Research Article pubs.acs.org/journal/ascecg
Biodiesel Profile Stabilization and Microbial Community Selection of Activated Sludge Feeding on Acetic Acid as a Carbon Source Dhan Lord Fortela,† Rafael Hernandez,*,†,‡ Andrei Chistoserdov,§ Mark Zappi,†,‡ Rakesh Bajpai,†,‡ Daniel Gang,∥ Emmanuel Revellame,‡ and William Holmes‡ †
Department of Chemical Engineering, University of Louisiana at Lafayette, Lafayette, Louisiana 70504, United States Energy Institute, University of Louisiana at Lafayette, Lafayette, Louisiana 70504, United States § Department of Biology, University of Louisiana at Lafayette, Lafayette, Louisiana 70504, United States ∥ Department of Civil Engineering, University of Louisiana at Lafayette, Lafayette, Louisiana 70504, United States ‡
ABSTRACT: This work studied the dynamics of the biodiesel profile [as lipid-derived fatty acid methyl esters (FAMEs)] and microbial community diversity in activated sludge microbial consortia during the accumulation of lipid on short chain fatty acid (SCFA) as a carbon source. The model carbon source used was acetic acid buffered with sodium acetate; the nitrogen source was ammonium sulfate, and the feeding was done in fed-batch mode in bioreactors. The significant variability of FAMES in the grab-activated sludge was verified prior to bioreactor runs. Results showed that cultivation on acetic acid stabilized the profile of the FAMEs of activated sludge. This stabilization was likely due to proliferation of budding yeasts as confirmed by fungal diversity analysis. The dominant yeasts in the lipid-enhanced activated sludge were in the genera Williopsis. A confirmation that the quality of biodiesel improves through homogenization of the profile during the accumulation of lipid in activated sludge feeding on acetic acid reinforces the potential of SCFAs as alternative carbon sources in organic waste-to-bioenergy conversion. KEYWORDS: Microbial oil, Fatty acid methyl esters, Short chain fatty acids, DNA analysis, Budding yeasts
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INTRODUCTION The oil content of wastewater-activated sludge microbial consortia can be enhanced by cultivation in a medium with a high carbon-to-nitrogen molar ratio (C/N).1 A C/N of at least 70 induces the accumulation of lipid in activated sludge microorganisms.2 The microbial oil can then be converted to biodiesel, which is characterized as fatty acid methyl esters (FAMEs), via transesterification.3 A key aspect of the commercialization of this technology is the cost of carbon sources, mainly glucose and xylose from biomass hydrolysis, to maintain a high C/N. The findings of a previous study by Fortela et al.4 showed that short chain fatty acids (SCFAs), such as acetic acid, could be alternative carbon sources for enhancing the accumulation of oil in activated sludge. Because SCFAs can be produced by anaerobic digestion of organic wastes, the integration of anaerobic and aerobic digestion could be applied to enhance the accumulation of oil in activated sludge generated by wastewater treatment operations, potentially reducing the cost of sugar production. The consistency of lipid composition, and consequently the operation and quality of biodiesel production, will be essential in the scale-up of this integrated system. The composition of FAMEs derived from microbial oil depends significantly on the species of microorganisms.5 The © XXXX American Chemical Society
microbial diversity of raw activated sludge generated during wastewater treatment operations depends on the concentration of biochemical oxygen demand (BOD), chemical oxygen demand (COD), and many other key nutrients, cofactors, and operational parameters.6 Previous studies by Revellame et al.7 found significant variability in the profile of FAMEs of lipids extracted from activated sludge collected from various wastewater treatment facilities during different seasons of the year. Though variability in the profile of FAMEs of accumulated lipids can be affected by reaction conditions during transesterification as well as the biodiesel refining operations, the homogenization of FAMEs via microbial selection during cultivation on SCFAs will be advantageous. Mondala et al.1 discovered that the microbial diversity of activated sludge can be significantly reduced when the mixed consortium is grown under a relatively high glucose concentration. This suggests the possible influence of the carbon source on microbial selection of activated sludge microorganisms. This study evaluated the impact of acetic acid as a carbon source on the consistency of the profile of FAMEs of lipids that Received: May 25, 2016 Revised: October 10, 2016
