Continuous fermentative hydrogen production from coffee drink manufacturing wastewater by applying UASB reactor

international journal of hydrogen energy 35 (2010) 13370–13378

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Continuous fermentative hydrogen production from coffee drink manufacturing wastewater by applying UASB reactor Kyung-Won Jung a, Dong-Hoon Kim b, Hang-Sik Shin a,* a b

Department of Civil and Environmental Engineering, KAIST, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea Department of Civil and Environmental Engineering, University of Windsor, 401 Sunset Ave., Essex Hall, Windsor, Ontario, Canada

article info

abstract

Article history:

The feasibility of continuous H2 production from coffee drink manufacturing wastewater

Received 9 November 2009

(CDMW) was tested in two different types of reactors: a completely-stirred tank reactor

Accepted 18 November 2009

(CSTR) and an up-flow anaerobic sludge blanket reactor (UASBr). While the performance in

Available online 29 December 2009

CSTR was limited, it was significantly enhanced in UASBr. The maximum H2 yield of 1.29 mol H2/mol hexoseadded was achieved at HRT of 6 h in UASBr operation. Non-hydro-

Keywords:

genic, lactic acid was the dominant in CSTR, while butyric and caproic acids in UASBr. As

CSTR

caproic acid is generated by consuming acetic and butyric acids, all of which are related to H2

UASBr

production, the presence of caproic acid in the broth also indicates H2 production, yielding

LAB

1.33 mol H2/glucose. It was speculated that the enhanced performance in UASBr was

Granule

attributed to the high concentration of biomass over 60,000 mg VSS/L in the blanket zone,

Caproic acid

which provided insufficient substrate for indigenous lactic acid bacteria (LAB) to survive. The

Lactic acid

abundance of LAB in CDMW was confirmed by natural fermentation of CDMW. That is without the addition of external inoculum, CDMW was mainly fermented into lactic acid under mesophilic condition. For the first time ever, H2 producing granules (HPG) with diameters of 2.1 mm were successfully formed by using actual waste as a substrate. ª 2009 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.

1.

Introduction

The extensive use of fossil fuels has caused not only global warming, but also economic and diplomatic problems due to their limited reserves and unequal distribution. Thus, developing sustainable energy chains based on the contemporary sunlight-like application of biofuels and hydrogen from renewable sources is indispensable. H2 offers tremendous potential as a sustainable energy source because it produces only water when it combusts, generating an energy yield (122 kJ/g) 2.75 times higher than hydrocarbon fuels [1]. In addition, H2 can be easily converted to electricity through fuel cells [2]. Numerous ways exist to produce H2, but the steam reforming of hydrocarbons is widely used due to its cost

advantage. However, the reaction occurs at high temperature and pressure condition which require a lot of external energy from fossil fuels, thereby rendering the process environmentally unfriendly. On the other hand, in nature, various microorganisms have capabilities to produce H2 from different sources. Algae and Rhodobacter sp. can generate H2 from water and simple organic substances, respectively, with the help of light [3,4]. Anaerobic bacteria, such as Clostridium sp. and Bacillus sp., can generate H2 from various organics without light. Although the former way depending on light warrants higher H2 yield than the latter one, called fermentative H2 production (FHP), only FHP is considered to be practically applicable owing to its rapid reaction rate, technical simplex, and the treatment of organic pollutants [5,6].

* Corresponding author. Tel.: þ82 42 350 3613; fax: þ82 42 350 8640. E-mail address: [email protected] (H.-S. Shin). 0360-3199/$ – see front matter ª 2009 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2009.11.120

