Growth,lipid content,productivity, and fatty acid composition of tropical microalgae for scale‐up production

Biomass and lipid productivity, lipid content, and quantitative and qualitative lipid composition are critical parameters in selecting microalgal species for commercial scale‐up production. This study compares lipid content and composition, and lipid and biomass productivity during logarithmic, late logarithmic, and stationary phase of Nannochloropsis sp., Isochrysis sp., Tetraselmis sp., and Rhodomonas sp. grown in L1‐, f/2‐, and K‐medium. Of the tested species, Tetraselmis sp. exhibited a lipid productivity of 3.9–4.8 g m^?2 day^?1 in any media type, with comparable lipid productivity by Nannochloropsis sp. and Isochrysis sp. when grown in L1‐medium. The dry biomass productivity of Tetraselmis sp. (33.1–45.0 g m^?2 day^?1) exceeded that of the other species by a factor 2–10. Of the organisms studied, Tetraselmis sp. had the best dry biomass and/or lipid production profile in large‐scale cultures. The present study provides a practical benchmark, which allows comparison of microalgal production systems with different footprints, as well as terrestrial systems. Biotechnol. Bioeng. 2010;107: 245–257. © 2010 Wiley Periodicals, Inc.     

Application of organic mono-phase and organic–aqueous two-liquid-phase systems in microalgal conversion of androst-4-en-3,17-dione by Nostoc muscorum

Organic mono-phase and organic–aqueous two-phase systems were applied for 17-carbonyl reduction of androst-4-en-3,17-dione to testosterone by whole cells of the microalga Nostoc muscorum (Nostocaceae). To investigate the correlation between solvent hydrophobicity and biotransformation yield in mono- and biphasic systems, a range of 16 organic solvents with log P_octanol values (logarithm of the solvent partition coefficient in the n-octanol/water system) between ? 1.1 and 8.8 were examined. Organic solvents with log P_octanol values greater than 7, such as hexadecane and tetradecane, provided the best biocompatibility with the bioconversion by algal cells. The data also indicated that the highest yields were obtained using organic–aqueous (1:1, v/v) biphasic systems. The optimum volumetric phase ratio, reaction temperature and substrate concentration were 1:1, 30°C and 0.5 mg mL^?1, respectively. Under the mentioned conditions a fourfold increase in biotransformation yield (from 7.8±2.3 to 33.4±1.8%) was observed.     

Nutritional Evaluation of Australian Microalgae as Potential Human Health Supplements

This study investigated the biochemical suitability of Australian native microalgal species Scenedesmus sp., Nannochloropsis sp., Dunaliella sp., and a chlorophytic polyculture as nutritional supplements for human health. The four microalgal cultures were harvested during exponential growth, lyophilized, and analysed for proximate composition (moisture, ash, lipid, carbohydrates, and protein), pigments, and amino acid and fatty acid profiles. The resulting nutritional value, based on biochemical composition, was compared to commercial Spirulina and Chlorella products. The Australian native microalgae exhibited similar, and in several cases superior, organic nutritional properties relative to the assessed commercial products, with biochemical profiles rich in high-quality protein, nutritious polyunsaturated fats (such as α-linolenic acid, arachidonic acid, and eicosapentaenoic acid), and antioxidant pigments. These findings indicate that the microalgae assessed have great potential as multi-nutrient human health supplements.     

Synthesis of propyl gallate by mycelium-bound tannase from <Emphasis Type="Italic">Aspergillus niger</Emphasis> in organic solvent

Aspergillus niger with mycelium-bound tannase activity was employed to investigate the synthesis of propyl gallate from gallic acid and 1-propanol in organic solvents. The effects of various organic solvents (log P: −1.0 to 6.6) on the enzymatic reactions showed that benzene (log P: 2.0) was the most suitable solvent. The water content and protonation state of mycelium-bound enzyme both had significant effects on the activity of tannase. The maximum molar conversion (65%) was achieved with 7.3% (v/v) 1-propanol and 5.56 mM gallic acid at stirring speeds of 200 rev/min, 40 °C in presence of anhydrous sodium sulfate and PEG-10,000. Enzyme specificity for the alcohol portion (C₁–C₈) of the ester showed that the optimum synthesis was observed with alcohols ranging from C₃ to C₅.     

