1. PHENOLIC COMPOUNDS WITH ANTIRADICAL ACTIVITY FROM THE CORK BOILING ANAEROBIC DIGESTION Isabel Paula Marques 1, Luis Gil 1, Francesco La Cara 2 1 LNEG – Instituto Nacional de Energia e Geologia IP, U. Bioenergia, Lisboa, Portugal 2 Istituto di Biochimica delle Proteine – CNR, Napoli, Italy Result and Discussion Cork boiling wastewater as substrate CBW is an aqueous and complex effluent of the cork industry that presents an acid pH (5.8) and an organic potential of 6.5 kg COD m -3, being the greatest amount in the soluble form (soluble COD = 6.2 kg m -3 ). This wastewater has unbalance composition, low biodegradability and deep dark colour. It is rich in phenolic compounds (about 1 kg m -3 ) and is deprived of nitrogen content (0.04 kg m -3 ). ParameterCBW* pH5.8 ± 0.0 Total COD (kg m -3 )6.5 ± 0.1 Soluble COD (kg m -3 )6.2 ± 0.1 BOD (kg m -3 )- TS (kg m -3 )5.13 ± 0.08 VS (kg m -3 )4.05 ± 0.04 TSS (kg m -3 )0.58 ± 0.11 VSS (kg m -3 )0.15 ± 0.07 Conductivity (mS cm -1 )1.5 ± 0.1 Total Phenols (kg m -3 )1.20 ± 0.00 Total Nitrogen (kg m -3 )0.04 ± 0.00 *values are expressed as an average ± standard deviation of three replicates Gas production The methane production was assessed along 44 days. No lag phases were observed in the presence of CBW. Batch experiments of 3 kg COD m -3 the maximum methane production (15 mg COD-CH 4 batch -1 ) was attained after 15 d and it was maintained stable until the experiment end. Batch experiment of 6 kg COD m -3, most of the methane was produced in the first 15 d, having evolved to 27 mg COD-CH 4 batch -1 by the end. CBW can be regarded as an interesting substrate for a production of carrier gas energy (biogas) through a biotransformation process. More diluted wastewater (3 kg COD m -3 ) shows higher conversion efficiency than the original substrate (6 kg COD m -3 ): 0.142 vs 0.126 m 3 CH 4 kg -1 COD added. ParameterAD3AD6 CBW (kg m -3 )36 CH 4 Yield (m 3 CH 4 kg -1 COD)0.142 ± 0.0140.126 ± 0.016 AD - Anaerobic digestion assay: at 3 kg COD m -3 (AD3) and 6 kg COD m -3 of CBW (AD6) Phenolic compounds (µg mL -1 ) CBWAD0AD3AD6 Gallic acid, GA19.54.17 ± 0.2615.47 ± 4.0822.43 ± 3.97 Protocatechuic acid, PCA8.50.64 ± 0.095.07 ± 1.599.82 ± 1.33 Caffeic acid, CA2.140.59 ± 0.441.15 ± 0.211.77 ± 0.36 Vanillic acid, VA2.0tr1.15 ± 0.371.91 ± 0.36 Syringic acid, SAtrndtr1.1 ± 0.99 Ellagic acid, EA96.52.49 ± 0.3922.3 ± 3.4841.8 ± 8.49 p-coumaric acid, p-CA4.11.1 ± 0.181.77 ± 0.282.47 ± 0.40 Ferulic acid, FA6.51.02 ± 0.081.55 ± 0.375.6 ± 0.98 o-coumaric acid, o-CA3.8tr1.17 ± 0.324.51 ± 0.33 Trans-cinnamic acid, t-CA3.72.16 ± 0.262.1 ± 0.2863.13 ± 0.63 CBW – Cork boiling wastewater, reactor influent; AD - Anaerobic digestion assay: blank (AD0), at 3 kg COD m -3 (AD3) and 6 kg COD m -3 of CBW (AD6); nd: not detected, tr: traces Phenolic compounds with antiradical activity Ten phenolic compounds - gallic acid, protocatechuic acid, caffeic acid, vanillic acid, syringic acid, ellagic acid, p-coumaric acid, ferulic acid, o-coumaric acid and trans-cinnamic acid - were identified. The major components are the ellagic (96.5 µg mL -1 ) and gallic (19.5 µg mL -1 ) acids. Concerning the gallic, protocatechuic, vanillic and syringic acids (benzoic acids family) they increased in treated samples (6 kg COD m -3 assay). On the contrary, the phenolic compounds belonging to the hydroxy-cinnamic acids family (caffeic acid, p-coumaric acid, ferulic acid, o-coumaric acid and trans-cinnamic acid, showed in both assays, 3 and 6 kg COD m -3 ) decreased. The exception concerns the o-coumaric