**5.1.2 Three-phase olive mill wastewaters (3POMWW)**

Anaerobic digestion is a promising alternative for the treatment of 3POMWW. It allows for the disposal of these wastewaters achieving considerable organic material removals and producing renewable energy in the form of biogas, which could be used as an energy source in the olive oil mill itself.

Certain components of 3POMWW such as poly-phenols, pH, oil, etc. may inhibit the AD process. Martín et al., (1991) obtained methane yields of 260 mL CH4 g-1 COD for 3POMWW. Borja et al. (1995b) improved the methane production using a pre-treatment stage with *Geotrichum candidum*, *Azotobacter Chroococcum* and *Aspergillus terreus*. The latest study reported methane yield coefficients of 300 (*Geotrichum*-pretreated 3POMWW), 315 (*Azotobacter*-pretreated 3POMWW) and 350 (*Aspergillus*-pretreated 3POMWW) mL CH4 g-1 COD against the 260 mL CH4 g-1 COD obtained for the untreated 3POMWW.

3POMWW have a low nitrogen content which limits the AD process due to the fact that the microorganisms need this element for their metabolism. In this way, co-digestion with rich nitrogen substrates may improve the biodegradability of the mixture. Azbar et al., (2008) studied the co-digestion of 3POMWW with laying hen litter obtaining a significant improvement in the biodegradability of 3POMWW. Co-digestion with liquid cow manure [20% 3POMWW, 80% liquid cow manure (v:v)] also showed good results in terms of COD and volatile solids removal (Dareioti et al., 2010).

Another option is the combination of catalytically oxidized olive mill wastewaters (by Fenton's reagent) plus anaerobic digestion. El-Gohary et al. (2009) found that the digestion of catalytically oxidized 3POMWW followed by a classical upflow anaerobic sludge blanket reactor (UASB) and a hybrid UASB as a post-treatment step is a promising alternative.

Other treatments envisage the combination of an initial liquid-liquid extraction with ethyl acetate for exploitation of the phenol content, followed by aerobic or anaerobic digestion of the phenolic extracted 3POMWW (Khoufi et al., 2006).

The use of sand filtration and subsequent treatment with powered activated carbon in batch systems has also been studied as a pre-treatment. This pre-treatment allowed COD removal efficiencies of 80%-85% for an HRT of 5 days and at an OLR of 8 g COD L-1 d-1. A methane yield of 300 mL biogas g-1 COD removed was achieved (Sabbah et al., 2004).

The separation of the digestion phases, hydrolytic-acidogenic reactor and methanogenic reactor, in two completely independent reactors can also be considered as a way to improve the AD digestion of these wastes. Bertín et al. (2010) studied different acidogenic configurations of biofilm reactors using ceramic filters or granular activated carbon with good results.

The latest research studies report AD as a promising technology for the treatment of 3POMWW, leading to sustainable waste treatment and an environmentally friendly solution.

Olive Oil Mill Waste Treatment: Improving the Sustainability

of the Olive Oil Industry with Anaerobic Digestion Technology 285

Fig. 4. DGGE analysis of the diversity of bacterial communities at different OLRs studied in one stage anaerobic digestion of 2POMSW (Rincón et al., 2008a). The position of the major electrophoretic bands corresponding to the 16S rRNA gene of the identified bacteria are indicated. A, B, C, D, E, F, G and H are the increasing OLRs studied in g COD L-1 d-1: 2.3 (A),

Other studies in two-stages at thermophilic scale reported 2POMSW as an ideal substrate for biohydrogen and methane production. These studies used diluted 2POMSW (1:4) with tap water achieving 18.5 ± 0.4 mmol CH4 g-1 total solid added (TS). Experiments for biohydrogen production followed by methane production, generated 1.6 mmol H2 g-1 TS added and 19.0 mmol CH4 g-1 TS in the methanogenic stage (Gavala et al., 2005). Mesophilic bio-hydrogen production from 3POMSW has shown to be feasible at mesophilic temperature resulting in 2.8-4.5 mmol H2 per gram of carbohydrates consumed in the reactor (Koutrouli et al., 2006). Methane production in these assays achieved a maximum value of 1.13 L CH4 L-1 d-1 at 10 days of HRT. Hydrogen is a renewable energy source and one of the most attractive applications is the conversion of

