evaluation of methane production and macronutrient degradation in the anaerobic co-digestion of...

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Evaluation of methane production and macronutrient degradation in the anaerobic co-digestion of algae biomass residue and lipid waste Stephen Park, Yebo Li Department of Food, Agricultural and Biological Engineering, The Ohio State University, Ohio Agricultural Research and Development Center, 1680 Madison Ave., Wooster, OH 44691-4096, USA article info Article history: Received 17 December 2011 Received in revised form 26 January 2012 Accepted 29 January 2012 Available online 6 February 2012 Keywords: Anaerobic digestion Co-digestion Microalgae Methane Macronutrient abstract Algae biomass residue was co-digested with lipid-rich fat, oil, and grease waste (FOG) to evaluate the effect on methane yield and macronutrient degradation. Co-digestion of algae biomass residue and FOG, each at 50% of the organic loading, allowed for an increased loading rate up to 3 g VS/L d, resulting in a specific methane yield of 0.54 L CH 4 /g VS d and a volumetric reactor productivity of 1.62 L CH 4 /L d. Lipids were the key contributor to methane yields, accounting for 68–83% of the total methane poten- tial. Co-digestion with algae biomass residue fractions of 33%, 50%, and 67% all maintained lipid degra- dations of at least 60% when the organic loading rate was increased to 3 g VS/L d, while synergetic effects on carbohydrate and protein degradation were less evident with increased loading. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction As microalgal lipid-derived fuel appears to be a promising alter- native to petroleum-based liquid fuel (Singh et al., 2011), the utiliza- tion or disposal of post-extraction microalgal biomass, or algae biomass residue (ABR), is a critical issue when considering the com- mercialization of the fuel production process. ABR, which accounts for approximately 65% of the harvested biomass (Subhadra and Edwards, 2011), can generate additional energy through anaerobic digestion. By integrating anaerobic digestion as an on-site operation with algal lipid production, the efficiency of converting photosyn- thetically captured energy into more accessible forms can be increased. In addition, the residual effluent from the anaerobic digester can be used as a nitrogen and phosphorus source for micro- algal growth, thus reducing the costs for nutrients as well as treating the waste stream. Accordingly, the synergetic integration of anaero- bic digestion with microalgae production has been previously explored to improve the commercial potentials of both technologies (Golueke and Oswald, 1959; Mussgnug et al., 2010; Ras et al., 2011; Sialve et al., 2009; Zamalloa et al., 2011). Species-dependent nutri- ent proportions of carbohydrates (5–23%), proteins (6–52%), and lipids (7–23%) (Brown et al., 1997) affect the potential of microalgae as a substrate for anaerobic digestion. Based on species and nutrient composition, the theoretical methane potential of unprocessed whole algae is estimated to be approximately 0.47–0.80 L CH 4 /g VS d (Sialve et al., 2009). How- ever, studies on the semi-continuous digestion of whole algae typ- ically report lower specific methane yields (SMY) ranging from 0.09 to 0.65 L CH 4 /g VS d, and suggest inhibitory factors including high cell wall resistance to bacterial attack and ammonia toxicity derived from high concentrations of protein (Golueke et al., 1957; Golueke and Oswald, 1959; Yen and Brune, 2007). The recal- citrance of the algal cell wall structure can be overcome by adding a pretreatment step prior to subjecting the substrate to hydrolytic, acetogenic, and methanogenic microorganisms. Thermal pretreat- ment of microalgae at 100 °C for 8 h was proposed to improve the methane production efficiency by 33% (Chen and Oswald, 1998). Samson and LeDuy (1983b) also showed an improvement with pretreatments including ultrasonic lysis, disintegration, and thermochemical treatments with acid and alkali. However, exces- sive energy input to maximize the methane conversion might neg- atively impact the economic feasibility of this technology. Issues that stem from high protein concentrations in the sub- strate can be moderated through co-digestion, a less energy- demanding alternative that can improve the anaerobic digestion performance by adding a secondary substrate that supplies addi- tional nutrients which the initial substrate lacks. The combination of two or more substrates creates a synergistic effect by alleviating the preexisting nutrient imbalance and, in turn, attenuating the inhibition that would otherwise occur during digestion of the indi- vidual substrate. In the anaerobic digestion of microalgal biomass, which generally contains superfluous nitrogen, the addition of a carbon-rich co-substrate may facilitate the methane conversion 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2012.01.160 Corresponding author. Tel.: +1 330 263 3855. E-mail address: [email protected] (Y. Li). Bioresource Technology 111 (2012) 42–48 Contents lists available at SciVerse ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech

