Adding Fe0 powder to enhance the anaerobic conversion of propionate to acetate

Biochemical Engineering Journal 73 (2013) 80–85

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Adding Fe0 powder to enhance the anaerobic conversion of propionate to acetate Xusheng Meng, Yaobin Zhang ∗ , Qi Li, Xie Quan Key Laboratory of Industrial Ecology and Environmental Engineering (MOE), School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024, PR China

a r t i c l e

i n f o

Article history: Received 3 September 2012 Received in revised form 31 January 2013 Accepted 3 February 2013 Available online 14 February 2013 Keywords: Propionate Fe0 Acetate Acetogenesis Homoacetogenesis Anaerobic

a b s t r a c t Propionate is an unfavorable substrate for the anaerobic digestion because it is thermodynamically difficult to be decomposed into acetate. An attempt to enhance the decomposition of propionate by adding Fe0 powder (10 g) into an acidogenic reactor (A1) with propionate as the sole carbon source was made in this study. The results showed that the propionate conversion rate (67–89%) in A1 were higher than that in a reference reactor (43–77%) without dosing of Fe0 (A2). The enhanced conversion of propionate caused both chemical oxygen demand removal (COD) (57–79%) and acetate production (178–328 mg/L) in A1 to increase significantly. Although Fe0 contributed the H2 production chemically, the H2 content of A1 was less than that of A2. The reason was ascribed to the enhanced utilization of H2 for the homoacetogenesis. It was calculated that the Gibbs free energy in the decomposition of propionate was decreased by about 8.0–10.2% with the dosing of Fe0 . Also, the activities of enzymes related to the acetogenesis were enhanced by 2–34-folds. Fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) analysis indicated that Fe0 increased the abundance of microbial communities, especially propionate-utilizing bacteria and homoacetogenic bacteria. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Propionate is a main intermediate in the stage of hydrolysis/fermentation, the degradation of which is thermodynamically unfavorable [1,2]. According to the calculation on the Gibbs free energy, the conversion of propionate to acetate does not occur until the partial pressure of hydrogen decreases below an extremely low level (<10 Pa) [1,3], but the partial pressure of hydrogen in practice usually exceeds this range. As a result, the accumulation of propionate is often observed in the anaerobic digesters, which may destroy the pH balance between acidogenesis and methanogenesis, further deteriorating the digestion [4,5]. Thus, the propionate production and/or propionic-type fermentation occurring in the acidogenesis stage should be reduced during the anaerobic digestion. Anaerobic digestion includes three fermentation types: propionic-type, butyric-type and acetic-type [3,6]. A way to relieve the accumulation of propionate is to reduce the propionictype fermentation. The fermentation type is closely related to oxidative–reductive potential (ORP) [7–9]. It is believed that the propionic-type fermentation is a facultative anaerobic process,

∗ Corresponding author. Tel.: +86 411 8470 6460; fax: +86 411 8470 6263. E-mail addresses: [email protected], [email protected] (Y. Zhang). 1369-703X/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2013.02.004

occurring at ORP higher than −278 mV [7]. Wang et al. [8] reported that the fermentation was shifted from the propionic-type to the butyric-type and acetic-type under lower ORP level. In our previous study [5], Fe0 powder was added into an acidogenic reactor to decrease ORP and reduce the propionate accumulation, and the results showed that the concentration of propionate dropped from 416 to 225 mg/L and the concentration of acetate increased from 222 to 408 mg/L. It suggested that more organics was not necessary to undergo the propionate stage and could directly be hydrolyzed and fermentated into acetate due to the addition of Fe0 . Alternatively, the accumulation of propionate could probably be reduced by accelerating the decomposition of propionate. It was assumed that Fe0 was likely to enhance the conversion of propionate to acetate because Fe0 could serve as an electron donor in the microbial metabolism [10] and promote a number of key enzymes activities in the acetogenesis process [5,11]. For example, pyruvate–ferredoxin oxidoreductase (POR) that contains Fe S clusters is a crucial enzyme to catalyze the decomposition of propionate, which meant that appropriate addition of Fe0 was likely to enhance its activity [11]. So far the effects of Fe0 on the conversion of propionate to acetate remained unknown. Thus, Fe0 powder was dosed in an acidogenic reactor with propionate as the sole carbon source to attempt to accelerate the decomposition of propionate and reduce its accumulation in this study.

