Exploring optimal conditions for thermophilic fermentative hydrogen production from cassava stillage

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 5 ( 2 0 1 0 ) 6 1 6 1 e6 1 6 9

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Exploring optimal conditions for thermophilic fermentative hydrogen production from cassava stillage Gang Luo a, Li Xie a,b,*, Zhonghai Zou a, Wen Wang a, Qi Zhou a,b a b

Key Laboratory of Yangtze River Water Environment, Ministry(Tongji University), Siping Road no 1239, Shanghai 200092, PR China UNEP-Tongji University Institute of Environment for Sustainable Development, Siping Road no 1239, Shanghai 200092, PR China

article info

abstract

Article history:

This study investigated the effects of seed sludges, alkalinity and HRT on the thermophilic

Received 18 January 2010

fermentative hydrogen production from cassava stillage. Five different kinds of sludges

Received in revised form

were used as inocula without any pretreatment. Though batch experiments showed that

25 March 2010

mesophilic anaerobic sludge was the best inoculum, the hydrogen yields with different

Accepted 28 March 2010

seed sludges were quite similar in continuous experiments in the range of 82.9e92.3 ml H2/ gVS without significant differences which could be attributed to the establishment of

Keywords:

Uncultured Thermoanaerobacteriaceae bacterium-dominant microbial communities in all

Thermophilic hydrogen production

reactors. It is indicated that results obtained from batch experiments are not consistent

Cassava stillage

with those from continuous experiments and all the tested seed sludges are good sources

Seed sludge

for continuous thermophilic hydrogen production from cassava stillage. The influent

Alkalinity

alkalinity of 6 g NaHCO3/L and HRT 24 h were optimal for hydrogen production with

Hydraulic retention time (HRT)

hydrogen yield of 76 ml H2/gVS and hydrogen production rate of 3215 ml H2/L/d. Butyrate was the predominant metabolite in all experiments. With the increase in alkalinity of more than 6 g/L, the concentration of VFA/ethanol increased while hydrogen yield decreased due to the higher concentration of acetate and propionate. The decrease in HRT resulted in the higher hydrogen production rate but lower hydrogen yield. Variation of hydrogen yields were quite correlated with butyrate/acetate (B/A) ratio with different influent alkalinities, however, butyrate was important parameter to justify the hydrogen yields with various HRTs. ª 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.

1.

Introduction

The use of cassava in the production of bioethanol has recently gained worldwide attention, especially in Asia, due to the increasing demands for renewable sources [1,2]. During fermentation and distillation processes, however, a large amount of stillage (i.e. cassava ethanol wastewater) is generated containing high levels of organic pollutants and suspended solids (SS) [3]. It has been estimated that a single mid-sized ethanol facility generated stillage pollution levels similar to those found in the sewage of cities populated by

500 000 people [4]. The produced cassava stillage is perceived as one of the serious pollution problems with high strength of organics. Recent advances on dark fermentative hydrogen production provide a promising way to treat cassava stillage. The dark fermentative hydrogen production process is simple and energy-saving compared with traditional hydrogen production process. Previous studies also have reported hydrogen production via dark fermentation from various wastes, such as palm oil mill effluent [5] and household solid waste [6], food wastes [7,8], and cheese

* Corresponding author. Tel.: þ86 21 65982692; fax: þ86 21 65986313. E-mail address: [email protected] (L. Xie). 0360-3199/$ e see front matter ª 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2010.03.126

