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

Bioresource Technology 102 (2011) 4995–5003

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Anaerobic Co-Digestion of table olive debittering & washing Effluent, cattle manure and pig manure in batch and high volume laboratory anaerobic digesters: Effect of temperature Ioannis S. Zarkadas ⇑, George A. Pilidis University of Ioannina, Department of Biological Applications and Technologies, Laboratory of Environmental Chemistry, 45110 Ioannina, Greece

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Article history: Received 6 August 2010 Received in revised form 19 January 2011 Accepted 20 January 2011 Available online 28 January 2011 Keywords: Anaerobic digestion Wastewater management Cattle and pig manure Biogas production Table olive process wastewater

a b s t r a c t The prospective of table olive debittering & washing Effluent (DWE) as feed stock wastewater for anaerobic digestion (AD) systems was investigated in batch and continuous systems together with cattle and pig manures. While DWE considered unsuitable for biological treatment methods due to its unbalanced nature, the co-digestion of the wastewaters resulted in a 50% increase in the methane production/gram volatile solidsadded (CH4/gVSadded), accompanied by 30% phenol reduction and 80% total organic carbon removal (TOC). pH increase during the co-digestion period was not identified as an inhibitory factor and 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-overloading state. Under thermophilic conditions a 7% increase on the TOC removal efficiency was achieved when compared to the mesophilic systems while, under mesophilic conditions phenolic compounds reduction was 10% higher compared to the thermophilic systems. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Olive (Olea Europea) is an evergreen tree mainly grown around the Mediterranean Sea. It is widely cultivated for producing olive fruit used either as a nutritious product, or for olive oil extraction. Production of table olives is a seasonal activity in Greece, with an annual production of 65.000 tonnes in about 75 different factories. 70% of this production is taking place near the sea coast (Kyriacou et al., 2005). During the process, large volumes of wastewaters with high pH levels, moderate COD (10 g/l) and polyphenolic content (150–400 mg/l) are being generated. The table olive production is based in 3 manufacturing stages. During the first stage the olive fruit is treated with NaOH (1, 5–2% by weight in water) in order to remove its bitter taste. It is then washed in order to remove the excess NaOH (Second stage) and finally, the third stage is the lactic fermentation of the oil fruit in brine which helps in preparing a tasteful product (Parinos et al., 2007) The volume of wastewaters produced per tonne of final olive product varies between 3.9 and 7.5 m3. This wastewater is usually dumped into water courses or sent to evaporation ponds, leading

Abbreviations: DWE, debittering and washing effluent; PM, pig manure; CM, cattle manure; TOC, total organic carbon; VFA, volatile fatty acids; TS, total solids; VS, volatile solids; C/N, carbon to nitrogen ratio; HRT, hydraulic retention time; COD, chemical oxygen demand; UV, ultraviolet radiation. ⇑ Corresponding author. Tel.: +30 2651007348; fax: +30 26510 07274. E-mail addresses: [email protected], [email protected] (I.S. Zarkadas). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.01.065

to environmental contamination through runoff and leaching (Beltran-Heredia et al., 2000). The addition of NaOH during the debittering process combined with the high polyphenolic content render this wastewater unsuitable 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 researchers have proposed the combined usage of a chemical method that is followed by a biological treatment step (Beltran-Heredia et al., 2000; Benitez et al., 2002). Nearly all of these methods are using the same idea, based on the application of a strong oxidising agent in order to oxidise the non or low biodegradable compounds, followed by a biological step, in order to reduce the organic load of the wastewater. For this purpose, strong oxidisers are applied to the wastewater, including hydrogen peroxide, Fenton reagent, ozone, ferric iron, or the combined usage of a UV radiation source together with one of the above oxidizing agents. However, many problems arise due to insufficient TC reduction, even when advanced oxidation methods are applied, and nearly always, a biological treatment method must be followed (Beltran et al., 1999; Justino et al., 2009). This need for a further treatment step renders the chemical methods as ineffective, by increasing the production cost of the final product, while requiring skilled operators and the acquisition and application of expensive chemicals. On the other hand, due to seasonal production of table olives lasting only between September and November every year (Aggelis et al., 2001), the construction of a biological treatment system with

