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Production of biofuels, limonene and pectin from citrus wastes
Bioresource Technology 101 (2010) 4246–4250
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Production of biofuels, limonene and pectin from citrus wastes Mohammad Pourbafrani a,b,*, Gergely Forgács a,b, Ilona Sárvári Horváth b, Claes Niklasson a, Mohammad J. Taherzadeh b a b
Chemical Reaction Engineering, Chalmers University of Technology, 412 96 Göteborg, Sweden School of Engineering, University of Borås, 501 90 Borås, Sweden
a r t i c l e
i n f o
Article history: Received 16 November 2009 Received in revised form 14 January 2010 Accepted 20 January 2010 Available online 9 February 2010 Keywords: Citrus waste Ethanol Biogas Limonene Pectin
a b s t r a c t Production of ethanol, biogas, pectin and limonene from citrus wastes (CWs) by an integrated process was investigated. CWs were hydrolyzed by dilute-acid process in a pilot plant reactor equipped with an explosive drainage. Hydrolysis variables including temperature and residence time were optimized by applying a central composite rotatable experimental design (CCRD). The best sugar yield (0.41 g/g of the total dry CWs) was obtained by dilute-acid hydrolysis at 150 °C and 6 min residence time. At this condition, high solubilization of pectin present in the CWs was obtained, and 77.6% of total pectin content of CWs could be recovered by solvent recovery. Degree of esterification and ash content of produced pectin were 63.7% and 4.23%, respectively. In addition, the limonene of the CWs was effectively removed through flashing of the hydrolyzates into an expansion tank. The sugars present in the hydrolyzates were converted to ethanol using baker’s yeast, while an ethanol yield of 0.43 g/g of the fermentable sugars was obtained. Then, the stillage and the remaining solid materials of the hydrolyzed CWs were anaerobically digested to obtain biogas. In summary, one ton of CWs with 20% dry weight resulted in 39.64 l ethanol, 45 m3 methane, 8.9 l limonene, and 38.8 kg pectin. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction World production of citrus fruits is over 88 million tons per year (Marin et al., 2007). Almost half of these fruits is squeezed to juice, and the remainder including peel, segment membranes and other by-products is considered as citrus wastes (CWs) (Wilkins et al., 2007a). These CWs can be dried and used as raw material for pectin extraction or pelletized for animal feed (Mamma et al., 2008). However, a large fraction of CWs is still deposited every year. This deposition is not favored due to both economic and environmental arguments such as high transportation costs, lack of disposal sites, and the land-filling material having high organic content (Tripodo et al., 2004). CWs contain different carbohydrate polymers, which makes them interesting sources for production of biogas and ethanol (Gunaseelan, 2004; Mizuki et al., 1990; Pourbafrani et al., 2007; Wilkins et al., 2007b). The main obstacle to using CWs as a substrate for biogas production is the presence of limonene in CWs. This component is very toxic for digesting microorganisms and decreases the biogas yield (Mizuki et al., 1990). Limonene is also a
* Corresponding author. Address: Chemical Reaction Engineering, Chalmers University of Technology, 412 96 Göteborg, Sweden. Tel.: +46 33 435 43 61; fax: +46 33 436 4008. E-mail address: [email protected] (M. Pourbafrani). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.01.077
strong inhibitor for microorganisms in ethanol production (Pourbafrani et al., 2007; Wilkins et al., 2007b). Therefore, this component should be separated from the CWs prior to digestion or fermentation steps. It should also be noticed that digesting bacteria can hydrolyze the carbohydrate polymers in CWs and convert them finally to biogas, while hydrolysis by enzymes or chemicals is necessary to convert these polymers to sugars and then ferment the sugars by e.g. baker’s yeast to ethanol. Two alternative processes for production of ethanol from CWs based on enzymatic hydrolysis have been previously introduced (Stewart et al., 2005; Wilkins et al., 2007b). In the first alternative (Stewart et al., 2005), CWs were hydrolyzed using a mixture of enzymes (cellulose, pectinase and b-glucosidase). Then, limonene was removed from hydrolyzate by filtration and ethanol was produced from fermentable sugars. In the second alternative (Wilkins et al., 2007b), limonene was partly released using steam stripping. Then, carbohydrate polymers were hydrolyzed and fermented to ethanol through a simultaneous saccharification and fermentation (SSF) process. In both alternatives, non-fermentable sugars and the residue of solid polymers were dried to be used as cattle feed (Stewart et al., 2005). Application of these alternatives is hampered by the high cost of enzyme and the slow rate of hydrolysis reactions (Grohmann et al., 1995). In addition, mechanical pretreatment of biomass, and high demand for energy in distillation and drying processes, might considerably increase the cost of the process.
