To determine the optimum light intensity per cell required for rapid growth regardless of cell density, continuous cultures of the microalga Chlorella zofingiensis were grown with a sufficient supply of nutrients and CO2 and were subjected to different light intensities in the range of 75-1000 μE m(-2) s(-1). The cell density of culture increased over time for all light conditions except for the early stage of the high light condition of 1000 μE m(-2) s(-1). The light intensity per cell required for the high specific growth rate of 0.5 day(-1) was determined to be 28-45 μE g-ds(-1) s(-1). The specific growth rate was significantly correlated to light intensity (y=0.721×x/(66.98+x), r(2)=0.85, p<0.05). A high specific growth rate was maintained over a range of light intensities (250-1000 μE m(-2) s(-1)). This range of light intensities suggested that effective production of C. zofingiensis can be maintained outdoors under strong light by using the optimum specific light intensity.
Light quality and the intensity are key factors which render microalgae as a potential source of biodiesel. In this study the effects of various intensities of blue light and its photoperiods on the growth and lipid content of Chlorella vulgaris were investigated by using LED (Light Emitting Diode) in batch culture. C. vulgaris was grown for 13 days at three different light intensities (100, 200 and 300 μmol m(-2)s(-1)). Effect of three different light and dark regimes (12:12, 16:08 and 24:00 h Light:Dark) were investigated for each light intensity at 25°C culture temperature. Maximum lipid content (23.5%) was obtained due to high efficiency and deep penetration of 200 μmol m(-2)s(-1) of blue light (12:12 L:D) with improved specific growth (1.26 d(-1)) within reduced cultivation time of 8 days. White light could produce 20.9% lipid content in 10 days at 16:08 h L:D.
The aim of this study is to identify the optimum thermal conversion of Chlorella vulgaris with neuro-evolutionary approach. A Progressive Depth Swarm-Evolution (PDSE) neuro-evolutionary approach is proposed to model the Thermogravimetric analysis (TGA) data of catalytic thermal degradation of Chlorella vulgaris. Results showed that the proposed method can generate predictions which are more accurate compared to other conventional approaches (>90% lower in Root Mean Square Error (RMSE) and Mean Bias Error (MBE)). In addition, Simulated Annealing is proposed to determine the optimal operating conditions for microalgae conversion from multiple trained ANN. The predicted optimum conditions were reaction temperature of 900.0 °C, heating rate of 5.0 °C/min with the presence of HZSM-5 zeolite catalyst to obtain 88.3% of Chlorella vulgaris conversion.
Cell immobilization on the magnetic nanoparticles (MNPs) and magnetic harvesting is a novel approach for microalgal cells separation. To date, the effect of these nanoparticles on microalgal cells was only studied over a short period of time. More studies are hence needed for a better understanding of the magnetic harvesting proposes or environmental concerns relating to long-term exposure to nanoparticles. In this study, the impact of various concentrations of MNPs on the microalgal cells growth and their metabolic status was investigated over 12 days. More than 60% reduction in mitochondrial activity and pigments (chlorophyll a, chlorophyll b, and carotenoids) content occurred during the first 6 days of exposure to ≥50 µg/mL nanoparticles. However, more than 50% growth inhibitory effect was seen at concentrations higher than 400 µg/mL. Exposure to MNPs gradually induced cellular adaptation and after about 6 days of exposure to stress generating concentrations (˂400 µg/mL) of IONs, microalgae could overcome the imposed damages. This work provides a better understanding regarding the environmental impact of MNPs and appropriate concentrations of these particles for future algal cells magnetic immobilization and harvesting.