A
DOI: 10.1021/acssuschemeng.6b01140 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
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pH-monitoring function of the bioreactors and gel-filled pH probe (405-DPAS-SC pH probe, Mettler Toledo). The working volume for each reactor was 4 L. The medium used for the fed-batch bioreactor experiments is a synthetic wastewater (SWW) based on the recipe used by Mondala et al.2 (per liter of deionized water): 0.15 g of gelatin, 0.21 g of starch, 0.07 g of yeast extract, 0.01 g of casamino acids, 1.5 g of NaH2PO4, 1 g of K2HPO4, and 5 mL of a solution of trace minerals. The solution of trace minerals has the following composition (per liter of deionized water): 0.5 g of ethylenediaminetetraacetic acid (EDTA), 3 g of MgSO4·7H2O, 0.5 g of MnSO4·H2O, 1 g of NaCl, 0.1 g of FeSO4· 7H2O, 0.1 g of anhydrous CaCl2, 0.1 g of ZnSO4·7H2O, and 0.01 g of CuSO4·5H2O. Prior to each culture run, the grab sample of activated sludge was centrifuged to separate the solids. The liquid phase was discarded and replaced with the SWW. The amount of recovered solids from the grab sludge sample was estimated using the volume displacement method to adjust the concentration of the solids of the synthetic wastewater sludge with a set target nominal concentration of solids of 13−14 g/L in all the runs. The SCFA model substrate for all runs was acetic acid, and the source of N was ammonium sulfate added at the start of each run to set the initial C/N at a nominal level of 70. The feeding of acetic acid buffered with equimolar sodium acetate occurred at 12 h intervals. The target nominal acetic acid loading in the bioreactor per feeding was 1.5 g/L. This was done by loading 6 g of acetic acid combined with 8.2 g of sodium acetate per feeding in the 4 L culture. The initial C/N was set by adding 0.19 g of ammonium sulfate to the 4 L culture at the start of cultivation. Each fed-batch setup was run for 120 h. These run conditions were based on a previous work by Fortela et al.4 that observed high biodiesel yields in the bioreactor cultivations that were run under these conditions. The batch (at the start) feeding of the source of N resulted in a high C/N (at least 70) per feeding of the source of C at 12 h intervals. This cultivation mode induces lipid accumulation while minimizing the inhibition effect of the acid source of C.4,8 Chemical Analysis. The target response was the profile of FAMEs, but other parameters were measured to make sure the runs were within the set experimental conditions. The cell biomass concentration was measured gravimetrically. The culture samples were centrifuged at 3000g for 20 min, and then the cell pellets were washed with 20 mL of a 2.5% saline solution and stored in a −80 °C freezer for 24 h. The frozen cell pellets were then freeze-dried for 48 h and the dried solids weighed (FreeZone Freeze-dry Systems, Labconco). The results were reported as the biomass dry mass concentration (grams per liter). Accelerated solvent extraction (ASE) was applied to extract biomassassociated lipids using an automated extractor (ASE 300, Dionex). The solvent system consisted of chloroform and methanol [65% (v/v) chloroform to 35% (v/v) methanol]. The lipid extracts were recovered after the evaporation of chloroform and methanol by purging nitrogen at 60 °C (TurboVap LV, Biotage). Gravimetric lipid yields were determined by weight difference and are reported as the weight percent of biomass cell dry weight (% CDW). The profile of FAMEs of the lipid extracts was determined using a gas chromatography−flame ionization detector (GC−FID) system (Agilent 6890N GC, Agilent Technologies; column, Stabilwax-DA, Restek Co.; carrier gas, helium; FID at 260 °C) as detailed by Mondala et al.2 The lipid FAMEs were produced using H2SO4-catalyzed transesterification of the activated sludge lipid extracts in methanol at 60 °C for 2 h. The mix standard of FAMEs contained C8−C24 methyl esters (Sigma-Aldrich): methyl octanoate (C8:0), methyl decanoate (C10:0), methyl laurate (C12:0), methyl tetradecanoate (C14:0), methyl palmitate (C16:0), methyl palmitoleate (C16:1), methyl octadecanoate (C18:0), methyl cis-9-octadecanoate (C18:1), methyl linoleate (C18:2), methyl linolenate (C18:3), methyl arachidate (C20:0), methyl docosanoate (C22:0), methyl erucate (C22:1), and methyl lignocerate (C24:0). The composition of FAMEs was reported as the weight percent of the esterifiable lipid [% (w/w) esterifiable lipid]. Esterifiable lipid [% (w/w) lipid content] was the total amount of FAMEs.
