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Anaerobic digestion of crude glycerol as sole substratein mixed reactorMiroslav Hutňan a , Nina Kolesárová a & Igor Bodík a

a Institute of Chemical and Environmental Engineering, Faculty of Chemical and FoodTechnology , Slovak University of Technology , Radlinského 9, 812 37 , Bratislava , SlovakRepublicAccepted author version posted online: 30 May 2013.Published online: 20 Jun 2013.

To cite this article: Miroslav Hutňan , Nina Kolesárová & Igor Bodík (2013) Anaerobic digestion of crude glycerol as solesubstrate in mixed reactor, Environmental Technology, 34:13-14, 2179-2187, DOI: 10.1080/09593330.2013.804581

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Environmental Technology, 2013Vol. 34, Nos. 13–14, 2179–2187, http://dx.doi.org/10.1080/09593330.2013.804581

Anaerobic digestion of crude glycerol as sole substrate in mixed reactor

Miroslav Hutnan∗, Nina Kolesárová and Igor Bodík

Institute of Chemical and Environmental Engineering, Faculty of Chemical and Food Technology, Slovak University of Technology,Radlinského 9, 812 37 Bratislava, Slovak Republic

(Received 5 February 2013; final version received 3 May 2013 )

Utilization of crude glycerol (CG) from the biodiesel industry in the production of biogas offers a perspective of furtherenergy generation, which may result into the drop of biodiesel costs on the developing world market. This contributionis focused on anaerobic treatment of CG as a single substrate in mixed laboratory reactors. Experiences from long-termoperation of mixed reactors processing either untreated or acidulated CG are discussed. The possibility of cofermentation ofwashing water (WW) from biodiesel production with CG was also attempted. It was demonstrated that long-term mesophilicanaerobic treatment of CG as the only substrate is possible. Except for nitrogen, and possibly phosphorus, the addition ofother nutrients is unnecessary. Processing of both non-acidulated and acidulated CG in laboratory mixed reactors inoculatedwith suspended sludge resulted in a stable operation with high specific methane production (0.328 L/g chemical oxygendemand (COD) for non-acidulated CG and 0.345 L/g COD for acidulated CG), regarding organic loading rate of up to 4 gCOD/(L·d). Due to the considerable content of dissolved inorganic salts in CG it is recommended to dilute this substratewith water to prevent the accumulation of salts and inhibition of the biomass activity. WW was proved to be a problematicsubstrate for anaerobic cofermentation with CG because its addition to the reactor caused a decrease in the pH value andbiogas production.

Keywords: anaerobic digestion; biodiesel; biogas; crude glycerol; mixed reactor

IntroductionIn order to replace fossil fuels by renewable energy sources,utilization of biofuels and renewable energy sources hasbeen incorporated in the national and international legalstandards and the government programmes of developedcountries.[1] The EU in the Directive 2009/28/EC hasdefined a programme of replacing 20% of total energy con-sumption with renewable energy sources and 10% of theconsumption of liquid fuels with biofuels by 2020.

Biodiesel is a liquid fuel based on methyl esters oflong-chain fatty acids and it is usually produced by base-catalyzed transesterification of vegetable oils, animal fatsor waste oils with methanol.[1,2] The most important by-product generated by this process is crude glycerol (CG).It is a heavier separate liquid phase composed mainly ofglycerol. Utilization of this by-product in the productionof biogas offers a perspective of further energy genera-tion used on site or redistributed. Proper valorization ofby-products can have a strong influence on the economicand environmental status of biofuel itself.[3]

In general, about 10 kg of CG are produced for every100 kg of biodiesel. The composition and characteristics ofCG depend on the source of oil used for the productionof biodiesel and on the processing technology.[4] CG gen-erated by the most common homogeneous base-catalyzed

