Microalgae can produce various natural products such as pigments, enzymes, unique fatty acids and vitamin that benefit humans. The objective of the study is to study the bioaccessibility of carotenoids (β-carotene and lycopene) and vitamin E (α- and β-tocopherol) of Nannochloropsis oculata and Chaetoceros calcitrans. Analyses were carried out for both the powdered forms of N. oculata and C. calcitrans, and the dried extract forms of N. oculata and C. calcitrans. In vitro digestion method together with RP-HPLC was used to determine the bioaccessibility of carotenoids and vitamin E for both forms of microalgae. Powdered form of N. oculata had the highest bioaccessibility of β-carotene (28.0 ± 0.6 g kg-1), followed by dried extract N. oculata (21.5 ± 1.1 g kg-1), dried extract C. calcitrans (16.9 ± 0.1 g kg-1), and powdered C. calcitrans (15.6 ± 0.1 g kg-1). For lycopene, dried extract of N. oculata had the highest bioaccessibility of lycopene (42.6 ± 1.1 g kg-1), followed by dried extract C. calcitrans (41.9 ± 0.6 g kg-1), powdered C. calcitrans (39.7 ± 0.1 g kg-1) and powdered N. oculata (32.6 ± 0.7 g kg-1). Dried extract C. calcitrans had the highest bioaccessibility of α-tocopherol (72.1 ± 1.2 g kg-1). However, β-tocopherol was not detected in both dried extract and powdered form of C. calcitrans. In conclusion, all samples in their dried extract forms were found to have significantly higher bioaccessibilities than their powdered forms. This may be due to the disruption of the food matrix contributing to a higher bioaccessibility of nutrients shown by the dried extract forms.
Chlorella is one of the common microalgae found in a wide range of habitats, including Antarctica. Chlorella UMACC 234 is an interesting isolate in the collection of Antarctic microalgae in the University of Malaya algae culture collection (UMACC) as it grows well at temperatures much higher than the ambience. The alga was isolated from snow samples collected from Casey, Antarctica. This study investigates the influence of nitrogen source on the growth, biochemical composition and fatty acid profile of Chlorella UMACC 234. The cultures were grown in Bold’s Basal Medium with 3.0 mM NaNO3, NH4Cl or urea. The cultures grown on NaNO3 attained the highest specific growth rate (μ = 0.43 day–1) while the specific growth rates of those grown on NH4Cl and urea were not significantly different (p > 0.05). The urea-grown cells produced the highest amounts of lipids (25.7% dry weight) and proteins (52.5% dry weight) compared to those grown on other nitrogen sources. The cell numbers attained by the cultures grown at NaNO3 levels between 0.3 and 3.0 mM were similar but decreased markedly at 9.0 mM NaNO3. The fatty acids of Chlorella UMACC 234 were dominated by saturated fatty acids, especially 16:0 and 18:0. The percentage of polyunsaturated fatty acids was very low, especially in cells grown on urea (0.9% total fatty acids). Characterisation of the growth and biochemical composition of this Antarctic Chlorella is important to our studies on the relationship of Chorella isolates from tropical, temperate and polar regions, especially in terms of phylogeny and stress adaptation.
