Chlorella for protein and biofuels: from strain selection to outdoor cultivation in a Green Wall Panel photobioreactor
Chlorellais one of the few microalgae employed for human consumption. It typically has a high protein content, but it can also accumulate high amounts of lipids or carbohydrates under stress conditions and, for this reason, it is of interest in the production of biofuels. High production costs and energy consumption are associated with its cultivation. This work describes a strategy to reduce costs and environmental impact ofChlorellabiomass production for food, biofuels and other applications.
The growth of four Chlorellastrains, selected after a laboratory screening, was investigated outdoors in a low-cost 0.25 m2 GWP-II photobioreactor. The capacity of the selected strains to grow at high temperature was tested. On the basis of these results, in the nitrogen starvation trials the culture was cooled only when the temperature exceeded 40°C to allow for significant energy savings, and performed in a seawater-based medium to reduce the freshwater footprint. Under nutrient sufficiency, strain CH2 was the most productive. In all the strains, nitrogen starvation strongly reduced productivity, depressed protein and induced accumulation of carbohydrate (about 50%) in strains F&M-M49 and IAM C-212, and lipid (40 - 45%) in strains PROD1 and CH2. Starved cultures achieved high storage product productivities: 0.12 g L−1 d−1 of lipids for CH2 and 0.19 g L−1 d−1 of carbohydrates for F&M-M49. When extrapolated to large-scale in central Italy, CH2 showed a potential productivity of 41 t ha−1 y−1 for biomass, 16 t ha−1 y−1 for protein and 11 t ha−1 y−1 for lipid under nutrient sufficiency, and 8 t ha−1 y−1 for lipid under nitrogen starvation.
The environmental and economic sustainability ofChlorellaproduction was enhanced by growing the organisms in a seawater-based medium, so as not to compete with crops for freshwater, and at high temperatures, so as to reduce energy consumption for cooling. All the four selected strains are good candidates for food or biofuels production in lands unsuitable for conventional agriculture.Chlorellastrain CH2 has the potential for more than 80 tonnes of biomass, 32 tonnes of protein and 22 tonnes of lipid per year under favourable climates.
Chlorella(Chlorophyta, Trebouxiophyceae), one of the most studied microalgae, is commercially cultivated by more than 70 companies in the world . The annual production of Chlorella biomass exceeds 2,000 tonnes [1, 2], mostly used for dietary supplements and nutraceuticals, with a minor share destined to the cosmetic market and aquaculture . Chlorella is commercially produced under photoautotrophic conditions, mainly in open ponds (both raceway and circular) [3, 4], or heterotrophically in fermenters . The largest closed system used for autotrophic production at commercial scale is the 700 m3 tubular photobioreactor operated by Roquette Klötze GmbH & Co. KG (Klötze, Germany), which produces annually about 100 tonnes of high quality Chlorella biomass for the health food market .
When molecular data became available it was clear that different green microalgae with similar morpho-physiological characters had been classified as ‘Chlorella’. The taxonomy of the genus is still under revision . C. vulgaris and C. pyrenoidosa are the two most cultivated at commercial scale. The latter species still has an uncertain taxonomic collocation [6, 7]. Chlorella thrives in fresh or brackish waters, but several marine strains are also known. In this respect, it is important to avoid confusion with the so-called ‘marine chlorella’, a much researched organism in the 1980s because of its high eicosapentaenoic acid (EPA) content, which was later correctly identified as Nannochloropsis sp. .
Chlorellais one of the few microalgae (together with Dunaliella, Haematococcusand Arthrospira) largely employed for human consumption. It has a high protein content with a balanced amino acid composition [9, 10], besides a good content of vitamins, minerals, pigments  and short-chain polyunsaturated fatty acids, including oleic and linoleic acids [11, 12]. Some strains are also a good source of lutein . Chlorella is recognized as a safe food ingredient worldwide [14, 15], mainly due to its long history of human consumption as a food supplement and nutraceutical [7, 9, 16, 17]. In vivo studies on its potential as food and protein source have been carried out mainly in the past [18, 19], when legislation concerning trials on people was less restrictive . More recently, Chlorella biomass has been proposed as a food ingredient: as colouring agent for traditional butter cookies , as additive for fermented milk and yoghurt to enhance the viability of bacterial probiotics [21, 22] and incorporated in pasta products to increase their nutritional quality . A Chlorella protein hydrolysate has also been tested as a food additive . The food and feed markets require large quantities of biomass produced at low cost (less than 1 € kg−1) . Currently, algae production costs are higher than 4 - 5 € kg−1 and, although recent economic analyses foresee a decrease to 1 - 2 € kg−1, the commercialization ofChlorella as a food commodity is not mature yet.
