Causative microorganisms for musty odor occurrence in the eutrophic Lake Kasumigaura

The musty odor phenomenon in Lake Kasumigaura, Japan (a eutrophic lake that is used for various purposes, including water supply source) was examined. The causative microorganisms responsible for the production of the odor compounds, geosmin and/or 2-methylisoborneol (MIB), and the odor release mechanism were studied in vitro and in situ. The numbers of the filamentous P. tenue in the lake water column were closely correlated with the concentration of MIB, but not geosmin. The isolated monoxenic P. tenue in culture produced only MIB. Geosmin concentration was well correlated with the number of actinomycetes in the sediment. Forty isolates of actinomycetes from the sediment around the water supply intake tower produced both geosmin and MIB in culture. Furthermore, the average ratio of production of geosmin to MIB was 1.40–1.0. Actinomycetes in the sediment played an important role in geosmin production. We concluded that geosmin and MIB from actinomycetes in the sediment, and MIB from P. tenue in the water column were jointly responsible for the severe musty odor occurrence of the waters supply source at Lake Kasumigaura.     

Mutagenesis by ethidium bromide and N-methyl-N′-nitro-N-nitrosoguanidine in off-flavour compound producing strains ofStreptomyces

Various species of actinomycetes and cyanobacteria can impart earthy/musty off-flavours to drinking water supplies and to pond-raised fish and other aquatic food animals. The genetic determinants for production of the most common off-flavour compounds [geosmin and 2-methylisoborneol (MIB)] have not been extensively studied. An attempt has been rrlade to study the genetics of production of these compounds was demonstrated by DNA-curing analysis. The effects of two curing agents [ethidium bromide (EB) and N-methyl-N′-nitro-N-nitrosoguanidine (NTG)] on tha loss of linear plasmid DNA and generation of bald mutants (no aerial mycelia) inStreptomyces halstedii andStereptomyces violaceusniger which produce geosmin and MIB, respectively, were observed. Production of earthy/musty odour was not eliminated, but was reduced by 55–95% in the plasmid cured strain. Data suggested that off-flavour production is likely chromosomally-encoded in theseStreptomyces isolates.     

A SYSTEMATIC STUDY OF GIGAR-TINACEAE FROM PACIFIC NORTH AMERICA BASED ON MOLECULAR AND MORPHOLOGICAL EVIDENCE

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.     

Isolation of bacteria capable of growth with 2-methylisoborneol and geosmin as the sole carbon and energy sources

Using a relatively simple enrichment technique, geosmin and 2-methylisoborneol (MIB)-biodegrading bacteria were isolated from a digestion basin in an aquaculture unit. Comparison of 16S rRNA gene sequences affiliated one of the three isolates with the Gram-positive genus Rhodococcus, while the other two isolates were found to be closely related to the Gram-negative family Comamonadaceae (Variovorax and Comamonas). Growth rates and geosmin and MIB removal rates by the isolates were determined under aerated and nonaerated conditions in mineral medium containing either of the two compounds as the sole carbon and energy source. All isolates exhibited their fastest growth under aerobic conditions, with generation times ranging from 3.1 to 5.7 h, compared to generation times of up to 19.1 h in the nonaerated flasks. Incubation of the isolates with additional carbon sources caused a significant increase in their growth rates, while removal rates of geosmin and MIB were significantly lower than those for incubation with only geosmin or MIB. By fluorescence in situ hybridization, members of the genera Rhodococcus and Comamonas were detected in geosmin- and MIB-enriched sludge from the digestion basin.     

