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Lipid profiling in sewage sludge
Water Research 116 (2017) 149e158
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Lipid profiling in sewage sludge Fenfen Zhu a, *, Xuemin Wu a, Luyao Zhao b, Xiaohui Liu c, Juanjuan Qi d, Xueying Wang c, Jiawei Wang e a
School of Environment and Natural Resources, Renmin University of China, 100872 Beijing, China Shougang Institute of Technology, 100043 Beijing, China c School of Life Sciences, Tsinghua University, 100084 Beijing, China d Development and Reform Commission of Bomi County in Linzhi Area, 860300, China e Research Center, Beijing Drainage Group, 100124 Beijing, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 November 2016 Received in revised form 15 March 2017 Accepted 15 March 2017
High value-added reutilization of sewage sludge from wastewater treatment plants (WWTPs) is essential in sustainable development in WWTPs. However, despite the advantage of high value reutilization, this process must be based on a detailed study of organics in sludge. We used the methods employed in life sciences to determine the profile of lipids (cellular lipids, free fatty acids (FFAs), and wax/gum) in five sludge samples obtained from three typical WWTPs in Beijing; these samples include one sludge sample from a primary sedimentation tank, two activated sludge samples from two Anaerobic-Anoxic-Oxic (A2/ O) tanks, and two activated sludge samples from two membrane bioreactor tanks. The percentage of total raw lipids varied from 2.90% to 12.3%. Sludge from the primary sedimentation tank showed the highest concentrations of lipid, FFA, and wax/gum and the second highest concentration of cellular lipids. All activated sludge contained an abundance of cellular lipids (>54%). Cells in sludge can from plants, animals, microbes and so on in wastewater. Approximately 14 species of cellular lipids were identified, including considerable high value-potential ceramide (9567e38774 mg/kg), coenzyme (937e3897 mg/ kg), and some phosphatidylcholine (75e548 mg/kg). The presence of those lipid constituents would thus require a wider range of recovery methods for sludge. Both cellular lipids and FFAs contain an abundance of C16eC18 lipids at high saturation level, and they serve as good resources for biodiesel production. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Sewage sludge Lipid profile Cellular lipids Free fatty acid Wastewater treatment Biodiesel
1. Introduction Recovery of energy or value-added material from wastewater attracts strong interest in sustainable wastewater treatment and resource utilization (Hao et al., 2015). Sewage sludge is a byproduct of wastewater treatment and a highly potential raw material for resource recovery because it contains considerable amount of organic compounds (Hao et al., 2015). In China, wastewater treatment plants (WWTPs) produce over 3 107 t of sludge (with 80% moisture content) yearly (Kroiss, 2004), and this production rate is expected to increase as a result of the progressively stringent regulatory standards for wastewater effluent, together with the growing number of WWTPs. The average organic matter content of sewage sludge in China is 38.4%; of which, 55% are carbohydrates, 20% are proteins, and approximately 20% are lipids (Li
* Corresponding author. E-mail address: [email protected] (F. Zhu). http://dx.doi.org/10.1016/j.watres.2017.03.032 0043-1354/© 2017 Elsevier Ltd. All rights reserved.
et al., 2003; Yeong et al., 2013; Siddiquee and Rohani, 2011) Although the current treatment methods for sewage sludge, such as anaerobic digestion, incineration, and composting, present many merits, they cannot recover sufficient energy or value-added materials to render sludge treatment cost effective (Hao et al., 2015). Municipal sewage sludge has been proposed to be a potential feedstock in diesel production (Mondala et al., 2009; Revellame et al., 2010; Zhu et al., 2014; Revellame et al., 2012a; Kwon et al., 2012), and this approach presents several potential advantages over the existing treatment methods. First, lipid concentrations in sewage sludge are considerably high, and extraction of such lipids will facilitate biodiesel production at an impressive ratio of up to 27.4% (Mondala et al., 2009; Dufreche et al., 2007). We have successfully conducted lipid extraction and in situ transesterification of sewage sludge, which indicated that most lipids, including phospholipids, can be transformed into biodiesel, and the quality of which was affected by sludge source (Zhu et al., 2014; Qi et al., 2016). Second, sewage sludge is abundant; being a solid waste,
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sewage sludge can be used as free or low-cost raw materials for diesel production. Third, sensitivity analysis indicated that if 10% (weight) biodiesel yield is attained, activated sludge can become economically competitive with petroleum (Kwon et al., 2012; Revellame et al., 2011). In addition, liquid fuel can be transported more easily than gas fuel (e.g., biogas). Detailed understanding of lipid profile in sewage sludge is critically important in production of biodiesel or other valuable products, such as nutrients, cosmetics, and health products. The cellular lipid species in sludge include triglycerides (TG), diglycerides (DG), monoglycerides, phospholipids, free fatty acids (FFAs), and sterol (Siddiquee and Rohani, 2011; Dufreche et al., 2007; Olkiewicz et al., 2014; Kargbo, 2010; Jarde et al., 2005). Phospholipids account for approximately 24%e25% of cell dry mass (Siddiquee and Rohani, 2011; Olkiewicz et al., 2014; Kargbo, 2010). Moreover, up to 36.8 wt% of dry sludge consists of fatty acids, sterol, and some aliphatic fractions (Jarde et al., 2005). Fatty acids in sludge are predominantly C10eC18 (Dufreche et al., 2007). Huynh et al. (2010) have studied the profiles of neutral lipid and fatty acids. However, information on lipids in sewage sludge remain limited, and quantitative analysis of lipids is generally lacking. To further understand the lipid profile in sewage sludge, we obtained sludge samples from three typical WWTPs in Beijing, China and analyzed these samples using the methods used in life sciences. Based on the characteristics of different lipids, three different extraction/pretreatment methods (two Ultra Performance Liquid Chromatography(UPLC)/Mass(MS)/Mass(MS) operation conditions þ weighing) were performed to measure three groups of lipids, namely, cellular lipids, FFA, and wax/gums (possibly including fat, grease, and steroid). Cellular lipids are the most complicated lipids, which should be from the cells of plants, animals, microbes and so on that deposit in the sludge from wastewater, including triglycerides (TG), diglycerides (DG), phosphatidylcholine (PC), ceramide (Cer), and coenzyme (Co). 2. Materials and methods 2.1. Sewage sludge Five sewage sludge samples (total solid (TS): 1.0% ± 0.5%) were collected from three municipal WWTPs in Beijing, China. The capacity of WWTP-I is 106 t/d, and the main technology employed is anaerobic-anoxic-oxic (A2/O). The capacity of WWTP-II is 6 105 t/ d, and this plant employs two parallel technology flows, namely, membrane bio-reactor (MBR) and A2/O. The capacity of WWTP-III is 105 t/d, and MBR is mainly employed in this plant. The main operation conditions in winter season are listed in Table 1, and the technology flows in these WWTPS are presented in Fig. 1. Among the existing WWTPs in China, the plants utilizing the A2/O process showed the largest design treatment capacity of 33.2% (Qiu et al., 2010; Jin et al., 2014). Moreover, MBR has become an increasingly attractive option in wastewater treatment and water reclamation processes with a projected annual growth rate of 20%e30% in the next 5 years (Sun et al., 2011; Wang et al., 2014).
The five sludge sampling points include the primary sedimentation tank of WWTP-I, A2/O tank of WWTP-I, MBR tank of WWTPII, A2/O tank of WWTP-III, and MBR tank of WWTP-III. Sampling points in A2/O and MBR tanks were located at the end of the aerobic tanks (refer to Fig. 1 for the schematic of the three WWTPs). The amount of sample from each sampling point is sufficient to obtain at least nine samples from the same sampling point for pretreatments and measurements. Three samples for extraction and analysis of cellular lipids, three samples for extraction and analysis of FFA, and the other three samples for extraction of wax and gums. Elements in sludge were analyzed by X-ray fluorescence (XRF-1800, Shimadzu, Kyoto, Japan), and results are shown in Supplementary Information I. All chemicals used in this study were chromatographic grade and used without further purification. The chemicals were purchased from Technology Development Co., Ltd., and standards were purchased from Sigma Co. Solutions were prepared using water purified by a Millipore Milli Q UV Plus System. 2.2. Extraction 2.2.1. Pretreatment A sludge sample (Total solid concentration (TS): 1.0% ± 0.5%) was centrifuged at 3000 rpm for 10 min to obtain dewatered sludge (TS: 14.0% ± 0.5%). Subsequently, the dewatered sludge was freeze-dried via a two-step process: first, the sludge was centrifuged and frozen for 2 days at 20 C, and second, the frozen sludge was freeze-dried in an automatic vacuum freeze dryer (Model: FD-1A-50) for 2 d. The dried sludge was crushed using mortar and pestle, homogenized, and stored in a freezer prior to use. 2.2.2. Extraction of cellular lipids Extraction was performed according to the method described by Folch et al. (1957) with modification. Three grams of dried sample was weighed directly into 100 mL centrifuge tubes with Teflonlined screw caps. Citric acid buffer (0.15 M, pH ¼ 7.3) was added to obtain a total volume of 2 mL; 7.5 mL of 1:2 methanolchloroform was subsequently added into the solution. The samples were extracted on a rotating oscillator for 2 h. 1:2 methanolchloroform (6 mL) and citric acid buffer (4.5 mL) were subsequently added into the tubes, and the solution was mixed overnight on a vortex mixer for phase separation. The samples were centrifuged at 4000 rpm for 10 min and then the lipid-containing organic phase (top) was transferred into 10 mL Pyrex tubes and allowed to evaporate completely under a stream of nitrogen. 2.2.3. FFA extraction Extraction was performed according to the method described by Bligh and Dyer (1959) with modification. Three grams of dried sludge was weighed directly into 50 mL centrifuge tubes with Teflon-lined screw caps. Subsequently, 1.5 mL of citric acid buffer (0.15 M, pH ¼ 7.3), 1.9 mL of chloroform, and 3.75 mL of methanol were successively added and then vortex oscillated (alternatively, the mixture can be homogenized by ultrasound for 10 min). The
Table 1 Main operation parameters of three WWTPs.
Primary sedimentation tank A2/O tank of WWTP(I) MBR tank of WWTP(II) A2/O tank of WWTP(III) MBR tank of WWTP(III)
CODinlet (mg/L)
BODinlet (mg/L)
F/M (kgBOD/kgVSS)
SRT (d)
HRT (h)
389 362 646 497 579
182 / 373 287 336
/ 0.103 0.0669 0.104 0.0404
/ 19.3 25.5 16.2 24.4
/ 12 14 15 16.5
Legend: F/M means how much does microbe will eat or “Feeding/microbes”.
