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.     

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.     

Lipid accumulation and CO2 utilization of Nannochloropsis oculata in response to CO2 aeration

In order to produce microalgal lipids that can be transformed to biodiesel fuel, effects of concentration of CO(2) aeration on the biomass production and lipid accumulation of Nannochloropsis oculata in a semicontinuous culture were investigated in this study. Lipid content of N. oculata cells at different growth phases was also explored. The results showed that the lipid accumulation from logarithmic phase to stationary phase of N. oculata NCTU-3 was significantly increased from 30.8% to 50.4%. In the microalgal cultures aerated with 2%, 5%, 10% and 15% CO(2), the maximal biomass and lipid productivity in the semicontinuous system were 0.480 and 0.142 g L(-1)d(-1) with 2% CO(2) aeration, respectively. Even the N. oculata NCTU-3 cultured in the semicontinuous system aerated with 15% CO(2), the biomass and lipid productivity could reach to 0.372 and 0.084 g L(-1)d(-1), respectively. In the comparison of productive efficiencies, the semicontinuous system was operated with two culture approaches over 12d. The biomass and lipid productivity of N. oculata NCTU-3 were 0.497 and 0.151 g L(-1)d(-1) in one-day replacement (half broth was replaced each day), and were 0.296 and 0.121 g L(-1)d(-1) in three-day replacement (three fifth broth was replaced every 3d), respectively. To optimize the condition for long-term biomass and lipid yield from N. oculata NCTU-3, this microalga was suggested to grow in the semicontinuous system aerated with 2% CO(2) and operated by one-day replacement.     

Effect of nitrate feeding strategies on lipid and biomass productivities in fed-batch cultures of Nannochloropsis gaditana

Controlled nitrate feeding strategies for fed-batch cultures of microalgae were applied for the enhancement of lipid production and microalgal growth rates. In particular, in this study, the effect of nitrate feeding rates on lipid and biomass productivities in fed-batch cultures of Nannochloropsis gaditana were investigated using three feeding modes (i.e., pulse, continuous, and staged) and under two light variations on both lipid productivity and fatty acid compositions. Higher nitrate levels negatively affected lipid production in the study. Increasing the light intensity increased the lipid contents of the microalgae in all three fed-batch feeding modes. A maximum of 58.3% lipid- to dry weight ratio was achieved when using pulse-fed cultures at an illumination of 200 μmol photons m⁻² s⁻¹ and 10 mg/day of nitrate feeding. This condition also resulted in the maximum lipid productivity of 44.6 mg L⁻¹ day⁻¹. The fatty acid compositions of the lipids consisted predominantly of long-chain fatty acids (C:16 and C:18) and accounted for 70% of the overall fatty acid methyl esters. Pulse feeding mode was found to significantly enhance the biomass and lipid production. The other two feeding modes (continuous and staged) were not ideal for lipid and biomass production. This study demonstrates the applicability of pulse feeding strategies in fed-batch cultures as an appropriate cultivation strategy that can increase both lipid accumulation and biomass production.     

囊状黄丝藻在不同初始氮浓度条件下特殊的油脂积累规律

对不同初始氮浓度条件下囊状黄丝藻(Tribonema utriculosum SAG22.94)的生长状况、油脂含量和脂肪酸组成与含量进行研究。结果显示,囊状黄丝藻在氮浓度为3.0 mmol/L时,获得生物质浓度最高,为6.39 g/L;氮浓度为18.0 mmol/L时获得总脂和总脂肪酸含量最高,分别为细胞干重的44.62%和42.21%;上述3个指标单位体积的产率均在氮浓度3.0 mmol/L时达到最高值,分别为0.538、0.209和0.206 g·L~(-1)·d~(-1)。在4种初始氮浓度条件下,囊状黄丝藻油脂和脂肪酸含量可随着氮浓度增加而增加。脂肪酸含量分析结果显示,该藻的主要脂肪酸为豆蔻酸(C14∶0)、棕榈酸(C16∶0)、棕榈油酸(C16∶1ω7)、花生四烯酸(C20∶4ω6)和二十碳五烯酸(C20∶5ω3,EPA)。其中棕榈油酸含量最高,占总脂肪酸含量的36.53%~50.08%。研究结果表明囊状黄丝藻在不同初始氮浓度条件下具有特殊的油脂积累规律,是一株具有重要应用价值的产油丝状微藻。     