M. Pourbafrani et al. / Bioresource Technology 101 (2010) 4246–4250
A process based on dilute-acid hydrolysis of CWs can be considered as another alternative (Grohmann et al., 1995; Talebnia et al., 2008). However, the data are limited to the lab-scale experiments and it is difficult to scale up the process to industrial scale. In both studies (Grohmann et al., 1995; Talebnia et al., 2008), mechanical pretreatment was used before hydrolysis and the experiments were carried out in low solid/liquid ratio (Grohmann et al., 1995). Furthermore, dilute-acid hydrolysis showed low yields of sugars from the carbohydrate polymers. The aim of the current work was to introduce a new process for production of ethanol and biogas from CWs. The CW was pretreated with dilute-acid explosion process to hydrolyze the CWs and also to get rid of limonene. The resultant slurry was then centrifuged, and the liquid part was fermented to ethanol and distilled. The stillage from the distillation column and the remained solids were mixed and digested to biogas. An industrial configuration was suggested for this process. 2. Methods 2.1. Citrus wastes composition The CWs used in this work was the residue of orange obtained from Brämhults juice factory (Borås, Sweden) and stored frozen at 20 °C until use. Total dry content of CW was determined by drying at 110 °C for 48 h and it was 20.00 ± 0.80% w/w. The composition of the CWs used in this work as percentage of dry matter was: glucose 8.10 ± 0.46; fructose 12.00 ± 0.21; sucrose 2.80 ± 0.15; pectin 25.00 ± 1.20; protein 6.07 ± 0.10; cellulose 22.00 ± 1.95; hemicellulose 11.09 ± 0.21; ash 3.73 ± 0.20; lignin 2.19 ± 0.04 and limonene 3.78 ± 0.30. 2.2. Dilute-acid hydrolysis A 10-L high-pressure reactor (Process & Industriteknik AB, Sweden) was used for dilute-acid hydrolysis. The CWs was diluted with distilled water to obtain 2 kg slurry with 15% total solid content. Sulfuric acid (98%) was added to the slurries to reach final concentration of 0.5% v/v. The slurries were then hydrolyzed at various temperatures of 130, 150 or 170 °C with different residence times of 3, 6 and 9 min according to the experimental design (Table 1). A central composite rotatable design (CCRD) was used to design the experimental setup and to optimize hydrolysis variables including temperature and time (Talebnia et al., 2008). The reactor was heated with direct injection of 60-bar steam, provided by a power
Table 1 Experimental design of hydrolysis temperature and time in dilute-acid hydrolysis of CWs and the total sugars yield of each hydrolysis (actual and predicted values based on the model in Eq. (1)). Test no.
1 2 3 4 5 6 7 8 9 10 11 12 13 a
Variables
YTSa
YTSa
T (°C)
Time (min)
Actual
Predicted
130 170 130 170 130 170 150 150 150 150 150 150 150
3 3 9 9 6 6 3 9 6 6 6 6 6
26.49 33.73 30.41 33.00 28.60 36.85 35.65 37.55 42.05 40.54 41.09 41.58 41.40
25.38 33.73 29.40 33.10 30.71 36.74 36.75 38.45 40.93 40.93 40.93 40.93 40.93
YTS = yield of total sugars.
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plant located in Borås, Sweden. The hydrolyzed slurry was then explosively discharged to an atmospheric pressure expansion tank to cool down. The materials were then centrifuged at 11,500g for 10 min to separate solid from the hydrolyzate supernatant. The solid residue was washed four times with 200 ml distilled water to separate the possible remaining sugars. The washing water was added to the hydrolyzate supernatant, which was neutralized and fermented. All experiments were duplicated and the standard deviations were less than 3%. 2.3. Yeast strain and fermentation The yeast Saccharomyces cerevisiae ATCC 96581, obtained from LGC standards (Sweden), was used in the fermentation experiment. The strain was maintained on agar plates made from yeast extract (10 g/l), soy peptone (20 g/l), and agar (20 g/l) with D-glucose (20 g/l) as an additional carbon source. The inoculum culture was grown in 250-ml cotton-plugged conical flasks on a shaker at 30 °C for 24 h. The liquid volume was 100 ml and the growth medium was a defined synthetic medium (Taherzadeh et al., 1996) including 50 g/l glucose as carbon and energy source. Anaerobic fermentation was carried out by adding 50 ml of inoculum culture to the hydrolyzate media in a bioreactor (Biostat A., B. Braun Biotech, Germany). The liquid volume of the reactor was 1 l. Temperature, stirring rate and pH were controlled at 30 °C, 200 rpm and 5, respectively. Nitrogen gas was steadily sparged at the rate of 600 ml/min in order to assure anaerobic conditions inside the reactor. 2.4. Pectin recovery and analysis The hydrolyzate supernatant (after the centrifugation) was filtered two times by filter paper to completely remove insoluble materials. The pH was then increased from 1.2 to 2.2 and an equal volume of 96% ethanol was added to precipitate pectin from the solution at room temperature within 4 h. The precipitate was separated by centrifugation at 180g for 60 min and washed five times with ethanol (45%) according to a previous procedure (Faravash and Ashtiani, 2007), and then dried at 50 °C. The degree of esterification (DE) of the pectin was determined by the Fourier transform infrared (FTIR, Nicolet Instrument Corporation, USA) as described before (Faravash and Ashtiani, 2007). A spectral resolution of 4 cm1 with 100 scan was applied to obtain the peak position and peak area with precise accuracy. The ash content of pectin was measured by heating the pectin at 660 °C for 8 h (Faravash and Ashtiani, 2007). 2.5. Digestion The stillage was obtained by heating the fermented hydrolyzate at 96 °C in order to evaporate ethanol. The stillage was then mixed with the solid residue out of the hydrolysis reactor, which was already centrifuged and washed. The mixture was then neutralized and used as ‘‘substrate” for digestion. Volatile solid (VS) of this substrate was measured by the loss on ignition of the dried sample at 550 °C. It was adjusted to 3 g VS/100 g substrate by adding distilled water. The active inoculum was supplied from a municipal waste digester (Borås, Sweden) operating at 55 °C. Two-liter glass bottles with a thick rubber septum were used as reactors (Hansen et al., 2004). Each reactor was fed with 200 g substrate (3% VS) and 400 g inoculum (about 1% VS), and flushed with a gas containing 80% N2 and 20% CO2 to ensure anaerobic conditions (Hansen et al., 2004). The reactors were incubated at 55 °C for 50 days, while shaking twice a day. Three blanks with only water and inoculum were used to measure the methane produc-
