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Separation of microalgae of various sizes and shapes is an important process that enables subsequent production of useful compounds. Herein, the separation of microalgae was accomplished using a magnetite-containing gel (42 μm) packed into a column. An algal suspension was injected into the top of the gel bed, after which water was passed through the column. The pressure generated during the process caused the lower domain of the gel bed to deform, resulting in narrowed gaps between the gel beads. When a suspension of Nannochloropsis sp. (0.0069-0.69 g L-1) was loaded and water was passed through the column at an applied pressure of 0.01-0.10 MPa, the majority of microalgae were captured within the upper domain of the gel bed, while only 20% were captured within the lower domain. The amount of Nannochloropsis sp. captured was expressed by an ordinary differential equation to determine the capture coefficient, K, and the maximum capture amount, Qmax. As pressure increased, gel gaps narrowed, K increased, and Qmax decreased because of a reduction in the number of effective capture sites upon compaction of the gel. When a mixed suspension of Anabaena sp., Monoraphidium sp., and Desmodesmus sp. (0.069 g L-1 each) was injected into the gel bed at an applied pressure of 0.01 MPa, only Anabaena sp. was captured at the bottom of the gel bed. This device can be applied for the separation of microalgae in rivers and the sea.

Mikroalge rodova Nannochloropsis i Isochrysis predstavljaju sirovinu koja se može koristiti za proizvodnju energije. Cilj ovog rada bio je opisati uzgoj mikroalgi rodova Nannochloropsis i Isochrysis u različitim tehnološkim sustavima te laboratorijskim metodama istražiti njihove kemijske i fizikalne karakteristike za preliminarnu procjenu energetskog potencijala za proizvodnju bioplina i biodizela. Analizom uzoraka mikroalgi Nannochloropsis oculata, utvrđene su srednje vrijednosti sadržaja vode 95,28 %, sadržaja pepela 76,5 %, sadržaja masti 0,3271 %. Salinitet otopine uzoraka mikroalge N. oculata iznosio je 37,114 ppt. Utvrđena je kemijska potrošnja kisika (KPK), od ostataka mikroalgi nakon proizvodnje biodizela, koja je iznosila 1244 mg/L. Analizom uzoraka mikroalgi Isochrysis galbana, utvrđene su srednje vrijednosti sadržaja vode 95,42 %, sadržaja pepela 77,91 %, sadržaja masti 0,3395 %. Salinitet otopine uzoraka mikroalge I. galbana je iznosio 36,516 ppt. Utvrđena je kemijska potrošnja kisika (KPK), od ostataka mikroalgi nakon proizvodnje biodizela, koja je iznosila 999 mg/L. Može se zaključiti da postoji potencijal u proizvodnji biogoriva iz mikroalgi, te da je moguća integrirana kružna proizvodnja. Međutim, potrebno je optimizirati sustav proizvodnje kako bi prinosi bili što veći, a troškovi proizvodnje što manji. Nannochloropsis and Isochrysis microalgae represent a raw material that can be used for energy production. The aim of this research was to describe the cultivation of the Nannochloropsis and Isochrysis microalgae in different technological systems and to determine their chemical and physical characteristics using laboratory methods for a preliminary assessment of the energy potential for the production of biogas and biodiesel. Analysing the Nannochloropsis samples, the average values of water content was measured at 95.28 %, ash content at 76.5 %, fat content at 0.3271 % were determined. The solution salinity of the Nannochloropsis samples was measured at 37.114 ppt. The chemical oxygen consumption (COD) of microalgae residues after biodiesel production was determined, which was 1244 mg/L. Through the analysis of the Isochrysis galbana samples, the average values of water content was measured at 95.42 %, ash content at 77.91 %, fat content at 0.3395 % were determined. The solution salinity of the I. galbana samples was measured at 36,516 ppt. The chemical oxygen consumption (COD) of microalgae residues after biodiesel production was determined, which was 999 mg/L. It can be concluded that there is potential in the production of biofuel from microalgae, and that integrated circular production is possible. However, it is necessary to optimize the production system so that yields are as high as possible and production costs are as low as possible.

Lipid mesophases are lyotropic liquid crystalline systems which differ from liposomes and other globular aggregates in dilute regimes due to their inner ordering. It is known that natural lipids enable to obtain a rich variety of nanosystems and many of them have been proposed as delivery agents for bioactive compounds. Due to their packing parameters, several classes of lipids found in natural sources are able to self-assemble into nonlamellar structures. Among lipids occurring in plants and algae, triglycerides display this tendency. In the present study we examine new nanosystems built with lipids extracted from the marine microalga Nannochloropsis sp and their use as carriers for lipophilic antioxidants. The antioxidants studied, curcumin and tocopherol were encapsulated with high rate in the carriers. The physico-chemical characterization of plain and loaded vectors showed their structure and localization site, as well as the structure-functionality relationship related to potential drug delivery. The results show that the cargo molecules play an active role in driving the interactions which characterize the overall structure of the aggregates. The systems studied showed several coexisting mesophases, the most predominant structure being of cubic symmetry.

Nannochloropsis sp. is a green alga that is widely used in the aquaculture industry as a feed in Malaysia, but genetic engineering studies of this alga are still underexplored even though there is a growing interest in microalgae genetic engineering for various industrial purposes. This study aims to investigate the efficiency of three transformation methods normally done on microalgae, namely polyethylene glycol (PEG), electroporation, and glass beads on Malaysian indigenous Nannochloropsis sp. using two commercially available plasmids, pUC19 and pGEM-T easy vector as well as an amplicon of ampicillin resistance (AMPR) gene. In this study, out of three transformation methods tested, positive transformants of Nannochloropsis sp. were successfully obtained via electroporation method. Further verification via polymerase chain reaction (PCR) and sequencing confirmed that the electroporation method was found to be the sole successful method in producing transgenic lines of our locally isolated Nannochloropsis sp. Results from this study proved the efficiency of electroporation for delivery of transgene to this green alga which has been reported to be tedious. The described method also provides the gateway for developing Nannochloropsis sp. as a delivery system to aquatic organism due to its importance in the industry.

Microalgae contain a significant amount of carbohydrates that can be converted further to produce valuable compounds. To extract carbohydrates from microalgae, sub-critical water extraction (SWE) is a viable and novel method. Compared to other existing chemical and biological extraction methods, SWE is more economical, effective, and efficient in terms of process conditions. This process uses high temperature and pressure of water at just below supercritical conditions to keep water at a liquid state. This study aims to investigate the factors that affect the extraction yield of carbohydrates from microalgae Nannochloropsis sp. using the SWE process. The extraction was carried out at a temperature of 160 – 320 oC, a duration of 5 – 25 min, and microalgal biomass loading of 5 - 25% w/v. The kinetics study was conducted in an extraction time of 5 – 25 min at 200 oC and 5% (w/v) biomass loading. A simplified kinetic model based on a consecutive reaction was used to describe carbohydrate production and decomposition via SWE. It was found that the maximum yield of total carbohydrate was 18.04 g/100 g which was obtained at 200 oC, 10 min, and 5% (w/v) microalgal biomass loading. The carbohydrate concentration was decreased as the temperature, extraction time and microalgal biomass loading increased. This result indicated that the SWE is a promising extraction method for carbohydrate recovery from microalgae, and the factors investigated here had a significant effect on the extraction process. The proposed kinetic model was also able to capture the experimental data well within the range of operating conditions studied in this work.

