Physiological responses of the green microalga Acutodesmus dimorphus to temperature induced oxidative stress conditions.

Temperature is the most critical factor that directly affects the physiological functioning and metabolic activities of any organism. With rising global temperature, understanding the heat stress response of an organism is critically important. In the present study, we investigated differences in the early changes occurring upon heat stress in the green microalga Acutodesmus dimorphus, a potential strain for biofuel production. The cells were heat-stressed at 45 and 50°C for 24 h and the temporal response of cells in terms of growth, pigments content, levels of oxidative stress biomarkers i.e., reactive oxygen species (ROS) and the response of enzymatic and non-enzymatic antioxidant scavengers were evaluated. The results revealed that after 24 h of heat stress at 45°C, the accumulations of chlorophyll a and carotenoids remained stable; all three ROS increased with the higher activities of various enzymatic and non-enzymatic antioxidants. On the contrary, at a higher temperature of 50°C, the accumulations of chlorophyll a, carotenoids and non-enzymatic antioxidants reduced drastically while the accumulations of all three ROS and the response of enzymatic antioxidants were significantly higher than those at 45°C. These results suggest that the cells utilize several stress acclimatization mechanisms to cope up the heat stress. There was a dramatic difference in the physiological changes and cellular antioxidant mechanism upon heat stress at 45 and 50°C. The cellular defense response of A. dimorphus gets impaired after heat stress at 50°C but remains active at 45°C, exhibiting the heat resistance and, thus, the thermotolerance.

Microalgal Target of Rapamycin (TOR): A Central Regulatory Hub for Growth, Stress Response and Biomass Production.

Target of rapamycin (TOR) is an evolutionarily conserved protein kinase that plays an important role in the regulation of cell growth and the sensing of nutrient and energy status in eukaryotes. In yeasts and mammals, the roles of TOR have been very well described and various functions of TOR signaling in plant lineages have also been revealed over the past 20 years. In the case of microalgae, the functions of TOR have been primarily studied in the model green alga Chlamydomonas reinhardtii and were summarized in an earlier single review article. However, the recent development of tools for the functional analysis of TOR has helped to reveal the involvement of TOR in various functions, including autophagy, transcription, translation, accumulation of energy storage molecules, etc., in microalgae. In the present review, we discuss recent novel findings relating to TOR signaling and its roles in microalgae along with relevant information on land plants and also provide details of topics that must be addressed in future studies to reveal how TOR regulates various physiological functions in microalgae.

Accelerated triacylglycerol production without growth inhibition by overexpression of a glycerol-3-phosphate acyltransferase in the unicellular red alga Cyanidioschyzon merolae.

Microalgae accumulate triacylglycerols (TAGs), a promising feedstock for biodiesel production, under unfavorable environmental or stress conditions for their growth. Our previous analyses revealed that only transcripts of CmGPAT1 and CmGPAT2, both encoding glycerol-3-phosphate acyltransferase, were increased among fatty acid and TAG synthesis genes under TAG accumulation conditions in the red alga Cyanidioschyzon merolae. In this study, to investigate the role of these proteins in TAG accumulation in C. merolae, we constructed FLAG-fused CmGPAT1 and CmGPAT2 overexpression strains. We found that CmGPAT1 overexpression resulted in marked accumulation of TAG even under normal growth conditions, with the maximum TAG productivity increased 56.1-fold compared with the control strain, without a negative impact on algal growth. The relative fatty acid composition of 18:2 in the TAGs and the sn-1/sn-3 positions were significantly increased compared with the control strain, suggesting that CmGPAT1 had a substrate preference for 18:2. Immunoblot analysis after cell fractionation and immunostaining analysis demonstrated that CmGPAT1 localizes in the endoplasmic reticulum (ER). These results indicate that the reaction catalyzed by the ER-localized CmGPAT1 is a rate-limiting step for TAG synthesis in C. merolae, and would be a potential target for improvement of TAG productivity in microalgae.

