Metabolites 2-methylisoborneol Research Articles

Heterologous expression of mtf and mtc genes of Pseudanabaena foetida var. intermedia is sufficient to produce 2-methylisoborneol in Escherichia coli.

Microbial volatile metabolite 2-methylisoborneol (2-MIB) causes odor and taste issues in drinking water, making it unappealing for human consumption. It has been suggested that 2-MIB biosynthesis consists of two main steps, namely, methylation of geranyl diphosphate into 2-methyl geranyl diphosphate by geranyl diphosphate methyl transferase (GPPMT) and subsequent cyclization into 2-MIB by 2-MIB synthase (MIBS). Pseudanabaena foetida var. intermedia is a 2-MIB-producing cyanobacterium whose GPPMT and MIBS enzymes are encoded by adjacent mtf and mtc genes. The present study identified a 2-MIB-related gene cluster composed of cnbA, mtf, mtc, and cnbB genes in P. foetida var. intermedia. The two homologous cyclic nucleotide-binding protein genes, cnbA and cnbB, were detected adjacent to the mtf and mtc genes, respectively. The nucleotide sequence of the cnbA-mtf-mtc-cnbB gene cluster showed 99.55% identity with 2-MIB synthesis-associated gene cluster of Pseudanabaena sp. dqh15. RT-PCR results revealed that mtf and mtc genes are co-expressed, while cnbA and cnbB genes are expressed independently in P. foetida var. intermedia. To investigate whether only mtf and mtc genes are sufficient for 2-MIB synthesis, the two-gene unit (mtf-mtc) was introduced into Escherichia coli strain JM109 via overexpression vector pYS1C. Gas chromatograph-mass spectrometry results showed that the E. coli strain transformed with mtf-mtc was able to produce 2-MIB. The intracellular 2-MIB level in P. foetida var. intermedia was higher than the extracellular 2-MIB level, while the transformed E. coli strain showed an opposite trend. Growth inhibition was observed in the 2-MIB-producing transformed E. coli strain. IMPORTANCE Contamination of drinking water with odiferous microbial metabolite 2-MIB is a worldwide concern. Removal of 2-MIB from drinking water burdens the water purification process. Therefore, it is important to search for alternative methods, such as suppressing the production of 2-MIB by aquatic microorganisms. For that, it is necessary to expand the current knowledge about the mechanism of 2-MIB synthesis at the genetic level. This study revealed that mtf and mtc genes of the 2-MIB-related gene cluster are transcribed as a single unit in P. foetida var. intermedia, and the expression of both mtf and mtc genes is essential and sufficient for 2-MIB synthesis in E. coli heterologous gene expression system.

Study of the removal capacity of 2-MIB and geosmin by nanofiltration membranes pretreated in water and 50% (v/v) aqueous ethanol solution

Nanofiltration membranes are highly effective in removing low-molecular weight compounds, which include the secondary metabolites 2-methylisoborneol (2-MIB) and 1,10-dimethyl trans-9-decalol (Geosmin), produced by cyanobacteria and difficult to remove by conventional treatment processes. Considering that high retention and permeate flux are important characteristics in the process, this study aimed to evaluate the efficiency of the NF90 membrane pretreated with water and 50% (v/v) ethanol solution in the retention of 2-MIB and Geosmin, considering the application of low constant working pressure values of 4, 7, and 10 bar and evaluating its permeability to water and metabolite retention capacity. Retention was evaluated from a concentration of 100 ng L−1 of 2-MIB and Geosmin for 120 min of filtration time. The occurrence of fouling was also evaluated, noting that there was no fouling. At the three pressure values considered, membranes pretreated in 50% (v/v) ethanol solution showed a higher permeate flux (91.4 L m-2 h-1 at 225.4 L m-2 h-1) than that observed for membranes treated in water (34.08 L m-2 h-1 at 59.14 L m-2 h-1). As for retention, no significant differences were observed between the membranes, with removals of 93 and 99% being obtained for membranes pretreated in 50% (v/v) ethanol solution and water, respectively. It can be observed that the pretreatment conserved the efficiency in the retention of compounds and provided an improvement in the physical and chemical characteristics of the membrane, allowing the achievement of permeate fluxes greater than those observed with the membrane pretreated in water.

