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Procedia Environmental Sciences 18 (2013) 515 - 521
2013 International Symposium on Environmental Science and Technology (2013 ISEST)
Degradation of Extracellular Polymeric Substances (EPS) in anaerobic digestion of dewatered sludge
Xiaohu Dai, Fan Luo, Lingling Dai, Bin Dong*
School of Environmental Science and Engineering, Tongji University, 1239 Siping Road, Shanghai 200092, China
Abstract
The degradation of extracellular polymeric substances based on high-solid anaerobic digestion was investigated by gel filtration chromatography (GFC) and three-dimensional excitation and emission matrix (EEM) The organic matters of Extracellular Polymeric Substances (EPS) in the sludge decreased 24% and shifted from the tightly bound EPS (TB-EPS) fraction to the Slime fraction during anaerobic digestion. The raw sludge and the digestate had opposite molecular weights (MW) distribution in EPS fractions. From the fluorescence spectra result, tyrosine-like substances were converted to tryptophan-like substances, and more humic-like substance in Slime fration was produced with the reduction of the protein-like substance during anaerobic digestion.
© 2013 The Authors. Published by Elsevier B.V.
Selection and peer-review under responsibility of Beijine Institute of Teehnology. Keywords: Extracellular polymeric substances (EPS); Degradation; Anaerobic digestion; High solid
1. Introduction
Large amount of sludge produced from wastewater treatment plants worldwide have received lots of attention [1]. The sludge is composed of microorganisms, extracellular polymeric substances (EPS), colloids, mineral particles and ionic components such as divalent cations [2]. EPS are a major component of organic substances (carbohydrates and proteins being major constituents with humic substances, uronic acids and nucleic acids in smaller quantities) for keeping the floc together in a three-dimensional matrix due to bridging with multivalent cations [3-5]. EPS have a crucial role within bacterial consortia and play a key role in the physico-chemical characteristics of sludge flocs. Hence, the anaerobic digestion process is based on EPS degradation by bacteria contained in flocs under anaerobic conditions.
* Corresponding author. Tel.: +86 21 65981794; fax: +86 21 65983602. E-mail address: tj_dongbin@163.com.
1878-0296 © 2013 The Authors. Published by Elsevier B.V.
Selection and peer-review under responsibility of Beijing Institute of Technology.
doi: 10.1016/j.proenv.2013.04.069
The anaerobic digestion process is commonly used in sludge treatment and thought to be effective in sludge stabilization by converting a part of its organic matter into biogas which is a renewable energy source. A survey of primary mesophilic anaerobic digestion (MAD) facilities which were considered to be well operated found the feedstocks were food wastes [6-8], agricultural wastes [9], and organic fraction of municipal solid wastes [10]. Recently, interest in high-solid anaerobic digestion is increasing, probably due to its advantages of smaller reactor volume, lower energy requirements for heating, less material handling compared with the traditional low-solid anaerobic digestion [11-12]. However, the sludge flocs are highly centralized and the concentration of EPS is increased in dewatered sludge. In this case, the degradation of EPS is especially important in high-solid anaerobic digestion.
Several researches had investigated the degradation of total EPS in anaerobic digestion with low-solid including excess sludge and actived sludge [13], and most of the reports proposed that the EPS can be classified as supernatant, Slime, loosely bound EPS (LB-EPS) and tightly bound EPS (TB-EPS) fractions, whereas the first fraction (supernatant) did not existed in the dewatered sludge. However, little information is available about the degradation of different EPS fractions when dewatered sludge has been investigated as feedstocks in high-solid anaerobic digestion.
In anaerobic digestion based on high-concentration of EPS, the change of cations and the degradation of organic matters in different EPS fractions were investigated.
2. Methods
2.1. Substrates and inoculums
Samples of dewatered sewage sludge were taken from Anting Waste Water Treatment Plant, located at Shanghai City, where the sludge was obtained by collecting primary and excess sludge and dewatered with the aid of a high-molecular flocculants. The mesophilic seed sludge was collected from an anaerobic digester of previous experiments. Characteristics of dewatered sludge and inoculums are listed in Table 1.
Table 1. Characteristics of the substrate and inoculums.
Parameters pH TS (%, w/w) VS/TS
Dewatered sludge 7.7±0.1 17.1%±0.2 60.5%±0.5
Inoculums 7.9±0.1 15.5%±0.2 41.2%±0.5
2.2. Reactor operation
Six liters of the inoculums (TS: 15.5%) was firstly put into a horizontal-type cylindrical reactor, with liquid working volume of 6.0 L, and purged with N2 for 10 min in order to provide anaerobic conditions. There was no substrate injection and no digestate discharge for three days in the reactor as an adaptation period. The raw sludge (dewatered sludge) was stored at 4 "Cand heated to 35 °C before everyday feeding. And then the reactor was operated semi-continuously (once-a-day draw-off and feeding) with dewatered sludge at 35X2 ± 1 °C, and equipped with helix-type stirrers, which were set at a rate of 60 rpm (rotations per minute) with 2 min stirring and 10 min break continuously. Volumes of produced biogas were measured by wet gas meters every day. In further operation, SRT was decreased to 30 days at a fixed solid content of 16% TS. Operation characteristics of anaerobic reactor are shown in Table 2.
