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Lipid-enhancement of activated sludges obtained from conventional activated sludge and oxidation ditch processes
Bioresource Technology 148 (2013) 487–493
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Lipid-enhancement of activated sludges obtained from conventional activated sludge and oxidation ditch processes Emmanuel D. Revellame 1, Rafael Hernandez ⇑,1, W. Todd French, William E. Holmes 1, Allison Forks, Robert Callahan II Renewable Fuels and Chemicals Laboratory, Dave C. Swalm School of Chemical Engineering, Mail Stop 9595, Mississippi State University, MS 39762, USA
h i g h l i g h t s This study demonstrated lipid-enhancement of activated sludges via fermentation. Significant increase in the lipid content of the sludges was observed. Triacylglycerides comprised more than 50% of total lipids from enhanced sludge. Regardless of the source, the resulting sludges have similar fatty acid profile.
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Article history: Received 25 June 2013 Received in revised form 23 August 2013 Accepted 25 August 2013 Available online 5 September 2013 Keywords: Wastewater bacteria Biofuel Lipid-accumulation
a b s t r a c t Lipid-enhancement of activated sludges was conducted to increase the amount of saponifiable lipids in the sludges. The sludges were obtained from a conventional activated sludge (CAS) and an oxidation ditch process (ODP). Results showed 59–222% and 150–250% increase in saponifiable lipid content of the sludges from CAS and ODP, respectively. The fatty acid methyl ester (FAMEs) obtained from triacylglycerides was 57–67% (of total FAMEs) for enhanced CAS and 55–73% for enhanced ODP, a very significant improvement from 6% to 10% (CAS) and 4% to 8% (ODP). Regardless of the source, the enhancement resulted in sludges with similar fatty acid profile indicating homogenization of the lipids in the sludges. This study provides a potential strategy to utilize existing wastewater treatment facilities as source of significant amount of lipids for biofuel applications. Published by Elsevier Ltd.
1. Introduction Wastewater treatment operations involve biological processes, which can be separated broadly into two categories: fixed film and suspended growth systems. Fixed film systems include trickling filters and rotating biological contactors while suspended growth systems include various modifications of the activated sludge process (Spellman, 1999). All biological processes produce solids (also referred to as ‘‘biosolids’’ or sludge) that need to be treated prior to disposal. Due to ecological, economic, social and legal factors, sludge treatment and disposal is considered to be the bottleneck of most wastewater treatment plants (WWTPs) (Pérez-Elvira et al., 2006). Thus, sludge minimization and more importantly, its utilization for production of high-value products could greatly benefit WWTPs.
⇑ Corresponding author. Tel.: +1 337 482 6062; fax: +1 337 482 1220. E-mail address: [email protected] (R. Hernandez). 1 Present address: Department of Chemical Engineering, University of Louisiana at Lafayette, Lafayette, LA 70504, USA. 0960-8524/$ - see front matter Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.biortech.2013.08.158
The utilization of waste sludge as source of feedstock for biofuel production has been the subject of several studies (Dufreche et al., 2007; Mondala et al., 2009; Revellame et al., 2010; Siddiquee and Rohani, 2011). These studies have focused on biodiesel production from the saponifiable lipids present in activated sludge. Due to relatively low concentration of such lipids, activated sludge is not economically competitive at current prices of petroleum-based fuels. Recent study showed that raw activated sludge contains significant amount of unsaponifiable compounds (i.e. hydrocarbons, and sterols) in addition to saponifiable lipids [i.e. free fatty acids (FFAs), monoacylglycerides (MAGs), diacylglycerides (DAGs), triacylglycerides (TAGs), phospholipids (PLs), wax esters (WEs), steryl esters (SEs) and polyhydroxyalkanoates (PHAs)] (Revellame et al., 2012). Unsaponifiable lipids could be important precursors for a variety of applications/ products, but are unwanted in biodiesel production technology. The viable utilization of wastewater sludge for biodiesel production is constrained by several factors: (1) Low saponifiable lipid yield. (2) Differences in the types of wastewater being treated (i.e. domestic, food, agricultural, clinical and industrial).
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Nomenclature CAS DAGs FAMEs FFAs HB HV MAGs ODP
Conventional activated sludge Diacylglycerides Fatty acid methyl esters Free fatty acids Hydroxybutyric acid Hydroxyvaleric acid Monoacylglycerides Oxidation ditch process
(3) Differences in configurations of existing WWTPs (i.e. conventional activated sludge, oxidation ditch, trickling filter, rotating biological contactors). (4) Differences in microbial populations involved in biological treatment. These factors could result in huge differences in yields and speciation of lipidic materials present in wastewater sludge. In turn, this would require specific fuel conversion strategy for each particular factor combination. This scenario is not desirable especially for low capacity WWTPs. To address these factors, researchers have looked at several strategies. These include utilization of pre-treated wastewater sludge as substrate for yeast-based biodiesel (Seo et al., 2013), and addition of oil-accumulating microbial consortium to the indigenous microorganisms in the wastewater (Hall et al., 2011). In a recent study, Mondala et al. (2012) proposed a modification of existing WWTPs that can possibly address these issues. The proposed concept involved an additional lipid-accumulation unit where the waste activated sludge from a WWTP, is subjected to an environmental condition (stressed condition) that facilitates lipid production. Results of their batch fermentation experiments using glucose and ammonium sulfate as carbon and nitrogen sources, respectively, showed a maximum lipid yield of 17.5 ± 3.9% (cell dry weight) can be obtained at a glucose loading of 60 g/L with a corresponding carbon–nitrogen mass ratio of 70:1. At this fermentation condition, they obtained a biodiesel yield of 10.2 ± 2.0% (cell dry weight) (Mondala et al., 2012). However, they applied the enhancement on sludge from a conventional sludge process alone. For sustainable utilization of WWTPs as source of lipid feedstock, the enhancement needs to be applied on sludges from other treatment configuration. It is well documented that bacteria can synthesize lipid storage compounds (i.e. acylglycerides) under stressful conditions (i.e. nitrogen, oxygen and nutrient limitation) provided that there is an excessive supply of carbon source (Alvarez, 2006; Alvarez et al., 1997; Alvarez and Steinbüchel, 2002). Commonly, nitrogen limitation is the physiological stress that is being used to channel metabolic fluxes to lipid accumulation (Courchesne et al., 2009; Ratledge and Wynn, 2002). Studies on the biodiesel production from activated sludge taken from a municipal WWTP indicated that a yield of around 3–6% (dry weight) could be obtained from this feedstock (Dufreche et al., 2007; Mondala et al., 2009; Revellame et al., 2010). Based on the economic analysis conducted by Revellame et al. (2011), a yield of more than 10% (sludge dry weight) is necessary for this feedstock to be economically viable (Revellame et al., 2011). Lipid-enhancement, as depicted in Fig. 1, can be one strategy to achieve the required biodiesel yield; wherein a portion of the wastewater input to the treatment plant is used as carbon and nutrient source. However, to induce lipid-accumulation, additional carbon and nutrient sources might be needed. This could affect the economics of this feedstock negatively, but might
PHAs PLs SEs TAGs WEs WWTP
Polyhydroxyalkanoates Phospholipids Steryl esters Triacylglycerides Wax esters Wastewater treatment plant
be compensated by using relatively inexpensive carbon sources (i.e. lignocellulosic materials). In the United States alone, approximately 1.3 billion tons per year of lignocellulosic biomass could be used sustainably for biofuel production (Kosa and Ragauskas, 2011). In this study, lipid-enhancements were applied on activated sludges from conventional activated sludge (CAS) and oxidation ditch process (ODP) configurations. Activated sludge process modifications include CAS, step aeration, completely mix, pure oxygen, contact stabilization, extended aeration and ODP (Spellman, 1999). Among these modifications, CAS and ODP were chosen since they are the most commonly used ones in the United States. According to US EPA, 23% of all WWTPs employing the activated sludge process (1690) utilize CAS. On the other hand, ODP comprises 40% (including extended aeration systems) (US EPA, 2010). ODP is fundamentally similar to extended aeration system (Arceivala and Asolekar, 2007). CAS and ODP were also chosen based on their operational requirement; CAS requires primary treatment (clarifier) while ODP does not. The main purpose of primary treatment in a WWTP is for removal of settleable and floatable solids. It is also in this section where oil and grease are skimmed along with other floatable materials (Spellman, 2009). The amount of lipids (oil, grease, fats and fatty acids) in most municipal wastewater sums up to about 30–40% of its total chemical oxygen demand. Studies on their fate in biological waste treatment indicated that in addition to biodegradation, they are also adsorbed by the biomass (Chipasa and Me˛drzycka, 2006). Thus, the absence of primary treatment could have a significant impact on the amount and speciation of lipids present in the sludges after lipid-enhancement. This study was conducted to address some of the factors mentioned earlier that prevent viable utilization of wastewater sludge for biofuel production. Particularly, this work seeks to increase the amount of saponifiable lipids in sludges from two WWTP configurations: CAS and ODP. Furthermore, this study aims to determine the effect of enhancement on the speciation of lipids in the sludges. For all the experiments, glucose and ammonium sulfate were used as carbon and nitrogen sources, respectively. This study provides initial and to date the only report on solidifying the concept of WWTPs as source of feedstock for biofuel applications. 2. Experimental section 2.1. Activated sludge collection and preparation Three batches of activated sludges were collected from WWTP in Tuscaloosa, AL, USA. (Hilliard Fletcher municipal WWTP) and Tupelo, MS, USA. They were collected in the months of October, November and February (coded O, N and F, respectively) during the plants’ normal operations. The Tuscaloosa plant utilizes CAS treatment configuration, while the Tupelo plant utilizes ODP. Samples were collected in 1-L plastic containers from the return
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Fig. 1. Modification (red rectangle) of WWTPs for lipid-enhancement of activated sludge: RAS (return activated sludge), AS (activated sludge). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
activated sludge line of CAS and from the effluent of the oxidation ditch unit of ODP and were transported in ice-bath. The collected samples from a plant were mixed and homogenized after which a portion was transferred in 1-L Thermo Scientific Nalgene Culture Vessel (Fisher Scientific, Pittsburgh, USA) maintained with aeration at ambient temperature.
