Anaerobic digestion of glycerol derived from biodiesel manufacturing

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    Kinetic constants

    glycbatco thiculf glsinwas

    phenomenon was observed at the highest load. 2009 Elsevier Ltd. All rights reserved.

    e useand thl., 200comp

    ble ene

    matic growth of the biodiesel industry has created a surplus of glyc-erol that has resulted in a dramatic 10-fold decrease in crudeglycerol prices over the last few years and generated environmentalconcerns associated with contaminated glycerol disposal (Yazdaniand Gonzalez, 2007; Sandun et al., 2007).

    At present, due to its properties, pure glycerol has more than2000 different applications (Elvers et al., 1990). However, the pro-

    properties for use in fuel or solvents (Karinen and Krause, 2006). The microbial conversion (fermentation) of glycerol to 1,3-pro-

    panediol, which can be used as a basic ingredient of polyesters(Barbirato et al., 1998; Ito et al., 2005).

    Other products such as butanol (Biebl, 2001), propionic acid(Bories et al., 2004), ethanol and formate (Jarvis et al., 1997),succinic acid (Lee et al., 2001), dihydroxyacetone (Bories et al.,1991; Claret et al., 1994), polyhydroxyalkanoates (Koller et al.,2005), or hydrogen and ethanol (Ito et al., 2005) were alsoobtained using glycerol as a carbon source.

    * Corresponding author. Tel.: +34 957 218586; fax: +34 957 218625.

    Bioresource Technology 100 (2009) 56095615

    Contents lists availab

    T

    elsE-mail address: a92siloj@uco.es (J.. Siles Lpez).ful emissions and non-toxic fuel, these have drawn much attentionrecently (Ito et al., 2005).

    During the biodiesel production process, oils/fats (triglycerides)aremixedwithmethyl alcohol and alkaline catalysts to produce freefatty-acid esters, with glycerol as a primary by-product (Chi et al.,2007). The production of 100 kg of biodiesel yields approximately10 kg of impure glycerol, with 5590% glycerol (Hazimah et al.,2003). Glycerol is generated as a by-product not only when biodie-sel fuels are produced chemically, but also when they are manufac-tured enzymatically (Du et al., 2003; Vicente et al., 2004) and duringthe productionof bioethanol (Yazdani andGonzalez, 2007). The dra-

    posed as a solution for the economic viability of this product. Severalstrategies based on chemical and biological transformations arebeing pursued to convert glycerol intomore valuable products (Yaz-dani and Gonzalez, 2007). An example of some of these includes:

    The conversion of glycerol into propylene glycol and acetone,through thermo-chemical processes (Chiu et al., 2006; Dasariet al., 2005).

    The etherication of glycerol with either alcohols (e.g. methanolor ethanol) or alkenes (e.g. isobutene) and the production ofoxygen-containing components, which could have suitableBiodegradability

    1. Introduction

    Concern has recently risen over thcost, their sustained availabilitywarming and pollution (Hansen et ahave various advantages such as abased fuel, renewable fuel, a favoura0960-8524/$ - see front matter 2009 Elsevier Ltd. Adoi:10.1016/j.biortech.2009.06.017of fossil resources, theireir impact on global5). Since biodiesel fuelslement to petroleum-rgy balance, less harm-

    duction of crude glycerol exceeds the present commercial demandfor puried glycerol. Furthermore, the purication of glycerol (witha view to being sold) that is generated during biodieselmanufactur-ing is not a viable option for the biodiesel industry (Chi et al., 2007).Although crude glycerol can be burnt, with the consequent ener-getic advantages, the setting up of bioreneries that co-produceproducts of higher economic value alongwith biofuels has beenpro-Glycerol-containing wasteAnaerobic digestion

    option for revalorising this available, impure and low priced by-product derived from the surplus of bio-diesel companies. The organic loading rate studied was 0.210.38 g COD/g VSS d, although an inhibitionAnaerobic digestion of glycerol derived f