X. Meng et al. / Biochemical Engineering Journal 73 (2013) 80–85

2. Materials and methods

2500

2.1. Experimental reactors

2000

2.2. Sludge and wastewater The seed sludge obtained from the sedimentation tank of a municipal sewage plant in Dalian (China). After removing large debris, the volatile suspended sludge (VSS) was 3.1 g/L and total suspended sludge (TSS) was 12.9 g/L. Each reactor was inoculated with the seed sludge of 1 L. The acidogenic reactors were fed with an artificial wastewater with a fixed COD of 3500 mg/L. Sodium propionate, NH4 Cl and KH2 PO4 were added as the carbon, nitrogen and phosphorus sources, respectively, to give a COD:N:P ratio of 200:5:1. The trace elements were added according to Liu et al. [5]. The pH of influent wastewater was adjusted to 6.0 by 1:1 HCl solution. 2.3. Analysis methods TSS, VSS and COD were conducted in accordance with Standard Methods for the Examination of Water and Wastewater [13]. The pH was recorded using a pH analyzer (Sartorius PB-20, Germany). The concentration of Fe2+ was determined by ortho phenanthroline spectrophotometry at 510 nm (UV-721, Techcomp, China). The composition of biogas was analyzed by a gas chromatograph (GC14C/TCD, Shimadzu, Japan) and the concentrations of acetate and propionate were determined by another gas chromatograph (GC2010/FID, Shimadzu, Japan) [14]. The dehydrogenase activity was analyzed by the method of Nybroe et al. [15]. The activity of POR was determined according to the reported method [16]. The activities of acetate kinase (AK) and phosphotransacetylase (PTA) were assayed with the method of Yan et al. [17]. FISH was used to determine the abundance of microbes in the acidogenic reactors. FISH was conducted according to the method of Wu et al. [18]. Fluorescence labels of the oligonucleotide probes used in this study included a Cy3-labeled Wol223 and MPOB222 (propionic-utilizing bacteria, red), and a FITC-labeled AW (homoacetogenic bacteria, green). 4 ,6 -Diamidino-2-phenylindole (DAPI) was used for characterization of the entire bacterium (blue). The samples were observed under a confocal laser scanning microscope (FV1000, Olympus, Japan). The obtained FISH images were imported to Image-Pro Plus 6.0 for analyzing of the relative abundance of microorganisms. The genomic DNA of the sample was extracted using an extraction kit (Bioteke Corporation, Beijing, China) according to the manufacturer’s instructions. A primer combined GM341F with DS907R was used to selectively amplify the 16S rDNA sequences of eubacteria. A 40-nucleotide GC clamp was added to the forward primer at the 5 -end to improve the detection of sequence variation in amplified DNA fragments by subsequent DGGE. The polymerase chain reaction (PCR) products obtained were applied to DGGE analysis, which was conducted by the Dcode system (Bio-Rad). The concrete steps of DGGE analysis for bacteria were conducted

A1 A2

COD(mg/L)

Fe0 powder (10 g, purity >98%, 0.2 mm in diameter, a BET surface area of 0.05 m2 /g) was added into A1 with a working volume of 2 L (ϕ 100 mm × 280 mm) (Fig. S1). The control reactor A2 was the same as A1 but without dosing Fe0 powder. The hydraulic retention times (HRTs) of A1 and A2 were decreased stepwise from 6 to 4 h then to 2 h and finally recovered to 4 h again. During the experiment at the HRT of 6 h, 2-bromoethanesulfonate (BES, 40 mM), a specifically methanogenic inhibitor [12], was added into A1 and A2 every three days. The acidogenic reactors were operated in an upflow mode at 35 ◦ C.

81

1500 HRT=6h

HRT=4h

HRT=2h

HRT=4h

1000

500

0 0

10

20

30

40

50 60 time/day

70

80

90

100

Fig. 1. Effluent COD from the acidogenic reactors under different HRTs. Each reactor was sampled three times in a given day for determination, namely, each point in the figure was the average value from three samples. Error bars represent standard deviations of triplicate tests.

