Endophytic Fungal Strains of Soybean for Lipid Production

Lipids created via microbial biosynthesis are a potential raw material to replace plant-based oil for biodiesel production. Oleaginous microbial species currently available are capable of accumulating high amount of lipids in their cell biomass, but rarely can directly utilize lignocellulosic biomass as substrates. Thus this research focused on the screening and selection of new fungal strains that generate both lipids and hydrolytic enzymes. To search for oleaginous fungal strains in the soybean plant, endophytic fungi and fungi close to the plant roots were studied as a microbial source. Among 33 endophytic fungal isolates screened from the soybean plant, 13 have high lipid content (>20 % dry biomass weight); among 38 fungal isolates screened from the soil surrounding the soybean roots, 14 have high lipid content. Also, five fungal isolates with both high lipid content and promising biomass production were selected for further studies on their cell growth, oil accumulation, lipid content and profile, utilization of various carbon sources, and cellulase production. The results indicate that most strains could utilize different types of carbon sources and some strains accumulated >40 % of the lipids based on the dry cell biomass weight. Among these promising strains, some Fusarium strains specifically showed considerable production of cellulase, which offers great potential for biodiesel production by directly utilizing inexpensive lignocellulosic material as feedstock.     

Isolation and evaluation of oil-producing microalgae from subtropical coastal and brackish waters

Microalgae have been widely reported as a promising source of biofuels, mainly based on their high areal productivity of biomass and lipids as triacylglycerides and the possibility for cultivation on non-arable land. The isolation and selection of suitable strains that are robust and display high growth and lipid accumulation rates is an important prerequisite for their successful cultivation as a bioenergy source, a process that can be compared to the initial selection and domestication of agricultural crops. We developed standard protocols for the isolation and cultivation for a range of marine and brackish microalgae. By comparing growth rates and lipid productivity, we assessed the potential of subtropical coastal and brackish microalgae for the production of biodiesel and other oil-based bioproducts. This study identified Nannochloropsis sp., Dunaniella salina and new isolates of Chlorella sp. and Tetraselmis sp. as suitable candidates for a multiple-product algae crop. We conclude that subtropical coastal microalgae display a variety of fatty acid profiles that offer a wide scope for several oil-based bioproducts, including biodiesel and omega-3 fatty acids. A biorefinery approach for microalgae would make economical production more feasible but challenges remain for efficient harvesting and extraction processes for some species.     

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.     

The effect of physicochemical conditions and nutrient sources on maximizing the growth and lipid productivity of green microalgae

Biodiesel is a renewable fuel produced mostly from edible and non‐edible vegetables, by transesterification of neutral lipids (triacylglycerols). However, vegetable oil‐based biodiesel production competes with food crops for arable land, increasing food prices and leading to biodiversity loss. The production of biodiesel from oleaginous microorganisms – particularly microalgae – has attracted attention due to the higher lipid productivity of these organisms, when compared with vegetables. Several environmental factors – including light, temperature, pH and the presence of nutrients (particularly nitrogen, phosphorus and iron) – influence directly the ability of microalgae to produce and store triacylglycerols and other lipids, and also modulate microalgal growth. Although some environmental factors affect several species in a similar manner, differential responses between species are frequent, highlighting the importance of identifying optimal cultivation conditions for each species, to balance growth and lipid productivity for biodiesel production. Here, we reviewed the particular influence of the physicochemical and nutritional factors on the growth and lipid productivity of different green oleaginous microalgae species.     

Molecular Identification and Comparative Evaluation of Tropical Marine Microalgae for Biodiesel Production

Marine microalgae have emerged as important feedstock for liquid biofuel production. The identification of lipid-rich native microalgal species with high growth rate and optimal fatty acid profile and biodiesel properties is the most challenging step in microalgae-based biodiesel production. In this study, attempts have been made to bio-prospect the biodiesel production potential of marine and brackish water microalgal isolates from the west coast of India. A total of 14 microalgal species were isolated, identified using specific molecular markers and based on the lipid content; seven species with total lipid content above 20% of dry cell weight were selected for assessing biodiesel production potential in terms of lipid and biomass productivities, nile red fluorescence, fatty acid profile and biodiesel properties. On comparative analysis, the diatoms were proven to be promising based on the overall desirable properties for biodiesel production. The most potential strain Navicula phyllepta MACC8 with a total lipid content of 26.54 % of dry weight of biomass, the highest growth rate (0.58 day^?1) and lipid and biomass productivities of 114 and 431 mgL^?1 day^?1, respectively, was rich in fatty acids mainly of C16:0, C16:1 and C18:0 in the neutral lipid fraction, the most favoured fatty acids for ideal biodiesel properties. The biodiesel properties met the requirements of fuel quality standards based on empirical estimation. The marine diatoms hold a great promise as feedstock for large-scale biodiesel production along with valuable by-products in a biorefinery perspective, after augmenting lipid and biomass production through biochemical and genetic engineering approaches.     