international journal of hydrogen energy 35 (2010) 13370–13378

In FHP, numerous studies were conducted on optimizing operation parameters including pH, temperature, inoculum preparation, organic loading rate, HRT, etc. [7–13]. However, most of them focused on synthetic wastewater, in spite of the fact that FHP has a potential of bioremediation. Actual pollutants such as industrial wastewater and organic solid waste could be treated along with H2 production. However, there may be harmful materials and indigenous microorganisms in the actual waste which could minimize H2 production potential. According to Noike et al. [14], continuous H2 production from bean curd manufacturing waste was impeded by the inhibitory effect of indigenous lactic acid bacteria (LAB). It was successful when the feed was thermally treated before being fed to the reactor. Similarly, in order to suppress the activity of indigenous microorganisms in food waste, it was alkali-pretreated at pH 12 for one day [15]. The completely-stirred tank reactor (CSTR) was the most frequently used reactor type because it is simple to operate. However, it is very sensitive to environmental shock, which limits the increase of the organic loading rate (OLR) [16]. To overcome these weaknesses, the upflow anaerobic sludge blanket reactor (UASBr) was recently introduced to FHP. With the formation of dense H2 producing granules, its application has enormously improved the H2 production stability at high OLR [17–19]. After 2–4 months of operation, the granules, with diameters ranging from 0.5 to 2.0 mm, were successfully formed and accumulated in the reactor bed zone. Stable H2 production with high yield was achieved even at over 100 g COD/L/d of OLR. However, research on the application of UASBr to treat actual wastewater for H2 production has been limited. The coffee industry is one of the biggest industries in the world and produces a large amount of high strength organic wastewater. As the wastewater has high energy content, it is ideal to recover bioenergy, especially H2, during the treatment process. In the present work, therefore, coffee drink manufacturing wastewater (CDMW) was chosen as a substrate for FHP and fed to two types of reactors, CSTR and

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UASBr. Although the performance in CSTR was limited, it was significantly enhanced by applying UASBr.

2.

Materials and methods

2.1.

Seed sludge and substrate

The seed sludge was taken from an anaerobic digester in a local wastewater treatment plant. It was heat-treated at 90  C for 20 min to inactivate non-hydrogen producing bacteria and to harvest spore-forming anaerobic bacteria such as Clostridium sp. [20]. The CDMW used in this study was the gatherings of returned goods and the wastewater generated during the mixing process of crude coffee, starch, lactose, and sugar. It was a high-strength organic wastewater (184 g COD/L), mostly comprised of carbohydrates (71%). The soluble portion (SCOD/ TCOD) was 78%, and total kjeldahl nitrogen (TKN) concentration was 3046 mg N/L. The substrate concentration was adjusted to 20 g COD/L (based on carbohydrate) by dilution, while no supplements were additionally added.

2.2.

CSTR and UASBr operation

The schematic diagram of CSTR and UASBr are indicated in Fig. 1. In this study, a CSTR with a working volume of 5.0 L (325 mm high by 140 mm ID) was seeded with the heat-treated sludge, equivalent to 30% of the working volume, and filled with the substrate. It was purged with N2 gas for 5 min to provide an anaerobic condition and agitated at 200 rpm. The initial and operation pH was maintained at 8.0  0.1 and 5.5  0.2, respectively, using pH sensors, pH controllers, and 3 N KOH. Continuous operation was delayed until 5.83 L of H2, equivalent to 0.5 mol H2/mol hexoseadded, was produced in the batch operation. At day 10, 1.5 L of mixed liquor of CSTR was transferred to UASBr (working volume 3.5 L; 690 mm high by 65 mm ID) as an inoculum. As accurate pH control is impossible in UASBr, 5.0 g/L of NaHCO3 was added externally to

Fig. 1 – Schematic diagrams of (A) CSTR and (B) UASBr.

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provide a buffer capacity. The HRT was progressively decreased from 12 to 6 h and 8–4 h in CSTR and UASBr operation, respectively. All experiments were conducted in a temperature controlled room at 35  1  C and gas production was monitored using a wet gas meter.

2.3.

