Bioresource Technology 102 (2011) 4995–5003

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Bioresource Technology

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Anaerobic Co-Digestion of table olive debittering & washing Effluent, cattlemanure and pig manure in batch and high volume laboratory anaerobicdigesters: Effect of temperature

Ioannis S. Zarkadas ⇑, George A. PilidisUniversity of Ioannina, Department of Biological Applications and Technologies, Laboratory of Environmental Chemistry, 45110 Ioannina, Greece

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

Article history:Received 6 August 2010Received in revised form 19 January 2011Accepted 20 January 2011Available online 28 January 2011

Keywords:Anaerobic digestionWastewater managementCattle and pig manureBiogas productionTable olive process wastewater

0960-8524/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.biortech.2011.01.065

Abbreviations: DWE, debittering and washing efflcattle manure; TOC, total organic carbon; VFA, volatilVS, volatile solids; C/N, carbon to nitrogen ratio; HRCOD, chemical oxygen demand; UV, ultraviolet radiat⇑ Corresponding author. Tel.: +30 2651007348; fax

E-mail addresses: izarkada@cc.uoi.gr, gpilidis@uoi.

The prospective of table olive debittering & washing Effluent (DWE) as feed stock wastewater for anaer-obic digestion (AD) systems was investigated in batch and continuous systems together with cattle andpig manures. While DWE considered unsuitable for biological treatment methods due to its unbalancednature, the co-digestion of the wastewaters resulted in a 50% increase in the methane production/gramvolatile solidsadded (CH4/gVSadded), accompanied by 30% phenol reduction and 80% total organic carbonremoval (TOC). pH increase during the co-digestion period was not identified as an inhibitory factorand all reactors were able to withstand this operational condition change. Moreover, no volatile fatty acid(VFA) accumulation was observed, indicating that the reactors were not operating under stress-overload-ing state. Under thermophilic conditions a 7% increase on the TOC removal efficiency was achieved whencompared to the mesophilic systems while, under mesophilic conditions phenolic compounds reductionwas 10% higher compared to the thermophilic systems.

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1. Introduction to environmental contamination through runoff and leaching

Olive (Olea Europea) is an evergreen tree mainly grown aroundthe Mediterranean Sea. It is widely cultivated for producing olivefruit used either as a nutritious product, or for olive oil extraction.

Production of table olives is a seasonal activity in Greece, withan annual production of 65.000 tonnes in about 75 different facto-ries. 70% of this production is taking place near the sea coast(Kyriacou et al., 2005). During the process, large volumes of waste-waters with high pH levels, moderate COD (10 g/l) and polypheno-lic content (150–400 mg/l) are being generated. The table oliveproduction is based in 3 manufacturing stages. During the firststage the olive fruit is treated with NaOH (1, 5–2% by weight inwater) in order to remove its bitter taste. It is then washed in orderto remove the excess NaOH (Second stage) and finally, the thirdstage is the lactic fermentation of the oil fruit in brine which helpsin preparing a tasteful product (Parinos et al., 2007)

The volume of wastewaters produced per tonne of final oliveproduct varies between 3.9 and 7.5 m3. This wastewater is usuallydumped into water courses or sent to evaporation ponds, leading

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uent; PM, pig manure; CM,e fatty acids; TS, total solids;T, hydraulic retention time;ion.: +30 26510 07274.gr (I.S. Zarkadas).

(Beltran-Heredia et al., 2000).The addition of NaOH during the debittering process combined

with the high polyphenolic content render this wastewaterunsuitable as a single feedstock for biological treatment systems.(Kyriacou et al., 2005; Parinos et al., 2007).

As a way to treat these wastewaters, a number of researchershave proposed the combined usage of a chemical method that isfollowed by a biological treatment step (Beltran-Heredia et al.,2000; Benitez et al., 2002). Nearly all of these methods are usingthe same idea, based on the application of a strong oxidising agentin order to oxidise the non or low biodegradable compounds, fol-lowed by a biological step, in order to reduce the organic load ofthe wastewater. For this purpose, strong oxidisers are applied tothe wastewater, including hydrogen peroxide, Fenton reagent,ozone, ferric iron, or the combined usage of a UV radiation sourcetogether with one of the above oxidizing agents. However, manyproblems arise due to insufficient TC reduction, even when ad-vanced oxidation methods are applied, and nearly always, a biolog-ical treatment method must be followed (Beltran et al., 1999;Justino et al., 2009). This need for a further treatment step rendersthe chemical methods as ineffective, by increasing the productioncost of the final product, while requiring skilled operators and theacquisition and application of expensive chemicals.