accumulated in the activated sludge microorganisms. The study tested the hypothesis that growing activated sludge using SCFAs to achieve a high C/N reduces the microbial diversity of the consortium and results in a more specialized microbial mixture for lipid accumulation. The success in enhancing the lipid content of activated sludge by feeding SCFAs at a high C/ N4 is a step forward in the large-scale realization of activated sludge microbial oil technology, but homogeneity of the biodiesel end product must be achieved to avoid additional costs for downstream processing. This could be achieved by the conversion of activated sludge microbes into a more a robust, stable, and consistent consortium for oil accumulation using a selected carbon source.
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MATERIALS AND METHODS
Sample-Activated Sludge. The sampling of activated sludge from the aeration units of WWTPs was designed for two objectives: (1) preliminary screening of the variation of the profile of FAMEs in microbial oil of grab samples as a function of time and processing plant and (2) cultivation under acetic acid fed-batch feeding in bioreactors. The wastewater treatment plants (WWTPs) were in the city of Lafayette, LA. The following types of aerobic biological treatments were used: East WWTP (30°12′59″N 92°00′03″W), oxidation ditch; South WWTP-East Section (30°11′35″N 92°01′29″W), conventional activated sludge; South WWTP-West Section (30°11′35″N 92°01′29″W), conventional activated sludge; and Ambassador Caffery Parkway WWTP (30°09′49″N 92°03′28″W), conventional activated sludge. Table 1 summarizes the sampling of activated sludge. Samples
Table 1. Grab-Activated Sludge Samples from WWTPs in Lafayette, LA sample/run code
source
sampling date
For Objective 1: Preliminary Screening of the Variation of the Profile of FAMEs TP1 East WWTP February 22, 2015 TP2 East WWTP February 28, 2015 TP3 East WWTP March 8, 2015 TP4 East WWTP June 21, 2015 TP5 East WWTP July 2, 2015 TP6 East WWTP July 11, 2015 PP1 East WWTP September 10, 2015 PP2 South WWTP-East Section September 10, 2015 PP3 South WWTP-West Section September 10, 2015 PP4 Ambassador Caffery Parkway September 10, 2015 WWTP For Objective 2: Cultivation under Acetic Acid Fed-Batch Feeding in Bioreactors AS1 East WWTP September 18, 2015 AS2 South WWTP-East Section Octobrt 7, 2015 AS3 Ambassador Caffery Parkway Octobrt 14, 2015 WWTP
TP1−TP6 were taken at various time points from a single WWTP, and samples PP1−PP4 were taken from various plants on the same day. Sample AS1−AS3 were collected specifically for the fed-batch lipid accumulation experiments. Fed-Batch Cultivation. Activated sludges (AS1−AS3) were cultivated in bioreactors to evaluate the changes in the FAMEs and the microbial community profile under aerobic feeding of acetic acid. Fed-batch cultures in the synthetic wastewater were performed in two 5 L bioreactors (BIOFLO 3000, New Brunswick ScientificEppendorf). The following operational conditions were maintained in all the bioreactor runs: temperature of 25 °C, agitation at 200 rpm, and aeration at 1 vvm (volume of air per volume of culture per minute). The pH was not controlled but monitored using the built-in B
