Molecular and Structural Characterizations of Lipases from Chlorella by Functional Genomics.

Microalgae have been poorly investigated for new-lipolytic enzymes of biotechnological interest. In silico study combining analysis of sequences homologies and bioinformatic tools allowed the identification and preliminary characterization of 14 putative lipases expressed by Chlorella vulagaris. These proteins have different molecular weights, subcellular localizations, low instability index range and at least 40% of sequence identity with other microalgal lipases. Sequence comparison indicated that the catalytic triad corresponded to residues Ser, Asp and His, with the nucleophilic residue Ser positioned within the consensus GXSXG pentapeptide. 3D models were generated using different approaches and templates and demonstrated that these putative enzymes share a similar core with common α/ß hydrolases fold belonging to family 3 lipases and class GX. Six lipases were predicted to have a transmembrane domain and a lysosomal acid lipase was identified. A similar mammalian enzyme plays an important role in breaking down cholesteryl esters and triglycerides and its deficiency causes serious digestive problems in human. More structural insight would provide important information on the enzyme characteristics.

Optimization of lipids' ultrasonic extraction and production from Chlorella sp. using response-surface methodology.

BACKGROUND: Three steps are very important in order to produce microalgal lipids: (1) controlling microalgae cultivation via experimental and modeling investigations, (2) optimizing culture conditions to maximize lipids production and to determine the fatty acid profile the most appropriate for biodiesel synthesis, and (3) optimizing the extraction of the lipids accumulated in the microalgal cells. METHODS: Firstly, three kinetics models, namely logistic, logistic-with-lag and modified Gompertz, were tested to fit the experimental kinetics of the Chlorella sp. microalga culture established on standard conditions. Secondly, the response-surface methodology was used for two optimizations in this study. The first optimization was established for lipids production from Chlorella sp. culture under different culture conditions. In fact, different levels of nitrate concentrations, salinities and light intensities were applied to the culture medium in order to study their influences on lipids production and determine their fatty acid profile. The second optimization was concerned with the lipids extraction factors: ultrasonic's time and temperature, and chloroform-methanol solvent ratio. RESULTS: All models (logistic, logistic-with-lag and modified Gompertz) applied for the experimental kinetics of Chlorella sp. show a very interesting fitting quality. The logistic model was chosen to describe the Chlorella sp. kinetics, since it yielded the most important statistical criteria: coefficient of determination of the order of 94.36%; adjusted coefficient of determination equal to 93.79% and root mean square error reaching 3.685 cells · ml- 1. Nitrate concentration and the two interactions involving the light intensity (Nitrate concentration × light intensity, and salinities × light intensity) showed a very significant influence on lipids production in the first optimization (p < 0.05). Yet, only the quadratic term of chloroform-methanol solvent ratio showed a significant influence on lipids extraction relative to the second step of optimization (p < 0.05). The two most abundant fatty acid methyl esters (≈72%) derived from the Chlorella sp. microalga cultured in the determined optimal conditions are: palmitic acid (C16:0) and oleic acid (C18:1) with the corresponding yields of 51.69% and 20.55% of total fatty acids, respectively. CONCLUSIONS: Only the nitrate deficiency and the high intensity of light can influence the microalgal lipids production. The corresponding fatty acid methyl esters composition is very suitable for biodiesel production. Lipids extraction is efficient only over long periods of time when using a solvent with a 2/1 chloroform/methanol ratio.

Effects of nutritional conditions on growth and biochemical composition of Tetraselmis sp.

BACKGROUND: This study aimed to maximize biomass concentration, biomass productivity and biochemical composition of the marine microalga Tetraselmis sp. METHODS: In the current study, Box-Behnken Design was used to model the effect of NaNO3, NaH2PO4, metals and vitamins in the F/2 medium on the growth, total chlorophylls, carotenoids and starch contents. The total chlorophylls content was quantified by spectrophotometry. The FT-IR spectroscopy was used to estimate the biochemical compositions of Tetraselmis sp. grown under both optimized medium culture for starch production and standard culture medium. RESULTS: Finalized NaNO3 (1.76 mM), NaH2PO4 (0.018 mM), metals (1500 µL.L-1) and vitamins (312.5 µL.L-1) concentrations, generated an increase in biomass concentration up to 5.72 g.L-1 which contributed to an increase about 2.4-fold than that of the standard conditions of biomass productivity (408.57 mg.L-1.day-1). The maximum value of carotenoids content (0.3 mg.g DW-1) was achieved at the highest level of all factors. The total chlorophylls content reached also its maximum (5.18 mg.g DW-1) at high nitrate (1.76 mM), phosphate (0.054 mM), metals and vitamins concentrations, while the maximum starch content (42% DW) was achieved with low nitrate and phosphate concentrations (0.58 mM and 0.027 mM) and with metals and vitamins limitations. Thus, the nitrogen, phosphorus, metals and vitamins limitations led to divert the metabolism for the starch biosynthesis. CONCLUSIONS: The high biomass concentration productivity and starch production make Tetraselmis sp. strain a good candidate for biotechnological applications.