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Bioresource Technology 111 (2012) 42–48

Contents lists available at SciVerse ScienceDirect

Bioresource Technology

journal homepage: www.elsevier .com/locate /bior tech

Evaluation of methane production and macronutrient degradationin the anaerobic co-digestion of algae biomass residue and lipid waste

Stephen Park, Yebo Li ⇑Department of Food, Agricultural and Biological Engineering, The Ohio State University, Ohio Agricultural Research and Development Center, 1680 Madison Ave.,Wooster, OH 44691-4096, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 17 December 2011Received in revised form 26 January 2012Accepted 29 January 2012Available online 6 February 2012

Keywords:Anaerobic digestionCo-digestionMicroalgaeMethaneMacronutrient

0960-8524/$ - see front matter � 2012 Elsevier Ltd. Adoi:10.1016/j.biortech.2012.01.160

⇑ Corresponding author. Tel.: +1 330 263 3855.E-mail address: [email protected] (Y. Li).

Algae biomass residue was co-digested with lipid-rich fat, oil, and grease waste (FOG) to evaluate theeffect on methane yield and macronutrient degradation. Co-digestion of algae biomass residue andFOG, each at 50% of the organic loading, allowed for an increased loading rate up to 3 g VS/L d, resultingin a specific methane yield of 0.54 L CH4/g VS d and a volumetric reactor productivity of 1.62 L CH4/L d.Lipids were the key contributor to methane yields, accounting for 68–83% of the total methane poten-tial. Co-digestion with algae biomass residue fractions of 33%, 50%, and 67% all maintained lipid degra-dations of at least 60% when the organic loading rate was increased to 3 g VS/L d, while synergeticeffects on carbohydrate and protein degradation were less evident with increased loading.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

As microalgal lipid-derived fuel appears to be a promising alter-native to petroleum-based liquid fuel (Singh et al., 2011), the utiliza-tion or disposal of post-extraction microalgal biomass, or algaebiomass residue (ABR), is a critical issue when considering the com-mercialization of the fuel production process. ABR, which accountsfor approximately 65% of the harvested biomass (Subhadra andEdwards, 2011), can generate additional energy through anaerobicdigestion. By integrating anaerobic digestion as an on-site operationwith algal lipid production, the efficiency of converting photosyn-thetically captured energy into more accessible forms can beincreased. In addition, the residual effluent from the anaerobicdigester can be used as a nitrogen and phosphorus source for micro-algal growth, thus reducing the costs for nutrients as well as treatingthe waste stream. Accordingly, the synergetic integration of anaero-bic digestion with microalgae production has been previouslyexplored to improve the commercial potentials of both technologies(Golueke and Oswald, 1959; Mussgnug et al., 2010; Ras et al., 2011;Sialve et al., 2009; Zamalloa et al., 2011). Species-dependent nutri-ent proportions of carbohydrates (5–23%), proteins (6–52%), andlipids (7–23%) (Brown et al., 1997) affect the potential of microalgaeas a substrate for anaerobic digestion.

Based on species and nutrient composition, the theoreticalmethane potential of unprocessed whole algae is estimated to be

ll rights reserved.

approximately 0.47–0.80 L CH4/g VS d (Sialve et al., 2009). How-ever, studies on the semi-continuous digestion of whole algae typ-ically report lower specific methane yields (SMY) ranging from0.09 to 0.65 L CH4/g VS d, and suggest inhibitory factors includinghigh cell wall resistance to bacterial attack and ammonia toxicityderived from high concentrations of protein (Golueke et al.,1957; Golueke and Oswald, 1959; Yen and Brune, 2007). The recal-citrance of the algal cell wall structure can be overcome by addinga pretreatment step prior to subjecting the substrate to hydrolytic,acetogenic, and methanogenic microorganisms. Thermal pretreat-ment of microalgae at 100 �C for 8 h was proposed to improvethe methane production efficiency by 33% (Chen and Oswald,1998). Samson and LeDuy (1983b) also showed an improvementwith pretreatments including ultrasonic lysis, disintegration, andthermochemical treatments with acid and alkali. However, exces-sive energy input to maximize the methane conversion might neg-atively impact the economic feasibility of this technology.

