Enhanced fermentative hydrogen production from cassava stillage by co-digestion: The effects of different co-substrates

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Enhanced fermentative hydrogen production from cassava stillage by co-digestion: The effects of different co-substrates Wen Wang, Li Xie*, Gang Luo, Qi Zhou Key Laboratory of Yangtze River Water Environment, Tongji University, 1239 Siping Road, Shanghai 200092, PR China

article info

abstract

Article history:

In this study, an efficient strategy to increase the hydrogen yield from cassava stillage (CS),

Received 6 November 2012

by its co-digestion with organic wastes, was developed. The effects of different co-

Received in revised form

substrates on hydrogen yield were evaluated and compared, with increases obtained in

19 February 2013

all co-digestion reactors. The maximal hydrogen yield was achieved by CS co-digestion

Accepted 2 April 2013

with cassava excess sludge (CES), which resulted in a 46% increase over the yield from

Available online 24 April 2013

CS alone. Improved hydrolysis and acidification performances, and thus increased hydrogen production, were additional benefits of co-digestion, especially with CES. Also,

Keywords:

the presence of a co-substrate promoted butyrate over lactate generation. The most

Hydrogen production

influential advantage of co-substrate addition was the enhanced pH buffering capacity,

Co-digestion

which correlated linearly with hydrogen yield (R2 ¼ 0.98). Moreover, the co-substrates

Cassava stillage

addition resulting in more favorable carbohydrate-COD/protein-COD ratio, C/N and C/P

Alkalinity

ratios, all of which also correlated linearly with hydrogen yield (R2 ¼ 0.86, 0.71, and 0.75 respectively). Polymerase chain reactionedenaturing gradient gel electrophoresis analysis of the role of microorganisms indicated that the addition of even small amounts of cosubstrate altered the bacterial communities within the reactors, enriching the proportion of hydrogen-producing bacteria. A species detected only in the co-digestion reactors was shown to be related to Clostridium cellulosi, which is an efficient hydrogen-producer, while some bacteria not involved in hydrogen production and present in the CS alone reactor were not detectable in any of the co-substrate-containing reactors. Copyright ª 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

1.

Introduction

The continued use of fossil fuels to meet the majority of the world’s energy demand is threatened by their association with an increased concentration of carbon dioxide in the atmosphere and thus with global warming [1,2]. Moreover, the shortage of fossil fuels has made it necessary to find alternative energy resources that are environmentally friendly and renewable. Biological processes that produce energy from

biomass are attractive replacement technologies since biomass plays a key role in the global carbon cycle of the biosphere. Furthermore, large amounts of biomass are available, especially in the form of organic residues, such as solid municipal wastes, agricultural residues, food industry wastes, and manures [3]. In the production of energy from biomass, anaerobic biomethane and biohydrogen production are biological processes that can be exploited as highly efficient technologies allowing simultaneous energy production and waste reduction [4,5].

Abbreviations: CS, cassava stillage; CES, cassava excess sludge; PM, pig manure; CM, cow manure; WAS, waste activated sludge. * Corresponding author. Tel.: þ86 21 65982692; fax: þ86 21 65986313. E-mail addresses: [email protected], [email protected] (L. Xie). 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.2013.04.004

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Biohydrogen from anaerobic digestion is an especially well-suited energy route due to the low levels of pollutants generated during combustion and the high energy density [6,7]. Several organic residues have been investigated for efficient biohydrogen production by anaerobic digestion, including wheat straw [8], sweet sorghum [9], potato waste [10], olive pulp [11], garbage slurry [12], molasses [13], grass silage [14], household solid waste [15], and cassava stillage [16]. The major criteria for the selection of organic residues to be used for biohydrogen production are availability, cost, carbohydrate content, and biodegradability [17]. Recently, improved hydrogen production was achieved in co-digestions of different organic residues. Among the reported advantages of co-digestion were the dilution of potentially toxic compounds, an improved balance of nutrients, and synergistic effects on microorganisms present in the reactions [18,19]. However, thus far the mechanisms underlying the improved hydrogen production are unclear, although several have been proposed. Kim et al. [20] proposed supplementary micronutrients to encourage the growth of H2-producing bacteria as the main reason by adding digester sludge to the tofu waste. Zhu et al. [21] postulated that a co-digestion consisting of municipal food waste and sewage sludge had an improved buffering capacity. According to Kim et al. [22], co-digestion food waste with sewage sludge resulted in a more favorable carbohydrate-COD/protein-COD for hydrogen production. In a previous study, we also found that an improved pH buffering capacity and balanced C/N ratio could account for the enhanced hydrogen production achieved in a co-digestion [23]. Nevertheless, to date hydrogen production has only been investigated in co-digestions with two organic residues, whereas comparisons among different types of residues as cosubstrates with carbohydrate-rich organic residues have not been carried out. Consequently, the reasons for the improved hydrogen production offered by the above authors and in other studies are likely to be neither comprehensive nor sufficient. To better understand the favorable conditions achieved by the addition of a co-substrate and thus to elucidate the mechanisms underlying the enhanced hydrogen yield, we evaluated a series of co-digestions in which carbohydrate-rich cassava stillage (CS) was mixed with one of four common and easily obtainable wastes: cassava excess sludge (CES), pig manure (PM), cow manure (CM), and waste activated sludge (WAS). Hydrogen production and liquid metabolite transformation were evaluated in these co-digestions and then analyzed with the aim of explaining the positive effects on hydrogen production. Variations in the microbial community contained in the different co-digestion systems were analyzed by PCR-DGGE. In addition, to maximize bioenergy generation, the effluents of the hydrogen reactors were further used in second-stage methane production.

