Development of microalga Scenedesmus dimorphus mutant with higher lipid content by radiation breeding

In this study, a high lipid-accumulating mutant strain of the microalgae Scenedesmus dimorphus was developed via radiation breeding. To induce mutant strain, S. dimorphus was gamma-irradiated at doses from 100 to 800 Gy, and then a mutant (Sd-Pm210) with 25 % increased lipid content was selected using Nile red staining methodology. Sd-Pm210 showed morphological changes and had higher growth rate compared to the wild type. From random amplified polymorphic DNA analysis, partial genetic modifications were also observed in Sd-Pm210. In comparisons of lipid content between wild type and Sd-Pm210 using thin-layer chromatography, the content of triacylglycerol was markedly higher in the Sd-Pm210 strain. The total peak area of fatty acid methyl ester was shown to have about 1.4-fold increase in Sd-Pm210, and major fatty acids were identified as palmitic acid, oleic acid, linoleic acid, and linolenic acid. To define the metabolic changes in the mutant strain, 2-dimensional electrophoresis was conducted. Several proteins related to lipid synthesis and energy metabolisms were overexpressed in the mutant strain. These results showed that radiation breeding can be utilized for the development of efficient microalgae strains for biofuel production.     

Enhancement of acid resistance of Scenedesmus dimorphus by acid adaptation

When using flue gas as carbon source for microalgae cultivation, the resulting acidic environment caused by SO _X and NO _X can inhibit microalgal growth. In this study, Scenedesmus dimorphus acquired increased acid resistance by prior exposure to sublethal acid stress; a process defined as acid adaptation. Among the five algal species tested, S. dimorphus showed the highest level of acid tolerance to extreme acid challenge (exposure to pH 3.0). Non-adapted and acid-adapted exponential algal cells were used as inocula for tubular photobioreactors aerated with 2 % CO₂. Previously adapted at pH 4.0 for 1 h, S. dimorphus developed highest growth rate under extreme acidic condition, and the maximum biomass concentration and specific growth rate at pH 3.0 (3.638?±?0.074 g?L^?1 and 1.037?±?0.008 d^?1, respectively) were respectively 14.22 and 10.79 % higher than those of non-adapted cells. Moreover, acid-adapted cells could tolerate lower pH of 2.5, at which the growth of non-adapted cells was totally inhibited. All the results indicated that acid adaptation was an effective approach for the acid resistance enhancement of microalgae.     

Comparative life cycle assessment of autotrophic cultivation of Scenedesmus dimorphus in raceway pond coupled to biodiesel and biogas production

Life cycle assessment (LCA) of indigenous freshwater microalgae, Scenedesmus dimorphus, cultivation in open raceway pond and its conversion to biodiesel and biogas were carried out. The LCA inventory inputs for the biogas scenario was entirely based on primary data obtained from algal cultivation (in pilot scale raceway pond), harvesting, and biogas production; while only the downstream processing involved in biodiesel production namely drying, reaction and purification were based on secondary data. Overall, eight scenarios were modeled for the integrated process involving: algae-based CO₂ capture and downstream processing scenarios for biodiesel and biogas along with impact assessment of nutrient addition and extent of recycling in a life cycle perspective. The LCA results indicated a huge energy deficit and net CO₂ negative in terms of CO₂ capture for both the biodiesel and biogas scenarios, majorly due to lower algal biomass productivity and higher energy requirements for culture mixing. The sensitivity analysis indicated that variability in the biomass productivity has predominant effect on the primary energy demand and global warming potential (GWP, kg CO₂ eq.) followed by specific energy consumption for mixing algal culture. Furthermore, the LCA results indicated that biogas conversion route from microalgae was more energy efficient and sustainable than the biodiesel route. The overall findings of the study suggested that microalgae-mediated CO₂ capture and conversion to biodiesel and biogas production can be energy efficient at higher biomass productivity (> 10 g m⁻² day⁻¹) and via employing energy-efficient systems for culture mixing (< 2 W m⁻³).

     

Growth and lipid accumulation properties of a freshwater microalga Scenedesmus sp. under different cultivation temperature

Microalgal lipid is a promising feedstock for biodiesel production. Effect of cultivation temperature on the growth and lipid accumulation properties of a freshwater microalga Scenedesmus sp. LX1 was studied. Scenedesmus sp. LX1 could grow in a wide range of temperature (10～30°C), and the growth activation energy was 49.3 kJ·mol(-1). The optimal temperature to produce microalgal biomass and lipid was 20°C, and after 15 days of batch cultivation the productivities of 313.3 g biomass·(g P)(-1), 112 g lipid (g P)(-1) and 14.7 g TAGs·(g P)(-1) were obtained. The content of polyunsaturated fatty acids decreased with the increase of cultivation temperature. The reactive oxygen species (ROS) levels at 10°C and 20°C were higher than that under higher temperature. For the first time the cultivation temperature, ROS level, specific growth rate and lipid content per microalgal biomass were correlated together.     