    Jos ngel Siles Lpez *, Mara de los ngeles MartnAntonio Martn MartnDepartamento de Qumica Inorgnica e Ingeniera Qumica, Facultad de Ciencias, Univekm 396, 14071 Crdoba, Spain

    a r t i c l e i n f o

    Article history:Received 6 March 2009Received in revised form 1 June 2009Accepted 6 June 2009Available online 9 July 2009

    Keywords:Biodiesel manufacturing

    a b s t r a c t

    The anaerobic digestion of1010 g/kg, was studied innon-granular sludge. Due tthis alkaline catalyst as agrically viable, a volume orevalorisation of glycerol umethane yield coefcient

    Bioresource

    journal homepage: www.ll rights reserved.m biodiesel manufacturing

    antos, Arturo Francisco Chica Prez,

    d de Crdoba, Campus Universitario de Rabanales, Edicio C-3, Ctra. Madrid-Cdiz,

    erol derived from biodiesel manufacturing, in which COD was found to beh laboratory-scale reactors at mesophilic temperature using granular ande high KOH concentration of this by-product, H3PO4 was added to recovertural fertilizer (potassium phosphates). Although it would not be econom-ycerol was distilled and utilised as reference substrate. The anaerobicg granular sludge achieved a biodegradability of around 100%, while the0.306 m3 CH4/kg acidied glycerol. Anaerobic digestion could be a good

    le at ScienceDirect

    echnology

    evier .com/locate /bior tech

  • TecAnaerobic digestion is another possible way to revalorise abun-dant and low-priced glycerol streams. This process may be denedas the biological conversion of organic material to a variety of endproducts including biogas whose main constituents are methaneand carbon dioxide (Gujer and Zehnder, 1983; Olthof andOleszkiewick, 1982; Speece, 1983; Wheatley, 1990). The advanta-ges of anaerobic digestion include low levels of biological sludge,low nutrient requirements, high efciency and the production ofmethane, which can be used as an energy source. The stoichiome-try of the anaerobic digestion of glycerol can be summarised as fol-lows (Christensen and McCarty, 1975; McCarty, 1975):

    C3H8O3 aNH3 ! bCH4 cCO2 dC5H7NO2 eNH4HCO3The products of the reaction are methane, carbon dioxide, bio-

    mass and ammonic bicarbonate, where a, b, c, d and e are stoichi-ometric coefcients. In order to obtain the value of thesecoefcients and determine the methane yield of the anaerobicdigestion of glycerol, a mass balance was made taking into consid-eration a biomass yield of anaerobic bacteria of 0.05 (w/w). Thevalues of a, b, c, d and e were found to be 0.663, 1.648, 0.526,0.041 and 0.622 mol, respectively. In contrast, when an electronbalance was carried out assuming that every electron was usedin methane generation, b reached a value of 1.750. A simple calcu-lation showed that the theoretical methane yield is 94.2%, which isa useful compound due to its caloric power (Lower Caloric Power):35,793 kJ/m3, equivalent to 9.96 kW h/m3. Revalorising glycerol isof special interest as it gives the highest reduced carbon with thecost advantage of anaerobic processes. The aim of this work was

    Nomenclature

    Alk alkalinity (mg CaCO3/L)CODremoved removed chemical oxygen demand (mg/L)CODsoluble soluble chemical oxygen demand (mg/L)COD STO added chemical oxygen demand (mg/L)CODtotal total chemical oxygen demand (mg/L)G cumulative methane volume (mL)Gm cumulative methane volume at innite time (mL)GT experimental maximum methane volume (mL)KG specic methane production kinetic constant (L/g VSS h)K 0G apparent kinetic constant (1/h)MS total mineral solids (mg/L)MSS mineral suspended solids (mg/L)OLR organic loading rate (g COD/g VSS d; kg COD/m3 d)

    5610 J.. Siles Lpez et al. / Bioresourceto evaluate the performance and the stability of anaerobic diges-tion process of glycerol-containing waste derived from biodieselmanufacturing. The study was carried out in six batch labora-tory-scale reactors at mesophilic temperature (35 C).