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whey wastewater [9]. Anaerobic sludge, sludge compost, sewage sludge and soil have been widely used as inoculums for the fermentative hydrogen production process since they contain various bacteria and hydrogen-producing bacteria can be obtained after appropriate enrichment [10]. However, inoculum sources were observed to have significant effects on the hydrogen yield and metabolic distribution [11e16]. Heat pretreated sewage sludge was shown to have the highest hydrogen yield from xylose than other heat pretreated biological sludge collected from distillery, food and paper mill wastewater treatment plants [14]. Akutsu et al. [12] compared eight different heat pretreated inocula for hydrogen production from starch, and the highest hydrogen yield was obtained by mesophilic digested activated sludge. In addition, two different fermentation patterns (butyratetype and ethanol-type fermentation) corresponding to different inoculums were observed. It is noteworthy that all the above results were obtained from batch experiments. Though batch experiment is easily conducted compared with continuous experiment, whether the result reflects the same case in continuous operation still needs further confirmation. Until now, only Akutsu et al. [17] reported the effects of seed sludges on continuous hydrogen production from starch. They found thermophilic digested activated sludge was the most efficient seed sludge with highest hydrogen yield than the other three kinds of thermophilic sludge and one compost. It is obvious that thermophilic sludge is so available in practice because most anaerobic reactors were operated under mesophilic condition. Moreover, most of the above studies focused on the pure substrates and the results may be not suitable for actual wastewater containing complex components. Therefore, more easily obtained sludge should be evaluated for their hydrogen production capacity from actual wastewater. We previously reported that mesophilic anaerobic sludge without any pretreatment was successfully used as seed sludge for thermophilic hydrogen production from cassava stillage and whether the result is applicable for other sludge under such condition is still unknown [18]. Besides, influent alkalinity influences the fermentation pH which further affects process efficiency in hydrogen production, distribution of metabolic products [19,20]. Increase in the alkalinity of cassava stillage was observed to lead to the increase of hydrogen production in our previous study [18]. To what extent alkalinity affects hydrogen production from cassava stillage has not been systemically investigated. To optimize the hydrogen production process, HRT is also an important parameter. With appropriate HRT, efficient hydrogen production could be achieved which will make the hydrogen production process more applicable [21,22]. In light of the above considerations, we evaluated five easily obtained inocula without any pretreatment (i.e. three kinds of mesophilic anaerobic sludge, one waste activity sludge and one thermophilic anaerobic sludge) for thermophilic hydrogen production from cassava stillage. Both batch and continuous experiments were conducted to see whether the results are consistent. After that, the alkalinity of the cassava stillage and HRT of the reactor were also studied by the efficient seed sludge to investigate their effects on hydrogen production and optimize the whole hydrogen production process.

2.

Material and methods

2.1.

Seed inocula

Five inocula from different sources were used for hydrogen production: Mesophilic anaerobic sludge (MAS) obtained from UASB treating cassava stillage, pH 7.5, TS 70 g/L, VS 42 g/L; Thermophilic digested cassava stillage (TDC) from ASBR, pH 7.3, TS 32 g/L, VS 19.8 g/L; Mesophilic digested cow manure (MDCO) from CSTR, pH 7.2, TS 25 g/L, VS 14.5 g/L; Mesophilic digested chicken manure (MDCH) from CSTR, pH 7.8, TS 28 g/L, VS 16 g/L; Waste activated sludge (WAS) from municipal wastewater treatment plant, pH 6.9, TS 14.8 g/L, VS 11.5 g/L. All the inocula were inoculated without any pretreatment.

2.2.

Feed stock

Cassava stillage used in this study was got directly from cassava ethanol plant (Jiangsu, China). The main characteristics of cassava stillage were as follows: pH 4.1, total solids (TS) 49.7 g/L, volatile solids (VS) 42.3 g/L, total carbohydrate 29.2 g/L, soluble carbohydrate 6.9 g/L, and total chemical oxygen demand (TCOD) 60 g/L. After collection, the cassava stillage was stored at 4  C before usage.

2.3.