layer chromatography and gas chromatography. The protocol was designed to analyze for hydrocarbons, PHAs, FFAs, MAGs, DAGs, TAGs, WEs, SEs, sterols and PLs. The detailed experimental procedures are presented elsewhere (Revellame et al., 2012).
2.2. Lipid-enhancement
The evaluation of using WWTPs as source of feedstock for biofuels and oleochemicals production was conducted by previous workers (Revellame et al., 2012). However, the profiles of compounds that may be obtained from activated sludge may vary with respect to wastewater type and treatment process configurations. This could affect the consistency of resulting target products (i.e. biodiesel) and might require different downstream processing strategy for each WWTP, which is undesired if the ultimate goal is to utilize all wastewater sludge countrywide as lipid feedstock for biofuel production.
The lipid-enhancement was conducted for seven (7) days using a synthetic wastewater as cultivation medium. Glucose and ammonium sulfate were used as carbon and nitrogen sources, respectively. The detailed composition of the synthetic wastewater, fermentation parameters and conditions can be found elsewhere (Mondala et al., 2012). A carbon loading of 60 g/L (as glucose) and a nitrogen loading of 1.62 g/L (as ammonium sulfate) were used giving a C:N mass ratio of 70:1. Prior to fermentation, the medium was autoclaved at 121 °C and 240 kPa for 20 min. Fermentation experiments were conducted using two 5-L BIOFLO 310 Bioreactors (New Brunswick Scientific, Edison, NJ, USA). Six hundred milliliters (600 mL) of activated sludge was inoculated to 2.4 L of sterile medium giving a total cultivation volume of 3 L. No initial pH adjustments were done and throughout the experiments, only the cultivation temperature was monitored and controlled at 25 ± 1 °C. 2.3. Biomass recovery, extraction and analyses Samples (35 mL) were taken at the start and conclusion of the fermentation experiments. These samples were used for the determination of biomass and lipid concentrations. Samples were centrifuged at 3000 rpm for 20 min and the supernatants were discarded. The concentrated solids were frozen at -18 °C and freeze-dried in a Freezone 6 Bulk Tray freeze dry system (Labconco, Kansas City, MO, USA) to determine biomass concentration. The dried solids were then subjected to Bligh & Dyer extraction to determine the gravimetric lipid content. The remainder of the fermentation broth was recovered and was subjected to centrifugation followed by extraction. Extraction details can be found elsewhere (Revellame et al., 2012). Analyses of different lipidic material present in the extracts were conducted utilizing the protocol developed by Revellame et al. (2012), which utilizes a series of analytical techniques and procedures including precipitation, solid phase extraction, thin
3. Results and discussion
3.1. Polyhydroxyalkanoates The results of the analyses of the Bligh & Dyer extract from raw and enhanced activated sludges are presented in Tables 1 and 2. The PHAs analysis of the sludges showed significant reduction after enhancement, which could be accounted to the dilution effect discussed in Section 3.2. For both raw and enhanced sludges, only two hydroxyacid monomers were detected which are hydroxybutyric (HB) and hydroxyvaleric (HV) acids. PHA production in activated sludge is more affected by influent characteristics than activated sludge operating conditions (Takabatake et al., 2002). Since the influent for both plants is domestic wastewater, this could have dictated the speciation of PHA monomers in the sludges. For the raw sludge from CAS, the ratio of HB:HV on the average was 1.20 while for raw sludge from ODP, a ratio of about 2.00 was obtained. The ratio of PHA monomers is greatly affected by concentrations and types of volatile fatty acids as well as other carbon sources present in the influent wastewater (Yan et al., 2006). Unlike CAS, ODP does not have primary treatment unit(s). Although both plants are treating domestic wastewater, the absence of primary treatment as for the case of ODP could have caused the differences on the ratio of PHA monomers obtained. Production of biodiesel from PHAs was shown to be not economical (Zhang et al., 2009). However, due to their biodegradability, PHAs has many other possible uses such as packaging films, disposable commodity plastics and many medical applications
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Table 1 Composition of lipid extract from CAS raw and enhanced activated sludge. O
Sludge collection date Aeration basin temperature, °C Initial biomass concentration, mg/mL D Biomass concentration, mg/mL Bligh & Dyer extract yield, wt.% of dry biomass PHAs, wt.% of extract Hydrocarbons, ppm (based on weight of extract) Fatty alcohols (from WEs), wt.% of extract Sterols (from SEs), wt.% of extract Free sterols, wt.% of extract TAGs, wt.% of extract FAMEs (from WEs and SEs), wt.% of extractb FAMEs (from TAGs), wt.% of extract FAMEs (from FFAs, MAGs and DAGs), wt.% of extract FAMEs (from PLs), wt.% of extract Total FAMEs yield, wt.% of extract Total FAMEs yield, wt.% of biomass a b
N
F
Raw
Enhanced
Raw
6-Oct-10 24.5
14-Oct-10 25 ± 1
29-Nov-10 7-Dec-10 20.8 25 ± 1 4.5 ± 0.9 8.6 ± 0.3 9.1 ± 0.2 19.7 ± 0.3 1.8 ± 0.7 1.2 ± 0.3 5.3 ± 2.6 – 0.4 ± 0.1 – 0.14 ± 0.01 – 1.4 ± 1.0 – 1.2 ± 0.2 17.8 ± 0.8 1.9 0.5 1.2 17.9 6.9 9.0 9.7 2.0 19.7 ± 1.1 29.4 ± 1.8 1.8 ± 0.1 5.8 ± 0.4
9.6 ± 0.8 3.0 ± 1.1 2.4 ± 1.0 1.2 ± 0.1 0.26 ± 0.03 2.0 ± 0.1 2.1 ± 0.6 3.0 2.1 8.3 7.7 21.1 ± 0.4 2.0 ± 0.2
N
F
3.5 ± 0.2 5.7 ± 0.6 8.3 ± 1.0 2.6 ± 0.8 3.3 ± 1.9 0.5 ± 0.1 0.18 ± 0.06 1.4 ± 0.1 1.76 ± 0.02 1.8 1.8 5.3 11.6 20.5 ± 1.0 1.7 ± 0.2
15.6 ± 0.4 2.0 ± 0.1 –a – – – 11.4 ± 1.0 1.0 11.4 3.7 1.0 17.1 ± 1.2 2.7 ± 0.2
Enhanced
Raw
Enhanced
15-Feb-11 15.6
23-Feb-11 25 ± 1 3.2 ± 0.3 5.3 ± 0.2 15.1 ± 0.3 0.7 ± 0.2 – – – – 18.2 ± 0.2 1.1 18.3 10.6 1.9 31.9 ± 1.2 4.8 ± 0.2
Below detection limit. Includes FAMEs initially present in the samples.