Salinity induced oxidative stress alters the physiological responses and improves the biofuel potential of green microalgae Acutodesmus dimorphus.

The main aim of the present study was to analyze salinity stress induced physiological and biochemical changes in a freshwater microalgae Acutodesmus dimorphus. During single-stage cultivation, the accumulations of lipids and carbohydrates increased with an increase in an initial salinity of the culture medium. The carbohydrate and lipid accumulations of 53.30±2.76% and 33.40±2.29%, respectively, were observed in 200mM NaCl added culture. During two-stage cultivation, salinity stress of 200mM was favorable for the growth up to 2days, as suggested by higher biomass, lower levels of oxidative stress biomarkers and no significant changes in the biochemical composition of the cells. Extending the stress to 3days significantly increased the lipid accumulation by 43% without affecting the biomass production. This study, thus, provides the strategy to improve the biofuel potential of A. dimorphus along with presenting the physiological adaptive mechanisms of a cell against salinity stress.

Nitrogen starvation-induced cellular crosstalk of ROS-scavenging antioxidants and phytohormone enhanced the biofuel potential of green microalga Acutodesmus dimorphus.

BACKGROUND: Microalgae accumulate a considerable amount of lipids and carbohydrate under nutrient-deficient conditions, which makes them one of the promising sustainable resources for biofuel production. In the present study, to obtain the biomass with higher lipid and carbohydrate contents, we implemented a short-term nitrogen starvation of 1, 2, and 3 days in a green microalga Acutodesmus dimorphus. Few recent reports suggest that oxidative stress-tolerant microalgae are highly efficient for biofuel production. To study the role of oxidative stress due to nitrogen deficiency, responses of various stress biomarkers like reactive oxygen species (ROS), cellular enzymatic antioxidants superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and non-enzymatic scavengers proline and polyphenols were also evaluated. Further, the endogenous levels of phytohormones abscisic acid (ABA) and indole-3-acetic acid (IAA) were also determined to study their response to nitrogen deficiency. RESULTS: We observed that nitrogen starvation of 2 days is effective to produce biomass containing 29.92% of lipid (comprising about 75% of neutral lipid) and 34.80% of carbohydrate, which is significantly higher (about 23 and 64%, respectively) than that of the control culture. Among all nitrogen-starved cultures, the accumulations of ROS were lower in 2 days starved culture, which can be linked with the several folds higher activities of SOD and CAT in this culture. The accumulations of proline and total polyphenols were also significantly higher (about 4.7- and 1.7-folds, respectively, than that of the control) in 2 days nitrogen-starved culture. The levels of phytohormones once decreased significantly after 1 day, increased continuously up to 3 days of nitrogen starvation. CONCLUSION: The findings of the present study highlight the interaction of nitrogen starvation-induced oxidative stress with the signaling involved in the growth and development of microalga. The study presents a comprehensive picture of the adaptive mechanisms of the cells from a physiological perspective along with providing the strategy to improve the biofuel potential of A. dimorphus through a short-term nitrogen starvation.

Surface-Active Ionic Liquid Cholinium Dodecylbenzenesulfonate: Self-Assembling Behavior and Interaction with Cellulase.

The conventional sodium dodecylbenzenesulfonate (NaDBS) has been converted into an efficient and nontoxic anionic surface-active ionic liquid, cholinium dodecylbenzenesulfonate (Cho[DBS]). We have investigated its self-assembling behavior, interaction with the enzyme cellulase, and ecotoxicity. The surface-active properties at the air-liquid interface and the aggregation behavior of Cho[DBS] in water have been determined using tensiometry, isothermal titration calorimetry (ITC), conductometry, and dynamic light scattering (DLS). The enzyme activity was observed using dinitro salicylic acid analysis. The enhanced enzyme activity was explained through active-site exfoliation and structural constancy of cellulase in the micellar medium using the results from fluorescence, circular dichroism, DLS, and ITC. The nontoxic nature was confirmed by toxicity analysis on the freshwater microalgae Scenedesmus sp.

