Non-invasive methods for measuring lipid content in live microalgal cultures are critically needed for algal biofuel research and development. A non-destructive method requiring minimal sample preparation was developed utilizing liquid state ¹H NMR for quantifying triacylglycerides (TAGs) in live algae cultures. ¹H NMR and ¹H HR-MAS spectra of live algae cells show outstanding correlation with published chemical shifts for TAGs. ¹H NMR spectra of Chlamydomonas reinhardtii whole cells and isolated lipid bodies were compared with a standard oleic TAG ¹H spectrum, demonstrating that only lipid body TAGs were observed. A “model” TAG was derived, providing a proton count and molecular weight for conversion of TAG ¹H NMR integrals to volumetric TAG or fatty acid methyl ester (FAME) equivalent concentrations, which were correlated to FAME concentrations by gas chromatography (FAME-GC) at several time points. A customized NMR flow cell was subsequently constructed, allowing real-time, continuous measurements of multiple cultures.
The review highlights the correlation between removal of various eco-toxins by micro algae and the production of high-value products thereof. It appraises established and novel strategies for micro algal cultivation, downstream processing methods for product recovery, and recent progress in algal generation of the green energy carriers biogas and biohydrogen micro algae. The suitability of selected micro algal species for various final products, and the potential of different strains for abating environmental problems are discussed.
Due to the fact that low cell densities and moderate growth rates are known as the major obstacles towards a broad market penetration of micro algal products, the article shows how high cell densities and reasonable volumetric productivities can be obtained. Here, the article deals with the improvements of process design and nutrient supply regimes that are needed to achieve these goals.
As demonstrated by an integrated case study, mixotrophic cultivation results in increased biomass concentration in a first cultivation step for some micro algal strains like Nannochloropsis oculata. In a second step, the fresh active algal biomass accumulates desired products via CO2 fixation, e.g. from industrial effluent gases, as the sole carbon source. This can be realized by a novel, two-stage, continuously operated closed photo-bioreactor system. After cell harvest and optimized product recovery, the value-added conversion of residual algal biomass for generation of green energy carriers, e.g. in biogas plants, constitutes another focal point of the ongoing research.
Transesterification is the most common method for producing biodiesel. Known as a suitable substitute to diesel fuel, the synthesis involves renewable sources as feedstock. Application of both organic and inorganic solvents in biodiesel production has been widely established. However, as the properties of conventional solvents are perpetually hazardous to human and environment, utilization of greener alternative is a better option. Among the various types of solvents available, ionic liquid seems prevalent. An ionic liquid is a combination of cations and anions, has low or negligible vapor pressure, and exists as liquid at temperature below 100 °C. The prospect of ionic liquids as green solvents in chemical processes is increasing in recent years, especially in biodiesel synthesis. This paper highlighted the properties of ionic liquids that emphasized their versatility as solvents, and the use of switchable ionic liquids as green solvents is also presented. The roles of ionic liquids in biodiesel synthesis are discussed, focusing on their pertinent capability as solvents, particularly as catalysts for transesterification reaction. Since the cost of ionic liquid may be an issue, a brief discussion about the recyclability of ionic liquids is also included.
In recent years herbicides and heavy metals have surfaced as the most significant environmental pollutants due to rapid industrialization and an increase in population worldwide. While quite effective and efficient, some of the treatment methods, for the removal of these environmental pollutants, involve high operating and maintenance cost along with producing toxic byproducts. Thus, biosorbents have become an attractive alternative to other well established methods. In this study red algae, Gracilaria verrucosa was investigated as a potential biosorbent for the phenoxyalkanoic acid herbicide 2,4-D and the heavy metal Cr(VI). The sorption capacity was found to be 22.3 mg g− 1 and 113.2 mg g− 1 respectively. In order to establish the efficiency of G. verrucosa as a biosorbent, sorption tests were conducted with different environmental and process parameters. This study verified that the acid treated biomass, indicates a gradual 47% and 21% increment for 2,4-D and Cr(VI) sorption compared with alkali, formaldehyde, and alcohol treatment. The sorption tests of the red algae G. verrucosa; offer two times more heavy metal removal when compared with low cost biosorbents. Two and three parameter equilibrium models were used to describe the sorption process. Experimental and modeling studies indicate that the sorption process of the investigated sorbents is physisorption with monolayer and endothermic characteristics. Also, surface properties of G. verrucosa were investigated and the effects of the surface active groups were identified. For 2,4 D sorption hydroxyl, carboxyl, and amine and for Cr(VI) sorption hydroxyl, carbonyl, and amino groups were identified as the important surface active groups.