Effects of light and temperature on the odor production of 2-methylisoborneol-producing Pseudanabaena sp. and geosmin-producing Anabaena ucrainica (cyanobacteria)

Frequent off-flavor events caused by geosmin and 2-methylisoborneol (MIB) have attracted research on the main producers, cyanobacteria. This study evaluated the effects of light and temperature on the odor production of MIB-producing Pseudanabaena sp. Lauterborn and geosmin-producing Anabaena ucrainica (Schhorb.) Watanabe. The maximum MIB production and lowest growth rate (indicated by the chlorophyll a (Chl a)) were observed at 35 °C compared with that at 10 °C and 25 °C. Cultures grown under a light intensity of 60 μmol photons m⁻² s⁻¹ demonstrated the highest MIB production and minimum growth rate, whereas the minimum MIB production and maximum growth rate were obtained under 10 μmol photons m⁻² s⁻¹. Similar patterns were observed for geosmin production. A. ucrainica had the highest geosmin production and lowest Chl a concentration under 10 °C and 60 μmol photons m⁻² s⁻¹. Moreover, greater proportions of geosmin and MIB were released into extracellular under growth-inhibiting temperatures and light intensities. An inverse correlation between odor production and the cell growth rate was suggested, and this relationship may reflect the competition for substrates of odor and Chl a synthesis. Thus, the accumulation of geosmin and MIB was probably the result of decreased cellular metabolic activity in cyanobacteria.     

Abundance of actinobacteria and production of geosmin and 2-methylisoborneol in Danish streams and fish ponds

Occurrence of the odours geosmin and 2-methylisoborneol (MIB) in freshwater environments indicates that odour-producing organisms are commonly occurring. In the present study, we assumed actinomycetes to be a major source of the odours. Seasonal concentrations of odours and abundance of Actinobacteria, which includes actinomycetes and other G+ and high GC bacteria, were determined in one oligotrophic and two eutrophic freshwater streams, as well as in aquacultures connected to these streams, in Denmark. Concentrations of geosmin and MIB ranged from 2 to 9 ng l(-1) and were lowest in the winter. Passage of stream water in the aquacultures increased the amount of geosmin and MIB by up to 55% and 110%, respectively. Densities of actinobacteria were determined by fluorescence in situ hybridization with catalyzed reporter deposition (CARD-FISH) technique and were found to make up from 4 to 38 x 10(7) cells l(-1), corresponding to 3-9% of the total bacterial populations. The lowest densities of actinobacteria occurred in the winter. Filamentous bacteria targeted by the FISH probe made up about 2.7-38% (average was 22%) of the actinobacteria and were expected to be actinomycetes. Combined microautoradiography and CARD-FISH demonstrated that 10-38% (incorporation of 3H-thymidine) and 41-65% (incorporation of 3H-leucine) of the actinobacteria were metabolically active. The proportion of active actinobacteria increased up to 2-fold during passage of stream water in the aquacultures, and up to 98% of the cells became active. Sequencing of 16S rRNA genes in 8 bacterial isolates with typical actinomycete morphology from the streams and ponds demonstrated that most of them belonged to the genus Streptomyces. The isolated actinomycetes produced geosmin at rates from 0.1 to 35 aggeosmin bacterium(-1)h(-1). MIB was produced at similar rates in 5 isolates, whereas no MIB was produced by three of the isolates. Addition of the odours to stream water demonstrated that indigenous stream bacteria were capable of reducing the odours, and that enrichment with LB medium stimulated the degradation. Our study shows that bacterial communities in freshwater include geosmin- and MIB-producing actinobacteria. However, the mechanisms controlling production as well as degradation of the odours in natural waters appear complex and require further research.     

Increasing the sensitivity of piezoelectric odour sensors based on molecularly imprinted polymers

The molecular imprinting technique has been used to create sensors with a predetermined selectivity for molecules in the gas phase. Based on previous results, piezoelectric quartz crystals were first coated with a layer of nylon, to provide increased sensitivity, and then a layer of a polymer imprinted with 2-methylisoborneol (MIB) was applied to endow the device with selectivity. The response of the 'imprinted' sensor was significantly higher than the response of the 'non-imprinted' sensor at MIB concentrations above 10 ppb. Our new sensor is thus 20 times more sensitive than our previous device. At concentrations greater than 10 ppb, the response of the 'non-imprinted' sensor to geosmin, another odorant often accompanying MIB, was significantly higher than the response of the 'imprinted' sensor. A number of other odorants were examined and, as in our previous work, the responses of the 'non-imprinted' sensors were found to be to greater than the responses of the 'imprinted' sensors.     

