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Enhanced anaerobic digestibility of waste activated sludge by plant-derived biosurfactant
Accepted Manuscript Title: Enhanced anaerobic digestibility of waste activated sludge by plant-derived biosurfactant Author: Aijuan Zhou Haichao Luo Cristiano Varrone Youzhao Wang Wenzong Liu Aijie Wang Xiuping Yue PII: DOI: Reference:
S1359-5113(15)00258-5 http://dx.doi.org/doi:10.1016/j.procbio.2015.04.023 PRBI 10423
To appear in:
Process Biochemistry
Received date: Revised date: Accepted date:
14-3-2015 24-4-2015 30-4-2015
Please cite this article as: Zhou A, Luo H, Varrone C, Wang Y, Liu W, Wang A, Yue X, Enhanced anaerobic digestibility of waste activated sludge by plant-derived biosurfactant, Process Biochemistry (2015), http://dx.doi.org/10.1016/j.procbio.2015.04.023 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Enhanced anaerobic digestibility of waste activated sludge by plant-derived biosurfactant
Aijie Wang 2,5 and Xiuping Yue 1,*
College of Environmental Science and Engineering, Taiyuan University of Technology,
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1
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Taiyuan, China 2
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Aijuan Zhou 1, Haichao Luo 2, Cristiano Varrone 3, Youzhao Wang 4, Wenzong Liu 5,
State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of
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Technology (SKLUWRE, HIT), Harbin, China
Section for Sustainable Biotechnology, Aalborg University Copenhagen. Denmark
4
School of Mechanical Engineering and Automation, Northeastern University, Shenyang,
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3
China
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Beijing, China
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Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences,
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5
*Corresponding author: College of Environmental Science and Engineering, Taiyuan University of Technology, Taiyuan 030024, China E-mail: [email protected]; Tel./fax: +86 0351-6010214;
Abstract
To improve anaerobic digestibility of waste activated sludge (WAS), a novel treatment was explored by a plant-derived biosurfactant (saponin biosurfactant (SB)). SB showed positive effects on WAS hydrolysis and acidogenesis with increasing concentration, but was insignificant at dosage > 0.20 g SB /g total suspended solids (TSS). Soluble protein
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and carbohydrate concentrations reached 2029 ± 28 and 343 ± 52 mg chemical oxygen demand (COD)/L (0.20 g SB/g TSS, 48 h), which were 4.77-fold and 5.87-fold higher than that without treatment. Volatile fatty acids (VFAs) bioproduction showed a 3.51-
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fold increase in 72 h. Moreover, acetic acid increased already after 2 h with SB treatment, but after 8 h in the control, indicating that SB accelerated syntrophic acetogenesis from
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butyric acid degradation to acetic acid. Additionally, methane production was enhanced
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at lower dosage (< 0.05 g SB/g TSS) and inhibited at higher dosage (> 0.10 g SB/g TSS), with 63.0 and 20.3 mL/g volatile suspended solids (VSS) respectively, while it was 46.5
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mL/g VSS in the control. The results obtained are in good agreement with previous ones obtained with chemosynthetic surfactants. Moreover, SB has advantages of easy
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production and sustainability of supply, which should promote the implementation for
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Keywords:
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WAS treatment.
Waste activated sludge (WAS); Anaerobic digestibility; Extracellular polymeric
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substances (EPS); Saponin; Plant-derived biosurfactant
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1. Introduction Waste activated sludge (WAS) is the main solid waste produced in the wastewater
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treatment processes. About 13 million tons of WAS (dry weight) are produced in China every year [1], which is larger than that produced in the whole EU (10 million tons) [2].
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In 2008 the main methods to deal with WAS in China were land application (44.8%),
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landfill (31.0%), and incineration (3.5%), which all had their own environmental sideeffects [3]. Moreover, there were even 13.8% without any treatment. Recently, the
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conversion of WAS into high-value products and/or cheap energy resource is starting to be considered with increasing interest, rather than being discarded as a waste [4-6].
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Microbial conversion from WAS to valuable products is an innovative, cost-effective and
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promising way to gain social and economic benefits.
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Anaerobic digestion is widely applied for sludge reutilization, which basically follows the four steps: hydrolysis, acidogenesis, acetogenesis and methanogenesis [7],
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where hydrolysis is known as the rate-limiting step [3]. Pretreatment is a prerequisite constituent for WAS hydrolysis, within an industrially acceptable time frame. Various pretreatment methods were previously studied, including ultrasonic [8, 9], microwave [10], alkaline [11], thermal [12], and so on. Nowadays, surfactants are regarded as an alternative strategy for WAS pretreatment, due to their unique characteristics [13]. Jiang et al., for instance, investigated the effect of chemically synthetic surfactants, sodium dodecylsulphate (SDS) and sodium dodecyl benzene sulfonate (SDBS), on volatile fatty acids (VFAs) production from WAS fermentation [14, 15]. Luo et al. [16] and Zhou et al. [17] studied the feasibility of VFAs production from WAS by a pretreatment of microbial
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biosurfactant (rhamnolipid). Huang et al. (2015) tested the effects of three types of biosurfactants (surfactin, rhamnolipid and saponin) on VFA production during sludge anaerobic fermentation [18]. Data showed that hydrolysis and acidogenesis were all
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clearly enhanced with the pretreatments of surfactants. However, having a low biodegradability, most chemical surfactants would accumulate in the treatment system as
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anaerobic digestion of WAS has not been recognized yet.
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pollutants [19]. Moreover, as to our knowledge, the effect of surfactant on the whole
Most of the organic matter in WAS is embedded in its cells and extracellular
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polymer matrix. The biochemical composition of extracellular polymeric substances (EPS) is a critical factor to explore the destruction mechanisms of WAS enhanced by
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pretreatment [20]. The majority of dissolved organic matter (DOM) is represented by
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soluble microbial products originated from bound EPS, which is ubiquitous in wastewater
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and sludge [21]. Thus, characterization of EPS and DOM would be useful for understanding the WAS destruction mechanisms after pretreatment. Excitation–emission
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matrix (EEM) fluorescence spectroscopy, coupled with parallel factor analysis (PARAFAC), is an established tool for organic matter fingerprinting in aqueous systems [22]. The resulting EEM provides the fluorescence characteristics of samples over the range of excitation and emission wavelengths scanned simultaneously, without destruction the samples.
Due to the particular properties of low toxicity and biodegradable nature, biosurfactants are more desirable in environmental applications [23]. In fact, they do not cause secondary pollution, even if they leak and/or discharge into the ecosystem. Based on the considerations above, saponin (SB), as a well-known plant-derived biosurfactant,
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which has the advantage of easy production, lower price, sustainable supply and possible reuse, was used as a promising pretreatment to enhance anaerobic digestion of WAS in this study. The effect of SB dosage on hydrolysis, acidification and methanogenesis was
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assayed. Hydrolytic enzyme activities were also studied. The mechanism of enhanced anaerobic digestibility by SB was investigated. The different fractions of DOM and EPS
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dimensional EEM fluorescence spectroscopy with PARAFAC.
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from the sludge flocks (before and after SB treatment) were characterized using three-
2. Material and methods
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2.1 Characteristics of WAS and SB
WAS was collected from Taiping municipal wastewater treatment plant (Harbin
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City, Heilongjiang Province, China) and concentrated by settling at 4 °C for 24 h, prior to
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tests. Its characteristics were as follows: pH 6.7 ± 0.5, total suspended solids (TSS) 19710
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± 398 mg/L , volatile suspended solids (VSS) 14070 ± 172 mg/L , soluble protein 175 ± 27 mg chemical oxygen demand (COD)/L , soluble carbohydrate 12 ± 2 mg COD/L,
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SCOD (soluble COD) 912 ± 77 mg/L, TCOD (total COD) 22074 ± 498 mg/L and VFAs 244 ± 58 mg COD/L. Quillaja Bark saponin biosurfactant was obtained from Sangon Biotech. Co. (Shanghai, China), with a sapogenin content of 25%, and used without further purification.
2.2 Batch fermentations set-up Batch experiments were conducted in 500 mL serum bottles filled with 350 mL WAS each. The specific saponin dosages of 0.05, 0.10, 0.20, 0.25 and 0.30 g/g TSS was applied to the sludge. Blank tests were conducted without SB, in parallel. Nitrogen gas
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was flushed to remove oxygen, and all bottles were capped, sealed, and stirred in an airbath shaker (100 rpm) at 35 ± 1 °C for 8 d. VFAs concentration and gas composition were analyzed every day. The concentrations of hydrolysis products (soluble proteins and
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carbohydrates), inorganics released (ammonia and phosphorus) and hydrolytic enzymes activities (protease and α-glucosidase) were analyzed at the fermentation time of 48 h.
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The production rate (k) of methane production was calculated by fitting the exponential
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growth model. All the fermentation experiments were carried out in triplicate.
