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Microalgae from the Selenastraceae as emerging candidates for biodiesel production: a mini review

Yee W

World J Microbiol Biotechnol, 2016 Apr;32(4):64.
PMID: 26931604 DOI: 10.1007/s11274-016-2023-6

Over the years, microalgae have been identified to be a potential source of commercially important products such as pigments, polysaccharides, polyunsaturated fatty acids and in particular, biofuels. Current demands for sustainable fuel sources and bioproducts has led to an extensive search for promising strains of microalgae for large scale cultivation. Prospective strains identified for these purposes were among others, mainly from the genera Hematococcus, Dunaliella, Botryococcus, Chlorella, Scenedesmus and Nannochloropsis. Recently, microalgae from the Selenastraceae emerged as potential candidates for biodiesel production. Strains from the Selenastraceae such as Monoraphidium sp. FXY-10, M. contortum SAG 47.80, Ankistrodesmus sp. SP2-15 and M. minutum were high biomass and lipid producers when cultivated under optimal conditions. A number of Selenastraceae strains were also reported to be suitable for cultivation in wastewater. This review highlights recent reports on potential strains from the Selenastraceae for biodiesel production and contrasts their biomass productivity, lipid productivity as well as fatty acid profile. Cultivation strategies employed to enhance their biomass and lipid productivity as well as to reduce feedstock cost are also discussed in this paper.
Feasibility of various carbon sources and plant materials in enhancing the growth and biomass productivity of the freshwater microalgae Monoraphidium griffithii NS16

Yee W

Bioresour Technol, 2015 Nov;196:1-8.
PMID: 26210717 DOI: 10.1016/j.biortech.2015.07.033

In order to assess the feasibility of various carbon sources and plant materials in increasing the growth rate and biomass productivity of Monoraphidium griffithii, ten carbon sources as well as six plant materials were tested in mixotrophic cultures with or without aeration. It was found that glucose, fructose, maltose, sodium acetate and mannitol were potential carbon sources for growth enhancement of M. griffithii. Supplementation of culture medium with these carbon sources resulted in approximately 1-4-fold increase in cell density compared to control in a small scale culture. In a larger scale mixotrophic culture with aeration, 0.05% mannitol and 0.1% fructose resulted in a decent 1-1.5-fold increase in final cell density, approximately 2-fold increase in growth rate and 0.5-1-fold increase in dry biomass weight. Findings from this study suggests that glucose, fructose, maltose and mannitol were potential organic carbon sources for mixotrophic culture of M. griffithii.
Assessment of factors affecting Agrobacterium-mediated genetic transformation of the unicellular green alga, Chlorella vulgaris

Cha TS, Yee W, Aziz A

World J Microbiol Biotechnol, 2012 Apr;28(4):1771-9.
PMID: 22805959 DOI: 10.1007/s11274-011-0991-0

The successful establishment of an Agrobacterium-mediated transformation method and optimisation of six critical parameters known to influence the efficacy of Agrobacterium T-DNA transfer in the unicellular microalga Chlorella vulgaris (UMT-M1) are reported. Agrobacterium tumefaciens strain LBA4404 harbouring the binary vector pCAMBIA1304 containing the gfp:gusA fusion reporter and a hygromycin phosphotransferase (hpt) selectable marker driven by the CaMV35S promoter were used for transformation. Transformation frequency was assessed by monitoring transient β-glucuronidase (GUS) expression 2 days post-infection. It was found that co-cultivation temperature at 24°C, co-cultivation medium at pH 5.5, 3 days of co-cultivation, 150 μM acetosyringone, Agrobacterium density of 1.0 units (OD(600)) and 2 days of pre-culture were optimum variables which produced the highest number of GUS-positive cells (8.8-20.1%) when each of these parameters was optimised individually. Transformation conducted with the combination of all optimal parameters above produced 25.0% of GUS-positive cells, which was almost a threefold increase from 8.9% obtained from un-optimised parameters. Evidence of transformation was further confirmed in 30% of 30 randomly-selected hygromycin B (20 mg L(-1)) resistant colonies by polymerase chain reaction (PCR) using gfp:gusA and hpt-specific primers. The developed transformation method is expected to facilitate the genetic improvement of this commercially-important microalga.
Fulltext Inclusion of 2-Mercaptoethanol in Lysis Buffer Could Interfere with Isolation of High Molecular Weight DNA from Freshwater Microalgae

Yee W, Kumar JN, Muthusamy PD

Indian J Microbiol, 2018 Mar;58(1):109-113.
PMID: 29434405 DOI: 10.1007/s12088-017-0698-5

