Waste Management xxx (2015) xxx–xxx

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Organic fraction of municipal solid waste as a suitable feedstock for the production of lipid by oleaginous yeast Cryptococcus aerius Hossein Ghanavati a, Iraj Nahvi a,⇑, Keikhosro Karimi b,c a

Department of Microbiology, Faculty of Sciences, University of Isfahan, Isfahan, Iran Department of Chemical Engineering, Isfahan University of Technology, Isfahan 84156-83111, Iran c Industrial Biotechnology Group, Institute of Biotechnology and Bioengineering, Isfahan University of Technology, Isfahan 84156-83111, Iran b

a r t i c l e

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Article history: Received 24 July 2014 Accepted 9 December 2014 Available online xxxx Keywords: Organic fraction of municipal solid waste Dilute acid pre-hydrolysate Enzymatic hydrolysate Detoxification Yeast fermentation Lipid

a b s t r a c t The detoxified pre-hydrolysate and enzymatic hydrolysate of OFMSW were used as substrates for lipid production by Cryptococcus aerius. Factorial experimental designs were employed for the optimization of dilute acid pre-hydrolysis, detoxification by over-liming, enzymatic hydrolysis, and lipid production. OFMSW pre-hydrolysis with 3% H2SO4 for 45 min was found to be the optimal treatment, resulted in total sugar concentration of 65.5 g/L (32.8% yield, based on grams of total reducing sugar per gram of OFMSW). The optimal detoxification conditions of the pre-hydrolysate by over-liming was incubation at 30 °C and pH 11 for 24 h, resulted in the reduction of total nitrogen, total phenolic compounds, and furans by 51.3%, 45.1%, and 100%, respectively. The residual solid was subjected to enzymatic hydrolysis, and the highest sugar concentration of 30.5 g/L was obtained. At optimal conditions, the yeast cultivation on the detoxified pre-hydrolysate and enzymatic hydrolysate resulted in the lipid production of 3.9 g/L (12.8% yield, based on g lipid per g consumed sugar) and 4.3 g/L (17.1% yield, based on g lipid per g consumed sugar), respectively. The elemental analysis showed the presence of heavy metals including iron (925 mg/l), zinc (59 mg/l), lead (4.7 mg/l), and nickel (3.5 mg/l) in the pre-hydrolysate, which were significantly reduced by the over-liming detoxification. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Nowadays, it is necessary to find alternative ways to meet increasing energy demands. Currently, biodiesel is one of the most attractive alternatives for fossil fuels, because of its unique characteristics, i.e., nontoxicity, biodegradability, and greenhouse gas emissions (Tao et al., 2010; Tsigie et al., 2012; Zhao et al., 2012). However, its development and application have been hindered by low availability and high price of feedstocks. Nowadays, main feedstocks are oil crops, waste cooking oil, and animal fat (Alptekin et al., 2014) that practically cannot provide even a small portion of existing demand for transport fuels. Furthermore, the use of traditional raw materials, e.g., vegetable oils, for biodiesel Abbreviations: MSW, municipal solid waste; OFMSW, organic fraction of MSW; ROFMSW, retained solid of OFMSW; NPH, non-detoxified pre-hydrolysate; DPH, detoxified pre-hydrolysate; TRS, total reducing sugars; TPC, total phenolic compounds; HMF, hydroxymethylfurfural; DCW, dry cell weight; OL, over-liming; pHOL, pH in detoxification process by OL; DOL, duration time in detoxification by OL; TOL, temperature in detoxification by OL. ⇑ Corresponding author. Tel.: +98 9132709633; fax: +98 3137932456. E-mail address: [email protected] (I. Nahvi).

production results in the shortage of edible oils and leads to the food versus energy conflict (Yousuf, 2012). Therefore, there is an increasing interest in exploring new and economically feasible sources of lipids for biodiesel production. Lipids production through fermentation using oleaginous microorganisms, which are able to produce considerable amounts of lipids (higher than 20% of their weight), are suggested as an alternative to crop-based oils (Tsigie et al., 2012). Microbial lipid, namely single cell oil (SCO), produced by oleaginous microorganisms, is considered as a promising feedstock for renewable diesel production due to several advantages, e.g., the accumulation of large quantities of lipids, short life cycle, less labor required, independent of season and climate, easier to scale-up, and non-arable land usage (Huang et al., 2011; Yousuf, 2012; Zhao et al., 2012). In addition, the fatty acid profile of microbial oils is quite similar to that of conventional vegetable oils (Hu et al., 2009; Huang et al., 2012; Yu et al., 2011). At present, the high fermentation cost of SCOs, in which 70–85% belongs to the raw material, is the major obstacle for its industrial development and applications (Tao et al., 2010), making the microbial oils less economically competitive (Tsigie et al., 2012).

http://dx.doi.org/10.1016/j.wasman.2014.12.007 0956-053X/Ó 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Ghanavati, H., et al. Organic fraction of municipal solid waste as a suitable feedstock for the production of lipid by oleaginous yeast Cryptococcus aerius. Waste Management (2015), http://dx.doi.org/10.1016/j.wasman.2014.12.007

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H. Ghanavati et al. / Waste Management xxx (2015) xxx–xxx

