use of coffee mucilage as a new substrate for hydrogen production in anaerobic co-digestion with...

Bioresource Technology xxx (2014) xxx–xxx

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Bioresource Technology

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Use of coffee mucilage as a new substrate for hydrogen productionin anaerobic co-digestion with swine manure

http://dx.doi.org/10.1016/j.biortech.2014.02.1010960-8524/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding author at: Environmental Engineering Research Center,Universidad de los Andes, Bogotá, Colombia. Tel.: +57 1 3324312; fax: +57 13324313.

E-mail address: [email protected] (M.A. Hernández).

Please cite this article in press as: Hernández, M.A., et al. Use of coffee mucilage as a new substrate for hydrogen production in anaerobic co-digestioswine manure. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech.2014.02.101

Mario Andrés Hernández a,b,⇑, Manuel Rodríguez Susa a, Yves Andres b

a Environmental Engineering Research Center, Universidad de los Andes, Bogotá, Colombiab Ecole des Mines de Nantes, GEPEA UMR CNRS 6144, Nantes, France

h i g h l i g h t s

� Biohydrogen production using coffee mucilage and swine manure was achieved.� C/N ratio around 50 could support the increase in organic load keeping stability.� Repetitive batch cultivation is useful to detect changes in the pathway.� Butyric and acetic pathways are the main routes in biohydrogen production.

a r t i c l e i n f o

Article history:Received 20 December 2013Received in revised form 15 February 2014Accepted 17 February 2014Available online xxxx

Keywords:BiohydrogenCo-digestionC/N ratioCoffee mucilageRepetitive batch cultivation

a b s t r a c t

Coffee mucilage (CM), a novel substrate produced as waste from agricultural activity in Colombia, thelargest fourth coffee producer in the world, was used for hydrogen production. The study evaluated threeratios (C1–3) for co-digestion of CM and swine manure (SM), and an increase in organic load to improvehydrogen production (C4). The hydrogen production was improved by a C/N ratio of 53.4 used in C2 andC4. The average hydrogen production rate in C4 was 7.6 NL H2/LCMd, which indicates a high hydrogenpotential compare to substrates such as POME and wheat starch. In this condition, the biogas compositionwas 0.1%, 50.6% and 39.0% of methane, carbon dioxide and hydrogen, respectively. The butyric and aceticfermentation pathways were the main routes identified during hydrogen production which kept a Bu/Acratio at around 1.0. A direct relationship between coffee mucilage, biogas and cumulative hydrogen vol-ume was established.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Biohydrogen production from renewable resources such aswastes is a second-generation renewable energy. Their productioncan be accomplished through several biological methods as darkfermentation. Anaerobic digestion has been considered able to con-vert the complex carbon sources of wastes into hydrogen (Sarataleet al., 2013). This substrate kind has several rewards and chal-lenges related to heterogeneous compositions, microorganisms, in-ert material and nutrients (Kapdan and Kargi, 2006; Ntaikou et al.,2010). In general, raw materials contain three types of macromol-ecule; carbohydrates, proteins and lipids. In lignocellulosicmaterials, lignin should be considered in addition with carbohy-drates as cellulose and hemicellulose. The dark fermentation

involves hydrolysis and acetogenic steps where carbohydrateshave been reported as a main substrate for hydrogen production(Kapdan and Kargi, 2006; Lin and Lay, 2004; O-Thong et al.,2008; Sreela-or et al., 2011). According to operating conditionsand microorganisms, the metabolic route can be addressed toseveral products as hydrogen, carbon dioxide, VFAs, ethanol, succi-nate, lactate and other metabolites (Liu et al., 2008; Saint-Amanset al., 2001; Temudo et al., 2007). These products, except hydrogenand carbon dioxide, remain in liquid phase limiting high CODremoval efficiencies (Lee et al., 2010). In fact, biohydrogen throughdark fermentation of complex substrates has been reported as auseful first stage for a subsequent methane production process(Lee et al., 2010; Wang et al., 2013).