2-mercaptoethanol (2-ME), alongside polyvinylpyrrolidone is commonly used in plant DNA extractions to deal with polyphenols, which could interfere with extraction and downstream applications. 2-ME is also commonly used to denature proteins and nucleases, especially RNAses. On the contrary, we found that the presence of 2-ME in lysis buffer interfered with DNA extraction from 12 strains of freshwater microalgae, resulting in DNA with poor integrity. We also found that the TNES-urea buffer, commonly used for preservation and DNA extraction from fish, appears as effective as the SDS and CTAB buffer for some microalgae strains. Results from our study suggests that the inclusion of 2-ME in DNA extraction protocols may be detrimental for isolation of good quality DNA from freshwater microalgae, and therefore recommend eliminating it or testing varying concentrations of 2-ME when developing species-specific extraction protocols for microalgae.
Effects of Excess and Limited Phosphate on Biomass, Lipid and Fatty Acid Contents and the Expression of Four Fatty Acid Desaturase Genes in the Tropical Selenastraceaen Messastrum gracile SE-MC4

Anne-Marie K, Yee W, Loh SH, Aziz A, Cha TS

Appl Biochem Biotechnol, 2020 Apr;190(4):1438-1456.
PMID: 31782088 DOI: 10.1007/s12010-019-03182-z

In this study, the effects of limited and excess phosphate on biomass content, oil content, fatty acid profile and the expression of three fatty acid desaturases in Messastrum gracile SE-MC4 were determined. It was found that total biomass (0.67-0.83 g L-1), oil content (30.99-38.08%) and the duration for cells to reach stationary phase (25-27 days) were not considerably affected by phosphate limitation. However, excess phosphate slightly reduced total biomass and oil content to 0.50 g L-1 and 25.36% respectively. The dominant fatty acids in M. gracile, pamitic acid (C16:0) and oleic acid (C18:1) which constitute more than 81% of the total fatty acids remained relatively high and constant across all phosphate concentrations. Reduction of phosphate concentration to 25% and below significantly increased total MUFA, whereas increasing phosphate concentration to ≥ 50% and ≥ 100% significantly increased total SFA and PUFA content respectively. The expression of omega-3 fatty acid desaturase (ω-3 FADi1, ω-3 FADi2) and omega-6 fatty acid desaturase (ω-6 FAD) was increased under phosphate limitation, especially at ≤ 12.5% phosphate, whereas levels of streoyl-ACP desaturase (SAD) transcripts were relatively unchanged across all phosphate concentrations. The first isoform of ω-3 FAD (ω-3 FADi) displayed a binary upregulation under limited (≤ 12.5%) and excess (200%) phosphate. The expression of ω-6 FAD, ω-3 FAD and SAD were inconsistent with the accumulation of oleic acid (C18:1), linoleic acid (C18:2) and alpha-linolenic acid (C18:3), suggesting that these genes may be regulated indirectly by phosphate availability via post-transcriptional or post-translational mechanisms.
Cinnamic acid, coumarin and vanillin: Alternative phenolic compounds for efficient Agrobacterium-mediated transformation of the unicellular green alga, Nannochloropsis sp

Cha TS, Chen CF, Yee W, Aziz A, Loh SH

J Microbiol Methods, 2011 Mar;84(3):430-4.
PMID: 21256888 DOI: 10.1016/j.mimet.2011.01.005

The use of acetosyringone in Agrobacterium-mediated gene transfer into plant hosts has been favored for the past few decades. The influence of other phenolic compounds and their effectiveness in Agrobacterium-mediated plant transformation systems has been neglected. In this study, the efficacy of four phenolic compounds on Agrobacterium-mediated transformation of the unicellular green alga Nannochloropsis sp. (Strain UMT-M3) was assessed by using β-glucuronidase (GUS) assay. We found that cinnamic acid, vanillin and coumarin produced higher percentages of GUS positive cells as compared to acetosyringone. These results also show that the presence of methoxy group in the phenolic compounds may not be necessary for Agrobacterium vir gene induction and receptor binding as suggested by previous studies. These findings provide possible alternative Agrobacterium vir gene inducers that are more potent as compared to the commonly used acetosyringone in achieving high efficiency of Agrobacterium-mediated transformation in microalgae and possibly for other plants.
Fulltext A simple and inexpensive physical lysis method for DNA and RNA extraction from freshwater microalgae