Experimental design and procedure

Batch experiments were firstly conducted to compare the effects of inocula for hydrogen production from cassava stillage. Five identical serum bottles were used as the reactors with working volume of 200 ml. 140 ml of cassava stillage was mixed with certain inoculum and distilled water to make the final volume 200 ml and inoculum VS concentration 10 g/L. Some of the inoculums were condensed by centrifugation before inoculation due to their low VS concentration. The initial pH value of the mixed solution in each bottle was adjusted to 6 by 2 N NaOH or 2 N HCl. Nitrogen gas was purged into bottles for 5 min to provide anaerobic condition. The capped bottles with rubber stoppers were placed in a reciprocating water bath shaker and rotated at 150 rpm with the preset temperature of 60  C. The evolved biogas was collected by gas bag. The amount of biogas was determined periodically using syringe and at the same time the composition of the biogas was measured. The above experiments were carried out in triplicate. After the hydrogen production ceased in the batch experiments, the reactors were switched to semi-continuous mode and operated as continuously stirred tank reactor (CSTR). Equal amount of digested and fresh cassava stillage were removed and added to the reactor periodically and the HRT was maintained for 72 h NaHCO3 was used to increase the alkalinity of cassava stillage and the concentration was 6 g/L. The analysis of the microbial community was also conducted when steady-state was achieved in all the experiments. After optimal seed sludge was found, the effects of alkalinity addition to cassava stillage (4 g/L, 6 g/L, 8 g/L and 10 g/L) and HRT (16 h, 24 h, 48 h, 72 h) on the hydrogen and VFA/ethanol production were investigated to optimize the fermentation process. In this study, all data were obtained at a steady-state

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of each operation condition. Steady-state was defined by a sustained biogas production within 10% deviation, and in this period, parameters including pH, volatile fatty acids (VFA), and hydrogen production were determined for three consecutive days, creating six replicate data points for every measured parameter for the experimental condition under examination.

2.4.

Analytical methods

The collected samples were centrifuged at 3500 rpm for 10 min, and filtrated through 0.45 mm filters to determine soluble components. TS, VS and TCOD were analyzed in duplicate in accordance with Standard APHA Methods [23]. Total and soluble carbohydrates were determined using phenolesulfuric acid method [24]. The concentrations of ethanol and VFA (C2eC5) were determined by gas chromatograph (HP6890II, USA) equipped with a flame ionization detector and analytical column CPWAX52CB (30 m  0.25 mm  0.25 mm). The biogas composition was determined using a gas chromatograph (Shimadzu GC-14B, Japan) equipped with thermal conductivity detector and a stainless steel column packed with Carbosieve S II (Ø 3.2 mm  2 m). The detailed information about detection of VFA/ethanol and biogas composition were described in our previous publication [18]. Sludge samples for microbial community analysis were centrifuged (12 000  g, 4  C, 5 min) and the solids were separated. DNA was extracted according to the method described in [25]. 16S rDNA fragments were amplified by polymerase chain reaction (PCR) with the primers GC-357F and 517R and the amplification was preformed according to Muyzer et al.’s report [26]. Denaturing gradient gel electrophoresis (DGGE) was performed using the Bio-Rad D gene system (Bio-Rad, Hercules, USA). Polyacrylamide gels were made with denaturing gradients ranging 35e60%, for the bacterial community. Conditions for electrophoresis operation were also selected according to the publication of [24]. The dominant bands were excised from the gel and then were immersed in 50 mL of sterilized water. DNA was recovered from the gels by freezeethawing more than three times. The recovered DNA was then reamplified by PCR and the sequencing of the PCR products was finished by the Bio Asia Company Shanghai Ltd.

3.

Results and discussion

3.1. Thermophilic hydrogen production with different seed sludges 3.1.1.

Batch experiments

Impacts of five different inocula were firstly evaluated on thermophilic hydrogen production from cassava stillage in batch experiments. Hydrogen was successfully obtained in all experiments. The cumulative hydrogen production was varied with different inoculums as shown in Fig. 1. The highest cumulative hydrogen production was obtained by MAS (504 ml), while the other four sludge gave relatively low hydrogen production with the sequence of TDC (494 ml), WAS (470 ml), MDCH (335 ml), and MDCO (302 ml). The

Cumulative hydrogen production (ml)

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600 500 400 300 200 100 0 TDC