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tion originating from the inoculum. All the digestion experiments were carried out in triplicate. 2.6. Analytical methods An Aminex HPX-87P ion-exchange column (Bio-Rad, USA) was used at 85 °C for measuring sucrose, glucose, galactose, arabinose and fructose concentrations. Ultra-pure water was used as eluent at a flow rate of 0.4 mL/min. Ethanol, succinic acid and glycerol concentrations were determined on an Aminex HPX-87H column (Bio-Rad, USA) at 60 °C using 5 mM H2SO4 at a flow rate of 0.6 ml/min. A refractive index (RI) detector (Waters 2414, Milipore, Milford, USA) and UV absorbance detector at 210 nm (Waters 2487) were used in series. Succinic acid was analyzed from UV chromatograms while the residue of metabolites was quantified from the RI chromatograms. Releasing the limonene from CW was carried out using a mixture of cellulase, pectinase and b-glucosidase enzymes (Pourbafrani et al., 2007; Talebnia et al., 2008). The concentration of limonene was determined by addition of n-heptane (99% purity) to the hydrolyzate with a ratio of 1/5 and centrifugation at 3500g for 30 min to extract the oil. The resulting supernatant was then analyzed by a GC–MS (Hewlett–Packard G1800C, Agilent, Palo Alto, CA) where the carrier gas was helium. The temperature was initially 50 °C and was increased to 250 °C at the rate of 15 °C/min and maintained at this temperature for 3 min (Pourbafrani et al., 2007). Gas samples from the digesting reactors were taken by a 0.25 ml glass syringe (VICI, Precision Sampling Inc., USA) equipped with pressure lock. The methane and carbon-dioxide content were analyzed by a gas chromatograph (Perkin–Elmer AutoSystem, USA). The carrier gas was nitrogen and the temperature of the oven was maintained at 60 °C. Data treatment was carried out according to a previous publication (Hansen et al., 2004). The cellulose, hemicellulose, ash and lignin content of CW were measured as described previously (Ververis et al., 2007). Pectin content was extracted by alkaline hydrolysis at 95 °C for 1 h and precipitated by adding ethanol (Ranganna, 1987). Protein content was measured according to the Kjeldahl method.
perature in the linear and quadratic forms and time in quadratic form are significant (p = 0.00) for the yields of sugars. The maximum sugar yield of about 41% can be obtained by conducting the hydrolysis experiment for 6 min reaction time at 150 °C. Therefore, hydrolysis at 150 °C and 6 min residence time was used for the rest of the experiments including limonene analysis, pectin recovery, fermentation and digestion. The conversions of hemicelluloses and cellulose to their monomer sugars were 69.48 ± 0.90 and 49.11 ± 0.75%, respectively. No pectin was converted to galacturonic acid and the carbon balance value for hydrolysis reactor input and output was 0.99 ± 0.01. 3.2. Limonene content of the hydrolyzate Hydrolysis at 150 °C for 6 min by dilute-acid followed by explosive pressure reduction (flashing) resulted in drastic decrease of limonene in the hydrolyzates. The limonene content of hydrolyzate was 0.0035% w/v. Considering dilution of the slurry of CW during the hydrolysis by 65.30% due to steam condensation, it can be calculated that 99% of the limonene content of CW was removed through the flashing in the flash drum. Limonene removed can be recovered by condensation of vapor outlet of the flash drum. Concentration of limonene left in hydrolyzate (0.0035% w/v) is lower than the minimum inhibitory concentration of limonene of 0.01% w/v reported for S. cerevisiae (Winniczuk and Parish, 1997). 3.3. Pectin recovery Dilute-acid hydrolysis can be considered the first step in a method for pectin recovery. The hydrolysis at 150 °C for 6 min resulted in solubilization of 83.5% of pectin present in CW, while still 16.5% of the pectin remained in the solid part of the hydrolyzate. This high solubilization is due to the applied high temperature and the low pH during the hydrolysis (Aravantinoszafiris and Oreopoulou, 1992). Precipitation of pectin content of the hydrolyzate liquid resulted in recovery of pectin by a total of 77.6% of pectin content of CWs. The degree of esterification and the ash content of recovered pectin were 63.7 (±0.98) and 4.23 (±0.08)%, respectively.
3. Results and discussion
3.4. Fermentation of CW hydrolyzate to ethanol
3.1. Dilute-acid hydrolysis
The hydrolyzed CWs was supplemented with nutrients and fermented anaerobically by baker’s yeast. The concentration of different sugars prior to inoculation was 15.17, 10.88, 2.91 and 4.01 g/l for glucose, fructose, galactose and arabinose, respectively. The yeast strain was not able to ferment arabinose, but it could assimilate the hexoses. The fermentation was completed in 24 h, in which all the fermentable sugars were consumed and ethanol was produced. Ethanol yield based on total fermentable sugar consumption was 0.43 ± 0.02 g/g. Glycerol, biomass and succinic acid were the identified by-products, which had yields of 0.10 ± 0.01, 0.070 ± 0.008 and 0.0060 ± 0.0004 g/g, respectively. The carbon balance in the fermentation was 1.02 ± 0.03.
