Improving of lipid productivity of the biodiesel promising green microalga <Emphasis Type="Italic">Chlorella pyrenoidosa</Emphasis> via low-energy ion implantation

High lipid content in microalgae is an essential parameter for adopting of microalgal biomass as a feedstock for biodiesel. Mutation is one approach to obtain desired algal strain with high lipid production. In this study, a mutant strain of Chlorella pyrenoidosa was isolated using 1.5?×?10¹⁵ ions cm^?2 s^?1 of N⁺ ion beam implantation technique, which has been widely used in mutagenesis of agricultural crops. N⁺ implantation slightly improved the growth of the mutant over the corresponding wild strain with significant increase in lipid content (32.4 % higher than the wild strain), which resulted in significant increase in lipid productivity by 35 %. In addition, ion implantation mutagenesis of C. pyrenoidosa resulted in 21.4 % decrease in total saturated fatty acids (SFAs) compared to the wild type, with a noticeable increase in polyunsaturated fatty acids (PUFAs). The increase in PUFAs was due mainly to stimulation of hexadecadienoic acid (C16:2) and octadecadienoic acid (C18:2) production. However, the SFA content of wild and mutant strains was 31.7 and 24.9 % of total fatty acids, respectively, highlighting the oxidative stability of biodiesel produced by both strains according to the European standards. Cultivation of C. pyrenoidosa mutant in selenite enrichment medium for five successive cultivation experiments showed insignificant changes in biomass productivity, lipid content, and lipid productivity alongside the study period, which confirms the genetic stability of the produced mutant. The present study confirmed the feasibility of generation of microalgae mutants with significant high lipid production using ion beam implantation.     

Content of fatty acids ofChlorella kessleri in a deep tank fermentation

Chlorella kessleri cultivated in a deep tank contained 4.8% of non-polar lipid; 51% of this fraction represents saturated fatty acids, 7% unsaturated fatty acids. Our investigation of the fatty acids profile demonstrated even- and odd-numbered saturated and unsaturated fatty acids ranging from C₁₂ to C₂₀. Unlike in otherChlorella species, stearic acid was the dominant fatty acid found. Also shown was an elevated C_16:0 fatty acid content and a reduced level of unsaturated fatty acids.     

Lipid and total fatty acid productivity in photoautotrophic fresh water microalgae: screening studies towards biodiesel production

Microalgae are considered as a promising feedstock for biomass production. The selection of the most suitable species is based on several key parameters such as lipid and fatty acid productivity. In the present study, the growth of different microalgae strains was examined in freshwater media for photoautotrophs suited for large-scale applications to identify the most suitable medium for each species. In the optimal medium, Scenedesmus obliquus showed the highest biomass productivity measured as increase of cell dry weight (0.25 g cellu dry weight (CDW) L^?1 day^?1), while Botryococcus braunii showed the highest lipid and total fatty acid content (430 and 270 mg g^?1 CDW, respectively) among the tested species. Regarding lipid and total fatty acid productivity, S. obliquus was the most lipid and total fatty acid productive strain with 41 and 18 mg L^?1 day^?1 during the exponential phase, respectively. Additionally, the proportion of saturated and monounsaturated fatty acids increased with duration of the incubation in S. obliquus, while polyunsaturated fatty acids decreased. These results nominate S. obliquus as a promising microalga in order to serve as a feedstock for renewable energy production.     

Optimization of biomass and fatty acid productivity of Scenedesmus obliquus as a promising microalga for biodiesel production