2.

Materials and methods

2.1.

Feedstock and inoculum

CS and CES were obtained from a cassava ethanol plant in Jiangsu province. PM and CM were collected from a pig farm and a dairy farm in Shanghai, respectively. WAS was

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withdrawn from the secondary sedimentation tank of a municipal wastewater treatment plant in Shanghai. Following their collection, the feedstocks were stored at 4  C. Their characteristics are detailed in Table 1. As inoculum, thermophilically digested CS from a continuously stirred tank reactor (CSTR) in the same cassava ethanol plant was used directly.

2.2.

Experimental design and procedure

Five sterilized identical 300-ml bottles with a working volume of 200 ml were used as hydrogen-production reactors. Prior to the co-digestion, the feedstocks were concentrated or diluted as needed to achieve a concentration of volatile solids (VS) in all five reactors of 30 g/l, to facilitate comparisons. Based on our previous research results [23,24], VSCS/VSco-substrate ratio of 7:1 was adopted. Each of the reactors was inoculated with 20 ml of digested CS, after which 180 ml of CS was added to the first reactor. For the co-digestions, 157.5 ml of CS and 22.5 ml of the co-substrates CES, PM, CM, and WAS were added to the remaining four reactors, respectively. The operation mode was changed to semi-continuous after hydrogen production ceased in the first batch, with the reactor operated as a CSTR. The reactors were fed every 24 h with 100 ml of fresh substrate (100 ml CS in the first reactor, 87.5 ml CS and 12.5 ml co-substrate in reactors 2e5) and an equivalent amount of the digestate was removed, corresponding to a hydraulic retention time of 2 d. The initial pH was adjusted to 5.5 with 2 N NaOH or 2 N HCl immediately after each feeding. In the co-digestions, the changes in pH after hydrogen production likely reflected substrate-related differences in the buffering capacities. Effluent from each hydrogen reactor was fed into a new reactor (total volume 300 ml, working volume of 200 ml) for subsequent methane production. Five methane reactors were also operated as CSTRs for 35 days, removing 20 ml of digestate and adding 20 ml of hydrogen reactor effluents every 24 h. The pH in each methane reactor was not adjusted. In the above experiments, the capped reactors with rubber stoppers were placed in a reciprocating water bath shaker at a temperature of 60  C and rotated at 120 rpm. Liquid and gas samples were collected periodically and sent for analysis.

2.3.

Analytical methods

2.3.1.

Chemical analysis

Total and volatile solids (TS and VS), total and soluble COD (TCOD and SCOD), carbohydrate, protein, ethanol, and volatile fatty acids (VFAs, C2eC5), biogas production, and biogas composition were determined as described in our previous publication [23]. Lactate was quantified using a high-performance liquid chromatography (HPLC) system (HP 1200; Agilent, USA) equipped with an Aminex HPX-87H column (Bio-Rad, USA) at 54  C and a refractive index detector. The eluting solution was 5 mM H2SO4 at a flow rate of 0.6 ml/min.

2.3.2.

PCR-DGGE analysis

Polymerase chain reactionedenaturing gradient gel electrophoresis (PCR-DGGE) was used to study the microbial

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Table 1 e Characteristics of feedstock.

pH TS VS Total alkalinity (mg/g-VS as CaCO3) TCOD (mg/g-VS) SCOD (mg/g-VS) Carbohydrate (mg/g-VS) Protein (mg/g-VS) TN (mg/g-VS) NHþ 4 -N (mg/g-VS) TP (mg/g-VS) C/N ratio (element analysis) N (% of TS) C (% of TS) H (% of TS)

CS

CES

PM

CM

WAS

4.08  0.01 60.5  5.9 g/l 49.4  2.1 g/l 0.0  0.0 2048  20 658  12 120.6  3.4 49.7  1.7 25.8  0.4 1.2  0.0 19.4  0.0 21.7  0.1 1.5  0.0 32.1  0.0 5.5  0.0

7.55  0.01 88.5  4.4 g/g 60.0  3.0 g/g 117.5  1.6 1945  19 48  3 4.1  0.3 9.7  0.5 70.9  0.3 8.1  0.1 113.8  0.1 7.2  0.0 4.5  0.0 32.2  0.0 5.3  0.1

7.40  0.02 159.9  0.6 g/g 125.1  0.5 g/g 91.3  2.1 1115  33 290  5 8.5  0.9 57.4  1.2 64.3  0.8 26.9  0.1 138.9  0.3 7.5  0.0 4.4  0.0 32.8  0.1 6.7  0.0

6.95  0.01 154.8  1.9 g/g 131.9  1.2 g/g 40.8  1.0 1312  16 285  5 20.0  1.1 78.0  1.2 32.0  0.1 3.2  0.0 55.1  0.1 11.4  0.0 3.50  0.0 39.8  0.2 6.3  0.0