The citrus wastes from orange juice production were hydrolyzed with 0.5% v/v sulfuric acid at 130–170 °C for 3–9 min, and the results are summarized in Table 1. The maximum sugar yield, 42.05%, was achieved at 150 °C and 6 min. However, the more realistic value is the average of results of the 9th to 13th experiments (Table 1), with a sugar yield value of 41.33 ± 0.56%. Increasing the temperature and time to more than their optimal values results in a decrease of the total liberated sugars (Table. 1). This is most likely due to decomposition of hexose sugars (mainly fructose) to hydroxymethylfurfural (Grohmann et al., 1995; Talebnia et al., 2008). A second order mathematical model was fitted to obtained sugar yields: 2
3.5. Anaerobic digestion of CW hydrolyzate to biogas
2
YTS ¼ 418:75 þ 5:66T þ 7:57t 0:018T 0:37t 0:019tT ð1Þ where t and T are time (min) and temperature (°C), respectively. There is good agreement between values predicted by Eq. (1) and actual values of sugar yield (Table 1). The fitness of the model was checked by regression coefficient (R2), which was 0.97. It means that only 3% of the total variations are not explained by the model. Considering the coefficients of operating variables, tem-
The substrate for digestion had TS and VS contents of 4.6% and 4.3%, respectively. The cumulative methane yield was 0.280 ± 0.01 l/g VS after 10 days and reached a constant level of 0.363 ± 0.02 l/g VS after 30 days. It results in a carbon balance of 1.04 ± 0.03 for the digestion. More than 90% of the maximum produced methane was achieved between 15 and 20 days. Compositions of methane and carbon dioxide in the produced biogas were 41% and 59% (v/v), respectively. The ultimate yield of meth-
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Citrus Waste
Sulfuric Acid
Water
Hydrolysis Reactor
Steam
Expansion Tank
Condenser/Decanter
Limonene
Hydrolysate Solid
Liquid Filter
Biogas Digester
Biogas
Precipitator
Pectin Depleted Residue
Fermenter
Pectin
Dryer
Dried Pectin
Ethanol
Distillation
Stillage Fig. 1. Block flow diagram for production of ethanol, biogas, pectin and limonene from CW.
ane can be compared with the value of 0.455–0.486 l/g VS reported by Gunaseelan (2004). 3.6. Overall process A method for commercial treatment of CW to yield different value-added products including ethanol, biogas, limonene and pectin is presented in this paper. A simplified block flow diagram of the process is shown in Fig. 1. By applying this process, 39.64 l ethanol, almost 45 m3 pure methane, 8.9 l limonene, and up to maximum 38.8 kg pectin can be produced per ton of the wet CW. It is an integrated process, in which the ethanol produced in the process can be used for pectin recovery, and the produced methane can be utilized in a steam boiler to generate steam required for distillation and hydrolysis (Fig. 1). 4. Conclusion In this work, a new process is presented to produce ethanol, biogas and limonene from CWs. Depending on the market and
profitability of the process, pectin can be recovered as a by-product from the process. Simplicity of the process and low price of biomass compared to other ethanol processes from lignocelluloses make this process unique and favorable. However, further economic optimizations are required to investigate the profitability of the process. Acknowledgements The authors are grateful to the Foundation of Swedbank in Sjuhärad, Brämhults Juice AB and Sjuhärad Association of Local Authorities (Sweden) for financial support of this work. References Aravantinoszafiris, G., Oreopoulou, V., 1992. The effect of nitric acid extraction variables on orange pectin. J. Sci. Food Agric. 60, 127–129. Faravash, R.S., Ashtiani, F.Z., 2007. The effect of pH, ethanol volume and acid washing time on the yield of pectin extraction from peach pomace. Int. J. Food Sci. Technol. 42, 1177–1187. Grohmann, K., Cameron, R.G., Buslig, B.S., 1995. Fractionation and pretreatment of orange peel by dilute acid hydrolysis. Bioresour. Technol. 54, 129–141.