Chlorella is a popular microalga with robust physiological and biochemical characteristics, which can be cultured under various conditions. The exploration of the small RNA content of Chlorella could improve strategies for the enhancement of metabolite production from this microalga. In this study, stress was introduced to the Chlorella sorokiniana culture to produce high-value metabolites such as carotenoids and phenolic content. The small RNA transcriptome of C. sorokiniana was sequenced, focusing on microRNA (miRNA) content. From the analysis, 98 miRNAs were identified in cultures subjected to normal and stress conditions. The functional analysis result showed that the miRNA targets found were most often involved in the biosynthesis of secondary metabolites, followed by protein metabolism, cell cycle, and porphyrin and chlorophyll metabolism. Furthermore, the biosynthesis of secondary metabolites such as carotenoids, terpenoids, and lipids was found mostly in stress conditions. These results may help to improve our understanding of regulatory mechanisms of miRNA in the biological and metabolic process of Chlorella species. It is important and timely to determine the true potential of this microalga species and to support the potential for genetic engineering of microalgae as they receive increasing focus for their development as an alternative source of biofuel, food, and health supplements.
This study investigated acclimation ability of native Chlorella sorokiniana (CS-N) and commercial Chlorella sorokiniana (CS-C) in palm oil mill effluent (POME), their metabolic profile and feasibility of effluent recycling for dilution purpose. Maximum specific growth rate, µmax and lag time, λ of the microalgae were evaluated. Result shows both strains produced comparable growth in POME, with µmax of 0.31 day-1 and 0.30 day-1 respectively, albeit longer λ by the CS-C. However, three cycles of acclimation was able to reduce λ from eight days to two days for CS-C. Metabolic profiling using principal component analysis (PCA) shows clear cluster of acclimatized strains to suggest better stress tolerance of CS-N. Finally, a remarkable µmax of 0.57 day-1 without lag phase was achieved using acclimatized CS-N in 40% POME concentration. Acclimation has successfully shortened the λ and dilution with final effluent was proved to be feasible for further improvement of the microalgae growth.
This study was undertaken to investigate the effects of different nitrate concentrations in culture medium on oil content and fatty acid composition of Chlorella vulgaris (UMT-M1) and Chlorella sorokiniana (KS-MB2). Results showed that both species produced significant higher (p<0.05) oil content at nitrate ranging from 0.18 to 0.66 mM with C. vulgaris produced 10.20-11.34% dw, while C. sorokiniana produced 15.44-17.32% dw. The major fatty acids detected include C16:0, C18:0, C18:1, C18:2 and C18:3. It is interesting to note that both species displayed differentially regulated fatty acid accumulation patterns in response to nitrate treatments at early stationary growth phase. Their potential use for biodiesel application could be enhanced by exploring the concept of binary blending of the two microalgae oils using developed mathematical equations to calculate the oil mass blending ratio and simultaneously estimated the weight percentage (wt.%) of desirable fatty acid compositions.
One of the main limitations of productivity in photobioreactor is the inefficient conversion of the available light into biomass. Photoautotrophic cells such as microalgae only absorb a small fraction of supplied illumination due to limitation of its photosystem's (PS) absorbing rate. However, phenomenon of Flashing Light Effect (FLE) allows microalgae to utilize strong light exceptionally through intermittent exposure. Exposure of strong light at correct frequency of light and dark photoperiod would allow two pigment-protein complexes, PSI and PSII to be at the equilibrium mid-point potential to allow efficient light conversion. Narrow range of optimum frequency is crucial since overexposure to strong light would injured photosynthetic apparatus whereas longer dark period would contributed to loss of biomass due to triacylglycerol metabolism. The behaviour of microalgae towards various illumination conditions of FLE was determined at batch Photobioreactor (PBR) by varying the aeration flow rate: 16.94, 33.14 and 49.28 mL sec(-1) which yield, respectively the light exposure time of 3.99, 1.71 and 1.1 seconds per cycle. Maximum cell density in FLE-PBR was significantly higher at the exponential phase as compared to the continuously illuminated culture (p = 5.62 x 10(-5), a = 0.05) under the flow rate of 25.07 mL sec(-1). Maximum cell density yield of FLE-PBR and continuously illuminated PBR was, respectively 3.1125 x 10(7) and 2.947 x 10(7) cells mL(-1). Utilization of FLE as an innovative solution to increase the efficiency of microalgae to convert light into chemical energy would revolutionize the microalgae culture, reduce the time for cultivation and produce higher maximum biomass density.