6.76  0.03 172.0  7.1 g/g 113.7  4.4 g/g 93.6  0.7 1750  9 197  2 9.9  0.4 29.0  0.9 93.9  0.6 17.7  0.1 110.2  0.2 5.8  0.1 5.4  0.0 31.3  0.0 5.2  0.0

community structure in the mixed culture. When hydrogen production had reached steady-state, 100 ml of the mixed culture was withdrawn from each of the five reactors, dispersed, and washed twice with 1 phosphate buffere saline. The cells were concentrated by centrifugation at 5000 g for 5 min and then resuspended in 50 ml of sterile MilliQ water. Genomic DNA was extracted using the Omega D562501 soil DNA kit (Omega Bio-Tek, USA). The extracted DNA was quality checked by agarose gel electrophoresis and then stored at 20  C for subsequent use as a template in PCR amplifications. The w200 bp fragment of the V3 region was PCR-amplified with the primers 518-r (50 - ATT ACC GCG GCT GCT GG -30 ) and 357-f (50 - CC TAC GGG AGG CAG CAG -30 ) [25]. PCR was performed in a 50-ml (total volume) reaction mixture containing 50 mM KCl, 20 mM TriseHCl (pH 8.4), 5 mM MgCl2, 200 mM of each deoxynucleotide triphosphate, 1 ml of Taq polymerase (2 U/ml), 10 pmol of each primer, and 1 ml of DNA extract. The thermal cycling program used for amplification was as follows: pre-denaturation at 94  C for 4 min; 30 cycles of denaturation at 94  C for 0.5 min, annealing at 56  C for 1 min, elongation at 72  C for 0.5 min, and post-elongation at 72  C for 7 min. The reactions were subsequently cooled to 4  C. PCR products were stored at 4  C and analyzed on 1.5% agarose before DGGE. DGGE analysis of the PCR amplicons was performed with the Dcode Universal Mutation Detection System (Bio-Rad, USA) with 8% (v/v) polyacrylamide gels and a denaturant gradient of 30e60%. A 100% denaturing solution consisted of 7 M urea and 40% formamide. PCR products (20 ml) were loaded into each gel lane and subjected to electrophoresis for 4 h at 180 V in 1 TAE buffer at 60  C. The DGGE gels were then stained with SYBR Green I, photographed with UV transillumination, and documented using the GelDoc system (BioRad, USA). The target bands were excised from the DGGE gels, washed once in TriseEDTA buffer, and incubated in 20 ml of the same buffer overnight at 4  C. The eluted DNA was re-amplified with primers 518-r/357-f under the same conditions as described above. The PCR products were then sent for sequencing (Sangon Bio-tech, China).

Sequence similarity searches were performed using the Basic Local Alignment Search Tool (BLAST) and used to search the National Center for Biotechnology Information sequence database (http://www.ncbi.nlm.nih.gov/BLAST/).

2.3.3.

Data analysis

In this study, all data were obtained when each operation condition had reached steady-state, which was defined as sustained biogas production within 10% deviation. Analysis of variance (ANOVA) at 0.05 level was used to analyze the data.

3.

Results and discussion

3.1. Comparison of hydrogen production with different co-substrates The five hydrogen fermentation reactors were operated for approximately 35 days. Their performances are shown in Fig. 1a and those at steady-state are summarized in Fig. 1b. The parameters of each reactor were calculated from the last 20 days of the steady-state period. Based on the total VS added, the highest hydrogen yield (HY) 57.8 ml/g-total-VS added was obtained in the codigestion of CS with CES, which was 28% higher than in the digestion consisting of CS alone (P < 0.05). With PM and WAS as co-substrates, HY was also enhanced, by 11% and 10% respectively (P < 0.05). However, when CS was co-digested with CM, the HY was 3% lower than CS digested alone (P < 0.05). It was previously shown that carbohydrate-rich wastes yield 20 times more hydrogen than fat- or proteinrich wastes [26,27]. Since the carbohydrate content in the CES, PM, CM and WAS used in this study was only 3%e16% of that in the CS (Table 1), hydrogen production was largely dependent on the CS utilization rate. Compared with the first reactor, in which CS was digested without a co-substrate, in the four co-digestion reactors 12.5% of the CS was replaced with co-substrates. Accordingly, HYs calculated on the basis of total VS added cannot accurately reflect the entire enhanced hydrogen production capacity. Furthermore, the HYs from the co-substrates themselves were negligible, as

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0 0

3

6

9

12

15

18

21

24

27

30

33

36

Hydrogen yield (mL H /g-VS added)

b

glycerol, long-chain fatty acids, amino acids, and oligo- and monosaccharides. These smaller products are subsequently consumed and degraded into VFAs/ethanol/lactate during the acidification step, which accompanies hydrogen generation. The hydrolysis and acidification of carbohydrates and proteins therefore correlate with hydrogen production. Thus, carbohydrate and protein degradation efficiencies were used in the current study as an indicator of the degree of hydrolysis and acidification taking place in the reactors. Fig. 2 describes the influence of the different co-substrates on carbohydrate and protein degradation efficiencies. In all five reactors, carbohydrate degradation efficiencies (>60%) were much higher than those of protein degradation (11%), consistent with the well-documented high potential of carbohydrate-rich wastes in intensive biohydrogen production [3,30]. As seen in Table 1, the carbohydrate concentration of CS is 6e30 times higher than that of the co-substrates. The differences in the efficiencies of carbohydrate and protein degradation provide further evidence that in this study hydrogen was mainly generated from CS, via processes stimulated and maximized by co-substrate addition. In addition, a clear linear correlation (R2 ¼ 0.98) between carbohydrate degradation efficiency and HY was determined (Fig. 2). The highest carbohydrate degradation efficiency of

a

Carbohydrate Protein

CS+CES

CS+PM

CS+CM

CS+WAS

Fig. 1 e Time courses of hydrogen production (a) and average hydrogen yield at steady states (b).

