Towards the commercialization of microalgal production: the role of environmental factors in production of triterpenoids from Botryococcus braunii

Production of hydrocarbons and biofuels from microalgae is an emerging field that shows great potential for carbon neutral production. The green alga Botryococcus braunii is widely recognized as a potential carbon-neutral source of hydrocarbons and biofuels. However a number of issues hamper its commercial development including, low growth rates and biomass production, low hydrocarbon yield, problems associated with scaling up of production, harvesting of biomass, and recovery of hydrocarbons and/or biofuel. This study addressed these issues using a series of laboratory-scale and large-scale experiments. Examination of the effects of three growth-media on biomass and hydrocarbon production in two B. braunii Race-B strains, Kossou-4 and Overjuyo-3, previously reported to produce high yields of hydrocarbons including triterpenoids showed that growth of B. braunii in Blue-green-11 medium (BG11) resulted in significantly higher biomass (dry-weight) compared to Jaworski’s medium (JM) and Bold-base-3-nitrogen medium (BBM-3N) after 15 days for both strains. Oil production was also significantly higher in BG11 whether measured as oil weight or absorbance. Examination of the effects of nutrients (nitrogen, iron) and environmental conditions (temperature, light intensity, photoperiod) on biomass and hydrocarbon production in Kossou-4 and Overjuyo-3 grown in BG11 showed highest biomass and oil production was obtained at nitrogen concentration 750mg l-1, iron concentration 6mg l-1, 25 oC, and 135μmol m-2 s-1 with photoperiod of 16h light/8h dark. Culturing the strains in modified BG11 containing optimized nutrients resulted in up to ~10.6 fold increase in biomass from 0.16g l-1 and 0.31g l-1 in normal BG11 to 1.74g l-1 and 2.17g l-1 in optimized BG11 and growth conditions for Kossou-4 and Overjuyo-3 respectively. This was accompanied by ~8-10 fold increase in oil production compared to that in normal BG11, with final oil yields of 264.2mg l-1 (Kossou-4) and 220.6mg l-1 (Overjuyo-3). Growth of Overjuyo-3 and Kossou-4 in 500L open tanks with 24h light (54μmol photons m-2s-1) at 25°C in modified BG11 for 60 days were then carried out. Maximum growth was reached after 40 days with Overjuyo-3 producing more biomass (3.05g l-1) than Kossou-4 (2.55g l-1). However, Kossou-4 produced more hydrocarbons (29% of dry weight) compared to Overjuyo-3 (20% of dry weight). The final stage investigated the feasibility of harvesting large-scale B. braunii using co-culture with a fungus to induce flocculation. Co-culture with the fungus Aspergillus fumigatus resulted in a flocculation efficiency of over 90% through co-pelletisation with both Overjuyo-3 and Kossou-4. Investigation of the ratio of fungus to algal broth found that 1:40 was optimal, resulting in large-scale flocculation efficiencies of 97% for Kossou-4 and 98% for Overjuyo-3 after 12h following inoculation. To determine the effects of co-culture with A. fumigatus potential energy yields, pyrolysis was conducted. Proximate analysis showed the proportions of bio-oil, bio-char and bio-gas were very similar for the pure Kossou-4 biomass and the co-cultured pellets resulting from harvest using A. fumigatus. Similar results were found for Overjuyo-3 confirming that the co-culture harvest method did not compromise the end product. This research demonstrates for the first time that large-scale production using bio-flocculation for harvesting of high value triterpenoids for commercial application is feasible using these B.braunii Race-B strains

An optimized method for the bio-harvesting of microalgae, Botryococcus braunii, using Aspergillus sp. in large-scale studies

The use of fossil fuels which are derived from non-renewable sources has been linked to global warming, adverse human health effects and environmental pollution. Consequently, there is a need to develop alternative sources of fuel that are renewable and more environment-friendly. Biofuel (biodiesel), produced from microalgae such as Botryococcus braunii is an alternative energy source, that is renewable (because algae can be cultured as needed), more biodegradable with lower global warming potential compared to fossil fuels. However, the use of microalgae is hampered by high costs associated with the production and harvesting of microalgal biomass in large-scale studies. In this article; • A robust and cost-effective method was developed for harvesting B. braunii • Optimized Aspergillus sp.: B. braunii ratio (1:40) was used to bio-flocculate up to 97% of cultured microalgae in both small and large-scale studies (250 L) • No damage to the harvested microalgal biomass (validated by pyrolysis) was observed with the harvested biomass being suitable for any desired downstream application. Method name: Bio-harvesting of B. braunii, Keywords: Bio-harvesting, Flocculation, Microalgae, Aspergillus, Large-scal