On the other hand, due to seasonal production of table oliveslasting only between September and November every year (Aggeliset al., 2001), the construction of a biological treatment system with

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exclusive purpose the treatment of DWE is not considered as eco-nomically viable and effective management method. This is due tothe fact that microorganisms require long adaptation periods in or-der to reach exponential growth phase firstly and stationarygrowth phase latter, where maximum removal of the organic loadis possible in short time.

A wastewater treatment method that has been applied to DWEwith some success is the anaerobic digestion (AD). AD offers theadvantages of reducing the organic loading of wastewaters, whileproducing fuel gas containing 55–70% methane (Rasi et al., 2007),which is considered as a component of sustainable development.Very few researches have examined the actual effects that ADhas to the organic loading of this wastewater and the efficient re-moval of the polyphenols. Aggelis et al. (2001) examined theanaerobic degradation of polyphenols and the reduction of the or-ganic load of DWE, concluding that anaerobic digestion was able toremove only 12% of the polyphenols and 49% of the organic load,while inhibition of the process was identified possible due toVFA and phenolic compounds accumulation.

For overcoming problems related to the accumulation of inhib-itory compounds in addition to the problems experienced byAggelis et al. (2001), co-digestion could be applied for the simulta-neous treatment of DWE with other locally available wastewaters.Although co-digestion of multiple wastewater streams is mainlyanticipated for resolving the problem of toxic and inhibitory com-pounds accumulation, provides additional advantages by encour-aging synergetic phenomena between the microorganisms,providing nutrient balance (resulting in increasing biogas produc-tion yields) (Cecchi et al., 1996) and sharing costs associated withtreatment between different operations. However, the co-digestionfacilities if not planned correctly present their own limitations dueto logistical problems of transporting large volumes of wastewa-ters within great distances, something that results in increasedcosts and adds onto the overall complexity of the process.

Pressure has been applied to farmers for proper application ofmanures on soil in a way to protect surface and undergroundwaters from nitrate pollution with the implementation of the ECNitrates Directive. While protection of the environment is a waytowards sustainable development, the requirements of the direc-tive are rarely fulfilled by the farmers due to the nature of theEuropean farming industry, which is composed by a large numberof low intensity farms. These farmers are most of the times unableto withstand the capital investment and operational cost of a man-ure treatment facility or they do not own the land required for theapplication of the produced manures in an environmental friendlymanner. This incapability of small farms to fulfil the requirementsof the directive provides the basis for the development of centra-lised treatment systems (as the ones in Denmark) for accommodat-ing the needs of the farmers as well as of other locally producedwastewaters.

Common wastewaters in Greece requiring management thatcan be used for combined treatment to the DWE includes (a)the cattle manure which is probably the most abundant of allagricultural wastewaters and produced in more than 28.000 dif-ferent farms and (b) the fattening pig manures, which are pro-duced in about 42.000 pig farms (El.STAT., 2000). Very few ofthese farms have in place a wastewater treatment system andmost of the farmers rely on the application of the wastewatersonto the ground as fertilisers and in a way to remove the problemfrom their farm and transfer it into the local environment. How-ever, The soil application of manures in an untreated form it hasgreat consequences on the environmental quality including path-ogenic contamination of ground and underground waters some-thing that could result in health risks to local communities dueto contamination of the potable water sources. There are manypublished cases of groundwater pollution due to mistreatment

of manures including the case of Walkerton, Canada in 2000where in a small community of 5000 residents more than 2300people became ill including 7 fatalities due to the consumptionof water contaminated with Escherichia coli O157:H7 (Unc andGoss, 2004). While the loss of life due to the consumption of con-taminated water is the most important consequence of the mis-application of manures, the economic impact could also becomeoverwhelming for local communities both due to the need forsafeguarding the water quality as well as for treating the avail-able waters ready for human consumption. A number of re-searches are presenting that the AD process could besuccessfully employed for the reduction of the pathogens presentin the wastewater mixtures by at least 2 log under mesophiliccondition in a HRT of 12 days (Horan et al., 2004) while underthermophilic conditions a 2 log reduction of pathogens could beachieved within 2 h after the introduction of the wastewaters intothe treatment vessel (Popat et al., 2010). This capability of AD toreduce the pathogens from the wastewaters when combined withthe biogas production and nitrate removal efficiency renders ADas a promising and attractive wastewater management methodwith multiple advantages compared to other treatment methods.

The aim of this work was to investigate the way that a combi-nation of usual agro-industrial wastes known to pose certain diffi-culties as single feed stocks in anaerobic digestion systems, can betreated together in a single anaerobic digestion system, aiming atthe reduction of environmental contamination, as well as to pro-duce a sustainable fuel.