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Fig. 1. Schematic diagram of a municipal wastewater treatment plants.
mixture was oscillated by a fully automatic flip oscillator for 2 h at 250 rpm in the dark and then centrifuged for 5 min at 4000 rpm.
The filtrate was collected. Afterwards, 7.6 mL of 2:1 methanolchloroform was added into the remaining sludge residue,
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extracted on a vortex oscillator for 2 h, oscillated in horizontal oscillator for 2 h, and centrifuged. The filtrate was collected and mixed with a previously obtained filtrate. 2:1 methanol-chloroform (6 mL) and citric acid buffer (4.8 mL) were subsequently added into the tubes containing the filtrate and then the solution was vortex oscillated and allowed to stand overnight for phase separation. The chloroform solution containing FFA was transferred into Pyrex tubes, dried under nitrogen steam in draught cupboard, and stored in refrigerator. 2.2.4. Wax and gum extraction The amounts of wax and gum were determined using the removal method as described previously (Huynh et al., 2010; Rajam et al., 2005). Lipid was first extracted from dried sludge through Soxhlet extraction. Five grams of the extracted sludge lipid was dissolved in acetone and maintained at 60 C for 1 h to obtain a clear solution. The solution was cooled to room temperature and stored in a refrigerator at 5 C for 24 h to allow the waxes to crystallize. The insoluble fraction that is rich in waxes was separated through centrifugation under 8000 rpm at 5 C. The solid and liquid components were subsequently separated through vacuum filtration. The entire process of dewaxing and degumming was repeated using the collected filtrate as substrate, which was stored at 5 C for 24 h. The process was repeated to separate the filtrate and filter residue. After removal of the acetone, the filtered residue was weighed together with former separated solid. 2.3. Lipid analysis Wax and gum were weighed only and no further analysis was performed. By contrast, FFA and the cellular lipids were extensively analyzed by a Q Exactive LC-MS/MS (Thermo Fisher Scientific) system. 2.3.1. Analysis of cellular lipids The extracted lipid was re-dissolved by chloroform just before analysis. Analysis was performed according to the method described by Tang et al. (2016). A UPLC system was coupled to a QExactive orbitrap mass spectrometer (Thermo Fisher, CA) equipped with a heated electrospray ionization probe. Lipid extracts were separated using a Hypersil GOLD C18 100 2.1 mm, 1.9 mm column (Thermo Fisher, CA) connected to a Thermo Fisher Scientific Autosampler and to a UPLC pump. A binary solvent system was used, in which mobile phase A consisted of ACN:H2O (60:40) and 10 mM ammonium acetate, whereas mobile phase B comprised IPA:ACN (90:10) and 10 mM ammonium acetate. Separations were performed for over 30 min under the conditions listed in Supplementary information II. A flow rate of 250 mL/min was used in the analysis, and the column and sample tray were stored at 45 C and 10 C, respectively. Data with a mass range of mass/ charge (m/z) 240e2000 and m/z 200e2000 were respectively acquired in the positive and negative ion modes through datadependent MSMS acquisition. Full scan and fragment spectra were collected with resolutions of 70,000 and 17,500, respectively. The source parameters were as follows: spray voltage: 3000 V; capillary temperature: 320 C; heater temperature: 300 C; sheath gas flow rate: 35; and auxiliary gas flow rate: 10. Lipidomics identification was performed using the analytical software Lipid Search 4.0 (Thermo Fisher, CA). Fig. 2 showed the base peak chromatograms under positive and negative ion modes of one sample, respectively. Depending on their specific molecular characteristics, different lipid species showed distinct intensity responses under positive or negative ion modes. For example, triglycerides (TG), diglycerides (DG), and phosphatidylcholine (PC) displayed better responses under positive ion
mode, whereas phosphatidylethanolamine (PE), phosphatidylinositol (PI), and cardiolipin (CL) were detected under negative ion mode. Thus, the lipids were examined in both ion modes. Supplementary information III presented the legend and reference. Quantitative analysis was performed using the area external standard method, and the standards used were as follows: 18:1 cardlolipin (CL), 16:0e18:1 phosphatidylglycerol (PG), 16:0e18:1 phosphatidylethanolamine (PE), 18:0e18:2 phosphatidylcholine (PC), 16:0e18:1 phosphatidylinositol (PI), 16:0e16:0e18:0 triglycerides (TG), C18 (d18:1/18:0) Ceramide (Cer), 16:0 diglycerides (DG). Other lipids, such as sphingosine (So), lysophosphatidylcholine (LPC), coenzyme (Co), (O-acyl)-1-hydroxy fatty acid (OAHFA), dimethylphosphatidylethanolamine (dMePE), phosphatidylmethanol (PMe), and PG were quantitatively analyzed using a semiquantitative method according to the ratio of the relative amounts of response area of lipids (refer to the Supplementary Information IV for the relative amount of each lipid determined under the two ion modes using the area ratio method). 2.3.2. FFA analysis The extracted lipid was re-dissolved by chloroform just before analysis. The analysis was performed by a UPLC-MS/MS. The conditions for UPLC were as follows: Hypersil GOLD C18 100 2.1 mm, 1.9 mm column (Thermo Fisher, CA); column temperature, 45 C; velocity, 250 mL/min; sample volume, 2 mL; mobile phase, A phase (ACN: H2O ¼ 60:40 and 10 mM ammonium acetate) and B phase (IPA: ACN ¼ 90:10 and 10 mM ammonium acetate). Separations were performed for over 30 min under the conditions listed in Supplementary information II. Mass condition: negative ion mode; spray voltage, 3000 V; capillary temperature, 320 C; heater temperature, 300 C; sheath gas flow rate, 35 arb; auxiliary gas flow rate, 10; and mass range, m/z 200e2000 (R 70,000); fragment spectra were collected with resolutions of 17,500. The standards were analyzed first before the unknown samples were examined. Arachidonic acid-d8 was used as standard samples in quantitative analysis using the area external standard method. 3. Results and discussion 3.1. Cellular lipids The cellular lipid contents of sludge obtained under different sewage treatments were extensively analyzed (Fig. 3). The amounts of total cellular lipids were 41,656 ± 15,897, 26,079 ± 4410, 23,006 ± 10,361, 66,397 ± 18,351, and 16,779 ± 7670 mg/kg in primary sedimentation tank of WWTP-I, A2/O tank of WWTP-I, MBR tank of WWTP-II, A2/O tank of WWTP-III, and MBR tank of WWTP-III, respectively. Fourteen species, namely, ceramide (Cer), phosphatidylcholine (PC), cardiolipin (CL), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), triglycerides (TG), diglycerides (DG), sphingosine (So), dimethylphosphatidylethanolamine (dMePE), (O-acyl)-1-hydroxy fatty acid (OAHFA), coenzyme (Co), lysophosphatidylcholine (LPC), and phosphatidylmethanol (PMe), were identified in the sewage sludge. Every species has more than 1 kinds of different molecules. For example there are 209 kinds of different TG molecules and 28 kinds of different Cer molecules in the sludge from primary sedimentation tank of WWTP-I. TG concentration in the sludge from primary sedimentation tank of WWTP-I is the sum of the concentration of all the TG molecules in that sludge sample so did the concentration of other lipid species. Liu et al. (2008) and Wong et al. (2005) have reported that microbial community in activated sludge is significantly influenced by process types, operation conditions, and scale, among others. Given that phospholipid was once used to identify microbial community
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Fig. 2. Chromatogram under positive and negative ion mode of one sludge sample.
in earth, lipid species should be related to microbial community, which in turn is influenced by WWTP operation. The lipid species in the sludge obtained from a primary sedimentation tank should be strongly affected by the influent. Among the cellular lipids, Cer (9567 ± 5095e38774 ± 11,683 mg/kg), So (3230 ± 1053e7833 ± 711 mg/kg), and Co (937 ± 352e3897 ± 3038 mg/kg) are the most abundant species. This finding suggests that Cer, So, and Co are important components
both of the microorganisms in bioreactors and the organisms in the influent. The amounts of the other species are as follows: TG (223 ± 64e837 ± 187 mg/kg), DG (47±4e585 ± 216 mg/kg), PC (75 ± 36e548 ± 269 mg/kg), CL (155 ± 59e763 ± 367 mg/kg), PE (186 ± 70e1103 ± 643 mg/kg), and PG (70 ± 22e347 ± 167 mg/kg). A considerable amount of other cellular lipids, such as PC, was observed. Thus, utilization or reutilization of lipids in sewage sludge may be explored; for instance, Cer may be extracted to be
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Fig. 3. Concentration of cellular lipids of sludge from different sewage treatment processes.
