Enhancement of sludge granulation in anaerobic acetogenesis by addition of nitrate and microbial community analysis

Biochemical Engineering Journal 95 (2015) 104–111

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Enhancement of sludge granulation in anaerobic acetogenesis by addition of nitrate and microbial community analysis Yang Li, Yaobin Zhang ∗ , Zibin Xu, Xie Quan, Shuo Chen Key Laboratory of Industrial Ecology and Environmental Engineering, Ministry of Education, School of Environmental Science and Technology, Dalian University of Technology, Dalian 116,024, China

a r t i c l e

i n f o

Article history: Received 18 September 2014 Received in revised form 29 November 2014 Accepted 21 December 2014 Available online 26 December 2014 Keywords: Granulation Batch processing Wastewater treatment Microorganism morphology Bioreaction

a b s t r a c t Sludge granulation is a key factor to sustain anaerobic systems operating efficiently and steadily. Nitrate as a H2 consumer was added into anaerobic digesters to investigate its effects on the sludge granulation. The results showed that adding nitrate increased the sludge granule size by 289%, 325% and 790% with acetate, propionate and butyrate as substrates, respectively. Butyrate was preferable to the denitrifying bacteria because it was capable of releasing more electrons available for denitrification during acetogenesis. The analyses of fluorescence in situ hybridization, scanning electron microscope, and denaturing gradient gel electrophoresis indicated that denitrifying bacteria and volatile fatty acid (VFA)-oxidizing bacteria in the butyrate digester were richer than those in the other digesters. Taken together, addition of nitrate accelerated the decomposition of VFA and simultaneously improved the granulation of anaerobic process.

1. Introduction Anaerobic digestion is a widely-applied technology to decompose organics and simultaneously produce biogas from wastewaters and sludge [1]. Hydrogen partial pressure in the anaerobic system is required to be maintained at a quite low range to allow the anaerobic process continuously happening [2]. Hydrogen is produced from non-methanogenic microorganisms metabolizing the fermentation products (especially organic acids) and can be consumed by hydrogenotrophic methanogens with the reduction of CO2 into CH4 [3,4]. When the anaerobic system suffers from a load or acidic shock, hydrogenotrophic methanogens may be inhibited so as to decrease the hydrogen utilization [5]. It will cause the organic-acid decomposition to stop, leading to a high concentration of organic acid accumulation, further deteriorating the final methanogenesis [6,7]. Denitrification as an electron-accepting process is considered as an alternative to consume the hydrogen produced in anaerobic digestion [8]. If the hydrogen produced from volatile fatty acid (VFA)-oxidizing bacteria is consumed by denitrifying bacteria, both anaerobic digestion and denitrification is expected to be accelerated. Barber and Stuckey observed that denitrification accelerated

∗ Corresponding author. Tel: +86 411 84706460; fax: +86 411 84706263. E-mail address: [email protected] (Y. Zhang). http://dx.doi.org/10.1016/j.bej.2014.12.011 1369-703X/© 2014 Elsevier B.V. All rights reserved.

© 2014 Elsevier B.V. All rights reserved.

the conversion of propionate and butyrate significantly and Lens et al. reported that addition of nitrate (2.3 gNO3 − /g acetate) considerably improved the acetate removal efficiency of sulfidogenic reactors [8,9]. Granules have regular and dense structure with superior settling properties and high biomass retention, which is a key factor to withstand high-strength organic loads in anaerobic system and maintain its operation steady [10,11]. Anaerobic granular aggregate is composed of three layers [14,15]. In the outmost layer of the granules, hydrolytic and/or acidogenic bacteria are predominant, whereas the internal core of the granules prevailingly consists of methanogens. The syntrophic communities including hydrogen-producing bacteria and hydrogen-consuming bacteria are located in the middle layer. Recently, enhancement of granulation in anaerobic reactors by addition of nitrate has been reported [11–13,19,30,35]. Hendriksen and Ahring cultivated active denitrifying/methanogenic granules with big size and high metabolic activities [19]. Wang et al. and Green et al. also observed the enhanced granulation in a denitrification (upflow anaerobic sludge bed) UASB reactor [30,31]. In the presence of nitrate, the interspecies electron transfer between acetogenesis and denitrification is likely to improve the digestion rates and provide a protection for the inner methanogens, and then accelerate the formation of compact aggregates [4]. Up to now, few researches had investigated the effect on the granulation with different VFA as carbon sources in denitrification.