Nowadays, microalgae are discussed as a promising feedstock for biodiesel production. The present study examines the possibility of enhancement of fatty acid productivity of Scenedesmus obliquus by modifications of the culture medium composition. The effect of different concentrations of sodium bicarbonate, salinity, potassium nitrate, glycerol and sugarcane molasses on the enhancement of biomass and esterified fatty acids production was studied. NaHCO₃ caused an increase in the biomass productivity at low concentrations (0.5 g L^?1), while negatively affected fatty acid productivity at all tested concentrations. Increase of salinity enhanced both biomass and fatty acid productivity. The optimum NaCl concentration and sea water ratio were 0.94 g L^?1 and 25 % which resulted in 56 and 39 % increase in fatty acid productivity, respectively. Nitrogen deficiency showed increase in fatty acid content by 54 % over control but fatty acid productivity was decreased as a result of growth inhibition. Nitrogen-free cultures and cultures treated with ?50 % concentrations of KNO₃ showed 96 and 42 % decrease in EFA productivity, respectively, as compared with the control. Addition of 0.05 and 0.1 M of glycerol increased the biomass productivity by 6 and 5 %, respectively but showed no significant effect on fatty acid productivity as a result of decrease in fatty acid content. Finally, usage of sugarcane molasses stimulated both biomass and fatty acid content. The increase in fatty acid productivity was 32, 65 and 73 % above the control level at 1, 3 and 5 g L^?1 of sugarcane molasses, respectively.     

Lipid productivity and fatty acid composition-guided selection of Chlorella strains isolated from Malaysia for biodiesel production

The need to develop biomass-based domestic production of high-energy liquid fuels (biodiesel) for transportation can potentially be addressed by exploring microalgae with high lipid content. Selecting the strains with adequate oil yield and quality is of fundamental importance for a cost-efficient biofuel feedstock production based on microalgae. This work evaluated 29 strains of Chlorella isolated from Malaysia as feedstock for biodiesel based on volumetric lipid productivity and fatty acid profiles. Phylogenetic studies based on 18S rRNA gene revealed that majority of the strains belong to true Chlorella followed by Parachlorella. The strains were similarly separated into two groups based on fatty acid composition. Of the 18 true Chlorella strains, Chlorella UMACC187 had the highest palmitic acid (C16:0) content (71.3?±?4.2 % total fatty acids, TFA) followed by UMACC84 (70.1?±?0.7 %TFA), UMACC283 (63.8?±?0.7 %TFA), and UMACC001 (60.3?±?4.0 %TFA). Lipid productivity of the strains at exponential phase ranged from 34.53 to 230.38 mg L^?1 day^?1, with Chlorella UMACC050 attaining the highest lipid productivity. This study demonstrated that Chlorella UMACC050 is a promising candidate for biodiesel feedstock production.     

Lipid productivity, settling potential and fatty acid profile of 11 microalgal species grown under nitrogen replete and limited conditions

Microalgae are a promising alternative source of oil for biodiesel production. Identification of a species with desirable characteristics is a key component towards achieving economic feasibility for the process. This has been compromised by a lack of data allowing effective interspecies comparison. Eleven species of microalgae, selected on the basis of available literature data, were tested for lipid productivity, gravity sedimentation and the suitability of their fatty acid profiles for biodiesel production. The response to nitrogen limitation was species-specific. Lipid yields and productivity were higher at 150?mg?L^?1 nitrate than at 1,500?mg?L^?1 for all species tested except Spirulina platensis. The Chlorophyta, particularly Chlorella vulgaris and Scenedesmus, had the highest growth rates and showed the greatest increase in lipid content in response to nitrogen limitation. Cylindrotheca fusiformis, S. platensis, Scenedesmus and Tetraselmis suecica had the fastest settling rates and highest biomass recoveries after 24?h of gravity sedimentation. For most species, the fuel would need to be blended or culture conditions to be optimised to achieve the correct lipid profile in order for microalgal fuel to meet the European standards for biodiesel production (EN 14214). The most promising species overall were the freshwater algae Scenedesmus and C. vulgaris and the marine algae C. fusiformis and Nannochloropsis.     

Lipid production by microalgae <Emphasis Type="Italic">Chlorella protothecoides</Emphasis> with volatile fatty acids (VFAs) as carbon sources in heterotrophic cultivation and its economic assessment

Volatile fatty acids (VFAs) that can be derived from food wastes were used for microbial lipid production by Chlorella protothecoides in heterotrophic cultures. The usage of VFAs as carbon sources for lipid accumulation was investigated in batch cultures. Culture medium, culture temperature, and nitrogen sources were explored for lipid production in the heterotrophic cultivation. The concentration and the ratio of VFAs exhibited significant influence on cell growth and lipid accumulation. The highest lipid yield coefficient and lipid content of C. protothecoides grown on VFAs were 0.187 g/g and 48.7 %, respectively. The lipid content and fatty acids produced using VFAs as carbon sources were similar to those seen on growth and production using glucose. The techno-economic analysis indicates that the biodiesel derived from the lipids produced by heterotrophic C. protothecoides with VFAs as carbon sources is very promising and competitive with other biofuels and fossil fuels.     