used in cosmetics production (Ramirez et al., 2008; Zhang et al., 2003). Sludge from A2/O tank of WWTP-III contained the highest ceramide (Cer) concentration (38,773.3 mg/kg) followed by that in the sludge from the primary sedimentation tank of WWTP-I (28,223.3 mg/kg). Sludge from the primary sedimentation tank contains the highest concentration of coenzyme (Co) (3896.7 mg/ kg), which is nearly double or triple that in activated sludge from bioreactors, except in the activated sludge from A2/O tank of WWTP-III. Thus, the sludge from the primary sedimentation tank and sludge from A2/O tank of WWTP-III are more promising for reutilization. As shown in Fig. 3 and Supplementary Information V, the decreasing amount of total glyceride (triglycerides (TG)þ diglycerides (DG)) in the sludge samples is as follows: sludge from primary settling tank (1422 mg/kg) > sludge from A2O tank of WWTP-III (1225 mg/kg) > sludge from MBR tank of WWTP-III (419 mg/kg) > sludge from A2/O tank of WWTP-I (329 mg/ kg) > sludge from MBR tank of WWTP-III (274 mg/kg). This result is consistent with the fact that lipids in primary sedimentation sludge are mainly released from animal or plant residues in the influent, and these residues contain more glyceride than microorganisms. Moreover, the sludge from the primary sedimentation tank contained a minimum amount of glycerol phospholipid (phosphatidylcholine (PC)þ cardiolipin (CL)þ phosphatidylethanolamine (PE)þ phosphatidylglycerol (PG)) (486 mg/kg). To determine the potential of using cellular lipids for biodiesel production, we performed transesterification reaction by using phosphatidylcholine (PC) (58.8% pure). Hexadecanoic acid methyl ester (C17H34O2), 9,12Octadecadienoic acid methyl ester (C19H34O2), 9-Octadecenoic acid methyl ester (C19H36O2) were identified by GCMS-QP2010 in the final product with nothing else which means it is 100% pure FAME (refer to Supplementary Information VI). Revellame et al. (2012b) has proposed the cracking theory of phospholipid in such kind of reaction and we proposed the theoretical reaction formula in
Supplementary Information VI which is also similar to the traditional transesterification reaction of triglyceride. . We subsequently checked the relative concentration of C16eC18 in fatty acyl groups (triglycerides (TG), diglycerides (DG), phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), cardiolipin (CL), ceramide (Cer), and phosphatidylcholine (PC)), and results are shown in Table 2. The relative amount of C16eC18 in all species, except PI, is higher than 90%. Considering that the concentration of saturated lipids is also important in producing biodiesel of better quality with higher cetane number (Dufreche et al., 2007), we determined the relative amount of the saturated fatty acid groups C16 and C18 in TG, DG, PI, PE, PG, CL, Cer, and PC (Table 3). The decreasing order of the average percentage of the saturated lipids is as follows: PI > Cer > TG > PG > PC > PE > CL > DG. The relative amount of most species is higher than 20%, and this result is possibly related to the characteristics of the source of the lipids. Lipids in sludge from primary sedimentation tank are mainly released from animal and plant residues, whereas the lipids in activated sludge are mainly released from microorganisms. Given the complexity of the influent source and the influencing factors of microbial community structure, it can be deduced that food consumption structure, cooking oil, cosmetics consumption tendency, and operation parameters of treatment units, among others, will have good impact on profiles of cellular lipids in sludge. 3.2. FFA Table 4 shows the detailed profile of FA9eFA32 (Given that the distribution of FA33eFA37 in FA is less than 0.1%, information about them was illustrated in Supplementary Information VII). FFA concentration is 0.11%e1.22%, consistent with the results of Jarde et al. (2005). When the concentration of FFA is higher than 1%, alkaline catalyst is not suitable for sewage sludge to make biodiesel at least not suitable in the first step (Sureshkumar et al., 2008; Mata et al., 2010).
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Table 2 Relative amount of fatty acyl group which had C16 and C18 from TG, DG, PI, PE, PG, CL, Cer and PC of sludge from five sewage treatment processes. Relative amount of fatty acyl group which had C16 and C18
Sludge from primary sedimentation tank of WWTP(I)
Sludge from A2/O tank Sludge from MBR tank Sludge from A2/O tank Sludge from MBR tank of WWTP(I) of WWTP(II) of WWTP(III) of WWTP(III)
Cer PC CL PE PG PI TG DG
89.3% 99.8% 100.0% 97.7% 97.4% 100.0% 94.4% 75.5%
95.5% 97.2% 100.0% 88.2% 98.0% 28.4% 99.4% 100.0%
88.8% 98.8% 100.0% 91.6% 96.7% 42.8% 97.8% 100.0%
87.3% 99.9% 100.0% 95.4% 99.3% 67.0% 93.2% 84.3%
90.6% 99.4% 100.0% 93.9% 94.7% 38.9% 94.8% 100.0%
Table 3 Relative amount of saturated fatty acids group of C16 and C18 in TG, DG, PI, PE, PG, CL, Cer and PC of sludge from five sewage treatment processes. Relative amount of saturated fatty acids Sludge from primary group of C16 and C18 sedimentation tank of WWTP(I)
Sludge from A2/O tank Sludge from MBR tank Sludge from A2/O tank Sludge from MBR tank of WWTP(I) of WWTP(II) of WWTP(III) of WWTP(III)
Cer PC CL PE PG PI TG DG
52.0% 41.7% 21.3% 30.7% 39.2% 70.8% 40.2% 0.0%
45.5% 37.1% 20.7% 15.5% 31.3% 50.0% 45.3% 24.6%
The sludge from primary sedimentation tank of WWTP-I contained the highest FFA content (1.22%), whereas the other sludge samples contained very low FFA concentrations (0.07%e0.20%). FFA is possibly a good substrate for microorganisms and is easily assimilated so that low amount of FFA remains in the activated sludge (Chipasa and Medrzycka, 2008). More than 24 kinds of FFA were identified, and the FA18 and FA16 series are the most abundant at 22.0%e42.9% and 36.7%e49.4%, respectively. FA16 and FA18 content both contribute to more than 64% of FA in sludge samples. The saturation level of (FA16 þ FA18) is higher than 40%, which is very favorable for production of good biodiesel. Moreover, (FA16 þ FA18) contributes to nearly 80% of FA in the sludge from primary sedimentation tank of WWTP-I. At this point, sewage sludge is suitable as raw material for biodiesel production. Similar to that of the cellular lipids, the profile of FFA is influenced by the influent source, such as cooking oil, cosmetics, and temperature. 3.3. Wax and gum Table 5 shows the results for wax and gum. Sludge from primary sedimentation contains the highest wax and gum concentrations because they are easily captured by the primary sedimentation tank. When a WWTP does not have a primary sedimentation tank, the activated sludge from the bioreactors may contain increased concentration of wax and gums. However, despite the increased concentration of wax and gums, they can be assimilated relatively easily. 3.4. Total lipid concentration The total amount of the combined cellular lipids, FFA, and wax and gum constitute 89.5%e98.1% of the total raw lipid extracted through Soxhlet extraction (Table 5), indicating that the lipids in sewage sludge mainly consist of cellular lipids, FFA, and wax and gum. The other lipid components possibly include little oils, grease, fat, and sterol (Jarde et al., 2005; Dufreche et al., 2007). Moreover, the unknown proportion of the lipid components is low. Being one of the important contributors of COD/BOD, lipid
56.3% 42.1% 25.6% 27.0% 42.8% 67.8% 38.3% 0.0%
48.4% 36.7% 21.3% 24.9% 40.0% 57.9% 35.1% 13.9%
45.0% 35.6% 25.1% 24.2% 41.8% 74.6% 37.9% 0.0%
concentration in sludge should be related to the unit function of wastewater treatment. Primary sedimentation tank mainly captures suspended organic compounds, including/containing lipids, whereas a bioreactor tank (A2O tank/MBR tank) digests resolvable organic compounds, which are reduced by using microorganisms. As a result, the captured organic compounds, including lipids, were stored in the sludge in primary sedimentation tank. By contrast, the organic compounds, including lipids, which flowed into the bioreactor tank, were digested by microorganisms. Variation in raw lipid concentration can be explained by the traditional mechanism of a bioreactor, wherein BOD loading, sludge retention time (SRT), and hydraulic retention time are important factors influencing the digestion/reduction of organic compounds. For example, BOD loading in A2/O tank of WWTP-III is higher than that in MBR tank of WWTP-III (Table 1); additionally, the former has shorter SRT than the latter. As a result, raw lipid concentration in sludge from A2/O tank of WWTP-III is approximately 2.7 times that of the lipid concentration in sludge from MBR tank of WWTPIII. The microorganisms in A2/O tank of WWTP-III assimilated the organic compounds as cellular lipids, considerably increasing the concentration of cellular lipids. The shorter SRT and higher BODinlet in A2/O tank of WWTP-III than in A2/O tank of WWTP-I and the similar BOD loading in both tanks indicate that the former contains higher lipid concentration.
4. Conclusions Our results showed that the three main groups of lipids in sewage sludge are the cellular lipids, free fatty acid (FFA), and wax and gum. Although both the cellular lipids and FFA contain various constituents, they contain an abundance of C16eC18 and display a good saturation level, rendering these lipid species good resources for biodiesel production; thus, separating one lipid from other lipid to produce biodiesel is unnecessary. Moreover, the presence of considerable amounts of ceramide (Cer), coenzyme (Co) and some phosphatidylcholine (PC) renders the cellular lipids as potential raw materials for other value-added products, such as cosmetics and nutriment, by extracting those specific lipids out.
