We isolated fifty-two strains from the marine aquaculture ponds in Malaysia that were evaluated for their lipid production and ammonium tolerance and four isolates were selected as new ammonium tolerant microalgae with high-lipid production: TRG10-p102 Oocystis heteromucosa (Chlorophyceae); TRG10-p103 and TRG10-p105 Thalassiosira weissflogii (Bacillariophyceae); and TRG10-p201 Amphora coffeiformis (Bacillariophyceae). Eicosapentenoic acid (EPA) in three diatom strain was between 2.6 and 18.6 % of total fatty acids, which were higher than in O. heteromucosa. Only A. coffeiformi possessed arachidonic acid. Oocystis heteromucosa naturally grew at high ammonium concentrations (1.4-10 mM), whereas the growth of the other strains, T. weissflogii and A. coffeiformi, were visibly inhibited at high ammonium concentrations (>1.4 mM-NH4). However, two strains of T. weissflogii were able to grow at up to 10 mM-NH4 by gradually acclimating to higher ammonium concentrations. The ammonium tolerant strains, especially T. weissflogii which have high EPA contents, were identified as a valuable candidate for biomass production utilizing NH4-N media, such as ammonium-rich wastewater.
Reserve lipids of microalgae are promising for biodiesel production. However, economically feasible and sustainable energy production from microalgae requires optimization of cultivation conditions for both biomass yield and lipid production of microalgae. Biomass yield and lipid production in microalgae are a contradictory problem because required conditions for both targets are different. Simultaneously, the mass cultivation of microalgae for biofuel production also depends extremely on the performance of the microalgae strains used. In this study a green unicellular microalgae Chlorella sorokiniana (DS6) isolated from the holding tanks of farm wastewater treatment plant using multi-step screening and acclimation procedures was found high-lipid producing facultative heterotrophic microalgae strain capable of growing on dairy farm effluent (DFE) for biodiesel feedstock and wastewater treatment. Morphological features and the phylogenetic analysis for the 18S rRNA identified the isolated strains. A novel three stage cultivation process of facultative strain of C. sorokiniana was examined for lipid production.
Ocean acidification, due to increased levels of anthropogenic carbon dioxide, is known to affect the physiology and growth of marine phytoplankton, especially in polar regions. However, the effect of acidification or carbonation on cellular metabolism in polar marine phytoplankton still remains an open question. There is some evidence that small chlorophytes may benefit more than other taxa of phytoplankton. To understand further how green polar picoplankton could acclimate to high oceanic CO2, studies were conducted on an Antarctic Chlorella sp. Chlorella sp. maintained its growth rate (∼0.180 d-1), photosynthetic quantum yield (Fv/Fm = ∼0.69) and chlorophyll a (0.145 fg cell-1) and carotenoid (0.06 fg cell-1) contents under high CO2, while maximum rates of electron transport decreased and non-photochemical quenching increased under elevated CO2. GCMS-based metabolomic analysis reveal that this polar Chlorella strain modulated the levels of metabolites associated with energy, amino acid, fatty acid and carbohydrate production, which could favour its survival in an increasingly acidified ocean.
In the present study, a novel oleaginous Thraustochytrid containing a high content of docosahexaenoic acid (DHA) was isolated from a mangrove ecosystem in Malaysia. The strain identified as an Aurantiochytrium sp. by 18S rRNA sequencing and named KRS101 used various carbon and nitrogen sources, indicating metabolic versatility. Optimal culture conditions, thus maximizing cell growth, and high levels of lipid and DHA production, were attained using glucose (60 g l⁻¹) as carbon source, corn steep solid (10 g l⁻¹) as nitrogen source, and sea salt (15 g l⁻¹). The highest biomass, lipid, and DHA production of KRS101 upon fed-batch fermentation were 50.2 g l⁻¹ (16.7 g l⁻¹ day⁻¹), 21.8 g l⁻¹ (44% DCW), and 8.8 g l⁻¹ (40% TFA), respectively. Similar values were obtained when a cheap substrate like molasses, rather than glucose, was used as the carbon source (DCW of 52.44 g l⁻¹, lipid and DHA levels of 20.2 and 8.83 g l⁻¹, respectively), indicating that production of microbial oils containing high levels of DHA can be produced economically when the novel strain is used.
