Bioprospecting for hyper-lipid producing microalgal strains for sustainable biofuel production

Global petroleum reserves are shrinking at a fast pace, increasing the demand for alternate fuels. Microalgae have the ability to grow rapidly, and synthesize and accumulate large amounts (approximately 20-50% of dry weight) of neutral lipid stored in cytosolic lipid bodies. A successful and economically viable algae based biofuel industry mainly depends on the selection of appropriate algal strains. The main focus of bioprospecting for microalgae is to identify unique high lipid producing microalgae from different habitats. Indigenous species of microalgae with high lipid yields are especially valuable in the biofuel industry. Isolation, purification and identification of natural microalgal assemblages using conventional techniques is generally time consuming. However, the recent use of micromanipulation as a rapid isolating tool allows for a higher screening throughput. The appropriate media and growth conditions are also important for successful microalgal proliferation. Environmental parameters recorded at the sampling site are necessary to optimize in vitro growth. Identification of species generally requires a combination of morphological and genetic characterization. The selected microalgal strains are grown in upscale systems such as raceway ponds or photobireactors for biomass and lipid production. This paper reviews the recent methodologies adopted for site selection, sampling, strain selection and identification, optimization of cultural conditions for superior lipid yield for biofuel production. Energy generation routes of microalgal lipids and biomass are discussed in detail.     

光生物反应器培养微藻研究进展

微藻具有固定CO2和净化有机废水的能力,在环保、食品饲（饵）料、医药和生物能源开发等领域备受关注,但规模化培养及其产业化仍是研究的难点,亟待解决。就常用于大规模培养微藻的光生物反应器的特点和结构进行了综述。其中,封闭式微藻光生物反应器能够较好地调控藻种的培养条件、不易遭受污染,藻种的纯度容易控制,但培养规模小,生产成本较高;而开放式微藻光生物反应器无法精确控制藻种生长环境,但生产规模大、产量高、生产成本低,因此应用广泛。最佳的方法是综合两者优点,即首先利用封闭式微藻光生物反应器进行中试放大,大量繁殖藻种,然后投入开放式微藻光生物反应器内进行大规模商业生产,此方法有望成为微藻光生物反应器的发展方向,以期为微藻大规模培养提供参考借鉴。     

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.     

Biofuel production from microalgae as feedstock: current status and potential

Algal biofuel has become an attractive alternative of petroleum-based fuels in the past decade. Microalgae have been proposed as a feedstock to produce biodiesel, since they are capable of mitigating CO₂ emission and accumulating lipids with high productivity. This article is an overview of the updated status of biofuels, especially biodiesel production from microalgae including fundamental research, culture selection and engineering process development; it summarizes research on mathematical and life cycle modeling on algae growth and biomass production; and it updates global efforts of research and development and commercialization attempts. The major challenges are also discussed.     

Life-cycle assessment of microalgae culture coupled to biogas production

Due to resource depletion and climate change, lipid-based algal biofuel has been pointed out as an interesting alternative because of the high productivity of algae per hectare and per year and its ability to recycle CO₂ from flue gas. Another option for taking advantage of the energy content of the microalgae is to directly carry out anaerobic digestion of raw algae in order to produce methane and recycle nutrients (N, P and K). In this study, a life-cycle assessment (LCA) of biogas production from the microalgae Chlorella vulgaris is performed and the results are compared to algal biodiesel and to first generation biodiesels. These results suggest that the impacts generated by the production of methane from microalgae are strongly correlated with the electric consumption. Progresses can be achieved by decreasing the mixing costs and circulation between different production steps, or by improving the efficiency of the anaerobic process under controlled conditions. This new bioenergy generating process strongly competes with others biofuel productions.     