Y. Li et al. / Biochemical Engineering Journal 95 (2015) 104–111 Table 1 Experimental settings. Number

R1

R2

R3

R4

R5

R6

Nitrate Substrate

+ Acetate

+ Propionate

+ Butyrate

− Acetate

− Propionate

− Butyrate

+ means the reactor with addition of nitrate. − means the reactor without addition of nitrate.

It was assumed that types of VFA had a significant effect on sludge granulation due to the fact that different VFA had different capacities to produce electron donor for denitrification. This study aimed to remit the accumulation of organic acids and enhance the sludge granulation by adding nitrate in anaerobic process. The relationship between anaerobic granulation and denitrification with different VFA as substrates was investigated. We hope to provide an efficient method to enhance the anaerobic treatment of the high-strength organic wastewater. 2. Materials and methods 2.1. Sludge and wastewater Sludge was acquired from a secondary sedimentation tank of a local municipal wastewater treatment plant based on the activated sludge process in Dalian, China. The sludge was cultured in a batch anaerobic reactor with a glucose solution (chemical oxygen demand [COD]: 1000 mg/L) as substrate. The sludge was not exposed to nitrate prior to adding into the digesters. The ratio of volatile suspended solids to total suspended solids (VSS/TSS) of the sludge was 0.65. An artificial wastewater consisted of a single VFA like acetate, propionate and butyrate in turn was used as the influent. NH4 Cl and KH2 PO4 were used as nitrogen and phosphorus sources, respectively (at mass ratio of COD:N:P 200:5:1). The trace elements including Zn, Mn, Cu, Co, Ni etc., were added according to reference [20]. 2.2. Batch experiments Several same conical flasks with a volume of 250 mL were used as anaerobic digesters. 100 mL sludge with 25 g/L of TSS was added into each digester. 150 mL artificial wastewater with nitrate and a type of VFA (sodium salt of acetate, propionate or butyrate, respectively) was added into each digester as shown in Table 1. The experiment was divided into three stages according to the different composition in the feeding liquid: (1) NO3 − -N of 130 mg/L and VFA of 1000 mg/L (1–30 cycles); (2) NO3 − -N of 260 mg/L and VFA of 1000 mg/L (31–65 cycles); (3) NO3 − -N of 260 mg/L and VFA of 3000 mg/L (66–95 cycles). The digesters were operated in a semi-continuous (i.e., sequencing batch) mode with a cycling time of 24 h. They were placed on a constant temperature breeding shaker (ZHWY-2102C, China) at 35 ± 1 ◦ C and 120 rpm. After each cycle, the treated liquid was completely poured out and replaced using fresh artificial wastewater. The batch experiments were continuously conducted for 95 cycles. The concentrations of nitrate and VFA were analyzed 3 h after the beginning of each cycle, and the sludge size and biogas content were measured after each cycle. The digesters were flushed with N2 for 20 min to remove dissolved oxygen, and then sealed with plugs before each cycle. 2.3. Analytical methods NO3 − -N, pH, TSS and VSS were measured according to the standard methods [27]. The concentrations of VFA, including acetate,

