Alkali-treated sewage sludge as a seeding source for hydrogen fermentation of food waste leachate

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Alkali-treated sewage sludge as a seeding source for hydrogen fermentation of food waste leachate Dong-Hoon Kim a, Mo-Kwon Lee a, Kyung-Won Jung b, Mi-Sun Kim a,c,* a

Clean Fuel Department, Korea Institute of Energy Research, 102 Gajeong-ro, Yuseong-gu, Daejeon 305-343, Republic of Korea b Department of Civil and Environmental Engineering, KAIST, 373-1 Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea c Division of Renewable Energy Engineering, University of Science and Technology, 217 Gajeong-ro, Yuseong-gu, Daejeon 305-350, Republic of Korea

article info

abstract

Article history:

In the present work, alkali-treated sewage sludge (SS) was used as a seeding source for H2

Received 18 January 2013

fermentation of food waste leachate (FWL). The role of alkaline treatment of SS was to

Received in revised form

suppress the activity of non-H2-producers in SS and also to enhance the solubility of SS.

19 May 2013

The effect of pretreatment pH and FWL:SS ratio on H2 production was crucial, by changing

Accepted 22 May 2013

the pH conditions and selecting the dominant species. High pretreatment pH and high SS

Available online 19 June 2013

content resulted in high initial pH conditions. The highest H2 yield of 2.1 mol H2/mol

Keywords:

these conditions, the initial pH was 7.9, and cultivation pH was maintained within the

Hydrogen

reported optimum range of 5.5e6.5 during fermentation. It was found that pretreatment pH

hexoseadded was achieved at pretreatment pH 10 and a mixing ratio of FWL:SS ¼ 3:5. At

Food waste leachate

9 was not strong enough to suppress the activity of non-H2-producers in SS, in particular,

Sewage sludge

lactic acid bacteria (LAB). Microbial analysis results confirmed that LAB such as Lactobacillus

Alkaline treatment

sp. and Enterococcus sp. were the dominant species at pretreatment pH 9 while Clostridium

Lactic acid bacteria

sp., the main anaerobic H2-producers, were dominant at pretreatment pH 10.

Clostridium sp

Copyright ª 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

1.

Introduction

Although the most economical sources of hydrogen gas (H2) are still coal and natural gas, it is expected that H2 production using renewable energy sources may become mandatory in the near future, due to the upcoming depletion of fossil fuels and rising concerns over greenhouse gas emissions [1]. Biohydrogen production technology is an attractive way that producing H2 from organic wastes or water. In particular, dark fermentation considered the most practically applicable

process because of its fast H2 production rate, simplicity in operation, and capacity for degradation of problematic organic wastes [2]. In worldwide, the production of food waste accounts for 15e63% of municipal solid waste, and finding a proper way to treat food waste is now a critical issue [3]. The daily production of food waste is 0.28 kg per capita in Korea, accounting for 27.7% of total municipal wastes in 2009 [4]. Since 2005, when landfilling of food waste was prohibited, over 90% of food waste has been made into compost or fertilizer. However,

* Corresponding author. Clean Fuel Department, Korea Institute of Energy Research, 102 Gajeong-ro, Yuseong-gu, Daejeon 305-343, Republic of Korea. Tel.: þ82 42 860 3554; fax: þ82 42 860 3739. E-mail address: [email protected] (M.-S. Kim). 0360-3199/$ e see front matter Copyright ª 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ijhydene.2013.05.120