∗Corresponding author. Email: miroslav.hutnan@stuba.sk

transesterification, separated from biodiesel by settling,contains approximately 50–60% of glycerol, 12–16% ofalkalies (in form of alkali soaps and hydroxides), 15–18%of methyl esters, 8–12% of methanol and 2–3% of water.[5]In some biodiesel production plants, CG is treated withstrong mineral acids (usually phosphoric or hydrochloricacid) in the process of acidulation in which mainly the long-chain fatty acids are removed from CG and returned intothe biodiesel production process. This type of CG containsabout 80% of glycerol.[5] CG can be subsequently treatedby distillation to remove methanol and water. Engagingmore advanced and complex technologies, CG can be puri-fied to over 99% of glycerol.[6] Despite the wide applicationof pure glycerol in pharmaceutical, food and cosmeticindustries, refining of CG to a high purity is quite expensive,especially for small and medium biodiesel producers.[7]Moreover, given the continuing trend of the constructionof new biodiesel production plants, the amount of glycerolproduced has increased causing the decrease in the priceof glycerol.[8] Because of the surplus of CG on the mar-ket and high costs of its purification, various alternatives ofutilization of this by-product have been investigated.[9–11]

The high energy content and high portion of readilydegradable organic substances make CG an interesting sub-strate for anaerobic degradation. Besides the production of

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methane, the advantages include low nutrient requirements,energy savings, generation of low quantities of sludge andexcellent waste stabilization.

Several research teams have recently focused on the pos-sibility of anaerobic treatment of CG.[12–18] Most of thestudies have been aimed at using CG as a co-substrate inother substrates’ processing. Our experience indicates thatother suitable substrates are often not available at the loca-tion of CG production. Hence, the main objective of ourresearch was to examine anaerobic processing of CG as thesingle substrate.

Previous experience with microbiological treatment ofCG has suggested some specific requirements and inhibitioneffects that must be taken into account, e.g. high concen-tration of inorganic salts in the CG originating from thecatalyst and neutralizing agents used in biodiesel produc-tion: mainly sodium and potassium cations from the catalystand chlorides, sulphates or phosphates anions from the neu-tralization or acidulation processes. The concentration ofsoluble salts can range between 5% and 15%.[19] Thesesalts can be accumulated in the reactor and they have anegative effect on the activity of methanogenic microor-ganisms. The effect of impurities in CG on the fermentationprocesses was reported in the work.[20] There is a grad-ual reduction in the yield of 1,3-propanediol during theaddition of K2SO4 (concentration of 2.5–10 g/L) and a con-stant reduction in the 1,3-propanediol yield was observedfor all concentrations of KCl (concentration of 2.5–10 g/L).Monovalent salts have a swelling effect on the membrane athigh concentrations, weakening the Van der Waals forcesbetween the lipid tails in the membrane.[21] This affectsthe energy barrier within the lipid layer leading to alter-ations in biochemical processes such as substrate transportthrough the membrane. This effect is more significant forKCl as it contains a monovalent cation and a monova-lent anion, and is reflected in the reduction in the yieldof 1,3-propanediol compared with K2SO4 at lower con-centrations. Inhibition of an anaerobic digestion processwas reviewed in [22]. The IC50 value for sodium inhi-bition of methanogenesis at mesophilic temperature hasbeen reported to range between 5.6 and 53 g/L, dependingon the adaptation period, antagonistic/synergistic effects,substrate and reactor configuration. The IC50 value foracetate-utilizing microorganisms was found to be 28.9 g/Lof potassium.[23] The inhibitory effect of salts does notusually become an issue during single batch experimentsdue to their low concentration.[17,18,24] Inhibition canbe prevented by dilution of salts with a larger amountof water [25,26] or by cofermentation of a small shareof CG with other substrates.[14,16,27] In some cases,the salts were removed prior to the anaerobic treatmentof CG.[28] Since the accumulation of salts from CG inthe reactor is a gradual process which depends on thecomposition of CG and the organic loading rate (OLR)of the reactor, the inhibitory effect may not occur evenwithin several months of operation.[13] Assuming gradual

acclimatization of microorganisms, biomass might be ableto tolerate relatively high concentrations of salts.