MAS

MDCO

MDCH

WAS

Fig. 1 e Cumulative hydrogen production of different seed sludges in batch experiments. TDC, Thermophilic digested cassava stillage; MDCO, Mesophilic digested cow manure; MDCH, Mesophilic digested chicken manure; WAS, Waste activated sludge; and MAS, Mesophilic anaerobic sludge.

corresponding hydrogen yields for all seed sludges were calculated and listed in Table 1. The hydrogen yields were significantly different in the batch experiments by the analysis of variation (ANOVA, p < 0.05). Such differences were expected because most batch studies have shown that different seed sludges exhibited different hydrogen-producing bioactivities [15,16]. Therefore, it seems necessary to find a suitable inoculum for efficient hydrogen production. However, whether MAS was still the best inoculum after long-term accumulation in continuous experiment was doubt since stable microbial community could not be established only after one batch cultivation. For example, Baghchehsaraee et al. [27] found that hydrogen production fluctuated a lot during five repeated batches by activated sludge and the microbial community was changed in different batches. Therefore, further evaluation on the hydrogen production from different seed sludges in continuous experiments was necessary. On the other hand, methane was only detected with little amount (less than 1% of total biogas) in the experiments seeded by MAS and TDC, which demonstrated that pretreatment of the inocula was unnecessary under thermophilic condition to inhibit methanogens for hydrogen production from cassava stillage. Similar results were obtained by Morimoto et al. [15]. In their study, three different kinds of untreated sludges (palm oil mill effluent sludge and two kinds of sludge composts) were used for batch hydrogen production under thermophilic condition and no methane was observed. They thought both lower pH and thermophilic condition were the reasons for the inhibition of methanogens. However, in our study, thermophilic anaerobic sludge (TDC) was studied and the methane was still negligible, indicating that thermophilic condition may not be the main reason for the inhibition of methanogens. The results should be more attributable to the pH decrease from initial 6 to the final 4.76e5.27 (Table 1) since the acidic pH condition would lead to the inhibition of methane production [28]. The amount and component of VFA/ethanol reflect the metabolism of hydrogen-producing anaerobes in different experiments. As presented in Table 1, the highest concentration of VFA/ethanol (7170 mg/L) was obtained by MAS that was consistent with the highest hydrogen production. The

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Table 1 e Hydrogen production performances by different seed sludges. TDC

MAS

MDCO

MDCH

WAS

Batch experiment Hydrogen yield (ml H2/gVS) pH VFA/ethanol (mg/L)

83.5  3.8 4.80  0.01 4550  254

85.2  4.2 5.27  0.02 7170  452

50.9  2.5 4.95  0.01 4130  321

56.6  2.1 4.88  0.01 3725  172

79.5  3.5 4.76  0.02 4320  331

Continuous experiment Hydrogen yield (ml H2/gVS) pH VFA/ethanol (mg/L)

87.9  3.3 5.49  0.03 10 084  735

82.9  4.3 5.49  0.03 10 481  399

92.0  2.9 5.52  0.01 9930  451

84.0  3.7 5.51  0.01 10 647  644

92.3  6.7 5.46  0.01 10 574  239

amounts of VFA/ethanol obtained from TDC and WAS were only 4550 mg/L and 4320 mg/L which were 36% and 40% lower than MAS. However, the hydrogen yields were similar with that of MAS, which may be attributable to the distribution of VFA/ethanol (Fig. 2). Though more butyrate was produced from MAS, the acetate and propionate concentrations were also higher (Fig. 2), which may lead to the similar hydrogen yield. Previous study demonstrated that the production of acetate and propionate could consume hydrogen by the following reactions [29e31]:

2CO2 þ 4H2 / CH3COOH þ 2H2O

(1)

C6H12O6 þ 2H2 / 2CH3CH2COOH þ 2H2O

(2)

6000 Ethanol Acetate Propionate Butyrate

Concentration (mg/L)