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there is little demand for these products due to their low quality, and furthermore, huge amounts (50e70% of food waste by volume) of secondary wastewater, called “food waste leachate (FWL)”, are generated during the process [5]. On the other hand, the daily production of sludge cake from 433 municipal wastewater treatment plants is approximately 8,295 tons, and the production continues to increase with the increasing number of wastewater treatment plants. Currently, more than half of FWL and sludge cake is disposed by ocean dumping. However, as these wastes have high organic content, energy generation such as H2 along with their degradation seems ideal. H2-producing inoculum has often been obtained from anaerobic digester sludge after the pretreatment such as heat, acid-, or alkali-treatment [6]. The role of pretreatment was to suppress the activity of non-H2-producers while cultivating H2-producers. The main H2-producers, Clostridium sp., are spore-forming bacteria, and the spores are resistant to harsh environmental conditions. Sewage sludge (SS) is another potential seeding source for H2 production since it contains various microbial species including anaerobic bacteria [7]. In order to obtain H2-producers from SS, pretreatment should be applied, and this procedure would also increase the hydrolysis rate of SS, which is considered a rate-limiting step in anaerobic digestion of SS [8]. In the present work, it was attempted to utilize alkalitreated SS as a seeding source for H2 fermentation of FWL. The amount of FWL addition was fixed at 30% of the reactor volume while it was varied in case of alkali-treated SS, 30%, 50%, and 70% of the reactor volume. The level of alkalitreatment pH was varied from pH 9 to 12. As the pretreatment pH and SS addition volume was varied, the initial pH was varied, which would cause the different H2 production performance. This is the first feasibility test on using FWL as a substrate for biological H2 production.

2.

Materials and methods

2.1.

Feedstock

The H2 production experiments were carried out using a mixture of FWL and SS as a substrate without extra seed. The concentration of total solids (TS), volatile solids (VS) carbohydrate, total chemical oxygen demand (TCOD), soluble COD (SCOD), total nitrogen (TN), and pH of the FWL was 120.6 g/L, 104.9 g/L, 54.5 g COD/L, 136.3 g COD/L, 36.1 g COD/L, 5.4 g-N/L, and 4.4, respectively. The concentration of TS, VS, carbohydrate, TCOD, SCOD, TN, and pH of the SS was 19.2 g/L, 16.2 g/L, 0.8 g COD/L, 26.0 g COD/L, 1.3 g COD/L, 1.7 g-N/L, and 6.9, respectively.

2.2.

Experimental conditions

A batch fermenter with a 250 mL total volume (100 mL working volume) was used for the batch experiment. In total, 12 different batch experimental sets were prepared depending on the pretreatment pH and the mixing ratio of FWL:SS. The amount of FWL injection was fixed at 30 mL, while the amount of alkali-treated (at pH 9, 10, 11, and 12) SS was 30, 50, and 70 mL. Alkali-pretreatment was conducted for 10 h using 6 N

KOH. Although NaOH has been widely used for alkalitreatment, KOH was used in this study because sodium is known to inhibit fermentative H2 production [9]. The remaining volume was filled with tap water, and purged with N2 gas to provide an anaerobic condition. Each batch fermenter was equipped with pH sensor, and was agitated using magnet bar at 150 rpm. Produced biogas and pH were monitored 2e10 h interval. The tests were carried out in triplicate and results were averaged. All experiments were conducted in a temperature-controlled room at 351  C.

2.3.

Analytical methods

Measured biogas production was adjusted to the standard conditions of temperature (0  C) and pressure (760 mmHg) (STP). H2 content in the biogas was determined by a gas chromatography (GC, Gow Mac series 580) using a thermal conductivity 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 stainlesssteel 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. TS, VS, COD, TN, and ammonia were measured according to Standard Methods [10]. The concentration of carbohydrate was determined by the colorimetric method [11].

2.4.