Specific requirements on the nutrients addition (espe-cially ammonium nitrogen) are connected with the process-ing of CG, because their concentrations in the substrateare not sufficient. In all the experiments focused on sin-gle processing of CG, nutrients (nitrogen, phosphorus andin some cases also micronutrients) were added. In caseof cofermentation, nutrients were supplied through thecosubstrate.[16,24,29]

Another possible by-product of biodiesel production iswater from raw biodiesel washing. During the conventionalprocess (alkali-catalyzed transesterification), about 20 L ofwashing water (WW) is discharged (or more in case of acidpre-treatment) for every 100 L of biodiesel produced.[30]WW (also referred to as biodiesel wastewater) is a viscousliquid with an opaque white colour similar to aqueous soap.It contains significant amounts of methanol, glycerol andsoaps. Methyl esters bound with soap, NaOH or KOH fromthe catalyst, sodium or potassium salts and trace mono-, di-and triglycerides bound with the soap are also contained inWW.[31,32]

Like the CG, WW has also quite a high content ofdegradable organic substances,[15,30–32] which makes it aperspective source of carbon for microbiological processes.However, some issues have to be considered. WW is alka-line due to the significant level of residual KOH, and itcontains a high level of oil and grease. Nutrients for micro-bial growth are not abundant in this substrate and it alsocontains a considerable amount of salts. Together, thesecomponents inhibit the growth of most microorganisms,making natural degradation of WW difficult.[30] Focus-ing on anaerobic degradation, long-chain fatty acids, whichare present in a high concentration in WW, can inhibit thedigestion process.[22] To reduce this effect, pre-treatmentof oily wastewater by electrocoagulation with a subsequentanaerobic treatment has been proposed.[15,32]

In this work, the possibility of separate treatment of GCin laboratory mixed reactors was studied. Non-acidulatedGC was used in the experiments. When studying acidulatedCG, the possibility of its cofermentation with WW was alsoinvestigated.

Materials and methodsCG was treated in two mixed anaerobic laboratory reactors,each of them with a working volume of 4 L, in a room with astable temperature of 37◦C; hence, they were operated undermesophilic condition. Suspended sludge from the municipalwastewater treatment plant Devínska Nová Ves (Slovakia)was used for inoculation. CG was dosed into the anaerobicreactors once a day through a filler hole. Considering thelack of nutrients in CG, ammonium nitrogen (N-NH4), inform of an NH4Cl solution (20 g/L of N-NH4), and phos-phate phosphorus (P-PO4), in form of a KH2PO4 solution(2 g/L of P-PO4), were added into the reactors. No other

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Table 1. Selected characteristics of two different kinds of CGused as a substrate in laboratory mixed reactors.

COD Total Total Density DISParameter (g/L) N (mg/L) P (mg/L) pH (kg/m3) (g/L)

CG 1 1600 2060 720 10.4 1052 21.3CG 2 1200 1413 2920 2.95 1180 19.9

nutrients were supplied. The content of the reactors wasperiodically stirred (15 min per hour). The produced bio-gas was led through a bubbler filled with water that servedas a water locker and indicator of biogas production. Theamount of produced biogas was measured by wet labora-tory gas metres. Depending on the dose of CG and WW, thehydraulic retention time was 160–1000 days.

Two different kinds of CG (untreated and acidulated)were processed in the laboratory reactors during theirlong-term operation. The samples of CGs and WW weresupplied from the largest biodiesel production plant inSlovakia, company MEROCO, whose production capac-ity is 150,000 tonne of biodiesel per year. Average valuesof CGs’ primary characteristics are listed in Table 1. CG 1was separated in the process of biodiesel production by set-tling without further treatment, providing a glycerol contentof about 55%. CG 2 was treated by acidulation resulting inthe glycerol content of about 80%.

During the long-term processing of CG 2, WW was usedas a co-substrate. Basic parameters of this material wereas follows: chemical oxygen demand (COD) of 114 g/L;pH 1.8; concentration of dissolved inorganic salts (DIS) of16.5 g/L; total nitrogen of 544 mg/L and total phosphorusof 2994 mg/L.