105

propionate, butyrate, were determined using a gas chromatograph (Shimadzu, GC-2010/FID, Japan). The composition of biogas was analyzed by a gas chromatograph (Shimadzu, GC-14C/TCD), Japan). The size distribution of sludge was measured and calculated by Malvern Mastersizer 2000 laser particle size analyzer (Worcestershire, UK) according to the method by Liu et al. [16]. Morphology of granules was examined by a scanning electron microscopy (SEM, Quanta 200 FEG). The granules samples were firstly immobilized using 205% (w/v) glutaraldehyde in Sorenson’s phosphate buffer, and dehydrated using ethanol with gradient concentrations (10%, 25%, 50%, 75%, 90% and 100%, 15 min per step). And then the samples were dried using carbon dioxide at their critical point. At last, the samples were sputter coated with gold and observed using the SEM. 2.4. DNA extraction, PCR amplification and denaturing gel gradient electrophoresis After the experiment (for 95 cycles), approximately 1 mL the sludge samples were collected from the homogenized sludge in each digester. The genomic DNA of the sample was extracted using an extraction kit (Bioteke Corporation, Beijing, China) according to the manufacturer’s instructions, before which the sludge samples were firstly washed with phosphate-buffered saline (pH 7.4). The general primers of 341f (5 -CCT ACG GGA GGC AGC AG-3 ) which contained a 40 bp GC clamp at the 5 -end (forward primer) and 907r (5 -CCG TCA ATT CMT TTR AGT TT-3 , reverse primer) were used to selectively amplify the 16 S ribosomal RNA sequences of Eubacteria [17]. Polymerase chain reaction (PCR) amplification was conducted on a thermal cycler (Thermal Cycler DiceTM; BioRad Co., Ltd., Hercules, CA, USA) with a touchdown PCR method as following program (initial denaturation at 95 ◦ C for 5 min followed by 30 cycles of denaturation at 94 ◦ C for 30 s, primer annealing at 50 ◦ C for 1 min and primer extension at 72 ◦ C for 2 min, and final extension at 72 ◦ C for 10 min). The PCR products obtained were applied to (denaturing gradient gel electrophoresis) DGGE analysis using the Dcode system (BioRad Co., Ltd., USA). A denaturing gradient of 30–60% denaturant (100% denaturant was a mixture of 7 mM urea and 40% [v/v] formamide) acrylamide gel (6%, w/v) was applied. Electrophoresis was conducted at 60 ◦ C and at a constant voltage of 200 V for 5 h in 1 × TAE buffer. The gels were then stained with SYBR Gold (Dalian TaKaRa, China) in 1 × TAE buffer for 40 min, after which the UV thansillumination image of the gel was then photographed using the Gel Doc 2000 System (BioRad Laboratories, USA). Selected DGGE bands were excised and re-amplified by PCR using the primers described above without the GC clamp. The obtained gene sequences were screened against the GenBank database using the BLAST program to identify the most similar sequences. The DGGE analysis above was conducted according to the reference of Zhang et al. [17]. 2.5. Fluorescence in situ hybridization Fluorescence in situ hybridization (FISH) was used to determine the abundance of VFA-oxidizing bacteria. The homogenized sludge samples collected from different digesters were harvested by centrifugation (110 × 100 g for 15 min at 4 ◦ C) and fixed in 4% freshly prepared paraformaldehyde solution for 2 h at 4 ◦ C. Then the samples were washed twice with phosphate-buffered saline (PBS, 130 mM NaCl, 10 mM sodium phosphate buffer, pH 7.2) and air dried at room temperature. After that the samples were dehydrated by successive passages through 50%, 80%, and 100% ethanol (three times). Hybridization steps were performed at 46 ◦ C for 1.5 h with buffer (0.9 M NaCl, 20 mM Tris-HCl [pH 7.2], 0.01 sodium dodecyl sulfate and 35% formamide) containing 50 ng probe per microliter and then washed with buffer (15 min at 48 ◦ C).

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Y. Li et al. / Biochemical Engineering Journal 95 (2015) 104–111

Influent nitrate-N Butyrate as substrate

Propionate as substrate Acetate as substrate

250 Nitrate-N (mg/L)

VFA 1000 mg/L

VFA 1000 mg/L

VFA 3000 mg/L

200 150

Table 2 Effect of denitrification on degradation of different VFAs. Error bars represent standard deviations (SD) of seven measurements. VFA (mg/L)

NO3 − -N (mg/L)

Acetate ± SD (mg/L)

