Mario R. Tredici

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.

PHOTOBIOLOGY OF MICROALGAE MASS CULTURES: UNDERSTANDING THE TOOLS FOR THE NEXT GREEN REVOLUTION

Several obstacles and limitations currently prevent the industrial exploitation of microalgae for feed, food and biofuel production. Photobioreactors (closed systems for algae cultivation) suffer from high-energy expenditures for mixing and cooling, while cultures in large-scale open ponds, which have a more favorable net energy ratio, are unstable ecosystems in which maintaining selected strains for long periods is difficult. Techniques for supplying nutrients and CO2, for mixing and for harvesting and processing the biomass in an energy-efficient manner are still under study and development. Despite these impediments and although microalgae are not superior to higher plants in terms of photosynthetic efficiency and productivity, microalgal cultures remain one of the most attractive sources of feed, food and next-generation biofuels since microalgae can be grown in saline or seawater on nonarable lands, can use fertilizers with an almost 100% efficiency, are able to attain much higher oil and protein yields than traditional crops and, being endowed with high growth rates, are easier to be improved via genetic and metabolic engineering.

From open ponds to vertical alveolar panels: the Italian experience in the development of reactors for the mass cultivation of phototrophic microorganisms

The need to develop new concepts in reactor design and the growing interest inSpirulina prompted our group to abandon open ponds in the seventies and to focus interest mainly on closed systems. Two substantially different closed photobioreactors have been developed and are at present under investigation in our Research Centre: the tubular photobioreactor (made of rigid or collapsible tubes) and the recently devised vertical alveolar panel (VAP) made of 1.6-cm-thick Plexiglas alveolar sheets.The technical characteristics of the two systems are described and discussed in relation to the main factors which regulate the growth of oxygenic photosynthetic microorganisms in closed reactors.

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.

A vertical alveolar panel (VAP) for outdoor mass cultivation of microalgae and cyanobacteria

A vertical alveolar panel (VAP) for microalgal mass cultivation has been devised and developed. VAP reactors with a surface area of 0·5−2·2 m2 were constructed from Plexiglas alveolar sheets 1·6 cm in thickness and used from May 1989 to February 1990 to grow Anabaena azollae and Spirulina platensis in the climatic conditions of Central Italy. The VAP has proven to be well suited to the outdoor mass cultivation of cyanobacteria, allowing operation at high cell concentrations (4–7 g liter−1) and achieving high biomass productivity even in winter. The high surface-to-volume ratio (80m−1), its flexible orientation with respect to the suns rays, effective mixing and O2 removal through air bubbling and a good control of environmental and nutritional conditions seem to be the major advantages of the system.

Polyphasic study of Antarctic cyanobacterial strains

We isolated 59 strains of cyanobacteria from the benthic microbial mats of 23 Antarctic lakes, from five locations in two regions, in order to characterize their morphological and genotypic diversity. On the basis of their morphology, the cyanobacteria were assigned to 12 species that included four Antarctic endemic taxa. Sequences of the ribosomal RNA gene were determined for 56 strains. In general, the strains closely related at the 16S rRNA gene level belonged to the same morphospecies. Nevertheless, divergences were observed concerning the diversity in terms of species richness, novelty, and geographical distribution. For the 56 strains, 21 operational taxonomic units (OTUs, defined as groups of partial 16S rRNA gene sequences with more than 97.5% similarity) were found, including nine novel and three exclusively Antarctic OTUs. Sequences of Petalonema cf. involvens and Chondrocystis sp. were determined for the first time. The internally transcribed spacer (ITS) between the 16S and the 23S rRNA genes was sequenced for 33 strains, and similar groupings were observed with the 16S rRNA gene and the ITS, even when the strains were derived from different lakes and regions. In addition, 48 strains were screened for antimicrobial and cytotoxic activities, and 17 strains were bioactive against the gram‐positive Staphylococcus aureus, or the fungi Aspergillus fumigatus and Cryptococcus neoformans. The bioactivities were not in coincidence with the phylogenetic relationships, but rather were specific to certain strains.

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.

As integrated culture system for outdoor production of microalgae and cyanobacteria

Cultivation of microalgae in closed photobioreactors suffers from high temperatures, whereas in open raceway ponds the optimal temperature is seldom reached. With an integrated device coupling a raceway pond with a suitably sized and positioned closed system (alveolar panel), the heat accumulated in the panel was efficiently transferred to the pond and a near-optimum temperature regimen for Arthrospira platensis was maintained, with no need for any additional cooling device. The productivity obtained in the integrated system was higher than the sum of the productivities (g reactor−1 day−1) of the pond and panel systems operating separately.