    2. Methods

    2.1. Experimental set-up

    The experimental set-up used for the anaerobic digestion of thebiodiesel-derived glycerol consisted of six 1-L Pyrex reactors withfour connections to allow for the loading of feedstock, the ventila-tion of the biogas, the injection of inert gas (nitrogen) to maintainthe anaerobic conditions and the removal of efuent. The contentof the reactors was magnetically stirred and temperature wasmaintained by means of a thermostatic jacket containing waterat 37 C. The volume of methane produced during the processwas measured by using 1-L BoyleMariotte reservoirs connectedto each reactor. To remove the CO2 produced during the process,tightly closed bubblers containing a NaOH solution (6 N) were con-nected between the two elements. The methane volume displacedan equal measurable volume of water from the reservoir.

    The reactors were inoculated with methanogenically-activegranular biomass obtained from a full-scale anaerobic reactor usedto treat brewery wastewater from the Heineken S.A. Factory (Jaen,Spain) and non-granular sludge from a full-scale anaerobic reactorused to treat urban wastewater in Jerez de la Frontera (Cadiz,Spain). The granular sludge contained 37,500 mg VSS/L and31,875 mg MSS/L, while the non-granular sludge contained28,400 mg VSS/L and 20,330 MSS/L. The inocula were selected onthe basis of their high methanogenic activity (Field et al., 1988)with values ranging from 0.87 to 0.99 g COD/g VSS d.

    2.2. Glycerol

    The raw material used as substrate was the glycerol-containingwaste discharged after the biodiesel manufacturing process at theBIDA S.A. Factory in Fuentes de Andalucia (Seville, Spain). In general,thiswaste contained glycerol, water,methanol, salts and fatty acids.

    2.3. Raw material pre-treatment

    The substrate was previously treated in two different ways: (a)acidication with phosphoric acid and centrifugation in order to re-cover the catalyst used in the transesterication reaction (KOH) asagricultural fertilizer (potassium phosphates). Additionally, metha-nol and water were removed by vacuum distillation. We call thissubstrate acidied glycerol. (b) Acidication followed by distilla-

    r0 specic rate of methane production (mL CH4/g VSS h)Sremoved removed substrate (g COD)STO added substrate (g COD)t time (h)TS total solids (mg/L)TSS total suspended solids (mg/L)VA volatile acidity (mg acetic/L)VS total volatile solids (mg/L)VSS volatile suspended solids (mg/L)X biomass concentration (mg VSS/L)YCH4=Sremoved methane yield coefcient (mL CH4/g COD removed)

    hnology 100 (2009) 56095615tion (rectication). After the same acidication process, a rectica-tion at laboratory-scale (135140 C; 1.62.0 103 atm) wascarried out. Subsequently, the organic impurities in the distillatewere removed by liquid/liquid extraction with hexane, which waseliminated by vacuum distillation. We call this substrate distilledglycerol. Table 1 shows the characteristics and features of acidied,distilled and commercially available pure glycerol (Elvers et al.,1990). Due to its high COD, the acidied and distilled glycerol werediluted using distilled water until reaching 81.6 and 85.7 g COD/L,respectively, and neutralized by adding sodium hydroxide. Finally,several nutrients and alkalinity (NaHCO3) were added to the dis-tilled glycerol to provide the necessary nutrients for the appropriatemetabolism of the anaerobic microorganisms (DiStefano andAmbulkar, 2006). Table 2 shows the composition of the nutrientand trace element solutions added to the distilled glycerol.

    2.4. Anaerobic digesters. Experimental procedure

    The anaerobic reactors were initially loaded with 12 g VSS ofgranular sludge as inoculum, and the anaerobic digestion of

  • 2.6. Software

    Sigma-Plot software (version 9.0) was used to create graphs,perform the statistical analysis and t the experimental data pre-sented in this work.

    Technology 100 (2009) 56095615 5611acidied and distilled glycerol was studied. Another set of reactorswere then loaded with non-granular sludge and the anaerobicdigestion of acidied glycerol was studied. In all cases, the nutrientand trace element solutions described by Fannin (1987) and Fieldet al. (1988) were added when the sludge was loaded. Bothsolutions are very important for activating bacterial growth andmetabolism at the beginning of the process.

    In order to activate the biomass prior to the experiments, thereactors were rst fed with a synthetic solution composed of glu-

    Table 1Composition and features of the acidied, distilled and pure glycerol.