Enhanced lipid and biomass production by a newly isolated and identified marine microalga.

BACKGROUND: The increasing demand for microalgae lipids as an alternative to fish has encouraged researchers to explore oleaginous microalgae for food uses. In this context, optimization of growth and lipid production by the marine oleaginous V2-strain-microalgae is of great interest as it contains large amounts of mono-unsaturated (MUFAs) and poly-unsaturated fatty acids (PUFAs). METHODS: In this study, the isolated V2 strain was identified based on 23S rRNA gene. Growth and lipid production conditions were optimized by using the response surface methodology in order to maximize its cell growth and lipid content that was quantified by both flow cytometry and the gravimetric method. The intracellular lipid bodies were detected after staining with Nile red by epifluorescence microscopy. The fatty acid profile of optimal culture conditions was determined by gas chromatography coupled to a flame ionization detector. RESULTS: The phenotypic and phylogenetic analyses showed that the strain V2 was affiliated to Tetraselmis genus. The marine microalga is known as an interesting oleaginous species according to its high lipid production and its fatty acid composition. The optimization process showed that maximum cell abundance was achieved under the following conditions: pH: 7, salinity: 30 and photosynthetic light intensity (PAR): 133 µmol photons.m-2.s-1. In addition, the highest lipid content (49 ± 2.1% dry weight) was obtained at pH: 7, salinity: 37.23 and photosynthetic light intensity (PAR): 188 µmol photons.m-2.s-1. The fatty acid profile revealed the presence of 39.2% and 16.1% of total fatty acids of mono-unsaturated fatty acids (MUFAs) and poly-unsaturated fatty acids (PUFAs), respectively. Omega 3 (ω3), omega 6 (ω6) and omega 9 (ω9) represented 5.28%, 8.12% and 32.8% of total fatty acids, respectively. CONCLUSIONS: This study showed the successful optimization of salinity, light intensity and pH for highest growth, lipid production and a good fatty acid composition, making strain V2 highly suitable for food and nutraceutical applications.

Isolation of a novel amylase and lipase-producing Pseudomonas luteola strain: study of amylase production conditions.

An amylase and lipase producing bacterium (strain C2) was enriched and isolated from soil regularly contaminated with olive washing wastewater in Sfax, Tunisia. Cell was aerobic, mesophilic, Gram-negative, motile, non-sporulating bacterium, capable of growing optimally at pH 7 and 30°C and tolerated maximally 10% (W/V) NaCl. The predominant fatty acids were found to be C(18:1)ω7c (32.8%), C(16:1)ω7c (27.3%) and C16:0 (23.1%). Phylogenetic analysis of the 16S rRNA gene revealed that this strain belonging to the genus Pseudomonas. Strain C2 was found to be closely related to Pseudomonas luteola with more than 99% of similarity. Amylase optimization extraction was carried out using Box Behnken Design (BBD). Its maximal activity was found when the pH and temperature ranged from 5.5 to 6.5 and from 33 to 37°C, respectively. Under these conditions, amylase activity was found to be about 9.48 U/ml.

Tetraselmis species for environmental sustainability: biology, water bioremediation, and biofuel production.

With increasing demand of fossil fuels and water pollution and their environmental impacts, marine green microalgae have gained special attention in both scientific and industrial  fields. This is due to their fast growth in non-arable lands with high photosynthetic activity, their metabolic plasticity, as well as their high CO2 capture capacity. Tetraselmis species, green and eukaryotic microalgae, are not only considered as a valuable source of biomolecules including pigments, lipids, and starch but also widely used in biotechnological applications. Tetraselmis cultivation for high-value biomolecules and industrial use was demonstrated to be a non-cost-effective strategy because of its low demand in nutrients, such as phosphorus and nitrogen. Recently, phycoremediation of wastewater rich in nutrients, chemicals, and heavy metals has become an efficient and economic-alternative that allows the detoxification of waters and induces mechanisms in algal cells for biomolecules rich-energy synthesis to regulate their metabolic pathways. This review aims to shed light on Tetraselmis species for their different culture conditions and metabolites bioaccumulation, as well as their human health and environmental applications. Additionally, phycoremediation of contaminants associated to biofuel production in Tetraselmis cells and their different intracellular and extracellular mechanisms have also been investigated.

Conception of an environmental friendly O/W cosmetic emulsion from microalgae.