Cultivation of Scenedesmus dimorphus using anaerobic digestate as a nutrient medium

Bioprocess and Biosystems Engineering - In this study, the microalga Scenedesmus dimorphus was cultivated phototrophically using unsterilized anaerobic digestate as a nutrient medium. A bench-scale...     

Physiological responses of the green microalga Acutodesmus dimorphus to temperature induced oxidative stress conditions

Temperature is the most critical factor that directly affects the physiological functioning and metabolic activities of any organism. With rising global temperature, understanding the heat stress response of an organism is critically important. In the present study, we investigated differences in the early changes occurring upon heat stress in the green microalga Acutodesmus dimorphus, a potential strain for biofuel production. The cells were heat-stressed at 45 and 50°C for 24 h and the temporal response of cells in terms of growth, pigments content, levels of oxidative stress biomarkers i.e., reactive oxygen species (ROS) and the response of enzymatic and non-enzymatic antioxidant scavengers were evaluated. The results revealed that after 24 h of heat stress at 45°C, the accumulations of chlorophyll a and carotenoids remained stable; all three ROS increased with the higher activities of various enzymatic and non-enzymatic antioxidants. On the contrary, at a higher temperature of 50°C, the accumulations of chlorophyll a, carotenoids and non-enzymatic antioxidants reduced drastically while the accumulations of all three ROS and the response of enzymatic antioxidants were significantly higher than those at 45°C. These results suggest that the cells utilize several stress acclimatization mechanisms to cope up the heat stress. There was a dramatic difference in the physiological changes and cellular antioxidant mechanism upon heat stress at 45 and 50°C. The cellular defense response of A. dimorphus gets impaired after heat stress at 50°C but remains active at 45°C, exhibiting the heat resistance and, thus, the thermotolerance.     

Development of nitrogen supply strategy for Scenedesmus rubescens attached cultivation toward growth and lipid accumulation

Bioprocess and Biosystems Engineering - In this study, the microalgae Scenedesmus rubescens were cultivated under the following nitrogen sources, nitrogen concentrations, and nitrogen feeding times...     

Effects of different nitrogen, phosphorus, and iron concentrations and salinity on lipid production in newly isolated strain of the tropical green microalga, Scenedesmus dimorphus KMITL

The fatty acid composition, the effect of different concentrations of nitrogen (16.5-344 mg ?L^?1), phosphorus (9–45 mg? L^?1), iron (9–45 mg? L^?1) and salinity levels (0–20 psu) on lipid production in the green microalga Scenedesmus dimorphus KMITL, a new strain isolated from a tropical country, Thailand, were studied. The alga was isolated from a freshwater fish pond, and cultured in Chlorella medium by varying one parameter at a time. The main fatty acid composition of this strain was C16–C18 (97.52 %) fatty acids. A high lipid content was observed in conditions of 16.5 mg? L^?1-N, or 22 mg ?L^?1-P, or 45 mg ?L^?1-Fe, or 5 psu salinity, which accumulated lipids to 20.3?±?0.4, 19.4?±?0.2, 24.7?±?0.5, and 14.3?±?0.2 % of algal biomass, respectively. Increasing lipid content and lipid productivity was noted when the alga was cultured under high iron concentration and high salinity, as well as under reduced phosphorus conditions, whereas nitrogen limitation only resulted in an increased lipid content.     

Development of genetic transformation methodologies for an industrially-promising microalga: Scenedesmus almeriensis

The development of the microalgal industry requires advances in every aspect of microalgal biotechnology. In this regard, the availability of genetic engineering tools for industrially-promising species is key. As Scenedesmus almeriensis has promise for industrial use, we describe here an Agrobacterium-based methodology that allows stable genetic transformation of it for the first time, thus opening the way to its genetic manipulation. Transformation was accomplished using two different antibiotic resistance genes [hygromicine phophotransferase (hpt) and Shble] and it is credited by PCR amplification of both hpt/Shble and GUS genes and by the β-glucuronidase activity of transformed cells. Nevertheless, the single 35S promoter seems unable to direct gene expression to a convenient level in S. almeriensis as suggested by the low GUS enzymatic activity. Temperature was critical for the transformation efficiency.     

