Liliana Rodolfi

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. Biotechnol. Bioeng. 2009;102: 100–112.

Production of eicosapentaenoic acid by Nannochloropsis sp. cultures in outdoor tubular photobioreactors

Autotrophic microalgae cultures have been proposed as an alternative source of EPA, a nutritionally important polyunsaturated fatty acid that plays a key role in the prevention and treatment of several human diseases and disorders. The technology currently available is however, considered commercially not viable because of the low degree of control of algae cultures in outdoor open ponds. The use of closed reactors could overcome these limitations and bring EPA production by microalgae closer to becoming a reality. In this study, we have demonstrated the feasibility of outdoor cultivation of Nannochloropsis sp. in tubular reactors and the potential of this eustigmatophyte as an alternative source of EPA. Nannochloropsis sp. was cultivated in NHTRs of different sizes (from 10.2 to 610 1) from spring to autumn under the climatic conditions of central Italy. EPA productivity essentially reflected the productivity of the culture and reached its maximum in May-June (mean monthly value: 32 mg l− 1 day− 1). Although the fatty acid composition of the biomass varied significantly during the cultivation period, EPA content remained rather stable around the value of 4% of dry biomass. The transfer of the cultures from laboratory to outdoor conditions, the exposure to natural light-dark cycles, along with lowering the salt concentration from 33 g l− 1 (seawater salinity value) to 20 g l− 1, factors that caused lasting modifications in the fatty acid content and composition of Nannochloropsis sp., did not significantly affect the EPA content of the biomass.

Oil production by the marine microalgae Nannochloropsis sp. F&M-M24 and Tetraselmis suecica F&M-M33.

Nannochloropsis sp. F&M-M24 and Tetraselmis suecica F&M-M33 were cultivated outdoors in Green Wall Panels under nutrient deficiency to stimulate oil synthesis. Under nitrogen deprivation, Nannochloropsis attained average biomass and lipid productivities of 9.9 and 6.5 g m(-2) day(-1), respectively. Starved Tetraselmis cultures achieved a biomass productivity of about 7.6 g m(-2) day(-1) and a lipid productivity of 1.7 g m(-2) day(-1). Lipids represented 39.1% and 68.5% of non-starved and starved Nannochloropsis biomass, respectively. Starvation did not increase lipid content in Tetraselmis biomass. Important differences in lipid classes and in fatty acid composition were observed under the different cultivation conditions for both microalgae.

Growth medium recycling in Nannochloropsis sp. mass cultivation

During cell division Nannochloropsis releases the thick and multilayered parent cell wall [Phycologia 35 (1996) 253]. The excretion of autoinhibitory substances in Nannochloropsis cultures has been also reported [J. Appl. Phycol. 11 (1999) 123]. Both wall remains and autoinhibitors may negatively affect culture growth and limit the recycling of the exhaust culture medium, a necessity in commercial microalgae plants to reduce production costs. The effect of medium recycling on growth and productivity of Nannochloropsis sp. cultures grown in 120 l annular reactors was investigated. The use of exhaust medium replenished with nutrients decreased significantly culture productivity. The partial removal of the cell walls alleviated, but did not solve the problem. In addition, medium recycling caused a massive formation of cell aggregates accompanied by a progressive deterioration of the culture. The structure of these aggregates was investigated by transmission electron microscopy. The images showed that the aggregates were held together by cell wall remains, which entrapped cells, bacteria and debris resulting from cell decay. Thus, in high density Nannochloropsis cultures, cell walls might play a key role in reducing productivity, favoring contamination and making the biomass unsuitable as aquaculture feed.

Mass cultivation of Nannochloropsis sp. in annular reactors

A study was made on the mass cultivation of Nannochloropsis sp. in newly designed annular reactors operated under natural, artificial or combined illumination. The annular reactor consists of two 2-m-high Plexiglas cylinders of different diameter placed vertically one inside the other so as to form an annular culture chamber. Artificial illumination is supplied by lamps placed inside the inner cylinder. Two annular reactors of different diameter (50 and 91 cm), light path (4.5 and 3.0 cm) and illuminated surface area (5.3 and 9.3 m2) were experimented with. The effect of two different artificial light sources (fluorescent tubes and metal halide lamps) on culture productivity was investigated in both systems. The highest productivity on a per reactor basis (about 34 g (d. wt) reactor−1 24 h−1) was achieved with the larger reactor illuminated by a 400-W metal halide lamp. From February to May a 91-cm reactor illuminated only with natural light was operated in parallel with a 91-cm reactor subjected to combined illumination. Under natural illumination productivity increased from 16.6 g (d. wt) reactor−1 d−1 in February to 34.1 g (d. wt) reactor−1 d−1 in May. Under combined illumination productivity was 41.3 g (d. wt) reactor−1 d−1 in February and increased up to 48.3 g (d. wt) reactor−1 d−1 in May. Although the culture exposed to combined illumination always attained higher yields, the productivity gap between the two cultures decreased gradually along the season as solar radiation and minimum night temperatures increased. A 1200-L plant made of ten 50-cm annular reactors was set up and operated for two years with combined illumination yielding an average of 270 g of dry Nannochloropsis sp. biomass per day. More than 2000 L of concentrate suspension (50 g (d. wt) L−1) of Nannochloropsis sp. were produced and successfully used by fish hatcheries as live feed for rotifers and for rearing seabream larvae with the green-water technique. This study indicates that the annular reactor can be profitably used for long-term cultivation of Nannochloropsis in temperate climates. Besides reliability and ease of operation, the main advantage of the system is that it can be used under natural illumination, yet artificial light can be also supplied to maintain high productivity levels in winter or on cloudy days.