Batch test

The first batch test was prepared to investigate the fermentation characteristics of CDMW. Without the addition of external inoculum, only CDMW was added into a fermenter reaching a working volume of 200 mL, and an anaerobic condition was provided by N2 gas purging. Samples were taken using syringe at 5–10 h intervals to measure organic acids production. An additional batch test was conducted to compare the H2 productivity of different sludges: collected from the bottom layer (BLU) and effluent of UASBr (Eff). Into a fermenter, 30 mL of collected sludge and a certain amount of CDMW were added. Tap water was filled to reach working volume of 200 mL, and the substrate concentration was kept the same during the continuous operation. Then, N2 purging was applied to get an anaerobic condition. The batch fermenter has a total volume of 635 mL, and a pH sensor was installed at the top, which was connected to a pH panel so that the pH change could be monitored during fermentation. The initial and cultivation pH were manually maintained at 8.0  0.1 and 5.5  0.2, respectively, by the addition of 3 N KOH. The mixing rate was adjusted to 100 rpm using a stirring magnetic bar at the bottom. Produced gas was sampled using a 100 mL glass syringe at regular intervals to analyze H2 components and the total amount. All batch experiments were conducted in the water bath of 35  1  C. To describe the hydrogen production, cumulative H2 production curves were obtained using the modified Gompertz equation (1) [21].    0 R e ðl  tÞ þ 1 ; HðtÞ ¼ P  exp  exp P

(1)

where H(t) is the cumulative H2 production (mL) at cultivation time t (h); P, ultimate H2 production (mL); R0 , H2 production rate (mL/h); l, lag phase (h); and e, exp(1) ¼ 2.71828. H2 production was calculated from headspace measurements of gas composition and the total volume of biogas produced at each time interval using the mass balance Eq. (2).

VH;i ¼ VH;i1 þ CH;i VG;i  VG;i1 þ VH CH;i  CH;i1 ;

detector and a 1.8 m  3.2 mm stainless-steel column packed with molecular sieve 5A with N2 as a carrier gas. The contents of CH4, N2, and CO2 were measured using a GC of the same model noted previously with a 1.8 m  3.2 mm stainless-steel column packed with porapak Q (80/100 mesh) using helium as a carrier gas. The temperatures of injector, detector, and column were kept at 80, 90, and 50  C, respectively, in both GCs. Volatile fatty acids (VFAs, C2–C6) and lactic acid were analyzed by a high performance liquid chromatography (HPLC) (Finnigan Spectra SYSTEM LC, Thermo Electron Co.) with an ultraviolet (210 nm) detector (UV1000, Thermo Electron) and an 100 mm  7.8 mm Fast Acid Analysis column (Bio-Rad Lab.) using 0.005 M H2SO4 as a mobile phase. Aliphatic alcohol was determined using another HPLC (DX-600, Dionex) with an electrochemical detector (ED50A, Dionex) and a 250 mm  9 mm IonPac ICE-ASI column (Dionex) using 50 mM HClO4 as mobile phase. The liquid samples were pretreated with a 0.45 mm membrane filter before injection to both HPLC. COD and VSS were measured according to Standard Methods [22]. Sucrose concentration was determined by the colorimetric method of Dubois et al. [23]. The sizes of the granules or flocs were analyzed with the free UTHSCSA Image Tool program, the software developed in the University of Texas Health Science Center at San Antonio, Texas. A sample of sludge (0.2 mL) was spread over a petry dish and fixed within a transparent 25 g-gelatin/L gelatin solution (5 mL). After the gelatin solidified, the sample dishes were placed over the scanner surface. The software gives imformation of the area, particle number, diameter, and other characteristics of the particles in the digital image [24].

3.

Results and discussion

3.1.

CSTR performance

Fig. 2 shows the daily variations of H2 yield in the CSTR. Generally, H2 yield is a good indicator of the effectiveness of H2 production. The theoretical H2 yield is 2.0 mol H2/mol hexoseadded when butyric acid is the main by-product in

(2)

where VH;i and V are the cumulative hydrogen gas volumes at the current (i) and previous (i  1) time intervals, VG;i and VG;i1 , total biogas volumes in the current and previous time intervals; CH;i and CH;i1 , the fractions of hydrogen gas in the headspace of the bottle measured using gas chromatography in the current and previous intervals; and VH , the total volume of headspace in the reactor [8].

