The depletion of fossil fuels has attracted global attention to alternative and renewable sources (Kang et al. 2017). Studies on renewable energy from biological sources have focused on several topics ranging from carbon-neutral material development to fuel transportation. Biodiesel generated from crops is potentially renewable and carbon neutral but cannot realistically satisfy even a small fraction of the existing demand for transport fuels owing to food shortages and the need for vast cultivation areas (Chisti 2007; Mathur et al. 2022). Various issues limit the production of bioenergy from wood during preconditioning. In contrast, microalgae are an attractive energy source because they do not compete with food crops and have higher energy yields per area than terrestrial crops (Clarens et al. 2010; Neupane 2023).
Microalgae are photosynthetic microorganisms that convert carbon dioxide into potential biodiesels. They play important roles in carbon and nitrogen cycles in several terrestrial environments. Microalgae can be used as a biodiesel source via the transesterification of algae-derived triglycerides with an alcohol, such as methanol, by a catalyst. However, this process is typically expensive (Chisti 2007; Mathur et al. 2022). Fatty acids are common components of complex lipids and differ according to chain length and the presence, number, and position of double bonds in the hydrocarbon chain (Burdge and Calder 2015). Fatty acid methyl esters obtained via transesterification are used as biodiesels. However, microalgae also produce alkanes, which can be used as biodiesels without transesterification (Zhang et al. 2013; Hayashi et al. 2015). The biological function hypothesized of the hydrocarbons blended into the lipid bilayers may enhance permeability, flexibility, and fluidity against curvature and oxidative stresses but it is still unclear of alkane in cyanobacteria (Hong et al. 2016). We demonstrated that KNUA012 belonged to
According to previous studies, the
Sequences of the 16S ribosomal RNA gene were compared with those of
Phylogenetic relationships of
The tree is constructed to scale with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the maximum composite likelihood method (Tamura et al. 2004), and evolutionary analyses were conducted using MEGA5 (Tamura et al. 2011).
AD (Gen-Bank accession no. KU341741) was amplified from the genomic DNA of Korean domestic
To determine the fatty acid composition, lipids were extracted and analyzed using gas chromatography/mass spectrophotometry (Bligh and Dyer 1959).
We compared individual data points using Student's
Global warming and petroleum depletion have prompted researchers to search for renewable energy sources. Microalgae autotrophically produce alkanes (CnHn) via an AD that can be used directly as a biodiesel (Regalbuto 2009). Commercial alkanes such as pentadecane (C15H32), 8-heptadecane (C17H34), and heptadecane (C17H36) could be directly used as gasoline, diesel and jet fuel (Lin and Lin 2011; Zhang et al. 2013; Hayashi et al. 2015; Kang et al. 2017; Mathur et al. 2022). To produce alkanes in microalgae, two alkane biosynthesis genes are needed to produce AAR and AD (Schirmer et al. 2010). AD catalyzes the decarbonylation of fatty aldehydes, and fatty acid intermediates are produced via the photosynthetic energy of the microalgae (Marsh and Waugh 2013; Vélez et al. 2015; Gao et al. 2020). Alkane-producing genes have not previously been reported in microalgae isolated in Korea but have been observed in microalgae from other countries. We sought to identify alkane-producing genes in Korean domestic
Several microalgae produce polysaccharides and small quantities of proteins, lipids, and glycoproteins (Wingender et al. 1999; Khattar et al. 2010; Spain and Funk 2022). Depending on the location, biofuel constituents, and gas vacuoles occur as capsular polysaccharides with the polymer loosely attached to the cell surface (Trabelsi et al. 2009; Khattar et al. 2010). Microbial biofuel constituents and gas vacuoles are produced by prokaryotes (Parikh and Madamwar 2006; Zou et al. 2006; Chi et al. 2007; Duan et al. 2008; Khattar et al. 2010; Cheah and Chan 2022). Microbial polysaccharides play a crucial role in several microbial traits, including pathogenesis, symbiotic ability, biofuel production, and stress resistance (Parikh and Madamwar 2006; Khattar et al. 2010; Spain and Funk 2022). The Korean domestic
a) Light microscopy images of
b) Growth curves of
Microalgae can produce straight-chain alkanes/alkenes from fatty acyl-ACP via the alkane biosynthesis pathway using the enzymes AAR and AD (Tillman et al. 1999; Hayashi et al. 2015; Kudo et al. 2019; Kittel et al. 2023). AAR reduces fatty acyl-ACP to fatty aldehyde, which is reduced to alkanes/alkenes by AD. In previous studies, the presented alkane operons in
SDS polyacrylamide gel images of the aldehyde decarbonylase (AD) protein purified from
a) M – marker, lane 1 – protein purified from
GC/MS results showing the alkanes and major fatty acids present in
Peak no. | Component name | pET28 empty vector (% w/w) | KNUA012 (% w/w) | pET28-AD (% w/w) |
---|---|---|---|---|
1 | Pentadecane | 0.87 ± 0.52 | 2.40 ± 0.85 | 1.78 ± 0.38 |
2 | Dodecanoic acid methyl ester | 0.18 ± 0.05 | 0.54 ± 0.21 | 0.25 ± 0.08 |
3 | 8-Heptadecene | 0.54 ± 0.07 | 0.62 ± 0.23 | 1.09 ± 0.31 |
4 | Heptadecane | 1.07 ± 0.17 | 1.15 ± 0.42 | 1.86 ± 0.52 |
5 | Methyl Z-11-tetradecenoate | 21.7 ± 1.52 | 22.1 ± 1.03 | 23.6 ± 1.59 |
6 | Tetradecanoic acid methyl ester | 11.2 ± 1.31 | 11.2 ± 1.07 | 15.7 ± 1.41 |
7 | 9-Hexadecenoic acid methyl ester | 2.62 ± 0.84 | 21.3 ± 1.51 | 8.63 ± 1.52 |
8 | Palmitoleic acid methyl ester | 2.35 ± 0.53 | 5.37 ± 1.03 | 3.82 ± 0.82 |
9 | Hexadecanoic acid methyl ester | 2.06 ± 0.51 | 21.7 ± 1.85 | 7.82 ± 1.31 |
10 | 9-Octadecenoic acid methyl ester | 3.64 ± 0.85 | 9.31 ± 1.37 | 6.83 ± 1.46 |
11 | Octadecanoic acid methyl ester | 0.84 ± 0.06 | 1.04 ± 0.52 | 1.83 ± 0.82 |
Error bars indicate ± SD values of three independent experiments.
w/w – dry cell weight/lipid yield
The alkanes and major fatty acid components of
Analysis of the GC peaks.
a) GC/MS total ion chromatogram of standard fatty acid methyl esters. 1 – Tridecanoic acid methyl ester, 2 – pentadecanoic acid methyl ester, 3 – heptadecanoic acid methyl ester, 4 – nonadecanoic acid methyl ester, and 5 – henelcosanoic acid methyl ester.
b) GC peak results for the fatty acids extracted from
Herein, we demonstrated the potential of Korean domestic