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Promotion effects of ultrasound on sludge biodegradation by thermophilic bacteria Geobacillus stearothermophilus TP-12
Biochemical Engineering Journal 105 (2016) 281–287
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Regular article
Promotion effects of ultrasound on sludge biodegradation by thermophilic bacteria Geobacillus stearothermophilus TP-12 Wan-Qian Guo a,∗ , He-Shan Zheng a , Shuo Li a , Shih-Hsin Ho a , Shan-Shan Yang a , Xiao-Chi Feng a , Jo-Shu Chang a,b,∗ , Xiang-Jing Wang c , Nan-Qi Ren a a
State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan c Research Center of Life Science and Biotechnology, Northeast Agricultural University, Harbin 150030, China b
a r t i c l e
i n f o
Article history: Received 5 February 2015 Received in revised form 25 September 2015 Accepted 1 October 2015 Available online 9 October 2015 Keywords: Sludge Waste treatment Biodegradation Thermophiles Protease Low-frequency ultrasound
a b s t r a c t Sludge biodegradation using thermophilic bacteria is a promising method for sludge treatment. In order to further enhance the efficiency of sludge reduction and hydrolysis, low-frequency ultrasound was used to promote this process. We isolated a thermophilic strain that is effective in secreting extracellular protease to hydrolyze sludge. Then the key ultrasound parameters were selected using the response surface methodology method. After 12 h treatment using thermophilic bacteria with short-time ultrasound promotion, volatile suspended solids (VSS) reduction ratio was achieved 32.8%, which is 41.4% higher than that without ultrasound promotion. Meanwhile the contents of soluble chemical oxygen demand (SCOD), protein and carbohydrate were increased by 20.2%, 16.8% and 15.9%, respectively. The composition of dissolved organic matter of sludge products evaluated by excitation-emission matrix spectroscopy demonstrated the promotion effect and eliminated the possibility of the direct sludge degradation caused by ultrasound treatment. Low-frequency ultrasound could effectively promote the thermophilic bacteria hydrolysis to achieve higher sludge biodegradation ratio without directly degrading the raw sludge. The promoted process of sludge biodegradation can further reduce the environmental risk and make sludge to be more readily usable. © 2015 Published by Elsevier B.V.
1. Introduction Increasing volumes of sludge generated from sewage treatment plants severely threatens environmental safety and human health [1]. The existing methods of sludge disposal, such as landfill internment, incineration, land application, etc., have potential environmental risks and obvious cost issues [2,3]. Thus, finding the more cost-effective and environmentally-friendly alternatives for sludge degradation has become a research direction that is receiving more attention [2,4]. Due to the special complex organism structure and particular microbial characteristic of sludge, main organic matters are wrapped in its extracellular polymeric substances (EPS) [5]. Thus, the essence and difficulty of sludge degradation is floc fragmentation and hydrolysis. To release wrapped organic matter
∗ Corresponding authors at: State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China. E-mail address: [email protected] (W.-Q. Guo). http://dx.doi.org/10.1016/j.bej.2015.10.002 1369-703X/© 2015 Published by Elsevier B.V.
and increase dissolved organic matter (DOM) content for further anaerobic digestion and utilization, currently, many sludge degradation methods have been developed to hydrolyze EPS and inner microorganism, such as ozonation [6], peracetic acid oxidation [7], sonication [8], alkali addition [9], mechanical and sludge thickening [10], and ozonation combined with sonication [11]. Due to high costs and serious secondary pollution caused by these physical and chemical approaches, biodegradation has aroused increasing attention, especially in sludge hydrolysis using thermophilic bacteria [12]. Sludge biodegradation by thermophilic bacteria is a technology of adding thermophilic bacteria solution which contains secreted thermophilic hydrolytic enzymes into sludge for hydrolysis at a thermophilic condition (ranging from 40 to 80 ◦ C) [13]. Sludge pretreated with thermophilic bacteria has drawn more interests for volatile fatty acids (VFAs) and hydrogen production [14,15]. Although this method seems superior to sludge biodegradation, it has not been extensively introduced to sludge treatment because of its relatively longer processing period and low degradation ratio compared to the physical-chemistry methods which is not feasible for large-scale commercial process [16]. To date, few
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Table 1 Characteristics of raw sludge and mixure of sludge with bacteria solution. Parameter
Raw sludge
Mixure of sludge with bacteria solution (9:1)
TSS (mg L−1 ) VSS (mg L−1 ) TCOD (mg L−1 ) SCOD (mg L−1 ) Soluble protein (mg L−1 ) Soluble carbohydrate (mg L−1 ) pH
14137.0 9872.0 14934.0 73.0 58.7 15.2 7.0
14371.0 10388.0 15326.0 1414.3 480.2 77.6 7.2
studies have been conducted to explore the promotion methods for enhancing sludge hydrolysis. A pretreatment method combining freezing/thawing with thermophilic bacteria was employed in pretreating sludge in order to achieve a higher degradation ratio [17]. However, the freezing step will also result in huge economic costs and practical resistance in commercial scale. Therefore, developing a cost-effective and convenient method to accelerate the thermophilic enzyme secretion and improve the enzyme hydrolysis efficiency is vital and urgent towards practical application. Low-frequency ultrasound (20–100 kHz) has been reported to have positive effects on microbial productivity and enzyme hydrolysis [18,19]. For instance, Song et al. [20] reported that enzymatic hydrolysis of alkaline protease from Bacillus licheniformis was greatly accelerated by ultrasound irradiation, and the final conversion ratio was significantly improved. Through the studies on stability of enzymes, including a-amylase [21], lactase [22], horse radish peroxidise and alkaline phosphatase [23], the active sites of enzyme were demonstrated not destroyed during the sonication process. In order to investigate the promotion effect of low-frequency ultrasound on bacterial activation and sludge solubilization products, the distributions of DOM was also analyzed using three-dimensional excitation-emission matrix (EEM) fluorescence spectroscopy. As a time-saving and accurate tool, EEM was extensively used to determine DOM which exhibits fluorescent emission characteristics [24]. This method can be regarded as an overall “fingerprint” of DOM that covers vitamins, NADH, protein-like and humic acid-like substances [25]. In this study, a strain that secretes high-efficiency thermophilic extracellular protease was first isolated under the specific growing conditions using selective culture mediums. This thermophilic strain was cultured as bacteria solution and then added into the raw sludge. Meanwhile the promotion effect of low-frequency ultrasound on sludge degradation process was comprehensively studied. The optimal parameters of ultrasound application were determined via the response surface methodology (RSM). Finally, three-dimensional EEM fluorescence spectroscopy was used to characterize the DOM distribution to evaluate the promotion effect of low-frequency ultrasound on sludge reduction and hydrolysis efficiency. 2. Materials and methods 2.1. Thermophilic strain isolation and identification For isolation of thermophilic bacteria which produce protease, the sludge was cultured in a shaking bath at 60 ◦ C for 36 h. The remainder liquor containing thermophilic bacteria was diluted using distilled water at a ratio of 10:1. A volume of 0.1 mL−1 sample of every level was screened on skim milk agar medium. After 24 h of cultivation at 60 ◦ C, the strains with transparent circles appeared were determined as protease producing ones. The colony with the biggest transparent hydrolytic zone on skim milk agar medium was selected and cultured in a soluble starch agar medium for 24 h. This procedure was repeated at least ten times to ensure a pure strain. The isolated colony was cultured on a standard LB agar medium,