A quantitative analysis of microalgal lipids for optimization of biodiesel and omega‐3 production

The lipid characteristics of microalgae are known to differ between species and change with growth conditions. This work provides a methodology for lipid characterization that enables selection of the optimal strain, cultivation conditions, and processing pathway for commercial biodiesel production from microalgae. Two different microalgal species, Nannochloropsis sp. and Chlorella sp., were cultivated under both nitrogen replete and nitrogen depleted conditions. Lipids were extracted and fractionated into three major classes and quantified gravimetrically. The fatty acid profile of each fraction was analyzed using GC–MS. The resulting quantitative lipid data for each of the cultures is discussed in the context of biodiesel and omega‐3 production. This approach illustrates how the growth conditions greatly affect the distribution of fatty acid present in the major lipid classes and therefore the suitability of the lipid extracts for biodiesel and other secondary products. Biotechnol. Bioeng. 2013; 110: 2096–2104. © 2013 Wiley Periodicals, Inc.     

Different co-occurring bacteria enhance or decrease the growth of the microalga Nannochloropsis sp. CCAP211/78

Marine photosynthetic microalgae are ubiquitously associated with bacteria in nature. However, the influence of these bacteria on algal cultures in bioreactors is still largely unknown. In this study, eighteen different bacterial strains were isolated from cultures of Nannochloropsis sp. CCAP211/78 in two outdoor pilot-scale tubular photobioreactors. The majority of isolates was affiliated with the classes Alphaproteobacteria and Flavobacteriia. To assess the impact of the eighteen strains on the growth of Nannochloropsis sp. CCAP211/78, 24-well plates coupled with custom-made LED boxes were used to simultaneously compare replicate axenic microalgal cultures with addition of individual bacterial isolates. Co-culturing of Nannochloropsis sp. CCAP211/78 with these strains demonstrated distinct responses, which shows that the technique we developed is an efficient method for screening the influence of harmful/beneficial bacteria. Two of the tested strains, namely a strain of Maritalea porphyrae (DMSP31) and a Labrenzia aggregata strain (YP26), significantly enhanced microalgal growth with a 14% and 12% increase of the chlorophyll concentration, respectively, whereas flavobacterial strain YP206 greatly inhibited the growth of the microalga with 28% reduction of the chlorophyll concentration. Our study suggests that algal production systems represent a ‘natural’ source to isolate and study microorganisms that can either benefit or harm algal cultures.     

Isolation of new toluene-tolerant marine strains of bacteria and characterization of their solvent-tolerance properties

Aims:  To isolate and characterize new marine bacteria capable of tolerating high concentrations of organic solvents, and to understand the toxic effects of these chemicals on marine bacteria. Methods and Results:  Five marine bacteria able to tolerate 0·1% (v/v) toluene were isolated and characterized on the basis of their growth and survival rates in the presence of different organic solvents. The toluene-tolerant marine bacteria identified in this study could not grow in the presence of 0·1% (v/v) of several organic solvents with a log P_ow higher than that of the toluene (which in theory should be less toxic than toluene). The mechanisms underlying solvent tolerance were explored. Conclusions:  Isolates of four different genera were identified as toluene-tolerant. Toxicity of a second phase of an organic solvent toward these isolates could not be predicted on the basis of the solvents’ log P_ow. Significance and Impact of the Study:  To improve the biodegradation rate of some water-insoluble compounds, double-phase bioreactors can be used. This type of bioreactor will require strains able to grow in a salt-containing environment and able to tolerate a second phase of an organic solvent.     

Relation between bacterial strain resistance to solvents and biodesulfurization activity in organic medium

Microorganisms used in biodesulfurization of petroleum products have to withstand high concentrations of hydrocarbons. The capacities of seven desulfurizing strains of Rhodococcus to be active in the presence of solvents were evaluated. Octanol and toluene (log P=2.9) were selected as toxic solvents. The effect of the solvents was determined by measuring either inhibition of growth or the decrease in respiratory activity of the cells. Differences among strains in their resistance to solvent responses were observed, but these variations were dependent on the test used. Resistance to solvents was then compared to the capacity of the different strains to retain biodesulfurization activity in the presence of hexadecane. Inhibition of desulfurization by high concentrations of hexadecane was found to be well correlated to the sensitivity of the strains to respiration inhibition by toluene, but not to growth inhibition. This result also showed that the respirometric test was a rapid and reliable test to select solvent-resistant strains for use as resting cells in biocatalysis processes, such as biodesulfurization, in organic media.     