DOI: 10.1021/acssuschemeng.6b01140 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
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Figure 1. Profile and correlation of activated sludge grab samples. (a) Profiles of FAMEs of grab-activated sludge samples taken at various periods from a single WWTP. (b) Pairwise correlation coefficient of the profiles of FAMEs of grab-activated sludge samples taken at various periods from a single WWTP. (c) Profiles of FAMEs of grab-activated sludge samples taken from various WWTPs at the same time. (d) Profiles of FAMEs of grabactivated sludge samples taken from various WWTPs at the same time. The concentration of residual acetic acid (grams per liter) was measured using liquid chromatography (Agilent 1100 HPLC system, Agilent Technologies) equipped with an organic acid column (Rezex ROA-Organic Acid; maintained at 40 °C using a double-pipe heat exchanger-water bath system; Phenomenex) and an ultraviolet detector (signal and bandwidth of 210 and 16 nm, respectively; reference and bandwidth of 360 and 100 nm, respectively; Agilent 1100 diode array detector, Agilent Technologies). The eluent phase was 0.005 N H2SO4. Prior to analysis, the liquid samples were filtered through a 0.45 μm syringe filter (PTFE 25 mm, 0.45 μm, Omicron Scientific) and placed in 2 mL amber autosampler vials. The initial concentration of ammonium (milligrams per liter) was measured using an ammonium test kit (TNT 832, Hach Co.) to verify the initial C/N in the fed-batch cultures. Microbial Community Analysis. The profiles of the fungal community in the fed-batch (AS1−AS3) culture samples were determined using DNA sequencing and analysis. DNA samples were extracted from the microbial cultures of the initial (raw) and final (lipid-enhanced) sludge cultures using the PowerSoil DNA Isolation Kit (MoBio Laboratories Inc.). The DNA isolates were sequenced by an external laboratory (MR-DNA Molecular Research LP, Shallowter, TX) using the bTEFAP Illumina 20k ITS1-2 fungal assay. The data from sequencing were processed by the laboratory’s ribosomal and functional gene analysis pipeline, and the final operational taxonomic units (OTUs) were taxonomically classified using BLASTn against a curated database derived from GreenGenes, Ribosomal Database
Project-II (RDPII), and the National Center for Biotechnology Information (NCBI). Statistical Analysis. The pattern similarities of the profiles of FAMEs from the various microbial lipid extracts were evaluated using the pairwise correlation coefficient (r) given by the following formula for the X data set and Y data set pair of variables containing n values: n
r=
∑i = 1 (xi − x ̅ )(yi − y ̅ ) n
n
∑i = 1 (xi − x ̅ )2 ∑i = 1 (yi − y ̅ )2 Σni=1
(1) Σni=1 yi.
xi, and y ̅ = (1/n) A Fisher z where xi ∈ X, yi ∈ Y, x̅ = (1/n) transformation was done on r prior to performing hypothesis testing against the population correlation coefficient (ρ) at a significance level of 0.05.