shown in on our previous work [23] and by other authors [28,29]. Therefore, HYs were also calculated based on the CScontributed VS, as shown in Fig. 1b. In all four co-digestion reactors, the HYs (ml/g-CS-VS added) were larger than by CS alone, ranging from a 46% increase in the CES co-digested reactor to an 11% increase in the CM co-digestion reactor. These results are consistent with synergistic and complementary effects by co-digestion. It should be pointed out that despite the addition of only a small amount of co-substrate (the ratio of CS to the cosubstrates was 7:1), hydrogen production from CS was significantly enhanced in the four reactors. This result suggests that the co-substrates contribute important components to the fermentation process. As the main purpose of this study was to maximize hydrogen production, in the following section the potential reasons for the enhanced HYs in all of the co-digestion reactors are discussed.

CS+CES

CS

CS+PM

CS+CM

CS+WAS

70

b Hydrogen yield (g-total-VS added)

CS

Degradation efficiency (%)

R = 0 .9 7 5 2

CS+CES

65

60

CS+PM 55

R =0.8591

CS+WAS

50

CS

CS+CM 45

3.2. Effect of different co-substrates on hydrolysis and acidification performances During the anaerobic biodegradation of complex organic matter, the main components, i.e., lipids, proteins, and carbohydrates, are depolymerized during the hydrolysis step to

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70

0.75

g-carbyhydrate-COD/ g-protein-COD

Fig. 2 e Carbohydrate and protein degradation efficiency (a) and linear regression analysis between carbyhydrate-COD/ protein-COD ratio and hydrogen yield (b).

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3.3. Effect of different co-substrates on the composition of acidification metabolites The addition of different co-substrates also influenced the composition of the generated acidification metabolites, mainly VFAs, ethanol, and lactate. Butyrate and lactate were the two most abundant metabolites in the five reactors (Fig. 3), while iso-butyrate, n-valerate and iso-valerate were not detected in any of the samples. Among the large range of end products generated by the various routes of microbial metabolism, butyrate accumulates with a theoretical production of 2-mol H2/mol-hexose, while lactate is involved in a zerohydrogen-balance pathway, as shown in Eq. (1) and Eq. (2): C6 H12 O6 /CH3 CH2 CH2 COOH þ 2CO2 þ 2H2

(1)

C6 H12 O6 /2CH3 CHOHCOOH þ 2CO2

(2)

kitchen waste were used as the substrates [13,31e33], it seems that a lower lactate and a higher butyrate fraction act to increase hydrogen production. In the co-digestion of CS with CES (Fig. 4), butyrate was the dominant metabolite (55%), with slightly lower amounts when WAS (52%) and PM (49%) were the co-substrates, whereas lactate was the major metabolite when CS was either digested alone (49%) or co-digested with PM (48%). In fact, the butyrate concentration was linearly associated with HY (R2 ¼ 0.97, Fig. 3). The distribution of acidification metabolites also indicated that the addition of suitable co-substrates, by promoting butyrate production, leads to significantly higher hydrogen production. Among the co-substrates examined in this work, the best results were obtained with CES.

3.4. Effect of different co-substrates on the pH and alkalinity of the system Large amounts of volatile fatty acids are generated during the hydrogen fermentation process when organic residues with a high carbohydrate content are used. However, this causes a drop in pH and consequently the breakdown of the reactor if it is not properly maintained. Since carbohydrate-rich organic residues such as municipal food [21,34,35] and food industry [20,36] wastes are of low alkalinity and low nitrogen content,

5.35

a

effluent pH

5.30

5.25

pH

75% was consistent with the highest HY, obtained by the codigestion of CS with CES, with lower but nonetheless highly improved efficiencies over that of CS alone (60%) obtained with WAS, PM and CM. These observations suggested that the co-digestion of CS with CES, WAS, PM, and CM allowed a better hydrolysis and acidification performance and that CES was an optimal co-substrate. In a previous study, we found that a favorable carbohydrate-COD/protein-COD ratio could explain the enhanced hydrogen production obtained by the co-digestion of CS with WAS [23]. This was also the explanation offered by Kim et al. [22], in their study examining food waste co-digested with sewage sludge. In the present work, in which different wastes were used as co-substrates, a linear correlation (R2 ¼ 0.86) between HY and the carbohydrate-COD/protein-COD ratio of the mixture in the reactors was shown (Fig. 2), strongly supporting the importance of this ratio in hydrogen production. Co-substrate addition, by enhancing the protein portion and thus decreasing the carbohydrate-COD/proteinCOD ratio, provided balanced nutrient conditions for hydrogen-producing bacteria and in turn enhanced hydrogen production.