To investigate the effect of SB treatment on the syntrophic acetogenesis and
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methanogenesis steps during anaerobic treatment of WAS, the following fermentation tests with synthetic wastewater containing respectively sodium butyrate (NaBu) and
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sodium acetate (NaAc), were conducted. Anaerobic sludge of an up-flow anaerobic
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sludge blanket (UASB) reactor from the Taiping WWTP in Harbin (China) was used as
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the inoculum at a feed ratio of 1:10 (v:v, 1 L synthetic wastewater with 100 mL anaerobic sludge). The solid concentrations of the anaerobic sludge were as follows: total solid (TS)
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35.1 ± 3.3 g/L, volatile solid (VS) 17.8 ± 4.7 g/L, and VSS 15.1 ± 2.2 g/L. The ratio of NaBu or NaAc to anaerobic sludge was 0.56 g /g VS. The SB dosage of 0.20 g/g Bu was applied in the syntrophic acetogenesis test, while the SB dosages of 0.05 and 0.20 g/g Ac were applied in the methanogenesis test. The reason for the specific SB dosages chose will discuss later. Simultaneously, blank tests were conducted without SB. The chemical composition of synthetic wastewater was: NaBu or NaAc (1.0 g/L), potassium phosphate (50 mM, pH 7.0), KCl (0.13 g/L), NH4Cl (0.31 g/L), Wolf’s trace element solution (0.5 mL/L) and vitamin solution (0.5 mL/L). All other operations were the same as described above.
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2.3 Analytical methods WAS samples were centrifuged at 10000 g for 10 min and filtered through a 0.45
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μm cellulose nitrate membrane filter and stored at 4 °C prior to analysis. The determinations of SCOD, TCOD, TSS, VSS, ammonia (NH4+-N) and phosphorus (PO43--
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P) were conducted according to standard methods [24]. The pH value was measured by a
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pH meter (Seven Multi, Mettler Toledo, Switzerland). According to a previous study, carbohydrates and proteins were measured by the phenol-sulfuric and bicinchoninic acid
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methods with glucose and bull serum albumin as the standards, respectively [25]. An Agilent 7890 gas chromatography (GC) was utilized to analyze the composition of the
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VFAs. The VFAs production was calculated as the sum of the measured acetic (HAc),
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propionic (HPr), n-butyric (n-HBu), iso-butyric (iso-HBu), n-valeric (n-HVa) and iso-
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valeric (iso-HVa) acids. Gas composition was analyzed using another GC (4890D, Agilent) [5]. Ultrasound protocol for EPS extraction of WAS samples were detailed
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elsewhere [26]. The WAS filtrate (0.45 μm cellulose nitrate membrane filter) was used for the analysis of DOM. The collected sediments were re-suspended to their original volume using a pH 7 buffer solution (1.3 mM Na3PO4, 2.7 mM NaH2PO4, 6 mM NaCl and 0.7 mM KCl). The suspensions were centrifuged again at 5000 g for 15 min. The supernatant represented the LB-EPS. Collected sediments were re-suspended again with buffer solution and then treated using ultrasound at 20 kHz and 480 W for 10 min. The extracted solutions were centrifuged at 20000g for 20 min. The supernatant represented the TB-EPS.
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The activity of protease and α-glucosidase enzymes was measured according to Goel et al. [27]. The related enzymatic assays were based on the release of a chromogen, following the hydrolysis of the following synthetic substrates: azocasein (Sigma A2765,
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MW = 25000), p-nitrophenyl-α-D-glucopyranoside (Sigma N1377, MW = 301). The analytical procedure of protease was as follows: Sludge samples (3 mL) were mixed with
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azocasein solution (1 mL, 0.5% (w/v)) and incubated 90 min in 37 ºC. The reaction was
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stopped by adding 2 mL 10% trichloroacetic acid (w/v). After centrifugation at 2500 g for 5 min, 2 mL supernatant were mixed with 2 mL NaOH (2 M) and read at an
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absorbance of 340 nm. The analytical procedure of α-glucosidase was as follows: Sludge samples (1 mL) were mixed with p-nitrophenyl-α-D-glucopyranoside solution (1 mL,
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0.1% (w/v)) and pH 7.6 Tris-HCl buffer solution (2 mL, 0.2 M), and incubated 60 min in
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37 ºC. The termination of the reaction was obtained by heating the sample in boiling
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water bath for 3 min. After centrifugation at 2500 g for 5 min, the supernatant absorbance was read at 410 nm. All the solutions used for enzyme assay were incubated at the
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desired temperature for 30 min, before additions of the sludge sample, to achieve incubation temperature.
Fluorescence excitation-emission matrix (EEM) was measured using a fluorescence
spectrometry (FP-6500, Jasco, Tokyo, Japan). Parallel factor analysis (PARAFAC) was used to model EEM fluorescence data in this study. With the fermentation time of 48 h, three sludge layers (DOM, LB-EPS and TB-EPS) were prepared and extracted in the different sludge samples: raw WAS (simplified as 0); blank test (simplified as 1) and SBpretreated test at SB dosage of 0.02g/g TSS (simplified as 2). DOM samples were simplified as DOM-0, DOM-1 and DOM-2, LB-EPS and TB-EPS were also named in an
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analogous way. Before PARAFAC analysis, the Raman scattering was removed by subtracting the pure distilled water spectrum from the sample spectrum, and the Rayleigh scattering was overcome by inserting a series of zero values in the region of no
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fluorescence (excitation wavelength << emission wavelength). The analysis of residuals (sum of squared error) in both, the excitation and emission dimensions, was used to find
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(MathWorks Inc., USA) was employed for handling EEM data.
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out the appropriate number of PARAFAC components. The software Matlab 2012a
3. Results and discussion
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3.1 Enhanced WAS hydrolysis
SB pretreatment showed a positive effect on the hydrolysis of particulate organic
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matter from sludge flocks. As proteins and carbohydrates are the main constituents of
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WAS, the hydrolysis of organics can be expressed by that of soluble proteins and
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carbohydrates in the WAS. The concentration of soluble proteins and carbohydrates increased with the increase of SB dosage, but did not show any further increase at SB
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dosage > 0.20 g/gTSS (Fig. 1A). After 48 h, soluble protein and carbohydrate concentrations (at SB dosage of 0.20 g/gTSS) were 2029 ± 28 and 343 ±52 mgCOD/L, which were 4.8-fold and 5.9-fold higher than that in the Blank, respectively. This was in accordance with the results of the corresponding activities of protease (218 ± 22 vs 58 ± 21 Eu) and α-glucosidase (16 ± 3 vs 9 ± 1 Eu), with and without SB treatment (Fig. 1B). Degradation of protein was also improved by SB treatment, with an increase in ammonia during the reaction. Taking 0.20 g SB/g TSS as example, the released ammonia concentration reached 29.2 mg/L, with an increase of 91.0% over that in the blank test. Significant amount of soluble phosphorus was also released with the treatment of SB: the
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activity of acid and alkaline phosphatases were slightly increased in the presence of SB (Fig. 1B), which hydrolyzed phosphate esters and released the phosphate groups into liquid. Cadoret et al. (2002) reported that a variable fraction of enzyme activities were
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associated to the easily extractable EPS from sludge flocks [28]. The reason for this might be that SB facilitates the detachment of aggregated cells and of EPS, while
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releasing immobilized enzymes. This was confirmed by the EEM spectroscopy analysis
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(as shown below).
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3.2 Characterization of SB-pretreated WAS by EEM spectra with PARAFAC Fig. 2 shows the three-dimensional EEM spectroscopy characterizing the component
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changes of WAS, in terms of DOM, LB-EPS and TB-EPS, under SB treatment (0.02 g/g
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TSS, 48 h). As shown in Fig. 3, by separating the spectra of the main components from
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overlapped EEM spectra, four components were found to be appropriate for PARAFAC analysis. The specific components were identified as tryptophan-like (Ex/Em 270/350,
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Com.1), tyrosine-like (Ex/Em 270/300, Com.2), other protein-like (Ex/Em 225/300, Com.3) and fulvic-like substrates (Ex/Em 300/440, Com.4) (Fig. 4) [4, 29]. The excitation and emission spectra (loadings) of the four organic fluorphores overlapped slightly and smoothly (Fig. 5). Additionally, excitation spectra had one or more maxima, but emission spectra only exhibited one emission maximum, which fitted the expected spectral properties of fluorophores well [30]. The DOM and EPS fluorescence spectra of three samples were similar in the peak locations, but had different fluorescence intensities (FI) (Fig. 2). The intensities of DOM were clearly enhanced by SB treatment. Tyrosine-like substance was the primary
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component (FI: 655), which was 11.6-fold higher than that in the un-pretreated WAS (FI: 52). Tryptophan-like and other protein-like substrates ranked second and third place. This also suggested the significance of dissolving of proteins, that were inside the microbial
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cell wall or enmeshed in EPS, into liquid phase under SB treatment. These results were in good agreement with practical measurements (Fig. 1A). Small amounts of fulvic-acid-
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like substance were found in both, DOM and EPS fluorescence spectra, which were
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mainly formed by the microbial degradation of dead organic matter [4]. It is believed that the disruption of EPS matrix could enhance the rate and extent of sludge biodegradation
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during anaerobic digestion [31]. Bound EPS exhibited a dynamic double-layer-like structure, the LB-EPS layer was characterized by loose structure and rheological
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property, which was easily detached by surfactant. On the contrary, the TB-EPS layer
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was tightly attached to the cell surface, in which matrix the organics enmeshed was hard
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to be disintegrated. As shown in Fig. 2, the FI of EPS peaks in EEM spectra were significantly decreased by SB treatment. Taking tyrosine-like substances into
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consideration, the concentration declined simultaneously in the LB-EPS and TB-EPS (FI: from 236 and 590 to 105 and 237). Thus, the SB doasge applied in this study could effectively decompose the LB-EPS layer, and further destroy the TB-EPS located in inner parts of sludge flocks. The same phenomenon was observed in the other three substrates. The detaching of EPS from WAS was evidently improved by SB treatment.