Basic chemical parameters such as dissolved COD, con-centrations of volatile fatty acids (VFA), DIS, N-NH4,P-PO4, pH, concentrations of total solids and volatile solids(VS) in the sludge and production of biogas and its com-position were regularly monitored during the long-termoperation. Analytical parameters were determined in thefiltered sludge samples according to the APHA standardmethods.[33] For the determination of total VFA, a three-point titration according to Kapp was applied.[34] Theconcentration of VFA is expressed as the equivalent ofacetic acid. The biogas composition was determined usinga GA 2000 Plus analyzer (Geotechnical Instruments, UK).The acquired specific biogas productions for GC1 and GC2were converted to normal conditions (temperature of 0◦C,pressure of 101 kPa),

The biomethane potential was measured at 37◦C bykinetic tests, while 0.5 L of anaerobic sludge (the samesludge that was used for the inoculation of the two mixedreactors) was mixed with samples of CG to achieve theinitial COD:VS (anaerobic sludge) ratio of 0.5 g/g. Themixture was diluted with tap water to 1 L, bubbled withnitrogen for 10 min, and the reactor was hermetically sealed.The amount of produced methane was measured in this

seven-day long experiment. From the methane productioncourse, the maximum specific methanogenetic activity andspecific methane production for both GC samples werecalculated.

Results and discussionAnaerobic treatment of CGFor the preliminary assessment of anaerobic degradabil-ity of the CGs used, kinetic tests of methanogenic activity(biomethane potential) were performed. Anaerobically sta-bilized sludge from a municipal wastewater treatment plantwas used as the inoculum in the tests, while CG was addedas the substrate.

In case of CG 1, the net methane production was0.310 L/g COD or 0.490 L/mL CG (reduced by themethane production of blank test), which corresponds to77.2% of the calculated theoretical production. The maxi-mum specific methanogenic activity achieved was 0.344 gCOD CH4/(g VS d). When CG 2 was used as the sub-strate, the net methane production was 0.350 L/g CODor 0.425 L/mL of CG. Over 90% of organic matter weredegraded and the maximum specific methanogenic activityreached 0.121 g COD CH4/(g VS d). Although the anaero-bic sludge, used for the tests of anaerobic degradability, wasnot adapted, high values of the degree of anaerobic degrad-ability as well as the maximum specific methanogenicactivity were obtained for both types of CG. Based onthese results, it can be concluded that CG is easily anaer-obically degradable. High anaerobic degradability of CGwas also achieved in [15]. In the batch reactors undermesophilic condition, CG biodegradation of almost 100%was achieved.

Processing of untreated CG in a mixed reactorUntreated CG (CG 1) was used as a substrate in the firststirred laboratory reactor. Given the considerable contentof higher fatty acids (according to the literature, their con-centration can be up to 20% [5,20]), COD of this kind of CGis higher (1600 g/L) than that of CG 2 (COD of 1200 g/L).The operation of the anaerobic reactor was initiated withan OLR of 1.6 g COD/(L·d) (dose of CG – 4 mL/d). Thisdose was gradually increased until maximum OLR of 8.0 gCOD/(L·d) was reached. The courses of OLR and specificbiogas production are shown in Figure 1(a).

During the initial 68-day period of the anaerobic lab-oratory reactor operation, the specific biogas productionincreased simultaneously with the increase in OLR up to5.6 g COD/(L·d). This could be a result of biomass adap-tation to the substrate. During this period, the performanceand operational parameters of the anaerobic reactor werestable. The specific biogas production was over 0.55 L/gof COD, concentration of VFAs was lower than 700 mg/L,dissolved COD was lower than 600 mg/L and pH in the

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reactor was around 7 (Figure 1(b) and 1(c)). Based on thesefacts as well as in order to accelerate the increase in OLR, thedose of the CG was increased faster (from 5.6 g COD/(L·d)to 8.0 g COD/(L·d) in 68th day). Another aim of this paperwas to simulate the effect of shock increase in OLR. Asa result, there was a sharp increase in the concentrationsof COD and VFA in sludge water and a decrease in thepH value below 6 were observed. The production of bio-gas also decreased significantly. Even attempts to adjustpH using NaHCO3 (5 g) led to no considerable improve-ment of the process. It took nearly three weeks until theanaerobic reactor catch up with this shock change, while

there was no substrate dosing during the first week andthen, the dose was gradually increased again. Higher valuesof specific biogas production during the period from 81stday to 111th day of the reactor operation were related to theconsumption of COD accumulated in the reactor after theshock change. Concentrations of COD and VFA graduallydecreased and the OLR in the reactor gradually increasedup to 5.6 g COD/(L·d) by the 139th day of the operation.At this OLR, however, the reactor overload was indicatedagain while the COD and VFA concentrations increased andthe biogas production decreased. Therefore, the OLR wasreduced to 4 g COD/(L·d) and this OLR remained unaltered

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for the rest of the operation. Given this OLR, the reactorperformance was stable for about 65 days with the specificbiogas production of 0.610 L/g COD (0.980 L/mL CG).