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.
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.
Global warming has become a serious issue nowadays as the trend of CO2 emission is increasing by years. In Malaysia, the electricity and energy sector contributed a significant amount to the nation's CO2 emission due to fossil fuel use. Many research works have been carried out to mitigate this issue, including carbon capture and utilization (CCUS) technology and biological carbon fixation by microalgae. This study makes a preliminary effort to screen native microalgae species in the Malaysian coal-fired power plant's surrounding towards carbon fixation ability. Three dominant species, including Nannochloropsis sp., Tetraselmis sp., and Isochrysis sp. were identified and tested in the laboratory under ambient and pure CO2 condition to assess their growth and CO2 fixation ability. The results indicate Isochrysis sp. as the superior carbon fixer against other species. In continuation, the optimization study using Response Surface Methodology (RSM) was carried out to optimize the operating conditions of Isochrysis sp. using a customized lab-scale photobioreactor under simulated flue gas exposure. This species was further acclimatized and tested under actual flue gas generated by the power plant. Isochrysis sp. had shown its capability as a carbon fixer with CO2 fixation rate of 0.35 gCO2/L day under actual coal-fired flue gas exposure after cycles of acclimatization phase. This work is the first to demonstrate indigenous microalgae species' ability as a carbon fixer under Malaysian coal-fired flue gas exposure. Thus, the findings shall be useful in exploring the microalgae potential as a biological agent for carbon emission mitigation from power plants more sustainably.
Bioprospecting for biodiesel potential in microalgae primarily involves a few model species of microalgae and rarely on non-model microalgae species. Therefore, the present study determined changes in physiology, oil accumulation, fatty acid composition and biodiesel properties of a non-model microalga Messastrum gracile SE-MC4 in response to 12 continuous days of nitrate-starve (NS) and nitrate-replete (NR) conditions respectively. Under NS, the highest oil content (57.9%) was achieved despite reductions in chlorophyll content, biomass productivity and lipid productivity. However, under both NS and NR, palmitic acid and oleic acid remained as dominant fatty acids thus suggesting high potential of M. gracile for biodiesel feedstock consideration. Biodiesel properties analysis returned high values of cetane number (CN 61.9-64.4) and degree of unsaturation (DU 45.3-57.4) in both treatments. The current findings show the possibility of a non-model microalga to inherit superior ability over model species in oil accumulation for biodiesel development.
In this study, the effects of limited and excess nitrate on biomass, lipid production, and fatty acid profile in Messastrum gracile SE-MC4 were determined. The expression of fatty acid desaturase genes, namely stearoyl-ACP desaturase (SAD), omega-6 fatty acid desaturase (ω-6 FAD), omega-3 fatty acid desaturase isoform 1 (ω-3 FADi1), and omega-3 fatty acid desaturase isoform 2 (ω-3 FADi2) was also assessed. It was found that nitrate limitation generally increased the total oil, α-linolenic acid (C18:3n3) and total polyunsaturated fatty acid (PUFA) contents in M. gracile. The reduction of nitrate concentration from 1.76 to 0.11 mM increased the total oil content significantly from 32.5 to 41.85% (dry weight). Palmitic (C16:0) and oleic (C18:1) acids as the predominant fatty acids in this microalgae constituted between 82 and 87% of the total oil content and were relatively consistent throughout all nitrate concentrations tested. The expression of SAD, ω-6 FAD, and ω-3 FADi2 genes increased under nitrate limitation, especially at 0.11 mM nitrate. The ω-3 FADi1 demonstrated a binary up-regulation pattern of expression under both nitrate-deficient (0.11 mM) and -excess (3.55 mM) conditions. Thus, findings from this study suggested that limited or excess nitrate could be used as part of a cultivation strategy to increase oil and PUFA content following media optimisation and more efficient culture methodology. Data obtained from the expression of desaturase genes would provide valuable insights into their roles under excess and limited nitrate conditions in M. gracile, potentially paving the way for future genetic modifications.