according to the illustration by Zhang et al. [19]. Some dominant DGGE bands were excised and reamplified by PCR using the primers described above without the GC clamp. The PCR products were then sequenced by TaKaRa Biotechnology Co., Ltd. (Dalian, China). The obtained gene sequences were compared with the reference microorganisms available in the GenBank by BLAST search. The sequences were aligned using ClustalX program and the phylogenetic tree was constructed by a distance method (neighbor-joining) using Mega software [20]. 2.4. Data analysis The Gibbs free energies (G) of propionate conversion and homoacetogenesis were calculated according to the Van’t Hoff equation as follows: G = G◦ + RT ln (products/reactants). The values of G◦ were calculated from the standard Gibbs energies of formation [21,22] using the equations under standard conditions (1 M, 105 Pa, 298.14 K) as follows: CH3 CH2 COO− (aq) + 2H2 O (l) = CH3 COO− (aq) + CO2 (g) + 3H2 (g)

G = +76.1 kJ/mol

(1)

4H2 (g) + 2CO2 (g) = CH3 COO− (aq) + H+ (aq) + 2H2 O (l) G = −55.1 kJ/mol

(2)

These two equations, namely acetification (or conversion of propionate) and homoacetogenesis, were the main processes in the acetogenesis stage, which could be combined into one equation (3): 4CH3 CH2 COO− (aq) + 2H2 O (l) + 2CO2 (g) = 7CH3 COO− (aq) + 3H+ (aq)

G = +139.1 kJ/mol

(3)

3. Results and discussion 3.1. Effluent COD under different HRTs It has been reported that the HRT was critical to successful operation of anaerobic system [23]. The acetogenesis was usually operated at a relatively low HRT to rush out methanogens for reducing their influence on acetogenesis [24]. To research the effects of Fe0 powder on the decomposition of propionate, A1 and A2 were operated at the HRTs ranging from 6 to 2 h, and then recovering to 4 h. The influent COD was fixed at about 3500 mg/L. As Fig. 1

X. Meng et al. / Biochemical Engineering Journal 73 (2013) 80–85

a

HRT (h)

6 4 2 0 a b

2+

pH

Fe

(mg/L)

A1

A2

A1

A2

7.58 ± 0.18 7.49 ± 0.22 6.66 ± 0.20 6.00

7.08 ± 0.22 6.81 ± 0.17 6.28 ± 0.14 6.00

28.99 21.08 15.65 ND

NDb ND ND ND

Hydraulic retention time. Undetectable.

shows, the effluent COD of A1 was always lower than that of A2, especially at the short HRT. At the HRT of 6 h, the effluent COD from A1 (750 mg/L) was less than that from A2 (1000 mg/L). While the HRT further went down to 2 h, the gap of effluent COD between A1 and A2 was expanded to about 800 mg/L. The higher COD removal of A1 was ascribed to the Fe0 dosed. The function of Fe0 could be only made by the reaction: Fe0 − 2e− = Fe2+ . Therefore, the Fe2+ leaching reflected the intensity of Fe0 function. From Table 1, the Fe2+ concentration dropped from 28.99 to 15.65 mg/L as the HRT decreased from 6 to 2 h, but at the same time the Fe2+ leaching rate increased from 4.8 to 7.8 mg/(L h). The variation of pH in Table 1 showed that the effluent acidity of the two reactors was gradually increased with the decrease of HRT. In other words, the acidity decreased with the reaction proceeded. On the contrary, according to Eq. (1), 1 mol of propionate produced 1 mol of acetate. Thus, with the reaction proceeded the acidity of effluent should be intensified due to the fact that the acidity of acetate is higher than that of propionate. The reason for the contradiction was ascribed that the part of acetate was further mineralized, which was confirmed from the CH4 percentage of biogas in Table 2. From the table, with ranging the HRT from 6 to 2 h, the CH4 percentage in the biogas changed from 46.02% to 17.84% for A1 and from 27.73% to 3.22% for A2. It could also be found that the methanogenesis was enhanced in A1 compared to A2, which made its pH less acidity (Table 1). There were many reports about the enhanced methanogenesis by Fe0 [25–27]. Xu et al. [26] found that the CH4 yield increased by 8.7% and the COD concentration decreased by 21.0% when Fe0 was added into the anaerobic sludge digester. Karri et al. [27] demonstrated that Fe0 was easily utilized by anaerobic mixed cultures to support the methanogenesis. It should be noted that the VFAs could not be utilized for methanogens until they were converted into acetate, formate, methyl-group containing compounds and CO2 /H2 [28]. The enhanced methanogenesis in A1 predicted that the conversion of propionate to acetate was accelerated in the presence of Fe0 . 3.2. Propionate conversion rate and acetate production under different HRTs The propionate conversion rate and acetate production in the effluent of A1 and A2 under different HRTs are shown in Fig. 2.

