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.
Biochemical methane potential of four species of Ulva and Gracilaria genus was assessed in batch assays at mesophilic temperature. The results indicate a higher specific methane production (per volatile solids) for one of the Ulva sp. compared with other macroalgae and for tests running with 2.5% of total solids (196 ± 9 L CH4 kg−1 VS). Considering that macroalgae can potentially be a post treatment of municipal wastewater for nutrients removal, co-digestion of macroalgae with waste activated sludge (WAS) was assessed. The co-digestion of macroalgae (15%) with WAS (85%) is feasible at a rate of methane production 26% higher than WAS alone without decreasing the overall biodegradability of the substrate (42–45% methane yield). The use of anoxic marine sediment as inoculum had no positive effect on the methane production in batch assays. The limiting step of the overall anaerobic digestion process was the hydrolysis.
In the present study, the use of immobilization technology to cultivate microalgae in entrapped matrix gel beads was demonstrated. Since the gel beads are denser in water, the beads can be easily collected through simple filtration method and hence, simplifying the overall separation process. Various parameters were investigated to optimize the growth rate of immobilized microalgae and the optimum conditions were obtained as: alginate to microalgae volume ratio of 0.3, Ca2+ concentration of 2%, organic nutrients concentration of 50 mL (equivalent to 13.09 mg/L nitrate), initial culture pH of 4 and photoperiod of 24 h. Using this optimum culture condition, 0.50 mg biomass/bead was attained on the 10th day of cultivation. Apart from that, this study also attempted to co-immobilize nutrients into microalgae beads in order to minimize free cell culture (microalgae cells that are released into the culture medium due to rupturing of beads) and to reduce water consumption. Through this approach, it was found that microalgae biomass yield increased to 0.67 mg/bead within a shorter culturing time (5 days) with insignificant amount of free cell culture detected. Furthermore, lipid extracted from immobilized microalgae biomass has high potential for biodiesel production due to the similarity of fatty acid profile with other oil bearing crops.
In order to develop feasible production processes for microalgal biodiesel, the isolation of high neutral lipid producing microalgae is crucial. Since the established Nile Red (NR) method for detection of intracellular lipids has been successful only for some microalgae, a more broadly applicable detection method would be desirable. Therefore, BODIPY 505/515, a lipophilic bright green fluorescent dye was tested for detection of intracellular lipids in Chlorella vulgaris, Dunaliella primolecta and Chaetoceros calcitrans. An optimum concentration of 0.067 μg ml−1 was determined for lipid staining in the microalgae. Compared to NR, BODIPY 505/515 was more effective in staining microalgae and showed resistance to photobleaching, maintaining its fluorescence longer than 30 min.
Future micro-algal biofuels will most likely be derived from open-pond production systems. These are by definition open to “invasion” by grazers, which could devastate micro-algal mass-cultures. There is an urgent requirement for methodologies capable of early detection and control of grazers in dense algal cultures. In this study a model system employing the marine alga Nannochloropsis oculata was challenged by grazers including ciliates, amoebae and a heterotrophic dinoflagellate. A FlowCAM flow-cytometer was used to detect all grazers investigated (size range <20–>80 μm in length) in the presence of algae. Detection limits were <10 cells ml−1 for both “large” and “small” model grazers, Euplotes vannus (80 × 45 μm) and an unidentified holotrichous ciliate (∼18 × 8 μm) respectively. Furthermore, the system can distinguish the presence of ciliates in N. oculata cultures with biotechnologically relevant cell densities; i.e. >1.4 × 108 cells ml−1 (>0.5 g l−1 dry wt.).
Microalgae-based bioenergy has gained extensive attention, but the consumption of non-renewable resource such as phosphorous is inevitable in the production of its feedstock. In this work, the minimal phosphorous consumption for algal biomass production of Scenedesmus sp. LX1 was investigated by monitoring the growth and nutrient uptake under two different cultivation modes: phosphorous-starvation and luxury-nutrient. The results showed that continuous nitrogen and phosphorous feeding in luxury-nutrient mode had no stimulating effect on biomass productivity at the nutrient level in this study, TN: 245 mg L−1, TP: 5.4 mg L−1. However, the sustained growth of biomass after the exhaust of phosphate in phosphorous-starvation mode led to significant increase in the biomass yield of phosphorous up to 160 g biomass/g -P, which was nearly six times more than that with nutrient feeding. To minimize phosphorous resource consumption in production of algal biomass, a phosphorous-starvation cultivation mode is proposed.