The present study aims to investigate the influence of Staphylococcus aureus, Escherichia coli and Enterococcus faecalis in public market wastewater on the removal of nutrients in terms of ammonium (NH4-) and orthophosphate (PO43) using Scenedesmus sp. The removal rates of NH4- and orthophosphate PO43- and batch kinetic coefficient of Scenedesmus sp. were investigated. The phycoremediation process was carried out at ambient temperature for 6 days. The results revealed that the pathogenic bacteria exhibited survival potential in the presence of microalgae but they were reduced by 3-4 log at the end of the treatment process. The specific removal rates of NH4- and PO43- have a strong relationship with initial concentration in the public market wastewater (R2 = 0.86 and 0.80, respectively). The kinetic coefficient of NH4- removal by Scenedesmus sp. was determined as k = 4.28 mg NH4- 1 log10 cell mL-1 d-1 and km = 52.01 mg L-1 (R2 = 0.94) while the coefficient of PO43- removal was noted as k = 1.09 mg NH4- 1 log10 cell mL-1 d-1 and km = 85.56 mg L-1 (R2 = 0.92). It can be concluded that Scenedesmus sp. has high competition from indigenous bacteria in the public market wastewater to remove nutrients, with a higher coefficient of removal of NH4- than PO43.
Production of Scenedesmus sp. biomass in chicken slaughterhouse wastewater (CSWW) is a promising alternative technique for commercial culture medium due to the high nutritional content of the generated biomass to be used as fish feeds. The current work deals with optimising of biomass production in CSWW using response surface methodology (RSM) as a function of two independent variables, namely temperature (10-30 °C) and photoperiod (6-24 h). The potential application of biomass yield as fish feeds was evaluated based on carbohydrate, protein and lipid contents. The results revealed that the best operating parameters for Scenedesmus sp. biomass production with high contents of carbohydrates, proteins and lipids were determined at 30 °C and after 24 h. The actual and predicted values were 2.47 vs. 3.09 g, 1.44 vs. 1.27 μg/mL, 29.9 vs. 31.60% and 25.75 vs. 28.44%, respectively. Moreover, the produced biomass has a high concentration of fatty acid methyl ester (FAME) as follows: 35.91% of C15:1; 17.58% of C24:1 and 14.11% of C18:1N9T. The biomass yields have 7.98% of eicosapentaenoic acid (EPA, C20:5N3) which is more appropriate as fish feeds. The Fourier transform infrared (FTIR) analysis of biomass revealed that the main functional groups included hydroxyl (OH), aldehyde (=C-H), alkanes and acyl chain groups. Scanning electron micrograph (SEM) and energy-dispersive X-ray spectroscopic analysis (EDS) indicated that the surface morphology and element distribution in biomass produced in BBM and CSWW were varied. The findings have indicated that the biomass produced in CSWW has high potential as fish feeds.
The unceasing rise of greenhouse gas emission has led to global warming and climate change. Global concern on this phenomenon has put forward the microalgal-based CO2 sequestration aiming to sequester carbon back to the biosphere, ultimately reducing greenhouse effects. Microalgae have recently gained enormous attention worldwide, to be the valuable feedstock for renewable energy production, due to their high growth rates, high lipid productivities and the ability to sequester carbon. The photosynthetic process of microalgae uses atmospheric CO2 and CO2 from flue gases, to synthesize nutrients for their growth. In this review article, we will primarily discuss the efficiency of CO2 biosequestration by microalgae species, factors influencing microalgal biomass productions, microalgal cultivation systems, the potential and limitations of using flue gas for microalgal cultivation as well as the bio-refinery approach of microalgal biomass.
