Differential effects of nitrogen and sulfur deprivation on growth and biodiesel feedstock production of Chlamydomonas reinhardtii

Biodiesel production from microalgae is a promising approach for energy production; however, high cost of its process limits the use of microalgal biodiesel. Increasing the levels of triacylglycerol (TAG) levels, which is used as a biodiesel feedstock, in microalgae has been achieved mainly by nitrogen starvation. In this study, we compared effects of sulfur (S) and nitrogen (N) starvation on TAG accumulation and related parameters in wild-type Chlamydomonas reinhardtii CC-124 mt(-) and CC-125 mt(+) strains. Cell division was interrupted, protein and chlorophyll levels rapidly declined while cell volume, total neutral lipid, carotenoid, and carbohydrate content increased in response to nutrient starvation. Cytosolic lipid droplets in microalgae under nutrient starvation were monitored by three-dimensional confocal laser imaging of live cells. Infrared spectroscopy results showed that relative TAG, oligosaccharide and polysaccharide levels increased rapidly in response to nutrient starvation, especially S starvation. Both strains exhibited similar levels of regulation responses under mineral deficiency, however, the degree of their responses were significantly different, which emphasizes the importance of mating type on the physiological response of algae. Neutral lipid, TAG, and carbohydrate levels reached their peak values following 4 days of N or S starvation. Therefore, 4 days of N or S starvation provides an excellent way of increasing TAG content. Although increase in these parameters was followed by a subsequent decline in N-starved strains after 4 days, this decline was not observed in S-starved ones, which shows that S starvation is a better way of increasing TAG production of C. reinhardtii than N starvation.     

High temperature enhances lipid accumulation in nitrogen-deprived <Emphasis Type="Italic">Scenedesmus obtusus</Emphasis> XJ-15

This study investigated the changes in lipid and starch contents, lipid fraction, and lipid profile in the nitrogen-starved Scenedesmus obtusus XJ-15 at different temperatures (17, 25, and 33 °C). The optimal temperature for both growth and lipid accumulation under nitrogen-sufficient condition was found to be 25 °C. However, under nitrogen deprivation, the total and neutral lipids increased with increasing temperature, and achieved the highest lipid content of 47.60 % of dry cell weight and the highest TAG content of 79.66 % of total lipid at 33 °C. In the meantime, the stored cellular starch content decreased with the increasing temperature. Thus, high temperature induced carbon flux from starch toward TAG accumulation in microalgae during nitrogen starvation. In addition, the decreased polar lipids may also serve for TAG synthesis under high temperature, and high temperature further reduced the degree of the fatty acid unsaturation and favored a better biodiesel production. These results suggested that high-temperature stress can be a good strategy for enhancing biofuel production in oleaginous microalgae during nitrogen deficiency.     

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.     

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.     

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.     

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。     

Growth and neutral lipid synthesis in green microalgae: A mathematical model

Many green microalgae significantly increased their cellular neutral lipid content when cultured in nitrogen limited or high light conditions. Due to their lipid production potential, these algae have been suggested as promising feedstocks for biofuel production. However, no models for algal lipid synthesis with respect to nutrient and light have been developed to predict lipid production and to help improve the production process. A mathematical model is derived describing the growth dynamics and neutral lipid production of green microalgae grown in batch cultures. The model assumed that as the nitrogen was depleted, photosynthesis became uncoupled from growth, resulting in the synthesis and accumulation of neutral lipids. Simulation results were compared with experimental data for the green microalgae Pseudochlorococcum sp. For growth media with low nitrogen concentration, the model agreed closely with the data; however, with high nitrogen concentration the model overestimated the biomass. It is likely that additional limiting factors besides nitrogen could be responsible for this discrepancy.     