Mixotrophic and photoautotrophic cultivation of 14 microalgae isolates from Saskatchewan, Canada: potential applications for wastewater remediation for biofuel production

Northern regions are generally viewed as unsuitable for microalgal biofuel production due to unfavorable climate and solar insolation levels. However, these conditions can potentially be mitigated by coupling microalgal cultivation to industrial processes such as wastewater treatment. In this study, we have examined the biomass and lipid productivity characteristics of 14 microalgae isolates (Chlorophyta) from the Canadian province of Saskatchewan. Under both photoautotrophic and mixotrophic cultivation, a distinct linear trend was observed between biomass and lipid productivities in the 14 SK isolates. The most productive strain under cultivation in TAP media was Scenedesmus sp.-AMDD which displayed rates of biomass and fatty acid productivities of 80 and 30.7?mg?L^?1?day^?1, respectively. The most productive strain in B3NV media was Chlamydomonas debaryana-AMLs1b which displayed rates of biomass and fatty acid productivities of 51.7 and 5.9?mg?L^?1?day^?1, respectively. In 11 of the isolates tested, secondary municipal wastewater (MCWW) supported rates of biomass productivity between 21 and 33?mg?L^?1?day^?1 with Scenedesmus sp.-AMDD being the most productive. Three strains, Chlamydomonas debaryana-AMB1, Chlorella sorokiniana-RBD8 and Micractinium sp.-RB1b, showed large increases in biomass productivity when cultivated mixotrophically in MCWW supplemented with glycerol. High relative oleic acid content was detected in 10 of the 14 isolates when grown mixotrophically in media supplemented with acetate. There was no detectable effect on the fatty acid profiles in cells cultivated mixotrophically in glycerol-supplemented MCWW. These data indicate that biomass and lipid productivities are boosted by mixotrophic cultivation. Exploiting this response in municipal wastewater is a promising strategy for the production of environmentally sustainable biofuels.     

Fatty acids and global metabolites profiling of Dunaliella tertiolecta by shifting culture conditions to nitrate deficiency and high light at different growth phases

In this study, Dunaliella tertiolecta cells were shifted to nitrate-deficient (ND) and/or high light (HL) conditions at different growth phases. Profiling of global metabolites and fatty acids of D. tertiolecta cells were performed by shifting cultivation condition at exponential (EX) and stationary (ST) phases. ND and/or HL induced saturation of fatty acids by increasing saturated fatty acid and monounsaturated fatty acid and decreasing polyunsaturated fatty acid in D. tertiolecta cultures. The relative levels of metabolites like proline, valine, isoleucine, and myoinositol, which act as osmolytes, and ergosterol and glycerol, which are related to the osmoregulatory mechanism, decreased to a greater extent by ND than HL shifts. In addition, the effects of these shifts at the EX phase were observed to be more significant in most of the fatty acids. The results of this research might be applicable to the interpretation of metabolic synthesis and fatty acid production, and to the optimization of cultivation conditions for the use of D. tertiolecta and other microalgae as biofuels or nutraceutical sources.     