Fatty Acid Characterization and Biodiesel Production by the Marine Microalga <Emphasis Type="Italic">Asteromonas gracilis</Emphasis>: Statistical Optimization of Medium for Biomass and Lipid Enhancement

Lipid production is an important indicator for evaluating microalgal species for biodiesel production. In this study, a new green microalga was isolated from a salt lake in Egypt and identified as Asteromonas gracilis. The main parameters such as biomass productivity, lipid content, and lipid productivity were evaluated in A. gracilis, cultivated in nutrient-starved (nitrogen, phosphorous), and salinity stress as a one-factor-at-a-time method. These parameters in general did not vary significantly from the standard nutrient growth media when these factors were utilized separately. Hence, response surface methodology (RSM) was assessed to study the combinatorial effect of different concentrations of the abovementioned factor conditions and to maximize the biomass productivity, lipid content, and lipid productivity of A. gracilis by determining optimal concentrations. RSM optimized media, including 1.36 M NaCl, 1 g/L nitrogen, and 0.0 g/L phosphorus recorded maximum biomass productivity, lipid content, and lipid productivity (40.6 mg/L/day, 39.3%, and 15.9 mg/L/day, respectively) which agreed well with the predicted values (40.1 mg/L/day, 43.6%, and 14.6 mg/L/day, respectively). Fatty acid profile of A. gracilis was composed of C16:0, C16:1, C18:0, C18:3, C18:2, C18:1, and C20:5, and the properties of fuel were also in agreement with international standards. These results suggest that A. gracilis is a promising feedstock for biodiesel production.     

A quantitative analysis of microalgal lipids for optimization of biodiesel and omega‐3 production

The lipid characteristics of microalgae are known to differ between species and change with growth conditions. This work provides a methodology for lipid characterization that enables selection of the optimal strain, cultivation conditions, and processing pathway for commercial biodiesel production from microalgae. Two different microalgal species, Nannochloropsis sp. and Chlorella sp., were cultivated under both nitrogen replete and nitrogen depleted conditions. Lipids were extracted and fractionated into three major classes and quantified gravimetrically. The fatty acid profile of each fraction was analyzed using GC–MS. The resulting quantitative lipid data for each of the cultures is discussed in the context of biodiesel and omega‐3 production. This approach illustrates how the growth conditions greatly affect the distribution of fatty acid present in the major lipid classes and therefore the suitability of the lipid extracts for biodiesel and other secondary products. Biotechnol. Bioeng. 2013; 110: 2096–2104. © 2013 Wiley Periodicals, Inc.     

Nitrogen starvation strategies and photobioreactor design for enhancing lipid content and lipid production of a newly isolated microalga Chlorella vulgaris ESP-31: implications for biofuels

Microalgae are recognized for serving as a sustainable source for biodiesel production. This study investigated the effect of nitrogen starvation strategies and photobioreactor design on the performance of lipid production and of CO(2) fixation of an indigenous microalga Chlorella vulgaris ESP-31. Comparison of single-stage and two-stage nitrogen starvation strategies shows that single-stage cultivation on basal medium with low initial nitrogen source concentration (i.e., 0.313 g/L KNO(3)) was the most effective approach to enhance microalgal lipid production, attaining a lipid productivity of 78 mg/L/d and a lipid content of 55.9%. The lipid productivity of C. vulgaris ESP-31 was further upgraded to 132.4 mg/L/d when it was grown in a vertical tubular photobioreactor with a high surface to volume ratio of 109.3 m(2)/m(3) . The high lipid productivity was also accompanied by fixation of 6.36 g CO(2) during the 10-day photoautotrophic growth with a CO(2) fixation rate of 430 mg/L/d. Analysis of fatty acid composition of the microalgal lipid indicates that over 65% of fatty acids in the microalgal lipid are saturated [i.e., palmitic acid (C16:0) and stearic acid (C18:0)] and monounsaturated [i.e., oleic acid (C18:1)]. This lipid quality is suitable for biodiesel production.     