100

propionate conversion rate(%)

Table 1 Fe2+ concentration and pH value of the acidogenic reactors under different HRTs. Data are the averages of the values obtained during selected periods. Errors represent standard deviations of statistical analysis.

a

A1 A2

n=20

n=25

n=20

80

n=30

60

40

20

0

400

4

6

2

HRT

b

4

A1 A2

n=30

300

acetate(mg/L)

82

n=25 n=20

200

n=20

100

0 6

4

4

2

HRT (h) Fig. 2. Propionate conversion rate (a) and acetate production (b) of the acidogenic reactors under different HRTs. Data are the averages of the values obtained during selected periods. Error bars represent standard deviations of statistical analysis. n means measurement times.

The results indicated that the propionate conversion rate of A1 was always more than that of A2 under each HRT. It is interesting that the conversion rate of A1 (66.9%) at the HRT of 2 h was closed to that of A2 (61.5%) at the HRT of 4 h and the conversion rate of A1 (79.2%) at the HRT of 4 h approached to that of A2 (77.3%) at the HRT of 6 h. The results suggested that the propionate conversion was accelerated by about 30–50% with the dosing of Fe0 . When the HRT was recovered to 4 h, the results showed a good repeatability. At the stage of acetogenesis, propionate was converted to acetate. The accelerated propionate conversion implied that more acetate was produced. Accordingly, the acetate production of A1 was greater than that of A2 (Fig. 2b). But the acetate production of the two reactors gradually dropped with the increase of HRT. When the HRT raised from 2 to 4 h again, the acetate production of A1 reduced from 327.9 to 260.9 mg/L, and the acetate production of A2 decreased from 191.0 to 171.8 mg/L. The decrease of acetate production was because of the methanogenesis. To further

Table 2 Biogas composition of the acidogenic reactors under different HRTs. Data are the averages of the values obtained during selected periods. HRTa (h)

A1 (%) A2 (%) A1 with BES (%) A2 with BES (%) a b

6

4

2

H2

CO2

CH4

H2

CO2

CH4

H2

CO2

CH4

0.31 1.29 0.48 1.68

53.67 70.98 99.12 98.00

46.02 27.73 0.40 0.32

0.56 1.68 –b –

76.85 87.66 – –

22.59 10.56 – –

0.92 1.94 – –

81.24 94.84 – –

17.84 3.22 – –

Hydraulic retention time. No detect.

X. Meng et al. / Biochemical Engineering Journal 73 (2013) 80–85

83

0.45

1800

n=15

A1 A2

A1 with BES A2 with BES A1 A2

0.40

0.30

1200 U/mg-protein

concentration(mg/L)

0.35

600

0.25 0.20 0.15

n=5

n=15 0.10 0.05

0

acetate

n=5

n=5

n=5

PTA

AK

0.00

propionate

Dehydrogenase

POR

Fig. 3. Effluent acetate and propionate concentrations from the acidogenic reactors with BES at the HRT of 6 h. Data are the averages of the values obtained during selected periods. Error bars represent standard deviations of statistical analysis. n means measurement times.

Fig. 4. Activities of dehydrogenase, POR, PTA and AK in the acidogenic reactors. Data are the mean values obtained from the acidogenic reactors with and without BES. Error bars represent standard deviations of statistical analysis. n means measurement times.

investigate that the effects of Fe0 on the acetogenesis, it was necessary to eliminate the influences of methanogenesis on the acetate production.

homoacetogens do not out-compete methanogens due to their less favorable thermodynamic characteristics because the affinity of methanogens for H2 is 10–100 times higher than the affinity of homoacetogens [29]. However, when the methanogenesis was selectively inhibited, the H2 was prior to the utilization for the homoacetogenesis [12,30]. Consistently, Siriwongrungson et al. [31] also found that the homoacetogenesis instead of methanogenesis became the sink of hydrogen to accumulate acetate during anaerobic degradation of butyrate under suppressed methanogenesis.