Issues that stem from high protein concentrations in the sub-strate can be moderated through co-digestion, a less energy-demanding alternative that can improve the anaerobic digestionperformance by adding a secondary substrate that supplies addi-tional nutrients which the initial substrate lacks. The combinationof two or more substrates creates a synergistic effect by alleviatingthe preexisting nutrient imbalance and, in turn, attenuating theinhibition that would otherwise occur during digestion of the indi-vidual substrate. In the anaerobic digestion of microalgal biomass,which generally contains superfluous nitrogen, the addition of acarbon-rich co-substrate may facilitate the methane conversion

S. Park, Y. Li / Bioresource Technology 111 (2012) 42–48 43

process. For example, the addition of carbon-rich waste paper to amixture of Scenedesmus spp. and Chlorella spp. resulted in an im-proved methane yield induced by a balance between carbon andnitrogen in the feed, as well as increased cellulase activity (Yenand Brune, 2007). Soybean oil and glycerin, both rich in carbon,also proved to have positive effects on biogas yield when addedto algae harvested from wastewater treatment ponds (Salernoet al., 2009). Similar studies have been performed with cyanobac-teria, which are not classified in the same eukaryotic domain as al-gae, but share similar traits such as photosynthetic metabolismand high protein content (Samson and LeDuy, 1986). Co-digestionof Spirulina maxima with a carbon-rich sewage sludge at a 50% ratioincreased the SMY by over twofold (Samson and LeDuy, 1983a). Incontrast, combining swine manure and algal biomass, both withhigh nitrogen contents, did not result in significant performanceimprovement (González-Fernández et al., 2011).

When selecting the substrates to be used in co-digestion, it iscritical that the co-substrates be mutually propitious. An idealco-substrate for ABR should have a high carbon-to-nitrogen ratioto minimize the inhibitory effects of ammonia, and should syn-chronously benefit from the ancillary nutrients as well. Reducingammonia inhibition is a key issue in the digestion of ABR, becauseABR may experience more severe ammonia inhibition than wholealgae, due to higher protein content. Materials that contain signif-icant lipid proportions can be such an ideal co-substrate. Com-pared to carbohydrates and proteins, lipids have high methanepotential (Sialve et al., 2009), but their low alkalinity and bufferingcapacity make lipids more susceptible to inhibition caused byintermediate products such as long chain fatty acids (LCFAs) andvolatile fatty acids (VFAs). LCFAs, which are enzymatically de-tached from the glycerol backbone of triglycerides during anaero-bic digestion, are reported to retard microbial activity bydisorienting essential groups on the cell membrane (Galbraithand Miller, 1973). VFAs, in high concentrations, can be detrimentalto anaerobic digestion and thus are often used as process indica-tors (Ahring et al., 1995). Therefore, the co-digestion of ABR anda lipid-rich material would concurrently offset the carbon andnitrogen imbalance found in ABR as well as the lack of alkalinityin the lipids, creating a synergistic effect (Boubaker and Ridha,2007; Yen and Brune, 2007). The well-maintained nutrient balanceof the digester inflow could also allow a higher loading capacity,improving the economic feasibility of the system.

In this study, lipid-rich fat, oil, and grease waste (FOG)was hypothesized to be an effective co-substrate in the semi-continuous anaerobic digestion of the residue of Nannochloropsissalina, an algal species known for its potential as a biodiesel feed-stock due to its high lipid content and biomass productivity(Gouveia and Oliveira, 2009). The objectives of this study were to(1) evaluate the effects of ABR loading fraction and organic loadingrate (OLR) on methane production, expressed in both SMY, volu-metric reactor productivity (VRP), and methane content; and (2)identify the synergetic effects of co-digestion through the degrada-tion of carbohydrates, protein, and lipids.

Table 1Characteristics of substrates and inoculant.

Characteristic Unit ABR FOG Inoculant

Total solids %, w/w 14.5 ± 0.0 9.3 ± 0.1 10.1 ± 0.0Volatile solids % of TS, w/w 77.2 ± 0.2 85.3 ± 0.1 55.4 ± 0.1pH – 5.9 ± 0.0 4.4 ± 0.0 8.1 ± 0.0Total carbon % of TS, w/w 37.0 ± 0.0 58.1 ± 0.0 45.7 ± 0.0Total nitrogen % of TS, w/w 8.4 ± 0.0 3.5 ± 0.0 6.6 ± 0.0Carbon-to-nitrogen

ratio– 4.4 ± 0.0 16.5 ± 0.0 7.0 ± 0.0

Total carbohydrates % of TS, w/w 4.1 ± 0.4 0.8 ± 0.0 1.3 ± 0.0Total crude lipids % of TS, w/w 14.6 ± 0.1 75.4 ± 10.1 10.5 ± 4.8Total crude protein % of TS, w/w 47.9 ± 1.2 10.2 ± 1.0 50.9 ± 1.1Ammonia (NH3, NH4