5.20

5.15

5.10

Although high levels of lactate formation were obtained when carbohydrate-rich molasses, food waste, lactose, and

CS

CS+CES

CS+PM

CS+CM

CS+WAS

C S+PM

CS+CM

CS+WAS

600

6500 6000

b

lactate ethanol

5500

acetate propionate

5000

n-butyrate

R = 0 .9 6 5 9

4500 4000 3500 3000 2500 2000

Total alkalinity (mg/l as CaCO )

VFA/ ethanol/ lactate concentration (mg/L)

7000

effluent alkalinity

500

400

300

200

1500 100

1000 500 0

0

CS

CS+CES

CS+PM

CS+CM

CS+WAS

Fig. 3 e Effect of different co-substrates on the composition of VFAs/ethanol/lactate.

CS

CS+CES

Fig. 4 e pH (a) and alkalinity (b) of the effluent from the hydrogen reactors.

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chemical reagents, e.g., sodium and calcium hydroxides, are commonly added to these reactions [36,37]. However, the alkaline cations used for pH regulation in the fermentation process may inhibit anaerobic digestion [38]; indeed, the HY was increased with organic rather than inorganic nitrogen sources [39,40]. Recently, several reports showed that the codigestion of low-pH buffered organic residues with wastes of suitable alkalinity allowed both effective pH control and economic management, together improving anaerobic digestion efficiency [21,23,34]. CS contains carbohydrate-rich organic residues with low pH and alkalinity (almost zero), while the pH and total alkalinity of CES, PM, CM and WAS are much higher (Table 1). Although the initial pH of each reactor was adjusted to 5.5 with NaOH immediately after each feeding, the effluent pH in the five reactors ranged from 5.18 to 5.28 (P < 0.01), suggesting differences in the buffering capacities of the co-substrates (Fig. 4a). The higher HY was in accordance with the higher effluent pH, as seen in a comparison of Fig. 1b with Fig. 4a. Moreover, the total alkalinity of the effluent from the hydrogen reactors further confirmed the correlation between HY and the buffering capacity of the system. The highest total alkalinity was obtained when CS was co-digested with CES (Fig. 4b), which also corresponded to the highest hydrogen production. By contrast, the lowest total alkalinity and lowest HY were measured in the reactor in which CS was digested alone. The use of CM as co-substrate resulted in a comparatively low alkalinity, which corresponded to a reduced HY. The total alkalinity in the reactor containing the CS/CES codigestion was two-fold higher than that in the CS alone and CS/CM co-digested reactors. In agreement with these observations, there was a significant linear correlation (R2 ¼ 0.98) between HY and the total alkalinity of the co-digestion system (Fig. 4b). We therefore conclude that the most critical factor influencing the enhanced HY obtained by co-substrate addition, and especially the higher HY in the CS/CES co-digestion reactor, was the improved pH buffering capacity of the codigestions.

3.5. ratios

hydrogen production, because of its importance in the growth of hydrogen-producing bacteria. Yokoi et al. [40] reported that the largest amounts of hydrogen were obtained from starch in the presence of 0.1% polypeptone. Bisaillon et al. [41] also found that an appropriate level of nitrogen addition is beneficial to the growth of hydrogen-producing bacteria and thus to fermentative hydrogen production. Similarly, in our study, HYs and C/N ratios were linearly correlated (R2 ¼ 0.71 and 0.66 in the element analysis and TCOD/TN, respectively).

Effects of different co-substrates on the C/N and C/P

As a form of wastewater, CS has a low nitrogen concentration. The addition of CES, PM, CM, or WAS to the digestions increased the nitrogen concentration and thus lowered the C/ N ratio, as determined by the C/N ratio measured in the element analysis and calculated from the TCOD/TN (Table 2). Nitrogen is one of the most essential nutrients in efficient

Table 2 e Effect of different co-substrates on the C/N and C/P ratios. CS

CS þ CES

CS þ PM

C/N ratio (element analysis) 20.7  0.0 15.1  0.0 16.6  0.0 C/N ratio (TCOD/TN) 117.7  2.0 54.5  2.5 56.7  2.2 C/P ratio (TCOD/TP) 145.2  3.1 70.4  1.7 67.9  2.0

CS þ CM

CS þ WAS

18.1  0.0

14.2  0.1

69.6  2.5

54.4  1.7

104.4  1.8

68.8  1.5

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Fig. 5 e DGGE bands of microbial communities in five hydrogen production reactors at steady-states.

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Table 3 e Sequencing results of 16S rDNA fragments obtained from DGGE gels. Band 1 2 3 4 5 6 7 8 9 10 11 12 13

Organism affiliation

Identify

Phylum

Accession no.

Thermoanaerobacterium thermosaccharolyticum Thermohydrogenium sp. Flavobacteriaceae Myroides Enterobacteriaceae Escherichia Thermohydrogenium sp. Thermoanaerobacterium thermosaccharolyticum Thermoanaerobacterium thermosaccharolyticum Flavobacteriaceae Myroides Devosia sp. Clostridium cellulosi Thermoanaerobacterium thermosaccharolyticum Thermoanaerobacterium thermosaccharolyticum Flavobacteriaceae Myroides

99% 98% 94% 100% 98% 99% 99% 94% 94% 99% 98% 99% 93%

Firmicutes Firmicutes Bacteroidetes Proteobacteria Firmicutes Firmicutes Firmicutes Bacteroidetes Proteobacteria Firmicutes Firmicutes Firmicutes Bacteroidetes

AF247003.1 FR749965.1 NR_041042.1 AP012306.1 FR749965.1 AF247003.1 AF247003.1 NR_041042.1 AB627758.1 NR_044624.1 AF247003.1 AF247003.1 NR_041042.1

Co-digestion of CS with CES, PM, CM and WAS was also found to increase the total phosphate concentration and to decrease the C/P (TCOD/TP) ratio (Table 2). Phosphate is another essential factor in hydrogen production due to its nutrient value and buffering capacity. An increase in the phosphate concentration was previously shown to increase the hydrogen-producing capacity of hydrogen-producing bacteria [41,42]. In our study, the HY linearly correlated with the C/P ratio (R2 ¼ 0.75). Based on the above analysis, the increased nitrogen and phosphorus concentrations and improved C/N and C/P ratios offer further explanations for the enhanced HYs obtained with co-substrate addition.