Lipid accumulation and growth of Chlorella zofingiensis in flat plate photobioreactors outdoors

Culturing microalgae using natural sunlight is an effective way to reduce the cost of microalgae-based biodiesel production. In order to evaluate the feasibility of culturing Chlorella zofingiensis outdoors for biodiesel production, effects of nitrogen limitation and initial cell concentration on growth and lipid accumulation of this alga were investigated in 60 L flat plate photobioreactors outdoors. The highest μmax and biomass productivity obtained was 0.994 day(-1) and 58.4 mg L(-1)day(-1), respectively. The lipid content was much higher (54.5% of dry weight) under nitrogen limiting condition than under nitrogen sufficient condition (27.3%). With the increasing initial cell concentrations, the lipid contents declined, while lipid concentrations and productivities increased. The highest lipid content, lipid concentration, and lipid productivity obtained was 54.5%, 536 mg L(-1) and 22.3 mg L(-1)day(-1), respectively. This study demonstrated that it was possible to culture C. zofingiensis under outdoor conditions for producing biodiesel feedstock.     

Enrichment as a screening method for a high-growth-rate microalgal strain under continuous cultivation system

Microalgae are a promising feedstock for renewable biodiesel production. High productivity of biodiesel production from microalgae is directly related to growth rate as well as lipid content of cells. In the present study, an enrichment process in a continuous cultivation system was developed to screen a high-growth-rate microalga from a mixed culture of microalgal species; Chlorella vulgaris, Chlorella protothecoides, and Chlamydomonas reinhardtii were used as test organisms for our experiments. The time-dependent washout of mixed microalgal pool was executed to successfully enrich the C. reinhardtii, which exhibits the higher growth rate than C. vulgaris and C. protothecoides under turbidostat conditions within 75 h. The domination of C. reinhardtii in the mixed culture was validated by on-line monitoring of growth rate and flowcytometric analysis. For the time-efficient production of microalgal biomass, this screening process has a high potential to segregate the fast-growing microalgal strains from the pool of various uncharacterized microalgal species and random mutants.     

Effects of nitrogen sources on cell growth and lipid accumulation of green alga <Emphasis Type="Italic">Neochloris oleoabundans</Emphasis>

Microalgal lipids are the oils of future for sustainable biodiesel production. However, relatively high production costs due to low lipid productivity have been one of the major obstacles impeding their commercial production. We studied the effects of nitrogen sources and their concentrations on cell growth and lipid accumulation of Neochloris oleoabundans, one of the most promising oil-rich microalgal species. While the highest lipid cell content of 0.40 g/g was obtained at the lowest sodium nitrate concentration (3 mM), a remarkable lipid productivity of 0.133 g l⁻¹ day⁻¹ was achieved at 5 mM with a lipid cell content of 0.34 g/g and a biomass productivity of 0.40 g l⁻¹ day⁻¹. The highest biomass productivity was obtained at 10 mM sodium nitrate, with a biomass concentration of 3.2 g/l and a biomass productivity of 0.63 g l⁻¹ day⁻¹. It was observed that cell growth continued after the exhaustion of external nitrogen pool, hypothetically supported by the consumption of intracellular nitrogen pools such as chlorophyll molecules. The relationship among nitrate depletion, cell growth, lipid cell content, and cell chlorophyll content are discussed.     

Extraction of oil from microalgae for biodiesel production: A review

The rapid increase of CO(2) concentration in the atmosphere combined with depleted supplies of fossil fuels has led to an increased commercial interest in renewable fuels. Due to their high biomass productivity, rapid lipid accumulation, and ability to survive in saline water, microalgae have been identified as promising feedstocks for industrial-scale production of carbon-neutral biodiesel. This study examines the principles involved in lipid extraction from microalgal cells, a crucial downstream processing step in the production of microalgal biodiesel. We analyze the different technological options currently available for laboratory-scale microalgal lipid extraction, with a primary focus on the prospect of organic solvent and supercritical fluid extraction. The study also provides an assessment of recent breakthroughs in this rapidly developing field and reports on the suitability of microalgal lipid compositions for biodiesel conversion.     