In order to efficiently convert microalgae into value added products, a sustainable integrated algal biorefinery is needed. Generally, conversion of microalgae into biofuel involves several processing steps: cultivation, harvesting, dewatering, drying, oil extraction, and biofuel production. One of the main challenges in designing and optimizing an integrated algal biorefinery is determining the configuration which meets the requirements for key outputs as well as environmental and resource limits. In this work, a systematic fuzzy linear programming (FLP) approach for design and optimization of an integrated algal biorefinery which considers water footprint, land footprint, and carbon footprint is presented. A hypothetical case study is presented to illustrate the proposed approach.
Algae show great potential for use as a feedstock for energy applications, but the cost of production must be reduced to compete with traditional feedstocks. One method of doing so is to optimize the algae production photobioreactor. This study presents the method of obtaining, and results of, such an optimization using a model derived from computational fluid dynamics simulations.
A two-stage process, composed of growth under nutrient-rich conditions followed by cultivation under nitrogen starvation and controlled conditions of phosphate, light intensity, aeration, and carbon sources was applied for lipid production by the green alga Chlorella vulgaris. Using conditions without addition of nitrogen, 2 mg/L PO4-P, light intensity of 100 μmol/m2/s and 0.25 vvm of air, about 43% of dry cell weight accumulated as lipids after 12 h, which equates to a lipid productivity of 77.8 mg/L/d. In a medium containing 5 mg/L NO3-N and 2 mg/L PO4-P, and at a light intensity of 100 μmol/m2/s and 0.25 vvm of 2% CO2, about 53% of dry cell weight consisted of lipids after 24 h, representing a lipid productivity of 77.1 mg/L/d. The low amount of nutrients, moderate aeration and light intensity were helpful for increasing lipid productivity.
Oilseed meals that are by-products from oil production are potential resources for protein. The aim of this work is to investigate the use of enzymes in assisting in the extraction of protein from different oilseed meals, namely rapeseed, soybean, and microalgae meals. In addition, microalgae without prior oil removal was also tested. The extraction was performed varying temperature, pH, and type of enzyme. More protein was extracted at alkaline conditions, compared to acidic conditions. At alkaline pH, 80% protein of soybean meal and 15–30% protein of rapeseed and microalgae meals was extracted without enzyme addition. The addition of enzyme under this condition increased protein extraction yield to 90% for soybean meal and 50–80% for rapeseed and microalgae meals. Here, Protex 40XL, Protex P, and Protex 5L that work at alkaline pH assisted protein extraction particularly for rapeseed and microalgae meals. Microalgae without prior oil removal had the lowest protein extraction yield, illustrating that oil removal prior to protein extraction is beneficial for protein recovery. In general, protein extraction was influenced by pH, the type of biomass, and the addition of enzyme, but not by the type of enzyme that was used.
The effect of the bacterium Azospirillum brasilense jointly immobilized with Chlorella vulgaris or C. sorokiniana in alginate beads on total carbohydrates and starch was studied under dark and heterotrophic conditions for 144 h in synthetic growth medium supplemented with either d-glucose or Na-acetate as carbon sources. In all treatments, enhanced total carbohydrates and starch content per culture and per cell was obtained after 24 h; only jointly immobilized C. vulgaris growing on d-glucose significantly increased total carbohydrates and starch content after 96 h. Enhanced accumulation of carbohydrate and starch under jointly immobilized conditions was variable with time of sampling and substrate used. Similar results occurred when the microalgae was immobilized alone. In both microalgae growing on either carbon sources, the bacterium promoted accumulation of carbohydrates and starch; when the microalgae were immobilized alone, they used the carbon sources for cell multiplication. In jointly immobilized conditions with Chlorella spp., affinity to carbon source and volumetric productivity and yield were higher than when Chlorella spp. were immobilized alone; however, the growth rate was higher in microalgae immobilized alone. This study demonstrates that under heterotrophic conditions, A. brasilense promotes the accumulation of carbohydrates in two strains Chlorella spp. under certain time–substrate combinations, producing mainly starch. As such, this bacterium is a biological factor that can change the composition of compounds in microalgae in dark, heterotrophic conditions.