In order to specify the kind of feedstock, it could be related toenergy crops which showed a main constrain as demand of arableland. In contrast, the residues from the processing of agriculturalproducts do not suppose additional technical equipment or infra-structure (Eisentraut, 2010). Due to the ability of dark fermenta-tion to use complex substrates as livestock, crop residues, wastes

n with

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2 M.A. Hernández et al. / Bioresource Technology xxx (2014) xxx–xxx

and wastewater, there are several opportunities to developco-digestion of two or more substrates with supplementarycharacteristics which could be: carbon and nitrogen sources,soluble chemical oxygen demand (COD), pH, alkalinity and micro-organisms. In fact, livestock residues are rich in nutrients and havea high pH (Saratale et al., 2013), which could be useful to raise thepH of substrates rich in carbohydrates (Wang et al., 2013).

In Colombia, coffee production is extensive, making it the fourthlargest world producer, after Brazil, Vietnam and Indonesia. Coffeemucilage can be separated using mechanical demucilaging of theripe harvested beans which uses less water than traditional sepa-ration process (Chanakya and De Alwis, 2004). This crop residuehas a high carbohydrates concentration and their availability isyear-round with two main harvest periods (Avallone et al., 2000).In addition, livestock pig in Colombia has a growing of 6.6% be-tween 2011 and 2012. Swine manure has a high protein and lipidconcentration and nutrients. It has been widely used in anaerobicco-digestion as the support substrate during the treatment of otherwastes. The use of both wastes could be feasible due to their occur-rence in similar geographic regions. The both substrates blendscould be made through several ratios where the most common isC/N. The C/N ratio has been widely evaluated for several co-diges-tion processes and experimental conditions, but the substratesdiversity has given results over a wide range from 33 to 200 (Argunet al., 2008; Lin and Lay, 2004; O-Thong et al., 2008; Sreela-or et al.,2011).

Biohydrogen production has been developed through the use ofpure cultures such as Clostridium, Bacillus and Thermoanaerobacte-rium or mixed cultures which are capable to produce mainly vola-tile fatty acids (VFA), hydrogen and carbon dioxide (Kapdan andKargi, 2006; Liu et al., 2008; Temudo et al., 2007). These culturesrequire the use of simple substrates, synthetic wastes and pre-treatment of complex substrates. These special conditions limitsthe knowledge about the microorganism interaction with sub-stances into the wastes, which could influence the real efficiencyover time, the pure or mixed culture and the metabolic pathway(Jo et al., 2007; Kim et al., 2009; Noike et al., 2002). In fact, biohy-drogen of second generation could be affected by microorganismcontent in complex substrates changing patterns: metabolitesand products (Noike et al., 2002; Zhu, 2000). The evolution overtime is an important aspect due to the possible change from hydro-gen to methane or both compounds production limiting the hydro-gen yields. The use of repetitive batch cultivation could be useful toidentify possible changes in the metabolic pathway.

In order to advance in the knowledge of biohydrogen of secondgeneration the present study evaluated the biohydrogen productionfrom a co-digestion process of two complex substrates; swine man-ure, a classic substrate used in methane and hydrogen production,and coffee mucilage, a new substrate with few studies on anaerobicdigestion. The aims of this research were: to evaluate theperformance of biohydrogen production through repetitive batchcultivation and C/N ratio to establish the substrates ratio and themetabolic pathway which report the high hydrogen yield, and sec-ond to determine the viability of coffee mucilage as biohydrogenproducer’s substrate. In addition, the process was subjected to anorganic load increase to evaluate the reactor stability. The effect ofthe substrate ratio and organic load increase on VFA production,biogas composition and hydrogen production rate was assessed.

2. Methods

2.1. Methodology

The study evaluated hydrogen production of four conditions byanaerobic co-digestion; the first three were related to the blend

Please cite this article in press as: Hernández, M.A., et al. Use of coffee mucilageswine manure. Bioresour. Technol. (2014), http://dx.doi.org/10.1016/j.biortech

ratio between two substrates. The swine manure and coffee muci-lage (SM:CM) ratios chosen were 7:3, 5:5 and 3:7, which wereidentified as C1, C2 and C3, respectively. These substrates ratioswere established through total COD with an initial organic loadof ±6.5 kg COD/m3 d. The C/N ratios were 33.8, 53.4 and 77.4 forC1, C2 and C3, respectively, which were obtained without theaddition of external compounds. The fourth condition (C4) wasdeveloped for the best substrate ratio; the organic load wasdoubled to ±12.1 kg COD/m3 d. Each experiment was conductedover a period of at least 30 days. The study began with the highestcontent of swine manure and ended with the highest content ofcoffee mucilage in the feedstock. It was established to have an ini-tial environment of microorganisms and nutrients from SM. Thesubsequent CM addition was to look for the acclimatization ofmicroorganisms to carbohydrate.