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RESULTS Variation of FAMEs in Grab-Activated Sludge. The profiles of FAMEs of transesterified lipid extracts from grabactivated sludges are shown in Figure 1. Hypothesis testing on the pairwise profile correlation of FAMEs of samples taken at various times (Figure 1a,b) shows that there is significant temporal variation in the composition of microbial oil and biodiesel from activated sludges sourced from a single WWTP. The samples taken during the spring season significantly differ C
DOI: 10.1021/acssuschemeng.6b01140 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
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3g−i). These patterns indicate that the activated sludge samples acclimated at the initial stage of cultivation. The fluctuations in pH manifest the acid−base equilibrium between acetic acid and acetate ion. As acetic acid was consumed after the initial acclimation period, the amount of hydronium ions decreased, resulting in the shift of some acetate into acetic acid, hence producing some hydroxide ions and, thus, the observed increase in pH. Microbial Community Selection. To evaluate the possible cause of stabilization in the profiles of FAMEs, the microbial diversity of the initial and final cultures was analyzed. A previous study by Fortela et al.4 has suggested that most of the lipid-accumulating cellular bodies at the end of cultivation using acetic acid as the carbon source are within the size range of fungi. Thus, the fungal diversity assay was conducted as part of this study. The phylum-level fungal composition is shown in Figure 5. Even though there was significant diversity in the initial composition of the cultures, the dominant fungal phylum in the enhanced activated sludges at the end of cultivation was Ascomycota. This classification includes the Saccharomycetes (Figure 6), which was ultimately dominated by the genera Pichia, Galactomyces, Cyberlindnera, Candida, Williopsis, Ogatea, and Hyphopichia at the genus level in all three cultures. These belong to the order Saccharomycetales, which are the budding yeasts. These trends show that the microbial community of activated sludge undergoes selection during lipid accumulation by feeding on acetic acid.
from each other, while the samples taken during the summer season (TP4−TP6) correlate almost perfectly (r = 0.99). Hypothesis testing on the pairwise profile correlation of FAMEs of samples taken at various WWTPs at the same time (Figure 1c,d) shows that there is significant variation in the composition of FAMEs with respect to location. Stabilization of the Profile of FAMEs. During the biomass growth and lipid accumulation of activated sludge (Figure 2) by fed-batch feeding of acetic acid (with an
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DISCUSSION The observed significant variations of FAMEs from lipid extracts of grab-activated sludge samples from wastewater aerobic biological treatment units collected at various periods and from different WWTPs imply significant fluctuations in the composition of potential microbial oil and biodiesel from activated sludge sourced from WWTPs (Figure 1). The seasonal variation can be explained by the changes in the activities of communities discharging wastewaters to WWTPs, and the seasonal changes in local temperature. The composition of organic wastes in the wastewater affects the microbial community profile in the aerobic biological treatment units.6 There were also times with no significant variations within a season (TP4−TP6). This stability could be due to the consistency of day-to-day temperature profiles such as those during the summer (Figure 1a,b, TP4−TP6). The variation with respect to the source WWTP can be explained by the differences in the operation of aerobic biological treatments for each WWTP.6 Revellame et al.7 found significant differences in the profiles of FAMEs of activated sludge samples from a conventional activated sludge process and samples from an oxidation ditch process. Stabilization of the profile of FAMEs would be advantageous in the production and recovery of microbial oils and biodiesel from activated sludge. As observed above, stabilization of the profile of FAMEs can be a consequence of enhancing the lipid content of activated sludge by feeding acetic acid at a relatively high C/N (Figure 3). An almost perfect correlation of the profiles of FAMEs occurred after cultivation for 36 h (Figure 4). Selection of the microbial community, specifically yeasts, during cultivation provides an explanation for the stabilization of the profile of FAMEs during cultivation (Figures 5 and 6). The profile of FAMEs of raw activated sludge samples are typically dominated by free fatty acids (FFAs) that significantly vary from one activated sludge sample to another.7 As the lipids, which are
Figure 2. Different activated sludge (AS) samples fed with acetic acid under fed-batch cultivation: (a) biomass concentration, (b) lipid accumulation, and (c) total esterifiable lipid.