3.3 Improved VFAs bioproduction VFAs bioproduction was also improved during WAS fermentation, in the presence of SB (Fig. 6A). It sharply increased until 72 h, and fluctuated little with the further
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increase of fermentation time. At a fermentation time of 72 h, the specific VFAs yields were 1.6-fold, 2.5-fold, 3.5-fold, 4.0-fold and 3.8-fold higher than that in the control (1153 ± 28 mg/COD L) with an SB dosages of 0.05, 0.10, 0.20, 0.25 and 0.30 g/g TSS,
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respectively. As can be observed in Fig. 6A, VFAs yield increased with the increase of SB dosage but didn’t enhance substantially at a SB dosage >0.20 g/g TSS. Further
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investigation (at 72 h fermentation time) revealed that it followed a parabolic curve, in
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the range of 0.05~0.30 g SB/g TSS (YVFAs = -65241 X2SB dosage + 33819 XSB dosage +1267, R² = 0.997). This was true at any fermentation time (quadratic equations not shown).
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Increasing the SB dosage from 0 to 0.05, 0.05 to 0.10, 0.10 to 0.20, 0.20 to 0.25 and from 0.25 to 0.3 g/g TSS, the generated incremental VFAs were 1416, 884, 450, 414 and 0 g
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COD/L respectively. The reason behind this phenomenon maybe that SB could induce
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EPS (and other bacterial aggregations) to leave the attached cell surface, as confirmed by
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the 3D-EEM analysis. Furthermore, it could also enhanced aqueous solubility of nonaqueous phase organics, by reducing their surface/interfacial tension. A certain amount of
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EPS, and other bacterial aggregations, was present in the WAS fermentation system. Hence, as shown in Fig.1A, the concentration of detached soluble organics was much higher with the addition of initial 0.1 g SB /g TSS, which provided more substrate to the subsequent acidification step and lead to more VFAs production. Restated, the SB dosage from 0–0.05 g SB/g TSS showed the highest increment rate for VFAs. According to the composition analysis, the six VFAs increased by varying degrees, during SB treatment. Among them, HAc and HPr showed a relatively larger increase, the sum of their concentrations reaching 2752 mg COD/L, versus 763 mg COD/L in the control, after 72 h fermentation time (at SB dosage of 0.05 g SB/g TSS). The main three
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(individual) VFAs produced were HAc (32 ± 1.4%), HPr (23 ± 0.9%) and n-HBu (17± 1.1%), at a SB dosage of 0.05 g/g TSS (Fig. 6B). Clearly, the product spectrum can be affected by the pretreatment method. Jiang et al. [14, 15], for instance, observed that the
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order of the individual VFAs was: HAc > HPr > iso-HVa by SDBS (0.02g/g TSS, 6d), and HAc > iso-HVa > HPr by SDS (0.1 g/g TSS, 6d) chemical-synthesis surfactant
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pretreatments. While Luo et al. [16] showed that the main VFAs from WAS
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fermentation, pretreated by a microbial biosurfactant (rhamnolipid), were HPr > HAc > iso-HVa (0.3 g/g DS, 2d). On the other hand, the spectrum is also determined by the
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dosage concentration (Fig. 6C). With the increase of SB dosage from 0 to 0.20/g TSS, HAc concentration increased from 436 to 1434 mg COD/L. Correspondingly, the
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produced HBu and HVa also increased 6.7 and 4.4 times over that without SB treatment,
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respectively. Increased concentrations of HBu and HVa probably derived from the
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degradation of carbohydrates and proteins in the WAS, while HVa might be mainly associated with the fermentation of proteins, via reductive deamination of single amino
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acids or by oxidation–reduction between pairs of amino acids via the Stickland reaction [32].
3.4 Effect of SB treatment on syntrophic acetogenesis and methanogenesis As stated previously, SB pretreatment had a positive effect on the hydrolysis and
acidogenesis steps during WAS digestion. VFAs, as the products of acidogenesis, are the substrates for methanogenesis and can be easily metabolized to methane. We noted that (cumulative) methane production was improved at SB dosage of 0.05 g/g TSS and inhibited at SB dosage > 0.10 g/g TSS (Fig. 7A). The data obtained fitted the exponential
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growth model (YCH4=e-kt) and the corresponding production rate constants (k) were calculated. With a fermentation time of 192 h (t), the cumulative methane yield (YCH4) increased from 46.5 mL/gVSS in the blank (k=0.0201 h-1, R2=0.9912) to 63.0 mL/gVSS
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at SB dosage of 0.05 g/g TSS (k=0.0219 h-1, R2=0.9620), while it decreased to 20.3 mL/gVSS at dosage of 0.10 g/g TSS (k=0.0154 h-1, R2=0.9935). The same result was
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observed in the study with synthetic wastewater containing NaAc. Data obtained fitted
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the linear growth model (YCH4= constant +kt). As shown in Fig. 7B, the methane production slightly increased from 4.7 ± 0.5 mL/g Ac (k=0.1927 h-1, R2=0.9401), in the
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control, to 6.6 ± 0.3 mL/g Ac (k=0.2745 h-1, R2=0.9696), at SB dosage of 0.05 g/g Ac, while it was inhibited at a dosage of 0.20 g/g Ac (0.5 ± 0.0 mL/g Ac, k=0.0233 h-1,
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R2=0.9865) after 24 h fermentation time. That is, the methanogenesis was enhanced at
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lower SB dosage (< 0.10 g/g TSS) and inhibited at higher SB dosage (> 0.10 g/g TSS).
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Almost the same phenomenon was found by using chemically synthetic surfactants. Jiang et al. showed that the inhibition rate of methane production increased sharply from 50 %
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to 100 % with the SDS dosage increasing from 0.1 to 0.3 g/g (at fermentation time of 21 d) [15]. He also found that the methane yield decreased from 65 (blank) to 16 and 8 mL/g VSS, at dosages of 0.05 and 0.1 g SDBS /g TSS, respectively [14]. Our previous study also found that the cumulative methane production decreased sharply from 58.8 mL/g VSS, in the blank, to 2.0 mL/g VSS with the treatment of rhamnolipid (0.04 g/g TSS, 192 h) [17]. Conversely, the acid-forming bacteria were not impaired by surfactant pretreatment, thereby maximizing yield of VFAs (Fig. 6A). Fig. 7C shows HBu conversion into HAc and the consequent methane production, at SB dosage of 0.20 g/g Bu. HBu concentration sharply decreased in the first 10 hours of
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fermentation and then stayed relatively constant, both with or without SB treatment. HAc, as the product of HBu degradation via ß-oxidation pathway, increased correspondingly and was subsequently degraded into methane. The specific
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thermodynamic equation of HBu degradation by acetogenic bacteria in the syntrophic acetogenesis process is shown below [32]:
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HBu+2H2O→2HAc+2H2
(1)
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Apparently, SB treatment increased the HBu degradation rate, as it was rapidly converted into HAc (from 2 h onward), while 8h were need without SB treatment. The result of
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methane production from the converted HAc further proved this point. After 16 h fermentation, the methane yield was 2.7 ± 0.1 mL/g Bu in the SB-treated and 1.5 ± 0.1
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mL/g Bu in the control tests, respectively.
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3.5 Significance of SB pretreatment and potential implementation Saponin has a wide range of industrial applications as a surfactant [33]. This study
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successfully demonstrated the feasibility of SB pretreatment for the enhancement of anaerobic digestibility of WAS. Currently, many researchers have conducted studies for the VFAs recovery from WAS and investigated the acidogenic fermented sludge as the feedstock for subsequent biological nutrient removal [34], polyhydroxyalkanoates production [35], hydrogen production by MEC reactor [5], and so on. A comparison of different VFAs yield from WAS fermentation by surfactants pretreatment is given in Table 1. The VFAs yield of 211±7 mg COD/g VSS obtained with treatment of 0.05 g SB /g TSS is comparable with the 222 mg COD/g VSS reported by Luo et al., 2012, using rhamnolipid (0.30 g/g DS) at mesophilic conditions. Jiang et al.