Microalgal biomass generation by phycoremediation of dairy industry wastewater: An integrated approach towards sustainable biofuel production.

Dairy wastewater collected from local dairy industry was used as a growth media (without any pre-treatment) for the cultivation of microalgae Acutodesmus dimorphus. The level of COD reduced over 90% (from 2593.33±277.37 to 215±7.07mg/L) after 4days of cultivation; whereas, ammoniacal nitrogen was consumed completely (277.4±10.75mg/L) after 6days of cultivation. Dry biomass of 840 and 790mg/L was observed after 4 and 8days of cultivation, respectively, which is about 5-6 times more than that of BG-11 grown culture (149mg/L after 8days). This biomass contains around 25% lipid and 30% carbohydrate, which can be further converted into biodiesel and bioethanol, respectively. Theoretical calculations based on the recently reported conversion yield suggest that 1kg biomass of A. dimorphus might produce around 195g of biodiesel and 78g of bioethanol, which sums up to 273g of biofuels.

Applications of de-oiled microalgal biomass towards development of sustainable biorefinery.

In view of commercialization of microalgal biofuel, the de-oiled microalgal biomass (DMB) is a surplus by-product in the biorefinery process that needs to be exploited to make the process economically attractive and feasible. This DMB, rich in carbohydrates, proteins, and minerals, can be used as feed, fertilizer, and substrate for the production of bioethanol/bio-methane. Further, thermo-chemical conversion of DMB results into fuels and industrially important chemicals. Future prospects of DMB also lie with its conversion into novel biomaterials like nanoparticles and carbon-dot which have biomedical importance. The lowest valued application of DMB is to use it for adsorption of dyes and heavy metals from industrial effluents. This study reviews how DMB can be utilized for different applications and in the generation of valuable co-products. The value addition of DMB would thereby improve the overall cost economics of the microalgal bio-refinery.

Hydrolysate of lipid extracted microalgal biomass residue: An algal growth promoter and enhancer.

The present study demonstrates the utilization of the algal hydrolysate (AH) prepared from lipid extracted residual harmful bloom-forming cyanobacteria Lyngbya majuscula biomass, as a growth supplement for the cultivation of green microalgae Chlorella vulgaris. BG-11 replacements with AH in different proportions significantly affects the cell count, dry cell weight (DCW), biomass productivity (BP) and pigments concentration. Among all, 25% AH substitution in BG11 media was found to be optimum which enhanced DCW, BP and pigments content by 39.13%, 40.81% and 129.47%, respectively, compared to control. The lipid content (31.95%) was also significantly higher in the 25% AH replacement. The volumetric productivity of neutral lipids (ideal for biodiesel) and total protein content of the cells significantly increased in all AH substitutions. Thus, lipid extracted microalgal biomass residue (LMBR) hydrolysate can be a potential growth stimulating supplement for oleaginous microalgae C. vulgaris.

Comparative evaluation of chemical and enzymatic saccharification of mixotrophically grown de-oiled microalgal biomass for reducing sugar production.

For the commercialization of microalgal based biofuels, utilization of de-oiled carbohydrate rich biomass is important. In the present study, chemo-enzymatic hydrolysis of mixotrophically grown Scenedesmus sp. CCNM 1077 de-oiled biomass is evaluated. Among the chemical hydrolysis, use of 0.5M HCl for 45 min at 121°C resulted in highest saccharification yield of 37.87% w/w of de-oiled biomass. However, enzymatic hydrolysis using Viscozyme L at loading rate of 20 FBGU/g of de-oiled biomass, pH 5.5 and temperature 45°C for 72 h resulted in saccharification yield of 43.44% w/w of de-oiled biomass. Further, 78% ethanol production efficiency was achieved with enzymatically hydrolyzed de-oiled biomass using yeast Saccharomyces cerevisiae ATCC 6793. These findings of the present study show application of mixotrophically grown de-oiled biomass of Scenedesmus sp. CCNM 1077 as promising feedstock for bioethanol production.