2.2. Substrates

The feedstock was 3.5 L of a mixture between swine manureand coffee mucilage. Swine manure was collected from a pig barnat the Servicio Nacional de Aprendizaje (SENA) in Bogotá. It waspreserved at 4 �C in bags with approximately 1 kg and replacedevery 15 days with fresh manure to avoid degradation. Coffeemucilage was collected every 2 months during the mechanicaldemucilaging process on a farm close to Bogotá. This was sepa-rated physically from coffee grains, pulp and other thick solidsusing a No. 4 standard sieve. Coffee mucilage was divided into bagsof 1 L and preserved at �4 �C. The both substrates were dilutedwith tap water to achieve the COD required. The feedstock wasacclimated at room temperature during the preparation procedure.

2.3. Inoculum

The inoculum was obtained in a preliminary phase from thesame reactor using both substrates in a ratio of 7:3 (SM:CM). Themicroorganism selection was conducted through the variation oftemperature, retention time and organic load (Hernández andRodríguez, 2013). The first stage of this process involved a highorganic load of 12 kg COD/m3 d, the second a reduction in organicload to 6 kg COD/m3 d, the third an increase in temperature from35 to 55 �C, and the last modification was a change in the retentiontime from 3 to 1 day. Each stage lasted approximately 30 days. Thefirst two stages improved the hydrogen production but, at day 15,it decreased due to the change to methane production. In contrast,the last two stages were able to maintain the hydrogen productionover time. At this point, the acclimatization process was ended tobegin the experiments.

2.4. Experimental design

The experiments were carried out in a reactor with a workingvolume of 5.5 L and a total volume of 7.2 L. The feedstock wasadded after the extraction of the same volume of mixed liquor.The reactor was operated as an Anaerobic Batch Reactor (ABR) withthe free evolution of biogas to avoid a high hydrogen partial pres-sure (semi-batch system). In order to preserve the biomass, 2 L ofmixed liquor was left inside the reactor during each feeding pro-cess. The initial pH of 5.5 was established by adding either HCl(1.5 N) or NaOH (1.5 N) using automatic dosage pumps regulatedby a control system; after that, the pH evolved without control ina range of 5.15–5.5. The reactor was stirred constantly at200 rpm to prevent the sedimentation of the solids within, andto improve the hydrogen transfer to the gas phase. The tempera-ture of 55 �C was maintained by a heating jacket coupled with acontrol system. The initial and final pH measurements were madein the reactor after the feeding process and at the end of each

as a new substrate for hydrogen production in anaerobic co-digestion with.2014.02.101


M.A. Hernández et al. / Bioresource Technology xxx (2014) xxx–xxx 3

batch cycle, respectively. Biogas production was measured by aMilliGasCounter MGC (RITTER�) and collected in Tedlar� bags of5 L. The volatile fatty acids, biogas volume, hydrogen, carbon dioxideand methane concentrations were recorded during experiments.

2.5. Analytical methods

Hydrogen in biogas was measured online using a HY-OPTIMA700 H2Scan� with a quantification range between 0.5% and 100%(v/v). Methane and carbon dioxide were measured by an Infra-red Gas Analyzer (LANDTEC� – BioGas Check CDM) with a maxi-mum deviation of ±0.3% (v/v) for methane and ±3% (v/v) for carbondioxide. Volatile fatty acids were determined by gas chromatogra-phy (Hewlett Packard� 6890 series G1530A) equipped with a flameionization detector (FID). The operational temperatures of theinjection port, column oven and detector were 250, 250 and300 �C, respectively. Argon was used as the carrier gas with a flowrate of 0.9 mL/min. Carbohydrates and proteins were measured byDubois et al. (1956) and Bradford (1976) methods, respectively.The COD, VFA, Total Kjeldahl Nitrogen (TKN), alkalinity (ALK) andammonia were measured according to Standard Methods (APHA,2005).