Production of lipids in 10 strains of Chlorella and Parachlorella, and enhanced lipid productivity in Chlorella vulgaris

We tested 10 different Chlorella and Parachlorella strains under lipid induction growth conditions in autotrophic laboratory cultures. Between tested strains, substantial differences in both biomass and lipid productivity as well as in the final content of lipids were found. The most productive strain (Chlorella vulgaris CCALA 256) was subsequently studied in detail. The availability of nitrates and/or phosphates strongly influenced growth and accumulation of lipids in cells by affecting cell division. Nutrient limitation substantially enhanced lipid productivity up to a maximal value of 1.5 g l⁻¹ day⁻¹. We also demonstrated the production of lipids through large-scale cultivation of C. vulgaris in a thin layer photobioreactor, even under suboptimal conditions. After 8 days of cultivation, maximal lipid productivity was 0.33 g l⁻¹ day⁻¹, biomass density was 5.7 g l⁻¹ dry weight and total lipid content was more than 30% dry weight. C. vulgaris lipids comprise fatty acids with a relatively high degree of saturation compared with canola oil offering a possible alternative to the use of higher plant oils.     

Distribution and chemotaxonomic significance of acetylenic fatty acids in mosses of the dicranales

Thirty-eight moss species from four families of the order Dicranales were analysed for the fatty acid composition of their acyl lipids. In the Ditrichaceae and the Dicranaceae numerous species were found to contain acetylenic fatty acids in their triglycerides, 9,12,15-Octadecatrien-6-ynoic acid was the major component, often accounting for more than 80 mol%, whereas 9,12-octadecadien-6-ynoic acid was found in small amounts of less than 5 mol%. In some genera, all the species examined contained acetylenic fatty acids, e.g.Dicranella andDicranum, whereas in the genusCampylopus all five species tested were free of acetylenic compounds. Two genera, Ditrichum andDicranoweisia, were found to have a non-homogeous distribution of acetylenic fatty acids. The chemotaxonomic significance of the fatty acid composition in relation to morphological characters is discussed.     

Systematic investigation of biomass and lipid productivity by microalgae in photobioreactors for biodiesel application

We describe a methodology to investigate the potential of given microalgae species for biodiesel production by characterizing their productivity in terms of both biomass and lipids. A multi-step approach was used: determination of biological needs for macronutrients (nitrate, phosphate and sulphate), determination of maximum biomass productivity (the “light-limited” regime), scaling-up of biomass production in photobioreactors, including a theoretical framework to predict corresponding productivities, and investigation of how nitrate starvation protocol affects cell biochemical composition and triggers triacylglycerol (TAG) accumulation. The methodology was applied to two freshwater strains, Chlorella vulgaris and Neochloris oleoabundans, and one seawater diatom strain, Cylindrotheca closterium. The highest total lipid content was achieved with N. oleoabundans (25-37% of DW), while the highest TAG content was found in C. vulgaris (11-14% of DW). These two species showed similar TAG productivities.     

Seasonal variation in triacylglyceride fatty acids of dwarf mistletoes

Fatty acids in the triacylglyceride fraction of three species of dwarf mistletoe (Arceuthobium, Viscaceae) showed considerable seasonal variation in proportions of saturated acids and some differences in chain length for those fatty acids. Early summer aerial shoot tissue contained 34–49% saturated fatty acids, while the same species in late October had only 8–15% saturated fatty acids. Fatty acids with a chain length of C₂₀ or greater comprised 18–25% of the total in late June but only 1.5–6.4% in the autumn. Three other species analysed in only one season showed similar saturated fatty acids, and chain length patterns were also parallel to the seasonally varying species analysed in the same season. The ratio of linolenic to oleic acid remained constant with season but linolenic acid increased from less than 1% in June to 7–8% in October samples.     

Fatty acids of some cornaceae,hydrangeaceae, aquifoliaceae,hamamelidaceae and styracaceae

Seed, kernel or fruit oils of the Cornaceae (nine species). Hydrangea, Hamamelis, Ilex (Aquifoliaceae) and the Styracaceae (two species) were analysed for fatty acid composition. Special attention was paid to the occurrence of petroselinic acid (18: 1Δ6c). In the species investigated. C₁₈ acids were always present in greater quantities than C₁₆ fatty acids; C₂₀ and C₂₂ acids were only minor components. The Cornaceae show differing fatty acid patterns which correlate well with anatomical, morphological and other chemical data. In Cornus, Curtisia, Mastixia and Corokia linoleic acid predominates, whereas linoleic and linolenic acid form the major components in Davidia and Nyssa. 18 : 1Δ6c, an aralioid type, occurs in large amounts in Aucuba and Griselinia. Hamamelis, Hydrangea and Ilex show a common fatty acid pattern with linoleic acid as the dominant component in all cases. Classification currently based on morphological and anatomical differences between the two species of Styracaceae which were investigated here should include their different fatty acid compositions: in Halesia linoleic acid predominates over oleic acid, whereas in Styrax equal amounts of these two acids are found.     