The experiments were conducted both in mesophilic and ther-mophilic temperatures, in order to identify best operational condi-tions, having in mind that thermophilic digestion usually results inhigher biogas yields and better effluent quality, while mesophilicdigestion is considered as a more attractive method for anaerobicdigestion systems due to lower operational costs and higher stabil-ity of process.

2. Methods

2.1. Wastewaters and inoculum

All wastewaters used in this work were sourced locally i.e. lessthan 50 km from the facility where the experiments took place,from small to medium size operations. Fresh green olive DWEwastewater was obtained from the production facility of the Agri-cultural Cooperation of Peta, Arta (Epirus, Greece). PM and CMwere collected from local small shaded farms. A small sample fromeach wastewater was separated at the day of collection, while therest of the quantity was divided into 4 liters plastic containers andimmediately refrigerated to �20 �C until further utilization. Activeinoculum acquired from 4 anaerobic digesters of 50 l operating un-der steady state within the facilities of the University of Ioannina.The chemical analysis for the different wastewaters and the inocu-lum can be seen in Table 1.

2.2. Analytical methods

2.2.1. Biogas measurement and analysisBiogas production measurements for the continuous systems

were conducted in a 4 channel water displacement apparatus(Fig. 1) controlled by a built on-purpose microcomputer. The mainparts of the meter were the microcomputer (PLC) for controllingthe system, the electro-valves for allowing the transfer of the pro-duced gas into the different parts of the meter, the electronic waterlevel sensors and the electronic counters for counting the releases.During the operation, the produced biogas was transferred due topressure into a water filled 250 ml volumetric flask which had

Table 1Characteristics of the wastewaters and active inoculum.

DWE Cattle manure Pig manure Inoculum

Thermophilic Mesophilic

TKN mg/1 39 ± 7 2550 ± 96 5900 ± 140 830 ± 37 1050 ± 88pH 11,06 ± 0,4 7,12 ± 0,21 7,42 ± 0,42 6,96 ± 0,12 7,14 ± 0,02TS g/g 0,011 ± 0 0,135 ± 0,04 0,106 ± 0,07 0,023 ± 0 0,026 ± 0,01VS g/g 0,008 ± 0 0,111 ± 0,02 0,082 ± 0,05 0,016 ± 0 0,020 ± 0,01TOC mg/1 8820 ± 138 56060 ± 1054 63340 ± 1147 4170 ± 231 3800 ± 93C/N ratio 226/1 21.9/1 10.7/1 – –Acetic (mg/1) 28 ± 5 122 ± 11 55 ± 8 33 ± 6 46 ± 4Propionic (mg/1) 0 41 ± 4 31 ± 3 44 ± 2 33 ± 2Isobutyric (mg/1) 0 39 ± 6 99 ± 0 28 ± 2 29 ± 3Butyric (mg/1) 33 ± 4 56 ± 6 109 ± 9 32 ± 2 42 ± 4Isovaleric (mg/1) 0 34 ± 3 26 ± 0 56 ± 2 51 ± 0Valeric (mg/1) 42 ± 2 38 ± 3 54 ± 6 30 ± 2 41 ± 1Total VFA mg/1 103 330 374 223 242

Fig. 1. Schematic representation of the complete mix anaerobic digester type utilized for this experiment (not in scale).

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been set into a 1000 ml volumetric flask in reverse. When thewater was displaced from the 250 ml flask, it was transferred intothe 1000 ml flask where, an electronic water level sensor was acti-vated, giving the signal to counter to count the biogas release andto the electro valves to deny the transfer of biogas from the reactorto the meters, allowing the transfer of the biogas from the 250 mlflask into the atmosphere. The release time of the biogas from theflask to the atmosphere was set to 10 s. The metering apparatuswas calibrated to operate when 200 ml of biogas was collected intothe 250 ml flask.

Biogas production for the batch experiments was measuredwith a similar apparatus as the continuous meter with out the elec-trical and electronic parts. After each measurement, the biogas wasmanually released into the atmosphere in order to reduce pressureinside the vial.

Biogas analysis was performed with a Shimadzu GC 2014equipped with a thermal conductivity detector. A CARBOXEN1000 60/80 Column of 15ft � 1/8in was used. GC operational con-ditions were as follows: hold for 5 min at 35 �C and then heat up to220 �C at a rate of 20 �C/min. Injector temperature was set to150 �C and detector temperature was set to 220 �C. Carrier gaswas helium.