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Gunaseelan, V.N., 2004. Biochemical methane potential of fruits and vegetable solid waste feedstocks. Biomass Bioenergy 26, 389–399. Hansen, T.L., Schmidt, J.E., Angelidaki, I., Marca, E., Jansen, J.L., Mosbaek, H., Christensen, T.H., 2004. Method for determination of methane potentials of solid organic waste. Waste Manage. 24, 393–400. Mamma, D., Kourtoglou, E., Christakopoulos, P., 2008. Fungal multienzyme production on industrial by-products of the citrus-processing industry. Bioresour. Technol. 99, 2373–2383. Marin, F.R., Soler-Rivas, C., Benavente-Garcia, O., Castillo, J., Perez-Alvarez, J.A., 2007. By-products from different citrus processes as a source of customized functional fibres. Food Chem. 100, 736–741. Mizuki, E., Akao, T., Saruwatari, T., 1990. Inhibitory effect of citrus unshu peel on anaerobic-digestion. Biol. Wastes 33, 161–168. Pourbafrani, M., Talebnia, F., Niklasson, C., Taherzadeh, M.J., 2007. Protective effect of encapsulation in fermentation of limonene-contained media and orange peel hydrolyzate. Int. J. Mol. Sci. 8, 777–787. Ranganna, S., 1987. Handbook of Analysis and Quality Control for Fruit and Vegetable Products, second ed. McGraw Hill Higher Education. Stewart, D.S., Widmer, W.W., Grohmann, K., Wilkins, M.R., 2005. Ethanol Production from Citrus Processing Waste, US Patent Application 11/052,620. Taherzadeh, M.J., Liden, G., Gustafsson, L., Niklasson, C., 1996. The effects of pantothenate deficiency and acetate addition on anaerobic batch fermentation
of glucose by Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol. 46, 176– 182. Talebnia, F., Pourbafrani, M., Lundin, M., Taherzadeh, M., 2008. Optimization study of citrus wastes saccharification by dilute-acid hydrolysis. Bioresources 3, 108– 122. Tripodo, M.M., Lanuzza, F., Micali, G., Coppolino, R., Nucita, F., 2004. Citrus waste recovery: a new environmentally friendly procedure to obtain animal feed. Bioresour. Technol. 91, 111–115. Ververis, C., Georghiou, K., Danielidis, D., Hatzinikolaou, D.G., Santas, P., Santas, R., Corleti, V., 2007. Cellulose, hemicelluloses, lignin and ash content of some organic materials and their suitability for use as paper pulp supplements. Bioresour. Technol. 98, 296–301. Wilkins, M.R., Widmer, W.W., Grohmann, K., Cameron, R.G., 2007a. Hydrolysis of grapefruit peel waste with cellulase and pectinase enzymes. Bioresour. Technol. 98, 1596–1601. Wilkins, M.R., Widmer, W.W., Grohmann, K., 2007b. Simultaneous saccharification and fermentation of citrus peel waste by Saccharomyces cerevisiae to produce ethanol. Process Biochem. 42, 1614–1619. Winniczuk, P.P., Parish, M.E., 1997. Minimum inhibitory concentrations of antimicrobials against micro-organisms related to citrus juice. Food Microbiol. 14, 373–381.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Production of biofuels, limonene and pectin from citrus wastes Mohammad Pourbafrani a,b,*, Gergely Forgács a,b, Ilona Sárvári Horváth b, Claes Niklasson a, Mohammad J. Taherzadeh b a b
Chemical Reaction Engineering, Chalmers University of Technology, 412 96 Göteborg, Sweden School of Engineering, University of Borås, 501 90 Borås, Sweden
a r t i c l e
i n f o
Article history: Received 16 November 2009 Received in revised form 14 January 2010 Accepted 20 January 2010 Available online 9 February 2010 Keywords: Citrus waste Ethanol Biogas Limonene Pectin
a b s t r a c t Production of ethanol, biogas, pectin and limonene from citrus wastes (CWs) by an integrated process was investigated. CWs were hydrolyzed by dilute-acid process in a pilot plant reactor equipped with an explosive drainage. Hydrolysis variables including temperature and residence time were optimized by applying a central composite rotatable experimental design (CCRD). The best sugar yield (0.41 g/g of the total dry CWs) was obtained by dilute-acid hydrolysis at 150 °C and 6 min residence time. At this condition, high solubilization of pectin present in the CWs was obtained, and 77.6% of total pectin content of CWs could be recovered by solvent recovery. Degree of esterification and ash content of produced pectin were 63.7% and 4.23%, respectively. In addition, the limonene of the CWs was effectively removed through flashing of the hydrolyzates into an expansion tank. The sugars present in the hydrolyzates were converted to ethanol using baker’s yeast, while an ethanol yield of 0.43 g/g of the fermentable sugars was obtained. Then, the stillage and the remaining solid materials of the hydrolyzed CWs were anaerobically digested to obtain biogas. In summary, one ton of CWs with 20% dry weight resulted in 39.64 l ethanol, 45 m3 methane, 8.9 l limonene, and 38.8 kg pectin. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction World production of citrus fruits is over 88 million tons per year (Marin et al., 2007). Almost half of these fruits is squeezed to juice, and the remainder including peel, segment membranes and other by-products is considered as citrus wastes (CWs) (Wilkins et al., 2007a). These CWs can be dried and used as raw material for pectin extraction or pelletized for animal feed (Mamma et al., 2008). However, a large fraction of CWs is still deposited every year. This deposition is not favored due to both economic and environmental arguments such as high transportation costs, lack of disposal sites, and the land-filling material having high organic content (Tripodo et al., 2004). CWs contain different carbohydrate polymers, which makes them interesting sources for production of biogas and ethanol (Gunaseelan, 2004; Mizuki et al., 1990; Pourbafrani et al., 2007; Wilkins et al., 2007b). The main obstacle to using CWs as a substrate for biogas production is the presence of limonene in CWs. This component is very toxic for digesting microorganisms and decreases the biogas yield (Mizuki et al., 1990). Limonene is also a
* Corresponding author. Address: Chemical Reaction Engineering, Chalmers University of Technology, 412 96 Göteborg, Sweden. Tel.: +46 33 435 43 61; fax: +46 33 436 4008. E-mail address: [email protected] (M. Pourbafrani). 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.01.077
strong inhibitor for microorganisms in ethanol production (Pourbafrani et al., 2007; Wilkins et al., 2007b). Therefore, this component should be separated from the CWs prior to digestion or fermentation steps. It should also be noticed that digesting bacteria can hydrolyze the carbohydrate polymers in CWs and convert them finally to biogas, while hydrolysis by enzymes or chemicals is necessary to convert these polymers to sugars and then ferment the sugars by e.g. baker’s yeast to ethanol. Two alternative processes for production of ethanol from CWs based on enzymatic hydrolysis have been previously introduced (Stewart et al., 2005; Wilkins et al., 2007b). In the first alternative (Stewart et al., 2005), CWs were hydrolyzed using a mixture of enzymes (cellulose, pectinase and b-glucosidase). Then, limonene was removed from hydrolyzate by filtration and ethanol was produced from fermentable sugars. In the second alternative (Wilkins et al., 2007b), limonene was partly released using steam stripping. Then, carbohydrate polymers were hydrolyzed and fermented to ethanol through a simultaneous saccharification and fermentation (SSF) process. In both alternatives, non-fermentable sugars and the residue of solid polymers were dried to be used as cattle feed (Stewart et al., 2005). Application of these alternatives is hampered by the high cost of enzyme and the slow rate of hydrolysis reactions (Grohmann et al., 1995). In addition, mechanical pretreatment of biomass, and high demand for energy in distillation and drying processes, might considerably increase the cost of the process.
