Geosmin Synthesis Research Articles

Insights into the Seasonal Olfactory Mechanism of Geosmin in Raw Water of Huangpu River.

Since the 1990s, the raw water of Huangpu River in Shanghai, China, has intermittently encountered off-flavor contamination. In this work, the concentrations of typical odor, geosmin, in raw water of Huangpu River are found to shift along with the seasons. However, microbes recognized as the producer of geosmin such as Cyanobacteria and Actinobacteria are not consistent with the shift of geosmin. Cyanobacteria blooms in summer rather than winter, whereas Actinobacteria thrives in winter. Representational difference analysis (RDA) reveals that microbes associated with blooming algae have positive co-occurrence correlations with the concentrations of geosmin and nutrients in winter, whereas those within Cyanobacteria and Planctomycete are in a positive correlation with temperature and thrive in summer. This causes the concentration of geosmin in raw water to appear to depend on the abundance of Actinobacteria rather than that of Cyanobacteria. However, combining with the synthesis and storage properties of geosmin in algae, as well as the decomposition properties of algae with Actinobacteria, geosmin might be synthesized by Cyanobacteria in summer, which is stored in cells of Cyanobacteria and released only via the decomposition of Actinobacteria in winter. This potential olfactory mechanism of geosmin is quite different from that derived from pure culture of odor producers or correlation analysis of bacteria and odors; thus, providing insights into the mechanism of practical off-flavor events.

Effects of elevated nitrogen on the growth and geosmin productivity of Dolichospermum smithii.

Geosmin is one of the most common earthy-musty odor compounds, which is mainly produced by cyanobacteria in surface water. Nitrogen (N) is an important factor affecting the growth of cyanobacteria and its secondary metabolites production due to the eutrophication. In this study, we compared the effects of elevated N on the growth and geosmin productivity of Dolichospermum smithii NIES-824 (synonym Anabaena smithii NIES-824), aiming to better understand the mechanisms involved and give an important and fundamental knowledge to solve off-flavor problem. Results show that elevated N concentration promoted more chlorophyll a (Chl-a) production, whereas the geosmin synthesis decreased, revealing a possible competitive correlation between the Chl-a concentration and geosmin production of D. smithii NIES-824. The majority of geosmin (> 90%) was retained intracellularly during the 28 days of cultivation. The qRT-PCR analysis demonstrates that the expression level of the geosmin synthase gene (geoA) was constitutive and decreased at the higher N concentration during the exponential growth phase of cyanobacterial cells. Furthermore, the decrease of geoA expression during the decline phase suggested that geoA transcription was closely related to cell activity and isoprenoid productivity.

The diversity, origin, and evolutionary analysis of geosmin synthase gene in cyanobacteria

The sesquiterpene geosmin, mainly originating from cyanobacteria, is considered one of the problematic odor compounds responsible for unpleasant-tasting and -smelling water episodes in freshwater supplies. The biochemistry and genetics of geosmin synthesis in cyanobacteria is well-elucidated and the geosmin synthase gene (geo) has been cloned and characterized in recent years. However, understanding the diversity, origin, and evolution of geo has been hindered by the limited availability of geo sequences to date. On the basis of the cloned geo sequences from16 filamentous geosmin-producing cyanobacterial species, representing 11 genera in Nostocales and Oscillatoriales, the diversity and evolution of geo in cyanobacteria was systematically analyzed in this study. Homologous alignment revealed that geo is highly conserved among the examined cyanobacterial species, with DNA sequence identities >0.72. Phylogenetic reconstruction and codon bias analysis based on geo suggest that cyanobacterial geo form a monophyletic branch with a common origin and ancestor for cyanobacteria, actinomycetes, and myxobacteria. The global ratio of nonsynonymous/synonymous nucleotide substitutions (dN/dS) was 0.125, which is substantially <1 and indicates strong purifying selection in the evolution of cyanobacterial geo. To add to further interest, horizontal gene transfer of cyanobacterial geo in evolutionary history was confirmed by the discovery of an incongruent coevolutionary relationship between geo and housekeeping genes 16S rDNA and rpoC. The present study enhances the fundamental understanding of cyanobacterial geo in diversity and evolution, and sheds light on the development of molecular assays for detection and molecular ecology research of geosmin-producing cyanobacteria.