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.     

Dehydration of the off-flavor chemical 2-methylisoborneol by the R-limonene-degrading bacteria Pseudomonas sp. strain 19-rlim and Sphingomonas sp. strain BIR2-rlima

The terpene 2-methylisoborneol (MIB), a major cause of off-flavor in farm-raised catfish and drinking water, is transformed by various different terpene-degrading bacteria. Two of these, the R-limonene-degrading strains Pseudomonas sp. 19-rlim and Sphingomonas sp. BIR2-rlima, dehydrated MIB with the formation of odorless metabolites 2-methylenebornane and 4-methylcamphene. These metabolites which have a structural resemblance to camphor, could be further transformed by camphor-degrading bacteria to more oxidized products. The bacterial dehydrations demonstrated here may have application in removing MIB where it is a problem.     

First report of geosmin and 2-methylisoborneol (2-MIB) in Dolichospermum and Oscillatoria from Vietnam

Limnology - Cyanobacteria are the major producers of the taste and odour compounds geosmin and 2-methylisoborneol (2-MIB) in drinking water and fish worldwide. In recent years, the outbreaks of...     

Studies on the earthy-musty odours in natural water (IV). Mechanism of earthy-musty odour production of actinomycetes

Many reservoirs for water supply have been troubled with earthy-musty odour compounds—2-methylisoborneol (2-MIB) and geosmin. Both of these compounds are terpenoid and related to the metabolite of l-methionine. An experiment using [CD₃]methionine and [¹⁴CH₃]methionine showed that the C-2 methyl group in 2-MIB originate from l-methionine. In the incubation experiment with 10–1000 mg/l of l-methionine, 2-MIB and geosmin appeared in the earlier stages, in greater amounts than in the control. The maximum production of 2-MIB and geosmin increased considerably in the experiments with 10 and 100 mg/l of l-methionine. The effective time for l-methionine addition was after 1 d. Additions after 3 and 5 d were similar to the control. In the incubation experiment with 10–1000 mg/l of folic acid, 2-MIB and geosmin increased only during the 1000 mg/l addition. There seems to be little doubt that l-methionine takes part in the metabolism of 2-MIB and geosmin.     

Origin of (−)-Geosmin on Grapes: On the Complementary Action of Two Fungi, <Emphasis Type="Italic">Botrytis Cinerea</Emphasis> and <Emphasis Type="Italic">Penicillium Expansum</Emphasis>

One of the consequences of rot on grapes is the development of volatile compounds giving fungal, mouldy or earthy odours. Among these compounds, (−)-geosmin (trans-1,10-dimethyl-trans-9-decalol), a powerful aromatic compound with an earthy smell is a persistent defect in grape juice and wines made with at least partially rotten grapes. A microbiota analysis of rotten grapes containing (−)-geosmin was carried out on sites from four French regions from 1999 to 2002, to clarify the involvement in geosmin appearance of Streptomyces spp. and Penicillium spp., two types of microorganisms present on grape, that are known for their ability to produce geosmin. In earthy grapes, Botrytis cinerea was largely present. Different species of Streptomyces were also isolated, but their pH sensitivity was an extremely limiting parameter for their development on grape juice, grapes or stem, and consequently for their potentiality to generate geosmin in the vineyard. Penicillium expansum, producing geosmin on a model medium, was omnipresent. Penicillium carneum, which is also a geosmin producer, was represented by a single colony during the 4 years of this study. P. expansum alone was able to produce geosmin on a model medium but not on grapes. However, after 7 days’ pre-culture of some B. cinerea strains on grape juice, this juice became favourable to geosmin production by P. expansum. We demonstrated the necessary and complementary action of B. cinerea and P. expansum in geosmin production in grape juice and in crushed grape berries.     