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found that the maximum VFAs yields of 235 and 241 mg COD/g VSS were achieved by treatments of SDS and SDBS, at ambient temperature, respectively. Seemingly, the use of chemosynthetic surfactants for VFAs production presented some advantages compared to
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SB biosurfactant. However, although the treatment temperature of chemosynthetic surfactants was lower (21 vs 35 °C), the needed fermentation time was twice that with
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SB. Moreover, as stated in paragraph 3.4, cumulative methane production was improved
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at SB dosage of 0.05 g/g TSS, which was generated from VFAs metabolism. Hence, the acidification efficiency should consider this consumed part as well (CODmethane), which
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can be calculated using a conversion of 0.35 m3 methane/ kg COD, under standard conditions [36]. In the present study, methane yield was 9.2 mL /g VSS (that is 26 mg
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COD/g VSS) at a SB dosage of 0.05 g/g TSS for 72 h, while it was negligible under SDS
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and SDBS treatments. Thus, from an overall perspective of WAS digestion, SB treatment
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was able to match the results obtained in other researches in this field. During the process of anaerobic digestion, the excessive formation of foam can
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induce a negative impact on the efficiency of biogas production, especially for the processes that utilize renewable resources or biogenic waste material as feedstock [37]. Normally, the liquid phase is located in a thin film, which is present between the gas bubbles. However, foam can be produced only after a certain threshold concentration of a surface-active compound has been exceeded. Biosurfactants are known to be one kind of surface-active compounds [38]. Hence, the utilization of biosurfactants for the anaerobic treatment of WAS requires solutions to prevent this phenomenon. Previous studies showed that the application of anti-foaming agents is a common treatment method that could minimize or even avoid the formation of foam [37]. Detailed investigation on the
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suitable anti-foaming agent for the SB-treated WAS digestion, as well as the dosage optimization and the microbial communities evolution induced by the SB and antifoaming agent, would provide valuable insight into the viable utilization of SB for WAS
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treatments. Therefore, further research is advisable. 4. Conclusions
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The contribution of saponin biosurfactant (SB) for improving anaerobic digestibility
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of WAS was successfully demonstrated. SB addition increased WAS hydrolysis substantially, in terms of VFAs production, with the increase of SB dosage ranging from
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0-0.2 g/g TSS. The activities of hydrolytic enzymes were all improved, because SB favoured the release of immobilized enzymes in the EPS and other bacterial aggregations.
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From EEM spectra analysis, SB treatment could effectively decompose the LB-EPS
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layer, and further detach the TB-EPS layer. Soluble protein and carbohydrate
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concentrations were 4.77-fold and 5.87-fold higher than that without SB addition (0.20 g SB/g TSS, 48 h), respectively. The main three individual VFAs were HAc, HPr and n-
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HBu. Methane production was enhanced by 35% at lower concentration (0.05 g SB/g TSS), but decreased by 56% at higher SB dosage (0.10 g SB/g TSS), compared to control conditions. The results obtained in the present study, using plant-derived biosurfactants, are comparable with previous ones obtained with chemosynthetic surfactants. However, it must be underlined that biosurfactants, such as saponin, have the advantage of an easy production, lower prices, sustainability of supply and possibility of reuse over synthetic surfactants, which should promote their implementation for WAS treatment.
Acknowledgement
Page 17 of 34
This research was supported by National Natural Science Foundation of China (NSFC, Grant No. 51208496 and 51378330), by Science Fund for Creative Research Groups of the National Natural Science Foundation of China (Grant No.51121062), by
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National Science Foundation for Distinguished Young Scholars of China (Grant No. 51225802), by National High-tech R&D Program of China (863 Program, Grant No.
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2012AA051502), by ‘‘Hundred Talents Program’’ of the Chinese Academy of Sciences,
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by Chinese Academy of Sciences (135 Project, Grant No. YSW2013B06).
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[35] Cai M, Chua H, Zhao Q, Sin N, Ren J. Optimal production of polyhydroxyalkanoates (PHA) in activated sludge fed by volatile fatty acids (VFAs) generated
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[38] Kosaric N, Cairns WL. Biosurfactants and Biotechnology. NewYork: Marcel
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Page 23 of 34
Table 1 Comparison of VFAs yield from WAS fermentation by surfactants pretreatment VFAs yield (mg
time (h)
COD/g VSS)
Dosages
References 48
222
SDS
0.10 g/g TSS
144
235
SDBS
0.02 g/g TSS
144
241
Saponin
0.05 g/g TSS
72
211 ± 7
[16]
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0.30 g/g DS
[15] [14]
This study
Ac ce p
te
d
M
an
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Rhmnolipid
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Surfactants
Fermentation
Page 24 of 34
Figure captions Figure 1 Effect of SB dosage on hydrolysis products and inorganics released (A) and
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hydrolytic enzymes activities at SB dosage of 0.20 g/g TSS (B)
Figure 2 EEM fluorescence spectra and spectral intensity of components for DOM, LB-
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EPS and TB-EPS obtained from (1) blank and (2) SB-pretreated WAS with the
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fermentation time of 48 h. (0) The spectra from raw WAS were used as controls. Figure 3 The sum of squared error (residuals) in both the excitation and emission
an
dimensions for three different models.
Figure 4 Four components of DOM and EPSs decomposed by the PARAFAC approach:
M
(Com.1) tryptophan-like substances, Ex/Em (270/350); (Com.2) tyrosine-like
d
substances, Ex/Em (270/300); (Com.3) protein-like substances, Ex/Em
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(225/300); (Com.4) fulvic-like substrates, Ex/Em (300/440). Figure 5 The excitation and emission spectra (loadings) of four components derived from
Ac ce p
PARAFAC modeling: (Com. 1) tryptophan-like substances, (Ex/Em, 270/350); (Com. 2) tyrosine-like substances, Ex/Em (270/300); (Com. 3) protein-like substances, Ex/Em (225/300); (Com. 4) fulvic-like substrates Ex/Em (300/440).
Figure 6 Effect of SB dosage on VFAs production (A) and composition (B) Figure 7 Effect of SB dosage on methane production from WAS (A), methane production from HAc at SB dosages of 0.05 and 0.20 g/g Ac (B), HAc conversion and methane production from HBu degradation at SB dosage of 0.20 g/g Bu (C).
Page 25 of 34
Ac ce p
te
d
M
an
us
cr
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> Anaerobic digestibility of waste activated sludge (WAS) was enhanced by saponin (SB). > SB showed positive effects on WAS hydrolysis and acidogenesis. > Methanogenesis was enhanced at lower dosage and inhibited at higher dosage. > Hydrolytic enzymes activity was improved. > SB could decompose the LB-EPS layer, and further detach the TB-EPS layer of WAS.
Page 26 of 34
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te
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*Graphical Abstract
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Figure 1
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554
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Fig. 1 Effect of SB dosage on hydrolysis products and inorganics released (A) and
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hydrolytic enzymes activities at SB dosage of 0.20 g/g TSS (B)
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556
Ac
ce pt
ed
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558
25
Page 28 of 34
Figure 2
560
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ed
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559
Fig. 2 EEM fluorescence spectra and spectral intensity of components for DOM,
562
LB-EPS and TB-EPS obtained from (1) blank and (2) SB-pretreated WAS with the
563
fermentation time of 48 h. (0) The spectra from raw WAS were used as controls.
564 565
Ac
561
26
Page 29 of 34
Figure 3
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566
567
Fig. 3 The sum of squared error (residuals) in both the excitation and emission
569
dimensions for three different models.
ed
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568
570
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ce pt
571
27
Page 30 of 34
Figure 4
573
Ac
ce pt
ed
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572
574
Fig. 4 Four components of DOM and EPSs decomposed by the PARAFAC approach:
575
(Com.1) tryptophan-like substances, Ex/Em (270/350); (Com.2) tyrosine-like
576
substances, Ex/Em (270/300); (Com.3) protein-like substances, Ex/Em (225/300);
577
(Com.4) fulvic-like substrates, Ex/Em (300/440).
578 579 28
Page 31 of 34
Figure 5
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an
us
cr
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580
ed
581
Fig. 5 The excitation and emission spectra (loadings) of four components derived
583
from PARAFAC modeling: (Com. 1) tryptophan-like substances, (Ex/Em, 270/350);
584
(Com. 2) tyrosine-like substances, Ex/Em (270/300); (Com. 3) protein-like substances,
585
Ex/Em (225/300); (Com. 4) fulvic-like substrates Ex/Em (300/440).
587
Ac
586
ce pt
582
29
Page 32 of 34
Figure 6
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588
589
Fig. 6 Effect of SB dosage on VFAs production (A) and composition (B)
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590 591
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Figure 7
ed
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593
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Fig. 7 Effect of SB dosage on methane production from WAS (A), methane
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production from HAc at SB dosages of 0.05 and 0.20 g/g Ac (B), HAc conversion and
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methane production from HBu degradation at SB dosage of 0.20 g/g Bu (C).