Influence of Environmental Factors on the Production of MIB and Geosmin Metabolites by Bacteria in a Eutrophic Reservoir

AbstractOccurrences of odorous bacterial metabolites, 2‐methylisoborneol (MIB) and geosmin (GSM), in drinking water supply reservoirs are considered as a nuisance by the water industry and a source of complaints from customers. In Eagle Creek Reservoir, routine monitoring programs of MIB and GSM highlight intense odorous outbreaks during the spring season when high inflow discharges occur. Cyanobacteria have always been assumed to be source of these metabolites even if no known producers are present in raw water. A copper‐based algaecide is often used to terminate the metabolite production and the algal growth in the reservoir. The current study was designed to investigate and identify other biological sources involved in the biosynthesis of MIB and GSM metabolites as well as environmental factors that could be important triggers for the growth of bacterial producers. The community structure of the bacterioplankton was determined using a 16S rRNA gene sequencing technique, which showed that not only Cyanobacteria but Actinobacteria also were involved in the reservoir internal production. Planktothrix species was identified as the main source of GSM (p < 0.001) while Streptomyces (Actinobacteria) was very likely responsible of MIB (p < 0.01). Application of an algaecide disrupted GSM and the growth of Planktothrix but was less effective against MIB and Streptomyces. Statistical analyses revealed that MIB‐ and GSM‐causing bacteria were found abundant when the water was enriched with nitrogen, temperature cooler, and the water column mixed.

MIB and 2,4‐D Removal by Biofilters During Episodic Loading

Episodic occurrences of trace organic contaminants may affect their removal by biofilters. Removal of the naturally occurring algal metabolite 2‐methylisoborneol (MIB) and the herbicide 2,4‐dichlorphenoxyacetic acid (2,4‐D) by seven biofilters with an average empty bed contact time (EBCT) of 8.7 min is presented under a range of intermittent exposure conditions with continuous presence of dissolved organic matter. An average 70% removal of both MIB and 2,4‐D was found under constant exposure conditions over seven months. With the use of a pseudo‐first‐order model, much of the variability in removal during this time could be explained by EBCT changes. Microorganisms in the biofilters retained the capacity to biodegrade MIB and 2,4‐D after non‐exposure periods of up to five months. However, after nine months of non‐exposure, the MIB and 2,4‐D removals were lower than with the control biofilter, although the biofilter began to quickly reacclimate to these contaminants.

In vivo and in vitro genotoxicity assessment of 2-methylisoborneol, causal agent of earthy–musty taste and odor in water

The water eutrophication process by phosphorus and nitrogen allows cyanobacteria blooms which promote, among other effects, the generation and release of the metabolite 2-methylisoborneol (2-MIB) in the environment. This substance has been shown to be recalcitrant to conventional water treatment, degrading water quality. Considering the limited number of studies on the biological effects of 2-MIB in eukaryotic organisms, the present study assessed the genotoxicity of 2-MIB using the in vitro comet assay and cytokinesis block-micronucleus (CBMN-Cytome) assay on Chinese Hamster Ovary (CHO) cells and the in vivo Drosophila melanogaster Somatic Mutation and Recombination Test (SMART). The results showed that 2-MIB (125, 250 and 500µg/mL) was unable to induce gene and chromosome mutations or events associated with mitotic recombination in the SMART. Similarly, four different concentrations (7.5, 15, 30 and 60µg/mL) of 2-MIB did not induce increments in frequencies of micronuclei, nuclear buds, and nucleoplasmatic bridges in the CBMN-Cytome assay. In the comet assay, the positive results were restricted to the highest dose, 60µg/mL of 2-MIB. The results obtained may help evaluate the genotoxic profile of extracellular algal products.