5000 4000 3000 2000 1000

Continuous experiments

Fig. 3 presents the variations of hydrogen production with operation time in continuous experiments. The hydrogen production in the first several days fluctuated, however, stable hydrogen production was obtained only after 6 days operation which showed that hydrogen-producing reactor could be easily started up. In the whole continuous operation processes, no methane was detected, indicating that the initial batch operations of the reactors were necessary to suppress the activity of methanogens and stimulate the activity of hydrogen-producing bacteria. The hydrogen production performances in the steady-state were summarized and listed in Table 1. Though the highest hydrogen yield (92.3 ml/gVS) was obtained by WAS, the differences in hydrogen yields were not significant by different inocula (ANOVA, p > 0.05), which showed that all the tested sludges could be used as suitable seed sludge for hydrogen production from cassava stillage. It is deserved to notice that MDCO and MDCH gave relatively poor hydrogen yields in batch experiments but the hydrogen yields increased to the similar level as other inoculums in the continuous experiments. It could be attributed to the long-term accumulation of hydrogenproducing bacteria. During repeated batch cultivation, Yokoyama et al.[32] also found that the hydrogen production from glucose increased from about 600 ml/L substrate in the first batch to 1000 ml/L substrate in the seventh batch. The total and individual amounts of VFA/ethanol in the steady-state in all experiments were similar as shown in Table 1 and Fig. 4. The total amount of VFA/ethanol was as high as 10 000 mg/L and butyrate was still the dominant species

Hydrogen production rate (ml H /L/d)

In all experiments, butyrate was predominant and accounted for more than 75% of the total VFA/ethanol, indicating that butyrate-fermentation type was obtained regardless of the seed sludges. The results were different from previous studies [11,12]. Ethanol and butyrate-fermentation types were observed with different seed sludges under mesophilic conditions. We suspected that the difference was resulted from the substrate because the above studies used pure substrates (starch or L-Arabinose) while cassava stillage was a kind of complex waste containing various substrates and nutrients that may be favorable for butyrate production. The fermentation temperature may also lead to the difference since the present study was conducted under thermophilic condition.

3.1.2.

3000 TDC MAS MDCO MDCH WAS

2000

1000

0 0

2

4

6

8

10

12

14

Time (d)

0 TDC

MAS

MDCO

MDCH

WAS

Fig. 2 e VFA/ethanol distribution of different seed sludges in batch experiments.

Fig. 3 e Variations of hydrogen production rate with operation time in continuous experiments with different seed sludges.

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Concentration (mg/L)

10000

Ethanol

Acetate

Propionate

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Butyrate

8000 6000 4000 2000 0 TDC

MAS

MDCO

MDCH

WAS

Fig. 4 e VFA/ethanol distribution of different seed sludges in continuous experiments.

accounted for more than 80% of the total VFA/ethanol. The result was expected because butyrate-fermentation type was found in batch experiment and the production of VFA/ethanol and butyrate was enhanced after continuous operation. Propionate concentration of about 350 mg/L was detected during continuous operation, however it was not detected in batch experiments seeded with TAC, MDCH and WAS. The final fermentation pH in batch experiments by TAC, MDCH and WAS was between 4.7 and 4.9 which was lower than the effluent pH of about 5.5 in continuous experiment. The increased fermentation pH was reported to be favorable for propionate production [33]. The similar distribution and amount of VFA/ethanol indicated similar metabolic pathway of bacteria by various inocula. Sludge samples taken from each reactor at steady-state, as well as two raw seed sludges (rMAS and rWAS) representing anaerobic and aerobic sludges, were analyzed by PCReDGGE (Fig. 5). Each band on DGGE profile corresponded to a gene fragment of unique 16S rDNA sequences and thus represented a specific species in the microbial community. The staining intensity of a band indicated the relative abundance of the corresponding microbial species [34]. Numerous bands in lanes of rMAS and rWAS indicated diversity of microbial communities in inocula. Based on migration distance, intensities and similarity between the lanes on DGGE gel, the banding patterns of sludge samples after continuous cultivation, which were taken from steady-state, showed great differences with that of seed sludges. For example, band 1 was dominant in all the reactors, but it was not obvious in seed sludges of rMAS and rWAS. Mixed inocula from different sources were good for hydrogen production since they contained various bacteria, therefore, the hydrogen-producing bacteria could be selectively enriched after cultivation. The BLAST analysis of the gene sequence from band 1 showed 99% identity to Uncultured Thermoanaerobacteriaceae bacterium (accession number GU372417) and members of Thermoanaerobacteriaceae have been found as the key player in thermophilic hydrogen production by some other studies [5,35,36]. Our results from PCReDGGE and sequencing analysis demonstrated that seed sludge was not important parameter that affected thermophilic hydrogen production from cassava stillage because Uncultured Thermoanaerobacteriaceae bacterium-dominant microbial communities were established in all the reactors after continuous cultivation.