and then stored at 4 ◦ C. The isolated pure strain of the thermophilic bacteria was designated as strain TP-12. Medium composition (g L−1 ): The LB agar medium contain: yeast extact 5; peptone 10; NaCl 10; agar 20. The skimmed milk agar medium contained: NaCl 5; peptone 3; skimmed milk 3; agar 20. The composition of fluid nutrient medium: yeast extact 5; peptone 10; NaCl 10. The DNA was extracted from the 1 mL pure culture solution (LB medium, 60 ◦ C, 24 h) using DNA Isolation Kit (Watson, Shanghai, China). The resultant DNA was subjected to polymerase chain reaction (PCR) with primers of 8F (5 -AGAGTTTGATCCTGGCTCAG3 ) and 1492R (5 -GGTTACCTTGTTACGACTT-3 ). The 16S rRNA gene obtained from isolated bacteria was cloned, purified, amplified and sequenced by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). The sequencing was done in triplicate in order to get the correct sequence. In the phylogenetic analysis, the nucleotide sequence was compared with the sequences in the GenBank/EMBL/DDBJ nucleotide sequence databases by the Basic Local Alignment Search Tool (BLAST) program (http://www.ncbi.nlm.nih.gov/BLAST/). 2.2. Source and preparation of WAS Sewage sludge was obtained from the secondary sedimentation tank of Harbin Wenchang Sewage Treatment Plant (Harbin, China). The raw sludge was first filtrated through a 20-mesh sieve to separate large debris, then settled for 24 h and stored at 4 ◦ C. The characteristics of the sludge used in this study are shown in Table 1. 2.3. Single-factor experiments To study the promotion effect of different ultrasound density and exposure time on thermophilic bacteria, and select ranges of independent variables for optimizing parameters by RSM, single-factor experiments were carried out. Different ultrasound densities (0.04 W mL−1 , 0.08 W mL−1 , 0.12 W mL−1 , 0.16 W mL−1 , 0.20 W mL−1 ) at the same exposure times of 20 s and different ultrasound exposure times (5 s, 10 s, 15 s, 20 s, 25 s) at the same ultrasound density of 0.12 W mL−1 were performed. The lowfrequency ultrasound radiation was conducted using an ultrasound probe system (CY-5D, Ningbo Scientz Biotechnology Co., Zhejiang, China) that emitted 20 kHz ultrasound wave, and had a variable power from 0 to 200 W. The diameter of the horn used was 20 mm. For each sonication experiment, thermophilic bacteria solution (cultured for 48 h) was inoculated in sludge in a ratio of 1:9 (the mixture of bacteria solution with sludge was shown in Table 1). 700 mL mixture sample was seeded into the reactor, heating at 60 ◦ C in a solubilization for 12 h at 140 rpm, timing supplement the evaporation of water, and the ventilation was about 0.16 L min−1 . Simultaneously, an ultrasonic instrument equipped with a sonoprobe was dipped into sludge treatment samples for 2 mm at three hours intervals. Liu et al. [26] found that enzyme remained its high activity even some hours after irradiation was stopped and the enhancement effects would be disappeared after 24 h. So the ultrasound interval is three hours in this experiment.
W.-Q. Guo et al. / Biochemical Engineering Journal 105 (2016) 281–287 Table 2 RSM experimental ranges and levels of the independent variables. Independent variables
Range and levels
Ultrasound density (W ml−1 ), x1 Exposure time (s), x2
− 1.414 0.06 0.86
−1 0.08 5.00
0 0.12 15.00
1 0.16 25.00
1.414 0.18 29.14
2.4. Parameters selection by RSM Response surface methodology (RSM) was used to determine the optimal conditions of ultrasound treatment and to predict the best value of response. Single-factor-test was employed to determine the preliminary range (Tables 2 and 3), and then 13 experimental runs were carried out according to a central composite design with two factors and five levels to optimize the relationship between the ultrasound density (X1 ,W mL−1 ), the exposure time (X2 , s), and the most important response, the sludge reduction rate (Y, %). The relation between the coded values and real values is described and shown in Eq. (1): xi = (Xi − Xi ∗) /Xi
(1)
where xi and Xi are the coded value and uncoded value of the independent variable, respectively; Xi * is the uncoded value of the independent variable at the center point; and Xi is the step change of variable. The fit quality can be expressed by the correlation coefficient (R2 ), and the significance of the model can be demonstrated by F-value and Probability value (Prob > F). 2.5. Fluorescence spectroscopy Prior to the fluorescence analysis, the samples were centrifuged at 9391 × g for 10 min and filtered using 0.45 m polytetrafluoroethylene (PTFE) filters and then diluted 200 times with distilled water. The three-dimensional EEM spectra were measured using a fluorescence spectrophotometer (FP-6500, JASCO, Japan). EEM spectra were obtained as follows: Scanning emission (Em) spectra from 220 to 650 nm were obtained at 1 nm increments by varying the excitation (Ex) wavelength from 220 to 450 nm at 5 nm increments. The scan speed was set at 2000 nm min−1 , and the slit widths were 5 nm for both excitation and emission monochromators. The software Origin 8.1 (OriginLab, Los Angeles, USA) was employed for figure processing. 2.6. Analytical procedures During the sludge pretreatment period, the analyses of SCOD, COD, SCOD, pH, TSS, VSS were conducted in accordance with Standard Methods [27]. The concentrations of TSS and VSS were determined using a 35 mL sludge sample at 105 ◦ C for 4 h and 600 ◦ C for 2 h [9]. The soluble-carbohydrate was measured by the phenolsulfuric acid method [25]. The soluble-protein was measured using a modified BCA protein assay kit (Sangon Biotech Co., Ltd., Shanghai, China).
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obtained using the neighbor joining method shown in Fig. S2. This strain was deposited at China General Microbiological Cultures Collection Center (CGMCC) with the accession number CGMCC 8643. The performance of TP-12 on sludge hydrolysis was expressed by VSS degradation ratio and the SCOD, soluble protein and carbohydrate increment. VSS reduction ratio could reach 23.2% for 12 h of sludge hydrolysis by thermophilic bacteria TP-12. The maximum amounts of SCOD, soluble protein and carbohydrate were 4008.7 mg L−1 , 1398.8 mg L−1 and 220.9 mg L−1 , respectively.