3.3. Conversion of propionate to acetate with the inhibition of methanogenesis To further clarify the impacts of Fe0 dosed on the conversion of propionate to acetate, BES was added into A1 and A2 to inhibit the methanogenesis [12] and eliminate its influences on the conversion of propionate to acetate. To simplify the experiment, the experiment with BES was only conducted at the HRT of 6 h. With dosing of BES, the CH4 percentage in the biogas from the two reactors was only 0.32–0.4%, significantly lower than that of no-BES cases (e.g. 46.0% for A1 and 27.7% for A2 at the HRT of 6 h) (Table 2). It indicated that the methanogenesis including hydrogen-utilizing methanogenesis was significantly inhibited. As a result, with the suppression of BES, the H2 percentage in the biogas raised to 0.48% for A1 and to 1.68% for A2. The H2 content in reactor was the important factor in the anaerobic fermentation [3,8,16]. The hydrogen partial pressure must be maintained at an extremely low level to meet favorable thermodynamic condition for the conversion of propionate to acetate. Otherwise, the decomposition of propionate would reduce and even stop so as to destroy the acidic balance of anaerobic system. In other words, the increase of H2 content would hinder the propionate conversion. Accordingly, the propionate conversion rate decreased from 88.6% to 45.9% in A1 and from 77.3% to 30.9% in A2 with BES, respectively (Fig. 3). Chemically, the Fe2+ leaching (Fe0 + 2 H+ = Fe2+ + H2 ) (Table S1) would increase the H2 percentage of A1 which was unfavorable for the propionate conversion. However, less H2 percentage (0.48%) was found in the biogas from A1. It made the propionate conversion rate in A1 (45.9%) obviously higher than that in A2 (30.9%) with BES. The H2 percentage in A1 was lower than that in A2. From the hydrogen utilization pathway in the anaerobic fermentation, besides that H2 was used for the hydrogen-utilizing methanogenesis, homoacetogenesis is another approach able to utilize H2 plus CO2 as the energy source to synthesize acetate [12]. Therefore, according to the less H2 content from A1, it was reasonably assumed that the homoacetogensis was enhanced by the Fe0 so that more H2 was consumed. The reduced H2 content helped create a favorable environment for the conversion of propionate. The methanogenic archaea and homoacetogenic bacteria are the main H2 consumers in the anaerobic system. In most cases,

3.4. Changes of the Gibbs free energy in the acetification The conversion of propionate to acetate is not spontaneous in thermodynamics. It is the main reason for the propionate accumulation in the anaerobic process [2]. With the decrease of H2 content, it made this unfavorable reaction possible. Considering that the H2 was utilized for the homoacetogenesis, the total Eq. (3) together with the propionate conversion Eq. (1) and homoacetogensis Eq. (2) was obtained. The Gibbs free energy of Eq. (3) was calculated based on the contents of compounds at the HRT of 6 h. The Gibbs free energy in A1 was 26.29 kJ/mol, lower than that in A2 (29.20 kJ/mol). With dosing of BES, the Gibbs free energy in A1 was 31.15 kJ/mol, also lower than that in A2 (33.85 kJ/mol). It meant that the Gibbs free energy of the propionate decomposition was decreased by about 8.0% to 10.2%. The results suggested that the conversion of propionate to acetate became easier in thermodynamics due to the dosing of Fe0 . 3.5. The key enzymes activities in the conversion of propionate to acetate The accelerated conversion of propionate to acetate could partly be ascribed to the enhancement of enzyme activity. Dehydrogenase, POR, PTA and AK were the key enzymes in organic acid fermentation [13–15]. They participated in the following metabolic process: propionate −→

dehydrogenase

pyruvic acid−→acetyl-CoA−→acetyl phosphate−→acetic acid. POR

PTA

AK

The key enzymes activities in the reactors are shown in Fig. 4. Apart from POR, BES had little effect on the activities of these acidogenic enzymes in the same reactor. The reason was because BES was only an inhibitor to methanogenesis. As an exception, the activity of POR in A1 with BES was 3.5 times higher than the no-BES case, which was related to its chemical structure. POR