氮源种类及浓度对眼点拟微绿球藻生长密度、油脂产率和二十碳五烯酸含量的影响

氮源是影响微藻生长和油脂积累的重要因素,文中通过单因素试验比较了NaNO3、CO(NH2)2、NH4Cl、CH3COONH4及其浓度对眼点拟微绿球藻生长密度、生长速率、油脂产率、二十碳五烯酸(EPA)含量的影响。结果表明：NH4+更易被眼点拟微绿球藻利用,能更好地促进微藻生长和油脂积累;氮浓度的增加有利于微藻的生长和藻油脂肪酸的去饱和,但不利于微藻油脂的积累。在实验考察的氮源种类和浓度范围内,CH3COONH4是促进眼点拟微绿球藻生长和油脂积累、EPA生成的适宜氮源,其适宜的浓度为5.29 mmol/L。     

Effects of nitrogen supplementation of the culture medium on the growth, total lipid content and fatty acid profiles of three microalgae (Tetraselmis subcordiformis, Nannochloropsis oculata and Pavlova viridis)

To improve the properties of microalgae as sources for biodiesel production, three microalgae (Tetraselmis subcordiformis SHOU-S05, Nannochloropsis oculata SHOU-S14 and Pavlova viridis SHOU-S16) were cultured in media supplemented with different amounts of nitrogen (0, 0.22, 0.44, 0.88 and 1.76 mmol N·L^?1). The growth, total lipid contents, lipid classes and fatty acid profiles of the three microalgae were assayed after 10 days of cultivation. The results indicated that the specific growth rates of T. subcordiformis, N. oculata and P. viridis were lowest (0.014, 0.033 and 0.018, respectively) in the treatments without nitrogen supplementation and increased significantly with increasing nitrogen supplementation. The microalgae treated with 0.22 mmol N·L^?1 had the highest total lipid contents, which were 29.77, 35.85 and 32.10 % in T. subcordiformis, N. oculata and P. viridis, respectively. The total lipid contents as well as the proportions of neutral lipid in the three microalgae decreased significantly with increasing nitrogen supplementation between 0.22 and 1.76 mmol N·L^?1. The fatty acid profiles of the three microalgae were significantly different. The obvious characteristic of the fatty acid profile of T. subcordiformis was a high amount (17.68–22.22 %) of 18:3n3. However, EPA (C20:5n3) and C16 fatty acids were significantly high in N. oculata and P. viridis, respectively. N. oculata and P. viridis accumulated more 16-carbon fatty acids and fewer polyunsaturated fatty acids in nitrogen-limited media. It is therefore suggested that a limited nitrogen treatment is effective for improving the properties of P. viridis and N. oculata as sources for biodiesel.     

Comparative lipid profiling of two endophytic fungal isolates--Colletotrichum sp. and Alternaria sp. having potential utilities as biodiesel feedstock

Lipid accumulation abilities of two endophytic fungal isolates - Colletotrichum sp. and Alternaria sp. grown under optimum and nutrient-stress conditions were investigated and compared. Significant variations in lipid contents, ranging from 30% to 58% of their dry biomass were found in liquid culture using various carbon sources. Since, >50% of the total lipid was estimated to be neutral lipid for both the fungal species, predicted biodiesel properties were theoretically calculated based upon the determined fatty acid profiles; and the values were found to be comparable to those of commonly used plant oils for biodiesel production. The two endophytes grew successfully on the combined rice straw and wheat bran as substrate that was degraded by their secretory enzymes including cellulase [1.21-2.51 FPU/g dry substrate (gds)] in solid state fermentation and produced substantial amount of lipid (60.32-84.30 mg/gds). Our study highlights the potential utilities of these two novel endophytic fungi as biodiesel feedstock.     

Biosorption of chromium (Cr(III)/Cr(VI)) on the residual microalga Nannochloris oculata after lipid extraction for biodiesel production

In order to increase the economic feasibility of biodiesel production from microalgae, the residual biomass after biodiesel production can be utilized as biosorbent for heavy metal removal. In this study, biosorption of chromium by residual Nannochloris oculata after lipid extraction was investigated. Increased surface area of N. oculata was observed after lipid extraction. Cr(III) removal increased as the pH increased from 2 to 6, while Cr(VI) removal was highest at pH 2 and it decreased with the increase in pH. Cr(VI) was reduced to Cr(III) in the presence of biomass under acidic conditions; X-ray photoelectron spectroscopy revealed that the converted Cr(III) was bound to the biomass. Chromium removal was significantly enhanced at high chromium concentrations, which indicates that surface reactions may occur at high chromium/biomass ratios. FTIR study indicated that phosphate and carboxyl functional groups of the biomass were mainly responsible for chromium binding.     