1000 1000 1000 3000 3000

0 130 260 260 0

240.20 15.05 2.83 255.50 911.25

± ± ± ± ±

43.07 4.67 1.51 9.20 23.36

Propionate ± SD (mg/L) 315.15 253.38 92.45 457.45 839.05

± ± ± ± ±

10.99 31.90 5.54 23.96 31.93

Butyrate ± SD (mg/L) 136.26 26.06 3.25 234.36 441.15

± ± ± ± ±

17.97 4.39 1.38 28.92 24.02

100 50 0 0

10

20

30

40 50 Cycle

60

70

80

90

Fig. 1. Concentration of NO3 − -N in the effluent with different VFA as carbon sources. The influent VFA increased from 1000 mg/L to 3000 mg/L, and NO3 − -N increased from 130 mg/L to 260 mg/L. Error bars represent standard deviations of three measurements.

A cyanine 3 (cy3)-labeled Wol223 probe with a sequence of 5 ACGCAGACTCATCCCCGTG-3 and Synm700 probe with a sequence of 5 -ACTGGTNTTCCTCCTGATTTGTA-3 were used for characterization of propionate-oxidizing bacteria and butyrate-oxidizing bacteria (red), while a fluorescein isothiocyanate (FITC)-labeled EUB338 probe with a sequence of 5 -GCTGCCTCCCGTAGGAGT-3 was used for characterization of bacteria (green) [32]. The sludge hybridized was viewed by confocal laser scanning microscopy (Leica-SP2, Heidelberger, Germany). The FISH images obtained were imported to Image-Pro plus 6.0 for analysis of the relative abundance of the microorganisms. 3. Results and discussion 3.1. Mutual impact on denitrification and degradation of different individual VFA In order to investigate the mutual impact between denitrification and VFA degradation, the experiment on denitrification was conducted using three different types of VFA (acetate, propionate or butyrate) as carbon sources, respectively (R1, R2 and R3). At the influent VFA of 1000 mg/L and NO3 − -N of 130 mg/L, the effluent NO3 − -N with butyrate as substrate decreased promptly to ca. 2.2 mg/L only using 5 cycles (Fig. 1). The effluent NO3 − -N with propionate decreased to ca. 2.7 mg/L after 14 cycles. Acetate as substrate was the slowest, which took 18 cycles to decrease the effluent NO3 − -N to ca. 5.6 mg/L. When the NO3 − -N increasing to 260 mg/L (VFA fixed at 1000 mg/L), the effluent NO3 − -N decreased gradually over the time and finally stabilized at ca. 140.5, 120.2 and 50.0 mg/L for acetate, propionate and butyrate, respectively. Further increasing VFA to 3000 mg/L (NO3 − -N fixed at 260 mg/L), the NO3 − -N removal presented the same tendency in the order of butyrate > propionate > acetate. The results were similar to Xie et al. who observed propionate and butyrate were more preferable for denitrification than acetate [18]. Hendriksen and Ahring also found that butyrate as substrate presented a higher rate of denitrification than propionate and acetate [19]. The reason might be ascribed that the H2 produced in the acetogenesis of propionate and butyrate served as an additional electron donor for denitrification. In anaerobic digestion, butyrate and propionate could be converted into acetate along with producing H2 , while acetate was the final form in acetogenesis [36]. Furthermore, the acetogenesis of propionate was more unspontaneous (Gibbs free