Effects of temperature and light on the growth and geosmin production of Lyngbya kuetzingii (Cyanophyta)

The effects of temperature and light on the growth and geosmin production of Lyngbya kuetzingii were determined. Of the three temperatures tested, 10, 25 and 35°C, the maximal geosmin concentration and geosmin productivity were yielded at 10°C, while the highest chl a production was observed at 25°C. In the studies on light intensity, the maximal geosmin concentration and geosmin productivity were observed at 10 μmol m⁻² s⁻¹, while the highest chl a production was at 20 μmol m⁻² s⁻¹. It was suggested that more geosmin was synthesized with lower chl a demand. Meanwhile, the relative amounts of extra- and intracellular geosmin were investigated. Under optimum growth conditions (20 μmol m⁻² s⁻¹, 25°C; BG-11 medium), the amounts of extracellular geosmin increased as the growth progressed and reached the maximum in the stationary phase, while the intracellular geosmin reached its maximum value in the late exponential phase, and then began to decline. However, under the low temperature (10°C) or light (10 μmol m⁻² s⁻¹) conditions, more intracellular geosmin was synthesized and mainly accumulated in the cells. The proportions of extracellular geosmin were high, to 33.33 and 32.27%, respectively, during the stationary phase at 35°C and 20 μmol m⁻² s⁻¹. It was indicated that low temperature or light could stimulate geosmin production and favor the accumulation of geosmin in cells, while more intracellular geosmin may be released into the medium at higher temperatures or optimum light intensity.     

INTRACELLULAR LOCALIZATION OF THE TASTE/ODOR METABOLITE 2-METHYLISOBORNEOL IN OSCILLATORIA LIMOSA (CYANOPHYTA)1

The monoterpene derivative, 2-methylisoborneol (MIB), is produced by many blue-green algae and often is responsible for the “musty” taste/odor in aquaculturally raised finfish and potable water supplies. Although previous researchers have suggested that taste/odor metabolites are partitioned among cell constituents and that coregulation with pigment biosynthesis occurs, no structural evidence for these hypotheses exists. MIB was localized in cells of Oscillatoria limosa (Roth) Agardh at the ultrastructural level using standard gold-labeled antibody techniques. There was no apparent relationship between age of the cells and MIB synthesis; cells that were and were not undergoing active division had similar amounts of MIB label. There was no consistent partitioning of MIB label within cells. However, occasionally, specific label was observed along photosynthetic lamellae, suggesting a potential linkage of MIB synthesis and/or binding to the pigment systems.     

微波蒸馏-顶空固相微萃取-气质联用检测鱼体中土霉昧化合物

采用微波蒸馏-顶空固相微萃取-气质联用检测鱼体中常见的两种土霉味化合物,即2-甲基异茨醇(2-MIB)和土腥素(Geosmin).研究并优化了微波蒸馏萃取过程的关键参数(微波蒸馏时间、载气流量),结果表明微波蒸馏6min、载气流70 mL/min为土霉味化合物微波蒸馏萃取的最佳条件.在此优化的条件下,土霉味化合物能够充分地从鱼体中蒸馏出来,再采用顶空固相微萃取的方法使馏分中的土霉味化合物吸附于纤维涂层上,将其在250℃高温下解吸,并用GC-MS分析.基于此测定方法,鱼肉中2-甲基异茨醇和土腥素的检测限均达到0.1μg/kg,且其在1-20μg/kg的范围内线性关系良好,相关系数R分别达到0.987、0.995.因此,用该方法分析鱼体中痕量的(ppb级)的土霉味化合物,结果可靠.     

Variation of geosmin content in Anabaena cells and its relation to nitrogen utilization

The addition of the proper amount of ammonium to the culture medium containing nitrate as nitrogen source enhanced the growth rate of Anabaena viguieri. The amount of geosmin produced by these cells varied with the concentrations of ammonium added. A negative correlation between the amount of geosmin produced and of the growth rate of cells was revealed. This was also found in cells grown on various forms of nitrogen sources. Without supply of any nitrogen compound, this organism is capable of fixing gaseous nitrogen, and under these conditions the cells grew relatively slowly. However, they produced more geosmin (per unit protein mass) than cells grown in the presence of combined nitrogen. The isolation of heterocysts, in which nitrogen was fixed, showed that these cells produced higher amounts of geosmin than vegetative cells. The possible relation of nitrogen assimilation to the production of geosmin in the cells was discussed.     