M. Pourbafrani et al. / Bioresource Technology 101 (2010) 4246–4250
A process based on dilute-acid hydrolysis of CWs can be considered as another alternative (Grohmann et al., 1995; Talebnia et al., 2008). However, the data are limited to the lab-scale experiments and it is difficult to scale up the process to industrial scale. In both studies (Grohmann et al., 1995; Talebnia et al., 2008), mechanical pretreatment was used before hydrolysis and the experiments were carried out in low solid/liquid ratio (Grohmann et al., 1995). Furthermore, dilute-acid hydrolysis showed low yields of sugars from the carbohydrate polymers. The aim of the current work was to introduce a new process for production of ethanol and biogas from CWs. The CW was pretreated with dilute-acid explosion process to hydrolyze the CWs and also to get rid of limonene. The resultant slurry was then centrifuged, and the liquid part was fermented to ethanol and distilled. The stillage from the distillation column and the remained solids were mixed and digested to biogas. An industrial configuration was suggested for this process. 2. Methods 2.1. Citrus wastes composition The CWs used in this work was the residue of orange obtained from Brämhults juice factory (Borås, Sweden) and stored frozen at 20 °C until use. Total dry content of CW was determined by drying at 110 °C for 48 h and it was 20.00 ± 0.80% w/w. The composition of the CWs used in this work as percentage of dry matter was: glucose 8.10 ± 0.46; fructose 12.00 ± 0.21; sucrose 2.80 ± 0.15; pectin 25.00 ± 1.20; protein 6.07 ± 0.10; cellulose 22.00 ± 1.95; hemicellulose 11.09 ± 0.21; ash 3.73 ± 0.20; lignin 2.19 ± 0.04 and limonene 3.78 ± 0.30. 2.2. Dilute-acid hydrolysis A 10-L high-pressure reactor (Process & Industriteknik AB, Sweden) was used for dilute-acid hydrolysis. The CWs was diluted with distilled water to obtain 2 kg slurry with 15% total solid content. Sulfuric acid (98%) was added to the slurries to reach final concentration of 0.5% v/v. The slurries were then hydrolyzed at various temperatures of 130, 150 or 170 °C with different residence times of 3, 6 and 9 min according to the experimental design (Table 1). A central composite rotatable design (CCRD) was used to design the experimental setup and to optimize hydrolysis variables including temperature and time (Talebnia et al., 2008). The reactor was heated with direct injection of 60-bar steam, provided by a power
Table 1 Experimental design of hydrolysis temperature and time in dilute-acid hydrolysis of CWs and the total sugars yield of each hydrolysis (actual and predicted values based on the model in Eq. (1)). Test no.
1 2 3 4 5 6 7 8 9 10 11 12 13 a
Variables
YTSa
YTSa
T (°C)
Time (min)
Actual
Predicted
130 170 130 170 130 170 150 150 150 150 150 150 150
3 3 9 9 6 6 3 9 6 6 6 6 6
26.49 33.73 30.41 33.00 28.60 36.85 35.65 37.55 42.05 40.54 41.09 41.58 41.40
25.38 33.73 29.40 33.10 30.71 36.74 36.75 38.45 40.93 40.93 40.93 40.93 40.93
YTS = yield of total sugars.