2.2.2. TOC, VFA, TS- VS, Total Kjeldahl, Total Phenols and pH analysesVolatile Fatty Acids analysis was carried out by a GC equipped

with a flame ionization detector every third day. Samples werecentrifuged to 2761 G-units for 10 min and the supernatant wereanalyzed by a Shimadzu GC17A Gas Chromatograph equipped witha flame ionization detector (FID) and a Nukol, 15 � 0.53 ID, 0.5 lm(Supelco, USA) column. Helium was used as carrier gas. GC opera-tional conditions were as follows: oven temperature from 120 to220 �C in 10 min, injector temperature was set to 250 �C and detec-tor to 300 �C.

TOC analysis was conducted with a Shimadzu TOC-VCPH carbonanalyzer coupled to a solid state combustion unit- SSM-5000A, to-tal organic carbon was derived by deducting the inorganic carbonfrom the total carbon.

Total solids, volatile solids and pH were analyzed as describedby Standard Methods, (APHA, 1989).

For the determination of TKN, a 2 ml sample of wastewater wasdigested in a HACH Digesdahl apparatus together with 3 ml H2SO4

(98% v/v), while for the amendment of the digest the HACH method8075 was used. The concentration of TKN within the sample wasmeasured in a HACH DR/2010 Spectrophotometer at the wave-length of 460 nm. (Gohil and Nakhla, 2006).

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For the determination of the concentrations of total phenolswithin the wastewater mixture, the Folin–Ciocalteau’s methodwas used (Fluka analytical reagents), following the procedure indi-cated by Garcia et al. (2000).

2.3. Experimentation setup

The experiment was divided into 2 different stages. In the firststage experiments were conducted in duplicate batches, while inthe second stage anaerobic digesters of 50 liters volume wereemployed.

Maximum addition of DWE were limited to 40% per mixture,since further addition was considered as unsafe due to the unbal-anced nature of the wastewater for biological treatment systems(C/N ratio >250/1), the high initial pH, the toxic properties of thepolyphenols toward anaerobic bacteria and the limitations of avail-ability considering its seasonal variation.

For the batch anaerobic co-digestion experiments, five dupli-cated batches for thermophilic and mesophilic digestion were pre-pared from the same raw materials within 118 ml glass vials with aworking volume of 60 ml. forty milli liter of the working volumewas filed with active anaerobic inoculum and the rest 20 ml withmixtures of the different wastewaters. The different mixes pre-pared are presented in Table 2. In order to obtain anaerobic condi-tions immediately within the vials, nitrogen gas was fed within theliquid wastewater mixture for 5 min. Finally, the vials were sealedwith butyl rubber stopper and aluminum clips and incubated at55 �C and 35 �C in two different temperature controlled drycabinets.

In the second stage of the experiment 3 mixtures for mesophilicdigestion and 3 for thermophilic digestion were prepared (table 3).The mixes examined on the continuous systems were selectedbased on the findings of the batch experimentation results, consid-ering CH4/gVSadded production as the most important parameter.For the continuous system experimentation, 4 identical stainlesssteel complete mix anaerobic digesters (Fig. 1) were utilized. Theanaerobic digesters were constructed from double layer stainlesssteel while all welding were made with food grade tungsten arcwelding. The employed digesters had diameter of 35 cm and heightof 52 cm, with a total volume of 50 litres of which the 35 were theworking volume. Digesters operated at a constant feed rate of1.66 l/d to a HRT of 21 days while waste water mixtures was intro-duced into the systems 2 times per day at 12 h intervals. Heatingfor the digesters was provided by a microcomputer controlledwater bath of 40 liters. Mixing was provided from 4 direct currentmotors coupled to stainless steel propellers operating at 60 RPMfor 6 min per hour. The propeller was constructed in such a way

Table 2Batch experimental series for thermophilic and mesophilic digestion and analysis of each

Vial % pH TKN mg/1 TS%

CM PM DWE

Thermophilic 55 �C1 50 25 25 7,40 ± 0,14 1120 ± 52 52 40 30 30 7,48 ± 0,15 1150 ± 74 53 35 35 30 7,32 ± 0,08 I240 ± 26 54 30 30 40 7,43 ± 0.10 1070 ± 103 45 25 35 40 7,54 ± 0,06 1200 ± 99 4Control (100% Water) 7,05 ± 0,07 440 ± 65 2Mesophilic 35 �C1 50 25 25 7,46 ± 0.09 1100 ± 73 52 40 30 30 7,42 ± 0,11 1250 ± 94 53 35 35 30 7,36 ± 0,17 1350 ± 67 54 30 30 40 7,52 ± 0,05 1140 ± 40 45 25 35 40 7,49 ± 0,07 1010 ± 55 4Control (100% Water) 7,11 ± 0,02 560 ± 22 2

as to cause a moderate upward movement of the wastewater mix-ture within the digester resulting in re-suspension of the settledsolids. Feeding and removal of the treated wastewater was pro-vided by eight macerator stainless steel – plastic pumps. This typeof pumps were able to deal efficiently with high solid-fiber con-tent, as well as to provide a better feed texture, with few large solidparticles, facilitating mixing inside the digester , as well as provid-ing wider contact areas for the microorganisms.