Biochemical and kinetic evaluation of lipase and biosurfactant assisted ex novo synthesis of microbial oil for biodiesel production by Yarrowia lipolytica utilizing chicken tallow

The present study explores the production of biodiesel, a sustainable replacement for depleting fossil fuel by utilizing microbial oil, which was procured from Yarrowia lipolytica employing chicken tallow as the carbon substrate. Chicken tallow, yeast extract, and MgSO₄·7H₂O were screened for biomass production through Plackett–Burman design. Further, Box–Behnken design analysis was performed, and the optimal concentration of the medium variables was found to be 20 g/L of chicken tallow, 7.0 g/L of yeast extract, and 0.45 g/L of MgSO₄·7H₂O.The various parameters viz., pH (6), temperature (30 °C), RPM (150), inoculum volume (5%, v/v), and C/N ratio (100) were optimized for maximal biomass and lipid yield, and lipid content. Nile red-stained cells were observed for intracellular lipid bodies using fluorescence microscopy, and its fluorescence intensity was measured bythe flow cytometer. The dimorphic transition and substrate assimilation of Y. lipolytica were analyzed using scanning electron microscopy (SEM), and Fourier transform infrared spectroscopy (FT-IR). Batch kinetic studies revealed the concomitant synthesis of microbial lipid (4.16 g/L), lipase (43 U/mL), and biosurfactant (1.41 g/L). The GC-MS analysis of microbial oil presented the fatty acid profile as oleic acid (49.15%), palmitic acid (29.83%), stearic acid (11.43%), linoleic acid (3.83%), palmitoleic acid (3.77%), and myristic acid (1.32%).     

Candida zeylanoides as a new yeast model for lipid metabolism studies: effect of nitrogen sources on fatty acid accumulation

Lipid homeostasis is well-known in oleaginous yeasts, but there are few non-oleaginous yeast models apart from Saccharomyces cerevisiae. We are proposing the non-oleaginous yeast Candida zeylanoides QU 33 as model. The aim of this study was to investigate the influence of the carbon/nitrogen ratio and the type of nitrogen source upon oil accumulation by this yeast grown on shake flask cultures. The maximum biomass was obtained in yeast extract (2.39?±?0.19 g/l), followed by peptone (2.24?±?0.05 g/l), while the highest content of microbial oil (0.35?±?0.01 g/l) and the maximum lipid yield (15.63 %) were achieved with peptone. Oleic acid was the predominant cellular fatty acid in all culture media (>32.23 %), followed by linoleic (>15.79 %) and palmitic acids (>13.47 %). The highest lipid yield using glucose and peptone was obtained at the C/N ratio of 200:1.     

Biodiesel production from isolated oleaginous fungi Aspergillus sp. using corncob waste liquor as a substrate

The study documented the potential of isolated filamentous fungus Aspergillus sp. as whole cell biocatalyst for biodiesel production using Sabourauds dextrose broth medium (SDBM) and corncob waste liquor (CWL) as substrates. SDBM showed improvement in both biomass production (13.6 g dry weight/1000 ml) and lipid productivity (23.3%) with time. Lipid extraction was performed by direct (DTE) and indirect (IDTE) transesterification methods. DTE showed higher transesterification efficiency with broad spectrum of fatty acids profile over IDTE. CWL as substrate showed good lipid productivity (22.1%; 2g dry biomass; 48 h) along with efficient substrate degradation. Lipids derived from both substrates depicted high fraction of saturated fatty acids than unsaturated ones. Physical characteristics of fungal based biodiesel correlated well with prescribed standards. CWL derived biodiesel showed relatively good fuel properties (acid number, 0.40 mg KOH/g of acid; iodine value, 11 g I?/100 g oil; density, 0.8342 g/cm3) than SDBM derived biodiesel.     

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.     