Permeabilization of<Emphasis Type="Italic">Ochrobactrum anthropi</Emphasis> SY509 cells with organic solvents for whole cell biocatalyst

Permeabilization is known to overcome cell membrane barriers of whole cell biocatalysts. The use of organic solvents is advantageous in terms of cost, simplicity, and efficiency. In this study,Ochrobactrum anthropi SY509 was permeabilized with various organic solvents. Treatment with organic solvents resulted in lower permeability barriers due to falling out lipids of the cell membrane. Therefore, permeabilized cells showed higher enzyme activity with no cell viability. Among various organic solvents, 0.5% (v/v) chloroform was selected as the most efficient permeabilizing reagent. Changes in the cell membrane structure were observed and the residual amounts of phospholipids of the cell membrane were measured to investigate the mechanism of the improved permeability.     

Effects of organic solvents on membrane of<Emphasis Type="Italic">Taxus cuspidata</Emphasis> cells in

The effects of organic solvents (oleic acid and dibutyl phthalate) on viability and membrane integrity of Taxus cuspidata cells were investigated in two-liquid-phase suspension cultures. It has been found that the cell viability, electrical conductivity and concentration of malonyl dialdehyde did not change obviously when the content of oleic acid or dibutyl phthalate was 2% (v/v), but varied markedly when the contents of oleic acid or dibutyl phthalate were raised to 6% (v/v) or more, indicating that the organic solvents at higher concentrations severely affected the cell membrane permeability.     

An improved colony PCR procedure for genetic screening of <Emphasis Type="Italic">Chlorella</Emphasis> and related microalgae

A colony PCR technique was applied for both genomic and chloroplast DNA in the green microalgae Chlorella. Of five different lysis buffers, Chelex-100 was superior for DNA extraction, PCR and DNA storage. It also was insensitive to variations in cell density. The conditions established for an improved PCR formulation are applicable for screening of genetically-engineered transformants as well as bioprospecting of natural microalgal isolates. Besides multiple Chlorella species, we also demonstrate the efficacy of Chelex-100 for colony PCR with a number of other microalgal strains, including Chlamydomonas reinhardtii, Dunaliella salina, Nannochloropsis sp., Coccomyxa sp., and Thalassiosira pseudonana.     

Selective extraction of carotenoids from the microalga Dunaliella salina with retention of viability

Simultaneous production and selective extraction of beta-carotene from living cells of Dunaliella salina in a two-phase system of aqueous and organic phases has been investigated. Solvents with values of log P(octanol), which denotes hydrophobicity of a compound, ranging from 3 to 9 were used as organic phase. Viability and activity of Dunaliella salina in the presence of organic solvents were checked by microscopic observation and photosynthetic oxygen-production-rate measurements, respectively. Extraction ability of different solvents for both beta-carotene and chlorophyll was determined spectrophotometrically. In addition, beta-carotene contents of the cells growing in the aqueous phase and extracted beta-carotene by the different organic phases were quantified by the same method. Results showed that solvents having log P(octanol) > 6 can be considered biocompatible for this alga. Moreover, pigment extraction ability of a solvent is inversely dependent on its log P(octanol) value. By increasing the degenerative hydrophobicity the extraction ability for both chlorophyll and beta-carotene, decreases. However, this decrease is more profound for chlorophyll. Therefore, selective extraction of beta-carotene becomes feasible. Comparison of the total beta-carotene produced in the presence and in the absence of solvents shows that the presence of a second phase of biocompatible solvents in the culture media may induce the beta-carotene production pathway. The beta-carotene productivity per cell in a two-phase system with dodecane was the highest observed. Extraction ability of the biocompatible solvents dodecane, tetradecan, and hexadecane was similar.     

Evaluation of extraction methods for recovery of fatty acids from Botryococcus braunii LB 572 and Synechocystis sp. PCC 6803

Microalgae are a very diverse group of organisms that consist in both prokaryotic and eukaryotic forms. Some species of microalgae can be induced to overproduce particular fatty acids through simple manipulations of the physical and chemical properties of the culture medium. In this paper, the effect of different extraction techniques on the recovery of fatty acids from the freeze-dried biomass from two lipid-producing microalgal strains: Botryococcus braunii LB 572 (green algae) and Synechocystis sp. PCC 6803 (cyanobacteria) was examined. Five procedures were used: after conversion of the lipid material into the corresponding fatty acid methyl esters (FAMEs) via suitable derivatization reactions (extraction-transesterification) and direct transesterification of biomass to produce FAMEs (without the initial extraction step) that used differential types of catalysts and processing conditions. This study has shown that procedure 3, a one step practical procedure for lipid extraction and in situ methyl ester derivation could be used successfully for the determination of the fatty acid compositions of microalgae and cyanobacteria.     