3.6. Effect of different co-substrates on microbial community structures When the conditions in each reactor had reached steadystate, the bacterial community structure was sampled and subsequently analyzed by PCR-DGGE (Fig. 5). As expected, differences in the bacterial communities between the codigestion reactors and the CS-alone reactor were found. For example, band 10 was present in all four co-digestion reactors but absent in the CS-alone reactor, whereas the opposite was observed for bands 9 and 13. Sequencing results (Table 3) showed that band 10 was closely related (99% similarity) to Clostridium cellulosi, a hydrogen-producing anaerobic thermophile that decomposes cellulose, with hydrogen, carbon dioxide, butyrate, acetate, and ethanol as fermentation end products. C. cellulosi has an optimum growth range of 55e60  C and forms spherical terminal endospores. It is also an important bacterial presence in hydrogen production from glucose, cellulose, and cow manure [29,39,43]. Bands 9 and 13, which were not detectable in the co-digestion reactors, were closely related to Devosia sp. (93%) and to Flavobacteriaceae Myroides (94%) (Table 3); neither of these bacteria is involved in hydrogen production. These results imply that even the addition of small amounts of co-substrate altered the community structure of the whole system, with the growth and enrichment of hydrogen-producing bacteria and therefore improved hydrogen production in the reactors. In all five reactors, Thermoanaerobacterium thermosaccharolyticum (bands 1, 6, and 7) was the predominant bacterial

species (Table 3). This thermophile has an optimal growth temperature of 60  C and is able to convert carbohydrates, such as cellulose, starch, sucrose, and xylose, to hydrogen, with butyrate as the end soluble product [44e47]. Thermoanaerobacterium sp. and related genera have been shown to dominate many thermophilic fermentations operating in a temperature range of 55e60  C and within a pH range of 5.0e6.0 in which cellulose-rich wastewater [48], starch [49], and food waste [50] serve as feedstocks.

3.7. Effect of the different co-substrates on methane production Effluent from each hydrogen production reactor was further utilized for subsequent methane production. The methane yield for CS digested alone was 351.6 ml/g-total-VS added compared with 346.2, 361.5, 326.5, 343.3, and 346.6 ml/g-totalVS added in the co-digestions with CES, PM, CM, and WAS, respectively. Efficient organic compound degradation was achieved in all five reactors with respect to the degradations of TCOD (55%e63%), SCOD (>93%), and VS (62%e68%), all of which also correlated with methane production (data not shown). Thus, the subsequent coupling of the methane fermentation phase with the hydrogen fermentation phase greatly increased the efficiency of organic compound degradation.

4.

Conclusion

In this study, maximal HY from CS was obtained with CES as cosubstrate, resulting in a yield 46% higher than that from CS digested alone. Moreover, better hydrolysis and acidification were achieved in all of the examined co-digestions, i.e., with CES, WAS, PM, and CM. The highest carbohydrate degradation efficiency of 75% was measured in the CES co-substrate reactor, and it showed that co-digestion can increase the CS utilization rate. The distribution of acidification metabolites indicated that suitable co-substrate addition to CS promoted the butyrate production pathway, with more butyrate and less lactatedand thus significantly higher hydrogen productiondespecially in the presence of CES. An enhanced pH buffering capacity was identified as the most critical factor influencing HY, evidenced by the latter’s strong linear correlation (R2 ¼ 0.98) with alkalinity.

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 ) 6 9 8 0 e6 9 8 8

Improved carbohydrate-COD/protein-COD, C/N, and C/P ratios resulted from the addition of the co-substrates, and these improvements were also linearly correlated with HY (R2 ¼ 0.86, 0.71 and 0.75, respectively). As shown in the PCR-DGGE analysis, even a small amount of co-substrate was sufficient to alter the bacterial community structure of the whole system, with the enrichment in hydrogen-producing bacteria markedly improving hydrogen production behavior. Among the identified bacterial species, hydrogen-producing C. cellulosi was present only in the co-digestion reactors, whereas bacteria unrelated to hydrogen production but present in the CS alone reactor were not detectable. For the practical application of the co-digestion process to enhance H2 production, the process needs to be further optimized by changing pH, HRT and temperature.