2.4.

Analytical methods

H2 content in biogas was determined by a gas chromatography (GC, Cow Mac series 580) using a thermal conductivity

Fig. 2 – Daily variations of H2 yield at different HRTs in CSTR.

– 29,658 (95%) – 29,970 (96%)

25,912 (83%) 29,419 (94.2%) 28,790 (92.2%)

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16

15

12 10 11

–

21

23 –

– – –

1

3 –

–

10 9 11

–

10

9 –

–

14

12 –

–

11

12

glucose degradation [20]. H2 yield in the CSTR was limited in spite of high carbohydrate degradation (Table 1). It was slightly increased at HRT of 8 h, but was still less than 20% of the theoretical value. At HRT of 6 h, the biomass concentration was maintained, but the yield was drastically decreased. It was speculated that the low performance in CSTR might have come from the activity of non-HPB originated from seeding inoculum or the feed, especially LAB. Before conducting continuous operation, H2 production potential of CDMW was tested by batch operation, which showed over 1.40 mol H2/mol hexoseadded (data not shown). In batch operation, once seeding inoculum is heat-shocked, there is little time and substrate for injured non-HPB to survive, but as the feed are continuously fed the chance is high in continuous operation. Kim et al. [25] reported that the decrease of H2 production after transition from batch to continuous operation was due to the regrowth of non-HPB such as LAB and propionic acid bacteria (PAB) which were inhibited by heat-shock but not totally exterminated. Similarly, in this study, H2 yield reached 1.0 mol H2/mol hexoseadded, but it decreased substantially at further operation. At all HRTs, lactic acid was the main liquid-state metabolite with over 40% (Table 1). Fig. 3 clearly indicates the abundance

eff

8 6 4 6re UASBr

0.21 1.29 0.96 1.30

0.25 2.59 4.64 2.52

1762 340 4988 440

– 8692 18,703 8868

350 2149 1599 2166

– 18,477 – 18,496

–

46 32 27 15,992 18,265 17,769 267 533 333 5959 9409 8208 3694 1212 2480 0.07 0.31 0.34 12 8 6 CSTR

0.16 0.32 0.20

HLa (%) Total (mgCOD/L)

H2 (mgCOD/L) Biomasseff (mg-COD/L) Carbohydrate remaining (mg-COD/L) H2 production rate (L H2//L/H) H2 yield (mol H2/mol hexoseadded) HRT (HR) Reactor

Table 1 – Average gas phase parameters and COD balance of CSTR and UASB at various HRTs.

Biomass , biomass in the effluent. a HLa, lactic acid; HFo, formic acid; HAc, acetic acid; HPr, propionic acid; HBu, butyric acid; HVa, valeric acid; HCa, caproic acid; EtOH, ethanol.

–

27

26 –

19 29 30 3 8 11 10 12 10

HAc (%)

a

HPr (%)

a

HBu (%)

a

HVa (%)

Fig. 3 – Natural fermentation of coffee drink manufacturing wastewater under mesophilic condition.

a

Liquid-state metabolites

a

HCa (%)

a

EtOH (%)

a

Total by-products (mg-COD/L)

international journal of hydrogen energy 35 (2010) 13370–13378

Fig. 4 – EPS concentration and P/C ratio in CSTR.

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effect on forming HPG. Since there is a vigorous mixing in CSTR, it is much easier to remove dead cells in CSTR than UASBr operation. It was reported that EPS play an important role in granule formation and that they are comprised of mainly carbohydrates and protein. Interestingly, the key EPS component differed greatly between methanogens and HPB: protein for methanogens and carbohydrates for HPB [18,19,27–29]. During the initial period of CSTR operation, protein was the main part of EPS, but it decreased during further operation, indicating the wash-out of methanogens (Fig. 4). Although a thorough investigation on the above hypothesis remains for the future work, HPG with diameter over 0.5 mm was successfully obtained in UASBr operation within three weeks, which was faster than ever reported. Fig. 5 – Daily variations of H2 yield at different HRTs in UASBr.