3.2. Analysis of single-factor test for ultrasound enhanced-sludge degradation It was reported that VSS reduction ratio followed the first order reaction kinetics model during the thermophilic aerobic sludge digestion [28]. In the present study, VSS content was regarded as an indicator of sludge reduction. Firstly, the preliminary studies were performed by single-factor experiments in order to determine the appropriate parameters of ultrasound density and exposure time. The VSS reduction ratio of different ultrasound density and exposure time was presented (Fig. 1). As shown in Fig. 1, compared with the 23.2% VSS reduction ratio of sludge degradation using TP-12 without ultrasound treatment, an obvious increase of VSS reduction ratio was observed for the ultrasound treated one. The peak reduction ratio of VSS reached 30.4% at exposure time of 20 s, which is about 43.5% higher than the blank experiment. After determining the optimum ultrasound density of 0.12 W mL−1 , the subsequent test showed that when exposure time exceed 15 s, the ultrasound may exert a negative influence and the VSS reduction ratio began to decrease. The similar phenomenon of particular ultrasound effect was also observed in the previous studies, depicting that the intracellular metabolism was enhanced under short time irradiation but inhibited under long time irradiation. Duan et al. [29] demonstrated that the cell wall became thinner after moderate low-intensity ultrasound irradiation through transmission electron microscopy (TEM) observation, which lead to increased its permeability and promoted the mass transfer efficiency. In addition, the bacterial structure would be destroyed under high energy inputs of lowfrequency ultrasound. Through TEM observation, it is found that the cytoplasmic membrane was dislocated and the intracellular content was leaked under high power irradiation [30]. Moreover, the enzyme specific activities also might be affected, even the capability of enzyme secretion. These individual promotion effects mentioned above have already been separately demonstrated and applied in the relevant studies [31–34]. In this study, the performance of sludge hydrolysis using thermophilic bacteria was promoted by moderate low-frequency ultrasound. This promotion effect is a complicated process, and the optimum parameters for the cells or enzymes are different. However, the specific mechanism of ultrasound effect on various parts in this mixed system was extremely difficult to explicitly distinguish and define, hence, it would be investigated in-depth in the further study.
3. Results and Discussions 3.1. Information of strain isolation, identification and performance As shown in Fig. S1a, the thermophilic bacterial strain TP-12 isolated from sludge could generate transparent circle through thermophilic protease hydrolysis of protein on skimmed milk agar medium. TP-12 was a brevibacterium detected by scanning electron microscope (Fig. S1b) and it was identified as a strain belongs to Geobacillus stearothermophilus. The phylogenetic tree of TP-12 was
3.3. Response surface methodology analysis of optimal ultrasound parameters After the single-factor experiments, optimal parameters of ultrasonic density and exposure time were further explored by the center composite design (CCD) of RSM. VSS reduction ratio was considered as an indicator for parameters optimization. The design matrix included two independent variables of exposure time (X1 ) and ultrasonic density (X2 ), and the corresponding results obtained are shown in Table 3. Coded and actual responses were established
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Table 3 Response surface Central Composite Design (CCD) and experiments. Run
Actual variables
Coded variables
Sludge reduction ratio (%)
1 2 3 4 5 6 7 8 9 10 11 12 13
X1 0.12 0.12 0.12 0.16 0.18 0.12 0.12 0.08 0.08 0.12 0.12 0.16 0.06
X2 15.00 15.00 15.00 25.00 15.00 15.00 29.14 5.00 25.00 0.86 15.00 5.00 15.00
x1 0 0 0 +1 +1.414 0 0 −1 −1 0 0 +1 −1.414
30
Y 33.03 ± 1.65 33.03 ± 1.65 33.03 ± 1.65 26.29 ± 1.31 23.64 ± 1.18 33.03 ± 1.65 26.79 ± 1.34 22.75 ± 1.14 26.04 ± 1.30 22.12 ± 1.17 33.03 ± 1.65 28.20 ± 1.41 25.36 ± 1.29
40 (a)
VSS Reduction Ratio (%)
VSS Reduction Ratio (%)
35
x2 0 0 0 +1 0 0 +1.414 −1 +1 −1.414 0 0 0
25 20 15 10 5 0
35
(b)
30 25 20 15 10 5 0
0.00
0.04
0.08
0.12
0.16
0.20
0
Ultrasound Density (W mL-1)
5
10
15
20
25
Exposure Time (s)
Fig. 1. Variations in VSS solubilization ratio of (a) different ultrasound densities and (b) different exposure times.
by Eqs. (1) and (2), respectively: YCODED = 33.03 + 0.41x1 + 1.00x2 − 1.30x1 x2 − 3.93x1 2 − 3.95x2 2 (2)
YACTUAL = −19.77880 + 648.22584X1 + 1.67430X2 − 3.24275X1 X2 − 2455.87891X1 2 − 0.039519X2 2
(3)
where Y is the VSS reduction ratio, x1 and x2 are the coded value of ultrasound density and exposure time; X1 , X2 , are the ultrasound density and exposure time, respectively. To determine the optimal levels, the relationship between two variables was illustrated by the 3-D representation of response surface generated by the model, as shown in Fig. 2. The optimized conditions obtained by RSM in the validated experiments were ultrasound density of 0.122 W mL−1 , exposure time of 16.2 s. According to the estimation model, the VSS reduction ratio reached 33.1% under the optimal ultrasound promotion conditions. They were employed to test the predictive model. Considered the operability in actual run, we modified the optimal conditions as follows: exposure time of 16 s, ultrasound density of 0.122 W mL−1 . Under the modified conditions, the average VSS reduction ratio obtained from the triplicate tests of 32.8% was achieved. The value was closed to the simulated value of 33.1% predicted by the model equation. Our results showed that low frequency ultrasound could effectively promote VSS reduction and increase sludge degradation ratio. Statistical analysis of the model was performed in the form of analysis of variance (ANOVA) to examine significance and adequacy of second-order polynomial equation (Table 4). The quadratic regression model showed that the value of determination coefficient (R2 ) was 0.9314, which implies that 93.14% of the variations
could be explained by the fitted model. It indicates a good agreement between experimental and predicted values. In addition, the P-values are used as a tool to check the significance of each variable. The small P-values indicate the higher significance of the corresponding variable. In this study, the model obtained was significant (P < 0.05) suggesting the ultrasound treatment time and density could play a synergistic role in VSS reduction. In addition, the VSS reduction ratio obtained at optimized ultrasound conditions in this study was much higher than the previous report, which was about 15% at 12 h [16]. Chen and Pan [35] confirmed that in the reactor operation of sludge solubilization using thermophilic bacteria, lower sludge solid contents corresponded to the higher acidification rate which could help achieving the higher degradation efficiencies of VSS. However, in the present study, we believed the main reason for high VSS degradation is due to the promotion effect from low-frequency ultrasound.