Table 2 Composition of lipid extract from ODP raw and enhanced activated sludge. O
Sludge collection date Aeration basin temperature, °C Initial biomass concentration, mg/mL D Biomass concentration, mg/mL Bligh & Dyer extract yield, wt.% of dry biomass PHAs, wt.% of extract Hydrocarbons, ppm (based on weight of extract) Fatty alcohols (from WEs), wt.% of extract Sterols (from SEs), wt.% of extract Free sterols, wt.% of extract TAGs, wt.% of extract FAMEs (from WEs and SEs), wt.% of extractb FAMEs (from TAGs), wt.% of extract FAMEs (from FFAs, MAGs and DAGs), wt.% of extract FAMEs (from PLs), wt.% of extract Total FAMEs yield, wt.% of extract Total FAMEs yield, wt.% of biomass a b
Raw
Enhanced
Raw
Enhanced
Raw
Enhanced
6-Oct-10 26
14-Oct-10 25 ± 1
29-Nov-10 20
7-Dec-10 25 ± 1
15-Feb-11 15
23-Feb-11 25 ± 1
15.7 ± 0.5 3.0 ± 1.5 –a – – – 23.8 ± 0.8 0.7 23.9 12.1 3.3 40.0 ± 1.1 6.3 ± 0.3
4.9 ± 0.5 10.4 ± 0.6 8.1 ± 0.2 18.2 ± 0.7 1.9 ± 0.4 0.52 ± 0.04 2.7 ± 1.1 – 1.1 ± 0.1 – 0.27 ± 0.04 – 1.1 ± 0.2 – 1.7 ± 0.2 21.8 ± 0.2 2.1 0.8 1.7 21.9 18.6 2.8 4.9 4.7 27.3 ± 0.3 30.2 ± 1.2 2.2 ± 0.1 5.5 ± 0.3
4.3 9.9 ± 0.1 6.4 ± 1.3 4.3 ± 1.4 1.3 ± 0.2 0.84 ± 0.01 0.2 ± 0.1 1.0 ± 0.1 1.00 ± 0.01 2.1 1.0 17.7 7.0 27.8 ± 1.1 1.8 ± 0.4
4.0 ± 0.7 10.1 ± 0.3 7.3 ± 0.2 16.4 ± 0.2 3.1 ± 0.5 2.0 ± 0.9 4.2 ± 2.0 – 2.3 ± 1.0 – 0.3 ± 0.1 – 1.5 ± 0.3 – 2.0 ± 0.2 20.1 ± 1.0 3.2 0.3 2.0 20.2 15.6 14.2 5.0 2.3 25.8 ± 1.0 37.0 ± 2.2 1.9 ± 0.1 6.1 ± 0.4
Below detection limit. Includes FAMEs initially present in the samples.
(Revellame et al., 2012). Thus, the presence of PHAs in the enhanced sludges could possibly improve the overall economics of the process. 3.2. Unsaponifiable lipids Results indicated that the speciation of lipidic compounds present in raw sludges were similar regardless of the source. However, the concentrations of these compounds were significantly different between the two plants (Tables 1 and 2). Analyses of the extracts from lipid-enhanced sludges from the two plants showed undetected levels of hydrocarbons, WEs, SEs and free sterols. However, analysis indicated that there were fatty acid methyl esters (FAMEs) (a.k.a. biodiesel) initially present in the samples. This could be accounted from the Bligh & Dyer extraction procedure, where methanol was one of the extraction solvents. During the extraction, the high amount of methanol could have resulted in alcoholysis of lipids present in the samples. This was also observed by previous workers (Revellame et al., 2012).
The undetected levels of hydrocarbons, WEs, SEs and free sterols in lipid-enhanced activated sludges could be accounted to several factors. The most obvious one is the switch in carbon sources. The presence of hydrocarbons in the raw sludges can be attributed to microbially-degraded petroleum residue and are characteristics of petroleum-polluted sediments (Jardé et al., 2005). Since the enhancement of the sludges used glucose as sole carbon source, the input of petroleum products residue was eliminated and thus hydrocarbons were not detected in the samples. The same is true for WEs, SEs and free sterols. The WEs in the raw sludges could be due to possible presence of detergent fatty alcohols in the influent wastewaters of the two plants. Detergent fatty alcohols are considered bioavailable and might have been used by raw activated sludge microorganisms for WE synthesis (Mudge et al., 2008). The input of these detergent fatty alcohols was eliminated during enhancement and thus, WEs were not detected in the lipid-enhanced activated sludges. As for the case of SEs and free sterols, their presence in the raw sludges can be accounted mainly due to anthropogenic contributions, particularly human feces.
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have caused the shift in bacterial population to the ones that can survive under acidic environment. 3.3. Saponifiable lipids 3.3.1. Raw sludges Fig. 2 shows the fatty acid profile of the lipids in the raw sludges. It can be seen that within a plant, the profiles are significantly similar. Between the two plants considered, the fatty acids present ranges from C12:0 to C22:1. However, between the two plants the concentrations of fatty acids were significantly different particularly those of C16s and C18s fatty acids. The huge differences in fatty acid profiles of raw activated sludges from CAS and ODP could be attributed to the absence of primary treatment unit(s) of the ODP. As mentioned (Section 1), without primary treatment, oil and grease in the wastewater could be adsorbed by the biomass and eventually recovered during extraction. 3.3.2. Enhanced sludges Analysis of the lipid-enhanced sludges indicated that 57–67% and 55–73% of the total FAMEs in CAS and ODP, respectively, were coming from TAGs. This was a very significant improvement considering that the FAMEs from TAGs in raw activated sludges ranges about 6–10% and 4–8% for CAS and ODP, respectively (Tables 1 and 2). TAGs are the most attractive feedstock for biodiesel production because of how easily they can be chemically converted to biodiesel. In biodiesel technology, lipid feedstock quality is defined as the inverse of free fatty acid content (Haas and Foglia, 2005). The increase in TAGs content in the enhanced sludges means decrease in its free fatty acid content and thus, an improvement in its quality. On the other hand, however, the yield of total FAMEs (2.7–5.8% and 5.5–6.3% dry sludge weight for enhanced CAS and ODP sludges, respectively) were significantly lower than what previous workers obtained (10.2 ± 2.0% dry sludge weight) at the same cultivation condition (Mondala et al., 2012). This could be due to losses during sample preparation and handling or to inherent variability of raw activated sludge samples within a plant. Nevertheless, the total FAMEs of the enhanced activated sludges are significantly higher than that of the raw activated sludges (1.7–2.0% and 1.8–2.2% dry sludge weight for CAS and ODP sludges, respectively). 50
45
45
30 25 20
12 : C
C
C
:2
18
C
18 :
C
18 :
C
C
16 :
C
14 :
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18 :3 20 :0 C 22 To :0 ta l U C2 n k 2: 1 no w ns
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Cessation of such contributions resulted to undetected level of SEs and free sterols on the resulting enhanced sludge. However, one might ask what happened to these compounds (hydrocarbons, WEs, SEs and free sterols) initially present in the raw sludges, which were used as fermentation seeds. It is highly unlikely that the microorganisms consumed these compounds for growth instead of glucose. After the 7-day fermentation, the glucose concentration in the broth was still above 20 g/L (Mondala et al., 2012), which suggest that these compounds would still be present in the lipid-enhanced activated sludges. However, the enhancement resulted to dilution of these compounds to a level that cannot be detected. Another way to look at this dilution effect is by considering the change in biomass concentration as a result of enhancement. On the average, the biomass of CAS sludges increased by 6.5 mg/mL while that of ODP increased by 10.1 mg/mL (Tables 1 and 2). This indicates that an inert material initially present in the raw sludges will be diluted by 1:7.5 and 1:11.1 for CAS and ODP sludges, respectively. In spite of the above discussion, the ability of raw activated sludge microorganisms to synthesize hydrocarbons, WEs, SEs and free sterols cannot be neglected. Thus, their absence in the lipidenhanced activated sludges could be due to microbial population shift brought about by the changes in carbon source and/or cultivation condition. Recent study indicated that lipid-enhancement of activated sludge could result to significant changes in microbial population. The raw activated sludge contains bacterial population in phyla Proteobacteria [a-/b-/c-/d-/e-Proteobacteria (i.e. Rhodobacterales and Xanthomonadales)], Verrucomicrobia (class Verrucomicrobiae), Bacteriodetes (class Flavobacteria and Sphingobacteria), Firmicutes (class Clostridia) and Actinobacteria. At the end of the 7-day fermentation period, the pH of the broth decreased from 6.50 to 2.00 and 99.5% of bacterial population shifted to a-Proteobacteria. Sequencing showed that these bacteria are similar in characteristics to Acidomonas methanolica, an acidotolerant, gram-negative bacteria (Mondala et al., 2012; Urakami et al., 1989). Acidomonas methanolica have been previously isolated from WWTP (Yamashita et al., 2004). In a related study, Niu and co-workers also observed a shift in bacterial community towards a gram-negative population when activated sludge was applied to treat synthetic wastewater with glucose at 25 °C (Niu et al., 2012). In the present paper, however, the decrease in pH could
(b)
Fig. 2. Fatty acid profile of lipids extracted from raw activated sludge; (a) CAS (b) ODP.