3.2.

of LAB in CDMW. Although there was no external inoculum addition, CDMW was naturally fermented into lactic acid without lag period under mesophilic condition. In the end, lactic acid accounted for more than 70% of the total organic acids. Due to their unique metabolic characteristics and antibiotic function, LAB is enriched in many fermentation processes of milk, meats, cereals, and vegetables. Till now, 11 genera of LABs in foods were identified and two fermentation reactions were reported, none of which were related to H2 production [26]. Noike et al. [14] also observed a similar phenomenon when bean curd manufacturing waste was utilized without pretreatment. H2 production ceased after three days, and they attributed this to the inhibitory effect of indigenous LAB. Thermal pretreatment of whole feed at 50–90  C for 30 min made the continuous H2 production successful. However, pretreatment of the whole feed is not feasible in a real plant. In this study, 1.5 L of mixed liquor in the CSTR after ten days of operation was transferred to UASBr as an inoculum based on the hypothesis that this process could facilitate the HPG formation in UASBr. It was assumed that the presence of methanogens, whether active or inactive, could exert adverse

UASBr performance

For the first 10 days, the reactor was operated at HRT of 8 h as a start-up period, and then the HRT was decreased to 6 hr (Fig. 5). Since then, H2 yield gradually increased to 1.29 mol H2/ mol hexoseadded, which was the average value from day 37 to day 53 (Table 1). Although H2 production rate increased to 4.64 L H2/L/h when HRT decreased to 4 h, it resulted in a H2 yield drop with biomass wash-out. Hence, the HRT was increased to 6 h, and then, H2 yield recovered rapidly. In Table 2, the obtained H2 yield and its production rate at HRT of 6 h were compared to previous studies applying UASBr. The H2 yield obtained in this study was lower than previous results, but its production rate was comparable. In addition, to the best of our knowledge, this was the world record applying UASBr to actual wastewater for FHP, which could significantly enhance the economic benefit. Unlike CSTR, butyric and caproic acids were the main liquid-state by-products in UASBr operation (Table 1). Other researchers also observed the formation of caproic acid in UASBr operation under mesophilic condition [7,10,19]. There are four reactions responsible for caproic acid production as shown in Eq. (3)–(6). However, Eq. (4) might be the governing reaction considering the 2nd thermodynamic law and surrounding pH [31]. While DG value is negative for all reactions at low pH (<4.0), DG values in only Eqs. (3) and (4) are negative at pH between 4.0 and 5.0. In this study, effluent pH

Table 2 – Comparison of performance data obtained in this study with previous data applying UASBr. Substrate

CDMW Sucrose Sucrose Sucrose Sucrose Glucose

HRT (h)

Substrate concentration (g-COD/L)

H2 production yield (mol H2/mol hexose)

H2 production rate (L/L/h)

Reference

6 13 18 8 18 14 26.7

20a 10 5.33 20 5.33 10.67 4.85

1.29 1.68 3.0 1.58b 1.50 1.07 2.58

2.59 0.145 0.05 0.25 0.05 0.07 –

In this study [10] [17] [18] [19]

a g-Carbo. COD/L. b mmol H2/mol hexoseadded.

[30]