598 599
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595
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S1359-5113(15)00258-5 http://dx.doi.org/doi:10.1016/j.procbio.2015.04.023 PRBI 10423
To appear in:
Process Biochemistry
Received date: Revised date: Accepted date:
14-3-2015 24-4-2015 30-4-2015
Please cite this article as: Zhou A, Luo H, Varrone C, Wang Y, Liu W, Wang A, Yue X, Enhanced anaerobic digestibility of waste activated sludge by plant-derived biosurfactant, Process Biochemistry (2015), http://dx.doi.org/10.1016/j.procbio.2015.04.023 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Enhanced anaerobic digestibility of waste activated sludge by plant-derived biosurfactant
Aijie Wang 2,5 and Xiuping Yue 1,*
College of Environmental Science and Engineering, Taiyuan University of Technology,
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1
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Taiyuan, China 2
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Aijuan Zhou 1, Haichao Luo 2, Cristiano Varrone 3, Youzhao Wang 4, Wenzong Liu 5,
State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of
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Technology (SKLUWRE, HIT), Harbin, China
Section for Sustainable Biotechnology, Aalborg University Copenhagen. Denmark
4
School of Mechanical Engineering and Automation, Northeastern University, Shenyang,
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3
China
Ac ce p
Beijing, China
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Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences,
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5
*Corresponding author: College of Environmental Science and Engineering, Taiyuan University of Technology, Taiyuan 030024, China E-mail: [email protected]; Tel./fax: +86 0351-6010214;
Abstract
To improve anaerobic digestibility of waste activated sludge (WAS), a novel treatment was explored by a plant-derived biosurfactant (saponin biosurfactant (SB)). SB showed positive effects on WAS hydrolysis and acidogenesis with increasing concentration, but was insignificant at dosage > 0.20 g SB /g total suspended solids (TSS). Soluble protein
Page 1 of 34
and carbohydrate concentrations reached 2029 ± 28 and 343 ± 52 mg chemical oxygen demand (COD)/L (0.20 g SB/g TSS, 48 h), which were 4.77-fold and 5.87-fold higher than that without treatment. Volatile fatty acids (VFAs) bioproduction showed a 3.51-
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fold increase in 72 h. Moreover, acetic acid increased already after 2 h with SB treatment, but after 8 h in the control, indicating that SB accelerated syntrophic acetogenesis from
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butyric acid degradation to acetic acid. Additionally, methane production was enhanced
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at lower dosage (< 0.05 g SB/g TSS) and inhibited at higher dosage (> 0.10 g SB/g TSS), with 63.0 and 20.3 mL/g volatile suspended solids (VSS) respectively, while it was 46.5
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mL/g VSS in the control. The results obtained are in good agreement with previous ones obtained with chemosynthetic surfactants. Moreover, SB has advantages of easy
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production and sustainability of supply, which should promote the implementation for
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Keywords:
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WAS treatment.
Waste activated sludge (WAS); Anaerobic digestibility; Extracellular polymeric
Ac ce p
substances (EPS); Saponin; Plant-derived biosurfactant
Page 2 of 34
1. Introduction Waste activated sludge (WAS) is the main solid waste produced in the wastewater
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treatment processes. About 13 million tons of WAS (dry weight) are produced in China every year [1], which is larger than that produced in the whole EU (10 million tons) [2].
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In 2008 the main methods to deal with WAS in China were land application (44.8%),
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landfill (31.0%), and incineration (3.5%), which all had their own environmental sideeffects [3]. Moreover, there were even 13.8% without any treatment. Recently, the
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conversion of WAS into high-value products and/or cheap energy resource is starting to be considered with increasing interest, rather than being discarded as a waste [4-6].
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Microbial conversion from WAS to valuable products is an innovative, cost-effective and
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promising way to gain social and economic benefits.
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Anaerobic digestion is widely applied for sludge reutilization, which basically follows the four steps: hydrolysis, acidogenesis, acetogenesis and methanogenesis [7],
Ac ce p
where hydrolysis is known as the rate-limiting step [3]. Pretreatment is a prerequisite constituent for WAS hydrolysis, within an industrially acceptable time frame. Various pretreatment methods were previously studied, including ultrasonic [8, 9], microwave [10], alkaline [11], thermal [12], and so on. Nowadays, surfactants are regarded as an alternative strategy for WAS pretreatment, due to their unique characteristics [13]. Jiang et al., for instance, investigated the effect of chemically synthetic surfactants, sodium dodecylsulphate (SDS) and sodium dodecyl benzene sulfonate (SDBS), on volatile fatty acids (VFAs) production from WAS fermentation [14, 15]. Luo et al. [16] and Zhou et al. [17] studied the feasibility of VFAs production from WAS by a pretreatment of microbial
Page 3 of 34
biosurfactant (rhamnolipid). Huang et al. (2015) tested the effects of three types of biosurfactants (surfactin, rhamnolipid and saponin) on VFA production during sludge anaerobic fermentation [18]. Data showed that hydrolysis and acidogenesis were all
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clearly enhanced with the pretreatments of surfactants. However, having a low biodegradability, most chemical surfactants would accumulate in the treatment system as
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anaerobic digestion of WAS has not been recognized yet.
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pollutants [19]. Moreover, as to our knowledge, the effect of surfactant on the whole
Most of the organic matter in WAS is embedded in its cells and extracellular
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polymer matrix. The biochemical composition of extracellular polymeric substances (EPS) is a critical factor to explore the destruction mechanisms of WAS enhanced by
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pretreatment [20]. The majority of dissolved organic matter (DOM) is represented by
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soluble microbial products originated from bound EPS, which is ubiquitous in wastewater
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and sludge [21]. Thus, characterization of EPS and DOM would be useful for understanding the WAS destruction mechanisms after pretreatment. Excitation–emission
Ac ce p
matrix (EEM) fluorescence spectroscopy, coupled with parallel factor analysis (PARAFAC), is an established tool for organic matter fingerprinting in aqueous systems [22]. The resulting EEM provides the fluorescence characteristics of samples over the range of excitation and emission wavelengths scanned simultaneously, without destruction the samples.
Due to the particular properties of low toxicity and biodegradable nature, biosurfactants are more desirable in environmental applications [23]. In fact, they do not cause secondary pollution, even if they leak and/or discharge into the ecosystem. Based on the considerations above, saponin (SB), as a well-known plant-derived biosurfactant,
Page 4 of 34
which has the advantage of easy production, lower price, sustainable supply and possible reuse, was used as a promising pretreatment to enhance anaerobic digestion of WAS in this study. The effect of SB dosage on hydrolysis, acidification and methanogenesis was
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assayed. Hydrolytic enzyme activities were also studied. The mechanism of enhanced anaerobic digestibility by SB was investigated. The different fractions of DOM and EPS
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dimensional EEM fluorescence spectroscopy with PARAFAC.
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from the sludge flocks (before and after SB treatment) were characterized using three-
2. Material and methods
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2.1 Characteristics of WAS and SB
WAS was collected from Taiping municipal wastewater treatment plant (Harbin
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City, Heilongjiang Province, China) and concentrated by settling at 4 °C for 24 h, prior to
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tests. Its characteristics were as follows: pH 6.7 ± 0.5, total suspended solids (TSS) 19710
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± 398 mg/L , volatile suspended solids (VSS) 14070 ± 172 mg/L , soluble protein 175 ± 27 mg chemical oxygen demand (COD)/L , soluble carbohydrate 12 ± 2 mg COD/L,
Ac ce p
SCOD (soluble COD) 912 ± 77 mg/L, TCOD (total COD) 22074 ± 498 mg/L and VFAs 244 ± 58 mg COD/L. Quillaja Bark saponin biosurfactant was obtained from Sangon Biotech. Co. (Shanghai, China), with a sapogenin content of 25%, and used without further purification.
2.2 Batch fermentations set-up Batch experiments were conducted in 500 mL serum bottles filled with 350 mL WAS each. The specific saponin dosages of 0.05, 0.10, 0.20, 0.25 and 0.30 g/g TSS was applied to the sludge. Blank tests were conducted without SB, in parallel. Nitrogen gas
Page 5 of 34
was flushed to remove oxygen, and all bottles were capped, sealed, and stirred in an airbath shaker (100 rpm) at 35 ± 1 °C for 8 d. VFAs concentration and gas composition were analyzed every day. The concentrations of hydrolysis products (soluble proteins and
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carbohydrates), inorganics released (ammonia and phosphorus) and hydrolytic enzymes activities (protease and α-glucosidase) were analyzed at the fermentation time of 48 h.
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The production rate (k) of methane production was calculated by fitting the exponential
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growth model. All the fermentation experiments were carried out in triplicate.