High production costs are also the main limitation to another potential application of this microalga: biofuel production. In the last decade, algal biofuels have received a great deal of attention . Chlorella is among the algae of major interest for biofuels, since under stress and depending on the strain, it can accumulate large amounts of lipids  or synthesize starch [29, 30]. Research carried out under nitrogen or phosphorus starvation has shown significant lipid accumulation (up to about 50%) and high lipid productivities [31, 32]. Studies have also been carried out under nutrient replete conditions. Moheimani cultivated Chlorella sp. in a 120 L bag photobioreactor, obtaining a biomass productivity during summer of up to 0.28 g L−1 d−1 and a lipid content of about 25% . Přibyl et al., with Chlorella vulgaris in a 150 L, 6.6 m2 thin-layer open system, obtained maximum biomass and lipid productivities of 1.26 and 0.33 g L−1 d−1, respectively . Some Chlorella are also highly productive in starch, and thus potential substitutes of starch-rich terrestrial plants for bioethanol production. Brányiková et al. increased starch content of Chlorella up to 50% by applying sulfur limitation in an outdoor thin-layer open system .
The extracted (delipidated) Chlorellabiomass, still rich in proteins, carbohydrates, minerals and bioactive compounds, could provide raw materials for feed and food applications [36, 37]. This is important in view of recent analyses that have shown that to achieve a positive energy balance and produce economically viable biofuels, the residue after extraction must be used for co-products [38–40]. A different approach, which seems more practical and feasible, is targeting feed, food or chemicals as the first product. After the extraction of the valuable compound, recovery of the residual energy (and nutrients) of the spent biomass by alcoholic fermentation or anaerobic digestion could be carried out. Integrating food and fuel production processes, besides providing economic advantages, would lead to a higher environmental sustainability [41, 42]. However, the issue of matching markets of different sizes, such as that of biofuels and high-value products, must be considered.
Microalgae have several advantages over traditional crops. Their cultivation does not need fertile soil and they are very efficient in using nutrients, thus avoiding or limiting pollution of water bodies by unused fertilizers. Some algae can be cultivated in brackish, saline or seawater, thus they do not compete for dwindling freshwater resources. The use of wastewaters as a nutrient source is also an attractive possibility that can be considered when biofuels are the target. Microalgae cultures can be fed with CO2 from flue gases [42–46]; however, the need to supply CO2 to the culture should be seen as a limitation, compared to plants that absorb CO2 directly from the air, rather than an advantage. To make microalgal biomass economically competitive and sustainable, either for food or biofuels, the cost of the culture system, as well as operational costs, must be significantly reduced [44, 45]. In particular, mixing [26, 44, 45, 47, 48] and cooling [44, 45, 47] costs, which are very high in closed systems, need to be cut substantially by, for example, selecting strains with high buoyancy and able to grow at high temperatures [49, 50]. For sustainable microalgae cultivation, strains able to grow with high productivity in seawater or brackish water are required.
The aim of this work was to evaluate the performance (in terms of protein, carbohydrate and lipid content and productivity) of selectedChlorellastrains grown under conditions devised to reduce operational costs and increase the sustainability of the cultivation process. To reach this goal, outdoor growth experiments with four strains, selected after a thorough laboratory screening, were carried out in a low-cost photobioreactor, the Green Wall Panel (GWP), with reduced or without cooling in a seawater-based, instead of the standard freshwater-based, culture medium. Cultivation in nitrogen deprived media was finally tested to increase storage product accumulation (lipid or carbohydrate) and evaluate the potential of the selected strains for biofuel (biodiesel or ethanol) production.
ResultsLaboratory screening of nineChlorellastrains
Nine Chlorellastrains were cultivated in 300 mL bubble tubes in the laboratory to evaluate their productivity and biochemical composition in nutrient sufficient and nitrogen deprived growth media. Under nutrient sufficiency, batch and semi-continuous cultures were compared. With two exceptions (IRT2 and CH2), batch cultures achieved higher productivities (on average 0.68 versus 0.55 g L−1 d−1). The more productive batch cultures were those of strains MACH1, CH2, PROD1, IAM C-212 and PAVV2P2, all above 0.7 g L−1 d−1. In semi-continuous culture, only strain CH2 attained a high productivity (0.82 g L−1 d−1) (Table 1). Under nitrogen starvation (evaluated only in batch), the average biomass productivity decreased from 0.68 to 0.37 g L−1 d−1. The decrease, observed for all the strains, ranged from a minimum of 31% for MACH1 to a maximum of 75% for IAM C-212 (Table 1).