Subcritical water extraction (SCW) was used to extract oil from Chlorella pyrenoidosa. The operational factors such as reaction temperature, reaction time, and biomass loading influence the oil yield during the extraction process. In this study, response surface methodology was employed to identify the desired extraction conditions for maximum oil yield. Experiments were carried out in batch reactors as per central composite design with three independent factors including reaction temperature (170, 220, 270, 320, and 370°C), reaction time (1, 5, 10, 15, and 20 min), and biomass loading (1, 3, 5, 10, and 15%). A maximum oil yield of 12.89 wt.% was obtained at 320°C and 15 min, with 3% biomass loading. Sequential model tests showed the good fit of experimental data to the second-order quadratic model. This study opens the great potential of SCW to extract algal oil for use in algal biofuel production.
The biosynthesis of calcium carbonate (CaCO3) minerals through a metabolic process known as microbially induced calcium carbonate precipitation (MICP) between diverse microorganisms, and organic/inorganic compounds within their immediate microenvironment, gives rise to a cementitious biomaterial that may emerge as a promissory alternative to conventional cement. Among photosynthetic microalgae, Chlorella vulgaris has been identified as one of the species capable of undergoing such activity in nature. In this study, response surface technique was employed to ascertain the optimum condition for the enhancement of biomass and CaCO3 precipitation of C. vulgaris when cultured in Blue-Green (BG)-11 aquaculture medium. Preliminary screening via Plackett-Burman Design showed that sodium nitrate (NaNO3), sodium acetate, and urea have a significant effect on both target responses (p < 0.05). Further refinement was conducted using Box-Behnken Design based on these three factors. The highest production of 1.517 g/L C. vulgaris biomass and 1.143 g/L of CaCO3 precipitates was achieved with a final recipe comprising of 8.74 mM of NaNO3, 61.40 mM of sodium acetate and 0.143 g/L of urea, respectively. Moreover, polymorphism analyses on the collected minerals through morphological examination via scanning electron microscopy and crystallographic elucidation by X-ray diffraction indicated to predominantly calcite crystalline structure.
Subcritical water extraction (SWE) technology has been used for the extraction of active compounds from different biomass materials with low process cost, mild operating conditions, short process times, and environmental sustainability. With the limited application of the technology to microalgal biomass, this work investigates parametrically the potential of subcritical water for high-yield extraction of biochemicals such as carbohydrates and proteins from microalgal biomass. The SWE process was optimized using central composite design (CCD) under varying process conditions of temperature (180-374°C), extraction time (1-20 min), biomass particulate size (38-250 μm), and microalgal biomass loading (5-40 wt.%). Chlorella vulgaris used in this study shows high volatile matter (83.5 wt.%) and carbon content (47.11 wt.%), giving advantage as a feedstock for biofuel production. The results showed maximum total carbohydrate content and protein yields of 14.2 g/100 g and 31.2 g/100 g, respectively, achieved under the process conditions of 277°C, 5% of microalgal biomass loading, and 5 min extraction time. Statistical analysis revealed that, of all the parameters investigated, temperature is the most critical during SWE of microalgal biomass for protein and carbohydrate production.