An alternative strategy for enhancing lipid accumulation in chlorophycean microalgae for biodiesel production

Oleaginous microalgae are considered as important feedstocks for production of biodiesel. Under nutrient stress conditions, microalgae have the ability to accumulate higher amount of lipids, which can be transesterified for the production of biodiesel. In the present investigation, four different phosphate application strategies were examined in five green microalgae (Tetradesmus obliquus, Tetradesmus lagerheimii, Chlorella vulgaris, Chlorella minutissima, and Chlamydomonas sp.) to achieve higher lipid productivity. Effects of those strategies such as phosphate-sufficient (Control), phosphate-starved approach (PSA), biphasic phosphate-starved approach (BPSA), and sequential phosphate addition (SPA) were studied under batch culture mode. The BPSA emerging as the best in terms of lipid productivity consisted of two biomass harvesting phases, which would lead to an increase in the overall cost of biodiesel production. On the other hand, the SPA with a 1/200th dose of N 11 medium, i.e., 0.4 mg L^?1 of phosphate application in 3-day intervals, also resulted into higher lipid productivity which was equal to BPSA. Fatty acid composition of the biodiesel obtained from the microalgae was analyzed and the fuel characteristics were also evaluated. A profound (~14-fold) reduction in phosphorus requirements under the SPA mode with higher lipid productivity ensured qualitative biodiesel production and a lesser amount of phosphorus release, thus making the process eco-friendly.     

Influence of nitrogen stress on Isochrysis galbana strain U4, a candidate for biodiesel production

Lipid accumulation has been investigated in numerous microalgal species to assess their potential with respect to biodiesel production. The present work determines the effect of nitrogen stress on physiological and ultrastructural changes in Isochrysis galbana U4. This study is unique in showing the correlations between growth, lipid production, pigmentation and ultrastructural changes in Isochrysis cells undergoing nitrogen starvation. The continuation of algal growth after the complete depletion of external nitrogen was shown to be supported by internal nitrogen stores, possibly in the pyrenoid. Cell growth ceased and lipid accumulation was initiated after the internal store of nitrogen had become exhausted. The depletion of intracellular nitrogen reservoirs to critical thresholds initiated the onset of the stationary phase, a decline in chlorophyll content and the initiation of lipid and carotenoid accumulation. The most notable ultrastructural changes, upon nitrogen stress, were the accumulation of plastidial and cytoplasmic lipid bodies and the dismantling of the chloroplast. The size of the pyrenoid when external nitrogen became depleted was found to decrease significantly, up to four‐fold. This was attributed to the remobilization of nitrogen from Rubisco. The level of expression of heterochromatin was found to increase when cells were nitrogen starved. This is thought to favor long‐term dormancy in this species because aging cells have been noted to recover rapidly when returned to conditions favorable for growth. The observations of this study are consistent with the hypothesis that the responses of Isochrysis cells to nitrogen starvation are regulated by the internal reserves of nitrogen, and the depletion of these reserves is an important trigger for lipid accumulation in this species. The findings of this study also indicate that Isochrysis galbana U4 is a promising candidate for biodiesel lipid production.     

Comparative lipidomic studies of Scenedesmus sp. (Chlorophyceae) and Cylindrotheca closterium (Bacillariophyceae) reveal their differences in lipid production under nitrogen starvation

Microalgae are a promising resource for the highly sustainable production of various biomaterials (food and feed), high‐value biochemicals, or biofuels. However, factors influencing the valued lipid production from oleaginous algae require a more detailed investigation. This study elucidates the variations in lipid metabolites between a marine diatom (Cylindrotheca closterium) and a freshwater green alga (Scenedesmus sp.) under nitrogen starvation at the molecular species level, with emphasis on triacylglycerols using liquid chromatography–electrospray ionization mass spectrometry techniques. A comprehensive analysis was carried out by comparing the changes in total lipids, growth kinetics, fatty acid compositions, and glycerolipid profiles at the molecular species level at different time points of nitrogen starvation. A total of 60 and 72 triacylglycerol molecular species, along with numerous other polar lipids, were identified in Scenedesmus sp. and C. closterium, respectively, providing the most abundant triacylglycerol profiles for these two species. During nitrogen starvation, more triacylglycerol of Scenedesmus sp. was synthesized via the “eukaryotic pathway” in the endoplasmic reticulum, whereas the increase in triacylglycerol in C. closterium was mainly a result of the “prokaryotic pathway” in the chloroplasts after 96 h of nitrogen starvation. The distinct responses of lipid synthesis to nitrogen starvation exhibited by the two species indicate different strategies of lipid accumulation, notably triacylglycerols, in green algae and diatoms. Scenedesmus sp. and Cylindrotheca closterium could serve as excellent candidates for the mass production of biofuels or polyunsaturated fatty acids for nutraceutical purposes.     