Selective carotenoid accumulation by varying nutrient media and salinity in Synechocystis sp. CCNM 2501.

Nutrients are the deciding factors in the biological production of bioactive compounds. Various growth media like BG11, Zarrouk's and Chu's 10 were studied for carotenoid production in Synechocystis sp. CCNM 2501. Maximum carotenoid content (dry weight basis) was found in Zarrouk's medium (ZM, 7.99mgg(-1)) followed by BG11 (5.13mgg(-1)). Echinenone content was 4 times higher in ZM (3.81mgg(-1)) as compared to BG11 (0.95mgg(-1)) and Chu's 10 (0.77mgg(-1)). Being an economical medium, BG11 was selected for carotenoid production. Further, increase in salinity from 0 to 0.2M in BG11 medium increases total carotenoid content from 5.82 to 7.05mgg(-1) and later it declines to 6.23mgg(-1) (1M). 3 times more ß-carotene is produced at 1M salinity as compared to control BG11. The variation in carotenoid composition with change in nutrients/salinity can be a good strategy to enhance certain targeted carotenoids.

Bicarbonate supplementation enhanced biofuel production potential as well as nutritional stress mitigation in the microalgae Scenedesmus sp. CCNM 1077.

The aim of the present study was to find out the optimum sodium bicarbonate concentration to produce higher biomass with higher lipid and carbohydrate contents in microalgae Scenedesmus sp. CCNM 1077. The role of bicarbonate supplementation under different nutritional starvation conditions was also evaluated. The results clearly indicate that 0.6 g/L sodium bicarbonate was optimum concentration resulting in 20.91% total lipid and 25.56% carbohydrate along with 23% increase in biomass production compared to normal growth condition. Addition of sodium bicarbonate increased the activity of nutrient assimilatory enzymes, biomass, lipid and carbohydrate contents under different nutritional starvation conditions. Nitrogen starvation with bicarbonate supplementation resulted in 54.03% carbohydrate and 34.44% total lipid content in microalgae Scenedesmus sp. CCNM 1077. These findings show application of bicarbonate grown microalgae Scenedesmus sp. CCNM 1077 as a promising feedstock for biodiesel and bioethanol production.

Salinity induced oxidative stress enhanced biofuel production potential of microalgae Scenedesmus sp. CCNM 1077.

Microalgal biomass is considered as potential feedstock for biofuel production. Enhancement of biomass, lipid and carbohydrate contents in microalgae is important for the commercialization of microalgal biofuels. In the present study, salinity stress induced physiological and biochemical changes in microalgae Scenedesmus sp. CCNM 1077 were studied. During single stage cultivation, 33.13% lipid and 35.91% carbohydrate content was found in 400 mM NaCl grown culture. During two stage cultivation, salinity stress of 400 mM for 3 days resulted in 24.77% lipid (containing 74.87% neutral lipid) along with higher biomass compared to single stage, making it an efficient strategy to enhance biofuel production potential of Scenedesmus sp. CCNM 1077. Apart from biochemical content, stress biomarkers like hydrogen peroxide, lipid peroxidation, ascorbate peroxidase, proline and mineral contents were also studied to understand the role of reactive oxygen species (ROS) mediated lipid accumulation in microalgae Scenedesmus sp. CCNM 1077.

Enhanced biofuel production potential with nutritional stress amelioration through optimization of carbon source and light intensity in Scenedesmus sp. CCNM 1077.

Microalgal mixotrophic cultivation is one of the most potential ways to enhance biomass and biofuel production. In the present study, first of all ability of microalgae Scenedesmus sp. CCNM 1077 to utilize various carbon sources under mixotrophic growth condition was evaluated followed by optimization of glucose concentration and light intensity to obtain higher biomass, lipid and carbohydrate contents. Under optimized condition i.e. 4 g/L glucose and 150 µmol m(-2) s(-1) light intensity, Scenedesmus sp. CCNM 1077 produced 1.2g/L dry cell weight containing 23.62% total lipid and 42.68% carbohydrate. Addition of glucose shown nutritional stress ameliorating effects and around 70% carbohydrate and 25% total lipid content was found with only 21% reduction in dry cell weight under nitrogen starved condition. This study shows potential application of mixotrophically grown Scenedesmus sp. CCNM 1077 for bioethanol and biodiesel production feed stock.

