Journal of Microbiology

Review

Progress and challenges in CRISPR/Cas applications in microalgae: Quynh-Giao Tran, Trang Thi Le, Dong-Yun Choi, Dae-Hyun Cho, Jin-Ho Yun, Hong Il Choi, Hee-Sik Kim, Yong Jae Lee; J. Microbiol. 2025;63(3):e2501028. Published online March 28, 2025; DOI: https://doi.org/10.71150/jm.2501028

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Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technologies have emerged as powerful tools for precise genome editing, leading to a revolution in genetic research and biotechnology across diverse organisms including microalgae. Since the 1950s, microalgal production has evolved from initial cultivation under controlled conditions to advanced metabolic engineering to meet industrial demands. However, effective genetic modification in microalgae has faced significant challenges, including issues with transformation efficiency, limited target selection, and genetic differences between species, as interspecies genetic variation limits the use of genetic tools from one species to another. This review summarized recent advancements in CRISPR systems applied to microalgae, with a focus on improving gene editing precision and efficiency, while addressing organism-specific challenges. We also discuss notable successes in utilizing the class 2 CRISPR-associated (Cas) proteins, including Cas9 and Cas12a, as well as emerging CRISPR-based approaches tailored to overcome microalgal cellular barriers. Additionally, we propose future perspectives for utilizing CRISPR/Cas strategies in microalgal biotechnology.

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Advancing microbial engineering through synthetic biology
Ki Jun Jeong
Journal of Microbiology.2025; 63(3): e2503100. CrossRef

Journal Articles

Isolation and genome analysis of Winogradskyella algicola sp. nov., the dominant bacterial species associated with the green alga Dunaliella tertiolecta: Jaeho Song , Yeonjung Lim , Hye-Jin Jang , Yochan Joung , Ilnam Kang , Seong-Joo Hong , Choul-Gyun Lee , Jang-Cheon Cho; J. Microbiol. 2019;57(11):982-990. Published online October 28, 2019; DOI: https://doi.org/10.1007/s12275-019-9378-y

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Abstract

Microalgae and bacteria are known to be closely associated in diverse environments. To isolate dominant bacterial species associated with a green alga, Dunaliella tertiolecta, a photoreactor culture of the microalga was investigated using culture- based and culture-independent approaches. The bacterial community structure of the algal culture showed that the most abundant bacterial species under the culture conditions was related to the genus Winogradskyella. The closely related amplicon sequences, showing ≥ 99.5% 16S rRNA gene sequence similarity to one of the isolates, designated IMCC- 33238T, constituted > 49% of the bacterial community and was therefore regarded as the most dominant species in the algal culture. Strain IMCC33238T was characterized by Gramstaining- negative and orange-colored rods. Phylogenetic analyses of the 16S rRNA genes as well as whole genome sequences revealed that strain IMCC33238T belonged to Winogradskyella and shared more than 97.2% 16S rRNA gene sequence similarity with Winogradskyella species. The strain contained iso-C15:1 G, iso-C15:0, iso-C15:0 3-OH, and summed feature 3 (C16:1 ω6c and/or C16:1 ω7c) as major fatty acids and MK-6 as the predominant quinone. The polar lipids found in strain IMCC33238T were phosphatidylethanolamine, two unidentified aminolipids, and three unidentified lipids. The genome of strain IMCC33238T was 3.37 Mbp in size with 33.9 mol% G + C content and proteorhodopsin. Many genes encoding folate and vitamin production are considered to play an important role in the bacteria-algae interaction. On the basis of phylogenetic and phenotypic characteristics, strain IMCC33238T represents a novel species in the genus Winogradskyella, for which the name Winogradskyella algicola sp. nov. is proposed. The type strain is IMCC33238T (= KACC 21192T = NBRC 113704T).