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exclusive purpose the treatment of DWE is not considered as economically viable and effective management method. This is due to the fact that microorganisms require long adaptation periods in order to reach exponential growth phase firstly and stationary growth phase latter, where maximum removal of the organic load is possible in short time. A wastewater treatment method that has been applied to DWE with some success is the anaerobic digestion (AD). AD offers the advantages of reducing the organic loading of wastewaters, while producing 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 AD has to the organic loading of this wastewater and the efficient removal of the polyphenols. Aggelis et al. (2001) examined the anaerobic degradation of polyphenols and the reduction of the organic load of DWE, concluding that anaerobic digestion was able to remove only 12% of the polyphenols and 49% of the organic load, while inhibition of the process was identified possible due to VFA and phenolic compounds accumulation. For overcoming problems related to the accumulation of inhibitory compounds in addition to the problems experienced by Aggelis et al. (2001), co-digestion could be applied for the simultaneous treatment of DWE with other locally available wastewaters. Although co-digestion of multiple wastewater streams is mainly anticipated for resolving the problem of toxic and inhibitory compounds accumulation, provides additional advantages by encouraging synergetic phenomena between the microorganisms, providing nutrient balance (resulting in increasing biogas production yields) (Cecchi et al., 1996) and sharing costs associated with treatment between different operations. However, the co-digestion facilities if not planned correctly present their own limitations due to logistical problems of transporting large volumes of wastewaters within great distances, something that results in increased costs and adds onto the overall complexity of the process. Pressure has been applied to farmers for proper application of manures on soil in a way to protect surface and underground waters from nitrate pollution with the implementation of the EC Nitrates Directive. While protection of the environment is a way towards sustainable development, the requirements of the directive are rarely fulfilled by the farmers due to the nature of the European farming industry, which is composed by a large number of low intensity farms. These farmers are most of the times unable to withstand the capital investment and operational cost of a manure treatment facility or they do not own the land required for the application of the produced manures in an environmental friendly manner. This incapability of small farms to fulfil the requirements of the directive provides the basis for the development of centralised treatment systems (as the ones in Denmark) for accommodating the needs of the farmers as well as of other locally produced wastewaters. Common wastewaters in Greece requiring management that can be used for combined treatment to the DWE includes (a) the cattle manure which is probably the most abundant of all agricultural wastewaters and produced in more than 28.000 different farms and (b) the fattening pig manures, which are produced in about 42.000 pig farms (El.STAT., 2000). Very few of these farms have in place a wastewater treatment system and most of the farmers rely on the application of the wastewaters onto the ground as fertilisers and in a way to remove the problem from their farm and transfer it into the local environment. However, The soil application of manures in an untreated form it has great consequences on the environmental quality including pathogenic contamination of ground and underground waters something that could result in health risks to local communities due to contamination of the potable water sources. There are many published cases of groundwater pollution due to mistreatment

of manures including the case of Walkerton, Canada in 2000 where in a small community of 5000 residents more than 2300 people became ill including 7 fatalities due to the consumption of water contaminated with Escherichia coli O157:H7 (Unc and Goss, 2004). While the loss of life due to the consumption of contaminated water is the most important consequence of the misapplication of manures, the economic impact could also become overwhelming for local communities both due to the need for safeguarding the water quality as well as for treating the available waters ready for human consumption. A number of researches are presenting that the AD process could be successfully employed for the reduction of the pathogens present in the wastewater mixtures by at least 2 log under mesophilic condition in a HRT of 12 days (Horan et al., 2004) while under thermophilic conditions a 2 log reduction of pathogens could be achieved within 2 h after the introduction of the wastewaters into the treatment vessel (Popat et al., 2010). This capability of AD to reduce the pathogens from the wastewaters when combined with the biogas production and nitrate removal efficiency renders AD as a promising and attractive wastewater management method with multiple advantages compared to other treatment methods. The aim of this work was to investigate the way that a combination of usual agro-industrial wastes known to pose certain difficulties as single feed stocks in anaerobic digestion systems, can be treated together in a single anaerobic digestion system, aiming at the reduction of environmental contamination, as well as to produce a sustainable fuel. The experiments were conducted both in mesophilic and thermophilic temperatures, in order to identify best operational conditions, having in mind that thermophilic digestion usually results in higher biogas yields and better effluent quality, while mesophilic digestion is considered as a more attractive method for anaerobic digestion systems due to lower operational costs and higher stability of process. 2. Methods 2.1. Wastewaters and inoculum All wastewaters used in this work were sourced locally i.e. less than 50 km from the facility where the experiments took place, from small to medium size operations. Fresh green olive DWE wastewater was obtained from the production facility of the Agricultural Cooperation of Peta, Arta (Epirus, Greece). PM and CM were collected from local small shaded farms. A small sample from each wastewater was separated at the day of collection, while the rest of the quantity was divided into 4 liters plastic containers and immediately refrigerated to 20 °C until further utilization. Active inoculum acquired from 4 anaerobic digesters of 50 l operating under steady state within the facilities of the University of Ioannina. The chemical analysis for the different wastewaters and the inoculum can be seen in Table 1. 2.2. Analytical methods 2.2.1. Biogas measurement and analysis Biogas 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 main parts of the meter were the microcomputer (PLC) for controlling the system, the electro-valves for allowing the transfer of the produced gas into the different parts of the meter, the electronic water level sensors and the electronic counters for counting the releases. During the operation, the produced biogas was transferred due to pressure into a water filled 250 ml volumetric flask which had