A bacterial-based bioremediation product, LakeRelief™ by Novozymes (Waterguru LakeRelief, 2011), was tested in a series of experiments between October 2008 and March 2009 to evaluate its suitability as a short-term intervention technique to reduce algal blooms in the Swan–Canning River system. Results from fibreglass tank experiments (1100 L) suggested that the product did not actively attack and lyse algal cells. The product decreased NH4 and NOx concentrations in treated tanks, both aerated and non-aerated. Product application decreased PO4 concentrations in non-aerated tanks but not in aerated tanks. The product appeared to suppress algal growth in non-aerated tanks over short periods (several days). Algal growth regularly diminished after product application but reappeared shortly afterwards. Aeration had a negative effect on bacterial proliferation in the tanks, possibly through alteration of environmental conditions (e.g. water mixing). As a consequence of the environmental conditions in the tanks being counterproductive to the development of a representative microbial composition, several aspects regarding the product’s effectiveness could not be assessed satisfactorily in the tank experiments. The importance of long-term nutrient immobilisation into a well developed food web and the subsequent nutrient removal through removal of the top order organisms is highlighted.
A novel cultivation strategy called “sequential heterotrophy–dilution–photoinduction” was developed for efficient algal biomass and lipid production. Three Chlorella species were first cultivated heterotrophically to achieve high cell density, then the broth was diluted to suitable concentration (2–5 g/L) and transferred to light environment for photoinduction. With this strategy, the Chlorella intracellular protein and chlorophyll increased rapidly to 50.87% and 32.97 mg/g by a 12-h illumination, which were close to the level of cells cultivated photoautotrophically. Moreover, the lipid contents were increased by 84.57%, 70.65% and 121.59% within 24-h photoinduction for C. vulgaris, C. pyrenoidosa and C. ellipsoidea, respectively. Maximum lipid content as 26.11% of biomass and maximum lipid productivity of 89.89 mg/L/d was both accomplished by C. pyrenoidosa. Further outdoor experiments showed consistent patterns. Therefore, the proposed strategy provided an effective approach for microalgal biomass production to meet the urgent need for both health food and biodiesel.
A new arabinomannan has been isolated from the cell wall of the green algae Chlorella vulgaris by extraction with 0.1 M NaOH, dialysis and SEC fractionation. Mw was about 8000 u. Terminal, 2- and 5-O-linked arabinofuranosyl residues, and 2,6-O-linked mannopyranosyl residues were detected as the main constituents beside some minor mannose components by methylation analysis. Electrospray ionisation mass spectrometry (ESI–MS) with collision induced dissociation (CID) up to MS3 experiments of oligosaccharides obtained by partial methanolysis or partial acid hydrolysis indicated the presence of Man→Man- and Ara→Ara- as well as Ara→Man- and also Man→Ara-sequences. MS2 experiments gave evidence of a (1→6)-linked mannan, probably also including some (1→2)-linked mannosyl and 5-linked arabinofuranosyl residues. Mannooligosaccharides up to DP5 with mainly (1→6)-, but at higher DP also (1→2)-linkages were obtained by acid hydrolysis, and arabino oligomers up to DP4 could be detected after mild methanolysis. In accordance with results from methylation analysis and ESI–MS/CID t-β-Araf, 2-α-Araf, 5-α-Araf and 2,6-α-Manp were identified from homo- and heteronuclear 1D- and 2D NMR experiments in a molar ratio of ∼2:2:1:2. A highly branched structure is suggested with a 6- and 2-O-linked mannan main chain, comprising 5-Araf residues. Araf-β-[(1→2)-Araf-α-(1→]n side chains with an average chain length of 2 are linked to the mannan main chain. The configuration of Ara is d. The new polysaccharide shows structural similarities with the (lipo)arabinomannans of Mycobacterium tuberculosis.
The effect of storage temperature and time on lipid composition of Scenedesmus sp. was studied. When stored at 4 °C or higher, the free fatty acid content in the wet biomass increased from a trace to 62.0% by day 4. Using two-step catalytic conversion, algae oil with a high free fatty acid content was converted to biodiesel by pre-esterification and transesterification. The conversion rate of triacylglycerols reached 100% under the methanol to oil molar ratio of 12:1 during catalysis with 2% potassium hydroxide at 65 °C for 30 min. This process was scaled up to produce biodiesel from Scenedesmus sp. and Nannochloropsis sp. oil. The crude biodiesel was purified using bleaching earth. Except for moisture content, the biodiesel conformed to Chinese National Standards.