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Table 4 Concentration of FFA9-FFA32 in sludge samples. Component Absolute amount (mg/g) SFA Relative amount (%) FA(9:0) FA(10:0) FA(11:0) FA(12:0) FA(13:0) FA(13:1) FA(14:0) FA(14:1) FA(15:0) FA(15:1) FA(15:2) FA(16:0) FA(16:1) FA(16:2) SFA16 Saturation level of FA16 FA(17:0) FA(17:1) FA(17:2) FA(18:0) FA(18:1) FA(18:2) FA(18:3) FA(18:4) SFA18 Saturation level of FA18 FA16 þ FA18 Saturation level of (FA16 þ FA18) FA(19:0) FA(19:1) FA(19:2) FA(19:3) FA(19:4) FA(20:0) FA(20:1) FA(20:2) FA(20:3) FA(20:4) FA(20:5) FA(21:0) FA(21:1) FA(21:2) FA(21:5) FA(21:6) FA(22:0) FA(22:1) FA(22:2) FA(22:3) FA(22:4) FA(22:5) FA(22:6) FA(23:0) FA(23:1) FA(23:2) FA(24:0) FA(24:1) FA(24:2) FA(24:3) FA(24:5) FA(24:6) FA(25:0) FA(25:1) FA(25:2) FA(25:3) FA(26:0) FA(26:1) FA(26:2) FA(26:3) FA(26:4)
Sludge from primary sedimentation tank of WWTP(I)
Sludge from A2/O tank of Sludge from MBR tank of Sludge from A2/O tank of Sludge from MBR tank of WWTP(I) WWTP(II) WWTP(III) WWTP(III)
12.19 ± 0.89
1.22 ± 0.10
0.65 ± 0.14
2.01 ± 1.08
1.11 ± 0.40
0.18 0.08 0.02 0.67 0.08 0.00 3.85 0.11 0.74 0.07 0.00 33.17 3.42 0.15 36.74 90.3 0.80 0.32 0.01 9.17 31.91 10.15 0.81 0.01 42.88 21.4 79.62 53.2
0.52 0.20 0.06 0.37 0.14 0.02 1.94 0.36 3.07 0.73 0.01 20.26 28.10 1.06 49.42 41.0 1.19 1.50 0.06 8.62 14.93 5.84 1.15 0.11 22.03 39.1 71.45 40.4
0.55 0.33 0.04 0.24 0.07 0.02 2.22 0.18 1.40 0.32 0.01 28.56 10.25 0.35 39.16 72.9 0.93 0.68 0.04 14.52 17.92 6.75 0.65 0.03 25.35 57.2 64.51 66.8
0.37 0.12 0.04 0.25 0.12 0.01 3.52 0.19 0.74 0.19 0.01 39.28 7.20 0.38 46.86 83.8 0.68 0.42 0.02 12.73 16.10 8.21 0.87 0.03 25.21 50.5 72.07 72.2
0.67 0.21 0.04 0.25 0.08 0.02 2.20 0.17 1.33 0.32 0.01 29.20 11.47 0.35 41.02 71.2 0.90 0.70 0.03 13.58 17.07 6.43 0.76 0.04 24.3 55.9 65.32 65.5
0.06 0.18 0.01 0.00 0.00 0.47 0.94 0.35 0.14 0.07 0.03 0.04 0.01 0.00 0.00 0.01 0.51 0.25 0.05 0.06 0.08 0.05 0.02 0.06 0.01 0.00 0.33 0.06 0.01 0.00 0.02 0.01 0.05 0.01 0.00 0.01 0.11 0.01 0.00 0.00 0.00
0.20 0.37 0.03 0.01 0.01 0.61 0.42 0.23 0.15 1.05 0.48 0.13 0.03 0.01 0.01 0.01 1.13 0.13 0.07 0.02 0.08 0.40 0.30 0.25 0.03 0.01 1.20 0.10 0.01 0.00 0.01 0.00 0.26 0.02 0.00 0.04 0.64 0.06 0.01 0.01 0.00
0.14 0.25 0.01 0.01 0.01 1.33 0.50 0.17 0.05 0.27 0.25 0.17 0.02 0.01 0.01 0.01 2.44 0.24 0.04 0.02 0.03 0.04 0.06 0.45 0.03 0.01 2.61 0.15 0.02 0.01 0.02 0.01 0.57 0.04 0.01 0.03 1.46 0.13 0.03 0.01 0.01
0.06 0.16 0.01 0.00 0.00 1.01 0.52 0.18 0.12 0.29 0.20 0.12 0.01 0.00 0.00 0.00 1.56 0.17 0.03 0.02 0.03 0.08 0.05 0.24 0.02 0.00 1.37 0.09 0.01 0.00 0.02 0.01 0.28 0.01 0.00 0.03 0.65 0.05 0.01 0.01 0.00
0.12 0.27 0.01 0.00 0.00 1.36 0.51 0.16 0.09 0.41 0.31 0.18 0.02 0.01 0.01 0.01 2.43 0.22 0.05 0.02 0.05 0.24 0.10 0.46 0.04 0.01 2.67 0.16 0.02 0.01 0.03 0.01 0.58 0.03 0.01 0.06 1.32 0.12 0.02 0.01 0.01
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Table 4 (continued ) Component
Sludge from primary sedimentation tank of WWTP(I)
Sludge from A2/O tank of Sludge from MBR tank of Sludge from A2/O tank of Sludge from MBR tank of WWTP(I) WWTP(II) WWTP(III) WWTP(III)
FA(26:5) FA(27:0) FA(27:5) FA(28:0) FA(28:1) FA(28:2) FA(28:4) FA(28:6) FA(29:0) FA(29:1) FA(29:6) FA(30:0) FA(30:1) FA(30:2) FA(30:3) FA(30:5) FA(30:6) FA(31:0) FA(31:3) FA(31:5) FA(32:0) FA(32:1) FA(32:2) FA(32:3) FA(32:4) FA(32:5)
0.00 0.01 0.00 0.04 0.01 0.00 0.00 0.00 0.01 0.00 0.00 0.02 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.01 0.00 0.00 0.00 0.00
0.01 0.08 0.01 0.26 0.04 0.01 0.00 0.01 0.03 0.02 0.00 0.13 0.05 0.01 0.00 0.01 0.00 0.01 0.00 0.02 0.05 0.05 0.02 0.01 0.03 0.01
0.00 0.20 0.01 0.57 0.09 0.02 0.01 0.00 0.07 0.02 0.00 0.26 0.09 0.01 0.01 0.01 0.01 0.03 0.01 0.01 0.10 0.10 0.02 0.01 0.04 0.01
0.00 0.08 0.01 0.23 0.04 0.01 0.00 0.00 0.02 0.01 0.00 0.15 0.05 0.01 0.01 0.00 0.00 0.01 0.00 0.00 0.05 0.05 0.01 0.01 0.01 0.00
0.00 0.17 0.01 0.43 0.08 0.02 0.00 0.00 0.06 0.02 0.00 0.21 0.09 0.01 0.01 0.01 0.01 0.02 0.01 0.00 0.08 0.10 0.02 0.02 0.05 0.02
Table 5 Lipid content in three sludge samples (%). Lipid
Sludge from primary Sludge from A2/O sedimentation tank of WWTP(I) tank of WWTP(I)
Sludge from MBR tank of WWTP(II)
Cellular lipids FFA Wax and gum S(Major lipid in cells þ FFA þ Wax and gum) Raw lipid by soxhlet extraction S(Major lipid in cells þ FFA þ Wax and gum)/ Raw lipid by soxhlet extraction
4.17 ± 1.22 ± 6.68 ± 12.07 12.3 ± 98.1
2.30 0.07 1.37 3.74 4.20 89.0
1.59 0.09 0.26 2.1 (Ad.)
2.61 0.12 0.67 3.40 3.80 89.5
± 0.44 ± 0.01 ± 0.06 ± 0.10 (Ad.)
± 1.04 ± 0.01 ± 0.19 ± 0.30 (Ad.)
Sludge from A2/O tank of WWTP(III) 6.64 0.20 0.98 7.82 8.01 97.6
± 1.85 ± 0.11 ± 0.04 ± 1.10 (Ad.)
Sludge from MBR tank of WWTP(III) 1.68 0.11 1.00 2.79 2.90 96.2
± 0.77 ± 0.04 ± 0.09 ± 0.20 (Ad.)
Legend: “Ad.” means absolute deviation. Deviations without special mark are standard deviation.
Sludge from primary sedimentation tank contained the highest concentration of total lipids (>10%), FFA, and wax and gum, as well as the second highest concentration of cellular lipids, indicating that sludge from primary sedimentation tank display a high potential as raw material for biodiesel production or for other reutilizations. The methods or technologies used to strengthen the function of primary sedimentation tank are favorable for accumulation of lipids from influent; with the use of a proper catalyst, an increased quantity of biodiesel with better quality can be produced from sewage sludge. Author contributions F.Z. conceived the idea for this paper. X.W., L.Z. and J.Q. conducted the sampling, pretreatment and extraction experiments. X.L. and X.W conducted the UPLC/MS/MS analysis. F.Z. finally composed this paper. J.W. provided the operation data of three WWTPs. Acknowledgments The present study is supported by the National Natural Science Foundation of China (grant number 51308538); the Major Science and Technology Program for Water Pollution Control and Treatment (grant number 2013ZX07314-001-006-01); Fundamental
Research Funds for the Central Universities, and the Research Funds of Renmin University of China (grant number 15XNLD04). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.watres.2017.03.032. References Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37 (8), 911e917. Chipasa, K.B., Medrzycka, K., 2008. Characterization of the fate of lipids in activated sludge. J. Environ. Sci. 20 (5), 536e542. Dufreche, S., Hernandez, R., French, T., Sparks, D., Zappi, M., Alley, E., 2007. Extraction of lipids from municipal wastewater plant microorganisms for production of biodiesel. J. Am. Oil Chem. Soc. 84 (2), 181e187. Folch, J., Lees, M., Sloane, S., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226 (1), 497e509. Hao, X., Liu, R., Huang, X., 2015. Evaluation of the potential for operating carbon neutral WWTPs. Water Res. 87, 424e431. Huynh, L., Kasim, N., Ju, Y., 2010. Extraction and analysis of neutral lipids from activated sludge with and without sub-critical water pretreatment. Bioresour. Technol. 101 (22), 8891e8896. Jarde, E., Mansuy, L., Faure, P., 2005. Organic markers in the lipidic fraction of sewage sludges. Water Res. 39 (7), 1215e1232. Jin, L., Zhang, G., Tian, H., 2014. Current state of sewage treatment in China. Water Res. 66, 85e98. Kargbo, D., 2010. Biodiesel production from municipal sewage sludges. Energy &
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Fuels 24 (5), 2791e2794. Kroiss, H., 2004. What is the potential for utilizing the resources in sludge. Water Sci. Technol. 49 (10), 1e10. Kwon, E., Kim, S., Jeon, Y., Yi, H., 2012. Biodiesel production from sewage sludge: new paradigm for mining energy from municipal hazardous material. Environ. Sci. Technol. 46 (18), 10222e10228. Li, Y., Chen, T., Luo, W., Huang, Q., Wu, J., 2003. Contents of organic matter and major nutrients and the ecological effect related to land application of sewage sludge in China. Acta Ecol. Sin. 23 (11), 2464e2474. Liu, J., Yang, M., Qi, R., 2008. Comparative study of protozoan communities in fullscale MWTPs in Beijing related to treatment processes. Water Res. 42 (8e9), 1907e1918. Mata, T., Martins, A., Caetano, N., 2010. Microalgae for biodiesel production and other applications: a review. Renew. Sustain. Energy Rev. 14 (1), 217e232. Mondala, A., Liang, K., Toghiani, H., Hernandez, R., French, T., 2009. Biodiesel production by in situ transesterification of municipal primary and secondary sludges. Bioresour. Technol. 100 (3), 1203e1210. Olkiewicz, M., Caporgno, M., Fortuny, A., Stuber, F., Fabregat, A., 2014. Direct liquideliquid extraction of lipid from municipal sewage sludge for biodiesel production. Fuel Process. Technol. 128, 331e338. Qi, J., Zhu, F., Wei, X., Zhao, L., Xiong, Y., 2016. In situ transesterification of sewage sludge obtained from A2O and MBR processes for biodiesel production. Waste Manag. 49 (4), 212e220. Qiu, Y., Shi, H., He, M., 2010. Nitrogen and phosphorous removal in municipal wastewater treatment plants in China: a review. Int. J. Chem. Eng. 2010 (1687e806X), 1e10. Rajam, L., Soban Kumar, D.R., Sundaresan, A., Arumughan, C., 2005. A novel process for physically refining rice bran oil through simultaneous degumming and dewaxing. J. Am. Oil Chem. Soc. 82 (3), 213e220. Ramirez, R., Garay, I., Alvarez, J., Marti, M., Parra, J., Coderch, L., 2008. Supercritical fluid extraction to obtain ceramides from wool fibers. Sep. Purif. Technol. 63 (3), 552e557. Revellame, E., Hernandez, R., French, W., Holmes, W., Alley, E., 2010. Biodiesel from activated sludge through in situ transesterification. J. Chem. Technol. Biotechnol. 85 (5), 614e620.