+) g/L 15.2 ± 0.1 8.4 ± 2.1 –

2. Methods

2.1. Substrates and inoculant

The ABR used in this study was derived from N. salina, grown inphotobioreactors and harvested by centrifugation (TouchstoneResearch Laboratory, Triadelphia, WV, USA). The continuous cen-trifuge was operated under a relative centrifugal force of12,000 g and a volumetric throughput of 0.19 L/s. Lipids were ex-tracted from the harvested algal biomass by means of electric pul-sation, acid hydrolysis, and solvent recovery using hexane (SRS

Energy, Dexter, MI, USA) and the resulting ABR slurry was directlyused as an anaerobic digestion substrate. FOG was collected from alocal oil receiving facility (Recycling and Treatment Technologiesof Ohio, Painesville, OH, USA). Both substrates were stored intightly sealed plastic 19-L buckets at 4 �C prior to use. Effluent froma commercial scale digester fed with wastewater solids (SchmackBioEnergy, Akron, OH, USA) was used as an inoculant, providingthe microbial consortia to degrade the organic compounds to bio-gas. The acetotrophic methanogen content was found to beapproximately 2.1 � 108 cells/g VS, using the most-probable-num-ber method (Balch et al., 1979). The characteristics of the materialsused in the anaerobic co-digestion of algal biomass residue andFOG are listed in Table 1.

2.2. Operational procedures

Semi-continuous anaerobic digestion was performed in 1-Lglass bottles, which were capped with rubber stoppers havingtwo outlet ports – one connected to a polyvinyl fluoride gas bag(CEL Scientific Corp., Santa Fe Springs, CA, USA) and the other hav-ing a Luer taper cap for the anaerobic transfer of feed and effluent.The reactors were constantly agitated in an Innova 43R incubatedshaker (New Brunswick, Edison, NJ, USA) at 150 rpm and 37 �C. Tocreate an anaerobic environment, each reactor was initially filledwith 0.70 L of inoculant and subsequently flushed with nitrogengas for 2 min. The reactors were agitated and incubated at 37 �Cfor 3 days to acclimate the microbes to the environment beforeadding any substrate.

Once the microbes were acclimated and steady daily gas produc-tion was confirmed, the digesters were unloaded and loaded on adaily basis. A fixed amount of effluent as designed was removeddaily from each reactor and stored at �20 �C for further analysis.Each reactor was then fed with an equal amount of substrate usinga 50-mL polypropylene syringe with a Luer taper (BD, FranklinLakes, NJ, USA). Twenty different feeding formulas were prepared,each corresponding to one of four OLRs (2, 3, 4 and 6 g VS/L d)and one of five ABR loading fractions (0%, 33%, 50%, 67%, and 100%of the total organic load). The hydraulic retention times (HRT) ofthe reactors with OLRs of 2, 3, 4, and 6 g VS/L d were 40, 27, 20,and 13 days, respectively. Each treatment had two replicates. Inorder to ensure sufficient mixing in the reactor, the syringes werepurged five times before reactor unloading and after feeding. Thepolyvinyl fluoride bags connected to the reactors were removeddaily for biogas volume measurement and composition analysis,and the emptied bags were reconnected to the reactors afterwards.

2.3. Analytical procedures

2.3.1. Total solids, volatile solids, pH, total carbon and nitrogenTotal solids (TS) content, volatile solids (VS) content, and pH were

measured according to the Standard Methods for the Examination of

44 S. Park, Y. Li / Bioresource Technology 111 (2012) 42–48

Water and Wastewater (APHA, 2005). One gram of each sample wasplaced in a porcelain crucible and dried in a Thelco Model 18 oven(Precision Scientific, Chennai, India) at 105 �C for 4 h. After eachsample was brought to constant weight, it was cooled in a desiccatorand weighed to obtain TS content. The ash weight was obtained byigniting the sample in an Isotemp muffle furnace (Fisher Scientific,Dubuque, IA, USA) at 500 �C for 4 h, then cooled and weighed. VScontent was determined as the difference between the dry weightand ash weight. The pH of each reactor was measured using anAP110 portable pH meter, equipped with an Ag/AgCl reference elec-trode probe (Accumet, Singapore). A Vario MAX CNS elemental ana-lyzer (Elementar Analyseneyeteme GmbH, Hanau, Germany) wasused for the dry combustion assay of total carbon (ISO, 1995) and to-tal nitrogen (Sweeney, 1989).