Lipidi izolirani iz biomase mikroalgi Nannochloropsis sp. predstavljeni su kao perspektivna sirovina za proizvodnju biodizela. U ovom radu proveden je fototrofni šaržni uzgoj s pritokom supstrata u otvorenom sustavu na izvornoj f/2 podlozi uz dodatak natrijevog bikarbonata te fototrofni šaržni uzgoj s pritokom supstrata u barbotirajućoj koloni na izvornoj f/2 podlozi bez dodatnog izvora ugljika. Tijekom uzgoja praćene su promjene udjela proteina, ugljikohidrata i lipida u izdvojenoj biomasi kao i sastav pigmenata te profil masnih kiselina u ukupnim lipidima. Utvrđeno je da u periodu smanjene koncentracije fosfora, metabolizam mikroalge se usmjerava prema akumulaciji lipida i ugljikohidrata kao rezervnih makromolekula uz smanjenje udjela proteina. Posljednjeg dana šaržnog uzgoja u otvorenom bazenu postignuta je koncentracija biomase od 0,515 g Lˉ¹, a maksimalna produktivnost biomase od 0,8796 mg Lˉ¹ hˉ¹ postignuta je osamnaestog dana uzgoja. Šaržnim uzgojem u barbotirajućoj koloni koncentracija biomase na kraju uzgoja iznosila je 0,458 g Lˉ¹, a produktivnost sinteze biomase tada je bila 0,8297 mg Lˉ¹ hˉ¹. Kemijska flokulacija sa željezovim (III) kloridom potvrđena je kao alternativna metoda za izdvajanje stanica mikroalgi. Naposljetku, u radu je predstavljen elaborat tehničko-tehnološkog rješenja postrojenja za proizvodnju biodizela. Lipids isolated from biomass of microalgae Nannochloropsis sp. are presented as a promising feedstock for biodiesel production. Cultivation methods used in this paper were phototrophic fed-batch cultivation with substrate inflow in an open pond system on the original f/2 medium with sodium bicarbonate and phototrophic fed-batch cultivation with substrate inflow on the original f/2 medium without supplementation of carbon source in a bubble column. Changes in the content of proteins, carbohydrates and lipids were monitored in the separated algae biomass during cultivation. The composition of pigments and the profile of fatty acids in total lipids were as well monitored simultaneously. During the period of reduced phosphorus concentration it was confirmed that metabolic responses of microalgae lead to the accumulation of lipids and carbohydrates as reserve macromolecules and to a decrease in protein content. On the last day of fed-batch cultivation in open pond, biomass concentration of 0,515 g Lˉ¹ was achieved. The maximum productivity of 0,8796 mg Lˉ¹hˉ¹ was achieved eighteenth day of cultivation. Additionally, on the last day of fed-batch cultivation in bubble column, biomass concentration was 0,458 g Lˉ¹ and productivity of biomass synthesis was 0,8297 mg Lˉ¹ hˉ¹. Chemical flocculation with iron (III) chloride has been confirmed as an alternative method for microalgae cell harvesting. Lastly, technical-technological solution for a biodiesel production facility was presented in this paper.

Nannochloropsis sp. is a green alga that is widely used in the aquaculture industry as a feed in Malaysia, but genetic engineering studies of this alga are still underexplored even though there is a growing interest in microalgae genetic engineering for various industrial purposes. This study aims to investigate the efficiency of three transformation methods normally done on microalgae, namely polyethylene glycol (PEG), electroporation, and glass beads on Malaysian indigenous Nannochloropsis sp. using two commercially available plasmids, pUC19 and pGEM-T easy vector as well as an amplicon of ampicillin resistance (AMPR) gene. In this study, out of three transformation methods tested, positive transformants of Nannochloropsis sp. were successfully obtained via electroporation method. Further verification via polymerase chain reaction (PCR) and sequencing confirmed that the electroporation method was found to be the sole successful method in producing transgenic lines of our locally isolated Nannochloropsis sp. Results from this study proved the efficiency of electroporation for delivery of transgene to this green alga which has been reported to be tedious. The described method also provides the gateway for developing Nannochloropsis sp. as a delivery system to aquatic organism due to its importance in the industry., Graphical Abstract

Nannochloropsis sp. has received increased attention by researchers in recent years due to its complexity and abundance of lipid structures. The lipids of this microalgae species have been identified to contain large quantities of neutral lipids which are capable of producing raw materials for nutraceuticals, food additives and biofuels. The production of biodiesel has received the greatest attention as there is an increase in global demand for both more fuel and more environmentally sustainable methods to produce such resources. The greatest challenges facing industries to mass produce viable products from microalgae involve the degradation of the cell wall and extracting the fatty acid of interest due to high costs. Various studies have shown that the extraction lipids from the microalgae can greatly influence the overall fatty acid composition. Different extraction methods can re- sult in recovering higher quantities of either saturated fatty acids, monounsaturated fatty acids or polyunsatu- rated fatty acids. Biodiesel production requires higher quantities of saturated fatty acids and monosaturated fatty acids as increased quantities of polyunsaturated fatty acids result in oxidation which decreases the perfor- mance of the biodiesel. Whereas, polyunsaturated fatty acids are required in order to produce pharmaceuticals and food additives such as omega 3. This review will focus on how different in-situ extraction methods for lipid and fatty acid recovery, influence the fatty acid composition of various Nannochloropsis species (oculate, gaditana, salina and oceanica). The mechanical methods (microwave, ultrasonic and supercritical‑carbon dioxide) of extraction for Nannochloropsis sp. will be critically evaluated. The use of enzymes will also be addressed, for their ability to extract fatty acids in a more environmentally friendly manner. This paper will report on the viable byproducts which can be produced using different extraction methods.

