In Mediterranean countries, annual OMW production is estimated to be over 30 million m3, and around 1 million m3 of this quantity is produced in Turkey. This dark colored wastewater is a substantial pollutant not only because of its high organic matter content and recalcitrant compounds such as polyphenols, therefore its fairly high chemical oxygen demand (COD) and biological oxygen demand (BOD) values that can reach up to 200 g/l and 100 g/l, respectively. As a consequence, the disposal of such a pollutant waste material becomes an important environmental problem that needs to be solved urgently.
Wide ranges of technological treatments are available in olive-oil producing countries, which are trying to face the harmful effects of the treatment and disposal of OMW. Modification of the technology used in oil extraction is one way of reducing the hazardous effects of OMW disposal. Nevertheless, most of the treatment methods are focused on both bioremediation, by means of reducing the polluting effect of OMW, and biotransformation into valuable products. OMW contains high amounts of organic constituents like sugars, organic acids, polyalcohols and fats that can be utilized for photobiological hydrogen production by purple non-sulphur phototrophic bacteria, whereas some valuable by-products such as polyhydroxybutyrate (PHB) and carotenoid pigment are also produced (Eroglu et al., IJHE-29 (2004), 163-171). PHB has important industrial applications, particularly to construct biodegradable carriers for long-term dosages, either in the agriculture for herbicides and insecticides or in the medical field for drugs and also for surgical sutures. It is mostly synthesized during unfavorable growth conditions, particularly under stress conditions through the stationary phase of growth, as an intracellular carbon and energy storage material for the bacteria and accumulated as granules at different sites of cytoplasm. Carotenoid pigments are essential for photosynthesis, since they transfer nearly half of the absorbed light energy to bacteriochlorophyll, and are to such an extent functional as light harvesting pigments. It has been used commercially during cancer chemoprevention and also as a food colorant, natural antioxidant, or provitamin A source.
The aim of the present study is to investigate the effect of light/dark cycles on the production of PHB and carotenoid during photobiological hydrogen production process under both natural sunlight (outdoor) and artificial illumination (indoor) conditions. Olive mill wastewater was collected from a centrifugal olive-oil mill in Burhaniye-Balýkesir (West Anatolia). Hydrogen production medium was prepared by 2% OMW containing media. Initial pH was adjusted to 6.8 – 7.0 by the addition of NaOH, and then the medium was sterilized at 121 °C for 15 minutes by autoclaving. Indoor hydrogen production experiments were performed in jacketed glass-column photobioreactors (400 ml) by Rhodobacter sphaeroides O.U.001 (DSM 5864). The temperature was maintained at 32 ºC. Light / dark cycles were changed by turning the artificial light source on and off at the periods: 12 h light–12 h dark; 14 h light–10 h dark and 16 h light–8 h dark. Light intensity at the outer surface of the reactor was kept at 200 W/m2, under the illumination of tungsten lamp. The results were compared with a control experiment having continuous illumination. The photobioreactors were stirred at a rate of 300 rpm with a magnetic stirrer.
Outdoor hydrogen production experiments were performed in a flat plate biosolar reactor made of acrylic sheet having 5 mm thickness and an illuminated front area of 0.2 m2. The maximum culture medium that can be used was 6.5 L. The reactor was inclined 30° and facing south. In order to obtain continuous light data, Luxmeter was connected to a computer. It had a sensor placed at the upper left part of the reactor, through which light density data were collected every after 5 min. A heating blanket was placed at the back face of the reactor. Temperature was controlled and kept at 32 °C.
During the experiments, the evolved gas was collected and measured volumetrically in a graduated glass burette. The composition of the evolved gas was analyzed by a gas chromatograph. The bacterial cell concentration was detected by measuring the optical density (OD) at 660 nm with a spectrophotometer. At the end of the experiments, the solid matter was separated from the processed OMW by centrifugation at 10,000 x g for 15 minutes at 4 °C. The solid residue of the centrifugation process was inspected for both PHB and carotenoid content. The PHB was recovered from the solid matter by the method of Bowker. Carotenoid pigment was extracted from the solid matter with acetone.
From the indoor experiments, the final carotenoid amount was within the range of 0.27 – 0.13 mg per g wet weight of bacteria. The highest was obtained at the continuous illumination. The final PHB amount was within the range of 0.28 – 0.17 mg per g wet weight of bacteria. The highest PHB accumulation was observed at the 16 h light / 8 h dark cycle. That experiment also gaved the highest H2 production capacity (12.5 liter hydrogen per liter of olive mill wastewater). In the case of outdoor experiments, relatively higher amounts of by-product formation (0.78 mg carotenoid and 1.2 mg PHB per g wet weight of bacteria) was observed.
The present study showed that microorganisms tend to protect themselves from the high light intensity by forming carotenoid pigments and they produce PHB as energy storage material for dark periods. Therefore, it has been concluded that by-product formation support the feasibility of large scale outdoor biological hydrogen production from olive mill wastewater.