Production and release of 2-MIB in Pseudanabaena: Effects of growth phases on cell characteristics and 2-MIB yield.

2-methylisoborneol (2-MIB), a secondary metabolite produced by cyanobacteria, often causes a musty odour in water, threatening the safety of drinking water supplies. This study investigated the effects of the growth phases on the production of 2-MIB by Pseudanabaena. The effects of cell characteristics on the production and release of 2-MIB were also explored. The total 2-MIB concentration increased during the exponential phase and decreased during the declining phase, which was consistent with the changes in cell density. However, the total 2-MIB yield (1.12-1.27 fg cell-1) of Pseudanabaena did not significantly differ throughout the growth cycle (p > 0.05). Meanwhile, the extracellular 2-MIB yield increased significantly from 0.31 fg cell-1 in the exponential phase to 0.76 fg cell-1 in the declining phase (p < 0.05), and the corresponding proportion of extracellular 2-MIB improved from 25.13% to 59.16% (p < 0.05). The surge in extracellular 2-MIB during the declining phase could be attributed to the breaking of the Pseudanabaena filament, as indicated by the decrease in Dmean during cell ageing. The findings of this study contribute to a more inclusive comprehension and management of musty odour issues resulting from cyanobacteria in the water supply.

Investigating Taste and Odour Characteristics in a Drinking Water Source: A Comprehensive 3-Year Monitoring Study.

The monitoring of drinking water quality is a vital public health concern together with taste and odour (T&O) episodes, an emerging global problem causing a loss of public trust to the quality of water. Our objective was to monitor water quality of an important drinking water source and also the production dynamics of geosmin and 2-methylisoborneol (2-MIB) which cause taste and odour problems in the lake. The trophic status of the lake was classified as mesotrophic. 2-MIB was positively correlated temperature while geosmin was positively correlated with depth. Other physicochemical parameters related with water quality did not show significant correlation with geosmin and 2-MIB. The highest 2-MIB and geosmin concentrations were detected during the thermal stratification period in 2016 and 2018 by gas chromatography-mass spectrometry (GC-MS). Cyanobacteria and Actinobacteria were detected in geosmin & 2-MIB detected samples as potential taste and odour producers by PCR. Selected samples were analysed with metabarcoding and Planktothrix, Pseudanabaena, Cyanobium, Streptomyces, and Nocardioides were detected as potential geosmin & 2-MIB producers. Micrococcus, Rhodococcus, Acinetobacter, Comamonas, Novosphingobium, Sphingopyxis, Pseudomonas, Sphingomonas, Stenotrophomonas and Flavobacterium were identified as potential geosmin & 2-MIB degraders. The results highlighted the significant role of the autochthonous bacterial community, temperature and thermal stratification in the taste and odour dynamics of a drinking water source.

[Identification method of 2-methylisoborneol and geosmin in water by purge and trap-gas chromatography-mass spectrometry].

OBJECTIVE: To establishe an analysis and identification method for 2-methylisoborneol(2-MIB) and geosmin(GSM) in water using purge and trap-gas chromatography-mass spectrometry. METHODS: The samples were enriched and analyzed using a purge and trap system, followed by the separation on a DB-624(30 m×0.25 mm, 1.4 µm) chromatographic column. Quantification was performed using gas chromatography-mass spectrometry with the selected ion monitoring and internal standard calibration. RESULTS: The calibration curves for 2-MIB and GSM showed an excellent linearity in the range of 1 to 100 ng/L with R~2 values greater than 0.999. The detection limit and quantification limit for both 2-MIB and GSM were 0.33 ng/L and 1.0 ng/L, respectively. Spike recovery experiments were further carried on the source water and drinking water at three concentration levels. It showed that the average recoveries were from 82.0% to 111.0% for 2-MIB while 84.0% to 110% for GSM. Additionally, the test precision of 2-MIB and GSM ranged from 1.9% to 7.3% and 1.9% to 5.0%(n=6), respectively. The analysis of multiple samples including the local source water, treated water and distribution network water confirmed the existence of 2-MIB and GSM. CONCLUSION: Compared to the national standard(GB/T 5750.8-2023), the proposed method enables fully automated sample introduction and analysis without the extra pre-treatment. It provides the advantages of simplicity, good repeatability and high accuracy.

Functional Characterisation of Highly Conserved and Structurally Prominent Residues of 2-Methylisoborneol Synthase.