    Parameter Acidiedglycerol

    Distilledglycerol

    Pureglycerol

    Density at 20 C (g/mL) 1.044 1.260 1.261Refraction index at 20 C 1.4440 1.4728 1.4746COD (g/kg) 1010 1155 1217Dynamic viscosity at 50 C (mPa s) 57 150 152Colour Brown Colourless Colourless

    Table 2Composition of the nutrient and trace element solutions added to distilled glycerol.

    Nutrient solution Trace element solution

    Compound g/La Compound g/L

    NH4CI 0.200 MnCl24H2O 0.100K2HPO43H2O 0.100 CoCl26H2O 0.170KH2PO4 0.055 ZnCl2 0.100MgCl26H2O 0.200 CaCl2 0.200Resazurine 0.001 H3BO4 0.019FeCl24H2O 0.100 NiCl26H2O 0.050NaS9H2O 0.500 Na2MoO4H2O 0.100NaHCO3 5.000 Ad 10 mL of trace element solution per litre of diluted

    glycerol

    a Concentration of each compound per litre of diluted glycerol.

    J.. Siles Lpez et al. / Bioresourcecose, sodium acetate and lactic acid (GAL solution) at concentra-tions of 50 g/L, 25 g/L and 20.8 mL/L, respectively. During thisinitial period, the organic load added to the reactors was graduallyincreased from 0.25 to 1.00 g COD over a 16-day period. After thisprevious stage, biomass acclimatization was carried out. The reac-tors were fed with 1 g COD, in which the percentage of glycerolused in the COD was increased from 25% to 100% after four loads.During this acclimatization period, the volume of methane wasmeasured as a function of time. The maximum duration of each as-say was 48 h; the time interval required for the complete biometh-anization of each load. Once this preliminary acclimatization stepwas nished, a series of batch experiments were carried out usingboth sludge types in addition to the acidied and distilled glycerolas substrates. During each set of experiments, the organic loadadded to the reactors was gradually increased from 1.0 to 1.5and 2.0 g COD with distilled glycerol and from 1.0 to 1.5, 2.0 and3.0 g COD with acidied glycerol. In all cases, the volume of meth-ane was measured as a function of time and samples were takenand analysed before and after feeding. The duration of each exper-iment was equal to the time interval required for maximum gasproduction and COD removal. Each glycerol solution load was car-ried out at least in duplicate and the results expressed as means.

    2.5. Chemical analyses

    The following parameters were determined in the efuents ofeach load: pH, COD total, COD soluble, TS, MS, VS, TSS, MSS, VSS,volatile acidity (VA) and alkalinity (Alk). All analyses were carriedout in accordance with the Standard Methods of the APHA (APHA,1989).3. Results and discussion

    3.1. Stability

    The stability of the process is evaluated based on the evolutionof the pH, alkalinity, volatile acidity and volatile acidity/alkalinityratio (VA/Alk) during the anaerobic digestion process of the differ-ent substrates. Table 3 shows the mean value and standard devia-tion of the pH and volatile acidity/alkalinity ratio in the efuents ofthe reactors with the different substrates and sludge types. The pHwas approximately constant across experiments, with a meanvalue of 7.72 0.24 using acidied glycerol-granular sludge,7.74 0.30 using acidied glycerol-non-granular sludge and7.81 0.17 with distilled glycerol-granular sludge. These values re-mained within the optimal range for methanogenic bacteria(Fannin, 1987; Wheatley, 1990). On the other hand, the volatileacidity/alkalinity ratio was always found to be lower than 0.300.40, thus indicating that the process operated favourably withoutthe risk of acidication (Balaguer et al., 1992).