A Proteomic Approach to Understand the Role of the Outer Membrane Porins in the Organic Solvent-Tolerance of Pseudomonas aeruginosa PseA

Solvent-tolerant microbes have the unique ability to thrive in presence of organic solvents. The present study describes the effect of increasing hydrophobicity (log P_ow values) of organic solvents on the outer membrane proteome of the solvent-tolerant Pseudomonas aeruginosa PseA cells. The cells were grown in a medium containing 33% (v/v) alkanes of increasing log P_ow values. The outer membrane proteins were extracted by alkaline extraction from the late log phase cells and changes in the protein expression were studied by 2-D gel electrophoresis. Seven protein spots showed significant differential expression in the solvent exposed cells. The tryptic digest of the differentially regulated proteins were identified by LC-ESI MS/MS. The identity of these proteins matched with porins OprD, OprE, OprF, OprH, Opr86, LPS assembly protein and A-type flagellin. The reported pI values of these proteins were in the range of 4.94–8.67 and the molecular weights were in the range of 19.5–104.5 kDa. The results suggest significant down-regulation of the A-type flagellin, OprF and OprD and up-regulation of OprE, OprH, Opr86 and LPS assembly protein in presence of organic solvents. OprF and OprD are implicated in antibiotic uptake and outer membrane stability, whereas A-type flagellin confers motility and chemotaxis. Up-regulated OprE is an anaerobically-induced porin while Opr86 is responsible for transport of small molecules and assembly of the outer membrane proteins. Differential regulation of the above porins clearly indicates their role in adaptation to solvent exposure.     

Microalgae as a raw material for biofuels production

Biofuels demand is unquestionable in order to reduce gaseous emissions (fossil CO₂, nitrogen and sulfur oxides) and their purported greenhouse, climatic changes and global warming effects, to face the frequent oil supply crises, as a way to help non-fossil fuel producer countries to reduce energy dependence, contributing to security of supply, promoting environmental sustainability and meeting the EU target of at least of 10% biofuels in the transport sector by 2020. Biodiesel is usually produced from oleaginous crops, such as rapeseed, soybean, sunflower and palm. However, the use of microalgae can be a suitable alternative feedstock for next generation biofuels because certain species contain high amounts of oil, which could be extracted, processed and refined into transportation fuels, using currently available technology; they have fast growth rate, permit the use of non-arable land and non-potable water, use far less water and do not displace food crops cultures; their production is not seasonal and they can be harvested daily. The screening of microalgae (Chlorella vulgaris, Spirulina maxima, Nannochloropsis sp., Neochloris oleabundans, Scenedesmus obliquus and Dunaliella tertiolecta) was done in order to choose the best one(s), in terms of quantity and quality as oil source for biofuel production. Neochloris oleabundans (fresh water microalga) and Nannochloropsis sp. (marine microalga) proved to be suitable as raw materials for biofuel production, due to their high oil content (29.0 and 28.7%, respectively). Both microalgae, when grown under nitrogen shortage, show a great increase (~50%) in oil quantity. If the purpose is to produce biodiesel only from one species, Scenedesmus obliquus presents the most adequate fatty acid profile, namely in terms of linolenic and other polyunsaturated fatty acids. However, the microalgae Neochloris oleabundans, Nannochloropsis sp. and Dunaliella tertiolecta can also be used if associated with other microalgal oils and/or vegetable oils.     

Lipid Profile Remodeling in Response to Nitrogen Deprivation in the Microalgae Chlorella sp. (Trebouxiophyceae) and Nannochloropsis sp. (Eustigmatophyceae)

Many species of microalgae produce greatly enhanced amounts of triacylglycerides (TAGs), the key product for biodiesel production, in response to specific environmental stresses. Improvement of TAG production by microalgae through optimization of growth regimes is of great interest. This relies on understanding microalgal lipid metabolism in relation to stress response in particular the deprivation of nutrients that can induce enhanced TAG synthesis. In this study, a detailed investigation of changes in lipid composition in Chlorella sp. and Nannochloropsis sp. in response to nitrogen deprivation (N-deprivation) was performed to provide novel mechanistic insights into the lipidome during stress. As expected, an increase in TAGs and an overall decrease in polar lipids were observed. However, while most membrane lipid classes (phosphoglycerolipids and glycolipids) were found to decrease, the non-nitrogen containing phosphatidylglycerol levels increased considerably in both algae from initially low levels. Of particular significance, it was observed that the acyl composition of TAGs in Nannochloropsis sp. remain relatively constant, whereas Chlorella sp. showed greater variability following N-deprivation. In both algae the overall fatty acid profiles of the polar lipid classes were largely unaffected by N-deprivation, suggesting a specific FA profile for each compartment is maintained to enable continued function despite considerable reductions in the amount of these lipids. The changes observed in the overall fatty acid profile were due primarily to the decrease in proportion of polar lipids to TAGs. This study provides the most detailed lipidomic information on two different microalgae with utility in biodiesel production and nutraceutical industries and proposes the mechanisms for this rearrangement. This research also highlights the usefulness of the latest MS-based approaches for microalgae lipid research.     