Chlorella vulgaris, a unicellular microalgae, produces many intracellular phytochemicals namely carotenoids, tocopherols, ubiquinone and protein. Skin ageing which is induced by oxidative stress involves decreased extracellular matrix synthesis and increased expression of enzymes that degrade the collagenous matrix. The objective of this study was to determine the effect of C. vulgaris on the expression of genes encoded for collagen (COL) and matrix metalloproteinases (MMPs) which are involved in skin ageing. Human diploid fibroblasts (HDFs) were obtained from circumcision foreskin of 8-12 year-old boys. HDFs were cultured into 3 groups: untreated control cells, cells with stress-induced premature senescence (SIPS; cells were induced with H2O2 at passage 6 for 2 weeks) and SIPS treated with C. vulgaris (prolonged C. vulgaris treatment started at passage 4 and combined treatment with H2O2 at passage 6 for 2 weeks). Senescence-associated ß-galactosidase (SA ß-gal) was determined using senescent cells histochemical staining kit (Sigma, USA). Expression of COLI, COLIII, COLIV, MMPI, MMPII and MMPIII genes was quantitatively analysed with real-time RT-PCR method (iScript™ One Step real-time PCR with SYBR® Green; Biorad). HDFs treated with H2O2 (SIPS) exhibited senescent morphological features of flattening and enlarged with increased expression of SA ß-gal (p
Elevated temperature affects marine benthic algae by reducing growth and limits the transport of electron or carbon fixation which may reduce the ability of the cell to use light. This resulting excess light energy may cause photoinhibition. In this study, the photosystem II of the benthic microalgal communities from Casey, eastern Antarctic were relatively unaffected by significant changes in temperatures up to 8ºC, along with high PAR level (450 μmol photons m–2 s–1). Similarly, the community was able to photosynthesize as the temperature was reduced to –5ºC. Recovery from saturating and photoinhibiting irradiances was not significantly influenced by temperatures at both –5ºC and 8ºC. These responses were consistent with those recorded by past experiments on Antarctic benthic diatoms and temperate diatoms which showed that climate change did not have a significant impact on the ability of benthic microalgae to recover from photoinhibitory temperature stress.
Green microalga Ankistrodesmus convolutus Corda is a fast growing alga which produces appreciable amount of carotenoids and polyunsaturated fatty acids. To our knowledge, this is the first report on the construction of cDNA library and preliminary analysis of ESTs for this species. The titers of the primary and amplified cDNA libraries were 1.1×10(6) and 6.0×10(9) pfu/ml respectively. The percentage of recombinants was 97% in the primary library and a total of 337 out of 415 original cDNA clones selected randomly contained inserts ranging from 600 to 1,500 bps. A total of 201 individual ESTs with sizes ranging from 390 to 1,038 bps were then analyzed and the BLASTX score revealed that 35.8% of the sequences were classified as strong match, 38.3% as nominal and 25.9% as weak match. Among the ESTs with known putative function, 21.4% of them were found to be related to gene expression, 14.4% ESTs to photosynthesis, 10.9% ESTs to metabolism, 5.5% ESTs to miscellaneous, 2.0% to stress response, and the remaining 45.8% were classified as novel genes. Analysis of ESTs described in this paper can be an effective approach to isolate and characterize new genes from A. convolutus and thus the sequences obtained represented a significant contribution to the extensive database of sequences from green microalgae.
This study aimed to assess the inhibitory activities of methanol extracts from the microalgae Ankistrodesmus convolutus, Synechococcus elongatus, and Spirulina platensis against Epstein-Barr virus (EBV) in three Burkitt's lymphoma (BL) cell lines, namely Akata, B95-8, and P3HR-1. The antiviral activity was assessed by quantifying the cell-free EBV DNA using real-time polymerase chain reaction (PCR) technique. The methanol extracts from Ankistrodesmus convolutus and Synechococcus elongatus displayed low cytotoxicity and potent effect in reducing cell-free EBV DNA (EC(50)<0.01 µg/ml) with a high therapeutic index (>28000). After fractionation by column chromatography, the fraction from Synechococcus elongatus (SEF1) reduced the cell-free EBV DNA most effectively (EC(50)=2.9 µg/ml, therapeutic index>69). Upon further fractionation by high performance liquid chromatography (HPLC), the sub-fraction SEF1'a was most active in reducing the cell-free EBV DNA (EC(50)=1.38 µg/ml, therapeutic index>14.5). This study suggests that microalgae could be a potential source of antiviral compounds that can be used against EBV.