acid concentration that increased 19 % (6 kg COD m -3 assay). Ellagic acid presents a concentration decrease of the 57 % (6 kg COD m -3 assay). Note: Ellagic acid is distinguished from the other ten identified phenolic compounds in CBW due to the hydrophobicity and higher molecular weight (MW = 302.2 Da; compared to less than 200 Da of the others CBW phenolic compounds). It is the main soluble molecule contained in the CBW responsible for the membrane fouling (ultrafiltration) due to its strong hydrophobic interaction [Minhalma & de Pinho 2001]. The additional production of valuable and useful molecules for several industrial applications is another very interesting aspect provided by anaerobic digestion. A similar increase in the total phenolic compounds concentration and antiradical activity were simultaneously supplied by anaerobic digestion, indicating that compounds with a higher industrial interest were formed. Phenolic compounds are significant components for the human diet due to their potential antioxidant activity, their capacity to diminish oxidative stress-induced tissue damage resulted from chronic diseases, and their potentially utilization in cancer therapy [Khadem & Marles 2010]. Other experimental parameters Total phenolic compounds concentration existing in the original fluid was no largely changed by the anaerobic process: increase of about 18 % (AD6). Antiradical activity increase after anaerobic digestion (EC 50 =7.2 µg:8.74 µg): phenols composition varied over the process; they present an higher antiradical activity; important natural antioxidants are formed. ParameterCBWAD0AD3AD6 pH7.28.23 ± 0.0857.67 ± 0.0217.52 ± 0.064 VFA (kg m -3 )0.2150.185 ± 0.0360.178 ± 0.0480.137 ± 0.013 Total phenols (mg mL -1 )1.20.05 ± 0.000.90 ± 0.001.41 ± 0.021 Optical density (254 nm)43.58.41 ± 0.64029.08 ± 0.66543.96 ± 0.000 DPPH inhibition (%)74.078.90 ± 0.42476.07 ± 1.55680.80 ± 1.061 EC 50 * (antiradical activity)7.22.22 ± 0.0785.90 ± 0.0788.74 ± 0.064 Conductivity (mS cm -1 )1.5 4.0 ± 0.1064.6 ± 0.0005.4 ± 0.106 CBW: Cork boiling wastewater, reactor influent; Anaerobic digestion assay: blank (AD0), at 3 kg COD m -3 (AD3) and 6 kg COD m -3 of CBW (AD6); *antiradical activity was defined as the amount of antioxidant (expressed as µg of total polyphenols) necessary to decrease the initial DPPH concentration by 50 % (EC 50 = Efficient Concentration) References Colleran E, Concannon F, Golden T, Geoghrgan F, Crumlish B, Killilea E, Henry M, Coates J: Use of methagenic activity test to characterize anaerobic sludges, screen for anaerobic biodegradability and determine toxicity thresholds against individual anaerobic trophic groups and species. Water Science and Technology 1992, 25:31-40. La Cara F, Ionata E, Del Monaco G, Marcolongo L, Gonçalves MR, Marques IP: Olive mill wastewater anaerobically digested: Phenolic compounds with antiradical activity. Chemical Engineering Transactions 2012, 27:325–330. Minhalma M, de Pinho MN: Tannic-membrane interactions on ultrafiltration of cork processing wastewaters. Separation and Purification Technology 2001, 22-3:479–488. Khadem S, Marles RJ: Monocyclic phenolic acids; hydroxy- and polyhydroxybenzoic acids: occurrence and recent bioactivity studies. Molecules 2010, 15:7985-8005. Conclusions Anaerobic digestion can be regarded as a biological process that provides other profits than energetic and agricultural valorization of the industrial cork effluents. Industrial valuable molecules can be recovery under a multiple biochemical valorisation plan involving the anaerobic process. [Some of these compounds, as the ellagic acid, are molecules that are important components of the human diet due to their potential antioxidant activity, their capacity to diminish oxidative stress-induced tissue damage resulted from chronic diseases, and their potentially utilization in cancer therapy]. 