energy +76.1 kJ/mol) in thermodynamics than that of butyrate (Gibbs free energy +48.1 kJ/mol), which would delay the conversation of propionate [20]. Thus, butyrate was a more efficient electron source for denitrification than propionate. In order to investigate the effects of denitrification on VFA degradation, another three control digesters (R4, R5 and R6) were operated under the same conditions as R1, R2 and R3 but without addition of nitrate, respectively. As shown in Table 2, the VFA removal efficiencies increased with addition of nitrate. At VFA (acetate, propionate and butyrate) of 1000 mg/L and NO3 − N of 130 mg/L, the removal efficiencies of acetate, propionate and butyrate were 98.5%, 74.7% and 97.4%, respectively, while they were 76.0%, 68.5% and 86.4% in the absence of nitrate (i.e., R4, R5 and R6), respectively. With NO3 − -N increasing to 260 mg/L (VFA = 1000 mg/L), the removal efficiencies of acetate, propionate and butyrate further rose to 99.7%, 90.8% and 99.7%, respectively. The results indicated that dosing nitrate could accelerate the decomposition of the three types of VFA. Similar references reported that the effluent VFA concentration decreased from 600 to 100 mg/L by dosing 2.3 g/L nitrate [9]. Acetates produced in the decomposition of propionate and butyrate with and without nitrate were detected and shown in Fig. 2. From the figure, the acetate concentration with nitrate was less than that without nitrate. For example, at the influent VFA of 1000 mg/L and without nitrate, the acetate production were 75 mg/L for propionate and 70 mg/L for butyrate, respectively. While adding NO3 − -N of 260 mg/L, the acetate concentration decreased to 25 mg/L for propionate and 30 mg/L for butyrate, respectively. Considering that acetate is the production of acetogenesis of propionate and butyrate, less acetate content with nitrate addition suggested the faster consumption by denitrification. Furthermore, the biogas production (Fig. 3) without nitrate (R4, R5, R6) was significantly higher than those with nitrate (R1, R2, R3). For the butyrate test, in the absence of nitrate the percentage of CH4 and CO2 was 44.0% and 18.2% (in R6), respectively, while it turned into 10.5% and 6.6% (in R3) at the NO3 − -N of 130 mg/L. It was because the denitrifying bacteria competed for utilization of acetate and H2 to reduce the methanogens and consequently boosted denitrification. 3.2. Development of denitrifying granules with different electron donors Fig. 4(a) shows the mean size (mean ± standard deviation, n = 3) of anaerobic sludge after the anaerobic digestion for 95 cycles. The mean granule sizes in the control digesters (R4, R5 and R6) were 90.2 ± 4.5, 120.5 ± 6.8 and 135.0 ± 7.2 ␮m for acetate, propionate and butyrate, respectively. Comparatively, the granule sizes in the presence of nitrate (260 mg/L for NO3 − -N) were 350.8 ± 18.5 (acetate, R1), 510.2 ± 20.6 (propionate, R2) and 1200.5 ± 50.5 ␮m (butyrate, R3), respectively. The results indicated that the granulation was accelerated with addition of nitrate. This phenomenon was reported by Tiwari et al. and Jin et al. who observed denitrifying bacteria were able to accelerate formation of granules [12,13]. Remarkably, the granulation varied significantly with different VFA as substrates. The mean granule size in the acetate digester with

Y. Li et al. / Biochemical Engineering Journal 95 (2015) 104–111

(a)

1600 1400

Butyrate

Without N With N

1200 Granule size (µm)

107

1000 800 600

Propionate Acetate

400 200 0

(b) 8

Acetate Propionate Butyrate

Volume (%)

6 4 2 0 10

100

1000

Granularity (µm) Fig. 2. Acetate production with propionate (a) and butyrate (b) as substrates at the presence and absence of nitrate. Error bars represent standard deviations of three measurements.

70 60

Biogas (%)

50

Without nitrate

CH4 CO2

40 30 20

With nitrate

10 0 Acetate Propionate Butyrate Acetate Propionate Butyrate Fig. 3. The effect on production of biogas by denitrification. The influent VFA concentration was 1000 mg/L and NO3 − -N was 130 mg/L. Error bars represent standard deviations of six measurements.

nitrate (R1) increased by 289% compared with that in the acetate digester without nitrate (R4), while the granule size increased by 325% in the propionate digesters (R2 vs R5) and increased by 790% in the butyrate digesters (R3 vs R6), respectively. This implied that the propionate and butyrate were more favorable for the granulation. The granule size distributions on cycle 95 are shown in Fig. 4(b). It was found that over 90% of granules fed with butyrate had a size bigger than 200 ␮m, while about 75% of granules in the acetate digester were smaller than 200 ␮m. The granule size of the pro-

Fig. 4. (a) Mean granules size in different digesters at the end of the experiment on cycle 95. (b) Distribution of granules with different VFA as substrates with nitrate on cycle 95. Error bars represent standard deviations of six measurements.