2.4. Start-up of the digesters

The start-up seeding for the systems was provided by 4 anaer-obic digesters of 50 l volume operating under steady state. Theinoculum was removed from the 4 digesters into a 180 l plasticcontainer, where mixing was provided. This step was followed inorder to ensure the same operational conditions for all 4 digesters.25 litres of inoculum were returned to each digester at day 0. Feed-ing with 500 ml of raw cattle manure diluted in 500 ml of distilledwater was provided for this day. Starting from day 1, the digesterswere fed daily with 1.66 l of liquid wastewater of which 1.26 l con-sisted of cattle manure and 400 ml of water, in order to produce amixture with TS equal to 8 ± 0.5%, until the operational 35 litreswas achieved. At day 6, the working volume of 35 l was achievedand from this day onward, removal of 1.58 l per day of treatedwastewater prior to the introduction of new wastewater wasinitiated.

3. Results and discussion,

3.1. Batch experiments

Anaerobic digestion in low volume batches was chosen as a costeffective way to assess the actual biogas production potential ofthe different waste mixtures, as low volume batch experimenta-tion poses the advantages of simple maintenance, trouble free con-trol of the processes, and parallel digestion of a large number ofsamples in a small space.

3.1.1. Biogasification of wastewater mixturesThe methane production per batch reactor per gVSadded is de-

scribed in Fig. 2 for the mesophilic and Fig. 3 for the thermophilicoperational conditions. All experiments performed well withoutlong start up times or inhibition phenomena. However, the perfor-mance of the batches under the different temperature regionswhen methane production is regarded was different, where forthe thermophilic conditions biogas production was nearly overwithin the first 12–13 days while for the mesophilic batches

mixture.

VS OLR kg/m3 C/N ratio, of the produced mixture TOC mg/1

26 18,95/1 21230 ± 46023.2 19,72/1 22680 ± 57223.3 17,63/1 21870 ± 62020.3 20,19/1 21610 ± 42819.9 16/1 19200 ± 570

– 4,431/1 1950 ± 111

26 19,34/1 21280 ± 68323.8 16,65/1 20820 ± 49023.3 16,77/1 22640 ± 47820.3 18,93/1 21590 ± 62319.9 19,71/1 19910 ± 340

– 3,76/1 2110 ± 194

Table 3Digester operation parameters and feed characteristics.

Digester CM% PM% DWE% pH TOC mg/1 TKN mg/l C/N ratio of the feed TS% OLR TOC kg/m3 per day

Thermophilic digestion (HRT 21 d)2 50 25 25 7.62 ± 0,13 44350 ± 2670 2670 ± 198 16.6/1 9 2.13 30 30 40 7.71 ± 0,09 36210 ± 2100 2500 ± 142 14.4/1 8 1.74 25 35 40 7.96 ± 0,16 37310 ± 2320 2410 ± 97 15.4/1 7 1.76Mesophilic digestion (HRT 21 d)2 50 25 25 7.59 ± 0,07 43150 ± 2740 2590 ± 127 16.6/1 9 2.043 30 30 40 7.86 ± 0,11 37560 ± 2410 2320 ± 76 16.1/1 8 1.784 35 35 30 7.74 ± 0,14 41210 ± 3280 2780 ± 113 14.8/1 8 1.9

Fig. 2. Cumulative CH4 production/gVSadded versus VFA developmet for the mesophilic batches. (Average of the duplicate batches).

Fig. 3. Cumulative CH4 production/gVSadded versus VFA developmet for the thermophilic batches. (Average of the duplicate batches).

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methane production was continued until nearly the end of the21 days. This can partly be an effect of the slower metabolism ofthe mesophilic microorganisms, possibly under mesophilic condi-tions, HRT longer than 21 days might be necessary for increasedbiogas recovery (Aoki and Kawase 1991).