3.0 (B), 4.5 (C), 5.8 (D), 6.8 (E), 8.3 (F), 9.2 (G) and 11.0 (H).

hydrogen to electricity via fuel cells (Koutrouli et al., 2006)

#### **5.2 Two-phase olive mill wastes**

#### **5.2.1 Two-phase olive mill solid wastes (2POMSW)**

Borja et al. (2002) carried out an initial anaerobic digestibility study with four different dilutions of 2POMSW (20%, 40%, 60% and 80 %). The main findings showed that the performance of the reactor [in terms of COD removal (%)] is practically independent of the feed COD concentration. Studies with no-diluted 2POMSW were carried out by the same authors with similar results (Rincón et al., 2007). The methane yields obtained in these studies ranged between 200-300 mL CH4 g-1 COD removed. The 2POMSW is easily biodegradable by mesophilic anaerobic digestion and COD removal efficiencies up to 97 % may be achieved (Rincón et al., 2007).

Rincón et al. (2006, 2008a) studied the different microorganisms participating in the 2POMSW anaerobic digestion. For the determination of the microorganism population polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), cloning and sequencing techniques were employed. The results showed differences in the microbial communities, both bacterial and archaeal, with varying OLRs. Analysis of the microbial communities may be decisive in understanding the microbial processes taking place during 2POMSW decomposition in anaerobic reactors and optimizing their performance. During this experimental study the most frequently encountered microbial group were the Firmicutes (53.3% of analyzed sequences), represented mostly by members of the Clostridiales (Figure 4). Chloroflexi also represented an important bacterial group in the study (23.4% of sequences) and has been reported as a major constituent in anaerobic systems (Rincón, 2006). The Gamma-Proteobacteria (8.5% sequences, represented mainly by the genus *Pseudomonas*), Actinobacteria (6.4%) and Bacteroidetes (4.3%) are also significant components of the microbial communities during the anaerobic decomposition of 2POMSW (Figure 4). The major archaeal component detected for the 2POMSW anaerobic digestion was *Methanosaeta concilii* (formerly *Methanothrix soehngenii*) (Figure 5). Furthermore, results showed the existence of molecular diversity within the genus *Methanosaeta* in the anaerobic process under study (Rincón et al., 2006).

As has been explained in section 4.1, the anaerobic digestion process could be more stable if the hydrolytic-acidogenic and the methanogenic stages were physically separated. The microorganisms participating in this kind of biological treatment (bacteria and methanogenic archaea) have different requirements in terms of growing kinetic, optimal working conditions and sensitivity to environmental conditions. Studies in two stages allow for the enrichment of the different populations of microorganisms (Cha & Noike, 1997). The separation in the hydrolytic-acidogenic and the methanogenic steps showed improved results as compared to one simple AD stage. The acidification of the 2POMSW in an initial hydrolytic-acidogenic step achieved a high concentration of total volatile fatty acids 14.5 g L-1 (expressed as acetic acid) at an OLR as high as 12.9 g COD L-1 d-1 (Rincón et al., 2008b). After this initial acidification, the OLRs achieved in the methanogenic reactor were in the order of 22.0 g COD L-1 d-1 with COD and volatile solid removals of 94.3%-61.3% and 92.8%- 56.1%, respectively, for OLRs between 0.8 and 20.0 g COD L-1d-1. Methane yields of 268 mL CH4 g-1 COD removed were achieved (Rincón et al., 2009, 2010).

