone C2 were enriched from the seed sludge with the 4000 Ethanol Acetic acid Propionic acid Butyric acid

3500 3000

VFA (mg/L)

2500 2000

Fig. 5. DGGE profiles of sludge samples at different period of UASB operation.

increase of COD concentration, but at COD concentration of greater than 8000 mg/L (16e31 d), the two bacteria decreased due to failure of resisting high organic loading. After UASB reactor was operated for about 75 days, Oxalobacteraceae bacterium QD1 Table 3 Affiliation of DGGE fragments determined by the 16S rDNA sequences of various samples. Band

Affiliation

Access number

1 2 3

Oxalobacteraceae bacterium QD1 Uncultured bacterium clone 24a07 Uncultured Herbaspirillum sp. clone AV_8R-S-G10 Uncultured Sphingomonas sp. clone GASP-MA1S1_B04 Uncultured Burkholderiaceae bacterium clone 401F06 Uncultured bacterium clone C8W_32 Janthinobacterium sp. 7 Uncultured Janthinobacterium sp. clone VE8B07 Janthinobacterium sp. WPCB148 Clostridium sp. HPB-4 Uncultured beta proteobacterium clone A13W_77 Janthinobacterium sp. LeLB46 Undibacterium sp. EM 1 Variovorax paradoxus strain SJ100 Uncultured bacterium clone 198 Uncultured bacterium clone C2 Variovorax sp. CN3b Uncultured bacterium clone CD140 Glacier ice bacterium sp. glbI3 Uncultured bacterium clone ncd905g08c1 Uncultured bacterium clone Gven_F20

DQ388765 EF515406 EU341291

99 98 98

EF662327

100

AM420125

99

HM057645 GU213359 GQ179711

100 100 99

FJ006906 AY862513 HM057621

100 99 99

AB453874 GQ379228 GQ140335 EU531785 GU368356 GQ332345 DQ441391 EU978844 HM308888 GU118480

97 99 100 99 99 99 99 99 99 98

4 5 6 7 8 9 10 11

1500 1000 500 0 0

20

40

60

80

100

120

140

160

Time (d) Fig. 4. Effect of substrate concentration on soluble metabolites formation.

12 13 14 15 16 17 18 19 20 21

Similarity (%)

16

Y.-Y. Ning et al. / Renewable Energy 53 (2013) 12e17

Uncultured Herbaspirillum sp. clone Uncultured bacterium clone Gven F20 Uncultured bacterium clone ncd905g08c1 Band 16 Oxalobacteraceae bacterium QD1 Uncultured bacterium clone 198 Uncultured Burkholderiaceae bacterium 53 Uncultured β-proteobacterium clone A13W_77 Undibacterium sp. EM 1 Uncultured bacterium clone C2 Band 6 86 Band 5 Band 15 Uncultured bacterium clone C8W 32 Uncultured bacterium clone CD140 Band 21

53

63 Band 13 Band 20

99 Band 18 Band 1

β-proteobacteria

Band 11 Band 3 Band 8

100 48

Band 12 Band 7 99

Janthinobacterium sp. 7 Band 9

65 Uncultured Janthinobacterium sp. clone VE8B07 Janthinobacterium sp. WPCB148

100

Glacier ice bacterium sp. glbI3 Janthinobacterium sp. LeLB46 Band 19 Band 14 100 Variovorax paradoxus strain SJ100 Band 17 Variovorax sp. CN3b 99

Band 4

α-proteobacteria

Uncultured Sphingomonas sp. clone GASP-MA1S1_B04 Band 2

98

Uncultured bacterium clone 24a07 Band 10

100 99

Clostridia

Clostridium sp. HPB-4

0.02 Fig. 6. Phylogenetic tree of dominant microbial species based on 16S rDNA sequence: phylogenetic tree was constructed using Neighbor-joining algorithm. The marked band was reamplified 16S rDNA gene sequences from DGGE band. Numbers at each node indicate the supporting percentage by bootstrap analysis with 1000 iterations. The scale bar represents 0.02 substitutions per nucleotide position.

(band 1) appeared and increased with the increase of COD concentration, suggesting that the bacteria was also enriched and could resist higher organic loading. Bands 4 and 6 were affiliated to uncultured Sphingomonas sp. clone GASP-MA1S1_B04 and uncultured bacterium clone C8W_32, respectively, whose activity could be inhibited at high COD

concentration. It should be noted that the microbial community structure and diversity in UASB reactor were altered with variation in substrate concentration. However, after 75 days of UASB operation, the microbial community structure in the bioreactor tends to stabilize gradually with anaerobic granules formation being able to resist the change organic loading.

Y.-Y. Ning et al. / Renewable Energy 53 (2013) 12e17

4. Conclusions In this work, H2-producing granules with the ethanol-type fermentation could be cultivated in UASB reactor treating a sucrose-rich synthetic wastewater. The effect of substrate concentration on continuous H2 production was investigated. The maximum H2 production rate of 2.89 m3 H2/m3/d was achieved at 7000 mg COD/L (52e66 d) while the peak ethanol concentration of 2840 mg/L was observed at 5000 mg COD/L (16e31 d). The analysis by applying DGGE approach demonstrates that the substrate concentration had a considerable effect on the microbial community structure. After the mature H2-producing granules were formed, the microbial community structure in the bioreactor tends to stabilize. The dominant bacterium in the mature H2-producing granules were composed of Janthinobacterium sp. WPCB148, Clostridium sp. HPB-4, V. paradoxus strain SJ100, Variovorax sp. CN3b and Uncultured bacterium clone C2. Acknowledgments The authors wish to thank the Natural Science Key Foundation of the Anhui Higher Education Institutions (KJ2011A003), the Natural Science Foundation of Anhui province (1208085ME61), the scientific research project of Huainan mining group (HNKY- JT-JS2012-3), the Natural Science Foundation of China (51278001), the professional project of Characteristics of Anhui province (2011-8), and the NSFCeJST Joint Project (20610002) for the support of this study. References [1] Teng SX, Tong ZH, Li WW, Wang SG, Sheng GP, Shi XY, et al. Electricity generation from mixed volatile fatty acids using microbial fuel cells. Appl Microbiol Biotechnol 2010;87:2365e72. [2] Levin BD, Pitt L, Love M. Biohydrogen production: prospects and limitations to practical application. Int J Hydrogen Energy 2004;29:173e85. [3] Yu HQ, Mu Y. Biological hydrogen production in a UASB reactor with granules. Ⅱ: reactor performance in 3-year operation. Biotechnol Bioeng 2006;94: 988e95.

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