Revellame, E., Hernandez, R., French, W., Holmes, W., Alley, E., 2011. Production of biodiesel from wet activated sludge. J. Chem. Technol. Biotechnol. 86 (1), 61e68. Revellame, E., Hernandez, R., French, W., Holmes, W., Benson, T., 2012a. Lipid storage compounds in raw activated sludge microorganisms for biofuels and oleochemicals production. RSC Adv. 2 (5), 2015e2031. Revellame, E., Holmes, W., Benson, T., Forks, A., French, W., Hernandez, R., 2012b. Parametric study on the production of renewable fuels and chemicals from phospholipid-containing biomass. Top. Catal. 55 (3), 185e195. Siddiquee, M., Rohani, S., 2011. Experimental analysis of lipid extraction and biodiesel production from wastewater sludge. Fuel Process. Technol. 92 (12), 2241e2251. Sureshkumar, K., Velraj, R., Ganesan, R., 2008. Performance and exhaust emission characteristics of a CI engine fueled with Pongamia pinnata methyl ester (PPME) and its blends with diesel. Renew. Energy 33 (10), 2294e2302. Sun, F., Wang, X., Li, X., 2011. Change in the fouling propensity of sludge in membrane bioreactors (MBR) in relation to the accumulation of biopolymer clusters. Bioresour. Technol. 102 (102), 4718e4725. Tang, H., Wang, X., Xu, L., Ran, X., Li, X., Chen, L., Zhao, X., Deng, H., Liu, X., 2016. Establishment of local searching methods for orbitrap-based high throughput metabolomics analysis. Talanta 156e157, 163e171. Wang, Q., Zheng, X., Luo, M., 2014. The past, the present and the future of MBR market in USA (in Chinese). Ind. Water Treat. 34, 15e17. Wong, M., Mino, T., Seviour, R., 2005. In situ identification and characterization of the microbial community structure of full-scale enhanced biological phosphorous removal plants in Japan. Water Res. 39 (13), 2901e2914. Yeong, hwan. Seo, Il, gyu Lee, Jong, in. Han, 2013. Cultivation and lipid production of yeast Cryptococcus curvatus using pretreated waste active sludge supernatant. Bioresour. Technol. 135, 304e308. Zhu, F., Zhao, L., Jiang, H., Zhang, Z., Xiong, Y., Qi, J., Wang, J., 2014. Comparison of lipid content and biodiesel production from municipal sludge using three extraction methods. Energy & Fuels 28 (8), 5277e5283. Zhang, M., Xie, J., Zhou, Q., Chen, G., Liu, Z., 2003. On-line solid-phase extraction of ceramides from yeast with ceramide III imprinted monolith. J. Chromatogr. A 984 (2), 173e183.
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Lipid profiling in sewage sludge Fenfen Zhu a, *, Xuemin Wu a, Luyao Zhao b, Xiaohui Liu c, Juanjuan Qi d, Xueying Wang c, Jiawei Wang e a
School of Environment and Natural Resources, Renmin University of China, 100872 Beijing, China Shougang Institute of Technology, 100043 Beijing, China c School of Life Sciences, Tsinghua University, 100084 Beijing, China d Development and Reform Commission of Bomi County in Linzhi Area, 860300, China e Research Center, Beijing Drainage Group, 100124 Beijing, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 November 2016 Received in revised form 15 March 2017 Accepted 15 March 2017
High value-added reutilization of sewage sludge from wastewater treatment plants (WWTPs) is essential in sustainable development in WWTPs. However, despite the advantage of high value reutilization, this process must be based on a detailed study of organics in sludge. We used the methods employed in life sciences to determine the profile of lipids (cellular lipids, free fatty acids (FFAs), and wax/gum) in five sludge samples obtained from three typical WWTPs in Beijing; these samples include one sludge sample from a primary sedimentation tank, two activated sludge samples from two Anaerobic-Anoxic-Oxic (A2/ O) tanks, and two activated sludge samples from two membrane bioreactor tanks. The percentage of total raw lipids varied from 2.90% to 12.3%. Sludge from the primary sedimentation tank showed the highest concentrations of lipid, FFA, and wax/gum and the second highest concentration of cellular lipids. All activated sludge contained an abundance of cellular lipids (>54%). Cells in sludge can from plants, animals, microbes and so on in wastewater. Approximately 14 species of cellular lipids were identified, including considerable high value-potential ceramide (9567e38774 mg/kg), coenzyme (937e3897 mg/ kg), and some phosphatidylcholine (75e548 mg/kg). The presence of those lipid constituents would thus require a wider range of recovery methods for sludge. Both cellular lipids and FFAs contain an abundance of C16eC18 lipids at high saturation level, and they serve as good resources for biodiesel production. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Sewage sludge Lipid profile Cellular lipids Free fatty acid Wastewater treatment Biodiesel
1. Introduction Recovery of energy or value-added material from wastewater attracts strong interest in sustainable wastewater treatment and resource utilization (Hao et al., 2015). Sewage sludge is a byproduct of wastewater treatment and a highly potential raw material for resource recovery because it contains considerable amount of organic compounds (Hao et al., 2015). In China, wastewater treatment plants (WWTPs) produce over 3 107 t of sludge (with 80% moisture content) yearly (Kroiss, 2004), and this production rate is expected to increase as a result of the progressively stringent regulatory standards for wastewater effluent, together with the growing number of WWTPs. The average organic matter content of sewage sludge in China is 38.4%; of which, 55% are carbohydrates, 20% are proteins, and approximately 20% are lipids (Li
* Corresponding author. E-mail address: [email protected] (F. Zhu). http://dx.doi.org/10.1016/j.watres.2017.03.032 0043-1354/© 2017 Elsevier Ltd. All rights reserved.
et al., 2003; Yeong et al., 2013; Siddiquee and Rohani, 2011) Although the current treatment methods for sewage sludge, such as anaerobic digestion, incineration, and composting, present many merits, they cannot recover sufficient energy or value-added materials to render sludge treatment cost effective (Hao et al., 2015). Municipal sewage sludge has been proposed to be a potential feedstock in diesel production (Mondala et al., 2009; Revellame et al., 2010; Zhu et al., 2014; Revellame et al., 2012a; Kwon et al., 2012), and this approach presents several potential advantages over the existing treatment methods. First, lipid concentrations in sewage sludge are considerably high, and extraction of such lipids will facilitate biodiesel production at an impressive ratio of up to 27.4% (Mondala et al., 2009; Dufreche et al., 2007). We have successfully conducted lipid extraction and in situ transesterification of sewage sludge, which indicated that most lipids, including phospholipids, can be transformed into biodiesel, and the quality of which was affected by sludge source (Zhu et al., 2014; Qi et al., 2016). Second, sewage sludge is abundant; being a solid waste,
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sewage sludge can be used as free or low-cost raw materials for diesel production. Third, sensitivity analysis indicated that if 10% (weight) biodiesel yield is attained, activated sludge can become economically competitive with petroleum (Kwon et al., 2012; Revellame et al., 2011). In addition, liquid fuel can be transported more easily than gas fuel (e.g., biogas). Detailed understanding of lipid profile in sewage sludge is critically important in production of biodiesel or other valuable products, such as nutrients, cosmetics, and health products. The cellular lipid species in sludge include triglycerides (TG), diglycerides (DG), monoglycerides, phospholipids, free fatty acids (FFAs), and sterol (Siddiquee and Rohani, 2011; Dufreche et al., 2007; Olkiewicz et al., 2014; Kargbo, 2010; Jarde et al., 2005). Phospholipids account for approximately 24%e25% of cell dry mass (Siddiquee and Rohani, 2011; Olkiewicz et al., 2014; Kargbo, 2010). Moreover, up to 36.8 wt% of dry sludge consists of fatty acids, sterol, and some aliphatic fractions (Jarde et al., 2005). Fatty acids in sludge are predominantly C10eC18 (Dufreche et al., 2007). Huynh et al. (2010) have studied the profiles of neutral lipid and fatty acids. However, information on lipids in sewage sludge remain limited, and quantitative analysis of lipids is generally lacking. To further understand the lipid profile in sewage sludge, we obtained sludge samples from three typical WWTPs in Beijing, China and analyzed these samples using the methods used in life sciences. Based on the characteristics of different lipids, three different extraction/pretreatment methods (two Ultra Performance Liquid Chromatography(UPLC)/Mass(MS)/Mass(MS) operation conditions þ weighing) were performed to measure three groups of lipids, namely, cellular lipids, FFA, and wax/gums (possibly including fat, grease, and steroid). Cellular lipids are the most complicated lipids, which should be from the cells of plants, animals, microbes and so on that deposit in the sludge from wastewater, including triglycerides (TG), diglycerides (DG), phosphatidylcholine (PC), ceramide (Cer), and coenzyme (Co). 2. Materials and methods 2.1. Sewage sludge Five sewage sludge samples (total solid (TS): 1.0% ± 0.5%) were collected from three municipal WWTPs in Beijing, China. The capacity of WWTP-I is 106 t/d, and the main technology employed is anaerobic-anoxic-oxic (A2/O). The capacity of WWTP-II is 6 105 t/ d, and this plant employs two parallel technology flows, namely, membrane bio-reactor (MBR) and A2/O. The capacity of WWTP-III is 105 t/d, and MBR is mainly employed in this plant. The main operation conditions in winter season are listed in Table 1, and the technology flows in these WWTPS are presented in Fig. 1. Among the existing WWTPs in China, the plants utilizing the A2/O process showed the largest design treatment capacity of 33.2% (Qiu et al., 2010; Jin et al., 2014). Moreover, MBR has become an increasingly attractive option in wastewater treatment and water reclamation processes with a projected annual growth rate of 20%e30% in the next 5 years (Sun et al., 2011; Wang et al., 2014).