Borja et al. (2002) carried out an initial anaerobic digestibility study with four different dilutions of 2POMSW (20%, 40%, 60% and 80 %). The main findings showed that the performance of the reactor [in terms of COD removal (%)] is practically independent of the feed COD concentration. Studies with no-diluted 2POMSW were carried out by the same authors with similar results (Rincón et al., 2007). The methane yields obtained in these studies ranged between 200-300 mL CH4 g-1 COD removed. The 2POMSW is easily biodegradable by mesophilic anaerobic digestion and COD removal efficiencies up to 97 %

Rincón et al. (2006, 2008a) studied the different microorganisms participating in the 2POMSW anaerobic digestion. For the determination of the microorganism population polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), cloning and sequencing techniques were employed. The results showed differences in the microbial communities, both bacterial and archaeal, with varying OLRs. Analysis of the microbial communities may be decisive in understanding the microbial processes taking place during 2POMSW decomposition in anaerobic reactors and optimizing their performance. During this experimental study the most frequently encountered microbial group were the Firmicutes (53.3% of analyzed sequences), represented mostly by members of the Clostridiales (Figure 4). Chloroflexi also represented an important bacterial group in the study (23.4% of sequences) and has been reported as a major constituent in anaerobic systems (Rincón, 2006). The Gamma-Proteobacteria (8.5% sequences, represented mainly by the genus *Pseudomonas*), Actinobacteria (6.4%) and Bacteroidetes (4.3%) are also significant components of the microbial communities during the anaerobic decomposition of 2POMSW (Figure 4). The major archaeal component detected for the 2POMSW anaerobic digestion was *Methanosaeta concilii* (formerly *Methanothrix soehngenii*) (Figure 5). Furthermore, results showed the existence of molecular diversity within the genus *Methanosaeta* in the anaerobic process under study

As has been explained in section 4.1, the anaerobic digestion process could be more stable if the hydrolytic-acidogenic and the methanogenic stages were physically separated. The microorganisms participating in this kind of biological treatment (bacteria and methanogenic archaea) have different requirements in terms of growing kinetic, optimal working conditions and sensitivity to environmental conditions. Studies in two stages allow for the enrichment of the different populations of microorganisms (Cha & Noike, 1997). The separation in the hydrolytic-acidogenic and the methanogenic steps showed improved results as compared to one simple AD stage. The acidification of the 2POMSW in an initial hydrolytic-acidogenic step achieved a high concentration of total volatile fatty acids 14.5 g L-1 (expressed as acetic acid) at an OLR as high as 12.9 g COD L-1 d-1 (Rincón et al., 2008b). After this initial acidification, the OLRs achieved in the methanogenic reactor were in the order of 22.0 g COD L-1 d-1 with COD and volatile solid removals of 94.3%-61.3% and 92.8%- 56.1%, respectively, for OLRs between 0.8 and 20.0 g COD L-1d-1. Methane yields of 268 mL

CH4 g-1 COD removed were achieved (Rincón et al., 2009, 2010).

**5.2 Two-phase olive mill wastes** 

may be achieved (Rincón et al., 2007).

(Rincón et al., 2006).

**5.2.1 Two-phase olive mill solid wastes (2POMSW)** 

Fig. 4. DGGE analysis of the diversity of bacterial communities at different OLRs studied in one stage anaerobic digestion of 2POMSW (Rincón et al., 2008a). The position of the major electrophoretic bands corresponding to the 16S rRNA gene of the identified bacteria are indicated. A, B, C, D, E, F, G and H are the increasing OLRs studied in g COD L-1 d-1: 2.3 (A), 3.0 (B), 4.5 (C), 5.8 (D), 6.8 (E), 8.3 (F), 9.2 (G) and 11.0 (H).