MSW typically refers to the mixture of wastes collected in municipal areas generated by household such as vegetable and meat scraps, food and cooking wastes, paper and garden wastes, and also inorganic materials such as plastics, glasses, and metals, which is sent to landfills, incinerators, or recycling facilities. The US Environmental Protection Agency reported the generation of 251 million tons of MSW in 2012, of which 160 million tons were potential cellulosic biofuel feedstocks. In Iran, more than 7.2 million tons of MSW is generated annually, whereas the daily production of MSW in Tehran (Iran) is about 8000 tons (Mahmoodabadi et al., 2010). MSW has a nearly-zero price and a low transportation cost as a feedstock (Jones et al., 2007). The OFMSW have been employed as substrates for the production of some bio-products such as lactic acid (Ohkouchi and Inoue, 2007), single cell protein, poly bhydroxybutyrate, organic acids (Vasudevan et al., 2005), biogas (Davidsson et al., 2007), and bioethanol (Kim et al., 2011). Using OFMSW as a low-cost substrate for SCO production would be economically and environmentally attractive. However, a major part of MSW is lignocelluloses that are very resistant to bioconversions. Pretreatment plays an important role in the efficiency of lignocelluloses conversion into bioproducts by fermentation. Different processes, such as physical, physico-chemical, chemical, and biological, have been employed for the pretreatment of lignocellulosic materials (Sun and Cheng, 2002; Taherzadeh and Karimi, 2007a). The pretreatment process is designed to initiate the breakdown of the biomass structure, partially hydrolyze the carbohydrate polymers and making them accessible to enzymatic attack, and increase the rate of enzymatic hydrolysis (Sun and Cheng, 2002, 2005; Taherzadeh and Karimi, 2008). Lignocellulosic biomass, mainly consists of cellulose, hemicelluloses, and lignin and could be hydrolyzed to monosaccharides including xylose, glucose, and arabinose that are suitable carbon sources for microbial lipid production (Zhang et al., 2011; Zhao et al., 2012). In addition, other non-lignocellulosic compounds, e.g., lipids and proteins, can be hydrolyzed and used by microorganisms. Among various chemical pretreatment methods, dilute acid hydrolysis has been proven to be a fast and inexpensive method for producing sugars from lignocellulosic biomass (Sun and Cheng, 2005; Taherzadeh and Karimi, 2008; Zhao et al., 2012). This method can be used either as a pretreatment preceding enzymatic hydrolysis as well as the actual method of hydrolyzing a high portion of hemicellulose to the monomeric sugars. Thus, this method can have a highly positive effect on the overall process economy of bio-products production from lignocellulosic materials (Taherzadeh and Karimi, 2007a). Nevertheless, one problem associated with dilute acid hydrolysis is the formation of inhibitory compounds, which mainly include furfural, HMF, organic acids, phenolic compounds (Hu et al., 2009; Huang et al., 2011; Tao et al., 2010; Yu et al., 2011; Zhang et al., 2011; Zhao et al., 2012), and heavy metal ions (Mussatto and Roberto, 2004). The formation of these compounds is a major problem limiting the bioconversion processes of the hydrolysates. Thus, a detoxification process was suggested to remove these inhibitors from the pretreated lignocellulosic materials (Huang et al., 2011). The identification of these compounds and choice of the best detoxification method are important for improving the efficiency of fermentative processes. This detoxification method should be inexpensive, easy to integrate into the process, and able to remove the inhibitors. Several detoxification methods such as physical, chemical, and biological techniques that have been proposed to reduce the concentration of toxic compounds (Taherzadeh and Karimi, 2007a, 2011). Among the chemical methods, over-liming (OL) stands out because lime is a low-cost material with a high capacity to remove compounds. Cellulase enzymes, that are highly specific catalysts, carry out the enzymatic hydrolysis of cellulose to glucose. Compared with acid hydrolysis, enzymatic hydrolysis is milder and more specific;

however, it requires a pretreatment to improve the enzymatic digestibility. The main advantages of enzymatic hydrolysis are low corrosion problems, low utility consumption, and low toxicity of the hydrolysates (Edama et al., 2014; Taherzadeh and Karimi, 2007b). The aim of this study is to test an inexpensive and renewable raw material (OFMSW) for yeast oil production. To the best of our knowledge, this is the first report on lipid production from OFMSW hydrolysates using yeast fermentation. Over-liming was used for the removal of inhibitors in OFMSW dilute acid prehydrolysate. Dilute acid pre-hydrolysis, enzymatic hydrolysis, OL detoxification, and lipid production by yeast fermentation were conducted and optimized. 2. Material and methods 2.1. Preparation of OFMSW OFMSW was collected in the fall 2012 from MSW at a compost plant of Isfahan, Iran. All MSW of Isfahan city (Iran) is collected in this plant with the capacity of around 1200 tons per day. This MSW was composed of different materials including organic matter (e.g., kitchen and yard wastes), plastics, glasses, and metals. Inorganic matter was separated from MSW. Separation part of the processing performed in the compost plant by both mechanical equipments (shredder, magnet separator and drum sieves) and manually (plastics); the residual impurities were separated manually for more purification of organic matter. In this study, the organic matter was air-dried, milled, and screened through a 2 mm sieve. Then the prepared OFMSW was sealed in plastic bags and stored at room temperature for further use. 2.2. Yeast strain and media The yeast strain Cryptococcus aerius UIMC65 (KC137267.1, GenBank) was recently isolated in our laboratory and was used in the present work (Ghanavati et al., 2014). The yeast was maintained on slants of YPD agar (20 g/L yeast extract, 20 g/L peptone, 20 g/L glucose) at 4 °C. For activation, the yeast strain was streaked on new plates of YPD agar and incubated at 28 °C for 48 h. Then one loop of the grown colonies was inoculated into 250 ml Erlenmeyer flask containing 50 ml of enrichment medium (g/L): glucose (10), xylose (10), (NH4)2SO4 (3), KH2PO4 (1), MgSO4.7H2O (0.5), yeast extract (1), and peptone (1) (pH 5.5). The flask was incubated in a shaking incubator (180 rpm) at 28 °C for 48 h. Finally, the resultant cell pellet was inoculated (5% or 10% or 15% v/v) in 250 ml Erlenmeyer flasks containing 70 ml of autoclaved (110 °C for 10 min) prehydrolysate, detoxified pre-hydrolysate, or hydrolysate supplemented with (g/L): KH2PO4 (2.4), K2HPO4 (0.6) MgSO4
7H2O (0.5), and yeast extract (1) (pH 5.5). Then the media was incubated in a shaking incubator (180 rpm) at 28 °C. Medium without inoculation were used as controls for each sample in all experiments. 2.3. Dilute acid pre-hydrolysis A factorial experimental design was used to investigate the effect of the most effective parameters in the pre-hydrolysis process i.e., acid concentration and residence time. Sulfuric acid concentrations of 1%, 2%, 3%, 4% and 5% (v/v) were examined for 15, 30, 45 and 60 min at 121 °C. Therefore, twenty combinations were examined. In all treatments, OFMSW was suspended and stirred at room temperature in dilute sulfuric acid solution at a solid loading of 20% (w/ v). After the pre-hydrolysis, the liquid fraction was separated by centrifugation (5000 rpm for 10 min) and vacuum filtration, and stored at 4 °C prior to use. The pH of all NPHs was less than 2.