Pharmaceuticals and their metabolites are released into the environment by domestic, hospital, and pharmaceutical industry wastewaters. Conventional wastewater treatment technology does not guarantee effluents of high quality, and apparently clean water may be loaded with pollutants. In this study, we assess the performance and efficiency of free and immobilised cells of microalgae Nannochloropsis sp. in removing four pharmaceuticals, chosen for their occurrence or persistence in the environment. These are paracetamol, ibuprofen, olanzapine and simvastatin. The results showed that free microalgae cells remain alive for a longer time than the immobilised ones, suggesting the inhibition of cell proliferation by the polymeric matrix polyvinyl alcohol. Both cells, free and immobilised, respond differently to each pharmaceutical. The removal of paracetamol and ibuprofen by Nannochloropsis sp., after 24 h of culture, was significantly higher in immobilised cells. Free cells removed a significantly higher concentration of olanzapine than immobilised ones, suggesting a higher affinity to this molecule than to paracetamol and ibuprofen. The results demonstrate the effectiveness of Nannochloropsis sp. free cells for removing olanzapine and Nannochloropsis sp. immobilised cells for removing paracetamol and ibuprofen.

Fungicides and, for the first time, microalgal phenolic extracts (MPE) from Spirulina sp. and Nannochloropsis sp. were applied on maize culture media under field conditions to evaluate their ability to minimize Fusarium species development and fumonisin production. An in vitro assay against F. verticillioides was carried out using maize grains as the culture medium. An open-field experiment was carried out in Northwest Italy under natural infection conditions. The compared treatments were factorial combinations of two insecticide treatments (an untreated control and pyrethroid, used against European Corn Borer), four antifungal treatments (an untreated control, MPE from Spirulina sp., MPE from Nannochloropsis sp., and a synthetic fungicide), and two timings of the application of the antifungal compounds (at maize flowering and at the milk stage). The MPE compounds were capable of inhibiting fumonisin production in vitro more efficiently than tebuconazole. Insecticide application reduced the infection by Fusarium species and subsequent fumonisin contamination. However, fumonisins in maize fields were not significantly controlled by either fungicide or MPE application.

Recently, microalgal biomass has attached much attention due to the wide diversity of compounds synthesized from different metabolic pathways. This work attempts to study metabolites recovery from Nannochloropsis sp. biomass concentrated by centrifugation and flocculation. Carbohydrates were obtained using acid and alkaline hydrolysis required for cell disruption. Protein extraction was performed after alkaline pretreatment and lipids were recovery by acid hydrolysis- Soxhlet and alkaline hydrolysis- Soxhlet extraction routes. It was found that carbohydrates were recovered by acid hydrolysis in 41 % and 35.39 % for centrifuged and flocculated biomass, respectively, values higher than thus reported using alkaline hydrolysis. For protein extraction, centrifuged biomass exhibited higher recovery yield (55.48%) than flocculated biomass (38.40%). The lipid extraction route that achieved highest yield (43.45%) was acid hydrolysis with HCl followed by Soxhlet extraction with hexane. In addition, statistical analysis by T test suggested that flocculants affect negatively biomass culture, hence, efficiency of metabolites extraction.

A new approach to recover microalgae from aqueous medium using a bio-flotation method is reported. The method involves utilizing a Moringa protein extract - oil emulsion (MPOE) for flotation removal of Nannochloropsis sp. The effect of various factors has been assessed using this method, including operating parameters such as pH, MPOE dose, algae concentration and mixing time. A maximum flotation efficiency of 86.5% was achieved without changing the pH condition of algal medium. Moreover, zeta potential analysis showed a marked difference in the zeta potential values when increase the MPOE dose concentration. An optimum condition of MPOE dosage of 50ml/L, pH 8, mixing time 4min, and a flotation efficiency of greater than 86% was accomplished. The morphology of algal flocs produced by protein-oil emulsion flocculant were characterized by microscopy. This flotation method is not only simple, but also an efficient method for harvesting microalgae from culture medium.

Data of this article describes growth, survival rate, water quality and hemato-biochemical indices of Nile Tilapia (Oreochromis niloticus) fry. To collect the data, the Nile Tilapia fry was reared in 30 L glass aquarium (18 fish/ tank) for 56-days under controlled environmental condition. Feed was prepared with 25 and 50% replacement of commercial fish meal with Nannochloropsis sp. and Tetraselmis sp. microalgae, while no replacement was made for control feed. Initial and final body weight of fish was recorded to find the data of growth rate; survival rate was calculated from the initial and final live individuals recorded during the experiment; physico-chemical parameters were analyzed to collect water quality data; hemato-biochemical indices were collected using hematology analyzer and photometry. The data on growth, survival rate and hemato-biochemical indices were statistically significant (p < 0.05). Therefore, these data might contribute to the selection of marine microalgae to improve the water quality during fish farming which could enhance the growth and survivability of fish. In addition, the data of hemato-biochemical indices represent that feeding selected marine microalgae might result in the production of healthy and disease-free fish.

Harvesting microalgae from medium is a major challenge due to their small size and low concentrations. In an attempt to find a cost-effective and eco-friendly harvesting technique, mung bean (Vigna radiata) protein extract (MBPE) was used for flocculation of Nannochloropsis sp. The effects of parameters such as pH, flocculant dose, algae concentration, and mixing time were used to study the flocculation efficiency (FE) of MBPE. Optimum parameters of MBPE dosage of 20 mL L−1 and a mixing rate of 300 rpm for 6 min achieved a FE of >92% after 2 h of settling time. MBPE-aggregated microlga flocs were characterized by microscopy. Zeta potential values decreased with increasing flocculant dose, and the values obtained were −6.93 ± 0.60, −5.36 ± 0.64, and −4.44 ± 0.22 for doses of 10, 20, and 30 mL L−1, respectively. In conclusion, MBPE flocculants used in this study are safe, nontoxic, and pollution free, so they could be used for an effective, convenient, and rapid harvesting of microalgae in an eco-friendly approach. These methods are sustainable and could be applied in industrial scale for aquaculture nutrition.

In this article, microalgae Nannochloropsis sp. was used for fatty acid (FA) extraction, using a supercritical fluid-carbon dioxide (SF-CO2) extraction method. This study investigated the influence of different pre-treatment conditions by varying the grinding speed (200&ndash, 600 rpm), pre-treatment time (2.5&ndash, 10 min), and mixing ratio of diatomaceous earth (DE) and Nannochloropsis sp. biomass (0.5&ndash, 2.0 DE/biomass) on FAs extraction. In addition, the effect of different operating conditions, such as pressure (100&ndash, 550 bar), temperature (50&ndash, 75 &deg, C), and CO2 flow rate (7.24 and 14.48 g/min) on eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) recovery, was analyzed. Experimental data evidenced that, keeping constant the extraction conditions, the pre-treatment step enhanced the FAs extraction yield up to 3.4 fold, thereby the maximum extracted amount of FAs (61.19 mg/g) was attained with the pre-treatment with a ratio of DE/biomass of 1 at 600 rpm for 5 min. Moreover, by increasing both SF-CO2 pressure and temperature, the selectivity towards EPA was enhanced, while intermediate pressure and lower pressure promoted DHA recovery. The highest amount of extracted EPA, i.e., 5.69 mg/g, corresponding to 15.59%, was obtained at 75 &deg, C and 550 bar with a CO2 flow rate of 14.48 g/min, while the maximum amount of extracted DHA, i.e., ~0.12 mg/g, equal to 79.63%, was registered at 50 &deg, C and 400 bar with a CO2 flow rate of 14.48 g/min. Moreover, the increased CO2 flow rate from 7.24 to 14.48 g/min enhanced both EPA and DHA recovery.