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plant located in Borås, Sweden. The hydrolyzed slurry was then explosively discharged to an atmospheric pressure expansion tank to cool down. The materials were then centrifuged at 11,500g for 10 min to separate solid from the hydrolyzate supernatant. The solid residue was washed four times with 200 ml distilled water to separate the possible remaining sugars. The washing water was added to the hydrolyzate supernatant, which was neutralized and fermented. All experiments were duplicated and the standard deviations were less than 3%. 2.3. Yeast strain and fermentation The yeast Saccharomyces cerevisiae ATCC 96581, obtained from LGC standards (Sweden), was used in the fermentation experiment. The strain was maintained on agar plates made from yeast extract (10 g/l), soy peptone (20 g/l), and agar (20 g/l) with D-glucose (20 g/l) as an additional carbon source. The inoculum culture was grown in 250-ml cotton-plugged conical flasks on a shaker at 30 °C for 24 h. The liquid volume was 100 ml and the growth medium was a defined synthetic medium (Taherzadeh et al., 1996) including 50 g/l glucose as carbon and energy source. Anaerobic fermentation was carried out by adding 50 ml of inoculum culture to the hydrolyzate media in a bioreactor (Biostat A., B. Braun Biotech, Germany). The liquid volume of the reactor was 1 l. Temperature, stirring rate and pH were controlled at 30 °C, 200 rpm and 5, respectively. Nitrogen gas was steadily sparged at the rate of 600 ml/min in order to assure anaerobic conditions inside the reactor. 2.4. Pectin recovery and analysis The hydrolyzate supernatant (after the centrifugation) was filtered two times by filter paper to completely remove insoluble materials. The pH was then increased from 1.2 to 2.2 and an equal volume of 96% ethanol was added to precipitate pectin from the solution at room temperature within 4 h. The precipitate was separated by centrifugation at 180g for 60 min and washed five times with ethanol (45%) according to a previous procedure (Faravash and Ashtiani, 2007), and then dried at 50 °C. The degree of esterification (DE) of the pectin was determined by the Fourier transform infrared (FTIR, Nicolet Instrument Corporation, USA) as described before (Faravash and Ashtiani, 2007). A spectral resolution of 4 cm1 with 100 scan was applied to obtain the peak position and peak area with precise accuracy. The ash content of pectin was measured by heating the pectin at 660 °C for 8 h (Faravash and Ashtiani, 2007). 2.5. Digestion The stillage was obtained by heating the fermented hydrolyzate at 96 °C in order to evaporate ethanol. The stillage was then mixed with the solid residue out of the hydrolysis reactor, which was already centrifuged and washed. The mixture was then neutralized and used as ‘‘substrate” for digestion. Volatile solid (VS) of this substrate was measured by the loss on ignition of the dried sample at 550 °C. It was adjusted to 3 g VS/100 g substrate by adding distilled water. The active inoculum was supplied from a municipal waste digester (Borås, Sweden) operating at 55 °C. Two-liter glass bottles with a thick rubber septum were used as reactors (Hansen et al., 2004). Each reactor was fed with 200 g substrate (3% VS) and 400 g inoculum (about 1% VS), and flushed with a gas containing 80% N2 and 20% CO2 to ensure anaerobic conditions (Hansen et al., 2004). The reactors were incubated at 55 °C for 50 days, while shaking twice a day. Three blanks with only water and inoculum were used to measure the methane produc-
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tion originating from the inoculum. All the digestion experiments were carried out in triplicate. 2.6. Analytical methods An Aminex HPX-87P ion-exchange column (Bio-Rad, USA) was used at 85 °C for measuring sucrose, glucose, galactose, arabinose and fructose concentrations. Ultra-pure water was used as eluent at a flow rate of 0.4 mL/min. Ethanol, succinic acid and glycerol concentrations were determined on an Aminex HPX-87H column (Bio-Rad, USA) at 60 °C using 5 mM H2SO4 at a flow rate of 0.6 ml/min. A refractive index (RI) detector (Waters 2414, Milipore, Milford, USA) and UV absorbance detector at 210 nm (Waters 2487) were used in series. Succinic acid was analyzed from UV chromatograms while the residue of metabolites was quantified from the RI chromatograms. Releasing the limonene from CW was carried out using a mixture of cellulase, pectinase and b-glucosidase enzymes (Pourbafrani et al., 2007; Talebnia et al., 2008). The concentration of limonene was determined by addition of n-heptane (99% purity) to the hydrolyzate with a ratio of 1/5 and centrifugation at 3500g for 30 min to extract the oil. The resulting supernatant was then analyzed by a GC–MS (Hewlett–Packard G1800C, Agilent, Palo Alto, CA) where the carrier gas was helium. The temperature was initially 50 °C and was increased to 250 °C at the rate of 15 °C/min and maintained at this temperature for 3 min (Pourbafrani et al., 2007). Gas samples from the digesting reactors were taken by a 0.25 ml glass syringe (VICI, Precision Sampling Inc., USA) equipped with pressure lock. The methane and carbon-dioxide content were analyzed by a gas chromatograph (Perkin–Elmer AutoSystem, USA). The carrier gas was nitrogen and the temperature of the oven was maintained at 60 °C. Data treatment was carried out according to a previous publication (Hansen et al., 2004). The cellulose, hemicellulose, ash and lignin content of CW were measured as described previously (Ververis et al., 2007). Pectin content was extracted by alkaline hydrolysis at 95 °C for 1 h and precipitated by adding ethanol (Ranganna, 1987). Protein content was measured according to the Kjeldahl method.
perature in the linear and quadratic forms and time in quadratic form are significant (p = 0.00) for the yields of sugars. The maximum sugar yield of about 41% can be obtained by conducting the hydrolysis experiment for 6 min reaction time at 150 °C. Therefore, hydrolysis at 150 °C and 6 min residence time was used for the rest of the experiments including limonene analysis, pectin recovery, fermentation and digestion. The conversions of hemicelluloses and cellulose to their monomer sugars were 69.48 ± 0.90 and 49.11 ± 0.75%, respectively. No pectin was converted to galacturonic acid and the carbon balance value for hydrolysis reactor input and output was 0.99 ± 0.01. 3.2. Limonene content of the hydrolyzate Hydrolysis at 150 °C for 6 min by dilute-acid followed by explosive pressure reduction (flashing) resulted in drastic decrease of limonene in the hydrolyzates. The limonene content of hydrolyzate was 0.0035% w/v. Considering dilution of the slurry of CW during the hydrolysis by 65.30% due to steam condensation, it can be calculated that 99% of the limonene content of CW was removed through the flashing in the flash drum. Limonene removed can be recovered by condensation of vapor outlet of the flash drum. Concentration of limonene left in hydrolyzate (0.0035% w/v) is lower than the minimum inhibitory concentration of limonene of 0.01% w/v reported for S. cerevisiae (Winniczuk and Parish, 1997). 3.3. Pectin recovery Dilute-acid hydrolysis can be considered the first step in a method for pectin recovery. The hydrolysis at 150 °C for 6 min resulted in solubilization of 83.5% of pectin present in CW, while still 16.5% of the pectin remained in the solid part of the hydrolyzate. This high solubilization is due to the applied high temperature and the low pH during the hydrolysis (Aravantinoszafiris and Oreopoulou, 1992). Precipitation of pectin content of the hydrolyzate liquid resulted in recovery of pectin by a total of 77.6% of pectin content of CWs. The degree of esterification and the ash content of recovered pectin were 63.7 (±0.98) and 4.23 (±0.08)%, respectively.