3.4. Effect of low-frequency ultrasound on enhancing waste sludge hydrolysis In order to achieve the economic feasibility of the waste sludge in the subsequent treatment, determining its hydrolysis ability is crucial. The SCOD, soluble protein and carbohydrate comprise the principal parts of the sludge hydrolysis products (shown in Fig. 3). The contents of these three soluble components exhibited greatly increased under low-frequency ultrasound stimulation, which demonstrated that the treated sludge was hydrolyzed into more soluble matters through thermal hydrolysis enzymes secreted from TP-12. As shown in Fig. 3a–c, during this process, SCOD and soluble protein concentrations increased rapidly within the initial 8 h, reaching 4982.1 mg L−1 and 1634.1 mg L−1 , respectively. The concentrations declined in 8 h. The highest soluble carbohydrate concentration was obtained at the optimal HRT of 12 h. The
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Fig. 2. The response surface 3D plot showing the effects of ultrasound density and ultrasound time on VSS reduction ratio.
Table 4 Analysis of variance (ANOVA) results for the response surface quadratic mode. Source Model x1 x2 x1 x2 x1 2 x2 2 Residual Lack of fit Pure error Cor total
Statistics Sum of squares 207.12 1.32 7.94 6.73 107.41 108.64 15.26 15.26 0 222.38
df 5 1 1 1 1 1 7 3 4 12
Mean Square 41.42 1.32 7.94 6.73 107.41 108.64 2.18 5.09 0
F-Value 19 0.61 3.64 3.09 49.26 49.83
P-Value <0.0001 0.4612 0.0981 0.1224 0.0002 0.0002
Significant
-1
SCOD Conc. (mg L )
6000 5000
Ultrasound Contral
4000 3000 2000 1000
(a)
0 0
12
24
36
48
60
72
-1
Soluble Carbohydrate Conc. (mg L )
1800
-1
Soluble Protein Conc. (mg L )
Time (h)
Ultrasound Control
1500 1200 900 600 300
(b)
0 0
12
24
36 Time (h)
48
60
72
300 250 200 150 100 50
Ultrasound Control
(c)
0 0
12
24
36
48
60
72
Time (h)
Fig. 3. (a) Comparison of SCOD, (b) soluble protein, and (c) soluble carbohydrate concentrations over a 72 h during thermophilic aerobic digestion of sludge with or without ultrasound promotion.
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Fig. 4. EEM spectra of DOM fractions extracted from (a) the mixure of sludge and bacteria solution (9:1) at 0 h, (b) hydrolysis by thermophilic bacteria for 8 h (with ultrasound promotion), (c) hydrolysis by thermophilic bacteria treatment for 8 h (without ultrasound promotion).
respective maximum concentrations of SCOD, soluble protein and soluble carbohydrate were 13.0%, 15.9% and 16.8%, respectively, which are higher than the control. Better hydrolysis performance was observed under the ultrasound promoted tests compared with the control group, proving that the low-frequency ultrasound has a positive impact on sludge hydrolysis capability of thermophilic bacteria.
3.5. Characteristics of dissolved organic matters by EEM spectra Three-dimensional EEM spectroscopy which can collect multiple emission scans of DOM by changing excitation wavelengths was applied to characterize the samples before and after pre-treatment in this study. The sludge solubilization type can be revealed from the DOM categories and their corresponding contents. Nine EEM fluorescence spectra of DOM characterized by a broad band are shown in Fig. 4. There were only two peaks at Ex/Em of 280/340 and 220/340 nm in all examined samples. The peak at 280/340 nm was recognized to be related to soluble microbial byproduct-like (SMP) substances and to be associated with aromatic protein-like peak [36]. The other peak at the 220/340 nm was identified as the fluorescence of protein-like substance, such as tryptophan-like acid [37]. These two small molecular proteins obtained in this process were the sludge hydrolyzates. The color variation showed that these two peaks in Fig. 4b and c are significantly higher than those in Fig. 4a, depicting that in the process of sludge degradation, the contents of DOMs with fluorescence were also increased. Fluorescence intensities of both peaks in Fig. 4b were higher than those in Fig. 4c, illustrating that the sludge biodegradation process could produce a larger amount of DOM under low-frequency ultrasound promotion. This phenomenon is consistent with the result of the generic parameters above (SCOD, soluble protein, etc.) A previous study of sludge direct-disruption using high-density ultrasound showed that one humic acid, two protein and two amino acid EEM peaks were appeared in the products [25]. The ultrasound wave could thoroughly disrupt the microbial cell wall and EPS during the process, and these two sludge components were broken into various kinds of small molecular proteins and amino acids. However, in the low-frequency ultrasound conditions of this study, only two EEM peaks were obtained, and the peak number and variety was the same with the non-ultrasound treated test. The results inferred that the ultrasound under low-frequency with low-density used in this study did not cause a direct sludge disintegration. The increased DOM concentration was the consequence of ultrasound promotion.