Effects of 2-methylisoborneol (MIB), and ethanol on the expression and activity of cytochrome P450s from the channel catfish

Injection of 1 mg kg⁻¹ of 2-methylisoborneol (MIB) caused an increase in a CYP1A-like protein of approximately 56 kDa from the kidneys of juvenile channel catfish as determined by western blot analysis using antibodies raised against trout CYP1A1. Ethoxyresorufin O-deethylase activity from kidneys of MIB-injected fish was significantly elevated as compared to ethanol-injected controls. Static exposure of juvenile channel catfish to 10 ppm MIB caused a statistically significant induction of a CYP2K-like protein of approximately 53 kDa from the livers of treated catfish as determined by western blot analysis using antibodies raised against trout CYP2K1. Treatment of juvenile channel catfish with ethanol (1 ml kg⁻¹) reduced the expression of one kidney and two constitutive liver P450s, while increasing another kidney form. There was no difference in carbon tetrachloride-induced lipid peroxidation in livers after ethanol treatment. Thus, MIB and ethanol affect the expression of at least three P450 isoforms in channel catfish tissues.     

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.     

Synthesis and release of chondroitin sulphate and heparan sulphate proteoglycans from mouse macrophagesin vitro

Macrophages were obtained from the mouse peritoneal cavity and culturedin vitro. The cells were exposed to³⁵S-sulphate for 20 h, and labelled proteoglycans were recovered from both medium and cell fractions by sodium dodecylsulphate solubilization. The cell fraction contained both proteoglycans and glycosaminoglycans, whereas only intact proteoglycans could be recovered from the medium fraction. ³⁵S-Glycosaminoglycans isolated from cell and medium fractions by papain digestion were shown to contain approximately 25% heparan sulphate and 75% galactosaminoglycans comprising 55% chondroitin sulphate and 20% dermatan sulphate. The galactosaminoglycans were shown by paper chromatography to contain more than 95% 4-sulphated units. Pulse-chase experiments showed that approximately 80% of the cell-associated material was released within 6 h of incubation.³⁵S-Proteoglycans released did not bind to the macrophages, but were recovered in a soluble form from the culture medium.Abbreviations CSPG chondroitin sulphate proteoglycan - HSPG heparan sulphate proteoglycan - SDS sodium dodecylsulphate - DME Dulbecco's Minimum Essential Medium - GAG glycosaminoglycan     

Geosmin production in the cyanobacterium Oscillatoria brevis

The cyanobacterium Oscillatoria brevis (Kütz.) Gom., strain NIVA CYA 7, was used to investigate how geosmin production is related to the synthesis of chlorophyll a, phycobiliproteins and β-carotene under nitrogen (NH ₄ ⁺ ) and light limiting conditions. Chemostat samples were used to inoculate batch cultures that were treated with inhibitors of isoprenoid synthesis, norflurazon and dimethazone, and gabaculine that inhibits tetrapyrrole synthesis. Dimethazone decreased and norflurazon increased geosmin production under light limited conditions, as was expected due to their sites of action in the isoprenoid pathway. This effect was not so pronounced in nitrogen limited cultures due to the additional effect of increasing nitrogen deficiency during the experimental period. Norflurazon was the only inhibitor that uncoupled geosmin production completely from β-carotene formation which indicates a strikt coupling between geosmin and β-carotene biosynthesis. From the observed increase of geosmin production relative to pigment synthesis after norflurazon treatment it was suggested that isoprenoid precursors are directed to geosmin synthesis when the demand for pigment precursors is very low. Within the framework of this study the data strongly support the hypothesis of geosmin formation via the isoprenoid pathway in Oscillatoria brevis as was found for actinomycetes. This research was performed at the Department of Microbiology, University of Amsterdam, with financial support provided by the royal Norwegian Ministry of Foreign Affairs and the Royal Norwegian Council for Scientific and Industrial Research