Effect of nitrogen, salt, and iron content in the growth medium and light intensity on lipid production by microalgae isolated from freshwater sources in Thailand

Four green microalgae (TRG, KB, SK, and PSU) identified as Botryococcus spp. by morphological criteria were isolated from lakes and freshwater ponds in southern Thailand. In nitrogen-rich medium the strains achieved a lipid content of 25.8%, 17.8%, 15.8% and 5.7%, respectively. A combination of nitrogen deficiency, moderately high light intensity (82.5 μE m(-2) s(-1)) and high level of iron (0.74 mM) improved lipid accumulation in TRG, KB, SK, and PSU strains up to 35.9%, 30.2%, 28.4% and 14.7%, respectively. The lipid contents and plant oil-like fatty acid composition of the microalgae suggested their potential as biodiesel feedstock.     

Metabolomics integrated with transcriptomics and proteomics: Evaluation of systems reaction to nitrogen deficiency stress in microalgae

Microalgae have higher productivity of biomass than the conventional crops of fuel and are therefore, considered a potential biofuel source. Lipid, an important precursor of biodiesel, can be overproduced in microalgae by nitrogen deprivation. During nitrogen deficiency, radicals are overproduced, and the antioxidant levels are insufficient to counteract the radicals. Thus, the increase in cellular oxidative stress level, consequently acts as a stimulus for lipid accumulation. Lipid accumulation requires an excess of acetyl CoA and NADPH that is made possible by the following mechanism. Glycolysis upregulation overproduces pyruvate, which could be further transformed into acetyl CoA by the pyruvate dehydrogenase complex; while the upregulation of the oxidative pentose phosphate cycle generates a high amount of NADPH. In addition to lipid overproduction, the lack of nitrogen often causes the accumulation of carbohydrates in selected species of microalgae, which could be used to generate biogas and bioethanol from the defatted biomass. By providing details on the differential regulation of the biochemical pathways leading to lipid and carbohydrate accumulation in nitrogen starved microalgae, the review opens up new possibilities in the microalgal biofuel production.     

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.     

Microalgae for oil: strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor

Thirty microalgal strains were screened in the laboratory for their biomass productivity and lipid content. Four strains (two marine and two freshwater), selected because robust, highly productive and with a relatively high lipid content, were cultivated under nitrogen deprivation in 0.6-L bubbled tubes. Only the two marine microalgae accumulated lipid under such conditions. One of them, the eustigmatophyte Nannochloropsis sp. F&M-M24, which attained 60% lipid content after nitrogen starvation, was grown in a 20-L Flat Alveolar Panel photobioreactor to study the influence of irradiance and nutrient (nitrogen or phosphorus) deprivation on fatty acid accumulation. Fatty acid content increased with high irradiances (up to 32.5% of dry biomass) and following both nitrogen and phosphorus deprivation (up to about 50%). To evaluate its lipid production potential under natural sunlight, the strain was grown outdoors in 110-L Green Wall Panel photobioreactors under nutrient sufficient and deficient conditions. Lipid productivity increased from 117 mg/L/day in nutrient sufficient media (with an average biomass productivity of 0.36 g/L/day and 32% lipid content) to 204 mg/L/day (with an average biomass productivity of 0.30 g/L/day and more than 60% final lipid content) in nitrogen deprived media. In a two-phase cultivation process (a nutrient sufficient phase to produce the inoculum followed by a nitrogen deprived phase to boost lipid synthesis) the oil production potential could be projected to be more than 90 kg per hectare per day. This is the first report of an increase of both lipid content and areal lipid productivity attained through nutrient deprivation in an outdoor algal culture. The experiments showed that this marine eustigmatophyte has the potential for an annual production of 20 tons of lipid per hectare in the Mediterranean climate and of more than 30 tons of lipid per hectare in sunny tropical areas.     