Please cite this article in press as: Ghanavati, H., et al. Organic fraction of municipal solid waste as a suitable feedstock for the production of lipid by oleaginous yeast Cryptococcus aerius. Waste Management (2015), http://dx.doi.org/10.1016/j.wasman.2014.12.007

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2.4. Detoxification of NPH A full-factorial design was employed for obtaining the best conditions of detoxification by OL. The lime powder was added to the NPH with continuous stirring using a heater stirrer until the pH of NPH reach to 10, 11, or 12. Then the pH was held at the adjusted level for different periods of time (0, 1, and 24 h) at two different temperatures (30 and 60 °C). The treatments were run in two blocks in a shaker incubator at 180 rpm. Afterwards, NPHs pH was adjusted to 5.5 using concentrated sulfuric acid (1 M). Centrifugation (3000 rpm for 10 min) followed by 0.22 lm filtration was used to remove any precipitate formed before and after the pH adjustment. The DPHs were used for yeast cultivation immediately. 2.5. Enzymatic hydrolysis The pretreatment step had two major outputs, a liquor and a solid residue. The ROFMSW recovered after sulfuric acid pretreatment, was washed with distilled water until it was neutralized (pH 7) and subjected to enzymatic hydrolysis using cellulose (Celluclast 1.5 L, Novozyme, Denmark) and b-Glucosidase (Novozym 188, Novozyme, Denmark). The cellulase activity (52.5 FPU/ml) was determined according to the method provided by (Adney and Baker, 1996). b-glucosidase activity (240 IU/ml) was measured using by the method presented by Ximenes et al. (1996). The factors that affect the enzymatic hydrolysis of cellulose include substrate concentration, cellulase activity, the ratio of enzyme to substrate, and reaction conditions (Sun and Cheng, 2002; Taherzadeh and Karimi, 2007b). Therefore, the optimization of hydrolysis was carried out at two solid loadings (5% and 10% (w/ v)) in sodium citrate buffer (0.05 M, pH 4.8), two enzymes’ loadings (20 FPU of cellulase and 40 IU of b-glucosidase per g of solid residue (named X), and twice this amount (named 2X)) with or without the presence of tween 20 (0.2 g/L) as a surface active additive. Sodium azide (0.3% (w/v)) was also added as an antibacterial agent. Enzymatic hydrolysis of the pretreated biomass was carried out in 100 ml Erlenmeyer flasks (containing 20 ml solution) in a rotary shaker at 150 rpm and 50 °C for 72 h. At different times (12, 24, 48, 72 and 96 h), one ml of sample was taken and centrifuged, and the supernatant were analyzed for their sugar content. The enzymatic hydrolysis of untreated biomass (the enzymes and substrate blanks) was conducted as a control. The enzymatic hydrolysate with the highest concentration of total reducing sugars was supplemented with the required nutrients (mentioned in Section 2.2), autoclaved (110 °C for 10 min) and subjected to the yeast cultivation process for lipid production. 2.6. Analytical methods DCW was measured according to the method described by Pan et al. (2009). The residual sugar concentration was detected by spectrophotometric method with 3,5-dinitrosalicylic acid (DNS) reagent (Miller, 1959). The nitrogen content of liquids was measured by Kjeldahl and automatic distillation analyzer (Gerhardt, Vap30) according to Clesceri et al. (1998). The amount of carbon, hydrogen, nitrogen, and sulfur was measured by a CHNS analyzer (LECO). The amount of metal ions was analyzed by atomic adsorption spectrophotometer (Perkin Elmer) (Jahanshah et al., 2013). Sulfate was detected by turbidimetric method using barium chloride as a precipitant reagent (Clesceri et al., 1998). Total phenolic content was determined by Folin Ciocalteu reagent method according to Škerget et al. (2005). The glucan, xylan, galactan, acid soluble lignin, and acid insoluble lignin in biomass were analyzed according to the method presented by Sluiter et al. (2008). Protein contents were calculated with total nitrogen contents analysis and multiplication by 6.25 (Ohkouchi and Inoue, 2007).