    3.2. Methane yield coefcient and biodegradability of glycerol

    Methane yield coefcient was determined from the experimen-tal maximummethane volume produced (GT) and the nal and ini-tial COD, which were known in all loads. By tting (GT, CODremoved) value pairs to a straight line (Fig. 1), the methane yieldcoefcient coincides with the slope of the regression line andwas found to be 292 mL CH4/g COD removed (at 1 atm, 25 C) usinggranular sludge-acidied glycerol, 288 mL CH4/g COD removedusing non-granular sludge-acidied glycerol and 356 mL CH4/gCOD removed using granular sludge-distilled glycerol. Similar val-ues have been described in the literature. Gross and Lanting (1988),for example, studied the anaerobic digestion of wastewater gener-ated during the manufacturing of fuel ethanol from corn, whichcontained a highly soluble mixture of weak organic acids such asacetic, propionic, lactic and butyric acid. The organic strength ofthe wastewater typically ranged from 3000 to 5000 mg/L COD sol-uble. The methane yield coefcient was found to be 330 mL CH4/gCOD removed.

    According to Wheatley (1990), and considering the biomassgrowth and cell maintenance null, 382 mL of methane are theoret-ically produced (at 1 atm, 25 C) per gram of COD removed. Exper-imentally, the effectiveness of the process in each case was: 76%using granular sludge-acidied glycerol, 75% using non-granularsludge-acidied glycerol and 93% with granular sludge-distilledglycerol. When only taking into account these results, granularsludge-distilled glycerol would be the best option. Studying the re-moved COD percentage is as important as evaluating the methaneproduction coefcient in order to determine waste biodegradabil-ity. The high biodegradability of glycerol can be demonstrated by

    Table 3pH and volatile acidity/alkalinity ratio values in the efuents of the reactors obtainedfor the different substrates and sludge studied.

    Substrate and sludge type pH VA (eq acetic)/Alk(eq CaCO3)Acidied glycerol and granular sludge 7.72 0.24 0.05 0.02Acidied glycerol and non-granular sludge 7.74 0.30 0.03 0.01Distilled glycerol and granular sludge 7.81 0.17 0.05 0.01

  • plotting the amount of substrate removed against the substrateadded for each experiment. Fig. 2 shows these data in such a man-ner that the slope of the straight line denotes the percentage bio-degradability of the waste, which was found to be around 100%using granular sludge-acidied glycerol, 75% using non-granularsludge-acidied glycerol and 85% using granular sludge-distilledglycerol. These values remained constant throughout the loads.The biodegradability obtained with glycerol was high and similarto bioethanol wastewater, which achieved 86% biodegradability(Gross and Lanting, 1988).

    In all the cases, the reactors contained soluble and non-biode-gradable COD before adding the substrate. In the same gures,the initial non-biodegradable COD value was found to be 0.502 gCOD when granular sludge-acidied glycerol was used, 0.093 g

    COD when non-granular sludge-acidied glycerol was added and0.665 g COD for granular sludge-distilled glycerol.

    Taking into account both the methane production percentageand the waste biodegradability, the granular sludge-acidied glyc-erol would be the best choice. All the substrate concentrationwould be removed, thus producing methane and biomass growthand obtaining 100% biodegradability and 76% effectiveness in gen-erating methane. This fact would simultaneously ensure cell main-tenance and non-waste accumulation in the reactors.

    3.3. Kinetics of methane production

    Fig. 3 shows the variation in the volume of methane accumu-lated (G) as a function of time for the loads with the different sub-strates and sludge types. The variable concerned is the amount of

    Granular sludge-Acidified glycerol

    GT

    (mL C

    H 4 )

    200

    400

    600

    800

    1000

    YCH4/S removed = 292 mL CH4 / g COD removed

    Non-granular sludge-Acidified glycerol

    GT

    (mL C

    H 4 )

    0

    200

    400

    600

    800

    1000

    YCH4/S removed = 288 mL CH4 / g COD removed

    Granular sludge-Distilled glycerol

    800

    1000

    S re

    mov

    ed (g

    COD

    )

    m = 1.01 g COD removed / g COD STOr

    2 = 0.9946

    S re

    mov

    ed (g

    COD

    )

    m = 0.74 g COD removed / g COD STOr

    2 = 0.9542

    Granular sludge-Distilled glycerol3.0

    3.5

    x = 0.093 g COD

    0.5

    1.0

    1.5

    2.0

    2.5

    3.0

    3.5

    Non-granular sludge-Acidified glycerol

    3.5

    2.0

    1.5

    1.0

    2.5

    3.0

    0.5

    x = 0.502 g COD

    Granular sludge-Acidified glycerol

    5612 J.. Siles Lpez et al. / Bioresource Technology 100 (2009) 56095615S removed (g COD)0 1 2 3 4

    GT

    (mL C

    H 4 )