Prediction and Characterization of Small Non-Coding RNAs Related to Secondary Metabolites in Saccharopolyspora erythraea

Saccharopolyspora erythraea produces a large number of secondary metabolites with biological activities, including erythromycin. Elucidation of the mechanisms through which the production of these secondary metabolites is regulated may help to identify new strategies for improved biosynthesis of erythromycin. In this paper, we describe the systematic prediction and analysis of small non-coding RNAs (sRNAs) in S. erythraea, with the aim to elucidate sRNA-mediated regulation of secondary metabolite biosynthesis. In silico and deep-sequencing technologies were applied to predict sRNAs in S. erythraea. Six hundred and forty-seven potential sRNA loci were identified, of which 382 cis-encoded antisense RNA are complementary to protein-coding regions and 265 predicted transcripts are located in intergenic regions. Six candidate sRNAs (sernc292, sernc293, sernc350, sernc351, sernc361, and sernc389) belong to four gene clusters (tpc3, pke, pks6, and nrps5) that are involved in secondary metabolite biosynthesis. Deep-sequencing data showed that the expression of all sRNAs in the strain HL3168 E3 (E3) was higher than that in NRRL23338 (M), except for sernc292 and sernc361 expression. The relative expression of six sRNAs in strain M and E3 were validated by qRT-PCR at three different time points (24, 48, and 72 h). The results showed that, at each time point, the transcription levels of sernc293, sernc350, sernc351, and sernc389 were higher in E3 than in M, with the largest difference observed at 72 h, whereas no signals for sernc292 and sernc361 were detected. sernc293, sernc350, sernc351, and sernc389 probably regulate iron transport, terpene metabolism, geosmin synthesis, and polyketide biosynthesis, respectively. The major significance of this study is the successful prediction and identification of sRNAs in genomic regions close to the secondary metabolism-related genes in S. erythraea. A better understanding of the sRNA-target interaction would help to elucidate the complete range of functions of sRNAs in S. erythraea, including sRNA-mediated regulation of erythromycin biosynthesis.

Monitoring of geosmin producing Anabaena circinalis using quantitative PCR

Geosmin is one of the most commonly detected off-flavor chemicals present in reservoirs and drinking water systems. Quantitative real-time PCR (qPCR) is useful for quantifying geosmin-producers by focusing on the gene encoding geosmin synthase, which is responsible for geosmin synthesis. In this study, several primers and probes were designed and evaluated to detect the geosmin synthase gene in cyanobacteria. The specificity of primer and probe sets was tested using 21 strains of laboratory cultured cyanobacteria isolated from surface waters in Australia (18) and Taiwan (2), including 6 strains with geosmin producing ability. The results showed that the primers designed in this study could successfully detect all geosmin producing strains tested. The selected primers were used in a qPCR assay, and the calibration curves were linear from 5 × 101 to 5 × 105 copies mL−1, with a high correlation coefficient (R2 = 0.999). This method was then applied to analyze samples taken from Myponga Reservoir, South Australia, during a cyanobacterial bloom event. The results showed good correlations between qPCR techniques and traditional methods, including cell counts determined by microscopy and geosmin concentration measured using gas chromatography (GC) coupled with a mass selective detector (MSD). Results demonstrate that qPCR could be used for tracking geosmin-producing cyanobacteria in drinking water reservoirs. The qPCR assay may provide water utilities with the ability to properly characterize a taste and odor episode and choose appropriate management and treatment options.

Community of Environmental Streptomyces Related to Geosmin Development in Chinese Liquors

Diverse Streptomyces species act as geosmin producers in the Chinese liquor-making process. In this paper, the ecology of these Streptomyces species was analyzed using denaturing gradient gel electrophoresis (DGGE) of amplified Actinobacteria -specified rDNA. The result showed that Streptomyces were widely distributed during Daqu incubation, and multiple processing, geographic, and climate factors can affect their distribution and diversity. The genes associated with geosmin production were characterized in four geosmin-producing Streptomyces strains, all of which were isolated from geosmin-contaminated Daqu. On the basis of this information, a real-time PCR method was developed, enabling the detection of traces of Streptomyces in complex solid-state matrices. The primer was targeted at the gene coding for geosmin synthase (geoA). The real-time PCR method was found to be specific for geosmin-producing Streptomyces and did not show any cross-reactivity with geosmin-negative isolates, which are frequently present in the Chinese liquor-brewing process. Quantification of geoA in the Chinese liquor-making process could permit the monitoring of the level of geosmin producers prior to the occurrence of geosmin production. Comparison of the qPCR results based on the gene encoding geosmin synthase and Actinobacteria-specified rDNA showed that about 1-10% of the Actinobacteria carry the geosmin synthesis gene.