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Cells lipid was extracted, dried, and weighed according to the method described by Ghanavati et al. (2014). The lipids were transesterified to fatty acid methyl esters (FAME) by reacting with methanol having 1% (v/v) H2SO4 as a catalyst at 50 °C overnight. After neutralization with KHCO3, the FAMEs were extracted out by n-hexane (Christie, 1993) and analyzed by gas chromatography (GC) (Clarus 500, PerkinElmer). The GC was equipped with HP-5 capillary column (30 m  0.32 mm  0.25 lm film thickness) and a flame ionization detector (FID). Helium served as a carried gas by the flow rate of 35 ml/min and airflow of 400 ml/min. The oven temperature was programmed with an initial temperature of 60 °C held for 3 min followed by an increase of 20 °C/min to a final temperature of 250 °C. The injector temperature was held constant at 250 °C. One microliter of sample was injected with a split-less injector. Standards of saturated and unsaturated fatty acids were used for the identification and quantification of fatty acids in the lipid. Furfural and HMF were analyzed using HPLC equipped with a Perfectsil target C18 column (250  4.6 mm, 5 lm) and a UV detector. The sample was filtered through a 0.22 lm PVDF syringe filter before injection to remove impurities. The column temperature was 25 °C, and the mobile phase was acetonitrile:water (10:90) with the flow rate of 0.6 ml/min. The concentrations of D-glucose, D-xylose, D-galactose, and L-arabinose in the hydrolysates samples were determined by HPLC equipped with TSK gelamide-80 column (150  4.6 mm, 3 lm) and refractive index detector. The sample was filtered through a 0.22 lm PVDF syringe filter to remove impurities and injected into the HPLC. The mobile phase was acetonitrile:water (80:20) with flow rate of 0.6 ml/min. 2.7. Statistical analyses All experiments in this work were performed in duplicate, and mean values are presented. Experimental data were statistically analyzed using general linear model (GLM) procedure by SPSS 19 software to perform an analysis of variance (ANOVA) to identify the significant effects of factors as well as their interactions statistically. The data was adjusted using Duncan’s adjustment, and significant differences were evaluated at p-value of less than 5%. The homogeneity of variance (Levene’stest) and lack of fit were checked for all experimental data. 3. Results and discussion 3.1. Chemical composition of OFMSW OFMSW is a very complex and heterogeneous mixture in nature. Table 1 shows the chemical composition of OFMSW. As the compositional analyses show, the major part of this mixture is composed of chemicals that can be converted to utilizable sugars for yeast. Glucan (29.8% w/w) and xylan (20.9% w/w) are the dominant compounds of OFMSW. In addition, the OFMSW contained appreciable amounts of protein (6.75% w/w) and fat (1.3% w/w). As it shown in Table 1, the OFMSW contains a wide range of elements. The major elements detected were nitrogen, phosphorous, potassium, calcium, and magnesium with the contribution of 1.1%, 0.4%, 1.3%, 4.6% and 0.31%, respectively. 3.2. Dilute acid pre-hydrolysis 3.2.1. Pre-hydrolysis optimization The concentration of TRS, nitrogen, TPC, and furans (HMF and furfural) were measured in all NPHs (Table 2). According to the results, TRS and sugar yield obtained in the range of 23.4–65.6 g/L and 11.7–32.8% TRS/OFMSW, respectively. The difference of

Please cite this article in press as: Ghanavati, H., et al. Organic fraction of municipal solid waste as a suitable feedstock for the production of lipid by oleaginous yeast Cryptococcus aerius. Waste Management (2015), http://dx.doi.org/10.1016/j.wasman.2014.12.007

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Table 1 The composition of OFMSW and ROFMSW. Composition

Unit

OFMSW

ROFMSW

Glucan Xylan Galactan Arabinan Lignin (acid soluble) Lignin (acid insoluble) Ash Protein Fat Other extractives C H N S P K Ca Na Mg Mn Zn Fe Cu Pb Ni Cr Cd

%, w/w %, w/w %, w/w %, w/w %, w/w %, w/w %, w/w %, w/w %, w/w %, w/w %, w/w %, w/w %, w/w %, w/w %, w/w %, w/w %, w/w mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg

29.8 ± 0.2 20.9 ± 0.1 0.5 ± 0.1 0.3 ± 0.1 19.9 ± 0.2 6.4 ± 0.1 11.7 ± 0.2 6.8 ± 0.1 1.3 ± 0.3 2.4 ± 0.1 32.7 ± 0.5 5.1 ± 0.1 1.1 ± 0.0 0.2 ± 0.0 0.4 ± 0.2 1.3 ± 0.2 4.6 ± 0.6 5830 ± 420 3160 ± 180 150 ± 31 331 ± 52 5240 ± 220 162 ± 35 148 ± 17 24 ± 4 7±4 2±1

49.1 ± 1.1 4.7 ± 0.1 0 0 2.4 ± 0.1 27.8 ± 0.5 14.2 ± 0.4 1.6 ± 0.5 0 0.18 ± 0.0 43.4 ± 0.2 6 ± 0.0 0.3 ± 0.1 0.6 ± 0.0 0.2 ± 0.1 0.1 ± 0.0 0.3 ± 0.0 301 ± 40 250 ± 54 28 ± 11 28 ± 1 1225 ± 95 55 ± 30 97 ± 11 10.2 ± 4 4±1 1±1

sugar yield between minimum and maximum levels (21.1%) could justifies using an optimizing design for the dilute acid pre-hydrolysis process. Obtaining the highest sugar yield is the aim of prehydrolysis optimization design. The highest sugar concentration (65.6 g/L) and sugar yield (32.8% TRS/OFMSW) were obtained after a pretreatment at 3% acid for 45 min, and the acquired liquor obtained at this condition was subjected to detoxification for better fermentation. ANOVA outputs confirmed that the selected optimal condition had a statistically significant difference with other treatments. In addition, the significant effect of each factor (time and acid concentration) as well as the interaction terms on the prehydrolysis was followed. According to the model, time and acid concentration have significant effect on dependent variations of TRS (R2 = 0.99), TPC (R2 = 0.69), HMF (R2 = 0.88), and furfural (R2 = 0.94), but not on nitrogen (R2 = 0.22). Pre-hydrolysis of biomass at 121 °C were employed in some other researches (Sun and Cheng, 2005; Tsigie et al., 2011).