equimolar ratio of acetate to buffer), the time-series profiles of FAMEs of the lipid extracts were determined (Figure 3). To statistically measure the similarities of the profiles of FAMEs, hypothesis testing on the pairwise correlation coefficients of the profiles was performed (Figure 4). In general, the profiles of FAMEs of the three activated sludge samples converged to a specific profile at the end of cultivation in the following order [% (w/w) esterifiable lipid in AS1−AS3, respectively]: C18:1 (39, 48, and 47%) > C16:0 (26, 21, and 22%) > C18:2 (10, 12, and 14%) > C18:0 (15, 8, and 5%) > C16:1 (5, 7, and 7%) > C18:3 (1.5, 1.3, and 3%). The samples that started with dissimilar profiles of FAMEs (AS1 and AS2, and AS2 and AS3) gradually changed throughout the cultivation with almost perfect correlation after 36 h (Figure 4a,c). The samples that started with almost similar profiles of FAMEs (Figure 4b, AS1− AS3) approached almost perfect correlation between 12 and 60 h but ended with a correlation similar to that from the beginning. Considerable fractions of lipids were nonesterifiable (Figure 2c). The identification and quantification of these nonesterifiable lipids such as sterols are important in the context of recovery of resources from activated sludge7 but were outside the scope of this study. The consumption of acetic acid (Figure 3d−f) was accompanied by significant changes in the pH profile (Figure D
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Figure 3. Profiles of FAMEs of lipid extracts from activated sludge samples cultivated by fed-batch acetic acid (with an equimolar ratio of acetate to buffer) feeding: (a) AS1, sample from East WWTP; (b) AS2, sample from South WWTP-East Section; (c) AS3, sample from Ambassador Caffery Parkway WWTP; (d) residual acetic acid in AS1 cultivation; (e) residual acetic acid in AS2 cultivation; (f) residual acetic acid in AS3 cultivation; (g) pH in AS1 cultivation; (h) pH in AS2 cultivation; and (i) pH in AS3 cultivation.
mostly triacylglycerols (TAGs),9 accumulate in yeasts, the profile of FAMEs of the accumulated lipids (TAGs) starts to dominate the measured FAMEs as manifested in the homogenization of FAMEs at the end of the cultivation. The dominant FAME from lipids of known oleaginous yeasts (Table 2) is C18:1, which was also the dominant FAME at the end of the cultivation on acetic acid (Figure 3). Therefore, the selection of a fungal community triggered by acetic acid likely resulted in the stabilization of the profile of FAMEs. Selection of the microbial community in activated sludge during lipid accumulation was also observed in bacterial groups cultivated on glucose,1 but the fluorescence micrographs presented in a previous study by Fortela et al.4 strongly suggest that the cellular bodies responsible for lipid (mostly TAGs) accumulation were yeasts. Further studies can be conducted on the species-level identification of the dominant yeasts in the lipid-enhanced acid-fed activated sludge. This can be an opportunity to discover new oleaginous yeasts that can tolerate and use SCFAs as carbon sources for lipid accumulation. Table 2 shows values from the literature of some yeasts that belong to the dominant genera identified in the lipid-enhanced activated sludge in this study. These species were proven to have oleaginous properties.
An example of the dominant Williopsis genus is Williopsis saturnus, which was shown to be tolerant to high concentrations of acetic acid and to low pH levels (3.53).10 On the other hand, most lipid accumulation studies with Candida species have been limited to sugar and alcohol substrates. Lipid studies of Pichia have been focused on Pichia pastoris, which was successfully used as an expression system for the production of recombinant proteins.11 Attention can also be turned to the other dominant genera, Galactomyces, Cyberlindnera, Ogatea, and Hyphopichia, as they are not common subjects of lipid accumulation studies. Their dominance in an acid-fed lipid accumulation culture suggests they could be candidates for oleaginous microbe screening studies. The results from this suggested species-level identification can be useful not only in the area of anaerobically derived SCFAs-to-lipids conversion but also in other biomass conversion applications such as the utilization of lignocellulosic hydrolysate for biofuel production. Other researchers have been working on the screening of oleaginous yeast strains that tolerate lignocellulose degradation compounds. Chen et al.12 found that acetic acid among other organic acids in lignocellulose hydrolysate strongly inhibits Rhodotorula glutinis, Rhodotorula rubra, Rhodosporidium toruloides, Rhodosporidium toruloides, and Lipomyces starkeyi. Acid-resistant oleaginous E
DOI: 10.1021/acssuschemeng.6b01140 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
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Figure 4. Pairwise correlation coefficients of profiles of FAMEs of lipids from the activated sludge samples cultivated on acetic acid in fed-batch mode.