Table 2. Operation characteristics of anaerobic reactor.
Parameters pH TS (%, w/w) VS/TS HRT (d) VSreduction (%) Methane content(%) Methane Yield(Y)
Raw sludge Digestate 7.7±0.1 8.0±0.1 17.1%±0.2 16.4%±0.2 60.5%±0.5 44.5%±0.5 30 43±2 60.3±1 0.37±0.02
2.3. Fractionation protocol
EPS extractions were carried out using different slightly modified methods which are described in detail by several authors [14-15]. For all extractions studied, the EPS distributed in the bulk solution after sludge settlement called the supernatant fraction, whereas it did not existed in the dewatered sludge. Hence, the fractionation process in this study was just divided into three parts. In brief, a 5g amount of the sludge sample was suspended in 50mL deionized water. The mixture was stirred at 500 rpm for 10 min with a vortex mixer, and then the suspension was centrifuged at 14000g for 20 min at 4 °C, in order to separate the Slime fraction from the solid. Then the collected bottom sediments were re-suspended to their original volume using a NaCl solution (0.5% w/w) at 70 °C and mixing it for 10 min. After the mixture was centrifuged (15000g for 20 min at 4 °C), the organic matter in the bulk solution was the loosely bound EPS (LB-EPS). The collected sediments were re-suspended again by the aforementioned NaCl solution at room temperature and placed in a water bath at 60 °C for 30 min [16]. The organic matter in the bulk solution was the tightly bound EPS (TB-EPS). After all the EPS fractions being extracted, membranes of 0.45(j.m polytetrafluoroethylene (PTFE) were used to filter out the particulates present in the Slime, LB-EPS, and TB-EPS solutions.
2.4. Analytical methods
The molecular weights (MW) were determined by a Gel Filtration Chromatography (LC-10ATVP, SHIMADZU), which was equipped with a differential detector (RID-10A). The GFC was run with the Milli-Q water at a flow rate of 0.5ml/min.
The three-dimensional excitation and emission matrix (EEM) spectra of different EPS fractions were measured by a fluorescence spectroscopy (FluoroMax-4, HORIBA Jobin Yvon), and were collected with subsequent scanning emission spectra from 300 to 550 nm at 2 nm increments by varying the excitation wavelength from 200 to 400 nm at 2 nm increments. The FRI technique was performed to analyze the excitation-emission regions of EEM spectroscopy, and the percentage of fluorescence response (Pi,n =®in/<I>T,n) was calculated according to Chen et al [17], where Oin and 0T n were the normalized excitation-emission area volumes referring to value of region i and the entire region, respectively. We divided EEM spectra into six excitation -emission regions: (I) tyrosine region with Ex/Em 200-250/300330 nm; (II) tyrosine-like protein region with Ex/Em 250-400/300-330 nm; (III) tryptophan region with Ex/Em 200-250/330-400 nm; (IV) tryptophan-like protein region with Ex/Em 250-400/330-400 nm; (V) fulvic acid-like region with Ex/Em 200-250 /400-550 nm and (VI) humic acid-like region with Ex/Em 250-400/400-550 nm [18].
TS, VS, Total alkalinity (TA) and free ammonia-nitrogen (FAN) concentrations were analyzed using standard methods (APHA (American Public Health Association), 1995. Standard Methods for the Examination of Water and Wastewater, 19th ed. Washington, DC, USA.); The composition of biogas was determined by employing a gas chromatograph (GC) (Agilent Technologies 6890N, CA, USA) with a thermal conductivity detector equipped with Hayseq Q mesh and Molsieve 5A columns. The volatile fatty
acid (VFA) was analyzed by a GC (Agilent Technologies 6890N, CA, USA) with flame ionization detector. The VSreduction was calculated by the formula as reported by Koch et al [20].
3. Results and discussion
3.1. Characteristics of organics in different EPS fractions
The distribution of COD in the different fractions was manifested that the degradation rate of organic matters in total EPS was just 24%. It can be explained that the cell lysis had released the intracellular organics which benefited microbial reproduction and produced new EPS. In the different EPS fractions for the raw sludge, PN and PS were occurred mainly (57.3% and 61.5%, respectively) in TB-EPS fraction, with a minor percentage (12-26%) detected in Slime and LB-EPS fractions. However, the organic composition of sludge was greatly changed during anaerobic digestion. PN and PS were dispersed mainly (48.8% and 62.4%, respectively) in the Slime fraction, which indicated that most of the biopolymers incorporated in TB-EPS were digested, scattered and attached in Slime fraction as the biocolloids.