Acknowledgments This research was supported by the National Natural Science Fund of China (51178326), the National Key Technology R&D Programme (2012BAC17B02), and the National Hi-Tech Research and Development Program of China (863) (2011AA060903).

references

[1] Balat M. Bioethanol as a vehicular fuel: a critical review. Energ Source Part A 2009;31:1242e55. [2] Levin DB, Pitt L, Love M. Biohydrogen production: prospects and limitations to practical application. Int J Hydrogen Energ 2004;29:173e85. [3] Guo XM, Trably E, Latrille E, Carrere H, Steyer JP. Hydrogen production from agricultural waste by dark fermentation: a review. Int J Hydrogen Energ 2010;35:10660e73. [4] Demirel B, Scherer P, Yenigun O, Onay TT. Production of methane and hydrogen from biomass through conventional and high-rate anaerobic digestion processes. Crit Rev Env Sci Tec 2010;40:116e46. [5] Kalinci Y, Hepbasli A, Dincer I. Biomass-based hydrogen production: a review and analysis. Int J Hydrogen Energ 2009;34:8799e817. [6] Balat H, Kirtay E. Hydrogen from biomass-present scenario and future prospects. Int J Hydrogen Energ 2010;35:7416e26. [7] Li CL, Fang HHP. Fermentative hydrogen production from wastewater and solid wastes by mixed cultures. Crit Rev Env Sci Tec 2007;37:1e39. [8] Kaparaju P, Serrano M, Thomsen AB, Kongjan P, Angelidaki I. Bioethanol, biohydrogen and biogas production from wheat straw in a biorefinery concept. Bioresour Technol 2009;100:2562e8. [9] Antonopoulou G, Gavala HN, Skiadas LV, Angelopoulos K, Lyberatos G. Biofuels generation from sweet sorghum: fermentative hydrogen production and anaerobic digestion of the remaining biomass. Bioresour Technol 2008;99:110e9. [10] Zhu HG, Stadnyk A, Beland M, Seto P. Co-production of hydrogen and methane from potato waste using a two-stage anaerobic digestion process. Bioresour Technol 2008;99:5078e84. [11] Koutrouli EC, Kalfas H, Gavala HN, Skiadas IV, Stamatelatou K, Lyberatos G. Hydrogen and methane production through two-stage mesophilic anaerobic digestion of olive pulp. Bioresour Technol 2009;100:3718e23.

6987

[12] Ueno Y, Fukui H, Goto M. Operation of a two-stage fermentation process producing hydrogen and methane from organic waste. Environ Sci Technol 2007;41:1413e9. [13] Park MJ, Jo JH, Park D, Lee DS, Park JM. Comprehensive study on a two-stage anaerobic digestion process for the sequential production of hydrogen and methane from cost-effective molasses. Int J Hydrogen Energ 2010;35:6194e202. [14] Nizami AS, Murphy JD. Optimizing the operation of a twophase anaerobic digestion system digesting grass silage. Environ Sci Technol 2011;45:7561e9. [15] Liu DW, Liu DP, Zeng RJ, Angelidaki I. Hydrogen and methane production from household solid waste in the two-stage fermentation process. Water Res 2006;40:2230e6. [16] Luo G, Xie L, Zou ZH, Wang W, Zhou Q. Exploring optimal conditions for thermophilic fermentative hydrogen production from cassava stillage. Int J Hydrogen Energ 2010;35:6161e9. [17] Kapdan IK, Kargi F. Bio-hydrogen production from waste materials. Enzym Microb Technol 2006;38:569e82. [18] Lee M, Hidaka T, Hagiwara W, Tsuno H. Comparative performance and microbial diversity of hyperthermophilic and thermophilic co-digestion of kitchen garbage and excess sludge. Bioresour Technol 2009;100:578e85. [19] Li M, Zhao YC, Guo Q, Qian XQ, Niu DJ. Bio-hydrogen production from food waste and sewage sludge in the presence of aged refuse excavated from refuse landfill. Renew Energ 2008;33:2573e9. [20] Kim MS, Lee DY. Fermentative hydrogen production from tofu-processing waste and anaerobic digester sludge using microbial consortium. Bioresour Technol 2010;101:S48e52. [21] Zhu HG, Parker W, Basnar R, Proracki A, Falletta P, Beland M, et al. Biohydrogen production by anaerobic co-digestion of municipal food waste and sewage sludges. Int J Hydrogen Energ 2008;33:3651e9. [22] Kim SH, Han SK, Shin HS. Feasibility of biohydrogen production by anaerobic co-digestion of food waste and sewage sludge. Int J Hydrogen Energ 2004;29:1607e16. [23] Wang W, Xie L, Chen JR, Luo G, Zhou Q. Biohydrogen and methane production by co-digestion of cassava stillage and excess sludge under thermophilic condition. Bioresour Technol 2011;102:3833e9. [24] Wang W, Xie L, Luo G, Zhou Q, Lu Q. Optimization of biohydrogen and methane recovery within a cassava ethanol wastewater/waste integrated management system. Bioresour Technol 2012;120:165e72. [25] Muyzer G, Dewaal EC, Uitterlinden AG. Profiling of complex microbial-populations by denaturing gradient gelelectrophoresis analysis of polymerase chain reactionamplified genes-coding for 16s ribosomal-RNA. Appl Environ Microbiol 1993;59:695e700. [26] Lay JJ, Fan KS, Chang J, Ku CH. Influence of chemical nature of organic wastes on their conversion to hydrogen by heatshock digested sludge. Int J Hydrogen Energ 2003;28:1361e7. [27] Tawfik A, El-Qelish M. Continuous hydrogen production from co-digestion of municipal food waste and kitchen wastewater in mesophilic anaerobic baffled reactor. Bioresour Technol 2012;114:270e4. [28] Kotsopoulos TA, Fotidis IA, Tsolakis N, Martzopoulos GG. Biohydrogen production from pig slurry in a CSTR reactor system with mixed cultures under hyper-thermophilic temperature (70 degrees C). Biomass Bioenerg 2009;33:1168e74. [29] Yokoyama H, Waki M, Ogino A, Ohmori H, Tanaka Y. Hydrogen fermentation properties of undiluted cow dung. J Biosci Bioeng 2007;104:82e5. [30] Okamoto M, Miyahara T, Mizuno O, Noike T. Biological hydrogen potential of materials characteristic of the organic fraction of municipal solid wastes. Water Sci Technol 2000;41:25e32.