Improvement of heat and desiccation tolerance in <Emphasis Type="Italic">Heterorhabditis bacteriophora</Emphasis> through cross-breeding of tolerant strains and successive genetic selection

Genetic selection can be a powerful tool to increase beneficial traits in biological control agents. In this study the heat and desiccation tolerance of the entomopathogenic nematode Heterorhabditis bacteriophora Poinar (Rhabditidomorpha: Strongyloidea) were significantly increased by cross breeding tolerant parental strains and successive genetic selection. These strains originated from a prior screening among 60 strains for increased stress tolerance. During genetic selection, the selection pressure was constantly increased and only the most tolerant 10% of the nematode populations were propagated for further selection steps. Assessment of tolerance and selection for both traits was performed with and without prior adaptation to the stress conditions. Eleven selection steps were performed to increase heat tolerance. A final overall increase in mean heat tolerance of 5.5°C was achieved when nematodes had been adapted to heat stress. For non-adapted tolerance an increase of 3.0°C from 40.1°C to 43.1°C was recorded. For comparison, a commercial strain had a mean tolerated temperature after adaptation of 38.2°C and of 36.5°C without adaptation. For assessment of the desiccation tolerance the mean tolerated water activity (a_w-value) of a population was measured. Cross-breeding most tolerant strains reduced the a_w-value from 0.67 to 0.65 after adaptation and from 0.9 to 0.7 without prior adaptation. The following six selection steps could not increase the tolerance whether nematodes had been adapted to stress or not. In comparison, the commercial strain tolerated a mean a_w-value of 0.985 after adaptation and 0.951 without adaptation. Further investigation will have to assess trait stability and possible trade-off effects. This study is a first important step on the road towards domestication of the entomopathogenic nematode H. bacteriophora.     

Characterization of potato genotypes by chlorophyll fluorescence during plant aging in a Mediterranean environment

Field experiments were conducted in Sicily (south Italy) during two seasons to characterize by chlorophyll (Chl) fluorescence four genotypes (Spunta, Sieglinde, Daytona, and Ninfa) of potato (Solanum tuberosum L.) for off-season production during plant aging and to analyse the possible relation between Chl parameters and tuber yield. Chl fluorescence parameters [initial fluorescence (F₀), maximum fluorescence (F_m), F_v/F_m, time in which maximal fluorescence occurs (T_max)] gained from Kautsky kinetics and Chl content were measured weekly, from 5^th to 6^th leaf appearance to beginning of plant senescence in the first season and to full plant senescence in the second season. F₀ and F_v/F_m were the most reliable Chl fluorescence parameters for the definition of genotypic differences while Chl content and T_max were the most reliable Chl parameters to predict plant aging. Tuber yield was highly correlated with Chl content, T_max, F₀, and F_m.     

Advances in culture and genetic modification approaches to lipid biosynthesis for biofuel production and in silico analysis of enzymatic dominions in proteins related to lipid biosynthesis in algae

Biofuels have been shown to be a promising and highly attractive alternative for minimizing the use of fossil fuels, and microalgae have positioned themselves as potential candidates for production of lipids and other substances of commercial interest. We briefly review recent advances made in microalgae culture conditions and genetic manipulation for improving lipid yields for biofuel production – with both approaches showing similar yields of triacylglycerides, indicating that more work is required for improving lipid yield and accumulation in algae. Aiming at gaining knowledge of algae genetic manipulation and exploring future use of this information for modifying the lipid biosynthesis pathway, we investigated whether some characteristics of enzymes involved in lipid biosynthesis could relate to lipid yield and accumulation in algae. We made an in silico analysis of amino acid sequence of enzymatic domains and modeled tertiary structure of three proteins involved in the biosynthesis of lipids in microalgae: acetyl‐CoA carboxylase, Acyl‐CoA: diacylglycerol acyltransferase, and glycerol‐3‐phosphate acyltransferase. Our results suggest that, based on primary amino acid sequences and tertiary structure of proteins shared by certain algae, it is likely that these proteins may relate to lipid yield and accumulation, which makes bioinformatics a powerful tool for in silico study of proteins and for selecting genes involved in lipid biosynthesis that could be useful for heterologous transformation in algae with the long term objective of improving their yield, accumulation, and fatty acid composition by genetic engineering.     