<Emphasis Type="Italic">Neochloris oleabundans</Emphasis> UTEX #1185: a suitable renewable lipid source for biofuel production

Energy crises, global warming, and climatic changes call for technological and commercial advances in manufacturing high-quality transportation fuels from unconventional feedstocks. Microalgae is one of the most promising sources of biofuels due to the high yields attained per unit area and because it does not displace food crops. Neochloris oleabundans (Neo) microalga is an important promising microbial source of single-cell oil (SCO). Different experimental growth and lipid production conditions were evaluated and compared by using optical density (540 nm), dry-weight determination, and flow cytometry (FC). Best Neo average biomass productivity was obtained at 30°C under conditions of nitrogen-sufficiency and CO₂ supplementation (N+/30°C/CO₂), with an average doubling time of 1.4 days. The second and third highest productivities occurred with N-sufficient cultures without CO₂ supplementation at 26°C (N+/26°C) and at 30°C (N+/30°C), with doubling times of 1.7 and 2.2 days, respectively. Microbial lipid production was monitored by flow cytometry using Nile red (NR), a lipophilic fluorochrome that possesses several advantageous characteristics for in situ screening near real time (at line). Results showed maximum lipid content (56%) after 6 days of nitrogen depletion under nitrogen starvation without CO₂ supplementation (N−/30°C), followed by N−/30°C/CO₂ and N−/26°C conditions with 52% lipid content, after 5 and 6 days of N starvation, respectively. The adequate fatty acid profile and iodine value of Neo lipids reinforced this microalga as a good source of SCO, in particular for use as biodiesel.     

Single cell oil production from a newly isolated <Emphasis Type="Italic">Candida viswanathii</Emphasis> Y-E4 and agro-industrial by-products valorization

Microbial lipids have drawn increasing attention in recent years as promising raw materials for biodiesel and added-value compounds production. To this end, new oleaginous yeast, Candida viswanathii Y-E4 was isolated, characterized and used for single cell oil (SCO) production. Physiologic and nutritional parameters optimization was carried out for improved biomass and lipid production. Y-E4 strain was able to use a wide range of substrates, especially C5 and C6 sugars as well as glycerol and hydrophobic substrates. The fatty acid profile analysis showed that oleic acid was the main component produced using different substrates. Batch and fed-bath fermentation were conducted using glucose as carbon source. Lipid production rate is twice higher in fed-batch culture providing a lipid content of 50 % (w/w). To minimize the SCO production cost, C. viswanathii Y-E4 was evaluated for its capacity to use different agro-industrial by-products for microbial oil production and changes in the fatty acid profile were monitored.     

Cryptococcus curvatus O3酵母菌培养及产油脂特性

生物柴油的发展, 导致全球油脂供求紧张。微生物油脂的甘三酯组成与植物油类似, 发展微生物油脂可部分缓解植物油脂供应压力。本文研究了Cryptococcus curvatus O3酵母利用葡萄糖为碳源生长和积累油脂的特性。Cryptococcus curvatus O3酵母在培养过程中能适应间歇式碳源流加方式达到高密度培养的目的, 但在相同培养条件下, 不同氮源能影响其代谢过程中糖到油脂转化的脂肪系数。Cryptococcus curvatus O3酵母利用葡萄糖作为碳源在30°C下摇瓶发酵, 菌体生物量为51.8 g/L, 油脂含量达65.1%。脂肪酸组成分析结果表明, 菌油富含饱和及低度不饱和长链脂肪酸, 其中饱和脂肪酸之和占总脂肪酸组成的64%左右, 其脂肪酸组成类似于可可脂, 这些结果对于利用产油微生物转化生物质获取如类可可脂等具有高附加值油脂的研究具有重要意义。     

Effects of some inhibitors on the growth and lipid accumulation of oleaginous yeast Rhodosporidium toruloides and preparation of biodiesel by enzymatic transesterification of the lipid

Microbial lipid produced using yeast fermentation with inexpensive carbon sources such as lignocellulosic hydrolyzate can be an alternative feedstock for biodiesel production. Several inhibitors that can be generated during acid hydrolysis of lignocellulose were added solely or together into the culture medium to study their individual inhibitory actions and their synergistic effects on the growth and lipid accumulation of oleaginous yeast Rhodosporidium toruloides. When the inhibitors were present in isolation in the medium, to obtain a high cell biomass accumulation, the concentrations of formic acid, acetic acid, furfural and vanillin should be lower than 2, 5, 0.5 and 1.5 g/L, respectively. However, the synergistic effects of these compounds could dramatically decrease the minimum critical inhibitory concentrations leading to significant growth and lipid production inhibitions. Unlike the above-cited inhibitors, sodium lignosulphonate had no negative influence on biomass accumulation when its concentration was in the range of 0.5-2.0 g/L; in effect, it was found to facilitate cell growth and sugar-to-lipid conversion. The fatty acid compositional profile of the yeast lipid was in the compositional range of various plant oils and animal tallow. Finally, the crude yeast lipid from bagasse hydrolyzate could be well converted into fatty acid methyl ester (FAME, biodiesel) by enzymatic transesterification in a tert-butanol system with biodiesel yield of 67.2% and lipid-to-biodiesel conversion of 88.4%.     