3. Results and discussion

3.1. Hydrogen production by dark fermentation

3.1.1. Repetitive batch cultivation and C/N ratioThe reactor was operated for about 144 days with periods of 37,

30, 34 and 43 days for C1, C2, C3 and C4, respectively (Fig. 1). Allconditions, except the first, were influenced during 8 days by theprevious condition related to the solids retention time (SRT).Therefore, the statistical data analysis used the records after thistime for each condition. The C/N ratio of 33.8 in C1, despite of

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has been used for biohydrogen production, showed the lowesthydrogen production. It was associated with the complexity ofthe swine manure, which was the main substrate (C1), limitingthe availability of carbon for microorganism growth and activity.The hydrogen concentration was improved through the repetitivebatch cultivation along the C/N ratio. The increase of carbon diox-ide during repetitive batch cultivation was associated with the lim-itation of methanogenic step which transform it to methane.

As shown in Fig. 1b, the best C/N was 53.4 (C2) with a biohydro-gen production rate of 557.8 NmL H2/LWd. This condition had ahigh stability with a standard deviation of 10.4%. In C/N of 77.4the biohydrogen production rate increased despite of the high fluc-tuation which was associated with the low swine manure contentlimiting the basic components as it was the support substrate. Infact, the high availability of carbon source could be responsibleof other kinds of microorganisms growth or high hydrogen partialpressure changing the main metabolic pathway reached in C2.Although C2 was a favorable condition to hydrogen production;methanogenic activity showed a positive response after the inhibi-tion in C1. Nevertheless, an additional condition (C4) evaluated theincrease in organic load rate up to 12.1 kg COD/m3 d for the C/N ra-tio of 53.4 to improve the biohydrogen production and to controlmethanogenic activity. This action was successful to decrease themethane percentage while preserving biohydrogen concentration.

The average hydrogen production rate in C4 was1398.3 NmL H2/LWd with a standard deviation of about 6.9%, whichwas highly stable compared to the other conditions evaluated,even C2 (Fig. 1b). The approximate doubling of the organic loadfrom C2 to C4 produced a 2.5-fold increase in the hydrogen pro-duction rate. Hydrogen concentration had a stable behavior withan average of 39.0%, slightly higher than that found in C2, whichsuggests a limit in the hydrogen concentration through the useof these substrates. The hydrogen production rate along C/N ratiosand repetitive batch cultivation revealed that a time between 8 andat least 18 days must be considered to identify possible changes inthe metabolic route (Jo et al., 2007; Saint-Amans et al., 2001). In C4,the process required at least 15 days to reach the same stabilitylevels as C2, which confirmed that the increase in organic loadaffected the process evolution. In addition, C4 could required amicrobial population adaptation in term of microorganisms’number.

Hydrogen response was improved at the midpoint of the C/N ra-tios, which suggests a limitation with the increase of any substrate.The C/N ratio 53.4 was in the range of 33–74 found in other studieswhich used food waste/sludge and palm oil mill effluent, respec-tively (O-Thong et al., 2008; Sreela-or et al., 2011). Lin and Lay(2004) found the highest hydrogen production at a C/N ratio of47 using sucrose but, in the same experiments, there was a closeresult at a ratio of 130. This showed the difference between theuse of complex substrates and substrates rich in carbohydrateswith nutrient addition.

The high hydrogen production during the first days in C3 wasassociated with an increase in hydrogen partial pressure whichcould change the pathway to solventgenesis or other metabolites(Liu et al., 2008; Saint-Amans et al., 2001; Temudo et al., 2007).In addition, an increase in carbon dioxide partial pressure changesthe metabolites towards formation of succinic and propionic acids(Park et al., 2005; Tanisho et al., 1998). The strong VFA productionalso could change the pathway from hydrogen to solventgenesiswith ethanol, propanol, butanol and acetone as end products(Saint-Amans et al., 2001; Temudo et al., 2007).

The methanogenesis activity was strongly inhibited over timewhich confirmed that the mixed culture was controlled by micro-organisms other than methanogenic ones (Kim et al., 2004). Themethanogenic archaea, which were provided by the feedstock,were inactivated during the repetitive batch cultivation. Under




the best hydrogen production condition (C4), the methanogenicstep reached a maximum methane concentration of 0.2% in biogas.