2-Methylisoborneol is a widespread musty odourant that is produced by many bacteria including actinomycetes, cyanobacteria and myxobacteria. Two 2-methylisoborneol synthases (MIBS) that are phylogenetically distant to the known enzyme from Streptomyces coelicolor were found to be highly active for 2-methylisoborneol biosynthesis. Based on the enzyme structure and on an amino acid sequence alignment, the MIBS from S. coelicolor was extensively studied through site-directed mutagenesis.

Biosynthesis of 2-methylisoborneol is regulated by chromatic acclimation of Pseudanabaena.

Cyanobacteria can sense different light color by adjusting the components of photosynthetic pigments including chlorophyll a (Chl a), phycoerythrin (PE), and phycocyanin (PC), etc. Filamentous cyanobacteria are the main producer of 2-methylisoborneol (MIB) and many can increase their PE levels so that they are more competitive in subsurface layer where green light is more abundant, and have caused extensive odor problems in drinking water reservoirs. Here, we identified the potential correlation between MIB biosynthesis and ambient light color induced chromatic acclimation (CA) of a MIB-producing Pseudanabaena strain. The results suggest Pseudanabaena regulates the pigment proportion through Type III CA (CA3), by increasing PE abundance and decreasing PC in green light. The biosynthesis of MIB and Chl a share the common precursor, and are positively correlated with statistical significance regardless of light color (R2=0.68; p<0.001). Besides, the PE abundance is also positively correlated with Chl a in green light (R2=0.57; p=0.019) since PE is the antenna that can only transfer the energy to PC and Chl a. In addition, significantly higher MIB production was observed in green light since more Chl a was synthesized.

Mercury and Organic Pollutants Removal from Aqueous Solutions by Heterogeneous Photocatalysis with ZnO-Based Materials.

The removal of four Contaminants of Emerging Concern, namely bisphenol A, sulfamethoxazole, diclofenac and benzotriazole; two odorous compounds, geosmin and 2-methylisoborneol, frequently detected in recirculating aquaculture systems; and Hg(II) was investigated using ZnO-based materials doped or co-doped with Ce and Cu under simulated solar radiation. Photocatalysts were synthetized via a hydrothermal route and their efficiency was assessed by changing some operational parameters in different water matrices of increasing complexity. The mixture of contaminants was successfully degraded in just 1 h, while the complete mineralization was achieved in a few hours; experiments performed in an actual aquaculture water confirmed the efficiency and broad versatility of the synthesized materials.

The Ubiquitous Soil Terpene Geosmin Acts as a Warning Chemical.

Known as the smell of earth after rain, geosmin is an odorous terpene detectable by humans at picomolar concentrations. Geosmin production is heavily conserved in actinobacteria, myxobacteria, cyanobacteria, and some fungi, but its biological activity is poorly understood. We theorized that geosmin was an aposematic signal used to indicate the unpalatability of toxin-producing microbes, discouraging predation by eukaryotes. Consistent with this hypothesis, we found that geosmin altered the behavior of the bacteriophagous nematode Caenorhabditis elegans on agar plates in the absence of bacteria. Normal movement was restored in mutant worms lacking differentiated ASE (amphid neurons, single ciliated endings) neurons, suggesting that geosmin is a taste detected by the nematodal gustatory system. In a predation assay, geosmin and the related terpene 2-methylisoborneol reduced grazing on the bacterium Streptomyces coelicolor. Predation was restored by the removal of both terpene biosynthetic pathways or the introduction of C. elegans that lacked differentiated ASE taste neurons, leading to the apparent death of both bacteria and worms. While geosmin and 2-methylisoborneol appeared to be nontoxic, grazing triggered bacterial sporulation and the production of actinorhodin, a pigment coproduced with a number of toxic metabolites. In this system, geosmin thus appears to act as a warning signal indicating the unpalatability of its producers and reducing predation in a manner that benefits predator and prey. This suggests that molecular signaling may affect microbial predator-prey interactions in a manner similar to that of the well-studied visual markers of poisonous animal prey. IMPORTANCE One of the key chemicals that give soil its earthy aroma, geosmin is a frequent water contaminant produced by a range of unrelated microbes. Many animals, including humans, are able to detect geosmin at minute concentrations, but the benefit that this compound provides to its producing organisms is poorly understood. We found that geosmin repelled the bacterial predator Caenorhabditis elegans in the absence of bacteria and reduced contact between the worms and the geosmin-producing bacterium Streptomyces coelicolor in a predation assay. While geosmin itself appears to be nontoxic to C. elegans, these bacteria make a wide range of toxic metabolites, and grazing on them harmed the worms. In this system, geosmin thus appears to indicate unpalatable bacteria, reducing predation and benefiting both predator and prey. Aposematic signals are well known in animals, and this work suggests that metabolites may play a similar role in the microbial world.