Establishment of a real-time PCR method for quantification of geosmin-producing Streptomyces spp. in recirculating aquaculture systems

Geosmin and 2-methylisoborneol (MIB) have been associated with off-flavour problems in fish and seafood products, generating a strong negative impact for aquaculture industries. Although most of the producers of geosmin and MIB have been identified as Streptomyces species or cyanobacteria, Streptomyces spp. are thought to be responsible for the synthesis of these compounds in indoor recirculating aquaculture systems (RAS). The detection of genes involved in the synthesis of geosmin and MIB can be a relevant indicator of the beginning of off-flavour events in RAS. Here, we report a real-time polymerase chain reaction (qPCR) protocol targeting geoA sequences that encode a germacradienol synthase involved in geosmin synthesis. New geoA-related sequences were retrieved from eleven geosmin-producing Actinomycete strains, among them two Streptomyces strains isolated from two RAS. Combined with geoA-related sequences available in gene databases, we designed primers and standards suitable for qPCR assays targeting mainly Streptomyces geoA. Using our qPCR protocol, we succeeded in measuring the level of geoA copies in sand filter and biofilters in two RAS. This study is the first to apply qPCR assays to detect and quantify the geosmin synthesis gene ( geoA) in RAS. Quantification of geoA in RAS could permit the monitoring of the level of geosmin producers prior to the occurrence of geosmin production. This information will be most valuable for fish producers to manage further development of off-flavour events.

Identification and Expression Analyses of Putative Sesquiterpene Synthase Genes in Phormidium sp. and Prevalence of geoA -Like Genes in a Drinking Water Reservoir

The occurrence of taste and odor problems in drinking water supplies is a widespread phenomenon. From a Saxonian water reservoir we isolated a cyanobacterial species which was classified as Phormidium sp. Under laboratory conditions it produced an earthy-musty smell due to the synthesis of geosmin. The only genes shown to be involved in geosmin biosynthesis are cyc2 and geoA of Streptomyces. Based on the alignment of Cyc2 with a putative sesquiterpene synthase of Nostoc punctiforme, a degenerate primer pair was designed. By PCR, we could amplify two similar genes in Phormidium sp., which we named geoA1 and geoA2. Their expression was studied by reverse transcription-PCR. This revealed that both genes are expressed at 20 degrees C and a light-dark cycle of 12 h. Expression was not detectable at the end of a 24-h dark period. To analyze the prevalence of geoA1 and geoA2 in samples from the phytobenthos, we generated PCR fragments with the same degenerate primer pair. Fifty-five different sequences that might represent geoA variants were obtained. The GC content ranged from 42% to 67%, suggesting that taxonomically very different bacteria might contain such genes.

Impact of the Botrytis cinerea strain and metabolism on (−)-geosmin production by Penicillium expansum in grape juice

Geosmin, an off-flavour of some rotten grapes, has been implicated in wine defects. Botrytis cinerea and Penicillium expansum were the most common among the numerous microorganisms isolated from rotten grapes. P. expansum produces geosmin on model media but not healthy grape juice. However, geosmin synthesis by P. expansum was demonstrated in grape juice and on crushed grapes that had been pre-cultured with certain B. cinerea strains. 34 out of 156 B. cinerea strains ([bot +] phenotype) isolated from the centre of grape bunches were able to induce high geosmin production, up to 494 ng/l, by P. expansum in grape juice. A study of the impact of grape juice composition on geosmin synthesis by P. expansum revealed the importance of nitrogen composition, particularly amino-acid deficiency. Metabolism of amino acids by B. cinerea was shown to be favourable to geosmin synthesis by P. expansum. However, the amino-acid and ammonium concentrations in grape juices pre-cultured with B. cinerea [bot -] and [bot +] strains were very similar implying that other factors are involved as well. Indeed, an ethanol-precipitable fraction, probably a polysaccharide, synthesized by B. cinerea [bot -], but not [bot +] strains, inhibited geosmin production by P. expansum.