Increasing energy prices demand a renewable, carbon neutral, transport fuel that is environmentally and commercially sustainable. The interest in the production of microalgae as biofuels is increasing due to their high oil content, rapid biomass production and small foot print. In this research, marine microalgae Dunaliella tertiolecta (Chlorophyceae) and Thalassiosira pseudonana (Bacillariophyceae) were incubated in nitrogen (N)-replete medium, and then transferred to N-free medium for 15 and 11 days, respectively. Fluorescence induction and relaxation (FIRe) fluorometry and Fourier transform infrared spectroscopy (FTIR) were used to monitor the photosynthetic performance, lipid production and metabolic responses to changing N availability. Growth rates of D. tertiolecta and T. pseudonana were 0.84 ± 0.16 d−1 and 1.21 ± 0.09 d−1, respectively in N-replete medium. Upon transfer to N-free medium. The growth rates of T. pseudonana declined rapidly, while D. tertiolecta continued to grow for 5 days in N-free medium before growth declined slowly. The maximum quantum yield of photochemistry (Fv/Fm) remained high initially for D. tertiolecta but decreased immediately after transfer to N-free media for T. pseudonana. The functional absorption cross section for PSII (σPSII) increased, the time constant for QA reoxidation (τQa) and connectivity factor (p) decreased in parallel to the nutritional status of the microalgae. The relative protein and lipid content varied in response to N limitation, but carbohydrates did not change. Based on FTIR, D. tertiolecta and T. pseudonana produced 20–26% lipid when most stressed. The combination of photosynthetic efficiency and biomass composition monitoring provided evidence that metabolic strategies to changing nutrient status are species-specific.
The cost analysis of a real facility for the production of high value microalgae biomass is presented. The facility is based on ten 3 m3 tubular photobioreactors operated in continuous mode for 2 years, data of Scenedesmus almeriensis productivity but also of nutrients and power consumption from this facility being used. The yield of the facility was close to maximum expected for the location of Almería, the annual production capacity being 3.8 t/year (90 t/ha·year) and the photosynthetic efficiency being 3.6%. The production cost was 69 €/kg. Economic analysis shows that labor and depreciation are the major factors contributing to this cost. Simplification of the technology and scale-up to a production capacity of 200 t/year allows to reduce the production cost up to 12.6 €/kg. Moreover, to reduce the microalgae production cost to approaches the energy or commodities markets it is necessary to reduce the photobioreactor cost (by simplifying its design or materials used), use waste water and flue gases, and reduce the power consumption and labor required for the production step. It can be concluded that although it has been reported that production of biofuels from microalgae is relatively close to being economically feasible, data here reported demonstrated that to achieve it by using the current production technologies, it is necessary to substantially reduce their costs and to operate them near their optimum values.
This study was performed to investigate the applicability of submerged microfiltration as a first step of up-concentration for harvesting both a freshwater green algae species Chlorella vulgaris and a marine diatom Phaeodactylum tricornutum using three lab-made membranes with different porosity. The filtration performance was assessed by conducting the improved flux step method (IFM) and batch up-concentration filtrations. The fouling autopsy of the membranes was performed by scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX) and Fourier transform infrared spectroscopy (FTIR). The cost analysis was estimated based on the data of a related full-scale submerged membrane bioreactor (MBR). Overall results suggest that submerged microfiltration for algal harvesting is economically feasible. The IFM results indicate a low degree of fouling, comparable to the one obtained for a submerged MBR. By combining the submerged microfiltration with centrifugation to reach a final concentration of 22% w/v, the energy consumption to dewater C. vulgaris and P. tricornutum is 0.84 kW h/m3 and 0.91 kW h/m3, respectively.
A new process evaluation methodology of microalgae biodiesel has been developed. Based on four evaluation criteria, i.e. the net energy ratio (NER), biodiesel production costs, greenhouse gases (GHG) emission rate and water footprint, the model compares various technologies for each step of the process, from cultivation to oil upgrading. An innovative pathway (hybrid raceway/PBR cultivation system, belt filter press for dewatering, wet lipid extraction, oil hydrotreating and anaerobic digestion of residues) shows good results in comparison to a reference pathway (doubled NER, lower GHG emission rate and water footprint). The production costs are still unfavourable (between 1.94 and 3.35 €/L of biodiesel). The most influential parameters have been targeted through a global sensitivity analysis and classified: (i) lipid productivity, (ii) the cultivation step, and (iii) the downstream processes. The use of low-carbon energy sources is required to achieve significant reductions of the biodiesel GHG emission rate compared to petroleum diesel.
This work reports, for the first time, the determination of major and trace elements (Al, As, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, La, Mg, Mn, Mo, Na, Ni, P, Pb, S, Se, Sn, Sr, Ti, Tl, U, V, and Zn) in the fractions of the synthesis of fatty acid methyl esters (FAMEs). These include fresh microalgae, residual biomass, lipid fraction, crude FAMEs, insoluble fraction and purified FAMEs from microalgae Chlorella sp. A microwave-assisted digestion procedure in closed vessels was applied for sample digestion and subsequent element determination by inductively coupled plasma-based techniques. The proposed method was suitable for the multielement determination in FAMEs and its fractions obtained from microalgae. The element concentration was compared with results found in the literature and a careful discussion about the use of residual biomass for different applications was performed.