6988

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 ) 6 9 8 0 e6 9 8 8

[31] Zhang B, Cai WM, He PJ. Influence of lactic acid on the twophase anaerobic digestion of kitchen wastes. J Environ SciChina 2007;19:244e9. [32] Kim DH, Kim SH, Shin HS. Hydrogen fermentation of food waste without inoculum addition. Enzym Microb Technol 2009;45:181e7. [33] Collet C, Adler N, Schwitzguebel JP, Peringer P. Hydrogen production by Clostridium thermolacticum during continuous fermentation of lactose. Int J Hydrogen Energ 2004;29:1479e85. [34] Lee DY, Ebie Y, Xu KQ, Li YY, Inamori Y. Continuous H-2 and CH4 production from high-solid food waste in the two-stage thermophilic fermentation process with the recirculation of digester sludge. Bioresour Technol 2010;101:S42e7. [35] Siddiqui Z, Horan NJ, Salter M. Energy optimisation from codigested waste using a two-phase process to generate hydrogen and methane. Int J Hydrogen Energ 2011;36:4792e9. [36] Fezzani B, Ben Cheikh R. Two-phase anaerobic co-digestion of olive mill wastes in semi-continuous digesters at mesophilic temperature. Bioresour Technol 2010;101:1628e34. [37] Fountoulakis MS, Manios T. Enhanced methane and hydrogen production from municipal solid waste and agroindustrial by-products co-digested with crude glycerol. Bioresour Technol 2009;100:3043e7. [38] Kyazze G, Dinsdale R, Guwy AI, Hawkes FR, Premier GC, Hawkes DL. Performance characteristics of a two-stage dark fermentative system producing hydrogen and methane continuously. Biotechnol Bioeng 2007;97:759e70. [39] Ueno Y, Haruta S, Ishii M, Igarashi Y. Microbial community in anaerobic hydrogen-producing microflora enriched from sludge compost. Appl Microbiol Biotechnol 2001;57:555e62. [40] Yokoi H, Saitsu A, Uchida H, Hirose J, Hayashi S, Takasaki Y. Microbial hydrogen production from sweet potato starch residue. J Biosci Bioeng 2001;91:58e63.

[41] Bisaillon A, Turcot J, Hallenbeck PC. The effect of nutrient limitation on hydrogen production by batch cultures of Escherichia coli. Int J Hydrogen Energ 2006;31:1504e8. [42] Lay JJ, Fan KS, Hwang JI, Chang JI, Hsu PC. Factors affecting hydrogen production from food wastes by Clostridium-rich composts. J Environ Eng-Asce 2005;131:595e602. [43] Fang HHP, Zhang T, Liu H. Microbial diversity of a mesophilic hydrogen-producing sludge. Appl Microbiol Biotechnol 2002;58:112e8. [44] O-Thong S, Prasertsan P, Intrasungkha N, Dhamwichukorn S, Birkeland NK. Optimization of simultaneous thermophilic fermentative hydrogen production and COD reduction from palm oil mill effluent by Thermoanaerobacterium-rich sludge. Int J Hydrogen Energ 2008;33:1221e31. [45] Cann IKO, Stroot PG, Mackie KR, White BA, Mackie RI. Characterization of two novel saccharolytic, anaerobic thermophiles, Thermoanaerobacterium polysaccharolyticum sp nov and Thermoanaerobacterium zeae sp nov., and emendation of the genus Thermoanaerobacterium. Int J Syst Evol Micrbiol 2001;51:293e302. [46] Collins MD, Lawson PA, Willems A, Cordoba JJ, Fernandezgarayzabal J, Garcia P, et al. The phylogeny of the genus Clostridium-proposal of 5 new genera and 11 new species combinations. Int J Syst Bacteriol 1994;44:812e26. [47] Liu SY, Rainey FA, Morgan HW, Mayer F, Wiegel J. Thermoanaerobacterium aotearoense sp nov, a slightly acidophilic, anaerobic thermophile isolated from various hot springs in New Zealand, and emendation of the genus Thermoanaerobacterium. Int J Syst Bacteriol 1996;46:388e96. [48] Liu H, Zhang T, Fang HHP. Thermophilic H-2 production from a cellulose-containing wastewater. Biotechnol Lett 2003;25:365e9. [49] Zhang T, Liu H, Fang HHP. Biohydrogen production from starch in wastewater under thermophilic condition. J Environ Manage 2003;69:149e56. [50] Shin HS, Youn JH. Conversion of food waste into hydrogen by thermophilic acidogenesis. Biodegradation 2005;16:33e44.