Isolation and Characterization of Taiwanese Heterotrophic Microalgae: Screening of Strains for Docosahexaenoic Acid (DHA) Production

Marine heterotrophic microalgal species which are potentially rich in docosahexaenoic acid (DHA, C22:6n−3) have been found in Taiwan; however, there was a lack of detailed analysis and characterization of these indigenous algae which is needed for the development of commercial applications. Hence, the objective of this study was to screen DHA-rich heterotrophic microalgae species indigenous to Taiwan for commercial purposes. Heterotrophic microalgae from a variety of marine habitats were isolated, cultivated, and then identified according to their 18S rRNA gene sequences and morphological characteristics. A comparison was made of their fatty acid profiles, fatty acid content, and amount of biomass. For the strain with highest DHA yield, the optimal growth conditions were determined in order to establish the best fermentation conditions for scale-up. In this study, 25 heterotrophic microalgal strains were successfully isolated from marine habitats around Taiwan. All of the isolated strains showed a close phylogenic relationship with the Thraustochytriaceae family according to their 18S rRNA gene sequences. GC/MS analysis discerned seven distinctive fatty acid profiles of these strains, with the production of eicosapentaenoic acid (C20:5n−3) ranging from 0.02 to 2.61 mg L⁻¹, and DHA ranging from 0.8 to 18.0 mg L⁻¹. An Aurantiochytrium strain BL10 with high DHA production was subsequently chosen for further manipulation. Under optimal growth conditions it could produce up to 59.0 g of dry biomass per liter of culture, with dry biomass containing 73% total fatty acid and 29% DHA, revealing BL10 as an excellent source of microbial DHA.     

Heterotrophic cultivation of microalgae for pigment production: A review

Pigments (mainly carotenoids) are important nutraceuticals known for their potent anti-oxidant activities and have been used extensively as high end health supplements. Microalgae are the most promising sources of natural carotenoids and are devoid of the toxic effects associated with synthetic derivatives. Compared to photoautotrophic cultivation, heterotrophic cultivation of microalgae in well-controlled bioreactors for pigments production has attracted much attention for commercial applications due to overcoming the difficulties associated with the supply of CO₂ and light, as well as avoiding the contamination problems and land requirements in open autotrophic culture systems. In this review, the heterotrophic metabolic potential of microalgae and their uses in pigment production are comprehensively described. Strategies to enhance pigment production under heterotrophic conditions are critically discussed and the challenges faced in heterotrophic pigment production with possible alternative solutions are presented.     

Co-Cultivation of Fungal and Microalgal Cells as an Efficient System for Harvesting Microalgal Cells,Lipid Production and Wastewater Treatment

The challenges which the large scale microalgal industry is facing are associated with the high cost of key operations such as harvesting, nutrient supply and oil extraction. The high-energy input for harvesting makes current commercial microalgal biodiesel production economically unfeasible and can account for up to 50% of the total cost of biofuel production. Co-cultivation of fungal and microalgal cells is getting increasing attention because of high efficiency of bio-flocculation of microalgal cells with no requirement for added chemicals and low energy inputs. Moreover, some fungal and microalgal strains are well known for their exceptional ability to purify wastewater, generating biomass that represents a renewable and sustainable feedstock for biofuel production. We have screened the flocculation efficiency of the filamentous fungus A. fumigatus against 11 microalgae representing freshwater, marine, small (5 µm), large (over 300 µm), heterotrophic, photoautotrophic, motile and non-motile strains. Some of the strains are commercially used for biofuel production. Lipid production and composition were analysed in fungal-algal pellets grown on media containing alternative carbon, nitrogen and phosphorus sources contained in wheat straw and swine wastewater, respectively. Co-cultivation of algae and A. fumigatus cells showed additive and synergistic effects on biomass production, lipid yield and wastewater bioremediation efficiency. Analysis of fungal-algal pellet''s fatty acids composition suggested that it can be tailored and optimised through co-cultivating different algae and fungi without the need for genetic modification.     