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I.S. Zarkadas, G.A. Pilidis / Bioresource Technology 102 (2011) 4995–5003 Table 1 Characteristics of the wastewaters and active inoculum.

TKN mg/1 pH TS g/g VS g/g TOC mg/1 C/N ratio Acetic (mg/1) Propionic (mg/1) Isobutyric (mg/1) Butyric (mg/1) Isovaleric (mg/1) Valeric (mg/1) Total VFA mg/1

DWE

Cattle manure

Pig manure

39 ± 7 11,06 ± 0,4 0,011 ± 0 0,008 ± 0 8820 ± 138 226/1 28 ± 5 0 0 33 ± 4 0 42 ± 2 103

2550 ± 96 7,12 ± 0,21 0,135 ± 0,04 0,111 ± 0,02 56060 ± 1054 21.9/1 122 ± 11 41 ± 4 39 ± 6 56 ± 6 34 ± 3 38 ± 3 330

5900 ± 140 7,42 ± 0,42 0,106 ± 0,07 0,082 ± 0,05 63340 ± 1147 10.7/1 55 ± 8 31 ± 3 99 ± 0 109 ± 9 26 ± 0 54 ± 6 374

Inoculum Thermophilic

Mesophilic

830 ± 37 6,96 ± 0,12 0,023 ± 0 0,016 ± 0 4170 ± 231 – 33 ± 6 44 ± 2 28 ± 2 32 ± 2 56 ± 2 30 ± 2 223

1050 ± 88 7,14 ± 0,02 0,026 ± 0,01 0,020 ± 0,01 3800 ± 93 – 46 ± 4 33 ± 2 29 ± 3 42 ± 4 51 ± 0 41 ± 1 242

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

been set into a 1000 ml volumetric flask in reverse. When the water was displaced from the 250 ml flask, it was transferred into the 1000 ml flask where, an electronic water level sensor was activated, giving the signal to counter to count the biogas release and to the electro valves to deny the transfer of biogas from the reactor to the meters, allowing the transfer of the biogas from the 250 ml flask into the atmosphere. The release time of the biogas from the flask to the atmosphere was set to 10 s. The metering apparatus was calibrated to operate when 200 ml of biogas was collected into the 250 ml flask. Biogas production for the batch experiments was measured with a similar apparatus as the continuous meter with out the electrical and electronic parts. After each measurement, the biogas was manually released into the atmosphere in order to reduce pressure inside the vial. Biogas analysis was performed with a Shimadzu GC 2014 equipped with a thermal conductivity detector. A CARBOXEN 1000 60/80 Column of 15ft  1/8in was used. GC operational conditions were as follows: hold for 5 min at 35 °C and then heat up to 220 °C at a rate of 20 °C/min. Injector temperature was set to 150 °C and detector temperature was set to 220 °C. Carrier gas was helium.