After about 200 days of the operation, the concentrationsof dissolved COD and VFA started to increase rapidly andthe pH and specific biogas production decreased indicat-ing the deterioration of the anaerobic degradation process.Considering the content of 21.3 g/L of DIS in CG 1, thesesalts were accumulated in the reactor. At the concentrationof about 10–12 g/L, the salts had no negative impact on theperformance of the reactor. However, the rapid increase inthe DIS concentration around the 200th day of the operation(Figure 1(c)) probably resulted in the decline of the processparameters. Hence, CG was diluted with tap water at theratio of 1:1. After the stabilization of the DIS concentration,the processes of anaerobic degradation ran satisfactorilyagain. Since the 410th day of the operation, concentra-tions of COD and VFA in the reactor were higher, butthis did not cause any long-term deterioration of the biogasproduction. The increase in the COD and VFA concentra-tions is probably related to another slight increase in theDIS concentration in the sludge water partially inhibitingthe anaerobic processes. The average specific biogas pro-duction during the operation of the laboratory anaerobicreactor was 0.560 L/g COD (0.9 L/mL CG). The averageconcentration of methane in the biogas was 61.1% and theaverage concentration of carbon dioxide was 38.6%. Dueto the nature of CG, which does not contain any signifi-cant amount of sulphur or proteins, no hydrogen sulphidewas measured in biogas. This is another fact favouring theanaerobic treatment of CG.

Despite the high pH of the CG used, it was not necessaryto adjust the pH value in the anaerobic reactor except forthe response to the shock change during the period from the70th day of the operation, when pH was adjusted to 7 with anaddition of NaHCO3. During the rest of the operation time ofthe anaerobic reactor, the pH value was in the range suitablefor smooth running of methanogenesis (Figure 1(c)).

Concentrations of N-NH4 and P-PO4 in the sludge waterof the anaerobic reactor were regularly monitored. Duringthe initial 100 days of CG dosing, the concentration of N-NH4 was decreasing since the nitrogen supplied into theinoculum was gradually consumed. Considering the lack ofnutrients in GC (the ratio of COD:N:P of 500:0.65:0.23),N-NH4 (in form of an NH4Cl solution) and P-PO4 (in formof a KH2PO4 solution) were added into the reactor. Theaddition of nutrients was initiated after about 100 days ofthe laboratory reactor operation, while the sufficient dosewas determined in the sludge water. The amount of 100 mgof nitrogen was added into the reactor once or twice perweek and the average dose made up to 2 g/L CG. Nitro-gen in the ratio of 500:0.63 was added per the quantity ofCOD supplied, which together with the nitrogen presentin CG provided a sufficient ratio of COD:N of 500:1.28.Phosphorus was supplied only twice (each dose of 20 mg ofP-PO4), which was sufficient to stabilize its concentration.

The reactor operation demonstrated that the amount of phos-phorus contained in the GC used was sufficient for theanaerobic digestion process.

During 540 days of the laboratory reactor operation, thesludge concentration in the reactor increased from 10.3 g/Lto 40 g/L. No excess sludge had to be withdrawn duringthe entire period of the reactor operation. Only the amountof sludge necessary for analyzes of the maximum volumeof 50 mL per week was taken from the laboratory reac-tor. Given the amount of the withdrawn sludge, the sludgeretention time was longer than 550 days. Specific produc-tion of sludge was 0.087 g/mL CG, which corresponds to0.055 g/g COD added.

Although, provided a sufficient adaptation, efficientdegradation of CG can be expected at higher DIS concentra-tions assuring the conservative DIS concentration of up to10–15 g/L recommended for optimal operation of a stirredanaerobic reactor processing CG.

In order to maintain the DIS concentration below10 g/L, it is necessary to dilute CG with water at the ratio of1:2. Such dilution obviously leads to higher energy require-ments on heating the inflow to the reactor and to highervolume of excess sludge since it is extended by the vol-ume of water added. However, the high energy content ofCG, such an increase in the substrate or excess sludge vol-ume, does not mean any significant increase in the operatingcosts. Despite the dilution, the hydraulic retention time inthe reactor was approximately 130 days.