Figure 5. Relative abundance of activated sludge fungi at the phylum level in the culture samples before (raw) and after (enhanced) lipid accumulation.
study. The work of Mondala et al.2 showed that xylose can also be used as a carbon source for the lipid accumulation of activated sludge while achieving the same lipid enhancement performance as glucose (final lipid content of ∼17−22% CDW). The study also revealed the dominance of C18:1 fatty acid at the end of cultivation [∼45% (w/w) esterifiable lipid], and homogenization of the profile of FAMEs of the lipid extracts at the end of cultivation. A recent work by Yook et al.16 demonstrated that food wastewater (21 g of glucose/L, 8.2 g of acetic acid/L, 20.9 g of lactic acid/L, and 10.9 g of mannitol/L) can induce lipid enhancement in activated sludge (∼15−25% CDW). Though activated sludge lipid enhancement studies have been limited to the carbon sources discussed (acetic acid, glucose, xylose, and food wastewater), many other carbon sources may be feasible because of the presence of various
yeasts in activated sludge can be alternative yeast cultures for lignocellulose hydrolysate utilization. The observed stabilization of FAMEs in this study agrees with the results of previous works that used other carbon sources for activated sludge lipid accumulation. Revellame et al.7 used glucose as a carbon source for the bioreactor cultivation of activated sludges from an oxidation ditch and a conventional aeration chamber in wastewater treatment facilities. In addition to the observed enhancement of lipid contents (∼15−20% CDW), the profiles of FAMEs of the extracted lipids were homogenized from a highly varied initial profile of FAMEs of the two sludge types. The dominant fatty acid in the lipid-derived FAMEs in the final activated sludges was C18:1 [∼40−45% (w/w) esterifiable lipid], which was also the dominant fatty acid in the final cultivated sludges in this F
DOI: 10.1021/acssuschemeng.6b01140 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
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Figure 6. Taxonomic tree of dominant fungal classifications in the lipid-enhanced activated sludge cultures.
The idea of using acetic acid as the model carbon source in this study was motivated by the potential of integrating anaerobic production of SCFAs (dominated by acetic acid) from organic wastes with the aerobic lipid accumulation of activated sludge feeding on the waste-derived SCFAs.8 The anaerobic production of SCFAs is a treatment process for the disposal of organic wastes, typically optimized for producing biogas. This in situ production of SCFAs makes the proposed integration economically attractive. Previous studies reported that production of SCFAs from food wastes can be as low as US $0.60/kg of SCFAs when government subsidies are applied (in Korea in 2011).18,19 An extensive cost analysis for the lipid accumulation from SCFAs by activated sludge is outside the scope of this investigation. However, cost analyses of systems operating on pure culture oleaginous microorganisms may provide insights into activated sludge lipid enhancement technology. Park and colleagues estimated that the production of lipids by Cryptococcus albidus feeding on food waste-derived SCFAs costs US$1.05/kg of lipid,18 which is less costly than soybean oil, castor oil, and microbial lipid produced by C. albidus feeding on glucose.19 Other investigators have demonstrated that the cost of extraction of lipid from microorganisms contributes significantly to the cost of biodiesel generated from activated sludge. Dufreche et al.20 studied in situ transesterification of activated sludge lipids to biodiesel, which is a process that eliminates the lipid extraction step prior to transesterification, hence intensifying the biodiesel production stage and aiming to reduce costs. This study found that the intensified process costs US$3.11/gal of biodiesel (based on year 2007 prices). A comprehensive techno-economic analysis of the integrated organic wastes-to-SCFAs-to-biodiesel system would be a valuable addition to the literature. Our findings show that even with variations in the profiles of FAMEs from lipids of raw activated sludge, the enhancement of lipid content by feeding on acetic acid induced selection of a microbial community, specifically fungi, resulting in stabilization of the profile of FAMEs. The resulting fungal community was dominated by budding yeasts, most of which belong to genera of some established oleaginous yeasts. These findings could have practical significance in the scale-up of activated sludge microbial oil technology operating on a platform of SCFAs.