Over 50% of the energy losses associated with the conversion of solar energy into chemical energy during photosynthesis are attributed to kinetic constraints between the fast rate of photon capture by the light harvesting apparatus and the slower downstream rate of photosynthetic electron transfer. At full sunlight intensities, energy flux from the light harvesting antennae to the reaction centers may be 100-folds greater than the overall linear electron flow resulting in the dissipation of up to 75% of the captured energy as heat or fluorescence. One possible means to couple energy capture and photosynthetic electron transfer more efficiently is to reduce the optical cross-section of the light harvesting antennae. We show that by partially reducing chlorophyll b levels in the green alga, Chlamydomonas reinhardtii, we can tune the peripheral light harvesting antennae size for increased photosynthetic efficiency resulting in more than a two-fold increase in photosynthetic rate at high light intensities and a 30% increase in growth rate at saturating light intensities. Unlike chlorophyll b-less mutants which lack the peripheral light harvesting antennae; transgenics with intermediate sized peripheral antennae have the advantage that they can carry out state transitions facilitating enhanced cyclic ATP synthesis and have robust zeaxanthin–violaxanthin cycles providing protection from high light levels. It is hypothesized that the large antennae size of wild-type algae and land plants offers a competitive advantage in mixed cultures due to the ability of photosynthetic organisms with large light harvesting antennae to shade competing species and to harvest light at low flux densities.
Batch experiments were performed to study biomass growth rate, nutrient removal and carbon dioxide bio-fixation of the marine microalgae Chlorella stigmatophora. Four different cultures at different salinities were tested: wastewater (WW), synthetic wastewater (SWW), seawater (SW) and diluted seawater (DSW). Experimental results showed that Chlorella stigmatophora grew satisfactorily in all culture media, except in SWW where inhibition occurred. In all cases, biomass experimental data were fitted to the Verlhust Logistic model (R2 > 0.982, p < or = 0.05). Maximum biomass productivity (P(bmax)) and CO2 biofixation (P(vCO2)) were reached in the WW medium, 1.146g SSL(-1)day(-1) and 2.324g CO2L(-1)day(-1) respectively. The order of maximum specific growth rates (micro max) was WW >DSW>SW. In order to compare nitrogen and phosphorous removal kinetics, an estimation of the time required to reach the most restrictive concentration of total N and P in effluents as defined in the Directive 98/1565/CE (10 mg sigmaNL(-1) (T10(N)) and 1 mg sigmaPL(-1) (T1(p)) was performed. In the WW test T10(N) and T1(p) needed were of 45.15 and 32.27 hours respectively and at the end of the experimental the removal was in both 100%.
Nitrogen (N) and sulfur (S) have inter-related and distinct impacts on microalgal metabolism; with N starvation having previously been reported to induce elevated levels of the biodiesel feedstock material triacylglycerol (TAG), while S deprivation is extensively studied for its effects on biohydrogen production in microalgae. ( 1) (,) ( 2) We have previously demonstrated that N- and S-starved cells of Chlamydomonas reinhardtii display different metabolic trends, suggesting that different response mechanisms exist to compensate for the absence of those two elements. ( 3) We used C. reinhardtii CC-124 mt(-) and CC-125 mt(+) strains to test possible metabolic changes related to TAG accumulation in response to N and S deprivation, considering that gamete differentiation in this organism is mainly regulated by N. ( 4) Our findings contribute to the understanding of microalgal response to element deprivation and potential use of element deprivation for biodiesel feedstock production using microalgae, but much remains to be elucidated on the precise contribution of both N and S starvation on microalgal metabolism.
Dinoflagellate microalgae are an important source of marine biotoxins. Bioactives from dinoflagellates are attracting increasing attention because of their impact on the safety of seafood and potential uses in biomedical, toxicological and pharmacological research. Here we review the potential applications of dinoflagellate toxins and the methods for producing them. Only sparing quantities of dinoflagellate toxins are generally available and this hinders bioactivity characterization and evaluation in possible applications. Approaches to production of increased quantities of dinoflagellate bioactives are discussed. Although many dinoflagellates are fragile and grow slowly, controlled culture in bioreactors appears to be generally suitable for producing many of the metabolites of interest.