The development of eco-friendly cosmetic such as those from microalgae for skin regeneration and collagen synthesis has gained a great interest worldwide. Accordingly, the potential of microalgae biomass as source of anti-aging cosmetic cream with high antioxidant activity has been investigated. Stabilities and sensory characteristics of cosmetic creams supplemented with Spirulina, Tetraselmis sp. and Dunaliella sp. at 0.5, 1.5 and 2.5%, respectively, revealed a conservation of physico-chemical and preliminary stability properties of formulations. To analyze physico-chemical and textural parameters, accelerated stability study was evaluated under two thermal conditions (25 and 40 °C) during 90 days. Results showed that pH values of all formulations were within the limits of normal skin pH range under storage time at 25 and 40 °C. During this period, the colored creams showed a significant changes of a* and b* indices, reflecting the instability of microalgae colors. Microalgae modified the textural characteristics of emulsions. The Tetraselmis sp. containing-cream had the lowest (P < 0.05) values of hardness, springiness, and cohesiveness. The 0.5% Spirulina containing-cream had the best stable consistency and adhesiveness under time and temperature variations. It exhibited the best properties to be used for skin care products. Thanks to their high content in bioactive macromolecules, microalgae considerably improved the antioxidant activity of the new formulated skin creams.

Effect of heavy metals mixture on the growth and physiology of Tetraselmis sp.: Applications to lipid production and bioremediation.

Phycoremediation of heavy metals from contaminated waters by oleaginous microalgae is an eco-friendly and emerging trend. Different concentrations of toxic metals such as nickel (Ni), chromium (Cr) and cobalt (Co) were added in Tetraselmis sp. culture media. Mixture Design was used to model the effect of these metals on cell growth, lipid production and heavy metals removal. Tetraselmis sp. was identified as an outstanding Ni, Cr and Co accumulator with bioconcentration factors of 675.17, 584.9 and 169.81 within binary mixtures (Ni × Cr), (Cr × Co) and (Cr × Co) at 6 mg/L, respectively. Optimization studies showed that the highest cell growth (9.22 × 105 cells/mL), lipid content (31% Dry Weight) and metals removal (91%) were obtained with the optimum binary mixture Ni (54.45 %) and Cr (45.45 %). This work presents interesting results revealing the potential of Tetraselmis sp. for nickel removal up to 97 % combined to its potential for biodiesel production.

Production and structure prediction of amylases from Chlorella vulgaris.

Amylases are enzymes required for starch degradation and are naturally produced by many microorganisms. These enzymes are used in several fields such as food processing, beverage, and medicine as well as in the formulation of enzymatic detergents proving their significance in modern biotechnology. In this study, a three-stage growth mode was applied to enhance starch production and amylase detection from Chlorella vulgaris. Stress conditions applied in the second stage of cultivation led to an accumulation of proteins (75% DW) and starch (21% DW) and a decrease in biomass. Amylase activities were detected and they showed high production levels especially on day 3 (35 U/ml) and day 5 (22.5 U/ml) of the second and third stages, respectively. The bioinformatic tools used to seek amylase protein sequences from TSA database of C. vulgaris revealed 7 putative genes encoding for 4 α-amylases, 2 ß-amylases, and 1 isoamylase. An in silico investigation showed that these proteins are different in their lengths as well as in their cellular localizations and oligomeric states though they share common features like CSRs of GH13 family or active site of GH14 family. In brief, this study allowed for the production and in silico characterization of amylases from C. vulgaris.

Biodegradation and detoxification of bisphenol A by bacteria isolated from desert soils.

The endocrine-disrupting chemical bisphenol A (BPA) has attracted much attention because of its estrogenic activity and widespread environmental contamination. In this study, we investigated the BPA biodegradation abilities of various bacterial strains isolated from deserts and arid soils from southern Tunisia. Ten bacterial strains that belong to Pseudomonas putida, Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella sp. and Pantoea sp. showed high BPA removal potential in mineral salt medium (MSM) containing 1 mM BPA. BPA removal rates varied between strains and ranged from 36 to 97%. The strain G320 (P. putida) presented the highest BPA removal rate with 97% within 4 days at 30 °C. The half-life when increasing the BPA concentration to 3 mM was 2 days for strain G320, while total degradation was achieved within 8 days. BPA biodegradation products were identified by GC-MS, and their toxicity was assessed by an algal toxicity test. BPA detoxification was confirmed by evaluating the effect of its biodegradation metabolites on algal growth (dry weight), cells morphology and chlorophylls levels of Tetraselmis sp. strain V2. Results showed the interesting potential of desert soil's bacteria in BPA detoxification as well as the eventual use of the algal specie in toxicity assessment.

Optimal cultivation towards enhanced biomass and floridean starch production by Porphyridium marinum.