The actual methane production of these wastewater mixtureswas found to vary between 250–300 ml/gVSadded for the meso-philic (Fig. 2) and 270–350 ml/gVSadded for the thermophilicbatches (Fig. 3). Higher methane production was observed in batch3 for the thermophilic range which was containing a mixture of

wastewater comprised from 35% CM, 35% PM and 30% DWE in aC/N ration of 17.63. For the mesophilic digestion highest methaneproduction was observed for the batch 1, containing a mixture of50% CM, 25% PM and 25% DWE with a C/N ratio of 19.34. No inhi-bition was observed since there was a very small lag-adaptationphase at the starting of the process. For approximately 3 days, allsamples where the biogas production of the control vials (onlyinoculum and water) and the experimental vials was nearly thesame. This delayed response of the microorganisms could be dueto the adaptation need of the anaerobic microorganisms to the

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new feedstock, or to a slow hydrolysis of the organic matter pres-ent within the wastewater mixtures as described by Palatsi et al.(2010).

During the digestion period the concentrations of VFA de-creased from around 750 mg/l to about 100 mg/l for the mesophilicand to nearly non detectable levels for the thermophilic conditions.As it can be seen from Figs. 2 and 3, the reduction of VFAs for themesophilic and thermophilic digestion was different. For the mes-ophilic digestion the reduction was slow and even at the end of the21 days there was still available VFAs present within the digestatein concentrations of approximately 100 mg/l, while under thermo-philic conditions the VFAs were rapidly consumed and only about a25% of the concentrations available at day 1 was left at day 9. Thisdifference in the speed in which the VFAs was consumed betweenthe 2 different temperature regions is possibly the effect of thegreater activity of microorganisms under thermophilic conditions(Aoki and Kawase, 1991), but is could also be an effect of special-ized microorganisms thrive in higher temperatures (Kim et al.,2002).

VS reduction at the present study varied between 65–73% forthe mesophilic digestion and 70–77% for the thermophilic batches.It is possible that the percentage of VS reduction for the mesophilicdigestion could have been equal to the thermophilic digestionexperiments if the HRT have been extended, since even after the21 days of the experiment the mesophilic batches were still pro-ducing biogas on a daily basis, showing that the consumption ofthe organic load of the wastewater was not over. A similar reduc-tion of about 10% of the VS between the mesophilic and thermo-philic digestion is presented by Kim et al. (2002), where in acomparison of the treatment of dog food within different typesof anaerobic digesters, they found that there is an increase onthe VS reduction of around 10% (from 70 to 80%) in the thermo-philic digesters compared to the mesophilic systems.

It was found that under anaerobic conditions total organic car-bon loading of the wastewater mixtures is possible to be reducedby more that 80% when thermophilic conditions are applied andwhen the hydraulic retention time is sustained at 21 days. Againthe treatment of the wastewaters under thermophilic conditionswas able to remove a higher proportion of the available organiccarbon from the wastewaters varied between 5 and 7% when com-pared to the mesophilic systems. These results are coming inagreement with the results of other researchers (Perez et al.,2006), finding similar TOC removal efficiencies under anaerobicdigestion, varying between 80 and 94%.

One of the aims of this research was to assess how the phenoliccompounds of the DWE behave under anaerobic digestion condi-tions, however, for the batch experiment and due to the high dilu-tion of such effluent with other wastewaters that are notcontaining phenols, polyphenols was not able to be detected afterthe preparation of the mixtures and their introduction into thevials containing the active seed.

3.2. Continuous systems

The mixtures chosen for the mesophilic digestion were the 1, 4and 5 while 1, 3 and 4 used for the thermophilic digestion (as de-scribed in Tables 2 and 3). The mixtures were chosen only on thebasis of CH4 potential per g/VSadded, as the method could have anindustrial application where the methane production is the mostimportant criteria for accepting the waste mixture on which theindustrial digester will operate, as is providing the revenue for awastewater biogasification system.

3.2.1. Experimental results of continuous systemsThe procedure followed for the start-up of the digesters is pre-

sented in chapter 2.4. All digesters were inoculated with active

seed taken from anaerobic systems operating under steady stateand sustained only with cattle manure. From the 4 digesters em-ployed for this experiment, 3 systems were fed with mixtures ofthe wastewaters, while the control digester was fed only with cat-tle manure with water addition, in order to control TS levels.