Flocculants are foreign particles that aggregate suspended microalgae cells and due to cost factor and toxicity, harvesting of microalgae biomass has shifted towards the use of bioflocculants. In this study, mild acid-extracted bioflocculants from waste chicken's eggshell and clam shell were used to harvest Chlorella vulgaris that was cultivated using chicken compost as nutrient source. It was found that a maximum of 99% flocculation efficiency can be attained at pH medium of 9.8 using 60 mg/L of hydrochloric acid-extracted chicken's eggshell bioflocculant at 50 °C of reaction temperature. On the other hand, 80 mg/L of hydrochloric acid-extracted clam shell bioflocculant was sufficient to recover C. vulgaris biomass at pH 9.8 and optimum temperature of 40 °C. The bioflocculants and bioflocs were characterized using microscopic, zeta potential, XRD, AAS and FT-IR analysis. The result revealed that calcium ions in the bioflocculants are the main contributor towards the flocculation of C. vulgaris, employing charge neutralization and sweeping as possible flocculation mechanisms. The kinetic parameters were best fitted pseudo-second order which resulted in R2 of 0.99 under optimal flocculation temperature. The results herein, disclosed the applicability of shell waste-derived bioflocculants for up-scaled microalgae harvesting for biodiesel production.
Microalgal lipid production by Chlorella protothecoides using sugarcane bagasse hydrolysate was investigated in this study. First, maximum glucose and reducing sugar concentrations of 15.2 and 27.0 g/L were obtained in sugarcane bagasse hydrolysate (SCBH), and the effects of different percentages of glucose and xylose on algal cultivation were investigated. Afterwards, SCBH was used as a carbon source for the cultivation of C. protothecoides and higher biomass concentration of 10.7 g/L was achieved. Additionally, a large amount of fatty acids, accounting up to 16.8% of dry weight, were accumulated in C. protothecoides in the nitrogen-limited (0.1-1 mmol/L) culture. Although SCBH inhibited fatty acid accumulation to a certain degree and the inhibition was aggravated by nitrogen starvation, SCBH favored microalgal cell growth and fatty acid production. The present study is of significance for the integration of cost-effective feedstocks production for biodiesel with low-cost SCBH as well as environmentally friendly disposal of lignocellulosic wastes.
Sarcopenia is characterized by the loss of muscle mass, strength, and function with ageing. With increasing life expectancy, greater attention has been given to counteracting the effects of sarcopenia on the growing elderly population. Chlorella vulgaris, a microscopic, unicellular, green alga with the potential for various pharmaceutical uses, has been widely studied in this context. This study is aimed at determining the effects of C. vulgaris on promoting muscle regeneration by evaluating myoblast regenerative capacity in vitro. Human skeletal myoblast cells were cultured and underwent serial passaging into young and senescent phases and were then treated with C. vulgaris, followed by the induction of differentiation. The ability of C. vulgaris to promote myoblast differentiation was analysed through cellular morphology, real-time monitoring, cell proliferation, senescence-associated β-galactosidase (SA-β-gal) expression, myogenic differentiation, myogenin expression, and cell cycle profiling. The results obtained showed that senescent myoblasts exhibited an enlarged and flattened morphology, with increased SA-β-gal expression, reduced myogenic differentiation, decreased expression of myogenin, and an increased percentage of cells in the G0/G1 phase. Treatment with C. vulgaris resulted in decreased SA-β-gal expression and promotion of myogenic differentiation, as observed via an increased fusion index, maturation index, myotube size, and surface area and an increased percentage of cells that stained positive for myogenin. In conclusion, C. vulgaris improves the regenerative capacity of young and senescent myoblasts and promotes myoblast differentiation, indicating its potential to promote muscle regeneration.