Many reports have revealed that the abundance of microalgae in shrimp ponds vary with changes in environmental factors such as light, temperature, pH, salinity and nutrient level throughout a shrimp culture period. In this study, shrimp cultivation period was divided into three stages (initial = week 0-5, mid = week 6-10 and final = week 11-15). Physical and chemical parameters throughout the cultivation period were studied and species composition of microalgae was monitored. Physical parameters were found to fluctuate widely with light intensity ranging between 182.23-1278 μmol photon m(-2)s(-1), temperature between 29.56°C -31.59°C, dissolved oxygen (DO) between 4.56-8.21 mg/l, pH between 7.65-8.49 and salinity between 20‰-30‰. Ammonium (NH4 (+)-N), nitrite (NO2 (-)-N), nitrate (NO3 (-)-N), and orthophosphate (PO4 (3-)-P) concentrations in the pond at all cultivation stages ranged from 0.017 to 0.38 mg/l, 0.24 to 2.12 mg/l, 0.06 to 0.98 mg/l and 0.16 to 1.93 mg/l respectively. Statistical test (ANOVA) showed that there were no significant difference (p<0.05) in nutrients concentrations among the cultivation stages. All nutrients concentrations however were still in the tolerable level and safe for shrimp culture. The chlorophyll a contents were found to range from 5.03±2.17 to 32.61±0.35 μg/l throughout the cultivation period. A total of 19 microalgae species were found in the shrimp pond, with diatoms contributing up to 72% of the species followed by Chlorophyta (11%) and Cyanophyta (11%). However, weekly species abundance varied through the study period. At the initial stage, when there were no shrimps in the pond, Anabaena spp. and Oscillatoria spp. (Cyanophyta) were the dominant species, followed by Chlorella sp. and Dunaliella sp. (Chlorophyta). When shrimps were introduced into the pond, Amphora sp., Navicula sp. Gyrosigma sp. and Nitzschia sp. (diatoms) started to exist. At the middle and towards the final stage of the shrimp culture period diatoms were the dominant species. The Chlorophyta (Chlorella sp.) domination took place only twice, which was at week 2 and 13. The absence of some of the coastal water microalgae species in the shrimp pond was most likely due to the fact that they could not tolerate the physicochemical factors of harsh environment. In this study, Cylindrotheca closterium was regarded as the most tolerant species among the microalgae due to its ability to exist for 6 weeks out of the 15 weeks of cultivation.
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.
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.
An initial study on gene cloning and characterization of unicellular green microalga Ankistrodesmus convolutus was carried out to isolate and characterize the full-length cDNA of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) as a first step towards elucidating the structure of A. convolutus RbcS gene. The full-length of A. convolutus RbcS cDNA (AcRbcS) contained 28 bp of 5' untranslated region (UTR), 225 bp of 3' non-coding region, and an open reading frame of 165 amino acids consisting of a chloroplast transit peptide with 24 amino acids and a mature protein of 141 amino acids. The amino acid sequence has high identity to those of other green algae RbcS genes. The AcRbcS contained a few conserved domains including protein kinase C phosphorylation site, tyrosine kinase phosphorylation site and N-myristoylation sites. The AcRbcS was successfully expressed in Escherichia coli and a ~21 kDa of anticipated protein band was observed on SDS-PAGE. From the phylogenetic analysis of RbcS protein sequences, it was found that the RbcS of A. convolutus has closer genetic relationship with green microalgae species compared to those of green seaweed and green macroalgae species. Southern hybridization analysis revealed that the AcRbcS is a member of a small multigene family comprising of two to six members in A. convolutus genome. Under different illumination conditions, RT-PCR analysis showed that AcRbcS transcription was reduced in the dark, and drastically recovered in the light condition. Results presented in this paper established a good foundation for further study on the photosynthetic process of A. convolutus and other green algae species where little information is known on Rubisco small subunit.
