The OMW used in this study was obtained from a discontinuous olive oil processing plant located in Sousse-Tunisia, according to a process of extraction and pression then centrifugation the OMW was stored in a refrigerator for a year at 4˚C for other uses.

The cow manure used was given from a farm located in Monastir-Tunisia. It’s an under-product of manure, which has an intrinsic value as an organic improvement; these products are composed of the animal extretas associated with carbonic composition (straw, sawdust).

At first, these products are sieved and divided into samples of 4 g. They are mixed with 10 ml of distilled water in tight bottles. Finally, the bottles are incubated at 37˚C to start anaerobic fermentation.

Ethyl acetate, sodium bicarbonate, sodium hydroxide (NaOH), ethanol, sulfuric acid (H₂SO₄).

1) Solution of (sodium bicarbonate + acetic acid); production of CO₂ that used to escape gas from mixture in the bottle n˚3.

2) Double boiler.

3) Bottle in which we put the mixture to ferment.

4) Solution of sulfuric acid 0.1 N to catch NH₃.

5) Solution of (NaOH 5M + lime water) to catch CO₂ and H₂S.

6) Burette that leads to detecting the volume of recovered gas.

7) Empty pump.

8) Tap.

In the bottle n˚3:

There is a mixture of 100 ml of cow manure + 700 ml of OMW + sodium bicarbonate. We add the sodium bicarbonate to adjust pH between 7 and 8 [15] [16] . We paddle OMW by N₂ to have anaerobic middle. We put the mixture in the double boiler at the thermophilic zone [55˚C - 65˚C]. This bottle had a knuckle that connects the sample to outgoing pipes. One is connected to an Erlenmeyer that contains a mixture of bicarbonate and acetic acid (A solution that produces the CO₂). This mixture is used when we start the experience to provide anaerobic conditions (during 5 minutes). The second pipe is used to collect the gas produced during the anaerobic fermentation. These paddle into the solution of NaOH (5M) and Ca(OH)₂ to catch the CO₂, H₂S and NH₃ produced during the anaerobic fermentation. The other gas (O₂, H₂, CH₄) are recovered into the tight and upset burette.

Finally, the pump used at the beginning of the experience, helps to aspire the gas from the burette until the solution n˚5 (NaOH + CH₃COOH) reaches the upper of the graduated burette extremity. We let the mixture ferment in the bottle n˚3 for 10 hours.

The experience was finished when the production of the gas becomes too weak (<1 ml/1 hour).

During the fermentation, we measure every hour, the volume of the gas released in the burette. These volumes need us to calculate the number of biogas mole’s obtained.

A sample was taken from the fermentation middle, at the beginning and at the final of the fermentation on which the experimental test was carried out: determination of pH, the volume of the gas given off and the polyphenols content.

The procedure to obtain the phenolic extract was as follows. The samples were acidified with HCl (pH 2.0) and extracted three times with ethyl acetate (v/v) at ambient temperature.

The three organic fractions were combined and dried with anhydrous Na₂SO₄ for 30 - 40 rain.

The extract was concentrated to dryness in a rotavapour and redissolved with a methanol/water mixture (60/40). For the determination of total phenol content [17] the method used the Folin-Ciocalteau’s phenol reagent (from Merck, Darmstadt) involving the successive addition of 5 ml sodium carbonate (200 g·l⁻¹) and 2.5 ml Folin-Ciocalteau phenol reagent to a 50.0 ml sample. After 60.0 min at 20˚C the absorbance was measured at 725 nm against a distilled water and reagent blank [18] .

Spectrophotometric measurements were performed with a UV-1650 PC/vis spectrophotometer, using Cary WinUV “version 2.21” software (varian), on crude extracts after dilution (1:100) with (methanol/water).

Phenolics were identified on the basis of their absorbance and their concentration in comparison with standard curve. When necessary, co-injection and elution with standards were used to insure about the identity of the compounds.

Gas samples were taken with a syringe from the headspace of the serum bottle and analysed by gas chromatography with a SHIMADZU instrument chromatograph equipped with a flame ionization detector. The flame ionization detector was fitted with a 30 cm stainless steel column. N₂ was used as carrier gas at 28 ml/min with H: and air flows of 25 and 30 ml/min, respectively. The oven, injector and detector temperatures were 200˚C.

The measurement was carried out with a pH meter (METTLER DELTA 340), after diving the electrod into homogenous solution.

This biogas production is due to the nutriment’s nature and microorganisms present in the cow manure dissolved in OMW.

Then, to provide anaerobic digestion, we must regulate the pH between 7 and 8 [19] . We added sodium bicarbonate. The addition of the basis product hasn’t any effect on the production of the biogas [16] but the ion of HCO 3 − leads to the neutralization of the free organic acid and to accumulate the volatile fatty acids [20] .

Proteins → NH 3 + H 2 O → NH 4 + + OH −

CO 2 + OH → HCO 3 −

The chromatographic analysis of gas proves the presence of methane; the following chromatogram shows the pic of methane result of anaerobic digestion of OMW by cow manure (Figure 5 & Figure 6).

The OMW contains easily fermentable products like sugar and glucose, and also products hard to be degradable like fatty acids and polyphenols [21] ; these polyphenols have inhibitor effect on the anaerobic digestion [22] but in the presence of methanogenic activities, they can be partially degradabled at pH near to 8.5. Then the biogas production varies according to the nature of manure (methanogenic activity) and the nature of OMW.

We carried out the UV visible spectrophometric analysis to pure OMW under a wave length of 725 nm. We found that the polyphenol concentration is about 1751.2 ppm.

We did the same analysis with a sample of OMW treated with cow manure. We found that the polyphenol concentration is about 0 ppm.

This informs us that the fermentation by cow manure damaged about 100% of the total phenol.

We varied the incubation time four times (1, 20, 40, and 60) hours; we followed the variation of the production of the gas according to incubation time.

We observed that the volume of the gas increases between 15 and 21 hours, it reaches 15 ml after 20 hours; on the contrary, the value of pH stays constant (Figure 7).