Microbial analysis

To identify the microbial communities in each batch fermenter, the DNA at different operating conditions: (A) pH 9, FWL:SS ¼ 3:3; (B) pH 9, FWL:SS ¼ 3:5; (C) pH 10, FWL:SS ¼ 3:5; (D) pH 10, FWL:SS ¼ 3:7 were extracted using an Ultraclean Soil DNA Kit (Cat # 12800-50; Mo Bio Laboratory Inc., USA). The 16S rDNA fragments were stored at 20  C before being amplified by polymerase chain reaction (PCR). The region corresponding to positions 357F and 518R in the 16S rDNA of Escherichia coli was PCR-amplified using the forward primer EUB357f (50 CCTACGGGAGGCAGCAG-30 ) with a GC clamp (50 -CGCCCG CCGCGCCCCGCGCCCGGCCCGCCGCCCCCGCCCC-30 ) at the 50 end to stabilize the melting behavior of the DNA fragments and the reverse primer UNIV518r (50 -ATTACCGCGGCTGCTGG30 ). PCR amplification was conducted in an automated thermal cycler (MWG-Bio TECH, Germany) using the following protocol: initial denaturation for 5 min at 94  C, annealing for 40 s at 55  C, extension for 1.5 min at 72  C (25 cycles), followed by a final extension for 8 min at 72  C. PCR mixtures had a final volume of 50 ml of 10  PCR buffer, 0.8 mM MgSO4, 0.5 mM of each primer, 0.1 mM dNTP, 25 pg template, and 1 U polymerase. PCR products were electrophoresed on 2% (wt/vol) agarose gel in 1  TAE for 30 min for 50 V, and then checked with ethidium bromide staining to confirm the amplification. Denaturing gradient gel electrophoresis (DGGE) was carried out using a Dcode Universal Mutation Detection System (BioRad, USA) in accordance with the manufacturer’s instruments. PCR products were electrophoresed in 1  TAE buffer for 480 min at 70 V and 60  C on a polyacrylamide gel (7.5%) containing a linear gradient ranging from 40% to 60% denaturant. After electrophoresis, the polyacrylamide gel was

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 8 ( 2 0 1 3 ) 1 5 7 5 1 e1 5 7 5 6

stained with ethidium bromide for 30 min, and then visualized on a UV transilluminator. Most bands were excised from the DGGE polyacrylamide gel for 16S rDNA sequencing. DNA was eluted from the excised bands by immersion in 20 ml of Tris EDTA buffer (pH 8.0) for one day, and then PCR-amplified with the forward primer EUB357f without a GC clamp and the reverse primer UNIV518r. After PCR amplification, PCR products were purified using a Multiscreen Vacuum Manifold (MILLIPORE com., USA). All strands of the purified PCR products were sequenced with primers EUB357f by an ABIPRISM Big Terminator Cycle Sequencing Kit (Applied Biosystems, USA) in accordance with the manufacturer’s instructions. Search of the GenBank database was conducted using the BLAST program.

3.

Results and discussion

3.1. Effect of alkali pretreatment and FWL:SS ratio on H2 production In order to evaluate the feasibility of applying SS as not only a seeding source but also a co-digestible material with FWL, codigestion of FWL and pretreated SS was conducted at various conditions. CH4 production was not detected at any of the experimental conditions during the entire fermentation period. The solubility (SCOD/TCOD, %) of SS gradually increased from 5% to 26% with pretreatment pH increase. The increased solubility could result in enhanced CH4 production but not H2, since SS has negligible content of carbohydrate. As shown in Fig. 1, the overall trend showed that pretreatment conditions and the mixing ratio exerted significant effects on the H2 production performance. The reason that H2 yield was calculated only based on hexose not COD or VS was that carbohydrate has much higher (more than 20 times) H2 production potential than other nutrients including protein and lipid [12,13]. When the pH level for alkali pretreatment of SS was adjusted to pH 9, H2 yield increased with increasing the mixing ratio. Meanwhile, at pH 10, the highest H2 yield of 2.1 mol H2/mol hexoseadded was achieved at an FWL:SS mixing

H2 yield (mol H2/mol hexoseadded)

2.5 FWL:SS = 3:3 FWL:SS = 3:5 FWL:SS = 3:7

2.0

1.5

1.0

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ratio of 3:5. At pH 11 and 12, a gradual decrease of H2 yield was observed by increasing SS content. It has been widely accepted that pH is a critical factor for biological H2 production, since it can directly affect the hydrogenase activity and metabolic pathway. In particular, in a batch operation, H2 fermentation can be highly affected by initial pH as well as operational pH [14]. In this study, there was no pH control during the fermentation, and thus that initial and cultivation pH depended on the alkali pretreatment condition and FWL:SS mixing ratio. During dark fermentative H2 production, organic acids are accompanied with H2 produciton, which would cause pH drop, and thereby able to lowering down H2 production [15]. Thus, proper amount of SS addition, which is known to contain higher buffer capacity than FWL, could be beneficial to achieve high H2 yield. The level of pretreatment condition can also affect the bacterial community in mixed culture. It is well known that acid-base enrichment of seeding source enhances biological H2 production by shifting population dynamics [16]. Therefore, the difference in H2 production performance was thought to be due mainly to the pH condition and pretreatment efficiency; this would be further discussed in the following sections.