Fig. 5 e Microbial community of seed sludges before and after continuous cultivation.

The results in our study were different from the study of Akutsu et al. [17] who observed significant differences in hydrogen production from starch by continuous thermophilic fermentation process with different seed sludges. Such difference may be resulted from the different substrate because it has been proven that the effects of seed sludge on hydrogen production were associated with the fermentation substrate. For example, the effects of the seed sludge on the hydrogen production potentials were significant in the case of the starch medium, but insignificant with the glycerol medium as substrate [8]. Moreover, the organic loading rate (OLR) in our study was about 20 gCOD/(L d), which was much higher than the value of about 11.8 gCOD/(L d) in Akutsu et al.’s [12] study. The change of microbial community with the variation of OLR has been reported by some studies [35,36]. The higher OLR in our study selectively enrich similar Uncultured Thermoanaerobacteriaceae bacterium-dominant microbial communities from seed sludges containing various bacteria which resulted in the similar hydrogen production performances.

3.2. Effects of influent alkalinity on the hydrogen production The alkalinity of cassava stillage is a crucial parameter which could lead to different fermentation pH and further result in the variation of hydrogen production performance. Since

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Alkalinity (g/L) 4 5.14 6 5.49 8 5.69 10 6.07

 0.02  0.03  0.02  0.03

8806  525 10 411  318 12 816  498 14 484  699

9.5 13.7 6.3 4.0

HRT (h) 16 24 48 72

 0.01  0.01  0.01  0.03

6630  518 10 272  533 10 430  606 10 411  318

11.5 10.6 12.7 13.7

5.68 5.62 5.5 5.49

1200

60

900

40

600

20

300

0

0

VFA/Ethanol (mg/L)

8000 6000 4000 2000 0 4

6 8 Alkalinity (g/L)

10

Fig. 6 e Hydrogen production and VFA/ethanol distribution at different alkalinities.

than 5.0 is suitable for propionate production. In our study, the fermentation pH was between 5.0 and 6.1 and butyrate was always the predominant product while propionate was lower compared with the other products. The differences in the distribution of VFA/ethanol were probably due to the fermentation temperature. Both of the above studies were conducted under mesophilic condition. In another study, we also compared the hydrogen production under both thermophilic and mesophilic conditions and found that propionate was much higher in mesophilic condition (data not shown). It

Hydrogen yield (ml H /gVS)

100

4000

80

3000

60 2000 40 1000

20 0

0

8000 VFA/Ethanol (mg/L)

B/A

80

Hydrogen production rate (ml H /L/d)

Total VFA/ethanol (mg/L)

1500

10000

Table 2 e Summary of effluent characters under different alkalinities and HRTs. pH