After the introduction of the active seed into the digesters andthe initiation of the start up plan, the digesters showed an increaseon their daily biogas production, which peaked at around days 17–21. From this time onwards, daily biogas production was stabilizedwith little fluctuations between 27–29 liters and 32–35 litres perday for the mesophilic and the thermophilic digesters respectively.The digesters were left to operate under this state for a completeHRT time of 21 days, prior to the initiation of the co-digestionexperiment. At day 42, the co-digestion experiment initiated andwas followed from an increase on the daily biogas production,which was stabilized around 14–16 days later. After this time,the levels of daily produced biogas were stabilized for all digestersin both thermophic and mesophilic conditions, showing an accep-tance of the microorganisms to the new feed stock. The biogasyield of the co-digestion mixtures showed an 8–25% and 17–27%increase for the mesophilic and thermophilic digesters respectivelywhen compared to the control digesters (data not shown). Bare inmind that only reactors 2 and 3 were treating the same mixtures ofwastewaters while reactor 4 was used for mixture 3 and 5 for themesophilic and thermophilic digestion respectively, as theseshown in table 3. Reactor 2 (mixture 1) was in both cases theone having the largest daily biogas production, something thatwas expected as it was the one with the highest levels of volatilesolids inside the wastewater mixture.

Comparing the methane production of ml/day and per gVSadded

(Fig. 4), reactor 2 for both mesophilic and thermophilic digestionwas the one producing the lower amount of CH4/gVS availablewithin the systems. However, in both cases reactor 2 could be con-sidered more stable compared to the other 2 digesters, whichshowed adaptation problems to the new feedstock, especially afterday 73 for the mesophilic and 57 for the thermophilic digestion.The adaptation incapability of the microorganisms was identifiedby the reduction of the CH4 content within the biogas by 5–8%. Thisbecomes even more clear at the mesophilic systems (Fig. 4), whereCH4 production per gVSadded for all mixtures were less than 7%apart, while in the case of batch experiment the differences ofthe methane produced between the studied mixtures were in therange of 25%. This insufficiency of the microorganism to withstandthe feedstock alteration and the addition of DWE however was notaccompanied with accumulation of VFAs (Fig. 5) and their concen-trations were rarely exceeding 500 mg/l for all mixes. This showsthat at least acetogenic bacteria were also having difficulties toconvert the organic matter within the wastewater mixture intomethanogenic substrate.

For the present study, the mesophilic co-digestion of DWE, CMand PM showed a positive methane yield of 50–56 % per gramVSadded, while for the thermophilic digestion this increase was be-tween 50 and 61% compared to the control digesters fed only withcattle manure and water. Other researchers have presented similarresults as (Goberna et al., 2010) where the combined treatment ofthe cattle excreta and olive mill wastes was able to derive 179 ml/CH4/gVSadded when mesophilic digestion was used, while there wasan increase in the methane production of about 17% under thermo-philic conditions. Furthermore, (Gelegenis et al., 2007) studied theimpact of the addition of the olive mill wastewater on the meso-philic anaerobic digestion of diluted poultry manure, concludingthat the addition of 25% (V/V) of olive mill wastewater affectsthe increase of biogas production by about 20–25%, possibly dueto the stabilization of the nutrients inside the feed mixtures as wellas due to lower TS levels. On the other hand, Angelidaki and Ahring(1997) on their study found that the co-digestion of manures and

Fig. 4. CH4 ml/gVSadded production for mesophilic and thermophilic systems.

Fig. 5. Total VFA development over time for the continuous systems (g/l). (Average of three samples).

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olive mill wastewater in dilution of 25–75% can result in a 150% in-crease on the biogas yield, indicating higher production increasecompared to the present study.

The high initial pH value of the DWE resulted to the increase ofthe pH within all reactors employed for the co-digestion experi-ment, with reactor 3 for mesophilic digestion being the only onewhere the pH values bypass 8 before the end of the experimentalperiod, increasing the possibilities for process failure. However,no inhibition of the process was observed and all digesters wereable to withstand this change of the operational conditions with-out signs of failure. It is possible that pH correction during the pro-cess might be necessary when DWE is used as feed stock foranaerobic digestion systems for long periods of time.

The total phenols concentrations for all mixtures tested in bothmesophilic and thermophilic temperature regions did never exceed120 mg/l, which is a concentration threshold considered as low en-ough for not affecting the microorganisms. However the removalefficiency of polyphenols was very limited, varying between 22and 30% for the mesophilic and 10–17% for the thermophilic reac-tors. This low degradation efficiency of phenols under thermophilicconditions is a common phenomenon as described by Levén andSchnürer (2005) attributed to the selective growth of phenoldegrading microorganisms or as an effect of the greater biodiversitypresent in a mesophilic digester, where combined actions of differ-ent microorganisms could result in higher degree of phenolics

utilization. Similar results for phenolics degradation were pre-sented by (Dalis et al., 1996) with a 35% removal, however, other re-searches have achieved much higher removal efficiencies of phenolsunder anaerobic conditions between 50 and 80% (Marques, 2001;Boubaker et al., 2010). These increased removal efficienciesachieved by other researchers could be attributed to different typesof reactors, increased HRT, increased dilution, better adaptation ofthe microorganisms and better optimization of the digestionprocess.