To investigate the effect of SB treatment on the syntrophic acetogenesis and
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methanogenesis steps during anaerobic treatment of WAS, the following fermentation tests with synthetic wastewater containing respectively sodium butyrate (NaBu) and
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sodium acetate (NaAc), were conducted. Anaerobic sludge of an up-flow anaerobic
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sludge blanket (UASB) reactor from the Taiping WWTP in Harbin (China) was used as
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the inoculum at a feed ratio of 1:10 (v:v, 1 L synthetic wastewater with 100 mL anaerobic sludge). The solid concentrations of the anaerobic sludge were as follows: total solid (TS)
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35.1 ± 3.3 g/L, volatile solid (VS) 17.8 ± 4.7 g/L, and VSS 15.1 ± 2.2 g/L. The ratio of NaBu or NaAc to anaerobic sludge was 0.56 g /g VS. The SB dosage of 0.20 g/g Bu was applied in the syntrophic acetogenesis test, while the SB dosages of 0.05 and 0.20 g/g Ac were applied in the methanogenesis test. The reason for the specific SB dosages chose will discuss later. Simultaneously, blank tests were conducted without SB. The chemical composition of synthetic wastewater was: NaBu or NaAc (1.0 g/L), potassium phosphate (50 mM, pH 7.0), KCl (0.13 g/L), NH4Cl (0.31 g/L), Wolf’s trace element solution (0.5 mL/L) and vitamin solution (0.5 mL/L). All other operations were the same as described above.
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2.3 Analytical methods WAS samples were centrifuged at 10000 g for 10 min and filtered through a 0.45
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μm cellulose nitrate membrane filter and stored at 4 °C prior to analysis. The determinations of SCOD, TCOD, TSS, VSS, ammonia (NH4+-N) and phosphorus (PO43--
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P) were conducted according to standard methods [24]. The pH value was measured by a
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pH meter (Seven Multi, Mettler Toledo, Switzerland). According to a previous study, carbohydrates and proteins were measured by the phenol-sulfuric and bicinchoninic acid
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methods with glucose and bull serum albumin as the standards, respectively [25]. An Agilent 7890 gas chromatography (GC) was utilized to analyze the composition of the
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VFAs. The VFAs production was calculated as the sum of the measured acetic (HAc),
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propionic (HPr), n-butyric (n-HBu), iso-butyric (iso-HBu), n-valeric (n-HVa) and iso-
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valeric (iso-HVa) acids. Gas composition was analyzed using another GC (4890D, Agilent) [5]. Ultrasound protocol for EPS extraction of WAS samples were detailed
Ac ce p
elsewhere [26]. The WAS filtrate (0.45 μm cellulose nitrate membrane filter) was used for the analysis of DOM. The collected sediments were re-suspended to their original volume using a pH 7 buffer solution (1.3 mM Na3PO4, 2.7 mM NaH2PO4, 6 mM NaCl and 0.7 mM KCl). The suspensions were centrifuged again at 5000 g for 15 min. The supernatant represented the LB-EPS. Collected sediments were re-suspended again with buffer solution and then treated using ultrasound at 20 kHz and 480 W for 10 min. The extracted solutions were centrifuged at 20000g for 20 min. The supernatant represented the TB-EPS.
Page 7 of 34
The activity of protease and α-glucosidase enzymes was measured according to Goel et al. [27]. The related enzymatic assays were based on the release of a chromogen, following the hydrolysis of the following synthetic substrates: azocasein (Sigma A2765,
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MW = 25000), p-nitrophenyl-α-D-glucopyranoside (Sigma N1377, MW = 301). The analytical procedure of protease was as follows: Sludge samples (3 mL) were mixed with
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azocasein solution (1 mL, 0.5% (w/v)) and incubated 90 min in 37 ºC. The reaction was
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stopped by adding 2 mL 10% trichloroacetic acid (w/v). After centrifugation at 2500 g for 5 min, 2 mL supernatant were mixed with 2 mL NaOH (2 M) and read at an
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absorbance of 340 nm. The analytical procedure of α-glucosidase was as follows: Sludge samples (1 mL) were mixed with p-nitrophenyl-α-D-glucopyranoside solution (1 mL,
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0.1% (w/v)) and pH 7.6 Tris-HCl buffer solution (2 mL, 0.2 M), and incubated 60 min in
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37 ºC. The termination of the reaction was obtained by heating the sample in boiling
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water bath for 3 min. After centrifugation at 2500 g for 5 min, the supernatant absorbance was read at 410 nm. All the solutions used for enzyme assay were incubated at the
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desired temperature for 30 min, before additions of the sludge sample, to achieve incubation temperature.
Fluorescence excitation-emission matrix (EEM) was measured using a fluorescence
spectrometry (FP-6500, Jasco, Tokyo, Japan). Parallel factor analysis (PARAFAC) was used to model EEM fluorescence data in this study. With the fermentation time of 48 h, three sludge layers (DOM, LB-EPS and TB-EPS) were prepared and extracted in the different sludge samples: raw WAS (simplified as 0); blank test (simplified as 1) and SBpretreated test at SB dosage of 0.02g/g TSS (simplified as 2). DOM samples were simplified as DOM-0, DOM-1 and DOM-2, LB-EPS and TB-EPS were also named in an
Page 8 of 34
analogous way. Before PARAFAC analysis, the Raman scattering was removed by subtracting the pure distilled water spectrum from the sample spectrum, and the Rayleigh scattering was overcome by inserting a series of zero values in the region of no
ip t
fluorescence (excitation wavelength << emission wavelength). The analysis of residuals (sum of squared error) in both, the excitation and emission dimensions, was used to find
us
(MathWorks Inc., USA) was employed for handling EEM data.
cr
out the appropriate number of PARAFAC components. The software Matlab 2012a
3. Results and discussion
an
3.1 Enhanced WAS hydrolysis
SB pretreatment showed a positive effect on the hydrolysis of particulate organic
M
matter from sludge flocks. As proteins and carbohydrates are the main constituents of
d
WAS, the hydrolysis of organics can be expressed by that of soluble proteins and
te
carbohydrates in the WAS. The concentration of soluble proteins and carbohydrates increased with the increase of SB dosage, but did not show any further increase at SB
Ac ce p
dosage > 0.20 g/gTSS (Fig. 1A). After 48 h, soluble protein and carbohydrate concentrations (at SB dosage of 0.20 g/gTSS) were 2029 ± 28 and 343 ±52 mgCOD/L, which were 4.8-fold and 5.9-fold higher than that in the Blank, respectively. This was in accordance with the results of the corresponding activities of protease (218 ± 22 vs 58 ± 21 Eu) and α-glucosidase (16 ± 3 vs 9 ± 1 Eu), with and without SB treatment (Fig. 1B). Degradation of protein was also improved by SB treatment, with an increase in ammonia during the reaction. Taking 0.20 g SB/g TSS as example, the released ammonia concentration reached 29.2 mg/L, with an increase of 91.0% over that in the blank test. Significant amount of soluble phosphorus was also released with the treatment of SB: the
Page 9 of 34
activity of acid and alkaline phosphatases were slightly increased in the presence of SB (Fig. 1B), which hydrolyzed phosphate esters and released the phosphate groups into liquid. Cadoret et al. (2002) reported that a variable fraction of enzyme activities were
ip t
associated to the easily extractable EPS from sludge flocks [28]. The reason for this might be that SB facilitates the detachment of aggregated cells and of EPS, while
cr
releasing immobilized enzymes. This was confirmed by the EEM spectroscopy analysis
us
(as shown below).
an
3.2 Characterization of SB-pretreated WAS by EEM spectra with PARAFAC Fig. 2 shows the three-dimensional EEM spectroscopy characterizing the component
M
changes of WAS, in terms of DOM, LB-EPS and TB-EPS, under SB treatment (0.02 g/g
d
TSS, 48 h). As shown in Fig. 3, by separating the spectra of the main components from
te
overlapped EEM spectra, four components were found to be appropriate for PARAFAC analysis. The specific components were identified as tryptophan-like (Ex/Em 270/350,
Ac ce p
Com.1), tyrosine-like (Ex/Em 270/300, Com.2), other protein-like (Ex/Em 225/300, Com.3) and fulvic-like substrates (Ex/Em 300/440, Com.4) (Fig. 4) [4, 29]. The excitation and emission spectra (loadings) of the four organic fluorphores overlapped slightly and smoothly (Fig. 5). Additionally, excitation spectra had one or more maxima, but emission spectra only exhibited one emission maximum, which fitted the expected spectral properties of fluorophores well [30]. The DOM and EPS fluorescence spectra of three samples were similar in the peak locations, but had different fluorescence intensities (FI) (Fig. 2). The intensities of DOM were clearly enhanced by SB treatment. Tyrosine-like substance was the primary
Page 10 of 34
component (FI: 655), which was 11.6-fold higher than that in the un-pretreated WAS (FI: 52). Tryptophan-like and other protein-like substrates ranked second and third place. This also suggested the significance of dissolving of proteins, that were inside the microbial
ip t
cell wall or enmeshed in EPS, into liquid phase under SB treatment. These results were in good agreement with practical measurements (Fig. 1A). Small amounts of fulvic-acid-
cr
like substance were found in both, DOM and EPS fluorescence spectra, which were
us
mainly formed by the microbial degradation of dead organic matter [4]. It is believed that the disruption of EPS matrix could enhance the rate and extent of sludge biodegradation
an
during anaerobic digestion [31]. Bound EPS exhibited a dynamic double-layer-like structure, the LB-EPS layer was characterized by loose structure and rheological
M
property, which was easily detached by surfactant. On the contrary, the TB-EPS layer
d
was tightly attached to the cell surface, in which matrix the organics enmeshed was hard
te
to be disintegrated. As shown in Fig. 2, the FI of EPS peaks in EEM spectra were significantly decreased by SB treatment. Taking tyrosine-like substances into
Ac ce p
consideration, the concentration declined simultaneously in the LB-EPS and TB-EPS (FI: from 236 and 590 to 105 and 237). Thus, the SB doasge applied in this study could effectively decompose the LB-EPS layer, and further destroy the TB-EPS located in inner parts of sludge flocks. The same phenomenon was observed in the other three substrates. The detaching of EPS from WAS was evidently improved by SB treatment.