pionate digester was bigger than that of the acetate digester but smaller than that of the butyrate digester. The results were well in agreement with the granule sizes shown in Fig. 4(a). From Fig. 5, the SEM pictures presented the morphological characteristics of the granules obtained from the digesters at the end of the experiment. These pictures gave an overview of the granule with different VFA as substrate. It revealed that the structures of sludge in the digesters without nitrate (R4, R5 and R6) presented a loose and incomplete granular appearance, while those in the digesters dosed with nitrate (R1, R2 and R3) formed roughly full granules with compact and stable structure. In the butyrate digester, the granules were more compact and solid with presence of nitrate. Actually, denitrification could increase the pH of the anaerobic system, which benefited the anaerobic granulation partly because it might neutralize acidity strength which was harmful for sensitive anaerobic microbes in the granule such as methanogens [37]. However, the pH with different NO3 − was all slightly alkaline (7.09–8.85 for 1000 mg/L VFA, see Table S1) and even the pH with no NO3 − was >7. A little change of pH in slightly alkaline ranges had no obvious effects on the anaerobic granule. The pH with different VFA also showed no significant differences (<0.2, Table S1). Both denitrifying bacteria and VFA-oxidizing bacteria belonged to facultative anaerobic processes, occurring at a relatively higher oxidation–reduction potential (ORP) value [21]. Consequently, these two kinds of bacteria were inclined to distribute in the outer layer of granule. The denitrifying bacteria utilized the hydrogen produced from acetogenesis to drive the acetogenesis happen-

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Y. Li et al. / Biochemical Engineering Journal 95 (2015) 104–111

Fig. 5. SEM images of the granules in digesters with different VFA as substrates at the end of the experiment on cycle 95 with a magnification of 220 × .R1, R2 and R3 represented the denitrification digesters fed with acetate, propionate and butyrate, respectively. R4, R5 and R6 represented the non-nitrate digesters fed with acetate, propionate and butyrate, respectively.

ing more easily. The cooperation between VFA-oxidizing bacteria and denitrifying bacteria extended the food chain in the anaerobic sludge. The VFA-oxidizing bacteria interweaving with denitrifying bacteria built up a more solid shield for the inner methanogens, which helped the granule develop a compact structure. This was in agreement with Hulshoff et al. who believed the H2 -producing bacteria and H2 -consuming bacteria distributed in the outer of the granules might give a better protection for the methanogens in the inside [14]. Therefore, the granule sizes in the digesters with propionate and butyrate as substrates were bigger. Meanwhile, butyrate producing H2 was more spontaneous than propionate in thermodynamics [20], which served as an ideal electron donor for use by denitrifying bacteria. Correspondingly, butyrate made the granules grow better. Comparatively, acetate was the simplest type of VFA which was consumed directly by denitrifying bacteria or methanogens [9,36]. Thus, the granulation with acetate as substrate was smaller than that with propionate and butyrate.

3.3. Analysis of microbial communities FISH was used to analyze the effect of denitrification on the VFAoxidizing bacteria of the granules after 95 cycles of operation, and the results are shown in Fig. 6. According to the analysis conducted using Image-Pro Plus 6.0, the relative abundance (mean ± standard deviation, n = 3) of VFA-oxidizing bacteria in the denitrification digesters fed with propionate and butyrate (R2 and R3) were 63.0 ± 8.8% and 70.6 ± 6.0%, while they were only 28.2 ± 2.6% and 35.5 ± 3.6% in the propionate and butyrate digesters without nitrate (R5 and R6). FISH images showed that addition of nitrate increased the relative abundance of VFA-oxidizing bacteria, which could accelerate the conversion of VFA into acetate with producing more H2 for the reduction of nitrate. The accelerated hydrogen transfer shortened the distance between VFA-oxidizing bacteria and denitrifying bacteria, and therefore made the granulation effective. Besides, the abundance of VFA-oxidizing bacteria in the butyrate digester was more than that in the propionate and acetate digesters. It implied the electron transfer between butyrate-oxidizing bacteria and denitrifying bacteria was easier, which benefited to form