Waste utilization and biodiesel production by the green microalga Scenedesmus obliquus

Scenedesmus obliquus was cultivated in three types of waste discharges to couple waste treatment with biodiesel production. The lipid pool accumulation was boosted to 1.0 g liter(-1) against 0.1 g liter(-1) for the control. The waste-grown S. obliquus showed an increase in the content of the saturated fatty acid pool, which is desirable for good-quality biodiesel.     

Heavy metals removal by the microalga Scenedesmus incrassatulus in continuous cultures

The microalga Scenedesmus incrassatulus was grown under continuous regime in the presence of chromium(VI), cadmium(II) and copper(II), as single metal species and as mixtures of two or three metals, in a laboratory scale system. We used an artificial wastewater with low free ion activities (as determined by MINEQL+) due to the presence of EDTA (a strong chelating agent) but with total concentrations not suitable for acceptable environments. Chromium(VI) and cadmium(II) had positive interaction that increased the removal percentages of both these metals; we could not, however, detect any interaction with copper(II). S. incrassatulus was able to remove all the tested metals to some extent (25-78%), but bivalent metals were not removed as efficiently as reported in batch cultures, probably due to the high pH values there recorded. Chromium(VI) was more efficiently removed in continuous cultures than in batch culture, because the uptake of chromate could be favored by actively growing algae.     

Ultrahigh-cell-density heterotrophic cultivation of the unicellular green microalga Scenedesmus acuminatus and application of the cells to photoautotrophic culture enhance biomass and lipid production

Although production of biodiesels from microalgae is proved to be technically feasible, a commercially viable system has yet to emerge. High-cell-density fermentation of microalgae can be coupled with photoautotrophic cultivation to produce oils. In this study, by optimizing culturing conditions and employing a sophisticated substrate feed control strategy, ultrahigh-cell-density of 286 and 283.5 g/L was achieved for the unicellular alga Scenedesmus acuminatus grown in 7.5-L bench-scale and 1,000-L pilot-scale fermenters, respectively. The outdoor scale-up experiments indicated that heterotrophically grown S. acuminatus cells are more productive in terms of both biomass and lipid accumulation when they are inoculated in photobioreactors for lipid production as compared to the cells originally grown under photoautotrophic conditions. Technoeconomic analysis based on the pilot-scale data indicated that the cost of heterotrophic cultivation of microalgae for biomass production is comparable with that of the open-pond system and much lower than that of tubular PBR, if the biomass yield was higher than 200 g/L. This study demonstrated the economic viability of heterotrophic cultivation on large-scale microalgal inocula production, but ultrahigh-productivity fermentation is a prerequisite. Moreover, the advantages of the combined heterotrophic and photoautotrophic cultivation of microalgae for biofuels production were also verified in the pilot-scale.     

Use of the microalga Scenedesmus obliquus to remove cadmium cations from aqueous solutions

The ability of a wild strain of Scenedesmus obliquus, isolated from a heavy metal-contaminated environment, to remove Cd²⁺ from aqueous solutions was studied at several initial concentrations. Viable biomass removed metal to a maximum extent of 11.4 mg_Cd/g at 1 mg_Cd/l, with most Cd²⁺ being adsorbed onto the cell surface. A commercially available strain (ACOI 598) of the same microalga species was also exposed to the same Cd concentrations, and similar results were obtained for the maximum extent of metal removal. Heat-inactivated cells removed a maximum of 6.04 mg_Cd/g at 0.5 mg_Cd/l. The highest extent of metal removal, analyzed at various pH values, was 0.09 mg_Cd/g at pH 7.0. Both strains of the microalga tested have proven effective in removing a toxic heavy metal from aqueous solutions, hence supporting their choice for bioremediation strategies of industrial effluents.     