Yee W, Abdul-Kadir R, Lee LM, Koh B, Lee YS, Chan HY

3 Biotech, 2018 Aug;8(8):354.
PMID: 30105179 DOI: 10.1007/s13205-018-1381-1

In this work, a simple and inexpensive physical lysis method using a cordless drill fitted with a plastic pellet pestle and 150 mg of sterile sea sand was established for the extraction of DNA from six strains of freshwater microalgae. This lysis method was also tested for RNA extraction from two microalgal strains. Lysis duration between 15 and 120 s using the cetyltrimethyl ammonium bromide (CTAB) buffer significantly increased the yield of DNA from four microalgalstrains (Monoraphidium griffithii NS16, Scenedesmus sp. NS6, Scenedesmus sp. DPBC1 and Acutodesmus sp. DPBB10) compared to control. It was also found that grinding was not required to obtain DNA from two strains of microalgae (Choricystis sp. NPA14 and Chlamydomonas sp. BM3). The average DNA yield obtained using this lysis method was between 62.5 and 78.9 ng/mg for M. griffithii NS16, 42.2-247.0 ng/mg for Scenedesmus sp. NS6, 70.2-110.9 ng/mg for Scenedesmus sp. DPBC1 and 142.8-164.8 ng/mg for Acutodesmus sp. DPBB10. DNA obtained using this method was sufficiently pure for PCR amplification. Extraction of total RNA from M. griffithii NS16 and Mychonastes sp. NPD7 using this lysis method yielded high-quality RNA suitable for RT-PCR. This lysis method is simple, cheap and would enable rapid nucleic acid extraction from freshwater microalgae without requiring costly materials and equipment such as liquid nitrogen or beadbeaters, and would facilitate molecular studies on microalgae in general.
Occurrence and potential human health risk of pharmaceutical residues in drinking water from Putrajaya (Malaysia)

Praveena SM, Mohd Rashid MZ, Mohd Nasir FA, Sze Yee W, Aris AZ

Ecotoxicol Environ Saf, 2019 Sep 30;180:549-556.
PMID: 31128553 DOI: 10.1016/j.ecoenv.2019.05.051

Occurrence of pharmaceutical residues in drinking water has been widely reported in countries that have registered steady economic growth. This can exert concerns among the general consumers, prompting them to explore the potential human health risks associated with continuous exposure to pharmaceuticals. However, such an occurrence is rarely reported in developing or under-developed countries. To give more contexts, this study looked at the presence of nine pharmaceutical residues in drinking water (amoxicillin, caffeine, chloramphenicol, ciprofloxacin, dexamethasone, diclofenac, nitrofurazone, sulfamethoxazole, and triclosan) at Putrajaya residential area in Malaysia. Additionally, the potential health risks associated with contaminated drinking water were investigated. This study has found the presence of pharmaceutical residue concentrations up to 0.38 ng/L, with the highest concentration of caffeine (0.38 ng/L) and the lowest concentration of diclofenac (0.14 ng/L). In comparison, all the nine pharmaceutical residues were substantially lower than previously reported studies. In general, Hazard Quotient (HQ) values indicated that low potential health hazards were present for all age groups. Nevertheless, quantitative occurrences of pharmaceutical residues in drinking water will help guide future toxicological studies to examine other chronic effects, while canvassing for proper framework to look into the water risk management and regulation in Malaysia.
Influence of nitrogen availability on biomass, lipid production, fatty acid profile, and the expression of fatty acid desaturase genes in Messastrum gracile SE-MC4

Anne-Marie K, Yee W, Loh SH, Aziz A, Cha TS

World J Microbiol Biotechnol, 2020 Jan 07;36(1):17.
PMID: 31912247 DOI: 10.1007/s11274-019-2790-y

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.
A brief period of darkness induces changes in fatty acid biosynthesis towards accumulation of saturated fatty acids in Chlorella vulgaris UMT-M1 at stationary growth phase

Cha TS, Yee W, Phua PSP, Loh SH, Aziz A

Biotechnol Lett, 2021 Apr;43(4):803-812.
PMID: 33438120 DOI: 10.1007/s10529-021-03077-2

OBJECTIVE: The effects of a brief (3 days) and prolonged (6 days) period of incubation in darkness and light on the biomass content, lipid content and fatty acid profile in Chlorella vulgaris UMT-M1 were determined.
RESULTS: Three days of incubation in darkness increased saturated fatty acid (SFA) content from 34.0 to 41.4% but decreased monounsaturated fatty acid (MUFA) content from 36.7 to 29.8%. Palmitic acid (C16:0) content was increased from 23.2 to 28.9%, whereas oleic acid (C18:1) content was reduced from 35.4 to 28.8%. Total oil content was slightly decreased from 20.4 to 18.7% after 3 days of darkness, without a significant reduction in biomass compared to 3 days of incubation in light. Biomass and oil content was highest in cultures incubated for 6 days in light, however the stimulatory and inhibitory effects of darkness (or light) on SFA and MUFA content was no longer present at 6 days of incubation.
CONCLUSIONS: Findings from this study suggests that fatty acid composition in C. vulgaris could be modulated to favor either C16:0 or C18:1 by a brief period of either darkness or light incubation, prior to harvesting.