Biofuel potential of the newly isolated microalgae Acutodesmus dimorphus under temperature induced oxidative stress conditions.

Lack of control over temperature is one of the major issues in large scale cultivation of microalgae. Therefore, it is important to evaluate the effects of cultivation temperature on the growth and physiology of microalgae. In the present study, freshwater microalgae Acutodesmus dimorphus was grown at different temperature in continuous and two stage cultivation. Results revealed that during continuous cultivation A. dimorphus grows better at 35°C than at 25°C and 38°C. At 35°C, A. dimorphus produced 22.7% lipid (containing 59% neutral lipid) and 33.7% carbohydrate along with 68% increase in biomass productivity (23.53mg/L/day) compared to 25°C grown culture. Stress biomarkers like reactive oxygen species, antioxidant enzymes like catalase and ascorbate peroxidase and lipid peroxidation were also lowest in 35°C grown culture which reveals that A. dimorphus is well acclimatized at 35°C.

Dominance of cyanobacterial and cryptophytic assemblage correlated to CDOM at heavy metal contamination sites of Gujarat, India.

Industrial clusters of Gujarat, India, generate high quantity of effluents which are received by aquatic bodies such as estuary and coastal water. In the present study, microalgal assemblage, heavy metals, and physico-chemical variables were studied from different habitats. Principal component analysis revealed that biovolume of cyanobacterial and cryptophytic community positively correlated with the heavy metal concentration (Hg, As, Zn, Fe, Mo, Ni, and Co) and chromophoric dissolved organic matter (CDOM) under hypoxic environment. Green algae and diatoms dominated at comparatively lower nitrate concentration which was positively associated with Pb and Mn.

Lipid Extracted Microalgal Biomass Residue as a Fertilizer Substitute for Zea mays L.

High volumes of lipid extracted microalgal biomass residues (LMBRs) are expected to be produced upon commencement of biodiesel production on a large scale, thus necessitating its value addition for sustainable development. LMBRs of Chlorella variabilis and Lyngbya majuscula were employed to substitute the nitrogen content of recommended rate of fertilizer (RRF) for Zea mays L. The pot experiment comprised of 10 treatments, i.e., T1 (No fertilizer); T2 (RRF-120 N: 60 P2O5: 40 K2O kg ha(-1)); T3 to T6-100, 75, 50, and 25% N through LMBR of the Chlorella sp., respectively; T7 to T10-100, 75, 50, and 25% N through LMBR of Lyngbya sp., respectively. It was found that all LMBR substitution treatments were at par to RRF with respect to grain yield production. T10 gave the highest grain yield (65.16 g plant(-1)), which was closely followed by that (63.48 g plant(-1)) under T5. T10 also recorded the highest phosphorus and potassium contents in grains. T4 was markedly superior over control in terms of dry matter accumulation (DMA) as well as carbohydrate content, which was ascribed to higher pigment content and photosynthetic activity in leaves. Even though considerably lower DMA was obtained in Lyngbya treatments, which might have been due to the presence of some toxic factors, no reduction in grain yield was apparent. The length of the tassel was significantly higher in either of the LMBRs at any substitution rates over RRF, except T6 and T7. The ascorbate peroxidase activity decreased with decreasing dose of Chlorella LMBR, while all the Lyngbya LMBR treatments recorded lower activity, which were at par with each other. Among the Chlorella treatments, only T5 recorded significantly higher values of glutathione reductase activity over RRF, while the rest were at par. There were significant increases in carbohydrate and crude fat, respectively, only in T4 and T3 over RRF, while no change was observed in crude protein due to LMBR treatments. Apparently, there was no detrimental effect on soil properties, suggesting that both the LMBRs can be employed to reduce the usage of chemical fertilizers, thus promoting maize crop production in a sustainable manner.