Optimized conditions of sugarcane bagasse hydrolysis (121 °C for 45 min using 2.5% acid) resulted in the maximum total sugar concentration of about 21.4 g/L obtained by Tsigie et al. (2011). Although the OFMSW is more complex than bagasse in structure, similar optimal condition for hydrolysis was obtained; however, higher concentration of TRS was obtained by OFMSW as a substrate in the present work. 3.2.2. Chemical composition of ROFMSW The solid residue of dilute acid pretreatment of MSW (ROFMSW) was analyzed, and its chemical composition is reported in Table 1. Compared with OFMSW, ROFMSW contained higher amount of glucan (which was the dominant compound in its structure), accompanying with lower portion of xylan, acid soluble lignin, and protein. In addition, fat was not detected in the ROFMSW structure. The presence of elements and heavy metals (iron, zinc, copper, manganese, lead, chromium, cadmium, and nickel) in ROFMSW structure was significantly lower than those in OFMSW. Nitrogen, phosphorous, potassium, calcium, and magnesium content in the ROFMSW were reduced by 45.4%, 63.6%, 96.2%, 94.3% and 92.1%, respectively. The highest reduction of heavy metals in the ROFMSW occurred in zinc (91.5%), manganese (81.3%), and iron (76.6%). They were released to NPH during the dilute acid pre-hydrolysis, making some inhibitory effects on the yeast fermentation. 3.3. Detoxification of the NPH The yeast strain was able to grow on NPHs; however, this substrate was not suitable for high lipid production by the yeast due to the presence of high concentration of several inhibitors. Therefore, it was necessary to reduce the concentration of toxic compounds by a detoxification method. OL procedure is one of the efficient methods for decreasing the inhibitors in acid hydrolysates (Huang et al., 2012; Liang et al., 2012; Millati et al., 2002; Yu et al., 2011). It was used in the present study for reducing the inhibitors in NDH. 3.3.1. Optimization of the detoxification by over-liming For obtaining the best conditions of the detoxification by OL, the most influencing parameters, i.e., pH (pHOL), duration (DOL), and temperature (TOL) (Millati et al., 2002), were optimized. The NPH obtained at the optimal pre-hydrolysis conditions (using 3% acid

Table 2 The dilute acid pre-hydrolysis conditions and the concentration of TRS, nitrogen, TPC and furans (HMF and furfural) in NPHs. H2SO4 (%, v/v)

Time (min)

TRS (g/L)

Sugar yield (% TRS/OFMSW)

Nitrogen (g/L)

TPC (g/L)

Furfural (g/L)

HMF (g/L)

1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5

15 15 15 15 15 30 30 30 30 30 45 45 45 45 45 60 60 60 60 60

25.4 44.2 56.6 58.2 55.9 24.7 48.5 58.5 60.3 59.3 23.4 50.7 65.6 59.5 56.5 27.8 53.7 58.2 57.6 56.6

12.7 22.1 28.3 29.1 28.0 12.3 24.3 29.3 30.2 29.6 11.7 25.4 32.8 29.7 28.3 13.9 26.9 29.1 28.8 28.3

3.11 3.25 3.28 3.41 3.38 3.55 3.87 3.98 4.04 3.96 3.40 3.51 3.88 3.92 3.85 3.66 3.70 3.82 3.74 3.89

0.55 0.77 0.87 0.83 0.90 0.57 0.83 0.88 1.05 1.05 0.54 0.80 0.91 0.96 1.02 0.59 0.79 0.96 1.02 1.02

0.00 0.02 0.04 0.08 0.10 0.00 0.03 0.07 0.09 0.16 0.00 0.03 0.08 0.13 0.28 0.01 0.05 0.11 0.12 0.30

0.59 0.77 1.09 1.00 0.94 0.79 0.91 1.25 1.21 1.14 0.89 1.42 1.77 1.69 1.43 0.83 1.39 1.60 1.51 1.40

Please cite this article in press as: Ghanavati, H., et al. Organic fraction of municipal solid waste as a suitable feedstock for the production of lipid by oleaginous yeast Cryptococcus aerius. Waste Management (2015), http://dx.doi.org/10.1016/j.wasman.2014.12.007

H. Ghanavati et al. / Waste Management xxx (2015) xxx–xxx

at 121 °C for 45 min) was subjected to OL detoxification process. After detoxification, the concentration of TRS, TPC, furans, and nitrogen were measured for all DPHs, and the results are depicted in Table 3. In all treatments, the concentrations of HMF, furfural, and TPC were reduced between 0% and 100%, 9.9% and 100%, and 7.2% and 48.2%, respectively. It was observed that there was a significant difference between the minimum and maximum amounts of the analyzed compounds. In addition, OL process led to the reduction of nitrogen content (10.1–51.9%) and helped the single cell oil production with supply of a low nitrogen content substrate. By increasing the pHOL from 9 to 11, the concentration of furans, nitrogen, and TRS was reduced, while TPC was not significantly reduced. Furthermore, the concentration of all components was affected by DOL and TOL. Unlike other compounds, more reduction of TPC was detected in the detoxification at 60 °C. In addition, increasing DOL was increased the reduction of concentration of all compounds. TRS reduced from 56.5 g/L to a range between 23.65 and 48.8 g/L (13.6–50.9% reduction), which was a drawback of OL resulting in missing some of the fermentable monosaccharide. ANOVA results confirmed the significance of the factors (pHOL, DOL, and TOL) as well as the interaction terms on OL detoxification design. To determine the performance of the detoxification and its optimization, DPHs were subjected to fermentation, and the results are shown in Table 3. The lipid production yield (g produced lipid per g of consumed sugar * 100) was used as an indicator for selecting the optimal conditions. The optimal fermentability was found after detoxification at pHOL 11 and 30 °C for 24 h resulting in about 1.1 g/L lipid concentration and 6.9% yield (lipid/consumed sugar). The statistical analysis showed the significance effect of factors (pHOL, DOL, and TOL) as well as their interactions. The data shows that the lipid production was increased by increasing pHOL and DOL in the detoxification. In addition, it was shown that the treatment at lower temperature (30 °C) resulted in improved fermentability. In fact, by reducing the amount of inhibitors and nitrogen in DPHs, more lipid was accumulated in the cells.