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X. Meng et al. / Biochemical Engineering Journal 73 (2013) 80–85

contains Fe S clusters, suggesting that appropriate increase of Fe2+ concentration benefited to enhance its activity [5,11]. The Fe2+ concentration of A1 raised from 15.65 to 56.32 mg/L after adding BES (Tables 1 and S1) due to the increase of the organic acidity with BES (Tables 1 and S1). On the other hand, the activities of the enzymes in A1 were higher than those in A2 whether with or without BES. When BES was added into the two reactors, the dehydrogenase activity of A1 was 0.0211 ± 0.0035 units/mg-protein and that of A2 was only 0.0120 ± 0.0021 units/mg-protein. The POR activity of A1 was 0.3544 ± 0.0426 units/mg-protein, while that in A2 was only 0.0105 ± 0.0021 units/mg-protein. Thus, the POR activity in A1 was about 34 times higher than that in A2. The PTA activity in A1 was 0.0284 ± 0.0039 units/mg-protein and that in A2 was only one-tenth of A1. The activity of AK in A1 was 0.0369 ± 0.0067 units/mg-protein, which was about twice as much as that in A2 (0.0162 ± 0.0011 units/mg-protein). These results indicated that Fe0 dosing enhanced the key enzymes activities in the conversion of propionate to acetate. From Fig. 4, the enzymes activities were increased by Fe0 dosing, thus enhanced the conversion of propionate to acetate. Fe0 as electron donor can promote this oxidation process because of whether a reaction occurred or not was depended on the reaction thermodynamics, such as Gibbs free energy, which was not directly related with the ORP value. Certainly, the addition of Fe0 could decrease the ORP to improve some metabolic processes (acetic-type and butyric-type fermentation) and benefit the reaction dynamically. Furthermore, it was reported that the dehydrogenase and POR contained Fe S clusters and they presented the activities only under low ORP [11,32]. 3.6. FISH test FISH was used to analyze the specific microbial composition in the acidogenic reactors with BES and the results are shown in Fig. S2. According to the analysis by Image-Pro Plus 6.0, the abundance of propionate-utilizing bacteria (red) was 52.9% in A1, while that in A2 was only 11.3%. The results were corresponded with the acetate production of the two reactors, further proving that the Fe0 enhanced the conversion of propionate to acetate. The abundance of homoacetogenic bacteria (green) in A1 was 35.6%, also higher than that in A2 (8.5%), which was the microbially reason for the less H2 percentage in A1. The larger amount of propionicutilizing bacteria and homoacetogenic bacteria in A1 was ascribed to the presence of Fe0 . More propionate-utilizing bacteria and homoacetogenic bacteria accelerated the conversion of propionate to acetate. As it was well-known that the conversion of propionate to acetate is unfavorable in thermodynamics (G = +76.1 kJ/mol). POR and other enzymes, as biological catalysts, only catalyze spontaneous reactions in thermodynamics [33,34]. Therefore, the increased activity of enzymes did not necessarily make the propionate conversion happen. In this study, we found H2 produced in the conversion of propionate to acetate was more utilized by homoacetogenesis with dosing of Fe0 , which created

Fig. 5. DGGE fingerprints of microbial communities in the acidogenic reactors (A1 and A2: sample from A1 and A2 with BES at the HRT of 6 h, respectively).

a thermodynamically beneficial condition for the conversion of propionate. It was proved by the results of H2 content in the biogas, the value of G and FISH analysis. 3.7. PCR–DGGE analysis PCR–DGGE analysis of the sludge taken from the acidogenic reactors with BES was performed at the end of operation. Fig. 5 shows the DGGE fingerprints of microbial communities in the acidogenic reactors. In order to provide further insight into the microbial diversity, the predominant species extracted from DGGE bands were sequenced and compared with the published species in NCBI (Table 3). From Fig. 5, when adding BES to inhibit methanogenesis in acidogenic reactors, the number and strength of the bands in A1 was much different from that in A2. The band 1 was observed in A1 and did not appear in A2. The clone of band 1 showed 100% sequence similarity to Uncultured bacterium clone 8-3 (AM158303), which was a kind of sulfate reducing bacteria observed in the deep-sea sediments [35]. The sulfate reducing bacteria could relieve propionate accumulation in anaerobic digester. Other bands in A1 were stronger compared with those of A2, especially the band 2, 5 and 6. The clone of band 2 presented 99% sequence similarity to Prevotellaceae bacterium clone WR041 (AB298732), which was a characteristic fermentative bacterial in the methanogenic fermenter [36]. The clone of band 5 showed 97% sequence similarity to Uncultured bacterium clone ambient-uncontrolled-45

Table 3 Identity of dominant DGGE bands. Band

Phylogenetic affiliation (Accession number)