Scenedesmus spp. have been reported as potential microalgal species used for the lipid production. This study investigated the effects of light intensity (at three levels: 50, 250, and 400 μmol photons m(-2) s(-1)) on the growth and lipid production of Scenedesmus sp. 11-1 under N-limited condition. Carotenoid to chlorophyll ratio was higher when algae 11-1 grew under 250 and 400 μmol photons m(-2) s(-1) than that under 50 μmol photons m(-2) s(-1), while protein contents was lower. Highest biomass yield (3.88 g L(-1)), lipid content (41.1 %), and neutral lipid content (32.9 %) were achieved when algae 11-1 grew at 400 μmol photons m(-2) s(-1). Lipid production was slight lower at 250 μmol photons m(-2) s(-1) level compared to 400 μmol photons m(-2) s(-1). The major fatty acids in the neutral lipid of 11-1 were oleic acid (43-52 %), palmitic acid (24-27 %), and linoleic acid (7-11 %). In addition, polyunsaturated fatty acids had a positive correlation with total lipid production, and monounsaturated fatty acids had a negative one.
Algal mats can hinder the adhesion of the tube feet of sea urchins. This leads to the hypothesis that the restriction of sea urchin feeding activity by wave action can potentially be enhanced by the presence of algal mats, which will facilitate the survival of kelp recruits at sites with wave action in urchin barrens. To evaluate the potential anti-attachment effect of algal mats on sea urchins, a laboratory tank experiment was performed on the movement of Strongylocentrotus nudus sea urchins and their grazing on juvenile kelp plants at the center of 30×30 cm flat test substrates with or without a thin-layer microalgal mat at four levels of oscillatory flow (maximum orbital velocity: 10, 20, 30 and 40 cm s(-1)). The grazing loss of kelp slightly increased with increasing velocity up to 30 cm s(-1) in the absence of microalgal mats, while in contrast the loss substantially decreased at 30 cm s(-1) in their presence. Sea urchins were dislodged more frequently at 20 cm s(-1) or higher velocities in the presence of microalgal mats. Mats were frequently abraded by scraping by the adoral spines during urchin movement at high velocities (30 and 40 cm s(-1)) but were subject to no or only slight urchin grazing in most cases. The results indicate that the overall decrease in grazing loss of kelp within the microalgal mats was attributable to the anti-attachment effect on urchins during incursions rather than due to urchins grazing on the mats.
The green alga Chlorella zofingiensis can accumulate high level of oleic acid (OA, C18:1△(9)) rich oils in response to stress conditions. To understand the regulation of biosynthesis of fatty acid in particular OA at the molecular level, we cloned and characterized the stearoyl acyl carrier protein (ACP) desaturase (SAD) responsible for OA formation through desaturation of stearic acid (C18:0) from C. zofingiensis. Southern blot indicated that the C. zofingiensis genome contained a single copy of SAD, from which the deduced amino acid sequence shared high identity to the corresponding homologs from other microalgae and higher plants. The desaturation activity of SAD was demonstrated in vitro using C18:0-ACP as a substrate. Stress conditions such as high light (HL), nitrogen deficiency (N(-)), or combination of HL and N(-) (HL + N(-)) drastically up-regulated the transcripts of biotin carboxylase (BC, a subunit of ACCase) and SAD, and therefore induced considerably the cellular accumulation of total fatty acids including OA. Glucose (50 mM) gave rise to the similar up-regulation of the two genes and induction of fatty acid accumulation. The accumulation of intracellular reactive oxygen species was found to be associated with the up-regulation of genes. This is the first report of characterization of Chlorella-derived SAD and the results may contribute to understanding of the mechanisms involved in fatty acid/lipid biosynthesis in microalgae.
The use of molecular methods to investigate microalgal communities of natural and engineered freshwater resources is in its infancy, with the majority of previous studies carried out by microscopy. Inefficient or differential DNA extraction of microalgal community members can lead to bias in downstream community analysis. Three commercially available DNA extraction kits have been tested on a range of pure culture freshwater algal species with diverse cell walls and mixed algal cultures taken from eutrophic waste stabilization ponds (WSP). DNA yield and quality were evaluated, along with DNA suitability for amplification of 18S rRNA gene fragments by polymerase chain reaction (PCR). QiagenDNeasy(®) Blood and Tissue kit (QBT), was found to give the highest DNA yields and quality. Denaturant Gradient Gel Electrophoresis (DGGE) was used to assess the diversity of communities from which DNA was extracted. No significant differences were found among kits when assessing diversity. QBT is recommended for use with WSP samples, a conclusion confirmed by further testing on communities from two tropical WSP systems. The fixation of microalgal samples with ethanol prior to DNA extraction was found to reduce yields as well as diversity and is not recommended.