100

Hydrogen production rate (mlH /L/d)

there were no significant differences in hydrogen production performance with different seed sludges, the reactor seeded by MAS was selected to study the effects of influent alkalinity. As shown in Table 2, the fermentation pH increased from 5.14 to 6.07 when influent alkalinity increased from 4 g/L to 10 g/L. The highest hydrogen yield and hydrogen production rate were obtained at influent alkalinity 6 g/L, and the corresponded fermentation pH was about 5.5. Unlike the variation of hydrogen production, higher alkalinity had no inhibition to the production of VFA/ethanol and the total amount of VFA/ ethanol increased with the increase of initial pH from about 9000 mg/L to 15 000 mg/L (Fig. 6, Table 2). Butyrate-type fermentation was observed under all the operation conditions. Though the concentrations of total VFA/ethanol and butyrate were higher under alkalinity of 8 g/L and 10 g/L than alkalinity of 6 g/L, the hydrogen yields were relatively lower which may attributed to the production of more acetate and propionate under both conditions that can consume more hydrogen. Highest hydrogen yield at alkalinity 6 g/L accompanied with highest butyrate/acetate (B/A) ratio (Table 2) and similar results were also found in other studies revealing that higher B/A ratio corresponds to higher hydrogen yield [37]. It is obvious that pH around 5.5 was suitable for hydrogen production from cassava stillage under thermophilic condition, which was in agreement with several studies about the influence of pH on hydrogen production [38,39]. However, the amount and distribution of VFA/ethanol were much different from previous studies. The highest hydrogen yield was corresponded with the highest butyrate/acetate (B/A) ratio 13.7 in our study. The result is also different from studies on hydrogen production under thermophilic condition. The highest hydrogen production was obtained at pH 5.1 with B/A 1.98 when converting starch to hydrogen in UASB in Akutsu et al.’s study [40]. Even much lower B/A value of 0.8 was observed in anaerobic sequencing batch reactor for hydrogen production from palm oil mill effluent with the hydrogen yield of 2.24 mol H2/mol hexose [5]. The much higher B/A in our study is most probably due to the substrates used in our study because different seed sludges still exhibited similar distribution of VFA/ethanol. Moreover, Li et al. [33] reported that by controlling the fermentation pH at 4.2, 4.6 and 5.0, ethanoltype, butyrate-type and propionate-type fermentation were obtained. Hwang et al. [41] also demonstrated that pH higher

Hydrogen yield (mlH /gVS)

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6000 4000 2000 0 10

20

30

40

50

60

70

80

HRT (h)

Fig. 7 e Hydrogen production and VFA/ethanol distribution at different HRTs.

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Table 3 e Comparison of hydrogen yields with literatures. Substrate Cassava stillage Kitchen waste Household solid waste (HSW) Starch Glucose Glucose

Reactor CSTR Rotating drum CSTR CSTR CSTR MBR

Temperature 60 40 70 55 35 35



C C  C  C  C  C 

is obvious that thermophilic condition was suitable for hydrogen production considering its higher butyrate and lower propionate production even under higher fermentation pH (5e6) than mesophilic condition (lower than 5).

HRT

pH

Hydrogen yield

24 240 72 24 12 9

5.62 5.8 4.7 5.1 5.3 5.5

76 ml H2/gVS or 0.89 mol H2/mol hexose 71 ml H2/gVS 78.8 ml H2/gVS 2.82 mol H2/mol hexose 0.92 mol H2/mol hexose 0.86 mol H2/mol hexose

h h h h h h

This study [38] [5] [40] [33] [41]

hydrogen yields only under different influent alkalinities while butyrate itself could decide hydrogen yields under different HRTs with the same influent.

3.4. 3.3.