Effect of pH on neutral lipid and biomass accumulation in microalgal strains native to the Canadian prairies and the Athabasca oil sands

Algal biodiesel has been a subject of growing importance in the realm of renewable energy due to carbon capture properties and its potential for photosynthetic efficiency with high lipid output. This study identified five isolates of freshwater green algae, belonging to the Chlorellaceae, and measured the lipid classes and fatty acid profiles of these species to determine suitability for biodiesel production. To induce the greater accumulation of lipids, especially in the form of triacylglycerols (TAGs) desired for biodiesel, we examined the lipid accumulation in cells stressed by nitrogen limitation, sulfur deficiency, or pH stress. Increases in biomass were monitored in order to determine if adjusting pH incrementally over the course of the experiment had any effect on growth and lipid accumulation of several isolates. TAG accumulation was visually screened by Nile Red fluorescence and further assessed by gas chromatography. Lipid amounts were comparably equal or better for pH stress treatments than for standard nutrient-deprivation treatments. Incrementally adjusted pH over the course of growth triggered lipid accumulation comparable to constant pH stress treatments, yet biomass accumulation was equivalent to unstressed growth. One isolate obtained from the Athabasca oil-sands region of Alberta, OS4-2, is a good candidate for biodiesel production, having accumulated over 45 % of its dry weight as lipid, with over 80 % of the lipid as triacylglycerols, and contains an abundance of 18:1 fatty acids. This class of fatty acids improves the cold flow and oxidative stability of biodiesel and is ideal for biofuel used in a Canadian climate.     

Dynamic flux balance analysis of biomass and lipid production by Antarctic thraustochytrid Oblongichytrium sp. RT2316-13

Production of biomass and lipids in batch cultures of the Antarctic thraustochytrid Oblongichytrium sp. RT2316-13, is reported. The microorganism proved capable of producing nearly 67% docosahexaenoic acid (DHA) and 15% eicosapentaenoic acid (EPA) in its total lipid fraction. Biomass with a maximum total lipid content of 33.5% (wt/wt) could be produced at 15°C in batch culture using a medium containing glucose (20 g/L), yeast extract (10.5 g/L), and other minor components. A lower culture temperature (5°C) reduced biomass and lipid productivities compared to culture at 15°C, but enhanced the DHA and EPA content of the lipids by 6.4- and 3.3-fold, respectively. Both a simple minimally structured mathematical model and a more complex genome-scale metabolic model (GEM) allowed the fermentation profiles in batch cultures to be satisfactorily simulated, but the GEM provided much greater insight in the biochemical and physiological phenomena underlying the observed behavior. Unlike the simpler model, the GEM could be interrogated for the possible effects of various external factors such as oxygen supply, on the expected outcomes. In silico predictions of oxygen effects were consistent with literature observations for DHA producing thraustochytrids.     

皮状丝孢酵母B3利用木薯淀粉发酵生产微生物油脂

对皮状丝孢酵母B3以木薯淀粉水解液为碳源发酵生产微生物油脂培养条件进行了优化,并在2 L发酵罐中对菌体生长和油脂积累进行了考察。摇瓶实验表明,木薯淀粉水解液的浓度高于90 g/L时不利于菌体的生长和油脂积累,皮状丝孢酵母B3发酵生产微生物油脂的最适氮源及其浓度、最适C/N比和pH分别为酵母提取物3.0 g/L、116、6.0,在此条件下培养144 h菌体生物量、油脂产量和油脂含量分别达到15.2 g/L、6.22 g/L和40.9％;在2 L发酵罐中分批发酵44 h后菌体生物量、油脂产量和油脂含量分别达28.7 g/L、12.27 g/L和42.8％。以皮状丝孢酵母B3所产油脂制备生物柴油,其主要组成包括棕榈酸甲酯、硬脂酸甲酯、油酸甲酯、亚油酸甲酯等,且理化特性符合相关国家标准,可作为一种有潜力的化石燃料替代品。