Total organic carbon removal achieved for the present studyvaried between 72 and 77% for the mesophilic digesters and 74and 81% for the thermophilic digestion during the complete periodof the co-digestion experiment. No inhibition of the anaerobicdigestion process could be attributed possibly to the organic loadof the wastewater mixtures observed, while under thermophilicconditions the removal efficiency was increased only by about 5–7%, which was lower than expected as under thermophilic condi-tions a 10% increase is more usual compared to mesophilicconditions.

4.1. Batch and continuous system digestion

The anaerobic co-digestion of DWE, CM and PM performed inboth experimental batches and continuous systems was undermesophilic and thermophilic temperature regions. While no adap-

5002 I.S. Zarkadas, G.A. Pilidis / Bioresource Technology 102 (2011) 4995–5003

tation problems or inhibition of the process were observed in batchexperiments, in the continuous systems the CH4/gVSadded produc-tion was reduced by 7–30% compared to the values obtained dur-ing batch digestion. This reduction of the methane recovery couldnot be attributed to any of the monitored process indicators (VFA,pH, TKN and Phenols), neither to OLR of the feed as none of theseexcided the threshold limits as proposed by other researchers(Azbar et al., 2009; Angelidaki and Ahring, 1993). A possible expla-nation of the experienced problem could be the effects associatedto the accumulation of salts within the digesters. As mentioned be-fore, NaCl is used during fermentation of table olives prior to pack-ing in concentrations of about 5% v/v in water. DWE used for thepresent research were containing low volumes of fermentationwastewater (as brine reused in the process or added to the finalproduct for preservation purposes), it is possible that the additionof NaCl sourced from the DWE into the digesters resulted in osmo-tic pressure change within the systems and damage to microbio-logical processes. During the experimentation period, no analysisof the wastewaters for the concentrations of NaCl was conductedas considered not important. However, as other researchers havepresented (Fang et al., 2011; Lefebvre et al., 2007), the additionof sodium in AD systems in concentrations of 1 to 11 g/l could re-sult in 25% reduction in the biogas yield or even to the completeinhibition of the process. However, the final effect of the salt inthe process is related to the type of substrate and tolerance ofthe microorganisms. Considering that the mechanism that additionof salts in the anaerobic digestion inhibits the process is not clear,Lefebvre et al., 2007 proposed that the presence of NaCl in ananaerobic digestion system is affecting the kinetics of the microor-ganism, thus resulting in longer digestion times. It is very possiblethat reduced activity of the microorganisms as a result of a lowaccumulation of NaCl within the digesters might be the reason ofthe lower methane yields/gVSadded achieved for the continuousdigesters compared to the batch systems.

While mesophilic and thermophilic microorganisms bothshowed sub-optional growth with the addition of more than 25%DWE, thermophilic flora appear more tolerant to the effect of theinhibitory parameter and provided better results for both methaneproduction and organic load reduction. This is coming in agree-ment that possibly the observed inhibition resulted from slow pro-cess kinetics due to NaCl accumulation. Batch experiments in thefirst stage of this work revealed that under thermophilic conditionsmore than 80% of the total methane was recovered within 12 daysof the introduction of the active inoculum into the system. Thisprovided time (when HRT is sustained >21 d) to the microorgan-isms to achieve better results compared to the mesophilic systems,even in the case of sub-optional growth.

4.2. Mesophilic VS thermophilic digestion of DWE, CM and PM

Anaerobic digestion of industrial wastewaters is usually per-formed in mesophilic temperatures due to low costs related toheating and maintaining a digester at the 55oC. However, in thecase of the combined treatment of DWE, CM and PM, when DWEcomprised the 25–40% of the feedstock, thermophilic digestionprovided a more stable process, coupled to limited gains on meth-ane recovery (7–15%) compared to the mesophilic digestion, prob-ably as a result of the higher kinetics of the microorganisms.Further research it is required for optimization of the process incontinuous systems in order to reach the methane recovery ratesachieved in the batch systems

5. Conclusion

The present study demonstrates the feasibility of anaerobicdigestion under the combined treatment of DWE, CM and PM in

different mixes. More than 50% increase on methane productionper gVSadded was achieved with the co-digestion of these wastewa-ters compared to single cattle manure fed system. For all parameters(except phenolic compounds) monitored, thermophilic digestionprovided better removal efficiencies. 40% Addition of DWE in anaer-obic digestion systems could be considered safe for the process,however it could lead to the sub-optional growth of the microorgan-isms something that will result in lower methane recovery than themethane production potential of the wastewater mixture.

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