    0

    200

    400

    600

    YCH4/S removed = 356 mL CH4 / g COD removed

    Fig. 1. Variation of the experimental maximum methane volume produced (GT) (at

    1 atm, 25 C) with the COD consumed to obtain the methane yield coefcient of theprocess using granular sludge-acidied glycerol, non-granular sludge-acidiedglycerol and granular sludge-distilled glycerol.STO (g COD)0 1 2 3 4 5

    S re

    mov

    ed (g

    COD

    )

    m = 0.85 g COD removed / g COD STOr

    2 = 0.8925

    x = 0.665 g COD

    0.0

    0.5

    1.0

    1.5

    2.0

    2.5

    Fig. 2. Plot of the amount of substrate removed against the substrate added for all

    the experiments to obtain the biodegradability percentage of the by-product usinggranular sludge-acidied glycerol, non-granular sludge-acidied glycerol andgranular sludge-distilled glycerol.

  • substrate (g COD) added to the reactors. The results show that alarger volume of methane was produced when the substrate loadincreased and the time to complete the biodegradable fraction re-moval was 40 h for both sludge types.

    In order to characterize each set of experiments kinetically, andthus facilitate comparisons, the methane production rst order ki-netic model described by Borja et al. (1995) was used to t theexperimental data. According to this model, the volume of meth-ane accumulated (G) (mL, at 1 atm, 25 C) at a given time t (h) tsthe following equation:

    0

    tration of 25 4 mg PO34 =L was obtained. The occurrence of a sim-ilar inhibitory phenomenon has been described in the literature(Ito et al., 2005; Barbirato et al., 1998).

    G (m

    L CH

    4 )

    0

    400

    600

    800

    1000

    1200

    1400

    1.0 g COD 2.0 g COD 3.0 g COD

    G (m

    L CH

    4 )

    0

    200

    400

    600

    800

    1000

    1200

    1400

    1.0 g COD1.5 g COD 2.0 g COD3.0 g COD

    Non-granular sludge-Acidified glycerol

    Granular sludge-Distilled glycerol

    Time (h)0 10 20 30 40 50 60

    G (m

    L CH

    4 )

    0

    200

    400

    600

    800

    1000

    1200

    1400

    Granular sludge-Acidified glycerol

    1.0 g COD1.5 g COD 2.0 g COD

    Fig. 3. Variation of the methane volume accumulated (G) as a function of time forthe loads of 1.0, 1.5, 2.0 and 3.0 g COD using granular sludge-acidied glycerol, non-granular sludge-acidied glycerol and granular sludge-distilled glycerol.

    lar sludge, acidied glycerol-non-granular sludge and distilled glycerol-granular sludge.

    ) K 0G (h1) R2 KG (Lg1 h1)

    0.2075 0.0117 0.9676 0.03840.1858 0.0053 0.9909 0.03380.1316 0.0036 0.9798 0.0246

    0.1655 0.0177 0.9527 0.02030.1175 0.0151 0.9960 0.01780.1216 0.0091 0.9698 0.01900.0749 0.0059 0.9716 0.0128

    0.2006 0.0105 0.9548 0.0131

    J.. Siles Lpez et al. / Bioresource Technology 100 (2009) 56095615 5613G Gm1 expKG t 1where Gm is the maximummethane volume accumulated at an in-nite digestion time; G is zero at t = 0 and the rate of gas productionbecomes zero at t equal to innite.

    K 0G is an apparent kinetic constant for methane production (h1)

    which included the biomass concentration:

    K 0G KG X 2where KG is the specic methane production kinetic constant (L/gVSS h) and X is the biomass concentration (g VSS/L).