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Clostridium sp. was dominant microorganisms in UASBr. Besides, 6 bands among 22 bands were matched to Megasphaera sp., which were capable of producing not only H2 but also caproate [31–33]. In detail, Megasphaera cerevisiae (accession no. L37040), Megasphaera elsdenii (AF283705) and Mitsuokella multacida (X81878) were detected with 98%, 98%, and 90% similarity, respectively. This was the first report documenting the existence of caproate producing bacteria in FHP broth. Although the biomass concentration in the effluent of CSTR and UASBr was in the same range of approximately 5000 mg-VSS/L, the biomass concentration in the reactor was considerably higher in UASBr owing to its own configuration characteristics: longitudinal structure, gas/liquid/solid separator, and no mechanical mixing (Table 3). Half of the reactor consisted of a creamy sludge blanket whose biomass concentration was over 60,000 mg VSS/L. Owing to this thick biomass layer, most supplied carbohydrates were degraded as soon as the feed entered. Accordingly, there might be a slight chance that indigenous LAB from CDMW could survive and accumulate in the reactor. In order to prove this, an additional batch test was conducted comparing the H2 productivity of different biomass collected from the bottom layer of UASBr (BLU) and its effluent (Eff). As arranged in Table 4, there was significant difference in H2 production and liquid-state metabolites between the two cases. In using the biomass from BLU, a high H2 yield of 1.11 mol H2/mol hexoseadded, which was 3.3-fold of Eff, was achieved. In addition, butyric and caproic acids were the main liquid-state metabolites in using BLU, while lactic acid accounted for nearly 45% of the liquid-state metabolites in using effluent as a seeding source. Therefore, it was proven that the dominant species in the sludge blanket zone was comprised of H2 producing bacteria other than LAB. It could be concluded that the existence of a large amount of biomass at the reactor bottom was the main reason for the superior performance in UASBr treating actual wastewater.

Table 3 – Biomass and carbohydrate concentrations in UASBr at different sampling ports (HRT [ 6 h). Reactor type

Sampling Carbohydrate Biomass Biomasseff port (mg-COD/L) (mg-VSS/L) (mg-VSS/L)

UASB 1 (Top) (meso-) 2 3 (Middle) 4 5 (Bottom)

1420 1906 2214 2467 4943

29,433

5205

62,217 77,250

was 4.3–4.8 and absolute DG value in Eq. (4) was much higher than the value in Eq. (3). CH3 ðCH2 Þ2 COO /CH3 ðCH2 Þ4 COO þ CH3 COO 1 DG0 ¼ 0:11 kJ mol

(3)

CH3 ðCH2 Þ2 COO þ CH3 COO þ 2H2 þ Hþ /CH3 ðCH2 Þ4 COO 1

þ2H2 O DG0 ¼ 48:01 kJ mol

(4)

CH3 ðCH2 Þ2 COO þ 2CO2 þ 6H2 /CH3 ðCH2 Þ4 COO þ 4H2 O 1 DG0 ¼ 143:34 kJ mol

(5)

3CH3 COO þ 3H2 þ 2Hþ /CH3 ðCH2 Þ4 COO þ 4H2 O 1 DG0 ¼ 86:20 kJ mol

(6)

According to Eq. (4), 1 mol of butyric and acetic acids are consumed with 2 mol of H2 for the 1 mol of caproic acid production. If it is presumed that Clostridium sp. are the main acetic and butyric acid producers (Eqs. (7) and (8)), caproic acid production from glucose could be one of the H2 production reactions, yielding 1.33 mol H2/mol glucose (Eq. (9)). Interestingly, the H2 partial pressure was lower in UASBr than that of CSTR regardless of higher H2 yield. This might be linked to the abundant caproic acid production in the UASBr via Eq. (9), resulting in 1.5-fold higher CO2 production than H2. Glucose þ 2H2 O/2 Acetate þ 2CO2 þ 4H2

(7)

Glucose/Butyrate þ 2CO2 þ 2H2

(8)

3 Glucose/2 Caproate þ 6CO2 þ 4H2 þ 2H2 O

(9)

3.3. Image analysis: size distribution of granules in UASBr At different heights ([1], top (55 cm); [2], middle (28 cm); [3], bottom (3 cm)) of UASBr, samples were taken after 2, 4, 7 and 9 weeks of operation. Fig. 6 shows the particle size distribution of each sample. Particle separation was evident with reactor height: it was much denser at the bottom, and it became

Also, in order to detect dominant microorganism, bacterial diversity was monitored by polymerase chain reactiondenaturing gradient gel electrophoresis (PCR-DGGE) (data not shown). The results of 16S rDNA sequences showed that