用混合氮源在室外培养产油链带藻的可行性研究

为探索两株链带藻（Desmodesmus sp.T28-1和Desmodesmus sp.NMX451）在室外培养的最优氮源,首先在室内就不同氮源（尿素、硝酸钠、碳酸铵以及尿素和硝酸钠混合氮源）下微藻的生长和油脂积累做了研究,筛选出最优的混合氮源在室外进行了培养的可行性研究。室内研究结果表明两株链带藻在尿素下培养油脂含量最低,在铵氮下培养生物量最低。且NMX451在混合态氮下的油脂产率显著性的高于其他氮源下的油脂产率。对两株链带藻在混合氮源下的脂肪酸组分做进一步分析,结果表明油脂组分适合生物柴油生产要求,估算的生物柴油品质达到国际和国内生产标准。将两株链带藻置于室外140 L柱式反应器中用混合氮源进一步扩大培养,结果表明NMX451比T28-1的油脂含量和油脂产率高,生产成本更低,且脂肪酸组分更适宜生物柴油生产。研究表明用混合氮源在室外培养微藻是非常可行的培养方法,也说明NMX451比T28-1在生物柴油生产方面具有更好的潜力。     

pH对小球藻Chlorella sp. XQ-200419光合作用、生长和产油的影响

以一种生长快、油脂含量高的小球藻(Chlorella sp. XQ-200419)为实验材料, 利用测定净光合放氧速率的方法研究了pH对其光合作用的影响; 使用改良的BG-11培养基在微藻环形培养池模拟系统中进行分批培养, 培养周期为8d, 培养过程中使用 pH控制仪在线监测藻液的pH, 根据pH变化, 自动接通、关闭CO2通气管道, 将藻液pH分别控制在5.06.0, 7.08.0, 8.09.0, 9.010.0, 10.011.0内, 研究pH对生长速率、生物质面积产率、总脂含量和总脂面积产率的影响。主要结果如下: 藻液pH对小球藻Chlorella sp. XQ-200419光合放氧、生长速率、生物质产率、总脂含量和产率都有显著影响, 适宜的pH范围是7.09.0, 在此范围内, 光合放氧、生长速率、生物质产率、总脂含量和产率均保持较高水平, 且pH的影响不显著; pH低于7.0, 高于9.0, 其光合放氧、生长速率、生物质产率、总脂含量和产率都显著降低。这表明pH对小球藻Chlorella sp. XQ-200419光合作用的影响和对生长、产油的影响是一致的。pH 7.08.0, 小球藻的生物质平均面积产率和总脂平均面积产率都达到最大值, 分别是8.9 g/(m2d)和2269.5 mg/(m2d); 当藻液pH超过10.0, 生物质平均面积产率和总脂平均面积产率分别降低42.1%和60.0%。适合于小球藻生长的pH也有利于其积累油脂, 所以, pH对小球藻产油的影响是一种适宜模式, 而非胁迫模式。规模化培养小球藻Chlorella sp. XQ-200419, 通过补充CO2将藻液pH控制在7.09.0内, 可以获得高生物质产率和总脂产率。研究结果反映出pH对小球藻光合作用、生长和产油影响的规律, 也为规模化培养小球藻生产微藻油脂过程中合理控制藻液pH提供了依据。       

A symbiotic gas exchange between bioreactors enhances microalgal biomass and lipid productivities: taking advantage of complementary nutritional modes

This paper describes the association of two bioreactors: one photoautotrophic and the other heterotrophic, connected by the gas phase and allowing an exchange of O₂ and CO₂ gases between them, benefiting from a symbiotic effect. The association of two bioreactors was proposed with the aim of improving the microalgae oil productivity for biodiesel production. The outlet gas flow from the autotrophic (O₂ enriched) bioreactor was used as the inlet gas flow for the heterotrophic bioreactor. In parallel, the outlet gas flow from another heterotrophic (CO₂ enriched) bioreactor was used as the inlet gas flow for the autotrophic bioreactor. Aside from using the air supplied from the auto- and hetero-trophic bioreactors as controls, one mixotrophic bioreactor was also studied and used as a model, for its claimed advantage of CO₂ and organic carbon being simultaneously assimilated. The microalga Chlorella protothecoides was chosen as a model due to its ability to grow under different nutritional modes (auto, hetero, and mixotrophic), and its ability to attain a high biomass productivity and lipid content, suitable for biodiesel production. The comparison between heterotrophic, autotrophic, and mixotrophic Chlorella protothecoides growth for lipid production revealed that heterotrophic growth achieved the highest biomass productivity and lipid content (>22%), and furthermore showed that these lipids had the most suitable fatty acid profile in order to produce high quality biodiesel. Both associations showed a higher biomass productivity (10–20%), when comparing the two separately operated bioreactors (controls) which occurred on the fourth day. A more remarkable result would have been seen if in actuality the two bioreactors had been inter-connected in a closed loop. The biomass productivity gain would have been 30% and the lipid productivity gain would have been 100%, as seen by comparing the productivities of the symbiotic assemblage with the sum of the two bioreactors operating separately (controls). These results show an advantage of the symbiotic bioreactors association towards a cost-effective microalgal biodiesel production.     