Genes Associated with 2-Methylisoborneol Biosynthesis in Cyanobacteria: Isolation, Characterization, and Expression in Response to Light

The volatile microbial metabolite 2-methylisoborneol (2-MIB) is a root cause of taste and odor issues in freshwater. Although current evidence suggests that 2-MIB is not toxic, this compound degrades water quality and presents problems for water treatment. To address these issues, cyanobacteria and actinomycetes, the major producers of 2-MIB, have been investigated extensively. In this study, two 2-MIB producing strains, coded as Pseudanabaena sp. and Planktothricoids raciborskii, were used in order to elucidate the genetic background, light regulation, and biochemical mechanisms of 2-MIB biosynthesis in cyanobacteria. Genome walking and PCR methods revealed that two adjacent genes, SAM-dependent methyltransferanse gene and monoterpene cyclase gene, are responsible for GPP methylation and subsequent cyclization to 2-MIB in cyanobacteria. These two genes are located in between two homologous cyclic nucleotide-binding protein genes that may be members of the Crp-Fnr regulator family. Together, this sequence of genes forms a putative operon. The synthesis of 2-MIB is similar in cyanobacteria and actinomycetes. Comparison of the gene arrangement and functional sites between cyanobacteria and other organisms revealed that gene recombination and gene transfer probably occurred during the evolution of 2-MIB-associated genes. All the microorganisms examined have a common origin of 2-MIB biosynthesis capacity, but cyanobacteria represent a unique evolutionary lineage. Gene expression analysis suggested that light is a crucial, but not the only, active regulatory factor for the transcription of 2-MIB synthesis genes. This light-regulated process is immediate and transient. This study is the first to identify the genetic background and evolution of 2-MIB biosynthesis in cyanobacteria, thus enhancing current knowledge on 2-MIB contamination of freshwater.

Preliminary Pond Evaluation of a Natural‐Based Algicide for Managing Musty Off‐Flavor in Channel Catfish Aquaculture

AbstractEnvironmentally derived “off‐flavors” such as “earthy” and “musty” in cultured channel catfish Ictalurus punctatus are attributed to the presence of certain species of cyanobacteria (blue‐green algae) in ponds. The musty off‐flavor is caused by absorption of the metabolite 2‐methylisoborneol (MIB) into channel catfish flesh. Most channel catfish producers manage off‐flavor episodes by the application of algicides; however, these algicides have high environmental persistence and broad‐spectrum toxicity. Recent research has discovered several natural and natural‐based quinones that are effective and selective in reducing the abundance of the MIB‐producing cyanobacterium Planktothrix perornata. In this study, efficacy testing of one of these quinones, anthraquinone‐59, in a channel catfish pond found that it is effective in reducing the abundance of P. perornata and subsequently lowering the MIB levels in channel catfish.

The application of powdered activated carbon for mib and geosmin removal: predicting pac doses in four raw waters

Blooms of blue–green algae in reservoirs often produce the musty-earthy taste and odour algal metabolites 2-methylisoborneol (MIB) and geosmin. MIB and geosmin are not removed by conventional water treatment and their presence in the distribution system, even at low ng L −1 levels, can result in consumer complaints. Powdered activated carbon (PAC) can effectively remove MIB and geosmin when the correct dose is applied. The homogeneous surface diffusion model (HSDM) was used to predict PAC doses required to reduce MIB and geosmin concentrations to below 10 ng L −1 at four water treatment plants in Adelaide, South Australia. In jar tests, undertaken under treatment plant conditions, the predicted doses were found to produce water of the desired quality in three of the four waters. The poor predictions found in the fourth water, which had a considerably higher turbidity, were attributed to the incorporation of PAC in a larger, denser floc, leading to a reduced effective contact time of the adsorbent. It was found that higher doses of PAC were required for both compounds to produce acceptable quality water when turbidities rose above 26 NTU.

ALGAL‐RELATED TASTES AND ODORS IN PHOENIX WATER SUPPLY: PRELIMINARY REPORT

Frequent episodes of algal‐related tastes and odors (T & O) in drinking waters in metropolitan Phoenix, Arizona prompted initiation of a three‐year project in July 1999 to investigate the occurrence of T & O metabolites and to develop a comprehensive management strategy to reduce the problems in drinking water supplies in arid environments. Two metabolites, 2‐methylisoborneol (MIB) and geosmin, have been identified as compounds responsible for the earthy‐musty tastes and odors in water supplies. Both were detected in the water supply system, including source rivers, reservoirs, canal delivery system and water treatment plants. Higher concentrations of MIB and geosmin occurred in distribution canals than in the upstream reservoirs indicating that significant production of the T & O compounds occurs within the canal system. A baseline‐monitoring program has been established for the complex water supply system, with special emphasis on the canal system. Efforts are underway to investigate possible correlations between physical/chemical parameters, algal composition and biomass, with the occurrence of MIB and geosmin. In addition, several physical and chemical treatments are planned for the canal system to reduce algal growth and related MIB and geosmin concentrations.