With the drops in VSS content, the soluble organic matter released from the sludge EPS and inner microorganism were increased. For the subsequent disposal and utilization, more soluble organic matters were released into the fermentation broth and the greater amount of desired products was obtained. The similar promotion effects also have the benefits in biological hydrogen production from sludge [12,13]. Thus the sludge hydrolysis using the present method will be more promising for further sludge utilization in many aspects, such as methane fermentation and hydrogen production, which will broaden the raw material alternatives for bio-energy production. Therefore, in this research, the low-frequency ultrasound promotion was proved to be an effective method to increase the soluble substance contents as well as the sludge reduction ability using thermophilic bacteria. The above findings of promotion effects were consistent with the previous study, which revealed that the microbial activity of hydrogen production bacteria could be enhanced by low-frequency ultrasound without change of the fermentation type [38]. 4. Conclusions This study clearly demonstrates that low-frequency ultrasound could effectively promotes the process of sludge reduction and hydrolysis using by thermophilic bacteria Geobacillus stearothermophilus TP-12. While VSS reduction ratio was significantly increased, more DOMs were released from by decomposing sludge EPS and inner microorganism. The products with easy-degradable forms would benefit for the following treatment process. The fluorescent properties of DOM characterized by EEM indicated that the low-frequency ultrasound could promote this sludge biodegradation process, without changing the main paths and product categories. Overall, the low-frequency ultrasound stimulation on sludge thermophilic biodegradation was a promising approach in the future applications. Acknowledgements This research was supported by the National Nature Science Foundation of China (Grant Nos. 51121062 and 51008105). The authors also gratefully acknowledge the financial support by the State Key Laboratory of Urban Water Resource and Environment (QA201211), the State Key Laboratory of Urban Water Resource and Environment (2014TS06), the Department of Education Fund for Doctoral Tutor (20122302110054), the Special S&T Project on Treatment and Control of Water Pollution (2013ZX07201007-
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Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Regular article
Promotion effects of ultrasound on sludge biodegradation by thermophilic bacteria Geobacillus stearothermophilus TP-12 Wan-Qian Guo a,∗ , He-Shan Zheng a , Shuo Li a , Shih-Hsin Ho a , Shan-Shan Yang a , Xiao-Chi Feng a , Jo-Shu Chang a,b,∗ , Xiang-Jing Wang c , Nan-Qi Ren a a
State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China Department of Chemical Engineering, National Cheng Kung University, Tainan, Taiwan c Research Center of Life Science and Biotechnology, Northeast Agricultural University, Harbin 150030, China b
a r t i c l e
i n f o
Article history: Received 5 February 2015 Received in revised form 25 September 2015 Accepted 1 October 2015 Available online 9 October 2015 Keywords: Sludge Waste treatment Biodegradation Thermophiles Protease Low-frequency ultrasound
a b s t r a c t Sludge biodegradation using thermophilic bacteria is a promising method for sludge treatment. In order to further enhance the efficiency of sludge reduction and hydrolysis, low-frequency ultrasound was used to promote this process. We isolated a thermophilic strain that is effective in secreting extracellular protease to hydrolyze sludge. Then the key ultrasound parameters were selected using the response surface methodology method. After 12 h treatment using thermophilic bacteria with short-time ultrasound promotion, volatile suspended solids (VSS) reduction ratio was achieved 32.8%, which is 41.4% higher than that without ultrasound promotion. Meanwhile the contents of soluble chemical oxygen demand (SCOD), protein and carbohydrate were increased by 20.2%, 16.8% and 15.9%, respectively. The composition of dissolved organic matter of sludge products evaluated by excitation-emission matrix spectroscopy demonstrated the promotion effect and eliminated the possibility of the direct sludge degradation caused by ultrasound treatment. Low-frequency ultrasound could effectively promote the thermophilic bacteria hydrolysis to achieve higher sludge biodegradation ratio without directly degrading the raw sludge. The promoted process of sludge biodegradation can further reduce the environmental risk and make sludge to be more readily usable. © 2015 Published by Elsevier B.V.
1. Introduction Increasing volumes of sludge generated from sewage treatment plants severely threatens environmental safety and human health [1]. The existing methods of sludge disposal, such as landfill internment, incineration, land application, etc., have potential environmental risks and obvious cost issues [2,3]. Thus, finding the more cost-effective and environmentally-friendly alternatives for sludge degradation has become a research direction that is receiving more attention [2,4]. Due to the special complex organism structure and particular microbial characteristic of sludge, main organic matters are wrapped in its extracellular polymeric substances (EPS) [5]. Thus, the essence and difficulty of sludge degradation is floc fragmentation and hydrolysis. To release wrapped organic matter
∗ Corresponding authors at: State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China. E-mail address: [email protected] (W.-Q. Guo). http://dx.doi.org/10.1016/j.bej.2015.10.002 1369-703X/© 2015 Published by Elsevier B.V.
and increase dissolved organic matter (DOM) content for further anaerobic digestion and utilization, currently, many sludge degradation methods have been developed to hydrolyze EPS and inner microorganism, such as ozonation [6], peracetic acid oxidation [7], sonication [8], alkali addition [9], mechanical and sludge thickening [10], and ozonation combined with sonication [11]. Due to high costs and serious secondary pollution caused by these physical and chemical approaches, biodegradation has aroused increasing attention, especially in sludge hydrolysis using thermophilic bacteria [12]. Sludge biodegradation by thermophilic bacteria is a technology of adding thermophilic bacteria solution which contains secreted thermophilic hydrolytic enzymes into sludge for hydrolysis at a thermophilic condition (ranging from 40 to 80 ◦ C) [13]. Sludge pretreated with thermophilic bacteria has drawn more interests for volatile fatty acids (VFAs) and hydrogen production [14,15]. Although this method seems superior to sludge biodegradation, it has not been extensively introduced to sludge treatment because of its relatively longer processing period and low degradation ratio compared to the physical-chemistry methods which is not feasible for large-scale commercial process [16]. To date, few
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Table 1 Characteristics of raw sludge and mixure of sludge with bacteria solution. Parameter
Raw sludge
Mixure of sludge with bacteria solution (9:1)
TSS (mg L−1 ) VSS (mg L−1 ) TCOD (mg L−1 ) SCOD (mg L−1 ) Soluble protein (mg L−1 ) Soluble carbohydrate (mg L−1 ) pH
14137.0 9872.0 14934.0 73.0 58.7 15.2 7.0
14371.0 10388.0 15326.0 1414.3 480.2 77.6 7.2