The effect of media on biomass and oil production in Botryococcus braunii strains Kossou-4 and Overjuyo-3

The green algae Botryococcus braunii is widely recognized as a source of oil, including hydrocarbons. However, the slow rate of growth B. braunii hampers its commercial development. This study addresses this by examining the effects of three growth media on biomass and oil production in two B. braunii Race B strains, Kossou-4 and Overjuyo-3. Growth of B. braunii strains in BG11 medium resulted in significantly higher growth (2.3 - 4.2 and 2.9 - 6.0 fold increases in Kossou-4 and Overjuyo-3 respectively) compared to the JM and BBM-3N media after 15 days. A similar trend was obtained when biomass was measured indirectly using optical density (OD) and chlorophyll fluorescence. Oil production was also significantly higher in BG11 whether measured as oil weight or absorbance (ODs at 680 and 750 nm). However, the presence of extracellular oil was shown to increase absorbance values making OD measurements less reliable than dry weight assays. Maximum recovery of oil was recorded when hexane was used as solvent compared to hexane-isopropanol and heptane. These results suggest that BG11 is the best growth medium for these two strains under the conditions of this experiment

Biomineralization of Platinum by <i>Escherichia coli</i>

The widespread use of platinum in many industrial applications has led to its release into the environment at elevated concentrations with potential adverse effects on human and environmental health. However, the nature of interactions between mobile platinum complexes and the biotic components of the environment, which are increasingly being exposed to platinum, is poorly studied. The aim of this study was to assess the impact of Pt(IV)-chloride on the growth and activity of the well-characterized bacteria Escherichia coli. Bacterial survival and viability in the presence of different concentrations of Pt(IV)-chloride were assessed in liquid culture, while platinum retention was assessed using experimentation with sand-filled columns with the residual platinum concentration measured by atomic absorption spectroscopy. Bacterial biomineralization of platinum was studied with scanning electron microscopy. The results showed that E. coli tolerated PtCl4 at concentrations of up to 10,000 &#181;M over 21 days and remained viable after 112 days of incubation with PtCl4 at 10,000 &#181;M in sand columns. Overall, 74 wt.% and 50 wt.% of platinum was mineralized in E. coli and blank sand columns, respectively. The results of this study confirm that E. coli is capable of biomineralizing platinum. The results confirm that the interaction of platinum with bacteria is not limited to known metal-resistant bacterial species

Bio-harvesting and pyrolysis of the microalgae Botryococcus braunii

The microalgae Botryococcus braunii is widely recognized as a potentially important biofuel-feedstock whose commercial exploitation is limited by difficulties with its cultivation and harvesting. In this study, two B. braunii strains, Kossou-4 and Overjuyo-3 were successfully cultured at a 500 l-scale for 60-days. Harvesting by bio-flocculation with Aspergillus fumigatus at an optimum ratio of 1:40 of fungus to microalgal culture resulted in up to 98% recovery of biomass in the two strains. Ultimate analysis (C, N, H, S, ash, high heating value) and pyrolysis (analytical and preparative pyrolysis and GC–MS assays) showed that co-harvesting with fungi did not cause any impairment of the feedstock value of the microalgal biomass. This work represents the first report on the successful culturing and harvesting of these strains at a 500 l-scale using bio-flocculation. The use of A. fumigatus represents an efficient and economical method for the harvest of B. braunii for biofuel production

Thermogravimetric study and evolved gas analysis of new microalga using TGA-GC-MS

The growing concerns over the environmental challenges emanating from the use of fossil fuels continue to generate interest in finding competitive and sustainable alternatives. This study presents physicochemical characteristics, thermal decomposition profile and kinetics of a new Botryococcus sp. of microalga isolated from Endau-Rompin, Malaysia. The proximate and ultimate analyses were carried out using standard analytical techniques. Thermogravimetric study was conducted in nitrogen atmosphere using a thermogravimetric analyser coupled with gas chromatography-mass spectrometer. The result revealed that the feedstock has high volatile matter (86.74 wt%) and calorific value of 17.18 MJ/kg. The thermal decomposition of the alga sample proceeded via dehydration, decomposition of extractives, hemicellulose, other carbohydrates and lipid evaporation. The kinetics of the alga sample evaluated using a distributed activation energy model showed that the model sufficiently described the pyrolysis of the feedstock with activation energy of 52.72–159.16 kJ/mol. The chemical composition of the evolved gas revealed high content of hydrocarbons, products of carbohydrate and protein decomposition. This suggests that the alga sample is a good candidate for production of valuable precursors for biofuel processing and production of biochemicals