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50
45
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25 20
1 18 :2 C 18 :3 C 20 :0
0
C
C
18 :
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18 :
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12 : C
C
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C
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Fig. 3. Fatty acid profile of lipids extracted from enhanced activated sludge; (a) CAS (b) ODP.
Enhancement of the sludges resulted to homogenization of the lipidic compounds associated with microbial biomass (Tables 1 and 2). This is also evident in the fatty acid profiles of the lipids in enhanced sludges (Fig. 3). Regardless of the source of the raw sludges, the lipid-enhancement resulted to an almost similar fatty acid profile. Important biodiesel properties such as cold flow and cetane number are dictated by its fatty acid profile. Looking at Fig. 2, it is apparent that the biodiesel from raw activated sludges from CAS and ODP have different properties and mixing them together will produce a biodiesel with completely different properties. This is not attractive if the goal is to utilize countrywide generated wastewater sludge for biodiesel production. However, the results of this study indicated that enhancement of activated sludges could be a potential strategy to produce a homogeneous feedstock across the United States with improved lipid content. 4. Conclusion Lipid-enhancement of sludges from two WWTPs with different treatment configurations was conducted to increase the amount of saponifiable lipids in the sludges. Irrespective of the source, results showed increase in the amount of lipids in the enhanced sludges, most of which are TAGs. Furthermore, the fatty acid profiles of the enhanced sludges indicated homogenization of sludges from different WWTPs. Although only two wastewater treatment configurations were considered, this study serves as an initial step in the utilization of existing WWTPs as source of significant amount of lipids for biofuel applications. Acknowledgement This work was funded by the United States Department of Energy, Office of Energy Efficiency and Renewable Energy (Grant No. DE-FG36-06GO86025). References Alvarez, H.M., 2006. Bacterial triacylglycerols. In: Welson, L.T. (Ed.), Triglycerides and Cholesterol Research. Nova Science Publishers, Inc., New York, pp. 159–176. Alvarez, H.M., Pucci, O.H., Steinbüchel, A., 1997. Lipid storage compounds in marine bacteria. Appl. Microbiol. Biotechnol. 47, 132–139.
Alvarez, H.M., Steinbüchel, A., 2002. Triacylglycerols in prokaryotic microorganisms. Appl. Microbiol. Biotechnol. 60, 367–376. Arceivala, S.J., Asolekar, S.R., 2007. Wastewater Treatment for Pollution Control and Reuse, third ed. Tata McGraw-Hill Publishing Co. Ltd., New Delhi. Chipasa, K., Me˛drzycka, K., 2006. Behavior of lipids in biological wastewater treatment processes. J. Ind. Microbiol. Biotechnol. 33, 635–645. Courchesne, N.M.D., Parisien, A., Wang, B., Lan, C.Q., 2009. Enhancement of lipid production using biochemical, genetic and transcription factor engineering approaches. J. Biotechnol. 141, 31–41. 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, 181–187. Haas, M.J., Foglia, T.A., 2005. Alternative feedstocks and technologies for biodiesel production. In: Knothe, G., Krahl, J., Van Gerpen, J.H. (Eds.), The Biodiesel Handbook, first ed. AOCS Press, Champaign, IL, pp. 42–61. Hall, J., Hetrick, M., French, T., Hernandez, R., Donaldson, J., Mondala, A., Holmes, W., 2011. Oil production by a consortium of oleaginous microorganisms grown on primary effluent wastewater. J. Chem. Technol. Biotechnol. 86, 54–60. Jardé, E., Mansuy, L., Faure, P., 2005. Organic markers in the lipidic fraction of sewage sludges. Water Res. 39, 1215–1232. Kosa, M., Ragauskas, A.J., 2011. Lipids from heterotrophic microbes: advances in metabolism research. Trends Biotechnol. 29, 53–61. 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, 1203–1210. Mondala, A.H., Hernandez, R., French, T., McFarland, L., Santo Domingo, J.W., Meckes, M., Ryu, H., Iker, B., 2012. Enhanced lipid and biodiesel production from glucose-fed activated sludge: kinetics and microbial community analysis. AIChE J. 58, 1279–1290. Mudge, S.M., Belanger, S.E., Nielsen, A.M., 2008. Fatty Alcohols: Anthropogenic and Natural Occurrence in the Environment. Royal Society of Chemistry, Cambridge, UK. Niu, C., Geng, J., Ren, H., Ding, L., Xu, K., 2012. The cold adaptability of microorganisms with different carbon source in activated sludge treating synthetical wastewater. Bioresour. Technol. 123, 66–71. Pérez-Elvira, S.I., Nieto Diez, P., Fdz-Polanco, F., 2006. Sludge minimisation technologies. Rev. Environ. Sci. Bio/Technol. 5, 375–398. Ratledge, C., Wynn, J.P., 2002. The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms. Adv. Appl. Microbiol. 51, 1–51. Revellame, E., Hernandez, R., French, W., Holmes, W., Alley, E., 2010. Biodiesel from activated sludge through in situ transesterification. J. Chem. Technol. Biotechnol. 85, 614–620. Revellame, E., Hernandez, R., French, W., Holmes, W., Alley, E., Callahan II, R., 2011. Production of biodiesel from wet activated sludge. J. Chem. Technol. Biotechnol. 86, 61–68. Revellame, E.D., Hernandez, R., French, W., Holmes, W.E., Benson, T.J., Pham, P.J., Forks, A., Callahan II, R., 2012. Lipid storage compounds in raw activated sludge microorganisms for biofuels and oleochemicals production. RSC Adv. 2, 2015– 2031. Seo, Y., Lee, I., Han, J., 2013. Cultivation and lipid production of yeast Cryptococcus curvatus using pretreated waste active sludge supernatant. Bioresour. Technol. 135, 304–308.
E.D. Revellame et al. / Bioresource Technology 148 (2013) 487–493 Siddiquee, M.N., Rohani, S., 2011. Experimental analysis of lipid extraction and biodiesel production from wastewater sludge. Fuel Process. Technol. 92, 2241– 2251. Spellman, F.R., 2009. Handbook of Water and Wastewater Treatment Plant Operations, second ed. Taylor & Francis, Boca Raton. Spellman, F.R., 1999. Spellman’s standard handbook for wastewater operators, vol. 2. Technomic Publishing Co., Lancaster, PA. Takabatake, H., Satoh, H., Mino, T., Matsuo, T., 2002. PHA (polyhydroxyalkanoate) production potential of activated sludge treating wastewater. Water Sci. Technol. 45, 119–126. Urakami, T., Tamaoka, J., Suzuki, K.-I., Komagata, K., 1989. Acidomonas gen. nov., Incorporating Acetobacter methanolicus as Acidomonas methanolica comb. nov.. Int. J. Syst. Bacteriol. 39, 50–55.