    As it was previously mentioned, a larger amount of gas was pro-duced as the load increased. Moreover, the slopes of the curves alsodecreased with increasing time. This decrease in the slopes withtime can be explained by the gradual decrease in the concentrationof biodegradable substrate. Eq. (1) shows a good t with the exper-imental data. Thus, it is correct to apply the proposed kinetic modelto all the loads studied. The values of K 0G and Gm for each load werecalculated numerically from the experimental data obtained bynon-linear regression using SigmaPlot (version 9.0) (Borja et al.,1995). Table 4 shows the Gm and K

    0G values and their standard devi-

    ations obtained in the experiments. The standard deviations of Gmand K 0G values were lower than 5% and 15%, respectively, for all theloads studied. This suggests that the proposed model adequatelyts the experimental data.

    Once the biomass concentration values were determined, thevalues of the specic methane production kinetic constant, KG,were calculated using Eq. (2). Table 4 also shows these KG values.The specic constant, KG, decreased with substrate concentrationfor all the experiments, showing an inhibition process. Specically,in the case of acidied glycerol, the KG value decreased by 42%when loading 1.03.0 g COD using granular sludge and by 58%using non-granular sludge. Surprisingly, a 76% decrease was ob-tained when loading between 1.0 and 2.0 g COD of distilled glyc-erol. This marked decrease in the KG value when using distilledglycerol could be explained by the accumulation in the reactorsof the alkalinity that was added as a nutrient. In contrast, the inhi-bition observed when using acidied glycerol could be the result oftwo causes: the increasing load or the accumulation of phospho-rous due to pre-treatment or even both effects simultaneously. Inorder to avoid the phosphorous concentration effect, CaO wasadded to the reactors during the experiments until a nal concen-

    Table 4Gm, K

    0G and KG values obtained for all the loads studied using acidied glycerol-granu

    Substrate and sludge type Load (g COD) Gm (mL CH4

    Acidied glycerol and granular sludge 1.0 323 52.0 592 53.0 857 7

    Acidied glycerol and non-granular sludge 1.0 318 111.5 451 82.0 590 153.0 789 20

    Distilled glycerol and granular sludge 1.0 375 6

    1.5 578 52.0 766 152000.1372 0.0048 0.9784 0.01010.0829 0.0046 0.9697 0.0068

  • Moreover, the theoretical values of maximummethane produc-tion (Gm) were calculated using Eq. (1) and plotted against theircorresponding experimental values (Fig. 4). The deviations ob-tained were lower than 5% in practically all cases, again suggestingthat the proposed model can be used to accurately predict thebehaviour of the process and that the kinetic parameters obtainedrepresented the microorganism behaviour affecting the anaerobicdigestion of this by-product.

    In order to compare the different substrates and sludge types,the specic methane production rate, r0 (mL CH4/g VSS h), was de-

    ned as the volume of methane generated per gram of volatile sus-pended solid and per hour for each set of experiments. As can beobserved in Fig. 5, the r0 values were always higher when usinggranular sludge and acidied glycerol at the same time. The lowestvalues were reached when using distilled glycerol with a high alka-linity concentration.

    3.4. Organic loading rate (OLR)

    The organic loading rate (OLR) for each substrate and sludgetype was determined using the experimental data. The OLR (kgCOD/m3 d) values were not excessively high for any set of experi-ments (0.922.00 kg COD/m3 d) when compared to other biode-gradable substrates. For food, organic chemical, soft drink andbakery wastes, which range from 5000 to 11,000 mg/L in rawCOD, removals of 6893% were obtained at organic loadings of327 kg COD/m3 d (Jeris, 1983).

    The OLR values, expressed as g COD/g VSS d, take into accountboth the biomass concentration and the organic load, thus makingthis an interesting variable for comparing the results. The highestvalues were obtained using granular sludge-acidied glycerol:0.210.38 g COD/g VSS d. On the contrary, when non-granularsludge-acidied glycerol was used, the ORL reached a value rang-ing from 0.12 to 0.26 g COD/g VSS d and 0.075 to 0.080 g COD/gVSSd when granular sludge-distilled glycerol was used. Fig. 6shows the evolution of the specic methane production kineticconstant, KG (L/g VSSh), with the organic loading rate (g COD/gVSSd) for each substrate and sludge type studied. A strong de-crease in the KG value was observed when the distilled glycerolOLR was increased. Although the constant also decreased whenusing acidied glycerol, the kinetic constant slowdown was much