Table 2. Lipid Content and Fatty Acid Profiles of Select Oleaginous Yeasts FAME relative % (w/w) yeast species Candida curvata13 Candida diddensii14 Candida guilliermondii15 W. saturnusa,14 a
lipid content [% (w/w)]
16:0
16:1
18:0
18:1
18:2
18:3
58
32
−
15
44
8
−
37
19
3
5
45
17
5
61
22
13
6
58
2
−
28
16
16
−
45
16
5
5
Previously known as Hansenula saturnus.
yeasts, molds, and bacteria in activated sludge. Other works that focused on pure cultures of oleaginous microorganisms showed that the following carbon sources can be used: (1) glycerol, whey, ethanol, and starch by yeasts, (2) cellulose, glycerol, and fruit peelings by molds, and (3) molasses, valerate, and gluconate by bacteria.17 The consumption profile of acetic acid (Figure 3) demonstrates a positive effect of lipid accumulation. It addresses the concern about the COD increase of the spent media relative to the initial due to the loading of the carbon source. Though the residual level of acetic acid in the first two runs (AS1 and AS2) did not reach zero, the level of the third set of runs (AS3) reached zero starting at 36 h, a pattern also observed in a previous work by Fortela et al.4 that employed the same operating conditions on the feeding of acetic acid. Therefore, the lipid accumulation of activated sludge feeding on acetic acid could consume almost all of the acetic acid (COD) added. The ratio of organics fed via acetic acid and acetate ion (expressed as acetic acid and summed together) to the lipid and biodiesel produced was calculated and expressed as the mass ratio. The ratio values for AS1−AS3 cultivations were 27.3, 31.1, and 23.0 g of acetic acid/g of lipid, respectively, and 92.0, 120.3, and 103.5 g of acetic acid/g of biodiesel, respectively. These values indicate an improvement upon lipid enhancement in activated sludge. A work by Mondala et al.2 using glucose as the source of C achieved a conversion of around 100−140 g of glucose fed/g of lipid produced (values were calculated using the results of the study). G
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(17) Kosa, M.; Ragauskas, A. J. Lipids from heterotrophic microbes: advances in metabolism research. Trends Biotechnol. 2011, 29 (2), 53− 61. (18) Park, G. W.; Fei, Q.; Jung, K.; Chang, H. N.; Kim, Y.-C.; Kim, N.-j.; Choi, J.-d.-r.; Kim, S.; Cho, J. Volatile fatty acids derived from waste organics provide an economical carbon source for microbial lipids/biodiesel production. Biotechnol. J. 2014, 9 (12), 1536−1546. (19) Fei, Q.; Chang, H. N.; Shang, L.; Choi, J.-d.-r.; Kim, N.; Kang, J. The effect of volatile fatty acids as a sole carbon source on lipid accumulation by Cryptococcus albidus for biodiesel production. Bioresour. Technol. 2011, 102 (3), 2695−2701. (20) Dufreche, S.; Hernandez, R.; French, T.; Sparks, D.; Zappi, M.; Alley, E. Extraction of lipids from municipal wastewater plant microorganisms for production of biodiesel. J. Am. Oil Chem. Soc. 2007, 84 (2), 181−187.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Telephone: (+1)-337-4826062. Notes
The authors declare no competing financial interest.
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REFERENCES
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DOI: 10.1021/acssuschemeng.6b01140 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX