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US EPA, 2010. CWNS 2008 Report to Congress. (accessed 15.08.2013). Yamashita, S.-i., Uchimura, T., Komagata, K., 2004. Emendation of the genus Acidomonas Urakami, Tamaoka, Suzuki and Komagata 1989. Int. J. Syst. Evol. Microbiol. 54, 865–870. Yan, S., Tyagi, R.D., Surampalli, R.Y., 2006. Polyhydroxyalkanoates (PHA) production using wastewater as carbon source and activated sludge as microorganisms. Water Sci. Technol. 53, 175–180. Zhang, X., Luo, R., Wang, Z., Deng, Y., Chen, G.-Q., 2009. Application of (R)-3hydroxyalkanoate methyl esters derived from microbial polyhydroxyalkanoates as novel biofuels. Biomacromolecules 10, 707–711.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Lipid-enhancement of activated sludges obtained from conventional activated sludge and oxidation ditch processes Emmanuel D. Revellame 1, Rafael Hernandez ⇑,1, W. Todd French, William E. Holmes 1, Allison Forks, Robert Callahan II Renewable Fuels and Chemicals Laboratory, Dave C. Swalm School of Chemical Engineering, Mail Stop 9595, Mississippi State University, MS 39762, USA
h i g h l i g h t s This study demonstrated lipid-enhancement of activated sludges via fermentation. Significant increase in the lipid content of the sludges was observed. Triacylglycerides comprised more than 50% of total lipids from enhanced sludge. Regardless of the source, the resulting sludges have similar fatty acid profile.
a r t i c l e
i n f o
Article history: Received 25 June 2013 Received in revised form 23 August 2013 Accepted 25 August 2013 Available online 5 September 2013 Keywords: Wastewater bacteria Biofuel Lipid-accumulation
a b s t r a c t Lipid-enhancement of activated sludges was conducted to increase the amount of saponifiable lipids in the sludges. The sludges were obtained from a conventional activated sludge (CAS) and an oxidation ditch process (ODP). Results showed 59–222% and 150–250% increase in saponifiable lipid content of the sludges from CAS and ODP, respectively. The fatty acid methyl ester (FAMEs) obtained from triacylglycerides was 57–67% (of total FAMEs) for enhanced CAS and 55–73% for enhanced ODP, a very significant improvement from 6% to 10% (CAS) and 4% to 8% (ODP). Regardless of the source, the enhancement resulted in sludges with similar fatty acid profile indicating homogenization of the lipids in the sludges. This study provides a potential strategy to utilize existing wastewater treatment facilities as source of significant amount of lipids for biofuel applications. Published by Elsevier Ltd.
1. Introduction Wastewater treatment operations involve biological processes, which can be separated broadly into two categories: fixed film and suspended growth systems. Fixed film systems include trickling filters and rotating biological contactors while suspended growth systems include various modifications of the activated sludge process (Spellman, 1999). All biological processes produce solids (also referred to as ‘‘biosolids’’ or sludge) that need to be treated prior to disposal. Due to ecological, economic, social and legal factors, sludge treatment and disposal is considered to be the bottleneck of most wastewater treatment plants (WWTPs) (Pérez-Elvira et al., 2006). Thus, sludge minimization and more importantly, its utilization for production of high-value products could greatly benefit WWTPs.
⇑ Corresponding author. Tel.: +1 337 482 6062; fax: +1 337 482 1220. E-mail address: [email protected] (R. Hernandez). 1 Present address: Department of Chemical Engineering, University of Louisiana at Lafayette, Lafayette, LA 70504, USA. 0960-8524/$ - see front matter Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.biortech.2013.08.158
The utilization of waste sludge as source of feedstock for biofuel production has been the subject of several studies (Dufreche et al., 2007; Mondala et al., 2009; Revellame et al., 2010; Siddiquee and Rohani, 2011). These studies have focused on biodiesel production from the saponifiable lipids present in activated sludge. Due to relatively low concentration of such lipids, activated sludge is not economically competitive at current prices of petroleum-based fuels. Recent study showed that raw activated sludge contains significant amount of unsaponifiable compounds (i.e. hydrocarbons, and sterols) in addition to saponifiable lipids [i.e. free fatty acids (FFAs), monoacylglycerides (MAGs), diacylglycerides (DAGs), triacylglycerides (TAGs), phospholipids (PLs), wax esters (WEs), steryl esters (SEs) and polyhydroxyalkanoates (PHAs)] (Revellame et al., 2012). Unsaponifiable lipids could be important precursors for a variety of applications/ products, but are unwanted in biodiesel production technology. The viable utilization of wastewater sludge for biodiesel production is constrained by several factors: (1) Low saponifiable lipid yield. (2) Differences in the types of wastewater being treated (i.e. domestic, food, agricultural, clinical and industrial).
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Nomenclature CAS DAGs FAMEs FFAs HB HV MAGs ODP
Conventional activated sludge Diacylglycerides Fatty acid methyl esters Free fatty acids Hydroxybutyric acid Hydroxyvaleric acid Monoacylglycerides Oxidation ditch process
(3) Differences in configurations of existing WWTPs (i.e. conventional activated sludge, oxidation ditch, trickling filter, rotating biological contactors). (4) Differences in microbial populations involved in biological treatment. These factors could result in huge differences in yields and speciation of lipidic materials present in wastewater sludge. In turn, this would require specific fuel conversion strategy for each particular factor combination. This scenario is not desirable especially for low capacity WWTPs. To address these factors, researchers have looked at several strategies. These include utilization of pre-treated wastewater sludge as substrate for yeast-based biodiesel (Seo et al., 2013), and addition of oil-accumulating microbial consortium to the indigenous microorganisms in the wastewater (Hall et al., 2011). In a recent study, Mondala et al. (2012) proposed a modification of existing WWTPs that can possibly address these issues. The proposed concept involved an additional lipid-accumulation unit where the waste activated sludge from a WWTP, is subjected to an environmental condition (stressed condition) that facilitates lipid production. Results of their batch fermentation experiments using glucose and ammonium sulfate as carbon and nitrogen sources, respectively, showed a maximum lipid yield of 17.5 ± 3.9% (cell dry weight) can be obtained at a glucose loading of 60 g/L with a corresponding carbon–nitrogen mass ratio of 70:1. At this fermentation condition, they obtained a biodiesel yield of 10.2 ± 2.0% (cell dry weight) (Mondala et al., 2012). However, they applied the enhancement on sludge from a conventional sludge process alone. For sustainable utilization of WWTPs as source of lipid feedstock, the enhancement needs to be applied on sludges from other treatment configuration. It is well documented that bacteria can synthesize lipid storage compounds (i.e. acylglycerides) under stressful conditions (i.e. nitrogen, oxygen and nutrient limitation) provided that there is an excessive supply of carbon source (Alvarez, 2006; Alvarez et al., 1997; Alvarez and Steinbüchel, 2002). Commonly, nitrogen limitation is the physiological stress that is being used to channel metabolic fluxes to lipid accumulation (Courchesne et al., 2009; Ratledge and Wynn, 2002). Studies on the biodiesel production from activated sludge taken from a municipal WWTP indicated that a yield of around 3–6% (dry weight) could be obtained from this feedstock (Dufreche et al., 2007; Mondala et al., 2009; Revellame et al., 2010). Based on the economic analysis conducted by Revellame et al. (2011), a yield of more than 10% (sludge dry weight) is necessary for this feedstock to be economically viable (Revellame et al., 2011). Lipid-enhancement, as depicted in Fig. 1, can be one strategy to achieve the required biodiesel yield; wherein a portion of the wastewater input to the treatment plant is used as carbon and nutrient source. However, to induce lipid-accumulation, additional carbon and nutrient sources might be needed. This could affect the economics of this feedstock negatively, but might
PHAs PLs SEs TAGs WEs WWTP
Polyhydroxyalkanoates Phospholipids Steryl esters Triacylglycerides Wax esters Wastewater treatment plant