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International Journal of Systematic and Evolutionary Microbiology .2023;[Epub] CrossRef
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Application of high-salinity stress for enhancing the lipid productivity of Chlorella sorokiniana HS1 in a two-phase process: Ramesh Kakarla , Jung-Woon Choi , Jin-Ho Yun , Byung-Hyuk Kim , Jina Heo , Sujin Lee , Dae-Hyun Cho , Rishiram Ramanan , Hee-Sik Kim; J. Microbiol. 2018;56(1):56-64. Published online January 4, 2018; DOI: https://doi.org/10.1007/s12275-018-7488-6

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Abstract

Increased lipid accumulation of algal cells as a response to environmental stress factors attracted much attention of researchers to incorporate this stress response into industrial algal cultivation process with the aim of enhancing algal lipid productivity. This study applies high-salinity stress condition to a two-phase process in which microalgal cells are initially grown in freshwater medium until late exponential phase and subsequently subjected to high-salinity condition that induces excessive lipid accumulation. Our initial experiment revealed that the concentrated culture of Chlorella sorokiniana HS1 exhibited the intense fluorescence of Nile red at the NaCl concentration of 60 g/L along with 1 g/L of supplemental bicarbonate after 48 h of induction period without significantly compromising cultural integrity. These conditions were further verified with the algal culture grown for 7 days in a 1 L bottle reactor that reached late exponential phase; a 12% increment in the lipid content of harvested biomass was observed upon inducing high lipid accumulation in the concentrated algal culture at the density of 5.0 g DW/L. Although an increase in the sum of carbohydrate and lipid contents of harvested biomass indicated that the external carbon source supplemented during the induction period increased overall carbon assimilation, a decrease in carbohydrate content suggested the potential reallocation of cellular carbon that promoted lipid droplet formation under high-salinity stress. These
results
thus emphasize that the two-phase process can be successfully implemented to enhance algal lipid productivity by incorporating high-salinity stress conditions into the pre-concentrated sedimentation ponds of industrial algal production system.

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Research Support, Non-U.S. Gov'ts

NOTE] Quantification of Toxic Effects of the Herbicide Metolachlor on Marine Microalgae Ditylum brightwellii (Bacillariophyceae), Prorocentrum minimum (Dinophyceae), and Tetraselmis suecica (Chlorophyceae): Vinitha Ebenezer , Jang-Seu Ki; J. Microbiol. 2013;51(1):136-139. Published online March 2, 2013; DOI: https://doi.org/10.1007/s12275-013-2114-0

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Abstract: Toxic effects of the herbicide metolachlor (MC) were evaluated for three marine microalgae, Tetraselmis suecica (chlorophyte), Ditylum brightwellii (diatom), and Prorocentrum minimum (dinoflagellate). MC showed a significant reduction in cell counts and chlorophyll a levels. Median effective concentration (EC50) was calculated based on chlorophyll a levels after a 72-h MC exposure. EC50 values for T. suecica, D. brightwellii, and P. minimum were 21.3, 0.423, and 0.07 mg/L, respectively. These values showed that the dinoflagellate was most sensitive when exposed to the herbicide, at a concentration comparable to freshwater algae, suggesting its potential as an appropriate model organism for ecotoxicity assessments in marine environments.

Accumulation of Lipid Production in Chlorella minutissima by Triacylglycerol Biosynthesis-Related Genes Cloned from Saccharomyces cerevisiae and Yarrowia lipolytica: Hsin-Ju Hsieh , Chia-Hung Su , Liang-Jung Chien; J. Microbiol. 2012;50(3):526-534. Published online June 30, 2012; DOI: https://doi.org/10.1007/s12275-012-2041-5

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Abstract

Discovery of an alternative fuel is now an urgent matter because of the impending issue of oil depletion. Lipids synthesized in algal cells called triacylglycerols (TAGs) are thought to be of the most value as a potential biofuel source because they can use transesterification to manufacture biodiesel. Biodiesel is deemed as a good solution to overcoming the problem of oil depletion since it is capable of providing good performance similar to that of petroleum. Expression of several genomic sequences, including glycerol-3-phosphate dehydrogenase, glycerol-3-phosphate acyltransferase, lysophosphatidic acid acyltransferase, phosphatidic acid phosphatase, diacylglycerol acyltransferase, and phospholipid:diacylglycerol acyltransferase, can be useful for manipulating metabolic pathways for biofuel production. In this study, we found this approach indeed increased the storage lipid content of C. minutissima UTEX 2219 up to 2-fold over that of wild type. Thus, we conclude this approach can be used with the biodiesel production platform of C. minutissima UTEX 2219 for high lipid production that will, in turn, enhance productivity.