Microalgae are the major photosynthesizers on earth and produce important pigments that include chlorophyll a, b and c, β-carotene, astaxanthin, xanthophylls, and phycobiliproteins. Presently, synthetic colorants are used in food, cosmetic, nutraceutical, and pharmaceutical industries. However, due to problems associated with the harmful effects of synthetic colorants, exploitation of microalgal pigments as a source of natural colors becomes an attractive option. There are various factors such as nutrient availability, salinity, pH, temperature, light wavelength, and light intensity that affect pigment production in microalgae. This paper reviews the availability and characteristics of microalgal pigments, factors affecting pigment production, and the application of pigments produced from microalgae. The potential of microalgal pigments as a source of natural colors is enormous as an alternative to synthetic coloring agents, which has limited applications due to regulatory practice for health reasons.
Biodiesel with improved yield was produced from microalgae biomass under simultaneous cooling and microwave heating (SCMH). Nannochloropsis sp. and Tetraselmis sp. which were known to contain higher lipid species were used. The yield obtained using this novel technique was compared with the conventional heating (CH) and microwave heating (MWH) as the control method. The results revealed that the yields obtained using the novel SCMH were higher; Nannochloropsis sp. (83.33%) and Tetraselmis sp. (77.14%) than the control methods. Maximum yields were obtained using SCMH when the microwave was set at 50°C, 800W, 16h of reaction with simultaneous cooling at 15°C; and water content and lipid to methanol ratio in reaction mixture was kept to 0 and 1:12 respectively. GC analysis depicted that the biodiesel produced from this technique has lower carbon components (<19 C) and has both reasonable CN and IV reflecting good ignition and lubricating properties.
Effective optimization of microalgae-to-bioethanol process systems hinges on an in-depth characterization of key process parameters relevant to the overall bioprocess engineering. One of the such important variables is the biomass particle size distribution and the effects on saccharification levels and bioethanol titres. This study examined the effects of three different microalgal biomass particle size ranges, 35 μm ≤ x ≤ 90 μm, 125 μm ≤ x ≤ 180 μm, and 295 μm ≤ x ≤ 425 μm, on the degree of enzymatic hydrolysis and bioethanol production. Two scenarios were investigated: single enzyme hydrolysis (cellulase) and double enzyme hydrolysis (cellulase and cellobiase). The glucose yield from biomass in the smallest particle size range (35 μm ≤ x ≤ 90 μm) was the highest, 134.73 mg glucose/g algae, while the yield from biomass in the larger particle size range (295 μm ≤ x ≤ 425 μm) was 75.45 mg glucose/g algae. A similar trend was observed for bioethanol yield, with the highest yield of 0.47 g EtOH/g glucose obtained from biomass in the smallest particle size range. The results have shown that the microalgal biomass particle size has a significant effect on enzymatic hydrolysis and bioethanol yield.
Wavelength of light is a crucial factor which renders microalgae as the potential biodiesel. In this study, Tetraselmis sp. and Nannochloropsis sp. as famous targets were selected. The effect of different light wavelengths on growth rate and lipid production was studied. Microalgae were cultivated for 14 days as under blue, red, red-blue LED and white fluorescent light. The growth rate of microalgae was analyzed by spectrophotometer and cell counting while oil production under improved Nile red method. Optical density result showed the microalgae exhibited better growth curve under blue wavelength. Besides, Tetraselmis sp. and Nannochloropsis sp. under blue wavelength showed the higher growth rate (1.47 and 1.64 day(-1)) and oil production (102.954 and 702.366 a.u.). Gas chromatography analysis also showed that palmitic acid and stearic acid which were compulsory components for biodiesel contribute around 49-51% of total FAME from Nannochloropsis sp. and 81-83% of total FAME from Tetraselmis sp.