Biomass production and fatty acid profile of a Scenedesmus rubescens-like microalga

This investigation examined the effects of nitrogen–phosphate combined deficiency on the biomass yield, fatty acid methyl esters (FAME) production and composition from Scenedesmus rubescens-like microalga. A 15-day indoor culture was performed as a 3 × 3 factorial design (NaNO₃ levels: 3, 10 and 20 mM; KH₂PO₄ levels: 20, 50 and 150 μM). The algae grown under medium nitrogen concentration (10 mM) and high phosphate concentration (150 μM) reached the highest biomass (1223.5 ± 152.5 mg/L). Both nitrogen and phosphate had a significant influence on the FAME yield (P < 0.05 and P < 0.0001, respectively). The FAME yield from algae grown under low nitrogen (3 mM) and phosphate concentration (20 μM) increased throughout the experiment and the highest FAME yield (42.2 ± 2.5% of AFDW) as well as C16 and C18 content (95.8 ± 1.6% of AFDW) was achieved under these conditions. Algae grown under medium nitrogen concentration (10 mM) and low phosphate concentration (20 μM) had the highest FAME productivity (426.0 mg/L ± 135.0 mg/L). Thus, the lower nitrogen concentration (3 mM–10 mM) and low phosphate concentration (20 μM) would be an optimal combination tested to produce the most FAME from S. rubescens-like algae.     

Effect of acetate on growth and ammonium uptake in the microalga Scenedesmus obliquus

The effect of acetate on growth and rate of ammonium uptake in Scenedesmus obliquus (UTEX 78) was investigated under light-limiting conditions. Addition of acetate to autotrophic cells with a growth constant of 0.71 day⁻¹ resulted in an increase in the growth rate (mixotrophy, k = 1.3 day⁻¹), and in the presence of acetate, growth occurred in the dark (heterophy, k = 0.44 day⁻¹). The rate of ammonium uptake in autotrophy (17.8 amol cell⁻¹ min⁻¹) was similar to that in heterotrophy (17.4 amol cell⁻¹ min⁻¹) but was 3.7 times lower than that in mixotrophy (65.9 amol cell⁻¹ min⁻¹). In general, mixotrophic cells showed optimum ammonium uptake at the acetate concentration at which they were grown. In autotrophy, uptake of ammonium leveled off at about 12.5 μ M while no saturation was observed in mixotrophic cells. An increase in the rate of uptake of ammonium was observed in autotrophic cells within 1 h after the addition of acetate. The activity of isocitrate lyase (EC 4.1.3.1), a key enzyme for the regulation of the glyoxylate cycle responsible for acetate catabolism, showed a 3.9-fold increase in activity after 24 h in the dark in the presence of acetate. The level of isocitrate lyase activity in cells grown for 24 h in the dark in the presence of 0–20 m M acetate also increased as a function of acetate concentration.     

Optimization of culture medium for the continuous cultivation of the microalga Haematococcus pluvialis

The freshwater microalga Haematococcus pluvialis is one of the best microbial sources of the carotenoid astaxanthin, but this microalga shows low growth rates and low final cell densities when cultured with traditional media. A single-variable optimization strategy was applied to 18 components of the culture media in order to maximize the productivity of vegetative cells of H. pluvialis in semicontinuous culture. The steady-state cell density obtained with the optimized culture medium at a daily volume exchange of 20% was 3.77 · 10⁵ cells ml⁻¹, three times higher than the cell density obtained with Bold basal medium and with the initial formulation. The formulation of the optimal Haematococcus medium (OHM) is (in g l⁻¹) KNO₃ 0.41, Na₂HPO₄ 0.03, MgSO₄ · 7H₂O 0.246, CaCl₂ · 2H₂O 0.11, (in mg l⁻¹) Fe(III)citrate · H₂O 2.62, CoCl₂ · 6H₂O 0.011, CuSO₄ · 5H₂O 0.012, Cr₂O₃ 0.075, MnCl₂ · 4H₂O 0.98, Na₂MoO₄ · 2H₂O 0.12, SeO₂ 0.005 and (in μg l⁻¹]) biotin 25, thiamine 17.5 and B₁₂ 15. Vanadium, iodine, boron and zinc were demonstrated to be non-essential for the growth of H. pluvialis. Higher steady-state cell densities were obtained by a three-fold increase of all nutrient concentrations but a high nitrate concentration remained in the culture medium under such conditions. The high cell productivities obtained with the new optimized medium can serve as a basis for the development of a two-stage technology for the production of astaxanthin from H. pluvialis. Received: 10 September 1999 / Received revision: 2 December 1999 / Accepted: 3 December 1999     

Biodiesel production by the green microalga Scenedesmus obliquus in a recirculatory aquaculture system

Biodiesel production was examined with Scenedesmus obliquus in a recirculatory aquaculture system with fish pond discharge and poultry litter to couple with waste treatment. Lipid productivity of 14,400 liter ha(-1) year(-1) was projected with 11 cultivation cycles per year. The fuel properties of the biodiesel produced adhered to Indian and international standards.