35/12.5cm 3 37 ºC vials triplicate original CBW ------------------ diluted 50% AD6 = 6 kg COD m -3 ------------ AD3 = 3 kg COD m -3 Total phenols and HPLC analysis of phenolic compounds The CBW phenolic compounds were determined by a HPLC analysis performed with a method previously described [La Cara et al. 2012]. Briefly aliquots of CBW samples were diluted in acid methanol, incubated at 37 °C, centrifuged and the recovered supernatant was used for Folin-Ciocalteau polyphenols assay. HPLC/UV analysis of single phenolic compounds was performed utilizing a 250 × 4.6 mm (5 μm) C18 Hypersil column with a Thermo-Finnigan Surveyor HPLC system. Peak identifications were confirmed from retention times, UV spectroscopic data, and direct comparison to pure standards. The solvent flow rate was 0.9 mL min -1 and the mobile phase was a four-step linear solvent gradient system (0–30 min, 10 % B; 30–35 min, 55 % B; 35–40 min, 100 % B; 40-45 min, 100 % B) using 2% acetic acid in water as solvent A and 0.5 % acetic acid in 50 % acetonitrile as solvent B. Methods Biodegradability experiments Antiradical activity The antiradical activity of the raw and the digested CBW was determined by a modification of the method described by von Gadow [La Cara et al. 2012]. A methanolic solution of 6 x 10 -5 M DPPH (2,2-diphenyl-1-picrylhydrazyl) was added to the CBW samples mixed and placed in cuvettes and the decrease in absorbance at 515 nm was determined continuously until the value was stabilized. The inhibition percentage (IP) of the DPPH radical by the phenolic compounds of the CBW extracts was calculated according to the formula: IP = [(AC(0) - AC(t)) / AC(0)] x 100 where AC(0) is the absorbance of the control at t= 0 min and AC(t) is the absorbance of the reaction solution at the end monitoring time. The antiradical activity was defined as the amount of antioxidant (expressed as µg of total polyphenols) necessary to decrease the initial DPPH concentration by 50 % (EC 50 = Efficient Concentration). hand held pressure transducer - measure the gas production methane accumulated - gas chromatography (STP) Energy supply plays an important role in the modern world. Production of biogas from renewable sources, such as the agro-forestry industrial residues, is a promising mean to decrease the accumulation of greenhouse gas and alleviate pressure on fossil fuel inevitable depletion. Cork boiling wastewater (CBW) is an aqueous effluent of the cork industry and a serious environmental hazard. Because anaerobic digestion is a promising technology and its application to CBW has never been subject of research, experimental trials were conducted using a mesophilic anaerobic consortium. CBW is an interesting substrate. The improvement of several CBW biochemical features shows that the anaerobic digestion provides other profits: increase of the phenolic compounds antiradical activity amount; rise of the molecules concentration of the benzoic acids family (gallic, protocatechuic, vanillic and syringic acids). The multidisciplinary approach adopted in this study allows the valorisation of the CBW in terms of energy and valuable biomolecules productions for industrial applications.