Integration of algal biofuel production to wastewater anaerobic digestion infrastructure has the potential to increase biogas production, decrease high and variable internal nitrogen loads, and improve sludge digestibility and dewaterability. In this research, two species of microalgae, Spirulina platensis and Chlorella sp., were grown on sludge centrate and a centrate and nitrified wastewater effluent mixture. Harvested algae were co-digested with waste activated sludge (WAS) at varying ratios. High-growth (6.8 g m(-2) x d(-1)), nitrogen (36.5 g m(-3) x d(-1)), and phosphorus (6.5 g m(-3) x d(-1)) uptake rates were achieved with Chlorella on centrate. No growth was observed with S. platensis under the same conditions; however, both organisms grew well on the centrate and effluent mixture. Co-digestion of algae with WAS improved volatile solids reduction. Although co-digestion with S. platensis improved biosolids dewaterability, Chlorella had a slight negative effect on dewaterability compared to WAS alone. The efficiency of energy conversion from photons to biogas generated from Chlorella was estimated at 1.4%.
Microalgae, with the ability to mitigate CO(2) emission and produce carbohydrates and lipids, are considered one of the most promising resources for producing bioenergy. Recently, we discovered that autophagy plays a critical role in the metabolism of photosynthetic system and lipids production. So far, more than 30-autophagy related (ATG) genes in all subtypes of autophagy have been identified. However, compared with yeast and mammals, in silico and experimental research of autophagy pathways in microalgae remained limited and fragmentary.
In this article, we performed a genome-wide analysis of ATG genes in 7 microalgae species and explored their distributions, domain structures and evolution. Eighteen "core autophagy machinery" proteins, four mammalian-specific ATG proteins and more than 30 additional proteins (including "receptor-adaptor" complexes) in all subtypes of autophagy were analyzed. Data revealed that receptor proteins in cytoplasm-to-vacuole targeting and mitophagy seem to be absent in microalgae. However, most of the "core autophagy machinery" and mammalian-specific proteins are conserved among microalgae, except for the ATG9-cycling system in Chlamydomonas reinhardtii and the second ubiquitin-like protein conjugation complex in several algal species. The catalytic and binding residues in ATG3, ATG5, ATG7, ATG8, ATG10 and ATG12 are also conserved and the phylogenetic tree of ATG8 coincides well with the phylogenies. Chlorella contains the entire set of the core autophagy machinery. In addition, RT-PCR analysis verified that all crucial ATG genes tested are expressed during autophagy in both Chlorella and Chlamydomonas reinhardtii. Finally, we discovered that addition of 3-Methyladenine (a PI3K specific inhibitor) could suppress the formation of autophagic vacuoles in Chlorella.
Taken together, Chlorella may represent a potential model organism to investigate autophagy pathways in photosynthetic eukaryotes. The study will not only promote understanding of the general features of autophagic pathways, but also benefit the production of Chlorella-derived biofuel with future commercial applications.
The supercritical water gasification (SCWG) of biomass for H(2) production is analyzed in terms of process development and energetic self-sustainability. The conceptual design of a plant is proposed and the SCWG process involving several substrates (glycerol, microalgae, sewage sludge, grape marc, phenol) is simulated by means of AspenPlus™. The influence of various parameters - biomass concentration and typology, reaction pressure and temperature - is analyzed. The process accounts for the possibility of exploiting the mechanical energy of compressed syngas (later burned to sustain the SCWG reaction) through expansion in turbines, while purified H(2) is fed to fuel cells. Results show that the SCWG reaction can be energetically self-sustained if minimum feed biomass concentrations of 15-25% are adopted. Interestingly, the H(2) yields are found to be maximal at similar feed concentrations. Finally, an energy balance is performed showing that the whole process could provide a net power of about 150kW(e)/(1000kg(feed)/h).