Sequence similarity

Exist conditions

1 2 3 4 5 6 7

Uncultured bacterium clone 8-3 (AM158303) Prevotellaceae bacterium clone WR041 (AB298732) Uncultured bacterium clone:UASB8 (AB329648) Uncultured bacterium clone BXHA50 (GQ480004) Uncultured bacterium clone ambient-uncontrolled-45 (GU454906) Uncultured alpha proteobacterium clone RT03-3B-42 (AY475198) Selenomonas sp. enrichment culture clone yan03 (FJ386554)

100% 99% 99% 99% 97% 99% 100%

Deep-sea sediments Methanogenic fermenter of cattle waste Anaerobic sludge granules Anaerobic sewage treated process Waste activated sludge alkaline fermentation Metal-rich and acidic river sediments Sludge backflow of municipal wastewater treatment plant

X. Meng et al. / Biochemical Engineering Journal 73 (2013) 80–85

(GU454906), which could enhance short-chain VFAs accumulation in waste activated sludge alkaline fermentation [37]. These bacteria were helpful to raise short-chain VFAs production, and the operational taxonomic unit (OUT) of band 5 was 100% sequence divergence to band 1 (sulfate reducing bacteria) in phylogenetic relationship (Fig. S3). The clone of band 6 showed 99% sequence similarity to Uncultured alpha proteobacterium clone RT03-3B-42 (AY475198), which was observed in metal-rich and acidic river sediments [38]. The two OTUs of band 3 and 6 were closely related to the alpha proteobacterium (Fig. S3), existing in acidogenic reactors containing Fe2+ , which was in agreement with the present study. 4. Conclusions Fe0 powder was added into an acidogenic reactor for enhancing the conversion of propionate to acetate in this work. According to the experimental results, the propionate conversion was enhanced by the Fe0 dosed, accordingly raising the production of acetate and COD removal. Also, the homoacetogenesis was accelerated to reduce the H2 content, which helped create a favorable condition for the propionate decomposition. The dosing of Fe0 decreased the Gibbs free energy of the propionate decomposition, and improved the activities of enzymes related to acetogenesis. It was observed that the quantity and diversity of microbial communities were increased, especially bacteria responsible for homoacetogens and conversion of propionate. These results suggested that the accumulation of propionate could be alleviated via accelerating the conversion of propionate by dosing Fe0 . Acknowledgments The authors acknowledge the financial support from the National Crucial Research Project for Water Pollution Control of China (2012ZX07202006), the National Basic Research Program of China (21177015) and the New Century Excellent Talent Program of the Ministry of Education of China (NCET-10-028). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bej.2013.02.004. References [1] M. Tatara, T. Makiuchi, Y. Ueno, M. Goto, K. Sode, Methanogenesis from acetate and propionate by thermophilic down-flow anaerobic packed-bed reactor, Bioresour. Technol. 99 (2008) 4786–4795. [2] P.L. McCarty, D.P. Smith, Anaerobic wastewater treatment, Environ. Sci. Technol. 20 (1986) 1200–1206. [3] J.Z. Li, G.C. Zheng, J.G. He, S. Chang, Z. Qin, Hydrogen-producing capability of anaerobic activated sludge in three types of fermentations in a continuous stirred-tank reactor, Biotechnol. Adv. 27 (2009) 573–577. [4] M. Sbarciog, M. Loccufier, E. Noldus, Determination of appropriate operating strategies for anaerobic digestion systems, Biochem. Eng. J. 51 (2010) 180–188. [5] Y.W. Liu, Y.B. Zhang, X. Quan, Y. Li, Z.Q. Zhao, X.S. Meng, S. Chen, Optimization of anaerobic acidogenesis by adding Fe0 powder to enhance anaerobic wastewater treatment, Chem. Eng. J. 192 (2012) 179–185. [6] A.R. Barros, E.L. Silva, Hydrogen and ethanol production in anaerobic fluidized bed reactors: performance evaluation for three support materials under different operating conditions, Biochem. Eng. J. 61 (2012) 59–65. [7] N.Q. Ren, H. Chua, S.Y. Chan, Y.F. Tsang, Y.J. Wang, N. Sin, Assessing optimal fermentation type for bio-hydrogen production in continuous-flow acidogenic reactors, Bioresour. Technol. 98 (2007) 1774–1780. [8] L. Wang, Q. Zhou, F.T. Li, Avoiding propionic acid accumulation in the anaerobic process for biohydrogen production, Biomass. Bioenerg. 30 (2006) 177–182.

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