2.2.2. TOC, VFA, TS- VS, Total Kjeldahl, Total Phenols and pH analyses Volatile Fatty Acids analysis was carried out by a GC equipped with a flame ionization detector every third day. Samples were centrifuged to 2761 G-units for 10 min and the supernatant were analyzed by a Shimadzu GC17A Gas Chromatograph equipped with a flame ionization detector (FID) and a Nukol, 15  0.53 ID, 0.5 lm (Supelco, USA) column. Helium was used as carrier gas. GC operational conditions were as follows: oven temperature from 120 to 220 °C in 10 min, injector temperature was set to 250 °C and detector to 300 °C. TOC analysis was conducted with a Shimadzu TOC-VCPH carbon analyzer coupled to a solid state combustion unit- SSM-5000A, total organic carbon was derived by deducting the inorganic carbon from the total carbon. Total solids, volatile solids and pH were analyzed as described by Standard Methods, (APHA, 1989). For the determination of TKN, a 2 ml sample of wastewater was digested in a HACH Digesdahl apparatus together with 3 ml H2SO4 (98% v/v), while for the amendment of the digest the HACH method 8075 was used. The concentration of TKN within the sample was measured in a HACH DR/2010 Spectrophotometer at the wavelength of 460 nm. (Gohil and Nakhla, 2006).

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For the determination of the concentrations of total phenols within the wastewater mixture, the Folin–Ciocalteau’s method was used (Fluka analytical reagents), following the procedure indicated by Garcia et al. (2000). 2.3. Experimentation setup The experiment was divided into 2 different stages. In the first stage experiments were conducted in duplicate batches, while in the second stage anaerobic digesters of 50 liters volume were employed. Maximum addition of DWE were limited to 40% per mixture, since further addition was considered as unsafe due to the unbalanced nature of the wastewater for biological treatment systems (C/N ratio >250/1), the high initial pH, the toxic properties of the polyphenols toward anaerobic bacteria and the limitations of availability considering its seasonal variation. For the batch anaerobic co-digestion experiments, five duplicated batches for thermophilic and mesophilic digestion were prepared from the same raw materials within 118 ml glass vials with a working volume of 60 ml. forty milli liter of the working volume was filed with active anaerobic inoculum and the rest 20 ml with mixtures of the different wastewaters. The different mixes prepared are presented in Table 2. In order to obtain anaerobic conditions immediately within the vials, nitrogen gas was fed within the liquid wastewater mixture for 5 min. Finally, the vials were sealed with butyl rubber stopper and aluminum clips and incubated at 55 °C and 35 °C in two different temperature controlled dry cabinets. In the second stage of the experiment 3 mixtures for mesophilic digestion and 3 for thermophilic digestion were prepared (table 3). The mixes examined on the continuous systems were selected based on the findings of the batch experimentation results, considering CH4/gVSadded production as the most important parameter. For the continuous system experimentation, 4 identical stainless steel complete mix anaerobic digesters (Fig. 1) were utilized. The anaerobic digesters were constructed from double layer stainless steel while all welding were made with food grade tungsten arc welding. The employed digesters had diameter of 35 cm and height of 52 cm, with a total volume of 50 litres of which the 35 were the working volume. Digesters operated at a constant feed rate of 1.66 l/d to a HRT of 21 days while waste water mixtures was introduced into the systems 2 times per day at 12 h intervals. Heating for the digesters was provided by a microcomputer controlled water bath of 40 liters. Mixing was provided from 4 direct current motors coupled to stainless steel propellers operating at 60 RPM for 6 min per hour. The propeller was constructed in such a way

as to cause a moderate upward movement of the wastewater mixture within the digester resulting in re-suspension of the settled solids. Feeding and removal of the treated wastewater was provided by eight macerator stainless steel – plastic pumps. This type of pumps were able to deal efficiently with high solid-fiber content, as well as to provide a better feed texture, with few large solid particles, facilitating mixing inside the digester , as well as providing wider contact areas for the microorganisms. 2.4. Start-up of the digesters The start-up seeding for the systems was provided by 4 anaerobic digesters of 50 l volume operating under steady state. The inoculum was removed from the 4 digesters into a 180 l plastic container, where mixing was provided. This step was followed in order to ensure the same operational conditions for all 4 digesters. 25 litres of inoculum were returned to each digester at day 0. Feeding with 500 ml of raw cattle manure diluted in 500 ml of distilled water was provided for this day. Starting from day 1, the digesters were fed daily with 1.66 l of liquid wastewater of which 1.26 l consisted of cattle manure and 400 ml of water, in order to produce a mixture with TS equal to 8 ± 0.5%, until the operational 35 litres was achieved. At day 6, the working volume of 35 l was achieved and from this day onward, removal of 1.58 l per day of treated wastewater prior to the introduction of new wastewater was initiated. 3. Results and discussion, 3.1. Batch experiments Anaerobic digestion in low volume batches was chosen as a cost effective way to assess the actual biogas production potential of the different waste mixtures, as low volume batch experimentation poses the advantages of simple maintenance, trouble free control of the processes, and parallel digestion of a large number of samples in a small space. 3.1.1. Biogasification of wastewater mixtures The methane production per batch reactor per gVSadded is described in Fig. 2 for the mesophilic and Fig. 3 for the thermophilic operational conditions. All experiments performed well without long start up times or inhibition phenomena. However, the performance of the batches under the different temperature regions when methane production is regarded was different, where for the thermophilic conditions biogas production was nearly over within the first 12–13 days while for the mesophilic batches