3. Results and discussion
3.4. Fermentation of CW hydrolyzate to ethanol
3.1. Dilute-acid hydrolysis
The hydrolyzed CWs was supplemented with nutrients and fermented anaerobically by baker’s yeast. The concentration of different sugars prior to inoculation was 15.17, 10.88, 2.91 and 4.01 g/l for glucose, fructose, galactose and arabinose, respectively. The yeast strain was not able to ferment arabinose, but it could assimilate the hexoses. The fermentation was completed in 24 h, in which all the fermentable sugars were consumed and ethanol was produced. Ethanol yield based on total fermentable sugar consumption was 0.43 ± 0.02 g/g. Glycerol, biomass and succinic acid were the identified by-products, which had yields of 0.10 ± 0.01, 0.070 ± 0.008 and 0.0060 ± 0.0004 g/g, respectively. The carbon balance in the fermentation was 1.02 ± 0.03.
The citrus wastes from orange juice production were hydrolyzed with 0.5% v/v sulfuric acid at 130–170 °C for 3–9 min, and the results are summarized in Table 1. The maximum sugar yield, 42.05%, was achieved at 150 °C and 6 min. However, the more realistic value is the average of results of the 9th to 13th experiments (Table 1), with a sugar yield value of 41.33 ± 0.56%. Increasing the temperature and time to more than their optimal values results in a decrease of the total liberated sugars (Table. 1). This is most likely due to decomposition of hexose sugars (mainly fructose) to hydroxymethylfurfural (Grohmann et al., 1995; Talebnia et al., 2008). A second order mathematical model was fitted to obtained sugar yields: 2
3.5. Anaerobic digestion of CW hydrolyzate to biogas
2
YTS ¼ 418:75 þ 5:66T þ 7:57t 0:018T 0:37t 0:019tT ð1Þ where t and T are time (min) and temperature (°C), respectively. There is good agreement between values predicted by Eq. (1) and actual values of sugar yield (Table 1). The fitness of the model was checked by regression coefficient (R2), which was 0.97. It means that only 3% of the total variations are not explained by the model. Considering the coefficients of operating variables, tem-
The substrate for digestion had TS and VS contents of 4.6% and 4.3%, respectively. The cumulative methane yield was 0.280 ± 0.01 l/g VS after 10 days and reached a constant level of 0.363 ± 0.02 l/g VS after 30 days. It results in a carbon balance of 1.04 ± 0.03 for the digestion. More than 90% of the maximum produced methane was achieved between 15 and 20 days. Compositions of methane and carbon dioxide in the produced biogas were 41% and 59% (v/v), respectively. The ultimate yield of meth-
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Citrus Waste
Sulfuric Acid
Water
Hydrolysis Reactor
Steam
Expansion Tank
Condenser/Decanter
Limonene
Hydrolysate Solid
Liquid Filter
Biogas Digester
Biogas
Precipitator
Pectin Depleted Residue
Fermenter
Pectin
Dryer
Dried Pectin
Ethanol
Distillation
Stillage Fig. 1. Block flow diagram for production of ethanol, biogas, pectin and limonene from CW.
ane can be compared with the value of 0.455–0.486 l/g VS reported by Gunaseelan (2004). 3.6. Overall process A method for commercial treatment of CW to yield different value-added products including ethanol, biogas, limonene and pectin is presented in this paper. A simplified block flow diagram of the process is shown in Fig. 1. By applying this process, 39.64 l ethanol, almost 45 m3 pure methane, 8.9 l limonene, and up to maximum 38.8 kg pectin can be produced per ton of the wet CW. It is an integrated process, in which the ethanol produced in the process can be used for pectin recovery, and the produced methane can be utilized in a steam boiler to generate steam required for distillation and hydrolysis (Fig. 1). 4. Conclusion In this work, a new process is presented to produce ethanol, biogas and limonene from CWs. Depending on the market and
profitability of the process, pectin can be recovered as a by-product from the process. Simplicity of the process and low price of biomass compared to other ethanol processes from lignocelluloses make this process unique and favorable. However, further economic optimizations are required to investigate the profitability of the process. Acknowledgements The authors are grateful to the Foundation of Swedbank in Sjuhärad, Brämhults Juice AB and Sjuhärad Association of Local Authorities (Sweden) for financial support of this work. References Aravantinoszafiris, G., Oreopoulou, V., 1992. The effect of nitric acid extraction variables on orange pectin. J. Sci. Food Agric. 60, 127–129. Faravash, R.S., Ashtiani, F.Z., 2007. The effect of pH, ethanol volume and acid washing time on the yield of pectin extraction from peach pomace. Int. J. Food Sci. Technol. 42, 1177–1187. Grohmann, K., Cameron, R.G., Buslig, B.S., 1995. Fractionation and pretreatment of orange peel by dilute acid hydrolysis. Bioresour. Technol. 54, 129–141.
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