the bigger and more compact granules, just as the results of Fig. 4 and Fig. 5 shown. The microbial communities of granules were analyzed by the DGGE patterns of extracted DNA (Fig. 8). The variety and abundance of the microbial communities differed obviously in each digester. In the digesters with nitrate, the dominant bands with propionate and butyrate were significantly stronger than those with acetate, indicating the bacteria grew better with propionate or butyrate as substrate, in agreement with the results in Fig. 1. As shown in Table 3, the dominant bacteria from band 4 and 8 in the digesters with nitrate (R1, R2 and R3) were similar to Aestuariimicrobium kwangyangense strain R27 (99% similarity) and Tessaracoccus flavescens strain: SST-39 (99% similarity), respectively, which were coccoid-shaped and found to be a member of family Propionibacteriaceae that was capable of denitrification [28,29]. Some denitrifying bacteria also showed a rod shape, for example the bacteria from band 6 and 7 in the digesters with nitrate (R1, R2 and R3) were similar to Acidovorax caeni strain R-24608 (99% similarity) and Achromobacter denitrifying bacteria (99% similarity) strain DSM 30,026. These two kinds of bacteria were considered as a denitrifying species from activated sludge of an aerobic–anaerobic wastewater treatment [23,24]. Besides, bacteria from band 1 and 5 in all the digesters (with and with not nitrate) were similar to Pelobacter seleniigenes strain KM and Thermovirga lienii DSM 17,291 strain DSM 17,291, respectively, which had the ability to metabolize the short-chain fatty acid [25,26]. Also, band 12, 13 and 14, dominant in all digesters (with and without nitrate), were similar to Pelodictyon phaeoclathratiforme BU-1 strain BU-1, Thermosipho melanesiensis BI429 strain BI429, and Sphaerochaeta pleomorpha str. Grapes strain Grapes, respectively, which could use organic acid as carbon sources [33–35]. These bacteria from bands 1, 5, 12, 13 and 14 with the ability to metabolize the organic acids might just be the VFA-oxidizing bacteria in FISH images detected by Wol223 or Synm700 (Fig. 6). The results of DGGE above indicated that VFA-oxidizing bacteria and denitrifying bacteria became richer after adding nitrate. The hydrogen electron transfer would inevitably occur between these two species, which not only improved denitrification but also might reduce the hydrogen partial pressure beneficial for the acetogenesis

Y. Li et al. / Biochemical Engineering Journal 95 (2015) 104–111

109

Fig. 6. FISH images of the homogenized sludge in the digesters with different VFA as substrates. R1, R2 and R3 represented the denitrification digesters fed with acetate, propionate and butyrate, respectively. R4, R5 and R6 represented the non-nitrate digesters fed with acetate, propionate and butyrate, respectively. Simultaneously hybridized with Cy3-labeled propionate-oxidizing bacteria and butyrate-oxidizing bacteria domain probe (Wol223 in R2, R5 and Synm700 in R3, R6, red) and FITC-labeled Eubacterial domain probe (EUB338, green). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.) Table 3 Identity of dominant DGGE bands of bacteria. Band

Closest relative

Remarks

Sequence similarity %

Accession number

1

Pelobacter seleniigenes strain KM

99

NR 044032.1

2

Acidaminobacter hydrogenoformans strain glu 65

99

NR 028683.1

3

Solitalea koreensis strain R2A36-4

99

NR 044568.1

4

Aestuariimicrobium kwangyangense strain R27

99

NR 043972.1

5

Thermovirga lienii DSM 17,291 strain DSM 17,291

99

NR 074606.1

6

Acidovorax caeni strain R-24608

99

NR 042427.1

7 8

Achromobacter denitrificans strain DSM 30,026 Tessaracoccus flavescens strain:SST-39