References

Comparison of hydrogen yields with other studies

Effects of HRT on the hydrogen production

Under optimal alkalinity 6 g/L, HRT was further investigated to optimize the fermentation process. As shown in Fig. 7, the hydrogen yield decreased from 83 ml H2/gVS to 56.7 ml H2/ gVS with the decrease of HRT from 72 h to 16 h. It is deserved to notice that the hydrogen yield decreased about 20% when HRT decreased from 24 h to 16 h while only little decrease was found when HRT reduced to 48 h and 24 h. Our result demonstrated that hydrogen yield was positively related with HRT. This was possible considering that more substrate was solubilized and consumed under the longer HRT. Wang and Zhao [42] also got similar result that the hydrogen yield decreased from 71 ml H2/gVS to 49 ml H2/gVS with the decrease of HRT from 240 h to 96 h. Contrary with the variation of hydrogen yield, the hydrogen production rate increased from 1170 ml H2/L/d to 3600 ml H2/L/d with decrease of HRT from 72 h to 16 h. The increased hydrogen production rate was resulted from the higher loading rate under lower HRT. The higher hydrogen yield meant more energy was recovered from wastes while higher hydrogen production rate meant higher treatment efficiency which could reduce the volume of the reactor. But it is difficult to achieve the above two targets simultaneously. Considering the sudden decrease of hydrogen yield from HRT 24 h to 16 h, HRT 24 h was chosen as the optimal condition for hydrogen production from cassava stillage. The distribution of VFA/ethanol was not significantly affected by the variation of HRT. Butyrate was always dominant under all tested HRTs (Fig. 7b). Unlike alkalinity experiment, where changes of B/A indicated changes of hydrogen yields, butyrate itself was associated with the hydrogen yields in the HRT experiment while B/A ratio did not change significantly (Table 2). The results were different from Arooj et al. [37] who found that B/A ratio was the most important parameter to explain the performance of hydrogen production in CSTR from starch under different HRTs. However, butyrate/ propionate instead of B/A was proposed to be the governing factor in hydrogen-producing ASBR from starch in his another study [43]. It seemed that whether B/A could be a crucial parameter was determined by rector configuration. However, in this study, we further found that different substrates and cultivation conditions also could lead to different results. When using cassava stillage as substrate, B/A reflected

The optimal condition for hydrogen production from cassava stillage was influent alkalinity 6 g/L and HRT 24 h with the hydrogen yield 76 ml H2/gVS. The hydrogen yield was comparable with complex organic wastes (kitchen waste and household solid waste) as shown in Table 3. But the hydrogen yield in our study was much lower than 2.82 mol H2/mol hexose obtained from starch under thermophilic condition [44]. This could be attributed to the much lower soluble carbohydrate (6.9 g/L) than total carbohydrate (29.2 g/L) in cassava stillage as described previously. Most of the carbohydrate existed in the suspended solid which could not be utilized directly by hydrogen-producing bacteria. On the other hand, the lower hydrogen yield may possibly be due to the lack of microbial diversity that established under thermophilic condition in our study since only one dominant band was detected. However, the hydrogen yield was still comparable with those from pure glucose under mesophilic conditions [37,45] that demonstrated the priority of thermophilic fermentation.

4.

Conclusions

The present study showed that though there were significant differences in thermophilic hydrogen production from cassava stillage by different seed sludges in batch experiments, similar hydrogen production performances were obtained in continuous experiments when steady-states were achieved. Therefore, all the tested sludges could be used as suitable inoculum for thermophilic hydrogen production from cassava stillage and continuous experiment is more reliable than batch experiment to evaluate the effects of seed sludge on hydrogen production. The alkalinity of cassava stillage was an important parameter since it was associated with the fermentation pH and further influenced the hydrogen production and VFA/ethanol distribution. The alkalinity higher than 6 g/L led to the higher production of VFA/ethanol and butyrate but lower hydrogen production due to the hydrogen consumption with the increased concentrations of acetate and propionate. B/A ratio could be used to justify the trend of hydrogen yields under different alkalinities. The HRT had opposite effects on hydrogen yield and hydrogen production rate. The butyrate instead of B/A showed close correlation with hydrogen yields under different HRTs.

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Acknowledgements This research was financially supported by the Foundation of Key Laboratory of Yangtze River Water Environment, Ministry of Education (Tongji University), China (No. YRWEY1003), the Bayer Sustainable Development Foundation and Ministry of Science and Technology (2008DFA91000). The authors wish to thank the Taicang cassava ethanol plant for the raw cassava stillage and valuable practical experience.

references

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