The freshwater alga Chlorella, a highly productive source of starch, might substitute for starch-rich terrestrial plants in bioethanol production. The cultivation conditions necessary for maximizing starch content in Chlorella biomass, generated in outdoor scale-up solar photobioreactors, are described. The most important factor that can affect the rate of starch synthesis, and its accumulation, is mean illumination resulting from a combination of biomass concentration and incident light intensity. While 8.5% DW of starch was attained at a mean light intensity of 215 µmol/(m2 s1), 40% of DW was synthesized at a mean light intensity 330 µmol/(m2 s1). Another important factor is the phase of the cell cycle. The content of starch was highest (45% of DW) prior to cell division, but during the course of division, its cellular level rapidly decreased to about 13% of DW in cells grown in light, or to about 4% in those kept in the dark during the division phase. To produce biomass with high starch content, it is necessary to suppress cell division events, but not to disturb synthesis of starch in the chloroplast. The addition of cycloheximide (1 mg/L), a specific inhibitor of cytoplasmic protein synthesis, and the effect of element limitation (nitrogen, sulfur, phosphorus) were tested. The majority of the experiments were carried out in laboratory-scale photobioreactors, where culture treatments increased starch content to up to about 60% of DW in the case of cycloheximide inhibition or sulfur limitation. When the cells were limited by phosphorus or nitrogen supply, the cellular starch content increased to 55% or 38% of DW, respectively, however, after about 20 h, growth of the cultures stopped producing starch, and the content of starch again decreased. Sulfur limited and cycloheximide-treated cells maintained a high content of starch (60% of DW) for up to 2 days. Sulfur limitation, the most appropriate treatment for scaled-up culture of starch-enriched biomass, was carried out in an outdoor pilot-scale experiment. After 120 h of growth in complete mineral medium, during which time the starch content reached around 18% of DW, sulfur limitation increased the starch content to 50% of DW.

Flue gas generated by combustion of natural gas in a boiler was used for outdoor cultivation of Chlorella sp. in a 55 m 2 culture area photobioreactor. A 6 mm thick layer of algal suspension continuously running down the inclined lanes of the bioreactor at 50 cm s −1 was exposed to sunlight. Flue gas containing 6-8% by volume of CO 2 substituted for more costly pure CO 2 as a source of carbon for autotrophic growth of algae. The degree of CO 2 mitigation (flue gas decarbonization) in the algal suspension was 10-50% and decreased with increasing flue gas injection rate into the culture. A dissolved CO 2 partial pressure (pCO 2 ) higher than 0.1 kPa was maintained in the suspension at the end of the 50 m long culture area in order to prevent limitation of algal growth by CO 2 . NO X and CO gases (up to 45 mg m −3 NO X and 3 mg m −3 CO in flue gas) had no negative influence on the growth of the alga. On summer days the following daily net productivities of algae [g (dry weight) m −2 ] were attained in comparative parallel cultures: flue gas = 19.4-22.8; pure CO 2 = 19.1-22.6. Net utilization (η) of the photosynthetically active radiant (PAR) energy was: flue gas = 5.58-6.94%; pure CO 2 = 5.49-6.88%. The mass balance of CO 2 obtained for the flue gas stream and for the algal suspension was included in a mathematical model, which permitted the calculation of optimum flue gas injection rate into the photobioreactor, dependent on the time course of irradiance and culture temperature. It was estimated that about 50% of flue gas decarbonization can be attained in the photobioreactor and 4.4 kg of CO 2 is needed for production of 1 kg (dry weight) algal biomass. A scheme of a combined process of farm unit size is proposed; this includes anaerobic digestion of organic agricultural wastes, production and combustion of biogas, and utilization of flue gas for production of microalgal biomass, which could be used in animal feeds. A preliminary quantitative assessment of the microalgae production is presented.

Two variants of open photobioreactors were operated at surface-to-volume ratios up to 170 m −1 . The mean values for July and September obtained for photobioreactor PB-1 of 224 m 2 culture area (length 28 m, inclination 1.7%, thickness of algal culture layer 6 mm), operated in Tȓebon (49 • N), Czech Republic, were: net areal productivity, P net = 23.5 and 11.1 g dry weight (DW) m −2 d −1 ; net photosynthetic efficiency (based on PAR -Photosynthetic Active Radiation), η = 6.48 and 5.98%. For photobioreactor PB-2 of 100 m 2 culture area (length 100 m, inclination 1.6%, thickness of algal culture layer 8 mm) operated in Southern Greece (Kalamata, 37 • N) the mean values for July and October were: P net = 32.2 and 18.1 g DW m −2 d −1 , η = 5.42 and 6.07%. The growth rate of the alga was practically linear during the fedbatch cultivation regime up to high biomass densities of about 40 g DW L −1 , corresponding to an areal density of 240 g DW m −2 in PB-1 and 320 g DW m −2 in PB-2. Night biomass loss (% of the daylight productivity, P L ) caused by respiration of algal cells were: 9-14% in PB-1; 6.6-10.8% in PB-2. About 70% of supplied CO 2 was utilized by the algae for photosynthesis. The concentration of dissolved oxygen (DO) increased from about 12 mg L −1 at the beginning to about 35 mg L −1 at the end of the 100 m long path of suspension flow in PB-2 at noon on clear summer days. Dissipation of hydraulic energy and some parameters of turbulence in algal suspension on culture area were estimated quantitatively. AbbreviationsA size of the culture area (m 2 ) C O 2 concentration (DO) of dissolved oxygen (g O 2 m −3 ) C O 2 ,0 DO concentration at the beginning of culture area (g O 2 m −3 ) C O 2 ,L DO concentration at the end of culture area (g O 2 m −3 ) C O 2 *

The following bead mills used for disruption of the microalga Chlorella cells were tested: (1) Dyno-Mill ECM-Pilot, grinding chamber volume 1.5 L; KDL-Pilot A, chamber volume 1.4 L; KD 20 S, chamber volume 18.3 L; KD 25 S, chamber volume 26 L of Willy A. Bachofen, Basel, Switzerland, (2) LabStar LS 1, chamber volume 0.6 L of Netzsch, Selb, Germany, (3) MS 18, chamber volume 1.1 L of FrymaKoruma, Neuenburg, Germany. Amount of disrupted cells decreased with increasing Chlorella suspension feed rate and increased up to about 85% of the beads volume in the grinding chamber of the homogenizers. It also increased with agitator speed and number of passes of the algae suspension through the chamber. The optimum beads diameter was 0.3-0.5 mm in the homogenizers Dyno-Mill and LabStar LS 1 and 0.5-0.7 mm in the homogenizer MS 18. While the degree of the cell disruption decreased with increasing cell density in Dyno-Mill and LabStar, the cell disruption in the MS 18 increased. Depending on processing parameters, more than 90% of algae cells were disrupted by passing through the bead mills and bacteria count in algae suspension was reduced to about two orders.