Dewatering of algal suspension using microfiltration with cross flow in the presence of magnetite as a filter aid

Dewatering algal suspensions is an important step in the extraction of oil and other useful substances from algae. In this study, spherical Nannochloropsis sp. and ellipsoidal Monoraphidium sp. suspensions were dewatered in the presence of different amounts of 350-nm magnetite particles using a microfiltration membrane with 360-nm pores in cross-flow mode. Magnetite functions as a filter aid by reducing the deformation of the cake of filtered algae on the membrane and providing paths for water to flow through the filtration cake of algae. In the case of Nannochloropsis sp., the highest dewatering rate was obtained when the number ratio, defined based on the size and ideal density, between Nannochloropsis sp. and magnetite was 1:12.5, but the addition of magnetite had no observable effect on the filtration of ellipsoidal Monoraphidium sp. suspensions through the membrane. After dewatering, magnetite was effectively separated from the concentrated algal suspension by the application of a magnetic field in an open flow system. Magnetite has the potential to enhance dewatering performance using a cross-flow membrane system.     

Tolerance of β-diketone hydrolases as representatives of the crotonase superfamily towards organic solvents

Crotonase superfamily enzymes catalyze a wide variety of reactions, including hydrolytic C–C bond cleavage in symmetrical β‐diketones by 6‐oxo camphor hydrolase (OCH) from Rhodococcus sp. The organic solvent tolerance and temperature stability of OCH and its structurally related ortholog Anabaena β‐diketone hydrolase have been investigated. Both enzymes showed excellent tolerance toward organic solvents; for instance, even in the presence of 80% (v/v) THF or dioxane, OCH was still active. In most solvent mixtures, except methanol, the stereospecificity was conserved (>99% e.e. of product), hence neither the type of solvent nor its concentration appeared to have an effect on the stereoselectivity of the enzyme. Attempts to correlate the observed activities with log P, functional solvent group or denaturing capacity (DC) of the solvent were only successful in the case of DC for water miscible solvents. This study represents the first investigation of organic solvent stability for members of the crotonase superfamily. Biotechnol. Bioeng. 2011;108: 2815–2822. © 2011 Wiley Periodicals, Inc.     

Response of growth and fatty acid compositions of Nannochloropsis sp. to environmental factors under elevated CO2 concentration

Nannochloropsis sp. was grown with different levels of nitrate, phosphate, salinity and temperature with CO₂ at 2,800 μl l⁻¹. Increased levels of NaNO₃ and KH₂PO₄ raised protein and polyunsaturated fatty acids (PUFAs) contents but decreased carbohydrate, total lipid and total fatty acids (TFA) contents. Nannochloropsis sp. grew well at salinities from 22 to 49 g l⁻¹, and lowering salinity enhanced TFA and PUFAs contents. TFA contents increased with the increasing temperature but PUFAs contents decreased. The highest eicosapentaenoic acid (EPA, 20:5ω3) content based on the dry mass was above 3% under low N (150 μM NaNO₃) or high N (3000 μM NaNO₃) condition. Excessive nitrate, low salinity and temperature are thus favorable factors for improving EPA yields in Nannochloropsis sp.     

Selection of organic solvents for in situ extraction of fermentation products fromClostridium thermohydrosulfuricum cultures

Summary Fifteen organic solvents were examined to determine their biocompatibility for in situ extraction of fermentation products from cultures of the thermophilic anaerobeClostridium thermohydrosul furicum. Five solvents (hexadecane, isooctane, kerosene, oleyl alcohol, Shellsol TD) were found to be non-toxic toClostridium thermohydrosul furicum. Interfacial tensions, phase separation and partition coefficients for ethanol of the biocompatible solvents were compared. With the exception of kerosene, these solvents showed good separation from the aqueous phase. Oleyl alcohol had the highest partition coefficient for ethanol (K_D=0.34 at 65°C) and appears to be suitable for extractive ethanol fermentation.