The potential of autochthonous microbial culture encapsulation in a confine environment for phenols biodegradation Eyal Kurzbaum1, Hassan Azaizeh2,3, Ofir Menashe4 1Golan Research Institute, University of Haifa, P.O.B 97, Qatzrin, Israel 2Institute of Applied Research, The Galilee Society, P.O. Box 437, Shefa-Amr, 20200, Israel 3Tel Hai College, Upper Galilee 12208, Israel. 4Achi Rachov School of Engineering, Water Industries Engineering, Kinneret College on the sea of Galilee, Israel Abstract Methods Olive mill wastewater (OMWW) contains high concentrations of phenolic compounds that can induce a significant damage to the biological process of a wastewater treatment plant. In a newly macro-encapsulation procedure named the Small-Bioreactor Platform (SBP) technology as a method for a bioaugmentation treatment, we introduced autochthonous microbial cultures into 2.5 cm long microfiltration membrane capsules. In the present study, we aimed to explore the ability of the postulated technology to provide a protective environment for the microbial culture while facilitating the breakdown of phenolic compounds. This will allow a successful bioaugmentation treatment approach within domestic wastewater treatment plants in order to protect the natural biomass of the wastewater treatment plants from the OMWW's antimicrobial agents. Our results present the ability of encapsulated autochthonous bacterial cultures to biodegrade phenols as sole contaminants or in a blend of phenolic compounds within minimal salts medium and within raw OMWW. The biodegradation of four phenols types (phenol, tannic, gallic and caffeic acids) as a sole carbon source, showed a complete breakdown within 48 h for tannic and caffeic acids while gallic acid and phenol was consumed within 24 h. When the biodegradation rate of a high concentration phenols mixture (3000 mg/l) was tested, the encapsulated biomass succeeded to reduce the phenolic blend concentration to approximate 500 mg/L within 24 h. In addition, polyphenols compounds concentration in fresh OMWW was effectively reduced in the presence of the encapsulated cultures. (left) SBP capsule illustration. The SBP capsule contains several elements: the external barrier made of cellulose acetate microfiltration membrane, aquatic interface which include inside the exogenous inoculums and nutrients supplemental agar. )right) A photo of a SBP capsule which encases OMWW sludge inoculums. The SBP capsule size is 2.5 cm long and 0.8 cm in diameter. Illustration of a SBP capsule microfiltration membrane encasing the autochthonous bacterial culture within a given medium (mixed liquor). The SBP capsule's microfiltration membrane physically separates the introduced microbial culture inside the capsule from the outer microorganisms while enabling the trafficking across the membrane of the dissolved molecules and organic matter (such as phenols). Results HPLC-PAD profile of polyphenols within fresh OMWW Biodegradation rate of phenols by encapsulated autochthonous bacterial culture Biodegradation rate of high phenolic compounds blend Biodegradation of crude OMWW as organic matter and phenolic compounds Chromatographic profile of the fractions acquired by HPLC-PAD detected at the relative maxima of absorbance of polyphenols (280 nm). Key to peak identities: (1) hydroxytyrosol; (2) tyrosol; (3) caffeic acid; (4) vanillic acid; (5) ferulic acid; (6) p-coumaric acid; (7) verbascoside; (8) oleuropein; (9) rutin. Biodegradation rate of caffeic, tannic and gallic acid in the presence of encapsulated autochthonous culture. Sterile-medium as control shows no phenols degradation during three days of incubation. Biodegradation rate of phenolic compounds blend: caffeic, tannic and gallic acid at high concentration (initial total concentration of 3000 mg L-1) by encapsulated microbial culture within the SBP capsules. Biodegradation of sterilized OMWW by encapsulated microbial culture within the SBP capsules compared with control (without added bacteria). (A) Organic matter (COD); (C) phenolic compounds (measured as tannic acid equivalent). Comparison between encapsulated and suspended culture Conclusions The encapsulated autochthonous bacterial culture presented a rapid and significant biodegradation of three phenolic acids up to an initial concentration of 3000 mg L-1. The encapsulated bacterial culture reduced the initial COD and phenols content of olive mill wastewater (OMWW) with 30% and 21% within 4 days, respectively. This study shows that the addition of encapsulated bacterial cultures within SBP capsules presents a successful bio-augmentation treatment by reducing phenolic compounds within OMWW. Biodegradation rate of caffeic, tannic and gallic acid blend (total concentration 700 mg L-1) in the presence of encapsulated microbial culture (within the SBP capsules) and in the presence of bacterial suspension.