Energy costs of carbon dioxide concentrating mechanisms in aquatic organisms

Minimum energy (as photon) costs are predicted for core reactions of photosynthesis, for photorespiratory metabolism in algae lacking CO₂ concentrating mechanisms (CCMs) and for various types of CCMs; in algae, with CCMs; allowance was made for leakage of CO₂ from the internal pool. These predicted values are just compatible with the minimum measured photon costs of photosynthesis in microalgae and macroalgae lacking or expressing CCMs. More energy-expensive photorespiration, for example for organisms using Rubiscos with lower CO₂–O₂ selectivity coefficients, would be less readily accommodated within the lowest measured photon costs of photosynthesis by algae lacking CCMs. The same applies to the cases of CCMs with higher energy costs of active transport of protons or inorganic carbon species, or greater allowance for significant leakage from the accumulated intracellular pool of CO₂. High energetic efficiency can involve a higher concentration of catalyst to achieve a given rate of reaction, adding to the resource costs of growth. There are no obvious mechanistic interpretations of the occurrence of CCMs algae adapted to low light and low temperatures using the rationales adopted for the occurrence of C₄ photosynthesis in terrestrial flowering plants. There is an exception for cyanobacteria with low-selectivity Form IA or IB Rubiscos, and those dinoflagellates with low-selectivity Form II Rubiscos, for which very few natural environments have high enough CO₂:O₂ ratios to allow photosynthesis in the absence of CCMs.     

Fatty acids in green algae cultivated on a pilot-plant scale

Fatty acids fromChlorella vulgaris, Scenedesmus obliquus var.acutus and from a mixed culture of the two strains, Melnik, were converted to methyl esters, separated by gas chromatography, and identified by means of standards. The spectrum of fatty acids included both saturated and unsaturated acids (with odd and even numbers of carbon atoms) from C₁₂ to C₂₂. Fatty acids C_16:0, C_18:0 and C_20:3 were the major components in all cultures. Pure strains differed from the mixed culture in the production of C_18:1, C_12:0 and C_19:2 acids; the first of these was present in higher amounts in pure cultures only, the latter two being found in the mixed culture. The level of lipids was lower as compared to the literature data and their extractability was affected by the manner of preparation of algae and extraction conditions.     

Algal biofuels

The world is facing energy crisis and environmental issues due to the depletion of fossil fuels and increasing CO₂ concentration in the atmosphere. Growing microalgae can contribute to practical solutions for these global problems because they can harvest solar energy and capture CO₂ by converting it into biofuel using photosynthesis. Microalgae are robust organisms capable of rapid growth under a variety of conditions including in open ponds or closed photobioreactors. Their reduced biomass compounds can be used as the feedstock for mass production of a variety of biofuels. As another advantage, their ability to accumulate or secrete biofuels can be controlled by changing their growth conditions or metabolic engineering. This review is aimed to highlight different forms of biofuels produced by microalgae and the approaches taken to improve their biofuel productivity. The costs for industrial-scale production of algal biofuels in open ponds or closed photobioreactors are analyzed. Different strategies for photoproduction of hydrogen by the hydrogenase enzyme of green algae are discussed. Algae are also good sources of biodiesel since some species can make large quantities of lipids as their biomass. The lipid contents for some of the best oil-producing strains of algae in optimized growth conditions are reviewed. The potential of microalgae for producing petroleum related chemicals or ready-make fuels such as bioethanol, triterpenic hydrocarbons, isobutyraldehyde, isobutanol, and isoprene from their biomass are also presented.     