Microalgae have a wide range of applications in industries such as feed and aquaculture, functional food, chemicals, pharmaceuticals, cosmetics and biofuels. Despite their high potential, widespread large-scale cultivation of microalgae especially for low-value applications struggles to get off the ground. To increase the economic viability of bulk cultivation, production costs need to be reduced. Recycling growth medium not only allows salvaging the unused nutrients remaining in the water after harvesting, but also permits to reduce water demand and to avoid or reduce waste water treatment costs. To be able to implement a recycling method in a fully automated production system of microalgae, several challenges such as cleaning of spent medium, replenishment of nutrients and comprehending positive or negative effects on microalgal growth needed to be addressed. The present study explored a method to rejuvenate centrifuge supernatant (centrate) in a robust and inexpensive way, mapped nutrient consumptions and formulated dosing strategies for an automated replenishment, and gained understanding in the effect of medium recirculation on the cultivation of Nannochloropsis sp. and Tisochrysis lutea, separately and across species borders. In Chapter 2, a polishing treatment was sought for centrate originating from centrifugation of high density cultures of Nannochloropsis sp. to feed into a microfiltration setup generating contaminant-free growth medium. High pH-induced coagulation/flocculation (with NaOH or Ca(OH)2) and ‘rapid’ sand filtration (start exhaust flow rate of 9 m³·m-2·h-1) were investigated separately, and linked at optimal settings to reduce membrane fouling and increase microfiltration performance. Stand-alone sand filtration provided a meagre improvement in centrate filterability; ‘modified fouling index’ decreased 21 ± 21% after 55 min. High pH induced coagulation/flocculation with NaOH and Ca(OH)2 at pH 11.5 reached flocculation efficiencies of 90 ± 1% and 92 ± 4% respectively. When combining both techniques, the obtained flocculated centrates, however, resulted in rapid clogging of the sand bed. Consequently, only the combination of NaOH coagulation/flocculation at pH 10.5 and subsequent sand filtration was further investigated. This combination removed 78 ± 18% of particles and improved ‘modified fouling indices’ with 75 ± 19%. After 60 minutes filtration runs, the sand bed was backwashed with (acidified) water during 5 minutes to regenerate the sand bed. The results indicated that there is room for improvement by optimizing washing regimes of the sand filter. In any case, the suggested strategy will greatly decrease membrane fouling thus allowing prolonged recirculation of growth medium. The impact of medium recycling on a continuous monoculture of Nannochloropsis sp. cultivated at high density was assessed in Chapter 3. It was observed at two different scales (1.3 L laboratory-scale reactors and a 24 m³ outdoor pilot setup) that it is feasible to cultivate Nannochloropsis sp. with reused, microfiltered and replenished medium. Average yields on light of 0.8 ± 0.2 g·mol-1 PAR and 0.26 ± 0.08 g·mol-1 PAR were obtained at indoor laboratory and outdoor pilot scale setups, respectively. Additionally, nutrient consumption rates were documented in order to guide the replenishment of reused media. It turned out that, due to a disproportionate consumption of nutrients, the replenishment strategy had to be adjusted to limit accumulation or depletion of nutrients in the medium upon repeated medium recycling. The N/P feed concentration was therefore altered from 37/1.3 to 24.7/1.75 (mM/mM). For each recycle round, an important decrease in nutrient (56%), salt (66%) and water (80%) consumption was obtained. Unfiltered centrate too was applied successfully as reused growth medium. However, if one wishes to prevent unwanted contamination in the production of multiple monocultures on a large scale facility, the use of this unfiltered media source is, while cost-efficient, not recommended. When multiple monocultures are cultivated in a large-scale installation, mixing of filtered harvest broths sourced from these cultures will occur. While reusing this medium, allelopathic effects may appear and induce favourable growth responses for some genera but not others. In Chapter 4, the potential of reusing medium from two algae species (Nannochloropsis sp. and Tisochrysis lutea) for growing monocultures of the opposite algal strain was evaluated at three successive cultivation setups with increasing volume; 400 mL in turbidostat mode, 2.6 L and 220 L in semi-continuous mode. Whereas small scale tests were principally used to detect major differences in growth characteristics when using recycled media, pilot scale experiments provided important information on nutrient consumption and generated productivity data that can be extrapolated to large scale cultivation systems. No contamination or negative effect on productivity occurred during cultivation of either species with reused, microfiltered and replenished medium derived from the other species. Microfiltration and balanced nutrient supplementation of the recycled medium contributed to this result. Average yields on light of 1.6 ± 0.5 and 0.6 ± 0.2 g·mol-1 PAR were obtained for Nannochloropsis sp. and T. lutea respectively. By reusing medium, a significant reduction in water footprint (77%) and salt expenditure (71%) was obtained. At pilot scale, a total nutrient cost reduction of 68% was achieved. In conclusion, the results of this thesis led to the implementation of a medium recycling method in a fully automated production system of microalgae, which in turn contributes to the economic viability of a large-scale cultivation of microalgae. Overall, compared to no medium recirculation, a reduction of 67.5% in medium cost and 34.0% in total variable production cost, or € 7.3 kg-1algae, is accomplished when 80% of the medium is reused on a 0.38 ha, 108 m³ microalgae cultivation plant.

Microalgae of Nannochloropsis sp. present valuable source of bio-molecules (pigments, lipids, proteins) that have nutritional potential for the prevention and treatment of human diseases. Moreover, some species of Nannochloropsis are the promising sources of biofuels and excellent candidates for the replacement of classical biofuel crops. This review describes and compares the efficiency of different conventional and novel techniques that can be used for cell disruption and recovery of bio-molecules from Nannochloropsis sp. Classification of different extraction techniques includes chemical, enzymatic, mechanical and other physical methods. The detailed analysis of extraction efficiency assisted by pressure and temperature (subcritical and supercritical fluids, hydrothermal liquefaction), ultrasound, microwaves, and pulsed electric energy (pulsed electric fields and high voltage electrical discharges) is presented. The general discussion includes comparison between techniques, their effectiveness for cell disruption and selectivity of bio-molecules extraction from Nannochloropsis sp. The cost-effectiveness, benefits and limitations of different techniques are also analyzed.