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.     

Biomass productivity and productivity of fatty acids and amino acids of microalgae strains as key characteristics of suitability for biodiesel production

Microalgae are discussed as an alternative source for the production of biofuels. The lipid content compared to cultivation time of used species is the main reason for any choice of a special strain. This paper reviews more analytical data of 38 screened microalgae strains. After the cultivation period, total content of lipids was analysed. The extracted fatty acids were quantified as fatty acid methyl esters by GC analysis. The amino acids were analysed by HPLC. Chlorella sp., Chlorella saccharophila, Chlorella minutissima and Chlorella vulgaris were identified as species with the highest productivity of fatty acids relevant to transesterification reactions. The components were mainly linoleic acid, palmitic acid and oleic acid. To increase productivity of highly saturated fatty acids, cultivation parameters light intensity and temperature were varied. In this manner, the ideal conditions for biodiesel production were defined in this publication.     

生物柴油原料资源高油脂微藻的开发利用

生物柴油作为化石能源的替代燃料已在国际上得到广泛应用。至今生物柴油的原料主要来自油料植物, 但与农作物争地的情况以及较高的原料成本限制了生物柴油的进一步推广。微藻作为高光合生物有其特殊的原料成本优势, 微藻的脂类含量最高可达细胞干重的80%。利用生物技术改良微藻, 获得的高油脂基因工程微藻经规模养殖, 可大大降低生物柴油原料成本。介绍了国内外生物柴油的应用现状, 阐述了微藻作为生物柴油原料的优势, 对基因工程技术调控微藻脂类代谢途径的研究进展, 以及在构建工程微藻中面临的问题和应采取的对策进行了综述和展望。     

Optimization of aeration for biodiesel production by <Emphasis Type="Italic">Scenedesmus obliquus</Emphasis> grown in municipal wastewater

Despite the significant breakthroughs in research on microalgae as a feedstock for biodiesel, its production cost is still much higher than that of fossil diesel. One possible solution to overcome this problem is to optimize algal growth and lipid production in wastewater. The present study examines the optimization of pretreatment of municipal wastewater and aeration conditions in order to enhance the lipid productivity of Scenedesmus obliquus. Results showed that no significant differences were recorded in lipid productivity of S. obliquus grown in primary settled or sterilized municipal wastewater; however, ultrasound pretreatment of wastewater significantly decreased the lipid production. Whereas, aeration rates of 0.2 vvm significantly increased lipid content by 51 %, with respect to the non-aerated culture, which resulted in maximum lipid productivity (32.5 mg L^?1 day^?1). Furthermore, aeration enrichment by 2 % CO₂ resulted in increase of lipid productivity by 46 % over the CO₂ non-enriched aerated culture. Fatty acid profile showed that optimized aeration significantly enhanced monounsaturated fatty acid production, composed mainly of C18:1, by 1.8 times over the non-aerated S. obliquus culture with insignificant changes in polyunsaturated fatty acid proportion; suggesting better biodiesel characteristics for the optimized culture.     

Effects of outdoor cultures on the growth and lipid production of <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis> using closed photobioreactors

One of the principal challenges for large scale production of microalgae is the high costs of biomass production. Aiming for minimize this problem, microalgal biodiesel production should focus on outdoors cultures, using available solar light and allowing lower energy cost process. Testing species that proved to be common and easy to culture may be a good approach in this process. The present work reports indoor-outdoor cultures of Phaeodactylum tricornutum using different bioreactors types, using cell growth, biochemical composition, and the profiles of the fatty acids produced as the parameters to test the optimization processes. The results show that the use of outdoor cultures is a good choice to obtain P. tricornutum biomass with a good potential for biodiesel production. The microalgae produced reached better growth efficiency, major lipid content and showed an increment in the percentage of saturated fatty acids (required on the biodiesel production) respect indoor cultures. These results are important to show the relevance of using outdoor cultures as a way to improve the efficiency and the energetic balance of the biodiesel production with P. tricornutum algae.