The Recovery of Flavor Quality by Channel Catfish

Abstract Intensive production of channel catfish Ictalurus punctatus requires copious feeding. Nutrients from uneaten feeds and fish excreta contribute to the growth of microorganisms. Certain microbes that occur frequently in aquaculture systems produce the “muddy and earthy” off-flavor metabolites 2-methylisoborneol (MIB) and geosmin. Fish containing MIB or geosmin can exhibit an off-flavor that makes them unacceptable for harvest as food fish. Following the cessation of exposure to MIB or geosmin, fish depurate off-flavors. The minimum period required for fish to regain flavor quality is a critical concern for aquaculturists; scant information concerning this period prompted our investigation. Channel catfish were harvested from three commercial ponds, and fish from each pond were sorted into four size-classes (0.4–0.8 kg, 0.8–1.3 kg, 1.3–1.6 kg, and 1.6–2.3 kg) on 30 August (summer) and on 18 October (autumn) 1993. Each group of fish was transferred into individual 2,000-L fiberglass tanks, and each t...

EVALUATING THE RELATIONSHIP BETWEEN PHOTOPIGMENT SYNTHESIS AND 2‐METHYLISOBORNEOL ACCUMULATION IN CYANOBACTERIA

The relationship between photopigments and the terpene‐derived secondary metabolite, 2‐methylisoborneol (MIB), was analyzed in photoacclimated cultures of Pseudanabaena articulata Skuja throughout growth, during the diel cycle, and following chemical‐induced inhibition of the isoprenoid pathway. Accumulation of MIB coincided with the accumulation of lipophilic and phycobilin pigments during the early to mid‐exponential portion of the growth cycle with the greatest accumulation of MIB during the late‐exponential phase. Cellular release of MIB occurred as culture populations entered mid‐ to late‐logarithmic phase of growth and was greatest in irradiance‐stressed cultures. The greater correspondence of MIB accumulation with photopigments was seen in cultures transferred from a 12:12 h LD photoperiod alone and the consistent relationship between MIB and photopigment accumulation under varying irradiance suggested a photopigment‐dependent regulation for MIB synthesis. However, the consistent allocation of carbon into MIB during instances of phytofluene and tetrapyrrole biosynthetic inhibition within P. articulata and Oscillatoria perornata Skuja indicated that MIB accumulation is not limited by isopreniod‐carbon availability and does not appear to serve as an “overflow” product. Rather, MIB accumulation simply appears to reflect overall carbon accumulation resulting from increased cell metabolism.

鞭毛虫類Ｍｏｎａｓ ｇｕｔｔｕｌａによるカビ臭産生藻類Ｐｈｏｒｍｉｄｉｕｍ ｔｅｎｕｅの分解に及ぼす温度の影響

Biodegradation characteristics of cyanobacteria, Phormidium tenue and its metabolites, 2-methylisoborneol(MIB) by protozoan microflagellate, Monas guttula, and the effect of temperature on them were examined in batch culture experiment. The flagellate was found to predate on P.tenue favorably as a food source within a wide temperature range from 10°C to 30°C. Specific growth rate(μ) and generation time(td) of the flagellate were 4.6day-1 and 3.5h-1, respectively at 20°C and 30°C, whereas in the case of 10°C the lag values of μ and td were obtained by 1.9 fold in comparison with the removal ratio of 61% at the first cultivation day and that of 99% at the 2nd day at 30°C, while at 20°C the removal was only about 30% in the first day, but P.tenue could be perfectly reduced at the 2nd day. Although the reduction rate of the P.tenue was slow at the lower temperature of 10°C, the removal reached 98% in the 7th day. Removal rate of P.tenue by the flagellate increased with the increase in temperature and reached maximal value with 4.0h-1 at 30°C. The flagellate could contribute to the degradation of MIB, although the degradation was lower than that of P.tenue. It was found that the microflagellate could favorably predate on P.tenue and degrade its metabolite, MIB emitting musty odor in a wide range of temperature.