Hydrothermal liquefaction (HTL) is a technology for converting algal biomass into biocrude oil and high-value products. To elucidate the underlying kinetics for this process, we conducted isothermal and non-isothermal reactions over a broad range of holding times (10s-60min), temperatures (100-400°C), and average heating rates (110-350°Cmin(-1)). Biocrude reached high yields (⩾37wt%) within 2min for heat-source set-point temperatures of 350°C or higher. We developed a microalgal HTL kinetic model valid from 10s to 60min, including significantly shorter timescales (10s-10min) than any previous model. The model predicts that up to 46wt% biocrude yields are achievable at 400°C and 1min, reaffirming the utility of short holding times and "fast" HTL. We highlight potential trade-offs between maximizing biocrude quantity and facilitating aqueous phase recovery, which may improve biocrude quality.

The effect of biomass concentration on areal productivity and photosynthetic efficiency of Nannochloropsis sp. CCAP211/78 was studied in three outdoor pilot-scale photobioreactors: an open raceway pond (OPR), a horizontal tubular (HT) photobioreactor and a vertically stacked horizontal tubular (VT) photobioreactor. The reactors were operated continuously as turbidostat at different biomass concentrations. For all systems highest areal productivities were obtained on days with a high light intensity, while the highest photosynthetic efficiencies were obtained on days with a low light intensity. Ground areal biomass concentration exceeding 51 g m − 2 had a negative effect on the areal productivity and photosynthetic efficiency. No significant effect of biomass concentration on the productivity was found for the HT at ground areal biomass concentration lower than 51 g m − 2 . Also for the VT, no significant effect of biomass concentration was found with the exception of the highest biomass concentration of 2.0 g L − 1 (68 g m − 2 ) resulting in decreased productivity. For the open raceway pond the highest biomass concentration (0.5 g L − 1 or 94 g m − 2 ) resulted in significantly lower areal productivity, compared to the lower biomass concentration (0.25 g L 47 g m − 2 ). Highest areal productivities were obtained for OPR and VT, most likely due to more efficient light interception. In this study we observed that night biomass loss was coupled to net growth. At lower biomass concentrations and concomitant higher growth rates the specific biomass loss rate was higher. Microalgal specific light absorption coefficient was correlated to biomass concentration; higher biomass concentrations resulted in higher specific absorption coefficients, resulting in a steeper light gradient in the microalgal cultures.

Algal lipids have gained wide interest in various applications ranging from biofuels to nutraceuticals. Given their complex nature composed of different lipid classes, a deep knowledge between extraction conditions and lipid characteristics is essential. In this paper, we investigated the influence of different pretreatments on lipid extraction with supercritical CO2 by a lipidomic approach. Pretreatment was found to double the total extraction yield, thereby reaching 23.1 wt.% comparable to the 26.9 wt.% obtained with chloroform/methanol. An increase in acylglycerides was concurrently observed, together with a nearly doubling of free fatty acids indicative of partial hydrolysis. Moreover, an alteration in the distribution of glyco- and phospholipids was noted, especially promoting digalactosyldiglycerides and phosphatidylcholine as compared to monogalactosyldiglycerides and phosphatidylglycerol. At optimized conditions, supercritical CO2 extraction provided a lipid extract richer in neutral lipids and poorer in phospholipids as compared to chloroform/methanol, though with a very similar fatty acid distribution within each lipid class.

Toward attaining a sustainability and eco-friendly process, a green and low-cost solvent&mdash, brine (NaCl solution) is proposed, as microwave-assisted extraction (MAE) technique solvent to extract lipids from microalgae Nannochloropsis sp. The effect of NaCl concentration on the quantity and quality of the extracted lipid was assessed, while MAE parameters were optimized using response surface methodology (RSM). The content of fatty acid methyl esters (FAMEs) in the lipid was analyzed by using a gas chromatography&mdash, flame ionization detector (GC/FID). The highest lipid yield (16.1%) was obtained using 10% (w/v) brine at optimum extraction parameters of 5% (w/v) solid loading, 100 &deg, C, and 30 min. The lipid extraction yield via optimized MAE-brine technique was thrice better than that Soxhlet extraction did and only 2% less than Bligh and Dyer (B&amp, D) lipid extraction, which utilized harmful solvents. The proposed MAE-brine technique offered better quality lipids containing the highest amount of polyunsaturated fatty acids (PUFA) (44.5%) and omega-3 fatty acids (FAs) (43%). Hence, the MAE-brine solvent technique appears to be a promising extraction method for cheaper, greener, and faster extraction of a high-quality lipid for specialty food applications.

Algal lipids have gained wide interest in various applications ranging from biofuels to nutraceuticals. Given their complex nature composed of different lipid classes, a deep knowledge between extraction conditions and lipid characteristics is essential. In this paper, we investigated the influence of different pretreatments on lipid extraction with supercritical CO2 by a lipidomic approach. Pretreatment was found to double the total extraction yield, thereby reaching 23.1 wt.% comparable to the 26.9 wt.% obtained with chloroform/methanol. An increase in acylglycerides was concurrently observed, together with a nearly doubling of free fatty acids indicative of partial hydrolysis. Moreover, an alteration in the distribution of glyco- and phospholipids was noted, especially promoting digalactosyldiglycerides and phosphatidylcholine as compared to monogalactosyldiglycerides and phosphatidylglycerol. At optimized conditions, supercritical CO2 extraction provided a lipid extract richer in neutral lipids and poorer in phospholipids as compared to chloroform/methanol, though with a very similar fatty acid distribution within each lipid class.

Hydrothermal liquefaction of biomass produces a complex biocrude, which can be further upgraded to biofuel or chemicals, but there is a need for improved molecular understanding of product composition and reaction pathways. This study extensively characterizes semivolatile compounds in biocrudes from hydrothermal liquefaction (HTL) of microalgae (N. gaditana, C. vulgaris), macroalgae (L. hyperborea), residue (dried distillers grains with solubles), and lignocellulosic (M. × giganteus). The biocrudes were analyzed using 2D gas chromatography coupled to time-of-flight mass spectrometry with in situ silylation. A total of 73 fatty acids were detected of which C18 and C20 compounds were most diverse, while palmitic acid was the single most abundant fatty acid. Multiple fatty acid amides were detected in biocrude from N. gaditana while being almost absent in samples from other lipid- and protein-containing feedstocks. Several alkylated indoles and quinolines were observed in biocrudes from protein-containing f...

Industrial cultivation of microalgae is becoming increasingly important, yet the process is still hampered by many factors, including contamination and biofouling of the algal reactors. We characterized a subset of microorganisms occurring in the broth and different biofilm stages of industrial scale photobioreactors applied for the cultivation of Nannochloropsis sp. A total of 69 bacterial strains were isolated, belonging to at least 24 different species. In addition, a green microalga was isolated and identified as Chlamydomonas hedleyi. The effect of C. hedleyi and 24 of the bacterial isolates on the productivity of Nannochloropsis was evaluated through growth and biofilm assays. C. hedleyi was shown to reduce growth and induce biofilm formation in Nannochloropsis. These effects were however indirect as they could be attributed to the bacteria associated to C. hedleyi and not C. hedleyi itself. Although most bacterial strains reported no effect, several were able to induce biofilm formation.