Needs and perspectives of odour research in the aquatic sciences

Over more than four decades odour research in the aquatic sciences has increasingly focused on cyanobacteria and the common odour-causing compounds, geosmin and 2-methylisoborneol. Success in future research requires a long-term perspective. Key areas for investigation are secondary metabolites and cyanobacteria, regulatory mechanisms for geosmin and other compounds' synthesis; understanding their spatial and temporal distribution (particularly relating to the food web in a habitat); and molecular mechanisms for liberation of geosmin by microorganisms.

Biosynthetic origin of geosmin in red beets (Beta vulgaris L.).

Geosmin provides the characteristic but sometimes undesirable "earthy" flavor to red table beets. To date, it is not known whether geosmin is a byproduct of beet metabolism or synthesized by soil-borne microorganisms and taken up by the beets during maturation. Analysis of mature beet roots revealed that peels contained 6 times the amount of geosmin compared to the bodies and cores. Sterilized beet seeds were aseptically grown in a basal medium prior to analysis for the presence of geosmin. Using a headspace solid-phase microextraction (HSPME) method, the relative recovery of geosmin from beet seedling extracts was 72.0 +/- 4.2% with (-)-menthone as the internal standard. The presence of geosmin in aseptically grown beet seedlings was confirmed by gas chromatography-mass spectrometry using authentic geosmin as the standard. During aseptic growth, the concentration of geosmin in seedlings remained constant for up to 5 months but increased at 6 months. Geosmin added to the growth medium was not absorbed by the seedlings. These studies support the conclusion that red beets are capable of endogenous synthesis of geosmin.

Environmental and nutritional factors affecting geosmin synthesis by Anabaena SP.

A cyanobacterium isolated from a source-water reservoir during a spring odor and taste episode and identified as Anabaena sp. consistently produced geosmin during laboratory culture on modified BG-11 liquid medium. Maximal geosmin/biomass occurred at 20°C and a light intensity of 17 μE/m 2/s; geosmin/chl a values directly correlated with increasing light intensity ( r 2=0.95, P<0.01). It was concluded that at 20°C, increasing light intensity favors less chl a synthesis and higher geosmin synthesis; at 17 μE/m 2/s, increasing temperature stimulates chl a production (to 25°C) while repressing geosmin synthesis (above 20°C). Nutritional factors promoting biomass, chl a, and geosmin synthesis by Anabaena sp. were also investigated. For cultures grown at 17 μE/m 2/s and 20°C for 20 days, both ammonium-N and nitrate-N generally enhanced the growth of Anabaena sp. Nitrate-N promoted more chl a production ( r 2=0.99) than ammonium-N. Geosmin synthesis was directly correlated with ammonium-N concentrations ( r 2=0.89), with low nitrate-N (123.5 μg/l) favoring maximal geosmin production (2.8 μg/l). Increasing nitrate-N concentrations promoted a three-fold increase in chl a content with geosmin synthesis decreased by two-fold. Geosmin/mg biomass was directly related to ammonium-N concentration; high nitrate-N levels suppressed geosmin production. No geosmin was detected at or below 118 μg phosphate-phosphorus/l. Geosmin, dry weight biomass, and chl a production were correlated with increasing phosphorus (P) concentration ( r 2=0.76, 0.96 and 0.98, respectively). No geosmin was detected when copper was present in growth media at or above 6.92 μg Cu 2+/l (CuSO 4·5H 2O). Dry weight biomass and chl a production were negatively correlated with Cu 2+ ion concentrations.