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Isolation and Evaluation of Terrestrial Fungi with Algicidal Ability from Zijin Mountain, Nanjing, China: Guomin Han , Xiaoguang Feng , Yong Jia , Congyan Wang , Xingbing He , Qiyou Zhou , Xingjun Tian; J. Microbiol. 2011;49(4):562-567. Published online September 2, 2011; DOI: https://doi.org/10.1007/s12275-011-0496-4

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Abstract: Approximately 60 fungal isolates from Zijin Mountain (Nanjing, China) were screened to determine their algicidal ability. The results show that 8 fungi belonging to Ascomycota and 5 belonging to Basidiomycota have algicidal ability. Of these fungi, Irpex lacteus T2b, Trametes hirsuta T24, Trametes versicolor F21a, and Bjerkandera adusta T1 showed strong algicidal ability. The order of fungal chlorophyll-a removal efficiency was as follows: T. versicolor F21a > I. lacteus T2b > B. adusta T1 > T. hirsuta T24. In particular, T. versicolor F21a completely removed algal cells within 30 h, showing the strongest algicidal ability. The results also show that all 4 fungal species degraded algal cells through direct attack. In addition, most of the tested fungi from the order Polyporales of Basidiomycota exhibited strong algicidal activity, suggesting that most fungi that belong to this order have algicidal ability. The findings of this work could direct the search for terrestrial fungi for bloom control.

Isolation and Characterization of a Marine Algicidal Bacterium against the Harmful Raphidophyceae Chattonella marina: Yun Sook Kim , Dae-Sung Lee , Seong-Yun Jeong , Won Jae Lee , Myung-Suk Lee; J. Microbiol. 2009;47(1):9-18. Published online February 20, 2009; DOI: https://doi.org/10.1007/s12275-008-0141-z

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Abstract

A bacterial strain named AB-4 showing algicidal activity against Chattonella marina was isolated from coastal water of Uljin, Republic of Korea. The isolated strain was identified as Bacillus sp. by culture morphology, biochemical reactions, and homology research based on 16S rDNA. The bacterial culture led to the lysis of algal cells, suggesting that the isolated strain produced a latent algal-lytic compound. Amongst changes in algicidal activity by different culture filtrate volumes, the 10% (100 ?/ml) concentration showed the biggest change in algicidal activity; there, estimated algicidal activity was 95%. The swimming movements of Chattonella marina cells were inhibited because of treatment of the bacterial culture; subsequently, Chattonella marina cells became swollen and rounded. With longer exposure time, algal cells were disrupted and cellular components lost their integrity and decomposed. The released algicide(s) were heat-tolerant and stable in pH variations, except pH 3, 4, and 5. Culture filtrate of Bacillus sp. AB-4 was toxic against harmful algae bloom (HAB) species and nontoxic against livefood organisms. Bacillus sp. AB-4 showed comparatively strong activity against Akashiwo sanguinea, Fibriocapsa japonica, Heterosigma akashiwo, and Scrippsiella trochoidea. These results suggest that the algicidal activity of Bacillus sp. AB-4 is potentially useful for controlling outbreaks of Chattonella marina.

