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Cycle 6 (2017 Deadline)

Evaluation of algal treatment options for olive mill wastewater to produce energy and biofertilizer 

PI: Sami Sayadi,, Center of Biotechnology of Sfax, Tunisia
U.S. Partner: David Blersch, Auburn University, and Walter Mulbry, (Ret.) USDA/ARS
Project dates: December 2017 - November 2020

Project Overview:

 6-308_Apr-Jun 2018_Indoor microalgae cultivation in pretreated Olive Mill Wastew
Indoor micro-algae cultivation in pre-treated Olive Mill Wastewater samples [Photo courtesy of Prof. Sayadi]   
The conventional olive mill wastewater (OMW) treatment methods adopted in Tunisia are either costly or ineffective,causing environmental pollution. This project is aimed at developing an eco-friendly and cost-effective microalgal-based process for olive mill waste treatment and recycling. Such a process should result in the production of (1) treated and reusable OMW for ferti-irrigation use, contributing to resolving the water scarcity problem; (2) renewable biomass useful for clean energy production, particularly bio gas; and (3) bio-fertilizers for agricultural use, using the process by-products, bio char and sludge.

The goal of this project is to develop an eco-friendly technology for creating a closed loop process with zero waste. It encompasses several disciplines and couples physical and biological technologies to achieve the target objectives, including pyrolysis, physical adsorption, micro algae cultivation with OMW treatment and CO2 mitigation, anaerobic digestion, and composting. The project will also foster linkages among the R&D sector (universities and research institutes) with enterprises involved in waste production (farms and olive mills), enterprises producing devices necessary for treatments, enterprises interested in the use of by-products, and farmers. This approach will create a cooperative network for reducing agricultural waste impacts on the environment, exchanging know-how and market intelligence, and developing research capacity and new markets. In particular, the enterprises will be involved in the demonstration activities of proposed technologies. Through the project, valuable resources that are not currently utilized will be exploited and thus will generate local added value in Tunisia. Such an integrated eco-technology should facilitate the production of biogas from the produced micro-algal biomass for electrical and heat energy generation, as well as biodiesel production, the extraction of high-added-value molecules for cosmetics and nutraceutical applications, nutrient recovery in the form of sludge and biochar for soil amendments, and treated OMW for ferti-irrigation use. This eco-technology will also improve the quality of life for local residents living near the OMW discharge area by reducing the environmental footprint of the existing evaporation ponds, in particular reducing populations of mosquitoes and noxious odors from the ponds.

Beyond the technological aspects, the project will also involve the collection and assessment of data to support decision-makers for the promotion of sound strategies and best practices for social, economic, and legislative measures at the regional and national levels regarding agricultural waste management. It is expected to generate business opportunities and create jobs in small and innovative companies for commercializing the process outcomes. The research team will also strive to enhance the impacts of the project by organizing roundtables and training workshops for farmers to help them better exploit the anticipated research results with regard to ferti-irrigation and soil fertilization.

Summary of Recent Activities:
 During the reporting period of the project, the research group at CBS continued the R&D work in the frame of the first and second Work Packages. Two main tasks were essentially developed: Microalgae cultivation using the pretreated OMW and Characterization of microalgae biomass for potential high-valueadded molecules recovery and biodiesel production. In the frame of Olive Mill Wastewater pretreatment task, the investigation of the adsorption process using biochar as adsorbent was continued. Woody residues from tree pruning were used for biochar production.

The adsorption process efficiency was evaluated in term of total organic carbon reduction. The study covered the evaluation of the effects of operating factors and revealed the importance of OMW initial concentration and biochar production temperature on the process efficiency. However, the organic matter abatement using biochar could be considered low in comparison with activated carbon which was used in the previous study. As far as the microalgae cultivation and extraction of valuable compounds tasks are concerned, the microalgae strain Scenedesmus sp. was cultivated in a semi-continuous mode in a synthetic culture medium. Biomass productivities were in the range of 0.15-0.17 g/ Afterward, cultivation of this strain in 50% OMW pre-treated by Electro-coagulation (EC 50%) and in 100% of OMW pre-treated by adsorption on activated charcoal (CA 100%) was performed. The biochemical analysis of the obtained biomass was subsequently performed. For the culture carried out in the synthetic medium, Scenedemus sp. presented a large amount of proteins, increasing to 15.30% d.wt. at 0.10 1/day to remain constant at all the higher dilution rates. The lipid content ranged from 17.80 - 22.90 % and the lipid productivities were in the 28.30 - 37.40 mg/ range, with the lipid content peaking at 0.20 1/day. The fatty acid content was between 6.20- 9.40 % d.wt. and comprised 16.00 to 23.50% of SFA, 2.60 to 30.50% of MUFA and 51.60 to 58.10% of PUFA (of the total fatty acids). The Chlorophyceae Scenedesmus sp. contained high C18:3n3 contents in the 16.20- 31.90 % range. Characterization of biomass obtained from the cultures in the pretreated OMW was also microalgal biomass recovered under both culture conditions where up to 52% and 53.4% were recorded for biomass obtained from CA 100% and EC 50%, respectively. It is worth mentioning the presence as well of palmitic acid up to 16.5% using CA 100% and 15.6% using EC 50%. In addition, the obtained biomass was extracted using a solvent gradient namely hexane, ether, acetone, ethanol and water.

The total phenol content and the antioxidant activity of the different extracts were determined. The results of antioxidant activity showed that IC 50 of 713.10 μg/mL was obtained from hexane-extracted biomass. The total polyphenols content varied from 13.75 to 2.90 mg GAE/ g extract (Gallic Acid Equivalent) from biomass produced using EC medium whereas using CA, this content was in the range of 7.00-0.30 mg GAE/ g extract. In this work, extraction conditions were optimized to obtain microalgae extracts with maximal antioxidant potential. Lyophilized biomasses, cultivated in medium alone (Sc) or supplemented with ultrafiltrated olive mill wastewater (Sc-OMW), were subjected to extraction procedure using ethanol 70% as a green solvent and precellys homogenizer. The extraction was repeated three times and all extracts were combined, concentrated by liquid nitrogen, lyophilized and then stored until use.

The ability of extracts to scavenge free radicals was evaluated in vitro using DPPH assay. Sc-OMW extract exhibited higher DPPH scavenging ability with a half-maximal inhibitory concentration (IC50) of 0,159 mg/ml. HPLC analysis showed that phenolic compounds were more abundant in Sc-OMW ethanolic extract compared to Sc ethanolic extract.

In this period, the project partners, Prof. Walter Mulbry (USDA/ARS) and Dr. David Blersch (Auburn University) visited the CBS from the 8 to 17 July 2019. They participated in the workshop on microalgae biotechnology as keynote speakers. This event was organized in the frame of the project from 9 to 10 July 2019 in Syphax hotel in Sfax. The US partners also trained Ph.D. students, post-docs and engineers to construct turf scrubber’s reactors and showed them how to choose optimal operating culture condition in this type of reactors.
  6-308_Truck tankers discharging Olive Mill Wastewater in storage and evaporation
 Truck tankers discharging Olive Mill Wastewater in storage and evaporation ponds