African elephants can detect water from natural and artificial sources via olfactory cues.

Water is vital for mammals. Yet, as ephemeral sources can be difficult to find, it raises the question, how do mammals locate water? Elephants (Loxodonta africana) are water-dependent herbivores that possess exceptional olfactory capabilities, and it has been suggested that they may locate water via smell. However, there is no evidence to support this claim. To explore this, we performed two olfactory choice experiments with semi-tame elephants. In the first, we tested whether elephants could locate water using olfactory cues alone. For this, we used water from two natural dams and a drinking trough utilised by the elephants. Distilled water acted as a control. In the second, we explored whether elephants could detect three key volatile organic compounds (VOCs) commonly associated with water (geosmin, 2-methylisoborneol, and dimethyl sulphide). We found that the elephants could locate water olfactorily, but not the distilled water. Moreover, they were also able to detect the three VOCs associated with water. However, these VOCs were not in the odour profiles of the water sources in our experiments. This suggests that the elephants were either able to detect the unique odour profiles of the different water sources or used other VOCs that they associate with water. Ultimately, our findings indicate that elephants can locate water olfactorily at small spatial scales, but the extent to which they, and other mammals, can detect water over larger scales (e.g. km) remains unclear.

Evaluation of the MIB-producing potential based on real-time qPCR in drinking water reservoirs.

Cyanobacteria release 2-methylisoborneol (MIB) as a secondary metabolite. Here, we propose a reverse transcription quantitative real-time PCR (RT-qPCR) based method to evaluate the MIB-producing potential in source water by detecting the MIB-synthesis gene (mic). A MIBQSF/R primer set was designed based on 35 mic gene sequences obtained from 12 pure-cultured MIB-producing strains and 23 sequences from the NCBI database. This primer set successfully identified all known 43 MIB-producing cyanobacterial strains (12 from this study and 31 from the NCBI database), belonging to different genera, showing a wider coverage than previous primer sets. The efficiency of the method was proved by the amplification efficiency (E = 91.23%), R2 of the standard curve (0.999), the limit of detection (LOD, 5.7 fg µL-1), and the limit of quantification (LOQ, 1.86 × 104 gene copies µL-1). Further, the method was verified by the correlation between the mic gene abundance and MIB concentration 50 field samples from different reservoirs (R2 = 0.614, p < 0.001) and one reservoir (R2 = 0.752, p < 0.001), suggesting its potential as an alternative warning tool to evaluate the risk of MIB problems in source water.

Occurrences of 2-methylisoborneol and geosmin -degrading bacteria in a eutrophic reservoir and the role of cell-bound versus dissolved fractions.

As taste-and-odor outbreaks are common in surface waters worldwide, extensive studies have focused on the identification of microorganisms involved in the production of 2-methylisoborneol (MIB) and geosmin (GSM). However, fewer studies have tried to identify potential degraders in natural environments. Eagle Creek Reservoir, a temperate and eutrophic water body, experienced two major seasonal odorous outbreaks in 2013 with maximal concentrations of 99.1 (MIB) and 77.3 ng L-1 (GSM). Fractionation analyses of the odorous compounds showed that MIB was found more frequently in the dissolved fraction while GSM was mostly cell-bound. This difference likely impacts taste-and-odor (T&O) compound susceptibility to biodegradation by bacteria. Spearman relationships of epilimnetic samples collected between spring and early fall linked dissolved MIB occurrences to higher abundances of Bacteroidetes like Flavobacterium resistens, F. granuli, F. saliperosum (p < 0.001), F. kamogawaensis (p < 0.01) capable of MIB degradation. Occurrences of cell-bound GSM were correlated to two α-Proteobacteria Novosphingobium hassiacum (p < 0.001) and Sphingomonas oligophenolica (p < 0.01), both identified as potential degraders of GSM. The roles of Pseudomonas and Bacillus were ambiguous, and these genera might have been involved in both compound biodegradations (p < 0.05).

A critical review on geosmin and 2-methylisoborneol in water: sources, effects, detection, and removal techniques.