The main source of n-3 long-chain polyunsaturated fatty acids (LC-PUFA) in human nutrition is currently seafood, especially oily fish. Nonetheless, due to cultural or individual preferences, convenience, geographic location, or awareness of risks associated to fatty fish consumption, the intake of fatty fish is far from supplying the recommended dietary levels. The end result observed in most western countries is not only a low supply of n-3 LC-PUFA, but also an unbalance towards the intake of n-6 fatty acids, resulting mostly from the consumption of vegetable oils. Awareness of the benefits of LC-PUFA in human health has led to the use of fish oils as food supplements. However, there is a need to explore alternatives sources of LC-PUFA, especially those of microbial origin. Microalgae species with potential to accumulate lipids in high amounts and to present elevated levels of n-3 LC-PUFA are known in marine phytoplankton. This review focuses on sources of n-3 LC-PUFA, namely eicosapentaenoic and docosahexaenoic acids, in marine microalgae, as alternatives to fish oils. Based on current literature, examples of marketed products and potentially new species for commercial exploitation are presented. NPST Program of King Saud University [11-ENE1719-02]; SEABIOMED [PTDC/MAR/103957/2008]; Foundation for Science and Technology (FCT); Portuguese National Budget info:eu-repo/semantics/publishedVersion

A suite of multivariate chemometrics methods was applied to a mid-infrared imaging dataset of a eustigmatophyte, marine Nannochloropsis sp. microalgae strain. This includes the improved leader–follower cluster analysis (iLFCA) to interrogate spectra in an unsupervised fashion, a resonant Mie optical scatter correction algorithm (RMieS-EMSC) that improves data linearity, the band-target entropy minimization (BTEM) self-modeling curve resolution for recovering component spectra, and a multi-linear regression (MLR) for estimating relative concentrations and plotting chemical maps of component spectra. A novel Alpha-Stable probability calculation for microalgae cellular lipid-to-protein ratio Λ i is introduced for estimating population characteristics.

Non-photochemical quenching (NPQ) of chlorophyll fluorescence is thought to be an indicator of an essential regulation and photoprotection mechanism against high-light stress in photosynthetic organisms. In this report, special chemicals were used to perturb the kinetics of the ΔpH build-up and the xanthophyll cycle (XC) in Nannochloropsis sp. We found that NPQ was stimulated rapidly on exposure to high light and relaxed rapidly in darkness. The ΔpH could be obligatory for NPQ and ΔpH alone was not sufficient to induce NPQ. The XC, being strictly mediated by ΔpH, was also essential for NPQ. The results demonstrate that the mechanism of NPQ in Nannochloropsis sp. resembled that of diatoms.

Microalgae-derived lipids in the form of triacylglycerols (TAGs) are considered an alternative resource for the production of biofuels and food commodities. Large scale production of microalgal TAGs is currently uneconomical. The cost price could be reduced by improving the areal and volumetric TAG productivity. The economic value could be increased by enhancing the TAG quality. To improve these characteristics, the impact of light intensity, and the combined impact of pH and temperature on TAG accumulation were studied for Scenedesmus obliquus UTEX 393 under nitrogen starved conditions. The maximum TAG content was independent of light intensity, but varied between 18% and 40% of dry weight for different combinations of pH and temperature. The highest yield of fatty acids on light (0.263 g/mol photon) was achieved at the lowest light intensity, pH 7 and 27.5 °C.

Cyanobacterial toxins have been regarded by some researchers as allelopathic substances that could modulate the growth of competitors. Nevertheless, often the concentrations of toxins used are too high to be considered ecologically relevant. In this work we tested the hypothesis that microcystin-LR (MC-LR) and cylindrospermopsin (CYN) at ecologically relevant concentrations have no allelopathic effects on some species of phytoplankton. Extracts containing the toxins as well as pure MC-LR and CYN toxins were used to assess their effects on the growth rates of Nannochloropsis sp., Chlamydomonas reinhardtii, and Chlorella vulgaris. Cyanobacterial crude extracts induced more pronounced effects on growth rates than pure toxins. Microcystis aeruginosa and Aphanizomenon ovalisporum crude extracts containing MC-LR and CYN at 0.025–2.5 mg l−1 stimulated growth rates of microalgae, whereas A. ovalisporum crude extracts containing 2.5 mg l−1 of CYN strongly inhibited growth rates of microalgae after 4 and 7 days of exposure. MC-LR and CYN at environmentally occurring concentrations were unable to affect negatively the growth of microalgae, and therefore these molecules may play roles other than allelopathy in natural ecosystems.

Background For a commercially feasible microalgal triglyceride (TAG) production, high TAG productivities are required. The operational strategy affects TAG productivity but a systematic comparison between different strategies is lacking. For this, physiological responses of Nannochloropsis sp. to nitrogen (N) starvation and N-rich medium replenishment were studied in lab-scale batch and repeated-batch (part of the culture is periodically harvested and N-rich medium is re-supplied) cultivations under continuous light, and condensed into a mechanistic model. Results The model, which successfully described both strategies, was used to identify potential improvements for both batch and repeated-batch and compare the two strategies on optimized TAG yields on light (amount of TAGs produced per mol of supplied PAR photons). TAG yields on light, for batch, from 0.12 (base case at high light) to 0.49 g molph−1 (at low light and with improved strain) and, for repeated-batch, from 0.07 (base case at high light) to 0.39 g molph−1 (at low light with improved strain and optimized repeated-batch settings). The base case yields are in line with the yields observed in current state-of-the-art outdoor TAG production. Conclusions For continuous light, an optimized batch process will always result in higher TAG yield on light compared to an optimized repeated-batch process. This is mainly because repeated-batch cycles start with N-starved cells. Their reduced photosynthetic capacity leads to inefficient light use during the regrowth phase which results in lower overall TAG yields compared to a batch process. Electronic supplementary material The online version of this article (doi:10.1186/s13068-016-0475-4) contains supplementary material, which is available to authorized users.

In large-scale microalgal production in tubular photobioreactors, the build-up of O(2) along the tubes is one of the major bottlenecks to obtain high productivities. Oxygen inhibits the growth, since it competes with carbon dioxide for the Rubisco enzyme involved in the CO(2) fixation to generate biomass. The effect of oxygen on growth of Nannochloropsis sp. was experimentally determined in a fully controlled flat-panel photobioreactor operated in turbidostat mode using an incident photon flux density of 100 μmol photons m(-2) s(-1) and with only the oxygen concentration as variable parameter. The dissolved oxygen concentration was varied from 20 to 250% air saturation. Results showed that there was no clear effect of oxygen concentration on specific growth rate (mean of 0.48 ± 0.40 day(-1)) upon increasing the oxygen concentration from 20% to 75% air saturation. Upon further increasing the oxygen concentration, however, a linear decrease in specific growth rate was observed, ranging from 0.48 ± 0.40 day(-1) at a dissolved oxygen concentration of 75% air saturation to 0.18 ± 0.01 day(-1) at 250% air saturation. In vitro data on isolated Rubisco were used to predict the quantum yield at different oxygen concentrations in the medium. The predicted decrease in quantum yield matches well with the observed decrease that was measured in vivo. These results indicate that the effect of oxygen on growth of Nannochloropsis sp. at low light intensity is only due to competitive inhibition of the Rubisco enzyme. At these sub-saturating light conditions, the presence of high concentrations of oxygen in the medium induced slightly higher carotenoid content, but the increased levels of this protective antioxidant did not diminish the growth-inhibiting effects of oxygen on the Rubisco.