Many reports have revealed that the abundance of microalgae in shrimp ponds vary with changes in environmental factors such as light, temperature, pH, salinity and nutrient level throughout a shrimp culture period. In this study, shrimp cultivation period was divided into three stages (initial = week 0–5, mid = week 6–10 and final = week 11–15). Physical and chemical parameters throughout the cultivation period were studied and species composition of microalgae was monitored. Physical parameters were found to
fluctuate widely with light intensity ranging between 182.23–1278 µmol photon m–2s–1, temperature between 29.56ºC –31.59ºC, dissolved oxygen (DO) between 4.56–8.21 mg/l, pH between 7.65–8.49 and salinity between 20‰–30‰. Ammonium (NH4+-N), nitrite (NO2– -N), nitrate (NO3– -N), and orthophosphate (PO43– -P) concentrations in the pond at all cultivation stages ranged from 0.017 to 0.38 mg/l, 0.24 to 2.12 mg/l, 0.06 to 0.98 mg/l and 0.16 to 1.93 mg/l respectively. Statistical test (ANOVA) showed that there were no significant difference (p
Magnetic collection of the microalgae Chlorella sp. from culture media facilitated by low-gradient magnetophoretic separation is achieved in real time. A removal efficiency as high as 99% is accomplished by binding of iron oxide nanoparticles (NPs) to microalgal cells in the presence of the cationic polyelectrolyte poly(diallyldimethylammonium chloride) (PDDA) as a binder and subsequently subjecting the mixture to a NdFeB permanent magnet with surface magnetic field ≈6000 G and magnetic field gradient <80 T m(-1) . Surface functionalization of magnetic NPs with PDDA before exposure to Chlorella sp. is proven to be more effective in promoting higher magnetophoretic removal efficiency than the conventional procedure, in which premixing of microalgal cells with binder is carried out before the addition of NPs. Rodlike NPs are a superior candidate for enhancing the magnetophoretic separation compared to spherical NPs due to their stable magnetic moment that originates from shape anisotropy and the tendency to form large NP aggregates. Cell chaining is observed for nanorod-tagged Chlorella sp. which eventually fosters the formation of elongated cell clusters.
Outdoor mass culture of microalgae in the tropical area is important to minimize its production cost. This study evaluates the growth of Chaetoceros calcitrans in 120 L annular photobioreactors at indoor temperature (Treatment I, 25 +/- 2 degrees C) and outdoor tropical ambient temperature, (Treatment II, 30 +/- 6 degrees C). Each treatment was done in duplicates. For both treatments, C. calcitrans was first grown in starter columns of 10 L capacity for a period of 7 days at 25 +/- 2 degrees C. After 7 days, the 9 L culture was transferred to the annular photobioreactors and subsequently brought to a final volume of 100 L by adding 20 L fresh medium every 5 days. There was no significant difference (p > 0.05) in the dry weight of microalgae grown in natural light and those grown indoor. The results suggest that C. calcitrans can be grown in outdoor conditions, hence, saving time and microalgae production cost for the larviculture industry.
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.
Culturing of microalgae as an alternative feedstock for biofuel production has received a lot of attention in recent years due to their fast growth rate and ability to accumulate high quantity of lipid and carbohydrate inside their cells for biodiesel and bioethanol production, respectively. In addition, this superior feedstock offers several environmental benefits, such as effective land utilization, CO(2) sequestration, self-purification if coupled with wastewater treatment and does not trigger food versus fuel feud. Despite having all these 'theoretical' advantages, review on problems and issues related to energy balance in microalgae biofuel are not clearly addressed until now. Base on the maturity of current technology, the true potential of microalgae biofuel towards energy security and its feasibility for commercialization are still questionable. Thus, this review is aimed to depict the practical problems that are facing the microalgae biofuel industry, covering upstream to downstream activities by accessing the latest research reports and critical data analysis. Apart from that, several interlink solutions to the problems will be suggested with the purpose to bring current microalgae biofuel research into a new dimension and consequently, to revolutionize the entire microalgae biofuel industry towards long-term sustainability.