Biosorption of methylene blue by de-oiled algal biomass: equilibrium, kinetics and artificial neural network modelling.

The main objective of the present study is to effectively utilize the de-oiled algal biomass (DAB) to minimize the waste streams from algal biofuel by using it as an adsorbent. Methylene blue (MB) was used as a sorbate for evaluating the potential of DAB as a biosorbent. The DAB was characterized by SEM, FTIR, pHPZC, particle size, pore volume and pore diameter to understand the biosorption mechanism. The equilibrium studies were carried out by variation in different parameters, i.e., pH (2-9), temperature (293.16-323.16 K), biosorbent dosage (1-10 g L(-1)), contact time (0-1,440 min), agitation speed (0-150 rpm) and dye concentration (25-2,500 mg L(-1)). MB removal was greater than 90% in both acidic and basic pH. The optimum result of MB removal was found at 5-7 g L(-1) DAB concentration. DAB removes 86% dye in 5 minutes under static conditions and nearly 100% in 24 hours when agitated at 150 rpm. The highest adsorption capacity was found 139.11 mg g(-1) at 2,000 mg L(-1) initial MB concentration. The process attained equilibrium in 24 hours. It is an endothermic process whose spontaneity increases with temperature. MB biosorption by DAB follows pseudo-second order kinetics. Artificial neural network (ANN) model also validates the experimental dye removal efficiency (R2 = 0.97) corresponding with theoretically predicted values. Sensitivity analysis suggests that temperature and agitation speed affect the process most with 23.62% and 21.08% influence on MB biosorption, respectively. Dye adsorption capacity of DAB in fixed bed column was 107.57 mg g(-1) in preliminary study while it went up to 139.11 mg g(-1) in batch studies. The probable mechanism for biosorption in this study is chemisorptions via surface active charges in the initial phase followed by physical sorption by occupying pores of DAB.

Effects of different media composition, light intensity and photoperiod on morphology and physiology of freshwater microalgae Ankistrodesmus falcatus--a potential strain for bio-fuel production.

Media composition, light intensity and photoperiod significantly affect the algal growth and productivity and their optimization is important for the commercialization of microalgae based biofuels. In the present study, effects of different culture medium, light intensity and photoperiod were studied on growth, biomass productivity, and biochemical composition of a fresh water microalgae Ankistrodesmus falcatus in batch culture. The results revealed that A. falcatus could yield more than 35% of total lipid (containing around 65.74% neutral lipid) along with optimal growth (0.20 µ) and biomass productivity (7.9 mg/L/day) in the BG-11 medium under a light intensity of 60 µmol m(-2) s(-1) and 12:12 (Light: Dark) cycle. The highest total lipid yield of 67.2% (containing 72.68% of neutral lipid) was observed in Zarrouk's medium grown culture but with altered cell morphology and ultra-structural changes.

Nitrogen stress triggered biochemical and morphological changes in the microalgae Scenedesmus sp. CCNM 1077.

The aim of present study was to investigate the effects of nitrogen limitation as well as sequential nitrogen starvation on morphological and biochemical changes in Scenedesmus sp. CCNM 1077. The results revealed that the nitrogen limitation and sequential nitrogen starvation conditions significantly decreases the photosynthetic activity as well as crude protein content in the organism, while dry cell weight and biomass productivity are largely unaffected up to nitrate concentration of about 30.87mg/L and 3 days nitrate limitation condition. Nitrate stress was found to have a significant effect on cell morphology of Scenedesmus sp. CCNM 1077. Total removal of nitrate from the growth medium resulted in highest lipid (27.93%) and carbohydrate content (45.74%), making it a potential feed stock for biodiesel and bio-ethanol production. This is a unique approach to understand morphological and biochemical changes in freshwater microalgae under nitrate limitation as well as sequential nitrate removal conditions.