A life cycle assessment (LCA) and an energy balance analysis of marine microalgal biomass production were conducted to determine the environmental impacts and the critical points of production for large scale planning. The artificial lighting and temperature conditions of an indoor bubble column photobioreactor (bcPBR) were compared to the natural conditions of an equivalent outdoor system. Marine microalgae, belonging to the dinoflagellate and raphidophyte groups, were cultured and the results were compared with published LCA data obtained from green microalgae (commonly freshwater algae). Among the species tested, Alexandrium minutum was chosen as the target marine microalgae for biomass production under outdoor conditions, although there were no substantial differences between any of the marine microalgae studied. Under indoor culture conditions, the total energy input for A. minutum was 923 MJ kg−1 vs. 139 MJ kg−1 for outdoor conditions. Therefore, a greater than 85% reduction in energy requirements was achieved using natural environmental conditions, demonstrating the feasibility of outdoor culture as an alternative method of bioenergy production from marine microalgae. The growth stage was identified as the principal source of energy consumption for all microalgae tested, due to the electricity requirements of the equipment, followed by the construction material of the bcPBR. The global warming category (GWP) was 6 times lower in outdoor than in indoor conditions. Although the energy balance was negative under both conditions, this study concludes with suggestions for improvements in the outdoor system that would allow up-scaling of this biomass production technology for outdoor conditions in the Mediterranean.
Direct and continuous measurement of dissolved CO2 (dCO2) is crucial for intensive aquaculture, especially in shallow raceway systems (SRS). In this work the performance of a portable dissolved CO2 probe analyzer was tested for the effects of different aqueous solutions, pure oxygen injection and agitation. Laboratory results showed significant (p < 0.05) solution effects on probe performance for low (10–20 mg L−1) and high (30–50 mg L−1) dCO2 concentrations. Globally performance was better in deionized water, followed by marine fish farm water and artificial seawater. Accuracy and response time were the parameters most affected by the type of solution tested. Linearity was always observed (R2 = 0.995–0.999). The probe was sensitive to 1 mg L−1 dCO2 increments for concentrations <6 mg L−1 in artificial seawater. Pure oxygen injection did not affect probe readouts, and agitation was needed for better accuracy and response time. In real marine SRS with tanks in series dCO2 dynamics was revealed using the probe coupled to a developed flow cell. A prototype SRS was built and used to study dCO2 dynamics without endangering cultivated fish. Generally, results obtained indicate that the probe tested although precise, is better suited for discrete, single-point dCO2 monitoring, being a limited resource for the special needs of shallow raceway systems. As SRS represent a paradigm change in aquaculture, new water quality monitoring strategies and instrumentation are needed, especially for dCO2. Fiber optic sensors can be a solution for continuous, multipoint monitoring, thus contributing to the understanding of water quality dynamics in hyperintensive aquaculture systems.
An alternating current was used to generate an electric field to enhance the fluorescent labeling of microalgae cellular lipids with Nile red and LipidTOX. The decay of the fluorescence intensity of Chlorella vulgaris cells in 0 V/cm was more than 50% after 10 min, and the intensity variation was as high as 7% in 20 s. At 2000 V/cm, the decay rate decreased to 1.22% per minute and the intensity fluctuation was less than 1% for LipidTOX-labeled cells. For Spirulina sp. cells at 0 V/cm, the fluorescence intensity increased by 10% after 10 min, whereas at 2000 V/cm, labeling was more rapid and fluorescence intensity doubled. These results show that applying an electric field can improve the quality of fluorescence detection by alleviating decay and fluctuation or by enhancing signal intensity.
Microalgal biomasses have been produced industrially for a long history for application in a variety of different fields. Most recently, microalgae are established as the most promising species for biofuel production and CO2 bio-sequestration owing to their high photosynthesis efficiency. Nevertheless, design of photobioreactors that maximize solar energy capture and conversion has been one of the major challenges in commercial microalga biomass production. In this review, we systematically survey the recent developments in this field.
Flocculation induced by pH increase for harvesting microalgae and reuse of flocculated medium were evaluated. Increasing the medium pH value induced the highest flocculation efficiency of up to 90% for freshwater microalgae (Chlorella vulgaris, Scenedesmus sp., Chlorococcum sp.) with low/medium biomass concentrations and marine microalgae (Nannochloropsis oculata, Phaeodactylum tricornutum). The mechanism may be explained that Mg2+ in the growth medium hydrolyzed to form magnesium hydroxide precipitate, which coagulated microalgal cells by sweeping flocculation and charge neutralization. Additionally, this study revealed that the microalgal biomass concentrations and released polysaccharide (RPS) from microalgae could influence the flocculation efficiencies. Furthermore, neutralizing pH and then supplementing nutrients allowed the flocculated medium to maintain an approximate growth yield to that of the fresh medium in algal cultivation. These results suggest that the method presented here is effective, and allows the reuse of the flocculated medium, thereby contributing to the economic production from algae to biodiesel.