Mixotrophic metabolism was evaluated as an option to augment the growth and lipid production of marine microalga Tetraselmis sp. FTC 209. In this study, a five-level three-factor central composite design (CCD) was implemented in order to enrich the W-30 algal growth medium. Response surface methodology (RSM) was employed to model the effect of three medium variables, that is, glucose (organic C source), NaNO3 (primary N source), and yeast extract (supplementary N, amino acids, and vitamins) on biomass concentration, X(max), and lipid yield, P(max)/X(max). RSM capability was also weighed against an artificial neural network (ANN) approach for predicting a composition that would result in maximum lipid productivity, Pr(lipid). A quadratic regression from RSM and a Levenberg-Marquardt trained ANN network composed of 10 hidden neurons eventually produced comparable results, albeit ANN formulation was observed to yield higher values of response outputs. Finalized glucose (24.05 g/L), NaNO3 (4.70 g/L), and yeast extract (0.93 g/L) concentration, affected an increase of X(max) to 12.38 g/L and lipid a accumulation of 195.77 mg/g dcw. This contributed to a lipid productivity of 173.11 mg/L per day in the course of two-week cultivation.
There are numerous commercial applications of microalgae nowadays owing to their vast biotechnological and economical potential. Indisputably, astaxanthin is one of the high value product synthesized by microalgae and is achieving commercial success. Astaxanthin is a keto-carotenoid pigment found in many aquatic animals including microalgae. Astaxanthin cannot be synthesized by animals and provided in the diet is compulsory. In this study, the production of astaxanthin by the freshwater microalgae Chlorella sorokiniana and marine microalgae Tetraselmis sp. were studied. The relationship between growth and astaxanthin production by marine and freshwater microalgae cultivated under various carbon sources and concentrations, environmental conditions and nitrate concentrations was investigated in this study. Inorganic carbon source and low nitrate concentration favored the growth and production of astaxanthin by the marine microalgae Tetraselmis sp. and the freshwater microalgae Chlorella sorokiniana. Outdoor cultivation enhanced the growth of microalgae, while indoor cultivation promoted the formation of astaxanthin. The results indicated that supplementation of light, inorganic carbon and nitrate could be effectively manipulated to enhance the production of astaxanthin by both microalgae studied.
High cell density cultivation of microalgae via heterotrophic growth mechanism could effectively address the issues of low productivity and operational constraints presently affecting the solar driven biodiesel production. This paper reviews the progress made so far in the development of commercial-scale heterotrophic microalgae cultivation processes. The review also discusses on patentable concepts and innovations disclosed in the past four years with regards to new approaches to microalgal cultivation technique, improvisation on the process flow designs to economically produced biodiesel and genetic manipulation to confer desirable traits leading to much valued high lipid-bearing microalgae strains.
Exploitation of renewable sources of energy such as algal biodiesel could turn energy supplies problem around. Studies on a locally isolated strain of Dunaliella sp. showed that the mean lipid content in cultures enriched by 200 mg L(-1) myoinositol was raised by around 33% (1.5 times higher than the control). Similarly, higher lipid productivity values were achieved in cultures treated by 100 and 200 mg L(-1) myoinositol. Fluorometry analyses (microplate fluorescence and flow cytometry) revealed increased oil accumulation in the Nile red-stained algal samples. Moreover, it was predicted that biodiesel produced from myoinositol-treated cells possessed improved oxidative stability, cetane number, and cloud point values. From the genomic point of view, real-time analyses revealed that myoinositol negatively influenced transcript abundance of AccD gene (one of the key genes involved in lipid production pathway) due to feedback inhibition and that its positive effect must have been exerted through other genes. The findings of the current research are not to interprete that myoinositol supplementation could answer all the challenges faced in microalgal biodiesel production but instead to show that "there is a there there" for biochemical modulation strategies, which we achieved, increased algal oil quantity and enhanced resultant biodiesel quality.