2.3.2. Total carbohydratesTotal carbohydrate content was measured according to ASTM

standard method E1758-01 (ASTM, 2007), where the availablecarbohydrates were converted to their monomeric counterparts byacid hydrolysis and then quantified by high performance liquidchromatography (HPLC). An LC-20AB HPLC unit (Shimadzu, Colum-bia, MD, USA) equipped with an Aminex� HPX-87P column (Bio-RadLaboratories, Hercules, CA, USA) was used with a refractive indexdetector. Deionized water was used as the mobile phase at a flowrate of 0.6 mL/min. The temperature of the column and detectorwere maintained at 80 and 55 �C, respectively.

2.3.3. Ammonia–nitrogen and total crude proteinAmmonia–nitrogen (NH3–N) was determined by a procedure

adapted from EPA Method 350.2 (EPA, 1974) and AOAC Interna-tional Method 973.49 (Horwitz and Latimer, 2005). In order to lib-erate the ammonia, 50 mL of 6.0 N sodium hydroxide was added toeach sample diluted with deionized water. The ammonia was dis-tilled into 4% boric acid solution within a B-324 distillation unit(Büchi Labortechnik AG, Flawil, Switzerland) and titrated with0.01 N hydrochloric acid, using a DL 22 titrator (Mettler-ToledoInc., Columbus, OH, USA). Total Kjeldahl nitrogen (TKN) was ob-tained through digestion with 98% sulfuric acid in a Tecator diges-ter (FOSS, Eden Prairie, MN, USA), followed by distillation andtitration. Total crude protein was determined with AOAC Interna-tional Method 2001.11 (Horwitz and Latimer, 2005), in which theTKN was multiplied by a conversion factor of 6.25.

2.3.4. Total crude lipidsSolvent extractives were analyzed with a modified Bligh and

Dyer (1959) method (White et al., 1979). A liquid sample with avolume of 6.5 mL was mixed with 8 mL of chloroform and 16 mLof anhydrous methanol. If the original sample was solid, 6.5 mLof phosphate buffer (prepared with 8.7 g K2HPO4/L neutralizedwith 1 N HCl to pH 7.4) was added to 1.5 g of sample prior to mix-ing with chloroform and methanol. After vortex mixing for 5 min,each sample was allowed to partition for a minimum of 2 h. Anadditional 8 mL of chloroform and 8 mL of deionized water wereadded to the suspension, mixed and allowed to separate for 24 h.The upper aqueous phase was removed by suction, and the organicphase was recovered with a Gooch filter crucible, lined with filterpaper. The proportions of water:methanol:chloroform were main-tained to be 0.8:2:1 (v/v) for the single phase extraction and0.9:1:1 (v/v) after separation into the second phase. Solvents wereremoved from the lipids with an Isotemp 282A vacuum oven (Fish-er Scientific, Marietta, OH, USA) at 40 �C and recovered with a con-densing apparatus.

2.3.5. Biogas volume and compositionGas volume was measured daily by liquid displacement. Using a

Dyna-Pump Model #3 vacuum pump (Neptune Products Inc.,

Dover, NJ, USA), gas from each polyvinyl fluoride bag was trans-ferred to a custom-made, graduated glass tube (Adria ScientificGlassworks Inc., Geneva, OH). The glass tube was filled with amixed solution of 3% v/v H2SO4 and 20% w/v Na2SO4�10H2O to pre-vent the solubilization of carbon dioxide or methane. Proportionsof methane, carbon dioxide, oxygen, and nitrogen in the collectedbiogas were measured with a HP 6890 gas chromatograph (AgilentTechnologies, Santa Clara, CA) equipped with a 3.05 m, stainlesssteel, 45/60-mesh 13� molecular sieve column and a thermalconductivity detector. Helium was used as the carrier gas, with aflowrate of 5.2 mL/min. The temperatures of the injector anddetector were set to 150 and 200 �C, respectively. For each sampleinjection, the column temperature was held at 40 �C for 2 min,increased to 60 �C at a rate of 20 �C/min, and held for 5 min.

2.4. Statistical analysis

Using a method suggested by Yum and Pierce (1997), steadystate values for all response variables were determined by locatinga time point where the slope of the response versus time curve didnot statistically differ from zero. Comparisons for all steady statevalues were performed using the Tukey–Kramer method, whileanalysis of variance tests were performed with a significance levelof 0.05 using JMP� 9.0.0 (SAS Institute Inc., Cary, NC, USA).