3.1.2. pH and volatile fatty acids and alkalinity ratioSwine manure enhanced pH buffering capacity for all the C/N ra-

tio increasing the initial pH of coffee mucilage. The pH changed from5.6 to 6.0 with a low fluctuation between 0.1 and 0.2 for each condi-tion, the most basic condition was achieved during C1 due to thehigh manure content compared with the other conditions. The alka-linity changed from a low buffer capacity in the feedstock (1557 to996 mg CaCO3/L) to a range of 2007–2611 mg CaCO3/L. This wasdue to the output of carbon dioxide due to the decrease in its solu-bility changing the bicarbonate equilibrium (Valdez-Vazquezet al., 2005). The VFA/ALK ratio through the C/N ratio of each condi-tion was about 1.0, which is a representative value for hydrogenproduction instead of methane production. These ratios were ade-quate to inhibit methanogenic step which require values below 0.2.

In C3, the high VFA/ALK variation (0.8–1.3) was a no tolerablecondition even for hydrogen producers microorganisms wherethe high VFA concentration could represent inhibition. In fact,the best hydrogen production rate results were achieved whenthe VFA/ALK ratio was around or up to 1.0. Additionally, the meth-anogenic step was not able to be recovered even during the VFA/ALK ratios below 0.8 where the VFA production was low. It was re-lated to the quick change in this condition and the suggestedmetabolite production different than VFA which are required formethanogens.

The decrease in final pH which achieved values near to the lowset point could be responsible of the carbon dioxide increase from42.4% to 50.6% due to the volatilization of this compound. Insteadof different hydrogen production, the similar VFA concentrationssuggest the production of other metabolites during C1 and C3which were not related to hydrogen producing reactions. The in-crease in organic load generated a strong VFA response changingbiogas composition due to: the decrease in CO2 solubility associ-ated with low pH, the increase in hydrogen production caused bythe short time, the changes in Gibbs free energy and the declinein methane production due to the disruption of the process bythe buffer capacity failure (Voolapalli and Stuckey, 2001; Xinget al., 1997). Despite of that, the VFA/ALK ratio had the highest sta-bility related to the lowest standard deviation.

3.1.3. Carbohydrates and proteinsThe initial C/N ratios were between 33.8 and 77.4 which are

considered functional levels to develop a hydrogen production pro-cess (Argun et al., 2008; Lin and Lay, 2004; O-Thong et al., 2008;Sreela-or et al., 2011). The reduction in swine manure and the in-crease in coffee mucilage through the C/N ratios showed the clearinfluence of each one as a nitrogen and carbon source, respectively(Fig. 2). The high fluctuation in hydrogen production showed in C3

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was related to a large reduction in ammonia concentration, evendepletion. It was associated with the increase in the available car-bon source which improved the biomass synthesis, consumingammonia by several routes (Massé and Drost, 2000). The lower car-bohydrate degradation in C1 (79%) could indicate a limitation inthe mixed culture to degrade this macromolecule. In contrast,the other conditions achieved carbohydrates degradation up to90% which were similar carbohydrate removal of other substratestype (Kapdan and Kargi, 2006; O-Thong et al., 2008).

The ammonia increase in C1 was related to the deamination ofamino acids from proteins which could produce acetic, ammoniumand even hydrogen (Thauer et al., 1977). This route to hydrogenproduction has lower rates than that obtained through glucose,which could explain the low hydrogen production achieved dueto the high protein content of swine manure (Thauer et al., 1977;Zhu, 2000). C1 had the highest ammonia concentration whichwas lower than inhibition levels to this kind of process(Valdez-Vazquez et al., 2005). Meanwhile, in the other conditions,nitrogen was maintained at the same concentration with a slightammonia consumption associated with bacterial growth.

3.1.4. Effect of mucilage concentrationThe addition of coffee mucilage during the substrate variation

and the increase in organic load showed a positive response inhydrogen formation by dark fermentation. The concentration ofthis substrate was linked by a linear relationship to biogas andcumulative hydrogen production. At the beginning, the mucilageconcentration of 3.2 g COD/L produced just 27.4% of the total gasas hydrogen. The increase to 5.1, 7.6 and 9.8 g COD/L maintainedthe hydrogen concentration between 38.8% and 40.8% relatedto the coffee mucilage increase to 1841.6 mg/L in C3 and2551.2 mg/L in C4. These concentrations correspond to 7.4%,17.2%, 18.1% and 13.9% of the total COD in the feedstock for eachcondition, which are lower than those found in other studies(O-Thong et al., 2008). Nevertheless, mucilage and manure as com-plex substrates could have a particulate polysaccharide fraction(Chanakya and De Alwis, 2004; Zhu, 2000). Some monosaccharides,such as arabinose, galactose, xylose and ribose, which could in-volve several degradation routes with hydrogen production andother metabolites, are present in coffee mucilage (Chanakya andDe Alwis, 2004). These compounds were associated with the in-crease in biogas and cumulative hydrogen production. Althoughthe trends showed a stable behavior, it could not be predictedfor mucilage concentrations higher than 9.8 g COD/L as themetabolites generation like VFA could inhibit the process.