be compensated by using relatively inexpensive carbon sources (i.e. lignocellulosic materials). In the United States alone, approximately 1.3 billion tons per year of lignocellulosic biomass could be used sustainably for biofuel production (Kosa and Ragauskas, 2011). In this study, lipid-enhancements were applied on activated sludges from conventional activated sludge (CAS) and oxidation ditch process (ODP) configurations. Activated sludge process modifications include CAS, step aeration, completely mix, pure oxygen, contact stabilization, extended aeration and ODP (Spellman, 1999). Among these modifications, CAS and ODP were chosen since they are the most commonly used ones in the United States. According to US EPA, 23% of all WWTPs employing the activated sludge process (1690) utilize CAS. On the other hand, ODP comprises 40% (including extended aeration systems) (US EPA, 2010). ODP is fundamentally similar to extended aeration system (Arceivala and Asolekar, 2007). CAS and ODP were also chosen based on their operational requirement; CAS requires primary treatment (clarifier) while ODP does not. The main purpose of primary treatment in a WWTP is for removal of settleable and floatable solids. It is also in this section where oil and grease are skimmed along with other floatable materials (Spellman, 2009). The amount of lipids (oil, grease, fats and fatty acids) in most municipal wastewater sums up to about 30–40% of its total chemical oxygen demand. Studies on their fate in biological waste treatment indicated that in addition to biodegradation, they are also adsorbed by the biomass (Chipasa and Me˛drzycka, 2006). Thus, the absence of primary treatment could have a significant impact on the amount and speciation of lipids present in the sludges after lipid-enhancement. This study was conducted to address some of the factors mentioned earlier that prevent viable utilization of wastewater sludge for biofuel production. Particularly, this work seeks to increase the amount of saponifiable lipids in sludges from two WWTP configurations: CAS and ODP. Furthermore, this study aims to determine the effect of enhancement on the speciation of lipids in the sludges. For all the experiments, glucose and ammonium sulfate were used as carbon and nitrogen sources, respectively. This study provides initial and to date the only report on solidifying the concept of WWTPs as source of feedstock for biofuel applications. 2. Experimental section 2.1. Activated sludge collection and preparation Three batches of activated sludges were collected from WWTP in Tuscaloosa, AL, USA. (Hilliard Fletcher municipal WWTP) and Tupelo, MS, USA. They were collected in the months of October, November and February (coded O, N and F, respectively) during the plants’ normal operations. The Tuscaloosa plant utilizes CAS treatment configuration, while the Tupelo plant utilizes ODP. Samples were collected in 1-L plastic containers from the return
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489
Fig. 1. Modification (red rectangle) of WWTPs for lipid-enhancement of activated sludge: RAS (return activated sludge), AS (activated sludge). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
activated sludge line of CAS and from the effluent of the oxidation ditch unit of ODP and were transported in ice-bath. The collected samples from a plant were mixed and homogenized after which a portion was transferred in 1-L Thermo Scientific Nalgene Culture Vessel (Fisher Scientific, Pittsburgh, USA) maintained with aeration at ambient temperature.
layer chromatography and gas chromatography. The protocol was designed to analyze for hydrocarbons, PHAs, FFAs, MAGs, DAGs, TAGs, WEs, SEs, sterols and PLs. The detailed experimental procedures are presented elsewhere (Revellame et al., 2012).
2.2. Lipid-enhancement
The evaluation of using WWTPs as source of feedstock for biofuels and oleochemicals production was conducted by previous workers (Revellame et al., 2012). However, the profiles of compounds that may be obtained from activated sludge may vary with respect to wastewater type and treatment process configurations. This could affect the consistency of resulting target products (i.e. biodiesel) and might require different downstream processing strategy for each WWTP, which is undesired if the ultimate goal is to utilize all wastewater sludge countrywide as lipid feedstock for biofuel production.
The lipid-enhancement was conducted for seven (7) days using a synthetic wastewater as cultivation medium. Glucose and ammonium sulfate were used as carbon and nitrogen sources, respectively. The detailed composition of the synthetic wastewater, fermentation parameters and conditions can be found elsewhere (Mondala et al., 2012). A carbon loading of 60 g/L (as glucose) and a nitrogen loading of 1.62 g/L (as ammonium sulfate) were used giving a C:N mass ratio of 70:1. Prior to fermentation, the medium was autoclaved at 121 °C and 240 kPa for 20 min. Fermentation experiments were conducted using two 5-L BIOFLO 310 Bioreactors (New Brunswick Scientific, Edison, NJ, USA). Six hundred milliliters (600 mL) of activated sludge was inoculated to 2.4 L of sterile medium giving a total cultivation volume of 3 L. No initial pH adjustments were done and throughout the experiments, only the cultivation temperature was monitored and controlled at 25 ± 1 °C. 2.3. Biomass recovery, extraction and analyses Samples (35 mL) were taken at the start and conclusion of the fermentation experiments. These samples were used for the determination of biomass and lipid concentrations. Samples were centrifuged at 3000 rpm for 20 min and the supernatants were discarded. The concentrated solids were frozen at -18 °C and freeze-dried in a Freezone 6 Bulk Tray freeze dry system (Labconco, Kansas City, MO, USA) to determine biomass concentration. The dried solids were then subjected to Bligh & Dyer extraction to determine the gravimetric lipid content. The remainder of the fermentation broth was recovered and was subjected to centrifugation followed by extraction. Extraction details can be found elsewhere (Revellame et al., 2012). Analyses of different lipidic material present in the extracts were conducted utilizing the protocol developed by Revellame et al. (2012), which utilizes a series of analytical techniques and procedures including precipitation, solid phase extraction, thin
3. Results and discussion
3.1. Polyhydroxyalkanoates The results of the analyses of the Bligh & Dyer extract from raw and enhanced activated sludges are presented in Tables 1 and 2. The PHAs analysis of the sludges showed significant reduction after enhancement, which could be accounted to the dilution effect discussed in Section 3.2. For both raw and enhanced sludges, only two hydroxyacid monomers were detected which are hydroxybutyric (HB) and hydroxyvaleric (HV) acids. PHA production in activated sludge is more affected by influent characteristics than activated sludge operating conditions (Takabatake et al., 2002). Since the influent for both plants is domestic wastewater, this could have dictated the speciation of PHA monomers in the sludges. For the raw sludge from CAS, the ratio of HB:HV on the average was 1.20 while for raw sludge from ODP, a ratio of about 2.00 was obtained. The ratio of PHA monomers is greatly affected by concentrations and types of volatile fatty acids as well as other carbon sources present in the influent wastewater (Yan et al., 2006). Unlike CAS, ODP does not have primary treatment unit(s). Although both plants are treating domestic wastewater, the absence of primary treatment as for the case of ODP could have caused the differences on the ratio of PHA monomers obtained. Production of biodiesel from PHAs was shown to be not economical (Zhang et al., 2009). However, due to their biodegradability, PHAs has many other possible uses such as packaging films, disposable commodity plastics and many medical applications
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Table 1 Composition of lipid extract from CAS raw and enhanced activated sludge. O
Sludge collection date Aeration basin temperature, °C Initial biomass concentration, mg/mL D Biomass concentration, mg/mL Bligh & Dyer extract yield, wt.% of dry biomass PHAs, wt.% of extract Hydrocarbons, ppm (based on weight of extract) Fatty alcohols (from WEs), wt.% of extract Sterols (from SEs), wt.% of extract Free sterols, wt.% of extract TAGs, wt.% of extract FAMEs (from WEs and SEs), wt.% of extractb FAMEs (from TAGs), wt.% of extract FAMEs (from FFAs, MAGs and DAGs), wt.% of extract FAMEs (from PLs), wt.% of extract Total FAMEs yield, wt.% of extract Total FAMEs yield, wt.% of biomass a b
N
F
Raw
Enhanced
Raw
6-Oct-10 24.5
14-Oct-10 25 ± 1
29-Nov-10 7-Dec-10 20.8 25 ± 1 4.5 ± 0.9 8.6 ± 0.3 9.1 ± 0.2 19.7 ± 0.3 1.8 ± 0.7 1.2 ± 0.3 5.3 ± 2.6 – 0.4 ± 0.1 – 0.14 ± 0.01 – 1.4 ± 1.0 – 1.2 ± 0.2 17.8 ± 0.8 1.9 0.5 1.2 17.9 6.9 9.0 9.7 2.0 19.7 ± 1.1 29.4 ± 1.8 1.8 ± 0.1 5.8 ± 0.4
9.6 ± 0.8 3.0 ± 1.1 2.4 ± 1.0 1.2 ± 0.1 0.26 ± 0.03 2.0 ± 0.1 2.1 ± 0.6 3.0 2.1 8.3 7.7 21.1 ± 0.4 2.0 ± 0.2
N
F
3.5 ± 0.2 5.7 ± 0.6 8.3 ± 1.0 2.6 ± 0.8 3.3 ± 1.9 0.5 ± 0.1 0.18 ± 0.06 1.4 ± 0.1 1.76 ± 0.02 1.8 1.8 5.3 11.6 20.5 ± 1.0 1.7 ± 0.2
15.6 ± 0.4 2.0 ± 0.1 –a – – – 11.4 ± 1.0 1.0 11.4 3.7 1.0 17.1 ± 1.2 2.7 ± 0.2
Enhanced
Raw
Enhanced
15-Feb-11 15.6
23-Feb-11 25 ± 1 3.2 ± 0.3 5.3 ± 0.2 15.1 ± 0.3 0.7 ± 0.2 – – – – 18.2 ± 0.2 1.1 18.3 10.6 1.9 31.9 ± 1.2 4.8 ± 0.2
Below detection limit. Includes FAMEs initially present in the samples.