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

pH

TKN mg/1

TS%

VS OLR kg/m3

C/N ratio, of the produced mixture

TOC mg/1

25 30 30 40 40

7,40 ± 0,14 7,48 ± 0,15 7,32 ± 0,08 7,43 ± 0.10 7,54 ± 0,06 7,05 ± 0,07

1120 ± 52 1150 ± 74 I240 ± 26 1070 ± 103 1200 ± 99 440 ± 65

5 5 5 4 4 2

26 23.2 23.3 20.3 19.9 –

18,95/1 19,72/1 17,63/1 20,19/1 16/1 4,431/1

21230 ± 460 22680 ± 572 21870 ± 620 21610 ± 428 19200 ± 570 1950 ± 111

25 30 30 40 40

7,46 ± 0.09 7,42 ± 0,11 7,36 ± 0,17 7,52 ± 0,05 7,49 ± 0,07 7,11 ± 0,02

1100 ± 73 1250 ± 94 1350 ± 67 1140 ± 40 1010 ± 55 560 ± 22

5 5 5 4 4 2

26 23.8 23.3 20.3 19.9 –

19,34/1 16,65/1 16,77/1 18,93/1 19,71/1 3,76/1

21280 ± 683 20820 ± 490 22640 ± 478 21590 ± 623 19910 ± 340 2110 ± 194

% CM

PM

Thermophilic 55 °C 1 50 25 2 40 30 3 35 35 4 30 30 5 25 35 Control (100% Water) Mesophilic 35 °C 1 50 25 2 40 30 3 35 35 4 30 30 5 25 35 Control (100% Water)

DWE

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I.S. Zarkadas, G.A. Pilidis / Bioresource Technology 102 (2011) 4995–5003 Table 3 Digester operation parameters and feed characteristics. Digester

CM%

PM%

Thermophilic digestion (HRT 21 d) 2 50 25 3 30 30 4 25 35 Mesophilic digestion (HRT 21 d) 2 50 25 3 30 30 4 35 35

DWE%

pH

TOC mg/1

TKN mg/l

C/N ratio of the feed

TS%

OLR TOC kg/m3 per day

25 40 40

7.62 ± 0,13 7.71 ± 0,09 7.96 ± 0,16

44350 ± 2670 36210 ± 2100 37310 ± 2320

2670 ± 198 2500 ± 142 2410 ± 97

16.6/1 14.4/1 15.4/1

9 8 7

2.1 1.7 1.76

25 40 30

7.59 ± 0,07 7.86 ± 0,11 7.74 ± 0,14

43150 ± 2740 37560 ± 2410 41210 ± 3280

2590 ± 127 2320 ± 76 2780 ± 113

16.6/1 16.1/1 14.8/1

9 8 8

2.04 1.78 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).

methane production was continued until nearly the end of the 21 days. This can partly be an effect of the slower metabolism of the mesophilic microorganisms, possibly under mesophilic conditions, HRT longer than 21 days might be necessary for increased biogas recovery (Aoki and Kawase 1991). The actual methane production of these wastewater mixtures was found to vary between 250–300 ml/gVSadded for the mesophilic (Fig. 2) and 270–350 ml/gVSadded for the thermophilic batches (Fig. 3). Higher methane production was observed in batch 3 for the thermophilic range which was containing a mixture of

wastewater comprised from 35% CM, 35% PM and 30% DWE in a C/N ration of 17.63. For the mesophilic digestion highest methane production was observed for the batch 1, containing a mixture of 50% CM, 25% PM and 25% DWE with a C/N ratio of 19.34. No inhibition was observed since there was a very small lag-adaptation phase at the starting of the process. For approximately 3 days, all samples where the biogas production of the control vials (only inoculum and water) and the experimental vials was nearly the same. This delayed response of the microorganisms could be due to the adaptation need of the anaerobic microorganisms to the