3.1.5. COD balanceThe total COD removals from experiments were 2.8%, 2.9%, 4.5%

and 5.0% for C1, C2, C3 and C4, respectively. These were associatedwith the carbon dioxide released in biogas due to the other carbon

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amounts remain in the soluble products as is typical for hydrolysisand acetogenic steps. The COD balance showed that the amounts ofsoluble carbohydrates and soluble proteins consumed representthe 36.4%, 70.1%, 68.1% and 77.4% of the soluble metabolites pro-duced for C1, C2, C3 and C4, respectively (Fig. 3). The contributionto hydrogen production by soluble proteins was related to a max-imum of 5.2% in C1 while it was below 1.6% for the other two C/Nconditions. In fact, protein degradation decreased through the in-crease in the carbon source, the increase in organic load changedthe degradation of protein from 47% to just 29%. The remainingconcentration of VFA produced was associated with the degrada-tion of several carbohydrates present in coffee mucilage.

The short retention time used in the experiments was a limitingfactor to achieve high COD degradation. Therefore, the soluble CODwas considered the main group of compounds taking place in theproduction of hydrogen, carbon dioxide and metabolites. Indeed,at the beginning of all experimental conditions, the soluble CODcannot be represented just by carbohydrates, protein and initialVFA which means that there were other soluble components. Thesoluble COD which is not represented by carbohydrates, proteinand VFA was associated with the formation of several metabolitesthrough the degradation of particulate material present in bothcomplex substrates. As a result, other reactions could be relatedto pyruvate production which is an intermediate in the mainroutes to produce hydrogen (Ntaikou et al., 2010). The solubleCOD removal was increased from 6.7% up to 18.4% between C2and C4, reaching theoretical values for dark fermentation (Leeet al., 2010). The increased removal was associated with the addi-tional amounts of carbon dioxide produced in C4 during the hydro-gen production reactions.

3.2. Coffee mucilage as novel substrate for hydrogen production

3.2.1. Pathway behaviorHydrogen production by dark fermentation was linked to the

production VFA and ethanol (Fig. 4). Iso-butyric acid was taken into

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account in the total butyric acid concentration, as iso-butyric acidconcentration was negligible compared to the main VFA. Indepen-dently of the C/N ratio, acetic and butyric acid fermentation werethe main degradation pathways. The repetitive batch cultivationimproved the butyric-type fermentation more than the acetic-typefermentation. Ethanol and propionic acid were associated with thedeviation of hydrogen production, which occurred mainly in C/N of33.8. The VFA concentration improved with the increase in the C/Nratio, while the contribution to the soluble COD decreased from78.9% to 60.3%. The contribution of similar C/N ratios to solubleCOD was between 67.1% and 69.5% showing the stability of thisratio.

Two special cases were identified during the experiments; thecomplete depletion of acetic acid in C2 and the changes in thehydrogen production rate in C3. In the first case, at point 3, therewas an increase in butyric acid concentration with a simultaneousincrease in ethanol concentration. Despite of butyric acid is a for-warder of hydrogen production; the hydrogen production ratehad a slight decrease associated with the reaction between aceticacid and hydrogen to produce ethanol (Thauer et al., 1977). Inthe second case, the similar VFA concentrations in C3 and C2showed that the hydrogen increase during C3 was obtained by an-other degradation pathway. The fluctuation of VFA and hydrogenin C3 was related to the production of more reduced metabolites(Liu et al., 2008; Noike et al., 2002; Temudo et al., 2007; Thaueret al., 1977).