3.3.2. Chemical compositions of NPH and DPH The chemical compositions of NPH and DPH are depicted in Table 4. Glucose and xylose were the main monosaccharides in NPH and DPH. However, decreasing of all monosaccharides (glucose, xylose, arabinose, and galactose) was occurred by detoxification. In other words, the concentration of monosaccharides in the DPH was lower than NPH, and OL process resulted in the loss of 32% glucose, 46.5% xylose, 100% arabinose, and 100% galactose. The presence of heavy metals was one of the problems of dilute acid hydrolysis process, which caused the reduction of microbial activity. In fact, the toxicity of heavy metal ions inhibited the enzymes in the microorganism’s metabolic pathways (Mussatto and Roberto, 2004). However, heavy metals were eliminated by OL, making the DPH suitable for fermentation. The most abundant elements in NPH and DPH were nitrogen, potassium, calcium, iron, and phosphorous (Table 4). In NPH, the concentration of sulfate was very high which was responded to addition of H2SO4 in dilute acid hydrolysis step, and it was decreased (95.2%) in DPH by forming CaSO4 precipitate during OL process. The calcium was increased in DPH, which could be due to increasing Ca2+ ions by the addition of Ca(OH)2 in detoxification. Iron (925 mg/l) was the most abundant heavy metals in NPH while other heavy metals such as lead and cadmium presented in low concentrations (below 5 mg/l). As can be seen in Table 4, OL process resulted in declining the heavy metals and other elements concentration. The most reduction was belonged to copper (79.3%) and zinc (64.4%) in OL process. These concentration reductions could be responsible for better growth and oil production from DPH compared with NPH.

5

3.4. Enzymatic hydrolysis Biomass pretreatment prior to enzymatic hydrolysis is typically essential to enhance the accessibility of cellulase to cellulose (Yu et al., 2011). ROFMSW remained after the dilute acid pre-hydrolysis of OFMSW was subjected to enzymatic hydrolysis, and the effective parameters were optimized (Fig. 1). In all treatments, the major part of TRS was produced in the first 24 h of hydrolysis. The concentration of obtained TRS was in range of 13.9–33.2 g/L (13.9–33.2% yield TRS/ROFMSW) during 72 h hydrolysis. Although the highest TRS was obtained in the treatment with 10% dry ROFMSW with addition of 2 enzymes with the presence of tween 20, the treatment with 10% dry ROFMSW with X enzyme without tween 20 was selected as an optimal condition (30.5 g/L TRS) used for improving lipid. Lower enzyme consumption without significant sugar yield reduction was the reason for the selection of this condition as an optimal condition. In fact, the presence of tween 20 (as a surfactant) has no significant positive effects on the enzymatic hydrolysis of ROFMSW. Due to conversion of cellulose to glucose in the presence of cellulase enzymes, glucose (27.85 g/L) was the most abundant monosaccharide and xylose (2.33 g/L) present in lower quantities in the selected hydrolysis. Moreover, no galactose and arabinose was detected in the hydrolysates.

3.5. Improvement of the lipid production After optimizations of the NPH detoxification by OL and enzymatic hydrolysis of ROFMSW, selected DPH and hydrolysate were used as substrates for lipid production. The one factor experimental designs (data not shown) suggested that the most influencing factors affecting the lipid production were inoculum concentration and fermentation time. Thus, a factorial experimental design was adopted to evaluate the effects of the parameters on the lipid production. Table 5 shows the outputs of the DCW, lipid production, lipid yield, and lipid content of the yeast from DPH and hydrolysate as substrates. The highest lipid production (3.87 g/L) and lipid yield (12.8% lipid/consumed sugar) were obtained after 8 days fermentation with 10% inoculation, with DPH as a substrate. However, the maximum lipid (4.32 g/L) and lipid yield (17.1% lipid/consumed sugar) were obtained after 5 days fermentation with 5% inoculation on hydrolysate as substrate (Table 5). Therefore, the total value of lipid concentration was 8.19 g/L, meaning that 39.6 g lipid was produced from each kg of dry OFMSW. The statistical analyses showed that both lipid production and lipid yield were significantly influenced by each of the two factors (time and inoculation) as well as the interaction of them. Different studies focused on yeast lipid production by lignocellulosic hydrolysates, e.g., Cryptococcus curvatus, Rhodotorula glutinis, Rhodosporidium toruloides, Lipomyces starkeyi, and Yarrowia lipolytica on wheat straw hydrolysate (Yu et al., 2011), Y. lipolytica on sugarcane bagasse (Tsigie et al., 2011) and rice bran hydrolysates (Tsigie et al., 2012), Trichosporon cutaneum on corn stover hydrolysate (Huang et al., 2011), C. curvatus on sorghum bagasse hydrolysate (Liang et al., 2012) and R. glutinis on Populuseuram evicana leaves hydrolysate (Tao et al., 2010). However, this is the first study of lipid production from MSW by yeast fermentation. (Yu et al., 2011) reported that C. curvatus produced higher lipid concentration on non-detoxified (5.8 g/L) than the detoxified hydrolysates (4.2 g/L) of sugarcane bagasse; however, they did not analyzed the potential inhibitors of the substrate. Comparing the (Yu et al., 2011) and current work results indicated that the loss of sugars by detoxification in the substrate with mono-substrate streams has a higher negative impact than removal of the produced inhibitors.