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new feedstock, or to a slow hydrolysis of the organic matter present within the wastewater mixtures as described by Palatsi et al. (2010). During the digestion period the concentrations of VFA decreased from around 750 mg/l to about 100 mg/l for the mesophilic and to nearly non detectable levels for the thermophilic conditions. As it can be seen from Figs. 2 and 3, the reduction of VFAs for the mesophilic and thermophilic digestion was different. For the mesophilic digestion the reduction was slow and even at the end of the 21 days there was still available VFAs present within the digestate in concentrations of approximately 100 mg/l, while under thermophilic conditions the VFAs were rapidly consumed and only about a 25% of the concentrations available at day 1 was left at day 9. This difference in the speed in which the VFAs was consumed between the 2 different temperature regions is possibly the effect of the greater activity of microorganisms under thermophilic conditions (Aoki and Kawase, 1991), but is could also be an effect of specialized microorganisms thrive in higher temperatures (Kim et al., 2002). VS reduction at the present study varied between 65–73% for the mesophilic digestion and 70–77% for the thermophilic batches. It is possible that the percentage of VS reduction for the mesophilic digestion could have been equal to the thermophilic digestion experiments if the HRT have been extended, since even after the 21 days of the experiment the mesophilic batches were still producing biogas on a daily basis, showing that the consumption of the organic load of the wastewater was not over. A similar reduction of about 10% of the VS between the mesophilic and thermophilic digestion is presented by Kim et al. (2002), where in a comparison of the treatment of dog food within different types of anaerobic digesters, they found that there is an increase on the VS reduction of around 10% (from 70 to 80%) in the thermophilic digesters compared to the mesophilic systems. It was found that under anaerobic conditions total organic carbon loading of the wastewater mixtures is possible to be reduced by more that 80% when thermophilic conditions are applied and when the hydraulic retention time is sustained at 21 days. Again the treatment of the wastewaters under thermophilic conditions was able to remove a higher proportion of the available organic carbon from the wastewaters varied between 5 and 7% when compared to the mesophilic systems. These results are coming in agreement with the results of other researchers (Perez et al., 2006), finding similar TOC removal efficiencies under anaerobic digestion, varying between 80 and 94%. One of the aims of this research was to assess how the phenolic compounds of the DWE behave under anaerobic digestion conditions, however, for the batch experiment and due to the high dilution of such effluent with other wastewaters that are not containing phenols, polyphenols was not able to be detected after the preparation of the mixtures and their introduction into the vials containing the active seed. 3.2. Continuous systems The mixtures chosen for the mesophilic digestion were the 1, 4 and 5 while 1, 3 and 4 used for the thermophilic digestion (as described in Tables 2 and 3). The mixtures were chosen only on the basis of CH4 potential per g/VSadded, as the method could have an industrial application where the methane production is the most important criteria for accepting the waste mixture on which the industrial digester will operate, as is providing the revenue for a wastewater biogasification system. 3.2.1. Experimental results of continuous systems The procedure followed for the start-up of the digesters is presented in chapter 2.4. All digesters were inoculated with active