Table 2 Composition of lipid extract from ODP raw and enhanced activated sludge. O
Sludge collection date Aeration basin temperature, °C Initial biomass concentration, mg/mL D Biomass concentration, mg/mL Bligh & Dyer extract yield, wt.% of dry biomass PHAs, wt.% of extract Hydrocarbons, ppm (based on weight of extract) Fatty alcohols (from WEs), wt.% of extract Sterols (from SEs), wt.% of extract Free sterols, wt.% of extract TAGs, wt.% of extract FAMEs (from WEs and SEs), wt.% of extractb FAMEs (from TAGs), wt.% of extract FAMEs (from FFAs, MAGs and DAGs), wt.% of extract FAMEs (from PLs), wt.% of extract Total FAMEs yield, wt.% of extract Total FAMEs yield, wt.% of biomass a b
Raw
Enhanced
Raw
Enhanced
Raw
Enhanced
6-Oct-10 26
14-Oct-10 25 ± 1
29-Nov-10 20
7-Dec-10 25 ± 1
15-Feb-11 15
23-Feb-11 25 ± 1
15.7 ± 0.5 3.0 ± 1.5 –a – – – 23.8 ± 0.8 0.7 23.9 12.1 3.3 40.0 ± 1.1 6.3 ± 0.3
4.9 ± 0.5 10.4 ± 0.6 8.1 ± 0.2 18.2 ± 0.7 1.9 ± 0.4 0.52 ± 0.04 2.7 ± 1.1 – 1.1 ± 0.1 – 0.27 ± 0.04 – 1.1 ± 0.2 – 1.7 ± 0.2 21.8 ± 0.2 2.1 0.8 1.7 21.9 18.6 2.8 4.9 4.7 27.3 ± 0.3 30.2 ± 1.2 2.2 ± 0.1 5.5 ± 0.3
4.3 9.9 ± 0.1 6.4 ± 1.3 4.3 ± 1.4 1.3 ± 0.2 0.84 ± 0.01 0.2 ± 0.1 1.0 ± 0.1 1.00 ± 0.01 2.1 1.0 17.7 7.0 27.8 ± 1.1 1.8 ± 0.4
4.0 ± 0.7 10.1 ± 0.3 7.3 ± 0.2 16.4 ± 0.2 3.1 ± 0.5 2.0 ± 0.9 4.2 ± 2.0 – 2.3 ± 1.0 – 0.3 ± 0.1 – 1.5 ± 0.3 – 2.0 ± 0.2 20.1 ± 1.0 3.2 0.3 2.0 20.2 15.6 14.2 5.0 2.3 25.8 ± 1.0 37.0 ± 2.2 1.9 ± 0.1 6.1 ± 0.4
Below detection limit. Includes FAMEs initially present in the samples.
(Revellame et al., 2012). Thus, the presence of PHAs in the enhanced sludges could possibly improve the overall economics of the process. 3.2. Unsaponifiable lipids Results indicated that the speciation of lipidic compounds present in raw sludges were similar regardless of the source. However, the concentrations of these compounds were significantly different between the two plants (Tables 1 and 2). Analyses of the extracts from lipid-enhanced sludges from the two plants showed undetected levels of hydrocarbons, WEs, SEs and free sterols. However, analysis indicated that there were fatty acid methyl esters (FAMEs) (a.k.a. biodiesel) initially present in the samples. This could be accounted from the Bligh & Dyer extraction procedure, where methanol was one of the extraction solvents. During the extraction, the high amount of methanol could have resulted in alcoholysis of lipids present in the samples. This was also observed by previous workers (Revellame et al., 2012).
The undetected levels of hydrocarbons, WEs, SEs and free sterols in lipid-enhanced activated sludges could be accounted to several factors. The most obvious one is the switch in carbon sources. The presence of hydrocarbons in the raw sludges can be attributed to microbially-degraded petroleum residue and are characteristics of petroleum-polluted sediments (Jardé et al., 2005). Since the enhancement of the sludges used glucose as sole carbon source, the input of petroleum products residue was eliminated and thus hydrocarbons were not detected in the samples. The same is true for WEs, SEs and free sterols. The WEs in the raw sludges could be due to possible presence of detergent fatty alcohols in the influent wastewaters of the two plants. Detergent fatty alcohols are considered bioavailable and might have been used by raw activated sludge microorganisms for WE synthesis (Mudge et al., 2008). The input of these detergent fatty alcohols was eliminated during enhancement and thus, WEs were not detected in the lipid-enhanced activated sludges. As for the case of SEs and free sterols, their presence in the raw sludges can be accounted mainly due to anthropogenic contributions, particularly human feces.