The exposure to geosmin (GSM) and 2-methylisoborneol (2-MIB) in water has caused a negative impact on product reputation and customer distrust. The occurrence of these compounds and their metabolites during drinking water treatment processes has caused different health challenges. Conventional treatment techniques such as coagulation, sedimentation, filtration, and chlorination employed in removing these two commonest taste and odor compounds (GSM and 2-MIB) were found to be ineffective and inherent shortcomings. The removal of GSM and MIB were found to be effective using combination of activated carbon and ozonation; however, high treatment cost associated with ozonation technique and poor regeneration efficiency of activated carbon constitute serious setback to the combined system. Other shortcoming of the activated carbon adsorption and ozonation include low adsorption efficiency due to the presence of natural organic matter and humic acid. In light of this background, the review is focused on the sources, effects, environmental pathways, detection, and removal techniques of 2-MIB and GSM from aqueous media. Although advanced oxidation processes (AOPs) were found to be promising to remove the two compounds from water but accompanied with different challenges. Herein, to fill the knowledge gap analysis on these algal metabolites (GSM and 2-MIB), the integration of treatment processes vis-a-viz combination of one or more AOPs with other conventional methods are considered logical to remove these odorous compounds and hence could improve overall water quality.

Forecasting a 2-methylisoborneol outbreak in a brackish lake.

2-Methylisoborneol (2-MIB) is the primary cause of the earthy and musty odor produced by cyanobacteria, which deteriorates the quality of fishery products and tap water. Despite the need for controlling outbreaks, few studies have been conducted on 2-MIB in brackish lakes, where capture fisheries are active. This study examined the association between water quality and the outbreak of 2-MIB in a brackish lake using statistical analysis of long-term monitoring data and developed forecasting models for 2-MIB outbreaks. We investigated Lake Ogawara, which is a brackish lake with a cool-temperate climate in Japan, where 2-MIB outbreaks frequently occur between August and December. Logistic regression analyses were performed using the outbreak or non-outbreak of 2-MIB (2-MIB(+ / -)) as the dependent variable and water quality parameters as the independent variables. The results suggested that the density of 2-MIB-producing cyanobacteria was higher when (1) dissolved inorganic nitrogen concentrations were low under the relaxation of phosphorus limitation and/or (2) salinity or micronutrient concentrations were high. In addition, we successfully developed forecasting models with a high predictive power that determined 2-MIB(+ / -) in August-December using only two water quality parameters: dissolved inorganic phosphate and pH in April and total nitrogen/total phosphorous and salinity in May.

Ecological niche and in-situ control of MIB producers in source water.

Odor problems in source water caused by 2-methylisoborneol (MIB) have been a common issue in China recently, posing a high risk to drinking water safety. The earthy-musty odorant MIB has an extremely low odor threshold (4-16 ng/L) and is hard to remove via conventional processes in drinking water plants (DWP), and therefore could easily provoke complaints from consumers. This compound is produced by a group of filamentous cyanobacteria, mainly belonging to Oscillatoriales. Different from the well-studied surface-blooming Microcystis, filamentous cyanobacteria have specific niche characteristics that allow them to stay at a subsurface or deep layer in the water column. The underwater bloom of these MIB producers is therefore passively determined by the underwater light availability, which is governed by the cell density of surface scum. This suggests that drinking water reservoirs with relatively low nutrient contents are not able to support surface blooms, but are a fairly good fit to the specialized ecological niche of filamentous cyanobacteria; this could explain the widespread odor problems in source water. At present, MIB is mainly treated in DWP using advanced treatment processes and/or activated carbon, but these post-treatment methods have high cost, and not able to deal with water containing high MIB concentrations. Thus, in situ control of MIB producers in source water is an effective complement and is desirable. Lowering the underwater light availability is a possible measure to control MIB producers according to their niche characteristics, which can be obtained by either changing the water level or other measures.

Potentiometric E-Tongue System for Geosmin/Isoborneol Presence Monitoring in Drinkable Water.

A potentiometric E-tongue system based on low-selective polymeric membrane and chalcogenide-glass electrodes is employed to monitor the taste-and-odor-causing pollutants, geosmin (GE) and 2-methyl-isoborneol (MIB), in drinkable water. The developed approach may permit a low-cost monitoring of these compounds in concentrations near the odor threshold concentrations (OTCs) of 20 ng/L. The experiments demonstrate the success of the E-tongue in combination with partial least squares (PLS) regression technique for the GE/MIB concentration prediction, showing also the possibility to discriminate tap water samples containing these compounds at two concentration levels: the same OTC order from 20 to 100 ng/L and at higher concentrations from 0.25 to 10 mg/L by means of PLS-discriminant analysis (DA) method. Based on the results, developed multisensory system can be considered a promising easy-to-handle tool for express evaluation of GE/MIB species and to provide a timely detection of alarm situations in case of extreme pollution before the drinkable water is delivered to end users.