The Biotechnological Potential of Thraustochytrids

Thraustochytrids are common marine microheterotrophs, taxonomically aligned with heterokont algae. Recent studies have shown that some thraustochytrid strains can be cultured to produce high biomass, containing substantial amounts of lipid rich in polyunsaturated fatty acid (PUFA). It is also evident that cell yield and PUFA production by some thraustochytrid strains can be varied by manipulation of physical and chemical parameters of the culture. At present, fish oils and cultured phototrophic microalgae are the main commercial sources of PUFA. The possible decline of commercial fish stocks and the relatively complex technology required to commercially produce microalgae have prompted research into possible alternative sources of PUFA. The culture of thraustochytrids and other PUFA-producing microheterotrophs is seen as one such alternative. Indeed, several thraustochytrid-based products are already on the market, and research into further applications is continuing. Many fish and microalgal oils currently available have relatively complex PUFA profiles, increasing the cost of preparation of high-purity PUFA oils. In contrast, some of the thraustochytrids examined to date have simpler PUFA profiles. If these or other strains can be grown in sufficient quantities and at an appropriate cost, the use of thraustochytrid-derived oils may decrease the high expense currently involved with producing high-purity microbial oils. As more is learned about the health and nutritional benefits of PUFA, demand for PUFA-rich products is expected to increase. Results to date suggest that thraustochytrids could form an important part in the supply of such products. Received February 17, 1999; accepted June 25, 1999     

Microalgal-Bacterial Consortia as Future Prospect in Wastewater Bioremediation,Environmental Management and Bioenergy Production

In the recent years, microalgae have captured researchers’ attention as the alternative feedstock for various bioenergy production such as biodiesel, biohydrogen, and bioethanol. Cultivating microalgae in wastewaters to simultaneously bioremediate the nutrient-rich wastewater and maintain a high biomass yield is a more economical and environmentally friendly approach. The incorporation of algal–bacterial interaction reveals the mutual relationship of microorganisms where algae are primary producers of organic compounds from CO₂, and heterotrophic bacteria are secondary consumers decomposing the organic compounds produced from algae. This review would provide an insight on the challenges and future development of algal–bacterial consortium and its contribution in promoting a sustainable route to greener industry. It is believed that microalgal-bacterial consortia will be implemented in the near-future for sub-sequential treatment of wastewater bioremediation, bioenergy production and CO₂ fixation, promoting sustainability and making extraordinary advancement in life sciences sectors.     

关于石油母质来源的探讨

浮游微藻是海洋和湖泊中的初级生产力,生物量巨大,平均脂类含量高,是沉积岩中有机质的主要母质来源,对石油和天然气的形成贡献最大。对下辽河坳陷早第三纪的大量微体浮游藻类化石研究表明,该坳陷油气资源的形成和微体浮游藻类,特别是沟鞭藻的大量存在密切相关,浮游藻类是该坳陷盆地油气形成有机质来源的重要基础之一。在微藻热模拟成烃实验中引入成岩早期生物、地化因子的影响,使模拟实验更接近自然界的实际情况。对微藻来源的生物标志物研究表明,作为高盐环境重要标志物的2,6,10,14,18—五甲基二十烷也可能来源于藻类生物。在藻类热模拟产物中发现惹烯的存在,提示在解释油气藏的生物母质来源时必须特别谨慎。     