Off-Flavor Problems in Aquaculture

Foods produced in aquaculture are often more expensive than other sources of animal protein. To capture and maintain market share, they must therefore be of consistent, superior quality. This article reviews off-flavor problems in aquaculture that can adversely affect market demand, with emphasis on off-flavors acquired prior to harvest of the cultured animals. Odorous compounds responsible for preharvest off-flavors may be acquired from the water or diet, although flavor problems of dietary origin are uncommon in aquaculture. The most common preharvest off-flavors in aquaculture products are caused by geosmin and 2-methylisoborneol, two highly odorous, earthy-musty metabolites of aquatic microorganisms. Planktonic cyanobacteria in the order Hormogonales are principally responsible for synthesis of geosmin and 2-methylisoborneol in aquaculture ponds and other eutrophic aquatic ecosystems. The compounds are rapidly absorbed by fish and stored in lipid-rich tissues. Elimination of geosmin and 2-methylisoborneol is slower than uptake, and the rate of elimination is reduced as water temperature decreases and tissue lipid content increases. Management of off-flavors caused by aquatic microorganisms is difficult under commercial conditions. Abatement strategies have focused either on eliminating odor-producing cyanobacteria from culture systems or allowing the taints to depurate naturally by holding animals in clean water. Although flavor problems attributable to pollution are rare in aquaculture, accidental spills of petroleum products occasionally cause off-flavors. Odorous petroleum hydrocarbons are extremely lipophilic, so they are rapidly absorbed from the environment and then slowly eliminated. Management of pollution-related off-flavors is difficult because the accidents leading to flavor problems cannot be predicted.

Lipase-catalyzed Asymmetric Synthesis of Enantiomerically Pure (2S,4aS,8S)-4a,8-Dimethyl-2,3,4,4a,5,6,7,8-octahydro-2-naphthalenol.

Enantiomerically pure (2S,4aS,8S)-(+)-4a,8-dimethyl-2,3,4,4a,5,6,7,8-octahydro-2-naphthalenol (3), a key intermediate in the synthesis of natural geosmin (1), was prepared by enzymatic kinetic resolution. When racemic 3 was submitted to lipase (PS-30)-catalyzed asymmetric acetylation, employing vinyl acetate as the acyl donor, requisite product (+)-3 with a high enantiomeric excess was attained as the remaining alcohol. Recrystallization resulted in an enantiomerically pure sample.

Comparative physiology of geosmin production by Streptomyces halstedii and Anabaena sp.

Geosmin and 2-methylisoborneol (MIB) are microbial metabolites that can cause earthy or musty off-flavors in aquatic food animals as well as seasonal taste and odor episodes in drinking water. This paper compares effects of selected environmental factors on biomass and geosmin production by the actinomycete Streptomyces halstedii and the cyanobacterium Anabaena sp., isolated from an aquaculture pond and from a source-water reservoir, respectively. For S. halstedii, optimal biomass production occurred at pH 6-7 and 30°C, and optimal geosmin synthesis occurred at pH 9 and 35°C. Low concentrations of both nitrate- and ammonium-nitrogen favored geosmin production, with higher concentrations stimulating biomass production. For Anabaena sp., optimal biomass production at 20 days occurred at 15°C and a light intensity of 17 μE/m2/s; optimal geosmin synthesis occurred at 20°C and 17 μE/m2/s. Chlorophyll a (chl a)/biomass varied inversely with light intensity. Maximal geosmin/biomass occurred at 20°C (17 μE/m2/s), and geosmin/chl a varied inversely with temperature. It was concluded that at 20°C, increasing light intensity favors lower chl a and greater geosmin synthesis by Anabaena sp.; at 17 μE/m2/s, increasing temperature stimulates chl a production (to 25°C) but represses geosmin synthesis (above 20°C).

Comparative physiology of geosmin production by Streptomyces halstedii and anabaena sp.

Geosmin and 2-methylisobomeol (MIB) are microbial metabolites that can cause earthy or musty off-flavors in aquatic food animals as well as seasonal taste and odor episodes in drinking water. This paper compares effects of selected environmental factors on biomass and geosmin production by the actinomycete Streptomyces halstedii and the cyanobacterium Anabaena sp., isolated from an aquaculture pond and from a source-water reservoir, respectively. For S. halstedii, optimal biomass production occurred at pH 6-7 and 30°C, and optimal geosmin synthesis occurred at pH 9 and 35°C. Low concentrations of both nitrate- and ammonium-nitrogen favored geosmin production, with higher concentrations stimulating biomass production. For Anabaena sp., optimal biomass production at 20 days occurred at 15°C and a light intensity of 17 µE/m2/s; optimal geosmin synthesis occurred at 20°C and 17 µE/m2/s. Chlorophyll a (chi a)/biomass varied inversely with light intensity. Maximal geosmin/biomass occurred at 20°C (17 µE/m2/s), and geosmin/chl a varied inversely with temperature. It was concluded that at 20°C, increasing fight intensity favors lower chi a and greater geosmin synthesis by Anabaena sp.; at 17 µE/m2/s, increasing temperature stimulates chi a production (to 25°C) but represses geosmin synthesis (above 20°C).