3.3 Improved VFAs bioproduction VFAs bioproduction was also improved during WAS fermentation, in the presence of SB (Fig. 6A). It sharply increased until 72 h, and fluctuated little with the further
Page 11 of 34
increase of fermentation time. At a fermentation time of 72 h, the specific VFAs yields were 1.6-fold, 2.5-fold, 3.5-fold, 4.0-fold and 3.8-fold higher than that in the control (1153 ± 28 mg/COD L) with an SB dosages of 0.05, 0.10, 0.20, 0.25 and 0.30 g/g TSS,
ip t
respectively. As can be observed in Fig. 6A, VFAs yield increased with the increase of SB dosage but didn’t enhance substantially at a SB dosage >0.20 g/g TSS. Further
cr
investigation (at 72 h fermentation time) revealed that it followed a parabolic curve, in
us
the range of 0.05~0.30 g SB/g TSS (YVFAs = -65241 X2SB dosage + 33819 XSB dosage +1267, R² = 0.997). This was true at any fermentation time (quadratic equations not shown).
an
Increasing the SB dosage from 0 to 0.05, 0.05 to 0.10, 0.10 to 0.20, 0.20 to 0.25 and from 0.25 to 0.3 g/g TSS, the generated incremental VFAs were 1416, 884, 450, 414 and 0 g
M
COD/L respectively. The reason behind this phenomenon maybe that SB could induce
d
EPS (and other bacterial aggregations) to leave the attached cell surface, as confirmed by
te
the 3D-EEM analysis. Furthermore, it could also enhanced aqueous solubility of nonaqueous phase organics, by reducing their surface/interfacial tension. A certain amount of
Ac ce p
EPS, and other bacterial aggregations, was present in the WAS fermentation system. Hence, as shown in Fig.1A, the concentration of detached soluble organics was much higher with the addition of initial 0.1 g SB /g TSS, which provided more substrate to the subsequent acidification step and lead to more VFAs production. Restated, the SB dosage from 0–0.05 g SB/g TSS showed the highest increment rate for VFAs. According to the composition analysis, the six VFAs increased by varying degrees, during SB treatment. Among them, HAc and HPr showed a relatively larger increase, the sum of their concentrations reaching 2752 mg COD/L, versus 763 mg COD/L in the control, after 72 h fermentation time (at SB dosage of 0.05 g SB/g TSS). The main three
Page 12 of 34
(individual) VFAs produced were HAc (32 ± 1.4%), HPr (23 ± 0.9%) and n-HBu (17± 1.1%), at a SB dosage of 0.05 g/g TSS (Fig. 6B). Clearly, the product spectrum can be affected by the pretreatment method. Jiang et al. [14, 15], for instance, observed that the
ip t
order of the individual VFAs was: HAc > HPr > iso-HVa by SDBS (0.02g/g TSS, 6d), and HAc > iso-HVa > HPr by SDS (0.1 g/g TSS, 6d) chemical-synthesis surfactant
cr
pretreatments. While Luo et al. [16] showed that the main VFAs from WAS
us
fermentation, pretreated by a microbial biosurfactant (rhamnolipid), were HPr > HAc > iso-HVa (0.3 g/g DS, 2d). On the other hand, the spectrum is also determined by the
an
dosage concentration (Fig. 6C). With the increase of SB dosage from 0 to 0.20/g TSS, HAc concentration increased from 436 to 1434 mg COD/L. Correspondingly, the
M
produced HBu and HVa also increased 6.7 and 4.4 times over that without SB treatment,
d
respectively. Increased concentrations of HBu and HVa probably derived from the
te
degradation of carbohydrates and proteins in the WAS, while HVa might be mainly associated with the fermentation of proteins, via reductive deamination of single amino
Ac ce p
acids or by oxidation–reduction between pairs of amino acids via the Stickland reaction [32].
3.4 Effect of SB treatment on syntrophic acetogenesis and methanogenesis As stated previously, SB pretreatment had a positive effect on the hydrolysis and
acidogenesis steps during WAS digestion. VFAs, as the products of acidogenesis, are the substrates for methanogenesis and can be easily metabolized to methane. We noted that (cumulative) methane production was improved at SB dosage of 0.05 g/g TSS and inhibited at SB dosage > 0.10 g/g TSS (Fig. 7A). The data obtained fitted the exponential
Page 13 of 34
growth model (YCH4=e-kt) and the corresponding production rate constants (k) were calculated. With a fermentation time of 192 h (t), the cumulative methane yield (YCH4) increased from 46.5 mL/gVSS in the blank (k=0.0201 h-1, R2=0.9912) to 63.0 mL/gVSS
ip t
at SB dosage of 0.05 g/g TSS (k=0.0219 h-1, R2=0.9620), while it decreased to 20.3 mL/gVSS at dosage of 0.10 g/g TSS (k=0.0154 h-1, R2=0.9935). The same result was
cr
observed in the study with synthetic wastewater containing NaAc. Data obtained fitted
us
the linear growth model (YCH4= constant +kt). As shown in Fig. 7B, the methane production slightly increased from 4.7 ± 0.5 mL/g Ac (k=0.1927 h-1, R2=0.9401), in the
an
control, to 6.6 ± 0.3 mL/g Ac (k=0.2745 h-1, R2=0.9696), at SB dosage of 0.05 g/g Ac, while it was inhibited at a dosage of 0.20 g/g Ac (0.5 ± 0.0 mL/g Ac, k=0.0233 h-1,
M
R2=0.9865) after 24 h fermentation time. That is, the methanogenesis was enhanced at
d
lower SB dosage (< 0.10 g/g TSS) and inhibited at higher SB dosage (> 0.10 g/g TSS).
te
Almost the same phenomenon was found by using chemically synthetic surfactants. Jiang et al. showed that the inhibition rate of methane production increased sharply from 50 %
Ac ce p
to 100 % with the SDS dosage increasing from 0.1 to 0.3 g/g (at fermentation time of 21 d) [15]. He also found that the methane yield decreased from 65 (blank) to 16 and 8 mL/g VSS, at dosages of 0.05 and 0.1 g SDBS /g TSS, respectively [14]. Our previous study also found that the cumulative methane production decreased sharply from 58.8 mL/g VSS, in the blank, to 2.0 mL/g VSS with the treatment of rhamnolipid (0.04 g/g TSS, 192 h) [17]. Conversely, the acid-forming bacteria were not impaired by surfactant pretreatment, thereby maximizing yield of VFAs (Fig. 6A). Fig. 7C shows HBu conversion into HAc and the consequent methane production, at SB dosage of 0.20 g/g Bu. HBu concentration sharply decreased in the first 10 hours of
Page 14 of 34
fermentation and then stayed relatively constant, both with or without SB treatment. HAc, as the product of HBu degradation via ß-oxidation pathway, increased correspondingly and was subsequently degraded into methane. The specific
ip t
thermodynamic equation of HBu degradation by acetogenic bacteria in the syntrophic acetogenesis process is shown below [32]:
cr
HBu+2H2O→2HAc+2H2
(1)
us
Apparently, SB treatment increased the HBu degradation rate, as it was rapidly converted into HAc (from 2 h onward), while 8h were need without SB treatment. The result of
an
methane production from the converted HAc further proved this point. After 16 h fermentation, the methane yield was 2.7 ± 0.1 mL/g Bu in the SB-treated and 1.5 ± 0.1
d
M
mL/g Bu in the control tests, respectively.
te
3.5 Significance of SB pretreatment and potential implementation Saponin has a wide range of industrial applications as a surfactant [33]. This study
Ac ce p
successfully demonstrated the feasibility of SB pretreatment for the enhancement of anaerobic digestibility of WAS. Currently, many researchers have conducted studies for the VFAs recovery from WAS and investigated the acidogenic fermented sludge as the feedstock for subsequent biological nutrient removal [34], polyhydroxyalkanoates production [35], hydrogen production by MEC reactor [5], and so on. A comparison of different VFAs yield from WAS fermentation by surfactants pretreatment is given in Table 1. The VFAs yield of 211±7 mg COD/g VSS obtained with treatment of 0.05 g SB /g TSS is comparable with the 222 mg COD/g VSS reported by Luo et al., 2012, using rhamnolipid (0.30 g/g DS) at mesophilic conditions. Jiang et al.