Depuration of geosmin- and 2-methylisoborneol-induced off-flavors in recirculating aquaculture system (RAS) farmed European whitefish Coregonus lavaretus.

European whitefish Coregonus lavaretus has increasingly become an important species for aquatic food production, especially in the Nordic countries. Whitefish is produced in traditional cage and pond operations, and in recirculating aquaculture system (RAS) in which, unfortunately, off-flavors and odors, mostly caused by geosmin (GSM) and 2-methylisoborneol (MIB), can accumulate in fish flesh from the circulating water. GSM and MIB have very low human sensory detection limits and, therefore, often disliked by consumers even at low concentrations. In this study, concentrations of GSM and MIB in RAS farmed European whitefish were studied by an analytical method based on headspace solid phase microextraction and gas chromatography-mass spectrometry. Concentrations were determined in different parts of fish: fillet, neck, belly, and tail during a depuration period and in depuration water. The highest initial concentrations were on average 32 ng g-1 (GSM) and 24 ng g-1 (MIB) in European whitefish fillet and 128 ng L-1 (GSM) and 94 ng L-1 (MIB) in water, respectively. After a depuration period of 16 days, concentrations decreased to below the detection limits, indicating the importance of the depuration period.

An alternative method to quantify 2-MIB producing cyanobacteria in drinking water reservoirs: Method development and field applications.

2-Methylisoborneol (2-MIB) is a commonly detected cyanobacterial odorant in drinking water sources in many countries. To provide safe and high-quality water, development of a monitoring method for the detection of 2-MIB-synthesis (mibC) genes is very important. In this study, new primers MIBS02F/R intended specifically for the mibC gene were developed and tested. Experimental results show that the MIBS02F/R primer set was able to capture 13 2-MIB producing cyanobacterial strains grown in the laboratory, and to effectively amplify the targeted DNA region from 17 2-MIB-producing cyanobacterial strains listed in the literature. The primers were further coupled with a TaqMan probe to detect 2-MIB producers in 29 drinking water reservoirs (DWRs). The results showed statistically significant correlations between mibC genes and 2-MIB concentrations for the data from each reservoir (R2=0.413-0.998; p<0.05), from all reservoirs in each of the three islands (R2=0.302-0.796; p<0.01), and from all data of the three islands (R2=0.473-0.479; p<0.01). The results demonstrate that the real-time PCR can be an alternative method to provide information to managers of reservoirs and water utilities facing 2-MIB-related incidents.

Spatiotemporal dynamics of 2-methylisoborneol produced by filamentous cyanobacteria and associated driving factors in Lake Taihu, China.

The proliferation of filamentous cyanobacteria in lakes can result in the generation of odor-causing compounds, predominantly 2-methylisoborneol (2-MIB), which pose odor-related challenges. In an effort to elucidate the spatiotemporal dynamics of 2-MIB and related influencing factors in East Lake Taihu, monthly investigations were undertaken from April 2022 to March 2023. In addition to the monthly survey, a whole-lake survey was conducted during the high-temperature period from July to September. The monthly survey revealed a distinct unimodal fluctuation in the concentration of 2-MIB in East Lake Taihu, with an average concentration at 297.0 ng/L during the high-temperature period. During the high-temperature period, the filamentous cyanobacterial communities detected in East Lake Taihu consisted primarily of species belonging to genera Leptolyngbya, Oscillatoria, Planktothricoides, and Pseudanabaena. However, no significant correlations were found between their densities and 2-MIB concentration. In addition, the mic gene was predominantly detected in genera Pseudanabaena and Planktothricoides, with the latter being the primary contributor to 2-MIB production. Furthermore, a succession of cyanobacteria capable of producing 2-MIB was detected, with water temperature and radiation intensity being identified as the primary driving factors. The temporal variation of 2-MIB concentration within East Lake Taihu during the whole year was primarily modulated by factors such as water temperature, water transparency, dissolved oxygen, and chlorophyll-a. During the high-temperature period, the 2-MIB concentration in the alga-dominated zone of East Lake Taihu was approximately 1.7 times greater than that in the macrophyte-dominated zone, with nutrient and transparency being identified as the main influencing factors. Consequently, our findings are of great significance for monitoring the sources and variation of 2-MIB in shallow lakes, providing a scientific foundation and theoretical guidance for odor management.