In this study freely suspended and Ca-alginate immobilized C. vulgaris cells were used for the biosorption of Fe(II), Mn(II), and Zn(II) ions, from the aqueous solution. Experimental data showed that biosorption capacity of algal cells was strongly dependent on the operational condition such as pH, initial metal ions concentration, dosages, contact time and temperature. The maximum biosorption of Fe(II) 43.43, Mn(II) 40.98 and Zn(II) 37.43 mg/g was achieved with Ca-alginate immobilized algal cells at optimum pH of 6.0, algal cells dosage 0.6 g/L, and contact time of 450 min at room temperature. The biosorption efficiency of freely suspended and immobilized C. vulgaris cells for heavy metals removal from the industrial wastewater was validated. Modeling of biosorption kinetics showed good agreements with pseudo-second-order. Langmuir and D-R isotherm models exhibited the best fit of experimental data. The thermodynamic parameters (ΔG°, ΔH°, and ΔS°) revealed that the biosorption of considered metal ions was feasible, spontaneous and exothermic at 25-45°C. The SEM showed porous morphology which greatly helps in the biosorption of heavy metals. The Fourier transform infrared spectrophotometer (FTIR) and X-rays Photon Spectroscopy (XPS) data spectra indicated that the functional groups predominately involved in the biosorption were C-N, -OH, COO-, -CH, C=C, C=S and -C-. These results shows that immobilized algal cells in alginate beads could potentially enhance the biosorption of considered metal ions than freely suspended cells. Furthermore, the biosorbent has significantly removed heavy metals from industrial wastewater at the optimized condition.
A potentially novel actinobacterium isolated from forest soil, Streptomyces sp. KSF103 was evaluated for its insecticidal effect against several mosquito species namely Aedes aegypti, Aedes albopictus, Anopheles cracens and Culex quinquefasciatus. Mosquito larvae and adults were exposed to various concentrations of the ethyl acetate (EA) extract for 24 h. Considerable mortality was evident after the EA extract treatment for all four important vector mosquitoes. Larvicidal activity of the EA extract resulted in LC50 at 0.045 mg/mL and LC90 at 0.080 mg/mL for Ae. aegypti; LC50 at 0.060 mg/mL and LC90 at 0.247 mg/mL for Ae. albopictus; LC50 at 2.141 mg/mL and LC90 at 6.345 mg/mL for An. cracens; and LC50 at 0.272 mg/mL and LC90 at 0.980 mg/mL for Cx. quinquefasciatus. In adulticidal tests, the EA extract was the most toxic to Ae. albopictus adults (LD50 = 2.445 mg/mL; LD90 = 20.004 mg/mL), followed by An. cracens (LD50 = 5.121 mg/mL; LD90 = 147.854 mg/mL) and then Ae. aegypti (LD50 = 28.873 mg/mL; LD90 = 274.823 mg/mL). Additionally, the EA extract exhibited ovicidal activity against Ae. aegypti (LC50 = 0.715 mg/mL; LC90 = 6.956 mg/mL), Ae. albopictus (LC50 = 0.715 mg/mL; LC90 = 6.956 mg/mL), and An. cracens (LC50 = 0.715 mg/mL; LC90 = 6.956 mg/mL), evaluated up to 168 h post-treatment. It displayed no toxicity on the freshwater microalga Chlorella sp. Beijerinck UMACC 313, marine microalga Chlorella sp. Beijerinck UMACC 258 and the ant Odontoponera denticulata. In conclusion, the EA extract showed promising larvicidal, adulticidal and ovicidal activity against Ae. aegypti, Ae. albopictus, An. cracens, and Cx. quinquefasciatus (larvae only). The results suggest that the EA extract of Streptomyces sp. KSF103 has the potential to be used as an environmental-friendly approach in mosquito control. The current study would serve as an initial step toward complementing microbe-based bioinsecticides for synthetic insecticides against medically important mosquitoes.
The utilization of metal-based conventional coagulants/flocculants to remove suspended solids from drinking water and wastewater is currently leading to new concerns. Alarming issues related to the prolonged effects on human health and further pollution to aquatic environments from the generated nonbiodegradable sludge are becoming trending topics. The utilization of biocoagulants/bioflocculants does not produce chemical residue in the effluent and creates nonharmful, biodegradable sludge. The conventional coagulation-flocculation processes in drinking water and wastewater treatment, including the health and environmental issues related to the utilization of metal-based coagulants/flocculants during the processes, are discussed in this paper. As a counterpoint, the development of biocoagulants/bioflocculants for drinking water and wastewater treatment is intensively reviewed. The characterization, origin, potential sources, and application of this green technology are critically reviewed. This review paper also provides a thorough discussion on the challenges and opportunities regarding the further utilization and application of biocoagulants/bioflocculants in water and wastewater treatment, including the importance of the selection of raw materials, the simplification of extraction processes, the application to different water and wastewater characteristics, the scaling up of this technology to a real industrial scale, and also the potential for sludge recovery by utilizing biocoagulants/bioflocculants in water/wastewater treatment.