3. Results and discussion

3.1. Effects of co-digestion on methane yield and content

3.1.1. Methane yieldSMY, expressed in L CH4/g VS�d, is used to express how effec-

tively the substrate is converted to methane; while VRP, expressedin L CH4/L�d, shows how much methane can be produced per unitreactor working volume. The optimization of both response param-eters is important in maximizing the efficiency of the anaerobicdigestion process. The comparison of the steady state SMY andVRP resulting from varying ABR loading fractions and OLRs isshown in Fig. 1. All steady state values were achieved betweenone and two HRTs after initial feeding. Among all feed formulas,100% FOG had the highest SMY of 0.69 L CH4/g VS d at an OLR of2 g VS/L d. The SMY of 100% ABR was no higher than 0.13 LCH4/g VS d, implying FOG as a more readily digestible substratewith a higher methane potential. It was noted that the SMY of100% ABR was on the lower end of the spectrum when comparedto those reported for whole microalgae (Golueke et al., 1957; Gol-ueke and Oswald, 1959; Yen and Brune, 2007), mainly due to thedecreased energy content after removing lipids from the cells.For both substrates, increasing the OLR to higher than 2 g VS/L d re-sulted in digester failure – characterized by a steady state SMY va-lue of zero – indicating possible inhibition of the methanogeniccommunities when subject to overloading.

In comparison to the digestion of 100% FOG and 100% ABR, co-digestion allowed an increase in OLR up to 3 g VS/L d. At an OLR of3 g VS/L d, the co-digestion of 50% ABR and 50% FOG resulted in aSMY of 0.54 LCH4/gVSd, a significant improvement in digestionperformance compared to 100% FOG or 100% ABR at the same load-ing rate (p < 0.05). However, when the OLR was greater than 3 gVS/L d, the same inhibitory behavior as seen in the digestion of100% FOG or 100% ABR was observed. The SMY of 50% ABR at anOLR of 3 g VS/L d was 22% less than that of 100% FOG at 2 gVS/L d, but the VRP displayed a 17% increase from 1.38 to 1.62 LCH4/L d. These results suggest that scale-up of co-digestion mayallow an increased organic feed throughput and biogas productiv-ity without an additional increase in processing capacity. As shownin Fig. 1, the ABR loading fraction of 50% resulted in optimal

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Fig. 1. Steady state (a) SMY and (b) VRP at varying ABR loading fractions and OLR.

Fig. 2. Steady state methane content at varying ABR loading fractions and OLR.

S. Park, Y. Li / Bioresource Technology 111 (2012) 42–48 45

responses when increasing the OLR. When the OLR was increasedfrom 2 to 3 g VS/L d for a feed formula with more FOG (67%) thanABR (33%), there was a 76% and 64% decrease in SMY and VRP,respectively, similar to what was observed for 100% FOG. The in-crease in ABR loading fraction from 0% to 50% appeared to amelio-rate the adverse impact of overloading lipids. On the other hand,when the OLR was increased from 2 to 3 g VS/L d for a feed formulawith more ABR (67%) than FOG (33%), there was a 26% decrease inSMY and a 8% increase in VRP. The ameliorative effect of ABR addi-tion diminished when there was excess ABR. Thus, it was deter-mined that the optimal condition for the co-digestion of ABR andFOG with maximum throughput was in the vicinity of a 50% ABRloading fraction.

3.1.2. Methane contentIn addition to SMY and VRP, the methane content in the biogas

should be sufficiently high to ensure high process efficiency andminimize purification needs. High methane content in an anaero-bic digester implies a steady balance of methane and carbon diox-ide, which are products of methanogenesis and acetogenesis,respectively. On the contrary, low methane content implies someform of inhibition that diminishes the methanogenic activity with-in the microbial consortium. It is shown in Fig. 2 that there was anoptimal ABR loading fraction of 50% at a given OLR that resulted ingreater methane content than other feeding formulas. Co-digestionat 50% ABR may have promoted an optimal balance between meth-anogenesis and acetogenesis so that the production of methanerelative to that of carbon dioxide could be maximized. Overall,methane contents ranged from 33% to 69%. Digesters with meth-ane contents of 60% or greater had ABR loading fractions less than100% and OLRs less than 4 g VS/L d. Methane contents of 60% or

greater were also associated with the reactors with greater meth-ane production, having SMYs of at least 0.27 LCH4/gVSd and VRPsof at least 0.73 L CH4/Ld. Although the data is not shown, it wasnoted that there was a moderately positive correlation (R2 = 0.60)between methane content and digester pH. Similar to pH, methanecontent can be used as a means of indicating the health and stabil-ity of the anaerobic digestion process.