seed taken from anaerobic systems operating under steady state and sustained only with cattle manure. From the 4 digesters employed for this experiment, 3 systems were fed with mixtures of the wastewaters, while the control digester was fed only with cattle manure with water addition, in order to control TS levels. After the introduction of the active seed into the digesters and the initiation of the start up plan, the digesters showed an increase on their daily biogas production, which peaked at around days 17– 21. From this time onwards, daily biogas production was stabilized with little fluctuations between 27–29 liters and 32–35 litres per day for the mesophilic and the thermophilic digesters respectively. The digesters were left to operate under this state for a complete HRT time of 21 days, prior to the initiation of the co-digestion experiment. At day 42, the co-digestion experiment initiated and was 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 digesters in both thermophic and mesophilic conditions, showing an acceptance of the microorganisms to the new feed stock. The biogas yield of the co-digestion mixtures showed an 8–25% and 17–27% increase for the mesophilic and thermophilic digesters respectively when compared to the control digesters (data not shown). Bare in mind that only reactors 2 and 3 were treating the same mixtures of wastewaters while reactor 4 was used for mixture 3 and 5 for the mesophilic and thermophilic digestion respectively, as these shown in table 3. Reactor 2 (mixture 1) was in both cases the one having the largest daily biogas production, something that was expected as it was the one with the highest levels of volatile solids inside the wastewater mixture. Comparing the methane production of ml/day and per gVSadded (Fig. 4), reactor 2 for both mesophilic and thermophilic digestion was the one producing the lower amount of CH4/gVS available within the systems. However, in both cases reactor 2 could be considered more stable compared to the other 2 digesters, which showed adaptation problems to the new feedstock, especially after day 73 for the mesophilic and 57 for the thermophilic digestion. The adaptation incapability of the microorganisms was identified by the reduction of the CH4 content within the biogas by 5–8%. This becomes even more clear at the mesophilic systems (Fig. 4), where CH4 production per gVSadded for all mixtures were less than 7% apart, while in the case of batch experiment the differences of the methane produced between the studied mixtures were in the range of 25%. This insufficiency of the microorganism to withstand the feedstock alteration and the addition of DWE however was not accompanied with accumulation of VFAs (Fig. 5) and their concentrations were rarely exceeding 500 mg/l for all mixes. This shows that at least acetogenic bacteria were also having difficulties to convert the organic matter within the wastewater mixture into methanogenic substrate. For the present study, the mesophilic co-digestion of DWE, CM and PM showed a positive methane yield of 50–56 % per gram VSadded, while for the thermophilic digestion this increase was between 50 and 61% compared to the control digesters fed only with cattle manure and water. Other researchers have presented similar results as (Goberna et al., 2010) where the combined treatment of the cattle excreta and olive mill wastes was able to derive 179 ml/ CH4/gVSadded when mesophilic digestion was used, while there was an increase in the methane production of about 17% under thermophilic conditions. Furthermore, (Gelegenis et al., 2007) studied the impact of the addition of the olive mill wastewater on the mesophilic anaerobic digestion of diluted poultry manure, concluding that the addition of 25% (V/V) of olive mill wastewater affects the increase of biogas production by about 20–25%, possibly due to the stabilization of the nutrients inside the feed mixtures as well as due to lower TS levels. On the other hand, Angelidaki and Ahring (1997) on their study found that the co-digestion of manures and

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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).

olive mill wastewater in dilution of 25–75% can result in a 150% increase on the biogas yield, indicating higher production increase compared to the present study. The high initial pH value of the DWE resulted to the increase of the pH within all reactors employed for the co-digestion experiment, with reactor 3 for mesophilic digestion being the only one where the pH values bypass 8 before the end of the experimental period, increasing the possibilities for process failure. However, no inhibition of the process was observed and all digesters were able to withstand this change of the operational conditions without signs of failure. It is possible that pH correction during the process might be necessary when DWE is used as feed stock for anaerobic digestion systems for long periods of time. The total phenols concentrations for all mixtures tested in both mesophilic and thermophilic temperature regions did never exceed 120 mg/l, which is a concentration threshold considered as low enough for not affecting the microorganisms. However the removal efficiency of polyphenols was very limited, varying between 22 and 30% for the mesophilic and 10–17% for the thermophilic reactors. This low degradation efficiency of phenols under thermophilic conditions is a common phenomenon as described by Levén and Schnürer (2005) attributed to the selective growth of phenol degrading microorganisms or as an effect of the greater biodiversity present in a mesophilic digester, where combined actions of different microorganisms could result in higher degree of phenolics

utilization. Similar results for phenolics degradation were presented by (Dalis et al., 1996) with a 35% removal, however, other researches have achieved much higher removal efficiencies of phenols under anaerobic conditions between 50 and 80% (Marques, 2001; Boubaker et al., 2010). These increased removal efficiencies achieved by other researchers could be attributed to different types of reactors, increased HRT, increased dilution, better adaptation of the microorganisms and better optimization of the digestion process. Total organic carbon removal achieved for the present study varied between 72 and 77% for the mesophilic digesters and 74 and 81% for the thermophilic digestion during the complete period of the co-digestion experiment. No inhibition of the anaerobic digestion process could be attributed possibly to the organic load of the wastewater mixtures observed, while under thermophilic conditions the removal efficiency was increased only by about 5– 7%, which was lower than expected as under thermophilic conditions a 10% increase is more usual compared to mesophilic conditions. 4.1. Batch and continuous system digestion The anaerobic co-digestion of DWE, CM and PM performed in both experimental batches and continuous systems was under mesophilic and thermophilic temperature regions. While no adap-