Other studies in two-stages at thermophilic scale reported 2POMSW as an ideal substrate for biohydrogen and methane production. These studies used diluted 2POMSW (1:4) with tap water achieving 18.5 ± 0.4 mmol CH4 g-1 total solid added (TS). Experiments for biohydrogen production followed by methane production, generated 1.6 mmol H2 g-1 TS added and 19.0 mmol CH4 g-1 TS in the methanogenic stage (Gavala et al., 2005). Mesophilic bio-hydrogen production from 3POMSW has shown to be feasible at mesophilic temperature resulting in 2.8-4.5 mmol H2 per gram of carbohydrates consumed in the reactor (Koutrouli et al., 2006). Methane production in these assays achieved a maximum value of 1.13 L CH4 L-1 d-1 at 10 days of HRT. Hydrogen is a renewable energy source and one of the most attractive applications is the conversion of hydrogen to electricity via fuel cells (Koutrouli et al., 2006)

Olive Oil Mill Waste Treatment: Improving the Sustainability

**6. Conclusion** 

application is not a reality yet.

132, ISSN: 1359-5113.

ISSN: 0961-9534

**7. Acknowledgment** 

**8. References** 

7799

of the Olive Oil Industry with Anaerobic Digestion Technology 287

single unit like the previously explained MFC (section 4.3) is very promising. Preliminary

Three-phase olive mill wastewaters (3POMWW) and two-phase olive mill solid wastes (2POMSW) are the main wastes generated in the olive mill industry (1,200 L of 3POMWW per ton of milled olives and 800 kg of 2POMSW per ton of milled olives, respectively). The composition of 3POMWW and 2POMSW is very complex due to the vegetation water. Currently, the final destination of 3POMWW is mainly evaporation ponds and the final destination of 2POMSW is evaporation ponds and co-generation. Although the evaporation ponds are very simple constructions, failure in the insulation of the basin can contaminate the ground water and they generate putrid odors and insects during the decomposition processes. The co-generation processes have a high number of environmental

Anaerobic digestion is already successfully used for many agro-industrial residues, such as sugar beet pulp, potato pulp, potato thick stillage or brewer´s grains. This technology allows an efficient solids stabilisation and energy recovery. Both 2POMSW and 3POMWW have been shown to be promising substrates for anaerobic digestion, however full scale

The authors wish to express their gratitude to the Spanish "Ministerio de Educación y Ciencia" (Project REN 2001-0472/TECNO) and to the contracts JAE-Doc from "Junta para la

Albi Romero, M. A. & Fiestas Ros De Ursinos, J. A. (1960). Estudio del alpechin para su

Alburquerque, J. A.; Gonzálvez, J.; García, D. & Cegarra, J. (2006). Effects of bulking agent

Angenent, L. T.; Karim, K.; Al-Dahhan, M. H.; Wrenn, B. A. & Dominguez-Espinosa, R.

Azbar, N.; Keskin, T. & Yuruyen, A. (2008). Enhancement of biogas production from olive

aprovechamiento industrial. Ensayos efectuados para su posible utilización como

on the composting of "alperujo", the solid by-product of the two-phase centrifugation method for olive oil extraction. *Process Biochemistry*, Vol. 41, pp. 127-

(2004). Production of bioenergy and biochemicals from industrial and agricultural wastewater. *Trends in Biotechnology,* Vol. 22, pp. 477-485, ISSN: 0167-

mill effluent (OME) by co-digestion. *Biomass and Bioenergy*, Vol. 32, pp. 1195-1201,

studies of the treatment of 2POMWW have been reported by Fermoso et al. (2011).

disadvantages: nitrogen oxides production, emission of suspended ashes, etc.

Ampliación de Estudios del CSIC" co-financed by the European Social Funds.

fertilizante. *Grasas y Aceites,* Vol. 11, pp. 123-124, ISSN: 0017-3495

Fig. 5. DGGE analysis of the diversity of archaeal communities at different OLRs studied in one stage anaerobic digestion of 2POMSW (Rincón et al., 2006). The position of the major electrophoretic bands corresponding to the 16S rRNA gene of the identified archaea are indicated. A, B, C and D are the increasing OLRs studied in g COD L-1 d-1): 0.75 (A), 1.5 (B), 2.25 (C) and 3.0 (D).