Microalgae are important biological resources that have a wide range of biotechnological
applications. Due to their high nutritional value, microalgae such as Spirulina and Chlorella are being mass cultured for health food. A variety of high-value products including polyunsaturated fatty acids (PUFA), pigments such as carotenoids and phycobiliproteins, and bioactive compounds are useful as nutraceuticals and pharmaceuticals, as well as for industrial applications. In terms of environmental biotechnology, microalgae are useful for bioremediation of agro-industrial wastewater, and as a biological tool for assessment and monitoring of environmental toxicants such as heavy metals, pesticides and pharmaceuticals. In recent years, microalgae have attracted much interest due to their potential use as feedstock for biodiesel production. In Malaysia, there has been active research on microalgal biotechnology for the past 30 years, tapping into the potential of our
rich microalgal resources for high-value products and applications in wastewater treatment and assessment of environmental toxicants. A culture collection of microalgae has been established, and this serves as an important resource for microalgal biotechnology
research. Microalgal biotechnology should continue to be regarded as a priority area of research in this country.
Isolation of promoter sequences from known gene sequences is a tedious task in genome-related research. An efficient method of obtaining the promoter sequences is necessary in order to successfully use targeted promoters for genetic manipulations. Here, efficiency and usefulness of two PCR-based methods, namely: ligation-mediated PCR and thermal asymmetric interlaced (TAIL) PCR, for isolation of promoter sequences of the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) gene from green microalgae Ankistrodesmus convolutus (A. convolutus) were evaluated. The results showed that the amplification efficiency of TAIL-PCR was higher than that of the ligation-mediated PCR method, i.e. the amplified promoter fragments of 1.2 and 0.8 kb in length or promoter sequences of 813 and 606 bp (after eliminating the unreadable sequences). The use of TAIL-PCR described here presents a low cost and efficient strategy for the isolation of promoter sequences of known genes, especially in GC-rich regions, and species with little or no available genome information such as A. convolutus.
The availability of highly active homologous promoters is critical in the development of a transformation system and improvement of the transformation efficiency. To facilitate transformation of green microalga Ankistrodesmus convolutus which is considered as a potential candidate for many biotechnological applications, a highly-expressed native promoter sequence of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (AcRbcS) has been used to drive the expression of β-glucuronidase (gusA) gene in this microalga. Besides the determination of the transcription start site by 5'-RACE, sequence analysis revealed that AcRbcS promoter contained consensus TATA-box and several putative cis-acting elements, including some representative light-regulatory elements (e.g., G-box, Sp1 motif and SORLIP2), which confer light responsiveness in plants, and several potential conserved motifs (e.g., CAGAC-motif, YCCYTGG-motifs and CACCACA-motif), which may be involved in light responsiveness of RbcS gene in green microalgae. Using AcRbcS promoter::gusA translational fusion, it was demonstrated that this promoter could function as a light-regulated promoter in transgenic A. convolutus, which suggested that the isolated AcRbcS promoter was a full and active promoter sequence that contained all cis-elements required for developmental and light-mediated control of gene expression, and this promoter can be used to drive the expression of heterologous genes in A. convolutus. This achievement therefore advances the development of A. convolutus as an alternative expression system for the production of recombinant proteins. This is the first report on development of gene manipulation system for unicellular green alga A. convolutus.
Illumination factors such as length of photoperiod and intensity can affect growth of microalgae and lipid content. In order to optimize microalgal growth in mass culture system and lipid content, the effects of light intensity and photoperiod cycle on the growth of the marine microalgae, Nannochloropsis sp. were studied in batch culture. Nannochloropsis sp. was grown aseptically for 9 days at three different light intensities (50, 100 and 200 μmol m(-2) s(-1)) and three different photoperiod cycles (24:0, 18:06 and 12:12 h light:dark) at 23 °C cultivation temperature. Under the light intensity of 100 μmol m(-2) s(-1) and photoperiod of 18 h light: 6 h dark cycle, Nannochloropsis sp. was found to grow favorably with a maximum cell concentration of 6.5×10(7) cells mL(-1), which corresponds to the growth rate of 0.339 d(-1) after 8 day cultivation and the lipid content was found to be 31.3%.