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tation problems or inhibition of the process were observed in batch experiments, in the continuous systems the CH4/gVSadded production was reduced by 7–30% compared to the values obtained during batch digestion. This reduction of the methane recovery could not be attributed to any of the monitored process indicators (VFA, pH, TKN and Phenols), neither to OLR of the feed as none of these excided the threshold limits as proposed by other researchers (Azbar et al., 2009; Angelidaki and Ahring, 1993). A possible explanation of the experienced problem could be the effects associated to the accumulation of salts within the digesters. As mentioned before, NaCl is used during fermentation of table olives prior to packing in concentrations of about 5% v/v in water. DWE used for the present research were containing low volumes of fermentation wastewater (as brine reused in the process or added to the final product for preservation purposes), it is possible that the addition of NaCl sourced from the DWE into the digesters resulted in osmotic pressure change within the systems and damage to microbiological processes. During the experimentation period, no analysis of the wastewaters for the concentrations of NaCl was conducted as considered not important. However, as other researchers have presented (Fang et al., 2011; Lefebvre et al., 2007), the addition of sodium in AD systems in concentrations of 1 to 11 g/l could result in 25% reduction in the biogas yield or even to the complete inhibition of the process. However, the final effect of the salt in the process is related to the type of substrate and tolerance of the microorganisms. Considering that the mechanism that addition of salts in the anaerobic digestion inhibits the process is not clear, Lefebvre et al., 2007 proposed that the presence of NaCl in an anaerobic digestion system is affecting the kinetics of the microorganism, thus resulting in longer digestion times. It is very possible that reduced activity of the microorganisms as a result of a low accumulation of NaCl within the digesters might be the reason of the lower methane yields/gVSadded achieved for the continuous digesters compared to the batch systems. While mesophilic and thermophilic microorganisms both showed sub-optional growth with the addition of more than 25% DWE, thermophilic flora appear more tolerant to the effect of the inhibitory parameter and provided better results for both methane production and organic load reduction. This is coming in agreement that possibly the observed inhibition resulted from slow process kinetics due to NaCl accumulation. Batch experiments in the first stage of this work revealed that under thermophilic conditions more than 80% of the total methane was recovered within 12 days of the introduction of the active inoculum into the system. This provided time (when HRT is sustained >21 d) to the microorganisms 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 performed in mesophilic temperatures due to low costs related to heating and maintaining a digester at the 55oC. However, in the case of the combined treatment of DWE, CM and PM, when DWE comprised the 25–40% of the feedstock, thermophilic digestion provided a more stable process, coupled to limited gains on methane recovery (7–15%) compared to the mesophilic digestion, probably as a result of the higher kinetics of the microorganisms. Further research it is required for optimization of the process in continuous systems in order to reach the methane recovery rates achieved in the batch systems 5. Conclusion The present study demonstrates the feasibility of anaerobic digestion under the combined treatment of DWE, CM and PM in

different mixes. More than 50% increase on methane production per gVSadded was achieved with the co-digestion of these wastewaters compared to single cattle manure fed system. For all parameters (except phenolic compounds) monitored, thermophilic digestion provided better removal efficiencies. 40% Addition of DWE in anaerobic digestion systems could be considered safe for the process, however it could lead to the sub-optional growth of the microorganisms something that will result in lower methane recovery than the methane production potential of the wastewater mixture. References Aggelis, G.G., Gavala, H.N., Lyberatos, G., 2001. Combined and separate aerobic and anaerobic biotreatment of green olive debittering wastewater. Journal of Agricultural Engineering Research 80 (3), 283–292. Angelidaki, I., Ahring, B., 1997. Co-digestion of olive-oil mill wastewaters with manure, household waste or sewage sludge. Biodegradation 8, 221–226. Angelidaki, I., Ahring, K.B., 1993. 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