Page 15 of 34
found that the maximum VFAs yields of 235 and 241 mg COD/g VSS were achieved by treatments of SDS and SDBS, at ambient temperature, respectively. Seemingly, the use of chemosynthetic surfactants for VFAs production presented some advantages compared to
ip t
SB biosurfactant. However, although the treatment temperature of chemosynthetic surfactants was lower (21 vs 35 °C), the needed fermentation time was twice that with
cr
SB. Moreover, as stated in paragraph 3.4, cumulative methane production was improved
us
at SB dosage of 0.05 g/g TSS, which was generated from VFAs metabolism. Hence, the acidification efficiency should consider this consumed part as well (CODmethane), which
an
can be calculated using a conversion of 0.35 m3 methane/ kg COD, under standard conditions [36]. In the present study, methane yield was 9.2 mL /g VSS (that is 26 mg
M
COD/g VSS) at a SB dosage of 0.05 g/g TSS for 72 h, while it was negligible under SDS
d
and SDBS treatments. Thus, from an overall perspective of WAS digestion, SB treatment
te
was able to match the results obtained in other researches in this field. During the process of anaerobic digestion, the excessive formation of foam can
Ac ce p
induce a negative impact on the efficiency of biogas production, especially for the processes that utilize renewable resources or biogenic waste material as feedstock [37]. Normally, the liquid phase is located in a thin film, which is present between the gas bubbles. However, foam can be produced only after a certain threshold concentration of a surface-active compound has been exceeded. Biosurfactants are known to be one kind of surface-active compounds [38]. Hence, the utilization of biosurfactants for the anaerobic treatment of WAS requires solutions to prevent this phenomenon. Previous studies showed that the application of anti-foaming agents is a common treatment method that could minimize or even avoid the formation of foam [37]. Detailed investigation on the
Page 16 of 34
suitable anti-foaming agent for the SB-treated WAS digestion, as well as the dosage optimization and the microbial communities evolution induced by the SB and antifoaming agent, would provide valuable insight into the viable utilization of SB for WAS
ip t
treatments. Therefore, further research is advisable. 4. Conclusions
cr
The contribution of saponin biosurfactant (SB) for improving anaerobic digestibility
us
of WAS was successfully demonstrated. SB addition increased WAS hydrolysis substantially, in terms of VFAs production, with the increase of SB dosage ranging from
an
0-0.2 g/g TSS. The activities of hydrolytic enzymes were all improved, because SB favoured the release of immobilized enzymes in the EPS and other bacterial aggregations.
M
From EEM spectra analysis, SB treatment could effectively decompose the LB-EPS
d
layer, and further detach the TB-EPS layer. Soluble protein and carbohydrate
te
concentrations were 4.77-fold and 5.87-fold higher than that without SB addition (0.20 g SB/g TSS, 48 h), respectively. The main three individual VFAs were HAc, HPr and n-
Ac ce p
HBu. Methane production was enhanced by 35% at lower concentration (0.05 g SB/g TSS), but decreased by 56% at higher SB dosage (0.10 g SB/g TSS), compared to control conditions. The results obtained in the present study, using plant-derived biosurfactants, are comparable with previous ones obtained with chemosynthetic surfactants. However, it must be underlined that biosurfactants, such as saponin, have the advantage of an easy production, lower prices, sustainability of supply and possibility of reuse over synthetic surfactants, which should promote their implementation for WAS treatment.
Acknowledgement
Page 17 of 34
This research was supported by National Natural Science Foundation of China (NSFC, Grant No. 51208496 and 51378330), by Science Fund for Creative Research Groups of the National Natural Science Foundation of China (Grant No.51121062), by
ip t
National Science Foundation for Distinguished Young Scholars of China (Grant No. 51225802), by National High-tech R&D Program of China (863 Program, Grant No.
cr
2012AA051502), by ‘‘Hundred Talents Program’’ of the Chinese Academy of Sciences,
us
by Chinese Academy of Sciences (135 Project, Grant No. YSW2013B06).
an
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Page 23 of 34
Table 1 Comparison of VFAs yield from WAS fermentation by surfactants pretreatment VFAs yield (mg
time (h)
COD/g VSS)
Dosages
References 48
222
SDS
0.10 g/g TSS
144
235
SDBS
0.02 g/g TSS
144
241
Saponin
0.05 g/g TSS
72
211 ± 7
[16]
ip t
0.30 g/g DS
[15] [14]
This study
Ac ce p
te
d
M
an
us
Rhmnolipid
cr
Surfactants
Fermentation
Page 24 of 34
Figure captions Figure 1 Effect of SB dosage on hydrolysis products and inorganics released (A) and
ip t
hydrolytic enzymes activities at SB dosage of 0.20 g/g TSS (B)
Figure 2 EEM fluorescence spectra and spectral intensity of components for DOM, LB-
cr
EPS and TB-EPS obtained from (1) blank and (2) SB-pretreated WAS with the
us
fermentation time of 48 h. (0) The spectra from raw WAS were used as controls. Figure 3 The sum of squared error (residuals) in both the excitation and emission
an
dimensions for three different models.
Figure 4 Four components of DOM and EPSs decomposed by the PARAFAC approach:
M
(Com.1) tryptophan-like substances, Ex/Em (270/350); (Com.2) tyrosine-like
d
substances, Ex/Em (270/300); (Com.3) protein-like substances, Ex/Em
te
(225/300); (Com.4) fulvic-like substrates, Ex/Em (300/440). Figure 5 The excitation and emission spectra (loadings) of four components derived from
Ac ce p
PARAFAC modeling: (Com. 1) tryptophan-like substances, (Ex/Em, 270/350); (Com. 2) tyrosine-like substances, Ex/Em (270/300); (Com. 3) protein-like substances, Ex/Em (225/300); (Com. 4) fulvic-like substrates Ex/Em (300/440).
Figure 6 Effect of SB dosage on VFAs production (A) and composition (B) Figure 7 Effect of SB dosage on methane production from WAS (A), methane production from HAc at SB dosages of 0.05 and 0.20 g/g Ac (B), HAc conversion and methane production from HBu degradation at SB dosage of 0.20 g/g Bu (C).
Page 25 of 34
Ac ce p
te
d
M
an
us
cr
ip t
> Anaerobic digestibility of waste activated sludge (WAS) was enhanced by saponin (SB). > SB showed positive effects on WAS hydrolysis and acidogenesis. > Methanogenesis was enhanced at lower dosage and inhibited at higher dosage. > Hydrolytic enzymes activity was improved. > SB could decompose the LB-EPS layer, and further detach the TB-EPS layer of WAS.
Page 26 of 34
Ac ce p
te
d
M
an
us
cr
ip t
*Graphical Abstract
Page 27 of 34
Figure 1
cr
ip t
554
us
555
Fig. 1 Effect of SB dosage on hydrolysis products and inorganics released (A) and
557
hydrolytic enzymes activities at SB dosage of 0.20 g/g TSS (B)
an
556
Ac
ce pt
ed
M
558
25
Page 28 of 34
Figure 2
560
ce pt
ed
M
an
us
cr
ip t
559
Fig. 2 EEM fluorescence spectra and spectral intensity of components for DOM,
562
LB-EPS and TB-EPS obtained from (1) blank and (2) SB-pretreated WAS with the
563
fermentation time of 48 h. (0) The spectra from raw WAS were used as controls.
564 565
Ac
561
26
Page 29 of 34
Figure 3
an
us
cr
ip t
566
567
Fig. 3 The sum of squared error (residuals) in both the excitation and emission
569
dimensions for three different models.
ed
M
568
570
Ac
ce pt
571
27
Page 30 of 34
Figure 4
573
Ac
ce pt
ed
M
an
us
cr
ip t
572
574
Fig. 4 Four components of DOM and EPSs decomposed by the PARAFAC approach:
575
(Com.1) tryptophan-like substances, Ex/Em (270/350); (Com.2) tyrosine-like
576
substances, Ex/Em (270/300); (Com.3) protein-like substances, Ex/Em (225/300);
577
(Com.4) fulvic-like substrates, Ex/Em (300/440).
578 579 28
Page 31 of 34
Figure 5
M
an
us
cr
ip t
580
ed
581
Fig. 5 The excitation and emission spectra (loadings) of four components derived
583
from PARAFAC modeling: (Com. 1) tryptophan-like substances, (Ex/Em, 270/350);
584
(Com. 2) tyrosine-like substances, Ex/Em (270/300); (Com. 3) protein-like substances,
585
Ex/Em (225/300); (Com. 4) fulvic-like substrates Ex/Em (300/440).
587
Ac
586
ce pt
582
29
Page 32 of 34
Figure 6
us
cr
ip t
588
589
Fig. 6 Effect of SB dosage on VFAs production (A) and composition (B)
an
590 591
Ac
ce pt
ed
M
592
30
Page 33 of 34
Figure 7
ed
M
an
us
cr
ip t
593
ce pt
594
Fig. 7 Effect of SB dosage on methane production from WAS (A), methane
596
production from HAc at SB dosages of 0.05 and 0.20 g/g Ac (B), HAc conversion and
597
methane production from HBu degradation at SB dosage of 0.20 g/g Bu (C).
598 599
Ac
595
31
Page 34 of 34