MW distributions were investigated to further elucidate the organic nature of the different EPS fractions (Fig. 1). It was noted that for the raw sludge, there were four peaks at MW of 0.1, 0.3 and 10000 kDa presented in Slime, LB-EPS, and TB-EPS fractions, two more peaks detected in LB-EPS fraction, and three more peaks examined in TB-EPS fraction. These results revealed that the TB-EPS was more complex than the other two fractions, consistent with the results in Table 3 that PN and PS (the content of MW at 10-10000 kDa) contents in TB-EPS fraction were three-fold higher than those in the other EPS fractions. However, in the digestate, there were only two peaks at MW of 0.1 and 10000 kDa presented in the Slime, LB-EPS, and TB-EPS fractions, which were the same as the peaks of the raw sludge. This result can be explained that two substances at around 0.1 and 10000 kDa in the sludge were difficult to be digested in anaerobic digestion. The LB-EPS and TB-EPS fractions of the digestate have similar chemical components, and both of them were simpler than the Slime fraction.
(1) Raw sludge (2) Digestate
Fig. 1. Molecular weight distributions of different EPS fractions in raw sludge and digestate.
In this study three-dimensional EEM spectroscopy was applied for characterizing the EPS extracted from both the feed sludge and the digestate. Each EEM gave spectral information about the chemical compositions of EPS samples. Five peaks were readily identified from EEM fluorescence spectra of feed sludge and digestate in Fig. 2.
Table 3. Characteristics of different EPS fractions.
Raw sludge Digestate
Slime LB-EPS TB-EPS Slime LB-EPS TB-EPS
COD(mg/g) 857 532 19.44 17.66 675 698
Proteins(mg/g) 1.168 1.432 3.466 2.732 1.507 1.482
Polysaccharides(mg/g) 0.490 0.242 1.058 1.281 0.307 0.517
400 380 360 340 320 "È 300 m 280
260 -240 -
450 500
(1) Raw sludge
350 400 450 500
350 400 450 500
Em(nm)
(2) Digestate
450 500
Fig. 2. EEM fluorescence spectra of: (1) raw sludge; and (2) digestate.
Em(nm)
Em(nm)
E„(nm)
The first main peak was identified at excitation/emission wavelengths (Ex/Em) of 280-290/340-350 nm (peak A), while the second main peak was identified at Ex/Em of 220-240/340-370 nm (peak B). The two peaks have been described as protein-like peaks, in which the fluorescence is associated with the tryptophan (peak A) and tyrosine (peak B) respectively [21-24]. The other three peaks (peak C, peak D and peak F) were located around Ex/Em at 220-240/400-480, 360-380/400-480 and 300-320/400-480 described as humic-like substance [25]. A similar fluorescence signal was observed for both the feed sludge and the digestate, and their spectral characteristics were listed in Fig. 3.
Fig. 3 shows the FRI values of each region at various fractions both in the raw sludge and digestate. SMP of the Slime fraction in raw sludge were dominated by fluorescence in Regions II, III and V (28.0%, 20.6% and 20.0%, respectively), whereas the LB-EPS and the TB-EPS were mainly dispersed in Regions I, II and III, indicating that the majority of SMP fluorescence is attributed to protein-like substances.
Different changing trends of each region in various fractions between raw sludge and digestate were observed. The significant increases in FRI from 20.6% to 28.0% and from 20.0% to 30.1% were observed for Regions III and V in the Slime fraction, respectively. A slight increase was observed for Region III and IV in the LB-EPS and TB-EPS fractions. This result indicated that tyrosine-like substances were converted to tryptophan-like substances during the anaerobic digestion. The sum of FRI values for the four regions, regarded as protein-like region percentage, decreased during the anaerobic digestion and more humic-like substances were produced and accumulated in the Slime fraction.
a Slime a LB-EPS a TB-EPS b Slime b LB-EPS b TB-EPS
Fig. 3. FRI distribution of the six defined regions for different fractions in both (a) raw sludge and (b) digestate.
4. Conclusions
After anaerobic digestion, the concentration of organic matters in extracellular polymeric substances (EPS) decreased by 24%. Proteins and polysaccharides in the raw sludge were occurred mainly in tightly bound EPS (TB-EPS) fraction, while most of proteins and polysaccharides in the digestate were dispersed in Slime fraction. From the molecular weights distribution, the TB-EPS fraction of raw sludge was more complex than the other two fractions, whereas the Slime fraction of digestate was the most complex. Three fluorescent substances were readily identified in both the raw sludge and the digestate. The fluorescence regional integration (FRI) values of the substances indicated that, during anaerobic digestion, the tyrosine-like substances were converted to tryptophan-like substances, while more humic-like substances were produced and accumulated in the Slime fraction with the reduction of protein-like substance. The degradation of organic matters and the accumulation of soluble microbial products (SMP) in Slime fraction can result in the decomposition of floc structures.
Acknowledgements
This work was financially supported by the National Key Technologies R&D Program of China (2010BAC67B04) and the key projects of National Water Pollution Control and Management of China (2011ZX07316-004).
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