Table 4 – Average liquid gas phase parameters in an additional batch fermentation. Rm Carbohydrate H2 Seeding T.H.P H2 yield source (mL) (mol H2/mol (mL H2/h) degradation (%) (%) hexoseadded)

Bottom Effluent

517 160

1.11 0.34

12.03 9.78

96 88

27 20

Organic acids production Total HLaa HAca HPra HBua HVaa HCaa EtOHa organic (%) (%) (%) (%) (%) (%) (%) acids (mg-COD/L) 17,794 18,648

8.5 40.5

15.6 8.6

0.5 18.4

29.2 6.4

4.3 3.9

33.6 12.7

8.3 9.5

T.H.P., total hydrogen production. a HLa, lactic acid; HFo, formic acid; HAc, acetic acid; HPr, propionic acid; HBu, butyric acid; HVa, valeric acid; HCa, caproic acid; EtOH, ethanol.

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Fig. 6 – Particle size distributions of the H2 producing granules in UASBr after (A) 2 weeks of operation at HRT 8 h; (B) 4 weeks of operation at HRT 6 h; (C) 7 weeks of operation at HRT 6 h; (D) 9 weeks of operation at HRT 4 h.

obvious as the operation went on. The particle density in bottom layer was continuously increased till 7th week, but fluctuated in middle and top layers, indirectly indicating the stabilization period. Interestingly, at 9th week (HRT 4 h) when biomass wash-out occurred, the particle density was increased again in top and middle layers. The average particle size in the bottom layer was 0.38 mm, 0.67 mm, 2.0 mm, and 2.1 mm after 2, 4, 7, and 9 weeks of operation, respectively. By the 7th week, a round-shaped granule was clearly visible, and its diameter was comparable to the previous ones where synthetic wastewater was used. In using sucrose as

a substrate, Mu et al. [17], Chang et al. [18], and Fang et al. [29] and could obtain the granules with diameters of 1.0–3.5 mm, 0.43 mm, and 1.6 mm, respectively. As shown in Table 5, CDMW has abundant divalent cations which probably showed a positive effect on forming HPG. As microorganisms generally have negative surface charges, there are few chances for the microorganisms to aggregate in natural condition. However, the presence of EPS and divalent cations, such as Ca2þ/Fe2þ/Mg2þ, could bring about an effective and irreversible adhesion by altering the ionic state of the cell surface [34–37]. Among various divalent ions, calcium was

Table 5 – Divalent cations concentrations in CDMW. Component (Unit)

Na (mg/L)

Ca (mg/L)

Mg (mg/L)

K (mg/L)

Zn (mg/L)

Cu (mg/L)

Fe (mg/L)

Concentration

3094

7309

1171

2222

27.8

<0.1

1.94

international journal of hydrogen energy 35 (2010) 13370–13378

the most abundant ions in CDMW, and it was reported that the existence of calcium can promote HPG formation [38].

4.

Conclusions

Continuous H2 production from CDMW was attempted using CSTR and UASBr under mesophilic condition, and the following conclusions were drawn: 1. While the H2 production was limited in CSTR, it was significantly enhanced in UASBr. This was attributed to the high concentration of biomass in the blanket zone, where over 60,000 mg-VSS/L provides insufficient substrate for intrinsic LAB to survive. 2. Without external inoculum addition, CDMW was naturally fermented into lactic acid under mesophilic condition, which confirmed the abundant existence of LAB in the feed. 3. Lactic acid was the dominant liquid-state metabolite in CSTR, but butyric and caproic acids in UASBr. The presence of caproic acid in the broth also indicates H2 production, yielding 1.33 mol H2/glucose. 4. In UASBr operation, particle separation was evident with reactor height, and this became obvious as the operation went on. For the first time ever, HPG with a diameter of 2.1 mm was successfully formed in using actual waste as a substrate.

Acknowledgement This research was supported by the Ministry of Knowledge Economy (Grant No.: 2006-NBI-02P0130102007) through the New and Renewable Energy Development Program.

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