studies have been conducted to explore the promotion methods for enhancing sludge hydrolysis. A pretreatment method combining freezing/thawing with thermophilic bacteria was employed in pretreating sludge in order to achieve a higher degradation ratio [17]. However, the freezing step will also result in huge economic costs and practical resistance in commercial scale. Therefore, developing a cost-effective and convenient method to accelerate the thermophilic enzyme secretion and improve the enzyme hydrolysis efficiency is vital and urgent towards practical application. Low-frequency ultrasound (20–100 kHz) has been reported to have positive effects on microbial productivity and enzyme hydrolysis [18,19]. For instance, Song et al. [20] reported that enzymatic hydrolysis of alkaline protease from Bacillus licheniformis was greatly accelerated by ultrasound irradiation, and the final conversion ratio was significantly improved. Through the studies on stability of enzymes, including a-amylase [21], lactase [22], horse radish peroxidise and alkaline phosphatase [23], the active sites of enzyme were demonstrated not destroyed during the sonication process. In order to investigate the promotion effect of low-frequency ultrasound on bacterial activation and sludge solubilization products, the distributions of DOM was also analyzed using three-dimensional excitation-emission matrix (EEM) fluorescence spectroscopy. As a time-saving and accurate tool, EEM was extensively used to determine DOM which exhibits fluorescent emission characteristics [24]. This method can be regarded as an overall “fingerprint” of DOM that covers vitamins, NADH, protein-like and humic acid-like substances [25]. In this study, a strain that secretes high-efficiency thermophilic extracellular protease was first isolated under the specific growing conditions using selective culture mediums. This thermophilic strain was cultured as bacteria solution and then added into the raw sludge. Meanwhile the promotion effect of low-frequency ultrasound on sludge degradation process was comprehensively studied. The optimal parameters of ultrasound application were determined via the response surface methodology (RSM). Finally, three-dimensional EEM fluorescence spectroscopy was used to characterize the DOM distribution to evaluate the promotion effect of low-frequency ultrasound on sludge reduction and hydrolysis efficiency. 2. Materials and methods 2.1. Thermophilic strain isolation and identification For isolation of thermophilic bacteria which produce protease, the sludge was cultured in a shaking bath at 60 ◦ C for 36 h. The remainder liquor containing thermophilic bacteria was diluted using distilled water at a ratio of 10:1. A volume of 0.1 mL−1 sample of every level was screened on skim milk agar medium. After 24 h of cultivation at 60 ◦ C, the strains with transparent circles appeared were determined as protease producing ones. The colony with the biggest transparent hydrolytic zone on skim milk agar medium was selected and cultured in a soluble starch agar medium for 24 h. This procedure was repeated at least ten times to ensure a pure strain. The isolated colony was cultured on a standard LB agar medium,
and then stored at 4 ◦ C. The isolated pure strain of the thermophilic bacteria was designated as strain TP-12. Medium composition (g L−1 ): The LB agar medium contain: yeast extact 5; peptone 10; NaCl 10; agar 20. The skimmed milk agar medium contained: NaCl 5; peptone 3; skimmed milk 3; agar 20. The composition of fluid nutrient medium: yeast extact 5; peptone 10; NaCl 10. The DNA was extracted from the 1 mL pure culture solution (LB medium, 60 ◦ C, 24 h) using DNA Isolation Kit (Watson, Shanghai, China). The resultant DNA was subjected to polymerase chain reaction (PCR) with primers of 8F (5 -AGAGTTTGATCCTGGCTCAG3 ) and 1492R (5 -GGTTACCTTGTTACGACTT-3 ). The 16S rRNA gene obtained from isolated bacteria was cloned, purified, amplified and sequenced by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). The sequencing was done in triplicate in order to get the correct sequence. In the phylogenetic analysis, the nucleotide sequence was compared with the sequences in the GenBank/EMBL/DDBJ nucleotide sequence databases by the Basic Local Alignment Search Tool (BLAST) program (http://www.ncbi.nlm.nih.gov/BLAST/). 2.2. Source and preparation of WAS Sewage sludge was obtained from the secondary sedimentation tank of Harbin Wenchang Sewage Treatment Plant (Harbin, China). The raw sludge was first filtrated through a 20-mesh sieve to separate large debris, then settled for 24 h and stored at 4 ◦ C. The characteristics of the sludge used in this study are shown in Table 1. 2.3. Single-factor experiments To study the promotion effect of different ultrasound density and exposure time on thermophilic bacteria, and select ranges of independent variables for optimizing parameters by RSM, single-factor experiments were carried out. Different ultrasound densities (0.04 W mL−1 , 0.08 W mL−1 , 0.12 W mL−1 , 0.16 W mL−1 , 0.20 W mL−1 ) at the same exposure times of 20 s and different ultrasound exposure times (5 s, 10 s, 15 s, 20 s, 25 s) at the same ultrasound density of 0.12 W mL−1 were performed. The lowfrequency ultrasound radiation was conducted using an ultrasound probe system (CY-5D, Ningbo Scientz Biotechnology Co., Zhejiang, China) that emitted 20 kHz ultrasound wave, and had a variable power from 0 to 200 W. The diameter of the horn used was 20 mm. For each sonication experiment, thermophilic bacteria solution (cultured for 48 h) was inoculated in sludge in a ratio of 1:9 (the mixture of bacteria solution with sludge was shown in Table 1). 700 mL mixture sample was seeded into the reactor, heating at 60 ◦ C in a solubilization for 12 h at 140 rpm, timing supplement the evaporation of water, and the ventilation was about 0.16 L min−1 . Simultaneously, an ultrasonic instrument equipped with a sonoprobe was dipped into sludge treatment samples for 2 mm at three hours intervals. Liu et al. [26] found that enzyme remained its high activity even some hours after irradiation was stopped and the enhancement effects would be disappeared after 24 h. So the ultrasound interval is three hours in this experiment.
W.-Q. Guo et al. / Biochemical Engineering Journal 105 (2016) 281–287 Table 2 RSM experimental ranges and levels of the independent variables. Independent variables
Range and levels
Ultrasound density (W ml−1 ), x1 Exposure time (s), x2
− 1.414 0.06 0.86
−1 0.08 5.00
0 0.12 15.00
1 0.16 25.00
1.414 0.18 29.14
2.4. Parameters selection by RSM Response surface methodology (RSM) was used to determine the optimal conditions of ultrasound treatment and to predict the best value of response. Single-factor-test was employed to determine the preliminary range (Tables 2 and 3), and then 13 experimental runs were carried out according to a central composite design with two factors and five levels to optimize the relationship between the ultrasound density (X1 ,W mL−1 ), the exposure time (X2 , s), and the most important response, the sludge reduction rate (Y, %). The relation between the coded values and real values is described and shown in Eq. (1): xi = (Xi − Xi ∗) /Xi
(1)
where xi and Xi are the coded value and uncoded value of the independent variable, respectively; Xi * is the uncoded value of the independent variable at the center point; and Xi is the step change of variable. The fit quality can be expressed by the correlation coefficient (R2 ), and the significance of the model can be demonstrated by F-value and Probability value (Prob > F). 2.5. Fluorescence spectroscopy Prior to the fluorescence analysis, the samples were centrifuged at 9391 × g for 10 min and filtered using 0.45 m polytetrafluoroethylene (PTFE) filters and then diluted 200 times with distilled water. The three-dimensional EEM spectra were measured using a fluorescence spectrophotometer (FP-6500, JASCO, Japan). EEM spectra were obtained as follows: Scanning emission (Em) spectra from 220 to 650 nm were obtained at 1 nm increments by varying the excitation (Ex) wavelength from 220 to 450 nm at 5 nm increments. The scan speed was set at 2000 nm min−1 , and the slit widths were 5 nm for both excitation and emission monochromators. The software Origin 8.1 (OriginLab, Los Angeles, USA) was employed for figure processing. 2.6. Analytical procedures During the sludge pretreatment period, the analyses of SCOD, COD, SCOD, pH, TSS, VSS were conducted in accordance with Standard Methods [27]. The concentrations of TSS and VSS were determined using a 35 mL sludge sample at 105 ◦ C for 4 h and 600 ◦ C for 2 h [9]. The soluble-carbohydrate was measured by the phenolsulfuric acid method [25]. The soluble-protein was measured using a modified BCA protein assay kit (Sangon Biotech Co., Ltd., Shanghai, China).
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obtained using the neighbor joining method shown in Fig. S2. This strain was deposited at China General Microbiological Cultures Collection Center (CGMCC) with the accession number CGMCC 8643. The performance of TP-12 on sludge hydrolysis was expressed by VSS degradation ratio and the SCOD, soluble protein and carbohydrate increment. VSS reduction ratio could reach 23.2% for 12 h of sludge hydrolysis by thermophilic bacteria TP-12. The maximum amounts of SCOD, soluble protein and carbohydrate were 4008.7 mg L−1 , 1398.8 mg L−1 and 220.9 mg L−1 , respectively.