Please cite this article in press as: Ghanavati, H., et al. Organic fraction of municipal solid waste as a suitable feedstock for the production of lipid by oleaginous yeast Cryptococcus aerius. Waste Management (2015), http://dx.doi.org/10.1016/j.wasman.2014.12.007

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H. Ghanavati et al. / Waste Management xxx (2015) xxx–xxx

Table 3 The detoxification conditions, the concentration of TRS, TPC, furans (furfural and HMF) and nitrogen in all DPHs and the results of batch fermentation on DPHs. Detoxification conditions

DPH compounds

Results of fermentation

pH

Time (h)

Tem. (°C)

TRS (g/L)

Nitrogen (g/L)

TPC (g/L)

Furfural (g/L)

HMF (g/L)

DCW (g/L)

Lipid production (g/L)

Lipid yield (%, lipid/consumed sugar)

9 9 9 10 10 10 11 11 11 9 9 9 10 10 10 11 11 11

0 1 24 0 1 24 0 1 24 0 1 24 0 1 24 0 1 24

30 30 30 30 30 30 30 30 30 60 60 60 60 60 60 60 60 60

55.8 54.1 51.6 52.0 51.0 46.8 47.5 44.6 40.2 56.8 52.5 57.9 42.0 47.2 49.4 40.1 36.9 32.8

3.48 3.46 3.40 2.68 2.32 2.20 2.07 1.95 1.89 2.31 2.24 2.21 2.23 2.07 2.10 1.86 1.86 1.70

0.55 0.54 0.43 0.56 0.53 0.45 0.51 0.51 0.50 0.72 0.65 0.58 0.77 0.71 0.68 0.74 0.66 0.63

0.03 0.01 0.01 0.01 0.01 0.00 0.00 0.00 0.00 0.01 0.01 0.00 0.00 0.00 0.00 0.00 0.00 0.00

0.82 0.76 0.68 0.69 0.69 0.61 0.36 0.01 0.00 0.66 0.64 0.72 0.18 0.11 0.09 0.01 0.00 0.00

5.95 6.55 8.90 8.15 9.85 9.85 9.45 10.05 10.80 3.60 4.90 5.45 4.20 5.65 7.95 5.50 7.65 9.60

0.51 0.57 0.65 0.90 1.14 1.22 0.98 1.40 1.73 0.38 0.56 0.58 0.63 0.88 1.06 1.44 1.47 1.64

2.29 2.53 2.90 3.40 4.05 4.12 4.07 5.71 6.92 1.13 1.86 1.77 2.58 3.01 3.46 5.82 5.80 6.68

3.6. Fatty acid composition analysis The fatty acid compositional profiles of C. aerius lipid during the growth on NPH, DPH, hydrolysate, and synthetic medium (with glucose as carbon source) are depicted in Table 6. The results revealed that the extracted oils mainly consisted of long-chain fatty acids (C16 and C18). The predominant fatty acids found in all samples were palmitoleic (C16:1), stearic (C18:0), oleic (C18:1), and linoleic (C18:2) acids. These four fatty acids accounted for higher than 95% of the total fatty acids in all cultivations (Table 6). The results showed that oleic acid (C18:1) was the most abundant fatty acid ranged from 55.1% to 65.2%, whereas palmitoleic acid (C16:1) was the second most abundant fatty acid ranged from 14.7% to 22.9%. In addition, the capric (C10:0), myristic (C14:0), palmitic (C16:0), and arachidic (C20:0) acids were available in lower concentration. However, the profile of fatty acids changed in cultivation on different substrates. Myristic (C14:0) and arachidic (C20:0) acids was not observed in NPH and DPH, while it was appeared in cultivation on hydrolysate and synthetic medium. The results show that these two fatty acids created when no inhibitors existed in the media. With decreasing the inhibitors in the media, the content of capric and stearic acids decreased, and that of palmitoleic, oleic, and linoleic acid were increased. Furthermore, the fatty acid composition of most commonly used plant oils in biodiesel production (Huang et al., 2012) and also some yeast strains that used in other researches (Yu et al., 2011; Zhang et al., 2011) are shown in Table 6. Comparing the oil from C. aerius and other oils indicated that this single cell oil was similar to that of plant oils such as olive and palm that are suitable for biodiesel

Table 4 The composition of NPH and DPH. Parameter

Unit

NPH

DPH

Glucose Xylose Galactose Arabinose TRS N P K Ca Na Mn Mg Zn Fe Cu Pb Ni Cr Cd SO4 2

g/L g/L g/L g/L g/L g/L mg/l mg/l mg/l mg/l mg/l mg/l mg/l mg/l mg/l mg/l mg/l mg/l mg/l g/L

44.30 19.50 1.40 0.20 65.60 3.88 582.20 1532.50 825.20 1925.80 46.40 1.70 59.90 925.20 1.45 4.70 3.50 3.90 0.23 54.30

30.10 9.70 0.00 0.00 40.20 1.89 237.60 1322.40 2880.70 1634.00 30.40 1.50 21.70 542.00 0.30 3.50 2.00 1.70 0.10 2.60

5% ROFMSW + X enzyme 5% ROFMSW + X enzyme + Tween20 5% ROFMSW + 2X enzyme 5% ROFMSW + 2X enzyme + Tween20 10% ROFMSW+ X enzyme 10% ROFMSW + X enzyme + Tween20 10% ROFMSW + 2X enzyme 10% ROFMSW + 2X enzyme + Tween20

40 35 30

Sugar (g/L)

Maximum lipid yield, as high as 6.68 g/L, was obtained from the growth of Y. lipolytica strain Po1 g on detoxified sugarcane bagasse hydrolysate (Tsigie et al., 2011). In the study by (Liang et al., 2012), the cultivation of C. curvatus on sorghum bagasse hydrolysate resulted in the maximal lipid production of 2.6 g/L. Under the optimized dilute acid hydrolysis of P. euramevicana leaves, R. glutinis accumulated 4.62 g/L and 6.18 g/L lipid in 5 L bioreactor without detoxification and after detoxification, respectively (Tao et al., 2010). However, the oil production in other studies was higher in some cases. It is worth mentioning that pure and lignocellulosic materials were used in all of those studies, but in present work, very complex and heterogeneous feedstock (OFMSW) was employed as substrate for the yeast fermentation.