3.2. Effects of co-digestion on nutrient reduction

3.2.1. CarbohydratesAs shown in Fig. 3a, the co-digestion of ABR and FOG resulted in

carbohydrate reductions ranging between 71% and 88%. Whendigesting 100% ABR, an average of 83% of the carbohydrates wereconverted to degradation products, whereas the digestion of100% FOG had an average carbohydrate reduction of 53%. At a gi-ven loading rate, there was no evident increase (p > 0.05) in carbo-hydrate reduction when performing co-digestion; however, thedecrease in reduction with increasing OLR was less severe in thepresence of ABR (p < 0.05). The carbohydrate reduction decreasedby 27% when increasing the OLR of 100% FOG from 2 to 4 g VS/L d, while reactors that included some portion of ABR had reduc-tions of 14% or less when the OLR was increased from 2 to 4 gVS/L d. In a mixed substrate environment, microbial growth willtend to occur more in communities that favor the dominant sub-strate (Hobson et al., 1974). In the case of 100% FOG, the consump-tion of lipids may have been preferred over that of carbohydratesfor the microbes that were able to metabolize both substrates. Cou-pled with an increased removal of microbes due to a greater volu-metric displacement at higher OLRs, the adverse impact oncarbohydrate reduction was more evident in 100% FOG than inother feed formulas.

3.2.2. LipidsThe positive effect of co-digestion on the degradation of lipids

was more evident than on carbohydrates. Fig. 3b shows that themean lipid degradation during co-digestion was greater than thatfor either 100% FOG or 100% ABR at OLRs of 3 and 4 g VS/L d. At100% FOG, 70% of lipids were reduced at an OLR of 2 g VS/L d,but the degree of reduction drastically decreased by 41% whenthe OLR was increased to 4 g VS/L d. The lipid degradation fromthe digestion of 100% ABR was only as high as 42% at OLR of 2 gVS/L d, and was not significantly affected by OLR (p > 0.05). By

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0% ABR 33% ABR 50% ABR 67% ABR 100% ABR100% FOG 67% FOG 50% FOG 33% FOG 0% FOG

ig. 4. NH3–N in the digester effluent per unit mass of protein fed at varying ABRading fractions and OLR.

46 S. Park, Y. Li / Bioresource Technology 111 (2012) 42–48

combining the two substrates, high lipid degradations of at least60% were maintained while simultaneously allowing increasedOLRs of up to 3 g VS/L d. When the OLR was further increased to4 g VS/L d, there were drastic decreases in lipid reduction at ABRfractions of 33% and 67%, but the decrease was less in the digestionof 50% ABR, resulting in a lipid degradation of 59%. Consideringthat the FOG was comprised of 75% lipids and most of the chemicalenergy in the feedstock was derived from the lipids, it could beconcluded that maintaining the lipid degradation efficiency is thecritical factor to allow increased OLR in the anaerobic digesters.By performing co-digestion, an optimal nutrient and alkalinity bal-ance for lipolytic activity was obtained, hence increasing the lipidreduction.

3.2.3. ProteinsIn comparison with carbohydrates, proteins were a more signif-

icant factor in the nutrient composition, accounting for 48% of thetotal solids in ABR and 10% in that of FOG. In the anaerobic diges-tion process, proteins in the substrate are broken down into aminoacids, while proteins in the form of microbial biomass are con-stantly synthesized as a result of heterotrophic metabolism. Thus,the total crude protein content in the digester effluent cannotclearly explain the protein reduction from the substrate. However,ammonia is a major by-product of protein degradation, and can beused as an indicator to assess the amount of protein that was re-duced in the digester. The amount of NH3–N in the effluent per unitmass of protein fed was used to quantify the degree of protein deg-radation from the feed stream without taking into account themicrobial generation. Although the total amount of ammonia re-leased from 100% ABR was greater than that from 100% FOG, it isshown in Fig. 4 that feed with 100% FOG produced up to 2.4 timesmore ammonia per unit mass of protein than that with 100% ABR,implying that protein degradation was relatively more active inFOG than in ABR. It was also noted that increasing the OLR of

Flo

100% FOG from 3 to 4 g VS/L d decreased the amount of NH3–N inthe effluent per unit mass of protein fed by 38%, while for 100%ABR the increase in OLR did not result in any significant decreasein NH3–N (p > 0.05). Similar to carbohydrates, the reduction of pro-teins in the substrate was less affected by OLR when the proportionof FOG was less than 100%.