3.2. Analysis of single-factor test for ultrasound enhanced-sludge degradation It was reported that VSS reduction ratio followed the first order reaction kinetics model during the thermophilic aerobic sludge digestion [28]. In the present study, VSS content was regarded as an indicator of sludge reduction. Firstly, the preliminary studies were performed by single-factor experiments in order to determine the appropriate parameters of ultrasound density and exposure time. The VSS reduction ratio of different ultrasound density and exposure time was presented (Fig. 1). As shown in Fig. 1, compared with the 23.2% VSS reduction ratio of sludge degradation using TP-12 without ultrasound treatment, an obvious increase of VSS reduction ratio was observed for the ultrasound treated one. The peak reduction ratio of VSS reached 30.4% at exposure time of 20 s, which is about 43.5% higher than the blank experiment. After determining the optimum ultrasound density of 0.12 W mL−1 , the subsequent test showed that when exposure time exceed 15 s, the ultrasound may exert a negative influence and the VSS reduction ratio began to decrease. The similar phenomenon of particular ultrasound effect was also observed in the previous studies, depicting that the intracellular metabolism was enhanced under short time irradiation but inhibited under long time irradiation. Duan et al. [29] demonstrated that the cell wall became thinner after moderate low-intensity ultrasound irradiation through transmission electron microscopy (TEM) observation, which lead to increased its permeability and promoted the mass transfer efficiency. In addition, the bacterial structure would be destroyed under high energy inputs of lowfrequency ultrasound. Through TEM observation, it is found that the cytoplasmic membrane was dislocated and the intracellular content was leaked under high power irradiation [30]. Moreover, the enzyme specific activities also might be affected, even the capability of enzyme secretion. These individual promotion effects mentioned above have already been separately demonstrated and applied in the relevant studies [31–34]. In this study, the performance of sludge hydrolysis using thermophilic bacteria was promoted by moderate low-frequency ultrasound. This promotion effect is a complicated process, and the optimum parameters for the cells or enzymes are different. However, the specific mechanism of ultrasound effect on various parts in this mixed system was extremely difficult to explicitly distinguish and define, hence, it would be investigated in-depth in the further study.
3. Results and Discussions 3.1. Information of strain isolation, identification and performance As shown in Fig. S1a, the thermophilic bacterial strain TP-12 isolated from sludge could generate transparent circle through thermophilic protease hydrolysis of protein on skimmed milk agar medium. TP-12 was a brevibacterium detected by scanning electron microscope (Fig. S1b) and it was identified as a strain belongs to Geobacillus stearothermophilus. The phylogenetic tree of TP-12 was
3.3. Response surface methodology analysis of optimal ultrasound parameters After the single-factor experiments, optimal parameters of ultrasonic density and exposure time were further explored by the center composite design (CCD) of RSM. VSS reduction ratio was considered as an indicator for parameters optimization. The design matrix included two independent variables of exposure time (X1 ) and ultrasonic density (X2 ), and the corresponding results obtained are shown in Table 3. Coded and actual responses were established
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Table 3 Response surface Central Composite Design (CCD) and experiments. Run
Actual variables
Coded variables
Sludge reduction ratio (%)
1 2 3 4 5 6 7 8 9 10 11 12 13
X1 0.12 0.12 0.12 0.16 0.18 0.12 0.12 0.08 0.08 0.12 0.12 0.16 0.06
X2 15.00 15.00 15.00 25.00 15.00 15.00 29.14 5.00 25.00 0.86 15.00 5.00 15.00
x1 0 0 0 +1 +1.414 0 0 −1 −1 0 0 +1 −1.414
30
Y 33.03 ± 1.65 33.03 ± 1.65 33.03 ± 1.65 26.29 ± 1.31 23.64 ± 1.18 33.03 ± 1.65 26.79 ± 1.34 22.75 ± 1.14 26.04 ± 1.30 22.12 ± 1.17 33.03 ± 1.65 28.20 ± 1.41 25.36 ± 1.29
40 (a)
VSS Reduction Ratio (%)
VSS Reduction Ratio (%)
35
x2 0 0 0 +1 0 0 +1.414 −1 +1 −1.414 0 0 0
25 20 15 10 5 0
35
(b)
30 25 20 15 10 5 0
0.00
0.04
0.08
0.12
0.16
0.20
0
Ultrasound Density (W mL-1)
5
10
15
20
25
Exposure Time (s)
Fig. 1. Variations in VSS solubilization ratio of (a) different ultrasound densities and (b) different exposure times.
by Eqs. (1) and (2), respectively: YCODED = 33.03 + 0.41x1 + 1.00x2 − 1.30x1 x2 − 3.93x1 2 − 3.95x2 2 (2)
YACTUAL = −19.77880 + 648.22584X1 + 1.67430X2 − 3.24275X1 X2 − 2455.87891X1 2 − 0.039519X2 2
(3)
where Y is the VSS reduction ratio, x1 and x2 are the coded value of ultrasound density and exposure time; X1 , X2 , are the ultrasound density and exposure time, respectively. To determine the optimal levels, the relationship between two variables was illustrated by the 3-D representation of response surface generated by the model, as shown in Fig. 2. The optimized conditions obtained by RSM in the validated experiments were ultrasound density of 0.122 W mL−1 , exposure time of 16.2 s. According to the estimation model, the VSS reduction ratio reached 33.1% under the optimal ultrasound promotion conditions. They were employed to test the predictive model. Considered the operability in actual run, we modified the optimal conditions as follows: exposure time of 16 s, ultrasound density of 0.122 W mL−1 . Under the modified conditions, the average VSS reduction ratio obtained from the triplicate tests of 32.8% was achieved. The value was closed to the simulated value of 33.1% predicted by the model equation. Our results showed that low frequency ultrasound could effectively promote VSS reduction and increase sludge degradation ratio. Statistical analysis of the model was performed in the form of analysis of variance (ANOVA) to examine significance and adequacy of second-order polynomial equation (Table 4). The quadratic regression model showed that the value of determination coefficient (R2 ) was 0.9314, which implies that 93.14% of the variations
could be explained by the fitted model. It indicates a good agreement between experimental and predicted values. In addition, the P-values are used as a tool to check the significance of each variable. The small P-values indicate the higher significance of the corresponding variable. In this study, the model obtained was significant (P < 0.05) suggesting the ultrasound treatment time and density could play a synergistic role in VSS reduction. In addition, the VSS reduction ratio obtained at optimized ultrasound conditions in this study was much higher than the previous report, which was about 15% at 12 h [16]. Chen and Pan [35] confirmed that in the reactor operation of sludge solubilization using thermophilic bacteria, lower sludge solid contents corresponded to the higher acidification rate which could help achieving the higher degradation efficiencies of VSS. However, in the present study, we believed the main reason for high VSS degradation is due to the promotion effect from low-frequency ultrasound.