25 20 15 10 5 0 0

12

24

48

72

96

Time (h) Fig. 1. The profiles of TRS during incubation of enzymatic hydrolysis treatments.

production. In addition, compared to other yeast strains, the proportion of unsaturated fatty acid is higher in C. aerius produced lipid.

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H. Ghanavati et al. / Waste Management xxx (2015) xxx–xxx Table 5 The outputs of the DCW, lipid production, lipid yield and lipid content in both DPH and hydrolysate treatments. Inoculation (%)

Time (day)

5 5 5 5 5 5 10 10 10 10 10 10 15 15 15 15 15 15

4 5 6 7 8 9 4 5 6 7 8 9 4 5 6 7 8 9

Fermentation on DPH

Fermentation on hydrolysate

DCW (g/L)

Lipid (g/L)

Lipid content (%)

Lipid yield (%)

DCW (g/L)

Lipid (g/L)

Lipid content (%)

Lipid yield (%)

10.7 12.4 13.9 14.3 14.4 14.4 12.1 13.9 14.5 14.6 14.5 14.5 12.4 16.5 17.1 17.7 17.9 17.9

1.87 2.12 2.46 2.87 3.25 3.08 1.93 2.34 2.68 3.35 3.87 3.78 1.82 2.01 2.70 3.18 3.28 3.52

17.4 17.2 17.7 20.0 22.6 21.4 16.0 16.9 18.4 22.9 26.7 26.0 14.6 12.2 15.8 18.0 18.3 19.6

9.9 10.2 10.5 11.0 11.0 10.4 11.0 11.4 11.8 12.7 12.8 12.3 8.7 9.1 10.3 10.8 10.9 11.1

13.7 14.1 14.1 14.1 14.0 13.9 14.9 15.6 15.7 15.6 15.4 15.4 15.1 16.0 16.0 15.8 15.7 15.6

3.05 4.32 4.31 4.25 4.10 4.08 2.91 3.66 3.77 3.71 3.62 3.45 3.01 3.59 3.67 3.55 3.48 3.39

22.3 30.6 30.6 30.2 29.3 29.3 19.6 23.4 24.1 23.8 23.5 22.4 19.9 22.5 23.0 22.4 22.2 21.7

16.7 17.1 16.6 16.2 15.7 15.7 16.3 15.8 15.8 15.3 14.8 14.3 16.0 16.2 16.2 15.2 14.6 14.2

Table 6 Fatty acid profiles of C. aerius and other yeast strains stored lipid and also some plant oils. Lipid source

C10:0

C14:0

C16:0

C16:1

C18:0

C18:1

C18:2

C20:0

C22:0

Refs.

C. aerius UIMC65 growth on NPHs C. aerius UIMC65 growth on DPHs C. aerius UIMC65 growth on hydrolysate C. aerius UIMC65 growth on synthetic medium (with glucose and without any inhibitor)

0.93–2.25

0

1.33–3.14

18.49–21.3

11.26–15.57

55.07–60

1.11–3.6

0

0

This study

0.58–2.05

0

1.09–3.10

16.2–20.4

10.11–14.66

57.01–63.12

2.63–4.45

0

0

This study

0.28–0.56

0.15–0.55

1.24–2.77

18.81–22.93

7.17–10.89

59.09–65.17

5.57–8.5

0.28–0.37 0

This study

0.44–0.95

0.17–0.59

1.38–3.31

20.23–22.28

5.25–7.16

62.52–65.23

3.55–4.96

0.46–0.55 0

This study

C. curvatus growth on detoxified wheat straw hydrolysate C. curvatus growth on nondetoxified wheat straw hydrolysate R. glutinis growth on switch grass hydrolysate R. toruloides Y4 growth on glucose

NA

NA

27

NA

15.3

45

7.3

NA

NA

Yu et al. (2011)

NA

NA

25.9

NA

15.2

47.7

6.42

NA

NA

Yu et al. (2011)

0.1–1.7

1.1–2.3

8.2–16.9

0.4–1.7

9.7–22.3

26.9–44.7

8.5–12.7

0.8–3.9

0.3–3.5 Zhang et al. (2011)

NA

NA

20

NA

14.6

46.9

13.1

NA

NA

NA NA NA NA

ND ND 1.3 0.6–2.4

4–5 7–13 7–18.3 32–46.3

NA NA NA NA

1–2 2.5–3 1.4–3.3 4–6.3

55–63 30.5–43 55.5–84.5 37–53

20–31 39–52 4–19 6–12

NA NA NA NA

NA NA NA NA

Canola Corn Olive Palm

Huang et al. (2012)

NA: not available in the reference. ND: not detected.

4. Conclusion The OFMSW introduced as an inexpensive and widely available source for biodiesel production by yeast fermentation. The

optimization of dilute acid hydrolysis, detoxification by over-liming, enzymatic hydrolysis, and fermentation parameters resulted in production of appreciate amount of lipid by C. aerius. At the optimal condition, 3.87 and 4.32 g/L of lipid were obtained with

Please cite this article in press as: Ghanavati, H., et al. Organic fraction of municipal solid waste as a suitable feedstock for the production of lipid by oleaginous yeast Cryptococcus aerius. Waste Management (2015), http://dx.doi.org/10.1016/j.wasman.2014.12.007

8

H. Ghanavati et al. / Waste Management xxx (2015) xxx–xxx

detoxified pre-hydrolysate and enzymatic hydrolysate as substrate, respectively. Therefore, 8.19 g/L (39.6 g lipid from each kg of dry OFMSW) was produced by this robust strain.

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Please cite this article in press as: Ghanavati, H., et al. Organic fraction of municipal solid waste as a suitable feedstock for the production of lipid by oleaginous yeast Cryptococcus aerius. Waste Management (2015), http://dx.doi.org/10.1016/j.wasman.2014.12.007