3.2. pH variation at various alkali-treatment pH and FWL:SS ratio Generally, H2-producing bacteria, especially Clostridium sp., in dark fermentation show a vigorous H2 production performance at weak acidic conditions in a range of 5.0e6.5. However, in terms of initial pH, the optimal value has often been found to lie in a range of 7.0e9.0 [17,18]. Thus, initial pH exerts effects before bacteria are active, while operational pH affects bacteria when they are in an active stage. Fig. 2 shows the pH change profile during co-digestion of FWL and SS at various pretreatment conditions and mixing ratios. As shown clearly, the initial pH and pH variation were significantly influenced by the pretreatment pH and the mixing ratio. High pretreatment pH and high SS content resulted in high initial pH conditions. When the pretreatment pH was 9, the initial pH was below 7. And pH dropped below 5 during fermentation, resulting in a low H2 yield. Meanwhile, when pretreatment pH was 10, the initial pH was in a range of 7.0e8.5. Except for the FWL:SS ratio of 3:3, pH was maintained at 5.5  0.2, which was within the optimal pH range for dark fermentation. At pretreatment pH 10 and an FWL:SS ratio of 3:5, initial pH was 7.9, which is close to the reported optimal initial pH condition [14]. Accordingly, the highest H2 yield was achieved at this condition. When the SS was treated at pH 12, initial pH was above 8.0 and pH was maintained above 6.0. It was reported that the alcohol production rate was greater than the H2 production rate if the pH was higher than 6.1 [19]. Notably, at an FWL:SS ratio of 3:7, pH was initially 10.0, which resulted in a long lag period and low H2 yield.

0.5

3.3. Metabolic products at various alkali-treatment pH and FWL:SS ratio 0.0 pH 9

pH10

pH11

pH12

Pretreatment pH

Fig. 1 e Effect of pretreatment pH and the mixing ratio of FWL:SS on H2 production.

As shown in Fig. 3, lactate was the main metabolite at pretreatment pH 9 while butyrate and acetate were the main metabolites at pretreatment pH 10. It appears that pretreatment pH 9 was not strong enough to suppress the activity of

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(a)

(b)

9

9

pH 9, FWL:SS = 3:3 pH 9, FWL:SS = 3:5 pH 9, FWL:SS = 3:7

8

pH 10, FWL:SS = 3:3 pH 10, FWL:SS = 3:5 pH 10, FWL:SS = 3:7

8

7

pH

pH

7

6

6

5

5

4 0

5

10

15

4

20

0

10

20

Time (hr)

30

40

Time (hr)

(c)

(d)

10

10 pH 11, FWL:SS = 3:3 pH 11, FWL:SS = 3:5 pH 11, FWL:SS = 3:7

9

pH 12, FWL:SS = 3:3 pH 12, FWL:SS = 3:5 pH 12, FWL:SS = 3:7

9

8

pH

pH

8 7

7 6

6

5

4

5 0

10

20

30

40

0

Time (hr)

10

20

30

40

50

60

Time (hr)

Fig. 2 e pH change profile at various mixing ratio of FWL:SS and pH pretreatment level (a) pretreatment pH 9, (b) pretreatment pH 10, (c) pretreatment pH 11, and (d) pretreatment pH 12 (pH X, FWL:SS [ a:b indicates that food waste leachate (FWL) was mixed with the alkali-treated sewage sludge at pH X by a:b ratio on volume basis).

non H2-producers, especially lactic acid bacteria (LAB). Like our study, the significant decrease of lactate production was observed by alkaline shock above pH 10 [16,20,21]. It is well known that the production of butyrate and acetate is closely related to H2 production reaction, but all known lactate producing pathways are non-hydrogenic as shown in Eqs. (1)e(3) [20]. The effect of pH level on the pretreatment of seeding sources for H2 production was previously reported by Chen et al. [16]. Glucose/2 Lactate þ 2ATP

(1)

Glucose/Butyrate þ 2CO2 þ 2H2 þ 3ATP

(2)

Glucose þ 2H2 O/2Acetate þ 2CO2 þ 4H2 þ 4ATP

(3)

3.4.