The five sludge sampling points include the primary sedimentation tank of WWTP-I, A2/O tank of WWTP-I, MBR tank of WWTPII, A2/O tank of WWTP-III, and MBR tank of WWTP-III. Sampling points in A2/O and MBR tanks were located at the end of the aerobic tanks (refer to Fig. 1 for the schematic of the three WWTPs). The amount of sample from each sampling point is sufficient to obtain at least nine samples from the same sampling point for pretreatments and measurements. Three samples for extraction and analysis of cellular lipids, three samples for extraction and analysis of FFA, and the other three samples for extraction of wax and gums. Elements in sludge were analyzed by X-ray fluorescence (XRF-1800, Shimadzu, Kyoto, Japan), and results are shown in Supplementary Information I. All chemicals used in this study were chromatographic grade and used without further purification. The chemicals were purchased from Technology Development Co., Ltd., and standards were purchased from Sigma Co. Solutions were prepared using water purified by a Millipore Milli Q UV Plus System. 2.2. Extraction 2.2.1. Pretreatment A sludge sample (Total solid concentration (TS): 1.0% ± 0.5%) was centrifuged at 3000 rpm for 10 min to obtain dewatered sludge (TS: 14.0% ± 0.5%). Subsequently, the dewatered sludge was freeze-dried via a two-step process: first, the sludge was centrifuged and frozen for 2 days at 20 C, and second, the frozen sludge was freeze-dried in an automatic vacuum freeze dryer (Model: FD-1A-50) for 2 d. The dried sludge was crushed using mortar and pestle, homogenized, and stored in a freezer prior to use. 2.2.2. Extraction of cellular lipids Extraction was performed according to the method described by Folch et al. (1957) with modification. Three grams of dried sample was weighed directly into 100 mL centrifuge tubes with Teflonlined screw caps. Citric acid buffer (0.15 M, pH ¼ 7.3) was added to obtain a total volume of 2 mL; 7.5 mL of 1:2 methanolchloroform was subsequently added into the solution. The samples were extracted on a rotating oscillator for 2 h. 1:2 methanolchloroform (6 mL) and citric acid buffer (4.5 mL) were subsequently added into the tubes, and the solution was mixed overnight on a vortex mixer for phase separation. The samples were centrifuged at 4000 rpm for 10 min and then the lipid-containing organic phase (top) was transferred into 10 mL Pyrex tubes and allowed to evaporate completely under a stream of nitrogen. 2.2.3. FFA extraction Extraction was performed according to the method described by Bligh and Dyer (1959) with modification. Three grams of dried sludge was weighed directly into 50 mL centrifuge tubes with Teflon-lined screw caps. Subsequently, 1.5 mL of citric acid buffer (0.15 M, pH ¼ 7.3), 1.9 mL of chloroform, and 3.75 mL of methanol were successively added and then vortex oscillated (alternatively, the mixture can be homogenized by ultrasound for 10 min). The
Table 1 Main operation parameters of three WWTPs.
Primary sedimentation tank A2/O tank of WWTP(I) MBR tank of WWTP(II) A2/O tank of WWTP(III) MBR tank of WWTP(III)
CODinlet (mg/L)
BODinlet (mg/L)
F/M (kgBOD/kgVSS)
SRT (d)
HRT (h)
389 362 646 497 579
182 / 373 287 336
/ 0.103 0.0669 0.104 0.0404
/ 19.3 25.5 16.2 24.4
/ 12 14 15 16.5
Legend: F/M means how much does microbe will eat or “Feeding/microbes”.
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Fig. 1. Schematic diagram of a municipal wastewater treatment plants.
mixture was oscillated by a fully automatic flip oscillator for 2 h at 250 rpm in the dark and then centrifuged for 5 min at 4000 rpm.
The filtrate was collected. Afterwards, 7.6 mL of 2:1 methanolchloroform was added into the remaining sludge residue,
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extracted on a vortex oscillator for 2 h, oscillated in horizontal oscillator for 2 h, and centrifuged. The filtrate was collected and mixed with a previously obtained filtrate. 2:1 methanol-chloroform (6 mL) and citric acid buffer (4.8 mL) were subsequently added into the tubes containing the filtrate and then the solution was vortex oscillated and allowed to stand overnight for phase separation. The chloroform solution containing FFA was transferred into Pyrex tubes, dried under nitrogen steam in draught cupboard, and stored in refrigerator. 2.2.4. Wax and gum extraction The amounts of wax and gum were determined using the removal method as described previously (Huynh et al., 2010; Rajam et al., 2005). Lipid was first extracted from dried sludge through Soxhlet extraction. Five grams of the extracted sludge lipid was dissolved in acetone and maintained at 60 C for 1 h to obtain a clear solution. The solution was cooled to room temperature and stored in a refrigerator at 5 C for 24 h to allow the waxes to crystallize. The insoluble fraction that is rich in waxes was separated through centrifugation under 8000 rpm at 5 C. The solid and liquid components were subsequently separated through vacuum filtration. The entire process of dewaxing and degumming was repeated using the collected filtrate as substrate, which was stored at 5 C for 24 h. The process was repeated to separate the filtrate and filter residue. After removal of the acetone, the filtered residue was weighed together with former separated solid. 2.3. Lipid analysis Wax and gum were weighed only and no further analysis was performed. By contrast, FFA and the cellular lipids were extensively analyzed by a Q Exactive LC-MS/MS (Thermo Fisher Scientific) system. 2.3.1. Analysis of cellular lipids The extracted lipid was re-dissolved by chloroform just before analysis. Analysis was performed according to the method described by Tang et al. (2016). A UPLC system was coupled to a QExactive orbitrap mass spectrometer (Thermo Fisher, CA) equipped with a heated electrospray ionization probe. Lipid extracts were separated using a Hypersil GOLD C18 100 2.1 mm, 1.9 mm column (Thermo Fisher, CA) connected to a Thermo Fisher Scientific Autosampler and to a UPLC pump. A binary solvent system was used, in which mobile phase A consisted of ACN:H2O (60:40) and 10 mM ammonium acetate, whereas mobile phase B comprised IPA:ACN (90:10) and 10 mM ammonium acetate. Separations were performed for over 30 min under the conditions listed in Supplementary information II. A flow rate of 250 mL/min was used in the analysis, and the column and sample tray were stored at 45 C and 10 C, respectively. Data with a mass range of mass/ charge (m/z) 240e2000 and m/z 200e2000 were respectively acquired in the positive and negative ion modes through datadependent MSMS acquisition. Full scan and fragment spectra were collected with resolutions of 70,000 and 17,500, respectively. The source parameters were as follows: spray voltage: 3000 V; capillary temperature: 320 C; heater temperature: 300 C; sheath gas flow rate: 35; and auxiliary gas flow rate: 10. Lipidomics identification was performed using the analytical software Lipid Search 4.0 (Thermo Fisher, CA). Fig. 2 showed the base peak chromatograms under positive and negative ion modes of one sample, respectively. Depending on their specific molecular characteristics, different lipid species showed distinct intensity responses under positive or negative ion modes. For example, triglycerides (TG), diglycerides (DG), and phosphatidylcholine (PC) displayed better responses under positive ion
mode, whereas phosphatidylethanolamine (PE), phosphatidylinositol (PI), and cardiolipin (CL) were detected under negative ion mode. Thus, the lipids were examined in both ion modes. Supplementary information III presented the legend and reference. Quantitative analysis was performed using the area external standard method, and the standards used were as follows: 18:1 cardlolipin (CL), 16:0e18:1 phosphatidylglycerol (PG), 16:0e18:1 phosphatidylethanolamine (PE), 18:0e18:2 phosphatidylcholine (PC), 16:0e18:1 phosphatidylinositol (PI), 16:0e16:0e18:0 triglycerides (TG), C18 (d18:1/18:0) Ceramide (Cer), 16:0 diglycerides (DG). Other lipids, such as sphingosine (So), lysophosphatidylcholine (LPC), coenzyme (Co), (O-acyl)-1-hydroxy fatty acid (OAHFA), dimethylphosphatidylethanolamine (dMePE), phosphatidylmethanol (PMe), and PG were quantitatively analyzed using a semiquantitative method according to the ratio of the relative amounts of response area of lipids (refer to the Supplementary Information IV for the relative amount of each lipid determined under the two ion modes using the area ratio method). 2.3.2. FFA analysis The extracted lipid was re-dissolved by chloroform just before analysis. The analysis was performed by a UPLC-MS/MS. The conditions for UPLC were as follows: Hypersil GOLD C18 100 2.1 mm, 1.9 mm column (Thermo Fisher, CA); column temperature, 45 C; velocity, 250 mL/min; sample volume, 2 mL; mobile phase, A phase (ACN: H2O ¼ 60:40 and 10 mM ammonium acetate) and B phase (IPA: ACN ¼ 90:10 and 10 mM ammonium acetate). Separations were performed for over 30 min under the conditions listed in Supplementary information II. Mass condition: negative ion mode; spray voltage, 3000 V; capillary temperature, 320 C; heater temperature, 300 C; sheath gas flow rate, 35 arb; auxiliary gas flow rate, 10; and mass range, m/z 200e2000 (R 70,000); fragment spectra were collected with resolutions of 17,500. The standards were analyzed first before the unknown samples were examined. Arachidonic acid-d8 was used as standard samples in quantitative analysis using the area external standard method. 3. Results and discussion 3.1. Cellular lipids The cellular lipid contents of sludge obtained under different sewage treatments were extensively analyzed (Fig. 3). The amounts of total cellular lipids were 41,656 ± 15,897, 26,079 ± 4410, 23,006 ± 10,361, 66,397 ± 18,351, and 16,779 ± 7670 mg/kg in primary sedimentation tank of WWTP-I, A2/O tank of WWTP-I, MBR tank of WWTP-II, A2/O tank of WWTP-III, and MBR tank of WWTP-III, respectively. Fourteen species, namely, ceramide (Cer), phosphatidylcholine (PC), cardiolipin (CL), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), triglycerides (TG), diglycerides (DG), sphingosine (So), dimethylphosphatidylethanolamine (dMePE), (O-acyl)-1-hydroxy fatty acid (OAHFA), coenzyme (Co), lysophosphatidylcholine (LPC), and phosphatidylmethanol (PMe), were identified in the sewage sludge. Every species has more than 1 kinds of different molecules. For example there are 209 kinds of different TG molecules and 28 kinds of different Cer molecules in the sludge from primary sedimentation tank of WWTP-I. TG concentration in the sludge from primary sedimentation tank of WWTP-I is the sum of the concentration of all the TG molecules in that sludge sample so did the concentration of other lipid species. Liu et al. (2008) and Wong et al. (2005) have reported that microbial community in activated sludge is significantly influenced by process types, operation conditions, and scale, among others. Given that phospholipid was once used to identify microbial community
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Fig. 2. Chromatogram under positive and negative ion mode of one sludge sample.