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Fatty Acid Biosynthesis in Eukaryotic Photosynthetic Microalgae: Identification of a Microsomal Delta 12 Desaturase in Chlamydomonas reinhardtii: Xiaoyuan Chi , Xiaowen Zhang , Xiangyu Guan , Ling Ding , Youxun Li , Mingqing Wang , Hanzhi Lin , Song Qin; J. Microbiol. 2008;46(2):189-201. Published online June 11, 2008; DOI: https://doi.org/10.1007/s12275-007-0223-3

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Abstract

Polyunsaturated fatty acids (PUFAs) are important components of infant and adult nutrition because they serve as structural elements of cell membranes. Fatty acid desaturases are responsible for the insertion of double bonds into pre-formed fatty acid chains in reactions that require oxygen and reducing equivalents. In this study, the genome-wide characterization of the fatty acid desaturases from seven eukaryotic photosynthetic microalgae was undertaken according to the conserved histidine-rich motifs and phylogenetic profiles. Analysis of these genomes provided insight into the origin and evolution of the pathway of fatty acid biosynthesis in eukaryotic plants. In addition, the candidate enzyme from Chlamydomonas reinhardtii with the highest similarity to the microsomal Δ12 desaturase of Chlorella vulgaris was isolated, and its function was verified by heterologous expression in yeast (Saccharomyces cerevisiae).

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Effect of β-Glucosidase on the Heteroprophic Bacteria in Cheonho Reservoir: Kwag, No Tae , Han, Suk Kyun , Go, You Seok , Ahn, Tae Young; J. Microbiol. 1998;36(2):145-150.

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Abstract: Cheonho reservoir is a small and eutrophicated lake. The variation of BOD in the reservoir was correlated to the variation of chlorophyll-a. BOD was maximum (39.1㎍/L) at site 1 in July of 1995 when chlorophyll-a was the highest. β-Glucosidase activities ranged from 9 to 241 nM/h. β-Glucosidase activity varied in the following order:site 3>site 2>site 1>site 4. The β-Glucosidase activity of the site 4 was less than 50% of the other three sites. Organic matter was produced by algae at all sites but was not directly related with the increase of heterotrophic bacteria. β-Glucosidase activity showed a close correlation with the number of heterotrophic bacteria, indicating a tight coupling between algae and heterotrophic bacteria through agal organic production.

Inferring the Molecular Phylogeny of Chroococcalian Strains (Blue-green algae/Cyanophyta) from the Geumgang River, Based on Partial Sequences of 16S rRNA Gene: Wook Jae Lee , Kyung Sook Bae; J. Microbiol. 2002;40(4):335-339.

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Abstract: Partial sequences of 16S rRNA gene of five chroococcalian blue-green algal strains, Aphanothece nidulans KCTC AG10041, Aphanothece naegelii KCTC AG10042, Microcystis aeruginosa KCTC AG10159, Microcystis ichthyoblabe KCTC AG10160, and Microcystis viridis KCTC AG10198, which were isolated from water from the Geumgang River, were determined and were inferred their phylogenetic and taxonomic positions among taxa of order Chroococcales. Most taxa of Chroococcales whose partial 16S rRNA gene sequences were aligned in this study, are clustered with other related taxa. Aphanothece nidulans KCTC AG10041 and Aphanothece naegelii KCTC AG10042 made a cluster with other European species of these genera, which supported 100% of the bootstrap trees with a very high sequence similarity (97.4-99.4%) in this study. Three strains, Microcystis aeruginosa KCTC AG10159, M. ichthyoblabe KCTC AG10160, and M. viridis KCTC AG10198, formed a cluster with other Microcystis spp. supported 100 % of the bootstrap trees with a similarity of 97.0-99.9% except for two strains. However, this phylogentic tree made no resolution among the species of Microcystis spp. The topology of the tree reconfirmed the taxonomic status of three species of Microcystis, identified in this study based on the morphology, as three colonial types of Microcystis aeruginosa com. nov. Otsuka et al. (1999c). The genera of chroococcalian cyanophytes are heterogeneously clustered in these sequence analyses. We suggest that more molecular studies on the genera of Chroococcales with reference strains, widely collected from restricted geographic or environmental ranges, get accurate taxonomic or phylogenetic determinations.

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