PHOTOPHYSIOLOGICAL EVIDENCE OF NUTRIENT LIMITATION OF PLATELET ICE ALGAE IN MCMURDO SOUND, ANTARCTICA

Seasonally changing photophysiological and biochemical characteristics of sea ice microalgae are interpreted with respect to light availability and measurements of nutrient concentration made at high vertical resolution (12.5 cm) during a dense bloom in the platelet ice layer of McMurdo Sound during a 6-week study in austral spring of 1989. Platelet ice algae remained highly shade adapted throughout the spring as shown by their low photoadaptive index (E_k, 3.7–8.4 μmol photons·m⁻²·s⁻¹), low mean specific absorption coefficient (<0.009 m² mg⁻¹ Chl a), high optical cross-sectional area of photosystem II (σ_PSII, 3.0–8.2), and high molar ratio of fucoxanthin:chlorophyll a (mean = 1.62 ± 0.15 SD). Between 24 October and 8 November, the algae exhibited a photoacclimative response that was marked by a 30% decrease in photosynthetic efficiency (α^B), a 75% decrease in maximum photosynthetic rate (P^B/_m), and a 60% increase in σ_PSII. The photochemical conversion efficiency at photosystem II (F_v/F_m= ca. 0.5) and the quantum yield of photosynthesis (Ø_C= 0.062– 0.078 mol C mol⁻¹ photons) were ca. 80% of their maximal values. After 8 November, changes in algal photophysiology and biochemistry, which were inconsistent with a photoacclimation response, suggest that the platelet ice algae near the platelet/congelation ice interface became increasingly nutrient limited. The number of pennate diatoms increased threefold to 150 × 10⁹ cells m⁻³ between 8 and 14 November, then remained unchanged throughout the remainder of the field season. Following the increase in cell number, F_v/F_m, Ø_C, and C:Chla decreased by >40%, σ_PSII increased by 70%; and the biochemical ratios C:N and C:Si increased 25%–30%. Nutrient depletion was apparent from the high-resolution vertical profiles, but nutrient concentrations limiting algal growth were not observed. However, nutrient concentrations at the likely site of nutrient limitation near the platelet/congelation ice interface were not measured, indicating that higher resolution sampling is necessary to fully characterize this highly variable habitat.     

Phylogeny,morphology, and physiology of Micractinium strains isolated from shallow ephemeral freshwater in Antarctica

Cryotolerant eukaryotic microalgae were isolated from meltwater streams on Ardley Island and King George Island in Antarctica, and their morphological, molecular, and physiological characteristics were investigated. Owing to their simple morphology, distinctive characters were not observed with neither light microscopy nor transmission electron microscopy. However, molecular phylogenetic inferences drawn from the concatenated small subunit rRNA and internal transcribed spacer sequence data indicated that these microalgal strains belonged to the genus Micractinium. All the Micractinium strains showed cryotolerant properties, while their optimum growth temperature was around 20°C. Similar to other cryotolerant organisms, these Antarctic microalgae also contained a higher ratio of polyunsaturated fatty acids to saturated fatty acids. In this study, new Antarctic Micractinium spp. were discovered and added to the culture collection. These cryotolerant strains may serve as a promising source of nutritionally important linoleic (C_18:2 ω6) and α‐linolenic (C_18:3 ω3) acids.     

Quantitative trait loci for grain moisture at harvest and field grain drying rate in maize (Zea mays, L.)

Hybrids with low grain moisture (GM) at harvest are specially required in mid- to short-season environments. One of the most important factors determining this trait is field grain drying rate (FDR). To produce hybrids with low GM at harvest, inbred lines can be obtained through selection for either GM or FDR. Thus, a single-cross population (181 F _2:3-generation plants) of two divergent inbred lines was evaluated to locate QTL affecting GM at harvest and FDR as a starting point for marker assisted selection (MAS). Moisture measurements were made with a hand-held moisture meter. Detection of QTL was facilitated with interval mapping in one and two dimensions including an interaction term, and a genetic linkage map of 122 SSR loci covering 1,557.8 cM. The markers were arranged in ten linkage groups. QTL mapping was made for the mean trait performance of the F _2:3 population across years. Ten QTL and an interaction were associated with GM. These QTL accounted for 54.8 and 65.2% of the phenotypic and genotypic variation, respectively. Eight QTL and two interactions were associated with FDR accounting for 35.7 and 45.2% of the phenotypic and genotypic variation, respectively. Two regions were in common between traits. The interaction between QTL for GM at harvest had practical implications for MAS. We conclude that MAS per se will not be an efficient method for reducing GM at harvest and/or increasing FDR. A selection index including both molecular marker information and phenotypic values, each appropriately weighted, would be the best selection strategy.