Processing of acidulated CG in mixed reactorIn the second stirred reactor, CG l treated by acidulation wasused as the substrate (CG 2). The operation of the laboratoryreactor was started with the OLR of 1.2 g COD/(L·d) (doseof CG – 4 mL/d). The OLR was gradually increased up to3.6 g COD/(L·d). The courses of OLR and specific biogasproduction are shown in Figure 2(a). Since the 164th dayuntil the 280th day of the reactor operation, WW was alsofed into the anaerobic reactor, while the volume of WW wasequal to the volume of CG added, i.e. 12 mL/d.

Initial high specific biogas production, corresponding tothe OLR of 1.2 g COD/(L·d), was caused by the fact thatduring the start-up of the reactor operation, CG was notdosed every day. The quantity of biogas produced from asingle dose corresponded to that of a few days of produc-tion. Only after the biomass activity improved, dosing onthe daily basis was initiated. When CG was added everyday, the average specific biogas production correspond-ing to the OLR of 1.2 g COD/(L·d) was 0.470 L/g COD(0.56 L/mL CG). With OLR increasing up to 3 g COD/(L·d)(36th day of the reactor operation), the specific biogasproduction also increased, which was consistent with theprevious experiment. It is most likely that the result of grad-ual adaptation of biomass and improved homogenizationof the reactor content is due to the increased production ofbiogas. Stable operation of the reactor during this period

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was also indicated by the values of the process parametersmonitored (Figure 2(b) and 2(c)).

After about 50 days of the operation, OLR was increasedto 3.6 g COD/(L·d) and the performance of the reactorstarted to deteriorate. Overload of the reactor was indi-cated by an increase in the concentrations of COD and VFA(Figure 2(b)) and by a decrease in pH (Figure 2(c)) and spe-cific biogas production. Therefore, the OLR was reducedagain to 3 g COD/(L·d) (56th day). A decrease in the pHvalue in the reactor was observed already around the 40thday of the operation, caused by continuous dosing of CG

with low pH and not by the deterioration of other param-eters (COD, VFA and biogas production). The pH valuewas adjusted with a single dose of 2 g of NaHCO3. A dropin pH due to the overload of the reactor was also averted,thanks to the dose of NaHCO3 of the total amount of 6 g.Setting the OLR to 3 g COD/(L·d) resulted in stable reactoroperation for over 100 days, even despite a further increasein the OLR of up to 3.6 g COD/(L·d). The average specificbiogas production during this period was 0.680 L/g COD(0.810 L/mL CG). When considering normal conditions,the specific biogas productions were 0.537 L/g COD for

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CG 1 and 0.600 L/g COD for CG 2. The average methaneconcentration in biogas was 57.5% for acidulated CG (con-tent of methane was 61.1% in case of non-acidulated CG).Average concentrations of 42.3% of CO2, 30 ppm of H2 and9 ppm of H2S were measured in biogas. Hence, the aver-age specific methane production was 0.328 L/g COD forCG 1 and 0.345 L/g COD for CG 2. A quite high methaneproduction of 93.7% of the theoretical value for CG 1 and98.6% for CG 2 was achieved. Such high biodegradabil-ity was also reported in [15,28]. In batch reactors undermesophilic condition, CG biodegradability of almost 100%was observed. Vlassis et al. [35] achieved the specific bio-gas production 0.300 L/g of COD in a continuous stirredtank reactor at the OLR of 0.25 g COD/(L·d) when treatingthe diluted pure glycerol. Viana [26] assessed the anaerobicbiodegradability of several types of CG without any pre-treatment obtaining the biodegradability efficiency between65.9% and 85.6%.

Concentrations of N-NH4 and P-PO4 were regularlymonitored in the sludge water of the anaerobic reactor pro-cessing acidulated CG. Considering the ratio of COD:N:Pof 500:0.59:1.2 in CG 2, sufficient amount of phosphoruswas clearly present and it was not necessary to add thisnutrient as its concentration was significantly higher thanin untreated CG. This is related to the treatment processsince phosphoric acid is used for acidulation.