in earth, lipid species should be related to microbial community, which in turn is influenced by WWTP operation. The lipid species in the sludge obtained from a primary sedimentation tank should be strongly affected by the influent. Among the cellular lipids, Cer (9567 ± 5095e38774 ± 11,683 mg/kg), So (3230 ± 1053e7833 ± 711 mg/kg), and Co (937 ± 352e3897 ± 3038 mg/kg) are the most abundant species. This finding suggests that Cer, So, and Co are important components
both of the microorganisms in bioreactors and the organisms in the influent. The amounts of the other species are as follows: TG (223 ± 64e837 ± 187 mg/kg), DG (47±4e585 ± 216 mg/kg), PC (75 ± 36e548 ± 269 mg/kg), CL (155 ± 59e763 ± 367 mg/kg), PE (186 ± 70e1103 ± 643 mg/kg), and PG (70 ± 22e347 ± 167 mg/kg). A considerable amount of other cellular lipids, such as PC, was observed. Thus, utilization or reutilization of lipids in sewage sludge may be explored; for instance, Cer may be extracted to be
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Fig. 3. Concentration of cellular lipids of sludge from different sewage treatment processes.
used in cosmetics production (Ramirez et al., 2008; Zhang et al., 2003). Sludge from A2/O tank of WWTP-III contained the highest ceramide (Cer) concentration (38,773.3 mg/kg) followed by that in the sludge from the primary sedimentation tank of WWTP-I (28,223.3 mg/kg). Sludge from the primary sedimentation tank contains the highest concentration of coenzyme (Co) (3896.7 mg/ kg), which is nearly double or triple that in activated sludge from bioreactors, except in the activated sludge from A2/O tank of WWTP-III. Thus, the sludge from the primary sedimentation tank and sludge from A2/O tank of WWTP-III are more promising for reutilization. As shown in Fig. 3 and Supplementary Information V, the decreasing amount of total glyceride (triglycerides (TG)þ diglycerides (DG)) in the sludge samples is as follows: sludge from primary settling tank (1422 mg/kg) > sludge from A2O tank of WWTP-III (1225 mg/kg) > sludge from MBR tank of WWTP-III (419 mg/kg) > sludge from A2/O tank of WWTP-I (329 mg/ kg) > sludge from MBR tank of WWTP-III (274 mg/kg). This result is consistent with the fact that lipids in primary sedimentation sludge are mainly released from animal or plant residues in the influent, and these residues contain more glyceride than microorganisms. Moreover, the sludge from the primary sedimentation tank contained a minimum amount of glycerol phospholipid (phosphatidylcholine (PC)þ cardiolipin (CL)þ phosphatidylethanolamine (PE)þ phosphatidylglycerol (PG)) (486 mg/kg). To determine the potential of using cellular lipids for biodiesel production, we performed transesterification reaction by using phosphatidylcholine (PC) (58.8% pure). Hexadecanoic acid methyl ester (C17H34O2), 9,12Octadecadienoic acid methyl ester (C19H34O2), 9-Octadecenoic acid methyl ester (C19H36O2) were identified by GCMS-QP2010 in the final product with nothing else which means it is 100% pure FAME (refer to Supplementary Information VI). Revellame et al. (2012b) has proposed the cracking theory of phospholipid in such kind of reaction and we proposed the theoretical reaction formula in
Supplementary Information VI which is also similar to the traditional transesterification reaction of triglyceride. . We subsequently checked the relative concentration of C16eC18 in fatty acyl groups (triglycerides (TG), diglycerides (DG), phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), cardiolipin (CL), ceramide (Cer), and phosphatidylcholine (PC)), and results are shown in Table 2. The relative amount of C16eC18 in all species, except PI, is higher than 90%. Considering that the concentration of saturated lipids is also important in producing biodiesel of better quality with higher cetane number (Dufreche et al., 2007), we determined the relative amount of the saturated fatty acid groups C16 and C18 in TG, DG, PI, PE, PG, CL, Cer, and PC (Table 3). The decreasing order of the average percentage of the saturated lipids is as follows: PI > Cer > TG > PG > PC > PE > CL > DG. The relative amount of most species is higher than 20%, and this result is possibly related to the characteristics of the source of the lipids. Lipids in sludge from primary sedimentation tank are mainly released from animal and plant residues, whereas the lipids in activated sludge are mainly released from microorganisms. Given the complexity of the influent source and the influencing factors of microbial community structure, it can be deduced that food consumption structure, cooking oil, cosmetics consumption tendency, and operation parameters of treatment units, among others, will have good impact on profiles of cellular lipids in sludge. 3.2. FFA Table 4 shows the detailed profile of FA9eFA32 (Given that the distribution of FA33eFA37 in FA is less than 0.1%, information about them was illustrated in Supplementary Information VII). FFA concentration is 0.11%e1.22%, consistent with the results of Jarde et al. (2005). When the concentration of FFA is higher than 1%, alkaline catalyst is not suitable for sewage sludge to make biodiesel at least not suitable in the first step (Sureshkumar et al., 2008; Mata et al., 2010).
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Table 2 Relative amount of fatty acyl group which had C16 and C18 from TG, DG, PI, PE, PG, CL, Cer and PC of sludge from five sewage treatment processes. Relative amount of fatty acyl group which had C16 and C18
Sludge from primary sedimentation tank of WWTP(I)
Sludge from A2/O tank Sludge from MBR tank Sludge from A2/O tank Sludge from MBR tank of WWTP(I) of WWTP(II) of WWTP(III) of WWTP(III)
Cer PC CL PE PG PI TG DG
89.3% 99.8% 100.0% 97.7% 97.4% 100.0% 94.4% 75.5%
95.5% 97.2% 100.0% 88.2% 98.0% 28.4% 99.4% 100.0%
88.8% 98.8% 100.0% 91.6% 96.7% 42.8% 97.8% 100.0%
87.3% 99.9% 100.0% 95.4% 99.3% 67.0% 93.2% 84.3%
90.6% 99.4% 100.0% 93.9% 94.7% 38.9% 94.8% 100.0%
Table 3 Relative amount of saturated fatty acids group of C16 and C18 in TG, DG, PI, PE, PG, CL, Cer and PC of sludge from five sewage treatment processes. Relative amount of saturated fatty acids Sludge from primary group of C16 and C18 sedimentation tank of WWTP(I)
Sludge from A2/O tank Sludge from MBR tank Sludge from A2/O tank Sludge from MBR tank of WWTP(I) of WWTP(II) of WWTP(III) of WWTP(III)
Cer PC CL PE PG PI TG DG
52.0% 41.7% 21.3% 30.7% 39.2% 70.8% 40.2% 0.0%
45.5% 37.1% 20.7% 15.5% 31.3% 50.0% 45.3% 24.6%
The sludge from primary sedimentation tank of WWTP-I contained the highest FFA content (1.22%), whereas the other sludge samples contained very low FFA concentrations (0.07%e0.20%). FFA is possibly a good substrate for microorganisms and is easily assimilated so that low amount of FFA remains in the activated sludge (Chipasa and Medrzycka, 2008). More than 24 kinds of FFA were identified, and the FA18 and FA16 series are the most abundant at 22.0%e42.9% and 36.7%e49.4%, respectively. FA16 and FA18 content both contribute to more than 64% of FA in sludge samples. The saturation level of (FA16 þ FA18) is higher than 40%, which is very favorable for production of good biodiesel. Moreover, (FA16 þ FA18) contributes to nearly 80% of FA in the sludge from primary sedimentation tank of WWTP-I. At this point, sewage sludge is suitable as raw material for biodiesel production. Similar to that of the cellular lipids, the profile of FFA is influenced by the influent source, such as cooking oil, cosmetics, and temperature. 3.3. Wax and gum Table 5 shows the results for wax and gum. Sludge from primary sedimentation contains the highest wax and gum concentrations because they are easily captured by the primary sedimentation tank. When a WWTP does not have a primary sedimentation tank, the activated sludge from the bioreactors may contain increased concentration of wax and gums. However, despite the increased concentration of wax and gums, they can be assimilated relatively easily. 3.4. Total lipid concentration The total amount of the combined cellular lipids, FFA, and wax and gum constitute 89.5%e98.1% of the total raw lipid extracted through Soxhlet extraction (Table 5), indicating that the lipids in sewage sludge mainly consist of cellular lipids, FFA, and wax and gum. The other lipid components possibly include little oils, grease, fat, and sterol (Jarde et al., 2005; Dufreche et al., 2007). Moreover, the unknown proportion of the lipid components is low. Being one of the important contributors of COD/BOD, lipid
56.3% 42.1% 25.6% 27.0% 42.8% 67.8% 38.3% 0.0%
48.4% 36.7% 21.3% 24.9% 40.0% 57.9% 35.1% 13.9%
45.0% 35.6% 25.1% 24.2% 41.8% 74.6% 37.9% 0.0%
concentration in sludge should be related to the unit function of wastewater treatment. Primary sedimentation tank mainly captures suspended organic compounds, including/containing lipids, whereas a bioreactor tank (A2O tank/MBR tank) digests resolvable organic compounds, which are reduced by using microorganisms. As a result, the captured organic compounds, including lipids, were stored in the sludge in primary sedimentation tank. By contrast, the organic compounds, including lipids, which flowed into the bioreactor tank, were digested by microorganisms. Variation in raw lipid concentration can be explained by the traditional mechanism of a bioreactor, wherein BOD loading, sludge retention time (SRT), and hydraulic retention time are important factors influencing the digestion/reduction of organic compounds. For example, BOD loading in A2/O tank of WWTP-III is higher than that in MBR tank of WWTP-III (Table 1); additionally, the former has shorter SRT than the latter. As a result, raw lipid concentration in sludge from A2/O tank of WWTP-III is approximately 2.7 times that of the lipid concentration in sludge from MBR tank of WWTPIII. The microorganisms in A2/O tank of WWTP-III assimilated the organic compounds as cellular lipids, considerably increasing the concentration of cellular lipids. The shorter SRT and higher BODinlet in A2/O tank of WWTP-III than in A2/O tank of WWTP-I and the similar BOD loading in both tanks indicate that the former contains higher lipid concentration.
4. Conclusions Our results showed that the three main groups of lipids in sewage sludge are the cellular lipids, free fatty acid (FFA), and wax and gum. Although both the cellular lipids and FFA contain various constituents, they contain an abundance of C16eC18 and display a good saturation level, rendering these lipid species good resources for biodiesel production; thus, separating one lipid from other lipid to produce biodiesel is unnecessary. Moreover, the presence of considerable amounts of ceramide (Cer), coenzyme (Co) and some phosphatidylcholine (PC) renders the cellular lipids as potential raw materials for other value-added products, such as cosmetics and nutriment, by extracting those specific lipids out.