Culture of microalgae Chlorella minutissima for biodiesel feedstock production

Microalgae are among the most promising of non‐food based biomass fuel feedstock alternatives. Algal biofuels production is challenged by limited oil content, growth rate, and economical cultivation. To develop the optimum cultivation conditions for increasing biofuels feedstock production, the effect of light source, light intensity, photoperiod, and nitrogen starvation on the growth rate, cell density, and lipid content of Chlorella minutissima were studied. The fatty acid content and composition of Chlorella minutissima were also investigated under the above conditions. Fluorescent lights were more effective than red or white light‐emitting diodes for algal growth. Increasing light intensity resulted in more rapid algal growth, while increasing the period of light also significantly increased biomass productivity. Our results showed that the lipid and triacylglycerol content were increased under N starvation conditions. Thus, a two‐phase strategy with an initial nutrient‐sufficient reactor followed by a nutrient deprivation strategy could likely balance the desire for rapid and high biomass generation (124 mg/L) with a high oil content (50%) of Chlorella minutissima to maximize the total amount of oil produced for biodiesel production. Moreover, methyl palmitate (C16:0), methyl oleate (C18:1), methyl linoleate (C18:2), and methyl linolenate (C18:3) are the major components of Chlorella minutissima derived FAME, and choice of light source, intensity, and N starvation impacted the FAME composition of Chlorella minutissima. The optimized cultivation conditions resulted in higher growth rate, cell density, and oil content, making Chlorella minutissima a potentially suitable organism for biodiesel feedstock production. Biotechnol. Bioeng. 2011;108: 2280–2287. © 2011 Wiley Periodicals, Inc.     

Air-Drying of Cells,the Novel Conditions for Stimulated Synthesis of Triacylglycerol in a Green Alga,Chlorella kessleri

Triacylglycerol is used for the production of commodities including food oils and biodiesel fuel. Microalgae can accumulate triacylglycerol under adverse environmental conditions such as nitrogen-starvation. This study explored the possibility of air-drying of green algal cells as a novel and simple protocol for enhancement of their triacylglycerol content. Chlorella kessleri cells were fixed on the surface of a glass fibre filter and then subjected to air-drying with light illumination. The dry cell weight, on a filter, increased by 2.7-fold in 96 h, the corresponding chlorophyll content ranging from 1.0 to 1.3-fold the initial one. Concomitantly, the triacylglycerol content remarkably increased to 70.3 mole% of fatty acids and 15.9% (w/w), relative to total fatty acids and dry cell weight, respectively, like in cells starved of nitrogen. Reduction of the stress of air-drying by placing the glass filter on a filter paper soaked in H₂O lowered the fatty acid content of triacylglycerol to 26.4 mole% as to total fatty acids. Moreover, replacement of the H₂O with culture medium further decreased the fatty acid content of triacylglycerol to 12.2 mole%. It thus seemed that severe dehydration is required for full induction of triacylglycerol synthesis, and that nutritional depletion as well as dehydration are crucial environmental factors. Meanwhile, air-drying of Chlamydomonas reinhardtii cells increased the triacylglycerol content to only 37.9 mole% of fatty acids and 4.8% (w/w), relative to total fatty acids and dry cell weight, respectively, and a marked decrease in the chlorophyll content, on a filter, of 33%. Air-drying thus has an impact on triacylglycerol synthesis in C. reinhardtii also, however, the effect is considerably limited, owing probably to instability of the photosynthetic machinery. This air-drying protocol could be useful for the development of a system for industrial production of triacylglycerol with appropriate selection of the algal species.     