Identification of odorous compounds from Streptomyces avermitilis

The production of avermectin-type antibiotics and the odours of a strain ofStreptomyces avermitilis was investigated. With the increasing production of the total of avermectins (0–600 ug/ml), the synthesis of geosmin is also enhanced by more than one order of magnitude and, in contrast, the production of homologues of oxolone (dihydrofuranone) decreases in a similar range. In addition to these compounds, more than twenty other volatile chemical products have been identified by means GC-MS,i.e. pyrazine derivatives, acetoin and its homologues, aromatic esters, furan derivatives,etc.).

Geosmin Production and Excretion in a Planktonic and Benthic Oscillatoria

Geosmin and carotenoid contents for the benthic Oscillatoriabrevis and the planktonic Oscillatoriabornetii were examined under different growth conditions. The results obtained when the effects of light intensity, light quality and growth rate were examined indicate that geosmiri could be a by-product of the isoprenoid pathway leading to chlorophyll and carotenoids in Oscillatoria and not a overflow product of this pathway as earlier suggested. The connection between geosmin and pigment synthesis suggests that geosmin production by a cyanobacterial bloom might be lowest at the surface and increase with depth in the water column. Geosmin is also found to be an intracellular compound, which is released upon death and decay of the organisms.

Accumulation and Release of Geosmin during the Growth Phases of Anabaena circinalis (Kutz.) Rabenhorst

Anabaena circinalis (Kutz.) Rabenhorst was isolated during a taste and odor episode characterized by high concentrations of geosmin, with raw water containing up to 45 ng geosmin/L. This species has been successfully cultured with sustained geosmin synthesis on both sterile river water and defined medium. Gas chromatography of cellular extracts and closed-loop stripping of growth media indicated that this organism produces geosmin and not 2-methylisoborneol (MIB). With cultures we examined the changes in cell-associated geosmin, chlorophyll and released geosmin during exponential and stationary growth phases of Anabaena. Cell-associated geosmin samples were collected daily from cultures and filtered onto a polycarbonate filter, extracted in acetone, and quantified by capillary gas Chromatograph using flame ionization detection. Media-associated geosmin was quantified from algae-free filtrates from each culture concentrated by closed-loop stripping and analyzed as above. Cell-associated geosmin averaged at 8 × 10−6 ng geosmin/cell in the exponential phase and dropped to 2.5 × 10−6 ng geosmin/cell in the stationary phase of growth. The loss in cell-associated geosmin was accompanied by an increase in geosmin released into the media. Media-associated geosmin reached 12 ng/mL in the stationary phase. Apparently cell lysis in the stationary phase caused the release of cell-associated geosmin into the media. Cell-associated geosmin was closely correlated with filament number (average r2 from 4 experiments =0.96) during the exponential growth phase and was correlated with chlorophyll a (average r2 from 3 experiments =0.95).

Effects of farnesol and the off-flavor derivative geosmin on Streptomyces tendae

Effects of the sesquiterpene farnesol (3,7,11-trimethyl-2,6,10-dodecatrien-1-ol) and the sesquiterpene derivative geosmin (1,10-trans-dimethyl-trans-9-decalol) were investigated in a geosmin-producing actinomycete, Streptomyces tendae. Exposure to 300 microM farnesol reduced biomass (fresh matter) accumulation by 97% compared with biomass accumulation by controls, whereas an equal amount of geosmin did not affect biomass accumulation. Increasing exposure to farnesol corresponded with reduced optical density of the culture, reduced levels of geosmin, and reduced metabolic heat production compared with controls, while exogenous geosmin did not affect these parameters. Geosmin dissipated from unioculated medium more rapidly than farnesol, indicating that in addition to the lower toxicity of geosmin, the actual exposure to geosmin over time may be less than exposure to an equal amount of farnesol. Cultures grown on Actinomyces-B medium contained 99.5% less geosmin and were more sensitive to farnesol than those grown on Hickey-Tresner medium, indicating that geosmin synthesis was associated with reduced sensitivity to farnesol. Consumption of farnesyl moieties during geosmin synthesis may reduce the potential for farnesol-induced inhibition of growth and metabolism.