3.4. Effect of low-frequency ultrasound on enhancing waste sludge hydrolysis In order to achieve the economic feasibility of the waste sludge in the subsequent treatment, determining its hydrolysis ability is crucial. The SCOD, soluble protein and carbohydrate comprise the principal parts of the sludge hydrolysis products (shown in Fig. 3). The contents of these three soluble components exhibited greatly increased under low-frequency ultrasound stimulation, which demonstrated that the treated sludge was hydrolyzed into more soluble matters through thermal hydrolysis enzymes secreted from TP-12. As shown in Fig. 3a–c, during this process, SCOD and soluble protein concentrations increased rapidly within the initial 8 h, reaching 4982.1 mg L−1 and 1634.1 mg L−1 , respectively. The concentrations declined in 8 h. The highest soluble carbohydrate concentration was obtained at the optimal HRT of 12 h. The
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Fig. 2. The response surface 3D plot showing the effects of ultrasound density and ultrasound time on VSS reduction ratio.
Table 4 Analysis of variance (ANOVA) results for the response surface quadratic mode. Source Model x1 x2 x1 x2 x1 2 x2 2 Residual Lack of fit Pure error Cor total
Statistics Sum of squares 207.12 1.32 7.94 6.73 107.41 108.64 15.26 15.26 0 222.38
df 5 1 1 1 1 1 7 3 4 12
Mean Square 41.42 1.32 7.94 6.73 107.41 108.64 2.18 5.09 0
F-Value 19 0.61 3.64 3.09 49.26 49.83
P-Value <0.0001 0.4612 0.0981 0.1224 0.0002 0.0002
Significant
-1
SCOD Conc. (mg L )
6000 5000
Ultrasound Contral
4000 3000 2000 1000
(a)
0 0
12
24
36
48
60
72
-1
Soluble Carbohydrate Conc. (mg L )
1800
-1
Soluble Protein Conc. (mg L )
Time (h)
Ultrasound Control
1500 1200 900 600 300
(b)
0 0
12
24
36 Time (h)
48
60
72
300 250 200 150 100 50
Ultrasound Control
(c)
0 0
12
24
36
48
60
72
Time (h)
Fig. 3. (a) Comparison of SCOD, (b) soluble protein, and (c) soluble carbohydrate concentrations over a 72 h during thermophilic aerobic digestion of sludge with or without ultrasound promotion.
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Fig. 4. EEM spectra of DOM fractions extracted from (a) the mixure of sludge and bacteria solution (9:1) at 0 h, (b) hydrolysis by thermophilic bacteria for 8 h (with ultrasound promotion), (c) hydrolysis by thermophilic bacteria treatment for 8 h (without ultrasound promotion).
respective maximum concentrations of SCOD, soluble protein and soluble carbohydrate were 13.0%, 15.9% and 16.8%, respectively, which are higher than the control. Better hydrolysis performance was observed under the ultrasound promoted tests compared with the control group, proving that the low-frequency ultrasound has a positive impact on sludge hydrolysis capability of thermophilic bacteria.
3.5. Characteristics of dissolved organic matters by EEM spectra Three-dimensional EEM spectroscopy which can collect multiple emission scans of DOM by changing excitation wavelengths was applied to characterize the samples before and after pre-treatment in this study. The sludge solubilization type can be revealed from the DOM categories and their corresponding contents. Nine EEM fluorescence spectra of DOM characterized by a broad band are shown in Fig. 4. There were only two peaks at Ex/Em of 280/340 and 220/340 nm in all examined samples. The peak at 280/340 nm was recognized to be related to soluble microbial byproduct-like (SMP) substances and to be associated with aromatic protein-like peak [36]. The other peak at the 220/340 nm was identified as the fluorescence of protein-like substance, such as tryptophan-like acid [37]. These two small molecular proteins obtained in this process were the sludge hydrolyzates. The color variation showed that these two peaks in Fig. 4b and c are significantly higher than those in Fig. 4a, depicting that in the process of sludge degradation, the contents of DOMs with fluorescence were also increased. Fluorescence intensities of both peaks in Fig. 4b were higher than those in Fig. 4c, illustrating that the sludge biodegradation process could produce a larger amount of DOM under low-frequency ultrasound promotion. This phenomenon is consistent with the result of the generic parameters above (SCOD, soluble protein, etc.) A previous study of sludge direct-disruption using high-density ultrasound showed that one humic acid, two protein and two amino acid EEM peaks were appeared in the products [25]. The ultrasound wave could thoroughly disrupt the microbial cell wall and EPS during the process, and these two sludge components were broken into various kinds of small molecular proteins and amino acids. However, in the low-frequency ultrasound conditions of this study, only two EEM peaks were obtained, and the peak number and variety was the same with the non-ultrasound treated test. The results inferred that the ultrasound under low-frequency with low-density used in this study did not cause a direct sludge disintegration. The increased DOM concentration was the consequence of ultrasound promotion.
With the drops in VSS content, the soluble organic matter released from the sludge EPS and inner microorganism were increased. For the subsequent disposal and utilization, more soluble organic matters were released into the fermentation broth and the greater amount of desired products was obtained. The similar promotion effects also have the benefits in biological hydrogen production from sludge [12,13]. Thus the sludge hydrolysis using the present method will be more promising for further sludge utilization in many aspects, such as methane fermentation and hydrogen production, which will broaden the raw material alternatives for bio-energy production. Therefore, in this research, the low-frequency ultrasound promotion was proved to be an effective method to increase the soluble substance contents as well as the sludge reduction ability using thermophilic bacteria. The above findings of promotion effects were consistent with the previous study, which revealed that the microbial activity of hydrogen production bacteria could be enhanced by low-frequency ultrasound without change of the fermentation type [38]. 4. Conclusions This study clearly demonstrates that low-frequency ultrasound could effectively promotes the process of sludge reduction and hydrolysis using by thermophilic bacteria Geobacillus stearothermophilus TP-12. While VSS reduction ratio was significantly increased, more DOMs were released from by decomposing sludge EPS and inner microorganism. The products with easy-degradable forms would benefit for the following treatment process. The fluorescent properties of DOM characterized by EEM indicated that the low-frequency ultrasound could promote this sludge biodegradation process, without changing the main paths and product categories. Overall, the low-frequency ultrasound stimulation on sludge thermophilic biodegradation was a promising approach in the future applications. Acknowledgements This research was supported by the National Nature Science Foundation of China (Grant Nos. 51121062 and 51008105). The authors also gratefully acknowledge the financial support by the State Key Laboratory of Urban Water Resource and Environment (QA201211), the State Key Laboratory of Urban Water Resource and Environment (2014TS06), the Department of Education Fund for Doctoral Tutor (20122302110054), the Special S&T Project on Treatment and Control of Water Pollution (2013ZX07201007-
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