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Table 4 Concentration of FFA9-FFA32 in sludge samples. Component Absolute amount (mg/g) SFA Relative amount (%) FA(9:0) FA(10:0) FA(11:0) FA(12:0) FA(13:0) FA(13:1) FA(14:0) FA(14:1) FA(15:0) FA(15:1) FA(15:2) FA(16:0) FA(16:1) FA(16:2) SFA16 Saturation level of FA16 FA(17:0) FA(17:1) FA(17:2) FA(18:0) FA(18:1) FA(18:2) FA(18:3) FA(18:4) SFA18 Saturation level of FA18 FA16 þ FA18 Saturation level of (FA16 þ FA18) FA(19:0) FA(19:1) FA(19:2) FA(19:3) FA(19:4) FA(20:0) FA(20:1) FA(20:2) FA(20:3) FA(20:4) FA(20:5) FA(21:0) FA(21:1) FA(21:2) FA(21:5) FA(21:6) FA(22:0) FA(22:1) FA(22:2) FA(22:3) FA(22:4) FA(22:5) FA(22:6) FA(23:0) FA(23:1) FA(23:2) FA(24:0) FA(24:1) FA(24:2) FA(24:3) FA(24:5) FA(24:6) FA(25:0) FA(25:1) FA(25:2) FA(25:3) FA(26:0) FA(26:1) FA(26:2) FA(26:3) FA(26:4)
Sludge from primary sedimentation tank of WWTP(I)
Sludge from A2/O tank of Sludge from MBR tank of Sludge from A2/O tank of Sludge from MBR tank of WWTP(I) WWTP(II) WWTP(III) WWTP(III)
12.19 ± 0.89
1.22 ± 0.10
0.65 ± 0.14
2.01 ± 1.08
1.11 ± 0.40
0.18 0.08 0.02 0.67 0.08 0.00 3.85 0.11 0.74 0.07 0.00 33.17 3.42 0.15 36.74 90.3 0.80 0.32 0.01 9.17 31.91 10.15 0.81 0.01 42.88 21.4 79.62 53.2
0.52 0.20 0.06 0.37 0.14 0.02 1.94 0.36 3.07 0.73 0.01 20.26 28.10 1.06 49.42 41.0 1.19 1.50 0.06 8.62 14.93 5.84 1.15 0.11 22.03 39.1 71.45 40.4
0.55 0.33 0.04 0.24 0.07 0.02 2.22 0.18 1.40 0.32 0.01 28.56 10.25 0.35 39.16 72.9 0.93 0.68 0.04 14.52 17.92 6.75 0.65 0.03 25.35 57.2 64.51 66.8
0.37 0.12 0.04 0.25 0.12 0.01 3.52 0.19 0.74 0.19 0.01 39.28 7.20 0.38 46.86 83.8 0.68 0.42 0.02 12.73 16.10 8.21 0.87 0.03 25.21 50.5 72.07 72.2
0.67 0.21 0.04 0.25 0.08 0.02 2.20 0.17 1.33 0.32 0.01 29.20 11.47 0.35 41.02 71.2 0.90 0.70 0.03 13.58 17.07 6.43 0.76 0.04 24.3 55.9 65.32 65.5
0.06 0.18 0.01 0.00 0.00 0.47 0.94 0.35 0.14 0.07 0.03 0.04 0.01 0.00 0.00 0.01 0.51 0.25 0.05 0.06 0.08 0.05 0.02 0.06 0.01 0.00 0.33 0.06 0.01 0.00 0.02 0.01 0.05 0.01 0.00 0.01 0.11 0.01 0.00 0.00 0.00
0.20 0.37 0.03 0.01 0.01 0.61 0.42 0.23 0.15 1.05 0.48 0.13 0.03 0.01 0.01 0.01 1.13 0.13 0.07 0.02 0.08 0.40 0.30 0.25 0.03 0.01 1.20 0.10 0.01 0.00 0.01 0.00 0.26 0.02 0.00 0.04 0.64 0.06 0.01 0.01 0.00
0.14 0.25 0.01 0.01 0.01 1.33 0.50 0.17 0.05 0.27 0.25 0.17 0.02 0.01 0.01 0.01 2.44 0.24 0.04 0.02 0.03 0.04 0.06 0.45 0.03 0.01 2.61 0.15 0.02 0.01 0.02 0.01 0.57 0.04 0.01 0.03 1.46 0.13 0.03 0.01 0.01
0.06 0.16 0.01 0.00 0.00 1.01 0.52 0.18 0.12 0.29 0.20 0.12 0.01 0.00 0.00 0.00 1.56 0.17 0.03 0.02 0.03 0.08 0.05 0.24 0.02 0.00 1.37 0.09 0.01 0.00 0.02 0.01 0.28 0.01 0.00 0.03 0.65 0.05 0.01 0.01 0.00
0.12 0.27 0.01 0.00 0.00 1.36 0.51 0.16 0.09 0.41 0.31 0.18 0.02 0.01 0.01 0.01 2.43 0.22 0.05 0.02 0.05 0.24 0.10 0.46 0.04 0.01 2.67 0.16 0.02 0.01 0.03 0.01 0.58 0.03 0.01 0.06 1.32 0.12 0.02 0.01 0.01
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Table 4 (continued ) Component
Sludge from primary sedimentation tank of WWTP(I)
Sludge from A2/O tank of Sludge from MBR tank of Sludge from A2/O tank of Sludge from MBR tank of WWTP(I) WWTP(II) WWTP(III) WWTP(III)
FA(26:5) FA(27:0) FA(27:5) FA(28:0) FA(28:1) FA(28:2) FA(28:4) FA(28:6) FA(29:0) FA(29:1) FA(29:6) FA(30:0) FA(30:1) FA(30:2) FA(30:3) FA(30:5) FA(30:6) FA(31:0) FA(31:3) FA(31:5) FA(32:0) FA(32:1) FA(32:2) FA(32:3) FA(32:4) FA(32:5)
0.00 0.01 0.00 0.04 0.01 0.00 0.00 0.00 0.01 0.00 0.00 0.02 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.01 0.01 0.00 0.00 0.00 0.00
0.01 0.08 0.01 0.26 0.04 0.01 0.00 0.01 0.03 0.02 0.00 0.13 0.05 0.01 0.00 0.01 0.00 0.01 0.00 0.02 0.05 0.05 0.02 0.01 0.03 0.01
0.00 0.20 0.01 0.57 0.09 0.02 0.01 0.00 0.07 0.02 0.00 0.26 0.09 0.01 0.01 0.01 0.01 0.03 0.01 0.01 0.10 0.10 0.02 0.01 0.04 0.01
0.00 0.08 0.01 0.23 0.04 0.01 0.00 0.00 0.02 0.01 0.00 0.15 0.05 0.01 0.01 0.00 0.00 0.01 0.00 0.00 0.05 0.05 0.01 0.01 0.01 0.00
0.00 0.17 0.01 0.43 0.08 0.02 0.00 0.00 0.06 0.02 0.00 0.21 0.09 0.01 0.01 0.01 0.01 0.02 0.01 0.00 0.08 0.10 0.02 0.02 0.05 0.02
Table 5 Lipid content in three sludge samples (%). Lipid
Sludge from primary Sludge from A2/O sedimentation tank of WWTP(I) tank of WWTP(I)
Sludge from MBR tank of WWTP(II)
Cellular lipids FFA Wax and gum S(Major lipid in cells þ FFA þ Wax and gum) Raw lipid by soxhlet extraction S(Major lipid in cells þ FFA þ Wax and gum)/ Raw lipid by soxhlet extraction
4.17 ± 1.22 ± 6.68 ± 12.07 12.3 ± 98.1
2.30 0.07 1.37 3.74 4.20 89.0
1.59 0.09 0.26 2.1 (Ad.)
2.61 0.12 0.67 3.40 3.80 89.5
± 0.44 ± 0.01 ± 0.06 ± 0.10 (Ad.)
± 1.04 ± 0.01 ± 0.19 ± 0.30 (Ad.)
Sludge from A2/O tank of WWTP(III) 6.64 0.20 0.98 7.82 8.01 97.6
± 1.85 ± 0.11 ± 0.04 ± 1.10 (Ad.)
Sludge from MBR tank of WWTP(III) 1.68 0.11 1.00 2.79 2.90 96.2
± 0.77 ± 0.04 ± 0.09 ± 0.20 (Ad.)
Legend: “Ad.” means absolute deviation. Deviations without special mark are standard deviation.
Sludge from primary sedimentation tank contained the highest concentration of total lipids (>10%), FFA, and wax and gum, as well as the second highest concentration of cellular lipids, indicating that sludge from primary sedimentation tank display a high potential as raw material for biodiesel production or for other reutilizations. The methods or technologies used to strengthen the function of primary sedimentation tank are favorable for accumulation of lipids from influent; with the use of a proper catalyst, an increased quantity of biodiesel with better quality can be produced from sewage sludge. Author contributions F.Z. conceived the idea for this paper. X.W., L.Z. and J.Q. conducted the sampling, pretreatment and extraction experiments. X.L. and X.W conducted the UPLC/MS/MS analysis. F.Z. finally composed this paper. J.W. provided the operation data of three WWTPs. Acknowledgments The present study is supported by the National Natural Science Foundation of China (grant number 51308538); the Major Science and Technology Program for Water Pollution Control and Treatment (grant number 2013ZX07314-001-006-01); Fundamental
Research Funds for the Central Universities, and the Research Funds of Renmin University of China (grant number 15XNLD04). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.watres.2017.03.032. References Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37 (8), 911e917. Chipasa, K.B., Medrzycka, K., 2008. Characterization of the fate of lipids in activated sludge. J. Environ. Sci. 20 (5), 536e542. Dufreche, S., Hernandez, R., French, T., Sparks, D., Zappi, M., Alley, E., 2007. Extraction of lipids from municipal wastewater plant microorganisms for production of biodiesel. J. Am. Oil Chem. Soc. 84 (2), 181e187. Folch, J., Lees, M., Sloane, S., 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226 (1), 497e509. Hao, X., Liu, R., Huang, X., 2015. Evaluation of the potential for operating carbon neutral WWTPs. Water Res. 87, 424e431. Huynh, L., Kasim, N., Ju, Y., 2010. Extraction and analysis of neutral lipids from activated sludge with and without sub-critical water pretreatment. Bioresour. Technol. 101 (22), 8891e8896. Jarde, E., Mansuy, L., Faure, P., 2005. Organic markers in the lipidic fraction of sewage sludges. Water Res. 39 (7), 1215e1232. Jin, L., Zhang, G., Tian, H., 2014. Current state of sewage treatment in China. Water Res. 66, 85e98. Kargbo, D., 2010. Biodiesel production from municipal sewage sludges. Energy &
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