    Gm

    (mL CH4 )0 200 400 600 800 1000

    GT

    expe

    rimen

    tal (m

    L CH 4

    )

    0

    200

    400

    600

    800

    1000

    m = 0.9942Confidence intervals of 95%

    Fig. 4. Comparison between the experimental maximum methane production (GT)values and the theoretical values (Gm) predicted by Eq. (1).

    ol

    5614 J.. Siles Lpez et al. / Bioresource Technology 100 (2009) 560956151.0 g COD

    CH 4

    /g

    VSS

    h)

    6

    8

    10

    12

    14

    Granular sludge-Acidified glycerolNon-granular sludge-Acidified glycerGranular sludge-Distilled glycerolr' (m

    L

    0

    2

    4

    2.0 g COD

    Time (h)

    r' (m

    L CH 4

    /g

    VSS

    h)

    0

    5

    10

    15

    20

    25

    0 10 20 30 40 50 60

    Fig. 5. Variation of the specic methane production rate, r0 , as a function of time for all t1.5 g COD

    3.0 g COD

    70 0 10 20 30 40 50 60 70Time (h)

    he loads using granular and non-granular sludge and acidied and distilled glycerol.

  • Teclower. Additionally, this substrate has allowed the reactors to oper-ate in a stable way with higher OLR. Finally, according to economiccriteria, acidied glycerol is the target substrate. Given that thegranular sludge KG shows values that are twice as high as thenon-granular sludge, the glycerol anaerobic treatment could becarried out using granular sludge in addition to acidied glycerol.

    However, one option to improve the OLR could be to mix glyc-erol with another organic waste such as wastewater generatedduring the biodiesel manufacturing process and study its co-diges-tion. This research would form part of what is known as the Bior-enery concept; a relatively new term referring to the conversionof feedstock into a host of valuable chemicals and energy. Indeed,this is considered an important strategy for achieving sustainabledevelopment.

    4. Conclusions

    The results obtained through this research study reveal thatglycerol-containing waste derived from biodiesel production afterpre-treatment has a high level of anaerobic biodegradability andthat a substantial quantity of methane can be obtained this way.The highest methane yield coefcient was obtained using granularsludge-distilled glycerol, but a strong inhibition was observed

    OLR (g COD/g VSSd)

    KG (L

    /g VS

    Sh)

    Granular sludge-Acidified glycerolNon-granular sludge-Acidified glycerolGranular sludge-Distilled glycerol

    0.00

    0.01

    0.02

    0.03

    0.04

    0.05

    0.06

    0.0 0.1 0.2 0.3 0.4 0.5

    Fig. 6. Variation of the specic kinetic constant KG with the Organic Loading Rate(OLR), using granular and non-granular sludge and acidied and distilled glycerol.

    J.. Siles Lpez et al. / Bioresourcewhen the load was increased. The use of granular sludge and acid-ied glycerol was found to be the best option for revalorising glyc-erol anaerobically given that biodegradability was found to bearound 100% and the methane yield coefcient was 0.306 m3

    CH4/kg acidied glycerol (at 1 atm, 25 C).

    Acknowledgements

    The authors are very grateful to the BIDA S.A. Factory (Fuentesde Andaluca, Seville, Spain) and to the Spanish Ministry of Scienceand Innovation for funding Jos ngel Siles Lpez through ProjectCTM2005-01293 and Grant BES2006-14074. This study was co--nanced by the European Social Fund. We also wish to expressour gratitude to the laboratory technician Inmaculada BellidoPadillo for her help.

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    Anaerobic digestion of glycerol derived from biodiesel manufacturingIntroductionMethodsExperimental set-upGlycerolRaw material pre-treatmentAnaerobic digesters. Experimental procedureChemical analysesSoftware

    Results and discussionStabilityMethane yield coefficient and biodegradability of glycerolKinetics of methane productionOrganic loading rate (OLR)

    ConclusionsAcknowledgementsReferences

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