Microalgae lipids and oils are potential candidates for renewable biofuels and nutritional inventions. Recent studies from our lab have shown that two plant hormones, auxin and jasmonic acid, influence microalgae growth and fatty acid accumulation. Therefore, in this study, a high oil-producing strain Chlorella vulgaris UMT-M1 was selected for hormonal study using gibberellin (GA). Exogenous GA3 was applied to early stationary culture of C. vulgaris UMT-M1. Results showed that GA3 gradually increases the cell density of C. vulgaris to up to 42% on days after treatment (DAT)-8 and also capable of delaying the algal senescence. However, the increment in cell density did not enhance the total oil production albeit transient modification of fatty acid compositions was observed for saturated (SFA) and polyunsaturated (PUFA) fatty acids. This illustrates that GA3 only promotes cell division and growth but not the oil accumulation. In addition, application of GA3 in culture medium was shown to promote transient increment of palmitic (C16:0) and stearic (C18:0) acids from DAT-4 to DAT-6 and these changes are correlated with the expression of β-ketoacyl ACP synthase I (KAS I) gene.
Conventional microalgae oil extraction applies physicochemical destruction of dry cell biomass prior to transesterification process to produce fatty acid methyl esters (FAMEs). This report presents a simple and rapid direct transesterification (DT) method for FAMEs production and fatty acid profiling of microalgae using freshly harvested biomass. Results revealed that the FAMEs recovered from Chlorella vulgaris were 50.1 and 68.3 mg with conventional oil-extraction-transesterification (OET) and DT method, respectively. While for Messastrum gracile, the FAMEs recovered, were 49.9 and 76.3 mg, respectively with OET and DT methods. This demonstrated that the DT method increased FAMEs recovery by 36.4% and 53.0% from C. vulgaris and M. gracile, respectively, as compared to OET method. Additionally, the DT method recovered a significantly higher amount of palmitic (C16:0) and stearic (C18:0) acids from both species, which indicated the important role of these fatty acids in the membranes of cells and organelles. The DT method performed very well using a small volume (5 mL) of fresh biomass coupled with a shorter reaction time (~ 15 min), thus making real-time monitoring of FAMEs and fatty acid accumulation in microalgae culture feasible.
Mangrove-dwelling microalgae are well adapted to frequent encounters of salinity fluctuations across their various growth phases but are lesser studied. The current study explored the adaptive changes (in terms of biomass, oil content and fatty acid composition) of mangrove-isolated C. vulgaris UMT-M1 cultured under different salinity levels (5, 10, 15, 20, 30 ppt). The highest total oil content was recorded in cultures at 15 ppt salinity (63.5% of dry weight) with uncompromised biomass productivity, thus highlighting the 'trigger-threshold' for oil accumulation in C. vulgaris UMT-M1. Subsequently, C. vulgaris UMT-M1 was further assessed across different growth phases under 15 ppt. The various short, medium and long-chain fatty acids (particularly C20:0), coupled with a high level of C18:3n3 PUFA reported at early exponential phase represents their physiological importance during rapid cell growth. Accumulation of C18:1 and C18:2 at stationary growth phase across all salinities was seen as cells accumulating substrate for C18:3n3 should the cells anticipate a move from stationary phase into new growth phase. This study sheds some light on the possibility of 'triggered' oil accumulation with uninterrupted growth and the participation of various fatty acid types upon salinity mitigation in a mangrove-dwelling microalgae.