In addition to comparing the degree of protein degradation, itwas also important to observe the ammonia concentration withinthe digester. Elevated NH3–N concentrations in the range of 4051–5734 mg/L resulted in a 56.5% loss of methanogenic activity in thesemi-continuous anaerobic digestion of potato juice (Koster andLettinga, 1988). The NH3–N concentration in the digester effluentfrom the current study was 1756–4968 mg/L, suggesting the possi-bility of ammonia inhibition in some of the digesters. A highammonia concentration in the digester medium not only has atoxic effect to methanogenic archaea, but also decreases the deam-ination activity of proteolytic bacteria (Gallert et al., 1998). Thecombination of FOG and ABR only appeared to create a dilution ef-fect, rather than improving the degradation activity.

3.3. Assessment of co-digestion performance with theoretical methanepotential

3.3.1. Evaluation of specific methane yieldThe SMY of each feed formula was compared to its theoretical

methane potential, calculated based on the yield estimates of car-bohydrates, lipids, and proteins. Angelidaki and Sanders (2004)were able to obtain theoretical methane potentials of these sub-strate components based on the following equation adapted fromSymons and Buswell (1933):

CaHbNd þ4a� b� 2c þ 3d

4

� �H2O

! 4aþ b� 2c � 3d8

� �CH4 þ

4a� bþ 2c þ 3d4

� �CO2

þ dNH3 ð1Þ

Although the methane potentials from Eq. (1) do not take intoaccount the nutrients required for cell maintenance, the degreeof possible conversion can be assessed from the calculated values.The steady state values of SMY from Fig. 1 were compared to thecorresponding methane potential for each feed configuration,shown in Fig. 5. An OLR of 2 g VS/L d resulted in SMYs of at least90% of the calculated methane potential, with the exception of

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 33 50 67 100

The

oret

ical

met

hane

pot

entia

l (L

CH

4/g

VS

add

edd)

ABR loading fraction (% of total organic load)

Carbohydrates Protein Lipids

Fig. 5. Contributions of carbohydrates, protein and lipids on the theoreticalmethane potentials of various feed formulas.

S. Park, Y. Li / Bioresource Technology 111 (2012) 42–48 47

100% ABR, which had an SMY of 42% of the theoretical value. Thelow SMY values of 100% ABR could be explained by the resistanceof residual compounds in the cell wall to bacterial attack, whichmay still be present after lysis for lipid extraction (Golueke et al.,1957). When the OLR was increased to 3 g VS/L d, co-digestionwith 33% and 67% ABR reached 22% and 68% of the methane poten-tial, respectively, while the digestion of both 100% FOG and 100%ABR experienced a significant drop in SMY (p < 0.05). The feed withan ABR loading fraction of 50% showed an SMY that was 23% great-er than the theoretical methane potential, indicating a synergeticeffect caused by mixing ABR and FOG at an optimal ratio.

3.3.2. Contributions of carbohydrates, proteins, and lipids to themethane yield

Using the experimentally acquired nutrient concentrations andthe methane potentials from Eq. (1), the contributions of carbohy-drates, proteins, and lipids to the methane yield were calculated asshown in Fig. 5. Lipids were responsible for 94% and 46% of themethane potential in FOG and ABR, respectively. Even though lip-ids accounted for only 15% of the total solids in ABR, they ac-counted for a significant portion of the methane potential(p < 0.05) due to their relatively high energy content compared tocarbohydrates and proteins. For the co-digestion configurations,lipids accounted for 68–83% of the total methane potential. Maxi-mizing the lipid content will increase the potential methane yield,but excessive portions can induce LCFA and VFA inhibition, whichcan reduce the pH in the digester leading to decreased lipid degra-dation and methane production. On the other hand, increasing theprotein content can help increase the lipid degradation stabilitywith favorable alkalinity levels, but excessive amounts of ammoniafrom the protein can decrease the methanogenic activity.

4. Conclusions

Co-digestion of ABR and FOG resulted in improved methaneyields at OLR of 3 g VS/L d. VRPs of up to 1.62 L CH4/L�d showedthat co-digestion could increase reactor productivity while allow-ing for higher feed throughput. During co-digestion, the degrada-tion of carbohydrates and proteins did not change significantlywith increasing OLR. Lipid degradation during co-digestion wasgreater than that in the digestion of 100% FOG or 100% ABR at in-creased OLRs. Additional alkalinity provided by the 50% ABRhelped retain the lipid degradation efficiency when the OLR wasincreased up to 4 g VS/L d.

Acknowledgements

This project was financially supported by the United StatesDepartment of Energy (cooperative agreement no. DE-FE0002546). The authors would like to acknowledge Mrs. MaryWicks for providing a thorough review and valuable commentson this article. Special thanks to David Cain and Fuqing Xu forassisting in sample collection and analysis.

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