Evaluation of Nostoc Strain ATCC 53789 as a Potential Source of Natural Pesticides

ABSTRACT The cyanobacterium Nostoc strain ATCC 53789, a known cryptophycin producer, was tested for its potential as a source of natural pesticides. The antibacterial, antifungal, insecticidal, nematocidal, and cytotoxic activities of methanolic extracts of the cyanobacterium were evaluated. Among the target organisms, nine fungi (Armillaria sp., Fusarium oxysporum f. sp. melonis, Penicillium expansum, Phytophthora cambivora, P. cinnamomi, Rhizoctonia solani, Rosellinia, sp., Sclerotinia sclerotiorum, and Verticillium albo-atrum) were growth inhibited and one insect (Helicoverpa armigera) was killed by the extract, as well as the two model organisms for nematocidal (Caenorhabditis elegans) and cytotoxic (Artemia salina) activity. No antibacterial activity was detected. The antifungal activity against S. sclerotiorum was further studied with both extracts and biomass of the cyanobacterium in a system involving tomato as a host plant. Finally, the herbicidal activity of Nostoc strain ATCC 53789 was evaluated against a grass mixture. To fully exploit the potential of this cyanobacterium in agriculture as a source of pesticides, suitable application methods to overcome its toxicity toward plants and nontarget organisms must be developed.

Use of fresh and preserved Tetraselmis suecica for feeding Crassostrea gigas larvae

Abstract In an attempt to provide cost-effective alternative diets and to simplify hatchery–nursery procedures, substitutes for live microalgae in mariculture were tested and among them, concentrated microalgae have so far appeared to be the best alternative products for bivalves, though there are few experimental studies. The food quality of fresh and preserved Tetraselmis suecica for umboned Crassostrea gigas larvae was studied here and its potential as an algal feed for remote production was evaluated. Because it leads to lower growth and higher mortalities than the control diets (>60%), the use of T. suecica OR (strain Orbetello) for C. gigas larvae is not recommended as a monospecific diet. However, a binary diet that includes T. suecica OR is as efficient as the standard ternary PTC diet for C. gigas larvae. On the other hand, even though microalgal utilisation was not clearly affected by the initial size of the larvae, it seemed that old umboned larvae took full advantage of T. suecica OR. Its usefulness in C. gigas larval rearing is therefore limited, but its potential for oyster spat feeding has to be considered. Lastly, preservation at low temperature is an efficient method to maintain the food value of T. suecica OR.

Advances in microalgal culture for aquaculture feed and other uses.

Abstract: The state of the art of microalgae biotechnology, particularly focusing on new culture techniques and actual and potential uses of microalgae in human and animal nutrition, in cosmetics and pharmaceutics, and for environmental applications, is described. Some examples of the worlds largest commercial plants in the field are presented. For the future, it is possible to foresee a huge increase in the demand for cultured algae, in terms of both quantity and diversity. For example, aquaculture will require new animal species and, consequently, new microalgae to fulfil their nutritional needs will be necessary. The production of algae for high–value markets (aquaculture, food supplements, nutraceuticals, pharmaceuticals) will be developed through the search for, isolation and cultivation of new algal strains endowed with the activity of interest. Algal biomass might become an important source of biofuels, especially if its production will be carried out in low-cost photobioreactors and associated with wastewater treatment and greenhouse gas abatement.

Photobioreactors for Microalgal Biofuel Production

Many different PBR designs have been proposed for biofuel production, few of them have been tested at pilot-scale, none developed at the (large) scale necessary for a complete and correct evaluation. Thus the main issues that impact on the reactor’s performance (i.e., suitable construction materials, efficient mixing, heating/cooling, CO2 supply and oxygen removal), although explored at pilot level, still await evaluation at real scale. Although the main limitations of PBR are the high cost and the reduced scalability, with few exceptions, R&D on photobioreactor design is aimed at achieving high photosynthetic efficiencies and at pushing productivity beyond that currently attainable. The main strategies explored to this end are intensive mixing, light dilution via large external surfaces or internal light conducting structures. This chapter reviews and examined recent advances and innovations in photobioreactor design and operation.

Growth of the toxic dinoflagellate Alexandrium minutum (Dinophyceae) using high biomass culture systems

Toxic dinoflagellates are important in natural ecosystems and are ofglobal economic significance because of the impact of toxic blooms onaquaculture and human health. Both the organisms and the toxins they producehave potential for biotechnology applications. We investigated autotrophicgrowth of a toxic dinoflagellate, Alexandrium minutum, inthree different high biomass culture systems, assessing growth, productivityandtoxin production. The systems used were: aerated and non-aerated2-L Erlenmeyer flasks; 0.5-L glass aerated tubes; anda 4-L laboratory scale alveolar panel photobioreactor. A range ofindicators was used to assess growth in these systems. Alexandriumminutum grew well in all culture conditions investigated, with amarked increase in both biomass and productivity in response to aeration. Thehighest cell concentration (4.9 × 105 cellsmL−1) and productivity (2.6 ×104cells mL−1d−1) was achieved inthe aerated glass culture tubes. Stable growth of A.minutum in the laboratory scale alveolar panel photobioreactor wasmaintained over a period of five months, with a maximum cell concentration of3.3 × 105 cells mL−1, a meanproductivity of 1.4 × 104 cells mL−1d−1, and toxin production of approximately 20μg L−1 d−1 with weeklyharvesting.