Aquaculture activities influencing the generation of geosmin and 2-methylisoborneol: a case study in the aquaculture regions of Hongze Lake, China.

Contamination by odor substances such as geosmin (GSM) and 2-methylisoborneol (2-MIB) was examined in the cultured water from aquaculture farming in the region of the Hongze Lake in 2022, and some factors influencing residual levels of them in the water were analyzed. Geographically, high concentrations of GSM were located mainly in the north and northeast culture areas of the lake, while those of 2-MIB were found in the northeast and southwest. Analysis of the water in the enclosure culture revealed significant differences in the concentrations of GSM and 2-MIB among the cultured species. The mean concentrations of GSM in culture water were ranked in the order: crab > the four major Chinese carps > silver and bighead carp, and silver and bighead carp > crab > the four major Chinese carps for 2-MIB. The concentration of GSM was significantly higher at 38.99 ± 18.93 ng/L in crab culture water compared to other fish culture water. Significant differences were observed in GSM concentrations between crab enclosure culture and pond culture, while 2-MIB levels were comparable. These findings suggest that cultural management practices significantly affect the generation of odor substances. The taste and odor (T&O) assessment revealed that the residual levels of GSM and 2-MIB in most samples were below the odor threshold concentrations (OTCs), although high levels of GSM and 2-MIB in all water bodies were at 30.9% and 27.5%, respectively. Compared with the corresponding data from other places and the regulation guidelines of Japan, USA, and China, the region in the Hongze Lake is generally classified as a slightly T&O area, capable of supporting the aquaculture production scale.

Histology and transcriptomic analysis reveal the inflammation and affected pathways under 2-methylisoborneol (2-MIB) exposure on grass carp.

2-Methylisoborneol (2-MIB) is a common and widely distributed off-flavor compound in water. However, the toxic mechanisms of 2-MIB on aquatic organisms remain largely unexplored. In this study, grass carp larvae were exposed to different concentrations (0, 5, and 20 µg L-1) of 2-MIB for 96 h. The accumulation of 2-MIB in the dorsal muscle was measured. Histological analysis, ultrastructure observations, and transcriptomic sequencing were conducted on the liver tissues. The results showed that 2-MIB accumulated significantly in the fish muscle, with the accumulation increasing as the exposure concentration increased through gas chromatography-mass spectrometry (GC-MS) detection. Histological and ultrastructure observations indicated that 2-MIB caused concentration-dependent inflammatory infiltration and mitochondrial damage in the liver. Transcriptomic analysis revealed lipid metabolism disorders induced by exposure to 2-MIB in grass carp. Additionally, 5 µg L-1 2-MIB affected the neurodevelopment and cardiovascular system of grass carp larvae through extracellular matrix (ECM)-receptor interaction and focal adhesion pathway. Furthermore, several pathways related to the digestive system were significantly enriched, implying that 2-MIB may impact pancreatic secretion function, protein digestion and absorption processes. These findings provide new insights into the potential toxicological mechanisms of 2-MIB.

Effect of Culture Temperature on 2-Methylisoborneol Production and Gene Expression in Two Strains of Pseudanabaena sp.

The presence of the odorant 2-methylisoborneol (2-MIB) in drinking water sources is undesirable. Although 2-MIB production is known to be influenced by temperature, its regulation at the gene level and its relationship with Chlorophyll-a (Chl-a) at different temperatures remain unclear. This study investigates the impact of temperature on 2-MIB production and related gene expression in Pseudanabaena strains PD34 and PD35 isolated from Lake Paldang, South Korea. The strains were cultured at three temperatures (15, 25, and 30 °C) to examine cell growth, 2-MIB production, and mic gene expression levels. 2-MIB production per cell increased with higher temperatures, whereas mic gene expression levels were higher at lower temperatures, indicating a complex regulatory mechanism involving post-transcriptional and enzyme kinetics factors. Additionally, the relationship between Chl-a and 2-MIB involved in metabolic competition was analyzed, suggesting that high temperatures appear to favor 2-MIB synthesis more than Chl-a synthesis. The distinct difference in the total amount of the two products and the proportion of 2-MIB between the two strains partially explains the variations in 2-MIB production. These findings highlight the significant effect of temperature on 2-MIB biosynthesis in Pseudanabaena and provide a valuable background for gene data-based approaches to manage issues regarding 2-MIB in aquatic environments.