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.     

Effect of nitrate on lipid production by T. suecica, M. contortum, and C. minutissima

Microalgae are an alternative and sustainable source of lipids that can be used as a feedstock for biodiesel production. Nitrate is a good nitrogen source for many microalgae and affects biomass and lipid yields of microalgae. In this study, the effect of nitrate on cell growth and lipid production and composition in Monoraphidium contortum, Tetraselmis suecica, and Chlorella minutissima was investigated. Nitrate affected the production of biomass and the production and composition of lipids of the three microalgae tested. Increasing the nitrate concentration in the culture medium resulted in increased biomass production and higher biomass productivity. Furthermore, increasing the nitrate concentration resulted in a reduction in lipid content and productivity in M. contortum; however, the opposite effect was observed in T. suecica and C. minutissima cultures. C. minutissima and M. contortum lipids contain high levels of oleic acid, with values ranging from 26 to 45.7% and 36.4 to 40.1%, respectively. The data suggest that because of its high lipid productivity (13.79 mg L^?1 d^?1) and high oleic acid productivity (3.78 mg L^?1 d^?1), Chlorella minutissima is a potential candidate for the production of high quality biodiesel.     

二氧化碳减排产柴油微藻培养体系研究进展

污水资源化、二氧化碳减排及微藻生物柴油是当前能源与环境领域的前沿课题。以下围绕污水及烟道气资源化培养产油微藻的培养体系,就藻种、营养条件、培养方式、培养环境及微藻生物反应器等影响产油微藻培养的因素研究进展进行了综述。在综述的基础上提出：由于微藻具有特殊营养方式,通过藻种筛选、微藻营养条件和培养环境的优化以及高效光生物反应器和生产工艺等的创新,可利用污水进行产油微藻生产,以获得生物柴油等高附加值产品,实现微藻生物能源、污水资源化处理和CO2减排三者高度耦合的产油微藻生产体系,从而减少微藻培养费用及污水处理费用,因此,该体系具有重要的环境、社会、经济价值和商业化应用前景。     

Lipid productivity as a key characteristic for choosing algal species for biodiesel production

Microalgae are a promising alternative source of lipid for biodiesel production. One of the most important decisions is the choice of species to use. High lipid productivity is a key desirable characteristic of a species for biodiesel production. This paper reviews information available in the literature on microalgal growth rates, lipid content and lipid productivities for 55 species of microalgae, including 17 Chlorophyta, 11 Bacillariophyta and five Cyanobacteria as well as other taxa. The data available in the literature are far from complete and rigorous comparison across experiments carried out under different conditions is not possible. However, the collated information provides a framework for decision-making and a starting point for further investigation of species selection. Shortcomings in the current dataset are highlighted. The importance of lipid productivity as a selection parameter over lipid content and growth rate individually is demonstrated.     

Bioprocess engineering of microalgae to optimize lipid production through nutrient management

Microalgae have been used commercially as a feedstock for the production of high-value compounds, pigments, cosmetics, and nutritional supplements. In addition, because of their rapid growth rates, high photosynthetic efficiency, and high lipid and protein content, commodity products including biodiesel, feed supplements, and polyunsaturated fatty acids derived from algal biomass are of current interest. Since microalgae lack non-photosynthetic structures and float in water, they do not need massive amounts of structural cellulose found in land plants. Thus, under optimal culture conditions, some oleaginous species can allocate up to 70 % of their biomass to lipids. Lipid production and its regulation in microalgae are species-specific and influenced by environmental conditions. Various strategies have been developed to improve lipid productivity and fatty acid composition to meet specific production goals. Manipulation of physiochemical parameters, trophic modes, and nutrient levels, known as process engineering, is a simple approach that leads to desired alterations in the biochemical composition of algal biomass, including lipid quantity and quality. In this paper, we review the effects of manipulating biochemical parameters such as necessary nutrients (C, N, P, S, Fe, and Si), NaCl concentration, and pH of culture medium to optimize lipid content and profile in some algae strains with commercial potential.     

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.