The scalability of microalgae growth systems is a primary research topic in anticipation of the commercialization of microalgae-based biofuels. To date, there is little published data on the productivity of microalgae in growth systems that are scalable to commercially viable footprints. To inform the development of more detailed assessments of industrial-scale microalgae biofuel processes, this paper presents the construction and validation of a model of microalgae biomass and lipid accumulation in an outdoor, industrial-scale photobioreactor. The model incorporates a time-resolved simulation of microalgae growth and lipid accumulation based on solar irradiation, species specific characteristics, and photobioreactor geometry. The model is validated with 9 weeks of growth data from an industrially-scaled outdoor photobioreactor. Discussion focuses on the sensitivity of the model input parameters, a comparison of predicted microalgae productivity to the literature, and an analysis of the implications of this more detailed growth model on microalgae biofuels lifecycle assessment studies.

Background: Microalgae are a potential source of sustainable commodities of fuels, chemicals and food and feed additives. The current high production costs, as a result of the low areal productivities, limit the application of microalgae in industry. A first step is determining how the different production system designs relate to each other under identical climate conditions. The productivity and photosynthetic efficiency of Nannochloropsis sp. CCAP 211/78 cultivated in four different outdoor continuously operated pilot-scale photobioreactors under the same climatological conditions were compared. The optimal dilution rate was determined for each photobioreactor by operation of the different photobioreactors at different dilution rates. Results: In vertical photobioreactors, higher areal productivities and photosynthetic efficiencies, 19-24 g m-2 day-1 and 2.4-4.2 %, respectively, were found in comparison to the horizontal systems; 12-15 g m-2 day-1 and 1.5-1.8 %. The higher ground areal productivity in the vertical systems could be explained by light dilution in combination with a higher light capture. In the raceway pond low productivities were obtained, due to the long optical path in this system. Areal productivities in all systems increased with increasing photon flux densities up to a photon flux density of 30 mol m-2 day-1. Photosynthetic efficiencies remained constant in all systems with increasing photon flux densities. The highest photosynthetic efficiencies obtained were; 4.2 % for the vertical tubular photobioreactor, 3.8 % for the flat panel reactor, 1.8 % for the horizontal tubular reactor, and 1.5 % for the open raceway pond. Conclusions: Vertical photobioreactors resulted in higher areal productivities than horizontal photobioreactors because of the lower incident photon flux densities on the reactor surface. The flat panel photobioreactor resulted, among the vertical photobioreactors studied, in the highest average photosynthetic efficiency, areal and volumetric productivities due to the short optical path. Photobioreactor light interception should be further optimized to maximize ground areal productivity and photosynthetic efficiency.

The present work established an efficient staining method for fluorescence activated cell sorting (FACS) with Chlorococcum littorale maintaining cellular viability. The method was designed to detect high-lipid cells and to guarantee cellular viability. BODIPY505/515 (BP) was more suitable to FACS when compared to Nile red. The optimum concentrations were 0.4 μg ml−1 of BP, 0.1% DMSO or 0.35% ethanol. Both ethanol and DMSO were equally efficient and assured cellular viability after the staining and sorting. Here a method is presented to rapidly screen and sort lipid rich cells of C. littorale with FACS, which can be used to produce new inoculum with increased cellular lipid content.

Submitted by MAASA Secretaria (maasa@ufba.br) on 2015-08-31T19:17:13Z No. of bitstreams: 1 Nanochloropsis in Brackish Groundwater.pdf: 274293 bytes, checksum: 0daa202d6a477db56753fff16e12db56 (MD5) Approved for entry into archive by LIVIA FREITAS (livia.freitas@ufba.br) on 2015-11-25T14:17:54Z (GMT) No. of bitstreams: 1 Nanochloropsis in Brackish Groundwater.pdf: 274293 bytes, checksum: 0daa202d6a477db56753fff16e12db56 (MD5) Made available in DSpace on 2015-11-25T14:17:54Z (GMT). No. of bitstreams: 1 Nanochloropsis in Brackish Groundwater.pdf: 274293 bytes, checksum: 0daa202d6a477db56753fff16e12db56 (MD5) Previous issue date: 2014 The aim of this work was to study growth potential of the green microalgae Nannochloropsis sp. using brackish groundwater from a well in the semi-arid northeast region of Brazil as culture medium. The medium was supplemented with (%) 19.4, 22.0, 44.0 and 50.0% of municipal wastewater after UASB treatment as a low-cost nutrient source. The results showed that the culture tested was capable of growing in the brackish groundwater even at salinity levels as low as 2 ppt. Furthermore it was shown that municipal wastewater could be used as a sole nutrient source for Nannochloropsis sp. Brazilian Archives of Biology and Technology

In most microalgal species, triacyglycerols (TAG) contain mostly saturated and monounsaturated fatty acids, rather than PUFA, while PUFA-enriched oil is the form most desirable for dietary intake. The ability of some species to produce LC-PUFA-enriched oil is currently of specific interest. In this work, we investigated the role of sodium bicarbonate availability on lipid accumulation and n-3 LC-PUFA partitioning into TAG during batch cultivation of Pavlova lutheri. Maximum growth and nitrate uptake exhibit an optimum concentration and threshold tolerance to bicarbonate addition (similar to 9 mM) above which both parameters decreased. Nonetheless, the transient highest cellular lipid and TAG contents were obtained at 18 mM bicarbonate, immediately after combined alkaline pH stress and nitrate depletion (day nine), while oil body and TAG accumulation were highly repressed with low carbon supply (2 mM). Despite decreases in the proportions of EPA and DHA, maximum volumetric and cellular EPA and DHA contents were obtained at this stage due to accumulation of TAG containing EPA/DHA. TAG accounted for 74% of the total fatty acid per cell, containing 55% and 67% of the overall cellular EPA and DHA contents, respectively. These results clearly demonstrate that inorganic carbon availability and elevated pH represent two limiting factors for lipid and TAG accumulation, as well as n-3 LC-PUFA partitioning into TAG, under nutrient-depleted P. lutheri cultures.