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have caused the shift in bacterial population to the ones that can survive under acidic environment. 3.3. Saponifiable lipids 3.3.1. Raw sludges Fig. 2 shows the fatty acid profile of the lipids in the raw sludges. It can be seen that within a plant, the profiles are significantly similar. Between the two plants considered, the fatty acids present ranges from C12:0 to C22:1. However, between the two plants the concentrations of fatty acids were significantly different particularly those of C16s and C18s fatty acids. The huge differences in fatty acid profiles of raw activated sludges from CAS and ODP could be attributed to the absence of primary treatment unit(s) of the ODP. As mentioned (Section 1), without primary treatment, oil and grease in the wastewater could be adsorbed by the biomass and eventually recovered during extraction. 3.3.2. Enhanced sludges Analysis of the lipid-enhanced sludges indicated that 57–67% and 55–73% of the total FAMEs in CAS and ODP, respectively, were coming from TAGs. This was a very significant improvement considering that the FAMEs from TAGs in raw activated sludges ranges about 6–10% and 4–8% for CAS and ODP, respectively (Tables 1 and 2). TAGs are the most attractive feedstock for biodiesel production because of how easily they can be chemically converted to biodiesel. In biodiesel technology, lipid feedstock quality is defined as the inverse of free fatty acid content (Haas and Foglia, 2005). The increase in TAGs content in the enhanced sludges means decrease in its free fatty acid content and thus, an improvement in its quality. On the other hand, however, the yield of total FAMEs (2.7–5.8% and 5.5–6.3% dry sludge weight for enhanced CAS and ODP sludges, respectively) were significantly lower than what previous workers obtained (10.2 ± 2.0% dry sludge weight) at the same cultivation condition (Mondala et al., 2012). This could be due to losses during sample preparation and handling or to inherent variability of raw activated sludge samples within a plant. Nevertheless, the total FAMEs of the enhanced activated sludges are significantly higher than that of the raw activated sludges (1.7–2.0% and 1.8–2.2% dry sludge weight for CAS and ODP sludges, respectively). 50
45
45
30 25 20
12 : C
C
C
:2
18
C
18 :
C
18 :
C
C
16 :
C
14 :
12 :
C
16 :
(a)
0
0
18 :3 20 :0 C 22 To :0 ta l U C2 n k 2: 1 no w ns
0
1
5
0
5
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Cessation of such contributions resulted to undetected level of SEs and free sterols on the resulting enhanced sludge. However, one might ask what happened to these compounds (hydrocarbons, WEs, SEs and free sterols) initially present in the raw sludges, which were used as fermentation seeds. It is highly unlikely that the microorganisms consumed these compounds for growth instead of glucose. After the 7-day fermentation, the glucose concentration in the broth was still above 20 g/L (Mondala et al., 2012), which suggest that these compounds would still be present in the lipid-enhanced activated sludges. However, the enhancement resulted to dilution of these compounds to a level that cannot be detected. Another way to look at this dilution effect is by considering the change in biomass concentration as a result of enhancement. On the average, the biomass of CAS sludges increased by 6.5 mg/mL while that of ODP increased by 10.1 mg/mL (Tables 1 and 2). This indicates that an inert material initially present in the raw sludges will be diluted by 1:7.5 and 1:11.1 for CAS and ODP sludges, respectively. In spite of the above discussion, the ability of raw activated sludge microorganisms to synthesize hydrocarbons, WEs, SEs and free sterols cannot be neglected. Thus, their absence in the lipidenhanced activated sludges could be due to microbial population shift brought about by the changes in carbon source and/or cultivation condition. Recent study indicated that lipid-enhancement of activated sludge could result to significant changes in microbial population. The raw activated sludge contains bacterial population in phyla Proteobacteria [a-/b-/c-/d-/e-Proteobacteria (i.e. Rhodobacterales and Xanthomonadales)], Verrucomicrobia (class Verrucomicrobiae), Bacteriodetes (class Flavobacteria and Sphingobacteria), Firmicutes (class Clostridia) and Actinobacteria. At the end of the 7-day fermentation period, the pH of the broth decreased from 6.50 to 2.00 and 99.5% of bacterial population shifted to a-Proteobacteria. Sequencing showed that these bacteria are similar in characteristics to Acidomonas methanolica, an acidotolerant, gram-negative bacteria (Mondala et al., 2012; Urakami et al., 1989). Acidomonas methanolica have been previously isolated from WWTP (Yamashita et al., 2004). In a related study, Niu and co-workers also observed a shift in bacterial community towards a gram-negative population when activated sludge was applied to treat synthetic wastewater with glucose at 25 °C (Niu et al., 2012). In the present paper, however, the decrease in pH could
(b)
Fig. 2. Fatty acid profile of lipids extracted from raw activated sludge; (a) CAS (b) ODP.
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50
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Fig. 3. Fatty acid profile of lipids extracted from enhanced activated sludge; (a) CAS (b) ODP.
Enhancement of the sludges resulted to homogenization of the lipidic compounds associated with microbial biomass (Tables 1 and 2). This is also evident in the fatty acid profiles of the lipids in enhanced sludges (Fig. 3). Regardless of the source of the raw sludges, the lipid-enhancement resulted to an almost similar fatty acid profile. Important biodiesel properties such as cold flow and cetane number are dictated by its fatty acid profile. Looking at Fig. 2, it is apparent that the biodiesel from raw activated sludges from CAS and ODP have different properties and mixing them together will produce a biodiesel with completely different properties. This is not attractive if the goal is to utilize countrywide generated wastewater sludge for biodiesel production. However, the results of this study indicated that enhancement of activated sludges could be a potential strategy to produce a homogeneous feedstock across the United States with improved lipid content. 4. Conclusion Lipid-enhancement of sludges from two WWTPs with different treatment configurations was conducted to increase the amount of saponifiable lipids in the sludges. Irrespective of the source, results showed increase in the amount of lipids in the enhanced sludges, most of which are TAGs. Furthermore, the fatty acid profiles of the enhanced sludges indicated homogenization of sludges from different WWTPs. Although only two wastewater treatment configurations were considered, this study serves as an initial step in the utilization of existing WWTPs as source of significant amount of lipids for biofuel applications. Acknowledgement This work was funded by the United States Department of Energy, Office of Energy Efficiency and Renewable Energy (Grant No. DE-FG36-06GO86025). References Alvarez, H.M., 2006. Bacterial triacylglycerols. In: Welson, L.T. (Ed.), Triglycerides and Cholesterol Research. Nova Science Publishers, Inc., New York, pp. 159–176. Alvarez, H.M., Pucci, O.H., Steinbüchel, A., 1997. Lipid storage compounds in marine bacteria. Appl. Microbiol. Biotechnol. 47, 132–139.
Alvarez, H.M., Steinbüchel, A., 2002. Triacylglycerols in prokaryotic microorganisms. Appl. Microbiol. Biotechnol. 60, 367–376. Arceivala, S.J., Asolekar, S.R., 2007. Wastewater Treatment for Pollution Control and Reuse, third ed. Tata McGraw-Hill Publishing Co. Ltd., New Delhi. Chipasa, K., Me˛drzycka, K., 2006. Behavior of lipids in biological wastewater treatment processes. J. Ind. Microbiol. Biotechnol. 33, 635–645. Courchesne, N.M.D., Parisien, A., Wang, B., Lan, C.Q., 2009. Enhancement of lipid production using biochemical, genetic and transcription factor engineering approaches. J. Biotechnol. 141, 31–41. 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, 181–187. Haas, M.J., Foglia, T.A., 2005. Alternative feedstocks and technologies for biodiesel production. In: Knothe, G., Krahl, J., Van Gerpen, J.H. (Eds.), The Biodiesel Handbook, first ed. AOCS Press, Champaign, IL, pp. 42–61. Hall, J., Hetrick, M., French, T., Hernandez, R., Donaldson, J., Mondala, A., Holmes, W., 2011. Oil production by a consortium of oleaginous microorganisms grown on primary effluent wastewater. J. Chem. Technol. Biotechnol. 86, 54–60. Jardé, E., Mansuy, L., Faure, P., 2005. Organic markers in the lipidic fraction of sewage sludges. Water Res. 39, 1215–1232. Kosa, M., Ragauskas, A.J., 2011. Lipids from heterotrophic microbes: advances in metabolism research. Trends Biotechnol. 29, 53–61. 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, 1203–1210. Mondala, A.H., Hernandez, R., French, T., McFarland, L., Santo Domingo, J.W., Meckes, M., Ryu, H., Iker, B., 2012. Enhanced lipid and biodiesel production from glucose-fed activated sludge: kinetics and microbial community analysis. AIChE J. 58, 1279–1290. Mudge, S.M., Belanger, S.E., Nielsen, A.M., 2008. Fatty Alcohols: Anthropogenic and Natural Occurrence in the Environment. Royal Society of Chemistry, Cambridge, UK. Niu, C., Geng, J., Ren, H., Ding, L., Xu, K., 2012. The cold adaptability of microorganisms with different carbon source in activated sludge treating synthetical wastewater. Bioresour. Technol. 123, 66–71. Pérez-Elvira, S.I., Nieto Diez, P., Fdz-Polanco, F., 2006. Sludge minimisation technologies. Rev. Environ. Sci. Bio/Technol. 5, 375–398. Ratledge, C., Wynn, J.P., 2002. The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms. Adv. Appl. Microbiol. 51, 1–51. Revellame, E., Hernandez, R., French, W., Holmes, W., Alley, E., 2010. Biodiesel from activated sludge through in situ transesterification. J. Chem. Technol. Biotechnol. 85, 614–620. Revellame, E., Hernandez, R., French, W., Holmes, W., Alley, E., Callahan II, R., 2011. Production of biodiesel from wet activated sludge. J. Chem. Technol. Biotechnol. 86, 61–68. Revellame, E.D., Hernandez, R., French, W., Holmes, W.E., Benson, T.J., Pham, P.J., Forks, A., Callahan II, R., 2012. Lipid storage compounds in raw activated sludge microorganisms for biofuels and oleochemicals production. RSC Adv. 2, 2015– 2031. Seo, Y., Lee, I., Han, J., 2013. Cultivation and lipid production of yeast Cryptococcus curvatus using pretreated waste active sludge supernatant. Bioresour. Technol. 135, 304–308.
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