Microbial community

To assess the difference in the main metabolites between pH 9 and 10, bacterial diversity was monitored by PCR-DGGE. Fig. 4 illustrates the DGGE profiles of the 16S rDNA gene fragment amplified from the fermentation broth of four cases: (pH 9, FWL:SS ¼ 3:3); (pH 9, FWL:SS ¼ 3:5); (pH 10, FWL:SS ¼ 3:5); and (pH 10, FWL:SS ¼ 3:7). The major bands were excised and purified to determine the sequence. In Table 1, the results of sequence affiliation, accession number, and similarity are arranged. Although a PCR-DGGE analysis cannot provide quantitative information, it easily shows the distinct change of population dynamics, and it thus often applied in this field [14,22]. In this experiment, a significant difference in the microbial community was observed between pH 9 and 10. As expected, it

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14000 Lactate Acetate Butyrate Propionate

Organic acids (mg COD/L)

12000

10000

Table 1 e Affiliation of DGGE fragments determined by their 16S rDNA sequence. Band

Affiliation

Accession number

Determined (bp)

Similarity (%)

1

Lactobacillus gasseri Enterococcus lactis Lactobacillus amylovorus Uncultured bacterium Clostridium diolis Clostridium perfringens Clostridium bifermentans Clostridium glycolicum

GU417842

151/152

99

DQ255948

151/152

99

EF439704

148/150

98

e

e

e

DQ831125

120/123

97

EF589958

143/150

95

AB075769

123/124

99

EF153869

123/125

98

8000

2 6000

3 4000

4 2000

5 0 pH10,3:5

pH10,3:7

pH9,3:3

pH9,3:5

6

Pretreatment pH and mixing ratio of FWL:SS

7

Fig. 3 e Organic acids production at different pH and mixing ratio of FWL:SS (pH X, a:b indicates that food waste leachate (FWL) was mixed with the alkali-treated sewage sludge at pH X by a:b ratio on volume basis).

was confirmed that LAB such as Lactobacillus sp. and Enterococcus sp. were the dominant species at pretreatment pH 9 while Clostridium sp., known as the main H2-producing bacteria in dark fermentation, including Clostridium diolis, Clostridium perfringens, Clostridium bifermentans, and Clostridium glyolicum were the dominant species when pretreatment pH was 10. In dark fermentation, Lactobacillus sp. and Enterococcus sp. have often been observed with low H2 production [20,21,23]. These results coincided with the distribution of metabolites in at pretreatment pH 9, where the portion of lactate exceeded 80%.

8

4.

Conclusions

The effect of pretreatment pH and FWL:SS ratio on H2 production from FWL was crucial, by changing the pH conditions and selecting the dominant species. As the pretreatment pH and SS content increased, initial pH was increased and the pH was maintained high during fermentation. A proper amount of SS addition and pH pretreatment level could maximize the H2 production potential of FWL. The highest H2 yield of 2.1 mol H2/mol hexoseadded was achieved at pretreatment pH 10 and a mixing ratio of FWL:SS ¼ 3:5, under which initial pH was 7.9, and pH was maintained within the reported optimum range of 5.5e6.5 during fermentation. A microbial analysis clearly revealed that H2-producers, Clostridium sp., were dominantly existed at pretreatment pH 10, while non-H2 producers, Lactobacillus sp. and Enterococcus sp., were detected at pretreatment pH 9.

Acknowledgment This research was performed for the Hydrogen Energy R&D Center, one of the 21st Century Frontier R&D Programs, funded by the Ministry of Education, Science and Technology.

references

Fig. 4 e DGGE profiles of the 16S rDNA gene fragment after H2 fermentation of FWL (from left [ (pH 9, FWL:SS [ 3:3); (pH 9, FWL:SS [ 3:5); (pH 10, FWL:SS [ 3:5); (pH 10, FWL:SS [ 3:7)).

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