Analysis of bulk biochemical composition is a key in fundamental and applied studies of microalgae and is essential to understanding responses to different cultivation scenarios. Traditional biochemical methods for the quantification of lipids, carbohydrates and proteins are often time-consuming, often involve hazardous reagents, require significant amounts of biomass and are highly dependent on practitioner proficiency. This study presents a rapid and non-destructive method, utilising Fourier-Transform Infrared (FTIR) spectroscopy for the simultaneous determination of lipid, protein and carbohydrate content in microalgal biomass. A simple univariate regression was applied to sets of reference microalgal spectra of known composition and recognised IR peak integrals. A robust single-species model was constructed, with coefficients of determination r2>0.95, high predictive accuracy and relative errors below 5%. The applicability of this methodology is demonstrated by monitoring the time-resolved changes in biochemical composition of the marine alga Nannochloropsis sp. grown to nitrogen starvation. © 2013 Elsevier Ltd.

Biofuel production from microalgal biomass could be an alternative solution to conventional biofuels typically dependent on food and high land/water demanding crops. However, the economic and energetic viability of microalgal biofuels is limited by their harvesting processes. The finding of innovative, low cost and efficient harvesting method(s) is imperative. In this study, the Electro-Coagulation (EC) was studied as a process to harvest the marine Nannochloropsis sp. microalga. Several EC operational conditions were studied and the best EC recovery efficiency (>97%) was achieved using a current density of 8.3 mA cm −2 for 10 min. The quality of the recovered microalgal biomass was evaluated in terms of total lipids, fatty acid and pigment profile where no significant differences were observed after EC treatment. The energy requirements of the harvesting process were estimated and the combination of EC and centrifugation processes proved to decrease significantly the energy demand when compared with the individual process.

Nannochloropsis sp. is an oil-rich microalga. The lipid content of cells at stationary growth phase was much higher than that of cells at logarithm growth phase, but very little information focusing on the abundances of gene transcripts concerning lipid accumulation is available. In present study, the second generation Illumina sequencing was used to investigate the genes and pathways of Nannochloropsis sp. in order to identify those involved in lipid metabolism. In total, 29,203 unigenes were obtained from logarithm and stationary growth phases. The transcripts of some unigenes were differentially abundant at two growth phases. Among the unigenes obtained, about 195 unigenes were involved in lipid metabolism and 315 unigenes were putatively transcription factors. These genes are a very useful resource for the future studies of Nannochloropsis sp.

A photobioreactor (PBR) for microalgae culture is a highly efficient system for biomass production. In the present study, the performance of an airlift (ALR) (with a centric-tube column) and a bubble column (BC) photobioreactors were compared considering Nannochloropsis sp. growth. The experiments were carried out keeping average light intensity, temperature, volume culture, and CO2 supply constant, while cell concentration and pH level were measured and examined. Furthermore, a computational fluid dynamics (CFD) simulation in cfx, ansys 11.0, was developed using a multiphase flow model with an Eulerian approach to evaluate the hydrodynamic behavior of both systems. The results showed that a higher cell concentration (375 × 105 cell/ml) was obtained in the airlift PBR yielding a better growth rate than the bubble column PBR. In terms of hydrodynamic performance, the existence of the centric-tube in the airlift system shows a well-defined flow pattern, better light distribution cycle, and more effective mixing. These hydrodynamic characteristic of the airlift PBR may allow a better yield for the microalgae biomass production.

In this study, chitosan and nano-chitosan were used as flocculants agents for harvesting microalga Nannochloropsis sp. chitosan was modified to nano-chitosan by crosslinking with sodium tripolyphosphate. The effects of type and dosage of flocculants and the pH of the culture were investigated on biomass recovery. Optimum dosages for both bio-flocculants were found. The results showed that the dosage of flocculant consumption decreases by 40% and biomass recovery increases by 9% when nano-chitosan instead of chitosan is used as flocculant agent. Also, the recycled water from the harvesting process was reused which increases the growth of microalgae by about 7%. Finally, the cost analysis of harvesting process showed the feasibility of using nano-chitosan as flocculation agent.

Illumination factors such as length of photoperiod and intensity can affect growth of microalgae and lipid content. In order to optimize microalgal growth in mass culture system and lipid content, the effects of light intensity and photoperiod cycle on the growth of the marine microalgae, Nannochloropsis sp. were studied in batch culture. Nannochloropsis sp. was grown aseptically for 9 days at three different light intensities (50, 100 and 200 μmol m(-2) s(-1)) and three different photoperiod cycles (24:0, 18:06 and 12:12 h light:dark) at 23 °C cultivation temperature. Under the light intensity of 100 μmol m(-2) s(-1) and photoperiod of 18 h light: 6 h dark cycle, Nannochloropsis sp. was found to grow favorably with a maximum cell concentration of 6.5×10(7) cells mL(-1), which corresponds to the growth rate of 0.339 d(-1) after 8 day cultivation and the lipid content was found to be 31.3%.

The microalga Nannochloropsis sp. was used in this study, in a biorefinery context, as biomass feedstock for the production of fatty acids for biodiesel, biohydrogen and high added-value compounds. The microalgal biomass, which has a high lipid and pigment content (mainly carotenoids), was submitted to supercritical CO 2 extraction. The temperature, pressure and solvent flow-rate were evaluated to check their effect on the extraction yield. The best operational conditions to extract 33 g lipids /100 g dry biomass were found to be at 40 °C, 300 bar and a CO 2 flow-rate of 0.62 g/min. The effect of adding a co-solvent (ethanol) was also studied. When supercritical CO 2 doped with 20% (w/w) ethanol was used, it was possible to extract 45 g lipids /100 g dry biomass of lipids and recover 70% of the pigments. Furthermore, the remaining biomass after extraction was effectively used as feedstock to produce biohydrogen through dark fermentation by Enterobacter aerogenes resulting in a hydrogen production yield of 60.6 mL/g dry biomass .

International audience; Keywords: Ultrasound-assisted extraction (UAE) Bio-oil Response surface methodology (RSM) Nannochloropsis sp. Scanning electron microscopy (SEM)–gas chromatography (GC) a b s t r a c t In order to comply with criteria of green chemistry concepts and sustainability, a new procedure has been performed for solvent-free ultrasound-assisted extraction (UAE) to extract lipids from fresh Nannochlor-opsis oculata biomass. Through response surface methodology (RSM) parameters affecting the oil recovery were optimized. Optimum conditions for oil extraction were estimated as follows: (i) 1000 W ultrasonic power, (ii) 30 min extraction time and (iii) biomass dry weight content at 5%. Yields were calculated by the total fatty acids methyl esters amounts analyzed by GC–FID–MS. The maximum oil recovery was around 0.21%. This value was compared with the one obtained with the conventional extraction method (Bligh and Dyer). Furthermore, effect of temperature on the yield was also investigated. The overall results show an innovative and effective extraction method adapted for microalgae oil recovery, without using solvent and with an enable scaling up.