At the beginning of the operation, the concentration ofN-NH4 was sufficient since nitrogen was supplied to thesludge water in the sludge used as the inoculum. After about50 days of the operation, its concentration decreased below10 mg/L, and the addition of nitrogen was initiated. N-NH4was added into the reactor once or twice per week in thevolume of 2 mL in form of a 26% solution of NH4OH. Thisform of N-NH4 was chosen due to the low pH of acidulatedCG. Thanks to the addition of NH4OH, the necessary nutri-ents were supplied into the reactor and at the same time thepH value of the reaction mixture was adjusted. The aver-age dose of nitrogen when processing acidulated CG was2.64 g/L of CG 2. Nitrogen in the ratio of 500:1.11 per thequantity of the COD supplied was added, which togetherwith the nitrogen present in CG 2 provided a sufficient ratioof COD:N of 500:1.7.

In 443 days of the laboratory reactor operation, thesludge concentration in the reactor increased from 10.3 g/Lto 39 g/L. Similarly in the process with non-acidulated CG,no excess sludge was withdrawn during the entire period ofthe reactor operation except for the samples for analyzes(maximum of 50 mL per week). Given this amount of with-drawn sludge, the sludge retention time was higher than550 days. Specific production of sludge was 0.082 g/mLCG 2, which corresponds to 0.068 g/g COD added. Hence,the processing of 1 L of CG 2 resulted in the production ofapproximately 82 g of excess sludge.

As it was already mentioned, from the 164th day tothe 280th day, WW was also dosed into the reactor in

the same volume as CG 2. Given the COD of 114 g/L,its addition to CG 2 caused no significant increase in theOLR. However, low pH and a relatively high DIS concen-tration of WW caused problems during its cofermentationwith CG 2. The first obvious effect of WW dosing was aslight decrease in pH (Figure 2(c)). After about 10 daysof the operation, there was a sharp increase in the CODand VFA concentrations (Figure 2(b)) in the sludge water,which was followed by a further decrease in pH in the reac-tor. In order to adjust pH, 9 g of NaHCO3 and 5.68 g ofNaOH were gradually added into the reactor. Even thoughthe operational parameters in the reactor were stabilized,the specific biogas production was negatively affected bythe addition of WW, and it gradually decreased during thecofermentation, as it is shown in Figure 2(a); even afterabout 100 days after the end of the WW dosing, the origi-nal specific biogas production was not achieved. The natureof sludge also changed due to the WW dosing; it becameviscous and more difficult to filtrate. The inhibition of anaer-obic processes during the WW dosing was probably causedalso by the faster accumulation of DIS, which exceeded20 g/L during the cofermentation. The experiment focusedon the cofermentation of CG 2 with WW showed that thelatter is not very suitable for anaerobic degradation whentreated in its original form. It is a difficult substrate formicrobiological processing showing significant inhibitoryeffects. Adjustment of pH, dilution or treatment by coag-ulation, are recommended prior to anaerobic digestion ofWW.[15,30,36,37]

ConclusionsBased on the experiments performed it was concluded thatlong-term anaerobic treatment of CG as an only substrateis possible. Except for nitrogen, and possibly phosphorus,the addition of other nutrients is not necessary. Process-ing of both non-acidulated and acidulated CG in laboratorystirred reactors inoculated with suspended sludge resultedin a stable operation with high specific biogas production,regarding an OLR of up to 4 g COD/(L·d). Due to theconsiderable content of DIS in CG, it is recommendedto dilute this substrate with water to prevent the accu-mulation of salts and thus the inhibition of the biomassactivity. In case of anaerobic degradation of acidic CG,it is necessary to maintain optimal pH for proper activityof methanogenic microorganisms by adding NaHCO3 orNH4OH. Cofermentation of CG with WW from biodieselproduction was also tested in a laboratory stirred reactorwith suspended biomass. However, WW was shown tobe a problematic substrate for anaerobic degradation. Itsaddition to the reactor caused a decrease in the pH valueand biogas production. Due to its inhibitory effects, thissubstrate is clearly not suitable for anaerobic processingwithout being pretreated.

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AcknowledgementsThis contribution is the result of the project implementation:National Centre for Research and Application of RenewableEnergy Sources (ITMS: 26240120016), supported by the Research& Development Operational Programme funded by the ERDF.

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