Grow fermentatively on short-chain organic acids and respiration of nitrate, within the family Geobacteraceae A propionigenic bacterium as Propionibacter pelophilus, a new member of the ␤-subclass of the Proteobacteria Rod-shaped, has several properties including anaerobic growth, nitrate reduction. Short rod or coccoid-shaped, a member of family Propionibacteriaceae. Straight rods, has a fermentative type of metabolism of a limited number of organic acids, but not sugars. A thermophilic nitrate-reducing species. Rod-shaped, a denitrifying species from activated sludge of an aerobic-anaerobic wastewater treatment. A denitrifying species Coccoid-shaped, belonged to the family Propionibacteriaceae. A strains of denitrifying bacteria Rod-shaped, anaerobic carbohydrates bacteria thriving at high pH values A strain of anaerobic, syntrophic, propionate-oxidizing bacteria. Rod-shaped, a strain of green sulfur bacteria, acetate and propionate could serve as carbon sources Rod-shaped, A thermophilic, anaerobic bacterium, grow on carbohydrates. A fermentative and anaerobic bacterium.

99 99

NR 042021.1 NR 042550.1

99 99

NR 027224.1 NR 042317.1

99

NR 024989.1

99

NR 074365.1

99

NR 102981.1

99

NR 102964.1

9 10

Thauera phenylacetica strain B4P Alkaliflexus imshenetskii strain Z-7010

11

Smithella propionica strain LYP

12

Pelodictyon phaeoclathratiforme BU-1 strain BU-1

13

Thermosipho melanesiensis BI429 strain BI429

14

Sphaerochaeta pleomorpha str. Grapes strain Grapes

of VFA. Studies on the micromorphology of the granules suggested that colonies of VFA-oxidizing bacteria are closely linked with micro-colonies of hydrogen-consuming bacteria (namely denitrifying bacteria) allowing an efficient interspecies hydrogen transfer to speed up the degradation rates [14,38,39]. Together with the

FISH analysis, the VFA-oxidizing bacteria and denitrifying bacteria grew together to extend the food chain in the sludge to develop compact granules. Moreover, the intensity of bands in butyrate digester (R3) was obviously stronger than that in the other, which indicated that the two species with butyrate as substrate was more

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Y. Li et al. / Biochemical Engineering Journal 95 (2015) 104–111

Fig. 7. SEM images of the granules at the end of the experiment on cycle 95 with a magnification of 10,000 × .R1, R2 and R3 represented the denitrification digesters fed with acetate, propionate and butyrate, respectively. R4, R5 and R6 represented the non-nitrate digesters fed with acetate, propionate and butyrate, respectively.

regular. According to the report that parts of VFA-oxidizing bacteria were bacilli and some kinds of denitrifying bacteria were cocci [22], the result of SEM was consistent with those of FISH and DGGE images. These results further indicated that the VFA-oxidizing bacteria and denitrifying bacteria were the dominant bacteria in the granules. 4. Conclusion Adding nitrate to enhance the decomposition of VFA (acetate, propionate and butyrate) and the granulation of anaerobic process was proposed in this study. Butyrate was the most effective carbon source among the three VFA for granulation and denitrification due to the better electron transfer between VFA-oxidizing bacteria and denitrifying bacteria. The FISH, DGGE and SEM analysis indicated the denitrifying bacteria and VFA-oxidizing bacteria prevailed in the granulation, which was helpful to effectively accelerate the anaerobic sludge granule. Acknowledgments The authors acknowledge the financial support from the National Natural Scientific Foundation of China (51378087, 21177015). Fig. 8. DGGE fingerprints of microbial communities from different digesters at the end of the experiment on cycle 95. R1, R2 and R3 represented the denitrification digesters fed with acetate, propionate and butyrate, respectively. R4, R5 and R6 represented the non-nitrate digesters fed with acetate, propionate and butyrate, respectively.

abundant. Consequently, the bigger granules were formed in the butyrate digester. The granules were taken for a magnification (10,000 ×) of SEM at the end of the experiment. It showed different morphologies of microorganism among the six digesters. As shown in Fig. 7, amorphous microorganism prevailed the surface of the granules in the digesters with no addition of nitrate (R4, R5 and R6) irrespective of the type of VFA, whereas many coccus and bacilli were found on the surface of granules of the denitrification digesters (R1, R2 and R3). Notably, the coccus and bacilli in the denitrification digesters with propionate and especially butyrate were much denser and more

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