3.2.2. Ratios to hydrogen productionHydrogen production has two main reactions related to acetic

and butyric acids which produce 4 and 2 mol H2, respectively,while both produce 2 mol of carbon dioxide. The molar ratioCO2:H2 and the molar ratio butyric–acetic acid (Bu/Ac) suggest sev-eral pathways in C1 due to excess carbon dioxide (Fig. 5). In C1, theinitial hydrogen production was related to the acetic pathway dueto the Bu/Ac ratios below 1. The evolution through the repetitivebatch cultivation shifted to the butyric-type fermentation which

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was related to the environmental conditions such as substrates ra-tio, final pH, biogas production and hydrogen partial pressure. Theincrease in the C/N ratio improved the butyric acid productionwhich is a trend both similar and opposite to other studies (Argunet al., 2008; Lin and Lay, 2004; Sreela-or et al., 2011).

The Bu/Ac ratio in C2 could be explained through Eq. (1) (Kimet al., 2004) which have a ratio of 1.5 between butyric and aceticacids. The increase in organic load from C2 to C4 produced achange in both ratios showing a shared pathway between aceticand butyric acids. Meanwhile, the slight increase in the CO2:H2 ra-tio suggests other pathways linked to an increase in carbon diox-ide. In conclusion, although the Bu/Ac ratio showed a slightdomination of butyric-type fermentation, the acetic pathway wasstill the most significant route due to the efficiency of 4 mol ofhydrogen per reaction. In addition, the hydrogen yield achieved avalue of 1.7 and 2.5 mol H2/mol glucose for C2 and C4, respectively.

4Glucose! 2Acetate� þ 3Butyrate� þ 8CO2 þ 8H2 ð1Þ

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Time (d)

C1 C2 C3 C4

Fig. 5. Evolution of the molar ratio CO2:H2 (e) and the ratio between Bu/Ac (j)acids over the repetitive batch cultivation for each condition.


3.2.3. Coffee mucilage as a new substrateThe hydrogen production achieved using coffee mucilage can be

compared with values obtained from other substrates and microor-ganisms. In terms of waste volume, the hydrogen production hassimilar results than several studies which using from glucose tocomplex materials. The hydrogen production yield related to CODshowed high similarity with some results obtained for wastewaterwhich containing compounds such as starch and cellulose. The pro-duction reached is in a range of 34.1 to 206.7 mL H2/g COD (Pereraet al., 2012) which showed the feasibility of these compounds forhydrogen production. There was a yield of 68.2 mL H2/g COD(Perera et al., 2012) for cattle wastewater which showed a veryclose production to the 77.6 mL H2/g COD of this study.

The increase in the organic load rate improved hydrogen pro-duction to a rate higher than the achieved by Ozmihci et al.(2011) who obtained 652 mL H2/L/d using sugar from acid-hydro-lyzed wheat starch. However, another study by Sagnak et al. (2010)using the same substrate achieved a hydrogen production rate of1220 mL H2/L/d. In addition, O-thong et al. (2008) showed a hydro-gen production rate of 6.33 L H2/LPOME (Palm Oil Mill Effluent) with anorganic load of 85 g COD/L which was 4.4 fold that used in thisstudy for C4. Nevertheless, in these studies, external compoundswere used for the nitrogen and phosphate nutritional levels re-quired during dark fermentation. Likewise, different amounts ofiron and magnesium were added to improve the hydrogen produc-tion. Thus, the use of raw POME showed a reduction in hydrogenproduction to 4.2 L H2/LPOME with the same organic load. As wasshown, there is a limitation to compare results due to the differentkind of operating conditions, pre-treatment, substrates, mixed cul-ture and reactor configuration.

4. Conclusions

The use of coffee mucilage in co-digestion with swine manurewas a successful blend to biohydrogen production at a C/N ratio




of 53.4. The best results were achieved during organic load in-crease without affecting the stability of the process. The averagehydrogen production rate was 7.6 NL H2/LCMd which indicates ahigh hydrogen potential compare to substrates as POME and wheatand cassava starch. The methanogenesis stage was almost com-pletely inhibited despite the presence of a mixed culture and with-out any kind of pre-treatment. The butyric and acetic fermentationpathways were the main routes through the repetitive batchcultivation.

Acknowledgements

The authors would like to acknowledge the practical support ofthe members of the Environmental Laboratory at the Universidadde los Andes and the financial support of the Research Center ofthe Faculty of Engineering (CIFI), ECOS Nord C10A01 and COLCIEN-CIAS 057-2010.

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