Optimal conditions for maximal biomass and starch production by the marine red microalgae Porphyridium marinum were investigated. Box-Behnken Design was used to model the effect of light intensity, NaNO3 concentration and salinity on the growth of microalgae but also on their starch and protein contents. These three factors increased biomass production by 13.6% in optimized conditions. A maximum starch production (140.21 µg·mL-1), 30.6% higher than that of the control, was attained at a light intensity of 100 µmol photons·m-2·s-1, a NaNO3 concentration of 1 g·L-1 and a NaCl concentration of 20 g·L-1. FT-IR spectroscopy was used to estimate the biochemical composition (carbohydrate accumulation) of P. marinum and revealed significant changes (P < 0.05) depending on culture conditions. FT-IR analysis highlighted also that the culture conditions leading to highest starch production by P. marinum corresponded to lowest sulfated polysaccharide and protein contents.

Quality Characteristics and Functional and Antioxidant Capacities of Algae-Fortified Fish Burgers Prepared from Common Barbel (Barbus barbus).

INTRODUCTION: Algae have been used as natural ingredients to produce new canned fish burgers prepared from minced flesh of common barbel. In this research, the impact of the addition of Cystoseira compressa and Jania adhaerens at concentrations of 0.5, 1, and 1.5% w/v on the texture and sensory characteristics of fish burgers were investigated. RESULTS: Compared to controls, fish burgers containing 1% algae had better texture and sensory properties (P < 0.05). Also, these burger formulations had higher water and oil holding capacities as well as swelling ability, due to the important polysaccharides and dietary fibers contents of algae. In addition, algae-supplemented burgers were characterized as having low L⁎, a⁎, and b⁎ values, which made the color appear to be paler. Thanks to their high richness in pigments (chlorophylls and carotenoids) and polysaccharides, algae considerably enhance the antioxidant activities of the new ready-to-eat fish burgers. So, Cystoseira compressa and Jania adhaerens could be used as nutritious additives to produce new fish-based products.

Modelling Tetraselmis sp. growth-kinetics and optimizing bioactive-compound production through environmental conditions.

The aim of this study is to predict Tetraselmis cells growth-kinetic and to induce the synthesis of bioactive compounds (chlorophylls, carotenoids and starch) with high potential for biotechnological applications. Using the statistical criteria, the Baranyi-Roberts model has been selected to estimate the microalgae growth-kinetic values. The simultaneous effects of salinity, light intensity and pH of culture medium were investigated to maximize the production of total chlorophylls, carotenoids and starch. The optimal culture conditions for the production of these compounds were found using Box-Behnken Design. Results have shown that total chlorophyll and carotenoids were attained 21.6mg·g-1DW and 0.042mg·g-1DW, respectively. In addition, the highest starch content of 0.624g·g-1DW has been obtained at neutral pH with high irradiance (182µmolphotonsm-2 s-1) and low salinity (20). A highly correlation (R2 = 0.884) has been found between the gravimetric and flow cytometric measurements of chlorophyll content.

Cyanobacteria as source of marine bioactive compounds: Molecular specific detection based on Δ9 desaturase gene.

The blue-green microalga, Arthrospira sp., isolated from the sea of Kssour Essef in Mahdia (Tunisia), was purified and then identified both morphologically and genetically based on 16S rRNA gene sequence. Following physicochemical analysis, the prokaryotic microalga tested represented a competitive source of pigments and showed a considerable rate in protein (64%) which was confirmed by FTIR measurement. The lipid content (4%) was quantified by the gravimetric method and the intracellular lipid bodies were detected with the Nile red staining. Using gas chromatography coupled with flame ionization detector, the fatty acid profile revealed the presence of 27.4% and 32.88% of monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs), respectively. Given the richness of the isolated microalga in unsaturated fatty acids, we have developed a SYBR Green real time PCR method for the specific identification of Arthrospira sp. Δ9 desaturase gene. This current method will be of great value for carrying out high-throughput studies like cloning, heterologous expression and structure-function relationship analysis.

Efficacy of Bacillus subtilis V26 as a biological control agent against Rhizoctonia solani on potato.

The aim of this study is to evaluate the efficacy of the strain Bacillus subtilis V26, a local isolate from the Tunisian soil, to control potato black scurf caused by Rhizoctonia solani. The in vitro antifungal activity of V26 significantly inhibited R. solani growth compared to the untreated control. Microscopic observations revealed that V26 caused considerable morphological deformations of the fungal hyphae such as vacuolation, protoplast leakage and mycelia crack. The most effective control was achieved when strain V26 was applied 24h prior to inoculation (protective activity) in potato slices. The antagonistic bacterium V26 induced significant suppression of root canker and black scurf tuber colonization compared to untreated controls with a decrease in incidence disease of 63% and 81%, respectively, and promoted plant growth under greenhouse conditions on potato plants. Therefore, B. subtilis V26 has a great potential to be commercialized as a biocontrol agent against R. solani on potato crops.