High contents of very long-chain polyunsaturated fatty acids in different moss species

Key message

Mosses have high contents of polyunsaturated fatty acids. Tissue-specific differences in fatty acid contents and fatty acid desaturase (FADS)-encoding gene expression exist. The arachidonic acid-synthesizing FADS operate in the ER.

Abstract

Polyunsaturated fatty acids (PUFAs) are important cellular compounds with manifold biological functions. Many PUFAs are essential for the human diet and beneficial for human health. In this study, we report on the high amounts of very long-chain (vl) PUFAs (≥C₂₀) such as arachidonic acid (AA) in seven moss species. These species were established in axenic in vitro culture, as a prerequisite for comparative metabolic studies under highly standardized laboratory conditions. In the model organism Physcomitrella patens, tissue-specific differences in the fatty acid compositions between the filamentous protonema and the leafy gametophores were observed. These metabolic differences correspond with differential gene expression of fatty acid desaturase (FADS)-encoding genes in both developmental stages, as determined via microarray analyses. Depending on the developmental stage and the species, AA amounts for 6–31 %, respectively, of the total fatty acids. Subcellular localization of the corresponding FADS revealed the endoplasmic reticulum as the cellular compartment for AA synthesis. Our results show that vlPUFAs are highly abundant metabolites in mosses. Standardized cultivation techniques using photobioreactors along with the availability of the P. patens genome sequence and the high rate of homologous recombination are the basis for targeted metabolic engineering in moss. The potential of producing vlPUFAs of interest from mosses will be highlighted as a promising area in plant biotechnology.     

Cultivation of a new microalga,Micractinium reisseri,in municipal wastewater for nutrient removal,biomass, lipid,and fatty acid production

Coupling of advanced wastewater treatment with microalgae cultivation for low-cost lipid production was demonstrated in this study. The microalgal species Micractinium reisseri and Scenedesmus obliquus were isolated from municipal wastewater mixed with agricultural drainage. M. reisseri was selected based on the growth rate and cultivated in municipal wastewater (influent, secondary and tertiary effluents) which varied in nutrient concentration. M. reisseri showed an optimal specific growth rate (μ_opt) of 1.15, 1.04, and 1.01 1/day for the influent and the secondary and tertiary effluents, respectively. Secondary effluent supported the highest phosphorus removal (94%) and saturated fatty acid content (40%). The highest lipid content (40%), unsaturated fatty acid content, including monounsaturated and polyunsaturated fatty acids (66%), and nitrogen removal (80%) were observed for tertiary effluent. Fatty acids accumulating in the microalgal biomass (M. reisseri) were mainly composed of palmitic acid, oleic acid, linoleic acid, and a-linolenic acid. Cultivation of M. reisseri using municipal wastewater served a dual function of nutrient removal and biofuel feedstock generation.     

The effect of nitrogen limitation on lipid productivity and cell composition in Chlorella vulgaris

Chlorella vulgaris accumulates lipid under nitrogen limitation, but at the expense of biomass productivity. Due to this tradeoff, improved lipid productivity may be compromised, despite higher lipid content. To determine the optimal degree of nitrogen limitation for lipid productivity, batch cultures of C. vulgaris were grown at different nitrate concentrations. The growth rate, lipid content, lipid productivity and biochemical and elemental composition of the cultures were monitored for 20 days. A starting nitrate concentration of 170 mg L^?1 provided the optimal tradeoff between biomass and lipid production under the experimental conditions. Volumetric lipid yield (in milligram lipid per liter algal culture) was more than double that under nitrogen-replete conditions. Interpolation of the data indicated that the highest volumetric lipid concentration and lipid productivity would occur at nitrate concentrations of 305 and 241 mg L^?1, respectively. There was a strong correlation between the nitrogen content of the cells and the pigment, protein and lipid content, as well as biomass and lipid productivity. Knowledge of the relationships between cell nitrogen content, growth, and cell composition assists in the prediction of the nitrogen regime required for optimal productivity in batch or continuous culture. In addition to enhancing lipid productivity, nitrogen limitation improves the lipid profile for biodiesel production and reduces the requirement for nitrogen fertilizers, resulting in cost and energy savings and a reduction in the environmental burden of the process.